1H NMR spectra were recorded on a Jeol Eclipse 400 MHz spectrometer in CDCl3 with tetramethylsilane as reference by Acorn NMR Spectroscopy Service (Livermore, CA). UV spectra were recorded on a Hewlett-Packard Spectrophotometer. HPLC analysis was performed on a Varian Prostar instrument with a C-18 reversed phase column (Varian, Walnut Creek, CA). Resolution of racemic TFD-etomidate was performed on Chiracel OD-H analytical column using hexane; isopropanol 95/5 and UV detection at 220 nm. Tritiation by esterification with labeled ethanol was performed by American Radiochemical (St. Louis, MO). Mass spectral analyses were performed by AnaSpec, Inc (San Jose, CA). Elemental analyses were performed by Galbraith Laboratories (Knoxville, TN); all compounds were >95% pure. Optical rotation measurements were performed by Organix Inc (Woburn, MA) at 20°C on Jasco P-1010 polarimeter in a 10 cm cell at concentrations expressed as g/100 ml.
To a solution of 4-bromo-α-methylbenzyl alcohol (18.1 g, 0.09 mol) and t-butyl-dimethylsilyl chloride (14.65 g, 0.099 mol) in anhydrous dichloromethane (100 mL) at room temperature under argon was added drop-wise a solution of DBU (15.8 g, 15.5 mL, 0.104 mol) in anhydrous dichloromethane (100 mL). After stirring at room temperature for 1 h, the mixture was extracted successively with water (200 mL), 0.1 M HCl (200 mL), twice with saturated sodium bicarbonate solution (200 mL) and water (200 mL). The organic layer was separated and dried over sodium sulfate. After rotary evaporation, the product was purified by silica gel chromatography with hexane to yield 24.8 g (88% yield) of a colorless, liquid silane derivative 2. 1H NMR spectrum: (CDCl3) δ 7.41 and 7.20 (4H, AA//BB/ phenyl), 4.81 (q, 1H, methine), 1.37 (d, 3H, methyl), 0.90 (s, 9H, methyl), 0.01 (d, 6H, methyl). Calcd. for C14H23BrOSi: C, 53.33%; H, 7.53%. Found: C, 53:88%; H, 7.22%.
The fluoroketone was synthesized following the procedure described by Fishwick et al. 28
that converts bromo compounds to fluoroketone in high yield. The silyl-protected bromo compound 2
(23.7 g, 75 mmol) in anhydrous THF (200 m l) was cooled to −78°C in ether/dry ice bath and treated under argon by drop-wise addition of n-butyl lithium (54.6 mL of 1.6 M solution in hexane) over a period of 1 h. After stirring the solution at −78°C for 75 min, a solution of diethyl trifluoroacetamide 916. 9 g, 99.4 mmol) in anhydrous THF (50 mL) was added drop-wise over a period of 1 h. The mixture was stirred at −78°C for 75 min. The reaction mixture was quenched by adding 200 mL of saturated ammonium chloride solution without warming. The mixture was brought to room temperature overnight. Ether (400 mL) was added and the mixture extracted twice with water (200 mL). The ether layer was dried over magnesium sulfate. After evaporation, the residue was taken up in hexane and applied to a column of silica gel equilibrated with hexane. Elution with 10% dichloromethane/hexane (10:90) gave 21.5 g (89% yield) of the fluoroketone 3
H NMR spectrum: (CDCl3
) δ 8.04 and 7.51 (4H, AA/
phenyl), 4.94 (q, 1H, methine), 1.41 (d, 3H, methyl), 0.92 (s, 9H, methyl), 0.01 (d, 6H, methyl). Calcd. for C16
Si: C, 57.81%; H, 6.97%. Found: C, 58.09%; H, 7.14%.
(1-(4-(1-(tert-Butyl)dimethylsilyloxy)ethyl)phenyl)-2,2,2-trifluroethanone oxime (4)
The fluoroketone was converted to oxime as described by Shih & Bayley 29
. A mixture of the fluoroketone 3
(15.7 g, 48.9 mmol), hydroxylamine hydrochloride (4.1 g, 58.6 mmol) and anhydrous pyridine 925 mL) was heated at 75°C for 4 h. Ethanol (12 mL) was added and the mixture heated at 60°C for 2h. The solvent was removed by rotary evaporation, the residue taken in ether (200 mL) and extracted three times with 200 mL portions of water. After drying the ethereal layer over magnesium sulfate, the solvent was removed by rotary evaporation and the residue applied to a column of silica gel, equilibrated with hexane/dichloromethane (75:25). Elution with dichloromethane gave the oxime 4
H NMR spectrum: (CDCl3
) δ 7.45 and 7.44 (4H, AA/
phenyl), 4.92 (q, 1H, methine), 1.42 (d, 3H, methyl), 0.92 (s, 9H, methyl), 0.01 (6H, methyl). Calcd. for C16
Si: C, 55.31%; H, 6.96%; N, 4.03%. Found: C, 55.33%; H, 7.25%; N, 4.16%.
(1-(4-(1-(tert-Butyl)dimethylsilyloxy)ethyl)phenyl)-2,2,2-trifluroethanone o-tosyl oxime (5)
To a stirred, ice-cooled solution of the oxime 4 (11.7 g, 34.8 mmol), triethylamine (4.3 g, 5.9 mL, 42.2 mmol) and dimethyl aminopyridine (3868 mg, 3.7 mmol) in anhydrous dichloromethane (50 mL) was slowly added tosyl chloride (7.6 g, 39,8 mmol). After the addition was complete, the mixture was stirred at room temperature for 30 min, extracted three times with 50 mL portions of water, the organic layer dried over magnesium sulfate, the solvent removed by rotary evaporation and the residue purified on a column of silica gel, equilibrated with 20% dichloromethane in hexane. Elution with 50% dichloromethane in hexane yielded the tosylate 5 (13. 7 g, 94%). 1H NMR spectrum: (CDCl3) δ 7.90 (m, 2H, phenyl), 7.40 (m, 6 H, phenyl), 4.90 (q, 1H, methine), 2.48 (s, 3H, methyl), 1.41 (d, 3H, methyl), 0.92 (s, 9H, methyl), 0.01 (6H, methyl). Calcd. for C23H30F3NO4Si: C, 55.07%; H, 6.03%. Found: C, 54.92%; H, 6.03%.
A solution of the tosylate 5 (13.6 g, 30 mmol) in anhydrous ether (8 mL) was added to liquid ammonia (25 mL) at −78°C and stirred at −45 to −35°C for 6 h. The solution was slowly allowed to come to room temperature and stirred overnight. The mixture was taken in ether (75 mL), filtered, and the precipitate washed with ether. Rotary evaporation of the ethereal solution gave 9.36 g of a viscous residue of the diaziridine 6 which was taken to the next step without further purification
To a mixture of the crude diaziridine 6 (9.36 g, 28 mmol) and triethylamine (5 mL) in dichloromethane (20 mL), cooled in ice, was added solid iodine in small portions until a brownish color persisted (4.3 g iodine required). The mixture was diluted with ether (400 mL) and 10 % aqueous citric acid (200 mL). Sodium metabisulfite was added until the color of iodine was discharged. The ethereal layer was separated, washed with water and dried with magnesium sulfate. The ether was removed by rotary evaporation and the crude product purified on a silica gel column equilibrated with hexane. A faintly pale colored, liquid diazirine 7 (5.51 g, 59 %) was obtained. 1H NMR spectrum: (CDCl3) δ 7.35 and 7.13 (4H, AA//BB/ phenyl), 4.84 (q, 1H, methine), 1.38 (d, 3H, methyl), 0.9 (s, 9H, methyl), 0.01 (6H, methyl). Calcd. for C16H23F3N2OSi: C, 55.79%; H, 6.73%; N, 8.13%. Found: C, 55.26%; H, 6.79%; N, 8.30%.
The tert-butyldimehylsilyl protecting group of the diazirine 7
was replaced by a bromo group by the procedure of Aizpurua et al. 30
. A solution of the diazirine 7
(5.5 g, 16.5 mmol) in anhydrous dichloromethane (25 mL) was added to a suspension of triphenylphosphine dibromide (7.7 g, 18.1 mmol) in anhydrous dichloromethane (40 mL). The mixture was stirred at room temperature for 15h. The solution was diluted with dichloromethane (125 mL), extracted twice with 100 mL water, and dried over sodium sulfate. The product was purified on a silica gel column, equilibrated with hexane to yield the bromo compound 8
(4.2 g, 87% yield). Because of its instability, the bromo compound was taken immediately to the next step without further purification.
A solution of the bromo compound 8 (4.1 g, 14 mmol) in methanol (100 mL) was saturated with ammonia and the solution kept for 72 h in a closed vessel. The solution was rotary evaporated and the residue taken up in ether (100 mL) and shaken with 1 M NaOH to breakup any hydrochloride. The ether layer was separated, washed with brine, and dried over sodium sulfate. The crude product was purified by chromatography on a column of silica gel, equilibrated with 10% ether in dichloromethane. Elution with the equilibration solvent containing 10% methanol provided a pale colored amine 9 (2.1 g, 65% yield). 1H NMR spectrum: (CDCl3) δ 7.39 and 7.16 (4H, AA//BB/ phenyl), 4.12 (q, 1H, methine), 1.61 (s, 2H, amino), 1.36 (d, 3H, methyl). Calcd. for C10H10F3N3: C, 52.40%; H, 4.40%; N, 18.33%. Found: C, 52.03%; H, 4.48%; N, 17.66%.
Ethyl 2-(1-(4-(3-((trifluoromethyl)-3H-dazirin-3-yl)phenyl)ethylamino)butanethanoate (10)
To a solution of the amine 9 (2 g, 8.9 mmol) and triethylamine (1.23 mL, 8.9 mmol) in anhydrous dimethylformamide (8 mL), cooled in an ice bath, was slowly added ethyl chloroacetate (1.085 g, 948 μL, 8.86 mmol). After the addition was complete, the ice bath was removed and the solution stirred at room temperature for 48 h. The mixture was diluted with ether (30 mL), filtered, and the precipitate washed with ether. The ethereal layer was extracted three times with 30 mL portions of ether and dried over magnesium sulfate The crude product was purified on a silica gel column, equilibrated with dichloromethane. Washing with the equilibrium solvent followed by elution with the equilibration solvent containing 10% ether yielded the pale liquid glycine ester derivative 10 (2.26 g, 81 % yield). 1H NMR spectrum: (CDCl3) δ 7.36 and 7.2 (4H, AA//BB/ phenyl), 4.15 (q, 2H, methylene), 3.81 (q, 1H, methine), 3.21 (q, 2H, methylene), 1.35 (d, 3H, methyl), 1.24 (t, 3H, methyl). Calcd. for C14H16F3N3O2: C, 53.33%; H, 5.11%; N, 13.33%. Found: C, 52.76%; H, 5.22%; N, 12.72%.
Ethyl 2-(N- (1-(4-(3-(trifluromethyl)-3H-diazirin-3-yl)phenyl)ethyl)methanamido)ethanoate (11)
Formylation of 10
was performed with formic anhydride by the procedure of Waki & Meienhofer 31
. A solution of 2 M formic acid in dichloromethane (8 mL) was added drop-wise with stirring and with cooling by ice bath to a solution of diisopropylcarbodiimide 1.01 g, 8 mmol) in anhydrous dichloromethane (10 mL). After stirring for 5 min, the mixture was added over a period of 30 min to an ice-cooled solution of the glycine ester 10
(1.26 g, 4 mmol) in anhydrous pyridine (10 mL). The solution was stirred at ice-bath temperature for 4 h. After removal of the solvent by rotary evaporation, the residue was suspended in ether and the insoluble residue removed by centrifugation. The crude product was purified by chromatography on a silica gel column, equilibrated with dichloromethane. Elution with the equilibration solvent, containing 10% ether yielded 1.3 g (95%) of pale colored, viscous formyl derivative 11
H NMR spectrum showed splitting of signal in a ratio of 0.74:0.26, especially in formyl, methyl and methylene protons adjacent to the nitrogen atom, indicating the presence of cis-trans isomers in that ratio. 1
H NMR: (CDCl3
) δ 8.41 and 8.18 (1H, formyl), 7.35 and 7.2 (4H, AA/
phenyl), 5.81 and 4.87 (q, 1H, methine), 4.10 and 4.05 (q, 2H, methylene), 4.15 (m, 2H, methylene), 1.57 (m, 3H, methyl;), 1.20 (m, 3H, methyl). Calcd. for C15
: C, 52.48%; H, 4.70%; N, 12.24%. Found: C, 52.75%; H, 4.77%; N, 11.68%.
2-Mercapto-1-(1-4-(3-((trifluoromethyl)-3H-diazirin-3-yl)phenyl)ethyl)-1H-imidazol-5-yl propanoate (12)
Ring closure of the formyl compound 11
to mercapto imidazole derivative and subsequent oxidative desulfuration was performed by the procedure of Jones et al. 32
as modified by Godefroi et al. 1
. Sodium ethoxide was freshly prepared by adding anhydrous ethanol (181 μl, 3.1 mmol) to 34% paraffinic suspension of sodium (210.4 mg suspension, containing 71.5 mg, 3.1 mmol, sodium) in anhydrous tetrahydrofuran (2 mL) under argon. To this suspension was added at 10°C ethyl formate (676 μl, 8.4 mmol), followed by the formyl derivative 11
(961 mg, 2.8 mmol). The reaction mixture was stirred at room temperature overnight. The suspension was rotary evaporated, the residue extracted with a mixture of xylene (9 mL) and water (3 mL), the aqueous layer separated, and the xylene layer washed with water. The aqueous layer was acidified with 12.1 M conc. HCl (0.57 mL, 6.85 mmol). Potassium thiocyanate (293 mg) was then added, and the suspension stirred at room temperature for 48 h. The mixture was extracted with chloroform, the organic layer separated and rotary evaporated to yield the mercapto derivative 12,
which was oxidatively desulfurized in the next step without further purification.
Ethyl 1-(1-(4-(3-((trifluoromethyl)-3H-diazirin-3-yl)phenyl)ethyl)-1H-imidazole-5-carboxylate (TFD-etomidate, 13)
To a stirred solution of sodium nitrite (12.5 mg), concentrated nitric acid (1.06 mL, 14.8 mmol) in water (5 mL), cooled to 10°C, was slowly added a solution of the thiol compound 12 in chloroform (5 mL). The solution was then stirred at room temperature for 1.5 h. Solid sodium bicarbonate (0.75 mg) was carefully added. The chloroform layer was separated, extracted with brine and the organic layer dried over magnesium sulfate. Rotary evaporation yielded crude product (585 mg). The product was purified by silica gel chromatography with dichloromethane containing 10% ether to yield white crystalline solid diazirinyl etomidate 13 (360 mg, 36% based on the formyl derivative). TLC, Silica gel: dichloromethane/ether 90:10 v/v, single spot, Rf 0.17. HPLC (Zorbax SB-C18 column, gradient A=0.1% TFA, B=acetonitrile), 10–100% B in 30 min. One peak, retention time 18 min 38 sec. 1H NMR spectrum: (CDCl3) δ 7.78 ((s, 1H, imidazole CH), 7.76 (s, 1H, imidazole CH), 7.26 and 7.14 (4H, AA//BB/ phenyl), 6.36 (q, 1H, methine), 4.24 (m, 2H, methylene), 1.85 (d, 3H, methyl), 1.30 (t, 3H, methyl). UV spectrum (methanol) λmax 358 nm, ε = 345 M−1 cm−1. Calcd. for C16H15F3N4O2: C, 54.55 %; H, 4.29%; N 15.90%. Found: C, 54.81 %; H, 4.43%; N 16.01%. Mass spectral analysis: (ESI +ve) M/Z: calculated for (C16 H15 F3 N4 O2 +H)+ 353, found 353. Judged by HPLC analyses the purity is 99%.
Resolution of racemic TFD-etomidate: chromatography of TFD-etomidate on an analytical Chiracel OD-H column with hexane:isopropanol 95:5 at a flow rate of 0.9 ml/min resolved the racemic mixture into S and R enantiomers that eluted at 12.5 and 15.5 min, respectively.
Synthesis of R-and S-enantiomers of TFD-etomidate by Mitsunobu reaction
S- and R- 3-(4-(1-(tert-butyl)dimethylsilyloxy)ethyl)phenyl)-3-((trifluoromethyl)-3H-diazirine (7)
The S- and R- enantiomers of 7 were synthesized starting with S- and R-4-bromo-α-methylbenzyl alcohol 1 (Aldrich Chemicals lot certification: [α]20D = −38.2 and +38.8 degrees (C=1%, CHCl3) for the S and R enantiomer, respectively) as described in the earlier section
S- and R-1-(4-(3H-diazirin-3-yl)-2,2,2-trifluoroethyl)ethanol (14)
The silyl-protected S- and R-diazirine derivatives 7 were deprotected as follows: To a solution of S- or R-7 (2.8 g, 8.13 mmol), in anhydrous THF (8 mL) was added a solution of 1 M tetrabutylammonium fluoride in THF (12 ml) at 0°C. The resulting solution was stirred at room temperature for 17 h, then 20 mL of a saturated solution of ammonium chloride was added, the THF layer separated and the aqueous layer extracted twice with 10 mL portions of ether. The combined organic extracts were washed with saturated NaCl solution and dried over magnesium sulfate. The crude product was purified by chromatography on silica gel with hexane/dichloromethane (75:25) followed by elution with dichloromethane to yield the S-diazirinylalcohol 15 (1.76 g, 94 % yield). 1H NMR spectrum: (CDCl3) 7.42 and 7.18 (4H, AA//BB/ phenyl), 4.92 (m, 1H, methine), 1.82 (hydroxyl), 1.48 (t, 3H, methyl). The optical rotations of (S)- and (R) -14 ([α]20D = −27.3 and +28.0 degrees (C=1%, ethanol) for the S- and R-enantiomer, respectively) indicated that the diazirinyl alcohol retained the chirality of the starting bromoalcohol 1.
Mitsunobu reaction between ethyl-1H
-imidazole carboxylate and (S)-14 was carried out as described by Zolle et al. for the synthesis of chiral metomidate19
. A solution of (S)-14 (253 mg, 1.1 mmol) in anhydrous THF (2 ml) was added dropwise to a stirred solution of ethyl-imidazole carboxylate (154 mg, 1.1 mmol) and triphenylphosphine (345 mg, 1.3 mmol) in anhydrous THF (2 mL) under an atmosphere of argon at −30°C. A solution of tert-butylazodicarboxylate (304 mg, 1.3 mmol) in anhydrous THF (2 mL) was added slowly and the temperature allowed to increase gradually to 0°C in 2 h. The mixture was then stirred at room temperature for 18 h. The THF was removed by rotary evaporation, the residue taken up in ether (5 mL) and stirred for 4 h. The precipitate was filtered off and washed 3 times with 2 mL portions of ether. The crude product obtained after rotary evaporation was taken up in dichloromethane (4 mL) and applied to a column of silica gel (20 g), equilibrated with dichloromethane. After washing with dichloromethane, the product R-TFD-etomidate (195 mg) was eluted with 10% ether. The product was further purified by preparative TLC on an 1 mm thick silica gel plate with ethylacetate:hexane 50/50 to yield R-TFD-product (150 mg). 1
H NMR spectrum: (CDCl3
) δ 7.78 (s, 1H, imidazole CH), 7.76 (s, 1H, imidazole CH), 7.26 and 7.14 (4H, AA/
phenyl), 6.36 (q, 1H, methine), 4.24 (m, 2H, methylene), 1.85 (d, 3H, methyl), 1.30 (t, 3H, methyl). Rotation measurement [α]20
D = +41.4 degrees (C=1%, Ethanol)) indicated that there was a complete reversal of chirality compared to the starting alcohol, confirming the result obtained by Zolle et al. 19
. Analytical chromatography on Chiracel OD-H column indicated that there was less than 0.5% racemization.
S-TFD-etomidate was synthesized by Mitsunobu reaction between ethyl-1H-imidazole carboxylate and (R)- 14 by a procedure similar to that described for the synthesis of R-TFD-etomidate. The NMR spectrum of the product was identical to that obtained with R-TFD-etomidate or racemic TFD-etomidate. [α]20D = +44.0 degrees (C=0.7%, Ethanol)).
Synthesis of [3H]TFD-etomidate
Unlabeled TFD-etomidate was heated with a solution of sodium hydroxide in ethanol at 60°C for 30 min to obtain the de-esterified intermediate. The hydrolyzed derivative was then re-esterified with [3H]ethanol using diisopropylcarbodiimide, dimethylaminopyridine in anhydrous dichloromethane to produce [3H]TFD-etomidate with a specific activity of 40 Ci/mmol.
Solubility and partition properties
To determine solubility, TFD-etomidate (5 mg) was stirred in 0.01 M Tris-HCl buffer, pH 7.4 (1 mL) for 24 h. After centrifugation of the suspension, aliquots were removed from the supernatant and analyzed on an HPLC C-18 reverse phase column (Varian, Walnut Creek, CA). The concentration of the probe in solution was calculated from the peak emerging at the calibrated retention time for the probe. To determine octanol/water partition coefficients, TFD-etomidate or etomidate (4 mg) were stirred in a two phase mixture of octanol (0.4 mL) and 0.01 M Tris-HCl buffer, pH 7.4 (2 mL) for 24 h. Aliquots were removed from the separated phases and applied to the HPLC column. Concentrations of the probe in the two phases were calculated from the peaks emerging at the calibrated retention time for the probe.
General Anesthetic Potency Xenopus laevis
tadpoles (Xenopus One, Dextor, Michigan) in the pre-limb-bud stage (1–2 cm in length) were housed in large glass jars filled with Amquel+ (Kordon, div. of Novalek, Inc, Hayward, CA) treated tap water. Stock solutions of the test compound were made in ethanol. With prior approval of the MGH Subcommittee on Research Animal Care, general anesthetic potency was assessed in the tadpoles as follows. Groups of 5 tadpoles were placed in foil-covered 100 mL beakers containing varying dilutions of the test compound in 2.5 mM Tris HCl at pH 7.4 under low levels of ambient light. The final concentration of ethanol did not exceed 5 mM, a concentration that does not contribute to anesthesia 33
. Every 10 minutes tadpoles were individually flipped using the hooked end of a fire-polished glass pipette until a stable response was reached (usually up to 40 minutes). Anesthesia was defined as the point at which the tadpoles could be placed in the supine position, but failed to right themselves after 5 seconds (loss of righting reflex, LoRR). All animals were placed in a recovery beaker of Amquel+ treated tap water and monitored for 30–60 minutes for fatality or full recovery. The quantal concentration response curves were analyzed by the method of Waud 34
using an Excel macro kindly provided by N.L. Harrison, A. Jenkins and S.P. Singh (Weill Medical College of Cornell University).
Electrophysiology of GABAA, Torpedo nicotinic ACh, and 5-HT3A receptors
With prior approval by the Massachusetts General Hospital Subcommittee on Research Animal Care, oocytes were obtained from adult, female Xenopus laevis
and prepared using standard methods and as previously described 35, 36
. In vitro
transcription from linearized cDNA templates and purification of subunit specific cRNAs was carried out using Ambion mMessage Machine RNA kits and spin columns. For GABAA
receptor studies, oocytes were injected with ~100 ng total mRNA (α1, β2, γ2L) mixed at a ratio of 1:1:1 transcribed from human GABA receptor subunit cDNAs in pCDNA3.1. β2M286W GABAA
R mRNA was prepared as described previously 21
. For Torpedo
nAChR ((1 1 1) studies, oocytes were injected with ~25 ng total mRNA mixed at a ratio of 2α:1β:1γ:1δ as previously described 36
. For human 5-HT3A
studies, oocytes were injected with ~50 ng of cRNA 35
All two-electrode voltage clamp experiments were done at room temperature, with the oocyte transmembrane potential clamped at −50 mV and with continuous oocyte perfusion with ND96 (100 mM NaCl, 2 mM KCl, 10 mM Hepes, 1 mM EGTA, 1 mM CaCl2, 0.8 mM MgCl2, pH 7.5) at ~2 mL/min. TFD-etomidate was dissolved in DMSO at a concentration of 10 mM just prior to use. Intravenous grade etomidate at a concentration of 2 mg/mL (8.2 mM) in 35% propylene glycol was obtained from Ben Venue Labs (Bedford, OH). A small stock of S-etomidate was solubilized at 1 mg/mL in 35% propylene glycol. Stocks were further diluted in ND96 to achieve the desired concentration.
GABAA and nAChR Dose-Response Studies
All agonist and agonist plus drug applications were 15–20 s in duration; oocytes were washed ~ 3 min between each application. Currents were amplified using an Oocyte Clamp OC-725C amplifier (Warner Instrument Corp), digitized using a Digidata 1322A (Axon Instruments, Foster City, CA), and analyzed using Clampex/Clampfit 8.2 (Axon Instruments) and OriginPro 6.1 software. Dose response data were fit by nonlinear least squares regression to the Hill (logistic) equations of the general form:
where X is the concentration of the activating ligand, IGABA,max
is the maximally evoked current, EC50
is the concentration of X eliciting half of its maximal effect, and n is the Hill coefficient of activation. Inhibition experiments were fit with logistic equations of the form:
5-HT3AR Concentration–Response Studies
Currents were amplified with a GeneClamp 500B amplifier (Molecular Device, Inc., Sunnyvale, CA) and signals were acquired using Axon’s pClamp 9.0 software. Preceding and following each current recording at a desired 5-HT test concentration, a maximum control current (for normalization) was obtained by perfusing the oocyte with ND96 buffer for 9 s followed by a 15 s exposure to 100 μM 5-HT to obtain a peak response followed by a 5-minute recovery period. Test traces were obtained by first perfusing the oocyte with ND96 buffer for 9 s followed by pre-exposing the oocyte to the etomidate derivative for 30 s, co-exposure of the etomidate derivative and 100 μM 5-HT for 30 s followed by a recovery period of 5 min. Peaks were measured and test currents recorded as percent of the average of the flanking maximum control currents. Data were analyzed using Igor Pro 4.07 (Wavemetrics Inc., Lake Oswego, OR) and concentration-response data fitted to the Hill Equation:
where I is the peak current evoked by the agonist, IC50
is the concentration of anesthetic, which inhibits the peak current to half of the control peak current, and n is the Hill coefficient. The time resolution was considered insufficient to analyze inactivation or desensitization.
Allosteric Regulation of the GABAA Receptor Ligand binding
Fresh whole bovine brain was placed on ice, and the cortex was rapidly removed, gray matter resected, and immersed in 0.32 M sucrose, and frozen at −80 °C. The frozen cerebral cortex was thawed and homogenized in 0.32 M sucrose, 10 mM phosphate buffer (pH 7.4). This homogenate was centrifuged (650 × g, 10 min, 4°C), and its supernatant was centrifuged again at 150,000g for 48 min. The pellet was resuspended in distilled water and recentrifuged at 150,000g for 48 min, and this pellet was washed with 10 mM phosphate buffer (pH 7.4) twice, centrifuged, and finally resuspended in 10 mM phosphate buffer (pH 7.4) and stored frozen at −80°C. Before use, the frozen suspension was thawed, centrifuged, and washed again with 10 mM phosphate buffer (pH 7.4); the pellet was resuspended with assay buffer (10 mM phosphate buffer (pH 7.4), 135 mM KCl, and 1 mM EDTA). Diluted membranes (400 μL) were incubated in a final volume of 0.5 mL for 1 h at 4°C with [3H]flunitrazepam (1 nM, 85.2 Ci/mmol, PerkinElmer Life Sciences; final protein concentration, 1 mg/mL) or [3H]muscimol (5 nM, 20 Ci/mmol, PerkinElmer Life Sciences; final protein concentration, 0.25 mg/mL). Nonspecific binding was determined by carrying out incubations in the presence of 7.5 μM flurazepam for [3H]flunitrazepam binding and 1 mM GABA for [3H]muscimol binding. The enhancement by etomidate or TFD-etomidate was measured in parallel at various concentrations (0.01, 0.1, 0.3, 1, 3, 10, 30 and 100 μM) in triplicate. Samples were filtered on GF/B glass fiber filters under suction, the filters washed with 3 mL of assay buffer twice, transferred to scintillation vials and subjected to scintillation counting after addition of 2.5 mL scintillation fluid (Ecolume, ICN).
5-HT3A receptor antagonist binding
5-HT3AR rich membranes, expressed in HEK 293 cells, corresponding to 200 pmoles of binding sites, were incubated in triplicate for 2 hours at room temperature with 0.5 nM [3H]GR65630 (Perkin Elmer, Waltham, MA) with or without etomidate or TFD-etomidate. Nonspecific binding was determined in the presence of 1 μM quipazine maleate (Sigma-Aldrich, St. Louis, MO). GF/B glass fiber filters (Whatman) were pre–incubated in 0.5% Poly(ethyleneimine) solution (P3143, Sigma-Aldrich) for an hour. Samples were filtered under vacuum and washed twice with 7 mL of cold HEPES/EDTA buffer. Filters were dried under a lamp for 1 hr, and [3H]GR65630 was determined by scintillation counting in 5 mL of Liquiscint (National Diagnostics, Atlanta, GA).
Photoincorporation of [3H]TFD-etomidate into the nAChR
nAChR-rich membranes, prepared as described in Middleton & Cohen 37
from Torpedo californica
electric organs (Aquatic Research Consultants, San Pedro, CA), were resuspended at 2 mg protein/mL in Torpedo
physiological saline (250 mM NaCl, 5 mM KCl, 3 mM CaCl2
, 2 mM MgCl2
, and 5 mM sodium phosphate, pH 7.0) supplemented with 1 mM oxidized glutathione. Aliquots (75 μ
L, 150 pmol ACh binding sites) were incubated at room temperature for 40 min with 0.3 μM [3
H]TFD-etomidate (40 Ci/mmol) in the absence or presence of other drugs. The samples were then transferred to a 96-well polyvinyl chloride microtiter plate and irradiated on ice with a 365 nm UV lamp (Model EN-16, Spectronics Corporation, Westbury, NJ) for 30 minutes at a distance of less than 2 cm. Electrophoresis sample buffer (12.5 mM Tris-HCL, 2% SDS, 8% sucrose, 1% glycerol, 0.01% bromophenol blue, pH 6.8) was added to the photolabeled samples, and the polypeptides were resolved on 1.5 mm thick, 8% polyacrylamide/0.33% bis-acrylamide gels. Following electrophoresis, the polypeptides were visualized by staining with Coomassie Blue R-250 (0.25% w/v in 45% methanol and 10% acetic acid). [3
H]TFD-etomidate photoincorporation into the membrane polypeptides subunits was visualized by fluorography (Amplify, Amersham Biosciences GE Healthcare) with exposure to film (Kodak BIOMAX XAR Film) for 4–6 weeks, and 3
H incorporation into individual polypeptide bands excised from the stained gel was quantified by liquid scintillation counting 37