P-trifluoromethyldiazirinyl-etomidate: A potent photoreactive general anesthetic derivative of etomidate that is selective for ligand-gated cationic ion channels

DOI: 10.1021/jm100498u

Source and publish data:

Journal of Medicinal Chemistry p. 6432 - 6444 (2010)

Update date:2022-07-30

Topics:

Authors:

Husain, S. Shaukat

Stewart, Deirdre

Desai, Rooma

Hamouda, Ayman K.

Li, S. Guo-Dong

Kelly, Elizabeth

Dostalova, Zuzana

Zhou, Xiaojuan

Cotten, Joseph F.

Raines, Douglas E.

Olsen, Richard W.

Cohen, Jonathan B.

Forman, Stuart A.

Miller, Keith W.

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Article abstract of DOI:10.1021/jm100498u

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) ((α1) ₂β1Δ1γ1) and 5-HT_3AR (serotonin type 3A receptor), causing significant inhibition at anesthetic concentrations. S- but not R-TFD-etomidate enhanced currents elicited from inhibitory α1β2γ2L GABA_ARs by low concentrations of GABA, but with a lower efficacy than R-etomidate, and site-directed mutagenesis suggests they act at different sites. [³H]TFD-etomidate photolabeled the α-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.

Full text of DOI:10.1021/jm100498u

6432 J. Med. Chem. 2010, 53, 6432–6444

DOI: 10.1021/jm100498u

p-Trifluoromethyldiazirinyl-etomidate: A Potent Photoreactive General Anesthetic Derivative of

Etomidate That Is Selective for Ligand-Gated Cationic Ion Channels

S. Shaukat Husain,^‡Deirdre Stewart,^‡Rooma Desai,^‡Ayman K. Hamouda,^§S. Guo-Dong Li, Elizabeth Kelly,^‡

Zuzana Dostalova,^‡Xiaojuan Zhou,^‡Joseph F. Cotten,^‡Douglas E. Raines,^‡Richard W. Olsen, Jonathan B. Cohen,^§

Stuart A. Forman,^‡and Keith W. Miller*^,‡,#

^‡Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, 32 Fruit Street, Boston, Massachusetts 02114,

^#Department of Biological Chemistry and Molecular Pharmacology , and Department of Neurobiology, 220 Longwood Avenue, Harvard Medical

School, Boston, Massachusetts 02115, and Department of Molecular and Medical Pharmacology, Geffen School of Medicine, University of

California at Los Angeles, Los Angeles, California 90095

Received April 22, 2010

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) ((R1)₂β1δ1γ1) and

5-HT_3AR (serotonin type 3A receptor), causing significant inhibition at anesthetic concentrations.

S- but not R-TFD-etomidate enhanced currents elicited from inhibitory R1β2γ2L GABA_ARs by low

concentrations of GABA, but with a lower efficacy than R-etomidate, and site-directed mutagenesis

suggests they act at different sites. [³H]TFD-etomidate photolabeled the R-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.

Introduction

currents by increasing the receptor’s open state probability,

resulting in a shift in the agonist concentration-response curve

to lower concentrations.^7,8At higher concentrations, etomi-

date can activate GABA_ARs directly and also acts as a nega-

tive allosteric inhibitor on the cationic ligand-gated excitatory

receptors, 5-HT_3AR and nAChR.^5,6

Etomidate (R-2-ethyl 1-(phenylethyl)-1H-imidazole-5-

carboxylate) is oneof the most potent general anesthetic drugs

in clinical use. It causes anesthesia with a half effective con-

centration of ∼2 μM.^1,2Etomidate interacts with members of

the Cys-loop ligand-gated ion channel superfamily of recep-

tors, which include subtypes activated byγ-aminobutyric acid

(GABA),^aglycine, serotonin (5-HT), and acetylcholine (ACh).

GABA_AR is the most sensitive to etomidate,^3-6which acts

as a positive allosteric mediator, enhancing GABA-induced

Because of the above selectivity, which it shares with

another high affinity anesthetic, propofol,⁷more progress

has been made in understanding the mechanism of etomi-

date’s action than for most other general anesthetics. In

R1β1γ2L GABA_ARs, a residue on the β-subunit 15 residues

from the conserved charge at the cytoplasmic end of the

channel (M2 15⁰N265) is a determinant of sensitivity to eto-

midate.^3,9,10It is located contralateral to the ion channel,

facing the interior of the subunit’s four transmembrane helix

bundle, a region that is hypothesized to be a binding pocket

for inhalation anesthetics on other subunits.¹¹Mutations at

this position in knock-in mice can ablate etomidate’s and

propofol’s anesthetic action, while having little effect on that

of inhalation anesthetics.^12-14However, this residue is not

part of an anesthetic binding pocket because it changes eto-

midate’s efficacy much more than its affinity,¹⁵and propofol

is unable to protect cysteines substituted at this position from

chemical modification.¹⁶

*Corresponding author. Phone: (617) 726-8985. Fax: (617) 724-8644.

E-mail: k_miller@helix.mgh.harvard.edu.

^aAbbreviations: 5-HT, serotonin; 5-HT_3AR, 5-HT receptor, subtype

3A; ACh, acetylcholine; azietomidate, R-2-(3-methyl-3H-diaziren-3-yl)-

ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate; BzBzl-etomidate,

R-4-benzoylbenzyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylate; DBU,

1,8-diazabicyclo[5.4.0]-undec-7-ene; EC₅₀, concentration required for

50% of full effect; [³H]EBOB, [³H]ethynylpropylbicycloorthobenzoate;

etomidate, R-2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate; LORR,

loss of righting reflexes; GABA, γ-amino butyric acid; GABA_AR, Type

A GABA receptor chloride channel; IC₅₀, concentration required for

50% of full inhibitory effect; n_H= Hill coefficient; nAChR, nicotinic

acetylcholine receptor ion channel; octanol, octan-1-ol; SD, standard

deviation; TDBzl-etomidate, R-4-[3-(trifluoromethyl])-3H-diazirin-3-yl]-

benzyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate; TFD-etomidate,

p-trifluoromethyldiazirinyl-etomidate or ethyl 1-(1-(4-(trifluoromethyl)-

3H-diazirin-3-yl)phenylethyl)-1H-imidazole-5-carboxylate; THF, tetra-

hydrofuran; TFA, trifluoroacetic acid; VDAC, voltage-dependent an-

ion channel.

A definitive way to locate etomidate’s binding site(s) is to

use derivatives of etomidate that are photoactivable. One

such agent, azietomidate or [2-(3-methyl-3Hdiaziren-3-yl)ethyl

pubs.acs.org/jmc

Published on Web 08/12/2010

2010 American Chemical Society

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6433

Scheme 1. Photoactivable Derivatives of the General Anesthetic Etomidate

1-(1-phenylethyl)-1H-imidazole-5-carboxylate], in which an

ethoxy group has been replaced by a 3-azibutoxy group,

exhibits potency and pharmacology very similar to the parent

compound in GABA_ARs, tadpoles and rodents.⁵Furthermore,

in etomidate-resistant knock-in mice bearing a N265M muta-

tion in the β3-subunit of the GABA_Areceptor, azietomidate’s

potency is attenuated in parallel with that of etomidate.¹⁷

Azietomidate photolabels GABA_ARs from bovine cortex in

an agonist-dependent manner, and two residues, which ap-

peared from pharmacological analysis to be part of a single

binding pocket, were identified, one on the RM1 and the other

on the βM3 transmembrane helix. Homology modeling suggests

that these residues are located not within the intra-subunit four

helix bundle but in the interface between the R-andβ-subunits.¹⁸

To further test the subunit interface site hypothesis, we

adopted the long-term goal of devising a set of two etomidate

derivatives that have photoactivable moieties on opposite ends

of the molecule. We have to date investigated the pharmacology

of two more ester substituents. Both benzophenone- and aryl

diazirine-derivatives of etomidate (Scheme 1) proved to be

potent general anesthetics that modulate GABA_AR function.^5,6

In this manuscript, we describe the synthesis and initial

pharmacologic characterization of ethyl 1-(1-(4-(trifluoro-

methyl)-3H-diazirin-3-yl)phenylethyl)-1H-imidazole-5-carbo-

xylate (trifluoromethyldiazirinyl-etomidate), or TFD-etomi-

date (Schemes 1 and 2). In this derivative, the TFD moiety

modifies etomidate’s benzyl group and is thus complementary

to TDBzl-etomidate, in which the same moiety is substituted

at etomidate’s ethoxy group. We determined that TFD-eto-

midate, like etomidate, is a potent general anesthetic that

interactswithandmodulatesthe GABA_AR, TorpedonAChR,

and 5-HT_3AR. However, we found it has a unique selectivity

for cation conducting Cys-loop receptors relative both to eto-

midate and to the other previously developed etomidate photo-

label derivatives.

etomidate analogue was the protected diazirine, 3-(4-(1-(tert-

butyl)dimethylsilyloxy)ethyl)phenyl)-3-((trifluoromethyl)-

3H-diazirine (7). Its synthesis involved protection of the hydro-

xyl group of the starting compound 4-bromo-R-methylbenzyl

alcohol (1) with the acid labile tert-butyldimethyl silyl group,

conversion of the bromo group of the protected derivative to

trifluoromethylketone (3), and subsequent conversion of the

keto group via a series of reactions to the diazirinyl functional

group. Synthesis of TFD-etomidate from the silyl-protected

diazirine (7) was performed by two alternate routes: Using the

first route, the silyl protecting group was replaced by the

bromo group, which after reaction with ammonia gave the

aryl amine (9). TFD-etomidate was synthesized from the aryl

amine utilizing the route originally developed by Godefroi

et al.¹for the synthesis of etomidate. Since the synthetic

sequence involved reaction at the chiral center during conver-

sion of the bromo compound to the amine, the final product

was a racemate. The racemic mixture of TFD-etomidate was

resolved by chromatography on Chiracel OD-H column.

After the above synthesis of TFD-etomidate was completed,

Zolle et al¹⁹described conditions for Mitsunobu coupling

between chiral precursor alcohols with methyl 1H-imida-

zole-5-carboxylate. This reaction yielded enantiomerically

pure analogues of etomidate having a reversed chirality

compared to that of the starting alcohol. Following these

conditions and starting with R- or S- diazirinyl alcohols

(14), produced by reaction of the silyl-protected diazirine

derivative (7) that we had already synthesized, application

of the Mitsunobu reaction provided R- and S- enantiomers

of TFD-etomidate.

Results

Solubility and Partition Coefficient. The saturated solubility

of TFD-etomidate when thoroughly equilibrated in 0.01 M

Tris-HCl buffer, pH 7.4, was 58 μM. It should be noted that

higher concentrations were easily achieved in fresh solutions

with crystalline solid only precipitating the next day. Thus, in

some physiological experiments herein freshly made super-

saturated solutions were used.

Chemistry

The synthesis of TFD-etomidate is outlined in Scheme 2.

The key intermediate for the preparation of the photoactivatable

6434 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17

Scheme 2. Synthesis of TFD-etomidate^a

Husain et al.

^a(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, dichlor-

omethane; (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.

The octanol/Tris buffer partition coefficient of TFD-

etomidate was 27 000. In comparison, we found the parti-

tion coefficient for etomidate to be 330 under the same

conditions. This compares to a previously reported value,

determined in unbuffered water, of 776.²⁰Thus, substitution

of etomidate’s aromatic ring with a trifluoromethyldiazir-

inyl group results in an increase in the partition coefficient

by almost 2 orders of magnitude. The partition coefficient

for TFD-etomidate is comparable to those for TDBzl-

etomidate and BzBzl-etomidate, which have values of

19 000 and 25 000, respectively.⁶Enantioselectivity is not

expected in physical properties, and because of the limited

quantities of the enantiomers, their solubility was not

examined.

General Anesthetic Potency. With institutional approval,

the general anesthetic potency of TFD-etomidate was as-

sayed in tadpoles. TFD-etomidate induced a reversible gen-

eral anesthetic state that resembled that of etomidate.

Tadpoles that had lost their righting reflexes showed a brief

response to a light touch and even twitched spontaneously,

behaviors that differ from conventional general anesthetics.

The onset of and recovery from anesthesia was somewhat

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6435

slower than for etomidate, with the fraction anesthetized

becoming constant after 30-40 min; however, the potency of

the two agents was comparable. The concentration-depen-

dence of LoRR was examined in groups of five animals at

seven concentrations between 0.25 and 25 μM. The EC₅₀and

number of animals for S- and R-TFD-etomidate was 4.9 (

0.15 μM (45 animals; all errors herein are standard devia-

tions unless otherwise stated) and 17.1 ( 0.35 μM (47

animals) respectively, comparable to two prior studies with

R- and S-etomidate that gave EC₅₀’s of 2.3 ( 0.13 and 25 (

2.2 μM, respectively.^2,5Only animals that fully recovered in

fresh water overnight were included in the analysis. At 20 μM,

2 of 5 animals died but none of 10 at 22.5 and 25 μM.

Action on GABA_AReceptor Physiology. In human R1β2γ-

2L GABA_ARs expressed in Xenopus oocytes at low (10 μM)

and maximal (0.1-10 mM) concentrations of GABA, race-

mic TFD-etomidate (5 μM) elicited much smaller enhance-

ments of currents than etomidate at the same concentration.

GABA concentration response curves (( drugs) were deter-

mined at fixed concentration of anesthetic equal to twice the

EC₅₀for tadpole anesthesia, a concentration generally con-

sidered to represent clinical anesthesia. The GABA concen-

tration-response curve was shifted to the left 1.8-fold by 5

μM TFD-etomidate but 18-fold by the same concentration

of R-etomidate (data not shown). Similar experiments were

done with the R- and S-enantiomers of TFD-etomidate. At

twice the tadpole EC₅₀, S-TFD-etomidate (10 μM) shifted

the GABA concentration response curve to the left 2.6-fold

without a change in the maximum current (Figure 1A),

whereas R-TFD-etomidate (34 μM) shifted the GABA con-

centration response curve 1.2-fold to the right and decreased

the maximum current (Figure 1B). At 10 μM GABA, 10-

100 μM R-TFD-etomidate inhibited the response up to 20%.

These observations suggested that TFD-etomidate and eto-

midate might have different sites of action. To test this hypoth-

esis, we employed the mutant GABA receptor R1β2M286Wγ2L

that lacks etomidate sensitivity.²¹For wild type (R1β2γ2L)

GABA receptors, in the same oocyte 3 μM GABA currents

were increased 2.2- and 3.0-fold by 5 and 10 μM S-TFD-

etomidate, respectively, compared to an increase of 3.7-fold

with 5 μM R-etomidate (Figure 1C, top line of traces). Using

the mutant GABA receptor in a similar experiment, the

action of 5 and 10 μM S-TFD was similar to that of the wild

type receptor, a 1.8- and 2.2-fold increase respectively in 1 μM

GABA currents (1 and 3 μM GABA elicit a current one-

tenth of I_max, that is, EC₁₀, in mutant and wild type receptors,

respectively), whereas 5 μM etomidate caused no change

(Figure 1C, bottom line of traces). Furthermore, TFD-eto-

midate (0-30 μM) caused a linear increase in 3 μM GABA

currents in wild type receptors of up to 2.5-fold that was not

attenuated by the presence of 1 μM etomidate (the slope was

0.05 ( 0.001 in each case). These results are consistent with

the separate site hypothesis.

Figure 1. Etomidate and TFD-etomidate modulation of GABA-

induced currents through human wild type R1β2γ2L 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 re-

cordings were normalized to the maximal GABA response. (A)

Data shown for the experiment with S-TFD-etomidate represent

experiments on three different oocytes. Nonlinear least-squares

fitting gave (EC50 in μM and Hill coefficient, n_H): for the GABA

control (9), 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 (2), 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 concentra-

tion of reagent needed. For the GABA control (9), EC₅₀= 25 ( 1,

n_H= 1.4 ( 0.1, I_max= 1.01 ( 0.01. In the presence of 34 μM R-

TFD-etomidate(1), 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 (R1β2γ2L) GABA_Areceptors using

5 and 10 μM S-TFD-etomidate and then 5 μM R-etomidate. The

bottom line of current traces shows the same series of applications

of drugs on 1 μM GABA currents elicited from the mutant

(R1β2M286Wγ2L) GABA receptor.

TFD-etomidate, like etomidate,⁵could directly activate

currents in the absence of GABA (data not shown), but once

again its action was weaker than etomidate’s. Saturated con-

centrations of TFD-etomidate induced a current that was

1.3% of that elicited by 10 mM GABA, whereas the compar-

able figure for 30 μM etomidate was 27%. The effect with

TFD-etomidate was too small to make it worth addressing

the question of enantioselectivity.

5 nM [³H]muscimol binding to cerebral cortex membranes were

examined (Figure 2). Etomidate enhanced [³H]flunitrazepam

binding at concentrations up to 10 μM with a subsequent

decrease (Figure 2A). The enhancement of binding was detect-

able at 0.1 μM etomidate and reached a plateau at ∼10 μM with

Allosteric Modulation of GABA_AReceptor. To compare

their allosteric action, the effects of various concentrations of

etomidate and TFD-etomidate on 1 nM [³H]flunitrazepam or

6436 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17

Husain et al.

Figure 2. TFD-etomidate does not interact allosterically with GA-

BA_Areceptor 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.

a ∼1.2-fold maximal enhancement of [³H]flunitrazepam bind-

ing. The concentration dependence of etomidate enhancement

could be fit to a logistic curve, yielding half-effect concentra-

tions of 0.94 ( 0.45 μM. In contrast, TFD-etomidate failed

to show modulation of [³H]flunitrazepam binding: it caused

neither enhancement nor inhibition in the concentration

range tested (Figure 2A).

Etomidate caused ∼3-fold maximal enhancement of

[³H]muscimol binding at a concentration of 10 μM and a

subsequent decrease at higher concentrations (Figure 2B).

The concentration dependence of enhancement could be fit to

a logistic curve, yielding a half-effect concentration of 0.42 (

0.21 μM. In comparison, TFD-etomidate failed to show any

modulation of [³H]muscimol binding. There was no en-

hancement or inhibition in the concentration range tested

(Figure 2B).

Inhibition of 5-HT_3AReceptor by TFD-etomidate. Figure 3

shows that in oocytes expressing 5-HT_3ARs, both racemic

TFD-etomidate and R-etomidate inhibited currents evoked

by 100 μM 5-HT. The magnitude of this inhibition increased

with anesthetic concentration and upon simultaneous termi-

nation of anesthetic and agonist, a surge current was recorded

(inset). Although the inhibitory actions of TFD-etomidate

and etomidate were qualitatively similar, TFD-etomidate

inhibited currents with an IC₅₀that was 7-fold lower than

etomidate and with a Hill coefficient of -1 rather than -2 for

etomidate (see legend to Figure 3). At a fixed concentration of

9 μM, S- and R-TFD-etomidate caused equal inhibition of

29 ( 11 (n = 9) and 34 ( 11% (n = 13) of control, respecti-

vely, compared to the racemic compound 28 ( 11% (n=4) in

Figure 3. Actions on the 5-HT_3Areceptor 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 con-

centration. The IC₅₀’s 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 (gray) 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^-1for etomidate and TFD-etomidate, respectively.

The inset shows a representative current trace obtained upon

application of 10 μM TFD-etomidate alone.

a parallel experiment. The action of etomidate was also with-

out marked stereoselectivity (not shown).

In addition to inhibiting currents, both TFD-etomidate

and R-etomidate evoked a small current prior to the applica-

tion of 5-HT as shown on the inset in Figure 3B. The peak

amplitudes of such directly activated currents are plotted as a

percentage of the current evoked by 100 μM 5-HT versus

anesthetic concentration. In general, the amplitude of this

directly activated current increased with anesthetic concen-

tration, reaching 1.7% and 1.3% of that evoked by 100 μM

5-HT at 20 μM TFD-etomidate and 300 μM etomidate,

respectively. This effect did not saturate, and a linear fit of

the data revealed that the slope of this relationship was 10-

fold greater for TFD-etomidate than for etomidate (see

legend Figure 3). S- and R-TFD-etomidate (9 μM) activated

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6437

Figure 4. TFD-etomidate is a more potent inhibitor of ACh-in-

duced currents than etomidate. Oocytes expressing wild type Tor-

pedo 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 (O), IC₅₀

21 ( 3 μM, n_H= 0.9 ( 0.1 (3 oocytes); for TFD-etomidate, IC₅₀

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 Coomas-

sie 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 tetra-

caine (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 R, β, γ, and δ subunits, rapsyn (Rsn), the R subunit of

the Na^þ/K^þ-ATPase (RN/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 R, β, γ, and

δ subunit gel bands was determined by liquid scintillation counting

(average and range for two photolabelings performed in parallel).

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 (1), IC₅₀= 11.3 ( 1.6 μM,

_H= 0.7 ( 0.1, and for S-TFD-etomidate (2): 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.

equally (0.4%), but the effect is small compared to the errors

and so we cannot rule out enantioselectivity.

5-HT_3AReceptor Antagonist Binding. TFD-etomidate

(6.25-70 μM) and etomidate (20-300 μM) caused no con-

sistent changes in displaceable [³H]GR65630 binding (data

not shown). Control [³H]GR65630 binding was 33 ( 0.85

pmol/mg. Pooled data revealed no significant changes in

[³H]GR65630 binding (unpaired t test, p > 0.01).

Action on nACh Receptor. When TFD-etomidate was

coapplied with acetylcholine to voltage clamped oocytes

expressing Torpedo nAChR receptors, it inhibited acetylcho-

line-induced currents (Figure 4). Peak currents elicited by

10 μM ACh (acetylcholine has EC₅₀≈ 25 μM for Torpedo)

were inhibited by increasing amounts of TFD-etomidate

with an IC₅₀value of 4.1 ( 0.5 μM. Etomidate behaved simi-

larly but was less potent, having an IC₅₀of 21 ( 3 μM. Sur-

prisingly, the R and S enantiomers of TFD-etomidate inhi-

bited 10 μM ACh-induced currents in an enantioselective

fashion; R- and S-TFD-etomidate had IC₅₀’s of 11.3 ( 1.6

and 4.7 ( 0.5 μM, respectively (Figure 4). TFD-etomidate in

the range from 10 to 100 μM showed no direct activation in

the absence of acetylcholine of either mouse muscle or

Torpedo nAChRs (data not shown).

cytoplasmic aspect of the nAChR, calelectrin, a 37 kDa

cytoplasmic protein that associates with membranes in a

Ca^2þ-dependent manner, and polypeptides from contami-

nating membranes from the noninnervated surface of the

electrocyte (90 kDa R subunit of the Na^þ/K^þ-ATPase) and

from mitochondria (34 kDa voltage-dependent anion chan-

nel (VDAC)).²²

For membranes photolabeled with [³H]TFD-etomidate in

the absence of other drugs, there was ³H incorporation into

each of the nAChR subunits, with labeling of the nAChR

R subunit most prominent, along with labeling of VDAC and

the Na^þ/K^þ-ATPase R subunit. For membranes labeled in

the presence of the agonist carbamylcholine, labeling in the

nAChR R subunit was increased by ∼50%, and labeling of

the β, γ, and δ subunits increased by ∼30%. To determine

whether nAChR subunit labeling was inhibited by drugs

known to bind within the nAChR ion channel, membranes

were also photolabeled with [³H]TDF-etomidate in the pre-

sence of tetracaine, which binds selectively in the ion channel

in the closed state, or with proadifen or phencyclidine, drugs

that bind in the ion channel with highest affinity when the

nAChR is in the desensitized state (i.e., equilibrated with

Photoincorporation of [³H]TFD-etomidate into the Torpe-

do nAChR. Photoincorporation of [³H]TFD-etomidate into

Torpedo nAChR-rich membranes was evaluated by SDS-

PAGE followed by fluorography (Figure 5A) or by liquid

scintillation counting of excised gel bands (Figure 5B). In the

Torpedo membrane preparation, nAChRs comprise about

10-20% of the protein, and other prominent polypeptides

include rapsyn, a 43 kDa protein that associates with the

6438 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17

Husain et al.

agonist). The carbamylcholine-enhanced labeling in each

nAChR subunit was inhibited by phencyclidine, but not by

proadifen. For membranes photolabeled in the absence of

agonist, tetracaine did not affect the level of nAChR subunit

photolabeling. Proadifen reduced ³H incorporation into

VDAC, as had been seen previously for nAChR-rich mem-

branes photolabeled by [³H]azietomidate.²³

Discussion

General Anesthetic Properties. TFD-etomidate was a re-

versible general anesthetic in tadpoles and was approxi-

mately equipotent to the parent general anesthetic, etomi-

date. However, S-TFD-etomidate was 3.5-fold more potent

than R-TFD-etomidate, whereas R-etomidate is 11-fold more

potent than S-etomidate.^2,5Remarkably, inserting the triflu-

oromethyl diazirinyl group in etomidate’s benzene ring

reversed the enantioselectivity while having little effect on

potency. Godefroi et al (1965) reported several active eto-

midate derivatives bearing para substituents in the aryl ring,

but none with as large a substituent as used herein.

Addition of the trifluoromethyl diazirinyl group increased

the octanol-water partition coefficient by 35-fold over that

of etomidate, but less than 2-fold over that of TDBzl-eto-

midate, which bears a substitution in the ester moiety.⁶The

potency of a general anesthetic can be predicted with fair

accuracy by the empirical Meyer-Overton rule, which states

that the product of the anesthetic EC₅₀and the octanol/water

partition coefficient is constant. Figure 6 shows such a cor-

relation for 20 agents (see legend) whose potencies cover

almost 7 orders of magnitude. Of the enantiomeric pairs of

etomidate derivatives, the potencies of R-TFD-etomidate,

S-etomidate and S-azietomidate are relatively well predicted

by the Meyer-Overton rule. However, all the R-enantio-

mers of the ester derivatives of etomidate are more potent

than predicted, suggesting a favorable specific interaction.^5,24

However, with TFD-etomidate the trend is reversed. The

more active enantiomer lies closer to the line, suggesting that

the least active enantiomer experiences an unfavorable inter-

action with its target.

Actions on Ligand-Gated Ion Channels at Anesthetic Con-

centrations. The puzzling reversal of the enantioselectivity

for general anesthesia noted above is partially explained by

the action on GABA_ARs. Briefly, S- but not R-TFD-etomi-

date enhanced responses to submaximal GABA concentra-

tions. In contrast, both R- and S-etomidate cause a large

enhancement of GABA-induced ion currents, with the S-

enantiomer being less potent and slightly less efficacious.⁵

However, at the excitatory 5-HT_3ARs and nAChRs, where

etomidate has little action at clinical concentrations, TFD-

etomidate inhibits in the clinical concentration range. Thus,

at twice the LoRR EC₅₀, a concentration corresponding to

clinical anesthesia, TFD-etomidate causes an ∼1.2-fold left-

shift in the GABA concentration-response curve compared

to an 18-fold left-shift for etomidate. In contrast, TFD-

etomidate causes 33% inhibition of 5-HT_3AR currents and

S- and R-TFD-etomidate cause 62% and 30% inhibition of

nAChR currents respectively, whereas the equivalent num-

bers for R-etomidate for 5-HT_3AR and nAChRs are 4 and

25%, respectively.

Figure 6. Does the Meyer-Overton rule correctly predict TFD-

etomidate’s anesthetic potency? The EC₅₀concentration for loss of

righting reflexes (LoRR) in tadpoles is plotted against the octanol/

water partition coefficient for 25 general anesthetics. The conven-

tional general anesthetics are shown as black circles, and the

photoactivable general anesthetics are shown 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 (( stan-

dard 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-(þ)-azie-

tomidate; 18, BzBzl-etomidate; 19, TDBzl-etomidate; 20, S-TFD-

etomidate; 21, R-TFD-etomidate; 22, R-(þ)-etomidate; 23, R-(þ)-

etomidate azide; 24, S-(-)-etomidate azide; 25, S-(-)-etomidate.

Data from refs 5, 6, 20, 24, 38, and 39.

subunit compositions, TFD-etomidate did not have any allos-

teric actions on agonist binding (Figure 2A,B).

To test whether TFD-etomidate binds to the etomidate

site but has low efficacy or whether it acts at a different site,

independent of etomidate, we first examined the ability of

TFD-etomidate to enhance currents elicited by low GABA

concentrations in the presence and absence of a fixed con-

centration of etomidate. We found that the concentration-

dependence of enhancement of GABA currents by TFD-

etomidate is unaffected by the presence of a fixed concentra-

tion of 1 μM etomidate. Second, we used a GABA_AR that

contains a methionine to tryptophan mutation on a residue

that has been photolabeled by azietomidate.¹⁸In the R1β2M-

286Wγ2L GABA_AR, which is relatively insensitive to eto-

midate,²¹TFD-etomidate exerted the same action as it did in

wild type R1β2γ2L GABA_ARs, whereas azietomidate and

TDBzl-etomidate were inactive like etomidate. Both pieces

of evidence point to TFD-etomidate having a separate site of

action from etomidate and the other photolabels.

Thus, TFD-etomidate occupies a separate site that does

not interact allosterically with the etomidate site at the R-β

subunit interface of the transmembrane domain. This site is

also distinct from those for flunitrazepam (at the γ-R

subunit interface of the N-terminal domain) and muscimol

(at the β-R subunit interface of the N-terminal domain) and

does not interact allosterically with them (Figure 2).

Actions on GABA_ARs. S-TFD-etomidate’s action on GABA-

induced ion currents in R1β2γ2L GABA_ARs (Figure 1A) were

modest compared to etomidate’s.⁵In membranes from cerebral

cortex, which would contain GABA_AR with a wider range of

TFD-etomidate’s weak actions on GABA_ARs is not

shared by TDBzl-etomidate (Table 1), which has a similar

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6439

Table 1. Pharmacology of Etomidate and Its Trifluorodiazirine Derivatives

general

anesthesia

^LoRREC₅₀(μM)

enhancement of EC₁₀

GABA currents at

twice ^LoRREC₅₀

enhancement of

flunitrazepam

binding at ^LoRREC₅₀

inhibition of

5 HT₃R IC₅₀(μM)

inhibition of

AChR IC₅₀(μM)

agent

R-etomidate

S-etomidate

2.3^b

25^b

9^b

120%^c

21^a

9^b

R-TFD-etomidate

S-TFD-etomidate

R-TDBzl-etomidate

17^a

inhibits^a

1.8 ^a

5.4^c

none^a

110%^c

9^a

11^a

4.7^a

4.9^a

0.7^c

none (>91)^c

enhances at g10 μM^d

^aThis work. ^bFrom Husain et al, 2003.^{5 c}From Husain et al, 2006.^{6 d}Nirthanan et al, 2008.²⁷

moiety substituted at the ester end of the etomidate back-

bone, nor by BzBzl-etomidate, with a benzophenone group

at the ester position. Both these agents enhance currents

robustly. Thus, it is unlikely that the bulky substituent per se

is responsible for TFD-etomidate’s weak actions. Most

likely, the orientation that etomidate adopts in its binding

site allows bulky groups to be tolerated at the ester but not

the benzoyl end of the drug. Indeed, when iodine was sub-

stituted at the para position of the phenyl ring, the IC₅₀of

etomidate’s inhibition of [³H]ethynylpropylbicycloortho-

benzoate ([³H]EBOB) binding to rat cortical membranes in

the presence of 1 μM GABA decreased some 20-fold.⁴⁰[³H]-

EBOB binds to the resting state of GABA_ARs and ligands

that enhance GABA binding therefore decrease its binding.

The iodo-substitution also abolished the ability to modulate

in the absence of GABA. These data are broadly consistent

with the role of trifluoromethyldiazirine substitution re-

ported herein. Such substitutions did not affect binding to

11β-hydroxylase,¹⁹and it will be interesting to see if TFD-

etomidate also binds to this target.

TDBzl-etomidate’s action on nAChRs. TDBzl-etomidate,

which incorporates the same photoreactive aryl diazirine at

the opposite end of the etomidate structure (Scheme 1), causes

potentiation of currents elicited at submaximal ACh concen-

trations (Table 1). In this case, TDBzl-etomidate photoincor-

porates at two distinct sites. The first is an inhibitory site

within the lumen of the ion channel, and the second, a novel

site, most likely the enhancing site, is at the interface bet-

ween the alpha and gamma subunits in the transmembrane

domain.²⁷

Photoincorporation of TFD-etomidate into nAChRs. [³H]-

TFD-etomidate photoincorporated into all nAChR subu-

nits equally in the resting state. Photolabeling was enhanced

in the agonist-bound desensitized state, ruling out the

agonist-binding site as a site of photolabeling. Photolabeling

of the agonist site has previously been observed with

azietomidate²³but not TDBzl-etomidate.²⁷The location of

the desensitization-enhanced photolabeling is likely in the

channel, because phencyclidine, a drug that binds in the ion

channel with highest affinity when the nAChR is in the

desensitized state, antagonized such labeling.

The pharmacological specificity of [³H]TFD-etomidate

labeling at the subunit level differs qualitatively from that

seen for [³H]TDBzl-etomidate, for which the most promi-

nent effect was a doubling of R-subunit photolabeling in the

presence of PCP that was not seen in the presence of

agonist.²⁷For [³H]TDBzl-etomidate, the PCP-enhanced

photolabeling of the R-subunit resulted from labeling of

the novel binding site referred to above in the nAChR

transmembrane domain at an interface between subunits.

Suggestively, the magnitude of the enhancement of [³H]TFD-

etomidate photolabeling is comparable to that seen for

[³H]TDBzl-etomidate at its novel binding site, but much

more detailed studies will be required to determine if this is so

and to delineate the other sites.

Conclusion. TFD-etomidate is a general anesthetic with an

unusual pharmacologic profile (Table 1). Although behavi-

orally it appears to be comparable to etomidate in its general

anesthetic action, its actions on the Cys-loop ligand-gated

ion channels were often unexpected. Thus, further explora-

tions of its pharmacology may yield novel information.

We designed TFD-etomidate to put the reactive group at

the opposite end of etomidate from that in TDBzl-etomidate

with the goal of further exploring the binding pocket that

azietomidate has revealed in the R-β subunit interface of

GABA_ARs. Although we obtained some useful structure-

activity relationship information about this pocket because

the agent appears not to interact with it, our original goal is

thereby frustrated. At the same time, this very observation

enhances TFD-etomidate’s usefulness for exploring other

unknown sites of its general anesthetic action, so that in the

end it may prove more broadly useful than we expected when

devising it for a more focused purpose.

Action on nAChRs and 5-HT_3ARs. In contrast to its weak

action on GABA_ARs, TFD-etomidate is the only etomidate

derivative that we have studied that inhibits nAChRs and

5-HT_3ARs in the clinical concentration range (see above, and

Figures 3 and 4).

Although etomidate derivatives do activate currents in

GABA_ARs,^8,25to our knowledge general anesthetics have

not previously been reported to exert this action on a cation-

conducting member of the Cys-loop ligand-gated ion chan-

nel superfamily. It was thus a surprise to find that TFD-

etomidate alone induces 5-HT_3AR currents (Figure 3B). This

partial agonism was weak. At saturating concentrations,

TFD-etomidate only activated ∼2% of the maximum cur-

rent that 5-HT activates. Etomidate also activated currents.

Its concentration-dependence was a tenth of, and its efficacy

comparable to, that of TFD-etomidate. This effect was not

observed in nAChRs. In contrast, etomidate, alone, can elicit

GABA currents up to 20%.⁵

More detailed kinetic measurements will be required to

understand how TFD-etomidate can activate the 5-HT_3A

without inhibiting it. One possibility is that it induces a dif-

ferent open state from the one stabilized by agonist, and that

this state has a lower affinity for TFD-etomidate than the

regular channel. However, in GABA_ARs anesthetics and ago-

nists are thought to induce a similar open state.²⁶In GABA_ARs,

the etomidate site that enhances GABA-induced currents is

thought to be isosteric with that which activates currents,⁸but it

is difficult to see how this would be the case for 5-HT_3ARs.

However, we can rule out a mechanism whereby TFD-etomi-

date binds to the agonist site, because even saturating concen-

trations do not displace the 5-HT_3AR antagonist, GR65630.

One further clue to the location of the site of TFD-

etomidate’s activation of 5-HT_3ARs above comes from

6440 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17

Husain et al.

Experimental Section

(d, 6H, methyl). Calcd. for C₁₆H₂₃F₃O₂Si: C, 57.81%; H,

6.97%. Found: C, 58.09%; H, 7.14%.

Materials. Anhydrous dichloromethane, diisopropylcarbo-

diimide, p-(dimethylamino)pyridine, and Merck silica gel 60 A,

230-400 mesh, were obtained from Aldrich (Milwaukee, WI).

R-(þ)-Etomidate was a kind gift from Organon Laboratories

(Newhouse, Lancashire, Scotland). R-(þ)-Etomidate used in

GABA_Areceptor electrophysiology studies was purchased from

Bedford Laboratories (Bedford, OH) as a 2.0 mg/mL solution in

35% propylene glycol/water (v/v). All other chemicals were

from Sigma (St. Louis, MO). cDNAs for the R1, β2 and γ2L

subunits of human GABA_Areceptors in pCDM8 vectors were

gifts from Dr. Paul J. Whiting (Merck Sharp & Dohme Research

Laboratories, Essex, UK).

(1-(4-(1-(tert-Butyl)dimethylsilyloxy)ethyl)phenyl)-2,2,2-trifl-

uoroethanone oxime (4). The fluoroketone was converted to

oxime as described by Shih & Bayley.²⁹A mixture of the fluo-

roketone 3 (15.7 g, 48.9 mmol), hydroxylamine hydrochloride

(4.1 g, 58.6 mmol), and anhydrous pyridine (25 mL) was heated

at 75 °C for 4 h. Ethanol (12 mL) was added and the mixture

heated at 60 °C for 2 h. The solvent was removed by rotary eva-

poration, the residue taken in ether (200 mL) and extrac-

ted three times with 200 mL portions of water. After the

ethereal layer was dried over magnesium sulfate, the solvent

was removed by rotary evaporation and the residue applied to

a column of silica gel, equilibrated with hexane/dichloro-

methane (75:25). Elution with dichloromethane gave the oxime

4. ¹H NMR spectrum: (CDCl₃) δ 7.45 and 7.44 (4H, AA^//BB^/

phenyl), 4.92 (q, 1H, methine), 1.42 (d, 3H, methyl), 0.92 (s, 9H,

methyl), 0.01 (6H, methyl). Calcd. for C₁₆H₂₄F₃NO₂Si: 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-trifl-

uoroethanone o-Tosyl Oxime (5). To a stirred, ice-cooled solu-

tion 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) were 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 was

dried over magnesium sulfate, the solvent was removed by

rotary evaporation and the residue was purified on a column

of silica gel, equilibrated with 20% dichloromethane in hexane.

Elution with 50% dichloromethane in hexane yielded the tosy-

late 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%.

Analytical Chemistry. H NMR spectra were recorded on a

Jeol Eclipse 400 MHz spectrometer in CDCl₃with tetramethylsi-

lane as reference by Acorn NMR Spectroscopy Service (Livermore,

CA). UV spectra were recorded on a Hewlett-Packard spectro-

photometer. 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 per-

formed 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.

(1-(4-Bromophenyl)ethoxy)(tert-butyl)dimethylsilane (2). To a

solution of 4-bromo-R-methylbenzyl alcohol (18.1 g, 0.09 mol)

and t-butyl-dimethylsilyl chloride (14.65 g, 0.099 mol) in anhy-

drous dichloromethane (100 mL) at room temperature under

argon was added dropwise a solution of DBU (15.8 g, 15.5 mL,

0.104 mol) in anhydrous dichloromethane (100 mL). After being

stirred 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

3-(4-(1-(tert-Butyl)dimethylsilyloxy)ethyl)phenyl)-3-((trifluo-

romethyl)-diaziridine (6). 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 tempera-

ture and stirred overnight. The mixture was taken in ether (75 mL),

filtered, and the precipitate was 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.

(88% yield) of a colorless, liquid silane derivative 2. H 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%.

3-(4-(1-(tert-Butyl)dimethylsilyloxy)ethyl)phenyl)-3-((trifluo-

romethyl)-3H-diazirine (7). To a mixture of the crude diaziridine

6 (9.36 g, 28 mmol) and triethylamine (5 mL) in dichloro-

methane (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

(1-(4-(1-(tert-Butyldimethylsilyloxy)ethyl)phenyl)-2,2,2-tri-

fluoroethanone (3). The fluoroketone was synthesized following

the procedure described by Fishwick et al.²⁸that converts bromo

compounds to fluoroketone in high yield. The silyl-protected bromo

compound 2 (23.7 g, 75 mmol) in anhydrous THF (200 ml) was

cooled to -78 °C in ether/dry ice bath and treated under argon

by dropwise addition of n-butyl lithium (54.6 mL of 1.6 M

solution in hexane) over a period of 1 h. After the solution was

stirred at -78 °C for 75 min, a solution of diethyl trifluoroace-

tamide (16.9 g, 99.4 mmol) in anhydrous THF (50 mL) was

added dropwise 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 with-

out 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. ¹H NMR

spectrum: (CDCl₃) δ 8.04 and 7.51 (4H, AA^//BB^/phenyl), 4.94

(q, 1H, methine), 1.41 (d, 3H, methyl), 0.92 (s, 9H, methyl), 0.01

obtained. H 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%.

3-(4-(1-(Bromoethyl)phenyl)-3-((trifluoromethyl)-3H-diazirine

(8). The tert-butyldimehylsilyl protecting group of the diazirine

7 was replaced by a bromo group by the procedure of Aizpurua

et al.³⁰A solution of the diazirine 7 (5.5 g, 16.5 mmol) in

anhydrous dichloromethane (25 mL) was added to a suspen-

sion of triphenylphosphine dibromide (7.7 g, 18.1 mmol) in

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6441

anhydrous dichloromethane (40 mL). The mixture was stirred at

room temperature for 15 h. The solution was diluted with

dichloromethane (125 mL), extracted twice with 100 mL of

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.

1-(4-(3-((Trifluoromethyl)-3H-diazirin-3-yl)phenyl)ethanamine

(9). A solution of the bromo compound 8 (4.1 g, 14 mmol) in

methanol (100 mL) was saturated with ammonia and the solu-

tion was kept for 72 h in a closed vessel. The solution was rotary

evaporated and the residue was 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 chromatog-

raphy 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)ethy-

lamino)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 was stirred at room temperature for 48 h. The mixture

was diluted with ether (30 mL), filtered, and the precipitate

was 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 equili-

brium 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 C₁₄H₁₆F₃-

N₃O₂: 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-(trifluoromethyl)-3H-diazirin-3-yl)phenyl)-

ethyl)methanamido)ethanoate (11). Formylation of 10 was per-

formed with formic anhydride by the procedure of Waki &

Meienhofer.³¹A solution of 2 M formic acid in dichloromethane

(8 mL) was added dropwise with stirring and with cooling by ice

bath to a solution of diisopropylcarbodiimide 1.01 g, 8 mmol in

anhydrous dichloromethane (10 mL). After being stirred 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 inso-

luble residue was removed by centrifugation. The crude product

was purified by chromatography on a silica gel column, equili-

brated with dichloromethane. Elution with the equilibration

solvent, containing 10% ether yielded 1.3 g (95%) of a pale

colored, viscous formyl derivative 11. ¹H 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.

¹H NMR: (CDCl₃) δ 8.41 and 8.18 (1H, formyl), 7.35 and 7.2

(4H, AA^//BB^/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 C₁₅H₁₆F₃N3O₃: 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 pro-

cedure of Jones et al.³²as modified by Godefroi et al.¹Sodium

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 suspen-

sion 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 was ex-

tracted with a mixture of xylene (9 mL) and water (3 mL), the

aqueous layer was separated, and the xylene layer was 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 was stirred at room temperature

for 48 h. The mixture was extracted with chloroform, the

organic layer was 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 and extracted with brine

and the organic layer was 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 s. H 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) λ_max358 nm, ε = 345 M^-1cm^-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 þve)

M/Z: calculated for (C16 H15 F3 N4 O2 þH)þ 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 Mit-

sunobu 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-R-methyl-

benzyl alcohol 1 (Aldrich Chemicals lot certification: [R]²⁰D =

-38.2 and þ38.8 deg (C = 1%, CHCl₃) for the S and R enantio-

mer, 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 was separated and the aqueous layer was

extracted twice with 10 mL portions of ether. The combined

organic extracts were washed with saturated NaCl solution and

6442 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17

Husain et al.

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 ([R]²⁰D = -27.3 and þ28.0 deg

(C = 1%, ethanol) for the S- and R-enantiomer, respectively)

indicated that the diazirinyl alcohol retained the chirality of the

starting bromoalcohol 1.

length) were housed in large glass jars filled with Amquelþ

(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 five 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 min tadpoles were individually flipped using the

hooked end of a fire-polished glass pipet until a stable response

was reached (usually up to 40 min). Anesthesia was defined as

the point at which the tadpoles could be placed in the supine

position, but failed to right themselves after 5 s (loss of righting

reflex, LoRR). All animals were placed in a recovery beaker of

Amquelþ treated tap water and monitored for 30-60 min for

fatality or full recovery. The quantal concentration response

curves were analyzed by the method of Waud³⁴using an Excel

macro kindly provided by N. L. Harrison, A. Jenkins, and S. P.

Singh (Weill Medical College of Cornell University).

Electrophysiology of GABA_A, 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 des-

cribed.^35,36In vitro transcription from linearized cDNA tem-

plates and purification of subunit specific cRNAs was carried

out using Ambion mMessage Machine RNA kits and spin

columns. For GABA_Areceptor studies, oocytes were injected

with ∼100 ng of total mRNA (R1, β2, γ2L) mixed at a ratio of

1:1:1 transcribed from human GABA receptor subunit cDNAs

in pCDNA3.1. β2M286W GABA_AR mRNA was prepared as

described previously.²¹For Torpedo nAChR ((R1)₂β1δ1γ1) stud-

ies, oocytes were injected with ∼25 ng of total mRNA mixed at a

ratio of 2R:1β:1γ:1δ as previously described.³⁶For human

5-HT_3Astudies, 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 Laboratories (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.

(R)-TFD-etomidate (13). Mitsunobu reaction between ethyl-

1H-imidazole carboxylate and (S)-14 was carried out as de-

scribed by Zolle et al. for the synthesis of chiral metomidate.¹⁹

solution of (S)-14 (253 mg, 1.1 mmol) in anhydrous THF (2 mL)

was added dropwise to a stirred solution of ethyl-imidazole car-

boxylate (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 was 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, and the residue

was 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 ethyla-

cetate/hexane 50/50 to yield R-TFD-product (150 mg). 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). Rotation measurement [R]²⁰D = þ41.4 deg

(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.¹⁹Analytical chromatography on

Chiracel OD-H column indicated that there was less than 0.5%

racemization.

(S)-TFD-etomidate (13). S-TFD-etomidate was synthesized

by Mitsunobu reaction between ethyl-1H-imidazole carboxy-

late 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. [R]²⁰D = þ44.0 deg (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 an-

hydrous 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-etomi-

date 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.

GABA_Aand 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 regres-

sion to the Hill (logistic) equations of the general form:

I_X=I_GABA,max¼ ðI_X,max=I_GABA,maxÞꢀð1=ð1 þ ðEC₅₀=½XꢁÞ ÞÞ

where X is the concentration of the activating ligand, I_GABA,max

is the maximally evoked current, EC₅₀is the concentration of X

eliciting half of its maximal effect, and n is the Hill coefficient of

activation. Inhibition experiments were fit with logistic equa-

tions of the form:

I ¼ 1 - ð½Xꢁ =ðIC₅₀þ ½Xꢁ ÞÞ

General Anesthetic Potency. Xenopus laevis tadpoles (Xenopus

One, Dextor, Michigan) in the prelimb-bud stage (1-2 cm in

5-HT_3AR Concentration-Response Studies. Currents were

amplified with a GeneClamp 500B amplifier (Molecular Device,

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6443

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-min

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, coexposure 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 concentra-

tion-response data fitted to the Hill Equation:

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. Ali-

quots (75 μL, 150 pmol ACh binding sites) were incubated at

room temperature for 40 min with 0.3 μM [³H]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 min 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 photo-

labeled samples, and the polypeptides were resolved on 1.5 mm

thick, 8% polyacrylamide/0.33% bis-acrylamide gels. Follow-

ing electrophoresis, the polypeptides were visualized by staining

with Coomassie Blue R-250 (0.25% w/v in 45% methanol and

10% acetic acid). [³H]TFD-etomidate photoincorporation into

the membrane polypeptides subunits was visualized by fluoro-

graphy (Amplify, Amersham Biosciences GE Healthcare) with

exposure to film (Kodak BIOMAX XAR Film) for 4-6 weeks,

and ³H incorporation into individual polypeptide bands excised

from the stained gel was quantified by liquid scintillation

counting.³⁷

I ¼ I_max=½1 þ ðIC₅₀=½IC₅₀=agonistꢁÞ ꢁ

where I is the peak current evoked by the agonist, IC₅₀is 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 inactiva-

tion or desensitization.

Allosteric Regulation of the GABA_AReceptor Ligand Binding.

Fresh whole bovine brain was placed on ice, and the cortex was

rapidly removed, the gray matter was resected, 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 (650g, 10 min,

4 °C), and its supernatant was centrifuged again at 150000g for

48 min. The pellet was resuspended in distilled water and

recentrifuged at 150000g 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 concen-

tration, 1 mg/mL) or [³H]muscimol (5 nM, 20 Ci/mmol, Perki-

nElmer Life Sciences; final protein concentration, 0.25 mg/mL).

Nonspecific binding was determined by carrying out incuba-

tions in the presence of 7.5 μM flurazepam for [³H]flunitraze-

pam 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 scintilla-

tion counting after addition of 2.5 mL of scintillation fluid

(Ecolume, ICN).

5-HT_3AReceptor Antagonist Binding. 5-HT_3AR rich mem-

branes, expressed in HEK 293 cells, corresponding to 200 pmol

of binding sites, were incubated in triplicate for 2 h at room tem-

perature 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 preincubated in 0.5% Poly(ethyleneimine)

solution (P3143, Sigma-Aldrich) for an hour. Samples were

filtered under a vacuum and washed twice with 7 mL of cold

HEPES/EDTA buffer. Filters were dried under a lamp for 1 h,

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 &

Cohen³⁷from Torpedo californica electric organs (Aquatic

Research Consultants, San Pedro, CA), were resuspended at 2 mg

Acknowledgment. This research was supported by a grant

from the National Institute for General Medical Sciences to

K.W.M. (GM 58448) and by the Department of Anesthesia,

Critical Care and Pain Medicine, Massachusetts General

Hospital. We thank Dr. Niall Hamilton, Organon Labora-

tories, UK, for a kind gift of R-(þ)-etomidate.

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