DeriVatiVes of Etomidate for Photolabeling
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16 4823
tion.13 However, further studies at higher drug concentrations
are required to determine photolabeling efficiency when binding
sites are fully occupied, and direct identification of the labeled
amino acids will be necessary to determine the actual side chain
preferences for these novel etomidate photoprobes. When
extrapolating to future photolabeling studies of GABAA recep-
tors, the higher affinity of the new agents will lead to greater
occupancy at a given concentration, likely resulting in higher
fractions of specific photoincorporation.
Conclusion. We have synthesized and characterized the
pharmacology of two potent new general anesthetics that are
photoactivable etomidate derivatives able to insert into the
nAcChoR in an allosterically regulated manner. These agents
also enhance agonist-stimulated currents with high selectivity
and potency in their likely physiological target, the GABAA
receptor, a member of the same superfamily as the nAcChoR.
Thus, they have the necessary pharmacological and photochemi-
cal properties to be useful in identifying the site of etomidate-
induced anesthesia.
of 4 (37 mg, 45% yield). The product was converted to crystalline
solid hydrochloride by acidification with HCl in ether. NMR
(CDCl3) δ 1.95 (d, 3H), 5.36 (s, 2H), 6.36 (q, 1H), 7.28 (m, 4H,
aromatic), 7.35 (m, 3H, aromatic), 7.46 (m, 2H, aromatic), 7.65
(m, 1H), 7.75 (m, 5H), 8.32 (s, 1H), 9.29 (s, 1H). UV spectrum
(methanol) λmax 355 nm, ꢀ ) 310 M-1 cm-1. Elemental analysis.
Calcd for C21H18ClF3N4O2‚H2O: C, 53.79%; H, 4.30%; N, 11.95%.
Found: C, 53.08%; H, 4.26%; N, 11.53%.
Preparation of 4(Hydroxymethyl)benzophenone (6). 4-(Bro-
momethyl)benzophenone 5 (2.75 g, 10 mmol) in dioxane (25 mL)
was mixed with a suspension of calcium carbonate powder (5 g,
50 mmol) in water (25 mL) and refluxed for 20 h. The solvent was
removed by rotary evaporation, and the residue was suspended in
dichloromethane (30 mL) and carefully treated with 1 M HCl until
the residue dissolved. The organic layer was separated, washed with
water, and dried over magnesium sulfate. The solvent was removed
by rotary evaporation, and the residue was purified on a column of
silica with 10% ether in dichloromethane, yielding the hydroxy
compound 6 (0.65 g, 31% yield). NMR (CDCl3) δ 4.72 (s, 2H),
7.64 (m, 2H), 6.36 (q, 1H), 7.18 (m, 2H), 7.30 (m, 3H), 7.53 (m,
4H), 7.65 (m, 1H), 7.75 (m, 9H). UV spectrum (methanol) λmax
330 nm, ꢀ ) 206 M-1 cm-1. Elemental analysis. Calcd for
C14H12O2: C, 79.23%; H, 5.70%. Found: C, 79.41%; H, 5.81%.
Preparation of R-4-Benzoylbenzyl-1-(1-phenylethyl)-1H-imi-
dazole-5-carboxylate (7). The benzobenzyl derivative 7 was
synthesized from hydroxybenzophenone 2 (48 mg, 0.22 mmol) and
R-1-(1-phenylethyl)-1H-imidazole-5-carboxylic acid (0.2 mmol) by
a procedure similar to that described for the preparation of TDBzl-
etomidate, 4. The reaction mixture was purified by silica gel
chromatography and by HPLC, yielding 23 mg (25% yield) of
BzBzl-etomidate 7. NMR (CDCl3) δ 1.90 (d, 3H), 5.36 (s, 2H),
6.36 (q, 1H), 7.18 (m, 2H), 7.30 (m, 3H), 7.53 (m, 4H), 7.65 (m,
1H), 7.75 (m, 5H), 8.18 (s, 1H). UV spectrum (methanol) λmax 331
nm, ꢀ ) 215 M-1 cm-1. Elemental analysis. Calcd for C26H23-
ClN2O3‚H2O: C, 67.17%; H, 5.42%; N, 6.02%. Found: C, 67.64%;
H, 5.47%; N, 6.09%.
Experimental Section
Materials and Methods. Anhydrous dichloromethane, diiso-
propylcarbodiimide, p-(dimethyamino)pyridine, and Merck silica
gel 60 A, 230-400 mesh were obtained from Aldrich (Milwaukee,
WI). 4-[3-(Trifluoromethyl])-3H-diazirin-3-yl]benzoic acid was
obtained from Bachem (King of Prussia, PA). R(+)-etomidate was
a kind gift from Organon Laboratories (Newhouse, Lancashire,
Scotland). R(+)etomidate used in GABAA receptor electrophysi-
ology 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, â2, and γ2L subunits of human GABAA receptors in
pCDM8 vectors were gifts from Dr. Paul J. Whiting (Merck Sharp
& Dohme Research Labs, Essex, U.K.).
Preparation of R-[3H]-4-[3-(Trifluoromethyl])-3H-diazirin-
3-yl]benzyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylate (4).
R-Etomidate was tritiated to a specific activity of 20 Ci/mmol and
converted by alkaline hydrolysis to R-[3H]1-(1-phenylethyl)-1H-
imidazole-5-carboxylic acid as described earlier.11 A mixture of
R-[3H]1-(1-phenylethyl)-1H-imidazole-5-carboxylic acid (8.8 mCi),
4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl alcohol 2 (23 µmol),
(dimethylamino)pyridine (10 µmol), and (dimethylamino)pyridine
hydrochloride (10 µmol) in anhydrous dichloromethane (0.6 mL)
was stirred with diisopropylcarbodiimide (20 µL of 1 M solution)
for 24 h at room temperature. The reaction mixture was purified
by chromatography on a silica gel column equilibrated with
dichloromethane and eluted successively with dichloromethane and
with dichloromethane containing 5% ether.
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).
Preparation of 4-[3-(Trifluoromethyl)-3H-diazirin-3-yl]benzyl
Alcohol (2). A 1 M solution of diborane in THF (2.4 mL) was
added dropwise over a period of 40 min to a solution of
4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoic acid 1 (92 mg) in
anhydrous THF (1 mL) under argon at -5 °C. Stirring was
continued for 5 h at -5 °C. The solution was slowly quenched by
adding 10% acetic acid in methanol (1 mL). The mixture was rotary
evaporated, and the residue was dissolved in dichloromethane and
applied to a column of silica gel, equilibrated with dichloromethane.
Elution was performed successively with dichloromethane and a
mixture of 2.5% ether in dichloromethane, yielding the alcohol 2
(68 mg, 78% yield). NMR (CDCl3) δ 4.63 (s, 2H), 7.23 (d, 2H,
aromatic), 7.46 (d, 2H, aromatic). UV spectrum (methanol) λmax
360 nm, ꢀ ) 309 M-1 cm-1. Elemental analysis. Calcd for
C9H7F3N2O: C, 50.00%; H, 3.27%; N, 12.96%. Found: C, 49.29%;
H, 3.28%; N, 12.44%.
Preparation of R-1-(1-Phenylethyl)-1H-imidazole-5-carboxylic
Acid (3). The carboxylic acid derivative 3 was prepared from R-2-
ethyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylate (etomidate) by
alkaline hydrolysis as described previously.11
Preparation of R-4-[3-(Trifluoromethyl)-3H-diazirin-3-yl]-
benzyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylate (4). A mix-
ture of R-1-(1-phenylethyl)-1H-imidazole-5-carboxylic acid 3 (0.2
mmol), 4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl alcohol 2 (48
mg, 0.2 mmol), and (dimethylamino)pyridine (24 mg, 0.2 mmol)
in anhydrous dichloromethane (0.5 mL) was stirred with diisopro-
pylcarbodiimide (47 µL, 0.3 mmol) overnight at room temperature.
The reaction mixture was purified by silica gel chromatography
with 5% diethyl ether in dichloromethane, yielding an oily residue
Preparation of R-[3H]4-Benzoylbenzyl-1-(1-phenylethyl)-1H-
imidazole-5-carboxylate (7). Tritiated benzoylbenzyl derivative of
etomidate was synthesized by a procedure similar to that described
for the synthesis of labeled trifluoromethyldiazirinyl-etomidate, 4.
General Anesthetic Potency. With institutional approval, general
anesthetic potency was assessed in prelimb bud Xenopus tadpoles,
1.5-2 cm in length (Xenopus 1, Inc., Dexter, MI). Groups of five
tadpoles were placed in covered 100 mL glass beakers or square
slide-staining dishes in oxygenated aqueous solutions buffered with
2.5 mM Tris HCl at pH 7.4 under low levels of ambient light.
Agents were added from stock solutions in ethanol. The final
concentration of ethanol did not exceed 5 mM, a concentration that
does not contribute to anesthesia.31 Tadpoles were tipped manually
with a flame polished pipet, and failure to right after 5 s was defined
as loss of righting reflexes (LORR). The response stabilized within
30-40 min, and measurements were made at 50-60 min. All
animals were placed in freshwater and observed the next day for
toxicity. The quantal concentration response curves were analyzed
by the method of Waud32 using an Excel macro kindly provided
by N. L. Harrison, A. Jenkins, and S. P. Singh (Weill Medical
College of Cornell University).