M -Azipropofol (AziP m) a photoactive analogue of the intravenous general anesthetic propofol

Article abstract of DOI:10.1021/jm1004072

Propofol is the most commonly used sedative-hypnotic drug for noxious procedures, yet the molecular targets underlying either its beneficial or toxic effects remain uncertain. In order to determine targets and thereby mechanisms of propofol, we have synthesized a photoactivateable analogue by substituting an alkyldiazirinyl moiety for one of the isopropyl arms but in the meta position. m-Azipropofol retains the physical, biochemical, GABA_A receptor modulatory, and in vivo activity of propofol and photoadducts to amino acid residues in known propofol binding sites in natural proteins. Using either mass spectrometry or radiolabeling, this reagent may be used to reveal sites and targets that underlie the mechanism of both the desirable and undesirable actions of this important clinical compound.

Full text of DOI:10.1021/jm1004072

J. Med. Chem. 2010, 53, 5667–5675 5667
DOI: 10.1021/jm1004072
m-Azipropofol (AziPm) a Photoactive Analogue of the Intravenous General Anesthetic Propofol
Michael A. Hall,‡ Jin Xi,† Chong Lor,§ Shuiping Dai,§ Robert Pearce,§ William P. Dailey,‡ and Roderic G. Eckenhoff*,†
†Department of Anesthesiology and Critical Care, and ‡Department of Chemistry, University of Pennsylvania School of Medicine, Philadelphia,
Pennsylvania, and §Department of Anesthesiology, University of Wisconsin, Madison, Wisconsin
Received March 31, 2010
Propofol is the most commonly used sedative-hypnotic drug for noxious procedures, yet the molecular
targets underlying either its beneficial or toxic effects remain uncertain. In order to determine targets
and thereby mechanisms of propofol, we have synthesized a photoactivateable analogue by substituting
an alkyldiazirinyl moiety for one of the isopropyl arms but in the meta position. m-Azipropofol retains
the physical, biochemical, GABAA receptor modulatory, and in vivo activity of propofol and photo-
adducts to amino acid residues in known propofol binding sites in natural proteins. Using either mass
spectrometry or radiolabeling, this reagent may be used to reveal sites and targets that underlie the
mechanism of both the desirable and undesirable actions of this important clinical compound.
Introduction
blood flow and metabolic rate1,6 and disfavoring those targets
underlying propofol’s toxicities. Most notable of these toxi-
cities would be respiratory depression and cardiac dysfunc-
tion, including bradycardia and hypotension,7 and also EEG
activation and even seizures.8,9 Here we describe the synthesis
and characterization of a propofol analogue with an alkyl-
diazirinyl group in place of one of the isopropyl arms.
Propofol (2,6-diisopropylphenol) is the most widely used
intravenous general anesthetic agent for induction and main-
tenance of anesthesia as well as sedation for procedures and in
the critically ill patient.1 Although more potent than the inhaled
anesthetics, propofol’s micromolar EC50 and lipophilicity still
predict only brief interactions with its in vivo targets. This is
considered clinically useful because it allows for short dura-
tion of action once administration is stopped. Unfortunately,
these transient interactions make the molecular targets of
general anesthetics difficult to study. The use of photoaffinity
labeling has emerged as a promising technique for investigat-
ing these otherwise fleeting interactions.2
Results
An obvious position for introduction of a photoactivatable
group into propofol would be as part of one of the existing
isopropyl groups in propofol itself. However, we recognized
that these positions, by virtue of being ortho to the phenolic
hydroxyl group, would likely lead to intramolecular reaction
of the active position with the hydroxyl group rather than the
desired intermolecular reaction with proteins. Therefore, we
designed our propofol photoaffinity probe to contain the
photoactivatable group in a meta position relative to the
hydroxyl group. This design would preclude any intramole-
cular reaction of the reactive site with the phenolic hydroxyl
group.
A molecular target that is thought to contribute to propo-
a
fol’s ability to induce anesthesia is the GABAA receptor, a
ligand-gated chloride channel responsible for the majority of
inhibitory signaling in the brain. Propofol both activates
GABAA receptors and potentiates the activity of receptors
activated by GABA; these activities correlate with its in vivo
anesthetic potency.3-5 However, the precise molecular inter-
actions between propofol and the GABAA receptor remain
unclear. Photoactive compounds that mimic propofol’s me-
chanism of action would allow a greater understanding of the
mechanisms.
In addition to studies directed at known propofol targets, a
propofol photoaffinity label could be used for identifying
other molecular targets of this drug, both desirable and un-
desirable. These studies would facilitate medicinal chemistry
aimed at favoring targets underlying effects such as induction
of anesthesia, anticonvulsant effects, and reduction of cerebral
Synthesis of m-Azipropofol (AziPm, 1). Synthesis of 1 is
shown is Scheme 1. Cumene (2) is treated with bromine in the
presence of a catalytic amount of iodine to give known
bromo compound 3, which is treated with magnesium fol-
lowed by N-trifluoroacetylpyrrolidine to give the previously
reported trifluoroacetylbenzene 4. Nitration conditions pro-
vide nitro compound 5, which is reduced under catalytic
hydrogenation conditions to give aniline 6. Conversion of 6
*To whom correspondence should be addressed. Address: Department
of Anesthesiology and Critical Care, 311a John Morgan Building, 3620
Hamilton Walk, Philadelphia, PA 19104. Phone: 215-662-3705. Fax: 215-
349-5078. E-mail: Roderic.eckenhoff@uphs.upenn.edu.
a Abbreviations: AziPm, m-azipropofol; 1-AMA, 1-aminoanthra-
cene; HSAF, horse spleen apoferritin; GABAA, γ-aminobutyric acid
receptor type A, EEG, electroencephalograph; ITC, isothermal titration
calorimetry; MEM, minimal essential media.
r
2010 American Chemical Society
Published on Web 07/02/2010
pubs.acs.org/jmc

5668 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Hall et al.
Scheme 1
Table 1. Physicochemical Properties
MW density, g/mL log P a dipole,a D HSAF KD, μM
1 244
propofol 178
1.12
0.96
3.93
3.79
2.12
1.70
19
10
a Calculated; see Experimental Procedures. log P = octanol/water
partition coefficient.
Table 2. Binding Parameters
1
propofol
fluorescence
competition
4.8 (3.9-6.5) 12 (9, 15)
1b
-1.0
(-0.8, -1.2)
fluorescence
competition
ITC
ITC
affinitya
Hill slope 1b
21 (20-22) 23 (17, 32)
-1.2
(-0.9, -1.6)
a Mean (95% CI), in μM. b Data fitted to single class binding site,
so Hill slope fixed at 1.
Table 2 and in Figures 1 and 2. There existed good agreement
across methods and across the two compounds. Propofol
had a 2- to 4-fold higher affinity for HSAF than did 1. In the
photo-occlusion experiments, a 40% reduction in 1-AMA
fluorescence was noted when compared to control HSAF
samples that were identically treated but without the addi-
tion of 1 (Figure 3). Combined, these results support the
proposition that 1 binds the anesthetic pocket of HSAF11
rather than inner filter effects explaining the competition
results.
In Vivo Anesthetic Potency. Both 1 and propofol showed
anesthetic activity in X. laevis tadpoles by reversibly extin-
guishing both spontaneous and elicited (startle reflex) move-
ment. The results of these assays are summarized in Table 3
and presented in Figure 4. The Hill slopes and EC50 values
for the two compounds had overlapping 95% confidence
intervals for all measurements, indicating similar potencies
in tadpoles. The only difference noted during the trials was
that propofol had more rapid kinetics; it reached its maxi-
mum effect in about half of the time that it took 1 to reach
steady state. Also, it was found that neither compound had a
concentration that immobilized 100% of the tadpoles (loss of
startle response) reversibly. This suggested a very narrow
therapeutic ratio for both compounds, which was confirmed
to be less than 2. The EC50 reported here for propofol in
Xenopus tadpoles agrees with previously reported values,
despite subtle differences in methodology.4
Electrophysiological Studies. We compared effects of pro-
pofol and 1 on R1β2γ2L GABAA receptors expressed in
HEK293 cells. At 0.3 and 3 μM, both compounds reversibly
enhanced responses of receptors activated by 3 μM GABA
(in fold changes, propofol 5.1 ( 3 and 9.3 ( 7.1; 1 1.8 ( 0.1
and 1.7 ( 0.4; p<0.05 for all). In addition, propofol but not
1 directly activated receptors; i.e., current flow was produced
even in the absence of GABA. At the highest concentration
tested (30 μM), propofol strongly activated receptors. By
contrast, 30 μM 1 did not directly activate receptors, and
receptor activation in the combined presence of 30 μM 1 and
GABA was reduced or eliminated. However, upon removal
of 1 and GABA, there was a modest “rebound” or tail cur-
rent. This pattern of responses has been suggested to arise
from direct channel block by anesthetics.12
to phenol 7 is accomplished using nitrous acid and boiling
aqueous acid. Subsequent protection of the free phenol group
using the tert-butyldimethylsilyl protecting group provides 8.
Introduction of the diazirine group into ketone 8 closely fol-
lowed a method previously described for a similar ketone.10
Thus, conversion to oxime 9 and oxime tosylate 10 followed
standard procedures. Treating 10 with ammonia produces
diaziridine 11, which is oxidized with iodine and triethylamine
to produce diazirine 12. Deprotection of the tert-butyldi-
methylsilyl group occurs under standard fluoride conditions
to give 1 in an overall yield of 18% starting with cumene.
Physicochemical Properties. The physicochemical proper-
ties for both 1 and propofol are summarized in Table 1. The
octanol/water partition coefficients of 1 (log P) and propofol
were calculated to be 3.93 and 3.79, respectively. Density of 1
was determined to be 1.12 g/mL. The calculated molecular
dipole for 1 was 2.12 D, while the dipole for propofol, cal-
culated the same way, was 1.70 D. The UV absorption spec-
trum demonstrates a prominent diazirine peak at 368 nm
with an extinction coefficient (Σ368) of 670/M. Because of this
absorption profile, all photolysis was done with a 350 nm UV
lamp. The rate of disappearance of the diazirine during
irradiation in a cuvette, as measured by changes in the UV
spectrum, has a t1/2 of 34 min (95% CI=31-37 min). The
maximum concentration of 1 achievable in distilled water
was found to be 185 μM.
Photolabeling. In order to confirm the ability of 1 ability to
act as a photolabel, HSAF was incubated in buffer with and
without saturated (185 μM) 1 and exposed to 350 nm illu-
mination for 20 min. Concentration and trypsinization were
followed by nano-LC/MS to identify peptides and residues
Binding Assays. Equilibrium binding of 1 to horse spleen
apoferritin (HSAF) was determined by two methods, ITC
and fluorescence competition, and the results are provided in

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5669
Figure 1. Isothermal titration calorimetry of the HSAF interaction with either 1 (left) or propofol (right), using sequential titrations.
More sigmoid shaped curve with propofol indicates higher affinity, consistent with the values given in Table 2.
Figure 3. Photo-occlusion experiment. In order to confirm that
fluorescence competition was not due to inner filter or other
nonspecific interactions between 1-AMA, 1, and HSAF, the protein
was photoadducted by 1/UV, washed, and then evaluated for
1-AMA binding via fluorescence enhancement. The photoadducted
sample had significantly reduced 1-AMA binding compared to the
UV control, consistent with adduction in the 1-AMA anesthetic
binding site.
Figure 2. Fluorescence competition with 1-aminoanthracene. Ti-
tration of the HSAF/1-AMA combination with either 1 (filled
symbols) or propofol (open symbols) produced inhibition of fluor-
escence consistent with competition for binding. Lines represent
nonlinear least-squares fits to variable slope Hill functions. Values
are given in Table 2.
On the basis of previous investigations into the anesthetic
potency of various alkyl-substituted phenols,13 we chose to
substitute the alkyldiazirine for one of the isopropyl groups
while leaving the other isopropyl in the 2-position on the ring.
This decision produced the possibility of synthesizing a
series of azi-propofols, varying in which position on the ring
carried the diazirine group. While the ortho-azi-propofol
would demonstrate the greatest steric similarity to propofol,
the concern that the compound would internally rearrange
after diazirine decomposition instead of photolabeling made
this a less attractive initial objective. The rearrangement of
similar aromatic compounds into the corresponding ortho-
quinone methanides has been described.14 With this potential
for decomposition via an intramolecular pathway, we were
concerned about the effectiveness as a photolabel. Therefore,
that had been modified by 216 Da (1 without the dinitrogen).
We detected peptides covering 42.9% of the apoferritin
sequence. The HSAF L chain demonstrated clear evidence
of two adducted peptides. MS/MS sequencing indicated that
the adducts were located at leucine-81 and leucine-24, both
of which are known to be lining residues in the previously
identified propofol binding cavity (see Supporting Informa-
tion and Figure 6).11
Discussion and Conclusions
In order to create a photolabel analogue of propofol, we
initially decided to incorporate an alkyldiazirine functional
group after the success of creating the smaller azi-isoflurane.2

5670 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Table 3. Tadpole Studies Results
Hall et al.
1,
endpoint
propofol,
endpoint
spontaneous movement
startle reflex
spontaneous movement
startle reflex
EC50 a (μM)
Hill slope
1.1 (0.9-1.4)
2.8 (1.6-4.0)
3.1 (2.7-3.5)
2.9 (1.7-4.1)
1.1 (0.9-1.3)
2.7 (1.6-3.9)
2.8 (2.6-3.0)
3.0 (2.4-3.7)
a Values in parentheses is the 95% confidence interval.
impaired movement (<3 μM), with 1 being less potent. This is
consistent with previous work on propofol analogues where
loss of bulk from the 6-position or transfer of bulk from the
ortho to meta or para resulted in loss of potency.4 It is of
interest that this lower potency at GABAA receptors is also
reflected in the HSAF affinity but not in the in vivo potency.
Previous work has shown that the relationship between
GABAA enhancement in heterologous expression systems
and tadpole immobilization only explains about half the
variation,4 so it is possible that additional molecular targets of
propofol and 1 are contributing to immobilization in the
tadpole.
Alternatively, the ability of high concentration 1 to block
the GABAA complex (Figure 5) may indicate the recruitment
of additional, low affinity sites within this ion channel because
of the electrophysiological signature for channel blockade at
high (lethal) concentrations. Such a functionally antagonistic
site would be expected to reduce the coagonist actions of the
propofol site, as was observed. Finally, it is unlikely that
receptor blockade underlies lethality, since both propofol and
1 were similarly lethal in tadpoles. Nevertheless, the ability of
1 to block receptors may provide a new tool for understanding
the origin of channel block at high concentrations, a feature of
other anesthetics.12,16,17
Figure 4. In vivo dose/response relationships. Tadpoles were eval-
uated with two end points: spontaneous (left set of curves, squares)
and elicited (right set of curves, circles) movement. Filled symbols
represent 1, and open symbols are propofol data. No significant
differences between the two drugs could be detected for either end
point.
after ruling out the ortho compound and considering that
previously reported13 structural analogues of the meta- and
para-compounds retained anesthetic potency, we chose to
synthesize the meta-compound 1 first.
Subsequent attempts to synthesize the para-azi-propofol
(AziPp) using the synthetic strategy outlined in Scheme 1
failed in the final, deprotecting step. We hypothesized thatthis
occurred because the base-catalyzed deprotection creates a
phenoxide intermediate. The phenoxideisconjugated withthe
diazirine in such a way that the diazirine decomposes imme-
diately; evolved N2 gas was observed during the addition of the
tetrabutylammonium fluoride. Attempts to synthesize AziPp
using a methoxymethyl protecting group, which is removed
under acid conditions, were only marginally more successful.
Any synthesized AziPp was seen to rapidly decompose when
isolation was attempted. Diazirines conjugated with phenols
are known to be very unstable under basic conditions,15 and
withthis conjugationpotentially lowering the pKa of the mole-
cule below that of phenol, we expect there to be significant
decomposition and loss of photolabeling functionality when
AziPp is added to the pH 7.4 phosphate buffered saline in
which the HSAF experiments are performed. Hence, 1 was
synthesized as described herein.
From a comparison of 1 to propofol, the diazirine exchange
for the isopropyl adds 66 Da to the molecular weight and in-
creases the dipole considerably, from 1.70 to 2.12 D, but still
makes the compound marginally more hydrophobic, with
log P changing from 3.79 to 3.93. Even with these changes, in
vitro and in vivo binding studies indicate that 1 is a highly ana-
logous and similarly potent anesthetic when compared to
propofol. The 1/HSAF interaction was found to be of slightly
lower affinity compared to propofol/HSAF, but their EC50
values for anesthetizing tadpoles were not different. Both
compounds also enhanced agonist-stimulated activity of the
GABAA receptor complex at concentrations near those that
The photolabeling experiment demonstrates that both 1
and propofol are recognized as similar by a protein and that 1
has the ability to act as a photoaffinity label. The competition
and occlusion studies had already suggested that 1 binds in the
propofol-binding cavity of HSAF. The photolabeling experi-
ment identified that the 1 binding site was within labeling
distance of two residues, leucine-81 and leucine-24, which are
adjacent to each other and contribute to the lining of the pre-
viously identified propofol binding site of HSAF.11 This con-
firms that 1 binds within the same cavity that propofol (and
inhaled anesthetics) binds, making this compound a promis-
ing photolabel for studies of propofol binding in other poten-
tialdrug targets. Wecannotruleout thatadduction took place
in 57% of the protein sequence for which peptides were not
identified, but we consider this unlikely because only a single
propofol site has been found with crystallography11 and the
ITC and 1-AMA competition data for 1 are analogous to the
data for propofol and give no indication of additional binding
sites. The fact that affinity of the 1/HSAF interaction pre-
dicted the lowered efficacy for GABAA enhancement is fur-
ther evidence that the HSAF site bears strong architectural
and physicochemical similarityto the site underlying allosteric
enhancement in GABAA receptors.
Current application of 1 requires detection using mass
spectrometry. While this is useful for detecting adducted
peptides within proteins, it is less useful for detecting novel
targets in complex mixtures or anatomic regions of the brain
or heart that might have a high density of targets. Thus, a
future direction for this work involves incorporating a radio-
active moiety into 1. For example, [3H]1 could be synthesized
by iodination of the aromatic ring followed by catalytic

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5671
Figure 5. Electrophysiology in GABAA receptors. Excised patches containing R1β2γ2L receptors were exposed to 3 μM GABA alone (left
column) or together with propofol (middle column) or 1 (right column) for 500 ms. At 0.3 and 3 μM, both compounds enhanced responses to
GABA. At a high concentration (30 μM), propofol directly activated channels, whereas 1 blocked channels.
Octanol/water partition coefficients were calculated using
XLOGP3.18
Electronic structure calculations at the ab initio RHF/6-
31þG(d,p) geometry optimized level demonstrate two confor-
mations with 160 cal/mol difference as the lowest energy con-
formations.19 These two conformations differ in rotation of
180° about the bond between the diazirine carbon and the aro-
matic ring such that the plane of the ring bisects the diazirine
double bond in both conformations. This indicates relatively
free rotation of the diazirine group relative to the aromatic ring.
The dipole moment calculated for each of these conformations
was found to be 1.99 and 2.25 D. With total energies so close,
we assumed that these conformations would contribute equally
to the total dipole at room temperature, giving 1 a dipole of 2.12 D.
The same calculations for propofol demonstrated fairly free
rotation about bonds connecting each isopropyl group to the
aromatic ring, creating several minima with different individual
dipole moments. Consideration of a Maxwell-Boltzmann dis-
tribution for the total energy of each conformation at room
temperature allowed the calculation of an average dipole of 1.70
D, as the individual dipoles ranged between 1.67 and 1.80 D.
Binding Studies. Fluorescence Competition. The affinity of 1
for HSAF was determined by adding increasing amounts of 1 to
a solution of HSAF and a constant amount of 1-aminoanthra-
cene (1-AMA).20 All solutions were prepared in pH 7.4 phos-
phate buffered saline. In 500 μL quartz cuvettes, 7 μM HSAF
and 7 μM 1-AMA were combined with increasing concentra-
tions of 1 (0-110 μM). The fluorescence of 1-AMA in each of
these solutions was determined with 380 nm excitation while
monitoring emission between 400 and 800 nm. The fluorescence
curves were corrected by subtracting the baseline fluorescence
curves of 1-AMA and HSAF. The fluorescence intensity vs
concentration data were fitted to variable slope Hill models to
obtain the IC50 and Hill slope. The KD was then calculated using
the Cheng-Prusoff equation to correct the IC50 for the presence
of the 1-AMA competitor.20
Figure 6. Adducted HSAF residues. Shown is the HSAF anesthetic
binding site with propofol bound (yellow), from PDB code 3F33.
Residues photolabeled by 1 (L24 and L81 from each of two
monomers) (pink) are immediately adjacent to propofol. Mass
spectra are provided in the Supporting Information.
hydrogenation to replace the iodine with tritium, using [3H]2
gas as the source.
Experimental Procedures
Physicochemical Properties. The density of 1 was calcu-
lated from the slope of the volume/mass relationship of this low-
volatility compound. The UV spectrum and extinction coeffi-
cient of the diazirine absorption were first determined from
a methanolic solution of 1 of known concentration, and then
maximal water solubility was calculated from the extinction
coefficient. The rate of photolysis was determined from a 160 μM
solution in distilled water in a 1 cm path length quartz cuvette in
contact with a 350 nm UV light for 30 min, taking UV spectra to
monitor the disappearance of the diazirinyl peaks every 3 min.
Photo-Occlusion. To ensure that the measured effect of 1 on
the fluorescence of 1-AMA in HSAF was indeed caused by
competition for the anesthetic-binding pocket of HSAF rather
than an inner-filter effect of 1, a photo-occlusion experiment

5672 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Hall et al.
was performed. In 1 mL quartz cuvettes, 5 μM HSAF with or
without 140 μM 1 in pH 7.4 phosphate buffered saline was
irradiated with 350 nm light (Rayonet RPR-3500 lamp) at 2 mm
distance for 20 min. Both samples were then passed through a
PD-10 buffer exchange column to remove any remaining soluble
1 or unadducted photolysis products. HSAF solutions were re-
turned to the quartz cuvettes, and baseline fluorescence curves
were obtained for the 1 sample, the irradiated control, and a con-
trol that had not been irradiated. There was no significant cha-
nge in fluorescence between the two controls, indicating a lack of
appreciable UV damage to the HSAF. To each of the irradiated
HSAF samples was added 1-AMA (3 μM final concentration),
and fluorescence was recorded. Occlusion of the anesthetic
(1-AMA) binding site by adducted 1 is reflected by a significant
loss of fluorescence in the 1/UV sample.
(37°C, 5% CO2) in culture medium consisting of minimum essential
medium (MEM) with L-glutamine and Earle’s salts (Gibco), sup-
plemented with MEM amino acids solution (0.1 mM), sodium
pyruvate (1 mM), 1% penicillin/streptomycin, and 10% fetal
bovine serum (Harlan). For transfection, cDNAs for rat GA-
BAA receptor R1, β2, and γ2L subunits were subcloned into the
multiple cloning site of the mammalian expression vector
pUNIV.21 For receptor expression, cells were plated onto 60 mm
culture dishes (Corning). After incubation for 24-48 h, cells
were co-transfected with rat R1β2γ2L (1:1:10) and 100 ng of
pCEP4-EGFP (Clontech) using Lipofectamine 2000 (Invitrogen),
then replated onto a 12 mm circle cover glass in 35 mm Petri
dishes (Falcon) 12-16 h after transfection. Transfected cells
were identified using a mercury arc lamp and a GFP filter cube
(Chroma Technology).
Isothermal Titration Calorimetry. The thermodynamics of 1’s
interaction with HSAF was assessed by isothermal titration
calorimetry using a Microcal, Inc. VP ITC (Northampton, MA;
http://www.microcalorimetry.com) and compared to that of
propofol. The ITC consists of a matched pair of sample and re-
ference vessels (1.43 mL) enclosed in an adiabatic enclosure and
a rotating stirrer-syringe for titrating aliquots of the ligand solu-
tion into the sample vessel. The sample cell contained 5 μM
HSAF, and the reference cell contained water. Saturated photo-
label (185 μM) was loaded in the syringe (volume of 0.28 mL) for
injecting into the sample. Because of the low solubility of these
ligands, sequential titrations were employed to achieve sufficient
molar ratios for reliable model fitting, and these titrations were
linked using ConCat32 software (Microcal, Inc., Northampton,
MA). In addition to these sequential titrations, control titra-
tions were performed, including ligand into buffer, buffer into
protein, and buffer into buffer, which were then used to correct
the experimental titration, ligand into protein. Origin 5.0 (Microcal
Software, Inc., Northampton, MA) was used to fit thermo-
dynamic parameters to the heat profiles using a single binding
site class.
Tadpole Studies. In vivo activity studies were performed in
Xenopus tadpoles. Groups of 10 tadpoles were placed in 30 mL
of pond water containing various concentrations of 1 or propo-
fol. Tadpoles were incubated in the pond water with compound
for 30 min to allow for full equilibration, regardless of the
progress toward immobilization. At the end of the 30 min, the
anesthetic effect was assessed using two end points. The first was
loss of spontaneous motion, defined as the percentage of tad-
poles at each dose that did not swim, right themselves, or twitch
in a 30 s observation. The second end point was loss of elicited
movement to a single, sharp tap on the lid of their dish. Tadpoles
that did not twitch or swim in response to this stimulus were
scored as anesthetized. The water in the dish was then replaced
with fresh pond water, and recovery was observed over the next
24 h.
Photolabeling. Small aliquots of pH 7.0 phosphate buffer
containing ∼5 μM HSAF with and without saturated (185 μM)
1 were placed in 300 μL, 1 mm path length quartz cuvettes and
exposed to 350 nm light (Rayonet RPR-3500 lamp) at <2 mm
distance for 20 min. Control samples received only UV irradia-
tion. Samples were washed and concentrated with 50 kDa
centrifuge filters and then trypsinized and injected into a
nano-LC/MS (10 cm C18 capillary column) to separate and
identify the digested peptides. Eksigent NanoLC proteomics
experiments were run at 200 nL/min for 60 min with gradient
elution. Nanospray was used to spray the separated peptides
into LTQ (Thermo Electron). Xcalibur was used to acquire the
raw data, and modified (photolabeled) peptides were identified
using Sequest. Mass spectrometry equipment and software were
available in the Proteomics Core Facility at the University of
Pennsylvania.
Electrophysiological Recordings. Recordings were performed
at room temperature on the stage of an inverted microscope.
Solutions were applied to excised outside-out patches or whole
cells using a custom-designed 12-barrel application system con-
structed of Teflon (28 gauge) and polyimide tubing (Cole-
Parmer). The tubes were sealed together and glued onto a micro-
scope slide using UV curing glue (Norland optical adhesive) and
secured in place using epoxy. The barrels were individually con-
nected to solution reservoirs (10 mL glass syringes) using Teflon
tubing and valves. The application system was mounted on a
motorized scanning stage (Corvus). An open-tip solution ex-
change time constant of 20-30 ms was typically achieved. The
recording chamber was perfused continuously with HEPES-
buffered saline containing the following (mM): NaCl 145, KCl
5, MgCl2 1, CaCl2 1.8 HEPES 10; pH 7.4. Recording electrodes
were fabricated from KG-33 glass (Garner Glass Company)
using a multistage puller (model P-87, Sutter Instruments). The
tips were fire-polished; open tip electrode resistance was typi-
cally 4-7 MΩ when filled with standard recording solution.
Recording pipettes were filled with the following (mM): KCl
130, EGTA 5, HEPES 10, MgCl2 1, MgATP 5; pH 7.2. All
recordings were obtained at a holding potential of -40 mV using
an Axopatch 200A amplifier (Molecular Devices). GABA solu-
tions were prepared fresh daily from powder and were diluted to
the desired concentration (3 μM) in HEPES-buffered saline
solution. Propofol and 1 solutions were prepared by diluting
stock solutions containing 0.53 mM propofol and 0.18 mM azi-
propofol in HEPES-buffered saline solution.
Data Analysis. Ionic currents were analyzed using Clampfit
10 (Molecular Devices) and Origin 7.5 (OriginLab, Northampton,
MA). Peak currents were measured relative to baseline and
normalized to the peak current elicited by a pulse of 3 μM
GABA. Data are presented as mean ( SD.
General Synthetic Procedures. All reagents and solvents were
used as received from commercial sources unless otherwise
noted. 1H, 13C, and 19F NMR spectra were recorded on a
Bruker DMX 360 MHz nuclear magnetic resonance spectro-
1
meter. H, 13C, and 19F NMR spectra are in the Supporting
Information. Determination of the final purity of 1 used capil-
lary GC (30 m ꢀ 0.25 mm DB-5 column, 135 °C injector, 200 °C
detector, column temperature of 100 °C for 25 min, then 10 °C/
min to 200 °C) using flame ionization detection. Under these
conditions 1 has a retention time of ∼25 min and was shown to
be >98% pure by integration.
2-Isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol (1).
To a round-bottom flask with a magnetic stir bar was added
466.8 mg (1.30 mmol) of 12 and 5 mL of dry THF. The solution
was cooled in an ice bath, and 1.45 mL (1.45 mmol) of TBAF
(1.0 M in THF) was added dropwise via syringe over 5 min. The
reaction mixture was allowed to warm and stir for 20 min at
room temperature. The mixture was poured into 20 mL of satu-
rated NH4Cl and extracted with methylene chloride. The or-
ganic layers were combined, washed with water, and dried with
Na2SO4. Solvent was removed under reduced pressure, and the
residue was purified on silica gel (20:1 hexanes/ethyl acetate).
Electrophysiology. HEK293 Cell Culture and GABAA Recep-
tor Expression. HEK293 cells (CRL 1573, American Type Cul-
ture Collection) were maintained in standard culture conditions

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5673
Evaporation of the solvent yielded 279.1 mg (88%) of 1 as a pale-
yellow oil which was one spot by TLC. 1H NMR: δ 7.25 (d, J =
10.6 Hz, 1H), 6.72 (d, J = 8.1 Hz, 1H), 6.60 (bs, 1H), 4.97 (s,
1H), 3.21 (septet, J = 6.9 Hz, 1H), 1.25 ppm (d, J = 6.9 Hz, 6H).
19F NMR: δ -65.27 ppm. 13C NMR: δ 152.9, 136.5, 127.7,
127.1, 122.1 (q, J = 274 Hz), 119.1, 113.3, 28.2 (q, J = 40 Hz),
27.0, 22.2 ppm. UV spectrum: λmax = 282, 368 nm. HRMS (ESI
neg): m/z calcd for C11H10F3N2O (M - H)-, 243.0745; found,
243.0746.
1-Bromo-4-isopropylbenzene (3). A 500 mL round-bottom
flask with a magnetic stir bar was filled with 10.0 g (39.4 mmol)
of iodine crystals and 80.0 g (667 mmol) of isopropylbenzene.
The solution was cooled in an ice bath, and 110.0 g (688 mmol)
of bromine was added over 15 min with good stirring. A solution
of 60.0 g (1.07 mol) of potassium hydroxide in 200 mL of H2O
was added to the mixture. The reaction mixture was steam-
distilled for 12 h, with periodic addition of fresh water. The
organic layer of the distillate was dried over MgSO4 and then
distilled at atmospheric pressure to give 108.9 g (82%) of a clear,
colorless liquid, bp 217 °C (lit. bp 216 °C).22 1H NMR: δ 7.44
(2H, d, J = 8.4 Hz), 7.13 (2H, d, J = 8.3), 2.85 (1H, septet),
1.27 ppm (6H, d, J = 6.9 Hz).
2,2,2-Trifluoro-1-(4-isopropylphenyl)ethanone (4). In a three-
neck round-bottom flask with a magnetic stir bar, 23.28 g (117
mmol) of 3 was dissolved in 115 mL of dry THF. To this solution
was added 2.87 g (118 mmol) of magnesium metal. The reaction
vessel was fitted with a condenser topped with a nitrogen line
and an addition funnel containing 15.66 g (101 mmol) of 2,2,2-
trifluoro-1-pyrrolidin-1-ylethanone in 24 mL of dry THF. The
pot was heated slowly to reflux, and heating was continued for
20 min after the magnesium began to react to ensure complete
consumption of 3. The flask was cooled in an ice/salt bath for
25 min, during which time a fine, white precipitate formed. The
amide solution was added dropwise over 30 min at 0 °C to the
well stirred solution. After the addition was complete, the ice/
salt bath was removed, and the mixture was stirred at room
temperature for 1 h. The reaction was quenched with 30 mL of
saturated aqueous NH4Cl solution and then vacuum-filtered to
leave a clear, golden liquid. After the mixture was dried over
MgSO4, solvent was removed under reduced pressure. Distilla-
tion under aspirator pressure yielded 16.20 g (74%) of 4, bp13
95 °C (lit. bp15 95 °C).23 1H NMR: δ 8.03 (2H, d, J = 7.6 Hz),
7.41 (2H, d, J = 7.7 Hz), 3.05 (1H, septet), 1.31 ppm (6H, d,
J = 6.9 Hz). 19F NMR: δ -71.73 ppm.
temperature for 2 days. The mixture was filtered through Celite,
and the residue was rinsed with 95% ethanol. The combined
ethanol and acetic acid were removed under reduced pressure to
give 6 in quantitative yield. 1H NMR: δ 7.37 (1H, s), 7.49 (1H, d,
J = 8.1 Hz), 7.30 (1H, d), 3.60 (2H, broad s), 2.95 (1H, septet),
1.31 ppm (6H, d, J = 6.8 Hz). 19F NMR: δ -71.49 ppm. 13C
NMR: δ 180.4 (q, J = 34 Hz), 144.2, 141.0, 128.3, 126.1, 120.9,
116.8 (q, J = 295 Hz), 116.1, 28.1, 21.7 ppm. HRMS (CIþ): m/z
calcd for C11H13NOF3(M þ Hþ) 232.0949; found 232.0939.
2,2,2-Trifluoro-1-(3-hydroxy-4-isopropylphenyl)ethanone (7).
A large beaker with a magnetic stir bar and thermocouple was
filled with 20.44 g (88.4 mmol) of 6 and a solution of 31.0 mL of
concentrated H2SO4 and 24.0 mL of water. This mixture was
cooled to 0 °C, and a solution of 9.16 g (133 mmol) of NaNO2 in
23.0 mL of H2O was added dropwise over the course of 90 min,
taking care to keep the temperature of the mixture below 4 °C.
Once the addition was complete, this mixture was poured in
several small additions over 30 min through a condenser into a
round-bottom flask containing a refluxing solution of 68 mL of
concentrated H2SO4 and 90 mL of H2O. Additional water was
used to rinse out the beaker. Once the addition was complete, the
mixture was allowed to reflux for an additional 15 min before
being poured into 1 L of cold water, at which point a dark oil
separated. The mixture was extracted with ether, and the ether
was washed with water, dried over MgSO4, and removed at
reduced pressure to leave 16.14 g (79%) of crude 7. This black oil
was taken to the next step without further purification. An
analytical sample was prepared by multiple sublimations under
reduced pressure, mp 58-60 °C. 1H NMR: δ 7.64 (1H, d), 7.48
(1H, s), 7.38 (1H, d), 3.35 (1H, sep), 1.31 ppm (6H, d). 19F NMR:
δ -71.59 ppm. 13C NMR: δ 180.2 (q, J = 34 Hz), 153.4, 144.2,
128.4, 127.2, 123.4, 116.7 (q, J = 295 Hz), 115.9, 27.6, 22.0 ppm.
HRMS (CIþ): m/z calcd for C11H11F3O2Na (M þ Na), 255.0609;
found, 255.0605.
1-[3-(tert-Butyldimethylsilanyloxy)-4-isopropylphenyl]-2,2,2-
trifluoroethanone (8). To a round-bottom flask with a magnetic
stir bar was added 10.78 g (46.4 mmol) of 7, 250 mL of dry THF,
12.2 g (94.5 mmol) of diisopropylethylamine, and 16.92 g (112.4
mmol) of tert-butyldimethylchlorosilane. This mixture was
stirred for 24 h under nitrogen atmosphere at room temperature.
The mixture was poured into 500 mL of H2O and extracted with
methylene chloride. The combined organic phase was washed
with water, and volatiles were removed under vacuum. Crude 8
was purified through a short column of silica, eluting with pure
hexanes to give 7.62 g (47%) of 8 as an oil. 1H NMR: δ 7.67 (1H,
bd), 7.50 (1H, bs), 7.38 (1H, d, J = 8 Hz), 3.40 (1H, septet), 1.25
2,2,2-Trifluoro-1-(4-isopropyl-3-nitrophenyl)ethanone (5). In a
beaker with a magnetic stir bar, an amount of 44.50 g of con-
centrated H2SO4 was added dropwise to 16.18 g (74.9 mmol) of 4
at -10 °C over 25 min, during which time a red precipitate
formed. While the temperature was maintained below -5 °C, a
mixture of 10.84 g of concentrated HNO3 and 20.66 g of con-
centrated H2SO4 was added dropwise over 25 min. The ice/salt
bath was replaced with an ice/water bath, and the mixture was
allowed to stir for 1 h. The reaction mixture was poured onto
200 g of ice and was extracted with ether (3 ꢀ 100 mL). The ether
fractions were combined and washed with saturated aqueous
NaHCO3 until no more CO2 was generated. The ether layer was
dried over anhydrous MgSO4, and the solvent was removed
under reduced pressure. The resulting oil was distilled under
(6H, d, J = 7 Hz), 1.06 ppm (9H, s). 19F NMR: δ -71.2 (s). 13
C
NMR: δ 179.8 (q, J = 34 Hz), 153.3, 148.4, 128.3, 126.9, 123.4,
118.9, 116.9 (q, J = 290 Hz), 27.2, 25.7, 22.2, 18.2 ppm. HRMS-
(CIþ): m/z calcd for C17H26F3O2Si (Mþ), 347.1654; found,
347.1653.
1-[3-(tert-Butyldimethylsilanyloxy)-4-isopropylphenyl]-2,2,2-
trifluoroethanone Oxime (9). To a round-bottom flask with a
magnetic stir bar was added 6.87 g (19.8 mmol) of 8, 1.75 g (25.2
mmol) of hydroxylamine hydrochloride, and 50 mL of pyridine.
The mixture was heated in an oil bath to 60 °C and stirred for 4 h
under nitrogen atmosphere. Volatiles were then removed under
vacuum. The remaining crude product was partitioned between
methylene chloride and water, and the organic phase was
subsequently washed with water. Solvent was removed under
vacuum to leave 6.82 g (95%) of 9 as an approximately 1:1 mix-
ture of oxime diastereomers as determined by NMR. The crude
product was taken to the next step without further purification.
An analytical sample was prepared by flash chromatography on
silica gel (10:1 hexanes/ethyl acetate, Rf ≈ 0.3). 1H NMR: δ 9.4
(1H, bs), 7.32 (0.6H, d, J = 8 Hz), 7.27 (0.4H, d, J = 8 Hz), 7.18
(0.6H, bd), 7.09 (0.4H, bd), 7.06 (0.6H, bs), 6.93 (0.4H, bs), 3.37
(1H, m), 1.24 (6H, m), 1.06 (9H, s), 0.28 ppm (6H, s). 19F NMR:
δ -62.3 (1F, s), -66.3 ppm (1.2F, s). 13C NMR: δ 152.8, 152.7,
147.8, 147.4, 147.3, 147.0, 142.2, 142.0, 127.8, 126.5, 126.4,
1
aspirator pressure to give 17.17 g (88%) of 5, bp15 140 °C. H
NMR: δ 8.39 (1H, s), 8.23 (1H, d), 7.72 (1H, d, J = 8.4 Hz), 3.47
(1H, septet), 1.36 ppm (6H, d, J = 6.8 Hz). 19F NMR: δ -72.39
ppm. 13C NMR: δ 178.6 (q, J = 37 Hz), 150.3, 150.1, 133.0, 128.9,
128.4, 125.4, 116.4 (q, J = 289 Hz), 29.2, 23.1 ppm. HRMS (CIþ):
m/z calcd for C11H11NO3F3( M þ H) 262.0691; found 262.0691.
1-(3-Amino-4-isopropylphenyl)-2,2,2-trifluoroethanone (6). A
round-bottom flask with a magnetic stir bar was filled with
26.50 g (101.5 mmol) of 5 dissolved in 160 mL of glacial acetic
acid. To this solution was added 68 mg of 30% Pt on asbestos
along with 511 mg of 10% activated Pd on carbon. The mix-
ture was stirred under a 1 atm hydrogen atmosphere at room

5674 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Hall et al.
123.5, 122.3, 121.6, 120.0, 119.2, 118.5, 118.0, 116.9, 26.8, 26.7,
25.8, 25.7, 22.6, 22.5, 18.2, -4.2, -4.3 ppm. HRMS(CIþ): m/z
calcd for C17H27F3NO2Si (Mþ), 362.1763; found 362.1780.
1-[3-(tert-Butyldimethylsilanyloxy)-4-isopropylphenyl]-2,2,2-
trifluoroethanone Oxime Tosylate (10). A round-bottom flask
with a magnetic stir bar was filled with 5.75 g (15.9 mmol) of 9
and 110 mL of methylene chloride. To this stirred solution was
added 93 mg (0.76 mmol) of 4-(N,N-dimethylamino)pyridine,
3.18 g (16.7 mmol) of p-toluenesulfonyl chloride, and 2.20 g
(21.8 mmol) of triethylamine. The mixture was stirred for 24 h
under nitrogen atmosphere at room temperature. The reaction
mixture was then partitioned between water and methylene chlo-
ride, and the organic phase was subsequently washed with addi-
tional water. Volatiles were removed under vacuum to leave 7.50 g
(91%) of crude 10 as a 2:3 mixture of diastereomers as deter-
mined by NMR. This crude product was taken to the next step
without further purification. An analytical sample was prepared
by flash chromatography on silica gel (20:1 hexanes/ethyl ace-
tate) and was followed by TLC in 10:1 hexanes/ethyl acetate,
Rf = 0.4, to give 10 as a thick gum. 1H NMR: δ 7.95 (2H, m), 7.4
(2H, m), 7.32 (0.6H, d), 7.28 (0.4H, d), 7.07 (1H, m), 6.97 (0.6H,
m), 6.91 (0.4H, m), 3.38 (1H, m), 2.47 (3H, m), 1.24 (3H, m), 1.07
(9H,m), 0.31 (3.4H, s), 0.26 ppm (2.6H, s) 19F NMR: δ -61.2
(2F), -66.1 ppm (3F). 13C NMR: δ 153.5, 153.2, 152.9, 152.8,
146.1, 145.9, 143.8, 143.6, 131.7, 131.4, 129.9, 129.8, 129.1, 126.8,
126.7, 125.6, 122.2, 121.8, 119.1, 118.5, 115.9, 31.6, 26.8, 26.8, 25.7,
22.6, 22.4, 21.65, 18.3, 18.2, 14.0, -4.3 ppm. HRMS(CIþ): m/z
calcd for C24H33F3NO4SSi (M þ Hþ), 516.1852; found 516.1835.
3-[3-(tert-Butyldimethylsilanyloxy)-4-isopropylphenyl]-3-tri-
fluoromethyldiaziridine (11). A solution of 7.50 g (14.5 mmol) of
10 dissolved in 15.0 g of diethyl ether was added to excess liquid
ammonia at -78 °C and stirred vigorously as it was allowed to
come to room temperature over the course of 5 h. More ether
was added to the reaction mixture, and the combined organic
phase was washed with water. The ether solution was dried over
Na2SO4, and solvent was removed under reduced pressure to
leave 5.13 g (98%) of crude 11. The crude product could be taken
to the next step without further purification. An analytical sam-
ple was prepared by flash column chromatography (hexanes/
ethyl acetate = 20:1) followed by TLC in the same solvent sys-
tem with an Rf of 0.25 to give 11 as a thick oil. 1H NMR: δ 7.26
(1H, d, J = 6.6 Hz), 7.20 (1H, d, J = 6.6 Hz), 7.065 (1H, s), 3.34
(1H, m, J = 5.7 Hz), 2.75 (1H, broad s), 2.20 (1H, broad d, J =
5.7 Hz), 1.24 (6H, d), 1.06 (9H, s), 0.28 ppm (6H, s). 19F NMR: δ
-75.43 ppm. 13C NMR: δ 152.78, 141.25, 129.60, 128.17,
126.56, 123.5 (q, J = 278 Hz), 120.45, 117.78, 57.66 (q, J =
36 Hz), 26.61, 25.74, 25.73, 22.64, 22.61, 18.25, -4.22, -4.27 ppm.
HRMS (CIþ): m/z calcd for C17H28F3N2OSi (Mþ), 361.1923;
found, 361.1926.
3-[3-(tert-Butyldimethylsilanyloxy)-4-isopropylphenyl]-3-tri-
fluoromethyl-3H-diazirine (12). A round-bottom flask with a
magnetic stir bar was filled with 5.35 g (14.8 mmol) of crude 11,
40 mL of methylene chloride, and 5.8 g (22.9 mmol) of iodine.
The mixture was cooled in an ice bath, and 4.57 g (45.2 mmol) of
triethylamine was added dropwise. The ice bath was removed,
and the mixture was allowed to stir at room temperature for
45 min. It was poured into 650 mL of 1 M NaOH and was stirred
vigorously for 15 min. The mixture was extracted with methyl-
ene chloride. The organic layer was washed with water and eva-
porated under reduced pressure. The resultant crude product
was flushed through a short column of silica with pure hexanes
to give 3.10 g (58%) of 12 as a yellow oil. 1H NMR: δ 7.22 (1H, d,
J = 8.1 Hz), 6.70 (1H, s), 6.63 (1H, d, J = 8.1 Hz), 3.30 (1H, sep,
J = 6.9 Hz), 1.19 (6H, d, J = 6.9 Hz), 1.04 (9H, s), 0.27 ppm (6H,
s). 19F NMR: δ -65.67 ppm. 13C NMR: δ 153.1, 141.1, 127.2,
126.8, 122.2 (q, J = 275 Hz), 118.9, 116.4, 28.2 (q, J=40 Hz),
26.6, 25.7, 22.5 18.2, -4.3 ppm. HRMS(CIþ): m/z calcd for
C17H26F3N2OSi (Mþ), 359.1767; found, 359.1762. The column
was then flushed with an equal volume of 10:1 hexanes/ethyl
acetate to recover 1.78 g of unreacted 11.
Acknowledgment. The authors declare no conflicts of inte-
rest. This work was supported by NIH Grants GM055876
(R.G.E.), MH84836 (R.G.E.), and NS056411 (R.P.) and the
Wisconsin Institutes for Discovery Seed Grant (R.P.).
Supporting Information Available: NMRspectraofcompounds
and mass spectra of protein. This material is available free of
charge via the Internet at http://pubs.acs.org.
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