P -(4-azipentyl)propofol: A potent photoreactive general anesthetic derivative of propofol

Article abstract of DOI:10.1021/jm200943f

We synthesized 2,6-diisopropyl-4-[3-(3-methyl-3H-diazirin-3-yl)propyl] phenol (p-(4-azipentyl)propofol), or p-4-AziC5-Pro, a novel photoactivable derivative of the general anesthetic propofol. p-4-AziC5-Pro has an anesthetic potency similar to that of pro

Full text of DOI:10.1021/jm200943f

Article
pubs.acs.org/jmc
p-(4-Azipentyl)propofol: A Potent Photoreactive General Anesthetic
Derivative of Propofol
Deirdre S. Stewart,†,⊥ Pavel Y. Savechenkov,∥,⊥ Zuzana Dostalova,† David C. Chiara,§ Rile Ge,†
Douglas E. Raines,† Jonathan B. Cohen,§ Stuart A. Forman,† Karol S. Bruzik,∥ and Keith W. Miller*,†,‡
†Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, 32 Fruit Street, Boston, Massachusetts
02114, United States
§
‡Department of Biological Chemistry and Molecular Pharmacology and Department of Neurobiology, 220 Longwood Avenue,
Harvard Medical School, Boston, Massachusetts 02115, United States
∥College of Pharmacy, University of Illinois at Chicago, 833 S. Wood Street (M/C 781), Chicago, Illinois 60612-7231, United States
ABSTRACT: We synthesized 2,6-diisopropyl-4-[3-(3-methyl-3H-diazirin-3-yl)propyl]phenol
(p-(4-azipentyl)propofol), or p-4-AziC5-Pro, a novel photoactivable derivative of the general
anesthetic propofol. p-4-AziC5-Pro has an anesthetic potency similar to that of propofol. Like
propofol, the compound potentiates inhibitory GABAA receptor current responses and
allosterically modulates binding to both agonist and benzodiazepine sites, assayed on
heterologously expressed GABAA receptors. p-4-AziC5-Pro inhibits excitatory current
responses of nACh receptors expressed in Xenopus oocytes and photoincorporates into native
nACh receptor-enriched Torpedo membranes. Thus, p-4-AziC5-Pro is a functional general anesthetic that both modulates and
photoincorporates into Cys-loop ligand-gated ion channels, making it an excellent candidate for use in identifying propofol
binding sites.
mechanisms of action leads to the question of whether or not
they act at the same site on the GABAA receptor. [3H]-
Azietomidate photolabeling assays on bovine brain GABAA
receptors support the idea that etomidate and propofol may act
through different sites.11 Recent crystallographic studies on a
bacterial homologue of the pentameric receptors from
Gloebacter violaceus (GLIC) have identified an intrasubunit
binding site for propofol and volatile anesthetics, in contrast to
the intersubunit binding site identified for etomidate in the
GABAA receptor.10,12
The development of propofol-based photoaffinity reagents is
critical for identifying residues that interact with this important
drug in mammalian receptors. One such reagent has been
synthesized, 2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-
yl]phenol (m-TFD-propofol), a propofol analogue with an
alkyldiazirinyl group placed at the meta-position of the phenyl
ring.13 To further investigate the nature of propofol’s binding
site, we have now synthesized 2,6-diisopropyl-4-[3-(3-methyl-
3H-diazirin-3-yl)propyl]phenol (p-(4-azipentyl)propofol), or p-
4-AziC5-Pro (6), an analogue that keeps the core propofol
structure intact by adding the reactive diazirine group at the
para-position of the phenol ring (Scheme 2). Here, we describe
its synthesis and initial pharmacologic characterization. It
functions as a general anesthetic and interacts with the GABAA
and nACh receptor ligand-gated ion channels. Importantly, the
p-4-AziC5-Pro derivative differs from the previously described
photoreactive propofol (m-TFD-propofol) in the location of
INTRODUCTION
■
Propofol is the most widely used intravenous general anesthetic
for induction and maintenance of anesthesia.1 Its advantages
include both rapid-onset and rapid-recovery from anesthesia
accompanied with relatively few of the common side effects of
general anesthetics, such as postoperative nausea and vomiting.
Many lines of evidence suggest that one of the main
pharmacological actions for general anesthetics is the
potentiation of the type A γ-aminobutyric acid (GABAA)
receptor’s inhibitory response.2−7 GABAA receptors are a
member of the Cys-loop superfamily of pentameric ligand gated
ion channels that also includes glycine, serotonin (5-HT3), and
nicotinic acetylcholine (nACh) receptors. In addition to
intravenous and volatile anesthetics, GABAA receptors are
modulated by a number of other classes of drugs, including
neurosteroids, benzodiazepines, and ethanol.8,9
Identifying the binding sites of these different classes of drugs
on the GABAA receptor is of major interest for understanding
their mechanisms of action and for further drug development.
Although mutagenesis and genetic experiments have been
successful for identifying residues involved in the action of
general anesthetics, the use of affinity labels is more likely to
identify residues involved in a binding pocket. Experiments
using a photoreactive analogue of etomidate ([3H]-
azietomidate) identified two residues (αMet236 and
βMet286) as contributing to an intersubunit etomidate binding
site, only one of which had been implicated by earlier
mutagenesis experiments.10 Both propofol and etomidate can
potentiate the GABAA receptor’s current response and can
directly activate the receptor, activities that correlate with the
drugs’ in vivo anesthetic potency. The similarity in these drugs’
Received: August 1, 2011
Published: October 26, 2011
© 2011 American Chemical Society
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a
Scheme 1
a
(i) TFAA, AlCl3, DCM; (ii) TBDMSCl, imidazol, DMF; (iii) NH2OH, HCl, Py, MeOH; (iv) (a) TsCl, Py, DCM; (b) NH3, MeOH; (c) I2, TEA,
MeOH.
a
Scheme 2
a
(i) Succinic anhydride, AlCl3; (ii) MeOH, SOCl2; (iii) NaBH4; (iv) TEA, BOP, N,O-dimethylhydroxylamine; (v) TBDMSCl, imidazole; (vi) MeLi;
(vii) (a) NH3, MeOH, NH2OSO3H; (b) I2, TEA, MeOH; (viii) CsF, EtOH.
a
Scheme 3
a
(i) Ethylene glycol, (MeO)3CH, TsOH; (ii) KOH−H2O; (iii) TEA, BOP, NH(Me)OMe; (iv) MeLi, THF; (v) (a) NH3, MeOH, NH2OSO3H; (b)
I2, TEA; (vi) TFA−H2O; (vii) TBDMSCl, imidazole; (viii) NaBH4 or NaBT4; (ix) (a) CsF; (b) Et3SiH, TFA.
the reactive group, opening the possibility of exploring the
binding site in more detail.
omethyldiazirines have been described in the literature.14 On
the basis of the existing QSAR of propofol’s anesthetic activity,
the introduction of the trifluoromethyldiazirine moiety on the
aromatic ring of propofol could be best achieved at the 4-
position, since substitutions at this position do not significantly
alter the anesthetic properties of propofol.15 An alternative
RESULTS
■
Synthesis of Diazirine Analogues of Propofol. A large
number of photoaffinity reagents based on 1-aryl-1-trifluor-
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Figure 1. GABAA receptor current enhancement. (A, B) Two electrode voltage clamp was used to measure currents from oocytes injected with
human α1β2γ2L GABAA receptor subunits. Currents were elicited with 3 μM GABA (∼EC5) in the presence of increasing amounts of 6 (A) or
propofol (B). Solid lines represent the nonlinear least-squares fit of the data. For 6: EC50 = 4.5 ± 2.7 μM, nH = 1.4 ± 1.3, Imax = 22 ± 6% (four
oocytes). For propofol: EC50 = 5.2 ± 2.1 μM, nH = 1.7 ± 1.2, Imax = 16 ± 3% (two oocytes). Insets show current traces elicited by 3 μM GABA
(black bars) plus increasing amounts of drug (1, 3, 10, 30, 100 μM 6 and 1, 3, 10, 30, 100, 300 μM propofol) (gray bars). (C, D) GABA
concentration−response curves shift to the left in the presence of drugs. For plotting, currents were normalized to IGABA,max and [GABA] was
normalized to the control GABA EC50 for each experiment to minimize the effect of the spread of EC50 values of the different oocytes.38 The data
were fit as described above and in the Experimental Section for 6 (C): EC50 = 0.99 ± 0.11, nH = 1.0 ± 0.1 for GABA alone (solid symbols) and EC50
= 0.12 ± 0.03, nH = 0.9 ± 0.2 in the presence of 6 μM 6 (open symbols) (three oocytes). For propofol (D): EC50 = 1.35 ± 0.2, nH = 0.9 ± 0.1 for
GABA alone (solid symbols) and EC50 = 0.12 ± 0.04, nH = 1.1 ± 0.3 in the presence of 4.4 μM propofol (open symbols) (four oocytes).
strategy involves the removal of one of the isopropyl groups
and modification of the 3-position.13
short nonpolar spacer. The synthetic expediency and the
necessity to maintain a nonpolar environment at the bottom of
the ring suggested a three-carbon linker.
As our first synthetic target, we selected 4-trifluoromethyl-
diazirynylpropofol (1, 4-TFD-Pf) (Scheme 1). The intermedi-
ate 5 was synthesized in a sequence of Friedel−Crafts acylation,
phenol protection with dimethyl-tert-butylsilyl group, and
assembly of the diazirine group by a routine procedure.
However, the final deprotection of the diazirine 5 failed to
produce a stable product. We have tried without success a
variety of deprotecting conditions, including TBAF, TBAF−
acetic acid, HF, pyridine−HF, TsOH, and HCl. The rapid
decomposition of the target compound 1 is most likely due to
the electronic effect of the hydroxy group in the para-position
stabilizing the benzylic carbene. Indeed, syntheses of 4-
hydroxyphenyldiazirine have been previously reported, but no
detailed synthetic procedures were provided.16 In contrast, the
isomeric m-hydroxyphenyldiazirines are stable.17
The synthesis of the diazirine 6 shown in Scheme 2 started
with an acylation of propofol with succinic anhydride to give
the key intermediate 7. The next steps involved esterification of
the carboxyl group and an unusual direct reduction of the
ketoester 8 into the acid 11. Interestingly, such a reduction has
not been previously reported, and the model NaBH4 reduction
of the keto group in 4-acetylpropofol stops at the stage of a
corresponding alcohol. We hypothesize that the initially formed
alcohol 9 is first converted into the lactone 10 and that the
activation of the hydroxyl group by an ester enables further
reduction of the C−O bond. Thus, the ketone group in 8 is
converted into a methylene group in 11 via a one-step
procedure and under very mild conditions. The carboxylic acid
11 was next converted into the corresponding ketone 12 using
a Weinreb procedure,18 and the subsequent diazirination by the
modified method of Church and Weiss19 followed by
deprotection of the phenol afforded the target diazirine 6.
To avoid this source of instability, we redesigned the
photoactivatable analogue of propofol to separate the diazirine
moiety from the phenyl ring. In the new analogue 6 (Scheme
2), the photoaffinity label is linked to the propofol core via a
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For the purpose of radiolabeling of 6 with tritium, an
alternative synthesis was developed (Scheme 3) in which the
introduction of the label was achieved by the reduction of the
ketone (e.g., step iii in Scheme 2), performed at a later stage of
synthesis. Thus, the keto group in the ester 8 was protected as
1,3-dioxolane to give the ketal 14. The subsequent one-carbon
elongation of the ketone 15 and assembly of the diazirine 16
were performed analogously as described in Scheme 2. The
keto group was deprotected with TFA and the phenol
protected with TBDMS to form the diazirine 18. The
protection of phenol with the TBDMS group was necessary
to minimize decomposition of the reducing agent (NaBH4 or
NaBT4) by the acidic phenolic OH group during the
radiolabeling reduction step. The reduction of the ketone 18
with sodium borohydride afforded the alcohol 19, and the
subsequent further reduction with triethylsilane and desilylation
produced the final product 6. The tritiation at the level of 12
Ci/mmol was achieved by substituting NaBT4 for NaBH4 in
step viii-a.
the maximal GABA response with an of EC50 of 50 ± 5 μM
(two oocytes).
Modulation of Ligand-Binding to GABAA Receptors. To
assess allosteric action, the effects of 6 were compared to those
of propofol for modulating the binding of 1.5 nM [3H]-
flunitrazepam (benzodiazepine site) or 3 nM [3H]muscimol
(agonist site) to α1β3γ2 GABAARs (Figure 2).
Pharmacological Properties. Solubility and Partition
Coefficient. The saturated aqueous solubility of 6 in 0.01 M
Tris-HCl, pH 7.4, was 73 ± 4 μM (all errors herein are
standard deviations unless otherwise stated). At a concentration
close to saturation, the compound was stable at 4 °C in the
aforementioned buffer for at least 2.5 days and eluted as a single
fraction at 81.5% acetonitrile when analyzed using reverse
phase HPLC. The octanol/water partition coefficient was
estimated to be 15 600 ± 1200. For comparison, the saturated
concentration of pure propofol in water is 1 ± 0.02 mM, and
the octanol/water partition coefficient is 4300 ± 280.20
Anesthetic Activity. The anesthetic potency of 6 was
assayed in tadpoles. The concentration dependence of loss of
righting reflexes (LoRR) was examined in groups of five
animals at seven concentrations between 1.0 and 20 μM (ten
animals per concentration). Anesthesia was reversible, the
animals recovering in fresh solution overnight. The one death
was not at the highest concentration, so it was likely unrelated
to the agent. Only animals that fully recovered in fresh water
overnight were included in the analysis. The EC50 determined
with 69 animals was 3.2 ± 0.55 μM and the slope was 1.7 ± 0.4.
Thus, 6 is a general anesthetic with potency comparable to that
of propofol, which has an EC50 of 2.2 ± 0.2 μM.20
Enhancement of GABAA Receptor Channel Function.
Human α1β2γ2L GABAA receptor currents (at EC5) expressed
in Xenopus oocytes were potentiated with increasing amounts of
6 (Figure 1A) with an EC50 of 4.5 ± 2.7 μM, compared to 5.2
± 2.0 μM for propofol (Figure 1B). At maximal concentrations
of 6 (100 μM), current responses were enhanced ∼20-fold
(Figure 1A), similar to ∼15-fold seen with 30−100 μM
propofol (Figure 1B).
GABA concentration response curves (±drugs) were
determined at a fixed concentration of anesthetic equal to
twice the EC50 for tadpole anesthesia, a concentration generally
considered to represent clinical anesthesia, and were shifted to
the left ∼9-fold by 6 μM 6 (Figure 1C) and ∼11-fold by 4.4
μM propofol (Figure 1D).
Figure 2. [3H]Flunitrazepam and [3H]muscimol binding to GABAA
receptors. The specific [3H]flunitrazepam (1.5 nM) and [3H]-
muscimol (3 nM) binding to membrane suspensions of α1β3γ2
GABAA receptors heterogeneously expressed in HEK293 cells was
evaluated at various concentrations of 6 or propofol. The percent
enhancement of binding of three measurements are plotted as the
mean ± standard deviation.
Specific binding of the benzodiazepine, [3H]flunitrazepam,
was enhanced by both agents between 0.1 and 40 μM (Figure
2A). Above this concentration, propofol caused a rapid
decrease in binding up to 400 μM whereas 6 caused no further
change. Compound 6 was less efficacious, causing a ∼150%
enhancement compared to ∼200% for propofol. The
concentration dependence and efficacy for enhancement of
binding of the agonist [3H]muscimol by both agents were very
similar to that exhibited by [3H]flunitrazepam, although
propofol’s efficacy was even higher than 6's and its effect
reached a maximum at somewhat lower concentrations (Figure
Like propofol, 6 directly activated GABAA receptor currents
in the absence of GABA (data not shown). The direct
activation data were also fit with the nonlinear least-squares
regression to the Hill equation, and 6 produced currents of 10
± 3% of the GABA response with an EC50 of 15 ± 3 μM (three
oocytes), whereas propofol activated the receptor 45 ± 10% of
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2B). Again, propofol caused a sharp decrease in binding above
30 μM, but 6 did not.
Because the action of propofol was biphasic, we did not
attempt to fit the data to a function. In the case of 6, the
enhancement is quite small when compared to the errors, but
the data could be fit to a mass action curve with half effect
concentrations of ∼0.5 μM.
Inhibition of Torpedo nACh Receptors. In oocytes
expressing Torpedo nACh receptors, currents elicited by 10
μM ACh (∼EC20) were inhibited by increasing amounts of 6
with IC50 = 4.0 ± 1.2 μM compared to IC50 = 7.3 ± 1.4 μM for
propofol (Figure 3). The difference in the shapes of the current
Figure 4. Torpedo nACh receptor photoincorporation. nACh
receptor-rich membranes were photolabeled with 1.7 μM [3H]6 as
described in the Experimental Section, and the polypeptides were then
resolved by SDS−PAGE. (A) Polypeptides were visualized by
Coomassie Blue stain (lane 1, a representative lane), and 3H
distribution was determined by fluorography (lanes 2−9, 14-day
exposure) for aliquots photolabeled in the absence of additional drugs
(lanes 2 and 9) in the presence of 100 μM tetracaine (lane 3), 100 μM
phencyclidine (lanes 4 and 7), 300 μM propofol (lanes 5 and 6), and 1
mM carbamylcholine (Carb, lanes 6−8). Indicated on the left are the
stained bands corresponding to the nACh receptor subunits (α, β, γ,
and δ), rapsyn (Rsn), the α subunit of the Na+/K+-ATPase, calelectrin
(37K), and the voltage dependent anion channel (VDAC).39 (B)
Figure 3. Torpedo nACh receptor current inhibition. Oocytes
expressing wild type Torpedo nACh receptors were tested with 10
μM ACh (∼EC20) and then with 10 μM ACh plus increasing amounts
of propofol or the propofol derivative. The current traces in the top
right inset show one oocyte’s current response to 10 μM ACh plus
increasing amounts of propofol (0.1, 0.3, 1, 3, 10, 30, 100, 300 μM).
The effect of 6 on 10 μM ACh currents is shown in the bottom left
inset (1, 3, 10, 30, 100 μM). Nonlinear least-squares analysis of the
curves yielded the following: for propofol (open symbols), IC50 = 7.3
± 1.4 μM, nH = 0.8 ± 0.1 (three oocytes); for 6 (solid symbols), IC50
= 4.0 ± 1.2, nH = 0.9 ± 0.2 (three oocytes). Currents were normalized
to the 10 μM ACh response.
3
Quantification by liquid scintillation counting of the H incorporation
into the gel bands containing the nACh receptor subunits.
[3H]6 photoincorporation were observed when propofol
(∼20% increase) or phencyclidine (∼20% decrease) was
added to the carbamylcholine-treated samples. For the other
nACh receptor subunits, the presence of agonist, propofol, or
channel blockers modulated photolabeling by <20%.
traces (insets in Figure 3), with increasing amounts of
compound, suggests that 6 equilibrates more slowly than
propofol or possibly stabilizes the desensitized state. This
hypothesis will be addressed in the future with rapid perfusion
patch clamp kinetic studies.
Photoincorporation into Torpedo nACh Receptor-En-
riched Membranes. Photoincorporation was assessed by
SDS−PAGE followed by fluorography (Figure 4A) and liquid
scintillation counting of excised gel bands (Figure 4B). For
nACh receptor-rich membranes photolabeled in the absence of
other drugs, there was photoincorporation in each nACh
receptor subunit, with the α-subunit labeled most prominently,
along with photolabeling of voltage dependent anion channel
(VDAC) and the Na+/K+-ATPase α-subunit. In the absence of
agonist, drugs that bind with high affinity with the nACh
receptor ion channel either in the resting closed channel state
(tetracaine) or in the desensitized state (phencyclidine) had
little effect on nACh receptor subunit photolabeling. However,
propofol alone increased α subunit photolabeling by ∼70%. For
nACh receptors desensitized in the presence of agonist
(carbamylcholine), α-subunit photolabeling was increased by
100% compared to the resting state. Small additional effects on
DISCUSSION
■
Synthesis. We have synthesized 6, a novel, photoreactive
anesthetic with potency similar to that of propofol. Initial
attempts to synthesize a propofol analogue with the photo-
reactive group at the para position, 4-TFD-propofol (Scheme
1), were unsuccessful. Therefore, we designed another strategy
for obtaining a propofol derivative. To avoid the instability
caused by the electronic effect of the hydroxy group in the para-
position stabilizing the benzylic carbene, we redesigned the
photoactivatable analogue of propofol by separating the
diazirine moiety from the phenyl ring by linking to the
propofol core via a short nonpolar, three-carbon spacer. The
reactive aliphatic diazirine forms a carbonium ion that reacts
preferentially with acidic side chains and with nucleophilic
residues (tyrosine and methionine) but not with aliphatic side
chains. Successful synthesis of another propofol derivative, m-
TFD-propofol, with the photoreactive group linked directly to
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Table 1
general
anesthesia
enhancement of EC5
GABA currents at
LoRREC50
EC50 for EC5
enhancement of GABAA
currents (μM)
direct
activation
(%)
shift in GABA
dose response
curve
enhancement of
[3H] muscimol
binding
enhancement of
[3H] flunitrazepam
binding
agent
LoRREC50 (μM)
c
a
b
a
a
a
a
propofol
2.2
5.2
4×
5×
30−50
67
11
2.4×
2×
b
d
a
d
2.8
1.9
d
1.9
a
a
a
a
a
a
a
6
3.2
4.5
8×
10
9
1.6×
ND
1.6×
b
e
b
b
e
e
e
m-TFD-
propofol
3.1
ND
1.4×
none
ND
ND
a
b
c
d
e
This work. From Hall et al., 2010.13 From Tonner and Miller, 1995.37 From Krasowski et al., 2001.2 ND = not reported.
the benzene group at the meta position has been reported,
suggesting that it is possible to synthesize a number of
photoreactive propofol analogues that vary in the location, size,
and type of the reactive group and yet maintain their anesthetic
properties.13 Such an array of compounds will allow for a set of
tools to aid in identifying and characterizing the residues
involved in propofol’s binding sites.
General Anesthetic Properties. Compound 6 acts as a
reversible general anesthetic in tadpoles and was approximately
equipotent to the parent general anesthetic, propofol. Propofol
was initially discovered during a screen of 97 alkylphenols in
mice and rabbits showing that 2,6-dialkylphenols, particularly
2,6-di-sec-alkylphenols, were the most potent general anes-
thetics, while more sterically hindered analogues were inactive
as anesthetics.21 The anesthetic activity of 6 is consistent with
later studies showing that substitutions at the para-position are
usually well tolerated.22,23 The potency of 6 is predicted by its
octanol/water partition coefficient, in line with the empirical
Meyer−Overton rule.
Effects on GABAA Receptors. The apparent affinity of
GABA-elicited currents was enhanced by 6 with a potency
similar to that of propofol. At twice their general anesthetic
EC50 concentrations in tadpoles, 6 (6 μM) and propofol (4.4
μM) shifted the GABA-induced current concentration−
response curve to the left by comparable amounts of 9- and
11−-fold, respectively (Figure 1C,D). This occurred without
any dramatic systematic change in the maximum current
elicited at high concentrations of GABA.
The concentration dependence of potentiation of currents
elicited by a subsaturating EC5 concentration of GABA for both
agents was comparable. Their EC50 values were similar and
comparable to their general anesthetic value (Table 1). The
efficacy of the compounds was also similar (Figure 1A,B). The
concentration dependence of potentiation is consistent with the
results observed by Krasowski et al. in their study of 27
propofol derivatives where the introduction of the trifluor-
omethyldiazirine moiety on the aromatic ring of propofol could
be best achieved at the 4-position, since substitutions at this
position do not significantly alter the anesthetic properties of
propofol.2 In addition, they noted that maximum potentiation
of GABA currents varied from 60% to 240%, with propofol
exhibiting the greatest efficacy. In a study of 14 active propofol
derivatives, two that were para-substituted had 2.5- and 4-fold
lower efficacy than propofol for potentiating currents elicited by
low concentrations of GABA.2,15 In another study, out of 14
propofol derivatives substituted in the para-position, only 4
enhanced GABA currents but with efficacy similar to
propofol’s.22
discussed above, the active para-substituted agents all directly
activated with efficacies 30−100% of propofol’s. Compound 6
also directly activated currents through the GABAA receptor to
10% of the maximal GABA response. In contrast, m-TFD-
propofol at 30 μM was reported to have no direct agonist
activity.13 While halogen and aromatic 4-substitution appear to
preserve efficacy for potentiation and activation,22 the weak
efficacy of the propofol photolabel derivatives adds to the small
body of evidence suggesting that alkyl groups at the 3-, 4-, and
5-positions result in low efficacy.2
Like propofol, 6 enhanced the binding of [3H]muscimol and
[3H]flunitrazepam to HEK293 cell membranes expressing
α1β3γ2 GABAA receptors. The subunits are arranged reading
clockwise as α1β3α1β3γ2. Muscimol binds at the two agonist
binding sites in the α−β subunit interfaces, whereas
flunitrazepam binds in the γ−α subunit interface. Both sites
are in the receptor’s extracellular ligand-binding domain.
Compound 6 potentiated both muscimol and flunitrazepam
binding to similar extents, 70% and 80%, respectively, with half
effect concentrations of ∼0.5 μM, well below the EC50 for
general anesthesia. Propofol was more efficacious and had a
similar concentration dependence except that at higher
concentrations it caused a sharp decrease in [3H]ligand binding.
Effects on nACh Receptors. The only readily available
and abundant biological source for a Cys-loop receptor is the
electric organ from the Torpedo ray, thus making the nACh
receptor the most convenient member of this family for
assessing a new photolabel. In this work, we photolabeled the
nACh receptor in the presence of various drugs that have
known sites of action and allosteric effects, thus enabling an
assessment of photolabeling efficacy and specificity. The nACh
receptor has five subunits (2α,1β,1γ,1δ) that exhibit homolo-
gous secondary structure. A large extracellular agonist-binding
domain is followed by a transmembrane domain of four α-
helices, M1−M4, with a large intracellular loop separating M3
and M4. The five M2 helices line the central ion-conducting
pore and are the target of many drugs.
Compound 6 most efficiently photolabeled the nACh
receptor α-subunit, and that photolabeling was enhanced
when the receptor was in the desensitized state (Figure 4).
For photolabeling at 1.7 μM [3H]6, the 3000 cpm of
carbamylcholine-enhanced [3H] photoincorporation in the
nACh receptor α-subunit indicates photolabeling of 0.2% of
α-subunits, an efficiency of incorporation similar to that seen
for [3H]TDBzl-etomidate or [3H]azietomidate24 and sufficient
to allow for identification of photolabeled nACh receptor
amino acids.25,26 This photolabeling pattern was similar to that
seen for azioctanol27 and azietomidate.25 Thus, carbamylcho-
line, an agonist that stabilizes the desensitized state, significantly
increased the photoincorporation of 6 into the nACh receptor
α-subunit. Propofol also enhanced α-subunit photolabeling in
In addition to enhancing GABA-induced currents, propofol
alone is a very effective partial agonist, eliciting currents up to
30−70% of the maximal GABA response.2,22 In the studies
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monitored by UV at 280 nm (aromatic absorbance). The analysis
indicated purity greater than 97%.
both the absence and presence of agonist. The propofol
enhancement of α-subunit photolabeling in the absence of
agonist may occur because propofol can stabilize the nACh
receptor in the desensitized state. However, in the presence of
agonist the receptor is already desensitized so that when
propofol further enhances photoincorporation, a second
distinct site for propofol may be invoked.
We also examined the effects of two known nACh receptor
channel blockers on [3H]6 photolabeling: tetracaine, which
binds with high affinity to the channel when the nACh receptor
is in the resting, closed channel state,28 and phencyclidine,
which binds with high affinity when nACh receptors are in the
desensitized state.29,30 The 6 binding site appears distinct from
that of tetracaine, which had no effect on photolabeling in the
resting state. However, the partial inhibition by phencyclidine
of photolabeling suggests that there may be some 6 binding
within the nACh receptor ion channel in the desensitized state.
Further photolabeling studies on a larger scale are required to
directly identify the nACh receptor amino acids that are
photolabeled.
4-Trifluoroacetylpropofol (2). Aluminum chloride (4.00 g, 30.0
mmol) was suspended in dichloromethane (150 mL) and cooled to
−48 °C. Trifluoroacetic anhydride (3.06 mL, 4.62 g, 22 mmol) in
dichloromethane (20 mL) was added dropwise to the stirred
suspension. The resulting suspension was stirred at −48 °C for 30
min, and a solution of 2,6-diisopropylphenol (3.56 g, 20.0 mmol) in
dichloromethane (20 mL) was added dropwise. The reaction mixture
was stirred at the same temperature for 3 h and allowed to warm to
room temperature overnight. The reaction mixture was poured onto a
mixture of ice and 2.0 M HCl (150 mL each) and stirred for 1 h. The
layers were separated, and the organic phase was washed with H2O (2
× 100 mL), saturated NaHCO3, and brine (100 mL each), dried over
MgSO4, and concentrated. The residue was chromatographed on silica
gel using ethyl acetate−hexanes, 1:10, as eluent to yield pure 2 as a
low-melting solid (3.18 g, 58%). 1H NMR (360 MHz, CDCl3): δ 7.81
(s, 2H, Harom), 3.26 (sept, 2H, J = 6.8 Hz, CH-i-Pr), 1.24 (d, 12H, J =
6.8 Hz, CH3). 13C NMR (90.6 MHz, CDCl3): δ 179.2 (q, 2JC‑F = 33.5
1
Hz, CO), 158.5, 135.8, 126.7, 117.6, 117.6 (q, JC‑F = 291.0 Hz),
30.0, 22.1. 19F NMR (338.8 MHz, CDCl3): δ −71.7 (s). HRMS (ESI)
calcd for C14H16F3O2: [M − H] 273.1108. Found: 273.1111.
O-tert-Butyldimethylsilyl-4-trifluoroacetylpropofol (3). The
mixture of the ketone 2 (1.00 g, 3.65 mmol), tert-butyldimethylsilyl
chloride (660 mg, 4.38 mmol), imidazole (497 mg, 7.30 mmol), and
dry DMF (5 mL) was stirred for 12 h at 20 °C. This mixture was
added to hexane (50 mL), washed with water (50 mL), and the
aqueous layer was extracted with hexane (2 × 20 mL). The combined
organic phases were washed with water (3 × 30 mL), dried over
MgSO4, and concentrated under vacuum. Column chromatography on
silica (1−3% of ether in hexanes as eluent) afforded the protected
On the nACh receptor, 6 inhibited currents with the same
potency and efficacy as propofol. The current traces observed
with increasing amounts of 6 suggest a role for desensitization
in its inhibitory response, consistent with the photolabeling
results that show increased labeling in the desensitized state.
CONCLUSIONS
■
Compound 6 is a novel, photoreactive derivative of the
anesthetic propofol. In most respects it acts like propofol. It
causes general anesthesia at a similar concentration and
modulates and activates heterologously expressed GABAA
receptors. This propofol derivative also functions as a
photoaffinity agent, photoincorporating into nACh receptors
in a pharmacologically specific fashion. Compared to a
previously developed photoactivable propofol derivative m-
TFD-propofol, 6 has a different photoselectivity for amino acid
residues. The two photoactivable derivatives of propofol have
similar GABAA receptor pharmacology, but they differ in detail,
and a comparison may allow for a more detailed mapping of
binding sites just as it has in the case of a pair of etomidate
derivatives containing an aliphatic diazirine, azi-etomidate, and
an aromatic diazirine, TDBzl-etomidate.31
1
ketone 3 as a colorless solid (1.02 g, 72%, mp 41−44 °C). H NMR
(CDCl3): δ 7.84 (unresolved q, 2H, 5J = 0.9 Hz, CHarom), 3.33 (m, 2H,
J = 6.9 Hz, CH-i-Pr), 1.22 (d, 12H, J = 6.9 Hz, CH3-i-Pr), 1.06 (s, 9H,
2
t-Bu), 0.27 (s, 6H, Si-CH3). 13C NMR (CDCl3): δ 179.5 (q, JC‑F
=
34.1 Hz, CO), 156.6, 140.3, 126.6 (unresolved q, JC‑F = 2.1 Hz),
1
123.8, 117.0 (q, JC‑F = 291.8 Hz), 26.8, 26.0, 23.0, 19.0, −3.2 (CH3-
Si). 19F NMR (CDCl3): δ −70.6 (s). HRMS (ESI) calcd for
C20H32F3O2Si: [M + H] 389.2218. Found: 389.2129.
1-[4-(tert-Butyldimethylsilyloxy)-3,5-diisopropylphenyl]-
2,2,2-trifluoroethanone Oxime (4). The ketone 3 (1.00 g, 2.57
mmol), hydroxylamine hydrochloride (357 mg, 5.15 mmol), and
pyridine (0.31 mL, 3.9 mmol) were refluxed with stirring in methanol
(4 mL) for 2 h. The resultant clear solution was partitioned between
water and 10% ethyl acetate in hexanes (40 mL each). The aqueous
layer was extracted with the same solvent (2 × 20 mL). The combined
organic phases were washed with water (3 × 20 mL), brine (20 mL)
and dried over MgSO4. Evaporation and chromatography on silica
(ethyl acetate−hexanes, 1:20) afforded oxime 4 as a mixture of two
EXPERIMENTAL SECTION
1
■
isomers with 1:0.74 ratio as a low-melting solid (999 mg, 96%). H
Materials. [3H]Muscimol (3-hydroxy-5-aminomethylisoxazole,
[methylene-3H(N)]) (22.46 Ci/mmol) and [3H]flunitrazepam (fluni-
trazepam, [methyl-3H]) (75.7 Ci/mmol) were from Perkin-Elmer
(Waltham, MA). GABA (γ-aminobutyric acid), flurazepam dihydro-
chloride, propofol (2,6-diisopropylphenol, 97%), and all other
chemicals and reagents, as well as solvents, including anhydrous
THF, were purchased from Sigma-Aldrich (St. Louis, MO) and were
used as received, without further purification. cDNAs for the α1, β2,
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.).
NMR (CDCl3): δ 9.49 (s, 1.7H, NOH), 7.38 (s, 2H, CHarom), 7.23 (s,
1.5H, CHarom), 3.42 − 3.29 (m, ∼3.5H, CH-i-Pr), 1.21 (d, 21H, J = 6.8
Hz, CH3-i-Pr), 1.08 (2 s, 16H, t-Bu), 0.26 and 0.24 (each s, 10.5H,
CH3-Si). 19F NMR (CDCl3): δ −62.3 (s, 1.26F), −65.7 (s, 1.74F).
HRMS (ESI) calcd for C20H33F3NO2Si: [M + H] 404.2227. Found:
404.2233.
3-[4-(tert-Butyldimethylsilyloxy)-3,5-diisopropylphenyl]-3-
trifluoromethyl-3H-diazirine (5). The oxime 4 (100 mg, 0.248
mmol) and p-toluenesulphonyl chloride (95 mg, 0.50 mmol) were
dissolved in dry dichloromethane (1 mL) and cooled with stirring on
an ice bath, and pyridine (0.2 mL) was added dropwise. After 1 h at 0
°C the reaction mixture was warmed to room temperature and stirred
for a further 24 h, diluted with ether (3 mL), washed with water (2 × 1
mL) and saturated NaH2PO4 (1 mL), dried over MgSO4, and
concentrated. The resultant colorless oil was dissolved in methanolic
ammonia (7 M, Aldrich), and the resultant yellow solution was stirred
at room temperature for 2 days. The reaction mixture was then
concentrated under vacuum, dissolved in methanol/triethylamine
mixture (10:1, 3 mL), and cooled to 0 °C with stirring. Finely
dispersed iodine (63 mg, 0.248 mmol) was added in small portions
Analytical Chemistry. 1H, 13C, and 19F NMR spectra were
recorded on a Bruker Avance spectrometer at 400, 100, and 376 MHz,
respectively, unless otherwise noted, with TMS as an internal standard.
HRMS experiments were performed with Q-TOF-2TM (Micromass).
TLC was performed with Merck 60 F254 silica gel plates. Purity of the
final compound was assessed by HPLC analysis with a Synergy Hydro-
Rp column (4 μm, 4.60 mm × 150 mm) using methanol−water with
0.05% trifluoroacetic acid; the gradient applied was 50% MeOH at the
start to 100% MeOH over 18 min, then isocratic. Elution was
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over 15 min until the mixture remained persistently brown. The excess
iodine was quenched with 5% Na2S2O3 (1 mL). The reaction mixture
was diluted with water (15 mL) and extracted with DCM (3 × 5 mL).
The combined organic phases were washed with water and brine (10
mL each) and dried over Na2SO4. Evaporation under vacuum and
chromatography on silica gel (ethyl acetate−hexanes, 1:100) afforded
the diazirine 5 (32 mg, 32%) as a low-melting solid. 1H NMR
(CDCl3): δ 6.85 (s, 2H, Harom), 3.30 (sept, 2H, J = 6.9 Hz, CH-i-Pr),
1.16 (d, 12H, J = 6.9 Hz, CH3-i-Pr), 1.03 (s, 9H, t-Bu), 0.20 (s, 6H, Si-
CH3). 13C NMR (CDCl3): δ 150.6, 140.0, 122.4 (q, 1JC‑F = 274.8 Hz),
122.1, 121.8, 28.6 (q, 2JC‑F = 40.0 Hz), 26.7, 26.0, 23.1, 18.9, −3.3 (Si-
CH3). 19F NMR (CDCl3): δ −65.2 (s) HRMS (ESI) calcd for
C20H31F3N2OSi: 400.2158. Found: 373.2178 (M+ − N2 + H+,
C20H32F3OSi, 373.2180).
Attempted Desilylation of Diazirine 5. In a typical experiment,
TBAF (1 mL, 1 M in THF) was cooled to 0 °C and mixed with acetic
acid (mixtures from 1 to 5 equiv of AcOH were used). Diazirine 5 (5.0
mg, 0.012 mmol) was dissolved in this TBAF−AcOH−THF mixture
with stirring, and the reaction was monitored by TLC. After
completion, the reaction mixture was poured onto crushed ice (10
mL) and extracted with ether (2 × 5 mL). After aqueous workup, TLC
indicated that the initially formed compound decomposed into a
complex mixture of several new products.
(COOMe), 155.0, 133.8, 129.3, 124.3 (CHarom), 51.8 (OCH3), 33.0
(CH2), 28.2 (CH2), 27.1 (CH-i-Pr), 22.6 (CH3-i-Pr). HRMS (ESI)
calcd for C17H23O4: [M − H] 291.1602. Found: 291.1616.
4-(4-Hydroxy-3,5-diisopropylphenyl)butyric Acid (11). Into
a stirring solution of the ketoester 8 (1.00 g, 3.4 mmol) in ethanol (40
mL) was added an excess of sodium borohydride in several portions
(total 1.287 g, 34 mmol) over 1 h. The reaction was quenched with
acetone (10 mL) and water (200 mL). The reaction mixture was
washed with dichloromethane (3 × 30 mL), acidified with HCl to pH
4, and extracted with dichloromethane (5 × 30 mL). The acidic extract
was washed with water and brine (50 mL each) and dried over
MgSO4. Crystallization from hexane afforded acid 11 as colorless
crystals (638 mg, 71%), mp 95−96 °C. 1H NMR (CDCl3): δ 11.5−9.0
(br s, 1H, CO2H), 6.87 (s, 2H, CHarom), 5.0−4.4 (br s, 1H, ArOH),
3.16 (sept, 2H, J = 6.9 Hz, CH-i-Pr), 2.62 (t, 2H, J = 7.5 Hz, CH2),
2.41 (t, 2H, J = 7.5 Hz, CH2), 1.96 (quint, 2H, J = 7.5 Hz, CH2), 1.28
(d, 12H, J = 6.9 Hz, CH3-i-Pr). 13C NMR (CDCl3): δ 180.2 (CO),
148.2, 133.8, 133.1, 123.4 (CHarom), 34.9 (CH2), 33.6 (CH2), 27.2
(CH-i-Pr), 26.7 (CH2), 22.8 (CH3-i-Pr). HRMS (ESI) calcd for
C16H23O3: [M − H] (263.1653). Found: 263.1662.
5-[4-(tert-Butyldimethylsilyloxy)-3,5-diisopropylphenyl]-
pentan-2-one (12). A mixture of acid 11 (300 mg, 1.135 mmol),
N,O-dimethylhydroxylamine hydrochloride (221 mg, 2.27 mmol), and
(benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluoro-
phosphate (753 mg, 1.7 mmol) was stirred in dry dichloromethane
(3 mL) under argon for 5 min at room temperature and cooled on an
ice bath. Triethylamine (0.47 mL, 3.4 mmol) was added dropwise. The
reaction mixture was left at room temperature overnight, diluted with
diethyl ether (20 mL), washed with water (2 × 15 mL), 0.5 M HCl,
10% Na2CO3, and brine (10 mL each), and dried over MgSO4.
Concentration under vacuum afforded crude Weinreb amide as a
reddish solid. The above was added to a solution of tert-
butyldimethylsilyl chloride (257 mg, 1.7 mmol) and imidazole (155
mg, 2.27 mmol) in dry DMF (0.5 mL) and stirred for 24 h. The
resulting emulsion was partitioned between ether and water (15 mL
each). The organic layer was washed with water, 0.1 M HCl, saturated
NaHCO3, and brine (10 mL each) and dried over MgSO4. The
solution was filtered, concentrated and the residue dried under
vacuum. This product was dissolved in dry THF (3 mL) under argon,
cooled with a dry ice−acetone bath, and a solution of methyllithium
(2.1 mL, 1.6 M in ether, 3 equiv) was added dropwise. After being
stirred at −78 °C for 2 h the reaction mixture was quenched with a
mixture of methanol and ethyl acetate (0.5 mL each) in THF (2 mL),
followed by saturated aqueous NH4Cl (10 mL). The reaction mixture
was then warmed to room temperature, extracted with ether (2 × 10
mL), and the combined organic phase was washed with water (2 × 10
mL), brine (10 mL) and dried over MgSO4. Concentration followed
by chromatography on silica gel (ethyl acetate−hexanes, 1:50)
afforded pure 12 as a colorless oil (337 mg, 79%). 1H NMR
(CDCl3): δ 6.84 (s, 2H, Harom), 3.29 (sept, 2H, J = 6.9 Hz, CH-i-Pr),
2.56 (t, 2H, J = 7.5 Hz, CH2), 2.47 (t, 2H, J = 7.5 Hz, CH2), 2.14 (s,
3H, CH3-CO), 1.90 (quint, 2H, J = 7.5 Hz, CH2), 1.17 (d, 12H, J =
6.9 Hz, CH3-i-Pr), 1.04 (s, 9H, tert-butyl), 0.20 (s, 6H, Si-CH3). 13C
NMR (CDCl3): δ 209.2 (CO), 147.2, 138.8, 134.3, 123.3 (CHarom),
53.4, 43.2, 34.9, 30.0, 26.5, 26.1, 25.6, 23.5, 18.9, −3.3 (CH3-Si).
HRMS (ESI) calcd for C23H41O2Si [M + H] 377.2870. Found:
377.2861.
4-(4-Hydroxy-3,5-diisopropylphenyl)-4-oxobutyric Acid
(7). Finely powdered succinic anhydride (6.73 g, 67.3 mmol) was
added in small portions to a stirring suspension of AlCl3 (29.9 g, 224
mmol) in dry dichloromethane (300 mL). The reaction mixture was
stirred at room temperature for 30 min, cooled to −78 °C, and the
solution of 2,6-diisopropylphenol (10 g, 56 mmol) in dichloromethane
(40 mL) was added dropwise over 30 min. The resultant yellowish
suspension was allowed to warm up overnight. The progress of the
reaction was monitored with 1H NMR. The reaction mixture was
cooled to −10 °C, poured onto crushed ice (500 g), and warmed to
room temperature with stirring (1 h). Organic and aqueous phases
were separated, the aqueous layer was extracted with dichloromethane
(4 × 100 mL), the combined organic phases were washed with brine
(200 mL) and dried over MgSO4. Concentration followed by drying
under vacuum gave a dark-brown oil which was added to a solution of
KOH (12 g, 214 mmol) in methanol/water, 1:1 (100 mL), and stirred
for 2 h. The resultant solution was diluted with water (0.5 L), acidified
to pH 10 with HCl, and washed with ether (3 × 100 mL). The
aqueous layer was acidified to pH 3 and extracted with dichloro-
methane (5 × 100 mL). The combined extracts were washed with
water and brine (100 mL each), dried over MgSO4, and concentrated.
Crystallization from ethyl acetate−hexane, 1:9, gave the acid 7 as off-
white crystals (3.00 g, 19%), mp 146−147.5 °C. 1H NMR (CDCl3): δ
12 − 9 (br s, 1H, COOH), 7.77 (s, 2H, CHarom), 5.6−5.3 (br s, 1H,
ArOH), 3.33 (t, 2H, J = 6.6 Hz, CH2), 3.18 (sept, 2H, J = 6.9 Hz, CH-
i-Pr), 2.82 (t, 2H, J = 6.6 Hz, CH2), 1.31 (d, 12H, J = 6.9 Hz, CH3).
13C NMR (CDCl3): δ 197.1 (CO), 178.7 (COOH), 154.8, 133.7,
129.3, 124.4 (CHarom), 32.8 (CH2), 28.3 (CH2), 27.2 (CH-i-Pr), 22.5
(CH3-i-Pr). HRMS (ESI) calcd for C16H27O3: [M + H] 279.1591.
Found: 279.1594.
Methyl 4-(4-Hydroxy-3,5-diisopropylphenyl)-4-oxobuta-
noate (8). Methanol (40 mL) was cooled to −60 °C, and thionyl
chloride (2.4 mL, 33 mmol) was added dropwise over 5 min. The
resultant solution was slowly warmed to 0 °C, and the ketoacid 7 (3.00
g, 10.8 mmol) was added at once. The reaction mixture was left
stirring overnight at room temperature. The resulting clear solution
was diluted with dichloromethane (100 mL), cooled to −5 °C, and
stirred with cold water (100 mL) for 5 min. The layers were separated.
The aqueous phase was extracted with dichloromethane (2 × 30 mL),
the combined organic phases were washed with water (2 × 50 mL),
brine (50 mL), dried over MgSO4 and concentrated. Column
chromatography on silica gel (ethyl acetate−hexanes, 1:10 to 1:5)
afforded the ketoester 8 as colorless crystals. Yield 2.84 g (90%), mp
3-{3-[4-(tert-Butyldimethylsilyl)-3,5-diisopropylphenyl]-
propyl}-3-methyl-3H-diazirine (13). The ketone 12 (50 mg, 0.133
mmol) was dissolved in a mixture of methanol−ether (1:1, 0.5 mL)
and cooled to −40 °C. A solution of ammonia in methanol (1 mL, ∼7
M) was added dropwise with stirring. The reaction mixture was
warmed to −30 °C and stirred at this temperature for 6 h. The
solution of hydroxylamine-O-sulfonic acid (23 mg, 0.20 mmol) in
methanol (0.4 mL) was added dropwise, and the resulting clear
solution was left at −20 °C for 5 days with occasional stirring (the
reaction progress was monitored by TLC). After completion, the
reaction mixture was evaporated in vacuo, dissolved in a mixture of
methanol (2 mL) and triethylamine (0.06 mL, 0.4 mmol), and cooled
to −10 °C. A solution of iodine (34 mg, 0.133 mmol) in
1
74 − 76 °C. H NMR (CDCl3): δ 7.77 (s, 2H, CHarom), 5.82 (s, 1H,
ArOH), 3.73 (s, 3H, OCH3), 3.33 (t, 2H, J = 6.7 Hz, CH2), 3.19 (sept,
2H, J = 6.9 Hz, CH-i-Pr), 2.78 (t, 2H, J = 6.6 Hz, CH2), 1.29 (d, 12H,
J = 6.9 Hz, CH3). 13C NMR (CDCl3): δ 197.3 (CO), 173.8
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dichloromethane (1 mL) was added dropwise until the mixture
remained persistently light-brown in color. The excess iodine was
quenched with 5% solution of Na2S2O3 (0.5 mL). The mixture was
diluted with water (15 mL) and extracted with ether (3 × 10 mL). The
combined organic phases were washed with water and brine (10 mL
each) and dried over Na2SO4. Concentration and chromatography on
silica gel using 1−2% ethyl acetate in hexanes as an eluent afforded
pure diazirine 13 as a colorless oil (30 mg, 58%). 1H NMR (CDCl3): δ
6.83 (s, 2H, Harom), 3.30 (sept, 2H, J = 6.9 Hz CH-i-Pr), 2.53 (t, 2H, J
= 7.4 Hz, CH2), 1.58−1.44 (m, 2H, CH2), 1.44−1.37 (m, 2H, CH2),
1.18 (d, 12H, J = 6.9 Hz, CH3-i-Pr), 1.05 (s, 9H, t-Bu), 1.04 (s, 3H, α-
azi-CH3) 0.21 (s, 6H, Si-CH3). 13C NMR (CDCl3): δ 147.2, 138.8,
134.5, 123.1 (CHarom), 35.1 (CH2), 34.0 (CH2), 26.5, 26.1, 26.0
(CH2), 25.8 (CN2), 23.4, 19.9 (α-azi-CH3), 18.9 (Si-CMe3), −3.3 (Si-
CH3). HRMS (ESI) calcd for C23H41N2OSi: [M + H] 389.2983.
Found: 389.2993.
2,6-Diisopropyl-4-[3-(3-methyl-3H-diazirin-3-yl)propyl]-
phenol (6). Method A (Scheme 2). Diazirine 13 from the previous
step (30 mg, 0.077 mmol) was deprotected with cesium fluoride (101
mg, 0.67 mmol) in ethanol or TBAF−acetic acid (1:2 molar ratio, 1 M
TBAF in THF + neat acetic acid, 1 mL, overnight) to obtain crude 6.
Column chromatography on silica (ethyl acetate−hexanes, 1:20)
afforded pure diazirine 6 as a colorless oil (19 mg, 52% from ketone
12). 1H NMR (CDCl3): δ 6.85 (s, 2H, Harom), 4.66 (br s, 1H, ArOH),
3.16 (sept, 2H, J = 6.9 Hz, CH-i-Pr), 2.53 (t, 2H, J = 7.5 Hz, CH2),
1.55−1.45 (m, 2H, CH), 1.45−1.37 (m, 2H, CH2), 1.29 (d, 12H, J =
6.9 Hz, CH3-i-Pr), 1.04 (s, 3H, α-azi-CH3). 13C NMR (CDCl3): δ
148.1, 133.6, 133.5, 123.3 (CHarom), 35.1 (CH2), 33.9 (CH2), 27.2
(CH-i-Pr), 26.2 (α-azi-CH2), 25.8 (CN2), 22.8 (CH3-i-Pr), 19.9 (α-
azi-CH3). HRMS (ESI) calcd for C17H25N2O: [M − H] 273.1972.
Found: 273.1976.
Method B. Compound 6 was also synthesized by another route
permitting introduction of tritium label by a reduction of the carbonyl
group in ketone 18 (Scheme 3). The solution of the ketone 18 (100
mg, 0.248 mmol) in absolute ethanol (1 mL) was stirred with sodium
borohydride (47 mg, 1.24 mmol) for 1 h at room temperature. After
completion of the reaction (TLC), cesium fluoride (188 mg, 1.24
mmol) was added and stirring continued for another 24 h (TLC).
Then the reaction mixture was divided between hexane−ethyl acetate
(5:1) and water phases (10 mL each). The aqueous phase was
additionally extracted with the same solvent (2 × 5 mL), and the
combined organic phases were washed with brine and dried over
MgSO4. The resulting solution was filtered, concentrated in vacuo, and
dissolved in a mixture of dichloromethane (3 mL) and triethylsilane
(0.5 mL). The reaction mixture was cooled to −30 °C. The solution of
trifluoroacetic acid (0.4 mL) in dichloromethane (0.6 mL) was added
dropwise, and the mixture was warmed to room temperature (0.5 h).
After the usual aqueous workup, chromatography on silica gel (ethyl
acetate−hexanes, 1:20) afforded the target compound 10 (61 mg,
90%) as a colorless oil, identical with that obtained via Scheme 2.
3H-2,6-Diisopropyl-4-[3-(3-methyl-3H-diazirin-3-yl)propyl]-
phenol. The tritium labeled 6 with specific radioactivity of 12 Ci/
mmol was synthesized as described above by ViTrax, Placentia, CA.
This compound was stored at −20 °C as a solution in ethanol
containing 1 mCi/mL.
Methyl 3-(2-(4-Hydroxy-3,5-diisopropylphenyl)-1,3-dioxo-
lan-2-yl)propanoate (14). The solution of the ketoester 8 (2.00
g, 6.84 mmol), ethylene glycol (5 mL), trimethyl orthoformate (2
mL), and p-toluenesulfonic acid monohydrate (66 mg, 0.34 mmol) in
dichloromethane (5 mL) was stirred at room temperature for 48 h
(the reaction was monitored by 1H NMR). The reaction was
quenched with triethylamine (1 mL), and the resulting solution was
partitioned between ether and water (40 mL each). The organic phase
was washed with water, brine (20 mL each), dried over MgSO4,
evaporated, and chromatographed on silica (ethyl acetate−hexanes,
1:10, 1% triethylamine) to afford dioxolane 14 as a viscous colorless oil
that crystallized upon storage in a refrigerator (1.43, 62%, mp 84−86
°C). 1H NMR (CDCl3): δ 7.14 (s, 2H, Harom), 4.88 (br s, 1H, ArOH),
4.06−3.96 (m, 2H, OCH2), 3.86−3.76 (m, 2H, OCH2), 3.67 (s, 3H,
OCH3), 3.16 (sept, 2H, J = 6.9 Hz), 2.50−2.42 (m, 2H, CH2), 2.28−
2.20 (m, 2H, CH), 1.29 (d, 12H, J = 6.9, Me2CH). 13C NMR
(CDCl3): δ 174.1 (CO), 149.7, 133.8, 133.3, 120.8 (CHarom), 109.8
(O-C-O), 64.6 (OCH2), 51.5 (OCH3), 35.6 (CH2), 28.7 (CH2), 27.3
(CH-i-Pr), 22.7 (CH3-i-Pr). HRMS (ESI) calcd for C19H29O5: [M +
H] 337.2010. Found: 337.2014.
4-(2-(4-Hydroxy-3,5-diisopropylphenyl)-1,3-dioxolan-2-yl)-
butan-2-one (15). Ester 14 (1.80 g, 5.35 mmol) was dissolved in
methanol (20 mL), and a solution of KOH (2.0 g, 36 mmol) in water
(10 mL) was added at once. The reaction mixture was stirred at room
temperature for 2 h (the progress was monitored by TLC), poured
into 100 mL of water, and carefully acidified with 10% H3PO4 to pH 3.
The resulting emulsion was extracted with dichloromethane (5 × 20
mL). The extracts were combined and added with triethylamine (3
mL), dried over MgSO4, evaporated, and dried at 0.1 mmHg and 100
°C for 1 h. The residual solid was cooled to 0 °C (with stirring under
argon) and mixed with a dichloromethane solution (20 mL) of
triethylamine (2.24 mL, 3 equiv), N,O-dimethylhydroxylamine hydro-
chloride (0.783 g, 8.03 mmol), and (benzotriazol-1-yloxy)tris-
(dimethylamino)phosphonium hexafluorophosphate (3.55 g, 8.03
mmol). The resulting dark-red suspension was stirred overnight.
After the completion of the reaction (monitored by TLC, ether−
hexane, 1:1, with small amount of 7 M ammonia in methanol), the
reaction mixture was stirred with water (20 mL) and diluted with
dichloromethane (100 mL). The layers were separated. The organic
layer was washed with brine (50 mL), dried over MgSO4, evaporated,
and filtered through a short silica gel column (eluted with ethyl
acetate/hexanes, 1:3, 1% triethylamine). The concentration of eluate
afforded a dark-red oil (2.5 g). This product was dissolved in
anhydrous THF (40 mL), cooled to −78 °C with vigorous stirring,
and a solution of methyllithium (10 mL, 1.6 M in ether, 3 equiv) was
added dropwise. The solidified reaction mixture was warmed to −20
°C for 5 min, cooled to −78 °C, and quenched with ethyl acetate (10
mL), ethyl acetate−acetic acid, 1:1 (5 mL), and water (50 mL). The
usual workup (as above), followed by chromatography on silica gel
(ethyl acetate−hexanes, 1:10, 1% triethylamine) afforded ketoketal 15
as a viscous colorless oil (1.07 g, 62%) (crystallized upon storage at 4
1
°C, mp 67−72 °C). H NMR (CDCl3): δ 7.12 (s, 2H, Harom), 5.11
(br s, 1H, ArOH), 4.05−3.95 (m, 2H, OCH2), 3.85−3.75 (m, 2H,
OCH2), 3.18 (sept, 2H, J = 6.9 Hz, CH-i-Pr), 2.55 (t, 2H, J = 7.5 Hz,
CH2), 2.20 (t, 2H, J = 7.5 Hz, CH2), 2.14 (s, 3H, CH3CO) 1.27 (d,
12H, J = 6.9, CH3-i-Pr). 13C NMR (CDCl3): δ 208.8 (CO), 149.8,
133.8, 133.5, 120.7 (CHarom), 110.0 (O-C-O), 64.5 (OCH2), 38.2
(CH2), 34.7 (CH2), 29.8, 27.2, 22.8 (CH3-i-Pr). HRMS (ESI) calcd
for C19H29O4: [M + H] 321.2060. Found: 321.2067.
2,6-Diisopropyl-4-(2-(2-(3-methyl-3H-diazirin-3-yl)ethyl)-
1,3-dioxolan-2-yl)phenol (16). Diaziridination of ketoketal 15 was
accomplished by a procedure similar to that used in the synthesis of 13
(Scheme 2). The crude product was purified by silica gel
chromatography using ethyl acetate−hexanes, 1:10, with 1% of
triethylamine as eluent. Starting from 1.00 g of 15, 330 mg of
diazirine 16 (32%) was obtained as a colorless oil that slowly
crystallized upon storage at 4 °C, mp 30−35 °C. 1H NMR (CDCl3): δ
7.10 (s, 2H, CHarom), 4.85 (br s, 1H, ArOH), 4.03−3.93 (m, 2H,
OCH2), 3.83−3.73 (m, 2H, OCH2), 3.16 (sept, 2H, J = 6.9 Hz),
1.84−1.76 (m, 2H, CH2), 1.48−1.40 (m, 2H, CH2), 1.29 (d, 12H, J =
6.9, CH-i-Pr), 0.99 (s, 3H, α-azi-CH3). 13C NMR (CDCl3): δ 149.7,
133.9, 133.3, 120.7 (CHarom), 109.9 (O-C-O), 64.5 (OCH2), 34.9
(CH2), 28.8 (CH2), 27.3 (CH-i-Pr), 25.7 (CN2), 22.7 (CH3-i-Pr), 19.7
(α-azi-CH3). HRMS (ESI) calcd for C19H28N2O3: [M + H] 333.2173.
Found: 333.2178.
1-(4-Hydroxy-3,5-diisopropylphenyl)-3-(3-methyl-3H-diazir-
in-3-yl)propan-1-one (17). Diazirine 16 (200 mg, 0.6 mmol) was
dissolved in dichloromethane (3 mL), cooled to −20 °C, and
trifluoroacetic acid (0.5 mL) was added dropwise with stirring. The
reaction mixture was allowed to warm to 0 °C, and water (1 mL) was
added. Stirring continued for 3 h (TLC was used to monitor the
reaction). Water (5 mL) was added, and the layers were separated.
The aqueous phase was extracted with dichloromethane (2 mL), and
the combined organic phases were washed with water and brine (5 mL
each; the product is unstable under basic conditions) and dried over
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MgSO4. Evaporation of solvent under vacuum left pure 17 as white
crystals (163 mg, 94%), mp 87−90 °C. H NMR (CDCl3): δ 7.71 (s,
oocytes were injected with ∼25 ng of total mRNA mixed at a ratio of
2α/1β/1γ/1δ as previously described.34
1
2H, CHarom), 5.5−5.3 (br s, 1H, ArOH), 3.19 (sept, 2H, J = 6.9 Hz,
CH-i-Pr), 2.80 (t, 2H, J = 7.4 Hz, CH2), 1.87 (t, 2H, J = 7.4 Hz, CH2),
1.31 (d, 12H, J = 6.9, CH3-i-Pr), 1.11 (s, 3H, α-azi-CH3). 13C NMR
(CDCl3): δ 197.6 (CO), 154.8, 133.7, 129.5, 124.3, 32.1, 28.8, 27.2,
25.7, 22.6, 20.1. HRMS (ESI) calcd for C17H23N2O2: [M − H]
287.1765. Found: 287.1770.
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. Compound 6 and propofol were
dissolved in DMSO at 10 mM just prior to use. Stocks were further
diluted in ND96 to achieve the desired concentration.
GABAA and nACh Receptor Concentration Response and
Current Inhibition Studies. All agents were applied for 15−20 s;
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 Instru-
ments, Foster City, CA), and analyzed using Clampex/Clampfit 8.2
(Axon Instruments) and OriginPro 6.1 software. Concentration−
response data were fit by nonlinear least-squares regression to the Hill
(logistic) equation of the following general form:
1-(4-(tert-Butyldimethylsilyloxy)-3,5-diisopropylphenyl)-3-
(3-methyl-3H-diazirin-3-yl)propan-1-one (18). The diazirine 17
(100 mg, 0.347 mmol) was dissolved in the preformed mixture of tert-
butyldimethylsilyl chloride (157 mg, 1.04 mmol), imidazole (47 mg,
0.69 mmol), and dry DMF (0.5 mL). After being stirred for 12 h at
room temperature, the reaction mixture was partitioned between ether
and water (10 mL each). The organic phase was washed with brine,
dried over MgSO4, evaporated under vacuum, and chromatographed
on silica gel (2−5% of ethyl acetate in hexanes as eluent) to afford 18
as a colorless solid (120 mg, 86%), mp 48−50 °C. 1H NMR (CDCl3):
δ 7.69 (s, 2H, Harom), 3.32 (sept, 2H, J = 6.9 Hz, CH-i-Pr), 2.81 (t, 2H,
J = 7.5 Hz, CH2), 1.86 (t, 2H, J = 7.5 Hz, CH2), 1.21 (d, 12H, J = 6.9,
CH3-i-Pr), 1.12 (s, 3H, α-azi-CH3), 1.04 (s, 9H, tert-butyl), 0.23 (s,
6H, Si-CH3). 13C NMR (CDCl3): δ 197.6 (CO), 154.2, 139.5,
130.6, 124.0 (CHarom), 32.1 (CH2), 28.8 (CH2), 26.7, 26.0, 25.6
(CN2), 23.2 (CH3-i-Pr), 20.1 (α-azi-CH3), 18.9 (Si-CMe3), −3.3 (Si-
CH3). HRMS (ESI) calcd for C23H39N2O2Si: [M + H] 403.2775.
Found: 403.2377.
Solubility and Partition Properties. The solubility of 6 in 0.01
M Tris-HCL, pH 7.4, was determined by stirring excess compound in
the buffer for 24 h, centrifuging at 10000g, and analyzing the average
fraction area by reverse phase HPLC. To determine octanol/water
partition coefficients of 6, the compound was stirred in a two-phase
mixture of octanol and water. Aliquots were removed from the
separated phases and applied to an HPLC Proto 300 C-18 5 μm
reverse-phase column (Higgins Analytical, Inc., Mountain View, CA).
The UV detector was set with an absorbance wavelength of 220 nm.
The mobile phase consisted of 100% acetonitrile, 0.05% TFA with a
flow rate of 1 mL/min, and the average fraction area in each phase was
estimated.
General Anesthetic Potency. Xenopus laevis tadpoles (Xeno-
pus1, INC., Dexter, MI) in the pre-limb-bud stage (1−2 cm in length)
were housed in large glass tanks filled with Amquel Plus (Kordon,
Division 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.32 Every 10 min tadpoles were individually flipped using
the hooked end of a fire-polished glass pipet until a stable response
was reached (usually at 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 (LoRR). All animals were placed in a
recovery beaker of Amquel Plus treated tap water and monitored for
recovery overnight. Each animal was assigned a score of either 0
(awake) or 1 (lost righting reflex), and the individual points were fit to
a logistic equation by nonlinear least squares.
Electrophysiology of GABAA and Torpedo nACh Receptors.
With prior approval by the Massachusetts General Hospital
Subcommittee on Research Animal Care, oocytes were obtained
from adult, female Xenopus laevis (Xenopus1, INC., Dexter, MI) and
prepared using standard methods as previously described (see below).
In vitro transcription from linearized cDNA templates and purification
of subunit specific cRNAs were carried out using Ambion mMessage
Machine RNA kits and spin columns. For GABAA receptor studies,
oocytes were injected with ∼100 ng of total mRNA (α1, β2, γ2L)
mixed at a ratio of 1:1:2 transcribed from human GABA receptor
subunit cDNAs in pCDNA3.1.33 For Torpedo nACh receptor studies,
where [X] is the concentration of the activating ligand, IGABA,max is the
maximally evoked current, EC50 is the concentration of X eliciting half
of its maximal effect, and nH is the Hill coefficient of activation.
Inhibition experiments were fit with logistic equations of the following
form:
Allosteric Regulation of GABAA Receptor Ligand Binding/
Radioligand Binding Assays. α1β3γ2 GABAA receptors were
expressed in HEK293S-TetR cells. Homogenized cell membranes
were prepared as described previously,35 and 200 μg of α1β3γ2
GABAA receptor membrane protein was resuspended in 500 μL of
assay buffer (10 mM phosphate buffer (pH 7.4), 200 mM KCl, and 1
mM EDTA). The suspension was equilibrated with radioligands (3
nM [3H]muscimol or 1.5 nM [3H]flunitrazepam) and various
concentrations of 6 or propofol at 4 °C for 1 h. The nonspecific
binding was determined in the presence of 1 mM GABA or 1 mM
flurazepam for [3H]muscimol and [3H]flunitrazepam binding,
respectively. Next, the suspension was filtered on GF/B glass fiber
filters (Whatman, Schleicher & Schuell, Maidstone, U.K.) that were
pretreated in 0.5% w/v poly(ethyleneimine) for 1 h. After receptor
application, filters were washed under vacuum with 7 mL of cold assay
buffer and dried under a lamp for 30 min. Subsequently, they were
equilibrated in Liquiscint (Atlanta, GA) and counted (Tri-Carb 1900,
liquid scintillation analyzer, Perkin-Elmer/Packard, Waltham, MA).
Photoincorporation of [3H]6 into the nACh Receptor. nACh
receptor-rich membranes, prepared from Torpedo californica electric
organs as described,36 were resuspended at 2 mg of protein/mL in
Torpedo physiological saline (250 mM NaCl, 5 mM KCl, 3 mM CaCl2,
2 mM MgCl2, and 5 mM sodium phosphate, pH 7.0) supplemented
with 1 mM oxidized glutathione. Aliquots (100 μL, 160 pmol of ACh
binding sites) were equilibrated in glass test tubes for 30 min with 1.7
μM [3H]6 (12 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 for 30 min with a 365 nm UV
lamp (Spectronics Corporation EN-16 lamp) at a distance of ∼2 cm.
After irradiation, the samples were pelleted and solubilized in
electrophoresis sample buffer, and equal aliquots were resolved on
two 1.5 mm thick 8% acrylamide/0.33% bis-acrylamide gels. The gels
were stained with Coomassie Blue R-250, and one was then treated
with Amplify for fluorography and exposed to film (Biomax XAR,
Kodak) for 2 weeks. For the second gel, the nAChR subunit bands
were excised and quantified by liquid scintillation counting by
extracting in 10% TS-2 tissue solubilizer (Research Products
International Corp, Mt. Prospect, IL) and 90% ecoscint A (National
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3
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: (617) 726-8985. Fax: (617) 724-8644. E-mail:
k_miller@helix.mgh.harvard.edu.
Author Contributions
⊥Both of these authors contributed equally to this work.
ACKNOWLEDGMENTS
■
This research was supported by a grant from the National
Institute for General Medicine to K.W.M. (Grant GM 58448)
and by the Department of Anesthesia, Critical Care and Pain
Medicine, Massachusetts General Hospital.
ABBREVIATIONS USED
■
ACh, acetylcholine; EC50, concentration required for 50% of
full effect; GABA, γ-aminobutyric acid; IC50, concentration
required for 50% of full inhibitory effect; LoRR, loss of righting
reflexes; nH, Hill coefficient; nACh, nicotinic acetylcholine; p-4-
AziC5-Pro, p-(4-azipentyl)propofol or 2,6-diisopropyl-4-[3-(3-
methyl-3H-diazirin-3-yl)propyl]phenol; PCP, phencyclidine;
Rsn, rapsyn; TBDMS, tert-butyldimethylsilyl; TBPS, tert-butyl
bicyclophosphorothionate; TFA, trifluoroacetic acid; VDAC,
voltage-dependent anion channel
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preliminary structure−activity considerations of a series of alkylphe-
nols as intravenous anesthetic agents. J. Med. Chem. 1980, 23, 1350−
1357.
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