Comparative molecular field analysis and synthetic validation of a hydroxyamide-propofol binding and functional block of neuronal voltage-dependent sodium channels

Article abstract of DOI:10.1016/j.bmc.2008.11.069

Voltage gated sodium channels represent an important therapeutic target for a number of neurological disorders including epilepsy. Unfortunately, medicinal chemistry strategies for discovering new classes of antagonist for trans-membrane ion channels have

Full text of DOI:10.1016/j.bmc.2008.11.069

Bioorganic & Medicinal Chemistry 17 (2009) 7056–7063
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Comparative molecular field analysis and synthetic validation
of a hydroxyamide-propofol binding and functional block of neuronal
voltage-dependent sodium channels
d
Milton L. Brown a,b,c, , Hilary A. Eidam , Mikell Paige a,c, Paulianda J. Jones d, Manoj K. Patel e
*
a Departments of Oncology, 3970 Reservoir Road, Georgetown University Medical Center, Washington, DC 20057, United States
b Departments of Neuroscience, 3970 Reservoir Road, Georgetown University Medical Center, Washington, DC 20057, United States
c Departments of Drug Discovery Program, 3970 Reservoir Road, Georgetown University Medical Center, Washington, DC 20057, United States
d Department of Chemistry, University of Virginia, Charlottesville, VA 22904, United States
e Department of Anesthesiology, University of Virginia, Charlottesville, VA 22904, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 April 2008
Revised 18 November 2008
Accepted 20 November 2008
Available online 3 December 2008
Voltage gated sodium channels represent an important therapeutic target for a number of neurological
disorders including epilepsy. Unfortunately, medicinal chemistry strategies for discovering new classes
of antagonist for trans-membrane ion channels have been limited to mostly broad screening compound
arrays. We have developed new sodium channel antagonist based on a propofol scaffold using the ligand
based strategy of comparative molecular field analysis (CoMFA). The resulting CoMFA model was corre-
lated and validated to provide insights into the design of new antagonists and to prioritize synthesis of
these new structural analogs (compounds 4 and 5) that satisfied the steric and electrostatic model. Com-
Keywords:
Amides
Alcohols
Sodium channels
Hippocampal neurons
Propofol
pounds 4 and 5 were evaluated for [3H]-batrachotoxinin-A-20-
yielding IC50’s of 22 and 5.7 M, respectively. We further examined the potency of these two compounds
to inhibit neuronal sodium currents recorded from cultured hippocampal neurons. At a concentration of
50 M, compounds 4 and 5 tonically inhibited sodium channels currents by 59 7.8% (n = 5) and
a
-benzoate ([3H]-BTX-B) displacement
l
l
70 7.5% (n = 7), respectively. This clearly demonstrates that these compounds functionally antagonize
native neuronal sodium channel currents. In summary, we have shown that CoMFA can be effectively
used to discover new classes of sodium channel antagonists.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
cent studies suggest that propofol effects on Na channels could
play a major role in its mechanism of action. Propofol has been
Voltage-gated sodium (Na) channels play an important role in
the generation and conduction of central and peripheral action
potentials. They are composed of a pore forming
shown to displace [3H]-BTX-B binding from sodium channels in
rat synaptoneurosomes7 and inhibit veratridine-evoked sodium in-
flux in rat synaptoneurosomes.8 In electrophysiology studies, pro-
pofol has been shown to inhibit sodium currents from isolated rat
neurohypophysial nerve terminals.9
a subunit and
auxiliary b subunits that modulate gating properties of the chan-
nel.1 To date nine
a isoforms have been cloned together with three
b subunits.2 Clinically important drugs that antagonize sodium
channels as a mechanism of their action include anticonvulsants,
anti-arrhythmic and anesthetics.
Recently, new propofol analogs and models have been designed
to optimize binding to GABA(A) channels and also for solubil-
ity.10,11 Since there is strong evidence for a role of Na channels in
the mechanism of action of propofol, we have developed a 3D
Propofol (2,6-diisopropylphenol, Fig. 1) is a clinically useful
intravenous anesthetic used for both the induction and mainte-
nance of general anesthesia. In addition it is also used in the treat-
ment of status epilepticus.3 It is widely known for its quick
anesthetic onset and titratability. The primary mechanism of ac-
tion of propofol is unknown but is thought to include both ligand
gated channels such as GABA and glycine and also voltage gated
ion channels including sodium and calcium channels.4–6 More re-
O
NH
HN
O
HO
Phenytoin
Propofol
* Corresponding author. Tel.: +1 202 687 8603.
E-mail address: mb544@georgetown.edu (M.L. Brown).
Figure 1. Structures of propofol and phenytoin.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.069

M. L. Brown et al. / Bioorg. Med. Chem. 17 (2009) 7056–7063
7057
QSAR model for propofol binding to site 2 of the Na channel. We
present a general anesthetic/anticonvulsant pharmacophore in
the voltage gated sodium channel and validate it with the design
and synthesis of two new compounds. Functional antagonism
was also assessed on neuronal sodium channels natively expressed
in cultured hippocampal neurons.
pounds had a greater neuronal antagonist effect than the intrave-
nous anesthetic propofol, which blocked the sodium current by
43 12% (n = 3).
3. Conclusion
We have demonstrated that CoMFA modeling can be an effec-
tive medicinal chemistry tool for designing and prioritizing the
synthesis of new ion channel antagonists. The CoMFA model was
able to accurately predict the [3H]-BTX-B displacement IC50 for
propofol only when it was placed and aligned to the anesthetic
CoMFA site. Using a hydroxyamide motif that interacts with the
anticonvulsant site we were able to envision and design a new li-
gand (compounds 4 and 5) that linked the two potential pharma-
cophores. Both compounds 4 and 5, designed to bridge the two
regions had low IC50 for [3H]-BTX-B displacement and demon-
strated functional antagonism of hippocampal neuronal sodium
channel currents.
2. Results and discussion
[3H]-BTX-B displacement measures the affinity of a compound
to site 2 on the sodium channel protein and is a useful screening
test.6,22,23 All compounds in our study were tested at 40
l
M in
duplicate assays. Propofol was also tested as a benchmark and
found to inhibit 16.6% at 40 M. Compound 5 was found to be
the most effective sodium channel blocker at 71.4% at 40
l
l
M
based on [3H]-BTX-B displacement, with the remaining com-
pounds ranging from 6.8% to 67.1% inhibition (Table 1, structure
in Fig. 2). This rank order was predicted by the CoMFA model.
The steric map (Fig. 3) shows that a six carbon linker is needed
to branch the anesthetic and anticonvulsant binding sites. By
aligning each molecule to either the anesthetic or anticonvulsant
site, we were able to obtain a CoMFA predicted IC50 for [3H]-
BTX-B displacement for each (see Table 1). The anticonvulsant
site is also termed the hydantoin site based upon previous stud-
ies utilizing this CoMFA.12
4. Experimental
4.1. Molecular modeling: conformational analysis
The exact training set for the CoMFA was used directly from
our previously reported work.12 The X-ray coordinates for propo-
fol were utilized in this study.13 Test compounds 2 and 5 were
constructed from the propofol crystal structure by adding the
appropriate atoms within the build edit mode of SYBYL (Tripos
Inc.) This modified structure was energy-minimized with the Tri-
pos force field,14 without solvent, using default bond distances
and angles and neglecting electrostatics. Minimization was com-
pleted by aggregating (using the SYBYL/AGGREGATE module)
only the X-ray structure atoms and allowing the modified por-
tion to minimize. For internal consistency with our previous
model, we used only the R-configuration for 2 and 5. To deter-
mine the low-energy conformations for 2 and 5, we utilized
GRIDSEARCH on rotatable bonds over 360° in one degree incre-
ments. The atomic charges for all analogs were calculated using
AM1 (MOPAC).
Based upon recent literature reports, we investigated whether
propofol was interacting with this hydantoin site or the smaller
site, termed as the anesthetic site in this study (Fig. 3). Propo-
fol’s predicted IC50 for [3H]-BTX-B displacement was 60.3
lM
for the anesthetic site and 371.5
which was consistent with its 16.6% inhibition at 40
ingly, compound 2’s IC50 was predicted as 29.5 M for the anes-
thetic site and 741.3 M for the hydantoin site, again falling in
line with our predictions based on its experimental value of
24.5% inhibition at 40 M. Compounds 4 and 5 were only pre-
dicted with the propofol core in the anesthetic site. The CoMFA
l
M for the anticonvulsant site,
l
M. Accord-
l
l
l
predicted IC50’s for compounds 4 and 5 were 51.3 and 16.2
Our experimentally determined values were 22.0 and 5.7
respectively (Table 1).
lM.
M,
l
With these results in hand, we turned to evaluating the func-
tional effects of compounds 4 and 5 on sodium currents expressed
4.2. Molecular alignment
in rat hippocampal neurons. At a concentration of 50
pound significantly blocked the Na current by 70 7.5%
(P < 0.05; n = 7). Compound at the same concentration of
50 M blocked the Na current by 59 7.8% (P < 0.05; n = 5). Cur-
lM com-
5
Propofol was utilized as a training molecule and was fit by over-
lapping the aryl ring atoms of propofol with the phenyl ring of phe-
nytoin in the anticonvulsant pharmacophore (see Fig. 3). A second
fit was obtained by fitting the phenyl ring over the C7 of the alkyl
side chain in the anesthetic pharmacophore (see Fig. 3). Similarly,
the designed hydroxyamides 2 and 5 were aligned such that the
OH group was superimposed with N1 and the carboxyamide was
aligned with C4 and N3. The n-alkyl groups at R1 in the test set
approximated a fully extended conformation following energy
minimization, which was arbitrarily selected for this study.
4
l
rent trace recordings are shown in Figure 4A and B. The effects of
both compounds were fully reversible on washout. In agreement
with our [3H]-BTX-B data, compound 5 was more effective than
compound 4 at blocking the sodium current. Finally, both com-
Table 1
CoMFA predictions and 3H-BTX-B displacement
Compound Alignment Predicted BTX
Observed 3H-BTX-B displacement
IC50 Single concentration
M)
4.3. CoMFA calculations
site
IC50 (lM)
(l
tested @ 40
16.6
lM
CoMFA, using default parameters except where noted, was cal-
culated in the QSAR option of SYBYL 6.8 on a Silicon Graphics com-
puter with an Octane II R12000 dual processor. The CoMFA grid
spacing was 2.0 Å in the x, y, and z directions, and the grid region
was automatically generated by the CoMFA routine to encompass
all molecules with an extension of 4.0 Å in each direction. A sp3
carbon and a charge of +1.0 were used as probes to generate the
interaction energies at each lattice point. The default value of
30 kcal/mol was used as the maximum electrostatic and steric en-
ergy cutoff.
Propofol
Propofol
Anesthetic
Hydantoin
Anesthetic
60.3
371.5
43.7
1
1
2
2
3
3
4
5
6.8
24.5
55.1
Hydantoin 1380.4
Anesthetic
Hydantoin
Anesthetic
Hydantoin
Both
29.5
741.3
34.7
354.8
51.3
22.0
5.7
67.1
71.4
Both
16.2

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M. L. Brown et al. / Bioorg. Med. Chem. 17 (2009) 7056–7063
A
Anesthetic Pharmacophore
Anticonvulsant Pharmacophore
B
HO CF3
NH2
HO CF3
( )
n
NH2
O
HO
+
HO
O
Anticonvulsant
Compound 1
Propofol
Anesthetic
Compound 4 (n=3)
Compound 5 (n=5)
Figure 2. (A) Model of general anesthetic and anticonvulsant pharmacophore; (B) strategy of compound design.
Figure 3. Stereoviews (relaxed) of the electrostatic and steric CoMFA fields for selected compounds. (A) Propofol aligned to the anesthetic site; (B) compound 2 aligned to the
hydantoin site; (C) compound 4; (D) compound 5.
4.4. Partial least squares (PLS) regression analysis
vored positive electrostatic (contoured in blue) effects and at
20% for disfavored steric (contoured in yellow) and favored
negative electrostatic (contoured in red) effects, as shown in
Figure 3. Based on the alignment of our low energy conform-
ers of test set ligands (propofol, and analogs 2 and 5) to this
model, we predicted the sodium channel binding activities
(Table 1).
In this study, we used the final non-cross-validated CoMFA
model previously published.12 The relative steric (0.624) and
electrostatic (0.376) contributions to the final model were
contoured as the standard deviation multiplied by the coeffi-
cient at 80% for favored steric (contoured in green) and fa-

M. L. Brown et al. / Bioorg. Med. Chem. 17 (2009) 7056–7063
7059
lowed for introduction of the fluorine yielding 17a. Several unsuc-
cessful attempts were made to form the Grignard reagent from the
iodide and bromine (formed with CBr4, Ph3P and Hunig’s base from
15a). Finally, a lithium–halogen exchange with n-BuLi and addition
of 6 allowed for formation of the ketone 17a. The hydroxyamide 4
was then formed under the above mentioned conditions (Scheme
4). Compound 5 was synthesized in the same manner utilizing
1,6-hexanediol as the starting diol (Scheme 4).
A
B
50 µM compound 4
4.6. [3H]-BTX-B displacement
100 pA
Batrachotoxin is an alkaloidal steroid released through colorless
or milky secretions from the granular glands of frogs from the
genus Phyllobates that binds to site 2 in the NVSC. Inhibition of spe-
cifically bound [3H]-BTX-B has served as a facile method to evalu-
ate whether a compound has interaction with site 2 on the sodium
channel protein.7
Washout
Control
1 ms
4.7. Cultured hippocampal neurons
50 µM compound 5
Rat hippocampal cultures were prepared according to the meth-
od described by Banker et al.21 Briefly, hippocampi were dissected
from 18-day-old rat embryos, dissociated by trypsin, and triturated
with a Pasteur pipette. The neurons were plated on coverslips
250 pA
coated with poly-L-lysine in minimal essential medium with 10%
Washout
Control
1 ms
horse serum at an approximate density of 25,000/cm2. Once the
neurons had attached to the substrate, they were transferred to a
dish containing a glial monolayer and maintained for up to four
weeks in serum-free minimal essential medium with N2
supplement.
Figure 4. Tonic block of Na current by compounds 4 (A) and 5 (B) at 50 lM in
cultured hippocampal neurons. Currents were elicited by a step depolarization to
À10 mV for 20 ms from a holding potential of À90 mV. Block of currents was fully
reversible on washout.
4.8. Electrophysiology studies
4.5. Chemistry
Sodium channel currents were recorded from cultured neurons
using the whole-cell configuration of the patch clamp recording
technique with an Axopatch 200B amplifier (Axon Instruments,
The synthesis of compound 1 was previously described in 99%
yield (Scheme 1).15 Compound 2’s synthesis commenced with bro-
mination of propofol with bromine in carbon tetrachloride in 92%
yield.16 The phenol 7 was then protected with a MOM (methoxy-
methyl) group using MOMCl and NaH in 96% yield.17 The trifluoro-
methyl ketone was obtained from formation of the corresponding
Grignard reagent from bromide 8 and adding 2,2,2-trifluoro-1-
piperidin-1-yl-ethanone 6 to introduce the fluorine in 99% yield
(Scheme 2).18 Ketone 9 was converted to the trimethylsilyl ether
with trimethylsilyl cyanide (TMSCN). Subsequent hydrolysis with
15% HCl generated the cyanohydrin. Conversion of the correspond-
ing cyanohydrin to the hydroxyamide was accomplished under
acidic conditions, by saturating with HCl gas and allowing the mix-
tures to stand at room temperature for 16 h.19 Ketone 9 was also
exposed to Bucherer–Burg conditions to synthesize the hydantoin
in 93% yield.20 The MOM group was then deprotected with the use
of BF3ÁOEt2 in a 3:1 solvent mixture of CH2Cl2:Me2S in 95% yield to
yield 3 (Scheme 3).
Foster City, CA). All voltage protocols were applied using PCLAMP
8
software (Axon, USA) and a Digidata 1322A (Axon, USA). Currents
were amplified and low pass filtered (2 kHz) and sampled at
33 kHz. Borosilicate glass pipettes were pulled using a Brown-
Flaming puller (model P-87, Sutter Instruments Co, Novato, CA)
and heat polished to produce electrode resistance of 1.5–2.0 M
X
when filled with pipette solution containing (in mM) CsF 140,
MgCl2 2, EGTA 1, Na-HEPES 10 (pH adjusted to 7.3 with CsOH). Cul-
tured neurons were superfused with solution containing (in mM)
NaCl 30, Choline Cl 110, KCl 3, CaCl2 1, CdCl2 0.1, MgCl2 2, HEPES
10, TEA-Cl 30 (pH adjusted to 7.3 with CsOH). On establishing
whole-cell configuration, neurons were held at À90 mV for 5 min
to account for equilibrium gating shifts. Currents were elicited
from a holding potential of À90 mV with a step to À10 mV for
25 ms. Capacitive and leak currents were corrected for using a P/
4 protocol. All experiments were performed at room temperature
(20–22 °C).
Data analysis was performed using Clampfit software (v8, Axon
Instruments, CA, USA) and Origin (v6, Microcal Software, MA, USA).
Statistical analyses were performed using the standard one-way
ANOVA followed by Tukey’s post hoc test (SigmaStat, Jandel). Aver-
aged data are presented as means standard error of the mean
(S.E.M.). Statistical significance was set at P < 0.05.
The synthesis of compound 4 was accomplished by starting
with the mono-TBS protection of 1,4-butanediol. Halogenation
with I2, Ph3P and imidazole yielded alkyl iodide 13a. Subsequent
reaction of 13a with the Grignard reagent of 8 to introduced the
long aliphatic chain. Deprotection of 14a with TBAF and formation
of the alkyl iodide 16a in the same manner as mentioned above al-
O
HO CF3
CN
HO CF3
O
1. TMSCN, TiCl4,
NH2
conc. HCl,
CF3
HCl gas,
1,4-dioxane,
99%
CH2Cl2
2. H2O, 99%
1
Scheme 1. Synthesis of compound 1.

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M. L. Brown et al. / Bioorg. Med. Chem. 17 (2009) 7056–7063
Br
Br
CCl4, Br2,
92%
MOMCl, NaH,
DMF, 96%
Mg, I2, THF
then
HO
HO
MOMO
O
N
(6)
CF3
7
8
99%
O
HO CF3
CN
HO CF3
O
1. TMSCN, KCN,
conc. HCl,
NH2
CF3
HCl gas,
1,4-dioxane,
81%
18-crown-6,
CH2Cl2
2. 15% HCl, THF, 93%
MOMO
MOMO
HO
2
10
9
Scheme 2. Synthesis of compound 2.
O
O
NH
NH
O
HN
HN
O
CF3
O
CF3
(NH4)2CO3, KCN,
CF3
BF3*OEt2,
50% EtOH, 650C, 93%
(3:1) CH2Cl2:Me2S,
95%
MOMO
MOMO
HO
3
9
11
Scheme 3. Synthesis of compound 3.
TBSCl, DMF,
I2, Ph3P, Imidazole,
OTBS
n
OH
n
OTBS
n
I
HO
HO
Imidazole
CH2Cl2
13a, 86%
13b, 83%
n = 3, 12a, 83%
n = 5, 12b, 78%
OTBS
OH
n
Br
Mg, I2, THF,
n
TBAF, THF,
then 13
MOMO
MOMO
MOMO
15a, 94%
15b, 96%
14a, 97%
14b, 93%
8
O
I2, Ph3P, Imidazole,
CH2Cl2
I
n
n-BuLi, Et2O,
then 6
CF3
n
MOMO
MOMO
17a, 66%
17b, 56%
16a, 76%
16b, 78%
HO CF3
HO CF3
1. TMSCN, KCN,
NH2
conc. HCl,
CN
n
n
18-crown-6,
CH2Cl2
2. 15% HCl, THF
HCl gas,
1,4-dioxane
O
MOMO
HO
18a, 99%
18b, 96%
4, 80%
5, 91%
Scheme 4. Synthesis of compounds 4 and 5.
4.9. Chemistry
specified otherwise. Chemical shifts are reported in ppm relative
to resonances of the solvent, CDCl3 (unless specified otherwise):
7.25 ppm (s) in the 1H spectra and 77.08 ppm (t) in the 13C spectra.
19F NMR spectra were measured on a Varian 300 MHz instrument,
externally referenced with TFA (À76.6 ppm). IR spectra were re-
corded on a FT-IR Impact 400 D. High-resolution mass spectrome-
try was performed at the University of Illinois at Urbana-
All reactions requiring anhydrous conditions were performed in
flame-dried glassware under an atmosphere of argon or nitrogen.
Melting points were determined with an Electrothermal Mel-Temp
melting point apparatus and are uncorrected. 1H and 13C NMR
spectra were measured on a Varian 300 or 500 MHz NMR, unless

M. L. Brown et al. / Bioorg. Med. Chem. 17 (2009) 7056–7063
7061
Champaign School of Chemical Science. Combustion analysis was
performed by Atlantic Microlabs, Inc. Norcross, GA. Concentration
in vacuo refers to high vacuum (0.35 mmHg). Concentration refers
to a rotary evaporator with a water aspirator. Anhydrous THF,
diethyl ether, and dichloromethane were purified by pressure fil-
tration through activated alumina. Flash chromatography was per-
formed on silica gel (Merck grade 9385, 230–400 mesh, 60 Å).
(25 mL) was added dropwise over 30 min. The mixture was re-
fluxed for 2 h and then allowed to cool to room temperature. The
mixture was cooled to 0 °C and 6 (5.0 g, 27.7 mmol) in dry THF
(5 mL) was added dropwise over 30 min. The mixture was brought
to room temperature and allowed to stir for 2 h. The reaction was
quenched with satd aq NH4Cl (5 mL), filtered, dried over MgSO4, fil-
tered and concentrated to yield the crude product. Purification was
performed on a flash column (8:1 hexanes/EtOAc), collecting all
fractions with a component of Rf = 0.38 to yield the pure ketone
as a yellow oil (8.9 g, 99% yield). 1H NMR: d 1.28 (d, J = 7.2 Hz,
12H), 3.56 (m, 2H), 3.62 (s, 3H), 5.03 (s, 2H), 7.92 (s, 2H); 13C
4.10. General procedure A: preparation of cyanohydrins from
ketones
1
The ketone (1.0 equiv) was dissolved in a minimal amount of
dry CH2Cl2. Trimethylsilyl cyanide (TMSCN, 2.2 equiv) and ZnI2
(1.0 equiv), (or KCN and 18-crown-6, 10 mg each for every
1.0 mmol of ketone) were added. The mixture was stirred at room
temperature overnight (16 h). The CH2Cl2 was evaporated in vacuo,
and a minimal amount of dry THF was added. The mixture was
cooled to 0 °C and 15% HCl (5 mL) was added and then stirred at
room temperature for 2 h. The solution was combined with H2O
and extracted with Et2O (3 Â 25 mL), dried over MgSO4, filtered,
and concentrated to yield the cyanohydrin. The cyanohydrins were
used in general procedure C without further purification.
NMR: d 23.2, 26.8, 57.1, 100.5, 116.8 (q, JCF = 290.2 Hz), 126.5,
2
127.0, 143.1, 159.1, 179.2 (q, JCF = 37.5 Hz); EI-MS: m/z 318.3.
4.15. 2-(3,5-Diisopropyl-4-methoxymethoxy-phenyl)-3,3,3-
trifluoro-2-hydroxy propionitrile (10)
General procedure A was employed with 9 (1.0 g, 3.1 mmol),
TMS-CN (0.9 mL, 6.9 mmol), KCN (40 mg) and 18-crown-6
(40 mg). The cyanohydrin was obtained as a yellow oil (1.0 g,
93% yield), and used without further purification.
4.16. 3,3,3-Trifluoro-2-hydroxy-2-(4-hydroxy-3,5-diisopropyl-
phenyl)-propionamide (2)
4.11. General procedure B: preparation of
from cyanohydrins
a-hydroxyamides
General procedure B was employed with 10 (1.0 g, 2.8 mmol)
and concd HCl (0.5 mL). The hydroxyamide was obtained as a
white solid (750 mg, 81% yield). mp = 139–142 °C; IR (KBr):
The cyanohydrin was dissolved in 1,4-dioxane (2 mL). The mix-
ture was cooled to 0 °C, and previously cooled concd HCl (0.2 mL
for every 1 mmol of cyanohydrin) was added. HCl gas was then
passed through the reaction mixture for 45 min at 0 °C. The mix-
ture was allowed to stand at room temperature overnight (16 h).
The mixture was extracted with EtOAc (3 Â 25 mL), dried over
1653 cmÀ1
;
1H NMR: d 1.26 (d, J = 6.6 Hz, 12H), 3.15 (sept,
J = 6.9 Hz, 2H), 4.65 (s, 1H), 5.01 (s, 1H), 5.92 (s, 1H), 6.19 (s, 1H),
7.31 (s, 2H); 13C NMR: d (CD3OD, 75 MHz) 173.6, 152.6, 136.3,
127.6, 125.7 (1JCF = 284 Hz), 122.8, 79.6 (2JCF = 27.3 Hz), 28.0,
23.3; 19F NMR: d À73.6; EI-MS: 319.3 (m/z); HRMS (EI) calcd for
C15H20F3NO3 319.1395, found 319.1388. Anal. Calcd for
C15H20F3NO3: C, 56.42; H, 6.31; N, 4.39. Found: C, 56.63; H, 6.26;
N, 4.40.
MgSO4, filtered, and concentrated to yield the crude
a-hydroxya-
mide. Purification was performed on a flash column (1:1 hex-
anes/EtOAc), collecting all fractions with a component of Rf = 0.28
to yield the pure a-hydroxyamide.
4.12. 4-Bromo-2,6-diisopropyl-phenol (7)
4.17. 5-(3,5-Diisopropyl-4-methoxymethoxy-phenyl)-5-
trifluoromethyl-imidazolidine-2,4-dione (11)
See Ref. 24.
To a stirring solution of 50% ethanol (60 mL) were added 9
(4.2 g, 13.2 mmol), KCN (1.7 g, 26 mmol), and (NH4)2CO3 (5.0 g,
53 mmol). The solution was warmed to 65 °C for 29 h., using a
3% KOH trap. The precipitate was filtered, and the filtrate was acid-
ified (pH 2) using concd HCl. The resulting solid was filtered, and
the filtrate was made basic (pH 8) using 3% KOH. This was concen-
trated to one-half volume and filtered again. The combined solids
were purified by flask chromatography (10:1 CH2Cl2/acetone), col-
lecting all fractions containing a component of Rf = 0.26 to yield the
hydantoin as a white solid (4.7 g, 93% yield). mp = 194–196 °C; 1H
NMR: d (DMSO-d6) 9.89 (s, 1H), 7.55 (s, 2H), 4.90 (s, 2H), 3.49 (s,
3H), 3.29 (sept, J = 6.9 Hz, 2H), 1.15 (d, 12H); 13C NMR: d (CD3OD,
500 MHz) 170.3, 158.2, 154.9, 143.7, 142.8, 127.8, 124.6 (q,
4.13. 5-Bromo-1,3-diisopropyl-2-methoxymethoxy-benzene
(8)
To a solution of 7 (1.0 g, 3.9 mmol) in dry DMF (5 mL) was
added NaH (172 mg, 4.3 mmol, 60% dispersion) at room tempera-
ture. Stirring at this temperature continued until the evolution of
hydrogen ceased (30 min). MOMCl (0.35 g, 4.3 mmol) was then
added over 5 min and stirring was continued for an additional
2 h. Excess NaH was then destroyed with cautious addition of
methanol (5 mL). The reaction mixture was diluted with ether,
washed with water, followed by brine. The organic layer was dried
over MgSO4, filtered and concentrated. Purification was performed
on a flash column (6:1 hexanes/EtOAc), collecting all fractions with
a component of Rf = 0.69 to yield the product as a white solid.
(6.75 g, 96% yield) mp = 34–36 °C; 1H NMR: d 1.21 (d, J = 6.9 Hz,
12H), 3.31 (m, 2H), 3.61 (s, 3H), 4.90 (s, 2H), 7.19 (s, 2H); 13C
NMR: d 23.7, 26.9, 57.4, 100.4, 118.1, 127.2, 144.3, 151.1; EI-MS:
m/z 300.1 (M+H+), 302.1 (M+2H+).
2
1JCF = 118 Hz), 124.0, 123.9, 101.8, 69.5 (q, JCF = 17.5 Hz), 57.7,
28.2, 24.1; ESI-MS: m/z 386.7 (MÀH+).
4.18. 5-(4-Hydroxy-3,5-diisopropyl-phenyl)-5-trifluoromethyl-
imidazolidine-2,4-dione (3)
To a solution of 11 (0.5 g, 1.28 mmol) in 3:1 v/v of CH2Cl2:Me2S
(80 mL) at 0 °C was added BF3ÁOEt2 (0.25 mL, 1.93 mmol). The reac-
tion mixture was stirred at 0 °C for 30 min. The reaction was
quenched with satd aq NaHCO3 (5 mL), diluted with EtOAc, washed
with satd aq NaHCO3 and brine. The organic layer was dried over
MgSO4, filtered and concentrated. Purification was performed on
a flash column (2:1 hexanes/EtOAc), collecting all fractions with
4.14. 1-(3,5-Diisopropyl-4-methoxymethoxy-phenyl)-2,2,2-
trifluoro-ethanone (9)
Crushed magnesium (0.94 g, 38.8 mmol), I2 (2–3 crystals) and a
small portion of bromide 8 was heated until the reaction began to
occur. The remaining bromide 8 (10.0 g, 33.2 mmol) in dry THF

7062
M. L. Brown et al. / Bioorg. Med. Chem. 17 (2009) 7056–7063
a component of Rf = 0.60 to yield the product as a yellow solid
(420 mg, 95% yield). mp: 215–217 °C; 1H NMR: d 7.27 (s, 2H),
6.56 (s, 1H), 5.12 (s, 1H), 5.03 (s, 1H), 3.16 (m, 2H), 1.15 (d, 12H);
13C NMR: d (CD3OD, 125 mHz) 170.7, 158.4, 153.4, 136.9,124.3
(q, JCF = 270 Hz), 123.1, 122.9, 122.6, 69.7 (q, JCF = 35 Hz), 38.6,
28.1, 23.3; ESI-MS: m/z 342.6 (MÀH+).
a flash column (8:1 hexanes/EtOAc), collecting all fractions with
a component of Rf = 0.10 to yield the pure alcohol.
4.25. 4-(3,5-Diisopropyl-4-methoxymethoxy-phenyl)-butan-1-
ol (15a)
1
2
General procedure
D
was employed with 14a (1.0 g,
4.19. tert-Butyl-(4-iodo-butoxy)-dimethyl-silane (13a)
2.45 mmol) and TBAF (1.0 M in THF, 2.94 mL, 2.94 mmol). The
alcohol was obtained as a colorless oil (678 mg, 94% yield). 1H
NMR: d 6.92 (s, 2H), 4.92 (s, 2H), 3.66 (t, J = 6.0 Hz, 2H), 3.63
(s, 3H), 3.34 (m, 2H), 2.60 (t, J = 7.5 Hz, 2H), 1.61–1.72 (m, 4H),
1.23 (d, J = 7.2 Hz, 12H); 13C NMR: d 149.7, 141.3, 138.5, 123.7,
100.2, 62.6, 57.2, 35.6, 32.5, 27.7, 26.6, 23.9; ESI-MS: (m/z)
311.8 (M+H2O).
See Ref. 25.
4.20. tert-Butyl-(6-iodo-hexyloxy)-dimethyl-silane (13b)
See Ref. 26.
4.21. General procedure C: Grignard reaction of aromatic
bromide and aliphatic iodide
4.26. 6-(3,5-Diisopropyl-4-methoxymethoxy-phenyl)-hexan-1-
ol (15b)
Crushed Mg (1.5 equiv), 2–3 I2 crystals and a small portion of
the bromide was heated until the reaction began to occur. The
remaining bromide (1.2 equiv) in dry THF (25 mL) was added drop-
wise over 30 min. The mixture was refluxed for 2 h. and then al-
lowed to cool to ambient temperature. The mixture was cooled
to 0 °C and the iodide (1.0 equiv) and CuI (10 mol %) in dry THF
(5 mL) was added dropwise over 30 min. The mixture was brought
to ambient temperature and allowed to stir for 2 h. The reaction
was quenched with saturated aqueous NH4Cl (5 mL), filtered, dried
over MgSO4, filtered and concentrated to yield the crude product.
Purification was performed on a flash column (20:1 hexanes/
EtOAc), collecting all fractions with a component of Rf = 0.70 to
yield the pure product.
General procedure D was employed with 14b (8.8 g, 20.1 mmol)
and TBAF (1.0 M in THF, 24.2 mL, 24.2 mmol). The alcohol was ob-
tained as a colorless oil (6.2 g, 96% yield). 1H NMR: d 6.90 (s, 2H),
4.92 (s, 2H), 3.63 (s, 3H), 3.33 (septet, J = 6.9 Hz, 2H), 2.56 (t,
J = 8.1 Hz, 2H), 1.54–1.65 (m, 4H), 1.38–1.45 (m, 4H), 1.29 (d,
J = 6.9 Hz, 12H); 13C NMR: d 149.6, 141.2, 138.8, 123.6, 100.1,
62.5, 57.1, 35.7, 32.5, 31.5, 29.1, 26.5, 25.5, 23.8; EI-MS: (m/z)
322.4.
4.27. General procedure E: halogenation of alcohol to iodide
Imidazole (3.75 equiv) and Ph3P (1.8 equiv) were added to a
solution of alcohol (1.0 equiv) in dry CH2Cl2 (120 mL). Iodine
(1.75 equiv) in dry CH2Cl2 (20 mL) was added dropwise. The mix-
ture was stirred for 30 min, quenched with satd aq Na2S2O3
(5 mL). The mixture was then cooled to 0 °C and 30% H2O2 was
added dropwise to oxidize the unreacted Ph3P (reaction monitored
by the disappearance of Ph3P by TLC). The mixture was extracted
with EtOAc (3 Â 40 mL), washed with brine (30 mL), dried over
MgSO4, filtered and concentrated. Purification was performed on
a flash column (8:1 hexanes/EtOAc), collecting all fractions with
a component of Rf = 0.90 to yield the iodide.
4.22. tert-Butyl-[4-(3,5-diisopropyl-4-methoxymethoxy-
phenyl)-butoxy]-dimethyl-silane (14a)
General procedure C was employed with magnesium (534 mg,
22.0 mmol), 8 (5.3 g, 17.6 mmol), 13a (4.6 g, 14.6 mmol) and CuI
(279 mg, 1.46 mmol). The aromatic derivative was obtained as a
colorless oil (5.3 g, 89% yield). 1H NMR: d 6.95 (s, 2H), 4.95 (s,
2H), 3.70 (t, J = 6.6 Hz, 2H), 3.66 (s, 3H), 3.38 (septet, J = 6.9 Hz,
2H), 2.62 (t, J = 7.8 Hz, 2H), 1.60–1.75 (m, 4H), 1.27 (d, J = 5.7 Hz,
12H), 0.95 (s, 9H), 0.10 (s, 6H); 13C NMR: d 149.7, 141.3, 138.7,
123.9, 100.3, 63.0, 57.2, 35.5, 32.5, 27.7, 26.7, 25.9, 23.9, 18.3, -
5.3; ESI-MS: (m/z) 426.0 (M+H2O).
4.28. 5-(4-Iodo-butyl)-1,3-diisopropyl-2-methoxymethoxy-
benzene (16a)
General procedure E was employed with imidazole (590 mg,
8.6 mmol), Ph3P (1.1 g, 4.1 mmol), 15a (680 g, 2.3 mmol) and io-
dine (1.0 g, 4.0 mmol). The iodide was obtained as a colorless oil
(710 mg, 76% yield). 1H NMR: d 6.91 (s, 2H), 4.93 (s, 2H), 3.64 (s,
3H), 3.35 (m, 2H), 3.23 (m, 2H), 2.60 (t, J = 7.2 Hz, 2H), 1.85–1.94
(m, 2H), 1.71–1.79 (m, 2H), 1.24 (d, J = 6.6 Hz, 12H); 13C NMR: d
149.9, 141.5, 137.9, 123.7, 100.3, 57.2, 34.6, 33.1, 32.2, 26.6, 23.9,
6.8; EI-MS: (m/z) 404.2.
4.23. tert-Butyl-[6-(3,5-diisopropyl-4-methoxymethoxy-
phenyl)-hexyloxy]-dimethyl-silane (14b)
General procedure C was employed with magnesium (191 mg,
7.8 mmol), 8 (2.0 g, 6.7 mmol), 14b (1.9 g, 5.6 mmol) and CuI
(106 mg, 0.56 mmol). The aromatic derivative was obtained as a
colorless oil (2.3 g, 93% yield). 1H NMR: d 6.96 (s, 2H), 4.97 (s,
2H), 3.64–3.70 (m, 5H), 3.40 (m, 2H), 2.62 (t, J = 7.8 Hz, 2H),
1.43–1.68 (m, 4H), 1.29 (d, J = 5.7 Hz, 12H), 0.96 (s, 9H), 0.12 (s,
6H); 13C NMR: d 149.7, 141.3, 138.9, 123.9, 100.3, 63.2, 57.1,
35.8, 32.8, 31.6, 29.2, 26.6, 25.9, 25.6, 23.9, 18.3, -5.3; APCI-MS:
(m/z) 437.0 (M+H+).
4.29. 5-(6-Iodo-hexyl)-1,3-diisopropyl-2-methoxymethoxy-
benzene (16b)
General procedure E was employed with imidazole (1.2 g,
17.4 mmol), Ph3P (2.2 g, 8.4 mmol), 15b (1.5 g, 4.65 mmol) and
iodine (2.1 g, 8.1 mmol). The iodide was obtained as a colorless
oil (1.6 g, 78% yield). 1H NMR: d 6.90 (s, 2H), 4.91 (s, 2H), 3.63
(s, 3H), 3.33 (septet, J = 6.9 Hz, 2H), 3.20 (t, J = 7.2 Hz, 2H), 2.56
(t, J = 7.8 Hz, 2H), 1.85 (m, 2H), 1.62 (m, 2H), 1.35–1.48 (m,
4H), 1.23 (d, J = 6.9 Hz); 13C NMR: d 149.7, 141.3, 138.6, 123.7,
100.2, 57.2, 35.7, 33.4, 31.3, 30.3, 28.2, 26.6, 23.9, 7.1; EI-MS:
(m/z) 432.1.
4.24. General procedure D: deprotection of the TBS group
A solution of TBS-protected alcohol (1.0 equiv) in dry THF
(20 mL) was cooled to 0 °C, followed by the addition of TBAF
(1.0 M in THF, 1.2 equiv). The reaction mixture was allowed to
warm to room temperature and stir for 4 h. The reaction was
quenched with satd aq NH4Cl, extracted with Et2O, dried over
MgSO4, filtered and concentrated. Purification was performed on

M. L. Brown et al. / Bioorg. Med. Chem. 17 (2009) 7056–7063
7063
4.30. General procedure F: trifluoromethyl ketone formation
J = 3.3 Hz, 2H), 1.37 (t, J = 3.3 Hz, 2H), 1.26 (d, J = 6.6 Hz, 12H);
1
13C NMR: d 169.6, 147.9, 134.5, 133.4, 124.0 (q, JCF = 171 Hz),
2
A solution of iodide (1.0 equiv) in dry Et2O was cooled to À78 °C
and n-BuLi (1.6 M in hexanes, 2.0 equiv) was added dropwise. The
reaction mixture was slowly allowed to warm to 0 °C over 2 h and
then cooled back down to À78 °C. A solution of 6 (1.3 equiv) in dry
Et2O (5 mL) was slowly added dropwise over 30 min. The reaction
was allowed to warm to 0 °C and then quenched with satd aq
NH4Cl. The Et2O layer was washed with satd aq NH4Cl (10 mL),
brine (30 mL), dried over MgSO4, filtered and concentrated. Purifi-
cation was performed on a flash column (20:1 hexanes/EtOAc), col-
lecting all fractions with a component of Rf = 0.50 to yield the pure
ketone.
123.3, 96.1 (q, JCF = 21 Hz) 35.6, 32.8, 29.0, 28.9, 27.2, 22.8; 19F
NMR: d À77.32; EI-MS: 375.4 m/z. Anal. Calcd for C19H28F3NO3: C,
60.79; H, 7.52; N, 3.73. Found: C, 60.80; H, 7.42; N, 3.43.
4.36. 2-Hydroxy-8-(4-hydroxy-3,5-diisopropyl-phenyl)-2-
trifluoromethyl-octanoic acid amide (5)
General procedure
B was employed with 18b (24 mg,
0.056 mmol) and concd HCl (2.0 mL). The hydroxyamide was ob-
tained as a white solid (20.5 mg, 91% yield). mp = 105–107 °C; 1H
NMR: d 6.84 (s, 2H), 6.09 (d, J = 4.6 Hz, 2H), 4.68 (s, 1H), 4.05 (s,
1H), 3.14 (septet, J = 6.9 Hz, 2H), 2.52 (t, J = 8.4 Hz, 2H), 1.90 (t,
J = 8.4 Hz, 2H), 1.58 (t, J = 3.3 Hz, 2H), 1.37 (t, J = 3.3 Hz, 2H), 1.26
(d, J = 6.6 Hz, 12H); 13C NMR: d 169.6, 147.9, 134.5, 133.4, 124.0
4.31. 6-(3,5-Diisopropyl-4-methoxymethoxy-phenyl)-1,1,1-
trifluoro-hexan-2-one (17a)
1
(q, JCF = 171 Hz), 123.3, 35.6, 32.6, 31.7, 29.7, 29.3, 29.1, 27.2,
22.8, 22.1; 19F NMR: d À77.45; EI-MS: m/z 403.2; HRMS (EI) calcd
for C21H32F3NO3 403.2334, found 403.2328; Anal. Calcd for
C21H32F3NO3: C, 62.51; H, 7.99; N, 3.47. Found: C, 62.61; H, 7.68;
N, 3.17.
General procedure
F was employed with 16a (708 mg,
1.75 mmol), n-BuLi (1.6 M in hexanes, 2.2 mL, 3.6 mmol) and 6
(412 mg, 2.28 mmol). The ketone was obtained as a colorless oil
(306 mg, 66% yield). 1H NMR: d 6.90 (s, 2H), 4.91 (s, 2H), 4.31 (t,
J = 5.1 Hz, 2H), 3.62 (s, 2H), 3.32 (septet, J = 6.9 Hz, 2H), 2.55 (t,
J = 7.5 Hz, 2H), 1.27–1.37 (m, 4H), 1.22 (d, J = 6.9 Hz, 12H); 13C
Acknowledgments
2
NMR: d 167.7 (q, JCF = 30 Hz), 149.6, 141.3, 139.0, 129.8 (q,
1JCF = 156 Hz), 123.8, 100.3, 57.3, 35.9, 31.6, 29.5, 29.4, 26.6, 24.0;
19F NMR: d À67.82; EI-MS: (m/z) 374.1.
The authors would like to thank NIH Grant RO1 CA105435-01
(M.L.B. & M.K.P.) and the Jeffress Trust Fund (M.L.B.) for financial
support. We would like to acknowledge Dr. Jaideep Kapur and Ash-
ley Renick (Dept. Neurology, University of Virginia) for providing
the cultured hippocampal neurons. We would also like to thank
the Cardiovascular Research Center at the University of Virginia
(M.K.P) and the Georgetown Drug Discovery Program (M.L.B.).
4.32. 8-(3,5-Diisopropyl-4-methoxymethoxy-phenyl)-1,1,1-
trifluoro-octan-2-one (17b)
General procedure
F was employed with 16b (330 mg,
0.76 mmol), n-BuLi (1.6 M in hexanes, 1.0 mL, 1.56 mmol) and 6
(109 mg, 0.60 mmol). The ketone was obtained as a colorless oil
(172 mg, 56% yield). 1H NMR: d 6.89 (s, 2H), 4.91 (s, 2H), 3.62 (s,
2H), 3.53 (s, 3H), 3.32 (septet, J = 6.9 Hz, 2H), 2.71 (t, J = 7.2 Hz,
2H), 2.55 (t, J = 8.1 Hz, 2H), 1.64–1.69 (m, 4H), 1.38 (m, 4H), 1.22
References and notes
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2
(d, J = 6.3 Hz, 12H); 13C NMR: d 191.6 (q, JCF = 36 Hz), 149.7,
1
141.4, 138.6, 123.7, 116.1 (q, JCF = 207 Hz), 100.3, 57.2, 36.3,
35.7, 31.2, 28.9, 28.5, 26.6, 23.9, 22.3; 19F NMR: d À79.09; EI-MS:
(m/z) 402.3.
4.33. 6-(3,5-Diisopropyl-4-methoxymethoxy-phenyl)-2-
hydroxy-2-trifluoromethyl-hexanenitrile (18a)
11. Krasowski, M. D.; Hong, X.; Hopfinger, A. J.; Harrison, N. L.; Cooke, A.;
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A was employed with 17a (200 mg,
0.53 mmol), TMSCN (0.18 mL, 1.34 mmol), KCN (5 mg) and 18-
crown-6 (5 mg). The cyanohydrin was obtained as a yellow oil
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4.34. 8-(3,5-Diisopropyl-4-methoxymethoxy-phenyl)-2-
hydroxy-2-trifluoromethyl-octanenitrile (18b)
15. Choudhury-Mukherjee, I.; Schenck, H. A.; Cechova, S.; Pajewski, T. N.; Kapur, J.;
Ellena, J.; Cafiso, D. S.; Brown, M. L. J. Med. Chem. 2003, 46, 2494–2501.
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General procedure
A was employed with 17b (23 mg,
0.057 mmol), TMSCN (0.02 mL, 0.14 mmol), KCN (10 mg) and 18-
crown-6 (10 mg). The cyanohydrin was obtained as a yellow oil
(24 mg, 96% yield), and was used without further purification.
20. Ware, E. Chem. Rev. 1950, 46, 403–470.
4.35. 2-Hydroxy-6-(4-hydroxy-3,5-diisopropyl-phenyl)-2-
trifluoromethyl-hexanoic acid amide (4)
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B was employed with 18a (212 mg,
0.53 mmol) and concd HCl (4.0 mL). The hydroxyamide was ob-
tained as a orange oil (158 mg, 80% yield). mp = 92–94 °C; 1H
NMR: d 6.84 (s, 2H), 6.09 (d, J = 4.6 Hz, 2H), 4.68 (s, 1H), 4.05 (s,
1H), 3.14 (septet, J = 6.9 Hz, 2H), 2.52 (t, J = 8.4 Hz, 2H), 1.58 (t,
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