Asymmetric synthesis and evaluation of a hydroxyphenylamide voltage-gated sodium channel blocker in human prostate cancer xenografts

Article abstract of DOI:10.1016/j.bmc.2011.08.061

Voltage-gated sodium channels are known to be expressed in neurons and other excitable cells. Recently, voltage-gated sodium channels have been found to be expressed in human prostate cancer cells. α-Hydroxy-α- phenylamides are a new class of small molecules that have demonstrated potent inhibition of voltage-gated sodium channels. The hydroxyamide motif, an isostere of a hydantoin ring, provides an active scaffold from which several potent racemic sodium channel blockers have been derived. With little known about chiral preferences, the development of chiral syntheses to obtain each pure enantiomer for evaluation as sodium channel blockers is important. Using Seebach and Frater's chiral template, cyclocondensation of (R)-3-chloromandelic acid with pivaldehyde furnished both the cis- and trans-2,5-disubsituted dioxolanones. Using this chiral template, we synthesized both enantiomers of 2-(3-chlorophenyl)-2-hydroxynonanamide, and evaluated their ability to functionally inhibit hNa_v isoforms, human prostate cancer cells and xenograft. Enantiomers of lead demonstrated significant ability to reduce prostate cancer in vivo.

Full text of DOI:10.1016/j.bmc.2011.08.061

Bioorganic & Medicinal Chemistry 20 (2012) 2180–2188
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Asymmetric synthesis and evaluation of a hydroxyphenylamide voltage-gated
sodium channel blocker in human prostate cancer xenografts
Gary C. Davis a,b, Yali Kong b, Mikell Paige b, Zhang Li b, Ellen C. Merrick c, Todd Hansen a,b, Simeng Suy b,
Kan Wang b, Sivanesan Dakshanamurthy b, Antoinette Cordova b, Owen B. McManus f, Brande S. Williams d,
Maksymilian Chruszcz e, Wladek Minor e, Manoj K. Patel c, Milton L. Brown b,
⇑
a University of Virginia, Department of Chemistry, Charlottesville, VA 22901, USA
b Georgetown University Medical Center, Drug Discovery Program, 3970 Reservoir Road, New Research Building, Rm. EP07, Washington, DC 20057, USA
c University of Virginia, Department of Anesthesiology, Charlottesville, VA 22908, USA
d Merck Research Laboratories, Rahway, NJ 07065, USA
e University of Virginia, Department of Molecular Physiology and Biological Physics, USA
f Johns Hopkins Ion Channel Center, Department of Neuroscience, Johns Hopkins, University, Baltimore, MD 21205, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Voltage-gated sodium channels are known to be expressed in neurons and other excitable cells. Recently,
Received 28 July 2011
Revised 26 August 2011
Accepted 28 August 2011
Available online 1 September 2011
voltage-gated sodium channels have been found to be expressed in human prostate cancer cells.
a-
Hydroxy- -phenylamides are a new class of small molecules that have demonstrated potent inhibition
a
of voltage-gated sodium channels. The hydroxyamide motif, an isostere of a hydantoin ring, provides
an active scaffold from which several potent racemic sodium channel blockers have been derived. With
little known about chiral preferences, the development of chiral syntheses to obtain each pure enantio-
mer for evaluation as sodium channel blockers is important. Using Seebach and Frater’s chiral template,
cyclocondensation of (R)-3-chloromandelic acid with pivaldehyde furnished both the cis- and trans-2,5-
disubsituted dioxolanones. Using this chiral template, we synthesized both enantiomers of 2-(3-chloro-
phenyl)-2-hydroxynonanamide, and evaluated their ability to functionally inhibit hNav isoforms, human
prostate cancer cells and xenograft. Enantiomers of lead demonstrated significant ability to reduce pros-
tate cancer in vivo.
Keywords:
Ion channel blocker
Prostate cancer
Frater–Seebach
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
of various types of cancer, such as prostate, colon, and glioma.4–7
Specifically, the voltage-gated sodium channel has been shown to
Voltage-gated sodium channels are important transmembrane
proteins critical in the generation and propagation of signals in
electrically excitable tissues like muscles, the heart, and nerves.
play a role in cancer cell proliferation, migration, and adhesion.8
However, the signaling pathways involved in cancer progression
are yet to be elucidated.9
They are comprised of a pore forming
a
-subunit, and auxiliary b-
Previously, Brown et al.10 described the design and synthesis of
2-(3-chlorophenyl)-2-hydroxynonanamide and its effect on so-
dium channels.11 Given the increasing focus of the effect of stereo-
chemistry on compound efficacy and toxicity, we desired to
subunits.1 To date, nine
a
-subunit isoforms have been cloned along
with four auxiliary b-subunit isoforms.2 These nine sodium chan-
nel isoforms are classified by their sensitivity to the neurotoxin
tetrodotoxin (TTX). There are six TTX-sensitive isoforms: Nav1.1,
Nav1.2, Nav1.3, Nav1.4, Nav1.6, and Nav1.7; and three TTX-resistant
isoforms: Nav1.5, Nav1.8, and Nav1.9.3 Pharmacological modulation
of voltage-gate sodium channels has proven clinically beneficial
for the treatment of pain, epilepsy, depression, and cardiac
arrhythmias.3
synthesize each enantiomer of 3-chlorophenyl-a-hydroxyamide.
The chiral template described by Seebach and Frater12 utilizes
cis-2,5-disubstituted 1,3-dioxolan-4-ones, as scaffolds for the ste-
reocontrol of alkylations, aldol additions, Michael additions, nucle-
ophilic additions, and Mannich reactions.13–18 Cyclocondensation
of a mandelic acid with pivaldehyde gives the 2,5-disubstituted
dioxolanone, and alkylation is directed by the tert-butyl group.
Using (R)-3-chloromandelic acid we observed both cis- and trans-
2,5-disubstituted dioxolanones. In this study, we employed the
use of both isomers to utilize this asymmetric strategy to synthe-
The role of ion channels in cancer is an emerging field. Recent
studies have demonstrated that voltage-gated ion channels could
play a role in the onset, proliferation and malignant progression
size each enantiomer of 3-chlorophenyl-a-hydroxyamide (( )-1)
to evaluate the chiral preference for inhibiting sodium channel
isoforms.
⇑
Corresponding author. Tel.: +1 202 687 8603; fax: +1 202 687 0617.
E-mail address: mb544@georgetown.edu (M.L. Brown).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.061

G. C. Davis et al. / Bioorg. Med. Chem. 20 (2012) 2180–2188
2181
the less polar solvent would result in a slower reaction rate with
increased selectivity. However, allowing this reaction to run over-
night resulted in no conversion of the starting material. Blay et al.
reported that inverse addition and the addition of 3 equiv of HMPA
to these reactions resulted in increased diastereoselectivity.17 The
inverse addition protocol calls for the addition of LDA to a pre-
mixed solution of the dioxolanone, heptyl iodide, and HMPA in
THF at ꢀ78 °C. Indeed, when we treated the cis-dioxolanone 3 with
LDA in THF–HMPA and heptyl iodide at ꢀ78 °C the alkylated prod-
uct 4 was obtained as a 1:10 ratio of diastereomers. Under these
conditions the major product had the heptyl chain trans to the
tert-butyl group.
2. Chemistry
2.1. Synthesis of compounds 1 and 2
Racemic ( )-1 was synthesized using the previously described
procedure shown in Scheme 1.4 Grignard addition of heptylmagne-
sium bromide into 3-chlorobenzonitrile followed by acidic hydro-
lysis gave the desired ketone 2. Treatment of the ketone with
TMSCN, followed by acidic hydrolysis furnished the racemic 2-(3-
chlorophenyl)-2-hydroxynonanamide (( )-1).
2.2. Synthesis of compounds (R)-(ꢀ)-1 and (S)-(+)-1
The surprising results provided by the THF–HMPA system led us
to consider what effect the leaving group of the electrophile would
have on this reaction. We attempted the reaction with heptyl
iodide, heptyl triflate, and heptyl bromide, as summarized in
Table 2. Our hypothesis was that reactivity of the electrophile
would strongly affect the diastereoselectivity of the alkylation
product. We predicted that using heptyl triflate would result in
an increased reaction rate, allowing for better selectivity. However,
addition of the triflate gave a 1:4.5 mixture of stereoisomers by
1H-NMR. Reaction with heptyl bromide was incomplete, and gave
a 1:1.5 mixture of stereoisomers, thus suggesting that the heptyl
iodide was the most suitable heptyl source for our synthesis result-
ing in a 1:10 mixture stereoisomers. Our most satisfactory result, a
diastereoselectivity of 6:94, was obtained using heptyl iodide and
carrying out the reaction at ꢀ78 °C for 3 h, followed by quenching
the reaction at that same temperature.
In Scheme 2, commercially available (R)-(ꢀ)-3-chloromandelic
acid was condensed with pivaldehyde to give cis isomer dioxola-
nones 3 with a 5:1 (cis:trans) selectivity. Interestingly, this same
reaction using the unsubstituted mandelic acid gives almost exclu-
sively the cis-isomer. The cis and trans isomers are easily separable
by column chromatography and both were isolated with high pur-
ity. Alkylation of 3 was carried out under various conditions sum-
marized in Table 1. Initially we tried to make the enolate with
freshly prepared LDA at ꢀ78 °C in THF. After the complete addition
of the dioxolanone the temperature was raised to 0 °C for 10 min,
and then lowered back to ꢀ78 °C. Heptyl iodide was then added
dropwise at ꢀ78 °C. This procedure is known as the direct addition
method. Initial alkylation of the dioxolanone in THF using this
addition method gave a 2:1 (cis:trans) mixture of the alkylated
product. Previously, a 1:3 cyclohexane/ether solvent mixture was
shown to promote diastereoselectivity in Li enolate reactions.19
Applying these conditions to our system however, resulted in no
reaction. With this in mind we ran the reaction in Et2O, hoping that
Separation of the diastereomers by column chromatography
was accomplished using an ether-hexanes gradient. Treatment of
alkyl dioxolanone 4, with concentrated ammonium hydroxide in
ethanol furnished (R)-(ꢀ)-2-(3-chlorophenyl)-2-hydroxynonana-
mide ((R)-(ꢀ)-1) directly in modest yields as a 91:1 ratio of enan-
tiomers as determined by chiral HPLC. This series of reactions
proceeds with retention of stereochemistry giving the (R)-enantio-
mer as the main product. By utilizing the synthesized (S)-(+)-3-
chloromandelic acid 5 and completing the same synthetic steps
as we previously accomplished with the commercially available
(R)-(ꢀ)-3-chloromandelic acid, we synthesized (S)-(+)-2-(3-chloro-
O
O
HO
NH2
C7H15
CN
a, b
c, d
C7H15
Cl
Cl
Cl
phenyl)-2-hydroxynonanamide ((S)-(+)-1) as
a 95:5 ratio of
2
( )-1
enantiomers as determined by chiral HPLC (scheme 2). The
(+)-enantiomer was confirmed by X-ray crystallography to have
the S-configuration (see Supplementary data) and the ligand
coordinates used as a starting geometry in our docking studies.
Scheme 1. Synthesis of racemic 2-(3-chloro-phenyl)-2-hydroxy-nonanoic acid
amide. Reagents and conditions: (a) (i) C7H15MgBr, THF, 0 °C to rt. overnight; (ii)
1 N HCl; (b) TMSCN, KCN, 18-c-6, CH2Cl2; (c) concd HCl, HCl (gas); (d) 0 °C.
O
O
NH2
OH
C7H15
O
OH
O
O
c
b
d
O
C7H15
OH
O
O
24%
57%
86%
Cl
Cl
Cl
Cl
3
4
(R)-(-)-1
a
O
O
NH2
O
OH
O
O
c
b
d
OH
C7H15
O
C7H15
OH
O
O
70%
71%
60%
Cl
Cl
Cl
Cl
5
6
7
(S)-(+)-1
Scheme 2. Synthesis of optically pure (R)- and (S)-2-(3-chloro-phenyl)-2-hydroxy-nonanoic acid amide. Reagents and conditions: (a) (1) AcCl, MeOH, 40 °C, 87%; (2) p-
nitrobenzoic acid, PPh3, DEAD, THF, 86%; (3) NaN3, MeOH, 40 °C, 72%; (4) 5% NaOH, 40 °C, 96%; (b) Pivaldehyde, TfOH, pentane, Dean–Stark; (c) LDA, C7H15I, THF–HMPA,
ꢀ78 °C; (d) 7 N NH3, MeOH, rt.

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G. C. Davis et al. / Bioorg. Med. Chem. 20 (2012) 2180–2188
Table 1
A
10
R-ICM-I-136
1 ms
B
S-ICM-I-136
Effect of solvent and addition method on diastereoselectivity of alkylation
µM
10 µM
Entry
1
Solvent
THF
Addition method
Direct
dr (cis:trans)
2:1
1
µ
M
1 ms
1 µM
2
3
THF–cyclohexane
Et2O
Direct
Direct
NR
NR
control
control
4
6
THF–HMPA
THF
Inverse
Inverse
1:10
NR
C
Racemic ICM-I-136
Legend. Heptyl source = C7H15I.
10
µ
µ
M
1 ms
Table 2
1
M
Effect of electrophile on diastereoselectivity
control
Figure 2. Effects of racemate and enantiomers of ICM-I-136 on hNav1.2.
Entry
Heptyl source
dr (cis:trans)
1
2
ꢁ
4
C7H15OTf
C7H15Br
1:4.5
1:1.5 + SM
1:10
C7H15
C7H15
I
I
6:94⁄
lectivity. The racemic mixture (IC50 = 1.81
(IC50 = 1.88 M) were similarly active against the Nav1.7 isoform,
while the (S)-(+)-1 enantiomer (IC50 = 2.62 M) was weaker as a
l
M) and (R)-(ꢀ)-1
Legend. Reaction held at ꢀ78 °C for 3–4 h and quenched immediately.
l
l
blocker. We observed that the (S)-(+)-1 enantiomer was preferred
by hNav1.5 and (R)-(ꢀ)-1 was more active against hNav1.7. DPH
was not active on either hNav1.5 or hNav1.7 at concentrations less
O
O
HO
NH2
NH
than 100 lM.
HN
C7H15
The effects of the racemic mixture and enantiomers of com-
pound 1 were also examined by patch clamp electrophysiology
on the human Nav channel isoform, Nav1.2, stably expressed in hu-
man embryonic kidney cells (HEK 293). Nav currents were elicited
by step depolarizations from a holding potential of ꢀ60 to +10 mV
for 25 ms at 15 s intervals. Sodium currents were record during a
control drug free condition, after 5 min of drug perfusion and fol-
O
Cl
A
1
Figure 1. Phenytoin (DPH) and 2-(3-chloro-phenyl)-2-hydroxy-nonanoic acid
amide (( )-1).
lowing washout. At 1
rent by 67.4 5.3% (n = 4) and by 94.3 0.6% (n = 3) at 10
contrast, (S)-(+)-1 and the racemic mixture were significantly less
potent than the (R)-(ꢀ)-1 enantiomer at 1 M, (P <0.05). (S)-(+)-1
inhibited the sodium channel current by 34.9 2.3% (n = 3) at
M and by 91.5 0.5% (n = 3) at 10 M. The racemic mixture
inhibited the sodium channel current by 31.9 2.9% (n = 3) at
M and by 87.0 3.1% (n = 3) at 10 M (Fig. 2). All drug effects
were fully reversible on washout.
l
M, (R)-(ꢀ)-1 inhibited the Nav channel cur-
lM. In
Table 3
l
hNav 1.5 and hNav 1.7 IC50’s for racemic, (R)-, (S)-2-(3-chloro-phenyl)-2-hydroxy-
nonanoic acid amide, and DPH
1
l
l
a
a
Compound
hNav 1.5 IC50
(lM)
hNav 1.7 IC50 (lM)
( )-1
5.78 1.2
7.43 1.5
4.78 1.0
>100
1.81 0.4
1.88 0.4
2.62 0.5
>100
1
l
l
R-(ꢀ)-1
S-(+)-1
DPH
3.2. Homology and structural modeling
a
Fluorescent based assay.
In an effort to rationalize the enantioselective effects of the so-
dium channel blockers with the sodium channel pore, and to
understand the differential activity and binding event that occurs
with the drug for the R and S configuration, we predicted the
structure for the open and the closed Nav1.7 channel. The sodium
channel pore was developed by aligning the pore-forming residues
15–101 of the X-ray structure of the open form of the KcsA
potassium channel, with residues 235–410 of domain I, residues
690–799 of domain II, residues 1123–1275 of domain III, and
residues 1445–1551 of domain IV of the sodium channel. KcsA
potassium channel residues 22–124 were used to model the P-loop
regions with the N- and C-terminal residues of these segments. The
orientations of the four domains were modeled by aligning sodium
channel domains I–IV with MthK channel chains A–D. The BTX
binding site location, as identified by mutational studies, is on
the pore-facing side of the S6 helices from domains I, III, and
IV.21 Upon analysis of the homology model structure, the IS6, IIIS6
and IVS6 segments, and the residues that form the drug binding
site are all conserved, and are mainly hydrophobic. We predicted
both the open and the closed channel of the Nav channel based
on the MthK and KcsA potassium channels as a template. In the
3. Results
3.1. Na channel activity
Using a FRET based assay to measure sodium channel depen-
dent changes in membrane potential, ( )-1, (R)-(ꢀ)-1, (S)-(+)-1
and DPH (Fig. 1) were evaluated for their isoform specific sodium
channel blocking effects and the IC50 values are shown in Table
3. Two isoforms of the channel were examined: hNav1.5, a cardiac
sodium channel associated with arrhythmias, and hNav1.7, an iso-
form found in the peripheral nervous system.20 Using a fluorescent
based assay, compound (S)-(+)-1 exhibited the greatest activity for
the hNav1.5 isoform of the channel with an IC50 of 4.78 lM. One
enantiomer possessed slightly greater activity than the other, and
the racemic mixture was observed to have intermediate activity.
Evaluation of compound (R)-(ꢀ)-1 gave an IC50 = 7.43
l
M and eval-
uation of ( )-1, the racemic mixture, gave an IC50 = 5.78
l
M. These
three compounds were more potent blockers of Nav1.7 channels
compared with Nav1.5 channels, but displayed a different stereose-

G. C. Davis et al. / Bioorg. Med. Chem. 20 (2012) 2180–2188
2183
Figure 3. Binding model of compound 1 and Nav1.7. (A) Proposed binding model of R-(ꢀ)-1 with Nav1.7. (B) Proposed binding model of S-(+)-1 with Nav1.7. The sodium
channel is represented as helices (cyan), ball and stick model representation for critical binding site residues (yellow), and compound 1 (atom color) with H-bond denoted by
dotted lines.
closed channel model, F1579 and Y1586 in IVS6 were oriented to-
ward the pore because of their possible interaction with LA drugs.
In the open channel model, the bends in the S6 helices were pro-
duced at the serine sites corresponding to the glycine residues
found in the MthK open channel structure. Both the R and S config-
uration of compound 1 were docked using AutoDock 4.022 and
FlexX incorporated in Sybyl 8.0. However, the docked poses gener-
ated by both programs show different interactions with the S6 he-
lix residues in comparison to the mutation data.23–25 To be
consistent with respect to the mutation studies and previous
known interactions of lidocaine analogs,26 the docked positions
were remodeled using step-by-step manual docking with con-
strained molecular dynamics (MD) simulations followed by mini-
mization. In the restrained MD simulations, the optimum H-bond
and hydrophobic distance constraints were set between the pore
forming residues and the ligand. The residues such as F1283,
F1579, L1582, F1283, V1583, Y1586 in IVS6, and T1279, L1280 in
IIIS6, and L788, F791, L792, in IIS6 and I433, N434, L437 in IS6,
and the selectivity filter residues D400, E755, K1237 in the do-
mains of I–IV P-loops were identified as participants in the puta-
tive binding site for our compounds.
difference in sodium channel activity we observe for the R versus
the S enantiomer of our compound.
3.3. Gene activity
All four compounds were also counter-screened for human
ether-a-go-go-related gene (hERG) activity against the radio-ligand
MK-0499. Blockade of hERG K+ channels is widely regarded as the
predominant cause of drug-induced QT prolongation.27 As seen in
Table 4, the racemic mixture and the enantiomers did not signifi-
cantly inhibit hERG below 10 lM.
3.4. Cell assays
We have an interest in evaluating the role of the Nav channels in
prostate cancer. From our previous work we have demonstrated
that sodium channel blockers have marked effects on prostate can-
cer cell proliferation.4 In this present work we have implicated sev-
eral isoforms of the channel to be involved with prostate cancer
cell proliferation. CWR22rv-1 whole cell lysate extracts were eval-
uated for expression of hNav1.5 and hNav1.7 by Western blot anal-
A structural model of Nav1.7 predicted interaction with com-
pounds (R)-(ꢀ)-1) and (S)-(+)-1 is shown in Figure 3. Our binding
model suggest that compound 1 interacts with that residues
F1283, F1579, L1582, V1583, Y1586 in IVS6, and T1279, L1280 in
IIIS6, and L788, F791, L792, in IIS6 and F430, I433, L437 in IS6
and suggest amino acids that may contribute to potential binding
interactions. In fact some of these residues are found to be impor-
tant in alanine mutation experiments.22–24 As seen in Figure 3,
strong hydrophobic contacts were noticed between 1 and F1283,
F1579, L1582, V1583, Y1586 L1280, L788, F791, L792, I433, and
L437. Enantiomeric selectivity could be rationalized for the (R)-
(ꢀ)-1 isomer which may be driven by strong H-bonding between
the amide functionality and T1279 (Fig. 3A). Modeling studies sug-
gest that this interaction is only observed for the R enantiomer, and
not present for the S enantiomer (Fig. 3B). This could explain the
ysis. Both a-subunits were detected at 260 kDa with each antibody
(see Supplementary data). Specific bands were also detected at
lower molecular weights and are likely degradation products. Pre-
treating with their respective specific oligomer epitope control
antigen before antibody addition eliminated the signal of both
the 260 kDa band as well as the lower molecular weight bands.
Table 4
% Inhibition for hERG assay
Compound
% Inhibition
0.3
lM
1
lM
3
lM
10
l
M
30 lM
( )-1
15 3.0
16 3.2
10 2.0
17 3.4
26 5.2
25 5.0
20 4.0
15 3.0
20 4.0
33 6.6
28 5.6
51 10.2
57 11.4
65 13.1
17 3.4
78 15.6
90 18.0
90 18.0
40 8.0
R-(ꢀ)-1
S-(+)-1
DPH
9
1.8
Figure 4. (R)-(ꢀ)-1 induces cell death of human CWR cells at 25
lM after 24 h.

2184
G. C. Davis et al. / Bioorg. Med. Chem. 20 (2012) 2180–2188
5. Experimental section
CT
)-I
1000
800
600
400
200
(
10mg/kg
(R)-(-)-1 10 mg/kg
(S)-(+)-I 10 mg/kg
5.1. Chemistry
5.1.1. Computational chemistry
Multiple sequence alignment of the S6 transmembrane residues
from domains I, III, and IV was carried out using PSI-BLAST and
CLUSTALW.29 Homology modeling of the S5, the P-loops, and S6
from all four domains was accomplished using open MthK channel
X-ray structure (PDB: 1lnq) as a template. Non-homologous re-
gions in the longer P-loops of domains I and III, which correspond
to putative glycosylation sites, were deleted. The P-loops and the N
and C termini were modeled based on homologous segments of the
KcsA channel structure (PDB: 1bl8). Sodium channel sequences
were aligned versus the MthK channel using ClustalW, and the
structure was modeled employing the program Modeler 8.1. To
avoid close contacts of the side chain atoms with each other, differ-
ent rotamer states of the residues were considered to find the state
with minimal clashes but favorable interactions. Local minimiza-
tion of side chain atoms was also performed. Docking studies be-
tween the ligand and the sodium channel were carried out using
the program AUTODOCK 4.022 with all the parameters set to default.
Molecular dynamics simulations were carried out using AMBER
8.030 with default parameters.
0
0
10
20
30
Day
Figure 5. Effects of ( )-1 and enantiomers of 1 on human prostate xenograft PC3.
Legend. Compounds were dosed ip once a day every other day for 26 days. Studies
with N = 6.
With the identification of both sodium channels in human pros-
tate cancer cell line CWR22rv-1, we evaluated ( )-1, (R)-(ꢀ)-1, and
(S)-(+)-1 for their effects on prostate cancer cell growth. We found
that compound (R)-(ꢀ)-1 showed the greatest effect on CWR pro-
liferation (Fig. 4). A concentration of 25 lM induced approximately
25% cell death after 24 h, while compounds ( )-1 and (S)-(+)-1
showed marginal effects after 24 h. After 72 h, compound (R)-
(ꢀ)-1 induced cell death in 60% of the human prostate cancer cells,
while compounds ( )-1 and (S)-(+)-1 induced 25% and 40% cell
death, respectively.
5.1.2. General remarks
Chemicals were purchased from Aldrich Chemical Company,
and were used without any further purification. Dry solvents were
dried over 4 Å molecular sieves prior to use. Air-sensitive reactions
were carried out in oven-dried glassware under an N2 atmosphere.
Flash column chromatography separations were done on a Biotage
SP1 system monitoring at 254 nm. All NMR spectra were recorded
on a Varian 400 spectrometer, operating at 400 MHz for 1H and
100 MHz for 13C NMR. Optical rotations were taken on a Belling-
ham & Stanley ADP220 polarimeter using a 25 mm cell. Chiral
HPLC analysis was carried out on a Shimadzu LCMS-2010EV using
a ChiralPak AS column monitoring at 254 nm.
3.5. Animal studies
We administered intraperitoneally (ip) 10 mg/kg of racemic ( )-
1 and each enantiomer in mice bearing PC3 xenografts (Fig. 5). This
dose was administered qd every other day for 24 days and effects
on reducing the tumor volume were measured. We observed a sta-
tistical reduction in the prostate tumor volume after day 15. Treat-
ment with the racemic and both enantiomers resulted in a 62%
decrease in tumor volume at a 10 mg/kg dose and a qd dosing
schedule. This demonstrates for the first time that Nav blockers
(such as ( )-1 or its enantiomers) can significantly reduce the size
of prostate tumors in vivo and tumors that are androgen
insensitive.
5.1.3. (2R,5R)-2-(tert-Butyl)-5-(3-chlorophenyl)-1,3-dioxolan-4-
one (3)
To
a
suspension of R-(ꢀ)-3-chloromandelic acid (6.0 g,
32.15 mmol) in dry pentane was added pivaldehyde (4.2 mL,
38.59 mmol), followed by the addition of triflic acid (0.11 mL,
1.29 mmol) at room temperature. A Dean–Stark trap was attached
added to the flask, and the reaction mixture was warmed to 36 °C
and allowed to reflux for 6 h. The mixture was cooled to room tem-
perature and 8% aqueous NaHCO3 solution was added and the reac-
tion was concentrated to remove pentane. The slurry was filtered
and concentrated to give the product as a 5:1 mixture of diastereo-
mers. The diastereomers were separated by flash column chroma-
tography eluting with 1:5 EtOAc/hexanes to give 4.26 g of pure
product as a fluffy white solid. mp 84–86 °C; 1H NMR (400 MHz,
CDCl3, d): 1.06 (s, 9H), 5.19 (s, 1H), 5.30 (d, J = 1 Hz, 1H), 7.33–
7.34 (m, 3H), 7.44 (m, 1H). 13C NMR (100 MHz, CDCl3, d): 20.1,
4. Conclusions
The relationship between voltage-gated ion channels and can-
cer represents an exciting new area of investigation, and the syn-
thesis of active chiral inhibitors for selected isoforms may
provide potential therapeutic opportunities. We have successfully
demonstrated that by using the Seebach and Frater chiral template,
both enantiomers of 2-(3-chlorophenyl)-2-hydroxynonanamide
can be synthesized. Our biological evaluation of compounds ( )-
1, (R)-(ꢀ)-1, and (S)-(+)-1, shows a preference for the R enantiomer
over the S enantiomer in CWR22rv-1 cells. Our model of (R)-(ꢀ)-1
docked in the sodium channel shows a critical hydrogen bond
interaction of the amide group with the T1279 residue in the sur-
rounding protein. This interaction is not present with (S)-(+)-1, and
is a potential reason for its reduced sodium channel activity.
Finally, our xenograft studies provide evidence that treatment
with Nav blockers (such as ( )-1 or its enantiomers) can signifi-
cantly reduce the size of prostate tumors in vivo including tumors
that are androgen insensitive. Since 80–90% of prostate cancer pa-
tients develop androgen-independent tumors 12–33 months after
androgen ablation, these findings may have significant clinical po-
tential for this phenotype.28
34.2, 91.1, 121.6, 127.3, 128.1, 134.2, 136.1, 173.5. ½a D25
ꢀ72.0 (c
ꢂ
1.00, CHCl3).
5.1.4. S-(+)-3-Chloromandelic acid (5)
To a round bottom flask was added 50 mL of MeOH and three
drops of AcCl, 1.61 g (8.60 mmol) of (R)-3-chloromandelic acid
was then added in one portion. The mixture was allowed to stir at
rt. overnight. The reaction was then poured in to 150 mL of H2O con-
taining 10 mL of saturated NaHCO3 and extracted with CH2Cl2
(3 ꢃ 30 mL). The combined organic fractions were washed with
brine, dried (Na2SO4), filtered and concentrated to give 1.53 g

G. C. Davis et al. / Bioorg. Med. Chem. 20 (2012) 2180–2188
2185
(87%) ofthe product as a thick viscous oil which was sufficientlypure
for the next step. 1H NMR (400 MHz, CDCl3, d): 3.73 (s, 3H), 3.78 (d,
J = 6 Hz, 1H), 5.13 (d, J = 5 Hz, 1H), 7.26–7.28 (m, 3H), 7.40–7.41 (m,
1H). 13C NMR (100 MHz, CDCl3, d): 53.4, 72.4, 125.0, 127.0, 128.8,
1.88–2.06 (m, 2H), 5.35 (s, 1H), 7.26–7.30 (m, 2H), 7.50–7.54 (m,
1H), 7.62–7.63 (m, 1H). 13C NMR (100 MHz, CDCl3, d): 14.3, 22.8,
23.7, 23.8, 29.2, 29.5, 31.9, 35.2, 38.9, 82.3, 109.1, 123.4, 125.4,
128.3, 129.7, 134.5, 140.5, 173.5. ½a D22:4
ꢀ40 (c 1.00, CH2Cl2).
ꢂ
130.1, 134.7, 140.3, 173.7. ½a D23
ꢀ168 (c 1.00, CHCl3).
ꢂ
To a stirred solution of 0.350 g (1.74 mmol) of (R)-3-chloroman-
delic acid methyl ester in dry THF was added 0.913 g (3.48 mmol)
of PPh3 and 0.582 g (3.48 mmol) of p-nitrobenzoic acid. The solu-
tion was cooled to 0 °C and 1.31 mL (3.48 mmol) of a 40% wt. solu-
tion of DEAD was slowly added. The reaction mixture was allowed
to stir at rt. for 6 h under N2. THF was then removed in vacuo and
the crude product partitioned between H2O and EtOAc. The com-
bined organic layers were washed with brine, dried (Na2SO4), fil-
tered and concentrated. The residue was purified by flash column
chromatography eluting with 1:19 EtOAc/hexanes then 1:9
EtOAc/hexanes to give 0.523 g (86%) of the product as a sticky yel-
low oil. 1H NMR (400 MHz, CDCl3, d): 3.74 (s, 3H), 6.14 (s, 1H),
7.34–7.36 (m, 2H), 7.42–7.45 (m, 1H), 7.53–7.54 (m, 1H), 8.25 (s,
4H). 13C NMR (100 MHz, CDCl3, d): 53.2, 74.8, 123.9, 126.1, 127.9,
130.0, 130.5, 131.3, 134.5, 135.1, 135.4, 151.1, 164.0, 168.5.
The prepared nitroester (4.58 g, 22.85 mmol) was added to
7.43 g (114.23 mmol) of NaN3 and the mixture was heated for
1 h at 40 °C. The solvent was then removed and the residue puri-
fied by flash column chromatography eluting with 1:5 EtOAc/hex-
anes to give 3.291 g (72%) of a white solid. 1H NMR (400 MHz,
CDCl3, d): 3.53 (d, J = 6 Hz, 1H), 3.76 (s, 3H), 5.13 (d, J = 5 Hz, 1H),
7.28–7.30 (m, 3H), 7.40–7.41 (m, 1H). 13C NMR (100 MHz, CDCl3,
d): 53.4, 72.3, 125.0, 126.9, 128.9, 130.1, 134.7, 140.3, 173.8.
To a 3.091 g (15.41 mmol) of (S)-3-chloromandelic acid methyl
ester was added 100 mL of 5% NaOH. The reaction mixture was
heated to 40 °C for one and then allowed to cool to rt. The reaction
was then acidified with 1 N and extracted with EtOAc (3 ꢃ 20 mL).
The combined organic fractions were dried (Na2SO4), filtered and
concentrated in vacuo. The solid residue was recrystallized from
hot toluene to give 2.768 g (96%) of a white crystalline solid 5.
1H NMR (400 MHz, DMSO-d6, d): 5.05 (s, 1H), 7.32–7.36 (m, 3H).
13C NMR (100 MHz, DMSO-d6, d): 72.4, 126.0, 127.0, 128.2, 130.7,
5.1.7. R-(ꢀ)-2-(3-Chlorophenyl)-2-hydroxy-nonanoic acid
amide ((R)-(ꢀ)-1)
To a flame dried round bottom flask was added the dioxolanone
4 (0.948 g, 2.69 mmol) in 5 mL or dry MeOH. To this was added
20 mL of 7 N NH3 in MeOH dropwise. This was allowed to stir over
16 h while monitoring by TLC (1:1 hexanes/EtOAc). The reaction
mixture was then poured into water and extracted with CH2Cl2
(4 ꢃ 10 mL). The combined organic layers were then washed with
brine, dried (Na2SO4), filtered and concentrated. The residue was
purified by flash column chromatography eluting with 1:1 hex-
anes/EtOAc to yield 0.659 g (86%). mp 78–79 °C; 1H NMR
(400 MHz, CDCl3, d): 0.85 (t, J = 7 Hz, 3H), 1.23–1.29 (m, 10H),
1.94–2.01 (m, 1H), 2.16–2.22 (m, 1H), 3.19 (s, 1H), 5.65 (br s,
1H), 6.47 (br s, 1H), 7.23–7.28 (m, 2H), 7.44–7.47 (m, 1H), 7.58–
7.59 (m, 1H). 13C NMR (100 MHz, CDCl3, d): 14.3, 22.8, 23.5, 29.3,
29.8, 32.0, 39.6, 78.8, 123.9, 125.9, 128.1, 129.9, 134.6, 144.6,
176.3. ½a 2D3:1
ꢀ24 (c 0.50, CHCl3).
ꢂ
5.1.8. (2S,5S)-2-(tert-Butyl)-5-(3-chlorophenyl)-5-heptyl-
1,3-dioxolan-4-one (7)
Prepared in the same manner as (2R,5R)-2-(tert-butyl)-5-(3-
chlorophenyl)-5-heptyl-1,3-dioxolan-4-one using (2S,5S)-2-(tert-
butyl)-5-(3-chlorophenyl)-1,3-dioxolan-4-one.
1.414 g
yield
(71%). 1H NMR (400 MHz, CDCl3, d): 0.84 (t, J = 7 Hz, 3H), 0.96 (s,
9H), 1.16–1.35 (m, 10H), 1.88–2.06 (m, 2H), 5.35 (s, 1H), 7.26–
7.30 (m, 2H), 7.50–7.54 (m, 1H), 7.62–7.63 (m, 1H). 13C NMR
(100 MHz, CDCl3, d): 14.3, 22.8, 23.7, 23.8, 29.2, 29.5, 31.9, 35.2,
38.9, 82.3, 109.1, 123.4, 125.4, 128.3, 129.7, 134.5, 140.5, 173.5.
5.1.9. S-(+)-2-(3-Chlorophenyl)-2-hydroxy-nonanoic acid amide
((S)-(+)-1)
Prepared in the same manner as (R)-(ꢀ)-2-(3-chlorophenyl)-2-
hydroxy-nonanoic acid amide using (2S,5S)-2-(tert-butyl)-5-(3-
chlorophenyl)-5-heptyl-1,3-dioxolan-4-one. 0.683 g yield (60%)
mp 78–79 °C; 1H NMR (400 MHz, CDCl3, d): 0.85 (t, J = 7 Hz, 3H),
1.23–1.29 (m, 10H), 1.94–2.01 (m, 1H), 2.16–2.22 (m, 1H), 3.19
(s, 1H), 5.65 (br s, 1H), 6.47 (br s, 1H), 7.23–7.28 (m, 2H), 7.44–
7.47 (m, 1H), 7.58–7.59 (m, 1H). 13C NMR (100 MHz, CDCl3, d):
14.3, 22.8, 23.5, 29.3, 29.8, 32.0, 39.6, 78.8, 123.9, 125.9, 128.1,
133.5, 143.3, 174.2. ½a D23
ꢂ
+123 (c 3.00, H2O).
5.1.5. (2S,5S)-2-(tert-Butyl)-5-(3-chlorophenyl)-1,3-dioxolan-4-
one (6)
Prepared in the same manner as (2R,5R)-2-(tert-butyl)-5-(3-
chlorophenyl)-5-heptyl-1,3-dioxolan-4-one (3) using (S)-(+)-3-
chloromandelic acid 5. Mp 40–42 °C; 1H NMR (400 MHz, CDCl3,
d): 1.06 (s, 9H), 5.24 (s, 1H), 5.30 (d, J = 1 Hz, 1H), 7.33–7.34 (m,
3H), 7.44 (m, 1H). 13C NMR (100 MHz, CDCl3, d): 21.1, 35.2, 91.6,
129.9, 134.6, 144.6, 176.3. ½a D23:1
ꢂ
+24 (c 0.50, CHCl3).
122.9, 127.3, 128.1, 133.5, 137.1, 173.5. ½a D25
ꢂ
ꢀ4.0 (c 1.00, CHCl3).
5.2. X-ray crystallography
5.1.6. (2R,5R)-2-(tert-Butyl)-5-(3-chlorophenyl)-5-heptyl-1,3-
dioxolan-4-one (4)
Both (ICM-1-136-1) (S)-2-(3-chloro-phenyl)-2-hydroxy-nona-
noic acid amide and (ICM-1-136-2) (R)-2-(3-chloro-phenyl)-2-hy-
droxy-nonanoic acid amide were crystallized from 70% ethanol
using slow evaporation method at room temperature. The diffrac-
tion data were collected at 100 K using a Rigaku R-axis Rapid dif-
To a flame-dried round bottom flask equipped with a magnetic
stir bar was added diisopropylamine in THF under N2. The flask
was cooled to -78 °C and BuLi was added in one portion. The cool-
ing bath was removed and replaced with an ice-water bath. Mean-
while, (2R,5R)-2-(tert-butyl)-5-(3-chlorophenyl)-1,3-dioxolan-4-
one, heptyl iodide, and HMPA were dissolved in THF and place in
a separate flame dried round bottom under an N2 atmosphere.
The flask was cooled to ꢀ78 °C and the previously prepared LDA
was added dropwise over 15 min. The reaction mixture was main-
tained at a constant temperature of ꢀ78 °C for 3 h at which time it
was quenched with saturated NH4Cl solution. The product was
then extracted with Et2O, the organic layers combined, dried
(Na2SO4), filtered and concentrated in vacuo. The crude 96:4 mix-
ture of diastereomers was separated by flash column chromatogra-
phy carefully eluting with 1% Et2O in hexanes. 1H NMR (400 MHz,
CDCl3, d): 0.84 (t, J = 7 Hz, 3H), 0.96 (s, 9H), 1.16–1.35 (m, 10H),
fractometer, equipped with a Mo K
a radiation source (60 kV,
40 mA). The radiation was monochromatized with graphite mono-
chromator. HKL-200031,32 was used for control of the data collec-
tion as well as for data reduction. The structure was solved and
refined by the HKL-3000SM system33 which is integrated with
SHELXS, SHELXL34 and O.35 Absolute configurations of both com-
pounds were determined using anomalous dispersion. Details of
data collection, processing and refinement (see Supplementary
Table 1). Interestingly both compounds crystallized with two
molecules in asymmetric unit (see Supplementary Fig. 1). The
molecules of the (+)-2-(3-chloro-phenyl)-2-hydroxy-nonanoic acid
amide forming crystals have two different conformations of the
aliphatic chains.

2186
G. C. Davis et al. / Bioorg. Med. Chem. 20 (2012) 2180–2188
5.3. Biology
tions (in triplicate per treatment point) for 24, 48, 72 h. After treat-
ment, cells were harvested by trypsinization and fixed in 70%
ethanol. The fixed cells were then stained with propidium iodide
5.3.1. Cell culture for western blots
The CWR22rv-1 cell line was obtained from the American Tis-
sue Culture Collection (Manassas, VA). All cell lines were main-
tained in RPMI-1640 with L-glutamine (CellGro, Lawrence, KS)
supplemented with 5% heat-inactivated fetal bovine serum (Sigma,
St. Louis, MO). LNCaP media was additionally supplemented with
0.1 nM DHT (Sigma, St. Louis, MO). Cells were seeded into Corning
T-75 flasks (Fisher, Pittsburg, PA) and incubated at 37 °C, 5% CO2,
and 100% relative humidity. Cultures were subcultured once per
week via trypsinization.
(50 lg/mL) after treatment with RNase (5 lg/mL). The stained cells
were analyzed for DNA content using FACSsort (Becton Dickinson).
Cell cycle fractions were quantified with Cell Quest (Becton Dickin-
son) or ModFit LT (Verity Software House).
5.3.3. Fluorescent based sodium channel assay
A functional fluorescent assay for Nav1.5 and Nav1.7 channel
activity was performed as previously described.37 Tissue culture
media and CC2-DMPE and DiSBAC2 were purchased from Invitro-
gen Corporation, Carlsbad CA, and pluronic acid from Molecular
Probes, Eugene, OR. HEK293 cells stably transfected with either
hNaV1.5 or hNaV1.7 were plated at approximately 11,000–20,000
Western protocols were adapted from Collins et al.36 Cells were
trypsin-harvested, washed and flash frozen prior to lysis. Lysates
were prepared in modified radioimmunoprecipitation (RIPA) buf-
fer (Sigma, St. Louis, MO) plus 50 mM Tris–HCl, 5 mM EDTA,
150 mM NaCl, 30 mM NaPPi, 50 mM NaF, 1 mM Na orthovanadate,
1% Triton-X 100, 0.1% SDS, 0.5% Na deoxycholate, 1 mM phenylm-
ethylsulphonyl fluoride (SIGMA, St. Louis, MO) and 1% protease
inhibitor cocktail (SIGMA, St. Louis, MO) for 2 h. Insoluble debris
was removed by centrifugation at 15,000g for 45 min. Total protein
was determined using the Bradford method (Bio-Rad, Hercules,
CA). Equivalent amounts of protein from different lysate samples
cells/well in flat bottom, poly-D-lysine coated black-wall, clear-bot-
tom, 384-well plates (Becton Dickinson, Bedford, MA), and incu-
bated overnight at 37 °C in a 10% CO2 atmosphere in growth
medium. Cells were washed with 0.03 mL of Dulbecco’s phosphate
buffered saline (D-PBS) containing calcium and magnesium. Cells
were then incubated with 0.025 mL of a solution containing
10 lM CC2-DMPE and 0.02% pluronic acid in D-PBS with calcium
and magnesium, supplemented with 10 mM glucose, and 10 mM
Hepes–NaOH, pH 7.4. After incubation in the dark for 45 min at
25 °C, cells were washed twice with 0.03 mL of (in mM): 165 NaCl,
4.5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, 10 Hepes–NaOH, pH 7.4
(VIPR-S). Afterwards, cells were incubated in the dark at 25 °C for
(30 lg/well) were denatured by boiling for 5 min and were re-
solved against Seeblue2 (Invitrogen, Carlsbad, CA) by SDS–PAGE
using 4% tris-glycine gels (Invitrogen, Carlsbad, CA) in Tris-Gly
SDS buffer (Biorad) at 85 V for 30 min and then 125 V for 2 h. Pro-
tein was transferred to methanol-pretreated PVDF membranes at
4 °C in Tris-glycine transfer buffer (Bio-Rad, Hercules, CA) at 30 V
for 16 h. Membranes were washed in 0.1% PBS-Tween, blocked
for 1 h in blocking buffer (50 mM Tris–Cl, 150 mM NaCl, and
10 g/L BSA in diH20), and subsequently incubated with either
625 ng/mL (1:2000) human Nav1.5 antibody pretreated with 1:10
blocking peptide in blocking buffer with 500 mg/L NaN3 at 4 °C
45 min with 0.025 mL of a solution containing 5 lM DiSBAC2(3)
in VIPR-S, in the absence or presence of test compound. At the
end of the incubation period, the plate was placed in a FLIPR TETRA
instrument (MDS Analytical Technologies, Sunnyvale CA), illumi-
nated at 390–420 nm, and fluorescence emissions were recorded
at approximately 1 Hz at 460 and 580 nm. Following a 7 s baseline
reading, 0.025 mL of VIPR-S containing 10
lM veratridine for
for 16 h or 7
lg/mL (1:1000) rat Nav1.7 (13/15 sequence homology
Nav1.5 or 20 M veratridine for Nav1.7 was added to each well,
l
to human) pretreated with 1:1 blocking peptide under similar con-
ditions. Membranes were washed four times for 15 min in 0.1%
PBS-Tween, blocked for 30 min in blocking buffer, and incubated
in 75 ng/mL (3:40,000) horse radish peroxidase-conjugated goat
anti-rabbit secondary antibody in blocking buffer for 1 h. Mem-
branes were washed in dH2O. After the last of four washes for
15 min with 0.1% PBS-Tween, the blots were developed using the
ECL chemiluminescence system (Amersham, Piscataway, NJ) and
visualized by exposure to Biomax MR Film (Kodak, Rochester,
NY). Membranes were washed in dH2O and then washed twice
for 5 min with 0.1% PBS-Tween. Blots were then stripped with
stripping buffer (0.375 M Tris–HCl, 12% SDS, 60 mM HCl, pH 6.8)
for 1 h at 56 °C and again washed in dH2O and then washed twice
for 5 min with 0.1% PBS-Tween. Membranes were blocked for 1 h
in blocking buffer and subsequently incubated with either
625 ng/mL (1:2000) human Nav1.5 antibody without blocking pep-
tide pretreatment in blocking buffer with 500 mg/L NaN3 at 4 °C for
and the emissions of both dyes were recorded for an additional
33 s. The change in fluorescence resonance energy transfer (FRET)
ratio (F/F0) was calculated as:
F=F0 ¼ ððA460=A580Þ=ðI460=I580ÞÞ
where A and I represent the readings after or before addition of
veratridine, respectively, at the indicated wavelengths. For I, the
readings from 2 to 5 s were averaged, and for A, readings from 3 s
after the signal had reached a plateau level (usually within 30–
40 s) were also averaged. Triplicate measurements were performed
for each experimental condition and the data were averaged.
5.3.4. HERG binding assay
MK-499, a potent hERG blocker, was used in a ligand binding
assay to evaluate the binding of test compounds to hERG potas-
sium channels as previously described.38 Test compounds were
incubated with 0.05 nM of radiolabeled MK-499 ([35S]MK-499;
specific Activity = 1,279,010 lCi/lmol) in an assay buffer solution
16 h or 7 lg/mL (1:1000) rat Nav1.7 without blocking peptide pre-
(70 mM NaCl, 60 mM KCl, 10 mM HEPES/NaOH (pH 7.4), 2 mM
MgCl2, 1 mM CaCl2) also containing membranes isolated from
HEK-293 cells stably expressing hERG channels in 96-well polypro-
pylene deep well assay plates at room temperature (25 °C) for
>90 min. Assay plates were rinsed three time with buffer
(130 mM NaCl, 10 mM HEPES/NaOH (pH 7.4), 2 mM MgCl2, 1 mM
CaCl2) and the solution transferred to a Packard FilterMate Univer-
sal Harvester apparatus and filtered using PerkinElmer UniFilter-
96 GF/C 96-well white microplates pre-soaked with 0.3% BSA
(10 mL/L of 30% BSA). Plates were dried overnight at 37 °C, or for
2 h at 56 °C. The bottom of the plates were sealed and 0.025 mL
of Microscint 0 was added to each well. The plates were top sealed
and counted for 2 min/well in a Packard TopCount Scintillation
treatment in blocking buffer with 500 mg/L NaN3 at 4 °C for 16 h.
The western procedure was repeated as described above.
5.3.2. Cell culture and treatment
CWR22Rv1 cells (ATCC, Manassas, VA) were seeded in 6 well
tissue culture plate at density of 300,000 cells/well and maintained
in RPMI 1640 (Mediatech, Herndon, VA) containing 10% fetal bo-
vine serum, 2.5 mM
(100 IU/mL and 100
L
-glutamine, and penicillin-streptomycin
l
g/mL, respectively) at 37 °C with 5% CO2.
Cells were then switched to serum free RPMI media overnight prior
to drug treatment. Compounds were dissolved in 100% dimethyl-
sufoxide (DMSO) and diluted to the desired concentrations in
serum free RPMI. Cells were treated with indicated drug concentra-

G. C. Davis et al. / Bioorg. Med. Chem. 20 (2012) 2180–2188
2187
counter. Test compounds were evaluated in a five point titration
( )-1, R-(ꢀ)-1 or S-(+)-1 or vehicle control respectively once every
other day for 4 weeks. At the same time, the tumor size of each
mouse was measured by caliper and calculated by the formula:
Length ꢃ width ꢃ height/2.
format using half-log steps from 0.3 to 30 lM. Percent inhibition
of [35S]MK-499 was calculated relative to high control values (no
unlabeled MK-499 added) and values obtained in the presence of
1 lM unlabeled MK-499. Less than 5% of the added radioactivity
was retained on the filters. Assays were performed in triplicate.
5.3.9. Data analysis
Statistical analyses were performed using the standard one-way
ANOVA or ANOVA on ranks followed by a Tukey or Dunn’s post hoc
test. Data is reported as mean S.E.M.
5.3.5. Nav electrophysiology studies
Sodium currents were recorded using the whole-cell configura-
tion of the patch clamp recording technique with an Axopatch 200
amplifier (Axon Instruments, Foster City, CA). All voltage protocols
were applied using pCLAMP 9 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 P87, Sutter Instru-
ments Co, Novato, CA) and heat polished to produce electrode
Acknowledgments
This work was supported by NIH grant CA105435-04, Merck
Academic Development Award, and the Georgetown University
Drug Discovery Program. The authors would also like to thank
Dr. H.A. Hartmann for the HEK 293 cells stably expressing hNav1.2
and the shared resources at the Lombardi Comprehensive Cancer
Center.
resistances of 1.5–3.0 MX when filled with the following electrode
solution (in mM); CsCl 130, MgCl2 1, MgATP 5, BAPTA 10, HEPES 5
(pH adjusted to 7.4 with CsOH). Cells were plated on glass cover-
slips and superfused with solution containing the following com-
position; (in mM) NaCl 130, KCl 4, CaCl2 1, MgCl2 5, HEPES 5, and
glucose 5 (pH adjusted to 7.4 with NaOH). Compounds were pre-
pared as 100 mM stock solutions in dimethyl sulfoxide (DMSO)
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.08.061.
and diluted to desired concentration (1 or 10 lM) in perfusion
solution. All experiments were performed at room temperature
(20–22 °C). After establishing whole-cell, a minimum series resis-
tance compensation of 75% was applied and cells were held at
ꢀ100 mV for 5 min to account for equilibrium gating shifts. So-
dium currents were evoked by stepping to +10 mV from a holding
potential of ꢀ60 mV for 25 ms at 15 s intervals. After control
recordings, test compounds were applied for 5 min to allow for
bath equilibration. Tonic block was assessed by comparing peak
sodium current in drug free conditions to peak current when drug
was present. Data analysis was performed using Clampfit software
(v9, Axon Instruments, CA, USA) and Origin (v6, Microcal Software,
MA, USA).
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Balb/c mice and Athymic Balb/c Nude mice were purchased
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4–6 per cage with microisolater tops and provided food (Furina
mice chow) and water ad libitum. The light cycle was regulated
automatically (12 hours light/dark cycle) and temperature was
maintained at 23 1 °C. All animals were allowed to acclimate to
this environment fro one week prior to experimental manipula-
tions. The Georgetown University Animal Care and Use Committee
approved all animal studies in accordance with the guideline
adopted by the National Institute of Health.
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