Discovery and evaluation of nNav1.5 sodium channel blockers with potent cell invasion inhibitory activity in breast cancer cells

Article abstract of DOI:10.1016/j.bmc.2018.04.003

Voltage-gated sodium channels (VGSC) are a well-established drug target for anti-epileptic, anti-arrhythmic and pain medications due to their presence and the important roles that they play in excitable cells. Recently, their presence has been recognized in non-excitable cells such as cancer cells and their overexpression has been shown to be associated with metastatic behavior in a variety of human cancers. The neonatal isoform of the VGSC subtype, Na_v1.5 (nNa_v1.5) is overexpressed in the highly aggressive human breast cancer cell line, MDA-MB-231. The activity of nNa_v1.5 is known to promote the breast cancer cell invasion in vitro and metastasis in vivo, and its expression in primary mammary tumors has been associated with metastasis and patient death. Metastasis development is responsible for the high mortality of breast cancer and currently there is no treatment available to specifically prevent or inhibit breast cancer metastasis. In the present study, a 3D-QSAR model is used to assist the development of low micromolar small molecule VGSC blockers. Using this model, we have designed, synthesized and evaluated five small molecule compounds as blockers of nNa_v1.5-dependent inward currents in whole-cell patch-clamp experiments in MDA-MB-231 cells. The most active compound identified from these studies blocked sodium currents by 34.9 ± 6.6% at 1 μM. This compound also inhibited the invasion of MDA-MB-231 cells by 30.3 ± 4.5% at 1 μM concentration without affecting the cell viability. The potent small molecule compounds presented here have the potential to be developed as drugs for breast cancer metastasis treatment.

Full text of DOI:10.1016/j.bmc.2018.04.003

Accepted Manuscript
Discovery and Evaluation of nNav1.5 Sodium Channel Blockers with Potent
Cell Invasion Inhibitory Activity in Breast Cancer Cells
Shilpa Dutta, Osbaldo Lopez Charcas, Samuel Tanner, Frédéric Gradek,
Virginie Driffort, Sébastien Roger, Katri Selander, Sadanandan E. Velu, Wayne
Brouillette
PII:
S0968-0896(17)32218-6
https://doi.org/10.1016/j.bmc.2018.04.003
BMC 14286
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 November 2017
25 March 2018
1 April 2018
Please cite this article as: Dutta, S., Lopez Charcas, O., Tanner, S., Gradek, F., Driffort, V., Roger, S., Selander, K.,
Velu, S.E., Brouillette, W., Discovery and Evaluation of nNav1.5 Sodium Channel Blockers with Potent Cell
Invasion Inhibitory Activity in Breast Cancer Cells, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/
10.1016/j.bmc.2018.04.003
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Discovery and Evaluation of nNav1.5 Sodium Channel Blockers with Potent Cell Invasion
Inhibitory Activity in Breast Cancer Cells
Shilpa Dutta1,2, Osbaldo Lopez Charcas3, Samuel Tanner1,2, Frédéric Gradek3, Virginie Driffort3,
Sébastien Roger3,4, Katri Selander5, Sadanandan E. Velu1,2* and Wayne Brouillette1,2*
1Department of Chemistry, University of Alabama at Birmingham, 901 14th Street South,
Birmingham, AL 35294-1240, USA
2Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, 1025
18th Street South, Birmingham, AL 35294-1240, USA
3Inserm UMR1069, Nutrition, Croissance et Cancer, Université de Tours, Faculté de médecine,
10 Boulevard Tonnellé, 37032 Tours, Cedex, France
4Institut Universitaire de France, 1, rue Descartes, 75231 Paris Cedex 05, France
5Division of Hematology and Oncology, Department of Medicine, University of Alabama at
Birmingham, 1808 7th Ave S, Birmingham, AL 35294-0012, USA
*Co-corresponding authors: Phone: (205) 934 4747 (Brouillette); Fax: (205) 934-2543; Email:
wbrou@uab.edu; Phone: (205) 975 2478 (Velu); Fax: (205) 934-2543; Email: svelu@uab.edu

Abstract:
Voltage-gated sodium channels (VGSC) are a well-established drug target for anti-
epileptic, anti-arrhythmic and pain medications due to their presence and important roles that
they play in excitable cells. Recently, their presence has been recognized in non-excitable cells
such as cancer cells and their overexpression has been shown to be associated with metastatic
behavior in a variety of human cancers. The neonatal isoform of the VGSC subtype, Nav1.5
(nNav1.5) is overexpressed in the highly aggressive human breast cancer cell line, MDA-MB-
231. The activity of nNav1.5 is known to promote the breast cancer cell invasion in vitro and
metastasis in vivo, and its expression in primary mammary tumors has been associated with
metastasis and patient death. Metastasis development is responsible for the high mortality of
breast cancer and currently there is no treatment available to specifically prevent or inhibit breast
cancer metastasis. In the present study, a 3D-QSAR model is used to assist the development of
low micromolar small molecule VGSC blockers. Using this model we have designed,
synthesized and evaluated five small molecule compounds as blockers of nNav1.5-dependent
inward currents in whole-cell patch-clamp experiments in MDA-MB-231 cells. The most active
compound identified from these studies blocked sodium currents by 34.9 ± 6.6 % at 1 μM. This
compound also inhibited the invasion of MDA-MB-231 cells by 30.3 ± 4.5 % at 1 μM
concentration without affecting the cell viability. The potent small molecule compounds
presented here have the potential to be developed as drugs for breast cancer metastasis treatment.

Key Words: Breast cancer, nNav1.5, voltage-gated sodium channel, sodium current, cancer cell
invasion, metastasis.
List of abbreviations:
CDCl3
Deuterated chloroform
CH2Cl2
13C-NMR
CoMFA
Methylene dichloride
Carbon-13 nuclear magnetic resonance
Comparative molecular field analysis
CuBr.Me2S Copper bromide dimethyl sulfide
DMEM
DMSO
3D-QSAR
Et3N
Dulbecco's modified eagle medium
Dimethyl sulfoxide
3-Dimensional quantitative structure activity relationship
Triethylamine
EtOAc
ER
Ethyl acetate
Estrogen receptor
FBS
fetal bovine serum
HER-2
1H-NMR
IC50
Human epidermal growth receptor 2
Hydrogen-1 nuclear magnetic resonance
Inhibition concentration at 50%
INa
Sodium currents
MeOH
MTS
Methanol
[3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium]

NaBH(OAc)3 Sodium triacetoxyborohydride
nNav1.5
Na2SO4
NH3
Neonatal Nav1.5 isoform
Sodium sulfate
Ammonia
NHE1
NMR
PBS
Na+/H+ exchanger type 1
Nuclear magnetic resonance
Phosphate buffered saline
Intracellular pH
pHi
ppm
Parts per million
PR
Progesterone receptor
Physiological saline solution
Retention factor
PSS
Rf
THF
TMS
TLC
TTX
UV
Tetrahydrofuran
Tetramethylsilane
Thin layer chromatography
Tetrodotoxin
Ultraviolet
VGSC
Voltage-gated sodium channels

1.
Introduction
Despite the increased awareness and advances in treatment, breast cancer remains as the
leading cause of cancer death in women.1-2 Though the survival rate for patients diagnosed with
localized breast cancer has improved significantly, it is the metastatic form of the disease that is
mainly associated with mortality.3-4 Metastasis development has a complex mechanism which
involves the acquisition of the ability by cancer cells to degrade and migrate through
extracellular matrices (ECM) to distant tissues.5-7 The biggest hurdle in controlling breast cancer
associated mortality is the lack of proper treatment for metastasis targeting and prevention.8-9
Thus, there is an urgent need to identify new druggable therapeutic targets for the treatment and
prevention of breast cancer metastasis. One such promising anti-metastasis drug target is the
voltage-gated sodium channels (VGSC).10-14 VGSCs have long been identified and characterized
in excitable cells in which they are responsible for the initiation and propagation of action
potentials.15-16 In recent years, VGSC expression has been found to be profoundly and aberrantly
enhanced in some non-excitable cells, including a variety of aggressive human cancers of
epithelial origin such as lung, prostate, ovarian, colon and breast cancer, and this overexpression
is associated with cancer cell invasiveness.11, 17-22 Voltage-gated sodium channels are abnormally
expressed in tumors, often as neonatal isoforms.23 These channels are not expressed in the non-
aggressive cancer cells or normal tissue, providing selectivity in targeting metastatic cancer cells.
Their expression levels and activities are related to the aggressiveness of the metastatic form of
the cancer giving us the prospect of developing drugs that could specifically inhibit the neonatal
VGSC leaving the adult isoform fully functional in the excitable cells.15
Nine different VGSC isoforms have been identified, and are classified as Nav1.1-
Nav1.9.24-25 The major VGSC isoform expressed in aggressive tumor biopsies and metastatic

breast cancer cells, such as triple-negative [estrogen receptors (ER-), progesterone receptor (PR-
), human epidermal growth receptor 2 (HER-2-)] MDA-MB-231 cells,22 is nNav1.5, a neonatal
splice variant showing a substitution of seven amino acids in the first domain of the channel
compared to the adult isoform.26 In a normal scenario, VGSCs open and inactivate within a few
milliseconds under membrane depolarization but in cancer cells nNav1.5 remains in a partially
activated and non-inactivated state at the resting membrane potential of cells (around -40mV,
because of a window of voltage between -60 and -20 mV), resulting in a continuous inward flow
of Na+ ions (called window current).22 Literature reports strongly support the involvement of
VGSC in breast cancer cell invasiveness; where overexpression of nNav1.5 (SCN5A gene) and
low expression of auxiliary β4-subunit (SCN4B gene) promote the development of metastasis.10,
27-29 Metastasis development depends on numerous abilities acquired by highly aggressive cancer
cells, one of which is their ability to degrade ECM mediated by various extracellular proteases
such as matrix metalloproteinases and cysteine cathepsins.30-32 Investigations on the mechanistic
role of nNav1.5 in metastasis revealed that nNav1.5 along with the Na+/H+ exchanger type 1
(NHE1) and caveolin-1 are all colocalized in the invasive structures of MDA-MB-231 cells,
called invadopodia.28 NHE1, often overexpressed and over-activated in breast cancer cells, is the
central regulator of intracellular and perimembrane pH augmenting the ECM proteolysis.33
nNav1.5 and NHE1 are functionally coupled to enhance H+ efflux, acidifying the extracellular
pH.28 The extracellular matrix proteolytic activity in MDA-MB-231 cells is induced by acidic
cysteine cathepsins B and S, which are released as soluble proforms in the extracellular
microenvironment and are activated in an acidic pH.27, 31 Thus, nNav1.5 displays a persistent
activity at the membrane potential of breast cancer cells, which is responsible for the increased
ECM proteolysis and cancer cell invasiveness. Since the functional activity, and not simply the

presence, of nNav1.5 channels is required to promote breast cancer cell invasiveness, nNav1.5
constitute a promising target for the breast cancer metastasis drug discovery.34 It is important to
note that numerous clinically used drugs target various subtypes of VGSCs in excitable tissues. 35
These drugs have acceptable toxicity as find use as local anesthetics, antiarrhythmics,
anticonvulsants, and for treating neuropathic pain.36 Similar clinical utility can also be
anticipated for nNav1.5 blockers for the treatment of breast cancer, particularly since nNav1.5 is
a neonatal variant not normally found in normal adult tissues.
Designing ligands for VGSCs has been, and continues to be, difficult since detailed
structural information of the drug binding site for this integral membrane protein remain largely
unclear. In recent years, we have developed a highly predictive, comprehensive 3D-QSAR
model for the design of VGSC ligands using Comparative Molecular Field Analysis (CoMFA).37
This 3D-QSAR model samples the differences in steric and electrostatic fields surrounding each
ligand in the training set, in 3D space, and correlates these changes with its biological activity.38
The training set for this model utilized several classes of drugs targeting sodium channels,
including local anesthetics, anticonvulsants and antiarrhythmics.37 A total of 67 compounds were
used to train this comprehensive 3D-QSAR model, which well covered 3D space and spanned
over 4 orders of magnitude in biological activity. This model was used to design a large number
of new small organic molecules, not yet synthesized or evaluated, with predicted VGSC
activities near 100 nM. Several of these were then synthesized and evaluated for VGSC binding
activity against a mixed population of VGSCs. Potency predictions by this model have been
highly accurate for all compounds that were evaluated. Here we synthesized five compounds
(Fig. 1) predicted to have low nanomolar VGSC binding and evaluated these for the inhibition of
nNav1.5 currents and inhibition of invasion using MDA-MB-231 cells.

Figure 1: Compounds evaluated for the inhibition of nNav1.5 currents in breast cancer MDA-
MB-231 cells.
2.
2.1. Chemistry:
Two straightforward approaches were employed for the synthesis of the target
Results and Discussion:
compounds 1-5. Compounds 1-3 were synthesized by N-alkylation of the suitable amines by
treatment with 4,4-di-(4-fluorophenyl)butyl chloride (6) in the presence of Et3N in DMSO at
80oC for 7-8 h as outlined in Scheme 1. Compound 1 was prepared by alkylation of 3-(N-
piperidino)propylamine with compound 6 in DMSO in 60% yield. Alkylation of 2-
phenethylamine with compound 6 yielded compound 2 in 58% yield and a similar alkylation of
3-phenylpropylamine with compound 6 afforded the target compound 3 in 60% yield.
Scheme 1: Synthesis of compounds 1, 2 and 3.

Compounds 4 and 5 were synthesized via a reductive amination procedure as outlined in
Schemes 2 and 3, respectively. The synthesis of compound 4 began with the reduction of 4-(4-
fluorophenyl)-4-oxobutanoic acid (7) using borane / tert-butylamine complex and AlCl3 in
anhydrous CH2Cl2 resulting in the formation of 4-(4-fluorophenyl)butan-1-ol (8) in 50% yield.
Compound 8 was oxidized with PCC in CH2Cl2 to furnish 4-(4-fluorophenyl)butan-1-al (9) in
52% yield.39 The aldehyde (9) was then subjected to reductive amination using 3-(N-
piperdino)propylamine in the presence of NaBH(OAc)3 in anhydrous CH2Cl2 resulting in the
formation of compound 4 in 30% yield.
Scheme 2: Synthesis of compound 4
The synthesis of compound 5 started with the preparation of the Grignard reagent 11 by
refluxing a solution of neopentyl bromide (10) in anhydrous THF with Mg metal in the presence
of iodine. The resulting neopentyl magnesium bromide was reacted with acrolein via Michael
addition using CuBr-Me2S, TMSCl, and HMPA in anhydrous THF at -100oC to provide (5,5-
dimethylhex-1-enyloxy)trimethylsilane (12) with 61% yield.40 The next two steps, removal of
the TMS group from compound 12 and the reductive amination were carried out as a one pot due
to the instability of the intermediate aldehyde, 5,5-dimethylhexanal (13). The TMS group was
removed by stirring the compound 12 with KF in MeOH at 0oC, and the aldehyde 13 thus
obtained was reacted with 3-(N-piperidino)propylamine in MeOH in the presence of
NaBH(OAc)3 to afford the compound 5 in 50% yield.

Scheme 3: Synthesis of compound 5
2.2. Biological evaluation:
The mechanistic studies so far on the involvement of nNav1.5 in breast cancer
progression reveal that they promote cell invasion without playing any role in cell proliferation.
Therefore, the cytotoxicity of compounds was first evaluated to avoid misinterpretation of the
effects of compounds on invasion or channel currents. Cell viability was evaluated using a
colorimetric MTS assay. All the biological evaluations were carried out using the highly
aggressive human breast cancer cell line, MDA-MB-231. Compounds were tested at a range of
concentrations from 1.0 to 25 μM, as compared to a control containing no drug. All solutions
contained 2% PBS and 0.2% DMSO. Cell viability was assessed after 24 hours of drug
treatment. Maximum non-toxic concentrations were determined for each compound and none of
the compounds affected cell viability at concentrations <5 μM (Table 1). Compound 1 displayed
a maximum non-toxic concentration of 5 μM while compound 4 displayed least cytotoxicity with
a maximum non-toxic concentration of 25 μM. Maximum non-toxic concentrations for
compounds 2, 3 and 5 were found to be about 10 μM. For compounds 1 and 4, these values are
much higher than the concentrations required (0.1 μM and 1 μM) to produce significant nNav1.5
current blockade and cell invasion inhibition (Table 1, Figs. 2A and 3A).

Table 1: Cell viability, nNav1.5 current blockade and invasion inhibition of compounds 1-5 in
MDA-MB-231 cells
Maximum
% Blockade of INa peak
currentsb
% Inhibition of cell
invasionc
Compd No nontoxic
concentrationa
1
2
3
4
5 M
34.9 ± 6.6 % at 1 M (n = 6)
30 % at 10 M (n = 2)
30 % at 10 M (n = 2)
25.0 ± 3.3 % at 1 M (n = 21) 28.0 ± 5.7 % at 1 M
30 % at 10 M (n = 2)
30.3 ± 4.5 % at 1 M
NEe
NEe
10 M
10 M
>25 M d
10 M
30 M
NEe
5
TTX 22
> 95%
31.5 ± 3.8 % at 30 µM
a. Determined by five measurements, b. Evaluated in ‘n’ cells and expressed as mean ± SEM,
c. Average of seven repeats, expressed as mean ± SEM, d. Maximum concentration tested, e.
Not evaluated.
The inhibition of sodium currents (INa inhibition) in individual MDA-MB-231 cells by
compounds at 10 μM concentration was initially measured in physiological saline solution (PSS)
using whole-cell patch-clamp experiments conducted according to a reported protocol.27 In this
study, cells were continuously perifused with PSS control solution (containing the same
percentage of DMSO, as used in the solvent for the test compounds) or the test compounds. After
5-10 min perifusion of the compounds, depolarization steps and INa-V protocols were run. At 10
μM, all five compounds showed some reduction in peak sodium currents. Compounds 1 and 4
were most active exhibiting 50 % and 60 % INa inhibition at 10 μM concentration, while
compounds 2, 3 and 5 showed relatively less INa inhibition of 30% (Table 1 and Figs. 2A and
3A). As the compounds 1 and 4 were better blockers, they were further evaluated at a range of
concentrations from 0.1 µM to 100 µM to examine the dose response relationship on INa
inhibition as well as to determine the IC50 values. Results of these studies are summarized in
Figs. 2 and 3.

Both compounds 1 and 4 demonstrated a slow, dose-dependent inhibition of the maximal
current elicited for a membrane depolarization from -100 to -5 mV, which was not reversible (for
compound 1) or weakly reversible (for compound 4) after wash-out (Figs. 2B and 3B).
Compound 1 exhibited an IC50 value of 4.8 ± 1.2 µM (n = 3-6 cells)whereas compound 4
exhibited an IC50 value of 5.9 ± 0.9 µM (n = 17-21 cells), (Figs. 2C and 3C, respectively). In
addition, the effect of the two compounds was studied on the activation and inactivation kinetics
of the sodium current (Figs. 2D and 3D). Indeed, compound 1 (at 10 µM) significantly slowed
down both activation and inactivation kinetics of INa measured for a membrane depolarization
from -100 to -5 mV (Figs. 2A and 2D), while compound 4 only reduced kinetics of activation
(Figs. 3A and 3D). The effect of Compounds 1 and 4 tested at a concentration of 10 µM on the
full INa-Voltage relationship (from -100 to +60 mV) is shown in figures 2E and 3E, respectively.
The voltage of half-inactivation (V1/2-inactivation) significantly shifted to hyperpolarized values
from –71.6 ± 0.4 mV in control condition, to –88.6 ± 3.4 mV in presence of compound 1 (n = 6
cells, p<0.01, Fig. 2F), while half-voltage of activation (V1/2-activation) was unchanged.
Compound 4 also shifted V1/2-inactivation to hyperpolarized values from –66.0 ± 2.5 mV (in
control condition) to –77.5 ± 3.0 mV (n= 15 cells, p<0.01, Fig. 3F). In addition, compound 4
also showed a light hyperpolarizing shift in V1/2-activation from –30.7 ± 0.9 mV (in control
condition) to –36.8 ± 2.8 mV (n = 15 cells, p<0.001, Fig. 3F).
These results indicate that the lead compounds 1 and 4 exhibited significant blockade of
nNav1.5 currents in MDA-MB-231 cells in low micromolar concentrations. These effects were
observed to be weakly reversible. Furthermore, these two compounds induced significant
leftward shift in the inactivation-voltage relationship suggesting that they could also reduce the
window of voltage, and therefore reduce the persistent window current associated with cancer

cell invasiveness. The observed nNav1.5 current blockade for compounds 1 and 4 were
compared to the positive control tetrodotoxin (TTX), which is a known sodium channel blocker
(>95% blockade at 30 μM).22 Compounds 1 and 4 displayed similar activity to TTX with 25 - 35
% blockade at 1 μM, 50-60% blockade at 10 μM. However, it required about 100 μM of
compounds 1 and 4 to produce a >95% blockade of channel current. The observed nNav1.5
current blockade by compounds 1 and 4 are unrelated to any effects on cell viability as their
maximum cell cytotoxicity concentrations were much higher at 5 μM and 25 μM respectively.
Based on these experimental results, compounds 1 and 4 were selected for further evaluation in
cell invasion assays.
0 .1
1 0 0
A
C
0
B
1
1 .0
0 .8
0 .6
0 .4
0 .2
0 .0
8 0
6 0
4 0
2 0
0
1 0 0
1 0
1 0 0
5 0 p A
1 0
5
m s
1
0 .1
C o n tro l
-1 0 0
-5 m V
1 0 -8
1 0 -7
1 0 -6
1 0 -5
1 0 -4
1 0 -3
0
4
8
1 2
1 6
[C o m p o u n d 1 ] (M )
Tim e (m in)
D
E
F
4
2
1 .0
0 .8
0 .6
0 .4
0 .2
0 .0
1 .0
0 .8
0 .6
0 .4
0 .2
0 .0
*
2 .0
1 .8
1 .6
1 .4
1 .2
1 .0
0 .8
0 .6
0 .4
0 .2
0 .0
1 .7 5
1 .5 0
1 .2 5
1 .0 0
0 .7 5
0 .5 0
0 .2 5
0 .0 0
*
V m (m V )
-1 0 0 -8 0 -6 0 -4 0 -2 0
C ontrol
0
2 0
4 0
6 0
0
-2
-4
V m (m
V )
0 .2
0 .0
-1 0 0 -8 0 -6
0
-4 0 -2 0
0
2 0 4 0
-6
C o n tro l
C o m p o u n d
C o m p o u n d
1
-0 .2
-0 .4
-0 .6
-0 .8
-1 .0
1
-8
-1 0
-1 2
-1 2 0 -1 0 0
-8 0
-6 0
-4 0
-2 0
0
V m (m V )
Figure 2: Effects of compound 1 on voltage-gated sodium currents from MDA-MB-231
cells. A) representative recordings showing steady-state block of NaV1.5 currents by increasing
concentrations (from 0.1 to 100 µM) of compound 1. Whole-cell patch-clamp recordings were
obtained in MDA-MB-231 cells and sodium currents were evoked by 30 ms duration voltage
steps to ─5 mV from a holding potential of ─100 mV, applied every 2 s. The dotted line
represents the zero current level. B) Time course of NaV1.5 channels block by compound 1.
Peak currents generated for a depolarization step from -100 to -5 mV were normalized to control
amplitude (before drug exposure), and defined as the fraction of INa remaining. C)
Concentration-response relationships for the effect of compound 1 on NaV1.5 channels. Percent
blocked current was calculated from peak current measurements from step voltages from -100 to

─5 mV in the presence of compound 1 concentrations indicated in the figure. Data from these
experiments were fitted using a Hill equation (IC50 = 4.8 ± 1.2 µM; Hill’s slope = 0.56 ± 0.09).
D) Compound 1 slows down the activation and inactivation kinetics of NaV1.5 currents.
Currents like those illustrated in panel A were fitted with two exponentials functions, one for the
activation and the other for the inactivation of the current at ─5 mV, and the respective time
constants (τ) were plotted. Columns, means from six cells before (control) and after a steady-
state block (compound 1, at 10 µM). *, statistical significance with a Student’s t test (P < 0.05)
represent mean values. E) I-V curves in absence and presence of 10 µM of compound 1. The
inset shows the normalized current-voltage relationship obtained from same cells, where values
were normalized to the maximal current recorded in each condition. F) Activation- of peak Na+
conductance (filled circles) and steady-state inactivation-voltage relationships (filled squares) of
NaV1.5 channels from MDA-MB-231 cells in absence and presence of 10 µM compound 1.
Smooth lines are fits to Boltzmann functions and V1/2 values were calculated for each condition.
While the V1/2-activation voltage were not significantly different, –33.0 ± 1.2 mV in control
condition and to – 34.9 ± 2.8 mV in presence of compound 1, the V1/2-inactivation voltage was
significantly shifted to hyperpolarized values from – 71.6 ± 0.4 mV in control condition, to –
88.6 ± 3.4 mV in presence of compound 1 (p<0.01, Mann-Whitney Rank Sum test). All data
were obtained from 6 cells.
0 . 1
1 0 0
A
C
1
0
B
1 0
1 .0
0 .8
0 .6
0 .4
0 .2
0 .0
1 0 0
8 0
6 0
4 0
2 0
0
w a s h o u t
1 0 0 p A
1 0
1 0 0
5
m s
1
W
a s h o u t
0 .1
C o n tro l
-5 m V
1 0 -8
1 0 -7
1 0 -6
1 0 - 5
1 0 - 4
1 0 -3
-1 0 0
0
4
8
1 2
T im e (m in)
1 6
2 0
2 4
[C o m p o u n d 4 ] (M )
D
E
F
4
2
1 .0
0 .8
0 .6
0 .4
0 .2
0 .0
1 .0
0 .8
0 .6
0 .4
0 .2
0 .0
1 .6
1 .4
1 .2
1 .0
0 .8
0 .6
0 .4
0 .2
0 .0
3 .0
2 .5
2 .0
1 .5
1 .0
0 .5
0 .0
*
V m (m V )
-1 0 0 -8 0 -6 0 -4 0 -2 0
C o n trol
0
2 0 4 0
6 0
0
-2
-4
-6
-8
0
0
.2
.0
V m (m
V )
C o m p o u n d
4
- 1 0 0 - 8 0 - 6 0 - 4 0 - 2 0
0
2
0
4 0
-0 .2
-0 .4
-0 .6
-0 .8
-1 .0
C o n tr o l
C o m p o u n d
-1 0
4
-1 2
-1 4
-1 2 0 -1 0 0
-8 0
-6 0
-4 0
-2 0
0
V m (m V )
Figure 3: Effects of compound 4 on voltage-gated sodium currents from MDA-MB-231
cells. A) Representative recordings showing steady-state block of NaV1.5 currents by increasing
concentrations (from 0.1 to 100 µM) of compound 4. Whole-cell patch-clamp recordings were
obtained in MDA-MB-231 cells and sodium currents were evoked by 30 ms duration voltage
steps to ─5 mV from a holding potential of ─100 mV, applied every 2 s. The dotted line
represents the zero current level. B) Time course of NaV1.5 channels block by compound 4.

Peak currents generated for a depolarization step from -100 to -5 mV were normalized to control
amplitude (before drug exposure), and defined as the fraction of INa remaining, only a slight
fraction of the initial current amplitude was recovered after drug washing (17.4 ± 3.1 %). C)
concentration-response relationships for the effect of compound 4 on NaV1.5 channels. Percent
blocked current was calculated from peak current measurements from step voltages to ─5 mV in
the presence of increasing concentrations of compound 4 (n = 22). Data from these experiments
were fitted using a Hill equation (IC50 = 5.9 ± 0.9 µM; Hill’s slope = 0.72 ± 0.06). D)
Compound 4 slows down the activation but not the inactivation kinetics of NaV1.5 currents.
Currents like those illustrated in panel A were fitted with two exponentials functions, and the
respective time constants (τ) were plotted. Columns, means from 17 cells before (control) and
after a steady-state block (compound 4, at 10 µM). *, statistical significance with a Student’s t
test (P < 0.05). E) I-V curves in absence and presence of 10 µM compound 4. The inset shows
the normalized current-voltage relationship obtained from same cells, where values were
normalized to the maximal current recorded in each condition. F) Activation of peak Na+
conductance (filled circles) and steady-state inactivation (filled squares) of NaV1.5 channels from
MDA-MB-231 cells in absence and presence of 10µM compound 4. Smooth lines are fits to
Boltzmann functions and V1/2 values were calculated for each condition. V1/2-activation voltage
values were significantly different and compound 4 induced a light hyperpolarizing shift from –
30.7± 0.9 mV in control condition to – 36.8 ± 2.8 mV in presence of compound 4 (p<0.001,
Mann-Whitney Rank Sum test). The V1/2-inactivation voltage was also significantly shifted to
hyperpolarized values from – 66.0 ± 2.5 mV in control condition, to – 77.5 ± 3.0 mV in presence
of compound 4 (p<0.01, Mann-Whitney Rank Sum test). These data were obtained from 15 cells.
Compounds 1 and 4 were then evaluated for their ability to inhibit invasion of MDA-
MB-231 cells using Matrigel-coated inserts in a trans-well invasion assay.41 Matrigel (Becton
Dickinson matrix) mimics the extracellular matrix and basement membrane that metastatic cells
must cross to enter the blood stream and travel to lymph nodes or invade distant organs. The
ability of cancer cells to invade is measured based on the number of cells that can invade the
matrigel and migrate through the pores across the membrane. The triple negative MDA-MB-231
cell line is one of the most invasive/ metastatic cell lines available to study breast carcinoma.
Compounds 1 and 4 were evaluated in the invasion assay at 0.1 μM and 1 μM along with the
control compound TTX at 30 μM. The results of the invasion assay are presented in Fig. 4. As

shown, both 1 and 4 exhibited 20% inhibition of invasion at 0.1 μM in comparison to the control,
whereas, at 1 µM, both compound 1 and 4 showed a significant reduction of invasion (30%),
similar level to what is observed for TTX at 30 µM (Fig. 4 and Table1). These results suggest
that our lead compounds 1 and 4 are very effective in inhibiting cell invasion producing an effect
similar to TTX at a much lower concentration. Furthermore, the lead compounds 1 and 4 are
more drug-like and have simpler chemical structures than TTX. In recent studies, VGSC-
blocking antiepileptic drug phenytoin and antiarrhythmic drug ranolazine inhibited the invasion
of metastatic MDA-MB-231 cells in vitro and reduced breast cancer metastasis in vivo without
much neuro-muscular or cardiac toxicity demonstrating good therapeutic index.42-44 Both these
drugs (with Na+ current blockade and invasion inhibition at 50 M) are considerably less active
than our lead compounds (Na+ current blockade and invasion inhibition at 1M) in vitro.
Therefore, we expect our lead compounds to be more potent in vivo and exhibit better therapeutic
index than phenytoin and ranolazine.
Figure 4: Effects of compounds 1 and 4 on MDA-MB-231 cancer cells invasive properties.
Effect of compounds 1 and 4 tested at 0.1 and 1 µM concentrations on MDA-MB-231 human
breast cancer cell invasiveness, as compared to the effect of the sodium channel inhibitor
tetrodotoxin (TTX, 30 µM). Results were obtained from 7 independent experiments, and are
expressed as relative invasion (mean ± SD), normalized to the control condition (CTL) obtained

without drug but with the maximal solvent dose (DMSO 0.2%). *, indicates a statistical
difference at p<0.05 (Mann-Whitney Rank Sum test).
3.
Conclusions:
The presence of nNav1.5 in aggressive breast cancer cells and their association with
invasive behavior is well established. Here, we report the discovery of small, potent drug-like
blockers of nNav1.5 channel. A 3D-QSAR model is used to assist the discovery of low
micromolar small molecule nNav1.5 blockers. Using this model we have designed, synthesized
and evaluated five small molecule compounds as blockers of nNav1.5-dependent inward
currents in whole-cell patch-clamp experiments in MDA-MB-231 cells. The identified lead
compounds 1 and 4 inhibited peak sodium currents in MDA-MB-231 cells by 35 % and 25 %
respectively at 1 μM. These two compounds also produced 30 % reduction of the invasion of
MDA-MB-231 cells at 1 μM without affecting the cell viability. The lead compounds are very
effective in inhibiting cell invasion producing an effect similar to what is observed for TTX at a
much lower concentration. Our lead compounds are much more drug-like and have simpler
chemical structures compared to TTX. In addition, our lead compounds are more active in
inhibiting breast cancer cell invasion than the known VGSC-blocking drugs such as ranolazine
and phenytoin. Compounds 1 and 4, unlike ranolazine and phenytoin, are developed to
specifically target the nNav1.5 and represent new and more potent class of compounds that may
be further developed in to drugs for the treatment of breast cancer metastasis.
4.
Experimental Procedures
4.1. Cell viability assay: Cytotoxicity of the compounds was tested in a MTS assay. Cells
were seeded in 96 well plates at 104 cells/well in growth media (5 % FBS in DMEM) and

o
incubated for 24 h at 37 C in 5 % CO2. Various concentrations of test compounds (1, 5, 10 and
25 μM) or vehicle control (0.25 % DMSO in PBS) were added and the cells were incubated for
an additional 24 h. For each test concentration, 5 wells were allotted. After 24 h of incubation
proliferation was determined using MTS. 20 μL of MTS were added to each well and the cells
were incubated for an additional 30 min. UV absorbance was recorded at 490 nm. The
cytotoxicity of the test compounds was compared to the vehicle control (0.25 % DMSO in PBS)
and results were compiled as the mean of five repeats using the GraphPad Prism software.
4.2.
Cell invasion inhibition assay: Cell invasion experiments were carried out in 24 well
plates containing BD Matrigel® invasion chambers (BD Biosciences). The invasion chambers
o
were hydrated for 2 h in the incubator at 37 C with 200 μL (5 % FBS in DMEM) in the upper
insert and 800 μL (10 % FBS in DMEM) in the lower well that acted as a chemoattractant.
MDA-MB-231 cells (4 × 104) were seeded in the upper chamber. Various concentrations of the
test compounds (0.1 and 1 μM) or vehicle control (0.2% DMSO in PBS) were added in both the
lower and upper chambers to maintain uniformity of concentration by 1:100 dilutions. After
incubating the cells for 24 h at 37 oC, under 5 % CO2, invaded cells were fixed and stained using
a Hema kit. The migrated cells were counted under the microscope by selecting five regions on
the whole membrane. Results were compiled as a mean of three repeats using graphPad Prism
software. The results are given as a mean ± SD.
4.3. Electrophysiology: A whole cell patch clamp technique was used for recording the
sodium channel activity in MDA-MB-231 cells. 30,000 cells were seeded into a 35 mm petri
dish with the growth media (5% FBS in DMEM). Na+ currents were recorded under voltage-

clamp mode using an Axopatch 200B patch clamp amplifier (Axon Instruments, USA),
compensating for cell capacitance and series resistance by 60 %. The P/2 sub pulse protocol was
applied to correct the linear component of capacitance and cell leak. Borosilicate glass was used
to pull the patch pipettes at a resistance of 3-5 MΩ. Analog signals were filtered at 5 kHz and
sampled at 10 kHz via a 1440A Digidata converter and analyzed using pCLAMP software
(v10.4, Axon Instruments). Cells were studied in ruptured, whole-cell, voltage-clamp mode of
the patch-clamp technique. Before starting the experiment growth medium was removed and
replaced with physiological saline solution (PSS, composition given below). In this experiment
the tip of the micro capillary (perifusion system) delivering the PSS or the test compound was
placed almost on top of the cell, so that the cells were continuously perifused with the respective
solutions. The stock solution for test compounds was prepared at 10 mM in 100% DMSO and
the test concentration used in the assay was 1 μM and 10 μM (0.2 % DSMO in PSS). Sodium
currents were recorded by depolarizing the cells from a holding potential of -100 mV to a
maximal test pulse of -5 mV for 30 ms every 500 ms. The protocol used to build the current-
voltage (INa-V) curve was as follows. From a holding potential of -100 mV, the membrane was
stepped to a potential of +60 mV, with 5 mV increments, for 50 ms at a frequency of 2 Hz. The
physiological saline solution (PSS) contained (in mM): NaCl 140, KCl 4, MgCl2 1, CaCl2 2,
NaH2PO4 0.33, D-Glucose 11.1, and HEPES 10, and was adjusted to pH 7.4. The intrapipette
solution (in mM) contained: KCl 130, NaCl 15, CaCl2 0.37, MgCl2 1, Mg-ATP 1, EGTA 1, and
HEPES 10, and was adjusted to pH 7.2.
4.4. General Methods for Synthesis: Solvent evaporations were carried out in vacuo using a
rotary evaporator. Thin layer chromatography (TLC) was performed on silica gel plates with

fluorescent indicator (Dynamic Adsorbents, Inc., Aluminum backed TLC, 20 X 20 cm F-254,
200 µm). Spots were visualized by UV light (254 and 365 nm) or staining agents such as
ninhydrin, KMnO4 and iodine. Purification by column and flash chromatography was carried out
using silica gel (32-63 µm) from Dynamic Absorbent in the solvent systems indicated. The
amount (weight) of silica gel for column chromatography was in the range of 50-100 times the
amount (weight) of the crude compounds being separated. The NMR spectra were recorded on a
Bruker DPX 300 spectrometer. Chemical shifts are reported in ppm relative to TMS or CDCl3 as
internal standard. The values of chemical shifts (δ) and coupling constants J were given in parts
per million and in Hz, respectively. Mass spectra were recorded on a MicroMass Platform LCC
instrument. HRMS were obtained on a Waters AutoSpec-UltimaTM NT mass spectrometer with
an EI source. Anhydrous solvents used for reactions were purchased in Sure-SealTM bottles from
Aldrich chemical company. THF was freshly distilled over sodium/benzophenone. Other
reagents were purchased from Aldrich, Alfa Aesar or Acros and used as received.
4.5. Synthesis of compounds 1-3, General procedure: To a solution of alkyl amine (1
equiv) in DMSO (10mL), a solution of alkyl chloride (1 equiv.) in DMSO (5 mL) and Et3N (1
equiv.) were added. The reaction mixture was heated at 80-90oC under a N2 atmosphere for 8 h.
TLC examination (MeOH / NH3 saturated CH2Cl2, 1:19) indicated the completion of the
reaction. The reaction mixture was diluted with EtOAc (60mL), washed with 1N NaOH (3 × 20
mL), water (2 × 20 mL), brine (20 mL) and dried over Na2SO4. The drying agent was filtered off
and the filtrate was concentrated under vacuum to obtain the crude product which was purified
by column chromatography over Si gel using MeOH/ NH3 saturated CH2Cl2 (1:49) to obtain pure
compounds 1-3 in 58 – 60 % yield.

4,4-Bis(4-fluorophenyl)-N-(3-(piperdin-1-yl)propyl)butan-1-amine (1): Prepared from 3-(N-
piperidino)propylamine (2.50 g, 17.6 mmol) and 4,4-di-(4-fluorophenyl)butyl chloride 6 (4.93 g,
17.6 mmol) to afford compound 1 (4.00 g, 60%) as a liquid (MeOH/NH3 saturated CH2Cl2, 1:19,
1
Rf = 0.40). H-NMR (CDCl3) δ 1.34 – 1.46 (m, 4H), 1.52 (quint, 4H, J = 5.3 Hz), 1.65 (quint,
2H, J = 7.5 Hz), 2.01 (q, 2H, J = 8 Hz), 2.20 – 2.42 (m, 8H), 2.60 (t, 4H, J = 7.5 Hz), 3.85 (t, 1H,
13
J = 7.5 Hz), 6.89 – 6.99 (m, 4H), 7.10 – 7.19 (m, 4H); C-NMR (CDCl3) δ 24.4, 26.0, 27.0,
28.5, 33.7, 49.0, 49.7, 49.8, 54.7, 57.9, 115.1 (115.4, F- coupling), 129.0 (129.1, F- coupling),
140.5 (140.6, F- coupling), 159.7 (162.9, F- coupling); MS (ES+) m/z 387 (M+H); HRMS
measured for C24H32F2N2: [M+H]+ 386.2535 (found), 386.2534 (calc).
4,4-Bis(4-fluorophenyl)-N-(3-phenylpropyl)butan-1-amine
(2):
Prepared
from
3-
phenylpropylamine (0.240 g, 1.78 mmol), and 4,4-di-(4-fluorophenyl)butyl chloride, 6 (0.500 g,
1.78 mmol) to afford compound 2 (0.230 g, 60.6%) as a thick liquid (MeOH/NH3 saturated
1
CH2Cl2, 1:19, Rf = 0.50). H-NMR (CDCl3) δ 1.47 (quint, 3H, J = 7.5 Hz), 1.84 (t, 2H, J = 7.5
Hz), 1.98 (q, 2H, J = 7.8 Hz), 2.63 (quint, 6H, J = 7.1 Hz), 3.82 (t, 1H, J = 7.8 Hz), 3.92 (bs,
13
1H), 6.90 – 7.10 (m, 4H), 7.08 – 7.21 (m, 7H), 7.22 – 7.30 (m, 2H); C-NMR (CDCl3) δ 27.6,
30.7, 33.5 (2C), 49.0, 49.3, 49.7, 115.3, 115.6, 126.1, 128.5, 128.6, 129.2, 129.3, 140.43, 140.5,
141.6, 159.9, 163.1; MS (ES+) m/z 380 (M+H); HRMS measured for C25H27F2N: [M+H]+
379.2116 (found), 379.2112 (calc).
4,4-Bis(4-fluorophenyl)-N-phenylethylbutan-1-amine (3): Prepared from 2-phenylethylamine
(0.996 g, 8.22 mmol) and 4,4-di-(4-fluorophenyl)butyl chloride 1 (2.31 g, 8.22 mmol) to afford
compound 3 (1.74 g, 58.0%) as a liquid (40% ethyl acetate / 55% hexanes / 5% Et3N, Rf = 0.35).
1H-NMR (CDCl3) δ 1.37 -1 .44 (m, 2H), 1.63 (bs, 1H), 1.97 (t, 2H, J = 7.4 Hz), 2.59 – 2.65 (m,
2H), 2.78 (t, 2H, J = 7.0 Hz), 2.83 (q, 2H, J = 7.7 Hz), 3.82 (q, 1H, J = 8.2 Hz), 6.90 – 6.97 (m,

4H), 7.09 – 7.21 (m, 7H), 7.22 – 7.29 (m, 2H); 13C-NMR (CDCl3) δ 28.5, 33.8, 36.4, 49.8, 49.9,
51.2, 115.4 (115.5, F-coupling), 126.4, 128.7, 128.9, 129.2, 129.3, 140.1 (140.7, F-coupling),
160.8 (162.2, F-coupling); MS (ES+) m/z 366 (M+H); HRMS measured for C24H25F2N: [M+H]+
365.1954 (found), 365.1955 (calc).
4-(4-Fluorophenyl) butan-1-ol (8): A suspension of AlCl3 (4.00 g, 30.0 mmol) in anhydrous
CH2Cl2 (100 mL) was stirred under a N2 atmosphere at 0 oC for 10 min followed by the addition
of borane tert-butylamine complex (5.20 g, 60.0 mmol). The reaction mixture was allowed to stir
at 0 oC till it gave a clear solution (10-15 mins). To the clear reaction mixture a solution of 4-(4-
fluorophenyl)-4-oxobutanoic acid 7 (2.00 g, 10 mmol) in anhydrous CH2Cl2 (10 mL) was added.
The resulting mixture was stirred at 0oC for 2h, and TLC examination (EtOAc/hexanes, 1:4, Rf =
0.30) indicated completion of the reaction. The reaction mixture was quenched by the addition of
0.1 N HCl (50 mL). It was extracted with EtOAc (3 × 50 mL); the combined organic layer was
washed with 0.1 N HCl (2 × 50 mL), brine (50 mL), and dried over Na2SO4. The drying agent
was filtered off and the filtrate was concentrated under vacuum to obtain the crude product. The
crude product was purified by column chromatography over Si gel using EtOAc / hexanes (1:4)
1
to afford 4-(4-fluorophenyl)butan-1-ol 4 (0.880 g, 52.3% yield) as a colorless liquid. H-NMR
(CDCl3) δ 1.56 – 1.73 (m, 4H), 2.14 (bs, 1H), 2.61 (t, 2H, J = 7.4 Hz), 3.65 (t, 2H, J = 6.3 Hz),
6.91- 7.00 (m, 2H), 7.08 – 7.16 (m, 2H); 13C-NMR (CDCl3) δ 27.9, 32.3, 35.0, 62.9, 115.0
(115.3), 129.8 (129.9), 138.0 (138.1), 159.8 (163.0).
4-(4-Fluorophenyl)butan-1-al (9): PCC (7.13 g, 33.1 mmol) was ground with silica gel (7.13 g)
and suspended in anhydrous CH2Cl2 (100 mL) under a N2 atmosphere at room temperature. After
stirring for 10-15 mins, a solution of 4-(4-fluorophenyl)butan-1-ol (8) (0.857 g, 5.09 mmol) in
anhydrous CH2Cl2 (10 mL) was added to the suspension. After 6 h of stirring at room

temperature, TLC examination (EtOAc/hexanes, 1:4, Rf = 0.50) indicated completion of the
reaction. The reaction mixture was filtered through celite and washed with CH2Cl2 (200 mL).
The filtrate was concentrated under vacuum to obtain the crude product. The crude product was
purified by column chromatography over silica gel, using EtOAc / hexanes (1:4) to afford 4-(4-
fluorophenyl)butan-1-al (9) (0.444 g, 52.5% yield) as a colorless liquid. 1H-NMR (CDCl3) δ 1.92
(pent, 2H, J = 7.7 Hz), 2.44 (td, 2H, J1 = 7.2 Hz, J2 = 1.5 Hz), 2.62 (t, 2H, J = 7.7 Hz), 6.91 –
7.01 (m, 2H), 7.08 – 7.16 (m, 2H), 9.74 (t, 1H, J = 1.5 Hz); 13C-NMR (CDCl3) δ 23.9, 34.3, 43.1,
115.1 (115.4, F-coupling), 129.9, 137.0, 159.9 (163.1, F-coupling), 202.2.
4-(4-Fluorophenyl)-N-(3-(piperdin-1-yl)propyl)butan-1-amine (4): 3-(N-Piperidino)propyl
amine (0.380 g, 2.67 mmol) was dissolved in anhydrous CH2Cl2 (25 mL) under a N2 atmosphere.
4-(4-Fluorophenyl)butan-1-al (9) (0.444 g, 2.67 mmol) was added to the reaction mixture. After
stirring the reaction mixture for 30 mins, NaBH(OAc)3 (1.13 g, 5.34 mmol) was added. After
stirring the reaction mixture for 5 h at room temperature, TLC examination (MeOH/NH3
saturated CH2Cl2, 1:19, Rf = 0.40) indicated completion of the reaction. The reaction mixture
was concentrated under vacuum and taken up into EtOAc (60 mL); the organic layer was washed
with 1 N NaOH (3 × 30 mL), water (30 mL), brine (30 mL) and dried over Na2SO4. The drying
agent was filtered off and the filtrate was concentrated under vacuum to obtain the crude product.
The crude product was purified by column chromatography over Si gel, using MeOH/NH3
saturated CH2Cl2 (3:97) to afford 4-(4-fluorophenyl)-N-(3-(piperdin-1-yl)propyl)butan-1-amine
1
(4) (0.224 g, 30.0% yield). H-NMR (CDCl3) δ 1.35 - 1.45 (m, 2H), 1.46 - 1.61 (m, 7H), 1.62 -
1.73 (m, 3H), 2.27 - 2.43 (m, 6H), 2.60 (quint, 6H, J = 7.4 Hz), 6.89 - 6.98 (m, 2H), 7.06 - 7.14
(m, 2H); 13C-NMR (CDCl3) δ 24.6, 26.2, 27.0, 29.5, 29.7, 35.1, 49.2, 49.9, 54.8, 58.1, 115.0
(115.2, F-coupling), 129.8 (129.9, F-coupling), 138.1 (138.2, F-coupling), 159.7 (163.0, F-

coupling); MS (ES+) m/z 293 (M+H); HRMS measured for C18H29FN2: [M+H]+ 292.2324
(found), 292.2315 (calc).
Neopentyl magnesium bromide (11): In an oven dried three-neck flask connected to a
condenser and dropping funnel was placed dry magnesium turnings (1.60 g, 66.0 mmol) under a
N2 atmosphere. The magnesium was further flame dried for half an hour under N2 before adding
a crystal of I2 and anhydrous THF (20 mL). A solution of neopentyl bromide 10 (10.0 g, 66.0
mmol) in THF (20 mL) was slowly added to the reaction mixture through a dropping funnel.
Disappearance of the coloration and beginning of effervescence indicated the onset of the
reaction. No external heating was applied until the complete addition of neopentyl bromide.
After refluxing for 3 h, the heating was stopped and the concentration of Grignard reagent
generated was determined to be 1.48 M by titration against a solution of isopropanol in xylene.
This Grignard reagent was used in the next step.
((5,5-Dimethylhex-1-en-1-yl)oxy)trimethylsilane (12): 1.48 M Neopentyl magnesium bromide
o
(7) (40 mL) was dissolved in anhydrous THF (150 mL) and was cooled to -100 C followed by
the addition of CuBr.Me2S (0.510 g, 2.50 mmol) and HMPA (21.1 g, 118 mmol) under a N2
atmosphere. After stirring for 10 mins, a solution of acrolein (2.76 g, 49.2 mmol) and
chlorotrimethylsilane (10.7 g, 98.4 mmol) in THF (50 mL) were added to the reaction mixture
via a cannula. After stirring the reaction mixture for 4 h at -100 oC under a N2 atmosphere, Et3N
(9.95 g, 98.4 mmol) and silica gel (30.0 g, 4 times the expected weight of the product) was
added. After stirring for 10 min at room temperature, hexanes (400 mL) was added. The slurry
was filtered off and the filtrate was concentrated under vacuum to obtain the crude product. The
o
crude product was purified by vacuum distillation (5 T, 66 C) and (5,5-dimethylhex-1-
enyloxy)trimethylsilane (12) (6.00 g, 61.0%) was obtained as colorless liquid (hexanes, Rf =

1
0.50); H-NMR (CDCl3) δ 0.16 (s, 9H), 0.86 (s, 9H), 1.14 – 1.23 (m, 2H), 1.78 – 1.88 (m, 2H),
4.96 (dt, 1H, J1 = 12Hz, J2 = 7.4 Hz), 6.18 (dt, J1 = 12 Hz, J2 = 1.2 Hz); 13C-NMR (CDCl3) δ 0.2,
22.9, 29.6, 30.4, 45.3, 113.0, 139.2.
5,5-Dimethyl-N-(3-(piperidin-1-yl)propyl)hexan-1-amine (5): Compound 12 (0.782 g, 3.97
mmol) was dissolved in anhydrous MeOH (15 mL) under a N2 atmosphere. The reaction mixture
o
was cooled to 0 C and KF (0.396 g, 6.82 mmol) was added. After stirring the reaction mixture
for 15 min, TLC examination (EtOAc/hexanes, 1:19, Rf = 0.60) indicated a complete conversion
to 5,5-dimethylhexanal (13), which was directly added via cannula to the solution of 3-(N-
piperidino)propylamine (0.566 g, 3.97 mmol) in anhydrous MeOH (70 mL) under a N2
atmosphere. After stirring the reaction mixture for 30 min, NaBH(OAc)3 (1.68 g, 7.94 mmol)
was added to the reaction mixture. After 5 h of stirring at room temperature, TLC examination
(MeOH/NH3 saturated CH2Cl2, 1:19, Rf = 0.40) indicated the completion of reaction. The
reaction mixture was concentrated under vacuum, the residue taken up in EtOAc (50 mL) and
washed with 1 N NaOH (3 × 30 mL). The organic layer was washed with water (30 mL)
followed by brine (30mL) and dried over Na2SO4. The drying agent was filtered off and the
filtrate was concentrated under vacuum to obtain the crude product. The crude product was
purified by column chromatography over silica gel using MeOH / NH3 saturated CH2Cl2 (3:97)
to afford 5,5-dimethyl-N-(3-(piperidin-1-yl)propyl)hexan-1-amine (5) (0.500 g, 49.5%) as a clear
1
light yellow liquid. H-NMR (CDCl3) δ 0.84 (s, 9H), 1.10 – 1.31 (m, 4H), 1.35 – 1.49 (m, 4H),
1.55 (quint, 4H, J = 5.4 Hz), 1.68 (quint, 2H, J = 7.2 Hz), 2.27 – 2.39 (m, 6H), 2.53 – 2.65 (m,
4H); 13C-NMR (CDCl3) δ 22.6, 24.7, 26.2, 27.3, 29.6, 30.5, 31.3, 44.4, 49.2, 50.3, 54.9, 58;
HRMS measured for C16H34N2: [M+H]+ 254.2731 (found), 254.2722 (calc).
5.
Acknowledgements

Funding from NIH (1UL1RR025777, R03DE025058-0) is acknowledged. The authors
wish to acknowledge the financial support by the Collaborative Programmatic Development
Grant from the UAB Comprehensive Cancer Center. SD would like to acknowledge the graduate
fellowship from the UAB chemistry department. SR was supported by the INSERM, the Région
Centre-Val de Loire, the “Ligue Nationale contre le Cancer”, the “Association CANCEN”, and
the “Prix Ruban Rose Avenir 2017”.
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