Synthesis and evaluation of hermitamides A and B as human voltage-gated sodium channel blockers

Article abstract of DOI:10.1016/j.bmc.2011.05.043

Hermitamides A and B are lipopeptides isolated from a Papau New Guinea collection of the marine cyanobacterium Lyngbya majuscula. We hypothesized that the hermitamides are ligands for the human voltage-gated sodium channel (hNa_V) based on their

Full text of DOI:10.1016/j.bmc.2011.05.043

Bioorganic & Medicinal Chemistry 19 (2011) 4322–4329
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of hermitamides A and B as human
voltage-gated sodium channel blockers
Eliseu O. De Oliveira a, , Kristin M. Graf b, , Manoj K. Patel c, Aparna Baheti c, Hye-Sik Kong a,
Linda H. MacArthur d, Sivanesan Dakshanamurthy a, Kan Wang a, Milton L. Brown a, Mikell Paige a,
⇑
a Drug Discovery Program, Department of Oncology, Georgetown University Medical Center, Washington, DC 20057, USA
b Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA
c Department of Anesthesiology, University of Virginia, Charlottesville, VA 22904, USA
d Department of Neuroscience, Georgetown University Medical Center, Washington, DC 20057, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Hermitamides A and B are lipopeptides isolated from a Papau New Guinea collection of the marine cya-
nobacterium Lyngbya majuscula. We hypothesized that the hermitamides are ligands for the human volt-
age-gated sodium channel (hNaV) based on their structural similarity to the jamaicamides. Herein, we
describe the nonracemic total synthesis of hermitamides A and B and their epimers. We report the ability
Received 24 March 2011
Revised 19 May 2011
Accepted 23 May 2011
Available online 30 May 2011
of the hermitamides to displace [3H]-BTX at 10
lM more potently than phenytoin, a clinically used
sodium channel blocker. We also present a potential binding mode for (S)-hermitamide B in the BTX-
binding site and electrophysiology showing that these compounds are potent blockers of the hNav1.2
voltage-gated sodium channel.
Keywords:
Sodium channel blockers
Hermitamide A
Hermitamide B
Lyngbic acid
Ó 2011 Elsevier Ltd. All rights reserved.
Lyngbya majuscula
1. Introduction
Recently, the jamaicamides A, B, and C (1–3) (Fig. 1) were iso-
lated from the dark green strain of L. majuscula collected at Hec-
The human voltage-gated sodium channels (hNav) comprise a
family of nine known subtypes of membrane-bound proteins
(hNav1.1–1.9), which play critical physiological roles in the body
and are therefore important targets in medicine.1 Their blockade
by specific agents has been the basis for the discovery of important
anticonvulsants, anesthetics, and antiarrhythmic drugs.2 Despite
the success of these agents in the clinic, the sodium channel re-
mains a challenging target for drug discovery.3 In an effort to dis-
cover novel ligands for these receptors, we have begun a program
to synthesize and evaluate the secondary metabolites of the mar-
ine cyanobacterium Lyngbya majuscula as a source for sodium
channel inhibitors.
tor’s Bay in Jamaica.6 These lipopeptides were shown to block
sodium channel activity in a cell-based screening assay developed
Several lipopeptidic secondary metabolites isolated from L.
majuscula have shown hNaV activity. For example, the cyclic
lipopetides antillatoxin and antillatoxin-B are sodium channel acti-
vators with potencies comparable to the brevetoxins.4 The mixed
polyketide–peptide kalkitoxin is a potent sodium channel blocking
agent.5
⇑
Corresponding author. Tel.: +1 202 687 5341; fax: +1 202 687 0617.
E-mail address: map65@georgetown.edu (M. Paige).
These authors have equally contributed to the paper.
Figure 1. The metabolites jamaicamides A, B, and C (1–3), and the hermitamides A
and B (4–5) isolated from L. majuscula.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.05.043

E. O. De Oliveira et al. / Bioorg. Med. Chem. 19 (2011) 4322–4329
4323
by Manger et al. which involves measuring the end-point of mito-
chondrial dehydrogenase activity in neuroblastoma cells.7
water solvent system afforded 7 in 41% yield.14 Finally, the total
syntheses of 4 and 5 were accomplished in excellent yields by
coupling 7 with phenethylamine (77%) or tryptamine (88%),
respectively, via diisopropylcarbodiimide in the presence of
1-hydroxybenzotriazole (Scheme 1).
The hermitamides A (4) and B (5), had been previously reported
as metabolites isolated from a Papua New Guinea collection of L.
majuscula.8 A careful analysis of the jamaicamide and the hermita-
mide structures reveal several similarities. For example, each class
contains a 14-carbon aliphatic chain, an E olefin between carbon-4
and carbon-5, a 2-carbon chain linker between the amide nitrogen
By following the same strategy depicted in Scheme 1, we have
also synthesized the racemates of hermitamides 4 and 5 and their
corresponding R enantiomers, (R)-4 and (R)-5. The R enantiomers
of the hermitamides were synthesized from R-8, which was de-
rived from addition of allyltributylstannane to octanal in the pres-
ence of (S)-(ꢀ)-1,10-bi-2-naphthol ((S)-BINOL). The racemic
hermitamides were synthesized from racemic 8, which was de-
rived from addition of an ethereal solution of allylmagnesium bro-
mide to octanal. The method also proved to be robust for scale up.
Compound 5 was obtained from octanal in seven steps on a four-
gram scale using N-(3-dimethylaminopropyl)-N0-ethylcarbodiim-
ide hydrochloride (EDC) in a solution of methylene chloride for
the last step. All reaction details and yields for the intermediate
compounds are reported in the experimental section of this paper.
atom and a region of unsaturation (a p-system) (see Fig. 2). On the
basis of these structural similarities, and on the analysis of two po-
tential modes of binding of the jamaicamides to the sodium chan-
nel in relation to that of kalkitoxin (6) (see Fig. 3), we hypothesized
that hermitamides 4 and 5 might also behave as channel sodium
blockers.
Herein, we report the first enantioselective total synthesis of
both enantiomers of hermitamides 4 and 5, hermitamide mediated
displacement of tritiated batrachotoxin ([3H]-BTX) from sodium
channels, and functional block of hNav1.2 sodium channel currents
in the presence of the hermitamides as measured by patch clamp
electrophysiology. A computational homology model of site 2 of
the hNaV1.2 channel and a binding model of hermitamide B (5)
to this construct is presented.
2.2. Pharmacology
2.2.1. Pharmacophore analysis
The hermitamide structures resemble the jamaicamides in that
both are lipopeptidic metabolites that contain a lipophilic side-
chain, a peptide linkage, and a carbon linker to an aromatic moiety
2. Results and discussion
2.1. Synthesis of hermitamides A and B
(the p-system) (Fig. 2). However, the carbon skeletal arrangement
of the jamaicamides is much more complicated for both the ali-
phatic chain and the aromatic moiety, which is a highly unsatu-
rated alkaloid with one stereocenter and a tri-substituted olefin.
In fact, the proposed pharmacophore seems also to be shared
with other L. majuscula metabolites, such as the sodium channel
blocker kalkitoxin. Examination of possible binding modes of jamai-
camide C in relation to kalkitoxin highlights an interesting feature.
By aligning each molecule pivoting around the amide bond, a poten-
tial second mode of binding for jamaicamide C is revealed. In this
The syntheses of hermitamides 4 and 5 have attracted the inter-
est of several organic synthesis groups. The configuration of the re-
mote stereocenter bearing the methoxy group in compounds 4 and
5 was confirmed by relay synthesis of the respective hermitamides
from (S)-lyngbic acid ((S)-7), which was isolated with the hermita-
mides.8 The preparation of the enantiopure lyngbic acid has been
accomplished by ring-opening of a chiral epoxide,9 lipase resolu-
tion,10 and asymmetric allylation of the requisite aldehyde.11
A
racemic total synthesis of hermitamides 4 and 5 and a formal syn-
thesis of the nonracemic compounds have also been reported.12
Although these methods have proved to be valuable to obtain
synthetic intermediates, to the best of our knowledge, completion
of the asymmetric total synthesis of hermitamides 4 and 5 has not
been reported. We have devised a simpler and efficient synthetic
route to hermitamides 4 and 5 based on three key synthetic strat-
egies as follows: (i) generation of the asymmetric center by Keck
allylation of octanal; (ii) sterospecific formation of the E olefin by
Johnson–Claisen rearrangement; and (iii) condensation of the
resulting lyngbic acid with the appropriate amines via carbodiim-
ide chemistry.
The synthesis of nonracemic lyngbic acid (7) depicted in
Scheme 1 commenced with the asymmetric allylation of octanal
with allyltributylstannane mediated by a titanium–binol complex
to give alcohol 8 in 40% yield and greater than 95:5 er as previously
reported.13 Treatment of 8 with sodium hydride in dimethylform-
amide followed by methylation with methyl iodide, afforded the
methyl ether 9 in 92% yield. Osmium dihydroxylation of 9 followed
by in situ oxidative cleavage of the intermediate with sodium per-
iodate under Johnson–Lemieux conditions yielded the aldehyde 10,
which was used in the next step without further purification. Addi-
tion of vinylmagnesium bromide in tetrahydrofuran to 10 afforded
the corresponding allylic alcohol 11 as a mixture of diastereomers
in 51% yield. The Johnson–Claisen rearrangement of 11 was then
effected by treatment with trimethylorthoacetate in the presence
of a catalytic amount of propionic acid. The mixture was heated
at reflux to allow the methanol generated to be distilled off after
which the methyl ester of lyngbic acid (12) is obtained in 63% yield.
Saponification of 12 with lithium hydroxide in a tetrahydrofuran-
conformation the carbon chain linking the p-system to the nitrogen
is shortened from 7 to 2 carbon atoms (Fig. 3). If this conformation is
active for the binding of jamaicamide C to the sodium channel, then
hermitamides 4 and 5 are hypothesized to be potential blocking
agents, because they share the following structural features: both
present a 14-carbon aliphatic chain bearing an E olefin between car-
bons C4 and C5, a peptide linkage, and a p-system connected to the
nitrogen atom separated by a 2-carbon chain linker (Fig. 3). The her-
mitamides are structurally simpler than the jamaicamides. If the
hermitamides present sodium channel blocking activity, the facility
in which these natural products can be synthesized will enable
large-scale preparation of this class of sodium channel blockers for
further biological testing in vitro and in vivo.
2.2.2. In vitro displacement of [3H]-BTX by hermitamides A and
B
In order to determine the binding of hermitamides 4 and 5 to
sodium channels, we tested the natural products, the correspond-
ing R enantiomers, and the racemic hermitamides for their ability
Figure 2. The proposed pharmacophore hermitamides A and B (4 and 5) .

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E. O. De Oliveira et al. / Bioorg. Med. Chem. 19 (2011) 4322–4329
Figure 3. Pharmacophore analysis of the jamaicamides, kalkitoxin and the hermitamides.
Scheme 1. Asymmetric synthesis of hermitamides A and B. Reagents and conditions: (a) allyltributylstannane, (R)-BINOL (20 mol %), TiCl2(O-i-Pr)2 (20 mol %), CH2Cl2, 4 Å MS,
ꢀ20 °C (40%); (b) (i) NaH, DMF, (ii) MeI (92%); (c) OsO4, NaIO4; (d) vinylmagnesium bromide, THF (51%, two steps); (e) CH3C(OCH3)3, n-PrCO2H, reflux (63%); (f) LiOH, H2O–
THF (41%); (g) DCC, 1-hydroxybenzotriazole, R-NH2 [R-NH2 = phenethylamine (77%) or tryptamine (88%). Obs.: EDC in methylene chloride has also been used in a scaled up
synthesis of (S)-5 (75%)].
to displace [3H]-BTX in rat forebrain membranes. Rat sodium chan-
nels exhibit a high degree of homology with human sodium chan-
nels (>95%),15 and the sodium channel is expressed highly in the
forebrain. Batrachotoxin (BTX) is a lipid-soluble neurotoxin that
specifically labels site 2 of the voltage-gated sodium channels. This
site overlaps with the local anesthetic binding site 9. Ligands that
bind to site 9 include anesthetics, anticonvulsants and antiarrthy-
mics. As a positive control, phenytoin, a clinically used sodium
channel blocker that is known to displace [3H]-BTX was also
included.
toin. Hermitamide B (5) was the most active natural product,
followed by hermitamide A (4), and then lyngbic acid (7). Surpris-
ingly, the remote methoxy stereocenter does not appear to have
much effect on the affinity of these compounds.
2.2.3. In vitro electrophysiology study via patch clamping
The ability of the hermitamides to displace [3H]-BTX suggests
that both bind to the sodium channel at the same site as BTX. In
order to test whether these compounds were functionally active
as sodium channel blockers, we tested the effects of the hermita-
mides on human embryonic kidney cells (HEK 293) stably express-
ing hNav1.2 sodium channels using the two-microelectrode
voltage clamp technique.
The assays were run at 10 lM concentration and recorded as
percent displacement of [3H]-BTX. As shown in Table 1, hermita-
mides 4 and 5 showed potencies equal to or greater than pheny-

E. O. De Oliveira et al. / Bioorg. Med. Chem. 19 (2011) 4322–4329
4325
As shown in Table 2 and Figure 4, hermitamides 4 and 5 indeed
function as sodium channel blockers. Hermitamide A (4) blocks
structural features that might govern the binding of hermitamide
B to the hNav receptors, and to use this information for future drug
design, a model of the human voltage-gated sodium channel was
predicted by homology and hermitamide B was docked to this
model.
ꢁ50% sodium channel current at 1
(5) was a more potent blocker, eliciting ꢁ80% blockade at 1
with a 50% blockage occurring somewhere between 1 M and
lM, whereas hermitamide B
lM
l
100 nM concentration. Apparently, the aromatic region of these
compounds is important for activity, with the indole group of her-
mitamide B being advantageous over the simple phenyl ring of
hermitamide A. Interestingly, the remote methoxy stereocenter
does not appear to have much influence on the ability of these
compounds to inhibit channel current. This result is in line with
our results in Table 1, where hermitamide B displaces [3H]-BTX
more efficiently than hermitamide A.
2.3.1. Homology model of the hNav channel
The sodium channel was modeled using the MthK (PDB: 1lnq)
potassium channel as a template. In this model, the S6 helical
bends were produced at the serine sites, which correspond to the
glycine residues in the MthK structure. A detailed description of
how this model was built is found in the experimental section of
this paper.
2.3. Molecular modeling
2.3.2. Docking of (S)-hermitamide B to the homology model of
the hNav channel
Since hermitamide B (5) displaced [3H]-BTX in a competitive
binding assay, this suggests that it binds to the sodium channel
at site 9. Site 9 overlaps with site 2, which is also known as the lo-
cal anesthetic (LA) binding site. In order to gain insight into the
Hermitamide B (5) was docked into our homology model using
the program FlexX incorporated in Sybyl 8.0. Comparison with an
initially predicted binding model of BTX using the same homology
model suggests that the binding mode of hermitamide B differs
from BTX at its interactions with the S6 helix residues. In order
to provide consistent results, the docked position of hermitamide
B was remodeled using a step-by-step manual docking methodol-
ogy with restrained molecular dynamic (MD) simulations followed
by energy minimization. In the restrained MD simulations, the
optimum van der Waals and H-bond distance constraints was set
between the ligand and the pore forming residues.
The following residues were identified as important for the
binding of hermitamide B to the sodium channel: F1283, F1579,
L1582, V1583, Y1586 in IVS6; T1279, L1280 in IIIS6; L788, F791,
L792, in IIS6; I433, N434, L437 in IS6; and selectivity filter residues
D400, E755, K1237 in the domains of I-IV P-loops. Mutation exper-
iments and computational modeling studies further support that
several of these residues participate in the binding of BTX and local
anesthetics.16–18 As shown in Figure 5, binding is driven mainly by
a hydrophobic interaction with residue K1237, and H-bonds be-
tween the amide group of hermitamide B with N434 and Y1586.
Strong hydrophobic contacts were also predicted between her-
mitamide B and F1283, F1579, L1582, V1583, Y1586 L1280, L788,
F791, L792, I433, and L437. The higher affinity of hermitamide B
for the sodium channel over hermitamide A may be due to a puta-
tive stacking interaction of the indole moiety with F791, and favor-
able van der Waals contact with L437 and L788. In addition, our
Table 1
In vitro displacement of [3H]-BTX by hermitamides A and B and lyngbic acid
Compounds
rac-Lyngbic acid
% [3H]-BTX displacement at 10 Ma
l
(rac)-7
(S)-7
(R)-7
(rac)-4
(S)-4
(R)-4
(rac)-5
(S)-5
(R)-5
10
7.35
7.77
(S)-Lyngbic acid
(R)-Lyngbic acid
rac-Hermitamide A
(S)-Hermitamide A
(R)-Hermitamide A
rac-Hermitamide B
(S)-Hermitamide B
(R)-Hermitamide B
Phenytoin
11.91
15.71
15.89
13.79
36.05
29.02
20.31
19.78
a
This screen has an error of approximately 20%.
Table 2
Electrophysiology effects of the hermitamides on hNav1.2
Compound
10
l
M
1
l
M
100 nM
<10 (3)
15.5 3.7 (4)
<10 (3)
24.3 2.9 (4)
17.2 2.4(4)
28.8 3.0 (4)
(rac)-4
(S)-4
90.2 2.5 (3)
94.4 1.3 (4)
49.7 3.8 (4)
74.4 2.1 (4)
58.7 1.3 (4)
88.4 2.9 (4)
87.8 2.2(4)
80.7 2.2(4)
<10 (3)
(R)-4
(rac)-5
(S)-5
100 (1)
100 (3)
(R)-5
Phenytoin
10
Values represent percentage block (%) SEM. Values in parenthesis represent n
number of experiments.
Figure 4. Pharmacological block of sodium currents by hermitamides at hNav1.2
channels expressed in HEK 293 cells. All drugs were tested at 1
l
M concentration
Figure 5. Model showing (S)-hermitamide B docked into the BTX binding site of
hNav channel.
(scale bar represents 1 ms). ⁄drug response.

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E. O. De Oliveira et al. / Bioorg. Med. Chem. 19 (2011) 4322–4329
predicted model shows no interactions between the sodium chan-
nel and the methoxy group. This is in agreement with our biolog-
ical evaluations showing that the stereochemistry of the methoxy
group did not significantly contribute to the activity of the com-
pounds (Tables 1 and 2).
4. Experimental
4.1. Chemistry
4.1.1. General procedures
Commercially available reagents and solvents were purchased
from Sigma–Aldrich, Fisher Scientific, EMD and TCI America. 1H
and 13C NMR spectra were recorded on a Varian 400 MR model
NMR instrument. Optical rotation measurements were performed
on a Bellingham & Stanley ADP220 Digital Polarimeter. Flash chro-
matographic purifications were done on Biotage SP1 chromatogra-
phy instrument.
3. Conclusion
Blockers of the human voltage-gated sodium channel are widely
used in the clinic as anticonvulsants, anesthetics, and antiarrhyth-
mics. However, several challenges remain regarding the sodium
channel in drug discovery. For example, selectivity for sodium chan-
nel subtypes as well as selectivity for the sodium channels over
potassium and calcium channels continue to be a hindrance in fur-
ther development of drugs for this target. In order to discover new
structural classes as a template for future drug discovery efforts,
we have synthesized and evaluated the natural products hermita-
mides A and B isolated from the marine cyanobacteria L. majuscula.
We have reported for the first time the asymmetric total syn-
thesis of these compounds by employing a simple and effective
synthetic strategy, that is, Keck allylation of octanal, sterospecific
formation of an E olefin by Johnson–Claisen rearrangement, and
coupling of the resulting lyngbic acid with the appropriate amines
via carbodiimide chemistry.
We have also demonstrated that hermitamides A and B are
inhibitors of the human voltage-gated sodium channel. Our
hypothesis for their activity was derived from an envisioned com-
mon pharmacophore that is shared with other known sodium
channel blockers. By examining two of the potential modes of
binding of jamaicamide C in relation to kalkitoxin, both of which
are known sodium channel blockers, we reasoned that the her-
mitamides share a very similar structural framework as the jamai-
camides and therefore should also block the sodium channel. Our
results show that the hermitamides efficiently displace [3H]-BTX,
suggesting that they bind to either site 2 or site 9 of the sodium
channel. Hermitamides A and B also proved to be more potent at
displacing [3H]-BTX from the sodium channel compared to the
clinical agent phenytoin. We then evaluated the functional block-
ing activity of the hermitamides on hNaV1.2 channels by patch
clamp techniques. Indeed both hermitamides A and B were found
to be potent inhibitors of sodium channel current in HEK-293 cells
stably expressing hNaV1.2.
Initial docking studies of hermitamides A and B to our voltage-
gated sodium channel homology model, built on the published
MthK potassium channel crystal structure, suggests that the bind-
ing is governed mainly by the hydrophobic interaction of the lipo-
philic side chain of the hermitamides with residue K1237, and by
the H-bond interaction between the amide group and residues
N434 and Y1586. The model also suggests that the slightly higher
potency of hermitamide B in displacing [3H]-BTX compared to her-
mitamide A can be explained by putative stacking interactions of
the indole moiety of hermitamide B with residues F791, and favor-
able van der Waals contact with residues L437 and L788.
Cyanobacteria are a rich source of biologically active secondary
metabolites. The hermitamides were isolated from a collection of L.
majuscula cyanobacteria isolated in Papua New Guinea. However
their function in nature remains unknown. We have shown that
these compounds are very potent sodium channel blockers and
we present a molecular modeling suggesting a potential binding
mode of these natural products to the sodium channel. Caco-2 per-
meability studies and preliminary in vivo pain studies in our labo-
ratory suggest that optimization of the physicochemical properties
of these compounds may allow for translation to bioavailable
drugs. And, we believe these natural products provide an interest-
ing new scaffold for drug discovery.
4.1.2. Syntheses
4.1.2.1. rac-undec-1-en-4-ol—((rac)-8). To a solution of 1.2 mL
(7.8 mmol) of octanal in 30 mL of ether was added 15.6 mL of a
1.0 M solution of allylmagnesium bromide in ether. The reaction
was allowed to stir at room temperature for 18 h and then
quenched with saturated NH4Cl. The product was extracted with
ether (2 ꢂ 20 mL), dried over Na2SO4 and filtered. The solvent
was removed under reduced pressure and the remaining residue
was purified by flash chromatography to give 0.79 g (60%) of the
allylic alcohol. 1H NMR (400 MHz, CDCl3): d 5.80–5.70 (m, 1H),
5.04–4.99 (m, 2H), 3.53 (m, 1H), 2.52 (br s, 1H), 2.20–2.05 (m,
2H), 1.38–1.34 (m, 2H), 1.25–1.15 (m, 10H), 0.80 (t, J = 7.1 Hz,
3H); 13C NMR (100 MHz, CDCl3): d 133.6, 116.3, 70.6, 42.5, 37.5,
32.7, 30.6, 30.2, 26.7, 23.7, 15.2.
4.1.2.2. (S)-1-undecen-4-ol—((S)-8). To a flame dried flask under
a N2 atmosphere was added 5.0 g of 4 Å molecular sieves and
100 mL of anhydrous CH2Cl2, followed by 1.9 g (7.8 mmol) of Ti-
Cl2(i-PrO)2 and 2.2 g (7.8 mmol) of (R)-(+)-1,10-bi-2-naphthol. The
solution immediately turned red and was stirred at room temper-
ature for 2 h. To this solution was added 17.9 mL (58.3 mmol) of
allyltributyltin. The reaction was cooled to ꢀ20 °C and 5.0 g
(39.0 mmol) of octanal was added. The solution was allowed to stir
for 48 h at ꢀ20 °C. The solution was filtered through a plug of Cel-
ite and the solvent was removed under reduced pressure. The
remaining residue was purified by flash chromatography eluting
with 1:9 EtOAc–hexanes to give 2.5 g (38%) of allylic alcohol (S)-
8. 1H NMR (400 MHz, CDCl3): d 5.85–5.75 (m, 1H), 5.11–5.07 (m,
2H), 3.60 (m, 1H), 2.29–2.22 (m, 1H), 2.14-2.07 (m, 1H), 1.91 (br
s, 1H), 1.44–1.41 (m, 2H), 1.32–1.22 (m, 10H), 0.85 (t, J = 6.5 Hz,
3H); 13C NMR (100 MHz, CDCl3): d 133.6, 116.8, 70.8, 42.6, 37.6,
32.8, 30.6, 30.3, 26.7, 23.8, 15.4. ½a D23
ꢃ
ꢀ7.7 (c 1.04, CHCl3); lit.13
½
a 2D5
ꢃ
ꢀ6.5 (c 1.04, CHCl3).
4.1.2.3. (4S)-methoxy-undec-1-ene—((S)-9). To
a solution of
2.5 g (14.7 mmol) of alcohol (S)-8 in 50 mL of DMF was added
0.90 g (22.0 mmol) of NaH (60% dispersion in mineral oil) and
1.4 mL (22.0 mmol) of CH3I. The reaction was refluxed for 10 h.
The solution was cooled to room temperature, taken up in EtOAc,
and washed with saturated LiCl (3ꢂ). The organic phase was dried
over Na2SO4, filtered, and the solvent was removed under reduced
pressure. The resulting residue was purified by flash chromatogra-
phy eluting with 1:9 EtOAc–hexanes to yield 2.5 g (92%) of the
titled compound. 1H NMR (400 MHz, CDCl3): d 5.85–5.75 (m, 1H),
5.08-5.01 (m, 2H), 3.32 (s, 3H), 3.18 (quin, J = 5.8 Hz, 1H), 2.24
(m, 2H), 1.46–1.40 (m, 2H), 1.30–1.20 (m, 10H), 0.86 (t, J = 6.9 Hz,
3H); 13C NMR (100 MHz, CDCl3): d 133.6, 115.7, 80.4, 56.9, 38.6,
34.3, 32.8, 30.8, 30.3, 26.4, 23.8, 15.4. ½a D22
ꢀ4.0 (c 2.00, CHCl3).
ꢃ
4.1.2.4. Methyl(4E,7S)-methoxy-tetradecenoate—((S)-12). To a
biphasic solution of 2.5 g (13.5 mmol) of alkene (S)-9 in 30 mL of
1:1 Et2O–H2O was added 2.2 mL (5 mol %) of OsO4 (2.5 wt % solu-

E. O. De Oliveira et al. / Bioorg. Med. Chem. 19 (2011) 4322–4329
4327
tion in t-BuOH). At room temperature, 6.4 g (29.7 mmol) of NaIO4
was added dropwise over 30 min. The solution was stirred for an
additional 18 h. The reaction was taken up in EtOAc and the aque-
ous phase was removed. The organic phase was dried over Na2SO4,
filtered, and the solvent was removed under reduced pressure to
give (3S)-methoxydecanal ((S)-10) as a yellow oil. No further puri-
fication was performed.
To a solution of aldehyde (S)-10 in 50 mL of THF was added
27 mL of a 1.0 M solution of vinylmagnesium bromide solution in
THF. The reaction was stirred at room temperature for 3 h and then
quenched with saturated NH4Cl. The product was extracted with
EtOAc (3 ꢂ 30 mL), dried over Na2SO4, filtered, and the solvent
was removed under reduced pressure. The resulting residue was
purified by flash chromatography eluting with 1:5 EtOAc–hexanes
to afford 1.5 g (51% over two steps) of 3-hydroxy-5S-methoxydo-
dec-1-ene ((S)-11) as a mixture of diastereomers.
To vinyl alcohol (S)-11 in 6 mL of trimethyl orthoacetate was
added one drop of propionic acid. The flask was fitted with a distil-
lation head, the solution was heated to 100 °C for 1.5 h, and the
resulting evolution of MeOH was removed by distillation. The sol-
vent was then removed under reduced pressure and the remaining
residue was purified by flash chromatography eluting with 1:5
EtOAc–hexanes to afford 1.2 g (63%) of methyl ester (S)-12. 1H
NMR (400 MHz, CDCl3): d 5.47 (m, 2H), 3.66 (s, 3H), 3.32 (s, 3H),
3.12 (quin, J = 5.8 Hz, 1H), 2.24–2.16 (m, 4H), 2.18 (m, 2H), 1.30–
1.23 (m, 2H), 1.19–1.09 (m, 10H), 0.87 (t, J = 6.9 Hz, 3H); 13C
NMR (100 MHz, CDCl3): d 172.5, 130.2, 127.5, 81.6, 58.0, 53.1,
38.2, 36.0, 35.3, 33.8, 31.8, 31.3, 30.0, 27.4, 24.9, 16.5.
Anal. Calcd for C23H37NO2: C, 76.83; H, 10.37; N, 3.90. Found: C, 76.60;
H, 10.46; N, 3.89.
4.1.2.7. 2-(1H-indol-3-yl)-ethyl (7S,4E)-methoxytetradecena-
mide—((S)-5). To a solution of 0.18 g (0.68 mmol) of carboxylic
acid (S)-7 in 10 mL of CH2Cl2 was added 0.12 mL (0.75 mmol) of
diisopropylcarbodiimide and 0.10 g (0.75 mmol) of 1-hydroxyben-
zotriazole. After stirring at room temperature for 10 min, 0.11 g
(0.68 mmol) of tryptamine was added. The reaction was stirred
at room temperature for 15 h. The solvent was then removed under
reduced pressure and the residue was taken up in EtOAc. The solu-
tion was washed with 1 N HCl (5 ꢂ 20 mL) followed by saturated
NaHCO3 (1 ꢂ 20 mL). The organic phase was dried over Na2SO4, fil-
tered, and the solvent was removed under reduced pressure. The
resulting residue was purified by flash chromatography eluting
with 1:3 EtOAc–hexanes to give 0.24 g (88%) of (S)-5. 1H NMR
(400 MHz, CDCl3): d 8.89 (br s, 1H), 7.58 (d, J = 7.8 Hz, 1H), 7.35
(d, J = 8.1 Hz, 1H), 7.19 (t, J = 8.1 Hz, 1H), 7.10 (t, J = 7.9 Hz, 1H),
6.97 (s, 1H), 5.80 (br s, 1H), 5.44 (m, 2H), 3.57 (q, J = 6.6 Hz, 2H),
3.32 (s, 3H), 3.15 (quin, J = 5.7 Hz, 1H), 2.95 (t, J = 6.8 Hz, 2H),
2.33–2.28 (m, 2H), 2.18-2.14 (m, 4H), 1.45–1.41 (m, 2H), 1.32-
1.22 (m, 10H), 0.89 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d 170.4, 135.0, 129.5, 126.2, 126.1, 121.1, 120.8, 118.1, 117.5,
111.6, 110.5, 80.5, 56.7, 40.5, 37.2, 37.1, 34.1, 32.7, 30.7, 30.2,
29.6, 26.3, 26.2, 23.7, 15.4. ½a D24
ꢃ
ꢀ2.0 (c 2.00, CHCl3); lit.8
½ ꢃ
a 2D6
ꢀ4.9 (c 0.15, CHCl3). Anal. Calcd for C25H38N2O2: C, 75.33; H,
9.61; N, 7.03. Found: C, 74.86; H, 9.67; N, 6.92.
4.1.2.8. (R)-1-undecen-4-ol—((R)-8). Prepared using the same
method employed for the preparation of compound (S)-8, with
the exception that (S)-BINOL was used as a catalyst.
4.1.2.5. (7S,4E)-methoxytetradecenoic acid—((S)-7). To a solu-
tion of 1.1 g (4.1 mmol) of methyl ester (S)-12 in 20 mL of 1:1
THF–H2O was added 0.49 g (20.3 mmol) of LiOH. The solution
was allowed to stir at room temperature for 18 h. The solution
was then acidified with 1 N HCl and the product was extracted
with EtOAc (2 ꢂ 30 mL). The organic phase was dried over Na2SO4,
filtered, and the solvent was removed under reduced pressure. The
remaining residue was purified by flash chromatography eluting
with 1:1 EtOAc–hexanes to afford 0.43 g (41%) of carboxylic acid
(S)-7. 1H NMR (400 MHz, CDCl3): d 11.04 (br s, 1H), 5.44 (m, 2H),
3.29 (s, 3H), 3.12 (quin, J = 5.8 Hz, 1H), 2.39–2.29 (m, 4H), 2.15
(m, 2H), 1.42–1.37 (m, 2H), 1.28–1.19 (m, 10H), 0.83 (t,
J = 6.9 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 176.5, 129.0, 126.4,
80.7, 56.7, 37.1, 34.8, 34.1, 32.7, 30.7, 30.2, 28.7, 26.3, 23.8, 15.4.
Yield = 33%. 1H NMR (400 MHz, CDCl3): d 5.81–5.70 (m, 1H),
5.05–5.00 (m, 2H), 3.55 (m, 1H), 2.54 (br s, 1H), 2.22–2.04 (m,
1H), 2.07 (m, 1H), 1.39–1.35 (m, 2H), 1.27-1.17 (m, 10H), 0.80 (t,
J = 6.9 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 133.6, 116.4, 70.7,
42.5, 37.5, 32.7, 30.5, 30.2, 26.7, 23.7, 15.3. ½a D23
ꢃ
+7.7 (c 1.04,
CHCl3); lit.13
½
a 2D5
+6.5 (c 1.04, CHCl3).
ꢃ
4.1.2.9. (4R)-methoxy-undec-1-ene—((R)-9). Prepared using the
same method employed for the preparation of compound (S)-9.
Yield = 80%. 1H NMR (400 MHz, CDCl3): d 5.71–5.61 (m, 1H),
4.93–4.86 (m, 2H), 3.17 (s, 3H), 3.02 (quin, J = 5.8 Hz, 1H), 2.10
(m, 2H), 1.34–1.30 (m, 2H), 1.19–1.09 (m, 10H), 0.75 (t, J = 6.9 Hz,
3H); 13C NMR (100 MHz, CDCl3): d 133.3, 115.3, 80.1, 56.4, 38.4,
34.1, 32.6, 30.6, 30.1, 26.1, 23.6, 15.1.
½
a 2D7
ꢃ
ꢀ10.9 (c 1.1, CHCl3); lit.23
½
a 2D6
ꢃ
ꢀ11.1 (c 3.9, CHCl3). Anal. Calcd
for C15H28O3: C, 70.27; H, 11.01. Found: C, 69.98; H, 11.12.
4.1.2.6. Phenethyl (7S,4E)-methoxytetradecenamide—((S)-4). To a
solution of 0.18 g (0.68 mmol) of carboxylic acid (S)-7 in 10 mL of
CH2Cl2 was added 0.12 mL (0.75 mmol) of diisopropylcarbodiimide
and 0.10 g (0.75 mmol) of 1-hydroxybenzotriazole. After stirring at
room temperature for 10 min, 0.090 mL (0.68 mmol) of phenethyl-
amine was added. The reaction was stirred at room temperature for
15 h, during which a white precipitate formed. The solvent was then
removed under reduced pressure and the residue was taken up in
EtOAc. The solution was washed with 1 N HCl (3 ꢂ 20 mL) followed
by saturated NaHCO3 (1 ꢂ 20 mL). The organic phase was dried over
Na2SO4, filtered, and the solvent was removed under reduced pres-
sure. The remaining residue was purified by flash chromatography
eluting with 1:3 EtOAc–hexanes to give 0.19 g (77%) of (S)-4. 1H
NMR (400 MHz, CDCl3): d 7.27-7.13 (m, 5H), 5.82 (br s, 1H), 5.40
(m, 2H), 3.45 (q, J = 6.9 Hz, 2H), 3.26 (s, 3H), 3.09 (quin, J = 5.7 Hz,
1H), 2.76 (t, J = 7.1 Hz, 2H), 2.29–2.25 (m, 2H), 2.17–2.11 (m, 4H),
1.40–1.33 (m, 2H), 1.30–1.22 (m, 10H), 0.83 (t, J = 6.8 Hz, 3H); 13C
NMR (100 MHz, CDCl3): d 170.2, 137.4, 129.6, 127.4, 127.3, 126.2,
125.2, 80.4, 56.7, 41.3, 37.2, 37.1, 36.5, 34.1, 32.7, 30.7, 30.2, 29.6,
4.1.2.10. Methyl (4E,7R)-methoxy-tetradecenoate—((R)-12). Pre-
pared using the same method employed for the preparation of
compound (S)-12.
Yield = 51%. 1H NMR (400 MHz, CDCl3): d 5.33 (m, 2H), 3.51 (s,
3H), 3.17 (s, 3H), 2.99 (quin, J = 5.8 Hz, 1H), 2.25–2.18 (m, 4H),
2.04 (m, 2H), 1.30–1.26 (m, 2H), 1.18–1.09 (m, 10H), 0.74 (t,
J = 6.9 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 173.0, 130.1, 127.3,
80.4, 56.1, 51.0, 36.0, 33.6, 33.0, 31.5, 31.8, 29.4, 29.0, 27.6, 24.9,
22.3, 13.7.
4.1.2.11. (7R,4E)-methoxytetradecenoic acid—((R)-7). Prepared
using the same method employed for the preparation of compound
(S)-7.
Yield = 65%. 1H NMR (400 MHz, CDCl3): d 10.23 (br s, 1H), 5.48
(m, 2H), 3.32 (s, 3H), 3.15 (quin, J = 5.8 Hz, 1H), 2.44–2.31 (m,
4H), 2.18 (m, 2H), 1.44–1.38 (m, 2H), 1.31–1.20 (m, 10H), 0.87 (t,
J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 176.8 128.9, 126.6,
80.7, 56.9, 37.2, 34.9, 34.3, 32.8, 30.8, 30.3, 28.7, 26.4, 23.9, 15.5.
½
a 2D3
ꢃ
+4.2 (c 1.91, CHCl3). Anal. Calcd for C15H28O3: C, 70.27; H,
26.3, 23.7, 15.4. ½a D23
ꢃ
ꢀ8.9 (c 0.45, CHCl3); lit.8 ½a D26
ꢀ9.3 (c 0.45, CHCl3).
ꢃ
11.01. Found: C, 70.13; H, 11.21.

4328
E. O. De Oliveira et al. / Bioorg. Med. Chem. 19 (2011) 4322–4329
4.1.2.12. Phenethyl (7R,4E)-methoxytetradecenamide—((R)-
4). Prepared using the same method employed for the prepara-
tion of compound (S)-4.
tem were performed in which the sodium channel was constrained
by a force constant of 100 kcal/mol/Å.2 After minimization, a 10 ps
simulation was used to gradually raise the temperature of the sys-
tem to 298 K while the complex was constrained by a force con-
stant of 20 kcal/mol/Å. Another 20 ps equilibration run was used
where only the backbone atoms of the complex were constrained
by a force constant of 5 kcal/mol/Å. Final production run of
100 ps was performed with no constraints. When applying con-
straints, the initial complex structure was used as a reference
structure. The PME method10 was used and the time step was
5 fs, and a neighboring pairs list was updated every 30 steps.
Yield = 74% for (R)-4. 1H NMR (400 MHz, CDCl3): d 7.26–7.12 (m,
5H), 5.93 (br s, 1H), 5.40 (m, 2H), 3.42 (q, J = 6.9 Hz, 2H), 3.25 (s,
3H), 3.09 (quin, J = 5.8 Hz, 1H), 2.75 (t, J = 7.1 Hz, 2H), 2.28–2.24
(m, 2H), 2.16–2.10 (m, 4H), 1.43-1.35 (m, 2H), 1.28-1.18 (m,
10H), 0.83 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 170.2,
137.4, 129.5, 127.4, 127.2, 126.1, 125.1, 80.4, 56.7, 41.2, 37.1,
37.0, 36.4, 34.1, 32.6, 30.6, 30.2, 29.6, 26.3, 23.7, 15.3. ½a D23
ꢃ
+8.9
(c 0.45, CHCl3). Anal. Calcd for C23H37NO2: C, 76.83; H, 10.37; N,
3.90. Found: C, 76.87; H, 10.57; N, 3.92.
4.3. [3H]-BTX displacement assay
4.1.2.13. 2-(1H-indol-3-yl)-ethyl (7R,4E)-methoxytetradecena-
mide—((R)-5). Prepared using the same method employed for
the preparation of compound (S)-5.
The [3H]-BTX binding assays were performed by Novascreen
Biosciences (Hanover, MD). Briefly, rat forebrain membranes
(10 mg tissue/well) were incubated with [3H]-BTX (30–60 Ci/
mmol). Reactions are carried out in 50 mM HEPES (pH 7.4) contain-
ing 130 mM choline chloride at 37 °C for 60 min. The reaction was
terminated by rapid vacuum filtration of the reaction contents onto
glass fiber filters. Radioactivity trapped onto the filters was deter-
mined and compared with control values to ascertain any interac-
tions of the test compound with the Na+ channel site 2 binding site.
Yield = 96% for (R)-5. 1H NMR (400 MHz, CDCl3): d 8.97 (br s,
1H), 7.58 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.18 (t,
J = 7.9 Hz, 1H), 7.10 (t, J = 7.7 Hz, 1H), 6.97 (s, 1H), 5.84 (br s, 1H),
5.44 (m, 2H), 3.57 (q, J = 6.6 Hz, 2H), 3.33 (s, 3H), 3.16 (quin,
J = 5.7 Hz, 1H), 2.95 (t, J = 6.8 Hz, 2H), 2.33–2.29 (m, 2H), 2.18–
2.14 (m, 4H), 1.45–1.39 (m, 2H), 1.37–1.29 (m, 10H), 0.90 (t,
J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 170.4, 135.0, 129.5,
126.1, 126.0, 121.1, 120.7, 118.1, 117.5, 111.6, 110.5, 80.5, 56.7,
40.5, 37.2, 37.1, 34.1, 32.7, 30.6, 30.2, 29.6, 26.3, 26.2, 23.7, 15.4.
Aconitine (1 lM) was used as a positive control (Sigma Aldrich,
Inc., St. Louis, MO).
½
a 2D4
+2.0 (c 2.00, CHCl3). Anal. Calcd for C25H38N2O2: C, 75.33; H,
9.61; N, 7.03. Found: C, 74.80; H, 9.65; N, 7.02.
ꢃ
4.4. Sodium hNav1.2 channel electrophysiology
4.2. Modeling of the hNav channel with hermitamide B ((S)-5)
Human embryonic kidney cells (HEK) cells stably expressing
human Nav1.2 were a kind gift from Dr. H.A. Hartmann (University
of Baltimore, Maryland, USA) and were grown in DMEM/F12 media
(Invitrogen, Corp, CA, USA) supplemented with 10% fetal bovine
Modeling of the Sodium Channel: Multiple sequence alignment of
the S6 transmembrane residues from domains I, III, and IV was car-
ried out using PSI-BLAST and CLUSTALW. Homology modeling of S5,
the P-loops, and S6 from all four domains used the MthK channel X-
ray structure (PDB: 1lnq) as a template. Since LAs bind to the inacti-
vated form of domains the potassium channel were aligned, that is,
S5 and S6 transmembrane segments based on both homology and
secondary structure prediction. Non-homologous regions in the
longer P-loopsof domains I and III, whichcorrespondto putative gly-
cosylation sites, were deleted. The P-loops, N and C termini were
modeled based on homologous segments of the KcsA channel struc-
ture (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 side chain atom con-
tacts, different rotamerstates of theresidue were considered and the
one with minimal contacts, but a favorable interaction was chosen.
Local side chain atom minimization was also performed.
Docking of (S)-hermitamide B: Initial docking studies between
the ligand and the sodium channel were carried out using the pro-
gram FlexX. After consistent manual intervention, a final model
was arrived. The structure of the Nav–hermitamide B complex
was then refined by molecular dynamics simulation using the Am-
ber 9 program suite19 with the PARM98 force-field parameter. The
charge and force field parameters of hermitamide B was obtained
using the most recent Antechamber module in the Amber 9 pro-
gram, where hermitamide B was minimized at the MP2/6-31G⁄ le-
vel. The SHAKE algorithm20 was used to keep all bonds involving
hydrogen atoms rigid. Weak coupling temperature and pressure
coupling algorithms21 were used to maintain constant temperature
and pressure, respectively. Electrostatic interactions were calcu-
lated with the Ewald particle mesh method22 with a dielectric con-
stant at 1Rij and a nonbonded cutoff of 12 Å for the real part of
electrostatic interactions and for van der Waals0 interactions. The
total charge of the system was neutralized by addition of a chloride
ion. The system was solvated in a 14 Å cubic box of water where
the TIP3P model8 was used. 3000 steps of minimization of the sys-
serum, penicillin (100 U/ml), streptomycin (100
(500 g/ml; Sigma, MO, USA). Cells were grown in a humidified
atmosphere of 5% CO2 and 95% air at 37 °C.
lg/ml) and G418
l
Sodium currents were recorded using the whole-cell configura-
tion of the patch clamp recording technique with an Axopatch 200
amplifier (Molecular Devices). All voltage protocols were applied
using pCLAMP 9 software and a Digidata 1322A (Molecular De-
vices). 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 Instruments Co, Nova-
to, CA) and heat polished to produce electrode resistances of 0.5–
1.5 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 coverslips and super-
fused with solution containing the following composition; (in mM)
NaCl 130, KCl 4, CaCl2 1, MgCl2 5, HEPES 5, and glucose 5 (pH ad-
justed to 7.4 with NaOH).
Compounds were prepared as 100 mM stock solutions in
DMSO and diluted to the desired concentration in perfusion
solution. The maximum DMSO concentration used was 0.1%
and had no effect on current amplitude. All experiments were
performed at room temperature (20–22 °C). After establishing
whole-cell, a minimum series resistance compensation of 75%
was applied. Sodium currents were elicited by a depolarizing
step from a holding potential of ꢀ60 to +10 mV for a duration
of 25 ms at 15 s intervals. Compounds were applied after a
3 min control period and continued until a steady state current
amplitude was observed. All data represent percentage mean
block standard error of the mean (SEM).
Acknowledgments
This publication was made possible by Grant Number
R21NS061069 from the NIH-NINDS. Its contents are solely the

E. O. De Oliveira et al. / Bioorg. Med. Chem. 19 (2011) 4322–4329
4329
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