Synthesis of skeletal analogues of saxitoxin derivatives and evaluation of their inhibitory activity on sodium ion channels NaV1.4 and Na V1.5

Article abstract of DOI:10.1002/chem.201101058

Skeletal analogues of saxitoxin (STX) that possess a fused-type tricyclic ring system, designated FD-STX, were synthesized as candidate sodium ion channel modulators. Three kinds of FD-STX derivatives 4 a-c with different substitution at C13 were synthesized, and their inhibitory activity on sodium ion channels was examined by means of cell-based assay. (-)-FD-STX (4 a) and (-)-FD-dcSTX (4 b), which showed moderate inhibitory activity, were further evaluated by the use of the patch-clamp method in cells that expressed Na_V1.4 (a tetrodotoxin-sensitive sodium channel subtype) and Na_V1.5 (a tetrodotoxin-resistant sodium channel subtype). These compounds showed moderate inhibitory activity towards Na_V1.4, and weaker inhibitory activity towards Na_V1.5. Uniquely, however, the inhibition of Na _V1.5 by (-)-FD-dcSTX (4 b) was "irreversible".

Full text of DOI:10.1002/chem.201101058

DOI: 10.1002/chem.201101058
Synthesis of Skeletal Analogues of Saxitoxin Derivatives and Evaluation of
Their Inhibitory Activity on Sodium Ion Channels NaV1.4 and NaV1.5**
Ryoko Shinohara,[a] Takafumi Akimoto,[a] Osamu Iwamoto,[a] Takatsugu Hirokawa,[b]
Mari Yotsu-Yamashita,[c] Kaoru Yamaoka,[d] and Kazuo Nagasawa*[a]
Abstract: Skeletal analogues of saxi-
toxin (STX) that possess a fused-type
tricyclic ring system, designated FD-
STX, were synthesized as candidate
sodium ion channel modulators. Three
kinds of FD-STX derivatives 4a–c with
different substitution at C13 were syn-
thesized, and their inhibitory activity
on sodium ion channels was examined
by means of cell-based assay. (À)-FD-
STX (4a) and (À)-FD-dcSTX (4b),
which showed moderate inhibitory ac-
tivity, were further evaluated by the
use of the patch-clamp method in cells
that expressed NaV1.4 (a tetrodotoxin-
sensitive sodium channel subtype) and
NaV1.5
(a
tetrodotoxin-resistant
sodium channel subtype). These com-
pounds showed moderate inhibitory ac-
tivity towards NaV1.4, and weaker in-
hibitory activity towards NaV1.5.
Uniquely, however, the inhibition of
NaV1.5 by (À)-FD-dcSTX (4b) was
Keywords: inhibitors
nels · patch-clamp method · saxitox-
ins sodium structure–activity
relationships
· ion chan-
·
·
AHCTUNGTREG“NNUN irreversible”.
Introduction
genes that encode a subunits (NaV1.1–NaV1.9) and four
genes that encode b subunits have been identified. The a
subunits can be classified into two groups on the basis of
their sensitivity to tetrodotoxin (TTX), the pufferfish toxin.
There are six TTX-sensitive (TTX-s) a subunits (i.e.,
NaV1.1–NaV1.4, NaV1.6, and NaV1.7) and three TTX-resist-
ant (TTX-r) a subunits (i.e., NaV1.5, NaV1.8, and NaV1.9),
which are only blocked by high concentrations of TTX.[3,4]
Recently, a related protein (NaXX) has been recognized as a
tenth member of the group.[5] These subtypes have quite
similar structures, but the expression of the a subunits is
strongly cell-type- and tissue-specific. Therefore, each of
these subtypes is believed to have unique properties.
Indeed, mutation studies have thrown some light on the
functions of these channels. For example, mutation of
NaV1.4 is associated with primary periodic paralysis,[6]
whereas mutation of NaV1.5 leads to cardiovascular syn-
dromes.[7] Consequently, these channels are considered to be
good candidates for new drug targets. In this context,
NaVCh-subtype-selective small-molecular modulators from
natural sources have been explored, as well as synthetic
compounds. Synthetic studies have led to some subtype-se-
lective compounds.[8] For example, A-803467 is an NaV1.8-
selective blocker developed by an Abbott research
group[8c,9] for possible application in pain control. However,
more selective modulators with a variety of action mecha-
nisms are still of great interest.
Voltage-gated sodium channels (NaVCh) are transmembrane
proteins that provide inward current carried by sodium ions,
and they contribute to the control of membrane excitability,
as well as the propagation of action potentials along axons.[1]
Sodium channels within neurons are composed of a single a
subunit, which forms the voltage-sensing and ion-selective
pore, and one or more auxiliary b subunits, which are pro-
posed to serve a number of functions, including modulation
of a-subunit function and targeting/anchoring the channels
at specific sites in the plasma membrane.[2] To date, nine
[a] R. Shinohara, T. Akimoto, Dr. O. Iwamoto, Prof. Dr. K. Nagasawa
Tokyo University of Agriculture and Technology
Department of Biotechnology and Life Science
2-24-16 Naka-cho, Koganei, Tokyo 184-8588 (Japan)
Fax : (+81)42-388-7295
E-mail: knaga@cc.tuat.ac.jp
[b] Dr. T. Hirokawa
Computational Biology Research Center
National Institute of Advanced Industrial
Science and Technology, 2-4-7 Aomi, Koto-ku
Tokyo 135-0064 (Japan)
[c] Prof. Dr. M. Yotsu-Yamashita
Tohoku University, Graduate of Agricultural Science
1-1 Tsutsumidori-Amamiyamachi, Aoda-ku
Sendai 981-8555 (Japan)
[d] Prof. Dr. K. Yamaoka
Hiroshima International University
555-36 Kurose-Gakuendai, Higashi-Hiroshima
Hiroshima 739-2695 (Japan)
Saxitoxin (STX) (1a) is a naturally occurring shellfish
neurotoxin that blocks ion influx to TTX-s NaVChs.[10] STX
(1a) is an indispensable tool for studying NaVCh-related
electrophysiology, and its derivatives are also promising can-
didates for NaVCh-subtype-selective modulators. STX (1a)
and its analogues are believed to bind to the P-loop region
of the ion-selective filter in NaVCh in a similar manner to
[**] NaVCh denotes voltage-gated sodium channels. NaV1.4 is a tetrodo-
toxin-sensitive sodium channel subtype; NaV1.5 is a tetrodotoxin-re-
sistant sodium channel subtype.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201101058.
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FULL PAPER
TTX (i.e., in a reversible manner).[11] Recently, a binding
model of STX with the P-loop domain was proposed based
on molecular docking studies, and it was suggested that the
two guanidinium groups, the carbamoyl group at C13 and
the hydrated ketone at C12, have critical roles in the bind-
ing.[12] On the other hand, most naturally occurring STX an-
alogues isolated to date are functionalized at C11, C12, C13,
N1, and N7, and these modifications have a significant influ-
ence on the NaVCh-inhibitory activity.[13] Thus, structure–ac-
tivity relationship (SAR) studies of STX have been focused
on those positions,[14] especially C13, with the aim of devel-
oping NaVCh-subtype-selective modulators.
We have recently reported a total synthesis of STX (1a;
see Scheme 1) and some of its naturally occurring deriva-
tives.[15] In the course of our synthetic studies of these com-
pounds, we unexpectedly obtained a fused-type tricyclic
compound (+)-3 during the oxidation of 2 with 2-iodoxy-
benzoic acid (IBX; Scheme 2).[15a] An X-ray analysis showed
compound 3 to have a conical shape similar to that of STX
(see Scheme 1), and compound 3 was expected to be able to
access the channel conical vestibule in a similar manner to
STX. Thus, this fused-type tricyclic skeleton can be regarded
as a new skeletal analogue of STX, and we named it FD-
STX. The FD-STX structure has almost the same functional
groups as STX at positions that interact with NaVCh, al-
though the angles and/or directions of functional groups are
slightly different from those of STX.[16] We considered that
these small structural differences of FD-STX compared with
STX might represent an opportunity to acquire new subtype
Scheme 2. Synthesis of fused-type tricyclic compound (+)-3.
selectivity and/or might result in a distinctive interaction
mode of FD-STX with NaVChs.
In this report, we describe the synthesis of FD-STX deriv-
atives (À)-4a–c functionalized at C13, which correspond to
the skeletal analogues of (+)-STX (1a), (+)-dcSTX (1b),
and (+)-doSTX (1c), respectively (Scheme 3). The NaVCh-
inhibitory activity of these FD-STX derivatives was initially
examined by cell-based assay, and (À)-FD-STX (4a) and
(À)-FD-dcSTX (4b) were further evaluated by means of the
patch-clamp method in cells that express NaV1.4 or NaV1.5.
The results are of particular interest because no SAR stud-
ies that focus on the STX skeleton itself have been reported
so far.
Scheme 3. Structures of (À)-FD-STXs 4a–c, skeletal analogues of
(+)-STXs 1a–c.
Results and Discussion
Synthesis of (À)-FD-STX and its derivatives (4a–c):
Scheme 4 and Scheme 5 illustrate the synthesis of 4a–c. We
first focused on the synthesis of (À)-FD-dcSTX (4b)
(Scheme 4).[15] 1,3-Dipolar cycloaddition reaction between
chiral nitrone 5 and nitro alkene 6 at 408C gave the endo
adduct of isoxazolidine 7 as a single diastereomer. The ste-
reochemistry at C5 was epimerized with 1,8-diazabicyclo-
AHCTUNGTREG[NNUN 5.4.0]undec-7-ene (DBU) in dichloromethane at 08C
(95:5), and selective reduction of the nitro group was carried
out with zinc powder in acetic acid to give hydroxylamine 8
Scheme 1. Structures of saxitoxin (STX) and its derivatives, and a model
of their binding to the sodium ion channel.[12c]
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K. Nagasawa et al.
Scheme 4. Synthesis of (À)-FD-dcSTX (4b): a) 408C; b) DBU, CH2Cl2, À408C; c) AcOH, then Zn, À408C, 72% (3 steps); d) CbzCl, K2CO3, THF/
H2O=3:1, 08C, 83%; e) NaOAc, TiCl3/HCl, Zn, CH2Cl2/MeOH=2:1, À508C; f) BocN=C
ACHTUNGTRNE(UNGN SMe)NHBoc (16), HgCl2, Et3N, DMF, 82% (2 steps);
g) ClCH2SO2Cl, iPr2EtN, CH2Cl2, 87%; h) TBAF, THF, 08C, 93%; i) H2, Pd(OH)2/C, MeOH; j) CbzN=CACTHUNGTRNEUNG(SMe)NHCbz (17), HgCl2, Et3N, DMF, 85%
(2 steps); k) NaOMe, THF/MeOH, 08C, 65%; l) IBX, DMSO, 708C, 24%; m) H2, Pd(OH)2/C, MeOH; n) 3n HCl, 78% (2 steps).
Scheme 5. Synthesis of (À)-FD-STX (4a): a) Ac2O, pyridine (py), 508C, 96%; b) DDQ, CH2Cl2, H2O, 94%; c) trichloroacetyl isocyanate, CH2Cl2, then
K2CO3, MeOH, 34%; d) IBX, DMSO, 708C, 20%; e) H2, Pd(OH)2/C, MeOH; f) TFA, CH2Cl2, 82% (2 steps). Synthesis of (À)-FD-doSTX (4c): g) TFA,
CH2Cl2; h) H2, Pd/C, MeOH, 92% (2 steps).
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Skeletal Analogues of Saxitoxin Derivatives
FULL PAPER
in 72% yield from (+)-5. The amino group of hydroxyla-
mine in 8 was protected with a carbobenzoxy (Cbz) group
using Cbz chloride in the presence of potassium carbonate
Evaluation of NaVCh-inhibitory activity of (À)-FD-STXs
4a–c: Next, the NaVCh-inhibitory activity of 4a–c was evalu-
ated in terms of inhibition of the cytotoxicity of ouabain
and veratridine in mouse neuroblastoma cell line Neuro-
2a.[20] In this cell-culture assay, Neuro-2a is treated with a
sodium channel activator, veratridine, in the presence of
ouabain, an inhibitor of Na+/K+ ATPase. This serves to
block sodium ion efflux, and decreases the cell viability.
NaVCh inhibition by TTX, STX, and related compounds an-
tagonizes this effect, and rescues the cells in a dose-depen-
dent manner. The results obtained with 4a–c are summa-
À
(83%), then the two N O bonds in 9 were reduced with
À
À
TiCl3 NaOAc and zinc powder in aqueous MeOH HCl to
give diamine 10. Next, a tert-butoxycarbonyl (Boc)-protect-
ed guanidine group was introduced with bisACTHNUGRTENUNG(Boc)-2-methyl-
2-thiopseudourea (16) in the presence of mercury(II) chlo-
ride and triethyl amine to give guanidine 11 in 82% yield
from 8. Cyclization of guanidine in 11 was carried out by
treatment with monochloromethanesulfonyl chloride and
diisopropylethylamine to give 12 in 87% yield, and this
product was subsequently converted into 13 in three steps:
1) deprotection of the triisopropylsilyl (TIPS) group with
tetra-n-butylammonium fluoride (TBAF) in tetrahydrofuran
(THF) at 08C, 2) deprotection of the Cbz group with hydro-
gen in the presence of Pearlmanꢁs catalyst,[17] and 3) guany-
A
showed a concentration-dependent inhibitory effect on the
cytotoxicity of ouabain and veratridine, and their IC50 values
were determined to be (7.3Æ2.7) and (15Æ5.4) mm, respec-
tively (synthetic (+)-dcSTX (1b),[15c] used as a control,
showed an IC50 value of (82Æ17) nm). On the other hand,
(À)-FD-doSTX (4c) showed no inhibitory activity even at
concentrations over 500 mm. Since (À)-FD-STX (4a) and
(À)-FD-dcSTX (4b) were inhibitory, these compounds were
further evaluated by means of the patch-clamp method with
cells that express NaV1.4 and NaV1.5, which are representa-
tive members of the TTX-s and TTX-r channel subtypes, re-
spectively.
lation with bisACHTUNGTRENNUNG(Cbz)-2-methyl-2-thiopseudourea (17) in the
presence of mercury(II) chloride in 79%.[18] One of the Cbz
groups of 13 was removed with sodium methoxide in THF/
methanol to give 14 in 65% yield.[19] Construction of the tri-
cyclic fused-type guanidine was carried out by oxidation of
alcohol 14 with IBX (4 equiv) in DMSO at 708C to give 15
in 24% yield. In this reaction, oxidation of the alcohol at
C12 takes place first, then the C4 position is oxidized
through the enamine–IBX complex.[15] Finally, (À)-FD-
dcSTX (4b) was obtained by deprotection of the Cbz and
Boc groups on guanidines by hydrogenolysis in the presence
of Pearlmanꢁs catalyst, followed by treatment with 3n HCl
in 78% yield from 15 (i.e., two steps).
Table 1. NaVCh-inhibitory activity of FD-STXs 4a–c in Neuro-2a cells.[21]
Compounds
IC50 (mean ÆSD)
(7.3Æ2.7) mm
(15Æ5.4) mm
>500 mm
(82Æ17) nm
n
(À)-FD-STX (4a)
(À)-FD-dcSTX (4b)
(À)-FD-doSTX (4c)
(+)-dc-STX (1b)
G
3
3
3
3
AHCTUNGTRENNUNG
The synthesis of (À)-FD-STX (4a) is illustrated in
Scheme 5. Synthesis of (À)-FD-STX (4a) was attempted
firstly from the tricyclic compound 15 by deprotection of the
p-methoxybenzyl (MPM) group with 2,3-dichloro-5,6-dicya-
no-1,4-benzoquinone (DDQ). However, the desired reaction
did not occur, and decomposition of the starting material
was observed. The difficulty of this deprotection reaction
presumably arises from steric effects at C13. Thus, function-
alization at C13 was carried out before construction of the
tricyclic FD-STX ring system. After protection of the hy-
droxyl group of bis-guanidine 13 at C12 with acetic anhy-
dride, the MPM group in the resulting compound was suc-
cessfully removed with DDQ to give alcohol 19 in 90%
yield (two steps). A carbamoyl group was introduced at C13
by treating the alcohol 19 with trichloroacetyl isocyanate,
followed by hydrolysis with potassium carbonate in MeOH,
thereby affording carbamate 20 in 34% yield. Then, the
fused-type tricyclic skeleton was constructed under IBX oxi-
dation conditions to give 21 in 20% yield. Finally, deprotec-
tion of the Cbz and Boc groups was carried out under the
same conditions as described for the synthesis of 4b to give
(À)-FD-STX (4a) in 82% yield (two steps).
AHCTUNGTRENNUNG
The sodium ion channel a subunits of the rat skeletal
muscle channel rNaV1.4 and rat heart channel rNaV1.5 were
each transiently transfected into HEK293 cells, and electro-
physiological recording was performed with a whole-cell
patch clamp. In this experiment, the sodium ion channels
were activated by applying 10-millisecond voltage steps
from a holding potential of À120 mV every 5 s, which in-
AHCTUNGTRENNUNG
duces peak sodium currents (INa).[22,23] Figure 1 shows time
courses of changes in INa, the effects of inhibitors 4a and 4b,
and the recovery upon washout of these compounds. In the
case of NaV1.4, sodium ion current was decreased by the ad-
dition of 10 mm (À)-FD-STX (4a) or (À)-FD-dcSTX (4b),
and reached an equilibrium peak current (Iinhibitor 4a or 4b).
Recovery of sodium ion current was observed upon washout
of the inhibitor, which indicates that the ligands were re-
moved from the channel (Figure 1a and b). This is a typical
reversible inhibition, like that of TTX and STX.[24] The IC50
values of 4a and 4b were (3.8Æ0.9) and (16Æ8.6) mm, re-
spectively (Table 2),[25] and paralleled the results of the cell-
based assay. In the case of NaV1.5, the sodium ion current
was decreased by the addition of 1 mm 4a or 4b, but recov-
ery of the current following washout was observed only in
the case of (À)-FD-STX (4a) (Figure 1c); the IC50 value of
4a was (118Æ8.1) mm (Table 2).[25] On the other hand, recov-
(À)-FD-doSTX (4c) was synthesized by deprotection of
fused-type tricyclic guanidine (À)-3, which was obtained
from methyl crotonate (22) and the chiral nitrone (+)-5 as
previously reported (Scheme 5).[15a,b]
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K. Nagasawa et al.
Figure 1. Time courses of inhibition by (À)-FD-STXs and recovery following washout. a) Effect of (À)-FD-STX (4a) (10 mm) on rNaV1.4. b) Effect of
(À)-FD-dcSTX (4b) (10 mm) on rNaV1.4. c) Effect of (À)-FD-STX (4a) (1 mm) on rNaV1.5. d) Effect of (À)-FD-dcSTX (4b) (1 mm) on rNaV1.5.
their inhibitory effect on sodium ion channels was evaluated
Table 2. Inhibitory activity of (À)-FD-STXs 4a and 4b towards NaV1.4
by means of cell-based assay and the patch-clamp method.
(À)-FD-STX (4a) and (À)-FD-dcSTX (4b) showed IC50
values of (7.3Æ2.7) and (15Æ5.4) mm, respectively, in cell-
based assay. These compounds inhibited NaV1.4 (a represen-
tative tetrodotoxin-sensitive NaVCh subtype) and NaV1.5 (a
representative tetrodotoxin-resistant NaVCh subtype) in
cells that express those channels. Uniquely, (À)-FD-dcSTX
(4b) showed “irreversible” inhibition of NaV1.5; it is the
first STX- or TTX-related molecule to do so. Further work
to elucidate the mechanism of the “irreversible” interaction
between (À)-FD-dcSTX (4b) and NaV1.5 is in progress.
and NaV1.5.
Compounds
IC50 (Nav1.4)
n
IC50 (Nav1.5)
n
A
ACHTUNGTRENNUNG
(À)-FD-STX (4a)
G
3
4
E
3
4
(À)-FD-dcSTX (4b)
N
ACHTUNGTRENNUNG
ery of the sodium ion current did not occur when (À)-FD-
dcSTX (4b) was washed out (Figure 1d), thus indicating that
the inhibition of NaV1.5 by (À)-FD-dcSTX (4b) was “irre-
versible.” Although the inhibitory action of 4b against
NaV1.5 was quite weak, with an IC50 value of (182Æ16) mm
(Table 2), this is the first observation of “irreversible” inhi-
ACHTUNGTRENNUNGbition of NaV1.5 by any STX- or TTX-related molecule. The
interaction mechanism of 4b and NaV1.5 has not yet been
clarified, but 4b may be a good lead compound for the de-
velopment of NaVCh-subtype-selective modulators.
Experimental Section
General: Flash chromatography was performed on silica gel 60 (spheri-
cal, particle size 40–100 mm; Kanto), Chromatorex NH (particle size 75–
150 mm; Fuji Silysia). Optical rotations were measured using a JASCO P-
2200 polarimeter with the sodium D line. 1H and 13C NMR spectra were
recorded using a JEOL JNM ECX 400 or JNM-ECA 500 spectrometer.
The spectra are referenced internally according to residual solvent signals
Conclusion
In summary, we have synthesized a novel skeletal analogue
of saxitoxin, FD-STX. Three FD-STX derivatives 4a–c,
which differ in substitution at C13, were synthesized, and
of CDCl3 (1H NMR, d=7.26 ppm; 13C NMR, d=77.0 ppm); CD3OD
13
(1H NMR, d=3.30 ppm; C NMR, d=49.0 ppm); or 4% CD3CO2D
À
D2O (1H NMR, d=4.79 ppm (D2O); 13C NMR, d=179.0 ppm
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Skeletal Analogues of Saxitoxin Derivatives
FULL PAPER
(CD3CO2D)). Mass spectra were recorded using a JEOL JMS-T100X
spectrometer in ESI-MS mode with methanol as solvent.
1H), 3.92 (d, J=10.9 Hz, 1H), 3.89 (dd, J=6.3, 6.9 Hz, 1H), 3.77 (s, 3H),
3.59 (dt, J=8.0, 10.9 Hz, 1H), 3.50 (m, 2H), 3.31 (dd, J=6.9, 9.7 Hz,
1H), 2.35 (m, 1H), 1.96 (dd, J=6.9, 13.8 Hz, 1H), 1.49 (s, 9H), 1.43 (s,
9H), 0.97 ppm (m, 21H); 13C NMR (125 MHz, CDCl3): d=161.3, 159.1,
157.6, 156.5, 150.2, 136.4, 130.2, 129.6, 128.4, 128.0, 127.9, 113.6, 82.3,
79.5, 73.0, 72.5, 69.8, 66.8, 55.2, 53.0, 46.8, 32.1, 28.1, 28.0, 17.8, 12.0 ppm;
HRMS (ESI): m/z calcd for C43H68N4O10SiNa: 851.4569 [M+Na+];
found: 851.4602.
Isoxazolidine 8: A mixture of nitrone (+)-5 (10.4 g, 40.5 mmol) and
nitro-olefin 6 (9.49 g, 42.5 mmol) was stirred for 30 min at 408C without
any solvents. The reaction mixture was diluted with CH2Cl2 (150 mL) and
cooled to À408C. DBU (6.28 mL, 44.6 mmol) was added to the solution,
and the resulting mixture was stirred for 1 h at the same temperature.
AcOH (11.6 mL, 203 mmol) and Zn powder (freshly activated with dilut-
ed aqueous HCl, 10.6 g, 162 mmol) were added to the reaction mixture,
and the resulting mixture was warmed to 08C over 5 h. The reaction mix-
ture was then filtered through a pad of Celite, and the filtrates were di-
luted with CH2Cl2 (150 mL) and washed with H2O and brine. The com-
bined organic layer was dried over MgSO4, filtered, and concentrated in
vacuo. The residue was purified by column chromatography on silica gel
(hexane/EtOAc=15:1 to 5:1) to give hydroxylamine 8 (13.5 g, 72%).
Spectral data for 8: [a]D16 = +1.3 (c=2.3 in CHCl3); 1H NMR (400 MHz,
CDCl3): d=7.25 (d, J=8.2 Hz, 2H), 6.86 (d, J=8.2 Hz, 2H), 5.47 (br,
1H), 4.48 (s, 2H), 4.23 (dt, J=2.3, 5.0 Hz, 1H), 4.12 (q, J=6.0 Hz, 1H),
3.79 (s, 3H), 3.72 (m, 2H), 3.58 (m, 2H), 3.3, (m, 2H), 2.06 (m, 1H), 1.73
(m, 1H), 1.04 ppm (m, 21H); 13C NMR (100 MHz, CDCl3) : d=159.3,
129.6, 129.5, 113.8, 77.9, 77.8, 77.4, 73.4, 71.9, 67.9, 55.8, 55.2, 34.4, 17.9,
12.0 ppm; HRMS (ESI): m/z calcd for C24H42N2O5SiNa: 489.2761
[M+Na+]; found: 489.2758.
Cyclic guanidine 12: Chloromethanesulfonyl chloride (5.72 mL,
47.6 mmol) was added to a solution of guanidine 11 (9.90 g, 11.9 mmol)
and iPr2NEt (12.3 mL, 71.4 mmol) in CH2Cl2 (60 mL) at 08C under an N2
atmosphere. After stirring for 12 h, the reaction was quenched with satu-
rated aqueous NaHCO3, and the organic layer was extracted with
CH2Cl2. The combined organic layer was dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by column chromatogra-
phy on silica gel (hexane/EtOAc=10:1 to 8:1) to give cyclic guanidine 12
(8.35 mg, 87%). Spectral data for cyclic guanidine 12: [a]1D8 = +70.9 (c=
2.0 in CHCl3); 1H NMR (400 MHz, CDCl3): d=7.33 (m, 5H), 7.20 (d,
J=8.24 Hz, 2H), 6.82 (d, J=8.7 Hz, 2H), 5.09 (d, J=12.2 Hz, 1H), 5.05
(br, 1H), 4.99 (d, J=12.2 Hz, 1H), 4.45 (d, J=10.3 Hz, 1H), 4.42 (m,
1H), 4.29 (d, J=10.3 Hz, 1H), 4.25 (m, 1H), 3.99 (m, 1H), 3.80 (m,1H),
3.78 (s=3H), 3.71 (dd, J=3.7, 10.1 Hz, 1H), 3.58 (m, 1H), 3.38 (dd, J=
7.3, 9.6 Hz, 1H), 3.18 (d, J=11.0 Hz, 1H), 1.76 (m, 2H), 1.49 (s, 9H),
1.48 (s, 9H), 0.98 ppm (m, 21H); 13C NMR (100 MHz, CDCl3): d=159.6,
159.0, 155.6, 151.6, 135.9, 130.4, 129.6, 128.6, 128.3, 128.2, 113.4, 82.6,
78.1, 74.1, 72.9, 71.4, 68.6, 67.2, 59.4, 55.2, 54.5, 46.0, 32.6, 28.5, 28.1, 17.9,
12.0 ppm; HRMS (ESI): m/z calcd for C43H67N4O9Si: 811.4677 [M+H+];
found: 811.4678.
Isoxazolidine 9: CbzCl (4.01 mL, 28.1 mmol) was added to a solution of
5a-hydroxylamine 8 (10.1 g, 21.6 mmol) and K2CO3 (15.0 g, 108 mmol) in
THF/H2O (1:1, 60 mL) at 08C. After stirring for 10 min at 08C, methanol
(150 mL) was added to the solution, and resulting mixture was warmed
to room temperature. The reaction mixture was then concentrated in
vacuo, and the resulting material was diluted with EtOAc/H2O and ex-
tracted with EtOAc. The combined organic layer was dried over MgSO4,
filtered, and concentrated in vacuo. The residue was purified by column
chromatography on silica gel (hexane/EtOAc=8:1 to 4:1) to give N-Cbz-
Bis(guanidine) 13: TBAF (3.23 g, 12.4 mmol) was added to a solution of
cyclic guanidine 12 (8.35 g, 10.3 mmol) in a solution of THF (50 mL) at
08C. After stirring for 1 h, the reaction mixture was quenched with satu-
rated aqueous NH4Cl and extracted with EtOAc. The organic layer was
dried over MgSO4, filtered, and concentrated in vacuo. The residue was
purified by column chromatography on silica gel (hexane/EtOAc; 3:1 to
EtOAc) to give alcohol (6.27 g, 93%). Pd(OH)2/C (5%, 33.3 mg) was
added to a solution of alcohol (665 mg 1.02 mmol) in methanol (15 mL),
and the mixture was vigorously stirred under an H2 atmosphere (balloon)
at room temperature for 3 h. The reaction mixture was filtered through a
pad of Celite, and the filtrates were concentrated in vacuo to give amine
(538 mg). HgCl2 (331 mg, 1.22 mmol), Et3N (425 mL, 3.05 mmol), and 1,3-
hydroxylamine 9 (10.7 g, 83%). Spectral data for isoxazolidine 9: [a]D16
=
+4.5 (c=2.9 in CHCl3); 1H NMR (400 MHz, CDCl3): d=7.35 (m, 5H),
7.19 (d, J=8.7 Hz, 2H), 6.79 (d, J=8.7 Hz, 2H), 5.20 (d, J=12.4 Hz,
1H), 5.15 (d, J=12.4 Hz, 1H), 4.90 (dd, J=4.6, 5.0 Hz, 1H), 4.44 (d, J=
11.0 Hz, 1H), 4.40 (d, J=11.0 Hz, 1H), 4.34 (m, 1H), 4.12 (dt, J=3.2,
6.0 Hz, 1H), 3.97 (dd, J=2.8, 3.2 Hz, 1H), 3.83 (dd, J=5.5, 11.0 Hz, 1H),
3.77 (s, 3H), 3.63 (dd, J=3.2, 11.0 Hz, 1H), 3.40 (ddd, J=6.0, 12.6,
9.6 Hz, 1H), 3.16 (ddd, J=4.1, 6.9, 12.6 Hz, 1H) 2.02 (m, 1H), 1.75 (m,
1H), 1.01 ppm (m, 21H); 13C NMR (100 MHz, CDCl3): d=159.5, 156.0,
136.0, 129.8, 128.5, 128.2, 113.9, 77.4, 76.3, 75.2, 73.9, 67.8, 67.1, 66.7, 55.2,
33.9, 17.9, 17.8, 11.9 ppm; HRMS (ESI): m/z calcd for C32H48N2O7SiNa:
623.3129 [M+Na+]; found: 623.3137.
bisACTHNUTRGNE(NUG Cbz)-2-methyl-2-thiopseudourea (17) (437 mg, 1.22 mmol) were
added to a solution of amine (538 mg) in DMF (10 mL) at room temper-
ature for 2 h. The reaction mixture was diluted with EtOAc, and the re-
sulting solution was filtered through a pad of Celite. The filtrates were
washed with H2O and brine twice, and the organic layer was dried over
MgSO4, filtered, and concentrated in vacuo. The residue was purified by
column chromatography on Chromatorex NH (hexane/EtOAc=3:2 to
1:2) to give bis(guanidine) 13 (718.2 mg, 85%, two steps). Spectral data
for bis(guanidine) 13: [a]2D1 = +122.8 (c=0.46 in MeOH); 1H NMR
(400 MHz, CDCl3): d=11.59 (s, 1H), 8.97 (d, J=6.41 Hz, 1H), 7.35 (m,
10H), 7.22 (d, J=8.42 Hz, 2H), 6.82 (d, J=8.42 Hz, 2H), 5.48 (brs, 1H),
5.16 (dd, J=11.91, 17.40 Hz, 2H), 5.13 (dd, J=12.36, 45.34 Hz, 2H), 4.62
(m, 1H), 4.51 (t, J=5.50, 1H), 4.39 (dd, J=11.45, 27.93 Hz, 2H), 3.90
(dd, J=7.33, 19.00 Hz, 1H), 3.77 (dd, J=4.12, 9.62 Hz, 1H), 3.73 (s, 3H),
3.68 (m, 1H), 3.47 (dd, J=8.24, 9.62 Hz, 1H), 3.17 (dd, J=4.12, 7.79 Hz,
1H), 3.09 (m, 1H), 2.01 (m, 1H), 1.67 (m, 1H), 1.53 (s, 9H), 1.49 ppm (s,
9H); 13C NMR (100 MHz, CDCl3): d=162.73, 159.61, 155.96, 153.58,
136.42, 134.40, 130.17, 129.56, 129.09, 128.83, 128.67, 128.18, 127.87,
113.93, 83.86, 78.85, 74.93, 73.33, 69.34, 68.79, 68.69, 67.43, 55.36, 54.95,
45.17, 30.13, 28.53, 28.06 ppm; HRMS (ESI): m/z calcd for C43H55N6O11:
831.3928 [M+H+]; found: 831.3896.
Guanidine 11: TiCl3/HCl (20% aq, 14.0 mL, 21.5 mmol; Kanto Chemical)
was added to a solution of N-Cbz hydroxylamine 9 (10.7 g, 17.9 mmol),
NaOAc (4.40 g, 53.7 mmol), and freshly activated Zn powder (3.51 g,
53.7 mmol) in CH2Cl2/MeOH (2:1, 100 mL) at À508C under Ar atmos-
phere. After stirring for 24 h, the reaction was neutralized by an excess
amount of Na2CO3 solid at À508C. Then the dark-green suspension was
warmed to room temperature and vigorously stirred under O2 atmos-
phere for 30 min. The light-gray suspension was extracted with EtOAc,
and the extracts were washed with water and brine. The organic layer
was dried over MgSO4, filtered, and concentrated in vacuo to give dia-
mine 10 (10.8 g). HgCl2 (4.37 mg, 16.1 mmol) was added to a solution of
crude diamine 10 (10.3 g), bisACTHUNRTGNEUNG(Boc)-2-methyl-2-thiopseudourea (16)
(4.68 g, 16.1 mmol), and Et3N (7.48 mL, 53.7 mmol) in DMF (40 mL) at
room temperature under Ar atmosphere. After stirring for 1 h, the reac-
tion mixture was diluted with EtOAc (150 mL) and filtered through a
pad of Celite. The filtrates were washed with H2O and brine. The organic
layer was dried over MgSO4, filtered, and concentrated in vacuo. The res-
idue was purified by column chromatography on silica gel (hexane/
EtOAc=15:1 to 6:1) to give guanidine 11 (12.1 g, 82%, two steps). Spec-
tral data for guanidine 11: [a]1D7 = +0.6 (c=2.6 in CHCl3); 1H NMR
(500 MHz, CDCl3): d=10.31 (s, 1H), 7.33 (m, 5H), 7.21 (d, J=8.6 Hz,
2H), 6.83 (d, J=8.6 Hz, 2H), 5.69 (br, 1H), 5.44 (d, J=9.7 Hz, 1H), 5.09
(d, J=12.0 Hz, 1H), 5.04 (d, J=12.0 Hz, 1H), 4.47 (d, J=2.9 Hz, 1H),
4.42 (d, J=12.0 Hz, 1H), 4.39 (d, J=9.7 Hz, 1H), 4.33 (d, J=12.0 Hz,
Mono-Cbz-guanidine 14: NaOMe (9.0 mg, 0.167 mmol) was added to a
solution of bis(guanidine) 13 (139 mg, 0.167 mmol) in THF/MeOH (1:1,
4 mL) at 08C under an N2 atmosphere, and the mixture was stirred for
5 h. Saturated aqueous NH4Cl was added to the reaction mixture, and or-
ganic layer was extracted with EtOAc. The extracts were washed with
brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue
was purified by column chromatography on Chromatorex NH (hexane/
Chem. Eur. J. 2011, 17, 12144 – 12152
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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12149

K. Nagasawa et al.
EtOAc=3:1 to 1:2) to give mono-Cbz-guanidine 14 (75.7 mg, 65%).
Spectral data for mono-Cbz-guanidine 14: [a]1D4 = +202.3 (c=1.0 in
MeOH); 1H NMR (500 MHz, CD3OD): d=7.17 (m, 5H), 7.08 (d, J=
8.70 Hz, 2H), 6.67 (d, J=8.70 Hz, 2H), 4.98 (dd, J=12.82, 39.38 Hz,
2H), 4.32 (d, J=10.53 Hz, 1H), 4.19 (m, 2H), 4.10 (d, J=3.21 Hz, 1H),
3.67 (m, 3H), 3.63 (s, 3H), 3.41 (m, 1H), 3.20 (m, 2H), 1.75 (m, 1H),
1.61 (m, 1H), 1.38 (m, 18H); 13C NMR (125 MHz, CD3OD): d=162.07,
160.11, 159.89, 152.55, 138.36, 129.96, 128.86, 128.17, 128.03, 113.94, 83.39,
78.93, 73.32, 73.02, 71.32, 67.93, 60.84, 55.11, 49.34, 46.76, 32.09, 28.42,
27.87 ppm; HRMS (ESI): m/z calcd for C35H49N6O9: 697.3561 [M+H+];
found: 697.3557.
Carbamate 20: Trichloroacetyl isocyanate (19 mL, 0.16 mmol) was added
to a solution of alcohol 19 (60 mg, 0.080 mmol) in CH2Cl2 (1 mL) at 08C
under an N2 atmosphere. The reaction was stirred for 1 h. Then K2CO3
and methanol were added at 08C and warmed to room temperature. The
reaction mixture was stirred for 3 h, quenched with H2O, and extracted
with EtOAc twice. The combined organic layer was dried over MgSO4,
filtered, and concentrated in vacuo. The residue was purified by prepara-
tive TLC (EtOAc, four times) to give carbamate 20 (16.6 mg, 34%).
Spectral data for carbamate 20: [a]2D2 = +52.3 (c=0.32 in MeOH);
1H NMR (400 MHz, CD3OD): d=7.30 (m, 5H), 5.04 (dd, J=3.66,
12.82 Hz, 2H), 4.25 (m, 2H), 3.58 (m, 4H), 3.28 (m, 2H), 1.84 (m, 1H),
1.69 (m, 1H), 1.41 ppm (m, 18H); 13C NMR (100 MHz, CD3OD): d=
162.6, 160.2, 159.1, 154.6, 152.8, 138.8, 129.4, 128.6, 84.2, 79.7, 73.3, 68.3,
64.8, 59.8, 55.6, 47.2, 45.5, 32.8, 28.8, 28.7 ppm; HRMS (ESI): m/z calcd
for C28H42N7O9: 620.3044 [M+H+]; found: 620.3074.
Fused-type bicyclic guanidine 15: IBX (117 mg, 0.416 mmol) was added
to a solution of mono-Cbz-guanidine 14 (48 mg, 0.0694 mmol) in DMSO
(1 mL), and the resulting suspension was stirred for 10 min at room tem-
perature. Then the reaction mixture was warmed to 708C and stirred for
6 h. The reaction was quenched with 10% aqueous Na2S2O3 and saturat-
ed aqueous NaHCO3, and the solution was diluted with EtOAc. The or-
ganic layer was dried over MgSO4, filtered, and concentrated in vacuo.
The residue was purified by column chromatography on Chromatorex
NH (hexane/EtOAc; 1:2 to pure EtOAc) to give fused-type bicyclic gua-
nidine 15 (11.8 mg, 24%). Spectral data for fused-type bicyclic guanidine
Fused-type bicyclic guanidine 21: IBX (62 mg, 0.223 mmol) was added to
a solution of 20 (23 mg, 0.0371 mmol) in DMSO (1 mL), and the resulting
suspension was stirred for 10 min at room temperature. Then, the reac-
tion mixture was warmed to 708C and stirred for 6 h. The reaction was
quenched with 10% aqueous Na2S2O3 and saturated aqueous NaHCO3,
and the solution was diluted with EtOAc. The organic layer was dried
over MgSO4, filtered, and concentrated in vacuo. The residue was puri-
fied by column chromatography on Chromatorex NH (hexane/EtOAc;
1:8 to EtOAc/MeOH; 10:1) to give 21 (4.6 mg, 20%). Spectral data for
fused-type bicyclic guanidine 21: [a]2D3 =À7.4 (c=0.1 in MeOH);
1H NMR (400 MHz, CD3OD): d=7.34 (m, 5H), 5.02 (d, J=1.37 Hz,
2H), 4.53 (dd, J=5.5, 11.9 Hz, 2H), 4.28 (dd, J=8.24, 11.91 Hz, 2H),
4.02 (d, J=3.96 Hz, 1H), 3.63 (m, 3H), 2.37 (m, 1H), 2.19 (m, 1H), 1.48
(m, 9H), 1.44 ppm (m, 9H); 13C NMR (100 MHz, CD3OD): d=164.5,
160.6, 159.3, 153.0, 149.5, 138.6, 130.0, 129.5, 86.9, 86.2, 83.9, 80.8, 68.1,
65.3, 59.8, 45.8, 33.5, 29.3, 28.9 ppm; HRMS (ESI): m/z calcd for
C28H40N7O10: 634.2836 [M+H+]; found: 634.2884.
1
15: [a]1D5 = +16.9 (c=0.47 in MeOH); H NMR (500 MHz, CD3OD): d=
7.20 (m, 7H), 6.79 (d, J=8.59 Hz, 2H), 4.94 (d, J=1.72 Hz, 2H), 4.61 (m,
1H), 4.51 (d, J=10.88 Hz, 1H), 4.39 (d, J=10.88 Hz, 1H), 4.00 (d, J=
1.72 Hz, 1H), 3.90 (dd, J=7.45, 9.74 Hz, 1H), 3.72 (m, 1H), 3.68 (s, 3H),
3.52 (m, 1H), 3.44 (m, 1H), 2.27 (m, 1H), 2.08 (m, 1H), 1.34 ppm (m,
18H); 13C NMR (125 MHz, CD3OD): d=160.95, 160.37, 153.10, 150.41,
138.66, 131.02, 130.75, 129.38, 128.86, 114.79, 86.42, 85.32, 82.96, 80.05,
74.15, 69.82, 67.48, 59.99, 55.69, 45.08, 33.10, 28.77, 28.27 ppm; HRMS
(ESI): m/z calcd for C35H47N6O10: 711.3353 [M+H+]; found: 711.3400.
(À)-FD-dcSTX (4b): Pd(OH)2/C (2.5 mg) was added to a solution of 15
(4.8 mg, 0.00676 mmol) in methanol (500 mL), and the suspension was
vigorously stirred under an H2 atmosphere (balloon) at room tempera-
ture. After 30 min, the reaction mixture was filtered through a pad of
Celite. The filtrates were concentrated in vacuo to give bis-Boc-bis-
aminal (3.1 mg). HCl (3n, 500 mL) was added to the resulting bis-Boc-
bis-aminal at 08C. After stirring for 1 h at room temperature, the reac-
tion mixture was concentrated in vacuo to give FD-dcSTX (4b) as a
2HCl salt (1.7 mg, 78%). Spectral data for FD-dcSTX (4b): [a]1D5 =À63.9
(À)-FD-STX (4a): Pd(OH)2/C (3.0 mg) was added to a solution of 21
(7.0 mg, 0.011 mmol) in methanol (600 mL) under an N2 atmosphere, and
the suspension was vigorously stirred under an H2 atmosphere (balloon)
at room temperature. After 7 h, the reaction mixture was filtered through
a pad of Celite. The filtrates were concentrated in vacuo to give bis-Boc-
bis-aminal (5.3 mg). TFA (500 mL) was added to the resulting bis-Boc-
bis-aminal (5.3 mg) in CH2Cl2 (1 mL) at room temperature. Methanol
was added to the reaction mixture, and it was concentrated in vacuo to
give FD-STX (4a) as a 2TFA salt (4.8 mg, 82%). Spectral data for FD-
STX (4a): [a]2D3 =À34.9 (c=0.32 in MeOH); 1H NMR (400 MHz, D2O):
d=4.39 (dd, J=8.70, 10.99 Hz, 1H), 4.17 (dd, J=4.58, 10.99 Hz, 1H),
4.05 (s, 1H), 3.95 (dd, J=4.58, 8.70 Hz, 1H), 3.68 (t, J=10.7 Hz, 1H),
3.45 (m, 1H), 2.47 (m, 1H), 2.33 ppm (dd, J=7.79, 13.28 Hz, 1H);
13C NMR (100 MHz, 4% CD3CO2D in D2O): d=159.1, 153.0, 152.1, 85.6,
81.7, 65.5, 52.5, 45.3, 43.2, 31.4 ppm; HRMS (ESI): m/z calcd for
C10H18N7O4: 300.1420 [M+H+]; found: 300.1390.
1
(c=0.11 in MeOH); H NMR (400 MHz, D2O): d=4.06 (s, 1H), 3.82 (m,
3H), 3.70 (t, J=10.07 Hz, 1H), 3.48 (dd, J=10.07, 17.40 Hz, 2H), 2.50
(m, 1H), 2.34 ppm (m, 1H); 13C NMR (100 MHz, 4% CD3CO2D in
D2O): d=152.91, 152.16, 85.53, 81.49, 62.52, 55.24, 45.78, 43.00,
31.44 ppm; HRMS (ESI): m/z calcd for C9H17N6O3: 257.1362 [M+H+];
found: 257.1319.
Alcohol 19: Ac2O (2 mL) and pyridine (2 mL) were added to bis-guani-
dine 13 (230 mg, 0.277 mmol) at room temperature. The reaction mixture
was warmed to 508C and stirred for a further 3 h, then cooled to room
temperature, quenched with toluene, and concentrated in vacuo. The re-
sulting residue was purified by column chromatography on Chromatorex
NH (hexane/EtOAc; 6:1 to 3:1) to give acetate (245 mg, 96%). DDQ
(À)-FD-doSTX (4c): TFA (0.5 mL) was added to a solution of (À)-3
(12.1 mg, 0.0199 mmol) in CH2Cl2 (1 mL) at 08C. After being stirred for
1 h at room temperature, the reaction mixture was concentrated under
reduced pressure to give bis-Cbz-bis-aminal. Pd/C (catalytic amount) was
added to a solution of bis-Cbz-bis-aminal in methanol (1 mL), and the re-
action mixture was stirred at room temperature under an atmosphere of
hydrogen (balloon). After 4 h, the reaction mixture was filtered through
a pad of Celite, and the filtrates were acidified with 10% HCl and con-
centrated in vacuo to give FD-doSTX (4c) (5.7 mg, 92%, two steps).
Spectral data for 4c: [a]2D0 =À62.7 (c=0.9 in MeOH); 1H NMR
(400 MHz, D2O): d=3.88 (d, J=1.0 Hz, 1H), 3.75 (dd, J=1.0, 7.1 Hz,
1H), 3.63 (dd, J=9.6, 9.6 Hz, 1H), 3.42 (ddd, J=7.6, 9.6, 10.1 Hz, 1H),
2.43 (ddd, J=9.6, 10.1, 13.6 Hz, 1H), 2.27 (dd, J=7.6, 13.6 Hz, 1H),
1.38 ppm (d, J=7.1 Hz, 3H); 13C NMR (100 MHz, CD3OD): d = 152.4,
151.4, 85.2, 81.8, 49.4, 48.4, 42.6, 30.9, 19.3 ppm; HRMS (ESI): m/z calcd
for C9H17N6O2: 241.1413 [M+H]; found: 241.1434.
(377 mg, 1.66 mmol) was added to
a solution of acetate (144 mg,
0.166 mmol) in CH2Cl2/H2O (2:1, 4.5 mL) at room temperature. The reac-
tion mixture was stirred for 5 h, saturated aqueous NaHCO3 was added,
and the resulting solution was extracted with EtOAc three times. The
combined organic layer was dried over MgSO4, filtered, and concentrated
in vacuo. The residue was purified by column chromatography on Chro-
matorex NH (hexane/EtOAc; 6:1 to 1:1) to give alcohol 19 (117 mg,
94%). Spectral data for alcohol 19: [a]2D3 = +171.7 (c=0.3 in MeOH);
1H NMR (400 MHz, CDCl3): d=11.76 (s, 1H), 8.65 (d, J=8.70 Hz, 1H),
7.36 (m, 10H), 5.23 (dd, J=11.91, 20.15 Hz, 2H), 5.11 (dd, J=12.82,
16.94 Hz, 2H), 4.48 (m, 1H), 4.08 (m, 1H), 4.00 (d, J=11.91 Hz, 1H),
3.90 (t, J=8.70, 1H), 3.65 (m, 3H), 3.52 (t, J=9.63 Hz, 1H), 2.05 (m,
5H), 1.49 ppm (m, 18H); 13C NMR (100 MHz, CDCl3): d=170.83,
163.55, 156.44, 153.82, 136.63, 134.44, 128.82, 128.59, 128.52, 127.87, 83.51,
80.11, 75.41, 68.63, 67.33, 64.91, 51.36, 46.46, 28.77, 28.38, 28.20,
21.16 ppm; HRMS (ESI): m/z calcd for C37H49N6O11: 753.3459 [M+H+];
found: 753.3496.
Cell culture: HEK293 was grown in Dulbeccoꢁs modified Eagleꢁs
medium (DMEM; Life Technologies, Grand Island, NY) supplemented
with 10% fetal bovine serum (30 units; BioWhittaker, Walkersville, MD)
and penicillin-streptomycin (30 mgmLÀ1; Gibco, Grand Island, NY), re-
12150
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 12144 – 12152

Skeletal Analogues of Saxitoxin Derivatives
FULL PAPER
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Inhibitory activity to cytotoxicity of ouabain and veratridine: Two daysꢁ
worth of cultured cells (Neuro-2a) were harvested with a trypsin/EDTA
solution (0.5%/0.2%) (Sigma) in phosphate buffer saline (PBS), and
cells were suspended at a density of 5ꢂ105 cells mLÀ1 in growth medium.
This suspension (100 mL) was plated into each well of 96-well plates and
cultured at 378C for 24 h. The test medium (100 mL), which contained
FD-STXs at various concentrations with 1:3 serial dilutions, and ouabain
(200 mm) (Sigma) and veratridine (100 mm) (Sigma) were poured into
each well. After incubation at 378C for 24 h, the number of viable cells
was determined using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan).
Cell Counting Kit-8 solution (5 mL) was added to each well, and the mix-
ture was incubated at 378C for 3 h, and then absorbance at 450/655 nm
was measured using a microplate reader (Model 550, Bio-Rad, Hercules,
CA). Three wells were used for one concentration of FD-STXs, and the
same experiments were performed three times for each compound. The
cell viability of the well that contained the test medium in the presence
of only ouabain and veratridine (in the absence of FD-STXs) was taken
as 0%, and that of the well that contained the medium in the absence of
all toxins was taken as 100%. The IC50 were determined from the Hill
plot.
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City, CA, USA); more than 80% of the series resistance was compensat-
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Acknowledgements
Financial support for this work was provided in part by a grant-in-aid for
scientific research from JSPS (no. 203130). O.I. is grateful for financial
support from JSPS in the form of a Pre-doctoral Fellowship for Young
Scientists.
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[21] See the Supporting Information for calculation of IC50 values.
[22] The voltage that provided a peak current (maximum active poten-
tial) for rNaV1.4 and rNaV1.5 was À10 mV.
[23] The holding potentials of NaV1.4 and NaV1.5 are À100 and
À120 mV, respectively.
[24] Under the same conditions, (+)-dc-STX (1b) showed inhibitory ac-
tivities against NaV1.4 and NaV1.5 with IC50 values of (29Æ1.8) nm
and (1.2Æ0.1) mm, respectively. Compound (+)-dc-STX (1b) inter-
acted reversibly with both channels.
[25] The IC50 values were determined according to the following equa-
tion; IC50 =[inhibitor 4](Iinhibitor4/Imax)/(1À(Iinhibitor4/Imax)), in which Imax
is the peak current under control conditions and Iinhibitor4 is the equi-
librium peak current in the presence of an inhibitor 4: J. L. Penzotti,
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[16] The structure of (+)-STX (1a) and compound 3, which is a synthetic
intermediate for FD-doSTX (4c), were superimposed by MOE.
Please see Figure S1 in the Supporting Information.
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Received: April 7, 2011
Revised: July 20, 2011
Published online: September 16, 2011
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12152
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