Polyacetylenes from Sardinian Oenanthe fistulosa: A molecular clue to risus sardonicus

Article abstract of DOI:10.1021/np8007717

An investigation of Oenanthe fistulosa from Sardinia afforded oenanthotoxin (1a) and dihydrooenanthotoxin (1b) from the roots and the diacetylenic epoxydiol 2 from the seeds. The absolute configuration of 1a and 1b was established as R by the modified Mos

Full text of DOI:10.1021/np8007717

962
J. Nat. Prod. 2009, 72, 962–965
Polyacetylenes from Sardinian Oenanthe fistulosa: A Molecular Clue to risus sardonicus
Giovanni Appendino,*,†,‡ Federica Pollastro,†,‡ Luisella Verotta,§ Mauro Ballero,‡ Adriana Romano,⊥ Paulina Wyrembek,¶
Katarzyna Szczuraszek,¶ Jerzy W. Mozrzymas,*,¶ and Orazio Taglialatela-Scafati*,⊥
Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, UniVersita` del Piemonte Orientale, Viale Ferrucci 33,
28100 NoVara, Italy, Consorzio per lo Studio dei Metaboliti Secondari (COSMESE), Viale S. Ignazio 13, 09123 Cagliari, Italy, Dipartimento di
Chimica Organica e Industriale, UniVersita` di Milano, Via Venezian 21, 20133 Milano, Italy, Dipartimento di Chimica delle Sostanze Naturali,
UniVersita` di Napoli Federico II, Via Montesano 49, 80131 Napoli, Italy, and Laboratory of Neuroscience, Department of Biophysics,
Wroclaw Medical UniVersity, ul. Chalubinskiego 3, 50-368 Wroclaw, Poland
ReceiVed December 4, 2008
An investigation of Oenanthe fistulosa from Sardinia afforded oenanthotoxin (1a) and dihydrooenanthotoxin (1b) from
the roots and the diacetylenic epoxydiol 2 from the seeds. The absolute configuration of 1a and 1b was established as
R by the modified Mosher’s method, and the structure of 2 by chemical correlation with (+)-(3R,8S)-falcarindiol.
Oenanthotoxin (1a) and dihydrooenanthotoxin (1b) were found to potently block GABAergic responses, providing a
molecular rationale for the symptoms of poisoning from water-dropwort (Oenanthe crocata) and related plants. These
observations bear relevance for a series of historical and ethnopharmacological observations on the identification of the
Sardonic herb and the molecular details of the facial muscular contraction caused by its ingestion (risus sardonicus).
Plants from the genus Oenanthe (Umbelliferae family) are among
the most poisonous species of the European flora.1 They bear a
general resemblance to parsnip and carrot and are still a cause of
often fatal human poisonings.1 Sardinian Oenanthe species have a
special ethnopharmacological relevance, being considered the most
likely candidate for the (in)famous sardonic herb, a neurotoxic plant
used in pre-Roman Sardinia for the ritual killing of elderly people.2
According to the ancient historians, elderly people unable to support
themselves were intoxicated with the sardonic herb and then killed
by dropping from a high rock or by beating to death.2 The facial
muscular contraction induced by the sardonic herb mimicked a
smile, and the expression risus sardonicus (sardonic smile) to
indicate a sinister smile is well documented in the Latin and Greek
literature and in most modern European languages. It even found
its way into the mainstream medical lingo as the hallmark of
lockjaw (trismus), the spasm of the muscles of mastication.3
The poisonous constituents of Oenanthe species are a series of
polyacetylenic alcohols exemplified by oenanthotoxin (1a).4 Sur-
prisingly, the absolute configuration at the stereogenic carbon
(C-14) of oenanthotoxin is still unknown, and little information on
the molecular details of its neurotoxic activity has been reported.
As part of a study on poisonous Sardinian plants,5 we have
investigated O. fistulosa L. (tubular water-dropwort), a species that,
despite its very broad distribution in Europe, had so far been
overlooked in terms of phytochemical studies. Large amounts of
the diacetylene epoxydiol 2 were obtained from the seeds, while
the roots gave, along with the widespread acetylene falcarindiol
(3), oenanthotoxin (1a) and dihydroenanthotoxin (1b). The absolute
configuration of 1a and 1b was clarified by application of the
modified Mosher method, while the activity of oenanthotoxin was
profiled against GABA receptors, a target involved in the action
of many convulsivant toxins of plant origin.6
amounts (ca. 0.80% of dried plant material) of (+)-(3R, 8S)-
falcarindiol (3)7 and a mixture of oenanthotoxin (1a) and its
dihydroderivative (1b) (0.070%).4 Compounds 1a and 1b were
easily separated by preparative HPLC and identified by comparison
with authentic samples of (+)-oenanthotoxin and (+)-dihydrooe-
nanthotoxin obtained, in over 10-fold higher yield, from the roots
of O. crocata, a more common species in Sardinia (see Experi-
mental Section). A complete assignment of the 1H NMR resonances
of both 1a and 1b is reported in the Experimental Section.
Oenanthotoxin (1a) is isomeric with cicutoxin (4) from water
hemlock (Cicuta Virosa L.), with the only difference between these
C-17 acetylenic toxins being the position of a single double bond,
located next to the secondary oxymethine in cicutoxin and adjacent
to the primary hydroxyl in oenanthotoxin.4b The absolute config-
uration at C-14 of cicutoxin was established as R by application of
a CD exciton chirality method on its p-methoxybenzoate, taking
advantage of the allylic nature of the stereogenic center, and was
confirmed by application of the Mosher method.8 Since the
stereogenic center of oenanthotoxin is not allylic, the Mosher
method9 was used to assign its absolute configuration.
To achieve this aim, two aliquots of oenanthotoxin (1a) were
dissolved in dry pyridine and allowed to react overnight with DMAP
and (R)- or (S)-MTPA chloride, respectively, affording the (S)- and
(R)-MTPA diesters 5a and 5b, respectively. Analysis of the ∆δ(S-R)
values of the protons neighboring the oxygenated methine according
An acetone extract from the underground parts of O. fistulosa
was fractionated by gravity column chromatography to afford large
* To whom correspondence should be addressed. Tel: +39 0321
375744 (G.A.); +48 71 7841550 (J.W.M.); +39 081 678509 (O.T.-S.). Fax:
+39 0321 375621 (G.A.); +48 71 784 1399 (J.W.M.); +39 081 678552
(O.T.-S.). E-mail: appendino@pharm.unipmn.it (G.A.); mozrzy@
biofiz.am.wroc.pl (J.W.M.); scatagli@unina.it (O.T.S.).
† Universita` del Piemonte Orientale.
‡ Consorzio per lo Studio dei Metaboliti Secondari.
§ Universita` di Milano.
⊥ Universita` di Napoli Federico II.
¶ Wroclaw Medical University.
10.1021/np8007717 CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/26/2009

Notes
Journal of Natural Products, 2009, Vol. 72, No. 5 963
convulsant site of GABAA receptors and causing death by inducing
convulsions and respiratory paralysis.13 In addition, oenanthotoxin has
also been reported to dramatically affect cationic (calcium and sodium)
currents in excitable membranes.14 We found that both oenanthotoxin
and dihydrooenanthotoxin could potently block the GABAergic
responses in neuronal cell cultures, with an EC50 value in the low
micromolar range for both compounds. Currents elicited by exogenous
GABA were recorded in the whole-cell configuration by rapid
application of 3 µM GABA at the membrane voltage of -40 mV,
and a typical GABA-evoked response, recorded in these conditions,
is presented in Figure 2 (left). The effect of 1a and 1b on current
amplitude was assessed by dividing the current amplitude measured
in the presence of this drug by the amplitude value in control conditions
measured from the same cell. Oenanthotoxin (1a) clearly inhibited the
GABAergic responses in a dose-dependent manner (Figure 2A). A
tentative fitting of the logistic equation (y ) 1/(1 + (c/Ec50)h)) to the
amplitude dose-dependence for oenanthotoxin shown in Figure 2B
yielded a EC50 value of 1.39 µM (h ) 0.59). Dihydrooenanthotoxin
(1b) exhibited a similar inhibitory activity on currents elicited by 3
µM GABA, but was slightly more potent (EC50 ) 0.835 µM, h )
0.58). The effect of both test compounds was reversible, although at
least 10 min of wash was required to restore the control responses.
Compound 1b can be viewed both as 2,3-dihydrooenanthotoxin and
as 12,13-dihydrocicutoxin (cf. 1a and 4), showing that, in polyacety-
lenic toxins, the presence of allylic double bonds is redundant for
activity and that the conjugated system required for the activity is
shorter than previously postulated,13 encompassing four, and not five,
unsaturations.
Unlike other plant toxins, the convulsant polyacetylenes from
water dropwort and related plant do not evoke unpleasant taste
(bitter) or chemesthetic (burning) sensations, and the roots of O.
crocata, an exceedingly poisonous plant, have a paradoxical
sweetish and pleasant taste and odor.15 The large concentration of
falcarindiol, a bitter compound,16 and the lower contents of
polyacetylene toxins in O. fistulosa when compared to O. crocata
could presumably underlie the observation that the former species
has not yet been associated with human or animal poisoning. The
name Oenanthe signifies “wine flower”, because the plant produces
a state of stupefaction similar to drunkenness.1,2 This, as well as
locked jaws (risus sardonicus), has been documented in human
poisoning from O. crocata,1,2 and there is little doubt that herba
sardonica of the ancient medical literature should be identified with
O. crocata,2 a plant that, within the Mediterranean area, is common
only in Sardinia.2 The results of our investigation provide a further
rationale for this identification, proposing a molecular mechanism
for the risus sardonicus described by the ancient authors.
Figure 1. Application of the modified Mosher’s method for
secondary alcohols on the MTPA esters of oenanthotoxin (5a and
5b) and dihydrooenanthotoxin (6a and 6b). ∆δ(δS-δR) values are
given in ppm.
to the Mosher model9 (Figure 1) allowed the assignment of the R
configuration at C-14 of 1a. The same methodology was applied to
dihydrooenanthotoxin (1b), which, by analysis of the ∆δ(S-R) values
between its (S)- and (R)-MTPA diesters 6a and 6b (Figure 1), turned
out to have the same R configuration at C-14. In both cases,
concomitant MTPA esterification at C-1 did not exert any influence
on the anisotropic effect of the phenyl group of the MTPA moiety
bound to the C-14, located 13 carbons away from the primary hydroxyl.
The seeds of O. fistulosa were totally devoid of oenanthotoxin-like
polyacetylenes and contained rather large amounts of the diacetylenic
epoxydiol 2. The structure elucidation of this compound (C17H24O3,
HRMS) was helped greatly by comparison with falcarindiol (3),7 since
the two compounds showed similar spectroscopic features.7 Thus, 2
differed from 3 only in the epoxidation of the disubstituted double
bond, as evident from the replacement of the two mutually coupled
olefin protons of 3 with two oxymethine signals [δ 3.06 (dd, J ) 7.1,
4.0 Hz, H-9) and 2.80 (dt, J ) 5.9, 4.0 Hz, H-10]. Corresponding
13
changes were observed in the C NMR spectrum (upfield shift of
the C-9 and C-10 methines, now resonating at δ 57.9 and 57.4,
1
respectively). Full assignment of H and 13C NMR resonances of 2
was achieved through analysis of 2D NMR (COSY, HSQC, and
gHMBC) data, which confirmed that 2 is an epoxidized derivative of
falcarindiol. To further confirm this assignment and establish the
configuration of the epoxide ring, the reaction of (+)-(3R,8S)-
falcarindiol (3)7 with peracids was investigated. Enines can be
chemoselectively epoxidized, and treatment of falcarindiol with meta-
chloroperoxybenzoic acid (MCPBA) under controlled conditions
(monitoring of the conversion by 1H NMR analysis, see Experimental
Section) gave the epoxide 2 in satisfactory (66%) yield and as the
only reaction product. This was identical in terms of both spectroscopic
properties and optical rotation to the natural product isolated from O.
fistulosa. Prolonged reaction times or the addition of NaHCO3 to buffer
the acidity of the medium led to extensive degradation of the reaction
product. The epoxidation of allylic alcohols is a syn addition, and the
peroxy acid is directed by hydrogen bonding to the allylic hydroxyl,
which, in the case of secondary alcohols, reacts in the conformation
that minimizes allylic strain (A(1,3)-strain) between the alkyl group
bound to the oxymethine and the distal substituent of the adjacent
double bond (C-7 and C-11, respectively, in the case of the epoxidation
of 3).10 Taking into account that the stereogenic double bond of
falcarindiol has a cis configuration, these considerations lead to a
9S,10R configuration for the epoxide ring of 2.11 A compound with
the planar structure of 2 and spectroscopic features compatible with
those of 2 was described from Panax quinquefolium.12 However, the
optical rotation was different [+ 107 (MeOH) for the epoxydiol from
O. fistulosa and +87 (MeOH) for that from P. quinquefolium], and in
the absence of an authentic sample, the identity of the two compounds
could not be established.
Neurotoxins have emerged as powerful neurochemical tools,17,18
and the scarce attention given so far to the C-17 neurotoxic ace-
tylenes can best be explained by their chemical instability, especially
in the highly purified form required for biological studies. Thus,
oenanthotoxin (1a) and dihydrooenanthotoxin (1b), just like many
other polyacetylenes, easily turned into highly colored and insoluble
polymeric materials upon storage, even at low temperature (-18
°C). To overcome this problem, we have developed a flash
chromatography protocol to separate oenanthotoxin (1a) and
dihydroenanthotoxin (1b) from a crude extract of O. crocata and
found that these compounds, while being highly unstable in the
solid state, can be conveniently stored for at least 6 months in
benzene or DMSO solution at 4 °C (see Experimental Section).
This might foster interest in this fascinating class of compounds,
with its unique capacity of dramatically perturbing calcium and
sodium ionic currents in excitable membranes14 and specifically
interacting with ligand-gated ion channels like the GABAA receptor.
Experimental Section
General Experimental Procedures. Optical rotations (CHCl3) were
Polyacetylene neurotoxins are believed to act like biological
analogues of the sesquiterpene lactone picrotoxin, binding to the
measured at 589 nm on a Perkin-Elmer 192 polarimeter equipped with
1
a sodium lamp (λ ) 589 nm) and a 10 cm microcell. H (500 MHz)

964 Journal of Natural Products, 2009, Vol. 72, No. 5
Notes
Figure 2. Oenanthotoxin (1a) and dihydrooenanthotoxin (1b) inhibit GABA-evoked responses in a dose-dependent manner. (A) Typical
current traces evoked by rapid application of 3 µM GABA at -40 mV in control conditions (left), in the presence of 10 µM 1a (middle),
and in the presence of 10 µM 1b (right). (B) Dose-dependent effect of 1a on GABA-evoked current amplitude relative value. (C) Dose-
dependent effect of 1b. Each data point in B and C was calculated from at least 3 values. Asterisks above the bars indicate statistically
significant difference.
and 13C (125 MHz) NMR spectra were measured on a Varian INOVA
spectrometer. Chemical shifts were referenced to the residual solvent
signal (CDCl3: δH 7.26, δC 77.0). Homonuclear 1H connectivities were
determined by the COSY experiment. One-bond heteronuclear 1H-13C
connectivities were determined with the HSQC experiment. Two- and
three-bond 1H-13C connectivities were determined by HMBC experi-
nathotoxin (1b), respectively. These compounds were obtained as colorless
foams and could not be stored in this form. However, solutions in
hydrocarbon solvents (benzene, toluene) or DMSO could be stored for at
least 6 months at 4 °C without any apparent (1H NMR analysis) degradation
or development of a reddish color.
Oenanthotoxin (1a): amorphous foam; [R]D25 +34 (c 0.5, CHCl3);
1H NMR (500 MHz, CDCl3) δ 6.71 (1H, dd, J ) 15.5, 11.0 Hz, H-9),
6.39 (1H, dt, J ) 15.0, 5.0 Hz, H-2); 6.12 (1H, dd, J ) 15.0, 11.0 Hz,
H-10), 5.87 (1H, overlapped H-3), 5.86 (1H, overlapped, H-11); 5.57
(1H, d, J ) 15.5 Hz, H-8), 4.25 (2H, d, J ) 5.0 Hz H2-1), 3.60 (1H,
m, H-14), 2.30 (1H, m, H-12a), 2.20 (1H, m, H-12b), 1.78 (1H, m,
H-13a), 1.50 (1H, overlapped, H-13b), 1.49 (1H, overlapped, H-15a),
1.40 (1H, m, H-15b), 1.30 (2H, m, H2-16), 0.92 (3H, d, J ) 7.3, Hz
H3-17); ESIMS (positive ion) m/z 281 [M + Na]+.
1
ments optimized for a 2,3J of 7 Hz. Through-space H connectivities
were evidenced using a ROESY experiment with a mixing time of 500
1
ms. Two- and three-bond H-13C connectivities were determined by
gradient 2D HMBC experiments optimized for a 2,3J of 9 Hz. Low-
and high-resolution EIMS spectra (70 eV) were performed on a VG
Prospec (Fisons) mass spectrometer. ESIMS spectra were performed
on a LCQ Finnigan MAT mass spectrometer. Silica gel 60 (70-230
mesh) was used for gravity column chromatography. Reactions were
monitored by TLC on Merck 60 F254 (0.25 mm) plates, which were
visualized by UV inspection and/or staining with 5% H2SO4 in ethanol
and heating. Organic phases were dried with Na2SO4 before evaporation.
Plant Material. Oenanthe fistulosa was collected near San Basilio
(CA, Sardinia, Italy) on September 14, 2007. The plant was identified
by M.B., and a voucher specimen (171007) is held at the University
of Cagliari. O. crocata was collected near Bonacardo (Abbasanta, NU,
Italy) in May 8, 2007, and a voucher specimen (OCR080506BO) is
held at the University of Cagliari.
Dihydrooenanthotoxin (1b): amorphous foam; 1H NMR (500 MHz,
CDCl3) δ 6.65 (1H, dd, J ) 15.5, 10.6 Hz, H-9), 6.12 (1H, dd, J )
15.0, 11.0 Hz, H-10), 5.84 (1H, dt, J ) 15.0, 5.0 Hz, H-11), 5.50 (1H,
d, J ) 15.5 Hz, H-8), 3.77 (2H, t, J ) 5.0 Hz, H2-1), 3.60 (1H, m,
H-14), 2.45 (2H, t, J ) 5.0 Hz, H2-3), 2.30 (1H, m, H-12a), 2.21
(1H, m, H-12b), 1.79 (2H, t, J ) 5.0 Hz, H2-2), 1.75 (1H, m, H-13a),
1.50 (1H, overlapped, H-13b), 1.49 (1H, overlapped, H-15a), 1.40 (1H,
m, H-13b), 1.30 (2H, m, H2-16), 0.93 (3H, d, J ) 7.3 Hz, H3-17);
ESIMS (positive ion): m/z 283 [M + Na]+.
Extraction and Isolation. (A) Powdered, dried underground parts of
O. fistulosa (608 g) were extracted with acetone (2 × 2 L) at room
temperature, affording 7.94 g of a dark oil, which was fractionated by
gravity column chromatography on silica gel (n-hexane-EtOAc, 9:1 to
6:4, as solvent gradient system). Fractions eluted with n-hexane-EtOAc
(9:1) were further purified by HPLC (n-hexane-EtOAc, 85:15) to yield
falcarindiol (3, 344 mg). Fractions eluted with n-hexane-EtOAc (6:4) were
further purified by HPLC (n-hexane-EtOAc, 65:35) to yield oenathotoxin
(1a, 68 mg) and dihydrooenanthotoxin (1b, 67 mg). (B) Powdered, dried
seeds of O. fistulosa (379 g) were extracted with acetone (2 × 2 L) at rt,
affording 21.3 g of a yellowish oil, which was fractionated by gravity
column chromatography on silica gel (n-hexane-EtOAc, 9:1 to 6:4, as
solvent gradient system). Fractions eluted with n-hexane-EtOAc (7:3) were
further purified by HPLC (n-hexane-EtOAc, 55:45) to yield epoxyfal-
carindiol (2, 432 mg). (C) Dried underground parts of O. crocata (95 g)
were extracted with acetone (2 × 1 L) to afford 3.1 g of a brownish oil,
which was purified with the Biotage SP1 system with a silica gel (10 g)
FLASH 12+M column and a n-hexane-EtOAc gradient of increasing
polarities (flow rate 9 mL/min, UV detector 316 nm; fractionation mode:
volume 9 mL/fraction). Fractions 29-31 and 34-36 (n-hexane-EtOAc,
6:4) afforded about 88 mg of oenanthotoxin (1a) and 89 mg of dihydrooe-
9-Epoxyfalcarindiol (2): colorless oil, [R]D25 +107 (c 0.9, MeOH);
IR (liquid film) νmax 3400, 3120, 2080, 1451, 1293, 1238, 1164, 983
1
cm-1; H NMR (500 MHz, C6D6) δ 5.78 (1H, ddd, J ) 17.0, 10.2,
5.45 Hz, H-2), 5.37 (1H, brd, J ) 17.0 Hz, H-1a), 5.03 (1H, brd, J )
10.3 Hz, H-1b), 4.67 (1H, d, J ) 5.4 Hz, H-3), 4.20 (1H, d, J ) 7.1
Hz, H-8), 3.06 (1H, dd, J ) 7.1, 4.0 Hz, H-9), 2.80 (1H, dt, J ) 5.9,
4.0 Hz, H-10), 1.53 (2H, brm, H-11a,b), 1.45-1.30 (10H, m, H-12a,b
+ H-13a,b + H-14a,b + H-15a,b + H-16a,b), 1.01 (3H, t, J ) 7.1 Hz,
H-17); 13C NMR (125 MHz, CDCl3) δ 136.1 (d, C-2), 116.2 (t, C-1),
79.3 (s, C-7), 79.1 (s, C-4), 70.3 (s, C-5), 70.1 (s, C-6), 63.2 (d, C-3),
61.0 (d, C-8), 57.9 (d, C-9), 57.4 (d, C-10), 31.9 (t, C-15), 27.6
(t, C-11), 29.4, 29.3 (t, C-13 and C-14) 26.6 (t, C-12), 22.8 (t, C-16),
14.1 (q, C-17). HREIMS m/z 276.1737 [M]+ (calcd for C17H24O3,
276.1725).
MTPA Esters of Oenanthotoxin and Dihydrooenanthotoxin.
Oenanthotoxin (1a, 2.5 mg) was dissolved in 0.5 mL of dry pyridine
and treated with (-)-R-MTPA chloride (20 µL) and N,N-dimethylami-
nopyridine (DMAP, a spatula tip), then maintained at room temperature
under stirring overnight. After removal of the solvent, the reaction
mixture was purified by HPLC on a SI60 column (eluent n-hex-
ane-EtOAc, 9:1), affording the (S)-MTPA diester 1c in a pure state

Notes
Journal of Natural Products, 2009, Vol. 72, No. 5 965
(3.5 mg, 53% yield). Using (+)-S-MTPA chloride, the same procedure
afforded the (R)-MTPA diester 1d in the same yield. This procedure
was repeated for dihydrooenanthotoxin (1b, 2.0 mg) to obtain the (S)-
MTPA diester 1e and (R)-MTPA diester 1f in the same yield.
Oenanthotoxin-1-O-14-O-(S)-MTPA diester (5a): amorphous
The cells in which series resistance showed a clear tendency to increase
during recordings were not considered in the analysis. Recordings in the
whole-cell mode were started at least 3 min after establishing the whole-
cell mode. This time was sufficient to stabilize the recording conditions.
The agonist was applied using the RSC-200 multibarrel rapid perfusion
system (Bio-Logic, Grenoble, France). With this system, in the whole-
cell configuration, the solution exchange occurred within 30-100 ms.
Before each control recording, cells were washed with normal external
solution for at least 3 min. In studies aiming at assessment of the impact
of considered test compounds [oneantotoxin (1a), dihydrooneantotoxin
(1b)] on GABA-elicited responses, these compounds were present both
in the washing solution (for at least 3 min before agonist application) and
in the agonist-containing saline. For acquisition and data analysis, pClamp
9.2 software was used (Molecular Devices Corporation). To avoid the data
scatter due to cell-to-cell variability, the effect of studied drugs was assessed
by calculating the relative amplitude values with respect to the controls
recorded from the same cell. For the analysis of currents, recorded in the
whole-cell configuration, the current signals were low-pass filtered at 3
kHz with a Butterworth filter and sampled at 10 kHz using the analog-
to-digital converter Digidata 1322A (Molecular Device Corporation) and
stored on a computer hard disk. Data are expressed as mean ( SEM. A
paired Student t test was used to assess the significance of differences
between considered data sets. All experiments were performed at room
temperature, 22-24 °C.
1
solid; H NMR (500 MHz, CDCl3) δ 7.45 and 7.35 (MTPA phenyl
protons), 6.66 (H-9, dd, J ) 15.5, 10.6 Hz), 6.28 (H-2, dt, J )
15.0, 5.0 Hz), 6.09 (H-10, dd, J ) 15.0, 10.6 Hz), 5.84 (H-3,
overlapped), 5.82 (H-11, overlapped), 5.57 (H-8, d, J ) 15.5 Hz),
5.10 (H-14, m), 4.86 (H2-1, d, J ) 5.0 Hz), 3.55 (MTPA OCH3, s),
2.16 (H-12a, overlapped), 2.12 (H-12b, overlapped), 1.81 (H-13a,
m), 1.73 (H-15a, m), 1.65 (H-13b, m), 1.58 (H-15b, m), 1.29 (H2-
16, m), 0.88 (H3-17, d, J ) 7.3 Hz); FABMS (glycerol matrix,
positive ions) m/z 691 [M + H]+.
Oenanthotoxin-1-O-14-O-(R)-MTPA diester (5b): amorphous
1
solid; H NMR (500 MHz, CDCl3) δ 7.55 and 7.53 (MTPA phenyl
protons), 6.69 (H-9, dd, J ) 15.5, 10.6 Hz), 6.26 (H-2, dt, J ) 15.0,
5.0 Hz), 6.16 (H-10, dd, J ) 15.0, 10.6 Hz), 5.91 (H-11, overlapped),
5.86 (H-3, overlapped), 5.57 (H-8, d, J ) 15.5 Hz), 5.10 (H-14, m),
4.86 (H2-1, d, J ) 5.0 Hz), 3.58 (MTPA OCH3, s), 2.35 (H-12a, m),
2.23 (H-12b, m), 1.98 (H-13a, m), 1.68 (H-13b, m), 1.61 (H-15a, m),
1.57 (H-15b, m), 1.28 (H2-16, m), 0.88 (H3-17, d, J ) 7.3 Hz); FABMS
(glycerol matrix, positive ions) m/z 691 [M + H]+.
Dihydrooenanthotoxin-1-O-14-O-(S)-MTPA diester (6a): amor-
1
phous solid; H NMR (500 MHz, CDCl3) δ 7.45 and 7.35 (MTPA
Acknowledgment. P.W., K.S., and J.W.M. were supported by a
Wellcome Trust International Senior Research Fellowship in Biomedical
Science (grant no. 070231/Z/03/Z).
phenyl protons), 6.64 (H-9, dd, J ) 15.5, 10.6 Hz), 6.09 (H-10, dd, J
) 15.0, 10.6 Hz), 5.83 (H-11, dt, J ) 15.0, 5.0 Hz), 5.45 (H-8, d, J )
15.5 Hz), 5.20 (H-14, m), 4.39 (H2-1, m), 3.55 (MTPA OCH3, s), 2.36
(H2-3, t, J ) 6.5 Hz), 2.25 (H-12a, overlapped), 2.21 (H-12b,
overlapped), 1.91 (H2-2, m), 1.75 (H-15a, m), 1.71 (H-13a, m), 1.60
(H-15b, m), 1.56 (H-13b, m), 1.30 (H2-16, m), 0.90 (H3-17, d, J ) 7.3
Hz); FABMS (glycerol matrix, positive ions) m/z 693 [M + H]+.
Dihydrooenanthotoxin-1-O-14-O-(R)-MTPA diester (6b): amor-
phous solid; 1H NMR (500 MHz, CDCl3) δ 7.55 and 7.53 (MTPA phenyl
protons), 6.70 (H-9, dd, J ) 15.5, 10.6 Hz), 6.11 (H-10, dd, J ) 15.0,
10.6 Hz), 5.85 (H-11, dt, J ) 15.0, 5.0 Hz), 5.48 (H-8, d, J ) 15.5 Hz),
5.20 (H-14, m), 4.39 (H2-1, m), 3.58 (MTPA OCH3, s), 2.36 (H2-3,
overlapped), 2.35 (H-12a, overlapped), 2.28 (H-12b, overlapped), 1.91
(H2-2, m), 1.90 (H-13a, m), 1.68 (H-15a, overlapped), 1.67 (H-13b,
overlapped), 1.54 (H-15b, m), 1.27 (H2-16, m), 0.90 (H3-17, d, J ) 7.3
Hz); FABMS (glycerol matrix, positive ions) m/z 693 [M + H]+.
Epoxidation of Falcarindiol. To a solution of falcarindiol (85 mg,
0.36 mmol) in dry CH2Cl2 (3 mL) was added 85% m-chloroperbenzoic
acid (MCPBA, 73 mg, 0.36 mmol, 1 molar equiv). The course of the
reaction was followed by 1H NMR analysis of a CDCl3 solution
containing an equimolecular mixture of falcarindiol and MCPBA. After
stirring 2 h at rt, the reaction was worked up by dilution with CH2Cl2
and the addition of silica gel (1 g) impregnated with a 5% water solution
of Na2S2O3. After stirring a few minutes, the slurry was filtered and
the filtrate was dried (Na2SO4) and evaporated, to afford 60 mg (66%)
of 9-epoxyfalcarindiol (2), spectroscopically (1H and 13C NMR) identical
References and Notes
(1) (a) Bruneton, J. Toxic Plants Dangerous to Humans and Animals;
Lavoisier Publishing: Paris, 1999; pp 105-107. (b) Teuscher, E.;
Lindequist, U. Biogene Gifte: Biologie, Chemie, Pharmakologie;
Fischer Verlag: Stuttgart, 1994; pp 30-34.
(2) For a comprehensive discussion, see: Ribichini, S. Il Riso Sardonico.
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2000.
(3) Farrar, J. J. J. Neurol. Neurosurg. Psychiat. 2000, 69, 292–301.
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Pharmacol. 1949, 1, 377–381. (b) Anet, E. F. L. J.; Lythgoe, B.; Silk,
M. H.; Trippet, S. J. Chem. Soc 1953, 62, 309–322.
(5) Appendino, G.; Prosperini, S.; Valdivia, C.; Ballero, M.; Colombano,
G.; Billington, R. A.; Genazzani, A. A.; Sterner, O. J. Nat. Prod. 2005,
68, 1213–1217.
(6) Bowery, N. G.; Smart, T. G. Br. J. Pharmacol. 2006, 147, S109–
S119.
(7) Ratnayake, A. S.; Hemscheidt, T. Org. Lett. 2002, 4, 4667–4669.
(8) Ohta, T.; Uwai, K.; Kikuchi, R.; Nozoe, S.; Oshima, Y.; Sasaki, K.;
Yoshizaki, F. Tetrahedron 1999, 55, 120878–12098.
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Soc. 1991, 113, 4092–4096.
(10) Hoveyda, A. H.; Evans, D. A.; Fu, G. C. Chem. ReV. 1993, 93, 1307–
1370.
(11) While falcarindiol (3) has an 8S configuration, the oxygenation of C-9
changes the priority of the substituents around C-8, resulting in an 8R
configuration for this carbon in 2.
(12) (a) Fujimoto, Y.; Satoh, M.; Takeuchi, N.; Kirisawa, M. Chem Pharm.
Bull. 1991, 39, 521–523. (b) Deng, S.; Chen, S. N.; Yao, P.; Nikolic,
D.; van Breemen, R. B.; Bolton, J. L.; Fong, H. H. S.; Farnsworth,
N. R.; Pauli, G. F. J. Nat. Prod. 2006, 69, 536541..
(13) Uwai, K.; Ohashi, K.; Takaya, Y.; Ohta, T.; Tadano, T.; Kisara, K.;
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Toxicol. 1978, 12, 417–426.
25
to the natural product and having [R]D +101 (c 1.1, MeOH).
Cell Culture for Electrophysiological Recordings. Neuronal cell
culture was prepared as previously described in detail by Andjus et
al.19 Briefly, P1-P3-day-old Wistar rats were decapitated. This
procedure is in accordance with the regulation of the Polish Animal
Welfare Act. Hippocampi were removed, manually sliced, treated with
trypsin, mechanically dissociated, centrifuged twice at 40 g, plated in
Petri dishes, and cultured. Experiments were performed on cells between
10 and 17 days in culture.
Electrophysiological Recordings. Currents were recorded in the
whole-cell configuration of the patch-clamp technique using the Axopatch
200B amplifier (Molecular Instruments, Sunnyvale, CA) at a holding
potential (Vh) of -40 mV. The intrapipet solution contained (in mM) CsCl
137, CaCl2 1, MgCl2 2, 1,2-bis(2-aminophenoxy)ethane-N,N,N′-tetraacetic
acid (BAPTA) 11, ATP 2, and HEPES 10 (pH 7.2 with CsOH). The
composition of the standard external solution was (in mM) NaCl 137, KCl
5, CaCl2 2, MgCl2 1, glucose 20, and HEPES 10 (pH 7.2 with NaOH).
For the whole-cell recordings, patch pipets had a resistance of 2.5-4.0
MΩ when filled with internal solution. The whole-cell recordings were
included in the statistics when the access resistance was below 10 MΩ.
(18) Habermehl, G. G. Neurotoxins from Plants and Fungi. In Natural and
Synthetic Neurotoxins; Academic Press: New York, 1993; pp 257-
276.
(19) Andjus, P. R.; Stevic-Marinkovic, Z.; Cherubini, E. J. Phys. (Paris)
1997, 504, 103–112.
NP8007717