Expeditious synthesis of polyacetylenic water hemlock toxins and their effects on the major GABAA receptor isoform

Article abstract of DOI:10.1039/c7cc09801d

Classical synthetic approaches to highly unsaturated polyene/yne natural products rely on iterative cross-coupling of linear fragments. Herein, we present an expeditious and unified approach to the unsaturated backbone of polyacetylenes via domino cuprate addition/4π-electrocyclic ring opening of a stereodefined cyclobutene intermediate. This sets the stage for a detailed biological assessment of the role of Virol A and Cicutoxin as inhibitors of GABA induced chloride currents, providing further insight into the interaction of these highly potent toxins towards the GABA_A receptor, including the structure-activity relationship of the derivatives.

Full text of DOI:10.1039/c7cc09801d

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Expeditious synthesis of polyacetylenic water
hemlock toxins and their effects on the major
Cite this:DOI: 10.1039/c7cc09801d
GABAA receptor isoform†
Received 27th December 2017,
Accepted 29th January 2018
Martin Berger,‡a Yong Chen,‡a Konstantina Bampali,b Margot Ernstb and
a
Nuno Maulide
*
DOI: 10.1039/c7cc09801d
rsc.li/chemcomm
Classical synthetic approaches to highly unsaturated polyene/yne As a result, the unsaturated core of Virol A was assembled by
natural products rely on iterative cross-coupling of linear fragments. the cross coupling of four smaller units, including an enantio-
Herein, we present an expeditious and unified approach to the pure alcohol as an early-stage building block. The first total
unsaturated backbone of polyacetylenes via domino cuprate addition/ synthesis of Cicutoxin was reported by Gung in 2009, using a
4p-electrocyclic ring opening of a stereodefined cyclobutene inter- very similar cross-coupling strategy (Fig. 1b).10 The previously
mediate. This sets the stage for a detailed biological assessment of unsuccessful building block diyne 4 was a viable partner in this
the role of Virol A and Cicutoxin as inhibitors of GABA induced specific case and, as before, the synthesis started from an
chloride currents, providing further insight into the interaction of
these highly potent toxins towards the GABAA receptor, including
the structure–activity relationship of the derivatives.
Water hemlock (Cicuta species) is regarded as one of the most
poisonous plants in Northern America and Central Europe.1
The reason for this unusual toxicity is conjugated, polyunsaturated
diols, such as Virol A (1) and Cicutoxin (2) (Fig. 1). The ingestion of
even small parts of the plant can cause seizures and lead to death,
and numerous cases with lethal outcome have been reported. Due
to the high risk of confusion with different plant species, water
hemlock still poses a serious danger to all forms of livestock.2
The highly conjugated backbone of these compounds
imparts on them considerable light- and air-sensitivity. This
notwithstanding, the isolation of Cicutoxin (2) was reported as
early as 19153 and its structural determination along with the
first (low-yielding) racemic total synthesis waited until 1953.4,5
The stereochemical assignment of Cicutoxin (2) and Virol A (1)
along with their relative toxicity was first reported much later in
1999,6 followed by the first asymmetric total synthesis of Virol A
(1) by Uwai in the same year. This approach made extensive use
of palladium-catalyzed cross coupling (Fig. 1a).7–9 However, a
convergent cross coupling using the diyne building block 4
(vide infra) failed to give a useful yield due to polymerization.
a
¨
Institute of Organic Chemistry, University of Vienna, Wahringer Straße 38,
1090 Vienna, Austria. E-mail: nuno.maulide@univie.ac.at
b Department of Molecular Neurosciences, Center for Brain Research/Medical
University of Vienna, Spitalgasse 4, 1090 Vienna, Austria
† Electronic supplementary information (ESI) available: Experimental and spectral
data. See DOI: 10.1039/c7cc09801d
Fig. 1 (a) Structure of Virol A (1) and previous synthesis via Sonogashira
cross-coupling; (b) Cicutoxin (2) and previous synthesis by cross-coupling
and (c) our approach via a cuprate-addition/4p-electrocyclic ring opening
cascade.
‡ These authors contributed equally.
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enantiopure alcohol building block. In 2011, Bru¨ckner reported at room temperature. In this event, the main pathway for the
an elegant approach using Stille cross-coupling of an advanced decomposition of 5 appeared to be its desired ring opening,
stannane intermediate.11 This use of iterative transition metal- which already took place in trace amounts during the reaction
catalyzed cross-coupling reactions of linear fragments reflects the itself (Scheme 1, top). Pleasingly, the crude product underwent full
general trend in the field of polyene natural product synthesis.12
conversion into 6 upon standing for 12 h at room temperature.
Herein, we present a concise total synthesis of the water Diene–diyne 6, isolated in 82% yield, was formed exclusively as the
hemlock toxins Virol A (1) and Cicutoxin (2). Our strategy provides (E,E)-isomer as expected for the conrotatory ring opening of the
a unified approach to both natural products through a concise trans-cyclobutene precursor 5. Straightforward amidation of 6 led
route.13 We also investigate the influence of the absolute to Weinreb amide 7, the pivotal common intermediate for both
configuration and the number of –OH groups on the GABAA toxins 1 and 2. Amide 7 can be conveniently stored at room
inhibitory effects of Virol A (1) and Cicutoxin (2).
temperature for several weeks without noticeable degradation. In
Synthesis: Our group has contributed to the chemistry of order to obtain Virol A 1, enone 8 was prepared by simple
strained bicyclo[2.2.0]lactones such as 3 in recent years,14,15 treatment of 7 with pentyllithium. The addition of LiCl was
and we envisaged an assembly of the key unsaturated core essential in order to obtain a reasonable yield of 67%. Presumably
structure of the toxins by domino ring opening/4p-electrocyclic owing to its lowered LUMO, 8 was prone to facile visible light-
ring opening of bicyclo lactone 3 to stereoselectively afford the mediated photoisomerization of the central olefin from (E) to (Z).13
all-trans polyacetylenic carboxylic acid 6 (Fig. 1c). In this context, Enantioselective reduction of 8 using (R)-CBS afforded the desired
the entire unsaturated core of Virol A 1 could be constructed in a THP-protected (S)-Virol A 9 in 76% yield and an (S):(R) ratio of
single step utilizing the logical building block 4 – a diyne which 495 : 5.§ 17 Final deprotection with pyridinium para-toluene-
was not applicable in prior synthesis of 1. Furthermore, key sulfonate (PPTS) afforded Virol A 1, which was found to be stable
intermediate 6 would also afford an advanced intermediate upon storage at À20 1C.
en route to the higher homologue Cicutoxin 2, uniting both
With both racemic and enantioenriched samples of 1 in
syntheses.
hand, we then focused on the synthesis of the more chemically
In the synthetic direction, cupration16 of known diyne 47 labile Cicutoxin 2 (Scheme 1, bottom). Taking into account the
followed by the addition of lactone 3 at À78 1C afforded the photolability of 8, reduction of the Weinreb amide 7 to the
desired trans-cyclobutene 5, which was found to be metastable corresponding enal with DIBAL and immediate homologation
Scheme 1 Top: Synthesis of Virol A (1). Legend: (a) 4 (2.0 equiv.), MeLi (2.0 equiv.), CuCN (2.0 equiv.), THF, À78 1C to r.t., 4 h. (b) Weinreb salt (1.5 equiv.),
EDCI (1.2 equiv.), HOBt (1.2 equiv.), Et3N (2.0 equiv.), DCM, r.t., 12 h. (c) Pentyllithium (3.0 equiv.), LiCl (5.0 equiv.), Et2O, À40 1C, 40 min. (d) (R)-(+)-CBS
(2.0 equiv.), BH3 * Me2S (2.0 equiv.), THF, À50 1C, 1.5 h. (e) PPTS (1.0 equiv.), MeOH, 35 1C, 2 h. Bottom: Synthesis of Cicutoxin 2 and condition-dependent
formation of methoxy analog 12 during THP-deprotection. Legend: (a) DIBAL-H (1.5 equiv.), toluene, À78 1C, 1 h. (b) C3H7COCH2PO(OEt)2 (2.0 equiv.),
Ba(OH)2 (4.0 equiv.), THF/H2O, r.t., 1 h. (c) (S)-(À)-CBS (2.0 equiv.), BH3 * Me2S (2.0 equiv.), THF, À50 1C, 1.5 h. (d) PTSA (0.5 equiv.), MeOH, r.t., 1.5 h.
(e) PPTS (1.0 equiv.), MeOH, 45 1C, 2 h.
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of the unsaturated system via HWE olefination (performed
directly on the crude mixture) were carried out under strict
exclusion of oxygen and light to yield enone 10 in reproducible
52% yield over two steps, as a single isomer. Enantioselective
reduction of 10 using (S)-CBS afforded the desired THP-
protected (R)-Cicutoxin 11 in 76% yield and an (R) : (S) ratio of
495 : 5. Deprotection was achieved with p-toluene sulfonic
acid (PTSA) at room temperature yielding Cicutoxin 2. The
attempted unravelling using PPTS at elevated temperature led
to the isolation of a considerable amount of the methoxy
derivative 12. We found that 11 can be quantitatively converted
to the methoxy analog 12 simply by extending the reaction time.
As such a substitution was not observed during the unmasking
of 9 en route to Virol A, it can be ultimately ascribed to extended
delocalization of a putative heptadienyl carbenium ion.
Owing to the late-stage chirality introduction in our synthetic
plan, racemic samples of 1 and 2 can be prepared by simple
reduction with NaBH4 instead of CBS-reduction from advanced
intermediates 8 and 10.§
Evaluation of compound effects on the major GABAA receptor
isoform: It is interesting to note that the toxicity of these diols is
reported to increase with the number of conjugated double
bonds.13 A study by Uwai et al. suggests a correlation of the
inhibition of GABA-induced chloride currents with the acute
toxicity of Cicutoxin13 and Virol A.18
We carried out a full characterization of a1b3g2 GABAA
receptors, which are the major GABAA receptor isoforms in
the adult mammalian brain.19 Two-Electrode Voltage Clamp
Electrophysiology was employed to test the compound effects
on currents elicited by an EC20 concentration of GABA on the
receptors expressed in Xenopus laevis oocytes. Subsequently,
their half maximal inhibitory concentration values (IC50 values)
were determined.
As shown in Fig. 2, co-application of racemic Virol A or
Cicutoxin inhibits GABA-evoked currents in a1b3g2 GABAA receptors.
A similar inhibition for the enantioenriched compounds was
observed, suggesting that the detected activity is independent
of the enantiopurity of the diol (Fig. 2). Chiral recognition,
therefore, does not appear to affect the interaction of these
compounds with the receptors.
Fig. 2 Inhibition of the control chloride current (100%) by racemic (a and c)
and enantioenriched (b and d) Virol A (1) and Cicutoxin (2) suggesting similar
inhibitory efficacy independent of the configuration. Concentration-
dependent inhibition of the GABA EC20 current at a1b3g2 receptors for
racemic (a and c) and enantioenriched (b and d) Virol A and Cicutoxin, with
IC50 values of 0.93 mM and 1.4 mM for Virol A and 2.4 mM and 1.7 mM
for Cicutoxin, respectively. (e) shows concentration-dependent effects of
14-O-Me-Cicutoxin rac-12 on the GABA EC20 current at a1b3g2 receptors.
The compound was measured one day after synthesis. The dotted line is
used to visualize the baseline of 100% of control current, which corresponds
to no modulatory activity. Data are depicted as mean Æ SEM (n = 4).
Interestingly, our 14-O-methylated Cicutoxin analogue 12
did not inhibit the GABA-induced chloride current (Fig. 2e).
Our data imply the secondary alcohol of the toxins 1 and 2 to be
absolutely essential for the inhibitory effect on the tested
receptor. Thus, the sole mode of action by the primary alcohol can
be ruled out. In agreement with this, Cicutol, the 14-deoxyanalog of
2, is reported to lack the capability of prolonging action potentials.20
The simple presence of an oxygen atom as a hydrogen atom acceptor
at the allylic position 14 seems to be, in the case of a methoxy-group,
insufficient to cause inhibition. As for the case of Virol A (1) and
Cicutoxin (2), it is tempting to propose that capability for hydrogen
bond donation is required for the interaction with the GABAA
receptor complex.21
ring opening of a stereodefined cyclobutene intermediate. Our
strategy stands in contrast to classical paradigms of polyene
synthesis that rely on extensive cross-coupling of small building
blocks and offers a more general solution to the preparation of
structurally related polyenic natural products. We generated the
targets in racemic and enantioenriched forms along a novel
analogue, enabling a detailed biological characterization of their
role as inhibitors of GABA-induced chloride currents on the
major GABAA receptor isoform in the adult mammalian brain.
The observation that both –OH groups are mandatory for the
inhibitory effect is in line with the idea that the effect on GABAA
receptors contributes to the compounds’ toxicity.
We have presented an approach to the common unsaturated
backbone of water hemlock polyacetylene natural products
Virol A and Cicutoxin by a domino cuprate addition/electrocyclic
This research was funded by the Austrian Research Fund
(FWF) Doctoral Program ‘‘Molecular Drug Targets’’ (W1232)
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and Stand-alone grant P27194. We are grateful to the University
¨
of Vienna for continued support of our research. Dr H. Kahlig
(U. Vienna) is acknowledged for expert NMR analysis. We are
grateful to R. Holzinger (Med. U. Vienna) for help with the
Oocyte work.
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Conflicts of interest
There are no conflicts to declare.
´
M. T. Oliveira, D. Audisio, F. Frebault and N. Maulide, Angew. Chem.,
Notes and references
§ See the ESI† for details.
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