A unified approach to the synthesis of both enantiomers of anatoxin-a and homoanatoxin-a cyanotoxins

Article abstract of DOI:10.1016/j.tet.2018.04.057

Anatoxin-a and homoanatoxin-a are highly neurotoxic compounds produced by cyanobacteria, principally during surface water-blooms (SWBs). Owing to their powerful biological activity and unique structural characteristics, these natural alkaloids have been t

Full text of DOI:10.1016/j.tet.2018.04.057

Accepted Manuscript
A unified approach to the synthesis of both enantiomers of anatoxin-a and
homoanatoxin-a cyanotoxins
Luis G. Addante-Moya, Consuelo Agulló, Guillermo Quiñones-Reyes, Josep V.
Mercader, Antonio Abad-Fuentes, Antonio Abad-Somovilla
PII:
S0040-4020(18)30446-0
10.1016/j.tet.2018.04.057
TET 29472
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 7 February 2018
Revised Date: 13 April 2018
Accepted Date: 17 April 2018
Please cite this article as: Addante-Moya LG, Agulló C, Quiñones-Reyes G, Mercader JV, Abad-
Fuentes A, Abad-Somovilla A, A unified approach to the synthesis of both enantiomers of anatoxin-a
and homoanatoxin-a cyanotoxins, Tetrahedron (2018), doi: 10.1016/j.tet.2018.04.057.
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Graphical Abstract
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Title:
A unified approach to the synthesis of both enantiomers of anatoxin-a and
homoanatoxin-a cyanotoxins
Authors:
Luis G. Addante-Moya,a Consuelo Agulló,a Guillermo Quiñones-Reyes,a,1 Josep V. Mercader,b
Antonio Abad-Fuentes,b Antonio Abad-Somovilla,a,*
a Organic Chemistry Department, Chemistry Faculty, Valencia University, Dr Moliner 50,
46100−Burjassot, Valencia, Spain
b Institute of Agrochemistry and Food Technology (IATA), Spanish National Research Council
(CSIC), Agustí Escardino 7, 46980−Paterna, Valencia, Spain
* Corresponding author
E-mail address: antonio.abad@uv.es
1 Present address:
Unidad Académica de Ciencias Químicas, Universidad de Zacatecas, Carretera Zacatecas-
Guadalajara Km 6, Ejido la Escondida, Zacatecas, México
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Abstract:
Anatoxin-a and homoanatoxin-a are highly neurotoxic compounds produced by
cyanobacteria, principally during surface water-blooms (SWBs). Owing to their powerful
biological activity and unique structural characteristics, these natural alkaloids have been the
subject of extensive research work in both pharmacological and synthetic studies. In this
contribution we report a simple and efficient synthetic approach for the preparation of both
the natural and unnatural enantiomers of these cyanotoxins, [(+)-1 and (+)-2] and [(−)-1 and
(−)-2] respectively. Key features of this approach include: i) construction of the azabicyclic
homotropane framework in both enantiomeric forms from cis-5,6-epoxycyclooctene, based
on a microwave mediated epoxide ring opening reaction by a chiral benzyl amine followed
by a transannular amine-alkene cyclization; ii) elaboration of the characteristic methyl or
ethyl enone by means of a Sonogashira cross-coupling reaction of an enol-triflate with a C2
or C3 terminal alkyne, followed by a chemo- and regio-selective hydration of the resulting
conjugated enyne group.
Keywords:
Homotropane; Epoxide ring opening–transannular cyclization; Sonogashira coupling;
Regioselective enyne hydration
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1. Introduction
(+)-Anatoxin-a [(+)-1], initially called “Very Fast Death Factor (VFDF)” due to its ability to
induce death within a few minutes after intraperitoneal injection in mice,1 and structurally
related (+)-homoanatoxin-a [(+)-2] are cyanobacterial neurotoxins produced by several
species of both benthic and planktonic cyanobacteria (formerly known as blue-green
algae).2,3 They are potent neuromuscular blocking agents which act as a nicotinic
(cholinergic) agonist by binding to neuronal nicotinic acetylcholine receptors (nAChR) and
blocking the electrical signals between nerve cells, which may result in respiratory paralysis,
asphyxiation and ultimately death. (+)-Anatoxin-a is approximately 50 times more potent
than (−)-nicotine and 20 times more potent than acetylcholine.4,5 This makes (+)-anatoxin-a
one of the most potent agonists of nicotinic acetylcholine receptors and the smallest toxic
alkaloid so far characterized. (+)-Homoanatoxin shares almost identical toxicological
properties.6 Both neurotoxins are mainly produced in cyanobacterial surface water blooms
and are responsible of several incidents of fatal poisoning of cattle, domestic animals, and
wildlife, as well as adverse human health effects.7,8 Currently, different countries have begun
to take action by developing regulations and implementing controls for these toxins in
drinking and recreational waters.9
Figure 1. Anatoxin-a and homoanatoxin-a enantiomer structures
Owing to their powerful biological activity and unique structural characteristics – the
bicyclic homotropane moiety is exclusive of these natural alkaloids10 – anatoxin-a, and to a
lesser extent, homoanatoxin-a, have been the subject of extensive research work in both
pharmacological11,12,13 and synthetic studies.14,15,16 Concerning anatoxin-a, numerous
synthetic strategies have been described so far involving different approaches for the
construction of the 9-azabicyclo[4.2.1]nonane ring system, including: ring expansion of
tropanes, transannular cyclization of amino-cyclooctenes derivatives, intramolecular
cyclization of iminium cations, intramolecular alkylation reaction, cycloaddition reactions,
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enyne metathesis, and carbenoid insertion into amide C-N bonds.14,17,18 Most of these
syntheses are racemic, but some of them are enantiospecific leading to (+)-anatoxin-a19
and/or, in some cases, to the unnatural enantiomeric form, (−)-anatoxin-a,19b,19e,19f,9i,20 in
high enantiomeric purity. In contrast, only two racemic syntheses have been described so far
for homoanatoxin-a. In the first one, published before its isolation from natural sources,
homoanatoxin was prepared as a synthetic analogue of (±)-anatoxin-a via alkylation of the
methyl carbonyl moiety of its N-Boc derivative.21 In the second one, the synthesis of racemic
homoanatoxin-a was completed using a cyclization reaction between an in-situ generated
enol silyl ether and a sulfonyliminium cation as the key step for the construction of the
azabicyclic ring system.22
In the context of a project aimed at developing an immunoassay for (+)-anatoxin-a,23 we
required anatoxin-a and homoanatoxin-a in both enantiomeric forms. Here, we describe the
development of a unified synthetic approach for the preparation of these compounds, which
is one of the simplest strategies published so far. This approach relies on the previous work
of Carroll et al.24 for the construction of the chiral 9-azabicyclo[4.2.1]nonane ring system and
on a new strategy for the elaboration of the methyl and ethyl enone moieties. According to
the retrosynthetic analysis shown in Scheme 1, the enone group of both anatoxin-a and
homoanatoxin-a can be generated through a regioselective hydration of the appropriate
conjugate enyne moiety, in turn available from a carbonyl group through coupling of the
corresponding enol triflate derivative with a C2 or C3 alkyne. As previously described, the
azabicyclic ketone framework can be prepared in each of the two required enantiomeric
forms from commercially available prochiral cis-5,6-epoxycyclooctene and (R)-(+)-α-
methylbenzylamine through an epoxide ring opening–transannular cyclization sequence.
Scheme 1. Retrosynthetic approach to both enantiomers of anatoxin-a (1) and homoanatoxin-a (2)
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2. Results and discussion
As mentioned above, the preparation of the (1R,6R) and (1S,6S) enantiomers of the 9-
aza-bicyclo[4.2.1]nonane ring system (homotropane) was based on the work developed by
Carroll et al. for the preparation of this moiety during the synthesis of nicotinic acceptors of
acetylcholine based on the pyrido[3,4-b]homotropane skeleton.24 According to this
approach, the preparation of the homotropane skeleton of enantiomeric azabicyclic ketones
(−)-11 and (+)-11, which are common intermediates for the preparation of anatoxin-a and
homoanatoxin-a in their natural and unnatural forms, respectively, started from the
prochiral epoxide 9-oxabicyclo[6.1.0]non-4-ene (3), whose desymmetrization was carried
out by bimolecular nucleophilic opening of the oxirane ring with chiral (R)-(+)-α-
methylbenzylamine (4) using a modification of the original procedure involving microwave
irradiation, with simultaneous external air-cooling, of a methanolic solution of equimolecular
amounts of 3 and 4 (Scheme 2). These conditions substantially shorten the reaction times
previously reported for this transformation, providing in only 16 h a nearly 1:1 mixture of
diastereoisomeric amino-alcohols 5 and 6, readily separable in a high degree of
diastereomeric purity via a combination of crystallization and column chromatography.
Preparation of the homotropane skeleton corresponding to each enantiomeric series
was undertaken in parallel from the corresponding diastereoisomeric amino-alcohol. First,
the construction of the nitrogen bridge was carried out by means of an intramolecular
cyclization reaction between the amino nitrogen and the double bond promoted by Hg2+.
The initially formed mercurial intermediate was reduced in situ with sodium borohydride to
afford amino-alcohols 7 or 8 from 5 or 6, respectively, in high yield. It is important to note
that, in contrast to the originally described procedure, the aminomercuration reaction
requires only 30 min at 0 °C to complete; an increase in the reaction time and/or
temperature prior to the demercuration step with sodium borohydride results in a
significant decrease in the yield of the azabicyclic alcohol. Subsequent oxidation of the
secondary hydroxyl group of amino-alcohols 7 and 8 to the corresponding ketone under
Swern conditions followed by amine protecting-group exchange through hydrogenolysis of
the benzylamino moiety in the presence of di-tert-butyl dicarbonate provided N-Boc ketones
(−)-11 and (+)-11. All spectroscopic data of both enantiomeric N-Boc ketones correspond
with those previously described for the racemic compound.18 It should be noted that, as
earlier observed for this and other N-Boc derivatives of the azabicyclic system,19b peak
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doubling or broadening is observed in the NMR spectra owing to the presence of rotational
isomers of the carbamate moiety (about a 2−3:1 mixture of rotamers).
Scheme 2. Synthesis of (1R,6R) and (1S,6S) enantiomeric homotropane frameworks
Once the homotropane skeleton was available in both enantiomeric forms, the
elaboration of the characteristic methyl enone group of (+)- and (−)-anatoxin-a was
addressed. First, the carbonyl group of N-Boc ketones (−)-11 and (+)-11 was transformed
into an enyne moiety. This was done via the Pd(0)-catalyzed Sonogashira cross-coupling
reaction of the corresponding enol triflate, readily obtained by trapping of the kinetic
potassium enolate with Comins' reagent under standard conditions, with
trimethylsilylacetylene, a convenient liquid acetylene surrogate (Scheme 3).25 The terminal
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alkyne group of thus obtained trimethylsilyl enynes (−)-13 and (+)-13 was unmasked upon
treatment with base in MeOH at room temperature, to afford enynes (−)-14 and (+)-14 in
65% and 60% overall yield, respectively, for the three step sequence.
Scheme 3. Synthesis of (+)- and (-)-anatoxin-a trifluoroacetate and hydrochloride salts
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After completing the formation of the enyne moiety, the triple bond was hydrated to
generate the corresponding ketone carbonyl group. This transformation was effected
chemo- and regioselectively by treatment of the enyne compound with a methanolic
solution of mercury(II) oxide and boron trifluoride etherate in the presence of catalytic
amounts of trichloroacetic acid (0.2−0.3 equivalents relative to HgO). Under these
conditions, the triple bond hydration reaction proceeds very efficiently, providing, after
aqueous work up and purification by column chromatography, the corresponding methyl
enone, (−)-15 or (+)-15, with an excellent yield. The effectiveness of this catalytic system
(Hennion-Nieuwland catalysts)26,27 for the hydration of the triple bond was much higher than
that of other catalyst systems tested, e.g. HCO2H-H2O, PdCl2/MeCN-H2O, Pt2Cl4(C2H4)2/THF-
H2O or NaAuCl4/MeOH-H2O.28 The hydration reaction proceeds through the initial formation
of a dimethyl ketal and/or methyl enol ether, from the addition of a single molecule of
MeOH to the triple bond, that are hydrolyzed to the ketone carbonyl group during the
aqueous work up. In fact, small percentages of dimethyl ketal and/or enol-ether
1
intermediates could be identified in the H NMR spectra of the hydration crude product of
some reaction batches. The physical and spectral data of enantiomeric enones (−)-15 and
(+)-15, which are the N-Boc derivatives of natural and unnatural anatoxin-a, respectively,
were identical to those previously reported.19b The enantiomeric purity of both N-Boc
derivatives of anatoxin-a was found to be >99%, based on chiral HPLC analysis (see
experimental part).
Finally, the synthesis of both enantiomers of anatoxin-a was completed by removal of
the N-Boc protecting group in acidic medium. Thus, treatment of (−)-15 and (+)-15 with a 1:1
mixture of trifluoroacetic acid and CH2Cl2 at room temperature directly afforded the
corresponding trifluoroacetate salts, (+)-1∙TFA and (−)-1∙TFA, respectively. Alternatively,
treatment of (−)-15 and (+)-15 with 4M HCl in dioxane at room temperature gave the
equivalent hydrochlorides, (+)-1∙HCl and (−)-1∙HCl, whose physical and spectroscopic data
were in agreement with those previously reported in the literature.19b,19i,19m
The synthesis of homoanatoxin-a in each of the enantiomeric forms was carried out
according to a synthetic route similar to that used for the preparation of anatoxin-a, which
was based on the Sonogashira reaction of the same intermediate enoltriflate, (+)-12 and (−)-
12 – in this case with propyne – followed by hydration reaction of the triple bond (Scheme
4). Apparently, as shown by TLC analysis of the reaction mixture, the reaction between the
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enoltriflate (+)-12 and propyne occurred quite efficiently using both propyne gas or a
commercial 4% solution of propyne in DMF under relatively similar conditions to those
described for the synthesis of anatoxin-a. However, in contrast to the analogous reaction of
the same enoltriflate with trimethylsilylacetylene, the initially isolated cross-coupling
product experimented a rapid degradation after work up and exposition of the crude extract
to the air. In fact, after a few hours, TLC of the crude extract showed the nearly complete
transformation of the initially formed enyne into a mixture of compounds from which a
major, more polar compound, was isolated. It was tentatively characterized as epoxide (i) on
the basis of its molecular formula and NMR data (Figure 2); in particular, the CH-epoxide
1
signal in the H NMR spectrum (a broad doublet at δ 3.17 ppm) and the signals of the
epoxide carbon atoms in the 13C NMR spectrum (C-2 and C-4 at ꢀ 57.4 and 62.1 ppm,
respectively). We speculate with the possibility that the formation of epoxide (i) could be
related to the use of a large excess of alkyne in this Sonogashira reaction, which could favor
the formation of complexes of copper with propyne or hexa-2,4-diyne, formed via a Glaser-
Hay homocoupling of propyne,29 that could be extracted within the organic phase and
catalyze the reaction of the enyne moiety with oxygen.30 In fact, this transformation was
substantially reduced by including a washing step with a strong copper complexing agent
such as EDTA during the work up of the reaction mixture. With this modification, the cross-
coupling reaction of enoltriflates (+)-12 or (−)-12 with propyne afforded the corresponding
enynes, (−)-16 or (+)-16, respectively, in about 75% yield after column chromatography.
With enantiomeric enynes (−)-16 and (+)-16 in hand, the synthesis of both enantiomers
of homoanatoxin-a was readily completed following the same procedure that was used for
the above described synthesis of structurally related anatoxin-a enantiomers. Thus, chemo-
and regioselectively hydration of the triple bond of enynes (−)-16 and (+)-16 using the
Hennion-Nieuwland catalytic system afforded N-Boc ethyl enones (−)-17 and (+)-17,
respectively, whose treatment with a 1:1 mixture of trifluoroacetic acid and CH2Cl2 at room
temperature gave the corresponding trifluoroacetate salts of natural and unnatural
homoanatoxina-a, (+)-2∙TFA and (−)-2∙TFA respectively, in 67−70% overall yield from the
starting enol triflates. The spectroscopic NMR data of both homoanatoxin-a enantiomers
were practically identical and consistent with those previously described for the natural
product31 and the synthetic racemic form.21,22
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Scheme 4. Synthesis of (+)- and (-)-homoanatoxin-a trifluoroacetate salts
Figure 2
3. Conclusions
In summary, we have successfully established a simple approach for the preparation of
both natural and unnatural enantiomers of the cyanotoxins anatoxin-a [(+)-1 and (−)-1] and
homoanatoxin-a [(+)-2 and (−)-2]. This approach is based on two synthetic transformations:
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first, the construction of the homotropane skeleton in both enantiomeric forms from cis-5,6-
epoxycyclooctene, via a microwave mediated epoxide ring opening reaction by a chiral
amine and a transannular amine-alkene cyclization; second, the elaboration of the required
methyl or ethyl enone moiety through a Sonogashira cross-coupling reaction of an enol-
triflate with a C2 or C3 terminal alkyne followed by chemo- and regio-selective hydration of
the resulting conjugated enyne group.The synthetic sequences for the preparation of the
enantiomers of anatoxin-a and homoanatoxin-a from initially prepared diastereomeric
amino-alcohols (−)-5 and (+)-6 comprised only eight and seven steps, respectively, and were
accomplished in all cases with an overall yield ranging from 37% to 42%. These strategies
represent one of the most simple ways so far described for the preparation of (+)- and (−)-
anatoxin-a and the first reported synthesis of (+)- and (−)-homoanatoxin-a.
4. Experimental Section
4.1. General Information
All organic solvents required in anhydrous conditions were dried and distilled prior to
use using standard techniques.32 CH2Cl2 was distilled over CaH2 under nitrogen and THF and
toluene were distilled over Na/benzophenone. MeOH was dried and stored over 3Å
molecular sieve. Other solvents and reagents were obtained from commercial sources and
used without purification. Operations with air-and/or moisture-sensitive reagents were
performed under an inert atmosphere of dry N2 or Ar, using syringes or cannulas, glassware
dried at 130°C and freshly distilled solvents and dried according to the techniques described
above. Microwave-assisted chemistry was performed in a laboratory monomode microwave
reactor (CEM corporation, model Discover). Reactions were monitored by thin-layer
chromatography (TLC) on precoated silica plates (0.25 mm layer thickness, Silica Gel 60 F254)
using UV light as the visualizing agent and ethanolic phosphomolybdic acid or aqueous ceric
ammonium molybdate solutions and heat as developing agents. Except for cases where the
1
crude product showed a sufficiently high purity (greater than 95% by H-NMR), the
synthesized compounds were purified by flash column chromatography using silica gel 60
(particle size 0.043–0.063 mm). Melting points were determined on a Büchi M-560 or Kofler
hot-stage apparatus and are uncorrected. Optical rotations were recorded on a Perkin Elmer
Mod. 343 polarimeter at a temperature of 20 °C, using a 1 dm cell and the solvent specified
in each case; concentrations of the solutions are expressed in g/100 mL. Proton, carbon and
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13
19
fluorine nuclear magnetic resonance (1H, C and F NMR) spectra were recorded at room
temperature (rt) on a Bruker DPX300 spectrometer operating at 300.1, 75.5 and 282 MHz,
respectively, or on a Bruker DRX500 spectrometer operating at 500.1, 125.8 and 470.5 MHz,
respectively. Chemical shifts (δ) are expressed in ppm recorded using the residual solvent as
the internal reference in all cases. Carbon substitution degrees were established by DEPT
pulse sequences. A combination of COSY and HSQC experiments was used in most cases for
1
13
the assignment of H and C chemical shifts. Infrared (IR) spectra were measured without
prior preparation using the total attenuated reflectance (ATR) technique on a Thermo
Scientific Nicolet i510 FTIR Spectrometer from ThermoFisher Scientific (IR band intensities: w
= weak, m = medium, s = strong). High resolution mass spectra (HRMS) were recorded by the
electrospray (ES) ionization mode, which was obtained with a Q-TOF premier mass
spectrometer with an electrospray source (Waters, Manchester, UK).
4.2. Preparation of the (1R,6R) and (1S,6S) enantiomeric form of the homotropane
framework: Synthesis of N-Boc homotropanones (−)-11 and (+)-11
4.2.1. (1R,8R,Z)-8-(((R)-1-phenylethyl)amino)cyclooct-4-en-1-ol [(−)-5] and (1S,8S,Z)-8-(((R)-1-
phenylethyl)amino)cyclooct-4-en-1-ol [(+)-6)]
A solution of (Z)-9-oxabicyclo[6.1.0]non-4-ene (3, 2.00 g, 16.1 mmol) and (R)-(+)-α-
methylbenzylamine (4, 2.1 mL, 16.5 mmol, 1.05 equiv) in anhydrous MeOH (15 mL) placed in
a sealed microwave quartz vessel was irradiated with microwave (300 W, 110 °C) for 16h
with simultaneous cooling of the reaction vessel with compressed air. The reaction mixture
was cooled to rt and concentrated to dryness at reduced pressure. The resulting oily residue
(3.273 g) was dissolved in hexane (7−8 mL) and kept in the fridge overnight. Then, the
formed crystals of (+)-6 formed (0.500 g) were separated and washed with cold hexane, the
filtrate and washing were concentrated under vacuum and the obtained residue was
chromatographed on silica gel, using CH2Cl2-acetone mixtures from 95:5 to 70:30 as eluent,
to afford, in order of elution, (−)-5 (1.120 g, 34%) as a viscous oil, followed by (+)-6 (0.613 g,
34.2% taking into account the crystals initially obtained) as a white solid.
Characterization data of (−)-5: [α]D −20.1 (c 3.36, CHCl3) {Lit.24 [α]D −20.1}; IR (ATR) νmax
(cm–1) 3350 (br, m), 3017 (w), 2926 (m), 1450 (m), 1104 (m), 1048 (m), 761 (s), 735 (s), 702
(s); 1H NMR (300 MHz, CDCl3) δ 7.35−7.15 (5H, m, Ph), 5.70−5.58 (1H, m, H-4), 5.46−5.35 (1H,
m, H-5), 3.72 (1H, q, J = 6.6 Hz, CHMe), 3.29 (1H, ddd, J = 8.9, 7.4, 3.0 Hz, H-1), 2.54 (1H,ddd,
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J = 11.3, 7.4, 4.3 Hz, H-8), 2.35−2.10 (4H, m, H-6, H2-3, H-2), 1.98−1.86 (1H, ddt, J = 14.0, 7.0,
4.8 Hz, H’-6), 1.78−1.65 (1H, m, H-7), 1.36 (1H, m. H‘-2), 1.31 (3H, d, J = 6.6 Hz, Me), 1.24 (1H,
m, H’-7); 13C NMR (75 MHz, CDCl3) δ 145.5 (C-1 Ph), 130.8 (C-4), 128.7 (C-3/C-5 Ph), 128.2 (C-
5), 127.5 (C-4 Ph), 126.6 (C2/C-6 Ph), 71.8 (C-1), 58.8 (C-8), 57.9 (CHMe), 35.0 (C-2), 33.5 (C-
7), 23.5 (Me), 23.1 (C-3), 22.9 (C-6); HRMS (TOF MS ES+) calcd for C16H24NO [M+H]∙+
246.1852, found 246.1859.
Characterization data of (+)-6: [α]D +102.8 (c 1.38, CHCl3), mp 88.6–89 °C (crystallized
from hexane) {Lit.24 [α]D +104.5, mp 91–92 °C}; IR (ATR) νmax (cm–1) 3369 (br, m), 2929 (m),
1
1639 (w), 1454 (w), 1218 (w), 1028 (w), 754 (s), 704(s); H NMR (300 MHz, CDCl3) δ 7.35–
7.20 (5H, m, Ph), 5.56–5.38 (2H, m, H-4, H-5), 3.90 (1H, q, J= 6.6 Hz, CHMe), 3.29 (1H, ddd, J=
8.8, 7.5, 3.1 Hz, H-1), 2.43−2.27 (2H, m, H-6, H-8), 2.1−1.90 (5H, m, H-7, H2-3, H’-6), 1.34 (3H,
13
d, J = 6.6 Hz, Me), 1.37−1.30 (1H, m, H’-7), 1.07 (1H, m, H’-2); C NMR (75 MHz, CDCl3) δ
144.7 (C-1 Ph), 130.6 (C-4), 128.6 (C-3/C-5 Ph), 128.1 (C-4 Ph), 127.3 (C-5), 127.1 (C2/C-6 Ph),
71.7 (C-1), 57.8 (C-8), 56.4 (CHMe), 34.6 (C-2), 32.8 (C-7), 25.0 (Me), 23.1 (C-3), 23.0 (C-6);
HRMS (TOF MS ES+) calcd for C16H24NO [M+H]∙+ 246.1852, found 246.1859.
4.2.2. (1R,2R,6R)-9-((R)-1-phenylethyl)-9-azabicyclo[4.2.1]nonan-2-ol [(−)-7]
A solution of amino alcohol (−)-5 (0.654 g, 2.665 mmol) in THF (5.3 mL) was added
dropwise to a stirred suspension of Hg(OAc)2 (1.00 g, 3.145 mmol, 1.18 equiv) in a 1:1
mixture of THF-H2O (16 mL) at 0 °C. The mixture was stirred at the same temperature for 30
min and the resulting transparent and pale yellow resulting mixture was treated with a 3M
aqueous solution of NaOH (3.1 mL). Then, the obtained cloudy, dark-orange mixture was
treated with a solution of NaBH4 (120 mg, 3.158 mmol, 1.18 equiv) in the same NaOH
solution (6.3 mL) at 0 °C. After stirring at this temperature for a few minutes the reaction
mixture turned black and the stirring was continued at rt until completion of the reaction
(about 30 min), as confirmed by TLC (CHCl3-CH3OH 95:5 as eluent). The reaction mixture was
allowed to stand, the metallic mercury was separated by decantation and the liquid phase
was extracted with Et2O. The combined organic layers were washed with brine, dried over
anhydrous MgSO4 and concentrated at reduced pressure to give a residue that was purified
by chromatography, using CHCl3 as eluent, to give azabicyclic alcohol (−)-7 (0.556 g, 85%) as
a low melting point solid. [α]D −9.5 (c 1.13, CHCl3) {Lit.24 [α]D −9.2}; IR (ATR) νmax (cm–1) 3295
(br, m), 2927 (s), 1452 (m), 1370 (m), 1081 (m), 1025 (s), 875 (m), 700 (s); 1H NMR (300 MHz,
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CDCl3) δ 7.40−7.15 (5H, m, Ph), 3.86−3.80 (1H, m, H-2), 3.79 (1H, q, J = 6.5 Hz, CHMe), 3.48
(1H, br t, J = 8.3 Hz, H-1), 3.32 (1H, br t, J = 6.5 Hz, H-6), 2.08 (1H, tdd, J = 12.6, 9.0, 5.7 Hz, H-
5), 1.92−1.35 (8H, m, H2-3, H2-4, H-5, H2-7, H-8), 1.32 (3H, d, J = 6.5 Hz, Me), 1.25−1.15 (1H,
13
m, H’-8); C NMR (75 MHz, CDCl3) δ 147.0 (C-1 Ph), 128.3 (C-3/C-5 Ph), 127.3 (C-2/C-6 Ph),
126.7 (C-4 Ph), 74.4 (C-2), 64.5 (C-1), 61.4 (CHMe), 59.2 (C-6), 36.0 (C-8), 33.5 (C-5), 33.2 (C-
3), 23.3 (C-7), 22.9 (Me), 20.4 (C-4); HRMS (TOF MS ES+) calcd for C16H24NO [M+H]∙+
246.1852, found 246.1845.
4.2.3. (1S,2S,6S)-9-((R)-1-phenylethyl)-9-azabicyclo[4.2.1]nonan-2-ol [(+)-8]
This diastereomer was prepared (658 mg, 95%) in an analogous way as described for
(−)-7 employing the (1S,8S,Z)-isomer (+)-6 (687 mg); (+)-8 was obtained as a colorless viscous
oil; [α]D +19.3 (c 1.05, CHCl3), {Lit.24 [α]D +20.5}; IR (ATR) νmax (cm–1) 3285 (br, w), 2924 (s),
1
1450 (m), 1048 (m), 761 (s), 733 (s), 702 (s); H NMR (300 MHz, CDCl3) δ 7.38–7.28 (4H, m,
Ph), 7.24–7.17 (1H, m, Ph), 3.85–3.77 (1H, m, H-2), 3.78 (1H, q, J = 6.5 Hz, CHMe), 3.49 (1H,
br t, J = 8.2 Hz, H-1), 3.31 (1H, br t, J = 6.4 Hz, H-6), 2.01 (1H, tdd, J = 12.5, 9.1, 5.5 Hz, H-5),
1.91−1.36 (8H, m, H2-3, H2-4, H-5, H2-7, H-8), 1.32 (3H, d, J = 6.5 Hz, Me), 1.16 (1H, m, H’-8);
13C NMR (75 MHz, CDCl3) δ 146.9 (C-1 Ph), 128.3 (C-3/C-5 Ph), 127.3 (C-2/C-6 Ph), 126.8 (C4
Ph), 73.8 (C-2), 66.1 (C-1), 61.3 (CHMe), 57.4 (C-6), 36.4 (C-8), 33.5 (C-5), 33.4 (C-3), 23.0 (C-
7), 22.7 (Me), 20.3 (C-4); HRMS (TOF MS ES+) calcd for C16H24NO [M+H]∙+ 246.1852, found
246.1851.
4.2.4. (1R,6R)-9-((R)-1-Phenylethyl)-9-azabicyclo[4.2.1]nonan-2-one [(−)-9]
DMSO (0.446 mL, 6.28 mmol) was added dropwise to a solution of oxalyl chloride (0.266
mL, 3.14 mmol) in anhydrous CH2Cl2 (6.8 mL) at −78 °C under nitrogen. After stirring at this
temperature for 10−15 min, a solution of alcohol (−)-7 (0.513 g, 2.09 mmol) in CH2Cl2 (3.5
mL) was added and the mixture was stirred for an additional 30 min before the addition of
Et3N (1.75 mL, 12.56 mmol). The reaction mixture was stirred at −78 °C for 1 h and then
allowed to slowly warm to rt, quenched by the addition of a saturated aqueous solution of
Na2CO3 (5 mL), diluted with water and extracted with Et2O. The combined organic layers
were washed with brine, dried over anhydrous MgSO4 and concentrated under vacuum to
give an oily residue that was chromatographed on silica gel, using CH2Cl2 as eluent, to give
ketone (−)-9 (0.452 g, 88%) as a colorless oil. [α]D −21.1 (c 1.99, CHCl3) {Lit.24 [α]D −20.7}; IR
(ATR) νmax (cm–1) 2969 (m), 2929 (m), 1700 (s), 1456 (m), 1374 (m), 1121 (s), 1072 (s), 704 (s);
15

ACCEPTED MANUSCRIPT
1H NMR (300 MHz, CDCl3) δ 7.40−7.15 (5H, m, Ph), 3.80 (1H, q, J = 6.6 Hz, CHMe), 3.67 (1H,
br dd, J = 10.2, 1.8 Hz, H-1), 3.62 (1H, br dd, J = 5.8, 2.5 Hz, H-6), 3.02 (1H, ddd, J = 14.0, 13.1,
3.0 Hz, H-3), 2.31 (1H, br dd, J = 14.0, 5.5 Hz, H’-3), 2.25−2.10 (1H, m, H-8), 1.90−1.67 (4H, m,
H’-8, H-7, H-5, H-4), 1.67−1.45 (3H, m, H’-7, H’-5, H’-4), 1.34 (3H, d, J = 6.6 Hz, Me); 13C NMR
(75 MHz, CDCl3) δ 218.6 (C-2), 145.7 (C-1 Ph), 128.5 (C-3/C-5 Ph), 127.3 (C-2/C-6 Ph), 127.2
(C-4 Ph), 70.0 (C-1), 61.6 (C-6), 60.7 (CHMe), 42.6 (C-3), 35.5 (C-5), 30.9 (C-8), 27.2 (C-7), 22.7
(Me), 20.1 (C-4); HRMS (TOF MS ES+) calcd for C16H22NO [M+H]∙+ 244.1696, found 244.1687.
4.2.5. (1S,6S)-9-((R)-1-Phenylethyl)-9-azabicyclo[4.2.1]nonan-2-one [(+)-10]
This diastereomer was prepared (505 mg, 79%) in an analogous way as described for
(−)-9 employing the (1S,2S,6S)-isomeric amino-alcohol (+)-8 (644 mg); (+)-10 was obtained as
a colorless viscous oil. [α]D +106.9 (c 0.49, CHCl3) {Lit.24 [α]D +107}; IR (ATR) νmax (cm–1) 2969
1
(w), 2928 (s), 1698 (s), 1454 (m), 767 (w), 704 (m); H NMR (300 MHz, CDCl3) δ 7.36–7.19
(5H, m, Ph), 3.94 (1H, q, J = 6.6 Hz, CHMe), 3.74 (1H, dd, J = 10.1, 2.4 Hz, H-1), 3.58–3.50 (1H,
m, H-6), 2.83 (1H, ddd, J = 15.2, 12.8, 3.4 Hz, H-3), 2.40 (1H, br dd, J = 15.1, 5.7 Hz, H’-3),
2.26–2.15 (1H, m, H-8), 1.96−1.50 (7H, m, H2-4, H2-5, H2-7, H’-8), 1.34 (3H, d, J = 6.5 Hz, Me);
13C NMR (75 MHz, CDCl3) δ 218.5 (C-2), 145.5 (C-1 Ph), 128.6 (C-3/C-5 Ph), 127.2 (C-2/C6 Ph),
127.1 (C-4 Ph), 70.2 (C-1), 59.7 (C-6), 59.4 (CHMe), 43.2 (C-3), 34.2 (C-5), 30.2 (C-8), 27.9 (C-
7), 22.8 (Me), 20.0 (C-4); HRMS (TOF MS ES+) calcd for C16H22NO [M+H]∙+ 244.1696, found
244.1687.
4.2.6. tert-Butyl (1R,6R)-2-oxo-9-azabicyclo[4.2.1]nonane-9-carboxylate [(−)-11]
A mixture of benzyl amine (−)-9 (0.280 g, 1.15 mmol), di-tert-butyl dicarbonate (0.296
mL, 1.29 mmol) and Pd(OH)2 on charcoal (50% moisture, 20% loading, 50 mg) in EtOAc (5
mL) was stirred under a pressure of about 60 psi of hydrogen at rt for 14 h. The mixture was
filtered through a short pad of celite using EtOAc for washing. The filtrate and washing were
combined and washed with 5% aqueous NaHCO3 solution and brine and dried over
anhydrous MgSO4. Evaporation of the solvent in vacuo led to (−)-11 (0.270 g, 98%) as a
viscous colorless oil that solidifies upon standing in the refrigerator. The compound thus
obtained was pure by NMR and was used in the next step without further purification. Mp
72.1–73.0 °C (crystallized from cold Hexane-EtOAc) {Lit.18 for (±)-11, mp 49–51 °C}; [α]D −54.2
(c 0.77, CHCl3); IR (ATR) νmax (cm–1) 2935 (m), 2972 (m), 1693 (s), 1395 (s), 1171 (m), 1108
1
(m), 774 (w); H NMR (300 MHz, CDCl3) (a mixture of rotamers, only signals of the major
16

ACCEPTED MANUSCRIPT
rotamer are given) δ 4.52 (1H, br d, J = 8.4 Hz, H-1), 4.25 (1H, br dd, J = 10.5, 2.3 Hz, H-6),
2.60–2.45 (1H, m, H-3), 2.45−2.25 (2H, m, H’-3, H-5), 2.25−2.00 (2H, m, H-7, H-8), 1.98−1.85
13
(1H, m, H’-5), 1.82−1.60 (4H, m, H2-4, H’-7, H’-8), 1.43 (9H, s, CMe3); C NMR (75 MHz,
CDCl3) δ 215.3 (C-2), 153.0 (CO Boc), 80.4 (CMe3), 65.0 (C-1), 56.6 (C-6), 41.8 (C-3), 33.0 (C-8),
30.0 (C-5), 28.5 (CMe3), 26.9 (C-7), 19.4 (C-4); HRMS (TOF MS ES+) calcd for C13H22NO3
[M+H]∙+ 240.1594, found 240.1587.
4.2.6. tert-Butyl (1S,6S)-2-oxo-9-azabicyclo[4.2.1]nonane-9-carboxylate [(+)-11]
This enantiomer (261.2 mg, 97%) was prepared in an analogous manner as described for
(−)-11 employing the (1S,6S)-isomer (+)-10 (273.9 mg); (+)-11 was obtained as a semisolid
that solidifies upon standing in the refrigerator. Mp 71.5–72.5 °C (crystallized from cold
Hexane-EtOAc) [α]D +54.6 (c 0.88, CHCl3). Its spectroscopic data correspond with those of
enantiomer (−)-11.
4.3. Synthesis of the natural and unnatural enantiomer of anatoxin-a [(+)-1 and (–)-1]
4.3.1. tert-Butyl (1R,6R)-2-(((trifluoromethyl)sulfonyl)oxy)-9-azabicyclo[4.2.1]non-2-ene-9-
carboxylate [(+)-12]
A 0.5 M solution of potassium bis(trimethylsilyl)amide (KHMDS) in toluene (3.8 mL, 1.9
mmol, 1.5 equiv) was added dropwise to a solution of ketone (−)-11 (276 mg, 1.252 mmol) in
anhydrous THF (15 mL) at −78 °C under nitrogen. The mixture was stirred under this
conditions for 1 h and then treated with
a
solution of N-(5-chloro-2-
pyridyl)bis(trifluoromethanesulfonimide) (Comins’ Reagent) (763 mg, 1.94 mmol, 1.55 equiv)
in THF (1 mL). After stirring at −78 °C for 30 min, the reaction mixture was quenched by the
addition of saturated aqueous ClNH4 (4 mL), diluted with water (60 mL) and extracted with
Et2O. The combined organic phases were washed with brine, dried over anhydrous MgSO4
and concentrated under reduced pressure to afford a brownish oil that was purified by
chromatography on silica gel, using hexane-EtOAc 9:1 as eluent, to give enol triflate (+)-12
(351.2 mg, 82%) as a white solid. Mp 63.1–64.2 °C (crystallized from hexane-EtOAc); [α]D
+19.5 (c 1.31, CHCl3); IR (ATR) νmax (cm–1) 2972 (w), 1679 (s), 1400 (s), 1366 (s), 1243 (m),
1207 (s), 1168 (s), 1138 (s), 1138 (s), 10210 (m), 884 (m), 827 (m); 1H NMR (300 MHz, CDCl3)
(a mixture of rotamers, only signals of the major rotamer are given) δ 5.76 (1H, br t, J = 6.2
Hz, H-3), 4.51 (1H, br d, J = 7.9 Hz, H-1), 4.38 (1H, m, H-6), 2.29−2.20 (2H, m, H2-4), 2.25−2.10
17

ACCEPTED MANUSCRIPT
(3H, m, H-5, H-7, H-8), 2.10−2.00 (1H, m, H’-5), 1.80−1.58 (2H, m, H’-7, H’-8), 1.46 (9H, s,
CMe3); 13C NMR (75 MHz, CDCl3) δ 154.6 (C-2), 153.1 (CO), 120.8 (C-3), 118.6 (q, J = 320.2 Hz,
CF3), 80.5 (CMe3), 58.9 (C-1), 54.9 (C-6), 31.1 (C-8), 30.5 (C-5), 28.5 (C-7), 28.3 (CMe3), 19.8
19
(C-4); F NMR (282 MHz, CDCl3) δ −74.3 (s); HRMS (TOF MS ES+) calcd for C14H21F3NO5S
[M+H]∙+ 372.1087, found 372.1088.
4.3.2. tert-Butyl (1S,6S)-2-(((trifluoromethyl)sulfonyl)oxy)-9-azabicyclo[4.2.1]non-2-ene-9-
carboxylate [(−)-12]
The preparation of this enantiomer (312.7 mg, 73%) was carried out in an analogous
manner to that described for (+)-12 employing the (1S,6S)-enantiomer (+)-11 (346.7 mg);
(−)-12 was obtained as a white solid. Mp 65.3–65.6 °C (crystallized from hexane-EtOAc); [α]D
−18.5 (c 1.04, CHCl3). Its spectroscopic data correspond with those of enantiomer (+)-12.
4.3.3.
tert-Butyl
(1R,6R)-2-((trimethylsilyl)ethynyl)-9-azabicyclo[4.2.1]non-2-ene-9-
carboxylate [(−)-13]
A mixture of enol triflate (+)-12 (174 mg, 0.468 mmol), PdCl2(PPh3)2, (34 mg, 48.6 µmol,
0.1 equiv), CuI (4.6 mg, 24.2 µmol, 0.05 equiv) and trimethylsilylacetylene (0.264 mL, 184
mg, 1.872 mmol, 4 equiv) in anhydrous DMF (3 mL) was exhaustively degassed by ultrasound
under nitrogen atmosphere. Et3N (0.196 mL 1.407 mmol, 3 equiv) was added to the resulting
yellowish suspension to give a clear solution that was stirred for 1 h at rt (during this time,
the color of the reaction mixture changed from light yellowish to orange, then brown, and
finally black). After this time, the solution was diluted with water and then extracted with
Et2O. The combined organic layers were washed with a 5% aqueous solution of LiCl and
brine, dried over anhydrous MgSO4 and concentrated under vacuum. The crude product was
purified by chromatography, eluting first with hexane and then with hexane-EtOAc 9:1, to
give alkyne (−)-13 (128 mg, 86%) as a slightly colored amorphous solid. [α]D −64.0 (c 1.05,
CHCl3); IR (ATR) νmax (cm–1) 2989 (s), 2363 (s), 2343 (s), 1685 (m), 1408 (m), 1069 (s), 845 (m),
1
756 (s); H NMR (300 MHz, CDCl3) (a mixture of rotamers, only signals of the major rotamer
are given) δ 6.13 (1H, t, J = 6.2 Hz, H-3), 4.48 (1H, br d, J = 8.5 Hz, H-1), 4.35 (1H, m, H-6),
2.31−2.20 (1H, m, H2-4), 2.22−1.97 (3H, m, H-5, H-7, H-8), 1.95−1.83 (1H, m, H’-5), 1.75−1.50
(2H, m, H’-7, H’-8), 1.46 (9H, s, CMe3), 0.16 (9H, s, SiMe3); 13C NMR (75 MHz, CDCl3) δ 153.4
(CO), 138.0 (C-3), 131.7 (C-2), 106.6 (C-1’), 92.6 (C-2’), 79.6 (CMe3), 59.8 (C-1), 55.6 (C-6),
18

ACCEPTED MANUSCRIPT
31.4 (C-8), 31.3 (C-5), 28.8 (C-7), 28.6 (CMe3), 24.3 (C-4), 0.15 (SiMe3); HRMS (TOF MS ES+)
calcd for C18H30NO2Si [M+H]∙+ 320.2040, found 320.2042.
4.3.4.
tert-Butyl
(1S,6S)-2-((trimethylsilyl)ethynyl)-9-azabicyclo[4.2.1]non-2-ene-9-
carboxylate [(+)-13]
This enantiomer (126.3 mg, 84.4%) was prepared in an analogous manner as described
for (−)-13 employing the (1S,6S)-isomer (−)-12 (379.1 mg); (+)-13 was obtained as a pale
colored low melting point solid, [α]D +66.3 (c 0.99, CHCl3). Its spectroscopic data correspond
with those of enantiomer (−)-13.
4.3.5. tert-Butyl (1R,6R)-2-ethynyl-9-azabicyclo[4.2.1]non-2-ene-9-carboxylate [(−)-14]
A mixture of trimethyl(alkynyl)silane (−)-13 (239 mg, 0.748 mmol), K2CO3 (516 mg, 3.740
mmol, 5 equiv) in MeOH (6.5 mL) was stirred at rt for 1 h. The reaction mixture was poured
into water and extracted with Et2O, the organic phase was washed with brine, dried over
anhydrous MgSO4 and concentrated at reduced pressure. The residue was purified by silica
gel column chromatography, eluting first with hexane and then with hexane-EtOAc 9:1, to
obtain alkyne (−)-14 (216 mg, 92%) as a pale yellow oil that solidified upon freezing. [α]D
−35.8 (c 1.01, CHCl3); IR (ATR) νmax (cm–1) 3311 (w), 2976 (m), 2935 (w), 1693 (s), 1408 (s),
1
1367 (m), 1173 (m), 1119 (m; H NMR (300 MHz, CDCl3) δ (a mixture of rotamers, only
signals of the major rotamer are given) δ 6.15 (1H, t, J = 6.1 Hz, H-3), 4.50 (1H, br d, J = 8.0
Hz, H-1), 4.35 (1H, m, H-6), 2.88 (1H, s, H-2’), 2.32−2.23 (2H, m, H2-4), 2.23−2.05 (3H, m, H-5,
H-7, H-8), 1.93−1.83 (1H, m, H’-5), 1.75−1.65 (1H, m, H’-7), 1.65−1.55 (1H, m, H’-8), 1.46 (9H,
13
s, CMe3); C NMR (75 MHz, CDCl3) δ 153.5 (CO), 138.3 (C-3), 130.1 (C-2), 85.1 (C-1’), 79.7
(CMe3), 76.1 (C-2’), 60.0 (C-1), 55.3 (C-6), 31.8 (C-8), 31.2 (C-5), 29.2 (C-7), 28.6 (CMe3), 24.2
(C-4); HRMS (TOF MS ES+) calcd for C15H22NO2 [M+H]∙+ 248.1645, found 248.1645.
4.3.6. tert-Butyl (1S,6S)-2-ethynyl-9-azabicyclo[4.2.1]non-2-ene-9-carboxylate [(+)-14]
This enantiomer (198.9 mg, 97%) was prepared in an analogous way as described for
(−)-14 employing the (1S,6S)-isomer (+)-13 (264.7 mg); (+)-14 was obtained as an oil; [α]D
+35.5 (c 1.02, CHCl3). Its spectroscopic data correspond with those of enantiomer (−)-14.
4.3.7. tert-Butyl (1R,6R)-2-acetyl-9-azabicyclo[4.2.1]non-2-ene-9-carboxylate [(−)-15]
19

ACCEPTED MANUSCRIPT
Freshly distilled BF3∙Et2O (23 µL, 0.184 mmol, 0.3 equiv) was dropwise added to a
solution of Cl3CCO2H (0.89 mg, 5.49 µmol, 9∙10−3 equiv) in anhydrous MeOH (2 mL) at rt
under nitrogen. The resulting solution was added dropwise via syringe to a stirred
suspension of HgO (64 mg, 0.294 mmol, 0.5 equiv) in MeOH (10 mL) under the same
conditions and, after stirring for a few minutes, a solution of enyne (−)-14 (147.5 mg, 0.596
mmol) in anhydrous MeOH (4 mL) was added and the mixture vigorously stirred at rt for
about 1.5 h. After completion of the reaction (TLC, hexane-EtOAc 8:2), the orange
suspension was poured into cold water (about 15 mL), stirred at rt for 10−15 min and
extracted with Et2O. The combined organic layers were washed with brine, dried and
concentrated to leave a residue that was purified by silica gel chromatography, eluting with
CH2Cl2 and then with CHCl3, to give N-Boc methyl enone (−)-15 (147 mg, 93%) as a colorless
oil. The enantiomeric purity was determined by HPLC analysis (Daicel Chiralpak AD-H
(250x4.6 mm), hexane/2-propanol 99/1, flow rate=1 mL/min, λ = 241 nm; retention time:
26.4 min [(1S,6S)-enantiomer, <1%] and 30.6 min [(1R,6R)-enantiomer, >99%]. [α]D −60.4 (c
0.96, CHCl3), −46.7 (c 1.02, CH2Cl2) {Lit19g. [α]D −46.8 (CH2Cl2); Lit.19b [α]D −47.2 (CH2Cl2)}; IR
(ATR) νmax (cm–1) 2974 (w), 2933 (w), 1689 (s), 1665 (s), 1633 (m), 1391 (s), 1365 (s), 1169 (s),
933 (w), 772 (w); 1H NMR (300 MHz, CDCl3) (a mixture of rotamers, only signals of the major
rotamer are given) δ 6.82 (1H, t, J = 6.0 Hz, H-3), 5.13 (1H, br d, J = 9.2 Hz, H-1), 4.41 (1H, m,
H-6), 2.50−2.40 (2H, m, H2-4), 2.37−2.25 (1H, m, H-8), 2.29 (3H, s, Me), 2.22−2.04 (2H, m, H-
13
5, H-7), 1.75−1.58 (3H, m, H’-5, H’-7, H’-8), 1.37 (9H, s, CMe3); C NMR (75 MHz, CDCl3) δ
197.9 (COMe), 153.3 (CO Boc), 150.5 (C-2), 142.3 (C-3), 79.5 (CMe3), 55.8 (C-6), 53.2 (C-1),
31.6 (C-8), 30.5 (C-5), 28.9 (C-7), 28.6 (CMe3), 25.5 (Me), 24.3 (C-4); HRMS (TOF MS ES+)
calcd for C15H24NO3 [M+H]∙+ 266.1751, found 266.1747.
4.3.8. tert-Butyl (1S,6S)-2-acetyl-9-azabicyclo[4.2.1]non-2-ene-9-carboxylate [(+)-15]
This enantiomer (190 mg, 94%) was prepared in an analogous manner as described
above for (−)-15 employing the (1S,6S)-isomer (+)-14 (188.1 mg); (+)-15 was obtained as a
slightly yellowish oil. The enantiomeric purity was determined by HPLC analysis (same
conditions as above for (−)-15; retention time: 26.4 min [(1S,6S)-enantiomer, >99%] and 30.6
min [(1R,6R)-enantiomer, <1%]. [α]D +60.0 (c 0.96, CHCl3), [α]D +43.8 (c 0.99, CH2Cl2), {Lit.19b
[α]D +51.9 (c 0.79, CH2Cl2)}. The spectroscopic data of (+)-15 correspond with those of
enantiomeric (−)-15.
20

ACCEPTED MANUSCRIPT
4.3.9. (+)-Anatoxin-a trifluoroacetate [(+)-1·TFA] and hydrochloride [(+)-1·HCl] salts
CF3CO2H (1.5 mL) was added dropwise to a solution of (−)-15 (106 mg, 0.40 mmol) in
anhydrous CH2Cl2 (1.5 mL) and the mixture was stirred at rt under nitrogen until
consumption of the starting material, as showed by TLC using CHCl3-MeOH-HCO2H 90:10:1
as eluent. After about 1 h, the solvents were evaporated under reduced pressure and the
residue kept under vacuum overnight to afford (+)-1∙TFA (112 mg, nearly quantitative yield)
as a viscous oil with a purity higher than 95% according to the 1H NMR spectra. [α]D +10.6 (c
0.45, CHCl3); IR (ATR) νmax (cm–1) 3686 (w), 3410 (br, m), 2989 (s), 2903 (s), 1660 (m), 1396
+
(m), 1069 (s); 1H NMR (400 MHz, CDCl3) δ 9.60 and 8.83 (1H each, each br s, NH2 ), 7.15 (1H,
ddd, J = 8.2, 4.4, 1.3 Hz, H-3), 5.24 (1H, br t, J = 6.9 Hz, H-1), 4.30 (1H, m, H-6), 2.61−2.55 (2H,
m, H2-4), 2.50 (1H, ddd, J = 12.9, 9.2, 3.1 Hz, H-8), 2.43−2.32 (1H, m, H-7), 2.29 (1H, s, Me),
2.21−2.12 (1H, m, H-5), 1.96 (1H, m, H’-7), 1.89 (1H, ddd, J = 14.2, 6.3, 2.8 Hz, H’-8), 1.85 (1H,
13
m, H’-5); C NMR (75 MHz, CDCl3) δ 196.9 (CO), 146.6 (C-3), 144.2 (C-2), 59.0 (C-6), 52.5 (C-
1), 30.4 (C-8), 28.0 (C-5), 27.6 (C-7), 25.0 (Me), 23.6 (C-4); 19F NMR (282 MHz, CDCl3) δ −76.32
(s); HRMS (TOF MS ES+) calcd for C10H16NO [M+H-CF3CO2H]+ 166.1226, found 166.1222.
Alternatively, a 4M solution of HCl in dioxane (220 µL, 0.88 mmol) was added to a
solution of (−)-15 (25.6 mg, 0.097 mmol) in anhydrous dioxane (300 µL) and the mixture was
stirred at rt for 1.30 h. The excess HCl and dioxane were removed under nitrogen steam,
then benzene was added and the solvent removed under reduced pressure (2 mL x 3). The
residue obtained was kept under high vacuum (5.0x10‒3 Torr) overnight to give (+)-1∙HCl
(19.2 mg, 99%) as a white powder which showed NMR spectral data identical to those
previously reported (see Supplementary Data File).19b,19c,19m This powder was suspended in a
95:5 mixture of Et2O and MeOH (2.2 mL) at 50-60 °C and MeOH was added to obtain a cleair
solution (about 200 µL). The solution was kept at rt for 3 h and then at 4 °C overnight. The
formed white prism crystals were separated and washed with a little Et2O and dried under
vacuum to give crystalline (+)-1∙HCl. Mp 156.5‒158.5 °C (with darkening), [α]D +40.8 (c, 0.49
EtOH) {Lit.19c mp 152-153 °C, Lit.19b [α]D +43.2 (c 0.67, EtOH); Lit.19m mp, 151-153 °C, [α]D
+37.3 (c, 2.08, EtOH)}.
4.3.10. (−)-Anatoxin-a trifluoroacetate [(−)-1·TFA] and hydrochloride salts [(−)-1·HCl]
The trifluoroacetate (17.8 mg, 99%) and hydrochloride (44.1 mg, 99%) salts of this
enantiomer were prepared in an analogous manner as described above for the equivalent
21

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salts of (+)-anatoxin-a employing the (1S,6S)-isomer (+)-15 (18 mg and 59.0 mg,
respectively). (−)-1∙TFA was obtained as a viscous oil; [α]D −9.6 (c 1.44, CHCl3). (−)-1∙HCl was
obtained as a white foam; it forms large colorless prisms after recrystallization from cold
Et2O ‒ 10% MeOH, mp 157.5−159.5 oC (with darkening), [α]D –42.9 (c 0.64, EtOH) {Lit.19b [α]D
−46.3 (c 0.57 EtOH); Lit.20b [α]D −39.0 (c 0.55 EtOH)}. The spectroscopic data of the unnatural
enantiomer, (−)-1∙TFA and (−)-1∙HCl, correspond with those of the natural enantiomer (+)-
1∙TFA and (+)-1∙HCl, respectively (see Supplementary Data File).
4.4. Synthesis of the natural and unnatural enantiomer of homoanatoxin-a [(+)-2 and (+)-2]
4.4.1. tert-Butyl (1R,6R)-2-(prop-1-yn-1-yl)-9-azabicyclo[4.2.1]non-2-ene-9-carboxylate [(−)-
16]
A mixture of enol triflate (+)-12 (174 mg, 0.468 mmol), PdCl2(PPh3)2, (34 mg, 48.6 µmol,
0.1 equiv), CuI (14.7 mg, 77.44 µmol, 0.16 equiv) and DMF (6 mL), contained in a Büchi 'Tiny
Clave' reactor equipped with a magnetic stirring bar, was exhaustively degassed by vacuum-
filling nitrogen cycles. Et3N (0.653 mL, 4.690 mmol, 10 equiv) was added to the resulting
yellowish suspension to give a clear solution that was first cooled at −78 °C and degassed
again by vacuum (200 mmHg)-filling nitrogen cycles. Then, the mixture was stirred and
homogenized (during this time the reaction mixture color changed from light yellowish to
orange, then brown, and finally black), and then cooled at −78 °C and propyne gas was
condensed (about 1/3 of the total volume, >10 equiv). Finally, the reaction mixture was
allowed to reach rt and then stirred overnight. The reactor was opened to the atmosphere
and the excess of propyne allowed to evaporate. Then, the resulting solution was diluted
with water and extracted with Et2O. The combined organic layers were washed with a 5%
aqueous solution of LiCl, a 2M aqueous solution of EDTA and brine, dried over anhydrous
MgSO4 and a small spoon of Dowex© M4195 resin, filtered and concentrated under vacuum.
The crude product was purified by chromatography, eluting first with hexane and then with
hexane-EtOAc 9:1, to give alkyne (−)-16 (83.7 mg, 75%) as a viscous light yellow oil. [α]D
−36.4 (c 0.60, CHCl3). IR (ATR) νmax (cm–1) 2969 (w), 2931 (w), 2363 (s), 2343 (s), 1698 (m),
1
1408 (m), 1365 (w), 789 (m), 758 (s); H NMR (300 MHz, CDCl3) (a mixture of rotamers, only
signals of the major rotamer are given) δ 5.95 (1H, t, J = 6.0 Hz, H-3), 4.46 (1H, br d, J = 7.8
Hz, H-1), 4.33 (1H, m, H-6), 2.30–2.18 (2H, m, H2-4), 2.18–2.05 (3H, m, H-5, H-7, H-8), 1.93
13
(3H, s, Me), 1.92–1.80 (1H, m, H’-5), 1.77–1.55 (2H, m, H’-7, H’-8), 1.46 (9H, s, CMe3); C
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NMR (75 MHz, CDCl3) δ 153.6 (CO Boc), 134.8 (C-3), 131.2 (C-2), 84.4 (C-1’), 80.9 (C-2’), 79.4
(CMe3), 60.5 (C-1), 55.2 (C-6), 32.2 (C-8), 31.2 (C-5), 29.5 (C-7), 28.6 (CMe3), 23.9 (C-4), 4.33
(Me); HRMS (TOF MS ES+) calcd for C16H24NO2 [M+H]∙+ 262.1802, found 262.1798.
A more polar compound, characterized as epoxide i, was aisolated from some reaction
batches (see the results and discussion part for details). Epoxide i (tert-butyl (1R,2R,4S,7S)-2-
(prop-1-yn-1-yl)-3-oxa-10-azatricyclo[5.2.1.02,4]decane-10-carboxylate)
showed
the
1
following spectroscopic properties: H NMR (300 MHz, CDCl3) (a mixture of rotamers, only
signals of the major rotamer are given) δ 4.40 (1H, d, J = 8.7 Hz, H-1), 4.11 (1H,dddd, J = 5.6,
2.7, 1.7, 1.7 Hz, H-7), 3.17 (1H, br d, J = 4.9 Hz, H-4), 2.30−2.17 (2H, m, H-9, H-5), 2.08 (1H, m,
H-6), 2.05−1.85 (3H, m, H´-5, H-8, H´-9), 1.84 (3H, s, Me), 1.54 (1H, m, H´-8), 1.50 (9H, s,
13
CMe3), 1.38 (1H, m, H´-6); C NMR (75 MHz, CDCl3) δ 154.0 (CO Boc), 79.9 (C-1’), 79.5
(CMe3), 78.5 (C-2’), 62.1 (C-4), 57.7 (C-1), 57.4 (C-2), 54.3 (C-7), 28.6 (C-9), 28.6 (CMe3), 27.7
(C-6), 25.1 (C-8), 22.8 (C-5), 3.9 (Me); HRMS (TOF MS ES+) calcd for C12H16NO3 [M+H-C4H8]∙+
222.1125, found 222.1126; calcd for C16H27N2O3 [M+NH4]∙+ 295.2016, found 295.2016.
4.4.2. tert-Butyl (1S,6S)-2-(prop-1-yn-1-yl)-9-azabicyclo[4.2.1]non-2-ene-9-carboxylate [(+)-
16]
This enantiomer (151.3 mg, 75%) was prepared in an analogous manner as described for
(−)-16 employing the (1S,6S)-isomer (−)-12 (188.1 mg); (+)-16 was obtained as a slightly
yellowish oil; [α]D +37.9 (c 0.60, CHCl3). The spectroscopic data of enantiomer (+)-16
correspond with those of (−)-16.
4.4.3. tert-Butyl (1R,6R)-2-propionyl-9-azabicyclo[4.2.1]non-2-ene-9-carboxylate [(−)-17]
Freshly distilled BF3∙Et2O (23 µL, 0.184 mmol, 0.3 equiv) was dropwise added to a
solution of Cl3CCO2H (0.89 mg, 5.49 µmol, 9∙10−3 equiv) in anhydrous MeOH (2 mL) at rt
under nitrogen. The resulting solution was added dropwise via syringe to a stirred
suspension of HgO (64 mg, 0.294 mmol, 0.5 equiv) in MeOH (10 mL) under the same
conditions and, after stirring for a few minutes, a solution of enyne (−)-16 (147.5 mg, 0.596
mmol) in anhydrous MeOH (4 mL) was added and the mixture vigorously stirred at rt for
about 1.5 h. After completion of the reaction (TLC, hexane-EtOAc 8:2), the orange
suspension was poured into cold water and processed as described above for (−)-15. The
crude product was purified by silica gel chromatography, eluting with CH2Cl2 and then with
23

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CHCl3, to afford N-Boc ethyl enone (−)-17 (144 mg, 91%) as a slightly yellowish oil. The
enantiomeric purity was determined by HPLC analysis (Daicel Chiralpak AD-H (250x4.6 mm),
hexane/2-propanol 99/1, flow rate=1 mL/min, λ = 235 nm; retention time: 16.6 min [(1S,6S)-
enantiomer, <1%] and 22.6 min [(1R,6R)-enantiomer, >99%). [α]D −36.5 (c 0.80, CHCl3); IR
(ATR) νmax (cm–1) 2933 (w), 2976 (w), 1672 (s), 1393 (s), 1365 (m),1169 (s), 1110 (s), 752 (s);
1H NMR (300 MHz, CDCl3) (a mixture of rotamers, only signals of the major rotamer are
given) δ 6.80 (1H, t, J = 6.0 Hz, H-3), 5.12 (1H, br d, J = 9.3 Hz, H-1), 4.43–4.37 (1H, m, H-6),
2.77–2.55 (2H, m, CH2Me), 2.46–2.36 (2H, m, H2-4), 2.37–2.26 (1H, m, H-8), 2.20–2.02 (2H,
m, H-5, H-7), 1.73−1.58 (3H, m, H’-5, H’-7, H’-8), 1.36 (9H, s, CMe3), 1.10 (3H, t, J = 7.2 Hz,
CH2Me). 13C NMR (75 MHz, CDCl3) δ 200.7 (CO), 153.3 (COBoc),149.9 (C-2), 140.6 (C-3), 79.4
(CMe3), 55.8 (C-6), 53.4 (C-1), 31.7 (C-8), 30.5 (CH2Me), 30.4 (C-5), 28.9 (C-7), 28.6 (CMe3),
24.2 (C-4), 9.00 (CH2Me);HRMS (TOF MS ES+) calcd for C16H25NO3 [M+H]∙+ 280.1907, found
280.1901.
4.4.4. tert-Butyl (1S,6S)-2-propionyl-9-azabicyclo[4.2.1]non-2-ene-9-carboxylate [(+)-17]
This enatiomer (189.6 mg, 94%) was prepared in an analogous manner as described for
(−)-17 employing the (1S,6S)-isomer (+)-16 (188.1 mg); (+)-17 was obtained as a slightly
yellowish oil. The enantiomeric purity was determined by HPLC analysis (the same conditions
as above for (−)-17; retention time: 16.6 min [(1S,6S)-enantiomer, >99%] and 22.6 min
[(1R,6R)-enantiomer, <1%]. [α]D +35.6 (c 0.60, CHCl3). Its spectroscopic data correspond with
those of enantiomer (−)-17.
4.4.5. (+)-Homoanatoxin-a trifluoroacetate salt [(+)-2·TFA]
CF3CO2H (400 µL) was added dropwise to a solution of (−)-17 (25 mg, 0.089 mmol) in
anhydrous CH2Cl2 (400 µL) and the mixture was stirred at rt under nitrogen until
consumption of the starting material, as showed by TLC using CHCl3-MeOH-HCO2H 90:10:1
as eluent. After about 1 h, the solvents were evaporated under reduced pressure and the
residue kept under vacuum overnight to afford (+)-2∙TFA (26 mg, 99%) as a viscous yellowish
oil, that was shown to be almost pure by NMR. [α]D +13.2 (c 0.91, CHCl3); IR (ATR) νmax (cm–1)
3665 (w), 3406 (br, m), 2974 (s), 2903 (m), 1668 (s), 1409 (m), 1383 (m), 1195 (s), 1134 (s),
1080 (s), 1069 (s), 724 (w); 1H NMR (300 MHz, CDCl3) δ 9.27 and 8.68 (1H each, each br s,
+
NH2 ), 7.17 (1H, t, J = 6.0 Hz, H-3), 5.29 (1H, m, H-1), 4.35 (1H, m, H-6), 2.66 (2H, m, CH2Me),
2.63−2.46 (3H, m, H2-4, H-8), 2.45−2.35 (1H, m, H-7), 2.18−2.07 (1H, m, H-5), 1.97 (1H, m, H’-
24

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13
7), 1.97−1.82 (2H, m, H’-5, H’-8), 1.07 (3H, t, J = 7.2 Hz, CH2Me); C NMR (75 MHz, CDCl3) δ
199.5 (CO), 145.1 (C-3), 143.6 (C-2), 59.1 (C-6), 52.9 (C-1), 30.4 (C-8), 30.0 (CH2Me), 28.0(C-
5), 27.6 (C-7), 23.5 (C-4), 8.4 (CH2Me); 19F NMR (282 MHz, CDCl3) δ −76.21 (s); HRMS (TOF
MS ES+) calcd for C11H18NO [M+H-CF3CO2H]∙+ 180.1383, found 180.1376.
4.4.6. (−)-Homoanatoxin-a trifluoroacetate salt [(−)-2·TFA]
This enantiomer (15 mg, 99%) was prepared in an analogous way as described for (+)-
2∙TFA employing the (1S,6S)-enatiomer (+)-17 (15.6 mg); (−)-2∙TFA was obtained as a viscous
yellowish oil; [α]D −15.3 (c 0.74, CHCl3). Its spectroscopic data correspond with those of
enantiomer (+)-2∙TFA.
Acknowledgements
This work was supported by the Spanish Ministerio de Ciencia e Innovación (AGL2015-
64488-C2-1/2-R) and cofinanced by FEDER funds. G. Q. was a recipient of a predoctoral
fellowship from the “Subsecretaría de Educación Superior” of the Mexican government. We
thank the Central Support Service for Experimental Research (SCSIE) of University of Valencia
for NMR and HRMS experiments.
Appendix A. Supplementary data
1
13
Supplementary data related to this article (copies of H and C NMR spectra) can be
found at the Supplementary Data File.
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