A six-step total synthesis of α-thujone and: D 6-α-thujone, enabling facile access to isotopically labelled metabolites

Article abstract of DOI:10.1039/c6cc05376a

The short synthesis of α-thujone relies on the functionalization of the readily available dimethylfulvene. Furthermore, the three main metabolites of the natural product were also synthesized. Since d₆-acetone can be used as a starting material, the route developed allows for the facile incorporation of isotopic labels which are required for detecting and quantifying trace amounts via GC/MS analysis.

Full text of DOI:10.1039/c6cc05376a

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A six-step total synthesis of a-thujone
and d6-a-thujone, enabling facile access
to isotopically labelled metabolites†
Irene Thamm,a Johannes Richers,b Michael Rychlika and Konrad Tiefenbacher*cd
Cite this:DOI: 10.1039/c6cc05376a
Received 28th June 2016,
Accepted 2nd September 2016
DOI: 10.1039/c6cc05376a
www.rsc.org/chemcomm
The short synthesis of a-thujone relies on the functionalization of
the readily available dimethylfulvene. Furthermore, the three main
metabolites of the natural product were also synthesized. Since
d6-acetone can be used as a starting material, the route developed
allows for the facile incorporation of isotopic labels which are
required for detecting and quantifying trace amounts via GC/MS
analysis.
A great variety of plants produce the monoterpenes a-thujone
(1a) and b-thujone (2, Fig. 1), which therefore are present in
diverse herbal products.1 The most famous product containing
thujone is certainly absinth, produced from wormwood. It had
been a popular spirit drink in the 19th century but later was
prohibited due to concerns about its toxicity.2 It was connected
to severe health problems, including hallucinations, depressions,
Fig. 1 Structures of a-thujone (1a), b-thujone (2) and the major metabolites
of a-thujone: 7-OH-a-thujone (3a), 2-OH-a-thujone (4a), and 4-OH-a-
thujone (5a).
convulsions, blindness and mental deterioration. More recent these products meet the requirements and additionally to better
studies propose that most of these effects were caused by alcohol detect trace amounts of a-thujone and its major metabolites,
intoxication.2 Nevertheless, thujone is neurotoxic and was shown access to isotopically labelled derivatives is required. Although
to inhibit the gamma-aminobutyric acid A (GABAA) receptor, the structure of the bicyclic monoterpene was elucidated in 1900
which leads to excitations and convulsions at higher concentra- by Semmler,6 only one total synthesis has been reported so far.7
tions in animal studies.3 a-Thujone (1a) was shown to be more Oppolzer et al. prepared enantioenriched a-thujone over twelve
active than its b-isomer 2. The metabolism of thujone was steps from commercially available materials, utilizing an elegant
investigated both in vitro and in vivo. 7-OH-a-thujone (3a) is palladium-catalyzed cyclization strategy. The route, however,
clearly the major metabolite in in vitro studies.3 In vivo studies, does not allow for a facile introduction of inexpensive isotopic
however, point to 2-OH-a-thujone (4a) and 4-OH-a-thujone (5a) as labels. Therefore, we developed and herein describe a novel
the main metabolites.4
six-step access to a-thujone, which enables the introduction of
The manufacture of thujone containing products is permitted isotopic labels from inexpensive d6-acetone. The synthesized
again in the European Union but maximum limits have been d6-thujone 1b thereafter is also functionalized to the most
imposed.5 To ensure accurate quantitation, to assess whether important metabolites 3b, 4b and 5b.
Our synthetic strategy is based on a late-stage oxidation of
alcohol 6a/6b (Scheme 1), followed by a regio- and diastereo-
a
¨
¨
Analytical Food Chemistry, Technische Universitat Munchen, Alte Akademie 10,
selective methylation at position C4. Such an approach seemed
attractive since alcohol 6a/6b should be directly accessible from
cyclopentenol 7a/7b via Simmons–Smith cyclopropanation.8
Cyclopentenol 7a/7b can be traced back to the known dimethyl-
fulvenes 8a and 8b, which are synthesized from cyclopentadiene
and acetone.9 Therefore, the inexpensive d6-acetone can function
85354 Freising, Germany
b
¨
¨
Department of Chemistry, Technische Universitat Munchen, Lichtenbergstraße 4,
85747 Garching, Germany
c Department of Chemistry, University of Basel, St. Johannsring 19, CH-4056 Basel,
Switzerland. E-mail: konrad.tiefenbacher@unibas.ch
d
¨
Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26,
CH-4058 Basel, Switzerland. E-mail: tkonrad@ethz.ch
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6cc05376a as the source of the isotopic labels.
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utilizing 2-iodoxybenzoic acid (IBX) as an oxidant,12 ketones
10a/10b were obtained in 77% and 88% yield, respectively.
The final alkylation step required careful optimization of the
reaction conditions. It was found that multi-alkylated products
could not be separated by flash chromatography. Therefore, the
reaction conditions were optimized to produce 1a/1b selectively.
The use of 1.0 eq. of potassium bis(trimethylsilyl)amide and
methyl iodide, as well as the addition of N,N0-dimethyl-
propyleneurea, facilitated this task. Thujones 1a and 1b were
obtained in 75% and 62% yield, respectively.
Although an optically active material is not required for use
as an internal standard in GC/MS or LC/MS analysis, it may be
helpful for other applications. Therefore, we investigated the
possibility of performing the hydroboration of the cyclopenta-
dienes 9a in an enantioselective fashion. Attempts with diisopino-
campheylborane13 only lead to very low levels of enantiomeric
enrichment (11% ee). It was therefore decided to turn to kinetic
resolution. Pancreatin was utilized for the kinetic resolution of a
structurally related cyclopentenol,14 and also proved to be effective
in this case. Kinetic resolution provided acetate (+)-11a in high
optical purity (95% ee) and acceptable conversion (35%) (Scheme 3).
Alcohol (+)-7a, the enantiomer required, was not accessible
efficiently via kinetic resolution. Time-consuming repetitions
of kinetic resolutions were required to increase the conversions and
enantiomeric excess. It turned out to be advantageous to work with
acetate (+)-11 and invert its stereocenter via the Mitsunobu reaction.
This three-step procedure delivered (+)-7a in high optical purity
(95% ee). This material was converted to optically active thujone
(ꢀ)-1a (95% ee) as described in Scheme 2 (see the ESI†).
Scheme 1 Retrosynthetic analysis of a-thujone (1a) and its deuterated
derivative 1b.
Our synthesis commenced with the formation of dimethyl-
fulvene (Scheme 2). The preparation of 8a followed a procedure
described by Little et al., utilizing pyrrolidine as a base.9a
This procedure was not suitable for the preparation of the
d6-dimethylfulvene 8b, since the deuterium label was partially
removed, presumably via enamine formation. Therefore, the
procedure introduced by the group of Gajewski was utilized to
prepare d6-dimethylfulvene 8b.9b Using n-butyllithium as a base,
a deuteration degree of 95.5% was achieved. The dimethyl-
fulvenes formed were reduced with lithium aluminium hydride
to yield a mixture of cyclopentadienes. It is known that related
mixtures can be converted convergently to a single alcohol product
via the hydroboration/oxidation sequence.10 Indeed, such a proce-
dure yielded, after purification by chromatography, alcohols 7a
and 7b in 34% and 27% yield over three steps, respectively.
Cyclopropanation was performed utilizing the Furukawa modi-
fication of the Simmons–Smith reaction.11 After oxidation
After having developed a concise route to thujone (1a) and
its isotopically labelled derivative 1b, we turned to the pre-
paration of the most important metabolites. The oxidation of
1a to 7-OH-a-thujone (3a) was described in the literature
utilizing ozone as the oxidant.15 However, these conditions
lead to considerable overoxidation and a reduced yield of 3a
(47%). After screening several oxidants, we found that methyl-
(trifluoromethyl)dioxirane16 (TFDO, 12) led to a clean conversion
to 3a (Scheme 4). For the synthesis of the 4-hydroxy derivative 5a,
we followed the procedure of the group of Casida.17 First, the
Scheme 3 Kinetic resolution of (ꢁ)-3-isopropylcyclopent-3-en-1-ol and
inversion of the chiral center via Mitsunobu reaction.
Scheme 2 Synthesis of thujone 1a/1b.
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Scheme 4 Synthesis of the hydroxylated main metabolites.
3 K. M. Hold, N. S. Sirisoma, T. Ikeda, T. Narahashi and J. E. Casida,
Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 3826.
enolacetate is formed by refluxing thujone in isopropenyl acetate
under acidic conditions. In contrast to the literature, we observed
the 2,3-enol acetate 13a as the main product (13a :14a = 8 :2).
Therefore, we decided to utilize this mixture to obtain both the
2-hydroxy and the 4-hydroxy derivative at once. The inseparable
mixture was oxidized with 3-chloroperoxybenzoic acid. In both
cases epoxidation was preferred from the convex bottom face,
delivering after migration of the acetate group the desired diastereo-
isomers 15 and 16. The isomers were separated by chromatography,
and subsequently deprotected to yield the metabolites 4 and 5,
respectively. Deprotection of 15 was successful only under acidic
conditions, as regular basic hydrolysis led to decomposition of the
material.
¨
4 K. M. Hold, N. S. Sirisoma and J. E. Casida, Chem. Res. Toxicol., 2001,
14, 589–595.
5 European Council Directive No. 88/388/EEC, 22 June 1988 http://ec.
europa.eu/food/fs/sfp/addit_flavor/flav09_en.pdf (accessed on
23.11.2015).
6 F. W. Semmler, Ber. Dtsch. Chem. Ges., 1900, 33, 275–277.
7 W. Oppolzer, A. Pimm, B. Stammen and W. E. Hume, Helv.
Chim. Acta, 1997, 80, 623.
8 (a) H. E. Simmons and R. D. Smith, J. Am. Chem. Soc., 1958, 80, 5323;
(b) H. Lebel, J.-F. Marcoux, C. Molinaro and A. B. Charette, Chem.
Rev., 2003, 103, 977.
9 (a) K. J. Stone and R. D. Little, J. Org. Chem., 1984, 49, 1849;
(b) J. J. Gajewski, G. C. Paul, M. J. Chang and A. M. Gortva, J. Am.
Chem. Soc., 1994, 116, 5150.
10 S. Collins, Y. Hong, M. Kataoka and L. Nguyen The, J. Org. Chem.,
1990, 55, 3395.
11 J. Furukawa, N. Kawabata and J. Nishimura, Tetrahedron Lett., 1966,
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13 H. C. Brown and B. Singaram, J. Org. Chem., 1984, 49, 945.
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Chem. – Eur. J., 2012, 18, 11046; (b) O. R. Ludek and C. Meier,
Synthesis, 2003, 2101.
In summary, we have developed a concise route to a-thujone,
which relies on the functionalization of dimethylfulvene. The
synthesis allows for the facile incorporation of inexpensive isotopic
labels by utilizing d6-acetone as a starting material. Furthermore,
the three main metabolites of a-thujone were prepared.
15 J. P. Kutney, K. Piotrowska, Y.-H. Chen, K.-P. N. Cheng, Z. Gao and
S. J. Rettig, Can. J. Chem., 1990, 68, 1698.
Notes and references
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2 D. W. Lachenmeier, J. Emmert, T. Kuballa and G. Sartor, Forensic 17 N. S. Sirisoma, K. M. Hold and J. E. Casida, J. Agric. Food Chem.,
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2001, 49, 1915.
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