Iso-seco-tanapartholides: Isolation, synthesis and biological evaluation

Article abstract of DOI:10.1002/ejoc.200901016

The isolation, identification and total synthesis of two plant-derived inhibitors of the NF-κB signaling pathway from the iso-seco-tanapartholide family of natural products is described. A key step in the efficient reaction sequence is a late-stage oxidat

Full text of DOI:10.1002/ejoc.200901016

SHORT COMMUNICATION
DOI: 10.1002/ejoc.200901016
Iso-seco-tanapartholides: Isolation, Synthesis and Biological Evaluation
Edward F. Makiyi,[a] Raquel F. M. Frade,[b] Tomas Lebl,[a] Ellis G. Jaffray,[b]
Susan E. Cobb,[a] Alan L. Harvey,[c] Alexandra M. Z. Slawin,[a] Ronald T. Hay,*[b] and
Nicholas J. Westwood*[a]
Keywords: Natural products / Total synthesis / Terpenoids / Cleavage reaction / Inflammation
The isolation, identification and total synthesis of two plant-
derived inhibitors of the NF-κB signaling pathway from the
iso-seco-tanapartholide family of natural products is de-
scribed. A key step in the efficient reaction sequence is a
late-stage oxidative cleavage reaction that was carried out in
the absence of protecting groups to give the natural products
directly. A detailed comparison of the synthetic material with
samples of the natural products proved informative. Bio-
logical studies on synthetic material confirmed that these
compounds act late in the NF-κB signaling pathway.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
reaction of sesquiterpene lactone derived dienes with oxy-
gen followed by fragmentation of the resulting cyclic perox-
ides to form the seco or iso-seco structures.[5,7]
Introduction
The search continues for bioactive compounds that can
act as leads for drug discovery or as tools for biological
studies. Whilst the use of large chemical libraries is of im-
portance in this research area,[1] approaches based on natu-
ral products remain powerful.[2] Here we describe the total
synthesis of plant-derived inhibitors of the NF-κB signaling
pathway. This pathway plays a key role in inflammation,
immunology and cancer. Compounds that modulate its ac-
tivity have been linked to the treatment of several diseases.[3]
In the initial phase, bioactive plant-derived natural-prod-
uct extracts were identified by using an NF-κB-based re-
porter-gene assay.[4] Bioactivity-guided fractionation of one
of the extracts resulted in the isolation of what was believed
to be the relevant components. Spectroscopic analysis led
to structural assignment of the compounds as members of
a natural-product family that includes the seco-[5] and iso-
seco-tanapartholides (Figure 1).[6] These interesting struc-
tures are believed to be biosynthesised by a Diels–Alder
Figure 1. Members of the iso-seco-tanapartholide family of natural
products isolated from plants of the genus Artemisia and Achillea.
The total synthesis of members of this natural-product
family was then carried out to confirm their structure and
biological activity. A key step in our short and efficient se-
quence was a late-stage oxidative cleavage reaction that was
carried out in the absence of protecting groups to give the
natural products directly. A comparison of the previously
reported spectroscopic data associated with the natural
products proved interesting.
This study was completed by showing that the synthetic
[a] School of Chemistry and Biomedical Sciences Research Com- samples of the iso-seco-tanapartholides are indeed inhibi-
plex, University of St. Andrews,
tors of the NF-κB signaling pathway. We also present ex-
periments that have enabled us to propose a mode of action
for these inhibitors.
North Haugh, St. Andrews, Fife, KY16 9ST, United Kingdom
Fax: +44-1334-462585
E-mail: njw3@st-andrews.ac.uk
[b] Wellcome Trust Centre for Gene Regulation and Expression,
College of Life Sciences, University of Dundee,
Dow Street, Dundee, DD1 5EH, United Kingdom
E-mail: rthay@dundee.ac.uk
Results and Discussion
[c] Strathclyde Institute of Pharmacy and Biomedical Sciences,
University of Strathclyde,
High-throughput screening of plant-derived extracts was
carried out by using an HeLa57A cell line that expresses an
NF-κB-dependent reporter.[4,8] Failure to trigger this re-
27, Taylor Street, Glasgow, G4 ONR, United Kingdom
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.200901016.
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Figure 2. Sections of the 1H NMR spectrum of (a) the bioactive sample purified from extracts of the plant Tanacetum parthenium; signals
corresponding to two compounds are visible for the C3 proton and one each of the C2 and C13 protons; (b) synthetic sample of 1
(dotted line) and (c) synthetic sample of 2 (full line) corresponding to the major compound present in the sample from Tanacetum
parthenium.
porter on addition of phorbol 12-myristate 13-acetate indi- ried out at an earlier stage in the synthesis, a considerable
cated that the tested extract inhibited the NF-κB signaling increase in the total number of steps would be required. It
pathway. Whilst several extracts were active, follow-up stud- was therefore planned that the syn-diol unit in 7 would be
ies focused on an extract from the plant Tanacetum parthen- introduced by dihydroxylation of the γ,δ-double bond in
ium.[9] This extract was selected due to the robust nature of 8. This dihydroxylation was expected to proceed with high
the bioactivity observed despite concerns that natural prod- selectivity for the β-face of 8 providing the β-syn-diol 9
ucts with the same biological activity have been isolated (Figure 3). This assumption was based on other work from
previously from this plant, including the much studied par- our laboratory in which epoxidation of 8 with m-CPBA fur-
thenolide.[10] Three rounds of bioactivity-guided fraction- nished almost exclusively the β-epoxide.[8] With β-syn-diol
ation resulted in the isolation of an enriched fraction that 9 in hand it was expected that diastereoselective reduction
retained the desired activity.[8] Detailed spectroscopic analy- of the C3 ketone and installation of the C11–C13 exo-meth-
sis of this fraction[8] led to its assignment as the natural ylene group would lead to substrates suitable for oxidative
product, iso-seco-tanapartholide (reported structure 1,[6a,6b] cleavage studies.
Figure 1), and our plans then focused on how an authentic
sample of 1 might be prepared. However, in contrast to
previous reports, our analysis showed that the sample of 1
we had isolated was in fact a mixture of two compounds.
Figure 2a shows the observed signals assigned to the C3
proton and one each of the C2 and C13 protons and clearly
shows that two signals are present for each proton. The two
compounds present in the purified extract from Tanacetum
parthenium were tentatively assigned the epimeric structures
1 and 2 (Figure 1).
Spectroscopic methods alone could not be used to assign
the stereochemistry at C3 in the two proposed structures.
The remote nature of the C3 stereocenter and the flexibility
inherent in this structure contribute to the analysis chal-
lenges, as does the fact that it was not possible in our hands
to obtain pure samples of the two epimers at this stage.
Figure 3. Retrosynthetic analysis of iso-seco-tanapartholide.
This raised additional concerns about the existing structural
assignments within the iso-seco-tanapartholide family and
provided a further motivation for preparing authentic sam- carbonyl reduction reaction (Scheme 1).[11] However, for
ples of both 1 and 2. practical reasons, we were forced to revise this by carrying
Our initial plan involved the use of 9 directly in the C3
Retrosynthetically, we envisaged accessing the seco struc- out the reduction on a protected version of 9, the β-syn-
ture in 1 by oxidative cleavage of the C1,C10 syn-diol func- diol 10. Reduction of the C3 carbonyl group in 10 was ex-
tionality in 7 in the absence of protection of the C3 alcohol pected to occur under steric control with approach of the
and αβ-unsaturated lactone functional groups (Figure 3). reducing agent occurring from the opposite face to the two
We reasoned that if the oxidative cleavage reaction was car- protected alcohols giving 7 after removal of the silyl groups.
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Iso-seco-tanapartholides
As pure samples of both 1 and 2 were required, it was pro- silyl ethers 19 and 20 that were readily separable. The pure
posed that epimerisation at C3 by using Mitsonobu proto- diastereoisomers 19 and 20 were then carried through the
cols would be possible late in the synthesis; however, this subsequent steps independently.
proved unnecessary. It was planned to access the 5,7,5 guai-
The next challenge was to introduce the exocyclic α-
anolide system in 8 by photochemical rearrangement of the methylene group. Reaction of the lithium enolate generated
eudesmanolide (–)-α-santonin 11 to give 12 (Figure 3).[12] from 19 with diphenyl diselenide gave exclusively 21
The overall plan, if successful, would provide rapid access (Scheme 2A).[15] The stereochemical outcome of this reac-
to authentic samples of both 1 and 2.
tion results from pseudoaxial approach of the electrophile
and was confirmed by nOe studies on 21.[8] Oxidation of
the selenide in 21 with concomitant elimination gave 22 as
the only product due to the anti relationship between the
C11 selenoxide and the C7 proton preventing formation of
the C7–C11 double bond (Scheme 2A). Global desilylation
by using TBAF gave triol 7 in excellent yield, and – pleas-
ingly – 7 was then converted into 1 in 86% yield on treat-
ment with lead tetraacetate at 0 °C in dichloromethane.
Interestingly, attempts to carry out this transformation by
using sodium periodate returned only starting material,
probably due to the fact that again the O1–C1–C10–O10
torsional angle in 7 is too large and inflexible to enable
formation of the required cyclic periodate ester. Whilst lead
tetraacetate is known to cleave diols that cannot form the
corresponding cyclic ester intermediate, the rates of these
reactions are extremely slow.[16] Therefore, as cleavage of 7
is complete within just 20 min, this reaction probably oc-
Scheme 1. Dihydroxylation and reduction of the C3 carbonyl
group.
In practice, acid-mediated elimination of acetic acid from curs via the cyclic intermediate that can be formed in this
12,[13] obtained from 11,[12] provided 8 in accordance with
literature precedent. Subsequent dihydroxylation of 8 gave
syn-diol 9 as the major component in a 3:1 mixture with
13, with the reaction occurring exclusively at the γ,δ-double
bond in 8. For ease of handling, the diol functionality in
both 9 and 13 was protected before reduction of the C3
carbonyl group. Attempts to protect the diol unit in 9 and
13 as acetonides proved unsuccessful returning only the
starting materials. This lack of reactivity could be rational-
ised when the X-ray structures of subsequent intermediates
were obtained.[14] In the cases of 15–17, the seven-mem-
bered ring adopts conformations in which the hydroxy
groups are positioned such that the O1–C1–C10–O10 tor-
sional angles are 45°, 74° and 55°, respectively. The rela-
tively rigid nature of this framework presumably stops the
oxygen atoms moving from their ground state positions to
adopt the required conformation for successful aceton-
isation. However, protection of 9 and 13 was achieved by
treatment with TMSOTf yielding a 3:1 mixture of bis(TMS)
ethers 10 and 14. Reaction of 10 and 14 with sodium
borohydride provided two major isomers 15 and 16 that,
despite being difficult to separate from each other, were
readily separable from minor quantities of the other two
possible isomers 17 and 18 (ratio 15+16/17+18 = 85:15;
95% yield). When this purification was repeated on a larger
scale, pure samples of 15, 16 and 17 were isolated and their
structures assigned by X-ray analysis. Alcohol 15, the major
product from the reduction of 10, and likewise alcohol 16,
the major product from the reduction of 14, result from
hydride addition to the less hindered face of the respective
ketones, as expected. As 15 and 16 could not be separated
easily, reaction of the mixture with TBSOTf resulted in tri- tection and oxidative cleavage.
Scheme 2. Installation of the exocyclic double bond, global depro-
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R. T. Hay, N. J. Westwood et al.
SHORT COMMUNICATION
case due to the larger size of lead compared to iodine.[16]
Biological analysis[4,20] of synthetic 1 and 2 confirmed
When the reaction of 7 with lead tetraacetate was run for that they inhibited the TNFα activation of NF-κB [IC50(1)
extended times or at higher temperatures over-oxidation of = 7.7Ϯ0.6 µ; IC50(2) = 4.7Ϯ0.2 µ; Figure 4A] over a
1 was observed.[17]
concentration range similar to that observed for our iso-
In an analogous sequence of reactions, the trisilyl-pro- lated material.[8] To establish the mode of inhibition, cells
tected triol 20 (Scheme 1) was transformed via the selenide were treated with the NF-κB activator TNFα and levels of
23[8] into 24, a compound that contained the required exo- the NF-κB inhibitor IκBα determined by western blotting.
cyclic α-methylene group (Scheme 2B). Subsequent de- In response to TNFα, IκBα is rapidly degraded and then
silylation of 24 gave 25 which, in an analogous manner to resynthesized as the IκBα gene is NF-κB-dependent (Fig-
the conversion of 7 into 1, underwent rapid and clean oxi- ure 4B; DMSO vector). However, in the presence of 1 and
dative cleavage to yield 2 (72% over 4 steps). Comparison 2, IκBα was degraded, but resynthesis of IκBα was not ob-
1
of the H and 13C NMR spectra of synthetic 1 and 2 with served. This indicates that 1 and 2 do not prevent IκBα
that reported for iso-seco-tanapartholide isolated from degradation but block the transcriptional activity of NF-
plants of the genus Artemisia[6] proved interesting. The κB. Immunofluorescence studies were also consistent with
spectra for both 1 and 2 correlated very closely with those this assumption.[8] In addition, we determined the effect of
reported for the natural product. In the absence of authen- 1 and 2 on the DNA binding activity of NF-κB. Recombi-
tic material from these sources, further insight was gained nant-purified DNA-binding domains of the p50 and p65
by comparing the optical rotation of the natural material[6] subunits of NF-κB were incubated with 1 and 2 and a gel
with those of synthetic 1 and 2.[18] Despite the small values electrophoresis DNA-binding assay performed.[8] Both 1
involved, it appears that the relative and absolute stereo- and 2 inhibited NF-κB-DNA binding in a dose-dependent
chemistry assigned to iso-seco-tanapartholide from Artemi- fashion only when a thiol-based reducing agent was absent
sia was correct (as in 1).
from the assay. The observed loss of biological activity of 1
However, the situation was complicated when a compari- and 2 in the presence of a thiol is consistent with a mode
son of synthetic 1 and 2 with our material from T. parthen- of action for these compounds in which covalent modifica-
ium and a sample from a plant of the genus Achillea[19] was tion of cysteine residues in either the p50 or p65 subunit of
carried out. NMR studies (Figure 2) showed that the bioac- NF-κB occurs. In addition, a close analogue of 1 in which
tive material we had isolated from T. parthenium was indeed the exocyclic α-methylene group was replaced by a methyl
a mixture of the two epimers 1 and 2 with the major isomer group [C11-(S)] did not inhibit TNFα activation of NF-κB,
isolated from this plant having the same relative stereo- again suggestive of a role for the exocyclic α-methylene
chemistry as 2 [compare Figure 2a and c (full line)]. In ad- group in 1 as an electrophile. Interestingly, covalent modifi-
dition, comparison of our authentic samples of 1 and 2, cation of cysteine by inhibitors of NF-κB activation, includ-
with material isolated from a plant of the genus Achillea[19] ing the sesquiterpene lactones, has been proposed previous-
gave an analogous result, again confirming that the major ly.[4b,21] In particular, a computational model of the pro-
isomer present in this sample was epi-iso-seco-tanaparthol- posed covalent binding mode of the sesquiterpene lactone,
ide 2. Further optical rotation comparisons[8,18] also sup- helenalin, to Cys38 and Cys120 of the p65 subunit of NF-
ported our conclusion that the major isomer isolated from κB has been described.[22] Attempts to overlap the structure
Achillea had the same absolute stereochemistry as 2. For a of the iso-seco-tanapartholides onto this model suggest that
more detailed comparison of synthetic 1 and 2 with the the C3 functional group would be expected to point away
previous literature reports of their isolation, see Supporting from the protein. If correct, this would provide an explana-
Information.
tion for the observed disruption of the NF-κB-DNA inter-
Figure 4. Bioactivity of 1 and 2: (a) NF-κB-dependent reporter gene assay; (b) IκBα western blot analysis after TNFα activation.
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Iso-seco-tanapartholides
[6] a) Iso-seco-tanapartholide was assigned structure 1, see: S. Hu-
neck, C. Zdero, F. Bohlmann, Phytochemistry 1986, 25, 883–
889; b) J. A. Marco, J. F. Sanz-Cereva, E. Manglano, F. San-
cenon, A. Rustaiyan, M. Kardar, Phytochemistry 1993, 34,
1561–1564 {[α]2D0(iso-seco-tanapartholide) = +3.3 (c = 6.1, sol-
vent not mentioned)}; c) S. Oksuz, Phytochemistry 1990, 29,
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[7] F. Bohlmann, C. Zdero, Phytochemistry 1982, 21, 2543–2549.
[8] See Supporting Information for further details.
[9] It was also confirmed that the active fraction did not target the
reporter protein, firefly luciferase, by using an in vitro assay
(data not shown).
action by both 1 and 2, in a manner that is independent of
the C3 stereochemistry.
Conclusions
The bioactivity-guided fractionation of an extract from
Tanacetum parthenium by using an NF-κB-dependent lucif-
erase reporter gene assay is described. The purified extract
was shown to contain two natural products from the iso-
seco-tanapartholide family. Synthesis of authentic samples
of these two natural products by using an efficient oxidative
cleavage reaction clarified the structures of the compounds
isolated from a series of plants. This resulted in the isolation
and synthesis of the natural product epi-iso-seco-tanapar-
tholide (2) as well as iso-seco-tanapartholide (1). Biological
[10] L. M. Bedoya, M. J. Abad, P. Bermejo, Curr. Signal Transduc-
tion Ther. 2008, 3, 82–87.
[11] As expected, this led to the C3 epimer of 7 as the major prod-
uct.
studies on synthetic material confirmed that these com- [12] D. H. R. Barton, P. De Mayo, M. Shafiq, J. Chem. Soc. 1957,
929–935.
pounds act late in the NF-κB signaling pathway.
[13] P. Metz, S. Bertels, R. Fröhlich, J. Am. Chem. Soc. 1993, 115,
12595–12596.
Supporting Information (see footnote on the first page of this arti-
cle): Details of the Strathclyde natural-product extract collection,
screening results, bioactivity-guided purification, structural assign-
ment, experimental procedures, characterisation data for all new
compounds, comparison studies of synthetic 1 and 2 with pre-
viously reported natural products and material isolated from T.
parthenium and Achillea.
[14] CCDC-734445 (for 15), -734446 (for 16), -734447 (for 17), and
734449 (for S1)[8] contain the supplementary crystallographic
data for this paper. These data can be obtained free of charge
from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
[15] P. A. Grieco, M. Meyers, R. S. Finkelhor, J. Org. Chem. 1974,
39, 119–120.
[16] R. Criegee, E. Hoger, G. Huber, P. Kruck, F. Marktscheffel, H.
Schellenberger, Justus Liebigs Ann. Chem. 1956, 599, 81–125.
It cannot be ruled out that lead tetraacetate rapidly cleaves 7
and 25 without forming a cyclic ester intermediate and that the
speed of the reaction results from release of steric congestion
on cleavage of the C1–C10 bond. We thank a reviewer for rais-
ing this possibility.
Acknowledgments
We would like to thank The Scottish Funding Council, SULSA
and The Royal Society (NJW) for funding and Professor Mike Fer-
guson and Drs. C. Botting, A. McCarthy, and S. Fujihara for ad-
vice.
[17] In this reaction a compound tentatively assigned as the C3 oxo
derivative of 1 was formed.
[18] [α]2D0(1) = +2.9 (c = 0.8, CHCl3); [α]2D0(2) = –6.5 (c = 0.2
CHCl3).
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Received: September 6, 2009
Published Online: October 7, 2009
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