A Route to the Thapsigargins from (S)-Carvone Providing a Substrate-Controlled Total Synthesis of Trilobolide, Nortrilobolide, and Thapsivillosin F

Article abstract of DOI:10.1002/anie.200353140

An entirely substrate-controlled total synthesis of three members of the thapsigargin family (e.g. trilobolide) is achieved starting from (S)-carvone. The synthesis is linear in nature but is achieved in high yield (> 90% per step). The route permits late

Full text of DOI:10.1002/anie.200353140

Communications
Natural Products
A Route to the Thapsigargins from (S)-Carvone
Providing a Substrate-Controlled Total Synthesis
of Trilobolide, Nortrilobolide, and
Thapsivillosin F**
Steven F. Oliver, Klemens Högenauer, Oliver Simic,
Alessandra Antonello, Martin D. Smith, and
Steven V. Ley*
Investigations into the biologically active components of the
Mediterranean plant species Thapsia led to the isolation of
thapsigargin (1) and fifteen closely related guaianolides,
[*] Prof. S. V. Ley, Dr. S. F. Oliver, Dr. K. Högenauer, Dr. O. Simic,
A. Antonello, Dr. M. D. Smith
University Chemical Laboratory
University of Cambridge
Lensfield Road
Cambridge, CB2 1EW (UK)
Fax: (+44)1223-336442
E-mail: svl1000@cam.ac.uk
[**] This work was supported by a Wellcome Trust Research Fellowship
(to S.F.O.), a Marie Curie Fellowship (to K.H.), the Deutsche
Forschungsgemeinschaft (to O.S.), The Royal Society (to M.D.S.),
and Novartis Research Fellowships (to S.V.L.). We thank Prof. S. B.
Christensen for providing natural product samples for reference
purposes and Dr. John Davis for X-ray crystallographic analyses.
Early synthesis efforts by Dr. Helen Gold, Dr. Alison Woolford, and
Emily Balskus are also gratefully acknowledged.
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DOI: 10.1002/anie.200353140
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Angewandte
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collectively termed “thapsigargins”.[1] The members of this
family of sesquiterpene lactones have a highly oxygenated
tricyclic framework containing seven or eight stereogenic
centers and are functionalized with a range of different acyl
groups derived from acetic, butyric, angelic, and octanoic
acids. The trilobolide subfamily (2–4) lack the acyloxy
substituent at C2 and it is the first total synthesis of these
Despite the prevalence of the thapsigargins in the fields of
molecular biology and oncology (a scan of the past 10 years'
literature reveals almost 5000 hits for the keyword “thapsi-
gargin”), the synthetic community has been slow to reflect its
significance. To date, no total or partial synthesis of any
member of this family has been reported. Using degrada-
tion,[1] Christensen and co-workers have carried out a number
of modifications on the periphery of the natural substance to
investigate some of the important structure–activity relation-
ships. However, no modification of the core has been possible
using this strategy. A flexible synthetic route to the thapsi-
gargins would allow the preparation of simplified analogues
and permit the investigation of detailed structure–activity
relationships, which would not be otherwise possible.
Our general approach to the thapsigargins is outlined in
Scheme 1. To allow access to all 16 natural compounds in the
series and a number of potential analogues, our plan leads to
the common intermediate A (PG = tert-butyldiphenylsilyl
(TBDPS)) as the point of late-stage divergent functionaliza-
tion of the cyclopentane ring prior to sequential introduction
of the various ester functionalities required. We envisaged the
installation of the two quarternary centers at C7 and C11 by a
stereoselective cis-dihydroxylation of butenolide B. Assembly
of this lactone should be possible from the a-hydroxy ketone
C, which we planned to generate by an enol ether metathesis/
oxidation sequence from diene D. Cyclopentane E (PG =
tetrahydropyranyl (THP)), has already served as an inter-
mediate in a synthesis of two other guaianolides: cladantho-
lide and estafiatin.[9]
The synthesis begins with the stereoselective epoxidation
of commercially available (S)-(+)-carvone with basic hydro-
gen peroxide (Scheme 2).[10] Opening of the resulting oxirane
with lithium chloride in the presence of trifluoroacetic acid
(TFA)[11] led exclusively to chlorohydrin 5, which was
protected to yield a diastereomeric mixture of the THP
ethers 6. Treatment of 6 with sodium methoxide effected a
regio- and stereoselective Favorskii rearrangement to give
methyl ester 7 as the sole product. At this point, the
protecting group was exchanged to the more robust TBDPS
group (7!8). It should be noted that the Favorskii rearrange-
three thapsigargins that we report herein.
Interest in the thapsigargins arose after they were
recognized to be potent histamine liberators[2] and selective
and irreversible inhibitors of the ubiquitous sarco-endoplas-
mic reticulum Ca2+ ATP dependent pumps (SERCAs) up to
subnanomolar concentrations.[3] When applied to intact cells,
thapsigargin (1) is able to penetrate the cell where it binds to
and locks the SERCA into a conformation that has a poor
affinity for Ca2+ and ATP.[4] Trilobolide (2) (KD = 1.0 nm)
binds only 2.5 times less tightly to SERCA than 1 (KD =
0.4 nm).[5] Thapsigargins demonstrate a remarkable specificity
for the SERCA isozymes, and have hence become powerful
tools to manipulate and study intracellular Ca2+-dependent
signalling pathways.[6] Thapsigargin (1) has also been shown to
restore apoptotic function in cancer cell lines.[7] This has
recently led to the development of a potential treatment for
prostate cancer using a prodrug conjugate of 1 that is cleaved
and activated by prostate-specific antigen.[8]
Scheme 1. The synthesis plan. PG=protecting group, MOM=methoxymethyl, TES=triethylsilyl.
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mixture could be separated by column chromatography to
give the C10 epimeric alcohols 12a and 12b.
Continuing the synthesis, alcohol 12a was oxidized with
tetra-n-propylammonium perruthenate (TPAP)[13] and N-
morpholine-N-oxide (NMO) (Scheme 4) and the resulting
aldehyde was treated with the lithium anion of ethyl vinyl
Scheme 2. Synthesis of left-hand fragment 9. Reagents and conditions:
a) 1. H2O2, NaOH, MeOH, 108C, 88%, 2. LiCl, TFA, THF, RT, 95%;
b) DHP, PPTS cat., CH2Cl2, RT, 87%; c) NaOMe, MeOH, 08C, 95%,
d.r.>95:5; d) 1. PPTS cat., MeOH, 408C, 84%; 2. TBDPSCl, imidazole,
DMF, RT, 98%; e) 1. LAH, THF, 08C; 2. NaH, PMBCl, DMF, RT.
TFA=trifluoroacetic acid, DHP=dihydropyran, PPTS=pyridinium
p-toluenesulfonate, THP=tetrahydropyranyl, TBDPS=tert-butyl-
diphenylsilyl, PMB=p-methoxybenzyl.
Scheme 4. Synthesis of the a,b-unsaturated lactone 17. Reagents and
conditions: a) 1. TPAP cat., NMO, 4 Š MS, CH2Cl2, RT, 90%, 2.
ment with the TBDPS-protected alcohol was also investi-
gated, but the yields and selectivities were inferior to the THP
case. Reduction of ester 8, followed by protection of the
primary alcohol gave 4-methoxybenzyl ether 9.
As testament to the efficiency of this chemistry, no column
chromatography was necessary in this opening eight-step
sequence, allowing the preparation of more than 50 g of 9 per
batch.
=
CH2 CHOEt, tBuLi, THF, ꢀ788C, 96%, d.r. >95:5; b) TESCl, imida-
zole, DMF, RT, 95%; c) 2.5 mol% Grubbs' dihydroimidazolidine Ru
cat., CH2Cl2, reflux, 92%; d) K2OsO2(OH)4 cat., K3Fe(CN)6, NaHCO3,
MeSO2NH2, K2CO3, tBuOH, H2O, RT, 90%, d.r. =16:1; e) 1. HO2CCH-
(Me)P(O)(OEt)2, EDCI, CH2Cl2, RT; 2. NaH, THF, reflux, 79% over two
steps.
Osmylation and in situ diol cleavage provided the ketone
10 (Scheme 3). Addition of allyl magnesium bromide resulted
in the formation of an inseparable diastereomeric mixture of
homoallylic alcohols (d.r. = 3.5:1), with preference for the
Felkin product.[12] After MOM protection of the tertiary
alcohol (!11a,b) and removal of the PMB group, this
ether to give alcohol 13 as a single diastereoisomer. The
stereochemical outcome of this addition is again consistent
with the Felkin–Anh model. TES protection gave enol ether
14, which was subjected to the key ring-closing metathesis
reaction using only 2.5 mol% of Grubbs' dihydroimidazoline
ruthenium catalyst[14] to form the required cyclic enol ether 15
in remarkable yield.[15]Osmylation in the absence of a chiral
ligand led predominantly to the desired a-hydroxy ketone
epimer 16 (d.r. = 16:1), resulting from attack on the concave
face.[16] The C8 alcohol of 16 was then esterified with 2-
(diethoxyphosphoryl)propionic acid, and a subsequent intra-
molecular Horner–Wadsworth–Emmons reaction resulted in
the formation of butenolide 17.
Attempts to effect the intended syn-dihydroxylation of
lactone 17 proved unsuccessful, most likely due to a
combination of unfavorable steric and electronic factors.
After some experimentation, we found that carrying out the
osmium-mediated dihydroxylation of a reduced derivative
was possible (Scheme 5). Thus, formation of diol 18 by lithium
borohydride reduction of lactone 17 was followed by orthog-
onal protection of the primary and secondary alcohol
functions to give olefin 19. At this stage, the dihydroxylation
proceeded smoothly to give diol 20 as a single diaster-
eoisomer.[17] Concomitant removal of TES and acetate
protecting groups from 20 afforded the tetraol 21.
Scheme 3. Synthesis of the C10 epimeric alcohols 12a and 12b.
Reagents and conditions: a) 1. OsO4 cat., NMO, acetone, H2O, RT; 2.
NaIO4, RT, 74% over four steps; b) 1. AllylMgBr, THF, ꢀ788C, 99%,
d.r.=3.5:1; 2. MOMCl, DIPEA, DMAP cat., CH2Cl2, RT, 88%; c) DDQ,
aq. pH 7 phosphate buffer, CH2Cl2, RT, 93%. DIPEA=N,N-diisopropyl-
ethylamine, DDQ=2,3-dichloro-5,6-dicyano-1,4-benzoquinone,
DMAP=4-(N,N-dimethylamino)pyridine.
The carbon framework was completed by a highly
selective TPAP oxidation that first oxidized the primary
alcohol of 21, facilitated intermediate lactol formation, and
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Scheme 5. Completion of the tricyclic framework. Reagents and condi-
tions: a) LiBH4, THF, reflux, 92%; b) 1. Ac2O, DMAP, 2,6-lutidine,
CH2Cl2, RT, 95%, 2. MOMCl, DIPEA, DMAP, CH2Cl2, RT, 92%; c) 20
mol% K2OsO2(OH)4, quinuclidine, K2CO3, MeSO2NH2, K3Fe(CN)6,
tBuOH, H2O, RT, d.r.>95:5; d) K2CO3, MeOH, RT, 85% over two
steps; e) 10 mol% TPAP, 40 equivalents NMO, 4 Š MS, MeCN, RT,
73%.
Scheme 6. Synthesis of ketone 28. Reagents and conditions: a) Amber-
lyst-15, acetone, RT, 85%; b) 1. TBAF, THF, RT, 98%, 2. TPAP cat.,
NMO, 4 Š MS, CH2Cl2, RT, 93%; c) TMSCl, Et3N, DMF, 1208C, 88%;
d) dimethyldioxirane, acetone, CH2Cl2, 08C, 99%, d.r.>95:5;
e) TMSCl, Et3N, DMF, 1508C, 90%; f) 10 mol% PhSeBr, CH2Cl2, 08C
to RT, 94%.
effected a second oxidation to lactone 22 in a single trans-
formation. The 26-step sequence to the core 22 has been
carried out on multigram scale (to make > 10 g of 22) with a
minimum of chromatography. It is intended that this inter-
mediate will also serve as a precursor for a number of
designed analogues.
Removal of the MOM groups in 22 with simultaneous
acetonide formation gave 23 (Scheme 6). Of particular note
here is the observation that the acetonide protecting group
imparted remarkable chemical stability and crystallinity to all
tetracyclic products in the sequence thereafter. Consequently,
unmasking of the C3 hydroxy group and TPAP oxidation
furnished crystalline ketone 24, which provided proof for the
correct configuration of all stereocenters.
Generation of the C4,C5 a,b-unsaturation from 24 either
by a direct (IBX) or indirect (via enol ether) approach was
unsuccessful due to an inherent preference for enolization to
the less substituted (C2) side. However, a previous model
study carried out within our laboratory[18] had shown that
enolization in the desired sense could be achieved after
installation of the C2 hydroxy group.
Accordingly, ketone 24 was transformed into silyl enol
ether 25 which was oxidized with dimethyldioxirane to afford
a-silyloxy ketone 26. X-ray crystal structure analysis of 26
confirmed that oxygen incorporation occurred exclusively
from the convex face. Submitting ketone 26 to TMSCl/Et3N at
1508C afforded the tetrasubsitituted silyl enol ether 27 in
excellent yield.
bromide gave enone 28 as the sole product, in which the C2
silyloxy group has been lost. The exact mechanism of this
formal elimination reaction is still subject to investigation and
will be reported later.
Ketone 28 was then stereoselectively reduced with sodium
borohydride (d.r. = 4:1) to give alcohol 29 (Scheme 7). To
distinguish the five hydroxyl groups in the molecule, the end-
game strategy had been designed to minimize protecting
group manipulation and take advantage of the inherent order
of reactivity of the alcohols in a concise fashion. Accordingly,
the C3 hydroxy group was first esterified with angelic acid
under Yamaguchi conditions[19] to provide 29 before unmask-
ing the tertiary alcohols with TBAF to yield 30. Selective
acylation of the C10 hydroxy group was then achieved with
isopropenyl acetate mediated by polymer-supported tosic
acid. Removal of the acetonide led to triol 31. Finally,
esterification with (S)-2-methylbutyric anhydride occurred
exclusively at the secondary alcohol of C8 to provide
trilobolide (2). Reaction of 31 with butyric anhydride and
senecioic anhydride also furnished nortrilobolide (3) and
thapsivillosin F (4), respectively. All spectral and analytical
characteristics of synthetic 2–4 are in full accordance with the
data obtained from the samples from natural sources.
A range of standard conditions was then tested for the
oxidation of enol ether 27 to its corresponding a,b-unsatu-
rated ketone. This screening process led to the discovery of an
unprecedented catalytic selenium reaction that provides a
direct route to the trilobolide series of the thapsigargins.
Treatment of 27 with a catalytic quantity of phenylselenyl
In summary, the total synthesis of three thapsigargins has
been achieved. Stereocontrol during this synthesis relies
entirely upon efficient substrate control with all seven
stereocentres of the natural product core set from the initial
stereochemistry in (+)-carvone. The synthesis is robust
(average yield of 90.3% per step), amenable to scale up,
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[6] M, Treiman, C. Caspersen, S. B. Christensen, Trends Pharma-
col. Sci. 1998, 19, 131 – 135, and references therein.
[7] Y. Furuya, P. Lundmo, A. D. Short, D. L. Gill, J. T. Isaacs,Cancer
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[8] S. R. Denmeade, C. M. Jakobsen, S. Janssen, S. R. Khan, E. S.
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[10] K. C. Nicolaou, J. Y. Xu, S. Kim, J. Pfefferkorn, T. Ohshima, D.
Vourloumis, S. Hosokawa, J. Am. Chem. Soc. 1998, 120, 8661–8673.
[11] J. S. Bajwa, R. C. Anderson, Tetrahedron Lett.1991, 32, 3021–3024.
[12] The configuration of C10 and all subsequently formed stereo-
centers was unambiguously proven by X-ray analysis of 24 and
26 (vide infra).
[13] S. V. Ley, J. Norman, W. P. Griffith, S. P. Marsden, Synthesis
1994, 7, 639 – 666.
[14] M. Scholl, S. Ding, C. W. Lee, R. H. Grubbs, Org. Lett. 1999, 1,
953 – 956.
[15] Slow addition of catalyst was required to obtain high yield at low
catalyst loading. For examples of enol ether metathesis reactions
with Grubbs' Ru catalysts see: J. D. Rainier, J. M. Cox, S. P.
Allwein, Tetrahedron Lett. 2001, 42, 179–181; A. Okada, T .
Ohshima, M. Shibasaki, Tetrahedron Lett. 2001, 42, 8023–8027;
V. K. Aggarwal, A. M. Daly, Chem. Commun. 2002, 21, 2490–2491.
[16] Force field calculations suggest that the OTES group blocks the
convex face. Using AD mix a, 16 could be obtained as a single
diastereomer (matched case).
[17] An inconsequential side product resulting from protecting group
migration under the reaction conditions was also observed. As
its deprotection also gave tetraol 21, the separation from 20 was
not necessary.
[18] E. P. Balskus, University of Cambridge, unpublished results.
[19] B. Hartmann, A. M. Kanazawa, J. P. Depres, A. E. Greene,
Tetrahedron Lett. 1991, 32, 5077 – 5080.
Scheme 7. Completion of the synthesis of trilobolide, nortrilobolide,
and thapsivillosin F. Reagents and conditions: a) 1. NaBH4, MeOH,
08C, 86%, d.r.=4:1, 2. angelic acid, Et3N, 2,4,6-trichlorobenzoyl chlo-
ride, toluene, 808C, 76%; b) TBAF, THF, RT, quant; c) 1. isopropenyl
acetate, PS-TsOH, CH2Cl2, RT, 68%, 2. HCl (aq), MeOH, 408C; d) (S)-
2-methylbutyric anhydride, DMAP, CH2Cl2, RT, 78% over two steps;
e) butyric anhydride, DMAP, CH2Cl2, RT, 72% over two steps; f) sene-
cioic anhydride, DMAP, CH2Cl2, RT, 73% over two steps. TBAF=tetra-
butylammonium fluoride.
and its divergent nature will allow the syntheses of other
thapsigargins as well as a number of analogues. After
biological assessment of these compounds, further insight
into the remarkable biological properties of this substance
class should be possible.
Received: October 22, 2003 [Z53140]
Keywords: asymmetric synthesis · natural products ·
.
terpenoids · total synthesis
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