Enantioselective Synthesis of (+)-Chamaecypanone C: A Novel Microtubule Inhibitor

Article abstract of DOI:10.1002/anie.200805486

A bicycle built for tubulin: The Total synthesis of (+)- chamaecypanone C has been achieved by using a tandem retro-Diels-Alder cascade reaction (see scheme). Initial biological studies demonstrate that (+)- chamaecypanone C is an inhibitor of tubulin assembly and binds at the colchicine site.

Full text of DOI:10.1002/anie.200805486

Communications
DOI: 10.1002/anie.200805486
Natural Product Synthesis
Enantioselective Synthesis of (+)-Chamaecypanone C: A Novel
Microtubule Inhibitor**
Suwei Dong, Ernest Hamel, Ruoli Bai, David G. Covell, John A. Beutler, and John A. Porco, Jr.*
A number of bicyclo[2.2.2]octenone-containing natural prod-
ucts have been isolated from the heartwood of Chamaecy-
paris obtusa var. formosana (Scheme 1), including the Diels–
[2]
Alder (DA) adducts[1] obtunone (1)
and chamaecypa-
none C (2),[3] as well as the [4+2] dimer (+)-3.[2,4] (+)-2 has
been shown to exhibit potent cytotoxicity against several
human cancer cells, including human oral epidermoid carci-
noma (KB; IC50 = 190 nm).[3] The biosynthesis of 2 is pro-
posed[3] to occur through endo [4+2] cycloaddition between
cyclohexa-2,4-dienone 4 (Scheme 2) and 1,3-bisarylcyclo-
penta-1,3-diene 5, and subsequent oxidation to an enone—
in accordance with literature reports of cyclopentadienes as
biosynthetic precursors to natural products.[1b] An alternative
biosynthetic possibility involves the corresponding cyclo-
pentadienone 6 as the dienophile, and which may also be
considered in light of known biosyntheses that involve
reactive cyclopentadienones.[5] Herein, we report a concise
synthesis of both enantiomers of chamaecypanone C (2). The
synthesis involves a retro-Diels–Alder/Diels–Alder cascade
of dimer 3, which is obtained by utilizing copper-mediated
asymmetric oxidative dearomatization.[6] Also presented are
biological studies which document that the cytotoxic action of
(+)-chamaecypanone C involves mitotic arrest as a conse-
quence of its binding in the colchicine site of tubulin.
Scheme 1. Representative bicyclo[2.2.2]octenone-containing natural
products.
[*] S. Dong, Prof. Dr. J. A. Porco, Jr.
Scheme 2. Plausible biosyntheses of (+)-chamaecypanone C ((+)-2).
Department of Chemistry, Center for Chemical Methodology and
Library Development (CMLD-BU), Boston University
590 Commonwealth Avenue, Boston, MA 02215 (USA)
Fax: (+1)617-358-2847
E-mail: porco@bu.edu
Inspired by literature reports on tandem retro-DA/DA
reactions of dimers derived from ortho-quinols and masked
ortho-benzoquinones (MOBs),[7,8] we first evaluated reactions
between the readily accessible dimer (À)-3[6] and N-phenyl-
maleimide (7) under thermolytic reaction conditions in
different solvents (Table 1). Although reactions in toluene
(Table 1, entry 1) and chlorobenzene (Table 1, entry 2) gen-
erated the desired cycloadduct 8 in moderate to good
conversion (12 h), reactions in mesitylene at 1508C gave
both excellent conversion and yield of isolated 8 in 1.5 hours
(Table 1, entries 3 and 4).
By using these optimized reaction conditions, a number of
representative dienophiles were thermolyzed in the presence
of dimer (À)-3 in mesitylene (Table 2). Thermolysis reactions
with methyl vinyl ketone (9; Table 2, entry 1), 2,3-dihydro-
furan (10; Table 2, entry 2), and indene (11; Table 2, entry 3)
successfully generated bicyclo[2.2.2]octenones 12–14 in good
to excellent yields. These results underscore the reactivity of
ortho-quinols as both “normal” and “inverse-demand”
Dr. J. A. Beutler
Molecular Targets Development Program
National Cancer Institute at Frederick, MD (USA)
Dr. E. Hamel, Dr. R. Bai
Toxicology and Pharmacology Branch
National Cancer Institute at Frederick, MD (USA)
Dr. D. G. Covell
Screening Technologies Branch, Developmental Therapeutics
Program, Division of Cancer Treatment and Diagnosis
National Cancer Institute at Frederick, MD (USA)
[**] Financial support from the National Institutes of Health (GM-
073855 and P50 GM067041), Wyeth Pharmaceuticals, Merck
Research Laboratories, and the NCI intramural research program is
gratefully acknowledged. We thank the Developmental Therapeutics
Program at the NCI for the 60-cell assay, ThalesNano, Inc.
(Budapest, Hungary) for assistance with the continuous-flow
hydrogenation reactor, and Prof. Dr. Corey Stephenson (Boston
University) for helpful discussions.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200805486.
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acetylated to afford 16 and 17.[9] Hydrolysis of 16 (aq. NaOH/
Table 1: Optimization of the Retro-DA/DA Cascade.
MeOH) afforded optically pure ent-1.[2,10] Furthermore,
cyclopentadiene dimer 18 (Table 2, entry 5) was very reactive,
and afforded the [4+2] adduct 19 as a single diastereomer in
nearly quantitative yield.[7a] In contrast, use of cyclopentadie-
none dimer 20 (Table 2, entry 6) produced 21 only in
moderate yield, probably as a result of side reactions
(including decarbonylation) of 20 at high temperature.[11]
Based on our ability to trap (6S)-4 with a number of
dienophiles, we proceeded to evaluate both cyclopentadienes
and cyclopentadienones for the synthesis of 2. Accordingly,
we targeted a single starting material for the preparation of
both precursors. Starting from the known bisarylcyclopentene
derivative 22,[12] allylic oxidation using selenium dioxide
afforded alcohol 23 as the major product (50% yield) along
with a small amount of enone 24 (Scheme 3). Although diaryl
cyclopentadienes[13] were detected by GC-MS analysis under
acid-catalyzed dehydration conditions (cat. MP-TsOH, tolu-
ene, 1108C, 1 h),[14] all attempts to isolate pure product 25, or
trap it with reactive dienophiles (e.g. maleic anhydride,
tetracyanoethylene) failed. Moreover, thermolysis of the
crude mixture from either dehydration of 23 or base-
promoted elimination of the derived mesylate derivative
with dimer 3 also did not afford the desired cycloadduct 26.
Alternatively, allylic alcohol 23 could be efficiently
converted into cyclopentenone 24 using IBX as the oxidant[15]
(Scheme 4). After extensive experimentation, it was found
that oxidation of 24 using DDQ[16] in the presence of dimer 3
afforded the desired cycloadduct 27 in good yield. The
endo configuration of 27 was unambiguously assigned by
NOE experiments.[10] The transformation presumably pro-
ceeds through the initial formation of the reactive cyclo-
pentadienone 28 from cyclopentenone 24.[17] Unfortunately,
all efforts to isolate either the cyclopentadienone monomer or
derived dimers have thus far failed in control experiments.
Finally, treatment of 27 with BBr3 effected smooth demethy-
lation to afford (À)-chamaecypanone C ((À)-2; 86%). To the
best of our knowledge, this is the first example of the
generation of a 2,4-diarylcyclopentadienone and its usage in
natural product synthesis.[18] The instability and high reac-
tivity of the diarylcyclopentadienone intermediate[19] is likely
due to the relief of antiaromaticity upon cycloaddition as
suggested by Harmata et al.[20]
Entry
Solvent
T [8C]
Dienophile 7
t [h]
Conv. [%][a]
[equiv]
1
2
3
4
toluene
110
130
150
150
5
5
5
3
12
12
1.5
1.5
69
92
chlorobenzene
mesitylene
mesitylene
>99 (98)[b]
>99 (97)[b]
1
[a] Conversion is based on H NMR analysis of 8 and starting material
(À)-3. [b] Yield of isolated 8.
Table 2: Tandem retro-DA/DA reactions using bicyclooctenone (À)-3.[a]
Entry Dienophile
Equiv Cycloadduct
t
Yield
[h] [%][b]
1
2
5
3
92
20
12 84
3
10
10
3
4
99
42
4[d]
4
4
39
98
5
6
5
In a similar manner, we prepared (+)-chamaecypanone C
((+)-2; Scheme 5). Hydrogenation of 29 quantitatively gen-
erated 2,4-disubstituted phenol 30. An asymmetric hydroxyl-
ation/a-ketol rearrangement/dimerization sequence[6] afford-
ed (+)-dimer 3 in moderate yield over two steps (> 99% ee),
and which was further elaborated into (+)-chamaecypa-
none C (53%, over 2 steps from enone 24). Synthetic (+)-2
was confirmed as being identical to natural chamaecypano-
2.5
5
57
[a] Reaction conditions: dimer (À)-3, dienophile, mesitylene, 1508C.
[b] Yield of isolated product after column chromatography. [c] Approx-
1
ne C by comparison of H and 13C NMR spectra, the mass
1
imately 6% of an inseparable minor product was detected by H NMR
spectroscopy. [d] Acetylation was required for product separation.
spectrum, IR, and [a]D data, thus confirming its absolute
configuration.[10]
dienes. The observed regioselectivity for products 12–14 is in
agreement with those reported for related cyclohexadienones
(MOBs).[7c,d] Treatment of (À)-3 with b-myrcene (15; Table 2,
entry 4) smoothly generated an inseparable 1:1 mixture of
ent-obtunone (1) and a decalin product, both of which were
Both enantiomers of 2 were tested in the National Cancer
Instituteꢀs 60-cell single dose assay at 10À5 m. Confirming
earlier studies on the natural product,[3] (+)-2 inhibited tumor
cell growth by an average of 71%, while (À)-2 had no effect.
(+)-2 was then tested in a dose response format, where it
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displayed robust selectivity with
a mean
GI50 value of 0.21 mm. COMPARE analysis of
the data[21] at the total growth inhibition level
suggested that (+)-2 might act through inter-
ference with tubulin function, as high correla-
tions were seen to the data for seven estab-
lished tubulin inhibitors.[10] Examination of this
hypothesis by using an in vitro tubulin poly-
merization assay[22] found this to be the case,
with an IC50 value of 2.0 Æ 0.1 mm, while (À)-2
had no effect at 40 mm. (+)-2 was also tested for
inhibition of colchicine binding,[23] where it was
found to have moderate activity at 50 mm with
5 mm [3H]colchicine and 1 mm tubulin. Finally,
we confirmed that (+)-2 had an effect on cells
consistent with its inhibitory effects on the
assembly of tubulin. A cytotoxic concentration
of (+)-2 arrested cells in mitosis concordant
with inhibition of cell growth (Figure 1) and
caused the disassembly of the intracellular
microtubule network (Figure 2).[24]
Scheme 3. Reagents and conditions: a) SeO2 (0.5 equiv), TBHP (2 equiv), DCE, 608C,
2 h, 50% (23), and 5% (24); b) cat. MP-TsOH, toluene, 1108C, 1 h; or Martin sulfurane,
CH2Cl2, RT, 0.5 h; c) (À)-3 (0.2 equiv), mesitylene, 1508C. DCE=1,2-dichloroethane,
MP=macroporous polymer, TBHP=tert-butyl hydroperoxide, Ts=4-toluenesulfonyl.
In conclusion, we have accomplished the
total syntheses of (+)- and (À)-chamaecypa-
none C. The key transformation involved a Diels–Alder
cycloaddition between a diarylcyclopentadienone, which
was generated in situ, and a chiral ortho-quinol derived
from a retro-Diels–Alder reaction of its dimeric form. Initial
biological studies indicate that (+)-chamaecypanone C is a
potent tumor cell growth inhibitor[3] that acts primarily
through inhibition of tubulin polymerization. Further studies
Scheme 4. Reagents and conditions: a) IBX (2.0 equiv), toluene/
DMSO (1m, 2:1), 508C, 30 min, 90%; b) (À)-3 (1.5 equiv), DDQ
(2.0 equiv), o-dichlorobenzene, 1508C, 1 h, 61%; c) BBr3 (8.0 equiv)
CH2Cl2, À788C to RT, 4 h, 86%. DDQ=2,3-dichloro-5,6-dicyanobenzo-
quinone, DMSO=dimethyl sulfoxide, IBX=o-iodoxybenzoic acid.
Figure 1. Human Burkitt lymphoma CA46 cells, obtained from the
American Type Tissue Collection, were grown in a suspension culture
for 24 hours at 378C in a humidified, 5% CO2 atmosphere. The
medium was RPMI 1640 supplemented with 5% fetal bovine serum.
Initially, the culture medium contained 20000 cellsmLÀ1. For determi-
nation of the cell growth, the increase in cell number was determined
with the cells counted in a model Z1 particle counter (Beckman
Coulter). For determination of mitotic cells, cells were harvested by
centrifugation, briefly swollen in a hypotonic solution, fixed on a glass
slide, and stained with Giemsa. The percentage of cells with con-
densed chromosomes was then determined.
Scheme 5. Reagents and conditions: a) H-Cube (Pd/C), H2 (40 bar),
MeOH (0.03m), 508C, 0.5 mLminÀ1, quantitative; b) LiOH·H2O
(1.0 equiv), EtOH/toluene, azeotrope; Cu(CH3CN)4PF6 (2.2 equiv),
(À)-sparteine (2.3 equiv), 3 ꢀ molecular sieves, O2, THF, À788C, 16 h;
c) benzene, reflux, 12 h, 47% (over 2 steps). H-Cube=continuous-flow
hydrogenation reactor, THF=tetrahydrofuran.
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[17] For syntheses of cyclopentadienones from cyclopentenones, see:
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Figure 2. Disruption of the intracellular microtubule network by cha-
maecypanone C. Potorus tridactylis PtK2 kidney epithelial cells were
obtained from the American Type Culture Collection and were cultured
in minimal essential medium supplemented with 10% fetal bovine
serum, 1 mm glutamine, and 1 mm sodium pyruvate. The cells were
grown to confluence, disrupted by trypsinization, and seeded at about
35000 cells into each compartment of a chambered coverglass system
(Nunc) with A) no compound or B) 0.5 mm of (+)-chamaecypanone C
added to the culture medium. After growth for 16 h at 378C in a
humidified, 5% CO2 atmosphere, the cells on the coverglass were
fixed with acetone at À208C, washed with phosphate-buffered saline,
and stained with a DNA stain and with a monoclonal antibody to b-
tubulin conjugated to the fluorescent dye Cy3 (Sigma product C4585:
instructions provided by the manufacturer were followed). The cover-
glass was mounted on a slide with antifade mounting solution and
examined in a Nikon Eclipse E800 microscope with a 100-times oil
objective and by using appropriate epifluorescence accessories.
Images were captured with a Spot digital camera. The scale bar shown
in A) represents 20 mm.
[18] For representative syntheses of cyclopentadienones, see: a) P. A.
Wender, T. J. Paxton, T. J. Williams, J. Am. Chem. Soc. 2006, 128,
14814 – 14815, and references therein; for preparation and
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615; for a total synthesis of the 2,3-substituted cyclopentadie-
none-derived natural product manzamenone A, see Ref. [5].
[19] The presumed cyclopentadienone 28 was also successfully
on the preparation of (+)-chamaecypanone C analogues by
using a retro-DA/DA cascade process, as well as biological
evaluation of these compounds, are currently in progress and
will be reported in due course.
Received: November 10, 2008
Published online: January 12, 2009
trapped with dimethyl acetylenedicarboxylate to afford
a
tetrasubstituted aromatic product.[10] For examples of cyclo-
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Keywords: cycloaddition · cyclopentadienone ·
microtubule inhibitor · natural product · total synthesis
.
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