Anticancer agents from the Australian tropical rainforest: Spiroacetals EBC-23, 24, 25, 72, 73, 75 and 76

Article abstract of DOI:10.1002/chem.200901525

EBC-23, 24, 25, 72, 73, 75 and 76 were isolated from the fruit of Cinnamomum laubatii (family Lauraceae) in the Australian tropical rainforests. EBC-23 (1) was synthesized stereoselectively, in nine linear steps in 8% overall yield, to confirm the reported relative stereochemistry and determine the absolute stereochemistry. Key to the total synthesis was a series of Tietze - Smith linchpin reactions. The novel spiroacetal structural motif, exemplified by EBC-23 (1), was found to inhibit the growth of the androgen-independent prostate tumor cell line DU145 in the mouse model, indicating potential for the treatment of refractory solid tumors in adults.

Full text of DOI:10.1002/chem.200901525

FULL PAPER
DOI: 10.1002/chem.200901525
Anticancer Agents from the Australian Tropical Rainforest: Spiroacetals
EBC-23, 24, 25, 72, 73, 75 and 76
Lin Dong,[a] Heiko Schill,[a] Rebecca L. Grange,[a] Achim Porzelle,[a] Jenny P. Johns,[b]
Peter G. Parsons,[b] Victoria A. Gordon,[c] Paul W. Reddell,[c] and Craig M. Williams*[a]
Abstract: EBC-23, 24, 25, 72, 73, 75
and 76 were isolated from the fruit of
Cinnamomum laubatii (family Laura-
ceae) in the Australian tropical rainfor-
ests. EBC-23 (1) was synthesized ste-
reoselectively, in nine linear steps in
8% overall yield, to confirm the re-
ported relative stereochemistry and de-
termine the absolute stereochemistry.
Key to the total synthesis was a series
of Tietze–Smith linchpin reactions. The
novel spiroacetal structural motif, ex-
emplified by EBC-23 (1), was found to
inhibit the growth of the androgen-in-
dependent prostate tumor cell line
DU145 in the mouse model, indicating
potential for the treatment of refracto-
ry solid tumors in adults.
Keywords: anticancer agents · Aus-
tralian tropical rainforests · natural
products · total synthesis
Introduction
In the course of undertaking a screening program in the
north east Australian tropical rainforest to identify biologi-
cally active candidates as potential treatments for cancer, a
group of novel spiroketals were found in the fruit of Cinna-
momum laubatii (family Lauraceae). Herein, we report the
isolation, structure elucidation and biological activity of
EBC-23, 24, 25, 72, 73, 75 and 76 (Figure 1) along with the
total synthesis of EBC-23 (1).[1]
Figure 1. EBC-23 (1) and related family members.
[a] Dr. L. Dong, Dr. H. Schill, Dr. R. L. Grange, Dr. A. Porzelle,
Dr. C. M. Williams
School of Chemistry and Molecular Biosciences
University of Queensland
Results and Discussion
Brisbane, 4072, Queensland (Australia)
E-mail: c.williams3@uq.edu.au
Both the frozen exocarp and seed from the fruit of Cinna-
momum laubatii were extracted. The exocarp gave EBC-23
(1), EBC-24 (3), EBC-25 (4), EBC-72 (5), EBC-73 (8),
EBC-75 (6) and EBC-76 (7), whereas EBC-23 (1), EBC-24
(3) and EBC-25 (4) were also obtained from the seed.
[b] J. P. Johns, Dr. P. G. Parsons
EcoBiotics Limited, PO Box 1
Yungaburra, 4884, Queensland (Australia)
www.ecobiotics.com.au
[c] Dr. V. A. Gordon, Dr. P. W. Reddell
Queensland Institute of Medical Research
PO Royal Brisbane Hospital
LRESIMS analysis of EBC-23 (1) proved difficult, howev-
er, derivatization with acetic anhydride provided the diace-
tyl derivative (2), which displayed a single signal on LR-
Brisbane, 4029, Queensland (Australia)
AHCTUNGTRENNUNG
ESIMS analysis ([M+Na]+, m/z 559) confirming the LR-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200901525.
AHCTUNGTRENNUNG
ESIMS ([M+Na]+, m/z 475) molecular ion. IR absorption
Chem. Eur. J. 2009, 15, 11307 – 11318
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11307

bands at 3448 and 1729 cmꢀ1 indicated hydroxy and lactone
carbonyl groups. HRESIMS analysis of 1 ([M+Na]+, m/z
475.3024), in conjunction with a remarkably well deconvo-
luted 13C NMR spectrum in the alkyl region, established a
molecular formula of C26H44O6. The 1H NMR (Table 1)
spectrum displayed characteristic resonances for a saturated
long chain (dH 1.2) and associated primary methyl (dH 0.9,
J=7.2 Hz, t), a one proton coupled AB system (dH 2.4), five
oxygenated methines (dH 3.8, 4.1, 4.4, 4.5, and 5.0), and two
olefinic protons (dH 6.2 and 6.9). The 13C NMR (Table 2)
spectra revealed 23 of the 26 carbons, which in combination
with DEPT measurements were found to be one methyl, 13
methylenes, 7 methines (2 olefinic and 5 oxygenated), one
spiroacetal and one a,b-unsaturated lactone carbonyl
carbon. Further evaluation of the a,b-unsaturated lactone
1
moiety with H,1H COSY, HSQC and HMBC experiments
suggested a syn-disubstituted six-membered a,b-unsaturated
lactone of type 9. Comparison of 9 with the known natural
product osumundalactone[2] 10 and the synthetic syn-
isomer[3] 11 confirmed, along with the NOESY spectrum,
the presence of fragment 9 (i.e., two olefinic dd patterns for
10, and only one dd and one d pattern for 11 matching that
of 1) (Figure 2).
1
Key H,1H COSY and HMBC correlations, in conjunction
with the ACD/labs software,[4] established the position of
the AB system (C7) in relation to the lactone ring and the
spiroacetal carbon (C8). In turn the spiroacetal carbon (C8)
initiated the connectivity and substitution of the tetrahydro-
Table 1. Proton-NMR data for EBC-23, 24, 25, 72, 75, and 76.
EBC-23
(750 MHz)
EBC-24
(500 MHz)
EBC-25
(500 MHz)
EBC-72
(500 MHz)
EBC-75
(500 MHz)
EBC-76
ACHTUNGTRENN(UNG 500 MHz)
G
G
E
G
U
3-H
4-H
5-H
6.21 (d, 1H,
J=10.0 Hz)
6.89 (dd, 1H,
J=10.0, 5.2 Hz)
4.51 (dd, 1H,
J=5.2, 4.5 Hz)
6.20 (d, 1H,
J=9.9 Hz)
6.88 (dd, 1H,
J=9.9, 5.2 Hz)
4.50 (dd, 1H,
J=5.2, 4.6 Hz)
6.17 (dd, 1H,
J=9.9, 0.4 Hz)
6.86 (dd, 1H,
J=9.9, 5.2 Hz)
4.51 (ddd, 1H,
J=5.2, 4.6, 0.4 Hz)
6.18 (dd, 1H,
J=9.9, 0.5 Hz)
6.87 (dd, 1H,
J=9.9, 5.1 Hz)
4.52 (ddd, 1H,
J=5.2, 4.6, 0.4 Hz)
6.20 (dd, 1H,
J=9.9, 0.3 Hz)
6.88 (dd, 1H,
J=9.9, 5.2 Hz)
4.52 (ddd, 1H,
J=5.2, 4.6,
0.3 Hz)
6.23 (d, 1H,
J=9.9 Hz)
6.91 (dd, 1H,
J=9.9, 5.2 Hz)
4.52 (dd, 1H,
J=5.2, 4.6 Hz)
6-H
5.04 (ddd, 1H,
J=6.9, 4.5, 2.5 Hz)
5.03 (ddd, 1H,
J=6.9, 4.6, 2.5 Hz)
5.00–5.05 (m, 1H)
5.01–5.05 (m, 1H)
5.03 (ddd, 1H,
J=6.9, 4.6,
2.5 Hz)
5.06 (ddd, 1H,
J=6.9, 4.6, 2.4 Hz)
2.54 (a, dd, 1H,
J=14.9, 6.9 Hz)
2.30 (b, dd, 1H,
J=14.9, 2.5 Hz)
1.98–2.05 (m, 2H)
2.53 (a, dd, 1H,
J=14.9, 6.9 Hz)
2.28 (b, dd, 1H,
J=14.9, 2.5 Hz)
1.99–2.03 (m, 2H)
2.49 (a, dd, 1H,
J=14.6, 6.9 Hz)
2.22 (b, dd, 1H,
J=14.7, 2.7 Hz)
1.93–1.98 (m, 2H)
2.50 (a, dd, 1H,
J=14.6, 7.0 Hz)
2.22 (b, dd, 1H,
J=14.6, 2.6 Hz)
1.90–1.99 (m, 2H)
2.54 (a, dd, 1H,
J=14.9, 6.8 Hz)
2.22 (b, dd, 1H,
J=14.9, 2.5 Hz)
1.99–2.03 (m,
2H)
4.07–4.13 (m,
1H)
1.74–1.80 (m,
1H)
2.56 (a, dd, 1H,
J=14.9, 6.9 Hz)
2.31 (b, dd, 1H,
J=14.9, 2.5 Hz)
2.01–2.05 (m, 2H)
10-H
11-H
4.09–4.13 (m, 1H)
1.75–1.80 (m, 1H)
4.07–4.13 (m, 1H)
1.73–1.80 (m, 1H)
4.04–4.11 (m, 1H)
1.70–1.76 (m, 1H)
4.05–4.11 (m, 1H)
1.71–1.77 (m, 1H)
4.09–4.16 (m, 1H)
1.79 (dddd, 1H,
J=14.0, 3.2, 1.8,
1.7 Hz)
1.47–1.52 (m, 1H)
4.35–4.40 (m, 1H)
1.45–1.53 (m, 1H)
4.33–4.40 (m, 1H)
1.36–1.44 (m, 1H)
1.37–1.44 (m, 1H)
1.47–1.52 (m,
1H)
4.33–4.40 (m,
1H)
1.51 (ddd, 1H,
J=14.0, 11.7, 2.8 Hz)
4.39 (dddd, 1H,
J=16.0, 6.7, 2.2,
2.0 Hz)
13-H
4.17 (dddd, 1H,
J=11.6, 9.2, 4.3,
2.2 Hz)
4.18 (dddd, 1H,
J=11.6, 9.2, 4.3,
2.2 Hz)
15-H
16-H
1.56–1.65 (m, 2H)
3.76–3.82 (m, 1H)
1.54–1.66 (m, 2H)
3.75–3.81 (m, 1H)
5.31–5.45 (m, 2H)
1.90–2.00 (m, 4H)
1.47–1.55 (m, 2H)
1.47–1.56 (m, 2H)
1.54-1.66 (m,
2H)
3.74–3.82 (m,
1H)
5.30–5.45 (m,
2H)
1.90–1.99 (m,
4H)
1.55–1.66 (m, 2H)
4.95–5.05 (m, 1H)
4.96–5.05 (m, 1H)
5.28–5.45 (m, 2H)
1.90–1.99 (m, 4H)
3.78–3.84 (m, 1H)
5.32–5.47 (m, 2H)
1.93–2.01 (m, 4H)
A
H
A
H
w-H
0.86 (t, 3H,
J=7.1 Hz)
1.42–1.47 (m, 1H)
1.35–1.42 (m, 2H)
1.20–1.33 (m, 21H)
0.94 (t, 3H,
J=7.4 Hz)
1.34–1.47 (m, 2H)
1.19–1.34 (m, 18H)
0.85 (t, 3H,
J=6.9 Hz)
1.76–1.87 (m, 1H)
1.58 (dt, 1H,
J=14.5, 4.5 Hz)
1.17–1.31 (m, 22H)
0.94 (t, 3H,
J=7.4 Hz)
1.77–1.85 (m, 1H)
1.56–1.63 (m, 1H)
1.17–1.34 (m, 14H)
0.93 (t, 3H,
J=7.4 Hz)
1.35–1.40 (m,
1H)
1.40–1.47 (m,
2H)
0.96 (t, 3H,
J=7.4 Hz)
1.36–1.45 (m, 3H)
1.22–1.36 (m, 21H)
alkyl-H
1.19–1.34 (m,
13H)
acetyl-H
OH
2.00 (s, 3H)
3.11 (d, 1H,
J=9.2 Hz)
2.00 (s, 3H)
3.11 (d, 1H,
J=9.2 Hz)
3.05 (d, 1H,
J=9.2 Hz) 2.96 (s,
1H)
3.06 (d, 1H,
J=9.2 Hz) 2.96 (s,
1H)
3.07 (brs, 2H)
3.07 (d, 1H,
J=9.1 Hz) 2.98 (s,
1H)
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Anticancer Agents
FULL PAPER
Table 2. Carbon-NMR data for EBC-23, 24, 25, 72, 75, and 76.
EBC-23
(125 MHz)
EBC-24
(125 MHz)
EBC-25
(125 MHz)
EBC-72
(125 MHz)
EBC-75
(125 MHz)
EBC-76
ACHTUNGTRENN(UNG 125 MHz)
G
G
E
G
N
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-10
C-11
C-12
C-13
C-15
C-16
C-(w-4)
C-(w-3)
C-(w-2)
C-(w-1)
C-w
161.0
124.6
138.6
68.9
78.8
47.7
106.6
38.8
64.2
37.7
67.6
42.2
71.8
161.0
124.5
138.6
68.8
78.8
47.7
106.5
38.7
64.2
37.7
67.7
42.2
71.8
32.5
129.3
131.8
25.6
161.2
124.2
139.1
68.6
79.0
47.5
106.4
38.7
64.3
37.5
64.3
34.6
71.7
161.3[a]
124.2
139.1
68.7
79.1
47.6
106.5
38.7
64.4
37.6
64.4
34.6
71.7
32.6
129.3
131.9
25.6
161.1
124.6
138.7
68.9
78.8
47.7
106.6
38.8
64.2
37.7
67.7
42.2
71.8
32.6
129.4
131.9
25.6
14.0
161.0
124.6
138.6
68.9
78.8
47.7
106.6
38.8
64.2
37.8
67.8
42.2
71.9
32.6
129.4
131.9
25.6
14.0
14.1
14.0
14.1
14.0
alkyl-C
37.7, 31.9, 29.67 (2),
37.7, 29.66, 29.63,
39.8, 31.9, 29.64,
39.9, 29.66, 29.53 (3), 37.7, 29.68, 29.66,
37.8, 31.9, 29.70,
29.65 (3), 29.63, 29.61, 29.62, 29.59 (2), 29.56, 29.62, 29.61 (2), 29.60, 29.50, 29.16, 25.1
29.61, 29.55, 29.51, 9.68 (3), 29.64 (2), 29.62,
29.58, 29.3, 25.4,
22.7
29.49, 29.15, 25.4
29.54, 29.49, 29.3,
25.1, 22.6
29.2, 25.5
29.54, 29.2, 25.5
acetyl-C
170.5, 21.4
170.6, 21.4
[a] Not visible in 13C spectrum, chemical shift obtained from HMBC spectrum.
and H16 (dH 3.8) defined both configurations at C11 and
C16 (i.e., H16b).
To ascertain the proposed configuration at C8, geometry
optimizations at the B3LYP/6-31G* level of theory[8] were
undertaken for isomers having the proposed and inverted
configuration at C5/6, C11, and C16.[9] To estimate the effect
of anomeric stabilization, geometry optimizations were also
performed on a set of isomers having the inverted configura-
tion at C8. It became apparent, that the isomer shown in
Figure 3 possessed the lowest energy of all studied isomers.
Each isomer with the inverted configuration at C8 was pre-
dicted to have a higher energy than the corresponding
epimer with the proposed configuration, although analysis
of the results was complicated by possible hydrogen bonding
between the hydroxyl group on C16 to the ketone part of
the enlactone moiety and the fact that inverting the six-
membered ring would lead to another chair-like conformer,
again benefiting from anomeric stabilization. Interestingly,
inversion at C11 and/or C16 was predicted to lead to the
Figure 2. Comparison of suspected syn-configuration 9 to that of osumun-
dalactone 10 and syn-synthetic isomer 11.
pyran ring supported by the carbon chemical shift value (dC
106.5) suggesting a [6.5]-[5] anomeric[6] over a [6.6]-[7] spiro-
ketal system. The remaining correlations in the 1H,1H
COSY, HSQC and HMBC spectra ascribed the location of
the hydroxyl substituted (C16) long chain alkyl group at po-
sition C13 of the tetrahydropyran ring (Figure 3). NOESY
and 1D NOE experiments suggested H5 (dH 4.5) correlated
with H13 (dH 4.4), which in turn correlated with H16 (dH
3.8). This indicated that the stereochemistry at the spiroace-
tal carbon (C8) was EE and the side chain hydroxyl was a
configured. Even though a syn correlation was present be-
tween H13 (dH 4.4) and H12b
(dH 1.8) the corresponding cor-
relation to H11 (dH 4.1) was
ambiguous. When NMR meas-
urements were performed on a
much more diluted sample a
sharp doublet (dH 3.05) and
sharp singlet (dH 2.95) ap-
peared for the two hydroxyl
groups. Consequently, this de-
velopment with NOE correla-
tions between H13 (dH 4.4),
the C11 hydroxyl hydrogen
Figure 3. HMBC correlations between the proton (tail) and the carbon (head), COSY correlations and signifi-
cant NOE correlations.
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C. M. Williams et al.
same increase in relative energy for both possible configura-
tions at C5/6, respectively.
With the elucidation of the parent molecule complete
other EBC-23 family members (namely 24, 25, 72, 73, 75
and 76) were easily identified and elucidated. The main dif-
ferences in structure from that of EBC-23 (1) were attribut-
ed to side chain length, acetylation and points of unsatura-
tion. Side chain length information could be garnered from
LRESIMS (and HRESIMS), whereas acetylation could be
Figure 4. Possible biosynthetic pathway to EBC-23 (1).
1
deduced from H NMR chemical shift data and absolute lo-
lactone (18) as the right-hand fragment. The synthetic ap-
proach to the left-hand fragment 16 must contain sufficient
stereochemical flexibility in the event that the relative ste-
reochemical assignment of 1 is incorrect, or structure–activi-
ty studies (SAR) are required. For this task multiple options
are available, for example, via asymmetric aldol chemistry,
reiterative[16] Bartlett iodo carbonate cyclization (i.e., 19),[17]
or again Tietze[13]–Smith[14] linchpin methodology to access
ketone 23 from dithiane 20 and epoxides 21 and 22
(Scheme 1).
cation inferred from the absence of H16 (dH 3.8) seen in
EBC-23 (1). Unsaturation occurred two carbons from the
end of the chain in all cases [that is, EBC-24 (3), EBC-72
(5), EBC-75 (6) and EBC-76 (7)] as realized from HMBC
correlations. In only one case [that is, EBC-73 (8)] was aro-
matic functionality observed as part of the side chain. This
was confirmed as a meta-substituted benzodioxole, due to
the signature aromatic splitting pattern, and the dioxy-
methylene NMR chemical shift (dH 5.9; dC 100).
For the purpose of confirm-
ing the relative and determin-
ing absolute stereochemistry,
and reaffirming the biological
activity, a synthetic campaign
was initiated to concisely con-
struct both enantiomers of
EBC-23 (1). In this light it was
beneficial to postulate a bio-
synthetic pathway. The EBC-
23 (1) structural motif could
be imagined by extension of
that known from the related
polyketide lactones already
isolated from the Lauraceae
family.[10] If a precursor such as
12, which closely resembles
passifloricin B albeit missing
one hydroxyl group,[11] under-
goes regioselective oxidation,
followed by elimination, allylic
oxidation
EBC-23
and
(1)
cyclization,
realized
Scheme 1. Retrosynthetic analysis of EBC-23 (1).
is
(Figure 4). Starting with the re-
gioselective oxidation, in the aforementioned sequence,
would be pivotal so as to prevent Michael addition of hy-
droxyl groups to the enlactone, as observed in related natu-
ral products.[12]
Synthetic studies began by targeting the all-R isomer. To
access the left-hand fragment 16 scalable amounts of enan-
tiopure epoxide 24 were required.[18] Initially Jacobsen epox-
ide resolution methodology[19] was utilized, but this was later
superseded by the method of Lepoittevin,[20] which provided
24 in large quantities and in high enantiomeric excess start-
ing from (R)-epichlorohydrin (25) (Scheme 2).
In consideration of the above postulated biosynthetic
origin retrosynthetic analysis was best initiated by ring open-
ing of the spiroacetal moiety arriving at a straight chain po-
lyketide 13. Keto functionality seen in 13 serves as a point
of fusion, as shown in 14, via an acyl equivalent (15). A
Tietze[13]–Smith[14] linchpin convergence strategy with epox-
ides 16 and 17 would satisfy this criteria, although subse-
quent ring-closing metathesis would be required to install
the lactone.[15] A slight deviation of this approach could di-
rectly utilize the fully constructed, and suitably substituted,
Scheme 2. a) CH
3A
NaOH, MeOH, THF, H2O, RT, 3 h, 91%. THF=Tetrahydrofuran,
MeOH=Methanol.
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Anticancer Agents
FULL PAPER
In the first instance the reiterative[16] Bartlett iodo carbon-
ate cyclization[17] approach was applied to the synthesis of
the left-hand fragment 29. Reaction of epoxide 24 with vinyl
magnesium bromide followed by treatment with di-tert-butyl
dicarbonate afforded 26, which on treatment with iodine
bromide gave iodo carbonate 27. Treatment of carbonate 27
with base followed by tert-butyldimethylsilyl chloride pro-
duced epoxide 28. Reiteration of this process facilitated con-
version of 28 into the desired material 29 (Scheme 3).
one carbon unit linker. Epoxide 29 was reacted with the
anion of TBS-dithiane 37, which underwent the first addi-
tion step of the linchpin reaction followed by the Brook re-
arrangement.[23] Albeit through much effort the second step
of the linchpin reaction, that is, addition of lactone 36, failed
to afford the advanced intermediate 38 with no recovery of
starting material. If the reaction was quenched after the ini-
tial step substituted dithiane 39 could be obtained in high
yield, however, deprotonation with any number of strong
bases and subsequent addition of lactone 36 also failed to
deliver the advanced intermediate 38, again with no recov-
ery of starting material (Scheme 5). Although dithianes
have been used extensively in natural product total synthe-
sis[24] it was suspected that the failed reactions above were
the result of dithiane S-alkylation of lactone 36 as opposed
to C-alkylation. However, spectroscopic evidence to support
these suspicions were unable to be obtained due to complete
decomposition.
Scheme 3. a) CH2CHMgBr, CuI, THF, ꢀ308C to RT, 1.5 h, 98%; b)
Boc2O, NEt3, DMAP, CH2Cl2, RT, 12 h, 99%; c) IBr, toluene/CH2Cl2,
ꢀ788C to RT, 1 h, quant.; d) K2CO3, MeOH, ꢀ788C to RT, overnight,
64%; e) TBSCl, NEt3, DMAP, DMF, RT, 8 h, 88%; TBS=tert-butyldime-
thylsilyl; Boc=tert-butyloxycarbonyl; DMAP=4-N,N-dimethylaminopyr-
idine; DMF=N,N-dimethylformamide.
Work by OꢁDoherty et al.[21] disclosing the synthesis of hy-
droxylactone 30, via transformations 31, 32,[22] 33, 34 to 35,
suggested access to a derivative that resembled the right
hand fragment lactone 18, that is bromide 36, might be ach-
ievable (Scheme 4). Repetition of the OꢁDoherty sequence
proceeded smoothly except in the case of hemiacetal 34,
which required the use of Celite as an additive in the Jones
oxidation. Conversion of 30, via a Finkelstein reaction of
the corresponding mesylate with lithium bromide, afforded
the target right hand fragment 36 in excellent yield (90%)
(Scheme 4). Treatment of 30 with carbon tetrabromide/tri-
phenylphosphine was less efficient.
Scheme 5. a) nBuLi, TBSCl, THF, ꢀ608C to RT, 6 h, (90%); b) 37,
nBuLi, THF, RT, 20 min; then 29, Et2O, ꢀ308C to RT, overnight; TBS=
tert-butyldimethylsilyl.
Since the initial step in the linchpin reaction (Scheme 5)
with epoxide 29 was working well but the coupling to lac-
tone 36 was not, the alternative retrosynthetic strategy
based on ring-closing metathesis was investigated. To test
this approach allyl epoxide 17 (Scheme 1) was required, but
at the same time it was clear that the 10-step sequence lead-
ing to epoxide 29 was long-winded and tedious for scale-up.
At this juncture a further application of the Tietze–Smith
linchpin reaction, applied to the synthesis of the left-hand
fragment (i.e., epoxide 16), was deployed.
Investigation into the convergence of the two halves (i.e.,
29 and 36) concentrated on the use of dithiane (20) as the
The one-pot linchpin reaction of TMS-dithiane (40) with
epoxide 24, and then (R)-epichlorohydrin (25), afforded the
disubstituted dithiane 41 in 61% yield. TBS-dithiane (37)
normally outperforms 40 in linchpin reactions (i.e., affording
higher yields),[25] but in this study O-TMS protection fortui-
tously underwent simultaneous deprotection when the keto
group was unmasked [HgACHTNUGTRNEUGN(ClO4)2], giving hydroxyketone 42
Scheme 4. a) NaOMe, MeOH, RT, 4 h, quant.; b) FeCl3·6H2O, MeCN,
RT, 1 h, 82%; c) TBSCl, imidazole, CH2Cl2, 08C to RT, 45 min, 87%; d)
NBS, NaOAc, NaHCO3, THF, 08C, 1 h, 89%; e) Jones reagent, Celite,
acetone, RT, 1 h, quant.; f) NaBH4, CeCl3, CH2Cl2/MeOH, ꢀ788C to RT,
1.5 h, 90%; g) TBSOTf, 2,6-lutidine, CH2Cl2, RT, 17 h, 75%; h) HF, H2O/
MeCN, 08C, 1 h, 76%; i) MsCl, CH2Cl2, 08C, 15 min, 98%; j) LiBr, ace-
tone, reflux, 96 hr, 92%; TBS=tert-butyldimethylsilyl; NBS=N-bromo-
succinimide; Tf=trifluoromethanesulfonyl; Ms=methansulfonyl.
in high yield. syn-Selective reduction,[26] followed by ketal
protection,[27] proceeded smoothly in 74% yield (over 2
steps) affording the desired left-hand fragment 43 [namely
(R,R,R)-enantiomer of 16] (Scheme 6).
For the second linchpin coupling enantiopure allyl epox-
ide 17 (Scheme 1) was required (i.e., 44) (Scheme 7).
Chem. Eur. J. 2009, 15, 11307 – 11318
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11311

C. M. Williams et al.
Scheme 6. a) 40, nBuLi, THF, RT, 20 min; then 24, Et2O, ꢀ308C, 1 h;
then HMPA, (R)-epichlorohydrin (25), ꢀ508C to RT, overnight, 61%; b)
Scheme 8. a) 37, nBuLi, THF, RT, 30 min; then 43, Et2O, ꢀ308C, 1 h;
then HMPA, 44, ꢀ508C to RT, overnight, 78%; b) CH2CHCOCl,
CH2Cl2, NEt3, 08C, 2 h, 82%; h) 56, MW, 1508C, 150 W, toluene, 3 h,
65%. MW=microwave irradiation.
HgACHTUNGTRENNUNG(ClO4)2·H2O, CaCO3, THF, H2O, RT, 6 min, 79%; c) Et2BOMe,
NaBH4, MeOH, THF, ꢀ788C, 4 h; d) Me2C
ACHTUGNTRENNUN(G OMe)2, PPTS, CH2Cl2, RT,
5 h, 74% over 2 steps (de>95:5, ee 92%); HMPA=hexamethylphospho-
rous triamide, PPTS=p-toluenesulfonic acid.
tion Hoveyda–Grubbs (55) and second-generation Hovey-
da–Grubbs (56) (Figure 5) have also been reported to effect
the desired ring-closure,[33] but these too failed under
normal conditions. When microwave irradiation[34] was ap-
plied to the system, however, lactone 52 was obtained in
65% yield, but only using the Hoveyda–Grubbs second-gen-
eration catalyst (56) (Scheme 8). Even though RCM reac-
tions are routinely performed in the presence of dithiane
ring systems,[35] it is believed the difficulties encountered
with the present RCM reactions are most likely attributed
to the close proximity of sulfur atoms to the reaction centre
allowing sulfur atom co-ordination to ruthenium thus pre-
venting catalyst turnover.[36]
Acquisition of this material was achieved through direct
transformation of the known epoxide 45 (derived from di-
vinyl carbinol 46),[28] via Mitsunobu inversion (47),[29] subse-
quent hydrolysis and protection (Scheme 7). Unfortunately,
Scheme 7. a) (+)-DIPT, Ti
G
80%; b) DIAD, PPh3, p-NO2PhCO2H, THF, RT, 3 h,>95%; c) K2CO3,
MeOH, H2O, RT, 2 h, 98%; d) TBSCl, Imidazole, CH2Cl2, 08C to RT,
1.5 h, 66%, ee 90%; e) (S)-ibuprofen (>99% ee), DCC, CH2Cl2, RT, 2 h,
59%; f) see ref. [31]. DIPT=diisopropyl tartrate, DIAD=diisopropyla-
zodicarboxylate, DCC=dicyclohexylcarbodiimide.
Figure 5. Ring-closing metathesis reaction catalysts.
however, the Mitsunobu reaction proceeded with slight
A
profen derivative 48 (Scheme 7). This stereochemical issue
could be avoided by substituting p-nitrobenzoic acid with p-
methoxyphenol in the Mitsunobu reaction, but it was discov-
ered that the change from a silyl protecting group (i.e., 44)
to an aromatic ether (i.e., 49[31]) completely prevented the
linchpin coupling shown in Scheme 8.
Gratifyingly, the long awaited key linchpin reaction of 43
and 44, with the anion of dithiane 37, gave the desired cou-
pled product 50 in high yield (78%). Reaction of 50 with
acryloyl chloride proceeded smoothly affording acrylate 51
in 82% yield (Scheme 8).
Much to our surprise, and contrary to literature prece-
dent, ring-closing metathesis (RCM) required substantial in-
vestigation to facilitate the formation of the desired lactone
ring system (52). Although Grubbs first-generation catalyst
53 has been reported to form lactones of this type with hy-
droxy substitution in the g-position,[32] all attempts with this
catalyst failed. Grubbs second-generation (54), first-genera-
Having established the carbon backbone in lactone 52, de-
protection cyclisation strategies could now be probed. The
dithiane deprotection approach, investigated first, proceed-
ed without incident giving ketone 57 in 95% yield. Concom-
itant deprotection–cyclisation of ketone 57 with a variety of
acids (e.g. aq HF,[37] CSA[37]) only afforded trace amounts of
the target 58 (Scheme 9). On recommendation from a won-
derful review by Pihko et al.,[38] direct methods for conver-
sion of 52 to 58 (i.e., treatment with HF·py,[39] aq HF[40] or
HCl[41,42]) were then investigated, which unfortunately pro-
vided little improvement. Following the work of Nakata,[43]
however, TBS and acetonide deprotection performed with
hydrogen fluoride pyridine complex afforded the polyol 59,
which on treatment with CAN[44] revealed the target in 54%
yield over two steps (substituting CAN for PIFA[45] failed).
Considering the effort required to drive cyclisation with acid
promoters, and the fact that CAN performed this task ex-
tremely well, suggests that formation of the spiroacetal was
11312
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ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 11307 – 11318

Anticancer Agents
FULL PAPER
Scheme 9. a) HgACHTUNGTRENNUNG(ClO4)2·H2O, CaCO3, THF, H2O, RT, 20 min, 95%; b) aq
HF (50%), CH3CN, CH2Cl2, RT, 4 h, <5%; c) CSA, CH2Cl2, MeOH,
14 h, 08C to RT, <5%; d) aq HF, CH3CN, CH2Cl2, RT, 16 h; e) CAN,
CH3CN, H2O, RT, 0.5 h, 54% (over 2 steps). CSA=camphorsulfonic
acid, CAN=cerium ammonium nitrate.
Figure 6. Inhibition of the growth of DU145 prostate tumors in nu-/nu-
mice treated with EBC-23 (1: black, controls in grey) i.p. daily from day
6 to day 33 (100 mg/mouse days 6–9, then 200 mg/mouse). Significance in
a permutation test including all time points p=0.005 (n=12 tumors per
group).
mediated by metal (namely cerium) templated preorganisa-
tion as precedented by Smith[41] and Evans.[37,46]
Direct comparison of the optical rotation of both the syn-
thetic material 58 (ꢀ16.6) and the natural material 1
material. Interestingly, however, the opposite enantiomer
also displayed strong activity, but less than the natural (and
matching synthetic) material.
ACHTUNGTRENNUNG(+16.3) revealed that the wrong enantiomer had been syn-
thesized. Repeating the synthesis with ent-43 and ent-44,
however, afforded synthetic mate-
EBC-23 (1) has the ability to inhibit the growth of the an-
drogen-independent prostate tumor cell line DU145 in the
mouse model indicating that it has potential for the treat-
ment of refractory solid tumors in adults. Both spiroace-
tals[47] and g-oxygen-d-lactones[48] of type 9 have independ-
ently demonstrated potent cancer related pharmacological
activities. It is therefore no surprise that these surfactant-
like molecules combining both spiroacetal and enlactone
moieties deliver potent efficacy, further optimized by a con-
venient lipophilic side chain to potentially control metabo-
lism and enable cell membrane permeability. This is the
wonder of mother nature (biodiversity) in that she not only
oversees and orchestrates plant evolution, but also plays me-
dicinal chemist for mankind.[49]
rial 60 (+15.9), matching both the
relative and absolute stereoconfi-
guration of the natural material
EBC-23 (1).
Inhibition of cell growth by
EBC-23 family members was
quantitated after six days of treat-
ment in culture. The results (Table 3) showed significant in-
hibitory activity against a range of tumor cell types, with
normal cells (NFF) being less susceptible. EBC-23 (1) was
selected for further evaluation in a mouse model.
Xenografts of the human prostate tumor cell line DU-145
in immunodeficient mice were treated daily with EBC-23
(1) (Figure 6). Tumor growth in the treated mice was inhibit-
ed compared with the solvent-only controls, and no side ef-
fects were observed. Considerable variation in tumor size
occurred within each group but comparison with an iterative
method (D statistic) which included measurements at all
time points showed that growth was significantly inhibited
in the group treated with EBC23 (1). Synthetic EBC-23 (1)
displayed the same biological profile as that of the natural
Conclusions
In conclusion, two linchpin reactions were key to the concise
total synthesis of EBC-23, in both enantiopodes (58 and 60),
achieved in nine linear steps in 8% overall yield. The total
syntheses unequivocally proved the proposed relative and
absolute stereoconfiguration. Suitable flexibility built into
the synthesis allows multiple
stereochemical arrangements
poised for further biological
evaluation of this unique struc-
ture class, reports of which will
appear in due course.
Table 3. Inhibition IC50 [mgmLꢀ1]* of human cell growth by EBC compounds.
EBC-23
EBC-24
EBC-25
EBC-72
EBC-73
EBC-75
EBC-76
NFF
1.8
0.2
0.45
0.48
0.8
0.4
0.4
0.3
0.75
0.48
1.1
0.8
1.8
>2
1.6
0.75
1.5
1.5
0.85
1.3
1.3
1.0
1.3
>2
0.98
MM96L
MCF7
DU145
0.4
1.8
1.7
[*] Dose required to inhibit growth to 50% of the untreated cells. NFF=normal fibroblasts; MM96L=mela-
noma; MCF7=breast carcinoma; DU145=prostate cancer.
Chem. Eur. J. 2009, 15, 11307 – 11318
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11313

C. M. Williams et al.
(S)-2-Tridecyloxirane (ent-24):[20] To a solution of (S)-1-chloro-pentade-
can-2-ol (10.8 g, 41.3 mmol) in THF (55 mL) and MeOH (152 mL) was
Experimental Section
added
a solution of sodium hydroxide (2.09 g, 52.2 mmol) in H2O
General: 1H and 13C NMR spectra were recorded on Bruker AV300
(300.13 MHz; 75.47 MHz), AV400 (400.13 MHz; 100.62 MHz), DRX500
(500.13 MHz; 125.76 MHz) and AV750 (749.41 MHz) instruments in deu-
(35 mL). The mixture was stirred at room temperature for 3 h. The THF
and MeOH were then removed in vacuo and additional H2O (150 mL)
was added. The crude product was extracted into Et2O (3ꢂ100 mL) and
the combined organic phase was washed with brine (200 mL), dried
(MgSO4) and concentrated in vacuo. Flash chromatography (PE/EA
20:1) afforded the title compound as a colorless oil (8.44 g, 91%).
1H NMR (CDCl3, 500 MHz): d = 0.86 (t, J=6.9 Hz, 3H), 1.18–1.36 (m,
20H), 1.36–1.47 (m, 2H), 1.47–1.54 (m, 2H), 2.43 (dd, J=5.2, 2.8 Hz,
1H), 2.72 (dd, J=5.2, 4.0 Hz, 1H), 2.88 ppm (tdd, J=5.2, 4.0, 2.8 Hz,
1H); 13C NMR (CDCl3, 125 MHz): d = 14.1, 22.7, 26.0, 29.3, 29.4, 29.5,
29.62, 29.63, 29.64, 29.7, 31.9, 32.5, 47.1, 52.4 ppm; MS (EI): m/z (%): 226
(2) [M+], 213 (3), 111 (8), 96 (26), 82 (38), 71 (48), 55 (56), 43 (100).
teriochloroform (CDCl3) or hexaACHTNUGTRENUNGdeuteriobenzene (C6D6) unless other-
wise stated. GC/MS data were recorded on a Shimadzu GC-17 A Ver.3,
mass-spectrometer: MS QP5050 A, ionisation at 70 eV, column: DB-5 ms
30 m ꢂ 0.25 mm, carrier-gas: He, total flow 32.2 mLminꢀ1, column flow
1.3 mLminꢀ1, injector temperature: 2508C, standard program: 2 min at
1008C, followed by a temperature increase of 168Cminꢀ1 and held at
2508C for 10 min. Low-resolution electrospray ionization mass spectrom-
etry measurements (LRESIMS) were recorded in positive ionization
mode on a Bruker Esquire HCT (High Capacity 3D ion trap) instrument
with a Bruker ESI source. High resolution electrospray ionization (HRE-
SIMS) accurate mass measurements were recorded in positive mode on a
Bruker MicrOTOF-Q (quadrupole-Time of Flight) instrument with a
Bruker ESI source. Accurate mass measurements were carried out with
external calibration using sodium formate as a reference calibrant. Micro-
analyses were performed by the University of Queensland Microanalyti-
cal Service. High- and low-resolution EI mass spectral data were ob-
tained on a Finnigan MAT900. Microwave irradiation was conducted
with a CEM Discover microwave in 10 or 80 mL pressurized vials. IR
spectra were measured on a Perkin–Elmer FT-IR spectrometer (Spec-
trum 2000) with Smiths detection (DuraSamplerIR II). Optical rotations
were measured at the sodium D line (589 nm) using a 1 dm quartz cell
on a Perkin–Elmer 241MC polarimeter. Tetrahydrofuran (THF) and di-
ethyl ether (Et2O) were dried with sodium/benzophenone. Dichlorome-
thane (CH2Cl2) was dried with calcium hydride (CaH2). Diethyl ether,
petroleum ether (PE) and ethyl acetate (EA) for column chromatogra-
phy were distilled before use. Flash chromatography was undertaken on
silica gel (Flash Silica gel 230–400 mesh).
(R)-2-Tridecyloxirane (24): The title compound was prepared via the
above procedure. 1H and 13C NMR were identical to that above.
Epoxide 41: Under an argon atmosphere nBuLi (7.60 mL, 1.39m solution
in hexanes, 10.6 mmol) was added to a solution of 2-trimethylsilyl-1,3-di-
thiane (40) (2.13 g, 11.1 mmol) in anhydrous THF (20 mL) and the solu-
tion was stirred at room temperature for 20 min. The solution was then
transferred via cannula to a suspension of (R)-2-tridecyloxirane (24)
(2.01 g, 8.88 mmol) in anhydrous Et2O (80 mL) at ꢀ308C. The mixture
was stirred at ꢀ308C for one hour and then cooled to ꢀ508C. Anhydrous
HMPA (2.00 mL, 11.5 mmol) and (R)-epichlorohydrin (25) (1.80 mL,
23.0 mmol) were added in quick succession and the mixture was slowly
warmed to RT overnight. The reaction was quenched with sat. NH4Cl so-
lution (40 mL) and was extracted with Et2O (2ꢂ60 mL). The combined
organic phase was washed with 10% LiCl solution (2ꢂ40 mL), sat.
NH4Cl solution (40 mL), H2O (40 mL) and brine (40 mL), dried (MgSO4)
and concentrated in vacuo. Flash chromatography (PE/EA/TEA
30:1:0.6) yielded the title compound as a colorless oil (2.57 g, 61%). [a]D20
= +15.9 (c=0.012, CHCl3); 1H NMR (CDCl3, 300 MHz): d = 0.12 (s,
9H), 0.80 (t, J=6.8 Hz, 3H), 1.24 (s, 22H), 1.47 (m, 2H), 1.91–1.95 (m,
2H), 2.11–2.18 (m, 3H), 2.24 (dd, J=14.7, 4.4 Hz, 1H), 2.51–2.54 (dd, J=
5.2, 2.7 Hz, 1H), 2.74–2.84 (m, 5H), 3.17–3.23 (m, 1H), 4.05–4.10 ppm
(m, 1H); 13C NMR (CDCl3, 75 MHz): d = 0.7, 14.0, 22.6, 24.6, 24.9, 26.1,
26.2, 29.3, 29.6, 29.8, 31.8, 38.9, 41.9, 45.4, 45.6, 46.6, 49.0, 51.5, 69.7 ppm;
MS (ESI): m/z: 497 [M+Na]+; HRMS: m/z: calcd for C25H50NaO2S2Si:
497.2914, found: 497.2902.
Isolation: The fruit of Cinnamomum laubatii (1.0 kg) was homogenized
in methanol, and the concentrated extract partitioned between water and
ethyl acetate. The ethyl acetate extract was chromatographed on silica
gel with petroleum spirit (b.p. 40–608C), ethyl acetate and methanol.
Fractions that inhibited the growth of the human melanoma cell line
MM96L were further fractionated by HPLC on an Agilent Series 1100
HPLC Prep system using a reverse phase Agilent C18 column (30 mm ꢂ
250 mm ꢂ 10 mm). Isolates were eluted with a 70–100% methanol/water
gradient in the order EBC73 (8) (10.7 mg, 0.0011%), EBC75 (6) (55 mg,
0.0055%), EBC72 (5) (10 mg, 0.0010%), EBC23 (1) (196 mg, 0.020%),
EBC24 (3) (43.5 mg, 0.0044%), EBC25 (4) (44.6 mg, 0.0045%) and
EBC76 (7) (20 mg, 0.0020%).
Epoxide ent-41: The title compound was prepared via the above proce-
dure. [a]2D0 =+14.7 (c=0.0097, CHCl3); 1H and 13C NMR were identical
to that above; MS (ESI): m/z: 497 [M+Na]+; HRMS: m/z: calcd for
C25H50NaO2S2Si: 497.2914, found: 497.2916.
AHCTUNGTERG(NNUN 2R,6R)-6-Hydroxy-1-oxiranyl-4-nonadecanone (42): Calcium carbonate
(S)-1-Chloro-pentadecan-2-ol: 1-Bromododecane (4.80 mL, 20.0 mmol)
and iodine (one crystal) were added to a stirred suspension of magnesium
turnings (0.673 g, 27.7 mmol) in anhydrous THF (80 mL) under an argon
atmosphere. The mixture was refluxed for 5 h and then cooled to room
temperature. This solution of dodecylmagnesium bromide was cannulated
into a stirred suspension of (S)-epichlorohydrin (1.30 mL, 16.6 mmol)
and copper (I) iodide (0.363 g, 1.91 mmol) in anhydrous THF (35 mL) at
ꢀ788C. The mixture was stirred for 90 min., during which time the tem-
perature increased to ꢀ458C. The reaction was quenched with sat.
NH4Cl solution (100 mL) and the layers were separated. Additional
product was extracted into Et2O (2ꢂ75 mL) and the combined organic
phase was washed with sat. NH4Cl solution (100 mL) and brine
(100 mL). Drying (MgSO4) followed by concentration in vacuo provided
a crude oil, which after flash chromatography (PE/EA 10:1) furnished
the title compound as a white solid (3.28 g, 75%). 1H NMR (CDCl3,
300 MHz): d = 0.86 (t, J=7.0 Hz, 3H), 1.24 (s, 21H), 1.38–1.56 (m, 3H),
2.10 (brs, 1H), 3.45 (dd, J=11.0, 7.1 Hz, 1H), 3.61 (dd, J=11.0, 3.3 Hz,
1H), 3.73–3.83 ppm (m, 1H); 13C NMR (CDCl3, 75 MHz): d = 14.1, 22.7,
25.5, 29.3, 29.5, 29.55, 29.64, 29.7, 31.9, 34.2, 50.6, 71.5 ppm; MS (EI): m/z
(%): 227 (3) [M+ꢀCl], 213 (28), 168 (4), 125 (7), 111 (14), 97 (27), 83
(28), 69 (30), 57 (41), 43 (100).
(1.06 g, 10.6 mmol) followed by mercury (II) perchlorate hydrate (1.06 g,
2.66 mmol) were added to a solution of epoxide 41 (0.497 g, 1.05 mmol)
in THF/H2O 4:1. The suspension was stirred for 6 min at room tempera-
ture. Sat. NaHCO3 solution (45 mL) was then added and the mixture was
filtered through a pad of Celite. After the Celite had been washed thor-
oughly with E2O the layers of the filtrate were separated and the organic
layer was washed with sat. NaHCO3 solution (45 mL), H2O (45 mL) and
brine (45 mL). Drying (MgSO4) followed by concentration in vacuo pro-
vided the title compound in sufficient purity for use in the next step as a
white solid (246 mg, 79%). [Note: this material was found to be unstable
to silica gel.] 1H NMR (CDCl3, 300 MHz): d = 0.85 (t, J=6.7 Hz, 3H),
1.23 (brs, 21H), 1.37–1.46 (m, 3H), 2.48 (dd, J=4.9, 2.6 Hz, 1H), 2.56–
2.70 (m, 4H), 2.81(t, J=6.9 Hz, 1H), 3.24 (dddd, J=6.4, 4.9, 4.0, 2.6 Hz,
1H), 3.99–4.07 ppm (m, 1H); 13C NMR (CDCl3, 75 MHz): d = 14.1, 22.6,
25.4, 29.3, 29.48, 29.53, 29.6, 31.9, 36.5, 46.5, 46.7, 47.5, 49.8, 67.5,
209.0 ppm; MS (ESI) 335 [M+Na]+; HRMS: m/z: calcd for C19H36O3:
663.4955, found: 663.4758.
AHCTUNGTERG(NNUN 2S,6S)-6-Hydroxy-1-oxiranyl-4-nonadecanone (ent-42): The title com-
pound was prepared via the above procedure. 1H and 13C NMR were
identical to that above; MS (ESI) 335 [M+Na]+; HRMS: m/z: calcd for
C19H36NaO3: 335.2557, found: 335.2544.
(R)-1-Chloro-pentadecan-2-ol: The title compound was prepared via the
above procedure except using (R)-epichlorohydrin (25). All spectral data
were identical to those for (S)-1-chloro-pentadecan-2-ol.
Epoxide 43: To a solution of hydroxyketone 42 (31.2 mg, 0.1 mmol) in
anhydrous THF (0.8 mL) and anhydrous MeOH (0.2 mL) at ꢀ708C
11314
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Chem. Eur. J. 2009, 15, 11307 – 11318

Anticancer Agents
FULL PAPER
under argon, was added dropwise diethylmethoxyborane (0.11 mL,
0.11 mmol, 1m), and the resulting mixture was stirred for 15 min. Then
sodium borohydride (4.2 mg, 0.11 mmol) was added, the mixture was
stirred for 4 h, followed by the addition of acetic acid (0.1 mL). The reac-
tion mixture was diluted with EtOAc, washed with aqueous NaHCO3,
and brine (3 mL), dried over Na2SO4 and concentrated in vacuo. The res-
idue thus obtained was azeotroped several times with MeOH until the
hydrolysis of the boronate was complete. The crude product was used
Nitroester 47: To (R)-1-[(S)-oxiran-2-yl]prop-2-enol (45) (460 mg,
4.6 mmol), triphenylphosphine (2.41 g, 9.2 mmol) and p-nitrobenzoic acid
(1.54 g, 9.2 mmol) in anhydrous THF (35 mL) at 08C under an argon at-
mosphere, was slowly added diisopropylazodicarboxylate (1.3 mL,
9.2 mmol) at 08C. The reaction was stirred for 15 min. while warming up
to room temperature, then stirred for 3 h at RT. The reaction mixture
was diluted with Et2O (30 mL), washed with aqueous NaHCO3 (2ꢂ
30 mL), and brine (30 mL), dried over Na2SO4 and concentrated in
vacuo. After flash chromatography on silica gel (PE/EA 3:1) the nitroest-
er (1.12 g, 98%) was obtained as a yellow oil. [a]2D0 =ꢀ24.2 (c=0.074,
CHCl3); 1HNMR (CDCl3, 300 MHz): d = 2.71–2.74 (dd, J=4.8, 2.6 Hz,
1H), 2.88 (dd, J=4.8, 4.0 Hz, 1H), 3.27 (ddd, J=6.3, 4.0, 2.6 Hz, 1H),
5.24–5.51 (m, 3H), 5.91 (dd, J=10.6, 6.3 Hz, 1H), 8.11–8.38 ppm (m,
4H); MS (ESI): m/z: 272 [M+Na]+; HRMS: m/z: calcd for
C12H11NNaO5: 272.0529, found: 272.0525.
1
without purification. H NMR (CDCl3, 300 MHz): d = 0.82 (t, J=6.9 Hz,
3H), 1.22 (s, 22H), 1.74 (m, 1H), 1.42–1.64 (m, 5H), 2.47 (m, 1H), 2.74
(m, 1H), 3.06 (m, 1H), 3.84 (m, 1H), 4.10 ppm (m, 1H); 13C NMR
(CDCl3, 75 MHz): d = 14.1, 22.7, 25.3, 29.3, 29.58, 29.63, 31.9, 38.2, 40.2,
42.8, 46.6, 50.0, 71.3, 73.0 ppm.
To the above diol (37 mg, 0.12 mmol) dissolved in anhydrous CH2Cl2
(1 mL) under an argon atmosphere, was added pyridium p-toluenesulfo-
nate (one crystal) and dimethoxypropane (60 mL, 0.5 mmol). The mixture
was then stirred for 4 h at room temperature. The filtrate was washed
with NaHCO3 solution (3 mL, 5%), and brine (3 mL), dried over Na2SO4
and concentrated in vacuo. Flash chromatography on silica gel (PE/EA
20:1) afforded the title compound [30 mg, 80% (two steps)] as a white
solid (dr 93:7).[50] [a]D20 =+5.0 (c=0.012, CHCl3); 1H NMR (CDCl3,
500 MHz): d = 0.86 (t, J=12.93 Hz, 3H), 1.21–1.29 (m, 22H), 1.32–1.36
(m, 1H), 1.36 (s, 3H), 1.41 (s, 3H), 1.47–1.50 (m, 1H), 1.52 (t, J=2.4 Hz,
1H), 1.54 (t, J=2.5 Hz, 1H), 1.64–1.77 (m, 2H), 2.49 (dd, J=5.1, 2.6 Hz,
1H), 2.74 (dd, J=5.1, 4.1 Hz, 1H), 3.02 (td, J=2.9, 1.9 Hz, 1H), 3.74–
3.83 (m, 1H), 3.99 (dtt, J=11.7, 5.9, 2.5 Hz, 1H); 13C NMR (CDCl3,
75 MHz): d = 14.1, 19.8, 22.7, 24.9, 29.3, 29.57, 29.64, 29.7, 30.2, 31.9,
36.4, 36.6, 38.8, 46.8, 48.9, 66.4, 68.9, 98.4; MS (ESI): m/z: 377 [M+Na]+;
HRMS: m/z: calcd for C22H42NaO3: 377.3026, found: 377.3020.
Nitroester ent-47: The title compound was prepared via the above proce-
dure. 1H and 13C NMR were identical to that above; HRMS: m/z: calcd
for C12H11NNaO5: 272.0529, found: 272.0525.
tert-Butyldimethyl{(S)-1-[(S)-oxiran-2-yl]allyloxy}silane (ent-44)
(S)-1-[(S)-oxiran-2-yl]prop-2-enol: Nitroester ent-47 (794 mg, 3.19 mmol)
and potassium carbonate (484 mg, 3.5 mmol) were suspended in MeOH
(30 mL) and H2O (2.0 mL) at RT. After stirring for 2 h the reaction flask
was placed in a Kugelrohr distillation apparatus to remove excess MeOH
[vacuum controlled (558C/80 Torr), cooling the outer collection flask
with dry ice]. The residue was washed with Et2O/n-pentane 1:1, then con-
centrated on a rotary evaporator (408C/540 Torr) to afford the title com-
pound (312 mg, 98%) as colorless oil. [a]2D0 =ꢀ5.1 (c=0.027, CHCl3);
1H NMR (CDCl3, 300 MHz): d
= 2.41 (brs, 1H), 2.72 (dd, J=4.9,
2.7 Hz, 1H), 2.80 (t, J=4.4 Hz, 1H), 3.03 (ddd, J=5.0, 4.1, 2.7 Hz, 1H),
3.97 (t, J=5.2 Hz, 1H), 5.18–5.41 (m, 2H), 5.90 ppm (ddd, J=17.3, 10.5,
5.5 Hz, 1H); 13C NMR (CDCl3, 75 MHz): d = 44.7, 54.7, 72.5, 116.8,
136.2 ppm.
Epoxide ent-43: The title compound was prepared via the above proce-
dure. [a]2D0 =ꢀ5.1 (c=0.011, CHCl3); 1H and 13C NMR were identical to
that above; MS (ESI): m/z: 377 [M+Na]+; HRMS: m/z: calcd for
C22H42NaO3: 377.3026, found: 377.3020.
(R)-1-[(R)-oxiran-2-yl]prop-2-enol: All spectral data were identical to
those above.
(R)-1-[(S)-Oxiran-2-yl]prop-2-enol (45): Freshly activated, powdered mo-
lecular sieves (3 ꢃ, 2.50 g) were suspended in anhydrous CH2Cl2 (55 mL)
in a 100 mL two-necked flask under an argon atmosphere. The suspen-
sion was then cooled to ꢀ208C, and d-DIPT (0.78 mL, 873 mg,
(S)-1-[(S)-Oxiran-2-yl]prop-2-enol (400 mg, 4 mmol) and imidazole
(656 mg, 9.63 mmol) were dissolved in anhydrous CH2Cl2 (10 mL) under
an argon atmosphere. tert-Butyldimethylsilyl chloride (1.12 g, 7.43 mmol)
was added in one portion at 08C (using 6 mL of anhydrous CH2Cl2 to
rinse). The reaction was stirred for 90 min. while slowly warming up to
RT, at which time TLC showed complete consumption of starting materi-
al. The reaction was quenched by adding NaHCO3 solution (10 mL) and
the product was extracted into Et2O (2ꢂ10 mL). The combined organic
phase was washed with NaHCO3 solution (10 mL) and NH4Cl solution
(10 mL), dried over Na2SO4, and concentrated (rotavap, 508C/200 Torr).
After flash chromatography on silica gel (pentane/Et2O 30:1) the title
3.73 mmol), TiACHTUNGTRENNUNG(OiPr)4 (1.18 mL, 1.13 g, 3.96 mmol), and tBuOOH (~5.5m
in decane, 7.20 mL, 39.6 mmol) were added consecutively. The mixture
was stirred at the same temperature for 15 min before adding freshly dis-
tilled [Kugelrohr, 1008C, discontinuous (tap on/off) vacuum] 1,4-penta-
dien-3-ol (46) (2.35 g, 27.9 mmol, rinse with ~3 mL of CH2Cl2). The flask
was sealed and placed in the freezer (ꢀ208C) for 9 d with occasional
shaking. The reaction was quenched by adding triethylphosphite
(2.50 mL, 2.39 g, 14.4 mmol) to the cold flask. After stirring for ca. 20–
30 min without a cold bath, saturated Na2SO4-solution (1.5 mL) was
added and the resulting mixture was stirred for 3 h at room temperature
open to the atmosphere. Celite (15 g) was added to the reaction. After
stirring for another 30 min the resulting slurry was filtered through a pad
of Celite which was washed thoroughly with CH2Cl2. The combined fil-
trate was dried over MgSO4, filtered and concentrated (rotavap, 200 Torr,
508C, ca. 10 min). The title compound was isolated by flash chromatogra-
phy [50 g SiO2, 5ꢂ5 cm, pentane ! pentane/ether 1:1; Rf (tBuOOH/
tBuOH)=0.69, Rf (starting material)=0.60, Rf (DIPT)=0.48, Rf (prod-
uct)=0.31; all in PE/EA 1:1] to give the title compound (60–70%, conta-
minated with 5–15% of tartrate and ca. 6% of another diastereomer)
which was used without further purification in the Mitsunobu reaction. If
necessary, the epoxyalcohol can be further purified by distillation (Kugel-
rohr distillation using a vacuum controller; 808C at 100 Torr to remove
volatiles, then cooling the receiver flask with dry ice and reduce pressure
(to 30 Torr) and increasing the temperature (to 1308C) to obtain the title
compound as a colorless oil (1.57 g, 56%). 1H NMR (CDCl3, 300 MHz) d
= 2.74 (dd, J=5.0, 4.0 Hz, 1H), 2.79 (ddd, J=5.0, 3.0, 0.44 Hz, 1H), 3.08
(d, J=4.0, 3.0 Hz, 1H), 4.29–4.36 (m, 1H), 5.38 (dt, J=17.3, 1.4 Hz, 1H),
5.45 (dt, J=10.5, 1.4 Hz, 1H), 5.83 ppm (ddd, J=17.3, 10.5, 6.3 Hz, 1H);
The 13C NMR spectrum was in agreement with that reported previous-
ly.[28]
compound (ent-44) (565 mg, 66%) was obtained as a colorless oil. [a]D20
=
1
ꢀ25.7 (c=0.055, CHCl3); H NMR (CDCl3, 300 MHz): d = 0.04 (s, 3H),
0.08 (s, 3H), 0.89 (s, 9H), 2.57–2.59 (dd, J=4.9, 2.7 Hz, 1H), 2.74–2.77
(dd, J=4.9, 4.0 Hz, 1H), 2.91–2.95 (m, 1H), 3.85 (td, J=6.3, 1.6 Hz, 1H),
5.13 (td, J=10.5, 1.6 Hz, 1H), 5.31 (td, J=17.2, 1.6 Hz, 1H), 5.81 ppm
(m, 1H); 13C NMR (CDCl3, 75 MHz): d = ꢀ5.0, ꢀ4.9, 25.7, 44.2, 55.2,
75.0, 115.8, 136.4 ppm.
tert-Butyldimethyl{(R)-1-[(R)-oxiran-2-yl]allyloxy}silane (44): The title
compound was prepared via the above procedure. [a]2D0 =+24.2 (c=
0.022, CHCl3); 1H and 13C NMR were identical to that above; MS (ESI):
m/z: 237 [M+ Na]+; HRMS: m/z: calcd for C11H22NaO2Si: 237.1281,
found: 237.1283.
Second linchpin reaction (ent-50):
A solution of TBS-dithiane (37)
(777 mg, 3.32 mmol) in anhydrous THF (10 mL), under an argon atmos-
phere, was treated with nBuLi (2.4 mL, 3.32 mmol, 1.39m) at 08C and
stirred at room temperature for 20 min. This solution was transferred via
cannula to a pre-cooled solution of epoxide (ent-43) (966 mg, 2.72 mmol)
in anhydrous Et2O (20 mL) under an argon atmosphere at ꢀ308C. The
reaction mixture was then stirred at ꢀ308C for 1 h and cooled to ꢀ508C
before anhydrous HMPA (1.17 mL, 5.2 mmol) and a solution of vinylep-
oxide (ent-44) (700 mg, 3.26 mmol) in Et2O (20 mL) were added succes-
sively. The reaction was then allowed to warm up to room temperature
with stirring overnight. The reaction was quenched by adding NH4Cl so-
Chem. Eur. J. 2009, 15, 11307 – 11318
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
11315

C. M. Williams et al.
lution (20 mL) at RT. The aqueous phase was extracted with Et2O
(20 mLꢂ3) and the combined organic extracts were washed with LiCl so-
lution (20 mLꢂ2, 10%), NH4Cl solution (20 mL), brine, dried over
Na2SO4 and concentrated in vacuo. Subjecting the residue to flash chro-
matography on silica gel (PE/EA 60:1) afforded the first generation di-
thiane addition by-product (320 mg, 20%) as a colorless oil. Changing
the solvent system (PE/EA 30:1) afforded the desired product (ent-50)
(1.7 g, 78%) as a colorless oil. [a]2D0 =ꢀ13.4 (c=0.025, CHCl3); 1H NMR
(CDCl3, 500 MHz): d = 0.03 (s, 3H), 0.06 (s, 6H), 0.10 (s, 3H), 0.85–0.88
(m, 12H), 0.88 (s, 9H), 1.08–1.15 (m, 1H), 1.23 (s, 23H), 1.35 (s, 3H),
1.40 (s, 3H), 1.42–1.49 (m, 2H), 1.62–1.67 (m, 1H), 1.74–1.79 (m, 1H),
1.89–1.95 (m, 2H), 1.98 (dd, J=15.1, 5.7 Hz, 1H), 2.07 (d, J=5.0 Hz,
2H), 2.37 (dd, J=15.2, 4.7 Hz, 1H), 2.75–2.82 (m, 4H), 2.94 (brs, 1H),
3.73–3.76 (m, 1H), 3.93–3.96 (m, 1H), 3.98–4.03 (m, 2H), 4.18 (qd, J=
5.2, 4.9 Hz, 1H), 5.17–5.27 (m, 2H), 5.82 ppm (ddd, J=17.2, 10.6, 6.3 Hz,
1H); 13C NMR (CDCl3, 125 MHz): d = ꢀ4.9, ꢀ4.3, ꢀ4.0, ꢀ3.6, 14.1,
18.0, 18.2, 19.9, 22.7, 24.7, 25.0, 25.9, 26.1, 26.2, 26.6, 29.3, 29.59, 29.60,
29.66, 29.68, 30.3, 31.9, 36.5, 37.4, 42.0, 45.7, 47.3, 52.0, 66.0, 66.8, 69.10,
71.7, 98.2, 117.0, 137.8 ppm; MS (ESI): m/z: 825 [M+Na]+; HRMS: m/z:
calcd for C43H86NaO5S2Si2: 825.5347, found: 825.5284.
Lactone 52: The title compound was prepared via the above procedure.
[a]2D0 =ꢀ59.8 (c=0.018, CHCl3); 1H and 13C NMR were identical to that
above; HRMS: m/z: calcd for C44H84NaO6S2Si2: 851.5140, found:
851.5134.
EBC-23 (60): To a solution of ent-52 (130 mg, 0.157 mmol) in CH2Cl2
(5.3 mL) at 08C was added a solution of aqueous hydrogen fluoride
(0.8 mL, 50%) dissolved in acetonitrile (2.6 mL). The reaction mixture
was then stirred for 8 h at RT. Additional aqueous hydrogen fluoride
(0.8 mL, 50%) was added to the mixture and stirring continued for an-
other 8 h. The reaction was quenched by adding NaHCO3 solution
(5 mL) and was extracted with Et2O (2ꢂ5 mL). The combined organic
phase was washed with NaHCO3 solution (5 mL), dried over Na2SO4,
and concentrated. Treatment of above mixture in 75% aqueous acetoni-
trile (12 mL) with CAN (353 mg, 0.028 mmol) at room temperature for
30 min, followed by dilution with water (10 mL) and extraction with
ether (2ꢂ10 mL). The filtrate was washed with brine (5 mL), dried over
Na2SO4 and concentrated in vacuo. The residue was subjected to flash
chromatography on silica gel (petroleum ether/EtOAc 1:1) affording
EBC-23 (1) (38 mg, 54%) as a white solid. [a]2D0 =+16.3 (c=0.034,
CHCl3); 1H NMR (CDCl3, 500 MHz): d = 0.84 (t, J=13.97 Hz, 3H),
1.23 (s, 23H), 1.47–1.52 (m, 2H), 1.56–1.65 (m, 3H), 1.75–1.79 (m, 1H),
1.97–2.05 (m, 2H), 2.27–2.31 (dd, J=15.0, 2.6 Hz, 1H), 2.51–2.56 (dd, J=
Second linchpin reaction (50): The title compound was prepared via the
1
above procedure. [a]2D0 =+12.8 (c=0.017, CHCl3); H and 13C NMR were
identical to that above; MS (ESI): m/z: 826 [M+Na]+; HRMS: m/z:
14.9, 6.8 Hz, 1H), 3.05ACTHNUTRGENUG(N ws, 1H), 3.76–3.81 (m, 1H), 4.11 (m, 1H), 4.34–
4.39 (m, 1H), 4.49–4.51 (t, J=4.77 Hz, 1H), 5.02–5.04 (ddd, J=6.9, 4.5,
2.6 Hz, 1H), 6.20–6.22 (d, J=9.90 Hz, 1H), 6.87–6.90 ppm (dd, J=9.9,
calcd for C43H86NaO5S2Si2: 825.5347, found: 825.5320.
Acrylate (ent-51): Acryloyl chloride (9 mg, 8 mL, 0.1 mmol) was added
dropwise to a solution of alcohol (ent-50) (40 mg, 0.05 mmol) in anhy-
drous CH2Cl2 (1 mL) and anhydrous triethylamine (20 mg, 28 mL,
0.20 mmol) at 08C under an argon atmosphere. The mixture was then
stirred for 2 h at 08C. After this time the mixture was concentrated in
vacuo and the residue loaded directly onto a silica gel column. Flash
chromatography (PE/EA 30:1) afforded the title compound (35 mg,
5.1 Hz, 1H); 13C NMR (CDCl3, 125 MHz): d
= 14.1, 22.7, 25.4, 29.3,
29.59, 29.62, 29.64, 29.66, 29.67, 31.9, 37.7, 38.7, 42.2, 47.7, 64.2, 67.8, 68.9,
71.8, 78.8, 106.6, 124.6, 138.6, 161.0 ppm; HRMS: m/z: calcd for
C26H44NaO6: 475.3030, found: 475.3037.
ent-EBC-23 (60): The title compound was prepared via the above proce-
1
dure. [a]2D0 =ꢀ16.6 (c=0.027, CHCl3); H and 13C NMR were identical to
colorless oil. [a]D20 =ꢀ19.2 (c=0.011, CHCl3); 1H NMR
that above; HRMS: m/z: calcd for C26H44NaO6: 475.3030, found:
82%) as
a
475.3034.
(CDCl3, 300 MHz): d = 0.06 (s, 3H), 0.07 (s, 3H), 0.11 (s, 3H), 0.13 (s,
3H), 0.82–0.88 (m, 21H), 1.23 (s, 23H), 1.34 (s, 3H), 1.39 (s, 3H), 1.42–
1.52 (m, 2H), 1.59–1.80 (m, 2H), 1.81–1.94 (m, 3H), 2.10–2.21 (m, 1H),
2.24–2.32 (m, 1H), 2.37 (dd, J=11.0, 4.4 Hz, 1H), 2.61–2.87 (m, 5H),
3.70–3.82 (m, 1H), 3.91–4.05 (m, 1H), 4.15–4.33 (m, 2H), 5.14 (dt, J=
10.5, 1.6 Hz, 1H), 5.19–5.25 (m, 1H), 5.25–5.34 (m, 1H), 5.73–5.86 (m,
2H), 6.03–6.20 (m, 1H), 6.38 ppm (dd, J=17.3, 1.5 Hz, 1H); 13C NMR
(CDCl3, 75 MHz): d = ꢀ5.0, ꢀ4.8 ꢀ4.1, ꢀ3.6, 14.1, 18.0, 18.2, 19.9, 22.7,
24.6, 25.0, 25.9, 26.05, 26.10, 26.6, 29.3, 29.59, 29.64, 29.67, 30.3, 31.9, 36.5,
37.3, 38.7, 45.9, 47.1, 52.4, 66.2, 67.1, 69.1, 72.1, 73.5, 98.2, 116.6, 128.9,
130.6, 136.2, 165.3 ppm; MS (ESI): m/z: 879 [M+Na]+; HRMS: m/z:
calcd for C46H88NaO6S2Si2: 879.5453, found: 879.5450.
Assays: Inhibition of cell growth was quantitated by dilution of com-
pounds into cell cultures seeded the previous day in 96-well plates (3000–
5000 cells/well in RPMI 1640 medium containing 10% fetal calf serum).
After 6 days the cultures were fixed in ethanol and stained with sulfo-
rhodamine B for comparison with untreated controls (absorbance at
540 nm, measured on an ELISA reader).
For the mouse xenograft model, 2 million DU145 human prostate cancer
cells were injected subcutaneously into each of four sites on the flanks of
nude (BALB/c nu-/nu-) mice. Tumor growth was monitored with callipers
and volume calculated with the formula: length ꢂ (breadth)2/2.
Acrylate 51: The title compound was prepared via the above procedure.
[a]2D0 =+12.3 (c=0.011, CHCl3); 1H and 13C NMR were identical to that
above; MS (ESI): m/z: 879 [M+Na]+; HRMS: m/z: calcd for
C46H88NaO6S2Si2: 879.5453, found: 879.5449.
Acknowledgements
Lactone ent-52: To a degassed solution of acrylate (ent-51) (238 mg,
0.278 mmol) in anhydrous toluene (3 mL) was added Hoveyda–Grubbꢁs
2nd generation catalyst (17 mg, 0.03 mmol, 10 mol%) in a microwave
vessel under an argon atmosphere. After microwave irradiation [T =
1508C; Hold: 4 h; power: 150 W] the mixture was concentrated in vacuo.
The residue was subjected to flash chromatography on silica gel (PE/EA
10:1) affording the title compound (150 mg, 65%) as a colorless oil.
[a]2D0 =+48.3 (c=0.013, CHCl3); 1H NMR (CDCl3, 400 MHz): d = 0.08
(s, 6H), 0.13 (s, 6H), 0.86 (m, 21H), 1.09–1.18 (m, 1H), 1.23 (s, 22H),
1.35 (s, 3H), 1.41 (s, 3H), 1.44ꢀ1.45 (m, 2H), 1.64 (ddd, J=14.0, 7.5,
4.1 Hz, 1H), 1.77 (ddd, J=14.1, 7.5, 4.1 Hz, 1H), 1.81–1.89 (m, 3H), 1.99
(dd, J=14.6, 6.8 Hz, 1H), 2.41–2.44 (m, 3H), 2.69 (ddd, J=14.6, 6.8,
2.9 Hz, 1H), 2.74–2.92 (m, 3H), 3.76–3.79 (m, 1H), 4.02–4.03 (m, 1H),
4.22–4.23 (m, 1H), 4.25–4.27 (dd, J=5.1, 2.9 Hz, 1H), 4.76–4.80 (m, 1H),
6.04 (d, J=9.6 Hz, 1H), 6.80 ppm (dd, J=9.6 Hz, 5.3 Hz, 1H); 13C NMR
(CDCl3, 75 MHz): d = ꢀ4.4, ꢀ4.0, ꢀ3.9, ꢀ3.6, 14.1, 18.0, 18.1, 19.8, 22.6,
24.5, 24.9, 25.9, 26.0, 26.1, 26.5, 29.3, 29.6, 30.3, 36.5, 37.4, 40.5, 45.4, 47.1,
51.9, 64.4, 65.8, 67.0, 69.1, 78.7, 98.2, 122.3, 144.6, 163.3 ppm; MS (ESI):
m/z: 851 [M+Na]+; HRMS: m/z: calcd for C44H84NaO6S2Si2: 851.5140,
found: 851.5129.
We thank Dr. L. Lambert (Centre for Magnetic Resonance) for assis-
tance with NMR experiments; Dr. A. Savchenko for attempts to crystal-
lise EBC-23 (1); Dr. S. Tennant and Dr. E. Lacey (Microbial Screening
Technologies) for providing non-stereodescriptive lead structures (using
ACD/Labs software) and initial isolation in some instances; Prof. W.
Kitching (University of Queensland) for constructive discussions; EcoBi-
otics Ltd and the University of Queensland for financial support.
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ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 11307 – 11318

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