Simplified silvestrol analogues with potent cytotoxic activity

Article abstract of DOI:10.1002/cmdc.201400024

The complex natural products silvestrol (1) and episilvestrol (2) are inhibitors of translation initiation through binding to the DEAD-box helicase eukaryotic initiation factor 4A (eIF4A). Both compounds are potently cytotoxic to cancer cells in vitro, an

Full text of DOI:10.1002/cmdc.201400024

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DOI: 10.1002/cmdc.201400024
Simplified Silvestrol Analogues with Potent Cytotoxic
Activity
Bill C. Hawkins,[a, b] Lisa M. Lindqvist,[a, b] Duong Nhu,[a, b] Phillip P. Sharp,[a, b] David Segal,[a, b]
Andrew K. Powell,[c] Michael Campbell,[c] Eileen Ryan,[c] Jennifer M. Chambers,[d]
Jonathan M. White,[d] Mark A. Rizzacasa,[d] Guillaume Lessene,[a, b] David C. S. Huang,[a, b] and
Christopher J. Burns*[a, b, d]
The complex natural products silvestrol (1) and episilvestrol (2)
are inhibitors of translation initiation through binding to the
DEAD-box helicase eukaryotic initiation factor 4A (eIF4A). Both
compounds are potently cytotoxic to cancer cells in vitro, and
1 has demonstrated efficacy in vivo in several xenograft cancer
models. Here we show that 2 has limited plasma membrane
permeability and is metabolized in liver microsomes in
a manner consistent with that reported for 1. In addition, we
have prepared a series of analogues of these compounds
where the complex pseudo-sugar at C6 has been replaced
with chemically simpler moieties to improve drug-likeness. Se-
lected compounds from this work possess excellent activity in
biochemical and cellular translation assays with potent activity
against leukemia cell lines.
Introduction
The cyclopenta[b]benzofuran family of natural products, exem-
plified by silvestrol (1) and rocaglamide (4) (Figure 1), possess
potent cytotoxicity against a range of cancer cell lines.[1–3] Iso-
lated from plants of the Aglaia genus and known variously as
flavaglines or aglains, their complex stereochemically dense
structure has led to numerous synthetic studies and total syn-
theses.[4,5] The most potent compound of this class, silvestrol
(1), has been shown to inhibit the initiation phase of transla-
tion through binding to the DEAD-box helicase eukaryotic ini-
tiation factor 4A (eIF4A), in turn disrupting formation of the
protein complex eIF4F.[6] A consequence of protein synthesis
inhibition by 1 is most notably apparent in a rapid reduction
in levels of short-lived proteins such as the pro-survival protein
Mcl-1. The loss of Mcl-1 and related proteins has been postu-
lated as the primary mechanism of cell death elicited by silves-
trol,[7] although more recent studies have demonstrated that
[a] Dr. B. C. Hawkins, Dr. L. M. Lindqvist, D. Nhu, Dr. P. P. Sharp, Dr. D. Segal,
Dr. G. Lessene, Prof. D. C. S. Huang, Dr. C. J. Burns
Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade
Parkville, VIC 3052 (Australia)
Figure 1. Chemical structures of silvestrol (1) and analogues, and rocagla-
mide (4).
E-mail: burns@wehi.edu.au
[b] Dr. B. C. Hawkins, Dr. L. M. Lindqvist, D. Nhu, Dr. P. P. Sharp, Dr. D. Segal,
Dr. G. Lessene, Prof. D. C. S. Huang, Dr. C. J. Burns
Department of Medical Biology, The University of Melbourne
VIC 3010 (Australia)
multiple mechanisms of cytotoxicity are induced upon com-
pound treatment.[8,9] A variety of molecular mechanisms under-
pinning the cytotoxicity of rocaglamide and analogues have
also been identified.[10–13]
[c] Dr. A. K. Powell, Dr. M. Campbell, Dr. E. Ryan
Centre for Drug Candidate Optimisation
Monash Institute of Pharmaceutical Sciences
Monash University
Silvestrol (1) has been reported to be particularly potent
against leukemia cell lines[7] and has demonstrated activity in
vivo in solid tumour xenograft studies both alone and in com-
bination with doxorubicin.[6,14] Despite this promising anticanc-
er activity and unusual mechanism of action, there are limited
structure–activity relationship (SAR) studies on 1, with most fo-
cusing on the functionally less active, and structurally simpler,
Parkville, Victoria 3052 (Australia)
[d] Dr. J. M. Chambers, Dr. J. M. White, Dr. M. A. Rizzacasa, Dr. C. J. Burns
School of Chemistry, The Bio21 Institute, The University of Melbourne
VIC 3010 (Australia)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201400024.
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rocaglates.[3,15] The naturally occurring episilvestrol (2), which is
the C-5’’’ epimer of silvestrol, is comparable to 1 in a range of
cytotoxicity and protein translation assays, whereas 2’’’,5’’’-die-
pisilvestrol (4) is significantly weaker.[16–19] These data indicate
that the dioxanyloxy pseudo-sugar moiety[20] of 1 has a signifi-
cant beneficial impact on the activity of 1 and 2 presumably
through discrete binding interactions with their molecular tar-
get(s).
Figure 2. Structure of episilvestric acid (5).
In this paper, we disclose our preliminary SAR studies on
changes to the pseudo-sugar moiety of silvestrol (1) to im-
prove drug-likeness and ease of synthesis. Several compounds
potently inhibit protein synthesis and display promising activi-
ty against a panel of human leukemia cell lines.
across a lipid membrane. No compound diffusion across the
artificial membrane could be detected over 4 h by our analyti-
cal methods, whilst controls performed as expected; however,
a significant loss of material from the donor chamber was ap-
parent (see Supporting Information). As stability control sam-
ples indicated that 2 was stable under the assay conditions,
the loss is likely due to redistribution into the lipid membrane,
consistent with the amphiphilic nature of the compound. As 2
differs from 1 only in the chirality at C5’’’, we anticipate that
1 would also exhibit this apparent low cellular permeability,
given that this chirality inversion would have minimal impact
on the compound’s physicochemical properties.
Results
Stability and permeability studies with 2
Published cytotoxicity data for silvestrol (1) and episilvestrol (2)
clearly indicate that these compounds are more potent (IC50 <
1 nm) than compounds where the dioxanyloxy group is absent
or replaced with a methyl ether (IC50 =10–100 nm),[16] con-
firmed by our own data (vide infra). Nonetheless, the presence
of the dioxanyloxy moiety reduces the compounds’ drug-like-
ness considerably by contributing to the high molecular
weight and polar surface area (PSA), as well as increasing its
structural complexity. In addition, the presence of two acetal
moieties in the dioxanyloxy groups of 1 and 2 could lead to
chemical instability whilst the high polarity of the group would
be expected to limit cellular penetration of the compounds.
We have found 2 to be stable in pH 3, 5 and 7 isotonic
aqueous buffers, and in simulated gastric fluid, over 24 h at
378C, with no appreciable acetal or ester hydrolysis apparent
over this timeframe (see Supporting Information). This stability
to acidic conditions is in contrast to metabolic stability, which
was assessed using human and mouse liver microsomes,
where episilvestrol exhibited a moderate rate of clearance (14–
18 mLminꢀ1 mgꢀ1 protein; Table 1). In these experiments, the
compound was shown to be metabolized to the acid 5
(Figure 2) in the absence of cofactors with both preparations,
suggesting contribution of non-NADPH-mediated metabolism
to the overall degradation rate, presumably via microsomal es-
terase activity. Importantly, no cleavage of the dioxanyloxy
group under these conditions was observed (Table 1).
Chemistry
We have prepared a series of analogues of silvestrol (1) where
the dioxanyloxy moiety has been replaced by benzylic and het-
erocyclic moieties. The cyclopenta[b]benzofuran framework of
these compounds were prepared in racemic form using a pho-
tocycloaddition reaction of 3-hydroxyflavones with cinnamates
originally developed by Porco and co-workers.[22] The requisite
3-hydroxyflavones 7 were prepared using the classical Algar–
Flynn–Oyamada reaction[23] or via lithiation of flavones 6 fol-
lowed by quenching with trimethylborate[24] and subsequent
oxidation and hydrolysis of the intermediate boronate
(Scheme 1). The photocycloaddition of hydroxyflavones 7 with
methyl cinnamate followed by an a-ketol rearrangement and
chromatographic separation of the unwanted exo-isomer, gen-
erated b-keto esters 8. Anti-selective reduction of the b-keto
ester function[25] afforded cyclopenta[b]-benzofurans 9 as the
major product. The benzyl or p-methoxybenzyl ether of 9 was
removed by hydrogenolysis using Pearlman’s catalyst to give
phenols 10, which in turn were alkylated with various alkyl hal-
ides to furnish the desired series of analogues 11.
To assess the effect the dioxanyloxy moiety has on cellular
penetration, we used parallel artificial membrane permeability
assay (PAMPA)[21] to measure the passive permeability of 2
The success of the hydrogenolysis of 9 was highly depen-
dent on the batch of Pearlman’s catalyst used with certain
batches repeatedly furnishing ethers 13 (Scheme 2)
on extended reaction times (>6 h), a reaction that
could be suppressed by buffering with acetic acid.
These ethers therefore presumably arise from hydro-
genolysis of the benzyl group and subsequent base-
induced elimination of the tertiary hydroxyl and sol-
volysis of the intermediate quinone methide. The
identity of the products 13a/13b from this unusual
reaction was confirmed by spectroscopic analysis and
a single crystal X-ray structure determined on the de-
Table 1. In vitro stability of episilvestrol in mouse and human microsomal prepara-
tions.
Species
Degradation
half-life [min][a]
In vitro CLint
[mLminꢀ1 mgꢀ1 proteinꢀ1
]
Metabolites detected
[b]
Human
Mouse
125ꢁ23
98ꢁ9
14ꢁ2
18ꢁ2
episilvestric acid
episilvestric acid
[a] Data are the mean ꢁSD (n=3). [b] Data are the mean ꢁSD (n=3).
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conditions and the resultant
a-hydroxyketone 15 stereoselec-
tively reduced to trans-diol 16a
with NMe4BH(OAc)3 or the syn-
diol 16b with NaBH4 (Scheme 3).
We also prepared methyl roca-
glate 17 following literature pro-
cedures[28] for use as an assay
benchmark. Hydroxamate 18 has
been reported to possess potent
cytotoxicity and inhibitory activi-
ty in translation assays.[29,30] We
prepared 18 and 19 (the hydrox-
amate analogue of 11 f), from
the corresponding methyl esters
by hydrolysis to the respective
acids and coupling with O-meth-
ylhydroxylamine under conven-
tional conditions (Scheme 4).
Assessment of biological
activity
All compounds prepared in this
work were profiled for effects on
protein translation in biochemi-
cal and cellular assays (see Sup-
porting Information for tabulat-
ed data). The biochemical assay
Scheme 1. Reagents and conditions: a) LDA, THF, B(OMe)3, AcOH, H2O2. b) methyl trans-cinnamate, hu, MeOH,
CH3CN. c) Me4NBH(OAc)3, AcOH, CH3CN. d) H2, Pd(OH)2, Pd/C, MeOH. e) alkyl halide, K2CO3, TBAI, DMF.
consisted of a translation assay in rabbit reticulocyte
lysates using an expression construct allowing for de-
termination of effects on cap-dependent translation,
as previously described.[31] In addition, the effect on
protein synthesis in cells was determined in a sepa-
rate assay measuring the metabolic incorporation of
radioactive methionine and cysteine, with the corre-
lation between the biochemical and cellular inhibi-
tion of translation giving a determination of com-
pound permeability into the cell. The effect of the
compounds on the proliferation and viability of
mouse embryonic fibroblasts (MEFs) and, for the
most active compounds, against selected leukemia
cell lines was also evaluated.
The effect of the compounds in the in vitro transla-
tion inhibition assays is presented in Figure 4 along
with data for episilvestrol (2) and methyl rocaglate
(18). As depicted in Figure 4, the compounds display
varying degrees of selectivity for cap-dependant
translation inhibition, with compounds 9b, 11 c, 11 f
and 19 showing the greatest potency and selectivity.
The effects that 2 and the compounds prepared
here have on translation in cells and on cellular proliferation
and viability are shown in Table 2. Compounds that show in-
hibition of translation in the cellular assay at 35% of vehicle
control or greater all show potent inhibition of MEF prolifera-
tion, and conversely those compounds that are weak in the
cellular translation assay are also less potent against MEF via-
Scheme 2. Reagents and conditions: a) H2, Pd(OH)2, Pd/C, R1OH. b) (14a) p-Br-benzoyl
chloride, Et3N, CH2Cl2. c) (14b and 14c) alkyl halide, K2CO3, TBAI, DMF.
rived p-bromobenzoate derivative 14a of 13a (Figure 3). Se-
lected compounds from this series, a functionalization that has
hitherto not been widely explored for the cyclopenta[b]benzo-
furans, were also profiled in our assays (vide infra).
Following published work for related cyclopenta[b]benzofur-
ans,[26,27] b-keto ester 8b was decarboxylated under Krapcho
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Figure 4. Biochemical assessment of cap-dependent activity and selectivity
determined for compounds at 50 mm (for assay details see Experimental Sec-
tion).
Figure 3. Thermal ellipsoid plot for one of the molecules of 14a. The ellip-
soids are at the 30% probability level. The heptane solvate molecule has
been removed for clarity.
in disease-relevant cells. For comparison, the recently de-
scribed hydroxamate 18 was included in the assay, as was 19,
the hydroxamate derivative of 11 f and one of the most potent
compounds prepared in this
work. The data is tabulated in
Table 3 and demonstrates that
both 11 f and its hydroxamate
derivative 19 show excellent ac-
tivity against these cell lines.
Discussion
The natural products silvestrol
(1) and episilvestrol (2) are the
most potent and selective inhibi-
tors of the DEAD-box helicase
eIF4A identified to date.[6,9] By
virtue of their activity against
Scheme 3. Reagents and conditions: a) LiCl, DMSO, 1008C. b) (for 16a) Me4NBH(OAc)3, AcOH, CH3CN. c) (for 16b)
NaBH4, MeOH.
eIF4A, these compounds potent-
ly inhibit translation in a cap-de-
pendent manner. Furthermore,
the compounds are strongly cy-
totoxic in vitro against a range of cell types, and in vivo studies
with 1 have shown promising anticancer activity against sever-
al tumour models xenografted into mice.[6,14] Despite this
promising activity profile, in depth pharmacological studies
with 1 and 2 have been hampered by limited availability of
the natural products from either the natural source or by total
synthesis.
There is strong biochemical evidence for the binding of
these compounds to eIF4A,[6] and recent affinity chromatogra-
phy studies with episilvestrol derivatives have identified only
eIF4AI/II as the molecular targets of the compound.[19] Further-
more, a very recent paper has identified key amino acids in-
volved in the binding of silvestrol to the yeast homolog of
eIF4A, TIM2; however, the precise binding mode of the com-
pound remains unknown.[32] Importantly, minor changes to the
pseudo-sugar moiety of 1 and 2 have profound effects on
compound activity, clearly indicating that this moiety is impor-
tant to the compounds’ interaction with eIF4A. Despite this,
most SAR studies on compounds of this class have focused on
Scheme 4. Reagents and conditions: a) i) LiOH, MeOH, THF. ii) MeONH2·HCl,
EDC, HOBt, Et3N, CH2Cl2.
bility. The exception is rocaglaol derivative 16a and hydroxa-
mate 19, which appear weaker in the translation assay but dis-
play sub-micromolar potency in the cellular proliferation assay.
Three of the most potent compounds from the studies
above (9b, 11 c and 11 f) were profiled against a panel of
human leukemia cell lines to determine if the activity observed
in the translation assays and against MEFs would be apparent
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stable under simulated physiological conditions, the com-
pound was susceptible to hydrolysis to the corresponding acid
(5) with both human and mouse liver microsomes. This data
mirrors earlier findings for silvestrol (1).[34]
Table 2. Cellular activity of silvestrol analogues.
Compd
Rel. translation rate at 1 mm
[% vehicle treated control][a]
IC50 [nm] of cell viability
and cytotoxicity[b]
A series of analogues of silvestrol (1)/episilvestrol (2) were
then prepared where the chemically complex pseudo-sugar
moiety was replaced with less polar benzylic and heterocyclic
species. The effect these compounds have on translation was
determined in a biochemical assay, and the data indicate that
most compounds exhibit inhibitory effects on cap-dependent
translation with varying degrees of selectivity over global non-
specific inhibition of translation (Figure 4). Inhibition of transla-
tion was also apparent in the cellular translation assay, and im-
portantly there is a good correlation between the biochemical
and cellular assays (Figure 5), indicating that the compounds
2
9a
9b
9c
3.35ꢁ0.31
27.7ꢁ1.63
34.4ꢁ1.72
81.0ꢁ3.02
116 ꢁ36.2
35.6ꢁ2.42
36.0ꢁ2.80
28.0ꢁ3.60
79.6ꢁ5.09
97.3ꢁ6.99
17.6ꢁ0.54
17.9ꢁ0.60
ND
97.0ꢁ4.85
88.6ꢁ1.93
102ꢁ5.87
63.2ꢁ4.91
85.9ꢁ5.17
11.4ꢁ0.36
4.78ꢁ0.15
93.5ꢁ3.96
0.66ꢁ0.12
123ꢁ7.25
53.1ꢁ3.26
1360ꢁ283
303ꢁ112
248ꢁ9.03
81.5ꢁ24.2
32.2ꢁ5.51
182.8ꢁ93.48
987ꢁ141
12.3ꢁ6.55
10.7ꢁ4.24
>10000
9d
11 a
11 b
11 c
11 d
11 e
11 f
11 g
14a
14b
14c
15
16a
16b
17
18
19
>10000
>10000
2070ꢁ207
142.9ꢁ23.0
659ꢁ107
2.43ꢁ0.32
0.96ꢁ0.06
44.9ꢁ7.2
[a] Translation inhibition in MEFs at 1 mm after 1 h (% vehicle ꢁSEM). ND:
not determined. [b] Inhibition of proliferation of MEFs. Data are the mean
ꢁSEM (n=3–4).
modifications to the cyclopenta[b]benzofuran moiety where
typically a methyl ether replaces the pseudo-sugar. The recent
disclosure by Infinity Pharmaceuticals, where a comprehensive
SAR exploration of the pseudo-sugar moiety was undertaken,
is the only report of which we are aware that has systematical-
ly investigated the pseudo-sugar region of the molecule in re-
lation to translation inhibition and cellular activity.[33] In the
work reported here, we have chosen to explore structurally
simple replacements of the pseudo-sugar moiety with benzylic
and heterocyclic functionality that can be readily introduced
and that improve drug-likeness.
Figure 5. Comparison of cap-dependant translation inhibition in the bio-
chemical and cellular assays.
possess acceptable cellular permeability. The exception is com-
pound 19 which displays good activity in the biochemical
assay but is weak in the cellular translation assay. The reason
for this anomalous activity is unclear, though notably the com-
pound shows potent cytotoxic activity against MEFs and leuke-
mia cell lines. Compounds lacking a methoxy group at the C8
position of the cyclopenta[b]benzofuran (9c and 9d) are signif-
icantly weaker in both biochemical and cellular translation
assays. The importance of substitution at the C8 position for
potent cellular activity has been reported previously in studies
of rocaglates.[27] Likewise, compounds 14a–c that bear an
ether at C3a of the tricyclic nucleus were also inactive in the
biochemical and cellular translation assays and were also inac-
tive against MEF viability indicating the importance of a hydro-
gen bond donor at this site. We are aware of only
Initially, we investigated the passive, transcellular permeabili-
ty of episilvestrol (2) using an in vitro PAMPA model, where we
observed the diffusion was limited and there was apparent re-
tention of the compound in the lipid membrane. These results
are consistent with the high PSA for 2 and amphiphilic nature
of the compound, indicating that cellular potency of com-
pounds of this class should be improved by improving cellular
permeability. We have also shown that whilst 2 is chemically
one report of cyclopenta[b]benzofurans with this
same substitution, and notably these compounds
Table 3. Antiproliferative activity (IC50 [nm]) of selected silvestrol analogues against
leukemia cell lines.[a]
were inactive when assessed in cellular cytotoxicity
assays.[35]
Cell line
2
9b
11 f
11c
18
19
Compounds with good potency in the cellular
translation assay (>35% inhibition of DMSO control)
also showed strong cytotoxicity against MEFs, the
only exception being rocaglaol analogue 16a which
was weak in the cellular translation assay but cyto-
toxic against MEFs (IC50 =143 nm). These data may in-
dicate that 16a possesses activities that contribute
CCRF-CEM 1.3ꢁ0.3 109.0ꢁ18.9 58.7ꢁ14.1 137.3ꢁ35.3 3.7ꢁ0.8
28.4ꢁ5.8
HL-60
K562
Molt4
1.7ꢁ0.1 130.7ꢁ2.2 70.7ꢁ1.2
1.7ꢁ0.1 135.0ꢁ3.1 77.3ꢁ1.5
168.7ꢁ7.7
155.7ꢁ2.9
57.3ꢁ5.0
3.6ꢁ1.2[b] 36.7ꢁ4.3
4.4ꢁ0.8
2.1ꢁ0.1
29.2ꢁ2.4
13.3ꢁ1.2
13.9ꢁ3.3
1.0ꢁ0.2
53.0ꢁ3.5 27.3ꢁ1.3
91.3ꢁ10.1 41.7ꢁ5.7
RPMI-8226 1.3ꢁ0.1
100.3ꢁ14.8 1.8ꢁ0.2
[a] Data are the mean ꢁSEM (n=3), except [b] where n=2.
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to its cytotoxicity in addition to effects on translation. Previous
studies have shown that the natural product rocaglaol (20)
and a synthetic analogue (21) have potent activity against
a range of cancer cell lines, though activity against translation
was not determined (Figure 6).[27,36,37] In our series, the pres-
similar potency against the DEAD-box helicase eukaryotic ini-
tiation factor 4A (eIF4A) therefore have great value to allow
further study of translation inhibition as a therapeutic ap-
proach in the treatment of cancer. The recent work from Porco
and co-workers, where hydroxamate 18 was shown to exhibit
similar activity to 1 in both protein synthesis and cellular cyto-
toxicity assays,[29,30] and the disclosure from researchers from
Infinity Pharmaceuticals who dem-
onstrated that nitrile derivative 23
(Figure 7) also possesses potent cy-
totoxic and eIF4A inhibitory activi-
ty,[33] indicates that the pseudo-
sugar moiety of 1/2 is not essential
for potent activity in compounds
of this class. Data for compounds
Figure 6. Rocaglaol (20) and known[27] synthetic analogues (21, 22).
prepared in this work indicates
that replacing the pseudo-sugar
with chemically less complex func-
tionality at C6 of the cyclopenta[b]-
benzofuran nucleus also leads to
Figure 7. Rocaglamide ana-
logue disclosed in ref. [33].
ence of the benzyl ether in 16a may be detrimental to effects
on translation initiation in cells compared to the methyl ether
present in 20 and 21. Indeed, syn-diol 16b is only moderately
active in all our assays whereas syn-diol 22 disclosed by Dꢁ-
saubry and co-workers is reported to be extremely potent
against a range of cancer cell lines.[27]
highly active translation initiation inhibitors with potent activi-
ty in cells and most notably significant cytotoxicity against leu-
kemia cell lines, albeit at reduced potencies compared to the
parent compounds and hydroxamate 18. It should be noted,
however, that the most active compounds tested all show sig-
nificant potency against mouse embryonic fibroblasts (MEFs),
indicating that these compounds are highly cytotoxic and may
therefore possess a narrow therapeutic window. Further study
with these simplified analogues will allow assessment of their
activity in disease-relevant models as well as determination of
on-target toxicity and tolerability, and thus therapeutic
window.
Three of the most active compounds (9b, 11 c and 11 f)
from our translation assays, along with episilvestrol (2), the
known hydroxamate 18 and the methyl hydroxamate deriva-
tive (19) of 11 f were tested against a panel of leukemia cell
lines to assess their activity against leukemia cell lines, which
have been reported to be highly sensitive to silvestrol (1).[7]
These data demonstrate that our most potent compounds do
indeed possess significant activity against leukemia cell lines
(IC50 values in the range of 12–135 nm), with the acute lympho-
blastic leukemia cell line Molt4 being the most sensitive to our
compounds. Importantly, the activity reported above is for the
compounds tested as racemates. Data reported for silvestrol
(1) and episilvestrol (2) indicates that the potent antiprolifera-
tive and cytotoxic activity resides in a single diastereomer,[10b]
therefore we anticipate the activity reported for our com-
pounds underestimates their likely bioactivity. Nonetheless, the
activity observed is notably weaker when compared to the ac-
tivity of 2 in the same assay (IC50 values approx. 1 nm), indicat-
ing that the moieties we have introduced at C6, in place of the
pseudo-sugar of 1 and 2, do not fully replicate the binding in-
teractions that the parent compounds make with their tar-
get(s). The most potent compound identified in this work is
hydroxamate 19 supporting the earlier findings from the Porco
group that the hydroxamate moiety confers additional potency
to compounds of this class.[29,30]
Experimental Section
All NMR spectra were measured at 300 K with CDCl3 as the solvent,
unless otherwise indicated, on a Bruker Avance DRX 300 (1H NMR
at 300 MHz, 13C NMR at 75 MHz). Chemical shifts (d) are reported in
ppm and referenced to the appropriate solvent peak. MS (ES) was
carried out on a Finnigan LCQ advantage MAX. HRMS (ESI) was
performed on a Waters Q-TOF high-resolution mass spectrometer.
The purity of all compounds described below was determined by
HPLC as >95% on a Waters 2525 Binary Gradient Module HPLC
coupled to a Waters 2996 Photodiode Array Detector (@254 nm)
with a Xbridge (C18, 4.6ꢂ100 mm 5 micron column) eluting with
H2O/MeCN (90:10!0:100, 1% formic acid). Analytical thin-layer
chromatography (TLC) was performed on Merck silica gel 60 F254
aluminum-packed plates and visualized with UV (254 nm) or by
staining with permanganate (potassium permanganate 1% v/w,
potassium carbonate 20% v/w, and sodium hydroxide 1% v/w in
H2O). Flash chromatography was performed with silica gel 60 (par-
ticle size 0.040–0.063 mm). All non-aqueous reactions were per-
formed in oven-dried glassware under an atmosphere of dry nitro-
gen. Anhydrous solvents were dried using an automated solvent
purification system (MBraun SPS, Garching, Germany) based upon
a technology originally described by Grubbs et al.[38] All commercial
reagents were used as received.
Conclusions
Despite the promising preliminary data published on the activ-
ity of silvestrol (1) and episilvestrol (2), extensive biochemical
and in vivo studies have been problematic due to their limited
availability and poor metabolic stability and permeability.
Structurally simpler, synthetically accessible analogues with
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to
a
solution of the known benzyl ether 9a[5c] (15.4 mg,
Synthesis
0.027 mmol) in MeOH (1 mL). The reaction was vigorously stirred
under a hydrogen atmosphere for 3 h and then filtered through
Celite. After removal of the solvent, the crude material was purified
by flash chromatography (60% EtOAc/cyclohexane) to afford the
known phenol 11[5c] (11.9 mg, 92%) as a white solid.
3-Hydroxy-5-methoxy-7-(4-methoxybenzyloxy)-2-(4-methoxy-
phenyl)-4H-chromen-4-one (7): A solution of nBuLi in hexanes
(17.6 mL, 1.70m, 30.0 mmol) was added dropwise to a solution of
diisopropylethylamine (4.24 mL, 30.0 mmol) in tetrahydrofuran
(THF; 10 mL) at ꢀ788C. The generated lithium diisopropylamide
(LDA) was allowed to warm to 08C for 15 min before being cooled
to ꢀ788C. A solution of 5-methoxy-7-(4-methoxybenzyloxy)-2-(4-
methoxyphenyl)-4H-chromen-4-one (6; 9.00 g, 21.5 mmol) in THF
(100 mL) was cannulated to the LDA solution, while maintaining
the bath at ꢀ788C. After 5 min, a solution of trimethylborate
(2.7 mL, 24.0 mmol) in THF (20 mL) was added, and the mixture
was stirred for 40 min at ꢀ788C. Glacial AcOH (2.1 mL, 36.0 mmol)
was added followed by 30% H2O2 (2.7 mL). The reaction was al-
lowed to warm to RT for 1 h and quenched with saturated
NaHCO3. EtOAc (150 mL) was then added, and the aqueous layer
was extracted with EtOAc (2ꢂ150 mL); the combined organic frac-
tions were washed with H2O (2ꢂ200 mL) and brine (200 mL), dried
(MgSO4) and concentrated in vacuo to provide the crude hydroxy-
flavone which was purified by flash chromatography (50% EtOAc/
cyclohexane) to yield the desired hydroxyflavone 7 as an orange
If reaction times were extended (6 h), the side-product 13a was
isolated exclusively as a white solid (10.6 mg, 80%): 1H NMR
([D6]acetone): d=2.38 (s, 3H), 3.53 (s, 3H), 3.71 (s, 3H), 3.90 (m,
1H), 3.91 (s, 3H), 4.13 (d, J=14.2 Hz, 1H), 4.24 (d, J=2.5 Hz, 1H),
5.02 (dd, J=2.5, 7.2 Hz, 1H), 6.24 (d, J=1.5 Hz, 1H), 6.29 (d, J=
1.5 Hz, 1H), 6.71 (d, J=8.6 Hz, 2H), 6.80–6.83 (m, 2H), 7.02–7.04
(m, 3H), 7.20 (bd, J=7.7 Hz, 2H), 8.85 ppm (s, 1H); 13C NMR
([D6]acetone) d=50.8, 51.6, 51.9, 55.3, 56.1, 80.7, 92.4, 93.4, 100.3,
100.7, 103.6, 113.1, 127.1, 128.3, 128.5, 128.8, 129.0, 138.4, 159.2,
159.6, 162.2, 162.9, 170.3 ppm; MS (ES+): m/z (%): 493 (100)
[M+H]+.
In an analogous procedure, ethyl ether 13b was obtained when
1
using EtOH as the solvent (46 mg, 58%): H NMR ([D6]acetone): d=
0.67 (t, J=7.0 Hz, 3H), 2.38–2.44 (m, 1H), 2.63–2.68 (m, 1H), 3.63 (s,
3H), 3.75 (s, 3H), 3.83 (dd, J=7.9, 14.4 Hz, 1H), 3.87 (s, 3H), 4.21 (d,
J=14.4 Hz, 1H), 4.46 (s, 1H), 5.17 (d, J=7.9 Hz, 1H), 6.06 (d, J=
1.6 Hz, 1H), 6.28 (d, J=1.6 Hz, 1H), 6.40 (bs, 1H), 6.71 (d, J=8.4 Hz,
2H), 6.80 (d, J=7.2 Hz, 2H), 7.09–6.98 (m, 3H), 7.17 ppm (bs, 2H);
13C NMR ([D6]acetone): d=15.1, 50.8, 51.5, 55.3, 56.1, 60.1, 81.0,
92.5, 93.3, 100.2, 100.5, 113.1, 127.1, 128.2, 128.7, 128.8, 129.1,
138.4, 159.1, 159.6, 162.0, 162.7, 170.3 ppm; MS (ES+): m/z (%):
507 (100) [M+H]+.
1
crystalline solid (1.46 g, 16%): H NMR: d=3.83 (s, 3H), 3.88 (s, 3H),
3.96 (s, 3H), 5.07 (s, 2H), 6.41 (s, 1H), 6.63 (s, 1H), 6.92 (d, J=
8.5 Hz, 2H), 7.03 (d, J=8.9 Hz, 2H), 7.39 (d, J=8.5 Hz, 2H),
8.16 ppm (d, J=8.9 Hz, 2H); MS (ES+): m/z (%) 419 (100) [M+H]+.
rac-Methyl (1R,2R,3S,3aR,8bS)-1,8b-dihydroxy-8-methoxy-6-((4-
methoxybenzyl)oxy)-3,3a-diphenyl-2,3,3a,8b-tetrahydro-1H-cy-
clopenta[b]benzofuran-2-carboxylate (9b): Methyl trans-cinna-
mate (3.42 g, 21.1 mmol) was added to a solution of 7 (702 mg,
1.68 mmol) in CH3CN (27 mL) and MeOH (17 mL). After degassing
for 5 min, the mixture was irradiated (450 W Hanovia UV lamp,
Pyrex filter) at ꢀ108C under a nitrogen atmosphere for 10 h. The
solvent was removed in vacuo, and the crude was purified by flash
chromatography (20% EtOAc/cyclohexane followed by 50%
EtOAc/cyclohexane) to yield a bright orange foam which was dis-
solved in MeOH (30 mL), and to the ensuing solution was added
a NaOMe solution (7.0 mL, 0.5m, 3.5 mmol). The reaction was
heated to reflux for 40 min and was quenched with sat. NH4Cl.
EtOAc (3ꢂ30 mL) was added, and the organic phase collected,
combined, dried (MgSO4), filtered and concentrated in vacuo to
provide b-keto ester 8b as a mixture of keto–enol tautomers as
a brown glassy oil which could be used in subsequent reactions
without further purification (706 mg, 71%).
General procedure for the alkylation of phenol derivatives:
K2CO3 (6.9 mg, 0.05 mmol) followed by the alkyl halide (0.05 mmol)
and tetrabutylammonium iodide (TBAI; 1.0 mg, 0.003 mmol) were
added to a solution of the phenol (namely, one of either 10, 13;
0.022 mmol) in DMF (0.5 mL). The reaction mixture was stirred at
RT for 16 h before H2O (2 mL) and EtOAc (2 mL) was added. The or-
ganic layer was collected and the aqueous layer extracted with
EtOAc (2ꢂ5 mL). The organic fractions were combined and
washed with H2O (3ꢂ5 mL) and brine and dried (MgSO4) and con-
centrated in vacuo to afford a crude residue, which was subjected
to flash silica gel chromatography. Elution with EtOAc/cyclohexane
(10:90–50:50) provided the alkylated cyclopentabenzofuran.
rac-Methyl (1R,2R,3S,3aR,8bS)-1,8b-dihydroxy-8-methoxy-6-((3-
methoxybenzyl)oxy)-3a-(4-methoxyphenyl)-3-phenyl-2,3,3a,8b-
tetrahydro-1H-cyclopenta[b]benzofuran-2-carboxylate (11a): Col-
ourless foam (3 mg, 56%): 1H NMR: d=3.65 (s, 3H), 3.71 (s, 3H),
3.85 (s, 3H), 3.87 (s, 3H), 3.90 (dd, J=6.5, 13.9 Hz, 1H), 4.31 (d, J=
13.9 Hz, 1H), 5.02 (dd, J=1.2, 6.5 Hz, 1H), 5.06 (s, 2H), 6.21 (d, J=
1.9 Hz, 1H), 6.35 (d, J=1.9 Hz, 1H), 6.67 (d, J=9.0 Hz, 2H), 6.86–
6.93 (m, 3H), 7.00–7.02 (m, 2H), 7.05–7.07 (m, 3H), 7.11 (d, J=
9.0 Hz, 2H), 7.33 ppm (dd, J=8.0, 8.0 Hz, 1H); MS (ES+): m/z: 599
[M+H]+; HRMS (ES+): m/z [M+H]+ calcd for C35H35O9: 599.2281,
found: 599.2284.
A solution of tetramethylammonium triacetoxyborohydride (1.02 g,
3.87 mmol) in CH3CN (20 mL) and AcOH (380 mL, 6.64 mmol) was
stirred under argon for 5 min. A solution of keto–enol tautomers
8b (381 mg, 0.64 mmol) in CH3CN (12 mL) was cannulated, and the
mixture was stirred at RT for 16 h. The reaction was quenched with
sat. NH4Cl and 0.5m sodium tartrate. The usual workup with CH2Cl2
and purification by flash chromatography (40% EtOAc/cyclohex-
ane) gave the endo isomer (ꢁ)-9b as a colorless oil (172 mg, 45%):
1H NMR: d=3.65 (s, 3H), 3.70 (s, 3H), 3.83 (s, 3H), 3.85 (s, 3H), 3.90
(dd, J=6.6, 14.2 Hz, 1H), 4.32 (d, J=14.2 Hz, 1H), 5.00 (s, 2H), 5.02
(d, J=6.6 Hz, 1H), 6.19 (d, J=1.9 Hz, 1H), 6.36 (d, J=1.9 Hz, 1H),
6.68 (d, J=9.0 Hz, 2H), 6.86–6.90 (m, 2H), 6.94 (d, J=8.8 Hz, 2H),
7.05–7.07 (m, 3H), 7.11 (d, J=9.0 Hz, 2H), 7.38 ppm (d, J=8.8 Hz,
2H); MS (ES+): m/z: 599 [M+H]+.
rac-Methyl (1R,2R,3S,3aR,8bS)-1,8b-dihydroxy-8-methoxy-6-((2-
methoxybenzyl)oxy)-3a-(4-methoxyphenyl)-3-phenyl-2,3,3a,8b-
tetrahydro-1H-cyclopenta[b]benzofuran-2-carboxylate (11b): Col-
1
ourless oil (4 mg, 65%): H NMR: d=3.67 (s, 3H), 3.71 (bs, 1H), 3.74
(s, 3H), 3.89 (s, 3H), 3.88–3.96 (m, 4H), 4.33 (d, J=14.1 Hz, 1H),
5.05 (d, J=6.5 Hz, 1H), 5.15 (s, 2H), 6.26 (d, J=2.0 Hz, 1H), 6.40 (d,
J=2.0 Hz, 1H), 6.70 (d, J=9.0 Hz, 2H), 6.86–6.90 (m, 2H), 7.09–6.95
(m, 5H), 7.13 (d, J=9.0 Hz, 2H), 7.35 (dt, J=1.8, 7.8 Hz, 1H),
7.48 ppm (dd, J=1.8, 7.8 Hz, 1H); MS (ES+): m/z (%): 599 (51)
[M+H]+.
rac-Methyl (1R,2R,3S,3aR,8bS)-1,6-dihydroxy-8,8b-dimethoxy-3a-
(4-methoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopen-
ta[b]benzofuran-2-carboxylate (13a): Pd(OH)2 (20 wt%, 4.0 mg,
0.0057 mmol) and Pd/C (10 wt%, 8.0 mg, 0.0075 mmol) was added
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rac-Methyl (1R,2R,3S,3aR,8bS)-1,8b-dihydroxy-8-methoxy-3a-(4-
methoxyphenyl)-3-phenyl-6-(pyridin-3-ylmethoxy)-2,3,3a,8b-tet-
rahydro-1H-cyclopenta[b]benzofuran-2-carboxylate (11c): Col-
ourless foam (9 mg, 74%): 1H NMR: d=3.65 (s, 3H), 3.72 (s, 3H),
3.87 (s, 3H), 3.87–3.94 (m, 1H), 4.30 (d, J=14.2 Hz, 1H), 5.05 (m,
3H), 6.21 (d, J=1.9 Hz, 1H), 6.33 (d, J=1.9 Hz, 1H), 6.67 (d, J=
9.0 Hz, 2H), 6.85–6.88 (m, 2H), 7.04–7.11 (m, 3H), 7.10 (d, J=9.0 Hz,
2H), 7.36 (bs, 1H), 7.78 (d, J=7.7 Hz, 1H), 8.53 ppm (bs, 2H); MS
(ES+): m/z: 570 [M+H]+; HRMS (ES+): m/z [M+H]+ calcd for
C33H32NO8: 570.2128, found: 570.2130.
tetrahydro-1H-cyclopenta[b]benzofuran-2-carboxylate
(14a):
Et3N (13.9 mL, 0.100 mmol) followed by 4-bromobenzoyl chloride
(11.0 mg, 0.050 mmol) were added to a solution of phenol 13a
(20.4 mg, 0.0414 mmol) in CH2Cl2 (1 mL) at 08C. The reaction was
warmed to RT and stirred for a further 14 h and was then
quenched with saturated aq NaHCO3 (2 mL). The aqueous layer
was extracted with CH2Cl2 (2ꢂ5 mL), and the organic fractions
were combined, washed with H2O (10 mL), saturated NaHCO3
(10 mL), brine, dried (MgSO4) and concentrated in vacuo. The
crude residue was subjected to flash chromatography (40–60%
EtOAc/cyclohexane) and provided compound 14a as a white crys-
rac-Methyl (1R,2R,3S,3aR,8bS)-6-((3-fluorobenzyl)oxy)-1,8b-dihy-
droxy-8-methoxy-3a-(4-methoxyphenyl)-3-phenyl-2,3,3a,8b-tet-
rahydro-1H-cyclopenta[b]benzofuran-2-carboxylate (11d): Col-
1
talline solid (23.2 mg, 83%): H NMR: d=2.50 (s, 3H), 3.62 (s, 3H),
3.77 (s, 3H), 3.83 (dd, J=7.4, 14.3 Hz, 1H), 3.95 (s, 3H), 4.16 (d, J=
14.3 Hz, 1H), 5.17 (d, J=7.4 Hz, 1H), 6.50 (d, J=1.8 Hz, 1H), 6.70–
6.75 (m, 5H), 7.05–7.09 (m, 3H), 7.16 (bd, J=7.9 Hz, 2H), 7.70 (d,
J=8.6 Hz, 2H), 8.09 ppm (d, J=8.6 Hz, 2H); MS (ES+): m/z (%): 697
(62) [M+Na]+. Recrystallisation from CH2Cl2/heptanes provided
crystals suitable for x-ray crystallography.
1
ourless oil (3 mg, 30%): H NMR: d=3.67 (s, 3H), 3.73 (s, 3H), 3.90
(s, 3H), 3.92 (dd, J=6.7, 14.2 Hz, 1H), 4.33 (d, J=14.2 Hz, 1H), 5.05
(d, J=6.7 Hz, 1H), 5.10 (s, 2H), 6.23 (d, J=2.0 Hz, 1H), 6.35 (d, J=
2.0 Hz, 1H), 6.70 (d, J=9.0 Hz, 2H), 6.88–6.91 (m, 2H), 7.07–7.11 (m,
4H), 7.13 (d, J=9.0 Hz, 2H), 7.19–7.23 (m, 2H), 7.39–7.42 ppm (m,
1H); MS (ES+): m/z (%): 587 (100) [M+H]+.
Single crystal X-ray structure determination: Intensity data were
collected with an Oxford Diffraction SuperNova CCD diffractometer
using MoKa or CuKa radiation, the temperature during data collec-
tion was maintained at 130.0(1) using an Oxford Cryosystems cool-
ing device. The structure was solved by direct methods and differ-
ence Fourier synthesis.[39] Thermal ellipsoid plots were generated
using the program ORTEP-3[40] integrated within the WINGX[41] suite
of programs. The asymmetric unit consists of two enantiomerically
related molecules of 14a and a molecule of heptane solvate. The
crystal was inversion twinned with Flack parameter refined to
0.36(1). Crystal data for 14a: 2(C35H30BrO9). (C7H18) M=1451.21, T=
130.0 K, l=1.54180, Monoclinic, space group C2, a=19.6904(2),
b=15.5914(1), c=24.3002(3) ꢃ, b=113.469(1)8. V 6843.0(1) ꢃ3, Z=
4, 1cald =1.409 MgMꢀ3 m (CuKa) 2.107 mmꢀ1, F(000)=3016, crystal
size 0.44ꢂ0.19ꢂ0.15 mm3, 25796 reflections measured, 11508 in-
dependent reflections [R(int)=0.0174], the final R was 0.0267 [I>
2s(I)] and wR(F2) was 0.0726 (all data).
rac-Methyl
(1R,2R,3S,3aR,8bS)-1,8b-dihydroxy-6-((4-isopropyl-
benzyl)oxy)-8-methoxy-3a-(4-methoxyphenyl)-3-phenyl-
2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-carboxylate
(11e): Colourless oil (5 mg, 46%): 1H NMR: d=1.29 (d, J=7.0 Hz,
6H), 2.96 (dq, J=7.0 Hz, 1H), 3.67 (s, 3H), 3.73 (s, 3H), 3.91 (s, 3H),
3.92 (dd, J=6.5, 14.4 Hz, 1H), 4.34 (d, J=14.4 Hz, 1H), 5.04–5.06
(m, 3H), 6.23 (d, J=1.9 Hz, 1H), 6.39 (d, J=1.9 Hz, 1H), 6.70 (d, J=
9.0 Hz, 2H), 6.89–6.91 (m, 2H), 7.07–7.12 (m, 3H), 7.13 (d, J=
9.0 Hz, 2H), 7.31 (d, J=8.3 Hz, 2H), 7.41 ppm (d, J=8.3 Hz, 2H);
MS (ES+): m/z: 611 [M+H]+.
rac-Methyl (1R,2R,3S,3aR,8bS)-1,8b-dihydroxy-8-methoxy-3a-(4-
methoxyphenyl)-3-phenyl-6-(pyrimidin-2-ylmethoxy)-2,3,3a,8b-
tetrahydro-1H-cyclopenta[b]benzofuran-2-carboxylate
(11 f):
1
White solid (4 mg, 63%): H NMR: d=3.67 (s, 3H), 3.73 (s, 3H), 3.89
(s, 3H), 3.91 (dd, J=6.8, 14.2 Hz, 1H), 4.31 (d, J=14.2 Hz, 1H), 5.04
(d, J=6.8 Hz, 1H), 5.33 (s, 2H), 6.33–6.35 (m, 2H), 6.69 (d, J=
9.0 Hz, 2H), 6.85–6.89 (m, 2H), 7.02–7.10 (m, 3H), 7.12 (d, J=
9.0 Hz, 2H), 7.31 (d, J=4.9 Hz, 1H), 8.82 ppm (d, J=4.9 Hz, 2H);
MS (ES+): m/z (%): 571 (100) [M+H]+.
rac-(3S,3aR,8bR)-8b-Hydroxy-8-methoxy-6-((4-methoxybenzy-
l)oxy)-3,3a-diphenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]ben-
zofuran-1-one (15): LiCl (39.5 mg, 0.93 mmol) followed by H2O
(33.2 mL, 1.85 mmol) were added to a solution of b-keto esters 8b
(370 mg, 0.618 mmol) in DMSO (3 mL). The reaction mixture was
heated at 1008C for 14 h before being cooled to RT and diluted
with EtOAc (10 mL) and H2O (10 mL). The aqueous phase was ex-
tracted with EtOAc (2ꢂ10 mL), and the combined organic fractions
were washed with H2O (3ꢂ10 mL), brine, dried (MgSO4) and con-
centrated in vacuo to afford a crude residue which was subjected
to flash silica gel chromatography. Elution with 20–30% EtOAc/cy-
clohexanes provided ketone 15 as a light yellow oil (85.1 mg,
26%): 1H NMR: d= 2.99–3.05 (m, 2H), 3.70 (s, 3H), 3.80 (s, 3H),
3.82 (s, 3H), 3.85–3.93 (m, 1H), 5.02 (s, 2H), 6.18 (d, J=2.0 Hz, 1H),
6.42 (d, J=2.0 Hz, 1H), 6.67 (d, J=8.8 Hz, 2H), 6.93–6.98 (m, 6H),
7.10–7.12 (m, 3H), 7.38 ppm (d, J=8.8 Hz, 2H); MS (ES+): m/z (%):
539 (100) [M+H]+.
rac-Methyl (1R,2R,3S,3aR,8bS)-8b-ethoxy-1-hydroxy-8-methoxy-
6-((2-methoxy-benzyl)oxy)-3a-(4-methoxyphenyl)-3-phenyl-
2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzo-furan-2-carboxylate
(14b): Light yellow solid (12 mg, 93%): 1H NMR: d=0.69 (t, J=
7.0 Hz, 3H), 2.38–2.44 (m, 1H), 2.63–2.68 (m, 1H), 3.59 (s, 3H), 3.74
(s, 3H), 3.78 (dd, J=7.4, 14.5 Hz, 1H), 3.85 (s, 3H), 3.88 (s, 3H),
4.09–4.13 (m, 2H), 5.07 (s, 2H), 5.11 (d, J=7.4 Hz, 1H), 6.24 (d, J=
1.9 Hz, 1H), 6.38 (d, J=1.9 Hz, 1H), 6.67–6.72 (m, 4H), 6.89–6.92
(m, 1H), 7.04 (m, 5H), 7.15 (bs, 2H), 7.34 ppm (dd, J=8.4, 8.4 Hz,
1H); MS (ES+): m/z (%): 628 (100) [M+H]+.
rac-Methyl (1R,2R,3S,3aR,8bS)-8b-ethoxy-1-hydroxy-8-methoxy-
3a-(4-methoxyphenyl)-3-phenyl-6-(pyridin-4-ylmethoxy)-
2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-carboxylate
(14c): Light yellow solid (9 mg, 93%): 1H NMR: d=0.68 (t, J=
7.0 Hz, 3H), 2.38–2.44 (m, 1H), 2.60–2.68 (m, 1H), 3.59 (s, 3H), 3.74
(s, 3H), 3.77 (dd, J=7.4, 14.2 Hz, 1H), 3.90 (s, 3H), 4.09–4.15 (m,
2H), 5.12 (bs, 3H), 6.24 (d, J=2.0 Hz, 1H), 6.32 (d, J=2.0 Hz, 1H),
6.60–6.69 (m, 4H), 6.99–7.09 (m, 3H), 7.14 (bs, 2H), 7.38 (d, J=
4.6 Hz, 2H), 8.61 ppm (s, 2H); MS (ES+): m/z (%): 598 (100) [M+
H]+.
rac-(1R,3S,3aR,8bS)-8-Methoxy-6-((4-methoxybenzyl)oxy)-3,3a-di-
phenyl-1,2,3,3a-tetrahydro-8bH-cyclopenta[b]benzofuran-1,8b-
diol (16a): A solution of tetramethylammonium triacetoxyborohy-
dride (93.5 mg, 0.356 mmol) and AcOH (35 mL, 0.58 mmol) in
CH3CN (1.5 mL) was stirred for 5 min. A solution of ketone 15
(20.8 mg, 0.039 mmol) in CH3CN (1 mL) was cannulated, and the
mixture was stirred at RT for 16 h. The reaction was quenched with
saturated aq NH4Cl (2 mL) and sodium/potassium tartrate (2 mL,
0.5m). CH2Cl2 (5 mL) was added, the organic layer was collected
and the aqueous layer was extracted with CH2Cl2 (2ꢂ5 mL). The
rac-Methyl
(1R,2R,3S,3aR,8bS)-6-((4-bromobenzoyl)oxy)-1-hy-
droxy-8,8b-dimethoxy-3a-(4-methoxyphenyl)-3-phenyl-2,3,3a,8b-
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combined organic phases were washed with H2O (2ꢂ10 mL), brine
(10 mL), dried (MgSO4) and concentrated in vacuo to provide
a crude residue which was subjected to flash chromatography. Elu-
tion with 20–40% EtOAc/cyclohexane provided compound 16a as
a white solid (15.9 mg, 76%): 1H NMR: d=2.22 (ddd, J=1.2, 6.9,
14.1 Hz, 1H), 2.76 (dt, J=6.4, 14.1 Hz, 1H), 3.73 (s, 3H), 3.85 (s, 3H),
3.91 (s, 3H), 4.02 (dd, J=6.4, 14.1 Hz, 1H), 4.83 (d, J=6.4 Hz, 1H),
5.03 (s, 2H), 6.24 (d, J=2.0 Hz, 1H), 6.39 (d, J=2.0 Hz, 1H), 6.70 (d,
J=9.0 Hz, 2H), 6.97 (d, J=8.8 Hz, 2H), 7.02–7.03 (m, 2H), 7.10–7.16
(m, 5H), 7.39 ppm (d, J=8.8 Hz, 2H); MS (ES+): m/z: 563 [M+Na]+
; HRMS (ES+): m/z [M+Na]+ calcd for C33H32O7Na: 563.2040,
found: 563.2039.
6.98–7.04 (m, 5H), 7.50 (t, J=6.0 Hz, 1H), 8.88 (d, J=6.0 Hz, 2H),
11.14 ppm (s, 1H); MS (ES+): m/z (%): 586 (100) [M+H]+; HRMS
(ES+): m/z [M+Na]+ calcd for C32H31N3O8: 608.2009, found:
608.2003.
Biology
In vitro translation assays: In vitro translation assays were per-
formed in rabbit reticulocyte lysate (Promega, Alexandria, Australia)
for 1 h at 308C as previously described.[19] Firefly luciferase was
translated in a cap-dependent manner whereas Renilla luciferase
production was driven by HCV IRES-mediated translation, which
occurs independent of eIF4A, from capped pSP/(CAG)33/FF/HCV/
Ren.pA51 mRNA. Compounds were tested at 50 mm with a final
concentration of 135 mm KCl. The Dual Glo Luciferase Assay kit
(Promega) was used to measure luciferase activity.
rac-(1S,3S,3aR,8bS)-8-Methoxy-6-((4-methoxybenzyl)oxy)-3,3a-di-
phenyl-1,2,3,3a-tetrahydro-8bH-cyclopenta[b]benzofuran-1,8b-
diol (16b): NaBH4 (19.3 mg, 0.510 mmol) was added to a solution
of ketone 15 (27.4 mg, 0.051 mmol) in MeOH (3 mL). The solution
was stirred at RT for 16 h before the solvent was removed in
vacuo, and the crude residue subjected to flash chromatography.
Elution with 20–40% EtOAc/cyclohexanes provided compound
Translation in mouse embryonic fibroblasts (MEFs): Protein synthesis
inhibition by silvestrol (1) and its analogues was performed as pre-
viously described in mouse embryonic fibroblasts.[19] Briefly, cells
were treated with 1 mm compound for 1 h, and 35S-labeled methio-
nine and cysteine was added in the last 20 min. Cells were then
washed and lysed. Proteins in the cell extract were precipitated
with trichloroacetic acid, and the radiolabeled protein/total protein
content calculated.
1
16b as a light yellow solid (16.9 mg, 61%): H NMR: d=2.34–2.45
(m, 1H), 2.61–2.70 (m, 1H), 3.48 (dd, J=7.0, 14.0 Hz, 1H), 3.73 (s,
3H), 3.85 (s, 3H), 3.83–3.86 (m, 1H), 4.83 (dd, J=7.8, 7.8 Hz, 1H),
5.02 (s, 2H), 6.20 (d, J=1.9 Hz, 1H), 6.35 (d, J=1.9 Hz, 1H), 6.71 (d,
J=9.0 Hz, 2H), 6.95 (d, J=8.8 Hz, 2H), 7.02–7.04 (m, 2H), 7.08–7.15
(m, 3H), 7.22 (d, J=9.0 Hz, 2H), 7.40 ppm (d, J=8.8 Hz, 2H); MS
(ES+): m/z (%): 563 (100) [M+Na]+; HRMS (ES+): m/z [M+Na]+
calcd for C33H32O7Na: 563.2040, found: 563.2041.
Cellular viability assays: Cell viability and proliferation assays were
performed as previously described[9,42] using the CellTiter-Glo Lumi-
nescent Cell Viability Assay (Promega). Briefly, cells (MEFs or leukae-
mia lines) were seeded in opaque 96-well plates and treated with
11 point dilution of indicated compounds, incubated at 378C/10%
CO2 for 72 h and analysed via the manufacturer’s instructions.
rac-(1R,2R,3S,3aR,8bS)-1,8b-Dihydroxy-N,8-dimethoxy-3a-(4-me-
thoxyphenyl)-3-phenyl-6-(pyrimidin-2-ylmethoxy)-2,3,3a,8b-tet-
rahydro-1H-cyclopenta[b]benzofuran-2-carboxamide
(19):
LiOH·H2O (37 mg, 0.87 mmol) was added to a stirred solution of
pyrimidine 12 f (42.0 mg, 0.087 mmol) in THF (2.5 mL) and H2O
(0.5 mL) at 208C. The mixture was heated at 508C under N2 for 4 h.
The reaction mixture was cooled to RT and adjusted to ꢂpH 3
with HCl (1m). The aqueous phase was extracted with EtOAc (3ꢂ
3 mL), and the combined extracts were dried (Na2SO4), filtered and
concentrated in vacuo. The crude material was subjected to flash
chromatography, elution with 0–30% MeOH/CH2Cl2 gave rac-
(1R,2R,3S,3aR,8bS)-1,8b-dihydroxy-8-methoxy-3a-(4-methoxyphen-
yl)-3-phenyl-6-(pyrimidin-2-ylmethoxy)-2,3,3a,8b-tetrahydro-1H-cy-
clopenta[b]benzofuran-2-carboxylic acid as a white solid (16.0 mg,
Chemical stability determination: Stability experiments were con-
ducted at a nominal episilvestrol (2) concentration of 10 mm in si-
mulated gastric fluid (SGF; USP23, p2053), pH 3 (50 mm isotonic
phosphate buffer), pH 5 (50 mm isotonic acetate buffer) and pH 7
(50 mm isotonic phosphate buffer) media at 378C. Samples were
analysed by LC–MS at the beginning of the study and after 1, 4
and 8 h by diluting 1:20 with quench solution (prepared at a ratio
of 0.9 (50 mm phosphate buffer, pH 7) to 1 (CH3CN)) containing ve-
rapamil as an internal standard.
LC–MS analysis was conducted using a Waters Acquity UPLC cou-
pled to a Waters Micromass Quattro Ultima Pt triple quadrupole
mass spectrometer. Separation was performed using a Supelco As-
centis Express RP-C18 (50ꢂ2.1 mm) column maintained at
a column temperature of 408C.
1
36%): H NMR (600 MHz, [D6]DMSO): d=3.57 (s, 3H), 3.76 (dd, J=
13.8, 5.4 Hz, 1H), 4.07 (d, J=13.8 Hz, 1H), 4.64 (d, J=5.4 Hz, 1H),
5.00 (s, 1H), 5.25 (s, 2H), 6.16 (d, J=1.8 Hz, 1H), 6.27 (d, J=1.8 Hz,
1H), 6.55 (d, J=9.0 Hz, 2H), 6.85 (d, J=7.2 Hz, 2H), 6.95–6.96 (m,
3H), 7.00–7.02 (m, 2H), 7.47 (t, J=4.8 Hz, 1H), 8.85 ppm (d, J=
4.8 Hz, 2H); MS (ES+): m/z (%): 557 (100) [M+H]+.
Microsomal stability: The metabolic stability assay was performed
by incubating episilvestrol (2; 1 mm) with human (XenoTech,
Lenexa, KS, USA) or mouse (BD Gentest, Woburn, MA, USA) liver
microsomes at 378C and 0.4 mgmLꢀ1 protein concentration. The
metabolic reaction was initiated by the addition of a NADPH-re-
generating system and quenched at various time points over the
60 min incubation period by the addition of CH3CN containing ve-
rapamil as an internal standard. Control samples (devoid of
NADPH) were included (and quenched at 2, 30 and 60 min) to
monitor for potential degradation and formation of metabolites in
the absence of cofactors. Incubations were conducted in triplicate.
Sample analysis by LC–MS was conducted as described above.
EDC (10.0 mg, 0.0501 mmol) followed by HOBt·H2O (10.9 mg,
0.0728 mmol) were added to a stirred solution of acid generated
as described above (17.0 mg, 0.0305 mmol) in CH2Cl2 (4 mL). After
stirring for 5 min, methoxyamine hydrochloride (12.2 mg,
0.144 mmol) was added followed by Et3N (20.1 mL, 14.6 mmol). The
reaction was stirred at 208C for 16 h then quenched with HCl (1m)
and diluted with H2O (2 mL). The aqueous phase was separated,
extracted with EtOAc (2ꢂ3 mL), and the organic phases were com-
bined and dried (MgSO4), filtered and concentrated in vacuo. The
crude material was subjected to flash chromatography, elution
with 0–20% MeOH/CH2Cl2 provided compound 19 as a white solid
(9.5 mg, 56%): 1H NMR ([D6]DMSO): d=3.49 (s, 3H), 3.61 (s, 3H),
3.73 (s, 3H), 4.17 (d, J=12 Hz, 1H), 4.53–4.56 (m, 1H), 4.69 (d, J=
3 Hz, 1H), 5.03 (s, 1H), 5.28 (s, 2H), 6.21 (d, J=1.8 Hz, 1H), 6.30 (d,
J=1.8 Hz, 1H), 6.59 (d, J=9.0 Hz, 2H), 6.88 (d, J=8.0 Hz, 2H),
Permeability assay: Standard 96-well PVDF filter plates were coated
with
a solution of lipid material (dioleoyl-3-phosphocholine
(DOPC)) to prepare the artificial membrane. The filter plate was
sandwiched with a 96-well acceptor plate filled with transport
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Acknowledgements
Prof. Jonathan Baell is thanked for useful discussions, Dana
Buczek is acknowledged for analytical support and H. Ierino for
technical assistance. Dr. David Owen is thanked for assistance in
establishing the photochemical reactor and Dr. John Flygare for
samples of key intermediates and silvestrol. This work is support-
ed by scholarships, fellowships and grants from the Australian
National Health and Medical Research Council (Early Career Fel-
lowship to L.M.L.; Research Fellowship to D.C.S.H.; program
grants: 461221 and 1016701; Independent Research Institutes In-
frastructure Support Scheme, grant 361646); the Victorian State
Government Operational Infrastructure Support (OIS) Grant; the
Australian Cancer Research Foundation; the Canadian Institutes
of Health Research (Postdoctoral Fellowship to L.M.L., who is also
a Bisby Fellow); Cancer Therapeutics CRC (D.N., J.M.C., M.A.R.);
and Dyson Bequest funding (Dunn Fellowship to C.J.B.).
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