Gedunin, a novel Hsp90 inhibitor: Semisynthesis of derivatives and preliminary structure-activity relationships

Article abstract of DOI:10.1021/jm8007486

Gedunin (1), a tetranortriterpenoid isolated from the Indian neem tree (Azadirachta indica), was recently shown to manifest anticancer activity via inhibition of the 90 kDa heat shock protein (Hsp90) folding machinery and to induce the degradation of Hsp90-dependent client proteins similar to other Hsp90 inhibitors. The mechanism of action by which gedunin induces client protein degradation remains undetermined, however, prior studies have demonstrated that it does not bind competitively versus ATP. In an effort to further probe the mechanism of action, 19 semisynthetic derivatives of gedunin were prepared and their antiproliferative activity against MCF-7 and SkBr3 breast cancer cells determined. Although no compound was found to exhibit antiproliferative activity more effective than the natural product, functionalities critical for antiproliferative activity have been identified.

Full text of DOI:10.1021/jm8007486

J. Med. Chem. 2008, 51, 6495–6502
6495
Gedunin, a Novel Hsp90 Inhibitor: Semisynthesis of Derivatives and Preliminary
Structure-Activity Relationships
Gary E. L. Brandt,† Matthew D. Schmidt,‡ Thomas E. Prisinzano,†,‡ and Brian S. J. Blagg*,†
Department of Medicinal Chemistry, The UniVersity of Kansas, 1251 Wescoe Hall DriVe, 4070 Malott Hall, Lawrence, Kansas 66045-7582,
and DiVision of Medicinal and Natural Products Chemistry, The UniVersity of Iowa, Iowa City, Iowa 52232
ReceiVed June 18, 2008
Gedunin (1), a tetranortriterpenoid isolated from the Indian neem tree (Azadirachta indica), was recently
shown to manifest anticancer activity via inhibition of the 90 kDa heat shock protein (Hsp90) folding
machinery and to induce the degradation of Hsp90-dependent client proteins similar to other Hsp90 inhibitors.
The mechanism of action by which gedunin induces client protein degradation remains undetermined,
however, prior studies have demonstrated that it does not bind competitively versus ATP. In an effort to
further probe the mechanism of action, 19 semisynthetic derivatives of gedunin were prepared and their
antiproliferative activity against MCF-7 and SkBr3 breast cancer cells determined. Although no compound
was found to exhibit antiproliferative activity more effective than the natural product, functionalities critical
for antiproliferative activity have been identified.
Introduction
DMAG), the major concern is related to the redox-active
quinone moiety. Quinones, which are good Michael acceptors
and catalysts for free-radical generation, can complicate treat-
ment by exhibiting toxicity unrelated to Hsp90 inhibition.12
Other Hsp90 inhibitors, e.g., novobiocin, coumarin analogues,
and purine analogues, target either the N- or C-terminal
nucleotide binding domains, but little toxicological data have
been reported to date. Since Hsp90 is known to interact with
various cochaperones during the protein folding cycle, there may
be additional mechanisms through which this chaperone can
be modulated and thus provide an alternative to these well-
established inhibitors.
Gedunin (1, Figure 1), a tetranortriterpenoid isolated from
the Indian neem tree (Azadirachta indica), has demonstrated
the ability to exhibit antimalarial, insecticidal, and most recently
anticancer activity.13-15 The antitumor activity of gedunin was
explored through the use of the connectivity map.16 Lamb et
al. found, via high connectivity scores with GDA, 17-AAG,
and 17-DMAG, that gedunin exhibited its antiproliferative
activity through Hsp90 modulation. In a subsequent article,
however, gedunin was determined to interact with Hsp90 via a
mechanism that does not involve competitive inhibition of
ATP,17 and therefore, the interaction of gedunin with Hsp90 is
distinct from that of GDA and related analogues. While gedunin
induces Hsp90-dependent client protein degradation similar to
other Hsp90 inhibitors, the natural product was unable to
displace GDA in a fluorescence polarization assay with purified
Hsp90 at concentrations up to 100 µM. As a structurally and
mechanistically distinct regulator of the Hsp90 protein folding
machinery, gedunin represents a lead compound that may
possess unique Hsp90 modulatory attributes.
The development of resistance to chemotherapeutic agents
is a major obstacle to the successful treatment of cancer.1-4
Whether because of the overexpression of efflux pumps, protein
mutagenesis, or activation of alternative cell signaling pathways,
resistance results from the genetic plasticity/instability of cancer
cells.5,6 Cancer therapies often target a single oncogenic protein,
and it is the same genetic plasticity/instability that allows cancer
to select for the most adaptable mutation in order to circumvent
drug effects. This type of resistance may be avoided, however,
if multiple cancer signaling pathways are targeted simulta-
neously with a single therapeutic agent. The family of 90 kDa
heat shock proteins (Hsp90a) has emerged as a drug target that
mediates multiple signaling nodes as a novel strategy to combat
cancer.5-7
Hsp90 is a molecular chaperone that is responsible for the
conformational maturation of many nascent polypeptides, as well
as denatured proteins, into their biologically active forms. To
date, over 100 client proteins have been identified (see the Web
page maintained by D. Picard, http://www.picard.ch/DP/
downloads/Hsp90interactors.pdf) many of which are essential
to oncogenic development, cancer cell survival, and the
development of drug resistance.5-11 As a target for chemo-
therapy, Hsp90 is well validated and several Hsp90 targeting
drugs are currently in clinical trials. There are, however,
drawbacks associated with the current drugs that target Hsp90.
For drugs based on the structure of geldanamycin (GDA), e.g.,
17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-
dimethylaminoethylamino-17-demethoxygeldanamycin (17-
* To whom correspondence should be addressed. Telephone: 785-864-
4495. Fax: 785-864-5326. E-mail: bblagg@ku.edu.
To date, no structure-activity relationships have been
established between Hsp90 and gedunin. The ease of acquiring
gedunin and its 7-desacetyl derivative from neem oil and the
functionality present on the natural products make them well-
suited for semisynthetic derivatization for the development of
more efficacious compounds. In this article, 19 analogues of
gedunin were prepared through semisynthetic procedures aimed
at elucidating the role of the R,ꢀ-unsaturated ketone, exploring
the sensitivity of the ketone binding pocket to steric bulk, and
†
The University of Kansas.
The University of Iowa.
‡
a Abbreviations: 17-AAG, 17-allylamino-17-demethoxygeldanamycin;
ATP, adenosine triphosphate; 17-DMAG, 17-dimethylaminoethlamino-17-
demethoxygeldanamycin; DCM, dichloromethane; GDA, geldanamycin;
HER2, human estrogen receptor 2; HPLC, high performance liquid
chromatography; Hsp90, 90 kDa heat shock protein; MCF-7, human breast
adenocarcinoma cell line; MTS/PMS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, innersalt/phena-
zine methosulfate; Raf, mitogen-activated protein kinase kinase kinase;
SKBr3,Sloan-Ketteringbreastcancercellline;TLC,thinlayerchromatography.
10.1021/jm8007486 CCC: $40.75
2008 American Chemical Society
Published on Web 09/25/2008

6496 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Brandt et al.
Figure 1. Chemical structures of GDA, gedunin (1), and novobiocin.
Scheme 2a
the effects of various substituents at the 7-position. All analogues
were evaluated for antiproliferative activity against MCF-7 and
SKBr-3 breast cancer cell lines, and a few compounds were
subsequently analyzed by Western blot analysis of Hsp90-
dependent client proteins. The results from these studies are
reported herein.
Chemistry
Commercially available neem oil presents an affordable
resource for a rich supply of gedunin. 1 kg of neem oil from
Neem King was extracted with methanol. The methanol extracts
were combined and washed with n-hexanes, and the remaining
insoluble dark-brown resin was purified by flash column
chromatography using a gradient of 100% hexanes to 100%
ethyl acetate. Fractions containing gedunin were pooled and
subjected to successive columns, each using a tighter gradient
of hexanes and ethyl acetate. Gedunin-containing fractions that
appeared clean by thin layer chromatography (TLC) were pooled
and concentrated under reduced pressure to provide a pale-
yellow resin that was crystallized from ethyl acetate to give
3 g of a white crystalline solid, which contained two products
a Reagents and conditions: (a) NaOH, H2O2, acetone.
N-oxide gave vicinal diol 6, which upon treatment with 2,2-
dimethoxypropane and p-toluenesulfonic acid yielded the cor-
responding acetonide 7. In contrast, hydrogenation of the double
bond was accomplished by use of palladium on carbon and
hydrogen gas to afford 8. Standard Luche conditions surprisingly
gave the over-reduced product, 9. After considerable investment,
chemoselective reduction of the ketone was finally accomplished
via Merwein-Pondorf-Verley conditions enlisting aluminum
isopropoxide and isopropanol to afford 10a. The allylic alcohol
of 10a was acetylated under standard conditions to give 10b.
Treatment of gedunin with excess sodium borohydride without
a lanthanide gave the corresponding alcohol, 11. Oxime ether
derivatives of gedunin, 12a-c, were synthesized by treatment
of the natural product with substituted hydroxylamines. Com-
pound 13, the product resulting from the treatment of gedunin
with hydroxylamine, is a further demonstration of unique
behavior demonstrated by the lactone moiety of gedunin.
1
by TLC and HPLC. H and 13C NMR data show all peaks
previously described for gedunin and desacetylgedunin.13 These
two compounds were obtained in their pure forms by a final
isocratic purification using n-hexanes/dichloromethane/diethyl
ether in a ratio of 5:3:2.
Compounds 3a-f and 4 were synthesized from 7-de-
sacetylgedunin (2) as outlined in Scheme 1. 7-Desacetylgedunin
(2) was treated with potassium tert-butoxide and reacted with
the requisite alkyl halide to afford ethers 3a-d. Standard
acylation procedures were used to generate 3e. Compound 3f
was produced by treatment of 7-desacetylgedunin with trichlo-
roacetyl isocyanate followed by basic methanolic workup.
Pyridinium chlorochromate oxidation of 7-desacetylgedunin
provided the corresponding ketone 4.
Results and Discussion
Without confirmation of the site to which gedunin binds
Hsp90, we set out to synthesize 19 derivatives of the natural
product in order to determine the importance of specific
functional groups on the tetracyclic core, as well as to probe
the chemical space surrounding these ring systems. Gedunin
(1, Figure 1) presents a variety of chemical surfaces, each of
which offers the potential of chemo- or regioselective modifica-
tion. The red numbers in Figure 1 indicate functional groups
The synthesis of compounds 6-13 are given in Schemes 2-4.
Treatment of 7-desacetylgedunin with sodium hydroxide and
hydrogen peroxide resulted in epoxide 5. Dihydroxylation of
olefin 1 using osmium tetroxide and N-methylmorpholine
Scheme 1a
a Reagents and conditions: (a) alkyl iodide, KOtBu, THF, 0°C; (b) propionyl chloride, DMAP, Et3N, CH2Cl2, 0 °C; (c) trichloroacetyl isocyanate, CH2Cl2;
(d) PCC, CH2Cl2.

Gedunin, a NoVel Hsp90 Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6497
Scheme 3a
a Reagents and conditions: (a) NMO, OsO4, acetone, H2O, 0 °C; (b) 1,2-dimethoxypropane, pTsOH, acetone; (c) 10% Pd/C, MeOH; (d) NaBH4, CeCl3,
i
MeOH/CHCl3; (e) PrOH, Al(OiPr)3, toluene, 70 °C; (f) acyl chloride, DMAP, Et3N, CH2Cl2; (g) NaBH4, EtOH; (h) alkoxyamine hydrochloride, pyridine,
70 °C.
Table 1. IC50 Values with Standard Deviations of Gedunin Analogues
focused upon by these studies. We sought to investigate steric
limitations and electronic properties of substituents at the
in MCF-7 and SKBr3 Breast Cancer Cell Linesa
compd
MCF-7
SKBr3
7-position and the R,ꢀ-unsaturated ketone moiety (carbons 1,
2, and 3). Because the goal of this project was to generate
preliminary structure-activity relationships, products of non-
stereospecific reactions were not separated into their diastereo-
merically pure forms.
1
2
8.84 ( 0.03
28.96 ( 4.48
38.88 ( 0.36
14.79 ( 0.87
37.27 ( 2.20
>100
3.22 ( 0.6
21.09 ( 5.95
12.99 ( 0.85
12.06 ( 1.27
18.14 ( 0.99
>100
3a
3b
3c
3d
3e
3f
4
5
6
7
Scheme 4a
>100
>100
9.79 ( 0.69
10.90 ( 0.41
>100
7.05 ( 1.00
4.49 ( 0.98
>100
>100
>100
>100
>100
8
9
>100
>100
47.72 ( 1.59
54.31 ( 6.78
>100
82.38 ( 17.62
47.70 ( 1.61
>100
10a
10b
11
12a
12b
12c
13
>100
>100
>100
>100
a Reagents and conditions: (a) hydroxylamine hydrochloride, pyridine,
70 °C.
>100
>100
>100
>100
33.17 ( 2.18
26.49 ( 0.93
a IC50 values are determined in µM from nonlinear dose-response curve
using GraphPad Prism based on data from standard antiproliferative assay
using MTS/PMS as a visualizing reagent. Each experiment was repeated
three times in triplicate.
Compounds 1, 2, 3a-f, 4-9, 10a,b, 11, 12a-c, and 13 were
evaluated for antiproliferative activity against MCF-7 and SKBr3
cells alongside geldanamycin, which is a previously reported
Hsp90 inhibitor that binds to the N-terminal ATP-binding
pocket. The antiproliferative activities for these compounds are
listed in Table 1. While many of the prepared compounds retain
activity, none of the derivatives are more active than the natural
product, however, some important preliminary structure-activity
relationships have been identified.
3e, a pronounced decrease in antiproliferative activity resulted
in a decrease or increase of the size of the substituent from an
ethyl ether (3b) to either a methyl ether or an n-propyl ether
(3a and 3c) and complete loss in activity was produced by
incorporation of the benzyl ether, as evidenced by compound
3d. A complete loss in activity was also noted upon incorpora-
tion of the propionate ester as in 3e. The n-propyl ether in 3c
may retain activity because of its less rigid nature compared to
Substituents at the 7-position are placed in an environment
that appears to be sensitive to the effects of steric bulk that
deviates from the native ligand. From compounds 3a through

6498 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Brandt et al.
Figure 2. Western blot analysis of compounds 1, 3f, and 4 in SKBr3 breast cancer cells after 24 h of incubation at given concentrations.
the propionate ester 3e. Also, preliminary molecular modeling
results suggest that the carbonyl oxygen present on the propi-
onate is tilted out of alignment with the carbonyl oxygen of the
natural product, suggesting that this moiety, when present, is
important for binding.
because aside from the additional projection of steric bulk and
the increase in hydrophobicity from the methyl group on the
oxime ether, 12a appears to include the requirements for
successful binding at other positions of the molecule, including
the R,ꢀ-olefin, a hydrogen bond acceptor at the 3-position, and
the appropriately fitting substituent at the 7-position. Neither
12b nor 12c exhibited antiproliferative activity which also
parallels this trend.
In order to demonstrate the retention of Hsp90 inhibition
despite structural manipulation, the most active compounds in
this series were evaluated to determine their ability to induce
Hsp90-dependent client protein degradation. 1, 3f, and 4 were
subjected to Western blot analysis for the detection of two
known Hsp90 client proteins, Raf and HER2, and the results
are presented in Figure 2. Both compounds 3f and 4 demonstrate
client protein degradation similar to 1 and GDA, which further
supports their activity stems from modulation of the Hsp90
molecular chaperone.
From the initial data set, it also appears that the binding site
for substituents at the 7-position is not sensitive to a change in
the electronic nature of the substituent. Compound 3f, which
incorporates a carbamate as a nonbulky hydrogen bond acceptor/
donor, exhibited an IC50 value almost identical to that of the
natural product. The most active analogue found in the 7-posi-
tion series is compound 4, the 7-oxo derivative, further
demonstrating the tolerance of this binding site to small,
hydrogen bond acceptors rather than small hydrophobic moieties
as in 3a. These results support two reasonable conclusions in
regard to the effect of differing substituents at the 7-position:
(1) the binding site complementary to substituents at this position
may provide a hydrogen bond donor that resides in a shallow
pocket. This may explain why hydrogen bond acceptors exhibit
the most potent antiproliferative activities, while decreasing or
increasing steric bulk from that possessed by the native ligand
without the presence of a hydrogen bond acceptor decreases
activity. (2) Substituents placed at the 7-position of the molecule
are positioned in an area critical to the overall conformation of
the molecule, thereby exhibiting significant impact on binding.
This latter observation explains why even small changes in the
size of substituents, e.g., the additional methyl group of 3e
compared to 1, cause a decrease in antiproliferative activity,
which is supported by preliminary molecular modeling (data
not shown).
The antiproliferative activity of the R,ꢀ-unsaturated ketone
series also provides key information regarding the importance
of this functional group. Compounds lacking the 1,2-olefin of
gedunin manifest IC50 values greater than 100 µM (i.e., 6-8
and 11). At first inspection, these data might suggest that the
R,ꢀ-unsaturated ketone is acting as a Michael acceptor; however,
antiproliferative activity of related compounds in this series
suggest otherwise. For example, compound 9, which contains
the reduced ketone and lactone moieties, retains antiproliferative
activity. Although 9 exhibits activity 5- to 10-fold less potent
than the natural product, evidence suggests that the R,ꢀ-
unsaturated ketone is not acting as a Michael acceptor, as a far
less potent compound would be expected. Support of this
reasoning is substantiated with compound 10a, which resulted
from chemoselective reduction of the R,ꢀ-unsaturated ketone.
10a manifests antiproliferative activitiy 5 times less potent than
gedunin in both MCF-7 and SKBr3 cells. Compound 12a also
retains some activity and is only 3-4 times less potent than
the natural product and incorporates an electron-rich oxime,
which serves as a poor Michael acceptor.
Conclusions
Preliminary structure-activity relationships with gedunin, a
novel Hsp90 inhibitor, have been completed. We have shown
that the R,ꢀ-unsaturated ketone, while required for antiprolif-
erative activity, does not behave as a Michael acceptor and may
therefore evade toxicity associated with this type of motif. We
have also shown that the antiproliferative activity of this series
of compounds is reduced by a decrease or increase in steric
bulk at the 7-position compared to gedunin, suggesting that the
natural product contains the optimal substituents at this position.
Also, it appears that activity of compounds within the 7-position
series is not affected by alteration of the electronic nature.
Of further note, the most active compounds, 3f and 4, may
present scaffolds that exhibit advantages over the natural product
1. The acetate moiety present in 1 is quite labile in physiological
systems, as it may be rapidly cleaved by endogenous, promiscu-
ous esterases. The desacyl derivative of gedunin has significantly
lower antiproliferative activity, and this may prevent derivatives
of gedunin containing the acetate moiety from producing clinical
significance. Conversely, the carbamate of 3f and the ketone of
4 represent more stable functional groups that can withstand
robust physiological environments. The carbamate of 3f also
has the added feature of lowering the lipophilicity of the natural
product, which also increases its solubility and perhaps physi-
ological relevance.
In summary, two lead compounds have been discovered on
the basis of the structure of gedunin, which have similar activity
to the natural product and improved physiological properties.
Further studies based on these lead structures are underway in
an effort to increase the potency of these potential chemothera-
peutic agents. The results from such studies will be reported in
due course.
Compounds 10b, 12a-c, and 13 also provide valuable insight
into the binding site of the R,ꢀ-unsaturated ketone. This binding
pocket appears sensitive to steric bulk and/or intolerant to
hydrophobic moieties. Compound 10b, the acetylated version
of 10a, exhibits no activity. Compound 12a, the O-methyloxime,
also manifests no antiproliferative activity. This is interesting
Experimental Section
General Methods. Unless otherwise indicated, all reagents were
purchased from commercial suppliers and are used without further
purification. All stir bars and glassware were flame-dried, and

Gedunin, a NoVel Hsp90 Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6499
glassware was flushed with argon immediately prior to use. The
1H and 13C NMR spectra were recorded at 500 and 125 MHz,
respectively, on a Bruker DRX 500 using CDCl3 as solvent, δ values
Hz, 1H), 3.10 (dt, J ) 2.3, 6.2 Hz, 1H), 3.42 (dt, J ) 1.6, 6.5 Hz,
1H), 3.55 (s, 1H), 5.52 (s, 1H), 5.77 (d, J ) 10.2 Hz, 1H),
6.27-6.29 (m 1H), 7.02 (d, J ) 10.2 Hz, 1H), 7.31-7.35 (m, 2H).
13C NMR (125 MHz, CDCl3): δ 10.1, 14.0, 16.8, 17.5, 18.9, 19.9,
20.5, 22.3, 25.4, 26.5, 37.3, 37.8, 39.0, 43.0, 43.2, 43.7, 56.4, 68.9,
69.1, 76.8, 77.4, 108.9, 119.7, 124.7, 140.1, 141.9, 156.9, 167.0,
203.6. HRMS (*ESI + pos) (m/z): [M + H] calcd for C29H39O6,
483.2747; found, 483.2737. HPLC tR ) 15.90 min. Purity ) 100%.
(1S,3aS,41R,4a1S,5R,10aR,12aS)-5-(Benzyloxy)-1-(furan-3-yl)-
4a1,7,7,10a,12a-pentamethyl-4a1,5,6,6a,7,10a,10b,11,12,12a-decahy-
dronaphtho[2,1-f]oxireno[2,3-d]isochromene-3,8(1H,3aH)-dione (3d).
Compound 3d was synthesized from 2 using general procedure A
and benzyl bromide and catalytic tetrabutylammonium iodide to
afford 14 mg (59%) as a colorless solid. 1H NMR (500 MHz,
CDCl3): δ 1.02 (s, 3H), 1.04 (s, 3H), 1.07 (s, 3H), 1.12 (s, 3H),
1.14 (s, 3H), 1.42-1.46 (m, 1H), 1.59-1.68 (m, 2H), 1.68-1.72
(m, 1H), 1.80-1.85 (m, 1H), 2.02 (dt, J ) 3, 14.7 Hz, 1H), 2.32
(dd, J ) 2.2, 13.1 Hz), 2.41 (dd, J ) 5.9, 12.7 Hz, 1H), 3.14 (d, J
) 2.2 Hz, 1H), 3.67 (s, 1H), 4.25 (d, J ) 10.2 Hz, 1H), 4.45 (d, J
) 10.2 Hz, 1H), 5.51 (s, 1H), 5.75 (d, J ) 10.2 Hz, 1H), 6.25 (d,
J ) 1.0 Hz, 1H), 6.99 (d, J ) 10.2 Hz, 1H), 7.24-7.30 (m, 2H),
7.35-7.21 (m, 5H). 13C NMR (125 MHz, CDCl3): δ 14.0, 16.9,
17.6, 19.1, 19.9, 20.5, 25.5, 26.6, 37.3, 37.7, 39.1, 43.2, 43.3, 43.7,
56.3, 69.0, 69.6, 76.4, 77.3, 109.0, 119.6, 124.7, 127.1, 127.5, 127.5,
127.7, 127.7, 135.8, 140.1, 141.9, 156.9, 166.9, 203.4. HRMS (*ESI
+ pos) (m/z): [M + H] calcd for C33H39O6, 531.2747; found,
531.2762. HPLC tR ) 16.08 min. Purity ) 97.8%.
1
in ppm (TMS as internal standard), and J (Hz) assignments of H
resonance coupling. Column chromatography was performed with
silica gel (40-63 µm particle size) from Sorbent Technologies
(Atlanta, GA). Analytical HPLC was carried out on an Agilent 1100
series capillary HPLC system with diode array detection at 254
nm (compounds 5-8, 10a, and 11 were detected at 214.15 nm) on
an Agilent Eclipse XDB-C18 column (4.6 mm × 150 mm, 5 µm)
with isocratic elution in 70% acetonitrile and 30% H2O at a flow
rate of 5.0 mL/min.
Gedunin (1). Spectral data correspond with that previously
reported. See Supporting Information.
Desacetylgedunin (2). Spectral data correspond with that previ-
ously reported. See Supporting Information.
General Procedure A. A solution of 2 (20 mg, 0.045 mmol, 1
equiv) in anhydrous THF (450 µL) was stirred at 0 °C under argon
atmosphere. Potassium tert-butoxide (45 µL, 0.09 mmol, 2 equiv)
was added dropwise and the mixture stirred for 30 min before alkyl
iodide (10 equiv) was added. The resulting mixture was stirred at
0 °C for 1.5 h and then quenched by the addition of H2O (1 mL).
The organic layer was removed, and the aqueous layer extracted
with DCM (3 × 5 mL). The combined organic layers were dried
(Na2SO4), filtered, and concentrated. The residue was purified via
SiO2 chromatography (5:3:2 hexanes/DCM/Et2O) to yield the
desired compound.
(1S,3aS,41R,4a1S,5R,10aR,12aS)-1-(Furan-3-yl)-5-methoxy-
4a1,7,7,10a,12a-pentamethyl-4a1,5,6,6a,7,10a,10b,11,12,12a-decahy-
dronaphtho[2,1-f]oxireno[2,3-d]isochromene-3,8(1H,3aH)-dione (3a).
Compound 3a was synthesized from 2 using general procedure A
(1S,3aS,41R,4a1S,5R,10aR,12aS)-1-(Furan-3-yl)-4a1,7,7,10a,12a-
pentamethyl-3,8-dioxo-1,3,3a,4a1,5,6,6a,7,8,10a,10b,11,12,12a-tet-
radecahydronaphtho[2,1-f]oxireno[2,3-d]isochromen-5-yl Propi-
onate (3e). A solution of 2 (20 mg, 0.045 mmol, 1 equiv),
dimethylaminopyridine (catalytic), and Et3N (35 µL, 0.248 mmol,
5.5 equiv) in 450 µL of anhydrous DCM was stirred under argon
atmosphere at 0 °C until completely dissolved. Propionyl chloride
(20 µL, 0.225 mmol, 5 equiv) was added dropwise to the solution
and then stirred for 5 h before the reaction was quenched by addition
of H2O (1 mL). The organic layer was removed and the aqueous
layer extracted with DCM (3 × 5 mL). The combined organic layers
were dried (Na2SO4), filtered, and concentrated. The resulting
yellow oil was purified via SiO2 chromatography (4:3:3 hexanes/
DCM/Et2O) to yield 17 mg (76%) 3e as a colorless solid. 1H NMR
(500 MHz, CDCl3): δ 0.98 (s, 3H), 1.00 (t, J ) 7.6 Hz, 3H), 1.00
(s, 3H), 1.12 (s, 3H), 1.17 (s, 3H), 1.36 (s, 3H), 1.66-1.97 (m,
4H), 2.09 (dd, J ) 2.4, 13.4 Hz, 1H), 2.20-2.38 (m, 1H), 2.30
(dq, J ) 2.6, 7.7 Hz, 1H), 2.40-2.46 (m, 2H), 2.42 (dq, J ) 2.6,
7.7 Hz, 1H), 3.48 (s, 1H), 4.52 (m, 1H), 5.52 (s, 1H), 5.79 (d, J )
10.2 Hz, 1H), 6.26 (m, 1H), 7.02 (d, J ) 10.2 Hz, 1H), 7.34 (m,
1H). 13C NMR (125 MHz, CDCl3): δ 10.5, 14.1, 15.2, 17.7, 18.9,
19.9, 21.3, 23.3, 25.3, 27.3, 38.9, 39.6, 40.2, 42.8, 44.1, 46.1, 57.6,
69.4, 72.7, 78.0, 110.0, 114.0, 120.5, 126.0, 141.2, 143.1, 157.0,
165.1, 166.9, 204.1. HRMS (*ESI + pos) (m/z): [M + H] calcd
for C29H37O7, 497.2539; found, 497.2542. HPLC tR ) 13.66 min.
Purity ) 97.1%.
1
and iodomethane to afford 13 mg (64%) as a colorless solid. H
NMR (500 MHz, CDCl3): δ 1.01 (s, 3H), 1.04 (s, 3H), 1.10 (s,
3H), 1.13 (s, 3H), 1.13 (s, 3H), 1.44-1.52 (m, 2H), 1.67-1.75
(m, 2H), 1.82-1.90 (m, 2H), 2.23 (dd, J ) 2.1, 13.2 Hz, 1H), 2.38
(dd, J ) 5.9, 12.7 Hz, 1H), 2.85 (d, J ) 2.1 Hz, 1H), 3.25 (s, 3H),
3.55 (s, 1H), 5.52 (s, 1H), 5.76 (d, J ) 10.2 Hz, 1H), 6.27 (d, J )
0.6 Hz, 1H), 7.01 (d, J ) 10.2, 1H), 7.30-7.35 (m, 2H). 13C NMR
(125 MHz, CDCl3): δ 14.1, 16.7, 17.5, 18.9, 19.2, 20.5, 25.4, 26.4,
37.3, 37.7, 39.0, 43.1, 43.2, 43.5, 54.6, 56.4, 69.1, 77.4, 78.2, 108.9,
119.7, 124.7, 140.1, 141.9, 156.8, 167.1, 203.5. HRMS (*ESI +
pos) (m/z): [M + H] calcd for C27H35O6, 455.2434; found, 455.2419.
HPLC tR ) 10.09 min. Purity ) 100%.
(1S,3aS,41R,4a1S,5R,10aR,12aS)-5-Ethoxy-1-(furan-3-yl)-
4a1,7,7,10a,12a-pentamethyl-4a1,5,6,6a,7,10a,10b,11,12,12a-decahy-
dronaphtho[2,1-f]oxireno[2,3-d]isochromene-3,8(1H,3aH)-dione (3b).
Compound 3b was synthesized from 2 using general procedure A
and 1-iodoethane to afford 7 mg (33%) as a colorless solid. 1H
NMR (500 MHz, CDCl3): δ 1.00 (s, 3H), 1.03 (s, 3H), 1.08 (s,
3H), 1.12 (s, 3H), 1.15 (s, 3H), 1.15 (t, J ) 12.7 Hz, 3H), 1.46-1.50
(m, 1H), 1.53-1.57 (m, 1H), 1.61-1.65 (m, 1H), 1.69-1.73 (m,
1H), 1.81-1.89 (m, 2H), 2.24 (dd, J ) 2.2, 13.1 Hz, 1H), 2.26
(dd, J ) 5.8, 12.8 Hz, 1H), 2.95 (d, J ) 2.1 Hz, 1H), 3.14 (dq J )
7.0, 8.7 Hz, 1H), 3.55 (s, 1H), 3.57 (dq, J ) 7.0, 8.7 Hz, 1H), 5.53
(s, 1H), 5.77 (d, J ) 10.2 Hz, 1H), 7.02 (d, J ) 10.3 Hz, 1H),
7.31-7.35 (m, 2H). 13C NMR (125 MHz, CDCl3): δ 14.0, 14.5,
16.8, 17.4, 18.9, 20.0, 20.5, 25.4, 26.4, 37.3, 37.8, 39.0, 43.0, 43.2,
43.7, 56.4, 62.8, 69.2, 77.1, 77.4, 109.0, 119.7, 124.7, 140.1, 141.9,
156.8, 167.1, 203.6. HRMS (*ESI + pos) (m/z): [M + H] calcd
for C28H37O6, 469.2590; found, 469.2581. HPLC tR ) 12.71 min.
Purity ) 100%.
(1S,3aS,41R,4a1S,5R,10aR,12aS)-1-(Furan-3-yl)-4a1,7,7,10a,12a-
pentamethyl-3,8-dioxo-1,3,3a,4a1,5,6,6a,7,8,10a,10b,11,12,12a-tet-
radecahydronaphtho[2,1-f]oxireno[2,3-d]isochromen-5-yl Carbam-
ate (3f). A solution of 2 (20 mg, 0.045 mmol, 1 equiv) in 450 µL
of anhydrous DCM under argon atmosphere was stirred at 0 °C.
Trichloroacetyl isocyanate (10.7 µL, 0.09 mmol, 2 equiv) was added
dropwise, and the solution was allowed to warm to room temper-
ature and stirred for 3 h, at which point the reaction was quenched
by the addition of H2O (1 mL). The organic layer was collected
and the aqueous layer extracted with DCM (3 × 5 mL). The
combined organic extracts were concentrated and then dissolved
in 1 mL of 0.1 M K2CO3 in MeOH, and the mixture was stirred
for 1.5 h at ambient temperature. The reaction was quenched by
the addition of H2O (1 mL). The reaction mixture was extracted
with DCM (3 × 5 mL). The combined organic layers were dried
(Na2SO4), filtered, and concentrated. The resulting colorless oil was
purified via SiO2 chromatography (4:3:3 hexanes/DCM/Et2O) to
(1S,3aS,41R,4a1S,5R,10aR,12aS)-1-(Furan-3-yl)-4a1,7,7,10a,12a-
pentamethyl-5-propoxy-4a1,5,6,6a,7,10a,10b,11,12,12a-decahy-
dronaphtho[2,1-f]oxireno[2,3-d]isochromene-3,8(1H,3aH)-dione (3c).
Compound 3c was synthesized from 2 using general procedure A
1
and 1-iodopropane to afford 5 mg (23%) as a colorless solid. H
NMR (500 MHz, CDCl3): δ 0.86 (t, J ) 7.4 Hz, 3H), 1.01 (s, 3H),
1.04 (s, 3H), 1.08 (s, 3H), 1.13 (s, 3H), 1.16 (s, 3H), 1.46-1.50
(m, 1H), 1.50-1.61 (m, 3H), 1.62-1.67 (m, 1H), 1.70-1.74 (m,
1H), 1.84-1.88 (m, 1H), 1.90-1.95 (m, 1H), 2.25 (dd, J ) 2.2,
13.1 Hz, 1H), 2.42 (dd, J ) 6.0, 12.8 Hz, 1H), 2.94 (d, J ) 2.2
1
yield 21 mg (97%) 3f as a colorless solid. H NMR (500 MHz,

6500 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Brandt et al.
CDCl3): δ 1.01 (s, 3H), 1.03 (s, 3H), 1.09 (s, 3H), 1.16 (s, 6H),
1.49-1.53 (m, 1H), 1.65-1.68 (m, 1H), 1.778-1.81 (m, 2H),
1.85-1.93 (m, 2H), 2.15 (dd, J ) 2.5, 12.7 Hz, 1H), 2.37 (dd, J )
6.1, 12.7 Hz, 1H), 3.66 (s, 1H), 4.45 (s, 1H), 4.67 (br s, 2H), 5.54
(s, 1H), 5.79 (d, J ) 10.2 Hz, 1H), 6.25-6.27 (m, 1H), 7.02 (d, J
) 10.2 Hz, 1H), 7.32-7.35 (m, 2H). 13C NMR (125 MHz, CDCl3):
δ 15.0, 18.0, 18.5, 19.8, 21.3, 23.6, 26.0, 27.2, 38.6, 39.5, 40.0,
42.9, 44.1, 45.9, 56.9, 69.7, 73.5, 78.2, 109.9, 120.5, 126.0, 141.2,
143.1, 155.1, 157.1, 167.4, 204.2. HRMS (*ESI + pos) (m/z): [M
+ H] calcd for C27H34N1O7, 484.2335; found, 484.2320. HPLC tR
) 3.59 min. Purity ) 98.7%.
chromatography (7:6:6 hexanes/DCM/Et2O) to yield 97 mg (89%)
of 6 as a colorless solid. H NMR (500 MHz, CDCl3): δ 0.99 (s,
1
3H), 1.02 (s, 3H), 1.07 (s, 3H), 1.18 (s, 3H), 1.19 (s, 3H), 1.48-1.51
(m, 1H), 1.60-1.63 (m, 2H), 1.79-1.82 (m, 2H), 2.03 (s, 3H),
2.16 (dd, J ) 4.1, 11.5 Hz, 1H), 2.48 (s, 1H), 2.96 (dd, J ) 6.4,
12.1 Hz, 1H), 3.42 (s, 1H), 3.86 (d, J ) 2.3 Hz, 1H), 3.89 (d, J )
2.8 Hz, 1H), 4.44 (br s, 1H), 4.60-4.62 (m, 1H), 5.54 (s, 1H),
6.25-6.27 (m, 1H), 7.32-7.34 (s, 1H). 13C NMR (125 MHz,
CDCl3): δ 13.5, 15.3, 15.7, 17.5, 19.9, 20.1, 22.0, 22.8, 24.6, 35.7,
37.9, 40.2, 40.8, 41.0, 45.5, 55.7, 69.0, 70.2, 72.5, 75.9, 77.5, 109.0,
119.6, 140.1, 141.9, 166.6, 169.1, 213.0. HRMS (*ESI + pos) (m/
z): [M + H] calcd for C28H37O9, 517.2438; found, 517.2421. HPLC
tR ) 4.71 min. Purity ) 100%.
(1S,3aS,41R,4a1R,10aS,12aS)-1-(Furan-3-yl)-4a1,7,7,10a,12a-
pentamethyl-6,6a,7,10a,10b,11,12,12a-octahydronaphtho[2,1-f]ox-
ireno[2,3-d]isochromene-3,5,8(1H,3aH,4a1H)-trione (4). A solution
of 2 (20 mg, 0.045 mmol, 1 equiv) in 450 µL of anhydrous DCM
was stirred at ambient temperature. Pyridinium chlorochromate
(29.1 mg, 0.135 mmol, 3 equiv) was added in one portion, and the
reaction mixture was stirred overnight before the solution was
filtered through a plug of silica using diethyl ether as eluent. The
colorless solution was dried (Na2SO4), filtered, and concentrated.
The resulting colorless oil was purified via SiO2 chromatography
(5:3:2 hexanes/DCM/Et2O) to yield 19 mg (96%) of 4 as a colorless
(1S,3aS,41R,4a1S,5R,10aR,12aS)-1-(Furan-3-yl)-9,10-dihydroxy-
4a1,7,7,10,10,10a,12a-heptamethyl-3,8-dioxohexadecahydronaph-
tho-9,11-dioxol[2,1-f]oxireno[2,3-d]isochromen-5-yl Acetate (7). A
solution of 6 (10.5 mg, 0.02 mmol, 1 equiv) in 100 µL of acetone
was stirred at room temperature. 2,2-Dimethoxypropane (11 µL,
0.088 mmol, 4.4 equiv) was added followed by the addition of
catalytic p-toluenesulfonic acid. After the mixture was stirred for
3 h, solvent was blown dry and residue was taken up in 300 µL of
DCM and washed with sodium bicarbonate and brine. The organic
layer was dried (Na2SO4), filtered, and concentrated. The resulting
colorless oil was purified via SiO2 chromatography (5:3:2 hexanes/
DCM/Et2O) to yield 10 mg (90%) of 7 as a colorless solid. 1H
NMR (500 MHz, CDCl3): δ 0.98 (s, 3H), 1.02 (s, 3H), 1.13 (s,
3H), 1.22 (s, 3H), 1.23 (s, 3H), 1.48 (s, 3H), 1.48-1.51 (m, 1H),
1.50 (s, 3H), 1.56-1.63 (m, 3H), 1.71-1.73 (m, 1H), 1.83 (dt, J
) 3.7, 14.9 Hz, 1H), 2.05 (s, 3H), 2.42 (dd, J ) 3.6, 13.7 Hz, 1H),
2.95 (dd, J ) 6.2, 12.4 Hz, 1H), 3.48 (s, 1H), 4.05 (d, J ) 7.5 Hz,
1H), 4.31 (d, J ) 7.5 Hz, 1H), 4.45 (dd, J ) 1.9, 3.4, 1H), 5.55 (s,
1H), 6.27 (d, J ) 1.0 Hz, 1H), 7.32-7.35 (m, 2H). 13C NMR (125
MHz, CDCl3): δ 13.8, 14.5, 15.7, 16.9, 20.1, 20.4, 22.5, 22.9, 24.5,
24.9, 28.4, 32.6, 36.7, 37.8, 39.5, 40.4, 44.0, 52.4, 55.7, 69.1, 72.3,
77.4, 81.1, 109.0, 109.4, 119.6, 140.1, 141.9, 166.7, 168.9, 210.9.
HRMS (*ESI + pos) (m/z): [M + Na] calcd for C31H40O9Na,
579.2570; found, 579.2548. HPLC tR ) 14.17 min. Purity ) 96.1%.
(1S,3aS,41R,4a1S,5R,10aR,12aS)-1-(Furan-3-yl)-4a1,7,7,10a,12a-
pentamethyl-3,8-dioxohexadecahydronaphtho[2,1-f]oxireno[2,3-
d]isochromen-5-yl Acetate (8). A solution of 1 (20 mg, 0.041 mmol,
1 equiv) and 10% Pd/C (2 mg) in 1 mL of methanol was stirred at
room temperature under an atmosphere of argon. The reaction flask
was purged with H2 repeatedly. The fully H2 purged flask was
stirred at room temperature for 5 h, after which the solution was
run through a plug of silica using ether as eluent. The organic filtrate
was dried (Na2SO4), filtered, and concentrated. The resulting
colorless oil was purified via SiO2 chromatography (5:3:2 hexanes/
DCM/Et2O) to yield 7 mg (35%) of 8 as a colorless solid. 1H NMR
(500 MHz, CDCl3): δ 0.96 (s, 3H), 1.00 (s, 3H), 1.05 (s, 3H), 1.17
(s, 3H), 1.42 (s, 3H), 1.48-1.53 (m, 2H), 1.63-1.71 (m, 4H),
1.77-1.81 (m, 1H), 1.81-1.83 (m, 1H), 1.84-1.86 (m, 1H), 2.24
(dd, J ) 6.9, 12.1 Hz, 1H), 2.38-2.40 (m, 1H), 2.50-2.52 (m,
1H), 3.45 (s, 1H), 4.44-4.47 (m, 1H), 5.54 (s, 1H), 6.25-6.26
(m, 1H), 7.31-7.33 (m, 2H). 13C NMR (125 MHz, CDCl3): δ 14.0,
14.7, 16.4, 17.0, 19.9, 20.1, 22.7, 24.8, 25.0, 32.7, 36.3, 37.8, 37.9,
40.9, 43.0, 45.6, 46.7, 55.7, 68.78, 72.7, 77.3, 108.9, 119.5, 140.1,
142.0, 166.6, 169.0, 214.9. HRMS (*ESI + pos) (m/z): [M + H]
calcd for C28H37O7, 485.2539; found, 485.2549. HPLC tR ) 6.35
min. Purity ) 97.6%.
1
solid. H NMR (125 MHz, CDCl3): δ 1.07 (s, 3H), 1.07 (s, 3H),
1.09 (s, 3H), 1.15 (s, 3H), 1.29 (s, 3H), 1.41 (dt, J ) 3.9, 9.8 Hz,
1H), 1.71-1.77 (m, 2H), 1.91-1.95 (m, 1H), 2.10-2.17 (m, 2H),
2.34 (dd, J ) 3.2, 14 Hz, 1H), 2.86 (t, J ) 14.4 Hz, 1H), 3.80 (s,
1H), 5.4 (s, 1H), 5.85 (d, J ) 10.2 Hz, 1H), 6.29 (dd, J ) 0.7, 1.8
Hz, 1H), 7.03 (d, J ) 10.2 Hz, 1H), 7.32-7-34 (m, 1H), 7.34-7.36
(m, 1H). 13C NMR (500 MHz, CDCl3): δ 16.2, 16.4, 18.8, 19.6,
19.9, 25.7, 31.2, 35.7, 36.7, 35.7, 36.7, 38.6, 44.2, 46.6, 52.4, 52.6,
53.5, 64.5, 77.0, 108.8, 119.2, 125.4, 140.0, 142.1, 154.9, 165.8,
202.2, 207.1. HRMS (*ESI + pos) (m/z): [M + H] calcd for
C26H31O6,439.2121; found, 439.2129. HPLC tR ) 5.45 min. Purity
) 100%.
(1S,3aS,41R,4a1S,5R,10aR,12aS)-1-(Furan-3-yl)-5-hydroxy-
4a1,7,7,10a,12a-pentamethyl-4a1,5,6,6a,7,10a,10b,11,12,12a-decahy-
dronaphtho[2,1-f][9,10]dioxireno[2,3-d]isochromene-3,8(1H,3aH)-
dione (5). A solution of 2 (20 mg, 0.045 mmol, 1 equiv) in 450 µL
of acetone was stirred at 0 °C. A solution of 8% aqueous sodium
hydroxide (85 µL) and a solution of 30% aqueous hydrogen
peroxide (52 µL) were added. The reaction mixture was allowed
to warm to room temperature. After the mixture was stirred
overnight, 2 N hydrochloric acid (1 mL) was added. The reaction
mixture was extracted with DCM (3 × 5 mL), and the combined
organic layers were dried (Na2SO4), filtered, and concentrated. The
resulting colorless oil was purified via SiO2 chromatography (5:
3:2 hexanes/DCM/Et2O) to yield 14 mg (68%) of 5 as a colorless
1
solid. H NMR (500 MHz, CDCl3): δ 0.92 (s, 3H), 0.96 (s, 3H),
1.00 (s, 3H), 1.04 (s, 3H), 1.22 (s, 3H), 1.45-1.52 (m, 2H), 1.63
(dd, J ) 4.1, 8.1 Hz, 1H), 1.71-1.77 (m, 2H), 1.92-1.96 (m, 1H),
2.64 (dd, J ) 2.6, 13.7 Hz, 1H), 2.72 (dd, J ) 6.6, 12.7 Hz, 1H),
3.32 (d, J ) 4.6 Hz, 1H), 3.46 (s, 1H), 3.48 (d, J ) 4.5 Hz, 1H),
3.85 (s, 1H), 5.53 (s, 1H), 6.29 (s, 1H), 7.33 (s, 1H), 7.32-7.34
(m, 2H). 13C NMR (125 MHz, CDCl3): δ 14.5, 14.8, 16.4, 17.7,
19.8, 25.0, 26.0, 26.5, 35.9, 36.1, 37.4, 38.2, 42.2, 43.2, 55.6, 57.0,
62.1, 68.5, 68.9, 77.4, 109.0, 119.6, 140.2, 141.9, 167.2, 210.7.
HRMS (*ESI + pos) (m/z): [M + H] calcd for C26H33O7, 457.2226;
found, 457.2220. HPLC tR ) 6.40 min. Purity ) 98.6%.
(1S,3aS,41R,4a1S,5R,10aR,12aS)-1-(Furan-3-yl)-9,10-dihydroxy-
4a1,7,7,10a,12a-pentamethyl-3,8-dioxohexadecahydronaphtho[2,1-
f]oxireno[2,3-d]isochromen-5-yl Acetate (6). A solution of 1 (100
mg, 0.21 mmol, 1 equiv) in 1.78 mL of fully degassed acetone
was stirred under argon atmosphere at 0 °C. A 4% solution of
osmium tetroxide in water (72 µL, 0.011 mmol, 0.05 equiv) and a
1 M solution of N-methylmorpholine N-oxide in deionized water
(315 µL, 0.315 mmol, 1.5 equiv) were added. After the mixture
was stirred overnight, saturated aqueous sodium sulfite was added
(4 mL) and the reaction mixture was stirred for 1 h at room
temperature. The reaction mixture was extracted 4 times with ethyl
acetate. The combined organics were dried (Na2SO4), filtered, and
concentrated. The resulting black oil was purified via SiO2
(1S,3aS,41R,4a1S,5R,10aS,12aS)-1-(Furan-3-yl)-3,8-dihydroxy-
4a1,7,7,10a,12a-pentamethyl-1,3,3a,4a1,5,6,6a,7,8,10a,10b,11,12,12a-
tetradecahydronaphtho[2,1-f]oxireno[2,3-d]isochromen-5-yl Ac-
etate (9). A solution of 1 (500 mg, 1.04 mmol, 1 equiv) in 4 mL of
a 2:1 mixture of methanol and chloroform was stirred under argon
atmosphere at 0 °C. Cerium trichloride hexahydrate (740 mg, 2.08,
2 equiv) was added followed by the slow addition of sodium
borohydride (39.33 mg, 1.04 mmol, 1 equiv). The reaction mixture
was stirred for 3 min and quenched by the addition of a solution
of 1% acetic acid in water. The reaction mixture was extracted 3
times with DCM. The combined organic layers were dried
(Na2SO4), filtered, and concentrated. The resulting colorless crystals

Gedunin, a NoVel Hsp90 Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6501
were dissolved in minimal DCM and purified via SiO2 chroma-
tography (6:7:7 hexanes/DCM/Et2O) to yield 473 mg (93%) of 9
as a colorless solid. Major diastereomer: 1H NMR (500 MHz,
CDCl3) δ 0.74 (s, 3H), 0.83 (s, 3H), 0.95 (s, 3H), 1.02 (s, 3H),
1.16 (s, 3H), 1.45-1.80 (m, 7H), 2.05 (s, 3H), 2.30 (dd, J ) 5.4,
12.9 Hz, 1H), 3.00-3.10 (m, 2H), 3.21 (d, J ) 1.8 Hz, 1H), 3.84
(bs, 1H), 4.56-4.60 (m, 1H), 4.92 (s, 1H), 5.05 (d, J ) 10.2 Hz,
1H), 5.28 (d, J ) 10.2, 1H), 5.80 (m, 1H), 6.20 (m, 1H), 7.23 (m,
1H), 7.27 (m, 1H). 13C NMR (125 MHz, CDCl3): δ 14.6, 16.1,
17.2, 18.5, 18.7, 20.3, 22.2, 25.6, 26.5, 35.6, 35.7, 338.4, 40.9, 41.9,
45.0, 57.5, 68.5, 71.4, 73.4, 73.4, 76.2, 86.8, 109.2, 121.9, 125.1,
136.3, 125.1, 136.3, 139.6, 141.3, 168.8. HRMS (*ESI + pos) (m/
z): [M + Na] calcd for C28H38O7Na, 509.2515; found, 509.2500.
HPLC tR ) 6.12 min. Purity ) 100%.
mixture was stirred for 45 min. The reaction mixture was quenched
by the addition of 1% aqueous acetic acid. The reaction mixture
was extracted 3 times with chloroform. The combined organic layers
were dried (Na2SO4), filtered, and concentrated. The resulting
colorless oil was purified via SiO2 chromatography (4:3:3 hexanes/
DCM/Et2O) to yield 17 mg (85%) 11 as a colorless solid. Major
1
diasteromer: H NMR (500 MHz, CDCl3) δ 0.69 (s, 3H), 0.81 (s,
3H), 0.85 (s, 3H), 0.94 (s, 3H), 1.18 (s, 3H), 1.15-1.36 (m, 2H),
1.45-1.75 (m, 9H), 2.05 (s, 3H), 2.10-2.21 (m, 1H), 2.96 (d, J )
10.4, 1H), 3.17-3.20 (m, 1H), 3.22 (s, 1H), 4.56-4.59 (m, 1H),
4.92 (s, 1H), 5.06 (d, J ) 10.4, 1H), 6.21 (m, 1H), 7.22-7.26 (m,
1H), 7.26-7.28 (m, 1H). 13C NMR (125 MHz, CDCl3): δ 14.2,
16.8, 17.2, 18.5, 20.4, 22.3, 25.4, 25.7, 26.5, 26.7, 26.5, 26.7, 35.7,
36.8, 37.3, 37.4, 43.3, 45.0, 47.0, 52.4, 57.8, 72.2, 73.8, 77.5, 86.8,
109.3, 122.4, 139.6, 141.3, 168.9. HRMS (*ESI + pos) (m/z): [M
+ Na] calcd for C28H40O7Na, 511.2672; found, 511.2681. HPLC
tR ) 4.23 min. Purity ) 98.3%.
(1S,3aS,41R,4a1S,5R,10aS,12aS)-1-(Furan-3-yl)-8-hydroxy-
4a1,7,7,10a,12a-pentamethyl-3-oxo-1,3,3a,4a1,5,6,6a,7,8,10a,10b,11,
12,12a-tetradecahydronaphtho[2,1-f]oxireno[2,3-d]isochromen-5-
yl Acetate (10a). A solution of 1 (500 mg, 1.04 mmol, 1 equiv) in
1.3 mL of toluene was stirred at ambient temperature under argon
atmosphere. Isopropyl alcohol (687 mg, 11.44 mmol, 11 equiv)
and aluminum triisopropoxide (127.5 mg, 0.624, 0.6 equiv) were
added, and the reaction mixture was heated at 70 °C for 20 h. After
being allowed to cool to ambient temperature, the reaction mixture
was quenched by the addition of 1 N HCl (4 mL) and ethyl acetate
(4 mL) and stirred for 1.5 h. The organic layer was washed with
water and concentrated. The resulting colorless oil was purified
via SiO2 chromatography (3:1:1 hexanes/DCM/Et2O) to yield 284
mg (56%) as a colorless solid. 1H NMR (500 MHz, CDCl3): δ
0.75 (s, 3H), 0.83 (s, 3H), 1.02 (s, 3H), 1.04 (s, 3H), 1.16 (s, 3H),
1.44-1.48 (m, 1H), 1.57-1.61 (m, 1H), 1.59-1.63 (m, 1H),
1.66-1.69 (m, 2H), 1.82-1.86 (m, 1H), 1.90 (dd, J ) 2.6, 11.7
Hz, 1H), 2.05 (s, 3H), 2.29 (dd, J ) 6.4, 12.6 Hz, 1H), 3.42 (s,
1H), 3.84 (d, J ) 8.3, 1H), 4.45 (bs, 1H), 5.29 (d, J ) 10.5 Hz,
1H), 5.53 (s, 1H), 5.78 (d, J ) 10.6 Hz, 1H), 6.26 (s, 1H), 7.33 (s,
2H). 13C NMR (125 MHz, CDCl3): δ 15.4, 17.3, 17.5, 18.5, 19.6,
21.2, 22.6, 26.0, 27.4, 36.5, 38.8, 39.7, 42.0, 42.7, 45.8, 56.8, 65.9,
69.9, 73.9, 78.4, 109.9, 120.6, 126.5, 136.7, 141.2, 143.0, 167.8,
170.0. HRMS (*ESI + pos) (m/z): [M + H] calcd for C28H37O7,
485.2539; found, 485.2540. HPLC tR ) 4.31 min. Purity ) 98.1%.
(1S,3aS,41R,4a1S,5R,10aS,12aS)-1-(Furan-3-yl)-4a1,7,7,10a,12a-
pentamethyl-3-oxo-1,3,3a,4a1,5,6,6a,7,8,10a,10b,11,12,12a-tet-
radecahydronaphtho[2,1-f]oxireno[2,3-d]isochromene-5,8-diyl Di-
acetate (10b). A solution of 10a (10 mg, 0.0206 mmol, 1 equiv),
dimethylaminopyridine (catalytic), and Et3N (15 µL, 0.103 mmol,
5 equiv) in anhydrous THF (250 µL) was stirred under argon
atmosphere at 0 °C. Acetyl chloride (8 µL, 0.103 mmol, 5 equiv)
was added dropwise, and the reaction mixture was stirred overnight
while allowing to warm to ambient temperature. The reaction
mixture was quenched by the addition of water (500 µL). The
organic layer was collected, and the aqueous layer was washed with
DCM (3 × 5 mL). The combined organic layers were dried
(Na2SO4), filtered, and concentrated. The resulting yellow oil was
purified via SiO2 chromatography (5:3:2 hexanes/DCM/Et2O) to
yield 9 mg (83%) of 10b as a colorless solid. 1H NMR (500 MHz,
CDCl3): δ 0.74 (s, 3H), 0.83 (s, 3H), 1.02 (s, 3H), 1.07 (s, 3H),
1.15 (s, 3H), 1.46-1.50 (m, 1H), 1.59-1.61 (m, 1H), 1.66-1.72
(m, 2H), 1.81 (m, 2H), 1.90 (d, J ) 15.0 Hz, 1H), 2.03 (s, 3H),
2.05 (s, 3H), 2.31 (dd, J ) 6.4, 12.7 Hz, 1H), 3.42 (s, 1H), 4.45
(bs, 1H), 5.07 (d, J ) 1.8 Hz, 1H), 5.17 (dd, J ) 1.4, 9.7 Hz, 1H),
5.52 (s, 1H), 5.82 (d, J ) 10.5 Hz, 1H), 6.26 (s, 1H), 7.33 (s, 2H).
13C NMR (125 MHz, CDCl3): δ 14.4, 16.4, 17.3, 17.4, 18.4, 20.2,
21.4, 25.0, 26.4, 34.5, 37.7, 38.7, 41.0, 41.6, 44.8, 55.7, 68.8, 72.7,
77.4, 77.7, 108.9, 119.5, 121.8, 136.6, 140.1, 142.0, 166.6, 169.0,
170.4. HRMS (*ESI + pos) (m/z): [M + H] calcd for C30H39O8,
527.2645; found, 527.2654. HPLC tR ) 16.63 min. Purity ) 96.2%.
(1S,3aS,41R,4a1S,5R,10aS,12aS)-1-(Furan-3-yl)-3,8-dihydroxy-
4a1,7,7,10a,12a-pentamethylhexadecahydronaphtho[2,1-f]oxireno[2,3-
d]isochromen-5-yl Acetate (11). A solution of 1 (20 mg, 0.041
mmol, 1 equiv) in 450 µL of ethanol was stirred at ambient
temperature under argon atmosphere. Sodium borohydride (10 mg,
0.25 mmol, 6 equiv) was added in one portion, and the reaction
General Procedure B. A solution of 1 (20 mg, 0.041 mmol, 1
equiv) was stirred in pyridine (450 µL) at room temperature.
Hydroxylamine or appropriate hydroxylamine derivative (2 equiv)
was added, and the reaction mixture was stirred at 70 °C in a sealed
tube overnight. The reaction mixture was diluted with toluene, and
solvents were condensed in vacuo. The coevaporation procedure
was repeated 2 more times. The resulting oil was dissolved in DCM
and washed with saturated aqueous sodium bicarbonate (2 × 5 mL).
The organic layer was collected, and the aqueous layer was re-
extracted with dichloromethane (2 × 5 mL). The combined organic
layers were dried (Na2SO4), filtered, and concentrated. The resulting
oil was purified via SiO2 chromatography (6:2:2, hexanes/DCM/
Et2O) to yield the desired oxime.
(1S,3aS,41R,4a1S,5R,10aS,12aS)-1-(Furan-3-yl)-8-(methoxyimino)-
4a1,7,7,10a,12a-pentamethyl-3-oxo-1,3,3a,4a1,5,6,6a,7,8,10a,10b,11,
12,12a-tetradecahydronaphtho[2,1-f]oxireno[2,3-d]isochromen-5-
yl Acetate (12a). Compound 12a was synthesized from 1 using
general procedure B and methoxyhydroxylamine hydrochloride to
afford 13 mg (62%) as a colorless solid. 1H NMR (500 MHz,
CDCl3): δ 1.05 (s, 3H), 1.06 (s, 3H), 1.07 (s, 3H), 1.08 (s, 3H),
1.16 (s, 3H), 1.46-1.50 (m, 1H), 1.63-1.65 (m, 1H), 1.67-1.71
(m, 1H), 1.77 (dd, J ) 1.8, 13.4, 1H), 1.76-1.78 (m, 1H), 1.90
(dd, J ) 2.2, 12.7, 1H), 1.89-1.93 (m, 1H), 2.32 (dd, J ) 6.0,
12.8 Hz, 1H), 3.45 (s, 1H), 3.79 (s, 3H), 4.45-4.49 (m, 1H), 5.54
(s, 1H), 6.24-6.28 (m, 1H), 6.33 (d, J ) 10.4 Hz, 1H), 6.49 (d, J
) 10.4, 1H), 7.32-7.34 (m, 1H). 13C NMR (125 MHz, CDCl3): δ
14.0, 16.7, 17.2, 18.0, 20.1, 21.9, 23.2, 25.0, 28.7, 36.3, 37.7, 38.8,
38.9, 41.5, 45.4, 55.8, 60.6, 68.9, 72.5, 77.3, 108.9, 113.3, 119.5,
140.1, 142.0, 145.4, 157.4, 166.6, 168.9. HRMS (*ESI + pos) (m/
z): [M + H] calcd for C29H38N1O7, 512.2649; found, 512.2635.
HPLC tR ) 21.37 min. Purity ) 100%.
(1S,3aS,41R,4a1S,5R,10aS,12aS)-8-(Ethoxyimino)-1-(furan-3-yl)-
4a1,7,7,10a,12a-pentamethyl-3-oxo-1,3,3a,4a1,5,6,6a,7,8,10a,10b,11,
12,12a-tetradecahydronaphtho[2,1-f]oxireno[2,3-d]isochromen-5-
yl Acetate (12b). Compound 12b was synthesized from 1 using
general procedure B and ethoxyhydroxylamine hydrochloride to
afford 17 mg (79%) as a colorless solid. 1H NMR (500 MHz,
CDCl3): δ 1.05 (s, 3H), 1.06 (s, 3H), 1.08 (s, 3H), 1.16 (s, 3H),
1.18 (s, 3H), 1.18 (t, J ) 6.9 Hz, 3H), 1.60-1.75 (m, 5H),
1.75-1.92 (m, 2H), 2.02 (s, 3H), 2.33 (dd, J ) 5.9, 12.8 Hz, 1H),
3.45 (s, 1H), 4.03 (q, J ) 7 Hz, 2H), 4.47 (s, 1H), 5.54 (s, 1H),
6.26 (m, 1H), 6.32 (d, J ) 10.3 Hz, 1H), 6.52 (d, J ) 10.3 Hz,
1H), 7.33 (m, 2H). 13C NMR (125 MHz, CDCl3): δ 14.6, 15.0,
17.7, 18.2, 19.1, 21.1, 22.9, 24.2, 26.1, 29.6, 37.4, 38.7, 39.9, 40.0,
42.5, 46.5, 56.9, 69.4, 70.0, 73.6, 78.3, 109.9, 114.5, 120.5, 141.2,
143.0, 146.0, 158.1, 163.1, 164.0. HRMS (*ESI + pos) (m/z): [M
+ Na] calcd for C30H39NO7Na, 548.2624; found, 548.2616. HPLC
tR ) 28.90 min. Purity ) 100%.
(1S,3aS,41R,4a1S,5R,10aS,12aS)-8-(Benzyloxyimino)-1-(furan-
3-yl)-4a1,7,7,10a,12a-pentamethyl-3-oxo-1,3,3a,4a1,5,6,6a,7,8,10a,
10b,11,12,12a-tetradecahydronaphtho[2,1-f]oxireno[2,3-d]isoch-
romen-5-yl Acetate (12c). Compound 12c was synthesized from 1
using general procedure B and benzyloxyhydroxylamine hydro-
chloride to afford 10 mg (42%) as a colorless solid. 1H NMR (500

6502 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Brandt et al.
MHz, CDCl3): δ 1.05 (s, 6H), 1.06 (s, 3H), 1.07 (s, 3H), 1.15 (s,
3H), 1.60-1.75 (m, 5H), 1.78-1.88 (m, 2H), 2.02 (s, 3H), 2.31
(dd, J ) 6.05, 12.8 Hz, 1H), 3.45 (s, 1H), 4.46 (t, J ) 1.6 Hz, 1H),
5.02 (s, 2H), 5.53 (s, 1H), 6.26 (m, 1H), 6.32 (d, J ) 10.4 Hz,
1H), 6.55 (d, J ) 10.4 Hz, 1H), 7.25-7.28 (m, 5H), 7.28-7.29
(m, 2H). 13C NMR (125 MHz, CDCl3): δ 14.0, 16.7, 17.2, 18.1,
20.1, 21.9, 23.2, 24.5, 28.6, 36.4, 37.6, 38.8, 38.9, 41.4, 45.3, 55.8,
68.9, 72.5, 74.9, 77.3, 108.9, 113.5, 119.4, 126.6, 126.8, 127.2,
127.2, 127.3, 137.0, 140.1, 142.0, 145.3, 157.8, 166.6, 169.0. HRMS
(*ESI + pos) (m/z): [M + H] calcd for C35H42N1O7, 588.2961;
found, 588.2915. HPLC tR ) 15.06 min. Purity ) 96.3%.
from Calbiochem (La Jolla, CA). Antibodies for actin, HER2, and
Raf were from Calbiochem (La Jolla, CA).
Acknowledgment. This work described was supported by
the University of Kansas. The authors also thank support from
the American Foundation for Pharmaceutical Education (G.E.L.B.
and M.D.S.) and the Madison and Lila Self Graduate Fellowship
Program (G.E.L.B.).
1
Supporting Information Available: H NMR, 13C NMR, and
HPLC results of compounds 1-13. This material is available free
of charge via the Internet at http://pubs.acs.org.
(1S,3aS,41R,4a1S,5R,10aS,12aS)-1-(Furan-3-yl)-3,8-bis(hydroxy-
imino)-4a1,7,7,10a,12a-pentamethyl-1,3,3a,4a1,5,6,6a,7,8,10a,10b,11,
12,12a-tetradecahydronaphtho[2,1-f]oxireno[2,3-d]isochromen-5-
yl Acetate (13). Compound 13 was synthesized from 1 using general
procedure B and hydroxylamine hydrochloride to afford 7 mg (34%)
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