Synthesis and biological evaluation of synthetic viridins derived from C(20)-heteroalkylation of the steroidal PI-3-kinase inhibitor wortmannin

Article abstract of DOI:10.1039/b405431h

A series of viridin analogs was prepared from wortmannin by nucleophilic ring opening at C(20) and evaluated against the signaling kinases PI-3-kinase and mTOR. Several subnanomolar enzyme inhibitors with orders of magnitude selectivity for PI-3-kinase and strong cytotoxic activity against four cancer cell lines were identified. Among the ten most promising derivatives, six demonstrated lower liver toxicity and greater promise for inhibition of tumor cell growth than the lead structure wortmannin.

Full text of DOI:10.1039/b405431h

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Synthesis and biological evaluation of synthetic viridins derived
from C(20)-heteroalkylation of the steroidal PI-3-kinase inhibitor
wortmannin
Peter Wipf,*a Daniel J. Minion,a Robert J. Halter,a Margareta I. Berggren,b Caroline B. Ho,c
Gary G. Chiang,c Lynn Kirkpatrick,d Robert Abrahamc and Garth Powisb
a Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA
b Arizona Cancer Center, University of Arizona, 1515 North Campbell Avenue, Tucson,
AZ 85724, USA
c The Burnham Institute, La Jolla, CA 92037, USA
d ProlX Pharmaceuticals, Tucson, AZ 85701, USA
Received 14th April 2004, Accepted 11th May 2004
First published as an Advance Article on the web 14th June 2004
A series of viridin analogs was prepared from wortmannin by nucleophilic ring opening at C(20) and evaluated
against the signaling kinases PI-3-kinase and mTOR. Several subnanomolar enzyme inhibitors with orders of
magnitude selectivity for PI-3-kinase and strong cytotoxic activity against four cancer cell lines were identified.
Among the ten most promising derivatives, six demonstrated lower liver toxicity and greater promise for inhibition
of tumor cell growth than the lead structure wortmannin.
Phosphoinositide-3-kinases (PI-3-kinases) represent an ubi-
quitously expressed enzyme family that catalyzes the addition
of a phosphate molecule to the 3-position of the inositol ring of
phosphoinositides (Fig. 1).1
Viridin was also found to exhibit antibiotic activity against
certain plant pathogens; however, co-metabolites viridiol and
demethoxyviridiol exhibited phytotoxic activity, rendering the
parent fungal species unsuitable for biocontrol. Wortmannin
and its 11-desacetoxy analog were subsequently identified as
potent antiinflammatory agents.8 In the 1970’s, researchers at
Sandoz AG observed that all of the highly active analogs
also exhibited a high degree of acute toxicity rendering them
unsuitable for clinical development.9 Nearly 20 years later, it
was discovered that most of the natural viridins are mechanism-
based nanomolar inhibitors of PI-3-kinase and since then they
have served as important pharmacological probes for the
characterization of the cellular functions of these enzymes.10
Proteolytic mapping, followed by site-directed mutagenesis
of all candidate amino acid residues within the region,
revealed Lys-802 to be the target for the covalent binding of
wortmannin.11 This lysine residue resides in the ATP binding
site of the p110 catalytic subunit and has a crucial role in
the phosphotransfer reaction. The irreversible inhibition of
PI-3-kinase occurs by the formation of an enamine following
the attack of the lysine on the furan ring at C(20) of
wortmannin (Fig. 3). These findings were later supported
through structure–activity relationship studies.12 Wymann et al.
also proposed a model for the noncovalent interactions of
wortmannin with its PI-3-kinase binding site on the basis of the
first X-ray crystallographic structures of PI-3-kinase inhibitors
bound to the ATP binding pocket.13
More recently, wortmannin’s selectivity has been called into
question as it has been shown to inhibit other serine/threonine
kinases of the PI-3-kinase family, such as mTOR and
DNA-dependent protein kinase, with IC50’s of 2 and 4 µM
respectively, in intact cells (the IC50 is considerably lower for
the isolated enzymes, 250 nM for mTOR and 16 nM for
DNA-PK).14,15 Functionally, mTOR is involved in the control
of protein synthesis through activation of a number of targets
including p70 S6 kinase. Recently, a direct link between mTOR
and the PI-3-kinase-AKT signalling pathway in transformed
cells has been established.16 Wortmannin is therefore expected
to affect other kinases with homologies within the PI-3-kinase
Lys-802 region. The natural product has also been reported to
be an inhibitor of myosin light chain kinase (MLCK) with an
IC50 of 0.17 µM and a membrane-bound form of PI-4-kinase at
Fig. 1 Structure of phosphatidylinositol (PtdIns).
PI-3-kinase is found in cellular complexes with almost all
ligand activated growth factor receptor and oncogene protein
tyrosine kinases. PI-3-kinase activity has been found to increase
in response to platelet-derived growth factor (PDGF), insulin,
insulin-like growth factor 1 (IGF-1), colony stimulating factor 1
(CSF-1), nerve growth factor (NGF), hepatocyte growth factor
(HGF), stem cell growth factor (Steel), and epidermal growth
factor (EGF).2 PI-3-kinase activity is found to be elevated in
cells transformed by v-src, v-ros, v-yes, and v-abl as well as the
polyomavirus middle T antigen/c-src complex.3,4 PI-3-kinase is
also regulated by and associated with members of the non-
receptor family of tyrosine-kinases (i.e. lck, fyn, lyn, and c-yes)
in response to cellular activators that stimulate these enzymes.
Approximately 5% of cellular phosphoinositides (PtdIns) are
phosphorylated at the 4-position (PtdIns-4-P), and another 5%
are phosphorylated at both 4- and 5-positions (PtdIns-4,5-P2).
However, less than 0.25% of the total inositol-containing lipids
are phosphorylated at the 3-position, consistent with them
having only specific regulatory functions instead of a structural
function.4
The viridin family of natural products consists of a small
group of pentacyclic steroidal antifungal antibiotics isolated as
metabolites from a variety of fungi (Fig. 2).5–7 Unlike other nat-
urally occurring steroids, all members of this family possess a
furan ring fused between C(4) and C(6). Both viridin and
wortmannin were originally characterized as antifungal agents.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 9 1 1 – 1 9 2 0
1911

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Fig. 2 General molecular scaffold and some specific examples of natural viridin-class steroidal antibiotics. IC50 values for PI-3-kinase inhibition are
listed in parentheses.
Fig. 3 Proposed mechanism of action of PI-3-kinase inhibition by wortmannin.
high nanomolar concentrations.17,18 Aside from selectivity and
toxicity issues, another obstacle to the use of wortmannin and
demethoxyviridin as clinical candidates is their instability. Both
compounds, when stored as aqueous solutions at either 37 or
0 ЊC at neutral pH, are subject to decomposition by hydrolytic
opening of the furan ring. This chemical instability is much
more pronounced in demethoxyviridin than wortmannin,
mirroring their relative potency.
Extensive SAR studies in the mid 1990’s by the medicinal
chemistry group at Eli Lilly and Company confirmed that
the electrophilicity of the furan ring was key to the inhibitory
activity of wortmannin.12,19,20 The reaction of wortmannin with
nucleophiles at the C(20)-position followed by furan ring open-
ing provided compounds with a range of biological activities,21
but its acute toxicity which included hepatotoxicity and
biological instability prevented its clinical development as an
antitumor agent.
As part of an interdisciplinary program in drug discovery
that is based on the use of natural products as lead structures
for chemical library synthesis,22 we selected the viridins as a
platform for the development of selective kinase inhibitors.
Our specific objectives for this class of compounds were to
develop synthetic analogs that possess modified A- and E-rings
with increased chemical stability and structurally simplified
C/D-ring systems while maintaining biological activity and
improving selectivity. We now report a first series of analogs
that originate from nucleophilic modification of the furan ring
in wortmannin.
A
library of 94 C(20)-substituted synthetic viridins
was constructed via the addition of nucleophiles including
primary and secondary amines, anilines, and amino acids
to the natural product wortmannin (Table 1). Additionally, a
series of five C(20) thioether analogs was prepared by reaction
of wortmannin with both alkyl and aryl mercaptans in the
presence of triethylamine.
The synthetic viridin library compounds were screened for
their ability to inhibit the enzymes PI 3-kinase and mTOR.
Samples were also submitted for the National Cancer Institute
three-cell prescreen consisting of MCF-7 (Breast), NCI-H460
(Lung), and SF-268 (CNS) cell lines (data reported as percent
of growth of the treated cells when compared to the untreated
control cells). In addition, growth inhibition of human MCF-7
breast cancer cells was measured over 4 days using the MTT
assay. The results of these assays are summarized in Table 2.
A wide range of activities was observed in these assays, with
many of the compounds exhibiting potent PI-3-kinase activity
at concentrations equal to or lower than wortmannin. Perhaps
more important, however, was the increased in vitro growth
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 9 1 1 – 1 9 2 0
1912

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Table 1 Synthetic viridins obtained by C(20) nucleophilic opening of wortmannin
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 9 1 1 – 1 9 2 0
1913

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Table 1 (Contd.)
Nu =
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 9 1 1 – 1 9 2 0
1914

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Table 1 (Contd.)
Nu =
Fig. 4 Cytotoxicity of wortmann and synthetic viridins in the NCI 60 human tumor cell line panel. Cell growth was measured over a 48 h exposure
to drug and total protein measured with sulforhodamine D and the response pattern expressed as a mean graph. Growth inhibition was measured as
the drug concentration resulting in a 50% reduction in the net protein increase in control cells during the drug incubation (IC50).
inhibition towards MCF-7 cells observed for many analogs vs.
wortmannin. Numerous derivatives caused 50% inhibition in
the nanomolar range, whereas the IC50 of wortmannin was
found to be around 10 µM.
Data from the NCI human tumor cell line panel shows
that as the synthetic viridins increase in cytotoxic potency
their selectivity for different cell lines increases (Fig. 4).23
Wortmannin and PX-889 which are the least potent
compounds show very little selectivity among the different cell
lines. PX-868 which has intermediate potency shows increased
selectivity while PX-867 and PX-881 which are among the
most potent analogs exhibit a high degree of selectivity among
different cell lines.
probably in part due to their improved chemical stability. In
addition, the C(20) substituents can lead to high affinity toward
the enzyme by serving as leaving groups upon attack by K802 or
by providing a better fit in the ATP binding pocket. Three
potential interactions may be envisioned to contribute to the
latter effect: (1) favorable hydrophobic interactions between
the substitutent and the binding site, (2) the introduction of an
additional hydrogen bond from the diosphenol hydroxide to the
enzyme, and (3) changes in the conformations of the A, B, C,
and D rings as a consequence of the opening of the furan (E)
ring that provide a tighter fit to the binding site.
The mean purity of the library compounds was found to be
61% by LC-MS total ion count (TIC) analysis. Ten compounds,
PX-866, PX-867, PX-868, PX-870, PX-871, PX-880, PX-881,
The high level of activity of the synthetic derivatives is
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 9 1 1 – 1 9 2 0
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Table 2 Preliminary biological assays
PI-3-K: % Enzyme activity remaining at:
PI-3-K
mTOR
MCF-7
NCI 3-cell
Compound
0.1 nM
91.4
0.5 nM
49.3
1.0 nM
38.4
10 nM
IC50/nM
IC50/µM
IC50/µM
Mean% vs. control
Wortmannin
6.1
77.5
77.2
80.9
100
42.9
25.8
9.0
73.5
37.1
46.6
48.9
100
43.5
100
35.4
81.2
96.6
100
65.5
100
49.8
16.9
59.1
91.4
47.8
44.8
39.3
5.4
0.80
>10
0.1
9.0
1
27
39
23
31
34
23
31
36
28
17
15
25
45
30
2
3
9.0
4
>10
>10
>10
>10
8.0
8.0
0.1
0.1
8.0
10.0
9.0
9.0
10.0
>10
10.0
0.2
10.0
10.0
0.4
5
>0.5
1.5
0.8
>0.5
0.9
0.3
0.5
0.8
2.8
1
6
7
80.4
48.1
63.3
40.1
0.75
8
9
PX-890
10
11
12
13
14
47
0.50
0.1
15
>0.5
>0.5
>0.5
0.8
34
34
32
30
28
22
8
16
17
18
19
20
>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
0.7
21
22
0.2
<1
39
26
31
25
35
28
29
29
36
41
25
20
42
30
29
19
23
3
14
39
48
53
<1
14
<1
13
18
38
7
23
>10
>10
>10
6.0
8.0
>10
8.0
0.10
>10
>10
>10
4.0
9.0
5.0
24
26
26
27
100
49
106
95.2
31
4.50
0.10
28
89.7
100
29
30
10.0
93.4
85.5
38.0
14.9
56.2
40.7
33.2
41.5
9.4
48.8
85.2
80.8
84.9
77.5
34.7
119.5
138.5
1.9
16.5
97.9
6.0
4.9
4.1
63.7
80.4
54.1
3.1
2.9
48.6
7.0
0.9
>0.5
31
32
33
34
0.5
4.4
0.9
0.9
0.8
35
36
37
5.0
38
>10
10.0
0.2
2.0
2.0
0.10
1.0
8.0
6.0
0.1
8.0
8.0
8.0
8.0
1.0
1.0
2.0
8.0
0.2
8.0
0.4
1.0
0.3
1.5
2.0
3.0
3.0
0.8
3.0
3.0
39
87.6
82.7
94.6
69.4
111.5
6.0
0.5
0.4
PX-880
40
>0.5
>0.5
>0.5
>0.5
>0.5
0.4
>0.5
1.1
0.5
0.4
0.8
0.2
0.5
1.8
0.6
>0.5
1.1
0.5
0.5
>0.5
0.9
41
42
43
PX-889
44
45
46
13.2
47
48
49
100
100
75.5
100
67.2
70.5
100
43.8
58.9
48.0
0.9
2.4
50
51
26
13
20
<1
19
<1
26
2
34
36
74
83
52
53
PX-867
54
82.5
100
69.8
44.8
37.8
37.4
0.8
0.4
PX-882
55
PX-866
56
100
75.4
61.7
100
62.8
62.7
100.9
4.5
51.4
0.5
0.3
1.0
6.3
2.7
57
58
49.4
71.4
3.3
45.9
61.6
76.7
42.9
100
59
60
67.8
61.6
37.3
0.3
61
49
70
70
42
60
45
31
79
62
63
5.0
64
0.80
1.00
1.00
1.00
1.00
65
66
73.9
12.4
95.2
67
68
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 9 1 1 – 1 9 2 0
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Table 2 (Contd.)
PI-3-K: % Enzyme activity remaining at:
Compound
PI-3-K
mTOR
MCF-7
NCI 3-cell
0.1 nM
0.5 nM
1.0 nM
10 nM
IC50/nM
IC50/µM
IC50/µM
Mean% vs. control
69
19.6
30.5
44.8
2.3
1.00
1.00
1.00
0.90
1.00
5.00
2.00
3.00
1.50
5.00
0.40
1.00
5.00
3.00
1.50
>10
5.00
1.50
2.00
0.7
69
75
88
27
30
13
17
20
71
17
20
29
33
28
70
71
72
69.7
47.8
48.6
39.5
40.4
31.5
0.5
0.1
PX-868
73
2.0
66.8
55.0
10.5
18.6
3.3
74
75
55.8
30.3
27.5
0.2
76
77
91.7
75.1
28.1
51.2
67.2
15.3
39.2
60.6
9.5
0.5
5.0
0.1
78
7.2
79
11.0
23.1
26.4
25.0
58.3
51.6
53.0
48.0
80
81
82
83
84
85
86
PX-881
PX-871
87
0.1
0.1
0.4
0.1
1.0
0.1
8.00
>10
0.5
>10
0.5
37
35
<1
22
<1
88
89
PX-870
PX-882, PX-889, and PX-890, were prepared on a 10 mg to 4 g
scale and were subjected to advanced screening. The purity of
these compounds was consistently >95% (NMR). This re-assay
confirmed the promising activities toward PI-3-kinase found
in the library screen (Table 3). Since toxicity is a major con-
cern with wortmannin, we also focused on myelosuppression
(a specific decrease in lymphocyte count) with this selection of
compounds. A high lymphocyte toxicity is often a surrogate for
successful clinical inhibition of tumor cell growth. Groups of
three C57BL6 mice were administered wortmannin at doses
of 1, 2, or 3 mg kgϪ1 or the analogs at 1, 3, 9, or 18 mg kgϪ1 by
the intraperitoneal route daily for 4 days. The animals were
killed 24 h after the last dose and differential blood counts and
serum chemistry were determined.24 Compounds exhibiting
higher cancer cell and lymphocyte toxicity levels relative to
wortmannin were selected for further scale up and in vivo anti-
tumor testing. The in vivo pharmacological profile of PX-866,
PX-867, PX-868, PX-880, and PX-881, and PX-889 will be
discussed elsewhere.21
In conclusion, although much information has been gained
on the biological function of the viridin class of steroidal anti-
biotics, there are still many aspects of its cellular action which
are not fully understood. In spite of extensive prior medicinal
chemistry evaluation of wortmannin and its analogs, clinical
efficacy has remained elusive.25 We have prepared 99 derivatives
of wortmannin by nucleophilic opening at C(20) and evaluated
their IC50’s against the signaling kinases PI-3 kinase and
mTOR. This focused library of synthetic viridins furnished sev-
eral subnanomolar enzyme inhibitors with orders of magnitude
selectivity for PI-3-kinase and high cytotoxic activity against
4 cancer cell lines. Among the ten most promising derivatives,
six, namely PX-866, PX-867, PX-868, PX-880, PX-881, and
PX-889, demonstrated lower liver toxicity and greater promise
for inhibition of tumor cell growth than the lead structure
wortmannin. Furthermore, this series of derivatives displayed
far higher selectivity for PI-3-kinase relative to mTOR, when
compared to the parent compound wortmannin. The synthesis
of these derivatives can be scaled up to a multigram level, and
in vivo testing has been initiated with the goal of identifying
a suitable clinical candidate. The results of these advanced
pharmacological studies will be reported in due course.
Experimental
General procedure A: construction of a library of C(20) amine
adducts of wortmannin
Stock solutions of the amino acids and amines were prepared at
0.06 M in DMSO as well as a stock solution of wortmannin at
0.1 M DMSO. The amino acids that were not soluble at this
concentration were diluted to a known concentration in H2O
and warmed at 60 ЊC until fully dissolved. Amino acids that
were not soluble at concentrations of 0.04 M 2 : 1 DMSO–H2O
were sonicated and used as suspensions. One equiv. of NEt3
from a stock solution was added to solutions of hydrochloride
salts or amines or amino acids. The array was assembled in a
1 mL deep-well plate and wortmannin solution was added
(42 µL, 4.2 µmol) to each well containing the appropriate amine
or amino acid (1.3 eq., 5.4 µmol). The wells were then diluted to
267 µL with DMSO (final concentration 0.0157 M). The plate
was covered with foil and heated at 58 ЊC for 2 h in an oven
and then allowed to stand at room temperature overnight.
Volatiles were removed in vacuo using a SpeedVac© apparatus
and daughter plates were prepared consisting of 1.0 µmol
(theoretical) of compound in DMSO and were kept frozen
until biological testing. Product verification and purity were
established through LCMS analysis (APCI, positive mode,
85 : 15 MeOH–H2O). LCMS samples were prepared by diluting
5 µL of the reaction mixture with MeOH–H2O (1.0 mL, 95 : 5).
Purity was determined through integration of the mass spectral
total ion count (TIC) trace. Complete product formation for
these reactions was confirmed by thin layer chromatography.
Samples were submitted regardless of purity.
General procedure B: synthesis of C(20) thiol adducts of
wortmannin
Stock solutions of the thiols were prepared at 1.0 M in CH2Cl2.
Stock solutions of wortmannin and NEt3 were prepared at 0.2
M and 0.25 M in CH2Cl2, respectively. To a 10 × 75 mm culture
tube fitted with a flea bar was added wortmannin solution (100
µL, 20 µmol), the appropriate thiol (40 µL, 40 µmol), and NEt3
(5 µL, 1.25 µmol) in 200 µL of CH2Cl2. The reactions were
stirred at room temperature for 3–40 h and monitored by TLC.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 9 1 1 – 1 9 2 0
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Table 3 Activity of scaled and purified synthetic viridins
PI-3K inhibition
IC50/nM
mTOR inhibition
IC50/µM
Cytotoxicity MCF-7 cells
IC50/µM
Lymphocyte toxicity
relative to wortmannin
Compound
Wortmannin
PX-866
PX-867
PX-868
PX-870
PX-871
PX-880
PX-881
PX-882
PX-889
PX-890
0.3
0.5
1.1
0.1
1.0
0.1
10
0.1
0.4
5
0.3
>10
>10
>3
>3
>3
2.0
>10
>3
>3
>3
5.0
0.5
0.1
1.0
0.4
8.0
0.2
ND
0.5
8.1
0.2
1.0
2.2
2.4
0.9
1.1
0.7
1.1
1.9
0.8
1.5
0.6
10
The products were purified via chromatography on SiO2
(hexanes–ethyl acetate 1 : 1). Product verification and purity
was established through 1H NMR and via LCMS (EIϩ, 65 : 35
MeOH–H2O). The compounds were recrystallized using
hexanes–ethyl acetate to give yellow solids (a modification of
this procedure for hindered thiols such as naphthyl, t-butyl, or
i-propyl used CHCl3 as the reaction solvent and the reaction
was heated to 55 ЊC. The reaction was much faster and no
bisadduct was observed).
0.86 (s, 3 H); 13C NMR δ 217.5, 178.9, 169.9, 164.9, 153.9,
151.3, 137.6, 137.1, 129.2, 89.4, 82.1, 73.3, 67.7, 59.3, 55.2, 49.2
(2 C), 42.6, 42.4, 37.8, 36.3, 25.6 (2 C), 24.5, 22.4, 21.0, 16.3;
MS (EI) m/z (rel. intensity) 499 (Mϩ, 1), 439 (2), 365 (7), 167
(35), 149 (100); HRMS (EI) calculated for C27H33NO8 499.2206,
found 499.2191.
Acetic acid 4-[(3-dimethylaminopropylamino)methylene]-6-
hydroxy-1ꢀ-methoxymethyl-10ꢁ,13ꢁ-dimethyl-3,7,17-trioxo-
1,3,4,7,10,11ꢁ,12,13,14ꢀ,15,16,17-dodecahydro-2-oxa-cyclo-
penta[a]phenanthren-11-yl ester (PX-868)
Acetic acid 4-diallylaminomethylene-6-hydroxy-1-ꢀ-methoxy-
methyl-10ꢁ,13ꢁ-dimethyl-3,7,17-trioxo-1,3,4,7,10,11ꢁ,12,13,-
14ꢀ,15,16,17-dodecahydro-2-oxa-cyclopenta[a]phenanthren-
11-yl ester (PX-866)
To a solution of wortmannin (10.7 mg, 25.0 µmol) in CH2Cl2
(125 µL) was added a freshly prepared 0.2 M stock solution of
3-dimethylaminopropylamine (138 µL, 27.5 µmol) in CH2Cl2.
The reaction mixture was stirred at room temperature for 15
min. The solvent and excess amine were removed in vacuo to
give PX-868 (11.6 mg, 21.8 µmol, 87%) as an orange oil: [α]D
Ϫ500 (c 0.008, CH2Cl2, 23 ЊC); IR (KBr) 3306, 3259, 1743,
1669, 1633, 1623, 1573, 1222 cmϪ1; 1H NMR δ 10.04–10.00 (m,
1 H), 8.54 (d, 1 H, J = 14.0 Hz), 5.99 (dd, 1 H, J = 7.9, 3.3 Hz),
4.31 (dd, 1 H, J = 7.0, 2.1 Hz), 3.76 (q, 2 H, J = 6.5 Hz), 3.26 (s, 3
H), 3.26–3.15 (m, 2 H), 2.99–2.84 (m, 2 H), 2.62–2.51 (m, 1 H),
2.40–2.17 (m, 5 H), 2.24 (s, 6 H), 2.04 (s, 3 H), 1.88 (dd, 1 H,
J = 13.6, 3.4 Hz), 1.82–1.76 (m, 1 H), 1.80 (t, 2 H, J = 6.7 Hz),
1.52 (s, 3 H), 0.82 (s, 3 H); 13C NMR δ 218.0, 178.5, 170.2,
165.7, 159.4, 151.0, 137.3, 137.0, 129.3, 88.4, 81.1, 77.8, 73.4,
67.2, 59.3, 56.7, 50.0, 48.6, 45.6, 42.4, 42.3, 38.6, 36.7, 28.4,
26.4, 22.5, 21.1, 16.8; MS (EI) m/z (rel. intensity) 530 (Mϩ, 100),
488 (18), 470 (34); HRMS (EI) calculated for C28H38N2O8
530.2628, found 530.2609.
To a 0 ЊC solution of wortmannin (0.89 g, 2.08 mmol) in
CH2Cl2 (10 mL) was added a freshly prepared 0.2 M stock
solution of freshly distilled (from KOH) diallylamine (10.8 mL,
2.16 mmol) in CH2Cl2. The reaction mixture was warmed to
room temperature and stirred for 1 h. The solvent and excess
amine were removed in vacuo, providing PX-866 (1.09 g, 2.08
mmol, quant.) as an orange solid: [α]D Ϫ630 (c 0.0015, CH2Cl2,
23 ЊC); IR (KBr) 3391, 1743, 1695, 1685, 1622, 1569, 1222,
1111, 1100 cmϪ1; 1H NMR δ 8.20 (s, 1 H), 6.81 (s, 1 H), 6.06 (dd,
1 H, J = 7.4, 4.8 Hz), 5.85 (br s, 1 H), 5.62 (br, 1 H), 5.44–5.04
(m, 4 H), 4.48 (dd, 1 H, J = 7.2, 1.9 Hz), 4.05–3.60 (m, 4 H), 3.26
(s, 3 H), 3.27–3.20 (m, 1 H), 3.16 (dd, 1 H, J = 10.9, 7.2 Hz),
3.00–2.90 (m, 2 H), 2.59 (dd, 1 H, J = 19.4, 8.6 Hz), 2.40 (dd,
1 H, J = 14.4, 7.7 Hz), 2.35–2.07 (m, 2 H), 2.07 (s, 3 H), 1.83 (dd,
1 H, J = 14.4, 4.7 Hz), 1.54 (s, 3 H), 0.86 (s, 3 H); 13C NMR
δ 217.0, 178.5, 169.6, 164.8, 156.3, 151.5, 139.0, 136.9, 132.2,
131.3, 127.7 (2 C), 119.2, 89.0, 81.9, 73.1, 67.6, 59.1, 50.9 (2 C),
48.9, 42.3, 42.2, 37.5, 36.0, 24.6, 22.2, 20.8, 16.1; MS (EI) m/z
(rel. intensity) 525 (Mϩ, 11), 466 (17), 391 (15), 350 (14), 323
(13), 266 (17), 239 (17), 60 (100); HRMS (EI) calculated for
C29H35NO8 525.2363, found 525.2386.
Acetic acid 6-hydroxy-1ꢀ-methoxymethyl-10ꢁ,13ꢁ-dimethyl-
3,7,17-trioxo-4-phenylsulfanylmethylene-1,3,4,7,10,11ꢁ,12,13,-
14ꢀ,15,16,17-dodecahydro-2-oxa-cyclopenta[a]phenanthren-
11-yl (PX-870)
Acetic acid 6-hydroxy-1ꢀ-methoxymethyl-10ꢁ,13ꢁ-dimethyl-
3,7,17-trioxo-4-pyrrolidin-1-yl-methylene-1,3,4,7,10,11ꢁ,12,13,-
14ꢀ,15,16,17-dodecahydro-2-oxa-cyclopenta[a]phenanthren-
11-yl (PX-867)
To a solution of wortmannin (15.0 mg, 35.0 µmol) in CH2Cl2
(500 µL) was added thiophenol (10 µL, 118 µmol) and a drop of
NEt3. The reaction mixture was stirred at room temperature for
20 h under N2. The solvent and excess thiol were removed
in vacuo and the product was purified by chromatography on
SiO2 (hexanes–ethyl acetate 9 : 1, then 1 : 1) to give PX-870 (9.7
To a 0 ЊC solution of wortmannin (290.4 mg, 0.678 mmol) in
CH2Cl2 (3.4 mL) was added a freshly prepared 0.2 M stock
solution of freshly distilled (from KOH) pyrrolidine (3.75 mL,
0.75 mmol) in CH2Cl2. The reaction mixture was warmed to
room temperature and stirred for 1 h. The solvent and excess
amine were removed in vacuo, providing PX-867 (338.0 mg,
0.677 mmol, quant.) as an orange solid: [α]D Ϫ390 (c 0.0073,
CH2Cl2, 23 ЊC); IR (KBr) 3337, 1740, 1684, 1617, 1570, 1261,
1
mg, 18 µmol, 51%) as an orange oil: H NMR δ 9.08 (s, 1 H),
7.60–7.57 (m, 2 H), 7.47–7.39 (m, 3 H), 7.34 (s, 1 H), 6.02 (dd, 1
H, J = 7.6, 3.5 Hz), 4.50 (dd, 1 H, J = 7.4, 1.4 Hz), 3.30 (dd, 1 H,
J = 11.1, 1.5 Hz), 3.28 (s, 3 H), 3.25–3.16 (m, 1 H), 3.02–2.95 (m,
1 H), 2.89–2.80 (m, 1 H), 2.65–2.52 (m, 1 H), 2.37 (dd, 1 H,
J = 15.0, 7.9 Hz), 2.31–2.22 (m, 2 H), 2.07 (s, 3 H), 1.91 (dd, 1 H,
J = 15.1, 3.4 Hz), 1.57 (s, 3 H), 0.82 (s, 3 H); 13C NMR δ 217.4,
179.9, 169.9, 162.6, 160.6, 152.6, 141.3, 137.2, 136.8, 130.4 (2
C), 129.7 (2 C), 128.7, 124.0, 114.4, 82.1, 73.6, 67.2, 59.5, 49.8,
42.9, 42.3, 38.4, 36.5, 25.3, 22.4, 21.1, 16.8; MS (EI) m/z (rel.
intensity) 538 (Mϩ, 1), 355 (100), 295 (21), 110 (39); HRMS (EI)
calculated for C29H30O8S 538.1661, found 538.1649.
1
1221, 1099, 1018 cmϪ1; H NMR δ 8.29 (s, 1 H), 6.72 (s, 1 H),
6.07 (dd, 1 H, J = 6.9, 4.8 Hz), 4.47 (dd, 1 H, J = 7.0, 1.9 Hz),
3.80–3.70 (m, 2 H), 3.25 (s, 3 H), 3.25–3.14 (m, 2 H), 3.02–2.90
(m, 2 H), 2.69 (br s, 1 H), 2.58 (dd, 1 H, J = 19.1, 8.4 Hz), 2.39
(dd, 1 H, J = 14.6, 7.8 Hz), 2.32–2.08 (m, 2 H), 2.06 (s, 3 H),
1.99–1.95 (m, 5 H), 1.84 (dd, 1 H, J = 14.5, 4.2 Hz), 1.56 (s, 3 H),
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 9 1 1 – 1 9 2 0
1918

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Acetic acid 6-hydroxy-4-isopropylsulfanylmethylene-1ꢀ-meth-
oxymethyl-10ꢁ,13ꢁ-dimethyl-3,7,17-trioxo-1,3,4,7,10,11ꢁ,12,-
13,14ꢀ,15,16,17-dodecahydro-2-oxa-cyclopenta[a]phenanthren-
11-yl (PX-871)
δ 217.3, 178.9, 169.9, 164.7, 158.3, 151.7, 138.8, 137.1, 134.9,
129.0 (3 C), 128.6, 128.1 (2 C), 88.7, 82.2, 73.4, 67.9, 64.3, 59.4,
49.1, 42.7, 42.5, 37.8 (2 C), 36.3, 25.2, 22.5, 21.1, 16.3; MS (EI)
m/z (rel. intensity) 549 (Mϩ, 14), 489 (37), 415 (15), 120 (23), 91
(100); HRMS (EI) calculated for C31H35NO8 549.2363, found
549.2340.
To a solution of wortmannin (15.0 mg, 35.0 µmol) in CHCl3
(500 µL) was added 2-propanethiol (16.0 µL, 177 µmol) and a
drop of NEt3. The reaction mixture was stirred at room
temperature for 20 h under N2. The solvent and excess thiol
were removed in vacuo and the product was purified by chroma-
tography on SiO2 (hexanes–ethyl acetate 9 : 1, then 1 : 1) to give
Acetic acid 4-[(dibenzylamino)methylene]-6-hydroxy-1ꢀ-meth-
oxymethyl-10ꢁ,13ꢁ-dimethyl-3,7,17-trioxo-1,3,4,7,10,11ꢁ,12,-
13,14ꢀ,15,16,17-dodecahydro-2-oxa-cyclopenta[a]phenanthren-
11-yl ester (PX-882)
1
PX-871 (14.5 mg, 28.7 µmol, 82%) as a yellow oil: H NMR
δ 9.00 (s, 1 H), 7.27 (s, 1 H), 6.01 (dd, 1 H, J = 7.9, 3.4 Hz), 4.44
(d, 1 H, J = 7.2 Hz), 3.31–3.19 (m, 2 H), 3.26 (s, 3 H), 3.14 (dd,
1 H, J = 11.1, 7.7 Hz), 3.00–2.96 (m, 1 H), 2.89–2.83 (m, 1 H),
2.62–2.54 (m, 1 H), 2.36 (dd, 1 H, J = 15.0, 7.9 Hz), 2.33–2.25
(m, 2 H), 2.05 (s, 3 H), 1.90 (dd, 1 H, J = 15.0, 3.4 Hz), 1.54 (s, 3
H), 1.46 (d, 3 H, J = 6.8 Hz), 1.44 (d, 3 H, J = 6.9 Hz), 0.83 (s, 3
H); 13C NMR δ 217.5, 180.0, 170.0, 162.5, 160.7, 152.6, 141.0,
137.0, 124.7, 113.8, 81.8, 73.6, 67.2, 59.5, 49.8, 42.9, 42.3, 40.7,
38.4, 36.6, 25.3, 23.7, 23.2, 22.4, 21.1, 16.8; MS (EI) m/z (rel.
intensity) 504 (Mϩ, 18), 370 (100), 355 (82), 313 (65), 323 (31),
295 (32), 266 (33), 239 (43), 165 (29), 152 (35), 129 (35), 115
(40), 91 (53); HRMS (EI) calculated for C26H32O8S 504.1818,
found 504.1824.
To a solution of wortmannin (10.7 mg, 25.0 µmol) in CH2Cl2
(125 µL) was added a freshly prepared 0.1 M solution of diben-
zylamine (450 µL, 45 µmol) in CH2Cl2. The reaction mixture
was stirred at room temperature for 6 h. The solvent was
removed in vacuo and the product was purified via chromato-
graphy on SiO2 (hexanes–ethyl acetate, 1 : 9) to give PX-882
(14.2 mg, 22.6 µmol, 90%) as an orange oil: 1H NMR δ 8.51 (s, 1
H), 7.43–7.31 (m, 8 H), 7.05 (br, 2 H), 6.69 (br, 1 H), 6.00 (dd, 1
H, J = 5.6, 1.4 Hz), 4.58–4.49 (m, 3 H), 4.37–3.34 (m, 1 H),
4.03–4.01 (m, 1 H), 3.27 (s, 3 H), 3.26–3.23 (m, 1 H), 3.19–3.15
(m, 1 H), 2.97–2.90 (m, 2 H), 2.55 (dd, 1 H, J = 19.5, 8.8 Hz),
2.37 (dd, 1 H, J = 14.3, 8.7 Hz), 2.30–2.22 (m, 1 H), 2.06 (s, 3 H),
1.77 (dd, 1 H, J =16.8, 4.9 Hz), 1.34 (s, 3 H), 0.78 (s, 3 H); 13
C
NMR δ 217.2, 178.9, 169.9, 165.7, 157.1, 152.1, 139.8, 137.1,
135.3, 135.2, 129.2 (2 C), 128.9 (2 C), 128.5 (4 C), 128.0 (3 C),
89.2, 82.6, 73.4, 68.1, 61.3, 59.5, 51.8, 49.1, 42.8, 42.6, 37.7,
36.3, 24.3, 22.5, 21.2, 16.2; MS (EI) m/z (rel. intensity) 625 (Mϩ,
1), 371 (11), 106 (19), 91 (100); HRMS (EI) calculated for
C37H39NO8 625.2676, found 625.2657.
Acetic acid 6-hydroxy-1ꢀ-methoxymethyl-10ꢁ,13ꢁ-dimethyl-
3,7,17-trioxo-4-(phenethylaminomethylene)-1,3,4,7,10,11ꢁ,12,-
13,14ꢀ,15,16,17-dodecahydro-2-oxa-cyclopenta[a]phenanthren-
11-yl (PX-880)
To a solution of wortmannin (10.7 mg, 25.0 µmol) in CH2Cl2
(125 µL) was added a freshly prepared 0.2 M stock solution
of phenethylamine (138 µL, 27.5 µmol) in CH2Cl2. The reaction
mixture was stirred at room temperature for 1 h. The solvent
and excess phenylethylamine were removed in vacuo to give
PX-880 (12.9 mg, 23.4 µmol, 94%) as an orange oil: [α]D Ϫ410
(c 0.0026, CH2Cl2, 23 ЊC); IR (KBr) 3332, 3266, 1741, 1670,
Acetic acid 6-hydroxy-1ꢀ-methoxymethyl-10ꢁ,13ꢁ-dimethyl-
3,7,17-trioxo-4-(phenylaminomethylene)-1,3,4,7,10,11ꢁ,12,13,-
14ꢀ,15,16,17-dodecahydro-2-oxa-cyclopenta[a]phenanthren-
11-yl (PX-889)
To a solution of wortmannin (12.7 mg, 30.0 µmol) in CH2Cl2
(125 µL) was added a freshly prepared 0.4 M solution of aniline
(100 µL, 40 µmol) in CH2Cl2. The reaction mixture was stirred
at room temperature for 18 h. The solvent was removed
in vacuo, and the product was purified by chromatography on
SiO2 (hexanes–ethyl acetate 1 : 1, then 1 : 99) to give PX-889
(11.3 mg, 21.7 µmol, 72%) as a yellow oil: [α]D Ϫ210 (c 0.00012,
CH2Cl2, 23 ЊC); IR (KBr) 3420, 1743, 1677, 1623, 1574, 1219,
1123 cmϪ1; 1H NMR δ 11.58 (d, 1 H, J = 13.5 Hz), 9.11 (d, 1 H,
J = 13.5 Hz), 7.41 (t, 2 H, J = 7.9 Hz), 7.33 (s, 1 H); 7.20–7.16
(m, 3 H), 6.03 (dd, 1 H, J = 7.9, 3.3 Hz), 4.41 (dd, 1 H, J = 7.6,
1.6 Hz), 3.33–3.28 (m, 1 H), 3.39 (s, 3 H), 3.23 (dd, 1 H, J = 11.1,
7.7 Hz), 3.02–2.85 (m, 2 H), 2.65–2.52 (m, 1 H), 2.38 (dd, 1 H,
J = 15.0, 7.9 Hz), 2.39–2.23 (m, 2 H), 2.06 (s, 3 H), 1.90 (dd, 1 H,
J = 15.0, 3.3 Hz), 1.58 (s, 3 H), 0.84 (s, 3 H); 13C NMR δ 217.8,
178.9, 170.0, 165.8, 151.5, 150.7, 139.3, 138.3, 137.1, 130.0 (2
C), 127.3, 125.1, 117.2 (2 C), 92.1, 81.6, 73.4, 67.2, 59.4, 49.9,
42.5, 42.3, 38.5, 36.6, 26.2, 22.5, 21.0, 16.7; MS (EI) m/z (rel.
intensity) 521 (Mϩ, 100), 461 (26), 387 (65), 372 (58), 359 (37),
344 (39), 330 (66); HRMS (EI) calculated for C29H31NO8:
521.2050, found: 521.2043.
1
1644, 1621, 1576, 1223 cmϪ1; H NMR δ 9.95–9.87 (m, 1 H),
8.41 (d, 1 H, J = 13.8 Hz), 7.36–7.20 (m, 5 H), 7.07 (br, 1 H),
5.98 (dd, 1 H, J = 8.1, 3.3 Hz), 4.31 (dd, 1 H, J = 7.5, 1.7 Hz),
3.68–3.60 (m, 2 H), 3.27 (s, 3 H), 3.26–3.21 (m, 1 H), 3.16 (dd,
1 H, J = 11.0, 7.6 Hz), 2.97 (t, 2 H, J = 7.1 Hz), 2.98–2.80 (m,
2 H), 2.63–2.51 (m, 1 H), 2.35 (dd, 1 H, J = 15.1, 7.8 Hz), 2.33–
2.24 (m, 2 H), 2.04 (s, 3 H), 1.88 (dd, 1 H, J = 15.1, 3.3 Hz), 1.50
(s, 3 H), 0.82 (s, 3 H); 13C NMR δ 218.0, 178.5, 170.1, 165.8,
159.2, 151.0, 137.5, 137.4, 136.9, 128.9 (5 C), 127.1, 88.6, 81.1,
73.3, 67.2, 59.3, 51.7, 49.9, 42.3 (2 C), 38.5, 37.3, 36.6, 26.3,
22.5, 21.0, 16.8; MS (EI) m/z (rel. intensity) 549 (Mϩ, 6), 415
(11), 129 (15), 105 (100); HRMS (EI) calculated for C31H35NO8
549.2363, found 549.2347.
Acetic acid 4-[(benzylmethylamino)methylene]-6-hydroxy-1ꢀ-
methoxymethyl-10ꢁ,13ꢁ-dimethyl-3,7,17-trioxo-1,3,4,7,10,11ꢁ,-
12,13,14ꢀ,15,16,17-dodecahydro-2-oxa-cyclopenta[a]phenan-
thren-11-yl ester (PX-881)
To a 0 ЊC solution of wortmannin (272.4 mg, 0.635 mmol) in
CH2Cl2 (3.2 mL) was added a freshly prepared 0.2 M solution
of freshly distilled (from KOH) N-methylbenzylamine (3.5 mL,
0.70 mmol) in CH2Cl2. The reaction mixture was warmed to
room temperature and stirred for 1 h. The solvent and excess
amine were removed in vacuo, providing PX-881 (348.6 mg,
0.634 mmol, quant.) as an orange solid: [α]D Ϫ835 (c 0.0014,
CH2Cl2, 23 ЊC); IR (neat) 1742, 1685, 1618, 1589, 1575, 1224
cmϪ1; 1H NMR δ 8.36 (br s, 1 H), 7.36–7.27 (m, 5 H), 6.60 (br s,
1 H), 6.10–6.00 (m, 1 H), 4.68–4.63 (m, 1 H), 4.53–4.47 (m, 2
H), 3.25 (s, 3 H), 3.25–3.11 (m, 2 H), 2.99–2.84 (m, 2 H), 2.71
(br, 2 H), 2.55 (dd, 1 H, J = 19.5, 8.9 Hz), 2.38 (dd, 1 H, J = 14.4,
7.6 Hz), 2.32–2.05 (m, 2 H), 2.05 (s, 3 H), 1.85 (br s, 1 H), 1.80
(dd, 1 H, J = 14.5, 4.7 Hz), 1.52 (s, 3 H), 0.82 (s, 3 H); 13C NMR
Acetic acid 4-[(carbamoylmethylamino)methylene]-6-hydroxy-
1ꢀ-methoxymethyl-10ꢁ,13ꢁ-dimethyl-3,7,17-trioxo-1,3,4,7,10,-
11ꢁ,12,13,14ꢀ,15,16,17-dodecahydro-2-oxa-cyclopenta[a]-
phenanthren-11-yl ester (PX-890)
To a culture tube charged with wortmannin (20.0 mg, 46.7
µmol) was added a homogeneous solution of glycinamide
hydrochloride (6.4 mg, 58 µmol) and NEt3 (8.0 µL, 58 µmol) in
dry DMF (1 mL). The reaction mixture was stirred at room
temperature for 1 h. The solvent was removed by a stream of N2
gas and the product was purified by chromatography on SiO2
(CH2Cl2–MeOH 9 : 1) to give PX-890 (14.6 mg, 29.1 µmol,
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 9 1 1 – 1 9 2 0
1919

View Article Online
1
11 M. P. Wymann, G. Bulgarelli-Leva, M. J. Zvelebil, L. Pirola,
62%) as a yellow oil: H NMR δ 10.02–9.94 (m, 1 H), 8.50 (d,
B. VanHaese-broeck, M. D. Waterfield and G. Panayotou, Mol. Cell.
Biol., 1996, 16, 1722.
12 B. H. Norman, J. Paschal and C. J. Vlahos, Bioorg. Med. Chem.
Lett., 1995, 5, 1183.
13 E. H. Walker, M. E. Pacold, O. Perisic, L. Stephens, P. T. Hawkins,
M. P. Wymann and R. L. Williams, Mol. Cell, 2000, 6, 909.
14 G. J. Brunn, J. Williams, C. Sabers, G. Wiederrecht, J. C. Lawrence
and R. T. Abraham, EMBO J., 1996, 15, 5256.
15 S. Boulton, S. Kyle, L. Yalcintepe and B. W. Durkacz,
Carcinogenesis, 1996, 17, 2285.
16 A. Sekulic, C. C. Hudson, J. L. Homme, P. Yin, D. M. Otterness,
L. M. Karnitz and R. T. Abraham, Cancer Res., 2000, 60, 3504.
17 S. Nakanishi, S. Kakita, I. Takahashi, K. Kawahara, E. Tsukuda,
T. Sano, K. Yamada, M. Yoshida, H. Kase, Y. Matsuda,
Y. Hashimoto and Y. Nonomura, J. Biol. Chem., 1992, 267, 2157.
18 S. Corvera and M. P. Czech, Trends Cell. Biol., 1998, 8, 442.
19 J. A. Dodge, H. U. Bryant, J. Kim, W. F. Matter, B. H. Norman,
U. Srinivasan, C. J. Vlahos and M. Sato, Bioorg. Med. Chem. Lett.,
1995, 5, 1713.
1 H, J = 13.7 Hz), 7.26 (s, 1 H), 6.51 (br s, 1 H), 6.22 (br s, 1 H),
5.99 (dd, 1 H, J = 7.8, 3.1 Hz), 4.32 (dd, 1 H, J = 6.5, 2.7 Hz),
4.12 (d, 2 H, J = 6.2 Hz), 3.25 (s, 3 H), 3.25–3.12 (m, 2 H),
2.98–2.81 (m, 2 H), 2.61–2.51 (m, 1 H), 2.38–2.20 (m, 3 H), 2.04
(s, 3 H), 1.87 (dd, 1 H, J = 15.0, 3.1 Hz), 1.52 (s, 3 H), 0.81 (s, 3
H); 13C NMR δ 217.9, 178.8, 170.5, 170.1, 166.0, 159.6, 151.1,
138.0, 137.1, 127.6, 90.3, 81.2, 73.3, 67.2, 59.3, 52.0, 49.9, 42.2
(2 C), 38.5, 36.6, 26.1, 22.5, 21.1, 16.8; MS (EI) m/z (rel. inten-
sity) 502 (Mϩ, 9), 442 (15), 368 (18), 167 (20), 146 (69), 60 (100);
HRMS (EI) calculated for C25H30N2O9 502.1951, found
502.1940.
Acknowledgements
This work was supported by the National Institutes of Health
(U19CA-52995). DJM gratefully acknowledges support from
T32 GM08424. The authors thank the National Cancer
Institute for supplying natural wortmannin.
20 B. H. Norman, C. Shih, J. E. Toth, J. E. Ray, J. A. Dodge,
D. W. Johnson, P. G. Rutherford, R. M. Schultz, J. F. Worzalla and
C. J. Vlahos, J Med. Chem., 1996, 39, 1106.
21 R. M. Schultz, R. L. Merriman, S. L. Andis, R. Bonjouklian,
G. B. Grindey, P. G. Rutherford, A. Gallegos, K. Massey and
G. Powis, Anticancer Res., 1995, 15, 1135.
References and notes
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2 R. Kapeller and L. C. Cantley, BioEssays, 1994, 16, 565.
3 L. Rameh and L. C. Cantley, J. Biol. Chem., 1999, 274, 8347.
4 H. Yano, S. Nakanishi, K. Kimura, N. Hanai, Y. Saitoh, Y. Fukui,
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5 J. R. Hanson, Nat. Prod. Rep., 1995, 12, 381.
6 P. Wipf and A. D. Kerekes, J. Nat. Prod., 2003, 66, 716.
7 For recent noteworthy total syntheses of wortmannin and viridin,
respectively, see: (a) T. Mizutani, S. Honzawa, S. Tosaki and
M. Shibasaki, Angew. Chem. Int. Ed., 2002, 41, 4680; (b)
E. A. Anderson, E. J. Alexanian and E. J. Sorensen, Angew. Chem.,
Int. Ed., 2004, 43, 1998.
8 D. Wiesinger, H. U. Gubler, W. Haefliger and D. Hauser, Experentia,
1974, 30, 135.
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1920