Gateway synthesis of daphnane congeners and their protein kinase C affinities and cell-growth activities

Article abstract of DOI:10.1038/nchem.1074

The daphnane diterpene orthoesters constitute a structurally fascinating family of natural products that exhibit a remarkable range of potent biological activities. Although partial activity information is available for some natural daphnanes, little information exists for non-natural congeners or on how changes in structure affect mode of action, function, potency or selectivity. A gateway strategy designed to provide general synthetic access to natural and non-natural daphnanes is described and utilized in the synthesis of two novel members of this class. In this study, a commercially available tartrate derivative was elaborated through a key late-stage diversification intermediate into B-ring yuanhuapin analogues to initiate exploration of the structure-function relationships of this class. Protein kinase C was identified as a cellular target for these agents, and their activity against human lung and leukaemia cell lines was evaluated. The natural product and a novel non-natural analogue exhibited significant potency, but the epimeric epoxide was essentially inactive.

Full text of DOI:10.1038/nchem.1074

ARTICLES
PUBLISHED ONLINE: 19 JUNE 2011 | DOI: 10.1038/NCHEM.1074
Gateway synthesis of daphnane congeners
and their protein kinase C affinities and
cell-growth activities
Paul A. Wender , Nicole Buschmann, Nathan B. Cardin, Lisa R. Jones, Cindy Kan, Jung-Min Kee,
*
John A. Kowalski and Kate E. Longcore
The daphnane diterpene orthoesters constitute a structurally fascinating family of natural products that exhibit a
remarkable range of potent biological activities. Although partial activity information is available for some natural
daphnanes, little information exists for non-natural congeners or on how changes in structure affect mode of action,
function, potency or selectivity. A gateway strategy designed to provide general synthetic access to natural and non-
natural daphnanes is described and utilized in the synthesis of two novel members of this class. In this study, a
commercially available tartrate derivative was elaborated through a key late-stage diversification intermediate into B-ring
yuanhuapin analogues to initiate exploration of the structure–function relationships of this class. Protein kinase C was
identified as a cellular target for these agents, and their activity against human lung and leukaemia cell lines was
evaluated. The natural product and a novel non-natural analogue exhibited significant potency, but the epimeric epoxide
was essentially inactive.
he daphnane diterpene orthoesters (DDOs) constitute a struc-
A major subset of DDOs, depicted in Fig. 1, is made up of at least
turally fascinating and synthetically challenging class of 73 congeners that differ in the functionality at C12, the orthoester
natural products, which collectively exhibit a remarkably side chain, A-ring oxidation and the substitution at C20. This sub-
T
broad range of biological activities and selectivities1–11. Plants that family includes yuanhuapin (1), which was first isolated in 1986
contain DDOs have been used medicinally for over 2000 years12, from the flowers of Daphne genkwa19, one of the 50 fundamental
and more than 140 unique members have been identified to herbs in Chinese traditional medicine. In addition to possessing
date. Many DDOs are exceptional, but relatively unexplored, anticancer activity20, yuanhuapin also displays structural features
leads for the treatment of cancer, diabetes, neurodegenerative dis- shared by many biologically active DDOs. Significantly, several of
eases and neuropathic pain. Some DDOs, such as resiniferatoxin these DDOs display over a 1000-fold greater activity against A549
(RTX), have advanced into clinical trials13–15
. Significantly, human lung cancer cells relative to that of MRC-5 normal lung epi-
however, the study of daphnanes is hampered by their scarce thelial cells5. By initially targeting yuanhuapin analogues, we sought
supply and high cost (generally .$50 per milligram), often exacer- to begin to identify the structural requirements for the activities of
bated by geopolitical issues in accessing sources, and further these and related daphnane leads, their biological targets and their
limited by their synthetic inaccessibility and the paucity of modes of action. This study would thus complement and sup-
methods for their selective modification. RTX is the only DDO plement the natural DDO library by addressing structural issues
to be synthesized to date16; however, it lacks the key functionalities that cannot be investigated with the known natural products. As
(for example, C5, C12 and C18 oxygenation) needed to investigate evident from studies on taxotere, halichondrin and our own bryo-
fully the therapeutic potential of the DDOs. More significantly, statin, prostratin and octaarginine drug-delivery programmes17,
and a major point of increasing emphasis, is that these natural such information can be used to design simpler and more effective
products are neither evolved nor optimized for human use.
clinical leads. A first issue of importance in this inaugural DDO
Thus, although they represent promising leads, little (if any) study was to investigate the role of the B-ring epoxide common to
information exists on the activity of structural analogues needed the majority of members of the DDO family.
to design less complex and potentially therapeutically superior
Our retrosynthetic analysis (Fig. 1) focused on the DDO class
agents. We disclose herein a ‘gateway strategy’ designed to address rather than on a single synthetic target, with the ultimate goal of
two interconnected goals: (1) to develop a synthetic route to establishing a systematically varied library of natural and non-
enable general access to DDOs and, more importantly, their as-yet natural members. Step economy arises in this strategy through the
unexplored analogues from a common differentially protected pre- development of a single synthetic route to an advanced intermediate
cursor, and (2) to exploit this capability to investigate the structural that, on late-stage differential diversification, would provide access
basis for their activities and their therapeutic potential. As in our to the greatest number of targets in short parallel sequences. The
other function-oriented synthesis programmes (for example, highly oxygenated intermediate 5 serves this gateway function,
phorbol, bryostatin and prostratin)17,18, these studies are expected and allows for differential modification of key conserved functional-
to inform the design and synthesis of structurally simpler targets ities (orthoester, oxygens at C3, C4, C5 and C20) and retention or
with activities comparable to or better than those of the deletion of oxygens at other sites. Importantly, gateway structure
natural products.
5 would enable access to a majority of daphnanes and their as-yet
Departments of Chemistry and Chemical and Systems Biology, Stanford University, Stanford, California 94305, USA. e-mail: wenderp@stanford.edu
*
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615

NATURE CHEMISTRY DOI: 10.1038/NCHEM.1074
ARTICLES
PMP
OH
12
X
R1
12
C
O
O
O
OAc
O
O
11
11
O
11
18
H
18
H
11
O
O
H
OH
OH
1
O
OH
H
O
H
C
R2
Ph
A
O
A
OBn
O
3
A
OBn
B
HO
B
4
5
5
TBSO
O
O
O
O
O
HO
O
HO
HO
O
HO
X
O
HO
O
20
OH
OR3
OH
20
Ph
Br
OTBS
O
Major subset of DDOs
(X=H, 73 congeners)
PMP
1
2
3
X=α-epoxide
X=β-epoxide
X=olefin
4
5
6
General precursor
O
O
O
O
O
O
OMe
OMe
O
O
O
11
11
O
11
OK
C
O
OBn
+
O
O
O
11
OBn
OBn
OH
O
B O
Ph
TBSO
O
OH
HO
O
Br
OTBS
OTBS
10
11
9
8
7
Figure 1 | Retrosynthetic analysis for a major subset of natural and non-natural DDOs. Introduction of the orthoester and functionalization of C3, C12, C18
and C20 from the general precursor 5 was designed to allow divergent access to a broad family of natural and non-natural DDOs. Assembly of the A-ring of
this general precursor was envisioned to arise from cyclization of appropriately functionalized enyne 7. An intramolecular oxidopyrylium cycloaddition was
planned to provide the BC core from the alkylated kojic acid derivative 9, which would be accessed from the readily available potassium salt of kojic acid (10)
and tartrate derivative 11. TBS ¼ t-butyldimethylsilyl; PMP ¼ p-methoxyphenyl.
unexplored analogues. Access to 5 was expected to draw on a palla-
With the ether-bridged oxidopyrylium cycloadduct 8 confor-
dium-catalysed cyclization of 7, whose allyl and alkynyl appendages mationally constrained and facially biased, the approach of incoming
would be introduced stereoselectively by exploiting the confor- reagents in subsequent steps was expected to and, indeed, did occur
mationally fixed and facially biased oxabicyclic (BC) core of 8. exclusively from the a-face. Thus, a-allyl addition to the C10
This BC core would be assembled convergently from commercially ketone in 8 gave the C10 b-alcohol, which on treatment with
available kojic acid and a tartrate-derived bromide via a novel thionyl bromide underwent syn 1,3-transposition to give the C5
Claisen rearrangement of 9 followed by a diastereoselective oxido- b-bromide 16. Treatment of this silylenol ether with tetrabutyl-
pyrylium [5 þ 2] cycloaddition. The absolute stereochemistry of 9 ammonium fluoride (TBAF) resulted in exclusive a-protonation at
would ultimately derive from the D-dimethyltartrate derivative 11.
C10 to provide the corresponding crystalline bromoketone (see
Supplementary Information for X-ray crystallography). Addition of
phenylacetylide to this ketone proceeded in excellent yield and with
Results
Our synthesis began with the conversion of 11 into the correspond- a-face selectivity to provide the tricyclic core precursor 7.
ing C2-symmetric diol 12 (ref. 21) (Fig. 2). Subsequent desymmetri- Subsequent palladium-catalysed cyclization29 was best achieved with
zation through monobenzyl ether formation afforded the secondary formic acid30 and polymethylhydrosiloxane (PMHS) to reveal
allylic alcohol 13, from which the primary allylic bromide 14 was the complete daphnane tricyclic core (6) in only 11 steps from
obtained readily. This fragment, which contained eight carbons of tartrate. Rhodium-catalysed cyclization31 provided the tricyclic core
the target, was then joined convergently to the six-carbon fragment with the opposite selectivity for the C2 methyl epimer (see
of kojic acid by alkylation of potassium kojate. On heating neat, the Supplementary Information for details). The step economical route
O-linked product 9 was converted, after bis-silyl ether formation, to the complete ABC daphnane-tricycle 6 represents a significant
into the C-linked Claisen product 15 in good isolated yield and advancement relative to the 20 synthetic operations employed to
with excellent diastereoselectivity. This is a relatively underexplored access an analogous intermediate in the synthesis of RTX16.
type of Claisen rearrangement in which stereoinduction is con- Significantly, 6 is oxidized more highly (C5, C12 and C18) than
trolled by a stereocentre external to rather than within the six- the corresponding RTX intermediate, which is vital for subsequent
centred transition state. We propose that the preferred transition functionalization towards the broader family of DDOs and
state of the Claisen rearrangement reported herein can be explained functional analogues.
with the ‘inside alkoxy’ effect, already described for Diels–Alder and
Global ozonolysis of the tricyclic core 6 then afforded the corre-
1,3-dipolar cycloadditions22. Heating the Claisen product 15 (micro- sponding crystalline ketoaldehyde (see Supplementary Information
wave) effected silyl migration to form the oxidopyrylium intermedi- for X-ray crystallography), which was reduced using Luche con-
ate23–27, which underwent cycloaddition to give the BC core 8. ditions. Zinc-mediated reductive fragmentation of the bridging
Conventional heating also provided the desired product, but extended ether was followed by vanadium-catalysed epoxidation of the
reaction times were required. Additionally, a one-flask procedure C5,C6 alkene, which proceeded with concomitant opening of
to effect the Claisen rearrangement/oxidopyrylium [5 þ 2] cyclo- the epoxide by the C9 alcohol to provide overall substitution of
addition was developed, but the two-flask procedure was adopted the C5 bromide by oxygen with retention of C5 stereochemistry.
for material throughput considerations (see Supplementary A number of allylic oxidations were attempted to install the
Information for details). As a result of the conformational preferences desired oxygenation without reformation of the bridging ether,
of the tether substituents, only one diastereomer of the BC core was but it was found that this functionality provided facial selectivity
expected and observed28. Significantly, only six linear steps are during installation of the isopropenyl unit (see below).
required to produce 8 from commercially available starting materials. Differential protection of the oxygens in 17 provided 18, which
616
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1074
ARTICLES
O
O
O
Br
O
OTBS
OMe
OMe
O
OH
11
OR
11
OBn
d
a
c
e
11
OBn
OBn
11
O
O
O
11
O
OH
OTBS
O
O
O
O
O
O
O
O
12 R=H
11
14
9
15
b
13 R=Bn
f
O
O
O–
10
O
11
7
O
O
O
11
O
10
11
11
TBSO
O
g, h
O
i, j
8
10
5
8
+ O
B
TBSO
OBn
OBn
O
OBn
OBn
7
O
O
Ph
TBSO
HO
OTBS
OTBS
Br
Br
OTBS
OTBS
7
16
8
k
PMP
PMP
O
O
H
O
C
O
O
OH
H
O
11
O
11
O
OH
11
18
H
O
11
9
OH
OBn
13
H
C
p–s
l–o
t, u
A
A
OBn
OBn
OBn
3
O
4
O
O
5
5
5
O
O
O
O
O
O
HO
Ph
HO
HO
HO
O
O
Br
20
O
OTBS
OTBS
O
PMP
PMP
6
17
18
5
Figure 2 | Synthesis of the complete daphnane skeleton (5). Conditions: (a) diisobutylaluminium hydride, toluene, –78 8C, then divinylzinc, –78 8C to room
temperature (r.t.) (6:1 ratio of diastereomers), 80%; (b) NaH, BnBr, dimethylformamide (DMF), 0 8C to r.t., 86%; (c) PPh3, CBr4, CH2Cl2, 0 8C to r.t., 86%;
(d) 10, i-PrOH, 80 8C, 78%; (e) neat, 122 8C, then TBS-Cl, imidazole, CH2Cl2, r.t. (10:1 ratio of diastereomers), 74%; (f) 1,2-dichlorobenzene, 250 8C,
microwave, 91%; (g) Mg, I2, allyl bromide, Et2O, 0 8C, 93%; (h) SOBr2, pyridine, Et2O, –40 8C, 82%; (i) TBAF, AcOH, tetrahydrofuran (THF), 0 8C, 84%;
†
†
(j) PhCCH, MeLi LiBr, THF, –78 8C to r.t., 92%; (k) tris(dibenzylideneacetone)dipalladium CHCl3 (1 mol%), PMHS (10 equiv.), HCOOH (3 equiv.), PhCH3, r.t.,
†
80%; (l) O3, CH2Cl2/MeOH, –78 8C, then thiourea, 89%; (m) NaBH4, CeCl3 7H2O, CH2Cl2/MeOH, –78 8C to r.t., 79%; (n) Zn, NH4Cl, EtOH, 55 8C, 89%;
(o) vanadyl acetylacetonate, t-BuOOH, CH2Cl2, r.t., 84%; (p) TBS-Cl, imidazole, CH2Cl2, r.t.; (q) triphosgene, pyridine, CH2Cl2, r.t., 86% (two steps);
(r) TBAF, THF, 0 8C to r.t.; (s) (PMP)CH(OMe)2, TsOH, CH2Cl2, r.t., 93% (two steps); (t) Dess–Martin periodinane, NaHCO3, CH2Cl2, r.t.; (u) isopropenyl
lithium, CeCl3, THF, –78 8C, 49% (two steps).
on oxidation at C13 and treatment of the resulting ketone with iso- orthoester formation initiated by the C14 ester. Gratifyingly, heating
propenyl lithium in the presence of cerium trichloride gave the bis-benzoate 22 to 200 8C (microwave) in the presence of tosylic acid
desired b-adduct 5 in 49% over two steps. This gateway intermedi- (TsOH) produced the orthoester, and subsequent acetylation
ate, available in 21 steps, incorporates the complete daphnane skel- provided 23 in 44% yield (two steps). Removal of the C3 silyl
eton with a fully differentiated and rich array of functionality.
ether using tris(dimethylamino)sulfonium difluorotrimethylsilicate
Although a C11 oxymethyl substituent is found in some daph- (TASF)32 (use of TBAF led to significant cleavage of the primary
nanes and will be exploited in future diversification studies, this benzoate) was followed by oxidation of the resulting alcohol to
inaugural study focused on accessing C11 methyl targets. the ketone33, thus completing the A-ring. Methanolysis of the C20
Accordingly, polycycle 5 was converted into pentaol 19, whose benzoate followed by hydrolysis of the acetonide34 provided des-
primary hydroxyl groups were replaced simultaneously with epoxy-yuanhuapin (3). Model studies of the epoxidation using
iodides (Fig. 3). The cyclic carbonate (step (d)) was essential, and phorbol-12,13-dibutyrate demonstrated that a reagent-controlled
failure to protect the C13 alcohol resulted in C13,C18 ether for- epoxidation35 can overcome the inherent facial bias of the seven-
mation during triflation. Subsequent zinc-mediated reduction dif- membered B-ring of this substrate36 and deliver principally the
ferentially converted the C11 iodomethyl to the desired methyl a-epoxide. However, because direct epoxidation of 3 provided the
group and cleaved the bridging B-ring ether to afford exo-alkene b-epoxide of yuanhuapin (2), optimization of this process was
20. Introduction of the B-ring allylic acetate to the C4,C5 acetonide deferred to conserve material for critical assays and because a small
intermediate derived from 20 was initiated by allylic bromination, sample of authentic yuanhuapin was in hand for assay comparisons.
which chemoselectively converted the exo-alkene with transposition Evidence suggests that the C5 alcohol and/or the oxidation of the
into an allylic bromide without interference from the carbonate- A-ring play important roles in determining the stereochemical
deactivated isopropenyl group. The resultant primary allylic outcome of the epoxidation of des-epoxy-yuanhuapin.
bromide was then displaced with acetate to give alkene 21.
The synthetic availability of 2 and 3 made possible for the first
Removal of both the acetate and carbonate groups was followed time the start of a comparative analysis of factors that contribute
by esterification of the C14 and C20 alcohols, which served to to daphnane activity, including the unexplored role of the ubiqui-
protect the C20 alcohol and set the stage for another key challenge, tous C6,C7 a-epoxide, and identification of their biological
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617

NATURE CHEMISTRY DOI: 10.1038/NCHEM.1074
ARTICLES
PMP
OH
OH
H
OH
C
O
OH
O
11
O
11
11
O
18
H
H
O
O
H
O
C
OH
C
O
A
O
O
A
O
O
a–e
f–h
i–k
A
O
OBn
OH
3
OH
B
4
4
7
TBSO
5
5
TBSO
O
HO
O
O
TBSO
HO
6
O
O
HO
HO
O
20
OAc
20
20
OH
20
O
PMP
5
19
20
21
l,m
OAc
O
OH
OAc
O
OAc
O
11
11
12
14
H
H
O
OH
H
O
H
O
Ph
Ph
Ph
A
t
p–s
A
O
n,o
OBz
A
A
O
O
OH
3
B
B
B
TBSO
TBSO
O
O
O
O
O
O
HO
HO
HO
O
HO
OBz
OBz
20
20
OH
OH
2
3
23
22
Figure 3 | Completion of des-epoxy- and C6,C7-epi-yuanhuapin. Conditions: (a) K2CO3, MeOH/H2O, 50 8C, 76%; (b) TBS-trifluoromethanesulfonate,
2,6-lutidine, CH2Cl2, 0 8C, 97%; (c) lithium naphthalenide, THF, –30 8C, 88%; (d) triphosgene, pyridine, CH2Cl2, r.t., 94%; (e) 4:1:1 AcOH:THF:H2O, r.t.,
82%; (f) trifluoromethanesulfonic anhydride, 2,6-lutidine, CH2Cl2, –78 8C; (g) tetrabutylammonium iodide, CH3CN, 65 8C, 93% (two steps); (h) Zn, NH4Cl,
EtOH, 160 8C, microwave, 77%; (i) 2,2-dimethoxypropane, pyridinium p-toluenesulfonate, CH2Cl2, 40 8C, 86%; (j) N-bromosuccinimide, (BzO)2, NaHCO3,
CCl4/PhH, 70 8C; (k) KOAc, 18-crown-6, CH3CN, r.t., 52% (two steps, 39% recovered starting material (RSM)); (l) lithium aluminium hydride, THF, 0 8C to
r.t., 75%; (m) BzCl, 4-dimethylaminopyridine (DMAP), Et3N, CH2Cl2, r.t., 90%; (n) TsOH, MeOH, DMF, 200 8C, microwave, 49% (11% RSM); (o) Ac2O,
DMAP, CH2Cl2, r.t., 90%; (p) TAS-F, DMF, r.t., 89%; (q) 2-iodoxybenzoic acid, DMSO/PhCH3, 70 8C, 97%; (r) K2CO3, MeOH, r.t.; (s) LiBF4, H2O/CH3CN,
70 8C, 29% (two steps); (t) VO(O-i-Pr)3, t-BuOOH, (1S,2S)-N,N′-dihydroxy-N,N-bis(3,3,3-triphenylpropionyl)-1,2-cyclohexadiamine, CH2Cl2/PhH, 4 8C, 81%.
targets. Although yuanhuapin was reported originally to be a DNA essentially inactive. Consistent with the role of PKC in these cellular
topoisomerase I inhibitor37, several DDOs are known to activate assays, coadministration of 1 or 3 with Go¨6983 (ref. 42), a broad-
protein kinase C (PKC), a family of serine/threonine kinases spectrum PKC inhibitor, abrogated growth inhibition in both
involved in myriad biological processes through its role in signal A549 and K562 cells.
transduction. We therefore tested the systematically varied triad
1–3 in a cell-free PKC binding assay. Significantly, we found that
Discussion
yuanhuapin is a highly potent (subnanomolar) ligand for PKC A gateway strategy was developed to access daphnane-inspired
(Table 1), a target that has implications for treating diseases includ- targets, which in turn led to the identification of a cellular mediator
ing cancer38, Alzheimer’s39 and human immunodeficiency virus of their activity. For this inaugural study, the previously unexplored
AIDS40,41. In particular, 3 exhibits single-digit nanomolar affinity yuanhuapin analogues 2 and 3 were selected for evaluation as they
to PKC. In striking contrast, C6,C7-epi-yuanhuapin (2) displays make possible a comparative systematic analysis of the role of a com-
nearly three orders of magnitude lower affinity than that of the monly encountered B-ring epoxide in DDO activity, including
a-epoxide. Additionally, the potency trends observed in this cell- whether the epoxide is required or could be replaced with a less
free assay for the three compounds were mirrored in cellular complex and more stable alkene. The synthesis employed a novel
assays (Table 1). Yuanhuapin (1) was reported to have growth-inhi- Claisen rearrangement to transfer tartrate-derived chirality to a
bition activity in A549 cells (human lung carcinoma)20; therefore, an pro-C11 centre, which in turn controlled stereoselectivity in one
evaluation of the triad of targets in this cell line was conducted. In of the most complex [5 þ 2] oxidopyrylium cycloadditions
addition, all three orthoester-containing compounds were assayed studied to date. The facial bias of the resulting oxygen-bridged
for activity in K562 (human chronic myelogenous leukaemia) B-ring cycloadduct was then used to control the introduction of
cells based on the reported activities of three naturally occurring C10 and C4 stereochemistry. Further elaboration, including con-
DDOs in this cell line (gnidimacrin, huratoxin and mezerein were struction of the A-ring and completion of the carbon skeleton, pro-
screened by the Developmental Therapeutics Program National vided 5. This advanced intermediate can be used as a starting point
Cancer Institute/National Institutes of Health). Significantly, des- for step economical access to a range of targets required for under-
epoxy-yuanhuapin (3) and yuanhuapin (1) inhibited cell prolifer- standing the structural basis for daphnane activity, which in turn
ation in both cell lines, although the unnatural b-epoxide (2) was would allow for the design of simpler, but potentially more active,
agents. Congeners 1–3 were identified as ligands for PKC, with 1
and 3 exhibiting subnanomolarand single-digit nanomolaraffinities,
respectively, a new finding that tracks with cell growth inhibition
Table 1 | Biological evaluation of 1, 2 and 3.
PKC affinity, Ki (nM)*
Cellular growth inhibition†
activity and is pertinent to ongoing synthetic and mode-of-action
studies. Furthermore, identification of the profound effect that
B-ring functionality has in determining PKC-binding affinities
across this series is consistent with a model in which oxygens
A549 EC50 (nM)
K562 EC50 (nM)
1‡
2
0.48+0.07
343+6
1.6+0.1
150+30
.10,000
1500+60
7+1
.10,000
87+5
present along the southern edge make contact with PKC43,44
.
3
Installation of the b-epoxide, as in 2, significantly perturbs the
location of the hydroxymethyl relative to 1, whereas replacement
of the epoxide with an alkene, as in 3, preserves the spatial arrange-
ment of oxygens present in the natural product (global minimum
*Ki values determined in duplicate experiments. †half-maximum effective concentration (EC50
)
values determined in triplicate experiments. All errors shown are standard errors of the mean. ‡A
sample of yuanhuapin 1 was provided by J-M. Yue, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai, China.
618
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1074
ARTICLES
´
25. Domingo, L. R. & Zaragoza, R. J. Toward an understanding of the mechanisms
of the intramolecular [5 þ 2] cycloaddition reaction of g-pyrones bearing
tethered alkenes. A theoretical study. J. Org. Chem. 65, 5480–5486 (2000).
conformations of 1–3 were determined with MM3* Monte Carlo
conformation searches). This finding demonstrates how the
gateway strategy disclosed herein enables elucidation of the struc-
tural requirements of DDOs for PKC activation and begins a sys-
tematic approach to simplified and improved agents.
´
26. Zaragoza, R. J., Aurell, M. J. & Domingo, L. R. The role of the transfer group in
the intramolecular [5 þ 2] cycloadditions of substituted b-hydroxy-g-pyrones: a
DFT analysis. J. Phys. Org. Chem. 18, 610–615 (2005).
27. Singh, V., Krishna, U. M., Vikrant, V. & Trivedi, G. K. Cycloaddition of
oxidopyrylium species in organic synthesis. Tetrahedron 64, 3405–3428 (2008).
28. Wender, P. A. et al. Studies on oxidopyrylium [5 þ 2] cycloadditions: toward a
general synthetic route to the C12-hydroxy daphnetoxins. Org. Lett. 8,
5373–5376 (2006).
Methods
Full experimental details for the synthesis of all new compounds, including
procedures, spectral data and characterization, are given in the Supplementary
Information. Protocols for the cell-free competitive binding assay and growth
inhibition experiments are also given in the Supplementary Information.
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Received 1 February 2011; accepted 18 May 2011;
published online 19 June 2011
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Acknowledgements
This research was supported by the National Institutes of Health (CA31841). Additional
funding was provided by the Alexander von Humboldt Foundation (N.B.), Stanford
Graduate Fellowships from the Office of the Vice Provost for Graduate Education (N.B.C.)
and the Office of Technology Licensing (K.E.L.), Bristol-Myers Squibb Graduate Fellowship
in Organic Chemistry (J.A.K.), Amgen Graduate Fellowship (C.K.) and Eli Lilly Graduate
Research Fellowships (N.B.C., J.A.K., J.M.K.). J-M. Yue is thanked for supplying a sample of
yuanhuapin for biological evaluation. P.L. Boudreault performed epoxidation studies on
phorbol-12,13-dibutyrate. L. Cegelski is acknowledged for providing access to tissue culture
space and equipment. K. Cimprich contributed A549 and K562 cells. X-ray crystallography
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Author contributions
P.A.W., N.B., N.B.C., L.R.J., C.K., J.M.K., J.A.K. and K.E.L. conceived and designed the
experiments. N.B., N.B.C., L.R.J., C.K., J.M.K., J.A.K. and K.E.L. performed the experiments
and analysed the data. P.A.W., N.B.C. and K.E.L. co-wrote the paper. All authors
commented on the manuscript.
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Additional information
The authors declare no competing financial interests. Supplementary information and
chemical compound information accompany this paper at www.nature.com/
naturechemistry. Reprints and permission information is available online at http://www.
nature.com/reprints/. Correspondence and requests for materials shouldbe addressed to P.A.W.
˜
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