Total synthesis and cytoprotective properties of dykellic acid

Article abstract of DOI:10.1021/jm801169s

Small molecule inhibitors of apoptosis hold considerable promise for the treatment of a host of diseases, including neurodegeneration, myocardial infarction, and stroke. Many compounds that delay or prevent apoptotic death either reduce the amount of cellular reactive oxygen species (ROS) or are direct inhibitors of caspases. With the goal of using small molecules to identify novel antiapoptotic targets, we have investigated the cytoprotective activity of the natural product dykellic acid. Described herein is the first total synthesis of dykellic acid, the synthesis of several dykellic acid derivatives, and the evaluation of these compounds in assays related to cell death. We have found that dykellic acid protects cells from death as induced by etoposide and rotenone. Further experiments strongly suggest that dykellic acid does not scavenge ROS or directly inhibit caspase enzymes, and analysis of synthetic derivatives establishes key functional groups of the molecule that are essential for its cytoprotective activity.

Full text of DOI:10.1021/jm801169s

J. Med. Chem. 2009, 52, 117–125
117
Total Synthesis and Cytoprotective Properties of Dykellic Acid
Christina M. Thompson, Catherine A. Quinn, and Paul J. Hergenrother*
Department of Chemistry, Roger Adams Laboratory, UniVersity of Illinois, Urbana, Illinois 61801
ReceiVed September 17, 2008
Small molecule inhibitors of apoptosis hold considerable promise for the treatment of a host of diseases,
including neurodegeneration, myocardial infarction, and stroke. Many compounds that delay or prevent
apoptotic death either reduce the amount of cellular reactive oxygen species (ROS) or are direct inhibitors
of caspases. With the goal of using small molecules to identify novel antiapoptotic targets, we have
investigated the cytoprotective activity of the natural product dykellic acid. Described herein is the first
total synthesis of dykellic acid, the synthesis of several dykellic acid derivatives, and the evaluation of
these compounds in assays related to cell death. We have found that dykellic acid protects cells from death
as induced by etoposide and rotenone. Further experiments strongly suggest that dykellic acid does not
scavenge ROS or directly inhibit caspase enzymes, and analysis of synthetic derivatives establishes key
functional groups of the molecule that are essential for its cytoprotective activity.
Introduction
of the antioxidant vitamin E have no increase in life span.14 In
addition, the recent failure of free radical scavenger disufenton
sodium (NXY-059) in large clinical trials for treatment of stroke
has led to a rethinking of the manner in which neuroprotection
clinical trials are performed,15 although certainly some antioxi-
dants may still hold promise as therapeutics.16 Multiple potent
caspase inhibitors are available,17 and both caspase-1 inhibitors
and pan-caspase inhibitors are being evaluated in clinical trials
for arthritis, liver disease, and protection of organs during
transplant.18 However, there is some concern that inhibition of
caspases may be too late in the apoptotic pathway to prevent
cellular demise.19 In this school of thought, the “point of no
return” for apoptotic cell death is believed to be much earlier
than caspase activation, likely depolarization of the mitochon-
drial membrane and the concomitant release of proapoptotic
proteins.20 By this logic, for an antiapoptotic agent to have
maximal therapeutic benefit it would need to work upstream of
caspase activation.
As a means to discover novel antiapoptotic targets, we sought
to identify cytoprotective agents that operate through mecha-
nisms not based on the scavenging of ROS or the direct
inhibition of caspases. In this regard, dykellic acid (1, Scheme
1), a product of the fermentation broth of soil fungus Wester-
dykella multispora, attracted our attention because of its
compact, densely functionalized structure and its reported ability
to inhibit etoposide-induced apoptosis in HL-60 (human leu-
kemia) cells.21 Other interesting biological properties of dykellic
acid have been described, including its ability to inhibit cell
migration22 and interfere with NF-κB transcriptional activity.23
Dykellic acid has also been reported to decrease the overall
caspase-3-like activity in cells treated with camptothecin,24
though on the basis of its structure, it seems unlikely that
dykellic acid is a direct caspase inhibitor. We thus set out to
synthesize dykellic acid, assess its protective effects in several
cell culture models of apoptosis, and conduct experiments
designed to clarify its mechanism of action.
Apoptosis is an energy-dependent pathway used by higher
eukaryotes to selectively remove damaged or unwanted cells.
The misregulation of this pathway has negative consequences
for the organism, as abnormally low levels of apoptosis allow
cells to proliferate unchecked and elevated levels of apoptosis
can damage healthy tissue. As such, compounds that modulate
apoptosis have great potential as therapeutics.1 Specifically,
inducers of apoptosis can be powerful anticancer agents,2 and
compounds that inhibit apoptosis could be useful in the treatment
of neurodegeneration,3 sepsis,4 osteoarthritis,5 myocardial inf-
arction,6 ischemic injury/stroke,7 and many other disease states
where cytoprotection is sought.
Apoptosis is classically believed to be initiated through one
of two major pathways: (1) the intrinsic pathway that involves
the mitochondria; (2) the extrinsic pathway in which apoptosis
is initiated through death receptors on the cell surface. The
intrinsic pathway can be stimulated by DNA damaging agents
or other insults that generate reactive oxygen species (ROSa),8
while death through the extrinsic pathway is initiated by a host
of death receptor ligands including FasL and TNFR.9 There is
considerable cross-talk between these pathways; thus, most
proapoptotic signals ultimately lead to the release of apoptogenic
factors from the mitochondria.10 Both the intrinsic and extrinsic
pathways funnel to the activation of caspases, cysteine proteases
that cleave a multitude of cellular substrates and execute the
cell death program.11
Targeted approaches to the inhibition of apoptosis that have
focused on the scavenging of ROS or the inhibition of caspases
have revealed that these strategies can effectively halt apoptosis
in certain cell culture models.12,13 Unfortunately, large retro-
spective studies have shown that individuals taking high doses
* To whom correspondence should be addressed. Phone: 217-333-0363.
Fax: 217-244-8024. E-mail: hergenro@uiuc.edu.
a Abbreviations: ROS, reactive oxygen species; HWE, Horner-Wads-
worth-Emmons; MBH, Morita-Baylis-Hillman; MNNG, N-methyl-N′-
nitro-N-nitrosoguanidine; TNFR, tumor necrosis factor R; PARP-1, poly-
(ADP-ribose)polymerase-1; Z-VAD-fmk, Cbz-VAD-fluoromethyl ketone;
pNA, p-nitroaninline; H2DCF, 2,7-dichlorodihydrofluorescein; DHE, dihy-
droethidium; DCF, 2,7-dichlorofluorescein; TACE, tumor necrosis factor
R converting enzyme; MMP, matrix metalloproteinases; SDM, standard
deviation from the mean.
Described herein is the first total synthesis of dykellic acid,
the synthesis of several dykellic acid derivatives, and the
biological evaluation of these compounds. Our results show that
dykellic acid strongly protects cells from apoptosis as induced
by two distinct proapoptotic stimuli, etoposide and rotenone.
On the basis of the evaluation of dykellic acid derivatives, it
10.1021/jm801169s CCC: $40.75
2009 American Chemical Society
Published on Web 12/10/2008

118 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Scheme 1. Retrosynthetic Analysis of Dykellic Acid
Thompson et al.
70% yield; this reaction has been performed on as much as a
72 g (0.75 mol) scale. Longer reaction times did not significantly
improve the yield, while temperatures above 0 °C led to
increased amount of side products. To the best of our knowledge
2,4-hexadienal has not previously been used in the MBH
reaction. Commercial 2,4-hexadienal is provided as a mixture
of trans,trans and trans,cis isomers (about 85:15, respectively),
leading to a corresponding mixture of isomers in the product.
As shown in Scheme 3, independent synthesis of isomerically
pure trans,trans-2,4-hexadienal (4a) from trans,trans-2,4-hexa-
dienol32 followed by the MBH reaction with methyl acrylate
also provided alcohol 7 as an isomeric mixture, presumably
because of unproductive 1,6 addition of DABCO to the
aldehyde.
Alcohol 7 was converted to its triethylsilyl ether 8 in 95%
yield before being subjected to reduction by DIBAL-H, which
provided alcohol 9 in 80% yield (Scheme 2). At this time the
trans,cis isomer of 9 was removed by chromatography with 25%
AgNO3-impregnated silica gel.33 Loading 1 g of alcohol 9 onto
100 g of AgNO3-impregnated silica gel yielded 35% pure isomer
9a and 50% of isomerically impure alcohol 9, which could be
purified again through this procedure. Repeating this process
several times afforded an overall yield of 55% of 9a. After this
separation, isomerically pure 9a was oxidized using Dess-Martin
periodinane34 to provide aldehyde 10 in 79% yield (Scheme
2).
In preparation for the key olefination, phosphonate 3 was
synthesized as shown in Scheme 4. Commercially available
1-bromo-3-propanol was protected as its tert-butyldimethylsilyl
ether, which was then subjected to Finkelstein’s conditions to
exchange the bromide for an iodide. Iodide 11 was subsequently
used to alkylate hexafluorodiethyl phosphonate 6, providing
target phosphonate 3 in 86% yield.
appears that the diene side chain and the carboxylic acid moiety
are critical to the cytoprotective effect of dykellic acid. Further,
dykellic acid does not inhibit caspases in vitro nor does it
scavenge ROS in cells. These data imply that dykellic acid exerts
its antiapoptotic effect through an alternative mechanism, one
that allows it to protect cells from diverse proapoptotic stimuli.
Results and Discussion
With both main subunits of dykellic acid in hand, attention
was turned to the critical HWE reaction. In an effort to obtain
the desired Z-olefin, Still’s modification of the HWE reaction
was utilized. Initially, aldehyde 2 (generated from an acid-
catalyzed deprotection of 10) was used as the substrate in the
HWE reaction with phosphonate 3 (Scheme 5, eq 1). It was
envisioned that olefination would be followed by rapid intramo-
lecular cyclization to provide lactone 12. Unfortunately, under
a variety of reaction conditions (bases, solvents, additives,
temperatures), the reaction between 2 and 3 produced only very
low (<15%) yields of the desired product (Scheme 5, eq 1).
However, utilization of the TES-protected substrate 10 in this
reaction provided the trisubstituted olefin 13 in 58% yield with
a Z/E ratio (around the new double bond) of 10:1 (Scheme 5,
eq 2).
With the final carbon-carbon bond in place, the two silylether
protecting groups of 13 were removed using an AcOH/H2O
mixture, which also catalyzed the spontaneous cyclization to
the desired lactone 14 (Scheme 6). Lactone 14 was found to be
unstable and was thus immediately oxidized to aldehyde 15
using Dess-Martin periodinane. Treatment of aldehyde 15 with
sodium chlorite35 furnished dykellic acid (1) in a 60% yield.
The spectroscopic data for this synthetic dykellic acid match
the data of the isolated natural product21 in all respects (see
Supporting Information).
Retrosynthetic Analysis. A concise and flexible route to
dykellic acid was desired to allow synthesis of both the parent
natural product and a series of analogues. In the retrosynthetic
analysis (Scheme 1), two key carbon-carbon bond forming
reactions were envisioned: a Horner-Wadsworth-Emmons
(HWE) reaction to unite aldehyde 2 with phosphonate 3 and a
Morita-Baylis-Hillman (MBH) reaction between 2,4-hexadi-
enal (4) and methyl acrylate (5). The Still modification of the
HWE olefination was chosen because of the reliable Z-selectivity
it offers in the formation of the desired trisubstituted double
bond,25 while the MBH reaction is a facile method for the
formation of allylic alcohols;26,27 as applied in Scheme 1, this
reaction would allow for the assembly of a large fragment of
dykellic acid in a single step. As dykellic acid was isolated from
Westerdykella multispora as the racemate,21 no attempt was
made to synthesize the compound as a single enantiomer.
Total Synthesis of Dykellic Acid. The MBH reaction creates
new carbon-carbon bonds between R,ꢀ-unsaturated esters,28
amides,29 and nitriles,30 and appropriate aldehyde electrophiles.
This method is most suited to alkylaldehydes and arylaldehydes,
though there is some precedent for R,ꢀ-unsaturated aldehydes.31
When R,ꢀ-unsaturated aldehydes are utilized in the MBH
reaction, longer reaction times and lower yields (relative to their
saturated counterparts) are typically observed.27 As the first step
toward the synthesis of dykellic acid, 2,4-hexadienal (4) and
methyl acrylate (5) were used as partners for the MBH reaction
(Scheme 2). After a variety of temperatures, times, and tertiary
amines were screened, it was determined that the reaction
proceeded best at 0 °C for 3 days in the presence of 1,4-
diazabiclyclo[2.2.2]octane (DABCO), generating alcohol 7 in
Synthesis of Dykellic Acid Derivatives. With a synthetic
route to dykellic acid in place, derivatives in which various
functional groups were systematically deleted/altered were
synthesized. These derivatives (shown in Figure 1) were
designed to probe the relationship of compound structure to
biological activity. Specifically, the importance of the diene side

Dykellic Acid
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 119
Scheme 2. Synthesis of Aldehyde 10
Scheme 3. Reaction of Isomerically Pure
trans,trans-2,4-Hexadienal with Methyl Acrylate under the
MBH Conditions Provides a Mixture of Product Isomers
of cell death. These models consisted of pretreatment of U-937
(human lymphoma cell line) cells with dykellic acid for 5 h,
followed by application of a cellular insult and measurement
of cell death after 24 h by propidium iodide staining and cell
flow cytometry. U-937 cells are a promonocytic cell line that
grows rapidly as a suspension (nonadherent) in tissue culture
flasks and are commonly used in cytoprotection assays.36-38
The ability of dykellic acid to protect U-937 cells from a diverse
array of cellular insults was explored through the use of rotenone
(a mitochondrial complex I inhibitor that induces apoptosis39
and is commonly used in cell culture models of Parkinson’s
disease40), etoposide (a proapoptotic topoisomerase inhibitor and
anticancer chemotherapeutic utilized as a cell culture model of
damage induced by chemotherapeutics),41 H2O2 (induces ne-
crotic cell death and used as a model of oxidative damage),42
and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) (a DNA
alkylating agent that can induce a variety of types of cell
death).43,44
Scheme 4. Synthesis of Phosphonate 3
Scheme 5. Olefination of Aldehydes 2 and 10
For these experiments, U-937 cells were incubated with a
range of dykellic acid concentrations for 5 h and then subjected
to the various toxins. After 24 h, the viability of the U-937 cells
was assessed by propidium iodide staining and cell flow
cytometry. The level of protection observed is expressed as
percent protection, calculated from the percent of cells alive
when compared to control cells treated with either vehicle alone
or toxin alone (for more details, see the Supporting Information).
As shown by the graphs in Figure 2, dykellic acid is able to
protect cells in a dose-dependent fashion from etoposide- and
rotenone-induced cell death. Interestingly, dykellic acid is only
minimally protective against MNNG-induced cell death and not
protective against H2O2-induced cell death (see Supporting
Information for graphs).
Cytoprotective Effects of Dykellic Acid Derivatives. The
various dykellic acid derivatives synthesized (Figure 1) were
evaluated for their ability to protect U-937 cells from
etoposide-induced cell death. As shown in Figure 3, com-
pound 19, the isomerically impure form of dykellic acid, is
as protective as 1. Removal of the γ,δ-unsaturation from the
diene chain (17) provides a compound that remains protective,
whereas the compound with the diene side chain fully
saturated (18) has no protective effect. All compounds in
which the carboxylic acid is modified (16 and 22), or removed
entirely (20, 21, and 23) are not protective, and in fact some
of these compounds are toxic to U-937 cells (see Supporting
Information). From the data, it appears that minimally the
R,ꢀ-unsaturation in the diene side chain and the carboxylic
acid functionality of dykellic acid are critical to its cytopro-
tective properties.
chain and the pendent acid were probed through the synthesis
and evaluation of compounds 16-23.
The carboxylic acids in 1 and 18 were converted to the
corresponding methyl esters using trimethylsilyldiazomethane
(TMSCHN2), producing derivatives 16 and 22, respectively.
Derivatives 17 and 18 were synthesized by following the same
route to 1 but with substitution of 2,4-hexadienal with hex-2-
enal or hexanal in the first step of the synthesis. Lactones 20,
21, and 23 (lacking the carboxylic acid side chain) were
produced through a HWE reaction with phosphonate 6 and the
appropriate aldehyde (see Supporting Information for full routes
and details).
Cytoprotective Effects of Dykellic Acid. To define the
generality of the cytoprotective effect of dykellic acid, this
compound was evaluated in several different cell culture models

120 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Thompson et al.
Scheme 6. Deprotection, Cyclization, and Oxidation Provides Dykellic Acid
Dykellic Acid Reduces Cellular Levels of Caspase-3/-7
Activity. Caspases are cysteine proteases that are key players
in the cascade of events leading to apoptotic death.45 Caspases
exist in the cell as low activity zymogens (procaspases) that
are activated in response to a variety of proapoptotic signals.
Caspases-8 and -9 are initiator caspases that directly catalyze
the hydrolysis of procaspase-3 and -7, converting them to
the active executioner caspases, caspases-3 and -7.46 Once
activated, these enzymes then cleave a variety of cellular
substrates. A common means to assess apoptotic death is
through the measurement of caspase-3/-7 activity in the lysate
of cells that have been treated with a proapoptotic agent.47
Thus, as an initial experiment to determine the mechanism
by which dykellic acid exerts its cytoprotective effect, U-937
cells were pretreated with a range of dykellic acid concentra-
tions for 5 h and then treated with etoposide (to a final
concentration of 1.5 µM) for 24 h. After 24 h the cells were
lysed and the amount of caspase-3/-7 activity was determined
by monitoring the cleavage of the peptidic substrate Ac-
DEVD-pNA. Active caspase enzymes catalyze the hydrolytic
release of p-nitroaniline (pNA) from this substrate, a reaction
that can conveniently be monitored at 405 nm. A control
with the pan-caspase inhibitor Cbz-VAD-fluoromethyl ketone
(Z-VAD-fmk)17 was also included in the experiment. As
shown in Figure 4, caspase activity is not detectable in the
lysates from Z-VAD-fmk treated cells, and high concentra-
tions of dykellic acid also significantly reduce the amount
of cellular caspase activity in this assay.
molecule caspase inhibitors are also known.49 The data presented
in Figure 4 indicate that dykellic acid exerts its cytoprotective
effect either by inhibiting an upstream cascade that leads to
caspase activation or through direct inhibition of caspases. To
test whether dykellic acid itself directly inhibits caspases in vitro,
full Michaelis-Menten (velocity vs [substrate]) curves were
obtained for recombinantly expressed and purified caspases-3,
-6, -7, and -8 in the presence of various concentrations of
dykellic acid (see Supporting Information for full curves). The
enzymes’ activity was assessed by monitoring the caspase-
mediated hydrolytic release of p-nitroaniline (pNA) from
tetrameric peptide substrates specific for each enzyme.50 Thus,
the Ac-DEVD-pNA substrate was used for caspase-3 and -7,
Ac-VEID-pNA was used for caspase-6, and Ac-IETD-pNA was
used for caspase-8. The data from these experiments show that
dykellic acid has no effect on the ability of these caspases to
process their substrate in vitro, even up to dykellic acid
concentrations of 100 µM. For example, we measured the kcat/
KM values for caspase-3, -6, -7, -8 as 308, 245, 80, and 38 M-1
s-1, respectively; in the presence of 100 µM dykellic acid we
obtained similar values of 292, 246, 78, and 41 M-1 s-1
,
respectively. Hence, dykellic acid is not an inhibitor of caspase-
3, -6, -7, or -8 in vitro and is unlikely to be a direct inhibitor of
any of the caspase enzymes
Effect of Dykellic Acid on Levels of Cellular ROS. General
antioxidants are often cytoprotective because of the central role
of ROS in mitochondria-mediated apoptosis.13 We therefore
sought to establish if dykellic acid acts directly as a ROS
scavenger in cells. Two different ROS sensitive dyes were
employed for this assay, 2,7-dichlorodihydrofluorescein
(H2DCF) and dihydroethidium (DHE). H2DCF is converted to
2,7-dichlorofluorescein (DCF) in the presence of intracellular
Dykellic Acid Is Not a Direct Inhibitor of Caspase-3,
-6, -7, or -8. One means by which compounds can delay cell
death is through direct inhibition of caspases. The vast majority
of caspase inhibitors are peptidic in nature,48 although small
Figure 1. Derivatives of dykellic acid.

Dykellic Acid
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 121
quench cellular ROS is also consistent with the inability of this
compound to protect against H2O2-induced cell death, as H2O2-
treatment results in the direct and rapid generation of cellular
ROS.54 Thus, the combined data suggest that dykellic acid exerts
its cytoprotective effect on cells in culture through some less
common mechanism, one not involving direct caspase inhibition
or ROS-scavenging.
The protective properties of dykellic acid were evaluated
against four different toxins: etoposide, rotenone, MNNG, and
hydrogen peroxide. Etoposide inhibits topoisomerase II, a
phosphodiesterase that catalyzes the hydrolysis of double
stranded DNA, allowing relaxation of supercoiled DNA. By
preventing topoisomerase II from re-ligating the double strand
break, etoposide turns topoisomerase II into a destructive DNA
cleaving enzyme.55 While the pathway between etoposide-
induced DNA damage and apoptotic induction is not entirely
understood, caspase-2, -3, and -8 have been shown to be
important links for stimulation of the mitochondrial apoptotic
pathway and the execution of apoptosis.56 The pesticide rotenone
is a potent inhibitor of complex I, an enzyme in the mitochon-
drial electron transport chain. This inhibition in turn causes the
production of ROS, which causes cellular stress and ultimately
leads to death, again through an apoptotic pathway in U-937
cells.39,57
The other two toxins tested, MNNG and H2O2, are funda-
mentally different from etoposide and rotenone in that in U-937
cells they cause mainly caspase independent cell death or
necrosis. MNNG is a DNA alkylating agent that causes caspase-
independent cell death through poly(ADP-ribose)polymerase-1
(PARP-1) activation.58 Upon DNA alkylation PARP-1 is
activated, inducing synthesis of the toxic poly(ADP-ribose)
biopolymer59 and causing depletion of cellular energy stores,
both of which lead to rapid death without the activation of
caspase enzymes.60 Hydrogen peroxide has been shown to
induce necrotic death in U-937 cells.61 When hydrogen peroxide
is added to cells, peroxide radicals oxidize not only DNA but
also proteins and lipids, starting a host of enzymatic cascades
that lead to cell death.35,37
Figure 2. Dykellic acid protects cells against two different types of
insult. U-937 cells were treated with dykellic acid for 5 h before (A)
etoposide was added to a final concentration of 1.5 µM or (B) rotenone
was added to a final concentration of 5 µM. Cell death was assessed
24 h later by propidium iodide staining and cell flow cytometry. Error
bars represent SDM from n ) 3, with each trial performed in triplicate.
peroxides,51 whereas DHE provides a sensitive readout on
intracellular levels of superoxide radicals through oxidation by
superoxide anion radials to form ethiduim.52 U-937 cells were
treated with dykellic acid at varying concentrations for 5 h, after
which time rotenone was added and the levels of cellular ROS
were measured. R-Tocopherol, a well established antioxidant,53
was used as a positive control. As shown in Figure 5,
R-tocopherol significantly decreases ROS levels in rotenone-
treated cells (as measured by the fluorescence of both DCF and
ethidium). However, dykellic acid had no effect in this assay,
even at concentrations of 100 µM, strongly suggesting that
dykellic acid does not alter intracellular levels of ROS.
Comparing the mechanisms of these toxins allows some
conclusions to be drawn about how dykellic acid might be
protecting cells from death. Dykellic acid is most protective
against the toxins that effect cell death through caspase-
dependent apoptosis, specifically through the mitochondria, and
is less protective against those that cause necrotic cell death/
caspase-independent cell death. On the basis of the data
presented herein, we postulate that dykellic acid inhibits
apoptosis at some point between the ROS-mediated initiation
of apoptosis and caspase activation. Importantly, by inhibiting
cell death before activation of the executioner caspases, dykellic
acid has the potential to protect cells before they have passed
the “point of no return” down the cell death pathway, although
it remains to be seen if dykellic acid acts on targets upstream
or downstream of mitochondria membrane depolarization.
Compounds that inhibit apoptosis by acting on targets before
caspase activation have intriguing potential as therapeutic agents.
Although small molecules that inhibit apoptosis without directly
affecting cellular ROS levels or caspase activity are less
common, they are not without precedent; for example, geldana-
mycin and herbimycin A inhibit death in U-937 cells through
induction of Hsp70 synthesis,36,62 and small molecule inhibitors
of poly(ADP-ribose)polymerase-1 (PARP-1) are known to be
protective in certain cell culture and animal models of neuro-
degeneration and stroke.63
Discussion
The data presented herein show that dykellic acid protects
U-937 cells from death as induced by rotenone and etoposide.
Although cells treated with dykellic acid show markedly reduced
levels of caspase-3/-7-like activity, our data indicate that dykellic
acid is not a direct inhibitor of caspase-3, -6, -7, or -8 in vitro,
suggesting that the compound acts to suppress caspase activation
in some upstream portion of the apoptotic cascade. As ROS is
a key player in apoptotic induction, we examined the effect of
dykellic acid on ROS production. Interestingly, the levels of
intracellular ROS were not altered in the presence of dykellic
acid. The fact that dykellic acid does not appear to directly

122 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Thompson et al.
Figure 3. An intact diene side chain is essential to the full cytoprotective activity of dykellic acid. Cells were treated with compound 18, 19, or
20 for 5 h before etoposide was added to a final concentration of 1.5 µM. Cell death was assessed 24 h later by propidium iodide staining and cell
flow cytometry. Error bars represent SDM from n ) 3, with each trial performed in triplicate.
Figure 4. Dykellic acid reduces caspase-3/-7-like activity in etoposide-treated cells. U-937 cells were treated with Z-VAD-fmk (100 nM) or
varying concentrations of dykellic acid for 5 h, and etoposide was then added to a final concentration of 1.5 µM. After 24 h the cells were transferred
to caspase assay buffer and lysed, and the caspase substrate Ac-DEVD-pNA was added to a final concentration of 200 µM. The amount of pNA
produced per minute was measured at 405 nm. All wells contained 1% of the vehicle, DMSO. The caspase-3/-7-like activity of cells treated with
vehicle plus etoposide was normalized to 1.0, while the activity of cells treated with just vehicle (not shown) was normalized to 0. Error bars
represent SDM from a single experiment performed in triplicate; these data are representative of three experiments. Statistical significance relative
to vehicle control is indicated: (/) P < 0.02; (//) P < 0.001.
The structure-activity relationship established through syn-
thesis and evaluation of dykellic acid derivatives provides
insights into the mode of action of this compound and suggests
opportunities for the enhancement of efficacy. Given the
importance of the R,ꢀ-unsaturation in the diene side chain to
its cytoprotective activity and the preponderance of electrophilic
sites on dykellic acid, a key outstanding question is whether
dykellic acid is covalently modifying its biological target.
However, the δ,γ-double bond of the diene side chain can be
eliminated with minimal loss of activity, suggesting that
chemical modifications could be made to this side chain in an
effort to improve biological activity or to identify the cellular
target. With a tractable and flexible route dykellic acid in place,
such studies are now feasible. Members of the gelastatin family
of natural products are structurally related to dykellic acid.
Gelastatins and some derivatives have been reported as inhibitors
of matrix metalloproteinases (MMP) and tumor necrosis factor
R converting enzyme (TACE),64 and dykellic acid itself is
known to inhibit the expression and activity of MMP-9 in cells.23
Whether or not there is a direct connection between this MMP
inhibition and dykellic acid’s cytoprotective effect, at this point,
is unclear.
In summary, we have achieved the first total synthesis of
dykellic acid through an efficient and flexible route whose
longest linear sequence is eight chemical steps from com-
mercially available starting materials. As shown herein, dykellic
acid protects U-937 cells from cellular insults that induce
caspase-dependent apoptosis but is minimally protective against
insults that induce other forms of cell death. Interestingly, this
cytoprotection is mediated through neither the scavenging of

Dykellic Acid
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 123
(6) Webster, K. A. Programmed death as a therapeutic target to reduce
myocardial infarction. Trends Pharmacol. Sci. 2007, 28, 492–499.
(7) Green, A. R.; Shuaib, A. Therapeutic strategies for the treatment of
stroke. Drug DiscoVery Today 2006, 11, 681–693.
(8) (a) Danial, N. N.; Korsmeyer, S. J. Cell death: critical control points.
Cell 2004, 116, 205–219. (b) Kuwana, T.; Newmeyer, D. D. Bcl-2-
family proteins and the role of mitochondria in apoptosis. Curr. Opin.
Cell Biol. 2003, 15, 691–699.
(9) (a) Ashkenazi, A.; Dixit, V. M. Death receptors: signaling and
modulation. Science 1998, 281, 1305–1308. (b) Falschlehner, C.;
Emmerich, C. H.; Gerlach, B.; Walczak, H. TRAIL signalling:
decisions between life and death. Int. J. Biochem. Cell Biol. 2007,
39, 1462–1475. (c) Schmitz, I.; Kirchhoff, S.; Krammer, P. H.
Regulation of death receptor-mediated apoptosis pathways. Int. J. Bio-
chem. Cell Biol. 2000, 32, 1123–1136.
(10) (a) Khosravi-Far, R.; Esposti, M. D. Death receptor signals to
mitochondria. Cancer Biol. Ther. 2004, 3, 1051–1057. (b) Kroemer,
G.; Reed, J. C. Mitochondrial control of cell death. Nat. Med. 2000,
6, 513–519.
(11) (a) Fischer, U.; Janicke, R. U.; Schulze-Osthoff, K. Many cuts to ruin:
a comprehensive update of caspase substrates. Cell Death Differ. 2003,
10, 76–100. (b) Timmer, J. C.; Salvesen, G. S. Caspase substrates.
Cell Death Differ. 2007, 14, 66–72.
(12) (a) Cai, S. X.; Guan, L.; Jia, S.; Wang, Y.; Yang, W.; Tseng, B.;
Drewe, J. Dipeptidyl aspartyl fluoromethylketones as potent caspase
inhibitors: SAR of the N-protecting group. Bioorg. Med. Chem. Lett.
2004, 14, 5295–5300. (b) Jaeschke, H.; Farhood, A.; Cai, S. X.; Tseng,
B. Y.; Bajt, M. L. Protection against TNF-induced liver parenchymal
cell apoptosis during endotoxemia by a novel caspase inhibitor in mice.
Toxicol. Appl. Pharmacol. 2000, 169, 77–83.
(13) (a) Kim, D. S.; Woo, E. R.; Chae, S. W.; Ha, K. C.; Lee, G. H.; Hong,
S. T.; Kwon, D. Y.; Kim, M. S.; Jung, Y. K.; Kim, H. M.; Kim, H. K.;
Kim, H. R.; Chae, H. J. Plantainoside D protects adriamycin-induced
apoptosis in H9c2 cardiac muscle cells via the inhibition of ROS
generation and NF-kappaB activation. Life Sci. 2007, 80, 314–323.
(b) Wang, X.; Perez, E.; Liu, R.; Yan, L. J.; Mallet, R. T.; Yang,
S. H. Pyruvate protects mitochondria from oxidative stress in human
neuroblastoma SK-N-SH cells. Brain Res. 2007, 1132, 1–9.
(14) Miller, E. R., 3rd; Pastor-Barriuso, R.; Dalal, D.; Riemersma, R. A.;
Appel, L. J.; Guallar, E. Meta-analysis: high-dosage vitamin E
supplementation may increase all-cause mortality. Ann. Intern. Med.
2005, 142, 37–46.
Figure 5. Dykellic acid does not quench intracellular reactive oxygen
species. U-937 cells were treated with R-tocopherol (100 µM) or a
range of dykellic acid concentrations for 5 h, at which point rotenone
was added to a final concentration of 20 µM. After 40 min dihydro-
ethidium or dichlorodihydrofluorescein diacetate (H2DCF) was added
and cellular fluorescence was assessed by cell flow cytometry. The 5,
10, 50, and 100 µM dykellic acid data points are not statistically
different from the vehicle plus rotenone control. Statistical significance
versus rotenone treated vehicle control is noted: (/) P < 0.016 Error
bars represent SDM from n ) 3, with each trial performed in triplicate.
cellular ROS nor the direct inhibition of caspases. The
structure-activity relationship established through the synthesis
of key dykellic acid derivatives sets the stage for the optimiza-
tion of dykellic acid’s cytoprotective activity and for studies
aimed at identifying the precise biological target of this
compound. There are a multitude of disorders caused by
premature cell death; dykellic acid’s potency and nonstandard
mechanism of cytoprotection make it an interesting candidate
for evaluation in models of these disease states.
(15) (a) Donnan, G. A. The 2007 Feinberg lecture: a new road map for
neuroprotection. Stroke 2008, 39, 242–248. (b) Savitz, S. I.; Fisher,
M. Future of neuroprotection for acute stroke: in the aftermath of the
SAINT trials. Ann. Neurol. 2007, 61, 396–402.
Acknowledgment. We thank Margaret Wong for performing
initial experiments directed toward the synthesis of 10. This
research was supported by the University of Illinois, Urbana-
Champaign, and by the Alfred P. Sloan Foundation (Sloan
Foundation Fellowship to P.J.H.).
(16) Jiang, Z. G.; Lu, X. C.; Nelson, V.; Yang, X.; Pan, W.; Chen, R. W.;
Lebowitz, M. S.; Almassian, B.; Tortella, F. C.; Brady, R. O.;
Ghanbari, H. A. A multifunctional cytoprotective agent that reduces
neurodegeneration after ischemia. Proc. Natl. Acad. Sci. U.S.A. 2006,
103, 1581–1586.
(17) Callus, B. A.; Vaux, D. L. Caspase inhibitors: viral, cellular and
chemical. Cell Death Differ. 2007, 14, 73–78.
Supporting Information Available: Materials and methods,
NMR spectra of all new compounds, and supporting figures. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(18) (a) Baskin-Bey, E. S.; Washburn, K.; Feng, S.; Oltersdorf, T.; Shapiro,
D.; Huyghe, M.; Burgart, L.; Garrity-Park, M.; van Vilsteren, F. G.;
Oliver, L. K.; Rosen, C. B.; Gores, G. J. Clinical trial of the pan-
caspase inhibitor, IDN-6556, in human liver preservation injury. Am. J.
Transplant. 2007, 7, 218–225. (b) Linton, S. D. Caspase inhibitors: a
pharmaceutical industry perspective. Curr. Top. Med. Chem. 2005, 5,
1697–1717.
References
(19) (a) Kovesdi, E.; Czeiter, E.; Tamas, A.; Reglodi, D.; Szellar, D.; Pal,
J.; Bukovics, P.; Doczi, T.; Buki, A. Rescuing neurons and glia: is
inhibition of apoptosis useful? Prog. Brain Res. 2007, 161, 81–95.
(b) Youle, R. J.; Strasser, A. The BCL-2 protein family: opposing
activities that mediate cell death. Nat. ReV. Mol. Cell Biol. 2008, 9,
47–59.
(20) (a) Chang, L. K.; Putcha, G. V.; Deshmukh, M.; Johnson, E. M., Jr.
Mitochondrial involvement in the point of no return in neuronal
apoptosis. Biochimie 2002, 84, 223–231. (b) Keeble, J. A.; Gilmore,
A. P. Apoptosis commitmentstranslating survival signals into deci-
sions on mitochondria. Cell Res. 2007, 17, 976–984. (c) Kroemer, G.
Mitochondrial control of apoptosis: an introduction. Biochem. Biophys.
Res. Commun. 2003, 304, 433–435.
(21) Lee, H.-J.; Lee, C.-H.; Chung, M.-C.; Chun, H.-K.; Rhee, J.-S.; Kho,
Y.-H. Dykellic acid, a novel apoptosis inhibitor from Westerdykella
multispora F50733. Tetrahedron Lett. 1999, 40, 6949–6950.
(22) Heo, J. C.; Park, J. Y.; Woo, S. U.; Rho, J. R.; Lee, H. J.; Kim, S. U.;
Kho, Y. H.; Lee, S. H. Dykellic acid inhibits cell migration and tube
formation by RhoA-GTP expression. Biol. Pharm. Bull. 2006, 29,
2256–2259.
(1) Alam, J. J. Apoptosis: target for novel drugs. Trends Biotechnol. 2003,
21, 479–483.
(2) (a) Makin, G.; Dive, C. Recent advances in understanding apoptosis:
new therapeutic opportunities in cancer chemotherapy. Trends Mol.
Med. 2003, 9, 251–255. (b) Meng, X. W.; Lee, S. H.; Kaufmann, S. H.
Apoptosis in the treatment of cancer: a promise kept? Curr. Opin.
Cell Biol. 2006, 18, 668–676.
(3) (a) Pattison, L. R.; Kotter, M. R.; Fraga, D.; Bonelli, R. M. Apoptotic
cascades as possible targets for inhibiting cell death in Huntington’s
disease. J. Neurol. 2006, 253, 1137–1142. (b) Vila, M.; Przedborski,
S. Targeting programmed cell death in neurodegenerative diseases.
Nat. ReV. Neurosci. 2003, 4, 365–375. (c) Waldmeier, P. C. Prospects
for antiapoptotic drug therapy of neurodegenerative diseases. Prog.
Neuropsychopharmacol. Biol. Psychiatry 2003, 27, 303–321. (d)
Waldmeier, P. C.; Tatton, W. G. Interrupting apoptosis in neurode-
generative disease: potential for effective therapy. Drug DiscoVery
Today 2004, 9, 210–218.
(4) Wesche-Soldato, D. E.; Swan, R. Z.; Chung, C. S.; Ayala, A. The
apoptotic pathway as a therapeutic target in sepsis. Curr. Drug Targets
2007, 8, 493–500.
(5) Kim, H. A.; Blanco, F. J. Cell death and apoptosis in osteoarthritic
cartilage. Curr. Drug Targets 2007, 8, 333–345.
(23) Woo, J. H.; Park, J. W.; Lee, S. H.; Kim, Y. H.; Lee, I. K.; Gabrielson,
E.; Lee, S. H.; Lee, H. J.; Kho, Y. H.; Kwon, T. K. Dykellic acid

124 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Thompson et al.
inhibits phorbol myristate acetate-induced matrix metalloproteinase-9
expression by inhibiting nuclear factor kappa B transcriptional activity.
Cancer Res. 2003, 63, 3430–3434.
M. F.; Sanchez-Reus, M. I.; Andres, D.; Cascales, M.; Benedi, J.
Neuroprotective effect of fraxetin and myricetin against rotenone-
induced apoptosis in neuroblastoma cells. Brain Res. 2004, 1009, 9–
16.
(24) Lee, S. H.; Youk, E. S.; Lee, H. J.; Kho, Y. H.; Kim, H. M.; Kim,
S. U. Dykellic acid inhibits drug-induced caspase-3-like protease
activation. Biochem. Biophys. Res. Commun. 2003, 302, 539–544.
(25) (a) Maryanoff, B. E.; Reitz, A. B. The Wittig olefination reaction and
modifications involving phosphoryl-stabilized carbanions. Stereochem-
istry, mechanism, and selected synthetic aspects. Chem. ReV. 1989,
89, 863–927. (b) Still, W. C.; Gennari, C. Direct synthesis of
Z-unsaturated esters. A useful modification of the Horner-Emmons
olefination. Tetrahedron Lett. 1983, 24, 4405–4408.
(26) Basavaiah, D.; Rao, A. J.; Satyanarayana, T. Recent advances in the
Baylis-Hillman reaction and applications. Chem. ReV. 2003, 103, 811–
892.
(27) Drewes, S. E.; Roos, G. H. P. Synthetic potential of the tertiary-amine-
catalysed reaction of activated vinyl carbanions with aldehydes.
Tetrahedron 1988, 44, 4653–4670.
(41) (a) Kapiszewska, M.; Cierniak, A.; Elas, M.; Lankoff, A. Lifespan of
etoposide-treated human neutrophils is affected by antioxidant ability
of quercetin. Toxicol. in Vitro 2007, 21, 1020–1030. (b) Rusovici, R.;
Ghaleb, A.; Shim, H.; Yang, V. W.; Yun, C. C. Lysophosphatidic
acid prevents apoptosis of Caco-2 colon cancer cells via activation of
mitogen-activated protein kinase and phosphorylation of Bad. Biochim
Biophys Acta 2007, 1770, 1194–1203. (c) Salido, M.; Gonzalez, J. L.;
Vilches, J. Loss of mitochondrial membrane potential is inhibited by
bombesin in etoposide-induced apoptosis in PC-3 prostate carcinoma
cells. Mol. Cancer Ther. 2007, 6, 1292–1299.
(42) (a) Bechoua, S.; Dubois, M.; Dominguez, Z.; Goncalves, A.; Nemoz,
G.; Lagarde, M.; Prigent, A. F. Protective effect of docosahexaenoic
acid against hydrogen peroxide-induced oxidative stress in human
lymphocytes. Biochem. Pharmacol. 1999, 57, 1021–1030. (b) Fatokun,
A. A.; Stone, T. W.; Smith, R. A. Hydrogen peroxide-induced
oxidative stress in MC3T3-E1 cells: the effects of glutamate and
protection by purines. Bone 2006, 39, 542–551.
(28) Hoffmann, H. M. R.; Rabe, J. Preparation of 2-(1-hydroxyalkyl)acrylic
esters; simple three-step synthesis of mikanecic acid. Angew. Chem.,
Int. Ed. Engl. 1983, 22, 795–796.
(43) Lee, M. W.; Kim, W. J.; Beardsley, D. I.; Brown, K. D. N-Methyl-
N′-nitro-N-nitrosoguanidine activates multiple cell death mechanisms
in human fibroblasts. DNA Cell Biol. 2007, 26, 683–694.
(44) Yu, S. W.; Wang, H.; Poitras, M. F.; Coombs, C.; Bowers, W. J.;
Federoff, H. J.; Poirier, G. G.; Dawson, T. M.; Dawson, V. L.
Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by
apoptosis-inducing factor. Science 2002, 297, 259–263.
(29) (a) Faltin, C.; Fleming, E. M.; Connon, S. J. Acrylamide in the Baylis-
Hillman reaction: expanded reaction scope and the unexpected
superiority of DABCO over more basic tertiary amine catalysts. J.
Org. Chem. 2004, 69, 6496–6499. (b) Wei, G.; Weiwei, W.; Ningjuan,
F.; Zhengang, W.; Chizhong, X. Synthesis of alpha-substituted N-aryl
acrylamide derivatives through Baylis-Hillman reaction. Synth. Com-
mun. 2005, 35, 1239–1251.
(30) Singh, V.; Yadav, G. P.; Maulik, P. R.; Batra, S. Synthesis of
substituted 3-methylene-2-pyridones from Baylis-Hillman derivatives
and its application for the generation of 2-pyridone substituted
spiroisoxazolines. Tetrahedron 2008, 64, 2979–2991.
(31) (a) Areces, P. C.; Carrasco, E.; Mancha, A.; Plumet, J. Tandem
ꢀ-elimination-Morita-Baylis-Hillman reaction in R,ꢀ-unsaturated
sugar aldehydes. Synthesis 2006, 6, 946–948. (b) Drewes, S. E.; Emslie,
N. D.; Khan, A. A.; Roos, G. H. P. 1,2-Addition of activated vinyl
carbanions to, ꢀ-unsaturated carbonyls. Synth. Commun. 1989, 19, 959–
964. (c) Heerden, F. R. v.; Huyser, J. J.; Holzapfel, C. W. Preparation
of multifunctional stereodefined dienes. Synth. Commun. 1994, 24,
2863–2872. (d) Hsu, J.-C.; Yen, Y.-H.; Chu, Y.-H. Baylis-Hillman
reaction in [bdmim][PF6] ionic liquid. Tetrahedron Lett. 2004, 45,
4673–4676.
(32) Zapf, C. W.; Harrison, B. A.; Drahl, C.; Sorensen, E. J. A Diels-
Alder macrocyclization enables an efficient asymmetric synthesis of
the antibacterial natural product abyssomicin C. Angew. Chem., Int.
Ed. 2005, 44, 6533–6537.
(33) Williams, C. M.; Mander, L. N. Chromatography with silver nitrate.
Tetrahedron 2001, 57, 425–447.
(34) Dess, D. B.; Martin, J. C. Readily accessible 12-I-5 oxidant for the
conversion of primary and secondary alcohols to aldehydes and
ketones. J. Org. Chem. 1983, 43, 4155–4156.
(35) Bal, B. S.; Childers, W. E.; Pinnick, H. W. Oxidation of [alpha],[beta]-
unsaturated aldehydes. Tetrahedron 1981, 37, 2091–2096.
(36) Demidenko, Z. N.; Vivo, C.; Halicka, H. D.; Li, C. J.; Bhalla, K.;
Broude, E. V.; Blagosklonny, M. V. Pharmacological induction of
Hsp70 protects apoptosis-prone cells from doxorubicin: comparison
with caspase-inhibitor- and cycle-arrest-mediated cytoprotection. Cell
Death Differ. 2006, 13, 1434–1441.
(37) Kanupriya; Prasad, D.; Ram, M. S.; Kumar, R.; Sawhney, R. C.;
Sharma, S. K.; Ilavazhagan, G.; Kumar, D.; Banerjee, P. K. Cytopro-
tective and antioxidant activity of Rhodiola imbricata against tert-
butyl hydroperoxide induced oxidative injury in U-937 human
macrophages. Mol. Cell. Biochem. 2005, 275, 1–6.
(38) (a) Sestili, P.; Martinelli, C.; Bravi, G.; Piccoli, G.; Curci, R.; Battistelli,
M.; Falcieri, E.; Agostini, D.; Gioacchini, A. M.; Stocchi, V. Creatine
supplementation affords cytoprotection in oxidatively injured cultured
mammalian cells via direct antioxidant activity. Free Radical Biol.
Med. 2006, 40, 837–849. (b) Young, J.; Wahle, K. W.; Boyle, S. P.
Cytoprotective effects of phenolic antioxidants and essential fatty acids
in human blood monocyte and neuroblastoma cell lines: surrogates
for neurological damage in vivo. Prostaglandins, Leukotrienes Essent.
Fatty Acids 2008, 78, 45–59.
(45) (a) Hengartner, M. O. The biochemistry of apoptosis. Nature 2000,
407, 770–776. (b) Taylor, R. C.; Cullen, S. P.; Martin, S. J. Apoptosis:
controlled demolition at the cellular level. Nat. ReV. Mol. Cell Biol.
2008, 9, 231–241.
(46) (a) Riedl, S. J.; Shi, Y. Molecular mechanisms of caspase regulation
during apoptosis. Nat. ReV. Mol. Cell Biol. 2004, 5, 897–907. (b)
Stennicke, H. R.; Jurgensmeier, J. M.; Shin, H.; Deveraux, Q.; Wolf,
B. B.; Yang, X.; Zhou, Q.; Ellerby, H. M.; Ellerby, L. M.; Bredesen,
D.; Green, D. R.; Reed, J. C.; Froelich, C. J.; Salvesen, G. S. Pro-
caspase-3 is a major physiologic target of caspase-8. J. Biol. Chem.
1998, 273, 27084–27090.
(47) (a) Benjamin, C. W.; Hiebsch, R. R.; Jones, D. A. Caspase activation
in MCF7 cells responding to etoposide treatment. Mol. Pharmacol.
1998, 53, 446–450. (b) Kim, Y. M.; Chung, H. T.; Kim, S. S.; Han,
J. A.; Yoo, Y. M.; Kim, K. M.; Lee, G. H.; Yun, H. Y.; Green, A.;
Li, J.; Simmons, R. L.; Billiar, T. R. Nitric oxide protects PC12 cells
from serum deprivation-induced apoptosis by cGMP-dependent inhibi-
tion of caspase signaling. J. Neurosci. 1999, 19, 6740–6747.
(48) (a) Goode, D. R.; Sharma, A. K.; Hergenrother, P. J. Using peptidic
inhibitors to systematically probe the S1′ site of caspase-3 and caspase-
7. Org. Lett. 2005, 7, 3529–3532. (b) Micale, N.; Vairagoundar, R.;
Yakovlev, A. G.; Kozikowski, A. P. Design and synthesis of a potent
and selective peptidomimetic inhibitor of caspase-3. J. Med. Chem.
2004, 47, 6455–6458. (c) Yoshimori, A.; Sakai, J.; Sunaga, S.;
Kobayashi, T.; Takahashi, S.; Okita, N.; Takasawa, R.; Tanuma, S.
Structural and functional definition of the specificity of a novel
caspase-3 inhibitor, Ac-DNLD-CHO. BMC Pharmacol. 2007, 7
8.
(49) (a) Choong, I. C.; Lew, W.; Lee, D.; Pham, P.; Burdett, M. T.; Lam,
J. W.; Wiesmann, C.; Luong, T. N.; Fahr, B.; DeLano, W. L.;
McDowell, R. S.; Allen, D. A.; Erlanson, D. A.; Gordon, E. M.;
O’Brien, T. Identification of potent and selective small-molecule
inhibitors of caspase-3 through the use of extended tethering and
structure-based drug design. J. Med. Chem. 2002, 45, 5005–5022. (b)
Chu, W.; Rothfuss, J.; d’Avignon, A.; Zeng, C.; Zhou, D.; Hotchkiss,
R. S.; Mach, R. H. Isatin sulfonamide analogs containing a Michael
addition acceptor: a new class of caspase 3/7 inhibitors. J. Med. Chem.
2007, 50, 3751–3755. (c) Kravchenko, D. V.; Kuzovkova, Y. A.; Kysil,
V. M.; Tkachenko, S. E.; Maliarchouk, S.; Okun, I. M.; Balakin, K. V.;
Ivachtchenko, A. V. Synthesis and structure-activity relationship of
4-substituted 2-(2-acetyloxyethyl)-8-(morpholine-4-sulfonyl)pyrrolo[3,4-
c]quinoline-1,3- diones as potent caspase-3 inhibitors. J. Med. Chem.
2005, 48, 3680–3683.
(50) (a) Smith, G. K.; Barrett, D. G.; Blackburn, K.; Cory, M.; Dallas,
W. S.; Davis, R.; Hassler, D.; McConnell, R.; Moyer, M.; Weaver,
K. Expression, preparation, and high-throughput screening of caspase-
8: discovery of redox-based and steroid diacid inhibition. Arch.
Biochem. Biophys. 2002, 399, 195–205. (b) Talanian, R. V.; Quinlan,
C.; Trautz, S.; Hackett, M. C.; Mankovich, J. A.; Banach, D.; Ghayur,
T.; Brady, K. D.; Wong, W. W. Substrate specificities of caspase
family proteases. J. Biol. Chem. 1997, 272, 9677–9682.
(51) (a) Gomes, A.; Fernandes, E.; Lima, J. L. Fluorescence probes used
for detection of reactive oxygen species. J. Biochem. Biophys. Methods
2005, 65, 45–80. (b) Li, L.; Jiang, L.; Geng, C.; Cao, J.; Zhong, L.
The role of oxidative stress in acrolein-induced DNA damage in
(39) Lee, J.; Huang, M. S.; Yang, I. C.; Lai, T. C.; Wang, J. L.; Pang,
V. F.; Hsiao, M.; Kuo, M. Y. Essential roles of caspases and their
upstream regulators in rotenone-induced apoptosis. Biochem. Biophys.
Res. Commun. 2008, 371, 33–38.
(40) (a) Egea, J.; Rosa, A. O.; Cuadrado, A.; Garcia, A. G.; Lopez, M. G.
Nicotinic receptor activation by epibatidine induces heme oxygenase-1
and protects chromaffin cells against oxidative stress. J. Neurochem.
2007, 102, 1842–1852. (b) Imamura, K.; Takeshima, T.; Kashiwaya,
Y.; Nakaso, K.; Nakashima, K. D-beta-Hydroxybutyrate protects
dopaminergic SH-SY5Y cells in a rotenone model of Parkinson’s
disease. J. Neurosci. Res. 2006, 84, 1376–1384. (c) Molina-Jimenez,

Dykellic Acid
HepG2 cells. Free Radical Res. 2008, 42, 354–361. (c) Vowells, S. J.;
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 125
de Murcia, J.; Susin, S. A. Sequential activation of poly(ADP-ribose)
polymerase 1, calpains, and Bax is essential in apoptosis-inducing
factor-mediated programmed necrosis. Mol. Cell. Biol. 2007, 27, 4844–
4862.
Sekhsaria, S.; Malech, H. L.; Shalit, M.; Fleisher, T. A. Flow
cytometric analysis of the granulocyte respiratory burst: a comparison
study of fluorescent probes. J. Immunol. Methods 1995, 178, 89
97.
(61) Sestili, P.; Martinelli, C.; Bravi, G.; Piccoli, G.; Curci, R.; Battistelli,
M.; Falcieri, E.; Agostini, D.; Gioacchini, A. M.; Stocchi, V. Creatine
supplementation affords cytoprotection in oxidatively injured cultured
mammalian cells via direct antioxidant activity. Free Radical Biol.
Med. 2006, 40, 837–849.
(62) Riordan, F. A.; Bravery, C. A.; Mengubas, K.; Ray, N.; Borthwick,
N. J.; Akbar, A. N.; Hart, S. M.; Hoffbrand, A. V.; Mehta, A. B.;
Wickremasinghe, R. G. Herbimycin A accelerates the induction of
apoptosis following etoposide treatment or gamma-irradiation of bcr/
abl-positive leukaemia cells. Oncogene 1998, 16, 1533–1542.
(63) (a) Chiarugi, A. Poly(ADP-ribosyl)ation and stroke. Pharmacol. Res.
2005, 52, 15–24. (b) Fossati, S.; Cipriani, G.; Moroni, F.; Chiarugi,
A. Neither energy collapse nor transcription underlie in vitro neuro-
toxicity of poly(ADP-ribose) polymerase hyper-activation. Neurochem.
Int. 2007, 50, 203–210. (c) Moroni, F. Poly(ADP-ribose)polymerase
1 (PARP-1) and postischemic brain damage. Curr. Opin. Pharmacol.
2008, 8, 96–103.
(64) (a) Cho, J.-H; Ko, S. Y. Synthesis and MMP-inhibitory activity of
gelastatin analogues. HelV. Chim. Acta 2002, 85, 3994–3999. (b) Chun,
K.; Park, S. K.; Kim, H. M.; Choi, Y.; Kim, M. H.; Park, C. H.; Joe,
B. Y.; Chun, T. G.; Choi, H. M.; Lee, H. Y.; Hong, S. H.; Kim, M. S.;
Nam, K. Y.; Han, G. Chromen-based TNF-alpha converting enzyme
(TACE) inhibitors: design, synthesis, and biological evaluation. Bioorg.
Med. Chem. 2008, 16, 530–535. (c) Kim, E. J.; Ko, S. Y. Synthesis
of arylidene-substituted gelastatin analogues and their screening for
MMP-2 inhibitory activity. Bioorg. Med. Chem. 2005, 13, 4103–4112.
(d) Lee, H. J.; Chung, M. C.; Lee, C. H.; Chun, H. K.; Rhee, J. S.;
Kho, Y. H. Gelastatins, new inhibitors of matrix metalloproteinases
from Westerdykella multispora F50733. Ann. N.Y. Acad. Sci. 1999,
878, 635–637. (e) Park, S. K.; Han, S. B.; Lee, K.; Lee, H. J.; Kho,
Y. H.; Chun, H.; Choi, Y.; Yang, J. Y.; Yoon, Y. D.; Lee, C. W.;
Kim, H. M.; Choi, H. M.; Tae, H. S.; Lee, H. Y.; Nam, K. Y.; Han,
G. Gelastatins and their hydroxamates as dual functional inhibitors
for TNF-alpha converting enzyme and matrix metalloproteinases:
synthesis, biological evaluation, and mechanism studies. Biochem.
Biophys. Res. Commun. 2006, 341, 627–634.
(52) (a) Bindokas, V. P.; Jordan, J.; Lee, C. C.; Miller, R. J. Superoxide
production in rat hippocampal neurons: selective imaging with
hydroethidine. J. Neurosci. 1996, 16, 1324–1336. (b) D’Agostino,
D. P.; Putnam, R. W.; Dean, J. B. Superoxide (*O2-) production in
CA1 neurons of rat hippocampal slices exposed to graded levels of
oxygen. J. Neurophysiol. 2007, 98, 1030–1041. (c) Shimoni, Y.; Chen,
K.; Emmett, T.; Kargacin, G. Aldosterone and the autocrine modulation
of potassium currents and oxidative stress in the diabetic rat heart.
Br. J. Pharmacol. 2008, 154, 675–687.
(53) Traber, M. G.; Atkinson, J. Vitamin E, antioxidant and nothing more.
Free Radical Biol. Med. 2007, 43, 4–15.
(54) Zhang, S.; Lin, Y.; Kim, Y. S.; Hande, M. P.; Liu, Z. G.; Shen, H. M.
c-Jun N-terminal kinase mediates hydrogen peroxide-induced cell death
via sustained poly(ADP-ribose) polymerase-1 activation. Cell Death
Differ. 2007, 14, 1001–1010.
(55) Bromberg, K. D.; Burgin, A. B.; Osheroff, N. A two-drug model for
etoposide action against human topoisomerase IIalpha. J. Biol. Chem.
2003, 278, 7406–7412.
(56) Hande, K. R. Topoisomerase II inhibitors. Update Cancer Ther. 2006,
1, 3–15.
(57) Sherer, T. B.; Betarbet, R.; Testa, C. M.; Seo, B. B.; Richardson, J. R.;
Kim, J. H.; Miller, G. W.; Yagi, T.; Matsuno-Yagi, A.; Greenamyre,
J. T. Mechanism of toxicity in rotenone models of Parkinson’s disease.
J. Neurosci. 2003, 23, 10756–10764.
(58) Bai, P.; Hegedus, C.; Erdelyi, K.; Szabo, E.; Bakondi, E.; Gergely,
S.; Szabo, C.; Virag, L. Protein tyrosine nitration and poly(ADP-ribose)
polymerase activation in N-methyl-N-nitro-N-nitrosoguanidine-treated
thymocytes: implication for cytotoxicity. Toxicol. Lett. 2007, 170, 203–
213.
(59) Andrabi, S. A.; Kim, N. S.; Yu, S. W.; Wang, H.; Koh, D. W.; Sasaki,
M.; Klaus, J. A.; Otsuka, T.; Zhang, Z.; Koehler, R. C.; Hurn, P. D.;
Poirier, G. G.; Dawson, V. L.; Dawson, T. M. Poly(ADP-ribose) (PAR)
polymer is a death signal. Proc. Natl. Acad. Sci. U.S.A. 2006, 103,
18308–18313.
(60) (a) Heeres, J. T.; Hergenrother, P. J. Poly(ADP-ribose) makes a date
with death. Curr. Opin. Chem. Biol. 2007, 11, 644–653. (b) Moubarak,
R. S.; Yuste, V. J.; Artus, C.; Bouharrour, A.; Greer, P. A.; Menissier-
JM801169S