Synthesis and biological evaluation of polyenylpyrrole derivatives as anticancer agents acting through caspases-dependent apoptosis

Article abstract of DOI:10.1021/jm100619x

A class of polyenylpyrroles and their analogues were designed from a hit compound identified in a fungus. The compounds synthesized were evaluated for their cell cytotoxicity against human non-small-cell lung carcinoma cell lines A549. Two compounds were found to exhibit high cytotoxicity against A549 cells with IC₅₀ of 0.6 and 0.01 μM, respectively. The underlying mechanisms for the anticancer activity were demonstrated as caspases activation dependent apoptosis induction through loss of mitochondrial membrane potential, release of cytochrome c, increase in B-cell lymphoma-2-associated X protein (Bax) level, and decrease in B-cell lymphoma-2 (Bcl-2) level. The two compounds were nontoxic to normal human lung Beas-2b cells (IC₅₀ > 80 μM), indicating that they are highly selective in their cytotoxicity activities. Furthermore, one compound showed in vivo anticancer activity in human-lung-cancer-cell-bearing mice. These results open promising insights on how these conjugated polyenes mediate cytotoxicity and may provide a molecular rationale for future therapeutic interventions in carcinogenesis.

Full text of DOI:10.1021/jm100619x

J. Med. Chem. 2010, 53, 7967–7978 7967
DOI: 10.1021/jm100619x
Synthesis and Biological Evaluation of Polyenylpyrrole Derivatives as Anticancer Agents Acting
through Caspases-Dependent Apoptosis
Zhanxiong Fang,†,^ Pei-Chun Liao,‡,^ Yu-Liang Yang,§ Feng-Ling Yang,§ Yi-Lin Chen,‡ Yulin Lam,*,† Kuo-Feng Hua,*,‡,
and Shih-Hsiung Wu*,§
†
Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore, ‡Institute of Biotechnology,
National Ilan University, Ilan, Taiwan, §Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, and Graduate Institute of Drug
Safety, School of Pharmacy, China Medical University, Taichung, Taiwan. ^Zhanxiong Fang and Pei-Chun Liao contributed equally to this work.
Received May 20, 2010
A class of polyenylpyrroles and their analogues were designed from a hit compound identified in a
fungus. The compounds synthesized were evaluated for their cell cytotoxicity against human non-small-
cell lung carcinoma cell lines A549. Two compounds were found to exhibit high cytotoxicity against A549
cells with IC50 of 0.6 and 0.01 μM, respectively. The underlying mechanisms for the anticancer activity
were demonstrated as caspases activation dependent apoptosis induction through loss of mitochondrial
membrane potential, release of cytochrome c, increase in B-cell lymphoma-2-associated X protein (Bax)
level, and decrease in B-cell lymphoma-2 (Bcl-2) level. The two compounds were nontoxic to normal
human lung Beas-2b cells (IC50>80 μM), indicating that they are highly selective in their cytotoxicity
activities. Furthermore, one compound showed in vivo anticancer activity in human-lung-cancer-cell-
bearing mice. These results open promising insights on how these conjugated polyenes mediate
cytotoxicity andmay providea molecular rationale forfuture therapeutic interventions in carcinogenesis.
Introduction
major pathways. One is the death receptor (extrinsic) pathway
involving death receptor and death ligand interaction, such as
Fas receptor (Fasa) and other members of the tumor-necrosis
factor (TNF) receptor family. These receptors activate cas-
pase-8 and subsequently caspase-3, the major caspases parti-
cipating in the execution phase of apoptosis.2 Another
apoptotic pathway is the mitochondrial (intrinsic) pathway,
which is activated by the release of proapoptotic factors from
mitochondria intermembrane space such as cytochrome c.3
The released cytochrome c interacts with apoptotic protease
activating factor 1 (Apaf-1) and activates caspase-9, which in
turn proteolytically activates downstream caspase-3.4 Acti-
vated caspase-3 cleaves many substrates, including poly ADP-
ribose polymerase (PARP), a DNA repair enzyme that leads
to inevitable cell death. Recently, novel molecules that induce
mitochondrial pathways of caspase activation have been
developed in cancer chemotherapy.5 Our interest in investi-
gating natural products for their potential therapeutic effects
has recently spurred us to examine the influences of conju-
gated polyenes on anticancer properties.
Conjugated polyenes is an interesting class of widely occur-
ring natural products, as they have been shown to possess
excellent biological properties such as antibacterial, antifun-
gal, and antitumor activities.6 Presently, some conjugated
polyenes that are sold commercially include rapamycin and
fumigillin.7 In 2006, Capon and co-workers published a report
on the isolation and structure elucidation of several polyenyl-
furans and polyenylpyrroles from the soil microbe Gymnoas-
cus reessii.8 In that study, they discovered three new conju-
gated polyenes, 12E-isorumbrin 1e, gymnoconjugatins A and B,
alongside rumbrin and auxarconjugatin A 1b which were
isolated previously (Figure 1).9 1b and 1e were subsequently
Cancer, being one of the leading causes of death globally, is
a disease of worldwide importance. Although anticancer
drugs have played a major role in the success stories in cancer
treatment, there are still many types of cancers where effective
molecular therapeutics are nonexistent. Hence, there is an
impetus to identify and develop more potent therapeutic
agents for cancer.
Activation of apoptotic pathways is a key method by which
anticancer drugs kill tumor cells.1 It is well-known that anti-
cancer drugs can stimulate apoptotic signaling through two
*To whom correspondence should be addressed. For Y.L.: phone,
(65) 6516-2688; fax, (65) 6779-1691; e-mail, chmlamyl@nus.edu.sg. For
K.-F.H.: phone, þ886-3-935-7400, extension 585; fax, þ886-3-9311526;
e-mail, kfhua@niu.edu.tw. For S.-H.W.: phone, þ886-2-2785-5696, ext.
ension 7101; fax, þ886-2-2653-9142; e-mail, shwu@gate.sinica.edu.tw.
a Abbreviations: Bax, B-cell lymphoma-2-associated X protein; Bcl-2,
B-cell lymphoma-2; Fas, Fas receptor; TNF, tumor-necrosis factor; Apaf-1,
apoptotic protease activating factor 1; PARP, poly ADP-ribose polymerase;
NS-1, nonstructural protein 1; DMP, Dess-Martin periodinane; DMSO,
dimethylsulfoxide; THF, tetrahydrofuran; TEA, triethylamine; DMF,
dimethylformamide; DIBAL, diisobutylaluminum hydride; IBX, iodoxy-
benzoic acid; NCS, N-chlorosuccimide; Pd2dba3, tris(dibenzylideneacetone)-
dipalladium(0); TBAF, tetrabutylammonium fluoride; NMP, N-methyl-2-
pyrrolidone; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end
labeling; PI, propidium iodide; Z-VAD-fmk, carbobenzoxyvalylalanyla-
spartyl[O-methyl]fluoromethylketone; ROS, reactive oxygen species;
NAC, N-acetylcysteine; DIDS, 4,40-diisothiocyano-2,20-stilbenedisul-
fonic acid; DiOC2(3), 3,30-diethyloxacarbocyanine iodide; dATP, deoxy-
adenosine triphosphate; Bak, Bcl-2 homologous antagonist killer; PTP,
permeability transition pore; IMM, inner mitochondrial membrane;
OMM, outer mitochondrial membrane; Bid, Bcl-2 homology-3 interact-
ing domain death agonist; sc, subcutaneous; TMS, tetramethylsilane;
H2DCFDA, dichlorodihydrofluorescein diacetate RPMI 1640, Roswell
Park Memorial Institute 1640; PBS, phosphate buffered saline; PVDF,
polyvinylidene fluoride.
r
2010 American Chemical Society
Published on Web 10/22/2010
pubs.acs.org/jmc

7968 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Fang et al.
Figure 1. Structures of auxarconjugatin, 12E-isorumbrin, and related polyenes.
Scheme 1. Retrosynthesis of Auxarconjugatin and Its Analogues 1a-n
found to possess potent cytotoxicity properties against non-
structural protein (NS-1) cell line, while earlier studies by
Yamagishi and co-workers9a have demonstrated that rumbrin
was able to provide cytoprotection against cell death caused
by calcium overload. Other related polyenylpyrroles that had
been isolated previously include 12E-bromoisorumbrin, 12E-
dechloroisorumbrin, auxarconjugatin B 1a, auxarconjugatin
C, and malbranpyrroles.10 Unlike 1b and 1e, 12E-bromoisor-
umbrin, 12E-dechloroisorumbrin, and gymnoconjugatins A
and B were absent of cytotoxicity activity, implying the im-
portance of the 3-chloropyrrole moiety in effecting cytotoxicity
in cancer cell lines.
Thus far, the main source of conjugated polyenes has been
from the isolation of fungi or bacteria. The typically small
quantitiesthatcanbeobtained via thesesources often limit the
extent of biological work that can be carried out. To address
this limitation as well as to provide access to structurally
diverse analogues of these compounds, it would be useful to
develop a synthetic strategy that allows conjugated polyenes
to be synthesized expediently. To the best of our knowledge,
there is presently only one reported synthesis of gymnoconju-
gatins A and B11 and there is no literature describing the
synthesis of polyenylpyrroles such as 1b and 1e. This, together
with the promising cytotoxicity properties of 1b and 1e,
prompted us to synthesize a class of polyenylpyrroles and
their analogues where the 3-chloropyrrole is replaced with
other 2- or 3-chlorosubstituted aromatic rings. Hence, we
hereindescribe the synthesis of these polyenyl compoundsand
their in vitro and in vivo antitumor activity against human
non-small-celllung carcinomacelllinesA549. The mechanism
of the active compounds’ action on death of these cells was
also examined.
Stille and Suzuki coupling reactions.12 With this strategy in
mind, we proceeded with the synthesis of 1.
Pyrones 2a-i were prepared from the respective 4-hydroxy-
6-methylpyran-2-ones 5a-c13 (Scheme 2). Methylation of the
hydroxyl group on 5a-c was achieved using dimethyl sulfate
to afford 6a-c. The oxidation of 6a-c to 7a-c was modified
from a procedure reported earlier.11 Instead of conventional
heating in a sealed tube, we applied microwave irradiation
which resulted in shorter reaction times with improved yields.
To introduce diversity at the R2 position for 2, compounds
7a-c were treated with MeMgBr or EtMgBr followed by
oxidation of the resulting alcohol using Dess-Martin period-
inane (DMP) to afford 7d-g. Attempts to convert the alde-
hyde moiety on 7a and 7b directly to a vinyl iodide group via
Takai olefination failed to provide the desired compound.
Hence, to synthesize 2, compounds 7a-g were first converted
to vinyl dibromides 8a-g via Corey-Fuchs olefination fol-
lowed by reduction using dimethylphosphite.14 This afforded
2a-g in excellent yields and E/Z ratio greater than 20:1.
Further treatment of compounds 2a and 2b with a mixture
of aqueous HBr and acetic acid gave the demethylated
products 2h and 2i, respectively.
The second coupling partner, pyrrole 4a, was synthesized
from commercially available 2-methyl-1-pyrroline 9(Scheme3).
The conversion of 9 to compound 11 was adapted from an
earlier report.15 Using THF instead of CCl4 as a solvent for
the chlorination of 9 led to a more than 200-fold increase in
reaction rate to provide 10 which was used directly for the
synthesis of 11 in excellent yield. Initial attempts to reduce 11
directly to the aldehyde 12 in a single step by using diisobu-
tylaluminum hydride (DIBAL) failed, and the fully reduced
alcohol was obtained as the major product. To obtain 12, we
therefore attempted to reduce 11 completely to the alcohol
with LiAlH4 and then oxidize the alcohol to the aldehyde 12
with DMP. Unfortunately, the addition of DMP led to the
immediate decomposition of the alcohol. This could be
attributed to the polymerization of pyrrole in the presence
of the acetic acid which was formed as a byproduct of the
DMP oxidation.16 Addition of sodium bicarbonate17 and
pyridine18 to neutralize the acetic acid byproduct did not
resolve the problem. Thus, to circumvent this problem, we
Results and Discussion
Chemistry. The retrosynthetic route of auxarconjugatin
and its analogues 1 (Scheme 1) was modified from the
synthesis of gymnoconjugatin.11 Disconnection of the tetra-
ene gave three fragments: pyrone 2, the central butadiene
connector 3, and vinyl bromide 4. It had been shown earlier
that the hetero-bis-metallated butadiene 3 could be used for
the synthesis of an extended polyene chain via sequential

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 7969
Scheme 2. Synthesis of Pyrones 2a-ia
a Reagents and conditions: (a) Me2SO4, K2CO3, DMSO, room temp; (b) SeO2, dioxane, 150-160 ꢀC; (c) (i) R2MgBr in Et2O, THF, room temp;
(ii) DMP, CH2Cl2, room temp; (d) CBr4, PPh3, CH2Cl2, room temp; (e) dimethylphosphite, TEA, DMF, room temp; (f) aq HBr, AcOH, 90 ꢀC.
Scheme 3. Synthesis of 4a-da
a Reagents and conditions: (a) NCS, THF, 55 ꢀC; (b) (i) NaOMe, MeOH, 0 ꢀC to room temp; (ii) aq HCl; (c) (i) LiAlH4, THF, -20 ꢀC to room temp;
(ii) IBX, NaHCO3, DMSO, room temp; (d) NaH, MsCl, THF, room temp; (e) (i) CuO, I2, pyridine, EtOH, reflux; (ii) K2CO3, reflux; (f) (i) LiAlH4, THF,
0 ꢀC; (ii) DMP, CH2Cl2, room temp; (g) CBr4, PPh3, CH2Cl2, room temp; (h) dimethylphosphite, TEA, DMF, room temp.
tried 2-iodoxybenzoic acid (IBX) which gratuitously gave 12
in moderate yields. The addition of excess sodium bicarbon-
ate to the reaction mixture to neutralize the acidic conditions
further improved the yield of 12. With compound 12 in our
hands, we proceeded to synthesize the corresponding vinyl
iodide via Takai olefination. However, in the course of
drying the vinyl iodide, polymerization occurred and a dark
tar was obtained. This problem was subsequently resolved by
first protecting 12 with a mesyl group whose electron-with-
drawing property served to stabilize the pyrrole for subse-
quent transformations.
Earlier studies have shown that the 3-chloropyrrole group
plays an important role in the cytotoxicity effects of 1b and 1e.
When the chloro group was substituted with a bromo or
hydrogen or when the 3-chloropyrrole moiety was substituted
with a furan ring, the activity was drastically reduced.11,19 To
study other ring systems besides pyrrole, we replaced 3-chloro-
pyrrole with other 2- or 3-chloro substituted aromatic rings.
Compound 15 was prepared by the oxidation of 2-acetyl-3-
chlorothiophene 14 (Scheme 3).20 Reduction of 15 with LiAlH4
followed by oxidation with DMP gave compound 16. Corey-
Fuchs olefination of 13, 16, 17a, and 17b gave the corre-
sponding vinyl dibromides 18a-d, and subsequent reduction
of 18a-d with dimethylphosphite afforded 4a-d. Com-
pound 4a and 4b were obtained as a ∼2:1 mixture of E and
Z stereoisomers, but for 4c and 4d, the E isomer was obtained
in greater than 9:1 ratio.
To establish if the cytotoxic effects of 1a would be affected
if the 3-chloropyrrole group was replaced by a methyl group
(Scheme 4), compound 21 was synthesized. This synthesis
involved the Takai olefination of 2,4-hexadienal with 1921 to
afford 20 which was then reacted with 2b via Suzuki coupling
to provide 21.
Pyrones 2a-i was treated with 3 via Stille coupling to afford
trienes 22a-i. Suzuki coupling of 22a-i with 4a followed by
treatment with tetrabutylammonium fluoride (TBAF) to re-
move the mesyl group afforded 1a-i (Scheme 5). Compounds
1j-n, bearing other aromatic rings besides 3-chloropyrrole,

7970 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Scheme 4. Synthesis of 21a
Fang et al.
a Reagents and conditions: (a) 2,4-hexadienal, CrCl2, LiI, THF, room temp; (b) 2b, Pd2dba3, AsPh3, aq KOH, THF, room temp.
Scheme 5. Synthesis of 1a-na
a Reagents and conditions: (a) 3, Pd2dba3, AsPh3, NMP, room temp; (b) (i) 4a, Pd2dba3, AsPh3, aq KOH, THF, room temp; (ii) TBAF, THF, room
temp; (c) 4a-d, Pd2dba3, AsPh3, aq KOH, THF, room temp.
Table 1. Cytotoxicity of Conjugated Polyenes against Human Lung Cancer A549 Cellsa
compd
R1
R2
R3
Ar
IC50 (μM)
auxarconjugatin B, 1a
auxarconjugatin A, 1b
H
H
Me
Me
Me
Me
Me
Me
Me
H
3-chloropyrrol-2-yl
3-chloropyrrol-2-yl
3-chloropyrrol-2-yl
3-chloropyrrol-2-yl
3-chloropyrrol-2-yl
3-chloropyrrol-2-yl
3-chloropyrrol-2-yl
3-chloropyrrol-2-yl
3-chloropyrrol-2-yl
3-chlorothiophen-2-yl
3-chlorothiophen-2-yl
2-chlorophenyl
0.6
1.2
Me
n-Bu
H
H
1c
1d
H
5.0
2.5
Me
Me
Et
Me
H
12E-isorumbrin, 1e
1f
Me
Me
n-Bu
H
5.2
5.6
1g
1h
1i
0.01
>20
>20
>20
>20
>20
>20
>20
>20
Me
H
H
H
1j
H
Me
Me
Me
Me
Me
Me
1k
1l
Me
Me
Me
Me
Me
H
H
1m
1n
21
H
3-chlorophenyl
3-chloro-1-mesylpyrrol-2-yl
H
H
H
a IC50 value expressed as the mean value of triplicate wells from at least three experiments.
1
were synthesized in a similar manner. Interestingly, the H
NMR of crude 1a-n showed that the E stereoisomer of
the respective compound was present in 75-85%. As the
E stereoisomers of 4a, 4b, and 4c,d were present in about
66%, 66%, and >90%, respectively, this indicated that isom-
erization could have occurred during the coupling process.
Table 1 shows the 15 auxarconjugatin analogues 1a-n and
21 synthesized.
that these compounds are more potent against A549 cell lines
than antitumor drugs like Gleevec (IC50 = 2-3 μM) and cis-
platin (IC50=64 μM).22 The loss of activity in compounds
1j-n, where other chloro-substituted aromatic rings were
present instead of 3-chloropyrrole, supported our hypothesis
that the latter group played an important role in effecting
cytotoxicity. In addition, the lack of cytotoxicity in com-
pounds 1h and 1i also illustrated the importance of a methyl
group at the R3 position.
Biological Results. Cytotoxicity. Compounds 1a-n and 21
were evaluated for their cytotoxicities against the human
lung cancer cell line A549 after 48 h of treatment. As shown
in Table 1, the two most potent compounds are 1a and 1g
with IC50 values of 0.6 and 0.01 μM respectively, indicating
To further explore the selectivity of the compounds against
A549 cells, the respective compound 1 with IC50 value less
than 1 μM was further examined against Beas-2b cells which
were derived from normal human bronchial epithelial cells.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 7971
Figure 2. Compounds 1a and 1g were noncytotoxic to normal human lung cells. (A) A549 cells (5 ꢀ 103 cells) and (B) normal human lung cells
Beas-2b (5 ꢀ 103 cells) were seeded in 96-well plates and treated with compounds 1a and 1g (0-80 μM) or vehicle (0.1% DMSO) for 48 h. Cell
viability was measured by proliferation assay. The data are expressed as the mean ( SE of three separate experiments.
Figure 3. Compounds 1a and 1g induced apoptosis in human lung cancer cells. (A) A549 cells were treated with compounds 1a or 1g (0-5 μM)
or vehicle (0.1% DMSO) for 48 h. The cell population in the sub-G1 phase was determined by flow cytometry after PI-staining of nuclei. The
data are expressed as the mean ( SE of three separate experiments. (B) A549 cells were treated with 1a or 1g (5 μM) for various time points, and
the DNA breaks were analyzed by flow cytometry based TUNEL assay. One of three repeated experiments is shown.
As can be seen in Figure 2, we found that compounds 1a and
1g, despite being very potent against A549 cells (0.6 and 0.01
μM, respectively) (Figure 2A), were found to be noncytotoxic
toward Beas-2b cells at up to 80 μM (Figure 2B). These results
indicated that compounds 1a and 1g have the potential to be
developed asanticancer agents becauseof theirhighselectivity
against A549 cells.
lyzed by flow cytometry based TUNEL assay in 1a- and 1g-
treated A549 cells. We found that at 5 μM, 1a and 1g induced
DNA breaks from 24 h treatment and then dramatically
increased cell population with DNA breaks. The TUNEL
positive cells for 1a- and 1g-treated A549 cells increased to
42% and 47%, respectively (Figure 3B). In addition, auto-
phagic cell death was not observed in 1a- and 1g-treated A549
cells (Supporting Information).
Apoptosis Induction in Human Non-Small-Cell Lung Car-
cinoma. To gain further insight into the mode of action of these
compounds, two assays targeting hallmarks of apoptosis,
namely, the cell cycle analysis and terminal deoxynucleotidyl
transferase dUTP nick end labeling (TUNEL) assay, were
performed. A549 cells were treated with 1a or 1g (0.31-5 μM)
for 48 h, and the cell cycle distribution was determined by flow
cytometry after propidium iodide (PI) staining of the nuclei.
The results obtained (Figure 3A) show that the cells in sub-G1
phase increased in a concentration-dependent manner after
treatment with 1a or 1g. These results are also consistent with
the data obtained from the cytotoxicity assay in that 1g is
more potent against A549 cells than 1a. The concentrations of
the compounds 1a and 1g required to elicit hypoploidy are
higher than the IC50 values for cell proliferation assays. This is
because the concentrationatnanomolar range may inhibitcell
proliferation but the concentration at micromolar range may
induce cell apoptosis. To confirm the proapoptotic activity of
compounds 1a and 1g, DNA breaks phenomenon was ana-
Caspases Activation in Apoptosis. To investigate whether
compounds 1a and 1g induced apoptosis via caspases-de-
pendent pathway, the effects of pan caspase inhibitor carbo-
benzoxyvalylalanylaspartyl[O-methyl]fluoromethylketone
(Z-VAD-fmk) on compounds 1a and 1g treated cells were
tested. As shown in Figure 4, Z-VAD-fmk dramatically bloc-
ked sub-G1 phase increase in 1a- and 1g-treated cells. In
contrast, reactive oxygen species (ROS) inhibitors NAC and
DIDS (N-acetylcysteine (NAC), antioxidant; 4,40-diisothio-
cyano-2,20-stilbenedisulfonic acid (DIDS), anion channel
inhibitor which blocks ROS release from mitochondria)
did not reduce the sub-G1 phase increase in 1a- and 1g-trea-
ted cells (Figure 4). These results imply that compounds 1a
and 1g induced A549 apoptosis through a caspases-dependent
pathway. The role of oxidative stress in apoptosis has been
controversial. Although ROS have been generally regarded to
be proapoptotic in nature by showing a protective effect of
antioxidants on apoptosis in various cell types,23 it has been

7972 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Fang et al.
recently suggested that ROS may also play an antiapoptotic
and protective role.24 Our results also clearly indicate that
antioxidative property of NAC can exert a proapoptotic effect
in compounds 1a and 1g induced A549 apoptosis.
Induction of Mitochondrial Death Pathway. Mitochondria
play an important role in cell death by changing its outer and
inner membrane permeability and thus leading to cytochrome
c release and caspases activation.25 To explore whether com-
pounds 1a and 1g induced apoptosis via the mitochondrial
signaling pathway, the mitochondrial membrane potential
alteration was determined using a mitochondria-specific fluo-
rescence dye, 3,30-diethyloxacarbocyanine iodide (DiOC2(3)).
A549 cells treated with 5 μM 1a or 1g were found to demon-
strate a loss of fluorescence intensity with time (Figure 5A),
indicating that 1a and 1g induced a loss of mitochondrial
membrane potential. Activation of the mitochondrial death
pathway can also be identified by the release of mitochondrial
cytochrome c. After cytochrome c is released from the mito-
chondria, it can bind to deoxyadenosine triphosphate (dATP)
and Apaf-1 and activate caspase-9 and caspase-3.26 We thus
investigated the release of cytochrome cfrom the mitochondria
into the cytosol by Western blotting. Cytosolic cytochrome c
was detected by varying the exposure of A549 cells to 1a and
1g, and the levels of cytochrome c that remained in the mito-
chondria was observed to decrease concomitantly (Figure 5B).
Cytochrome c release from mitochondria is a critical step in
apoptosis, and earlier investigations had shown that ionizing
radiation (IR) and etoposide induced the release of cyto-
chrome c from mitochondria in two distinct stages.27 At the
early stage, low levels of cytochrome c are released from
mitochondria and activate caspases 9 and 3. In contrast, the
late stage cytochrome c release resulted in a drastic loss of
mitochondrial cytochrome cand was associated with a reduction
Figure 4. Compounds 1a and 1g induce caspases-dependent apo-
ptosis in human lung cancer cells. A549 cells were treated with 1a or
1g (5 μM) or vehicle (0.1% DMSO) in the presence or absence of
Z-VAD-fmk (20 μM), NAC (10 mM), or DIDS (10 μM) for 36 h.
The cell population in the sub-G1 phase was determined by flow
cytometry after PI-staining of nuclei. The data are expressed as the
mean ( SE of three separate experiments.
Figure 5. (A) Compounds 1a and 1g induce mitochondria membrane potential lost and cytochrome c release. A549 cells were treated with
1a or 1g (5 μM) or vehicle (0.1% DMSO) for the time as indicated, followed by DiOC2(3) staining. The mitochondrial membrane potential
was analyzed by flow cytometry. One of three repeated experiments is shown. (B) Compound 1a or 1g (5 μM) or vehicle (0.1% DMSO) for 36 or
48 h. The cytochrome c in mitochondrial fraction and cytosolic fraction was measured by Western blot. One of three repeated experiments
is shown.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 7973
Figure 6. (A, B) Effects of compounds 1a and 1g on caspases activation and mitochondrial protein expression. A549 cells were treated with 1a
or 1g (0-10 μM) or vehicle (0.1% DMSO) for 36 h. (A) The expression of pro-caspase 3, pro-caspase 9, PARP, and actin was measured by
Western blot. One of three repeated experiments is shown. (B) The expression of Bcl-2, Bax, Bak, and actin were measured by Western blot. One
of three repeated experiments is shown. (C) Effects of compounds 1g on the expression of p53 and p21 protein expression. A549 cells were
treated with compound 1g (5 μM) or vehicle (0.1% DMSO) for 0-16 h. The expression of p53, p21, and actin were measured by Western blot.
One of three repeated experiments is shown.
of the ATP levels and mitochondrial transmembrane potential.
After accumulation, this protein is progressively degraded by
caspase-like proteases.28 Using immunoblotting, we did not
detect earlier cytochrome c release before 32 h. This could
probably be due to the very small amount of cytochrome c
released. However, compound 1g induced decrease of mito-
chondrial cytochrome c was similar to 1a but the amount of
cytosolic cytochrome c that was induced by 1g was lesser than
that induced by 1a. This may be attributed to the greater
cytosolic degradation of cytochrome c in 1g-treated cells
compared to 1a-treated cells.
Caspases are known to cleave into a shorter active form
upon activation.29 As shown in Figure 6A, 1a and 1g (1.25-
10 μM) induced a decrease of procaspase-3 and procaspase-9
in a concentration-dependent manner. The activation of
caspase-3 was further confirmed by detecting the degrada-
tion of PARP which is cleaved by active caspase-3 during
apoptosis. In contrast, we did not observe the cleavage of
caspase-8 (data not shown). Bcl-2 family proteins including
Bcl-2, Bax, and Bcl-2 homologous antagonist killer (Bak) are
the critical regulatory proteins for mitochondrial mediated
cell death.30 The amount of antiapoptotic protein, Bcl-2, was
observed to decrease dramatically after treatment with 1a or
1g (Figure 6B). In contrast, the expression of proapoptotic
protein, Bak, increased in the presence of 1a or 1g. The other
proapoptotic protein, Bax, was not affected by 1a or 1g
(Figure 6B). These data confirm that 1a and 1g induced cell
death through the mitochondrial death pathway.
Apoptotic signals that are transduced in response to stresses
converge mainly on the mitochondria. Upon stimulation by
death signals, a series of biochemical events is induced that
results in the permeabilization of the outer mitochondrial
membrane and release of cytochrome cand other proapoptotic
molecules. A transmembrane channel, called the permeability
transition pore (PTP), is formed at the contact sites between
the inner mitochondrial membrane (IMM) and outer mito-
chondrial membrane (OMM).31 Bcl-2 family proteins are
shown to interact with the PTP complex proteins.32 Apoptotic
signals activate proapoptotic Bcl-2 members such as Bax, Bak,
and Bcl-2 homology-3 interacting domain death agonist (Bid),
resulting in a disturbed balance between pro- and antiapopto-
tic Bcl-2 family proteins. As a consequence, OMM integrity is
lost because of the oligomerization of proapoptotic Bcl-2
members in the OMM.33 This results in the permeabilization
of the OMM, loss of mitochondrial membrane potential, and
the release of proteins from the intermembrane space.

7974 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Fang et al.
apoptosis signaling pathway is a promising strategy for the
development of novel chemotherapeutic molecules.37 In our
efforts to develop potential chemotherapeutic agents from
natural products, we have herein provided the first reported
synthesis of a class of polyenylpyrrole natural products and
their analogues. The compounds were evaluated for the cell
cytotoxicity against human lung cancer cells A549. Two
compounds, 1a and 1g, displayed potent effects in the inhibi-
tion of tumor cell proliferation. Flow cytometric analysis
revealed that compounds 1a and 1g increase the sub-G1 cell
population and DNA breaks suggesting apoptosis. Induction
of apoptosis by these compounds in A549 cells followed the
steps commonly observed for the intrinsic pathway involving
mitochondrial damage. In vivo study revealed that compound
1g demonstrated anticancer activities in tumor bearing mice.
Further studies are presently ongoing to develop the com-
pounds as a potential anticancer therapeutic.
Experimental Section
Chemistry. General Procedures. All chemical reagents and
solvents were obtained from Sigma Aldrich, Merck, Lancaster,
or Fluka and were used without further purification. The micro-
wave-assisted reactions were performed using the Biotage initia-
tor microwave synthesizer. Analytical thin layer chromatography
was carried out on precoated silica plates (Merck silica gel 60,
F254) and visualized with ultraviolet light or stained with phos-
phomolybdic acid stain. Flash column chromatography was
performed with silica (Merck, 70-230 mesh). The purities of
the compounds were determined via HPLC using a Shimadzhu
LCMS-IT-TOF system with a Phenomenex Luma C18 column
(50 mm ꢀ 3.0 mm, 5 μm). Detection was conducted at 254 nm,
and integration was obtained with a Shimadzhu LCMS solution
software. Compounds used in the biological assays have purities
of at least 95%. 1H NMR and 13C NMR spectra were measured
on a Bruker ACF 300 or AMX 500 Fourier transform spectro-
meter. Chemical shifts were reported in parts per million (δ)
relative to the internal standard of tetramethylsilane (TMS). The
signals observedweredescribed as follows: s (singlet), d (doublet),
t (triplet), q (quartet), dd(doublet of doublets), m (multiplet). The
number of protons (n) for a given resonance was indicated as nH.
Mass spectra were performed on a Finnigan/MAT LCQ mass
spectrometer under electron spray ionization (ESI) or electron
impact (EI) techniques. The purities of the tested compounds
were established by HPLC and were found to be >95% pure.
General Procedure for the Synthesis of 6a-c. To a mixture of
K2CO3 (1.73 g, 12.5 mmol) and the corresponding pyrone 5
(5.00 mmol) in DMSO (10 mL) was added dimethyl sulfate
(0.693 g, 5.50 mmol). The mixture was stirred at room tempera-
ture for 1 h and poured into water (60 mL). The mixture was
extracted with EtOAc, and the combined organic extract was
washed with saturated NaCl solution, dried over MgSO4, con-
centrated, and purified by column chromatography.
4-Methoxy-6-methyl-2H-pyran-2-one (6a). The residue was
purified using flash chromatography (EtOAc/hexane=2:1) to
afford 6a (0.588 g, 86%) as a white solid. 1H NMR (500 MHz,
CDCl3) δ 5.74 (d, J = 1.3 Hz, 1H), 5.36 (d, J = 1.9 Hz, 1H), 3.75
(s, 3H), 2.16 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 171.2,
164.8, 161.9, 100.2, 87.2, 55.7, 19.7. HRMS (EI): calcd for
C7H8O3, 140.0473; found 140.0472.
3-Butyl-4-methoxy-6-methyl-2H-pyran-2-one (6c). The resi-
due was purified using flash chromatography (EtOAc/hexane =
1:2) to afford 6c (0.725 g, 74%) as a white solid. 1H NMR (500
MHz, CDCl3) δ 5.97 (s, 1H), 3.82-3.81 (d, J = 3.2 Hz, 3H),
2.39-2.34 (m, 2H), 2.21 (d, J = 3.8 Hz, 3H), 1.41-1.40 (m, 2H),
1.32-1.28 (m, 2H), 0.89-0.85 (m, 3H); 13C NMR (125 MHz,
CDCl3) δ 165.8, 165.4, 160.8, 105.6, 94.9, 56.1, 30.1, 22.9, 22.6,
20.2, 13.9. HRMS (EI): calcd for C11H16O3, 196.1099; found
196.1098.
Figure 7. (A) Inhibition of human lung cancer xenografts growth
in vivo by compound 1g. A549 tumor-bearing mice were adminis-
tered sc with vehicle control (ꢀ) or 3 mg/kg compound 1g (0) on
days 0-4 for 5 days. The figure shows the relative tumor volume of
vehicle and compound 1g-treated groups. (B) Immunohistochem-
ical staining with activated caspase-3 was performed in tumor
tissues from control and 3 mg/kg 1g-treated mice (on day 28 after
inoculation). Scale bar, 100 μm.
Bcl2 family gene transcription is regulated by a number
of transcription factors. Previous report demonstrated that
both Bcl2 and Bax are transcriptional targets for the tumor
suppressor protein p53. P53 suppresses the activation of the
Bcl-2 promoter by the Brn-3a POU family transcription
factor.34 The expression of Bax has been found to be up-
regulated at the transcriptional level by p53,35 and the Bax
gene promoter was shown to contain four p53-binding sites
that could be specifically transcriptionally transactivated by
p53.36 Our data revealed that protein levels of p53 and p21
were increased after 2 and 8 h treatment with compound 1g
(Figure 6C). These suggest that p53 activation may be involved
in our system and regulates Bcl-2 family protein expression.
Antitumor Activity In Vivo. To evaluate the antitumor
activity of compound 1g in vivo, human lung cancer xeno-
grafts were established by subcutaneous (sc) injection of
approximately 1ꢀ107 A549 cells on the backs of nude mice.
After the tumor has reached about 100 mm3 in size, the mice
were randomized into vehicle control and treatment groups
(six animals each) and were given a dailyscinjection ofeither 0
(vehicle control group) or 3 mg/kg 1g (treatment groups) for
5 successive days. Results obtained show that 1g inhibited
the growth of tumor (Figure 7A). The human lung tumor
tissues with in vivo 1g treatment also displayed an increase in
activated caspase-3 protein expression (relative to the vehicle
control group) as evidenced by immunohistochemistry
(Figure 7B). Histological examination showed that there
were no observable histological changes in various normal
tissues including brain, liver, and kidney in the 1g-treated
mice. Additionally the changes in body weight of the
1g-treated mice were similar to that of the vehicle treated
mice (data not shown).
Conclusion
Induction of apoptosis is considered a possible mechanism
of most of the chemotherapeutic agents, and targeting the

Article
General Procedure for the Synthesis of 7a-c. SeO2 (1.11 g,
10.0 mmol) was added to the corresponding pyrone 6 (2.00
mmol) in 1,4-dioxane (4 mL). The reaction mixture containing
6b or 6c was heated at 160 ꢀC for 15 min while 6a was heated at
150 ꢀC for 15 min using microwave irradiation in a sealed tube.
Then the mixture was allowed to cool, saturated NaHCO3
solution was added, and the mixture was extracted with CH2Cl2.
The combined organic extract was dried over MgSO4, concen-
trated, and purified by column chromatography.
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 7975
164.6, 164.1, 157.7, 135.2, 108.0, 98.1, 94.3, 56.3, 29.8, 23.2, 22.6,
22.5, 13.8. HRMS (ESI) [M þ Na]þ: calcd for C13H17Br2O3
378.9544; found 378.9532.
General Procedure for the Synthesis of 2a-g. To a solution of
the corresponding pyrone 8 (0.80 mmol) and triethylamine
(0.364 g, 3.60 mmol) in DMF (1.5 mL) was added dimethylphos-
phite (0.352 g, 3.20 mmol). The reaction mixture was stirred at
room temperature for 1 h. Water was added to the mixture and
extracted with Et2O. The combined organic extract was washed
with saturated NaCl solution, dried over MgSO4, concentrated
under reduced pressure, and purified by column chromatography.
(E)-6-(2-Bromovinyl)-4-methoxy-2H-pyran-2-one (2a). The res-
idue was purified using flash chromatography (EtOAc/hexane =
1:2) to afford 2a (0.181 g, 98%) as a white solid. 1H NMR (500
MHz, CDCl3) δ 7.30-7.27 (d, J = 13.9 Hz, 1H), 6.63-6.61 (d,
J = 13.3 Hz, 1H), 5.83 (d, J = 2.5 Hz, 1H), 5.49 (d, J = 2.6 Hz,
1H), 3.80 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 170.5, 163.1,
156.3, 128.2, 116.3, 101.4, 89.5, 56.0. HRMS (EI): calcd for
C8H7O3Br, 229.9579; found 229.9585.
(E)-6-(1-Bromoprop-1-en-2-yl)-3-butyl-4-methoxy-2H-pyran-
2-one (2g). The residue was purified using flash chromatography
(EtOAc/hexane=1:8) to afford 2g (0.232 g, 96%) as a white
solid. 1H NMR (500 MHz, CDCl3) δ 7.32 (d, J = 1.3 Hz, 1H),
6.20 (s, 1H), 3.89 (s, 3H), 2.42-2.39 (t, J = 7.6 Hz, 2H), 2.06 (d,
J = 1.3 Hz, 1H), 1.46-1.39 (m, 2H), 1.35-1.28 (m, 2H), 0.90-
0.87 (t, J = 7.3 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 165.2,
163.9, 157.2, 131.8, 114.8, 108.4, 93.5, 56.2, 30.0, 23.2, 22.6, 15.7,
13.9. HRMS (EI): calcd for C13H17O3Br, 300.0361; found
300.0357.
4-Methoxy-6-oxo-6H-pyran-2-carbaldehyde (7a). The residue
was purified using flash chromatography (EtOAc/hexane/
CH2Cl2 =1:3:6) to afford 7a (0.188 g, 61%) as a pale brown
1
solid. H NMR (500 MHz, DMSO-d6) δ 9.46 (s, 1H), 7.14 (d,
J = 1.9 Hz, 1H), 6.01 (d, J = 1.9 Hz, 1H), 3.89 (s, 3H); 13C NMR
(125 MHz, DMSO-d6) δ 184.3, 169.0, 161.2, 153.7, 112.6, 94.6,
57.1. HRMS (EI): calcd for C7H6O4, 154.0266; found 154.0265.
5-Butyl-4-methoxy-6-oxo-6H-pyran-2-carbaldehyde (7c). The
residue was purified using flash chromatography (EtOAc/
hexane/CH2Cl2=1:6:12) to afford 7c (0.319 g, 76%) as a pale
brown solid. 1H NMR (500 MHz, CDCl3) δ 9.55 (s, 1H), 6.99
(s, 1H), 3.96 (s, 3H), 2.50-2.46 (t, J = 7.6 Hz, 2H), 1.47-1.41
(m, 2H), 1.35-1.31 (m, 2H), 0.91-0.88 (t, J = 7.3 Hz, 3H); 13
C
NMR (125 MHz, CDCl3) δ 183.3, 163.3, 162.3, 152.4, 115.9,
101.8, 56.7, 29.7, 23.9, 22.6, 13.8. HRMS (EI): calcd for
C11H14O4, 210.0892; found 210.0893.
General Procedure for the Synthesis of 7d-g. 3 M MeMgBr or
3 M EtMgBr in Et2O (0.733 mL, 2.20 mmol) was added drop-
wise to the corresponding pyrones 7a-c (2.00 mmol) in THF.
The mixture was allowed to stir at room temperature for 30 min
before quenching with saturated NH4Cl solution. The mixture
was extracted with CH2Cl2, and the combined organic extract was
washed with saturated NaCl solution and dried over MgSO4. The
solvent was removed under reduced pressure to afford a brown
residue. Dess-Martin periodinane (DMP) (1.02 g, 2.40 mmol)
was added to the residue dissolved in CH2Cl2 (5 mL), and the
reaction mixture was stirred at room temperature for 1 h. Subse-
quently, saturated NaHCO3 solution (5 mL) and 15% Na2S2O3
solution (5 mL) were added and the mixturewas allowed to stirfor
an additional 15 min. Then the mixture was extracted with CH2Cl2
and the combined organic extract was dried over MgSO4, con-
centrated and purified by column chromatography.
6-Acetyl-3-butyl-4-methoxy-2H-pyran-2-one (7g). The residue
was purified using flash chromatography (EtOAc/hexane = 1:3)
to afford 7g (0.327 g, 73%) as a pale yellow solid. 1H NMR (500
MHz, CDCl3) δ 6.99 (s, 1H), 3.89 (s, 3H), 2.46 (s, 3H), 2.43-2.40
(t, J = 7.6Hz, 2H), 1.41-1.36 (m, 2H), 1.31-1.23 (m, 2H), 0.85-
0.82 (t, J = 7.3 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 191.4,
163.9, 162.7, 153.1, 114.1, 98.0, 56.5, 29.6, 25.7, 23.6, 22.5, 13.7.
HRMS (ESI) [M þ Na]þ: calcd for C12H16O4Na 247.0946; found
247.0942. HRMS (ESI) [M þ H]þ: calcd for C12H16O4Na,
247.0946; found 247.0942.
Synthesis of Methyl 3-Chloro-1H-pyrrole-2-carboxylate (11).
2-Methyl-1-pyrroline 9 (0.831 g, 10.0 mmol) was added to a
suspension of N-cholorosuccinimide (10.7 g, 80.0 mmol) in
THF (25 mL), and the reaction mixture was heated at 55 ꢀC for
20 min. Then the reaction mixture was cooled to room tempera-
ture, water was added, and the mixture was extracted with hexane.
The combined organic extract was concentrated under reduced
pressure to afford 10 which was directly used for the next step.
Compound 10 was dissolved in MeOH (10 mL) and cooled to
0 ꢀC. 3 M NaOMe in MeOH (20 mL, 60.0 mmol) was added
dropwise over 5 min, and thereafter, the reaction mixture was
warmed to room temperature and stirred for an additional 30 min.
The mixture was acidified using 2 M HCl and extracted with
EtOAc. The combined organic extract was washed with saturated
NaCl solution, dried over MgSO4, concentrated and the residue
purified using flash chromatography (EtOAc/hexane = 1:4) to
1
afford 11 (1.50 g, 94%) as a yellow solid. H NMR (500 MHz,
CDCl3) δ 9.56 (br, 1H), 6.86 (s, 1H), 6.23 (s, 1H), 3.89 (s, 3H); 13
C
NMR (125 MHz, CDCl3) δ 160.9, 122.0, 119.2, 118.2, 111.9, 51.6.
HRMS (EI): calcd for C6H6NO2Cl, 159.0087; found 159.0088.
Synthesis of 3-Chloro-1H-pyrrole-2-carbaldehyde (12). 2 M
LiAlH4 in THF solution (4.80 mL, 9.60 mmol) was added drop-
wise to 11 (1.28 g, 8.00 mmol) in THF (15 mL) at -20 ꢀC.
The reaction mixture was warmed to 0 ꢀC and stirred for
30 min before quenching with EtOAc (5 mL). Water (20 mL)
and 2 M aqueous NaOH (20 mL) were added to the reaction
mixture, and the solid formed was filtered and washed with
EtOAc. The filtrate was extracted with EtOAc and the com-
bined organic extract was washed with saturated NaCl solution
and then concentrated to obtain the crude alcohol product.
2-Iodoxybenzoic acid (5.60 g, 20.0 mmol) was dissolved in
DMSO (20 mL) before the addition of NaHCO3 (4.03 g, 48.0
mmol) and the crude alcohol product. The mixture was stirred at
room temperature for 16 h and quenched with 0.5 M aqueous
NaOH (150 mL). The solid was filtered and washed with EtOAc,
and the filtrate was extracted with EtOAc. The combined organic
extract was washed with saturated NaCl solution, dried over
MgSO4, concentrated and the residue was purified using flash
chromatography (EtOAc/hexane = 1:3) to afford 12 (0.777 g,
75%) as a pale brown solid. 1H NMR (500 MHz, CDCl3) δ 10.7
(br, 1H), 9.63 (s, 1H), 7.10-7.08 (m, 1H), 6.29-6.28 (m, 1H); 13C
General Procedure for the Synthesis of 8a-g. CBr4 (0.464 g,
1.40 mmol) in CH2Cl2 (4 mL) was added to a solution of the cor-
responding pyrone 7 (1.00 mmol) and PPh3 (0.734 g, 2.80 mmol)
in CH2Cl2 (8 mL). The mixture was stirred at room temperature
for 30 min, and thereafter, the solvent was removed under reduced
pressure and the residue was purified by column chromatography.
6-(2,2-Dibromovinyl)-4-methoxy-2H-pyran-2-one (8a). The res-
idue was purified using flash chromatography (EtOAc/CH2Cl2 =
1:40) to afford 8a (0.285 g, 92%) as a white solid. 1H NMR (500
MHz, CDCl3) δ 7.09, (s, 1H), 6.35 (d, J = 2.6 Hz, 1H), 5.53 (d,
J = 1.9 Hz, 1H), 3.81 (s, 3H); 13C NMR (125 MHz, CDCl3) δ
170.2, 162.8, 155.4, 128.5, 103.5, 97.3, 89.9, 56.1. HRMS (EI):
calcd for C8H6O3Br2, 307.8684; found 307.8678.
3-Butyl-6-(1,1-dibromoprop-1-en-2-yl)-4-methoxy-2H-pyran-2-one
(8g). The residue was purified using flash chromatography (EtOAc/
hexane = 1:6) to afford 8g (0.338 g, 89%) as a white solid. 1H NMR
(500 MHz, CDCl3) δ 6.39 (s, 1H), 3.85 (s, 3H), 2.39-2.36 (t, J =
7.6 Hz, 2H), 2.10 (s, 3H) 1.44-1.38 (m, 2H), 1.33-1.25 (m, 2H),
0.87-0.85 (t, J = 7.3 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ

7976 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Fang et al.
NMR (125 MHz, CDCl3) δ 177.8, 127.7, 126.3, 125.3, 111.6.
HRMS (EI): calcd for C5H4NO2Cl, 128.9981; found 129.9977.
Synthesis of 3-Chloro-1-(methylsulfonyl)-1H-pyrrole-2-car-
baldehyde (13). 60% NaH in mineral oil (0.132 g, 3.30 mmol)
was added to 12 (0.388 g, 3.00 mmol) in THF portionwise. After
evolution of hydrogen gas had ceased, methanesulfonyl chloride
(0.378 g, 3.30 mmol) was added and the reaction mixture was
stirred at room temperature for 20 min. Subsequently, the sol-
vent was removed and the residue was purified by flash chro-
matography (EtOAc/hexane = 1:3) to afford 13 (0.573 g, 92%)
as a pale brown solid. 1H NMR (500 MHz, CDCl3) δ 9.83
(s, 1H), 7.58-7.57 (d, J = 3.2 Hz, 1H), 6.37 (d, J = 3.2 Hz, 1H),
3.68 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 176.8, 132.5,
129.2, 127.2, 112.2, 42.9. HRMS (EI): calcd for C6H6NO3ClS,
206.9757; found 206.9755.
139.5, 135.4, 133.9, 123.5, 101.5, 88.9, 83.3, 55.9, 24.7. HRMS
(ESI) [M þ Na]þ: calcd for C18H23O5BNa, 353.1536; found
353.1522.
3-Butyl-4-methoxy-6-((2E,4E,6E)-7-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)hepta-2,4,6-trien-2-yl)-2H-pyran-2-one (22g).
22g (48.8 mg, 61%) was obtained as an orange solid. 1H NMR
(500 MHz, CDCl3) δ 7.20-7.10 (m, 2H), 6.73-6.68 (dd, J =
12.0 Hz, 15.1 Hz, 1H), 6.62-6.57 (dd, J = 10.7 Hz, 14.5 Hz,
1H), 6.21 (s, 1H), 5.74-5.70 (d, J = 17.7 Hz, 1H), 3.91 (s, 3H),
2.46-2.43 (t, J = 7.6 Hz, 2H), 2.01 (s, 3H), 1.49-1.44 (m, 2H),
1.38-1.29 (m, 2H), 1.25 (s, 12H), 0.93-0.90 (t, J = 7.6 Hz, 3H);
13C NMR (125 MHz, CDCl3) δ 165.5, 164.3, 159.3, 148.9, 139.3,
131.1, 131.2, 127.6, 107.9, 93.1, 83.3, 56.0, 30.2, 24.7, 23.2, 22.6,
13.9, 12.6. HRMS (ESI) [M þ Na]þ: calcd for C23H33O5BNa,
423.2319; found 423.2334.
General Procedure for the Synthesis of 18a-d. CBr4 (0.345 g,
1.04 mmol) in CH2Cl2 (2 mL) was added to a solution of PPh3
(0.545 g, 2.08 mmol) and 13, 16, 17a, or 17b (0.800 mmol) in
CH2Cl2 (4 mL). The mixture was stirred at room temperature
for 30 min, concentrated, and purified by column chromatog-
raphy.
3-Chloro-2-(2,2-dibromovinyl)-1-(methylsulfonyl)-1H-pyrrole
(18a). The residue was purified using flash chromatography
(EtOAc/hexane=1:10) to afford 18a (0.194 g, 85%) as a pale
brown solid. 1H NMR (500 MHz, CDCl3) δ 7.40 (s, 1H), 7.19-
7.18 (d, J = 3.2 Hz, 1H), 6.33-6.32 (d, J = 3.8 Hz, 1H), 3.13
(s, 3H); 13C NMR (125 MHz, CDCl3) δ 126.1, 124.7, 122.1,
119.3, 113.4, 98.5, 42.9. HRMS (EI): calcd for C7H6NO2Br2ClS,
360.8175; found 360.8177.
General Procedure for the Synthesis of 4a-d. To a solution of
18 (0.735 mmol) and triethylamine (0.334 g, 0.331 mmol) in DMF
(1.5 mL) was added dimethylphosphite (0.323 g, 0.294 mmol),
and the reaction mixture was stirred at room temperature for 1 h.
Thereafter, water was added to the mixture and extracted with
Et2O. The combined organic extract was washed with saturated
NaCl solution, dried over MgSO4, concentrated under reduced
pressure, and purified by column chromatography.
General Procedure for the Synthesis of 1a-i. Pd2dba3 (2.7 mg,
3.0 μmol) and AsPh3 (4.6 mg, 15 μmol) was added to THF
(1 mL) followed by 4a (56 mg, 0.195 mmol) and 1.8 M aqueous
KOH (0.167 mL, 0.300 mL). The respective compound 22 (0.150
mmol) was dissolved in THF (0.5 mL) and added dropwise to
the reaction mixture over 5 min with stirring. The reaction
mixture was stirred for 20 min at room temperature and
quenched with saturated NH4Cl. EtOAc was added, and the
mixture was washed thrice with water followed by saturated
NaCl solution. The organic extract was dried over MgSO4 and
concentrated under reduced pressure. The residue obtained was
dissolved in THF (1 mL), and 1 M TBAF in THF (0.300 mL,
0.300 mmol) was added. The mixture was stirred at room
temperature for 30 min, and thereafte, EtOAc was added and
the mixture was washed thrice with water followed by saturated
NaCl solution. The organic extract was dried over MgSO4,
concentrated under reduced pressure, and purified by column
chromatography.
Auxarconjugatin B (1a). The residue was purified using flash
chromatography (acetone/hexane/CH2Cl2 = 1:5:10 to afford
1a (36.8 mg, 74%) as a red solid. 1H NMR (500 MHz, DMSO-
d6) δ 11.45 (br, 1H), 7.07-7.02 (dd, J = 11.4 Hz, 15.1 Hz, 1H),
6.90-6.89 (m, 1H), 6.79-6.69 (m, 2H), 6.63-6.58 (dd, J = 11.4
Hz, 14.5 Hz, 1H), 6.54-6.37 (m, 3H), 6.30-6.27 (d, J = 15.1 Hz,
1H), 6.23-6.22(d, J=1.9 Hz, 1H), 6.14-6.13 (m, 1H), 5.59-
5.58 (d, J = 2.5 Hz, 1H), 3.81 (s, 3H); 13C NMR (125 MHz,
DMSO-d6) δ 170.7, 162.6, 158.4, 138.8, 136.8, 135.1, 131.1,
130.5, 126.0, 124.7, 121.6, 120.8, 120.5, 111.9, 109.1, 100.6, 88.4,
56.3. HRMS (ESI) [M - H]-: calcd for C18H15NO3Cl, 328.0740;
found 328.0738.
3-Butyl-6-((2E,4E,6E,8E)-9-(3-chloro-1H-pyrrol-2-yl)nona-
2,4,6,8-tetraen-2-yl)-4-methoxy-2H-pyran-2-one (1g). The re-
sidue was purified using flash chromatography (acetone/hexane/
CH2Cl2 = 1:8:16) to afford 1g (49.2 mg, 82%) as a red solid. 1H
NMR (500 MHz, DMSO-d6) δ 11.45 (br, 1H), 7.06-7.04 (d, J =
10.7 Hz, 1H), 6.90-6.89 (m, 1H), 6.80-6.69 (m, 3H), 6.64-6.44
(m, 4H), 6.13-6.12 (m, 1H), 3.94 (s, 3H), 2.33-2.30 (t, J=7.3
Hz, 2H), 2.05 (s, 3H), 1.40-1.34 (m, 2H), 1.30-1.23 (m, 2H),
0.89-0.86 (t, J = 7.3 Hz); 13C NMR (125 MHz, DMSO-d6) δ
166.0, 163.1, 159.0, 138.5, 136.3, 131.6, 131.1, 127.9, 126.0, 125.8,
124.8, 120.5, 120.4, 111.8, 109.1, 105.1, 93.7, 56.7, 29.7, 22.7, 22.0,
13.8, 12.3. HRMS (ESI) [M þ Na]þ: calcd for C23H26NO3ClNa,
422.1499; found 422.1510.
Reagents Used in Biological Assays. Roswell Park Memorial
Institute 1640 (RPMI 1640) medium was obtained from GIBCO
BRL (Gaithersburg, MD). DiOC2(3) was obtained from Molec-
ular Probes (Eugene, OR). Anti-caspase-3, anti-caspase-8, anti-
caspase-9, anti-PARP, anti-Bcl-2, anti-Bax, anti-Bak, anti-
cytochrome c, and anti-actin antibody were purchased from
Chemicon (Temecula, CA). Anti-p53 and anti-p21 antibody
were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). All other chemicals were obtained from Sigma Chemical
Co. (St. Louis, MO). Stock solution was kept at -20 ꢀC and
freshly diluted to the desired concentrations with medium
2-(2-Bromovinyl)-3-chloro-1-(methylsulfonyl)-1H-pyrrole (4a).
The residue was purified using flash chromatography (EtOAc/
hexane = 1:10) to afford a pale brown solid 4a (0.147 g, 97%) as
1
a mixture of E/Z isomers in ∼2:1 ratio. H NMR (500 MHz,
CDCl3) (mixture of E/Z isomers) δ 7.41-7.38 (d, J = 13.9 Hz,
1H), 7.19-7.18 (d J = 3.2 Hz, 1H), 7.16-7.13 (m, 1.8H), 6.77-
6.76 (d, J = 7.6 Hz, 0.4H), 6.34-6.33 (d, J = 3.2 Hz, 0.4 H),
6.30-6.29 (d, J = 3.8 Hz, 1H), 3.14 (s, 3H), 3.08 (s, 1.3H); 13
C
NMR (125 MHz, CDCl3) (mixture of E/Z isomers) δ 125.2,
124.2, 122.8, 122.6, 122.0, 121.6, 118.8, 117.5, 115.5, 113.6, 113.3,
112.6, 42.8, 42.7. HRMS (EI): calcd for C7H7NO2BrClS,
282.9069; found 282.9069.
General Procedure for the Synthesis of 22a-i. Pd2dba3
(5.5 mg, 6.0 μmol) and AsPh3 (7.3 mg, 24 μmol) were added to
a mixture of the respective compounds 2 (0.20 mmol) and 3
(0.169 g, 0.36 mmol) in NMP (1 mL) and allowed to stir at room
temperature for 6 h. Then water was added and the mixture was
extracted with Et2O. The combined organic extract was washed
with saturated NaCl solution, dried over MgSO4, concentrated,
and the residue obtained was dissolved in CH3CN (15 mL) and
washed with pentane (15 mL ꢀ 5) to remove the tributyltin bro-
mide byproduct. Following that, CH3CN was removed under
reduced pressure and the residue was purified using a short
column (∼5 cm) of silica gel (EtOAc/hexane = 1:2).
4-Methoxy-6-((1E,3E,5E)-6-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-2-yl)hexa-1,3,5-trienyl)-2H-pyran-2-one (22a). 22a (48.2
mg, 73%) was obtained as an orange solid. 1H NMR (500 MHz,
CDCl3) δ 7.16-7.11 (dd, J = 10.7 Hz, 15.1 Hz, 1H), 7.06-7.00
(dd, J = 10.7 Hz, 17.8 Hz, 1H), 6.53-6.48 (dd, J = 10.8 Hz, 14.5
Hz, 1H), 6.43-6.38 (dd, J = 10.7 Hz, 14.5 Hz, 1H), 6.10-6.07
(d, J = 15.2 Hz, 1H), 5.84 (d, J = 1.9 Hz, 1H), 5.72-5.68 (d, J =
17.7 Hz, 1H), 5.45-5.44 (d, J = 2.5 Hz, 1H), 3.79 (s, 3H), 1.26 (s,
12H); 13C NMR (125 MHz, CDCl3) δ 170.8, 163.8, 158.3, 148.4,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 7977
immediately before use (the final concentration of DMSO in
culture medium was 0.1%).
in buffer A (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM
MgCl2, 1 mM Na-EDTA, 1 mM Na-EGTA, 1 mM dithio-
threitol, 0.1 mM phenylmethylsulfonyl fluoride containing
250 mM sucrose). After being chilled on ice for 30 min, the cells
were disrupted by 15 strokes of a glass homogenizer. The homo-
genate was centrifuged twice to remove unbroken cells and nuclei
(750g, 10 min, 4 oC). The supernatant was then obtained by
centrifugation at 10000g for 60 min at 4 ꢀC. The resulting pellets
were identified as the mitochondrial fraction, and supernatants
were identified as cytosolic fraction. All steps were performed on
ice or 4 ꢀC. Cytochrome c release into the cytosolic fraction for
each condition was assessed by Western blot analysis.
Antitumor Activity in Vivo. Xenograft mice were used as a
model system to study the cytotoxicity effect of compound 1g in
vivo. Femalecongenital athymic BALB/cnude(nu/nu) mice were
purchased from National Sciences Council (Taipei, Taiwan),
and all procedures were performed in compliance with the stan-
dard operating procedures of the Laboratory Animal Center of
National Ilan University (Ilan, Taiwan). All experiments were
carried out using 6-8 week old mice weighing 18-22 g. The
animals were sc implanted with 1 ꢀ 107 A549 cells into the back of
mice. When the tumor reached 80-120 mm3 in volume, animals
were divided randomly into control and test groups consisting of
six mice per group (day 0). Daily sc administration of compound
1g, dissolved in a vehicle of 20% Tween 80 in normal saline (v/v),
was performed from days 0 to 4 far from the inoculated tumor
sites (>1.5 cm). The control group was treated with vehicle only.
The mice were weighed three times a week up to days 21-28 to
monitor the effects, and the same time the tumor volume was
determined by measurement of the length (L) and width (W) of
the tumor. The tumor volume on day n (TVn) was calculated
as TV (mm3) = (L ꢀ W2)/2. The relative tumor volume on day
n (RTVn) versus day 0 was expressed according to the following
formula: RTVn = TVn/TV0. Xenograft tumors as well as other
vital organs of treated and control mice were harvested and fixed
in 4% formalin, embedded in paraffin, and cut in 4 mm sections
for histological study.
In Vitro Growth Inhibition Study. Human non-small-cell lung
carcinoma A549 cells and normal human bronchial epithelial
cell line Beas-2b cells were obtained from ATCC (Manassas,
VA). Cells were seeded at a density of 5000 cells in 100 μL of
RPMI 1640 medium with 10% (v/v) fetal bovine serum per well
in 96-well flat-bottom plates and incubated for 24 h at 37 ꢀC in a
5% CO2 incubator. Test compounds were dissolved in DMSO,
further diluted with the culture media to obtain an optimal
range of concentrations for treatments in triplicate per concen-
tration, and incubated at 37 ꢀC in a CO2 incubator for 48 h. All
treatment media contained the final DMSO concentration of
0.1%. Alamar Blue assay was used to determine the cytotoxicity
of the test compounds. The procedure was conducted following
the protocol described in the manufacturer’s instructions (AbD
Serotec, Oxford, U.K.).
Cell Cycle Determination. Aliquots of 5 ꢀ 105 cells was fixed
in 70% ethanol on ice for at least 2 h and centrifuged. The pellet
was incubated with RNase (200 μg/mL) and PI (10 μg/mL) at
room temperature for 30 min. DNA content and cell cycle
distribution were analyzed using a Becton Dickinson FACScan
Plus flow cytometer. Cytofluorometric analysis was performed
using a CellQuestR (Becton Dickinson, San Jose, CA) on a
minimum of 10 000 cells per sample.
TdT End-Labeling Assay. Apoptosis was assayed by a TdT-
mediated dUTP nick-end labeling assay. After treatments, 5 ꢀ 105
cells were fixed in 1% paraformaldehyde and 70% ethanol and
then washed with phosphate buffered saline (PBS) and resus-
pended in 50 μL of TdT reaction solution containing 0.2 M
sodium cacodylate, 25 mM Tris-HCl, 5 mM CoCl2, 0.25 mg/mL
bovine serum albumin, 10 units of terminal transferase, and
0.5 nM DIG-dUTP (all from Chemicon International, Inc.).
The TdT end-labeling reactions were carried out at 37 ꢀC for
30 min. The cells were rinsed with cold PBS, resuspended in
100 μL of fluorescein antibody solution (2.5 μg/mL anti-DIG
conjugate fluorescein, 4ꢀ saline sodium citrate buffer, 0.1%
Triton X-100, and 5% nonfat dry milk), and incubated in the
dark for 30 min. Subsequently, the cells were rinsed in PBS
containing 0.1% Triton X-100 and then treated with 1 mL of
propidium iodide (PI, 5 μg/mL in PBS) and RNase A (100 μg/
mL) for 30 min in the dark. An increase in fluorescence was
determined by the FACScan system.
Acknowledgment. This research was supported by grants
from National Science Council, Taiwan. Grant No.: NSC
98-2320-B-039-003-MY2 (K.-F. H.); NSC 098-2811-B-197-001
(K.-F. H.).
Measurement of Mitochondrial Membrane Potential (4ψm).
A reduction in mitochondrial membrane potential following treat-
ment with compound 1 was monitored by flow cytometry. The
4ψm was estimatedbystaining 5 ꢀ 105 cells with50nM DiOC2(3)
(Molecular Probes), a cationic lipophilic dye, for 15 min at 37 ꢀC
in the dark and then washed with cold PBS. Cells were subjected
to flow cytometry (excitation 488 nm; emission 525 nm; recorded
in FL-1). The fluorescence of DiOC2(3) is oxidation-dependent
and correlates with 4ψm. Cells without treatment were processed
as the control. Mitochondrial damage was recognized by the
presence of cells displaying “low” uptake of DiOC3.
Supporting Information Available: Additional details of
1
experimental procedures and H, 13C, and HRMS data of all
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
(1) (a) Kaufmann, S. H.; Earnshaw, W. C. Induction of apoptosis by
cancer chemotherapy. Exp. Cell Res. 2000, 256, 42–49. (b) Fulda, S.;
Debatin, K. M. Extrinsic versus intrinsic apoptosis pathways in anti-
cancer chemotherapy. Oncogene 2006, 25, 4798–4811.
(2) Thorburn, A. Death receptor-induced cell killing. Cell. Signalling
Western Blot Analysis. After treatment, cells were washed three
times with PBS and lysed in 100 μL of sample buffer (62.5 mM
Tris-HCl, pH 6.8, 2% SDS, 5% 2-mercaptoethanol, 0.002% bromo-
phenol blue, and 10% glycerol). The samples were boiled for 5 min
and then centrifuged at 12 000 rpm for 10 min. Protein extracts were
subjected to electrophoresis on an SDS-polyacrylamide gel and
then transferred onto polyvinylidene fluoride (PVDF) membrane.
The blots were blocked for 1 h in PBST (10 mM Tris-HCl, pH 7.4,
150 mM NaCl, 0.05% Tween-20) containing 5% nonfat dried milk
and then probed overnight with an appropriate dilution of the pri-
mary antibody. Reactions were detected with a suitable secondary
antibody conjugated to horseradish peroxidase and an enhanced
chemiluminescence kit (Millipore).
2004, 16, 139–144.
(3) Kuwana, T.; Newmeyer, D. D. Bcl-2-familyproteins and the role of
mitochondriain apoptosis. Curr. Opin. Cell Biol. 2003, 15, 691–699.
(4) Jiang, X.; Wang, X. Cytochrome C-mediated apoptosis. Annu.
Rev. Biochem. 2004, 73, 87–106.
(5) Bose, J. S.; Gangan, V.; Prakash, R.; Jain, S. K.; Manna, S. K. A
dihydrobenzofuran lignan induces cell death by modulating mito-
chondrial pathway and G2/M cell cycle arrest. J. Med. Chem. 2009,
52, 3184–3190.
(6) Thirsk, C.; Whiting, A. Polyene natural products. J. Chem. Soc.,
Perkin Trans. 1 2002, 999–1023.
(7) (a) Schreiber, S. L. Chemistry and biology of the immunophilins
and their immunosuppressive ligands. Science 1991, 251, 283–287.
(b) Rosen, M. K.; Schreiber, S. L. Natural products as probes in the study
of cellular functions. Investigation of immunophilins. Angew. Chem.,
Int. Ed. Engl. 1992, 31, 384–400. (c) Katznelson, H.; Jamieson, C. A.
Control of nosema disease of honeybees with fumagillin. Science 1952,
115, 70–71. (d) McCowen, M. C.; Callender, M. E.; Lawlis, J. F., Jr.
Cytosolic Cytochrome c Release. Cells with or without treat-
ment were harvested by centrifugation at 600g for 10 min. After
being washed once with ice-cold PBS, cell pellets were resuspended

7978 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Fang et al.
Fumagillin (H-3), a new antibiotic with amebicidal properties. Science
1951, 113, 202–203.
(21) Wuts, P. G. M.; Thompson, P. A. Preparation of halomethanebor-
onates. J. Organomet. Chem. 1982, 234, 137–141.
(22) Zhang, W.; Gao, W. Y.; Turner, S.; Ducatman, B. S. Gleevec (STI-
571) inhibits lung cancer call growth (A549) and potentiates the
cisplatin effect in vitro. Mol. Cancer 2003, 2, 1–9.
(23) Perez-Cruz, I.; Carcamo, J. M.; Golde, D. W. Vitamin C inhibits
FAS-induced apoptosis in monocytes and U937 cells. Blood 2003,
102, 336–343.
(24) Vaquero, E. C.; Edderkaoui, M.; Pandol, S. J.; Gukovsky, I.;
Gukovskaya, A. S. Reactive oxygen species produced by NAD-
(P)H oxidase inhibit apopotosis in pancreatic cancer cells. J. Biol.
Chem. 2004, 279, 34643–34654.
(25) (a) Bose, J. S.; Gangan, V.; Prakash, R.; Jain, S. K.; Manna, S. K. A
dihydrobenzofuran lignan induces cell death by modulating mito-
chondrial pathway and G2/M cell cycle arrest. J. Med. Chem. 2009,
52, 3184–3190. (b) Croninge, E.; Meeuwsen-De Boer, G. J.; De Graaf,
S.S. N.;Kamps, W. A.;DeBont, E. S.J. M. Vincristine induced apoptosis
inacute lymphoblasticleukemia cells: a mitochondrialpathway regulated
by reactive oxygen species. Int. J. Oncol. 2002, 21, 1339–1345.
(26) Adams, J. M.; Cory, S. Apoptosomes: engines for caspase activa-
tion. Curr. Opin. Cell Biol. 2002, 14, 715–720.
(27) Chen, Q.; Gong, B.; Almasan, A. Distinct stages of cytochrome c
release from mitochondria: evidence for a feedback amplification loop
linking caspase activation to mitochondrial dysfunction in genotoxic
stress induced apoptosis. Cell Death Differ. 2000, 7, 227–233.
(28) Bobba, A.; Atlante, A.; Giannattasio, S.; Sgaramella, P.; Calissano,
P.; Marra, E. Early release and subsequent caspase-mediated degra-
dation of cytochrome c in apoptotic cerebellar granule cells. FEBS
Lett. 1999, 457, 126–130.
(29) Park, J. B.; Schonen, N. N-Caffeoyltyramine arrests growth of
U937 and Jurkat cells by inhibiting protein tyrosine phosphoryla-
tion and inducing caspase-3. Cancer Lett. 2003, 202, 161–171.
(30) Pepper, C.; Hoy, T.; Bently, D. P. Bcl2/Bax ratios in chronic
lymphocytic leukaemia and their correlation with in vitro apopto-
sis and clinical resistance. Br. J. Cancer 1997, 76, 935–938.
(31) (a) Saelens, X.; Festjens, N.; Vande Walle, L.; van Gurp, M.; van
Loo, G.; Vandenabeele, P. Toxic proteins released from mitochon-
dria in cell death. Oncogene 2004, 23, 2861–2874. (b) van Gurp, M.;
Festjens, N.; van Loo, G.; Saelens, X.; Vandenabeele, P. Mitochondrial
intermembrane proteins in cell death. Biochem. Biophys. Res. Com-
mun. 2003, 304, 487–497.
(8) Clark, B. R.; Capon, R. J.; Lacey, E.; Tennant, S.; Gill, J. H.
Polyenylpyrroles and polyenylfurans from an Australian isolate of
the soil ascomycete Gymnoascus reessii. Org. Lett. 2006, 8, 701–704.
(9) (a) Yamagishi, Y.; Matsuoka, M.; Odagawa, A.; Kato, S.; Shindo,
K.; Mochizuki, J. Rumbrin, a new cytoprotective substance
produced by Auxarthron umbrinum. I. Taxonomy, production,
isolation and biological activities. J. Antibiot. 1993, 46, 884–887.
(b) Yamagishi, Y.; Shindo, K.; Kawai, H. Rumbrin, a new cytopro-
tective substance produced by Auxarthron umbrinum. II. Physico-
chemical properties and structure determination. J. Antibiot. 1993,
46, 888–891. (c) Hosoe, T.; Fukushima, K.; Takizawa, K.; Miyaji, M.;
Kawai, K.-I. Three pyrrolyloctatetraenyl-alpha-pyrones from Auxar-
thron conjugatum. Phytochemistry 1999, 52, 459–463.
(10) (a) Clark, B. R.; Lacey, E.; Gill, J. H.; Capon, R. J. The effect of
halide salts on the production of Gymnoascus reessii polyenylpyr-
roles. J. Nat. Prod. 2007, 70, 665–667. (b) Yang, Y.-L.; Liao, W.-Y.;
Liu, W.-Y.; Liaw, C. C.; Shen, C.-N.; Huang, Z.-Y.; Wu., S.-H. Discovery
of new natural products by intact-cell mass spectrometry and LC-SPE-
NMR: malbranpyrroles, novel polyketides from thermophilic fungus
Malbranchea sulfurea. Chem.;Eur. J. 2009, 15, 11573–11580.
(11) Coleman, R. S.; Walczak, M. C. Total synthesis of gymnoconju-
gatins A and B. J. Org. Chem. 2006, 71, 9841–9844.
(12) Coleman, R. S.; Walczak, M. C. Tandem Stille/Suzuki-Miyaura
coupling of a hetero-bis-metalated diene. rapid, one-pot assembly
of polyene systems. Org. Lett. 2005, 7, 2289–2291.
~
(13) (a) Cervello, J.; Marquet, J.; Moreno-Manas, M. Copper and
cobalt mediated regioselective alkylation of polyketide models:
methyl 3,5-dioxohexanoate and triacetic acid lactone. Tetrahedron
1990, 46, 2035–2046. (b) Nagawade, R. R.; Khanna, V. V.; Bhagwat,
S. S.; Shinde, D. B. Synthesis of new series of 1-aryl-1,4-dihydro-4-
oxo-6-methyl pyridazine-3-carboxylic acid as potential antibacterial
agents. Eur. J. Med. Chem. 2005, 40, 1325–1330.
(14) Abbas, S.; Hayes, C. J.; Worden, S. The “Hirao reduction”
revisited: a procedure for the synthesis of terminal vinyl bromides
by the reduction of 1,1-dibromoalkenes. Tetrahedron Lett. 2000,
41, 3215–3219.
(15) Abbaspour Tehrani, K.; Borremans, D.; De Kimpe, N. Synthesis
of 2-acyl-3-chloropyrroles: application to the synthesis of the
trail pheromone of the ant Atta texana. Tetrahedron 1999, 55,
4133–4152.
(32) Martinou, J. C.; Green, D. R. Breaking the mitochondrial barrier.
(16) Jolicoeur, B.; Chapman, E. E.; Thompson, A.; Lubell, W. D.
Pyrrole protection. Tetrahedron 2006, 62, 11531–11563.
(17) (a) Trost, B. M.; Gunzner, J. L.; Dirat, O.; Rhee, Y. H. Callipelto-
side A: total synthesis, assignment of the absolute and relative
configuration, and evaluation of synthetic analogues. J. Am. Chem.
Soc. 2002, 124, 10396–10415. (b) Harris, J. M.; O'Doherty, G. A. An
olefination approach to the enantioselective syntheses of several styr-
yllactones. Tetrahedron 2001, 57, 5161–5171.
Nat. Rev. Mol. Cell Biol. 2001, 2, 63–67.
(33) Kuwana, T.; Mackey, M. R.; Perkins, G.; Ellisman, M. H.; Latterich,
M.;Schneiter, R.;Green, D. R.;Newmeyer, D. D. Bid, Bax, and lipids
cooperate to form supramolecular openings in the outer mitochon-
drial membrane. Cell 2002, 111, 331–342.
(34) Budhram-Mahadeo, V.; Morris, P. J.; Smith, M. D.; Midgley,
C. A.; Boxer, L. M.; Latchman, D. S. p53 suppresses the activation
of the Bcl-2 promoter by the Brn-3a POU family transcription
factor. J. Biol. Chem. 1999, 274, 15237–15244.
€
(18) (a) Ruhmann, A.; Wentrup, C. Synthesis of a photoactivatable
9-Z-oleic acid for protein kinase C labeling. Tetrahedron 1994, 50,
3785–3796. (b) Caprio, V.; Brimble, M. A.; Furkert, D. P. Synthesis of
the novel 1,7,9-trioxadispiro[4.1.5.2]-tetradecane ring system present in
the spirolides. Tetrahedron 2001, 57, 4023–4034.
(35) Miyashita, T.; Krajewski, S.; Krajewska, M.; Wang, H. G.; Lin,
H. K.; Liebermann, D. A.; Hoffman, B.; Reed, J. C. Tumor
suppressor p53 is a regulator of bcl-2 and bax gene expression in
vitro and in vivo. Oncogene 1994, 9, 1799–1805.
(36) Miyashita, T.; Reed, J. C. Tumor suppressor p53 is a direct tran-
scriptional activator of the human bax gene. Cell 1995, 80, 293–299.
(37) (a) Hu, W.; Kavanagh, J. J. Anticancer therapy targeting the
apoptotic pathway. Lancet Oncol. 2003, 4, 921–729. (b) Thompson,
C. B. Apoptosis in the pathogenesis and treatment of disease. Science
1995, 267, 1456–1462.
(19) Clark, B. R.; Lacey, E.; Gill, J. H.; Capon, R. J. The effect of halide
salts on the production of Gymnoascus reessii polyenylpyrroles.
J. Nat. Prod. 2007, 70, 665–667.
(20) Yin, G.; Gao, M.; Wang, Z.; Wu, Y.; Wu, A. Direct conversion of
aromatic ketones to arenecarboxylic esters via carbon-carbon
bond-cleavage reactions. Bull. Chem. Soc. Jpn. 2008, 81, 369–372.