Article

pubs.acs.org/jnp

Dehydroleucodine, a Sesquiterpene Lactone from Gynoxys verrucosa,

Demonstrates Cytotoxic Activity against Human Leukemia Cells
Paola E. Ordóñez,†,§,∥ Krishan K. Sharma,‡ Laura M. Bystrom,‡ Maria A. Alas,‡ Raul G. Enriquez,⊥
Omar Malagón,§ Darin E. Jones,∥ Monica L. Guzman,*,‡ and Cesar M. Compadre*,†
†
Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, United States
‡
Division of Hematology/Oncology, Department of Medicine, Weill Cornell Medical College, New York, New York 10065, United
States
§
Departamento de Química, Universidad Técnica Particular de Loja, Loja, Ecuador
⊥
Instituto de Química, Universidad Nacional Autónoma de México, México DF, México
∥
Department of Chemistry, University of Arkansas at Little Rock, Little Rock, Arkansas 72205, United States
*
S Supporting Information

ABSTRACT: The sesquiterpene lactones dehydroleucodine (1) and leucodine (2) were isolated from Gynoxys verrucosa, a
species used in traditional medicine in southern Ecuador. The activity of these compounds was determined against eight acute
myeloid leukemia (AML) cell lines and compared with their activity against normal peripheral blood mononuclear cells.
Compound 1 showed cytotoxic activity against the tested cell lines, with LD50 values between 5.0 and 18.9 μM. Compound 2 was
inactive against all of the tested cell lines, demonstrating that the exocyclic methylene in the lactone ring is required for cytotoxic
activity. Importantly, compound 1 induced less toxicity to normal blood cells than to AML cell lines and was active against
human AML cell samples from five patients, with an average LD50 of 9.4 μM. Mechanistic assays suggest that compound 1 has a
similar mechanism of action to parthenolide (3). Although these compounds have significant structural differences, their
lipophilic surface signatures show striking similarities.

A cute myeloid leukemia (AML) is a fatal disease with an

overall five-year survival rate of 19.8%.1 In the United
States, over 12 000 adults are diagnosed with AML each year.2
The treatment regimens for AML patients have remained
relatively stagnant for the last three decades.1 Despite achieving
complete remission after aggressive multiagent chemotherapy
and allogeneic stem cell transplantation, most patients relapse
and die of this disease.3 AML is a genetically heterogeneous
disease consisting of leukemic stem, progenitor, and blast cell
populations. Common chemotherapeutic agents, such as
cytosine arabinoside (ara-C), target only actively cycling cells;
thus, they fail to eliminate quiescent leukemic stem cell (LSC)
populations.4 The failure to eliminate LSCs is thought to
provide a reservoir for disease relapse. To effectively target all
of the AML subpopulations, including leukemic stem and
progenitor cells, new chemotherapeutic drugs are urgently
required. Furthermore, common chemotherapy is significantly
toxic and can have adverse side effects.5
© 2016 American Chemical Society and
American Society of Pharmacognosy
Naturally occurring compounds are one of the most
promising sources of new drugs for cancer treatment. Their
large structural diversity and compatibility with biological
systems make them one of the best sources of therapeutic
agents for cancer treatment. A recent review reports that 49%
of the small molecules approved for cancer from the 1940s to
2014 are either natural products or directly derived from
natural products.6 Found in many plants, sesquiterpene
lactones (SLs) are a class of compounds exhibiting a wide
range of biological activities such as anti-inflammatory,
antiparasitic, antibacterial, and cytotoxic effects.7−11
Parthenolide (3), a SL extracted from the medicinal herb
feverfew (Tanacetum parthenium), possesses antileukemic
activity. Compound 3 induces several intracellular effects,
including the generation of reactive oxygen species and the
inhibition of the nuclear factor kappa-light-chain-enhancer of
Received: May 2, 2015
Published: April 8, 2016
691 DOI: 10.1021/acs.jnatprod.5b00383
J. Nat. Prod. 2016, 79, 691−696
Journal of Natural Products Article

activated B cells (NF-κB), which are critical for the survival of

AML stem and progenitor cells.4,12,13 However, the potential
clinical applications of 3 have been hindered because of its
limited stability,14 water solubility,15,16 and bioavailability.12
Recently it has been shown that other SLs can inhibit AML
stem and progenitor cell growth.8 However, the structural
characteristics of SLs required to target all of the AML
subpopulations, including stem cells, remain unclear. Thus, it
would be advantageous to discover and evaluate the
antileukemic potential of additional SLs for therapeutic
purposes and for mechanistic studies.

In this context, the activity of the extracts from Gynoxys Figure 1. ORTEP projections of the X-ray crystal structure of
verrucosa V.M. Badillo, a shrub from the family Asteraceae, was compound 2 with 50% probability ellipsoids.
tested against a battery of AML cells. The aerial parts of this
plant, commonly known as guángalo, are used in traditional Table 1. Cytotoxic Effects of Compounds 1−3 and the G.
medicine in southern Ecuador to treat skin infections and to verrucosa Extract, Expressed as LD50 Valuesa
promote wound healing by direct application to the skin.9,17
From the extract exhibiting activity against leukemia cells two G. verrucosa EtOAc extract (μg/ 1 2 3
mL) (μM) (μM) (μM)
SLs were isolated: dehydroleucodine (1), which shows
HL-60 4.3 14.1 >20 13.9
cytotoxic activity, and leucodine (2), which has no discernible
Kasumi-1 3.5 12.9 >20 3.6
activity. In this study it was also determined that 1 inhibited the
KG-1 7.1 18.7 >20 >20
activation of NF-κB in a manner similar to 3.

■
MOLM-13 3.1 12.6 >20 3.4
RESULTS AND DISCUSSION MV4-11 3.0 5.0 >20 8.6
THP-1 7.0 16.8 >20 >20
Compounds 1 and 2 were isolated from G. verrucosa by TUR 5.7 12.2 >20 7.4
following a previously reported procedure.9 The X-ray crystal U937 6.0 18.9 >20 8.4
structure of 2 was determined, demonstrating that its structure
is similar to that of compound 1, except for the presence of two
a
Tested range for pure compounds: 1.25−160 μM.
hydrogens at C-13 and C-12. The hydrogen attached to C-12 is
above the approximate plane of the seven-membered ring. could dramatically increase their specificity against particular
Therefore, the relative configuration of 2 is confirmed and it is cell populations.
consistent with the previously reported single X-ray diffraction When determining the potential use of a new chemo-
analysis of this compound.18 However, in this research the therapeutic agent in patients, it is desirable for the drug to
absolute configuration for molecule 2 was determined based on effectively target tumor cells without causing significant harm to
its Flack parameter (0.01(4)) as 8S, 9S, 10S, and 12S; a noncancerous cells. Thus, to better establish the potential of 1
perspective ORTEP plot is shown in Figure 1. as an antileukemic drug, its activity and that of compound 2
The results in Table 1 show the activity of the G. verrucosa were tested on normal bone marrow (n = 1) and peripheral
extract as well as compounds 1−3 against eight human blood (n = 3) mononuclear cells. Figure 2A displays the
leukemia cell lines. Although this is the first report of the viability of the normal samples after 48 h of treatment with 20
potential antileukemic activity of 1, its activity has been μM dehydroleucodine (1). Compound 3 and the G. verrucosa
evaluated in other models including KB (IC50 of 5.3 μM),19 extract were included as references, compound 3 being a well-
HeLa S3 (IC50 of 10.0 μM), and MCF-7 (IC50 of 5.0 μM) cell known anticancer compound.12,13 In a similar way to when
lines.20 Compound 1 has also shown an effect on the tested against AML cell lines 2 displayed no toxicity against
proliferation of B16 melanoma and Melan-A cells,21 and it normal cells. Importantly, Figure 2B shows that 1 was
has inhibited the activation of LAD2 mast cells.22 significantly more potent to the tested AML cell lines (n =
As shown in Table 1, compound 1 has cytotoxic activity 8) than to normal mononuclear cells (n = 4, p = 0.004). These
against multiple AML cell lines after 48 h of treatment, with data strongly suggest that 1 represents a promising chemo-
LD50 values ranging from 5.0 to 18.9 μM. Compound 2 was therapeutic agent.
inactive against all of the leukemia cell lines, demonstrating that To better understand the mechanism by which 1 kills AML
the exocyclic methylene in 1 is required for the observed cells, the intracellular effects upon treatment with compounds 1
activity. Table 1 shows that the activity profiles of 1 and 3 are and 2 were tested using quantitative PCR (Figure 3). Leukemia
similar. However, 3 was inactive against the KG-1 and THP-1 cells were treated with 20 μM of each test compound for 6 h
cell lines, while 1 was active against all the cell lines. These before RNA extraction. As the reported mechanism of action of
results show clearly that structural modification of the SLs 3 involves an increase in oxidative stress and inhibition of NF-
692 DOI: 10.1021/acs.jnatprod.5b00383
J. Nat. Prod. 2016, 79, 691−696
Journal of Natural Products Article

Figure 2. Compound 1 is significantly less potent in normal peripheral blood mononuclear cells (PBMNCs) than in leukemic cells. (A) Viability of
normal cells after treatment with 1 and its derivatives. Bone marrow and PBMNCs were isolated from healthy donors and treated for 48 h with 20
μM compound 1 or 3 or with 50 μM compound 2. Cells were also treated with 10 μg/mL G. verrucosa extract as a reference. Viability was evaluated
by annexin V and 7AAD staining using flow cytometry. Each line represents a distinct sample with viabilities calculated relative to an untreated
control. (B) Average viability of AML cell lines compared with normal BM/PBMNCs treated with 20 μM compound 1 after 48 h. The percent
viabilities of the eight cell lines from Table 1 at 20 μM compound 1 were averaged and graphed next to the average percent viability of the four
healthy donor samples. The significance between the two groups was calculated using the Mann−Whitney test, p = 0.004.

Figure 3. Compound 1 induced HMOX1 and HSPA1A and downregulated NF-κB. This graphic represents the fold changes of (A) HMOX1, (B)
HSPA1A, and (C) NFkB1A gene expression in MOLM-13 cells. Experiments were performed in triplicate. Lines represent the mean for each
specimen, with error bars representing the SD. The fold changes were calculated by the delta−delta Ct method. (D) DNA-binding ELISAs for NF-
κB (p65) activity for compounds 1−3. TNFα was used as a positive control. Experiments were performed in triplicate. Lines represent the mean for
each specimen, with error bars representing the SD. The fold changes were calculated by the delta−delta Ct method. (E) Fold change in gene
expression for known NF-κB target genes (CDK6, MYB). (F) Immunoblot for MOLM-13 cells after 6 h of treatment with either compound 1 or 3
at 5 or 10 μM. The blot was probed for phospho-p65 and p65 antibodies. Total p65 is shown as the loading control.

κB,12 the ability of 1 to perturb these pathways was studied.
Heme oxygenase 1 (HMOX1) and the primary stress-inducible
isoform of Hsp70 (HSPA1A) were investigated to determine
the activation of an oxidative stress response upon drug
treatment (Figure 3A and B, respectively). It was found that 1
upregulated HMOX1 and HSPA1A, as observed with 3,
693
suggesting that compound 1 can activate oxidative stress
responses as reported for compound 3.12 However, the higher
fold changes by treatment with 1 suggest that this compound
may induce a greater amount of oxidative stress than 3.
Furthermore, compound 2 moderately upregulated HMOX1
and HSPA1A.
DOI: 10.1021/acs.jnatprod.5b00383
J. Nat. Prod. 2016, 79, 691−696
Journal of Natural Products Article

Compound 3 has been shown to induce apoptosis on mented in SYBYLX 2.1.1 (Certara, St. Louis, MO, USA) was
leukemic stem cells and progenitor cells through inhibition of performed.
NF-κB.5 Thus, the levels of NFKB1A transcript upon drug Figure 5 compares the lipophilic surface signatures of
treatment (Figure 3C) were evaluated. Treatment with compounds 1 and 3, showing a strong and clearly delineated
compounds 1−3 resulted in downregulation of NFKB1A
transcription. To better understand their effects on the activity
of NF-κB, compounds 1 (the lead compound from G.
verrucosa), 2 (inactive against leukemia cells and lacking the
exocyclic methylene), and 3 (reported NF-κB inhibitor) were
studied using a DNA-binding ELISA assay to directly evaluate
the DNA-binding activity of NF-κB. From this analysis, it was
found that compounds 1 and 3 could inhibit NF-κB (Figure
3D), while compound 2 does not significantly affect its activity.
In addition, the levels of phospho-p65 and total p65 upon drug
treatment were evaluated using a Western blot analysis (Figure
3D). This activity was confirmed by evaluating other reported
NF-κB target genes (MYB and CDK6)23 (Figure 3E). The p65
transcription factor is involved in NF-κB heterodimer
formation, and the phosphorylation of p65 is essential for
NF-κB activation. Strikingly, 1 and 3 induced a significant
decrease in phospho-p65, as shown in Figure 3F, where total
p65 is shown as a control. Owing to its mechanistic similarities
to compound 3 and its ability to decrease phospho-p65,
compound 1 may be useful in the treatment of leukemic
progenitor and stem cells. Figure 5. (A) X-ray crystal structures of 1 and 3 and (B) their
The use of tumor-derived cell lines, such as those used to corresponding lipophilic surface signatures.
screen the activity of compound 1, presents many practical
advantages. However, these cell lines may not be representative
lipophilic area at the top of the convex part of the molecules
of the cell phenotypes encountered when evaluating potential
and a more hydrophilic surface at the bottom of the concave
drugs in vivo.24 Thus, to further establish the potential of
part of the structures. Interestingly, the convex lipophilic part of
compound 1 to treat AML, its cytotoxic activity was tested
the molecule is where the highest similarity occurs, and the
against primary leukemia cells isolated from seven different
concave part is where there are the greatest differences. The
AML patients. Figure 4 shows that compound 1 induced cell
implication of these findings is that the SL receptor should have
death in all of the primary samples tested, with an average LD50
a complementary lipophilic area, possibly explaining why these
of 9.4 μM. substantially different compounds appear to interact with the
same receptors.
Although, 1 is mechanistically very similar to 3, compound 1
has some advantages that bode well for its potential
development as an antileukemic drug. Compound 1 showed
cytotoxicity against AML cell lines and against human AML cell
samples from patients. As shown in Figure 2A, compound 1 is
slightly less toxic to normal cells than 3, but as shown in Table
1, compound 1 is more toxic than 3 to most of the AML cell
lines. Importantly, the epoxide ring that is responsible for the
instability of 328 is not present in compound 1. Additionally, 1
is present in high yield (0.35%), and it is easily isolated from G.
verrucosa as an abundant plant species.
Figure 4. Activity of dehydroleucodine (1) in primary samples
compared with cytosine arabinoside (Ara-C). Each symbol represents
a primary AML sample tested (compound 1, n = 7; Ara-C, n = 5).
■ EXPERIMENTAL SECTION
General Experimental Procedures. Column chromatography
was performed using 60−230 mesh silica gel. Preparative TLC was
performed on precoated silica gel 60 F254 plates (0.2 mm thick, EMD
Millipore, Billierica, MA, USA).
Compounds 1 and 3 have considerable structural differences. Plant Material. Gynoxys verrucosa was collected in June 2007 in the
For example, compound 3 has a 10-membered ring attached to locality of Yangana, in the Loja Province of Ecuador. The plant
a five-membered ring, while compound 1 has a five-membered material was identified by Vladimir Morocho, Ph.D., and voucher
ring attached to a seven-membered ring, which is attached to specimens (PPN-as-11) are deposited at the Herbarium of the
another five-membered ring. However, the fact that these Chemistry Department of the Universidad Técnica Particular de Loja,
Loja Ecuador, and at the Herbarium Reynaldo Espinoza of the
compounds appear to have the same mechanism of action Universidad Nacional de Loja.
suggests that they possess chemical similarities that are Extraction and Isolation. The dried aerial parts of G. verrucosa
recognizable by their receptors. To identify those similarities, were extracted with ethyl acetate at room temperature and
a surface signature analysis of both molecules using their X-ray concentrated under reduced pressure. To remove the chlorophylls,
crystal structures9,25 and the MOLCAD program as imple- the crude extract was filtered through a reversed-phase C18 column

694 DOI: 10.1021/acs.jnatprod.5b00383

J. Nat. Prod. 2016, 79, 691−696
Journal of Natural Products
with a mixture MeOH−H2O (85:15). The filtrate was fractioned by
column chromatography using silica gel and a hexane−EtOAc
gradient. Dehydroleucodine (1) was eluted in the hexane−EtOAc
(85:15) fraction and recrystallized from EtOAc as a white crystalline
solid. Leucodine (2) was isolated from the mother liquid using
preparative TLC hexane−EtOAc (70:30) and recrystallized from
EtOAc. The identification of these compounds was done by direct
comparison with an authentic sample.9
Single-Crystal X-ray Diffraction Analysis and Crystallo-
graphic Data for Compound 2. Crystallographic data for all of
the structures were deposited in the Cambridge Crystallographic Data
Centre, and the deposition number is listed below. The data can be
obtained free of charge at www.ccdc.cam.ac.uk (or from the
Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge
CB2 1EZ, UK; fax: (+44) 1223-336-033; or desposit@ccdc.cam.a-
c.uk).
Crystallographic data of 2: crystallization from ethyl acetate
yielded colorless crystals of 2. X-ray data were collected on a Bruker-
Nonius X8 Proteum CCD diffractometer using Cu Kα radiation. The
structures were solved using SHELXT and were refined using
SHELXL.26 Crystal data: C15H18O3, Mw = 246.29 g mol−1, space
group P212121, a = 7.5528(2) Å, b = 11.7831(3) Å, c = 14.1030(3) Å,
α = β = γ = 90.00°, V = 1255.10(5) Å3, T = 90 K, Z = 4, Dc = 1.303 g/
cm3, R1 = 0.0284 (wR2 = 0.0753); Flack parameter = 0.01(4).
Deposition number: CCDC 1047867.
Cell Lines and Cell Culture. Bone marrow and peripheral blood
samples were obtained from human donors with informed consent
under Weill Cornell Medical College IRB 0909010629 approval.
Mononuclear cells were isolated from the samples using Ficoll-Plaque
(Pharmacia Biotech, Piscataway, NY, USA) density gradient
separation. Cells were cultured in serum-free medium supplemented
with cytokines (50 ng/mL rhFLT-3 ligand, 50 ng/mL rhSCF, 20 ng/
mL rhIL-3, and 20 ng/mL rhIL-6) for 1 h before the addition of drugs.
The HL-60 (purchased 9/2010, ATCC), Kasumi-1 (purchased 4/
2011, ATCC), KG-1 (purchased 9/2010, ATCC), MOLM-13 (kind
gift from G. Chiosis-MSKCC 7/2010, 2/2014 authenticated; biosyn-
thesis), MV4-11 (purchased 9/2010, ATCC), THP-1 (purchased 9/
2010, ATCC), TUR (purchased 1/2010, ATCC), and U937
(purchased 12/2009, ATCC) cell lines were cultured in Iscove’s
modified Dulbecco’s medium (Life Technologies, Carlsbad, CA, USA)
supplemented with 10−20% fetal bovine serum, according to the
culture conditions indicated by ATCC, and were supplemented with
1% penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA) at
37 °C and 5% CO2.
Flow Cytometry and Cytotoxicity Assays. The cells were
seeded into 96-well plates and were maintained at a concentration of
0.5 million cells per mL. The samples were treated with compounds
1−3 at varying concentrations in triplicate. Cell counting for these
experiments was performed using the Invitrogen Cell Countess system
with Trypan Blue stain. Compound 3 was obtained from Biomol
(Plymouth Meeting, PA, USA). Cell viability was determined after
incubating for 48 h. The cells were stained with annexin V-fluorescein
isothiocyanate or phycoeritrine and 7-aminoactinomycin (7-AAD) all
from Molecular Probes-Invitrogen (Carlsbad, CA, USA) to detect
phosphatidylserine exposition and cell permeability, respectively. At
least 50 000 events were recorded per condition on either an LSR-II or
an LSR-Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA).
Data analysis was conducted using FlowJo 9.6 software for Mac OS X
(Tree Star, Ashland, OR, USA). Cells negative for annexin V and 7-
AAD were scored as viable. Analyses and graphs were produced using
the GraphPad Prism software (La Jolla, CA, USA).
RNA Extraction and Quantitative PCR. MOLM-13 cells were
seeded at 0.5 million cells per mL. Cells were treated for 6 h with 20
μM compounds 1−3 and then collected. Total RNA was extracted
using the Qiashredder and the Qiagen RNeasy Mini kits (Qiagen,
Hilden, Germany) according to the manufacturer’s protocol. Real-time
PCR was performed using the Taqman RNA-to-CT 1-Step kit
(Applied Biosystems, Waltham, MA, USA). The thermal cycling
conditions were as follows: one RT step (48 °C, 15 min), one enzyme
activation step (95 °C, 10 min), and 40 cycles of denaturation (95 °C,
695
Article
15 s) and annealing/extension (60 °C, 1 min). Quantitative PCR was
performed using probes for HMOX1 (Hs01110251_m1), HSPA1A
(Hs00359163_s1), and NFKB1 (Hs00765730_m1). GADPH
(Hs02758991_g1) was used as a housekeeping gene, which is an
internal control to normalize the variability in the expression levels. All
of the probes were provided by Applied Biosystems. Single-plex real-
time PCR was performed in triplicate in a StepOne Plus RealTime
PCR system (Applied Biosystems), and the PCR products were
analyzed with the StepOne Software (Applied Biosystems). The fold
changes were calculated using the 2-DDCT method described by
Livak and Schmittgen.27
Western Blotting. MOLM-13 cells were seeded at 0.5 million cells
per mL and were subjected to treatment with either 10 or 20 μM
compound 1 or 3 for 6 h. Cells were lysed in protein lysis buffer (50
mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1
mM EDTA, 5 mM β-glycerophosphate, 1 mM Na3VO4, 1 mM NaF,
and Set I protease inhibitor cocktail (EMD Millipore)) on ice for 30
min. Protein lysates were resolved on a 10% SDS-PAGE gel
(Invitrogen, Carlsbad, CA, USA) and transferred to an Immobilon-
FL polyvinylidene difluoride membrane (EMD Millipore). The
membrane was incubated with primary antibodies at 4 °C overnight,
then washed and incubated with IRDye 680 goat anti-rabbit or IRDye
800CW goat anti-mouse secondary antibodies (Li-COR, Lincoln, NE,
USA) at room temperature for 30 min. The membrane was then
washed with phosphate-buffered saline with 0.1% Tween 20X (Sigma-
Aldrich, St. Louis, MO, USA). The membrane was scanned using the
Odyssey infrared imaging system (Li-COR). Phospho-NF-κB p65
mouse primary antibody (Cell Signaling, Danvers, MA, USA) and NF-
κB p65 rabbit primary antibody (Cell Signaling) were used separately
to stain the membranes.
■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00383.
Crystallographic data (CIF)
■
AUTHOR INFORMATION
Corresponding Authors
*Tel (M. L. Guzman): +1 (212) 746-6838. Fax: +1 (212) 746-
8154. E-mail: mlg2007@med.cornell.edu.
*Tel (C. M. Compadre): +1 (501) 686-6493. Fax: +1 (501)
686-6057. E-mail: compadrecesarm@uams.edu.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This research was supported in part by the University of
Arkansas for Medical Sciences College of Pharmacy Research
Fund, the Irma T. Hirschl/Monique Weill-Caulier Trust and by
the Arkansas Science and Technology Authority, Project No.
15-B-01. P.E.O. is a recipient of a graduate student scholarship
from Ecuador’s Higher Education, Science, and Technology
Ministry (SENESCYT). M.L.G. is a recipient of the NIH
Director’s New Innovator Award Program, 1 DP2 OD007399-
01. We also thank Dr. V. Morocho of Universidad Técnica
Particular de Loja for his help identifying G. verrucosa.
■
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696 DOI: 10.1021/acs.jnatprod.5b00383

J. Nat. Prod. 2016, 79, 691−696