Evelynin, a cytotoxic benzoquinone-type retro -dihydrochalcone from Tacca chantrieri

Article abstract of DOI:10.1021/np100350s

A new benzoquinone-type retro-dihydrochalcone, named evelynin, was isolated from the roots and rhizomes of Tacca chantrieri. The structure was elucidated on the basis of the analysis of spectroscopic data and confirmed by a simple one-step total synthesis. Evelynin exhibited cytotoxicity against four human cancer cell lines, MDA-MB-435 melanoma, MDA-MB-231 breast, PC-3 prostate, and HeLa cervical carcinoma cells, with IC₅₀ values of 4.1, 3.9, 4.7, and 6.3 μM, respectively.

Full text of DOI:10.1021/np100350s

1590
J. Nat. Prod. 2010, 73, 1590–1592
Evelynin, a Cytotoxic Benzoquinone-Type retro-Dihydrochalcone from Tacca chantrieri
Jiangnan Peng,† Evelyn M. Jackson,† David J. Babinski,‡ April L. Risinger,† Gregory Helms,§ Doug E. Frantz,*,‡ and
Susan L. Mooberry*,†,⊥
Department of Pharmacology and Cancer Therapy & Research Center, UniVersity of Texas Health Science Center at San Antonio, San
Antonio, Texas 78229, Department of Chemistry, UniVersity of Texas at San Antonio, San Antonio, Texas 78249, and Department of Chemistry,
Washington State UniVersity, Pullman, Washington 99164
ReceiVed May 24, 2010
A new benzoquinone-type retro-dihydrochalcone, named evelynin, was isolated from the roots and rhizomes of Tacca
chantrieri. The structure was elucidated on the basis of the analysis of spectroscopic data and confirmed by a simple
one-step total synthesis. Evelynin exhibited cytotoxicity against four human cancer cell lines, MDA-MB-435 melanoma,
MDA-MB-231 breast, PC-3 prostate, and HeLa cervical carcinoma cells, with IC50 values of 4.1, 3.9, 4.7, and 6.3 µM,
respectively.
Natural products have long been an important source for new
drug discovery. When considering anticancer drugs, natural products
continue to play a prominent role. On the basis of recent reviews
from the Developmental Therapeutics Program of the National
Cancer Institute (USA), 63% of FDA-approved anticancer drugs
are natural products, semisynthetic compounds derived from a
natural product, and/or a synthetic product designed after a natural
product pharmacophore.1,2 During our search for new anticancer
agents from tropical plants, we found that the lipophilic extract
from the roots of Tacca chantrieri caused microtubule bundling
similar to the cellular effects of Taxol. We isolated the taccalono-
lides A and E, highly acetylated steroids from the roots and
rhizomes of T. chantrieri, and identified them as new microtubule-
stabilizing agents.3 Plants of the Tacca genus are native to tropical
areas, and T. chantrieri is distributed throughout tropical Asiatic
countries including Malaysia, Thailand, and southern China. The
rhizomes of various Tacca species have been used by many cultures
in traditional folk medicine to treat ailments including ulcers, high
blood pressure, and hepatitis.4 T. plantaginea is purported to have
analgesic and anti-inflammatory properties,5 and extracts of T.
leontopetaloides were used in Africa to control slugs, snails, and
roundworms.6
Over the last few decades, many classes of molecules have been
isolated from T. chantrieri including diarylheptanoid glucosides,4
steroidal saponins,7 steroidal glycosides,8–10 and withanolide glu-
cosides.11 Perhaps the most unique and well-studied class of
compounds isolated from Tacca sp. chantrieri, plantaginea, leon-
topetaloides, and integrifolia are the appropriately named taccalono-
lides.5,12,13 The taccalonolides are pentacyclic steroidal compounds
with microtubule-stabilizing properties and antitumor activity.3,14
In the process of isolating and purifying the white, amorphous
solid of the taccalonolides from T. chantrieri, we noticed a yellow
component that coextracted with the taccalonolides through the
process of supercritical fluid extraction and in subsequent hexane
and CH2Cl2 extractions. Solid-phase extraction chromatography
using successive CH2Cl2 and EtO2 extractions allowed successful
separation of the taccalonolides from the yellow compound. The
structure of the new compound was identified through spectroscopic
analysis, and biological evaluations show that it has cytotoxic
activity at low micromolar concentrations.
Figure 1. Structure of evelynin (1) and key HMBC correlations
shown with arrows.
Roots and rhizomes of T. chantrieri were extracted using
supercritical CO2 with MeOH. The extract was subjected to column
chromatography followed by SPE to yield taccalonolides A and E
and evelynin (1).
Evelynin (1) was obtained as a yellow powder, and the molecular
formula was determined as C19H20O7 by HRMS (calcd 360.1209,
measd 360.1229). The 1H NMR spectrum showed signals for four
methoxy groups at δ 3.98, 3.94, 3.93, and 3.80, two vicinal
methylene groups at δ 3.08 (2H, J ) 7.8 Hz) and 2.85 (2H, J )
7.8 Hz), and four aromatic protons at δ 7.59 (dd, J ) 8.4, 1.9 Hz),
7.53 (d, J ) 1.9 Hz), 6.87 (d, J ) 8.4 Hz), and 5.85 (s). The spin
pattern of the aromatic protons indicated a 1,3,4-trisubstituted
phenyl ring. The HMBC correlations of the two methoxy groups
at δ 3.94 and 3.93 with the aromatic carbons at δ 153.4 (C-13)
and 149.1 (C-12), respectively, indicated two of the substitutions
are O-methyl groups. The additional substitution was assigned as
a carbonyl functionality at δ 197.6 (C-9) by its HMBC correlations
with H-15 (δ 7.59) and H-11 (δ 7.53) (see Figure 1). A p-
benzoquinone moiety was suggested by two carbonyl signals at δ
178.2 (C-1) and 187.4 (C-4) and four aromatic signals at δ 157.5
(C-2), 154.8 (C-6), 132.3 (C-5), and 107.1 (C-3) in the 13C NMR
spectrum and was verified by the HMBC correlations shown in
Figure 1. Two methoxy substituents on the p-benzoquinone ring
were readily assigned by the HMBC correlations of CH3O/C-2 and
CH3O/C-6. The connection of the p-benzoquinone and the 3,4-
dimethoxylbenzoyl moiety via a CH2-CH2 bridge was evidenced
by the HMBC correlations between H2-7/C-4, C-5,C-6, C-9 and
H2-8/C-9, C-10. The structure of 1 was determined as depicted in
Figure 1, and a trivial name, evelynin, was given in honor of Evelyn
M. Jackson for her many years’ contribution to this program and
her recent retirement. Evelynin may, thus, be classified as a unique
benzoquinone-type retro-dihydrochalcone.
* To whom correspondence should be addressed. E-mail: Mooberry@
uthscsa.edu. Tel: (210) 567-4788. Fax: (210) 567-4300.
† Department of Pharmacology, University of Texas Health Science
Center at San Antonio.
To confirm the chemical structure and biological activities, 1
was synthesized via a simple one-step procedure as outlined in
Scheme 1. The approach involves the addition of 3-(3,4-dimethoxy-
benzoyl)propionic acid (2) to 2,6-dimethoxy-1,4-benzoquinone (3)
via the well-known silver(I)/persulfate-mediated decarboxylative
radical alkylation of quinones and related compounds.16,17 The
‡ University of Texas at San Antonio.
§ Washington State University.
⊥ Cancer Therapy & Research Center, University of Texas Health Science
Center at San Antonio.
10.1021/np100350s  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/17/2010

Notes
Journal of Natural Products, 2010, Vol. 73, No. 9 1591
Scheme 1. Synthesis of Evelynin via a Catalytic Radical Alkylation Approach
dimethoxybenzoyl)propionic acid (3.6 g, 15 mmol), and AgNO3 (17
mg, 1.0 mmol). The flask was fitted with an addition funnel (125 mL),
a thermocouple, and a nitrogen inlet valve. The flask was purged with
N2 for ∼20 min followed by the addition of degassed MeCN (75 mL)
and H2O (15 mL). The heterogeneous mixture was heated to 60 °C, at
which point a solution of NH4S2O8 (2.7 g, 12 mmol) in H2O (10 mL)
was added dropwise via the addition funnel over a period of ∼25 min.
After complete addition, the reaction was monitored by LC/MS for
product formation. The reaction was heated for a total of 16 h and
then cooled to room temperature. The mixture was extracted with
EtOAc (3 × 50 mL), and the combined organic extracts were washed
with H2O (2 × 50 mL) and 10% brine (20 mL). The organic layer was
dried over Na2SO4 and concentrated to give a crude dark yellow solid
(3.1 g). The product was partially purified via silica gel chromatography
using a mixture of EtOAc/CH2Cl2 (3:97) as eluent to give synthetic
evelynin (350 mg) in ∼90% purity. This was further purified via
recrystallization from hot EtOH to give pure evelynin as a light yellow
crystalline solid (180 mg, 5.0% yield). All spectroscopic data matched
that of natural evelynin (see above). Additional data on synthetic
evelynin: mp )165-168 °C; ESIMS (m/z) 361 (M + H), 383 (M +
Na).
Biological Assays. The MDA-MB-435 human melanoma cancer cell
line was obtained from the Lombardi Cancer Center (Georgetown
University, Washington, DC). The MDA-MB-231 breast, PC-3 prostate,
and HeLa cervical human cancer cell lines as well as the A-10
embryonic rat aortic smooth muscle cell line were purchased from
American Type Culture Collection (Manassas, VA). MDA-MB-435 and
MDA-MB-231 cells were cultured in Richter’s IMEM medium (In-
vitrogen, Carlsbad, CA) supplemented with 10% FBS and 25 µg/mL
gentamicin. HeLa and A-10 cells were cultured in Basal Medium Eagle
(Sigma, St. Louis, MO) with 10% FBS and 50 µg/mL gentamicin. PC-3
cells were grown in RPMI-1640 medium (Invitrogen, Carlsbad, CA)
supplemented with 10% FBS and 50 µg/mL gentamicin. The SRB
assay15 was used to determine the sensitivity of the cancer cell lines to
evelynin as previously described.14 Briefly, a range of concentrations
of synthetic evelynin was added to cells in triplicate and incubated for
48 h. The concentration of evelynin that caused 50% inhibition of cell
proliferation (IC50) was calculated from the linear portion of the log
dose-response curves. The mean IC50 value with standard deviation
was calculated from three independent experiments, each performed
in triplicate. Cellular microtubules were evaluated as previously
described.3
synthetic evelynin obtained via this procedure is spectroscopically
identical to the authentic sample and can be readily obtained in
high purity (>98.5% by LC/MS) via recrystallization from EtOH.
While the overall yield of this unoptimized approach is low (5%),
the simplicity of the one-step procedure from two commercially
available starting materials and its ability to provide multigram
quantities in a relatively short period of time (<1 day) more than
compensate for its inefficiency.
The IC50 value for synthetic evelynin in the MDA-MB-435
human melanoma cell line was determined to be 4.1 ( 0.3 µM.
Other cancer cell lines, including the MDA-MB-231 breast cancer
cell line, the PC-3 prostate cancer cell line, and the HeLa cervical
cancer cell line, showed similar sensitivities to evelynin, with IC50
values of 3.9 ( 0.1, 4.7 ( 0.4, and 6.3 ( 0.7 µM, respectively.
Synthesized evelynin was evaluated side by side with the tacca-
lonolides for effects on cellular microtubules in A-10 smooth muscle
cells and in HeLa cells. Unlike the taccalonolides, evelynin had no
observable effects on microtubule density or structure at concentra-
tions up to 40 µM, demonstrating that the cytotoxicity exhibited
by this compound was not a result of disruption of cellular
microtubules (Figure 1 in the Supporting Information).
Experimental Section
General Experimental Procedures. NMR spectra were recorded
on a Bruker Avance 600 MHz or a Varian QE 300 MHz instrument.
All spectra were measured and reported in ppm by using the residual
solvent (CDCl3) as an internal standard. The HRMS was measured using
a Thermo Scientific LTQ Orbitrap mass spectrometer. IR data were
obtained on a Bruker Vector 22 using a Specac Golden Gate ATR
sampler. The UV spectrum was measured on a Varian Cary 5000
UV-vis-NIR spectrophotometer. TLC was performed on aluminum
sheets (silica gel 60 F254, Merck KGaA, Germany). HPLC was
performed on a Waters Breeze HPLC system, and LC/MS was recorded
on an Agilent 1200 series HPLC connected to a 6130 series single-
quad mass spectrometer.
Biological Material. Tacca chantreiri plants were purchased from
a commercial grower. The roots and rhizomes were collected from
living plants grown in greenhouses and stored at 80 °C until lyophilized.
A voucher specimen was used for identification of T. chantrieri and
was deposited in the herbarium of the University of Hawaii.
Extraction and Isolation. Dried and pulverized rhizome and root
material (168.15 g) was extracted in several batches with supercritical
CO2 and MeOH. The crude extracts were washed with hexanes and
extracted with CH2Cl2. The extracts were applied to silica gel columns
(Biotage) for flash chromatography, and the yellow fractions, containing
evelynin and the taccalonolides, were dried. Extracts were dissolved
in CH2Cl2 for solid-phase extraction on silica cartridges (Burdick and
Jackson). Evelynin was eluted with CH2Cl2; 59 mg of material was
recovered and analyzed as described below.
Acknowledgment. This work was supported by NCI CA121138
(S.L.M.), DOD-CDMRP Postdoctoral Award BC087466 (A.L.R.), and
the NCI P30 CA054174 (S.L.M.). D.E.F. thanks the University of Texas
at San Antonio for a generous startup package.
Supporting Information Available: NMR spectra of compound 1.
This information is available free of charge via the Internet at http://
pubs.acs.org.
Evelynin (1): yellow powder; IR (film) νmax 3069, 2950, 1682,
References and Notes
1664, 1596, 1514, 1417, 1274, 1082, 1023 cm-1; UV λmax (log ε)
1
274 (3.97) nm; H NMR (600 MHz, CDCl3) δ 7.59 (dd, 8.4, 1.9,
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NP100350S