Synthesis and cancer chemopreventive activity of zapotin, a natural product from Casimiroa edulis

Article abstract of DOI:10.1021/jm060915+

An efficient method has been developed to synthesize zapotin (5,6,2′,6′-tetramethoxyflavone), a component of the edible fruit Casimiroa edulis, on a multigram scale. The synthesis utilizes a regioselective C-acylation of a dilithium dianion derived from a substituted o-hydroxyactophenone to afford a β-diketone intermediate that can be cyclized to zapotin in good overall yield, thus avoiding the inefficient Baker-Venkataraman rearrangement pathway. Zapotin was found to induce both cell differentiation and apoptosis with cultured human promyelocytic leukemia cells (HL-60 cells). In addition, the compound inhibits 12-O-tetrade-canoylphorbol 13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity with human bladder carcinoma cells (T24 cells), and TPA-induced nuclear factor-kappa B (NF-κB) activity with human hepatocellular liver carcinoma cells (HepG2 cells). These data suggest that zapotin merits further investigation as a potential cancer chemopreventive agent.

Full text of DOI:10.1021/jm060915+

350
J. Med. Chem. 2007, 50, 350-355
Synthesis and Cancer Chemopreventive Activity of Zapotin, a Natural Product from Casimiroa
edulis
Arup Maiti,† Muriel Cuendet,† Tamara Kondratyuk, Vicki L. Croy, John M. Pezzuto,‡ and Mark Cushman*
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, College of Pharmacy,
Nursing and Health Sciences, and The Purdue Cancer Center, Purdue UniVersity, West Lafayette, Indiana 47907
ReceiVed July 31, 2006
An efficient method has been developed to synthesize zapotin (5,6,2′,6′-tetramethoxyflavone), a component
of the edible fruit Casimiroa edulis, on a multigram scale. The synthesis utilizes a regioselective C-acylation
of a dilithium dianion derived from a substituted o-hydroxyactophenone to afford a â-diketone intermediate
that can be cyclized to zapotin in good overall yield, thus avoiding the inefficient Baker-Venkataraman
rearrangement pathway. Zapotin was found to induce both cell differentiation and apoptosis with cultured
human promyelocytic leukemia cells (HL-60 cells). In addition, the compound inhibits 12-O-tetrade-
canoylphorbol 13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity with human bladder
carcinoma cells (T24 cells), and TPA-induced nuclear factor-kappa B (NF-κB) activity with human
hepatocellular liver carcinoma cells (HepG2 cells). These data suggest that zapotin merits further investigation
as a potential cancer chemopreventive agent.
Introduction
evaluate the potential of zapotin (1) as a chemopreventive agent
in various biological systems and to devise and execute a
practical synthesis of zapotin (1) that would afford gram
quantities for more advanced biological testing.
Cancer is currently the second most common cause of death
in the United States, and it is likely to become the most common
in the near future.1 Chemoprevention is the use of either
synthetic drugs or natural products to inhibit, reverse, or suppress
the development of invasive malignant cancer, either by blocking
the DNA damage that initiates carcinogenesis or by arresting
or reversing the progression of premalignant cells in which DNA
damage has already started. Chemoprevention is one of the most
direct ways to reduce cancer-related morbidity and mortality.2
Several food-based chemopreventive agents have shown promise
in clinical trials.3 The medicinal value of zapote blanco, a fruit
of Casimiroa edulis Llave and Lex (Rutaceae) that is consumed
in many parts of the world, was first discovered by Aztecs, and
crude plant extracts of the seeds or leaves of Casimiroa edulis
were later found to affect blood pressure,4-6 cardiac activity,4-6
aortic muscular tone,7 and to possess anticonvulsant activity.8,9
Recently, zapotin (1), a polymethoxylated flavonoid isolated
Limited quantities of zapotin (1) are available from natural
sources as well as from several poor-yielding syntheses that
arebasedontheBaker-Venkataramanrearrangementpathway.11-14
In general, the Baker-Venkataraman flavone synthesis (Scheme
1) involves the O-acylation of o-hydroxyacetophenone 2 with
a benzoyl chloride 3 to afford an ester intermediate 4, which
rearranges to a â-diketone 5 under basic conditions. Cyclization
of the â-diketone 5 then affords the desired flavone 6. The main
disadvantages of this method of flavone synthesis are that it
involves an indirect rearrangement pathway to yield the â-dike-
tone intermediate 5, and it often results in the formation of
3-aroylflavones instead of the desired 3-unsubstituted flavones.15
In order to circumvent these difficulties, a direct synthesis of
the required â-diketone was investigated in which a dilithium
dianion of an appropriately substituted o-hydroxyacetophenone
would be directly acylated regioselectively on carbon, and the
resulting â-diketone would be cyclized to afford zapotin (1).16-18
Chemistry. In practice, the present synthesis of zapotin (1)
was executed in a straightforward manner without any unex-
pected difficulties as outlined in Scheme 2. Commercially
available 2-hydroxy-6-methoxyacetophenone (7) was subjected
to Elbs oxidation19 using sodium persulfate and aqueous sodium
hydroxide to yield the substituted acetophenone 8, followed by
regioselective methylation using anhydrous potassium carbonate
and dimethyl sulfate in acetone to afford 6-hydroxy-2,3-
dimethoxyacetophenone (9) in 53% yield in two steps. Inter-
mediate 9 is a known compound that has been synthesized
previously,19 but the reported reaction conditions were modified
as described in the Experimental Section. The yields of both
steps increase significantly when the mixtures are stirred for a
prolonged period at room temperature; however, over-oxidation
of the intermediate hydroquinone 8 was detected if the oxidation
was carried out for more than one week. The generation of the
dilithium dianion 10 of the acetophenone 9 was ensured by
treatment with four equivalents of lithium hexamethyldisily-
lazide in THF. Treatment of dilithium dianion 10 with com-
from zapote blanco seeds, has been found to be a nontoxic
inducer of cellular differentiation with cultured HL-60a promy-
elocytic cells.10 Zapotin (1), therefore, has the potential to inhibit
carcinogenesis. The goals of the present investigation were to
* To whom correspondence should be addressed. Tel 765-494-1465, fax
765-494-6790, e-mail cushman@pharmacy.purdue.edu.
† Contributed equally to this work.
‡ Current address: University of Hawaii at Hilo, College of Pharmacy,
Hilo, Hawaii 96720.
a Abbreviations: TPA, 12-O-tetradecanoylphorbol 13-acetate; ODC,
ornithine decarboxylase; T24 cells, human bladder carcinoma cells; NF-
κB, nuclear factor-kappa B; HepG2 cells, human hepatocellular liver
carcinoma cells; HL-60 cells, promyelocytic leukemia cells; DAPI, 4′,6-
diamidino-2-phenylindole; LiHMDS, lithium hexamethyldisilazide; BrdU,
5-bromo-2-deoxyuridine.
10.1021/jm060915+ CCC: $37.00 © 2007 American Chemical Society
Published on Web 12/23/2006

Synthesis and Cancer ChemopreVentiVe ActiVity of Zapotin
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 351
Scheme 1
stimuli including growth factors, steroid hormones, cAMP-
elevating agents, and tumor promoters.20,21 A number of factors
finely tune ODC activity, including the expression, stability,
and transcription rate of ODC messenger RNA, the stability
and translation rate of the ODC enzyme, and also the post-
translational modification of the enzyme.22 Since ODC activity
is essential for proliferation of normal cells, and, on the other
hand, is overexpressed in various cancer cell lines, it is now
recognized that inhibition of ODC may be a good strategy for
cancer chemoprevention and chemotherapy.23,24 Zapotin (1) was
therefore tested for inhibition of the induction of ODC activity
by TPA using human bladder carcinoma T24 cells and was
found to be active, with an IC50 of 3.4 ( 1.7 µM. This activity
is comparable to standard inhibitors of TPA-induced ODC
activity, such as apigenin (IC50 6.0 µM), menadione (IC50 8.3
µM), and deguelin (IC50 0.1 µM).
Scheme 2a
Inhibition of TPA-induced NF-κB Activity in HepG2 Cells.
Nuclear factor-κB (NF-κB) is an inducible transcription factor
for genes involved in cell survival, cell adhesion, inflammation,
differentiation, and growth.25-28 In normal cells, NF-κB present
in the cytoplasm binds to the inhibitory IκB proteins, which
blocks the nuclear localization sequences of NF-κB.29 Activation
of NF-κB by a variety of stimuli, such as carcinogens,
inflammatory agents, tumor promoters including cigarette
smoke, phorbol esters, and okadaic acid, promote degradation
of IκBR and thus unmasks the nuclear localization sequences,
permitting NF-κB to enter the nucleus and bind to a specific
sequence in DNA, which in turn results in transcription of
targeted genes. Various genes that are involved in tumor cell
invasion and angiogenesis have been found to be regulated by
NF-κB. During the past few years, several reports have shown
that activation of NF-κB promotes cell survival and proliferation,
and down regulation of NF-κB sensitizes the cell to apoptosis.
Thus, agents that suppress NF-κB activation can abrogate
carcinogenesis. This has encouraged a search for specific
inhibitors of NF-κB activation from natural sources, which might
lead to a good candidate for cancer chemoprevention. Zapotin
(1) was tested, and it displayed promising inhibition of TPA-
induced NF-κB activity in HepG2 cells stably transfected with
NF-κB-luciferase plasmid with an IC50 value of 7.6 ( 3.3 µM.
Induction of Differentiation by Zapotin (1) in HL-60 Cell
Line. The HL-60 cell line is used as a model system to
investigate the mechanism of cell differentiation, proliferation,
and cell death by inducers. Differentiation-inducing agents
suppress cancer cell self-renewal selectivity from normal stem
cell renewal by inducing terminal differentiation followed by
apoptosis. Inducers of terminal differentiation have shown
promising chemopreventive activity as suppressing agents that
act during the promotion-progression stages of carcinogenesis.
Previous studies performed in our laboratories demonstrated that
zapotin (1) was able to induce differentiation of HL-60 cells in
a concentration-dependent fashion without cytotoxicity.10 Zapo-
tin (1) induced 50% of the cells to differentiate at 0.2 µg/mL
(ED50 0.5 µM), compared to 10 µM required by apigenin and
30 µM by genistein to exert the same activity, representing a
20-60 fold increase in potency. In the current study, cells were
treated with various concentrations of zapotin (1) for 24, 48,
72, or 96 h and harvested after 4 days for evaluation of
enzymatic and cell membrane markers of differentiation.
a Reagents and conditions: (a) (1) NaOH, K2S2O8, 20 °C (7 d), (2) aq
HCl; (b) K2CO3, Me2SO4, Me2CO (7 d); (c) compound 9, LiHMDS, THF,
-78 °C to -10 °C, (3 h), (1.5 h); (d) Compound 11, -78 °C to 23 °C (25
h), (2) aq HCl; (e) H2SO4, AcOH, 95 °C to 100 °C (3.5 h).
mercially available 2,6-dimethoxybenzoyl chloride (11), fol-
lowed by acidification, afforded the â-diketone intermediate 12,
which was used without purification for cyclization to zapotin
(1). The crude intermediate 12 was heated at 100 °C in the
presence of glacial acetic acid containing 0.5% sulfuric acid
for 3.5 h to provide zapotin (1) on multigram scale and also in
high yield (82%). Thus, a short and economical synthesis of
zapotin (1) can be executed in 44% overall yield from a
commercially available starting material, which compares favor-
ably with the previously reported zapotin (1) syntheses that rely
on the Baker-Venkataraman rearrangement.11-14
Biological Results and Discussion
As a part of a comprehensive program for discovery of cancer
chemopreventive agents from natural sources, synthetic zapotin
(1) was tested in several in vitro systems and found to
demonstrate potential chemopreventive activity.
Inhibition of TPA-Induced ODC Activity in T24 Cells.
Ornithine decarboxylase (ODC) is the first and apparently the
rate-limiting enzyme for the biosynthesis of polyamines in
mammalian cells and is highly inducible by growth-promoting
Analysis of NBT (nitroblue tetrazolium)-reduction for evalu-
ation of superoxide formation demonstrated myeloid maturation
in HL-60 cells. The irreversibility of zapotin (1) effects on
growth and differentiation of HL-60 cells was tested using
withdrawal assays during a 4-day experiment. Withdrawal of

352 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Maiti et al.
Figure 3. Dose-dependent induction of apoptosis in HL-60 cells treated
with zapotin (1). Cells were treated with the indicated concentrations
for 24 h. Apoptosis was quantified by counting nuclei stained with
4′,6-diamidino-2-phenylindole (DAPI). Results represent the mean of
two independent studies. *Significantly different from control values
(p < 0.01).
Figure 1. Commitment toward differentiation of HL-60 cells is
obtained at 24 h of exposure to zapotin (1). In each case, total incubation
time was 4 days (96 h), and then cells were analyzed for differentiation
marker. Cells were treated with the specified concentrations of zapotin
(1), which was withdrawn after 24 (9), 48 (diagonal negative slope),
72 (0) or 96 (diagonal positive slope) h. For the 24, 48, and 72 h
exposures, cells were resuspended in fresh complete media for the
remaining time. Results are shown as the mean of duplicate samples.
Figure 4. Cell cycle effects of zapotin (1) in HL-60 cells. Cells were
treated with the indicated concentrations for 24 h, fixed in ethanol,
and stained with PI (propidium iodide) for flow cytometric analysis,
as described in the Experimental Section. Values are expressed as
percentage of total cells and represent the mean ( SD of three
determinations, for the following compartments of the cell cycle: G1
(9), S (diagonal negative slope), G2/M (0), and apoptotic peak sub G1
(diagonal positive slope). *Significantly different from control values
(p < 0.01).
Figure 2. Effect of zapotin (1) on the membrane phenotype of HL-60
cells. As described in the Experimental Section, cells were induced to
differentiate with the indicated concentrations of zapotin (1) using a
4-day protocol and then analyzed for the following membrane markers
of differentiation: CD11b (9), CD13 (diagonal negative slope), CD14
(0), and CD15 (diagonal positive slope). Results are expressed as the
specific mean fluorescence intensity (ratio of antigen antibody fluo-
rescence over isotype antibody fluorescence) and represent the mean
of two independent studies. *Significantly different from control values
(p < 0.05).
phenylindole) staining (Figure 3), which were significant with
doses of 3 µM and higher (p < 0.01). A time-dependent increase
of apoptosis was also observed when cells were treated with
12 µM zapotin (1) (data not shown).
zapotin (1) after 24 h of exposure resulted in the differentiation
of a similar percentage of cells as without withdrawal (Figure
1), while maintaining a higher cellular viability and density.
This indicates that there is no further need for the presence of
the compound after 24 h, at which time cells have become
committed to differentiate. However, cell density was reduced
by increasing the time of exposure to the drug.
Membrane phenotype was also analyzed using flow cytometry
with a set of four myeloid markers (CD11b, CD13, CD14, and
CD15). Zapotin (1) up-regulated CD11b, CD13, and CD14, and
down-regulated CD15 in HL-60 cells (Figure 2). Thus, zapotin
(1) induced a pattern of expression similar to that produced by
macrophage inducers, with down-regulation of CD15 (granu-
locytic marker) and up-regulation of CD13 and CD11b (granu-
locytic/monocytic markers).30
Zapotin (1) Causes Accumulation of HL-60 Cells in S
Phase. The impact of zapotin (1) on the cell cycle progression
of HL-60 cells was determined by flow cytometry. Cell cycle
analysis performed after 24-h incubations of HL-60 cells with
increasing concentrations of zapotin (1) showed that this agent
arrested the cells in the S phase of the cell cycle in a dose-
dependent manner (Figure 4). In the cells treated with 3 µM
zapotin (1), 61% of the cells were in S-phase as compared with
36% in the control population. A complete suppression of cells
in the G2/M phase of the cycle could be noted with concentra-
tions of zapotin (1) as low as 0.75 µM. Also, a dose-dependent
increase of the sub G1 peak, characteristic of apoptosis, was
evident.
To firmly establish this point, control and zapotin (1)-treated
cells were allowed to incorporate BrdU for 30 min prior to
harvest, and the level of incorporation due to new DNA
synthesis was determined by staining with fluorescently labeled
antibody to BrdU prior to analysis by flow cytometry. In Figure
5, total DNA content is presented along the x-axis and BrdU
Induction of Apoptosis by Zapotin (1) in HL-60 Cell Line.
After a 24 h treatment period, zapotin (1) induced dose-
dependent increases in apoptosis, as judged by the formation
of apoptotic bodies observed with DAPI (4′,6-diamidino-2-

Synthesis and Cancer ChemopreVentiVe ActiVity of Zapotin
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 353
Figure 5. Effects of zapotin (1) on BrdU incorporation in HL-60 cells. Cells were treated with DMSO (A), 0.75 µM (B), 1.5 µM (C), 3 µM (D),
6 µM (E), or 12 µM (F) of zapotin (1) and harvested for cell cycle analysis 24 h later. For 30 min prior to harvest, cells synthesizing DNA were
allowed to incorporate BrdU. Cells synthesizing DNA during this period were then labeled using fluorescein-modified antibody to BrdU and were
analyzed by flow cytometry.
under reduced pressure, and the residue was purified by chroma-
tography on silica gel (eluent: CHCl3) to give 3,6-dihydroxy-2-
methoxyacetophenone (8) as a pale greenish-yellow solid (3.2 g,
staining along the y-axis. After treatment with zapotin (1), cells
did continue to enter the early stages of S phase confirming
that zapotin (1) induces an S phase arrest.
1
60%): mp 95 °C (lit 94-97 °C).19 Rf ) 0.25 (SiO2, CHCl3); H
NMR (300 MHz, CDCl3) δ 12.0 (s, 1 H), 7.12 (d, J ) 9.2 Hz, 1
H), 6.67 (d, J ) 9.2 Hz, 1 H), 5.08 (s, 1 H), 3.81 (s, 3 H), 2.70 (s,
3 H). All other data are the same as reported.19
Conclusion
Zapotin (1) was tested for various biological activities and
found to be capable of mediating responses of sufficient
magnitude or selectivity to demonstrate preliminary chemopre-
ventive activity. To help facilitate more advanced biological
investigations, a novel, efficient, multigram synthesis of zapotin
(1) has been devised, which relies of the regioselective C-
acylation of a dilithium dianion generated from an o-hydroxy-
acetophenone.
6-Hydroxy-2,3-dimethoxyacetophenone (9). A mixture of 3,6-
dihydroxy-2-methoxyacetophenone (1.3 g, 7.1 mmol) and oven-
dried potassium carbonate (1.0 g, 7.6 mmol) in dry acetone was
stirred for 10 min. Dimethyl sulfate (0.6 mL, 7.1 mmol) was added
dropwise to the reaction mixture, and the mixture was stirred for 7
d at room temperature. The solvent was evaporated under reduced
pressure, and water (25 mL) was added to the residue. The mixture
was extracted with chloroform (3 × 100 mL), and the organic phase
was dried over Na2SO4 and evaporated under reduced pressure.
The residue was column chromatographed on silica gel eluting with
CHCl3 to afford compound 9 (1.26 g, 89% yield). Rf ) 0.75 (SiO2,
CHCl3); 1H NMR (300 MHz, CDCl3) δ 7.10 (d, J ) 9.3 Hz, 1 H),
6.66 (d, J ) 9.3 Hz, 1 H), 3.92 (s, 3 H), 3.81 (s, 3 H), 2.70 (s, 3
H). All other data are the same as reported.19
Experimental Section
NMR spectra were obtained at 300 MHz (1H) and 75 MHz (13C)
in CDCl3 using CHCl3 as internal standard. Flash chromatography
was performed with 230-400 mesh silica gel. TLC was carried
out using commercially available precoated glass silica gel plates
of 2.5 mm thickness. Melting points are uncorrected. Unless
otherwise stated, chemicals and solvents were of reagent grade and
used as obtained from commercial sources without further purifica-
tion. Tetrahydrofuran (THF) was freshly distilled from sodium/
benzophenone ketyl radical prior to use. Acetone was freshly
distilled from potassium carbonate prior to use.
3,6-Dihydroxy-2-methoxyacetophenone (8). A solution of
2-hydroxy-6-methoxyacetophenone (5.0 g, 30.1 mmol) in 10%
sodium hydroxide (57.5 g in 575 mL H2O) was added dropwise at
room temperature to an aqueous solution of potassium persulfate
(8.2 g, 30.3 mmol) in water (350 mL) with stirring for 7 d at 20
°C. The mixture was cooled in an ice bath, acidified to pH 5-6
with concd HCl, and left overnight at room temperature. The
unreacted starting material was removed by extraction with ethyl
acetate, and the aqueous solution was further acidified to pH 2,
and then heated for 4 h on a water bath after addition of solid
sodium sulfite (5.8 g, 46.0 mmol). The cooled solution was extracted
with chloroform (3 × 100 mL). The organic extract was evaporated
Zapotin (1). A solution of LiHMDS in THF (1 M, 20 mL, 20
mmol) was added to a well-stirred solution of acetophenone 9 (1.0
g, 5.1 mmol) in THF (10 mL) under argon at -78 °C over 15 min.
The reaction mixture was stirred at -78 °C for 1 h and at -10 °C
for 2 h and was cooled again to -78 °C, and a solution of 2,6-
dimethoxybenzoyl chloride 11 (1.38 g, 90% tech. grade, 6.2 mmol)
in THF (10 mL) was added in one portion. Stirring was continued
at -78 °C for 1 h and at room temperature for 24 h (until the
disappearance of the starting material by TLC). The reaction mixture
was poured into a mixture of ice (50 g) and concd HCl (5.4 mL)
and extracted with CHCl3 (3 × 100 mL). The solvent was
evaporated from the dried (Na2SO4) extracts, and the residue was
dried under vacuum for 24 h. A small portion of the crude product
was taken out and purified by column chromatography on silica
gel (eluent EtOAc-hexanes 3:1) to give compound 12: mp 119-
120 °C. Rf ) 0.55 (SiO2, EtOAc-hexanes 3:1); IR (neat) 2939,
1
2838, 1610, 1593, 1574, 1474, 1269, 1254, 1112 cm-1; H NMR

354 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Maiti et al.
(300 MHz, CDCl3) δ 7.30 (t, J ) 8.7 Hz, 1 H), 7.02 (d, J ) 8.7
Hz, 1 H), 6.87 (s, 1 H), 6.67 (d, J ) 8.7 Hz, 1 H), 6.58 (d, J ) 8.4
Hz, 2 H), 3.80 (s, 6 H), 3.78 (s, 6 H); 13C NMR (75 MHz, CDCl3)
δ 193.6, 178.2, 158.0, 155.9, 145.5, 131.5, 120.4, 114.1, 112.7,
105.8, 104.0, 61.5, 57.1, 56.0; EIMS (m/z, relative intensity) 360
(M+, 6), 329 (3), 222 (2), 180 (7), 165 (100), 150 (9), 137 (5), 122
(5), 107 (7); HRMS m/z calcd for (C19H20O7) 360.1209, found
360.1213. Anal. (C19H22O7) C, H. The rest of the residue was mixed
with glacial acetic acid (20.0 mL) and sulfuric acid (0.1 mL) and
heated at 95-100 °C under argon atmosphere for 3.5 h. Solvent
was removed under reduced pressure, and the residue was poured
into water (100 mL). The mixture was extracted with chloroform
(3 × 100 mL) and dried with Na2SO4, and the residue was
chromatographed with silica gel (eluent ethyl acetate-hexane, 3:1)
to yield pure zapotin (1, 1.4 g, 82%): mp 146-147 °C (lit.31 147-
148 °C). Rf ) 0.25 (SiO2, EtOAc-hexane 3:1); IR (neat) 2939, 2840,
Determination of TPA-Induced NF-κB Activity in HepG2
Cells. HepG2 cells stably transfected with NF-κB-luciferase plasmid
were treated with various concentrations of zapotin (1), and
determination of luciferase activity was performed as described
previously.34 In brief, transfected cells were incubated for 48 h in
96-well plates. After 6 h incubation with TPA (100 nM) and zapotin
(1), cells were analyzed for luciferase activity. Cells were washed
with PBS and lysed using 50 µL 1X Reporter Lysis Buffer
(Promega, Madison, WI) for 10 min, and the luciferase determi-
nation was performed according to the manufacturer’s protocol.
Data were expressed as the concentration required to inhibit
activation by 50% (IC50 value). Tumor necrosis factor (TNF)-R
was used as a standard inhibitor (IC50 15-25 ng/mL).33 IC50 values
were generated from the results of four serial dilutions of zapotin
(1) tested in duplicate. With the experimental conditions used, no
signs of overt cellular toxicity were observed.
Cell Differentiation Assays. HL-60 cells were tested using a
4-day protocol.35 In brief, cells in log phase (approximately 106
cells/mL) were diluted to 105 cells/ mL and preincubated overnight
(18 h) in 24-well plates to allow cell growth recovery. Then,
samples dissolved in DMSO were added, keeping the final DMSO
concentration at 0.1% (v/v). Control cultures were treated with the
same concentration of DMSO. After 4 days of incubation, cells
were analyzed to determine the percentage exhibiting functional
nitroblue tetrazolium (NBT) reduction, and cell surface markers
of differentiated cells, as described below.
1
1650, 1592, 1475, 1417, 1357, 1281, 1255, 1111 cm-1; H NMR
(300 MHz, CDCl3) δ 7.35 (t, J ) 8.7 Hz, 1 H), 7.25 (d, J ) 9.3
Hz, 1 H), 7.16 (d, J ) 9.3 Hz, 1 H), 6.59 (d, J ) 8.4 Hz, 2 H),
6.26 (s, 1 H), 3.94 (s, 3 H), 3.88 (s, 3 H), 3.75 (s, 6 H); 13C NMR
(75 MHz, CDCl3) δ 177.9, 158.7, 158.2, 152.2, 149.3, 147.4, 131.8,
119.0, 118.5, 114.9, 113.5, 110.9, 103.6, 61.5, 56.8, 55.7; EIMS
(m/z, relative intensity) 342 (M+, 50), 327 (100), 311 (7), 283 (5),
253 (8), 237 (3), 197 (3), 182 (5), 165 (37), 137 (83), 109 (26), 91
(18), 69 (19), 53 (14); HRMS m/z calcd for (C19H18O6) 342.1103,
found 342.1107. Anal. (C19H18O6) C, H.
Nitroblue Tetrazolium (NBT) Reduction. Evaluation of NBT
reduction was used to assess the ability of sample-treated cells to
produce superoxide when challenged with TPA. A 1:1 (v/v) mixture
of a cell suspension (106 cells) and TPA/ NBT solution [2 mg/mL
NBT and 1 µg/mL TPA in phosphate buffer saline (PBS)] was
incubated for 1 h at 37 °C. Then cells were smeared on glass slides
and counterstained with 0.3% (w/v) safranin O in methanol. Positive
cells reduce NBT yielding intracellular black-blue formazan deposits
and were quantified by microscopic examination of >200 cells.
Results were expressed as a percentage of positive cells.
Cell Culture. T24 and HL-60 cells were obtained from the
American Type Culture Collection (Rockville, MD). T24 cells were
cultured in MEM medium (Invitrogen, Carlsbad, CA) containing
10% heat-inactivated fetal bovine serum, non-essential amino acids,
1 mM sodium pyruvate (BioWhittaker, Walkersville, MD), 100
units penicillin/mL, 100 µg streptomycin/mL, and 250 ng ampho-
tericin B/mL (Gibco Invitrogen, Grand Island, NY). The HL-60
cell line was maintained in suspension culture using RPMI 1640
medium (Invitrogen) supplemented with 10% heat-inactivated fetal
bovine serum, 100 units penicillin/mL, and 100 µg streptomycin/
mL. HepG2 human hepatoma cells stably transfected with NF-κB-
luciferase plasmid32 were maintained in Ham’s F12 supplemented
with 10% heat-inactivated fetal bovine serum, 100 units/mL
penicillin G sodium, 100 µg/mL streptomycin sulfate, 1% MEM
amino acid, and 0.1% insulin. The cell lines were maintained in a
5% CO2 atmosphere at 37 °C and were routinely tested for
mycoplasma contamination.
Determination of TPA-Induced ODC Activity in T24 Cells.
T24 cells were treated with various concentrations of zapotin (1)
and determination of ODC activity was performed as described
previously.33 In brief, cells were plated at an initial density of 2 ×
105 cells per well in 24-well plates. After an 18 h preincubation, a
solution of zapotin (1) in DMSO was added in duplicate (5 µL,
0.5% final concentration) before the induction of ODC activity with
TPA (200 nM final concentration). After an additional 6 h
incubation, plates were washed twice with PBS and kept at -85
°C until tested. ODC activity was directly assayed by measuring
the release of [14C]CO2 from L-[1-14C]-ornithine HCl in the presence
of 190 µM nonradioactive ornithine HCl. The amount of radioactiv-
ity captured in NaOH-impregnated filter discs was determined by
scintillation counting in 24-well plates using a Wallac 1450
Microbeta liquid scintillation counter. Protein was determined
according to the Lowry procedure. Interfering dithiothreitol con-
tained in the reaction mixture was destroyed by adding chloramine
T (50 µL, 8 mg/mL) to each well (30 min incubation at RT),
followed by NaOH (50 µM, 5.7 M) to solubilize the protein. The
protein was measured in 96-well plates using an aliquot of the
reaction mixture and bovine serum albumin as a standard. The
optical density was measured at 660 nm using a BT2000 Micro-
kinetic Reader. The results were calculated as nmol [14C]CO2/h/
mg protein and expressed as a percentage in comparison with a
control treated with DMSO and TPA. Dose-response curves were
prepared, and the results were expressed as IC50 values in
micromolar concentrations. IC50 values were generated from the
results of four serial dilutions of zapotin (1) tested in duplicate.
Determination of Cell Surface Antigens. Cells (106), prewashed
with PBS, were resuspended in 100 µL diluent (PBS with 0.1%
sodium azide and 1% BSA) and incubated for 30 min at room
temperature with the monoclonal antibodies anti-CD-11b (Sigma,
St. Louis, MO), anti-CD13 (Caltag, Burlingame, CA), anti-CD14
(Sigma), and anti-CD15 (Caltag), conjugated with FITC. Cells were
washed with 20 volumes of diluent and resuspended in 0.5 mL of
2% paraformaldehyde for flow cytometry evaluation. Identical
samples were prepared using isotype antibodies to correct fluores-
cence due to nonspecific binding.
Quantification of Apoptosis. Cells were treated with various
concentrations of zapotin (1) for 24 h, or with 12 µM zapotin (1)
for various time intervals, washed with PBS, and fixed with
methanol-acetic acid 1:1 for 30 min at room temperature. Cells
were then treated with 4′,6-diamidino-2-phenylindole (DAPI, 1 µg/
mL) for 15 min at room temperature. DAPI staining of the nucleus
was observed by fluorescence microscopy. At least 100 cells were
counted for each sample. Dose-response curves showing the
percentage of apoptosis at different doses and times were con-
structed.
Cell Cycle Analysis. Cells (3 × 106) were treated with various
concentrations of zapotin (1) for 24 h and washed with PBS. Cells
were resuspended in 1 mL PBS + 9 mL ice-cold 70% EtOH and
stored at -20 °C. Just before analysis, samples were centrifuged
and cell pellets were resuspended in 2 mL of propidium iodide
solution (2 µg/mL propidium iodide, 100 µg/mL ribonuclease A
in PBS). Solutions were incubated at 37 °C for 1 h, placed on ice,
and analyzed by flow cytometry. At least 10 000 cells were counted
for each sample. The percentage of apoptotic cells was calculated
by measuring the area under the subdiploid (DNA < 2 N) peak in
the plot of cell number against cellular DNA content.
Alternatively, cells were exposed to 5-bromo-2-deoxyuridine
(BrdU) for 30 min prior to trypsinization to specifically label
S-phase cells. After fixation, cells were stained with fluorescein-
conjugated antibody to BrdU and counterstained with propidium

Synthesis and Cancer ChemopreVentiVe ActiVity of Zapotin
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 355
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