1294 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Brief Articles
Figure 4. Fluorescence microscopy images showing appear-
ance of apoptotic cells (MDA MB 468) following drug treatment
at 10 µM for 48 h: (A) control; (B) 5by; (C) 5dy; (D) 5jy.
Figure 3. AnnexinV-PI analysis in MDA MB 468 cells,
following 48 h of drug treatment at 10 µM, demonstrating early
apoptotic events (an increase in AnnexinV-FITC binding)
without concurrent increase in PI staining. Results are the
mean of three repeats ( SEM.
orange, spotted on glass slides, and visualized under a
fluorescence microscope. Cells showing shrinkage ac-
companied by chromatin condensation and marginal-
ization are defined as being apoptotic.
AnnexinV-FITC/Propidium Iodide Analysis of
Apoptosis. An early apoptotic event is the translocation
of phosphatidylserine (PS) from the inner to the outer
membrane leaflet of the cell. This can be detected using
Conclusions
fluorescein-labeled annexinV (annexinV-FITC), a Ca2+
-
In this study we have synthesized a novel family of
monohydroxylated (E)-stilbenes and studied their ability
to inhibit the growth and induce apoptosis in human
tumor cell lines. The potential of these hydroxylated
stilbenes as antitumor agents will probably not reside
in the potency of their growth-inhibitory activity per se.
Rather, the potential of these agents may manifest itself
in their ability to induce apoptosis in a selective manner
and inhibit a number of cancer relevant biological
targets, as is the case for resveratrol. We have observed
moderately potent growth-inhibitory effects most nota-
bly in the MDA MB 468 cell line, particularly for the
3-hydroxylated stilbene derivatives such as 5dy and 5jy.
This agent also produced the highest percentage of sub-
G0/1 cells in MDA MB 468 cell lines following drug
treatment, indicative of apoptotic potential, a finding
reinforced by fluorescence microscopy of apoptotic cells.
Our search for mechanistic targets underpinning the
antitumor activity of these novel agents will be reported
at a future date.
dependent phospholipid-binding protein with high af-
finity for PS. Combined with PI (used as an indicator
of cell integrity), a measure of percentage cell population
in early apoptosis can be achieved. Following treatment
of MDA MB 468 cells according to the AnnexV-FITC
detection kit protocol (Santa Cruz), cells were analyzed
by flow cytometry. AnnexinV-FITC fluorescence was
collected in FL1, and propidium iodide fluorescence was
collected in FL3. Cells showing increased FL1 fluores-
cence without a concurrent increase in FL3 fluorescence
are considered to be in early apoptosis. Where an
increase is seen in both fluorescence channels, the cells
are considered to be in late apoptosis or to have
undergone necrosis.
The results of the AnnexinV-FITC/PI analysis for
early apoptosis are shown in Figure 3. As for the flow
cytometric analysis (Figure 2), a subset of hydroxylated
stilbenes was chosen according to their growth inhibi-
tory properties in the MDA MB 468 human cancer cell
line. Figure 3 clearly indicates that the highest percent-
age of cells deemed to be in early apoptosis (increase in
AnnexinV-FITC binding without concurrent increase in
PI staining) was observed following treatment (10 µM,
48 h) with compounds 5dy, 5jy, and 5gx. Once again,
induction of apoptosis correlated with antiproliferative
activity in MDA MB 468 cells, particularly for the
relatively potent 3-hydroxylated stilbenes 5dy and 5jy.
Notably, the percentage of cells deemed to be in early
apoptosis at 10 µM for compound 5dy (20%) was much
higher than for resveratrol itself (4%) but much lower
than for the standard anticancer agent camptothecin
(37% at 250 nM); see Supporting Information for further
details.
Experimental Section
General Method for Parallel Synthesis of Hydroxy-
lated (E)-Stilbene MOM Ethers. Substituted phosphonic
acid diethyl esters (1a-j) (10 mmol) were dissolved in dry DMF
(10 mL). Sodium methoxide (20 mmol) and 18/6-crown ether
(2 mmol) were added, and the mixture was stirred at room
temperature for 5 min. Hydroxybenzaldehyde methoxymethyl
ethers (2x-z) (15 mmol) dissolved in dry DMF (5 mL) were
added dropwise at 0 °C, and the mixture was stirred at room
temperature for 1 h followed by heating to 120 °C for 5 h. The
reactions were quenched with water (20 mL), and the mixture
was extracted with Et2O (3 × 20 mL). The ether was evapo-
rated in vacuo, and the residues were redissolved in dichlo-
romethane (15 mL). Girard’s reagent T (6 mmol) and acetic
acid (60 mmol) were added, and the reaction stirred at room
temperature for 2 h. After quenching with water (15 mL), the
organic layers were collected, washed with brine (3 × 15 mL)
and aqueous Na2CO3 (3 × 15 mL), dried over MgSO4, and
evaporated in vacuo to afford the substituted methoxymethyl-
oxystyrylbenzenes (3ax-3jz) as oils.
Fluorescence Microscopy. Further confirmation of
the ability of the most potent hydroxylated stilbenes in
the series to induce apoptosis was obtained by analysis
of drug-treated MDA MB 468 cell populations (10 µM,
48 h) by fluorescence microscopy (Figure 4). Following
drug treatment the cells were resuspended in acridine
General Method for Synthesis of Hydroxylated (E)-
Stilbenes. The substituted hydroxylated (E)-stilbene meth-