Synthesis and biological activity of a fluorescent schweinfurthin analogue

Article abstract of DOI:10.1016/j.bmc.2009.04.071

Most of the natural schweinfurthins are potent and selective inhibitors of cell growth as measured by the National Cancer Institute's 60-cell line screen. Due to the limited supply of these natural products, we have initiated a program aimed at their synt

Full text of DOI:10.1016/j.bmc.2009.04.071

Bioorganic & Medicinal Chemistry 17 (2009) 4718–4723
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological activity of a fluorescent schweinfurthin analogue
Craig H. Kuder a, Jeffrey D. Neighbors b,c, Raymond J. Hohl a,c, David F. Wiemer a,b,
*
a Department of Pharmacology, University of Iowa, Iowa City, IA 52242-1294, USA
b Department of Chemistry, University of Iowa, Iowa City, IA 52242-1294, USA
c Department of Internal Medicine, University of Iowa, Iowa City, IA 52242-1294, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Most of the natural schweinfurthins are potent and selective inhibitors of cell growth as measured by the
National Cancer Institute’s 60-cell line screen. Due to the limited supply of these natural products, we
have initiated a program aimed at their synthesis. To date, this effort has led to the preparation of three
natural schweinfurthins and more than 40 analogues, and assays on these compounds have afforded
some understanding of structure–activity relationships in this family. Further development of schwein-
furthins as chemotherapeutic agents would benefit from characterization of their mechanism(s) of action.
This perspective led to development of a fluorescent schweinfurthin analogue that retains the differential
activity of the natural products, and yet has properties that facilitate its visualization within cells.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 15 January 2009
Revised 22 April 2009
Accepted 24 April 2009
Available online 6 May 2009
Keywords:
Schweinfurthin
Stilbene
Fluorescent
Cytotoxic
60-Cell assay
1. Introduction
(GI50’s of 11 nM, 10 nM, and 15 nM respectively, for schweinfurthin
A). However, development of schweinfurthins as cancer therapeu-
Higher plants have proven to be an excellent source of novel
compounds that are useful in the treatment of cancer.1 Among
the most notable successes in this area are the alkaloids vincristine
and vinblastine isolated from Catharanthus roseus,2 paclitaxel iso-
lated from Taxus brevifolia,3,4 and camptothecin isolated from Cam-
ptothecin acuminate.5 As part of an ongoing National Cancer
Institute (NCI) initiative to examine new plant extracts for poten-
tial chemotherapeutic agents, several closely related compounds
named schweinfurthins have been discovered. After an extract
from the Cameroonian plant Macaranga schweinfurthii displayed
potent and cell type selective growth inhibition (GI) in the NCI’s
60-cell line screen,6 the more active components of this extract
were isolated, characterized, and named schweinfurthin A and B7
(1 and 2, Fig. 1). A less active analogue was named schweinfurthin
C, and later studies yielded the closely related schweinfurthin D.8
Independent studies of Macaranga alnifolia later yielded schwein-
furthins E–H.9
Further study of the schweinfurthins as potential cancer thera-
peutics is encouraged by the substantial differential activity seen
among the cell lines of the NCI’s 60-cell line screen. In addition,
the unique pattern of activity can be considered a ‘fingerprint’
indicative of a possible novel mechanism of action.10 Finally, these
agents demonstrated very potent antiproliferative activity against
human-derived CNS cell lines such as SF-295, SF-539, and SNB-75
tics has been hindered by limited material due to difficulties in iso-
lation of significant additional quantities of the active compounds
from the original plant source. Therefore, we have undertaken the
total synthesis of these natural products. While installation of the
vicinal diol on the A-ring has proven to be elusive, analogues with-
out the C-3 hydroxyl group have been prepared and shown to main-
tain the growth inhibition and differential activity exhibited by the
natural schweinfurthin diols.6–9 Furthermore, compounds lacking
the C-3 hydroxyl group were isolated during the more recent stud-
ies of M. alnifolia,9 and already have been synthesized as single
enantiomers.11
OR
R'
O
3
2
A
B
C
OH
HO
H
D
OH
1 R = H
R' = OH
Schweinfurthin A
Schweinfurthin B
3-Deoxyschweinfurthin A
3-Deoxyschweinfurthin B
2 R = CH3 R' = OH
3 R = H
R' = H
4 R = CH3 R' = H
* Corresponding author. Tel.: +1 319 351 5905; fax: +1 319 335 1270.
E-mail address: david-wiemer@uiowa.edu (D.F. Wiemer).
Figure 1. Structures of some natural (1 and 2) and synthetic (3 and 4)
schweinfurthins.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.071

C. H. Kuder et al. / Bioorg. Med. Chem. 17 (2009) 4718–4723
4719
Two of the synthetic compounds, (R,R,R)-3-deoxyschweinfur-
thin A (3, 3dSA)12 and B (4, 3dSB),13 have excellent potency with
sub-micromolar mean GI50’s, while maintaining differential activ-
ity across the NCI’s 60-cell cancer screen. Since the inception of
the synthetic program, our group has prepared three natural schw-
einfurthins and more than 40 analogues. These compounds have
allowed us to explore some structure-function relationships within
the family, particularly as they pertain to the D-ring.14 To date
however, these analogues have not clarified the mechanism of ac-
tion, which remains essentially unknown.
Development of schweinfurthins as cancer therapeutics might
be accelerated by elucidation of their mechanism(s) of action. Fluo-
rescence microscopy often has been used to determine the locali-
zation of drugs15 or proteins, and potential interaction with other
cellular components.16 Synthesis of schweinfurthin analogues that
incorporate fluorescence properties could advance our current
understanding of schweinfurthin activity, as long as such com-
pounds retain their anti-proliferative activity. Here, we present
the synthesis and biological activity of a novel fluorescent schw-
einfurthin analogue.
During the synthesis of 3dSB, it was determined that many of
these stilbenes were fluorescent under UV light. For example,
3dSB itself has an absorption maximum of 331 nm and an emission
maximum of 391 nm (data not shown). The possibility that these
properties might provide information on the sub-cellular localiza-
tion of schweinfurthins immediately was recognized. After treat-
ment with 3dSB, SF-295 cells (a human-derived glioblastoma
multiforme cell line) were examined for potential UV fluorescence.
However, 3dSB failed to generate a significant fluorescent signal
above the autofluorescence of control cells (data not shown).
The limited utility of 3dSB as a fluorescent probe prompted a re-
view of our prior structure–activity data with the hope of finding a
position amenable to change and consistent with improved fluo-
rescent characteristics. Previous work has demonstrated the
importance of at least one free D-ring phenol for biological
activity.14 Consideration of the natural schweinfurthins indicates
the para position of the D-ring as a point where the structure might
be modified without loss of the desirable biological properties. For
example, 3dSB bears a 10-carbon geranyl substituent at this posi-
tion while schweinfurthin F carries only a 5-carbon prenyl substi-
tuent and both display comparable activity.11 Therefore,
a
literature review was performed with an eye toward identification
of accessible substitutions that might be made at this position to
afford new analogues with improved fluorescence properties.
Substituted stilbenes are among the most studied fluorescent
compounds.17,18 For example, Maddy et al. synthesized the fluores-
cent stilbene 4-acetamido-40-isothiocyanostilbene-2,20-disulfonic
acid in the mid 1960s.19 This compound then was shown to be
an excellent fluorescent label for the plasma membrane of erythro-
cytes, and was subsequently shown to penetrate the blood–brain
barrier.20 More recently a bis-stilbene was demonstrated to pene-
trate mouse brain and fluorescently label myelin.21 The idea of
attaching a second stilbene linkage at the para position of the D-
ring also was attractive from a synthetic standpoint, because it
fit well into our modular approach to the schweinfurthins. There-
fore the novel bis-stilbene 13 was identified as the initial target.
2. Chemical synthesis
Synthesis of compound 13 began with the known aryl bromide
5 (Scheme 1).22,23 Halogen–metal exchange on bromide 5 presum-
ably afforded a lithiated intermediate, which was subsequently al-
lowed to react with anhydrous dimethylformamide to afford the
new aldehyde 6 in acceptable yield. This aldehyde was treated
with the known phosphonate 714 under modified Horner–Wads-
worth–Emmons (HWE) conditions to give the protected stilbene
8. Removal of the silyl protecting group under standard conditions
gave the benzylic alcohol 9. A three step procedure then was used
to convert the alcohol 9 into the benzylic phosphonate 10. Thus,
treatment of the alcohol 9 with methanesulfonyl chloride and
OTBS
OTBS
OMOM
n-BuLi, DMF
-78 oC
OMOM
O
H
Br
56%
MOMO
MOMO
5
6
NaH, 15-crown-5, 0 ºC to rt
100%
OR
OMOM
(CH3CH2O)2P
O
O
(CH3CH2O)2P
1) TEA, MsCl, 0 oC
2) NaI, acetone
3) P(OCH2CH3)3
7
OMOM
OMOM
OMOM
OMOM
88% (3 steps)
MOMO
MOMO
10
TBAF, 0 oC to rt
100%
8 R = TBS
9 R= H
OCH3
O
26%
O
HO
H
H
OCH3
11
O
NaH, 15-crown-5
0 ºC to rt
OR
HO
H
OR
RO
TsOH, 60 ºC
79%
12 R = MOM
13 R= H
Scheme 1. Synthesis of fluorescent stilbene 13.

4720
C. H. Kuder et al. / Bioorg. Med. Chem. 17 (2009) 4718–4723
triethylamine affords the expected mesylate which can be
smoothly transformed into the iodide upon reaction with NaI. Dis-
placement of the iodide by the soft nucleophile triethyl phosphite
gives the desired benzylic phosphonate 10 in high overall yield. A
second HWE condensation with the nonracemic tricyclic aldehyde
1113 gives the protected schweinfurthin analogue 12. Final hydro-
lysis of the MOM protecting groups through treatment with cam-
phorsulfonic acid or toluenesulfonic acid gives the desired bis-
stilbene 13. This compound was highly fluorescent with an absor-
bance maximum of 330 nm, an emission maximum of 416 nm and
comparison to SF-295 cells (Fig. 3B). Treatment with 3dSB
(500 nM) or compound 13 (500 nM) does not induce a morpho-
logic change apparent at either 24 h or 48 h (Fig. 3B). The ab-
sence of change is consistent with previous studies which
indicate that A549 cells are less sensitive to schweinfurthin
treatment. Similar to the effects in SF-295 cells, Y-27632
(10 lM) induces ruffled F-actin at the edges of the A549 cells
(Fig. 3B). These findings further suggest that the structural
changes introduced in compound 13 do not alter the differential
activity observed with the schweinfurthins.
an extinction coefficient
e
of 31,200 (Fig. 2).
The bis-stilbene 13 represents the first compound that might fur-
ther understanding of the schweinfurthin localization through fluo-
rescence microscopy. Indeed, SF-295 cells treated with
concentrations of compound 13 as low as 100 nM could be visual-
ized at all time points tested (Fig. 4). Unlike previous experiments
withother schweinfurthins, thefluorescenceintreatedcells wassig-
nificantly greater than the background of control cells. As expected,
treatment of SF-295 cells with increasing concentrations of com-
pound 13 increased fluorescence intensity at all time points tested
(Fig. 3). The localization of compound 13 within cells appears largely
cytosolic with the most intense fluorescence in the perinuclear re-
gion. The fluorescent properties of compound 13 allow its visualiza-
tion above the background autofluorescence in control cells.26
3. Biological results and discussion
As noted above, the schweinfurthins display potent growth
inhibition and differential activity in the NCI’s 60-cell screen. In
this assay, the SF-295 cell line is among the most sensitive to schw-
einfurthin-induced growth inhibition, while the human-derived
lung adenocarcinoma A549 cells display only moderate growth
inhibition upon parallel treatment. Due to this difference in sensi-
tivity between SF-295 and A549 cells, these cell lines can be used
to investigate the schweinfurthin-like activity of synthetic ana-
logues such as compound 13. Indeed, investigation of cytotoxicity
with compound 13 demonstrates a significant toxicity in SF-295
cells (IC50ꢀ800 nM) while A549 cells are much less sensitive.
In addition to their growth inhibitory effects, schweinfurthins
induce drastic changes in cell morphology at time points beyond
24 h. In culture, SF-295 cells are large polygonal shaped cells with
multiple outstretched processes (Fig. 3A). Treatment of SF-295
cells with schweinfurthin analogues such as 3dSB causes morpho-
logic changes congruent with those induced by the natural prod-
ucts. These alterations are characterized by a decrease in cell
area and a reduction in F-actin, which results in spindle-shaped
cells with F-actin staining only at the periphery of the treated cells.
When SF-295 cells were treated for 24 h with compound 13
(500 nM) or 3dSB (500 nM), they exhibited morphologic character-
istics somewhat similar to control cells (Fig. 3A). However at 48 h
cells treated with 3dSB (500 nM) or compound 13 (500 nM) display
altered morphology. These changes appear to differ from those in-
4. Conclusions
In conclusion, these studies have led to the preparation of the
first schweinfurthin analogue with fluorescence properties that al-
low its cellular localization to be tracked by microscopy. This com-
pound maintains the activity of schweinfurthins in SF-295 cells,
and results in characteristic changes in cell morphology over time.
While the morphologic changes induced by schweinfurthins differ
from those induced by the ROCK inhibitor Y-27632, these findings
do not preclude the involvement of RhoA through other down-
stream mediators or other Rho family members such as Rac1 and
Cdc 42, which are also crucial for proper cell structure and move-
ment.27 The localization of compound 13 appears be cytosolic as
well as perinuclear. Unfortunately, compound 13 undergoes rapid
photobleaching and thus did not allow co-localization studies to
determine its potential concentration in organelles. Nevertheless,
incorporation of such a sizeable substituent at the para position
of the D-ring suggests that other substituents might be placed at
this site without significant loss of biological activity, and a series
of fluorescent analogues can be readily envisioned. Other useful
probes might include compounds linked to moieties such as biotin
or polyethylene glycol to aide in the examination of mechanism.28–
duced by the Rho kinase (or ROCK) inhibitor Y-27632 (10 l
M),24
which caused jagged F-actin at the periphery and long slender pro-
jections to form. Inhibition of ROCK disrupts RhoA signaling, which
is critical in the maintenance of cell shape and motility.25 In addi-
tion, ROCK inhibition induces changes readily detected at 24 h. At
equivalent concentrations compound 13 displays similar activity
to 3dSB in SF-295 cells, yet both compound 13 and 3dSB differ
from the activity of Y-27632.
In comparison to the SF-295 cells, the schweinfurthins are
much less active against the A549 cells. In control experiments,
the A549 cells are rectangular and cover less surface area in
30
Such probes may allow determination of binding partners, sig-
naling cascades, and/or localization. Studies in this vein are ongo-
ing and will be reported in due course.
5. Experimental procedures and methods
5.1. General experimental conditions
Tetrahydrofuran (THF) was distilled from sodium/benzophe-
none immediately prior to use, CH2Cl2 was freshly distilled from
CaH2, and DMF was dried over CaH2. All reactions in non-aqueous
solvents were conducted in oven-dried glassware under a positive
pressure of argon and with magnetic stirring. NMR spectra were
recorded at 300 MHz for 1H, and 75 MHz for 13C with CDCl3 as sol-
vent and (CH3)4Si (1H) or CDCl3 13C, 77.2 ppm) as internal stan-
(
dards unless otherwise noted. Silica gel (60 Å, 0.040–0.063 mm)
was used for flash chromatography. Yields refer to pure com-
pounds after chromatography. High resolution mass spectra were
obtained at the University of Iowa Mass Spectrometry Facility.
Figure 2. Absorbtion and emission spectra of compound 13.

C. H. Kuder et al. / Bioorg. Med. Chem. 17 (2009) 4718–4723
4721
Figure 3. SF-295 (A) and A549 (B) cells treated with indicated concentrations and intervals of Y-27632, 3dSB, and compound 13 were stained with fluorescent phalloidin as
an F-actin marker (green) and DAPI as nuclear stain (blue).
5.2. Aldehyde 6
organic phase was washed with brine, dried (MgSO4), and concen-
trated in vacuo. Final purification by column chromatography (6:1
to 2:1 hexanes/ethyl acetate) gave the stilbene 8 (228 mg, 100%) as
a clear oil: 1H NMR (CDCl3) d 7.61 (d, J = 17 Hz, 1H), 7.50 (d,
J = 17 Hz, 1H), 7.38–7.29 (m, 3H),7.03–7.00 (m, 1H), 6.93 (s, 2H),
5.34 (s, 4H), 5.30 (s, 2H), 4.80 (s, 2H), 3.60 (s, 6H), 3.59 (s, 3H),
1.05 (s, 9H), 0.02 (s, 6H); 13C NMR (CDCl3) d 157.4, 156.1 (2C),
142.3, 140.7, 132.0, 129.4, 120.4, 120.1, 115.1, 114.6, 114.1, 106.0
(2C), 94.8 (2C), 94.3, 64.7, 56.1 (2C), 55.9, 25.8 (3C), 18.3, ꢁ5.3
(2C). Anal. Calcd for C27H40O7Si: C, 64.30; H, 7.99. Found: C,
64.09; H, 8.02.
To a solution of the known bromide 522,23 (1.25 g, 3.0 mmol) in
THF (10 mL) at ꢁ78 °C was added n-BuLi (1.6 mL, 2.0 M in hexanes)
dropwise over 2 min. After 30 min, DMF was added (0.5 mL,
6.5 mmol, containing some CaH2 as a drying agent). The reaction
was allowed to progress for an additional 3 h, and quenched by
addition of saturated aq. NH4Cl. The aqueous phase was extracted
with EtOAc, and the combined organic phases were washed with
brine, dried (MgSO4), and concentrated in vacuo to afford a yellow
oil. Final purification by column chromatography afforded alde-
hyde 6 (1.09 g, 99%) as a clear oil: 1H NMR (CDCl3) d (10.5 s, 1H),
6.84 (s, 2H), 5.26 (s, 4H), 4.73 (s, 2H), 3.50 (s, 6H), 0.95 (s, 9H),
0.12 (s, 6H); 13C NMR (CDCl3) d 188.9, 159.6 (2C), 151.0, 114.7,
105.6 (2C), 94.8 (2C), 64.5, 56.4 (2C), 25.8 (3C), 18.3, ꢁ5.4 (2C);
HRMS (EI) calcd for C18H30O6Si (M+) 370.1812; found 370.1822.
5.4. Benzylic alcohol 9
Silyl ether 8 (227 mg, 0.45 mmol) was dissolved in THF (10 mL)
and the solution was cooled to 0 °C. To this solution was added
TBAF (0.6 mL, 1.00 M in THF), after 5 min the ice bath was re-
moved, and after 4 h the reaction was quenched by addition of
sat. NH4Cl. After extraction with ethyl acetate, the combined or-
ganic extracts were washed with water and brine, dried (MgSO4),
and concentrated in vacuo to give the desired benzylic alcohol 9
(176 mg, 100%) as a clear oil: 1H NMR (CDCl3) d 7.52 (d, J = 17 Hz,
1H), 7.40 (d, J = 17 Hz, 1H), 7.29–7.19 (m, 3H), 6.94–6.91 (m,1 H),
6.82 (s, 2H), 5.24 (s, 4H), 5.21 (s, 2H), 4.62 (s, 2H), 3.49 (s, 9H),
2.51 (br s, 1H); 13C NMR (CDCl3) d 157.4, 156.1 (2C), 141.9,
5.3. Stilbene 8
A suspension of NaH (290 mg, 7.8 mmol, 60% in oil), and 15-
crown-5 (1 drop, cat.) in THF (10 mL) was cooled to 0 °C. To this
was added a solution of aldehyde 6 (168 mg, 0.45 mmol) and phos-
phonate 7 (185 mg, 0.64 mmol) in THF (10 mL). The mixture was
allowed to warm to rt and stirred a total of 10 h. Water was added
dropwise, and the solution was extracted with EtOAc. The resulting

4722
C. H. Kuder et al. / Bioorg. Med. Chem. 17 (2009) 4718–4723
Figure 4. Fluorescence of compound 13 in SF-295 cells at indicated concentrations and time points.
140.5, 132.3, 129.4, 120.1, 120.1, 115.7, 114.7, 114.1, 106.7 (2C),
94.6 (2C), 94.3, 64.9, 56.2 (2C), 55.9.
JCP = 6.0 Hz, 2C), HRMS (EI) calcd for C25H35O9P (M+) 510.2019;
found 510.2018.
5.5. Phosphonate 10
5.6. Protected bis-stilbene 12
Methanesulfonyl chloride (0.15 mL, 1.9 mmol) was added to a
solution of benzylic alcohol 9 (176 mg, 0.45 mmol) and Et3N
(0.2 mL 1.4 mmol) in CH2Cl2 (10 mL). The reaction mixture was
allowed to warm to room temperature over 1 h, quenched by
addition of H2O, and extracted with CH2Cl2. The combined or-
ganic layers were washed with NH4Cl (sat), brine, dried (MgSO4),
and concentrated in vacuo. The resulting residue and NaI
(150 mg, 1.0 mmol) were stirred in acetone (10 mL) for 8 h.
The reaction mixture was concentrated in vacuo to afford a
red solid, which was dissolved in EtOAc. After the resulting yel-
low solution was washed with Na2S2O3 until the color faded, it
was washed with brine, dried (MgSO4) and concentrated in va-
cuo. The resulting yellow oil was added to triethyl phosphite
(2 mL) and the reaction mixture was heated at 80 °C for 24 h.
After the solution was allowed to cool to rt, the excess phosphite
was removed at high vacuum. The initial yellow oil was purified
by flash chromatography (1:2 hexanes/ethyl acetate to 100%
EtOAc) to afford phosphonate 10 (201 mg, 88%) as a clear oil:
1H NMR (CDCl3) d 7.53 (d, J = 16.7 Hz, 1H), 7.40 (d, J = 16.7 Hz,
1H), 7.28–7.21 (m, 3H), 6.97–6.94 (m, 1H), 6.80–6.79 (m, 2H),
5.26 (s, 4H), 5.23 (s, 2H), 4.12–4.02 (m, 4H), 3.52 (s, 9H), 3.13
(d, JHP = 21.8 Hz, 2H), 1.30 (t, J = 7.0 Hz, 6H); 13C NMR (CDCl3) d
157.4, 155.9 (d, JCP = 3.7 Hz, 2C), 140.6, 132.3 (d, JCP = 1.6 Hz),
132.0 (d, JCP = 8.0 Hz), 129.4, 120.1, 120.1, 115.3 (d, JCP = 4.1 Hz),
114.7, 114.1, 110.2 (d, JCP = 6.7 Hz, 2C), 94.8 (2C), 94.4, 62.1 (d,
JCP = 6.8 Hz, 2C), 56.2 (2C), 55.9, 33.5 (d, JCP = 138 Hz), 16.3 (d,
A suspension of NaH (80 mg, 20 mmol, 60% in oil), and 15-
crown-5 (1 drop, cat.) in THF (5 mL) was cooled to 0 °C. To this
mixture was added a solution of aldehyde 11 (60 mg, 0.20 mmol)
and phosphonate 10 (95 mg, 0.19 mmol) in THF (5 mL). The result-
ing mixture was allowed to warm to rt and stirred a total of 30 h.
Water was added dropwise, and the solution was extracted with
EtOAc. The resulting organic phase was washed with brine, dried
(MgSO4), and concentrated in vacuo. Final purification by column
chromatography (3:1 to 1:1 hexanes/ethyl acetate) gave the stil-
bene 12 (71 mg, 56%) as a green waxy solid oil: 1H NMR (CDCl3)
d 7.55 (d, J = 16.6 Hz, 1H), 7.43 (d, J = 16.6 Hz, 1H), 7.32–7.20 (m,
4H), 7.04–6.87 (m, 6H), 5.32 (s, 4H), 5.23 (s, 2H), 3.91 (s, 3H),
3.55 (s, 6H), 3.52 (s, 3H), 3.47–3.42 (m, 1H), 2.74–2.71 (m, 2H),
2.20–2.12 (m, 1H), 1.90–1.47 (m, 5H), 1.27 (s, 3H), 1.14 (s, 3H),
0.90 (s, 3H); 13C NMR (CDCl3) d 157.4, 156.3 (2C), 148.9, 142.7,
140.7, 137.8, 132.1, 129.5, 129.1, 128.6, 125.9, 122.6, 120.7,
120.3, 120.1, 125.7, 114.7, 114.2, 106.7, 106.4 (2C), 94.8 (2C),
94.4, 77.9, 77.1, 60.3, 56.3 (2C), 56.0, 46.7, 38.3, 37.6, 28.2, 27.3,
23.1, 19.8, 14.1; HRMS (EI) calcd for C39H48O9 (M+) 660.3298, found
660.3301.
5.7. Bis-stilbene 13
To a solution of stilbene 12 (16 mg, 0.024 mmol) in MeOH
(5 mL) was added p-toluenesulfonic acid (3 mg, 0.014 mmol). The
resulting solution was heated 60 °C for 5 h. The reaction was

C. H. Kuder et al. / Bioorg. Med. Chem. 17 (2009) 4718–4723
4723
quenched by addition of sat. NaHCO3, extracted with ethyl acetate,
and the organic phase was washed with brine and dried (MgSO4).
Concentration in vacuo, followed by final purification by column
chromatography (2:1 to 1:1 hexanes/ethyl acetate) afforded the
bis-stilbene 13 (10 mg, 79%) as a slightly yellow oil: 1H NMR
((CD3)2CO) d 7.19 d, J = 17 Hz, 1H), 7.55 (d, J = 17 Hz, 1H), 7.18 (t,
J = 7.9 Hz, 1H), 7.04–6.98 (m, 4H), 6.95 (d, J = 6.9 Hz, 2H), 6.90–
6.88 (m, 1H), 6.73–6.70 (m, 2H), 3.82 (s, 3H), 3.43–3.38 (m, 1H),
2.76–2.73 (m, 2 H), 1.85–1.63 (m, 6 H), 1.23 (s, 3H), 1.13 (s, 3H),
0.90 (s, 3H); 13C NMR ((CD3)2CO) d 158.5, 157.6 (2C), 150.2,
144.1, 142.1, 138.6, 131.5, 130.4, 129.7, 129.6, 126.5, 123.7,
121.8, 121.5, 118.7, 114.7, 113.0, 112.3, 108.3 (2C), 77.7, 77.6,
56.1, 47.2, 39.2, 38.7, 29.2, 27.9, 23.8, 20.4, 14.9; HRMS (EI) calcd
for C33H36O6 (M+) 528.2512; found 528.2523.
interval cells were washed three times in complete media to re-
move residual compound. Immediately following rinsing, cells
were mounted onto microscope slides and imaged using a Bio-
Rad Multi-photon microscope at the University of Iowa Central
Microscopy facility. Images were further processed using Im-
age-J software.
Acknowledgments
This project was supported by the Roy. J. Carver Charitable Trust
as a Research Program of Excellence, the Roland W. Holden Family
Program for Experimental Cancer Therapeutics, The Iowa Centers
for Enterprise, and the National Institute of Health
(IR41CA126020-01).
5.8. Cell culture
References and notes
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The SF-295 cells were maintained in RPMI 1640 media supple-
mented with 10% bovine fetal calf serum and penstreptamycin. The
A-549 cells were maintained in F-12 media with 10% fetal calf ser-
um and penstreptamycin. Both cell lines were incubated at 37 °C
and 5% CO2.
5.9. Cytotoxicity
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SF-295 and A549 cells were plated in 24-well plates. Upon 65%
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was added to all wells 44 h after compound treatment. At 48 h sol-
ubilization solution (HCl, triton X-100, Isopropyl alcohol) was
added to each well and incubated overnight with gentle agitation
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540 nm
and
650 nM
(reference
k)
was
measured
spectrophotometrically.
5.10. Cellular morphology
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Cells were plated on sterilized coverslips in 6-well plates and al-
lowed to reach 65% confluency before treatment with the indicated
compounds. Rock inhibitor Y-27632 was purchased from Calbio-
chem San Diego, CA. At the conclusion of the treatment interval,
cells were rinsed and fixed with 3.7% formaldehyde for 15 min.
After subsequent rinses in PBS, cells were permeabilized with
0.2% triton X-100 for 4 min and rinsed in PBS. Cells were then incu-
bated with 1% BSA for 30 min before incubation with phalloidin
stain (Molecular Probes, Eugene, OR) for 20 min. At the completion
of the staining interval, cells were rinsed and mounted using Vec-
tashield mounting media containing DAPI (Vector laboratories,
Inc., Burlingame, CA). Slides were imaged using a Bio-Rad Multi-
photon microscope and processed using Image-J software.
5.11. Fluorescence microscopy
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SF-295 cells were plated on sterilized coverslips in 6-well
plates and allowed to reach 65% confluency and then treated
for indicated intervals. At the conclusion of the treatment