Design, synthesis, and biological evaluation of (E)-styrylbenzylsulfones as novel anticancer agents

Article abstract of DOI:10.1021/jm701077b

Cell cycle progression is regulated by cyclins and cyclin-dependent kinases, which are formed at specific stages of the cell cycle and regulate the G1/S and G2/M phase transitions, employing a series of "checkpoints" governed by phosphorylation of their substrates. Tumor development is associated with the loss of these checkpoint controls, and this provides an approach for the development of therapeutic agents that can specifically target tumor cells. Here, we describe the synthesis and SAR of a novel group of cytotoxic molecules that selectively induce growth arrest of normal cells in the G1 phase while inducing a mitotic arrest of tumor cells resulting in selective killing of tumor cell populations with little or no effect on normal cell viability. The broad spectrum of antitumor activity in vitro and xenograft models, lack of in vivo toxicity, and drug resistance suggest potential for use of these agents in cancer therapy.

Full text of DOI:10.1021/jm701077b

86
J. Med. Chem. 2008, 51, 86–100
Design, Synthesis, and Biological Evaluation of (E)-Styrylbenzylsulfones as Novel Anticancer
Agents
M. V. Ramana Reddy,* Muralidhar R. Mallireddigari, Stephen C. Cosenza, Venkat R. Pallela, Nabisa M. Iqbal,
Kimberly A. Robell, Anthony D. Kang, and E. Premkumar Reddy*
Fels Institute for Cancer Research and Molecular Biology, Temple UniVersity School of Medicine, 3307 North Broad Street,
Philadelphia, PennsylVania 19140-5101
ReceiVed August 31, 2007
Cell cycle progression is regulated by cyclins and cyclin-dependent kinases, which are formed at specific
stages of the cell cycle and regulate the G1/S and G2/M phase transitions, employing a series of “checkpoints”
governed by phosphorylation of their substrates. Tumor development is associated with the loss of these
checkpoint controls, and this provides an approach for the development of therapeutic agents that can
specifically target tumor cells. Here, we describe the synthesis and SAR of a novel group of cytotoxic
molecules that selectively induce growth arrest of normal cells in the G1 phase while inducing a mitotic
arrest of tumor cells resulting in selective killing of tumor cell populations with little or no effect on normal
cell viability. The broad spectrum of antitumor activity in vitro and xenograft models, lack of in vivo toxicity,
and drug resistance suggest potential for use of these agents in cancer therapy.
Introduction
machinery. The genetic changes seen in a malignant cell are
primarily aimed at overriding this negative regulation and result
in the loss of one or both of the intrinsic checkpoints that are
normally used by their normal counterparts. While some of the
oncogenes, such as ras, force progression through G1, other
genes such as Rb, which are termed tumor suppressor genes,
function as “gatekeepers” of these restriction points.10,11 Cancer
is characterized by a loss of one or more tumor suppressor genes,
which enables a malignant cell to ignore all of the safeguards
that are aimed at preventing unwanted cell division.
Cancer is now believed to result from unlimited growth of a
given cell, which is often due to a block in the ability of cells
to undergo differentiation and/or apoptosis. Most of our
understanding of how cells grow and divide comes from the
study of cells grown in vitro. The cell cycle is typically divided
into four phases: G1, S, G2, and M. The periods associated
with DNA synthesis (S phase) and mitosis (M phase) are
separated by gaps called G1 and G2.1–6 The past 2 decades have
seen a series of discoveries that have provided us with a better
understanding of the complexity of the control mechanisms,
which ensure ordered progression of cell cycle. It is becoming
apparent that the order and timing of the cell cycle are critical
for accurate transmission of genetic information, and conse-
quently, a number of biochemical pathways have evolved to
ensure that initiation of a particular cell cycle event is dependent
on the accurate completion of the others. These biochemical
pathways have been termed “checkpoints”.1–6
Most normal cells, unless they have received a stimulus to
proliferate or differentiate, remain in a resting state, termed Go.
However, when the organism requires additional cells, extra-
cellular stimuli induce the cells to enter the G1 phase of the
cell cycle and become committed to cell division. It is at a late
point in the G1 phase of the cell cycle that a potentially dividing
cell reaches the “restriction point”, a time at which the cell must
determine whether the conditions are suitable for continued
proliferation.4,7,8 Provided that conditions are conducive to
proliferation, the cell proceeds past this checkpoint. An absolute
prerequisite for cell growth is the duplication of its genetic
material, which occurs during the S phase. Once the DNA has
been replicated, the cell “ascertains” whether this process has
been correctly executed during the second checkpoint during
G2, and provided that it has, the cell divides during mitosis, or
M phase.8,9 The ordered growth process seen in normal cells is
a result of regulatory control mechanisms that restrain cell cycle
An important rule associated with cell cycle progression (for
both normal and tumor cells) is the fact that once a cell crosses
the “restriction point” (which is the G1/S boundary), it has to
either divide into two daughter cells or die4 because of the fact
that most eukaryotic cells can exist in S, G2, and M phases of
the cell cycle for only a limited time. Most chemotherapeutic
agents, such as paclitaxel, that are currently used in cancer
therapy function by blocking cell cycle progression at a point
beyond the G1/S boundary (M phase in the case of paclitaxel),
resulting in the death of the tumor cell.12 A major problem with
many of the current drugs is their inability to discriminate
between normal and tumor cells. As a result, normal cells
undergoing active cell division also become blocked at the
mitotic phase of the cell cycle and enter programmed cell death
pathways, the effects of which are often manifested as the toxic
side effects seen in patients treated by these drugs. A second
problem appears to be the development of resistance to many
of the chemotherapeutic agents often due to overexpression of
drug transporters. Our quest was to design new chemical entities
that exhibit reduced toxicity in normal cells and are not
recognized by drug transporters that are overexpressed in drug-
resistant tumor cells. In this communication, we describe the
synthesis of a group of styrylbenzylsulfones that induce apop-
totic death of a wide variety of human tumor cell lines at
subnanomolar concentrations while exhibiting relatively low
toxicity to normal human cells. More importantly, these
compounds were found to be active against a wide variety of
* To whom correspondence should be addressed. For M.V.R.R.:
phone, (215) 707-7336; fax, (215) 707-1454; e-mail, rreddy@temple.edu.
For E.P.R.: phone, (215) 707-4307; fax, (215) 707-1454; e-mail,
reddy@temple.edu.
10.1021/jm701077b CCC: $40.75
2008 American Chemical Society
Published on Web 12/19/2007

(E)-Styrylbenzylsulfones as Anticancer Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 87
Scheme 1. Synthesis of (E)-Styrylbenzylsulfonesa
a Reagents and conditions: (a) NaOH, MeOH, 4 h, room temp, 94%; (b) 30% H2O2, CH3COOH, 18 h, room temp, 92%; (c) piperidine, benzoic acid,
toluene, 120 °C, 2–4 h, 48%; (d) CH3COOH, benzylamine, 118 °C, 2–8 h, 66%.
Scheme 2. Alternative Method for the Synthesis of (E)-Styrylbenzylsulfonesa
a Reagents and conditions: (a) NaOH, MeOH, 4 h, room temp, 93%; (b) 30% H2O2, CH3COOH, 24 h, room temp, 94%; (c) NaBH4, EtOH, 30 min, 0 °C,
88%; (d) p-toluenesulfonic acid, C6H6, 3 h, 80 °C, 65%.
human tumor cell lines that are resistant to the activity of many
of the cytotoxic agents.
condensation of 4 with aromatic aldehydes 5 in toluene in the
presence of catalytic amounts of piperidine and benzoic acid
yielded styrylbenzylsulfones, 6.14 Alternately, the condensation
between 4 and 5 was also carried out in glacial acetic acid in
the presence of a catalytic amount of benzylamine to obtain
6.13
Chemistry
The synthetic routes for the preparation of substituted
styrylbenzylsulfones are shown in Schemes 1 and 2. In Scheme
1 substituted benzyl bromides, 1, were reacted with thioglycollic
acid 2 in the presence of mild or strong bases to obtain
compound 3. Complete oxidation of 3, with hydrogen peroxide
in the presence of glacial acetic acid, gave 4.13 Knoevenagel
Monosubstituted styrylbenzylsulfones were also prepared as
shown in Scheme 2. Benzyl mercaptans with desired substitu-
ents, 7, were reacted with R-bromo substituted acetophenones,
8, in absolute alcohol in the presence of a base to obtain 9.

88 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Reddy et al.
Scheme 3. Synthesis of (E)-2,4,6-(Trimethoxystyryl)-3-hydroxy-4-methoxybenzylsulfonea
a Reagents and conditions: (a) TBDMS-THF, N,N-diisopropylethylamine, DMF, room temp, 12–16 h, 100%; (b) NaBH4, MeOH, 0 °C, 30 min, 73%;
(c) SOCl2, C6H6, 0 °C, 2 h, 70%; (d) HSCH2COOH, NaOH, MeOH, 3 h, room temp, 75%; (e) TBAF-THF, THF, room temp, 2 h, 50%; (f) 30% H2O2,
CH3COOH, 18 h, room temp, 60%; (g) piperidine, benzoic acid, toluene, 120 °C, 2–4 h, 63%.
Oxidation of 9 with hydrogen peroxide in glacial acetic acid
gave 10.13 Reduction of the carbonyl group in 10 with sodium
borohydride in ethanol gave corresponding alcohol 11.14
Elimination of water from 11 in refluxing solution of p-
toluenesulfonic acid in benzene afforded 6.15
To enhance the bioavailability and water solubility of (E)-
2,4,6-trimethoxystyryl-3-hydroxy-4-methoxybenzylsulfone 20,
its sodium phosphate prodrug was synthesized (Scheme 5). The
prodrug was synthesized in three steps starting from styrylben-
zylsulfone 20. Phosphorylation of the phenolic group in 20
employing dibenzyl phosphite under basic conditions gave
O-dibenzyl phosphate 27. Cleavage of benzyl groups with
bromotrimethylsilane in acetonitrile produced 3-O-phosphate
28.18 Treatment of the phosphate 28 with sodium hydroxide in
anhydrous ethylene glycol dimethyl ether yielded disodium
O-phosphate 29.
Schemes 3 and 4 depict the method used for the preparation
of 2,4,6-trimethoxystyryl-3-hydroxy-4-methoxybenzylsulfone 20
and its water-soluble disodium phosphate salt 29 (Scheme 5).
The synthesis of compound 20 is outlined in Scheme 3.
TBDMS protected isovanillin 13 was reduced to alcohol 14 with
sodium borohydride. The benzyl alcohol 14 was converted to
benzyl chloride 15 with SOCl2 and was subsequently converted
to 16 by condensing with thioglycollic acid. Deprotection of
TBDMS with tetrabutylammonium fluoride and subsequent
oxidation of 17 with hydrogen peroxide provided 18. Condensa-
tion of 18 with aldehyde 19 provided styrylbenzylsulfone 20.
Alternatively, 20 was also prepared as shown in Scheme 4.
Isovanillin 12 was reacted with 4- toluenesulfonyl chloride in
the presence of pyridine to obtain compound 21.16 Reduction
of 21 with sodium borohydride gave 22. Treatment of 22 with
thionyl chloride resulted in 23, which on condensation with
thioglycollic acid yielded benzylthioacetic acid 24. Oxidation
of 24 with hydrogen peroxide gave corresponding sulfonylacetic
acid 25. Knoevenagel type condensation of 25 with 2,4,6-
trimethoxybenzaldehyde 19 in the presence of a base produced
unsaturated sulfone 26. Removal of tosyl group by treating 26
with sodium hydroxide gave the styrylbenzylsulfone 20.
Structure–Activity Relationships (SAR)a
Following the synthesis of this group of compounds, their in
vitro cytotoxicity was assessed using four different human tumor
cell lines derived from human breast (BT20), prostate (DU145),
lung (H157), and colorectal (DLD1) cancers. The results of this
study are presented in Table 1. These studies show that the
cytotoxic activity of the styrylbenzylsulfones is completely
dependent on the nature and position of the substituents on the
two aromatic rings. In a majority of the compounds described
here, we have kept a methoxy group constant at the fourth
aAbbreviations: SAR, structure–activity relationship; MDR, multidrug
resistance; HFL, human lung fibroblasts; PARP, poly(ADP-ribose) poly-
merase-1; HUVEC, human vascular endothelial cells; NBT, nitroblue
tetrazolium.

(E)-Styrylbenzylsulfones as Anticancer Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 89
Scheme 4. Alternative Method for the Synthesis of (E)-2,4,6-(Trimethoxystyryl)-3-hydroxy-4-methoxybenzylsulfonea
a Reagents and conditions: (a) p-toluenesulfonyl chloride, pyridine, 70–80 °C, 2 h, 98%; (b) NaBH4, MeOH, 20 °C, 30 min, 97%; (c) SOCl2, C6H6, 15–20
°C, 2 h, 90%; (d) HSCH2COOH, NaOH, MeOH, 65 °C, 5 h, 93%; (e) 30% H2O2, CH3COOH, room temp, 18 h, 80%: (f) piperidine, benzoic acid, C6H6,
80 °C, 4–5 h, 65%; (g) 20% NaOH, MeOH, 65 °C, 3–4 h, 95%.
position on the aromatic ring of the benzyl moiety. A moderate
cytotoxic activity was seen when a fluorine atom was present
at the 4-position (6b) on the styryl aromatic ring. Changing the
fluorine atom at the 4-positon with a chlorine (6c), a nitro (6d),
a methoxy (6a), or an amino (6e) group gradually decreased
the activity of the molecules. When the position of the methoxy
group is changed from the 4- to the 2-position (6f) on the styryl
aromatic ring, the molecule partially recovered the lost activity.
The introduction of a chlorine atom in the 2-position (6g) in
6b retained cytotoxic activity, whereas a methoxy group in the
same position (6i) resulted in the loss of activity. Dimethyl
substitutions on the styryl aromatic ring with a methoxy group
at the 4-position on the benzyl aromatic ring (6h, 6k, and 6l)
resulted in the molecules possessing a low level of cytotoxicity.
Whereas the results are quite surprising for the molecules that
are disubstituted with methoxy groups (6j, 6m, 6n, 6o, and 6p)
on the styryl aromatic ring, the results obtained in cytotoxicity
assays using these compounds (6j, 6m, 6n, 6o, and 6p) clearly
show that the methoxy group, when present at the 2,6-positions
(6m), enhances the activity of the molecule by greater than 40-
fold when compared to other disubstituted methoxysulfones (6j,
6n, 6o, and 6p). Because the introduction of two methoxy groups
on the styryl aromatic ring enhanced the biological activity, we
have synthesized some trimethoxystyryl analogues to determine
if this further enhances their cytotoxic properties. Analysis of
these compounds (6q, 6r, 6s, and 6t) in the cell-killing assays
showed that 4-methoxybenzyl-2,4,6-trimethoxystyrylsulfone (6s)
is 20-fold more active than 6m, whereas the other trisubstituted

90 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Reddy et al.
Scheme 5. Method for the Synthesis of (E)-2,4,6-(Trimethoxystyryl)-3-O-phosphate Disodium 4-Methoxybenzylsulfonea
a Reagents and conditions: (a) CH3CN, triethylamine, CBr4, dibenzyl phosphite, KH2PO4, room temp, 2 h, 73%; (b) CH3CN, bromotrimethyl silane,
sodium thiosulfate, room temp, 45 min, 44%; (c) NaOH/H2O, ethyleneglycoldimethyl ether, room temp, 3 h, 98%.
styrylsulfones (6q, 6r, and 6t) were totally inactive at the highest
concentration (20 µM) tested. These results show that when the
2, 4, and 6 positions on the styryl aromatic ring are occupied
by methoxy groups, the molecules attain optimum biological
activity. To validate whether methyl groups at those positions
can replace the methoxy groups and retain the activity, we have
prepared 2,4,6-trimethylstyrylsulfone (6w), which was found
to be inactive in cell-killing assays. To further assess the
significance of the methoxy group on the 4-position of styryl
ring in 6m, we replaced the methoxy group at that site with a
hydroxy (6u) or a fluoro (6v) substituent. Both of these
replacements resulted in either a reduced level or a total loss of
activity. Once the methoxy subtituents are fixed at the 2, 4, and
6 positions of the styryl ring, to further enhance the activity of
the molecule, we then determined the effect of other substituents
on the benzyl aromatic ring. Replacing the methoxy group at
the 4-position on the benzylic aromatic ring of 6s by chloro
(6ae), nitro (6af), cyano (6ag), carboxy (6ah), and hydroxy (6ai)
resulted in molecules that substantially lost activity. These results
show that the methoxy group is indispensable at the 4-position
of the benzyl aromatic ring of 6s with respect to its biological
activity. To analyze the effect of the additional substituents on
the benzyl aromatic ring, we have synthesized a number of
analogues containing 4-methoxy-3-halo, nitro, cyano, carboxy,
methoxy (data not shown), hydroxy (6aa), 3,4,5-trimethoxy
(6ac), and 2,3,4-trimethoxy (6ad) benzylsulfones. Cytotoxicity
analyses of these analogues on four cancer cell lines showed
that the compound with the hydroxy substituent at position 3
(6aa) exhibited the best activity in the entire series. This
compound, 6aa, is almost 8- to 10-fold more active than 6s in
all four cell lines. Further, the introduction of a hydroxy group
at the third position not only enhanced the potency of the
molecule but also created a method to generate a water-soluble
analogue (6ab), which is critical for intravenous administration
of the compound. The conversion of the hydroxyl group in 6aa
to a disodium phosphate 6ab derivative did not alter the potency
of the molecule.
Biological Results and Discussion
In Vitro Antitumor Effects of 6s and 6aa Compounds.
We next tested the activity of two of the most active compounds
listed in Table 1 against 94 different human tumor cell lines,
and surprisingly, they were found to induce apoptosis of all of
these cell lines with very similar GI50 values (selected data
shown in Table 2). Some of these compounds (such as 6s, 6aa)
were also tested by the National Cancer Institute through its
Developmental Therapeutics Program (DTP) against their panel
of 60 human cancer cell lines.21 Their results showed that these
compounds exhibited broad-spectrum activity and inhibited the
growth of all of the tested cell lines, including drug-resistant
cell lines, at nanomolar concentrations. Notably, the GI50 and
LC50 values for many of these cell lines were similar, indicating
that they induced apoptosis in these cells. Statistical comparison
(using the NCI algorithm COMPARE) revealed that these drugs
are mitotic blockers of tumor cells.
6s and 6aa Compounds Are Highly Active against
Drug-Resistant Tumor Cell Lines. Development of resistance
to classical chemotherapeutic agents is widely observed in
patients who have not responded or have relapsed after first
round therapy and is the primary cause of treatment failure. In
the initial screening experiments, we observed that both our cell
panel and the NCI panel included several cell lines that are
multidrug resistant19,20 but were highly sensitive to the proapo-
ptotic effects of this series of compounds. To further investigate
the activity of these compounds against MDR positive tumor
types, we determined the IC50 values of 6s and 6aa using two
classical MDR positive cell lines. The results shown in Figure
1A show a 96 h dose response of the uterine sarcoma cell line
MES-SA and the multidrug resistant subline MES-SA/DX519

(E)-Styrylbenzylsulfones as Anticancer Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 91
Table 1. In Vitro Cytotoxicity of Styrylbenzylsulfones
IC50(µM)
compd
R
R1
BT20
>20
DU145
>20
H157
>20
DLD1
>20
6a
6b
6c
6d
6e
6f
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-F
4-Cl
4-NO2
4-NH2
2-OMe
2-Cl,4-F
1.5
2.0
5.0
1.25
2.5
7.5
2.5
3.5
7.5
2.0
3.0
5.0
>20
>20
>20
>20
5.0
2.5
3.5
2.0
5.0
2.5
7.5
4
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
6u
6v
6w
6x
6y
6z
6aa
6ab
6ac
6ad
6ae
6af
6ag
6ah
6ai
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OCF3
4-OMe,3-OH
4-OMe,3-OH
4-OMe,3-OH
4-OMe,3-OPO3Na2
3,4,5-(OMe)3
2,3,4-(OMe)3
4-Cl
4-NO2
4-CN
4-COOH
4-OH
2,4-(CH3)2
2-OMe,4-F
3,4-(OMe)2
3,5-(CH3)2
2,6-(CH3)2
2,6-(OMe)2
2,4-(OMe)2
2,5-(OMe)2
3,5-(OMe)2
2,4,5-(OMe)3
2,3,4-(OMe)3
2,4,6-(OMe)3
3,4,5-(OMe)3
2,6-(OMe)2,4-OH
2,6-(OMe)2,4-F
2,4,6-(CH3)3
2,4,6-(OMe)3
3,4,5-(OMe)3
2,6-(OMe)2,4-OH
2,4,6-(OMe)3
2,4,6-(OMe)3
2,4,6-(OMe)3
2,4,6-(OMe)3
2,4,6-(OMe)3
2,4,6-(OMe)3
2,4,6-(OMe)3
2,4,6-(OMe)3
2,4,6-(OMe)3
15
12
15
15
15
>20
>20
>20
10
>20
>20
>20
20
>20
>20
>20
15
>20
>20
>20
0.40
0.25
0.70
0.70
>20
>20
>20
>20
>20
0.020
>20
>20
>20
>20
>20
>20
>20
0.025
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
0.030
0.040
>20
>20
>20
>20
0.75
>20
0.5
>20
0.5
0.75
>20
>20
3
7.5
0.25
0.010
0.005
4
5
0.5
0.003
0.0025
6
4
10
0.5
0.004
0.007
>20
15
15
20
10
0.5
0.003
0.007
>20
15
15
15
20
15
20
>20
15
10
15
12.5
15
15
>20
12.5
7.5
15
7.5
10
7.5
15
17.5
20
treated with 6aa. This cell line has been shown to express high
levels of P-glycoprotein and is resistant to a number of drugs
including doxorubicin, paclitaxel, vincristine, vinblastine, eto-
poside, mitoxantrone, dactinomycin, and daunorubucin. The
activity of our compounds was then compared to the activity
of paclitaxel (MDR sensitive drug). Our results show that the
parental cell line was very sensitive to paclitaxel (IC50 ) 4 nM),
but the MDR positive subline was greater than 100-fold resistant
(IC50 ) 750 nM). When the two cell lines were treated with
6aa, both the parental and the MDR positive cell lines were
equally sensitive to the cell killing activity of the compound.
We also investigated as to whether atypical multidrug resistant
cells are sensitive to 6aa. For these studies, we employed the
parental leukemic cell line CEM and its MDR subline CEM/
C2.20 CEM/C2 was selected and subcloned for resistance to
camptothecin and has cross-resistance to etoposide, dactinimy-
cin, bleomycin, mitoxantrone, doxorubicin, and daunorubicin.
Our results show that the campothecin-resistant subline, CEM/
C2, was highly sensitive to the styrylbenzylsulfone series of
compounds, suggesting that these compounds do not share any
cross-resistance to classical MDR and atypical MDR cell lines
(Table 2).
Effect of 6aa and 6ab on Soft Agar Colony formation.
We next tested the antitumorigenic activity of 6aa and 6ab in
soft agar. For soft agar assays, we used three different cell lines,
BT20, DU145, and MIA-PaCa-2, representing breast, prostate,
and pancreatic cancers, respectively. In all cases we observed
complete inhibition of the growth of tumor cells in a dose-
dependent manner for the individual compounds (Figure 1B
shows data for MIA-PaCa-2). In these assays, paclitaxel was
used as a positive control, which showed a slightly lower
potency than 6aa and 6ab (Figure 1B).
Effects of 6s and 6aa on Cell Cycle Progression of
Normal and Tumor Cells. We next examined the effect of
these compounds on normal and tumor cell cycle progression
using FACS analysis. Figure 2A shows the effect of 6aa on
the cell cycle progression of human vascular endothelial cells
(HUVEC) and DU145 (prostate cancer) cells. The results of
this study show that the addition of the 6aa to HUVEC cells
resulted in a block of their cell cycle progression in the G1 phase,
causing growth arrest without a loss of viability. On the other
hand, tumor cells treated with this compound gradually ac-
cumulated in the G2/M phase of the cell cycle and appeared to
be unable to exit from this phase, leading to the activation of

92 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Reddy et al.
Table 2. Tumor Cell Killing Concentrations (IC50, µM) of 6s and 6aa
measured on alternative days, and the total length of the
experiment was 21 days. The results presented in Figure 3B
show that 6ab readily inhibited tumor growth in this xenograft
model system. Of the eight mice included in each group, 100%
of the control mice (placebo administered) showed a doubling
or tripling of the total tumor volume. On the other hand, the
majority of the mice administered with 6s or the phosphate salt
of 6aa showed growth arrest or a gradual reduction in their
tumor volume, suggesting that these compounds, with proper
formulation, can be valuable anticancer therapeutics.
In Vivo Toxicity Studies in Mice. To assess the in vivo
toxicity profile, 100 mg/kg 6aa was intravenously administered
into mice and its effect on the in vitro hematopoietic colony
formation of bone marrow cells was determined at 12, 24, and
48 h intervals following the injection of the drug. These studies
(Figure 4) show that there was no reduction in total, myeloid,
or lymphoid colony formation. Single-dose and repeat-dose (28
daily injections) toxicology studies and detailed biochemical
and cellular analysis of one of the water-soluble analogues (6ab)
revealed that unlike most other cytotoxic agents, this drug did
not cause hematotoxicity (no myleosuppression), liver damage,
or detectable neurotoxicity in these animals. High dose-
dependent drug levels were sustained in circulation, suggesting
that therapeutic levels could be achieved without overt toxicity.
Acute and Repeat Toxicity Studies in Rats. We have carried
out single-dose and repeat-dose (28 daily injections) toxicology
studies with one water-soluble form of this series (6ab) to assess
the safety of intravenously administered drug in rats. Detailed
biochemical and cellular analysis revealed that this drug did
not cause hematotoxicity (no myleosuppression), liver damage,
or any detectable neurotoxicity in these animal studies. High
dose-dependent drug levels were sustained in circulation,
suggesting that therapeutic levels could be achieved without
overt toxicity.
cell line
tumor type
6s
6aa
T47D
breast (ER+)
breast (ER+)
prostate (AR-)
prostate (AR+)
ovarian
ovarian
pancreatic
glioblastoma
NSCLC
NSCLC
SCLC
SCLC
gastric
gastric
gastric
colorectal
colorectal
colorectal
0.025
0.003
0.025
0.03
0.008
ND
0.008
0.04
0.03
0.02
0.015
0.008
0.02
0.008
0.01
0.015
0.04
ND
0.02
ND
0.04
0.03
ND
0.009
0.015
0.008
0.009
0.01
0.01
0.03
0.01
ND
0.006
0.004
0.007
0.008
0.006
0.006
0.004
0.007
0.007
0.01
0.007
0.005
0.007
0.006
0.005
0.009
0.008
0.009
0.008
0.007
0.007
0.009
0.004
0.005
0.006
0.007
0.004
0.006
0.005
0.01
MCF-7
DU145
PC-3
OV-CAR-3
Sk-OV-3
MIA-PaCa2
U87
H157
A549
H187
N417
AGS
RF1
RF48
COLO-205
DLD-1
HCT-116
HCT-15
SW480
SK-MEL-28
CEM
colorectal
colorectal
melanoma
leukemic
K562
CML
MOLT-4
Namalwa
Daudi
T-lymphoblastic: all
Burkitt’s lymphoma (B-cell)
Burkitt’s lymphoma (B-cell)
Burkitt’s lymphoma (B-cell)
sarcoma
resistant sarcoma
leukemic
resistant leukemic
ovarian
Raji
MES-SA
MESSA/DX5a
CEM
CEM/C2a
2008
0.01
0.005
0.004
2008/17/4a
resistant ovarian
ND
a These cell lines constitute multidrug-resistant cell lines and show up-
regulation of MDR and in the case of CEM/C2, additional mutations in the
Topo-2 gene.19,20
apoptotic pathways as judged by PARP [poly(ADP-ribose)
polymerase-1] cleavage,22 which is a marker for caspase
activation (Figure 2B). No PARP cleavage was observed in
HUVEC cells following similar treatment with 6aa compounds
(Figure 2B).
Conclusion
In this communication, we describe the synthesis of a group
of styrylbenzylsulfones that induce apoptotic death of a wide
variety of human tumor cell lines at subnanomolar concentra-
tions while exhibiting relatively low toxicity to normal human
cells. Our studies show that the cytotoxic activity of the
styrylbenzylsulfones is completely dependent on the nature and
position of the substituents on the two aromatic rings. In a
majority of the compounds described here, we have kept a
methoxy group constant at the fourth position on the aromatic
ring of the benzyl moiety. These structure–function studies show
that when the 2, 4, and 6 positions on the styryl aromatic ring
are occupied by the methoxy groups, the molecules attain
optimum biological activity (6s). This activity could be further
enhanced by the introduction of a hydroxyl group at the third
position of the benzylic ring (6aa and 6ab). Biological evalu-
ation of the activity of these compounds shows that these
compounds are highly active against a wide variety of human
tumor cell lines including those that are resistant to the activity
of many of the currently used chemotherapeutic agents.
The low toxicity profile, both in vitro and in vivo, and their
potent tumor inhibitory activity as seen in soft agar and nude
mouse xenograft assays point to the potential value of these
compounds as safe therapies for cancer, lacking many of the
side effects normally associated with current chemotherapeutic
agents. Recent studies with 6s, 6aa, and 6ab show that these
compounds altered the growth and cell cycle status of mantle
cell lymphoma cell lines and potently inhibited the expression
of several important proteins, including cyclin-dependent kinase
In Vivo Antitumor Effects of 6s and 6aa of Compounds. In
order to determine in vivo efficacy, we utilized the nude mouse
model system. A highly aggressive human estrogen negative
breast carcinoma cell line (BT20) was xenografted into athymic
nude mice. The animals were treated with either 50 mg/kg 6s
using a Q4D schedule or 25 mg/kg using a Q2D schedule. The
animals were treated when the tumors were approximately 70
mm3 in size. Figure 3A shows that intraperitonial (ip) injections
of 6s using either of the two schedules were able to inhibit the
growth of the tumors. The vehicle control treated tumors, on
average, increased in volume over the 22 day period by 5-fold
(62-335.5 mm3), while the Q2D 6s treated tumors increased
in volume by only 2.5-fold (67.5-165 mm3). The Q4D 6s
treated tumors also had significant but slightly less tumor growth
inhibition, whereby these tumors increased in average volume
by only 2.9-fold (74-217 mm3). 6s was well tolerated at these
doses, as determined by body weights and physical observations.
These studies show that 6s is efficacious against human tumor
xenografts while showing no signs of toxicity at the schedules
tested under this study.
Because 6s is poorly water-soluble, we synthesized 6ab,18
which was highly water-soluble and allowed intravenous
administration. To test the effects of 6ab in vivo, we used two
groups of mice. One group received the vehicle alone, while
the second group received the compound by intravenous (iv)
injection into the tail vein (Figure 3B). The tumor size was then

(E)-Styrylbenzylsulfones as Anticancer Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 93
Figure 1. Antitumor effects of 6aa. (A) 6aa inhibits the growth of parental (MES-SA) and paclitaxel-resistant (MES-SA/DX5) cell lines with
equal efficiency. The parental uterine sarcoma cells and the MDR positive (MES-SA/DX5) cells were plated into six-well dishes and treated with
various concentrations of 6aa and paclitaxel for 96 h. The number of viable cells from duplicate plates was determined by trypan blue exclusion.
(B) Shown are results from soft agar assays. MIA-PaCa-2 cells (1.0 × 105) were plated in soft agar containing various concentrations of each
compound in triplicate. After 3 weeks of growth, the plates were stained for 48 h using 0.05% nitroblue tetrazolium solution. Representative plates
were photographed using an Olympus stereoscope mounted with a Sony digital camera system (DKC5000, Sony Inc.).
4, p53, mouse double minute 2 (MDM2), and cyclin D.23 Since
room temperature, the precipitated product was filtered and washed
with 2-propanol. If solid was not formed, the reaction mixture was
diluted with ether and washed with saturated NaHCO3, dilute
hydrochloric acid, and water. The organic layer was dried over
sodium sulfate, filtered, and concentrated under vacuum to obtain
the desired crude product 6. The crude product was recrystallized
in 2-propanol to yield an analytically pure sample of 6. The
following (E)-styrylbenzylsulfones 6 were prepared using the above
procedure.
(E)-4-Methoxystyryl-4-methoxybenzylsulfone (6a). The title
compound was obtained from 4-methoxybenzylsulfonylacetic acid
and 4-methoxybenzaldehyde following the procedure as described
in method A. Yield, 51%; white solid, mp 150–152 °C. 1H NMR:
δ 3.65 (s, 3H, OCH3), 3.68 (s, 3H, OCH3), 4.07 (s, 2H, CH2), 6.36
(d, 1H, J ) 14.4 Hz, dCH), 6.71-6.76 (m, 4H, Ar-H), 7.09–7.22
(m, 5H, Ar-H + vinylic). Anal. (C17H18O4S) C, H.
6s, 6aa, and 6ab are highly effective in various combinations
with conventional chemotherapy,23 the lack of overt hemato-
toxicity of these compounds may be beneficial for testing novel
combinations for advanced cancers, including tumors resistant
to conventional chemotherapy. In addition, their safety profile
seen with normal hematopoietic cells suggests that these
compounds have a potential use in in vitro purging of tumor
cells from patient bone marrow for use in autologous bone
marrow transplantation. Clinical and preclinical studies currently
underway will reveal the best way to utilize these compounds
in cancer therapy.
Experimental Section
Chemistry. General Methods. All reagents and solvents were
obtained from commercial suppliers and used without further
purification unless otherwise stated. Solvents were dried using
standard procedures, and reactions requiring anhydrous conditions
were performed under N2 atmosphere. Reactions were monitored
by thin layer chromatography (TLC) on precoated silica gel F254
plates (Sigma-Aldrich) with a UV indicator. Column chromatog-
raphy was performed with Merck 70–230 mesh silica gel 60 Å.
Yields were of purified product and were not optimized. Melting
points were determined using an Electrothermal Mel-Temp 3.0
(E)-4-Fluorostyryl-4-methoxybenzylsulfone (6b). The title com-
pound was obtained from 4-methoxybenzylsulfonylacetic acid and
4-fluorobenzaldehyde following the procedure as described in
1
method A. Yield, 60%; white solid, mp 148–149 °C. H NMR: δ
3.81 (s, 3H, OCH3), 4.25 (s, 2H, CH2), 6.61 (d, 1H, J ) 14.0 Hz,
dCH), 6.91–7.45 (m, 9H, Ar-H + vinylic). Anal. (C16H15FO3S)
C, H.
(E)-4-Chlorostyryl-4-methoxybenzylsulfone (6c). The title com-
pound was obtained from 4-methoxybenzylsulfonylacetic acid and
4-chlorobenzaldehyde following the procedure as described in
1
micromelting point apparatus and are uncorrected. H NMR and
1
13C NMR spectra were obtained with a Bruker AM 300 and 400
MHz spectrometer. The chemical shifts are reported in parts per
million (δ) downfield using tetramethylsilane (Me4Si) as internal
standard and CDCl3 as the solvent except where indicated. Spin
multiplicities are given as s (singlet), d (doublet), br s (broad
singlet), m (multiplet), and q (quartet). Coupling constants (J values)
were measured in hertz (Hz). Combustion analyses were performed
with a Perkin-Elmer 2402 series II CHNS/O analyzer by Quantita-
tive Technologies Inc. (White House, NJ). All the compounds gave
satisfactory combustion analysis results (C, H, N within 0.4% of
calculated values). Benzylsulfonylacetic acids were prepared ac-
cording to the procedure reported in the literature.13
method A. Yield, 66%; white solid, mp 176–177 °C. H NMR: δ
3. 56 (s, 3H, OCH3), 4.00 (s, 2H, CH2), 6.40 (d, 1H, J ) 16.0 Hz,
dCH), 6.63–7.14 (m, 9H, Ar-H + vinylic). Anal. (C16H15ClO3S)
C, H.
(E)-4-Nitrostyryl-4-methoxybenzylsulfone (6d). The title com-
pound was obtained from 4-methoxybenzylsulfonylacetic acid and
4-nitrobenzaldehyde following the procedure as described in method
1
A. Yield, 56%; light-yellow solid, mp 179–181 °C. H NMR: δ
3.76 (s, 3H, OCH3), 4.24 (s, 2H, CH2), 6.78 (d, 1H, J ) 15.6 Hz,
dCH), 6.84 (m, 2H, Ar-H), 7.20–7.24 (m, 2H, Ar-H), 7.41 (d,
1H, J ) 15.6 Hz, CHd), 7.52 (d, 2H, J ) 13.3 Hz, Ar-H), 8.19
(d, 2H, J ) 11.1 Hz, Ar-H). Anal. (C16H15NO5S) C, H, N.
General Procedure for the Preparation of (E)-Styryl-
benzylsulfones (6). Method A (Scheme 1). A mixture of benzyl-
sulfonyl acetic acid 4 (10 mmol), araldehyde 5 (10 mmol), acetic
acid (10 mL), and a catalytic amount of benzylamine (150 µL)
was refluxed for about 2–8 h. After completion of the reaction (TLC
monitoring, CHCl3 on silica gel plate), with the contents cooled to
(E)-4-Aminostyryl-4-methoxybenzylsulfone (6e). In a 100 mL
round-bottomed flask fitted with condenser, 5% Pd/C (0.073 g) was
taken, and 20 mL of ethanol was added slowly. Compound 6d (0.5
g, 1.4 mmol) and hydrazine hydrate (1.24 g, 38.7 mmol) were added
to the contents of the flask, and the mixture was refluxed for 6 h.
Completion of the reaction was monitored by TLC (CHCl3 on silica

94 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Reddy et al.
Figure 2. Preferential tumor cell killing activity of 6aa. (A) Cell cycle analyses. Normal (HUVEC) and tumor cells (DU145) were treated with 20
nM 6aa and incubated in medium containing 10% fetal bovine serum. At 24 h intervals, the cells were fixed, stained with propidium iodide, and
analyzed for their DNA content by flow cytometry. (B) Induction of apoptosis in normal (HUVEC) and tumor cells (DU145) was assessed by
Western blot analysis of cell lysates treated with 6aa for 24, 48, and 72 h. The Western blots were probed with anti-PARP antibodies to assess the
cleavage of the protein.
gel plate). The reaction mixture was filtered through Celite and
concentrated to 50% volume. The concentrated mixture was poured
onto crushed ice, and the solid formed was filtered, washed with
cooled water, and dried to get the desired product 6e. Yield, 52%;
light-yellow solid, mp 164–168 °C. 1H NMR: δ 3. 80 (s, 3H,
OCH3), 4.22 (s, 2H, CH2), 4.04 (br s, 2H, NH2), 6.42 (d, 1H, J
)15.6 Hz, dCH), 6.60–7.31 (m, 9H, Ar-H + vinylic). Anal.
(C16H17NO3S) C, H, N.
(E)-2-Methoxystyryl-4-methoxybenzylsulfone (6f). The title
compound was obtained from 4-methoxybenzylsulfonylacetic acid
and 2-methoxybenzaldehyde following the procedure as described
in method A. Yield, 53%; white solid, mp 115–117 °C. 1H NMR:
δ 3.75 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 4.18 (s, 2H, CH2),
6.82–7.27 (m, 9H, Ar-H + vinylic), 7.56 (d, 1H, J ) 15.6 Hz,
CHd). Anal. (C17H18O4S) C, H.
1H, J ) 15.6 Hz, dCH), 6.83–7.41 (m, 7H, Ar-H), 7.68 (d, 1H,
J ) 15.6 Hz). Anal. (C16H14ClFO3S) C, H.
(E)-2,4-Dimethylstyryl-4-methoxybenzylsulfone (6h). The title
compound was obtained from 4-methoxybenzylsulfonylacetic acid
and 2,4-dimethylbenzaldehyde following the procedure as described
in method A. Yield, 55%; white solid, mp 126–128 °C. 1H NMR:
δ 2.21 (s, 3H, CH3), 2.27 (s, 3H, CH3), 3.74 (s, 3H, OCH3), 4.19
(s, 2H, CH2), 6.49 (d, 1H, J ) 15.4 Hz, dCH), 6.83–7.24 (m, 7H,
Ar-H), 7.55 (d, 1H, J ) 15.4 Hz). Anal. (C18H20O3S) C, H.
(E)-4-Fluoro-3-methoxystyryl-4-methoxybenzylsulfone (6i).
The title compound was obtained from 4-methoxybenzylsulfony-
lacetic acid and 4-fluoro-3-methoxybenzaldehyde following the
procedure as described in method A. Yield, 55%; white solid, mp
105–107 °C. 1H NMR: δ 3.69 (s, 3H, OCH3), 3.75 (s, 3H, OCH3),
4.20 (s, 2H, CH2), 6.55 (d, 1H, J ) 15.5 Hz, dCH), 6.82–7.31 (m,
8H, Ar-H + vinylic). Anal. (C17H17FO4S) C, H.
(E)-2-Chloro-4-fluorostyryl-4-methoxybenzylsulfone (6g). The
title compound was obtained from 4-methoxybenzylsulfonylacetic
acid and 2-chloro-4-fluorobenzaldehyde following the procedure
as described in method A. Yield, 56%; white solid, mp 154–155
(E)-3,4-Dimethoxystyryl-4-methoxybenzylsulfone (6j). The ti-
tle compound was obtained from 4-methoxybenzylsulfonylacetic
acid and 3,4-dimethoxybenzaldehyde following the procedure as
described in method A. Yield, 54%; white solid, mp 160–161 °C.
1
°C. H NMR: δ 3.75 (s, 3H, OCH3), 4.22 (s, 2H, CH2), 6.62 (d,

(E)-Styrylbenzylsulfones as Anticancer Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 95
6.64–7.05 (m, 7H, Ar-H), 7.55 (d, 1H, J ) 15.4 Hz). Anal.
(C18H20O5S) C, H.
(E)-3,5-Dimethylstyryl-4-methoxybenzylsulfone (6k). The title
compound was obtained from 4-methoxybenzylsulfonylacetic acid
and 3,5-dimethylbenzaldehyde following the procedure as described
in method A. Yield, 58%; white solid, mp 127–130 °C. 1H NMR:
δ 2.08 (s, 6H, 2XCH3), 3.57 (s, 3H, OCH3), 3.99 (s, 2H, CH2),
6.40 (d, 1H, J ) 15.5 Hz, dCH), 6.83–7.24 (m, 7H, Ar-H), 7.11
(d, 1H, J ) 15.5 Hz). Anal. (C18H20O3S) C, H.
(E)-2,6-Dimethylstyryl-4-methoxybenzylsulfone (6l). The title
compound was obtained from 4-methoxybenzylsulfonylacetic acid
and 2,6-dimethylbenzaldehyde following the procedure as described
1
in method A. Yield, 53%; white solid, mp 99–101 °C. H NMR:
δ 2.15 (s, 3H, CH3), 2.27 (s, 3H, CH3), 3.75 (s, 3H, OCH3), 4.13
(s, 2H, CH2), 6.41 (d, 1H, J ) 15.4 Hz, dCH), 6.83–7.35 (m, 7H,
Ar-H), 7.55 (d, 1H, J ) 15.4 Hz). Anal. (C18H20O3S) C, H.
(E)-2,6-Dimethoxystyryl-4-methoxybenzylsulfone (6m). The
title compound was obtained from 4-methoxybenzylsulfonylacetic
acid and 2,6-dimethoxybenzaldehyde following the procedure as
described in method A. Yield, 51%; white solid, mp 136–138 °C.
1H NMR: δ 3.81(s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.91 (s, 3H,
OCH3), 4.23 (s, 2H, CH2), 7.19 (d, 1H, J ) 15.4 Hz, dCH),
6.64–7.05 (m, 7H, Ar-H), 7.90 (d, 1H, J ) 15.4 Hz). 13C NMR:
δ 160.6, 160.3, 135.9, 132.9.9, 132.6, 126.4, 120.9, 114.5, 110.7,
104.0, 61.7, 56.2, 55.7. Anal. (C18H20O5S) C, H.
(E)-2,4-Dimethoxystyryl-4-methoxybenzylsulfone (6n). The ti-
tle compound was obtained from 4-methoxybenzylsulfonylacetic
acid and 2,4-dimethoxybenzaldehyde following the procedure as
described in method A. Yield, 59%; white solid, mp 161–162 °C.
1H NMR: δ 3.73 (s, 3H, OCH3), 3.77 (s, 6H, 2 × OCH3), 4.14 (s,
2H, CH2), 6.71 (d, 1H, J ) 15.5 Hz, dCH), 6.37-7.23 (m, 7H,
Ar-H), 7.42 (d, 1H, J ) 15.5 Hz). Anal. (C18H20O5S) C, H.
(E)-2,5-Dimethoxystyryl-4-methoxybenzylsulfone (6o). The ti-
tle compound was obtained from 4-methoxybenzylsulfonylacetic
acid and 2,5-dimethoxybenzaldehyde following the procedure as
described in method A. Yield, 54%; white solid, mp 105–107 °C.
1H NMR: δ 3.71 (s, 3H, OCH3), 3.75 (s, 3H, OCH3), 3.76 (s, 3H,
OCH3), 4.18 (s, 2H, CH2), 6.78–7.26 (m, 8H, Ar-H + vinylic),
7.52 (d, 1H, J ) 15.6 Hz). Anal. (C18H20O5S) C, H.
(E)-3,5-Dimethoxystyryl-4-methoxybenzylsulfone (6p). The
title compound was obtained from 4-methoxybenzylsulfonylacetic
acid and 3,5-dimethoxybenzaldehyde following the procedure as
described in method A. Yield, 62%; white solid, mp 119–121 °C.
1H NMR: δ 3.70 (s, 6H, 2 × OCH3), 3.71 (s, 3H, OCH3), 4.15 (s,
2H, CH2), 6.55 (d, 1H, J ) 15.5 Hz, dCH), 6.42–7.20 (m, 7H,
Ar-H), 7.23 (d, 1H, J ) 15.5 Hz). Anal. (C18H20O5S) C, H.
(E)-2,4,5-Trimethoxystyryl-4-methoxybenzylsulfone (6q). The
title compound was obtained from 4-methoxybenzylsulfonylacetic
acid and 2,4,5-trimethoxybenzaldehyde following the procedure as
described in method A. Yield, 66%; white solid, mp 146–148 °C.
1H NMR: δ 3.73 (s, 3H, OCH3), 3.78 (s, 6H, 2 × OCH3), 3.81 (s,
3H, OCH3), 4.20 (s, 2H, CH2), 6.86 (m, 4H, Ar-H), 7.00 (d, 1H,
J ) 15.6 Hz, dCH), 7.31 (d, 2H, J ) 8.9 Hz), 7.61 (d, 1H, J )
15.6 Hz, CHd). Anal. (C19H22O6S) C, H.
Figure 3. In vivo antitumor effects of 6s and 6ab. Female athymic
(NCr-nu/nu) mice were injected subcutaneously with (0.5–1) × 107
ER-negative human breast tumor cells (BT-20) in 0.2 mL of PBS and
the tumors allowed to grow to a size of 100–150 mm3 in about 14
days. The mice were then paired such that the pairs harbored equal
sized tumors, which were then used to test the therapeutic effects of 6s
and 6ab. (A) Of the pairs, the animals were treated with either 50 mg/
kg 6s following a Q4D schedule or 25 mg/kg using a Q2D schedule or
vehicle (DMSO) control. The tumor size was then measured on alternate
days in two dimensions and the volume determined using the following
equation: V ) (L(S2))π/6), where L is the longer and S is the shorter of
the two measurements. (B) 6ab was dissolved in PBS and was
administered intravenously (50 mg/kg) through the tail vein on every
alternate day. Tumor measurements were done as in Experimental
Section.
(E)-2,3,4-Trimethoxystyryl-4-methoxybenzylsulfone (6r). The
title compound was obtained from 4-methoxybenzylsulfonylacetic
acid and 2,3,4-trimethoxybenzaldehyde following the procedure as
described in method A. Yield, 54%; white solid, mp 154–156 °C.
1H NMR: δ 3.75 (s, 3H, OCH3), 3.79 (s, 6H, 2 × OCH3), 3.83 (s,
3H, OCH3), 4.18 (s, 2H, CH2), 6.76 (d, 2H, J ) 8.9 Hz, Ar-H),
6.86 (d, 2H, J ) 9.0 Hz, Ar-H), 6.99 (d, 1H, J ) 15.6 Hz, dCH),
7.31 (d, 2H, J ) 8.9 Hz), 7.61 (d, 1H, J ) 15.6 Hz, CHd). Anal.
(C19H22O6S) C, H.
(E)-2,4,6-Trimethoxystyryl-4-methoxybenzylsulfone (6s). The
title compound was obtained from 4-methoxybenzylsulfonylacetic
acid and 2,4,6-trimethoxybenzaldehyde following the procedure as
described in method A. Yield, 34%; white solid, mp 143–145 °C.
1H NMR: δ 3.80 (s, 3H, OCH3), 3.82 (s, 6H, 2 × OCH3), 3.85 (s,
3H, OCH3), 4.20 (s, 2H, CH2), 6.08 (s, 2H, Ar-H), 6.88 (d, 2H, J
) 9.2 Hz, Ar-H), 7.00 (d, 1H, J ) 15.6 Hz, dCH), 7.31 (d, 2H,
J ) 8.8 Hz), 7.81 (d, 1H, J ) 15.6 Hz, CHd). 13C NMR: δ 164.2,
Figure 4. Bone marrow toxicity profile of 6ab. To assess the toxicity
of 6ab, the phosphate salt of the drug was injected into mice (100
mg/kg) and bone marrow harvested from femur and tibia after 12, 24,
and 36 h following the injection of the drug. The bone marrow cells
were cultured in methylcellulose medium supplemented with a mixture
of stem cell factor, GM-CSF, IL-3, and erythropoietin for 1 week, and
colony forming units were determined.
1H NMR: δ 3.23 (s, 3H, OCH3), 3.55 (s, 3H, OCH3), 3.81 (s, 3H,
OCH3), 4.37 (s, 2H, CH2), 6.71 (d, 1H, J ) 15.4 Hz, dCH),

96 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Reddy et al.
161.8, 160.2, 135.9, 132.6, 123.0, 121.1, 114.4, 104.2, 90.9, 61.8,
56.1, 55.8, 55.6. Anal. (C19H22O6S) C, H.
7.05 (d, 1H, J ) 15.6 Hz, dCH), 7.85 (d, 1H, J ) 15.6 Hz, CHd).
Anal. (C19H22O7S) C, H.
(E)-3,4,5-Trimethoxystyryl-4-methoxybenzylsulfone (6t). The
title compound was obtained from 4-methoxybenzylsulfonylacetic
acid and 3,4,5-trimethoxybenzaldehyde following the procedure as
described in method A. Yield, 54%; white solid, mp 138–141 °C.
1H NMR: δ 3.76 (s, 3H, OCH3), 3.80 (s, 6H, 2 × OCH3), 3.83 (s,
3H, OCH3), 4.16 (s, 2H, CH2), 6.78 (d, 2H, J ) 9.1 Hz, Ar-H),
6.91 (d, 2H, J ) 8.9 Hz, Ar-H), 7.04 (d, 1H, J ) 15.6 Hz, dCH),
7.37 (d, 2H, J ) 8.8 Hz), 7.79 (d, 1H, J ) 15.6 Hz, CHd). Anal.
(C19H22O6S) C, H.
(E)-2,6-Dimethoxy-4-hydroxystyryl-4-methoxybenzylsul-
fone (6u). The title compound was obtained from 4-methoxyben-
zylsulfonylacetic acid and 2,6-dimethoxy-4-hydroxybenzaldehyde
following the procedure as described in method A. Yield, 58%;
white solid, mp 134–136 °C. 1H NMR: δ 3.47 (s, 6H, 2 × OCH3),
3.55 (s, 3H, OCH3), 3.98 (s, 2H, CH2), 5.77 (s, 2H, Ar-H), 6.63
(d, 2H, J ) 8.5 Hz, Ar-H), 6.73 (d, 1H, J ) 15.6 Hz, dCH), 7.05
(d, 2H, J ) 8.5 Hz), 7.55 (d, 1H, J ) 15.6 Hz, CHd). Anal.
(C18H20O6S) C, H.
(E)-2,6-Dimethoxy-4-hydroxystyryl-3-hydroxy-4-methoxyben-
zylsulfone (6z). The title compound was obtained from 3-hydroxy-
4-methoxybenzylsulfonylacetic acid and 2,6-dimethoxy-4-hydrox-
ybenzaldehyde following the procedure as described in method A.
Yield, 54%; white solid, mp 123–125 °C. 1H NMR: δ 3.77 (s, 6H,
2 × OCH3), 3.81 (s, 3H, OCH3), 4.28 (s, 2H, CH2), 6.10 (s, 2H,
Ar-H), 6.71–6.92 (m, 3H, Ar-H), 7.00 (d, 1H, J ) 15.5 Hz,
dCH), 7.59 (d, 1H, J ) 15.5 Hz, CHd). Anal. (C18H20O7S) C, H.
(E)-2,4,6-Trimethoxystyryl-3-hydroxy-4-methoxybenzylsul-
fone (6aa). The synthesis of the title compound was described in
the preparation of 20 (Scheme 3). Yield, 63%; white solid, mp
1
125–127 °C. H NMR: δ 3.83 (s, 6H, 2 × OCH3), 3.85 (s, 3H,
OCH3), 3.89 (s, 3H, OCH3), 4.16 (s, 2H, CH2), 5.60 (s, 1H, OH),
6.09 (s, 2H, Ar-H), 6.82–6.96 (m, 3H, Ar-H), 7.05 (d, 1H, J
)15.6 Hz, dCH), 7.85 (d, 1H, J ) 15.6 Hz, CHd). 13C NMR: δ
164.1, 161.9, 147.3, 145.9, 135.7, 123.4, 123.1, 122.2, 117.5, 111.1,
105.5, 104.4, 90.9, 62.0, 56.3, 56.1, 55.8. Anal. (C19H22O7S) C, H.
(E)-2,4,6-Trimethoxystyryl-31-O-phosphate Disodium 4-Meth-
oxybenzylsulfone (6ab). The synthesis of the title compounds was
described in the preparation of 29 (Scheme 5). Yield, 98%; white
solid, mp 152–154 °C. 1H NMR (D2O): δ 3.68 (s, 6H, 2 × OCH3),
3.71 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 4.35 (s, 2H, CH2), 5.92
(s, 2H, Ar-H), 6.91 (s, 2H, Ar-H), 6.97 (d, 1H, J ) 15.6 Hz,
Preparation of 4-Fluoro-2,6-dimethoxybenzaldehyde. Phos-
phorus oxychloride (1.8 mL, 19.3 mmol) was added slowly to a
well-stirred mixture of 1-fluoro-3,5-dimethoxybenzene (2.6 mL,
19.25 mmol) and N,N-dimethylformamide (2.5 mL, 20 mmol) while
the temperature was kept below -5 °C. Stirring was continued at
room temperature for 1.5 h and at 60 °C for another 2 h. Reaction
completion was monitored by TLC. The reaction mixture was
cooled and hydrolyzed with ice–water (60 mL). The resulting
suspension was neutralized by addition of 5 N NaOH and extracted
with ethyl acetate (2 × 30 mL). The aqueous phase was adjusted
to pH 10 by 5 N NaOH and re-extracted with ethyl acetate (2 ×
30 mL). The combined organic phases were washed with saturated
aqueous NaHCO3 solution (30 mL) and brine (30 mL) and dried
over anhydrous sodium sulfate. The dried solution was concentrated
to get the crude product, which on purification by column
chromatography afforded a colorless pure product. Yield, 75%;
dCH), 7.39 (s, 1H, Ar-H), 7.43 (d, 1H, J ) 15.6 Hz, CHd). 13
C
NMR (D2O): δ 164.4, 161.7, 151.1, 143.8, 136.8, 125.9, 123.4,
120.6, 120.2, 113.1, 103.4, 91.1, 61.0, 56.4, 56.2, 55.9. Anal.
(C19H21O10Na2PS) C, H.
(E)-2,4,6-Trimethoxystyryl-3,4,5-trimethoxybenzylsulfone (6ac).
The title compound was obtained from 3,4,5-trimethoxybenzylsul-
fonylacetic acid and 2,4,6-trimethoxybenzaldehyde following the
procedure as described in method A. Yield, 53%; white solid, mp
151–153 °C. 1H NMR: δ 3.81(s, 6H, 2 × OCH3), 3.83 (s, 6H, 2 ×
OCH3), 3.84 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 4.19 (s, 2H, CH2),
6.10 (s, 2H, ArH), 6.60 (s, 2H, ArH), 7.03 (d, 1H, J ) 15.6 Hz,
dCH), 7.83 (d, 1H, J ) 15.6 Hz, CHd). Anal. (C21H26O8S) C, H.
1
white solid, mp 79–81 °C. H NMR: δ 3.85 (s, 3H, OCH3), 3.89
(s, 3H, OCH3), 6.25 (S, 2H, Ar-H), 10.24 (s, 1H, CHO). Anal.
Calcd for C18H19FO5S: C, 58.70; H, 4.92. Found: C, 58.64; H, 4.91.
(E)-2,6-Dimethoxy-4-fluorostyryl-4-methoxybenzylsul-
fone (6v). The title compound was obtained from 4-methoxyben-
zylsulfonylacetic acid and 2,6-dimethoxy-4-fluorobenzaldehyde
following the procedure as described in method A. Yield, 55%;
white solid, mp 146–148 °C. 1H NMR: δ 3.47 (s, 6H, 2 × OCH3),
3.55 (s, 3H, OCH3), 3.98 (s, 2H, CH2), 5.77 (s, 2H, Ar-H), 6.63
(d, 2H, J ) 8.5 Hz, Ar-H), 6.73 (d, 1H, J ) 15.6 Hz, dCH), 7.05
(d, 2H, J ) 8.5 Hz), 7.55 (d, 1H, J ) 15.6 Hz, CHd). Anal.
(C18H19FO5S) C, H.
(E)-2,4,6-Trimethoxystyryl-2,3,4-trimethoxybenzylsulfone (6ad).
The title compound was obtained from 2,3,4-trimethoxybenzylsul-
fonylacetic acid and 2,4,6-trimethoxybenzaldehyde following the
procedure as described in method A. Yield, 52%; white solid, mp
1
94–96 °C. H NMR: δ 3.77, 3.83, 3.84, 3.86, 3.90 (s, 6 × 3H,
OCH3), 4.32 (s, 2H, CH2), 6.08 (s, 2H, aromatic), 6.67 (d, J ) 8.4
Hz, 1H, aromatic), 7.11 (d, J ) 15.6 Hz, 1H, dCH), 7.16 (d, J )
8.4 Hz, 1H, aromatic), 7.79 (d, J ) 15.6 Hz, 1H, CHd). Anal.
(C21H26O8S) C, H.
(E)-2,4,6-Trimethoxystyryl-4-chlorobenzylsulfone (6ae). The
title compound was obtained from 4-chlorobenzylsulfonylacetic acid
and 2,4,6-trimethoxybenzaldehyde following the procedure as
described in method A. Yield, 60%; white solid, mp 181–184 °C.
1H NMR: δ 3.83 (s, 2 × 3H, OCH3), 3.85 (s, 3H, OCH3), 4.22 (s,
2H, CH2), 6.09 (s, 2H, Ar-H), 6.99 (d,1H, J ) 15.5 Hz, dCH),
7.29 (s, 4H, Ar-H), 7.76 (d, 1H, J ) 15.5 Hz, CHd). Anal.
(C18H19ClO5S) C, H.
(E)-2,4,6-Trimethoxystyryl-4-nitrobenzylsulfone (6af). The
title compound was obtained from 4-nitrobenzylsulfonylacetic acid
and 2,4,6-trimethoxybenzaldehyde following the procedure as
described in method A. Yield, 60%; light-yellow solid, mp 179–184
°C. 1H NMR: δ 3.83 (s, 2 × 3H, OCH3), 3.86 (s, 3H, OCH3), 4.35
(s, 2H, CH2), 6.09 (s, 2H, Ar-H), 7.01 (d, 1H, J ) 15.5 Hz, dCH),
7.57 (d, 2H, J ) 9.0 Hz, Ar-H), 7.76 (d, J ) 15.5 Hz, 1H, CHd),
8.21 (d, 2H, J ) 9.0 Hz, Ar-H). Anal. (C18H19NO7S) C, H, N.
(E)-2,4,6-Trimethoxystyryl-4-cyanobenzylsulfone (6ag). The
title compound was obtained from 4-cyanobenzylsulfonylacetic acid
and 2,4,6-trimethoxybenzaldehyde following the procedure as
described in method A. Yield, 58%; white solid, mp 140–142 °C.
1H NMR: δ 3.81 (s, 3H, 2 × OCH3), 3.87 (s, 3H, OCH3), 4.21 (s,
2H, CH2), 6.00 (s, 2H, Ar-H), 6.78 (d, 2H, J ) 8.5 Hz, Ar-H),
7.07 (d, 1H, J ) 15.6 Hz, dCH), 7.29 (d, 2H, J ) 8.5 Hz, Ar-H),
7.86 (d, 1H, J ) 15.6 Hz, CHd). Anal. (C19H19NO5S) C, H, N.
(E)-2,4,6-Trimethylstyryl-4-methoxybenzylsulfone (6w). The
title compound was obtained from 4-methoxybenzylsulfonylacetic
acid and 2,4,6-trimethylbenzaldehyde following the procedure as
described in method A. Yield, 51%; white solid, mp 97–99 °C. 1H
NMR: δ 2.16 (s, 3H,CH3), 2.28 (s, 6H, 2 × CH3), 3.76 (s, 3H,
OCH3), 4.13 (s, 2H, CH2), 6.08 (s, 2H, Ar-H), 6.42 (d, 1H, J )
15.4 Hz, dCH), 6.82 (m, 4H, Ar-H), 7.56 (d, 1H, J ) 15.4 Hz,
CHd). Anal. (C19H22O3S) C, H.
(E)-2,4,6-Trimethoxystyryl-4-trifluoromethoxybenzylsul-
fone (6x). The title compound was obtained from 4-trifluo-
romethoxybenzylsulfonylacetic acid and 2,4,6-trimethoxybenzal-
dehyde following the procedure as described in method A. Yield,
1
52%; white solid, mp 133–135 °C. H NMR: δ 3.82 (s, 6H, 2 ×
OCH3), 3.87 (s, 3H, OCH3), 4.26 (s, 2H, CH2), 6.10 (s, 2H, Ar-H),
6.99 (d, 1H, J ) 15.6 Hz, dCH), 7.20–7.48 (m, 4H, Ar-H), 7.78
(d, 1H, J ) 15.6 Hz, CHd). Anal. (C19H19F3O6S) C, H.
(E)-3,4,5-Trimethoxystyryl-3-hydroxy-4-methoxybenzylsul-
fone (6y). The title compound was obtained from 3-hydroxy-4-
methoxybenzylsulfonylacetic acid and 3,4,5-trimethoxybenzalde-
hyde following the procedure as described in method A. Yield,
1
60%; white solid, mp 118–120 °C. H NMR: δ 3.83 (s, 6H, 2 ×
OCH3), 3.85 (s, 3H. OCH3), 3.89 (s, 3H. OCH3), 4.16 (s, 2H, CH2),
5.60 (s, 1H, OH), 6.09 (s, 2H, Ar-H), 6.82–6.96 (m, 3H, Ar-H),

(E)-Styrylbenzylsulfones as Anticancer Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 97
(E)-2,4,6-Trimethoxystyryl-4-carboxybenzylsulfone (6ah). The
title compound was obtained from 4-carboxybenzylsulfonylacetic
acid and 2,4,6-trimethoxybenzaldehyde following the procedure as
described in method A. Yield, 60%; white solid, mp 143–145 °C.
1H NMR: δ 3.83 (s, 6H, 2 × OCH3), 3.88 (s, 3H, OCH3), 4.11 (s,
2H, CH2), 6.01 (s, 2H, Ar-H), 6.68 (d, 2H, J ) 8.5 Hz, Ar-H),
6.97 (d, 1H, J ) 15.6 Hz, dCH), 7.13 (d, 2H, J ) 8.5 Hz, Ar-H),
7.76 (d, 1H, J ) 15.6 Hz, CHd). Anal. (C19H20O7S) C, H.
(E)-2,4,6-Trimethoxystyryl-4-hydroxybenzylsulfone (6ai). The
title compound was obtained from 4-tert-butoxybenzylsulfonylacetic
acid and 2,4,6-trimethoxybenzaldehyde following the method A
procedure. During the condensation the protective tert-butoxy group
cleaved to the hydroxy group. Yield, 52%; white solid, mp 141–143
°C. 1H NMR: δ 3.73 (s, 6H, 2 × OCH3), 3.77 (s, 3H, OCH3), 4.11
(s, 2H, CH2), 4.36 (s, 1H, OH), 6.01 (s, 2H, Ar-H), 6.68 (d, 2H,
J ) 8.5 Hz, Ar-H), 6.97 (d, 1H, J ) 15.6 Hz, dCH), 7.13 (d, 2H,
J ) 8.5 Hz, Ar-H), 7.76 (d, 1H, J ) 15.6 Hz, CHd). Anal.
(C15H20O6S) C, H.
1-(4-Chlorophenyl)-2-(4-methoxybenzylsulfonyl)ethanol (11b).
The title compound was obtained by the reduction of 10b with
sodium borohydride. Yield, 78%; white solid, mp 130–132 °C. 1H
NMR: δ 3.63 (dd, 2H, J )10.1 and 4.4 Hz), 3.93 (s, 3H, OCH3),
4.75 (dd, 2H, J ) 13.5 and 10.6 Hz), 5.26 (d, 1H, J ) 8.3 Hz,
CHOH), 6.17 (br s, 1H, OH), 7.10 (d, 2H, J ) 8.3 Hz, Ar-H),
7.51(d, 2H, J ) 8.3 Hz, Ar-H), 7.64 (dd, 4H, J ) 9.4 and 8.2 Hz,
Ar-H). Anal. Calcd for C16H17ClO4S: C, 56.39; H, 5.03. Found:
C, 56.34; H, 4.99.
Preparation of (E)-Styrylbenzylsulfones (6). p-Toluenesulfonic
acid (1 mmol) was added in one portion to a mixture of
ꢀ-hydroxybenzylsulfone 11 (5 mmol) in anhydrous benzene (25
mL) at room temperature and under N2 atmosphere. The temperature
was raised to 80 °C, and the mixture was refluxed for 3 h using a
Dean–Stark water separator. After completion of the reaction
monitored by TLC, the reaction mixture was concentrated under
reduced pressure and then quenched by the addition of water (25
mL). The aqueous layer was neutralized with a saturated aqueous
solution of sodium hydrogen carbonate and extracted with dichlo-
romethane (3 × 25 mL). The combined organic extracts were
washed with brine (2 × 25 mL), dried over Na2SO4, and filtered,
and the solvent was evaporated under reduced pressure to afford
crude product, which on recrystallization in 2-propanol afforded
the desired product 6 in excellent yield.
Method B. See Scheme 2.
Preparation of Phenacylbenzylsulfones (10). General Pro-
cedure. To a cooled solution of sodium hydroxide (100 mmol) in
absolute methanol (50 mL), taken in a 250 mL round-bottomed
flask, benzylthiol 7 (100 mmol) was added slowly through a
dropping funnel, and the reaction mixture was stirred for 5 min.
An appropriate phenacyl bromide 8 (100 mmol) was added in
portions to the contents of the flask and stirred for 3–4 h. After
completion of the reaction, the contents of the flask were poured
onto crushed ice and the compound formed was washed with ice-
cold water and dried to get phenacylbenzyl sulfide 9.
The above crude phenacylbenzyl sulfide 9 (50 mmol) in glacial
acetic acid (100 mL) was taken in a 250 mL round-botomed flask,
and 30% hydrogen peroxide (60 mL) was added in portions at
frequent intervals. Then the reaction mixture was kept at room
temperature for 24 h. The solid, if any formed, was separated by
filtration, and the filtrate was poured onto crushed ice. The
compound separated was filtered, washed with water, dried, and
added to the first crop, if any. The total product on recrystallization
from methanol afforded pure phenacylbenzylsulfones (10).
4-Methoxyphenacyl-4-methoxybenzyl sulfone (10a). The title
compound was obtained from 4-methoxybenzylthiol and 4-meth-
oxyphenacyl bromide followed by oxidation of the resultant
compound. Yield, 80%; white solid, mp 128–130 °C. 1H NMR: δ
3.78(s, 3H, OCH3), 3.88 (s, 3H, OCH3), 4.60 (s, 2H, CH2), 4.89 (s,
2H, CH2), 6.98 (d, 2H, J ) 8.7 Hz, Ar-H), 7.09 (d, 2H, J ) 8.9
Hz, Ar-H), 7.36 (d, 2H, J ) 8.7 Hz, Ar-H), 8.04 (d, 2H, J ) 8.9
Hz, Ar-H). Anal. Calcd for C17H18O5S: C, 61.06; H, 5.42. Found:
C, 61.09; H, 5.40.
(E)-4-Methoxystyryl-4-methoxybenzylsulfone (6a). The title
compound was obtained by dehydration of 11a as described in the
above procedure. Yield, 65%; white solid, mp 151–153 °C.
Analytical data are same as for 6a obtained by method A.
(E)-4-Chlorostyryl-4-methoxybenzylsulfone (6c). The title
compound was obtained by dehydration of 11b as described in the
above procedure. Yield, 69%; white solid, mp 175–177 °C.
Analytical data is same as for 6c obtained by method A.
Synthesis of (E)-2,4,6-Trimethoxystyryl-3-hydroxy-4-meth-
oxybenzylsulfone, 6aa. See Scheme 3.
Preparation of 3-[(tert-Butyldimethylsilyl)oxy]-4-methoxy-
benzaldehyde (13). To a cooled solution of 3-hydroxy-4-meth-
oxybenzaldehyde 12 (10.0 g, 65.7 mmol) in dry N,N-dimethylfor-
mamide (75 mL) was added diisopropylethylamine (16.99 g, 131.4
mmol). Before the addition of 1.0 M solution of tert-butyldimeth-
ylsilyl chloride in tetrahydrofuran (11.89 g or 78.9 mL, 78.85
mmol), the mixture was stirred under nitrogen for 10 min. After
complete addition over 30 min, the reaction mixture was left
overnight (12–16 h). Reaction completion was checked by TLC
(chloroform on silica gel plate). Water was added to the reaction
mixture, extracted with dichloromethane, and the organic layer was
washed with a saturated sodium bicarbonate solution and water and
dried. Removal of solvent in vacuo yielded as an oil, which was
subjected to filter column chromatography (eluant, chloroform) to
afford a yellow viscous oil 13 (100%). 1H NMR: δ 0.21 (s, 6H, 2
× CH3), 1.01 (s, 9H, 3 × CH3), 3.91 (s, 3H, OCH3), 6.94 (d, 1H,
J ) 8.5 Hz, 5H), 7.36 (d, 1H, J ) 2 Hz, 2H), 7.47 (dd, 1H, J )
8.5, 2 Hz, 6H), 9.89 (s, 1H, CHO). Anal. Calcd for C14H22O3Si: C,
63.12; H, 8.32. Found: C, 63.09; H, 8.30.
Preparation of 3-[(tert-Butyldimethylsilyl)oxy]-4-methoxy-
benzyl Alcohol (14). To a cooled solution of 3-[(tert-butyldim-
ethylsilyl)oxy]-4-methoxybenzaldehyde 13 (17.5 g, 65.7 mmol) in
methanol (100 mL) under nitrogen, sodium borohydride (2.98 g,
78.8 mmol) was added. The mixture was stirred at room temperature
for 30 min. After the reaction was complete as indicated by TLC
(chloroform on silica gel plate), ice was added to the reaction
mixture and extracted with ethyl acetate. The organic layer was
separated, washed with water, and dried. Removal of solvent in
vacuo yielded a yellow oil 14 (73.5%). 1H NMR: δ 0.18 (s, 6H, 2
× CH3), 1.00 (s, 9H, 3 × CH3), 1.90 (br s, 1H, OH), 3.82 (s, 3H,
OCH3), 4.56 (s, 2H, CH2OH), 6.94 (br s, 3H, Ar-H). Anal. Calcd
for C14H24O3Si: C, 62.64; H, 9.01. Found: C, 62.59; H, 9.03.
Preparation of 3-[(tert-Butyldimethylsilyl)oxy]-4-methoxy-
benzyl Chloride (15). To a cooled solution of 3-[(tert-butyldim-
ethylsilyl)oxy]-4-methoxybenzyl alcohol 14 (9.5 g, 35.4 mmol) in
benzene (50 mL) under nitrogen, thionyl chloride (6.32 g or 3.87
mL, 53.1 mmol) dissolved in benzene (5 mL) was added over 10
4-Chlorophenacyl-4-methoxybenzylsulfone (10b). The title
compound was obtained from 4-methoxybenzylthiol and 4-chlo-
rophenacyl bromide followed by oxidation of the resultant com-
pound. Yield, 82%; white solid, mp 141–143 °C. 1H NMR: δ 3.89
(s, 3H, OCH3), 4.50 (s, 2H, CH2), 4.79 (s, 2H, CH2), 7.06 (d, 2H,
J ) 8.3 Hz, Ar-H), 7.47 (d, 2H, J ) 8.4 Hz, Ar-H), 7.60 (dd,
4H, J ) 8.2, 9.4 Hz, Ar-H). Anal. Calcd for C16H15ClO4S: C,
56.72; H, 4.46. Found: C, 56.69; H, 4.42.
Preparation of ꢀ-Hydroxybenzylsulfones (11). General Pro-
cedure. To an ethanolic solution (20 mL) of phenacylbenzylsulfone
10 (10 mmol) maintained at 0 °C was added NaBH4 (10 mmol)
slowly under N2 atmosphere. The reaction mixture was maintained
at 0 °C for 0.5 h. After completion of the reaction, monitored by
TLC, the contents were poured onto crushed ice. The solid was
filtered, washed with water, and dried under vacuum to yield 11.
2-(4-Methoxybenzylsulfonyl)-1-(4-methoxyphenyl)ethanol
(11a). The title compound was obtained by the reduction of 10a
with sodium borohydride. Yield, 70%; white solid, mp 112–114
1
°C. H NMR: δ 3.43 (dd, 2H, J ) 9.9 and 4.6 Hz), 3.76 (s, 3H,
OCH3), 3.78 (s, 3H, OCH3), 4.47 (dd, 2H, J )15.7 and 13.6 Hz),
5.09 (m, 1H, CHOH), 6.00 (d, 1H, J ) 4.2 Hz, OH), 6.93 (d, 2H,
J ) 8.6 Hz, Ar-H), 6.99 (d, 2H, J ) 8.6 Hz, Ar-H), 7.35 (t, 4H,
J ) 8.8 Hz, Ar-H). Anal. Calcd for C17H20O5S: C, 60.69; H, 5.99.
Found: C, 60.73; H, 5.97.

98 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Reddy et al.
min, and the temperature was maintained at 0 °C for 2 h. The
completion of reaction was checked by TLC (chloroform on silica
gel plate). Ice was added to the reaction mixture, and the solution
was extracted with ethyl acetate. The organic layer was washed
with saturated bicarbonate solution and water and dried over
anhydrous sodium sulfate. Removal of the solvent in vacuo afforded
hyde (21). A mixture of 3-hydroxy-4-methoxybenzaldehyde 12 (5.0
g, 32 mmol) and p-toluenesulfonyl chloride (10.0 g, 52.5 mmol)
was dissolved in pyridine (12.5 mL, 154 mmol). The reaction
mixture was stirred for 5 min and maintained at 70–80 °C. The
clear reaction mixture became turbid and became a slurry. The
stirring was continued for 2 h at 70–80 °C. The reaction completion
was determined by TLC, and the contents of the flask were cooled
to room temperature and poured onto cold water. The white
crystalline solid formed was filtered, washed successively with 5
mL of 1:1 HCl/H2O, 5 mL of 5% NaOH solution, and water till
the filtrate was free from pyridine, and dried to constant weight to
get the desired product 21. Yield, 98%; white solid, mp 148–151
°C. 1H NMR: δ 2.40 (s, 3H, CH3), 3.71 (S, 3H, OCH3), 6.91–7.83
(m, 7H, Ar-H), 9.83 (s, 1H, CHO). Anal. Calcd for C15H14O5S:
C, 58.81; H, 4.61. Found: C, 58.79; H, 4.59.
Preparation of 3-[(p-Toluenesulfonyl)oxy]-4-methoxybenzyl
Alcohol (22). To a cooled solution of 3-[(p-toluenesulfonyl)oxy]-
4-methoxybenzaldehyde 21 (9.0 g, 29 mmol) in methanol (25 mL)
was added sodium borohydride (0.55 g, 14.5 mmol) in methanol
(2.5 mL) over a period of 5–10 min, maintaining the temperature
around 15–20 °C. The reaction mixture was maintained at that
temperature for a further 30 min, and TLC was used to check for
completion of the reaction. Water was added to the reaction mixture,
and the solid formed was filtered, washed with water, and dried to
afford 22. Yield, 97%; white solid, mp 88–90 °C. 1H NMR: δ 2.41
(s, 3H, CH3), 3.59 (s, 3H, OCH3), 4.57 (s, 2H, CH2), 6.80–7.79
(m, 7H, Ar-H). Anal. Calcd for C15H16O5S: C, 58.43; H, 5.23.
Found: C, 58.49; H, 5.19.
1
15. Yield, 98.5%; yellow oil. H NMR: δ 0.16 (s, 6H, 2 × CH3),
1.00 (s, 9H, 3 × CH3), 3.80 (s, 3H, OCH3), 4.44 (s, 2H, CH2),
6.70–7.01 (m, 3H, Ar-H). Anal. Calcd for C14H23ClO2Si: C, 58.62;
H, 8.08. Found: C, 58.69; H, 8.03.
Preparation of 3-[(tert-Butyldimethylsilyl)oxy]-4-methoxy-
benzylthioacetic Acid (16). To a solution of sodium hydroxide
(2.79 g, 69.7mmol) in methanol (30 mL) was added mercaptoacetic
acid (3.21 g or 2.42 mL, 34.9 mmol) slowly and stirred under
nitrogen for 10 min. 3-[(tert-Butyldimethylsilyl)oxy]-4-methoxy-
benzyl chloride 15 (10.0 g, 34.9 mmol) was added slowly to the
reaction mixture and stirred at room temperature for 3 h. Reaction
completion was checked by TLC (chloroform on silica gel plate).
The reaction mixture was then poured onto ice and neutralized with
concentrated HCl. The resulting material was extracted with ethyl
acetate. The ethyl acetate solution was washed with water and dried
over anhydrous sodium sulfate. Removal of the solvent in vacuo
1
afforded 16. Yield, 75%; white solid, mp 57–59 °C. H NMR: δ
0.18 (s, 6H, 2 × CH3), 1.02 (s, 9H, 3 × CH3), 3.34 (s, 2H, CH2),
3.84 (s, 3H, OCH3), 4.04 (s, 2H, CH2), 6.80–7.01 (m, 3H, Ar-H).
Anal. Calcd for C16H26O4SiS: C, 56.10; H, 7.65. Found: C, 56.08;
H, 7.61.
Preparation of 3-Hydroxy-4-methoxybenzylthioacetic Acid
(17). To a cooled solution of 3-[(tert-butyldimethylsilyl)oxy]-4-
methoxybenzylthioacetic acid 16 (8.75 g, 25.5 mmol) in tetrahy-
drofuran (40 mL) was added 1.0 M solution of tetra-n-butylam-
monium fluoride in tetrahydrofuran (6.68 g or 25.54 mL, 25.5
mmol) slowly and stirred under nitrogen for 2 h at room temper-
ature. The progress of the reaction was monitored by TLC (9:1
chloroform/methanol on silica gel plate). Water was added to the
reaction mixture and extracted with ethyl acetate. The organic layer
was washed with water and dried. Removal of the solvent in vacuo
yielded a semisolid that was subjected to column chromatography
(initial with chloroform and finally with ethyl acetate) to afford
the pure product 17. Yield, 50%; white solid, mp 128–130 °C. 1H
NMR: δ 3.34 (s, 2H, CH2), 3.84 (s, 3H, OCH3), 4.04 (s, 2H, CH2),
6.80–7.01 (m, 3H, Ar-H). Anal. Calcd for C10H12O4S: C, 52.62,
H, 5.30. Found: C, 52.58, H, 5.35.
Preparation of 3-[(p-Toluenesulfonyl)oxy]-4-methoxybenzyl
Chloride (23). To a cooled solution of 3-[(p-toluenesulfonyl)oxy]-
4-methoxybenzyl alcohol 22 (8.0 g, 26 mmol) in benzene (25 mL)
was added thionyl chloride (1.9 mL, 26 mmol) slowly over 5–10
min, maintaining the temperature around 15–20 °C. The reaction
was maintained at those conditions for 2 h, and TLC was used to
check for the completion. The flask was connected to a high vacuum
through a trap containing formic acid under mild heating to remove
excess thionyl chloride. The slurry was formed after complete
removal of thionyl chloride and benzene, filtered, washed with
hexane, and dried to afford 23. Yield, 90%; white solid, mp 102–105
1
°C. H NMR: δ 2.43 (s, 3H, CH3), 3.61 (s, 3H, OCH3), 4.50 (s,
2H, CH2), 6.80–7.79 (m, 7H, Ar-H). Anal. Calcd for C15H15ClO4S:
C, 55.13; H, 4.63. Found: C, 53.20; H, 4.59.
Preparation of 3-[(p-Toluenesulfonyl)oxy]-4-methoxyben-
zylthioacetic Acid (24). To a solution of sodium hydroxide (1.72
g, 42.9 mmol) in methanol (30 mL) was added mercaptoacetic acid
(1.5 mL, 21.5 mmol) in portions and stirred under nitrogen
atmosphere for 10 min. 3-[(p-toluenesulfonyl)oxy]-4-methoxybenzyl
chloride 23 (7.0 g, 21.5 mmol) was then added slowly to the reaction
mixture and stirred at reflux temperature for 5 h. The reaction
mixture was then poured onto ice containing concentrated HCl.
The white crystalline solid was filtered, washed with water, and
dried to get the desired product 24. Yield, 93%; white solid, mp
Preparation of 3-Hydroxy-4-methoxybenzylsulfonylacetic Acid
(18). To a solution of 3-hydroxy-4-methoxybenzylthioacetic acid
17 (2.9 g, 12.7 mmol) in glacial acetic acid (15 mL) was added 6
mL of 30% hydrogen peroxide and stirred overnight (18 h). The
completion of the reaction was determined by TLC. The mixture
was then poured onto ice–water and extracted with ethyl acetate.
The organic layer was washed with water and dried. Removal of
the solvent in vacuo afforded pure product 18. Yield, 60%; white
1
solid, mp 164–165 °C. H NMR: δ 3.84 (s, 3H, OCH3), 4.04 (s,
1
2H, CH2), 4.29 (s, 2H, CH2), 6.85–7.11 (m, 3H, Ar-H). Anal. Calcd
for C10H12O6S: C, 46.15; H, 4.65. Found: C, 46.21; H, 4.69.
Preparation of (E)-2,4, 6-Trimethoxystyryl-3-hydroxy-4-
methoxybenzylsulfones (20, 6aa). A mixture of 3-hydroxy-4-
methoxybenzylsulfonylacetic acid 18 (1.9 g, 7.3 mmol), 2,4,6-
trimethoxybenzaldehyde 19 (1.58 g, 8.0 mmol), benzoic acid (0.134
g, 1.1 mmol), and piperidine (0.081 g, 0.95mmol) in toluene (50
mL) was refluxed for 2–3 h with continuous removal of water using
a Dean–Stark water separator. Reaction completion was determined
by TLC (9:1 chloroform/methanol on silica gel plate). The reaction
mixture was then cooled to room temperature, and water was added
and extracted with ethyl acetate. The organic layer was washed
with saturated sodium bicarbonate solution, dilute hydrochloric acid,
and water and dried. Removal of the solvent in vacuo yielded a
crude product, which on recrystalization from 2-propanol resulted
in pure product 20/6aa.
116- 120 °C. H NMR: δ 2.41 (s, 3H, CH3), 3.03 (s, 2H, SCH2),
3.61 (s, 3H, OCH3), 3.79 (s, 2H, CH2S), 6.80–7.81 (m, 7H, Ar-H).
Anal. Calcd for C17H18O6S2: C, 53.39; H, 4.74. Found: C, 53.33;
H, 4.71.
Preparation of 3-[(p-Toluenesulfonyl)oxy]-4-methoxybenzyl-
sulfonylacetic Acid (25). To a solution of 3-[(p-toluenesulfonyl)-
oxy]-4-methoxybenzylthioacetic acid 24 (7.0 g, 18.4 mmol) in
glacial acetic acid (35 mL) was added 21 mL of 30% hydrogen
peroxide and stirred overnight (18 h). The reaction mixture was
poured onto ice–water, and the solid that separated was filtered,
washed with cold water, and dried to get pure 25. Yield, 80%; white
solid, mp 142–146 °C. 1H NMR: δ 2.50 (s, 3H, CH3), 3.61 (s, 3H,
OCH3), 4.03 (s, 2H, SCH2), 4.60 (s, 2H, CH2S), 7.09–7.74 (m, 7H,
Ar-H), 13.4 (br s, 1H, OH). Anal. Calcd for C17H18O8S2: C, 49.27;
H, 4.38. Found: C, 49.22; H, 4.34.
Preparation of (E)-2,4,6-Trimethoxystyryl-3-[(p-toluenesulfo-
nyl)oxy]-4-methoxybenzylsulfone (26). A mixture of 3-[(p-tolu-
enesulfonyl)oxy]-4-methoxybenzylsulfonylacetic acid 25 (6.0 g,
14.5 mmol), 2,4,6-trimethoxybenzaldehyde 19 (2.85 g, 14.5 mmol),
Alternative Method for the Synthesis of (E)-2,4,6-Trimethox-
ystyryl-3-hydroxy-4-methoxybenzylsulfone 6aa (Scheme 4).
Preparation of 3-[(p-Toluenesulfonyl)oxy]-4-methoxybenzalde-

(E)-Styrylbenzylsulfones as Anticancer Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 99
benzoic acid (0.27 g, 2.2 mmol), and piperidine (0.19 mL, 1.9
mmol) in benzene (50 mL) was refluxed for 4–5 h with continuous
removal of water using a Dean–Stark water separator. The reaction
completion was checked by TLC (9:1 chloroform/methanol on silica
gel plate). The reaction mixture was then cooled to room temper-
ature, and the crystalline solid formed was filtered, washed with
cold benzene, and dried to get the desired product 26. Yield, 65%;
white solid, mp 159–166 °C. 1H NMR: δ 2.42 (s, 3H, CH3), 3.61(s,
3H. OCH3), 3.81 (s, 3H, 3 × OCH3), 4.21 (s, 2H, CH2), 6.06 (s,
2H, Ar-H), 6.80–7.74 (m, 7H, Ar-H), 7.01 (d, 1H, J )15.5 Hz,
dCH), 7.83 (d, 1H, J ) 15.5 Hz, CHd). Anal. Calcd for
C26H28O9S2: C, 56.90; H, 5.14. Found: C, 56.83; H, 5.11.
Preparation of (E)-2,4,6-Trimethoxystyryl-3-hydroxy-4-meth-
oxybenzylsulfone (6aa). A mixture of (E)-2,4,6-trimethoxystyryl-
3-[(p-toluenesulfonyl)oxy]-4-methoxybenzylsulfones 26 (5.0 g, 9.1
mmol), 50 mL (20%) of sodium hydroxide solution, and methanol
(50 mL) was taken in a round bottomed flask and refluxed until
the reaction mixture was clear without any turbidity (3–4 h). The
progress of the reaction was monitored by TLC. The reaction
mixture was cooled to room temperature and neutralized with cold
dilute HCl solution. The precipitate that separated after neutraliza-
tion was filtered, washed with water, and dried to get the crude
product, which on recrystallization from 2-propanol resulted in an
analytically pure sample of 6aa.Yield, 95%; white solid, mp
124–127 °C.
glycol dimethyl ether (125 mL) was added 2 N sodium hydroxide
(0.27 g dissolved in 13.66 mL of H2O, 6.8 mmol) and stirred for
3 h. The solid formed was filtered, washed with acetone (2 × 25
mL), and dried under vacuum to get the product 6ab. Yield, 98%;
1
white solid, mp 152–154 °C. H NMR (D2O): δ 3.68 (s, 6H, 2 ×
OCH3), 3.71 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 4.35 (s, 2H, CH2),
5.92 (s, 2H, Ar-H), 6.91 (s, 2H, Ar-H), 6.97 (d, 1H, J ) 15.6
Hz, dCH), 7.39 (s, 1H, Ar-H), 7.43 (d, 1H, J ) 15.6 Hz, CHd).
13C NMR (D2O): δ 164.4, 161.7, 151.1, 143.8, 136.8, 125.9, 123.4,
120.6, 120.2, 113.1, 103.4, 91.1, 61.0, 56.4, 56.2, 55.9. Anal.
(C19H21O10Na2PS) C, H.
Biology. Tissue Culture and Reagents. Paclitaxel was pur-
chased from Sigma. Cell lines were purchased from ATCC. Cell
lines were routinely grown in DMEM or RPM1 (CellGro)
supplemented with 10% fetal bovine serum (Atlas) and 1 unit/mL
penicillin-streptomycin (Gibco).
Cytotoxicity Assay. We have tested a number of tumor cell lines
using a dose response end point assay system. The cells were grown
in either DMEM or RPMI supplemented with 10% fetal bovine
serum and 1 unit/mL penicillin-streptomycin solution. The tumor
cells were plated into six-well dishes at a cell density of 1.0 × 105
cells/mL/well, and compounds were added 24 h later at various
concentrations. Cell counts were determined from duplicate wells
after 96 h of treatment. The total number of viable cells was
determined by trypan blue exclusion.
Synthesis of (E)-2,4,6-Trimethoxystyryl-3-O-phosphate Di-
sodium 4-Methoxybenzylsulfone (6ab) (Scheme 5). Prepara-
tion of (E)-2,4,6-Trimethoxystyryl-3-O-bis(benzyl)phosphoryl-
4-methoxybenzyl Sulfone (27). To a stirred solution of (E)-2,4,6-
trimethoxystyryl-3-hydroxy-4-methoxybenzylsulfone 6aa (3.8 g, 9.6
mmol) in acetonitrile (48 mL) under nitrogen atmosphere was added
carbon tetrabromide (3.88 g, 11.72 mmol) and triethylamine (1.46
g, 14.4 mmol), and stirring was continued for 10 min. Dibenzyl
phosphite (3.20 g, 11.6 mmol) dissolved in acetonitrile (32 mL)
was added to the reaction mixture slowly. After the addition, the
reaction mixture was stirred for 2 h and TLC was used to check
for completion of the reaction. The phosphorylation was terminated
by dropwise addition of potassium dihydrogen phosphate (20 mL,
0.5 M) to the reaction mixture over 10 min. The solution was then
extracted with ethyl acetate (3 × 60 mL). The organic extracts were
combined and washed with water, dried, and concentrated in vacuo.
The thick liquid obtained after concentration was purified on silica
column using chloroform/methanol with increasing polarity. The
purified product was concentrated in vacuo to afford pure dibenzyl
ester 27. Yield, 73%; semisolid. 1H NMR: δ 3.68 (s, 6H, 2 ×
OCH3), 3.71(s, 3H, OCH3), 3.74 (s, 3H, OCH3), 4.07 (s, 2H, CH2),
4.96–5.04 (m, 4H, OCH2), 5.98 (s, 2H, Ar-H), 6.60–7.42 (m, 14H,
Ar-H + vinylic), 7.71 (d, 1H, J ) 15.6 Hz, CHd). Anal. Calcd
for C33H35O10PS: C, 60.54; H, 5.39. Found: C, 60.48; H, 5.44.
Preparation of (E)-2,4,6-Trimethoxystyryl-3-O-phosphoryl-
4-methoxybenzylsulfone (28). To a stirred solution of the above
dibenzyl ester 27 (4.36 g, 6.7 mmol) in anhydrous dichloromethane
(40 mL) under nitrogen at 0 °C was added bromotrimethylsilane
(2.14 g, 14.1 mmol). The stirring was continued for 45 min at
the same temperature, and TLC was used to check for completion
of the reaction. Sodium thiosulfate (1%, 50 mL) was added to the
reaction mixture, and stirring was continued for an additional 5
min. The separated aqueous phase was extracted with ethyl acetate
(3 × 25 mL). The organic extracts were concentrated in vacuo to
afford the crude phosphoric acid 28, which was purified on a silica
column using chloroform/methanol with increasing polarity. The
purified product was concentrated in vacuo to afford pure acid 28.
Yield, 44.3%; white solid, mp 202–205 °C. 1H NMR (DMSO-d6):
δ 3.78(s, 6H, 2 × OCH3), 3.85(s, 3H, OCH3), 3.86 (s, 3H, OCH3),
4.34 (s, 2H, CH2), 6.30 (s, 2H, Ar-H), 7.02 (m, 2H, Ar-H), 7.12
(d, 1H, J ) 15.6 Hz, dCH), 7.32 (s, 2H, OH), 7.52 (s, 1H,Ar-H),
7.61 (d, 1H, J ) 15.6 Hz, CHd). Anal. Calcd for C19H23O10PS: C,
48.10; H, 4.89. Found: C, 48.14; H, 4.92.
Soft Agar Assay. The soft agar plates were prepared as described
by Cosenza et al.24 Briefly, Noble bottom agar (0.8%) was plated
onto 60 mm tissue culture plates. Exponentially growing MIA-
PaCa-2 cells (1.0 × 105) were mixed with growth medium with
various concentrations of each compound and mixed with Noble
agar to a final concentration of 0.4%. Each concentration was plated
in triplicate. The top agar was allowed to solidify, and the plates
were then incubated at 5% CO2 at 37 °C for 3 weeks. The plates
were then stained with 0.05% nitroblue tetrazolium (NBT) solution,
and representative plates were photographed using an Olympus
stereoscope mounted with a Sony digital camera system (DKC5000,
Sony Inc.).
Flow Cytometry. Human prostate tumor cells, DU145 cells, and
normal diploid human lung fibroblasts, HFL-1 cells, were grown
in DMEM (Cellgro) supplemented with 10% fetal bovine serum
and 1 unit/mL penicillin-streptomycin. The cells were plated onto
100 mm2 dishes at a cell density of 1.0 × 106 cells/dish, and 24 h
later, they were treated with 2.5 µM of the compound. The cells
were harvested 24, 48, and 72 h after treatment. The cells were
removed from the plate by trypsin digestion and combined with
the nonattached cells found in the medium. The cell pellets were
washed in phosphate buffered saline (PBS) and fixed in ice cold
70% ethanol for at least 24 h. The fixed cells were then washed
with room temperature PBS and stained with propidium iodide (50
µg/mL) and RNase A (0.5 mg) for 30 min at 37 °C. The stained
cells were then analyzed on a Becton-Dickinson (BD) (FACScan)
flow cytometer and the data analyzed by cell cycle analysis software
(Modfit, BD).
PARP Western. BT20 cells were plated at a density of 3.0 ×
106 cells per 150 mm2 plate and treated 24 h later with either DMSO
or 6aa. The cells were collected at the indicated time points, and
cell pellets were frozen. The frozen cell pellets were lysed in 1%
NP40/PBS lysis buffer containing protease inhibitors. Equal
amounts of total cellular protein were then resolved on a 10%
SDS-polyacrylamide gel. The gels were transferred onto nitrocel-
lulose paper (S/S), hybridized with anti-PARP antibodies (BD) and
developed using ECL (Perkin-Elmer, MA) solution.
Nude Mouse Assay. Female athymic (NCR-nu/nu, Taconic)
nude mice were injected with (0.5–1.0) × 107 BT20 cells
subcutaneously in the hind leg using a 1 mL tuberculin syringe
equipped with a 271/2 gauge needle. Approximately 14 days later,
mice were paired (N ) 8) and injected with 6ab or phosphate
buffered saline as the vehicle control. The intravenous injections
were performed in the mouse tail vein using a 1 mL tuberculin
syringe equipped with a 30 gauge needle. The animals were injected
following a Q2D ×3 schedule. Tumor measurements (two dimen-
Preparation of (E)-2,4,6-Trimethoxystyryl-31-O-phosphate
Disodium 4-Methoxybenzylsulfone (6ab). To a stirred solution
of the above phosphoric acid 28 (1.35 g, 2.85 mmol) in ethylene

100 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Reddy et al.
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sions) were done three times per week using traceable digital vernier
calipers (Fisher). Tumor volume was calculated using the following
equation: V ) (L(S2)π/6, where L is the longer and S is the shorter
of the two dimensions. Body weight was determined during each
measurement. The animals were observed for signs of toxicity. The
time of tumor volume doubling was calculated and the T - C value
(difference in the average times post-treatment for tumors of
the treated groups to attain a doubling in volume compared to the
average of the control group) was determined. We did not observe
body weight loss of more than 10% in any group nor were there
any animal deaths. All studies were performed under the guidelines
of Temple University IACUC.
Bone Marrow Harvest and Colony Formation Assay. Bone
marrow was harvested from femur and tibia of CD-1 mice injected
with 200 µL of PBS or 6ab [10 mg/mL:100 mg/kg dose] at 12,
24, or 48 h before the sacrifice. The bone marrow cells were
cultured in methylcellulose medium supplemented with 50 ng/mL
rmStem cell factor, 10 ng/mL rmIL-3, 10 ng/mL rh IL-6, 200 µg/
mL human transfeffin, and 3 units/mL rhErythropoietin (Stem Cell
Technologies, Vancouver, British Columbia, Canada). Cultures were
seeded in duplicate using 35 mm plastic Petri dishes, and colony
forming units were determined after 1 week.
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Acknowledgment. This work was supported by grants from
the NIH (Grant R01CA-109820) and Onconova Therapeutics
Inc. We thank Dr. Stacey Baker for editorial assistance. We
also thank Dr. Stanley Bell, Onconova Therapeutics Inc., for
his valuable comments and suggestions.
Supporting Information Available: Elemental analysis data.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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