Structure-activity-relationship studies of conformationally restricted analogs of combretastatin A-4 derived from SU5416

Article abstract of DOI:10.1016/j.bmc.2006.06.017

A series of combretastatin A-4 analogs derived from the ATP competitive, VEGF receptor tyrosine kinase inhibitor, SU5416 were synthesized. The cytotoxic effects of the analogs were evaluated against PC-3 and MDA-MB-231 cancer cell lines, as well as their

Full text of DOI:10.1016/j.bmc.2006.06.017

Bioorganic & Medicinal Chemistry 14 (2006) 6492–6501
Structure–activity-relationship studies of conformationally
restricted analogs of combretastatin A-4 derived from SU5416
a
a
a
b
a
a
Bulbul Pandit, Yanjun Sun, Ping Chen, Dan L. Sackett, Zhigen Hu, Wendy Rich,
a
a
a
a,
*
Chenglong Li, Andrew Lewis, Kevin Schaefer and Pui-Kai Li
a
Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
b
Laboratory of Integrative and Medical Biophysics, National Institute of Child Health and Human Development,
National Institute of Health, Bethesda, MD 20892, USA
Received 16 March 2006; revised 31 May 2006; accepted 8 June 2006
Available online 24 July 2006
Abstract—A series of combretastatin A-4 analogs derived from the ATP competitive, VEGF receptor tyrosine kinase inhibitor,
SU5416 were synthesized. The cytotoxic effects of the analogs were evaluated against PC-3 and MDA-MB-231 cancer cell lines,
as well as their abilities to inhibit tubulin polymerization. Results are compared to those of compound 1, our lead compound
previously reported.
ꢀ
2006 Elsevier Ltd. All rights reserved.
1
. Introduction
blood vessels resulting in reduced blood flow to tumor
7
,8
and eventual hemorrhagic necrosis. The anti-tumor
and anti-vascular activities of CA-4 analogs have been
Microtubules are involved in cellular functions such as
cell transport, movement and separation of chromo-
somes during mitosis. Anti-microtubule agents are a class
of anti-tumor agents that target tubulin, the building
block of microtubules. Mechanistically, anti-microtubule
agents can be classified into three classes: (i) microtubule
stabilizing agents, (ii) vinca site binding agents and (iii)
colchicine site binding agents. Of the three classes of
anti-microtubule agents, colchicine site agents are the
only class of agents that do not have a representative drug
in clinical use for cancer. However, these agents are struc-
turally the most diverse group among the three classes of
tubulin binding agents. They include natural products as
well as synthetic small molecules (Fig. 1). Among all the
colchicine site agents, combretastatin A-4 (CA-4) has re-
ceived special attention in the last few years. In addition
to its potent cytotoxicity and inhibitory activity on tubu-
lin polymerization, CA-4 is one of the few anti-microtu-
bule agents reported to have selective vascular targeting
9
–12
demonstrated both in animal and human studies.
Many analogs of CA-4 have been designed to study the
structure–activity-relationship of the molecule in order
to enhance both the cytotoxic and selective vascular tar-
1
3–24
25,26
geting activity.
We have been using SU5416,
a
potent ATP competitive, VEGF receptor tyrosine kinase
inhibitor, as a template for the design of inhibitors for
other growth factor receptors. Recently, we discovered
and reported a 2-indolinone containing compound (com-
pound 1, Fig. 2) with potent growth inhibitory activities
on hormone-independent prostate (PC-3) and breast
(MDA-MB-231) cancer cell lines with IC values in
1
,2
5
0
2
7
low to subnanomolar range. Structurally, compound
1 resembles CA-4 (Fig. 2). In this paper, we report the
NCI 60 cell line screening and cell cycle analysis data
of compound 1 and the synthesis and biological activities
of its structurally related analogs (compounds 2–25).
3
,4
5
,6
activity. CA-4 and its water-soluble prodrug, combre-
tastatin A-4 phosphate (CA-4P), are selectively cytotoxic
to rapidly proliferating tumor vasculature than normal
2. Results and discussion
2
.1. Chemistry
Keywords: Combretastatins; SU5416; Antimicrotubule; Antitumor
agents; Cell cycle arrest; Colchicine.
*
Corresponding author. Tel.: +1 614 688 0253; fax: +1 614 688
556; e-mail: li.27@osu.edu
The 6-substituted 3-benzylideneindolin-2-ones (com-
pounds 1–25) were synthesized by coupling 6-substituted
8
0
968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.06.017

B. Pandit et al. / Bioorg. Med. Chem. 14 (2006) 6492–6501
6493
OH
O
H
OCH3
F
CH3
N
CH O
O
3
OCH3
N
O
H C
3
H
O
F
F
HN
S O
O
F
OCH3
OCH3
CH O
3
O
H
F
F
O
CH O
3
N N
O
O
CH O
OCH3
OCH3
3
H C
3
OCH3
Colchicine
Podophyllotoxin
A204197
T138067
OCH3
OCH3
OCH3
CH O
CH3O
CH3O
3
CH O
3
CH O
3
CH O
3
OH
OCH3
NH2
OCH3
OPO Na
OCH3
3
2
Combretastatin A-4
CA-4)
AC 7739
CA-4P
(
Figure 1. Examples of colchicine like agents and combretastatin A4 analogs.
OCH3
H CO
OCH3
mercially available except for 2,4,6-triethylbenzalde-
hyde, which was prepared according to the reported
29
procedure.
3
CH O
3
CH O
3
N
H
CH O
H
O
3
O
In general, the coupling reaction gave the E isomers as
the major product, and only in three cases (compounds
2, 5, and 12), Z isomers were also isolated. The E config-
urations of the compounds were assigned based upon
N
H
OH
OCH3
CA-4
N
CH O
3
H
SU 5416
Compound 1
0
0
the chemical shifts of the protons at the C-2 and C-6
positions in the phenyl ring at the C-3 position or 2D
NOE analysis. It has been demonstrated through NOE
4'
3'
R2
2
'
0
0
experiment that the chemical shift of C-2 and C-6 pro-
tons of three substituted benzylidenindolin-2-ones is
approximately 7.45–7.84 for the E isomers and 7.85–
R1 = H, OMe, OEt, OPr, OBu, OiPr, OiBu
R2 = mono, di and tri OMe, Me, Et
R3 = H, CH3
H
4
3
5
2
5
O
6
8.53 for the Z isomers.
N1
R3
R1
2
2
.2. Biological activity
.2.1. NCI cancer cell lines screen of compound 1. The
Figure 2. Structures of SU5416, CA-4, compound 1 and its analogs.
anti-proliferative profile of compound 1 was evaluated
at the National Cancer Institute (NCI) against 53
human cancer cell lines. The results are shown in
Table 1. Compound 1 was extremely potent with GI50
indolin-2-ones and substituted benzaldehydes in the
presence of a secondary amine, such as piperidine
(
Fig. 3). All the 6-alkoxyindolin-2-ones were prepared
2
8
according to the reported procedure. Indolin-2-one
and most of the substituted benzaldehydes were com-
(
http://dtp.nci.nih.gov/docs/compare/compare_method-
ology.html) below 10 nM towards 46 out of 53 human
cancer cell lines tested. The compound is active at
<
10 nM including all colon, CNS, prostate, and renal
cancer cell lines. An interesting observation from this
screen was that compound 1 was equally effective on
Adriamycin resistant NCI/ADR-RES cell line.
2.2.2. Effect of compound 1 on cell cycle. The effect of
compound 1 on the cell cycle progression of proliferat-
ing tumor cells was studied in a concentration-depen-
dent cell cycle analysis. Compound 1 caused a G /M
phase arrest in PC-3 cells following 24 h of incubation
2
(Fig. 4). Table 2 lists the percentage of cells arrested in
the G /M phase. Podophyllotoxin, a known anti-
mitotic, anti-microtubule agent, was used as control.
2
Figure 3. Synthesis of compounds 1–25.

6494
B. Pandit et al. / Bioorg. Med. Chem. 14 (2006) 6492–6501
Table 1. GI50 values on NCI cancer cell line screen of compound 1
Table 1 (continued)
CELL-LINE
GI50 (mol/l)
CELL-LINE
GI50 (mol/l)
ꢀ
8
Leukemia
BT-549
T-47D
<10
1.03 · 10
ꢀ8
ꢀ8
ꢀ8
ꢀ5
CCRF-CEM
MOLT-4
RPMI-8226
<10
<10
<10
NSCLC
2.2.3. Anti-proliferative activities of compounds 2–25. In
this study, we synthesized 24 new compounds and tested
them against hormone-independent prostate (PC-3) and
breast (MDA-MB-231/ATCC) cancer cells to elucidate
the structure–activity-relationship of their anti-prolifer-
ative activities (Tables 3–5). In addition, selected com-
pounds were also tested for their abilities to inhibit
tubulin polymerization (Table 6).
ꢀ
ꢀ
8
8
A549/ATCC
EKVX
HOP-62
<10
<10
ꢀ
7
HOP-92
2.5 · 10
ꢀ
ꢀ
8
8
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
<10
<10
ꢀ
6
2.54 · 10
ꢀ
ꢀ
8
8
<10
<10
0
0
0
Compound 1 contains the 3 ,4 ,5 -tri-OMe groups on
the benzylidene moiety, a common structural feature
of colchicine, CA-4, podophyllotoxin, and others. Our
first structure–activity-relationship study was to investi-
gate the substitution pattern of the tri-OMe groups in
compound 1. The location of the tri-OMe groups on
the benzylidene ring plays a critical role for the com-
pound to exhibit potent cytotoxicity. Rotating the
Colon cancer
COLO-205
HCC-2998
HCT-116
HCT-15
HT29
ꢀ8
ꢀ8
ꢀ8
ꢀ8
ꢀ8
ꢀ8
ꢀ8
<10
<10
<10
<10
<10
<10
<10
KM12
SW-260
0
0
0
0
0
0
3
,4 ,5 -tri-OMe groups in compound 1 to the 2 ,3 ,4
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
ꢀ8
ꢀ8
ꢀ8
ꢀ8
ꢀ8
ꢀ8
<10
<10
<10
<10
<10
<10
positions decreases the cytotoxicity by three orders of
magnitude (Table 3). It is interesting to note that a sim-
0
0
0
ilar substitution pattern (2 ,3 ,4 -tri-OMe to replace
0
0 0
3
ed in dramatic (5-fold) decrease in cytotoxicity. Inter-
,4 ,5 -tri-OMe) in a combretastatin analog also result-
30
0
0
0
estingly, transferring the 3 ,4 ,5 -tri-OMe groups in
0
0
0
0
0
0
Prostate cancer
PC-3
DU-145
compound 1 to the 2 ,4 ,5 (compound 4) or 2 ,4 ,6
(compound 6) positions retained the potent cytotoxicity
Table 3).
ꢀ
ꢀ
8
8
<10
<10
(
Melanoma
LOX IMVI
MALME-3M
M14
ꢀ
8
8
The substitution pattern of the trimethoxy groups is not
the only factor that determines the cytotoxicity of the
compounds. The substituents also play a significant role.
<10
<10
ꢀ
ꢀ
5
6
0 0 0
Substituting the 2 ,4 ,6 -tri-OMe groups in compound 6
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
1.33 · 10
ꢀ
ꢀ
8
8
<10
<10
0
0
0
0
0
0
with 2 ,4 ,6 -tri-Me or 2 ,4 ,6 -tri-Et groups results in a
decrease in cytotoxicity by 73–500-fold (Table 3). This
might be due to the loss of the hydrogen bonding ability
or altering the electronic character of the phenyl ring in
compounds 7 and 8. In addition, the N-1 hydrogen on
compound 1 is not critical for cytotoxicity. Substituting
the N-1 hydrogen with a methyl group (compound 9) re-
tains cytotoxicity with IC50 values of 31 and 37 nM on
PC-3 and MDA-MB-231 cells, respectively (Table 3).
ꢀ
4.64 · 10
<10
ꢀ8
Ovarian cancer
IGROV1
ꢀ
ꢀ
8
8
<10
<10
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
ꢀ5
1.4 · 10
ꢀ
ꢀ
8
8
<10
<10
ꢀ7
1.43 · 10
Our second study was to investigate the importance of
the number of methoxy groups on the benzylidene ring
with respect to the cytotoxicity. This study shows that
the tri-OMe group is optimum for activity. Reducing
the number of OMe groups from 3 (compound 1) to 2
(compounds 10–16) reduced the cytotoxicity (Table 4).
The reduction ranges from 6–500-fold in PC-3 cells
and 80–3400-fold in MDA-MB-231 cells. The most
dramatic reduction in cytotoxicity is observed when
Renal cancer
786-0
A498
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
8
8
8
8
8
8
8
<10
<10
<10
<10
<10
<10
<10
CAKI-1
RFX 393
SN 12C
TK-10
UO-31
Breast cancer
MCF 7
0
0
ꢀ8
ꢀ8
ꢀ8
ꢀ8
ꢀ8
the di-OMe groups are at 2 and 3 positions (500- and
2400-fold decrease in cytotoxicity in PC-3 and MDA-
MB-231 cells, respectively) (Table 4). As discussed in
the previous section, compound 3, an analog of com-
<10
<10
<10
<10
<10
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
0 0
pound 1 which also contains 2 and 3 OMe groups in
MDA-MB-435

B. Pandit et al. / Bioorg. Med. Chem. 14 (2006) 6492–6501
6495
0
30
60
90
120
150
0
30
60
90
120
150
Channels (FL2-A)
Channels (FL2-A)
0
30
60
90
120
150
0
30
60
90
120
150
0
30
60
90
120
150
Channels (FL2-A)
Channels (FL2-A)
Channels (FL2-A)
Figure 4. DNA profiles of PC-3 cells treated for 24 h with compound 1 and Podophyllotoxin.
Table 2. Percent of cells in
compound 1 and podophyllotoxin for 24 h
2
G /M phase after treatment with
the bulk tolerance at the 6 position by substituting
the OMe with alkoxy groups of varying sizes. The alk-
oxy groups contain both the straight chains (OEt,
compound 21; OPr, compound 22; and OBu, com-
pound 23) and the branched chain analogs (O-i-P-r,
compound 24; O-i-Bu, compound 25). In the straight
chain series, replacing the 6-OMe in compound 1 with
a OEt group retains the potent cytotoxicity. As the
length of the alkoxy group increases, the activity
decreases slightly. The extent of reduction is similar
if the alkoxy group is switched from straight to
branched chains.
a
Treated group
2
% G -M
Control
12.04
92.5
55.8
Podophyllotoxin 100 nM
Compound 1 10 nM
Compound 1 50 nM
Compound 1 100 nM
a
92.04
91.6
%
values are average of two separate experiments.
the benzylidene nucleus, exhibits a 3 orders of magni-
tude decrease in cytotoxicity as compared to compound
1
(Table 3). Based on these results, we postulate that the
To investigate whether the cytotoxicity of the com-
pounds has direct correlation with their abilities to
inhibit tubulin polymerization, selected compounds
were evaluated for inhibition of tubulin polymerization
using purified porcine tubulin (Table 6). The most
potent compound in this series in tubulin polymeriza-
tion is compound 1 with an IC50 of 4.5 lM. This is in
agreement with 1 being the most cytotoxic compound.
It should be noted that for the compounds with potent
cytotoxicity, there is a positive correlation between the
inhibition of tubulin polymerization and cytotoxicity
(Table 6). In general, compounds with trimethoxy
groups on the benzylidene ring exhibit the highest
inhibition.
0
0
substitution of OMe groups on the 2 and 3 positions is
detrimental to the cytotoxicity of the compounds.
Reducing the number of methoxy groups from 3 to 1
(
compounds 17–19) also decreases the cytotoxicity of
the compounds by 2–3 orders of magnitude (Table 4).
The order of cytotoxicity is based on the number of
OMe groups on benzylidene ring, with 3 > 2 > 1.
Next, we investigated the significance of the 6-OMe
group on the 2-indolinone ring of compound 1. Six
analogs of compound 1 (compounds 20–25) were syn-
thesized with different substituents at the 6 position.
The 6-OMe group is essential for potent cytotoxicity.
Replacing the 6-OMe group in compound 1 with H
(compound 20) resulted in the elimination of cytotox-
icity (Table 5). Our next approach was to investigate
In the previous section, we discussed about the existence
of E and Z isomers in some of the compounds

6496
B. Pandit et al. / Bioorg. Med. Chem. 14 (2006) 6492–6501
Table 3. Anti-proliferative activities of compounds 2–9
4'
3'
R2
2
'
H
4
3
5
6
O
N1
R3
CH O
3
a
PC-3 (IC50 nM)
a
MDA-MB-231 (IC50 nM)
Compound
R
2
R
3
E or Z isomer
0
0
0
1
3 ,4 ,5 -OCH
3 ,4 ,5 -OCH
3
3
3
3
3
3
H
H
H
H
H
H
H
H
CH
E
Z
E
E
Z
E
E
E
E
8 ± 2.3
34 ± 10
0.9 ± 0.07
4.0 ± 1.0
1100 ± 140
4.0 ± 0.6
110 ± 1.0
10 ± 0.1
0 0 0
2
0
0
0
3
2 ,3 ,4 -OCH
2 ,4 ,5 -OCH
5800 ± 60
6 ± 1.5
9 ± 1.4
0 0 0
4
0
0
0
0
0
0
0
0
0
0
0
0
5
2 ,4 ,5 -OCH
2 ,4 ,6 -OCH
6
22 ± 3.0
1600 ± 90
11400 ± 600
31 ± 7.0
13 ± 1.5
7
2 ,4 ,6 -CH
3
940 ± 80
2600 ± 100
37 ± 2.0
8
2 ,4 ,6 -CH
2
CH
3
0
0
0
9
3 ,4 ,5 -OCH
3
3
Podophyllotoxin
8 ± 0.5
a
Data are means of three or more experiments and are reported as means ± standard error of the mean (SEM).
Table 4. Anti-proliferative activities of compounds 10–18
4
'
3'
R2
2'
H
4
3
5
6
O
N1
H
CH O
3
a
PC-3 (IC50 nM)
a
MDA-MB-231 (IC50 nM)
Compound
R
2
E or Z isomer
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
3 ,4 ,5 -OCH
3
E
E
E
Z
E
E
E
E
E
E
E
8 ± 2.3
280 ± 20
740 ± 18
0.9 ± 0.07
90 ± 3.0
10
11
12
13
14
15
16
17
18
19
3 ,5 -OCH
3 ,4 -OCH
3
3
3
3
3
3
3
860 ± 113
3100 ± 67
2200 ± 339
84 ± 5.0
3 ,4 -OCH
2 ,3 -OCH
410 ± 155
4200 ± 600
50 ± 7.0
2 ,4 -OCH
2 ,5 -OCH
67 ± 12
75 ± 10
2 ,6 -OCH
640 ± 9.0
1060 ± 172
1400 ± 144
12800 ± 840
13 ± 1.5
180 ± 36
730 ± 80
800 ± 53
8650 ± 840
8 ± 0.5
0
2 -OCH
3
3
3
0
3 -OCH
0
4 -OCH
Podophyllotoxin
a
Data are means of three or more experiments and are reported as means ± standard error of the mean (SEM).
(compounds 2, 5, and 12). Compound 2 is the Z isomeric
form of compound 1 (Table 3). While compound 1 is a
analyses begin with pure compound 1 in solution
(Fig. 5). Since the degree and rate of isomerization may
be solvent dependent, the isomerization studies of com-
pounds 1 and 2 were also performed in the cell culture
medium (RPMI 1640) used in the cell cytotoxicity assay.
A similar result was obtained. Compound 2 slowly isom-
erized to compound 1 and attained a ratio of 3:1 (com-
pound 1 to compound 2) after 3 days (Fig. 6).
Interestingly, a similar process is also observed between
potent inhibitor of tubulin polymerization, compound
2
does not inhibit tubulin polymerization to any extent
Table 6). This might be due to stereochemical require-
(
ment for binding to tubulin and requires further investi-
gation. However, compound 2 still possesses potent
cytotoxicity in low nM range (Table 3). The cytotoxicity
of compound 2 may result from its isomerization to com-
pound 1 in solution. NMR analyses of compound 2 in
1
4,30
cis and trans isomers of CA-4.
1
solution (DMSO—in the presence of light between H
NMR analysis) showed that compound 2 gradually
isomerized to compound 1 and attained equilibrium after
2.2.4. Conclusion. In summary, a series of 3-benzylidene-
2-indolinone analogs have been prepared and tested for
cytotoxicity on tumor cells and for inhibition of tubulin
polymerization. The most potent of these analogs is 1.
7 days with a ratio of compound 1: compound 2 about
3:1. A similar ratio was also obtained if the NMR

B. Pandit et al. / Bioorg. Med. Chem. 14 (2006) 6492–6501
Table 5. Anti-proliferative activities of compounds 19–25
6497
OCH3 OCH3
CH O
3
H
4
3
5
O
6
N1
R1
H
a
PC-3 (IC50 nM)
a
MDA-MB-231 (IC50 nM)
Compound
R
1
E or Z isomer
1
OCH
H
3
E
E
E
E
E
E
E
8 ± 2.3
>30000
7 ± 2.0
0.9 ± 0.07
9400 ± 186
8.0 ± 2.0
70 ± 6.0
20
21
22
23
24
25
OCH
2
CH
3
O(CH
O(CH
2
)
)
2
CH
CH
3
35 ± 4.0
11 ± 3.0
35 ± 7.0
230 ± 50
13 ± 1.5
2
3
3
NT
OCH(CH
3
)
2
810 ± 110
11870 ± 330
8 ± 0.5
OCH CH(CH )
2
3 2
Podophyllotoxin
NT, not tested.
a
Data are means of three or more experiments and are reported as means ± standard error of the mean (SEM).
Table 6. Inhibition of tubulin polymerization by selected inhibitors
a
PC-3 (IC50 nM)
a
MDA-MB-231 (IC50 nM)
Compound
Inhibition of tubulin
a
polymerization (IC50 lM)
1
2
3
4
5
6
9
8 ± 2.3
34 ± 10
0.9 ± 0.07
4.0 ± 1.0
1100 ± 140
4.0 ± 0.6
110 ± 1
4.5 ± 0.8
> 40
> 40
5800 ± 60
6 ± 1.5
9 ± 1.4
11.3 ± 1.2
> 40
22 ± 3.0
31 ± 7.0
280 ± 20
13000 ± 840
>30000
10 ± 0.1
37 ± 2.0
90 ± 3.0
10.3 ± 0.3
13.4 ± 1.1
22.2 ± 2.1
> 40
1
1
2
0
7
1
8650 ± 840
9400 ± 200
8.0 ± 0.5
>40
1.2 ± 0.3
Podophyllotoxin
13 ± 1.5
a
Data are means of three or more experiments and are reported as means ± standard error of the mean (SEM).
1
20
00
120
1
00
1
8
0
0
0
0
8
6
4
2
0
0
0
0
0
6
4
2
0
0
20
45
Time (Hours)
67
88
0
1
2
3
4
5
6
7
Time (days)
Figure 6. Isomerization of pure compounds 1 (j) and 2 (Ç) in cell
culture media (RPMI 1640) with time. The result is presented as %
compound 1 vs time (h).
Figure 5. Isomerization study of compounds 1 (j) and 2 (Ç). Pure
compounds 1 and 2 are dissolved in deuterated DMSO and the rate of
isomerization was measured using NMR spectroscopy. The result is
presented as % compound 1 vs time (day).
cell cycle following 24-h incubation. It is clear from the re-
sults that two structural features namely the 6-OCH3
group in the indolinone ring and the trimethoxy groups
in thebenzylidenering areessentialforpotentcytotoxicity
and inhibition of tubulin polymerization. The study
NCI60celllinescreenshowedthat1isextremelycytotoxic
with GI50 values below 10 nM toward 45 out of 53 human
cancercelllinestested. ItcausedaG /Mphasearrestinthe
2

6498
B. Pandit et al. / Bioorg. Med. Chem. 14 (2006) 6492–6501
contributestotheknowledgeaboutthestructure–activity-
relationship of 3-benzylidene-2-indolinone analogs.
Ph), 6.47 (dd, J = 8.4, 2.4 Hz, 1H, Ph), 6.80 (s, 1H,
Ph), 7.25 (s, 1H, Ph), 7.49 (s, 1H, vinyl-H), 7.52 (d,
J = 8.4 Hz, 1H, Ph), 10.47 (s, 1H, NH); HRMS: calcd
+
Since the compounds are derived from the KDR inhib-
itor SU5416, studies are currently underway to examine
if the compounds also inhibit the receptor. Potentially,
some of the compounds could be dual antitubulin–anti-
angiogenic agents.
for [C H NO Na] : 364.11554; found: 364.11481.
5
1
9
19
3.4. (Z)-1,3-Dihydro-6-methoxy-3-(2,4,5-trimethoxyben-
zylidene)-1H-indol-2-one (5)
1
Yellow solid (39% yield): mp 236–238 ꢁC; H NMR
(
DMSO-d ): d 3.76 (s, 6H, –OCH ), 3.88 (s, 3H,
6
3
3. Experimental
–OCH ), 3.91 (s, 3H, –OCH ), 6.39 (d, J = 2.4 Hz, 1H,
3 3
Ph), 6.53 (dd, J = 8.4, 2.4 Hz, 1H, Ph), 6.44 (d,
J = 8.4 Hz, 1H, Ph), 7.43 (d, J = 8.4 Hz, 2H, Ph), 7.77
(s, 1H, vinyl-H), 8.81 (s, 1H, Ph), 10.45 (s, 1H, NH);
Chemicals and biochemicals were purchased from com-
mercial vendors and checked for purity by thin-layer
chromatography and NMR before use. Melting points
were determined on a Thomas Hoover capillary melting
point apparatus and are uncorrected. Proton NMR
spectra were obtained on Bruker (250 MHz) FT-NMR
instrument. High-resolution mass spectra were recorded
on a Micromass QTOF-Electrospray mass spectrometer
at The Ohio State University Campus Chemical Instru-
mentation Center. Analytical HPLC was performed
with reverse phase C18 column (Resolvex C18,
+
HRMS: calcd for [C H NO Na] : 364.11554; found:
1
9
19
5
364.11473.
3.5. (E)-1,3-Dihydro-6-methoxy-3-(2,4,6-trimethoxyben-
zylidene)-1H-indol-2-one (6)
1
Yellow solid (58% yield): mp 202–204 ꢁC; H NMR
(DMSO-d ): d 3.73 (s, 3H, –OCH ), 3.75 (s, 6H,
6
3
–OCH ), 3.87 (s, 3H, –OCH ), 6.37 (m, 4H, Ph), 6.70
3
3
4
.6 mm · 25 cm; Fisher Scientific, Pittsburgh, PA) using
(d, J = 8.4 Hz, 1H, Ph), 7.23 (s, 1H, vinyl-H), 10.37 (s,
1H, NH); HRMS: calcd for [C H NO Na] :
364.11554; found: 364.11451.
+
a Beckman ‘System Gold’ HPLC, Model 127 pump and
Model 166 detector.
1
9
19
5
3.1. Synthesis of (E)-1,3-Dihydro-6-methoxy-3-(2,3,4-tri-
methoxybenzylidene)-1H-indol-2-one (3)
3.6. (E)-1,3-Dihydro-6-methoxy-3-(2,4,6-trimethylbenzy-
lidene)-1H-indol-2-one (7)
1
A mixture of 6-methoxy-indolin-2-one (0.15 g, 0.92 mmol),
2
Yellow solid (32% yield): mp 218–220 ꢁC; H NMR
,3,4-trimethoxybenzaldehyde (0.36 g, 1.84 mmol), and
(DMSO-d ): d 2.10 (s, 6H, –CH ), 2.29 (s, 3H, –CH ),
6
3
3
piperidine (0.01 ml, 0.1 mmol) in ethanol (15 ml) was
stirred at 80 ꢁC for 2.5 h. The mixture was cooled to
room temperature and concentrated under reduced pres-
sure to form a yellow solid. The solid was washed with a
equal mixture of hexane–ethyl acetate (50 ml) and puri-
fied by silica gel chromatography (hexane/EtOAc/
3.71 (s, 3H, –OCH ), 6.33 (s, 2H, Ph), 6.39 (br s, 1H,
3
Ph), 6.98 (br s, 2H, Ph), 7.42 (s, 1H, vinyl-H), 10.53 (s,
1H, NH); HRMS: calcd for [C H NO Na] :
316.13080; found: 316.13217.
+
1
9
19
2
3.7. (E)-1,3-Dihydro-6-methoxy-3-(2,4,6-triethylbenzy-
lidene)-1H-indol-2-one (8)
MeOH, 10:10:1), yielding pure compound 3 (0.13 g,
1
4
3
–
0%): mp 181–183 ꢁC; H NMR (DMSO-d ): d 3.75 (s,
6
1
H, –OCH ), 3.80 (s, 3H, –OCH ), 3.82 (s, 3H,
OCH ), 3.88 (s, 3H, –OCH ), 6.42 (m, 2H, Ph), 6.95
Yellow solid (28% yield): mp 164–166 ꢁC; H NMR
3
3
(DMSO-d ): d 0.99 (t, J = 7.5 Hz, 6H, –CH ), 1.23 (t,
3
3
6
3
(
2
[
d, J = 8.7 Hz, 1H, Ph), 7.42 (s, 1H, vinyl-H), 7.45 (m,
H, Ph), 10.53 (s, 1H, NH); HRMS: calcd for
C H NO Na] : 364.11554; found: 364.11574.
J = 7.5 Hz, 3H, –CH ), 2.44 (m, 4H, –CH ), 2.60 (q,
J = 7.5 Hz, 2H, –CH ), 3.70 (s, 3H, –OCH ), 6.27 (m,
2 3
3 2
+
2H, Ph), 6.38 (d, J = 2.1 Hz, 1H, Ph), 7.03 (s, 2H, Ph),
.52 (s, 1H, vinyl-H), 10.53 (s, 1H, NH); HRMS: calcd
19
19
5
7
+
3.2. (Z)-1,3-Dihydro-6-methoxy-3-(3,4,5-trimethoxyben-
zylidene)-1H-indol-2-one (2)
for [C H NO Na] : 358.17775; found: 358.17904.
22 25 2
3.8. (E)-1,3-Dihydro-6-methoxy-1-methyl-3-(3,4,5-tri-
methoxybenzylidene)-1H-indol-2-one (9)
1
Yellow solid (28% yield): mp 229–231 ꢁC; H NMR
(
DMSO-d ): d 3.74 (s, 3H, –OCH ), 3.78 (s, 3H,
6 3
–OCH ), 3.84 (s, 6H, –OCH ), 6.41 (d, J = 2.4 Hz, 1H,
Ph), 6.57 (dd, J = 8.4, 2.4 Hz, 1H, Ph), 7.57 (d, J =
1
Yellow solid (42% yield): mp 252–254 ꢁC; H NMR
(CDCl ): d 3.27 (s, 3H, –CH ), 3.85 (s, 3H, –OCH ),
3
3
3
3
3
8
1
3
.4 Hz, 1H, Ph), 7.58 (s, 1H, vinyl-H), 7.94 (s, 2H, Ph),
0.53 (s, 1H, NH); HRMS: calcd for [C H NO Na] :
64.11554; found: 364.11441.
3.88 (s, 6H, –OCH ), 3.93 (s, 6H, –OCH ), 6.43 (m, 2H,
Ph), 6.89 (s, 2H, Ph), 7.62 (s, 1H, vinyl-H), 7.71 (d,
J = 8.1 Hz, 1H, Ph), HRMS: calcd for [C H NO Na] :
3 3
+
1
9
19
5
+
2
0
21
5
3
78.13119; found: 378.13202.
3.3. (E)-1,3-Dihydro-6-methoxy-3-(2,4,5-trimethoxyben-
zylidene)-1H-indol-2-one (4)
3.9. (E)-1,3-Dihydro-6-methoxy-3-(3,5-dimethoxybenzy-
lidene)-1H-indol-2-one (10)
1
Yellow solid (48% yield): mp 175–177 ꢁC; H NMR
1
(
DMSO-d ): d 3.71 (s, 3H, –OCH ), 3.75 (s, 3H,
Yellow solid: mp 156–158 ꢁC; H NMR (DMSO-d ): d
6
3
6
–
OCH ), 3.91 (s, 6H, –OCH ), 6.42 (d, J = 2.4 Hz, 1H,
3.76 (s, 3H, –OCH ), 3.78 (s, 6H, –OCH ), 6.42
3
3
3
3

B. Pandit et al. / Bioorg. Med. Chem. 14 (2006) 6492–6501
6499
(
d, J = 2.4 Hz, 1H, Ph), 6.48 (dd, J = 8.4, 2.4 Hz, 1H,
Ph), 6.58 (t, J = 2.4 Hz, 1H, Ph), 6.82 (d, J = 2.4 Hz,
H, Ph), 7.38 (s, 1H, vinyl-H), 7.53 (d, J = 8.4 Hz, 1H,
Ph), 10.55 (s, 1H, NH); HRMS: calcd for [C H NO -
–OCH ), 6.38 (m, 2H, Ph), 6.60 (m, 1H, Ph), 6.76
3
(d, J = 8.4 Hz, 2H, Ph), 7.25 (s, 1H, vinyl-H), 7.41
(t, J = 8.4 Hz, 1H, Ph), 10.43 (s, 1H, NH); HRMS:
calcd for [C H NO Na] : 334.10498; found:
2
+
1
8
17
4
18 17
4
+
Na] : 334.10498; found: 334.10575.
334.10509.
3.10. (E)-1,3-Dihydro-6-methoxy-3-(3,4-dimethoxyben-
zylidene)-1H-indol-2-one (11)
3.16. (E)-1,3-Dihydro-6-methoxy-3-(2-methoxybenzyl-
idene)-1H-indol-2-one (17)
1
1
Yellow solid (39% yield): mp 184–186 ꢁC; H NMR
Yellow solid (65% yield): mp 208–209 ꢁC; H NMR
(
DMSO-d ): d 3.76 (s, 3H, –OCH ), 3.80 (s, 3H,
(DMSO-d ): d 3.75 (s, 3H, –OCH ), 3.85 (s, 3H,
6
3
6
3
–
OCH ), 3.84 (s, 3H, –OCH ), 6.45 (m, 2H, Ph), 7.10
–OCH ), 6.40 (m, 2H, Ph), 7.06 (t, J = 8.1 Hz, 1H,
3
3
3
(d, J = 8.4 Hz, 1H, Ph), 7.31 (m, 2H, Ph), 7.40 (s, 1H,
vinyl-H), 7.65 (d, J = 2.4 Hz, 1H, Ph), 10.51 (s, 1H,
Ph), 7.14 (d, J = 8.1 Hz, 1H, Ph), 7.33 (d, J = 8.1 Hz,
1H, Ph), 7.44 (d, J = 8.1 Hz, 1H, Ph), 7.45 (s, 1H,
vinyl-H), 7.65 (d, J = 8.1 Hz, 1H, Ph), 10.54 (s, 1H,
+
NH); HRMS: calcd for [C H NO Na] : 334.10498;
found: 334.10575.
1
8
17
4
+
NH); HRMS: calcd for [C H NO Na] : 304.09441;
3
1
7
15
found: 304.09430.
3.11. (Z)-1,3-Dihydro-6-methoxy-3-(3,4-dimethoxyben-
zylidene)-1H-indol-2-one (12)
3.17. (E)-1,3-Dihydro-6-methoxy-3-(3-methoxybenzyl-
idene)-1H-indol-2-one (18)
1
Yellow solid (25% yield): mp 252–254 ꢁC; H NMR
1
(
DMSO-d ): d 3.77 (s, 3H, –OCH ), 3.82 (s, 6H,
Yellow solid (69% yield): mp 162–163 ꢁC; H NMR
6
3
–
J = 8.4, 2.4 Hz, 1H, Ph), 7.04 (d, J = 8.4 Hz, 1H, Ph),
OCH ), 6.40 (d, J = 2.4 Hz, 1H, Ph), 6.55 (dd,
(DMSO-d ): d 3.76 (s, 3H, –OCH ), 3.80 (s, 3H,
3
6
3
–OCH ), 6.44 (m, 2H, Ph), 7.02 (d, J = 8.4 Hz, 1H,
Ph), 7.24 (m, 2H, Ph), 7.42 (s, 1H, vinyl-H), 7.43 (t,
J = 8.4 Hz, 1H, Ph), 7.50 (d, J = 8.4 Hz, 1H, Ph),
3
7
7
1
.57 (d, J = 8.4 Hz, 1H, Ph), 7.57 (s, 1H, vinyl-H),
.77 (dd, J = 8.4, 2.4 Hz, 1H, Ph), 8.59 (d, J = 2.4 Hz,
H, Ph), 10.50 (s, 1H, NH); HRMS: calcd for
+
10.56 (s, 1H, NH); HRMS: calcd for [C H NO Na] :
304.09441; found: 304.09513.
1
7
15
3
+
[
C H NO Na] : 334.10498; found: 334.10405.
4
1
8
17
3.12. (E)-1,3-Dihydro-6-methoxy-3-(2,3-dimethoxyben-
zylidene)-1H-indol-2-one (13)
3.18. (E)-1,3-Dihydro-6-methoxy-3-(4-methoxybenzyl-
idene)-1H-indol-2-one (19)
1
1
Yellow solid (37% yield): mp 172–173 ꢁC; H NMR
Yellow solid (77% yield): mp 180–181 ꢁC; H NMR
(
DMSO-d ): d 3.73 (s, 3H, –OCH ), 3.75 (s, 3H,
(DMSO-d ): d 3.76 (s, 3H, –OCH ), 3.84 (s, 3H,
6
3
6
3
–
OCH ), 3.86 (s, 3H, –OCH ), 6.41 (m, 2H, Ph), 7.20
–OCH ), 6.45 (m, 2H, Ph), 7.07 (d, J = 8.4 Hz, 2H,
3
3
3
(
m, 3H, Ph), 7.30 (d, J = 8.4 Hz, 1H, Ph), 7.46 (s, 1H,
vinyl-H), 10.56 (s, 1H, NH); HRMS: calcd for
Ph), 7.41 (s, 1H, vinyl-H), 7.57 (d, J = 8.4 Hz, 1H,
Ph), 7.67 (d, J = 8.4 Hz, 2H, Ph), 10.51 (s, 1H, NH);
HRMS: calcd for [C H NO Na] : 304.09441; found:
+
C H NO Na] : 334.10498; found: 334.10575.
+
[
1
8
17
4
17 15
3
304.09511.
3.13. (E)-1,3-Dihydro-6-methoxy-3-(2,4-dimethoxyben-
zylidene)-1H-indol-2-one (14)
3.19. (E)-1,3-Dihydro-3-(3,4,5-trimethoxybenzylidene)-
H-indol-2-one (20)
1
1
Yellow solid (50% yield): mp 198–199 ꢁC; H NMR
1
(
DMSO-d ): d 3.75 (s, 3H, –OCH ), 3.86 (s, 6H, –OCH ),
Yellow solid (61% yield): mp 189–191 ꢁC; H NMR
(DMSO-d ): d 3.75 (s, 3H, –OCH ), 3.85 (s, 6H,
–OCH3), 6.84 (d, J = 7.5 Hz, 1H, Ph), 7.00 (t,
6
3
3
6.43 (m, 2H, Ph), 6.67 (m, 2H, Ph), 7.43 (m, 2H, Ph),
6
3
7.65 (d, J = 8.7 Hz, 1H, Ph), 10.48 (s, 1H, NH); HRMS:
calcd for [C H NO Na] : 334.10498; found: 334.10573.
+
J = 7.5 Hz, 1H, Ph), 7.21 (t, J = 7.5 Hz, 1H, Ph),
7.68 (d, J = 7.5 Hz, 1H, Ph), 7.77 (s, 1H, vinyl-
H), 8.02 (s, 2H, Ph), 10.59 (s, 1H, NH); HRMS:
18
17
4
3.14. (E)-1,3-Dihydro-6-methoxy-3-(2,5-dimethoxyben-
zylidene)-1H-indol-2-one (15)
+
calcd for [C H NO Na] : 334.10498; found:
1
8
17
4
334.10575.
1
Yellow solid (65% yield): mp 179–180 ꢁC; H NMR
TM
DMSO-d ): 3.73 (s, 3H, –OCH ), 3.75 (s, 3H, –OCH ),
(
3.20. (E)-1,3-Dihydro-6-ethoxy-3-(3,4,5-trimethoxyben-
zylidene)-1H-indol-2-one (21)
6
3
3
3
7
7
.79 (s, 3H, –OCH ), 6.44 (m, 2H, Ph), 7.05 (m, 2H, Ph),
3
.20 (d, J = 2.7 Hz, 1H, Ph), 7.39 (d, J = 8.4 Hz, 1H, Ph),
.44 (s, 1H, vinyl-H), 10.54 (s, 1H, NH); HRMS: calcd
1
Yellow solid (60% yield): mp 169–170 ꢁC; H NMR
+
for [C H NO Na] : 334.10498; found: 334.10460.
17
(DMSO-d ): d 1.32 (t, J = 6.9 Hz, 3H, –CH ), 3.74 (s,
18
4
6
3
3H, –OCH ), 3.82 (s, 6H, –OCH ), 4.02 (q, J = 6.9 Hz,
2H, –OCH ), 6.40 (d, J = 2.1 Hz, 1H, Ph), 6.46 (d,
3 3
3
.15. (E)-1,3-Dihydro-6-methoxy-3-(2,6-dimethoxyben-
zylidene)-1H-indol-2-one (16)
2
J = 8.4 Hz, 1H, Ph), 7.02 (s, 2H, Ph), 7.38 (s, 1H, Ph),
7.63 (d, J = 8.4 Hz, 1H, Ph), 10.48 (s, 1H, NH); HRMS:
1
+
Yellow solid (68% yield): mp 215–217 ꢁC; H NMR
DMSO-d ): d 3.73 (s, 3H, –OCH ), 3.74 (s, 6H,
calcd for [C H NO Na] : 378.13119; found:
20 21 5
(
378.13103.
6
3

6500
B. Pandit et al. / Bioorg. Med. Chem. 14 (2006) 6492–6501
3.21. (E)-1,3-Dihydro-6-propoxy-3-(3,4,5-trimethoxyben-
zylidene)-1H-indol-2-one (22)
100 ll of media and were allowed to attach overnight.
Varying concentrations of compounds were added to
the cells with a final volume per well of 200 ll. The treat-
1
Yellow solid (62% yield): mp 142–143 ꢁC; H NMR
ed cells were incubated under 37 ꢁC, 5% CO for 72 h.
2
(
DMSO-d ): d 1.02 (t, J = 6.7 Hz, 3H, –CH ), 1.78 (m,
Cell viability was determined by Promega CellTiter 96
Aqueous Non-Radioactive Cell Proliferation assay
(MTS). IC50 values were calculated using the SOFTmax
Pro plate reader program. Each compound was tested at
least three times in triplicate.
6
3
2
3
1
2
1
3
H, –CH ), 3.74 (s, 3H, –OCH ), 3.82 (s, 6H, –OCH ),
.95 (t, J = 6.9 Hz, 2H, –OCH ), 6.41 (d, J = 2.1 Hz,
2 3 3
2
H, Ph), 6.48 (dd, J = 8.6 Hz, 2.1 Hz, 1H, Ph), 7.03 (s,
H, Ph), 7.38 (s, 1H, Ph), 7.64 (d, J = 8.6 Hz, 1H, Ph),
0.51 (s, 1H, NH); HRMS: calcd for [C H NO Na] :
+
2
1
23
5
92.146842; found: 392.14629.
3.27. Tubulin assembly assay
3
.22. (E)-1,3-Dihydro-6-butoxy-3-(3,4,5-trimethoxyben-
zylidene)-1H-indol-2-one (23)
Porcine brain tubulin was purified according to the
1,32
3
reported procedure.
a 96-well half area (Costar) plate in PME buffer
The assay was carried out on
1
Yellow solid (65% yield): mp 182–183.5 ꢁC; H NMR
[0.1 M PIPES (pH 6.9), 1 mM MgCl , and 1 mM
2
(
DMSO-d ): d 0.91 (t, J = 7.2 Hz, 3H, –CH ), 1.41
EGTA] at a protein concentration of 1.5 mg/ml
(15 mM). The components of disassembly assay includ-
ed PME buffer, various drug dilutions, DMSO (final
concentration 10% v/v) and tubulin were added to 96-
well half-area plate kept on ice. Reaction was initiated
by simultaneous addition of GTP to a 1 mM final con-
centration to all of the wells. Polymerization was moni-
tored by the increase/change in absorbance at 351 nm
using a Spectra Plus micro plate reader. Podophyllotox-
in was used as a positive control in each assay.
6
3
(
–
m, 2H, –CH ), 1.68 (m, 2H, –CH ), 3.73 (s, 3H,
OCH ), 3.80 (s, 6H, –OCH ), 3.96 (m, 2H, –OCH ),
3 3 2
2 2
6
Ph), 7.02 (s, 2H, Ph), 7.38 (s, 1H, Ph), 7.64 (d,
J = 7.8 Hz, 1H, Ph), 10.53 (s, 1H, NH); HRMS: calcd
for [C H NO Na] : 406.16249; found: 406.16119.
.41 (d, J = 2.1 Hz, 1H, Ph), 6.49 (d, J = 7.8 Hz, 1H,
+
2
2
25
5
3.23. (E)-1,3-Dihydro-6-isopropoxy-3-(3,4,5-trimeth-
oxybenzylidene)-1H-indol-2-one (24)
1
Yellow solid (62% yield): mp 187.5–189 ꢁC; H NMR
3.28. Isomerization studies of compounds 1 and 2
(
DMSO-d ): d 1.26 (d, J = 6.0 Hz, 6H, –(CH ) ), 3.74 (s,
6 3 2
1
(a) Isomerization study using H NMR spectroscopy—
3
–
1
H, –OCH ), 3.82 (s, 6H, –OCH ), 4.59 (m, 1H,
OCH), 6.40 (d, J = 2.1 Hz, 1H, Ph), 6.46 (d, J = 8.4 Hz,
H, Ph), 7.02 (s, 2H, Ph), 7.38 (s, 1H, Ph), 7.64 (d,
3
3
pure compounds 1 and 2 (3–4 mg) were dissolved sepa-
rately in DMSO-d and placed in NMR tubes. NMR
6
J = 8.4 Hz, 1H, Ph), 10.48 (s, 1H, NH); HRMS: calcd
for [C H NO Na] : 392.146842; found: 392.14528.
spectra were then obtained in 1, 2, 4, 8, 12, 24 h and then
once daily for up to 10 days. The extent of isomerization
was measured by comparing the peak ratio of the vinyl
protons of compound 1 (7.40 ppm) and compound 2
(7.58 ppm). The results are plotted as % compound 1
versus time (day).
+
2
1
23
5
3.24. (E)-1,3-Dihydro-6-isobutoxy-3-(3,4,5-trimethoxy-
benzylidene)-1H-indol-2-one (25)
1
Yellow solid (57% yield): mp 107–109 ꢁC; H NMR
(
DMSO-d ): d 0.97 (d, J = 6.6 Hz, 6H, –(CH ) ), 1.24
(b) Isomerization study of compounds 1 and 2 in cell
culture media—compounds 1 and 2 (2.3 mg of com-
pound 1 and 2.5 mg of compound 2) were each dissolved
in 3 ml of DMSO. An aliquot (2.3 ml) of the DMSO
solutions (compounds 1 and 2) were added separately
6
3 2
(
–
m, 1H, –CH), 3.74 (s, 3H, –OCH ), 3.83 (s, 6H,
OCH ), 3.96 (d, J = 6.3 Hz, 2H, –OCH ), 6.41 (d,
3 2
3
J = 2.1 Hz, 1H, Ph), 6.49 (dd, J = 8.4 Hz, 2.1 Hz, 1H,
Ph), 7.03 (s, 2H, Ph), 7.38 (s, 1H, Ph), 7.65 (d,
J = 8.4 Hz, 1H, Ph), 10.52 (s, 1H, NH); HRMS: calcd
to RPMI media (19.77 ml). The concentrations of com-
pounds 1 and 2 in the media were 2.58 · 10
+
ꢀ5
for [C H NO Na] : 406.16249; found: 406.16233.
2
and
2
25
5
ꢀ
5
2
.8 · 10 M, respectively. The concentrations were cho-
sen due to the detection limit of the UV detector. The
media containing the compounds were placed in a 24-
well plate (2 ml per well). The plate was placed in an
3
.25. Cell lines and culture
Tumor cell lines (MDA-MB-231; prostate: PC-3) were
originally obtained from American type culture collec-
tion. PC-3 cells were cultured in complete RPMI 1640
incubator (37 ꢁC, 5% CO ). At set time intervals (0,
2
20, 45, 67, and 88 h) media were removed for HPLC
analysis. The media in each well (2 wells per time inter-
val per compound) were extracted with CH Cl
(
(
Gibco, Invitrogen, Grand Island, NY) + 10% FBS
GIBCO), 2 mM L-glutamine (GIBCO), and 1% genta-
2
2
2
micin (GIBCO). MDA-MB-231 cells were cultured in
DMEM F-12 + 10% FBS, 2 mM L-glutamine, and 1%
gentamicin.
(2· 1 ml). An analytical sample (10 ll) of the CH Cl
2
layer was injected into HPLC to determine the degree
of isomerization. HPLC grade water and methanol
(3:7) were used with a flow rate of 1 ml/min applied with
UV detection at 352 nm.
3
.26. Cell growth inhibition assay
Cells were grown in suspension cultures at 37 ꢁC in a
Comparison of compound 1 to 2 absorption peaks
showed percentage of each compound. The results are
plotted as % compound 1 versus time (hour).
humidified 5% CO atmosphere. Cells were plated into
9
2
6-well plates at a cell density of 1000 cells per well in

B. Pandit et al. / Bioorg. Med. Chem. 14 (2006) 6492–6501
6501
Acknowledgment
16. Wang, L.; Woods, K. W.; Li, Q.; Barr, K. J.; McCroskey,
R. W.; Hannick, S. M.; Gherke, L.; Credo, R. B.; Hui, Y.
H.; Marsh, K.; Warner, R.; Lee, J. Y.; Zielinski-Mozng,
N.; Frost, D.; Rosenberg, S. H.; Sham, H. L. J. Med.
Chem. 2002, 45, 1697.
This work is supported in part by The Intramural
Research Program of National Institute of Child
Health and Human Development, NIH.
17. Pettit, G. R.; Rhodes, M. R.; Herald, D. L.; Hamel, E.;
Schmidt, J. M.; Pettit, R. K. J. Med. Chem. 2005, 48, 4087.
18. Pettit, G. R.; Grealish, M. P.; Herald, D. L.; Boyd, M. R.;
Hamel, E.; Pettit, R. K. J. Med. Chem. 2000, 43, 2731.
19. Medarde, M.; Ramos, A. C.; Caballero, E.; Pelaez-
Lamamie de Clairac, R.; Lopez, J. L.; Gravalos, D. G.;
Feliciano, A. S. Bioorg. Med. Chem. Lett. 1999, 9, 2303.
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