Conformationally restricted analogs of Combretastatin A-4 derived from SU5416

Article abstract of DOI:10.1016/j.bmcl.2005.09.001

A series of compounds originally derived from the vascular endothelial growth factor receptor tyrosine kinase inhibitor, SU5416, was synthesized and evaluated. The most potent compound in this series, compound 7, structurally resembles the potent anti-mic

Full text of DOI:10.1016/j.bmcl.2005.09.001

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5382–5385
Conformationally restricted analogs of Combretastatin A-4
derived from SU5416
a,
Pui-Kai Li, Zili Xiao, Zhigen Hu, Bulbul Pandit, Yanjun Sun, Dan L. Sackett,
a
a
a
a
b
*
a
Karl Werbovetz, Andrew Lewis and Jayasekar Johnsamuel
a
a
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 Institutes of Health, Bethesda, MD 20892, USA
Received 24 May 2005; revised 6 September 2005; accepted 6 September 2005
Available online 6 October 2005
Abstract—A series of compounds originally derived from the vascular endothelial growth factor receptor tyrosine kinase inhibitor,
SU5416, was synthesized and evaluated. The most potent compound in this series, compound 7, structurally resembles the potent
anti-microtubule agent Combretastatin A-4, inhibited tubulin polymerization, and showed potent growth inhibitory activities on
both prostate and breast cancer lines with IC50 values in low to subnanomolar range.
ꢀ
2005 Elsevier Ltd. All rights reserved.
Microtubules are involved in cellular functions such as
cell transport, movement, and separation of chromo-
somes during mitosis. Microtubules are polymers of
a- and b-tubulin heterodimers. During mitosis, microtu-
bule polymerization and depolymerization are tightly
controlled and disruption of this process can cause cell
lotoxin. In addition, many small synthetic molecules
are reported to bind to the colchicine site. Some of the
representative agents are indanocine, oxazoline
5
,6
(A204197), and arylsulfonamide (T138067) (Fig. 1).
In our search for growth factor receptor kinase inhibi-
tors using SU5416, a potent ATP competitive vascular
endothelial growth factor receptor tyrosine kinase inhib-
1
cycle arrest and subsequently cell death. Mechanistical-
ly, anti-microtubule agents can be classified into three
classes: (i) microtubule stabilizing agents, (ii) Vinca site
binding agents, and (iii) colchicine site binding agents.
Taxanes (paclitaxel and docetaxel) are agents that
induce tubulin polymerization and stabilize microtu-
7
itor, as the lead structure, we recently discovered a
group of 2-indolinone containing compounds (Fig. 2)
with potent anti-proliferative activities in both prostate
and breast cancer cell lines.
2
,3
bules.
Vinca alkaloids (vincristine, vinblastine, and
vinorelbine) bind to b-tubulin and block the formation
of microtubules. Like Vinca alkaloids, colchicine site
binding agents also inhibit tubulin polymerization by
The syntheses of compounds 1 and 2 were carried out by
refluxing 6-methoxy-2-indolinone with 3- and 4-
hydroxybenzaldehydes, respectively (Fig. 3). 6-Meth-
oxy-2-indolinone was synthesized according to the
4
binding to the colchicine site on b-tubulin.
8
reported procedure. Demethylation of compounds 1
Currently, Taxanes and Vinca alkaloids are used clini-
cally for the treatment of cancers. Colchicine site agents
are the only class that do not have a representative drug
in clinical use for cancer. However, these agents are
structurally the most diverse group among the three
classes of tubulin binders and include natural products
such as colchicine, Combretastatin A-4, and podophyl-
and 2 with boron tribromide at 0 ꢁC yielded the respec-
tive compounds 5 and 6 (Fig. 3). The procedure for the
synthesis of compounds 3 and 4 was the same as for
compounds 1 and 2, except that the commercially avail-
able 2-indolinone was used as the reagent instead of 6-
methoxy-2-indolinone. Compounds 1–6 were purified
as E isomers. The E configurations of the compounds
were assigned based upon the chemical shifts of the pro-
0
0
0
tons at the C-2 and C -6 positions in the phenyl ring at
the C-3 position of compounds 1–6. It has been demon-
strated through NOE experiment that the chemical
*
Corresponding author. Tel.: +1 614 688 0253; fax: +1 614 688
556; e-mail: li.27@osu.edu
8
0
960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.09.001

P.-K. Li et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5382–5385
5383
OH
N
OCH3
N
O
CH O
3
O
R'O
HO
O
CH O
3
OH
CH3O
N
R
NH
O
H
CH O
3
CH O
3
HOC
N
OCH3
Ph
O
O
H
AcO
OBz
O
OH
CH3OOC
OH
N
OH
OCH3
OCH3
CH3CO
Paclitaxel: R = Ph, R'=Ac
Docetaxel: R=t-BuO, R'=H
Colchicine
Combretastatin A-4
Vincristine
OH
H
OCH3
F
CH3
N
O
O
H C
3
OCH3
OCH3
O
OH
CH3
H C
3
F
F
HN
S O
O
F
O
H
F
F
O
CH O
3
N N
OCH3
O
CH O
OCH3
3
CH O
3
H C
3
OCH3
O
NH2
T138067
Podophyllotoxin
Indanocine
A204197
Figure 1. Structures of Taxanes, Vinca alkaloid, and colchicine site binders.
4'
3'
R2
1
R
2
R
Compound #
5'
2'
1
'
6'
N
H
O
1
2
6 - OCH
6 - OCH
4
3
3
5
6
3
O
2
N
1N
H
R1
H
SU 5416
Figure 2. Structures of SU5416 and 3-(hydroxy-benzylidene)-2-indolinones (compounds 1–6).
4'
3'
4' 3'
OH
'
OH
2'
2
a
b
O
O
O
N
H
N
H
N
H
CH O
CH O
HO
3
3
Compound 1 3'-OH
Compound 2 4'-OH
Compound 5 3'-OH
Compound 6 4'-OH
6
-Methoxy-2-indolinone
Figure 3. Synthesis of compounds 1, 2, 5, and 6. Reagents and conditions: (a) 3 or 4-hydroxybenzaldehyde/piperidine/EtOH/reflux/2–3 h; (b) BBr
CH Cl
/0 ꢁC, rt/overnight.
3
/
2
2
Table 1. Inhibition of cancer cell proliferation and tubulin polymerization by compounds 1–6
1
R
2
R
Compound
PC-3 (IC50 lM)
MCF-7 (IC50 lM)
MDA-MB-231 (IC50 lM)
Inhibition of tubulin
polymerization (lM)
0
0
0
1
6-OCH
6-OCH
H
3
3 -OH
4 -OH
0.54 ± 0.2
0.64 ± 0.2
>50
0.39 ± 0.1
0.23 ± 0.1
31.4 ± 7.5
25.7 ± 5.2
8.6 ± 1.4
19.7 ± 3.8
>50
1.4 ± 1.0
2.6 ± 1.7
16.9 ± 7.3
17.5 ± 3.5
4.3 ± 0.7
9.4 ± 1.3
>50
9.1
18
2
3
3
3 -OH
4 -OH
>40
>40
>40
>40
>40
1.56
0
0
4
H
>50
5
6-OH
6-OH
—
3 -OH
4 -OH
19.5 ± 3.7
44.7 ± 6.8
>50
0
6
SU5416
Podophyllotoxin
—
—
—
0.013
Not tested
0.008
Cells (1000 cells/well) were treated with varying concentrations of compounds and cell-associated protein was determined using MTS assay. The IC50
values represent means of three experiments in triplicate. Tubulin polymerization studies were determined as described in Ref. 9.

5384
P.-K. Li et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5382–5385
Inhibiton of Tubulin Polymerization
dependent (MDA-MB-231) breast cancer cell lines. Cells
0
.1
were treated with test compounds for 72 h and cell via-
bility was determined by the MTS assay. As shown in
Table 1, compounds 1 and 2 are the most active mole-
cules against all three cell lines tested, suggesting that
the 6-OMe group is important for cytotoxicity. Removal
of the 6-OMe and replacement with a 6-H (3 and 4) de-
creased cytotoxicity significantly by 100-fold against
both PC-3 and MCF-7 cells, and by 6- to 10-fold against
MDA-MB-231 cells. Conversion of the 6-OMe to a
hydrophilic 6-OH group (compounds 5 and 6) caused
a 22- to 86-fold decrease in inhibition of the growth of
PC-3 and MCF-7 cells, and there was only a slight
reduction in inhibitory activity against MDA-MB-231
cell line (3- to 4-fold, Table 1). SU5416, the potent
VEGF receptor tyrosine kinase inhibitor with a 2-indoli-
none moiety, did not show anti-proliferative activity on
any of the three cell lines.
0
.09
.08
.07
.06
.05
.04
.03
.02
.01
0
0
0
0
0
0
0
0
0
0
0.5
1
1.5
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20
-
0.01
Time in minutes
Figure 4. Effect of compound 1 on the assembly of purified porcine
brain tubulin. The assembly of 1.5 mg/ml (15 lM) of purified bovine
tubulin was measured by a change in the absorbance at 351 nm at
To investigate the mechanism of the anti-proliferative
activities of the compounds, we examined the effects of
compounds on tubulin polymerization in vitro. Using
3
7 ꢁC in the absence-control (Ç) or presence of 5 lM ( ), 10 lM (·),
and 20 lM (m) of compound 1. Podophyllotoxin at 5 lM (j) was used
as a positive control.
*
GTP-induced assembly of purified porcine brain tubulin
9
(without microtubule-associated protein) as our assay,
we demonstrated that compound 1 inhibited tubulin
assembly in a dose-dependent manner (Fig. 4). Quanti-
tatively, we measured the extent of tubulin assembly
after 20 min incubation and determined that the IC50
of tubulin inhibition for compounds 1 and 2 were 9.1
and 18 lM, respectively (Table 1). Compounds with
substantially lower anti-proliferative effects on cancer
cells (compounds 3–6) have much lower inhibitory activ-
ities on tubulin assembly (IC50 > 40 lM). The IC50 of
0
0
shifts of C-2 and C-6 protons of 3-(substituted ben-
zyldenyl)indolin-2-ones were approximately 7.45–7.84
7
for the E isomers and 7.85–8.53 for the Z isomers.
Compounds 1–6 were examined for their anti-prolifera-
tive activities against three cancer cell lines which includ-
ed: androgen-independent prostate cancer cell lines (PC-
3
), and estrogen-dependent (MCF-7), and estrogen-in-
OCH
3
OH
H CO
3
3
CH O
H
H
O
N
3
CH O
CH O
3
H
OH
Combretastatin A-4
Compound 1
3
H CO
OCH
3
CH O
3
O
N
H
CH O
3
Compound 7
Figure 5. Illustration of the structural similarity between compound 1 and Combretastatin A-4, and the design of compound 7. The modeling work
was carried out on a SGI O workstation using SYBYL 6.9 modeling software (Tripos In., St. Louis, MO). The molecules 7 and Combretastain-A-4
were minimized using Tripos force field and Gasteiger–Huckel charges, using Powell method and an energy gradient of 0.005 kcal mol
2
ꢀ ꢀ1
1
˚
A .
Conformational search of Combretastain A-4 molecule with a 30ꢁ rotation increment of the C@C–C bonds produced 6 conformers in the energy
range of 44.34–34.59 kcal mol . Among the conformers, the lowest energy conformer of Combretastain A-4 was aligned with the compound 7 using
SYBYL 6.9.
ꢀ1

P.-K. Li et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5382–5385
5385
Table 2. Inhibition of cancer cell proliferation and tubulin polymerization by compounds 1 and 7
Compound
PC-3 (IC50 lM)
MCF-7(IC50 lM)
MDA-MB-231 (IC50 lM)
Inhibition of tubulin
polymerization (lM)
1
0.54 ± 0.2
0.0082
>50
0.39 ± 0.1
0.0007
>50
1.4 ± 1.0
0.0009
>50
9.1
4.5
7
SU5416
Podophyllotoxin
>40
1.56
0.013
Not tested
0.008
Cells (1000 cells/well) were treated with varying concentration of compounds and cell-associated protein was determined using MTS assay. The IC50
values represent means of three experiments in triplicate. Tubulin polymerization studies were determined as described in Ref. 9.
podophyllotoxin, a potent colchicine site agent, was
.5 lM under the same condition.
also exhibited potent inhibitory activities on tubulin
polymerization. The most potent compound in this
series, compound 7, structurally resembled the potent
anti-microtubule agent Combretastatin A-4.
1
By comparing the structures of compound 1 with those
of the colchicine site binders (Fig. 1), we observed a
structural similarity between compound 1 and Combre-
tastatin A-4, a potent inhibitor of tubulin polymeriza-
tion derived from the South African tree Combretum
References and notes
1
0
caffrum (Fig. 5). Thus, compound 7 was designed
as a rigid analog of Combretastatin A-4 (Fig. 5). In
addition, overlapping of the energy minimized struc-
tures of compound 7 and Combretastatin A-4 further
illustrates the structural similarity of the two molecules
1
. Wang, L. G.; Liu, X. M.; Kreis, W.; Budman, D. R.
Cancer Chemother. Pharmacol. 1999, 44, 355.
2. Wani, M. C.; Taylor, H. L.; Wall, M. E.; Coggon, P.;
McPhail, A. T. J. Am. Chem. Soc. 1971, 93, 2325.
3
4
5
. Schiff, P. B.; Fant, J.; Horwitz, S. B. Nature 1979, 277,
65.
. Uppuluri, S.; Knipling, L.; Sackett, D. L.; Wolff, J. Proc.
Natl. Acad. Sci. U.S.A. 1993, 90, 11598.
. Leoni, L. M.; Hamel, E.; Genini, D.; Shih, H.; Carrera, C.
J., et al. J. Natl. Cancer Inst. 2000, 92, 217.
6
(
Fig. 5).
Compound 7 was synthesized in a similar manner as
compound 1 by refluxing for 3 h of 6-methoxy-2-indoli-
none, 3,4,5-trimethoxybenzaldehyde, and piperidine
6
7
. Fahy, J.; Hill, B. T. Curr. Pharm. Des. 2001, 7, 1297.
. Sun, L.; Tran, N.; Tang, F.; App, H.; Hirth, P., et al.
J. Med. Chem. 1998, 41, 2588.
1
1
(
catalytic amount) in EtOH. The anti-proliferative
activities of compound 7 were examined on PC-3,
MCF-7, and MDA-MB-231 cancer cells via MTS assay.
Compound 7 exhibited extremely potent anti-prolifera-
tive activities against all three cell lines with IC50 values
of 8.2, 0.7, and 0.9 nM, respectively. In addition, it also
showed potent inhibitory activity against tubulin poly-
merization in purified porcine brain tubulin with an
IC50 of 4.5 lM (Table 2).
8. Quallich, G.; Morrissey, P. Synthesis 1993, 51.
9. Werbovetz, K. A.; Sackett, D. L.; Delfin, D.; Bhattach-
arya, G.; Salem, M.; Obrzut, T.; Rattendi, D.; Bacchi, C.
Mol. Pharmacol. 2003, 64, 1325.
1
0. Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts,
D. S., et al. Experientia 1989, 45, 209.
1. All the compounds showed satisfactory spectroscopic
1
1
data. Selected analytical data: Compound 7: H NMR
TM
(
OCH
300 MHz, DMSO-d
6
)
3.74 (3H, s, –OCH
), 3.81 (6H, s, OCH ), 6.43 (1H, d, J = 2.4 Hz, H-7),
3
), 3.78 (3H, s,
In conclusion, we have discovered a series of compounds
originally derived from the vascular endothelial growth
factor receptor tyrosine kinase inhibitor SU5416 with
potent anti-proliferative activities in both breast and
prostate cancer cell lines. In addition, the compounds
3
3
0
0
6.50 (1H, dd, J = 8.4, 2.4 Hz, H-5), 7.03 (2H, s, H-2 ,6 ),
7.40 (1H, s, vinyl-H), 7.67 (1H, d, J = 8.4 Hz, H-4), 10.54
+
Na] :
(1H, s, NH). HRMS: calculated for [C19
64.11554. Found: 364.11574.
H19NO
5
3