Design, synthesis, biochemical, and biological evaluation of nitrogen-containing trifluoro structural modifications of combretastatin A-4

Article abstract of DOI:10.1016/j.bmcl.2008.07.070

A new trifluorinated amino-combretastatin analogue, (Z)-2-(4′-methoxy-3′-aminophenyl)-1-(3,4,5-trifluorophenyl)ethene, prepared by chemical synthesis, was found to be a potent inhibitor of tubulin assembly (IC₅₀ = 2.9 μM), and cytotoxic against selected human cancer cell lines. This new lead compound is among the most active from a group of related structural modifications.

Full text of DOI:10.1016/j.bmcl.2008.07.070

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5146–5149
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, biochemical, and biological evaluation of nitrogen-containing
trifluoro structural modifications of combretastatin A-4
John J. Hall a, Madhavi Sriram a, Tracy E. Strecker a, Justin K. Tidmore a, Christopher J. Jelinek a,
G. D. Kishore Kumar a, Mallinath B. Hadimani a, George R. Pettit b, David J. Chaplin c, Mary Lynn Trawick a,
Kevin G. Pinney a,
*
a Department of Chemistry and Biochemistry, Baylor University, One Bear Place #97348, Waco, TX 76798-7348, USA
b Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State University, PO Box 872404, Tempe, AZ 85287-2404, USA
c Oxigene Inc., 230 Third Avenue, Waltham, MA 02451, USA
a r t i c l e i n f o
a b s t r a c t
A new trifluorinated amino-combretastatin analogue, (Z)-2-(40-methoxy-30-aminophenyl)-1-(3,4,5-tri-
Article history:
Received 22 May 2008
Revised 15 July 2008
Accepted 16 July 2008
Available online 24 July 2008
fluorophenyl)ethene, prepared by chemical synthesis, was found to be a potent inhibitor of tubulin
assembly (IC50 = 2.9
lM), and cytotoxic against selected human cancer cell lines. This new lead com-
pound is among the most active from a group of related structural modifications.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Combretastatin
Inhibitors of tubulin assembly
Anti-cancer agents
The combretastatin family of compounds was isolated from the
African bush willow tree [Combretum caffrum Kuntze (Combreta-
ceae)] by Pettit and colleagues.1,2 The stilbenoids combretastatin
A-4 (CA4)3 and combretastatin A-1 (CA1)4 are potent inhibitors
of tubulin assembly into microtubules resulting in both antimitotic
activity and tumor vascular disruption. The corresponding CA4 and
CA1 phosphate prodrugs, CA4P5,6 and CA1P,7 are examples of clin-
ically relevant vascular disrupting agents (VDAs). VDAs selectively
hinder blood flow by occluding tumor vasculature, resulting in hy-
poxia and ultimately necrosis of tumors. Both CA4P (ZybrestatTM)
and CA1P (OXi4503) are currently in human clinical trials.8 These
compounds function through a proposed mechanism involving
vascular damage that has been described.9,10
Structure–activity relationship (SAR) studies of the Z-stilbenoid
combretastatin molecular scaffold led to the discovery of biologi-
cally active CA4 analogues, where an amine is substituted at the
20-position10 and the 30-position11–15 (Fig. 1). A review of the liter-
ature revealed that fluoro-substitutions can be well tolerated on
the combretastatin scaffold,16–24 and that a 3,4,5-trifluorinated
derivative of CA4, first reported by Hadfield and co-workers, inhib-
its tubulin polymerization.16 Accordingly, a design paradigm cen-
tered on trifluorinated nitrogen-substituted combretastatin
derivatives was utilized for the preparation of eight new analogues.
Of significance is the (Z)-2-(40-methoxy-30-aminophenyl)-1-(3,4,5-
trifluorophenyl)ethene analogue 8 which is an excellent inhibitor
of tubulin polymerization, and is moderately inhibitory against
both the NCI-H460 lung cancer and the DU-145 prostate cancer
cell lines. The excellent biochemical and biological activity of
compound 8 is significant and establishes it as a new lead com-
pound among analogues bearing nitrogen substitution on the
trifluorinated combretastatin platform. The synthesis of these
nitrogen-containing fluorinated combretastatin analogues as well
as preliminary biochemical and biological results are presented
herein.
The synthesis of newanalogues 2–9 utilized a Wittig reaction
as the key synthetic transformation. Commercially available
3,4,5-trifluorobenzyl bromide was reacted with triphenylphos-
phine to form the corresponding phosphonium bromide salt 1
(Scheme 1), which was treated with NaH followed by
Br
Br (Ph)3P
F
PPh3 / CH2Cl2
Reflux, 22 h
98 % yield
F
F
F
F
F
1
* Corresponding author. Tel.: +1 254 710 4117; fax: +1 254 710 4272.
E-mail address: Kevin_Pinney@baylor.edu (K.G. Pinney).
Scheme 1. Synthesis of phosphonium bromide salt 1.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.070

J. J. Hall et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5146–5149
5147
Wittig salt 1010a was treated with n-BuLi to generate the
ylide, which was then reacted with 4-methoxy-2-nitrobenzalde-
hyde to afford a mixture of Z- and E-combretastatin isomers (2
and 3, respectively) isolated in a 2.7:1 ratio (Scheme 2). The Z-
alkenes were separated from their corresponding E isomers using
flash column chromatography. Reduction of nitrostilbene ana-
logues 2–5 to their corresponding amines 6–9 was achieved by
treatment with zinc and glacial acetic acid at room temperature.
Interestingly, reduction of a mixture of nitrostilbenes 4 and 5
using iron and acetic acid with toluene as solvent afforded the
corresponding stilbenes 8 and 9 in much lower yields (11%
and 20%, respectively).
H3CO
H3CO
H3CO
H3CO
OH
OH
CH3O
CH3O
OH
OCH3
OCH3
CA4
CA1
ONa
H3CO
H3CO
H3CO
H3CO
O
P
ONa
O
O
O
P
ONa
CH3O
CH3O
P
O
ONa
O
For purposes of comparison, 20-amino-CA4 and F3-CA4, both of
which are excellent inhibitors of tubulin assembly, were evaluated
for inhibition against two cancer cell lines, and found to be active.
The series of eight new trifluorinated nitrogen-containing combre-
tastatin derivatives (2–9) was evaluated for their cytotoxicity
against NCI-H460 (lung) and DU-145 (prostate) human cancer cell
lines using a standard SRB assay.25 In addition, selected analogues
were screened for their ability to inhibit tubulin polymerization.26
The amino derivative 8 was an excellent inhibitor of tubulin
ONa
OCH3 ONa
OCH3
CA4P
CA1P
H3CO
H3CO
H3CO
H3CO
NH2
CH3O
CH3O
NH2
OCH3
OCH3
2'-NH2-CA4
polymerization having an IC50 value of 2.9
cancer cell growth inhibition (GI50 = 0.11
GI50 = 0.072 g/mL) against the NCI-H460 and the DU-145 cancer
lM, and demonstrated
3'-NH2-CA4
lg/mL, and
l
F
F
H3CO
H3CO
cell lines, respectively. The trifluorinated Z-stilbenes with nitro
substitution in either the 20- (analogue 2), or 30- (analogue 4) posi-
tion, and an amino substituent in the 20-position (analogue 6)
showed no inhibition against the NCI-H460 and the DU-145 hu-
F
CH3O
OH
OCH3
NO2
OCH3
man cancer cell lines (Table 1) at a concentration of 5 lg/mL. The
corresponding nitrogen-containing E-fluoro combretastatins (3, 5,
7, and 9) were not cytotoxic against these cancer cell lines (Table
2). In general, combretastatin analogues of the E-configuration re-
ported in the literature are inactive in these assays.9,14,16,27
Although, the trifluorinated-30-amino stilbene 8 inhibited tubu-
lin polymerization at a concentration comparable to that of 30-ami-
no-CA4 (Table 1), its cytotoxicity was somewhat diminished. It is
interesting to note that the cytotoxicity of F3-CA4 is also less than
the corresponding parent compound CA4.
3'-NO2-CA4
F3-CA4
Figure 1. Combretastatin A4, A1, and related analogues.
4-methoxy-3-nitrobenzaldehyde to afford (Z/E)-2-(40-methoxy-30-
nitrophenyl)-1-(3,4,5-trifluorophenyl)ethene (4 and 5) isolated in
a 2.7:1 ratio, respectively (Scheme 2).
PPh3Br
O2N
OCH3
F
NO2
1. n-BuLi /THF/ -30 ºC
F
F
NO2
F
2. 3,4,5-trifluorobenzaldehyde
-30 ºC to r.t. 12 h
F
OCH3
1010a
F
OCH3
2 (45% yield)
3 (17% yield)
PPh3Br
NO2
OCH3
F
1. NaH / CH2Cl2 / 0 ºC
F
F
2. 4-methoxy-3-nitro-
benzaldehyde,
1.5 h, 0 ºC
F
F
F
F
F
1
NO2
OCH3
F
5 (13% yield)
R2
4 (35% yield)
R1
OCH3
F
F
Zn (dust) / glacial AcOH
1 h, r.t.
R1
F
2 - 5
or
F
R2
OCH3
F
F
7, R1 = NH2, R2 = H (59% yield)
9, R1 = H, R2 = NH2 (84% yield)
6, R1 = NH2, R2 = H (68% yield)
8, R1 = H, R2 = NH2 (65% yield)
Scheme 2. Synthesis of E and Z nitrogen-containing stilbene analogues (2–9).

5148
J. J. Hall et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5146–5149
Table 1
Acknowledgments
Inhibition of microtubule formation and cytotoxicity for
Z nitrogen-containing
trifluorostilbenes 2, 4, 6, and 8
The authors are grateful to Oxigene Inc. (Waltham, MA, grants
to M.L.T. and K.G.P.) and The Welch Foundation (Grant AA-1278
to K.G.P.) for generous financial support of this research, and the
National Science Foundation for funding both the Varian
500 MHz NMR spectrometer (Award CHE-0420802) and the Bruker
X8 APEX diffractometer (Grant CHE-0321214). The authors are
grateful to Prof. Charles M. Garner, and Mr. Cody Carson, graduate
student, Department of Chemistry and Biochemistry, Baylor Uni-
versity, for assisting with structural analysis of selected com-
pounds. The authors thank Dr. Alejandro Ramirez (Mass
Spectrometry Core Facility, Baylor University) for HRMS analysis.
The authors also thank H&B Packing (Waco, TX) for providing calf
brain.
F
R1
F
F
R2
OCH3
Compound
R1
R2
Tubulin Inhibition
IC50
NCI-H460
DU-145
GI50 (lg/mL)
(
l
M)
GI50
(lg/mL)
2
4
6
8
NO2
H
NH2
H
—
—
H
NO2
H
NH2
—
—
nd
>40
nd
>5
>5
>5
>5
>5
4.3
2.9
0.11
0.072
CA4
1.2a
2.6b
1.4c
4.5d
0.0006a
0.00068b
0.0012
0.20
0.0008a
0.00096b
0.0016
0.040
30-NH2-CA4
20-NH2-CA4
F3-CA4
—
—
—
—
Supplementary data
Detailed experimental syntheses, 1H NMR, 13C NMR, 19F NMR,
HRMS, and HPLC data have been made available. Supplementary
data associated with this article can be found, in the online version,
at doi:10.1016/j.bmcl.2008.07.070.
nd, not determined.
a
Ref. 27.
Ref. 14.
Ref. 10a.
b
c
d
Refs. 16 and 28.
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E nitrogen-containing
R2
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OCH3
F
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Compound
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R2
H
Tubulin Inhibition
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DU-145
IC50
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nd, not determined.
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biochemical and biological evaluations. The activity of compound
8 is impressive and warrants its further development as a potential
VDA.
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