Design, synthesis, and biological evaluation of combretastatin nitrogen-containing derivatives as inhibitors of tubulin assembly and vascular disrupting agents

Article abstract of DOI:10.1016/j.bmc.2005.12.033

A series of analogs with nitro or serinamide substituents at the C-2′-, C-5′-, or C-6′-position of the combretastatin A-4 (CA4) B-ring was synthesized and evaluated for cytotoxic effects against heart endothelioma cells, blood flow reduction to tumors in

Full text of DOI:10.1016/j.bmc.2005.12.033

Bioorganic & Medicinal Chemistry 14 (2006) 3231–3244
Design, synthesis, and biological evaluation of
combretastatin nitrogen-containing derivatives as inhibitors
of tubulin assembly and vascular disrupting agents
Keith A. Monk,a Rogelio Siles,a Mallinath B. Hadimani,a Benon E. Mugabe,a
J. Freeland Ackley,a Scott W. Studerus,a Klaus Edvardsen,b Mary Lynn Trawick,a
Charles M. Garner,a Monte R. Rhodes,c George R. Pettitc and Kevin G. Pinneya,*
aBaylor University, Department of Chemistry and Biochemistry and The Center for Drug Discovery,
One Bear Place # 97348, Waco, TX 76798, USA
bDepartment of Cell and Molecular Biology, Section for Tumor Immunology, University of Lund, BMC: 112, 22184 Lund, Sweden
cCancer Research Institute, Arizona State University, Tempe, AZ 85287-2404, USA
Received 21 October 2005; revised 16 December 2005; accepted 20 December 2005
Available online 25 January 2006
Abstract—A series of analogs with nitro or serinamide substituents at the C-20-, C-50-, or C-60-position of the combretastatin A-4
(CA4) B-ring was synthesized and evaluated for cytotoxic effects against heart endothelioma cells, blood flow reduction to tumors in
SCID mice, and as inhibitors of tubulin polymerization. The synthesis of these analogs typically featured a Wittig reaction between a
suitably functionalized arylaldehyde and an arylphosphonium salt followed by separation of the resultant E- and Z-isomers. Several
of these nitrogen-modified CA4 derivatives (both amino and nitro) demonstrate significant inhibition of tubulin assembly as well as
cytotoxicity and in vivo blood flow reduction. 20-Aminostilbenoid 7 and 20-amino-30-hydroxystilbenoid 29 proved to be the most
active in this series. Both compounds, 7 and 29, have the potential for further pro-drug modification and development as vascular
disrupting agents for treatment of solid tumor cancers and certain ophthalmological diseases.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
interfere with pro-angiogenic factors or their receptors
and a number of antiangiogenic agents that inhibit
neovascularization in tumors are undergoing clinical
evaluation. AvastinTM (bevacizumab) has recently been
approved for treatment of colorectal cancer in combina-
tion with chemotherapy.5 Bevacizumab is a recombinant
humanized monoclonal IgG1 antibody that binds to vas-
cular endothelial cell growth factor (VEGF), which is
overexpressed in tumors, and blocks VEGF interaction
with its corresponding receptors on the surface of endo-
thelial cells. Bevacizumab reduces microvascular growth
and inhibits progression of metastatic disease.
Tumor vasculature is an important and rapidly emerging
target for anticancer therapy because of the requirement
of proliferating tumor cells for a functional network of
blood vessels and the profound differences between tu-
mor-associated and normal blood vessels.1–4 Tumor ves-
sels are highly disorganized, have an incomplete
underlying basement membrane, and exhibit increased
permeability. While the endothelial cells of normal tissues
are largely quiescent, those of tumor vessels are activated
and are more responsive to angiogenic cell signaling. Two
different strategies have been developed for blocking tu-
mor growth and metastasis through targeting the endo-
thelial cells of the tumor-associated microvasculature.
Much of the focus of research in this area is on agents that
In vascular disruption, the aim is to effect a rapid and
extensive disruption of the pre-established tumor vascu-
lature.6–8 Antivascular strategies are characterized by
secondary tumor cell death and by extensive tumor
necrosis. Vascular targeting can be subdivided into
two groups: ligand-directed vascular targeting agents
and small-molecule vascular disrupting agents. Both
groups aim to exploit the differences between normal
Keywords: Combretastatin; Tubulin; Inhibitors of tubulin assembly;
Anti-cancer; Pro-drug constructs; Vascular disrupting agents.
*
Corresponding author. Tel.: +1 254 710 4117; fax: +1 254 710
4272; e-mail: Kevin_Pinney@baylor.edu
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.12.033

3232
K. A. Monk et al. / Bioorg. Med. Chem. 14 (2006) 3231–3244
tissue and tumor vessels. Ligand-directed antivascular
agents or biologics include agents that use antibodies
and peptides to target toxins, procoagulants, and pro-
apoptotic agents to specific cell surface receptors and
biomolecules that are localized predominantly in endo-
thelial cells associated with tumors.
phosphatase
CA4
CA1
CA4P
CA1P
COLCHICINE
VINBLASTINE
endothelial cell membrane
Microtubules/Associated Proteins
Depolymerization of MT
Proteins
GEFs
Two classes of compounds show promise in pre-clinical
and clinical development as small-molecule vascular dis-
rupting agents (VDAs). Although the exact mechanism
of action has not been established, the flavenoid, 5,6-
dimethylxanthenone-4-acetic acid, DMXAA (AS1404),
acts indirectly on tumor cells to induce synthesis and re-
lease of tumor nuclear transcription factor (NFjB)
resulting in the production of tumor necrosis factor
(TNF) and other cytokines which act on tumor endothe-
lial cells to effect vascular shutdown and apoptosis.9–11
The second class of compounds includes a rapidly grow-
ing number of small-molecule, tubulin-binding agents
that interfere with microtubule dynamics and acutely
disrupt the tumor vasculature. Microtubules are essen-
tial cytoskeletal elements formed from the polymeriza-
tion of subunit dimers composed of the closely related
GTP-binding proteins a- and b-tubulin. Microtubules
can switch to a fast shrinking state (catastrophe) or tran-
sit to a ‘rescue’ phase characterized by polymerization.
This nonequilibrium dynamics is called dynamic insta-
bility and is powered by GTP hydrolysis. The dynamics
of microtubule assembly are further regulated by a rep-
ertoire of microtubule-binding proteins and complexes.
Dynamic instability is essential to microtubule function
which includes cytoskeletal architecture, intracellular
transport, cell migration, wound healing, and mitotic
spindle development for chromosome segregation and
cell division.12
alpha, beta tubulin
dimers & oligomers
GTP
RhoA-GDP
GDP
(inactive)
RhoA-GTP
(active)
GAPs
RhoA kinase (SAPK2, mDia)
*increased actinomyosin contractility
*increase in cell rounding
*increase in cell detachment
*apoptosis
*disruption of cell/cell junctions
*increased permeability
*myosin phosphorylation
*increase in cell motility
*increase in stress fibers
*increase in focal adhesions
*membrane blebbing
Figure 1. Proposed mechanism of vascular disruption by tubulin-
binding agents. Microtubule (MT) depolymerization results in the
release of microtubule-associated proteins leading to activation of
RhoA and its downstream effectors such as RhoA kinase. The
resulting cytoskeletal rearrangements lead to rapid vascular shutdown.
H CO
3
H CO
3
OR
OR
H CO
3
H CO
3
Agents such as colchicine and vinblastine that result in
microtubule depolymerization are known to cause vas-
cular disruption, but at concentrations that are too close
to their maximum tolerated dose. The colchicine-in-
duced depolymerization of microtubules activates the
small guanosine nucleotide triphosphatase, RhoA,
which is an intracellular coordinator of the cytoskeletal
rearrangement of microtubules and actin.13 RhoA-GDP
is activated by guanosine nucleotide exchange factors
(GEFs) that promote the exchange of GTP for GDP.
While the exact mechanism by which microtubule depo-
lymerization activates RhoA has not yet been estab-
lished, binding to microtubules inhibits a number of
proteins such as GEF-H1. Upon dissociation from
microtubules, GEF-H1 can activate RhoA.14,15 RhoA-
GTP in turn can activate a number of downstream effec-
tors such as RhoA kinase which is able to phosphorylate
myosin resulting in increased actinomyosin contractili-
ty.16,17 (Fig. 1)
OCH
OCH
3
3
OR
OCH
OCH
3
3
CA4, R= H
CA1, R= H
CA1P, R= PO Na
2
CA4P, R= PO Na
3
2
3
Figure 2. Combretastatin natural products and synthetic phosphate
salts.
than their maximum tolerated dose.20–23 Both com-
pounds are currently in human clinical trials. Upon
exposure to cultured endothelial cells, CA4P was con-
verted to CA4 by phosphatases and characteristics of
RhoA activation such as phosphorylation of myosin
light chain, an increase in stress fibers (bundles of
crosslinked actin filaments) and focal adhesions were
observed: myosin light chain was phosphorylated, and
there was an increase in stress fibers (bundles of cross-
linked actin filaments) and focal adhesions. Inhibitors
of RhoA-kinase and RhoA abolished these effects. Some
cells also exhibited membrane blebbing, a specialized
form of membrane protrusion, which was associated
with decreased cell viability.24
Combretastatin A-4 (CA4),18 and combretastatin A-1
(CA1),19 first isolated from the South African bush wil-
low tree, Combretum caffrum, bind to tubulin at the col-
chicine site and are very effective inhibitors of tubulin
polymerization. These compounds and their water-solu-
ble phosphate pro-drugs (Fig. 2) are potent antimitotic
agents and are very active VDAs at doses much lower
Treatment of SCID mice bearing hemangioendothelio-
ma with a single dose of CA1P (OXi4503) reduced tu-
mor blood flow by 50% in 1 h, and tumor vessel

K. A. Monk et al. / Bioorg. Med. Chem. 14 (2006) 3231–3244
3233
H CO
3
Bromination at the benzylic positions of 4-methyl-2-
nitroanisole and 4-methyl-3-nitroanisole gave com-
pounds 1 and 2, as previously reported.41 A considerable
amount of dibromide was formed if the reaction was
carried out for more than 32 h. After treatment with
PPh3, triphenylphosphonium bromides 3 and 4 were ob-
tained with yields in excess of 90%. A Wittig reaction of
3,4,5-trimethoxybenzaldehyde with phosphonium salts 3
and 4 afforded a mixture of Z- and E-stilbenes. The Z-
isomers 5a and 6a were separated from their respective
E-isomers 5b and 6b by column chromatography. The
Z-isomer in a dichloromethane solution and in the pres-
ence of UV–vis light has a tendency to isomerize to its
respective E-isomer as previously reported.51,52 The
stereochemical assignment of the stilbene structures
was based on the typical values of the coupling con-
stants for Z (J ꢀ 12 Hz) and E (J ꢀ 16 Hz). The nitro
stilbene derivatives 5a and 6a were reduced to the free
amines 7 and 8 by refluxing with sodium hydrosulfite
in a 3:1 mixture of acetone/water. The serinamide resi-
due was incorporated by reacting 7 and 8 with
FMOC-L-Ser (Ac) in the presence of DCC and HOBt
to yield compounds 9 and 10, respectively, which upon
further reaction with sodium hydroxide formed serina-
mide derivatives 11 and 12. For comparison purposes,
AC-7700 14, a compound previously reported by Aji-
nomoto,46 and its analog 13 were prepared by reacting
intermediates 12 and 11 with a dioxane solution of
4 N HCl. Similarly, nitrogen-containing derivatives (C-
2, C-50, and C-60, 26–32, Scheme 2) were synthesized
from commercially available isovanillin (3-hydroxy-4-
methoxybenzaldehyde) to incorporate structural motifs
reminiscent of both CA4 and AC-7739.
H CO
3
H CO
3
H CO
3
O
OCH
OCH
3
3
NH HCl
2
N
OH
NH HCl
H
OCH
OCH
3
3
2
AC-7739
AC-7700
AVE-8062
Figure 3. Synthetic aza-CA4 analogs.
permeability increased markedly.25 Endothelial cells of
the tumor vasculature underwent apoptosis resulting in
blood flow shutdown and massive tumor necrosis.
CA4P is well along in clinical development both as a
VDA for cancer chemotherapy as well as a potential
treatment agent for certain ocular diseases such as wet,
age-related macular degeneration.23,26,27 OXi4503 has
recently entered human clinical trials.27–30 The robust
biological activity as evidenced through clinical success
coupled with the structural simplicity of the combretast-
atins has inspired the synthesis of a growing library of
combretastatin analogs.31–40 These analogs typically in-
clude A-ring, bridge-modified, and B-ring alterations.
From this library, nitrogen-substituted analogs have
emerged as among the most potent biologically. For
example, the synthetic analog substituting nitrogen ami-
no for oxygen hydroxyl at C-30 of the B-ring is extremely
active in terms of inhibition of tubulin assembly and
in vitro cytotoxicity against selected human cancer cell
lines (Fig. 3).41,42 This compound was originally de-
scribed in the patent literature by Ajinomoto Inc.43 and
then presented publicly in 1998 by the Ajinomoto group44
and by Pinney and co-workers45 independently. A serina-
mide-based pro-drug (AVE-8062) developed by Ajinom-
oto has been introduced in clinical development by
Aventis Inc.46,47 Herein, we describe a series of B-ring
nitrogen-substituted combretastatin analogs in which
the A-ring and ethylene bridge characteristic of CA4 are
kept intact.
Accordingly, isovanillin was nitrated using previously
reported methods for the preparation of benzaldehydes
17–19 (Scheme 2).53 Subsequent conversion to the
TBS-protected compounds 20–22 was accomplished
using TBS-chloride, providing one partner for the requi-
site Wittig reaction. The 3,4,5-trimethoxybenzylphos-
phonium bromide, prepared according to methodology
reported by Pettit and coworkers,54 was reacted with
sodium hydride to form a brightly colored ylide at
0 ꢁC. The TBS-protected Z- and E-stilbenes 23–25 were
isolated following a Wittig reaction of the ylide with
compounds 20–22. Minor amounts of the TBS-protect-
ed 60-nitrostilbenes 25a and 25b were isolated, as a con-
sequence of TBS-deprotection under the reaction
conditions to yield the phenolic Z-stilbene 28 and its
corresponding E-isomer. The Z-20-nitroalkene 23a and
Z-50-nitro analog 24a were converted to the free phenols
26 and 27 by reaction with TBAF. Reduction of the ni-
tro functionality of compounds 26, 27, and 28 was
accomplished using sodium dithionite as previously
discussed.
2. Results and discussion
2.1. Design and synthesis
Encouraged by the promising biological activity dis-
played by certain combretastatins and their synthetic
analogs, a series of seventeen new nitrogen-substituted
CA4 analogs has been prepared by total synthesis.48–50
These new analogs contain nitro, amino, amino hydro-
chloride, or serinamide pro-drug moieties at the C-20,
C-50, or C-60 position on the B-ring. Nitrogen substitu-
tion at C-30 of the B-ring has been previously repor-
ted41–45. In order to incorporate structural functionality
deemed significant for enhanced tubulin activity, the
molecules maintain the trimethoxyphenyl A-ring and
cis-ethylene bridge of CA4 as well as oxygen functional-
ity at C-40 in terms of either a methoxy or hydroxyl
group. The compounds are grouped by C-40-methoxy
analogs (Table 1), C-30-hydroxy-C-40-methoxy analogs
(Table 2), and C-30-methoxy-C-40-hydroxy analogs
(Table 3). The synthesis of the first series of CA4 ana-
logs and serinamide derivatives is presented in Scheme 1.
The Z-60-amino CA4 analog 31 was found to slowly
isomerize with prolonged reaction times. The Z-20-ami-
no CA4 analog 29 was converted to the hydrochloride
salt 32 using a 4 N HCl-dioxane solution.
Scheme 3 details the analogous synthesis of nitro-
substituted combretastatin analogs from commercially

3234
K. A. Monk et al. / Bioorg. Med. Chem. 14 (2006) 3231–3244
Table 1. Inhibition of tubulin polymerization, cytotoxicity, and blood flow reduction for compounds 8–13
H CO
3
R
R
1
H CO
3
H CO
3
2
OCH
3
Compounda
R1
R2
Tubulin inhibition,
IC50 (lM)
MTT, IC50 in vitro
cytotoxicity (lM)
In vivo blood flow
shutdown (%)
1 h
5 days
10 mg/kg
100 mg/kg
CA4
CA1
CA4P
CA1P
AC-7739
AC-7700
8
H
OH
OH
OH
1.2 (1.2)c
1.9 (2)d
na
nab
na
na
na
na
na
10
70
na
na
36
0
na
na
88
H
OPO3Na2
OPO3Na2
OPO3Na2
NH3Cl
NH-SerHCl
NH2
0.8
3.2
na
0.0020
0.0046
na
na
1.3 (1)e
11.3
2.6 (1.2)c
9.6
99
H
na
na
100
100
50
H
na
na
H
0.8
5.0
2.0
10.0
na
0.0080
0.0140
0.0080
0.050
na
12
H
NH2
NH-Ser
H
7
11
1.4
na
25
24
22.8
NH-Ser
NH-SerHCl
H
43
na
13
H
>40
a CA4, CA1, CA4P, CA1P, AC-7739, AC-7700, 8, and 12 have all been previously synthesized and reported in the literature. They are included here
for the purpose of comparison (see Refs. 18–21,41,42,46).
b na, not analyzed in this study.
c Ref. 41.
d Ref. 19.
e Ref. 57.
Table 2. Inhibition of tubulin polymerization, cytotoxicity, and blood flow reduction for compounds 26–31
H CO
3
H CO
3
R
R
R
1
3
H CO
3
OH
2
OCH
3
Compound
R1
R2
R3
Tubulin inhibition,
IC50 (lM)
MTT, IC50 in vitro
cytotoxicity (lM)
In vivo blood flow
shutdown (%)
1 h
5 days
10 mg/kg
100 mg/kg
26
27
28
29
30
31
NO2
H
H
H
1.5
2.0
>44.8
8
0.14
1.3
0
0
4
1.5
0
NO2
H
H
H
NO2
H
>40
2.5
>44.8
1.24
44.8
11.14
>1.4
0.017
1.35
1.4
0
NH2
H
H
27.3
0
59.2
1.5
0
NH2
H
H
NH2
>20
6.2
H
0
Table 3. Inhibition of tubulin polymerization, cytotoxicity, and blood flow reduction for compounds 42–47
H CO
3
H CO
3
R
3
R
1
H CO
3
R
2
OCH
3
OH
Compound
R1
R2
R3
Tubulin inhibition,
IC50 (lM)
MTT, IC50 in vitro
cytotoxicity (lM)
In vivo blood flow
shutdown (%)
1 h
5 days
10 mg/kg
100 mg/kg
42
43
44
45
46
47
NO2
H
H
H
>40
>40
>40
>40
>40
>40
naa
na
na
0
na
0
NO2
H
H
>44.8
na
na
>1.4
na
H
NO2
H
na
na
0
na
na
0
NH2
H
H
na
NH2
H
H
NH2
>44.8
na
>1.4
na
H
na
na
a na, not analyzed.

K. A. Monk et al. / Bioorg. Med. Chem. 14 (2006) 3231–3244
3235
Br
PPh3, Br
R1
CH3
R1
R2
R1
R2
b
a
c
R2
OCH3
OCH3
OCH3
R1=NO2, R2=H
R1=H, R2= NO2
3, R1=NO2, R2=H
4, R1=H, R2= NO2
1, R1=NO2, R2=H
2, R1=H, R2= NO2
H3CO
OCH3
R2
H3CO
H3CO
R1
R2
H3CO
d
H3CO
e
5a, 6a
R1
R2
OCH3
R1
H3CO
OCH3
OCH3
OCH3
OCH3
7, R1=NH2, R2=H
5a, R1=NO2, R2=H
6a, R1=H, R2= NO2
5b, R1=NO2, R2=H
6b, R1=H, R2= NO2
g
8, R1=H, R2= NH2
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
R1
R1
R2
R1
R2
R1
R2
H3CO
g
f
H3CO
OCH3
OCH3
OCH3
OCH3
R2
OCH3
OCH3
OCH3
OCH3
9, R1=NH-Fmoc-Ser(Ac), R2=H
10, R1=H, R2= NH-Fmoc-Ser(Ac)
13, R1=NHSerHCl, R2=H
14, AC7700, R1=H, R2= NH-SerHCl
11, R1=NH-Ser, R2=H
12, R1=H, R2= NH-Ser
15, R1=NH2HCl, R2=H
16, AC7739 ,R1=H, R2= NH2HCl
Scheme 1. Synthesis of the first series of CA4 analogs and serinamide derivatives. Reagents and conditions: (a) NBS, AIBN, CCl4, reflux; (b) PPh3,
dichloromethane, reflux; (c) NaH, 3,4,5-trimethoxybenzaldehyde, dichloromethane, 0 ꢁC to rt; (d) Na2S2O4, acetone/water (3:1), 50 ꢁC; (e) DCC,
HOBt-H2O, Fmoc-L-Ser(Ac), DMF (anhydrous), rt; (f) NaOH, dichloromethane/CH3OH (1:1); (g) 4 N HCl/dioxane, dichloromethane, rt.
H3CO
H3CO
R2
R1
CHO
R1
CHO
R1
R3
OCH3
OTBS
R3
R2
R3
R2
a
b
R3
R2
R1
H3CO
H3CO
H3CO
+
OH
OCH3
OTBS
OCH3
OTBS
OCH3
H3CO
17, R1=NO2, R2= R3=H
18, R1= R3=H, R2=NO2
19, R1= R2=H, R3=NO2
20, R1=NO2, R2= R3=H
21, R1= R3=H, R2=NO2
22, R1= R2=H, R3=NO2
23a, R1=NO2, R2= R3=H
24a, R1= R3=H, R2=NO2
25a, R1= R2=H, R3=NO2
23b, R1=NO2, R2= R3=H
24b, R1= R3=H, R2=NO2
25b, R1= R2=H, R3=NO2
H3CO
H3CO
H3CO
H3CO
e
d
c
H3CO
H3CO
H3CO
R3
R2
R1
R3
R2
R1
R3
R2
R1
23a-25a
H3CO
H3CO
OH
OH
OH
OCH3
OCH3
OCH3
32, R1=NH2-HCl, R2= R3=H
29, R1=NH2, R2= R3=H
30, R1= R3=H, R2=NH2
31, R1= R2=H, R3=NH2
26, R1=NO2, R2= R3=H
27, R1= R3=H, R2=NO2
28, R1= R2=H, R3=NO2
Scheme 2. Preparation of the second series of combretastatin analogs from isovanillin. Reagents and conditions: (a) TBS-Cl, DMAP, Et3N,
dichloromethane, 0 ꢁC; (b) NaH, dichloromethane, 3,4,5-trimethoxybenzyltriphenylphosphonium bromide, 0 ꢁC to rt; (c) TBAF, dichloromethane,
0 ꢁC; (d) Na2S2O4, acetone/water (3:1), 50 ꢁC; (e) 4 N HCl/dioxane, dichloromethane, rt.
available vanillin (3-methoxy-4-hydroxybenzaldehyde).
Reversal of the hydroxyl and methoxy functionalities
deemed important at C-30 and C-40 in this series of com-
pounds was undertaken to gain additional insight into
the relative significance of maintaining oxygen function-
ality at the 30- and 40-positions while incorporating addi-
tional nitrogen-containing groups. The synthetic
strategy adopted was similar to that used for com-
pounds described in Scheme 2. Nitration of vanillin
was accomplished by reported methodologies to yield
compounds 33–35.55 The Z-nitro stilbenes 42–44 were
prepared in moderate to good yield following protec-
tion, Wittig reaction, and deprotection. The nitro func-
tionality of compounds 42–44 was once again reduced

3236
K. A. Monk et al. / Bioorg. Med. Chem. 14 (2006) 3231–3244
H3CO
R2
R1
CHO
R1
CHO
R3
OTBS
OCH3
R3
R2
b
a
R3
R2
R1
H3CO
H3CO
R3
R2
R1
H3CO
H3CO
+
OCH3
OCH3
OTBS
OCH3
OTBS
OH
H3CO
33, R1=NO2, R2= R3=H
34, R1= R3=H, R2=NO2
35, R1= R2=H, R3=NO2
36, R1=NO2, R2= R3=H
37, R1= R3=H, R2=NO2
38, R1= R2=H, R3=NO2
39a, R1=NO2, R2= R3=H
40a, R1= R3=H, R2=NO2
41a, R1= R2=H, R3=NO2
39b, R1=NO2, R2= R3=H
40b, R1= R3=H, R2=NO2
41b, R1= R2=H, R3=NO2
H3CO
H3CO
d
c
H3CO
H3CO
H3CO
H3CO
R3
R2
R1
R3
R2
R1
39a-41a
OCH3
OCH3
OH
OH
45, R1=NH2, R2= R3=H
46, R1= R3=H, R2=NH2
47, R1= R2=H, R3=NH2
42, R1=NO2, R2= R3=H
43, R1= R3=H, R2=NO2
44, R1= R2=H, R3=NO2
Scheme 3. Synthesis of the third series of combretastatin analogs from vanillin. Reagents and conditions: (a) TBS-Cl, DMAP, Et3N,
dichloromethane, 0 ꢁC; (b) NaH, dichloromethane, 3,4,5-trimethoxybenzyltriphenylphosphonium bromide, 0 ꢁC to rt; (c) TBAF, dichloromethane,
0 ꢁC; (d) Na2S2O4, acetone/water (3:1), 50 ꢁC.
with sodium dithionite to give Z-amino combretastatin
analogs 45–47.
shutdown assays. The activity of 7 is especially notewor-
thy in that a substituent at the 30-position of CA4 ana-
logs is often required for significant cytotoxic
activity.56 Selected biological data for reference com-
pounds, CA4, CA1, CA4P, AC-7739, AC-7700, 8, and
12, and compound 13, the hydrochloride salt of 11,
are also included in Table 1.
2.2. Biological evaluation
Biological evaluation of these new nitrogen-modified
CA4 analogs features both an in vitro and in vivo com-
ponent. Initially, compounds are screened for their abil-
ity to inhibit tubulin assembly in a cell-free assay using
purified tubulin. In addition, selected compounds are
evaluated in terms of cytotoxicity (MTT assay) and for
their ability to selectively impair blood flow to tumors
in SCID mice. The combination of enhanced activity
toward tubulin and in vivo blood flow capability has
proven to be indicative of pre-clinical efficacy and predic-
tive of potential clinical success. Accordingly, these assays
are key components of our drug design and development
program as it relates to vascular disrupting agents.
Amino and nitro substitutions in the B-ring of CA4 ana-
logs in which the 30-hydroxyl remains unmodified are
summarized in Table 2. CA4 analogs with a nitro moiety
at the 20- or 50-position of the B-ring (26 and 27, respec-
tively) were very effective in their ability to inhibit tubu-
lin assembly with IC50 values of 1.5 and 2.0 lM,
respectively, but were much less active in the MTT
and in vivo blood flow shutdown assays compared to
CA4P. A nitro substituent at the 60-position 28 led to
loss of the ability to inhibit tubulin polymerization.
Amino substitution at the 20-position 29 of the B-ring
gave an active compound that inhibited tubulin assem-
bly with an IC50 value of 2.5 lM and showed significant
activity in the MTT and in vivo blood flow shutdown
assays (Fig. 4). Substitution of an amino group at the
50-position gave compound 30 that was much less active.
The compound with an amino substituent at the 60-posi-
tion 31 demonstrated moderate potency in the inhibition
of tubulin assembly (IC50 = 6.2 lM) and significant
activity in the MTT assay, but no activity in the in vivo
blood flow shutdown assay.
Three compounds, CA4, CA1, and AC-7739, and their
water-soluble pro-drug derivatives, CA4P, OXi4503,
and AVE-8062 (AC-7700), serve as benchmark reference
compounds in order to evaluate new VDA candidates in
a pre-clinical sense. CA4, CA1, and AC-7739 all inhibit
tubulin assembly with IC50 values in the range of
1–2 lM, while CA4P and OXi4503 demonstrate the
ability to shutdown tumor blood flow on the order of
80–100% at a dose of 100 mg/kg.
The effects on biological activity of B-ring, nitrogen
monosubstitutions in CA4 analogs are summarized in
Tables 1–3. Previous studies have shown that amino
substitution at the B-ring, 30-position of CA4 results in
analogs (AC-7739, and its serinamide pro-drug,
AVE-8062) that are very biologically active. Introduc-
tion of an amino group in the B-ring, 20-position of a
combretastatin analog in which the 30-hydroxyl has been
removed, gave a Z-stilbenoid compound 7 with an intact
A-ring that inhibits tubulin polymerization with an
IC50 = 1.5 lM which is close to those of CA4 and
AC-7739 as shown in Table 1. The Z-20-aminostilbenoid
product 7 and its corresponding serinamide pro-drug 11
were also very active in the MTT and in vivo blood flow
Amino and nitro substitutions at the 20-, 50-, and 60-po-
sitions of the B-ring of CA4 in which the 30-hydroxyl
and the 40-methoxy groups have been switched are sum-
marized in Table 3. None of these compounds, 42–47,
demonstrated any activity in the inhibition of tubulin
polymerization assay. Compounds 43 and 46 were also
evaluated in the MTT and in vivo blood flow shutdown
assays, but exhibited no activity in either of these assays.
2.3. Conclusions
We were very encouraged to find that the application of
existing structure–activity relationships led to the

K. A. Monk et al. / Bioorg. Med. Chem. 14 (2006) 3231–3244
3237
Figure 4. Inhibition of tubulin polymerization by compound 29. (A) Kinetics of inhibition of microtubule assembly by increasing concentrations of
compound 29. Tubulin (1.0 mg/mL in 1.0 M glutamate buffer, pH 6.6) was incubated with DMSO for controls 1 and 2, or with compound 29 at
concentrations of 0.5, 1.0, 2.0, 2.5, 4.0, and 5.0 lM for 15 min at 30 ꢁC and cooled to 0 ꢁC for 15 min on ice. GTP (0.4 mM final concentration) was
added to the reaction mixture in a quartz microcuvette. Baseline was established at 350 nm and after 100 s, tubulin polymerization was induced by a
temperature jump to 30 ꢁC, and after 1500 s, depolymerization was induced by a drop in the temperature to 0 ꢁC. Turbidity readings were collected
every 10 s. (B) The IC50 value was obtained from a plot of percent tubulin polymerization against log concentration (M) of compound 29 at 30 ꢁC.
Inset: column scatter graph of the absorbance against compound 29 concentration in the reaction mixture. IC50 = 2.5 lM, log
IC50 SE = ꢁ5.596 0.03.
development of a small library of new, synthetic, nitro-
gen-bearing CA4 analogs from which two compounds, 7
and 29, emerged as potentially viable pre-clinical candi-
dates with respect to their potent inhibition of tubulin
assembly and their significant in vivo blood flow shut-
down capability. In fact, these compounds are compara-
ble in many ways to AVE-8062 (AC-7700) which is
currently in human clinical development. Collectively,
this new nitrogen-modified CA4 compound library
enhances the overall structure–activity relationship pro-
file that is known for the combretastatins and related
analogs. One pro-drug congener 11 is also shown to
have significant in vivo activity, and it is anticipated that
further pro-drug modification of the analogs may result
in improved VDAs with enhanced bioavailability.
from EM Science and visualized using UV irradiation,
PMA (phosphomolybdic acid) staining, or I2. Gas chro-
matography (Hewlett Packard 5890 Series II with a
SE-54 column) and/or gas chromatography mass spec-
trometry (Hewlett Packard GCD system with electron
impact ionization) were also used to follow the reac-
tions, unless otherwise noted. Flash chromatography
was performed with the indicated solvents using silica
gel (230–400 mesh) purchased from EM Science.
Characterization of the products was accomplished by
NMR spectroscopy using Bruker DPX-300 (300 MHz
¨
for 1H and 75 MHz for 13C) or Bruker AMX-360
¨
1
(360 MHz for H and 90 MHz for 13C) spectrometers
in deuterated chloroform with 0.03% TMS as the inter-
nal standard unless otherwise specified. Chemical shifts
are expressed in ppm (d), and peaks are listed as singlets
(s), doublets (d), triplets (t), quartets (q), broad singlet
(br s), multiplet (m), or combinations of each, with cou-
pling constants (J) expressed in Hertz. All 13C spectra
reported are decoupled.
3. Experimental
3.1. General procedures
Reactions which involved air or moisture sensitive re-
agents were performed in oven-dried glassware under
an argon atmosphere, unless otherwise stated. Solvents
were transferred using cannulas or syringes, which were
stored in an oven prior to use. Before use solvents for
chromatography and isolation (dichloromethane, ethyl
acetate, hexanes, and acetone) were purified by distilla-
tion. Anhydrous dichloromethane and diethyl ether
were purchased from Aldrich Chemical Company or
Fischer Scientific and used as received. All other com-
mercially available reagents were purchased from Al-
drich Chemical Company or ACROS Chemical and
used as received unless otherwise stated.
3.2. Synthesis of key intermediates and analogs
3.2.1. 4-Methoxy-2-nitrobenzyl bromide (1). To a well-
stirred solution of 4-methyl-3-nitroanisole (7.45 g,
44.1 mmol) in CCl4 (70 mL), AIBN (0.740 g, 4.41 mmol)
and NBS (8.73 g, 48.6 mmol) were added. After the reac-
tion mixture was heated at reflux for 16 h under nitrogen
atmosphere, water was added and the product extracted
from the aqueous phase with dichloromethane. The
resultant organic phase was washed with brine, dried un-
der sodium sulfate, and the solvent evaporated yielding a
solid, which was recrystallized (hexanes) to afford 7.90 g
1
of the bromide in a 73% yield. H NMR (300 MHz,
CDCl3): d 7.52 (d, J = 2.7, ArH, 1H), 7.43 (d, J = 8.6,
ArH, 1H), 7.11 (dd, J = 8.6, 2.7, ArH, 1H), 4.77
All reaction mixtures were magnetically stirred and
monitored by TLC on silica plates that were obtained

3238
K. A. Monk et al. / Bioorg. Med. Chem. 14 (2006) 3231–3244
(s, benzylic CH2, 2H), 3.86 (s, OCH3, 3H). DEPT 135
NMR (75 MHz, CDCl3): d 133.6 (CH), 120 (CH), 110
(CH), 56.0 (CH3), 29.1 (CH2). EIMS: m/z (% rel intensi-
ty, ion) 247 (4, M+ +2), 245 (M+, 4), 166 (100), 108 (20).
3.2.7.
4-tert-Butyldimethylsilyloxy-3-methoxy-2-nitro-
benzaldehyde (36). Yield (63%) of a yellow solid. Rf
(20% EtOAc/hexanes) = 0.60. 1H NMR (CDCl3,
300 MHz): d 9.81 (s, 1H, CHO), 7.59 (d, 1H, J = 8.4,
ArH), 7.07 (d, 1H, J = 8.4, ArH), 3.92 (s, 3H, OCH3),
1.03 (s, 9H, t-Bu CH3), 0.29 (s, 6H, CH3). 13C NMR
(CDCl3, 75 MHz): d 185.8, 155.8, 143.4, 127.1, 122.2,
121.2, 62.3, 25.5, 18.3, ꢁ4.4. EIMS: m/z (% rel intensity,
ion): 311 (M+), 254 (base peak).
3.2.2.
4-Methoxy-2-nitrobenzyltriphenylphosphonium
bromide (3). Bromide 1 (12.0 g, 48.8 mmol) and triphe-
nylphosphine (14.2 g, 53.6 mmol) were dissolved in
dichloromethane (150 mL). The reaction mixture was
heated at reflux for 16 h at which point water was added
and the product extracted by using dichloromethane.
The organic phase was washed twice with brine and
dried over magnesium sulfate. The resultant solid was
washed three times with diethyl ether to afford 23.4 g
of the product in a 95% yield. 1H NMR (300 MHz,
CDCl3): d 7.95 (dd, J = 8.4, 2.4, ArH, 1H), 7.75 (m,
ArH, 3H), 7.61 (m, ArH, 12H), 7.39 (d, J = 2.3, ArH,
1H), 7.09 (dd, J = 8.6, 2.6, ArH, 1H), 5.90 (d,
J = 14.3, benzylic CH2, 2H), 3.78 (s, OCH3, 3H).
3.2.8.
4-tert-Butyldimethylsilyloxy-3-methoxy-5-nitro-
benzaldehyde (37). Yield (64%) of a pale yellow oil. Rf
(25% EtOAc/hexanes) = 0.38. 1H NMR (CDCl3,
300 MHz): d 9.87 (s, 1H, CHO), 7.87 (d, 1H, J = 1.9,
ArH), 7.55 (d, 1H, J = 1.9, ArH), 3.94 (s, 3H, OCH3),
0.98 (s, 9H, t-Bu CH3), 0.25 (s, 6H, CH3). 13C NMR
(CDCl3, 75 MHz): d 189.1, 152.4, 145.2, 128.7, 121.1,
111.7, 55.9, 25.5, 18.7, ꢁ3.9.
3.2.9.
4-tert-Butyldimethylsilyloxy-3-methoxy-6-nitro-
3.2.3. General procedure for the preparation of TBS-
protected aldehydes. To a 250 mL round-bottomed flask
containing a stir bar and the appropriate nitro-substitut-
ed benzaldehyde was added approximately 0.3 equiva-
lents of DMAP, and dichloromethane (ꢂ20.0 mL) was
transferred to the flask by syringe. The solution was stir-
red and cooled to 0 ꢁC in an ice-water bath. Triethyl-
amine (1.1 equivalents) was added, followed by 1.2
equivalents of TBS chloride. The reaction was moni-
tored by TLC, and following completion quenched with
20.0 mL of water. The product was extracted with
dichloromethane (2· 10 mL), and the organic phases
combined. The solvent was removed in vacuo, and the
product was purified by flash chromatography.
benzaldehyde (38). Yield (63%) of a yellow solid. Rf
(15% EtOAc/hexanes) = 0.51. 1H NMR (CDCl3,
300 MHz): d 10.43 (s, 1H, CHO), 7.58 (s, 1H, ArH),
7.41 (s, 1H, ArH), 3.97 (s, 3H, OCH3), 1.01 (s, 9H,
t-Bu CH3), 0.23 (s, 6H, CH3). 13C NMR (CDCl3,
75 MHz): d 187.9, 155.4, 149.3, 143.7, 126.2, 116.9,
110.7, 56.2, 25.5, 18.5, ꢁ4.5. EIMS: m/z (% rel intensity,
ion): 311 (M+), 254 (base peak).
3.2.10.
(Z)-2-(20-Nitro-40-methoxyphenyl)-1-(3,4,5-tri-
methoxyphenyl)ethene (5a). 3,4,5-Trimethoxybenzalde-
hyde (8.27 g, 41.7 mmol) and 4-methoxy-2-nitrobenzyl-
triphenylphosphonium bromide 3 (19.3 g, 37.9 mmol)
were dissolved in 150 mL of anhydrous dichlorometh-
ane, and the solution was stirred under nitrogen atmo-
sphere. After the reaction mixture was cooled at 0 ꢁC,
NaH (5.05 g, 200 mmol) was added and the mixture
was stirred for 16 h at room temperature at which point,
water (ꢂ10 mL) was added until bubbling stopped. The
product was isolated from the aqueous phase with
dichloromethane, and the organic phase was washed
with brine and dried over sodium sulfate. After evapora-
tion of the solvent, the product was purified by flash
chromatography (15% EtOAc/hexanes) to afford 5.29 g
of the product in a 41% yield. 1H NMR (300 MHz,
CDCl3): d 7.62 (d, J = 2.6, ArH, 1H), 7.27 (d, J = 8.8,
ArH, 1H) , 7.04 (dd, J = 8.7, 2.7, ArH, 1H), 6.83 (d,
J = 12.1, C@CH, 1H), 6.65 (d, J = 12.0, C@CH, 1H),
6.31 (s, ArH, 2H), 3.89 (s, OCH3, 3H), 3.83 (s, OCH3,
3H), 3.65 (s, OCH3, 6H). GC–MS m/z (% rel intensity,
ion): 345 (64, M+), 196 (93), 181 (100), 149 (25), 122
(32).
3.2.4. 3-tert-Butyldimethylsilyloxy-4-methoxy-2-nitro-
benzaldehyde (20). Yield (86%) of a tan solid. Rf (25%
1
EtOAc/hexanes) = 0.32. H NMR (CDCl3, 300 MHz):
d 9.77 (s, 1H, CHO), 7.49 (d, 1H, J = 8.6, ArH), 7.05
(d, 1H, J = 8.6, ArH), 3.96 (s, 3H, OCH3), 0.94 (s, 9H,
t-Bu CH3), 0.21 (s, 6H, CH3). 13C NMR (CDCl3,
75 MHz): d 156.1, 186.2, 156.1, 137.8, 125.2, 120.9,
111.7, 100.0, 55.9, 25.5, 18.5, ꢁ4.1. EIMS: m/z (% rel
intensity, ion): 311 (M+), 75 (base peak).
3.2.5.
3-tert-Butyldimethylsilyloxy-4-methoxy-5-nitro-
benzaldehyde (21). Yield (78%) of a colorless oil. Rf
(25% EtOAc/hexanes) = 0.77. 1H NMR (CDCl3,
300 MHz): d 9.91 (s, 1H, CHO), 7.89 (d, 1H, J = 2.0,
ArH), 7.57 (d, 1H, J = 2.0, ArH), 4.03 (s, 3H, OCH3),
1.05 (s, 9H, t-Bu CH3), 0.28 (s, 6H, CH3). 13C NMR
(CDCl3, 75 MHz): d 188.9, 151.5, 150.1, 145.5, 131.6,
123.4, 119.6, 62.0, 25.5, 18.2, ꢁ4.6. EIMS: m/z (% rel
intensity, ion): 311 (M+), 254 (base peak).
3.2.11. General Wittig reaction conditions for the prep-
aration of TBS-protected (Z)- and (E)-stilbenes. To
a 100 mL round-bottomed flask containing a stir
bar was added approximately 1.1 equivalents of
3,4,5-trimethoxybenzyltriphenylphosphonium bromide
and 25 mL of anhydrous dichloromethane. The flask
was cooled to 0 ꢁC in an ice-water bath, and 7–8 equiv-
alents of sodium hydride were cautiously transferred to
the flask forming the brightly colored ylide. A previously
prepared solution of TBS-protected aldehyde (1.0
3.2.6.
3-tert-Butyldimethylsilyloxy-4-methoxy-6-nitro-
benzaldehyde (22). Yield (61%) of a yellow solid. Rf
(25% EtOAc/hexanes) = 0.71. 1H NMR (CDCl3,
300 MHz): d 10.40 (s, 1H, CHO), 7.61 (s, 1H, ArH),
7.38 (s, 1H, ArH), 3.97 (s, 3H, OCH3), 1.00 (s, 9H,
t-Bu CH3), 0.22 (s, 6H, CH3). 13C NMR (CDCl3,
75 MHz): d 187.7, 168.7, 154.4, 150.3, 125.7, 119.9,
108.0, 56.3, 25.5, 18.5, ꢁ4.5.

K. A. Monk et al. / Bioorg. Med. Chem. 14 (2006) 3231–3244
3239
equivalent in 20 mL of dichloromethane) was trans-
ferred to the round-bottomed flask using a cannula.
The reaction mixture was allowed to warm from 0 ꢁC
to room temperature. The reaction was monitored by
TLC and cautiously quenched with 5 mL of water (after
approximately 5–8 h). The products were isolated by
extraction with dichloromethane and purified with flash
chromatography using gradient elution.
(d, 1H, J = 12.0, vinyl CH), 6.83 (s, 1H, ArH), 6.61 (d,
1H, J = 12.0, vinyl CH), 6.31 (s, 2H, ArH), 6.08 (s,
1H, ArH), 3.99 (s, 3H, OCH3), 3.81 (s, 3H, OCH3),
3.64 (s, 6H, OCH3). 13C NMR (CDCl3, 75 MHz): d
152.8, 150.2, 145.4, 140.2, 137.6, 131.4, 130.6, 129.6,
126.6, 117.1, 107.3, 106.5, 60.9, 56.5, 55.9. Anal. Calcd
for C18H19NO7: C, 59.83; H, 5.30; N, 3.88. Found: C,
59.97; H, 5.20; N, 3.91.
3.2.12. (Z)-2-(20-Nitro-30-tert-butyldimethylsilyloxy-40-
methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)ethene (23a).
Yield (47%) of a yellow liquid. Rf (25% EtOAc/hex-
3.2.17. (E)-2-(30-Hydroxy-40-methoxy-60-nitrophenyl)-1-
(3,4,5-trimethoxyphenyl)ethene (25b)ꢀ. Yield (33%) of a
yellow solid. Rf (25% EtOAc/hexanes) = 0.16. 1H
NMR (CDCl3, 300 MHz): d 7.64 (s, 1H, ArH), 7.62
(d, 1H, J = 15.9, vinyl CH), 7.22 (s, 1H, ArH), 6.90 (d,
1H, J = 15.9, vinyl CH), 6.75 (s, 2H, ArH), 6.15 (s,
1H, ArH), 3.99 (s, 3H, OCH3), 3.91 (s, 6H, OCH3),
3.88 (s, 3H, OCH3). 13C NMR (CDCl3, 75 MHz): d
153.5, 150.3, 145.6, 140.0, 138.6, 132.8, 132.5, 129.3,
123.8, 113.1, 107.8, 104.1, 61.0, 56.5, 56.2.
1
anes) = 0.41. H NMR (CDCl3, 300 MHz): d 6.77 (s,
2H, ArH), 6.61 (d, 1H, J = 12.0, vinyl CH), 6.41 (s,
2H, ArH), 6.38 (d, 1H, J = 12.0, vinyl CH), 3.82 (s,
3H, OCH3), 3.81 (s, 3H, OCH3), 3.67 (s, 6H, OCH3),
0.94 (s, 9H, t-Bu CH3), 0.19 (s, 6H, CH3). 13C NMR
(CDCl3, 75 MHz): d 152.8, 149.9, 144.3, 137.4, 137.2,
133.6, 131.6, 122.8, 122.6, 122.1, 112.4, 106.1, 60.9,
55.8, 55.6, 25.6, 18.5, ꢁ4.3.
3.2.18. (Z)-2-(20-Nitro-30-methoxy-40-tert-butyldimethyl-
silyloxyphenyl)-1-(3,4,5-trimethoxyphenyl)ethene (39a).
Yield (21%) of a yellow oil. Rf (25% EtOAc/hex-
3.2.13. (E)-2-(20-Nitro-30-tert-butyldimethylsilyloxy-40-
methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)ethene (23b).
Yield (25%) of a yellow solid. Rf (25% EtOAc/hex-
1
anes) = 0.53. H NMR (CDCl3, 300 MHz): d 6.88 (d,
1
anes) = 0.29. H NMR (CDCl3, 300 MHz): d 7.23 (d,
1H, J = 8.5, ArH), 6.81 (d, 1H, J = 8.5, ArH), 6.63 (d,
1H, J = 12.0, vinyl CH), 6.40 (s, 2H, ArH), 6.37 (d, 1H,
J = 12.0, vinyl CH), 3.90 (s, 3H, OCH3), 3.82, (s, 3H,
OCH3), 3.68 (s, 6H, OCH3), 1.01 (s, 9H, t-Bu CH3),
0.20 (s, 6H, CH3). 13C NMR (CDCl3, 75 MHz): d 152.9,
148.7, 146.4, 143.1, 137.6, 133.9, 131.3, 125.4, 123.8,
122.7, 122.1, 106.1, 61.9, 60.9, 55.8, 25.5, 18.2, ꢁ4.6.
1H, J = 8.4, ArH), 6.94 (d, 1H, J = 15.9, vinyl CH),
6.93 (d, 1H, J = 8.7, ArH), 6.73 (d, 1H, J = 15.9, vinyl
CH), 6.66 (s, 2H, ArH), 3.89 (s, 6H, OCH3), 3.87 (s,
3H, OCH3), 3.86 (s, 3H, OCH3), 0.95 (s, 9H, t-Bu
CH3), 0.21 (s, 6H, CH3). 13C NMR (CDCl3, 75 MHz):
d 153.4, 150.0, 143.6, 138.4, 137.1, 132.3, 131.7, 122.6,
120.2, 118.1, 112.8, 103.8, 61.0, 56.1, 55.6, 25.6, 18.5,
ꢁ4.2.
3.2.19. (E)-2-(20-Nitro-30-methoxy-40-tert-butyldimethyl-
silyloxyphenyl)-1-(3,4,5-trimethoxyphenyl)ethene (39b).
Yield (30%) of a yellow solid. Rf (25% EtOAc/hex-
3.2.14. (Z)-2-(30-tert-Butyldimethylsilyloxy-40-methoxy-
50-nitrophenyl)-1- (3,4,5-trimethoxyphenyl)ethene (24a).
Yield (33%) of a yellow oil. Rf (25% EtOAc/hex-
1
anes) = 0.45. H NMR (CDCl3, 300 MHz): d 7.33 (d,
1H, J = 8.7, ArH), 6.96 (d, 1H, J = 16.2, vinyl CH),
6.96 (d, 1H, J = 8.7, ArH), 6.75 (d, 1H, J = 16.2, vinyl
CH), 6.66 (s, 2H, ArH), 3.91 (s, 3H, OCH3), 3.90 (s,
6H, OCH3), 3.87 (s, 3H, OCH3), 1.03 (s, 9H, t-Bu
CH3), 0.24 (s, 6H, CH3). 13C NMR (CDCl3, 75 MHz):
d 153.4, 148.8, 145.7, 143.0, 138.6, 132.3, 132.1, 123.3,
123.0, 121.1, 119.7, 103.9, 61.9, 60.9, 56.1, 25.6, 18.2,
ꢁ4.5.
1
anes) = 0.75. H NMR (CDCl3, 300 MHz): d 7.31 (d,
1H, J = 2.0, ArH), 6.93 (d, 1H, J = 2.0, ArH), 6.62 (d,
1H, J = 12.0, vinyl CH), 6.43 (s, 2H, ArH), 6.43 (d, 1H,
J = 12.0, vinyl CH), 3.90 (s, 3H, OCH3), 3.84 (s, 3H,
OCH3), 3.73 (s, 6H, OCH3), 0.94 (s, 9H, t-Bu CH3),
0.06 (s, 6H, CH3). 13C (CDCl3, 75 MHz): d 153.3,
150.2, 145.2, 143.8, 137.7, 133.2, 132.1, 131.8, 127.3,
125.1, 117.6, 105.8, 61.8, 60.9, 56.0, 25.5, 18.1, ꢁ4.8.
3.2.20. (Z)-2-(30-Methoxy-40-tert-butyldimethylsilyloxy-
50-nitrophenyl)-1-(3,4,5-trimethoxyphenyl)ethene (40a).
Yield (40%) of a yellow liquid. Rf (25% EtOAc/hex-
3.2.15. (E)-2-(30-tert-Butyldimethylsilyloxy-40-methoxy-
50-nitrophenyl)-1 -(3,4,5-trimethoxyphenyl)ethene (24b).
Yield (24%) of a yellow solid. Rf (25% EtOAc/hex-
1
anes) = 0.50. H NMR (CDCl3, 300 MHz): d 7.27 (d,
1
anes) = 0.58. H NMR (CDCl3, 300 MHz): d 7.54 (d,
1H, J = 1.9, ArH), 6.90 (d, 1H, J = 1.9, ArH), 6.58 (d,
1H, J = 12.1, vinyl CH), 6.51 (s, 2H, ArH), 6.43 (d, 1H,
J = 12.1, vinyl CH), 3.84 (s, 3H, OCH3), 3.73 (s, 6H,
OCH3), 3.59 (s, 3H, OCH3), 0.95 (s, 9H, t-Bu CH3),
0.16 (s, 6H, CH3). 13C NMR (CDCl3, 75 MHz): d 153.2,
151.0, 142.6, 138.4, 137.6, 132.0, 131.1, 129.6, 127.7,
117.0, 115.2, 105.9, 60.9, 56.0, 55.4, 25.6, 18.6, ꢁ4.2.
3.2.21. (E)-2-(30-Methoxy-40-tert-butyldimethylsilyloxy-
50-nitrophenyl)-1-(3,4,5-trimethoxyphenyl)ethene (40b).
Yield (33%) of a yellow solid. Rf (25% EtOAc/
1H, J = 2.2, ArH), 7.15 (d, 1H, J = 2.2, ArH), 6.96 (d,
1H, J = 16.2, vinyl CH), 6.86 (d, 1H, J = 16.2, vinyl
CH), 6.73 (s, 2H, ArH), 3.95 (s, 3H, OCH3), 3.93 (s,
6H, OCH3), 3.88 (s, 3H, OCH3), 1.05 (s, 9H, t-Bu
CH3), 0.26 (s, 6H, CH3). 13C (CDCl3, 75 MHz): d
153.5, 150.8, 145.5, 144.3, 138.5, 133.5, 132.1, 130.6,
125.4, 122.9, 114.9, 103.8, 61.8, 61.0, 56.2, 25.6, 18.3,
ꢁ4.5.
3.2.16. (Z)-2-(30-Hydroxy-40-methoxy-60-nitrophenyl)-1-
(3,4,5-trimethoxyphenyl)ethene (28). Yield (11%) of a
yellow solid. Rf (35% EtOAc/hexanes) = 0.21. 1H
NMR (CDCl3, 300 MHz): d 7.74 (s, 1H, ArH), 6.85
ꢀ
TBS-deprotection occurred in the reaction.

3240
K. A. Monk et al. / Bioorg. Med. Chem. 14 (2006) 3231–3244
hexanes) = 0.37. 1H NMR (CDCl3, 300 MHz): d 7.45 (d,
1H, J = 1.9, ArH), 7.14 (d, 1H, J = 1.9, ArH), 6.96 (d,
1H, J = 16.2, vinyl CH), 6.89 (d, 1H, J = 16.2, vinyl
CH), 6.73 (s, 2H, ArH), 3.92 (s, 9H, OCH3), 3.88 (s,
3H, OCH3), 0.98 (s, 9H, t-Bu CH3), 0.21 (s, 6H, CH3).
13C NMR (CDCl3, 75 MHz): d 153.4, 151.8, 142.7,
138.8, 138.2, 132.3, 130.1, 129.3, 125.9, 114.3, 112.1,
103.6, 60.9, 56.1, 55.7, 25.6, 18.6, ꢁ4.2.
J = 12.1, vinyl CH), 6.47 (s, 2H, ArH), 6.42 (d, 1H,
J = 12.1, vinyl CH), 5.97 (br s, 1H, OH), 3.94 (s, 3H,
OCH3), 3.85 (s, 3H, OCH3), 3.72 (s, 6H, OCH3). 13C
NMR (CDCl3, 75 MHz): d 153.2, 150.1, 142.6, 139.8,
138.0, 133.9, 132.4, 131.3, 126.8, 120.7, 117.3, 106.1,
62.6, 61.0, 56.1. Anal. Calcd for C18H19NO7: C, 59.83;
H, 5.30; N, 3.88. Found: C, 59.80; H, 5.27; N, 3.86.
3.2.27. (Z)-2-(20-Nitro-30-methoxy-40-hydroxyphenyl)-1-
(3,4,5-trimethoxyphenyl)ethene (42). Yield (73%) of a
yellow oil. Rf (50% EtOAc/hexanes) = 0.10. H NMR
3.2.22. (Z)-2-(30-Methoxy-40-tert-butyldimethylsilyloxy-
60-nitrophenyl)-1-(3,4,5-trimethoxyphenyl)ethene (41a).
Yield (64%) of a yellow liquid. Rf (15% EtOAc/hex-
1
(CDCl3, 300 MHz): d 6.94 (s, 2H, ArH), 6.62 (d, 1H,
J = 11.9, vinyl CH), 6.40 (d, 1H, J = 11.9, vinyl CH),
6.37 (s, 2H, ArH), 3.92 (s, 3H, OCH3), 3.82 (s, 3H,
OCH3), 3.67 (s, 6H, OCH3). 13C NMR (CDCl3,
75 MHz): d 152.9, 148.8, 144.8, 139.1, 137.7, 133.9,
131.3, 126.4, 123.2, 122.2, 118.0, 106.2, 62.7, 60.9, 55.9.
1
anes) = 0.33. H NMR (CDCl3, 300 MHz): d 7.67 (s,
1H, ArH), 6.90 (d, 1H, J = 12.0, vinyl CH), 6.66 (s,
1H, ArH), 6.65 (d, 1H, J = 12.0, vinyl CH), 6.33 (s,
2H, ArH), 3.80 (s, 3H, OCH3), 3.64 (s, 6H, OCH3),
3.57 (s, 3H, OCH3), 0.99 (s, 9H, t-Bu CH3), 0.17 (s,
6H, CH3). 13C NMR (CDCl3, 75 MHz): d 155.1,
152.9, 144.3, 140.5, 137.5, 131.7, 130.6, 128.8, 127.2,
117.0, 113.9, 106.2, 60.9, 55.9, 55.7, 25.6, 18.4, ꢁ4.7.
3.2.28. (Z)-2-(30-Methoxy-40-hydroxy-50-nitrophenyl)-1-
(3,4,5-trimethoxyphenyl)ethene (43). Yield (62%) of an
orange oil. Rf (35% EtOAc/hexanes) = 0.33. H NMR
1
3.2.23. (E)-2-(30-Methoxy-40-tert-butyldimethylsilyloxy-
60-nitrophenyl)-1-(3,4,5-trimethoxyphenyl)ethene (41b).
Yield (26%) of a yellow solid. Rf (15% EtOAc/hex-
(CDCl3, 300 MHz): d 7.64 (d, 1H, J = 1.9, ArH), 7.03
(d, 1H, J = 1.9, ArH), 6.64 (d, 1H, J = 12.1, vinyl
CH), 6.49 (s, 2H, ArH), 6.45 (d, 1H, J = 12.1, vinyl
CH), 3.84 (s, 3H, OCH3), 3.74 (s, 6H, OCH3), 3.65 (s,
3H, OCH3). 13C NMR (CDCl3, 75 MHz): d 153.3,
149.2, 145.6, 137.7, 133.6, 132.1, 131.4, 128.2, 127.5,
118.2, 116.5, 105.9, 61.0, 56.4, 56.1. Anal. Calcd for
C18H19NO7: C, 59.83; H, 5.30; N, 3.88. Found: C,
60.44; H, 5.40; N, 3.78.
1
anes) = 0.22. H NMR (CDCl3, 300 MHz): d 7.59 (s,
1H, ArH), 7.05 (s, 1H, ArH), 6.89 (d, 1H, J = 15.9, vinyl
CH), 6.76 (s, 2H, ArH), 6.63 (d, 1H, J = 15.9, vinyl CH),
3.97 (s, 3H, OCH3), 3.91 (s, 6H, OCH3), 3.88 (s, 3H,
OCH3), 1.02 (s, 9H, t-Bu CH3), 0.21 (s, 6H, CH3). 13C
NMR (CDCl3, 75 MHz): d 155.4, 153.5, 144.5, 140.3,
138.6, 132.5, 132.2, 128.8, 124.5, 117.4, 109.7, 104.1,
61.0, 56.2, 55.9, 25.6, 18.4, ꢁ4.6.
3.2.29. (Z)-2-(30-Methoxy-40-hydroxy-60-nitrophenyl)-1-
(3,4,5-trimethoxyphenyl)ethene (44). Yield (73%) of a
yellow solid. Rf (50% EtOAc/hexanes) = 0.52. 1H
NMR (CDCl3, 300 MHz): d 7.75 (s, 1H, ArH), 6.90
(d, 1H, J = 12.0, vinyl CH), 6.66 (s, 1H, ArH), 6.64 (d,
1H, J = 12.0, vinyl CH), 6.30 (s, 2H, ArH), 5.80 (br s,
1H, OH), 3.80 (s, 3H, OCH3), 3.67 (s, 3H, OCH3),
3.64 (s, 6H, OCH3). 13C NMR (CDCl3, 75 MHz): d
152.9, 150.3, 144.9, 141.3, 137.5, 131.7, 130.7, 127.4,
127.0, 113.0, 111.1, 106.3, 60.9, 56.4, 55.9. Anal. Calcd
for C18H19NO7: C, 59.83; H, 5.30; N, 3.88. Found: C,
59.79; H, 5.25; N, 3.89.
3.2.24. General conditions for TBS-deprotection of (Z)-
stilbenes. To a 100 mL round-bottomed flask containing
a stir bar and the appropriate TBS-protected nitro-
substituted (Z)-stilbene was added dichloromethane
(ꢂ20 mL). The flask was cooled to 0 ꢁC in an ice-water
bath, and 1.1 equivalents of TBAF (1.0 M in THF) was
added. The reaction mixture was stirred and typically
complete after 5–10 min as determined by TLC. Water
(5 mL) was added and the nitro-substituted combretast-
atin analog was extracted with dichloromethane. The
products were purified with flash chromatography.
3.2.30. General procedure for the reduction of the nitro
group for combretastatin analogs. A solution (acetone/
water, 3:1 ratio) of a (Z)-nitro-substituted combretasta-
tin analog was stirred, and the contents were heated in a
water bath at 50 ꢁC for 30 min before adding sodium
dithionite (8–10 equivalents). The reaction was held at
50 ꢁC and monitored by TLC. Following completion,
the reaction was worked up by adding a 10% sodium
bicarbonate solution (ꢂ10 mL) and extracting with ethyl
acetate (3· 10 mL). The organic phases were combined,
dried with sodium sulfate, and concentrated under re-
duced pressure.
3.2.25. (Z)-2-(20-Nitro-30-hydroxy-40-methoxyphenyl)-1-
(3,4,5-trimethoxyphenyl)ethene (26). Yield (47%) of a
yellow oil. Rf (25% EtOAc/hexanes) = 0.10. H NMR
1
(CDCl3, 300 MHz): d 6.92 (d, 1H, J = 8.4, ArH), 6.78
(d, 1H, J = 8.4, 0.9, ArH), 6.67 (d, 1H, J = 12.0, vinyl
CH), 6.58 (d, 1H, J = 12.0, vinyl CH), 6.31 (s, 2H,
ArH), 3.92 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.64
(s, 6H, OCH3). 13C NMR (CDCl3, 75 MHz): d 152.8,
148.2, 144.0, 137.4, 135.5, 131.6, 131.1, 126.1, 125.9,
121.9, 115.2, 106.2, 60.9, 56.7, 55.9. Anal. Calcd for
C18H19NO7: C, 59.83; H, 5.30; N, 3.88. Found: C,
59.91; H, 5.30; N, 3.96.
3.2.31. (Z)-2-(20-Amino-40-methoxyphenyl)-1-(3,4,5-tri-
methoxyphenyl)ethene (7). Yield (38%). 1H NMR
(360 MHz, CDCl3): d 7.03 (d, J = 8.4, ArH, 1H), 6.51
(s, ArH, 2H,), 6.49 (d, J = 11.9, C@CH, 1H), 6.42 (d,
J = 12.0, C@CH, 1H), 6.30 (dd, J = 8.4, 2.5, ArH,
1H), 6.25 (d, J = 2.4, ArH, 1H), 3.80 (s, OCH3, 3H),
3.2.26. (Z)-2-(30-Hydroxy-40-methoxy-50-nitrophenyl)-1-
(3,4,5-trimethoxyphenyl)ethene (27). Yield (40%) of a
yellow solid. Rf (25% EtOAc/hexanes) = 0.17. 1H
NMR (CDCl3, 300 MHz): d 7.39 (d, 1H, J = 2.0,
ArH), 7.16 (d, 1H, J = 2.0, ArH), 6.61 (d, 1H,

K. A. Monk et al. / Bioorg. Med. Chem. 14 (2006) 3231–3244
3241
3.75 (s, OCH3, 3H), 3.64 (s, OCH3, 6H), 1.56 (br s, NH2,
1H). 13C NMR (75 MHz, CDCl3): d 160.1, 152.7, 144.9,
137.2, 132.3, 131.0, 130.6, 125.7, 116.1, 105.8, 104.3,
100.7, 60.9, 55.8, 55.2. DEPT 90 NMR (75 MHz,
CDCl3): d 131.4, 131.0, 126.1, 106.1, 104.7, 101.1. DEPT
135 NMR (75 MHz, CDCl3): d 131.4 (CH), 131.0 (CH),
126.1 (CH), 106.1 (CH), 104.7 (CH), 101.1 (CH), 61.3
(CH3), 56.2 (CH3), 55.6 (CH3). EIMS: m/z (% rel inten-
sity, ion) 315 (100, M+), 300 (80), 142 (15). Anal. Calcd
for C18H21NO4: C, 68.55 H, 6.71 N, 4.44. Found: C,
68.49, H, 6.71, N, 4.25.
146.2, 137.0, 133.8, 133.0, 132.2, 130.3, 128.7, 128.4,
110.2, 106.0, 102.3, 60.9, 56.0, 55.9.
3.2.37. (Z)-2-(30-Methoxy-40-hydroxy-60-aminophenyl)-
1-(3,4,5-trimethoxyphenyl)ethene (47). Yield (68%) of a
yellow oil. Rf (50% EtOAc/hexanes) = 0.26. H NMR
1
(CDCl3, 300 MHz): d 6.63 (s, 1H, ArH), 6.53 (s, 2H,
ArH), 6.49 (d, 1H, J = 12.0, vinyl CH), 6.42 (d, 1H
J = 12.0, vinyl CH), 6.34 (s, 1H, ArH), 3.81 (s, 3H,
OCH3), 3.67 (s, 3H, OCH3), 3.66 (s, 6H, OCH3). 13C
NMR (CDCl3, 75 MHz): d 152.7, 146.1, 139.7, 138.4,
137.3, 132.2, 130.5, 125.7, 114.3, 112.5, 105.9, 102.8,
60.8, 56.6, 55.8.
3.2.32. (Z)-2-(20-Amino-30-hydroxy-40-methoxyphenyl)-
1-(3,4,5-trimethoxyphenyl)ethene (29). Yield (98%) of a
yellow oil. Rf (35% EtOAc/hexanes) = 0.26. H NMR
1
3.2.38. General procedure for the preparation of hydro-
chloride salts of amino combretastatin analogs. To a
round-bottomed flask containing a stir bar and a
(Z)-amino combretastatin analog, anhydrous dichloro-
methane (ꢂ10 mL) was added. The solution was stirred
at room temperature and a solution of 4 N HCl/dioxane
(3–4 equivalents) was added. The contents were stirred
overnight and the solvent was removed to give a brown
oil. The product was recrystallized and the salt was
collected.
(CDCl3, 300 MHz): d 6.68 (d, 1H, J = 8.4, ArH), 6.52
(s, 2H, ArH), 6.52 (d, 1H, J = 12.0, vinyl CH), 6.46 (d,
1H, J = 12.0, vinyl CH), 6.32 (d, 1H, J = 8.4, ArH),
3.85 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.64 (s, 6H,
OCH3). 13C NMR (CDCl3, 75 MHz): d 152.7, 145.7,
137.3, 132.6, 132.2, 132.1, 131.0, 125.7, 120.1, 117.6,
105.9, 101.1, 60.9, 56.1, 55.7.
3.2.33. (Z)-2-(30-Hydroxy-40-methoxy-50-aminophenyl)-
1-(3,4,5-trimethoxyphenyl)ethene (30). Yield (15%) of a
brown oil. Rf (50% EtOAc/hexanes) = 0.26. H NMR
1
3.2.39. (Z)-2-(20-Methoxy-40-aminophenyl)-1-(3,4,5-tri-
methoxyphenyl)ethene hydrochloride (15). Yield (83%)
of a yellow solid. 1H NMR (300 MHz, CDCl3): d
10.05 (3H, br s, NH3+), 7.18 (1H, d, J = 8.6, ArH),
7.14 (1H, br s, ArH), 6.73 (1H, d, J = 8.2, ArH), 6.71
(1H, d, J = 11.8, C@CH), 6.56 (1H, d, J = 11.8, C@CH),
6.37 (2H, s, ArH), 3.79 (3H, s, OCH3), 3.74 (3H, s,
OCH3), 3.60 (6H, s, OCH3). Anal. Calcd for
C18H22NO4Cl: C, 61.45 H, 6.30 N, 3.98. Found: C,
61.16, H, 6.39, N, 3.91.
(CDCl3, 300 MHz): d 6.54 (s, 2H, ArH), 6.43 (d, 1H,
J = 12.1, vinyl CH), 6.39 (d, 1H, J = 12.1, vinyl CH),
6.35 (d, 1H, J = 1.8, ArH), 6.27 (d, 1H, J = 1.8, ArH),
3.84 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 3.71 (s, 6H,
OCH3). Anal. Calcd for C18H21NO5: C, 65.24; H,
6.39; N, 4.23. Found: C, 64.55; H, 6.39; N, 4.24.
3.2.34. (Z)-2-(30-Hydroxy-40-methoxy-60-aminophenyl)-
1-(3,4,5-trimethoxyphenyl)ethene (31). Yield (47%) of a
yellow oil. Rf (50% EtOAc/hexanes) = 0.20. H NMR
1
(CDCl3, 300 MHz): d 6.74 (s, 1H, ArH), 6.53 (s, 2H,
ArH), 6.49 (d, 1H, J = 12.0, vinyl CH), 6.40 (d, 1H,
J = 12.0, vinyl CH), 6.28 (s, 1H, ArH), 3.83 (s, 3H,
OCH3), 3.82 (s, 3H, OCH3), 3.66 (s, 6H, OCH3). 13C
NMR (CDCl3, 75 MHz): d 152.7, 146.7, 138.3, 137.3,
136.9, 132.1, 130.9, 125.6, 115.8, 115.1, 105.8, 99.7,
60.9, 55.9, 55.8.
3.2.40. (Z)-2-(20-Amino-30-hydroxy-40-methoxyphenyl)-
1-(3,4,5-trimethoxyphenyl)ethene hydrochloride (32).
Yield (75%) of a tan powder. 1H NMR (D2O,
300 MHz): d 6.82 (d, 1H, J = 8.7, ArH), 6.60 (d, 1H,
J = 8.7, ArH), 6.57 (d, 1H, J = 12.0, vinyl CH), 6.42
(d, 1H, J = 12.0, vinyl CH), 6.20 (s, 2H, ArH), 3.68 (s,
3H, OCH3), 3.51 (s, 3H, OCH3), 3.39 (s, 6H, OCH3).
13C NMR (D2O, 75 MHz): d 155.2, 150.7, 143.0,
139.3, 136.9, 135.1, 128.0, 126.6, 124.0, 119.2, 115.4,
109.5 63.9, 59.5, 58.7.
3.2.35. (Z)-2-(20-Amino-30-methoxy-40-hydroxyphenyl)-
1-(3,4,5-trimethoxyphenyl)ethene (45). Yield (39%) of a
yellow oil. Rf (50% EtOAc/hexanes) = 0.17. H NMR
1
(CDCl3, 300 MHz): d 6.82 (dd, 1H, J = 8.2, 0.9, ArH),
6.51 (d, 1H, J = 11.9, vinyl CH), 6.50 (s, 2H, ArH),
6.42 (d, 1H, J = 11.9, vinyl CH), 6.36 (d, 1H, J = 8.2,
ArH), 3.81 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 3.64
(s, 6H, OCH3). 13C NMR (CDCl3, 75 MHz): d 152.8,
148.2, 137.5, 134.3, 132.2, 131.2, 125.5, 125.5, 116.5,
105.8, 105.4, 60.9, 59.7, 55.8.
3.2.41. (Z)-1-(20-Amino-40-methoxyphenyl)-2-(3,4,5-tri-
methoxyphenyl)ethene-FMOC-L-serinamide (9). To a
well-stirred solution of free amine 7 (0.996 g, 3.07 mmol)
in anhydrous DMF (10 mL), DCC (0.767 g, 3.68 mmol),
FMOC(Ac) serine (1.37 g, 3.68 mmol), and HOBtÆH2O
(0.508 g, 3.68 mmol) were added and the mixture was
stirred at room temperature for 16 h at which point
the solid formed was filtered and the filtrate was washed
5 times with water and twice with brine. After the organ-
ic phase was dried over sodium sulfate, the solvent was
evaporated and the crude product of the reaction was
purified by flash chromatography (35% EtOAc/hexanes)
to afford 1.13 g of the product in a 55% yield. 1H NMR
(300 MHz, CDCl3): d 8.11 (1H, br s, NH), 7.93 (1H, d,
J = 2.2, ArH), 7.79 (2H, d, J = 7.4, ArH), 7.59 (2H, d,
J = 6.5, ArH), 7.40 (2H, t, J = 7.5, ArH), 7.34 (2H, d,
3.2.36. (Z)-2-(30-Methoxy-40-hydroxy-50-aminophenyl)-
1-(3,4,5-trimethoxyphenyl)ethene (46). Yield (97%) of a
yellow oil. Rf (50% EtOAc/hexanes) = 0.27. H NMR
1
(CDCl3, 300 MHz): d 6.56 (s, 2H, ArH), 6.42 (d, 1H,
J = 12.1, vinyl CH), 6.39 (d, 1H, J = 1.8, ArH), 6.38
(d, 1H, J = 12.1, vinyl CH), 6.30 (d, 1H, J = 1.8,
ArH), 3.83 (s, 3H, OCH3), 3.72 (s, 6H, OCH3), 3.67
(s, 3H, OCH3). 13C NMR (CDCl3, 75 MHz): d 152.8,

3242
K. A. Monk et al. / Bioorg. Med. Chem. 14 (2006) 3231–3244
J = 7.0, ArH), 7.14 (1H, d, J = 8.6, ArH), 6.71 (1H, dd,
J = 8.6, 2.5, ArH), 6.35 (1H, d, J = 11.7, C@CH), 6.31
(1H, d, J = 11.7, C@CH), 6.23 (2H, s, ArH), 5.28 (1H,
d, J = 6.6, CH2), 4.51 (1H, dd, J = 12.9, 6.0, CH2),
4.40 (2H, br s, CH2), 4.20 (1H, d, J = 5.5, CH), 4.09
(1H, m, CH), 3.80 (3H, s, OCH3), 3.73 (3H, s, OCH3),
3.48 (6H, s, OCH3), 1.96 (3H, s, CH3). 13C NMR
(75 MHz, CDCl3): d 170.8, 166.3, 159.5, 152.9, 143.5,
141.3, 137.8, 135.0, 132.9, 131.1, 129.9, 127.9, 127.8,
127.1, 124.8, 124.7, 123.9, 120.0, 119.9, 111.3, 105.6,
105.5, 63.5, 60.7, 60.4, 55.6, 55.4, 47.1, 20.5, 14.2.
3.3. Biological assays
3.3.1. Tubulin polymerization assay. Tubulin was puri-
fied from calf brain according to the method of Hamel
and Lin.58 This method is based on six selective poly-
merization/cold depolymerization cycles. Tubulin puri-
ty was determined by SDS–PAGE and by its ability to
polymerize. The purified tubulin was flash-frozen and
stored in liquid nitrogen. The tubulin concentration
was determined by a two-component59 analysis using
the absorption measured at 278 and 255 nm, and
the following extinction coefficients: for tubulin,
1.2 L/g-cm at 278 nm (Harrison et al.)60 and 0.65 L/g-
cm at 255 nm; for GTP, 12.17 · 103 and 7.66 · 103
M/cm at 255 and 278 nm, respectively. Tubulin poly-
merization was induced in the presence of organic acid
(sodium glutamate) and GTP by a temperature jump
to 30 ꢁC according to the method of Verdier-Pinard
et al.61,62 Polymerization was followed turbidimetrical-
ly at 350 nm on an Agilent 8453 spectrophotometer
equipped with kinetics program software, a jacketed
cell holder, and two circulating water baths. To ice-
chilled tubulin at 1.0 mg/mL in 1.0 M glutamate, pH
6.6 (10 lM), pure DMSO (4% v/v final concentration)
was added for the control or 2.5–1000 lM of the ana-
log to be analyzed in DMSO, giving a final concentra-
tion of 0.1–40.0 lM compound in a total volume of
200 ll of reaction mixture. The mixture was incubated
at 30 ꢁC for 15 min and cooled at 0 ꢁC for 15 min. An
aliquot of 10 mM GTP (final concentration of
0.4 mM) was added to the reaction mixture on ice,
mixed, and immediately transferred into a pre-cooled
250 ll quartz microcuvette. The baseline was estab-
lished at 350 nm at 0 ꢁC and after 100 s, polymeriza-
tion was initiated by a temperature jump to 30 ꢁC.
After 1500 s, the cell was cooled to 0 ꢁC to induce
depolymerization. Turbidity (absorbance) readings
were recorded every 10 s. IC50 values of the various
analogs were determined from the data using nonlinear
regression analysis with the Prism software (Graph-
Pad) 3.02 version.
3.2.42. (Z)-2-(20-Amino-40-methoxyphenyl)-1-(3,4,5-tri-
methoxyphenyl)ethane-L-serinamide (11). FMOC(Ac)-L-
serinamide 9 (0.131 g, 0.196 mmol) was dissolved in
6 mL of a mixture of dichloromethane/MeOH (1:1 ratio)
and 0.22 mL of an aqueous solution of 2 N sodium
hydroxide (0.0176 g, 0.44 mmol) was added. After the
reaction mixture was stirred at room temperature for
18 h, dichloromethane (1 mL) was added and the organ-
ic phase was washed once with water, twice with brine
and dried under sodium sulfate. After the solvent was
evaporated, the resulting oil was purified by normal-
phase preparative TLC (95% dichloromethane/MeOH)
to obtain 0.0529 g of the product in a 67% yield.
1H NMR (300 MHz, CD2Cl2): d 9.66 (1H, br s, NH),
7.95 (1H, d, J = 2.6, ArH), 7.15 (1H, d, J = 8.5, ArH),
6.66 (1H, dd, J = 8.5, 2.6, ArH), 6.61 (1H, d, J = 12.0,
C@CH), 6.49 (1H, d, J = 12.1, C@CH), 6.40 (2H, s,
ArH), 3.79 (3H, s, OCH3), 3.70 (3H, s, OCH3), 3.64
(1H, dd, J = 10.7, 5.5, CH2OH), 3.56 (1H, dd,
J = 10.5, 5.6, CH2OH), 3.57 (6H, s, OCH3), 3.30 (1H,
t, J = 5.4, CHNH2), 2.07 (3H, br s, OH, NH2). 13C
NMR (75 MHz, CD2Cl2): d 172.2, 159.8, 153.2, 138.1,
136.3, 132.8, 132.2, 130.4, 124.7, 120.4, 110.5, 106.3,
106.0, 65.2, 60.8, 57.0, 56.1, 55.7. DEPT 45 NMR
(75 MHz, CD2Cl2): d 132.5, 130.0, 124.3, 110.1, 105.9,
105.7, 64.9, 60.4, 56.7, 55.7, 55.4. DEPT 90 NMR
(75 MHz, CDCl3): d 132.9, 130.5, 124.7, 111.3, 106.2,
105.7, 56.8. DEPT 135 NMR (75 MHz, CD2Cl2): d
132.5 (CH), 130.0 (CH), 124.3 (CH), 110.1 (CH), 105.9
(CH), 105.7 (CH), 64.9 (CH2), 60.4 (CH3), 56.7 (CH),
55.7 (CH3), 55.4 (CH3).
3.3.2. MTT assay. The MTT cell proliferation assay was
used to quantify the cell viability, measuring cell sur-
vival and proliferation spectrophotometrically.63 This
colorimetric assay measures the reduction of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT), reflecting the changes in cell proliferation.
The mechanism of this assay involves the cleavage of
the tetrazolium ring by active mitochondria in living
cells, producing a dark blue formazan product which
can be quantified in a rapid colorimetric fashion. The
reduction of MTT can only occur with living cells con-
taining active mitochondria. Comparison of the cells
treated with the drug to an untreated control group
provides the relative cytotoxicity, reflecting the loss of
cell viability as MTT reduction decreases. Heart endo-
thelioma cells (MHEC5-T) from mice were exposed to
serial dilutions of the reported compounds, and cell via-
bility was determined after incubation at 37 ꢁC at 1 h
and at 5 days by the MTT method, yielding the drug
concentration which reduced cell viability by 50% of
the control (IC50).
3.2.43. (Z)-2-(20-Amino-40-methoxyphenyl)-1-(3,4,5-tri-
methoxyphenyl)ethene-L-serinamide hydrochloride (13).
To a well-stirred solution of L-serinamide 11 (0.220 g,
0.546 mmol) in methanol (5 mL), 0.57 mL of a dioxane
solution of 4 N hydrochloric acid (0.0832 g, 2.28 mmol)
was added dropwise. After the reaction mixture was stir-
red at room temperature for 22 h, the solvent was re-
moved and the yellow oil formed was triturated with
chloroform at 0 ꢁC forming a solid which was filtered
and rinsed with chloroform (3 mL). Purification of the
salt by reverse-phase preparative TLC (87% dichloro-
methane/MeOH) afforded 0.0505 g in a 21% yield of the
1
L-serinamide salt. H NMR (300 MHz, methanol-d6): d
7.37 (1H, d, J = 2.0, ArH), 7.10 (1H, d, J = 8.5, ArH),
6.72 (1H, dd, J = 8.5, 2.3, ArH), 6.57 (1H, d, J = 11.9,
C@CH), 6.48 (1H, d, J = 12.0, C@CH), 6.43 (2H, s,
ArH), 4.06 (1H, br s, OH), 3.78 (2H, m, CH2), 3.74
(3H, s, OCH3), 3.66 (3H, s, OCH3), 3.55 (6H, s, OCH3).

K. A. Monk et al. / Bioorg. Med. Chem. 14 (2006) 3231–3244
3243
12. Desai, A.; Mitchison, T. J. Annu. Rev. Cell Dev. Biol. 1997,
13, 83.
13. Zheng, Y. Annu. Rev. Cell Dev. Biol. 2004, 20, 867.
14. Krendel, M.; Zenke, F. T.; Bokoch, G. M. Nat. Cell Biol.
2002, 4, 294.
15. Zenke, F. T.; Krendel, M.; DerMardirossian, C.; King, C.
C.; Bohl, B. P.; Bokoch, G. M. J. Biol. Chem. 2004, 279,
18392.
16. Burridge, K.; Wennerberg, K. Cell 2004, 116, 167.
17. Bayliss, K. J.; Davis, G. F. J Biol. Chem. 2004, 279, 11686.
18. Pettit, G. R.; Singh, S. B.; Boyd, M. R.; Hamel, E.; Pettit,
R. K.; Schmidt, J. M.; Hogan, F. J. Med. Chem. 1995, 38,
1666.
19. Pettit, G. R.; Singh, S. B.; Niven, M. L.; Hamel, E.;
Schmidt, J. M. J. Nat. Prod. 1987, 50, 119.
20. Pettit, G. R.; Temple, C., Jr.; Narayanan, V. L.; Varma,
R.; Simpson, M. J.; Boyd, M. R.; Rener, G. A.; Bansal, N.
Anti-Cancer Drug Des. 1995, 10, 299.
21. Pettit, G. R.; Lippert, J. W. Anti-Cancer Drug Des. 2000,
15, 203.
22. Chaplin, D. J.; Pettit, G. R.; Parkins, C. S.; Hill, S. A. Br.
J. Cancer 1996, 27, S86.
3.3.3. Blood flow reduction. In vivo experiments were
performed in the MHEC5-T tumor model established
by the injection of cultured MHEC5-T cells into the
right flank of SCID mice.25 When the established tumor
reached the size of 300 mm3 (a mass without the devel-
opment of necrosis), mice were injected ip with doses
of the various compounds at 100 or 10 mg/kg. At 24 h
after injection, the animals were injected in the tail vein
with 0.25 mL of diluted FluoSphere beads (1:6 in phys-
iological saline). The mice were sacrificed 3 min thereaf-
ter, and cryosections at a thickness of 8 lm were
removed from the tumor, heart, liver, spleen, and kid-
ney. Three control animals were tested for blood flow
reduction in tumor and control tissues only after being
injected with the vehicle without any reduction in blood
flow. These cryosections were directly examined under a
fluorescent microscope, providing a blue fluorescence
from the injected microbeads. The results were quanti-
fied from three sections of three tumors in each group
and in each section, recording more than 70% of the
area using a microscopic digital camera at 100· magni-
fication. The computer program Stage Pro (Media
Cybernetics, MD) was used to control the picture
recording, and image analysis was performed using Im-
age Plus software (Media Cybernetics, MD).
23. Young, S.; Chaplin, D. J. Expert Opin. Investig. Drugs
2004, 13, 1171.
24. Kanthou, C.; Tozer, G. M. Blood 2002, 99, 2060.
25. Sheng, Y.; Hua, J.; Pinney, K. G.; Garner, C. M.; Kane,
R. R.; Prezioso, J. A.; Chaplin, D. J.; Edvardsen, K. Int. J.
Cancer 2004, 111, 604.
26. Chaplin, D. J.; Hill, S. A. Int. J. Radiat. Oncol. Biol. Phys.
2002, 54, 1491.
27. Web link: www.oxigene.com (September 2005).
28. Hill, S. A.; Tozer, G. M.; Pettit, G. R.; Chaplin, D. J.
Anticancer Res. 2002, 22, 1453.
Acknowledgments
The authors are grateful to Oxigene Inc. (Waltham,
MA, grants to K.E., M.L.T., C.M.G., and K.G.P.),
Baylor University Research Committee (grants to
M.L.T. and K.G.P.), and The Welch Foundation
(Grant Nos. AA-1278 to K.G.P. and AA-1395 to
C.M.G.) for generous financial support of this project.
One of us (G.R.P.) also thanks for financial support
Grant RO1 CA-90441-01-04 awarded by the Division
of Cancer Treatment and Diagnosis, National Cancer
Institute, DHHS; The Arizona Disease Control Re-
search Commission; Dr. Alec D. Keith; and The Robert
B. Dalton Endowment Fund. The authors thank H&B
Packing (Waco, TX) for providing calf brain.
29. Hua, J.; Sheng, Y.; Pinney, K. G.; Garner, C. M.; Kane,
R. R.; Prezioso, J. A.; Pettit, G. R.; Chaplin, D. J.;
Edvardsen, K. Anticancer Res. 2003, 23, 1433.
30. Holwell, S. E.; Cooper, P. A.; Grosios, K.; Lippert, J. W.,
III; Pettit, G. R.; Shnyder, S. D.; Bibby, M. C. Anticancer
Res. 2002, 22, 3933.
31. Hadimani, M. B.; Hua, J.; Jonklaas, M. D.; Kessler, R. J.;
Sheng, Y.; Olivares, A.; Tanpure, R. P.; Weiser, A.;
Zhang, J.; Edvardsen, K.; Kane, R. R.; Pinney, K. G.
Bioorg. Med. Chem. Lett. 2003, 13, 1505.
32. Pinney, K. G.; Jelinek, C.; Edvardsen, K.; Chaplin, D. J.;
Pettit, G. P. In Antitumor Agents from Natural Products;
Kingston, D., Newman, D., Cragg, G., Eds.; CRC Press:
Taylor and Francis Group: Boca Raton, Florida, 2005; pp
23–46.
33. Chaplin, D. J.; Garner, C. M.; Kane, R. R.; Pinney, K. G;
Prezioso, J. A. PCT Int. Application 2003,
WO2003035008 A2.
34. Cushman, M.; Nagarathnam, D.; Gopal, D.; Chakraborti,
A. K.; Lin, C. M.; Hamel, E. J. Med. Chem. 1991, 34, 2579.
35. Hamel, E. Med. Res. Rev. 1996, 16, 207.
36. Li, Q.; Sham, H. L. Expert Opin. Ther. Pat. 2002, 12,
1663.
37. Pettit, G. R.; Singh, S. B. U.S. Patent 5,569,786, 1996.
38. Pinney, K. G.; Pettit, G. R.; Mocharla, V. P.; Mejia, M.
P.; Shirali, A. U.S. Patent 6,350,777, 2000.
39. Hsieh, H. P.; Liou, J. P.; Mahindroo, N. Curr. Pharm.
Des. 2005, 11, 1655.
References and notes
1. Neri, D.; Bicknell, R. Nat. Rev. Cancer 2005, 5, 436.
2. Tozer, G. M.; Kanthou, C.; Baguley, B. C. Nat. Rev.
Cancer 2005, 5, 423.
3. Gaya, A. M.; Rustin, G. J. S. Clin. Oncol. 2005, 17, 277.
4. Thorpe, P. E. Clin. Can. Res. 2004, 10, 415.
5. Ferrara, N.; Hilian, K. L.; Novotry, W. Biochem. Biophys.
Res. Commun. 2005, 333, 328–335.
6. Siemann, D. W.; Chaplin, D. J.; Horsman, M. R. Cancer
2004, 100, 2491.
7. Siemann, D. W.; Bibby, M. C.; Dark, G. G.; Dicker, A. P.;
Eskens, F. A. L. M.; Horsman, M. R.; Marme, D.;
LoRusso, P. M. Clin. Cancer Res. 2005, 11, 416.
8. Szala, S. Curr. Cancer Drug Targets 2004, 4, 501.
9. Baguley, B. C.; Wilson, W. R. Expert Rev. Anticancer
Ther. 2002, 2, 593.
40. Lawrence, N. J.; McGown, A. T. Curr. Pharm. Des. 2005,
11, 1679.
41. Pinney, K. G.; Mejia, M. P.; Villalobos, V. M.; Rosen-
quist, B. E.; Pettit, G. R.; Verdier-Pinard, P.; Hamel, E.
Bioorg. Med. Chem. 2000, 8, 2417.
10. Baguley, B. C. Lancet Oncol. 2003, 4, 141.
11. Zhou, S.; Kestell, P.; Baguley, B. C.; Paxton, J. W. Invest.
New Drugs 2002, 20, 281.
42. Ohsumi, K.; Nakagawa, R.; Fukuda, Y.; Hatanaka, T.;
Morinaga, Y.; Nihei, Y.; Ohishi, K.; Suga, Y.; Akiyama,
Y.; Tsuji, T. J. Med. Chem. 1998, 41, 3022.

3244
K. A. Monk et al. / Bioorg. Med. Chem. 14 (2006) 3231–3244
43. Ohsumi, K.; Tsuji, T.; Morinaga, Y.; Ohishi, K. U.S.
Patent 5,525,632, 1996.
53. Perez, R. A.; Fernandez-Alvarez, E.; Nieto, O.; Piedrafita,
F. J. J. Med. Chem. 1992, 3, 4584.
44. Ohsumi, K.; Nakagawa, R.; Hatanaka, T.; Morinaga, Y.;
Nihei, Y.; Ohishi, K.; Akiyama, Y.; Tsuji, T. Book of
Abstracts, 215th ACS National Meeting, Dallas, 1998;
MEDI-195.
45. Mejia, del P. M.; Pinney, K. G. Book of Abstracts, 215th
ACS National Meeting, Dallas, 1998; ORGN-042.
46. Ohsumi, K.; Hatanaka, T.; Nakagawa, R.; Fukuda, Y.;
Morinaga, Y.; Suga, Y.; Nihei, Y.; Ohishi, K.; Akiyama,
Y.; Tsuji, T. Anti-Cancer Drug Des. 1999, 14, 539.
47. Hori, K.; Saito, S. Br. J. Cancer 2003, 89, 1334–1344.
48. Siles, R.; Hadimani, M.; Pinney, K. G. 58th Southwest
Regional Meeting of The American Chemical Society,
(Abstract No. 236), Austin, TX, Nov 4, 2002.
49. Trawick, M. L.; Studerus, S. W.; Mugabe, B. E.; Garner,
C. M.; Kane, R. R.; Munk, K. A.; Pinney, K.; Siles, R.;
Zhang, J. FASEB J. 2003, 17, A1314, Abstract.
50. Trawick, M. L.; Mugabe, B. E.; Monk, K. A.; Garner, C.
M.; Pinney, K. G.; Siles, R.; Arthesary, P. FASEB J. 2004,
18, A839, Abstract.
54. Pettit, G. R.; Moser, B. R.; Boyd, M. R.; Schmidt, J. M.;
Pettit, R. K.; Chapuis, J. C. Anti-Cancer Drug Des. 2001,
16, 185.
55. Raiford, L. C.; Stoesser, W. C. J. Am. Chem. Soc. 1928,
50, 2556.
56. Pettit, G. R.; Rhodes, M. R.; Herald, D. L.; Hamel, E.;
Schmidt, J. M.; Pettit, R. K. J. Med. Chem. 2005, 48, 4087.
57. Hatanaka, T.; Fujita, K.; Ohsumi, K.; Nakagawa, R.;
Fukuda, Y.; Nihei, Y.; Suga, Y.; Akiyama, Y.; Tsuji, T.
Bioorg. Med. Chem. Lett. 1998, 3371–3374.
58. Hamel, E.; Lin, C. M. Biochemistry 1984, 23, 4173.
59. Barbier, P.; Peyrot, V.; Dumortier, C.; D’Hoore, A.; Rener,
G. A.; Engelborghs, Y. Biochemistry 1996, 35, 2008.
60. Harrisson, C. M. H.; Page, B. M.; Keir, H. M. Nature
1976, 260, 470–476.
61. Verdier-Pinard, P.; Lai, J.-Y.; Yoo, H.-D.; Yu, J.; Mar-
quez, B.; Nagle, D. G.; Nambu, M.; White, J. D.; Falck, J.
R.; Gerwick, W. H.; Day, B. W.; Hamel, E. Mol.
Pharmacol. 1998, 53, 62.
51. Waldeck, D. H. Chem. Rev. 1991, 91, 415.
52. Pettit, G. R.; Singh, S. B. Can J. Chem. 1987, 65, 2390.
62. Hamel, E. Cell Biochem. Biophys. 2003, 38, 1–21.
63. Mosmann, T. J. Immunol. Methods 1983, 65, 55–63.