Design, synthesis, and biological evaluation of dibromotyrosine analogues inspired by marine natural products as inhibitors of human prostate cancer proliferation, invasion, and migration

Article abstract of DOI:10.1016/j.bmc.2010.08.057

Bioactive secondary metabolites originating from dibromotyrosine are common in marine sponges, such as sponges of the Aplysina species. Verongiaquinol (1), 3,5-dibromo-1-hydroxy-4-oxocyclohexa-2,5-diene-1-acetamide, and aeroplysinin-1 are examples of such

Full text of DOI:10.1016/j.bmc.2010.08.057

Bioorganic & Medicinal Chemistry 18 (2010) 7446–7457
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of dibromotyrosine analogues
inspired by marine natural products as inhibitors of human prostate
cancer proliferation, invasion, and migration
Asmaa A. Sallam a, Sindhura Ramasahayam b, Sharon A. Meyer b, Khalid A. El Sayed a,
⇑
a Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of Louisiana at Monroe, 1800 Bienville Drive, Monroe, LA 71201, USA
b Department of Toxicology, College of Pharmacy, University of Louisiana at Monroe, 1800 Bienville Drive, Monroe, LA 71201, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 July 2010
Revised 24 August 2010
Accepted 30 August 2010
Available online 29 September 2010
Bioactive secondary metabolites originating from dibromotyrosine are common in marine sponges, such
as sponges of the Aplysina species. Verongiaquinol (1), 3,5-dibromo-1-hydroxy-4-oxocyclohexa-2,5-
diene-1-acetamide, and aeroplysinin-1 are examples of such bioactive metabolites. Previous studies have
shown the potent antimicrobial as well as cytotoxic properties of verongiaquinol and the anti-angiogenic
activity of aeroplysinin-1. The work presented herein shows the design and synthesis of dibromotyro-
sine-inspired phenolic ester and ether analogues with anti-angiogenic, anti-proliferative and anti-migra-
tory properties and negligible cytotoxicity. Several analogues were synthesized based on docking
experiments in the ATP binding site of VEGFR2 and their anti-angiogenic potential and ability to inhibit
angiogenesis and prostate cancer proliferation, migration and invasion were evaluated using the chick
chorioallantoic membrane (CAM) assay, MTT, wound-healing, and CultrexÒ BME cell invasion assay mod-
els, respectively. Analogues with high docking scores showed promising anti-angiogenic activity in the
CAM assay. In general, ester analogues (5, 6, and 8–10) proved to be of higher anti-migratory activity
whereas ether analogues (11–14) showed better anti-proliferative activity. These results demonstrate
the potential of dibromotyrosines as promising inhibitory scaffolds for the control of metastatic prostate
cancer proliferation and migration.
Keywords:
Aeroplysinin-1
Angiogenesis
Dibromotyrosine
Invasion
Marine natural products
Migration
Proliferation
Prostate cancer
Verongiaquinol
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
(1), a 3,5-dibromo-1-hydroxy-4-oxocyclohexa-2,5-diene-1-acet-
amide, was shown to possess potent antimicrobial activity, against
a wide range of Gram positive and negative bacteria and fungi, as
well as cytotoxic properties.4,5 The cytotoxic effect of 1 was attrib-
uted to the formation of free radicals.2 The dienone structure pro-
vides the basis for this free radical formation. Previous reports of
the anti-angiogenic potential of aeroplysinin-1,6,8 isolated from
sponges of the Aplysina species, encouraged the molecular modeling
and docking studies of dibromotyrosines at the ATP binding site of
the vascular endothelial growth factor receptor 2 (VEGFR2) as one
of their possible molecular target(s) in an attempt to design and syn-
thesize a number of analogues with improved activity and low
cytotoxicity.
The role of VEGF/VEGFR axis in angiogenesis and cancer pro-
gression is well-documented.9–11 Angiogenesis is a tightly regu-
lated multistep process, whereby new capillaries sprout and
grow from existing blood vessels.9 In cancer, angiogenesis is crucial
for supplying growing tumors with a vasculature to provide nutri-
ents, oxygen, and remove waste products, as well as providing a
conduit for the dispersal of metastases.10,11 The tissue localization
of the VEGFRs and the tight control of angiogenesis makes VEGFR
modulation an attractive approach to selectively inhibit tumor
Marine sponges are extraordinary rich sources of biologically
active secondary metabolites, unrivaled by any other marine
organism.1 Brominated natural products are common in marine
sponges. The ecological function of these brominated natural prod-
ucts is not clear yet, but they may play a role in chemical defense
and deterrence.2 Sponges of the Aplysina species are characterized
by the presence of secondary metabolites that originate from
dibromotyrosine,1–3 such as aeroplysinin-1, fistularin-3, agelorins
A and B (Supplementary data, Fig. S1), verongiaquinol (1), as well
as many others. Many biological properties have been described
for these brominated secondary metabolites including antimicro-
bial activity,4,5 cytotoxicity against several tumor cell lines,2,4 inhi-
bition of the EGFR tyrosine kinase and anti-angiogenic
properties,6–8 which renders them an interesting class for further
investigation as potential anti-cancer scaffolds.
The structurally-simple dibromotyrosines, verongiaquinol and
aeroplysinin-1, inspired the work presented herein. Verongiaquinol
⇑
Corresponding author. Tel.: +1 318 342 1725; fax: +1 318 342 1737.
E-mail address: elsayed@ulm.edu (Khalid A. El Sayed).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.057

A. A. Sallam et al. / Bioorg. Med. Chem. 18 (2010) 7446–7457
7447
growth and metastasis, without affecting the surrounding normal
tissue.9 Although VEGF was initially thought to be an endothelial
cell-specific growth factor, recent reports have identified VEGF
receptors on non-endothelial cells.12 Liang et al., for example, have
shown that VEGF can induce proliferation and survival of breast
cancer cells expressing VEGFR2 (flk) through the MAPK/ERK signal-
ing pathway.12 Targeting the VEGF receptor and/or its downstream
signaling pathways would block the VEGF-dependent proliferation
and angiogenesis of tumor cells.
The work presented herein shows the design and synthesis of
dibromotyrosine analogues as anti-angiogenic agents and inhibi-
tors of human prostate cancer proliferation, invasion, and migra-
tion with negligible cytotoxicity.
human prostate cancer cell line, PC-3 and for anti-angiogenic activ-
ity in the chick chorioallantoic membrane (CAM) assay. In general,
ester analogues were more active as anti-migratory agents
whereas ether analogues proved to be more active in the prolifer-
ation assay. Also, consistent with in silico data, those analogues
with higher docking scores showed promising anti-angiogenic
activity in the CAM assay. Unlike verongiaquinol, none of the ana-
logues showed any evidence of cytotoxicity towards bone marrow
myeloid progenitor cells (GM-CFC). The GM-CFC assay was con-
ducted using a commercial colony forming assay (HALOÒ kit) to
assess the cytotoxic potential of various analogues against a non-
cancerous cell type often affected by cancer chemotherapeutics.
2.1. Chemistry
2. Results and discussion
Six new and one known ester analogues (4–10) were synthe-
sized via the DMAP-catalyzed nucleophilic addition reaction of 2
and 3 with alkyl, aryl and heterocyclic acid chlorides to generate
the corresponding esters in 37–82% yields (Scheme 1).15,16
Analogues were designed based on docking experiments at the
ATP binding site of the vascular endothelial growth factor receptor
2 (VEGFR2). Two intermediates, 2 and 3, from the synthetic scheme
of verongiaquinol (Supplementary data, Scheme S1) were selected
as starting scaffolds for the generation of new analogues. These
simple dibrominated phenolic scaffolds are common in drug dis-
covery programs. In fact, there is plenty of literature describing
brominated phenolic and polyphenolic compounds as promising
anti-cancer candidates currently under further development.13,14
Two sets of reactions, guided by docking studies, were carried
out (Schemes 1 and 2). The first reaction set included the esterifi-
cation of the phenolic hydroxyl group with a variety of alkyl and
aryl acid chlorides, using N,N-dimethylaminopyridine (DMAP) as
a catalyst (Scheme 1).15,16 The other reaction set included the
etherification of the same phenolic hydroxyl group using a variety
of alkyl and aryl bromides in the presence of NaH (Scheme 2).16,18
Analogues were then screened for biological activity in the MTT,
wound-healing and CultrexÒ BME cell invasion assays against the
The HREIMS of 4 showed an [M+H]+ peak at m/z 349.9166, sug-
gesting the molecular formula C10H9Br2NO3 and a possible C-1
acetyl ester analogue of 2. Further investigation of 1H and 13C
NMR data proved that 4 is a known natural product that was pre-
viously isolated from the marine sponge Suberea aff. Praetensa.17
The HREIMS, 1H and 13C NMR data of 5 (Tables 1 and 2) sug-
gested the molecular formula C15H11Br2NO3 and possible C-1 ben-
zoate ester analogue of 2. The upfield shift of C-1 (
D
d ꢀ4.4) as well
as the downfield shifts of C-4 ( d +6.0) and C-2/6 (
D
D
d +7.1), as
compared to the parent 2 (Table S1), provided the evidence for
C-1 benzoylation. Furthermore, the aromatic methine doublet H-
30 (dH 8.20) showed 3J-HMBC correlations with the quaternary car-
bonyl carbon C-10 (dC 163.6) and the aromatic carbon C-50 (dC
134.4), confirming the presence of an intact benzoyl moiety. Thus,
compound 5 was determined to be 2,6-dibromo-1-benzoyloxyben-
zene-4-acetamide.
The HREIMS, 1H and 13C NMR data of 6 (Tables 1 and 2) sug-
gested the molecular formula C17H15Br2NO3 and possible C-1 2-
(4-methoxyphenyl)acetic acid ester analogue of 2. The methoxy
protons singlet H3-100 (dH 3.77) showed a 3J-HMBC correlation with
the quaternary aromatic carbon C-60 (dC 159.1). The aromatic
methine doublet H-40 (d 7.29) showed 3J-HMBC correlations with
C-60 and methylene carbon C-20 (dC 40.0). The methylene protons
H2-20 (dH 3.87) showed 3J-HMBC correlations with the quaternary
carbonyl carbon C-10 (dC 168.7), the aromatic quaternary carbon
C-30 (dC 124.6) and the aromatic methane carbons C-40/80 (dC
130.8). Thus, compound 6 was determined to be 2,6-dibromo-1-
(p-methoxyphenylacetoxy)benzene-4-acetamide.
OR1
OH
Br
Br
O
Br
Br
O
DMAP/CH2Cl2
R1-Cl
Reflux, 2-12 h
R2
R1
O
O
O
O
R1=
O
The HREIMS, 1H and 13C NMR data of 7 (Tables 1 and 2) sug-
gested the molecular formula C12H12Br2O4 and possible C-1 acetyl
ester analogue of 3. The 3J-HMBC correlation between the methyl
protons H3-20 (dH 2.38) and the quaternary carbonyl carbon C-10
N
R2 = NH2; EtO-
Scheme 1. General synthetic scheme for ester analogues 4–10.
OR
OH
Br
Br
O
Br
Br
O
NaH/DMF
R-Br, rt, overnight
O
O
O
R =
Scheme 2. General synthetic scheme of ether analogues 11–14.

7448
A. A. Sallam et al. / Bioorg. Med. Chem. 18 (2010) 7446–7457
Table 1
1H NMR Data of compounds 5–10a
Position
dH
5
6
7
8
9
10
3/5
7
7.51, s
3.48, s
7.44, s
3.44, s
7.49, s
3.54, s
7.55, s
3.58, s
7.46, s
3.53, s
7.56, s
3.59, s
9
—
—
—
—
—
—
—
3.87, s
4.16, q (7.0)
1.26, dd (7.3)
—
2.38, s
—
—
—
—
—
4.18, q (7.3)
1.28, dd (7.4)
—
—
8.25, dd (7.0, 1.4)
7.54, dd (7.7, 1.5)
7.67, dd (7.7, 1.1)
7.54, dd (7.7, 1.5)
8.25, dd (7.0, 1.4)
—
4.15, q (7.0)
1.25, dd (7.0)
—
3.89, s
—
7.32, dd (8.8, 2.2)
6.89, dd (8.8, 2.2)
—
6.89, dd (8.8, 2.2)
7.32, dd (8.8, 2.2)
3.80, s
4.18, q (7.2)
1.28, dd (7.2)
—
—
8.04, dd (5.9, 1.2)
8.89, dd (5.9, 1.2)
—
8.89, d (5.9)
8.04, d (5.9)
—
—
10
10
20
30
8.20, d (8.0)
7.50, dd (7.7)
7.64, dd (7.3)
7.50, dd (7.7)
8.20, d (8.0)
—
—
40
7.29, d (8.4)
6.86, d (8.8)
—
6.86, d (8.8)
7.29, d (8.4)
3.77, s
50
60
70
80
—
—
100
—
—
a
In CDCl3, 400 MHz. Coupling constants (J) are in Hertz.
Table 2
13C NMR Data of compounds 5–10a
Position
dC
5
6
7
8
9
10
1
145.5, qC
117.9, qC
133.3, CH
135.7, qC
41.4, CH2
172.9, qC
—
145.3, qC
117.8, qC
133.3, CH
135.6, CH
41.5, CH2
172.5, qC
—
145.4, qC
145.6, qC
145.3, qC
117.6, qC
133.2, CH
134.7, qC
40.1, CH2
170.3, qC
61.5, CH2
14.2, CH3
168.2, qC
40.1, CH2
124.8, qC
130.9, CH
114.1, CH
159.0, qC
114.1, CH
130.9, CH
55.4, CH3
145.0, qC
117.4, qC
133.4, CH
135.3, qC
40.1, CH2
170.2, qC
61.5, CH2
14.2, CH3
161.9, qC
135.7, qC
123.5, CH
151.0, CH
—
2/6
3/5
4
7
8
117.6, qC
133.3, CH
134.7, qC
40.1, CH2
170.3, qC
61.5, CH2
14.2, CH3
167.4, qC
20.6, CH3
—
—
—
—
—
—
—
117.8, qC
133.3, CH
134.8, qC
40.1, CH2
170.4, qC
61.5, CH2
14.3, CH3
163.2, qC
128.4, qC
130.7, CH
128.8, CH
134.2, CH
128.8, CH
130.7, CH
—
9
10
10
20
30
40
50
60
70
80
90
—
—
163.6, qC
128.2, qC
130.6, CH
128.8, CH
134.4, CH
128.8, CH
130.6, CH
—
168.7, qC
40.0, CH2
124.6, qC
130.8, CH
114.1, CH
159.1, qC
114.1, CH
130.8, CH
55.3, CH3
151.0, CH
123.5, CH
—
—
—
—
qC = quaternary, CH = methine, CH2 = methylene, CH3 = methyl carbons.
a
In CDCl3, 100 MHz. Carbon multiplicities were determined by APT experiments.
(dC 167.4) supported the presence of the acetate ester. Thus, com-
pound 7 was determined to be 2,6-dibromo-1-acetoxybenzene-4-
acetic acid ethyl ester.
Table 3
1H NMR Data of compounds 11–14a
The HREIMS, 1H and 13C NMR data of 8 (Tables 1 and 2) sug-
gested the molecular formula C17H14Br2O4 and possible C-1 benzo-
ate ester analogue of 3. The assignment of benzoate ester was
based on HMBC data as previously described in 5. Thus, compound
8 was determined to be 2,6-dibromo-1-benzoyloxybenzene-4-ace-
tic acid ethyl ester.
Position
dH
11
12
13
14
3/5
7
9
7.43, s
7.47, s
7.42, s
3.51, s
4.16, q (7.2)
1.26, t (7.2)
4.01, t (6.4)
2.15, m
2.83, t (7.9)
—
7.17, d (8.8)
6.84, d (8.4)
—
6.84, d (8.4)
7.17, d (8.8)
—
3.79, s
7.42, s
3.50, s
4.16, q (7.2)
1.26, t (7.3)
4.00, t (6.8)
1.75, q (13.6)
1.92, m
0.98, d (6.6)
0.98, d (6.6)
—
—
—
—
—
—
—
—
—
—
3.51, s
3.53, s
4.16, q (7.0)
1.26, t (7.2)
4.54, d (7.3)
5.64, t (6.2)
—
2.10, m
2.15, m
5.12, m
4.17, q (7.2)
1.27, t (7.2)
5.01, s
10
10
The HREIMS, 1H and 13C NMR data of 9 (Tables 1 and 2)
suggested the molecular formula C19H18Br2O5 and possible C-1
60-methoxyphenylmethyl ester analogue of 3. HMBC further con-
firmed this assignment as previously described in 6. Thus, 9 was
determined to be 2,6-dibromo-1-(p-methoxyphenylacetoxy)ben-
zene-4-acetic acid ethyl ester.
The HREIMS of 10 showed a molecular ion peak at m/z 441.9269
suggesting the molecular formula C16H13Br2NO4 and possible C-1-
esterification of 3 with an isonicotinoyl moiety. The 1H, 13C NMR,
and HMBC data (Tables 1 and 2) were used to verify C-1 esterifica-
tion. The aromatic methine doublet H-30/70 (dH 8.04) showed a
3J-HMBC correlation with the quaternary carbonyl carbon C-10 (dC
161.9) as well as a COSY coupling with the H-40/60 doublet (dH
20
—
30
7.60, m
7.37, m
7.42, m
7.37, m
40
50
60
70
7.60, dd (6.6, 1.5)
80
1.97, m
2.05, m
5.08, m
—
1.67, s
1.74, s
1.60, s
1.59, s
—
—
—
—
—
—
—
—
90
100
110
120
130
140
150
—
—
—
—
a
In CDCl3, 400 MHz. Coupling constants (J) are in Hertz.

A. A. Sallam et al. / Bioorg. Med. Chem. 18 (2010) 7446–7457
7449
8.89). Thus, 10 was determined to be 2,6-dibromo-1-(isonicoti-
noyloxy)benzene-4-acetic acid ethyl ester.
Analysis of 1H and 13C NMR data (Tables 3 and 4) showed that
etherification primarily induced a downfield shift most signifi-
O
OH
OH
1
Br
O
Br
Br
Br
5
Br
Br
3
6
3
1
O
HO
7
O
8
7
8
NH2
O
10
NH2
9
Verongiaquinol (1)
2
3
OR1
1
Br
Br
O
O
O
O
O
6`
2`
1``
2`
N
1`
2`
2`
a
O
7
5`
b
d
c
R2
R1
a
R2
4
5
NH2
NH2
NH2
EtO
EtO
EtO
EtO
b
c
6
7
a
8
b
c
9
10
d
Four new ether analogues 11–14 were synthesized according to
Scheme 2.18 This scheme shows the base-catalyzed nucleophilic
substitution reaction16 in which the electrophilic center is the bro-
minated carbon of the alkyl or alkenyl bromides used. Reaction
yields ranged between 47% and 70%, with the aryl bromides giving
the higher yields.
cantly at the C-1 and the C-2/6 positions. Carbon C-1 was shifted
by d +3.8, while C-2/6 were shifted by d +8.9, as compared to
the parent 3 (Table S1). The intact trans, trans-farnesyl moiety
was confirmed by HMBC and 1H–1H COSY experiments. The
methylene proton doublet H2-10 (dH 4.54) showed 3J-HMBC corre-
lations with the aromatic oxygenated quaternary carbon C-1 and
D
D
13`
3`
H
4`
OR1
12`
15`
5`
1`
1`
Br
Br
10`
H
6`
6`
H
14`
f
e
4`
3`
O
O
1`
1``
2`
5`
2`
O
3`
g
h
R
e
f
g
h
11
12
13
14
The HREIMS data of 11 showed a molecular ion peak at m/z
the quaternary olefinic carbon C-30 (dC 152.4 and 143.1, respec-
581.0668, suggesting the molecular formula C25H34Br2O3 and
possible C-1-etherification of 3 with a trans, trans-farnesyl moiety.
tively). COSY correlations were observed between the olefinic
methine H-20 (dH 5.64) and both H2-10 and H3-130 (dH 4.54 and

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A. A. Sallam et al. / Bioorg. Med. Chem. 18 (2010) 7446–7457
Table 4
Table 5
13C NMR Data of compounds 11–14a
Docking scores for analogues 2–14 resulting
from docking at the ATP binding site of VEG-
FR2 (PDB: 3cjf) using SYBYL 8.1 Surflex-Dock
Position
dC
11
12
13
14
Compound
Total score
1
152.4, qC
118.7, qC
133.5, CH
132.4, qC
39.9, CH2
170.7, qC
61.4, CH2
14.3, CH3
70.1, CH2
119.0, CH
143.1, qC
39.7, CH2
26.3, CH2
123.8, CH
135.5, qC
39.8, CH2
26.8, CH2
124.4, CH
131.4, qC
25.8, CH3
16.7, CH3
16.1, CH3
17.8, CH3
152.0, qC
118.5, qC
133.6, CH
132.9, qC
39.9, CH2
170.6, qC
61.4, CH2
14.3, CH3
74.7, CH2
136.3, qC
128.6, CH
128.6, CH
128.5, CH
128.6, CH
128.6, CH
—
—
—
—
—
—
—
—
152.7, qC
118.3, qC
133.6, CH
132.4, qC
39.9, CH2
170.7, qC
61.4, CH2
14.3, CH3
73.0, CH2
32.1, CH2
31.4, CH2
133.9, qC
129.5, CH
113.9, CH
157.9, qC
113.9, CH
129.5, CH
—
152.7, qC
118.4, qC
133.5, CH
132.4, qC
39.9, CH2
170.7, qC
61.4, CH2
14.3, CH3
72.2, CH2
38.9, CH2
24.9, CH
22.8, CH3
22.8, CH3
—
—
—
—
—
—
—
—
—
Ligand
7.66
6.83
5.35
5.30
4.66
4.47
4.37
4.37
4.14
3.65
3.55
3.51
3.48
3.45
3.11
2/6
3/5
4
7
8
11
13
14
9
3
6
5
12
8
2
7
9
10
10
20
30
40
50
4
10
1
60
70
80
90
100
110
120
130
140
150
The HREIMS, 1H and 13C NMR data of 13 (Tables 3 and 4)
suggested the molecular formula C20H22Br2O4 and possible C-1 p-
methoxyphenylpropyl ether analogue of 3. The methylene proton
triplet H2-10 (dH 4.01) showed 3J-HMBC correlations with the oxy-
genated quaternary aromatic carbon C-1 and the aliphatic benzylic
methylene carbon C-30 (dC 152.7 and 31.4, respectively). The meth-
oxy protons singlet H3-100 (dH 3.79) showed a 3J-HMBC correlation
with the oxygenated quaternary aromatic carbon C-70 (dC 157.9).
The aromatic methine doublet H-50/90 (dH 7.17) showed 3J-HMBC
correlation with C-70 and C-30. They also showed COSY coupling
with the aromatic methine doublet H-60/H-80 (dH 6.84). Thus, 13
was determined to be 2,6-dibromo-1-(p-methoxyphenylpropyl-
oxy) benzene-4-acetic acid ethyl ester.
55.3, CH3
—
—
—
—
—
qC = quaternary, CH = methine, CH2 = methylene, CH3 = methyl carbons.
a
In CDCl3, 100 MHz. Carbon multiplicities were determined by APT experiments.
1.74, respectively). The olefinic proton H-60 (dH 5.12) showed COSY
coupling with both methylene protons H2-40 and H2-50 (dH 2.10 and
2.15, respectively), and virtual coupling with the methyl H3-140 (dH
1.60). The 3J-HMBC correlation between the methyl protons H3-130
and C-40 (dC 39.7) connected both segments together. 1H–1H COSY
and HMBC data further connected the rest of the farnesyl segment.
Hence, 11 was determined to be 2,6-dibromo-1-(trans, trans-farne-
syloxy) benzene-4-acetic acid ethyl ester.
The HREIMS, 1H and 13C NMR data of 14 (Tables 3 and 4) sug-
gested the molecular formula C15H20Br2O3 and possible C-1 iso-
pentyl ether analogue of 3. The methylene proton triplet H2-10
(dH 4.00) showed 3J-HMBC correlations with the oxygenated aro-
matic carbon C-1 and the aliphatic methine carbon C-30 (dC 152.7
and 24.9, respectively). The methyl proton doublet H3-40/H3-50
(dH 0.98) showed COSY coupling with proton H-30 (dH 1.92), which
was coupled to the methylene protons H2-20 (dH 1.75). The latter
The HREIMS, 1H and 13C NMR data of 12 (Tables 3 and 4) sug-
gested the molecular formula C17H16Br2O3 and possible C-1 benzyl
ether analogue of 3. The benzylic oxygenated methylene proton
singlet H2-10 (dH 5.01) showed 3J-HMBC correlations with the aro-
matic carbons C-1 and C-30/C-70 (dC 152.0 and 128, respectively).
Thus, 12 was determined to be 2,6-dibromo-1-(benzyloxy)-ben-
zene-4- acetic acid ethyl ester.
also showed H–1H COSY coupling with H2-10. Thus, compound
1
14 was determined to be 2,6-dibromo-1-(300-methylbutyloxy)ben-
zene-4-acetic acid ethyl ester.
Figure 1. (A) Important HB interactions of 11 at the ATP binding site of VEGFR2 (PDB 3cjf). (B) Overlaid ligand obtained from the crystal structure of VEGFR2 (green stick) and
the same ligand after the docking simulation (bold green).

A. A. Sallam et al. / Bioorg. Med. Chem. 18 (2010) 7446–7457
7451
Figure 2. Chick chorioallantoic membrane (CAM) assay. (A) Growing vascular network at day-five prior to treatment. (B) Vascular network of a control-treated egg after 48 h
(3ꢁ). (C) Vascular network of an egg treated with 13 (50
l
g/disc) after 48 h (3ꢁ).
Table 6
Table 7
Chick chorioallantoic membrane
IC50 Values of 2–14 using the MTT and WHA assays against PC-3
cells
(CAM) assay resultsa
Grade
Compounds
Compound
MTT (IC50
,
lM)
WHA (IC50, lM)
0
1
2
3
2, 3, 14
8, 9
4, 11, 13
5, 6, 10, 12
2
3
4
5
6
7
8
9
10
11
12
13
14
>100
>100
>100
>100
>100
>100
>100
>100
>100
16.5
>100
>100
74.8
12.2
18.9
>100
44.6
< 10
44.6
19.9
75.5
57.9
91.3
a
0: Number of blood vessels per
48 mm2 area around the treatment
disc >16. 3: Number of blood vessels
per 48 mm2 area around the treat-
ment disc <12.
29.7
40.0
49.2
DMSO
11
120
100
80
60
40
20
0
12
thermore, the ether oxygen is involved in a HB interaction with
the amide NH of Asp1044. Table 5 shows the docking scores of
analogues 1–14 versus the VEGFR2 ligand (N4-(3-methyl-1H-inda-
zol-6-yl)-N2-(3,4,5-trimethoxyphenyl)-pyrimidine-2,4-diamine).
The docking-scoring procedure was validated by docking the VEG-
FR2 ligand into the ATP binding site, resulting in a docked pose
close to that observed in the crystallographic structure22,23
(Fig. 1B).
In an attempt to gain an insight into the effect of the type of the
halogen at positions C-2 and C-6 on the biological activity, virtually
designed difluoro and dichloro analogues of 2–14 were docked into
the ATP binding site of VEGFR2 (Supplementary data, Table S3).
Generally, with few exceptions, difluoro substitution proved asso-
ciated with higher docking scores, which may imply superior activ-
ity versus the dibromo analogues. The difluoro substitution is
uncommon in natural products and therefore the focus of this
study remained on the dibromotyrosine analogues to mimic this
common marine natural products class.
13
14
Controls
10
50
100
Concentration (uM)
Figure 3. Anti-proliferative activity of ether analogues 11–14 against PC-3 cells.
Error bars indicate the SD of n = 3/dose.
2.2. Molecular docking studies
Analogues were docked into the ATP binding site of VEGFR2
(PDB code 3cjf) using Surflex-Dock interface implemented into
SYBYL 8.0.19–21 Surflex-Dock is a fully automatic flexible molecular
docking algorithm that combines the scoring function from the
Hammerhead docking system with a search engine that relies on
a surface-based molecular similarity method as a means to rapidly
generate suitable putative poses for molecular fragments.19,20
The high binding affinity of any active ligand with VEGFR2 is
usually satisfied through hydrogen bonding (HB) interactions with
the following amino acid residues: Lys866, Cys917, Asn921 and
Asp1044.22–24 Compound 11 satisfied at least two of these interac-
tions (Fig. 1A). Both ethyl ester oxygens are hydrogen bond accep-
tors (HBA) and are involved in reinforced ionic HB interactions
with the positively charged guanidinium cation of Arg840 and
protonated quaternary amino group of Lys866, respectively. Fur-
2.3. Biological activity
The anti-angiogenic potential of 4–14 was tested in the CAM as-
say. The anti-proliferative, anti-migratory and anti-invasive activi-
ties against the human prostate cancer cell line PC-3 were
evaluated using the MTT, wound-healing and CultrexÒ BME cell
invasion assays, respectively.
2.3.1. Chick chorioallantoic membrane (CAM) assay
The chick chorioallantoic membrane (CAM) assay is a widely
used in vivo model for the study of angiogenesis and represents
a simple functional assay to screen agents for anti-angiogenic

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A. A. Sallam et al. / Bioorg. Med. Chem. 18 (2010) 7446–7457
Figure 4. WHA using PC-3 cells after 24 h exposure to: (A) A 50
lM dose of 9. (B) A 50
lM dose of (Z)-5-(4-(ethylthio)benzylidene)-hydantoin (S-Ethyl) as a positive
control.31 (C) Vehicle (DMSO) treatment as a negative control.
100
90
80
70
60
50
40
30
20
10
DMSO
S-Ethyl
5
6
8
9
10
11
0
Controls
20
50
100
Concentration (uM)
Figure 5. Anti-migratory activity of the most active analogues against the human prostate cancer cell line, PC-3. Error bars indicate the SD of n = 3/dose.
activity.25 The main advantages of the CAM assay are its low-cost,
simplicity, reliability, and applicability to large-scale screening.26
VEGFR2 is expressed in CAM’s endothelial cells and the pericytes,
while VEGF is expressed in the chorionic epithelial cells.25 VEGF re-
lease promotes angiogenesis via initiating endothelial cells
expressing VEGFR2 in the CAM blood vessels.25 Therefore, modula-
tion of VEGFR2 will directly correlate with angiogenesis inhibition
in the CAM model. Analogues 4–14 as well as the parent com-
2.3.2. MTT Assay
The MTT assay allows for the measurement of cell viability and
proliferation of cell populations in a quantitative colorimetric fash-
ion by utilizing cellular ability to reduce the MTT reagent to insol-
uble purple formazan dye.27–29 In this assay, each analogue was
tested at three concentrations; 10, 50, and 100 lM (Fig. 3). The
concentration of each analogue that resulted in 50% cell growth
inhibition, IC50, was measured (Table 7). All ether analogues
showed lower IC50 values than any of the ester analogues (Fig. 3).
Consistent with in silico studies, the farnesyl analogue 11 showed
pounds 2 and 3 were tested at 1, 10, and 50 lg/disc doses in this
assay. Evaluation of the anti-angiogenic potential was based on
two criteria; the number of newly formed blood vessels as well
as the length of these blood vessels (Fig. 2). Analogues were then
graded using a 0–3 scale,26 0 grade being inactive and 3 showing
the highest activity. As predicted in silico (Table 5), analogues with
docking scores higher than 4.0 showed good activities within
grades 2 and 3 (Table 6), including the most active 5, 6, and 11–
13. The only outliers were compound 14 with a good docking score
of 5.30 but no activity (grade 0), and compounds 4 and 10, with
low docking scores of 3.48 and 3.45, respectively, but showed good
activity (grades 2 and 3, respectively). Such deviation could be
attributed to the activity of 4, 10, and 14 on unknown protein tar-
get(s) other than VEGFR2.
the most potent inhibition with an IC50 value of 16.5 lM. A closer
look at the structural features of the most active analogues sug-
gests that chain extension and unsaturation, as in 11, are associ-
ated with a better activity profile compared to those with shorter
saturated chains, as in 14. Electron donating substituents and long-
er chain linking the aromatic ring to the ether oxygen, as in 13,
afforded a better anti-proliferative activity profile versus the sim-
ple unsubstituted benzyl ether 12.
2.3.3. Wound-healing assay (WHA)
The wound-healing assay is a simple method for the study of
directional cell migration in vitro.30,31 Figure 4 shows cell migra-

A. A. Sallam et al. / Bioorg. Med. Chem. 18 (2010) 7446–7457
7453
140
120
100
80
60
40
20
0
DMSO
(+ve) control
5
6
8
9
10
11
13
Compound (50 uM)
Figure 6. Anti-invasive activities of 50 l
M dose of selected ester and ether analogues against the human prostate cancer cell line, PC-3 in the CultrexÒ BME cell invasion assay
kit. (Z)-5-(4-(ethylthio)benzylidene)imidazolidine-2,4-dione was used as a positive control.31 Error bars indicate the SD of n = 3/analogue.
Figure 7. GM-CFC HALOÒ assay results. ATP luminescence for culture wells with compounds was normalized to mean of concurrent vehicle for each compound. Compounds
were tested in two separate platings with vehicle means SEM of 46.2 3.9 (1, 10, and 11) and 113.0 9.7 (2, 3, 12, 13, and 14) pmol/well. Relative treatment means SEM
(n = 4) are shown.
tion across the wound inflicted in the PC-3 cell monolayer for the
vehicle and positive controls, and the active analogue 9. The con-
centration of each analogue that results in 50% inhibition of cell
side chains in compounds 4–6 and 7–10 had no observable influ-
ence on the activity pattern. In general, aromatic and heteroaro-
matic esters proved to be more active than aliphatic esters.
migration, IC50, was measured (Table 7). Interestingly, ester
analogues showed a better anti-migratory profile than ether ana-
logues, except for the farnesyl ether 11. Figure 5 shows the anti-
migratory effect of the most active analogues. Analogues 5, 6,
and 9–11 showed a much better activity profile than the positive
2.3.4. CultrexÒ BME cell invasion assay
The CultrexÒ BME cell invasion assay is an accelerated in vitro
screening process for compounds that influence cell migration
through extracellular matrices, which is a fundamental function
of cellular processes such as angiogenesis, embryonic development,
immune response, and metastasis of cancer cells.32,33 Those ester
control, (Z)-5-(4-(ethylthio)benzylidene)-hydantoin used at
a
50 M dose (reported IC50 51.4
l
l
M).31 The amide or ethyl ester

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A. A. Sallam et al. / Bioorg. Med. Chem. 18 (2010) 7446–7457
TLC plates (EMD Chemicals), using CHCl3–MeOH (9.8:0.2) or
(9:1) as developing systems. For column chromatography, Si Gel
60 (Natland International Corporation, 230–400 mm) was used,
and gradient n-hexane–EtOAc or CHCl3–MeOH solvent systems
were used. HPLC analyses were conducted on a Shimadzu HPLC
system (Columbia, MD) using a 5
lm C18 column (150 ꢁ 4.6 mm
id; Agilent, Santa Clara, CA) and isocratic elution (CH3OH/H2O;
80:20) with UV detection set at 230 nm to verify the purity of com-
pounds 4–14. A purity of >95% was established for compounds 4–
8, 10 and 14 by HPLC analysis. Compounds 9 and 11–13 showed a
purity of >90%. The 1H and 13C NMR spectra were recorded in CDCl3
and CD3OD, using (CH3)4Si as an internal standard, on a JEOL
Eclipse NMR spectrometer operating at 400 MHz for 1H NMR and
100 MHz for 13C NMR. The HREIMS experiments were conducted
at Louisiana State University on a 6200-TOF LC–MS (Agilent, Santa
Clara, CA) equipped with a multimode source (mixed source that
can ionize the samples alternatively by ESI and APCI).
4.2. Chemical reactions
Figure 8. GM-CFC on the 3rd day after incubation with vehicle (A) and 10
(B), 10 (C), and 11 (D).
lM of 1
4.2.1. Esterification of 2 and 3
analogues that showed the best IC50 values in the WHA (5, 6, and 8–
10), as well as the two most active ether analogues (11 and 13) were
tested at a single concentration of 50 lM for their ability to inhibit
the invasion of PC-3 cells (Fig. 6). Analogues showed a comparable
activity profile to the known anti-invasive positive control, (Z)-5-
(4-(ethylthio)benzylidene)-hydantoin,31 with compound 13 being
almost twofold more active than this positive control.
To solutions of 2 or 3 (0.148 mmol) in CH2Cl2 (5.0 mL), N,N-
dimethylaminopyridine (2 equiv), and different alkyl or aryl acid
chlorides (2 equiv) were added (Scheme 1).15,16 Each solution
was separately stirred and refluxed (60–70 °C) for 2–12 h. Each
reaction mixture was diluted with CH2Cl2, poured into water
(10.0 mL), shaken and collected. Each CH2Cl2 layer was then sha-
ken with 0.1 N HCl (2 ꢁ 10.0 mL) and dried over anhydrous
Mg2SO4. Crude mixtures were then purified on Si Gel 60 using gra-
dient CHCl3/MeOH system. Evaporation of fractions containing tar-
get compounds afforded 4–10 in 37–82% yields.
2.3.5. GM-CFC/HALOÒ assay
The phenol structure, rather than the free radical-producing
dienone observed in verongiaquinol, was selected as the starting
point for the synthesis of all analogues as a strategy to eliminate
the source of free radicals, thus eliminating the free radical-medi-
ated cytotoxicity. A clonogenic assay for rat bone marrow myeloid
progenitors (GM-CFC HALOÒ assay) was used as a model to test the
cytotoxic potential of verongiaquinol and analogues. In this assay,
the number of colonies formed in methylcellulose supplemented
with myeloid growth/differentiation factors (GM-CSF, IL-3 and
SCF) is monitored. Tested compounds 2–3, 11, and 13–14 showed
no cytotoxic effects and no inhibition of colony formation even at
4.2.2. Etherification of 3
Solutions of 3 (0.148 mmol) in dry DMF (1.0 mL) were added
drop-wise to cooled (5 °C) oil-free suspension of NaH (0.148 mmol)
in 1.0 mL dry DMF (Scheme 2). Different alkyl or aryl bromides
(0.148 mmol) were then added drop-wise to the mixture.16,18 After
H2 evolution ceased, solutions were warmed to room temperature
and stirred overnight. Each solution was then poured into 1 N
NaOH and the mixture was extracted with CHCl3 (3 ꢁ 10 mL).
Organic layer was then washed with brine (1 ꢁ 10 mL) and dried
over anhydrous Na2SO4. Crude mixtures were then purified on Si
Gel 60 using gradient CHCl3–MeOH or n-hexane–EtOAc systems,
which afforded 11–14 in 47–70% yields.
100 lM, whereas verongiaquinol was cytotoxic at 10 lM (Figs. 7
and 8). This clearly highlights the potential of dibromotyrosine
analogues as effective and non-toxic hits because cells used in this
assay are normal bone barrow cells. Therefore dibromotyrosines
may have excellent therapeutic potential for the control of meta-
static prostate cancer.
4.2.3. 2,6-Dibromo-1-acetoxybenzene-4-acetamide (4)
A reaction of 50 mg of 2 with 23.2 lL of acetyl chloride was car-
3. Conclusion
ried out to give 4, 47% yield: white powder; IR mmax (CH2Cl2) 2987,
1772, 1693, 1653, 1208, 1182, 896 cmꢀ1; HRESIMS m/z 349.9166
[M+H]+ (calcd for C10H10Br2NO3, 349.9027).13
Structurally simple dibrominated phenolic compounds inspired
by marine secondary metabolites offer interesting scaffolds for
anti-cancer drug discovery. We showed herein the synthesis and
biological evaluation of a series of ether and ester analogues of
dibromotyrosine intermediates from the synthetic scheme of
verongiaquinol. The results obtained demonstrate the potential of
these dibrominated scaffolds as effective anti-angiogenic and
inhibitors of prostate cancer proliferation and migration and are
appropriate for further optimization.
4.2.4. 2,6-Dibromo-1-benzoyloxybenzene-4-acetamide (5)
A reaction of 25 mg of 2 with 18.8
carried out to give 5, 72% yield: white powder; IR mmax (CH2Cl2)
2987, 1749, 1695, 1557, 1235, 896 cmꢀ1 1H and 13C NMR, see
Tables 3 and 4; HRESIMS m/z 413.9196 [M+3H]3+ (calcd for
15H14Br2NO3, 413.9340).
lL of benzoyl chloride was
;
C
4.2.5. 2,6-Dibromo-1-(p-methoxyphenylacetoxy)benzene-4-
acetamide (6)
4. Experimental
A reaction of 25 mg of 2 with 24.8 lL of 4-methoxyphenylacetyl
chloride was carried out to give 6, 59% yield: white powder; IR mmax
(CH2Cl2) 2987, 1770, 1695, 1248, 1155, 987 cmꢀ1; 1H and 13C NMR,
see Tables 3 and 4; HRESIMS m/z 457.9432 [M+3H]3+ (calcd for
4.1. General experimental procedures
IR spectra were recorded on a Varian 800 FT-IR spectrophotom-
C17H18Br2NO4, 457.9602).
eter. TLC analysis was carried on precoated Si Gel 60 F254 500 lm

A. A. Sallam et al. / Bioorg. Med. Chem. 18 (2010) 7446–7457
7455
4.2.6. 2,6-Dibromo-1-acetoxybenzene-4-acetic acid ethyl ester
(7)
on DELL desktop workstations equipped with a dual 2.0 GHz IntelÒ
XeonÒ processor running the Red Hat Enterprise Linux (version 5)
operating system.34 Conformations of each compound were gener-
ated using Confort™ conformational analysis. Energy minimiza-
A reaction of 50 mg of 3 with 21.0
l
L of acetyl chloride was car-
max (CH2Cl2) 2987, 1773,
ried out to give 7, 58% yield: clear oil; IR
m
1734, 1557, 1181, 896 cmꢀ1
;
1H and 13C NMR, see Tables 3 and 4;
tions were performed using the Tripos force field with a
HRESIMS m/z 380.9270 [M+3H]3+ (calcd for C12H15Br2O4, 380.9336).
distance-dependent dielectric and the Powell conjugate gradient
algorithm with a convergence criterion of 0.01 kcal/(mol A).35 Par-
tial atomic charges were calculated using the semi-empirical pro-
gram MOPAC 6.0 and applying the AM1.36
4.2.7. 2,6-Dibromo-1-benzoyloxybenzene-4-acetic acid ethyl
ester (8)
A reaction of 50 mg of 3 with 17.2
lL of benzoyl chloride was
carried out to give 8, 37% yield: clear oil; IR mmax (CH2Cl2) 2987,
4.3.1. Molecular docking
1773, 1734, 1421, 1181, 896 cmꢀ1 1H and 13C NMR, see Tables 3
;
Surflex-Dock Program version 2.0 interfaced with SYBYL 8.0 was
used to dock the compounds in the ATP binding site of VEGFR2.37
Surflex-Dock employs an idealized active site ligand (protomol) as
a target to generate putative poses of molecules or molecular frag-
ments. These putative poses were scored using the Hammerhead
scoring function.38,39 The 3D structure was taken from the Brook-
haven Protein Databank (PDB code: 3cjf).23
and 4; HRESIMS m/z 450.8912 [MꢀC2H4+K]+ (calcd for
C15H10Br2O4K, 450.8583).
4.2.8. 2,6-Dibromo-1-(p-methoxyphenylacetoxy)benzene-4-
acetic acid ethyl ester (9)
A reaction of 50 mg of 3 with 22.6
chloride was carried out to give 9, 82% yield: yellow oil; IR mmax
(CH2Cl2) 2987, 1769, 1734, 1551, 1272, 896 cmꢀ1 1H and 13C
NMR (Tables 3 and 4); HRESIMS m/z 484.9412 [M+H]+ (calcd for
19H19Br2O5, 484.9599).
lL of 4-methoxyphenylacetyl
;
4.4. Cell culture
C
Prostate cancer cell line, PC-3 was purchased from ATCC
(Manassas, VA). The cell line was grown in RPMI 1640 medium
(GIBCO-Invitrogen, NY) with 10% fetal bovine serum (FBS) and sup-
4.2.9. 2,6-Dibromo-1-(isonicotinoyloxy)benzene-4-acetic acid
ethyl ester (10)
plemented with glutamine (2 mmol/L), penicillin G (100
and streptomycin (100 g/mL) at 37 °C under 5% CO2.
lg/mL),
A reaction of 50 mg of 3 with 26.3 mg of isonicotinoyl chloride
l
hydrochloride, in the presence of 62
lL Et3N and 18.0 mg (1 equiv)
DMAP, was carried out to give 10, 86% yield: clear oil; IR mmax
(CHCl3) 2991, 1760, 1732, 1644, 1140, 891 cmꢀ1 1H and 13C
;
4.4.1. Preparation of various dilutions of analogues for cell
culture assays
A stock solution of each analogue was prepared by dissolving the
compound in DMSO at a concentration of 50 mM for all assays.
NMR, see Tables 3 and 4; HRESIMS m/z 441.9269 [M+H]+ (calcd
for C16H14Br2NO4, 441.9289).
4.2.10. 2,6-Dibromo-1-(trans, trans-farnesyloxy)benzene-4-
acetic acid ethyl ester (11)
About 2
um-free medium to obtain 100
rial dilutions were then conducted to obtain the desired
l
L of each stock solution was transferred to 998
lL of ser-
lM concentrations (0.2% DMSO). Se-
A reaction of 50 mg of 3 with 40.1
lL trans, trans-farnesyl bro-
mide was carried out to give 11, 86% yield: yellow oil; IR mmax
concentrations for each assay. The negative control was prepared
(CHCl3) 2928, 1732, 1545, 1456, 948, 869 cmꢀ1 1H and 13C NMR,
;
as follows: adding 2
say); adding 3 L DMSO to 1497
ing 1.2 L DMSO to 199 L serum-free media (cell invasion assay).
lL DMSO to 998
lL serum-free media (MTT as-
see Tables 5 and 6; HRESIMS m/z 581.0649 [M+2H+K]3+ (calcd
for C25H36Br2O3K, 581.0668).
l
lL serum-free media (WHA); add-
l
l
4.2.11. 2,6-Dibromo-1-(benzyloxy) benzene-4-acetic acid ethyl
ester (12)
4.5. MTT proliferation assay
A reaction of 50 mg of 3 with 17.6
out to give 12, 50% yield: clear oil, IR
lL benzyl bromide was carried
The anti-proliferative effect of analogues was tested in culture
on the human prostate cancer cell line, PC-3. Cell growth was mea-
sured using MTT kit (TACS™, TrevigenÒ, Inc.).27–29 Cells in expo-
nential growth were plated in a 96-well plate at a density of
8 ꢁ 103 cells per well, and allowed to attach for 24 h at 37 °C under
m
max (CHCl3) 2928, 1732, 1546,
1260, 967, 857 cmꢀ1; 1H and 13C NMR, see Tables 5 and 6; HRESIMS
m/z 448.9266 [M+Na]+ (calcd for C17H16Br2O3Na, 448.9364).
4.2.12. 2,6-Dibromo-1-(p-methoxyphenylpropyloxy)benzene-4-
acetic acid ethyl ester (13)
5% CO2. Complete growth medium was then replaced with 100 lL
of RPMI serum-free medium (GIBCO-Invitrogen, NY) containing
various doses (10, 50, and 100 M) of the specific test compound
and incubation resumed at 37 °C under 5% CO2 for 72 h. The cells
were then treated with MTT solution (20 L/well) and re-incu-
bated for 4 h. The color reaction was stopped by the addition of sol-
ubilization/stop solution (100 L/well), and incubation at 37 °C
A reaction of 50 mg of 3 with 25.9
bromide was carried out to give 13, 51% yield: clear oil, IR mmax
(CHCl3) 2938, 1733, 1612, 1545, 984, 842 cmꢀ1 1H and 13C NMR,
see Tables 5 and 6; HRESIMS m/z 506.9587 [M+Na]+ (calcd for
20H22Br2O4Na, 506.9783).
lL 4-methoxyphenylpropyl
l
;
l
C
l
was continued to ensure complete dissolution of the formazan
product. Absorbance of the samples was determined at k 570 nm
with an ELISA plate reader (BioTek, VT, USA). The IC50 value for
each compound was calculated by nonlinear regression (curve
fit) of log (concentration) versus the % survival, implemented in
GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA,
USA). The % cell survival was calculated as follows: % cell sur-
vival = (Abstreatment/AbsDMSO) ꢂ 100%.
4.2.13. 2,6-Dibromo-1-(300-methylbutyloxy)phenylacetic acid
ethyl ester (14)
A reaction of 50 mg of 3 with 17.72
lL 3-methylbutyl bromide
was carried out to give 14, 47% yield: clear oil; IR mmax (CHCl3)
2957, 1733, 1545, 1457, 978, 872 cmꢀ1 1H and 13C NMR, see
;
Tables 5 and 6; HRESIMS m/z 428.9676 [M+Na]+ (calcd for
C15H20Br2O3Na, 428.9677).
4.6. Wound-healing assay30,31
4.3. Molecular modeling
Three-dimensional structure building and all modeling were
performed using the SYBYL program package, version 8.0, installed
PC-3 cells were plated onto sterile 24-well plates and allowed to
form a confluent cell monolayer per well (>95% confluence) over-

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A. A. Sallam et al. / Bioorg. Med. Chem. 18 (2010) 7446–7457
night. Wounds were then inflicted in each cell monolayer using a
sterile 200 L pipette tip. Media was removed and cells were
washed twice with PBS and once with fresh serum-free media. Test
compounds at the desired concentrations (20, 50, and 100 M) in
fresh serum-free media were added to each well. The incubation
was carried out for 24 h under serum-free conditions, after which
media was removed and cells were washed, fixed and stained
using Diff-Quick™ staining (Dade Behring Diagnostics, Aguada,
Puerto Rico). Cells which migrated across the inflicted wound were
counted under the microscope in three or more randomly selected
fields (magnification: 400ꢁ).
Histopaque-1077 density gradient. Cells were collected at the
interface, washed and centrifuged (400g, 10 min), and resus-
pended in 0.25 mL of medium and counted using hemocytometer.
The GM-CFC 2 assay was performed using a HALO kit as per sup-
plier’s instructions (HemoGenix, Colorado Springs, CO). In brief,
l
l
20,000 mononuclear cells in 15
lL medium were mixed with
60 L methyl cellulose, 60 L FCS, 15 l
l
l
L growth factor mix (rat re-
combinant GM-CSF, IL-3 and SCF) and plated in 96-well plates.
After 5 days, GM-CFC colonies were measured as ATP lumines-
cence measured with luciferase and luciferin. ATP luminescence
was calibrated against a standard curve generated on the same
day. Dose means for all compounds were statistically compared
to vehicle with two-way ANOVA with Bonferroni post-hoc pair-
wise comparisons.
4.7. CultrexÒ BME cell invasion assay
Anti-invasive activities were measured using Trevigen’s Cult-
rexÒ BME cell invasion assay.32 About 50
lL of basement mem-
brane extract (BME) coat was added per well of the top chamber.
Supplementary data
After an overnight incubation at 37 °C in a 5% CO2 atmosphere,
Supplementary data (structures of the known bromotyrosine-
derived marine natural products listed in the article (Fig. S1), 1H
and 13C NMR data of 2 and 3, LRMS Data of 1–3, and Docking scores
of 2–14 and their virtual difluoro and dichloro analogues at the ATP
binding site of VEGFR2 (Tables S1–S3) as well as the verongiaqui-
nol synthetic scheme (Scheme 1)) associated with this article can
be found, in the online version, at doi:10.1016/j.bmc.2010.08.057.
50,000 PC-3 cells per 50
per well of the top chamber. 150
added to the lower chamber. Media contained 10% FBS and penicil-
lin/streptomycin as well as fibronectin (1 L/mL) and N-formyl-
Met-Leu-Phe (10 nM) as chemoattractants. Test compounds were
M) and 10 L of
lL serum-free RPMI medium were added
l
L of RPMI medium was then
l
prepared at 6ꢁ the desired concentration (300
l
l
each of the compounds was added per well of the top chamber.
Cells were incubated at 37 °C under 5% CO2 which allowed for cell
migration from the top to the lower chamber. After 24 h, the top
and bottom chambers were aspirated and washed with washing
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