A Novel Arylurea Fatty Acid That Targets the Mitochondrion and Depletes Cardiolipin to Promote Killing of Breast Cancer Cells

Article abstract of DOI:10.1021/acs.jmedchem.7b00701

Cancer cell mitochondria are promising anticancer drug targets because they control cell death and are structurally and functionally different from normal cell mitochondria. We synthesized arylurea fatty acids and found that the analogue 16-({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)hexadecanoic acid (13b) decreased proliferation and activated apoptosis in MDA-MB-231 breast cancer cells in vitro and in vivo. In mechanistic studies 13b emerged as the prototype of a novel class of mitochondrion-targeted agents that deplete cardiolipin and promote cancer cell death.

Full text of DOI:10.1021/acs.jmedchem.7b00701

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Brief Article
A novel aryl-urea fatty acid that targets the mitochondrion and
depletes cardiolipin to promote killing of breast cancer cells
Tristan Rawling, Hassan Choucair, Nooshin Koolaji, Kirsi Bourget, Sarah
E Allison, Yong-Juan Chen, Colin R. Dunstan, and Michael Murray
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00701 • Publication Date (Web): 18 Sep 2017
Downloaded from http://pubs.acs.org on September 19, 2017
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A novel aryl-urea fatty acid that targets the mitochondrion
and depletes cardiolipin to promote killing of breast cancer
cells.
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Tristan Rawling,*,† Hassan Choucair,‡ Nooshin Koolaji,‡ Kirsi Bourget,‡ Sarah E. Allison,‡ Yong-
Juan Chen,§ Colin R. Dunstan§ and Michael Murray‡
†School of Mathematical and Physical Sciences, Faculty of Science, University of Technology Sydney, Ultimo, New
South Wales, 2007, Australia,
‡Discipline of Pharmacology, School of Medical Sciences, Sydney Medical School, University of Sydney, New South
Wales, 2006, Australia, and
§School of Aerospace, Mechanical and Mechatronic Engineering, University of Sydney, New South Wales, 2006,
Australia.
KEYWORDS. Fatty acid, urea, cancer, mitochondria, cardiolipin.
ABSTRACT: Cancer cell mitochondria are promising anticancer drug targets because they control cell death and are
structurally and functionally different from normal cell mitochondria. We synthesized aryl-urea fatty acids and found that
the analogue 16({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)hexadecanoic acid (13b) decreased proliferation
and activated apoptosis in MDA-MB-231 breast cancer cells in vitro and in vivo. In mechanistic studies 13b emerged as the
prototype of a novel class of mitochondrion-targeted agents that deplete cardiolipin and promote cancer cell death.
prostaglandin E2 and the epoxyeicosatrienoic acids
INTRODUCTION
(EETs), respectively, that drive tumor proliferation and
promote metastasis.9 In contrast, certain ω-3 polyunsatu-
Oncology drugs target pathways in tumor cells that
promote proliferation or activate cell death.1 New classes
rated fatty acid metabolites are anti-tumorigenic. Zhang
of anti-cancer agents with novel mechanisms of action are
required to provide additional therapeutic alternatives in
cancer chemotherapy as sole agents or in combination
with other drugs. The cancer cell mitochondrion is a
promising anticancer drug target because of its role in
apoptotic cell death.2 Cytochrome c is stored within the
mitochondrial inter-membrane space and, in response to
proapoptotic stimuli, is released into the cytoplasm where
it activates executioner caspases such as caspase-3/7.3
Furthermore the tumor cell mitochondrion is functionally
and structurally distinct from those in non-cancerous
cells, which provides an opportunity for selective cytotox-
icity.2 The atypical phospholipid cardiolipin (CL) regu-
lates apoptosis by modulating the release of cytochrome c
from the mitochondrial membrane.4
et al. found that epoxides of docosahexaenoic acid inhib-
ited primary tumor growth and metastasis in tumor-
bearing mice.10 Similarly, the ω-3-17,18-epoxide metabolite
of eicosapentaenoic acid (ω-3-17,18-epoxy-EPA) inhibited
cell viability by arresting cell cycle progression and acti-
vating apoptosis.11 Development of fatty acid epoxides as
therapeutics is complicated by their in vivo instability,
including degradation by soluble epoxide hydrolase
(sEH).12 Indeed, coadministration of the sEH inhibitor
trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-
benzoic acid promotes in vivo anti-tumorigenic activity
of ω-3-epoxydocosahexaenoic acids,10 and adamantyl-urea
sEH inhibitors mimic the action of epoxides independent
of sEH inhibition, possibly by interacting directly with a
putative EET receptor.13 An alternate approach is re-
placement of the unstable epoxide moiety with bioisoster-
ic functionalities such as the corresponding ureas. Indeed,
this technique has been used to develop more metaboli-
cally stable mimetics of ω-314 and ω-615-19 epoxides, and
one such compound is a potential anti-arrhythmic agent
now in Phase 1 clinical trials.20 In addition to mimicking
the parent epoxides, these ureas may also inhibit sEH and
amplify the in vivo effects of endogenous epoxides.12
Interest in the capacity of lipids and lipid metabolites to
modulate cellular homeostasis is increasing.5,6 The al-
kylphospholipid analogues, typified by miltefosine and
perifosine, have found clinical application in the treat-
ment of amebal infections and high-risk neuroblastoma.7,8
Polyunsaturated fatty acids undergo biotransformation to
metabolites that modulate intracellular signalling and the
viability of cancer cells. Thus, the ω-6 arachidonic acid is
converted by cyclooxygenases and cytochromes P450 to
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(P<0.05) of control (Figure 2A) and longer-term treat-
ment for 48 h produced a more pronounced decrease to
30±5% (P<0.001; Figure S1A in Supporting Information).
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To substantiate the decrease in ATP viability in 13b-
treated cells we assessed the impact of the aryl-ureas on
cell cycle kinetics using flow cytometry. Treatment of
MDA-MB-231 cells with 13b (10 μM, 24 h) produced a
marked increase in the proportion of cells in subG1-phase
and decreases in cells in G0G1-, S- and G2M-phases (Figure
S1B in Supporting Information). In contrast, the inactive
analogues 2, 4b and 11b minimally altered cell cycle dis-
tribution (Figure S1C in Supporting Information). Using
crystal violet staining 13b, but not 4b or the disubstituted
analogues 11b and 12b, decreased cell adherence and via-
bility (Fig S1D in Supporting Information). This indicates
that 13b selectively decreases the viability of MDA-MB-231
cells by impairing energy production and cell cycle pro-
gression.
Figure 1. Chemical structures of the saturated fatty acid
epoxide 1 and alkyl urea 2.
Using ω-3-17,18-epoxy-EPA as a lead compound, we re-
cently reported the synthesis of the saturated ω-3-17,18-
epoxyeicosanoic acid (1, Figure 1). 1 retained the antipro-
liferative and proapoptotic activities of ω-3-17,18-epoxy-
EPA in breast cancer cells.21 Interestingly, Falck et al
found that functional epoxide mimetics retained at least
one double bond.14-15 Whether 1 exerts its anticancer ac-
tion by the same mechanism as ω-3-17,18-epoxy-EPA, or
functions by a different mechanism, is the focus of ongo-
ing investigation. The epoxide ring in 1 presents a meta-
bolic liability. We previously evaluated simple alkyl-urea
isosteres, including 2 (Figure 1), but these were relatively
non-potent and only minimally altered breast cancer cell
viability in vitro.21 In the present study we report the de-
velopment of aryl-urea analogues of 2 that also possess a
ω-terminal phenyl ring, a structural moiety used in stabi-
lized lipid analogues to prevent ω- and ω-1 hydroxyla-
tion.5 We found that analogue 13b markedly decreased
the viability of MDA-MB-231 breast cancer cells in vitro
and in vivo when administered to nude mice carrying
MDA-MB-231 xenografts. In mechanistic studies, we
found that 13b targeted the mitochondrion and decreased
CL content in tumor cells, which promoted apoptotic cell
death. Considered together, 13b has emerged as the pro-
totype of a new class of lipid-derived agents with poten-
tial anticancer activity.
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Scheme 1. Synthesis of aryl ureas 4b – 13b.
RESULTS
Reagents and conditions: (i) aryl isocyanate, rt, 2 h; (ii)
1.5M NaOH, ethanol, rt, 3 h.
Synthesis of aryl-urea fatty acids. A series of aryl-
ureas was prepared according to our previously reported
synthesis of the alkyl-urea 2 (Scheme 1).21 Briefly, the ethyl
ester-protected amino fatty acid 3 was reacted with aryl
isocyanates under anhydrous conditions to afford the
aryl-ureas 4a – 13a, which were readily purified on silica
gel in good yields. The ethyl ester protecting groups were
subsequently removed by base-catalysed hydrolysis, and
acidification of the reaction mixture resulted in precipita-
tion of carboxylic acids 4b – 13b that were isolated by fil-
tration in excellent yield and purity.
Activation of apoptosis by aryl-urea 13b. Because 13b
increased the proportion of cells in subG1-phase (20±3%
versus 1.4±0.03% in control) we tested its capacity to acti-
vate apoptosis in MDA-MB-231 cells. 13b (10 µM) strongly
increased the activity of the executioner caspases-3/7 after
24 h to 203±2% of control (P<0.001; Figure 2B), but the
other analogues were inactive, even after 48 h of treat-
ment (Figure 2B, Figure S2A in Supporting Information).
To further substantiate the capacity of 13b to produce
cell death, MDA-MB-231 cells were subjected to annexin
V-FITC/7-aminoactinomycin D (annexin V/7AAD) stain-
ing. Treatment of MDA-MB-231 cells with 13b for 48 h
increased the proportion of cells that exhibited early
apoptosis (annexin V staining alone, quadrant E4: 11±2%
of cells versus 6.6±1.4% in control, P<0.001; Figure 2C)
and late apoptosis (dual staining, quadrant E2: 20±4%
versus 5.9±0.9% in control, P<0.01; Figure 2C). The in-
crease in 7AAD-stained cells (quadrant E1; 5.9±2.1% versus
1.5±0.2% in control, P <0.001) is consistent with DNA
staining following cell membrane disruption. As expected,
Impaired ATP production by an aryl-urea in breast
cancer cells. The capacity of the urea-based analogues 2
and 4b – 13b to decrease ATP production was assessed in
the highly aggressive MDA-MB-231 breast cancer cell line.
Consistent with previous findings the alkyl urea 2,21 and
also aryl-ureas 4b – 12b, were inactive after 24 h or 48 h of
treatment (Figure 2A, Figure S1A in Supporting Infor-
mation), but analogue 13b, which carries a 4-chloro, 3-
trifluoromethyl substituted aromatic ring (Scheme 1),
effectively decreased ATP production. Thus, after 24 h,
13b (10 μM) decreased ATP production to 72±14%
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13b targets the mitochondrial membrane in MDA-
the proportion of unstained viable cells (quadrant E3) was
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lower after 13b treatment (62.4±5.9% versus 86.2±2.3% in
control, P<0.001; Figure 2C). In contrast, annexin V/7AAD
staining of MDA-MB-231 cells was unaltered after treat-
ment with the inactive analogues 2, 4b and 11b (Figure
S2B in Supporting Information).
MB-231 cells. The capacity of the active agent 13b to im-
pair ATP production and to activate caspase-3/7 suggest-
ed that the mechanism of action involves mitochondrial
disruption in MDA-MB-231 cells. This possibility was test-
ed using the membrane-permeable redox-active cationic
dye JC-1. JC-1 monomers fluoresce green in the cell cyto-
plasm but form aggregates that fluoresce red in the elec-
tronegative environment of the intact inner mitochondri-
al membrane. 13b treatment (10 µM, 24 h) decreased the
red:green fluorescence ratio in JC-1 stained cells (to 32±3%
of control; P<0.001; Figure 2D), consistent with mito-
chondrial impairment and disruption. By comparison,
inactive analogues, that minimally altered other end-
points of cell viability, did not affect the JC-1 ratio. In
accord with these findings, fluoresence microscopy in
cells that were treated with 13b (10 µM, 6 h), showed a
striking increase in JC-1 monomers (green) and a decrease
We tested the capacity of 13b to impair the viability of
other cell lines. As shown in Figure S2C (in Supporting
Information), 13b decreased ATP production in T47D,
MDA-MB-468 and MCF-7 breast cancer cells, especially
after longer-term treatments. 13b also increased caspase-
3/7 activity in T-47D cells, but not MDA-MB-468 or MCF-
7 cells (Figure S2D in Supporting Information). Im-
portantly, 13b did not impair the viability of well-
differentiated MCF-10A breast cells (Figure S2E in Sup-
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porting
Information).
in
JC-1
aggregates
(red;
Figure
2E).
Figure 2. (A) Effects of 2, 4b-11b and 13b (10 µM, 24 h) on ATP formation by MDA-MB-231 cells; basal ATP formation in
MDA-MB-231 cells at 24 h was 0.34±0.02 nmol/7.5 x 104 cells. (B) Effects of 2, 4b-11b and 13b on caspase-3 activity in MDA-
MB-231 cells (10 µM, 24 h); basal caspase-3 activity at 24 h was 1.5±0.2 Units/7.5 x 104 cells. (C) annexin V/7AAD staining in
MDA-MB-231 cells after treatment with 13b (10 µM; 48 h). Black: 13b-treated cells, Blue: control cells (CTL). (D) JC-1
red:green fluorescence ratio in MDA-MB-231 cells (10 µM; 24 h). (E) MDA-MB-231 cells were treated with DMSO (panels i-
iii) or 13b (10 µM, 6 h, panels iv-vi), washed twice with serum-free medium and then incubated with JC-1 in serum-free
medium (37°C, 20 min) and subjected to fluorescence microscopy; cells were counter-stained with Hoechst 33342. All data
are mean±SEM from three separate experiments. Different from DMSO-treated control: *P<0.001, †P<0.01, ‡P<0.05.
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The mitochondrial actions of 13b were assessed further. caspase-3/7 activity and apoptotic cell death (Figure 3C).
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CL is an atypical phospholipid that maintains the integri-
ty of the mitochondrial membrane; mitochondrial disrup-
tion releases CL and initiates apoptosis.4 Addition of 13b
to MDA-MB-231 cells decreased the mitochondrial con-
tent of CL and its precursor phosphatidylglycerol to
61±7% of control (Figure 3A); in contrast, CL was not de-
pleted by the inactive analogue 2. The decrease in mito-
chondrial CL/phosphatidylglycerol was selective because
no change in the plasma membrane phospholipid phos-
phatidylcholine was produced in 13b-treated cells (88±7
µg/1.05 x 107 cells, compared with 91±9 µg/1.05 x 107 cells
in control). Addition of the monounsaturated fatty acid
oleic acid has been reported to prevent cellular CL deple-
tion by the saturated palmitic acid (0.1 mM) in MDA-MB-
231 breast cancer cells.22 In the present study, oleic acid
As part of the present study we also assessed other poten-
tial mechanisms that could operate in 13b-mediated
MDA-MB-231 cell death. However, neither the Ca2+ chela-
tor BAPTA-AM (5, 25 µM; Figure S3B in Supporting In-
formation), or the mitochondrial permeability transition
pore inhibitor cyclosporin A (10, 20 µM; Figure S3C in
Supporting Information) altered the 13b-mediated de-
crease in ATP formation. We also found no evidence that
13b increased the production of reactive oxygen species in
MDA-MB-231 cells. Flow cytometry with 2’,7’-
dichlorofluorescin diacetate revealed no increase in pro-
oxidant stress after 2-6 h of treatment with 13b and co-
treatment with α-tocopherol or other antioxidants did
not prevent the 13b-mediated loss of cell viability (not
shown). Together, these findings strongly implicate the
loss of mitochondrial CL in the mechanism of 13b-
mediated cell killing. It is possible that alternate mecha-
nisms may operate but we found no evidence for effects of
13b on Ca2+ homeostasis, production of reactive oxygen
species or the mitochondrial permeability transition pore.
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protected
MDA-MB-231
cells
against
CL/phosphatidylglycerol depletion elicited by 13b (Figure
3B). In parallel, oleic acid treatment also prevented the
13b-mediated
increase
in
Figure 3. (A) 13b but not the inactive analogue 2, decreased the mitochondrial content of CL and its precursor phosphatidyl-
glycerol in MDA-MB-231 cells. (B) oleic acid protected MDA-MB-231 cells against CL/phosphatidylglycerol depletion elicited by
13b. (C) oleic acid prevented the 13b-mediated increase in caspase-3/7 activity and apoptotic cell death. (D) Dose-dependent
effects of 13b on the in vivo growth of MDA-MB-231 cell xenografts after intraperitoneal administration. Female nu/nu mice (7
weeks of age; n=5-8 per group) received 4x105 cells/100µl by subcutaneous injection into the 4th mammary fat pad. Body weights
and tumor sizes were measured. (E) Histological analysis showing the increase in TUNEL (apoptosis) and decrease in Ki67 (pro-
liferation) staining in sections from 13b-treated and control mice. DAPI (4′,6-diamidino-2-phenylindole) was used to stain nu-
clei. All data from cell experiments are mean±SEM from three separate experiments. Different from control: *P<0.001, †P<0.01,
‡P<0.05. Scale bar: 50 µm.
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staining in 13b-treated tumor sections compared with
In vivo activity of 13b in mice carrying MDA-MB-231
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breast cancer xenografts. 13b emerged from in vitro
testing as a novel molecule with potentially promising
anticancer properties. Further evaluation was undertaken
in animal models. In initial experiments 13b was found to
be well tolerated in Balb/c mice (2.5-40 mg/kg ip, 5 days
treatment). Weight gain was normal in 13b-treated ani-
mals and there were no other indications of toxicity.
control tumors is consistent with the evidence for in-
creased apoptosis in cells in vitro.
It has been proposed recently that the cancer cell mito-
chondrion could be a novel therapeutic target because the
organelle is structurally and functionally different from
those in normal cells.2 There are fewer mitochondria in
cancer cells and, whereas oxidative phosphorylation is the
major pathway of ATP generation in normal cells, in high-
ly aggressive tumors cells ATP generation occurs primari-
ly by aerobic glycolysis (the Warburg effect).23 Instead, the
cancer cell mitochondrion is adapted to increased pro-
duction of macromolecules required for rapid cell replica-
tion.23 The mitochondrion is also a critical regulator of
apoptotic cell death and in cancer cells these pathways
are frequently less responsive to cytotoxic agents. Thus,
the development of novel agents that selectively target
the mitochondrion in tumor cells could lead to the devel-
opment of new therapeutic strategies.2,23
The capacity of 13b to kill MDA-MB-231 cells in vivo was
assessed in xenografted nu/nu mice. As shown in Figure
3D, a dose-dependent decrease in tumor volume was not-
ed from 32 days of treatment. In addition, increased
TUNEL staining, consistent with activation of apoptosis,
and decreased staining for the proliferative marker Ki67,
were noted in isolated tumors (Figure 3E). Taken togeth-
er, 13b is active in vivo and decreases tumorigenesis from
MDA-MB-231 xenografts by inhibiting proliferation and
increasing apoptosis.
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DISCUSSION
The redox active dye JC-1 fluoresces red in cells whose
mitochondria have intact membranes and fluoresces
green when in the cytoplasm or when the mitochondrial
membrane potential is disrupted. Studies with JC-1
showed that the active analogue 13b, but not the inactive
analogues 2 and 4b-11b, targeted MDA-MB-231 cell mito-
chondrion and rapidly depolarized the inner membrane.
There is some evidence that other oncology drugs may
interact with mitochondria in breast cancer cells, but the
kinetics and selectivity of mitochondrial impairment ap-
pear to be somewhat different from that produced in
MDA-MB-231 cells by 13b. Thus, the estrogen receptor
antagonist tamoxifen (~1 µM) impaired the mitochondrial
membrane potential in MCF-7 tumor cells after ~1 h, but
also in normal mammary epithelial cells.24-25 Similar find-
ings were made with doxorubicin, however the duration
of treatment required for mitochondrial disruption was
relatively long at ~24-72 h.26-27 In comparison, the present
findings indicate that 13b disrupted the MDA-MB-231 cell
mitochondrion within 6 h (Figure 3E). We also found that
the multikinase inhibitor sorafenib disrupted the mito-
chondrion in these cells (not shown). Because 13b and
sorafenib contain the same aryl substitution pattern the
role of that feature in mitochondrial impairment should
now be evaluated in greater detail.
The development of novel well-tolerated anti-cancer
agents could provide valuable options in the treatment of
cancer patients. This study has identified aryl-urea 13b as
the first in a new class of potential anti-cancer agents
with activity against MDA-MB-231 and other tumor cells.
13b did not produce observable toxicity in mice or impair
the viability of well-differentiated breast-derived MCF10A
cells.
In previous studies, we reported that the saturated
epoxy fatty acid 1 was antiproliferative and proapoptotic
in MDA-MB-231 breast cancer cells.21 Replacement of the
epoxide group in 1 with an alkyl urea group produced
analogues like 2 that exhibited very low anti-cancer activi-
ty in cells.21 In the present study, we show that incorpora-
tion of a 3-trifluoromethyl, 4-chloro-substituted terminal
aromatic system into 2 produced the novel aryl-urea 13b
that had significant anticancer activity in vitro and in vivo.
In contrast, several other 4-mono- and 3,4-disubstituted
analogues were inactive.
Arrest of MDA-MB-231 cells in S- and G2M-phases by
13b, and the failure of cells to complete mitosis, is con-
sistent with observed impairments in cellular ATP pro-
duction. In addition to anti-proliferative actions 13b also
activated cell killing, as reflected by the increases in subG1
phase, caspase-3/7 activity and annexin V/7AAD staining.
Apoptosis appears to be the major initial death mecha-
nism that is activated in 13b-treated cells, although plas-
ma membrane disruption following prolonged treatment
enables DNA staining by 7AAD, and is consistent with
extensive tumor cell destruction.
In the present study 13b decreased the mitochondrial
content of CL and its precursor phosphatidylglycerol in
MDA-MB-231 cells. This was selective for CL because no
change in plasma membrane phosphatidylcholine content
was noted. Cellular CL depletion was mechanistically im-
portant because supplementation by oleic acid protected
MDA-MB-231 cells against 13b-mediated CL depletion and
apoptotic cell death. Alternate hypotheses for the actions
of 13b are possible. 13b is structurally similar to aryl-urea-
based sEH inhibitors that recapitulate the cellular effects
of fatty acid epoxides by slowing their degradation.12 13b
may also possess surface activity that could disrupt the
mitochondrial membrane. However these possibilities are
unlikely because the inactive analogues 4b – 12b are
structurally similar to 13b.
13b was assessed for in vivo efficacy in female nu/nu
mice that had been inoculated with MDA-MB-231 breast
cancer cells in mammary fat pads. Tumor volume was
decreased in a dose-dependent fashion during treatment
with 13b. In untreated tumors Ki67 staining was extensive
but was decreased in tumor sections from 13b-treated
mice, consistent with the antiproliferative actions of 13b
seen in vitro. In addition, the increase in positive TUNEL
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This study was supported by grants from the Australian Na-
tional Health and Medical Research Council (1031686 and
1087248).
Taken together, 13b has emerged as the first in a new
class of agents with activity against cancer cells produced
by targeting of the tumor cell mitochondrion and CL de-
pletion. Further studies are now warranted to define the
structural requirements that underlie the anti-cancer ac-
tivity of 13b analogues so that the therapeutic potential of
this novel class of agents may be optimized.
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ACKNOWLEDGMENT
We would like to thank Martin Conolly for his assistance
with the synthetic chemistry, Julie Dwyer and Sarah Cui for
technical assistance, and Prof Christine Clarke, The West-
mead Institute, Westmead Hospital, NSW, Australia, for the
generous gift of MCF10A cells.
CONCLUSION
9
We have developed the first member of a promising
new class of fatty acid-based anticancer agents. The active
analogue 13b depolarised the mitochondrial membrane in
MDA-MB-231 breast cancer cells leading to extensive cell
death. After intraperitoneal injection 13b was also effec-
tive in breast cancer cell killing in a mouse xenograft
model.
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ABBREVIATIONS
7AAD, 7-aminoactinomycin; CL, cardiolipin; EET, epoxyeico-
satrienoic acid; ω-3-EEA, sEH, soluble epoxide hydrolase, ω-
3-17,18-epoxyeicosanoic acid; JC-1, 5,5’,6,6’-tetrachloro-
1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine
iodide;
TUNEL, terminal deoxynucleotidyl transferase nick end la-
beling.
EXPERIMENTAL SECTION
The purity of all test compounds was confirmed to be ≥
95% by elemental analysis.
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To a suspension of the amine 3 (1.33 mmol) in anhy-
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ASSOCIATED CONTENT
Supporting Information. Information on general chemis-
try, analytical data for all compounds and methods for cell
culture, cell-based assays, flow cytometry, animal experi-
ments and statistics. This material is available free of charge
via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Dr Tristan Rawling, School of Mathematical and Physical
Sciences, Faculty of Science, University of Technology Syd-
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Email: Tristan.Rawling@uts.edu.au.
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hibit angiogenesis, tumor growth, and metastasis. Proc. Natl. Acad.
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Author Contributions
All authors contributed to manuscript writing and have given
approval to the final version of the manuscript.
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