Carbon Chain Length Modulates MDA-MB-231 Breast Cancer Cell Killing Mechanisms by Mitochondrially Targeted Aryl?Urea Fatty Acids

Article abstract of DOI:10.1002/cmdc.201900577

Targeting the tumor cell mitochondrion could produce novel anticancer agents. We designed an aryl?urea fatty acid (1 g; 16({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)hexadecanoic acid) that disrupted the mitochondrion and decreased MDA-MB-231 breast cancer cell viability. To optimize the aryl?ureas the present study evaluated mitochondrial targeting by 1 g analogues containing alkyl chains between 10–17 carbons. Using the dye JC-1, the C12?C17 analogues efficiently disrupted the mitochondrial membrane potential (IC₅₀s 3.5±1.2 to 7.6±1.1 μM) and impaired ATP production; shorter analogues were less active. 7-Aminoactinomycin D/annexin V staining and flow cytometry showed that these agents activated the killing mechanisms of necrosis and apoptosis to varying extents (7-aminoactinomycin D/annexin V staining ratios 4.3–6.0). Indeed, 1 g and its C17 analogue preferentially activated necrosis and apoptosis, respectively (ratios 2.1 and 16). Taken together, alkyl chain length is a determinant of mitochondrial targeting by aryl?ureas and can be varied to develop analogues that activate apoptosis or necrosis in a regulated fashion.

Full text of DOI:10.1002/cmdc.201900577

Accepted Article
Title: Carbon chain length modulates MDA-MB-231 breast cancer cell
killing mechanisms by mitochondrially targeted aryl-urea fatty
acids
Authors: Michael Murray, Ariane Roseblade, Yongjuan Chen, Kirsi
Bourget, and Tristan Rawling
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Carbon chain length modulates MDA-MB-231 breast cancer cell
killing mechanisms by mitochondrially targeted aryl-urea fatty
acids
Prof. Michael Murray*[a], Dr. Ariane Roseblade[b], Dr. Yongjuan Chen[a], Kirsi Bourget[a] and Dr. Tristan
Rawling[b]
[a]
Prof. M. Murray*, Dr. Y. Chen, K. Bourget
Discipline of Pharmacology, School of Medical Sciences, Faculty of Medicine and Health
University of Sydney
Camperdown, New South Wales, 2006, AUSTRALIA
*E-mail: michael.murray@sydney.edu.au
[b]
Dr. A. Roseblade, Dr. T. Rawling
School of Mathematical and Physical Sciences, Faculty of Science
University of Technology Sydney
Ultimo, New South Wales, 2007, AUSTRALIA
Supporting information for this article is given via a link at the end of the document
Abstract: Targeting the tumor cell mitochondrion could produce
novel anti-cancer agents. We designed an aryl-urea fatty acid (1g;
16({[4-chloro-3-
modulate homeostasis.[5] Cytochromes P450 (CYPs) have been
studied extensively for their roles in the oxidative
biotransformation of drugs and diverse foreign compounds,[6-9]
but CYPs are also active in endobiotic metabolism, including
PUFA epoxidation. Epoxyeicosatrienoic acids are produced by
the actions of CYPs on the ω-6 PUFA arachidonic acid and have
been shown to promote proliferation and protect cells against
deleterious stimuli;[10] in contrast, certain ω-3 PUFA epoxides
are anti-proliferative.[11,12] Thus, we found that the CYP-derived
ω-3-17,18-epoxide of eicosapentaenoic acid and its fully-
saturated synthetic analogue ω-3-17,18-epoxyeicosanoic acid
impaired the viability of breast cancer cells by decreasing
proliferation and activating apoptosis.[11,13] Epoxides of ω-3
docosahexaenoic acid (22:6) are also reported to prevent tumor
(trifluoromethyl)phenyl]carbamoyl}amino)hexadecanoic acid) that
disrupted the mitochondrion and decreased MDA-MB-231 breast
cancer cell viability. To optimize the aryl-ureas the present study
evaluated mitochondrial targeting by 1g analogues containing alkyl
chains between 10-17 carbons. Using the dye JC-1, the C12-C17
analogues efficiently disrupted the mitochondrial membrane potential
(IC50s 3.5±1.2 to 7.6±1.1 µM) and impaired ATP production; shorter
analogues were less active. 7-Aminoactinomycin D/annexin
V
staining and flow cytometry showed that these agents activated the
killing mechanisms of necrosis and apoptosis to varying extents (7-
aminoactinomycin D/annexin V staining ratios 4.3-6.0). Indeed, 1g
and its C17 analogue preferentially activated necrosis and apoptosis, progression.[12]
respectively (ratios 2.1 and 16). Taken together, alkyl chain length is
a determinant of mitochondrial targeting by aryl-ureas and can be
varied to develop analogues that activate apoptosis or necrosis in a
regulated fashion.
ω-3 Fatty acid epoxides are active in vivo provided they are
coadministered with a soluble epoxide hydrolase inhibitor to
protect the labile epoxide group from enzymic hydration and
degradation.[14,15] Alternately, bioisosteric replacement of the
epoxide moiety with urea has been used to generate epoxide
mimetics that retain biological activity in the absence of soluble
epoxide hydrolase inhibitors.[16,17] Thus, we recently reported the
development of an aryl-urea mimetic of ω-3-17,18-
epoxyeicosanoic acid, termed 1g (Figure 1, n=9), that targeted
the mitochondrion and decreased the viability of MDA-MB-231
breast cancer cells in vitro, and inhibited tumour growth in vivo in
mice carrying MDA-MB-231 cell xenografts.[18]
1g is a promising new lead compound for anti-cancer drug
development, but its optimisation requires information on the
structural requirements for activity. Structure-activity relationship
studies have identified two pharmacophoric groups in the drug
scaffold: the aryl system substituted with strongly electron-
withdrawing substituents,[18,19] and the carboxylate group that
can be replaced with bioisosteric hydroxamic acid, oxo-
thiadiazole or sulfonate moieties.[20] In 1g these two
pharmacophores are linked together by a saturated 16 carbon
Introduction
Molecules that kill cancer cells by novel mechanisms could lead
to the development of new anti-cancer agents and agents that
enable drug resistance to be overcome when used in
combination with conventional oncology drugs. The
mitochondrion controls energy metabolism and cell proliferation,
as well as cell death and survival.[1] These mechanisms are
regulated differently in tumor cells, which creates the opportunity
for the development of new anticancer agents that selectively
target cancer cell mitochondria to induce cell death.
ω-3 Polyunsaturated fatty acids (ω-3 PUFAs), such as
eicosapentaenoic acid (ω-3 C20:5), decrease tumorigenesis
while the corresponding ω-6 PUFA, like arachidonic acid,
promote tumour development.[2-4] Thus, ω-3 PUFA could be
utilized in novel anti-cancer drug development programs. In cells
PUFA undergo biotransformation to diverse lipid mediators,
including prostaglandins, leukotrienes and epoxides, that
alkyl chain. In the present study we explored
a chain
extension/contraction strategy to identify the optimal chain
length for antiproliferative activity. A series of 1g analogues with
10 (1a) to 17 (1h) carbon alkyl chains between the aryl urea and
1
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carboxylate groups were prepared (Figure 1), and their activity
was assessed in human MDA-MB-231 cells. The principal
findings to emerge were that analogues of intermediate and
longer chain length (C12-C17) effectively disrupted the
mitochondrial membrane potential, impaired ATP production and
promoted cell death.
JC-1 remains in its monomeric form and fluoresces green. We
assessed the capacity of alkyl chain length-modified aryl-ureas
based on 1g to disrupt the mitochondrial membrane potential in
MDA-MB-231 cells using JC-1 staining. Treatment of cells with
1g analogues of differing chain length (1 h) decreased the JC-1
red:green fluorescence ratio in cells, consistent with membrane
depolarisation. Efficient mitochondrial disruption was produced
by the analogues 1c to 1h (IC50 range 3.5±1.2 µM to 7.6±1.1
µM; Figure 2) whereas the analogues with the shortest alkyl
chains (1a and 1b) were much less effective (IC50s 99±1 and
113±1 µM, respectively). In accord with these findings,
fluorescence spectroscopy showed that treatment (10 µM, 4 h)
of MDA-MB-231 cells with all analogues, especially 1c to 1h,
decreased the red fluorescence intensity and increased green
fluorescence intensity (Figure 3; Figure S1).
Figure 1. Chemical structures of lead aryl-urea substituted fatty acid 1g (n=9)
and analogues of varying alkyl chain length.
Results
Synthesis of aryl-urea analogues with altered alkyl chain
length
The synthesis of 1g was reported previously.[18] The procedure
used to prepare the chain extension/contraction series was
dependent on the commercial availability of starting materials
(Scheme 1). Syntheses of analogues with 10, 11 and 12 carbon
chains (1a, 1b and 1c, respectively) commenced from
commercially available bromo-fatty acids, which had been ethyl
ester-protected (2-4).[21] In the first step 2-4 were reacted with
NaN3 to generate azides 6-8. Staudinger reduction of the azides
with triphenylphosphine and water yielded amines 10-12, which
were reacted with 4-chloro-3-(trifluoromethyl)phenyl isocyanate
to generate ureas 17a – 17c. Removal of the ester protecting
groups with NaOH gave 1a, 1b and 1c. The 15-carbon analogue
1f was prepared similarly from the ester-protected hydroxy-fatty
acid 5, which was converted to azide 9 under Mitsunobu
conditions.[22]
Due to the limited commercial availability of appropriate hydroxy-
or bromo-fatty acids 1d, 1e and 1h were prepared by an
alternate route that commenced from the nitriles 14-16. We
previously reported the synthesis of 14-16 by alkyl−alkyl Negishi
cross-coupling reactions catalyzed by the N-heterocyclic
carbene
[1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene](3-
chloropyridyl)palladium(II) dichloride.[21] Reduction of the nitrile
groups using nickel boride, which was generated in situ using
sodium borohydride and
a catalytic amount of nickel(II)
chloride,[21] produced crude amines that were immediately
reacted with 4-chloro-3-(trifluoromethyl)phenyl isocyanate to
give 17d, 17e and 17h. Subsequent hydrolysis of the ester
protecting groups gave 1d, 1e and 1h.
Alkyl chain length in aryl urea fatty acids influences
mitochondrial targeting in MDA-MB-231 cells
Scheme 1. Synthesis of 1g analogues in which the alkyl chain length was
varied. Reagents and conditions: (a) NaN3, DMSO, rt, 18 h; (b) PPh3,
diisopropyl azodicarboxylate, 0°C − rt, 30 min, diphenyl phosphoryl azide, rt,
4.5 h; (c) PPh3, rt, 8 h, then H2O, rt, 16 h; (d) 4-chloro-3-(trifluoromethyl)phenyl
isocyanate, anhydrous THF, rt, 2 h; (e) NiCl2·6H2O, NaBH4, 0°C − rt, 2 h, and
then 4-chloro-3-(trifluoromethyl)phenyl isocyanate; (f) 1.5M NaOH,
ethanol/water, rt, 4 h.
Using the membrane-permeable redox-active cationic dye JC-1
the aryl-urea 1g has been found previously to target the
mitochondrion in MDA-MB-231 cells.[18] In the electronegative
environment of the intact inner mitochondrial membrane JC-1
forms aggregates that fluoresce red. In cells containing
damaged mitochondria that have lost their membrane potential,
2
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with analogues from 1b to 1h (58.3-71.6% of total cells versus
80.7±0.3% of total in DMSO-treated control cells; Figure 5A,
Figure S3; Table 2); similar findings were produced by 48 h of
treatment (Table S1). In accord with these findings the active
analogues increased both 7AAD and annexin
V staining,
consistent with increases in early necrosis and apoptosis,
respectively, but to varying extents (Table 2). The most
pronounced increase in 7AAD-stained cells to ~5-fold of control
was effected by 1g itself, with lesser increases by 1b, 1c and 1f
and trends toward an increase in staining produced by other
analogues (Table 2).
In contrast with these findings, 1h did not increase 7AAD
staining over control. Instead, this analogue produced a strong
increase in annexin V staining to ~2.4-fold of control at 24 h
(22.6±2.6% versus 9.5±0.5% in control; Table 2, P<0.001);
smaller increases in annexin V staining were effected by shorter
chain analogues. Increased dual staining by annexin V/7AAD,
reflecting late necrosis and apoptosis, was produced by most
analogues after 24 h of treatment. These findings suggest that
the balance between the death mechanisms of apoptosis and
necrosis in 1g analogues varies according to alkyl chain length
(Figure 5B). Thus, although the shortest analogue (1a) had
relatively low activity, 1b to 1f activated both necrosis and
apoptosis, resulting in annexin V/7AAD staining ratios in the
range 4.3-6.0 at 24 h, relative to control (ratio 6.5; Table 2);
similar ratios were produced by 48 h of treatment (Table S1).
There was a noticeable difference in the apparent killing modes
of the two longest chain analogues from a pronounced activation
of necrosis for 1g, as evidenced by strong 7AAD staining and a
Figure 2. Mitochondrial targeting (JC-1 IC50s) by 1g analogues of varying alkyl
chain length. Data are means±SD obtained using 6-7 individual concentrations
in at least triplicate experiments
Impaired ATP production by 1g analogues in breast cancer
cells
The impact of alkyl chain length-modified aryl-ureas on the
viability of MDA-MB-231 cells was assessed using a series of
approaches. ATP production was impaired significantly by all
analogues (10 µM, 48 h), with the exception of 1a (Figure 4A).
Decreases were most pronounced for 1d, 1f and 1g to 25±2%,
40±11% and 39±5% of control (P<0.001). In the control breast
cell line MCF10A 1g did not impair ATP production or cell
viability.[18]
Because ATP is required for cell proliferation we next used flow
cytometry to evaluate the impact of the alkyl chain-modified aryl-
ureas on cell cycle kinetics. Significant decreases in the
population of cells in G0G1 phase, which reflects the proportion
of cycling cells, were produced by analogues with alkyl chains of
at least 13 carbons (range 54-67% versus 76% in control; Table
1; Figure 4B, Figure S2). All analogues, especially 1b and longer,
decreased the proportion of cells in G2M phase (4.4-6.8% versus
9.3% in control; Table 1, P<0.001). In addition, analogues 1c-1e
containing alkyl chains of 12-14 carbons in length increased the
proportion of cells accumulating in S-phase (9.6-11.4% versus
6.5% in control; Table 1, P<0.001). This suggests that
analogues with alkyl chains greater than 11 carbons decrease
cell viability in part by preventing completion of the cell cycle and
impairing mitosis.
smaller increase in annexin
V staining, to predominantly
apoptosis for 1h (strong annexin V and dual staining, with no
increase staining by 7AAD over control). This is shown by the
annexin V/7AAD staining ratios at 24 h of 2.1 and 16 for 1g and
1h, respectively (Table 2, Figure 5B); similar ratios were noted
after 48 h of treatment (Table S1).
Flow cytometry also provides an estimate of cells with low DNA
content (subG1 phase). As shown in Table 1, marked increases
in subG1 phase were effected by treatment with analogues
containing alkyl chains of 15-17 carbons (~5-fold of control;
P<0.001), while less pronounced increases were produced by
the C13 and C14 analogues to ~2-3-fold of control (P<0.001 and
P<0.01, respectively).
Figure 3. Fluorescence microscopy of MDA-MB-231 cells after treatment with
analogues 1a and 1h (C10 and C17 chains; 10 µM, 4 h) or DMSO (CTL) 16 h
after serum deprivation. After consecutive washes with serum-free medium,
cells were incubated with JC-1 and Hoechst 33258 (Hoechst) for 20 min,
according to the manufacturer’s instructions. Fluorescence images of JC-1
aggregates (red), JC-1 monomers (green) and nucleus staining (blue) on cells
were acquired with a fluorescence microscope (Olympus BX51, Notting Hill,
VIC, Australia). Images for analogues 1b-1g are shown in Fig S1
Activation of cell killing by chain length-modified aryl-ureas
The increase in subG1 phase suggests that longer chain
analogues promote cell killing, which depletes DNA content.
Mechanisms of cell killing were further assessed using staining
with annexin V-FITC/7-aminoactinomycin D (7AAD) in cells that
had been treated with 1a to 1h for 24 or 48 h. Decreases in the
unstained (live) cell population were noted after 24 h treatment
3
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pathway for ATP synthesis in normal cells, aerobic glycolysis
can predominate in aggressive tumor cells (the Warburg
effect).[23] Instead, the cancer cell mitochondrion is adapted for
the increased production of macromolecules that are necessary
for tumor cell replication.[1] The mitochondrion also has an
important role in cell death and survival; these pathways are
frequently over-active in cancer cells so that the cell killing
capacity of cytotoxic drugs is attenuated. New agents like 1g,
that selectively disupt the mitochondrion in tumor cells, while
leaving normal cell mitochondria undamaged, could target
dysregulated mechanisms in tumor cells and lead to new
therapeutic strategies.[1,2,23]
Through structure-activity relationship studies we have identified
two pharmacophoric groups in the aryl-urea fatty acid drug
scaffold. Effective agents contained a terminal aryl system with
strongly electron-withdrawing substituents.[18,19] In addition, the
carboxylate functionality is required for activity and can be
replaced with bioisosteric hydroxamic acid, sulfonic acid or
oxathiazole groups, but not by non-bioisosteric amide or ester
functionalities.[20] The present study evaluated the importance of
the alkyl chain length in the activity of 1g by preparing and
testing analogues with chains that varied from 10 to 17 carbons.
All of these aryl-urea fatty acid analogues disrupted the
mitochondrial membrane potential, as reflected by the shift in the
red:green fluorescence ratio with the dye JC-1. While the
shortest chain analogues (1a and 1b) were relatively non-potent
(IC50 ~100 µM), the intermediate and longer chain analogues (1c
to 1h) were much more effective (IC50s 3.5-7.6 µM). It has been
shown previously that other naturally occurring and synthetic
saturated and unsaturated lipids can penetrate the mitochondrial
membrane.[24] For example, α-eleostearic acid produced a loss
of mitochondrial membrane potential and activated mitochondrial
apoptosis in MDA-MB-231 breast cancer cells.[25] However, to
our knowledge, the present study is the first to formally relate
mitochondrial disruption to fatty acid structure in an homologous
series of lipid analogues.
The present aryl-urea fatty acids, especially 1d, 1f and 1g,
decreased cellular ATP production and arrested cell cycle
progression, which led to accumulation of cells in S phase and
the failure to complete mitosis. Longer chain analogues also
activated cell death mediated by the dual mechanisms of
apoptosis and necrosis, as reflected by increases in the
proportion of cells in subG1 phase and annexin V/7AAD staining.
Annexin V detects phosphatidylserine residues in the plasma
membrane that are externalized during apoptosis, while
disruption of the plasma membrane activates necrosis and
enables DNA staining by 7AAD. The annexin V/7AAD staining
ratio was used to quantify the relative importance of apoptosis
and necrosis. Analogues 1a to 1f produced ratios that were
similar to control, suggesting that both cell death pathways were
increased to similar extents. Interestingly, however, the ratios
produced by 1g and 1h were quite different (respectively 2.1 and
16 at 24 h, and 2.5 and 14 at 48 h) and are consistent with
pronounced activation of necrosis and apoptosis, respectively.
Taken together, these findings indicate that alkyl chain length
can be modified to optimise the anti-proliferative and cell killing
activities of 1g analogues in MDA-MB-231 breast cancer cells.
Our ongoing studies to identify the cellular target(s) of aryl-urea
fatty acids may eventually provide additional mechanistic
information to expand on the present findings.
Figure 4. (A) Effects of aryl-ureas 1a to 1h (10 µM, 48 h) on ATP formation by
MDA-MB-231 cells. Basal ATP formation was 0.63±0.02 nmol/7 x 104 MDA-
MB-231 cells/24 h. (B) Flow cytometric analysis of control (CTL) MDA-MB-231
cells and cells after treatment with aryl-ureas 1a and 1h (10 µM) for 48 h,
showing the decrease in G0G1 and increases in S and subG1 phases. All data
are mean±SEM from three separate experiments. Different from DMSO-
treated control: *P<0.05, **P<0.01, ***P<0.001. Histograms from cells treated
with 1b-1g are shown in Fig S2
Table 1. Alkyl chain length in 1g analogues modulates cell cycle kinetics in
MDA-MB-231 cells (48 h; 10 µM)
% cells
Analogue
subG1
G0G1
S
G2M
control
1a
5.97±0.49
75.7±1.0
6.50±0.40
5.47±0.33
7.23±1.19
9.60±0.86**
11.37±0.44***
10.13±0.73**
8.50±0.79
7.10±0.81
6.23±0.75
9.27±0.62
4.20±0.55
80.0±2.2
7.40±0.49**
6.47±0.38***
5.53±0.33***
6.78±0.20***
5.90±0.32***
5.30±0.70***
5.57±0.17***
4.37±0.33***
1b
7.37±1.03
76.3±2.1
1c
9.50±2.68
74.2±3.0
1d
20.63±2.24***
15.10±1.04*
30.63±4.49***
25.37±4.17***
31.73±2.71***
60.6±2.1***
67.2±1.2*
54.1±3.4***
59.6±3.3***
55.2±2.6***
1e
1f
1g
1h
Different from control: *P<0.05, **P<0.01, ***P<0.001
Discussion
The development of well-tolerated oncology agents with novel
mechanisms of action could provide therapeutic alternatives for
the treatment of cancer patients. The long-chain fatty acid aryl-
urea 1g was recently identified as the prototype of a new class
of agents that decrease the viability of MDA-MB-231 breast
cancer cells by mitochondrial targeting.[18] There are potential
novel drug targets in the cancer cell mitochondrion due to
structural and functional differences in the organelle from normal
cells.[1] Whereas oxidative phosphorylation is the primary
4
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It has been suggested that fatty acids may be useful adjunct
agents to augment the activity of chemotherapeutic drugs.
Previously we prepared a series of ω-3 monounsaturated fatty
acids of chain length C16-C22 that decreased proliferation and
increased apoptosis in MDA-MB-468 breast cancer cells that
overexpressed the enzyme cyclooxygenase-2 (COX-2).[21] In
COX-2-dependent breast and colorectal cancers the
administration of non-steroidal anti-inflammatory agents that
inhibit COX-2 activity has been found to be an effective
treatment, but toxicity has precluded their long-term use.[26,27] An
alternate approach could be co-treatment with anti-cancer lipids,
such as the ω-3 monounsaturated fatty acids, that may be better
tolerated. In designing such combination approaches the carbon
chain length in the lipid agent could be an important
consideration.[28]
The present findings suggest that chain length in aryl-ureas
determines not only the efficacy of mitochondrial targeting but
also the cell death mechanisms that are activated. Many
investigations have focused on apoptosis as a major mechanism
of tumor cell killing by oncology agents. More recently it has
emerged that other forms of cell death, such as necrosis, are
also activated by many established anti-cancer agents. Necrosis
is activated by anthaquinones such as doxorubicin, as well as
other cytotoxic agents like mitoxantrone, cisplatin and
melphalan.[29-31]
Necrotic
cells
are characterized
by
mitochondrial swelling and loss of plasma membrane integrity
without nuclear damage.[32] Recently, Shen et al. screened
~1400 compounds to identify their mode of cell killing, with many
agents activating either apoptosis or necrosis or both.[33]
A
number of fatty acids and fatty acid metabolites were evaluated
but the relationship between lipid structure and the mode of cell
killing remained unclear. The findings in the present study
suggest that the balance between the death mechanisms of
apoptosis and necrosis in aryl-urea analogues varies according
to chain length. Together, the present study with aryl-ureas
suggests that it may now be feasible to utilise aryl-ureas to
preferentially activate alternate death mechanisms in order to
optimise tumour cell killing.
Conclusion
Figure 5. (A) Annexin V-FITC/7AAD staining in control (CTL) MDA-MB-231
cells after treatment with 1a, 1g and 1h (10 µM) for 24 h. 7AAD-stained cells
(quadrant E1), annexin V-stained cells (quadrant E4), dual 7AAD and annexin
V-stained cells (quadrant E3) and unstained live cells (quadrant E2) are shown.
(B) Relationship between 7AAD stained and annexin V stained MDA-MB-231
cells by the aryl-ureas 1a to 1h (10 µM) for 24 h. Data were derived in three
separate experiments and representative images are shown. Cell staining
after treatment with 1b-1f are shown in Fig S3
In summary, we have prepared analogues of the novel anti-
cancer aryl-urea 1g in which the alkyl chain length was modified.
Analogues containing longer alkyl chains promoted cell killing
and decreased energy metabolism, whereas those with shorter
alkyl chains were less active. It also emerged that the longest
chain analogues - 1g and 1h - were not only effective in
mitochondrial targeting but also preferentially activated the cell
death mechanisms of necrosis and apoptosis, respectively.
These agents may prove useful in unravelling the relationships
between fatty acid chain length and selectivity in cell killing
mechanisms in aryl-ureas.
Table 2. Alkyl chain length in 1g analogues modulates cell cycle kinetics in
MDA-MB-231 cells (48 h; 10 µM) †
% cells
Analogue
7AAD
dual
live
annexin V
ratio
control
1a
1.43±0.09
1.73±0.37
3.40±0.50**
3.27±0.33**
2.67±0.49
2.60±0.49
3.43±0.67**
6.47±0.37***
1.43±0.09
8.7±0.6
80.7±0.3
9.5±0.5
6.50
5.95
4.33
5.90
5.66
5.69
5.39
2.07
15.80
Experimental Section
15.2±0.7**
17.8±1.76***
14.3±1.11***
10.9±0.03
72.7±2.4
10.3±2.3
14.7±0.8
19.3±2.2**
15.1±2.9
14.8±2.2
18.5±2.8**
13.4±0.7
22.6±2.6***
Chemistry
1b
64.2±1.6**
63.2±1.2***
71.6±2.4*
70.8±1.5*
64.0±3.4**
70.4±6.3*
58.3±4.3***
1c
General: 1g[18], ester 5[22], nitrile 14[34] and compounds 2, 3, 4, 15 and
16[21] were prepared as previously reported. All other reagents and
anhydrous solvents were purchased from Sigma Aldrich (Castle Hill,
NSW, Australia). Dry Column Vacuum Chromatography was used to
purify reaction products on silica gel with gradient elutions. TLC was
performed on silica gel 60 F254 plates. Melting points were measured on
a Stuart SMP10 melting point apparatus. 1H and 13C NMR spectra were
recorded on either a Varian 400-MR instrument or Agilent 500 MHz NMR.
Spectra were referenced internally to residual solvent (CDCl3; 1H δ 7.26,
13C δ 77.10. d6-DMSO; 1H δ 2.49, 13C δ 39.52). High resolution mass
spectrometry (HRMS) was performed on an Agilent Technologies 6510
Q-TOF LCMS.
1d
1e
12.0±1.16
1f
14.0±1.21**
13.9±1.83**
18.0±1.42***
1g
1h
Different from control: *P<0.05, **P<0.01, ***P<0.001
†7AAD, % cells stained by 7-aminoactinomycin D; dual, % cells stained by
both 7-aminoactinomycin D and annexin V; live, % unstained cells; annexin
V,
% cells stained by annexin V; ratio, ratio of cells stained by 7-
aminoactinomycin D to cells stained by annexin V
5
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10.1002/cmdc.201900577
ChemMedChem
FULL PAPER
General procedure for synthesis of azides from bromo esters
Ethyl 11-aminoundecanoate (11). White solid, 96% yield. 1H NMR (400
MHz, CDCl3): δ 4.10 (q, J = 7.2 Hz, 2H), 2.66 (t, J = 7.2 Hz, 2H), 2.26 (t,
J = 7.6 Hz, 2H), 1.59 (p, J = 7.6 Hz, 2H), 1.41 (p, J = 6.8 Hz, 2H), 1.20 –
1.35 (m, 15H). 13C NMR (100 MHz, CDCl3): δ173.91, 60.13, 42.19, 34.36,
33.73, 29.51, 29.43, 29.35, 29.21, 29.10, 26.84, 24.94, 14.22. HRMS
(ESI) m/z [M+H]+ calcd for C13H28NO2, 230.2114; found, 230.2110.
To a solution of the bromo ester (2-4, 20 mmol) in DMSO (75 mL) was
added sodium azide (60 mmol). The resulting suspension was stirred at
room temperature for 18 h. Ethyl acetate (250 mL) was added, and the
resulting mixture was extracted with water (3 x 350 mL). The organic
layer was dried and concentrated under reduced pressure to yield the
desired products as oils.
Ethyl 12-aminododecanoate (12). White solid, 96% yield. 1H NMR (400
MHz, CDCl3): δ 4.10 (q, J = 7.2 Hz, 2H), 2.65 (t, J = 6.8 Hz, 2H), 2.26 (t,
J = 7.6 Hz, 2H), 1.59 (p, J = 7.2 Hz, 2H), 1.41 (p, J = 6.8 Hz, 2H), 1.20 –
1.35 (m, 17H). 13C NMR (100 MHz, CDCl3): δ 173.90, 60.11, 42.24,
34.35, 33.86, 29.55, 29.49, 29.46, 29.39, 29.21, 29.10, 26.86, 24.94,
14.22. HRMS (ESI) m/z [M+H]+ calcd for C14H30NO2, 244.2271; found,
244.2271.
Ethyl 10-azidodecanoate (6). Pale yellow oil, 94% yield. 1H NMR (500
MHz, CDCl3): δ 4.12 (q, J = 7.0 Hz, 2H), 3.25 (t, J = 7.0 Hz, 2H), 2.28 (t,
J = 7.5 Hz, 2H), 1.50 – 1.65 (m, 4H), 1.20 – 1.40 (m, 13H). 13C NMR (125
MHz, CDCl3): δ 173.88, 60.15, 51.44, 34.33, 29.32, 29.11, 29.05, 29.04,
28.79, 26.65, 24.91, 14.23. HRMS (ESI) m/z [M+H]+ calcd for C12H24N3O2,
242.1863; found, 242.1866.
Ethyl 15-aminopentadecanoate (13). White solid, 86% yield. 1H NMR
(400 MHz, CDCl3): δ 4.10 (q, J = 7.2 Hz, 2H), 2.66 (t, J = 7.2 Hz, 2H),
2.26 (t, J = 7.6 Hz, 2H), 1.59 (p, J = 7.2 Hz, 2H), 1.39 (p, J = 7.2 Hz, 2H),
1.20 – 1.35 (m, 23H). 13C NMR (100 MHz, CDCl3): δ 173.89, 60.11,
42.19, 34.37, 33.73, 29.60 (2C), 29.59, 29.58, 29.55, 29.47, 29.42, 29.23,
29.1126.86, 24.96, 14.22. HRMS (ESI) m/z [M+H]+ calcd for C17H36NO2,
286.2741; found, 286.2736.
Ethyl 11-azidoundecanoate (7). Pale yellow oil, 93% yield. 1H NMR
(500 MHz, CDCl3): δ 4.11 (q, J = 7.0 Hz, 2H), 3.25 (t, J = 7.5 Hz, 2H),
2.28 (t, J = 7.5 Hz, 2H), 1.55 – 1.65 (m, 4H), 1.20 – 1.40 (m, 15H). 13C
NMR (125 MHz, CDCl3): δ 173.89, 60.14, 51.45, 34.35, 29.36, 29.78,
29.17, 29.08 (2C), 28.80, 26.66, 24.93, 14.23. HRMS (ESI) m/z [M+H]+
calcd for C13H26N3O2, 256.2020; found, 256.2019.
Ethyl 12-azidododecanoate (8). Pale yellow oil, 86% yield. 1H NMR
(500 MHz, CDCl3): δ 4.12 (q, J = 7.0 Hz, 2H), 3.24 (t, J = 7.0 Hz, 2H),
2.27 (t, J = 7.5 Hz, 2H), 1.55 – 1.65 (m, 4H), 1.20 – 1.40 (m, 17H). 13C
NMR (125 MHz, CDCl3): δ 173.89, 60.15, 51.46, 34.34, 29.37, 29.35 (2C),
29.33, 29.23, 29.10, 28.79, 26.67, 24.92, 14.23. HRMS (ESI) m/z [M+H]+
calcd for C14H28N3O2, 269.2176; found, 269.2176.
General procedure for synthesis of ureas from amines
To a solution of the amine (10-13, 1.50 mmol) in anhydrous THF (15 mL)
under
a
nitrogen
atmosphere
was
added
4-chloro-3-
(trifluoromethyl)phenyl isocyanate (1.65 mmol). The mixture was stirred
at room temperature for 2 h, and then concentrated under reduced
pressure. The residue was purified on silica gel by stepwise gradient
elution with dichloromethane/ethyl acetate (100:0 to 80:20), yielding the
products as solids.
Ethyl 15-azidopentadecanoate (9)
To a solution of triphenyl phosphine (2.633g, 10.04 mmol) in anhydrous
tetrahydrofuran (THF; 40 mL) at 0oC was added diisopropyl
azodicarboxylate (2.029g, 10.04 mmol) dropwise. The mixture was
stirred for 10 min, and then alcohol 5 (2.388g, 8.36 mmol) in THF (20 mL)
was added dropwise. After 30 min diphenyl phosphoryl azide (2.763g,
10.04 mmol) was added and the mixture was warmed to room
temperature and stirred for 4.5 h. Water (80 mL), diethyl ether (150 mL)
and brine (100 mL) was then added, and the ether layer was separated
and concentrated under reduced pressure. The residue was purified on
silica gel by stepwise gradient elution with dichloromethane/hexane
(20:80 to 100:0), yielding 1.516g (49%) of 9 as a pale yellow oil. 1H NMR
(500 MHz, CDCl3): δ 4.10 (q, J = 7.0 Hz, 2H), 3.23 (t, J = 7.0 Hz, 2H),
2.26 (t, J = 7.5 Hz, 2H), 1.55 – 1.65 (m, 4H), 1.20 – 1.40 (m, 23H). 13C
NMR (125 MHz, CDCl3): δ 173.92, 60.13, 51.47, 34.37, 29.57 (2C), 29.55,
29.50, 29.44, 29.42, 29.24, 29.12 (2C), 28.81, 26.67, 24.96, 14.23.
HRMS (ESI) m/z [M+H]+ calcd for C12H24N3O2, 312.2646; found,
312.2645.
Ethyl
10-({[4-chloro-3-
(trifluoromethyl)phenyl]carbamoyl}amino)decanoate (17a). White
solid, 82% yield. 1H NMR (500 MHz, CDCl3): δ 7.55-7.65 (m, 2H), 7.53
(dd, J = 9.0, 2.5 Hz, 1H), 7.33 (d, J = 9.0 Hz, 1H), 5.40 (s, 1H), 4.13 (q, J
= 7.0 Hz, 2H), 3.20 (t, J = 7.0 Hz, 2H), 2.30 (t, J = 7.0 Hz, 2H), 1.59 (p, J
= 7.0 Hz, 2H), 1.44 (p, J = 7.0 Hz, 2H), 1.35 – 1.18 (m, 13H). 13C NMR
(125 MHz, CDCl3): δ 174.68, 155.56, 138.25, 131.84, 128.53 (q, J = 31
Hz), 124.85, 122.85, 122.64 (q, J = 272 Hz), 117.81 (q, J = 6 Hz), 60.48,
40.16, 34.34, 29.79, 29.01, 28.89, 28.83, 28.81, 26.60, 24.83, 14.18.
HRMS (ESI) m/z [M+H]+ calcd for C20H41ClF3N2O3, 437.1813; found,
437.1813.
Ethyl
11-({[4-chloro-3-
(trifluoromethyl)phenyl]carbamoyl}amino)undecanoate (17b). White
solid, 62% yield. 1H NMR (500 MHz, CDCl3): δ 7.61 (d, J = 2.5 Hz, 1H),
7.55 (dd, J = 9.0, 2.5 Hz, 1H), 7.38-7.34 (m, 2H), 5.22 (t, J = 5.5 Hz, 1H),
4.13 (q, J = 7.0 Hz, 2H), 3.22 (q, J = 6.5 Hz, 2H), 2.30 (t, J = 7.5 Hz, 2H),
1.61 (p, J = 7.5 Hz, 2H), 1.44 (p, J = 7.5 Hz, 2H), 1.35-1.20 (m, 15H). 13
C
General procedure for synthesis of amines
NMR (125 MHz, CDCl3): δ 174.64, 155.31, 138.21, 131.87, 128.57 (q, J
= 31 Hz), 124.94, 122.91, 122.60 (q, J = 272 Hz), 117.88 (q, J = 6 Hz),
60.45, 40.26, 34.40, 29.86, 29.16, 29.00, 28.92, 28.89, 28.87, 26.66,
24.82, 14.10. HRMS (ESI) m/z [M+H]+ calcd for C22H43ClF3N2O3,
451.1970; found, 451.1969.
Azides 6 - 9 (4.81 mmol) and triphenyl phosphine (6.25 mmol) were
dissolved in anhydrous THF (30 mL) and stirred at room temperature for
8 h. Water (9.62 mmol) was then added, and the reaction was stirred for
16 h. The reaction was concentrated under reduced pressure and the
residue was purified on silica gel by stepwise gradient elution with
dichloromethane/methanol (95:5 to 40:60), yielding the desired products
as solids.
Ethyl
12-({[4-chloro-3-
(trifluoromethyl)phenyl]carbamoyl}amino)dodecanoate (17c). White
solid, 56% yield. 1H NMR (500 MHz, CDCl3): δ 7.60 (d, J = 2.5 Hz, 1H),
7.54 (dd, J = 9.0, 2.5 Hz, 1H), 7.38 (s, 1H), 7.34 (d, J = 9.0 Hz, 1H), 5.26
(t, J = 5.5 Hz, 1H), 4.13 (q, J = 7.5 Hz, 2H), 3.22 (q, J = 7.0 Hz, 2H), 2.30
(t, J = 7.0 Hz, 2H), 1.60 (p, J = 7.0 Hz, 2H), 1.47 (p, J = 7.0 Hz, 2H), 1.35
– 1.20 (m, 17H). 13C NMR (125 MHz, CDCl3): δ 174.59, 155.37, 138.18,
131.86, 128.53 (q, J = 31 Hz), 124.96, 122.93, 122.60 (q, J = 272 Hz),
117.86 (q, J = 6 Hz), 60.42, 40.30, 34.42, 29.91, 29.28, 29.16, 29.12,
29.08, 29.03, 28.94, 26.74, 24.89, 14.19. HRMS (ESI) m/z [M+H]+ calcd
for C22H45ClF3N2O3, 465.2126; found, 465.2125.
Ethyl 10-aminodecanoate (10). White solid, 71% yield. 1H NMR (400
MHz, CDCl3): δ 4.10 (q, J = 7.2 Hz, 2H), 2.65 (t, J = 7.2 Hz, 2H), 2.26 (t,
J = 7.6 Hz, 2H), 1.59 (p, J = 7.2 Hz, 2H), 1.41 (p, J = 7.2 Hz, 2H), 1.20 –
1.35 (m, 13H). 13C NMR (100 MHz, CDCl3): δ 173.90, 60.13, 42.17,
34.35, 33.73, 29.38 (2C), 29.17, 29.08, 26.81, 24.93, 14.22. HRMS (ESI)
m/z [M+H]+ calcd for C12H26NO2, 216.1958; found, 216.1955.
6
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10.1002/cmdc.201900577
ChemMedChem
FULL PAPER
Ethyl
15-({[4-chloro-3-
(17f).
General procedure for ester hydrolysis
(trifluoromethyl)phenyl]carbamoyl}amino)pentadecanoate
White solid, 80% yield. 1H NMR (500 MHz, CDCl3): δ 7.59 (d, J = 2.5 Hz,
1H), 7.52 (dd, J = 8.5, 2.5 Hz, 1H), 7.45 (s, 1H), 7.33 (d, J = 8.5 Hz, 1H),
5.35 (s, 1H), 4.13 (q, J = 7.0 Hz, 2H), 3.20 (t, J = 7.0 Hz, 2H), 2.30 (t, J =
7.5 Hz, 2H), 1.61 (p, J = 7.5 Hz, 2H), 1.47 (p, J = 7.0 Hz, 2H), 1.35 – 1.20
(m, 23H). 13C NMR (125 MHz, CDCl3): δ 174.54, 155.48, 138.15, 131.84,
128.56 (q, J = 31 Hz), 124.98, 121.54, 122.60 (q, J = 272 Hz), 117.93 (q,
J = 6 Hz), 60.39, 40.46, 34.46, 29.82, 29.42, 29.38 (4C), 29.28, 29.22,
29.13, 29.05, 26.83, 24.97, 14.19. HRMS (ESI) m/z [M+H]+ calcd for
The ester (17a - 17h, 0.80 mmol) was dissolved in ethanol (30 mL) at
40°C, and 1.5M NaOH (10 mL) was added dropwise. The reaction was
stirred for 4 h. The volume of the reaction solvent was reduced by half
under reduced pressure, and then acidified with 1M HCl to a pH of 1-2.
The resulting precipitate was then filtered and washed with H2O, followed
by H2O/ethanol (1:1), and dried on a Buchner funnel.
10-({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)decanoic
acid (1a). White solid, 78% yield. Mp = 110 – 111oC. 1H NMR (500 MHz,
DMSO-d6): δ 11.93 (s, 1H), 8.88 (s, 1H), 8.04 (d, J = 2.0 Hz, 1H), 7.45-
7.55 (m, 2H), 6.28 (t, J = 5.5 Hz, 1H), 3.05 (q, J = 7.0 Hz, 2H), 2.16 (t, J =
7.0 Hz, 2H), 1.35-1.50 (m, 4H), 1.20-1.30 (m, 10H). 13C NMR (125 MHz,
DMSO-d6): δ 174.93, 155.29, 140.68, 132.25, 126.96 (q, J = 31 Hz),
123.33 (q, J = 271 Hz), 122.66, 121.61, 116.45 (q, J = 6 Hz), 34.10,
30.04, 29.47, 29.34, 29.16, 29.15, 28.98, 26.77, 24.93. HRMS (ESI) m/z
[M+H]+ calcd for C18H24ClF3N2O3, 409.1500; found, 409.1501.
C25H51ClF3N2O3, 507.2596; found, 507.2596.
General procedure for synthesis of ureas from cyano esters
To a solution of NiCl2.6H2O (1.87 mmol) and a cyano ester (14-16, 0.93
mmol) in methanol (20 mL) at 0oC was added NaBH4 (0.361 g, 9.53
mmol) over 30 min. The black reaction mixture was allowed to warm to
room temperature, and was then stirred for 1.5 h. 1M HCl was added
until the black precipitate disappeared. The mixture was made alkaline
with concentrated NH4OH solution and extracted with dichloromethane (3
11-({[4-chloro-3-
x
30 mL). The combined extracts were dried over NaSO4 and
(trifluoromethyl)phenyl]carbamoyl}amino)undecanoic acid (1b).
White solid, 63% yield. Mp = 113 – 114oC. 1H NMR (500 MHz, DMSO-
d6): δ 11.93 (s, 1H), 8.90 (s, 1H), 8.04 (d, J = 2.0 Hz, 1H), 7.45-7.55 (m,
2H), 6.30 (t, J = 5.5 Hz, 1H), 3.06 (q, J = 7.0 Hz, 2H), 2.16 (t, J = 7.0 Hz,
2H), 1.35-1.50 (m, 4H), 1.20-1.30 (m, 12H). 13C NMR (125 MHz, DMSO-
d6): δ 174.95, 155.29, 140.68, 132.23, 126.95 (q, J = 31 Hz), 123.33 (q, J
= 271 Hz), 122.64, 121.62, 116.47 (q, J = 6 Hz), 34.18, 30.04, 29.42,
29.31, 29.18, 29.00, 26.78, 24.94. HRMS (ESI) m/z [M+H]+ calcd for
concentrated under reduced pressure. The residue was then dissolved in
anhydrous in anhydrous THF (15 mL) under a nitrogen atmosphere. 4-
Chloro-3-(trifluoromethyl)phenyl isocyanate (0.066 g, 0.93 mmol) was
added and the mixture was stirred at room temperature for 2 h, and then
concentrated. The residue was purified on silica gel by stepwise gradient
elution with dichloromethane/ethyl acetate (100:0 to 75:25), yielding the
desired products as white solids.
C19H26ClF3N2O3, 423.1657; found, 423.1657.
Ethyl
13-({[4-chloro-3-
(trifluoromethyl)phenyl]carbamoyl}amino)tridecanoate (17d). White
solid, 70% yield. 1H NMR (500 MHz, CDCl3): δ 7.60 (d, J = 2.5 Hz, 1H),
7.53 (dd, J = 8.5, 2.5 Hz, 1H), 7.41 (s, 1H), 7.33 (d, J = 8.5 Hz, 1H), 5.30
(t, J = 5.5 Hz, 1H), 4.13 (q, J = 7.0 Hz, 2H), 3.21 (t, J = 6.5 Hz, 2H), 2.31
(t, J = 7.5 Hz, 2H), 1.60 (p, J = 7.5 Hz, 2H), 1.47 (p, J = 7.5 Hz, 2H), 1.35
– 1.18 (m, 19H). 13C NMR (125 MHz, CDCl3): δ 174.59, 155.41, 138.18,
131.85, 128.54 (q, J = 31 Hz), 124.94, 122.93, 122.61 (q, J = 273 Hz),
117.88 (q, J = 6 Hz), 60.41, 40.31, 34.43, 29.92, 29.28, 29.27, 29.24,
29.13, 29.12, 29.05, 28.99, 26.75, 24.90, 14.19. HRMS (ESI) m/z [M+H]+
calcd for C23H47ClF3N2O3, 479.2283; found, 479.2285.
12-({[4-chloro-3-
(trifluoromethyl)phenyl]carbamoyl}amino)dodecanoic acid (1c).
White solid, 72% yield. Mp = 131 – 132oC. 1H NMR (500 MHz, DMSO-
d6): δ 11.95 (s, 1H), 9.11 (s, 1H), 8.05 (d, J = 2.0 Hz, 1H), 7.50-7.65 (m,
2H), 6.49 (t, J = 5.5 Hz, 1H), 3.04 (q, J = 7.0 Hz, 2H), 2.13 (t, J = 7.0 Hz,
2H), 1.50-1.60 (m, 4H), 1.15-1.25 (m, 14H). 13C NMR (125 MHz, DMSO-
d6): δ 175.18, 155.39, 140.82, 132.21, 127.27 (q, J = 31 Hz), 123.34 (q, J
= 271 Hz), 122.62, 121.50, 116.43 (q, J = 6 Hz), 34.39, 30.00, 29.37,
29.26, 29.24, 29.15, 29.13, 28.96, 26.74, 25.00. HRMS (ESI) m/z [M+H]+
calcd for C20H28ClF3N2O3, 437.1813; found, 437.1814.
Ethyl
14-({[4-chloro-3-
(17e).
13-({[4-chloro-3-
(trifluoromethyl)phenyl]carbamoyl}amino)tridecanoic
(trifluoromethyl)phenyl]carbamoyl}amino)tetradecanoate
acid
(1d).
White solid, 64% yield. 1H NMR (500 MHz, CDCl3): δ 7.60 (d, J = 2.5 Hz,
1H), 7.55 (dd, J = 8.5, 2.5 Hz, 1H), 7.34 (d, J = 8.5 Hz, 1H), 7.23 (s, 1H),
5.17 (t, J = 5.5 Hz, 1H), 4.13 (q, J = 7.0 Hz, 2H), 3.21 (q, J = 6.0 Hz, 2H),
2.29 (t, J = 7.5 Hz, 2H), 1.59 (p, J = 7.5 Hz, 2H), 1.44 (p, J = 7.5 Hz, 2H),
1.35 – 1.18 (m, 21H). 13C NMR (125 MHz, CDCl3): δ 174.50, 155.25,
138.14, 131.86, 128.57 (q, J = 31 Hz), 125.02, 122.98, 122.57 (q, J = 273
Hz), 117.91 (q, J = 6 Hz), 60.37, 40.37, 34.75, 29.96, 29.36, 29.34, 29.31,
29.30, 29.21, 29.17, 29.12, 29.02, 26.78, 24.93, 14.20. HRMS (ESI) m/z
[M+H]+ calcd for C24H49ClF3N2O3, 493.2439; found, 493.2437.
White solid, 59% yield. Mp = 135 – 136oC. 1H NMR (500 MHz, DMSO-
d6): δ 10.75 (s, 1H), 8.13 (d, J = 2.5 Hz, 1H), 7.96 (s, 1H), 7.61 (dd, J =
2.5, 9.0 Hz, 1H), 7.45 (d, J = 9.0 Hz, 1H), 3.01 (q, J = 7.0 Hz, 2H), 2.01 (t,
J = 7.0 Hz, 2H), 1.44 (q, J = 7.0 Hz, 2H), 1.36 (q, J = 7.0 Hz, 2H), 1.15-
1.25 (m, 16H). 13C NMR (125 MHz, DMSO-d6): δ 177.11, 156.06, 141.77,
131.96, 126.71 (q, J = 31 Hz), 123.33 (q, J = 272 Hz), 122.02, 120.59,
116.16 (q, J = 6 Hz), 30.05, 29.25, 29.14, 29.02, 28.99 (2C), 28.95, 28.85,
26.70, 26.01. HRMS (ESI) m/z [M+H]+ calcd for C21H30ClF3N2O3,
451.1970; found, 451.1969.
Ethyl
17-({[4-chloro-3-
(17h).
14-({[4-chloro-3-
(trifluoromethyl)phenyl]carbamoyl}amino)heptadecanoate
(trifluoromethyl)phenyl]carbamoyl}amino)tetradecanoic acid (1e).
White solid, 64% yield. Mp = 100 – 102oC. 1H NMR (500 MHz, DMSO-
d6): δ 8.93 (s, 1H), 8.06 (d, J = 2.5 Hz, 1H), 7.50-7.60 (m, 2H), 6.33 (t, J =
5.5 Hz, 1H), 3.07 (q, J = 6.5 Hz, 2H), 2.17 (t, J = 7.0 Hz, 2H), 1.35-1.50
(m, 4H), 1.20-1.30 (m, 18H). 13C NMR (125 MHz, DMSO-d6): δ 174.53,
154.85, 140.25, 131.79, 126.54 (q, J = 31 Hz), 122.88 (q, J = 271 Hz),
122.20, 121.15, 116.04, 33.96, 29.57, 29.00 (2C), 28.97 (2C), 28.89,
28.73 (2C), 28.53, 26.30, 24.50. HRMS (ESI) m/z [M+H]+ calcd for
White solid, 58% yield. 1H NMR (500 MHz, CDCl3): δ 7.60 (d, J = 2.5 Hz,
1H), 7.55 (dd, J = 8.5, 2.5 Hz, 1H), 7.35 (d, J = 9.0 Hz, 1H), 7.11 (s, 1H),
5.10 (t, J = 5.5 Hz, 1H), 4.13 (q, J = 7.0 Hz, 2H), 3.21 (q, J = 6.5 Hz, 2H),
2.30 (t, J = 7.5 Hz, 2H), 1.60 (p, J = 7.5 Hz, 2H), 1.48 (p, J = 7.5 Hz, 2H),
1.35 – 1.18 (m, 27H). 13C NMR (125 MHz, CDCl3): δ 174.44, 155.14,
138.09, 131.88, 128.51 (q, J = 32 Hz), 125.06, 123.03, 122.42 (q, J = 272
Hz), 117.98 (q, J = 6 Hz), 60.35, 40.42, 34.46, 30.02, 29.43, 29.41 (6C),
29.32, 29.24, 29.18, 29.09, 26.85, 24.99, 14.20. HRMS (ESI) m/z [M+H]+
calcd for C27H55ClF3N2O3, 535.2909; found, 535.2909.
C22H32ClF3N2O3, 465.2126; found, 465.2127.
7
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10.1002/cmdc.201900577
ChemMedChem
FULL PAPER
15-({[4-chloro-3-
Fluorescence microscopy: MDA-MB-231 cells were seeded in 12-well
plates and cultured for 24 h in DMEM medium supplemented with 10%
FBS and 1% penicillin/streptomycin. After serum deprivation for 16 h,
cells were treated with 1g analogues (10 µM, 4 h) or DMSO in serum-
free DMEM. Following two washes with serum-free medium, cells were
incubated with JC-1 and Hoechst 33258 (Sigma-Aldrich) for 20 min,
according to the manufacturer’s instructions. Images of JC-1 aggregates
(red), JC-1 monomers (green) and nuclear staining (blue) of cells were
acquired with a fluorescence microscope (Olympus BX51, Notting Hill,
VIC, Australia).
(trifluoromethyl)phenyl]carbamoyl}amino)pentadecanoic acid (1f).
White solid, 93% yield. Mp = 97-98 oC. 1H NMR (500 MHz, DMSO-d6): δ
11.93 (s, 1H), 8.90 (s, 1H), 8.04 (d, J = 2.5 Hz, 1H), 7.45-7.55 (m, 2H),
6.30 (t, J = 6.0 Hz, 1H), 3.05 (q, J = 6.5 Hz, 2H), 2.16 (t, J = 7.5 Hz, 2H),
1.35-1.50 (m, 4H), 1.20-1.30 (m, 20H). 13C NMR (125 MHz, DMSO-d6): δ
174.97, 155.30, 140.70, 132.24, 126.96 (q, J = 30 Hz), 123.32 (q, J = 271
Hz), 122.65, 121.61, 116.47 (q, J = 6 Hz), 34.14, 30.21, 29.74 (2C),
29.45, 29.43, 29.42, 29.35, 29.18 (2C), 28.99, 29.75, 24.95. HRMS (ESI)
m/z [M+H]+ calcd for C23H34ClF3N2O3, 479.2283; found, 479.2284.
17-({[4-chloro-3-
Statistical analysis: All data are expressed throughout as means ± SEM.
Data were analyzed by one-way ANOVA and the Fisher’s Partial Least-
Squares Difference test was used to detect differences between multiple
treatments. All experiments were replicated on 3-4 occasions.
(trifluoromethyl)phenyl]carbamoyl}amino)heptadecanoic acid (1h).
White solid, 82% yield. Mp = 79-80 oC. 1H NMR (500 MHz, DMSO-d6): δ
11.94 (s, 1H), 8.94 (s, 1H), 8.04 (d, J = 2.5 Hz, 1H), 7.50-7.60 (m, 2H),
6.33 (t, J = 5.5 Hz, 1H), 3.05 (q, J = 6.5 Hz, 2H), 2.15 (t, J = 7.0 Hz, 2H),
1.35-1.50 (m, 4H), 1.15-1.30 (m, 10H). 13C NMR (125 MHz, DMSO-d6): δ
174.99, 155.31, 140.72, 132.24, 126.94 (q, J = 31 Hz), 123.32 (q, J = 270
Hz), 122.65, 121.59, 116.49, 34.18, 30.02, 29.46 (4C), 29.43, 29.41,
29.40, 29.33, 29.18, 29.16, 28.99, 26.74, 24.96. HRMS (ESI) m/z [M+H]+
calcd for C25H38ClF3N2O3, 507.2596; found, 507.2597.
Acknowledgements
This study was supported by grants from the Australian National
Health and Medical Research Council (1031686 and 1087248).
We are grateful to Prof Christine Clarke of the Westmead
Institute for Medical Research (Westmead, NSW, Australia) for
the generous gift of MCF10A cells.
Cell based assays
Biochemicals: Dulbecco's Modified Eagle's Medium (DMEM), Fetal
Bovine Serum (FBS), trypsin/EDTA, penicillin and streptomycin,
phosphate-buffered saline (PBS) and biochemicals were obtained from
Sigma-Aldrich Chemical (Castle Hill, NSW, Australia). JC-1 was from
Cayman Chemical (Ann Arbor, MI, USA).
Contributor roles
Designed research (MM, TR), performed experiments (TR, AR,
YC, KB), analysed data (MM, AR, YC, KB, TR), drafted
manuscript (MM, TR)
Cell culture and viability assays: Human MDA-MB-231 breast cancer
cells were obtained from ATCC (Manassas, VA, USA) and grown at 37°C
in a humidified atmosphere of 5% CO2 in air in DMEM that was
supplemented with 10% FBS and 1% penicillin/streptomycin. The control
MCF10A cell line was a gift from Prof Christine Clarke (Westmead
Institute for Medical Research, Westmead, NSW, Australia). Confluent
cells (80-90%) were harvested using Trypsin/EDTA after washing with
PBS. Cells were treated with various concentrations of 1g analogues in
DMSO (final concentration 0.1%); control cells were treated with DMSO
alone. ATP formation was assessed using the CellTiter-Glo® assay
(Promega; Annandale, NSW, Australia) as described previously.[13]
Keywords: antitumor agents, apoptosis, breast cancer, fatty
acids, lipid drugs
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Entry for the Table of Contents
Alkyl chain was varied in the novel anti-cancer aryl-urea fatty acid 1g (16({[4-chloro-3-
(trifluoromethyl)phenyl]carbamoyl}amino)hexadecanoic acid). The C12-C17 analogues efficiently impaired the mitochondrial
membrane potential and ATP production while shorter analogues were less active. 1g and the C17 analogue also preferentially
activated necrosis and apoptosis, respectively. Thus, alkyl chain length is a determinant of mitochondrial targeting by these agents
and can be varied to develop analogues that activate apoptosis or necrosis in a regulated fashion.
10
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