Inhibitory effects of ethacrynic acid analogues lacking the α,β-unsaturated carbonyl unit and para-acylated phenols on human cancer cells

Article abstract of DOI:10.1016/j.bmcl.2010.12.074

A series of ethacrynic acid analogues, lacking the α,β- unsaturated carbonyl unit, was synthesized and subsequently evaluated for their ability to inhibit the migration of human breast cancer cells, Hs578Ts(i)8 as well as of human prostate cancer cells, C4-2B. These cell lines provide a good model system to study migration and invasion, since they represent metastatic cancer. Our studies show that ethacrynic acid analogues with methyl substituents at the aromatic ring demonstrate no inhibitory effect on the migration of both cancer cell lines, whereas a precursor in the synthesis of these ethacrynic acid analogues (II-1, a para-acylated m-cresol) is an excellent inhibitor of the migration of both cancer cell lines.

Full text of DOI:10.1016/j.bmcl.2010.12.074

Bioorganic & Medicinal Chemistry Letters 21 (2011) 912–915
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibitory effects of ethacrynic acid analogues lacking the
a,b-unsaturated
carbonyl unit and para-acylated phenols on human cancer cells
Zack E. Bryant a, Romy F. J. Janser a, Medina Jabarkhail a, Melissa S. Candelaria-Lyons b, Brittni B. Romero b,
Severine Van slambrouck b, , Wim F. A. Steelant b,c, Ingo Janser a, ,
⇑
a Laboratory of Organic Synthesis, Catalysis & Biochemistry (LOSCB), Department of Chemistry, New Mexico Institute of Mining and Technology, 801 Leroy Place, Socorro, NM
87801, USA
b Laboratory of Biochemical and Biomedical Research, Department of Chemistry, New Mexico Institute of Mining and Technology, Socorro, NM 87801, USA
c School of Science, Technology and Engineering Management, St. Thomas University, Miami Gardens, FL 33054, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of ethacrynic acid analogues, lacking the
a,b-unsaturated carbonyl unit, was synthesized and
Received 24 November 2010
Revised 9 December 2010
Accepted 15 December 2010
Available online 19 December 2010
subsequently evaluated for their ability to inhibit the migration of human breast cancer cells, Hs578Ts(i)8
as well as of human prostate cancer cells, C4-2B. These cell lines provide a good model system to study
migration and invasion, since they represent metastatic cancer. Our studies show that ethacrynic acid
analogues with methyl substituents at the aromatic ring demonstrate no inhibitory effect on the migra-
tion of both cancer cell lines, whereas a precursor in the synthesis of these ethacrynic acid analogues
(II-1, a para-acylated m-cresol) is an excellent inhibitor of the migration of both cancer cell lines.
Ó 2010 Elsevier Ltd. All rights reserved.
Ethacrynic acid (EA) (Fig. 1) is a loop diuretic and is used to
treat high blood pressure and the swelling caused by diseases like
liver, kidney, and congestive heart failure.1,2 EA possesses an
important aspect of cancer treatment is the prevention of metasta-
ses formation since the metastatic spread of primary tumors to dis-
tant sites constitutes the most lethal aspect of cancer. The exact
mechanisms of local invasion and the formation of metastases
are still not completely understood and remain an important field
of research.14 Intrigued by our very promising results, we were ea-
ger to further develop and evaluate biologically active ethacrynic
a
,b-unsaturated carbonyl unit (a Michael acceptor) and can be at-
tacked by nucleophiles (e.g., sulfur-atoms) at the b-carbon. This
,b-unsaturated carbonyl moiety is often employed in the design
a
of SH-enzyme inhibitors (e.g., cysteine proteases).3–6 EA inhibits
the enzyme by binding to the cysteinyl residue in the active site
by means of a Michael-like addition and is therefore a good gluta-
thione S-transferase P1-1 (GST P1-1) inhibitor.7 Several research
groups have synthesized EA analogues in order to increase the
inhibitory effect on GST P1-1. GST P1-1 is also of importance in
controlling multidrug resistance (MDR), proliferation and apopto-
sis processes and hence can be considered a target for cancer treat-
ment. Along this line, it has been demonstrated that EA exerts
anti-proliferative effects against tumor cells, but only at higher
acid analogues lacking the a,b-unsaturated carbonyl unit.
Our previous studies show that the most potent candidates to
inhibit the migration of human breast cancer cells, MCF-7/AZ, are
EA analogues with one or two methoxy groups attached to the aro-
matic system but lacking the
a,b-unsaturated carbonyl unit. We
hypothesized that the increase in electron density of the aromatic
system, caused by electron donating groups like the methoxy
group, is responsible for the anti-migratory properties of the corre-
sponding compounds.13 To further investigate this hypothesis, we
were interested in synthesizing EA analogues with one or two
methyl groups attached to the aromatic system. Methyl groups
are only very weak electron donating substituents in comparison
to the strong electron donating methoxy groups.
concentrations (60–100 l
M).2,8–10 Additionally, it has also been
shown that EA is cytotoxic towards primary chronic lymphocytic
leukemia (CLL) cells.11 However, the relative lack of potency and
the diuretic properties do not support the use of EA as a chemo-
therapeutic agent.12
The preparation of the various ethacrynic acid analogues,
Recently, we demonstrated that EA analogues lacking the
lacking the a,b-unsaturated carbonyl unit, was accomplished by
a,b-unsaturated carbonyl unit are able to inhibit the migration of
human breast cancer cells, MCF-7/AZ, at non toxic concentra-
tions.13 This is, insofar, a very promising result, because an
Cl
Cl
O
COOH
O
⇑
Corresponding author. Tel.: +1 575 835 6948; fax: +1 575 835 5364.
E-mail address: ijanser@nmt.edu (I. Janser).
These authors contributed equally.
Figure 1. The chemical structure of ethacrynic acid (EA).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.074

Z. E. Bryant et al. / Bioorg. Med. Chem. Lett. 21 (2011) 912–915
913
O
1.)
I
HOOC
NaOH,
O
O
COOH
Cl
n
Δ
OH
OH
O
n
n
2.) HCl
AlCl3, CS2
n=1,2
R
III
R
I
R
II
O
COOEt
O
BrCH2COOEt
K2CO3, acetone
n
R
IV
Scheme 1. Synthetic pathway for the synthesis of EA analogues.
OH
OH
OH
higher ratio of the para-acylated versus the ortho-acylated product
(with respect to the hydroxyl group), the reaction was carried out
at 0 °C. After separation of the obtained isomers on silica gel, the
para-acylated phenol-analogues II were treated with 2-iodocarb-
oxylic acid in the presence of sodium hydroxide to afford the
corresponding sodium salts. Acidification of these salts with hydro-
chloric acid yielded the corresponding compounds III. In order to
obtain the phenoxy acetic acid esters IV, the para-acylated phenols
II were treated with ethyl bromoacetate and refluxed in acetone in
the presence of potassium carbonate as the base.
I-3
I-2
I-1
Figure 2. Phenol I-1 and two substituted phenols I-2–3.
a two-step reaction shown in Scheme 1. The Friedel–Crafts acyla-
tion of the phenol I-1 or the substituted phenols I-2–3 (Fig. 2),
respectively, with acyl chloride, was performed in the presence
of powdered aluminum chloride (AlCl3) in carbon disulfide, to gen-
erate the corresponding acylated compounds II. In order to obtain a
In the present study, we used the Hs578Ts(i)8 breast cancer cell
line and the C4-2B prostate cancer cell line, which are ideal model
systems to study the effect of the synthesized compounds on
Table 1
The inhibitory effect of ethacrynic acid (EA)
Code
Structure
Inhibition (%)
Hs578Ts(i)8
OD80/IC20
(l
M)
Hs578Ts(i)8
C4-2B
None
C4-2B
10
Cl
Cl
O
COOH
EA
None
80
O
O
COOH
COOH
III-1
III-2
IV-1
IV-2
IV-3
None
None
None
None
23
None
None
None
None
19
>100
>100
>100
50
>100
>100
>100
>100
>100
O
O
O
O
COOEt
COOEt
O
O
O
O
COOEt
>100
O
O
O
II-1
II-2
65
86
28
17
>100
>100
>100
>100
>100
>100
OH
OH
25
O
II-3
None
OH
EA analogues lacking the
a,b-unsaturated carbonyl unit (III-1–2, IV-1–3), and para-acylated phenols (II-1–3) on the migration of human C4-2B prostate cancer cells and
human Hs578Ts(i)8 breast cancer cells and their respective observed cytotoxicities expressed in OD80/IC20 values.

914
Z. E. Bryant et al. / Bioorg. Med. Chem. Lett. 21 (2011) 912–915
migration and invasion of cancer cells, since they represent meta-
static cancer.15–17 The results from the in vitro cytotoxicity as-
says18–20 (Table 1) reveal that 24 h treatment of human
Hs578Ts(i)8 breast cancer cells and human C4-2B prostate cancer
cells with compounds II-1-3, III-1-2, IV-1, and IV-3 has no effect
Driven by these highly promising results, we also tested com-
pounds II-2 and II-3 for their ability to inhibit the migration of
the human C4-2B prostate cancer cells and the human Hs578Ts(i)8
breast cancer cells. Both compounds are able to inhibit the migra-
tion of the respective cancer cells, but only in a range of 17–28%
(Figs. 3 and 4). Although, compounds II-2 and II-3 are very similar
in structure to compound II-1 (they only differ in the amount of
methyl groups attached to the aromatic system) they are very dif-
ferent in their ability to inhibit the above mentioned cancer cells.
The data from the wound healing assays reveal that ethacrynic
on the cell viability, even at high concentrations of 100
compound IV-2 reduces the cell viability of the human C4-2B pros-
tate cancer cells by 20% at a concentration of 50 M. In contrast to
these results, concentrations of 10 (C4-2B) or 80 lM
lM. Only
l
lM
(Hs578Ts(i)8), respectively, result in a 20% reduction of the cell via-
bility of the corresponding cell line, when they are treated with
ethacrynic acid.
acid analogues, lacking the a,b-unsaturated carbonyl unit, and only
weak electron donating groups attached to the aromatic ring
(III-1–2 and IV-1–3), are not able to significantly inhibit the migra-
tion of either the human C4-2B prostate cancer cell line nor the
human Hs578Ts(i)8 breast cancer cell line. These results were ex-
pected, since we already tested several ethacrynic acid analogues
with no or one methyl group attached to the aromatic system,
which showed no potential to inhibit the migration of human
breast cancer cells, MCF-7/AZ.13 However, to our surprise and de-
light, the para-acylated m-cresol II-1 shows a very high activity to-
wards the inhibition of human C4-2B prostate cancer cells and
human Hs578Ts(i)8 breast cancer cells. Comparing these results
with the results obtained for compounds II-2 and II-3 (Table 1),
which are all very similar in structure, it becomes obvious that only
slight changes in the substitution pattern of the aromatic ring can
cause a significant change in the observed biological activity. It is
clear that compound II-1 is an excellent inhibitor for the migration
of the two human cancer cell lines, C4-2B and Hs578Ts(i)8, and it
will be worth to further investigate the activity of this compound
towards other cancer cell lines with an eye to the mechanism of
action.
In accordance with our expectations, the in vitro wound healing
assays17,21 for compounds III-1–2, IV-1–3 don’t show any substan-
tial effect on the migration of the two human cancer cell lines. In
stark contrast to these results, compound II-1 shows a significant
inhibition of the migration of both human cancer cell lines,
C4-2B and Hs578Ts(i)8. This compound inhibits the migration of
the human C4-2B prostate cancer cells by 65% and the migration
of the human Hs578Ts(i)8 breast cancer cells even by 86% (Figs.
3 and 4), without affecting the growth of these cell lines (data
not shown).
Migration Assay of C4-2B
18
16
14
12
10
8
In summary, we have synthesized five different EA analogues,
lacking the a,b-unsaturated carbonyl unit, which didn’t show any
*
6
pronounced activity towards the inhibition of the migration of
the two cancer cell lines, C4-2B (prostate cancer) and Hs578Ts(i)8
(breast cancer). In sharp contrast, the para-acylated m-cresol II-1
revealed a very pronounced activity towards the inhibition of both
cancer cell lines. This compound inhibits the migration of the
human C4-2B prostate cancer cells by 65% and the migration of
the human Hs578Ts(i)8 breast cancer cells even by 86%. Further
studies of these compounds against other cancer cell lines (e.g.,
the human breast cancer cells, MCF-7/AZ) are currently underway
in our laboratory.
4
2
0
Control (C4-2B), EA and its Analogues
Figure 3. Migration assay of the human prostate cancer cell line, C4-2B, in the
absence (control) or presence of ethacrynic acid (EA), its analogues (III-1–2,
⁄
IV-1–3), and para-acylated phenols (II-1–3), indicates p <0.05.
Acknowledgments
We thank New Mexico Tech for supplying the start-up funds for
I.J. and the US National Institutes of Health (P20 RR016480) under
the INBRE program of the National Center for Research Resources
(NCRR).
Migration Assay of Hs578Ts(i)8
70
60
50
40
30
20
References and notes
1. Zhao, G.; Yu, T.; Wang, R.; Wang, X.; Jing, Y. Bioorg. Med. Chem. 2005, 13, 4056.
2. Zhao, G.; Liu, C.; Wang, R.; Song, D.; Wang, X.; Lou, H.; Jing, Y. Bioorg. Med.
Chem. 2007, 15, 2701.
3. Schirmeister, T.; Kaeppler, U. Mini-Rev. Med. Chem. 2003, 3, 361.
4. Kaeppler, U.; Stiefl, N.; Schiller, M.; Vicik, R.; Breuning, A.; Schmitz, W.;
Rupprecht, D.; Schmuck, C.; Baumann, K.; Ziebuhr, J.; Schirmeister, T. J. Med.
Chem. 2005, 48, 6832.
10
*
5. Vicik, R.; Busemann, M.; Baumann, K.; Schirmeister, T. Curr. Top. Med. Chem.
2006, 6, 331.
0
6. Dude, M.-A.; Kaeppler, U.; Herb, M.; Schiller, M.; Schulz, F.; Vedder, B.;
Heppner, S.; Pradel, G.; Gut, J.; Rosenthal, P. J.; Schirmeister, T.; Leippe, M.;
Gelhaus, C. Molecules 2009, 14, 19.
7. Iersel, M. L.; Ploemen, J. P.; Struit, I.; Van Amersfoort, C.; Keyzer, A. E.;
Schefferlie, J. G.; Van Bladeren, P. J. Chem. Biol. Interact. 1996, 102, 117.
8. Zhao, G. S.; Wang, X. B. Curr. Med. Chem. 2006, 13, 1461.
9. Aizawa, S.; Ookawa, K.; Kudo, T.; Asano, J.; Hayakari, M.; Tsuchida, S. Cancer Sci.
2003, 94, 886.
Control (Hs578Ts(i)8), EA and its Analogues
Figure 4. Migration assay of the human breast cancer cell line, Hs578Ts(i)8, in the
absence (control) or presence of ethacrynic acid (EA), its analogues (III-1–2,
⁄
IV-1–3), and para-acylated phenols (II-1–3), indicates p <0.05.

Z. E. Bryant et al. / Bioorg. Med. Chem. Lett. 21 (2011) 912–915
915
10. Yang, X.; Liu, G.; Li, H.; Zhang, Y.; Song, D.; Li, C.; Wang, R.; Liu, B.; Liang, W.;
Jing, Y.; Zhao, G. J. Med. Chem. 2010, 53, 1015.
11. Twentyman, P. R.; Lambert, E.; Muller, M.; Rees, J. K. Leukemia 1992, 6, 726.
12. Jin, G.; Lu, D.; Yao, S.; Wu, C. C. N.; Liu, J. X.; Carson, D. A.; Cottam, H. B. Bioorg.
Med. Chem. 2009, 17, 606.
13. Janser, R. F. J.; Meka, R. K.; Bryant, Z. E.; Adogla, E. A.; Vogel, E. K.; Wharton, J. L.;
Tilley, C. M.; Kaminski, C. N.; Ferrey, S. L.; Van slambrouck, S.; Steelant, W. F. A.;
Janser, I. Bioorg. Med. Chem. Lett. 2010, 20, 1848.
14. Christofori, G. Nature 2006, 25, 444.
15. Hughes, L.; Malone, C.; Chumsri, S.; Burger, A. M.; McDonnell, S. Clin. Exp.
Metastasis 2008, 25, 549.
16. Van slambrouck, S.; Jenkins, A. R.; Romero, A. E.; Steelant, W. F. Int. J. Oncol.
2009, 34, 1717.
removed prior to the addition of the MTT reagent. Three independent
experiments were carried out for each concentration, to determine the mean
optical density (OD) referring to cell viability. OD80/IC20 values were
determined from the respective graphs. These values represent the
concentrations of the analogues, required for 20% inhibition in vitro or
leaving approximately 80% of the cells viable.
19. All compounds were tested in a concentration range of 1–100
toxicity of 50% was observed for any of the compounds, which means that the
concentration to kill 50% of the cells is a lot higher than 100 M. The highest
toxicity observed was 10–15% at 100 M, even after 72 h incubation.
lM, and no
l
l
20. Sigstedt, S. C.; Hooten, C. J.; Callewaert, M. C.; Jenkins, A. R.; Romero, A. E.;
Pullin, M. J.; Kornienko, A.; Lowrey, T. K.; Van Slambrouck, S.; Steelant, W. F. Int.
J. Oncol. 2008, 32, 1085.
17. Van slambrouck, S.; Hilkens, J.; Bisoffi, M.; Steelant, W. F. A. Int. J. Oncol. 2009,
35, 693.
21. Cells were grown in 6-well plates in the appropriate medium until confluence
and then washed twice with phosphate buffered saline (PBS).
A gap of
18. The cytotoxicities of ethacrynic acid and the analogues were tested in
accordance to Sigstedt et al.20 Mitochondrial dehydrogenase activities were
measured by an MTT-reagent. Cells were seeded in 96-well plates at an initial
approximately 1500 m (the wound) was created in the cell monolayer and
l
images were captured. Subsequently, 3 mL of medium, in the presence or
absence of ethacrynic acid or EA analogues, was added. The respective
concentrations of these solutions were determined in the 24 h MTT assay.
After 24 h of cell migration in order to close the wound, images were once
again captured, and compared to the respective previous images in order to
quantify the migration rate, which is expressed as migratory velocity (lm/h).
At least three independent experiments were performed. Student’s t-test was
used for the analysis of data with p <0.05 considered significant.
density of 1.5 Â 104 cells in 200
lL of the appropriate culture medium,
supplemented with 5% fetal bovine serum (FBS), 100 IU/mL penicillin,
100
atmosphere containing 5% CO2, cells were treated with eight different
concentrations (1, 5, 10, 20, 40, 60, 80, and 100 M) of EA and various EA
analogues in culture medium. After 24 h of incubation, 100 L medium was
lg/mL streptomycin. After 24 h of incubation, at 37 °C in a humidified
l
l