Ethacrynic acid analogues lacking the α,β-unsaturated carbonyl unit-Potential anti-metastatic drugs

Article abstract of DOI:10.1016/j.bmcl.2010.01.144

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, MCF-7/AZ. Several of the analogues were already active in the low micromolar range, whereas ethacrynic acid itself shows no potential to inhibit the migration of these cancer cells. Preliminary studies show that the presence of one or more methoxy groups at the phenyl ring of ethacrynic acid is important in order for the ethacrynic acid analogues to demonstrate an inhibitory effect on the migration.

Full text of DOI:10.1016/j.bmcl.2010.01.144

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1848–1850
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Ethacrynic acid analogues lacking the
unit—Potential anti-metastatic drugs
a,b-unsaturated carbonyl
Romy F. J. Janser a, , Ranjith K. Meka a, Zack E. Bryant a, Enoch A. Adogla a, Elizabeth K. Vogel a,
Jaimie L. Wharton a, Cynthia M. Tilley a, Catherine N. Kaminski b, Seth L. Ferrey b,
Severine Van slambrouck b, , Wim F. A. Steelant b, , Ingo Janser a, ,
*
a Laboratory of Organic Synthesis, Catalysis and 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
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 12 January 2010
Revised 28 January 2010
Accepted 29 January 2010
Available online 2 February 2010
subsequently evaluated for their ability to inhibit the migration of human breast cancer cells, MCF-7/
AZ. Several of the analogues were already active in the low micromolar range, whereas ethacrynic acid
itself shows no potential to inhibit the migration of these cancer cells. Preliminary studies show that
the presence of one or more methoxy groups at the phenyl ring of ethacrynic acid is important in order
for the ethacrynic acid analogues to demonstrate an inhibitory effect on the migration.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Ethacrynic acid
Metastases
Inhibition
Anti-cancer
Drug
Ethacrynic acid (EA) (Fig. 1), a loop diuretic, is used to treat high
blood pressure and the swelling caused by diseases like congestive
heart failure, liver failure, and kidney failure.1,2 EA possesses an
An important aspect of cancer treatment is the prevention of
metastases formation, because the metastatic spread of primary
tumors to distant sites constitutes the most lethal aspect of cancer.
Despite extensive research, the exact mechanisms of local invasion
and the formation of metastases are still not entirely understood.
The development and evaluation of biologically active compounds
will give additional insight into this process and would therefore
allow the development of new therapeutics that can effectively
slow down or even prevent the metastatic process and conse-
quently improve the treatment success of cancer patients.9
The metastatic process consists of a complex series of linked
sequential steps, involving detachment of cells within a primary
tumor and local area migration and invasion, followed by intrava-
sation of invading tumor cells into the blood and lymphatic vessels.
After circulating in the vascular system, tumor cells arrest and
extravasate at distant sites where they form secondary tumors or
metastases.10
a,b-unsaturated carbonyl unit which, structurally, can be consid-
ered as a Michael acceptor, an active moiety often employed in the
design of enzyme inhibitors. For example, EA is known to be a good
glutathion S-transferase P1-1 (GSTP1-1) inhibitor.3 As GSTP1-1 be-
longs to the SH-enzyme family, it is more than likely that the a,b-
unsaturated carbonyl unit of EA inhibits the enzyme by binding to
the cysteinyl residue in the active site by means of a Michael-like
addition.
Several research groups have synthesized EA analogues to test
their inhibitory properties on several enzymes. Jing and co-work-
ers showed that EA also has anti-proliferative effects towards tu-
mor cells but only at higher concentrations (60–100 l
M).2,4–6
In another series of experiments, researchers could demon-
strate that EA is cytotoxic towards primary chronic lymphocytic
leukemia (CLL) cells.7 However, EA is not ideal as a chemothera-
peutic agent for cancer treatment due to its diuretic properties
and relative lack of potency.8
Our results show that some analogues of EA, lacking the
a,b-
unsaturated carbonyl unit, are good to excellent inhibitors of the
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.2010.01.144

R. F. J. Janser et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1848–1850
1849
O
1.)
HOOC
I
O
O
COOH
Cl
OH
O
OH
NaOH, Δ
2.) HCl
AlCl3, CS2
R
R
R
IIIa-1-6
I-1-6
IIa-1-6
Scheme 1. Synthetic pathway for the synthesis of EA analogues.
Table 1
migration of human MCF-7/AZ breast cancer cells. Since migration
is a prerequisite of invasion, these EA analogues could be lead com-
pounds for drug development in order to prevent metastases
formation.
The inhibitory effect of ethacrynic acid (EA) and EA analogues, lacking the
a,b-
unsaturated carbonyl unit (IIIa-1–6), on the migration of human MCF-7/AZ breast
cancer cells and their respective observed cytotoxicities expressed in OD80/IC20
values
The preparation of the various ethacrynic acid analogues, lack-
ing the a,b-unsaturated carbonyl unit (IIIa-1–6), was accomplished
Code
Structure
Inhibition (%)
OD80/IC20 (
lM)
Cl
Cl
by a two-step reaction shown in Scheme 1. The Friedel–Crafts acyl-
ation reaction of the phenol I-1 or the substituted phenols I-2–6
(Fig. 2), respectively, with acyl chloride, was performed in the pres-
ence of powdered aluminum chloride (AlCl3) in carbon disulfide, to
generate compounds IIa-1–6, respectively. To prevent cleavage of
the methoxy groups of compounds I-4–6 by the Lewis-acid AlCl3,
the reactions were carried out at 0 °C. Additionally, at low temper-
atures, more of the para-acylated product was obtained with re-
spect to the hydroxyl group. The obtained intermediates IIa-1–6
were treated with 2-iodocarboxylic acid in the presence of sodium
hydroxide to give the corresponding sodium salts. Acidification of
these salts with hydrochloric acid yielded compounds IIIa-1–6.
The results from the in vitro cytotoxicity assay11,12 (Table 1)
show, that 24 h treatment of MCF-7/AZ cells with compounds
O
COOH
EA
None
>100
O
O
COOH
COOH
COOH
IIIa-1
IIIa-2
None
None
25
20
O
O
O
O
O
IIIa-3
IIIa-4
None
38
10
OMe
O
O
COOH
COOH
COOH
>100
IIIa-1, IIIa-2, and IIIa-3 at concentrations of 25
lM, 20 lM, and
MeO
O
10 M, respectively, results in a 20% reduction of cell viability.
l
IIIa-5
IIIa-6
29
52
>100
>100
Compound IIIa-1 is not substituted at the phenyl ring, whereas
compound IIIa-2 possesses a methyl substituent in ortho-position
to the phenolic oxygen atom and compound IIIa-3 possesses a
methyl group in meta-position with respect to the phenolic oxygen
atom.
O
OMe
O
O
OMe
In contrast, no significant cytotoxic effect could be observed for
compounds IIIa-4, IIIa-5, and IIIa-6, at concentrations lower or
equal to 100 lM. These three compounds have in common that
their phenyl rings are substituted with either one (IIIa-4, IIIa-5)
or two (IIIa-6) methoxy groups.
The in vitro wound healing assay13,14 shows very interesting
and promising results (Fig. 3). Compounds IIIa-1, IIIa-2, and IIIa-
3 show no substantial effect on the migration of the breast cancer
cells. In stark contrast to these results, compounds IIIa-4, IIIa-5,
and IIIa-6 show a significant inhibition of the migration of the can-
cer cells. Compound IIIa-4 inhibits the migration of the human
MCF-7/AZ breast cancer cells by 38%, compound IIIa-5 by 29%
and compound IIIa-6 even by 52%.
The data from the wound healing assay suggest that electron
donating groups, attached to the aromatic system of the EA ana-
logues, have a positive effect on the potency of the corresponding
compounds to inhibit the migration of human MCF-7/AZ breast
cancer cells. Additionally, these compounds do not show any ob-
servable cytotoxicity at concentrations up to 100 lM.
Electron donating groups, like the methoxy groups, increase
the electron density of the aromatic system by donating lone
pair electrons into the aromatic system by resonance, thereby
Figure 3. Migration assay of the human breast cancer cell line, MCF-7/AZ, in the
absence (control) or presence of ethacrynic acid (EA) and its analogues (IIIa-1–6).
OH
OH
OH
OH
OH
OH
MeO
OMe
OMe
OMe
I-2
I-5
I-1
I-3
I-6
I-4
Figure 2. Several substituted phenols I-1–6.

1850
R. F. J. Janser et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1848–1850
increasing its reactivity towards electrophiles. On the other
hand, methyl groups are weak activators, they can only donate
References and notes
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Jing, Y.; Zhao, G. J. Med. Chem. 2010, Publication Date (Web): January 7, 2010.
7. Twentyman, P. R.; Lambert, E.; Muller, M.; Rees, J. K. Leukemia 1992, 6, 726.
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electron density into the aromatic system through the
r
-bond,
resulting in only
electrophiles.
a slight increase in reactivity towards
It can be speculated that the strong increase of electron density
in the aromatic system is responsible for the anti-migratory prop-
erties of compounds IIIa-4, IIIa-5, and IIIa-6. In further investiga-
tions, other electron donating substituents will be attached to
the phenyl ring. These new compounds will subsequently be eval-
uated for their effect on the migratory capacity of human MCF-7/
AZ breast cancer cells.
Due to the relative lack of potency and its diuretic properties,
EA is not suitable as a drug in cancer treatment. Several of the ana-
logues mentioned above demonstrate anti-migratory activity to-
wards the migration of human MCF-7/AZ breast cancer cells at
non-toxic concentrations. Due to the considerable difference in
structure of the new analogues versus EA, it can be speculated that
these analogues do not possess a diuretic nature. These trends are
therefore worth to be studied further with an eye to drug
development.
9. Christofori, G. Nature 2006, 25, 444.
10. Fidler, I. J. Nat. Rev. Cancer 2003, 3, 453.
11. The cytotoxicities of ethacrynic acid and its analogues, lacking the
a,b-
unsaturated carbonyl unit, were tested in accordance to Sigstedt et al.12
Mitochondrial dehydrogenase activities were measured by an MTT-reagent.
Cells were seeded in 96-well plates at an initial 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
incubation, at 37 °C in a humidified 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 removed prior to the addition of the MTT reagent. Three
lg/mL streptomycin. After 24 h of
l
l
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.
In summary, we have synthesized EA analogues, lacking the
,b-unsaturated carbonyl unit, with enhanced potency, relative
a
to EA, towards the inhibition of migration of human MCF-7/AZ
breast cancer cells (Table 1 and Fig. 3). Differences in potency
among the various analogues may simply be due to the presence
of different substituents at the phenyl ring of the EA analogues.
Further studies with other EA analogues and various cancer cell
lines are underway.
12. 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.
13. 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
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
Acknowledgments
We thank New Mexico Tech for supplying the start-up funds for
Dr. I.J. The biomedical evaluation of this work was supported by
the US National Institutes of Health (RR-16480) under the INBRE
program of the National Center for Research Resources.
quantify the migration rate, which is expressed as migratory velocity (
lm/h).
At least three independent experiments were performed.
14. Van slambrouck, S.; Hilkens, J.; Bisoffi, M.; Steelant, W. F. A. Int. J. Oncol. 2009,
35, 693.