Amide derivatives of ethacrynic acid: Synthesis and evaluation as antagonists of Wnt/β-catenin signaling and CLL cell survival

Article abstract of DOI:10.1016/j.bmcl.2008.12.067

A series of amides of ethacrynic acid was prepared and evaluated for their ability to inhibit Wnt signaling and decrease the survival of CLL cells. Several of the most potent derivatives were active in the low micromolar range. Reduction of the α,β-unsatu

Full text of DOI:10.1016/j.bmcl.2008.12.067

Bioorganic & Medicinal Chemistry Letters 19 (2009) 606–609
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Amide derivatives of ethacrynic acid: Synthesis and evaluation as antagonists
of Wnt/b-catenin signaling and CLL cell survival
*
Guangyi Jin, Desheng Lu, Shiyin Yao, Christina C. N. Wu, Jerry X. Liu, Dennis A. Carson, Howard B. Cottam
Moores Cancer Center, University of California, San Diego, 3855 Health Sciences Drive, La Jolla, CA 92093-0820, 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 amides of ethacrynic acid was prepared and evaluated for their ability to inhibit Wnt signaling
and decrease the survival of CLL cells. Several of the most potent derivatives were active in the low micro-
molar range. Reduction of the a,b-unsaturated carbon–carbon double bond of EA abrogated both the inhi-
bition of Wnt signaling as well as the decrease in CLL survival. Preliminary mechanism of action studies
suggest that these derivatives covalently modify sulfhydryl groups present on transcription factors
important for Wnt/b-catenin signaling.
Received 22 October 2008
Revised 13 December 2008
Accepted 16 December 2008
Available online 24 December 2008
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
Chronic lymphocytic leukemia
Wnt/b-catenin signaling
Ethacrynic acid amides
Lef-1
Chronic lymphocytic leukemia (CLL), the most common adult
leukemia in the United States, is characterized by the accumulation
of mature-appearing, but functionally incompetent small lympho-
cytes. There is as yet no cure for this disease, nor has conventional
chemotherapy been definitively shown to prolong patient survival.
Lateinthediseasecourse, patientstypicallydeveloppronouncedbone
marrow dysfunction due to chemotherapy-induced toxicity and dis-
ease progression, making them intolerant to further treatment with
cytotoxic agents. Thus, it is necessary to develop new treatments that
target the molecular defects in CLL with minimal bone marrow toxic-
ities. The clonal expansion of B-lymphocytes in CLL is caused by an
abnormal balance between the signaling for survival and cell death.1
Wnt signaling pathways play a number of key roles in embry-
onic development and maintenance of homeostasis in mature tis-
sues. Wnt proteins are a large family of secreted glycoproteins
that activate signal transduction pathways to control a wide vari-
ety of cellular processes such as determination of cell fate, prolifer-
ation, migration, and polarity. Wnts are capable of signaling
through several pathways, the best-characterized being the canon-
ical b-catenin/Tcf-LEF mediated pathway. Canonical Wnts stabilize
b-catenin protein, which has implications in the genesis of many
human cancers. Indeed, growing evidence suggests that deregula-
tion of the Wnt/b-catenin pathway is directly linked to tumorigen-
esis.2,3 Recently, it has been demonstrated that the Wnt signaling
pathway is activated in CLL cells, and that uncontrolled Wnt/b-
catenin signaling may contribute to the defect in apoptosis that
characterizes this malignancy.4,5 Therefore, the Wnt/b-catenin sig-
naling molecules are attractive candidates for developing targeted
therapies for CLL.
Cell-based screens of libraries of natural products and synthetic
small molecules have provided useful tools for the study of complex
cellular processes. Indeed, a number of small molecules have been
identified that modulate Wnt/b-catenin signaling, including NSA-
IDs,6 quercetin,7 ICG-001,8 and others.9 To identify additional mole-
cules that regulate the canonical Wnt/b-catenin signaling pathway,
we employed a b-catenin dependent cell reporter system to screen
for specific antagonists of the Wnt/b-catenin signaling pathway
from a library of 960 known FDA-approved drugs (MicroSource
Gen-Plus library). One such molecule, ethacrynic acid (1, EA) was
identified among the few inhibitors. Ethacrynic acid, a once com-
monly used loop diuretic drug, was previously shown to be uniquely
cytotoxic toward primary CLL cells.10 However, EA is not ideal as a
chemotherapeutic agent for CLL treatment due to its diuretic prop-
erties and relative lack of potency. Therefore, as initial efforts to
optimize this lead, we report here the synthesis and in vitro evalu-
ation of some amide derivatives of EA for inhibition of Wnt signaling
and for decreasing the survival of cells from CLL patients.
The preparation of the amide derivatives of EA was accom-
plished by refluxing EA in benzene with thionyl chloride to form
the EA acyl chloride intermediate followed by reaction in pyridine
with desired amines (Scheme 1).
Thus, by this procedure over 40 compounds were prepared and
evaluated. Furthermore, to explore the contribution of the C–C
double bond of the a–b-unsaturated carbonyl function to the bio-
activity, we reduced the double bond of EA by catalytic hydrogena-
tion11 to afford EA-R (42). In addition, a few simple alkyl esters of
EA and a ‘‘truncated” decarboxylated version of EA (compound 43)
* Corresponding author. Tel.: +1 858 534 5424; fax: +1 858 534 5399.
E-mail address: hcottam@ucsd.edu (H.B. Cottam).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.067

G. Jin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 606–609
607
Cl
Cl
Cl
O
Cl
O
Cl
Cl
O
O
O
H2N
R
SOCl2 /Benzene
O
N
R
Cl
O
O
OH
O
H
EAamides
EA
EA acyl chloride
Scheme 1.
0.4
0.3
0.2
0.1
0.0
Cl
Cl
Cl
O
Cl
O
O
OH
O
O
42
43
EA
The mechanism of ethacrynic acid cytotoxicity has been attrib-
EA-R
12
uted to the drug’s known capacity to inhibit glutathione S-transfer-
ase (GST), causing increased cellular oxidative stress. However, a
recent study13 showed that the antioxidant N-acetyl-
L-cysteine
13
(NAC) protected ethacrynic acid-induced cell death with no effect
on cellular glutathione levels, whereas the free radical scavenger
3(2) tert-butyl-4-hydroxyanisole (BHA) did not repress ethacrynic
acid-induced cell death, suggesting the existence of additional or
alternative pathways that are altered by the drug. Since EA is clas-
0.01
0.1
1
10
100
Concentration (µM)
Figure 1. Comparison of EA and EA-R on CLL viability.18 This experiment was
performed as described in Ref. 18.
sified as an a,b-unsaturated ketone, its Wnt inhibition activities are
most likely due to the alkylation effects on Wnt proteins which are
comprised of cysteine-rich glycoproteins.14 Indeed, we found that
inhibition of Wnt signaling by EA can be blocked by adding N-acet-
yl-L-cysteine or 2-aminoethanethiol to the media prior to testing
(data not shown). Moreover, decreased survival of CLL cells and
inhibition of Wnt signaling by EA were completely abrogated after
were prepared. Although EA esters were reported to kill CLL cells at
low micromolar concentrations,12 those esters along with 43 were
also highly toxic to peripheral blood mononuclear cells (PBMC) in
our assays and were not studied further for Wnt and CLL activity
(data not shown).
Table 1
Inhibition of Wnt signaling and CLL survival by ethacrynic amides
Cl
O
Cl
O
N
R
O
H
Entry
R
CLL inhibition
Wnt inhibition
IC50 M)
Entry
R
CLL inhibition
EC50 M)
Wnt inhibition
EC50
(lM)
(
l
(
l
IC50
(lM)
COOH
1
Ethacrynic acid
9.9
32.7
21
22
14.9
>50
N
OH
O
NO2
2
3
4
5
4.1
6.4
3.7
4.5
>50
>25
>50
COOH
CO2Me
>50
23
24
25
4.1
11.38
6.58
2.63
OH
OH
O
N
N
4.76
8.5
2.8
NH2
OH
–OH
9.89
5.81
O
O
O
H
O
N
6
5.0
26
2.8
4.42
COOH
(continued on next page)

608
G. Jin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 606–609
Table 1 (continued)
Entry
R
CLL inhibition
EC50 M)
Wnt inhibition
Entry
R
CLL inhibition
EC50 M)
Wnt inhibition
IC50 M)
(
l
IC50
(l
M)
(
l
(l
O
SH
N
7
8
4.8
>50
27
28
1.8
2.93
5.06
CO2Et
O
O
7.8
6.84
3.9
3.2
CN
COOH
Cl
N
9
15.9
>50
29
30
3.62
N
H
O
N
Cl
HO
OMe
10
13.8
21.9
9.62
>25
>50
N
HOOC
HOOC
S
O
OMe
NH
OEt
11
>50
31
2.1
2.61
2.97
OMe
O
EtO
O
O
O
NH
O
12
13
14
15
16
17
2.5
1.5
6.4
3.2
4.88
4.86
32
33
34
35
36
37
2.1
13.8
7.4
O
O
N
S
>50
N
NO2
OH
>50
4.23
3.80
N
O
Cl
COOH
>50
>50
>50
>50
5.5
OH
COOH
>25
>25
>50
SO3H
COOH
COOH
8.0
3.7
2.93
CO2Et
COOMe
18
19
10.6
20.6
38
39
1.7
3.0
3.79
2.51
N
COOH
5.85
COOH
H
N
O
20
41
5.9
10.7
40
42
2.6
1.81
NHOH
OH
>25
>50
EA-R
>25
>50

G. Jin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 606–609
609
5. Lu, D.; Zhao, Y.; Tawatao, R.; Cottam, H. B.; Sen, M.; Leoni, L. M.;
Kipps, T. J.; Corr, M.; Carson, D. A. Proc. Natl. Acad. Sci. U.S.A. 2004,
101, 3118.
6. Lu, D.; Cottam, H. B.; Corr, M.; Carson, D. A. Proc. Natl. Acad. Sci. U.S.A. 2005, 102,
18567.
7. Park, C. H.; Hahm, E. R.; Lee, J. H.; Jung, K. C.; Yang, C. H. Biochem. Biophys. Res.
Commun. 2005, 331, 1222.
8. Teo, J.-L.; Ma, H.; Nguyen, C.; Lam, C.; Kahn, M. Proc. Natl. Acad. Sci. U.S.A. 2005,
102, 12171.
9. Barker, N.; Clevers, H. Nat. Rev. Drug Discov. 2006, 5, 997.
10. Twentyman, P. R.; Lambert, E.; Muller, M.; Rees, J. K. Leukemia 1992, 6,
726.
11. Woltersdorf, O. W., Jr.; DeSolms, S. J.; Schultz, E. M.; Cragoe, E. J., Jr. J. Med.
Chem. 1977, 20, 1400.
12. Zhao, G.; Liu, C.; Wang, R.; Song, D.; Wang, X.; Lou, H.; Jing, Y. Bioorg. Med.
Chem. 2007, 15, 2701.
13. Aizawa, S.; Ookawa, K.; Kudo, T.; Asano, J.; Hayakari, M.; Tsuchida, S. Cancer Sci.
2003, 94, 886.
14. Takahashi-Yanaga, F.; Sasaguri, T. J. Pharmacol. Sci. 2007, 104, 293.
15. Rossi, A.; Kapahi, P.; Natoli, G.; Takahashi, T.; Chen, Y.; Karin, M.; Santoro, M. G.
Nature 2000, 403, 103.
16. Wnt signaling inhibition method I: The human embryonic kidney cell line
HEK293 was grown and transfected with various expression plasmids
encoding proteins in the Wnt and b-catenin pathway, exactly as described
previously.6 They included the b-catenin regulated TOPFlash reporter gene
and expression plasmids for Wnt1, Wnt3, b-catenin, DSH, LRP6, as
described.
17. Wnt signaling inhibition method II: To determine the specificity of
compounds on Wnt/b-catenin pathway inhibition, CellSensor LEF/TCF-bla
SW480 cell-based assay (Invitrogen, Carlsbad, CA) was used according to
the supplier’s instructions, but modified for 96 well format. Cells were
plated at 25,000 cells/well in assay medium in 96-well black plates with
clear bottom (Corning) the day prior to compound treatment. Compounds
reduction of the
a–b double bond by hydrogenation (Fig. 1, EA-R
and Table 1, compound 42), suggesting that this Michael acceptor
function is essential for its activity.
Several compounds were found to effectively decrease CLL sur-
vival and antagonize Wnt signaling at low micromolar concentra-
tions (25, 29, 31, 37, 39 and 40). These results correlate with our
earlier findings that Wnt signaling genes are over-expressed and ac-
tiveinCLL.5 ItispossiblethatEAderivativesmightinhibitWntsignal-
ing by covalent modification of sulfhydryl groups of Wnt-dependent
genes such as Lef-1 (which is highly expressed in CLL) and this possi-
bility is the subject of ongoing studies in our laboratories.
Structure–activity trends among the amides in terms of Wnt sig-
naling inhibition revealed that aromatic-containing amides were
generally more active than aliphatic amides. Moreover, the larger
aromatic substitutions (benzothiazole, phthalimide, naphthyl car-
boxylicacid,etc.)showedgoodactivityinbothsystems.Itisnotewor-
thy that the IC50s for inhibition of Wnt signaling are consistently
lower than the EC50s for inhibition of CLL survival, except for most
of the aromatic carboxylic acids. This suggests that the active EA
derivatives may have some other target receptor in the cell, a target
that may impact CLL survival in addition to the Wnt signaling alone.
An example of a possible off-target receptor for the EA derivatives
might be inhibition of NF-jB activity through direct inhibition of
IKK-b, wherein the cysteine 179 in the activation loop of IKK-b can
be covalently modified by Michael acceptors.15 Two well known
examples of this are prostaglandin J2 and prostaglandin A1, both of
which contain the a,b-unsaturated carbonyl function, and thus the
EA derivatives may be acting in a similar manner.
were added to cells at a final concentration ranging from 33.3 to 0.5 lM,
incubated for 20 h and then combined with LiveBLAzerTM-FRET B/G
Substrate (CCF4-AM) for 2 h at room temperature. Fluorescence emission
values at 465 and 535 nm were obtained using
a standard fluorescence
In summary, we have synthesized amides of EA19–23 with
enhanced potency, relative to EA, toward the inhibition of Wnt sig-
naling and of CLL cell survival (Table 1 and Fig. 2 see Supplement).
Differences in the potency among the various derivatives may be
simply due to relative efficiency of compound delivery to cells
and their ability to access the nuclear compartment and make con-
tact with transcription factors important in Wnt signaling.
Further in vivo studies of these amide compounds are
underway.
plate reader and the 465/535 ratios were calculated for each treatment
(n = 2 for each data point). Results were normalized to untreated control
cells (set at 100%, n = 4), plotted as
% of control, and EC50 determined
using Prism 4.0a software (GraphPad).
18. Fresh CLL or peripheral blood mononuclear cells (PBMC) were plated at
2.5 Â 105 per well and treated with compounds for 48 h. Then 1/10 V of 5 mg/
mL MTT was added, and cells were incubated at 37 °C overnight. Finally, ½ V of
Lysis buffer was added to dissolve the insoluble purple formazan product,
incubated at 37 °C overnight, and OD at 570 nm was read and recorded.
19. General procedure for synthesis: To a mixture of 1 mmol of ethacrynic acid in
10 mL of benzene, 1 mL of thionyl chloride was added. The mixture was heated
at reflux for 1.5 h, solvent was removed in vacuo. Another 10 mL of benzene
was added and distilled off again. The residue was dissolved in a small volume
of benzene for the next step. The resulting ethacrynic chloride solution was
added dropwise to a solution of 1 mmol of amine in pyridine (10 mL) at 0 °C
with stirring. The reaction was stirred at ambient temperature for 3 h, the
solvent was distilled off in vacuo, the residue was dissolved in ethyl acetate,
and washed with water and brine. The organic layer was dried over anhydrous
MgSO4, and the residue was purified by silica gel column chromatography
(dichloromethane:methanol from 100:0 to 100:5) to obtain the pure EA
amides shown in Table 1.
Acknowledgments
We thank Michael Rosenbach, Haowen Zhou, Michael Chan, for
the technical assistance, and Nancy Noon for secretarial support.
This work was supported in part by the Leukemia and Lymphoma
Society SCOR grant, and Grants CA81534 and CA113318, both from
the National Institutes of Health.
20. Selected data for compound 4: 1H NMR (400 MHz, CDCl3) d 9.23 (br, 1H), 7.89 (d,
J = 8 Hz, 1H), 7.70 (d, J = 8 Hz, 1H), 7.21 (d, J = 8 Hz, 2H), 6.98 (d, J = 8 Hz, 2H),
6.20 (br, 1H), 5.98 (d, J = 8 Hz, 1H), 5.62 (d, J = 12 Hz, 1H), 4.79 (d, J = 12 Hz, 1H),
2.77 (s, 2H), 2.44 (q, J = 8 Hz, 2H), 1.17 (t, J = 8 Hz, 3H). MS (ESI) m/z: 422,
[M+H]+.
Supplementary data
21. Selected data for compound 6: 1H NMR (400 MHz, CDCl3) d 9.47 (br, 1H), 8.03 (d,
J = 8 Hz, 2H), 7.73 (d, J = 8 Hz, 2H), 7.21 (d, J = 8 Hz, 1H), 6.99 (d, J = 8 Hz, 1H),
5.98 (d, J = 8 Hz, 1H), 5.62 (d, J = 8 Hz, 1H), 4.81 (s, 2H), 2.90 (br, 1H), 2.45 (q,
J = 8 Hz, 2H), 1.17 (t, J = 8 Hz, 3H). MS (ESI) m/z: 423, [M+H]+.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.12.067.
22. Selected data for compound 37: 1H NMR (400 MHz, CDCl3) d 9.30 (br, 1H), 8.57
(s, 1H), 8.40 (s, 1H), 7.95(d, J = 8 Hz, 1H), 7.93 (d, J = 8 Hz, 1H), 7.86 (d, J = 8 Hz,
1H), 7.62 (d, J = 8 Hz, 1H), 7.22 (d, J = 8 Hz, 1H), 7.04 (d, J = 8Hz, 1H), 5.99 (s,
1H), 5.63 (s, 1H), 4.83 (s, 2H), 2.60 (br, 1H), 2.48 (q, J = 8 Hz, 2H), 1.17 (t,
J = 8 HZ, 3H). MS (ESI) m/z: 473, [M+H]+.
References and notes
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2. Peifer, M.; Polakis, P. Science 2000, 287, 1606.
3. Polakis, P. Genes Dev. 2000, 14, 1837.
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L.; Pickeral, O. K.; Rassenti, L. Z.; Powell, J.; Botstein, D.; Byrd, J. C.; Grever, M. R.;
Cheson, B. D.; Chiorazzi, N.; Wilson, W. H.; Kipps, T. J.; Brown, P. O.; Staudt, L.
M. J. Exp. Med. 2001, 194, 1639.
23. Selected data for compound 40: 1H NMR (400 MHz, CDCl3) d 10.70 (br, 1H),
10.38 (br, 1H), 9.00 (br, 1H), 7.64 (d, J = 1.6 Hz, 2H), 7.50 (d, J = 1.6 Hz, 2H), 7.32
(d, J = 8.4 Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 6.37 (s, 1H), 6.33 (s, 1H), 6.06 (s, 1H),
5.56 (s, 1H), 4.97 (s, 2H), 2.36 (q, J = 6.8 Hz, 2H), 1.07 (t, J = 7.6 Hz, 3H). MS (ESI)
m/z: 463, [M+H]+.