Ethacrynic acid as a lead structure for the development of potent urease inhibitors

Article abstract of DOI:10.1016/j.crci.2013.03.020

Ethacrynic acid and a series of its analogues were synthesized and subsequently evaluated for their inhibitory effect on jack bean urease. Ethacrynic acid showed, even at low concentrations, very potent inhibitory activity against the enzyme. For ethacrynic acid, the inhibition potential increased with increasing preincubation time of ethacrynic acid and enzyme, whereas for some other compounds a higher preincubation time lead to a significant reduction of their activity. We could demonstrate that the α,β-unsaturated carbonyl unit of our compounds is mandatory to inhibit the enzyme, possibly due to its ability to bind to cysteine residues in the active site of the jack bean urease.

Full text of DOI:10.1016/j.crci.2013.03.020

C. R. Chimie 16 (2013) 660–664
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´
Full paper/Memoire
Ethacrynic acid as a lead structure for the development of potent
urease inhibitors
b
b
Ingo Janser a, , Caitlyn M. Vortolomei , Ranjith K. Meka ,
*
Courtney A. Walsh b, Romy F.J. Janser b
a Department of Chemistry, Eastern Michigan University, 48197 Ypsilanti, MI, USA
b Department of Chemistry, New Mexico Institute of Mining and Technology, 87801 Socorro, NM, USA
A R T I C L E I N F O
A B S T R A C T
Article history:
Ethacrynic acid and a series of its analogues were synthesized and subsequently evaluated
for their inhibitory effect on jack bean urease. Ethacrynic acid showed, even at low
concentrations, very potent inhibitory activity against the enzyme. For ethacrynic acid, the
inhibition potential increased with increasing preincubation time of ethacrynic acid and
enzyme, whereas for some other compounds a higher preincubation time lead to a
Received 9 December 2012
Accepted after revision 28 March 2013
Available online 6 May 2013
Keywords:
significant reduction of their activity. We could demonstrate that the
a,b-unsaturated
a,b-unsaturated ketones
Ethacrynic acid
Inhibitor
carbonyl unit of our compounds is mandatory to inhibit the enzyme, possibly due to its
ability to bind to cysteine residues in the active site of the jack bean urease.
´
ß 2013 Academie des sciences. Published by Elsevier Masson SAS. All rights reserved.
Peptic ulcer
Urease
1. Introduction
However, without denaturation of the enzyme, only six of
the 15 cysteine residues are accessible for reagents. One of
Urease (EC 3.5.1.5) is an enzyme that catalyzes the
conversion of urea into ammonia and carbon dioxide [1,2].
While urea amidolyase breaks down urea in two steps,
urease does this in a single step [3]. Urease can be found in
a great variety of algae, bacteria, plants, and fungi [1,4,5].
Although, the structure, type and number of subunits, their
molecular weight and the amino acid sequence of the
ureases differ, the amino acid sequence of the active sites
are conserved and therefore, the mechanism of the enzyme
activity is the same for all ureases [5,6]. Urease isolated
from the plant jack bean is a homohexameric molecule
whose active site contains two nickel ions that are involved
in binding of substrates as well as of some of the inhibitors
[7,8]. Each jack bean urease subunit possesses 15 cysteine
residues, which makes urease a thiol-rich enzyme [6,9].
these residues, cysteine-592, is recognized to play a critical
role in the catalytic activity of the enzyme [10,11]. Ureases
are ubiquitous in nature and it is known that they are
directly associated with the formation of infection stones
and contribute to the pathogenesis of several infectious
diseases like e.g. pyelonephritis, an ascending urinary tract
infection [12]. Additionally, they are an important factor in
the pathogenesis of many clinical conditions [13,14].
Helicobacter pylori (H. pylori) produces high quantities of
urease and thereby large amounts of ammonia are formed
which makes it possible for the bacteria to inhabit the
stomach [12]. This high amount of ammonia in the
stomach is now accepted as the major cause of peptic
ulcers [2,15]. For this reason, urease inhibitors have
attracted a lot of attention as potent anti-ulcer drugs
[16]. Due to the diverse function of the enzyme, the study
of urease inhibition not only has medical but also
environmental and agronomic significance [6]. The devel-
opment of potent and specific inhibitors could lead to the
treatment of infections caused by urease-producing
* Corresponding author. Department of Chemistry, Eastern Michigan
University, 541 Science Complex, 48197 Ypsilanti, MI, USA.
E-mail address: ijanser@emich.edu (I. Janser).
1631-0748/$ – see front matter ß 2013 Acade´mie des sciences. Published by Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.crci.2013.03.020

I. Janser et al. / C. R. Chimie 16 (2013) 660–664
661
Fig. 1. Ethacrynic acid (IV-1) and seven of its analogues (IV-2-8). Compounds IV-1 and IV-2, IV-3 and IV-4, IV-5 and IV-6, as well as IV-7 and IV-8 are
considered as chain length analogues.
bacteria [17]. In 2003, Kawase et al. reported for the first
time that several -unsaturated ketones are inhibitors
for jack bean urease. The most potent -unsaturated
ketones were cyclic and of low-molecular weight, e.g. 2-
cyloheptene-1-one (IC50 = 0.16 mM), 2-cyclohexene-1-one
(IC50 = 0.69 mM), 2-cyclopentene-1-one (IC50 = 0.97 mM)
[12].
(n = 1) or butanoyl chloride (n = 2), respectively, was
performed in the presence of powdered aluminium
chloride (AlCl3) in carbon disulfide, as recently described
by us [22]. Compounds II-1-8 were purified by flash
column chromatography on silica gel and consecutively
refluxed in acetone for 48 hours in the presence of 1.2
equivalents of ethyl bromoacetate and two equivalents
potassium carbonate (K2CO3) to yield compounds III-1-8.
In the third step, an aldol condensation reaction, com-
pounds III-1-8 were refluxed in an ethanol/water (50/50)
mixture for 24 hours in the presence of two equivalents of
formaldehyde and 2.5 equivalents of K2CO3. Compounds
IV-1-8 were obtained by flash chromatography using a
hexanes/ethyl acetate/methanol mixture as the eluent.
a,b
a,b
To the best of our knowledge, no further report on other
a
,b
-unsaturated carbonyl compounds as potential inhi-
bitors of urease has been published. Ethacrynic acid, a loop
or high ceiling diuretic, possesses an -unsaturated
a,b
carbonyl unit and is used to treat high blood pressure and
edema [18,19]. We have recently synthesized several
analogues of ethacrynic acid lacking the
a,b-unsaturated
carbonyl unit and discovered that some of our analogues
possess a significant potency to inhibit the migration of
several cancer cell lines [20,21]. We herein report the
inhibitory effect of ethacrynic acid (IV-1) and seven of its
analogues (IV-2-8) (Fig. 1) on jack bean urease.
2.2. Urease inhibition assay
The assay mixture containing 50
(5 mg of type III urease dissolved in 0.5 mL phosphate
buffered saline [PBS]) and 1 L of the test compound
mL jack bean urease
m
2. Experimental
(dissolved in dimethyl sulfoxide [DMSO]) was preincu-
bated for 60, 120, 180, and 240 minutes at room
temperature in a 96-well assay plate. After preincubation,
2.1. General procedure for the preparation of ethacrynic acid
and its analogues
150 mL urea broth containing phenol red pH indicator
(9.68 g urea medium dissolved in 250 mL reverse osmosis
[RO] water) was added to each well. Once the substrate and
the indicator were added, a micro plate reader was used to
measure the initial absorbance for each well at 595 nm.
The results (change in absorbance per min) were processed
using SoftMax Pro software (Molecular Devices). Inhibition
The synthesis of ethacrynic acid (IV-1) and its seven
analogues (IV-2-8, Fig. 1) was accomplished by a three-
step reaction shown in Scheme 1. The Friedel–Crafts
acylation reaction of the phenol or the substituted phenols
I-1-4 (Scheme 1), respectively, with propanoyl chloride
Scheme 1. Synthesis of ethacrynic acid (IV-1) and seven of its analogues (IV-2-8).

662
I. Janser et al. / C. R. Chimie 16 (2013) 660–664
percentages were calculated using the formula 100–
(ODtestwell/ODcontrol) Â 100. In order to determine IC50
values, inhibition studies were carried out at various
concentrations of the test compound (1.0 mM, 0.5 mM,
0.4 mM, 0.3 mM, 0.2 mM, and 0.1 mM). All reactions were
performed in triplicate.
urease, were used. After an incubation time of 1 hour, the
inhibitory activity of the most potent compounds (IV-5-8)
was equal or better than that of hydroxyurea. However,
they are three to four times less active than acetohy-
droxamic acid. An early conclusion is that the chain length
of the acyl group does not seem to have a significant effect
on the inhibitory activity of the corresponding compound,
whereas the substitution pattern at the aromatic ring of
the ethacrynic acid seems to have a great influence on the
activity of the corresponding compound (chloro- versus
hydrogen- versus methoxy substituent).
3. Results and discussion
Compounds IV-1-8 (Fig. 1) all of which possess an a,b-
unsaturated carbonyl unit were subsequently tested
against jack bean urease. We initially tested the inhibitory
effects of our eight compounds (IV-1-8) at various
concentrations thereby keeping the concentration of
urease constant. Before adding the urea broth, we
preincubated the enzyme with the respective compound
for 60 minutes. The results of our enzyme assays are
shown in Table 1. Ethacrynic acid (IV-1) itself inhibits jack
bean urease by 28% (0.5 mM) or 43% (1.0 mM), respec-
tively (IC50 = 1.21 mM). Similar observations were made
for its chain length analogue (IV-2) whose inhibitory
activity of 26% (0.5 mM) or 41% (1.0 mM), respectively
(IC50 = 1.32 mM), is slightly lower than that observed for
ethacrynic acid (IV-1). For compound IV-3, we found that
it inhibits jack bean urease by 50% at a concentration of
0.5 mM and even by 65% at a concentration of 1.0 mM
(IC50 = 0.50 mM). Similar results were obtained for its
chain length analogue IV-4 (48% inhibition at a concen-
tration of 0.5 mM and 63% inhibition at a concentration of
1.0 mM). Even higher inhibitory activities could be found
for compound IV-5 and its chain length analogue IV-6.
These compounds are capable of inhibiting the enzyme
by 64% or 65%, respectively, at the lower concentration
of 0.5 mM and by 78% or 79%, respectively, at the
higher concentration of 1.0 mM (IV-5, IC50 = 0.15 mM;
IV-6, IC50 = 0.13 mM). Similar results were found for
compounds IV-7 and its chain length analogue IV-8.
Compound IV-7 inhibited jack bean urease by 62%
(0.5 mM) and by 76% at a concentration of 1.0 mM
(IC50 = 0.18 mM) and compound IV-8 showed 61% inhibi-
tion at a concentration of 0.5 mM and 75% inhibition at a
concentration of 1.0 mM (IC50 = 0.21 mM). As a control,
hydroxyurea (IC50 = 0.21 mM) and acetohydroxamic acid
(IC50 = 0.05 mM), both known inhibitors of jack bean
We were now interested if an increase in preincubation
time has an effect on the inhibition of the enzyme.
Therefore, we investigated the inhibitory activities of our
compounds at various preincubation times (1, 2, 3, and
4 hours). Much to our delight, the inhibitory activities of
ethacrynic acid (IV-1) and its chain length analogue (IV-2)
were significantly increased at higher incubation times.
The highest activity observed was after a preincubation of
4 hours. After this preincubation time, ethacrynic acid (IV-
1) inhibited the enzyme by 71% at a concentration of
1.0 mM (IC50 = 0.25 mM). Surprisingly, with increasing
preincubation time, the activities of the other compounds
(IV-5-8) did not increase as significantly as for ethacrynic
acid and its chain length analogue after an incubation time
of 4 hours (IV-5, IC50 = 0.08 mM; IV-6, IC50 = 0.05 mM, IV-7,
IC50 = 0.07 mM; IV-8, IC50 = 0.10 mM) (Table 1). The
observation we made for compounds IV-3 and IV-4 was
completely unexpected. The inhibitory activity of these
compounds decreased from 48% inhibition (0.5 mM, 1 hour
preincubation) to nearly no observable activity after a
preincubation of 4 hours. We speculated that compounds
IV-3 and IV-4 were degraded by the enzyme, which was
affirmed by the fact that we were able to detect the
presence of compounds IV-3 and IV-4 in the assay mixture
after the preincubation of 1 hour, but not after 4 hours of
incubation. The resulting product of the degradation
couldn’t be identified. As a control, after a preincubation
of 4 hours, we added more of compound IV-3 and IV-4 and
could observe a returning inhibitory activity. Why this
decomposition happens for compounds IV-3 and IV-4 and
not for the chloro- or methoxy substituted compounds is
not understood yet. It is noteworthy that all of the tested
compounds inhibit the urease reversibly. Adding more
urea to the assay mixture resulted in further activity of the
enzyme.
Table 1
In another experiment, we wanted to determine, if the
addition of sulfhydryl compounds, such as benzyl mer-
captan or cyclohexyl mercaptan, reduces the inhibitory
activity of our compounds or even prevents the inhibition
of jack bean urease. Sulfhydryl compounds are Michael
Inhibition of jack bean urease by ethacrynic acid (IV-1) and seven of its
analogues (IV-2-8) at different incubation times.
Compound
IC50 (mM)
1 ha
2 ha
3 ha
4 ha
donors and can attack the b-carbon of the a,b-unsaturated
IV-1 (Ethacrynic acid)
1.21
1.32
0.50
0.48
0.15
0.13
0.18
0.21
0.21
0.05
0.51
0.55
0.81
0.89
0.12
0.11
0.16
0.20
0.20
0.04
0.32
0.36
2.21
2.25
0.10
0.09
0.10
0.17
0.21
0.04
0.25
0.28
À
carbonyl unit, thereby preventing its reaction with
cysteine residues in the active site of the jack bean urease
The results from our enzyme assays show that one
equivalent of the corresponding sulfhydryl compound
(benzyl mercaptan or cyclohexyl mercaptan) affected the
inhibitory activity of ethacrynic acid IV-1 and its analogues
IV-3, IV-5, IV-7 significantly. We preincubated the enzyme
with the respective compounds IV-1, IV-3, IV-5, and IV-7 at
various concentrations in the presence of one equivalent of
IV-2
IV-3
IV-4
À
IV-5
0.08
0.05
0.07
0.10
0.22
0.05
IV-6
IV-7
IV-8
Hydroxyurea
Acetohydroxamic acid
a
Preincubation time.

I. Janser et al. / C. R. Chimie 16 (2013) 660–664
663
Table 2
and IV-4 (IC50 = 0.48 mM, 1 h preincubation) are also
capable of inhibiting jack bean urease. However, their
inhibitory activity is reduced with increasing incubation
time. It can be speculated that these compounds are
degraded by the enzyme. Further investigations to
account for these findings are necessary. Due to the
distinct inhibitory activities of compounds IV-5, IV-6, IV-
7, and IV-8, it can be speculated that the substituents
attached to the aromatic system (chloro- versus hydro-
gen- versus methoxy substituent) have a great influence
Effects of additives on the inhibitory activity of ethacrynic acid (IV-1) and
three of its analogues (IV-3, IV-5, and IV-7) against jack bean urease at
different incubation times.
Compound
Additives
IC50
(mM)
1 ha 4 ha
1.21 0.25
IV-1
None
(Ethacrynic acid)
Benzyl mercaptan (1 equiv)
2.65 0.50
Cyclohexyl mercaptan (1 equiv) 2.48 0.55
on the activity of the corresponding compound.
A
IV-3
IV-5
IV-7
None
0.50
1.15
À
À
possible explanation for these distinct inhibitory activ-
ities is that methoxy groups favor the Michael addition
reaction due to the fact that they activate the aromatic
system through a resonance effect. On the other hand, the
chloro substituents deactivate the ring through an
electron-withdrawing inductive effect and thereby pos-
sibly disfavoring the Michael addition reaction. Further
structure–activity relationship experiments have to be
performed in order to get a better understanding of the
influence of the substituents. The addition of sulfhydryl
compounds reduces the activity of ethacrynic acid and its
analogues significantly. This is insofar not surprising
since the sulfur of the sulfhydryl compound can attack
Benzyl mercaptan (1 equiv)
Cyclohexyl mercaptan (1 equiv) 1.11
None
0.15 0.08
0.35 0.20
Benzyl mercaptane (1 equiv)
Cyclohexyl mercaptan (1 equiv) 0.28 1.17
None
0.18 0.07
0.35 0.15
Benzyl mercaptane (1 equiv)
Cyclohexyl mercaptan (1 equiv) 0.34 0.16
Acetohydroxamic
acid
None
0.05 0.01
Benzylmercaptane (1 equiv)
0.05 0.01
Cyclohexyl mercaptan (1 equiv) 0.05 0.01
a
Preincubation time.
the
b-carbon of the a,b-unsaturated carbonyl unit,
thereby preventing its reaction with cysteine residues
in the active site of the jack bean urease. Testing
the corresponding sulfhydryl compound (benzyl mercap-
tan or cyclohexyl mercaptan, respectively) for 1 and for
4 hours and found that the inhibitory activity of the
compounds was reduced by approximately half of their
original activity (Table 2). As expected, the inhibitory
activity of our control (acetohydroxamic acid) was not
affected by the additives, no loss of activity could be
precursors of our compounds which lack the
unsaturated carbonyl unit (III-1-8) for their inhibitory
activity on urease reveals the importance of the
unsaturated carbonyl unit because none of them show a
measurable inhibition of the enzyme. Further investiga-
tions to design even more potent ethacrynic acid
analogues for the inhibition of jack bean urease are
currently underway in our laboratory.
a,b-
a,b-
observed. This is an indication that the
a,b-unsaturated
carbonyl unit is responsible for the inhibitory activity of
our compounds described above. In order to verify our
hypothesis, we tested all eight precursors (III-1-8), lacking
Acknowledgements
the
a,b-unsaturated carbonyl unit (Scheme 1) for their
The financial support of the US National Institutes of
Health (P20 RR016480) under the INBRE program of the
National Center for Research Resources (NCRR) is greatly
appreciated.
inhibitory activity on jack bean urease. As expected,
none of the tested compounds were able to measurably
inhibit the urease, not even at concentrations higher
than 1.0 mM.
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In summary, we have tested ethacrynic acid (IV-1)
and seven of its analogues (IV-2-8) for their potency
to inhibit jack bean urease. We could demonstrate that
the inhibitory activities of ethacrynic acid (IV-1) and
its chain length analogue (IV-2) were significantly
increased with higher incubation times. The highest
activity observed was after a preincubation of 4 hours
(IC50 = 0.25 mM or IC50 = 0.28 mM, respectively). How-
ever, the highest inhibitory activity was found for
compound IV-6 (IC50 = 0.05 mM), compound IV-7
(IC50 = 0.07 mM), compound IV-5 (IC50 = 0.08 mM), and
compound IV-8 (IC50 = 0.10 mM) after a preincubation
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