Catechol-based inhibitors of bacterial urease

Article abstract of DOI:10.1016/j.bmcl.2019.02.032

Targeted covalent inhibitors of urease were developed on the basis of the catechol structure. Forty amide and ester derivatives of 3,4-dihydroxyphenylacetic acid, caffeic acid, ferulic acid and gallic acid were obtained and screened against Sporosarcinia pasteurii urease. The most active compound, namely propargyl ester of 3,4-dihydroxyphenylacetic acid exhibited IC₅₀ = 518 nM andk_inact/K_i = 1379 M^?1 s^?1. Inhibitory activity of this compound was better and toxicity lower than those obtained for the starting compound – catechol. The molecular modelling studies revealed a mode of binding consistent with structure-activity relationships.

Full text of DOI:10.1016/j.bmcl.2019.02.032

Accepted Manuscript
Catechol-based inhibitors of bacterial urease
Aikaterini Pagoni, Theohari Daliani, Katarzyna Macegoniuk, Stamatia
Vassiliou, Łukasz Berlicki
PII:
S0960-894X(19)30116-7
https://doi.org/10.1016/j.bmcl.2019.02.032
BMCL 26312
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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25 February 2019
27 February 2019
Please cite this article as: Pagoni, A., Daliani, T., Macegoniuk, K., Vassiliou, S., Berlicki, L., Catechol-based
inhibitors of bacterial urease, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.
2019.02.032
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Catechol-based inhibitors of bacterial urease
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Aikaterini Pagoni, Theohari Daliani, Katarzyna Macegoniuk,
Stamatia Vassiliou and Łukasz Berlicki

Bioorganic & Medicinal Chemistry Letters
Catechol-based inhibitors of bacterial urease
Aikaterini Pagonia, Theohari Daliania, Katarzyna Macegoniukb, Stamatia Vassilioua* and Łukasz
Berlickib*
a Laboratory of Organic Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis, Zografou, 15701 Athens, Greece
b Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Targeted covalent inhibitors of urease were developed on the basis of the catechol structure.
Forty amide and ester derivatives of 3,4-dihydroxyphenylacetic acid, caffeic acid, ferulic acid
and gallic acid were obtained and screened against Sporosarcinia pasteurii urease. The most
Accepted
Available online
active compound, namely propargyl ester of 3,4-dihydroxyphenylacetic acid exhibited IC50
=
518 nM and
= 1379 M-1s-1. Inhibitory activity of this compound was better and
toxicity lower than those obtained for the starting compound – catechol. The molecular
modelling studies revealed a mode of binding consistent with structure-activity relationships.
Keywords:
Targeted covalent inhibitors
urease
2009 Elsevier Ltd. All rights reserved.
nickel-dependent enzyme
cysteine
irreversible inhibition
Targeted covalent inhibitors of enzymes are an emerging class
of biologically active compounds, which offer several key
advantages [1, 2]. In particular, higher potency, selectivity and
duration of action are expected in comparison to noncovalent
binders. Most typically, a cysteine residue is targeted, but other
amino acid residues are also explored in this context. Covalent
inhibition can lead to complete inactivation of the target, what is
particularly valuable in the case of antibacterial compounds.
There are numerous known antibiotics exhibiting such a mode of
action including beta-lactams and phosphomycin.
compounds, and (b) cysteine-binding structures. Hydroxamic
acids [17], phosphordiamidates [18], phosphonates [19],
phosphinates [20] and some heterocycles [21] represent the first
mentioned group of inhibitors, while unsaturated compounds
[22], ebselen derivatives [23], quinones [24] and heavy metal
ions [25] are members of the second set.
Our recent studies showed that covalent inhibitors of urease
could be highly active, but, surprisingly, most of the studied
compounds exhibited reversible mode of action [22,23,19].
Ebselen with Ki = 2.1 nM and acetylenedicarboxylic acid with Ki
= 42.5 nM are the most representative examples. Ciurli and co-
workers have recently shown that catechol is an irreversible
urease inhibitor that binds to the cysteine residue placed on the
movable flap [26]. The radical-based autocatalytic reaction
mechanism is proposed to explain this mode of action. The
studies are illustrated by the crystal structure of catechol-
inhibited urease from Sporosarcinia pasteurii (Figure 1).
Analysis this crystal structure indicates that catechol is a good
starting point for development of effective urease inhibitors.
Sufficient space around this molecule in the active site of the
enzyme gives a chance to introduce fragments that should
enhance the specific binding to the protein.
One of bacterial enzymes, which could be targeted by
covalent inhibitors is urease. Urease is a virulence factor of
Helicobacter pylori and Proteus spp., bacteria that commonly
infect gastroduodenal and urinary tracts, respectively [3,4]. This
enzyme catalyzes the reaction of urea hydrolysis to carbamate
and ammonia [5]. The released ammonia allows H. pylori
colonization of the stomach by creating a micro-environment
with elevated pH values [6] and, by degrading stomach mucosa
[7,8]. The enzymatic release of ammonia in the urinary tract
during infections involving Proteus spp. causes alkalinization of
urine and subsequently, rapid formation of urinary stones [9,10].
The active site of urease is built with two nickel ions
coordinated by six histidine residues and carbamoylated lysine (a
posttranslationally modified residue) [11,12]. During the reaction
the active site is closed by a movable loop that contains cysteine,
which could be important for inhibitor design. The substrate –
urea bound by nickel ions and a net of hydrogen bonds is
attacked by hydroxyl coordinated also by the two nickel ions.
In this article, we describe exploration of numerous
derivatives of catechol that contain carboxylic acid or ester/amide
functionalities as inhibitors of a bacterial urease. This class of
compounds was selected on the basis of previous studies which
shown that numerous carboxylic acid and their derivatives were
active against urease. Synthesis and evaluation of over forty
derivatives allowed to indicate compounds with good inhibitory
There are several classes of urease inhibitors [13-16], which
can be roughly divided to: (a) nickel ions coordinating

profile, that were further characterized by reactivity with
glutathione and toxicity evaluation.
Propargyl esters 12, 26 and 34 and phenyl azide were used in
a click chemistry approach to obtain triazole derivatives (method
E). Phenyl azide was prepared from aniline by diazotization with
sodium nitrite NaNO2 in acidic conditions followed by
displacement with sodium azide NaN3 in quantitative yield. 1,3-
Dipolar cycloaddition reaction of propargyl esters with
phenylazide in the presence of CuSO4·5H2O and sodium
ascorbate in t-BuOH:H2O (2:1) resulted in formation of triazolyl
derivatives (18, 30, 38) in very good yields (Scheme 1).
All obtained compounds were assayed against urease purified
from Sporosarcinia pasteurii using the phenol red method. Only
the derivatives of 3,4-dihydroxyphenylacetic acid exhibited
inhibitory properties similar to catechol. The starting acid 2, its
esters 3-18 and amides 19-24 showed irreversible inhibition with
IC50 in micromolar range. The series of aliphatic esters with
increasing number of carbon atoms (compounds 2-10) was
prepared and clear pattern of the structure-activity relationship
was observed. The activity increased from the acid 2 to the butyl
Figure 1. Crystal structure of catechol-inhibited S. pasteurii urease (PDB id.
5G4H) [26]. Protein is shown as green ribbon with movable flap marked in
orange. Nickel ions are shown as dark blue spheres, and catechol-modified
Cys322 residue as sticks.
ester 6 achieving level substantially higher than catechol (IC50
=
0.667 M for inhibitor 6) and then, further increase of chain
length caused slow decrease of activity up to inactive hexadecyl
ester 10. Highly active derivative was also found among
unsaturated esters — propargyl ester 12 exhibited IC50 = 0.518
M and kinact/Ki = 1379 M-1s-1 and was the most active in the
whole tested set. Cyclohexyl and phenyl-containing esters 13-18
showed moderate activity at the level similar to catechol. Amides
of 3,4-dihydroxyphenylacetic acid showed moderate activity, in
most cases lower than the corresponding esters (e.g. 11 vs 22, 15
vs 19, 17 vs 21).
Forty derivatives of 3,4-dihydroxyphenylacetic acid 2, caffeic
acid 25, ferulic acid 33 and gallic acid 41 (compounds 3−45)
were prepared, among which twenty six were synthesized for the
first time (Scheme 1). Diverse methods were used for the
synthesis of these derivatives. Esters 3-15, 26-28, 34-35 and 42-
43 were conveniently obtained in very good yields and purities
by alkylation (method A) using the corresponding halide and
sodium carbonate (Na2CO3) in hexamethyl phosphoramide
(HMPA) in the presence of catalytic amount of potassium iodide
(KI). Several esters were obtained using thionyl chloride and the
corresponding alcohol (method B, compounds 16−17, 29, 36-37,
44-45) in 1,2-dimethoxyethane (DME) as it results in fewer side
products compared to the frequently used 1,4-dioxane. Low to
moderate but utilizable yields (10–30%) were obtained. Amide
derivatives (compounds 19-21, 31-32 and 39-40) were
synthesized using a condensing agent such as EDC∙HCl (method
C) in good yields. Some derivatives (compounds 22-24) almost
unavailable by this method were prepared by SOCl2 acid
activation (method D).
Surprisingly, although caffeic acid 25 incorporates structural
fragment of catechol, the compound itself, as well as its various
derivatives (esters 26-30 and amides 31-32) did not exhibit any
inhibitory activity against urease. Similarly, O-methylated
analogues of caffeic acid – compounds 33-40 also did not show
any potency. Gallic acid 41 was inactive, but its phenyl-
containing esters gave moderate inhibition with IC50 equal to 972
and 206 M for compounds 43 and 44, respectively. However,
the inhibition by gallic acid esters was reversible, thus of a
different mode in comparison to catechol.
Table 1. Inhibitory activity of catechol derivatives against S.
pasteurii urease
No Structure
Synthetic
methoda
-
R
[μM]
[M-1s-1]
1
7.40 ± 0.08
346 ± 16
2
-H
-
240.4± 2.8
158.2 ± 2.3
106.1 ± 1.3
22.49 ± 0.30
0.667 ± 0.005
25.64 ± 0.31
62.4 ± 4.8
32.0 ± 2.4
44.0 ± 0.9
56.3 ± 3.9
123.5 ± 9.8
571.3 ± 7.1
101.3 ± 8.4
92.7 ± 6.4
27.56 ± 0.14
3
-CH3
(a)
(a)
(a)
(a)
(a)
(a)
(a)
(a)
(a)
(a)
4
-CH2CH3
5
-(CH2)2CH3
-(CH2)3CH3
-(CH2)4CH3
-(CH2)5CH3
-(CH2)7CH3
-(CH2)15CH3
-CH2CH=CH2
-CH2C≡CH
6
7
Scheme 1. (a) i) Νa2CO3, RX, cat. KI, HMPA, rt, 48h, ii)
HCl; (b) i) SOCl2, DME, 70°C 4h, ii) ROH, DME, 70°C, 16h;
(c) RNH2, HOBt∙H2O, EDC∙HCl, CH2Cl2, rt, 24h; (d) SOCl2,
50°C, 3h, ii) RNH2, Et3N, CH2Cl2, rt, 16h; (e) i) aniline, con.
HCl, NaNO2, NaN3, H2O 0°C 10 min, rt, 2h, ii) sodium
ascorbate, CuSO4∙5H2O, t-BuOH: H2O, rt, 24 h.
8
9
235.5 ± 3.6
NI
10
11
12
47.12 ± 0.32
0.518 ± 0.004
94.1 ± 35.3
1379 ± 26

13
14
15
16
17
18
-C6H11
(a)
(a)
(a)
(b)
(b)
(e)
50.12 ± 0.40
44.09 ± 0.30
6.54 ± 0.05
106.4 ± 1.1
7.85 ± 0.09
73.1 ± 0.9
169.2 ± 7.8
99.7 ± 6.9
243.3 ± 1.4
46.8 ± 2.5
379 ± 23
-CH2Ph
41
42
43
44
45
-H
-
NI
-(CH2)2Ph
-CH2C≡CH
-(CH2)2Ph
-(CH2)2(4-F-Ph)
-(CH2)2(4-NO2-Ph)
AHA
(a)
(a)
(b)
(b)
-
NI
972 ± 11
205.8 ± 1.7
NI
1106 ± 18b
-(CH2)2(4-F-Ph)
-(CH2)2(4-NO2-Ph)
143.6 ± 2.1b
113.6 ± 5.1
14.31 ± 0.85
3.30 ± 0.40b
a Synthetic methods are defined in Scheme 1.
b Reversible inhibitors, Ki [M] value is given.
19
20
21
22
23
24
-(CH2)2Ph
(c)
(c)
(c)
(d)
(d)
(d)
13.69 ± 0.10
667.0 ± 7.6
296.5 ± 2.2
89.20 ± 0.94
33.60 ± 0.25
11.33 ± 0.09
247.2 ± 1.7
12.12 ± 0.88
29.1 ± 1.8
76.4 ± 4.8
142 ± 12
-(CH2)2(3-F-Ph)
-(CH2)2(4-NO2-Ph)
-CH2CH=CH2
-CH(CH3)CH2CH3
-C6H11
The most active identified inhibitors (1, 2, 6, 12 and 17) were
further evaluated for their chemical reactivity with thiols using a
model substrate — glutathione (Table 2). Kinetic parameter
describing this reaction i.e. logkGSH was found to be at the similar
level of 2.09 – 2.42 for all tested compounds. Thus, differences
in observed inhibitory activities were not the result of variable
chemical reactivity of studied inhibitors, but are related to
specific interactions with the enzyme.
221.2 ± 1.2
Additionally, cytotoxicity of selected urease inhibitors
towards normal mouse fibroblasts (BALB/3T3) was also
measured (Table 2). As expected, catechol showed toxicity at
micromolar level very close to its inhibitory activity. The toxicity
of 3,4-dihydroxyphenylacetic acid and its derivatives was
variable. The acid 2 showed low toxicity (> 100 M), but it also
exhibited poor inhibitory activity against urease. Butyl and 2-(4-
nitropehyl)ethyl esters exhibited toxicity similar to catechol, but
for propargyl ester 12 this value was one order of magnitude
higher (LC50 = 53.2 M). Thus, significant improvement of
inhibition/toxicity profile was achieved — in case of compound
12, two orders of magnitude difference between inhibitory
activity and toxicity was observed.
25
26
27
28
29
30
-H
-
NI
NI
NI
NI
NI
NI
-CH2C≡CH
-(CH2)2Ph
(a)
(a)
(b)
(b)
(e)
-(CH2)2(4-F-Ph)
-(CH2)2(4-NO2-Ph)
Table 2. Chemical reactivity and cytotoxicity for selected
compounds
31
32
-(CH2)2Ph
(c)
(c)
NI
NI
No
Structure
[µM]
-(CH2)2(3-F-Ph)
R
1
2.24 ± 0.17
7.87 ± 2.58
33
34
35
36
37
38
-H
-
NI
NI
NI
NI
NI
NI
-CH2C≡CH
-(CH2)2Ph
(a)
(a)
(b)
(b)
(e)
2
-H
2.09 ± 0.17
> 100 µM;
22.7 ± 8.0%a
-(CH2)2(4-F-Ph)
-(CH2)2(4-NO2-Ph)
6
Bu
2.37 ± 0.19
2.21 ± 0.18
2.42 ± 0.19
5.6 ± 1.3
12
17
-CH2C≡CH
-(CH2)2(4-NO2-Ph)
53.2 ± 23.7
5.4 ± 1.1
a Percent of proliferation inhibition at 100 M.
The mode of binding of the most active compound 12 was
modelled using the catechol-inhibited urease crystal structure as
the starting point for calculations (Figure 2). There are two
possible points of attachment of thiol group on the ring – namely
the positions 2 and 5. Unfortunately, on the basis of the radical-
type reaction mechanism it is not possible to indicate the
preferred reaction point. Therefore, both possibilities were
modelled (Figure 2 and S1) and similar mode of binding was
found. The space between the movable flap and the rest of the
39
40
-(CH2)2Ph
(c)
(c)
NI
NI
-(CH2)2(3-F-Ph)

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Structure-activity relationships for phenyl substituted esters
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Substitution of phenyl ring with electron-withdrawing fluorine
atom decrease cation- interaction energy, what corresponds to
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between NO2 group and Arg369 instead of possible weak cation-
 interaction (Figure S3).
In summary, a new class of bacterial urease inhibitors based
on catechol molecule was discovered. Derivatives of 3,4-
dihydroxyphenylacetic acid exhibit irreversible inhibition of the
studied enzyme and selected compounds show better inhibitory
activity and lower toxicity in comparison to the reference
compound. Such mode of inhibition should be particularly
effective against microbial pathogens for which urease was
identified as virulence factor because it allows complete
inactivation of the target protein.
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The work was supported by statutory founds of Faculty of
Chemistry, Wrocław University of Science and Technology. The
Biovia Discovery Studio package was used under a Polish
country-wide license. The use of software resources (BIOVIA
Discovery Studio program package) of the Wrocław Centre for
Networking and Supercomputing is kindly acknowledged.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at @.