Xanthine oxidase inhibitory properties and anti-inflammatory activity of 2-amino-5-alkylidene-thiazol-4-ones

Article abstract of DOI:10.1016/j.cbi.2015.01.022

Thirty 2-amino-5-alkylidene-thiazol-4-ones were assayed for inhibitory activity against commercial enzyme xanthine oxidase (XO) in vitro and XO in rat liver homogenate as well as for anti-inflammatory response on human peripheral blood mononuclear cells (PBMCs). 4-((2-Benzylamino-4-oxothiazol-5(4H)-ylidene)-methyl)benzonitrile showed the most potent inhibitory effect against commercial XO (IC₅₀ = 17.16 μg/mL) as well as against rat liver XO (IC₅₀ = 24.50 μg/mL). All compounds containing the 4-cyanobenzylidene group or (indol-3-yl)methylene group at the position 5 of thiazol-4-one moiety were moderately potent inhibitors of commercial XO. The assayed compounds were docked into the crystal structures of XO enzyme complexes with three diverse inhibitors (PDB codes: 1FIQ, 1VDV, and 1V97) using OEDocking software. Our results strongly point to a correlation between the data on inhibitory activity against commercial XO and data on antioxidant activity of studied compounds, screened using a lipid peroxidation (LP) method. 2-(Benzylamino)-5-((thiophen-2-yl)methylene)thiazol-4(5H)-one showed the highest anti-inflammatory response on PBMCs, exerted most probably through the NF-κB inhibition. Studied 2-amino-5-alkylidene-thiazol-4-ones obey the "Rule of five" and meet all criteria for good solubility and permeability.

Full text of DOI:10.1016/j.cbi.2015.01.022

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Contents lists available at ScienceDirect
Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
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Xanthine oxidase inhibitory properties and anti-inflammatory activity of
2-amino-5-alkylidene-thiazol-4-ones
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Zaklina Smelcerovic a, Andrej Veljkovic b, Gordana Kocic b, Denitsa Yancheva c, Zivomir Petronijevic d,
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Marko Anderluh e, , Andrija Smelcerovic f,
⇑
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a Center for Biomedicinal Science, Faculty of Medicine, University of Nis, Bulevar Dr Zorana Djindjica 81, 18000 Nis, Serbia
b Institute of Biochemistry, Faculty of Medicine, University of Nis, Bulevar Dr Zorana Djindjica 81, 18000 Nis, Serbia
c Laboratory of Structural Organic Analysis, Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Build. 9, 1113 Sofia,
Bulgaria
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d Faculty of Technology, University of Nis, Bulevar oslobodjenja 124, 16000 Leskovac, Serbia
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Department of Pharmaceutical Chemistry, University of Ljubljana, Faculty of Pharmacy, Aškerceva 7, SI-1000, Slovenia
f Department of Chemistry, Faculty of Medicine, University of Nis, Bulevar Dr Zorana Djindjica 81, 18000 Nis, Serbia
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a r t i c l e i n f o
a b s t r a c t
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Article history:
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Thirty 2-amino-5-alkylidene-thiazol-4-ones were assayed for inhibitory activity against commercial
enzyme xanthine oxidase (XO) in vitro and XO in rat liver homogenate as well as for anti-inflammatory
response on human peripheral blood mononuclear cells (PBMCs). 4-((2-Benzylamino-4-oxothiazol-
5(4H)-ylidene)-methyl)benzonitrile showed the most potent inhibitory effect against commercial XO
Received 17 November 2014
Received in revised form 7 January 2015
Accepted 16 January 2015
Available online xxxx
(IC50 = 17.16 lg/mL) as well as against rat liver XO (IC50 = 24.50 lg/mL). All compounds containing the
4-cyanobenzylidene group or (indol-3-yl)methylene group at the position 5 of thiazol-4-one moiety were
moderately potent inhibitors of commercial XO. The assayed compounds were docked into the crystal
structures of XO enzyme complexes with three diverse inhibitors (PDB codes: 1FIQ, 1VDV, and 1V97)
using OEDocking software. Our results strongly point to a correlation between the data on inhibitory
activity against commercial XO and data on antioxidant activity of studied compounds, screened using
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Keywords:
2-Amino-5-alkylidene-thiazol-4-ones
Xanthine oxidase inhibition
Molecular docking
Anti-inflammatory activity
NF-jB inhibition
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a
lipid peroxidation (LP) method. 2-(Benzylamino)-5-((thiophen-2-yl)methylene)thiazol-4(5H)-one
showed the highest anti-inflammatory response on PBMCs, exerted most probably through the NF-
jB
inhibition. Studied 2-amino-5-alkylidene-thiazol-4-ones obey the ‘‘Rule of five’’ and meet all criteria
for good solubility and permeability.
Ó 2015 Published by Elsevier Ireland Ltd.
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1. Introduction
conditions [1]. ROS generated by XO activity may activate redox-
associated transcriptional factors, among them one of the most
important is NF-jB. NF-jB may function as the cellular checkpoint
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Xanthine oxidase (XO) is a highly versatile flavoprotein enzyme,
ubiquitous among species (from bacteria to human) and within the
various tissues of mammals [1]. It catalyzes the last two steps in
the purine degradation pathway prior to formation of uric acid,
that is, hydroxylation of hypoxanthine to xanthine, and then to uric
acid [2]. There is an overwhelming acceptance that XO serum lev-
els are significantly increased in various pathological states like
hepatitis, inflammation, ischemia–reperfusion, carcinogenesis and
aging, and that ROS generated in the enzymatic process are
involved in oxidative damage. Thus, the inhibition of this
enzymatic pathway could be beneficial in a number of mentioned
of metabolic stress conditions, such as hyperglycemia, oxidative,
nitrosative stress and hyperuricemia, which may reflect disease
processes associated with progression of autoimmune or autoin-
flammatory conditions [3].
2-Amino-5-alkylidene-thiazol-4-one is a privileged scaffold in
drug discovery [4,5] as its derivatives show a variety of biological
activities, such as antimicrobial [6,7], antioxidant [8], antiviral
[9], anti-inflammatory [10], and cardiotonic [11]. We have synthe-
sized a library of 30 diverse 2-amino-5-alkylidene-thiazol-4-ones
(1–30) [12] and recently we have investigated their antimicrobial
[7] and antioxidant activity [8], as well as cytotoxicity [7]. The anti-
oxidant activity of some of studied compounds was comparable
with activity of standard antioxidants (trolox, quercetin, caffeic
⇑
Corresponding authors. Tel.: +386
1 47 69 639; fax: +386 1 42 58 031
(M. Anderluh). Tel.: +381 18 457 0029; fax: +381 18 423 8770 (A. Smelcerovic).
E-mail addresses: marko.anderluh@ffa.uni-lj.si (M. Anderluh), a.smelcerovic@
yahoo.com (A. Smelcerovic).
acid and
L-ascorbic acid) [8]. The important feature of these
compounds is their low level of influence on cell viability, as
http://dx.doi.org/10.1016/j.cbi.2015.01.022
0009-2797/Ó 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Z. Smelcerovic et al., Xanthine oxidase inhibitory properties and anti-inflammatory activity of 2-amino-5-alkylidene-
thiazol-4-ones, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.01.022
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assayed by the HEK-293 metabolic activity assay [7]. As a part of a
detailed biological screening of our 2-amino-5-alkylidene-thiazol-
4-ones library, in the present study 1–30 were evaluated for inhib-
itory activity against commercial enzyme XO in vitro and XO in rat
liver homogenate as well as for anti-inflammatory response on
human peripheral blood mononuclear cells (PBMCs). The molecu-
lar docking studies were performed in order to examine the bind-
ing mode of studied compounds in the XO active site. Additionally,
the physico-chemical properties of studied compounds were calcu-
lated using Molinspiration tool [13].
contained the same amount of rat liver homogenate, appropriate
amount of DMSO, xanthine and TRIS–HCl buffer; (iii) control group
contained the same amount of rat liver homogenate, xanthine and
TRIS–HCl buffer adjusted to the same volume.
Corresponding blank samples were prepared for each group in
the same way as the test solutions (i–iii). The obtained inhibition
was calculated as a percent change of the control which involves
the effect of appropriate amount of DMSO. All samples were
assayed for XO inhibitory activity at concentration of 50 lg/mL.
Those showing greater than 50% inhibition at this concentration
were tested further to ascertain the corresponding IC50 values.
IC50 curves were generated using three concentrations of studied
compounds (50, 40 and 25 lg/mL). Allopurinol was used as posi-
tive control. All experiments were performed in triplicate and
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2. Materials and methods
2.1. Synthesis
averaged.
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The synthesis of the studied compounds (1–30) were performed
by an innovative one-pot tandem reaction using microwave-
assisted synthesis, as described in our previous study [12].
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2.3. In vitro experiments on PBMC
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Peripheral venous blood (350 mL) from 24 year old male
healthy volunteer was drawn between 8 and 9 h into sterile hepa-
rinized tubes and was processed within 2 h. PBMC were isolated
under sterile conditions by centrifugation in Ficoll Histopaque
1077 (Lymphoprep, Nycomed Pharma) according to the manufac-
turer instructions. Cell viability was above 90%, as determined
using Trypan blue stain exclusion. After washing in PBS, obtained
PBMC were resuspended in RPMI 1640 medium containing 10%
of FCS.
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2.2. Evaluation of xanthine oxidase inhibition
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2.2.1. Inhibition of commercial xanthine oxidase
Commercial bovine milk XO, purchased from Sigma–Aldrich,
was employed for in vitro evaluation of enzyme inhibition, by spec-
trophotometric measurement uric acid formation at 293 nm
(method slightly modified by [14]).
The inhibition was studied in a series of test-tubes with the
reaction mixture (total volume 2150 lL), prepared in a following
order: (i) test samples contained 0.01 units of XO, one of the studied
compounds (1–30) diluted in DMSO (the final concentration of
DMSO in the assay was 4.65% v/v), 232.5 lM of xanthine (Serva),
and 46.5 mM TRIS–HCl buffer (pH 7.8); (ii) solvent control samples
contained the same amount of XO, appropriate amount of DMSO,
xanthine and TRIS–HCl buffer; (iii) control samples contained the
same amount of XO, xanthine and TRIS–HCl buffer adjusted to
the same volume; (iv) test substrate samples were group of samples
which contained only XO in the reaction mixture, one of the stud-
ied compounds (1–30) diluted in DMSO, and TRIS–HCl buffer, in
order to test if the compounds 1–30 are XO substrates. The tubes
were allowed to incubate at 37 °C for 15 min, together with the
corresponding duplicate blank aliquots, where the enzyme was
omitted. After incubation, the reaction was stopped by adding
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2.4. Detection of NF-jB
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All chemical were purchased from Sigma while the antibodies
were purchased from Santa Cruz Biotechnology. Anti-inflamma-
tory activity of compounds 1–30 dissolved in DMSO (the final con-
centration of studied compounds was 1
concentration of DMSO in the assay was 0.1% v/v) was examined
via quantification of NF- B, as described in our previous papers
[3,14]. Washed PBMCs (each aliquot of 100
L contained 105 cells)
were distributed in 12-well plates and cultured for 4 h at 37 °C in
5% CO2. The 50- L aliquots of each sample were plated in 12 U-bot-
lg/mL, while the final
j
l
l
tom 96-well culture plates. The cells were fixed using 70% metha-
nol and permeabilized with 0.1% Triton PBS. They were incubated
with anti-NF-
ping at the C-terminus of NF-
j
B primary antibody (p65 C-20: sc-372 epitope map-
B p65), washed three times, and fur-
j
100 lL of perchloric acid; afterwards the XO was added in corre-
ther incubated with the FITC-conjugated secondary antibody. The
mean fluorescence intensity (MFI; logarithmic scale) of cell popu-
lations was determined and analyzed on a Victor™ multiplate
reader (Perkin Elmer-Wallace, Wellesley, MA). The results pre-
sented were obtained following subtraction of blank values that
were treated with primary and secondary antibodies only. The
fluorescence intensity of cells indicated for both up or down regu-
lated populations for each compound used, compared to control
samples with the appropriate amounts of DMSO, and calculated
as a percent change of the control PBMCs which involves the effect
of appropriate amount of DMSO. All experiments were conducted
in triplicate and averaged.
sponding blank samples in duplicate. The percentage of enzyme
inhibition was determined by measuring the difference in absor-
bance that correlates with uric acid formation; it was calculated
as a percentage of specimen absorbance vs. absorbance of the sol-
vent control samples which involves only the absorbance of DMSO.
All samples were assayed for XO inhibitory activity at concentra-
tions of 50 lg/mL. Those showing inhibition greater than 50% at
this concentration were tested in a broader concentration range
to allow calculation of IC50 values. IC50 curves were generated
using four concentrations of studied compounds (50, 40, 25 and
5 lg/mL). Allopurinol was used as positive control. All experiments
were performed in triplicate and averaged.
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2.5. Molecular docking studies
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2.2.2. Inhibition of rat liver xanthine oxidase
Inhibition of XO activity in rat liver homogenate was evaluated
using a spectrophotometric method (slightly modified by [14]).
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2.5.1. Ligand preparation
The molecules were built with ChemBioDraw Ultra 13.0 (Perk-
inElmer, Inc.) and their geometries optimized with ChemBio 3D
Ultra 13.0 (PerkinElmer, Inc.) using MM2 force field until a mini-
mum 0.100 Root Mean Square (RMS) gradient was reached. The
optimized structure was refined with GAMESS interface using the
semi-empirical AM1 method, QA optimization algorithm and
Gasteiger Hückel charges for all atoms for 100 steps. FRED requires
The reaction mixture (total volume 2200
cating the following test sample groups: (i) test sample group con-
tained 100 L of 10% rat liver homogenate, one of the studied
compounds (1–30) diluted in DMSO (the final concentration of
DMSO in the assay was 4.55% v/v), 454.5 M of xanthine (Serva),
and 45.5 mM TRIS–HCl buffer (pH 7.8); (ii) solvent control group
lL) was prepared by allo-
l
l
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a set of input conformers for each ligand, which were generated
with OMEGA (OMEGA version 2.5.1.4. OpenEye Scientific Software,
Santa Fe, NM. http://www.eyesopen.com), with maximum number
of conformations set to 100 [15,16]. All the other options were left
as default values.
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3. Results and discussion
3.1. Xanthine oxidase inhibition
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3.1.1. Assays on xanthine oxidase inhibition
19 of 30 studied 2-amino-5-alkylidene-thiazol-4-ones inhibit
commercial bovine milk XO with an IC50 below 50 g/mL (Table 1).
The inhibitory activity of 1–30 on XO were further tested in rat
liver homogenate and only 8 compounds inhibited XO with an
IC50 lower than 50 lg/mL (Table 2). 4-((2-Benzylamino-4-oxothia-
zol-5(4H)-ylidene)-methyl)benzonitrile (compound 14) showed
the most potent inhibitory effect against commercial XO
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2.5.2. Receptor preparation and docking protocol
The crystal structures of three XO enzyme complexes with
known inhibitors (PDB codes: 1FIQ, 1VDV, and 1V97) were taken
as starting points. The ligands were taken as reference structures
and all cofactors included were left as ‘‘receptor’’ to avoid overlap
with their binding sites. For each crystal structure, one grid box
was created with volumes of 13,277, 12,212, and 12,779 Å3, and
outer contours of 1932, 2056, and 1966 Å2 using Make Receptor
3.0.1.
The docking software FRED (OEDocking version 3.0.1. OpenEye
Scientific Software, Santa Fe, NM. http://www.eyesopen.com) was
used for docking studies with the default settings, and number of
poses, which was set to 50 [17]. The proposed five binding poses
with the highest rank of the docked inhibitors were evaluated
using final score and relative position to the native ligand. The
graphical representations of the calculated binding poses were
obtained using VIDA (VIDA version 4.2.1. OpenEye Scientific Soft-
ware, Santa Fe, NM. http://www.eyesopen.com).
(IC50 = 17.16
g/mL). All compounds containing the 4-cyanobenzylidene group
or (indol-3-yl)methylene group at the position 5 of thiazol-4-one
moiety inhibited commercial XO with IC50 values below 50 g/
lg/mL) as well as against rat liver XO (IC50 = 24.50 -
l
l
mL. Compounds with 4-cyanobenzylidene group (with the excep-
tion of compound 11 containing piperidinyl group as the 2-amino
substituent) inhibit rat liver XO with an IC50 lower than 50 lg/mL.
On the other hand, compounds with (indol-3-yl)methylene substi-
tuent at position 5 exhibited lower than the threshold inhibitory
activity (50%) against rat liver XO at concentrations of 50 lg/mL.
Allopurinol, a widely used XO inhibitor and drug to treat gout,
exhibited stronger inhibitory effect on commercial XO
(IC50 = 0.26 lg/mL) as well as rat liver XO (IC50 = 0.79 lg/mL) than
1–30.
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2.5.3. Validation of the docking protocol
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For all three diverse crystal structures, we have tried to repro-
duce the pose of the specific inhibitor (PDB codes: 1FIQ, 1VDV,
and 1V97). In each case, one of the top five docking poses of the
docked inhibitor were within 1.5 Å root mean square deviation
(RMSD) of the ligand crystal structure. In case of the 1VDV crystal
structure, the predicted top score pose was the one that best fitted
the crystal structure of the ligand, overlapping it almost com-
pletely, which offers the proof of the protocol validation (Fig. 1).
3.1.2. Binding pose prediction
All assayed compounds were docked into the crystal structures
of XO enzyme complexes with three diverse inhibitors (PDB codes:
1FIQ, 1VDV, and 1V97, [18–20]) using OEDocking software
(Release 3.0.1, OpenEye Scientific Software, Inc.). The structures
of known inhibitors vary significantly (Fig. 2), so we have used
them as three distinct starting points to allow higher pose variabil-
ity, as MakeReceptor (OEDocking version 3.0.1. OpenEye Scientific
Fig. 1. Crystal structure of the XO inhibitor (niraxostat (32); 1-[3-cyano-4-(neopentyloxy)phenyl]-1H-pyrazole-4-carboxylic acid, carbons shown as green sticks) in complex
with XO (PDB code: 1VDV), and top docked pose of the same inhibitor docked in the XO active site using FRED (shown as stick colored by atom type). (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Z. Smelcerovic et al., Xanthine oxidase inhibitory properties and anti-inflammatory activity of 2-amino-5-alkylidene-
thiazol-4-ones, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.01.022
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Table 1
In vitro screening of the synthesized compounds (1–30) for inhibitory activity against commercial XO.
Substituent at position 5
Substituent at position 2
Entry (Entrya) IC50
(lg/mL)
N
O
HN
N
N
N
CH
3
1 (8a)
2 (9a)
3 (10a)
4 (11a)
5 (12a)
43.45
26.67
>50
>50
>50
6 (13a)
>50
7 (14a)
25.82
8 (15a)
>50
9 (16a)
37.57
10 (17a)
39.41
S
11 (18a)
12 (19a)
13 (20a)
14 (21a)
15 (22a)
33.18
24.73
25.98
17.16
32.53
CN
16 (23a)
17 (24a)
18 (25a)
19 (26a)
20 (27a)
>50
>50
>50
31.77
>50
O
21 (28a)
21.43
22 (29a)
23.17
23 (30a)
27.03
24 (31a)
33.62
25 (32a)
44.08
H
N
26 (33a)
27.24
27 (34a)
>50
28 (35a)
>50
29 (36a)
39.77
30 (37a)
33.85
O
O
Standard XO inhibitor
Allopurinol
IC50 (lg/mL)
0.26
O
R'
5
S
N
2
R''
a
Entries as in the original article [12].
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294
295
296
297
298
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266
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268
269
270
271
272
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Software, Santa Fe, NM. http://www.eyesopen.com) builds the
docking grid based on the native inhibitor in the crystal structure.
As expected, the ‘‘receptors’’ constructed from 1VDV and 1V97
reproduced more credible validation results as a consequence of
larger inhibitor molecules that match the size of the assayed com-
pounds. 1VDV-based receptor was used for further studies, and the
results obtained indicate two possible binding modes for the
assayed compounds that depend upon the nature of the ‘‘amine’’
part of the molecule:
Compound 14 displayed the highest observed potency on iso-
lated enzyme, which suits before mentioned binding hypothesis.
Namely, compound 14 is predicted to follow the ‘‘amine-in’’ bind-
ing mode, with benzylamine moiety buried deeply into binding
site, while the polar cyano group of the 4-cyanobenzilidene moiety
makes contact with the solvent thus diminishing the entropic pen-
alty during binding (Fig. 3).
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3.2. Anti-inflammatory activity
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282
283
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ꢀ the ‘‘amine-out’’ mode; molecules with the tertiary (cyclic)
amine are oriented with the arylidene part toward the Arg880
of the XO binding pocket, while the amine part protrudes
toward solvent, and
ꢀ the ‘‘amine-in’’ mode; XO accommodates molecules with ben-
zylamine moiety so that the benzylamine moiety points to
Arg880.
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301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
2-(Benzylamino)-5-((thiophen-2-yl)methylene)thiazol-4(5H)-
one (9) showed the highest anti-inflammatory response on PBMCs,
exerted through the NF-
Also, 3, 5, 16 and 21 at concentrations of 1
potent NF- B downregulation. 14, which was the most potent
jB inhibition (Table 3).
lg/mL caused a
j
XO inhibitor in both used assays, showed a significant anti-inflam-
matory activity. Also, this compound showed very strong LP inhib-
itory effect [8], comparable to the effect of standard antioxidants.
The compounds containing the 2-(benzylamino) group, such as
compounds 9 and 14, are secondary amines in contrast to the other
studied compounds which are tertiary amines. Hydrogen atom of
secondary amino group enables two tautomeric forms that could
The scores obtained (not shown) do not match exactly the
potency observed on isolated XO enzyme, but might explain the
general tendency that emerged out of the inhibition data:
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ꢀ indole derivatives are generally potent inhibitors of the isolated
XO enzyme, due to high steric complementarity with XO bind-
ing site, if the molecule follows the ‘‘amine-out’’ binding mode,
ꢀ ‘‘amine-in’’ binding mode is predicted for compounds with ben-
zylamine fragment, where the arylidene moiety points toward
surface (Fig. 3).
play an important role for NF-jB inhibition. However, it is difficult
to get any clear SAR conclusions based on the results presented in
the Table 3. Song et al. [10] reported that some 2-amino-5-alkyl-
idene-thiazol-4-ones are potent cyclooxygenase-2 (COX-2) inhibi-
tors. Within their thiazolone series they investigated the effect of
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Table 2
In vitro screening of the synthesized compounds (1–30) for inhibitory activity against XO in rat liver homogenate.
Substituent at position 5
Substituent at position 2
Entry (Entrya) IC50
(lg/mL)
N
O
HN
N
N
N
CH
3
1 (8a)
2 (9a)
3 (10a)
4 (11a)
5 (12a)
47.71
42.02
>50
>50
>50
6 (13a)
>50
7 (14a)
>50
8 (15a)
>50
9 (16a)
40.87
10 (17a)
>50
S
11 (18a)
12 (19a)
13 (20a)
14 (21a)
15 (22a)
>50
33.98
29.25
24.50
48.16
CN
16 (23a)
17 (24a)
18 (25a)
19 (26a)
20 (27a)
>50
>50
>50
>50
>50
O
21 (28a)
>50
22 (29a)
>50
23 (30a)
>50
24 (31a)
>50
25 (32a)
>50
H
N
26 (33a)
48.56
27 (34a)
>50
28 (35a)
>50
29 (36a)
>50
30 (37a)
>50
O
O
Standard XO inhibitor
Allopurinol
IC50 (lg/mL)
0.79
O
R'
5
S
N
2
R''
a
Entries as in the original article [12].
NH
N
O
N
COOH
OH
N
O
N
N
N
N
N
OH
33
, inhibitor from 1V97
31, inhibitor from 1FIQ
32, inhibitor from 1VDV
Fig. 2. Structural formulae of the known XO inhibitors in complex with (PDB codes: 1FIQ, 1VDV, and 1V97).
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
the substituent at the position 2 of the thiazol-4-one moiety in
detail and found that the nature of the chemical bond between
the nitrogen atom and the substituents attached to it is critical
for potency and selectivity of COX-2 inhibition. Namely, an NAO
bond gives compounds which are potent and selective COX-2
inhibitors. NAH bond leads to weak COX-2 inhibition. An NAC
and NAN bonds lead to loss of potency [10].
and it is known that XDH conversion to XO may represent a feed-
forward mechanism for stimulation of ROS production [23].
Accordingly, we speculate that NF-
activity and ROS production.
jB may directly affect the XO
339
340
3.3. Physico-chemical properties and potential reactivity of studied
compounds
NF-
for maximal transcription of a wide array of pro-inflammatory
mediators. It is well known that ROS stimulate the NF- B pathway
in the cytoplasm through I B (inhibitor of kappa B) degradation.
Overexpression of the antioxidant proteins was shown to inhibit
NF- B activation [21]. We have previously proposed the electron
jB is a redox-associated transcription factor that is required
341
342
343
344
345
346
347
348
349
350
The above sections demonstrate the promising inhibitory activ-
ities of studied 2-amino-5-alkylidene-thiazol-4-ones toward com-
mercial and rat liver XO. However, in view of future medical
application, other important features should be also taken into
account – favorable pharmacokinetic behavior in living organisms,
providing the required bioavailability and transportation through
different membranes to the site of action, optimal process of
metabolism and elimination. For this reason preliminary screening
of molecular physico-chemical properties such as lipophilicity,
molecular size, flexibility and presence of hydrogen-donor and
j
j
j
transfer (SET) mechanism as the most probable one to explain
the observed antioxidant activity of 2-amino-5-alkylidenethiazol-
4-ones [8]. Accordingly, this might be one of the mechanisms of
NF-
NF-
j
j
B inhibition by studied compounds. Xu et al. [22] have found
B binding site on human xanthine dehydrogenase (XDH) gene,
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Z. Smelcerovic et al. / Chemico-Biological Interactions xxx (2015) xxx–xxx
A
B
O
H
N
O
N
N
S
S
NC
HN
N
14
21
Fig. 3. Two proposed binding modes for assayed 2-amino-5-alkylidene-thiazol-4-ones: (A) ‘‘amine-out’’ mode of 21, and (B) ‘‘amine-in’’ mode of 14. The figures present
docking results into 1VDV crystal structure with the known XO inhibitor rendered as green sticks and the docked molecule rendered as sticks colored by atom type. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 3
Effect of studied 2-amino-5-alkylidene-thiazol-4-ones (sample concentration 1
l
g/mL) on quantitative expression of NF-jB.
Substituent at position 5
Substituent at position 2
Entry (Entrya)
% of control PBMCs with 0.1% DMSO
N
HN
N
N
N
O
CH
3
1 (8a)
2 (9a)
3 (10a)
4 (11a)
5 (12a)
123.14 15.28
95.56 14.81
43.95 6.10
72.21 17.95
47.67 20.37
6 (13a)
70.43 17.32
7 (14a)
72.00 13.94
8 (15a)
91.55 24.16
9 (16a)
26.18 9.28
10 (17a)
91.95 23.77
S
11 (18a)
12 (19a)
13 (20a)
14 (21a)
15 (22a)
68.64 23.14
81.22 28.42
50.20 14.39
54.41 2.56
97.04 7.63
CN
16 (23a)
17 (24a)
18 (25a)
19 (26a)
20 (27a)
42.06 32.70
70.46 5.84
67.34 27.32
66.26 18.60
66.28 5.19
O
21 (28a)
41.69 10.80
22 (29a)
54.05 11.33
23 (30a)
74.02 12.43
24 (31a)
92.34 51.20
25 (32a)
64.92 27.93
H
N
26 (33a)
67.30 1.87
27 (34a)
51.60 5.39
28 (35a)
99.14 32.89
29 (36a)
66.75 17.85
30 (37a)
74.69 30.96
O
O
O
R'
5
S
N
2
R''
a
Entries as in the original article [12].
Please cite this article in press as: Z. Smelcerovic et al., Xanthine oxidase inhibitory properties and anti-inflammatory activity of 2-amino-5-alkylidene-
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7
Table 4
Calculated molecular properties of compounds 1–30 for assessment of the drug-likeness.
c
e
f
g
h
Compd. No.
Rule
mi logPa
<5
TPSAb
Natoms
MWd
<500
NON
<10
NOHNH
<5
Nviol.
Nrotb.
Voli
(<10)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
3.17
2.11
3.41
3.42
2.66
3.07
2.01
3.31
3.31
2.56
2.92
1.86
3.17
3.17
2.42
4.82
3.76
5.06
5.07
4.32
3.32
2.26
3.56
3.57
2.82
3.06
2.00
3.30
3.31
2.55
33
42
33
42
33
33
42
33
42
33
57
66
57
66
57
42
52
42
51
42
49
58
49
58
49
52
61
52
60
52
19
19
20
21
18
18
18
19
20
17
21
21
22
23
20
27
27
28
29
26
22
22
23
24
21
22
22
23
24
21
272
274
286
294
258
278
280
292
300
264
297
299
311
319
283
378
380
392
400
364
311
313
325
333
297
316
318
330
338
302
3
4
3
3
3
3
4
3
3
3
4
5
4
4
4
4
5
4
4
4
4
5
4
4
4
5
6
5
5
5
0
0
0
1
0
0
0
0
1
0
0
0
0
1
0
0
0
0
1
0
1
1
1
2
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
2
2
2
4
2
2
2
2
4
2
2
2
2
4
2
5
5
5
7
5
2
2
2
4
2
2
2
2
4
2
246
239
263
261
230
237
229
254
252
220
263
255
280
278
247
344
336
360
358
327
275
268
292
290
259
270
263
287
285
254
a
b
c
d
e
f
Octanol–water partition coefficient, calculated by the methodology developed by [13].
Polar surface area.
Number of nonhydrogen atoms.
Molecular weight.
Number of hydrogen-bond acceptors (O and N atoms).
Number of hydrogen-bond donors (OH and NH groups).
Number of ‘‘Rule of five’’ violations.
Number of rotatable bonds.
g
h
i
Molecular volume.
O
O
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
group 11–15 show the lowest mi logP values compared to the
other corresponding compounds, while 16–20, containing benzyl-
oxy group, are the most lipophilic. The largest derivatives 18 and
19 with molecular weight of about 400 are the only compounds
approaching the critical limit of 5.
The amine moiety show greater impact on the lipophilicity of
1–30. Smaller rings such as pyrrolidine and more polar ones such
as the morpholine significantly lower the lipophilicity of the mol-
ecules. The variation of the substitution pattern of the amine moi-
ety (secondary acyclic benzylamine fragment vs. tertiary cyclic
piperidinyl fragments) contributes to mild tuning of the lipophilic-
ity. On the other hand, all compounds containing benzylamine
moiety show higher conformational flexibility manifested through
their greater number of rotatable bonds.
The number of rotatable bonds is an important factor for the
efficient binding to receptors and channels as well as for the oral
bioavailability [25]. Molecules with more than 10 rotatable bonds
tend to show poor oral bioavailability. All studied 2-amino-5-alkyl-
idene-thiazol-4-ones, including the benzylamine containing deriv-
atives, satisfy this criterion and most of them have low
conformational flexibility with only 2 rotatable bonds. Another
descriptor for the oral bioavailability [25] and drug transport prop-
erties [26,27] is the polar surface area. It is expressed here as topo-
logical surface area (TPSA) which is a sum of the surface areas
occupied by the oxygen and nitrogen atoms and the hydrogens
attached to them. TPSA represents the hydrogen bonding capacity
R2-NH2
NH
NH
R1
R1
S
S
S
N
R2
rhodanine - electrophilic
thiocarbonyl moiety
2-amino-5-alkylidene-thiazol-4-ones
lack thiocarbonyl moiety
Fig. 4. The synthesis of 2-amino-5-alkylidene-thiazol-4-ones eliminates thiocar-
bonyl moiety as an electrophilic center of the rhodanine.
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
acceptors facilitate considerably the development of new pharma-
ceuticals and outline the usefulness of new drug candidates.
Physico-chemical properties of 2-amino-5-alkylidene-thiazol-
4-ones 1–30, calculated using Molinspiration tool [13], are shown
in Table 4. Data indicate that none of the compounds is above the
critical limits established by the Lipinski ‘‘Rule of five’’ [24]. Molin-
spiration methodology for calculation of mi logP implements frag-
ment-based contributions and correlation factors which makes it
robust and applicable to virtually all organic and organometallic
compounds. The mi logP values of 1–30 show favorable physico-
chemical profiles for oral bioavailability [24]. Variation of the type
of the ring (phenyl, thienyl, indolyl) attached to the alkylidene
group does not lead to a dramatic change in the lipophilicity of
the molecules studies, except when a polar (CN) or bulky (benzyl-
oxy) group is present in the ring. Thus the compounds with CN
Please cite this article in press as: Z. Smelcerovic et al., Xanthine oxidase inhibitory properties and anti-inflammatory activity of 2-amino-5-alkylidene-
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Z. Smelcerovic et al. / Chemico-Biological Interactions xxx (2015) xxx–xxx
of the molecules. Molecules with TPSA less than 140 Å2 are recog-
nized to have good intestinal absorption, and those with TPSA less
than 60 Å2 show good blood–brain barrier penetration [26,27]. As
could be seen in Table 4, all presented 2-amino-5-alkylidene-thia-
zol-4-ones are expected to have good intestinal absorption and
most of them also good blood–brain barrier penetration. Hydrogen
bonding capacity of the drug candidates is also described by the
number of H-bond donors and acceptors. The compounds in the
series show 3–6 H-bond acceptors. H-bond donors are present only
in the 2-amino-5-alkylidene-thiazol-4-ones containing indolyl and
benzylamine fragments. Molecular volumes of the compounds in
the series are less than 300 Å3 with a few exceptions due to the
large benzyloxy group in 16–20. Summarizing the physico-chemi-
cal properties of studied 2-amino-5-alkylidene-thiazol-4-ones, we
could conclude that they obey the ‘‘Rule of five’’ and meet all crite-
ria for good solubility and permeability.
Recent publication by Baell and Walters raised an interesting
issue on pan-assay interference compounds, or PAINS, and we
quote: ‘‘Repeated identification of the same types of molecule as
promising hits against different proteins is polluting the chemical
literature’’ [28]. We are aware that rhodanine derivatives with exo-
cyclic double bond are frequent PAINS by 2 alternative mecha-
nisms, i.e., they might act as covalent modifiers and metal
complexers [28,29]. Our series of 2-amino-5-alkylidene-thiazol-
4-ones indeed are rhodanine derivatives, and since we have
already published their activity on other targets [7,8], we were par-
ticularly interested to determine whether our compounds might
act as PAINS. Our data indicate that not all compounds act as XO
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
4. Conclusion
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
The present study identifies 2-amino-5-alkylidene-thiazol-4-
ones as a new class of XO inhibitors. The binding mode of studied
compounds with XO was studied by predicting binding pose with
molecular docking. 4-((2-Benzylamino-4-oxothiazol-5(4H)-yli-
dene)-methyl)benzonitrile (14) showed the most potent inhibitory
effect against commercial and rat liver XO, as well as a significant
anti-inflammatory activity. It was interesting to note that the most
potent compound has a different substitution pattern on thiazol-4-
one core (secondary vs. tertiary amine, substituted with benzyl-
amine moiety), which enables alternative tautomeric form of the
compound 14. Namely, all the compounds with benzylamine moi-
ety (4, 9, 14, 24, 29) have shown quite potent inhibition of the iso-
lated bovine XO. Furthermore, our data point out that 4-
cyanobenzylidene moiety at position 5 is optimal for XO inhibition
against both commercial and rat liver XO (11–15). We therefore
conclude that these two moieties are optimal for XO inhibition,
and that the compound 14, as well as several other studied com-
pounds, offer a good starting point toward drugs for the treatment
of gout and other excessive uric acid production disorders.
2-Amino-5-alkylidene-thiazol-4-ones under study obey the
‘‘Rule of five’’ and meet all criteria for good solubility and perme-
ability in such a way that they allow further structural modifica-
tion for achieving desired pharmacological properties combined
with appropriate pharmacokinetic behavior. Although we did not
assay our compounds in vivo, the assay on PBMCs requires com-
pounds to undergo passive diffusion prior action, so the observed
compound activity is the direct proof that compounds do possess
suitable physicochemical properties that enable passive diffusion.
Furthermore, our study suggests that 2-amino-5-alkylidene-thia-
zol-4-ones do not undergo the same mechanism of target covalent
modification as is the case for rhodanine-based compounds with
thiocarbonyl moiety, and therefore do not fall in PAINS category.
Additional research to obtain more potent XO inhibitors is in pro-
gress in our laboratories.
inhibitors below 50 lg/mL threshold, and that the results for 11–
15 correlate in two independent assays, namely in inhibition of
commercial XO and in rat liver homogenate assays. The latter is
performed in the tissue homogenate, where non-specific interac-
tions (aggregation-based mechanism, covalent modifier mecha-
nism, metal complexing mechanism) with numerous protein
components and/or metal ions of the homogenate would undoubt-
edly reduce the potency/activity of our compounds. Since this was
not the case, we believe that our study proves that the measured
activity is the consequence of XO inhibition.
493
494
Conflict of Interest
Carter et al. have reported similar compounds to covalently
modify the TNFRc1 in a light-dependent fashion [30]. We envis-
aged 2 functional groups in these molecules that could lead to
The authors declare that there are no conflicts of interest.
covalent modification, namely exocyclic double bond as
a
495
Transparency Document
Michael-type acceptor, and a thiocarbonyl moiety. The mechanism
of covalent binding via Michael addition for the present com-
pounds would probably result in significant difference in reactivity
depending on whether an electron donating and withdrawing
group is attached to the benzylidene group adjacent to 2-amino-
5-alkylidene-thiazol-4-one core. This was not found to be the case,
as both 4-cyanobenzylidene (11–15, electron withdrawing) and
indole-3-methylidene (21–25, electron donating) derivatives have
displayed similar potency in inhibiting commercial XO. It is how-
ever true that compounds can have similar IC50 values and very dif-
ferent mechanisms/modes of action, so a definite conclusion
cannot be drawn by comparing compounds electronic properties.
Since all the compounds reported by Carter et al. possess thiocar-
bonyl moiety, we believe it is responsible for covalent bond forma-
tion as it is known to react with nucleophiles via addition–
elimination mechanism [31]. Our compounds have rhodanine car-
bonyl sulfur substituted with an amine, as it was the synthetic
route by which our compounds were synthesized [12]. Therefore,
the absence of covalent modulatory activity of our compounds is
probably due to the elimination of the reactive part of the rhoda-
nine molecule, as depicted in the Fig. 4. Substitution of thiocarbon-
yl moiety with the primary or secondary amine could therefore be
an attractive approach to circumvent the undesired or even toxic
properties of the rhodanine-based compounds.
496
497
498
The Transparency document associated with this article can be
found in the online version.
499
Acknowledgments
Q3 500
Q4 501
502
The financial support of this work by Ministry of Education, Sci-
ence and Technological Development of the Republic of Serbia
(Grants Nos. OI 172044 and TR 31060), Slovenian Research Agency
(Grant No. P1-0208), and National Science Fund of Bulgaria (Con-
tract RNF01/0110) is gratefully acknowledged.
503
504
505
References
506
507
508
509
510
511
512
513
514
515
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Please cite this article in press as: Z. Smelcerovic et al., Xanthine oxidase inhibitory properties and anti-inflammatory activity of 2-amino-5-alkylidene-
thiazol-4-ones, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.01.022
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