Development of 2-(Substituted Benzylamino)-4-Methyl-1, 3-Thiazole-5-Carboxylic Acid Derivatives as Xanthine Oxidase Inhibitors and Free Radical Scavengers

Article abstract of DOI:10.1111/cbdd.12686

A series of 2-(substituted benzylamino)-4-methylthiazole-5-carboxylic acid was designed and synthesized as structural analogue of febuxostat. A methylene amine spacer was incorporated between the phenyl ring and thiazole ring in contrast to febuxostat in which the phenyl ring was directly linked with the thiazole moiety. The purpose of incorporating methylene amine was to provide a heteroatom which is expected to favour hydrogen bonding within the active site residues of the enzyme xanthine oxidase. The structure of all the compounds was established by the combined use of FT-IR, NMR and MS spectral data. All the compounds were screened in vitro for their ability to inhibit the enzyme xanthine oxidase as per the reported procedure along with DPPH free radical scavenging assay. Compounds 5j, 5k and 5l demonstrated satisfactory potent xanthine oxidase inhibitory activities with IC₅₀ values, 3.6, 8.1 and 9.9 μm, respectively, whereas compounds 5k, 5n and 5p demonstrated moderate antioxidant activities having IC₅₀ 15.3, 17.6 and 19.6 μm, respectively, along with xanthine oxidase inhibitory activity. Compound 5k showed moderate xanthine oxidase inhibitory activity as compared with febuxostat along with antioxidant activity. All the compounds were also studied for their binding affinity in active site of enzyme (PDB ID-1N5X).

Full text of DOI:10.1111/cbdd.12686

Chem Biol Drug Des 2016; 87: 508–516
Research Article
Development of 2-(Substituted Benzylamino)-4-Methyl-1,
3-Thiazole-5-Carboxylic Acid Derivatives as Xanthine
Oxidase Inhibitors and Free Radical Scavengers
Md Rahmat Ali1, Suresh Kumar1, Obaid Afzal1,
catalyses the hydroxylation of hypoxanthine to xanthine
and xanthine to uric acid (UA) with simultaneous produc-
tion of hydrogen peroxide and super oxide anions (1–3). In
last two steps of biosynthesis process, excessive produc-
Nishtha Shalmali1, Manju Sharma2 and
Sandhya Bawa1,*
1Department of Pharmaceutical Chemistry, Faculty of
tion of uric acid ultimately leads to the deposition of
Pharmacy, Jamia Hamdard, New Delhi 110062, India
2Department of Pharmacology, Faculty of Pharmacy,
Jamia Hamdard, New Delhi 110062, India
*Corresponding author: Sandhya Bawa,
drsbawa@rediffmail.com
monosodium hydrogen urate crystals in the joints of
humans which may lead to the hyperuricemic condition
called gout (4,5). The reduction of plasma levels of UA in
blood can prevent gout, which is possible with XO inhibi-
tors that block the production of UA from purine metabo-
lism (6). There is an irresistible acceptance that XO serum
levels are increased in several pathological conditions like
hepatitis, inflammation, ischaemia-reperfusion, cancer and
ageing. Thus, the selective inhibition of XO may outcome
for the broad spectrum therapeutics like gout, cancer,
inflammation, oxidative damage, cardiovascular and
A series of 2-(substituted benzylamino)-4-methylthia-
zole-5-carboxylic acid was designed and synthesized
as structural analogue of febuxostat.
A methylene
amine spacer was incorporated between the phenyl
ring and thiazole ring in contrast to febuxostat in which
the phenyl ring was directly linked with the thiazole
moiety. The purpose of incorporating methylene amine
was to provide a heteroatom which is expected to
favour hydrogen bonding within the active site resi-
dues of the enzyme xanthine oxidase. The structure of
all the compounds was established by the combined
use of FT-IR, NMR and MS spectral data. All the com-
pounds were screened in vitro for their ability to inhibit
the enzyme xanthine oxidase as per the reported pro-
cedure along with DPPH free radical scavenging assay.
Compounds 5j, 5k and 5l demonstrated satisfactory
potent xanthine oxidase inhibitory activities with IC50
values, 3.6, 8.1 and 9.9 lM, respectively, whereas com-
pounds 5k, 5n and 5p demonstrated moderate antioxi-
dant activities having IC50 15.3, 17.6 and 19.6 lM,
respectively, along with xanthine oxidase inhibitory
activity. Compound 5k showed moderate xanthine oxi-
dase inhibitory activity as compared with febuxostat
along with antioxidant activity. All the compounds were
also studied for their binding affinity in active site of
enzyme (PDB ID-1N5X).
ischaemic-reperfusion therapy (7,8). Allopurinol (1)
a
known XO inhibitor and an analogue of hypoxanthine is
widely used in treatment of gout for last many years.
However, in some cases, severe life-threatening side-
effects have been reported which include a toxicity syn-
drome dramatized by eosinophilia, vasculitis, rash hepatitis
and progressive renal failure due to purine backbone of
allopurinol and its analogs (9,10). Therefore, there is an
immense need of developing non-purine alternatives to
allopurinol with potent XO inhibitory activity and possess-
ing fewer adverse effects. In the year 2009, USFDA
approved Febuxostat (2), a thiazole derivative, since then
multitudinous non-purine inhibitors have attracted world-
wide attention. Outstanding potency and associated toxi-
cology profile was observed during clinical trials, and
diverse febuxostat analogues have been designed (11,12).
Recently, in June 2013 in Japan one of the drugs topirox-
ostat (3) (FYX-051) was approved for the treatment of gout
and hyperuricemia (13,14).
Key words: febuxostat, free radical, methylene amine, molec-
ular docking, xanthine oxidase
In addition, a somewhat higher incidence of toxicological
profile was observed in clinical study of above described
drugs along with large amount of superoxide anion genera-
tion by XO involved in the various pathological conditions.
Development of XO inhibitors with free radical scavengers
potential may prove to be promising agents to treat Gout.
This encouraged the researchers to develop structurally
diverse molecules without purine backbone with promising
XO inhibitory activity such as N-(1,3-diaryl-3-oxopropyl)
Received 17 June 2015, revised 19 October 2015 and
accepted for publication 25 October 2015
Xanthine oxidase (XO, E.C. 1.1.3.22) is a complex met-
alloflavoprotein in the purine scavenging pathway which
508
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12686

Xanthine Oxidase Inhibitory Activity of Aminothiazole
Figure 1: Chemical structures of reported xanthine oxidase
inhibitors.
Figure 2: Chemical modification of febuxostat and 2ꢁ
(3ꢁcyanoꢁ2ꢁisopropylindolꢁ5ꢁyl)ꢁ4ꢁmethylthiazoleꢁ5ꢁcarboxylic
amides (4) (15),1-acetyl-3,5-diaryl-4,5-di-hydrol(1H) pyra-
zoles (5) (16), Y-700 (6) (12), N-aryl-5-amino-4-cyanopyrazole
(7) (17), flavonoids (8) (18), anacardic acid (9) (19), thiazolo-
pyrazolyl 5-phenylisoxazole-3-carboxylic acid (10) (20),
naphthoflavones (11) (21), and pyrazolo[3,4-d] thiazolo[3,2-
a]pyrimidin-4-one (12) (22), 2-(3-cyano-2-isopropyl-1H-
indol-5-yl)-4-methylthiazole-5-carboxylic acid (13) (23),
derivatives (Figure 1). It was assumed worthwhile to ration-
ally design with pharmacophoric approaches to develop
structural analogous of febuxostat as non-purine XO inhibi-
tors with antioxidant activity for diminishing the associated
toxicities. In this article, we describe the synthesis and eval-
uation of 2-(benzylamino)-4-methyl-1,3-thiazole-5-car-
boxylic acid derivatives (Figure 2) as potent XO inhibitors
and free radical scavengers.
acid
for
development
of
2ꢁ(substituted
benzylamino)ꢁ
4ꢁmethylꢁ1,3ꢁthiazoleꢁ5ꢁ carboxylic acid derivatives (5aꢁp)
showing similarity in binding interaction within the active site of xanthine
oxidase.
Synthesis of ethyl 2-amino-4-methyl-1,3-thiazole-
5-carboxylate (3) (24)
To a mixture of anhydrous ethanol (100 mL) and thiourea
(0.098 mole), ethyl-2-chloro acetoacetate (0.097 mole)
was added drop-wise under constant stirring at room tem-
perature. Once addition of ethyl-2-chloro acetoacetate is
completed, reaction mixture was heated at 70–80 °C for
15 min. Then reaction mass was cooled to room tempera-
ture, and the solid precipitate was isolated, washed with
100 mL of cold ethanol and further washed with saturated
sodium bicarbonate solution to obtain white solid which
was finally dried under vacuum at 50 °C for 8 h.
Experimental
Materials and Method
Yield: 91%; m.p.: 170–174 °C; IR (KBr v max): 3222,
All the chemicals and reagent used were of LR grade and
purchased from S.D. fine Chemicals, Merk India, CDH
Spectrochem, etc. All reactions were monitored, and the
purity of the compounds was checked by thin layer chro-
matography (TLC) using precoated silica gel G plates at
254 nm under UV lamp/iodine vapours using toluene/ethyl
acetate/formic acid or benzene/acetone as eluent. Melting
points were determined by the open capillary method with
electric melting point apparatus and are uncorrected. IR
spectra were recorded on Bruker FT-IR spectrophotome-
ter, and 1H and 13C-NMR spectra were recorded on Bru-
ker DPX 400 MHz spectrophotometer (Bruker Bio Spin
Corporation, Billerica, MA, USA) using DMSO-d6 or CDCl3
as solvent. Mass spectra (ESI-MS) were obtained by
JEOL-AccuTOF JMS-T100LS mass spectrometer (JEOL
USA, Inc., Peabody, MA, USA), and elemental analysis
was performed on a Vario-EL III CHNOS. Elemental analy-
sis was ꢀ0.4%, that is within the theoretical values.
3051,1598, 1415, 1299, 698 cmꢁ1 1H-NMR (400 MHz,
;
DMSO-d6); d 1.36 (s, 3H, J = 7.2 Hz, CH3), 2.62 (s, 3H,
CH3), 4.32 (s, 2H, J = 7.2 Hz, CH2CH3), 5.09 (bs, 2H,
D2O exchangeable NH2); 13C-NMR (100 MHz, CDCl3);
13.17 (CH2CH3), 17.43 (thiazole CH3), 62.41(CH2CH3),
117.18, 156.06, 161.43(C=N), 165.55(C=O); ESI-MS: m/z
187.07 (M+H); Anal. Calcd. for C7H10N2O2S: C,45.15; H,
5.41; N, 15.04%. Found: C, 45.13; H, 5.42; N, 15.09%.
Synthesis of substituted ethyl 2-(benzylamino)-4-
methyl-1,3-thiazole-5-carboxylate (4a-p)
To a solution of substituted benzaldehyde (0.001 mole) in
10 mL of methanol, ethyl 2-amino-4-methyl-1,3-thiazole-5-
carboxylate (3) (0.0012 mole) was added and stirred at
room temperature. In the saturated reaction mixture,
50 mg iodine (0.0004 mole) was added with stirring at
room temperature till all iodine was dissolved. To the stir-
Chem Biol Drug Des 2016; 87: 508–516
509

Ali et al.
red solution, 55 mg (0.0014 mole) of sodium borohydride
was slowly added, further stirred at room temperature till
the completion of reaction. A solid precipitate obtained
was filtered, washed with water, dried and further recrys-
tallized with ethanol to give solid products (4a-p) (25,26).
The progress of the reaction and purity of the compound
were checked by TLC, using toluene:ethylacetate:formic
acid (5:4:1) as mobile phase.
computational study. Lipophilicity (c-log P), water solubility
(c-log S), molecular weight (MW), number of rotatable
bonds (NROTB) of Lipinski’s rule of five, drug likeness and
toxicity were calculated using online Molinspiration prop-
erty calculation toolkit and online OSIRIS Property explorer.
With the help of Osiris Property Explorer software, toxicities
were predicted which indicated that the synthesized com-
pounds would be free of mutagenicity, tumorigenicity, repro-
ductive side-effects and irritation (28–33). These all
synthesized compounds were also predicted for their phar-
macological activity by online PASS computer program (pre-
diction of activity spectra for substances) (34,35).
Ethyl 2-(benzylamino)-4-methyl-1,3-thiazole-5-
carboxylate (4a)
Yield: 83%; m.p.: 165–167 °C; IR (KBr v max): 3097,
2955, 1701, 1485, 1336, 1029, 789 cmꢁ1; 1H-NMR
(400 MHz, DMSO-d6); d 1.36 (d, 3H, J = 7.4 Hz, CH3),
2.69 (s, 3H, CH3), 4.30 (s, 2H, CH2), 4.36 (q, 2H,
J = 7.4 Hz, CH2CH3), 7.26- 7.45 (m, 5H, Ar-H), 9.55 (bs,
1H, D2O exchangeable NH); 13C-NMR (100 MHz, CDCl3);
13.91 (CH2CH3), 17.26 (thiazole CH3), 49.97(CH2NH),
61.01(CH2CH3), 115.18, 121.32, 127.17, 129.83, 140.10,
151.52, 162.43(C=N), 165.91(C=O); ESI-MS: m/z 277.13
(M+H); Anal. Calcd. for C14H16N2O2S: C, 60.85; H, 5.84;
N, 10.14%. Found: C, 60.89; H, 5.83; N, 10.16%.
Molecular docking studies
Molecular docking studies were carried out using Glide XP
Docking protocol in SCHRODINGER 9.4 (36,37). Co-crystal
structure of xanthine dehydrogenase (XDH) with febuxostat
(PDB entry-1N5X) was selected based on superior crystal
structure parameters and compared with other XO or XDH
structures. There was no difference seen in the binding
sites and co-crystal structures of XO and XDH. Using LIG-
PREP module within Maestro BUILD, ligand was prepared
by default setting. The tautomeric forms of ligands were
generated at physiological pH which includes keto and
enol forms of ligands. Thirty conformations for each ligand
were generated, and lowest energy conformers were used
for the docking analysis. Protein was prepared, optimized
and minimized by Protein Preparation Wizard using OPLS-
2005 force field. Active site for docking was defined as a
Synthesis of substituted 2-(benzylamino)-4-
methyl-1,3-thiazole-5-carboxylic acid acetamide
derivatives (5a-p) (27)
Substituted ethyl 2-(benzylamino)-4-methyl-1,3-thiazole-5-
carboxylate (4a-p) (0.075 mole) and potassium carbonate
(0.3 mole) were added to a mixture of methanol:water
(9:1). The solution was refluxed with stirring until the reac-
tion was completed. The clear solution was cooled and
neutralized with glacial acetic acid and stirred for 1 h.
Precipitate obtained was filtered, washed with water, dried
and recrystallized with ethanol. The progress of the reac-
tion and purity of the compounds were checked by TLC,
using benzene:acetone (8:2) as mobile phase.
ꢀ3
grid box of dimensions 25 9 25 9 25 A around the cen-
troid of the ligand assuming that the ligands to be docked
are of similar size as the co-crystallized ligand. Using Glide
XP module, the docking of molecules was carried out with
Epik state penalties for different ionizations and tautomeric
states. Different docking poses of ligands were generated
and analysed for interpretation of final results.
Xanthine oxidase inhibitory activity
2-(Benzylamino)-4-methyl-1,3-thiazole-5-
carboxylic acid (5a)
Xanthine oxidase (XO) assay of 2-(benzylamino)-4-methyl-
1,3-thiazole-5-carboxylic acid derivatives 5a-p was evalu-
ated using Bovine milk XO (grade 1, ammonium sulphate
suspension, purchased by Sigma Aldrich). UV–visible
spectrophotometer (EI 2371) was used for measuring uric
acid formation at 293 nm at 25 °C under aerobic condi-
tion. The reaction mixture containing 1 mL xanthine
(0.15 mM), 2.5 mL potassium phosphate buffer (50 mM,
pH 7.4) and 0.5 mL of XO solution (0.405 U/mL) was incu-
bated for 5 min at 25 °C. Inhibition of XO activity by vari-
ous inhibitors was measured by following the decrease in
the uric acid formation at 293 nM at 25 °C. The blank was
prepared without enzyme solution. Febuxostat was used
as positive control. The enzyme was preincubated for
5 min, with test compounds ranging from 6.25 to 100 lM
(six concentrations in triplicate) dissolved in DMSO (1% v/
v) (38,39), and the reaction started by the addition of xan-
thine. DMSO (1% v/v) did not interfere with the enzyme
Yield: 67%; m.p.:102–104 °C; IR (KBr v max): 3220, 3031,
1697, 1599, 1426, 1283, 1029, 809 cmꢁ1
;
1H-NMR
(400 MHz, CDCl3); d 2.28 (s, 3H, CH3), 4.36 (bs, 2H, CH2),
6.17 (bs, 1H, D2O exchangeable NH), 7.06–7.33 (m, 5H, Ar-
H), 10.06 (bs, 1H, COOH); 13C-NMR (100 MHz, CDCl3);
17.36 (thiazole CH3), 48.38 (CH2NH), 101.67, 118.55,
123.03, 129.47, 140.55, 148.38, 165.29 (C=O); ESI-MS: m/
z 248.11 (M+H); Anal. Calcd. for C12H12N2O2S: C, 58.05; H,
4.87; N, 11.28%. Found: C, 58.11; H, 4.86; N, 11.30%.
In silico bioactivity study
All the synthesized compounds were evaluated for their
oral bioavailability, physicochemical properties, toxicity and
online pharmacological activity by utilizing these online
software Osiris Property Explorer, Molinspiration and PASS
510
Chem Biol Drug Des 2016; 87: 508–516

Xanthine Oxidase Inhibitory Activity of Aminothiazole
activity. All the experiments were performed in triplicate,
and values were expressed as mean of three experiments
(40).
Xanthine oxidase activity was expressed as percentage
inhibition of XO, as:
XO Inhibition rate ð%Þ ¼
Abs.C ꢁ Abs.S
ꢂ 100;
Abs.C
where S and C represent the absorbance’s (Abs.) of test
compound and control, respectively. The IC50 values,
that is the lg concentration of inhibitor necessary for
50% inhibition, were determined using GraphPad (Prism)
software.
Figure 3: Synthetic scheme for 2ꢁ(substituted benzylamino)ꢁ
4ꢁmethylꢁ1,3ꢁthiazole ꢁ5ꢁcarboxylic acid derivatives (5aꢁp).
Reagent and conditions: (A) ethanol, reflux 70 °C (B) NaBH4,
iodine and methanol, stirring rt. (C) Potassium carbonate,
methanol: water (9:1) stirring with reflux.
DPPH free radical scavenging assay
The stable 1, 1-diphenyl-2-picryl hydrazyl radical (DPPH)
was used for the determination of free radical scavenging
activity of synthesized 2-(benzylamino)-4-methyl-1,3-thia-
zole-5-carboxylic acid derivatives 5a-p by Koleva et al.
method (41). Different concentrations of test compounds
(6.25–100 lM) in methanol were added separately to an
equal volume of 100 lM methanolic solution of DPPH, and
the reaction mixture was kept at room temperature for
15 min. The absorbance of the reaction mixture was
recorded at 515 nm using a UV–visible spectrophotome-
ter. The control sample contained DPPH and methanol
excluding test compounds. Ascorbic acid was used as
standard. Free radical scavenging (%) activity was calcu-
lated using the formula.
obtain substituted 2-(benzylamino)-4-methyl-1,3-thiazole-
5-carboxylic acid derivatives (5a-p) as target com-
pounds.
All synthesized compounds were characterized by com-
bined use of IR, 1H and 13C-NMR and mass spec-
troscopy. All spectral values were in accordance with
the assumed structures. IR spectral data displayed the
presence of carbonyl function of carboxylic acid which
appeared as stretching band at 1598 cmꢁ1
,
N-H
stretching band appeared at 3222 cmꢁ1, C-N stretching
at 1105 cmꢁ1 for compound 5a. In 1H NMR spectral
analysis of compound 4, the ester proton resonated as
a doublet and quartet at d 1.36 and 4.36 ppm, which
disappeared in spectrum of compound 5a, and a new
peak of carboxylic acid was observed at high down-field
Abs.C ꢁ Abs.S
Free radical scavenging ð%Þ ¼
ꢂ 100;
Abs.C
where C represents control and S represents sample reac-
tion mixture as described above in the method. The con-
centration of test compounds having 50% radical
scavenging activity (IC50) was calculated using Graph Pad
Software, Inc. (La Jolla, CA, USA).
values at
d 10.06 ppm (bs, 1H) integrating for one
proton. The signal due to NH proton was observed at d
6.17 ppm. The methylene group of -CH2NH- appeared
as broad singlet at d4.36 ppm. Furthermore, in 13C
NMR spectrum of the compound 5a, signal due to the
carbonyl carbon was observed at 165.29 ppm,
respectively. No ester signal was found in the
spectrum of compounds 5a, and a new signal for -
CH2NH- appeared at 48.38 ppm. The elucidation of
spectral data showed successful synthesis of desired
compounds.
Results and Discussion
Chemistry
The target compounds, 2-(benzylamino)-4-methyl-1,3-thia-
zole-5-carboxylic acid derivatives (5a-p), were synthesized
as outlined in Figure 3. Commercially available 2-chlor-
oethylacetoacetate was cyclized with thiourea to form
ethyl 2-amino-4-methyl-1,3-thiazole-5-carboxylate ester
(3) (24). Subsequently, reaction was carried out by sim-
ply stirring 1.0 equivalent of an substituted aldehyde and
1.2 equivalents of ethyl 2-amino-4-methyl-1,3-thiazole-5-
carboxylate in methanol in the presence of the NaBH4/
I2. This one-pot procedure involved iodine catalysed
in situ formation of an imine at room temperature which
was ultimately reduced with sodium borohydride. The
substituted benzyl amino esters (4a-p) were hydrolysed
(27) with potassium carbonate (after acidification) to
In-silico computational studies
Physicochemical properties
For development of XO inhibitors with enhanced phar-
macological profile, the prediction of physicochemical
properties is the most emphasizing parameter. Increased
lipophilicity with poor water solubility is one of the impor-
tant parameters. Using online Osiris Property explorer
Chem Biol Drug Des 2016; 87: 508–516
511

Ali et al.
and Molinspiration property calculation toolkit, the drug-
likeness characters such as lipophilicity (clogP), water
solubility (clogS), molecular weight (MW), number of
rotatable bonds (NROTB) and drug-likeness score of Lip-
inski’s rule of five (28–30) were calculated for the tar-
geted synthesized compounds (5a-p) (Table 1). The
solubility (clogS) of synthesized compounds was found
in an acceptable range (<ꢁ4). Moreover, the lipophilicity-
related clogP quantifying target-oriented drug-likeness
properties, drug potency, pharmacokinetics and toxicity
analysis has been used for many years. Compounds
with clogP value <5 have more favourable drug-likeness
profile (31,32). Among the series, all the compounds
were found with <5 clogP value which signify their appli-
cability for oral route of administration. Other physico-
chemical parameters of topological polar surface area
(TPSA) were also calculated for observing poorly mem-
brane permeable with less CNS bioavailability which are
useful for selecting oral drug candidates. Compounds
PASS prediction
Potential biological activities of a compound based on a
comparison of its chemical structure with a database of
available quantitative structure activity relationships (2D
QSAR) of over 250 000 compounds exhibiting more than
4000 kinds of biological activities including pharmacologi-
cal effects, mechanism of action, toxic and adverse
effects, interaction with metabolic enzymes and
transporters, influence on gene expressions can be
predicted by PASS online software (34,35). Keeping in
view the features of XOIs, PASS prediction of synthesized
compounds was performed to eliminate the synthesized
compounds with probability of activity (Pa) <0.400.
These compounds were selected for gout treatment which
have net probability of activity over inactivity (Pa-Pi),
greater than 0.400, which added the evidence in their
favour of being active (Table 1). Further it was proved by
in vitro screening of the synthesized compounds which
gave potential drug candidates. The PASS prediction
software helped in fastly recognizing a set of probable
compounds having much more reliable results of docking.
Poorly docked compounds, with either low dock scores or
poorly docked poses, were found to be inactive.
ꢀ
with TPSA values >60 A are commonly picked for oral
drug molecules (33). In case of present series of synthe-
sized compounds 5a-p, the TPSA values were found in
ꢀ
the range of 62.21–108.04 A, which impressed further
structural optimization for the development of new
derivatives. The drug-likeness properties were calculated
by Molinspiration property calculation toolkit, and it sug-
gested that compounds having zero or negative values
should not be considered as drug-like applicant. In dis-
tinction of all synthesized compounds influencing drug-
likeness scores >0, all compounds except 5h and 5i
showed maximum-likeness score in between 1.52 and
5.00 as depicted in Table 1. This online prediction soft-
ware helped in quickly identifying a set of probable
compounds and also useful for screening of compounds
for pharmacological activities.
Molecular docking studies
Enzyme–inhibitor interaction was exposed by molecular
docking studies on XO (PDB entry-1N5X) using Glide XP
Docking protocol in SCHRODINGER 9.4 (36,37,42). Molecular
docking variations include Vander Waals and electrostatic
interactions between active site and ligand which is the
basis of force field scoring. Molecular docking studies of
synthesized compounds with active site of XO were car-
ried out, and results were compared with febuxostat. The
most tightly bound part of febuxostat was its carboxylate
Table 1: In silico physicochemical properties for oral bioavailability and bioactivity of the test compounds as evaluated utilizing computa-
tional predictive software
Gout
H-bond
Comp. treatment
Dock score
Kcal/mol
H-bond Accep-
Rotatable Drug
Drug
ID
Pa
Pa-Pi
XP score c LogP c LogS donors
tors
Mol.Wt. bonds likeliness score TPSA
Toxicity
5a
5b
5c
5d
5e
5f
0.57 0.56
0.51 0.51
0.50 0.49
0.49 0.49
0.56 0.56
ꢁ10.54
ꢁ11.71
ꢁ11.71
ꢁ10.49
ꢁ10.24
ꢁ10.66
ꢁ11.82
ꢁ11.82
ꢁ10.60
ꢁ10.36
ꢁ11.71
ꢁ10.64
ꢁ11.82
ꢁ10.81
ꢁ12.31
ꢁ12.44
ꢁ12.36
ꢁ11.64
ꢁ12.00
ꢁ10.05
ꢁ10.27
2.32
2.92
2.42
3.04
2.66
1.97
2.25
1.39
3.04
2.92
2.66
3.53
1.97
2.25
2.18
1.62
ꢁ3.22
ꢁ3.96
ꢁ3.54
ꢁ4.06
ꢁ3.57
ꢁ2.93
ꢁ3.24
ꢁ3.68
ꢁ4.06
ꢁ3.96
ꢁ3.57
ꢁ4.70
ꢁ2.93
ꢁ3.24
ꢁ3.26
ꢁ2.63
2
2
2
2
2
3
2
2
2
2
2
2
3
2
2
3
4
248.30
282.75
266.29
327.20
262.33
264.30
278.33
293.30
327.20
282.75
262.33
317.19
264.30
278.33
308.35
280.30
4
4
4
4
4
4
5
5
4
4
4
4
4
5
6
4
2.8
4.54
2.81
1.52
2.07
3.64
3.89
ꢁ6.77
ꢁ0.81
3.29
2.68
3.91
2.78
3.09
5.0
0.85
0.79
0.83
0.69
0.79
0.89
0.86
0.42
0.50
0.78
0.81
0.67
0.87
0.85
0.86
0.90
62.21 NM, NC
62.21 NM, NC
62.21 NM, NC
62.21 NM, NC
62.21 NM, NC
82.44 NM, NC
71.45 NM, NC
108.04 SM
4
4
4
4
0.54 0.532 ꢁ11.71
4.75
4.75
5
5g
5h
5i
0.49 0.48
0.48 0.47
0.47 0.47
0.48 0.48
0.54 0.54
0.47 0.47
0.53 0.53
0.48 0.47
0.49 0.48
0.56 0.55
ꢁ10.52
ꢁ11.59
ꢁ10.70
ꢁ12.20
ꢁ12.32
ꢁ12.24
ꢁ11.53
ꢁ11.89
ꢁ9.53
4
62.21 NM, NC
62.21 NM, NC
62.21 NM, NC
62.21 NM, NC
82.44 NM, NC
71.45 NM, NC
80.68 NM, NC
102.67 NM, NC
5j
4
5k
5l
4
4
5m
5n
5o
5p
4.75
4.75
5.50
4.75
ꢁ10.27
3.83
Pa, Probability of being active; Pi, probability of being inactive; cLogP, lipophilicity; cLogS, water solubility; TPSA, topological polar surface
area; NM, non-mutagenic; NC, non-carcinogenic; SM, slightly mutagenic; NP, not predicted.
512
Chem Biol Drug Des 2016; 87: 508–516

Xanthine Oxidase Inhibitory Activity of Aminothiazole
group (43) which was also present in our inhibitors and in
docking studies that binds with same amino acids at same
places. It showed the binding pattern as the oxygen atoms
interacted with side chain of guanidinium group of Arg
ꢀ
880, hydrogen bonds (2.14 A) to the side chain and
ꢀ
hydroxyl with backbone amide of Thr1010 (1.68 A). The
ꢀ
carboxylate group of GlH802 (2.01 A) was located at N-3
of the thiazole ring, which led to formation of hydrogen
bond between the thiazole nitrogen and carboxylate side
chain. Whole thiazole ring was inserted between two
ꢀ
phenylalanine residues Phe914 (4.00 A) and Phe1009
ꢀ
(4.85 A) with PI-PI interaction; these interactions may sup-
port the stabilization of the binding positions and also help
in substrate recognition. The linker -CH2NH- introduced in
between benzyl and thiazole rings has hydrogen binding
pattern with -NH- and amino acid Ser876, which favours
the confirmation of expansion of lead molecules. The ben-
zylamino part of synthesized derivatives was introduced in
between Leu873 and Leu1014 with Asn768-nitrile bond,
and this introduction provided guidance for the alignment
of benzylamino part. Hydrophobic amino acids Leu 648,
Lys771, Thr803, Leu1014, Asn768, Pro1076, Leu873,
Phe1009, Phe 914, Val1011, Ser876, Arg880, Ser1008,
Thr1010, Ala1078 and Ala1079 appeared as birdcage
(Figure 4) for derivatives 5j, 5k and 5l mainly comprising of
hydrophobic interactions. The electron donating groups 2-
CH3, 4-OH, 2, 6-di-Cl and 2-OCH3 were surrounded by
amino acids Leu648, Lys 771. Significant docking score of
most active compounds was found to be ꢁ12.32, ꢁ12.24
and ꢁ12.20 for derivative 5j, 5k and 5l, respectively, when
compared with febuxostat ꢁ12.71 Kcal/mol. According to
molecular docking analysis of 5j, 5k and 5l, we may
suggest that presence of large aromatic thiazole rings in
Figure 4: Two-dimensional docking pose of compound 5k in the
active site of xanthine oxidase.
Figure 5: Three-dimensional docking pose of compound 5k in
the active site of xanthine oxidase.
Table 2: In vitro IC50 value of XO inhibitory
and free radical scavenging activities of
derivatives
Free radical
scavenging
inhibition (%) activity IC50 (lM)
XO inhibition In-vitro IC50 Free radical
Comp. ID Compound
(%)
(lM)
5a
5b
H
4-Cl
4-F
4-Br
4-CH3
4-OH
4-OCH3
4-NO2
2-Br
39
30
71
60
34
76
36
–
31
79
89
84
35
81
39
68
98
98.1 ꢀ 0.1
>100
69
29
23
23.8 ꢀ 0.5
>100
>100
5c
5d
5e
5f
30.3 ꢀ 0.3
43.2 ꢀ 0.51 18
>100
>100
63
29.3 ꢀ 0.47
47.3 ꢀ 0.35
25.0 ꢀ 0.88
ND
21.5 ꢀ 0.21 58
5g
5h
5i
>100
68
–
ND
>100
9.9 ꢀ 0.30
3.6 ꢀ 0.7
8.1 ꢀ 0.4
>100
32
39
84
33
59
>100
5j
2-Cl
59.0 ꢀ 0.90
15.3 ꢀ 0.52
61.7 ꢀ 0.45
34.6 ꢀ 0.68
19.6 ꢀ 0.12
49.1 ꢀ 0.24
17.6 ꢀ 0.87
ND
5k
5l
2- CH3
2,4-di-Cl
2-OH
2-OCH3
3,4-di-OCH3
3,4-OH
Febuxostat
5m
5n
5o
5p
–
14.7 ꢀ 0.32 73
ND 49
60.2 ꢀ 0.54 79
0.03 ꢀ 0.01 ND
–
Ascorbic acid ND
ND
96
13.4 ꢀ 0.50
ND, Not Determined, % inhibitor, calculated at 100 lM concentration; IC50 value ꢀ SEM.
Chem Biol Drug Des 2016; 87: 508–516
513

Ali et al.
the structure of 2-(benzylamino)-4-methyl-1,3-thiazole-5-
carboxylic acid derivatives which fit a position in the active
site of XO, favoured its binding and inhibition towards XO
(Figure 5).
Antioxidant potential of 2-(substituted benzylamino)-4-
methyl-1,3-thiazole-5-carboxylic acid derivatives was also
evaluated using DPPH assay (41). DPPH is a stable free
radical and accepts electron or hydrogen radical to
Biological activity
In vitro XO inhibitory activity of compounds 5a-p was
carried out spectrophotometrically by measuring uric acid
(38–40) levels at 295 nm (Table 2). Febuxostat was used
as a reference drug. Overall, most of the synthesized
2-(benzylamino)-4-methyl-1,3-thiazole-5-carboxylic
acid
derivatives exhibited XO inhibitory activity, but less than
that of febuxostat. Among the limited number of synthe-
sized 2-(substituted benzylamino)-4-methyl-1,3-thiazole-5-
carboxylic acid derivatives, XO inhibition potency can be
inferred due to the presence of thiazole rings with free
carboxylic acid in the synthesized derivatives which may
be responsible for comparatively potent XO inhibition.
Comparison of inhibitory effects among all synthesized
2-(benzylamino)-4-methyl-1,3-thiazole-5-carboxylic
acid
Figure 7: Percent inhibition of xanthine oxidase by 2-(substituted
derivatives, leading to an observation that the presence
of electron donating groups like methoxy, methyl and
hydroxyl at para followed by ortho position in 5c, 5f, 5j,
5k, 5l, and 5n, enhances the Xanthine oxidase inhibitory
activity. Within the series of synthesized compounds,
most of them exhibited potency levels in the micromolar
range; however, compounds 5a, 5b, 5g, 5i, 5m and 5o
were found to be less active in comparison with electron
donating group derivatives. The insertion of chlorine
atom at ortho and para position in 5l as electron with-
drawing group resulted into a general increase in the
potency of inhibitor’s in contrast to the insertion of elec-
tron-releasing groups in 5h, 5g and 5o. The presence
of methyl group at ortho position improved the inhibitory
property as compared to para position in 5k. Com-
pounds 5j, 5k and 5l emerged as most potent XO inhi-
bitor molecule among the series of synthesized
derivatives. IC50 values of all these compounds were
depicted in Table 2, and percentage inhibition has been
shown in Figures 6 and 7.
benzylamino)ꢁ4ꢁmethylꢁ1,3
derivatives (5a-p).
ꢁthiazoleꢁ5ꢁcarboxylic
acid
Figure 8: Percent inhibition pattern of DPPH free radical
scavengers (ascorbic acid, 5k and 5p) at different concentrations.
Figure 9: Percent inhibition of DPPH free radical scavenging
potential of 2ꢁ(substituted benzylamino)ꢁ4ꢁmethylꢁ1,3ꢁthiazoleꢁ5ꢁ
carboxylic acid derivatives (5a-p).
Figure 6: Percent inhibition pattern of xanthine oxidase inhibitors
(febuxostat, 5j, 5k and 5l) at different concentrations.
514
Chem Biol Drug Des 2016; 87: 508–516

Xanthine Oxidase Inhibitory Activity of Aminothiazole
become a stable diamagnetic molecule. Ascorbic acid was
used as a reference compound. Derivatives 5a, 5k, 5n
and 5p showed moderate free radical scavenging activity
on the basis of their IC50 values, and the presence of elec-
tron or hydrogen radical releasing groups OCH3, OH, CH3
and H in the structures of 5a, 5e, 5g, 5j, 5k, 5m, 5o and
5p may be responsible for reduction of DPPH free radical
that imparts scavenging activity to them. Although all syn-
thesized compounds possess moderate to most potent
free radical scavenging activity but, derivatives 5j, 5k, and
5l have potent XO inhibitory along with free radical scav-
enging, IC50 values of the compounds of interest (Table 2)
and percentage inhibition pattern have been shown in
Figures 8 and 9.
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In conclusion, a series of thiazole derivatives (5a-p) as
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Compounds 5k, 5j and 5l demonstrated satisfactory
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was found that several interactions with the various
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Acknowledgments
13. “New Drugs FY”. (2013) Pharmaceuticals and Medical
Devices Agency, Japan.
Authors are thankful to Department of Pharmaceutical
Chemistry, Jamia Hamdard, New Delhi, for providing nec-
essary facilities. Thanks are due to Jamia Hamdard, IIT
Delhi, and CDRI, Lucknow, for providing spectral data.
One of the authors (Md Rahmat Ali) is grateful to the UGC
(Moulana Azad National Fellowship), New Delhi, India, for
providing financial assistance.
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The authors declare nil conflict of interests.
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