Chemosensing, molecular docking and antioxidant studies of 8-aminoquinoline appended acylthiourea derivatives

Article abstract of DOI:10.1016/j.molstruc.2019.02.098

Acylthiourea derivatives, 1-benzoyl-3-(quinolin-8-yl)thiourea (1), 1-(furan-2-carbonyl)-3-(quinolin-8-yl)thiourea (2) and 1-(thiophene-2-carbonyl)-3-(quinolin-8-yl)thiourea (3) were synthesized and well characterized by using NMR (¹H and ¹

Full text of DOI:10.1016/j.molstruc.2019.02.098

Accepted Manuscript
Chemosensing, molecular docking and antioxidant studies of 8-aminoquinoline
appended acylthiourea derivatives
A. Kalaiyarasi, J. Haribabu, D. Gayathri, K. Gomathi, N.S.P. Bhuvanesh, R.
Karvembu, V.M. Biju
PII:
S0022-2860(19)30238-8
DOI:
https://doi.org/10.1016/j.molstruc.2019.02.098
MOLSTR 26255
Reference:
To appear in: Journal of Molecular Structure
Received Date: 24 November 2018
Revised Date: 11 February 2019
Accepted Date: 25 February 2019
Please cite this article as: A. Kalaiyarasi, J. Haribabu, D. Gayathri, K. Gomathi, N.S.P. Bhuvanesh, R.
Karvembu, V.M. Biju, Chemosensing, molecular docking and antioxidant studies of 8-aminoquinoline
appended acylthiourea derivatives, Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/
j.molstruc.2019.02.098.
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ACCEPTED MANUSCRIPT
Chemosensing, molecular docking and antioxidant studies of
8-aminoquinoline appended acylthiourea derivatives
A. Kalaiyarasia, J. Haribabua, D. Gayathrib, K. Gomathic, N. S. P. Bhuvaneshd,
R. Karvembua,*, V. M. Bijua,*
a Department of Chemistry, National Institute of Technology, Tiruchirappalli, 620015, India
b Centre of Advanced Study in Crystallography and Biophysics, University of Madras,
Guindy Campus, Chennai 600025, India
c Department of Biotechnology, Dr. MGR Educational and Research Institute University,
Maduravoyal, Chennai 600095, India
d Department of Chemistry, Texas A & M University, College Station, TX, 77842, USA
ABSTRACT
Acylthiourea derivatives, 1-benzoyl-3-(quinolin-8-yl)thiourea (1), 1-(furan-2-carbonyl)-3-
(quinolin-8-yl)thiourea (2) and 1-(thiophene-2-carbonyl)-3-(quinolin-8-yl)thiourea (3) were
synthesized and well characterized by using NMR (1H and 13C), FT-IR, UV-visible and mass
spectroscopy tools. The molecular structures of compounds 1 and 3 were confirmed by single
crystal X-ray crystallography. The fluorescence emission of the compounds (1-3) was studied
using fluorescence spectrophotometer. The sensing ability of the acylthiourea derivatives was
analyzed by colorimetric, UV-visible and fluorescence titrations. In silico anti-inflammatory,
antimalarial and anti-tuberculosis activities were investigated using molecular docking
studies. Acylthiourea derivatives also possessed good antioxidant activity.
Keywords: 8-aminoquinoline, acylthiourea, chemosensor, molecular docking, antioxidant,
antimicrobial
*Corresponding authors.
E-mail addresses: vmbiju@nitt.edu (V. M. Biju), kar@nitt.edu (R. Karvembu)

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1. Introduction
N-Acylthioureas are promptly synthesized from cheap starting materials in just a
single or two stages. Their poisonous quality is very low, and they are stable over fairly long
periods. Some derivatives of thiourea are biologically active; they show antifungal [1],
antitumor [2-4], antiviral, antibacterial [5-7], pharmacological [8], herbicidal and insecticidal
properties [5-7]. The aminoquinolines are significant precursors/intermediates in the
synthesis of some dyes and drugs. Strikingly, 8-aminoquinoline (8-AQ) derivatives, for
example, pamaqune and primaqune have pulled in much enthusiasm as chemotherapeutic and
prophylactic operators against the liver phases of Plasmodium vivax and Plasmodium
falciparum malarials [8-10]. Antimalarial action of 7-chloroquinolinyl thiourea was reported
by Mahajan et al. [11].
Numerous transition metal ions are pervasive in nature and they play out several
biological functions while in the meantime, overabundance of these ions in bio systems can
prompt sicknesses [12]. Hence, multifunctional sensors that create fluorescence or colour
response to various metal ions will have more potential in ecological applications [13].
Quinoline derivatives, especially 8-hydroxyquinoline and 8-aminoquinoline, are notable
fluorogenic chelator for transition metal ions. In view of the above, we report herein the
synthesis, and chemosensing, antioxidant and molecular docking studies of three acylthiourea
derivatives bearing quinoline moiety namely, 1-benzoyl-3-(quinolin-8-yl)thiourea (1), 1-
(furan-2-carbonyl)-3-(quinolin-8-yl)thiourea (2) and 1-(thiophene-2-carbonyl)-3-(quinolin-8-
yl)thiourea (3).
2. Experimental
2.1 Materials and methods
Required chemicals were purchased from Sigma Aldrich/Merck and utilized as
received. Solvents were purified by the standard methods. The melting points were
determined on Lab India Instrument and are uncorrected. FT-IR spectra were recorded as
KBr pellets on a Nicolet-iS5 spectrophotometer. UV-Vis spectra were recorded using a
Shimadzu-2600 spectrophotometer. NMR spectra were recorded in DMSO-d6 or CDCl3 using
TMS as an internal standard on a Bruker 500 MHz spectrometer. Thermo exactive Orbitrap
mass spectrometer was used to record mass spectra of the compounds. Fluorescence
experiments were carried out at room temperature using Fluromax-P high performance
research and analytical spectrofluorometer/phosphorimeter. The fluorescence of the
compounds with metal ions was observed in 10 µM solution in DMSO at the excitation

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wavelength of 343 nm. Life time of the compounds was measured using Horiba instrument.
The antioxidant potential of the acylthiourea derivatives was tested using the DPPH free
radical scavenging method [14]. Blood biocompatibility of the compounds was studied using
haemolysis method [15].
2.2 Synthesis
Benzoyl chloride, furan-2-carbonyl chloride or thiophene-2-carbonyl chloride (0.01
mol) in 50 mL of acetone was slowly added to the suspension of KSCN (0.971 g, 0.01 mol)
in 50 mL of anhydrous acetone. The resultant mixture was refluxed for 1 h and allowed to
cool at room temperature. 8-Aminoquinoline dissolved in 40 mL of acetone (1.442 g, 0.01
mol) was added dropwise and the solution was stirred for 2 h at room temperature. HCl (0.1
N, 600 mL) was added and the product separated was filtered off, washed with water and
dried in vacuo. Single crystals of acylthiourea derivatives for XRD analysis were grown at
room temperature from their CH3CN solutions.
2.2.1 1-Benzoyl-3-(quinolin-8-yl)thiourea (1)
Yield: 71%. Yellow solid. M.p.: 184 °C. UV-Vis (DMSO): λmax, nm (ε, dm3mol-1cm-
1) 281 (94832), 337 (64828). FT-IR (KBr): υ, cm-1 3312 (N−H), 3040 (H−N−C=S), 1677
(C=O), 1244 (C=S). 1H NMR (500 MHz, CDCl3): δ, ppm 14.47 (s, 1H, NH), 9.67 (d, J = 7.7
Hz, 1H, Ar-H), 9.17 (s, 1H, NH), 9.04 (d, J = 4.1 Hz, 1H, Ar-H), 8.25 (dd, J = 27.8, 8.0 Hz,
13
2H, Ar-H), 8.00 (d, J = 8.1 Hz, 1H, Ar-H), 7.79-7.46 (m, 5H, Ar-H). C NMR (125 MHz,
DMSO-d6): δ, ppm 177.65 (C=S), 168.19 (C=O), 149.66, 139.83, 137.20, 135.03, 133.57,
132.71, 129.29, 128.91, 128.34, 126.69, 124.95, 122.93 and 119.52 (aromatic carbons). ESI-
MS found m/z = 308.0852 [M+H]+, calcd. 307.3696.
2.2.2 1-(Furan-2-carbonyl)-3-(quinolin-8-yl)thiourea (2)
Yield: 63%. Yellow solid. M.p.: 176 °C. UV-Vis (DMSO): λmax, nm (ε, dm3mol-1cm-
1) 297 (41164), 337 (19056). FT-IR (KBr): υ, cm-1 3400 (N-H), 3148 (H-N-C=S), 1684
(C=O), 1270 (C=S). 1H NMR (500 MHz, CDCl3): δ, ppm 14.26 (s, 1H, NH), 9.68 (d, J = 7.7
Hz, 1H, Ar-H), 9.27 (s, 1H, NH, Ar-H), 9.01 (d, J = 4.1 Hz, 1H, Ar-H), 8.20 (d, J = 8.2 Hz,
1H, Ar-H), 7.88-7.13 (m, 5H, Ar-H), 6.70-6.45 (m, 1H, Ar-H). 13C NMR (126 MHz, CDCl3):
δ, ppm 175.34 (C=S), 155.88 (C=O), 149.16, 146.20, 145.32, 140.00, 136.36, 134.94, 128.10,
126.48, 124.30, 121.91, 118.85, 118.82 and 113.40 (aromatic carbons). ESI-MS found m/z =
298.0646 [M+H]+, calcd. 297.3317.

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2.2.3 1-(Thiophene-2-carbonyl)-3-(quinolin-8-yl)thiourea (3)
Yield: 59%. Yellow solid. M.p.: 178 °C. UV-Vis (DMSO): λmax, nm (ε, dm3mol-1cm-
1) 298 (36516), 337 (18584). FT-IR (KBr): υ, cm-1 3270 (N-H), 3062 (H-N-C=S), 1663
(C=O), 1244 (C=S). 1H NMR (500 MHz, CDCl3): δ, ppm 14.21 (s, 1H, NH), 9.65 (d, J = 7.7
Hz, 1H, Ar-H), 9.02 (s, 1H, NH), 8.21 (d, J = 8.2 Hz, 1H, Ar-H), 7.79 (d, J = 3.7 Hz, 1H, Ar-
H), 7.72-7.50 (m, 5H, Ar-H), 7.20 (t, J = 4.3 Hz, 1H, Ar-H), 13C NMR (126 MHz, CDCl3): δ,
ppm 175.47 (C=S), 159.99 (C=O), 149.25, 140.05, 136.42, 136.36, 134.89, 133.99, 130.67,
128.40, 128.11, 126.45, 124.39, 121.92, 118.99 (aromatic carbons). ESI-MS found m/z =
314.0417 [M+H]+, calcd. 313.3973.
2.3 X-ray crystallography
Crystal structures of the two quinolone based acylthiourea compounds (1 and 3) have
been confirmed by using X-ray diffraction. The single crystal data collection and structure
refinement details of the two quinolone based acylthiourea compounds (1 and 3) are provided
in Table 1. The data of acylthiourea compounds 1 and 3 were collected at 100 K by Bruker
Venture diffractometer (kappa geometry) utilizing a Cu-Iµs X-ray tube (Kα= 1.5418 Å).
Integrated intensity information for each reflection was obtained by reduction of the data
frames with the program APEX3 [16]. The integration method employed a three dimensional
profiling algorithm and all data were corrected for Lorentz and polarization factors as well as
for crystal decay effects. Finally, the data were merged and scaled to produce a suitable data
set. SADABS [17] was employed to correct the data for absorption effects. A solution was
obtained readily using XT/XS in APEX3 [18]. The H atoms were added at the calculated
positions in the final refinement cycles. All non-H atoms were refined with the anisotropic
thermal parameters. The structure was refined (weighted least squares refinement) on F2 to
convergence [18,19].
2.4 Sensing studies
The UV-Vis absorption spectra were acquired from the DMSO solutions of the
samples in a 1 cm quartz cell in the range of 200-700 nm at room temperature. Each sample
solution was prepared from 1 mM stock solution of the compounds in DMSO and diluted to
the desired concentration with DMSO. The 1 mM stock solution of the cations tested was
prepared by dissolving the salts such as sulphates of Mg(II), Zn(II), Cu(I), Cu(II) and Al(III),
acetates of Cd(II) and Hg(II), nitrates of Pb(II) and Co(II), and chlorides of Ni(II), Mn(II)
Fe(II), Fe(III), and Cr(III) in Milli-Q water.
,

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The fluorescence spectrum was obtained from the solution in 3.5 mL fluorescence
quartz cell with 1 cm optical path length at ambient temperature and an excitation wavelength
of 338 nm.
2.5 Molecular docking studies
In silico induced fit molecular docking studies were performed using Schrodinger-
Maestro [20] (Schrodinger LLC 2009, USA) to evaluate the binding mode of the compounds
with phospholipase A2, lactate dehydrogenase and decaprenylphosphoryl-D-ribose oxidase
enzymes. Energy minimized three dimensional structures of the compounds were generated
by Ligprep. Three dimensional coordinates of the enzymes were downloaded from RCSB
Protein Data Bank [21] followed by energy minimization in order to account for adding
hydrogen atoms, assigning proper bond orders, ionization states along with charge fixing.
Energy minimized compounds were subjected to induced fit molecular docking at the active
site of the enzymes and the best docked conformation was analysed in terms of docking
score, energy, hydrogen bonding and hydrophobic interactions. Protein-ligand interactions
are represented by Ligplot using PDBSUM [22]. Cartoon/surface view representations of
enzyme with ligand bound at the active site were generated with PyMol [23].
3. Results and discussion
3.1 Synthesis and characterization
Quinolin-8-yl thiourea derivatives (1-3) were prepared from acid chloride (furan-2-
carbonyl chloride / thiophene-2-carbonyl chloride / benzoyl chloride), potassium thiocyanate
and 8-aminoquinoline in dry acetone (Scheme 1) [24]. The acylthiourea compounds (1-3)
showed two bands at 281-298 and 337-338 nm in their UV-Visible spectra, which were
characteristic of π-π* and n-π* transitions respectively. In their FT-IR spectra, strong bands
appeared around 3400-3270 and 3148-3010 cm-1, which were attributed to amide N−H and
H−N−C=S respectively. The carbonyl and thiocarbonyl stretching bands appeared at 1684-
1
1663 and 1251-1244 cm-1 respectively. In the H NMR spectra of 1-3, the carbonyl attached
and thiocarbonyl attached NH protons were observed in the regions 14.47-14.21 and 9.68-
9.65 ppm respectively. Signals due to aromatic protons appeared at 9.04-6.64 ppm. 13C NMR
spectra showed signals at 175.6-175.3 and 168.1-155.5 ppm which were due to C=S and C=O
carbons respectively. The signals around 149.7-149.2 ppm were assigned to the aromatic
carbons (Fig S1-S6) [25].

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Crystal structures of the acylthiourea derivatives (1 and 3) were determined by XRD
technique. The crystals of two acylthiourea compounds 1 and 3 grown at room temperature
from their CH3CN solutions were found to be suitable for single crystal X-ray diffraction
analysis. Three dimensional molecular structures of two acylthiourea compounds (1 and 3)
are displayed in Figs. 1 and 2. Crystallographic data and refinement parameters are given in
Table 1. CIF files have been deposited in Cambridge structure database (CCDC 1875313 and
1875314). The selected bond lengths and angles of compounds 1 and 3 are provided in Table
2 and were comparable with the similar thiourea compounds reported [25]. Table 3 shows
torsion angles for compounds 1 and 3. An intra molecular hydrogen bonding was found
between carbonyl oxygen and thiocarbonyl attached NH (O…HN) with bond distance of
2.6304(15) (1) and 2.650(2) (3) Å (Table 4).
3.2 Visual detection, UV-Vis absorbance and fluorescence studies
The visual detecting ability of the compounds (1-3, 1 mM) towards cations (1 mM)
such as Mg(II), Zn(II), Cd(II), Hg(II), Pb(II), Co(II), Ni(II), Mn(II), Fe(II), Cu(I), Cu(II),
Fe(III), Al(III), and Cr(III) was analyzed by colorimetric method. Colour of compounds 1-3
was changed to pale yellow upon addition of Ni(II), Co(II), Zn(II), Cu(I), Fe(II) and Fe(III),
yellow on addition of Cu(II), and soiled yellow with Hg(II) ion. Figs. S7-S9 reveal colour
changes of compounds 1-3 in the absence and presence of various metal ions. The negligible
colour changes induced by the other metal ions tested indicated that compounds 1-3 could
selectively recognize a range of metal ions such as Ni(II), Co(II), Zn(II), Cu(I), Fe(II),
Fe(III), Cu(II) and Hg(II).
The sensing behaviour of compounds 1-3 towards cations was additionally examined
by UV-Vis spectroscopy. UV-Vis spectra were recorded while adding the metal ions (up to
100 µL from 1 mM solution) into compounds 1-3 (1 mM). Figs. S10-S12 illustrate the
spectral changes upon addition of different metal ions to the compounds (1-3). With Hg(II),
all the compounds showed an increase in absorbance along with red shift in n-π* transition.
Compounds 1-3 showed a new band around 400 nm when Cu(I), Ni(II), Zn(II) or Cu(II) ion
was added. This indicated that these ions formed complexes with the acylthiourea derivatives.
With the other metal ions tested, only negligible shifts in the absorbance values were
observed.
The steady-state fluorescence emission spectra of compounds 1-3 were recorded and
found to be similar. The maximum emission wavelength of the compounds was in the range
450-500 nm. A phenomenal red shift of emission wavelength was noticed for all the
compounds Fig S13. The fluorimetric behaviour of compounds 1-3 in DMSO was

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investigated upon addition of aqueous solution of metal ions such as Mg(II), Zn(II), Cd(II),
Hg(II), Pb(II), Co(II), Ni(II), Mn(II), Fe(II), Cu(I), Cu(II), Fe(III), Al(III) and Cr(III).
Interestingly, ten-fold increase in fluorescence intensity with red shift was seen with Zn(II)
ion. Marginal fluorescent enhancement accompanied by blue shift was observed with Hg(II),
Cd(II) and Mg(II) ions. Fluorescence quenching was observed while adding the other metal
ions. Hence, it was confirmed that the acylthiourea derivatives can act as fluorescent
chemosensors for Zn(II) ion. Moreover, Hg(II), Cd(II) and Mg(II) ions may also be detected
(Figs. 3-5).
Figs. S14-S16 demonstrate the fluorescence spectra of 1-3 in water/DMSO (3:97%
v/v), with different concentrations of Zn(II) ion. They were recorded with an excitation at 343
nm. Without Zn(II) ion, compounds 1-3 produced weak fluorescence around 450-500 nm. On
the introduction of Zn(II), another emission band at 520 nm was shown. Moreover, the
emission intensity gradually increased with the increasing Zn(II) ion concentration. Further,
the emission band was red-shifted with the increasing concentration of Zn(II) ions; red-shift
of 70 nm was observed with 2.5×10-6 M Zn(II). The detection limit for compounds 1-3 with
Zn(II) was found to be 6, 5.3 and 5.6 µM respectively [26]. These values are about 13 fold
lower than that prescribed in WHO guidelines (~76 µM) for drinking water, indicating that
compounds 1-3 can act as efficient fluorogenic chemosensor for Zn(II) recognition [27]. The
UV-Vis spectral changes ascribed to the coordination of Zn(II) with the acylthiourea
derivatives. After the compound binds with Zn(II), the intramolecular hydrogen bond in the
compound breaks, and the intramolecular electron transfer is forbidden which enhances the
fluorescence emission [28].
To determine the stoichiometry, solutions were prepared at different ratios of 1-3 and
Zn2+ (0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1), and their absorbance was measured at
343 nm. When Zn2+ was close to 40%, higher absorbance was noted with compounds 1, 2,
and 3 separately (Fig S17- S19). This demonstrated approximately 2:1 stoichiometry between
the compounds and Zn2+ (Scheme 2).
3.3 Molecular docking analysis
Thiourea derivatives possess wide range of biological properties like antioxidant, anti-
inflammatory, antibacterial, antiviral, antimalarial, etc. In silico screening of the compounds
has been carried out using molecular docking studies to reveal their biological properties and
binding mode at the active site of the enzymes. Three enzymes were chosen for the present in
silico studies; non pancreatic phospholipase A2, lactate dehydrogenase and

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decaprenylphosphoryl-D-ribose oxidase to screen the antiinflammatory, antimalarial and
antituberculosis potential, respectively, of the synthesized compounds.
3.3.1 Phospholipase A2
Phospholipase A2 (PLA2), an essential enzyme in lipid signalling, inflammation and
immune response, hydrolyzes the phospholipids to form fatty acid and lysophospholipids.
The released fatty acid being mostly arachidonic acid is the precursor of the eicosanoid
family and the increase in the concentration of eicosanoids leads to various inflammatory
responses. Compounds that inhibit PLA2 may act as potent anti-inflammatory compounds as
the inhibition of an enzyme leads to decrease in the concentration of eicosanoids. Structural
coordinates of human non-pancreatic secretoary PLA2 were downloaded from protein data
bank (PDB ID: 1DCY) [29]. Based on the induced fit docking results, it was clearly evident
that the compounds bound efficiently; comparable with the co-crystal ligand, indicating that
the compounds might possess good anti-inflammatory property. For comparing the docked
results of the compounds, 1-benzyl-5-methoxy-2-methyl-1H-indol-3-yl-acetic acid (co-
crystal ligand) was re-docked at the active site of PLA2. Docking energy, glide score and
active site interactions are listed in Table 5, which are comparable with those reported in the
literature [29, 30]. Hydrogen bonding and hydrophobic interactions at the active site of PLA2
are shown by Ligplot representation in Fig. 6. Cartoon representation of PLA2 with
compound 1 bound at the active site pocket is shown in Fig. 7.
3.3.2 Lactate dehydrogenase
Plasmodium falciparum lactate dehydrogenase, one of the potential molecular targets
for antimalarial drug designing, catalyzes the interconversion of pyruvate and lactate in the
glycolysis pathway with concomitant interconversion of NADH and NAD+. Three
dimensional structural coordinates of P. falciparum lactate dehydrogenase were downloaded
from protein data bank (PDB: 1LDG, [31]) and compounds 1, 2 and 3 were subjected to
molecular docking. Mefloquine [32] was used as a control. Docking energy and glide score of
mefloquine, compounds 1, 2 and 3, are listed in Table 5. Compound 3 showed better binding
energy, score and active site interactions when compared with mefloquine. Mefloquine
showed hydrogen bonds with residues Thr97 and Thr101, and hydrophobic interactions with
Gly29, Met30, Ile31, Gly99, Phe100, Val138, Thr139, Ser245, Pro246 and Tyr247.
Compound 3 was found to possess hydrogen bonds with Thr97, Thr101 and Asn140, and
hydrophobic interactions with Met30, Ile31, Gly99, Phe100, Val138, Leu163, Leu167,
His195, Ser245, Pro246 and Pro250 residues. Fig. 8 shows the Ligplot representation of

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hydrogen bonds and hydrophobic interactions with lactate dehydrogenase enzyme. Fig. 9
shows the surface view of the enzyme bound with compound 3.
3.3.3 Decaprenylphosphoryl-D-ribose oxidase
Flavo enzyme DprE1, decaprenylphosphoryl-D-ribose oxidase enzyme, catalyzes an
important epimerization step in the decaprenylphosphoryl-D-arabinose (DPA) pathway which
is essential for mycobacterial cell wall biogenesis, cell wall metabolism, cell growth and cell
survival. DprE1 is considered as one of the most vulnerable targets of mycobacterium
tuberculosis as it is essential for mycobacterial cell wall biogenesis. Based on the importance
of enzyme DprE1, the binding affinity of the three acylthiourea derivatives at the active site
of DprE1 was identified through in silico induced fit molecular docking studies. Three-
dimensional structural coordinates of DprE1 were downloaded from protein data bank (PDB
ID: 4FDO) [33] and energy minimized using protein preparation wizard in Schrodinger-
Maestro. Co-crystal ligand, 3-nitro-N[(1R)-1-phenylethyl]-5-(trifluoromethyl)benzamide, has
been re-docked at the active site of DprE1 to compare the binding energy, glide score and
active site interactions with those of the three compounds. Based on the docking energy, glide
score, hydrogen bonds and hydrophobic interactions (Table 5), it was observed that all the
compounds showed good docking energy and score as compared to the co-crystal ligand.
Interactions of the three compounds with the active site residues were found to be better than
those of co-crystal ligand. The active site interactions and binding modes of the compounds
were comparable with those of the reported compounds [33-35]. Fig. 10 shows the Ligplot
representation of active site interactions. Fig. 11 shows the cartoon representation of
compound 1 at the active site of DprE1.
3.4 DPPH assay
Wide range of metabolic reactions happening inside the system indirectly accumulates
reactive oxygen species (ROS) [36]. The mount up of ROS inside the body plays pathogenic
role by initiating various metabolic disorders. Even though anti-oxidative protective system
inside our body encounters these ROS, sometimes the defence network fails [37]. In this
scenario external supply of anti-oxidative compounds is needed to overcome the oxidative
stress.
The 8-aminiquinoline based acylthiourea compounds were evaluated for their
antioxidant potential using DPPH scavenging assay. The IC50 value of compounds 1, 2 and 3
was found to be 10 µg/mL. At the concentration of 500 µg/mL, more than 95 % of the
radicals were scavenged by the compounds. Further increase in concentration did not change

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the activity of the compounds significantly (Fig. 12). In our earlier work [34], the parent
acylthiourea compounds exhibited an IC50 value of 250 µg/mL while in the present study, the
8-aminoquinoline derivatives showed an IC50 of around 10 µg/mL. From the comparison, it
was inferred that 8-aminiquinoline derivatives had 25 times more antioxidant potential than
their parent compounds. The increase in antioxidant potency of 8-aminoquinoline compounds
may be related to their aromatic or heterocyclic structure. It was also reported in other works
that quinoline compounds possessed high antioxidant potential [38, 39].
3.5 Haemolysis assay
The acylthiourea compounds with six different concentrations were examined for
their toxicity against red blood cells (RBCs). Results were compared to the control cells
treated with triton X, which produced 100 % lysis. The results clearly suggested that the
acylthiourea derivatives did not induce any haemolysis. The study also proposes that these
compounds can be used for further biological studies as they do not produce any toxicity
against RBC (Fig. 13).
4. Conclusion
In the present study, three acylthiourea derivatives were synthesized and characterized
by spectroscopic techniques. Three dimensional crystal structures of the two compounds were
elucidated. The compounds showed multi-fold turn on fluorescent sensing towards Zn2+. In
silico molecular docking of the acylthiourea derivatives with PLA2, lactate dehydrogenase
and DprE1 showed interactions at the active site similar to the co-crystal ligands indicating
that the acylthiourea derivatives may possess antiinflammatory, antimalarial and
antituberculosis activities. The acylthiourea derivatives were subjected to in vitro DPPH
antioxidant and antihaemolytic assays and the results showed that the compounds possess
good antioxidant and antihaemolytic activities.
Acknowledgement
J. H. thanks the University Grants Commission for the fellowship (F1-17.1/2012-
13/RGNF-2012-13-ST-AND-18716).

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[39] P. Mahajan, M. Nikam, A. Asrondkar, A. Bobade, C. Gill,
J. Heterocycl. Chem. 54 (2017) 1415-1422
Compound
R
1
2
3
Scheme 1. Synthesis of compounds 1-3.
Fig. 1. Molecular struture of 1.

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Fig. 2. Molecular structure of 3.
Fig. 3. Fluorescence spectra of 1 with metal ions Mg(II), Zn(II), Cd(II), Hg(II), Pb(II), Co(II)
,
Ni(II), Mn(II), Mn(IV), Fe(II), Cu(I), Cu(II), Fe(III), Al(III) and Cr(III).

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Fig. 4. Fluorescence spectra of 2 with metal ions Mg(II), Zn(II), Cd(II), Hg(II), Pb(II), Co(II)
,
Ni(II), Mn(II), Mn(IV), Fe(II), Cu(I), Cu(II), Fe(III), Al(III) and Cr(III).
Fig. 5. Fluorescence spectra of 3 with metal ions Mg(II), Zn(II), Cd(II), Hg(II), Pb(II), Co(II)
,
Ni(II), Mn(II), Mn(IV), Fe(II), Cu(I), Cu(II), Fe(III), Al(III) and Cr(III).

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Scheme 2. Illustration of the interaction of acylthiourea with Zn2+.
Co-crystal
Compound 1

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Compound 2
Compound 3
Fig. 6. Ligplot representation showing hydrogen bonding and hydrophobic interactions at the
active site of non-pancreatic secretory PLA2 by co-crystal ligand ((1-benzyl-5-methoxy-2-
methyl-1h-indol-3-yl)acetic acid) and compounds 1-3.
Fig. 7. Compound 1 bound at the active site of non-pancreatic secretory PLA2.

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Co-crystal
Compound 1
Compound 2
Compound 3
Fig. 8. Ligplot representation showing hydrogen bonding and hydrophobic interactions at the
active site of lactate dehydrogenase by co-crystal ligand (1,4-dihydronicotinamide adenine
dinucleotide) and compounds 1-3.

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Fig. 9. Surface view of lactate dehydrogenase with compound 3 bound at the active site.

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Co-crystal
Compound 1
Compound 2
Compound 3
Fig. 10. Ligplot representation showing hydrogen bonding and hydrophobic interactions at
the active site of DprE1 by co-crystal ligand (3-nitro-N-[(1R)-1-phenylethyl]-5-
(trifluoromethyl)benzamide) and compounds 1-3.

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Fig. 11. Representation of DprE1 with compound 1 bound at the active site.
2
Gallic acid
1.5
1
1
2
3
DPPH
0.5
0
10
25
50
100
500
1000
Concentration µg/ml
Fig. 12. DPPH assay of the compounds (1-3).

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1.5
1
1
2
3
RBC + PBS
0.5
0
10
25
50
100
500 1000
Concentration µg/mL
Fig. 13. Haemolysis assay of the compounds (1-3).

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Table 1. Crystallographic data and refinement parameters
1
3
Chemical formula
Mr
C17H13N3OS
307.36
C15H11N3OS2
313.39
Crystal system, space group Monoclinic, P21/n
Monoclinic, P21/n
100.0
Temperature (K)
100.0
Unit cell dimensions
a = 10.2772(2) Å
b = 4.80670(10) Å
c = 28.6407(7) Å
α = 90°
a = 9.9317(5) Å
b = 4.8302(2) Å
c = 28.4859(14) Å
α = 90°
β = 92.1460(10)°
γ = 90°
β = 91.320(2)°
γ = 90°
V (Å3)
1413.84(5)
4
1366.17(11)
4
Z
Absorption coefficient ꢀ
1.444
3.548
(mm−1)
F(000)
640
648
Crystal size (mm)
0.434 × 0.201 × 0.058
0.424 × 0.36 × 0.138
Radiation type
Cu Kα radiation
1.54178
Wavelength (Å)
1.54178
Diffractometer
BRUKER VENTURE (KAPPA)9
Measured reflections
24273
2673
14854
2571
Independent reflections
Reflections with I > 2σ(I)
Rint
0.0366
0.0364
70.145, 3.103
θmax, θmin (°)
70.207, 3.088
−12<=h<=11,
−5<=k<=4,
−34<=l<=34
h
k
l
−12<=h<=12,
−4<=k<=5,
−33<=l<=34
Absorption correction
Refinement method
No. of reflections /
Semi-empirical from equivalents
Full-matrix least-squares on F2
2571 / 0 / 190
2673 / 0 / 199

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parameters
R indices [F2 > 2σ(F2)]
R indices (all data)
Goodness of fit on F2 (S)
ꢀρmax, ꢀρmin (e Å−3)
R1 = 0.0320, wR2 = 0.0790
R1 = 0.0366, wR2 = 0.0817
1.084
R1 = 0.0361, wR2 = 0.0892
R1 = 0.0380, wR2 = 0.0905
1.074
0.229 and −0.271
0.271 and −0.468
Table 2. Bond lengths (Å) and angles (°) for 1 and 3
1
3
S(1)−C(10) 1.6754(14)
O(1)−C(11) 1.2230(18)
1.6805(18)
1.223(2)
1.317(2)
1.368(2)
0.8800
N(1)−C(1)
N(1)−C(5)
N(2)−H(2)
N(2)−C(9)
1.3200(19)
1.3663(18)
0.8800
1.4095(18)
1.409(2)
1.337(2)
0.8800
N(2)−C(10) 1.3366(19)
N(3)−H(3) 0.8800
N(3)−C(10) 1.3982(18)
N(3)−C(11) 1.3812(18)
1.394(2)
1.382(2)
1.7256(18)
1.708(2)
S(2)−C(12)
S(2)−C(15)
-
-
Table 3. Torsion angles (°) for 1 and 3
1
3
N(1)-C(5)-C(9)-N(2)
C(9)-N(2)-C(10)-S(1)
C(9)-N(2)-C(10)-N(3)
C(10)-N(3)-C(11)-O(1)
C(11)-N(3)-C(10)-S(1)
C(11)-N(3)-C(10)-N(2)
N(3)-C(11)-C(12)-S(2)
0.75(19)
-0.1(2)
-179.18(13)
-3.6(2)
-179.36(12)
-0.2(2)
-
3.0(2)
1.0(3)
-178.49(16)
-2.4(3)
-178.99(14)
0.5(3)
-164.86(12)

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Table 4. Inter- and intra-molecular hydrogen bonds (Å and °) in 1 and 3
D-H...A
d(D-H) d(H...A) d(D...A)
<(DHA)
Compound 1
N(2)-H(2)...O(1)
N(2)-H(2)...N(1)
0.88
0.88
1.89
2.18
2.84
2.6304(15) 140.4
2.6356(17) 112.0
3.6340(12) 151.2
N(3)-H(3)...S(1)#1 0.88
Compound 3
N(2)-H(2)...O(1)
N(2)-H(2)...N(1)
N(3)-H(3)...S(1)#1
0.88
0.88
0.88
1.91
2.18
2.72
2.650(2)
2.641(2)
140.2
111.9
3.4597(14) 142.0
Symmetry transformations used to generate equivalent atoms:
#1 -x+1,-y+1,-z+1

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Table 5. Docking energy, glide score, hydrogen bonds and hydrophobic interactions at the
active site of the enzymes
Compounds
Energy
(kcal/mol)
Docking
score
Hydrogen
bonding
Hydrophobic interactions
interaction
1-benzyl-5-
methoxy-2-
methyl-1H-
indol-3-yl-acetic
acid
Ca2+, His47
Phe5, His6, Ala17, Ala18,
Tyr21, Gly22, Gly29, Val30,
Phe98
−50.552
−53.590
−7.551
Ca2+, Gly29,
Gly31, His47,
Lys62
Phe5, Cys28, Val30, Cys44,
Asp48, Tyr51, Lys52, Phe98
1
−7.398
Ca2+, Gly29,
Lys62
Phe5, Gly22, Val30, Gly31,
His47, Asp48, Phe98
Tyr21, Gly22, Val30, Cys44,
His47, Asp48, Tyr51, Glu55,
Lys62, Phe98
2
3
−49.329
−52.191
−8.076
−9.093
Ca2+, Gly29,
Gly31
Mefloquine
Thr97, Thr101
Gly29, Met30, Ile31, Gly99,
Phe100, Val138, Thr139,
Ser245, Pro246, Tyr247
−45.562
−51.620
−6.063
−6.230
Met30, Thr97,
Gly99
Gly29, Ile31, Asp53, Ala98,
Thr101, Val138
1
Met30, Ile31,
Thr97, Gly99
Thr97, Thr101,
Asn140
Gly27, Gly29, Asp53, Ala98,
Phe100, Thr101, Pro246
Met30, Ile31, Gly99, Phe100,
Val138, Leu163, Leu167,
2
3
−51.612
−58.492
−5.733
−7.016
His195,
Pro250
Pro116,
Lys134, Ser228, Gln336,
Val365, Phe 366, Lys367,
Cys387, Ala417, Lys418
Ser245,
Pro246,
3-nitro-N[(1R)-
1-phenylethyl]-
5-
(trifluoromethyl)
benzamide
1
Gly117
Ile131,
His132,
−41.76
−6.77
Gly117, Leu317 Trp16, Arg58, Tyr60, Trp230,
Phe320, Gly321, Leu363,
Cys387, Lys418
−57.295
−9.135
Gly117, His132, Ser228, Pro116, Val365,
2
3
−53.931
−56.500
−9.148
−7.733
Tyr415
Phe366
Tyr415, His132
Arg58,
Ile131,
Pro116,
Lys134,
Gly117,
Ser228,
Tyr314, Val365

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Highlights
ꢀ
ꢀ
Synthesis and characterization of 8-aminoquinoline based aroylthiourea compounds
Metal ion sensing ability of the compounds was studied by various spectroscopic tools
ꢀ Aroylthiourea derivatives showed good antioxidant activity
ꢀ Antimalarial, antiinflammatory and antituberculosis potentials were verified by molecular
docking