Understanding the binding of quinoline amines with human serum albumin by spectroscopic and induced fit docking methods

Article abstract of DOI:10.1080/07391102.2018.1496141

Seven new quinoline-based bioorganic compounds were prepared by solvent-free synthesis and characterized using spectral techniques. The binding of these compounds with human serum albumin (HSA) was investigated by multi-spectroscopic methods. The quenching of Trp fluorescence upon addition of these compounds to HSA confirmed their significant binding. The quenching analysis at three different temperatures revealed that the complex formation is static and the reaction is entropy driven, spontaneous, and exothermic. Hydrogen bonds and van der Waals forces mainly contributed in the interactions as confirmed by the negative ΔH and ΔS values as well as molecular docking. The results from the circular dichroism (CD) spectroscopy indicated the minimal conformational changes of the protein upon binding with these quinoline compounds. The specific binding site and mode of interactions with HSA were also modeled using induced fit molecular docking procedure and their binding site was found to be in the interface of domains II and III, which is similar to the binding of the drug iodipamide with serum albumin. Communicated by Ramaswamy H. Sarma.

Full text of DOI:10.1080/07391102.2018.1496141

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20
Understanding the binding of Quinoline Amines
with Human Serum Albumin by Spectroscopic and
Induced Fit Docking Methods
K.N. Vennila & Kuppanagounder P. Elango
To cite this article: K.N. Vennila & Kuppanagounder P. Elango (2018): Understanding the binding
of Quinoline Amines with Human Serum Albumin by Spectroscopic and Induced Fit Docking
Methods, Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2018.1496141
To link to this article: https://doi.org/10.1080/07391102.2018.1496141
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1
Understanding the binding of Quinoline Amines with Human Serum
Albumin by Spectroscopic and Induced Fit Docking Methods
K.N.Vennila and Kuppanagounder P. Elango*
Department of Chemistry, Gandhigram Rural Institute (Deemed to be University),
Gandhigram 624 302, India
*Corresponding author E-mail: drkpelango@rediffmail.com
Abstract
Seven new quinoline based bioorganic compounds were prepared by solvent free
synthesis and characterized using spectral techniques. The binding of these compounds
with Human Serum Albumin (HSA) was investigated by multi-spectroscopic methods.
The quenching of Trp fluorescence upon addition of these compounds to HSA
confirmed their significant binding. The quenching analysis at three different
temperatures revealed that the complex formation is static and the reaction is entropy
driven, spontaneous and exothermic. Hydrogen bonds and van der Waals forces mainly
contributed in the interactions as confirmed by the negative ΔH and ΔS values as well as
molecular docking. The results from the CD spectroscopy indicated the minimal
conformational changes of the protein upon binding with these quinoline compounds.
The specific binding site and mode of interactions with HSA was also modeled using
induced fit molecular docking procedure and their binding site was found to be in the
interface of domains II and III, which is similar to the binding of the drug iodipamide
with serum albumin.
Keywords: HSA; quinoline; fluorescence quenching; induced fit molecular docking

2
Introduction
Quinoline scaffold fused with various heterocycles always attract both synthetic and
medicinal chemists because of its diverse chemical and pharmacological properties
( Kumar et al., 2009). Compounds with this motif have been found to possess a broad
range of biological activities such as anti-cancer (Afzal et al., 2015; Chan et al., 2012),
anti-fungal (Musiol et al., 2006; Oliva et al., 2003), anti-malarial (Korotchenko et al.,
2015) anti-tuberculosis (Adhikari et al., 2010; Lilienkampf et al., 2009; Thomas et al.,
2011) anti-protozoal (Hayat et al., 2011) and anti-neoplastic (Heidary et al., 2014)
inhibition of epidermal growth factor receptor (EGFR) and human epidermal growth
factor receptor-2 (HER-2) kinases (Tsou et al., 2005). Few commercially available
drugs containing quinoline moiety are shown in Figure 1 in which chloroquine and
mefloquinone are still the most recommended for the treatment of Malaria. They are
also reported to inhibit anticancer targets such as EGFR & HER2 due to its unique
binding mode. Considering cell line inhibition, many quinoline derivatives are being
reported as MCF-7 breast cancer cell line inhibitors with micro to nano molar IC50
values (Jain et al., 2016). On the other hand, the clear understanding of interactions
between quinoline derivatives and proteins would offer a basis for the future design of
promising drugs (Wang et al., 2013). With this importance and as a part of our research
interest, here in this article we report on the synthesis, characterization and serum
binding properties of 4-aminoquinolines (1a-g). To exhibit all the above mentioned
bioactivities in vivo, the ligands should be transported to the desired site, which will be
done by the carriers, the serum albumin. Albumin is the main extra cellular protein and
is highly concentrated and abundant in blood plasma and hence the present study.

3
Figure 1.
Important quinoline containing drugs
It is the main cargo and a delivery vehicle for transporting the drug molecules to their
target organs and plays an unavoidable role in pharmacokinetic properties of drugs
(Varshney et al., 2010). Also the conformational adaptability of serum albumin extends
well beyond the immediate vicinity of the binding sites. Considering these facts, several
literatures have been reported accounting the serum albumin interactions with the
drug/drug like, herbicide, pesticide, dye compounds, biomolecules etc. (Bagoji et al,
2017, Roy et al., 2018; Salci & Toprak, 2017; Singh et al., 2017, Jafari et al., 2017).
Hence it is important to address this factor to detail the ligand-binding properties (Fanali
et al., 2012). In the current work, Human Serum Albumin (HSA) is selected as our
protein model because of its wide range of functions involving the transport and

4
delivery of fatty acids, porphyrins, bilirubin, tryptophan, thyroxine, drug molecules and
steroids. HSA consists of 585 amino acid residues and has more hydrophobic pockets
inside the protein and is flexible to adapt its shape by means of altering its secondary or
tertiary structure. HSA has three homologous domains (I, II and III) and each domain is
divided into two subdomains (A and B). Drug binding to HSA takes place mostly at
subdomain IIA and IIIA which are commonly referred to as Sudlow site I and II
respectively (Ghuman et al., 2005). Molecular interactions at these sites can be
monitored using intrinsic fluorescence properties of Trp214 (located in subdomain IIA).
Some drug molecules, which are known as subdomain IIIA binders, can bind to
subdomain IB to make it as a third major drug site (Zsila, 2013). Primary binding sites
for fatty acids are located in subdomain IA and IIIA of HSA.
The binding nature of biologically potent quinoline scaffold with HSA is not
known and the analysis may provide some important clues on its pharmacokinetics. In
this aspect, the binding interactions between newly synthesized aminoquinolines and
HSA were quantitatively analyzed based on spectroscopic characterizations such as
UV-Vis, fluorescence, circular dichroism spectrometry and molecular modeling and the
results are presented and discussed.
Materials and methods
Chemicals
All the chemicals used for the synthesis of compounds are of commercially available
reagent grade (Merck, India). Commercially available human serum albumin was
obtained from Sigma Aldrich, India and was used as received. Double distilled water
was used throughout the work and the second distillation was carried out using alkaline
permanganate. The stock solutions of HSA were prepared in an aqueous solution of
Tris-HCL buffer (pH 7.4) and the compounds in 10% DMSO in buffer (v/v).

5
Instrumentation
Nuclear magnetic resonance spectra were recorded in DMSO-d6 (1H NMR 300 MHz).
1
The H-NMR spectral data is expressed in the form: Chemical shift in units of ppm
(normalized integration, multiplicity, and the value of J in Hz). Electro spray ion mass
spectra (m/z) were recorded using LC/MSD TRAP XCT Plus (1200 Agilent). FT-IR
spectra were recorded on a JASCO (FT-IR 460 Plus, Japan) spectrometer with samples
prepared as KBr pellets. The absorption spectra were measured on a UV-Vis double
beam spectrophotometer using 10 mm path length quartz cuvette (JASCO V630, Japan).
Fluorescence measurements were performed with Cary Eclipse-Agilent Technologies
spectrophotometer model G9800A equipped with xenon lamp using a 1.0-cm quartz
cell. The excitation and emission slit width (5 nm) and the scan rate (250 nm) was kept
constant for all of the experiments. Circular dichroism (CD) measurements were
performed with a JASCO-810 spectrophotometer using a 1.0 mm path length quartz
cell. Molecular docking studies were carried out using Maestro 11(Schrödinger LLC)
installed in CentOS platform.
Procedures
General procedure for synthesis of compounds (1a-g)
To a stirred solution of 4-chloroquinaldine, R-NH2 (1eq) was slowly added at room
temperature and the reaction mixture was stirred at 180˚C for 4 h (Scheme 1). The
completion of the reaction was monitored using thin layer chromatography (TLC). Once
the reaction has been completed, the reaction mixture was cooled to RT and extracted
with ethyl acetate. The combined organic layer was dried over anhydrous sodium
sulfate and concentrated under reduced pressure. The reaction mixture was washed with
hexane and ethyl acetate (10%) to get the pure product. The LC-MS and FT-IR spectra
1
of all the compounds were given as Figure S1 and S2, respectively. The H and 13C

6
NMR spectral images for all the compounds were given as Figure S3 in supporting
information.
Scheme 1. Synthesis of 4-substituted quinoline amines
N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylquinolin-4-amine (1a). Compound
(1a) was prepared using the above mentioned procedure using 4-chloroquinaldine (100
mg, 0.56 mmol) and 2,3-dihydrobenzo [b][1,4]dioxin-6-amine to obtain (1a) as white
1
solid. M.p. 70-72˚C. FTIR (KBr) cm-1: 3406(N-H), 1203(C-O), 1065(C-N). H NMR
(DMSO-d6, 400 MHz, ppm): 8.672, s, 1H, 8.291-8.270 (d, J=-8.4Hz, 1H), 7.762-7.742
(d, J=8.0Hz, 1H), 7.641-7.605(t, J=7.2Hz, 1H), 7.444-7.409 (t, J=7.6,6.4Hz, 1H), 6.930-
6.908 (d, J=8.8Hz,1H) 6.835-6.812 (d, J=9.2Hz, 2H) 6.629,s,1H, 4.270,s,4H,
2.412,s,3H. 13C NMR (DMSO-d6, 100 MHz, ppm): 159.06, 149.17, 148.91, 144.13,
140.77, 134.20, 129.57, 128.79, 124.09, 122.22, 118.45, 117.98, 117.35, 112.99, 101.0,

7
64.65, 64.45, 25.59. LCMS m/z: [M+H]+calculated for C18H16N2O2: 292.34, found:
293.5.
N-(benzo[d][1,3]dioxol-5-ylmethyl)-2-methylquinolin-4-amine (1b). Compound (1b)
was prepared using the above mentioned procedure using 4-chloroquinaldine (50 mg,
0.28 mmol) and benzo[d][1,3]dioxol-5-ylmethanamine to obtain (1b) as white solid.
M.p. 136-140˚C. FTIR (KBr) cm-1: 3433(N-H), 1251(C-O), 1038(C-N). 1H NMR
(DMSO-d6, 400 MHz, ppm): 8.215-8.195 (d, J=-8 Hz, 1H), 7.698-7.676 (d, J=8.8Hz,
2H), 7.568,s,1H, 7.385-7.369(d, J=6.4Hz,1H), 6.960,s,1H, 6.869,s,1H, 6.292,s,1H,
5.974,s,2H, 4.438,s,2H, 2.380,s,1H. 13C NMR (DMSO-d6, 100 MHz, ppm):158.98,
150.23, 148.55, 147.86, 146.57, 133.37, 129.15, 128.83, 123.64, 121.85, 120.50,
118.06, 108.58, 107.92, 101.30, 99.24, 45.74, 40.60, 39.35, 25.73. LCMS m/z: [M+H]+
calculated for C18H16N2O2: 292.34, found: 293.5.
N-(3,4-dimethoxyphenyl)-2-methylquinolin-4-amine (1c). Compound (1c) was prepared
using the above mentioned procedure using 4-chloroquinaldine (50 mg, 0.28 mmol) and
3,4-dimethoxy aniline to obtain (1c) as ash color solid. M.p. 90-94˚C. FTIR (KBr) cm-1:
1
3423(N-H), 1233 (C-O), 1025 (C-N). H NMR (DMSO-d6, 400 MHz, ppm): 8.767,s,
1H, 8.329-8.310 (d, J= 7.6Hz, 1H), 7.772-7.751 (d, J=8.4Hz, 1H), 7.654-7.617 (t,
J=7.2Hz,7.6Hz, 1H), 7.458-7.422 (t J= 7.2Hz, 1H), 7.032-7.011(d, J=8.4Hz, 1H),
6.950,s,1H, 6.905-6.885 (d, J= 8Hz, 1H), 6.631,s,1H,3.788,s, 3H, 3.766,s,3H,
2.415,s,3H. 13CNMR (DMSO-d6, 100 MHz, ppm): 159.14, 149.77, 149.35,
149.02,146.32, 133.89, 129.50, 128.99, 124.02, 122.18, 118.43, 116.28, 112.87, 109.23,
100.96, 56.19, 56.00, 40.59, 40.38, 40.18, 39.97, 39.76, 39.55, 39.34, 25.63. LCMS
m/z: [M+H]+ calculated for C18H18N2O2: 294.35, found: 295.6.

8
N-(benzo[d][1,3]dioxol-5-yl)-2-methylquinolin-4-amine (1d). Compound (1d) was
prepared using the above mentioned procedure using 4-chloroquinaldine (50 mg, 0.28
mmol) and benzo[d][1,3]dioxol-5-amine to obtain (1d) as brown solid. M.p. 46˚C. FTIR
1
(KBr) cm-1: 3416(N-H), 1233(C-O), 1025(C-N). H NMR (DMSO-d6, 400 MHz, ppm):
8.707,s,1H, 8.296-8.276 (d,J=8Hz, 1H), 7.766-7.745 (d,J=8.4Hz, 1H), 7.644-7.607 (t,
J=7.6Hz,7.2Hz, 1H), 7.448-7.412 (t, J=7.2Hz, 2H) 6.989-6.944 (t, J=8Hz,1H) 6.814-
6.794(d, J=8Hz, 1H), 6.590,s,1H, 6.069,s,2H, 2.413,s,3H.13C NMR (DMSO-d6,
100MHz, ppm): 159.11, 149.36, 148.96, 148.32, 144.60, 134.92, 129.54, 128.85,
124.07, 122.20, 118.45, 117.49, 109.06, 106.15, 101.75, 101.11, 65.39, 25.61, 15.2.
LCMS m/z: [M+H]+ calculated for C17H14N2O2: 278.31, found: 279.5.
2-methyl-N-(pyridin-2-ylmethyl)quinolin-4-amine (1e). Compound (1e) was prepared
using the above mentioned procedure using 4-chloroquinaldine (30 mg, 0.0168 mmol)
and 2-(pyridin-2-yl)ethan-1-amine to obtain (1e) as light brown crystals. M.p. 110-
116˚C. FTIR (KBr) cm-1: 3456(N-H). 1H NMR (DMSO-d6, 400 MHz, ppm): 8.552-
8.540 (d, J= 4.8Hz, 1H), 8.104-8.083 (d,J=8.4Hz, 1H), 7.735-7.672 (m, 2H), 7.566-
7.528 (q, 1H), 7.363-7.315 (m, 2H), 7.256-7.226(q,1H), 7.173-7.147(t, J=5.2, 5.2Hz,
1H), 6.406,s,1H, 3.659-3.609 (q, 2H), 3.155-3.118 (t, J=7.6, 7.2Hz, 2H), 2.505-2.458
(d, J=18.8Hz, 3H).13C NMR (DMSO-d6, 100MHz, ppm): 159.55, 159.34, 150.50,
149.45, 148.01, 137.24, 129.41, 128.26, 123.96, 123.82, 122.20, 121.65, 117.79, 98.66,
42.71, 40.23, 40.02, 39.81, 39.60, 39.39, 39.18, 38.97, 36.63, 25.30. LCMS m/z:
[M+H]+ calculated for C17H17N3: 263.35, found: 264.5.
N-(2-methoxybenzyl)-2-methylquinolin-4-amine (1f). Compound (1f) was prepared
using the above mentioned procedure using 4-chloroquinaldine (50 mg, 0.28 mmol) and
(2-methoxy phenyl) methanamine to obtain (1f) as pale pink solid. M.p. 170-172˚C.
1
FTIR (KBr) cm-1: 3426(N-H). H NMR (DMSO-d6, 400 MHz, ppm): 8.234-8.213 (d,

9
J= 8.4Hz, 1H), 7.707-7.688 (d,J=7.6Hz, 1H), 7.614-7.556 (q, 2H), 7.394-7.357 (q, 1H),
7.261-7.219 (m, 1H), 7.181-7.162 (d, J=7.6Hz, 1H),7.049-7.029 (d, J= 8Hz, 1H), 6.881-
6.844 (t, J= 7.6, 7.2Hz, 1H), 6.194,s,1H, 4.490-4.476 (d, J= 5.6Hz, 2H), 3.888,s,3H,
2.360,s,3H. 13C NMR (DMSO-d6, 100MHz, ppm): 159.04,157.28, 150.64,148.17,
129.40, 128.62, 128.46, 127.64, 126.28, 123.85,,121.79, 120.73, 117.86, 111.11, 98.86,
55.85, 40.91, 40.38, 40.17, 39.96, 39.75, 39.55, 39.34, 39.13, 25.44. LCMS m/z:
[M+H]+ calculated for C18H18N2O: 278.36, found: 279.5.
N-(4-fluorobenzyl)-2-methylquinolin-4-amine (1g). Compound (1g) was prepared using
the above mentioned procedure using 4-chloroquinaldine (50 mg, 0.28 mmol) and (4-
fluorophenyl)methanamine to obtain (1g) as pale yellow solid. M.p. 70-74˚C. FTIR
(KBr) cm-1: 3406(N-H). 1H NMR (DMSO-d6, 400 MHz, ppm): 8.224-8.203 (d, J=
8.4Hz, 1H), 7.768-7.739 (t, J=6Hz, 1H), 7.702-7.681(d, J= 8.4Hz, 1H), 7.588-7.550
(q,1H), 7.445-7.355 (m, 3H), 7.178-7.133 (t, J= 9.2, 8.8Hz,1H), 6.271,s,1H, 4.534-
4.519 (d, J= 6Hz,2H), 2.369,s,3H.13C NMR (DMSO-d6, 100MHz, ppm): 162.85,
160.44, 158.99, 150.44, 148.08, 135.47, 135.44, 129.45, 129.33, 129.25, 128.38,
123.92, 121.80, 117.92, 115.70, 115.49, 99.22, 45.20, 40.39, 40.18, 39.98, 39.77, 39.56,
39.35, 39.14, 25.36. LCMS m/z: [M+H]+ calculated for C17H15FN2: 266.32, found:
267.5.
Fluorescence measurements
A 2 mL solution containing 10 µM HSA was titrated by successive additions of
compounds (1a-g) with concentrations ranging between 0 to 100 µM. These solutions
were allowed to stand for a minute to equilibrate. The fluorescence emission spectra
were then measured at room temperature with an excitation wavelength at 290 nm. The
appropriate blank corresponding to the buffer solution was subtracted to correct for
background fluorescence. The inner filter effect of the compounds on HSA quenching

10
has been corrected as reported ( Suganthi & Elango, 2017). The binding constants of
compounds (1a-g) to HSA were determined using the corrected emission intensity
values.
UV-Vis absorption spectra
The UV-Vis absorption spectra of HSA in the absence and presence of aminoquinolines
were measured over a wavelength range of 200-500 nm in the Tris-HCl buffer solution
(pH 7.4) at room temperature.
CD studies
The CD spectra of HSA incubated with compounds (1a-g) at 1:1 molar ratio were
recorded in the wavelength range of 200-500 nm under constant nitrogen flush using a
1.0 mm path length quartz cuvette. All observed spectra were baseline subtracted for
buffer solution and the α-helical content was calculated on the basis of molar ellipticity
value.
Molecular Docking
Structures of the compounds (1a-g) were sketched in using build panel and were
prepared for docking using Ligprep module (Madhavy Sastry et al., 2013) implemented
in Maestro 11 (Schrodinger LLC). The molecules were subjected to energy
minimization with OPLS-2005 force field (Shivakumar et al., 2010) to generate single
low energy 3-D structure for each input structure. For molecular docking studies, we
utilized X-ray crystal structure of Human serum albumin and its three dimensional
coordinates were obtained from Protein Data Bank (PDB ID: 5UJB). Protein was
prepared by removal of solvent molecules coupled with the addition of hydrogen atoms
using Prepwizard in Maestro. The missing side chains were built using prime and the
active site was visually inspected and the appropriate corrections were made for
tautomeric states of histidine residues, orientations of hydroxyl groups, and protonation

11
states of basic and acidic residues. The hydrogen atoms were minimized using
OPLS2005 force field, while constraining all the heavy atoms (non-hydrogen) to their
original positions. The protein with optimized hydrogen coordinates was finally saved
as a separate file to be used for docking. For all the molecules, induced fit docking
protocol implemented in Glide was used to carry-out the docking simulation.
In reality, HSA is one of the receptors which alter their binding site to conform
to the shape and binding mode of the ligand which is reflected in CD experiment. This
is often referred to as induced fit and is one of the main complicating factors in
structure-based drug design. Hence, Induced Fit Docking (IFD) protocol which treats
both the protein and ligand as flexible was used to generate new receptor conformations
and ligand binding modes (Sherman et al., 2006, Karthikeyan et al., 2017).
Results and Discussion
A series of 4-aminoquinoline derivatives were prepared and characterized by
spectral techniques such as NMR, LC-MS and FTIR. These biologically important
amino quinolines were tested for binding with transporter protein human serum albumin
using UV-Vis, Fluorescence and CD spectrometry techniques. The binding site of the
test compounds and their interactions with amino acid residues of HSA were revealed
by molecular docking method and the results were discussed.
Absorption spectral studies
UV-Vis absorption spectroscopy is one of the effective methods in detecting the
changes in the structural conformation of proteins upon binding to ligands. The
absorption spectra of HSA in the absence and presence of increasing concentrations of
the compounds are shown in Figure 2. The free HSA showed an absorption peak at 277
nm which is due to the polarity of the microenvironment around the tyrosine and
tryptophan residues of HSA and other one at 223 nm due to the backbone of the protein.

12
Upon addition of the compounds the absorption intensity of the peak at 277 nm was
found to increase with a small red-shift. This observation confirmed that all the
compounds interacted with HSA and also changes the polarity of the microenvironment
around the tyrosine and tryptophan residues of HSA and decreased the hydrophobicity
of the binding site (Seedher & Agarwal, 2010). The quenching of protein backbone
peak at 223 nm upon addition of the compounds suggested the quenching mechanism
was mainly a static quenching process (Kumar et al., 2016). The results also indicated
the formation of complex between the protein and the compounds in the ground state.
3.0
2.5
2.0
1.5
1.0
0.5
0.0
(1b)
(1a)
2.5
2.0
1.5
1.0
0.5
0.0
200
250
300
350
400
200
250
300
wavelength (nm)
wavelength (nm)
3.0
2.5
2.0
1.5
1.0
0.5
0.0
3.0
2.5
2.0
1.5
1.0
0.5
0.0
(1c)
(1d)
200
250
300
350
400
200
250
300
350
400
wavelength (nm)
wavelength (nm)

13
3.0
2.5
2.0
1.5
1.0
0.5
0.0
3.0
2.5
2.0
1.5
1.0
0.5
0.0
(1f)
(1e)
200
250
300
350
400
200
250
300
350
400
wavelength (nm)
wavelength (nm)
3.0
2.5
2.0
1.5
1.0
0.5
(1g)
0.0
200
250
300
350
400
wavelength (nm)
Figure 2. UV-Vis spectra of HSA- (1a-g) complex with increasing concentration from
0-50 µM with the Tris-HCl buffer solution (pH 7.4) at room temperature (298K)

14
Fluorescence quenching of HSA
The use of fluorescence spectroscopy to determine the quenching mechanism,
mode and strength of interaction of HSA with drugs can be found in recently published
reports, wherein the intrinsic fluorescent property of tryptophan is mainly utilized for
the study (Zhou et al., 2018, Kosiha et al., 2017, Kosiha, Parthiban & Elango, 2017;
Suganthi & Elango, 2017a; Suganthi & Elango, 2017b). When the HSA solution is
excited at 290 nm, the maximum emission intensity of tryptophan will be contributed
(Peng et al., 2016). In this study, the fluorescence spectra of HSA (10 µM) in the
absence and presence of increasing concentrations (0 to 100 µM) of the compounds
were recorded in Tris-HCl buffer solution (pH 7.4) at room temperature with excitation
at 290 nm. Pure HSA exhibited a strong fluorescence emission at 348 nm (Figure 3). It
is evident from the Figure 3 that with gradual increase in the concentration of these
compounds, there is a significant reduction in the fluorescence emission intensity of
HSA. A substantial red shift in the emission maximum (8-10 nm) was also observed
suggesting that the polarity of the protein environment was higher in the protein-ligand
complex when compared to pure HSA. The above observations indicated that there
exists a significant binding of these compounds to HSA with changes in the
microenvironment of tryptophan residues and conformation of protein structure.
In order to describe the quenching mechanism the fluorescence data is analyzed
by Stern-Volmer equation.
⁄
[ ]
푄
퐹0 퐹 = 1 + 퐾푠푣
(1)
Where 퐹0 and 퐹 are the fluorescence intensities in the absence and presence of the
[ ]
quencher 푄 and 퐾푠푣 is the Stern-Volmer quenching constant which measures the
⁄
[ ]
efficiency of quenching (Khan et al., 2008). A plot of 퐹0 퐹 versus 푄 , of all the
compounds 1a-g, are found to be linear (Figure 4) and the values of correlation

15
coefficient and 퐾푠푣 are collected in Table 1. The linear Stern-Volmer plots indicated the
occurrence of single type of quenching which may be static as per the absorption
spectral confirmation.
(1a)
(1b)
(1c)
(1d)
(1e)
5
(1f)
(1g)
4
3
2
1
0.00000
0.00003
0.00006
0.00009
[Q]
Figure 4. The Stern–Volmer plots of fluorescence titrations for
compounds (1a-g) with HSA
To ascertain the mechanism of the quenching phenomena, as representative
cases, the intrinsic fluorescence of HSA upon addition of 1a and 1g were measured at
298, 308 and 318 K and the thermodynamic parameters were calculated. The quenching
constants for 1a were found to be 4.72x104, 6.21 x104 and 5.30 x104 L mol-1 at 298, 308
and 318 K, respectively. For 1g, KSV values were found to be 1.22 x104, 3.05 x104 and
2.41 x104 L mol-1 at 298, 308 and 318 K, respectively (Figure S4). These values don’t

16
show any linear trend with increasing temperature. It is reported that, the static
quenching constant doesn’t necessarily decrease with an increase in temperature.
Therefore, in the present study the quenching of fluorescence of HSA by these
compounds occurs via static quenching mechanism (Hu, Liu, & Xiao, 2009; Tian et al.,
2015).
Table 1. Stern-Volmer constant 푲풔풗 , correlation coefficient r and standard deviation
S.D calculated from Stern-Volmer plot
Compounds
푲풔풗
r
S.D
(퐿 푚표푙−1)
4.72 x 104
3.90 x 104
4.33 x 104
5.76 x 104
0.59 x 104
0.91 x 104
1.22 x 104
0.999 0.002
0.990 0.001
0.989 0.001
0.993 0.003
0.999 0.010
0.998 0.006
0.994 0.013
1a
1b
1c
1d
1e
1f
1g
For a static quenching process, if it is assumed that there are similar and
independent binding sites in the protein molecules, the binding constant (퐾푎) and
number of binding sites (푛) can be calculated from the following equation.
[( [ ]
)⁄ ]
푙표ꢀ 퐹0 − 퐹 퐹 = 푙표ꢀ 퐾푎 + 푛 푙표ꢀ 푄
(2)

17
where 퐹0 and 퐹 are the fluorescence intensity of HSA in the absence and presence of
the ligands, respectively. The values of correlation coefficient and binding constants
thus calculated are given in Table 2. The number of binding sites 푛 was found nearly
equal to 1, indicating that there was only one binding site in HSA for the compounds
during their binding process and also indicates in the binding reaction, the molar ratio of
HSA and the compounds is 1:1. The magnitude of the binding constant values in the
order of 104- 105 M-1 obtained suggested that there is a significant interaction between
the compounds and the protein molecule. As representative cases, the binding constants
for the interaction of 1a and 1g with HSA were also calculated at three different
temperatures. The Ka values obtained are 13.80x104, 9.46 x104 and 4.99 x104 ( for 1a)
and 2.20 x103, 0.98 x103 and 0.65 x103 (for 1g) at 298, 308 and 318 K, respectively.
From these binding constant values, the thermodynamic parameters were evaluated
⁄
⁄
using van’t Hoff equation (ln 퐾푎 = −∆퐻 푅푇 + ∆푆 푅 , where 퐾푎 is the binding
constant, 푅 is the gas constant and 푇 is the temperature). For both these systems plot of
⁄
푙푛 퐾푎 versus 1 푇 was found to be linear (Figure S5). The enthalpy, entropy and free
energy changes obtained are: For 1a, ΔH = − 44 kJ mol-1; ΔS = − 48 J mol-1 and ΔG = −
58 kJ mol-1; For 1g, ΔH = − 48 kJmol-1; ΔS= − 98 J mol-1 and ΔG = − 77 kJ mol-1. The
observed ΔH < 0 and ΔS < 0 indicated that the main forces acting between HAS and
these compounds are hydrogen bonds and van der Waals forces and the interaction
reactions were exothermic (Ross & Subramanian, 1981). The negative Gibbs free
energy changes indicated that the interaction of these compounds with HSA is
spontaneous.

18
Table 2. Association constant 푲풂 , number of binding site n, Correlation coefficient r,
(
)
standard deviation S.D calculated from 푙표ꢀ( 퐹0 − 퐹 /퐹) versus log [푄]
Compounds
푲풂
풏
r
S.D
(퐿 푚표푙−1)
1.38 x 105
4.731x 104
0.907 x 105
1.05 x 105
0.138 x 104
0.118 x 104
0.22 x 104
1.118 0.999
1.032 0.997
1.118 0.997
1.084 0.999
0.0010
0.0102
0.0023
0.0011
0.0022
0.0111
0.0023
1a
1b
1c
1d
1e
1f
0.59
0.786 0.991
0.83 0.994
0.989
1g

19
(1b)
(1a)
400
200
0
400
200
0
300
350
400
450
500
350
400
450
500
Wavelength (nm)
Wavelength (nm)
(1c)
(1d)
400
400
200
200
0
0
350
400
450
500
350
400
450
500
Wavelength (nm)
Wavelength (nm)
(1e)
(1f)
400
200
0
400
200
0
350
400
450
500
350
400
450
500
Wavelength (nm)
Wavelength (nm)
(1g)
400
200
0
350
400
450
500
Wavelength (nm)

20
Figure 3. Fluorescence emission quenching of HSA (10 µM) upon addition of
compounds (1a-g) complex with increasing concentration from 0-100 µM in the
Tris-HCl buffer solution (pH 7.4) at room temperature (298 K)
Binding distance between HSA and 1a-g
The binding distance between the receptor and the ligands is related to the
critical energy transfer distance as given in Förster non-radiation energy transfer theory
(Suganthi & Elango, 2017). It is given by the equation
6
6⁄(
)
( ⁄ )
6
퐸 = 푅0 푅0 + 푟 = 1 − 퐹 퐹0
(3)
Where 퐸 is the energy transfer efficiency, 푅0 is the critical distance when the energy
transfer efficiency is 50%, and 푟 is the binding distance between donor and acceptor.
Also
6
−4
2
[
]
푅0 = 0.2108 푁 × 휑푑 × 퐾 × 퐽 ,
(4)
Where 퐾2 is the spatial orientation factor of the dipole, 푁 is the refractive index of the
medium, 휑푑 is the fluorescence quantum yield of the donor, and 퐽 is the overlap
integral of the fluorescence emission spectrum of the donor and the absorption spectrum
of the acceptor. The overlap of the absorption spectra of the test compounds and the
emission spectra of HSA is shown in Figure 5. The spectrum overlap integral is
calculated using fluortools (UV-Vis-IR spectral analysis software) with 푁 = 1.336, 휑푑 =
0.15, 퐾2 =2/3 and 푅0 and 푟 values calculated are collected in Table 3. All the
calculated 푟 values are less than 8 nm indicating the highest possibility of energy
transfer between HSA and the test compounds.

21
5
4
400
300
200
100
0
5
4
400
300
200
100
0
(1a)
(1b)
3
3
2
2
1
1
0
0
-1
-2
-3
-4
-1
-2
-3
-4
300
350
400
450
500
300
350
400
450
500
wavelength (nm)
wavelength (nm)
5
4
400
300
200
100
0
5
4
400
(1c)
(1d)
3
3
300
200
100
0
2
2
1
1
0
0
-1
-2
-3
-4
-1
-2
-3
-4
300
350
400
450
500
300
350
400
450
500
wavelength (nm)
wavelength (nm)
5
4
3
2
1
0
400
5
4
400
(1f)
(1e)
300
200
100
0
3
300
200
100
0
2
1
0
-1
-1
-2
-3
-4
-2
-3
-4
300
350
400
450
500
300
350
400
450
500
wavelength (nm)
wavelength (nm)

22
5
4
400
300
200
100
0
(1g)
3
2
1
0
-1
-2
-3
-4
300
350
400
450
500
wavelength (nm)
Figure 5. Spectral overlap of absorption and emission from
test compounds and HSA, respectively
Table 3. Donor-acceptor distance r between HSA and the compounds calculated using
FRET theory
푱 (cm3 L M-1)
1.386 × 1010
5.603 × 109
1.707 × 1010
1.415 × 1010
6.173 × 109
1.434 × 1010
1.845 × 1010
푬 (%)
0.17
푹ퟎ(nm)
0.853
0.724
0.872
0.845
0.736
0.847
0.883
풓 (nm)
1.10
1.09
1.34
1.26
1.19
1.54
1.55
Compounds
1a
1b
1c
1d
1e
1f
0.08
0.07
0.09
0.05
0.027
0.034
1g

23
Circular dichroism studies
The structural change of HSA by the binding of ligands can be determined by
CD measurements. CD spectra of pure HSA and that after the addition of the
compounds (1a-g) are given in Figure 6. The CD spectrum of pure HSA has two
negative peaks at 209 and 222 nm, which correspond to α–helical structure of the
protein owing to 휋 → 휋∗ and 푛 → 휋∗ transitions in the peptide bond in helices,
respectively (Wang et al., 2008). Changes in the ellipticity at 209 and 222 nm by the
addition of compounds are useful probes for visualizing changes in the protein’s α-
helical structure (Na Ji et al., 2015). The CD results are usually expressed as mean
residue ellipticity (MRE) with deg cm2 dmol-1 as units according to the following
equation (Berova et al. 2000).
푂푏푠푒푟푣푒푑 퐶퐷 (푚 deg)
푀푅퐸 =
(5)
퐶 ×푛×푙×10
푝
where 퐶푝 is the molar concentration of the protein, 푛 is the number of amino acid
residues and 푙 is the path length. The α-helicity of free and combined HSA were
calculated from MRE values using the following equation
훼 − ℎ푒푙ꢁ푥 (%) = {−푀푅퐸 −2340} × 100
(6)
222
30300
where 푀푅퐸222 is the observed MRE value at 222 nm. In this experiment, 퐶푝 = 1 µM, 푛
is 585, 푙 is 1.0 mm. With the above equations and experimental data, the α-helix % were
calculated and given in Table 4.

24
0
-20
-40
(HSA)
(1a)
(1b)
(1c)
(1d)
(1e)
(1f)
-60
-80
(1g)
-100
200 210 220 230 240 250 260 270 280
Wavelength (nm)
Figure 6. CD spectra in the absence and presence of test compounds
with HSA (10 µM) at 1:1 ratio concentration
It was suggested that the formation of HSA-ligands complex perturbs the
secondary structural elements of HSA where the ligands bound with the amino acid
residues of the main poly peptide chain, which confirms the interaction of the ligands
with protein. Also, the observation of minor increase in ellipticity (~30 Mdeg) at 222
nm indicated that the protein structure remains predominantly helix and decrease in α-
helicity suggested that there were slight perturbations in the conformation of α-helices
of the protein during binding of these compounds (Wei et al., 2014).

25
Table 4. Change in helical percentage in HSA upon addition of compounds calculated
using the absorbance value from CD spectrum at 222 nm.
푶풃풔풆풓풗풆풅 푪푫 (풎 퐝퐞퐠)
휶 − 풉풆풍풊풙 (%)
49
System
-101.466
-88.1414
-69.4206
-76.1836
-77.6536
-78.7693
-79.0823
-71.8374
Free HSA
HSA + 1a
HSA + 1b
HSA + 1c
HSA + 1d
HSA + 1e
HSA + 1f
HSA + 1g
42
31
35
36
37.
37
33
Binding site prediction
The induced fit docking studies of HSA with test compounds were carried out
initially to validate the docking algorithm. In this study, both the protein and the
compounds are treated flexible so as to predict the binding site exactly with the
conformational changes of HSA. All the test compound structures were prepared by
ligand preparation wizard and the protein was refined by protein preparation wizard by
adding the missing side chains and removal of waters. Also, the cocrystallized ligand
propofol in PDB structure 5UJB was redocked to HSA by setting the grid box length
equal to 40Å in order to test the ability of the program to redetermine the docking site as
site III in HSA which was originally observed in HSA-propofol crystallized complex.

26
Quinoline amines
IB
IA
IIIB
IIIA
IIA
IIB
Trp214
Figure 9. Binding site of all the synthesized quinoline amines in HSA.

27
Table 5. IFD results showing the highly scored pose of the docked orientation of the
test compounds and the bonded amino acid residues in the binding site.
Docking score
ΔGexp
Glide energy
(kcal/mol)
Compounds
ΔGcalc
Interacting residues
(kcal/mol)
(kcal/mol)
-7.003
Lys432, Asp451,
Arg218
-8.063
-61.189
1a
-6.370
-6.755
-6.842
-4.265
-4.185
-4.554
-6.693
-7.727
-8.419
-7.733
-7.724
-7.089
-57.479
-58.113
-38.585
-48.173
-58.450
-53.588
Arg197
1b
1c
1d
1e
1f
Tyr 452
Tyr452, Val455
Trp214, Arg218
Lys190
Tyr452, Lys432
1g
ΔGexp = − RT ln Ka

28

29
Figure 8. Ligands + Binding sites created from Maestro depicting
the type of interactions with HSA
As expected the docking energy (-76.975 kcal/mol) and the docking score (-12.168
kcal/mol) was found to be higher and at the same time, the RMSD value between the
crystal structure complex and the redocked complex was found 0.209 which indicated
the parameter settings and the program ability to determine the binding site is fine. The
binding site was correctly located and the interactions between propofol and HSA were
similar as in crystal structure. Hence the same procedure was followed for the docking
of the compounds under investigation and the results obtained are tabulated in Table 5.
The negative docking score and glide energy indicated the binding of these compounds
with HSA. All the seven compounds were found to be docked in the cleft formed by the
interface of the domains IIA and IIIA (Figure 7). This is referred as the Iodipamide (a
radiopaque drug used to diagnose certain medical problems) binding site which was