Exploring 3-hydroxyflavone scaffolds as mushroom tyrosinase inhibitors: synthesis, X-ray crystallography, antimicrobial, fluorescence behaviour, structure-activity relationship and molecular modelling studies

Article abstract of DOI:10.1080/07391102.2020.1805364

To explore new scaffolds as tyrosinase enzyme inhibitors remain an interesting goal in the drug discovery and development. In due course and our approach to synthesize bioactive compounds, a series of varyingly substituted 3-hydroxyflavone derivatives (1-23) were synthesized in one-pot reaction and screened for in?vitro against mushroom tyrosinase enzyme. The structures of newly synthesized compounds were unambiguously corroborated by usual spectroscopic techniques (FTIR, UV-Vis, ¹H-, ¹³C-NMR) and mass spectrometry (EI-MS). The structure of compound 15 was also characterized by X-ray diffraction analysis. Furthermore, the synthesized compounds (1-23) were evaluated for their antimicrobial potential. Biological studies exhibit pretty good activity against most of the bacterial-fungal strains and their activity is comparable to those of commercially available antibiotics i.e. Cefixime and Clotrimazole. Amongst the series, the compounds 2, 4, 5, 6, 7, 10, 11, 14 and 22 exhibited excellent inhibitory activity against tyrosinase, even better than standard compound. Remarkably, the compound 2 (IC₅₀ = 0.280 ± 0.010 μg/ml) was found almost sixfold and derivative 5 (IC₅₀ = 0.230 ± 0.020 μg/ml) about sevenfold more active as compared to standard Kojic acid (IC₅₀ =1.79 ± 0.6 μg/ml). Moreover, these synthetic compounds (1-23) displayed good to moderate activities against tested bacterial and fungal strains. Their emission behavior was also investigated in order to know their potential as fluorescent probes. The molecular modelling simulations were also performed to explore their binding interactions with active sites of the tyrosinase enzyme. Limited structure-activity relationship was established to design and develop new tyrosinase inhibitors by employing 2-arylchromone as a structural core in the future. Communicated by Ramaswamy H. Sarma.

Full text of DOI:10.1080/07391102.2020.1805364

Journal of Biomolecular Structure and Dynamics
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20
Exploring 3-hydroxyflavone scaffolds as
mushroom tyrosinase inhibitors: synthesis, X-
ray crystallography, antimicrobial, fluorescence
behaviour, structure-activity relationship and
molecular modelling studies
Jamshaid Ashraf , Ehsan Ullah Mughal , Amina Sadiq , Maryam Bibi , Nafeesa
Naeem , Anser Ali , Anam Massadaq , Nighat Fatima , Asif Javid , Muhammad
Naveed Zafar , Bilal Ahmad Khan , Muhammad Faizan Nazar , Amara
Mumtaz , Muhammad Nawaz Tahir & Masoud Mirzaei
To cite this article: Jamshaid Ashraf , Ehsan Ullah Mughal , Amina Sadiq , Maryam Bibi ,
Nafeesa Naeem , Anser Ali , Anam Massadaq , Nighat Fatima , Asif Javid , Muhammad Naveed
Zafar , Bilal Ahmad Khan , Muhammad Faizan Nazar , Amara Mumtaz , Muhammad Nawaz
Tahir & Masoud Mirzaei (2020): Exploring 3-hydroxyflavone scaffolds as mushroom tyrosinase
inhibitors: synthesis, X-ray crystallography, antimicrobial, fluorescence behaviour, structure-activity
relationship and molecular modelling studies, Journal of Biomolecular Structure and Dynamics,
DOI: 10.1080/07391102.2020.1805364
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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
https://doi.org/10.1080/07391102.2020.1805364
Exploring 3-hydroxyflavone scaffolds as mushroom tyrosinase inhibitors:
synthesis, X-ray crystallography, antimicrobial, fluorescence behaviour,
structure-activity relationship and molecular modelling studies
Jamshaid Ashrafa, Ehsan Ullah Mughala, Amina Sadiqb, Maryam Bibia, Nafeesa Naeema, Anser Alic, Anam
Massadaqc, Nighat Fatimad, Asif Javida, Muhammad Naveed Zafare, Bilal Ahmad Khanf, Muhammad Faizan
Nazara, Amara Mumtazg, Muhammad Nawaz Tahirh and Masoud Mirzaeii
b
aDepartment of Chemistry, University of Gujrat, Gujrat, Pakistan; Department of Chemistry, Govt. College Women University, Sialkot,
c
d
Pakistan; Department of Zoology, Mirpur University of Science and Technology, Mirpur, Pakistan; Department of Pharmacy, COMSATS
e
f
University Islamabad, Abbotabad, Pakistan; Department of Chemistry, Quaid-i-Azam University, Islamabad, Pakistan; Department of
Chemistry, University of Azad Jammu and Kashmir, Muzaffarabad, Pakistan; Department of Chemistry, COMSATS University Islamabad,
Abbottabad, Pakistan; Department of Physics, University of Sargodha, Sargodha, Pakistan; Department of Chemistry, Faculty of Science,
Ferdowsi University of Mashhad, Mashhad, Iran
g
h
i
Communicated by Ramaswamy H. Sarma
ABSTRACT
ARTICLE HISTORY
Received 28 June 2020
Accepted 30 July 2020
To explore new scaffolds as tyrosinase enzyme inhibitors remain an interesting goal in the drug dis-
covery and development. In due course and our approach to synthesize bioactive compounds, a series
of varyingly substituted 3-hydroxyflavone derivatives (1-23) were synthesized in one-pot reaction and
screened for in vitro against mushroom tyrosinase enzyme. The structures of newly synthesized com-
pounds were unambiguously corroborated by usual spectroscopic techniques (FTIR, UV-Vis, 1H-, 13C-
NMR) and mass spectrometry (EI-MS). The structure of compound 15 was also characterized by X-ray
diffraction analysis. Furthermore, the synthesized compounds (1-23) were evaluated for their anti-
microbial potential. Biological studies exhibit pretty good activity against most of the bacterial-fungal
strains and their activity is comparable to those of commercially available antibiotics i.e. Cefixime and
Clotrimazole. Amongst the series, the compounds 2, 4, 5, 6, 7, 10, 11, 14 and 22 exhibited excellent
inhibitory activity against tyrosinase, even better than standard compound. Remarkably, the com-
KEYWORDS
3-hydroxyflavones; Algar-
Flynn-Oyamada reaction;
antibacterial; antifungal;
tyrosinase enzyme
inhibition; molecular
docking studies
pound
2
(IC50
¼
0.280 0.010 lg/ml) was found almost sixfold and derivative
5
(IC50
¼
0.230 0.020 lg/ml) about sevenfold more active as compared to standard Kojic acid (IC50
¼1.79 0.6 lg/ml). Moreover, these synthetic compounds (1-23) displayed good to moderate activities
against tested bacterial and fungal strains. Their emission behavior was also investigated in order to
know their potential as fluorescent probes. The molecular modelling simulations were also performed
to explore their binding interactions with active sites of the tyrosinase enzyme. Limited structure-activ-
ity relationship was established to design and develop new tyrosinase inhibitors by employing 2-aryl-
chromone as a structural core in the future.
Abbreviations: AFO: Algar-Flynn-Oyamada; Ala: Alanine; Asn: Asparagine; CA: Candida albicans; CP:
Candida parapsilosis; DMSO: Dimethyl sulfoxide; E.coli: Escherichia coli; EI-MS: Electron impact mass
spectrometry; FTIR: Fourier transformed infrared; Glu: Glutamic acid; His: Histidine; H2O2: Hydrogen per-
oxide; MMFF: Merck molecular force field; NMR: Nuclear magnetic resonance; PA: Pseudomonas agari-
cus; Phe: Phenylalanine; PDB: Protein data bank; SA: Staphylococcus aureus; TLC: Thin-layer
chromatography; UV-Vis: Ultraviolet-visible spectroscopy; XRD: X-ray diffraction
Introduction
as the compounds possessing the C6-C3-C6 carbon frame-
work or more specifically a phenylbenzopyran skeleton, as
core structure (Figure 1) (Aherne & O’Brien, 2002; Lopez-
Lazaro, 2002; Tsao, 2010).
3-Hydroxyflavones or simply flavonols (Figure 1), also
known as 3-hydroxy-2-phenyl-4H-1-benzopyran-4-ones, are
the sub-category of the family of flavonoids that are ubiqui-
tously found in the plants, fruits, and vegetables, etc. (Strack
Flavonoids belong to a class of naturally occurring poly-
phenolic compounds which are widely distributed in the vas-
cular plant kingdom as secondary metabolites (Benavente-
ꢀ
Garcıa et al., 1997; Farhadi et al., 2019; Hollman & Katan,
1999; Kumar & Pandey, 2013; Middleton, 1996; Seleem et al.,
2017; Tschesche et al., 1979). Generally, they are recognized
CONTACT Ehsan Ullah Mughal
anser.zoology@must.edu.pk
ehsan.ullah@uog.edu.pk
Department of Chemistry, University of Gujrat, Gujrat, 50700, Pakistan; Anser Ali
Department of Zoology, Mirpur University of Science and Technology, Mirpur, 10250, AJK, Pakistan; Masoud Mirzaei
mirzaeesh@um.ac.ir
Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran.
Supplemental data for this article can be accessed online at https://doi.org/10.1080/07391102.2020.1805364.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
J. ASHRAF ET AL.
Figure 1. General structure of flavonoids (A) and 3-Hydroxyflavone (Flavonol) (B).
et al., 1994). Over the past decades, they have attained compounds, some of the flavonoid derivatives such as flavo-
increasing attention on account of their immense biological nols are considered to be the potent inhibitors of tyrosinase.
activities and incredible health benefits (Alluis et al., 2000; There is a significant correlation between the inhibitory
Walle et al., 2003; Yoshida et al., 2009). For example, they are potency of flavonols on mushroom tyrosinase and melanin
effective against cardiovascular diseases, inflammation synthesis in melanocytes (Promden et al., 2018).
ꢀ
(Garcıa-Lafuente et al., 2009), rheumatoid arthritis, diabetes,
In the past decade, many natural and synthetic tyrosinase
and asthma (Haslam, 1998; Rao et al., 2016). Recently, inhibitors have been evaluated, with many reported to also
researchers have reported a number of significant pharmaco- possess intrinsic antibacterial activity. Further, the enzyme
logical activities of these compounds (Cody et al., 1986; product melanin has been shown to compromise the activity
Hussin et al., 2009; Manthey et al., 2001; Ren et al., 2003; of traditional antibiotics. Due to the antibiotic resistance cri-
Rice-Evans et al., 1995). Additionally, they have been a sub- sis and the slow development of new antibiotics, tyrosinase
ject of interest because of their ability to display a wide inhibitors may have the potential for the development of
range of enzyme-modulatory properties against various novel antimicrobials or antibiotic adjuvants that enhance the
enzymes (Agullo et al., 1997; Akiyama et al., 1987; Cushman activity of incumbent drugs (Yuan et al., 2020). Recently,
et al., 1991; Ferriola et al., 1989; Gamet-Payrastre et al., 1999; tyrosinase inhibitors have been also evaluated for their anti-
Linassier et al., 1990; Liu et al., 2013; Mughal et al., 2019). bacterial activity, and some of them exhibit better potency
Both natural and synthetic flavones have been shown to compared with current antibiotics (Xia et al., 2014). It has
exhibit substantial biological activities including antimicrobial been found that bacterial melanin can protect bacteria from
and enzymes inhibition (Verma & Pratap, 2010).
UV radiation thereby increasing cell viability in the long term
Tyrosinase inhibitors have attracted the attention of medi- (Geng et al., 2008). In addition to protecting bacteria from
cinal chemists because of their ability to inhibit the synthesis UV radiation, melanin also chelates metals, increasing bacter-
of melanin pigment (Chen & Kubo, 2002; Maeda & Fukuda, ial survival under environmental stress (Garcia-Rivera &
1996). Their potential to decrease pigmentation gives them Casadevall, 2001; Nosanchuk & Casadevall, 2003). More
amazing applications in various fields, including the food importantly, melanin can neutralize antibiotics, increasing the
industry (Tsuji-Naito et al., 2007), cosmetics (Nihei & Kubo, inhibitory dose of antibiotics, and improving the viability
2003) and medicines (Maeda & Yoshizaki, 1991). Melanin is a of bacteria.
protein that has great importance for skin and hair color. It
In searching for promising mushroom tyrosinase inhibi-
also possesses photo screening effects, provides skin photo- tors, many flavonols derivatives have been isolated and eval-
protection, and prevents skin injury. Despite its advantages, uated for their inhibitory activity on mushroom tyrosinase
pigmentations can cause skin problems such as freckles, age from different natural sources. However, their isolation pro-
spots, and melanoma. Therefore, control of melanin synthesis cedure is lengthy, time-consuming, expensive, and boring as
could be an effective strategy to control pigmentation well. Also, natural sources are insufficient to meet the
related disorders. Tyrosinase is known as a rate limiting or requirement all over the world.
the key enzyme for melanin synthesis. It catalyzes the
Thus, laboratory synthesis may be considered as a substi-
hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine tute to the natural sources, in addition, it may also provide
(L-DOPA) and L-DOPA to dopaquinones (Cooksey et al., these compounds with structural variety.
1997) which cause abnormal melanin accumulation under
To the best of our knowledge, there is no much literature
€
unregulated conditions (Fairhead
& Thony-Meyer, 2012; available on the synthetic flavonols as competitive mush-
ꢀ
Hearing & Jimenez, 1989). Thus, tyrosinase inhibition is the room tyrosinase inhibitors except few examples (Kim et al.,
simplest and promising approach and tyrosinase inhibitors 2006; Promden et al., 2018; Qin et al., 2015; Zolghadri et al.,
can be used for the prevention of conditions related to the 2019). Furthermore, among the tyrosinase inhibitors, car-
hyperpigmentation of the skin, such as melasma and age bonyl-based compounds including aurones, chalcones, flava-
spots. In this context, a number of both natural and syn- nones, and pyrazole derivatives have gained attention owing
thetic polyphenols including flavonoids, phenolic acids, stil- to their interaction with the hydrophobic protein pocket
benes, and lignans have been reported as weak or potent nearby the binuclear copper active site of tyrosinase (Qin
tyrosinase inhibitors (Chang, 2009; Kim et al., 2006; Promden et al., 2015).
€
ꢁ
et al., 2018; Qin et al., 2015; S¸ohretoglu et al., 2018;
Encouraged by the potential pharmacological applications
Zolghadri et al., 2019). Among these polyphenolic of flavonols and promising tyrosinase inhibitory potential of

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
3
related structures, we have been motivated to design and
General procedure for the synthesis of 3-hydroxyflavone
synthesize these novel compounds as potent mushroom derivatives (1-23)
tyrosinase inhibitors, and by keeping in mind the enhanced
A mixture of o-hydroxyacetophenone (1.2mL, 10.0mmol) in
resistance of pathogens against antibiotics, it’s also necessary
to find new structurally varied flavonol-based drugs as antibi-
otics. Moreover, it is also desirous to study the inhibitory
effect of various substituents at different positions and ana-
lyse how these compounds bind with tyrosinase protein at a
molecular level. The experimental results were also verified
by molecular docking studies.
aqueous 30% sodium hydroxide solution was stirred in metha-
nol (20.0 mL) for 30min at ambient temperature followed by
the addition of substituted benzaldehyde (10.0 mmol). This
reaction mixture was allowed to stir for further 4.0 h at the same
temperature. The progress of the reaction was monitored by
comparative TLC. After the completion of the reaction (checked
by TLC), 20-Hydroxychalcone, formed in situ, was further oxida-
tively cyclized by adding 1.5 mL of 35% H2O2 to the same reac-
tion mixture which was then allowed to stir for additional 1.0 h.
Afterward, 10% aqueous HCl was added to the reaction mixture
in order to neutralize it, and as a result flavonols (3-hydroxyfla-
vones) were precipitated out. These precipitates were then fil-
tered, washed with water and subsequently with cold ethanol.
Residue obtained was collected, dried, and finally recrystallized
by ethanol (Mughal et al., 2019).
Materials and methods
All the chemicals were purchased from Merck (Germany) and
Sigma-Aldrich (USA) and used as delivered. Melting points
were measured on an Electrothermal melting point appar-
atus and are uncorrected. The IR spectra were recorded on a
Bio-Rad spectrophotometer. NMR spectra were measured on
a Bruker DRX 300 instrument (1H, 500 MHz, 13C, 125 MHz).
Accurate mass measurements were carried out with the
Fisons VG sector-field instrument (EI) and a FT-ICR mass spec-
trometer. The IR values are mentioned in ῡ units and NMR
chemical shift values were determined in ppm units.
Absorption spectra were recorded in very dilute solutions
prepared in different solvents such as Acetonitrile, Methanol,
DMSO on the Jasco UV-VIS V-670 instrument by using
QUARTZ cell. Fluorescence spectra were recorded using
Shimadzu 8101AFT-IR. Bruker Smart APEX-II CCD diffractom-
eter was used for single crystal XRD studies.
Biological evaluation studies
Antifungal activity
Antifungal activities of all the synthesized compounds were
determined by employing known “Well Diffusion Method”.
Two fungal strains were used one was Candida parapsilosis
(CP) (ATCC#22019) and the second one was the Candida albi-
cans (CA) (ATCC#9002). The standard drug used was
Clotrimazole, 1.0 mg of the Clotrimazole taken in 1.0 mL of
DMSO. In mm zone of inhibition was measured and this
zone of inhibition was the antifungal potential. The amount
of DMSO taken to prepare the sample was 1 mL and the
3.0 mg of the sample dissolved in this DMSO SDA plates
were used to spread the sample. These are pre-prepared
plates made up of Sabouraud dextrose agar. The spreading
of the sample on SDA plates was assisted by using a sterile
glass rod. The depth of the well prepared by using the sterile
cork bore and it was 8 mm and all of these wells were at the
appropriated distance from each other. Sample taken for
Experimental X-ray diffraction details
X-ray data were collected at 296 K on a Bruker Kappa APEX II
CCD diffractometer equipped with a graphite monochrom-
ator and a sealed molybdenum tube (k ¼ 0.71073 Å). The raw analysis was 80 mL, samples along with standards were
data were converted to F2 values with SAINT (Bruker, 2016)
poured on their appropriate wells. 24–46 h were utilized and
the prepared plates were incubated. The antifungal activity
recorded in mm units and it was calculated by the measur-
ing zone of inhibition (Mughal, Sadiq, et al., 2018).
while multiple measurements of equivalent reflections pro-
vided the basis for an empirical absorption correction as well
as a correction for any crystal deterioration during the data
collection (SADABS; Bruker, 2016). The structure was solved
by direct methods and refined by full-matrix, least ꢀ squares
procedures using the SHELXTL program package (Bruker,
2016). Hydrogen atoms were included as riding contributions
in idealized positions with isotropic displacement parameters
tied to those of the attached atoms. Satisfactory hydrogen
bond parameters were achieved with this model. The
PLATON software (Spek, 2015) was used to calculate bond
distances, bond angles, torsion angles, hydrogen bonds, and
other geometric parameters. The Mercury 3.7 software
(Macrae et al., 2006) was used to generate the figures and to
perform other calculations (Bruker, 2016; Macrae et al., 2006;
Spek, 2009, 2015). The crystal data and refinement of the
given in Table S1.
Antibacterial activity
Antibacterial activity against different strains of bacteria was
measured. The method used for measuring the antibacterial
potential was the disc diffusion method (Mughal, Sadiq,
et al., 2018). The bacterial strains used were Escherichia coli
(ATCC#25922), Pseudomonas aeruginosa (ATCC#9721), and
Staphylococcus aureus (ATCC#6538). The zone of inhibition
was measured as the antibacterial potential of the synthe-
sized compounds. Two controls were used one is positive
Cefixime and the other is negative DMSO. Different samples
of the compounds were prepared by dissolving 4 mg of the
sample into 1 mL of the DMSO. The lawn created on nutrient
agar plates by using bacterial strains having equivalent

4
J. ASHRAF ET AL.
Scheme 1. Synthesis of varyingly substituted 3-hydroxyflavone derivatives (1-23).
turbidity that is attained by using 0.5% of McFarland solu- expressed as the potential of samples against used bacter-
tion. The depth of the well was 8 mm and they were all
made at the appropriate distances from each other. The sam-
ple and the standard were poured into their appropriate
wells. The amount of sample used was 80 mL in a well and
along with the two control. At 37 ꢁC the prepared plates
were incubated for 24 h and zone of inhibition usually meas-
ial strains.
Enzyme inhibition activity
Mushroom anti-tyrosinase assay. The mushroom tyrosinase
enzyme was used to access in vitro anti-tyrosinase activity of
ured in mm and diameter of the zone of inhibition was the test compounds as described previously with some

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
5
Scheme 1. (Continued).

6
J. ASHRAF ET AL.
Figure 2. ORTEP diagram of the compound 15 showing 30% probability thermal ellipsoids and the atom-numbering scheme.
modifications (Ali et al., 2016; Seo et al., 2010). Briefly, 140 lL benzaldehydes in methanolic-sodium hydroxide solution to
of phosphate buffer (20 mM, pH 6.8), 20 lL of mushroom produce 20-hydroxychalcones as key intermediates. 20-
tyrosinase (30 U/ml), and 20 lL of test compound were mixed Hydroxychalcones, generated in situ, were then cyclized into
in the wells of a 96-well plate. After 10 min incubation at flavonols (1-23) through oxidative cyclization using 35%
room temperature, 20 lL (0.85 mM) of 3,4-dihydroxyphenyla- hydrogen peroxide (H2O2) under basic medium and same
lanine (L-DOPA) was added. The plate was incubated at solvent. All the synthesized target compounds (1-23) were
25 ꢁC for 20 min in dark. Later, the absorbance of dopa-
purified by recrystallization in ethanol and obtained in mod-
chrome was monitored at 400 nm using a plate reader (Bio
Tek ELX 808). Percentage inhibition was calculated by the
following formula:
erate to excellent yields, and are soluble in common solvents
for instance acetone, acetonitrile, methanol, chloroform, and
DMSO, etc. Subsequently, all the newly synthesized com-
pounds were characterized by FTIR, UV-Vis, and NMR spectro-
scopic techniques. In addition, their accurate molecular
masses were determined by electron ionization (EI) mass
spectrometry. All the spectral data unequivocally confirm the
structures of the newly synthesized molecules.
Furthermore, the single-crystal for the compound 15 was
grown through recrystallization in ethanol, and its molecular
structure was determined by single-crystal X-ray analysis
(Altaf et al., 2017; Bruker, 2016; Lal et al., 2016; Macrae et al.,
2006; Saleem et al., 2018; Spek, 2009, 2015) and is displayed
in Figure 2 and other details are given in supporting
information.
Inhibition ð%Þ ¼ ½ðBꢀꢀaÞ=Bꢂ ꢃ 100
where, A ¼ absorbance of enzyme with the test compound
and, B ¼ absorbance of the enzyme without test compound.
Later, the concentration of test compounds necessary to
achieve 50% tyrosinase inhibition (IC50) was determined by
the data analysis and graphing software, Origin.
Molecular docking studies
In order to carry out the docking studies, various tools are
available for the study of protein-ligand associations,
Autodock 1.5.6 was applied for their automation capability
(Ehsan U Mughal, Sadiq, et al., 2018). The crystal structure of
Agaricus bisporus tyrosinase (PDB ID: 2Y9X) was obtained,
from RCSB protein data bank (PDB), as a model. 3D grids of
tropolone in the binding pocket of tyrosinase was estimated
by using discovery studio 4.0. The protein structure was opti-
mized and side-chain hydrogens were added. Total Koll man
charges (ꢀ820.404) were added to the tyrosinase targeted
biological molecule.
The spectroscopic data of all the newly synthesized com-
pounds 2, 4, 11, 13, 14, 15, and 20 are given below.
However, the spectral data of the remaining compounds are
given in the literature (Bansal & Kaur, 2015; Ehsan et al.,
2016; Golub et al., 2011; Gunduz et al., 2012; Gupta et al.,
€
2012, 2014; Hofener et al., 2013; Kaur et al., 2017; Khanna
et al., 2015; Klymchenko et al., 2001; Mughal, Javid, et al.,
2018; Mughal et al., 2019; Nhu et al., 2015; Singh et al., 2017;
You et al., 2020).
Results and discussion
2-(3-(trifluoromethyl)phenyl)-3-hydroxy-4H-chromen-4-
one (2)
Chemistry
ꢁ
Bright-yellow crystalline solid; Yield: 76%; m.p. 178–180 C; Rf
3-Hydroxyflavones (1-23) were synthesized starting from 20- (Ethyl acetate: n-hexane 1:3) ¼ 0.8; UV-Vis kmax (Methanol) 5
hydroxyacetophenone and differently substituted benzalde- 255, 370 nm; FTIR (cmꢀ1): 3096, 1607, 1483, 1318, 1209, 1111,
1
hydes in one-pot synthesis through Algar-Flynn-Oyamada 1085, 964; H NMR (300 MHz, DMSO-d6): d 10.00 (bs, 1H, OH),
(AFO) reaction (Scheme 1). 20-Hydroxyacetophenone under- 8.57 (s, 1H, Ar–H), 8.51 (d, 1H, J ¼ 6.0 Hz, Ar–H), 8.14–8.10 (m,
went
Claisen-Schmidt
condensation
with
various 1H, Ar–H), 7.89–7.80 (m, 4H, Ar–H), 7.52–7.46 (m, 1H, Ar–H);

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
7
13C NMR (75 MHz, DMSO-d6): d 173.0, 154.8, 143.4, 140.1, 120.9, 120.4, 118.4, 117.1, 115.6, 114.2, 113.1, 21.3; accurate
134.3, 132.7, 131.4, 130.2, 130.0, 129.4, 126.4, 125.2, 125.0, mass (EI-MS) of [M]þꢄ: Calcd. for C24H17NO5S 431.08274;
124.5, 122.0, 118.8; accurate mass (EI-MS) of [M]þꢄ: Calcd. for found 431.08265.
C16H9F3O3 306.05037; found 306.05025.
2-(3-bromophenyl)-3-hydroxy-6-methyl-4H-chromen-4-
2-(2-(trifluoromethyl)phenyl)-3-hydroxy-4H-chromen-4-
one (4)
one (15)
Mustard-yellow crystalline solid; Yield: 87%; m.p. 179–181 ꢁC;
Dull-yellow solid; Yield: 70%; m.p. 187–189 ꢁC; Rf (Ethyl acet- Rf (Ethyl acetate: n-hexane 1:3)
¼
0.6; UV-Vis kmax
(Acetonitrile) 5 241, 332 nm; FTIR (cmꢀ1): 3228, 2115, 1604,
ate: n-hexane 1:3) ¼ 0.6; UV-Vis kmax (Methanol) 5 273,
320 nm; FTIR (cmꢀ1): 3297, 1688, 1443, 1367, 1285, 1243,
1153, 1103, 1034, 844; 1H NMR (300 MHz, DMSO-d6): d 9.98
(bs, 1H, OH), 8.70–8.50 (m, 2H, Ar–H), 7.85–7.65 (m, 4H,
Ar–H), 7.60–7.49 (m, 2H, Ar–H); 13C NMR (75 MHz, DMSO-d6):
d 173.1, 154.7, 143.6, 140.2, 135.0, 132.8, 131.1, 131.5, 130.1,
129.6, 127.0, 125.5, 125.1, 124.4, 122.1, 118.7; accurate mass
(EI-MS) of [M]þꢄ: Calcd. for C16H9F3O3 306.05037;
found 306.05021.
1
1568, 1554, 1487, 1463, 1282, 1137, 1093; H NMR (500 MHz,
CD2Cl2): d 9.67 (s, 1H, OH), 7.95 (s, 1H, Ar–H), 7.86 (s, 1H,
Ar–H), 7.78 (d, J ¼ 10.0 Hz, 1H, Ar–H), 7.59 (d, J ¼ 10.0 Hz, 1H,
Ar–H), 7.40–7.37 (m, 1H, Ar–H), 7.27–7.25 (m, 1H, Ar–H),
7.10–7.07 (m, 1H, Ar–H); 13C NMR (125 MHz, DMSO-d6): d
173.4, 153.4, 143.5, 140.0, 135.7, 134.6, 134.1, 132.8, 131.1,
130.3, 126.8, 124.3, 122.3, 121.4, 118.8, 20.8; accurate mass
(EI-MS) of [M]þꢄ: Calcd. for C16H11BrO3 329.98915;
found 329.98902.
2-(4-(diphenylamino)
one (11)
phenyl)-3-hydroxy-4H-chromen-4-
2-(3-bromophenyl)-3-hydroxy-6-chloro-4H-chromen-4-
one (20)
Mustard-yellow crystalline solid; Yield: 90%; m.p. 188–190 ꢁC;
Rf (Ethyl acetate: n-hexane 1:3) ¼ 0.7; UV-Vis kmax (Methanol)
5 293, 393 nm; FTIR (cmꢀ1): 3221, 1686, 1481, 1410, 1329,
Dark-yellow solid, Yield: 80%; m.p. 189–191 ꢁC; Rf (Ethyl acet-
ate: n-hexane 1:3) ¼ 0.8; UV-Vis kmax (Acetonitrile) 5 244,
334 nm; FTIR (cmꢀ1): 3246, 3055, 2902, 1625, 1606, 1567,
1
1154, 1134, 824; H NMR (300 MHz, CDCl3): d 9.74 (s, 1H, OH),
1
1387, 1259, 1210, 1139; H NMR (500 MHz, DMSO-d6): d 9.87
7.63–7.58 (m, 2H, Ar–H), 7.30–7.24 (m, 6H, Ar–H), 7.12–7.07
(m, 8H, Ar–H), 6.96 (d, J ¼ 9.0 Hz, 2H, Ar–H), 13C NMR
(75 MHz, CDCl3): d 181.5, 153.4, 146.1, 131.4, 129.7, 129.3,
126.3, 126.2, 125.0, 118.3, the other carbons are isochronous;
accurate mass (EI-MS) of [M]þꢄ: Calcd. for C27H19NO3
405.13649; found 405.13640.
(s, 1H, OH), 8.40 (s, 1H, Ar–H), 8.22 (d, J ¼ 5.0 Hz, 1H, Ar–H),
7.90 (s, 1H, Ar–H), 7.71 (d, J ¼ 10.0 Hz, 2H, Ar–H), 7.64, (dd,
J ¼ 10.0, 5.0 Hz, 1H, Ar–H), 7.54 (t, J ¼ 10.0 Hz, 1H, Ar–H), 2.45
(s, 3H, CH3); 13C NMR (125 MHz, CD2Cl2): d 172.5, 159.0, 139.0,
137.4, 135.5, 133.6, 132.2, 131.7, 130.4, 129.8, 128.3, 127.8,
126.5, 121.4, 120.1; accurate mass (EI-MS) of [M]þꢄ: Calcd. for
C15H8BrClO3 349.93453; found 349.93461.
2-(naphthalen-1-yl)-3-hydroxy-4H-chromen-4-one (13)
Light-yellow solid; Yield: 80%; m.p. 177–179 ꢁC; Rf (Ethyl acet-
ate: n-hexane 1:3) ¼ 0.8; UV-Vis kmax (Methanol) 5 251,
330 nm; FTIR (cmꢀ1): 3051, 1603, 1461, 1416, 1342, 1142, 670,
UV-visible spectroscopic analysis
The UV-visible analysis was carried out mostly in methanol.
In general, flavonols show two maxima in the range of
250–285 nm and 320–385 nm corresponding to benzoyl ring
(Ring A) and cinnamoyl ring (Ring B) respectively. The UV-vis
spectra of some of all the newly synthesized compounds
have been displayed in supporting information (SI).
1
571; H NMR (300 MHz, DMSO-d6): d 8.24–8.14 (m, 1H, Ar–H),
8.00 (dd, J ¼ 9.0, 3.0 Hz, 1H, Ar–H), 7.89–7.80 (m, 3H, Ar–H),
7.68 (dd, J ¼ 9.0, 3.0 Hz, 1H, Ar–H), 7.57 (ddd, J ¼ 9.0, 6.0,
3.0 Hz, 1H, Ar–H), 7.52–7.48 (m, 2H, Ar–H), 7.45–7.38 (m, 2H,
Ar–H); 13C NMR (75 MHz, DMSO-d6): d 173.4, 168.0, 158.7,
154.2, 140.3, 136.1, 133.4, 130.5, 127.8, 127.0, 124.7, 118.2;
accurate mass (EI-MS) of [M]þꢄ: Calcd. for C19H12O3
288.07864; found 288.07870.
Fluorescence behavior
Emission wavelengths were recorded for the synthesized 3-
hydroxyflavone derivatives (1-23) mostly in methanol and
acetonitrile solvents. The results are listed in Table 1.
3-Hydroxyflavone derivatives have great potential as fluor-
escence probes for bio-labelling in aqueous medium (Ehsan
et al., 2016; Gupta et al., 2012, 2014; Mughal, Javid, et al.,
2018; Singh et al., 2017). This property comes from its well-
separated dual emission bands in fluorescence spectra, origi-
2-(1-tosyl-1H-indol-3-yl)-3-hydroxy-4H-chromen-4-one (14)
Dirty-yellow crystalline solid, Yield: 78%; m.p. 244–246 ꢁC; Rf
(Ethyl acetate: n-hexane 1:3) ¼ 0.7; UV-Vis kmax (Methanol) 5
254, 297 nm; FTIR (cmꢀ1): 3356, 2190, 1656, 1447, 1394, 1161,
1130, 692; 1H NMR (300 MHz, DMSO-d6): d 9.89 (s, 1H, OH),
8.45 (s, 1H, Ar–H), 8.26 (s, 1H, Ar–H), 8.12–7.95 (m, 2H, Ar–H),
7.74 (d, J ¼ 9.0 Hz, 1H, Ar–H), 7.52 (d, J ¼ 9.0 Hz, 2H, Ar–H),
ꢅ
ꢅ
7.31–7.18 (m, 1H, Ar–H), 7.14 (d, J ¼ 9.0 Hz, 2H, Ar–H), nated from normal (N ) and photo tautomer forms (T ) of an
6.97–6.87 (m, 1H, Ar–H), 6.53 (d J ¼ 9.0 Hz, 1H, Ar–H), 6.36 excited state intramolecular proton transfer reaction (Figure 3).
(t, J ¼ 9.0 Hz, 1H, Ar–H), 2.28 (s, 3H, CH3); 13C NMR (75 MHz, The fluorescent behavior of these compounds has been
DMSO-d6): d 171.2, 167.7, 154.1, 150.5, 145.4, 141.0, 139.5, studied in various organic solvents using emission spectros-
138.5, 132.2, 131.8, 130.3, 128.6, 125.9, 124.6, 123.7, 121.1, copy, performed at room temperature. The synthesized

8
J. ASHRAF ET AL.
Table 1. Fluorescence analysis results.
compound 10 (Table 1). From the emission spectra of the
reported compounds, it can be concluded that the introduc-
tion of electron-donating groups at ring B significantly affects
the emission intensity without causing any major blue or red-
shift in the emission wavelength, and electron-withdrawing
groups offer reverse effect. The emission spectra of all the
compounds (1-23) have been shown in SI.
Compound
Number
Excitation Wavelength
Emission Wavelength
Values (nm) (Solvent)
Values (nm)
1
386
403, 531
(Methanol)
402, 540
(Methanol)
417, 533
(Methanol)
404, 546
(Methanol)
400, 538
(Methanol)
402, 537
(Methanol)
409, 529
(Methanol)
473, 602
(Methanol)
412, 532
(Methanol)
390, 698
(Methanol)
426, 541
(Methanol)
441, 591
(Methanol)
481
(Methanol)
423, 582
(Methanol)
411, 546
(Methanol)
470, 537
(Methanol)
2
380
395
330
363
365
373
373
355
370
403
371
340
307
342
335
362
358
372
344
336
320
380
3
4
Antimicrobial evaluation
5
6
The growing resistance of microorganisms to standard antibi-
otics encourages the search for novel antimicrobial agents.
In this context, the antibacterial activities of all the synthe-
sized compounds (1-23) were tested against a panel of one
Gram-positive Bacterium Staphylococcus aureus (SA,
ATCC#6538) and two Gram-negative Bacteria Escherichia coli
(E-coli, ATCC# 25922) and Pseudomonas aeruginosa (PA,
ATCC#9721) using conventional agar well diffusion method.
Similarly, anti-fungal activities of the same compounds were
evaluated against two fungi Candida albicans (CA,
ATCC#9002) and Candida parapsilosis (CP, ATCC# 22019)
using the previously mentioned method. Cefixime and
Clotrimazole were used as standard drugs. The results were
recorded for each tested compound as the average diameter
of inhibition zones (IZ) of bacterial or fungal growth around
the discs in mm. The antibacterial and antifungal results are
displayed in Tables 2 and 3 respectively.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
530
(Acetonitrile)
404, 536
(Acetonitrile)
402, 537
(Acetonitrile)
428, 535
(Acetonitrile)
549
(Acetonitrile)
528
(Acetonitrile)
Antibacterial activity
The antibacterial assay revealed that these compounds are
relatively more active against P. aeruginosa and E. coli than S.
aureus. Noteworthy, simple flavonol 1 is least active amongst
the series. However, by introducing various substituents on
flavonol scaffold led to an increased antibacterial activity.
This finding most probably resulted from the high hydropho-
bicity of such compounds. It is obvious that the compounds
4, 14, 19, 20, 21, 22 and 23, which contain mostly electron-
withdrawing groups, exhibited the highest activity against
both P. aeruginosa and E.coli, whereas the compounds 3, 15,
17 and 18 showed the highest activity against P. aeruginosa
(Table 2). Their activities were found to be comparable with
that of standard drug tested (Cefixime). In comparison, the
envisioned derivatives are more active against P. aeruginosa
than E. coli. Moreover, the other compounds among the ser-
ies manifested moderate to good activities against the tested
bacterial strains.
541
(Acetonitrile)
Figure 3. Conversion of simple flavonol (1) from normal electronic state to
excited electronic state.
compounds, when irradiated with ultraviolet radiation, were
observed to have fluorescence properties in the blue portion
of the visible spectrum. In comparison, emission intensities
showed that the alkyl groups have a surprising effect on the
emission strength of 3-hydroxyflavone derivatives. The alkyl
substitution at the aryl group (ring B) can be expected to have
some effect on the physico-chemical properties due to its elec-
tron-donating nature and if we replaced the 2-phenyl group of
3-hydroxyflavone with any heterocyclic compound having
extended conjugation, the product exhibited better fluores-
Antifungal activity
The lesser number of available harmless antifungal agents is
the driving force to search for new candidates. The promis-
ing antibacterial activity of the tested compounds has
encouraged us to test these compounds against two differ-
ent pathogenic fungi. In general, all the compounds
screened exhibited moderate to good activity against both
C. albicans and C. parapsilosis fungi (Table 3). However, these
derivatives were found comparatively more active against C.
cence properties than 3-hydroxyflavone, for example, the parapsilosis than C. albicans. For instance, amongst the series,

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
9
Table 2. Potential of 3-hydroxyflavone derivatives (1-23) against bacter- Table 4. Tyrosinase inhibitory efficacy of synthesized derivatives (1-23).
ial strains.
Compounds
Mushroom Tyrosinase Inhibition IC50 SEMa (mg/ml)
Zones of Inhibition (mm)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
4.268 0.182
0.280 0.010
n.d
0.30 0.0
Compound Number
PA
SA
E-COLI
ꢅ
ꢅ
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
7.2 0.2
14.2 0.7
20.3 0.5
22.2 0.5
16.6 0.6
14.2 0.6
15.4 0.6
14.1 0.5
17.2 0.4
16.2 0.3
18.7 0.2
14.1 0.5
18.1 0.2
25.1 0.4
24.1 0.4
9.4 0.2
21.1 0.2
24.2 0.3
25.2 0.3
19.2 0.1
25.4 0.6
25.2 0.6
NA
NA
9.6 0.6
14.6 0.7
12.7 0.6
20.5 0.2
16.8 0.6
14.7 0.8
14.5 0.7
14.7 0.8
16.7 0.4
20.5 0.2
18.5 0.7
16.5 0.6
19.1 0.4
26.1 0.3
24.5 0.4
19.1 0.3
19 0.1
16.6 0.7
23.5 0.4
19.4 0.6
16.6 0.3
18.7 0.3
NA
0.230 0.020
0.284 0.018
1.787 0.445
14.171 0.855
2.564 0.251
0.347 0.078
0.327 0.041
NA
NA
NA
20.7 0.
NA
11.1 0.1
NA
19.2 0.2
NA
ꢅ
n.d
2.90 0.032
0.370 0.025
13.912 3.339
19.595 0.515
4.844 0.347
4.437 0.032
35.189 2.384
5.783 0.642
7.226 0.589
0.799 0.008
2.236 0.008
1.79 0.6
17
18
19
20
21
22
NA
20.2 0.3
23.2 0.1
NA
NA
21.0 0.5
NA
NA
27.5 0.7
18 0.3
20 0.2
22.5 0.5
26.5 0.3
27.5 0.6
24.1 0.4
NA
23
Kojic Acidb
aIC50 values (mean standard error of the mean);
bStandard inhibitor for mushroom tyrosinase;
DMSO
Cefixime (1 mg/ml)
NA
28.7 0.3
ꢅ
n.d ¼ not determined.
30.5 0.3
ꢅ
NA ¼ No Activity.
Mushroom tyrosinase inhibition assay. In continuation of
our efforts on enzyme inhibition activity (Mughal, Javid,
et al., 2018; Mughal, Sadiq, et al., 2018; Mughal et al., 2019),
3-hydroxyflavones (1–23) were synthesized and evaluated,
in vitro, for their anti-tyrosinase activity. Kojic acid (IC50
¼1.79 0.6 lg/ml) was used as a reference compound. The
IC50 values of tested compounds are summarized in Table 4,
and these values are presented as the mean of three experi-
ments. The obtained results revealed that most of the com-
pounds displayed potent inhibition on mushroom tyrosinase
enzyme with IC50 values ranging from 0.230 0.020 lg/ml to
35.189 2.384 lg/ml except the compounds 3 and 12 whose
IC50 values were not determined (Table 4).
Table 3. Potential of 3-hydroxyflavone derivatives (1-23) against fun-
gal strains.
Zones of Inhibition (mm)
Compound Number
CA
CP
1
2
3
4
5
6
14.5 0.5
15.7 0.9
14.6 0.6
13.6 0.6
NA
11.8 0.5
NA
19.7 0.6
14.6 0.4
13.6 0.5
NA
ꢅ
NA
7
8
NA
NA
19.6 0.7
NA
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
DMSO
NA
NA
17.5 0.9
19.6 0.3
16.5 0.2
23.5 0.1
20.4 0.1
14.2 0.2
18.5 0.3
15.3 0.3
9.3 0.3
20.3 0.0
19.4 0.3
10.2 0.2
18.3 0.3
20.3 0.5
NA
16.5 0.4
14.6 0.8
21.5 0.3
20.5 0.2
22.7 0.2
21.1 0.4
19.1 0.6
17.3 0.5
20.2 0.4
21.2 0.6
21.5 0.2
NA
Structure-activity relationship of mushroom tyrosinase
inhibitory activity
The general structure of the flavonol nucleus is comprised of
a chromone moiety and an aryl ring (B) (Figure 1). All these
structural features are playing a significant role in inhibitory
activity, however, a little variation in the activity of these
compounds is attributed to variability in the positions and
nature of substituents on aryl ring (B) as well as the nature
of ring B. Limited SAR studies was established purely on the
basis of substitution pattern on the 3-hydroxyflavone scaffold
20.3 0.2
21.6 0.2
NA
Clotrimazole (1 mg/ml)
28.9 0.8
25.6 0.6
ꢅ
NA ¼ No Activity.
(Figure 4). In this connection, compound
1
(IC50
¼
4.268 0.182 mg/ml) bearing no substituent displayed the
least activity amongst the series. However, the substituted
derivatives demonstrated very good inhibitory activities in
comparison to flavonol 1 presenting that substitution on
rings A & B increases the inhibitory potential of these com-
pounds. Additionally, compounds 8, 15, 16, and 19 were
found to be less active against the envisioned enzyme. These
findings reflect that highly hydrophilic (i-e –COOH) and
the compounds 3, 7, 12, 13, 14, 15, 16, 18, 19, 20, 22, and
23 exhibited the highest activity against C. parapsilosis.
Although with respect to the standard drugs, all the eval-
uated compounds were found to show moderate activity, so
the result of all preliminary study indicated that the flavonol
moiety containing compounds represent a new class of
pharmacophore for broad-spectrum antibacterial and antifun-
gal activity.

10
J. ASHRAF ET AL.
Figure 4. Pharmacophoric features of 3-hydroxyflavones and tyrosinase inhibitor, structure-activity relationship (SAR) of the most potent compounds.
hydrophobic (–CH3 and iso-butyl) groups on both aryl rings tyrosinase enzyme. All these compounds are even more
(A & B) are accountable for their low activities because of active than the reference compound. Excitingly, the test
their less interaction with the enzyme.
It is noteworthy that the derivatives 2, 4, 5, 6, 10, 11, 14, 0.230 0.020 lg/ml) were found to be the most active,
and 22 demonstrated exceptional inhibitory activity against among all the synthesized analogs, as compared to the
compounds 2 (IC50 ¼ 0.280 0.010 lg/ml) and 5 (IC50 ¼

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
11
Table 5. Binding energies of selective modes against tyrosinase enzyme.
polar sulphonamide-like group in the place of ring B, also
showed outstanding activity against tyrosinase. The increased
activity by 14 might be due to its position and effective inter-
actions with the active pocket of the enzyme. Apart from that,
all other synthetic 3-hydroxyflavones demonstrated good to
moderate inhibitory activity against mushroom tyrosinase.
Overall, the results presented herein show that the nature and
pattern of substitution at both rings A and B enhance the
inhibitory potential of these compounds, and thus are
accountable to control it. Since all the proposed structures
have a common 3-hydroxyflavone skeleton in their scaffolds.
Compound No.
Lowest Binding Energy (Kcal molꢀ1
)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
ꢀ6.67
ꢀ7.98
ꢀ6.38
ꢀ7.42
ꢀ8.55
ꢀ8.47
ꢀ7.10
ꢀ7.27
ꢀ7.05
ꢀ7.05
ꢀ9.56
ꢀ6.63
ꢀ8.94
ꢀ8.59
ꢀ7.05
ꢀ7.30
ꢀ6.71
ꢀ6.55
ꢀ6.33
ꢀ7.10
ꢀ7.06
ꢀ6.31
ꢀ6.83
ꢀ4.44
Molecular modelling studies
In order to gain insight into the most probable binding con-
formation of 3-hydroxyflavone derivatives (1-23) with the
active site of an enzyme and confirm the experimental
results, the molecular docking studies were performed
against mushroom tyrosinase enzyme. In this connection, 2-
D structures of the compounds (1-23) were built up by using
Chemdraw and then converted into 3-D by using Chem
Pro3D software. The ligands were energetically minimized
according to Merck Molecular Force Field (MMFF) and
docked to the protein by using autodock1.5.6. By place-
ments, the method will check 100 different poses into the
active site pockets of 2Y9X and will result out of few best
placements in the form of a histogram. For each conformer
with the best (minimum) docking score was saved in prefer-
ences. Docking results are listed in Table 5. We have listed
only the most excellent conformers and the docking score
for each compound. The ligand developing most stable
Standard (Tropolone)
standard Kojic acid (IC50 ¼1.79 0.6 lg/ml). The derivative 2
possesses a highly electron-withdrawing group (–CF3) at
meta- position on ring B. This indicates that the structure 2
nicely fits and strongly interacts with the active site of the
envisioned enzyme. Interestingly, the analogous 4 (IC50
¼
0.30 0.0 lg/ml), having trifluoromethyl group –CF3) at the
ortho- position of ring B, was found comparatively less active
than its meta-isomer 2, but still more active than standard.
Furthermore, the flavonol
5 bears electron-withdrawing
group (–Cl) at 40 -position on ring B. Probably, this group
and its position are appropriate to agreeably interact with
the active pocket of the enzyme. The same explanation holds
true in case of next halogen-substituted compound 22 (IC50
¼ 0.799 0.008 lg/ml) in which fluoro group is present at
para-position on the ring B. Afterwards, the next most potent
derivative is compound 6 (IC50 ¼ 0.284 0.018 lg/ml) which
contains strong electron-withdrawing group (–NO2) at 40
-position on ring B. Interestingly, the meta nitro substituted
drug-receptor complex is the one which is having
a
minimum docking score. Validation of docking studies is
evaluated by RMSD between the experimentally docking-
observed and the x-ray ligand. The co-crystalline ligand was
detached from the active pockets of the targeted substrate
and the re-docked to calculate the RMSD value (Azam et al.,
2012; Ounthaisong
& Tangyuenyongwatana, 2017). The
RMSD value of the re-docking of the co-crystalline ligand is
0.358 A0, which depicts the validation of the docking process.
The drug-receptor complexes of some potent ligands were
analysed for various types of interactions such as hydrogen
bonding, hydrophobic type interactions, and van der Waals
interactions, etc.
compound
9
(IC50
¼
2.564 0.0251 mg/ml) exhibited
decreased activity as compared to its para analog 6. The
nitro group at meta position (ring B) might be creating steric
hindrance, thus results in reduced activity.
The ligand (5) with binding energy (ꢀ8.55 Kcal molꢀ1
)
Furthermore, the replacement of aryl ring B with other het-
erocyclic rings such as thiophene in compound 10 (IC50
¼
articulated its inhibitory characteristics against the targeted
biological specimen (2Y9X) via versatile kinds of associations.
The ligand (5) coordinates with Ala286, Val283, and Val248
amino acid residues of the active site of tyrosinase through
hydrophobic-p-alkyl type interaction. Delocalized p-electronic
cloud of aromatic ring (A) of this ligand build up hydropho-
bic p- p stacked force of attraction with His263. Similarly, the
alcoholic group (–OH) of the ring (C) engaged with the
Glu256 by conventional hydrogen bonding. His259 also
showed hydrogen bonding with the ligand 5 as shown in
Figures 5 and 6.
0.347 0.078 mg/ml) also displayed exceptional activity as
compared to the standard (Table 4). The tyrosinase inhibition
by compound 10 relied on the ability of a sulfur atom to
coordinate with the dinuclear copper in the active site of the
enzyme. Subsequently, the next synthetic analogue with com-
parable IC50 value is compound 11 (IC50 ¼ 0.327 0.041 mg/
ml). This compound also showed stronger inhibitory activity
against tyrosinase enzyme than the reference standard inhibi-
tor Kojic acid. This might be a result of the strong chelation of
electron-rich moiety (triphenylamine) of the derivative 11 with
the active electrophilic centre of the tyrosinase. Finally, the
Likewise, the ligand 14 could be an important inhibitor
analog 14 (IC50 5 0.370 0.025 mg/ml), possessing bulky and for the targeted biological receptor protein. Docking score

12
J. ASHRAF ET AL.
Figure 5. Putative binding associations of ligand 5 inside the active pockets of tyrosinase.
Figure 6. Associations of the ligand 5 against tyrosinase at 3D space. Box in the lower right corner shows the associations of specific amino acid residues. The 3D
ribbon attributes the enzyme-stick model along with the lowest energy conforms of the inhibitor 5 and amino acids of tyrosinase interacting with it.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
13
Figure 7. Putative binding associations of ligand 14 inside the active pockets of tyrosinase enzyme.
Figure 8. Associations of the ligand 14 against tyrosinase at 3D space. Box in the lower right corner shows the associations of specific amino acid residues. The 3D
ribbon attributes the enzyme-stick model along with the lowest energy conforms of the inhibitor 14 and amino acids of tyrosinase interacting with it.
(ꢀ8.59 Kcal molꢀ1) of the ligand 14 is due to some important hydrophobic p-p T-shaped and hydrophobic p-p Stacked
type of linkages such as it exhibits hydrogen bonding with type interactions with His263, Phe264, Ala286, Val283, and
Asn260 amino acid residues inside the active pockets of the His85 amino acid residues inside of activation loop of tar-
protein 2Y9X. This ligand expressed its inhibitory potential geted tyrosinase biological molecule as displayed in Figures
by possessing hydrophobic p-alkyl, hydrophobic p-r, 7 and 8.

14
J. ASHRAF ET AL.
Conclusions
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imum inhibition against tyrosinase with (IC50
¼
0.230 0.020 lg/ml) demonstrating that 5 was about seven-
fold more effective mushroom tyrosinase inhibitor as com-
pared to Kojic acid (IC50 ¼1.79 0.6 lg/ml). Limited SAR
studies showed that all structural features were contributing
to tyrosinase inhibition, however, flavonols with functional
groups such as F, Cl, NO2, CF3, triphenylamine, and sulphona-
mide moieties present at meta- and para-positions on ring B
were actively taking part in the activity. Additionally, the
antimicrobial assay of the synthetic derivatives (1-23)
showed good to moderate activities, however, none of the
compounds was active more than the standard Cefixime and
Clotrimazole. Moreover, the emission behavior of these syn-
thetic flavonols was also investigated. Results showed that
they emit light at longer wavelengths upon excitation
around 330 nm wavelength. To recognize the structural fea-
tures that were involved in the binding interactions with the
active site of tyrosinase, in silico study was also performed.
The above-mentioned results suggest that these molecules
can serve as fascinating candidates for the treatment of tyro-
sinase-related ailments and as lead compounds for the devel-
opment of potent new tyrosinase inhibitors and
antimicrobial agents. Hence, more results aiming at the
investigation of these potent compounds against test
enzyme and their mechanistic studies are underway in our
group and will be reported in due course in the future.
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Disclosure statement
No potential conflict of interest was reported by the author(s).
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Funding
This research work was financially supported by the Higher Education
Commission of Pakistan under the NRPU Project No. 6484. M.M.
acknowledges financial support from Ferdowsi University of Mashhad.
M.M. is also supported by the Iran Science Elites Federation and Zeolite
and Porous Materials Committee of Iranian Chemical Society.
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