Novel piperonal 1,3,4-thiadiazolium-2-phenylamines mesoionic derivatives: Synthesis, tyrosinase inhibition evaluation and HSA binding study

Article abstract of DOI:10.1016/j.ijbiomac.2018.02.050

A novel series of piperonal mesoionic derivatives (PMI 1–6) was synthesized. Tyrosinase inhibition in the presence of PMI-1, ?2, ?3, ?4, ?5 and ?6 as well as human serum albumin (HSA) binding studies with PMI-5 and PMI-6 were done by spectroscopic and the

Full text of DOI:10.1016/j.ijbiomac.2018.02.050

Accepted Manuscript
Novel piperonal 1,3,4-thiadiazolium-2-phenylamines mesoionic
derivatives: Synthesis, tyrosinase inhibition evaluation and HSA
binding study
Natália Drumond Lopes, Otávio Augusto Chaves, Márcia C.C.
de Oliveira, Carlos Mauricio R. Sant'Anna, Danilo Sousa-Pereira,
José Carlos Netto-Ferreira, Aurea Echevarria
PII:
S0141-8130(17)32734-4
DOI:
Reference:
https://doi.org/10.1016/j.ijbiomac.2018.02.050
BIOMAC 9104
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25 July 2017
6 February 2018
7 February 2018
Please cite this article as: Natália Drumond Lopes, Otávio Augusto Chaves, Márcia C.C.
de Oliveira, Carlos Mauricio R. Sant'Anna, Danilo Sousa-Pereira, José Carlos Netto-
Ferreira, Aurea Echevarria , Novel piperonal 1,3,4-thiadiazolium-2-phenylamines
mesoionic derivatives: Synthesis, tyrosinase inhibition evaluation and HSA binding study.
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ACCEPTED MANUSCRIPT
Novel piperonal 1,3,4-thiadiazolium-2-phenylamines mesoionic derivatives:
Synthesis, tyrosinase inhibition evaluation and HSA binding study
Natália Drumond Lopes,† Otávio Augusto Chaves,† Márcia C. C. de Oliveira, Carlos Mauricio R.
Sant’Anna, Danilo Sousa-Pereira, José Carlos Netto-Ferreira,* Aurea Echevarria,*
Departamento de Química, Universidade Federal Rural do Rio de Janeiro, BR-465 Km 7, 23970-
000 Seropédica-RJ, Brazil.
† These authors contributed equally to this work
*jcnetto.ufrrj@gmail.com (J.C.N-F.); echevarr@ufrrj.br (A.E.)
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Abstract
A novel series of piperonal mesoionic derivatives (PMI 1-6) was synthesized. Tyrosinase
inhibition in the presence of PMI-1, -2, -3, -4, -5 and -6 as well as human serum albumin (HSA)
binding studies with PMI-5 and PMI-6 were done by spectroscopic and theoretical methods. The
mesoionic compound PMI-5 is the most promising tyrosinase inhibitor with a noncompetitive
inhibitory mechanism and an IC50 = 124 µmol L-1. In accordance with the kinetic profile, molecular
docking results show that PMI-5 is able to interact favorably with the tyrosinase active site
containing the substrate molecule, L-DOPA, interacting with Val-247, Phe-263 and Val-282
residues. The spectroscopic results for the interaction HSA:PMI-5 and HSA:PMI-6 indicated that
these mesoionic compounds can associate with HSA in the ground state and energy transfer can
occur with high probability. The binding was moderate, spontaneous and can perturb significantly
the secondary structure of the albumin. The molecular docking results suggest that PMI-5 and
PMI-6 are able to be accommodated inside the Sudlow’s site I in HSA, interacting with
hydrophobic and hydrophilic amino acid residues.
Keywords: Mesoionic compounds, tyrosinase, human serum albumin.
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1. Introduction
Tyrosinase is the enzyme involved in melanin production and other pigments via oxidation
reaction of L-tyrosine. It is a dicopper-contained enzyme which catalyzes the melanin synthesis by
the action of monophenolase in the hydroxylation reaction of tyrosine and by the diphenolase action
in the L-DOPA oxidation to ortho-dopaquinone [1]. Tyrosinase plays an important role in
medicinal, agricultural and cosmetic fields. Tyrosinase inhibitors are used to treat
hyperpigmentation diseases, age spots and skin whitening among others, whereas tyrosinase
activators cause the increase of melanogenesis that can protect the skin from UV damage [2]. It has
also been suggested that tyrosinase contributes to neurodegeneration processes correlated to
Parkinson’s disease [3] and, thus, tyrosinase inhibitors has been studied as a possible treatment for
this kind of disease.
In recent years, many natural and synthetic compounds that can act as tyrosinase inhibitors or
activators have been reported [4-6]. However, only a few of them are in clinical use. Thus, new
investigations are necessary to lead to the discovery of novel compounds with activity towards this
enzyme. In searching for new synthetic compounds with anti-tyrosinase activity the 1,3,4-
thiadiazolium class was chosen because some other heterocyclic compounds, such as thiazoles [7],
oxazolones [8], pyrazoles [9], and triazolothiadiazoles [10], have shown interesting effects against
tyrosinase.
The 1,3,4-thiadiazolium-2-phenylamine chlorides are precursors of mesoionic compounds, a
special class of heterocyclic compounds with diverse biological activities, such as anti-
inflammatory, analgesic [11,12], antibacterial, antifungal [13,14], antiparasitic [15,16] and
antitumor [17-19]. These mesoionic compounds possess a positive charge in the heterocyclic ring
and contain a negatively charged atom on the exocyclic group; however, their overall neutrality
allows them to cross biological membranes and to interact with biomolecules such as proteins and
DNA [20].
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Human serum albumin (HSA) is the most abundant carrier and well-known protein in the
human circulatory system. HSA is composed of three structurally similar domains, i. e I-III, and
each domain consists of two subdomains (A and B) stabilized by 17 disulfide bridges [21]. Besides,
HSA contributes to the osmotic blood pressure and can play a dominant role in transportation,
disposition and efficacy of many biologically active compounds in the circulatory system [22]. A
crucial fact is that drugs and bioactive small molecules bind reversibly to albumin and by
consequence the study of the interaction of potential drugs with this protein is extremely important.
The interesting structure of the 1,3,4-thiadiazolium-2-phenylamine class of compounds and
the promising therapeutic applications therefrom led us to synthesize a new series of such
compounds (Figure 1). In the present work, the synthesis and characterization of novel piperonal
1,3,4-thiadiazolium-2-phenylamine mesoionic derivatives were described, as well as the
experimental and theoretical evaluation of their action as tyrosinase inhibitors. In order to
understand their transportation and delivery ability following interaction with HSA, the two best
tyrosinase inhibitors, namely PMI-5 and PMI-6, were investigated under physiological conditions
(PBS solution, pH = 7.4) at 296 K, 303 K and 310 K by spectroscopic techniques (fluorescence and
circular dichroism) and molecular docking.
PMI-1 R = H, R’ = H
PMI-2 R = H, R’ = NO2
PMI-3 R= H, R’ = Br
PMI-4 R = NO2, R’ = H
PMI-5 R = Cl, R’ = H
PMI-6 R = Br, R’ = H
Figure 1. Chemical structure and code of the synthetic piperonal 1,3,4-thiadiazolium-2-
phenylamine mesoionic derivatives.
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2. Experimental
2.1. General
Melting points were recorded on a Mel-Temp II capillary melting point apparatus and were
not corrected. IR spectra were recorded on a Bruker Vertex 70 spectrophotometer using potassium
13
bromide tablets. 1H and C NMR spectra were obtained in a Bruker NMR Ultrashield 400 MHz
spectrometer, with tetramethylsilane as the internal reference and DMSO-d6 as the solvent.
Elemental analyses were performed on a PerkinElmer 2400 CHN in the Laboratory of
Environmental Science at Universidade Estadual do Norte Fluminense (UENF). Reactions were
monitored by TLC on Merck silica-gel 60 F254. TLC spots were visualized by inspection of the
plates under UV light (254 and 365 mm). The microwave-assisted organic reactions were
performed in a Consul CM020BFANA, a domestic oven.
L-DOPA, EDTA and mushroom tyrosinase were obtained from Sigma-Aldrich Chemical
Company. Formation of dopachrome was determined by monitoring the absorbance at 475 nm with
a Shimadzu UV-VIS spectrophotometer UV Mini 1240 (Kyoto, Japan).
Commercially available HSA, warfarin, ibuprofen and PBS buffer (pH = 7.4) were obtained
from Sigma-Aldrich Chemical Company. One tablet of PBS dissolved in 200 mL of millipore water
yields a 0.01 mol L-1 phosphate buffer solution, containing 0.0027 mol L-1 potassium chloride and
0.137 mol L-1 sodium chloride, pH 7.4, at 298 K. Water used in all experiments was Millipore
water. DMSO and methanol (both spectroscopic grade) were obtained from Tedia Ltd.
2.2. Synthesis
2.2.1. General procedure for the microwave-promoted synthesis of 5-(3,4-methylenedioxy-6-R’)-
1,3,4-thiadiazolium-2-(4-R-phenyl)amine chlorides (PMI-1 to PMI-6):
The 5-(3,4-methylenedioxy-6-R’)-1,3,4-thiadiazolium-2-(4-R-phenyl)amine chlorides were
synthesized as previously reported [18]. Briefly, the substituted 3,4-methylenedioxy-6-R’-
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benzaldehydes (0.35 mmol) [23], and the N1-phenyl-N4-(4’-R-phenyl)-thiosemicarbazides (0.35
mmol) [24] were mixed in the presence of thionyl chloride and few drops of 1,4-dioxane. The
mixture was then submitted to microwave irradiation for 5 min in an open vessel. After this time,
the mixture was added to 1,4-dioxane and left to stand overnight at room temperature. The obtained
products were filtered, washed with ice-cold water and purified by recrystallization from
chloroform:ethanol (60:40, v/v).
2.2.2. 5-(3,4-methylenedioxy-phenyl)-1,3,4-thiadiazolium-2-phenylamine chloride (PMI-1)
Yellow solid; 92% yield; mp 286-287 °C [25]; IR (KBr) ν / cm-1 3053, 2902, 2642, 1604, 1566,
1
1498, 1448, 1311, 1242, 754 and 692; H NMR (400 MHz, DMSO-d6) δ 12,37 (s, 1H, NH), 7.72
(m, 2H, H-2’’, H-6’’), 7.62 (m, 4H, H-3’’, H-4’’, H-5’’, H-2’’’), 7.46 (t, 2H, J 7.6 Hz, H3’’’,
13
H5’’’), 7.15 (m, 3H, H-5’, H6’, H4’’’), 6.94 (s, 1H, H2’), 6.17 (s, 2H, OCH2O); C NMR (100
MHz, DMSO-d6) δ 164.4 (C5), 160.6 (C2), 152.7 (C3’), 148.3 (C4’), 138.9 (C1’’), 138.3 (C1’’’),
131.9 (C4’’), 130.9 (C3’’’), 130.0 (C3’’), 127.0 (C6’), 126.6 (C2’’), 124.6 (C4’’’), 118.9 (C2’’’),
116.5 (C1’), 109.8 (C2’), 109.7 (C5’), 103.2 (OCH2O).
2.2.3. 5-(3,4-methylenedioxy-6’-nitro-phenyl)-1,3,4-thiadiazolium-2-phenylamine chloride (PMI-2)
Yellow solid; 95% yield; mp 264-266 °C [25]; IR (KBr) ν / cm-1 3008, 1602, 1566, 1494, 1450,
1
1365, 1326, 1267, 752 and 688; H NMR (400 MHz, DMSO-d6) δ 12.75 (s, 1H, NH), 7.95 (s, 1H,
H5’), 7.65 (d, J 7.6 Hz, 2H, H2’’, H6’’), 7.58 (m, 5H, H3’’, H4’’, H5’’, H3’’’, H5’’’), 7.46 (t, 2H, J
7.9 Hz, H2’’’, H6’’’), 7.19 (t, J 7.4 Hz 1H, H4’’’), 6.38 (s, 2H, OCH2O); 13C NMR (100 MHz,
DMSO-d6) δ 163.2 (C5), 161.8 (C2), 152.9 (C6’), 151.7 (C3’), 142.8 (C4’), 138.8 (C1’’), 137.5
(C1’’’), 132.2 (C4’’), 130.6 (C3’’’), 130.0 (C3’’), 125.6 (C2’’), 124.7 (C4’’’), 119.0 (C2’’’), 114.6
(C1’), 111.2 (C5’), 106.7 (C2’), 105.6 (OCH2O).
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2.2.4. 5-(3,4-methylenedioxy-6’-bromo-phenyl)-1,3,4-thiadiazolium-2-phenylamine chloride (PMI-
3)
Yellow solid; 90% yield; mp 278-279 °C; IR (KBr) ν / cm-1 3012, 1604, 1568, 1498, 1469, 1244,
1
748 and 688; H NMR (400 MHz, DMSO-d6) δ 13.00 (s, 1H, NH), 7.64 (m, 7H, H2‘, H5‘, H2’’,
H3‘‘, H4’’, H5‘‘, H6’’), 7.46 (m, 4H, H2’’’, H3’’’, H5’’’, H6’’’), 7.18 (t, 1H, J 7.3 Hz, H4’’’), 6.21
(s, 2H, OCH2O); 13C NMR (100 MHz, DMSO-d6) δ 162.5 (C5), 162.2 (C2), 152.4 (C4’), 148.0
(C3’), 138.7 (C1’’’), 137.8 (C1’’), 132.1 (C4’’), 130.3 (C3’’), 130.0 (C2’’), 125.8 (C3’’’), 124.7
(C4’’’), 119.0 (C2’’’), 116.7 (C1’), 115.2 (C6’), 113.6 (C5’), 112.2 (C2’), 104.0 (OCH2O); Anal.
calcd. for C21H15BrClN3O2S: C, 51.60; H, 3.09; N, 8.60. Found: C, 51.73; H, 2.89; N, 8.72.
2.2.5. 5-(3,4-methylenedioxy-phenyl)-1,3,4-thiadiazolium-2-(4’’-nitrophenyl)amine chloride (PMI-
4)
Yellow solid; 89% yield; mp 243-244 °C; IR (KBr) ν / cm-1 2892, 1620, 1539, 1500, 1450, 1330
1
and 1243; H NMR (400 MHz, MeOD) δ 8.34 (d, 2H, J 9.0 Hz, H3’’’, H5’’’), 7.83 (d, 2H, J 9.0
Hz, H2’’, H6’’), 7.70 (m, 5H, H3’’, H4’’, H5’’, H2’’’, H6’’’), 7.22 (d, 1H, J 8.3, 1.5 Hz, H6’), 7.01
13
(d, 1H, J 8.3 Hz, H5’), 6.87 (s, 1H, J 1.5 Hz, H2’), 6.12 (s, 2H, OCH2O); C NMR (100 MHz,
MeOD) δ 166.2 (C5), 160.4 (C2),153.0 (C3’), 148.9 (C4’), 143.6 (C4’’’), 143.5 (C1’’’), 137.9
(C1’’), 131.6 (C4’’), 130.0 (C3’’), 126.9 (C6’), 125.7 (C2’’), 125.0 (C3’’’), 118.3 (C2’’’), 115.6
(C1’), 109.1 (C5’), 108.8 (C2’), 103.1 (OCH2O); Anal. calcd. for C21H15ClN4O4S: C, 55.45; H,
3.32; N, 12.32. Found: C, 55.61; H, 3.12; N, 12.43.
2.2.6.
5-(3,4-methylenedioxy-phenyl)-1,3,4-thiadiazolium-2-(4’’-chlorophenyl)amine
chloride
(PMI-5)
Yellow solid; 60% yield; mp 309-310 °C; IR (KBr) ν / cm-1 2730, 1616, 1562, 1494, 1469, 1330
and 1240; 1H NMR (400 MHz, MeOD) δ 7,65 (m, 7H, H2‘‘, H3‘‘, H4‘‘, H5‘‘, H6‘‘, H3‘‘‘, H5‘‘‘),
7.44 (d, 2H, J 8.8 Hz, H2‘‘‘, H6‘‘‘), 7.18 (dd, 1H, J 8.2, 1.9 Hz, H6‘), 6.99 (d, 1H, J 8.3 Hz, H5‘),
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13
6.85 (d, 1H, J 1.8 Hz, H2‘), 6.11 (s, 2H, OCH2O) ; C NMR (100 MHz, MeOD) δ 164.8 (C5),
160.8 (C2), 152.8 (C4’), 148.9 (C3’), 138.0 (C1’’’), 137.1 (C1’’), 131.5 (C4’’), 129.9 (C3’’), 129.5
(C6’), 129.2 (C1’), 126.6 (C2’’), 125.6 (C3’’’), 120.2 (C2’’’), 115.9 (C4’’’), 109.0 (C5’), 108.7
(C2’), 103.1 (OCH2O); Anal. calcd. for C21H15Cl2N3O2S: C, 56.77; H, 3.40; N, 9.46. Found: C,
56.81; H, 3.37; N, 9.52.
2.2.7.
5-(3,4-methylenedioxy-phenyl)-1,3,4-thiadiazolium-2-(4’’-bromophenyl)amine
chloride
(PMI-6)
Yellow solid; 65% yield; mp 280-281 °C; IR (KBr) ν / cm-1 3402, 1616, 1562, 1494, 1470, 1330
1
and 1242; H NMR (400 MHz, DMSO-d6) δ 12.95 (s, 1H, NH), 7.63 (m, 9H, H2’’, H3’’, H4’’,
H5’’, H6’’, H2’’’, H3’’’, H5’’’, H6’’’), 7.11 (d, 2H, J 9.8 Hz, H5’, H6’), 6.95 (s, 1H, H2’), 6.16 (s,
2H, OCH2O); 13C NMR (100 MHz, DMSO-d6) δ 164.7 (C5), 160.3 (C2), 152.0 (C4’), 148.3 (C3’),
138.3 (C1’’’), 132.8 (C4’’), 131.9 (C3’’), 130.5 (C6’), 127.0 (C2’’), 126.6 (C3’’’), 120.7 (C2’’’),
116.5 (C1’), 116.1 (C4’’’), 109.8 (C5’), 109.7 (C2’), 103.2 (OCH2O); Anal. calcd. for
C21H15BrClN3O2S: C, 51.60; H, 3.09; N, 8.60. Found: C, 51.74; H, 2.92; N, 8.72.
2.3. Tyrosinase assays
The mushroom tyrosinase activity was determined by the oxidation of L-DOPA (0.17
mmolL-1) into dopachrome in a phosphate buffer medium (50 mmol L-1, pH 6.8) in the presence of
EDTA (0.022 mmol L-1), tyrosinase (50-100 units) and different concentrations of each of the
mesoionic compounds under investigation. All experiments were done in triplicate. Formation of
dopachrome was determined by monitoring the absorbance at 475 nm at room temperature.
Ascorbic acid was used as standard. The % of inhibition was calculated applying the equation 1 for
all mesoionic compounds:
[(
-
) - (
-
)]
B30 B0 A30 A0
Inhibition % =
x 100
(1)
8
(
-
)
B30 B0

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where B0 and B30 are the absorbance values of the control sample at zero time and after 30 min of
reaction, respectively. A0 and A30 are the absorbance of the test samples at zero time and after 30
min of reaction, respectively. In the equation 1, the interference of possible absorbance of the
organic compounds was subtracted.
In order to calculate the IC50 for PMI-5 and PMI-6 (the most promising tyrosinase
inhibitors) the increase in absorption at 475 nm was determined for different inhibitor
concentrations. The IC50 value was calculated from the equation generated by polinomial fit of the
experimental data (Origin software, ANOVA statistical function).
The inhibitory mechanism for PMI-5 on mushroom tyrosinase for oxidation of L-DOPA
was determined from Lineweaver-Burk double reciprocal plots. The Lineweaver-Burk plot is a
widely method used to determine important terms in enzyme kinetics, such as 퐾m, 푉max and Ki. In
this analysis was used the same metodology for the IC50 determination, but with different
concentrations of L-DOPA (33; 66; 100; 200; 333; 466 and 666 mol mL-1) as substrate and
different concentrations of PMI-5 (0; 330; 460 and 660 mol mL-1). After incubation of 30 min at
310 K, the absorbance was measured at 475 nm. Dopachrome production was analyzed in absence
and presence of the inhibitors, in the same conditions of the inhibitory test, but the increase in
absorption at 475 nm was determined from 0 to 60 min, at 10 min intervals.
2.4. HSA binding studies
Fluorescence and UV-Vis spectra were measured on a Jasco J-815 fluorimeter using a quartz
cell (1 cm optical path) and employing a thermostatic cuvette holder Jasco PFD-425S15F. The
circular dichroism spectra were measured in a spectropolarimeter Jasco J-815 and employing the
same thermostatic cuvette holder as described above. All spectra were recorded with appropriate
background corrections.
To a 3.0 mL solution containing an appropriate concentration of HSA (1.00x10-5 mol L-1),
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successive aliquots from a stock solution of the corresponding mesoionic compounds, i. e PMI-5
and PMI-6 (1.00x10-3 mol L-1) were added, leading to final concentrations of 0.17; 0.33; 0.50; 0.66;
0.83; 0.99; 1.15 and 1.32x10-6 mol L-1. The addition was done manually by using a micro syringe.
The fluorescence spectra were measured in the 290-450 nm range, at 296 K, 303 K and 310 K, with
excitation wavelength at 280 nm. The UV-Vis spectra for solutions of PMI-5 and PMI-6 (1.00x10-5
mol L-1, in methanol) were measured in the 200-450 nm range at 310 K. In order to compensate for
the inner filter effect, fluorescence intensity values were corrected for the absorption of each studied
mesoionic derivative at the excitation and emission wavelength using equation 2 [26]:
Fcor = Fobs10[(A +A )/ 2]
ex
em
(2)
where Fcor and Fobs are the corrected and observed fluorescence intensity values, respectively; Aex
and Aem represent the experimental absorbance values at the excitation wavelength (280 nm)
(ε=14942 mol L-1cm-1 for PMI-5 and ε=16966 mol L-1cm-1 for PMI-6) and emission wavelength
(340 nm; ε=10454 mol L-1cm-1 for PMI-5 and ε=8363 mol L-1cm-1 for PMI-6), respectively.
Circular dichroism spectra were recorded in the 200-260 nm range, at 310 K, for free serum
albumin (1.00x10-6 mol L-1) and for serum albumin with the highest concentration of PMI-5 and
PMI-6 (2.22x10-6 mol L-1) used in the fluorescence study.
2.5. Molecular modeling
The crystallographic structures of tyrosinase from the mushroom Agaricus bisporus and of
HSA were obtained from the Protein Data Bank (PDB) with access codes 2Y9X [27] and 1N5U
[28], respectively. The mesoionic compounds’ structures were built and energy-minimized with the
Spartan'14 program (Wavefunction, Inc.) at the Density Functional Theory (DFT) level with the
Becke-3-Lee Yang Parr (B3LYP) functional, using the 6-31G* basis set [29].
Molecular docking for both protein structures was performed with the GOLD 5.2 program
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(CCDC). Hydrogen atoms were added to the protein according to the ionization and tautomeric
states inferred by the program [30]. The tyrosinase crystallographic structure is composed by four
chains (A, B, C and D) and each chain contains a dicopper center responsible for the tyrosinase
activity [27]. In order to propose a molecular explanation for the experimental tyrosinase inhibition
data, a 10 Å radius-spherical domain around the dicopper center in the chain D was defined for the
molecular docking calculations with the ligands (PMI-1,2,3,4,5 and 6). Chain D was defined for the
molecular docking studies due the best results obtained by the redocking studies with the
cocrystallized ligand in the 2Y9X structure, tropolone. As the studied mesoionic compounds
presented noncompetitive inhibition (see Results and Discussion section), firstly a tyrosinase:L-
DOPA complex was obtained by selecting the corresponding best pose of a molecular docking run
of the substrate inside the enzyme implemented with GOLD 5.2. This complex was then used for a
second molecular docking run with the PMI-1-6 ligands. On the other hand, from the fluorescence
quenching studies and site-specific drug displacements (see Results and Discussion section), the
main HSA cavity for PMI-5 and PMI-6 is Sudlow’s site I (subdomain IIA), where the Trp-214
residue can be found [31]. In order to identify the main amino acid residues that contribute to the
association of PMI-5 and PMI-6 with HSA, a 10 Å radius-spherical domain around the tryptophan
residue was defined and then molecular docking calculations with both ligands were carried out.
The score of each pose identified is calculated as the negative of the sum of a series of
energy terms involved in the protein-ligand interaction process, so that the more positive the score,
the better is the interaction. The number of genetic operations (crossover, migration, mutation) in
each docking run used in the searching procedure was set to 100,000. The program optimizes
hydrogen-bond geometries by rotating all hydroxyl and amino groups of the amino acid side chains.
The scoring function used to perform the tyrosinase study was GoldScore, the best scoring function
identified in the tropolone redocking study. For the HSA docking studies, ChemPLP was used,
which is the default function of the GOLD 5.2 program [32]. The figures of the docking poses with
the largest docking score value were generated with the PyMOL Delano Scientific LLC program
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[33].
3. Results and Discussion
3.1. Synthesis
The mesoionic compounds 5-(3,4-methylenedioxy-6-R’)-1,3,4-thiadiazolium-2-(4-R-
phenyl)amine chlorides derived from 6-substituted piperonal, where R’ = R = H (PMI-1), R’ = NO2,
R = H (PMI-2), R’ = Br, R = H (PMI-3), R’ = H, R = NO2 (PMI-4), R’ = H, R = Cl (PMI-5) and R’
= H, R = Br (PMI-6) were sinthesized by microwave assisted irradiation. Of these, PMI-3, PMI-4,
PMI-5 and PMI-6 are new compounds. The convergent strategy [18] involved the reaction of the
appropriate 3,4-methylenedioxy-6-R’-benzaldehyde23 and N1-R-phenyl-N4-thiosemicarbazide [24]
in the presence of SOCl2 as a Lewis acid catalyst (Scheme 1). Both, the substituted piperonal
aldehydes and thiosemicarbazides were also prepared in this work [23,24]. The reaction mixture
was irradiated in a microwave oven for 5 min at 100 kW. The products were obtained as fine
crystals in high-purity and satisfactory yields, 60 to 95%, after a short irradiation time.
Scheme 1. Reagents and conditions: (a) HNO3, rt, 30 min; (b) bromine, methanol, acetic acid drops, rt until complete
comsumption of piperonal, NaHSO4 (1 molL-1).
The synthezised compounds were fully characterized by IR, 1H and 13C NMR. The 1H and 13C
chemical shifts were assigned based on reported data to other 1,3,4-thiadiazolium-2-phenylamine
derivatives [18,25,34] and were consistent with the structures proposed in this work. The chemical
shifts of C2 and C5 in the heterocyclic ring appear at high frequencies compared with signals from
12

ACCEPTED MANUSCRIPT
the other carbon atoms. The assigned values of δ 160.3 to 162.2 and δ 162.5 to 166.2 for C2 and C5,
respectively, showed the coherent correlation with the electronic effects of the substituents. The
remaining aromatic carbons in the rings attached to N4 and N-exocyclic were assigned using
previous data from the literature [18,25,34].
3.2. Inhibition of mushroom tyrosinase
In order to make a previous test on the inhibitory potential of the mesoionic compounds on
the tyrosinase enzyme a screening test was performed (Table 1). The compounds PMI-1, PMI-2,
PMI-3, PMI-5 and PMI-6 were tested in concentrations of 66.6 and 666 mol L-1, with exception
of PMI-4, which was insoluble in this concentration. Therefore, PMI-4 was only assayed in a 10
fold more diluted solution (ca. 66.6 mol L-1 – Table 1).
Table 1. % Inhibition of tyrosinase by mesoionic compounds (66.6 and 666 mol L-1) in the
presence of the substrate L-DOPA.
Compound
Substituent
% Inhibition
% Inhibition
R
R'
H
66.6 µM
10
666 µM
H
H
23
29
22
-
PMI-1
PMI-2
PMI-3
PMI-4
PMI-5
PMI-6
NO2
Br
H
2.0
H
1.0
NO2
Cl
4.0
H
35
89
79
Br
H
10
For the two best tyrosinase inhibitors, PMI-5 and PMI-6, we determined IC50 values, which
were 124 mol L-1 and 358 mol L-1, respectively. PMI-5 showed the most promising tyrosinase
inhibition result (Figure S19 in supplementary information) and proved to be a highly effective
inhibitor of mushroom tyrosinase, being more efficient than ascorbic acid, the reference inhibitor
[35] (in this work, IC50 = 260 mol L-1).
13

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The enzymatic inhibition mechanism was also investigated for the mesoionic compound
PMI-5 by performing a kinetic study in the presence of tyrosinase with different concentrations of
the substrate L-DOPA. The corresponding Lineweaver-Burk and Hanes-Woolf plots are depicted in
Figures 2A and 2B, respectively.
80
0.30
B
A
70
60
50
40
30
20
10
0
0.25
0.20
0.15
0.10
0.05
0.00
-400
-200
0
200
400
600
800
-0.015
-0.010
-0.005
0.000
0.005
0.010
0.015
0.020
[L-DOPA] (mol L-1)
1/[L-DOPA] (mol L-1)-1
Figure 2. (A) Lineweaver-Burk plot and (B) Hanes-Woolf plot for tyrosinase inhibition, in the
absence (control ■) or in the presence of PMI-5 (♦ 466 molL-1 and ▲ 333 molL-1) with L-
DOPA as substrate.
The Lineweaver-Burk plot shows that PMI-5 acted apparently as a noncompetitive
inhibitor, and clearly not as a competitive inhibitor, because the values of Km and Vm decreased in
the presence of inhibitor. A way to distinguish between competitive and noncompetitive inhibitors
is employing the Hanes-Woolf plot [36]. In this plot a competitive inhibition exhibits parallel lines,
whereas there is an intersection between the lines in the noncompetitive mechanism. In addition, the
values of Km and Vm obtained in Hanes-Woolf plot confirmed the involvement of an noncompetitive
mechanism (Table 2). In the noncompetitive mechanism the inhibitor binds reversibly to the
complex formed between the enzyme and the substrate (ES complex) and does not bind to the free
enzyme.
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Table 2. Kinetic parameters obtained from the Lineweaver-Burk and Hanes-Woolf plots for PMI-5
in the catalytic oxidation of L-Dopa by tyrosinase.
Kinetic parameters
Inhibitor concentration
466 molL-1
333 molL-1
90.9
without inhibitor
Km (molL-1)
Vm (molL-1 min-1)
Km (molL-1)
Lineweaver-Burk
Hanes-Woolf
166.6
10.4
66.6
7.8
7.8
345.8
16.4
112.6
9.4
155.2
9.9
Vm (molL-1 min-1)
The in vitro method to evaluate the inhibitory effect of organic compounds on tyrosinase has
a standart time of 30 min [36]. After this time, the inhibition power of the test sample may decrease
or remain constant, conferring a stable inhibition mechanism. Based on this, the kinetics of the L-
DOPA oxidation by tyrosinase in the absence and presence of the PMI-5 was evaluated (Figure S20
in supplementary information). The result showed that the concentration of dopachrome produced
during the oxidative process was lower in the presence of inhibitor, therefore decreasing the
inhibition power, which confirms the noncompetitive mechanism.
3.3. Molecular docking studies for the interaction between tyrosinase and the mesoionic compounds
under study
As the experimental tyrosinase assay results showed that the mesoionics compounds under
study present a noncompetitive inhibitory mechanism, the potential inhibitors bind reversibly to the
complex formed between the enzyme and the substrate L-DOPA (ES complex) and does not to the
free enzyme. Based on these experimental informations, a molecular docking study was carried out
in order to offer a molecular level explanation on the tyrosinase inhibition by the mesoionic
compounds PMI 1-6. Figure 3A depicts the best docking pose for all mesoionic compounds in the
15

ACCEPTED MANUSCRIPT
presence of L-DOPA. As expected by the presence of the substrate molecule, the potential electron
donor piperonal and 1,3,4-thiadiazolium groups present in the ligand structures are not near enough
to coordinate with the dicopper center, suggesting that the tyrosinase inhibition by PMI 1-6 is not
related with the Cu2+ coordination ability. In fact, is one of the hydroxyl groups from L-DOPA that
is near enough to coordinate with the dicopper center, with a distance of 2.30 Å. The molecular
surface of tyrosinase (Figure 3A) suggests that L-DOPA is buried inside the enzyme’s active site,
and PMI 1-6 are able to interact with L-DOPA, but they are not totally buried inside the protein
active site.
The molecular docking results also suggest that the ligands PMI-5 and PMI-6, which
showed better experimental inhibition percentage than PMI-1, PMI-2, PMI-3, PMI-4, adopt very
similar poses inside the protein active site. Despite, molecular docking results also suggest that
PMI-4 (assayed in a 10 fold more diluted solution) could also interacts similar than PMI-5 and
PMI-6. The other mesoionic compounds are accomodated diferently inside the protein cavity,
resulting in different interactions between the ligands and the amino acid residues present inside the
active site. The different interaction pattern presented by PMI-5 and PMI-6 in comparison with the
remaining mesoionic compounds may be at least in part related to the observed differences in the
tyrosinase inhibition activities of these compounds.
Figure 3B presents the superposition of the best docking poses for PMI-5 and PMI-6 inside
the tyrosinase:L-DOPA active site, showing the main amino acid residues of the enzyme that
interact with these two ligands. The Phe-263 residue is interacting via t-stacking with the aromatic
ring of the piperonal group with a distance of 2.70 Å and the apolar amino acid residue Val-247
residue is interacting via hydrophobic interactions with the same aromatic ring, within distance of
2.60 Å. There is a hydrogen bond between the peptidic NH hydrogen of the Val-282 residue and the
exociclic nitrogen of the ligand, with a distance of 2.30 and 2.50 Å, for PMI-5 and PMI-6,
respectively. Beside these interactions, PMI-5 and PMI-6 are able to interact with the aromatic ring
16

ACCEPTED MANUSCRIPT
of the substrate L-DOPA via t-stacking, within distance of 2.50 Å.
Figure 3. (A) Molecular surface of tyrosinase and its interaction with the mesoionic compounds
PMI-1-6 in the presence of L-DOPA. (B) Superposition of the best docking poses for the
interaction of tyrosinase with PMI-5 and PMI-6 in the presence of L-DOPA (GoldScore function).
Tyrosinase is in cartoon representation; L-DOPA, selected amino acid residues and ligands in stick
representation are in pink, light pink, and beige, respectively. In the Fig. 3B, PMI-5 and PMI-6 are
represented in cyan and orange, respectively. Copper atoms are represented as spheres in brown.
Element colors: hydrogen: white; oxygen: red; nitrogen: dark blue; bromine: brownish red and
chloro: green.
3.4. HSA binding studies – Fluorescence Quenching Analysis
Fluorescence quenching can be used as a technique to measure the binding affinity between
macromolecules and ligands acting as quenchers. Quenching of tryptophan (Trp) fluorescence is
widely used as a tool to monitor changes in protein structure and to make inferences regarding local
structure and dynamics [37]. There is just one tryptophan residue (Trp-214) in the HSA structure,
which is located in subdomain IIA, buried inside the protein structure [38]. As can be seen in Figure
4, the fluorescence spectrum of HSA presents a strong emission with maximum at 340 nm (λexc
=
280 nm). After successive additions to the HSA solution of the two mesoionic compounds that
showed the highest inhibition % for tyrosinase, i. e PMI-5 and PMI-6 (see above), the
fluorescence emission resulting from the Trp-214 residue showed a clear decrease, indicating that
the likely location of the quencher molecule within the protein cavity must be close to the
tryptophan residue [39]. The absence of considerable changes in the position of the maximum
17

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emission of HSA is a clear evidence that the presence of PMI-5 or PMI-6 does not exert a great
influence on the polarity of the cavity microenvironment around the Trp residue [38].
A variety of molecular interactions can result in two possible quenching mechanism of a
fluorescent species, i. e dynamic or static. These interactions include ground-state complex
formation, collisional quenching, excited state reactions, molecular rearrangement and energy
transfer [40]. In general, Stern-Volmer analysis (equations 3A and inset in the Figure 4), and the
well known relationship between kq and Ksv (equation 3B) are useful in the estimation of the
accessibility of the quencher molecule to the tryptophan residue in proteins as well as in the
understanding of the mechanism involved in the quenching process [37]:
KSV
F0
F
[ ]
[ ]
kq =
(A)
=1+kqτ0 Q =1+KSV Q
(B)
(3)
τ0
where, F0 and F are the fluorescence intensities for HSA in the absence and in the presence of PMI-
5 or PMI-6, respectively; Ksv is the Stern-Volmer quenching constant; kq is the bimolecular
quenching rate constant of the HSA fluorescence, [Q] is the ligand concentration and τo is the HSA
lifetime in the absence of the quencher (10-8 s) [38].
1.9
1.6
296K
303K
310K
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
296K
303K
310K
(B)
(A)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.5
1.4
1.3
1.2
1.1
3.0x10-6
6.0x10-6
9.0x10-6
1.2x10-5
3.0x10-6
6.0x10-6
9.0x10-6
1.2x10-5
[PMI-6] (M)
[PMI-5] (M)
300
350
400
450
300
320
340
360
380
400
420
440
Wavelength (nm)
Wavelength (nm)
Figure 4. Fluorescence emission spectra forfree HSA and its fluorescence quenching by addition of
succesive aliquots of PMI-5 (A) and PMI-6 (B). HSA prepared in a PBS buffer solution (pH 7.4).
[HSA] = 1.00x10-5 mol L-1, [PMI-5] = [PMI-6] = 0.17; 0.33; 0.50; 0.66; 0.83; 0.99; 1.15 and
1.32x10-6 mol L-1, T = 310 K, λexc = 280 nm. Inset: Stern-Volmer plot for the fluorescence
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quenching of HSA by PMI-5 (A) and PMI-6 (B) at 296 K, 303 K and 310 K.
Table 3 shows the KSV and kq values obtained from the fluorescence quenching experiments
involving HSA:PMI-5 and HSA:PMI-6. Since the bimolecular quenching rate constants obtained
(kq ≈ 1012 M-1 s-1, Table 3) are three orders of magnitude larger than the diffusion rate constant (kdiff
≈ 5.00x109 M-1 s-1 in water, at 298 K) [41] and the KSV values decrease with the temperature
increasing, the probable mechanism of fluorescence quenching is static [42]. Static quenching is
due to the formation of an association in the ground-state between the fluorophore (albumin) and
the quencher (PMI-5 or PMI-6) [43].
To obtain information about the binding capacity in the interaction HSA:PMI-5 and
HSA:PMI-6, the modified Stern-Volmer binding constant (Ka) was calculated employing the
modified Stern-Volmer equation (equation 4) according to Figure 5:
F
1
1
0
=
+
(4)
F - F fKa[Q]
f
0
where, Fo and F are the fluorescence intensities of HSA without and with the ligand at 340 nm,
respectively; Ka is the modified Stern-Volmer binding constant; ƒ is the fraction of the initial
fluorescence that is accessible to the quencher and [Q] is the ligand concentration.
The Ka values for the association HSA:PMI-5 and HSA:PMI-6 are in the range of 104 M-1,
showing a moderate interaction between the ligands and HSA [44,45]. From the pharmacological
point of view, if drugs are metabolized and excreted from the body too fast because of low protein
binding they will be not able to provide their therapeutic effect. On the other hand, if drugs bind too
strongly to protein and are metabolized and excreted too slowly, the in vivo half-life of these drugs
can increase excessively, which may lead to undesired side effects. Therefore, a moderate binding
constant to serum albumin is expected for a possible drug candidate [46]. The decrease of Ka values
19

ACCEPTED MANUSCRIPT
with the increasing of temperature is in total agreement with the proposed static fluorescence
quenching mechanism discussed above [43].
11.3
11.26
(B)
(A)
12
10
8
14
12
10
8
11.24
11.22
11.20
11.18
11.2
11.1
11.0
3.2x10-3
3.3x10-3
3.3x10-3
3.4x10-3
3.2x10-3
3.3x10-3
3.3x10-3
3.4x10-3
1/T (K-1
)
1/T (K-1)
6
6
4
296K
303K
310K
296K
303K
310K
4
2
2
1x105
2x105
3x105
4x105
5x105
6x105
1x105
2x105
3x105
4x105
5x105
6x105
1/[PMI-5] (M-1)
1/[PMI-6] (M-1)
Figure 5. Modified Stern-Volmer plot for fluorescence quenching of HSA upon binding of PMI-5
(A) and PMI-6 (B) at 296 K, 303 K and 310 K. Inset: Van't Hoff plot of Ka values from modified
Stern-Volmer by each ligand at 296 K, 303 K and 310 K. [HSA] = 1.00x10-5 molL-1 and [PMI-5] =
[PMI-6] = 0.17; 0.33; 0.50; 0.66; 0.83; 0.99; 1.15 and 1.32x10-6 molL-1
The interaction forces between endogenous and exogenous agents with proteins (van der
Waals and electrostatic forces - including hydrogen bond and hydrophobic effects) can be related to
the thermodynamic parameters ΔG°, ΔH° and ΔS°. In this sense, if ΔH°<0 and ΔS°>0 there is an
indication that the main force operating is typically through electrostatic effect and hydrophobic
interactions [47].
The thermodynamic parameters ΔG°, ΔH°, ΔS°, which control the interaction HSA:PMI-5
and HSA:PMI-6 were obtained from the inset in Figures 5A and 5B, respectively, using the van´t
Hoff equation (5A) and the Gibbs free energy equation (5B) [42] and are collected in Table 3.
H0 S0
(B) G0  H0 TS0
(5)
ln Ka  

(A)
RT
R
where, ΔH°, ΔS°, ΔG° are the enthalpy, entropy and Gibbs free energy, respectively; R is the gas
20

ACCEPTED MANUSCRIPT
constant (R = 8.314x10-3 kJ mol-1 K-1), T is the temperature (296 K, 303 K and 310 K) and Ka the
modified Stern-Volmer binding constant.
Negative values for the Gibbs free energy change (ΔG°<0; Table 3) are in total accord with
the spontaneity of the binding process between HSA and PMI-5 or PMI-6. The negative and
positive values for enthalpy and entropy change, respectively, are contributing to the spontaneity of
the binding process and by consequence the association HSA:PMI-5 and HSA:PMI-6 is
enthalpically and entropically driven, suggesting hydrophobic and electrostatic interactions as the
main binding forces [48,49].
Table 3. Binding constants (KSV, kq and Ka) and thermodynamic parameters (ΔH°, ΔS° and ΔG°)
for the HSA:PMI-5 and HSA:PMI-6 complexes at 296 K, 303 K and 310 K.
Ligand T (K) Ksv x104 (molL-1)
kq x1012 (molL-1s-1) Ka x104 (molL-1) ΔH (kJ mol-1) ΔS (kJ mol-1 K-1) ΔG (kJ mol-1)
296
303
310
296
303
310
(6.14±0.05)
(5.49±0.09)
(4.35±0.15)
(4.26±0.08)
(4.05±0.10)
(2.81±0.16)
6.14
5.49
4.35
4.26
4.05
2.81
(7.80±0.05)
(7.49±0.05)
(7.13±0.06)
(8.08±0.06)
(7.22±0.05)
(5.96±0.08)
-4.93
-4.93
-4.93
-16.5
-16.5
-16.5
0.0770
0.0770
0.0770
0.0382
0.0382
0.0382
-27.7
-28.3
-28.8
-27.8
-28.1
-28.3
PMI-5
PMI-6
Obs.: r2 = 0.9997-0.9918 for all measurements listed above.
3.5. Energy transfer from HSA to PMI-5 and PMI-6
Fluorescence resonance energy transfer (FRET) is an electrodynamic phenomenon that
occurs between a donor molecule in its excited state and an acceptor molecule in the ground state.
Non-radiative energy transfer occurs when the electronic excitation energy of a donor chromophore
(Tryptophan in HSA) is transferred to an acceptor molecule (halogenated mesoionic compounds) by
a dipole-dipole interaction [50]. The efficiency of energy transfer (E) can be given by the equations
21

ACCEPTED MANUSCRIPT
6A and 6B [51].
6
(
)
F
R06
F0 R06  r6
R0 . 1- E
r6 =
E 1

(A)
(B)
(6)
E
where F0 and F are the donor fluorescence intensities in the absence and in the presence of an
acceptor, respectively; r is the distance between acceptor and donor; R0 is the critical distance for
transfer efficiency of 50%, which can be determined by equation 7:
R06 = 8.8x10-25 K 2n-4ΦJ
(7)
where K2 is the spatial orientation factor of the dipole (K2 = 2/3 for random orientation in fluid
solution), n is the averaged refraction index of the medium (usually resulting in a value of 1.336,
which is the average for water containing organic material), [52]  is the fluorescence quantum
yield of the donor (HSA) in the absence of quencher ( = 0.118) [53] and J is the overlap integral
between the emission spectrum of the donor and the absorption spectrum of the acceptor. J can be
calculated from Figure 8A and 8B and equation 8:
∞F






4 d

F

∑






4 
F() 
∑
∫
0
J 

∞F() d
(8)
∫
0
where F(λ) is the normalized fluorescence intensity of the donor at the wavelength λ and ε(λ) is the
extinction coefficient of the acceptor at λ.
Figure 6 shows the spectral overlap between the fluorescence emission spectrum of HSA
and the absorption spectrum of PMI-5 and PMI-6, indicating that there is a reasonable probability
of an energy transfer process from HSA to PMI-5 and PMI-6 [54]. The calculated values for the
distances between HSA (as donor) and each of the ligands (as acceptors) (r) shown in Table 4 is
between 2 and 8 nm, in full agreement with the rule 0.5R0 < r < 1.5R0, confirming the high
probability of occurrence of energy transfer between albumin and PMI-5 or PMI-6 [53].
22

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0.5
0.4
0.3
0.2
0.1
0.0
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.5
0.4
0.3
0.2
0.1
0.0
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Absorption PMI-6
Fluorescence Emission HSA
Absorption PMI-5
Fluorescence Emission HSA
(B)
(A)
300
320
340
360
380
400
420
440
300
320
340
360
380
400
420
440
Wavelength (nm)
Wavelength (nm)
Figure 6. Spectral overlap between the fluorescence emission spectrum of HSA and the absorption
spectra of PMI-5 (A) and PMI-6 (B), respectively, at 310 K. [HSA] = [PMI-5] = [PMI-6] =
1.00×10-5 molL-1 at 310 K.
Table 4. Parameters to estimate the spatial distance between the donor (HSA) and the acceptor
(PMI-5 and PMI-6) measured by the FRET theory.
Sample
PMI-5
PMI-6
J (cm3 molL-1)
1.55x10-14
R0 (nm)
2.64
r (nm)
2.99
1.20x10-14
2.53
2.82
3.6. Identification of the main binding site on HSA structure for the association with PMI-5 and
PMI-6
HSA crystal structure analysis revealed that its main drug binding sites are located in
subdomains IIA and IIIA, also known as Sudlow’s site I and II, respectively. A large hydrophobic
cavity is present in the IIA subdomain whereas a hydrophilic cavity is present in the subdomain
IIIA. In order to further identify the main binding site for PMI-5 and PMI-6 on the HSA structure,
23

ACCEPTED MANUSCRIPT
competitive binding experiments were carried out at 310 K using warfarin and ibuprofen as specific
site markers for the Sudlow’s site I and II, respectively [31,52,54].
The modified Stern-Volmer binding constant (Ka) values at 310 K for HSA:PMI-5 and
HSA:PMI-6 binding in the presence of warfarin was reduced by 96.8% (2.26x103 mol L-1) and
96.1% (2.30x103 mol L-1), respectively, as determined by using equation 4 (Figure S21 in
supplementary information). On the other hand, in the presence of ibuprofen the Ka values for
HSA:PMI-5 and HSA:PMI-6 binding were reduced by only 18.9% (5.78x104 mol L-1) and 20.5%
(4.74x104 mol L-1), respectively. As the Ka values in the absence and in the presence of ibuprofen
are practically the same, whereas they change significantly in the presence of warfarin, this is a
clear evidence that both PMI-5 and PMI-6 compete with warfarin for the same binding site in
HSA, i. e the subdomain IIA, where the Trp-214 residue can be found [49].
3.7. Perturbation on the HSA secondary structure induced by PMI-5 and PMI-6
To understand the alteration in the protein secondary structure after addition of the
mesoionic compounds PMI-5 and PMI-6 to its surroundings, Circular Dichroism spectroscopy
(CD) was employed due to its sensitive prediction [54]. HSA exhibited two negative bands in the
CD spectra: one at 208 nm that corresponds to the highest electronic energy transition (π-π*) and
another at 222 nm that corresponds to the lowest electronic energy transition (n-π*), both of which
are characteristic of the α-helix structure units of the protein [55]. As can be seen in Figures 7A
and 7B, a decrease in the intensity at 208 nm and 222 nm is clearly observed upon the adition of the
maximum concentration of the ligands used in the fluorescence quenching studies . These data
indicate a strong change on the secondary structure of HSA [56].
24

ACCEPTED MANUSCRIPT
0
0
-20
(B)
(A)
HSA
HSA
HSA:PMI-5
-20
HSA:PMI-6
-40
-60
-40
-60
-80
-80
-100
-120
-140
-100
-120
-140
200
210
220
230
240
250
260
200
210
220
230
240
250
260
Wavelength (nm)
Wavelength (nm)
Figure 7. Circular dichroism spectra of HSA in the absence and presence of the mesoionic
compounds PMI-5 (A) and PMI-6 (B) in a PBS buffer solution (pH=7.4). [HSA] = 1.00x10-6 molL-
1 and [PMI-5] = [PMI-6] = 1.32x10-5 molL-1.
In order to offer a quantitative decrease on the loss of helical structure (α-helix %) due to the
ligand binding, firstly the CD results were expressed in terms of significant molar residual
ellipticity (MRE) in deg.cm2/dmol, which were calculated according to equation 9:

MRE 
(9)
(10.n.l.CP )
where, θ is the observed ellipticity (mdeg); n is the number of amino acid residues (585 to HSA)
[57] l is the length of the optical cuvette (1.0 cm) and Cp is the molar concentration of HSA
(1.00x10-6 mol L-1).
The α-helix % is calculated as contents of either free HSA or complexed with each ligand
from the MRE values at 208 nm and 222 nm, using equations 10A and 10B:
(
)
(
)
- MRE208 - 4000
- MRE222 - 2340
(A)
(B)
(10)
α - helix% =
x 100
α - helix% =
x 100
(
)
33000 - 4000
30300
where, MRE208 and MRE222 are the significant molar residual ellipticities (deg cm2 dmol-1) at 208
nm and 222 nm, respectively.
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The calculated α-helix content of the secondary structure of HSA in the absence of the
ligands has its maximum at about 72.3% (at 208 nm) and 70.7% (at 222 nm) at 310K. In presence
of the ligand, i. e PMI-5 or PMI-6 (1.32x10-5 mol L-1), the calculated α-helix content was about
53.3% and 53.1% at 208 nm for HSA:PMI-5 and HSA:PMI-6, respectively. On the other hand,
under the very same conditions the calculated α-helix content was 52.4% and 54.1% at 222 nm for
HSA:PMI-5 and HSA:PMI-6, respectively. These results indicate that the ligands perturbate
significantly the secondary structure of albumin, possibly destroying the hydrogen bonding
networks of the amino acid residues of the main polypeptide chain due the high kinetic volume of
each ligand and its charged nature [58]. In the literature, interaction between serum albumin and
some thiadiazole derivatives, as well as 3-carboxyphenoxathiin, chalcone and some charged
surfactants (C14HDAB and C14DHAB) also suggested high perturbation on the secondary structure
of the protein, although still remains predominantly α-helix [58,59-61].
3.8. Molecular docking studies for the interaction between HSA and PMI-5 or PMI-6
From the experimental studies of HSA fluorescence quenching, it is clear that PMI-5 and
PMI-6 are binding next to the Trp-214 residue and did not show major differences in their binding
ability. Studies using molecular docking were performed to analyze the main intermolecular
interactions between PMI-5 and PMI-6 with the amino acid residues present in the binding site
(Sudlow’s site I). Figure 8 shows the superposition of the best poses obtained for the interaction of
PMI-5 and PMI-6 with HSA. The molecular docking results suggest that both molecules are able to
interact with hydrophobic and hydrophilic amino acid residues. One of the oxygen atoms of the
piperonal group of both ligands behaves as an acceptor for a weak hydrogen bond from the Arg-221
residue, with distances of 3.61 Å and 3.80 Å, for PMI-5 and PMI-6, respectively; while the
protonated amino group of Lys-198 residue is interacting by a hydrogen bond with one of the
nitrogen atoms present in the 1,3,4-thiadiazolium ring of the ligands with a distance of 2.67 Å, for
both ligands. On the other hand, the intrinsic fluorophore of albumin, the Trp-214 residue,
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participates of t-stacking interactions with the aromatic ring of each ligand with distances of 2.52 Å
and 2.65 Å, for PMI-5 and PMI-6, respectively. Finally, the molecular docking results also suggest
that ligands can have hydrophobic interactions with the side chains of the amino acids Leu-197, and
Leu-480 residues.
Overall, for HSA the molecular docking results suggest that both ligands are able to be
accommodated in the Trp-214 residue-containing site, interacting with the same amino acid
residues, with only small differences on the interaction distances, which is in accordance with their
similar experimental binding constants.
Figure 8. Superposition of the best docking pose for the interaction between HSA:PMI-5 and
HSA:PMI-6 (ChemPLP function). HSA structure is in cartoon representation; PMI-5, PMI-6 and
selected hydrophobic and hydrophilic amino acid residues in stick representation are in brown,
beige, cyan and yellow, respectively. Element colors: hydrogen: white; oxygen: red; nitrogen: dark
blue; bromine: carnelian and chloro: green.
4. Conclusions
A novel series of 1,3,4-thiadiazolium-2-phenylamine mesoionic compounds was
synthesized by microwave assisted irradiation. The two mesoionic compounds that showed the
highest tyrosinase inhibition were PMI-5 and PMI-6. Between them, PMI-5 is the most
promising tyrosinase inhibitor through a noncompetitive inhibitory mechanism and a IC50 = 124
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µmol L-1, being more efficient than the commercial inhibitor ascorbic acid (IC50 at 260 µmol L-1).
These results were supported by molecular docking studies of the compounds with the
tyrosinase:L-DOPA complex. The KSV and kq values obtained from Stern-Volmer plots indicated
that the intrinsic fluorescence quenching of HSA by PMI-5 and PMI-6 occurs through a static
mechanism. Therefore, an association HSA:PMI-5 and HSA:PMI-6 in the ground state can be
expected. Energy transfer from HSA to either PMI-5 or PMI-6 can occur with high probability.
The modified Stern-Volmer binding constant values (Ka≈104 mol L-1) results indicated that the
association HSA:PMI-5 and HSA:PMI-6 is moderate. Besides, CD studies show that upon
HSA:PMI-5 and HSA:PMI-6 association can significantly perturb the secondary structure of
albumin, possibly destroying the hydrogen bonding networks of the amino acid residues of the
main polypeptide chain. Thermodynamic parameters indicated that the association HSA:PMI-5
and HSA:PMI-6 is spontaneous (ΔG° < 0) and both are enthalpically and entropically driven. The
molecular docking results suggest that PMI-5 and PMI-6 are able to be accommodated in the
Trp-214-containing binding site of HSA, interacting with the same amino acid residues, with only
small differences on the distances between them and the HSA residues.
Conflict of interest
Authors declare no conflict of interest.
Acknowledgements
This research was supported by the Brazilian funding agencies: Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro
(FAPERJ).
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Author Contributions
N.D.L. and D.S.-P. synthesized the mesoionic derivatives, M.C.C.O. conducted tyrosinase
assays, O.A.C. conducted HSA spectroscopic experiments and molecular docking experiments for
HSA and tyrosinase, C.M.R.S. professor responsible for molecular docking analysis, J.C.N.-F.
professor responsible for spectroscopic analysis, A.E. professor responsible for synthesis.
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