Synthesis and biological evaluation of N-aryl-2-phenyl-hydrazinecarbothioamides: Experimental and theoretical analysis on tyrosinase inhibition and interaction with HSA

Article abstract of DOI:10.1016/j.bioorg.2018.07.035

A series of N-aryl-2-phenyl-hydrazinecarbothioamides have been investigated as possible inhibitors of tyrosinase, an enzyme involved in the development of melanomas. The hydrazinecarbothioamides 1–6 were synthesized from the reaction between phenylhydrazine and isothiocyanates, for which three different methods have been employed, namely stirring at room temperature, by microwave irradiation or by mechanochemical grinding. Quantitative yields were obtained for the later technique. Compound 4 showed the best value for tyrosinase inhibition (IC₅₀ = 22.6 μM), which occurs through an uncompetitive mechanism. Molecular docking results suggested that 4 can interact via T-stacking with the substrate L-DOPA and via hydrogen bonding and hydrophobic forces with the amino acid residues Ala-79, His-243, Val-247, Phe-263, Val-282, and Glu-321. The interaction between human serum albumin (HSA) and compound 4 occurs through a ground state association and does not perturb the secondary structure of the albumin as well as the microenvironment around Tyr and Trp residues. The binding is spontaneous, moderate and occurs mainly in the Sudlow's site I. Molecular docking results suggested hydrogen bonding, hydrophobic and electrostatic interactions as the main binding forces between the compound 4 and the amino acid residues Lys-198, Trp-214, Glu-449, Leu-452, and Leu-480.

Full text of DOI:10.1016/j.bioorg.2018.07.035

Accepted Manuscript
Synthesis and biological evaluation of N-aryl-2-phenyl-hydrazinecarbothioa-
mides: Experimental and theoretical analysis on tyrosinase inhibition and in-
teraction with HSA
Danilo Sousa-Pereira, Otávio Augusto Chaves, Camilla Moretto dos Reis,
Márcia C. C. de Oliveira, Carlos Maurício R. Sant'Anna, José Carlos Netto-
Ferreira, Aurea Echevarria
PII:
S0045-2068(18)30370-5
DOI:
https://doi.org/10.1016/j.bioorg.2018.07.035
YBIOO 2454
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
17 April 2018
20 July 2018
31 July 2018
Please cite this article as: D. Sousa-Pereira, O. Augusto Chaves, C. Moretto dos Reis, M. C. C. de Oliveira, C.
Maurício R. Sant'Anna, J. Carlos Netto-Ferreira, A. Echevarria, Synthesis and biological evaluation of N-aryl-2-
phenyl-hydrazinecarbothioamides: Experimental and theoretical analysis on tyrosinase inhibition and interaction
with HSA, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.07.035
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Synthesis and biological evaluation of N-aryl-2-phenyl-
hydrazinecarbothioamides: Experimental and theoretical analysis on
tyrosinase inhibition and interaction with HSA
a,†
a,†
a
Danilo Sousa-Pereira, Otávio Augusto Chaves, Camilla Moretto dos Reis, Márcia C. C.
a
a
a,b,*
de Oliveira, Carlos Maurício R. Sant’Anna, José Carlos Netto-Ferreira,
Aurea
a,*
Echevarria,
a
Departmento de Química, Universidade Federal Rural do Rio de Janeiro-UFRRJ, 23851-
70, Seropédica-RJ, Brazil.
9
b
Instituto Nacional de Metrologia, Qualidade e Tecnologia-INMETRO, Divisão de
Metrologia Química, 25250-020, Duque de Caxias-RJ, Brazil.
†
These authors contributed equally to this work
*
Corresponding authors: jcnetto@ufrrj.br (J. C. Netto-Ferreira) and echevarr@ufrrj.br(A.
Echevarria).
1

Abstract
A series of N-aryl-2-phenyl-hydrazinecarbothioamides have been investigated as
possible inhibitors of tyrosinase, an enzyme involved in the development of melanomas. The
hydrazinecarbothioamides 1-6 were synthesized from the reaction between phenylhydrazine
and isothiocyanates, for which three different methods have been employed, namely stirring
at room temperature, by microwave irradiation or by mechanochemical grinding. Quantitative
yields were obtained for the later technique. Compound 4 showed the best valuefortyrosinase
inhibition (IC50 = 22.6 µM), which occurs through an uncompetitive mechanism. Molecular
docking results suggested that 4 can interact viaT-stacking with the substrate L-DOPA and
via hydrogen bonding and hydrophobic forces with the amino acid residues Ala-79, His-243,
Val-247, Phe-263, Val-282, and Glu-321. The interaction between human serum albumin
(
HSA) and compound 4 occurs through a ground state association and does not perturb the
secondary structure of the albumin as well as the microenvironment around Tyr and Trp
residues. The binding is spontaneous, moderate and occurs mainly in the Sudlow’s site I.
Molecular docking results suggested hydrogen bonding, hydrophobic and electrostatic
interactions as the main binding forces between the compound 4 and the amino acid residues
Lys-198, Trp-214, Glu-449, Leu-452, and Leu-480.
Keywords:N-aryl-2-phenyl-hydrazinecarbothioamides; mechanochemical grinding; enzyme
tyrosinase; human serum albumin; spectroscopy; molecular docking.
2

1
. Introduction
Enzyme tyrosinase (EC 1.14.18.1), also known as polyphenol oxidase (PPO), is a
copper-containing enzyme widely distributed in fungi, plants, and animals. It can catalyze the
skin pigmentation and is directly related to pigmentation disorders in mammals [1].
Tyrosinase activity is related to its ability to catalyze the aerobic hydroxylation of L-tyrosine
to L-DOPA and the subsequent oxidation of the later to L-Dopaquinone, which leads to
melanin accumulation and hyper pigmentation [2]. Recently, it has been demonstrated that
various dermatological disorders, such as age spots, melasma freckle, and sites of actinic
damage can be caused by the accumulation of an excessive level of epidermal pigmentation
and not only by ultraviolet radiation. Thus, the inhibitory action on tyrosinase activity
minimizes the occurrence of dermatological disorders, mainly melanomas, becoming the
major target in the treatment of disorders related to the abnormal accumulation of melanin
[
2,3].
Carbothioamide derivatives were already tested as tyrosinase inhibitors, among them
a series of 1-(1-arylethylidene) thiosemicarbazide with the derivatives containing either a 4-
methoxyphenyl or a 2-thiophenyl group showed IC50 = 0.11 µM and 0.14 µM, respectively
[
4]. Besides 1-(1-(4-methoxyphenyl)-3-phenylallylidene) thiosemicarbazide exhibited IC50
=
0
.27 µM [5] and 4-methyl-1-(2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetyl)
thiosemicarbazide IC50 = 10.5 µM [6]. Recently, the (Z)-2-(3-(phenethylamino)-but-2-enoyl)-
hydrazine-carbothioamide showed potential tyrosinase inhibition (89%) through an
uncompetitive inhibitory mechanism and IC50 = 49 μM [3].
Human serum albumin (HSA) is the major protein in human bloodstream and
accounts for almost 60% of total plasma protein content. Serum albumin functional activity is
essential for maintaining normal tissue and organ homeostasis, but its function depends on
3

both its concentration and structure. HSA is the main transporting protein for ligands due to
its ability to bind to a broad range of both hydrophobic and hydrophilic molecules, including
drugs [7,8]. HSA has a molecular weight of 66.7 kDa and is composed of a single
polypeptide chain containing 585 amino acids,among them one tryptophan residue (Trp-214),
seventeen tyrosine residues (Tyr-30, -84, -138, -140, -148, -150, -161, -263, -319, -332, -334,
-
341, -353, -370, -401, -411, -497), a free thiol group of a cysteine residue (Cys-34) and
seventeen intramolecular disulfide bridges [9,10].
In the present work six hydrazinecarbothioamides (Fig. 1),i. eN-2-
diphenylhydrazinecarbothioamide (1); N-(4-nitrophenyl)-2-phenylhydrazinecarbothioamide
(
2); N-(4-chlorophenyl)-2-phenylhydrazinecarbothioamide (3); N-(4-bromophenyl)-2-
phenylhydrazinecarbothioamide (4); N-(4-methoxyphenyl)-2-phenylhydrazinecarbothioamide
5) and N-(p-tolyl)-2-phenyl-hydrazinecarbothioamide (6) were synthesized and
(
characterized. The experimental and theoretical evaluation of the action of these compounds
as tyrosinase inhibitors was then performed. In addition, as the distribution and metabolism
of many biologically active compounds in the body are correlated to their affinity for serum
albumin, the interaction between the most potential tyrosinase inhibitor,i.e.4, and HSA was
investigated under physiological conditions by multiple spectroscopic techniques (circular
dichroism, steady-state, time-resolved and synchronous fluorescence) combined with
molecular docking.
Fig. 1.Chemical structure for the N-aryl-2-phenyl-hydrazinecarbothioamides 1-6.
4

2
2
. Results and Discussion
.1. N-aryl-2-phenyl-hydrazinecarbothioamides synthesis
N-aryl-2-phenyl-hydrazinecarbothiamides 1-6 were obtained by an anhydrous
acylation reaction between phenylhydrazine and 4-X-phenyl-isothiocyanates employing
initially two very well knownsynthetic methods for these type of compounds: (I) stirring in
hot toluene for 60 min, and (II) under microwave irradiation for 30 min [11]. Further,
mechanochemistry [12] was used as a novel method (method III, in Scheme 1) for the
synthesis of hydrazinecarbothiamides, in which phenylhydrazine was milled in the presence
of 4-X-phenyl-isothiocyanates in a porcelain mortar under solvent-free conditions, at room
temperature, during a very short reaction time (2 min) (Scheme 1).
Scheme 1. Thesynthetic route for the preparation of compounds 1-6.
Reaction products were obtained in quantitative yields and high-purity using method
III when compared to the traditional procedure (I, 60 min, 68-95%) or to microwave
irradiation (II, 30 min, 66-83%). Method III allowed the synthesis of the target compounds in
solvent-free conditions and high purity, without the need for further recrystallization.
The methods II and III were aimed to obtain a reduction in the reaction time as well as
to fulfill some of the principles of Green Chemistry [13]. However, the
5

hydrazinecarbothiamidespreparation via the traditional methodology using toluene as the
solvent and under stirring at room temperature (I) was more efficient than the microwave-
assisted procedure (II), affording better yields on the formation of products. This result may
be due to the high volatility of the isothiocyanates employed as starting materials. Table 1
shows the product yields obtained in the preparation of 1-6. The hydrazinecarbothiamides
1
were fully characterized by H- and DEPT Q-NMR and IR spectroscopy, and identified by
comparison with spectroscopic data from the literature (Fig. 1S-6S in the supplementary
material) [14,15]. The chemical shift values for H-1 (δ7.38 to 7.53,  = 0.15) did not show
significant differences as a function of the different substituents on the N-4 linked ring.
However, the chemical shift values for H-2 and H-4 showed range of δ9.51 to 10.19 ( =
0
.51) and δ8.60 to 9.08 ( = 0.48), respectively, due the deshielding effects of withdraw
moieties NO , Cl and Br, when compared to the presence of donor electron groups OCH and
CH
2
3
3
.
Table 1
Yields and corresponding melting points(ºC) for the thiosemicarbazides 1-6obtained under methods I or II.
Yield (%) / Method
Thiosemicarbazides
X
mp(ºC)
Conventional
Microwave
Method (I; 60 min) Irradiation (II; 30 min)
1
2
3
4
5
6
H
NO
Cl
92
95
68
93
92
92
83
73
66
80
80
78
171-173
233-234
233-234
209-212
184-186
174-175
2
Br
OCH
3
CH
3
2
.2. Tyrosinase inhibitory assay. Experimental and theoretical approach
A preliminary screening employing colorimetric assays with the enzyme tyrosinase
showed that hydrazinecarbothiamides1-6 were able to inhibit the oxidation of L-DOPA, the
6

substrate for the diphenolase activity of mushroom tyrosinase, with inhibition percentage of
3%; 81%; 98%; 96%; 17% and 29% for hydrazinecarbothiamides1, 2, 3, 4, 5 and 6,
7
respectively. The IC50 values obtained for the more active compounds, i. e.1, 2, 3 and 4 are in
the order of 623.00; 189.00; 56.50 and 22.60 μM, respectively(Fig. 7S in the supplementary
material). Overall, the hydrazinecarbothiamides substituted with an electron acceptor group
showed better tyrosinase inhibition than the commercial standard ascorbic acid (IC50
66.00μM) [3]. In addition, hydrazinecarbothiamides4 showed the best inhibitory effect
among all compounds analyzed.
=
2
The inhibitory mechanism in the mushroom tyrosinase, as well as the kinetic
parameters associated to it, were determined in the presence of the two best inhibitors of
tyrosinase, 3 and 4, employing Hanes-Woof plots (Fig. 2 and Table 2). Since K
m
(Michaelis-
Menten constant) andV (maximum velocity in the presence of each inhibitor) in the absence
m
of the hydrazinecarbothiamides3 and 4 showed higher values than those in the presence of
these two inhibitors, at different concentrations, it can be concluded that the mechanism of
inhibition of tyrosinase by3 and 4is uncompetitive [3]. Thus, the potential inhibitors bind
reversibly to the enzyme-substrate (ES) complex, providing a ternary complex enzyme-
substrate-inhibitor (ESI). This result indicates that thehydrazinecarbothiamides3 and 4 do not
bind to the free enzyme, which allows an increase in the degree of inhibition as the
concentration of the substrate increases in the reaction medium.
Table 2
m m
Kinetic parameters (K and V ) for the tyrosinase activity in the absence and in the presence of different
concentrations of hydrazinecarbothiamides3 or 4 (0.00; 83.0; 50.0 and 33.3 µM).
Kinetic parameters Without3or4
3or4 (83.0 µM)
(2.13 or 1.23) x10
11.8 or 9.72
3or4 (50.0 µM)
(2.11 or 2.24) x10
13.1or 7.28
3or4 (33.3 µM)
(1.35 or 2.22) x10
20.1or 8.67
-3
-2
-2
-2
K
m
(M)
(3.00 or 2.79) x10
20.8 or 22.10
V
m
(M/min)
7

(A)
Noinhibitor
3.0M
0.0M
3.3M
180
8
5
3
120
60
0
-4 00
-2 00
0
200
400
600
-
1
[
L -D OPA](mol mL )
(B)
Noinhibitor
83.0M
120
50.0M
33.3M
80
40
0
-4 00
-2 00
0
200
400
600
-
1
[
L -D OPA](mol mL )
Fig. 2. Hanes-Woof plots for tyrosinase in the presence of L-DOPA and hydrazinecarbothiamide3 (A) and 4 (B).
[
L-DOPA] = 66.6; 100; 200; 333; 467 and 667 μM and [3] = [4] = 0.00; 33.3; 50.0 and 83.0 μM.
In order topropose a molecular level explanation on the tyrosinase inhibition by the
four best inhibitors (hydrazinecarbothiamides derivatives 1-4), a molecular docking study
was carried out assuming the ESI complex formation (uncompetitive inhibitory mechanism).
In this case, due to interaction between one of the phenolic hydroxyl group from the L-DOPA
2
+
structure (substrate) with Cu , the acidity of this group has increased and the molecular
docking calculations assumed a deprotonation state. Fig. 3 depicts the best docking pose for
all hydrazinecarbothiamides under study in the presence of L-DOPA. As expected, in the
8

presence of the substrate molecule the electron donor group present in the
hydrazinecarbothiamide structure is not near enough to coordinate with the dicopper center,
2
+
suggesting that the tyrosinase inhibition by 1-4 is not related to the Cu coordination ability.
In fact, one of the hydroxyl groups belonging to L-DOPA is near enough to coordinate with
the dicopper center, within a distance of 2.30 Å. The molecular docking results for the best
pose inside the protein pocket (Fig. 3A) suggest that the interactionsof 3 and 4with the active
site are essentially of a hydrophobic nature, involving T-stacking with the aromatic ring of L-
DOPA. On the other hand, inhibitors 1 and 2do not show significant intermolecular
interaction with this substrate. Thus, the low IC50 value for hydrazinecarbothiamides1 and 2
could probably be related to its low affinity for the substrate L-DOPA, while 3 and 4
presented similar IC50 values due to their high ability for this substrate.
Fig. 3B shows the main amino acid residues that interact with the two best
experimental tyrosinase inhibitors, i. ehydrazinecarbothiamides3 and 4, which are
superimposed on the figure for a comparison of their interaction profiles. Despite the fact that
the ligands can interact viaT-stacking with L-DOPA, one N-H group of the ligandsisa
potential hydrogen bond donor with the amino acid residue Glu-321, within an intermolecular
interaction distance of 2.73 Å. Molecular docking results also suggested hydrophobic
interactions between the structure of the ligands and Ala-79, His-243, Val-247, Phe-263, and
Val-282residues.
9

Fig. 3. (A) Superposition of the best docking poses for the interaction between tyrosinase and
hydrazinecarbothiamides1-4 in the presence of L-DOPA (GoldScore function). (B) Superposition between
hydrazinecarbothiamides3 and 4 and representation of the main amino acid residues which interact with these
inhibitors. Tyrosinase is in green cartoon representation; L-DOPA and selected amino acid residues are
represented in violet and cyan, respectively. Hydrazinecarbothiamides1-4 are represented in olive, salmon,
magenta, and orange, respectively. Copper atoms are represented as brown spheres. Black dots represent
interaction via hydrogen bonding. Element colors: hydrogen: white; oxygen: red; nitrogen: dark blue; bromine:
brownish red, chloro: dark green and sulfur: yellow.
10

2
2
.3. HSA binding studies
.3.1. Interaction between HSA and the hydrazinecarbothiamide 4
Steady-state fluorescence quenching is considered as an effective approach to
investigate the transport ability of HSA towards small molecules [16]. Fig. 4 depicts the
fluorescence emission of HSA and its quenching upon successive additions of the best
tyrosinase inhibitor under study (compound 4), at 310 K. Fluorescence quenching occurs in
the presence of 4 and there is no significant change in the position of the maximum emission
for HSA (λem = 340 nm) upon ligand binding. This is a clear indication that the ligand is able
to bind near the Trp-214 residue and that it does not perturb the microenvironment
surrounding this residue [8].
HSA
1,6
,5
,4
,3
,2
1,1
1
.6
.4
.2
.0
.8
.6
.4
.2
2
303K
310K
96K
1
1
1
1
1
1
1
HSA:4
0
-6
-6
-6
-5
3,0x10
6,0x10
9,0x10
1,2x10
0
[4 ](M)
0
Thiosemicarbazide4
0
3
00 320 340 360 380 400 420 440
Wavelength(nm)
Fig. 4. Steady-state fluorescence emission of hydrazinecarbothiamide4 and HSA without and in the presence of
successive additions of 4 at pH 7.4 and 310 K. Inset: Stern-Volmer plots for the interaction HSA:4 at 296 K,
-5
-5
3
03 K and 310 K. [HSA] = 1.0 x 10 M and [4] = 0.17; 0.33; 0.50; 0.66; 0.83; 0.99; 1.15; and 1.32 x 10 M.
11

Steady-state fluorescence quenching may be due to a dynamic and/or static
mechanism and a Stern-Volmer analysis can be used to identify which fluorescence
mechanism must be occurring (Eq. 1) [17]:
F0
[
]
[ ]
=
1+k τ Q =1+K Q
q 0
SV
(
1)
F
where F
presence of 4 at 340 nm, respectively. [Q] is the quencher concentration, KSV and k
Stern-Volmer constant and the bimolecular quenching rate constant, respectively. τ
HSA fluorescence lifetime in the absence of quenchers. The experimental average value
0
and F are the steady-state fluorescence intensities of HSA in the absence and
are the
is the
q
0
-9
measured in this work for the fluorescence lifetime of HSA in PBS was (5.75±0.10) x 10 s.
Table 3 shows the KSV and k values for the interaction HSA:4 at 296 K, 303 K, and
q
3
10 K. Since the KSV values decrease with the increase of the temperature and the k
q
values
9
−1 −1
are higher than the limiting diffusion rate constant in water (kdiff ≈7.40 × 10 M s ,
according to Smoluchowski-Stokes-Einstein theory at 298 K) [18],the main fluorescence
quenching mechanism must be static, which indicates that a ground state association between
HSA and 4must be present [19].
Time-resolved fluorescence is a sensitive technique which can be used to confirm if
the main fluorescence quenching of a protein occurs through a static and/or a dynamic
mechanism [20]. To get a further confirmation of the involvement of a static mechanism on
the association HSA:4, time-resolved fluorescence measurements were carried out for HSA
in the absence and presence of the maximum ligand concentration used in the steady-state
fluorescence experiments, at pH = 7.4. Fig. 8S in the supplementary material depicts the
fluorescence decays for HSA and HSA:4. Serum albumin showed two fluorescence lifetimes,
12

2
with different percentage contribution, i. e 5.75±0.10 ns (78%) and 1.80±0.11 ns (22%) (χ =
1
.105), which is fully in accordance with literature results [21]. After ligand binding there
was no significant change in the fluorescence lifetime for HSA: 5.71±0.10 ns (73%) and
2
1
.75±0.11 ns (27%) (χ = 1.128). Thus, since the fluorescence lifetime for HSA in the
absence and presence of 4did not change within the experimental error, it can be concluded
that, in this case,the fluorescence quenching occurs through a static mechanism [22], in
accordance with the ground state association HSA:4 described above. The same behavior was
previously described for other thiosemicarbazide derivatives [23,24].
Since steady-state and time-resolved fluorescence studies have shown that the
fluorescence quenching of HSA is due to a static mechanism involving the formation of a
non-fluorescence complex HSA:4, the number of binding sites (n) and the binding constant
(
K
b
) at 296 K, 303 K, and 310 K can be obtained by employing a double logarithmic plot
according to Eq. 2 [22]:


F0 - F 
  log K  nlog
log
Q
 
(
2)
b
F

where F
presence of 4, respectively.
The double logarithmic plot for HSA:4 (Fig. 9S, supplementary material) shows that n
values were approximately 1.00 in all temperatures (Table 3), which is a clear indication that
there is a single binding site for 4 on the HSA structure [25]. The K values are in the order of
0 M , indicating a moderate interaction between HSA and 4 [19,25,26]. Thus, compound 4
can be stored and transported by this protein in the human body [23].
0
and F are the steady-state fluorescence intensities of HSA in the absence and
b
4
-1
1
13

The interaction forces between drug and biomolecules include hydrogen bonds, van
der Waals forces, electrostatic and hydrophobic interactions, which can be demonstrated from
the thermodynamic parameters obtained from the van’t Hoff (Eq. 3A) and Gibb’s free energy
equations (Eq. 3B) [27]:
0
0
ΔH
ΔS
0
0
0
(
A)
ln K = -
+
(B)
ΔG = ΔH -TΔS
(3)
a
RT
R
where ΔH°, ΔS°, and ΔG° are the enthalpy, entropy and Gibbs’ free energy change,
respectively. T and R are the temperature (296 K, 303 K, and 310 K) and gas constant
−
1
−1
(
8.3145 Jmol K ), respectively. The van’t Hoff plot for HSA:4 is shown inFig. 10S in the
supplementary material and the thermodynamic parameters are given in Table 3. From the
negative and positive values of ΔG° and ΔS°, respectively, the binding HSA:4 is spontaneous
and entropically driven [19]. According to Ross and Subramanian proposal [28], for ΔH°> 0
and ΔS°> 0hydrophobic and electrostatic interactions can be suggested as the main binding
forces for this interaction.
Table 3
Binding constant values (KSV, k
q
, n, K
b
, ΔH°, ΔS° and ΔG°) for the interaction HSA:4 at 296 K, 303 K, and 310
K.
4
12
4
Code
T
K
SV (x10 )
k
q
(x10 )
n
K
b
(x10 )
ΔH°
ΔS°
ΔG°
-
1
-1 -1
-1
-1
-1 -1
-1
(K)
(M )
(M s )
(M )
(kJmol )
(kJmol K )
(kJmol )
296 4.47±0.07
7.78
0.98±0.10 3.42±0.28
-25.7
HSA:4
303 4.41±0.04
7.69
1.00±0.05 4.11±0.42 17.2± 1.3
0.145± 0.004
-26.7
310 4.12±0.03
7.16
1.01± 0.06 4.69±0.57
-27.8
2
2
2
Obs: r for KSV and k
q
: 0.9981-0.9997; r for n andK
b
0.9992-0.9998; r for ΔH°,ΔS° and ΔG° 0.9882.
14

2
.3.2. Perturbation of the HSA structure upon ligand binding
Circular dichroism spectroscopy (CD) is a valuable technique for studying changes in
the secondary structure of a protein after its interaction with small molecules. The CD
spectrum for HSA at physiological pH shows negative peaks at 208 nm (π→π* transition)
and 222 nm (n→π* transition), which are related to its α-helical content. The quantitative
secondary structure of albumin at 208 and 222 nm can be obtained using Eq. 4 [19]:
(
- MRE208 - 4000
)
(
- MRE222 - 2340) x 100
(
A) α - helix% =
x 100 (B)α - helix% =
(4)
(
33000 - 4000
)
30300
where MRE208 and MRE222 are the mean residue ellipticity at 208 and 222 nm. The % α-
helical content of the secondary structure of HSA without and in the presence of the
hydrazinecarbothiamide4 was about 68.1% and 67.7%, respectively, at 208 nm, whereas 66.7%
and 65.4% at 222 nm. The quantitative decrease of 0.40% (208 nm) and 1.30% (222 nm) in
the α-helical content observed in the CD results (Fig. 5A) indicatesthat there is no significant
conformational change in the secondary structure of HSA upon binding of the
hydrazinecarbothiamide4 [25].
Synchronous fluorescence spectroscopy (SFS) is widely used in protein-molecule
interactions to provide information about the molecular environment in the vicinity of the
fluorophore groups. In SFS, a simultaneous scanning of the excitation and emission
monochromators (Δλ) at 15 nm (for tyrosine) and 60 nm (for tryptophan) is performed [22].
Fig. 5B and 5C show SFS for Tyr and Trp residues for the association HSA:4 at pH = 7.4.
For both, Δλ = 15 and 60 nm, the maximum emission wavelength of Tyr and Trp residues
15

shows no obvious shifts, which indicates that the microenvironment around the Tyr and Trp
residues is not disturbed by the presence of the hydrazinecarbothiamide4 [22].
0
(A)
-
20
40
60
80
100
120
140
HSA
HSA:4
-
-
-
-
-
-
200
210
220 230
Wavelength(nm)
240
250
1
5
HSA
1
.5x10 (B)
15
1.2x10
HSA:4
14
9.0x10
14
6.0x10
14
3.0x10
240
260
280
Wavelength(nm)
300
320
HSA
(C)
15
2.0x10
HSA:4
15
1.5x10
15
1.0x10
14
5.0x10
240
260
280
300
320
Wavelength(nm)
Fig. 5.(A) CD spectra of HSA in the absence and bound to 4 at pH 7.4 and 310 K. SFS for HSA without and in
-
5
the presence of 4 at Δλ = 15 nm (B) and Δλ = 60 nm (C). [HSA] = 1.0 x 10 M and [4] = 0.17; 0.33; 0.50; 0.66;
-
5
0
.83; 0.99; 1.15; and 1.32 x 10 M.
16

2
.3.3. Investigation on the main binding site of 4 to HSA
HSA structure shows three major binding sites: smaller drugs mostly bind to drug
sites I (also known as Sudlow’s site I in subdomain IIA) or II (also known as Sudlow’s site II
in subdomain IIIA), while higher molecular weight ligands typically bind to site III
(
subdomain IB). All three sites share a common physical characteristic, i. ethe presence of a
hydrophobic pocket near multiple basic residues, which helps to explain the broad tolerance
of the protein to a very diverse group of ligands. It is well known that several compounds can
bind to site III of albumin; however, the number of molecules associated with sites I and II is
much higher than for site III, which indicates that the former sites are of more general interest
[
3,29].
In this sense, competition experiments in the presence of the site markers warfarin
site I), ibuprofen (site II) and digitoxin (site III) were performed to further identify the main
(
binding pocket for 4. Fig. 11S in the supplementary material shows the modified Stern-
Volmer plots for the association between HSA and 4 in the presence of each site marker. The
calculated binding constants (K ) in the presence of warfarin, ibuprofen, and digitoxin were
b
4
4
4
-1
(
2.63±0.20)x10 , (3.63±0.17)x10 , and (4.40±0.19)x10 M , respectively. Since the K
b
value
has changed significantly only in the presence of warfarin (43.9%), this is a clear indication
that the main binding site for hydrazinecarbothiamide4 is subdomain IIA, Sudlow’s site I
[
3,7],the exact site where the Trp-214 residue can be found
In order to identify the main amino acid residues as well as the main binding forces
involved in the association HSA:4, molecular docking calculations were carried out. Fig. 6
depicts the main amino acid residues that are able to interact with the ligand inside the protein
binding site.
17

Molecular docking results also suggested hydrophobic and electrostatic interactions as
the main binding forces, which is in accord to the experimental data.Additionally, hydrogen
bonding was also detected by theoretical calculations. The N-H group of the indole moiety of
Trp-214 is a potential hydrogen bond donor to one of the nitrogen atoms of the ligand
structure, within a distance of 2.40 Å, while the carboxylate group of Glu-449 is ahydrogen
bond acceptor fromone of the N-H groups of the ligand, within a distance of 2.00 Å.
Molecular docking also suggests an electrostatic interaction between the bromine group of
+
the ligand with the –NH
3
group from Lys-198 residue, within a distance of 2.30 Å. Finally,
hydrophobic interactions occur between the thiosemicarbazide 4 and Leu-452 and Leu-480
residues.
Fig. 6. Best score poses for the interaction HSA:4 inside Sudlow’s site I (ChemPLP function). Select amino acid
residues and hydrazinecarbothiamide 4carbon atoms are represented in cyan and orange, respectively. Black
dots represent interaction via hydrogen bonding. Hydrogen: white; oxygen: red; bromine: brownish red; sulfur:
yellow and nitrogen: dark blue.
18

3
. Conclusion
In summary, six hydrazinecarbothiamides were prepared using two known synthetic
methods and a novel method employing a solvent-free grinding. The later method strictly
follows the principles of Green Chemistry. The mechanochemical grinding under solvent-free
conditions resulted in quantitative yields, higher purity and in lower reaction time compared
to the conventional and microwave-assisted methods. Hydrazinecarbothiamide4 showed the
best tyrosinase inhibition (IC50 = 22.6 µM), even better than the usual standard, i. e
commercial ascorbic acid (IC50 = 266 µM). The inhibition mechanism of tyrosinase is
uncompetitive, therefore there is the formation of a ternary complex enzyme-substrate-
inhibitor (ESI). Molecular docking results suggest t-Stacking interaction as a critical force
between the two better tyrosinase inhibitors, i. e3 and 4, and the substrate L-DOPA. Finally,
molecular docking results also suggested that the hydrazinecarbothiamide4 can interact with
the amino acid residuesAla-79, His-243, Val-247, Phe-263, Val-282, and Glu-321 via
hydrogen bonding and hydrophobic forces. For the interaction HSA:4, the main fluorescence
quenching mechanism is via a static process, which results in a ground-state association. The
binding is spontaneous, moderate and entropically driven. The interaction does not perturb
significantly the secondary structure of the albumin as well as the microenvironment around
the Trp and Tyr residues. Competitive experiments indicated that Sudows’s site I is the HSA
main binding site for compound 4 and molecular docking results suggest hydrogen bonding,
hydrophobic and electrostatic interactions as the main binding forces between the ligand 4
and amino acid residues Lys-198, Trp-214, Glu-449, Leu-452, and Leu-480. Overall, in
addition to the fact that hydrazinecarbothiamide4 presented the best tyrosinase inhibition, it
can be easily transported and distributed by HSA in the human bloodstream.
19

4
. Experimental
4
.1. General information
The melting points of the products were determined on a Meltemp II apparatus and
1
were not corrected. Nuclear magnetic resonance ( H- and DEPTQ-NMR) spectra were
1
recorded on a Bruker Avance III spectrometer ( H, 500 MHz; DEPT Q, 125 MHz) using
tetramethylsilane (TMS) as the internal standard and acetone-d
6
or DMSO-d as the solvent.
6
Infrared spectra (KBr pellets) were recorded on a Bruker Vertex 70 spectrophotometer.
Elemental analyses were performed on a Perkin-Elmer Model 2400 instrument. The
microwave-assisted organic reactions were performed in a CEM Discovery System reactor.
Commercially available phenylhydrazine, 4-X-phenyl-isothiocyanates, tyrosinase
from mushroom lyophilized enzyme, (S)-2-amino-3-(3,4-dihydroxyphenyl)-propanoic acid
(
L-DOPA), 2,2',2'',2'''-(ethane-1,2-diylbis(azanetriyl))-tetraacetic acid (EDTA), ascorbic acid,
dimethyl sulfoxide (DMSO, spectroscopic grade), HSA, warfarin, ibuprofen, digitoxin and
PBS buffer were purchased from Sigma-Aldrich Chemical Company. Methanol
(
spectroscopic grade) and toluene were obtained from Tedia Ltd. One tablet of commercial
-2
-3
PBS dissolved in 200 mL of Milli-Q water yields 1.00 x 10 M phosphate buffer, 2.70 x 10
-1
M potassium chloride and 1.37 x 10 M sodium chloride.
4
.2. General procedure for the synthesis of hydrazinecarbothiamides (1-6)
Hydrazinecarbothiamides1-6 were obtained by an anhydrous acylation reaction
between phenylhydrazine and 4-X-phenyl-isothiocyanates through three different methods.
All hydrazinecarbothiamides were identified by melting point, FT-IR, NMR data and
elemental analyses [11,12].
20

Method I:Under Magnetic Stirring
The different aryl isothiocyanates (25 mmol) and phenylhydrazine (25 mmol) were
mixed in the presence of boiling toluene (20 mL). The reaction mixture was stirred for 60
minutes at room temperature and was monitored by TLC. The solid product was obtained by
filtration, washed with ice-cold toluene and recrystallized from ethanol [11].
Method II:Under Microwave Irradiation
The mixture containing the aryl isothiocyanate (25 mmol) and phenylhydrazine (25
mmol) was submitted to microwave irradiation for 30 min at 100 W, in the presence of
toluene. The completion of the reaction was monitored by TLC. The solid obtained was
filtered and washed with ice-cold toluene. The hydrazinecarbothiamides were recrystallized
from ethanol [11].
Method III:Under solvent-free grinding
The aryl isothiocyanates (0.74 mmol) and phenylhydrazine (0.74 mmol) were mixed
for 2 minutes in a porcelain mortar and pestle in the absence of any organic solvent, at room
temperature (monitored by TLC). The solid formed was obtained in high purity without
recrystallization.
4
.2.1. N-2-diphenylhydrazinecarbothioamide(1).White solid; m.p 173-171 °C; FT-
−
1
1
IR (KBr, υ cm ): 3282, 3212, 3170 (N-H), 1594, 1542, 1496 (C=C), 1207 (C=S); H NMR
6
(
500 MHz, acetone-d ) δ 9.66 (s, 1H, H-2), 8.70 (s, 1H, H-4), 7.73 (d, 2H, J = 7.78 Hz,H-
6
, H-10), 7.42 (s, 1H, H-1), 7.29 (t, 2H,J = 7.78 Hz,H-7, H-9), 7.25 (t, 2H, J = 7.78 Hz,
21

H-13, H-15), 7.12 (t, 1H, J = 7.28 Hz, H-8), 6.89 (t, 1H, J = 7.28 Hz, H-14), 6.87 (d,
H, J = 7.78 Hz, H-12, H-16). DEPT Q NMR (125 MHz, acetone-d ) δ 183.2 (C-3), 149.0
C-11), 140.5 (C-5), 130.3 (C-13, C-15), 129.2 (C-7, C-9), 126.0 (C-8), 125.2 (C-6, C-10),
2
6
(
1
1
22.0 (C-14), 114.6 (C-12, C-16). Anal. calcd for C13
7.27. Found: C, 64.22; H, 5.40; N, 17.23.
H
13
3
N S: C, 64.17; H, 5.39; N,
4
.2.2. N-(4-nitrophenyl)-2-phenylhydrazinecarbothioamide (2).Yellowsolid; m.p
−
1
2
34-233 °C; FT-IR (KBr, υ cm ): 3435, 3249, 3190 (N-H), 1600, 1568, 1504 (C=C), 1280
1
6
(
C=S), 1504 (C-NO ); H NMR (500 MHz, acetone-d ) δ 10.19 (s, 1H, H-2), 9.08 (s, 1H, H-
2
4
2
5
), 8.20 (t, 2H,J = 8.47 Hz,H-13, H-15), 8.20 (d, 2H, H-7, H-9), 7.50 (s, 1H, H-1), 7.27 (t,
H, J = 8.47 Hz, H-12, H-16), 6.89 (d, 2H, J = 9.60 Hz, H-6, H-10), 6.91 (t, 1H,J =
.65 Hz, H-14); DEPT Q NMR (125 MHz, acetone-d ) δ 183.0 (C-3), 148,8 (C-11), 146.8
6
(
C-5), 145.1 (C-8), 130.5 (C-13, C-15), 125.0 (C-7, C-9), 124.1 (C-6, C-10), 122.5 (C-14),
14.9 (C-12, C-16). Anal. calcd for C13 S: C, 54.16; H, 4.20; N, 19.43.
Found: C, 54.12; H, 4.25; N, 19.38.
1
H
12
N
4
O
2
4
.2.3. N-(4-chlorophenyl)-2-phenylhydrazinecarbothioamide (3).White solid; m.p 234-233
−
1
°
C; FT-IR (KBr, υ cm ): 3435, 3315, 3140 (N-H), 1600, 1554, 1508 (C=C), 1286 (C=S),
1
6
1
7
7
209, 758 (C-Cl); H NMR (500 MHz, acetone-d ) δ 9.79 (s, 1H, H-2), 8.85 (s, 1H, H-4),
.77 (d, 2H, J = 8.51 Hz, H-7, H-9), 7.45 (s, 1H, H-1), 7.31 (d, 2H, J = 8.51 Hz, H-6, H-10),
.25 (t, 2H, J = 7.72 Hz, H-13, H-15), 6.89 (t, 1H, J = 7.25 Hz, H-14), 6.88 (d, 2H, J = 7.88
6
Hz, H-12, H-16); DEPT Q NMR (125 MHz, acetone-d ) δ 183.2 (C-3), 148.9 (C-11), 139.3
(
C-5), 130.5 (C-8), 130.2 (C-13, C-15), 129.1 (C-7, C-9), 126.8 (C-6, C-10), 121.8 (C-14),
1
5
14.5 (C-12, C-16). Anal. calcd for C13
6.19; H, 4.39; N, 15.08.
H
12ClN S: C, 56.21; H, 4.35; N, 15.13. Found: C,
3
4
.2.4. N-(4-bromophenyl)-2-phenylhydrazinecarbothioamide (4). White solid; m.p 212-209
−
1
°
C; FT-IR (KBr, υ cm ): 3249, 3182 (N-H), 1597, 1585, 1489 (C=C), 1199 (C=S), 1072,
1
6
7
3
3
69 (C-Br); H NMR (500 MHz, acetone-d ) δ 9.81 (s, 1H, H-2), 8.85 (s, 1H, H-4), 7.74 (d,
H, J = 8.20 Hz, H-7, H-9), 7.46 (s, 1H, H-1), 7.46 (t, 2H, J = 8.51 Hz,H-6, H-10), 7.24 (t,
13
H,J = 7.57 Hz, H-13, H-15), 6.90 (t, 1H, H-14), 6.87 (d, 2H, J = 7.57 Hz, H-12, H-16); C
) δ 181.1 (C-3), 147.9 (C-11), 138.7 (C-5), 130.7 (C-7, C-9),
NMR (125 MHz, DMSO-d
6
22

1
28.9 (C-13, C-15), 127.2 (C-6, C-10), 119.9 (C-14), 116.9 (C-8), 113.1 (C-12, C-16). Anal.
calcd for C13 S: C, 48.46; H, 3.75; N, 13.04. Found: C, 48.42; H, 3.81; N, 12.98.
H12BrN
3
4
.2.5. N-(4-methoxyphenyl)-2-phenylhydrazinecarbothioamide (5).White solid;
−
1
m.p. 186-184 °C; IR (KBr, υ cm ): 3267, 3161, 3091 (N-H), 3028 (C-H), 1600, 1548, 1492
1
(
(
C=C), 1247 (C=S), 1170 (ArC-O), 1026 (Ar-O-C); H NMR (500 MHz, acetone-d
6
) δ 9.51
s, 1H, H-2), 8.60 (s, 1H, H-4), 7.53 (d, 2H, J = 9.64 Hz, H-12, H-16), 7.38 (s, 1H, H-1),
7
3
.25 (t, 2H, J = 9.64 Hz, H-13, H-15), 6.88 – 6.84 (m, 5H, H-6, H-7, H-9, H-10, H-14),
6
.76 (s, 3H, OCH
3
); DEPT Q NMR (125 MHz, acetone-d ) δ 183.6 (C-3), 158.5 (C-8), 149.2
(
C-11), 133.4 (C-5), 130.3 (C-13, C-15), 127.4 (C-6, C-10), 121.9 (C-14), 114.6 (C-12, C-
6), 114.4 (C-7, C-9), 56.0 (OCH ). Anal. calcd for C14 OS: C, 61.52; H, 5.53;
N, 15.37. Found: C, 61.57; H, 5.48; N, 15.43.
1
3
H
15
N
3
4
1
1
2
7
7
3
.2.6. N-(p-tolyl)-2-phenyl-hydrazinecarbothioamide (6).White solid; m.p 175-
−
1
74 °C; IR (KBr, υ cm ): 3437, 3275, 3186 (N-H), 3020 (C-H), 1627, 1550, 1490 (C=C),
1
6
269 (C=S); H NMR (500 MHz, acetone-d ) δ 9.57 (s, 1H, H-2), 8.63 (s, 1H, H-4), 7.57 (d,
H, J = 8.03 Hz, H-6, H-10), 7.38 (s, 1H, H-1), 7.25 (t, 2H, J = 7.78 Hz, H-13, H-15),
.09 (d, 2H,J = 7.78 Hz, H-7, H-9), 6.88 (t, 1H, J = 7.46 Hz,H-14), 6.88 (d, 2H, J =
.53 Hz, H-12, H-16), 2.28 (s, 3H; CH
), 148.7 (C-11), 137.5 (C-5), 135.2 (C-8), 129.9 (C-13, C-15), 129.4 (C-7, C-9), 125.0 (C-6,
). Anal. calcd for C14 S: C,
5.34; H, 5.87; N, 16.33. Found: C, 64.39; H, 5.83; N, 16.28.
3
); DEPT Q NMR (125 MHz, acetone-d ) δ 182.9 (C-
6
C-10), 121.6 (C-14), 114.2 (C-12, C-16), 20.8 (CH
3
H
15
N
3
6
4
4
.3. Tyrosinase inhibition assays
.3.1. Experimental assays
The UV-Vis absorption spectrum for tyrosinase in the activity measurements was
recorded on a Shimadzu UV-Vis spectrophotometer UV Mini 1240 (Kyoto, Japan), at room
3
0
temperature. Tyrosinase inhibition activity was measured by the method of Amorim et.al.
Firstly, stock solutions of hydrazinecarbothiamide derivatives1-6 (2.50 mM) were prepared
by dissolving them in DMSO. Then, each thiosemicarbazide was mixed with L-DOPA (0.17
mM), EDTA (0.022 mM) and tyrosinase (50–100 units), in PBS (pH=7.4). Due to the fact
23

thatthe hydrazinecarbothiamidesshowed different solubility in water, the concentration of the
compounds 1-6 used to determine the % inhibition was 666; 466; 333; 66.6; 167; and 167 µM,
respectively. When the tyrosinase solution was added to the mixture, the absorbance was
immediately measured at 475 nm and the formation of L-Dopaquinone was monitored for 30
min (Scheme 2) [31].
O
O
HO
HO
O
O
O / tyrosinase
OH
2
OH
NH2
NH2
EDTA/PBS
pH 7.4 t = 30 min
L-DOPA
L-Dopaquinone
Scheme 2. Oxidation of L-DOPA to L-Dopaquinone by tyrosinase enzyme.
The inhibition percentage was calculated according to the following equation (Eq. 5)
[
31]:
[
(B30 -B A30 A0)]
0) - (
-
(
5)
Inhibition % =
x 100
(
B30 -
0)
B
where B
0
and B30 are the absorbance of L-DOPA + tyrosinase at t = 0 min and t = 30 min,
and A30 are the absorbance of L-DOPA + tyrosinase + inhibitor at t = 0 min
respectively. A
0
and t = 30 min, respectively. In Eq. 1, the possible interference of the absorbance of organic
compounds was subtracted.
The IC50 value for the hydrazinecarbothiamides1, 2, 3 and 4 was calculated from the
equation generated by a polynomial fit of the experimental data (Origin software, ANOVA
statistical function). Commercial ascorbic acid (IC50 = 266 µM) was used as a positive
control [32]. The final inhibitor concentration to each assay which was used to calculate the
IC50 value was 66.66; 133; 200; 400; 533; and 667 µM.
24

The inhibitory mechanism of oxidation of L-DOPA on mushroom tyrosinase when in
presence of hydrazinecarbothiamides3 and 4 (the two most promising tyrosinase inhibitors)
was determined from Hanes-Woolf plots. In this analysis, different concentrations of L-
DOPA (66.6; 100; 200; 333; 467; and 667 M) as substrate and different concentrations
ofhydrazinecarbothiamides3 and 4 (0; 33.3; 50.0; and 83.0 M). L-Dopaquinone production
was analyzed in the absence and presence of the inhibitors after incubation for 30 min at 310
K and the absorbance was measured at 475 nm.
4
.4. Spectroscopic analysis of the interaction between HSA and hydrazinecarbothiamide 4
UV-vis and steady-state fluorescence spectra were measured on a Jasco J-815
spectropolarimeter using a quartz cell (1.0 cm optical path) equipped with a Peltier thermal-
-5
controlled cuvette holder Jasco PFD-425S15F. UV-vis spectrum of 4 (1.32 x 10 M, in PBS)
was measured in the 200-400 nm range at 310 K.Since, both HSA and
hydrazinecarbothiamide4 showed UV absorption in the 200-300 nm range(Fig. 12S in the
supplementary material), the effect probably observed on the UV-Vis absorption spectrum of
HSA upon ligand addition will be due to the contribution of the ligand absorption and not due
to ligand-protein complex formation. For this reason, other spectroscopic techniques were
employed, such as circular dichroism, steady state, time-resolved and synchronous
fluorescence to explore the structural change of protein and ligand-protein complex.
In order to compensate the inner filter effect, the steady-state fluorescence intensity
for HSA:4was corrected by the absorption of the ligand at excitation (λ = 280 nm) and
emission (λ = 340 nm) wavelengths (Fig. 12S in the supplementary material), using Eq. 6
[
33]:
25

(
6)
[
( A +A )/2]
ex em
F = F 10
cor
obs
where Fcor and Fobs are the corrected and the observed fluorescence intensity values,
respectively. Aex is the experimental absorbance value at the excitation wavelength in PBS
-1
-1
(
280 nm, ε = 7845 M cm ). Aem is the experimental absorbance value at the emission
-1
-1
wavelength in PBS (340 nm, ε = 1454 M cm ).
Steady-state fluorescence spectra were collected in the 290-450 nm range at 296 K,
3
03 K and 310 K, with λexc = 280 nm. Using a microliter syringe, successive aliquots of a
-5
stock solution of 4 were added to a 3.0 mL solution of HSA (1.00 x 10 M, at pH = 7.4),
leading to final ligand concentrations of 0.17; 0.33; 0.50; 0.66; 0.83; 0.99; 1.15; and 1.32 x
-
5
1
0 M.
Competitive binding studies were carried out using the HSA site markers warfarin,
ibuprofen and digitoxin. The proportion between HSA and each site marker was 1:1 (1.00 x
-5
1
0 M concentration, at pH = 7.4). Using a microliter syringe, successive aliquots of a stock
solution of hydrazinecarbothiamide4 were added to a 3.0 mL solution of HSA:site marker, at
3
10 K. The final ligand concentration after each addition was the same as described above.
Time-resolved fluorescence measurements were performed on an Edinburgh
Instruments fluorimeter model FL920 CD, equipped with an EPL laser (excitation
wavelength of 280 ± 10 nm; pulse width of 850 ps; energy per pulse of 1.8 µW). The
-5
fluorescence decay of a 1.0 x 10 M solution of HSA (3.0 mL at pH = 7.4) was monitored at
40 nm in the absence and presence of the maximum concentration of the ligand used in the
3
-5
steady-state fluorescence studies (1.32 x 10 M). The fluorescence decay lifetime for HSA
and HSA:4 was analyzed using the deconvolution software of the fluorimeter.
Synchronous fluorescence spectra (SFS) were performed in an Edinburgh Instruments
fluorimeter model Xe900. SFS were obtained by setting Δλ = 15 nm and 60 nm, for tyrosine
26

and tryptophan residues, respectively, at room temperature. Using a microliter syringe,
successive aliquots of a stock solution of hydrazinecarbothiamide4 were added to a 3.0 mL
solution of HSA. The final ligand concentrations were the same to those employed in the
steady-state fluorescence studies.
Circular dichroism (CD) spectra were recorded from 250 to 200 nm on a Jasco J-815
spectropolarimeter, equipped with a Peltier thermal-controlled cuvette holder. The spectra of
-5
a 1.00 x 10 M solution of HSA were recorded in the absence and in the presence of the
-5
maximum ligand concentration used in the steady-state fluorescence studies (1.32 x 10 M),
at 310 K. The spectra were collected and averaged over three scans. The CD signal reported
as ellipticity (in millidegrees) can be converted in mean residue ellipticity: [θ]MRW =
2
-1
θ
obs(0.1MRW)/(cl) (in deg cm dmol ), where θobs is the observed ellipticity in millidegrees,
MRW is the protein mean weight (molecular weight of the protein in daltons/number of
residues), c is the concentration in milligrams per milliliter, and l is the length of the light
path in centimeters.
4
.5. Molecular docking analysis for tyrosinase and HSA
The chemical structure of the hydrazinecarbothiamides under study, i. e1-6, and L-
DOPA were built and energy-minimized by Density Functional Theory (DFT) calculations
(
B3LYP potential) with basis set 6-31G*, available in the Spartan'14 program.
The molecular docking studies were performed with the GOLD 5.2 program (CCDC,
Cambridge Crystallographic Data Centre) and the crystallographic structure of tyrosinase
and HSA were obtained from Protein Data Bank, with access code 2Y9X [34] and 1N5U [10],
respectively. Hydrogen atoms were added to each protein structure according to the data
inferred by GOLD 5.2 program on the ionization and tautomeric states. For tyrosinase, a 10 Å
radius spherical cavity around the dicopper center was defined as the binding site for the
27

molecular docking calculations. On the other hand, for HSA molecular docking interaction
cavity in the protein was established with a 10 Å radius from the Trp-214 residue. The
scoring function used was ‘GoldScore’ and ‘ChemPLP’, for tyrosinase and HSA, respectively.
For furtherinformationseepreviouspublications [8,32,35].
Acknowledgments
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). The authors also acknowledge Prof. Dr. Nanci Camara
de Lucas Garden (Chemistry Institute - UFRJ) for the time-resolved and synchronous
fluorescence facilities.
References
[
1]Y. Wang, M.-M. Hao, Y. Sun, L.-F. Wang, H. Wang, Y.-J. Zhang, H.-Y. Li,P.-W.
Zhuang, Z. Yang, Synergistic promotion on tyrosinase inhibition by antioxidants,
Molecules23 (2018) 106-118.
[
2]N. Gençer, D. Demir, F. Sonmez, M. Kucukislamoglu, New saccharin derivatives as
tyrosinase inhibitors,Bioorg. Med. Chem. 20 (2012) 2811–2821.
[
3]O.A. Chaves, M.R.L. Santos, M.C.C. de Oliveira, C.M.R. Sant'Anna, R.C. Ferreira, A.
Echevarria, J.C. Netto-Ferreira, Synthesis, tyrosinase inhibition and transportation behavior
of novel β-enamino thiosemicarbazide derivatives by human serum albumin, J. Mol. Liq. 254
(
2018) 280-290.
[
4]J. Liu, W. Yi, Y. Wan, L. Ma, H. Song, 1-(1-Arylethylidene) thiosemicarbazide
derivatives: A new class of tyrosinase inhibitors,Bioorg. Med. Chem. 16 (2008) 1096-1102.
28

[
5]J. Liu, C. Chen, F. Wu, L. Zhao, Microwave-assisted synthesis and tyrosinase inhibitory
activity of chalcone derivatives, Chem. Biol. Drug Des. 82 (2013) 39–47.
[
6]I. Strelec, P. Burić, I. Janković, T. Kovač, M. Molnar, Inhibitory effect of coumarin
derivatives on apple (cv. Idared) polyphenol oxidase. Croat. J. Food Sci. Technol. 9 (2017)
7-65.
5
[
7]A.S. Abdelhameed, A.H. Bakheit, F.M. Almutairi, H. AlRabiah, A.A. Kadi, Biophysical
and in silico studies of the interaction between the anti-viral agents acyclovir and penciclovir,
and human serum albumin, Molecules 22 (2017) 1906-1920.
[
8]O.A. Chaves, A.P.O. Amorim, L.H.E. Castro, C.M.R. Sant'Anna, M.C.C. de Oliveira, D.
Cesarin-Sobrinho, J.C. Netto-Ferreira, A.B.B. Ferreira, Fluorescence and docking studies of
the interaction between human serum albumin and pheophytin, Molecules 20 (2015) 19526-
1
9539.
[
9]A. Szkudlarek, D. Pentak, A. Ploch, J. Pozycka, M. Maciazek-Jurczyk,In vitro
investigation of the interaction of tolbutamide and losartan with human serum albumin in
hyperglycemic states, Molecules 22 (2017) 2249-2277.
[
10]M. Wardell, Z. Wang, J.X. Ho, J. Robert, F. Ruker, J. Ruble, D.C.Carter, The atomic
structure of human methemalbumin at 1.9 Å,Biochem. Biophys. Res. Commun,291 (2002)
13–819.
8
[
11]C.M. dos Reis, D. Sousa-Pereira, R.O. Paiva, L.F. Kneipp, A. Echevarria,Microwave-
assistedsynthesisofnew N ,N -substituted thiosemicarbazones,Molecules16 (2011) 10668-
0684.
1
4
1
[
12]A.F.M. Fahmy, A.A. El-Sayed, M.M. Hemdan, A.I. Hassaballah, A.F. Mabied, Synthesis
of N-containing heterocycles via mechanochemical grinding and conventional techniques,
Asian J. Chem. 29 (2017) 2679-2686.
29