Palladium(ii) complexes bearing 1-iminothiolate-3,5-dimethylpyrazoles: Synthesis, cytotoxicity, DNA binding and enzymatic inhibition studies

Article abstract of DOI:10.1039/d0nj02825h

Four palladium(ii) compounds of general formulae [PdCl(Ln)(PPh3)] {L1 = 3,5-dimethylpyrazole-1-iminothiolate (1); L2 = 3,5-dimethyl-pyrazole-N-methyl-1-iminothiolate (2); L3 = 3,5-dimethylpyrazole-N-ethyl-1-iminothiolate (3); L4 = 3,5-dimethylpyrazole-N-phenyl-1-iminothiolate (4); and PPh3 = triphenylphosphine} have been synthesized. The novel synthesized compounds have been characterized by C, H and N elemental analysis, 1D (1H and 13C) and 2D (HSQC and HMBC) NMR, MS, FT-IR, and molar electrical conductivity measurements. The molecular structure of complex 3 has been solved by single-crystal X-ray crystallography. The stability of the complexes in solution was studied in a DMSO/D2O (7?:?3) solution after 48 h. The antiproliferative activity of all free ligands and the stable palladium complexes 2-4 was assayed using the human breast tumour cell line MCF-7, lung tumour cell line A549 and human fetal lung fibroblast cell line MRC-5. Complex 3 was more active than cisplatin against MCF-7 cells, whilst palladium compounds 2-4 exhibited no drug response towards A549 cells at concentrations <50 μM. The binding properties of compounds 2 and 3 to ct-DNA have been studied using circular dichroism and fluorescence spectroscopy. The topoisomerase IIα inhibition has been studied for complex 2 and 3. The ability of all complexes to inhibit the activity of cathepsin B and L has also been investigated in this work. Compound 4 inhibited more than 50% of the cathepsin B activity at a concentration of 10 μM. Docking simulations have been carried out to gain more information about the interaction of the complexes and cathepsin B.

Full text of DOI:10.1039/d0nj02825h

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Palladium(II) complexes bearing 1-iminothiolate-3,5-
dimethylpyrazoles: synthesis, cytotoxicity, DNA binding and
enzymatic inhibition studies
Thales Reggiani de Moura,*a Renan Diego Zanetti,a Debora Eduarda Soares Silva,a Renan Lira de
Farias,a Antonio Eduardo Mauro,a José Clayston Melo Pereira,a Aline Aparecida de Souza,b Fábio da
Silva Siqueira,b Wagner Alves de Souza Júdice,b Mauro Almeida Lima,c Fillipe Vieira Rocha,c Victor
Marcelo Deflond and Adelino Vieira de Godoy Netto#a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Four palladium(II) compounds of general formulae [PdCl(Ln)(PPh3)] {L1 = 3,5-dimethylpyrazole-1-iminothiolate (1); L2 = 3,5-
dimethyl-pyrazole-N-methyl-1-iminothiolate (2), L3
= 3,5-dimethylpyrazole-N-ethyl-1-iminothiolate (3); L4 = 3,5-
dimethylpyrazole-N-phenyl-1-iminothiolate (4); PPh3 = triphenylphosphine} have been synthesized. The novel synthesized
compounds have been characterized by C, H and N elemental analysis, 1D (1H and 13C) and 2D (HSQC and HMBC) NMR, MS,
FT-IR, and molar electrical conductivity measurements. The molecular structure of complex 3 has been solved by single-
crystal X-ray crystallography. The stability of the complexes in solution was studied in a DMSO/D2O (7:3) solution after 48h.
The antiproliferative assay of all free ligands and the stable palladium complexes 2-4 were assayed using the human breast
tumour cell line MCF-7, lung tumour cell line A549 and human fetal lung fibroblast cell line MRC-5. Complex 3 was more
active than cisplatin against MCF-7 cells whilst palladium compounds 2-4 exhibited no drug response towards A549 cells on
concentrations < 50 μM. The binding properties of compounds 2 and 3 on ct-DNA have been studied using circular dichroism
and fluorescence spectroscopy. The Topoisomerase IIα inhibition has been studied for complex 2 and 3. The ability of all
complexes to inhibit the activity of cathepsin B and L has also been investigated in this work. Compound 4 inhibited more
than 50% the cathepsin B activity at a concentration of 10 μM. Docking simulations have been carried out to gain more
information about the interaction of the complexes and cathepsin B.
In previous studies we have described the synthesis of
compounds of the type [PdX(tcz)(PPh3)]X (tcz
1. Introduction
=
4-
methylthiosemicarbazide, 4-phenylthiosemicarbazide; PPh3 =
triphenylphosphine; X = Cl, I, NCS).11 These compounds were
more cytotoxic than cisplatin against a panel of tumour cells
Over the last three decades, much research efforts have
been devoted to PdII complexes in the search for novel metal-
based drugs.1-8 Several biologically active PdII compounds have
been described in the literature and particular attention has
been drawn to complexes bearing N,S-chelating ligands, such as
with IC50 values below 10 M and showed a limited reactivity
towards the model nucleobase guanosine and DNA,11
suggesting that their mechanism of action may include not only
interaction with DNA but also binding to other pharmacological
targets. In fact, some of these PdII complexes induced the
thiosemicarbazides
and
thiosemicarbazones.9-13
The
incorporation of such ligands into PdII compounds is a well-
known strategy to enhance their kinetic stability in solution and
consequently to reach their pharmacological targets in
significant concentration.14
inhibition of human Topoisomerase II
 (hTop2) at 5 μM.
Moreover, complexes [PdI(tcz)(PPh3)]I induced the inhibition of
cathepsin B activity with IC50 values inferior to 10 μM.11 Similar
compounds of the general formulae [PdX(TSC)(PPh3)] (X = Cl,
NNN, NCS) and [PdI(TSC)(PPh3)]I (TSC = (2E)-2-[(2E)-3-phenyl-1-
ylidene]hydrazinecarbothioamide) demonstrated to be active
against MCF-7 cells with IC50 values ranging from 1.81–4.46 μM
and able to prevent hTop2 activity at 6.25–25 μM.12 These
findings have prompted us to investigate the influence of the
type N,S-chelating ligand on the antiproliferative activity and
ability to inhibit hTop2 and cathepsins.
a.UNESP – Univ Estadual Paulista, Instituto de Química, Departamento de Química
Geral e Inorgânica, 14800-060, Araraquara, SP, Brazil. Email:
thales4014@gmail.com, adelino.netto@unesp.br .
b.UMC –Univ de Mogi das Cruzes, Centro Interdisciplinar de Investigação
Bioquímica, 08701-970, Mogi das Cruzes, SP, Brazil.
c. UFSCar – Univ Federal de São Carlos, Departamento de Química, 13565-905, São
Carlos, SP, Brazil
d.USP – Univ de São Paulo, Instituto de Química de São Carlos, 13566-590, São
Carlos, SP, Brazil
†
Electronic Supplementary Information (ESI) available: Relevant geometrical
In this work, we sought to investigate the effects of
replacing thiosemicarbazides/thiosemicarbazones by 1-(N-
substituted)-thiocarbamoyl-3,5-dimethylpyrazoles
parameters for the complex, DNA Interaction (Spectroscopic Titration, Circular
Dichroism and Hoechst 33258) and Topoisomerase IIα experimental methods,
Docking studies and modelling calculation, vibrational (FTIR), NMR and ESI-MS
spectroscopic data see DOI: 10.1039/x0xx00000x
on
cytotoxicity and inhibition of hTop2 and cathepsins B and L. The
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Scheme 1
Synthetic pathways for the preparation of complexes 1–4 and numbering adopted for the signals assignment observed in the NMR spectra.
choice of using, such ligands was motivated by our previous ligand and triphenylphosphine in chloroform in a molar ratio of
studies on the cytotoxic activity observed for their palladium 1:1:1, respectively. Deprotonation of coordinated 1-
derivatives.15,16
In the framework of our current research on the achieved by washing the organic reaction media with water
coordination and biological chemistry of metal-based until the aqueous solution gives a pH 7. Washing the organic
compounds,11,12,15,16 we describe herein the synthesis and solution with water allowed to segregate the HCl produced,
characterization of the compounds [PdCl(Ln)(PPh3)] where L1 = avoiding the formation of cationic complexes.
3,5-dimethylpyrazole-1-iminothiolate (
pyrazole-N-methyl-1-iminothiolate
dimethylpyrazole-N-ethyl-1-iminothiolate
dimethylpyrazole-N-phenyl-1-iminothiolate
antiproliferative activity against human breast and lung tumour conditions brings the product decomposition in phenyl
cell lines (MCF-7 and A549, respectively) together with their isothiocyanate and 3,5-dimethylpyrazole. Taking into account
ability to induce the inhibition of hTop2 and cathepsin B and L that the synthesis of pyrazoles from 1,3-diketones with
thiocarbamoyl-3,5-dimethylpyrazoles HL1-HL3 has been
1
), L2 = 3,5-dimethyl-
Although the conventional methodology employing acid
(
2
),
L3
=
3,5- catalysis has been successful for obtaining HL1 HL3, several
-
(
3
), L4
=
3,5- attempts to prepare the N-phenyl-1-thiocarbamoyl-3,5-
(
4
).
Their dimethylpyrazole (HL4) have proved fruitless. The acid
have been evaluated in this work.
hydrazines involves the formation of hydroxypyrazoline
intermediates,19 we hypothesized that HL4 could be obtained in
situ upon coordination and further dehydration of its
corresponding hydroxypyrazoline HL4’. Thus, we have prepared
the intermediate HL4’ by the condensation reaction under
neutral conditions20 (see Scheme 1). The characterization of
HL4´ is discussed in the ESI file. The reaction between
[PdCl2(MeCN)2], HL4’ and PPh3 has been carried out in methanol
for 24 h. After evaporating the solvent almost to dryness, the
residue was mixed with chloroform. The resulting organic
mixture was washed with water until the aqueous phase gives
a neutral pH. This procedure has not yielded the corresponding
2. Results and discussion
2.1. Synthesis and characterization of the complexes
The ligands and their palladium(II) derivatives have been
synthetized via the process shown in Scheme 1. As reported
previously, 1-thiocarbamoylpyrazoles HL1
the acid-catalyzed condensation of thiosemicarbazide and 2,4-
pentadienone in water.11,15,17,18 Complexes
prepared from the reaction
-HL3 were obtained by
1
-
3
have been
between
bis(acetonitrile)dichloropalladium(II), the suitable pyrazolyl
pyrazoline-based complex, but the pyrazole complex
4
instead.
Table 1 Selected signals in 1H and 13C DEPTQ NMR, in ppm, for 1-4
Complex 1
Complex 2
Complex 3
Complex 4
Position
1
1H
13C
1H
13C
1H
13C
1H
13C
2.71s
15.5
2.69s
15.5
2.69s
15.5
2.74s
15.5
156.1d
154.6d
154.5d
155.8
2
3
-
-
-
-
(3JCP = 3.3)
111.2d
(3JCP = 3.3)
110.5d
(3JCP = 2.2)
110.4d
(3JCP = 3.3)
111.1d
5.99d
(5JHP = 2.0)
5.92d
(5JHP = 2.6)
5.91d
(5JHP = 1.8)
6.02d
(5JHP = 2.2)
(4JCP = 5.5)
164.3d
(4JCP = 5.5)
156.9d
(4JCP = 4.4)
154.4d
(4JCP = 5.5)
156.8d
6
-
-
-
-
7.73m
-
(3JCP = 5.5)
(3JCP = 5.5)
(3JCP = 5.5)
(3JCP = 4.4)
134.6d
134.8d
134.8d
134.7d
8
7.77m
8.38br
7.79m
3.06s
7.79m
(2JCP = 11.1)
(2JCP = 11.1)
(2JCP = 11.1)
(2JCP = 11.1)
3.27q
(3JHH = 7.2)
11
38.7
46.2
148.2
s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet; br = broad.
2 | J. Name., 2012, 00, 1-3
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The dehydration of the hydroxypyrazoline HL4’ was clearly
detected in the 1H NMR spectrum of
by the following
View Article Online
DOI: 10.1039/D0NJ02825H
4
evidences (see Fig. S28-S32 in ESI): i) the disappearance of the
doublets at 3.2 and 2.9 ppm (18.3 Hz) attributable to the
methylene protons at C4 of hydroxypyrazoline nucleus; ii) the
lack of the signal at 6.48 ppm assigned to -OH group at 5-
position; iii) the appearance of a new doublet at 6.01 ppm (4JP-H
= 2.2 Hz) related to H-4 atom of the pyrazolyl nucleus. The
dehydration of the pyrazoline ligand HL4’ has occurred in situ,
merely on the complex shape. This dehydration is expected to
occur by the H+ byproduct issued from the thioamide group
after coordination to palladium, catalyzing the final dehydration
to bring the pyrazole moiety. All four compounds were isolated
as air-stable and nonhygroscopic solids, soluble in chloroform
and DMSO and showed colours varying from yellow to orange.
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1
Complexes 1-4 were characterized by 1D (31P, H, DEPTQ)
Fig. 1 ORTEP representation of cis-[PdCl(N,S-L3)(PPh3)] (3), showing the labelling of
the atoms. Displacement ellipsoids are drawn at the 50% probability level.
and 2D (HSQC, HMBC) NMR and IR spectroscopy, CHN analyses,
molar conductance and X-ray crystallography (complex 3). The
CHN analyses of the complexes are consistent with the formulae
[PdCl(Ln)(PPh3)]. Molar conductivity results of all complexes in
DMSO (1,0 mmol L−1) were observed in the range of 2.23-4.50 S
cm2 mol−1, indicating that the complexes are non-electrolytes.
21
bond character of the CN bond on deprotonation of the
thiocarbamoyl moiety and S-coordination.26 Intense IR
absorptions of the coordinated PPh3 ligand were found at 1480
cm-1 (νCC), 1097 cm-1 (νP-C) and 747 cm-1 (νC-H).27
The ESI-MS study confirmed the molecular formula
proposed for all complexes. In the positive mode a main peak
assigned to the singly charged form of (M+) or its chloride-
eliminated species (M-Cl-) were obtained.
The obtained 1H/13C/31P-NMR spectra showed good purity
1
for all complexes. The H NMR spectra of 1-4 in CDCl3 showed
only one set of signals for coordinated L1
which evidence the existence of single species in solution. H
-
L4 and PPh3 ligands,
1
2.2. X-ray crystal structure of complex 3
NMR spectra of
1-4 indicated the deprotonation of the
Crystals suitable for X-ray diffraction studies were obtained in a
vapour diffusion system, using chloroform as solvent and
thioamide group with the lack of N-H signal and its coupling with
CH2 or CH3 (3JNH-CH = 4.95 and 5.50 Hz for L2 and L3 respectively).
Also, the shielding of H11 (L2: 3.24 ppm; L3: 3.74 ppm) and
deshielding of H1 (L2 and L3: 2.22 ppm) signals in comparison
with the free ligands strongly suggests the N,S-anionic
coordination mode in all cases. The coordination of PPh3 is
observed by the deshielding of phenyl signals in 1H and 13C NMR.
Long-range couplings 5JP-H3 (1.8-2.2 Hz) and 4JP-C3 (4.4-5.5 Hz) in
the spectra are typical of the formation of the H4–C–C=N–Pd–P
system and therefore the Pd-pyrazole-PPh3 complexes. Long-
range 31P-1H spin-spin couplings is dependent on the extent of
coplanarity of the linking bonds and are in agreement with the
expected highly planar complexes structure.22,23 The major
changes in NMR spectra are showed in Table 1. The 31P NMR
spectra of complexes in DMSO-d6 display a singlet in 32.51-
32.69 ppm. The chemical shifts are consistent with a structure
in which the phosphorus atom is trans to an iminic nitrogen
atom.22,24
methanol as antisolvent.28 The crystal structure of
3 has been
determined by single-crystal X-ray diffraction, and its ORTEP
representation with the atom labeling scheme is illustrated in
Fig. 1. Selected interatomic bond distances and angles with their
estimated standard deviations are shown in Table 2.
Complex
3 crystalizes in the orthorhombic space group
Pbca. The palladium(II) center is coordinated in slightly
distorted square-planar geometry with one deprotonated 3,5-
dimethylpyrazole-N-ethyl-1-iminothiolato acting as N,S-
bidentate ligand via its sulfur (Pd-S = 2.2542(11) Å) and pyridine-
Table 2 Selected geometric parameters (Å, °) for cis-[PdCl(N,S-L3)(PPh3)] (3)
Bond distances
Pd1—S1
2.2542(11)
Pd1—P1
2.2482(10)
Pd1—N1
Pd1—Cl1
Cl···H1−C1
Cl···H16−C16
2.121(3)
2.3376(11)
2.74 (Cl···C: 3.285(5))
2.79 (Cl···C: 3.353(4))
The infrared spectra of all compounds share the
characteristic absorption bands of the N,S-anionic coordination
mode in the complexes. Firstly, the lack of the strong and broad

NH band (3240-3325 cm-1) indicates the deprotonation of the
thiocarbamoyl moiety. Secondly, the shift of the absorption
Bond angles
S1—Pd1—P1
S1—Pd1—N1
N1—Pd1—Cl1
P1—Pd1—Cl1
P1—Pd1—N1
Cl1—Pd1—S1
C6—N3—C7
91.44(4)
83.83(9)
99.97(9)
84.75(4)
174.49(9)
176.18(4)
117.8(4)
band attributed to ring stretching mode (ring) to lower
frequency (1435-1437 cm–1) when compared with that one of
the free ligands (1574–1593 cm–1).25 Thirdly, the shift to higher
frequency of the thioamide I band (
1523 cm-1 in free ligands L2 L4 (1599 cm-1 for L1) to ca. 1578-1610
cm-1 (2086 cm-1 for complex
) because of the greater double
CN + NH) from ca. 1513-
-
1
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like nitrogen (Pd-N = 2.121(3) Å) atoms, forming a five-
membered metallocycle with a bite angle N(1)-Pd-S(1) of
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DOI: 10.1039/D0NJ02825H
Table 3 EC50(μM) for Cisplatin and Compounds 2−4
EC50 (M)
83.83(9)°. The chlorido group is oriented trans to the sulfur
atom, while the triphenylphosphine ligand is trans to the
coordinated nitrogen (Fig 1). The short exocyclic C(6)-N(3) bond
length of 1.258(6) Å is consistent with an enhanced double
bond character, confirming that the thiocarbamoylpyrazolyl
ligand is in the thiolate form.29,30 In addition, the C(6)-S(1) bond
distance of 1.741(4) Å is indicative of an increased single bond
nature. The Pd–N bond length of 2.121(3) Å is longer than the
predicted single bond value of 2.011 Å, based on the sum of
covalent radii for N(sp2) and Pd, 0.701 and 1.31 Å,
respectively,31 and is attributed to the trans effect of the
phosphine ligand. The Pd–P bond distance of 2.2482(10)Å is
smaller than the values found for a number of neutral
complexes of the type [PdCl(N,S-TSC)(PPh3)] (2.2526-2.2707 Å)
Compounds
MCF-7
47.57 ± 1.16
31.94 ± 3.35
>78
A549
69.37 ±4.32
71.795 ± 7.15
>78
MRC-5
26.31 ± 0.38
26.32 ± 0.15
>78
2
3
4
Cisplatin
>50
44.39 ± 0.17
19.86 ± 1.22
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decrease of the cytotoxicity against MCF-7. For instance,
complex (31.94 ± 3.35 μM) was less active than [PdCl(PPh3)(
cinnamaldehydethiosemicarbazonato)] (4.46 ± 1.23 μM)12 and
[PdCl(PPh3)(4-methyl-3-thiosemicarbazide)]Cl (5.92 0.59
3
±
μM).11 It is important to emphasize that this structure-activity
relationship is only preliminary taking into account the limited
number of PdII compounds synthesized.
In order to shed some light on the ability of compounds
2
; PY4MeTSC';
{N,S-TSC: MTPTSC 32 33 4MeBTSC34}.
and to interact with some possible pharmacological targets,
preliminary DNA binding studies and enzymatic inhibition
assays were performed.
3
2.3. Stability in solution
Time-dependent experiments in solution phase were
carried out aiming at evaluating the stability of palladium
complexes under biologically occurring conditions. The
2.5. DNA binding studies
UV–visible absorption spectroscopy is one of the most
employed methods for detecting the binding of metal
complexes with DNA by monitoring the spectral changes in the
CT bands upon addition of increasing concentrations of DNA.36
behavior of
1
-
4
in DMSO-d6 solution and phosphate buffered
-
saline solution ([H2PO4 ] = 10 mM, pH 7.3; deutered solvent: D2O
3:7 DMSO-d6) was studied by monitoring the changes in H/31P
1
NMR spectra at 10 min, 24 and 48 h after sample preparation
(Figs. S33-S39 in ESI). With exception of complex 1, all palladium
The interaction of
2 and 3 with DNA was firstly verified by
spectrophotometric titration using fixed concentrations of the
complexes and varying the concentration of the ct-DNA.
Successive additions of both complexes led to a hypochromism
(up to 14%) without significant red shift of the CT band at 258
nm as shown in Fig. 2a-b. The intrinsic constant Kb was
calculated through the Wolfe-Shimer37 relationship, with values
compounds demonstrated to be stable over a period of 48 h in
the presence of DMSO, and only a minor additional signal (less
than 5%, obtained by measuring the relative heights of their
corresponding 1H NMR signals) occurs in a phosphate buffered
saline solution. Taking into account the low stability of complex
1
in DMSO-d6 (see 31P NMR in Fig. S33, ESI), its biological effects
found in the order of 6.05x104 M-1 (log Kb 4.78) for
7.12x104 M-1 (log Kb 4.85) for
. Such Kb values are significantly
2 and
were not studied in this work.
3
lower than those ones found for ethidium bromide (4.94 x 105
M-1) and other classical metallointercalators (105 - 106 M-1).38,39
Aiming at providing additional evidence of the DNA binding
2.4. Antiproliferative assay in vitro of 2-4
The antiproliferative effect of complexes 2, 3, and 4 was
evaluated against the tumor cell lines MCF-7 and A549 using the
MTT cell survival assay,35 cisplatin was used as a positive
control. The EC50 values of the compounds were calculated and
mode of
2 and 3, circular dichroism (CD) and competitive
binding experiments were performed.
Circular dichroism (CD) is a powerful method to investigate
the interaction between metal complexes and DNA.40,41 As can
be seen in Fig. 2c-d, no appreciable changes in the typical CD
are listed in Table 3. The ligands L2
towards drug concentrations <200 μM for both tumor cell lines.
For breast tumor cells (MCF-7), compound displayed
medium EC50 value of 31.94 μM, being up to ca. 1.5 fold more
active than and cisplatin. Complex demonstrated to be
inactive towards MCF-7. With respect to the antiproliferative
effects on lung tumor cells (A549), compounds exhibited no
-L4 exhibited to be inactive
3
bands of ct-DNA with increasing concentration of of
2 and 3
were observed, indicating that B-DNA structure remains
unchanged upon the interaction of complexes and DNA.
2
4
Competitive binding experiments with Hoechst 33258 were
also carried out. Hoechst 33258 binds to the minor groove of
DNA, providing fluorescence at 458 nm when excited around
350 nm. The quenching of the intrinsic fluorescence of Hoechst-
bound DNA system with increasing concentration of metal-
based compounds can be employed to investigate whether the
metal complex can interact with DNA and displace the Hoechst
33258.42-44 As shown in Fig. 2e-f, the emission intensity of
Hoechst-bound DNA decreased by ca. 75% upon addition of
2-4
drug response at drug concentrations <50 μM, and therefore
were considered inactive. As a comparison, the effect of
complexes
was examined. The compounds
comparable EC50 values, whereas complex
2
-
4
on non-tumor human lung fibroblasts (MRC-5)
and and cisplatin showed
was inactive.
2
3
4
From the inspection of Table 3, it was noticed that the
replacement of thiosemicarbazides/thiosemicarbazones by 3,5-
dimethylpyrazole-N-substituted-1-iminothiolato ligands into
the molecular structure of [PdCl(N,S-ligand)(PPh3)] resulted in a
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Fig. 2 a,b) Absorption spectra of compounds 2 (a) and 3 (b) with increased amounts of DNA at pH 7.3 in tris-HCl buffer. The arrows show the decreasing of the absorbance with
respect to an increase in the DNA concentration (inset: Plot of [DNA]/(εa – εf) [DNA] vs at λ=258 nm); c,d) Circular dichroism spectra of ct-DNA in increased amounts of compound
2 (c) and compound 3 (d) at pH 7.3 in tris-HCl buffer; e,f) Fluorescence emission spectra (λexcitation= 340 nm) for the Hoechst 33258-DNA solution ([Hoechst]= 6 µM, [DNA]= 60 µM)
with increased amounts of complex 2 (e) and 3 (f) at pH 7.3 in tris-HCl buffer. The arrows show the decreasing of the fluorescence intensity with respect to an increase in the
compound concentration (inset: Plot of relative fluorescence intensity (F/F0, %) vs ratio [Complex]/[DNA] at λemission= 458 nm).
palladium complexes. The quenching constants (Ksv) values dimethylpyrazole-N-substituted-1-iminothiolato
calculated for compounds and
were 6.86x103 M-1 (log Ksv afforded inactive compounds towards hTop2.
3.84) and 5.36x103 M-1 (log Ksv 3.73), respectively. This finding
indicates that complexes and can interact with DNA and can 2.7. Preliminary studies on Cathepsin B and L inhibition
ligands
2
3
2
3
displace the Hoechst 33258 from the minor groove. It is
important to point out that other agents that bind elsewhere on
Cathepsin B and L belong to a family of cysteine peptidase
of the papain family. Besides their involvement in normal
physiological processes, abnormal cathepsin B or L activities are
linked in many serious diseases such as cancer, autoimmune
disorders and osteoporosis.48,49
the DNA can also replace Hoechst.45 Although
2 and 3 are able
to displace Hoechst, it seems reasonable to suggest that both
complexes are a less strong binder, since the Hoechst-bound
DNA fluorescence is not totally quenched even in presence of a
9-fold excess of the palladium complexes.
In this work, complexes 1−4 were initially evaluated for their
inhibitory activity against human cathepsin B and L in
2.6. Human topoisomerase II inhibition assay
Type II topoisomerases are a class of ubiquitous enzymes
that alter DNA topology by catalyzing the passing of an intact
DNA double helix through a transient double-stranded break
made in a second helix. Since hTop2 is highly up-regulated in
some cancer cells, it is considered to be the primary
pharmacological target for some of the most active drugs
currently available for the treatment of human malignancies, as
etoposide and doxorubicin.46 The ability of compounds
2 and 3
to induce the in vitro inhibition of hTop2 was also evaluated in
this work by agarose gel electrophoresis experiments (Fig. 3).
Circular pBR322 plasmid DNA was incubated with the
enzyme in the presence of 12.5, 25, 50 and 100 μM of the
complexes. None of the compounds was able to inhibit hTop2
in the tested concentrations. This findings may indicate that the
replacement of thiosemicarbazides/thiosemicarbazones from
Fig. 3 Topoisomerase IIα relaxation assay. C+ (supercoiled plasmid, topoisomerase and
ATP), C− (supercoiled plasmid) and different concentrations (μM) of complex 2 (left) and
3 (right)
the
structure
of
[PdCl(N,S-ligand)(PPh3)]
by
3,5-
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Fig. 4 Cathepsin L and B resisual activity in presence of 1-4 in 10 and 100 μM. *insoluble at 100 μM in 100 mM sodium acetate buffer as item 3.3
concentrations of 10 and 100 µM after 5 min of incubation (data 2.10. In silico approach
are reported in Fig. 4).
The 3-D molecular structures of
1
-
4
were built with chloride
At high concentration (100
induced a reduction of cathepsins B and L activity to ca. 30-55
% whilst complexes and were insoluble. With respect to the
inhibitory effects on cathepsins B and L at 10 M, a progressive
M), compounds 1 and 2
ligand given the previous stability studies. The geometry
optimized molecules were achieved out by means of the
QM/MM calculations.61,62 The root-mean-square deviation
(RMSD) retrieved from an overlay of heavy atoms from the
theoretical and molecular crystal structure of 3, which amounts
to 0.4884 Å, endorses the quality of the modelling. See ESI for
more details (Fig S2).
3
4

decrease on the residual enzymatic activity was observed
according to the R-substituent group at thioamide moiety,
following the order:
The active site of the two-folded cathepsin B covers an
active cysteine (Cys29), from the left domain, keeping in contact
with a right one histidine (His199) through an ion pair. Based on
cysteine protease studies,63-66 it is well-known that residues
localized in the S-site are responsible for the enzyme specificity,
notable S2 since Sn’ is restricted by the occluding loop. Herein
Cathepsin L: R = Et (97%) > H (91%) > Me (78%) > Ph (61%)
Cathepsin B: R = Et (98%) > H (91%) > Me (75%) > Ph (42%)
Compound
complexes tested at 10
B and L to ca. 58 and 39 %, respectively. It is worth mentioning
that inhibition of cathepsin B by was comparable to that of
observed for similar complexes of the type [PdI(tscz)(PPh3)]I
(tscz 4-methyl-3-thiosemicarbazide; 4-phenyl-3-
thiosemicarbazide) with IC50 < 10 μM.11
4
demonstrated to be the most active among all

M, inhibiting the activity of cathepsins
based on docking studies, the ranked binding modes for
were similar, exhibiting only slight changes in the position (Fig
5a-b). These complexes were not able to interact with active
Cys29, and is in unfavourable proximity to the side chain of
Met196 (N42…OMet196, d = 2.80 Å). However, the presence of
a N-methyl group at thioamide in favoured a hydrogen
1 and
4
2
=
1
The design of new cathepsin B inhibitors is of particular
interest since its aberrant activity is associated with tumor
invasion and metastasis.49,50 Thus, cathepsin B is considered to
be an attractive target for the control of tumor progression.
Taking into account that the active site of cathepsin B contains
a thiolate-imidazolium ion pair vital for the catalytic activity, a
strategy to inhibit the proteolytic activity of cathepsin B consists
of designing molecules able to establish reversible or
irreversible bonds with the reactive thiolato group.51,52 Due to
its high affinity for sulfur-based ligands, PdII center has been
investigated as potential cathepsin B inhibitor.53-60 As
hypothesized by Spencer et al.,59 the thiolato group is expected
to replace a labile ligand from the palladium complex whereas
the co-ligand(s) may establish additional attractive contacts
within the active site pocket. However, more detailed studies
are required in order to confirm the binding of a PdII center to
the catalytic cysteine residue.
2
bonding interaction with Met196. According to our model,
pyrazolyl nucleus of all complexes seemed to play an important
role for protein-ligand recognition in S-binding pocket by the
establishment of lone pair hydrophobic interactions with
Gly198 (Fig 5a-c) and Glu122 (Fig 5d). Likewise, as the nonpolar
character increases at N-terminal thioamide position, the ligand
conformations into the binding site changes. Thus,
afforded hydrophobic lone pair- interactions with side-chain
of Cys29 by the aromatic rings of triphenylphosphine. Only
complex established additional interactions with the side-
3 and 4

4
chains of Gln23 and Gly73, which were as important as for the
inhibitor conformation viewed from the crystal structure67 (Fig
5d and S1). Accordingly, these computational outcomes agree
well with the enhanced in vitro inhibition of cathepsin B by
compound 4.
In order to gain additional information about the possible
non-covalent interactions between complexes
cathepsin B, complementary theoretical studies have been
undertaken by molecular docking.
1-4 and
3. Experimental
All solvents and reagents were purchased commercially and
used without further purification unless otherwise noted. Calf
thymus DNA sodium salt (CT DNA) was obtained from Sigma
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Fig. 5 The best docking pose of the 1-4 at S-site from cathepsin B. The a-d poses are 1-4, respectively. Interacting residues are shown as sticks. The key side-chains are
shown as C-atom in light blue, O-atom in red, N-atom in royal blue, S-atom in yellow. The complexes are shown as C-atom in grey, Cl-atom in green, P-atom in orange
and Pd-atoms in cyan. Non-interacting H-atoms were omitted for clarity.
Aldrich. Cell lines of MCF-7 were obtained from Banco de The compound was stored in the freezer (T < 0o C). yield 137.1
Células do Rio de Janeiro (Rio de Janeiro, RJ, Brazil), A549 and mg (97%) of L4’. 1H NMR (600 MHz, CDCl3): δ = 8.97br ([1], NH),
MRC-5 were generously donated by Dr. Alzir Azevedo Batista, 7.51m (7.8 Hz [2H], ortho-phenyl), 7.37t (3J = 7.8 Hz [2H], meta-
from DQ - UFSCar (São Carlos, SP, Brazil), and by Dr. Fernando phenyl), 5.92t (3J = 7.8 Hz [1H], para-phenyl), 6.48br ([1H], OH),
Rogério Pavan, from FCF/UNESP (Araraquara, SP, Brazil), 3.22d (2J = 18.3 Hz [1H], CH2), 2.92d (2J = 18.3 Hz [1H], CH2) 2.09s
respectively. Mass spectra were recorded on a 3200QTRAP (AB ([3H], CH3, H1) 2.05s ppm ([3H], CH3, H5). DEPTQ-135 NMR (125
Sciex) spectrometer. The data of elemental analysis were MHz, CDCl3): δ = 172.9 (C=S), 155.0 (C2), 137.7 (C7), 128.7 (C9),
carried out on a Perkin Elmer 2400 series II. IR spectra of solid 126.1 (C10), 125.4 (C8), 94.2 (C4), 52.8 (C3), 27.6 (C5), 16.3 ppm
samples (KBr pellets) were obtained on a Nicolet IS5 Thermo (C1). IR (KBr): 3325 (νOH), 3250(νNH), 1593 (νCNring), 1513
Scientific spectrometer in the 4000–400 cm-1 region. The NMR (νCNthioamide), 1245 (δC-N), 1123 (δC-O), 1054 (νNNring + δrCH3)
spectra were recorded on a Bruker Avance III HD 600 801 cm-1 (νC-S). Calcd. for C12H15N3OS (249.3): C, 57.81; H, 6.06;
spectrometer with DMSO-d6 or CDCl3 as solvent at room N, 16.85; found: C, 57.45; H, 6.51; N, 17.17.
temperature. The chemical shifts are reported on the δ-scale in
parts per million relative to tetramethylsilane (TMS) as internal
standard for protons and relative to H3PO4 as an external
reference for the 31P nuclei
Chlorido(triphenylphosphine)(3,5-dimethylpyrazole-1-imino-
thiolate‐κ2N2,S)palladium(II)
hydrate,
[PdCl(L1)(PPh3)]·H2O
(complex 1). Triphenylphosphine (101.1 mg, 0.3854 mmol, 1.0
equiv.) and L1 (59.8 mg, 0.385 mmol, 1.0 equiv.) were treated
with [PdCl2(MeCN)2] (100.0 mg, 0.3852 mmol, 1.0 equiv.) in
chloroform/methanol (9:1, v/v, 10 mL). After stirring for 2 h, 10
mL of water was added to the reaction media. The organic layer
was washed three times with 10 mL of water until the aqueous
solution gives a pH 7. The organic layer was evaporated under
reduced pressure to a final volume of ca. 1 mL and pentane (30
mL) was added to precipitate the compound. The yellow solid
3.1. Synthesis
5-hydroxy-3,5-dimethyl-(N4-phenylthiosemicarbazone)pyrazoline
(L4’). 2,4-pentanedione (133.0 mg, 1.328 mmol, 1.1 equiv.) were
treated with 4-phenyl-3-thiosemicarbazide (201.8 mg, 1.207
mmol, 1.0 equiv.) in ethanol (20 mL) at room temperature. After
stirring for 14 h, the solvent was removed under reduced
pressure and the resulting residue was washed three times with
diethyl ether (3 x 3 mL). All the extracts were then combined
and filtered off. After removing the solvent under vacuum, a
viscous oil was obtained. Thin layer chromatography showed
just a single spot, indicating the presence of a single product.
was filtered off, washed with pentane, and finally dried under
1
vacuum to yield 143.5 mg (67%) of complex
1
. H NMR (600
MHz, CDCl3): δ = 8.34s ([1H], NH), 7.77ddd (3JP-H = 12.1 Hz, J =
3
7.15 Hz, J = 1.3 Hz, [6H], ortho-phenyl), 7.51m ([3H], para-
4
phenyl), 7.45m ([6H], meta-phenyl), 5.99d (4JP-H = 2 Hz, [1H], CH),
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2.71s ([3H], CH3), 2.55 ppms ([3H], CH3). DEPTQ-135 NMR (600 finally dried under vacuum to yield 186.4 mg (83%) of complex
View Article Online
1
MHz, CDCl3): δ = 164.3d (3JP-C = 5.5 Hz, N=C-S), 156.1d (3JP-C = 3.3
3
. H NMR (600 MHz, CDCl3): δ = 7.78mDO([I6: 1H0].,10o3r9t/hDo0N-pJh0e28n2y5lH),
Hz, C2), 144.4 (C4), 134.6d (2JP-C = 11 Hz, ortho-PPh3), 131.1d (4JP- 7.49m ([3H], para-phenyl), 7.44m (m, [6H], meta-phenyl), 5.91d
C = 2.2 Hz, para-PPh3), 128.5d (1JP-C = 57.5 Hz, ipso-PPh3), 128.2d (4JP-H = 1.8 Hz, [1H], CH), 3.27q (3J = 7.2 Hz, [2H], CH2) 2.69s ([3H],
(3JP-C = 12,1 Hz, meta-PPh3), 111.2d (4JP-C = 5.5 Hz, CH), 15.50 CH3 - pyrazole), 2.50s ([3H], CH3 - pyrazole), 1.15t ppm (3J = 7.2
(CH3), 14.44 ppm (CH3). 31P NMR: 32.55 ppm. IR (KBr): 3295 (νN- Hz, [3H], CH3). DEPTQ-135 NMR (600 MHz, CDCl3): δ = 154.5d
H), 1436 (vC-Nring), 2086 (vC-Nthioamide), 1481 (νC-CPPh3), 1273 (3JP-C = 3.3 Hz, C2), 154.4d (3JP-C = 5.5 Hz, N=C-S) 143.5 (C4),
(δC-N), 1051 (vNNring + δrCH3) 691 cm-1 (νCS). Calcd. for 134.8d (2JP-C = 11.1 Hz, ortho-PPh3), 131.1d (4JP-C = 2.2 Hz, para-
C24H23ClN3PPdS·H2O (558.4): C, 50.01; H, 4.37; N, 7.29; found: PPh3), 129.2d (1JP-C = 57.5 Hz, ipso-PPh3), 128.2d (3JP-C = 12,2 Hz,
C, 50.41; H, 4.01; N, 6.74. ESI-MS: base peak at m/z = 522 (M - meta-PPh3), 110.4d (4JP-C = 4.4 Hz, CH), 46.2 (CH2), 15.8 (CH3),
Cl-); molecular ion at m/z = 558. Conductivity data (10 mM in 15.5 (CH3 - pyrazole), 15.0 ppm (CH3 - pyrazole). 31P NMR: 32.57
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DMSO): ΛM = 2.62 ohm-1 cm2 mol−1
ppm. IR (KBr): 1436 (vCNring), 1600 (vCNthioamide), 1480 (νCCPPh3),
1253 (δC-N), 1046 (vNNring + δrCH3) 705 cm-1 (νCS). Calcd. for
C26H27ClN3PPdS·0.5H2O (586.4): C, 52.45; H, 4.74; N, 7.06;
found: C, 52.35; H, 4.64; N, 7.42. ESI-MS: base peak at m/z (M -
Cl-) = 550; molecular ion at m/z = 586. Conductivity data (10 mM
in DMSO): ΛM = 4.50 ohm-1 cm2 mol−1
Chlorido(triphenylphosphine)(3,5-dimethylpyrazole-N-methyl-1-
iminothiolate‐κ2N2,S)palladium(II), [PdCl(L2)(PPh3)] (complex 2).
Triphenylphosphine (70.8 mg, 0.2698 mmol, 1.0 equiv.) and L2
(54.7 mg, 0.270 mmol, 1.0 equiv.) were treated with
[PdCl2(MeCN)2] (70.0 mg, 0.2696 mmol, 1.0 equiv.) in
chloroform/methanol (7:1, v/v, 8 mL). After stirring for 2 h, 10
mL of water was added to the reaction media. The organic layer
was washed three times with 10 mL of water until the aqueous
solution gives a pH 7. The organic layer was evaporated under
reduced pressure to a final volume of ca. 1 mL and methanol (30
mL) was added to precipitate the compound. The yellow
crystalline solid was filtered off, washed with methanol, and
finally dried under vacuum to yield 137.1 mg (88%) of complex
Chlorido(triphenylphosphine)(3,5-dimethylpyrazole-N-phenyl-1-
iminothiolate‐κ2N2,S)palladium(II), [PdCl(L4)(PPh3)] (complex 4).
Triphenylphosphine (101.1 mg, 0.3854 mmol, 1.0 equiv.) and L4’
(96.1 mg, 0.385 mmol, 1.0 equiv.) were treated with
[PdCl2(MeCN)2] (100.0 mg, 0.3852 mmol, 1.0 equiv.) in
methanol (10 mL). After stirring for 24 h, the solution was
evaporated under reduced pressure to a final volume of circa 2
mL and 20 mL of chloroform were added. In a separation funnel,
the organic layer was washed three times with 10 mL of water
until the aqueous solution gives a pH 7. The resulting red
solution was then evaporated under reduced pressure to a final
volume of ca. 1 mL and pentane (20 mL) was added to
precipitate the compound. The brown solid was filtered off,
2
. 1H NMR (600 MHz, CDCl3): δ = 7.79ddd (3JP-H = 12.1 Hz, 3J = 7.15
4
Hz, J = 1.4 Hz, [6H], ortho-phenyl), 7.50m ([3H], para-phenyl),
7.44m ([6H], meta-phenyl), 5.92d (4JP-H = 2.6 Hz, [1H], CH), 3.06s
([3H], CH3 - thioamide) 2.69s ([3H], CH3 - pyrazole), 2.48s ppm
([3H], CH3 - pyrazole). DEPTQ-135 NMR (600 MHz, CDCl3): δ =
156.9d (3JP-C = 5.5 Hz, N=C-S), 154.6d (3JP-C = 3.3 Hz, C2 - pyrazole),
143.4 (C4 - pyrazole), 134.7d (2JP-C = 11 Hz, ortho-PPh3), 131.1d
(4JP-C = 3.3 Hz, para-PPh3), 129.2d (1JP-C = 57.5 Hz, ipso-PPh3),
128.2d (3JP-C = 11,1 Hz, meta-PPh3), 110.5d (4JP-C = 5.5 Hz, CH -
pyrazole), 38.7 (CH3 - thioamide) 15.5 (CH3 - pyrazole), 14.8 ppm
(CH3 - pyrazole). 31P NMR: 32.51 ppm. IR (KBr): 1437 (vCNring),
washed with pentane, and finally dried under vacuum to yield
1
65.0 mg (27%) of complex
4
. H NMR (600 MHz, CDCl3): ): δ =
7.72ddd (3JP-H = 12.1 Hz, 3J = 7.15 Hz, 4J = 1.3 Hz, [6H], ortho-PPh3),
7.45m ([3H], para-PPh3), 7.38m ([6H], meta-PPh3), 7.19m ([2H],
meta-phenyl), 6.99tt (3J = 7.3 Hz, 4J = 1.1 Hz, [1H], para-phenyl),
6.89m ([2H], ortho-phenyl), 6.01d (4JP-H = 2.2 Hz, [1H], CH,
pyrazole), 2.74s ([3H], CH3), 2.61s ppm ([3H], CH3). DEPTQ-135
NMR (600 MHz, CDCl3): δ = 156.8d (3JP-C = 4.4 Hz, N=C-S), 155.8
(C2), 148.2 (ipso-thioamide) 144.2 (C4), 134.8d (2JP-C = 11.1 Hz,
ortho-PPh3), 131.1d (4JP-C = 3.3 Hz, para-PPh3), 128.8d (1JP-C = 57.5
Hz, ipso-PPh3), 128.3 (meta-thioamide) 128.1d (3JP-C = 11,1 Hz,
meta-PPh3), 123.5 (para-thioamide) 121.7 (ortho-thioamide),
111.1d (4JP-C = 5.5 Hz, CH - pyrazole), 15.5 (CH3), 15.2 ppm (CH3).
31P NMR: 32.69 ppm. IR (KBr): 1435 (vCNring), 1578 (vCNthioamide),
1480 (νCCPPh3), 1273 (δC-N), 1050 (vNNring + δrCH3) 692 cm-1
(νCS). Calcd. for C30H27ClN3PPdS·0.5H2O (634.5): C, 56.00; H,
4.39; N, 6.53; found: C, 55.99; H, 3.82; N, 6.45. ESI-MS: base
peak at m/z (M - Cl-) = 599; molecular ion at m/z = 635.
Conductivity data (10 mM in DMSO): ΛM = 2.55 ohm-1 cm2
mol−1.
1610 (vCNthioamide), 1479 (νCCPPh3), 1262 (δC-N), 1034 (vNNring
+
δrCH3) 695 cm-1 (νCS). Calcd. for C25H25ClN3PPdS·0.5H2O (572.4):
C, 51.65; H, 4.51; N, 7.23; found: C, 51.46; H, 4.29; N, 7.28. ESI-
MS: base peak at m/z (M - Cl-) = 538; molecular ion at m/z = 572.
Conductivity data (10 mM in DMSO): ΛM = 2.23 ohm-1 cm2
mol−1.
Chlorido(triphenylphosphine)(3,5-dimethylpyrazole-N-ethyl-1-
iminothiolate‐κ2N2,S)palladium(II), [PdCl(L3)(PPh3)] (complex 3).
Triphenylphosphine (101.1 mg, 0.3854 mmol, 1.0 equiv.) and L3
(70.6 mg, 0.385 mmol, 1.0 equiv.) were treated with
[PdCl2(MeCN)2] (100.0 mg, 0.3852 mmol, 1.0 equiv.) in
chloroform/methanol (9:1, v/v, 10 mL). After stirring for 2 h, 10
mL of water was added to the reaction media. The organic layer
was washed three times with 10 mL of water until the aqueous
solution gives a pH 7. The organic layer was evaporated under
reduced pressure to a final volume of ca. 1 mL and methanol (30
mL) was added to precipitate the compound. The orange
crystalline solid was filtered off, washed with methanol, and
3.2. Cell proliferation
The free ligands and palladium complexes were assayed using
the human breast tumor cell line MCF-7, human lung tumor cell
line A549 and human fetal lung fibroblast cell line MRC-5. The
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Molecular Docking. The crystal structure of a mature Cathepsin
DOI: 10.1039/D0NJ02825H
B, co-crystallized with a dipeptidyl nitrile inhibitor, was obtained
cells were routinely maintained in Dulbecco's Modified Eagle's
medium (DMEM) supplemented with 10% fetal bovine serum
(FBS), gentamicin sulfate (50 mg L-1) and amphotericin B (2 mg
L-1) at 310 K in a humidified 5% CO2 atmosphere. Briefly, all cell
lines were prepared at a concentration of 1.5 × 104 cells/100 μL,
in complete medium, and plated on sterile 96 well plates for 24
h at 310 K in a humidified 5% CO2 atmosphere. The compounds
were added to the wells at different concentrations and
incubated for 48 h under the same conditions as described
above. The cell proliferation assay was performed compared to
the wells where the vehicle (0.5% DMSO) was added instead of
the tested compounds (at 0.5% DMSO). After incubation, the
culture medium of each well was removed and a solution
containing MTT (1 mg mL−1) was added (50 μL/well).35 The
plates were then kept at 310 K for 4 h and the formed crystals
were dissolved in isopropyl alcohol. The absorbance was read
on an ELISA plate reader (BioTek, Epoch) at a wavelength of 570
nm and the IC50 (concentration of compound that induced 50%
of cell death) value were determined using the GraphPad Prism
software (www.graphpad.com/). Each assay were performed in
triplicate.
View Article Online
from Protein Data Bank (PDB: 1GMY) and prepared for the
modelling work.71 Before docking, all hydrogen were added and
the binding site was fixed at x = 28.8380, y = 37.2531 and z =
37.2500 coordinates (Cys29, S-donor atom). The docking radius
was 15 Å and 50 runs were set to be calculated using GoldScore
fitness function, which is embedded in CCDC GOLD software.72,73
The docking results were evaluated using the top-ranked pose
and the ability of that to reproduce the original intermolecular
contacts as the co-crystallized inhibitor.
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Conclusions
The synthesis and spectroscopic characterization of novel
complexes of the type [PdCl(Ln)(PPh3)] {Ln = 1-iminothiolate-3,5-
dimethylpyrazoles} have been reported in this work. The
molecular structure of compound [PdCl(L3)(PPh3)] (L3 = 3,5-
dimethyl-pyrazole-N-ethyl-1-iminothiolate; complex
solved by single-crystal X-ray diffraction technique. The
cytotoxicity of against MCF-7 cells demonstrated to be
dependent on the substituent at N-thioamide position. Complex
displayed the most potent cytotoxic effect, with an IC50 value
of 31.94 ± 3.35 μM. In addition, compounds did not induce
3) was
2-4
3.3. Cathepsin B and L inhibition
3
Cathepsin B and L were expressed and purified according
1-4
Linnevers et al.68 The assays were performed in
a
inhibition of human topoisomerase II even at 100 μM,
suggesting that other targets may be involved. Preliminary
studies on the inhibition of cathepsins B and L activity showed
that their potency seems to be also dependent on the type of
spectrofluorometer Hitachi F-2700. The monitoring of the
enzyme activity was followed at wavelengths λex=360 nm and
λem=480 nm, under constant agitation, in 10 mm optical path
quartz cuvettes at final volumes of 1 mL. Cathepsin B and L was
incubated in 1000 µL of a preheated solution of sodium acetate
buffer (100 mM); 5 mM EDTA; 100 mM NaCl; 0.01% Triton X-
100 (only for cathepsin B); 20% glycerol, pH 5.5, 3 mM DTT, and
40 µM (cathepsin B) or 15 µM (cathepsin L) of Z-FR-AMC
(Benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin). The
measurement of the enzyme activity was performed monitoring
the product formation AMC (amino-4-methylcoumarin) by
hydrolysis of substrate Z-FR-AMC used as fluorogenic probe
after 5 minutes. The effects of the activity of the compounds on
the enzymes were performed at concentration of 10 and 100
μM.
R-substituent group at thioamide fragment. Compound
4 was
the most potent inhibitor among all tested PdII complexes,
resulting in a residual activity of 42% (cat B) and 61 % (cat L). In
silico studies indicated that only complex
4 established
additional interactions with the residues in the S-binding
pocket. These findings agree well with the obtained results from
in vitro inhibition assays. Thus, new palladium-based cathepsins
inhibitors may be designed from a careful selection of 3,5-
dimethyl-pyrazole-N-substitued-1-iminothiolate and phosphine
ligands.
Authors’ contributions
3.4. Computational Methodology
TRM conceived the study, synthesized and characterized all four
complexes and wrote the manuscript; RDZ performed all DNA
binding studies; DESS performed the experiments for EC50
determination; RLF performed the in silico experiments; MAL
and FVR performed the topoisomerase assay; VMD performed
the X-ray crystallography and structure elucidation of complex
Molecular Modelling. The 3-D molecular structures of the
complexes were handled as an in silico approach reported
recently, but slightly modified. 12,53 As follows, the structures of
1-4 were constructed and primarily minimized by using
molecular mechanics (MM) simulation in Biovia Discovery
Studio.69 Further, the semi-empirical PM7 Hamiltonian method
was employed to acquire minimal energy structures.70 The
quality of the outcomes has been proved by root-mean-square
deviation from the alignment between heavy atoms of the
theoretical and molecular crystal structure of 3, see ESI for more
details.
3
; AAS, FSS and WASJ performed the cathepsins assays; JCMP
wrote the manuscript; AVGN, AEM and WASJ participated in the
coordination of the study, analysis of data and helped draft the
manuscript. All authors read and approved the final version of
the manuscript.
Conflicts of interest
There are no conflicts of interest to declare.
This journal is © The Royal Society of Chemistry 20xx
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21 W. J. GEARY, Coord. Chem. Rev., 1971,
Acknowledgements
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22 V. V. Dunina, E. I. Turubanova, M. V. Livantsov, K. A. Lyssenko,
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23 C. E. Griffin and M. Gordon, Journal of the American Chemical
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We thank the São Paulo State University (UNESP), Institute of
Chemistry of Araraquara. This work was sponsored by FAPESP
(proc. VMD-2009/54011-8, WASJ-2014/02205-1, JCMP-
2015/12098-0, AVGN-2016/17711-5, WASJ-2016/25112-4,
FVR-2019/11242-1), CNPq (proc. AVGN-475322/2009-6 and
AVGN-422105/2016-3) and CAPES.
24 R. Ares, M. López-Torres, A, Fernández, S. Castro-Juiz, A.
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DOI: 10.1039/D0NJ02825H
Palladium(II) complexes bearing 1-iminothiolate-3,5-dimethylpyrazoles: synthesis,
cytotoxicity, DNA binding and enzymatic inhibition studies
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W. A. S. Júdice, M. A. Lima, F. V. Rocha, V. M. Deflon and A. V. G. Netto
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TABLE OF CONTENTS ENTRY:
This work describes the enzymatic inhibitory activity of four novel Pd(II) complexes towards
topoisomerase IIα and cathepsins B and L. In silico studies agree well with the enhanced in vitro
cathepsin B inhibition induced by compound 4 (58% at 10 μM)