New Water-Soluble Copper(II) Complexes with Morpholine-Thiosemicarbazone Hybrids: Insights into the Anticancer and Antibacterial Mode of Action

Article abstract of DOI:10.1021/acs.jmedchem.8b01031

Six morpholine-(iso)thiosemicarbazone hybrids HL¹-HL⁶ and their Cu(II) complexes with good-to-moderate solubility and stability in water were synthesized and characterized. Cu(II) complexes [Cu(L^1-6)Cl] (1-6) formed weak dimeric associates in the solid state, which did not remain intact in solution as evidenced by ESI-MS. The lead proligands and Cu(II) complexes displayed higher antiproliferative activity in cancer cells than triapine. In addition, complexes 2-5 were found to specifically inhibit the growth of Gram-positive bacteria Staphylococcus aureus with MIC₅₀ values at 2-5 μg/mL. Insights into the processes controlling intracellular accumulation and mechanism of action were investigated for 2 and 5, including the role of ribonucleotide reductase (RNR) inhibition, endoplasmic reticulum stress induction, and regulation of other cancer signaling pathways. Their ability to moderately inhibit R2 RNR protein in the presence of dithiothreitol is likely related to Fe chelating properties of the proligands liberated upon reduction.

Full text of DOI:10.1021/acs.jmedchem.8b01031

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Article
New Water-Soluble Copper(II) Complexes with
Morpholine-Thiosemicarbazone Hybrids: Insights
into the Anticancer and Antibacterial Mode of Action
Kateryna Ohui, Eleonora Afanasenko, Felix Bacher, Rachel Lim Xue Ting, Ayesha Zafar, Nuria Blanco-
Cabra, Eduard Torrents, Orsolya Domotor, Nora V. May, Denisa Darvasiova, Eva A Enyedy, Ana D.
Popovi#-Bijeli#, Johannes Reynisson, Peter Rapta, Maria Babak, Giorgia Pastorin, and Vladimir B. Arion
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01031 • Publication Date (Web): 03 Dec 2018
Downloaded from http://pubs.acs.org on December 5, 2018
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New Water-Soluble Copper(II) Complexes with Morpholine-Thiosemicarbazone
Hybrids: Insights into the Anticancer and Antibacterial Mode of Action
Kateryna Ohui,a,  Eleonora Afanasenko,a,  Felix Bacher, Rachel Lim Xue Ting, Ayesha Zafar, Núria
a
b
c
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Blanco-Cabra, Eduard Torrents, Orsolya Dömötör, Nóra V. May, Denisa Darvasiova, Éva A.
e
h
c
g
,i,j
Enyedy, Ana Popović-Bijelić, Jóhannes Reynisson, Peter Rapta, Maria V. Babak,* Giorgia
b
,a
Pastorin, Vladimir B. Arion*
aUniversity of Vienna, Institute of Inorganic Chemistry, Währinger Strasse 42, A-1090 Vienna, Austria,
bDepartment of Pharmacy, National University of Singapore, Singapore, 3 Science Drive 2, 117543
Singapore
cSchool of Chemical Sciences, University of Auckland, Auckland, New Zealand
dBacterial Infections: Antimicrobial Therapies, Institute for Bioengineering of Catalonia (IBEC), The
Barcelona Institute of Science and Technology, Barcelona, Spain
eDepartment of Inorganic and Analytical Chemistry, University of Szeged, Dóm tér 7. H-6720 Szeged,
Hungary
fResearch Centre of Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok körútja 2. H-
1
117, Budapest, Hungary
gInstitute of Physical Chemistry and Chemical Physics, Slovak Technical University of Technology,
Radlinského 9, 81237 Bratislava, Slovak Republic
hFaculty of Physical Chemistry, University of Belgrade, 11158 Belgrade, Serbia
iDepartment of Chemistry, National University of Singapore,3 Science Drive 2, 117543 Singapore
jDrug Development Unit, National University of Singapore,28 Medical Drive, 117546 Singapore

these co-authors contributed equally
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Keywords: Thiosemicarbazones, Cu(II)-TSC, solution equilibrium, stability constants,
spectroelectrochemistry, molecular modelling, antitumor activity, antibacterial activity, mechanism of
action, cellular accumulation, ribonucleotide reductase inhibition, ER stress, apoptosis, cell cycle
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Abstract
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Six morpholine-(iso)thiosemicarbazone hybrids HL ‒HL and their Cu(II) complexes with good-to-
moderate solubility and stability in water were synthesized and characterized. Cu(II) complexes
1‒6
[
Cu(L )Cl] (1‒6) formed weak dimeric associates in the solid state, which did not remain intact in
solution as evidenced by ESI-MS. The lead proligands and Cu(II) complexes displayed higher
antiproliferative activity in cancer cells than Triapine. In addition, complexes 2‒5 were found to
specifically inhibit the growth of Gram-positive bacteria S. aureus with MIC values at 2 to 5 µg/mL.
5
0
Insights into the processes controlling intracellular accumulation and mechanism of action were
investigated for 2 and 5, including the role of ribonucleotide reductase (RNR) inhibition, endoplasmic
reticulum (ER) stress induction and regulation of other cancer signaling pathways. Their ability to
moderately inhibit R2 RNR protein in the presence of DTT is likely related to Fe chelating properties of
the proligands liberated upon reduction.
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Introduction
Progress in modern anticancer therapy has led to significant improvements in survival rates of cancer
patients. However, even though more patients achieve remission nowadays, their life-span often remains
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short. One of the major causes of cancer death is the malignant process itself, which is associated with
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extensive formation of metastases, but it is not commonly mentioned that a significant number of
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immunocompromised cancer patients die due to infections, such as pneumonia and peritonitis. Despite
that cancer patients are very prone to develop infections during chemotherapy, their preventive antibiotic
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treatment is hampered by additional adverse effects. However, recent clinical evidence demonstrated
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that benefits of antibiotic prophylaxis of cancer patients outweighed its risks. The simultaneous
suppression of pathogenic microorganisms during anticancer chemotherapy could not only interrupt or
abolish tumor growth but eventually protect cancer patients from infection. Hence, the development of
novel drugs which exhibit dual anticancer and antibacterial properties in comparable concentration range
would affect the malignant process and simultaneously decrease the risk of patients’ death due to
infection, febrile neutropenia and bacteraemia.
Both cancer and bacterial cells share similar properties, such as high rate of proliferation, rapid
5
adjustment and quick spreading within the host and aggressive disease progression. In order to sustain
such rapid proliferation, cancer and bacterial cells have to support DNA replication and production of
RNA by increasing de novo nucleotide synthesis. Ribonucleotide reductase (RNR) is the key enzyme
that catalyzes the reduction of ribonucleotides to their corresponding deoxyribonucleotides, thereby
initiating DNA synthesis or repair, making it an important biomolecular target for drugs with anticancer
and antibacterial properties.6,7 RNR consists of a large subunit (NrdA or R1), which contains the
allosteric site that regulates and catalyzes substrate reduction, and a small subunit (NrdB or R2) with the
diferric-tyrosyl radical cofactor, essential for the catalytic activity of the RNR enzyme. Small molecules,
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that can sequester Fe(III) from the dinuclear metal center and/or scavenge the tyrosyl radical, inhibit R2
activity, thereby preventing de novo DNA synthesis in cancer cells and bacteria.
α-N-heterocyclic thiosemicarbazones (TSCs) are excellent transition metal chelators and have a broad
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range of activities, including anticancer and antibacterial properties, which are believed to be at least
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partially due to their RNR inhibition. To date, several TSC compounds, namely 3-amino-2-
pyridinecarboxaldehyde thiosemicarbazone (Triapine),9,10,11 di-2-pyridylketone 4-cyclohexyl-4-methyl-
3
-thiosemicarbazone (DpC)12,13 and (E)-N´-(6,7-dihydroquinolin-8(5H)-ylidene)-4-(pyridine-2-
1
4
yl)piperazine-1-carbothiohydrazide (COTI-2) are undergoing phase I and II clinical trials against
various types of cancer. The main caveat in the use of TSCs is their high toxicity which is reflected in a
number of side-effects in clinical trials involving Triapine.9,10,11 Many studies have shown that Cu(II)-
TSC complexes often display better selectivity than their TSC proligands, which can be attributed to the
induction of different intracellular signaling pathways.15
Despite advances in the design and synthesis of new TSCs and Cu(II)-TSC complexes over the years,
these compounds are facing the problem of low aqueous solubility, thereby hampering their further
development. Therefore, the synthesis of water-soluble and cytotoxic TSCs and Cu(II)-TSC complexes
requires a thoughtful selection of the functional groups that can be attached at the TSC backbone without
reducing their biological activity.16,17,18,19,20 With the aim to reach the optimal combination of aqueous
solubility and high cytotoxicity, we designed new (iso)thiosemicarbazone-morpholine hybrids and their
Cu(II) complexes. Notably, morpholine moiety was chosen since it confers excellent water solubility,
which typically translates into an improved pharmacological effect. Additionally, morpholine
derivatives possess a broad spectrum of biological activities, including anticancer and antibacterial
2
1
therapeutic potential. For example, commercial anticancer drugs Aprepitant and Gefinitib, as well as
antibacterial drugs Finafloxacin and Levofloxacin include a morpholine fragment in their structures.
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Cl
Cu
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N
N
S
S
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S
S
HL1
1
2
N
N
N
N
N
H
NH2
N
NH2
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Me
N
Me
N
H
N
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N
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O
Me
Me
Cl
Cu
N
S
N
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S
HL3
3
N
N
N
N
H
N
N
N
N
O
O
Cl
Cu
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N
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HL4
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N
N
N
N
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N
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H
N
O
O
O
O
O
O
O
O
Cl
Cu
N
N
N
HL5
N
H
N
H
N
N
H
Cl
Cu
N
SMe
NH2
NH
HL6
6
N
N
SMe
Chart 1. Line drawings of proligands HL1‒6 and Cu(II) complexes [Cu(L1‒6)Cl] (1‒6) reported in this work. All proligands
3
and Cu(II) complexes, but HL and 2, were investigated by single crystal X-ray crystallography.
Herein we report on the synthesis of water-soluble proligands HL1‒6 and Cu(II) complexes 1‒6 (Chart
1
), which were characterized by analytical and various spectroscopic techniques. X-ray diffraction
1
2
3
6
1
structures of HL , HL , HL ‒HL and 1, 3‒6 were established and solution equilibria studies for HL
1
1
and Cu(II) complex formation with HL by pH-potentiometry, UV‒vis, EPR and H NMR spectroscopy,
as well as electrochemistry were performed. Antiproliferative activity against human ovarian cancer
A2780 and cisplatin resistant A2780cis cell lines, non-cancerous HEK293 cell line, as well as
antibacterial activity against Gram-positive (S. aureus) compared to Gram-negative bacteria (P.
aeruginosa) were investigated and structure-activity relationships discussed. The processes controlling
cellular accumulation of 2 were investigated. Preliminary insights into the mode of action of 2 and 5,
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including mouse R2 RNR inhibitory potential, studied by molecular modeling and tyrosyl radical
quenching monitored by EPR spectroscopy, as well as by Western Blotting and flow cytometry are also
presented.
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Results
Synthesis and characterization of (iso)thiosemicarbazone-morpholine hybrids and their Cu(II)
complexes. The 5-methylmorpholine-pyridine-2-carboxaldehyde H was prepared in seven steps as
shown in Scheme 1 and the detailed synthesis is described in the Supporting Information. First pyridine-
2
,5-dicarboxylic acid A was converted into diester B that was further reduced to the diol C. The latter
was selectively oxidized with SeO to the aldehyde D. After protection of the aldehyde group the alcohol
2
E was converted into the chloride F that was further reacted with morpholine to give species G. Finally,
the hydrolysis of the methyl ester function in acidic conditions afforded the required aldehyde H for
condensation reactions with thiosemicarbazide, 4N-dimethyl-3-thiosemicarbazide, 4N-pyrrolydinyl-3-
thiosemicarbazide, 4N-morpholinyl-3-thiosemicarbazide, 4N-phenyl-3-thiosemicarbazide and S-
methylisothiosemicarbazide hydroiodide to give the hybrids HL1‒6 (Scheme 1), respectively. One- and
1
‒6
two-dimensional NMR spectra were in agreement with the proposed structures for HL , enabling the
1
13
assignment of all H and C resonances. The ESI mass spectra recorded in a positive ion mode showed
+
+
1‒6
strong peaks corresponding to [M+H] and [M+Na] ions, respectively. The proligands HL were
reacted with Cu(II) chloride dihydrate and triethylamine in 1:1:1 mole ratio in methanol to give
1
‒6
[
Cu(L )Cl] (1‒6) in 35‒91% yields. Positive ion ESI mass spectra showed strong peaks attributed to
1
‒6 +
[Cu(L )] ions. The structures of 1, 3‒6 were also established by single crystal X-ray diffraction (vide
infra).
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OH
OH
O
O
OH
O
(i)
(ii)
(iii)
HO
N
HO
O
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(iv)
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OH
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(
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(v)
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N
OMe
N
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O
MeO
MeO
MeO
H
G
F
E
Scheme 1. Synthesis of 5-methylmorpholine-pyridine-2-carboxaldehyde. Reagents and conditions: (i) thionyl chloride,
2
2
methanol, 0 °C → room temperature, 12 h; (ii) sodium borohydride, ethanol, acetone, potassium carbonate, chloroform, 0
°
C, 1 h → reflux, overnight;23 (iii) selenium dioxide, dioxane, water, 100 °C, 3 h;24 (iv) trimethyl orthoformate,
methanesulfonic acid, methanol, reflux, 48 h; (v) thionyl chloride, dichloromethane, ‒80 °C → room temperature, overnight;
vi) moprholine, triethylamine, THF/CH Cl 1:1, 50 °C, overnight, purification by column chromatography; (vii) HCl, water,
0 °C, overnight.
(
2
2
6
1
2
4‒6
X-ray Crystallography. The results of X-ray diffraction studies of HL , HL and HL are shown in
1
3
4
5
Figure S1, while those of [Cu(L )Cl] (1), [Cu(L )Cl] (3), [Cu(L )Cl] (4), [Cu(L )Cl] (5) and
1
6
[Cu(L )Cl(H O)] (1´), [Cu(L )Cl] (6) in Figures 1 and 2, respectively. Selected bond distances and
2
bond angles are quoted in Tables S1 and S2. The proligands adopted different isomeric configurations
in the solid state depending on substituents at the terminal nitrogen atom of the thiosemicarbazide moiety.
Complexes 1, 3, 4 and 5 form dimeric associates as shown in Figure 1 (co-crystallized solvent was
omitted for clarity). Each Cu(II) ion has a distorted square-planar coordination geometry. Intermolecular
contacts supporting the dimeric associates in the crystals are of different nature in 1 and 3‒5,
respectively. The presence of long intermolecular contacts Cu···Cl or Cu···Cl and Cu···S (see legend to
Figure 1) provides evidence of weak association of complexes in dimers, which most probably dissociate
in solution with formation of monomeric species. There was no evidence from ESI mass spectra on the
presence of dimeric species in solution.
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Figure 1. ORTEP views of weak dimeric associates of (a) [Cu(L )Cl] (1), (b), [Cu(L )Cl] (3), (c) [Cu(L )Cl]
2
(4) and (d)
5
[
Cu(L )Cl] (5).
1
Moreover, HL reacts with CuCl in aqueous solution with formation of five-coordinate complex
2
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6
[Cu(L )Cl(H O)] (1´·2H O) (Figure 2b). The monomeric square-planar complex [Cu(L )Cl] forms an
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infinite chain via weak coordination of the morpholine oxygen atom of one complex to the Cu(II) atom
of the next molecule as shown in Figure S2. The intermolecular Cu···O contact is of 2.603(3) Å.
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9
0
1
2
3
4
5
6
7
8
9
0
1
6
2
Figure 2. ORTEP views of (a) [Cu(L )Cl(H O)] (1´) and (b) [Cu(L )Cl] (6).
Solution Chemistry. In order to establish the presence of isomers of the proligands in aqueous solution
and proton dissociation processes, in which these can be involved solution equilibrium studies have been
performed. Likewise, the solution speciation of the copper(II) complexes, as well as their stability have
been investigated to elucidate the species, which is the most stable and abundant at physiological pH.
1
The proligand HL (Chart 1) was chosen for the detailed solution equilibrium studies since it has the
simplest structure and the best aqueous solubility among the proligands prepared. The presence of
1
different isomers in solution was excluded by measurements of H NMR spectra in 10% D O/90% H O
2
2
(Figure S3), which showed only one set of signals for the proligand in accordance to its low (C1)
molecular symmetry. The proligand most probably adopts the E configuration found in the solid state
(Figure S1). The same configuration was reported previously for the reference compound 2-
25
formylpyridine thiosemicarbazone in polar solvents. Proton dissociation processes were monitored in
1
aqueous solution by pH-potentiometric and H NMR titrations and three pK values were determined by
a
1
both methods (Table S3). According to the obtained pK values HL is mainly neutral (97% HL, 3%
a
+
1
H L ) at physiological pH (Figure S4). Inspection of H NMR spectra revealed stepwise deprotonation
2
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
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3
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
6
+
+
of three functional groups in the following order: NpyridiniumH → NmorpholiniumH → NhydrazineH (Figure
S3).
1
The solution speciation of the Cu(II) complexes with HL was characterized by the combined use of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
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7
8
9
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
pH-potentiometry, UV−vis spectrophotometry (via charge transfer (CT) and d-d bands) and EPR
spectroscopy. The spectral changes (Table S4, Figures 3a and S5) in the UV and visible regions
measured at 1:1 metal-to-ligand ratio show the high-extent formation of a Cu(II) complex already at
strongly acidic pH values (e.g. pH 1) and its stepwise deprotonation by increasing the pH. At pH 7.4 the
+
dominant species is [CuL] (Figure 3b).
(
a)
(B)
(b)
(
A)
1
.0
1
1
0
0
0
0
0
.2
.0
.8
.6
.4
.2
.0
0.6
0.4
0.2
0.0
1
.11
[
CuLH-1]
[CuL]+
CuLH]2+
[
0.8
0.6
0.4
0.2
0.0
3
.79
6
.37-7.48
[
CuLH
2
]3+
9.56-11.60
240
290
340
390
440
1
3
5
7
9
11

/ nm
pH
1
Figure 3. UV–vis spectra recorded for the Cu(II)–HL (1:1) system at various pH values (a), and concentration distribution
curves and measured absorbance values at 346 nm (♦) and 440 nm (■) for the same system (b). {cCu(II) = clig = 121 µM; T =
2
98 K; I = 0.10 M (KCl); l = 0.5 cm}.
Due to the high stability of the Cu(II) complexes formed, the cumulative constant (logβ) for the [CuL]+
species (Table S4) was determined via EDTA displacement studies (Figure S6). Then the pK and the
a
3
+
2+
logβ values for the other type of complexes ([CuLH ] , [CuLH] , [CuLH ]) were computed using each
2
-1
method and data were in a fairly good agreement (Table S4). It is worth noting that formation of a bis-
ligand complex [CuL ] at ligand excess was also confirmed by the UV−vis CT and EPR titrations. The
2
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5
5
5
6
d-d bands (Figure S5) recorded from pH 3.4 to 7.5 at 1:1 metal-to-ligand ratio revealed that the proton
2+
+
dissociation process of [CuLH] → [CuL] is accompanied by rather weak spectral changes as most
+
probably the non-coordinating morpholinium NH is deprotonating. In order to further characterize the
coordination modes of the various complexes in solution EPR parameters (Table S5) obtained by the
deconvolution of the recorded spectra (Figure S7) were analyzed. The isotropic g and A values of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
+
+
[
CuLH] and [CuL] are quite similar indicating the like coordination mode.
1
Comparison of the EPR parameters of the Cu(II) complexes of HL with those of 2-formylpyridine
2
5
26
2+
+
thiosemicarbazone and Triapine permitted to conclude that in the [CuLH] and [CuL] complexes a
−
typical coordination mode via the Npyridyl,N,S binding site is realized in accordance with the X-ray
3
+
diffraction data. [CuLH ] contains the diprotonated ligand in which the protons are attributed to the
2
2
+
non-coordinating N H and morpholinium NH moieties, while [CuLH ] is most probably a mixed
-1
hydroxido complex [CuL(OH)] formed by the deprotonation of water molecule coordinated in the fourth
equatorial position. At ligand excess besides the bis-ligand complex a minor dinuclear species
2+
[
Cu L H] was detected resulting in the weak exchange coupling between neighboring Cu(II) centers
2 3
smearing out the expected hyperfine structure.
Cu(II)-TSC complexes undergo quasi-reversible one-electron reduction at biologically accessible
potentials. In order to assess the redox properties of 1‒6, detailed electrochemical and spectroscopic
studies in various solvents were performed using cyclic voltammetry as well as EPR–
spectroelectrochemistry. Additionally, the UV‒vis spectra of 1‒5 were measured (Figure S8).
Electrochemical and spectroscopic data are summarized in Table S6. Almost reversible one-electron
cathodic reduction was observed for 1‒5 in DMSO with half-wave redox potentials from –0.77 V to –
+
0.81 V vs Fc /Fc (for comparison from –0.13 V to –0.17 V vs NHE) (Table S6 and Figure S9). In
general, the values of redox potentials of 1‒6 for the first reduction step decreased in the following rank
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2
2
2
2
2
2
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3
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3
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5
5
5
5
5
5
5
6
order (E (5) > E (2) = E (3) = E (4) > E (1) > E (6)). The second reduction step occurred at –
1
/2
1/2
1/2
1/2
1/2
1/2
+
1
.8 V vs Fc /Fc (–1.16 V vs NHE) and was less reversible indicating a ligand based reduction. This was
confirmed by the CVs of the corresponding proligands which exhibited the first reduction step at around
+
–1.9 V vs Fc /Fc (not shown). In aqueous solutions the reduction potentials shifted to the less negative
0
1
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3
4
5
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7
8
9
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3
4
5
6
7
8
9
0
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2
3
4
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6
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8
9
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
values and reduction was less reversible as shown for 2 in Figure S10. Complex 6 exhibited different
redox behavior with the lowest electrochemical reversibility and the most negative reduction potential
+
of –0.86 V vs Fc /Fc (–0.26 V vs NHE) (Figure S9c). Unlike the cathodic reduction, the anodic oxidation
+
of 1‒6 was irreversible with potential values in the region from 0.4 to 0.8 V vs Fc /Fc (0.68 V to 1.44 V
2
vs NHE). Similar response was observed for the corresponding proligand HL (Figure S11), indicating
ligand based oxidation in 2.
The biologically accessible reduction of Cu(II)-TSC complexes is Cu-centered. To investigate
whether the biologically accessible reduction is metal-centered, the reversible one-electron reduction of
5 was further studied by in situ UV‒vis-spectroelectrochemistry (Figure 4a). Upon cathodic reduction
at the first reduction peak two isosbestic points at 389 and 325 nm were detected. The spectral changes
of the S→Cu(II) charge transfer bands (~425 nm) clearly confirmed the reduction of Cu(II) to Cu(I).
Additionally, upon voltammetric reverse scan, reoxidation and a nearly full recovery of the initial optical
bands were observed, attesting the chemical reversibility of the cathodic reduction even at low scan rates
(Figure 4). To confirm the involvement of Cu(II) in the reduction processes, in situ EPR electrochemistry
of 1‒6 in nBu NPF /DMSO and water was performed, since metal-based reduction would result in the
4
6
formation of EPR-silent Cu(I) species (Figure 4b). As can be seen for electrochemical reduction of 2 in
nBu NPF /DMSO in the region of the first one–electron reduction step (see inset in Figure 4b), a
4
6
significant decrease of EPR signal was observed in accord with the formation of diamagnetic Cu(I) d10
complex. For aqueous solutions the reversibility was significantly reduced, implying a more complex
mechanism involving the release of the proligand. However, by decreasing the scan rate and going to
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5
5
5
5
5
5
5
5
6
the more positive potentials upon reverse scan, a partial recovery of the initial optical bands was also
observed in aqueous solutions as shown for 2 in Figure S12. Thus, electrochemical data indicated a likely
reduction of Cu(II)-TSC complexes to Cu(I) species with the subsequent release of the proligands.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
5
6
7
8
9
0
2
1
1
0
0
.8
.2
.6
.0
(a)
0
A
-
2
-
1.2
-1.0
-0.8
+
E vs Fc /Fc / V
-0.6
-0.4
-0.46
-
0.46
V
/
c
-1.16
+
/F
c
F
s
v
-0.46
3
00
350
400
450
500
550
600
650
700
E

/ nm
(b)
1.5
1.0
0.5
0.0
2
60 280 300 320 340 360 380 400
B / mT
300
350
400
450
500
550
600

/ nm
Figure 4. Spectroelectrochemistry of 2 in nBu
4
6
NPF /DMSO in the region of the first cathodic peak. (a) Potential dependence
of UV‒vis spectra with respective cyclic voltammogram (Pt-microstructured honeycomb working electrode, scan rate v = 5
−1
mV s ); (b) evolution of UV‒vis spectra in 2D projection in forward scan (Inset: EPR spectra measured at the first reduction
peak using Pt mesh working electrode).
Lead TSCs and their Cu(II) complexes exhibited marked antiproliferative activity in a nanomolar
concentration range. The in vitro anticancer activity of 1‒6 and their respective TSCs was determined
in ovarian carcinoma cells (A2780 and A2780cisR) and non-cancerous human embryonic kidney cells
1
6
(
HEK293) by the colorimetric MTT assay with an exposure time of 72 h. The IC values for HL ‒HL
50
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2
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2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and 1‒6 in comparison with Triapine, CuCl , Cu-Triapine and cisplatin are listed in Tables 1 and 2,
2
respectively, and concentration-effect curves are depicted in Figure S13.
Table 1. Cytotoxicity of proligands HL1‒6 and their n-octanol/water distribution coefficients (logD )
7
.4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
logD7.4d
Compound
IC [μM]
50
A2780
7.6  1.9
A2780cis
13  1
RFb
HEK293
9.1  1.7
SFc
HL1
HL2
1.7
1.2
+0.46  0.01
+0.51  0.02
+1.0  0.1
+0.37  0.04
≥2
0.010  0.001
0.008  0.002
0.07  0.01
0.31  0.08
157  21
0.035  0.006
0.028  0.006
0.76  0.06
1.1  0.2
3.5
3.5
10.9
3.5
2.7
4.6
0.030  0.005
0.022  0.005
0.35  0.05
0.67  0.06
89  2
3.0
2.8
5.0
2.2
0.6
0.6
HL3
HL4
HL5
HL6
426  44
+0.84  0.01
n.d.e
Triapine
0.67  0.22
1.1  0.1
0.39  0.05
a
5
0% inhibitory concentrations (IC50) in human ovarian carcinoma cell lines A2780 and A2780cisR and human embryonic
kidney cell line HEK293, determined by means of the MTT assay after 72 h exposure. Values are means ± standard deviations
obtained from at least three independent experiments, Resistance Factor (RF) is determined as IC50 (A2780cisR)/IC50 (A2780), c
b
d
Selectivity Factor (SF) is determined as IC50 (HEK293)/IC50 (A2780), Distribution coefficients between n-octanol and buffered
aqueous solution determined at physiological pH by UV‒vis spectroscopy. Values are means ± standard deviations obtained
e
from at least three independent experiments, n.d. – not determined.
1
6
With the exception of HL and HL , the proligands demonstrated marked antiproliferative activity in a
submicromolar to nanomolar concentration range in both A2780 and A2780cisR cells. The efficacy of
2
3
5
compounds HL , HL and HL in cisplatin-resistant A2780cisR cells decreased by factor 3.5 in
comparison to that in sensitive A2780 cells, whereas a 4.6- and 11-fold drop of activity was observed
4
1‒6
for Triapine and HL , respectively. The activity of proligands HL and Triapine increased in the
6
1
5
4
2
3
following rank order HL < HL < Triapine < HL < HL << HL  HL (Table 1).
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Cytotoxicity of Cu(II)-TSC complexes 1‒6, CuCl and cisplatin and their n-octanol/water
2
distribution coefficients (logD )
7
.4
a
logD7.4e
Compound
IC [μM]
Cellular
accumulation,
nmol Cu/
5
0
d
mg protein
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A2780
2.2  0.1
A2780cis
HEK293
RFb SFc
A2780
0.63  0.09
3.9  0.6
2.2  0.6
3.2  0.7
4.1  0.9
0.47  0.08
0.17  0.05 f
n.d.
1
13  4
16  2
5.9 7.3
–0.81  0.04
+0.30  0.01
+0.86  0.02
+0.12  0.01
+1.49  0.06
–0.23  0.01
n.d. g
2
0.012  0.002
0.26  0.02
0.08  0.01
0.009  0.002
43  3
0.030  0.003 0.032  0.008 2.5 2.7
3
0.60  0.05
0.26  0.04
0.78  0.20
0.24  0.02
2.3 3.0
3.3 3.0
4
5
6
0.017  0.002 0.020  0.003 1.9 2.2
62  6
82  3
72  0
187  37
n.d.
1.4 1.7
1.0 2.3
CuCl2
83  12
Cu-Triapineh
22.3
10.5
-
-
n.d.
1.3  0.1
29  0.3
4.6  0.3
–2.30  0.79i
Cisplatin
0.44  0.13
n.d.
-
a
5
0% inhibitory concentrations (IC50) in human ovarian carcinoma cell lines A2780 and A2780cisR and human embryonic
kidney cell line HEK293, determined by means of the MTT assay after exposure for 72 h. Values are means ± standard
b
deviations obtained from at least three independent experiments, Resistance Factor (RF) is determined as IC50 (A2780cisR)/IC50
c
Selectivity Factor (SF) is determined as IC50 (HEK293)/IC50 (A2780), d Cellular accumulation in A2780 cells, determined
(A2780),
by ICP-MS after 24 h exposure at concentration of 1 μM. Values are means ± standard deviations obtained from at least three
independent experiments, e Distribution coefficients between n-octanol and buffered aqueous solution determined at
physiological pH by UV‒vis spectroscopy. Values are means ± standard deviations obtained from at least three independent
f
g
h
2
experiments, Cellular accumulation of CuCl was detected at concentration of 2.5 μM, n.d. – not determined, the IC50
.
i
values (exposure for 72 h) were taken from the ref. 27, Log Po/w value was taken from the ref. 28.
As can be seen in Table 2, with the exception of 3, all Cu(II)-TSC complexes were equally or more
cytotoxic than the respective proligands. The activity of complexes 1‒6 increased in the following order
6
< 1 < 3 < 4 << 2 < 5, similar to the trend in electrochemical redox potentials for Cu-based reduction
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
+
step (Table S6). In order to compare the cytotoxicity of 1‒6 with that of [Cu(H O) ] , A2780 cells were
2
6
29
treated with aqueous solution of CuCl . In agreement with the literature, CuCl revealed
2
2
antiproliferative activity in the high micromolar range, in contrast to high activity of 1‒6, which should
be regarded as individual entities with their own biological, pharmacokinetic and metabolic profiles.
Complexes 1‒6 were up to ~50 times more cytotoxic than cisplatin in A2780 cells and up to ~270 times
more cytotoxic in A2780cisR cells. Additionally, the differences in cytotoxicity of 1‒6 in A2780 and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
6
A2780cisR cells were significantly lower than for HL ‒HL and cisplatin, indicating high potential of
Cu(II) complexes for the treatment of cisplatin-resistant tumors. The most active TSC proligands and
their respective Cu(II) complexes were more selective towards A2780 cells over HEK293 cells;
however, they did not show any selectivity towards A2780cisR cells over HEK293 cells. On the
contrary, Triapine was significantly more cytotoxic towards non-cancerous HEK cells than A2780 or
A2780cis cells.
Antiproliferative activity of Cu(II)-TSC complexes correlates with their cellular accumulation and
lipophilicity. The ability of anticancer drugs to penetrate biological membranes, tissues and barriers
underlies their biological, pharmacokinetic and metabolic properties. These properties can be predicted
3
0
by calculating physicochemical characteristics of drug candidates using molecular descriptors. In order
1
6
to estimate if the proligands HL ‒HL and corresponding Cu(II) complexes demonstrate drug-like
properties, they were evaluated with main stream molecular descriptors, such as molecular weight
(
MW), number of hydrogen bond donors and acceptors (HBD/HBA), n-octanol-water partition
coefficient (log P), polar surface area (PSA) and rotatable bonds (RB). The results are shown in Table
S7 in the Supporting Information. Based on the calculated physicochemical parameters, we determined
if novel compounds belonged to the drug-like chemical space, which is commonly defined by the well-
known Lipinski rule, or to the known drug space (KDS), which includes all small compounds in medical
3
1
use. As can be seen from Table S7, all compounds fell within the boundaries of drug-like chemical
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3
3
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3
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space with the exception of HL which demonstrated higher HBA value, referring to KDS space. Based
on these results, novel compounds are expected to be sufficiently cell permeable. Following the
calculations, we determined the lipophilicity of all compounds experimentally (logD , Tables 2 and 3).
7
.4
It is known that cancer cells accumulate hydrophilic compounds to a smaller extent relative to
hydrophobic compounds, thereby affecting their activity. Hence, the differences in the lipophilicity of
compounds might be related to the observed trends in cytotoxicity of 1‒6. In general, with the exception
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of morpholine derivative HL , proligands which were obtained by systematic substitution at the terminal
1
thioamide nitrogen, demonstrated higher lipophilicity when compared to that of HL . As expected,
proligands were more lipophilic than the corresponding Cu(II) species, which are positively charged at
physiological pH (Figure 3b). Among Cu(II)-TSC complexes, only the least cytotoxic complexes 1 and
6, as well as cisplatin, demonstrated negative logD values, indicating their higher hydrophilicity. The
7
.4
lipophilicity of 2‒5 increased in the following order 4 < 2 < 3 < 5, and correlated well with their
cytotoxicity in cancer cells, with the exception of 3. Subsequently, we determined the total cellular
accumulation of Cu in A2780 cells by ICP-MS upon 24 h exposure to 1‒6 in comparison with CuCl2
(
Table 4). The IC values from MTT assays with 72 h drug exposure varied from nanomolar to high
50
micromolar concentrations; therefore, in the cellular accumulation experiment cells were treated with 1
μM of compounds of interest for 24 h to minimize cell detachment. The cellular accumulation of all
Cu(II)-TSC complexes was significantly higher than for CuCl , indicating the role of lipophilic TSC
2
ligands in the delivery of the complexes into the cells. The accumulation increased in the order 6 < 1 <
3
< 4 < 2 < 5.
Cellular accumulation and efflux of Cu(II)-TSC complexes are energy- and/or temperature-
dependent processes. Dependence of cytotoxicity on lipophilicity of 1‒6 indicates that their cellular
accumulation at least partially occurs via passive diffusion. In order to gain additional insights into the
mechanisms controlling the accumulation of Cu(II)-TSC complexes, we investigated the effects of
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temperature and various inhibitors on total cellular accumulation of 2 with the results shown in Figure
. A2780 cells were treated with 2 at 3 μM for 30, 60 and 120 min at 37 and 4 C and intracellular Cu
content was measured by ICP-MS. Prolonged incubation at low temperatures may result in the changes
5
3
2
in membrane fluidity, decreasing membrane permeability and restricting drug uptake; therefore, the
shorter time point of 10 min was also included. The cellular accumulation of 2 dramatically decreased
at low temperature even after 10 min treatment, indicating the involvement of active carrier-mediated
transport or facilitated diffusion. Likewise, the decrease of cellular accumulation at low temperatures
has been reported for both Cu(II)-TSC complexes and metal-free TSCs.33,34 To further clarify whether
the uptake of 2 requires energy, the cellular ATP production was blocked by incubating cells in a saline
solution (HBSS), resulting in total starvation and rapid reduction of intracellular ATP content, and/or by
addition of oligomycin, leading to the inhibition of oxidative phosphorylation.
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Figure 5. Effects of temperature and inhibitors on the accumulation of 2 in A2780 cells. Intracellular content was determined
by ICP-MS: a) A2780 cells were treated with 2 (3 μM) at 37 or 4 C for the indicated time periods. b) A2780 cells were pre-
treated with oligomycin (5 μM), and/or pre-incubated in HBSS for 1 h, or pre-treated with cycloheximide (100 μM) for 4 h
and subsequently co-treated with 2 (3 μM) for 1 h. The presence of inhibitors did not show any effects on the intracellular
Cu level of untreated A2780 cells (data not shown). Statistical analysis was performed by two-tailed T-test using GraphPad
Prism software (GraphPad Software Inc., CA) with p < 0.05 considered as significant (* p < 0.05, ** p < 0.01).
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When A2780 cells were pre-treated with 5 μM of oligomycin for 1 or 4 h and subsequently co-treated
with 3 μM of 2, the intracellular Cu levels were not affected, even though the mitochondrial respiration
of the cancer cells was successfully inhibited (data not shown). However, when A2780 cells were starved
in HBSS for 1 h and subsequently treated with 3 μM of 2 for 1 h, an increase in intracellular Cu content
was observed. Co-treatment of 2 with 5 μM of oligomycin in HBSS did not result in a further increase
of intracellular Cu levels. The increase of Cu content in HBSS-starved cells might stem from the
inhibition of energy-dependent efflux processes. In order to assess the role of proteins in both cellular
accumulation and efflux of 2, A2780 cells were pre-treated with cycloheximide, which is a known
inhibitor of protein synthesis. Significant increase of intracellular Cu content was observed, similar to
HBSS starvation, providing further evidence for the involvement of protein-dependent efflux processes.
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Similar effects have been reported for related Cu(II)-TSC complexes. Accumulated data provide
evidence that cellular accumulation of 2 occurs via carrier-facilitated diffusion, which might co-exist
with passive permeation through the lipid bilayer.35 The efflux processes, however, are actively
mediated by proteins and upon inhibition of protein synthesis or energy production a strong increase of
intracellular Cu concentration was observed.
Cu(II)-TSC complexes demonstrated high antibacterial activity against S. aureus in a comparable
1
6
concentration range to ciprofloxacin. The antibacterial activity of HL ‒HL and 1‒6 on planktonic
cells of P. aeruginosa and S. aureus was investigated by determination of MIC and MIC values
5
0
100
(Table 3). Overall, compounds showed a higher antimicrobial activity against Gram-positive (S. aureus)
compared to Gram-negative bacteria (P. aeruginosa). The highest activity against S. aureus was
observed for 2‒5, which specifically inhibited its growth with MIC values ranging between 2 and 5
5
0
µg/mL and completely abolished its growth at 10 µg/mL (MIC ). This activity is comparable but
1
00
slightly lower that the antibacterial activity of a well-known benchmarked antibiotics ciprofloxacin
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(CPX). On the contrary, P. aeruginosa exhibited high resistance to most of the compounds tested with
2
the exception of HL and 2 with MIC values at 70 and 50 µg/mL, respectively.
5
0
Table 3. Antibacterial activity of HL1‒6 and 1‒6a
S. aureus (g/mL)
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P. aeruginosa (g/mL)
MIC50
MIC100
MIC50
MIC100
HL1
100
>400
NA
NA
HL2
10
10
100
10
NA
100
2
400
300
>400
300
NA
>500
10
50
100
NA
NA
NA
NA
NA
200
400
400
450
NA
2
HL3
100
NA
NA
NA
NA
70
HL4
HL5
HL6
1
2
3
3
10
100
100
200
200
0.5
4
5
10
5
3
10
6
NA
0.5
NA
1
Ciprofloxacin
aSee experimental section for details. NA denotes no antibacterial activity detected (>500 µg/mL).
6
Compounds HL and 6 based on S-methylisothiosemicarbazide were completely devoid of antibacterial
activity and no inhibition of bacterial growth was observed at the whole concentration range tested.
1
5
Interestingly, metal-free thiosemicarbazones HL ‒HL displayed relatively low antibacterial activity
MIC100 > 300 μg/mL), which was significantly enhanced upon coordination to Cu(II). Subsequently,
(
the mode of action of the most active compounds 2‒5 was evaluated in S. aureus and P. aeruginosa
using the Live/Dead viability assay. As shown in Figure 6, compounds 2‒5 greatly reduced cell
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replication, reflected by the lower number of cells compared to the untreated sample and high percentage
of PI-stained cells, indicating compromised cell membrane allowing the penetration of propidium iodide
and a bacteriostatic action of these compounds.
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Figure 6. Live/dead bacterial cells are shown by staining with green fluorescence indicating live cells and red fluorescence
indicating dead cells.
TSCs and their Cu(II) complexes are located in a close proximity to the active site of mouse R2
1
6
RNR protein. Next, the RNR inhibitory potential of the TSC proligands HL ‒HL and their
1
6
corresponding Cu(II) complexes 1‒6 was investigated. The proligands HL ‒HL and Cu(II) complexes
36
1
‒6 were docked into the crystal structure of mouse R2 RNR subunit (PDBid:1W68) using GOLD
3
7
software. To predict the binding of 1‒6, the GoldScore function was used and its parameters were
3
8
modified to include Cu since they are not in GOLD’s database. The scoring functions are presented in
Table S8. Overall, similar docking results were observed for all proligands and their Cu(II) complexes,
suggesting a plausible binding to the mouse R2 protein. The compounds were deeply embedded into the
pocket of the R2 subunit, close to the diferric center (Fe O), and the enzymatically essential tyrosyl
2
residue (Tyr177). The docked configuration of 5 in comparison with Triapine into the binding site is
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shown in Figure 7. An overlap with Triapine was predicted, resulting in a partial reproduction of a
hydrogen-bonding pattern (Figure S14, Table S8).
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Figure 7. The overlay of docked configurations of Cu(II) complex 5 (grey) and Triapine (green) in the binding site of the
mouse R2 RNR protein (PDB ID: 1W68). The surface is rendered. Blue and brown depict hydrophilic and hydrophobic areas,
respectively.
Lead TSCs and their Cu(II) complexes cause moderate inhibition of a mouse R2 RNR protein in
a reducing environment as a result of iron chelation. The effects of the most cytotoxic complexes 2
2
5
and 5, and the corresponding proligands HL and HL ,on the mouse R2 tyrosyl radical were investigated
by EPR spectroscopy at 30 K. The proligands and their Cu(II) complexes were incubated with the mouse
R2 RNR protein at 1:1 protein-to-compound mole ratio at 298 K in the presence or absence of the
reducing agent dithiothreitol (DTT), and the time-dependent reduction of the tyrosyl radical was
3
9
monitored. Tyrosyl radical reduction by the investigated compounds was observed only in the presence
2
5
of DTT. Upon incubation with HL , HL and 2, 5, 50% of tyrosyl radical was reduced within 90 s, after
which no further reduction was observed (Figure S15). Since the diferric center of the mouse R2 protein
is expected to be reduced upon addition of DTT,40,41 the ability of proligands HL and HL to form
2
5
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stable Fe(II) complexes was investigated by monitoring these reactions by UV‒vis measurements in
2
5
buffered aqueous solutions at pH 7.4. HL and HL ( = 312 and 320 nm, respectively) readily reacted
max
with FeSO ·7H O affording Fe(II) bis-ligand complexes ( = 373, 610 nm and max = 385, 645 nm,
4
2
max
respectively) at both 1:1 and 1:2 Fe-to-ligand mole ratios. Additionally, the formation of Fe(III)
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complexes with the subsequent reduction by DTT was investigated (Figure S15). HL and HL reacted
.
III
2
+
III
5
+
with FeCl 6H O (at 1:1 or 1:2 Fe-to-ligand mole ratios) to give [Fe (L ) ] and [Fe (L ) ] complexes
3
2
2
2
III
5
+
(
max = 378 nm, 612 nm and max = 386 nm, 635 nm, respectively). The formation of [Fe (L ) ] species
2
was confirmed by ESI-MS (Figure S16). The absorption spectra of reduced forms were almost identical
II
to those of Fe(II) bis-ligand species, and to the previously reported spectra of other Fe -TSC
complexes.42
2
HL and 2 induced unfolded protein response (UPR), antioxidant defense and cell cycle
perturbations. In order to get further insight into the mechanism of action of 2, Western Blotting of ER
stress-related protein markers, namely IRE1α, CHOP, BiP/GRP78 and p-ERK, was performed (Figure
8
). When A2780 cells were treated with increasing concentrations of 2 for 24 h, the dose-dependent
upregulation of IRE1α and CHOP proteins, decrease of ER stress chaperone BiP/GRP78, as well as
phosphorylation of ERK were observed as a response to ER stress. To assess the effects of 2 on the cell
cycle, we examined the expression of cyclin D1 and cyclin B1 which serve as cell cycle regulatory
switches in actively proliferating cells and are required for cell cycle progression in G and G phases,
1
2
respectively. Complex 2 displayed significant inhibition of cyclin D1 expression, indicating cell cycle
arrest at G /S phase, whereas no changes in cyclin B1 expression were detected. Subsequently, we
1
2
compared the effects of 2 and the respective proligand HL on the expression of p-ERK1/2 and the
2
marker of antioxidant defence NRF2. Both HL and 2 demonstrated phosphorylation of ERK1/2;
however, the effects on NRF2 were markedly different. Whereas no increase of NRF2 expression was
2
observed, when cells were treated with HL , complex 2 induced increased expression of NRF2 even at
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low concentration, indicating activated antioxidant defense, possibly as a result of ROS insult caused by
Cu(II) reduction.
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Figure 8. Western Blot analysis of various proteins. A2780 cells were treated with increasing concentrations of proligand
2
HL and complex 2 (corresponding to IC50 value from MTT experiment with exposure time of 72 h) for 24 h. Total lysates
were isolated and examined by Western Blot. Actin was used as a loading control.
2
HL and 2 induced dose-dependent apoptosis accompanied by PARP cleavage and XIAP
inhibition. To elucidate if observed cytotoxicity of new compounds described in this study was a result
of apoptosis induction, we conducted Annexin V/PI apoptosis assay. In brief, fluorochrome-labeled
Annexin V reagent is used for detection of membrane phosphatidylserine (PS), which translocates to the
cellular surface from the inner side of plasma membrane in the event of early apoptosis. Simultaneously,
propidium iodide (PI) is used for detection of later stages of apoptosis or necrosis, which are
characterized by loss of membrane integrity and accumulation of PI inside the cells. A2780 cells were
2
treated with 2IC and 6IC concentrations of HL and 2 (IC values were obtained from MTT
5
0
50
50
experiment in A2780 cells with the exposure time of 72 h). Results are illustrated in Figure 9. Upon
2
incubation of A2780 cells with increasing concentrations of HL and 2, the dose-dependent increase of
apoptotic cells was observed.
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Figure 9. Bar graphs showing the percentage of cell death due to apoptosis (left) and necrosis (right) in A2780 cells treated
2
with increasing concentrations of HL , 2 (2IC50 and 6IC50) and CuCl
2
and detected by Annexin V/PI apoptosis assay.
Statistical analysis was performed by two-tailed T-test using GraphPad Prism software (GraphPad Software Inc., CA) with
p < 0.05 considered as significant (* p < 0.05, ** p < 0.01).
At low concentrations both the proligand and Cu(II) complex induced a 2- to 3-fold increase of apoptotic
cells in comparison with untreated cells (7.8  1.8 %, 17.2  3.9 % and 22.5  5.3 % for untreated cells,
2
HL (2IC ) and 2 (2IC ), respectively). At high concentrations a further increase of apoptotic cells was
5
0
50
2
observed (28.1  4.0% and 24.4  7.8.% for HL (6IC ) and 2 (6IC )). No significant differences
5
0
50
2
between the apoptosis-inducing properties of the proligand HL and 2 were noticed. When CuCl was
2
incubated with the cells at the concentration, corresponding to Cu content in 2 at 2IC , no induction of
50
apoptosis was detected, which can be explained by poor cellular accumulation of CuCl . The proportion
2
of necrotic cells was negligible and no significant deviation from untreated cells was detected; therefore,
2
2
the induction of necrosis by HL and 2 was ruled out. Subsequently, the effects of HL and 2 on apoptotic
signaling in cells were investigated by Western Blotting. A2780 cells were treated with increasing
concentrations of the drug for 24 h, and the cleavage of poly (ADP-ribose) polymerase-1 (PARP) was
investigated (Figure 8). Cleavage of poly (ADP-ribose) polymerase-1 (PARP) through suicidal proteases
4
3
such as caspases has been widely accepted as an indicator of apoptosis. As can be seen in Figure 8,
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2
both HL and 2 revealed a dose-dependent PARP cleavage, indicating apoptosis induction in agreement
with the results of Annexin V/PI assay. Additionally, expression of X-linked inhibitor of apoptosis
(
XIAP) was markedly reduced in a dose-dependent manner upon treatment with increasing
concentrations of 2.
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Discussion
The diverse biological and chemical properties of Triapine and other TSCs, as well as their transition
metal complexes, have been extensively studied for decades.44,45,46,47,48,41,49,50,51 Their antiproliferative
and antibacterial activity varies greatly, from low nanomolar to high micromolar range, depending on
the substituents at the TSC backbone.16,20,52 Even though clinical trials demonstrated some benefits of
patient treatment with Triapine, its low aqueous solubility and high toxicity prevent it from further
clinical progress. In a continuous effort to improve the bioavailability and therapeutic profile of TSCs
and Cu(II)-TSC complexes, novel thiosemicarbazone hybrids with bioactive L-proline, homoproline,
piperazine or iminodiacetate moieties, using the molecular hybridization approach, have been designed
and synthesized.16,17,18,19,20 However, the improvement of the aqueous solubility was often accompanied
by a significant decrease in cytotoxicity. In this study, the attachment of N-substituted morpholine
moiety at the TSC backbone resulted in a simultaneous improvement of aqueous solubility and
cytotoxicity (up to 50 times in comparison with Triapine). To ensure the selectivity of novel TSCs
towards cancer cells over healthy cells, their cytotoxicity against A2780 cancer cells and noncancerous
HEK293 cells was compared. It was shown that new compounds were 2‒5 fold more toxic towards
cancer cells than healthy cells (Tables 1 and 2), whereas Triapine was more toxic towards noncancerous
cells, which is in agreement with its known high toxicity. The coordination of TSCs to Cu(II) resulted
in Cu(II)-TSC complexes with similar or improved antiproliferative activity. In general, when TSCs are
coordinated to Cu(II), an increase in cytotoxicity is observed;17,18,53 however, Cu(II)-Triapine complex
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was shown to be significantly less cytotoxic than Triapine itself. The activity of the Cu(II) complexes
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1‒6 was in a good correlation with their lipophilicity, cellular accumulation, as well as their
electrochemical redox potentials (Tables S6 and 2). Interestingly, metal-free TSCs exhibited only low
antibacterial properties against S. aureus, whereas Cu(II)-TSC complexes revealed significant
antibacterial activity similar to that of the commonly used antibiotic ciprofloxacin (Table 4). These
results indicate that coordination of TSCs to Cu(II) improved their therapeutic potential. It should be
noted that the replacement of N,N,S- with N,N,N-chelating moiety in 6 resulted in a drop of cytotoxicity
and antibacterial activity, which is in agreement with the previously reported data.20
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The antiproliferative activity of Triapine and other TSC proligands is often related to their Fe-chelating
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properties. To sustain their high proliferation rate, cancer cells rely on increased uptake of Fe from Fe-
transporting proteins; therefore, Fe chelation is a valuable therapeutic strategy for cancer treatment.
Based on UV–vis and ESI-MS experiments, it was demonstrated that irrespective of metal-to-ligand
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ratio, proligands HL and HL readily reacted with Fe(III) to form [Fe (L ) ] and [Fe (L ) ] species,
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which could be reduced to Fe(II) bis-ligand complexes by the reducing agent DTT. These results indicate
that morpholine-TSC hybrids, investigated in this study, may act as Fe chelators. Additionally,
spectroelectrochemical experiments revealed that under reducing conditions, the Cu(II)-TSC complexes
could undergo reversible reduction to Cu(I) species, with simultaneous release of proligands, suggesting
that these complexes may act as Fe chelators, also in cancer cells. In addition, the presence of the Cu(II)
ion in the cells may lead to the production of ROS in vivo, due to Cu(II)/Cu(I) redox cycling.55
It is well known that one of the mechanisms contributing to the cytotoxic effects of Fe chelators is
inhibition of the RNR enzyme, which is highly expressed in cancer cells, and essential for their DNA
synthesis. The mechanism of RNR inhibition by Triapine has been extensively studied by several
research groups.41,46,47,51,56 It has been proposed that Triapine interferes with the assembly of the
differic-tyrosyl radical cofactor in R2, necessary for the catalytic activity of RNR.46,56 Moreover, it has
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been shown that the presence of Fe is required for effective R2 inhibition,44 and that it is actually the
Fe(II)-Triapine species that is responsible for the R2-specific RNR inhibitory effect of
Triapine.41,46,47 51 58 The Fe(II)-Triapine complex may be formed by chelation of Fe from the differic
,
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cofactor in R2 in vitro, 41 and/or from intracellular iron pools in vivo. 56 The potent inhibition of
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human, and mouse R2 RNR in vitro by catalytic amounts of Fe(II)-Triapine has been proposed to
involve ROS. Namely, ROS had been spin-trapped, and detected by EPR spectroscopy, in the aerobic
reaction between human R2 RNR and Fe(II)-Triapine, which had implicated that O is important in
2
tyrosyl radical destruction, and that ROS may ultimately be responsible for the pharmacologic effects of
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Triapine in vivo. In another study of time-dependent tyrosyl radical reduction in mouse R2 by Triapine,
and its Zn, Ga, Cu, and Fe complexes, the requirement of O in R2 inhibition was also suggested.41 This
2
was based on the observation that substoichiometric amounts of Fe(III)-Triapine relative to R2 (protein-
to-complex mole ratio, 5:1) reduced only 20% tyrosyl radical in anaerobic conditions, compared to
1
00% in aerobic conditions. However, more recently, in a different experimental setup, namely in the
presence of a 10-fold excess of Fe(II)-Triapine over R2 protein, the role of ROS (and O ) in human R2
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inhibition was excluded. This study showed that the principle mechanism of human R2 RNR inhibition
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by Triapine, based on kinetic measurements of tyrosyl radical, and Fe loss, is direct radical quenching,
in an iron-loaded protein. Furthermore, it implied that Fe(II)-Triapine can rapidly reduce the tyrosyl
radical, while leaving the protein in the met-state.
In order to investigate if R2 RNR could be the potential biomolecular target for the novel morpholine-
TSC hybrids, and their Cu(II) complexes, molecular docking studies, and R2 RNR tyrosyl radical
reduction kinetic experiments, were performed. It has been suggested that the efficiency of R2 inhibition
4
1,
is likely to depend on the access of the inhibitors to the differic center of R2; 47 therefore, we estimated
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the likelihood of the binding of TSC proligands HL ‒HL and their corresponding Cu(II) complexes
‒6 to the surface of the R2 protein. It was demonstrated that new TSCs, and their Cu(II) complexes
1
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were deeply embedded into the pocket of the R2 protein and exhibited hydrogen bonding patterns similar
to Triapine (Figure 7). Based on the results of the molecular docking studies suggesting plausible binding
to the mouse R2 protein, the time-dependent tyrosyl radical reduction in the mouse R2 protein upon
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incubation with the most cytotoxic complexes, 2 and 5, and the corresponding proligands HL and HL ,
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at a 1:1 protein-to-compound ratio, was measured by EPR spectroscopy. Only in the presence of DTT
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(
which can reduce Fe(III) to Fe(II) in the R2 differic cofactor), HL and HL caused 50% tyrosyl
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radical reduction (Figure S15a). These results show that R2 inactivation by HL and HL is not as
efficient as with Triapine, which causes 100% tyrosyl radical loss in mouse R2 after 5 min (in the same
experimental conditions).41 The most plausible explanation for this observation is that different
2
5
mechanisms are likely to be involved in R2 inhibition by HL , HL and Triapine. The R2 inhibitory
activity of the corresponding Cu(II) complexes 2 and 5 was found to be similar to that of the proligands
(Figure S15a). This is likely due to the fact that in the presence of DTT, the reduction of Cu(II) to Cu(I)
leads to the release of the proligands, which is in agreement with the results of spectroelectrochemical
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5
experiments (vide supra). Since the UV–vis, and ESI-MS experiments showed that HL and HL act as
tridentate ligands, and form stable Fe(II)L complexes (Figure S15), it is possible to propose that the
2
mechanism of R2 inhibition by the TSC proligands involves Fe chelation from the differic cofactor.
Based on the extent of tyrosyl radical reduction, it is tempting to speculate that the subsequently formed
Fe(II) complexes are not potent inhibitors like the Fe(II)-Triapine complex. However, as this was not
the aim of this paper, it is reasonable to refrain from any conclusions prior to the detailed mechanistic
studies of mouse, human, and p53 R2 RNR inhibition by the morpholine-TSC hybrids. Moreover, a
separate study should be dedicated only to the comparison of the inhibitory potentials of selected TSCs,
and their Fe(II) complexes, in order to comprehensively understand the structure-activity relationship,
which in turn may explain the mechanism of R2 RNR inhibition. Finally, even though R2 inhibition can
be partially attributed to the marked anticancer activity of 2 and 5, other biomolecular targets are likely
to be involved (Figure 10).
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