Iuliana Besleaga1, Iryna Stepanenko1, Tatsiana V Petrasheuskaya2,3, Denisa Darvasiova4, Martin Breza4, Marta Hammerstad5, Małgorzata A Marć2,6, Alexander Prado-Roller1, Gabriella Spengler3,6, Ana Popović-Bijelić7, Eva A Enyedy2,3, Peter Rapta4, Anatoly D Shutalev8, Vladimir B Arion1. 1. Institute of Inorganic Chemistry, University of Vienna, Währinger Strasse 42, A-1090 Vienna, Austria. 2. Department of Inorganic and Analytical Chemistry, Interdisciplinary Excellence Centre, University of Szeged, Dóm tér 7, H-6720 Szeged, Hungary. 3. MTA-SZTE Lendület Functional Metal Complexes Research Group, University of Szeged, Dóm tér 7, H-6720 Szeged, Hungary. 4. Institute of Physical Chemistry and Chemical Physics, Faculty of Chemical and Food Technology, Slovak University of Technology in Bratislava, Radlinského 9, SK-81237 Bratislava, Slovak Republic. 5. Section for Biochemistry and Molecular Biology, Department of Biosciences, University of Oslo, P.O. Box 1066, Blindern, NO-0316 Oslo, Norway. 6. Department of Medical Microbiology, Albert Szent-Györgyi Health Center and Faculty of Medicine, University of Szeged, Dóm tér 10, 6725 Szeged, Hungary. 7. Faculty of Physical Chemistry, University of Belgrade, Studentski trg 12-16, 11158 Belgrade, Serbia. 8. N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 47 Leninsky Avenue, 119991 Moscow, Russian Federation.
Abstract
Three new thiosemicarbazones (TSCs) HL1-HL3 as triapine analogues bearing a redox-active phenolic moiety at the terminal nitrogen atom were prepared. Reactions of HL1-HL3 with CuCl2·2H2O in anoxic methanol afforded three copper(II) complexes, namely, Cu(HL1)Cl2 (1), [Cu(L2)Cl] (2'), and Cu(HL3)Cl2 (3), in good yields. Solution speciation studies revealed that the metal-free ligands are stable as HL1-HL3 at pH 7.4, while being air-sensitive in the basic pH range. In dimethyl sulfoxide they exist as a mixture of E and Z isomers. A mechanism of the E/Z isomerization with an inversion at the nitrogen atom of the Schiff base imine bond is proposed. The monocationic complexes [Cu(L1-3)]+ are the most abundant species in aqueous solutions at pH 7.4. Electrochemical and spectroelectrochemical studies of 1, 2', and 3 confirmed their redox activity in both the cathodic and the anodic region of potentials. The one-electron reduction was identified as metal-centered by electron paramagnetic resonance spectroelectrochemistry. An electrochemical oxidation pointed out the ligand-centered oxidation, while chemical oxidations of HL1 and HL2 as well as 1 and 2' afforded several two-electron and four-electron oxidation products, which were isolated and comprehensively characterized. Complexes 1 and 2' showed an antiproliferative activity in Colo205 and Colo320 cancer cell lines with half-maximal inhibitory concentration values in the low micromolar concentration range, while 3 with the most closely related ligand to triapine displayed the best selectivity for cancer cells versus normal fibroblast cells (MRC-5). HL1 and 1 in the presence of 1,4-dithiothreitol are as potent inhibitors of mR2 ribonucleotide reductase as triapine.
Three new thiosemicarbazones (n class="Chemical">TSCs) HL1-HL3 as triapine analogues bearing a redox-active phenolic moiety at the terminal nitrogen atom were prepared. Reactions of HL1-HL3 with CuCl2·2H2O in anoxic methanol afforded three copper(II)complexes, namely, Cu(HL1)Cl2 (1), [Cu(L2)Cl] (2'), and Cu(HL3)Cl2 (3), in good yields. Solution speciation studies revealed that the metal-free ligands are stable as HL1-HL3 at pH 7.4, while being air-sensitive in the basic pH range. In dimethyl sulfoxide they exist as a mixture of E and Z isomers. A mechanism of the E/Z isomerization with an inversion at the nitrogen atom of the Schiff baseimine bond is proposed. The monocationic complexes [Cu(L1-3)]+ are the most abundant species in aqueous solutions at pH 7.4. Electrochemical and spectroelectrochemical studies of 1, 2', and 3 confirmed their redox activity in both the cathodic and the anodic region of potentials. The one-electron reduction was identified as metal-centered by electron paramagnetic resonance spectroelectrochemistry. An electrochemical oxidation pointed out the ligand-centered oxidation, while chemical oxidations of HL1 and HL2 as well as 1 and 2' afforded several two-electron and four-electron oxidation products, which were isolated and comprehensively characterized. Complexes 1 and 2' showed an antiproliferative activity in Colo205 and Colo320 cancer cell lines with half-maximal inhibitory concentration values in the low micromolar concentration range, while 3 with the most closely related ligand to triapine displayed the best selectivity for cancer cells versus normal fibroblast cells (MRC-5). HL1 and 1 in the presence of 1,4-dithiothreitol are as potent inhibitors of mR2 ribonucleotide reductase as triapine.
Thiosemicarbazones
(n class="Chemical">TSCs) are known as biologically active compounds
with a broad spectrum of pharmacological properties, including anticancer
activity.[1−4] These properties can be modulated by coordination to physiologically
relevant metal ions.[5,6] In addition, as versatile ligands,
TSCs have tunable electronic and steric properties, which may have
a favorable effect on their pharmacological profile.[7−10] α-N-HeterocyclicTSCs such as 2-formylpyridineTSC (FTSC)
and 5-hydroxy-2-formylpyridineTSC were reported to possess anticancer
activity several decades ago,[11,12] and further optimization
resulted in the most well-known TSC, 3-aminopyridine-2-carboxaldehydeTSC (triapine). Triapine was tested in more than 30 clinical phase
I and II trials and currently is involved in a triapine-cisplatin-radiation
combination therapy in phase III trial.[13] Because of the documented side effects (e.g., methemoglobinemia)
of triapine and its unfavorable pharmacokinetic profile (e.g., short
plasma half-life),[14] the development of
novel TSCs with improved pharmaceutical properties and an established
mechanism of action is of high research interest. Notably, two other
TSCs, namely, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone
(DpC) and 4-(2-pyridinyl)-2-(6,7-dihydro-8(5H)-quinolinylidene)-hydrazide
(COTI-2), are currently undergoing a phase I evaluation as chemotherapeutic
agents.[8,15]
The iron-n class="Chemical">containing ribonucleotide
reductase (RNR) is considered
as one of the main targets for triapine and related α-N-pyridinecarboxaldehydeTSCs.[16−19] This enzyme catalyzes the reduction
of ribonucleotides to deoxyribonucleotides, and it is particularly
important in rapidly dividing cells, such as tumor cells, virally
infected cells, and invading bacteria. All these cells share similar
properties, such as high proliferation rates, quickly spreading within
the host, and aggressive disease progression.[20] A sustained proliferation requires an increased de novo nucleotide
synthesis for DNA replication, making RNR targeting a relevant strategy
in the treatment of cancer.[21,22] RNRs are free radical-containing
proteins. One way to control and modulate their reactivity is via
quenching the catalytically essential tyrosyl radical Y· located
in the small RNR subunit (R2 or NrdB).[23,24] The radical
scavengers and iron-chelating ligands, which are able to destroy the
diferric-tyrosyl radicalcofactor, with the aim to inhibit R2RNR,
are widely investigated in anticancer research.[25] In the case of triapine, it has been suggested that the
intracellularly formed, highly potent, redox-active ironcomplex either
leads to reactive oxygen species (ROS) formation, which are then responsible
for tyrosyl radical quenching, or that the iron(II)complex itself
is able to directly reduce the tyrosyl radical.[16] Besides triapine, several other R2RNR inhibitors such
as hydroxyurea, 3,4-dihydroxybenzohydroxamic acid (Didox), and 3,4,5-trihydroxybenzamidoxime
(Trimidox) have entered clinical trials.[26] Among other potential tyrosyl radical quenchers, p-alkoxyphenols (i.e., p-methoxyphenol, p-ethoxyphenol, p-propoxyphenol, and p-allyloxyphenol) and pyrogallol as well as 4-mercaptophenol were
identified.[27−29] The mechanism of RNR inhibition by the p-alkoxyphenols and pyrogallol was investigated by both experimental
techniques (electron paramagnetic resonance (EPR) and UV–visible
(UV–vis) spectroscopy) and theoretical tools (molecular docking
and molecular dynamics simulations). Among the aminophenols several
compounds were tested as anticancer agents, for example, the nonsteroidal
anti-inflammatory drug N-acetyl-p-aminophenol (acetaminophen), which showed antimelanoma activity
to prooxidant glutathione (GSH) depletion by the 3-hydroxy-1,4-quinone-imine-metabolite.[29,30] Fenretinide (a synthetic retinoid derivative) was introduced in
clinical trials for the treatment of breast, bladder, renal, and neuroblastoma
malignancies due to its antioxidant activities via scavenging radicals.[31]
It is also worth noting that a coordination
to n class="Chemical">copper(II) may significantly
augment the cytotoxic activity of TSCs.[6,10] Copper(II)
as an essential trace element is redox-active, biocompatible, and
less toxic than nonendogenous heavy metals. The redox metabolism of
cancer cells is different from that of healthy cells and is characterized
by increased copper levels in an intracellular environment.[32,33] Moreover, it was recently suggested that the copper(II)TSCcomplexes,
rather than any metal-free TSCs or their cellular metabolites, are
responsible for the biological effects in vitro and in vivo.[6] One of the reasons for the increased antiproliferative
activity of copper(II)complexes of TSCs and the selectivity for cancer
cells is considered to be the redox cycling between two oxidation
states (Cu2+ ↔ Cu+) in a biologically
accessible window of potentials (from −0.4 to +0.8 V vs normal
hydrogen electrode (NHE)) and ROS generation.[6,34] In
this context it is also remarkable that a copper-redox cycle mechanism
was found to be responsible for the oxidation of phenolic compounds
leading ultimately to reactive oxygen-dependent DNA damage.[35] The same authors suggested that singlet oxygen
or a singlet oxygen-like entity (e.g., a copper-peroxidecomplex)
rather than the free hydroxyl radical plays a role in DNA damage.[35] At the same time it is worth noting that the
idea that an efficient redox cycling of copper(II,I)complexes with
thiosemicarbazones can be involved in the anticancer mechanism has
been recently challenged[36] by showing that
the most resistant to reduction copper(II) thiosemicarbazonates were
the most cytotoxic. In addition, the complexes can also dissociate
fast, if the thiosemicarbazone has different affinities to copper(II)
and copper(I) and can lose the competition for copper(I) to metallothioneins
(MT) and glutathione (GSH).[37]
With
this background in mind we aimed at (i) attachment of a phenolic
moiety at atom N4 of thiosemicarbazide, (ii) investigation of solution
speciation, n class="Chemical">complex formation reactions of new TSCs with copper(II)
in solution, and synthesis of copper(II)complexes, (iii) investigation
of the reduction/oxidation of TSCscontaining this potentially redox
active group, namely, the 4-aminophenolic unit, and copper(II)complexes
thereof by electrochemical and spectroelectrochemical techniques and
by using chemical oxidants, for example, O2, p-benzoquinone (PBQ), 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ),
and phenyliodine(III) diacetate (PIDA), as two-electron/two proton
acceptors and Ag2O, along with an analysis of the reversibility
of the oxidation process and the number of participating electrons,
(iv) identification of the effects of phenolic unit and coordination
to copper(II) on the redox activity and cytotoxicity in vitro as well
as on the mR2RNR inhibition and estimation of their potency to act
as reductants for a tyrosyl radical with an apparent redox potential
of +1000 ± 100 mV versus NHE.[38]
In this work we report on the synthesis of new n class="Chemical">triapine derivatives HL–HL, which contain a potentially redox-active 4-aminophenolic
unit, and of copper(II)complexes Cu(HL)Cl (1), [Cu(L)Cl] (2′), and Cu(HL)Cl (3) (Chart ). The solution behavior of the new TSCs
(HL–HL), the mechanism typical for TSC E/Z isomerization, and the stability and redox properties
of both the metal-free ligands and copper(II)complexes (1, 2′, 3) were also investigated
by UV–vis spectrophotometry and UV–vis/EPR spectroelectrochemistry
and density functional theory (DFT) calculations. In addition, the
two- and four-electron oxidation products H and H, respectively, were prepared both electrochemically and by chemical
oxidation and used in a complex formation with copper(II). Several
oxidation products of HL (H, H, H, and H) were prepared by using different oxidation
agents. Likewise, copper(II)complexes with oxidized ligands 4–6 were obtained (see Chart and Scheme ). The isolated compounds were characterized
by analytical and spectroscopic methods (one-dimensional (1D) and
two-dimensional (2D) NMR, UV–vis, IR), electrospray ionization
(ESI) mass spectrometry (MS), cyclic voltammetry (CV), and single-crystal
X-ray diffraction (SC-XRD). The anticancer activity of the TSCs (HL–HL), their oxidized products (H, H, and H·CHCOOH), and the copper(II)complexes (1, 2′, and 3) was tested against two humancancer cell lines
(doxorubicin-sensitive Colo205 and the multidrug-resistant Colo320
humancolonic adenocarcinoma) and normal human embryonal lung fibroblast
cells (MRC-5) along with their mR2RNR inhibiting ability, and the
results are discussed.
Chart 1
TSCs and Their Copper(II) Complexes Studied
in This Worka
Chart 2
Oxidation Products of HL and HL and Copper(II) Complexes with
Oxidized Ligandsa
Scheme 1
Oxidation Products
of HL and HL along with Those of Copper(II)
Complexes
The bottom left panel shows
the oxidants used.
Underlined labels/numbers
indicate compounds studied by SC-XRD. The five-n class="Chemical">coordination of copper(II)
in 1 and 3 has not been confirmed by X-ray
crystallography.
Underlined labels/numbers
indicate compounds studied by SC-XRD, while the italic L denotes an oxidized ligand.
Oxidation Products
of HL and HL along with Those of Copper(II)
Complexes
The bottom left panel shows
the oxidants used.
Experimental
section
Chemicals
2-Formylpyridine, n class="Chemical">2-acetylpyridine, and CuCl2·2H2O were purchased from commercial suppliers
and used without further purification. 3-(tert-Butoxycarbonyl)amino-2-formylpyridine
and 4-(4-hydroxy-3,5-dimethylphenyl)thiosemicarbazide were synthesized
as reported previously.[39,40] KCl, KOH, HCl, and
dimethyl sulfoxide (DMSO) were obtained from Reanal. GSH, 2-morpholinoethanesulfonic
acid (MES), and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic
acid (HEPES) were purchased from Sigma-Aldrich and used without further
purification. Copper(II) stock solution was prepared by the dissolution
of CuCl2 in water, and its concentration was determined
by complexometry with ethylenediaminetetraacetic acid (EDTA). The
stock solutions of HL–HL in DMSO were prepared on a weight-in-volume
basis.
To a solution of
3-(tert-butoxycarbonyl)amino-2-formylpyridine (210
mg, 0.95 mmol) and n class="Chemical">4-(4-hydroxy-3,5-dimethylphenyl)thiosemicarbazide
(200 mg, 0.95 mmol) in a mixture of ethanol/water 3:1 (8 mL) was added
dropwise 12 M HCl (0.19 mL, 2.28 mmol). This solution was stirred
at room temperature for 1 h to give Boc-HL·HCl (C20H25N5O3S·HCl, positive ion ESI-MS for C20H25N5O3S (Mr = 415.51) (MeCN/MeOH+1% H2O): m/z 416.18 [Boc-HL3+H]+, negative
ion ESI-MS: m/z 414.02 [Boc-HL3–H]−). The Boc-deprotection of HL was completed at 85 °C for
7 h with monitoring by ESI-MS (positive ion ESI-MS for C15H17N5OS (Mr = 315.39)
(MeCN/MeOH + 1% H2O): m/z 316.12 [HL3+H]+, 338.11 [HL3+Na]+, negative ion ESI-MS: m/z 314.11 [HL3–H]−). After ethanol
evaporation, the solution was neutralized with a saturated solution
of NaHCO3 (pH = 8). The precipitate was collected and dried
in vacuo. Yield: 267 mg, 87.9%. Anal. Calcd for C15H17N5OS·0.25H2O (Mr = 319.90): C, 56.31; H, 5.51; N, 21.89; S, 10.02. Found:
C, 56.33; H, 5.34; N, 21.68; S, 10.29%. 1H NMR (600 MHz,
DMSO-d6, E isomer) δ,
ppm: 11.47 (s, 1H, H9), 9.70 (s, 1H, H11), 8.39
(s, 1H, H7), 8.21 (s, 1H, H18), 7.85 (dd, J = 4.3, 1.4 Hz, 1H, H6), 7.15 (dd, J = 8.3, 1.2 Hz, 1H, H4), 7.08 (dd, J =
8.3, 4.3 Hz, 1H, H5), 6.92 (s, 2H, H13+H17), 6.49 (s, 2H, H3′), 2.16 (s, 6H, H19+H20). 13C NMR (151 MHz, DMSO-d6, E isomer) δ, ppm:
176.13 (C10), 151.17 (C15), 149.23 (C7), 143.99 (C3), 137.25 (C6), 132.97 (C2), 130.59 (C12), 126.88 (C13+C17), 124.52 (C5), 123.83 (C14+C16),
122.34 (C4), 16.63 (C19+C20). 15N NMR (61 MHz, DMSO-d6, E isomer) δ, ppm: 321.53 (N1), 312.8 (N8), 174.57 (N9), 126.69 (N11), 71.10
(N3′). IR (ATR, selected bands, ṽmax): 3456.59, 3347.73, 3142.99, 3002.80, 1615.50, 1547.68,
1512.07, 1299.63, 1248.47, 1189.77, 1143.84, 861.56, 796.22, 685.36
cm−1. UV–vis (MeOH), λmax, nm (ε, M–1 cm–1): 299
(1374), 375 (2220), 448 sh. Single crystals of HL suitable for X-ray data collection were obtained
from the mother liquor.
Synthesis of the Copper(II) Complexes
Cu(HL1)Cl2·0.5H2O (1·0.5H2O)
CuCl2·2n class="Chemical">H2O (128 mg,
0.75 mmol) was added to HL (225
mg, 0.75 mmol) in anoxic methanol (10 mL) in a Schlenk tube and stirred
at room temperature under argon for 10 min. The reaction mixture was
allowed to stand at 4 °C overnight. The dark green precipitate
was filtered off under argon, washed with anoxic methanol, and dried
in vacuo. Yield: 294 mg, 88.4%. Anal. Calcd for C15H16N4OSCuCl2·0.5H2O (Mr = 443.84): C, 40.59; H, 3.86; N, 12.62; S,
7.22. Found: C, 40.73; H, 3.59; N, 12.63; S, 7.19%. Positive ion ESI-MS
for C15H16N4OSCuCl2 (MeCN/MeOH+1%
H2O): m/z 362.03 [Cu(HL1)2+–H]+, negative ion ESI-MS: m/z 395.99 [Cu(HL1)Cl+–2H]−. IR (ATR, selected bands, ṽmax): 3480.77, 2989.07, 1610.63, 1479.59,
1269.25, 1229.98, 1189.75, 1025.69, 774.69, 665.85 cm–1. UV–vis (MeOH), λmax, nm (ε, M–1 cm–1): 280 (16 800), 376
sh, 422 (18 160). Crystals of [Cu(L)Cl]·CHOH (1′·CHOH) (Mr = 398.37) suitable
for X-ray diffraction study were grown from an ∼20-fold-diluted
reaction mixture in a Schlenk tube under argon upon standing at 4
°C. A recrystallization of [Cu(HL)Cl] (1) in methanol in air afforded a minor amount of X-ray diffraction-quality
crystals of [Cu()Cl] (4).
[Cu(L2)Cl]·0.5H2O (2′·0.5H2O)
CuCl2·2n class="Chemical">H2O (129 mg,
0.76 mmol) was added to a solution of HL (238 mg, 0.76 mmol) in anoxic methanol (10 mL)
in a Schlenk tube. The reaction mixture was stirred at room temperature
under argon for 10 min and then allowed to stand at 4 °C overnight.
The greenish-brown precipitate was filtered off under argon, washed
with anoxic methanol, and dried in vacuo. Yield: 316 mg, 98.8%. Anal.
Calcd for C16H17N4OSCuCl·0.5H2O (Mr = 421.40): C, 45.60; H,
4.31; N, 13.30; S, 7.61. Found: C, 45.74; H, 4.03; N, 13.42; S, 7.56%.
Positive ion ESI-MS for C16H17N4OSCuCl
(MeCN/MeOH+1% H2O): m/z 376.04 [Cu(L2)]+, negative ion ESI-MS: m/z 410.00 [Cu(L2)Cl–H]−. IR (ATR, selected bands, ṽmax): 3341.84, 3223.12, 1609.18, 1547.35, 1483.22, 1452.56,
1303.41, 1202.82, 1019.61, 846.14, 701.29 cm–1.
UV–vis (MeOH), λmax, nm (ε, M–1 cm–1): 277 (11 835), 316 sh, 421 (12 953).
Crystals of [Cu(L)Cl] (2′) suitable for X-ray diffraction study were
obtained from an ∼20-fold-diluted reaction mixture under argon
in a Schlenk tube at 4 °C.
Cu(HL3)Cl2·0.25H2O (3·0.25H2O)
CuCl2·2n class="Chemical">H2O (114 mg,
0.67 mmol) was added to HL (210
mg, 0.67 mmol) in anoxic methanol (10 mL) in a Schlenk tube and stirred
at room temperature under argon for 10 min. The reaction mixture was
allowed to stand at 4 °C overnight. The green precipitate was
filtered off under argon, washed with anoxic methanol, and dried in
vacuo. Yield: 285 mg, 93.6%. Anal. Calcd for C15H17N5OSCuCl2·0.25H2O (Mr = 454.35): C, 39.65; H, 3.88; N, 15.41; S,
7.06. Found: C, 39.58; H, 3.79; N, 15.21; S, 6.98%. Positive ion ESI-MS
for C15H17N5OSCuCl2 (MeCN/MeOH+1%
H2O): m/z 377.04 [Cu(HL3)2+–H]+, negative ion ESI-MS: m/z 411.00 [Cu(HL3)Cl+–2H]−. IR (ATR, selected bands, ṽmax): 3422.07, 3340.63, 1647.85, 1569.29,
1480.67, 1223.63, 1185.74, 1023.07, 718.76, 660.61 cm–1. UV–vis (MeOH), λmax, nm (ε, M–1 cm–1): 262 (19 564), 288
(17 425), 462 (23 514). Crystals of [Cu(L)Cl]·CHOH, (3′·CHOH) (Mr =
413.38) suitable for X-ray diffraction study were grown from an ∼20-fold-diluted
reaction mixture in a Schlenk tube under argon at 4 °C.
Details about the synthesis and characterization of oxidized n class="Chemical">thiosemicarbazones
and their copper(II)complexes, X-ray data collection and refinement
(Tables S1–S3), elemental analysis,
UV–vis titrations, kinetic measurements, lipophilicity determination,
spectroelectrochemical studies, in vitro cell studies, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide (MTT) assays, and tyrosyl radical reduction in mouseR2RNR
protein as well as computational details are given in the Supporting Information (Sections 1 and 2).
Results and Discussion
Synthesis and Characterization of HL–HL
The new TSCs HL–HL were obtained by
n class="Chemical">Schiff base condensation
reactions of 4-(4-hydroxy-3,5-dimethylphenyl)thiosemicarbazide[40] with the corresponding aldehyde (HL, HL) or ketone (HL) in boiling
ethanol (HL, HL) or ethanol/water (3:1, HL) in the absence (HL and HL) or in
the presence of 12 M HCl (HL). The hydrochloric acid in this latter case was used for Boc-deprotection
of the intermediate Boc-HL. This deprotection reaction was monitored by ESI-MS (disappearance
of peaks attributed to [Boc-HL3+H]+ and [Boc-HL3–H]− ions) and completed at 85 °C
after 7 h, with yields ranging from 67 to 88%. The formation of HL–HL was confirmed by ESI mass spectra, which showed peaks
assigned to ions [HL1–3+H]+, [HL1–3+Na]+, and [HL1–3–H]−. One- and two-dimensional NMR spectra were in agreement
with the expected structures for HL–HL of C1 molecular symmetry. In addition, the spectra
indicated the presence of E and Z isomers in DMSO-d6, which is typical
for thiosemicarbazones,[41−43] with a significant predominance
of E isomers (E/Z = 23:1, 17:1, and 31:1 for HL–HL, respectively).
The assignment of E and Z isomers
was based on NMR spectra, including 1H, 1H nuclear
Overhauser effect spectroscopy (NOESY), which are presented in more
detail in the Supporting Information (see
also Schemes S1 and S2 and Tables S4–S6). It is noteworthy
that, in contrast to the E isomers of HL–HL, their Z isomers can form an intramolecular
hydrogen bond between the pyridinenitrogen and the NH-N group hydrogen,
resulting in an increase in the relative stability of these conformers.
Indeed, the DFT B3LYP/6-311++G (d,p) calculations for E- and Z-HL in a DMSO solution (the polarizable continuum model (PCM) solvation
model) showed that the most stable conformer of Z-HL lies lower in energy than
the most stable conformer of E-HL (ΔE = 1.45 kcal/mol;
ΔG = 0.76 kcal/mol at 298 K and 1 atm). The
calculations also demonstrate that E- and Z-HL are very close
in thermodynamic stability (ΔE = 0.90 kcal/mol
in favor of Z-HL, ΔG = 0.00 kcal/mol), and E-HL is slightly more stable
than Z-HL (ΔE = 0.84 kcal/mol, ΔG = 0.86 kcal/mol),
which can be explained by the presence of an intramolecular hydrogen
bond between the 3-NH2 group and the aldiminenitrogen
in E-HL. Thus,
the formation of HL–HL with a large predominance of the E isomers indicates that the reactions proceed under a kinetic
control. By using DFT B3LYP/6-311++G(d,p) calculations to understand
the interconversion between E and Z isomers of 2-formylpyridine and thiosemicarbazones as model compounds
we found out that an isomerization involving a tautomeric shift of
the thioamide N2H proton to the pyridinenitrogen followed by a rotation
around the formed C–N1 bond, as proposed previously,[44] is not favored energetically (see the Supporting Information for details). We believe
that the most plausible Z/E isomerization
pathway in thiosemicarbazones and semicarbazones involves an inversion
at the iminenitrogen.[45] The intrinsic
reaction coordinate (IRC) analysis for one of the aforementioned model
compounds revealed that the found transition state connects the desired
minima. However, the calculation data obtained show (for more details
see the Supporting Information) that the
Gibbs free energy barrier for the conversion of the most stable conformer
of the Z isomer into the E isomer
is relatively high (ΔG = 35.2 kcal/mol in the
gas phase, 35.4 kcal/mol in DMSO solution) (Figure ), which rejects the possibility of an interconversion
between the isomers at room temperature.
Figure 1
Electronic energy and
Gibbs free energy profiles (in kcal/mol)
for the transformation of the most stable conformer of (Z)-2-formylpyridine thiosemicarbazone into the most stable conformer
of (E)-2-formylpyridine thiosemicarbazone in DMSO
solution. Free energies (in parentheses) at 298 K and 1 atm.
Electronic energy and
Gibbs free energy profiles (in kcal/mol)
for the transformation of the most stable conformer of (Z)-n class="Chemical">2-formylpyridine thiosemicarbazone into the most stable conformer
of (E)-2-formylpyridinethiosemicarbazone in DMSO
solution. Free energies (in parentheses) at 298 K and 1 atm.
The redox activity of HL–HL in
the anodic region was validated
by cyclic voltammetry (vide infra). Their behavior as reductants is
also relevant for quenching the n class="Chemical">tyrosyl radical in the mR2-protein.
Therefore, attempts to perform an oxidation of HL and HL by electrolysis and by chemical oxidation were undertaken.
Oxidation
of TSCs
The oxidation of different organic
molen class="Chemical">cules with p-benzoquinone derivatives is well-documented
in the literature.[46] The reaction of HL with DDQ (2e–/2H+E° = +0.887 V vs NHE in an
acidic 0.1 M aqueous solution of p-TsOH)[47] in a 1:1 molar ratio resulted in two-electron
and four-electron oxidative cyclizations with the major formation
of H (60.9%) accompanied by a minor generation of H (<5%), both containing a 1,3,4-thiadiazole ring (Chart , Scheme ). The formation of the 1,3,4-thiadiazole
ring occurs via a nucleophilic attack of the sulfur atom to the carbon
atom of the aldimine bond of HL as evidenced by frontier molecular orbitals with the highest occupied
molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO)
located at opposite sides of the molecule (Figure ).
Figure 2
Frontier orbitals in HL:
(a) LUMO and (b) HOMO drawn at 0.1 au isosurface.
Frontier orbitals in HL:
(a) LUMO and (b) HOMO drawn at 0.1 au isosurface.The use of a double amount of DDQ led to the formation of the four-electron
oxidation product HL in 71.6% yield. The electrolysis of HL at 1000 mV inn class="Chemical">CH3CN versus Ag/AgCl resulted
in the same oxidation products (vide infra). Both compounds were characterized
by ESI mass spectra, which showed peaks at m/z 299.17 [HL1+H]+, 321.16 [HL1a′+Na]+, 297.18 [HL1a+H]+, 319.20 [HL1a+Na]+, and 296.94 [HL1a′–H]−. The more sterically hindered ketiminecarbon atom in HL was expected
to reduce the likelihood of the 1,3,4-thiadiazole ring formation.
The reaction of HL with DDQ
in a 1:1 molar ratio in methanol led to decomposition of the TSC with
formation of an unidentified species. When PBQ, a weaker oxidant (2e–/2H+E° = 0.643 V
vs NHE in an acidic 0.1 M aqueous solution of p-TsOH)
than DDQ, was used,[47] a two-electron oxidative
cyclization with the formation of a 1,2,4-triazole-3-thione ring (TAT
group, H) occurred, accompanied by desulfurization of HL and conversion into diphenolic species H (DP group).[48] The formation of H was confirmed by ESI mass spectra, where peaks corresponding to
[HL2b+H]+ (m/z 313.25), [HL2b+Na]+ (m/z 335.14), and [HL2b–H]− (m/z 310.99) were present. We suppose that the initial
step of the reaction of HL with
PBQ involves a one-electron oxidation of HL favored by the character of the HOMO of HL (see Figure S1) along with a NH deprotonation to give a highly conjugated N/S-centered
free radical (see Scheme S8 in Supporting Information). This radical intermediate transforms into triazole H in two
steps or undergoes a fragmentation affording 4-isothiocyanato-2,6-dimethylphenol.
The phenol reacts with HL via
an SE2 mechanism to form the corresponding thioamide followed
by a radical-promoted intermolecular transformation into indole H according to a Fukuyama-like indole synthesis[49] (for a more detailed discussion of the oxidation of HL with PBQ see the Supporting Information).
Other oxidation agents (lead
tetraacetate, phenyliodinen class="Chemical">(III) diacetate
(PIDA) with E° = +1.70 V vs Fc/Fc+ in ACN,[50] and silver(I) oxide) for N-alkyl(aryl)-aminocarbonyl-4-aminophenols,[51] were also used in an attempt to obtain the desired oxidation
products with a 1,4-benzoquinone imine moiety (see also Scheme S3, its accompanying explanation, and
Figure S2 in the Supporting Information). The exposure of HL to 1
equiv of PIDA furnished the two-electron oxidized product H and traces of the four-electron oxidized species H. As for HL and HL, the use
of a double amount of oxidant resulted in H as the main oxidation
product. ESI mass spectra showed peaks at m/z 313.21, 310.98 attributed to [HL2c′+H]+, [HL2c′–H]− as well as 311.12, 309.01 assigned
to [HL2c″+H]+, HL2c″–H]− in line
with the loss of two (H) or four (H) protons
when compared to original TSC HL (315.13 [HL2+H]+, 313.11 [HL2–H]−).
Characterization of Oxidized Organic Compounds
by NMR Spectroscopy
The formation of a 1,3,4-thiadiazole-ring
in H and H by an oxidation of HL resulted in the disappearance of peaks of the n class="Chemical">aldimine
CH proton (H7) and NH (H9) in H and H as well as of
the signal
of NH (H11) in H. The formation
of a 1,4-benzoquinone imine moiety in H was confirmed
also by the absence of the OH signal, which resonates
at 8.08–8.22 ppm in HL–HL, H (see
Scheme S4 and Tables S4–S6 in the Supporting Information). The ring-closure reaction resulted in a downfield
shift of the resonance signal of carbon C7, which was directly
involved in the 1,3,4-thiadiazole ring formation. The quaternary carbon
C7 in H and H resonates at 158.40 and
169.98 ppm, respectively, whereas the aldimine CH
carbon atom C7 in HL resonates at 142.51 ppm. Analogously, the involvement of the sulfur
atom in the 1,3,4-thiadiazole ring led to a downfield shift of the
signal of the carbon atom C10 (C=S)
to 166.77 ppm in H and to 171.58 ppm in H when compared to 176.55 ppm in HL.
The four-electron oxidation of HL to H with the formation of the imine
N(11)=C(12) bond resulted in strong downfield shift of the
resonance signal of n class="Chemical">carbon C12 of 1,4-benzoquinone moiety
of HL (162.21
ppm) when compared to that of carbon C12 of phenolic moiety
in HL–HL, H (130.18–132.53 ppm).
In addition, the formation of the carbonyl C(15)=O(18) bond
in H has a strong effect on the resonance of carbon atom C15, which is strongly downfield-shifted to 187.14 ppm when
compared to that in HL–HL and H at 148.97–151.17
ppm. Remarkable shifts of resonance signals for other atoms of the
1,4-benzoquinone moiety in H in comparison to the phenolic
moiety in HL–HL and H were also noticed (see
the Supporting Information and Scheme S5 therein).
The formation of the
benzothiazole ring in H is en class="Chemical">videnced
by the presence in the 1H NMR spectrum of one singlet of
the CH group and two singlets of methyl groups of
an unsymmetrical phenolic moiety with the intensity ratio of 1:3:3
as well as by one NH signal at 11.76 ppm in comparison
with a number of signals in the spectrum of HL (1(NH)/1(NH)/2(CH)/6(CH3)). Of the two proposed
tautomers for HL (A (N(11)H) and B (N(9)H); see Scheme S6 in the Supporting Information) the formation of the E isomer of form B in DMSO-d6 was evidenced by the cross-peak between protons of methyl (H7′) and NH (H9) groups in
the 1H, 1H NOESY spectrum. The DFT B3LYP/6-311++G(d,p)
calculations showed that the E isomer of tautomer A is less stable than the E isomer of tautomer B in a DMSO solution (ΔE = 1.58 kcal/mol;
ΔG = 1.01 kcal/mol at 298 K and 1 atm). We
found that, in contrast to HL–HL, the E/Z isomerization was observed for H. As expected in case of H·CHCOOH, where nitrogen atom N1 of the pyridine ring is protonated
and prevents the hydrogen-bond formation between H9 and
N1, which is present in the Z isomer of H, only one set of signals attributed to the E isomer was found. The neutral species H in DMSO-d6 and MeOH-d4 is
present as the E isomer, which converts slowly into
the Z isomer. The process is solvent-dependent. The E/Z equilibrium was reached in 6 d with
a molar ratio of E/Z isomers of
7.2:1 (DMSO-d6) and 3:1 (MeOH-d4) (see Figure S3 in the Supporting Information). The Z isomer of H in DMSO-d6 is characterized by
the downfield-shifted proton NH(9) due to the hydrogen
bond to the pyridinenitrogen atom and resonates at 15.00 ppm (the
same proton of the E isomer of H is seen
at 11.58 ppm). The Z/E isomerization of H was also studied in MeOH-d4 and methanol
by 1H NMR and UV–vis spectroscopy reaching 1:3.6
molar ratio in 14 d according to NMR spectra (for optical spectra
difference see Figure S4). The carbon atom
of the methyl group (C7′) in the E isomers of H·CHCOOH and H resonates at 12.55 and 12.56 ppm,
respectively, whereas in the Z isomer of H it resonates at 21.72 ppm. Note that these chemical shifts are consistent
with those calculated for E- and Z-H (8.29 and 23.26 ppm, respectively) by the gauge-independent
atomic orbital (GIAO) method at the WC04/6-311+G(2d,p) level of theory
using the DFT B3LYP/6-311++G(d,p) optimized geometries (DMSO solution,
the PCM solvation model). A similar difference in chemical shifts
of the CH3 group was also observed for the E (12.31 ppm) and Z isomers (21.73 ppm) of HL. The DFT calculation also demonstrated
that E and Z isomers of H have a quite similar stability in a DMSO solution (ΔG = 0.11 kcal/mol in favor of the E isomer;
298 K, 1 atm). As expected, the pyridine ring carbonatom C3 is also sensitive to the hydrogen-bond formation between H9 and N1 in the Z isomer of H. The C3 signal in the latter is markedly shifted (124.08
ppm) in comparison to C3 in the E isomer
(119.65 ppm). A full assignment of resonances was possible only for H·CHCOOH (the three quaternary carbons C12, C7, and
C17 were identified according to 1H, 13C HMBC; see Figure S5 in the Supporting Information).
The two-electron oxidation of H to Hwith the
formation of the quinone moiety is acn class="Chemical">companied by the downfield shift
of the resonance signal of carbonC15 at 184.43 ppm in
comparison to that of C15 in H·CHCOOH at 148.14 ppm, in E-H at 148.15 ppm, and in Z-H at 148.39 ppm. The lack of the NH signal confirms the formation
of the imine N(9)=C(10) bond (see Scheme S7 and Tables S4 and
S5 in the Supporting Information).
Synthesis
and Characterization of Copper(II) Complexes
The reaction
of HL–HL with CuCl2·2n class="Chemical">H2O in anoxic methanol under an argonatmosphere to preclude
an eventual oxidation of the ligands by air oxygen in a 1:1 molar
ratio at room temperature afforded green-brown solids of the formulas Cu(HL)Cl (1), [Cu(L)Cl] (2′), and Cu(HL)Cl (3) in almost quantitative yields. The formation of
these copper(II)complexes was confirmed by elemental analyses and
ESI mass spectra. The latter showed peaks attributed to [Cu(L1,3)–H]+, [Cu(L1,3)Cl–H]−, or [Cu(L2)]+ and [Cu(L2)Cl–H]−. XRD-quality single crystals
of [Cu(L)Cl] (1′–3′) were
grown from diluted by a factor of ca. 20 reaction mixtures under argon
upon standing at 4 °C. Under these conditions the deprotonation
of ligands HL and HL occurred. Attempts to crystallize 1, 2′, and 3 in air failed,
most likely because of an occurring oxidation of complexes by O2.
Synthesis of the Copper(II) Complexes with Oxidized Ligands
Upon a prolonged standing of a methanolic solution of n class="Chemical">Cu(HL)Cl (1) in air, a minor amount of crystals of [Cu()Cl] (4) formed, in which the ligand underwent
an oxidative dehydrogenation along with the intramolecular cyclization
via a C–S coupling reaction between phenolic carbon and thione
group into a five-membered thiazole ring, as confirmed by SC-XRD (vide
infra). Some rare examples of thiosemicarbazone cyclization with the
benzothiazole ring formation due to a coordination to copper(II) were
recently reported.[52,53] A direct complex formation reaction
between the prepared benzo[d]thiazol-6-ol H and copper(II) chloride produced [Cu(H)Cl] (6) under an
inert atmosphere. The same reaction in air was accompanied by a further
oxidation of H with the formation of benzo[d]thiazol-6-one (H) bound to copper(II). Complex 6 was characterized by the positive ion ESI mass spectrum
with a peak at m/z 374.08 attributed
to [Cu(L2c′)]+, whereas
the product obtained by an oxidation in air revealed a peak at m/z 373.06 assigned to [CuI(HL2c″)]+. The peak at m/z 373.06 was also seen when the reaction
mixture of H with CuCl2·2H2O was subjected to an ESI MS measurement.
The reactions of
copper(II) with the oxidized n class="Chemical">TSCs, namely, 1,3,4-thiadiazole-containing
species H and H, were monitored by ESI-MS experiments. When
CuCl2·2H2O was allowed to react with H and H in a 1:1 molar ratio, ESI mass spectra of
the reaction mixtures indicated the formation of complexes with metal-to-ligand
stoichiometry of 1:2, namely, [Cu(HL1a′)2]+ and [Cu(HL1a)2]+, respectively. Interestingly,
under varied reaction conditions (different solvents, air atmosphere,
and varied temperature and reaction time, see details in Table S7) the synthesis of copper(II)complex
of H resulted in a sequential oxidation of the two ligands, and
several oxidized products could be identified based on ESI-MS peaks
as [Cu(HL1a′)2]+ (m/z 659.16), [Cu(HL1a′)(HL1a)]+ (m/z 657.13), [Cu(HL1a)2]+ (m/z 655.18), [Cu(HL1a′)(CH3CN)]+ (m/z 402.10),
[Cu(HL1a)(CH3CN)]+ (m/z 400.10).
Moreover, attempts of the chromatographic separation of the obtained
compounds (on SiO2 with MeOH as eluent) led to a new species
[Cu(HL1a′)(HL1d)]+ (m/z 537.15),
in which one already oxidized ligand H in [Cu(HL1a)2]+ lost
the phenolic moiety. The complex formation of H in MeOH
under heating at 50 °C resulted in two species [Cu(HL1a)(HL1d)]+ (m/z 537.15) and
[Cu(HL1d)2]+ (m/z 419.08), whereas under prolonged heating
(36 h) only [Cu(HL1d)2]+ was detected, and the formation of complex [Cu(H)Cl] (5) was confirmed by SC-XRD.
The
potentially redox-active TSC ligands (HL, (L)−, and HL) in 1, 2′, and 3 proved to react slowly
with n class="Chemical">oxygen in air. Indeed, ESI mass spectra of methanolic solutions
of 1, 2′, or 3 after
a prolonged standing in air showed peaks with m/z shifted by 2 amu to lower masses in agreement with an
oxidative dehydrogenation required for the formation of two-electron
oxidation products.
To finally determine the redox status of
the 4-aminophenolic moiety,
the n class="Chemical">configurations adopted by the metal-free ligands in the solid
state and their protonation level in copper(II)complexes SC-XRD studies
were performed.
X-ray Crystallography of the Metal-Free Ligands HL–HL and Copper(II) Complexes 1′–3′
The results of X-ray diffraction studies of TSCs HL·CHOH, HL and HL are presented in Figure , while those of [n class="Chemical">Cu(L)Cl]·CHOH (1′·CHOH), [Cu(L)Cl] (2′), and [Cu(L)Cl]·CHOH (3′·CHOH) are in Figure .
The HL·CHOH crystallized in the triclinic centrosymmetric space group P1̅, while HL and HL crystallized in the
monoclinic space groups P21/c and P21/n, respectively.
All three metal-free ligands adopt an E configuration
in terms of the nomenclature used for the α-N-heterocyclicthiosemicarbazones[41] with
the iminenitrogen in the s-trans position to the
sulfur atom and the pyridine N1 atom. All TSCs crystallized in the
thione form with the C7–S bond length of 1.6839(15), 1.683(4)
and 1.695(2) Å, respectively. The distribution of electron density
in the dimethylphenolic moiety is typical for aromatic systems. The
C11–O bond length of 1.3780(19), 1.370(4), and 1.380(2) Å,
respectively, is also characteristic for phenols. The molecules of
the three proligands are not planar. The strong deviation of the phenolic
unit from the mean plane of the thiosemicarbazone fragment can be
estimated by a comparison of the torsion angle ΘC7–N4–C8–C13 of 88.7(2) and 78.4(4)° in the first two structures (Figure a,b) and ΘC7–N5–C8–C13 and ΘC22–N10–C23–C28 of 52.5(3) and 54.2(3)° in two crystallographically independent
molecules of HL (Figure c).
ORTEP views of 1′–3′ with thermal ellipsoids at the 50% probability level.
Selected bond
distances (Å), bond angles (deg) and torsion angles (deg) in 1′: Cu–N1 2.005(2), Cu–N2 1.962(2), Cu–S
2.2325(7), Cu–Cl 2.2507(7), C11–O1 1.370(4); N1–Cu–N2
81.77(9), N2–Cu–S 84.07(7), ΘC7–N4–C8–C13 −0.8(5); in 2′: Cu–N1 2.022(4),
Cu–N2 1.952(4), Cu–S 2.2636(16), Cu–Cl 2.2215(15),
C11–O1 1.370(6); N1–Cu–N2 80.76(17), N2–Cu–S
84.46(12), ΘC7–N4–C8–C13 −2.1(8);
in 3′: Cu–N1 2.025(2), Cu–N3 1.961(2),
Cu–S 2.2432(8), Cu–Cl 2.2636(8), C11–O1 1.374(4);
N1–Cu–N3 81.58(10), N3–Cu–S 83.40(7),
ΘC7–N5–C8–C13 9.4(5).
ORTEP views of HL–HL with thermal ellipsoids at the
50% probability level. Selected bond distances (Å) and torsion
angles (deg): (a) HL: C6–N2
1.280(2), N2–N3 1.3701(18), N3–C7 1.357(2), C7–S
1.6839(15), C7–N4 1.331(2), N4–C8 1.442(2), C11–O1
1.3780(19); ΘC7–N4–C8–n class="Gene">C13 –
88.7(2); (b) HL: C6–N2
1.287(4), N2–N3 1.374(4), N3–C7 1.363(4), C7–S
1.683(4), C7–N4 1.326(4), N4–C8 1.446(4), C11–O
1.370(4); ΘC7–N4–C8–C13 –
78.4(4); (c) HL: C4–N2
1.361(3), C6–N3 1.288(2), N3–N4 1.385(2), N4–C7
1.343(2), C7–S1 1.695(2), C7–N5 1.342(3), N5–C8
1.430(2), C11–O1 1.380(2); ΘC7–N5–C8–C13 52.5(3).
ORTEP views of 1′–3′ with thermal ellipsoids at the 50% probability level.
Selected bond
distances (Å), bond angles (deg) and torsion angles (deg) in 1′: Cu–N1 2.005(2), n class="Chemical">Cu–N2 1.962(2), Cu–S
2.2325(7), Cu–Cl 2.2507(7), C11–O1 1.370(4); N1–Cu–N2
81.77(9), N2–Cu–S 84.07(7), ΘC7–N4–C8–C13 −0.8(5); in 2′: Cu–N1 2.022(4),
Cu–N2 1.952(4), Cu–S 2.2636(16), Cu–Cl 2.2215(15),
C11–O1 1.370(6); N1–Cu–N2 80.76(17), N2–Cu–S
84.46(12), ΘC7–N4–C8–C13 −2.1(8);
in 3′: Cu–N1 2.025(2), Cu–N3 1.961(2),
Cu–S 2.2432(8), Cu–Cl 2.2636(8), C11–O1 1.374(4);
N1–Cu–N3 81.58(10), N3–Cu–S 83.40(7),
ΘC7–N5–C8–C13 9.4(5).
In contrast to the structures of HL and HL, the asymmetric
unit of HL n class="Chemical">consists of two molecules
associated in a centrosymmetric dimer via hydrogen-bonding interactions,
namely, N4–H···S2 [N4–H4 = 0.88 Å,
H4···S2 = 2.48 Å, N4···S2 = 3.3243(17)
Å] and N9–H···S1 [N9–H9 = 0.88°,
H9···S1 = 2.47 Å, N9···S1 = 3.3341(17)
Å]. A similar centrosymmetric association was recently reported
for acetylpyrazine 4-N-phenyl thiosemicarbazone.[54]
The copper(II)n class="Chemical">complexes 1′·CHOH and 3′·CHOH crystallized in the monoclinic
centrosymmetric space group P21/c, while 2′ crystallized
in the triclinic centrosymmetric space group P1̅
without any cocrystallized solvent. The copper(II) adopts a square-planar
coordination geometry in all three structures (Figure ). The thiosemicarbazones act as tridentate
monoanionic ligands binding to copper(II) via a pyridinenitrogen
atom, an azomethinenitrogen atom, and a thiolatesulfur atom. The
fourth coordination site in all complexes is occupied by the chlorido
coligand. Pertinent bond distances and bond angles are quoted in the
legend to Figure .
The same coordination geometry of a copper(II) bound by a monoanionic
thiosemicarbazone and a monodentate coligand was reported for [CuCl(mPip-FTSC–H)]·0.15CH3OH,[55] [Cu(L1)(μ-Cl)]Cl,
and [Cu(L2)(μ-Cl)]Cl·H2O, where ligands
L1 and L2 represent 3-methyl-5-oxo-1-phenyl-3-pyrazolin-4-carboxaldehyde
and 5-oxo-3-phenyl-3-pyrazolin-4-carboxaldehydeTSC, respectively.[56]
A comparison of the n class="Chemical">Cu(II) to TSC ligand
bond lengths in 1′ with those in the copper(II)complex with pyridine-2-carboxaldehydethiosemicarbazone[57] (Cu–N1 = 2.034(4),
Cu–N2 = 1.975(3), Cu–S = 2.278(1) Å) shows that
these are statistically significantly shorter in 1′. This difference is probably due to the formation of centrosymmetric
associates via intermolecular interactions with the shortest contact
Cu···Si = 2.760(2) Å and not due to
the presence of a phenolic moiety at N4. The interatomic repulsions
in the copper(II)complex with a 4 + 1 coordination geometry are expected
to be stronger than those in 1′, in which the
Cu(II) is four-coordinate. In another complex [CuLCl]2[Cu(pic)2] (with HL = pyridine-2-carboxaldehydethiosemicarbazone and
pic– = pyridine-2-carboxylate), in which the intermolecular
contacts are over 3 Å, the Cu(II) to TSC bond distances are shorter
and very similar to those in 1′ (Cu–N1
= 2.005(8), Cu–N2 = 1.942(9), Cu–S = 2.264(3) Å).[48] The metric parameters in the copper(II)-ligand
chromophore of [Cu(triapine-H)Cl](H3O)Cl (Cu–Npy = 2.031(8), Cu–Nhydrazine = 1.937(9),
Cu–S = 2.281(3) and Cu–Cl = 2.2493(5) Å) are statistically
the same as those in 3′, except Cu–S, which
is by ca. 0.04 Å (>12σ) shorter in 3′ than in the copper(II)complex with triapine. This is likely due
to different protonation states of the ligands in the two complexes,
even though the authors described the triapine ligand in its copper(II)complex as a monoanion with an extra proton at a cocrystallized water
molecule.[58]
Note that the organic
ligands in all three complexes are almost
planar inn class="Chemical">contrast to the situation described previously for the metal-free
ligands. The value of the torsion angle ΘC7–N4–C8–C13 for 1′·CHOH and 2′ (Figure a,b) increased from −88.7(2) and −78.4(4)°
in HL and HL to −0.8(5) and −2.1(8)°,
respectively. Analogously, the torsion angle ΘC7–N5–C8–C13 of 52.5(3) in HL becomes of
9.4(5)° in 3′·CHOH upon coordination to copper(II).
As for the
metal-free n class="Chemical">TSCs, the phenolic moiety remained intact
in all three complexes, namely, in its original oxidation state. The
distribution of electron density over the aromatic phenolic ring is
well-comparable to that in the TSCs.
X-ray Crystallography of
Oxidized Products
The results
of X-ray diffraction studies of oxidized organic species H, H, H, H, and H·0.5CHCl are displayed in Figure and Figure S6, while those of n class="Chemical">copper(II) complexes with oxidized ligands 4–6 are shown in Figure and Figure S7. The oxidized species H and H crystallize in
the monoclinic space groups P21/n and Cc, respectively. The molecule H is almost planar, while in H the moiety at N4 slightly
deviates from planarity. The dihedral angle ΘC7–N4–C8–C13 is of 5.8(3)°. Both contain a thiadiazole five-membered ring.
The distribution of electron density in them is very similar. In contrast,
the bond length distribution in the aryloxide moiety is quite different.
In the two-electron oxidized product H the distribution of electron
density is in agreement with that of the 3,5-dimethyl-1,4-aminophenolic
moiety, while in the four-electron oxidized species H the electron density agrees with that of the 3,5-dimethyl-1,4-benzoquinoneimine unit (see legend to Figure a,b). In particular, the C11–O1 bond length
in these two compounds is quite different at 1.3820(16) and 1.226(3)
Å, respectively. The X-ray diffraction structure of H confirmed
the two-electron oxidation of the original ligand HL and the formation of the TAT ring, while that
of H confirmed the further two-electron oxidation of H. The bond-length distribution in the molecule of H indicates
the presence of the benzo[d]thiazol-6-one moiety. The double-bond
character of N3–C7 1.296(4) indicates the formation of this
four-electron oxidation product from the two-electron oxidation product H by the loss of two electrons and two protons.
Figure 5
ORTEP views of two-electron
and four-electron oxidized species
of (a) H and (b) H, as well as of products that resulted
from an oxidation of HL, namely,
of (c) H and (d) H·0.5CHCl. Selected bond distances (Å) and torsion
angles (deg) in (a) H: C6–N2 1.3029(17), N2–N3
1.3739(15), C6–S 1.7405(14), C7–S 1.7382(13), C8–C9
1.3878(19), C9–C10 1.4003(18), C10–C11 1.3927(19), C11–C12
1.4006(19), C12–C13 1.3922(19), C11–O1 1.3820(16); ΘC7–N4–C8–C9 1.0(2); in (b) H: C6–N2 1.305(3), N2–N3 1.382(2), C6–S 1.727(2),
C7–S 1.734(2), C8–C9 1.458(3), C9–C10 1.341(3),
C10–C11 1.480(3), C11–C12 1.491(3), C12–C13 1.342(3),
C11–O1 1.226(3); ΘC7–N4–C8–C13 5.8(3); in (c) H: C6–N2 1.485(2), N2–N3 1.247(2),
N3–C7 1.472(2), C7–S 1.6465(18), C7–N4 1.325(2),
N4–C6 1.479(2), C11–O 1.3728(18), N4–C8 1.4409(19);
in (d) H·0.5CHCl: C6–N2 1.306(4), N2–N3 1.394(4), N3–C7 1.296(4),
C7–N4 1.388(4), N4–C8 1.311(4), C8–C9 1.444(4),
C9–C10 1.340(4), C10–C11 1.488(5), C11–O1 1.234(4),
C11–C12 1.496(4), C12–C13 1.349(4), C13–C8 1.461(4),
C13–S1 1.745(3).
Figure 6
ORTEP views of [Cu()Cl] (4), [Cu(H)Cl] (5), and [Cu(H)Cl] (6) with thermal
ellipsoids at the 50% probability level. Selected
bond distances (Å) and bond angles (deg) in (a) 4: Cu–N1 2.054(4), Cu–N2 1.956(4), Cu–N4 2.001(3),
Cu–Cl 2.2575(12), C11–O 1.370(5); N2–Cu–N1
80.02(15), N2–Cu–N4 78.59(14); in (b) 5: Cu–N1 2.0384(11), Cu–N2 2.0089(11), Cu–Cl
2.8116(3), N2–Cu–N1 99.32(4); in (c) 6:
Cu–N1 2.0329(16), Cu–N2 1.9854(16), Cu–N4 2.0358(16),
Cu–Cl1 2.2100(5), C11–O 1.374(2); N2–Cu–N1
78.00(16), N2–Cu–N4 79.31(6).
ORTEP views of two-electron
and four-electron oxidized species
of (a) H and (b) H, as well as of products that resulted
from an oxidation of HL, namely,
of (c) H and (d) H·0.5CHCl. Selected bond distances (Å) and torsion
angles (deg) in (a) H: C6–N2 1.3029(17), N2–N3
1.3739(15), C6–S 1.7405(14), C7–S 1.7382(13), C8–C9
1.3878(19), C9–n class="Gene">C10 1.4003(18), C10–C11 1.3927(19), C11–C12
1.4006(19), C12–C13 1.3922(19), C11–O1 1.3820(16); ΘC7–N4–C8–C9 1.0(2); in (b) H: C6–N2 1.305(3), N2–N3 1.382(2), C6–S 1.727(2),
C7–S 1.734(2), C8–C9 1.458(3), C9–C10 1.341(3),
C10–C11 1.480(3), C11–C12 1.491(3), C12–C13 1.342(3),
C11–O1 1.226(3); ΘC7–N4–C8–C13 5.8(3); in (c) H: C6–N2 1.485(2), N2–N3 1.247(2),
N3–C7 1.472(2), C7–S 1.6465(18), C7–N4 1.325(2),
N4–C6 1.479(2), C11–O 1.3728(18), N4–C8 1.4409(19);
in (d) H·0.5CHCl: C6–N2 1.306(4), N2–N3 1.394(4), N3–C7 1.296(4),
C7–N4 1.388(4), N4–C8 1.311(4), C8–C9 1.444(4),
C9–C10 1.340(4), C10–C11 1.488(5), C11–O1 1.234(4),
C11–C12 1.496(4), C12–C13 1.349(4), C13–C8 1.461(4),
C13–S1 1.745(3).
ORTEP views of [Cu()Cl] (4), [n class="Chemical">Cu(H)Cl] (5), and [Cu(H)Cl] (6) with thermal
ellipsoids at the 50% probability level. Selected
bond distances (Å) and bond angles (deg) in (a) 4: Cu–N1 2.054(4), Cu–N2 1.956(4), Cu–N4 2.001(3),
Cu–Cl 2.2575(12), C11–O 1.370(5); N2–Cu–N1
80.02(15), N2–Cu–N4 78.59(14); in (b) 5: Cu–N1 2.0384(11), Cu–N2 2.0089(11), Cu–Cl
2.8116(3), N2–Cu–N1 99.32(4); in (c) 6:
Cu–N1 2.0329(16), Cu–N2 1.9854(16), Cu–N4 2.0358(16),
Cu–Cl1 2.2100(5), C11–O 1.374(2); N2–Cu–N1
78.00(16), N2–Cu–N4 79.31(6).
The X-ray diffraction study of 4 (Figure a) revealed that the ligand
underwent an oxidative dehydrogenation acn class="Chemical">companied by the intramolecular
cyclization via a C–S coupling reaction between a phenolic
carbon and a thione group into a five-membered thiazole ring instead
of the expected oxidative dehydrogenation (two-electron oxidation
accompanied by the loss of two protons) of the 3,5-dimethyl-1,4-aminophenol
unit with formation of a 3,5-dimethyl-1,4-benzoquinone imine moiety
(see Chart , Scheme ). This intramolecular
sulfur arylation resulted in the change of coordination mode, so that
the thioether sulfur atom with diminished electron-donor properties
is not involved in the coordination to copper(II). This is in agreement
with the coordination chemistry of isothiosemicarbazones,[59] which as a rule do not use a sulfur atom for
coordination to first-row transition metals. In this context, it is
worth mentioning that the binding of isothiosemicarbazones to zinc(II)
and copper(II) via a thioether sulfur atom has been documented quite
recently,[60] when bulkier than chlorido
coligands, for example, iodido and bromido, were involved in coordination
to the metal. Complex 4 might be one of the products
of the oxidation of copper(II)complexes over time in methanol by
air oxygen. Some rare examples of a thiosemicarbazone cyclization
with the thiazole ring formation due to the coordination to copper(II)
were recently reported (iminodiacetate–thiosemicarbazones and N-phenylthiosemicarbazones).[52,53,61] The new ligand obtained by the intramolecular cyclization
in Cu(HL)Cl belongs to the class of biologically active
substituted 2-hydrazinylbenzothiazoles, which showed anticancer activity
themselves as well as upon coordination to different metals.[62−65] Two molecules of complex 4 are associated into a centrosymmetric
dimer via two intermolecular μ-chlorido bridges as shown in Figure S7.
The molecular structure of 5 shown in Figure b indicates a strongly tetragonally
distorted six-n class="Chemical">coordinate geometry of copper(II), in which two pyridine-thiadiazole
ligands act as bidentate and occupy the equatorial sites in a trans mutual arrangement and two quite weakly bound chlorido
coligands in axial positions. Taking into account the interatomic
Cu–Cl separation (2.8116(3) Å) the complex can also be
described as square-planar.
As in 4, the coordinated
ligand in 6 acts
as tridentate and binds to n class="Disease">copper(II) via atoms N1, N2, and N4. However,
while 4 is square-planar, 6 is very close
to square-pyramidal (τ5 = 0.16).[66] The organic ligand is monoanionic in 4, while
neutral in 6. An additional coordination of chlorido
coligands counterbalances the 2+ charge of the central atom.
To understand the difference in protonation states and reactivity
of the originally prepared n class="Chemical">complexes and those isolated upon crystallization
from diluted methanolic solutions equilibrium studies were performed
on the ligands and their copper(II)complexes.
Solution Equilibrium
Studies
Proton Dissociation Processes and Lipophilicity of the Ligands
Proton dissociation constants (pKa)
of drug molen class="Chemical">cules indicate the actual protonation state and the charge
at a given pH, and therefore pKa are important
parameters that affect the pharmacokinetic properties as well. The
N-terminally monosubstituted TSCs HL–HL belong to
the family of α-N-pyridyl TSCs; thus, they
possess the pyridinium (PyH)+ and the hydrazinic-NNH as
proton dissociable groups besides the phenolic moiety. Since these
TSCs and their copper(II)complexes have a limited water solubility,
the equilibrium studies were performed by UV–vis spectrophotometry
in a 30% (v/v) DMSO/H2O solvent mixture using relatively
low concentrations (50 μM). Representative UV–vis spectra
recorded for HL at various pH
values are shown in Figure a.
Figure 7
UV–vis absorption spectra recorded for proligand HL in the pH ranges of (a) 1.00–4.95
and (b) 5.49–11.82. cHL = 50 μM;
30% (v/v) DMSO/H2O; I = 0.1 M (KCl); T = 25 °C.
UV–vis absorption spectra recorded for proligand HL in the pH ranges of (a) 1.00–4.95
and (b) 5.49–11.82. cHL = 50 μM;
30% (v/v) n class="Chemical">DMSO/H2O; I = 0.1 M (KCl); T = 25 °C.
On the basis of the spectral changes two well-separated deprotonation
processes were observed between pH 2 and 11. The first proton dissociation
step taking place at pH < 5 is accompanied by a blue shift, and
the λmax is shifted from 362 to 322 nm. This deprotonation
step is attributed to the proton on the n class="Chemical">pyridinium nitrogen (PyH+). Upon an increase of the pH a new process occurred as evidenced
by a new band in the range of 350–450 nm (Figure b) and an isosbestic point
at 350 nm, namely, the deprotonation of the hydrazinicnitrogen. In
the strongly basic pH range (pH > 11.2) new broad bands appear
at
400–600 nm (Figure b) with irreversible spectral changes most likely due to an
oxidation of the TSC by the air oxygen.
Therefore, only two
pKa values could
be determined (Table ) based on the den class="Chemical">convolution of the UV–vis spectra recorded
at pH < 11.2 for HL (molar
absorbance spectra are seen in Figure S8a) as the oxidation hindered the accurate determination of the pKa for the aromatic OH group. Two pKa values were computed for HL from the UV–vis titration data (Figure S9) as well; however, only one pKa was obtained in the case of HL (Table ),
namely, that for the deprotonation of the PyH+, since the
proton dissociation of the hydrazinicnitrogen and the oxidation of
the TSC were partly overlapped. On the basis of the determined pKa values, it can be concluded that the presence
of the electron-donating methyl group in HL results in a significant increase of both pKa values when compared to that of HL. A similar behavior was reported for the
analogous 2-formylpyridine and 2-acetylpyridineTSC in our previous
work.[67] The pKa of the PyH+ group was also increased significantly by
the addition of the electron-donating amine group at the pyridine
ring, in agreement with data reported previously for the FTSC and
triapine.[68] All proligands are air-sensitive
in the strongly basic pH range (pH > 11). Concentration distribution
curves were computed for them at pH < 11 (see Figure S8b for HL) revealing
that their neutral forms predominate at a physiological pH.
Table 1
pKa Values
Determined by UV–vis Titrations in 30% (v/v) DMSO/H2O and log D7.4 (n-Octanol/Water)
Values of the TSCs HL–HL and Their Complexesa
method
HL1
HL2
HL3
pKa (PyH+)
UV–vis
3.01 ± 0.01
3.59 ± 0.02
3.95 ± 0.04
pKa (NNH)
UV–vis
10.55 ± 0.01
11.08 ± 0.02
nd
log D7.4 (proligand)
partitioning
+1.30 ± 0.03
+2.1 ± 0.1
+1.67 ± 0.01
log K′5.9 (complex)
EDTA displacement
9.67 ± 0.01
ndb
9.78 ± 0.01
log D7.4 (complex)
partitioning
–0.40 ± 0.06
ndb
–0.42 ± 0.03
kobs (min–1)
(complex) in 30% DMSO
UV–vis (with GSH)
0.033 ± 0.004
ndb
0.035 ± 0.004
kobs (min–1)
(complex) in 60% DMSO
UV–vis (with GSH)
0.021 ± 0.001
too slowc
0.024 ± 0.004
Conditional
stability constants
(log K′5.9) of the complexes determined
by UV–vis EDTA displacement studies in 30% (v/v) DMSO/H2O and rate constants (kobs) obtained
for the redox reaction of the complexes with GSH (pH = 7.4 (50 mM
HEPES); ccomplex = 25 μM; cGSH = 1.25 mM in 30% (v/v) DMSO/H2O); ccomplex = 12.5 μM; cGSH = 600 μM in 60% (v/v) DMSO/H2O) {T = 25 °C; I = 0.1 M (KCl)}.
Not determined (nd) due to
the bad
solubility of the complex under the conditions.
Rate constant could not be determined
due to the very slow redox reaction.
Conditional
stability n class="Chemical">constants
(log K′5.9) of the complexes determined
by UV–vis EDTA displacement studies in 30% (v/v) DMSO/H2O and rate constants (kobs) obtained
for the redox reaction of the complexes with GSH (pH = 7.4 (50 mM
HEPES); ccomplex = 25 μM; cGSH = 1.25 mM in 30% (v/v) DMSO/H2O); ccomplex = 12.5 μM; cGSH = 600 μM in 60% (v/v) DMSO/H2O) {T = 25 °C; I = 0.1 M (KCl)}.
Not determined (nd) due to
the bad
solubility of the complex under the n class="Chemical">conditions.
Rate constant n class="Chemical">could not be determined
due to the very slow redox reaction.
The solution stability of the proligands was monitored
at pH 7.4
by spectrophotometry. The UV–vis spectra recorded over 4 h
revealed no measurable spectral changes, suggesting that the oxidation
of these proligands does not take place (or just very slowly) in an
aqueous solution at a physiological pH. However, HL showed a certain level of slow den class="Chemical">composition
at pH 1.5, namely, a 6% absorbance decrease at 354 nm in ∼3
h (Figure S10), which is most likely the
consequence of the less extended conjugation in the molecule due to
the cleavage of the C=N Schiff base bond, as it was also reported
for 2-acetylpyridineTSC.[67] Thus, the rate
of this acid-catalyzed reaction is increased with the increasing number
of methyl groups present in the α-N-pyridyl
TSC.
Besides pKa values, lipophilicity
is
also an important pharmacological property of a drug, as it strongly
influences the ability of the n class="Chemical">compound to pass through biological
membranes. Therefore, distribution coefficients (log D7.4) were determined using the shake-flask method in an n-octanol-buffered aqueous solution at pH 7.4 (Table ). The log D7.4 values indicate the moderate lipophilic
character of the proligands. The substitution at the end nitrogen
atom of the thosemicarbazide moiety and the presence of a methyl group
at the Schiff base bond induce a somewhat higher lipophilicity. The
presence of the phenolic moiety undoubtedly increases the log D7.4 values compared to those of FTSC (+0.73),[67] AcTSC (+1.02)[67] and
triapine (+0.85).[69]
In summary, these
TSCs are stable in their neutral form in a quite
n class="Gene">broad pH range (including pH 7.4).
Solution Stability and
Redox Properties of the Copper(II) Complexes
The metaln class="Chemical">complexes
often undergo transformation processes upon
dissolution, such as protonation, deprotonation, or dissociation to
a metal-free ligand and metal ion depending on the pH, their concentration,
and the solution speciation. The knowledge of the actual chemical
form of the biologically active metalcomplexes in solution close
to physiologically relevant conditions is quite important to elucidate
the mechanism of action. Therefore, the solution stability of the
copper(II)complexes (Cu(HL)Cl, [Cu(L)Cl], and Cu(HL)Cl) was studied
by UV–vis spectrophotometry. The simple α-N-pyridyl TSCs (e.g., triapine, FTSC) generally form very stable monoligand
copper(II)complexes, and the species in which the monoanionic ligand
is coordinated via the (Npyridine,N,S–) mode predominates in a wide pH range at a 1:1 metal-to-ligand ratio.[68] At lower pH this type of complex is protonated,
and thus the neutral ligand is bound via (Npyridine,N,S)
donor atoms, while a mixed hydroxido complex with the (Npyridine,N,S–)(OH) coordination pattern is formed in the
basic pH range. On the basis of the close structural similarities
between HL–HL and the listed TSCs with a simpler scaffold,
the formation of the same type of complexes is feasible. UV–vis
titrations were performed with the complexes in a 30% (v/v) DMSO/H2O solvent mixture, and representative spectra are shown for Cu(HL)Cl in Figure . The spectra remain intact in a broad pH range (2.7–7.6),
and an absorption band is observed with λmax at 406
nm being typical for a S → Cu charge transfer. This finding
indicates the dominant presence of only one kind of complex, which
is most probably the species with the (Npyridine,N,S–) tridentate coordination mode. By decreasing the pH
the λmax is hypsochromically shifted to 322 nm. The
presence of the isosbestic point at 362 nm implies that only two species
are involved in this equilibrium. As the spectrum recorded at pH 1.01
significantly differs from that of the TSC, this equilibrium corresponds
to the protonation of the complex at the noncoordinating hydrazinicnitrogen (Chart S1) rather than to its
dissociation to the free metal ion and ligand. This process is not
completed when the pH decreases to 1, and a pKa value less than 1.5 could be estimated. When the pH is increased,
two overlapping processes are suggested to take place at pH > 8
via
the continuous bathochromic shift of the absorption maximum, and pKa values of 9.80 ± 0.01 and 11.02 ±
0.01 were computed. In this pH range most probably the coordinated
water molecule deprotonates, and a mixed hydroxido complex is formed
along with the deprotonation of the phenolic group of the bound ligand.
Similar spectral changes were monitored for Cu(HL)Cl, and
pKa < 1.5 was estimated for the process
in the acidic pH range as well.
Figure 8
UV–vis absorption spectra recorded
for complex Cu(HL)Cl in the pH range of 1.01–12.20 (solid
lines) and for HL at pH 1.01
(dashed gray line). ccomplex/HL = 50 μM;
30% (v/v) DMSO/H2O; I = 0.1 M (KCl); T =
25 °C.
UV–vis absorption spectra recorded
for n class="Chemical">complex Cu(HL)Cl in the pH range of 1.01–12.20 (solid
lines) and for HL at pH 1.01
(dashed gray line). ccomplex/HL = 50 μM;
30% (v/v) DMSO/H2O; I = 0.1 M (KCl); T =
25 °C.
However, the formation of precipitate
(significant baseline elevation
and absorbance decrease in the whole wavelength range) at pH >
8 hindered
the calculation of the proton dissociationn class="Chemical">constants of the complexes
from spectra collected in this pH range. Unfortunately, during the
titration of [Cu(L)Cl] the formation of a precipitate was observed already at the acidic
pH; thus, the deprotonation processes could not be evaluated.
The copper(II)–n class="Gene">TSC complexes are often redox-active under
physiological conditions, which has an impact on their cytotoxicity.
To investigate whether complexes [Cu(L)], [Cu(L)], and [Cu(L)] can be reduced by the most abundant low molecular
mass cellular reductant, GSH, spectrophotometric measurements were
performed on their direct reaction under strictly anaerobic conditions
at pH 7.4. First, the assay was performed in the presence of 30% DMSO
using a 25 μM complex concentration. However, the limited solubility
of [Cu(L)] did not allow the measurement. Therefore, the
assay was also performed in the presence of 60% DMSO at a lower (12.5
μM) concentration for all the three complexes. The spectral
changes are shown in Figure for [Cu(L)] and [Cu(L)] complexes in the presence
of a large excess of GSH in 30% (v/v) DMSO/H2O. After the
complexes were mixed with GSH, a well-detectable change is observed
due to the formation of ternary complexes via the coordination of
GSH as it was reported for several TSCcomplexes.[70,71] Then the spectral changes show the absorbance decrease at the λmax of the S → Cu charge transfer band of the complexes.
The final spectra show a strong similarity to those of HL and HL at λ > 310 nm suggesting the release of the TSCs. However,
in this case the reduction is responsible for the liberation of the
TSCs and copper(I), which forms complexes with GSH (that is in high
excess in the sample). Copper(I) favors a tetrahedral coordination
environment, while HL and HL as planar tridentate ligands cannot
satisfy these requirements and accommodate the cation. This contradiction
is a driving force for a complex destabilization, especially in the
presence of GSH, which can efficiently bind copper(I).[64] In addition, a one-electron reduction increases
the basicity of the coordinated TSCs facilitating their protonation
and dissociation from the copper(I).[72] Note,
however, that the process was reversible, as bubbling oxygen into
the samples regenerated the starting spectra. Complex [Cu(L)] behaved differently, as only minor spectral changes were seen upon
treatment with GSH in 60% (v/v) DMSO/H2O (Figure S11b). From the measured absorbance–time curves
rate constants (kobs) were calculated
(Table ). Similar
reduction rates for [Cu(L)] and [Cu(L)] complexes
were obtained, and somewhat lower kobs values were found in the presence of the higher fraction of DMSO.
Notably, ascorbate, which is a weaker reducing agent compared to GSH
and is found in higher concentration in the extracellular fluids,
was not able to reduce these complexes under the same conditions.
On the contrary, the more powerful reducing agent DTTcould reduce [Cu(L)], [Cu(L)], and [Cu(L)] in a very fast reaction.
The reduction was complete within several seconds (at 12.5 μM
complex and 600 μM DTTconcentrations in the presence of 60%
DMSO, Figure S11c,d). In this case, the
reaction was reversible upon exposure to O2 only for [Cu(L)].
Figure 9
Time-dependent changes of the UV–vis spectra of (a) Cu(HL)Cl and (b) Cu(HL)Cl in the presence of 50 equiv of GSH at pH
7.4 under anaerobic conditions. ccomplex = 25 μM; cGSH = 1.25 mM; pH =
7.40; 30% (v/v) DMSO/H2O; I = 0.1 M (KCl); T = 25 °C.
Time-dependent changes of the UV–vis spectra of (a) Cu(HL)Cl and (b) n class="Chemical">Cu(HL)Cl in the presence of 50 equiv of GSH at pH
7.4 under anaerobic conditions. ccomplex = 25 μM; cGSH = 1.25 mM; pH =
7.40; 30% (v/v) DMSO/H2O; I = 0.1 M (KCl); T = 25 °C.
Overall, the solution equilibrium data pron class="Chemical">vide further evidence
that the complex [Cu(L)] with
the coordinated monoanionic ligand predominates in a wide pH range.
In order to obtain a deeper insight into the observed behavior of
both metal-free ligands and their copper(II)complexes in the presence
of oxidants (atmospheric oxygen) and reductants (GSH and ascorbate)
spectroelectrochemical investigations were also performed.
Electrochemistry
and Spectroelectrochemistry
Cyclic
voltammograms of 1, 2′, and 3 in DMSO/n class="Chemical">n-Bu4NPF6 recorded with a glassy carbon (GC) working electrode at a scan rate
of 100 mV s–1 showed a redox activity in both cathodic
and anodic regions. Copper(II) undergoes an electrochemically irreversible
or quasi-reversible reduction to copper(I) at Epc = −0.83 V for 1 and −0.93 V versus
Fc+/Fc for both 2′ and 3 (Figure a). Notably,
the corresponding ligands are not redox-active in the cathodic region
(data not shown). An irreversible oxidation was observed for these
complexes, which was identified as a two-electron oxidation of the
TSCs with a release of two protons. A two-electron oxidation was confirmed
by a comparison of the reduction peak (one-electron Cu(II) →
Cu(I) redox process) and the oxidation peak of 2′ taken in equivalent amounts as shown in Figure b. In addition, an electrolysis of HL at 1000 mV versus Ag/AgCl in CH3CN in the presence of 0.2 M n-Bu4NPF6 generated a mixture of several products from which H and H were separated on silica. ESI-MS and 1H NMR spectra were identical with those of the products obtained
by an oxidation of HL with DDQ
as mentioned previously.
Figure 10
(a) Cyclic voltammograms of 0.5 mM 1 (black trace), 2′ (red trace), and 3 (green trace) in
DMSO/n-Bu4NPF6 at a GC working
electrode at the scan rate of 100 mV s–1; (b) comparison
of the reduction and the oxidation peak of 2′ (scan
rate of 100 mV s–1).
(a) Cyclic voltammograms of 0.5 mM 1 (black trace), 2′ (red trace), and 3 (green trace) in
DMSO/n class="Chemical">n-Bu4NPF6 at a GC working
electrode at the scan rate of 100 mV s–1; (b) comparison
of the reduction and the oxidation peak of 2′ (scan
rate of 100 mV s–1).
The oxidation peak of the TSC ligand was observed at Epa = +0.06 V for 1 and 2′ and at +0.04 V for 3, and it is negatively shifted
inn class="Chemical">comparison to the corresponding metal-free ligands (Epa = +0.21 V for HL, +0.24 V for HL, and +0.18
V for HL (all vs Fc+/Fc at a scan rate of 100 mV s–1)), as shown for 1 and its corresponding metal-free ligand HL in Figure a,b, respectively. There are also significant changes
in the shape and intensity of cyclic voltammograms upon the second
oxidation scan (see red traces in Figure a,b), which indicate a further oxidation
of the products obtained after the first oxidation in DMSO, in line
with the chemical oxidation of the compounds. Note that, in a proton-donating
solvent, the potentials of both reduction and oxidation processes
were shifted to the more positive values versus the internal potential
standard Fc+/Fc, and additionally, a broad reduction peak
appeared during the reverse scan in the cyclic voltammogram at a strongly
negatively shifted potential (Figure c). A distinct oxidation pattern of the corresponding
voltammograms in protic media is caused by the involvement of protons
in the process in accordance with chemical oxidations discussed previously
and the well-known reaction mechanism proposed for the quinone-like
systems.[70,71]
Figure 11
Cyclic voltammograms of 0.5 mM of (a) 1 and (b) the
corresponding ligand in DMSO/n-Bu4NPF6 and of (c) 1 in MeOH/LiClO4 at the
GC working electrode, at scan rate of 100 mV s–1.
Cyclic voltammograms of 0.5 mM of (a) 1 and (b) the
corresponding ligand inn class="Chemical">DMSO/n-Bu4NPF6 and of (c) 1 in MeOH/LiClO4 at the
GC working electrode, at scan rate of 100 mV s–1.
Similar redox behavior was observed
for the anodic oxidation of H in DMSO with several new
redox-active species, which appeared
upon the first and the sen class="Chemical">cond voltametric scans (Figure S12a). However, the oxidized 1,4-benzoquinone imine
species Hcan be reversibly reduced in the cathodic part (Figure S12b) with a voltammetric pattern characteristic
for the electrochemistry of quinones in aprotic media.[72] Moreover, EPR spectroelectrochemistry confirmed
the formation of an anion radical at the first reduction peak (see
inset in Figure S12b). A rich hyperfine
splitting and a g-value of 2.0046 points to the spin
delocalization and contribution of heteroatom (presumably nitrogen)
to the g-value.
To support the assignment of
the redox processes described previously,
EPR/UV–vis spectroelectrochemical measurements were performed,
and the results are shown for 1 in Figures and 13. The UV–vis spectrum of 1 exhibits two absorption
bands at 276 and 428 nm, where the first one is due to the absorption
of the TSC ligand, while the sen class="Chemical">cond one can be attributed to the ligand-to-metal
(S → Cu) charge transfer (LMCT).[73,74] Upon the cathodic
reduction of 1 in the region of the first reduction peak
a new broad absorption band at 331 nm appears with a simultaneous
decrease of the initial optical bands at 276 and 428 nm via an isosbestic
point at 302 nm (Figure ). An analogous spectroelectrochemical response was observed
for 2′ as shown in Figure S13. This observation is different from that encountered by
the reduction of the copper(II)–TSCcomplexes by GSH (vide
supra), which led to the liberation of the ligand and formation of
the copper(I)complex with GSH. In the spectroelectrochemical experiment
in the absence of strong Cu(I)complexing agents, such as GSH, the
TSC ligand may coordinate to Cu(I) and form a linear or tetrahedral
complex. Upon the voltammetric reverse scan, a nearly full recovery
of the initial optical bands was observed, which confirms the relatively
good stability of cathodically generated Cu(I)complex with HL and, thus, the chemical reversibility
of this redox process. Rare examples of four- and three-coordinate
copper(I)complexes with potentially tridentate and bidentate thiosemicarbazones
were reported previously.[75,76] The room-temperature
X-band EPR spectrum of 1 showed a typical signal for
d9 Cu(II) species, which decreased stepwise upon a cathodic
reduction at the first cathodic peak. This is in line with the metal-centered
reduction and formation of EPR-silent d10 Cu(I) species[10] (see inset in Figure b). EPR spectra of 1, 2′, and 3 measured in frozen n-Bu4NPF6/DMSO at 77 K show a characteristic
axial symmetry (g∥ > g⊥ > ge) implying a
square-planar coordination and the presence of one dominating species
in DMSO (Figure S14).
Figure 12
Spectroelectrochemistry
of 1 in n-Bu4NPF6/DMSO in the region of the first cathodic
peak: (a) potential dependence of UV–vis spectra with the corresponding
in situ cyclic voltammogram (Pt-microstructured honeycomb working
electrode, scan rate of 5 mV s–1); (b) evolution
of UV–vis spectra in 2D projection upon forward scan. (inset)
Evolution of EPR spectra measured at the first reduction peak using
a Pt mesh working electrode.
Figure 13
UV–vis
spectra measured simultaneously (a) upon anodic oxidation
of 1 in the region of the first anodic peak (inset: time
evolution of EPR spectra acquired at the first anodic peak) and (b)
upon the back scan (inset: the corresponding in situ cyclic voltammogram).
Spectroelectrochemistry
of 1 in n-Bu4NPF6/n class="Chemical">DMSO in the region of the first cathodic
peak: (a) potential dependence of UV–vis spectra with the corresponding
in situ cyclic voltammogram (Pt-microstructured honeycomb working
electrode, scan rate of 5 mV s–1); (b) evolution
of UV–vis spectra in 2D projection upon forward scan. (inset)
Evolution of EPR spectra measured at the first reduction peak using
a Pt mesh working electrode.
UV–vis
spectra measured simultaneously (a) upon anodic oxidation
of 1 in the region of the first anodic peak (inset: time
evolution of EPR spectra acquired at the first anodic peak) and (b)
upon the back scan (inset: the corresponding in situ cyclic voltammogram).The in situ cyclic voltammogram and simultaneously
recorded evolution
of UV–vis spectra upon an anodic oxidation of 1 inn class="Chemical">DMSO provide further evidence for the ligand-based irreversible
oxidation. Spectral changes accompanying the oxidation of 1 are shown in Figure . These changes are characteristic for the other two complexes 2′ and 3 as well. Note that, in the region
of the first oxidation peak, new optical bands at 295 and 356 nm appear
with a simultaneous decrease of the initial absorption with a maximum
at 428 nm (Figure a). However, the product formed upon oxidation is not reduced back
during the reverse voltammetric scan (Figure b), indicating the chemical irreversibility
of the redox process. In the EPR spectroelectrochemistry of 1 in DMSO/n-Bu4NPF6, no changes of the EPR signal were detected upon the oxidation at
the first anodic peak, providing evidence of the two-electron oxidation
process taking place on the TSC ligand.
The remarkable stability
of copper(II)n class="Chemical">complexes 1, 2′, and 3 at a physiological pH,
their moderate lipophilic character (log D7.4 = −0.4 to −0.42) and copper(II)/copper(I) redox activity
(Ered = −0.83 to −0.93 V
vs Fc+/Fc) in a biologically relevant window of redox potentials
(−0.4 to +0.8 V vs NHE or −1.04 to 0.16 V vs Fc/Fc+) prompted the investigation of their antiproliferative activity
in cancer cell lines.
Inhibition of Cell Viability and Apoptosis
Assay
Cytotoxicity of the TSCs, Their Oxidized Products and Copper(II)
Complexes
The in vitro cytotoxicity of the n class="Chemical">TSCs HL–HL, copper(II)complexes Cu(HL)Cl, [Cu(L)Cl], and Cu(HL)Cl, and oxidized TSCs H, H, and H·CHCOOH was tested
in the doxorubicin-sensitive Colo205 and the multidrug-resistant Colo320
humancolonic adenocarcinoma cell lines as well as in normal human
embryonal lung fibroblast cells (MRC-5) by the colorimetric MTT assay.
The data that resulted (expressed as the half-maximal inhibitory concentration
(IC50)) are collected in Table and compared with those for triapine, doxorubicin,
and CuCl2.
Table 2
In Vitro Cytotoxicity
(IC50 Values in μM) of Metal-Free Ligands HL–HL, Copper(II)
Complexes Cu(HL)Cl, [Cu(L)Cl], and Cu(HL)Cl, and, of the Oxidized Species H, H, and H·CHCOOH in Colo205, Colo320, and MRC-5 Cell Lines after
72 h of Exposure
IC50 (μM)
Colo205
Colo320
MRC-5
HL1
>100
6.32 ± 0.49
>100
HL2
>100
>100
>100
HL3
48.2 ± 6.8
>100
>100
Cu(HL1)Cl2
2.08 ± 0.12
2.21 ± 0.18
3.13 ± 0.17
[Cu(L2)Cl]
0.181 ± 0.039
0.159 ± 0.009
0.276 ± 0.049
Cu(HL3)Cl2
26.6 ± 1.6
27.6 ± 1.6
>100
HL1a′
>25
>25
>25
HL1a″
>25
>25
>25
HL2c′·CH3COOH
2.733 ± 0.059
0.188 ± 0.041
2.15 ± 0.10
CuCl2
19.7a
20.0a
24.5a
triapine
3.34 ± 0.12
4.21 ± 0.46
10.2 ± 1.3
doxorubicin
3.28a
3.12a
5.19a
Data are taken from ref (77).
Data are taken from ref (77).The metal-free ligands were either devoid of n class="Disease">cytotoxicity
or showed
a weak response; only HL and HL revealed a somewhat higher activity
against Colo320 and Colo205 cells, respectively, even though it was
inferior to that of triapine. Notably, the copper(II)complexes are
quite cytotoxic. So the effect of the copper(II)coordination is obvious
in all cases. Low IC50 values (0.16–2.2 μM)
were obtained for Cu(HL)Cl and [Cu(L)Cl] in both cancer cell lines (Colo205
and Colo320). To gain further insights into the cytotoxic behavior
of the compounds, apoptosis induction by lead compounds HL and [Cu(L)Cl] was investigated by a flow cytometry analysis
of multidrug-resistant Colo320 cells stained with Annexin-V-FITC and
propidium iodide (PI). The two compounds that displayed the highest
cytotoxicity against this cell line were tested at two concentrations
in the range of their IC50 values. 12H-Benzophenothiazine (M627) and cisplatin were used as positive controls.
The fluorescence of PI (FL3) was plotted versus Annexin-V fluorescence
(FL1) as shown in Figure for the positive controls and for the tested compounds at
a chosen concentration. The percentage of the gated events regarding
the early apoptosis, the late apoptosis and necrosis, and cell death
is quoted in Table S8. According to these
data, both compounds studied, HL and [Cu(L)Cl], can be considered as efficient apoptosis inducers.
Figure 14
Quantification of apoptosis
in Colo320 cells treated with HL and 2′ and
M627 and cisplatin (as positive controls) using the Annexin-V/PI double
staining assay. Colo320 cells were treated at the indicated concentration
of the drugs. The dual parametric dot plots that combine the Annexin-V
(FL1) and PI (FL3) fluorescence show the viable cell population in
the lower-left quadrant Annexin-V–/PI– (Q4), the early apoptotic cells in the lower-right quadrant Annexin-V+/PI–
(Q3), and the late apoptotic and necrotic cells in the
upper-right quadrant Annexin-V+/PI+ (Q2). (Number of
cells counted: 23 193 (M627), 20 262 (cisplatin), 33 193
(HL), and 19 312 (2′)).
Quantification of apoptosis
in Colo320 cells treated with HL and 2′ and
n class="Chemical">M627 and cisplatin (as positive controls) using the Annexin-V/PI double
staining assay. Colo320 cells were treated at the indicated concentration
of the drugs. The dual parametric dot plots that combine the Annexin-V
(FL1) and PI (FL3) fluorescence show the viable cell population in
the lower-left quadrant Annexin-V–/PI– (Q4), the early apoptotic cells in the lower-right quadrant Annexin-V+/PI–
(Q3), and the late apoptotic and necrotic cells in the
upper-right quadrant Annexin-V+/PI+ (Q2). (Number of
cells counted: 23 193 (M627), 20 262 (cisplatin), 33 193
(HL), and 19 312 (2′)).
The antiproliferative
activity of 1 and 2′ in the normal
cells (n class="CellLine">MRC-5) was only slightly lower than in Colo205
and Colo320 cells, indicating a quite moderate selectivity for cancer
cells. Complex Cu(HL)Cl was found to be less cytotoxic compared
to the other two complexes tested, and the IC50 values
are similar to those of the copper(II) chloride, while the selectivity
for cancer cells is obvious in this case (SI > 3). It is worth
mentioning
that the analogous α-N-pyridyl thiosemicarbazones,
that is, FTSC, AcTSC, and triapine, were reported to be cytotoxic
in the low micromolar concentration range against several humancancer
cells, the latter being the most potent among them (IC50 values reported for triapine: 0.4–2.6 μM (in good agreements
with the data quoted in Table 5), for FTSC: 1.9–10.6 μM,
for AcFTSC: 2.5–3.6 μM in SW480,[36] MES-SA,[36] MES-SA/Dx5,[36] HL60,[58] 41M,[80] SK-BR-3[80]).
Their Cu(II)n class="Chemical">complexes were reported to possess a similar or even
weaker cytotoxicitycompared to the metal-free ligands, in contrast
to complexes studied in the present work, which might indicate a distinct
mode of action. It is also of note that the two-electron oxidized
product H revealed a superior antitumor activity in the two cancer cell
lines over that of H and H. In agreement with this,
closely related 2-formyl- and 2-acetylpyridine 2-benzothiazolyl hydrazones
were shown to be potent cytotoxic drugs against a series of 17 murine
(e.g., L1210 lymphoid leukemia, P388 lymphocytic leukemia) and humancancer cells (e.g., HeLa cervix carcinoma, bone SOS, lung MB9812,
lung A549). In addition, these compounds showed selectivity for the
multidrug-resistant doxorubicin-selected uterine sarcoma cell line
MES-SA/Dx5 over parental or sensitive MES-SA cells.[78,79]
Tyrosyl Radical Reduction in mR2 RNR
The TSCs HL–HL and their n class="Chemical">copper(II) complexes 1, 2′, and 3 were found to effectively quench
the tyrosyl radical in mR2RNR in the presence of an external reductant
(DTT). The time-dependent tyrosyl radical reduction in mR2RNR by
equimolar concentrations of TSCs and their respective copper(II)complexes,
under reducing conditions, is shown in Figure . The mR2 inhibition potency follows the
order HL ≈ triapine > HL > HL. The coordination to copper(II) was found to increase
the
tyrosyl radical quenching potential for all TSCs, which is in agreement
with the observed lowering of IC50 values in all cancer
cell lines (Table ). Complex 1 was shown to be as efficient as triapine,[17] reducing 100% of the tyrosyl radical in 3 min.
Complexes 2′ and 3 exhibited comparable
reduction kinetics despite the fact that, among the investigated TSCs, HL was found to be most inefficient.
The favorable impact of the copper(II)coordination on the HL inhibitory activity is quite obvious, when
the ability to quench the tyrosyl radical by HL is compared to that of 2′. Interestingly,
the two-electron oxidized product of HL, namely, H·CHCOOH, is as potent as HL in tyrosyl radical reduction.
Figure 15
Tyrosyl radical reduction
kinetics in mouse R2 RNR protein by TSCs HL, HL, HL, and their corresponding
copper complexes 1, 2′, and 3 as well as by the two-electron oxidized product of HL (H·CHCOOH), in the presence of an external
reductant, measured at 30 K by EPR spectroscopy and compared to triapine.
The samples contained 20 μM mR2 in 50 mM HEPES buffer, pH 7.60/100
mM KCl, 20 μM compound in 1% (v/v) DMSO/H2O, and
2 mM DTT.
Tyrosyl radical reduction
kinetics inn class="Species">mouse R2RNR protein by TSCs HL, HL, HL, and their corresponding
coppercomplexes 1, 2′, and 3 as well as by the two-electron oxidized product of HL (H·CHCOOH), in the presence of an external
reductant, measured at 30 K by EPR spectroscopy and compared to triapine.
The samples contained 20 μM mR2 in 50 mM HEPES buffer, pH 7.60/100
mM KCl, 20 μM compound in 1% (v/v) DMSO/H2O, and
2 mM DTT.
The ability of HL–HL and 1, 2′, and 3 to quench the tyrosyl radicaln class="Chemical">correlates well
with their first anodic redox potentials (0.82–0.88 V vs NHE)
and (0.68–0.70 V vs NHE), respectively, which are well-compared
with redox potential of hydroxyurea (+0.724 V),83 which
reduced the tyrosyl radical in the R2 protein with an estimated redox
potential of 1.0 ± 0.1 V vs NHE.[36] Note, however, that hydroxyurea, a well-known inhibitor of RNR and
an anticancer drug,84 is a small molecule able to enter
the hydrophobic R2 protein pocket, where the tyrosyl radical is buried.
Finally, the two- and four-electron oxidized products of HL, namely, H and H, do not
have an effect on the tyrosyl radical in the absence of DTT and, interestingly,
cause an increase in the radical content in the presence of DTT (Figure S15).
Previously it has been shown
that the radical content inn class="Gene">mR2 may
be slightly increased in the presence of DTT, as the result of the
so-called radical reconstitution reaction,[17],85 in which the DTT–reduced diiron center in the
reaction with molecular oxygen is spontaneously oxidized through a
series of intermediate states, generating the active Fe(III)-O2–-Fe(III)/Tyr· cofactor. However, the radical
increase caused by HL and HL (in reducing conditions) is much greater than that observed
for DTT, providing evidence that the formation of the active iron/radical
site in mR2 is more efficient when the DTT–reduced form of
mR2 is oxidized by H or H, than by molecular oxygen
only.
Consistent with enzyme inhibition studies, which revealed
a potent
inhibition of n class="Gene">mR2 RNR, compounds HL, 1, and 2′ were found to
increase the population of the S-phase in SW480 cells.
Cell Cycle
Arrest
The perturbation effects of 10 μM HL, 1, and 2′ on the cell cycle progression of n class="CellLine">SW480 cells when compared to negative
control are shown in Figure and Table S9, while the effects
of 0, 1.0, and 10 μM are presented in Figure S16. It can be noted that the population of S-phase cells increased
after an incubation with HL (37.1%),
complex 1 (44.0), and 2′ (46.5) compared
with the negative control (29.8%). Gemcitabine (GC), a positive control,
showed a canonical G1/S-phase arrest at the concentration of 0.01
μM with 26.8% of cells in the G1 phase and 62.3% of cells in
the S phase compared to the negative control with 49.1% of cells in
the G1 phase and 29.8% of cells in the S phase (Figure S17). An increase in the population of the S-phase
cells by ca. 20% has been reported for a series of triapine analogues
at concentrations from 0.25 to 5.0 μM.[80] The S-phase arrest is characteristic for cells treated with triapine.[81]
Figure 16
Flow cytometry analysis for a cell cycle distribution
of SW480
cells induced by TSC HL (B)
and complexes 1 (C) and 2′ (D) at
the concentration of 10 μM for 24 h compared to the negative
control (DMSO) (A).
Flow cytometry analysis for a cell cycle distribution
of SW480
cells induced by n class="Gene">TSC HL (B)
and complexes 1 (C) and 2′ (D) at
the concentration of 10 μM for 24 h compared to the negative
control (DMSO) (A).
These data indicate
that there is a correlation between the ability
of the n class="Chemical">compounds tested to inhibit R2RNR and their ability to induce
an S-phase arrest. Nevertheless, the inhibition of RNR does not appear
to be the main mechanism underlying the antiproliferative activity
of both TSCs studied herein and their copper(II)complexes.
ROS Generation
Since metal-free n class="Chemical">TSCs that enter the
cells or are released from copper(I)complexes generated by a reduction
of their copper(II)counterparts can react in the cells with iron(II),
the redox activity of the [Fe(L)] complex, prepared by the reaction of
an anoxic aqueous solution of FeSO4·7H2O with a DMSO solution of HL at a 1:2 molar ratio, was investigated by EPR spin-trapping experiments.
To investigate whether this ferrous complex is able to generate ROS
in the aqueous environment by a Fenton reaction, which is supposed
to quench the tyrosyl radical of the mR2 enzyme, hydrogen peroxide
was added into the system in the presence of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin-trapping agent. A four-line EPR
signal characteristic for the ·OH-DMPO spin adduct was observed
(Figure , black
trace, EPR signal marked with circles).
Figure 17
Experimental EPR spectra
of Fe(II)/HL/DMPO/H2O2 in 5% (v/v) H2O–DMSO
(black line), in the system of Fe(II)/HL/DMPO in 5% (v/v) H2O–DMSO (blue line), and
in 30% (v/v) H2O–DMSO (red line). Initial concentrations: c(HL1) = 0.2 mM, c(FeSO4·7H2O) = 0.1 mM, c(DMPO)
= 20 mM, c(H2O2) = 10 mM.
Experimental EPR spectra
of Fe(II)/HL/n class="Chemical">DMPO/H2O2 in 5% (v/v) H2O–DMSO
(black line), in the system of Fe(II)/HL/DMPO in 5% (v/v) H2O–DMSO (blue line), and
in 30% (v/v) H2O–DMSO (red line). Initial concentrations: c(HL1) = 0.2 mM, c(FeSO4·7H2O) = 0.1 mM, c(DMPO)
= 20 mM, c(H2O2) = 10 mM.
Additionally a ·DMPO-OCH3 spin
adduct can be seen
in the n class="Chemical">corresponding EPR spectrum as a consequence of the reaction
of hydroxyl radicals with the DMSO solvent forming methyl radicals,
which react with molecular oxygen resulting in the generation of peroxomethyl
radicals serving as a source of •DMPO–OCH3 spin adducts (Figure , black trace, EPR signal marked with squares).[82] Only a trace amount of carbon-centered radicals
was detected for Fe(II)/HL/DMPO
in H2O–DMSO in the absence of H2O2 (Figure , blue and red traces, EPR signal marked with stars). In this case
DMSO acts as a HO· scavenger, generating reactive carbon-centered
radicals, which are trapped by DMPO. It is important to mention that
no radicals were formed in the system of HL/H2O2/DMPO/H2O–DMSO
(not shown), which indicates the crucial role of the Fe(II)complex
for ROS generation. Consequently complex [Fe(L)] is redox-active in the Fenton
reaction indicating the important role of the HL ligand for the observed antiproliferative activity
against cancer cell lines and its ability to quench the tyrosyl radical
in the mR2 protein. A direct reduction of the tyrosyl radical by iron(II)complexes with reported TSCs can also not be excluded.[16]
Conclusions
New triapine analogues
bearing a redox-active n class="Chemical">p-aminophenolic moiety and
their copper(II)complexes have been synthesized
and characterized by spectroelectrochemical and analytical techniques,
which confirmed the noninnocent identity of the latter. The crystal
structures of TSCs HL–HL and complexes [Cu(L)Cl] were
studied by SC-XRD revealing the tridentate (N,N,S)coordination mode
of the ligands. The presence of E and Z isomers of HL–HL with a predominance of the first
one in DMSO has been disclosed by 1D and 2D NMR spectroscopy. These
data along with DFT calculations on the model compound 2-formylpyridineTSC indicate that the Z/E isomerization
involves an inversion at the aldiminenitrogen atom, rather than a
tautomeric shift of the thioamide N2H proton to the pyridinenitrogen,
followed by a rotation around the C–N1 bond as suggested previously.[44] The relatively high Gibbs free energy barrier
(∼35.3 kcal/mol) for the Z/E conversion rules out the possibility of an isomerization at room
temperature, in agreement with time-dependent NMR data.
A two-electron
oxidative dehydrogenation of HL by a reaction with 1 equiv of n class="Chemical">DDQ afforded the
new species H containing a thiadiazole five-membered ring
formed via a nucleophilic attack of a thionesulfur atom on an aldiminecarbon atom. This is supported by frontier molecular orbitals (MOs)
with the HOMO and LUMO located at opposite parts of the molecule of HL. When 2 equiv of DDQ were used,
a further two-electron oxidation coupled with a two-proton loss occurred
at the 3,5-dimethyl-4-aminophenolic moiety to give the 3,5-dimethyl-1,4-benzoquinoneimine unit in H. Also note that the coordinated ligand HL is able to form a thiazole five-membered
ring in 4 via a sulfur attack on the carbon atom in position
2 or 6 of the 3,5-dimethyl-4-aminophenolic moiety. The arylated sulfur
atom has lost the competition in binding to copper(II) for an end
nitrogen atom due to the reduction of the electron-donating ability
of the sulfur atom. The oxidation of HL with PBQ in a 1:1 molar ratio furnished the two-electron oxidative
cyclization product H and the diphenolic species H. A tentative
mechanism of their formation is proposed. The pathway to H implies
the formation of the 4-isothiocyanato-2,6-dimethylphenol intermediate.
Treatments of HL with 1 and
2 equiv of PIDA afforded the two-electron oxidation product H and the four-electron oxidation product H, respectively.
In contrast to HL–HL, the Z/E isomerization was observed at room temperature for H. The isolation and investigation of oxidation products of
new TSCs was of interest also from the point of view of collecting
spectroscopic data that might be useful for an eventual analysis of
metabolites, which can be generated in vivo from the corresponding
TSCs and their copper(II)complexes.
Solution equilibrium studies
performed by UV–vis spectrophotometry
revealed the acidic pKa values (3.01–3.95)
of the n class="Chemical">pyridinium nitrogen and pKa values
greater than or equal to 10.55 for the hydrazinic-NNH and phenolic
(PhOH) moiety of the metal-free ligands. The latter are neutral and
stable at a physiological pH. However, they become air-sensitive upon
deprotonation of the OH group in the basic pH range. The formation
of high-stability monoligand copper(II)complexes was found in different
protonation states in solution; namely, coordination via (Npyridine,N,S)(H2O), (Npyridine,N,S–)(H2O), and (Npyridine,N,S–)(OH–) donor sets are probable. The complexes with
a (Npyridine,N,S–)(H2O) coordination
predominate in a wide pH range including pH 7.4. Conditional stability
constants determined for the [Cu(L)] and [Cu(L)] complexes by an EDTA UV–vis spectrophotometric competition
experiment show the somewhat higher stability of the [Cu(L)] complex. The attachment of a phenolic moiety undoubtedly increases
the lipophilicity of new Schiff bases and copper(II)complexes when
compared to triapine and its copper(II)complex. The new complexes
can be reduced by glutathione, the most abundant low molecular mass
reducing agent in a cell, in a reversible redox reaction. According
to the electrochemical studies complexes 1, 2′, and 3 can undergo a redox process in a biologically
accessible window (−0.4 to +0.8 V vs Fc+/Fc). These
findings suggest a possible role of the redox properties of the copper(II)complexes in their biological activity.
The metal-free ligands
and several oxidized products showed no
or only a moderate n class="Disease">cytotoxicity against doxorubicin-sensitive Colo205
and the multidrug-resistant Colo320 humancolonic adenocarcinoma cell
lines. Their copper(II)complexes revealed a high cytotoxic potency
when compared to that of the corresponding metal-free ligands. [Cu(L)Cl] showed the
highest cytotoxic activity with IC50 values in the low
micromolar concentration range and induced apoptosis, while Cu(HL)Cl has the highest selectivity for cancer cells over the normal
fibroblast MRC-5 cells. The highest antiproliferative activity of [Cu(L)Cl] is likely
due to the more negative reduction potential when compared to those
of 1 and 3 and low reduction rate in reaction
with GSH.[36] In addition, HL–HL and their copper(II)complexes were found to efficiently quench
the tyrosyl radical in mR2RNR in the presence of DTT as an external
reductant and increase the population of S-phase cells. The capacity
of HL to destroy the tyrosyl
radical is almost identical with that of triapine, which is by the
factor of 1000 a more potent R2RNR inhibitor than hydroxyurea, a
known clinical drug.[17] Thus, the copper(II)complexes reported herein deserve further investigation as potential
anticancer drugs.
Authors: Kimiko Ishiguro; Z Ping Lin; Philip G Penketh; Krishnamurthy Shyam; Rui Zhu; Raymond P Baumann; Yong-Lian Zhu; Alan C Sartorelli; Thomas J Rutherford; Elena S Ratner Journal: Biochem Pharmacol Date: 2014-08-15 Impact factor: 5.858
Authors: Christian R Kowol; Rene Eichinger; Michael A Jakupec; Markus Galanski; Vladimir B Arion; Bernhard K Keppler Journal: J Inorg Biochem Date: 2007-07-31 Impact factor: 4.155
Authors: Alice Santoro; Jenifer S Calvo; Manuel David Peris-Díaz; Artur Krężel; Gabriele Meloni; Peter Faller Journal: Angew Chem Int Ed Engl Date: 2020-03-18 Impact factor: 15.336
Authors: Sonja Hager; Veronika F S Pape; Vivien Pósa; Bianca Montsch; Lukas Uhlik; Gergely Szakács; Szilárd Tóth; Nikolett Jabronka; Bernhard K Keppler; Christian R Kowol; Éva A Enyedy; Petra Heffeter Journal: Antioxid Redox Signal Date: 2020-06-09 Impact factor: 8.401
Chart 1
Chart 2
Scheme 1
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Figure 11
Figure 12
Figure 13
Figure 14
Figure 15
Figure 16
Figure 17