Tamara R.
Todorović
a,
Jelena
Vukašinović
a,
Gustavo
Portalone
b,
Sherif
Suleiman
c,
Nevenka
Gligorijević
d,
Snezana
Bjelogrlić
d,
Katarina
Jovanović
d,
Siniša
Radulović
d,
Katarina
Anđelković
a,
Analisse
Cassar
c,
Nenad R.
Filipović
*e and
Pierre
Schembri-Wismayer
*c
aFaculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia
bDepartment of Chemistry, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy
cAnatomy Department, Faculty of Medicine and Surgery, University of Malta, Malta. E-mail: pierre.schembri-wismayer@um.edu.mt
dInstitute for Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia
eFaculty of Agriculture, University of Belgrade, Nemanjina 6, 11081 Belgrade, Serbia. E-mail: nenadf.chem@gmail.com
First published on 20th October 2016
Cobalt complexes with semi- and thiosemicarbazones of 8-quinolinecarboxaldehyde have been synthesized and characterized by X-ray diffraction analysis. These novel complexes and a previously synthesized cobalt complex with a selenium-based selenosemicarbazone ligand showed myeloid differentiation activity on all trans retinoic acid resistant HL-60 acute myeloid leukaemia cells. They also showed varying levels of cytotoxicity on five human tumor cell lines: cervix carcinoma cells (HeLa), lung adenocarcinoma cells (A549), colorectal adenocarcinoma cells (LS-174), breast carcinoma cells (MDA-MB-361), and chronic myeloid leukaemia (K562) as well as one normal human cell line: fetal lung fibroblast cells (MRC-5). Leukaemia differentiation was most strongly induced by a metal-free oxygen ligand and the selenium ligand, whilst the latter and the cobalt(II) complex with an oxygen ligand showed the strongest dose-dependent cytotoxic activity. In four out of five investigated tumor cell lines, it was of the same order of magnitude as cisplatin. These best compounds, however, had lower toxicity on non-transformed MRC-5 cells than cisplatin.
(Chalcogen)semicarbazones, condensation products of (chalcogen)semicarbazides and carbonyl compounds, have been a subject of interest in coordination chemistry for many years. Versatile modes of coordination of this class of ligands have been reported.16–19 They can coordinate to metal ions bidentately via chalcogen donor and imine nitrogen atoms, but the coordination ability may be extended if a parent carbonyl compound possesses other suitable donor atoms. This general class of compounds has been shown to possess a wide range of bioactive properties, including antitumor activity.20–37 In fact (chalcogen)semicarbazones with activities comparable to standard anticancer drugs, like cis-diammindichloridoplatinum(II) (cisplatin, CDDP), have been developed. A few comprehensive comparative studies of (chalcogen)semicarbazones and their complexes pointed out the importance of the chalcogen donor atom identity for biological activity.26,30,38 Among the three types of (chalcogen)semicarbazones, thiosemicarbazones have been studied to a greater extent than semicarbazones and selenosemicarbazones. Some results indicated that sulphur compounds are more active in comparison with oxygen analogues, which showed a rather limited spectrum of biological activities.24 These thiosemicarbazones are also more stable and safer to handle than their selenium analogues.37,39,40 Selenosemicarbazones and their metal complexes have been an area of our particular interest for years.31–47,41,42 Research suggests that the mechanisms of cytotoxicity of these compounds may include the production of reactive oxygen species and oxidative stress in the complexes with 2-quinolinecarboxaldehyde selenosemicarbazone.37
In the current study we used 8-quinolinecarboxaldehyde-based (chalcogen)semicarbazones as ligands (Scheme 1), providing detailed spectroscopic and structural characterization (X-ray diffraction, XRD) of the novel cobalt complexes with oxygen and sulphur-based ligands. Cobalt was chosen as the central metal ion since the coordination of (chalcogen)semicarbazones to cobalt often results in complexes with activities higher than the activity of standard anticancer drugs such as cisplatin.33,43–46 We analysed the effects of (chalcogen)semicarbazones and the corresponding cobalt complexes on the differentiation of high passage number (70+) HL-60 cells which are known to be poorly responsive to ATRA-induced differentiation.47 We further test the general cytotoxic effects of these compounds on a number of tumor cell lines and on normal cells and perform some analysis of the cell cycle perturbations induced.
Scheme 1 Schematic structure and atomic numbering scheme of (chalcogen)semicarbazone ligands: H8qaSC, H8qaTSC and H8qaSeSC. |
Synthesis of the cobalt(III) complex with the oxygen ligand H8qaSC was unsuccessful even when a stream of air was passed through the reaction mixture for 3 h. Similarly, in the case of related 2-formyl, 2-acetyl, and 2-benzoylpyridine N(4)-cyclohexylsemicarbazones, cobalt(II) complexes with neutral ligands were obtained, whereas with the analogous thiosemicarbazones, cobalt(III) complexes with the anionic form of the ligands were obtained.48 It is anticipated that oxidation reaction of cobalt(II) to cobalt(III) species consists of two main steps: (1) the reversible formation of a dioxygen adduct (μ-peroxo-bridged cobalt compound) and (2) its irreversible decomposition to related cobalt(III) complexes, where the redox rearrangement reactions can be classified as metal-centered oxidations and ligand-centered oxidative dehydrogenations.49,50 It is well known that the octahedral hexaaquacobalt(II) ion is stable to aerial oxidation, but data on standard reduction potential for a variety of cobalt(III) complexes showed the stabilization of the +3 oxidation state relative to the +2 as the ligands are changed from O- to N-donors.51 In the case of (chalcogen)semicarbazones derived from 8-quinolinecarboxaldehyde, it can be assumed that a combination of favorable thermodynamic and kinetic factors allows for facile synthesis of cobalt(III) complexes with heavier chalcogens, as opposed to the cobalt(II) complex with an O analogue.
H8qaTSC | 1 | 2 | |
---|---|---|---|
Crystal data | |||
Empirical formula | C11H10N4S | C22H20CoN8O2·2Cl·2(H2O) | C22H18CoN8S2·ClO4·C2H6SO |
Formula weight | 230.29 | 594.32 | 695.02 |
Crystal system | Monoclinic | Monoclinic | Triclinic |
Space group | P21/c | C2/c | P |
a, b, c (Å) | 8.9464 (9), 12.8728 (11), 9.7816 (7) | 21.752 (3), 10.0815 (9), 13.905 (2) | 10.3016 (6), 10.5421 (7), 14.5516 (10) |
α, β, γ (°) | 90, 95.947 (8), 90 | 90, 124.39 (2), 90 | 80.662 (6), 89.829 (5), 65.733 (6) |
V (Å3) | 1120.43 (17) | 2516.1 (8) | 1418.02 (17) |
Z | 4 | 4 | 2 |
μ (mm−1) | 0.27 | 0.94 | 0.97 |
Crystal size (mm) | 0.20 × 0.18 × 0.12 | 0.15 × 0.12 × 0.09 | 0.09 × 0.08 × 0.08 |
T min, Tmax | 0.697, 1.000 | 0.585, 1.000 | 0.955, 1.000 |
No. of measured, independent and observed [I > 2σ(I)] reflections | 16892 | 18343 | 20909 |
2551 | 2881 | 6165 | |
2116 | 2365 | 4897 | |
R int | 0.032 | 0.049 | 0.042 |
(sin θ/λ)max (Å−1) | 0.650 | 0.650 | 0.639 |
R[F2 > 2σ(F2)] | 0.039 | 0.042 | 0.046 |
wR(F2) | 0.110 | 0.103 | 0.126 |
S | 1.06 | 1.05 | 1.03 |
No. of reflections | 2551 | 2881 | 6165 |
No. of parameters | 157 | 188 | 389 |
Δ〉max, Δ〉min (e Å−3) | 0.21, −0.33 | 0.40, −0.26 | 0.53, −0.53 |
In the molecular structure of complex 1, a [Co(H8qaSC)2]2+ cation crystallizes with two chloride anions and two water molecules. The cobalt(II) ion lies on a twofold rotation axis, hence the asymmetric unit of 1 consists of a half of the complex cation, one chloride ion and one water molecule (Fig. 1a). In the complex cation, the cobalt(II) metal center is coordinated to two tridentate neutral H8qaSC ligands, giving rise to a chiral octahedral arrangement. Complex 1 is nonetheless a racemate, since it crystallizes in the centrosymmetric C2/c space group. Chelation occurs by means of quinoline (N1, N1i) and imine (N2, N2i) nitrogen atoms and by the oxygen donors (O1, O1i), where i = −x, y, 1.5 – z, in a mer geometry constrained by the ligand's planarity. Fig. 1b reports the molecular structure of [Co(H8qaSC)2]2+ in 1, while Table S1 (ESI‡) reports the coordination geometry. The coordinative bond lengths in 1 are similar to the respective bonds in analogous N-heteroaromatic semicarbazone cobalt(II) complexes: bis{1-[(E)-2-pyridinylmethylidene]semicarbazide}cobalt(II) diperchlorate monohydrate (CSD refcode ATUNEI)53 and bis[bis(2-pyridyl)ketone semicarbazonato-N,N′,N′′]cobalt(II) dinitrate monohydrate (CSD refcode WEJNOO).54 The crystal packing (Fig. S1 and Table S2, ESI‡) is based on a 3D hydrogen bond network involving terminal NH2 groups, N–H groups, chloride ions and crystal water molecules.
XRD shows that in the case of complex 2 two deprotonated H8qaTSC ligands are coordinated to the cobalt(III) ion, while the outer sphere of the complex consists of a perchlorate ion and one DMSO solvent molecule. The octahedral bis-chelate cation [Co(8qaTSC)2]+ possesses a mer geometry. Since the complex crystallizes in the centrosymmetric P space group, it is a racemic compound regardless of the chiral octahedral arrangement of cationic species in 2. Fig. 2 shows a perspective view of the complex cation in 2, while Table S1 (ESI‡) reports the most relevant bonding parameters. The monoanionic form of the H8qaTSC ligand coordinates the metal via the sulfur atom, the quinoline and the imine nitrogen atoms, with the formation of one six-membered and one five-membered chelate ring. All metal–donor atom bonds are close to the average corresponding bonds found in a search on quinoline thiosemicarbazone-cobalt systems performed through the Cambridge Structural Database (CCDC: 734053; 2016 release, v. 5.37 with updates: Nov15, Feb16).55 Besides electrostatic interactions between complex cations and counter ions, the crystal packing of complex 2 (Fig. S2 and Table S3, ESI‡) is based on hydrogen bonds and π–π stacking interactions of the quinoline rings. Complex 2 is isostructural with the cobalt(III) complex with the selenium ligand [Co(8qaSeSC)2]ClO4·DMSO (3).56
Fig. 2 Perspective view and labeling of the molecular structure of [Co(8qaTSC)2]+ in 2. Thermal ellipsoids are at the 50% probability level. |
1H and 13C NMR spectroscopy data (Fig. S5 and S6, respectively, ESI‡) confirm the tridentate coordination of the ligand H8qaTSC in its monoionic form in complex 2. The correlation of H–C2 with H–C9 and H–C7 in the 2D ROESY spectrum of complex 2 (Fig. S7, ESI‡), as previously noticed for the Co-selenosemicarbazone complex 3,56 is attributed to the octahedral geometry, which indicates that the geometry of complex 2 is preserved in the solution.
The electronic absorption spectra of the (chalcogen)semicarbazone ligands exhibited three bands in the region 350–220 nm, corresponding to the intra-ligand transitions associated with the azomethine, quinoline, and CX (X = O, S) portions of the ligands (Fig. S8, ESI‡). In the spectra of the complexes, the intense absorption bands attributed to the intra-ligand transitions within the coordinated ligand moiety and ligand-to-metal charge transfer transitions can be observed (Fig. S8, ESI‡). The aqueous solution behavior of complexes 1–3 with respect to hydrolysis was studied in DMSO/H2O 1:100 (v/v) solutions at ambient temperature over 24 h by UV-vis spectroscopy. Complexes 1–3 were stable, as can be seen from their electronic absorption spectra (Fig. S9, ESI‡).
As can be seen in Fig. 3, the NBT/MTT ratio, a spectroscopic screen for the state of differentiation,57 shows that H8qaSC and its cobalt(II) complex 1 and H8qaSeSC and its cobalt(III) complex 3 appear to induce some differentiation (of greatly varying extent) whilst both the thiosemicarbazone ligand H8qaTSC and its cobalt(III) complex 2 have relatively little effect. The selenium ligand H8qaSeSC, as well as the oxygen ligand H8qaSC, is effective at inducing differentiation at three days post-exposure even at 1 μM doses. After five days of exposure, H8qaSeSC and complex 1 and to a lesser extent 3 appear the better differentiating agents.
Data from the MTT assay acquired after 72 h incubation and used to calculate the NBT/MTT ratio were also employed to estimate the cytotoxic activity of the investigated compounds on HL60 cells. As it can be seen in Fig. S10A (ESI‡), ligands H8qaSC, H8qaSeSC and complex 3 were those whose activity reached IC50 concentrations. The IC50 value implies that the investigated treatment reduced the size of the treated population by 50% compared to non-treated control but does not obtain information on the particular mechanism responsible for this effect. In order to evaluate whether the cytotoxic activity or inhibition of proliferation was the underlying cause, cell cycle analysis on HL60 cells subjected to IC50 concentrations of H8qaSC, H8qaSeSC and 3 for 72 h was further performed to assess the percentages of cells accumulated at the sub-G1 subpopulation (Fig. S10B, ESI‡). Compound H8qaSeSC was the only one that notably, but still modestly, increased the percentage of dead cells, whereas H8qaSC and complex 3 barely raised the size of the sub-G1 fraction compared to control levels. These results clearly indicate that the investigated compounds did not exert cytotoxic activities on HL60 cells in a range of applied concentrations.
The ligand H8qaSeSC which showed the strongest induction of NBT activity, through spectrophotometry, caused the development of numerous granules in the HL60 cells, indicating a strong differentiation towards granulocytes, together with the loss of nucleoli in the non-granular cells (Fig. 4f). The same compound has also been shown to induce some markers of differentiation in solid tumour cancer stem cells.56
Despite H8qaSC, H8qaTSC and H8qaSeSC having a rather similar structure with a sulphur or selenium atom substituting an oxygen atom in the parent compound H8qaSC, it appears that these small modifications greatly vary the differentiation-inducing activity of H8qaSC, with selenium enhancing it and sulphur removing it. Selenium is an important trace element in the body and the inclusion of this atom may mimic a selenium-containing natural factor. Zinc-finger transcription factor PLZF is known to abnormally repress gene activity in HL-60,59 and selenium is a known inhibitor of zinc-finger transcription factors.60,61 Thus inhibiting PLZF-dependent gene repression may result in differentiation.59,61 This may well be the reason for the stronger differentiation induced by H8qaSeSC as opposed to H8qaSC. The complexing of the selenium atoms with the cobalt in complex 3 may also then reduce this zinc-finger interaction and explain why the complex is less effective. The cobalt complex 3 resulted in some changes in nuclear chromatin condensation as well as of nuclear cytoplasmic ratios and development of numerous pseudopodia; however, the occasional granular cells also appeared. This, together with clearly lower NBT activity, suggests that the differentiation induced by the complex is less than that induced by the metal-free ligand H8qaSeSC, which may relate to the selenium-induced effects being suppressed by the very tight binding within the complex.
Exposure to the oxygen ligand H8qaSC shows smaller sized cells with reduced nuclear cytoplasmic ratios as well as evidence of nuclear chromatin condensation also indicating a partial differentiation. It is interesting to note that its complex 1 showed only minimal differentiation, unlike its strong cytotoxic activity to tumour cells (see below). From cell cycle analysis (vide infra), complex 1 appears to damage DNA resulting in sub G1 fragmentation of DNA, possibly due to the cobalt interfering with DNA repair proteins, including zinc-finger factors.62,63 However, one should point out that complex 1 toxicity occurs at doses above 40 μM, whilst effects on differentiation were tested at doses of 1 to 10 μM. Thus whilst the DNA damage induced at higher doses may be cytotoxic, the little amount induced at these low doses may be differentiating without being cytotoxic.64 As in the case of complex 3, in complex 1 too, the complexation appears to reduce the efficacy of the metal-free semicarbazone ligand as a differentiating agent, whilst in this case, markedly increasing its cytotoxicity.
On the other hand, the H8qaTSC and its cobalt(III) complex 2 (latter not shown) both show little activity on the NBT screen and similarly show a morphology very similar to the undifferentiated cells, with large nuclear-cytoplasmic ratios, nucleoli and many mitoses. Again here, the stronger binding between the sulphur chalcogen donor atoms in the complex with cobalt(III) may reduce its ability to interact with repair proteins causing DNA damage.
One should point out that since these are abnormal leukaemia cells (and not normal haematopoietic progenitors) being differentiated, the variability in the morphology creates difficulty in understanding the nominal stage of (normal haematopoietic) differentiation induced by the various agents. For this reason, three independent medical observers reviewed the slides to score the indicative features of the differentiated cells (Table S4, ESI‡).
IC50 (μM) | ||||||
---|---|---|---|---|---|---|
HeLa | A549 | MDA-MB-361 | LS-174 | K562 | MRC-5 | |
a Values represent the mean ± SD from three independent experiments. | ||||||
H8qaSC | 36.5 ± 3.9 | >100 | >100 | >100 | >100 | >100 |
H8qaTSC | 75.6 ± 5.8 | >100 | >100 | >100 | >100 | >100 |
H8qaSeSC | 6.6 ± 1.4 | 53.1 ± 2.8 | 9.2 ± 4.4 | 14.4 ± 2.3 | 4.0 ± 0.2 | 30.3 ± 3.6 |
1 | 17.2 ± 1.6 | 24.9 ± 3.2 | 45.5 ± 3.4 | 32.9 ± 4.5 | 37.6 ± 0.3 | 39.5 ± 1.0 |
2 | 46.2 ± 3.6 | >100 | >100 | >100 | >100 | >100 |
3 | 33.2 ± 2.2 | >100 | 36.3 ± 0.4 | >100 | 31.3 ± 5.9 | >100 |
CDDP | 5.2 ± 0.3 | 26.2 ± 5.4 | 14.7 ± 1.2 | 22.4 ± 7.2 | 18.6 ± 3.3 | 12.1 ± 0.9 |
In the investigated series of compounds, the selenosemicarbazone ligand H8qaSeSC had the highest cytotoxicity. It showed a strong cytotoxic effect on HeLa, K562, LS-174 and MDA-MB-361 cells, in the range of the activity of CDDP. In fact for a number of these cell lines it appeared considerably more toxic than this standard. It is worth mentioning that the ligand H8qaSeSC had a lower toxicity on normal cells (MRC-5), than on most of the investigated tumor cell lines. The toxicity of H8qaSeSC on these untransformed MRC-5 cells was also considerably less than that of CDDP. The two other ligands, H8qaSC and H8qaTSC, had a low cytotoxicity on all investigated cell lines, reaching the IC50 in the investigated range of concentrations only on HeLa cells. Among the complexes, complex 1 with the semicarbazone ligand H8qaSC showed the highest cytotoxicity, possibly due to the cobalt(II) ion and its possible interaction with DNA repair proteins,65,66 which can also explain the DNA damage and sub-G1 cells seen in the cell cycle analysis. However, this toxicity was still considerably less than that of the metal-free selenium ligand on most cell lines. HeLa cells were the most sensitive to the action of complex 1, while the breast cancer cells (MDA-MB-361) were the most resistant. The cytotoxicity of this complex on the normal MRC-5 cells was two times lower than the activity on HeLa cells. The selenosemicarbazone complex 3 showed similar cytotoxicity on HeLa, MDA-MB-361 and K562 cells, but it was not cytotoxic to A549, LS-174 and normal cells (MRC-5) in the investigated concentration range. The thiosemicarbazone complex 2 had the lowest activity in the investigated series of complexes, reaching IC50 only on HeLa cells in the investigated concentration range.
The IC50 values (Table 2) indicate that the ligands generally show a cytotoxic activity in the following order: H8qaSeSC > H8qaSC > H8qaTSC, which is consistent with the literature data for related (chalcogen)semicarbazones.26,30 The order of activity for the complexes is: 1 > 3 > 2, where complexation increased the activity just in the case of complex 1. In this case cytotoxicity is most likely due to the metal, as the ligand H8qaSC is not active. Namely, it is known that cellular uptake of cobalt is genotoxic due to radical-mediated DNA damage and direct cobalt interference with DNA repair probably by substituting zinc ions from zinc-finger proteins.63,67 Also, cobalt(II) ions themselves induce generation of reactive oxygen species in a Fenton-like reaction,68 and can replace magnesium(II) ions in enzymatic physiological enzyme reactions, which strongly enhance DNA cleavage.69 It can be assumed that cobalt(II) complex 1 is involved in oxidative damage of DNA, which is further supported indirectly by the results of cell cycle analysis (vide infra). Selectivity toward cancer cells as compared to the normal cell line was noticed for the selenosemicarbazone ligand H8qaSeSC, its cobalt(III) complex 3, and cobalt(II) complex 1 with the semicarbazone ligand.
An initial assessment of mechanisms of cell death induced by these compounds was performed using Annexin-V and PI staining both with cytometry and microscopy. Results of the microscopy (Fig. S13, ESI‡) clearly confirm the patterns shown by the cell cycle analysis with H8qaSeSC and the complexes having the major effects and with complex 2 exhibiting effects more rapidly than the others. Further details of perturbations of the cell cycle and apoptosis induction (Fig. S14, ESI‡) are found in the ESI.‡
Footnotes |
† The authors declare no competing interests. |
‡ Electronic supplementary information (ESI) available: Experimental section. Selected geometrical parameters (Table S1); hydrogen bond and π–π stacking interaction parameters (Tables S2 and S3); packing diagrams (Fig. S1 and S2); IR spectra of 1 and 2 (Fig. S3 and S4); NMR spectra of 2 (Fig. S5–S7) and UV-vis spectra (Fig. S8 and S9); feature scoring to indicate signs of differentiation (Table S4); concentration–response curves and cell cycle on HL60 cells (Fig. S10); cell survival diagrams (Fig. S11); cell cycle distribution (Fig. S12); fluorescence micrograph (Fig. S13); flow cytometry dot plot diagrams (Fig. S14). CCDC 1401684–1401686. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6md00501b |
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