Strong Effect of Copper(II) Coordination on Antiproliferative Activity of Thiosemicarbazone- Piperazine and Thiosemicarbazone-Morpholine Hybrids†

In this study, 2-formylpyridine thiosemicarbazones and three different heterocyclic pharmacophores were combined to prepare thiosemicarbazone-piperazine mPip-FTSC (HL) and mPip-dm-FTSC (HL), thiosemicarbazone-morpholine Morph-FTSC (HL) and Morph-dm-FTSC (HL), thiosemicarbazone-methylpyrrole-2-carboxylate hybrids mPyrr-FTSC (HL) and mPyrr-dmFTSC (HL) as well as their copper(II) complexes [CuCl(mPipH-FTSC−H)]Cl (1+H)Cl, [CuCl(mPipH-dm-FTSC−H)]Cl (2+H)Cl, [CuCl(Morph-FTSC−H)] (3), [CuCl(Morph-dmFTSC−H)] (4), [CuCl(mPyrr-FTSC−H)(H2O)] (5) and [CuCl(mPyrr-dm-FTSC−H)(H2O)] (6). The substances were characterized by elemental analysis, oneand two-dimensional NMR spectroscopy (HL−HL), ESI mass spectrometry, IR and UV−vis spectroscopy and single crystal X-ray diffraction (1−5). All compounds were prepared in an effort to generate potential antitumor agents with an improved therapeutic index. In addition, the effect of structural alterations with organic hybrids on aqueous solubility and copper(II) coordination ability was investigated. Complexation of ligands HL and HL with copper(II) was studied in aqueous solution by pH-potentiometry, UV−vis spectrophotometry and EPR spectroscopy. Proton dissociation processes of HL and HL were also characterized in detail and microscopic constants for the Z/E isomers were determined. While the hybrids HL, HL and their copper(II) complexes 5 and 6 proved to be insoluble in aqueous solution, precluding antiproliferative activity studies, the thiosemicarbazone-piperazine and thiosemicarbazone-morpholine hybrids HL−HL, as well as copper(II) complexes 1−4 were soluble in water enabling cytotoxicity assays. Interestingly, the metal-free hybrids showed very low or even a lack of cytotoxicity (IC50 values > 300 μM) in two human cancer cell lines HeLa (cervical carcinoma) and A549 (alveolar basal adenocarcinoma), whereas their copper(II) complexes were cytotoxic showing IC50 values from 25.5 to 65.1 μM and 42.8 to 208.0 μM, respectively in the same human cancer cell lines after 48 h of incubation. However, the most sensitive for HL and complexes 1−4 proved to be the human cancer cell line LS174 (colon carcinoma) as indicated by the calculated IC50 values varying from 13.1 to 17.5 μM. Page 1 of 46 Dalton Transactions D al to n Tr an sa ct io ns A cc ep te d M an us cr ip t

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Dalton Transactions
Introduction Thiosemicarbazones (TSCs) are known as potent metal chelators with high affinity for first row transition metals. 1,2 TSCs and their metal complexes possess a variety of biological activities, such as antifungal, antiviral, antibacterial, antimalarial and anticancer. [3][4][5][6][7][8] The anticancer activity of α-Nheterocyclic TSCs (HCTs) has been known since the 1950s when 2-formylpyridine thiosemicarbazone (FTSC) showed antileukemic activity in a mice model. 9 To date, the best-studied HCT is 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP or Triapine). Several clinical phase I and II trials revealed that Triapine is ineffective against a variety of solid tumors but very promising against hematologic malignancies such as leukemia. [10][11][12][13][14][15][16][17][18] The outcome of a recent clinical phase II study including 37 patients with aggressive myeloproliferative neoplasms, with a response rate of 49% and complete remission in 24% of all patients, has recently been reported. 19 Ribonucleotide reductase (RNR), 20,21 an enzyme catalyzing the reduction of ribonucleotides to the corresponding 2'-deoxyribonucleotides, which is the rate determining step in DNA synthesis, 22 and topoisomerase IIα (Topo IIα), an enzyme that controls the DNA topology during cell division by introducing temporary double strand breaks have been considered as possible targets for this class of compounds. [23][24][25][26] New insights into the mechanism of action for RNR inhibiting HCTs and especially Triapine were recently reported. [27][28][29][30] The enzymes ATP binding pocket was suggested as major target for Topo IIα inhibiting HCTs. 31 The reaction of copper(II) with HCTs leading to square-planar complexes markedly enhances the Topo IIα inhibition rate. 32 2-Acetylpyridine thiosemicarbazones possess very high cytotoxicity in human cancer cell lines with IC 50 values in the nanomolar concentration range and the ability to destroy the tyrosyl radical of the mammalian RNR R2 protein under the slightly reducing conditions typical for tumors. 33,34 However, high general toxicity and, consequently, the low therapeutic index along with low aqueous solubility for these and other related thiosemicarbazones prompted us to design hybrid systems, based on thiosemicarbazones and other pharmacophores. Recently, we prepared proline-TSC hybrids (3-methyl-(S)-pyrrolidine-2-carboxylate-2-formylpyridine thiosemicarbazone (L-Pro-FTSC) and 3methyl-(R)-pyrrolidine-2-carboxylate-2-formylpyridine thiosemicarbazone (D-Pro-FTSC)) and their copper(II) complexes. 35 These new compounds are highly water soluble but exhibit very low cytotoxicity, most probably because of their very low lipophilicity. We decided to extend our work and use other pharmacophoric groups for attachment at the 6-position of the TSCs pyridine ring, in order to increase the lipophilicity and modulate the antiproliferative activity. We attached the sixmembered rings methylpiperazine and morpholine as well as methylpyrrole-2-carboxylate containing a five-membered planar heterocycle. It is well-known that the attachment of a piperazine moiety on a hydrophobic scaffold has a favourable effect on its water solubility, [36][37][38][39] moreover the piperazine heterocycle is found in a broad variety of biologically active compounds, some of which are currently used in clinical therapy. [40][41][42][43][44][45][46][47][48][49] Biologically active metal-based compounds containing a piperazine ring have also been reported. [50][51][52][53][54] Morpholine is another well-known water-solubilizing unit incorporated in structures of biologically active compounds, showing often favorable pharmacologic effects. [55][56][57] In particular, a morpholine moiety is also present in the approved anticancer drugs Gefitinib (against certain breast, lung and other cancers) and Carfilzomib (against multiple myeloma). 58,59 A series of TSCs with different substituents at position 4 of the pyridine ring was tested on mice bearing sarcoma 180 ascites cells. Intriguingly, the 4-morpholino-2formylpyridine thiosemicarbazone was the most effective compound, increasing the average survival time of tumor bearing mice from 13.8 to 38 days. 60 The methylpyrrole-2-carboxylate ring was chosen as third possible option since it resembles proline.
Herein, we report the synthesis of six new organic compounds, namely HL 1− − − −6 , representing three types of potential hybrid ligands for transition metals, as well as six copper(II) complexes all shown in Chart 1. The compounds were characterized by analytical and spectroscopic methods ( 1 H and 13 C NMR, UV−vis, IR) and X-ray diffraction (1− − − −5). Solution equilibria of the copper(II) complexes formed with HL 2 and HL 4 were studied by pH-potentiometry, UV−vis and EPR spectroscopy and the thermodynamic stability data were compared to those for other related hybrid and non-hybrid systems. The antiproliferative activity of four ligands and four copper(II) complexes has been assayed. The cytotoxicity of 1−4 is markedly lower than that of the parent 2-acetylpyridine and 2formylpyridine thiosemicarbazones, but significantly higher than that of thiosemicarbazone-proline hybrids and their copper(II) complexes making them pertinent for further development as potential anticancer drugs.

Experimental
Chemicals. 2,6-Dihydroxymethylpyridine, 4-methylpiperazine, morpholine and methylpyrrole-2carboxylate were purchased from Acros Organics. 2-Hydroxymethyl-6-chloromethylpyridine and 6chloromethylpyridine-2-carboxaldehyde were synthesized according to published protocols. 61 2-(Chloromethyl)-6-(dimethoxymethyl)pyridine was prepared as described previously. 35 Solvents were dried using standard procedures, if required. 62 64 The average water ionization constant pK w , is 13.76 ± 0.01, which corresponds well to the literature data. 65 The reproducibility of the titration points included in the calculations was within 0.005 pH. The pH-metric titrations were performed in the pH range 2.0 − 11.5. The initial volume of the samples was 5.0 mL. The concentration of the ligands was 2 mM and metal ion-to-ligand ratios of 1:1, 1:1.5, 1:2 and 1:3 were used. The accepted fitting of the titration curves was always less than 0.01 mL. Samples were deoxygenated by bubbling purified argon through them for ca. 10 min prior to the measurements and argon was also passed over the solutions during the titrations.
The protonation constants of the ligands were determined with the computer program HYPERQUAD. 66 PSEQUAD 67 was utilized to establish the stoichiometry of the complexes and to calculate the stability constants (logβ(M p L q H r )). β(M p L q H r ) is defined for the general equilibrium where n is the number of reflections and p is the total number of parameters refined.

Results and Discussion
These studies examined the effects of attachment of methylpiperazine, morpholine and methylpyrrole-2-carboxylate to the pyridine ring of the parent thiosemicarbazone on the aqueous solubility, lipophilicity, ability to form copper(II) complexes, their thermodynamic stability in aqueous solution, and antiproliferative activity in human cancer cell lines HeLa, A549 and LS174, as well as in nontumorigenic cell line MRC5.

Synthesis and Characterization of HL 1 −
− − −HL 6 . The organic hybrids were synthesized in six steps, as shown in Scheme S2. The first four steps were described in detail previously. 61,76 The key aldehydes were prepared in two steps. First, 2-(chloromethyl)-6- Chemistry.        Table 2. The identical N-terminally dimethylated α-N-pyridyl thiosemicarbazone moiety of the ligands is expected to have a relatively low pK a value for the N pyridyl H + and a significantly higher value for the N hydrazinic H functional group based on the proton dissociation constants of structurally similar HCTs, such as 2-formylpyridine N 4 ,N 4 -dimethylthiosemicarbazone (PTSC, pK 1 : 3.38 and pK 2 : 10.54) or 3-aminopyridine-2-carboxaldehyde N 4 ,N 4 -dimethylthiosemicarbazone (APTSC, pK 1 : 4.31 and pK 2 : 10.29). 81 Taking into account these data we attributed the pK 2 of Morph-dm-FTSC to the deprotonation of the morpholinium ion. It should also be noted that the assignment of the pK a values for the methylpiperazine-thiosemicarbazone hybrid is not so straightforward. The proton dissociation steps of the ligands studied were assigned to the different functional groups by careful analysis of the results of the 1 H NMR titrations and are shown in Schemes 1 and S3.   Figure 6A). The E isomer was also found to be the major species in DMSO-d 6 and its molar fraction (0.62) corresponds well to that found for aqueous solution (0.61) between pH ~7 and ~9, where the neutral HL form predominates. Based on the pH-dependence of the 1 H NMR signals ( Figure S2) microscopic proton dissociation constants could be computed for both Z and E isomers ( Table 2). Concentration distribution curves were calculated based on these data providing the macroscopic constants as well (Table 2), which are in good agreement with the results of the pH-potentiometry. The first deprotonation process was accompanied by significant changes of the chemical shifts of the C 6 H pyridine ring proton and C 14,15 H 3 terminal methyl protons. The morpholine (C 8,11 H 2 , C 9,10 H 2 ) and C 7 H 2 protons were very sensitive to the second deprotonation step, as were, also, the pyridine ring protons, while the chemical shifts of protons of the thiosemicarbazone moiety (C 12 H, C 14,15 H 3 ) remain unaltered during the process. In the pH-range where the third proton dissociation occurs the signals of the last mentioned protons were shifted exclusively. These observed changes strongly support the subsequent deprotonation steps of the N 4 pyridyl H + , N 5 morpholinium H + and N 2 hydrazinic H functional groups of both isomers of Morph-dm-FTSC as indicated in Scheme 1. On the other hand, marked differences are found between the pK a values of the Z and E isomers ( Table 2). Most probably the hydrogen bond between the pyridyl nitrogen and the N 2 hydrazinic H moiety in the H 2 L + , HL forms of the Z isomer is responsible for these differences. Namely, it decreases pK 1 of the Z isomer via stabilization of the conjugate base (H 2 L + ) as well as pK 2 due to the diminished π-electron density in the pyridine ring, which results in an easier deprotonation of the N 5 morpholinium H + group. The pK 3 of the Z isomer is higher than that of the E form, since the dissociation of the N 2 hydrazinic H functional group participating in the hydrogen bonding is more difficult.   The pH-dependent 1 H NMR spectra of mPip-dm-FTSC ( Figure S3) and the changes of the chemical shifts of the various protons ( Figure S4) were analyzed similarly. Data revealed that pK 1 corresponds to the deprotonation of pyridinium nitrogen. However only the macroscopic constant could be determined by pH-potentiometry (Table 2) as the 1 H NMR signals were fairly broadened in the pH range where this process takes place and data were not appropriate for calculation. The second deprotonation step is accompanied by significant electronic shielding effects in the case of the pyridine ring protons and a large upfield shift of the C 7 H 2 protons. The signals belonging only to the C 16 H 3 methyl protons are sensitive to the third proton dissociation process. These changes strongly indicate that pK 2 and pK 3 can be assigned to the deprotonation of the N 5 piperazinium H + and N 6 piperazinium H + groups, respectively (Scheme S3). Protons of the thiosemicarbazone moiety were found to be sensitive to the last deprotonation step in which the N 2 hydrazinic H releases the proton. Comparing the microscopic constants of the E and Z isomers of the methylpiperazine-thiosemicarbazone hybrid ( Table 2) it can be concluded that the lower pK 2 (N 5 piperazinium H + ) and higher pK 4 (N 2 hydrazinic H) values of the Z isomer are due to the presence of the hydrogen bond in the H 3 L 2+ and HL forms (see the explanations in the case of Morph-dm-FTSC vide supra). At the same time the isomerization has no effect on the pK 3 value since the N 6 piperazinium H + group is quite far from the CH 12 =N 1 double bond. The E isomer was found to be predominant in the whole pH range studied ( Figure S5).

Synthesis and Characterization of Copper(II) Complexes. By reaction of
It is worth noting that the pK a values of the N pyridyl H + functional group of the studied thiosemicarbazone-based hybrids are significantly lower compared to those of ligands PTSC,  Table 3.
The results indicate a slightly higher lipophilicity of the terminally dimethylated derivatives (HL 2 and HL 4 ) compared to that of the corresponding non-methylated ligands (HL 1 and According to the pK a values of the ligands studied (Table 2) Table   4. EPR spectra were recorded at various pH values at 1:1 and 1:2 metal-to-ligand ratios at room temperature and at 77 K; the fitted experimental and simulated isotropic spectra are depicted in Figures 8A,B and S7A (Table 4) taking into account the protonation of the ligands at these pH values, which were kept constant during subsequent data evaluation.  In the case of Morph-dm-FTSC, [CuL] + predominates between pH ~4 and ~10. This is clearly indicated by the unaltered UV-vis spectra in the wavelength range of both the d-d ( Figure 9) and CT ( Figure S10B) bands. EPR spectra were also intact in this particular pH range ( Figure   S7A). Based on the EPR parameters of [CuL] + ( Table 5, (Table 5). On the other hand the deprotonation of [CuL] + observed at pH > ~10, is accompanied by only minor changes of the UV-vis spectra (see changes at ~256 nm in Figure S10B). However, the decreasing ligand field (lower A 0 ) supports the was also calculated on the basis of the minor changes of the d-d bands of the UV-vis spectra and the pH-dependent EPR spectra ( Figure 8A). The data obtained by the three different methods are in good agreement (  (Figure 2), while the ligand field is slightly increased (somewhat higher A 0 ) due to the deprotonation of [CuLH] 2+ . These results strongly indicate that the process is assigned to the deprotonation of the N 6 of the methylpiperazine moiety which is not involved in the binding to copper(II). The observed UV-vis spectral changes ( Figure S10A) and EPR parameters (  Formation of merely mono-ligand copper(II) complexes for HL 2 and HL 4 was expected.
However at ligand excess (c L /c Cu > 2) bis-ligand complexes were detected mainly in the basic pH range. Formation of the neutral bis-ligand complexes [CuL 2 ] resulted in precipitation which hindered the accurate determination of their stability constants by pH-potentiometry and UV-vis spectrophotometry, although these were estimated by the EPR measurements (Table 4). The EPR data for this kind of complexes represent quite high g 0 and low A 0 values ( It is worth noting that the isotropic g and A values calculated by averaging the anisotropic values (g 0,calc and A 0,calc in Table 5) are in relatively good agreement with the corresponding values measured in solution, indicating that the coordination modes adopted by the ligands in solution are preserved upon freezing.
Representative concentration distribution curves were calculated by using the overall stability constants (average values obtained by the 3 methods) for the the copper(II) -mPip-dm-FTSC (HL 2 ) and copper(II) -Morph-dm-FTSC (HL 4 ) systems at 1:1 metal-to-ligand ratio to represent the complex formation processes in the pH range studied (Figure 10). It can be concluded that complexes [CuL] + predominate at physiological pH even at submicromolar concentrations, although 6% of the complex is protonated in the case of mPip-dm-FTSC.
In order to compare the copper(II) binding ability of mPip-dm-FTSC (HL 2 ) and Morph-dm-  Table   3 in order to characterize the hydro-lipophilic character of these species. Comparing these values to those of the metal-free ligands it can be concluded that the same lipophilicity trend is obtained. Namely, the terminal dimethylation results in somewhat increased values and complexes of the morpholine-thiosemicarbazone derivatives possess enhanced lipophilic character. Note that the copper(II) complexes are much more hydrophilic than the corresponding ligands since the positively charged [CuL] + species predominate at physiological pH. Complex 5 is much more lipophilic than the other complexes, although its log D 7.4 value cannot be determined exactly and compared to that of mPyrr-FTSC (HL 5 ).    83 The favorable effect of N 4 -dimethylation is also well-documented for other related α-heterocyclic thiosemicarbazones. 31 The coordination of FTSC to copper(II) was reported to increase or decrease the activity depending on the cell type. [85][86][87][88] In particular, [Cu(FTSC)Cl 2 ] revealed an increase of cytotoxicity by a factor of 3 in SW480 cells when compared to that of FTSC, while against HL60 cells the activity of FTSC and the copper(II) complex was very similar. 84 The proline-FTSC hybrids, we synthesized previously, 80  Comparison of IC 50 values for 3 and 4 indicates that terminal N 4 -dimethylation enhances the cytotoxicity in accord with the general trend observed in the literature. 31,83 In contrast, the effect is opposite, although also cell type dependent, if the activity of compounds 1 and 2 is compared. The observed divergent effects of terminal N 4 -dimethylation suggest that structural modifications at the pyridine ring (coupling to piperazine and morpholine moieties which increases the denticity of the ligands) play an important role in structure-activity relationships.

Conclusions
The index. Further experimental work to get insight into the mechanism of action of the prepared copper(II) complexes with hybrid ligands is required to ascertain whether they are really good candidates for further development as potential anticancer drugs.