Copper Thiosemicarbazone Complexes

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The anti-tumour potential of quinonoidal compounds is known since 1974 and several 1,2-napthaquinone derivatives behave as anti-cancer compounds.26 28 Recently, the synthesis and structural characterization of metal conjugates of 1,2-naphthaquinone (NQTS, Scheme 12.2) along with their anti-tumour activities against the MCF7 breast cancer cell line have been studied.29 Thiosemi-carbazonates have been intensively studied due to their inhibitory action on the DNA enzyme ribonucleotide diphosphate reductase and their selectivity towards hormone-responsive cancers.30 All synthesized compounds were evaluated for their anti-proliferative activities against the human breast cancer cell line MCF7 and their IC50 values (mM) determined. From the results obtained it is clear that the addition of the TSC-pharmacophore to the parent quinonoidal moiety considerably enhances the anti-proliferative activity of the quinone, and the Cu(II) compound has a low IC50 value (4.02 mM). This cytotoxic action could be attributed to its topoisomerase II inhibitory activity.

NQTS Scheme 12.2

A new salicylaldehyde thiosemicarbazone (H2-STSC, Scheme 12.3) has been synthesized as the main hydrolysis product of the parent compound helicin-thiosemicarbazone (H-TSC).31 The new H2STSC ligand, which bears a chain with the nucleophilic S, N centres and has anti-tubercolostatic activity, was characterized by IR and NMR spectroscopies and X-ray diffractometry, and its biological activity tested, in comparison with H-TSC. Although complexation assays of H2-STSC with several metals were performed, only the reaction of this ligand with CuSO4 was successful, leading to the solid complex [Cu(H-STSC)(OH2)]2SO4 • 2DMSO • 6H2O (I).

H2-STSC = salicyladehide thiosemicarbazone Scheme 12.3

The complex I has been tested in vitro on human leukaemic U937 cell lines, focusing the experiments on the activity with respect to cell proliferation inhibition and apoptosis induction. The results indicate that the complex inhibited about 40% of the cell proliferation at 0.3 and 0.5 mg/ml, but DNA fragmentation and apoptosis were not observed at these concentrations (Figure 12.2a).

A possible explanation of the unusual biological behaviour of the complex I can be found in the copper coordination geometry: the metal centre is substantially square planar, with the Cu atom displaced only 0.07 A from the coordination plane and an angle between the chelation planes of 7.6°. Instead, the

tf t

Figure 12.2 (a) Apoptosis assays on complex i. Lane c: positive control; lane 1: untreated control cells; lanes 2 and 3: H-TSC (20 and 30 mg/ml, respectively); lanes 4 and 5: complex i (0.3 and 0.5 mg/ml, respectively). (b) Lane l ladder; lane 1: cycloeximide; lane 2: untreated control cells; lane 3: DMSO; lane 4: [Cu(H3ut)Cl2] • 2H2O; lane 5: [Cu(H2ut)(OH2)Cl] • 2H2O; lane 6: [Cu(H2L)(OH2)Cl]Cl monomeric copper complexes [Cu(H2L)(OH2)Cl]Cl (II; H2L = pyridoxal-thiosemicarbazone) and [Cu(H2ut)(OH2)Cl] • 2H2O (III; H3ut = 5-formylura-cil-thiosemicarbazone), previously synthesized and able to induce apoptosis (Figure 12.2b), presented a different geometry.32 These and other compounds characterized in the same research inhibited cell proliferation but only compounds II and III caused DNA fragmentation when used at 40 mg/ml, inducing apoptosis.

There is a common fact that the two complexes II and III, active in inducing apoptosis, share: the copper coordination geometry. While in the complex I, as explained above, the metal centre is substantially square planar with the copper atom displaced 0.07 A from the coordination plane, complexes II and III showed the Cu atom displaced by 0.24 and 0.25 A, respectively, from the coordination plane, with interplanar angles of 10.2° and 11.1°, respectively. These important structural features are postulated as responsible of the apoptotic properties of the last two mentioned complexes.

Carboxamidrazones (a) (Scheme 12.4) bear structural similarities to heterocyclic thiosemicarbazones (b), and their copper conjugates show remarkable anti-tumour activities towards the hormone-responsive cancers. Thus, copper complexes of N'-(2-benzyloxy-benzylidiene) pyridine-2-carboxamidrazone (pcm) have shown to be highly effective against the human breast cancer cell line MCF7.33 The corresponding Cu(II)-pcm complex (1) was prepared as a green solid and suitable X-ray quality crystals were obtained.

Scheme 12.4

The ligand and the copper complex were tested for their anti-tumour activity against the human breast cancer cell line MCF7. While the cells treated with pcm were viable, those treated with complex (1) showed cell death at comparatively low concentrations inducing a direct cytotoxic action. The IC50 value for the copper complex is 3 mM, which is four times less than that of the ligand. Compared with earlier data and with those of analogous thiosemicarbazones,30

the present copper compound is found to exhibit higher anti-tumour activity which may be due to the generation of cytotoxic Cu(I) species through intracel-lular enzymatic reduction.34,35 Alternatively, the copper complexes have been shown to undergo affinity binding with estrogen receptors in the case of breast cancer cells producing anti-proliferative effects observed in the present case.36

Following research on the anti-cancer properties of carboxamidrazone derivatives, the biological action of two copper conjugates of the new ligands appc and atpc (Scheme 12.4) [Cu(appc)Cl2] (c) and [Cu(atpc)Cl2] (d), against the hormone-responsive cancer cell line, mouse melanoma B16F10, was studied.37 The complex is monomeric with appc molecule acting as a planar tridentate ligand. During metal complexation, the ligand utilizes two of its pyridyl nitrogens which are at the apex of the bipyramid and one diazine nitrogen which is part of the trigonal plane. Two unidentate chloride ions occupy the remaining positions of the trigonal plane in cis configuration.

Figure 12.3 shows the bar diagram of the anti-proliferative activities of copper complexes against the B16F10 cell line. The incubation of the cells with the parent ligands for 48 h is found to result in a decrease in the metabolic activity of these cells but not in a complete stop of the cell proliferation even at the highest dose employed (10 mM). The copper complexes on the other hand show a dose-dependent inhibition of the cell proliferation in the same concentration range indicating higher anti-proliferative activities. Complex c was found to be more potent (IC50 = 6.8 mM) than complex d (IC50 = 10 mM), suggesting that substitution on the N1 nitrogen of pyridyl-2-carbox-midrazone is only effective when the heteroatom is a nitrogen in a six-member aryl ring. For thiosemicarbazone ligands and their metal complexes, the presence of a thiocarbonyl sulphur in the side chain is known to increase the cytotoxicity.38

70 60

70 60

Figure 12.3 Inhibitory effect of complexes c and d on cell line B16F10 (y-axis: inhibition of cell proliferation, ICP)

Figure 12.3 Inhibitory effect of complexes c and d on cell line B16F10 (y-axis: inhibition of cell proliferation, ICP)

The results, however, clearly indicate that the heterocyclic sulphur in the carboxamidrazone side chain offers no advantage in terms of the anti-proliferative activity against B16F10 mouse melanomal cells. Thus, the five-coordinate copper complexes are consistently found to exhibit enhanced anti-proliferative activities against B16F10 mouse melanoma cells. On the other hand, copper conjugation to the carboxamidrazone ligands may facilitate their intracellular transportation resulting in their estrogen receptor blocking action as previously suggested.36

Other interesting thiosemicarbazone derivatives have been studied in a search for new anti-tumour active substances. Thus, copper complexes of glucosone-bTSC (Scheme 12.5) were isolated and their anti-tumour activity studied.39 The complexes appeared as crystalline substances of red colour, soluble in DMSO, DMF and some other organic solvents.

It was established that the synthesized copper complexes Ib-Id, possess low acute toxicities on white mongrel mice (LD100 >2000mg/kg), while complexes If and Ig are characterized by pronounced toxicity (LD100 = 80 and 100 mg/kg, respectively).

According to the chemotherapy tests developed on mice and rats, bTSC do not offer any protection against Walker's carcinosarcoma (WCS) and sarcoma 45. However, complexes Ib and Ic, in contrast to their ligands, exhibited somewhat more pronounced anti-tumour properties under the same conditions of testing with both sarcoma 45 {TGI (Tumour Growth Inhibition) = 54 and 53%, respectively, p < 0.05} and WCS (TGI = 47 and 50%, respectively, p < 0.05). Compound Ib retained the effect when tested on sarcoma 180 (TGI = 55%, p <0.05). Complex Ig inhibited WCS growth (TGI = 34%, p = 0.05).

A comparison of the results of these experiments to those for a bromobenzyl analogue tested on the same strains shows that the substitution of bromine for chlorine does not change the therapeutic effect of the ligand, but significantly decreases the anti-tumour effect of the complex, in particular for sarcoma 45.

I: R = C3H7 (a), /-C3H7 (b), C4Hg (c), CH3 (d), C^ (e, g), C4Hg (f);

R' = CH3 (a-e), (CHOH)3CH2OH (f, g); X=H (a-c, f), NO2 (d), Cl (e, g)

I: R = C3H7 (a), /-C3H7 (b), C4Hg (c), CH3 (d), C^ (e, g), C4Hg (f);

R' = CH3 (a-e), (CHOH)3CH2OH (f, g); X=H (a-c, f), NO2 (d), Cl (e, g)

Scheme 12.5

Thiosemicarbazones of heterocyclic aldehydes possess anti-tumour properties and can inhibit DNA synthesis.40 In the search for new anti-tumour drugs of this type, thiosemicarbazones of cyclodimers of 2-alkoxyacroleins, such as 2-formyl-2,5-dimethoxy-2,3-dihydro-4H-pyran thiosemicarbazones (Ligand 12.1), and its copper complex (Cu-I) were synthesized. The complex formation reaction was accompanied by the reduction of Cu(II) to Cu(I) under the action of the initial thiosemicarbazone. The acute toxicity (LD50 = 62.5mg/kg, ip) of Cu-I was determined on intact SHK mice. The DNA-inhibiting activity of the copper complex was studied in vivo on C3Ha mice with rapidly growing hepatoma 22I. In addition, its anti-tumour activity was studied in vitro on a CV-1 grivet kidney cell culture, using 6-mercaptopurine as the reference compound. In the in vivo tests, Cu-I (40 mg/kg) was introduced into mice and allowed to act for 3, 6, 9 or 24 h before analysis. For evaluating the selectivity of drug action, the DNA synthesis inhibition was determined both in tumour cells and in organ tissues (spleen and large intestine). The copper complex inhibited the inclusion of 14C-thymidine into tumour cells by 90 and 74% after 9 and 24 h, respectively; at the same time, DNA synthesis in the splenic and intestinal cells did not exceed 47% for a 24 h exposure. Therefore, the presence of copper accounts for an increase in both efficacy and selectivity of the anti-tumour action, through an increase of the number of single-strand scissions of DNA chains and decreased DNA-protein interactions in cell nuclei.

The results of the anti-tumour tests with compound Cu-I in a dose of 20-25 mg/kg indicate suppression of the growth of both RShM-5 and LLC tumours by 99% without any loss of the experimental animals. The pronounced anti-tumour activity of Cu-I was observed not only for intraperitoneal injection. Moreover, a significant effect was retained upon peroral administration of the drug: treatment with a 35 mg/kg dose of the complex inhibited the growth of RShM-5 by 81%.

Initial interest in 2-formylpyridine thiosemicarbazone (HL) and its substituted derivatives arose from their marked anti-tumour properties.41 More recently it has been shown that a dimer Cu(II) complex of HL is also active.42 In view of the interest in these substances, three modified ligands based on the HL type, 6-methyl-2-formylpyridine thiosemicarbazone (6HL), 2-formylpyri-dine-2'-methyl thiosemicarbazone (2'L) and (2-formylpyridine-40-methyl thiosemicarbazone) (4'HL) have been obtained (Scheme 12.6) and a selection of their Cu(II) complexes have been synthesized with N-donor ligands [2,2'-bipyr-idyl (bipy) and 4-dimethylaminopyridine (dmap)], as well as an S-donor ligand [pentafluorothiophenolate (pftp)] and O-donor ligands [paratrityl-phenolate

Ligand 12.1

Ligand 12.1

(ptp) and the monohydrogen phosphate ion (HPO42~)]. The complexes have been characterized by physical and spectroscopic techniques.43

Cytotoxicity testing (IC50) for selected compounds was carried out on the cell line HCT-8, a human colon adenocarcinoma as previously described, cells of which are generally resistant to intercalating drugs (relative to other cell lines).44 Results evidence that CuL+ is less active (IC50 = 14 mM) than the free ligand HL (IC50 = 4.7 mM). As ethylenediaminetetraacetic acid (EDTA) can quantitatively remove the Cu(II) from CuL+, the cytotoxicities of both HL and CuL+ were re-evaluated with EDTA present to ensure the activity of HL was not due also to the formation of cytotoxic metal chelates in the culture medium. The IC50 value of 3.8 mM for HL + EDTA is not significantly changed from the average value of 4.7 mM for HL; thus, the activity is due to the uncomplexed HL and not to metal complexes formed in the assay medium by accessible metal ions and HL or EDTA. The addition of EDTA to CuL+ has, however, increased the cytotoxicity when compared with CuL+ alone (IC50 values 7.6 and 14 mM, respectively), again showing the uncomplexed ligand to be more active than its Cu(II) complex. In contrast, but using WCS, the cytotoxicity of H2KTS (Figure 12.1) has been shown to be due to the in vivo formation of the complex CuKTS, as H2KTS has only no activity in animals maintained on a copper-deficient diet.45

The ligand 2'L and its complex Cu (2'L)2+ are also both inactive. As published, in vitro and in vivo activity for 2'L was lost compared to HL.46 The addition of the 2'-methyl group effectively traps the ligand in the thione form, rendering impossible the overall conjugation. The loss of activity for 2'L may therefore be due to the ligand being unable to have this delocalized form and/or its inability to coordinate as an anionic moiety.

HL R,=R2 = R3 = H

Scheme 12.6

The ligand 6-methyl-2-formylpyridine thiosemicarbazone, 6HL, and its complex, Cu (6L)+, are both inactive (IC50 > 20 mM). The general decrease in activities for the 6-methylated ligands was thought to be a steric effect due to the interference with the ring N chelation and partly electronic due to changes in the conjugated ligand system.

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