Synthesis, characterization, and biological activity of platinum II, III, and IV pivaloamidine complexes.

Imino ligands have proven to be able to activate the trans geometry of platinum(II) complexes towards antitumor activity. These ligands, like aromatic N-donor heterocycles, have a planar shape but, different from the latter, have still an H atom on the coordinating nitrogen which can be involved in H-bond formation. Three classes of imino ligands have been extensively investigated: iminoethers (HN=C(R)OR'), ketimines (HN=CRR'), and amidines (HN=C(R)NR'R″). The promising efficacy of the platinum compounds with amidines (activity comparable to that of cisplatin for cis complexes and much greater than that of transplatin for trans complexes) prompted us to extend the investigation to amidine complexes with a bulkier organic residue (R = t-Bu). The tert-butyl group can confer greater affinity for lipophilic environments, thus potentiating the cellular uptake of the compound. In the present study we describe the synthesis and characterization of pivaloamidine complexes of platinum(II), (cis and trans-[PtCl2(NH3){Z-HN=C(t-Bu)NH2}] and cis and trans-[PtCl2{Z-HN=C(t-Bu)NH2}2]), platinum(III) ([Pt2Cl4{HN=C(t-Bu)NH}2(NH3)2]), and platinum(IV) (trans-[PtCl4(NH3){Z-HN=C(t-Bu)NH2}] and trans-[PtCl4{Z-HN=C(t-Bu)NH2}2]). The cytotoxicity of all new Pt complexes was tested toward a panel of cultured cancer cell lines, including cisplatin and multidrug resistant variants. In addition, cellular uptake and DNA binding, perturbations of cell cycle progression, induction of apoptosis, and p53 activation were investigated for the most promising compound trans-[PtCl2(NH3){Z-HN=C(t-Bu)NH2}]. Remarkably, the latter complex was able to overcome both acquired and intrinsic cisplatin resistance.

Monofunctional platinum(II) complexes with potent tumor cell growth inhibitory activity: the effect of a hydrogen-bond donor/acceptor N-heterocyclic ligand.

In this paper we investigate the possibility of further increase the role of the N-donor aromatic base in antitumor Hollis-type compounds by conferring the possibility to act as a hydrogen-bond donor/acceptor. Therefore, we synthesized the Pt(II) complex cis-[PtCl(NH3 )2 (naph)]NO3 (1) containing the 1,8-naphthyridine (naph) ligand. The naphthyridine ligand is generally monodentate, and the second nitrogen atom can act as H-bond donor/acceptor depending upon its protonation state. The possibility of forming such an H-bond could be crucial in the interaction of the drug with DNA or proteins. Apart from the synthesis of the compound, in this study we evaluated its in vitro antitumor activity in a wide panel of tumor cell lines, also including cells selected for their sensitivity/resistance to oxaliplatin, which was compared with that of previously reported complex 2 ([PtI(2,9-dimethyl-1,10-phenanthroline)(1-methyl-cytosine)]I) and oxaliplatin and cisplatin as reference compounds. The cytotoxicity data were correlated with the cellular uptake and the DNA platination levels. Finally, the reactivity of 1 towards guanosine 5'-monophosphate (5'-GMP) and glutathione was investigated to provide insights into its mechanism of action.

Effects of redox modulation by inhibition of thioredoxin reductase on radiosensitivity and gene expression.

The thioredoxin system is a promising target when aiming to overcome the problem of clinical radiation resistance. Altered cellular redox status and redox sensitive thiols contributing to induction of resistance strongly connect the ubiquitous redox enzyme thioredoxin reductase (TrxR) to the cellular response to ionizing radiation. To further investigate possible strategies in combating clinical radiation resistance, human radio-resistant lung cancer cells were subjected to a combination of single fractions of γ-radiation at clinically relevant doses and non-toxic levels of a well-characterized thioredoxin reductase inhibitor, the phosphine gold(I) compound [Au(SCN)(PEt(3))]. The combination of the TrxR-inhibitor and ionizing radiation reduced the surviving fractions and impaired the ability of the U1810 cells to repopulate by approximately 50%. In addition, inhibition of thioredoxin reductase caused changes in the cell cycle distribution, suggesting a disturbance of the mitotic process. Global gene expression analysis also revealed clustered genetic expression changes connected to several major cellular pathways such as cell cycle, cellular response to stress and DNA damage. Specific TrxR-inhibition as a factor behind the achieved results was confirmed by correlation of gene expression patterns between gold and siRNA treatment. These results clearly demonstrate TrxR as an important factor conferring resistance to irradiation and the use of [Au(SCN)(PEt(3))] as a promising radiosensitizing agent.

Photoreactivity of 5-fluorouracil under UVB light: photolysis and cytotoxicity studies.

The photodegradation of the chemotherapeutic agent 5-fluorouracil (5-FU) under UVB light was studied both in aqueous and methanol solutions and in systemic and topical formulations. As monitored by HPLC, photodegradation in solution takes place in a concentration dependent manner; thus, the solution for parenteral administration (10(-1) M) showed negligible loss of the active principle. On the contrary, the commercial cream containing 5% of 5-FU showed low stability under UVB exposure. When dissolved either in water or methanol, 5-FU yields two photoproducts which have been characterized as two isomers coming from the addition of the solvent to the 5,6 double bond of the drug. As a consequence, photomodified 5-FU loses its antiproliferative activity on HCT-15 and HeLa cells. MS analysis showed that photoaddition occurred with nucleophilic amino acids, such as cysteine and serine, while susceptible amino acids (cysteine and methionine) were oxidized. In fact, high production of the superoxide anion under UVB light as well as photooxidation of BSA suggests protein photodamage as a mechanism of photosensitization. Indeed, some phototoxicity was shown in experiments on NCTC keratinocytes and MCF-7 resistant cells irradiated with UVB light. The interactions with these biological targets may contribute to skin phototoxicity and photoallergy induced by 5-FU in vivo.

DNA binding ellipticine analogues: synthesis, biological evaluation, and structure-activity relationships.

In connection with our interest in the synthesis and study of the biological properties of ellipticine analogues as anticancer agents, some 7H-pyrido[2,3-c]carbazoles (7H-PyC) and their corresponding tetrahydro derivatives (7H-THPyC) were synthesized. A common multistep pathway characterized by conventional reactions involving carbazole Fischer and Conrad-Limpach quinoline syntheses yielded the tetracyclic compounds. With the aim to improve the cytotoxic activity of the new 7H-PyC derivatives, we provided them with one or two diethylaminoethyl side chains. The new THPyCs, PyCs, and smaller pyrroloquinoline (PQ) derivatives were tested for their in vitro cytotoxic properties against several human tumor cell lines. Most of the compounds tested showed considerable cytotoxic activity, particularly compound 24, which, with two basic alkylamino chains, has IC(50) values in the sub-micromolar range, about one order of magnitude lower than those of the reference compound, ellipticine. Chemosensitivity tests on cisplatin-, mitoxantrone-, and multidrug-resistant (MDR) phenotypes indicated that compound 24 shows no cross-resistance; this suggests that, besides not being a potential MDR substrate, it might act as a mixed inhibitor of topoisomerases I and II. Flow cytometric and caspase-3 activation analyses revealed that 24 induces a caspase-3-dependent apoptotic cell-death mechanism. Linear dichroism and unwinding experiments suggested that the most active compounds act as DNA intercalators. For compound 24, an inhibitory concentration-dependent effect on topoisomerases II and I was demonstrated. Herein we discuss interesting structure-activity relationships with respect to molecular size and planarity, as well as the substitution and position of one side chain on the PyC nucleus, in comparison with corresponding smaller PQs.

Synthesis and antitumor activity of novel amsacrine analogs: the critical role of the acridine moiety in determining their biological activity.

A new series of N-[4-(2'-oxo-2H-pyrano[2,3-b]quinolin-5'-ylamino)-phenyl]-methanesulfonamides was prepared and analyzed as novel amsacrine-like derivatives. Our preliminary biological evaluation has shown that the replacement of the acridine moiety with the analogous 2-oxo-2H-pyrano[2,3-b]quinoline system drastically reduced both their anticancer activity and their propency to intercalate into double stranded DNA.

Synthesis, characterization and cytotoxic activity of substituted benzyl iminoether Pt(II) complexes of the type cis- and trans-[PtCl2{E-N(H)=C(OMe)CH2-C6H4-p-R}2] (R=Me, OMe, F). X-ray structure of trans-[PtCl2{E-N(H)=C(OMe)CH2-C6H4-p-F}2].

New substituted benzyl iminoether derivatives of the type cis- and trans-[PtCl(2){E-N(H)C(OMe)CH(2)-C(6)H(4)-p-R}(2)] (R=Me (1a, 2a), OMe (3a, 4a), F (5a, 6a)) have been synthesized and characterized by elemental analyses, FT-IR spectroscopy and NMR techniques. The iminoether ligands are in the E configuration, which is stable in solution and in the solid state, as confirmed by the (1)H NMR data. Complex trans-[PtCl(2){E-N(H)C(OMe)CH(2)-C(6)H(4)-p-F}(2)] (6a) was also characterized by an X-ray diffraction study. Complexes 1a-6a have been tested against a panel of human tumor cell lines in order to evaluate their cytotoxic activity. cis-Isomers were significant more potent than the corresponding trans-isomers against all tumor cell lines tested; moreover, complexes 1a and 5a showed IC(50) values from about 2-fold to 6-fold lower than those exhibited by cisplatin, used as reference platinum anticancer drug.

Cisplatinum and transplatinum complexes with benzyliminoether ligands; synthesis, characterization, structure-activity relationships, and in vitro and in vivo antitumor efficacy.

New benzyliminoether derivatives [PtCl2{N(H)=C(OMe)CH2Ph}2] of cis (1a, 1b) and trans (2a, 2b) geometry were prepared and characterized by means of elemental analysis, multinuclear NMR and FT-IR techniques, and X-ray crystallography; this latter was carried out for 1b. The cytotoxic properties of these new platinum(II) complexes were evaluated in terms of cell growth inhibition against a panel of different types of human cancer cell lines. cis-[PtCl2{E-N(H)=C(OMe)CH2Ph}2] (1a) was significantly more potent than cisplatin against all tumor cell lines tested, showing IC50 values from about 2- to 17-fold lower than the reference compound. Chemosensitivity tests performed on cisplatin-sensitive and -resistant cell lines have demonstrated that complex 1a is able to overcome cisplatin resistance. Analyzing the mechanism by which complex 1a led to cell death, we have found that it induced apoptosis in a dose-dependent manner, accompanied by the activation of caspase-3. The in vivo studies carried out using two transplantable tumor models (L1210 leukemia and Lewis lung carcinoma) showed that derivative 1a induced a remarkable antitumor activity in both tumor models, as measured by prolonged survival and reduced tumor mass compared to control groups.

Inhibition of thioredoxin reductase by auranofin induces apoptosis in cisplatin-resistant human ovarian cancer cells.

Cisplatin is an effective antitumor agent for the treatment of several carcinomas. However, the development of resistance to cisplatin represents a serious clinical problem. The effects of auranofin, a gold(I) compound clinically used as an antirheumatic agent, on cisplatin-sensitive (2008) and-resistant (C13*) cancer cells were studied. Auranofin is more effective than cisplatin in decreasing cell viability and its action is particularly marked in C13* cells, indicating that no cross-resistance occurs. Furthermore, auranofin is able to permeate C13* cells more efficiently than 2008 cells. Treatment with auranofin determines a consistent release of cytochrome c in both cell lines, while cisplatin is effective only in sensitive cells. Both auranofin and cisplatin induce apoptosis in 2008 cells, while in C13* cells only auranofin is effective. Apoptosis is accompanied by an increased production of hydrogen peroxide that, however, is inhibited by N-acetyl-L-cysteine. In resistant cells, H(2)O(2) production is counteracted by a large overexpression of thioredoxin reductase that constitutes the preferred target of the inhibitory action of auranofin. This specific effect of auranofin might rationalize its ability in overcoming cisplatin resistance in human ovarian cancer cells.

Gold(III) dithiocarbamate derivatives for the treatment of cancer: solution chemistry, DNA binding, and hemolytic properties.

Gold(III) compounds are emerging as a new class of metal complexes with outstanding cytotoxic properties and are presently being evaluated as potential antitumor agents. We report here on the solution and electrochemical properties, and the biological behavior of some gold(III) dithiocarbamate derivatives which have been recently proved to be one to 4 orders of magnitude more cytotoxic in vitro than the reference drug (cisplatin) and to be able to overcome to a large extent both intrinsic and acquired resistance to cisplatin itself. Their solution properties have been monitored in order to study their stability under physiological conditions; remarkably, they have shown to undergo complete hydrolysis within 1 h, the metal center remaining in the +3 oxidation state. Their DNA binding properties and ability in hemolyzing red blood cells have been also evaluated. These gold(III) complexes show high reactivity toward some biologically important isolated macromolecules, resulting in a dramatic inhibition of both DNA and RNA synthesis and inducing DNA lesions with a faster kinetics than cisplatin. Nevertheless, they also induce a strong and fast hemolytic effect (compared to cisplatin), suggesting that intracellular DNA might not represent their primary or exclusive biological target.

New water soluble pyrroloquinoline derivatives as new potential anticancer agents.

A new class of water soluble 3H-pyrrolo[3,2-f]quinoline derivatives has been synthesized and investigated as potential anticancer drugs. Water solubility profiles have been used to select the most promising derivatives. The novel compound 10, having two (2-diethylamino-ethyl) side chains linked through positions 3N and 9O, presents a suitable water solubility profile, and it was shown to exhibit cell growth inhibitory properties when tested against the in-house panel of cell lines, in particular those obtained from melanoma.

Gold dithiocarbamate derivatives as potential antineoplastic agents: design, spectroscopic properties, and in vitro antitumor activity.

At present, cisplatin (cis-diamminodichloroplatinum(II)) is one of the most largely employed anticancer drugs as it is effective in the treatment of 70-90% of testicular and, in combination with other drugs, of ovarian, small cell lung, bladder, brain, and breast tumors. Anyway, despite its high effectiveness, it exhibits some clinical problems related to its use in the curative therapy, such as a severe normal tissue toxicity (in particular, nephrotoxicity) and the frequent occurrence of initial and acquired resistance to the treatment. To obtain compounds with superior chemotherapeutic index in terms of increased bioavailability, higher cytotoxicity, and lower side effects than cisplatin, we report here on some gold(I) and gold(III) complexes with dithiocarbamate ligands (DMDT = N,N-dimethyldithiocarbamate; DMDTM = S-methyl-N,N-dimethyldithiocarbamate; ESDT = ethylsarcosinedithiocarbamate), which have been synthesized, purified, and characterized by means of elemental analyses, conductivity measurements, mono- and bidimensional NMR, FT-IR, and UV-vis spectroscopy, and thermal analyses. Moreover, the electrochemical properties of the designed compounds have been studied through cyclic voltammetry. All the synthesized gold complexes have been tested for their in vitro cytotoxic activity. Remarkably, most of them, in particular gold(III) derivatives of N,N-dimethyldithiocarbamate and ethylsarcosinedithiocarbamate, have been proved to be much more cytotoxic in vitro than cisplatin, with IC50 values about 1- to 4-fold lower than that of the reference drug, even toward human tumor cell lines intrinsically resistant to cisplatin itself. Moreover, they appeared to be much more cytotoxic also on the cisplatin-resistant cell lines, with activity levels comparable to those on the corresponding cisplatin-sensitive cell lines, ruling out the occurrence of cross-resistance phenomena and supporting the hypothesis of a different antitumor activity mechanism of action.

Erythrocyte aminolevulinic acid dehydratase inhibition by cis-platin.

The effect of cis-platin on erythrocyte aminolevulinic acid dehydratase (ALAD) activity was studied in vivo and in vitro. Young male Wistar rats were treated with a single i.p. injection of cis-platin at 2.5, 5.0, and 10.0mg/kg dose. In addition, a single i.p. injection of lead nitrate (1.0mg/kg dose) was administered as positive control. Experiments in vitro were also performed to elucidate the possible mechanism of action. The aminolevulinic acid dehydratase was almost completely inhibited in vitro from 0.5mM concentration, and the IC(50) was stated at 0.265 mM, 20 times higher than lead (IC(50) stated at 0.013 mM). Reduced glutathione, partially but significantly, reactivated in vitro the enzyme treated with cis-platin (0.5 and 5.0mM), whereas zinc showed a positive, significant effect with the higher dose (5.0mM) only. On the contrary, inhibition caused by lead (0.005 mM) was partially, but significantly restored by reduced glutathione, and, almost completely, by zinc. The experiments in vivo show that cis-platin causes a dose- and time-dependent inhibition of ALAD activity with 5.0 and 10.0mg/kg dose, until 66 and 33% of the control activity 96 h after treatment, respectively. The results show that erythrocyte ALAD is sensitive to cis-platin and suggest that the mechanism of enzyme inhibition is a direct interaction with sulfhydryl groups, whereas zinc site appears involved with the higher doses only. This mechanism appears different from lead that prevalently inhibits ALAD removing zinc from the enzyme, other than interacting with sulfhydryl groups.

Synthesis of a palladium(II)-dithiocarbamate complex: biological assay and nephrotoxicity in rats.

A new palladium(II)-dithiocarbamate complex, [Pd(ESDT)Cl](n), was synthesised and its chemical characteristics are discussed. This complex was examined for its cytotoxic properties in human tumour cell lines; for comparison, the cytotoxicity of cisplatin was evaluated under the same experimental conditions. In particular, Pd(II)-complex cytotoxicity on ovarian carcinoma C13 cells, resistant to cisplatin, showed that there seemed to be no cross-resistance between [Pd(ESDT)Cl](n) and cisplatin. The effects on the kidney were also studied. Biochemical investigation on urinary parameters showed that the effects after a single injection are similar to those of cisplatin, with an increase of urinary proteins and enzyme excretion in urine, and a significant decrease of glutamine synthetase activity in the renal tissue. In addition, the Pd(II)-complex caused a significant decrease of p-aminohippuric acid uptake in renal cortical slices relative to cisplatin. On the other hand, histopathological findings showed that the effects of the Pd(II)-complex are more severe and diffuse than the damage caused by cisplatin. Biochemical and histopathological findings show that the Pd(II)-complex affects the pars recta and pars convoluta, in contrast to cisplatin, which only affects the pars recta.

Photobiological properties of hydroxy-substituted flavothiones.

Flavothione (FT) and a series of 18 hydroxy- and methoxy-substituted flavothiones were screened for photobiological activity. The 5-hydroxy-substituted compounds (group 3) and the methoxy-substituted flavothiones were inactive. FT and the remaining hydroxy-substituted compounds, all displayed photobiological activity. Among these, the 3-hydroxy-substituted compounds (group 2) were the most efficient photosensitizers overall in spite of their concurrent fast photodegradation. FT and all other hydroxyflavothiones, not substituted in the 3- or 5-positions (group 1), were inefficient compared with group 2. Detailed photobiological tests were carried out for four flavothiones of groups 1 and 2. The biological tests included fungi, several strains of Escherichia coli, Salmonella typhimurium and mammalian cells. In addition, the ability of these flavothiones to perform lipid peroxidation was evaluated. FT and 6-hydroxyflavothione (group 1) induce DNA damage via H-atom abstraction from the lowest n, pi* triplet state of the thione (oxygen independent). For 3-hydroxy and 3,6-dihydroxyflavothione (group 2), both DNA and the membrane are targets. The mechanism likely involves both energy transfer and electron transfer from the lowest pi, pi* triplet state to oxygen, to form singlet oxygen and the superoxide anion. Some of these compounds could be considered as models for environmentally safe photopesticides.

Synthesis and biological evaluation of a new furo[2,3-h]quinolin-2(1H)-one.

A new furoquinolinone derivative, namely 4-hydroxymethyl-1,6,8-trimethylfuro[2,3-h]quinolin-2(1H)-one (HOFQ), was synthesized and its biological activity studied. By UVA activation, HOFQ induced strong antiproliferative effects in Ehrlich ascite cells, which lost their ability to transmit the tumor by transplantation. HOFQ exhibited poor genotoxicity and absence of skin phototoxicity. Actually, HOFQ sensitization forms DNA-protein cross-linkages but not interstrands cross-links. Therefore, HOFQ appears to be a new promising drug for PUVA photochemotherapy and photopheresis.