Francarosa Baccichetti

Platinum(II) and palladium(II) complexes with dithiocarbamates and amines: synthesis, characterization and cell assay

The [M(ESDT)Cl]n (M = Pd or Pt; ESDT = EtO2CCH2(CH3)NCS2, methylamino-acetic acid ethyl ester-dithiocarboxylate) species have been reacted with various amines (py, pyridine; PrNH2, n-propylamine; c-BuNH2, cyclobutylamine; en, ethylenediamine) in dichloromethane or chloroform with the aim to obtain mixed ligand complexes. The neutral complexes [M(ESDT)(L)Cl] (L = py, PrNH2 or c-BuNH2) and the ionic species ([M(ESDT)(L)2]Cl and [M(ESDT)(En)]Cl) have been isolated, and characterized by IR and proton NMR spectroscopies. The crystal structure of [Pd(ESDT)(PrNH2)Cl] has been determined by X-ray crystallography. The behaviour of the complexes in various solvents was described on the basis of the proton NMR spectra. The complexes and the dithiocarbamato intermediates have been tested for in vitro cytostatic activity against human leukemic HL-60 and HeLa cells.

4,5‘‐DIMETHYLANGELICIN: A NEW DNA‐PHOTOBINDING MONOFUNCTIONAL AGENT*

Abstract— 4,5′‐Dimethylangelicin is a new angular furocoumarin showing interesting photochemical and photosensitizing properties. In the dark it forms a complex with native DNA having higher values of the binding parameters than angelicin; by irradiation at 365 nm it is able to photobind with DNA several times faster than angelicin and in about the same degree as psoralen, without forming crosslinkages. It therefore behaves as a pure monofunctional reagent. The same high photobinding capacity with DNA is shown also in vivo in Ehrlich ascites tumor cells and bacterial E. coli cells.

DNA repair and recovery in Escherichia coli after psoralen and angelicin photosensitization

The correlation between DNA repair and recovery of biological functions was studied using three wild type strains of Escherichia coli and two skinphotosensitizing furocoumarins, psoralen and angelicin, which are well known specific reagents of the pyrimidine bases of DNA. In addition to mono-adducts psoralen is able to form a high number of inter-strand cross-links, while angelicin forms only mono-adducts. Both of these damages were repaired, in a short time, in the following way: at first DNA was cut into small pieces that were then rejoined into molecules of normal size, free from cross-links, while the furocoumarin residue was split from DNA almost quantitatively. Recovery of biological functions was studied performing photosensitization experiments in such a manner that the same amounts of psoralen or of angelicin were linked to bacterial DNA. DNA synthesis, tested just after the damage, was inhibited in a similar extent by both drugs. The same bacteria, however, showed a very different colony-forming capacity; angelicin was much less effective than psoralen with a D37 dose about 2.7 times higher. A similar picture was obtained studying DNA synthesis at different times after photosensitization: in the bacteria damaged by angelicin it was restored while no recovery was observed in cells photosensitized by psoralen. These results suggest that both mono-adducts and cross-links can be chemically repaired more or less in a quantitative measure, but that repair of cross-links in much less effective on cell recovery; this behaviour is very probably connected with the different repair mechanisms of mono-adducts and of cross-links.

FUROCOUMARIN SENSITIZATION INDUCES DNA-PROTEIN CROSS-LINKS

The capacity of some linear and angular furocoumarins to induce DNA‐protein cross‐links by UVA (320–400 nm) irradiation has been evaluated in Chinese hamster ovary cells. Two linear furocoumarins, psoralen and 8‐methoxypsoralen appeared to be capable of inducing DNA‐protein cross‐links to a noticeable extent. 4′‐Methylangelicin and 4,4′‐dimethylangelicin formed only reduced amounts of DNA‐protein cross‐links, while angelicin and 4,6,4′‐trimethylangelicin seemed to be unable to induce significant levels of this lesion. The biological significance of this damage remains to be elucidated, but it might have an important role in furocoumarin sensitization. In the examined compounds, the capacity for inducing DNA‐protein cross‐links appears to be a property of the skin phototoxic furocoumarins. This result suggests the hypothesis of a connection between this damage and the formation of skin erythemas.

Synthesis and antiproliferative activity of some variously substituted acridineand azacridine derivatives

A group of 9-substituted acridine and azacridine derivatives (m-AMSA analogues) were synthesised following classical procedures as potential antitumour agents with inhibitory effects on DNA topoisomerase II. Some were found to have noticeable cytotoxicity against human HL-60 and HeLa cells grown in culture. Their non-covalent interactions with calf thymus DNA have been studied using fluorescence quenching. We evaluated DNA damage produced by the tested compounds by means of DNA filter elution and protein precipitation techniques. Catalytic studies carried out with purified topoisomerase confirmed these agents as antitopoisomerase inhibitors. Chemotherapy of solid-tumour-bearing mice with tested compounds allowed an aza-analogue (compound IIIb), as potent as m-AMSA but less toxic towards the host, to be recognised.

6-Methylangelicins: new monofunctional photochemotherapeutic agents for psoriasis

The monofunctional furocoumarins, the 6‐methylangelicins, were tested for their antiproliferative activity with various animal models and for genotoxicity in micro‐organisms and in mammalian cells. The most active compound was 6,4,4′‐trimethylangelicin, which showed a high antiproliferative effect and reduced genotoxicity in comparison with 8‐methoxypsoralen (8‐MOP). Some of these compounds were also tested clinically by topical application on 17 patients with psoriasis. They appeared to be more active than 8‐MOP in clearing psoriasis without inducing skin phototoxicity. The methylangelicins also caused skin pigmentation.

DNA damage induced by 4,6,8,9-tetramethyl-2H-furo[2,3-h]quinolin-2-one, a new furocoumarin analog: biological consequences.

Abstract 4,6,8,9-Tetramethyl-2H-furo[2,3-h]quinolin-2-one (HFQ) and its isomer FQ (1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2-one) showed very strong antiproliferative activity in mammalian cells, about two times greater than 8-methoxypsoralen (8-MOP). Both compounds induced DNA–protein cross-links (DPC) but not interstrand cross-links. The FQ generated DPC in a biphotonic process, yielding a new kind of diadduct, whereas HFQ induced DPC by a monophotonic one, probably without its physical participation in the covalent bridge. These lesions gave different toxic responses. Sensitization of FQ led to extensive DNA fragmentation and to a number of chromosomal aberrations. Conversely, HFQ seemed to be completely inactive and 8-MOP gave intermediate results. A strict relationship between DPC formation and induction of chromosomal aberrations was observed. The HFQ did not induce light skin erythemas, whereas FQ was more phototoxic than 8-MOP, thus suggesting that FQ lesions, DPC in particular, may be implicated in skin phototoxicity. Ehrlich ascites cells, a transplantable mouse tumor, inactivated by furoquinolinone sensitization and injected into healthy mice, protected them from a successive challenge by viable tumor cells. This response appeared to be based on an immune mechanism. Comparable amounts of base substitution revertants were scored when testing furoquinolinones and 8-MOP in bacteria but no DPC were detected. This suggests that classic mutagenesis tests on bacteria are insufficient to give adequate information on furocoumarin genotoxicity. Given its features, HFQ can be regarded as an interesting new agent for psoralen plus UVA photochemotherapy and photopheresis.

4-Hydroxymethyl- and 4-methoxymethylfuro[2,3-h]quinolin-2(1H)-ones: synthesis and biological properties

4-Hydroxymethyl-1,6,8-trimethylfuro[2,3-h]quinolin-2(1H)-one (HOFQ) was prepared by a new profitable way, which allowed to synthesize also 4-methoxymethyl-1,6,8-trimethylfuro[2,3-h]quinolin-2(1H)-one (MOFQ), and 4-hydroxymethyl-6,8-dimethylfuro[2,3-h]quinolin-2(1H)-one (HOHFQ). Some biological activities of the three compounds were studied in comparison with 8-MOP. In the dark, they inhibited topoisomerase II, leading to a moderate antiproliferative activity in mammalian cells. The antiproliferative activity was also tested upon UVA irradiation in mammalian cells: all compounds showed higher activity than 8-MOP, without mutagenicity and skin phototoxicity, with the best results for HOFQ. Photobinding to DNA was investigated, demonstrating a different sequence specificity for these furoquinolinones in comparison with furocoumarins. For all these features, HOFQ and the other analogues appeared very promising photochemotherapeutic agents, whose mechanism of action will be further investigated.

Synthesis and photobiological properties of 4- hydroxymethyl-4'- methylpsoralen derivatives.

The synthesis and the photobiological activity of two new hydroxymethyl derivatives of psoralen namely 4‐hydroxymethyl‐4′‐methyl‐ and 4‐hydroxymethyl‐4′‐methyl‐8‐rnethoxypsoralen are described. Both compounds exhibited efficient photobinding to DNA and RNA. The DNA‐photobinding process was investigated using different nucleic acid structures such as double‐helical DNA, ribosomal RNA, bacterial DNA and DNA organized in the nucleosomal arrangement. The test derivatives were able to induce cross‐links to a similar extent as 8‐methoxypsoralen (8‐MOP), used as a reference photochemotherapeutic drug. In contrast to 8‐MOP, they produced relatively high levels of lO2. Most photobiological effects (DNA synthesis inhibition, T2 phage sensitization, inhibition of tumor transmitting capacity) showed a good correlation with the extent of covalent photoaddition. On the other hand, the new 4‐hydroxymethylpsoralens were unable to induce skin erythema, in striking contrast with 8‐MOP. Thus, neither cross‐linking of the nucleic acid nor 1O2 production were coupled with skin phototoxicity in this class of compounds. The new derivatives appear to represent an important beginning to development of new active photochemotherapeutic agents devoid of undesired phototoxic side effects.

Cytotoxicity and DNA damage induced by a new platinum(II) complex with pyridine and dithiocarbamate.

A new platinum(II) complex containing a pyridine nucleus and a dithiocarbamate moiety as ligands ([Pt(ESDT)(Py)Cl]) was evaluated for in vitro cytotoxicity in the cisplatin-sensitive human ovarian 2008 and in the isogenic-resistant C13* cell lines. In both cell types, a tumor cell growth inhibition greater than cisplatin and a complete lack of cross-resistance in C13* cells were found. Despite its molecular size, [Pt(ESDT)(Py)Cl] accumulation was much higher than cisplatin both in parent and resistant cells. Studying the mechanism of action in cell-free media, we established that [Pt(ESDT)(Py)Cl] more efficiently interacts with DNA in vitro compared to cisplatin maintaining a binding preference for GG rich sequences of DNA. On the contrary, DNA platination in vivo by [Pt(ESDT)(Py)Cl] was found lower than cisplatin. An analysis of the type of DNA lesions induced by [Pt(ESDT)(Py)Cl] suggests that the cytotoxic efficacy and the ability to overcome cisplatin resistance seem to be related to a different mechanism of interaction with DNA and/or with other key cellular components.