DNA binding by antitumor trans-[PtCl2(NH3)(thiazole)]. Protein recognition and nucleotide excision repair of monofunctional adducts

Antitumor effects of cis-diamminedichloroplatinum(II) (cisplatin) and the clinical inactivity of its trans isomer (transplatin) have been considered a paradigm for the classical structure-activity relationships of platinum drugs. However, several new analogues of transplatin which exhibit a different spectrum of cytostatic activity including activity in tumor cells resistant to cisplatin have been recently identified. Analogues containing the planar amine ligand of the general structure trans-[PtCl(2)(NH(3))(L)], where L = planar amine, represent an example of such compounds. DNA is believed to be the major pharmacological target of platinum compounds. To contribute to the understanding of mechanisms underlying the activation of trans geometry in transplatin analogues containing planar amine ligands, various biochemical and biophysical methods were employed in previous studies to analyze the global modifications of natural DNA by trans-[PtCl(2)(NH(3))(L)]. These initial studies have revealed some unique features of the DNA binding mode of this class of platinum drugs. As the monofunctional lesions represent a significant fraction of stable adducts formed in DNA by bifunctional antitumor trans-platinum compounds with planar ligands, we analyzed in the present work short DNA duplexes containing the single, site-specific monofunctional adduct of a representative of this class of platinum drugs, antitumor trans-[PtCl(2)(NH(3))(thiazole)]. It has been shown that, in contrast to the adducts of monodentate chlorodiethylenetriamineplatinum(II) chloride or [PtCl(NH(3))(3)]Cl, the monofunctional adduct of trans-[PtCl(2)(NH(3))(thiazole)] inhibits DNA synthesis and creates a local conformational distortion similar to that produced in DNA by the major 1,2-GG intrastrand CL of cisplatin, which is considered the lesion most responsible for its anticancer activity. In addition, the monofunctional adducts of trans-[PtCl(2)(NH(3))(thiazole)] are recognized by HMGB1 domain proteins and removed by the nucleotide excision repair system similarly as the 1,2-GG intrastrand CL of cisplatin. The results of the present work further support the view that the simple chemical modification of the structure of an inactive platinum compound alters its DNA binding mode into that of an active drug and that processing of the monofunctional DNA adducts of the trans-platinum analogues in tumor cells may be similar to that of the major bifunctional adducts of "classical" cisplatin.     

DNA-protein cross-linking by trans-[PtCl(2)(E-iminoether)(2)]. A concept for activation of the trans geometry in platinum antitumor complexes

The structure-pharmacological activity relationships generally accepted for antitumor platinum compounds stressed the necessity for the cis-[PtX(2)(amine)(2)] structure while the trans-[PtX(2)(amine)(2)] structure was considered inactive. However, more recently, several trans-platinum complexes have been identified which are potently toxic, antitumor-active and demonstrate activity distinct from that of conventional cisplatin (cis-[PtCl(2)(NH(3))(2)]). We have shown in the previous report that the replacement of ammine ligands by iminoether in transplatin (trans-[PtCl(2)(NH(3))(2)]) results in a marked enhancement of its cytotoxicity so that it is more cytotoxic than its cis congener and exhibits significant antitumor activity, including activity in cisplatin-resistant tumor cells. In addition, we have also shown previously that this new trans compound (trans-[PtCl(2)(E-iminoether)(2)]) forms mainly monofunctional adducts at guanine residues on DNA, which is generally accepted to be the cellular target of platinum drugs. In order to shed light on the mechanism underlying the antitumor activity of trans-[PtCl(2)(E-iminoether)(2)] we examined oligodeoxyribonucleotide duplexes containing a single, site-specific, monofunctional adduct of this transplatin analog by the methods of molecular biophysics. The results indicate that major monofunctional adducts of trans-[PtCl(2)(E-iminoether)(2)] locally distort DNA, bend the DNA axis by 21 degrees toward the minor groove, are not recognized by HMGB1 proteins and are readily removed from DNA by nucleotide excision repair (NER). In addition, the monofunctional adducts of trans-[PtCl(2)(E-iminoether)(2)] readily cross-link proteins, which markedly enhances the efficiency of this adduct to terminate DNA polymerization by DNA polymerases in vitro and to inhibit removal of this adduct from DNA by NER. It is suggested that DNA-protein ternary cross-links produced by trans-[PtCl(2)(E-iminoether)(2)] could persist considerably longer than the non-cross-linked monofunctional adducts, which would potentiate toxicity of this antitumor platinum compound toward tumor cells sensitive to this drug. Thus, trans-[PtCl(2)(E-iminoether)(2)] represents a quite new class of platinum antitumor drugs in which activation of trans geometry is associated with an increased efficiency to form DNA-protein ternary cross-links thereby acting by a different mechanism from 'classical' cisplatin and its analogs.     

Activation of trans geometry in bifunctional mononuclear platinum complexes by a piperidine ligand. Mechanistic studies on antitumor action

A paradigm for the structure-pharmacological activity relationship of bifunctional platinum antitumor drugs is that the trans isomer of antitumor cisplatin (transplatin) is clinically ineffective. To this end, however, several new complexes of the trans structure have been identified that exhibit cytotoxicity in tumor cells that is even better than that of the analogous cis isomers. We reported recently (Kasparkova, J., Marini, V., Najajreh, Y., Gibson, D., and Brabec, V. (2003) Biochemistry 42, 6321-6332) that the replacement of one ammine ligand by the heterocyclic ligand, such as piperidine, piperazine, or 4-picoline in the molecule of transplatin resulted in a radical enhancement of its cytotoxicity. We examined oligodeoxyribonucleotide duplexes bearing a site-specific cross-link of the transplatin analogue containing the piperidine ligand by biochemical methods. The results indicate that in contrast to transplatin, trans-(PtCl2(NH3)(piperidine)) forms stable 1,3-intrastrand cross-links in double-helical DNA that distort DNA and are not readily removed from DNA by nucleotide excision repair system. Hence, the intrastrand cross-links of trans-(PtCl2(NH3)(piperidine)) could persist for a sufficiently long time, potentiating its toxicity toward tumor cells. trans-(PtCl2(NH3)(piperidine)) also forms in DNA minor interstrand cross-links that are similar to those of transplatin so that these adducts appear less likely candidates for genotoxic lesion responsible for antitumor effects of trans-(PtCl2(NH3)(piperidine)). Hence, the role of structurally unique intrastrand cross-links in the anti-tumor effects of transplatin analogues in which one ammine group is replaced by a heterocyclic ligand may predominate.     

DNA adducts of antitumor trans-[PtCl2 (E-imino ether)2].

It has been shown recently that some analogues of clinically ineffective trans-diamminedichloroplatinum (II) (transplatin) exhibit antitumor activity. This finding has inverted the empirical structure-antitumor activity relationships delineated for platinum(II) complexes, according to which only the cis geometry of leaving ligands in the bifunctional platinum complexes is therapeutically active. As a result, interactions of trans platinum compounds with DNA, which is the main pharmacological target of platinum anticancer drugs, are of great interest. The present paper describes the DNA binding of antitumor trans-[PtCl(2)(E-imino ether)(2)] complex (trans-EE) in a cell-free medium, which has been investigated using three experimental approaches. They involve thiourea as a probe of monofunctional DNA adducts of platinum (II) complexes with two leaving ligands in the trans configuration, ethidium bromide as a probe for distinguishing between monofunctional and bifunctional DNA adducts of platinum complexes and HPLC analysis of the platinated DNA enzymatically digested to nucleosides. The results show that bifunctional trans-EE preferentially forms monofunctional adducts at guanine residues in double-helical DNA even when DNA is incubated with the platinum complex for a relatively long time (48 h at 37 degrees C in 10 mM NaCIO(4). It implies that antitumor trans-EE modifies DNA in a different way than clinically ineffective transplatin, which forms prevalent amount of bifunctional DNA adducts after 48 h. This result has been interpreted to mean that the major adduct of trans-EE, occurring in DNA even after long reaction times, is a monofunctional adduct in which the reactivity of the second leaving group is markedly reduced. It has been suggested that the different properties of the adducts formed on DNA by transplatin and trans-EE are relevant to their distinct clinical efficacy.     

Bending studies of DNA site-specifically modified by cisplatin, trans-diamminedichloroplatinum(II) and cis-[Pt(NH3)2(N3-cytosine)Cl]+

Duplex oligonucleotides containing a single intrastrand [Pt(NH3)2]2+ cross-link or monofunctional adduct and either 15 or 22 bp in length were synthesized and chemically characterized. The platinum-modified and unmodified control DNAs were polymerized in the presence of DNA ligase and the products studied on 8% native polyacrylamide gels. The extent of DNA bending caused by the various platinum-DNA adducts was revealed by their gel mobility shifts relative to unplatinated controls. The bifunctional adducts cis-[Pt(NH3)2[d(GpG)]]+, cis-[Pt(NH3)2[d(ApG)]]+, and cis-[Pt(NH3)2[d(G*pTpG*)]], where the asterisks denote the sites of platinum binding, all bend the double helix, whereas the adduct trans-[Pt(NH3)2[d(G*pTpG*)]] imparts a degree of flexibility to the duplex. When modified by the monofunctional adduct cis-[Pt(NH3)2(N3-cytosine)(dG)]Cl the helix remains rod-like. These results reveal important structural differences in DNAs modified by the antitumor drug cisplatin and its analogs that could be important in the biological processing of the various adducts in vivo.     

Recognition of DNA modified by trans-[PtClNH(4-hydroxymethylpyridine)] by tumor suppressor protein p53 and character of DNA adducts of this cytotoxic complex

trans-[PtCl(2)NH(3)(4-Hydroxymethylpyridine)] (trans-PtHMP) is an analogue of clinically ineffective transplatin, which is cytotoxic in the human leukemia cancer cell line. As DNA is a major pharmacological target of antitumor platinum compounds, modifications of DNA by trans-PtHMP and recognition of these modifications by active tumor suppressor protein p53 were studied in cell-free media using the methods of molecular biology and biophysics. Our results demonstrate that the replacement of the NH(3) group in transplatin by the 4-hydroxymethylpyridine ligand affects the character of DNA adducts of parent transplatin. The binding of trans-PtHMP is slower, although equally sequence-specific. This platinum complex also forms on double-stranded DNA stable intrastrand and interstrand cross-links, which distort DNA conformation in a unique way. The most pronounced conformational alterations are associated with a local DNA unwinding, which was considerably higher than those produced by other bifunctional platinum compounds. DNA adducts of trans-PtHMP also reduce the affinity of the p53 protein to its consensus DNA sequence. Thus, downstream effects modulated by recognition and binding of p53 protein to DNA distorted by trans-PtHMP and transplatin are not likely to be the same. It has been suggested that these different effects may contribute to different antitumor effects of these two transplatinum compounds.     

DNA binding mode of the cis and trans geometries of new antitumor nonclassical platinum complexes containing piperidine,piperazine, or 4-picoline ligand in cell-free media. Relations to their activity in cancer cell lines

The global modification of mammalian and plasmid DNAs by novel platinum compounds, cis- or trans-[PtCl(2)(NH(3))(Am)], where Am = NH(3), nonplanar heterocycle piperidine, piperazine, or aromatic planar heterocycle 4-picoline, was investigated in cell-free media using various biochemical and biophysical methods. These modifications have been compared with the activity of these new compounds in several tumor cell lines including those resistant to antitumor cis-diamminedichloroplatinum(II) (cisplatin). The results show that the replacement of the NH(3) group in cisplatin by the heterocyclic ligands does not considerably affect the DNA binding mode of this drug. Cytotoxicity studies have revealed that the replacement lowers the activity of the platinum compound in both sensitive and resistant cell lines. It has been suggested that the reduced activity of these analogues of cisplatin is associated with some features of the damaged DNA and/or its cellular processing. Alternatively, the reduced activity of the analogues of cisplatin might also be due to the factors that do not operate directly at the level of the target DNA, such as intracellular platinum uptake. In contrast to the analogues of cisplatin, the replacement of one ammine group by the heterocyclic ligand in its clinically ineffective trans isomer (transplatin) results in a radical enhancement of its activity in tumor cell lines. Importantly, this replacement also markedly alters the DNA binding mode of transplatin. The results support the view that one strategy of how to activate the trans geometry in bifunctional platinum(II) compounds including circumvention of resistance to cisplatin may consist of a chemical modification of the ineffective transplatin that results in an increased stability of its intrastrand cross-links in double-helical DNA and/or in an increased efficiency to form interstrand cross-links.     

Interstrand cross-linking reaction in triplexes containing a monofunctional transplatin-adduct.

Our aim was to determine whether a single transplatin monofunctional adduct, either trans-[Pt(NH3)2(dC)Cl]+ or trans-[Pt(NH3)2(dG)Cl]+ within a homopyrimidine oligonucleotide, could further react and form an interstrand cross-link once the platinated oligonucleotide was bound to the complementary duplex. The single monofunctional adduct was located at either the 5' end or in the middle of the platinated oligonucleotide. In all the triplexes, specific interstrand cross-links were formed between the platinated Hoogsteen strand and the complementary purine-rich strand. No interstrand cross-links were detected between the platinated oligonucleotides and non-complementary DNA. The yield and the rate of the cross-linking reaction depend upon the nature and location of the monofunctional adducts. Half-lives of the monofunctional adducts within the triplexes were in the range 2-6 h. The potential use of the platinated oligonucleotides to modulate gene expression is discussed.     

Preparation, cytotoxicity and interactions with nucleophiles of three isomeric transplatinum complexes containing methylpiperidine ligands

Three isomeric complexes, trans-[PtCl2(NH3)(2-methylpiperidine)], trans-[PtCl2(NH3)(3-methylpiperidine)] and trans-[PtCl2(NH3)(4-methylpiperidine)], were prepared and their cytotoxicities against six ovarian cancer cell lines, three sensitive and three resistant to cisplatin, were measured. There were no significant differences in the cytotoxicities of the three isomers against these cell lines. The interactions of the three complexes with reduced glutathione (GSH) and with ubiquitin (Ub), as a model protein, were studied. The trans-[PtCl2(NH3)(2-methylpiperidine)] reacted approximately twice as slowly with GSH as did the other two isomers. In the 1:1 interactions of the three complexes with ubiquitin (Mr = 8565 amu), trans-[PtCl2(NH3)(3-methylpiperidine)] and trans-[PtCl2(NH3)(4-methylpiperidine)] attained 100% modification while trans-[PtCl2(NH3)(2-methylpiperidine)] reached only less than 50% modification. Trans-[PtCl2(NH3)(2-methylpiperidine)] reacts significantly less efficiently with GSH and proteins than the other two isomers yet this is not reflected in the cytotoxicity values. These results indicate that for these complexes, in these cell lines, cytosolic detoxification probably does not play a dominant role in determining the cytotoxicity of the complexes.     

Mechanism of the formation of DNA-protein cross-links by antitumor cisplatin

DNA–protein cross-links are formed by various DNA-damaging agents including antitumor platinum drugs. The natures of these ternary DNA–Pt–protein complexes (DPCLs) can be inferred, yet much remains to be learned about their structures and mechanisms of formation. We investigated the origin of these DPCLs and their cellular processing on molecular level using gel electrophoresis shift assay. We show that in cell-free media cisplatin [cis-diamminedichloridoplatinum(II)] forms DPCLs more effectively than ineffective transplatin [trans-diamminedichloridoplatinum(II)]. Mechanisms of transformation of individual types of plain DNA adducts of the platinum complexes into the DPCLs in the presence of several DNA-binding proteins have been also investigated. The DPCLs are formed by the transformation of DNA monofunctional and intrastrand cross-links of cisplatin. In contrast, interstrand cross-links of cisplatin and monofunctional adducts of transplatin are stable in presence of the proteins. The DPCLs formed by cisplatin inhibit DNA polymerization or removal of these ternary lesions from DNA by nucleotide excision repair system more effectively than plain DNA intrastrand or monofunctional adducts. Thus, the bulky DNA–protein cross-links formed by cisplatin represent a more distinct and persisting structural motif recognized by the components of downstream cellular systems processing DNA damage considerably differently than the plain DNA adducts of this metallodrug.     

Study by HPLC-MS of the interaction of platinum antitumor complexes with potato carboxypeptidase inhibitor (PCI)

The interaction of the well-known antitumor drug cisplatin cis-[PtCl(2)(NH(3))(2)] and the compound trans-[PtCl(2)NH(3)(4-hydroxymethylpyridine)] with the small protein potato carboxypeptidase inhibitor (PCI) and a PCI mutant in which glycine-39 was substituted by methionine has been followed by HPLC/mass spectrometry. Our results showed that both Pt drugs were able to bind PCI through Met-39 and histidines in mutated PCI, whereas only the trans complex interacted significantly with wild PCI. In the cytotoxic studies, the monofunctional adduct PCI-Met-cisplatin was neither more active nor more selective than cisplatin itself when tested against three tumor cell lines with different number of EGF receptors. Those results suggested that the poor activity of the adduct could be just due to the small fraction of cisplatin which was decoordinated from the adduct and able to penetrate the tumor cells, as well as to the changes in the structure of the platinum drug after the loss of NH(3) groups upon binding PCI-Met.     

Comparison of the electronic properties,and thermodynamic and kinetic parameters of the aquation of selected platinum(II) derivatives with their anticancer IC<Subscript>50</Subscript> indexes

Three potential anticancer agents {trans-[PtCl(2)(NH(3))(thiazole)], cis-[PtCl(2)(NH(3))(piperidine)], and PtCl(2)(NH(3))(cyclohexylamine) (JM118)} were explored and compared with cisplatin and the inactive [PtCl(dien)](+) complex. Basic electronic properties, bonding and stabilization energies were determined, and thermodynamic and kinetic parameters for the aquation reaction were estimated at the B3LYP/6-311++G(2df,2pd) level of theory. Since the aquation process represents activation of these agents, the obtained rate constants were compared with the experimental IC(50) values for several tumor cells. Despite the fact that the processes in which these drugs are involved and the way in which they affect cells are very complex, some correlations can be deduced.     

Rates of platination of -AG- and -GA- containing double-stranded oligonucleotides: effect of chloride concentration

The kinetics of the reactions between 15N-labelled cisplatin and 14-base pair duplex oligonucleotides with either 5'-AG-3' or 5'-GA-3' groupings as the principal platination site are examined in the presence of 60-80 mM chloride by [1H,15N]HSQC 2D NMR spectroscopy. The presence of chloride at these concentrations results in a five-fold decrease in the rate of hydrolysis of cisplatin to cis-[PtCl(NH3)2(OH2)]+ and a two- to twenty-fold decrease in the rate of monofunctional adduct formation. The effects on the rate of closure from monofunctional to bifunctional adducts are less well established but some of these rates appear not to be significantly reduced by the presence of added chloride. The results provide a caution that the use of chloride to quench platination reactions may not be fully effective.     

Increased targeting of adenine-rich sequences by (2-amino-2-methyl-3-butanone oxime)dichloroplatinum(II) and investigations into its low cytotoxicity

Using assays based on the inhibition of restriction enzyme cleavage of plasmid and synthetic DNA, the complex (2-amino-2-methyl-3-butanone oxime)dichloroplatinum(II), [PtCl2(ambo)], has been shown to have an increased tendency for binding to adenine-rich sequences when compared to cis[PtCl2(NH3)2] (cisplatin). [PtCl2(ambo)] was found to form substantially fewer interstrand adducts than does cisplatin. The in vitro cytotoxicity of [PtCl2(ambo)] against a human bladder cancer cell line was determined and found to be more than two orders of magnitude lower than that of cisplatin, yet it was also found to be equally effective at passing into cells and binding to isolated DNA.     

Steric control of stereoselective interactions between the platinum(II) complex [PtCl2(1,4-diazacycloheptane)] and DNA: comparison with cis-[PtCl2(NH3)2] and [PtCl2(ethane-1,2-diamine)] using DNA binding and molecular modeling studies

The rate and extent of binding of [PtCl2(hpip)] (hpip=homopiperazine-1,4-diazacycloheptane) and cis-[PtCl2(NH3)2] to calf thymus DNA was measured using atomic absorption spectroscopy and it was found that [PtCl2(hpip)] bound both more rapidly and to a greater extent than did cis-[PtCl2(NH3)2]. The binding of [PtCl2(hpip)] and [PtCl2(en)] (en=ethane-1,2-diamine) to salmon sperm DNA and to synthetic, self-complementary 10-base-pair and 52-base-pair oligonucleotides was studied using enzymatic digestion and HPLC analysis of the products. [PtCl2(hpip)] forms approximately two-fold fewer GpG and ApG intrastrand adducts and concomitantly more monofunctional adducts than does [PtCl2(en)]. In the case of [PtCl2(hpip)], two GpG adducts, corresponding to the different orientations of the hpip ligand with respect to the DNA, were observed in a 1:3.3 ratio. The minor product corresponds to the orientation in which the bulkier propylene chain of the hpip ligand is adjacent to, and makes close contacts with, the floor of the major groove. When the reaction was repeated with a synthetic oligonucleotide decamer duplex, the ratio of the two forms was approximately 1:1.9 and with the 52-mer duplex it was 1:2.4, revealing an apparent systematic dependence of stereoselectivity on nucleotide size. Computer modeling of the two adducts formed by [PtCl2(hpip)] and those formed by [PtCl2(en)] and cis-[PtCl2(NH3)2] revealed that non-bonded interactions between the hpip ligand and the DNA were probably responsible for both the decreased proportion of GpG adducts formed by [PtCl2(hpip)] and the stereoselectivity exhibited in the formation of these adducts. This is the first case in which the stereoselectivity can be ascribed to steric factors alone.     

Influence of the position of substituents in the cytotoxic activity of trans platinum complexes with hydroxymethyl pyridines

The synthesis and chemical characterization of two trans platinum complexes, (1) trans-[PtCl(2)NH(3)(2-hydroxymethylpyridine)] and (2) trans-[PtCl(2)NH(3)(3-hydroxymethylpyridine)], are described. The structures and chemical behaviour of these compounds have been compared to those of their isomer (3) trans-[PtCl(2)NH(3)(4-hydroxymethylpyridine)] previously studied. X-ray structures of all of them were solved and some interesting differences were found. The values of the dihedral angle (85 degrees , 57 degrees and 42 degrees for 1, 2 and 3, respectively) demonstrate how important is the position of substituent from a structural point of view. Studies of circular dichroism (CD), electrophoretic mobility (EM) in agarose gel and atomic force microscopy (AFM) showed differences in the modifications caused by the three complexes on DNA. Studies of antiproliferative activity of complexes 1 and 2 against cell tumour lines (HL-60) and apoptosis assays have also been carried out, showing that 1 as well as 2 are far less active than the previously described complex 3 (IC(50)=19; 19 and 3 microM, respectively). This fact probes that slight modifications on the drug's design may generate significant differences in the final antitumour activity by modifying the DNA-drug adducts, performance of resistance mechanisms and all the factors that play a fundamental role in Pt complexes' cytotoxicity.     

Mono- and polynuclear complexes of Pt(II) with polypyridyl ligands. Synthesis, spectroscopic and structural characterization and cytotoxic activity

An array of poly- and mononuclear complexes of Pt(II) with polypyridyl ligands is reported. The framework complexes [(PtCl(2))(2)(bpp)(2)(micro-PtCl(2))](H(2)O)(2) [bpp=2,3-bis(2-pyridyl)pyrazine], [PtCl(2)(micro-tptz)PtClNCPh]Cl [tptz=2,4,6-tris(2-pyridyl)-1,3,5-triazine], and mononuclear PtCl(2)(NH(2)dpt) [NH(2)dpt=4-amino-3,5-bis(2-pyridyl)-1,2,4-triazole] have been prepared and structurally characterized. Both neutral and ionic complexes are present, with bifunctional and monofunctional Pt(II) moieties, whose size and shape enable them to behave as novel scaffolds for DNA binding. Pt(II) complexes were tested for their biological activity. Cell viability assay and flow cytometric analysis demonstrated that these complexes, particularly [PtCl(2)(micro-tptz)PtClNCPh]Cl, were effective death inducers in human colon rectal carcinoma HT29 cells and their cytotoxic activity was higher than that exerted by cisplatin. Morphological analysis of treated HT29 cells, performed by fluorescence microscopy after Hoechst 33258 staining, showed the appearance of the typical features of apoptosis. Moreover, our results suggested that mitochondria are involved in apoptosis induced by Pt(II) complexes in HT29 cells as demonstrated by dissipation of mitochondrial transmembrane potential.     

In vitro and in vivo antitumour activity and cellular pharmacological properties of new platinum-iminoether complexes with different configuration at the iminoether ligands

In order to widen our knowledge on antitumour trans-[PtCl2(iminoether)2] complexes, we have synthesised two new derivatives, trans-[PtCl2?E-HN = C(OEt)Me?2] (1) and trans-[PtCl2?Z-HN = C(OEt)Me?2] (2), which differ in the configuration of the iminoether ligands. Isomer 1 showed an in vitro cytotoxicity similar to that of cisplatin in a panel of human tumour cell lines (mean IC50 = 8 and 7.7 microM, respectively), whereas isomer 2 showed a lower activity (IC50 = 14.3 microM). Both 1 and 2 isomers overcame cisplatin resistance of ovarian cancer cell line A2780/Cp8. In agreement with the n-octanol/saline partition ratios, intracellular platinum content (and DNA platination) after a 2-h exposure to equimolar drug concentrations was in the order 1 > 2 > cisplatin, thus indicating that substitution of imminoethers for ammines determines a major lipophilicity and cellular uptake of the platinum drug. Both 1 and 2 showed a major toxic effect towards an excision repair-defective Drosophila strain, thus indicating cellular DNA as cytotoxic target. Finally, both 1 and 2 were active in vivo against the murine P388 system, but, contrary to the in vitro activity, isomer 2 was slightly more active than 1. On the whole, the results confirm the antitumour activity of trans-[PtCl2(iminoether)2] complexes, and indicate that the configuration of the iminoether ligands may affect the pharmacological properties of this class of complexes.     

Synthesis and characterization of trans-[Pt(NH3)2Cl2] adducts of d(CCTCGAGTCTCC).d(GGAGACTCGAGG)

The reaction of trans-diamminedichloroplatinum(II) (trans-DDP), the inactive isomer of the anticancer drug cisplatin, with the single-stranded deoxydodecanucleotide d(CCTCGAGTCTCC) in aqueous solution at 37 degrees C was monitored by reversed-phase HPLC. Consumption of the dodecamer follows pseudo-first-order reaction kinetics with a rate constant of 1.25 (4) x 10(-4) s-1. Two intermediates, shown to be monofunctional adducts in which Pt is coordinated to the guanine N7 positions, were trapped with NH4(HCO3) and identified by enzymatic degradation analysis. These monofunctional adducts and a third, less abundant, one are rapidly removed from the DNA by thiourea under mild conditions. When allowed to react further, the monofunctional intermediates formed a single main product that was characterized by 1H NMR spectroscopy and enzymatic digestion as the bifunctional 1,3-intrastrand cross-link trans-[Pt(NH3)2[d(CCTCGAGTCTCC)-N7-G(5),N7-G(7]]). Binding of the trans-[Pt(NH3)2]2+ moiety to the guanosine N7 positions decreases the pKa at N1 and leads to destacking of the intervening A(6) base. The double-stranded trans-DDP-modified and unmodified DNAs were obtained by annealing the complementary strand to the corresponding single strands and then studied by 31P and 1H NMR and UV spectroscopy. trans-DDP binding does not induce large changes in the O-P-O bond or torsional angles of the phosphodiester linkages in the duplex, nor does it significantly alter the UV melting temperature. trans-DDP binding does, however, cause the imino protons of the platinated duplex to exchange rapidly with solvent by 50 degrees C, a phenomenon that occurs at 65 degrees C for the unmodified duplex. A structural model for the platinated double-stranded oligonucleotide was generated through molecular dynamics calculations. This model reveals that the trans-DDP bifunctional adduct can be accommodated within the double helix with minimal distortion of the O-P-O angles and only local disruption of base pairing and destacking of the platinated bases. The model also predicts hydrogen bond formation involving coordinated ammine ligands that bridge the two strands.