Cis-platinum(II) complexes of carboxymethyl-dextran as potential antitumor agents. I. Preparation and characterization

Cis-diamminedichloro platinum (II) (cis-DDP) and cis-diamminediaquo platinum (II) nitrate (cis-aq) were demonstrated to form complexes with dextran (dex) substituted with carboxymethyl (CM) groups at an average substitution ratio of 1 mole CM per 2 mole glucose units of dextran. The complexes were formed by reacting each of the two platinum (II) derivatives with carboxymethyl-dextran (CM-dex) at room temperature (RT) or at 37 degrees C in an aqueous solution. The complexing rate depended on temperature, ratio of platinum (II) compounds to CM-dex in the reaction mixture, and time of reaction. Experiments were performed with two CM-dex preparations, derived from dex T-10 (Mr-10,000) and from dex T-40 (Mr-40,000). Soluble cis-DDP and cis-aq complexes formed with CM-dex T-10 and CM-dex T-40 could carry up to 15 mole or 60 mole of the platinum (II) compounds per 1 mole CM-dex, respectively but higher complexing ratios resulted in complex precipitation. Reactivity of cis-aq with CM-dex was higher than that of cis-DDP. NaCl interfered with complex formation, but did not cause dissociation of already formed complexes. The binding of cis-DDP and cis-aq to CM-dex is, however, reversible since the drugs could be exchanged by other acceptors of higher affinity to platinum (II) such as O-phyenlenediamine, or DNA.     

In vivo delayed rejection of tumors and inhibition of delayed - type hypersensitivity by HT-29 human colonic adenocarcinoma cell line

Summary Cis-diamminedichloroplatinum (II) (cis-DDP) and its structural analogue cis-diamminediaquoplatinum(II) nitrate (cis-aq) were complexed via an intermediate dextran carrier to antibodies specifically reactive with B lymphoma cells (38C-13). The potential use of these drugs in site-directed immunotargeting was evaluated. The two platinum(II) compounds were previously shown to form pharmacologically active complexes with carboxymethyl dextran (CM-dex). For the purpose of preparing drug-antibody complexes, CM-dex was first conjugated to idiotypic antibodies that recognize a specific membrane IgM on the B lymphoma cells. The conjugates were prepared by a modified water-soluble carbodiimide method in which N-hydroxysuccinimide was used to enhance the coupling reaction. The conjugation was followed by separation of the CM-dex-IgG conjugates from unconjugated CM-dex or IgG. The platinum(II) compounds were then complexed to the CM-dex-IgG resulting in complexes carrying up to 50 mole drug/mole IgG. Both cis-DDP and cis-aq complexes of CM-dex-antibody conjugates maintained most of the original cell-binding activity of the antibodies. An in vitro assay was used to demonstrate selective binding to tumor cells in which the target cells were treated with specific immune complexes and washed before culture. In this assay the specific complexes showed preferential cytotoxicity for the B lymphoma cells in comparison to the free drugs, drug CM-dex, or nonspecific immune complexes.     

Preparation, characterization, and antitumor activity of water-soluble aminoalkanol platinum(II) complexes

A new series of highly water-soluble aminoalkanol platinum(II) complexes have been synthesized and characterized by elemental analysis, conductance, IR, and 195Pt NMR. Preliminary in vitro and in vivo screening tests for antitumor activities of these complexes against L1210 murine leukemia were performed. In general, these compounds were far less cytotoxic than cisplatin and possessed only a moderate degree of antitumor activity.     

Synthesis and characterization of complexes of p-isopropyl benzaldehyde and methyl 2-pyridyl ketone thiosemicarbazones with Zn(II) and Cd(II) metallic centers. Cytotoxic activity and induction of apoptosis in Pam-ras cells.

It is known that metallic complexes of methyl 2-pyridyl ketone thiosemicarbazone (HL1) and p-isopropyl benzaldehyde thiosemicarbazone (HL2) may have potential antitumor activity. We have prepared complexes of HL1 and HL2 with Zn(II) and Cd(II). The cytotoxic activity shown by these compounds against cell lines sensitive and resistant to cis-diamminedichloroplatinum(II) (cis-DDP) indicates that coupling of HL1 and HL2 to Zn(II) and Cd(II) centers may result in metallic complexes with important biological properties since they display IC50 values in a microM range similar to that of the antitumor drug cis-DDP. Moreover, it is interesting to note that the Zn/HL2 complex exhibits specific cytotoxic activity against Pam-ras cells (cis-DDP resistant cells which over-express the H-ras oncogene) with an in vitro therapeutic index of 3.26 versus 0.78 for cis-DDP. Treatment of Pam-ras cells with the IC50 value of the Zn/HL2 compound induces a 'DNA ladder' (fragmentation of genomic DNA in nucleosome units) indicative of apoptosis in this ras-transformed cell line. In contrast, a 'DNA smear' (non-specific fragmentation of genomic DNA) is observed in Pam 212 normal cells treated with the IC50 of this compound. The analysis by circular dichroism (CD) spectroscopy of the interaction of the Zn/HL2 compound with calf thymus DNA (CT DNA) indicates that it produces stronger alterations on the double helix conformation than cis-DDP. So, these results suggest that Zn/HL2 may be considered a potential antitumor agent.     

Antitumor steroidal-cis-platinum(II)-o-catecholato conjugates: preliminary evaluation on breast cancer MCF-7 cells

Six new steroidal-cis-platinum(II)-o-catecholato complexes (5–9 and 15) were prepared by treatment of either [4-(2-aminoethyl)1,2-benzenediolato(2-)-O, O′]-bis(triphenylphosphine)platinum(II) or [3,4-dihydroxybenzenepropionic acid (2-)-O³, O⁴]-bis-(triphenylphosphine)platinum(II) with appropriate functionalized steroids. The biological effect of the air-stable conjugates on a human breast tumor cell line, MCF-7, was compared with that of cis-dichloro-diaminoplatinum(II) (cis-DDP). The activity of the new compounds proved to be of the same order of magnitude as cis-DDP.     

A cycloplatinated compound of p-isopropylbenzaldehyde thiosemicarbazone and its chloro-bridged derivative induce apoptosis in cis-DDP resistant cells which overexpress the H-ras oncogene.

cis-Diamminedichloroplatinum(II) (cis-DDP) is a widely used antitumour drug which produces important damage on the DNA inducing apoptosis in several cell lines. We have analyzed the cytotoxic activity of novel cyclometallated complexes of p-isopropylbenzaldehyde thiosemicarbazone (p-is.TSCN) and their dimeric chloro-bridged derivatives in murine keratinocytes transformed by the H-ras oncogene which are resistant to cis-DDP (Pam-ras cells). The data show that, in contrast with cis-DDP, the tetrameric cycloplatinated complex [Pt(p-is.TSCN)]4 and its dimeric chloro-bridged derivative [Pt(microCl)(p-is.TSCN)]2 have a good in vitro therapeutic index when comparing the cytotoxicity in Pam-ras cells to normal murine keratinocytes (Pam 212 cells) since they induce cell death in Pam-ras cells at drug concentrations significantly lower than those needed to kill Pam 212 cells. At equitoxic doses (IC90), both complexes produce characteristic features of apoptosis in Pam-ras cells together with a drastic decrease in levels of H-ras protein. These effects are not observed when the cells are treated with the IC90 of the cis-DDP drug nor the p-is.TSCN ligand. Altogether, these results suggest that the platinum compounds [Pt(p-is.TSCN)]4 and [Pt(microCl)(p-is.TSCN)]2 might have potential as antitumour agents in view of their specific induction of apoptosis in cis-DDP resistant cells.     

Pd(II)- and Pt(II)-cimetidine complexes. Crystal structure of trans-[Pt(N,S-cimetidine)(2)]Cl(2)(*)12H(2)O

The influence of cimetidine on patients under cisplatin treatment for cancer is controversial. It has moderate or no effects on several types of cancer and its effects on the nephrotoxicity induced by cisplatin are uncertain. To examine the binding properties and antiproliferative effects of the known anticancer noble metals, cimetidine (cim) was complexed to platinum(II) and palladium(II). The crystal structure of the Pt-cim compound shows two molecules of cimetidine coordinated to the metal through thioether sulfur and imidazolic nitrogen whereas spectroscopic studies in solution for Pd-cim reveal that the ratio of the metal to cimetidine is 1:1 with identical coordination environments. To determine the antitumor activity of the drugs, the interaction of the metallic complexes and free cimetidine with DNA was assessed. Their cytotoxic activity was compared with that of cisplatin.     

Modulation of platinum antitumor drug binding to DNA by linked and free intercalators

We report the DNA binding site preferences of the novel molecule AO-Pt, in which the anticancer drug dichloro(ethylenediamine)platinum(II) is linked by a hexamethylene chain to acridine orange. The sequence specificity of platinum binding was mapped by exonuclease III digestion of 165 and 335 base pair restriction fragments from pBR322 DNA. Parallel studies were carried out with the unmodified anticancer drugs cis-diamminedichloroplatinum(II) (cis-DDP) and dichloro(ethylenediamine)platinum(II), [Pt(en)Cl2]. Oligo(dG) sequences are the most prevalent binding sites for AO-Pt, with secondary binding occurring mainly at d(AG) sites. cis-DDP and [Pt(en)Cl2] bind less readily to the secondary sequences, with cis-DDP showing greater binding site selectivity than [Pt(en)Cl2]. The DNA intercalator ethidium bromide promotes binding of [Pt(en)Cl2] and cis-DDP to many sites containing d(CGG) and, to a lesser extent, d(AG) sequences. AO-Pt exhibits enhanced binding to these sequences without the need for an external intercalator. Unlinked acridine orange, however, does not promote binding of [Pt(en)Cl2] and cis-DDP to d(CGG) and d(AG) sequences. These results are discussed in terms of the sequence preferences, stereochemistry, and relative residence times of the intercalators at their DNA binding sites. By modulating local structure in a sequence-dependent manner, both linked and, in the case of ethidium, free intercalators can influence the regioselectivity of covalent modification of DNA by platinum antitumor drugs.     

Antitumor activity of phenylene bridged binuclear bis(imino-quinolyl)palladium(II) and platinum(II) complexes

Antitumor effects of a known bis(imino-quinolyl)palladium(II) complex 1 and its newly synthesized platinum(II) analogue 2 were evaluated against human breast (MCF-7) and human colon (HT-29) cancer cell lines. The complexes gave cytotoxicity profiles that were better than the reference drug cisplatin. The highest cytotoxic activities were pronounced in complex 2 across the two examined cancer cell lines. Both compounds represent potential active drugs based on bimetallic complexes.     

Synthesis and characterization of Pd(II) and Pt(II) complexes of p-isopropylbenzaldehyde N-protected thiosemicarbazones. Cytotoxic activity against ras-transformed cells.

Cytotoxicity tests in tumor cells sensitive to cis-DDP (HL-60, JURKAT, Hela and 3T3) and in tumor cells transformed by ras oncogenes and resistant to cis-DDP (Pam 212-ras) show that cyclometallated complexes 1a [Pd(p-is.TSCN-NHMe)]4, 2b [Pt(p-is.TSCN-NMe2)]4 and 4a [Pd(p-is.TSCN-NHex)]4 may be endowed with specific cytotoxic properties. In fact, these three novel metal-thiosemicarbazone compounds kill Pam 212-ras cells through apoptosis induction. These results, together with others recently published, indicate that the design and synthesis of metallated-thiosemicarbazone compounds may lead to the discovery of novel antitumor agents able to circumvent cis-DDP resistance, in particular tumor cell lines.     

Synthesis and characterization of novel palladium(II) complexes of bis(thiosemicarbazone). Structure, cytotoxic activity and DNA binding of Pd(II)-benzyl bis(thiosemicarbazonate).

The preparation of palladium(II) complexes of 3,5-diacyl-1,2,4-triazole bis(thiosemicarbazone) (H2L2), 2,6-diacylpyridine bis(thiosemicarbazone) (H2L3) and benzyl bis(thiosemicarbazone) (H2L4) is described. The new complexes [PdCl2(H2L2)] (1), [PdCl2(H2L3)] (2) and [PdL4].DMF (3) have been characterized by elemental analyses and spectroscopic studies (IR, 1H NMR and UV-Vis). The crystal and molecular structure of PdL4.DMF (L = bideprotonated form of benzyl bis(thiosemicarbazone)) has been determined by single-crystal X-ray diffraction: green triclinic crystal, a = 10.258(5), b = 10.595(5), c = 11.189(5) A, alpha = 97.820(5), beta = 108.140(5), gamma = 105.283(5) degrees, space group P1, Z = 1. The palladium atom is tetracoordinated by four donor atoms (SNNS) from L4 to form a planar tricyclic ligating system. The testing of the cytotoxic activity of compound 3 against several human, monkey and murine cell lines sensitive (HeLa, Vero and Pam 212) and resistant to cis-DDP (Pam-ras) suggests that compound 3 might be endowed with important antitumor properties since it shows IC50 values in a microM range similar to those of cis-DDP [cis-diamminedichloroplatinum(II)]. Moreover, compound 3 displays notable cytotoxic activity in Pam-ras cells resistant to cis-DDP (IC50 values of 78 microM versus 156 microM, respectively). On the other hand, the analysis of the interaction of this novel Pd-thiosemicarbazone compound with DNA secondary structure by means of circular dichroism spectroscopy indicates that it induces on the double helix conformational changes different from those induced by cis-DDP.     

Platinum complexes with anticancer potential and their evaluation by a colorimetric lambda prophage induction assay

A simple biochemical phage induction assay (BIA) showed significant activity with 90% of the antitumor platinum compounds tested and lack of activity for all Pd(II) compounds and Pt(II) cationic complexes, compounds that are expected to be inactive. Structure-activity relationships for a large number of chemicals can be studied simultaneously by this simple, rapid, inexpensive and quantitative biochemical assay. Fifty-three platinum complexes were tested, including a number of ethylenediamines synthesized for this work. The magnitude of inducing activity varied over a 25-fold range; differences among analogs reflected structural differences in a chemically consistent manner. Seven platinum complexes showed greater activity than that of cis-diamminedichloroplatinum(II) (cisplatin, cis-DDP), while other compounds appeared to be substantially less toxic. The assay was predictive for most compounds with very high or very low activity in vivo against L1210. For compounds with intermediate levels of activity, no correlation between inducing and antitumor activity was observed.     

Apoptosis induction and inhibition of H-ras overexpression by novel trans-[PtCl2(isopropylamine)(amine')] complexes

Hitherto, it has been generally accepted as a paradigm of the biochemical pharmacology of platinum antitumor drugs that a cis configuration of the leaving groups is necessary for antitumor activity of platinum compounds. However, it has been recently observed that certain trans-platinum complexes have both in vitro and in vivo antitumor activity. We previously reported the synthesis, characterization and cytotoxic activity against ras-transformed cells of several trans-[PtCl2LL'] complexes where L and L' are asymmetric aliphatic amines (L = dimethylamine and butylamine, L' = isopropylamine). The results reported in this paper show that the compounds trans-[PtCl2(isopropylamine)(dimethylamine)] and trans-[PtCl2(isopropylamine)(butylamine)] kill Pam 212-ras cisplatin resistant cells through apoptosis induction. Moreover, Western blot data show that both compounds inhibit overexpression of H-ras oncogene in Pam 212-ras cells. Altogether, these data indicate that, in contrast with cis-DDP, the apoptotic activity of these novel trans-Pt(II) compounds in ras-transformed cells is associated with their ability to abolish ras-overexpression.     

Synthesis characterization and cytotoxicity studies of platinum(II) complexes with reduced amino pyridine schiff base and its derivatives as ligands

A series of reduced amino pyridine Schiff base platinum(II) complexes were prepared as potential anticancer drugs, and characterized by NMR, IR spectroscopy, elemental analysis, and molar conductivity. UV and CD results showed the binding mode between these compounds and salmon sperm DNA may be intercalation. The cytotoxicity of these complexes was validated against A549, Hela, and MCF-7 cell lines by MTT assay. Some complexes exhibited better cytotoxic activity than cisplatin against Hela and MCF-7 cell lines.     

The interaction of platinum and anthrapyrazole antitumor drugs with mouse thymocytes studied by terbium fluorescence

The fluorescent lanthanide, terbium has been employed to study the effect of a series of platinum and anthracycline drugs and an anthrapyrazole (oxanthrazole) on terbium binding to mouse thymocytes. It was observed that terbium fluorescence intensity was markedly decreased by two platinum drugs (cis-dichlorodiammine platinum(II) (cis-DDP) and cis-dichloro-trans-dihydroxybis(isopropylammine) platinum(IV) (CHIP)) and an anthrapyrazole (oxanthrazole), but that the lipophylic derivative cis-diammine-1,1-cyclobutanedicarboxylate platinum(II) had a small but significant effect and the anthracyclines (at low concentrations) had no effect. The calcium channel blocker, verapamil also had no effect. The effect of cis-DDP was markedly dependent on ionic strength in contradistinction to CHIP. The decreases in phosphorescence decay produced by cis-DDP also showed a marked dependence on ionic strength. It is proposed that cis-DDP interacts with the membrane primarily by a charge effect, but that CHIP may produce a conformational change in the membrane. These data are interesting, since the lipophylic platinum drugs (CHIP and CBDCA) also increased significantly the amount of bound intracellular calcium, but all the drugs decreased mitogen-stimulated calcium uptake into mouse thymocytes.     

Induction of apoptosis and necrosis in lymphocytes by the cis-Pt(II) complex of 3-aminoflavone in comparison with cis-DDP

cis-Diamminedichloroplatinum(II) (cis-DDP) is one of the most widely administrated antitumor drugs. However, the use of cis-DDP is severely limited because of its toxic side effects. Therefore, efforts are concentrated on the development of improved platinum compounds with a broader activity spectrum and effectiveness in chemotherapy, but lower toxicity. Beneficial properties of flavonoids, e.g. their antitumor activity, encouraged scientists to synthesize cis-bis(3-aminoflavone)dichloroplatinum(II). Abilities of these compounds to induce apoptosis and necrosis were compared by use of trypan blue, fluorochrome staining (Hoechst 33258/propidium iodide double staining) and TUNEL assays. The cytotoxicity was evaluated by MTT. The results obtained show that the cis-Pt(II) complex of 3-aminoflavone is less toxic than cis-DDP. However, the former compound has a faster rate of apoptosis induction in lymphocytes than the latter. The cis-Pt(II) complex of 3-aminoflavone induces apoptosis in normal lymphocytes to a lesser degree and could be a potential antitumor drug.     

DNA-protein cross-linking in L1210 cells sensitive and resistant to cis-diamminedichloroplatinum (II)

The role of restricted cellular accumulation of cis-diamminedichloroplatinum(II) (cis-DDP) and altered repair of DNA-Pt-protein cross-links in the mechanism of L1210 murine leukemia cell resistance was examined. An immunochemical method was used to analyze the formation and removal of DNA-Pt-protein complexes in L1210 cells sensitive and resistant to cis-DDP. The accumulation of Pt into the cells and the binding of Pt to the DNA was measured by atomic absorption spectroscopy. The results demonstrated that both decreased accumulation of the drug and the rate of DNA-Pt protein cross-link removal may be important factors in L1210 cell resistance to cis-DDP.Abbreviations AAS atomic absorption spectroscopy - cis-DDP cis-diamminedichloroplatinum(II)     

Synthesis and cytotoxic activities of estrone and estradiol cis-dichloroplatinum(II) complexes

Sixteen platinum(II) complexes of estrone and estradiol were synthesized in this work to evaluate their cytotoxic activity against several cancer cell lines including estrogen dependent and independent ones. The synthesis of all the complexes was done in three steps. The reaction of steroids with dibromoalkanes was followed by a reaction of the bromoalkyl steroids with 2-(aminomethyl)pyridine or 2-(2-aminoethyl)pyridine. The last step was a reaction of steroidal diamino ligands with potassium tetrachloroplatinate to obtain the desired platinum(II) complexes. Cytotoxicity assays showed that most of the complexes prepared are active against the cancer cell lines used—CEM, U-2 OS, MCF7, MCF7 AL, MDA-MB-468, BT-474, BT-549, and BJ fibroblasts. The six-membered platinum complexes are more active than five-membered ones.     

Ligands of second generation platinum analogs decrease both platinum-induced DNA cross-linking and its ability to interact with 1-beta-D-arabinofuranosyl cytosine to potentiate cytotoxic efficacy

We evaluated the cytotoxic and DNA cross-linking (CL) ability of four second generation platinum coordination complexes (TNO-6, JM-89, JM-8 and JM-9) delivered alone or in combination with 1-beta-D-arabinofuranosyl cytosine (ara-C) to human colon cancer cells (LoVo). Cell survival varied markedly as a function of the particular substitution moiety. JM-8 and JM-9 were virtually ineffective, even at concentrations as high as 50 micrograms/ml. At that concentration cis-diamminedichloroplatinum(II) (cis-DDP) killed greater than 99.99% of the cells. JM-82 was slightly more active while TNO-6 was the only derivative with appreciably higher cytotoxic activity due to an abrogation of the shoulder region of the type C survival curve. The highest CL effect was observed for cis-DDP followed closely by TNO-6. Very little CL effects were demonstrated for the other three analogs JM-82, JM-8 and JM-9 when measured 6 h after treatment. The combination of cis-DDP and ara-C augmented 10-fold the cytotoxic activity of cis-DDP alone, an effect accompanied by an almost 2-fold increase in CL; every other analog failed to interact in a potentiating manner (either cytotoxicity, or CL at 6 h) with the antimetabolite. Thus, it appears clear that the associated moieties of the Pt coordination complex play a fundamental role in reducing the interaction of the analogs with DNA (as reflected by the decreased CL and cytotoxic effects produced by each agent alone) and in totally preventing their interaction with ara-C to yield a potentiating lethal effect.     

DNA targeted platinum complexes: synthesis, cytotoxicity and DNA interactions of cis-dichloroplatinum(II) complexes tethered to phenazine-1-carboxamides

A series of intercalator-tethered platinum(II) complexes PtLCl2 have been prepared, where L are the diamine ligands N-[2-[(aminoethyl)amino]ethyl]-phenazine-1-carboxamide, N-[3-[(2-aminoethyl)amino]propyl]-phenazine-1-carboxamide, N-[4-[(2-aminoethyl)amino]butyl]-phenazine-1-carboxamide and N-[5-[(aminoethyl)amino]pentyl]-phenazine-1-carboxamide. Measurements of the time-course of unwinding of supercoiled pUC19 plasmid DNA by the phenazine complexes PtLCl2 reveal that the presence of the intercalator leads to enhanced rates of DNA platination when compared with the complex Pt(en)Cl2. The platinum(II) complexes where the polymethylene linker chain contains three, four or five carbon atoms are considerably more cytotoxic against murine P388/W than either cisplatin, Pt(en)Cl2, or the metal-free ligands themselves.