Stereoselective metabolism of dexrazoxane (ICRF-187) and levrazoxane (ICRF-186)

A chiral HPLC method has been developed to separate razoxane (ICRF-159) in blood plasma into its enantiomers dexrazoxane (ICRF-187) and levrazoxane (ICRF-186). Dexrazoxane is clinically used as a doxorubicin cardioprotective agent and little is known of its in vivo metabolism. After intravenous administration of 20 mg/kg of razoxane to rats, the razoxane was eliminated from the plasma with a half-time of approximately 20 min. The levrazoxane:dexrazoxane ratio continuously increased with time to a value of 1.5 at 150 min, indicating that dexrazoxane is metabolized faster than levrazoxane. These results, confirmed with studies on liver supernatants, are consistent with the hypothesis that dihydropyrimidine amidohydrolase in the liver and kidney is responsible for the preferential metabolism of dexrazoxane in the rat compared to levrazoxane. It is possible that on a dose-per-dose basis marginally higher therapeutic levels of levrazoxane might be achieved in the heart tissue for a longer time compared to dexrazoxane due to dihydropyrimidine amidohydrolase-based metabolism in the liver and kidney. However, given the relatively small difference in elimination of the two enantiomers, it would be difficult to predict from this study whether or not dexrazoxane or levrazoxane might be more efficacious in reducing cardiotoxicity.     

The effect of the catalytic topoisomerase II inhibitor dexrazoxane (ICRF-187) on CC9C10 hybridoma viability and productivity

The effect of dexrazoxane on monoclonal antibody (Mab) production by CC9C10 hybridoma cells was investigated. Dexrazoxane is a catalytic inhibitor of DNA topoisomerase II. DNA topoisomerase II has a critical role in DNA metabolism and its inhibition by dexrazoxane can prevent completion of cytokinesis. Incubation of hybridomas with dexrazoxane was found to increase specific monoclonal antibody production by up to four-fold. However, due to the growth inhibitory effects of dexrazoxane the total Mab yield decreased by 40%. Under high density culture conditions(defined here as 10⁶ cells ml^-1) specific monoclonal antibody production increased by up to 37%, which was, however, accompanied by up to a 48% decrease in Mab yield. Hybridomasthat were incubated with dexrazoxane significantly increased in size due to the inhibition of cytokinesis. Dexrazoxane was also observed to induce a delayed apoptosis in the hybridomas. The caspase inhibitor Z-VAD-fmk slightly decreased the apoptotic effects of dexrazoxane. Preincubation with the caspase inhibitorZ-Asp-CH2-DCB had no effect on dexrazoxane-treated hybridomas, but it did have antiapoptotic effects on the untreated hybridomas which normally undergo a significant basal level of apoptosis. In conclusion, dexrazoxane-induced growth inhibition (which results in higher specific antibody production) and apoptosis inhibition (which results in prolonged viability) has the potential to significantly enhance the productivity of hybridoma cell cultures. This revised version was published online in August 2006 with corrections to the Cover Date.     

The iron chelating cardioprotective prodrug dexrazoxane does not affect the cell growth inhibitory effects of bleomycin

The clinical use of bleomycin is limited by a dose-dependent pulmonary toxicity. Bleomycin is thought to be growth inhibitory by virtue of its ability to oxidatively damage DNA through its complex with iron. Our previous preclinical studies showed that bleomycin-induced pulmonary toxicity can be reduced by pretreatment with the doxorubicin cardioprotective agent dexrazoxane. Dexrazoxane is thought to protect against iron-based oxygen radical damage through the iron chelating ability of its hydrolyzed metabolite ADR-925, an analog of ethylenediaminetetraacetic acid (EDTA). ADR-925 quickly and effectively displaced either ferrous or ferric iron from its complex with bleomycin. This result suggests that dexrazoxane may have the potential to antagonize the iron-dependent growth inhibitory effects of bleomycin. A study was undertaken to determine if dexrazoxane could antagonize bleomycin-mediated cytotoxicity using a CHO-derived cell line (DZR) that was highly resistant to dexrazoxane through a threonine-48 to isoleucine mutation in topoisomerase IIalpha. Dexrazoxane is also a cell growth inhibitor that acts through its ability to inhibit the catalytic activity of topoisomerase II. Thus, the DZR cell line allowed us to examine the cell growth inhibitory effects of bleomycin in the presence of dexrazoxane without the confounding effect of dexrazoxane inhibiting cell growth. The cell growth inhibitory effects of bleomycin were unaffected by pretreating DZR cells with dexrazoxane. These results suggest that dexrazoxane may be clinically used in combination with bleomycin as a pulmonary protective agent without adversely affecting the antitumor activity of bleomycin.     

In vivo inhibition of E. coli growth by a Ru(II)/Pt(II) supramolecule [(tpy)RuCl(dpp)PtCl2](PF6)

Supramolecular complexes consisting of ruthenium chromophores and a cisplatin unit represent an emerging class of bioactive molecules of interest as anti-cancer agents. Although the ability of Ru(II)/Pt(II) heteronuclear complexes to bind to DNA has been demonstrated, the in vivo activity of these complexes has not yet been reported. In the present work, we report the anti-bacterial activity of the complex [(tpy)RuCl(dpp)PtCl(2)](PF(6)) (where dpp=2,3-bis(2-pyridyl)pyrazine, tpy=2,2':6',2'-terpyridine). The impact on bacterial cell growth of exposure to different concentrations of [(tpy)RuCl(dpp)PtCl(2)](PF(6)) and cisplatin was studied. The bioactivity of this complex was found to be due to the presence of the cis-PtCl(2) moiety, as the monometallic synthon [(tpy)RuCl(dpp)](PF(6)) did not inhibit bacterial cell growth.     

Comparative studies on the mechanism of cytotoxic action of novel platinum II complexes with pyrazole ligands

In search for new platinum-based anticancer drugs, four cisplatin analogues, which contain pyrazole rings as non-leaving ligands, have been synthesized: cis-PtCl(2)(3,5-DM HMPz)(2), cis-PtCl(2)(Pz)(2), cis-PtCl(2)(ClMPz)(2), and cis-PtCl(2)(HMPz)(2), where Pz=pyrazole, H=hydroxyl, M=methyl. We tested their cytotoxicity, apoptosis induction ability, DNA damaging and modification properties comparing them in respect to the parent compound. The cytotoxic activity of these platinum pyrazole complexes toward the murine leukemia cell line was 2.9-3.8 times lower than actvity of cisplatin. The tested compounds varied in their mechanism of action by producing different DNA lesions. The most interesting compound seems to be the complex with chloromethyl groups at N1 of pyrazole rings, which exhibited the highest ability to form bifunctional adducts with DNA in vitro.     

Effects of the DNA intercalators 4'-(9-acridinylamino)methanesulfon-m-anisidide and 2-methyl-9-hydroxyellipticinium on topoisomerase II mediated DNA strand cleavage and strand passage

DNA topoisomerase II is believed to be the enzyme that produces the protein-associated DNA strand breaks observed in mammalian cell nuclei treated with various intercalating agents. Two intercalators--4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA, amsacrine) and 2-methyl-9-hydroxyellipticinium (2-Me-9-OH-E+)--differ in their effects on protein-associated double-strand breaks in isolated nuclei. m-AMSA stimulates their production at all concentrations, whereas 2-Me-9-OH-E+ stimulates at low concentrations and inhibits at high concentrations. We have reproduced these differential effects in experiments carried out in vitro with purified L1210 DNA topoisomerase II, and we have found that concentrations of 2-Me-9-OH-E+ above 5 microM prevent the trapping of DNA-topoisomerase II cleavable complexes irrespective of the presence of m-AMSA. It also stimulated topoisomerase II mediated DNA strand passage, again with or without inhibitory amounts of m-AMSA (this result suggests that extensive intercalation by 2-Me-9-OH-E+ destabilized the cleavable complexes). From these data, it is concluded that intercalator-induced protein-associated DNA strand breaks observed in intact eukaryotic cells and isolated nuclei are generated by DNA topoisomerase II and that intercalators can affect mammalian DNA topoisomerase II in more than one way. They can trap cleavable complexes and inhibit DNA topoisomerase II mediated DNA relaxation (m-AMSA and low concentrations of 2-Me-9-OH-E+) or destabilize cleavable complexes and stimulate DNA relaxation (high concentrations of 2-Me-9-OH-E+).     

Analysis of bisdioxopiperazine dexrazoxane binding to human DNA topoisomerase II alpha: decreased binding as a mechanism of drug resistance

Topoisomerase II is an ATP-operated clamp that effects topological changes by capturing a double stranded DNA segment and transporting it through another DNA molecule. Despite the extensive use of topoisomerase II-targeted drugs in cancer chemotherapy and the impact of drug resistance on the efficacy of treatment, much remains unknown concerning the interactions between these agents and topoisomerase II. To identify the interaction of the bisdioxopiperazine dexrazoxane (ICRF-187) with topoisomerase II, we developed a rapid gel-filtration assay and characterized the binding of ((3)H)-dexrazoxane to human topoisomerase II alpha. Dexrazoxane binds to human topoisomerase II alpha in the presence of DNA and ATP with an apparent K(d) of 23 microM and a stoichiometry of 1 drug molecule per enzyme dimer. Various N-terminal single amino acid substitutions in human topoisomerase II alpha that were previously shown to confer specific bisdioxopiperazine resistance either totally abolished drug binding or resulted in less efficient binding. The effect of the various mutations on drug binding correlated well with their effect on drug resistance in vivo and in vitro. Interestingly, an altered active site tyrosine mutant of human topoisomerase II alpha, which is incapable of carrying out DNA strand passage, was unable to bind dexrazoxane, which agrees with the drug's proposed mechanism of action late in the topoisomerase II catalytic cycle. The direct correlation between the level of drug binding and dexrazoxane resistance is consistent with a decreased drug binding mechanism of action for these dexrazoxane resistance conferring mutations.     

Topoisomerase II antagonism and anticancer activity of coordinated derivatives of [RuCl(2)(C(6)H(6))(dmso)

Topoisomerase II poisoning and anticancer activity by the organometallic compound [RuCl(2)(C(6)H(6))(dmso)] was shown by us in an earlier study [Biochemistry 38 (1999) 4382]. Since high concentrations of this complex were required to achieve either effects, we have synthesized four derivatives of this complex in which the dimethyl sulphoxide group on the ruthenium atom was replaced with pyridine, 3-aminopyridine, p-aminobenzoic acid, and aminoguanidine. Three of these molecules showed enhanced potency of topoisomerase II poisoning and consequently also showed higher anticancer activity in breast and colon carcinoma cells in vitro. Detailed analysis of the molecular action of these compounds on topoisomerase II activity was carried out using the classical relaxation and cleavage activity of the enzyme, which revealed that the compounds poison topoisomerase II by freezing the enzyme and enzyme-cleaved DNA in a ternary "cleavage complex". The cleavage complex is implicated in the anti-neoplastic activity of these compounds. DNA interaction studies showed that these compounds interact with DNA in much the same way as [RuCl(2)(C(6)H(6))(dmso)], by external binding of the DNA helix. This is unlike most other topoisomerase II poisons, which predominantly interact with DNA through intercalation with the double helix.     

Preparation and cell growth inhibitory activity of [PtR(2)L(2)] (R=polyfluorophenyl,L(2)=diene,cyclohexane-1,2-diamine (chxn) or cis-(dimethyl sulfoxide)(2)) and the X-ray crystal structure of [Pt(C(6)F(5))(2)(cis-chxn)

A range of [PtR(2)(chxn)] (R=C(6)F(5), o-HC(6)F(4), p-HC(6)F(4), p-MeOC(6)F(4) or 3,5-H(2)C(6)F(3); chxn=cyclohexane-1,2-diamine) and cis-[PtR(2)(dmso)(2)] (R=C(6)F(5), p-HC(6)F(4) or p-MeOC(6)F(4); dmso=dimethyl sulfoxide) complexes have been prepared from the corresponding [PtR(2)(diene)] (diene=cis,cis-cycloocta-1,5-diene (cod), hexa-1,5-diene (hex), norbornadiene (nbd) or dicyclopentadiene (dcy)) derivatives and have been spectroscopically characterized. A representative crystal structure of [Pt(C(6)F(5))(2)(cis-chxn)] was determined and shows a slightly distorted square planar geometry for platinum with chxn virtually perpendicular to the coordination plane. The biological activity against L1210 and L1210/DDP cell lines of these compounds together with the behaviour of other organoplatinum complexes, [PtR(2)L(2)] (L(2)=ethane-1,2-diamine (en) or cis-(NH(3))(2)) have been determined. Despite the use of relatively inert fluorocarbon anions as leaving groups, moderate-high cell growth inhibitory activity is observed. None of the fluorocarbon complexes displayed any cross resistance with cisplatin.     

Synthesis, characterization, and cytotoxic activity of copper(II) and platinum(II) complexes of 2-benzoylpyrrole and X-ray structure of bis[2-benzoylpyrrolato(N,O)]copper(II)

Copper(II) and platinum(II) complexes of 2-benzoylpyrrole (2-BZPH) were synthesized and characterized with IR, 1H and 13C NMR spectroscopies and coordination geometry with ligands arranged in transoid fashion. The crystal structure of [Cu(II)(2-BZP)2] was determined by X-ray diffraction. Death of complex treated Jurkat cells was measured by flow cytometry. The bis-chelate complexes [Cu(II)(2-BZP)2] and [Pt(II)(2-BZP)2] adopt square-planar coordination geometry with ligands, arranged in transoid fashion. Concentrations of 1-10 microM Platinum(II) complexes reduced cell survival from 100% to 20%, in contrast to the copper(II) complex which caused no cell death at a concentration of 10 microM. While the Pt(II) complexes may have damaged DNA to induce cell death, treatment with the Cu(II) complex did not induce Jurkat cell death.     

Cleavage of DNA induced by 9-anilinoacridine inhibitors of topoisomerase II in the malaria parasite Plasmodium falciparum

Due to resistance by Plasmodium falciparum, the most virulent strain of the four species of human malaria parasites, to most currently used antimalarial drugs, development of new effective antimalarials is urgently needed. Derivatives of 9-anilinoacridine, an antitumor drug, have been shown to inhibit P. falciparum growth in culture and to inhibit parasite DNA topoisomerase II activity in vitro. Using KCl-SDS precipitation assay to detect the presence of protein-DNA complexes within parasite cells, an indicator of DNA topoisomerase II inactivation, derivatives containing 3,6-diNH(2) substitutions with 1'-electron donating (NMe(2), CH(2)NMe(2), NHSO(2)Me, OH, OMe), and 1'-electron withdrawing (SO(2)NH(2)) groups produced protein-DNA complexes. However, the antimalarial pyronaridine, 9-anilinoazaacridine, did not generate protein-DNA complexes, although it was capable of inhibiting P. falciparum DNA topoisomerase II activity in vitro. These results should prove useful in future designs of novel antimalarial compounds directed against parasite DNA topoisomerase II.     

Cadmium is a catalytic inhibitor of DNA topoisomerase II

Cadmium (Cd²⁺) is a highly toxic and carcinogenic metal that is an environmental and occupational hazard. DNA topoisomerase II is an essential nuclear enzyme and its inhibition can result in the formation of genotoxic and recombinogenic DNA double strand breaks. In this study we showed that cadmium chloride strongly inhibited the DNA decatenation activity of human topoisomerase IIα in the low micromolar concentration range and that its inhibitory effects were reduced by glutathione. Because the activity of topoisomerase II is strongly inhibited by thiol-reactive compounds this result suggested that cadmium may be binding to critical topoisomerase II cysteine thiols. Cadmium, however, did not stabilize DNA-topoisomerase II covalent complexes, as measured by the lack of formation of DNA double strand breaks. Hence, it is not likely to be a topoisomerase II poison. Consistent with the idea that cadmium cytotoxicity may be modulated by glutathione levels, buthionine sulfoximine pretreatment to decrease glutathione levels resulted in a greatly increased cadmium-induced cytotoxicity in K562 cells. The results of this study suggest that cadmium may exert some of its cell growth inhibitory, and possibly its toxicity and carcinogenicity, by inhibiting topoisomerase IIα through reaction with critical cysteine thiols.     

Inhibition of topoisomerase II catalytic activity by two ruthenium compounds: a ligand-dependent mode of action

The ability of two structurally different ruthenium complexes to interfere with the catalytic activity of topoisomerase II was studied to elucidate their molecular mechanism of action and relative antineoplastic activity. The first complex, [RuCl2(C6H6)(dmso)], could completely inhibit DNA relaxation activity of topoisomerase II and form a drug-induced cleavage complex. This strongly suggests that the drug interferes with topoisomerase II activity by cleavage complex formation. The bi-directional binding of [RuCl2(C6H6)(dmso)] to DNA and topoisomerase II was verified by immunoprecipitation experiments which confirmed the presence of DNA and ruthenium in the cleavage complex. The second complex, Ruthenium Salicylaldoxime, could not inhibit topoisomerase II relaxation activity appreciably and also could not induce cleavage complex formation, though its DNA-binding characteristics and antiproliferation activity were almost comparable to those of [RuCl2(C6H6)(dmso)]. The results suggest that the difference in ligands and their orientation around a metal atom may be responsible for topoisomerase II poisoning by the first complex and not by the second. A probable mechanism is proposed for [RuCl2(C6H6)(dmso)], where the ruthenium atom interacts with DNA and ligands of the metal atom form cross-links with topoisomerase II. This may facilitate the formation of a drug-induced cleavage complex.     

DNA intercalation and inhibition of topoisomerase II. Structure-activity relationships for a series of amiloride analogs

Among its many properties, amiloride is a DNA intercalator and topoisomerase II inhibitor. Previous work has indicated that the most stable conformation for amiloride is a planar, hydrogen-bonded, tricyclic structure. To determine whether the ability of amiloride to intercalate into DNA and to inhibit DNA topoisomerase II was dependent on the ability to assume a cyclized conformation, we studied the structure-activity relationship for 12 amiloride analogs. These analogs contained structural modifications which could be expected to allow or impede formation of a cyclized conformation. Empirical assays consisting of biophysical, biochemical, and cell biological approaches, as well as computational molecular modeling approaches, were used to determine conformational properties for these molecules, and to determine whether they intercalated into DNA and inhibited topoisomerase II. Specifically, we measured the ability of these compounds to 1) alter the thermal denaturation profile of DNA, 2) modify the hydrodynamic behavior of DNA, 3) inhibit the catalytic activity of purified DNA topoisomerase II in vitro, 4) promote the topoisomerase II-dependent cleavage of DNA, and 5) inhibit functions associated with DNA topoisomerase II in intact cells. Results indicated that only those analogs capable of cyclization could intercalate into DNA and inhibit topoisomerase II. Thus, the ability of amiloride and the 12 analogs studied to intercalate into DNA and to inhibit topoisomerase II appears dependent on the ability to exist in a planar, hydrogen-bonded, tricyclic conformation.     

Stereoselective hydrolysis of ICRF-187 (dexrazoxane) and ICRF-186 by dihydropyrimidine amidohydrolase

The enzymatic ring-opening hydrolyses of the doxorubicin cardioprotective agents (+)-(S)-ICRF-187 (dexrazoxane), (?)-(R)-ICRF-186, and rac-ICRF-159 by the enzyme dihydropyrimidine amidohydrolase (DHPase) have been studied. ICRF-187 underwent enzymatic ring-opening hydrolysis by DHPase 4.5 times faster than did ICRF-186. It was also shown that DHPase opens only one ring of ICRF-186 and does not act on this one-ring open hydrolysis product, as has been observed for ICRF-187. Differences in the rates at which the two optical isomers are acted upon by DHPase suggest that they could have differing protective effects. © 1994 Wiley-Liss, Inc.     

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.     

The displacement of iron(III) from its complexes with the anticancer drugs piroxantrone and losoxantrone by the hydrolyzed form of the cardioprotective agent dexrazoxane

Piroxantrone and losoxantrone are new DNA topoisomerase II-targeting anthrapyrazole antitumor agents that display cardiotoxicity both clinically and in animal models. A study was undertaken to see whether dexrazoxane or its hydrolysis product ADR-925 could remove iron(III) from its complexes with piroxantrone or losoxantrone. Their cardiotoxicity may result from the formation of iron(III) complexes of losoxantrone and piroxantrone. Subsequent reductive activation of their iron(III) complexes likely results in oxygen-free radical-mediated cardiotoxicity. Dexrazoxane is in clinical use as a doxorubicin cardioprotective agent. Dexrazoxane presumably acts through its hydrolyzed metal ion binding form ADR-925 by removing iron(III) from its complex with doxorubicin, or by scavenging free iron(III), thus preventing oxygen-free radical-based oxidative damage to the heart tissue. ADR-925 was able to remove iron(III) from its complexes with piroxantrone and losoxantrone, though not as efficiently or as quickly as it could from its complexes with doxorubicin and other anthracyclines. This study provides a basis for utilizing dexrazoxane for the clinical prevention of anthrapyrazole cardiotoxicity.     

Synthesis, characterization, and cytotoxic studies of alpha-diimine/1,2-diamine platinum(II) and palladium(II) complexes of selenite and tellurite and binding of some of these complexes to DNA.

Eleven new complexes of formula [M(NN)(XO3)] (where M is Pd(II) or Pt(II); NN is 2,2'-bipyridine, 1,10-phenanthroline, 2,2'-dipyridylamine, ethylenediamine or (+-)trans-1,2-diaminocyclohexane, and XO3(2-) is SeO3(2-) or TeO3(2-)) have been synthesized. These water soluble complexes have been characterized by chemical analysis and conductivity measurements as well as ultraviolet-visible and infrared spectroscopy. In these complexes the selenite or tellurite ligand coordinates to platinum(II) or palladium(II) as bidentate with two oxygen atoms. These complexes inhibit the growth of P 388 lymphocytic leukemia cells, their targets are DNA. The selenite complexes invariably show I.D.50 values less than cisplatin. However, the I.D.50 values of the tellurite complexes are usually higher than cisplatin, except that of [Pd(dach)(TeO3)] which has comparable I.D.50 values, as compared to cisplatin. [Pt(bipy)(SeO3)] and [Pd(bipy)(SeO3)] have been interacted with calf thymus DNA and bind to DNA through a coordinate covalent bond.     

Dissecting the cell-killing mechanism of the topoisomerase II-targeting drug ICRF-193

Topoisomerase II is an essential enzyme that is targeted by a number of clinically valuable anticancer drugs. One class referred to as topoisomerase II poisons works by increasing the cellular level of topoisomerase II-mediated DNA breaks, resulting in apoptosis. Another class of topoisomerase II-directed drugs, the bis-dioxopiperazines, stabilizes the conformation of the enzyme where it attains an inactive salt-stable closed clamp structure. Bis-dioxopiperazines, similar to topoisomerase II poisons, induce cell killing, but the underlying mechanism is presently unclear. In this study, we use three different biochemically well characterized human topoisomerase IIalpha mutant enzymes to dissect the catalytic requirements needed for the enzyme to cause dominant sensitivity in yeast to the bis-dioxopirazine ICRF-193 and the topoisomerase II poison m-AMSA. We find that the clamp-closing activity, the DNA cleavage activity, and even both activities together are insufficient for topoisomerase II to cause dominant sensitivity to ICRF-193 in yeast. Rather, the strand passage event per se is an absolute requirement, most probably because this involves a simultaneous interaction of the enzyme with two DNA segments. Furthermore, we show that the ability of human topoisomerase IIalpha to cause dominant sensitivity to m-AMSA in yeast does not depend on clamp closure or strand passage but is directly related to the capability of the enzyme to respond to m-AMSA with increased DNA cleavage complex formation.     

Molecular structure,solution chemistry and biological properties of the novel [ImH][trans-IrCl(4)(Im)(DMSO)], (I) and of the orange form of [(DMSO)(2)H][trans-IrCl(4)(DMSO)(2)], (II), complexes

The new iridium(III) complex, imidazolium[trans(DMSO,imidazole)tetrachloroiridate(III)], (I) (DMSO=dimethyl sulfoxide), and the orange form of [(DMSO)(2)H][trans(DMSO)(2)tetrachloroiridate(III)], (II) have been prepared and characterized, both in the solid state and in solution, by X-ray diffraction and by various physicochemical techniques. Single crystal X-ray diffraction studies point out that complex (II) is isomorphous to the ruthenium(III) analogue, [(DMSO)(2)H][trans-RuCl(4)(DMSO)(2)], (III). Crystallographic data are the following: a=16.028(2) A, b=24.699(3) A, c=8.262(1) A, in space group Pbca (Z=8) for (imidazolium)[trans(DMSO,imidazole)tetrachloroiridate(III)], (I); and a=9.189(2) A, b=16.511(4) A, c=14.028(3) A, beta=100.82(2) degrees in space group P2/n (Z=4) for [(DMSO)(2)H][trans(DMSO)(2)tetrachloroiridate(III)], (II). Visible absorption spectra show that both complexes are stable for several days, at pH 7.4, at room temperature. No significant chloride hydrolysis is observed, even at high temperature (70 degrees C), over 24 h. The extreme stability of these iridium(III) complexes within a physiological buffer was further assessed by (1)H NMR; in addition, cyclic voltammetry measurements evidenced a high stability of the oxidation state +3. Preliminary biological studies show that both complexes do not bind appreciably bovine serum albumin nor inhibit significantly the proliferation of representative human tumor cell lines, suggesting that hydrolysis of coordinated chlorides is a crucial feature for the biological properties and the antitumor activity of the parent ruthenium(III) complexes.