New Platinum, Titanium, and Ruthenium Complexes with Different Patterns of DNA Damage in Rat Ovarian Tumor Cells S

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(CANCER RESEARCH 51.2943-2948. June I. 1991] New Platinum, Titanium, and Ruthenium Complexes with Different Patterns of DNA Damage in Rat Ovarian Tumor Cells S. FrÃ¼haufand \V. J. Zeller1

InstÃlateof Toxicology and Chemotherapy, GermÃ¡nCancer Research Center, 6900 Heidelberg, Federal Republic of Germany

ABSTRACT carboxylato-platinum(II) (carboplatin). Their antitumor effect is mainly ascribed to a reaction of the platinum molecule with DNA protein cross-links (DPC), DNA interstrand cross-links (ISCL), nucleophilic sites on the DNA (1). Major adducts are intra- and DNA single strand breaks following treatment of experimental strand cross-links mainly formed by the binding of cisplatin to ovarian tumor cells (O-342) with five new metal complexes (three plati two neighboring guanines. Other types of adducts are inter num, one titanium, one ruthenium compounds) were investigated at 6, 24, strand cross-links between two guanines in complementary and 48 h after drug exposure and compared with their Â¡nvitro growth DNA strands and DNA protein cross-links. ISCL2 and DNA inhibitory potential. m-Diamminedichloroplatinum(II) (cisplatin, DDP) served as reference drug. The following new compounds were tested: 18- protein cross-links can be demonstrated by the alkaline elution crown-6-tetracarboxybis-diammineplatinum(II) (CTDP), c/Ã®-aminotris- assay (2). There is a correlation between the extent of inter methylenephosphonato-diamimneplatinum(II) (AMDP), cis-diammi- strand cross-link formation and cisplatin cytotoxicity, whereas necyclohexano-aminotrismethylenephosphonato-platinum(II) (DAMP), DNA protein cross-links seem to be of minor importance in diethoxybis-(l-phenylbutane-l,3-dionato)-titanium(IV)(budotitane), and this respect (3). fruni-indazolium-tetrachlorobisindazole-ruthenate(IH)(IndCR). At equi- In order to obtain a better therapeutic ratio, new metal molar concentrations DNA cross-linking activity of the platinum agents complexes continue to be designed. Our interest is focused on decreased in the order cisplatin, CTDP, AMDP, DAMP; this was paralleled by growth inhibition in a cell proliferation assay. CTDP- new platinum, titanium and ruthenium complexes. Although induced interstrand cross-linking occurred more slowly compared to some of these new substances were already tested in animal tumor systems and clinical trials, little is known about their cisplatin (DDP) (6 h: CTDP, 73 Â± 15 versus DDP, 365 Â± 72 rad equivalents), but reached a peak similar to cisplatin 24 h after exposure mechanism of action. (CTDP, 317 Â±68 versus DDP, 392 Â±116 rad equivalents). At this time Structures of the new metal complexes investigated are shown point in contrast to DDP no DNA protein cross-links were observed for in Fig. 1: CTDP is a compound containing two dicarboxylato CTDP (total cross-links: CTDP 310 Â±71, DDP 1987 Â±436 rad equiv complexes. In vitro its antitumor activity is equal to cisplatin alents). Thus, at 24 h, CTDP was found to be distinctly less reactive to (4) but its in vivo toxicity is considerably lower.' AMDP is proteins than DDP, and it is suggested that CTDP might be similar Â¡n another agent which displayed a favorable therapeutic ratio in its toxicity pattern to the structurally related compound carboplatin which a rat osteosarcoma (5). A third new platinum compound is was also reported to be less reactive to protein than DDP. By 48 h, DAMP, a DACH derivative. Compounds with the DACH CTDP- and DDP-induced interstrand cross-links were 65 Â±21 and 180 moiety are of special interest since they were found to lack Â±33 rad equivalents, respectively. Although at a lower level, by 24 h, cross-resistance to cisplatin (6). AMDP showed a ratio of ISCL to total cross-links (179 Â±39 versus 213 The titanium complex DBT entered clinical trial in 1986. Â±31 rad equivalents), which was comparable to CTDP. The second Such bis-/i-diketonato compounds were shown to be active in biphosphonate complex DAMP was the least active platinum compound in terms of DNA damage, effecting only 16 Â±7 rad equivalents ISCL tumors not responding to cisplatin (7). It has become clear and 63 Â±28 rad equivalents total cross-links; similar to DDP, DAMP from structure activity investigations that their therapeutic ac displayed a higher DPC fraction at 24 h. The titanium complex diethox- tivity is rather connected with the ji-diketonato ligands than ybis-(l-phenylbutane-l,3-dionato)-tita-nium(IV) showed dose-dependent with the hydrolyzable leaving groups, e.g., the ethoxy groups in inhibition of cell proliferation, while no significant DNA damage could DBT (8). For cisplatin, on the other hand, these hydrolyzable be detected with the alkaline elution technique. These results, together leaving groups are known to be of major importance for its with observations from other authors, indicating that space-filling planar reaction with DNA (9). Research into interactions of DBT with aromatic ring systems are important for its antitumor activity, suggest as DNA and correlations with cell growth inhibition may therefore possible mechanism of action of diethoxybis-(l-phenylbutane-l,3-dion- help to elucidate the mechanism of action of this new class of ato)-titanium(IV) intercalation into the DNA. Following administration anticancer agents. of the ruthenium compound IndCR only few ISCL and DPC were Ruthenium complexes are another class of metal coordina observed with a maximum at 6 h (ISCL, 15 Â±5; total cross-links, 49 Â± tion compounds. As ruthenium compounds were found to at 14 rad equivalents); thereafter both lesions were declining. Further tack the same initial site of DNA (N-7 of guanine) as cisplatin studies on the mechanism of action of this class of antitumor agents should take into account that in hypoxic tumor tissue the Ru(III)-ion of (10) their mode of action might well be similar. Compounds IndCR might be reduced to Ru(II) which is known to be more reactive to with the general formula HB(RuB2CI4), B being a nitrogen DNA. heterocycle, showed better therapeutic activity than cisplatin in various transplantable rodent tumors (11). Out of a series of analogues, IndCR had the most favorable therapeutic index. INTRODUCTION

Prominent examples for transition metal complexes which 2The abbreviations used are: ISCL. DNA interstrand cross-links: DACH. 1.2- have successfully passed clinical trials are m-diamminedichlo- diamniine-cyclohe\aneplatinum(II): AMDP. m-aminotrismethylenephosphonato-diammineplatinum(ll) (fij[Pt(ATMP)(NHj)j]): CTDP. 18-crown-6-tetra- roplatinum(II) (cisplatin) and c/s-diammine-l.l-cyclobutanedi- carboxybis-diamniineplatinum(ll) ([18C6(COO)4]|Pt(NH3)2]2): DAMP, cis- diamminecyclohexano-aminotrismcthylenephosphonato-platinum(II) (cis- Received 4/16/90: accepted 3/21/91. [Pt(DACH)(ATMP]): DBT. diethoxybis-(l-phenylbutane-1.3-dionato)-tita- The costs of publication of this article were defrayed in part by the payment nium(IV) (budotitane) [Ti(bzac)2(OEt)2]: DDP. m-diamminedichloro- of page charges. This article must therefore be hereby marked advertisement in platinum(II) (cisplatin): IndCR. fra/a-inda/olium-tetrachlorobisinda/oleruthen- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ate(IIl)|lndH(Rulnd;Cl4)]. 1To whom requests for reprints should be addressed. ' B. K. Keppler. personal communication.

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studies final concentrations of 100, 10, and 1 ^M were investigated for P OH cisplatin, DBT, and IndCR 24 h after exposure; DBT was additionally o tested at 200 ^M; the effects of the three new platinum analogues CO NV \ -NHs CH, CTDP. AMDP, and DAMP were investigated at 100 Â¿IMonly.In a yNU, V PI second series, experiments were performed 6 and 48 h after exposure. NH, \ CÃ•Ã•Â¡ CHj OC Here only the most promising new compounds of each metal complex II PO,H,l POjH l - B O group (CTDP, DBT, and IndCR, all at 100 MM)were included for evaluation. Alkaline Elution. DNA protein cross-links, DNA interstrand, and DNA single strand breaks, were determined in vitro by using the alkaline elution assay according to the method of Kohn et al. (2), as described previously (15). In short, O-342 cells were incubated for 1 h with the test compounds in a 200. 100, 10, or 1 H.Msolution, respectively, washed 3 times with RPMI 1640, and incubated for another 6, 24, or 48 h in drug-free medium. After collection of cells by means of tryp- sinization (0.25% trypsin/5% EDTA). viability was found to be >96% for treated and untreated cells. After adjustment to 3 x IO6 cells/ml, alkaline elution was performed as follows. Medium (1 ml) containing 3 x 10* cells/ml was loaded onto polycarbonate filters (Nucleopore GmbH, TÃ¼bingen,Federal Republic of Germany) attached to a peri staltic pump with a flow rate of 0.3 ml/min. The cells were lysed with a buffer containing sodium dodecyl sulfate at pH 10. For determination of DNA interstrand cross-links and DNA single strand breaks, proteinase K (Sigma GmbH, Deisenhofen, Federal Republic of Germany) was added to the lysis buffer. Without this enzyme total cross-links consist ing of DNA interstrand plus DNA protein cross-links could be meas ured. After continuous washing procedures at pH 10, the DNA was eluted at a flow rate of 0.03 ml/min by using an alkali buffer (tetraeth- ylammonium hydroxide plus EDTA), pH 12.2. Ten fractions, 2ml each, were collected at 66-min intervals from each filter. Subsequently the Fig. I. Structures of metal complexes investigated: CTDP. (A); AMDP. (BY. filters of each holder were minced and incubated in 6 ml alkaline buffer DAMP, (C); DBT. (budotitane) (D): IndCR, (Â£). at 37Â°Cfor 2 h to force the remaining DNA into suspension. DNA contents of all 10 fractions as well as of the filter suspension were showing good therapeutic activity and lower toxicity than its determined fluorimetrically by using Hoechst dye 33258 (Serva). We congeners (12). used an autoanalyzer system consisting of a continuous flow analyzer The alkaline elution technique, using the ovarian tumor line (type 5100; Skalar Analytical, Breda, The Netherlands) with a table O-342, which serves in our laboratory to investigate cisplatin sampler holding 375 samples (sample processor SP100) and fluorimeter (type 6300) connected with a personal computer (Olivetti type M21). action (13), was considered an appropriate method to study In order to introduce a controlled level of DNA single strand breaks, a DNA damage. For all new complex compounds DNA protein specimen of cell was subjected to -y-irradiation (500 rads) in the dark cross-links, DNA interstrand cross-links and DNA single at 0Â°C("7Cs source, Gammacell 100; Atomic Energy of Canada, Ltd.; strand breaks were determined. Experiments were undertaken dose rate, 1616 rads/min). DNA single strand breaks, "no proteinase" at several time points after drug exposure to assess formation cross-links (DNA interstrand plus DNA protein cross-links) and "with proteinase" cross-links (DNA interstrand cross-links alone) were quan and stability of DNA lesions. Cisplatin served as reference compound. Additionally, growth studies of tumor cells after tified in terms of rad equivalents. Growth Studies. Aliquots of drug-exposed cells prepared for alkaline drug treatment were performed in order to study correlations elution were allowed to proliferate in drug-free medium for at least 3 with DNA damage. population doublings after treatment. They were harvested by trypsin- MATERIALS AND METHODS ization and counted by the Neubauer method. The percentage of viable cells was determined by trypan blue exclusion. Inhibition of prolifera Tumor Cell Culture. Ovarian tumor 342 (O-342) was induced in a tion was assessed by the ratio of cell numbers in treated cultures to female BDIXrat by a single i.p. injection of 100 mg/kgethylnitrosourea untreated cultures. Each determination was performed in triplicate. with a manifestation time of 422 days (14). Historically the primary tumor corresponded to a partly cystic, partly papillary growing granu losa cell tumor. Cell cultures were grown at 37Â°Cin 175-cm3 tissue RESULTS culture flasks (Greiner, Frickenhausen, Federal Republic of Germany) containing RPMI 1640 medium (Gibco, Karlsruhe, Federal Republic Formation of DNA cross-links at different time points post- of Germany) supplemented with 10% fetal calf serum, penicillin (100 exposure are given in Tables 1 and 2. When O-342 cells were units/ml), streptomycin (100 Mg/ml) (all Serva, Heidelberg. Federal incubated with 100 and 10 MMcisplatin, DNA protein and Republic of Germany), and 0.6% 4-(2-hydroxyethyl)-ethanesulfonic DNA interstrand cross-links were detectable 6 h after drug acid buffer (Gibco). Cultures utilized to assess drug effects were in exposure and reached a maximum at 24 h. By 48 h, cross-links exponential growth phase with a doubling time of about 24 h. were substantially reduced. At any time point and at all concen Drugs. Cis-diamminedichloroplatinum (Cisplatin-LÃ¶sung Behring) trations investigated, a major part of cross-links consisted of was purchased from Behringwerke, Marburg. Federal Republic of Ger DNA protein cross-links. Fig. 2 shows typical DNA elution many. All new test agents were synthesized and kindly supplied by Dr. B. K. Keppler, Institute of Inorganic Chemistry, University of Heidel patterns 24 h after exposure of cells to 100, 10, and 1 AIM berg. Purity was determined by elemental analysis. IR and 'H nuclear cisplatin and irradiation with 500 rads (5 Gy) obtained by magnetic resonance spectroscopy. Compounds were dissolved in phys alkaline elution without and with application of proteinase K iological saline; DBT required addition of polyvinylpyrrolidone (ratio, (no proteinase and with proteinase assay, respectively), to dis 1:11) prior to solution.3 For the alkaline elution assay and the growth criminate between DNA interstrand and DNA protein cross- 2944

Table 1 Formation and repair of tola! DNA cross-links (DNA interslrand plus DNA protein cross-links) induced by various metal complexes (mean rad creased in the order cisplatin, CTDP, AMDP, DAMP. CTDP equivalents Â±SE) was tested at different time points at a concentration of 100 O-342 cells in exponential growth were treated at 37Â°Cfor 1 h as described in MM.Six h after exposure to CTDP, DNA interstrand cross "Materials and Methods." Alkaline elution was performed after subsequent links (Table 2) were substantially lower as compared to equi- incubation in drug-free medium for 6. 24. and 48 h. respectively. molar concentrations of cisplatin (73 Â±15 versus 365 Â±72 rad Total cross-links equivalents, respectively). CTDP-induced interstrand cross Compound 6h 24 h 48 h links showed a peak at 24 h; at this time point the amount of PlatinumDDP"CTDPAMDPDAMPTitaniumDBT10010100100100100663 ISCL was similar to that of cisplatin (317 Â±68 versus 392 Â± Â±49(3)*164 Â±436(4)238 Â±103(4)42 116 rad equivalents, respectively). CTDP interstrand cross Â±2(4)219 + 51(4)310 Â±13(4)121 Â±23(5)Câ€”9Â± Â±71(4)213 Â±19(4)â€”â€”-6 links were less stable than cisplatin interstrand cross-links, Â±31(4)63 which is reflected by their rapid decrease up to 48 h (65 Â±21 Â±28(4)-5 for CTDP versus 180 Â±33 rad equivalents for cisplatin). Six, 24, and 48 h following exposure to 100 Mmconcentrations of 10(3)1987 Â±15 (4)402 Â±18 (3) both compounds, the quotient DNA interstrand cross-links/ total cross-links was 0.33, 1.02, 0.54 (CTDP) versus 0.55, 0.20, Ruthenium IndCR 100 49 Â±14(5) 29 Â±9(4) 7 Â±8(3) 0.45 (cisplatin). Thus at 24 h, CTDP produced distinctly less Â°DDP. cisplatin. DNA protein cross-links as compared to cisplatin (Fig. 4). * Numbers in parentheses, number of experiments. Similar to cisplatin, some CTDP-induced DNA single strand ' â€”,drug effects not evaluated. breaks could only be detected 48 h after drug exposure (100 MM,34 Â±8 rad equivalents). Although at a somewhat lower Table 2 Formation and repair of DNA interstrand cross-links induced hy various level, AMDP showed a ratio of ISCL to total cross-links (24 h: metal complexes (mean rad equivalents Â±SE) O-342 cells in exponential growth were treated at 37Â°Cfor 1 h as described in 179 Â±39 versus 213 Â±31 rad equivalents), which was com "Materials and Methods." Alkaline elution was performed after subsequent parable to CTDP (Fig. 4). DAMP, the second biphosphonate incubation in drug-free medium for 6, 24. and 48 h. respectively. complex investigated, was the least active platinum compound DNA interstrand cross-links in terms of DNA damage; by 24 h it effected only 16 Â±7 rad Compound 6h 24 h 48 h equivalents of ISCL and 63 Â±28 rad equivalents of total cross PlatinumDDPÂ°CTDPAMDPDAMPTitaniumDBTRutheniumIndCR100101100100100200100101100101365links at 100 MM(Tables 1 and 2). Similar to cisplatin, DAMP Â±72(4)*46 Â±116(4)63 Â±33(4)4 displayed a low ISCL/total cross-links quotient of 0.25 at 24 h Â±10(3)C73 Â±11(4)0 Â±3(4)â€”65 (Fig. 4). Neither AMDP nor DAMP produced measurable Â±8(4)317 Â±15(4)â€”â€”â€”- Â±68(4)179 Â±21(4)â€”â€”â€”-10 numbers of DNA single strand breaks. Â±39(5)16 Following incubation of O-342 cells with the titanium com Â±7(5)-6 pound DBT, no DNA protein cross-links or DNA interstrand cross-links could be demonstrated for the concentrations and Â±5rf(3)-22 time points investigated (Tables 1 and 2); negative cross-link 11 Â±3(4)â€”â€”15 Â±7(3)-25 Â±5(3)â€”â€”1 Â±3(3)-1Â±17(3)-49 values confirm the absence of this lesion. DNA single strand breaks did not occur either, except at a toxic dose level of 200 MMDBT which produced 87 Â±17 rad equivalents single strand Â±5(5)â€”â€”392 Â±30*(4)-18 2 Â±4(3)â€”â€” breaks by 24 h (not given in the tables); this may, however, be Â±16(4)-6 attributed to the reduced cell viability (<70%) prior to alkaline Â±28 (4)180 elution. Viability in all other DBT treatment groups and in Â°DDP, cisplatin. * Numbers in parentheses, number of experiments. controls ranged above 96% (cf. Table 2). ' â€”.drug effects not evaluated. The ruthenium complex IndCR showed a maximum of total d Mean viability of cells prior to alkaline elution below 70?. for all other cross-links (49 Â±14 rad equivalents) already 6 h after exposure compounds and concentrations mean viability was above 96%. (Table 1), which subsequently decreased (29 Â±9rad equivalents at 24 h and 7 Â±8 rad equivalents at 48 h). At 6 h the majority links. It is evident from this figure and Tables 1 and 2 that of cross-links were DNA protein cross-links (approximately DNA cross-linking effected by cisplatin is proportional to drug 70%); the minor fraction were DNA interstrand cross-links (15 concentration. Interestingly, at 24 h the elution rate of cisplatin- Â±5 rad equivalents at 6 h; 12 Â±4 rad equivalents at 48 h); 24 treated and irradiated cells at the highest concentration (100 h postexposure no DNA interstrand cross-links could be de ÃŸM)in the no proteinase assay was slower than that of controls tected. DNA single strand breaks were not observed for IndCR. which had not received irradiation (Fig. 2A). This is due to the Investigation of growth inhibition (Table 3) revealed that strong cross-linking effect of cisplatin which makes the double- CTDP was the most active new platinum complex, followed by treated DNA (cisplatin plus irradiation) to appear even more AMDP and DAMP. At equimolar concentrations, however, intact than the untreated one. No measurable numbers of DNA their growth inhibitory potential was inferior to that of cispla single strand breaks could be demonstrated at 6 and 24 h after tin. For the titanium compound, dose-dependent activity could drug exposure; 100 MMcisplatin at 48 h produced 28 Â±16 rad be demonstrated at concentrations higher than 10 MM. The equivalents of DNA single strand breaks, while no strand breaks ruthenium complex IndCR showed a steep increase in growth could be detected at 10 MM(not given in the tables). inhibition only at the highest concentration. Elution patterns 24 h after treatment with the three new platinum compounds CTDP, AMDP, and DAMP at a concen DISCUSSION tration of 100 MMin the no proteinase and the with proteinase assay are given in Fig. 3. DNA cross-linking activity of platinum It is evident from the data in Tables 1 to 3 that total cross- complexes including the reference compound cisplatin de linking (DNA interstrand plus DNA protein cross-links) and 2945

1001

-O- -O -O- -â‚¬>

Fig. 2. DNA alkaline clulion profiles of O- \ 342 cells exposed to various concentrations of cisplatin for I h and then incubated in drug- free medium for 24 h. A, cells that were ana lyzed for total cross-links (DNA interstrand cross-links plus DNA protein cross-links) without the addition of proteinase K; B, cells that were analyzed for DNA interstrand cross links along with proteinase K. A no proteinase: with proteinase: O Control _ O Control _ â€¢DPP,100 MM + 500 R â€¢DPP.100 uU -f 500 R â€¢DOP.IO M" * 500 R â€¢DPP.10 pM + 500 B V Control <â€¢500R â€¢DPP,1tiM + 500 R 7 Control + 500 R

345678 3 4 5 6 Fractions of elution Fractions of elution

Fig. 3. DNA alkaline elution profiles of O- 342 cells exposed to a concentration of 100 Â»Â¿Mcisplatin,CTDP, AMDP. and DAMP for 1 h and then incubated in drug-free medium for 24 h. A, cells that were analyzed for total cross-links (DNA interstrand cross-links plus DNA protein cross-links) without the addition of proteinase K: B, cells that were analyzed for DNA interstrand cross-links along with pro teinase K. A B no proteinase: with proteinase: : Control O Control _ â€¢DJ)P.100 Â¡iu + 500 R â€¢DOPADOjjM + 500 R A CTDP.100//U * 500_R_ ffl CTDP.100 nM -I-500 R 'A AMDP. 100 Â¡tM+ 500 R K AMDP, lOO^iM+_5Ã–OR B DAMP;IPO MM -t-500 R O DAMP.100 fÂ¿ut 500 R "7 Control + 500 R '." Control -t-500 R

3456789 34567 Fractions of elution Fractions of elufion

DNA interstrand cross-linking by cisplatin correlate with the activity; repeatedly, mainly DNA interstrand cross-links could growth inhibitory potential of this agent; kinetics of formation be demonstrated to correlate with cisplatin cytotoxicity (3, 17). and repair of DNA interstrand cross-links with a maximum at DNA cross-linking activity of the new platinum compounds 24 h after exposure paralleled observations by other investiga decreased in the order CTDP, AMDP, DAMP. These results tors (1). In agreement with others (3) we observed proteinase- correlate with their growth-inhibitory potential observed in the sensitive DNA protein cross-links to constitute about 80% of present in vitro studies and with results of ex vivo colony- total cross-links. The total amount of DNA protein cross-links forming assays using the bilayer soft agar method (4). For might yet be higher because proteinase K-resistant DNA protein CTDP, initial formation of DNA interstrand cross-links resem cross-links cannot be determined by this method ( 16). However, bled results reported for the structurally related dicarboxylato according to the literature this high fraction of DNA protein platinum complex carboplatin (16); cross-link formation 6 h cross-links appears to play a subordinate role for anticancer after exposure was slower than for cisplatin. CTDP interstrand 2946

mary bone tumors and bone mÃ©tastases.First in vivo studies yielded marginally better results for AMDP than cisplatin in a transplantable rat osteosarcoma (5). The present study of gen- otoxicity in O-342 ovarian tumor cells showed AMDP to 100-1 produce fewer DNA interstrand cross-links than cisplatin at equimolar dosage. At the time point investigated (24 h), AMDP induced a small proportion of DNA protein cross-links (Fig. 4). Comparison of both phosphonate-linked compounds (AMDP and DAMP) shows considerable differences in their reactivity to DNA and in cytotoxicity, which must be due to the amino ligands that are the only distinguishing feature between both agents. In vivo studies of structure-activity rela tionships of platinum compounds showed that drugs bearing the amino ligand NH, were 8.5 times more toxic on an equi o 'dlC molar basis than those bearing the DACH moiety (9). With regard to DNA interstrand cross-link formation, AMDP and la o DAMP differed at equimolar dosage by a factor of approxi mately 11. Reduced activity of DAMP versus AMDP is possibly due to replacement of the H atoms in the DACH moiety leading to reduced H bonding with DNA which according to Ref. 19 is of essential importance for antitumor activity and thus DNA interstrand cross-link formation. On the other hand, com pounds containing the DACH moiety were reported to be able to overcome acquired resistance to cisplatin (6). In vitro results ZZ3 with proteinase in a cisplatin-resistant subline of the tumor used for the present I no proteinase studies, however, do not support this latter observation (4).

DDP CTDP AMDP DAMP [100 MU] Several mechanisms of action have been proposed for tita Fig. 4. Relation of DNA-interstrand cross-links to DNA-protein cross-links in nium antitumor complexes. Investigations of structure-activity DNA of O-342 cells 24 h after treatment with the platinum complexes cisplatin. relationships of bis-ÃŸ-diketonato titanium antitumor agents CTDP. AMDP. and DAMP (100 MM).Columns, mean; bars. SE; n = 4 (cisplatin. (e.g., DBT; Fig. ID) yielded no dependence of therapeutic CTDP); n = 5 (AMDP. DAMP). activity on the hydrolyzable leaving ligands. Variation of the bis-ÃŸ-diketonato ligands rather showed space-filling planar ar Table 3 Proliferation ofO-342 cells following exposure to different metal complexes" omatic systems to be important for antitumor activity. The unsubstituted acetylacetone ligand was entirely inactive (8). For Cell count (% of Compound controls Â±SE) titanocene dihalides. a different group of titanium compounds, Platinum on the other hand, studies of structure-activity relationships DDP* 15 Â±2(4)c 100 revealed the importance of the leaving ability of the hydrolyz 10 22 Â±2 (7) able acido ligands; strongly bonded ligands did not show any 1 49 Â±5(4) CTDP 100 22 Â±4 (8) cancerostatic activity against Ehrlich ascites tumor (20). Con AMDP 100 27 Â±2 (9) sequently, more easily hydrolyzable titanocene dihalides could DAMP 100 48 Â±9(5) be demonstrated to form titanium nucleotide adducts, similar Titanium to cisplatin (21). Although in our experiments for DBT dose- DBT 200 6 Â±3 (3) dependent growth-inhibitory potential was observed, neither 100 26 Â±3(6) 10 Â»2Â±21(3) DNA protein cross-links nor DNA interstrand cross-links could 1 66 Â±13(3) be demonstrated at any time point or concentration investi Ruthenium gated. A synopsis of these results together with findings from IndCR 100 20 Â±2(4) structure-activity investigations suggests the mechanism of ac 10 112 Â±8(4) tion of bis-/3-diketonato titanium to be substantially different 1 107 Â±6(4) * Drug exposure for l h and subsequent proliferation in drug-free medium for from cisplatin and the titanocene dihalides. The importance of 72 h. planar aromatic ligands hints to intercalation into the DNA as * DDP, cisplatin. a possible mechanism of action, which was also observed for * Numbers in parentheses, number of experiments. other transition metal complexes with large, bidentate aromatic ligands (22). cross-links proved to be less stable than cisplatin-induced ISCL; The ruthenium compound IndCR containing a Ru(III) ion 24 h after CTDP exposure, no DNA protein cross-links were was found to produce DNA cross-links which were to a major observed. Interestingly, the structurally related platinum com part (70%) proteinase K sensitive; the maximum of cross-links plex carboplatin likewise showed a reduced ratio of DNA occurred shortly after incubation (6 h). Subsequently IndCR- protein cross-links to total cross-links (16) and less plasma induced cross-links decreased and were scarcely detectable at protein binding (18) than cisplatin. AMDP and DAMP, which 48 h. Other ruthenium(III) complexes such as [C1(NH3)5 were previously also termed ADP and DAP (4), were originally Ru'"]2+ were reported to bind covalently to DNA and RNA synthesized as "combination drugs" containing an osteotropic (10). They were shown to bind initially at the N-7 of guanine phosphonate and a cytostatically active platinum moiety. This which is a target for platinum compounds. Ruthenium com should warrant a more specific antitumor action against pri- pounds may also lead to DNA single strand breakage via 2947

Fenton's chemistry taking place at the metal ion to generate 7. Bischoff, Hâ€žBerger, M. R., Keppler. B. K., and SchmÃ¤hl.D. Efficacy of beta-diketonato complexes of titanium, zirconium and hafnium against chem radicals capable of strand cleavage or via catalytical hydrolysis ically induced autochthonous colonie tumors in rats. J. Cancer Res. Clin. of phosphodiester groups which constitute the backbone of Oncol., 113:446-450, 1987. 8. Keppler, B. K.. and Heim. M. E. Antitumor-active bis-beta-diketonato metal DNA (10, 23, 24). No single strand breaks were observed after complexes: budotitaneâ€”a new anticancer agent. Drugs Future, 13:637-652, IndCR exposure when viability of cells prior to alkaline elution 1988. was satisfactory (>90%). The discrepancy between low cross- 9. Cleare, M. J.. Hydes, P. C, Malerbi, B. W., and Watkins, D. M. Anti- tumour platinum complexes: relationships between chemical properties and linking potential in vitro as demonstrated here and more favor activity. Biochimie (Paris). 60: 835-850. 1978. able in vivo action of ruthenium analogues as reported by Ref. 10. Clarke, M. J. Ruthenium chemistry pertaining to the design of anticancer 12 may be accounted for by the following reason. Ruthen- agents. Prog. Clin. Biochem. Med., 10: 25-39. 1989. 11. Keppler, B. K., Rupp, W.. Juhl, U. M.. Endres. H., Niebl. R., and Balzer, ium(III) ion complexes are relatively stable, so that ligands are W. Synthesis, molecular structure and tumor-inhibiting properties of trans- retained for fairly long periods as long as this oxidation state bis(imidazole)tetraehlororuthenate(III) and its methyl-substituted deriva tives. Inorg. Chem., 26:4366-4370. 1987. is maintained. In hypoxic tumor tissue, Ru(III) becomes re 12. Berger. M. R.. GarzÃ³n, F. T.. Keppler. B. K., and SchmÃ¤hl.D. Efficacy of duced to Ru(II) which has little affinity for acido ligands and is new ruthenium complexes against chemically-induced autochthonous colo- more reactive to DNA (10, 25). rectal carcinoma in rats. Anticancer Res., 9 (N3): 761-765, 1989. 13. Zeller, W. J., and Chen, G. Chemosensitization of experimental ovarian Altogether, the data presented indicate that different metal tumors to cisplatin (DDP) by D.L-buthionine (S.Ã„J-sulfoximine (BSO) or 3- complexes may have completely different mechanisms of ac amino-benzamide (3-AB) in vivo. Proc. Am. Assoc. Cancer Res.. 31: 406, tion. Future structure-activity investigations may help to clarify 1990. 14. Zeller, W. J., FrÃ¼hauf,S.,Chen, G., Keppler. B. K., Frei, E., and Kaufmann, the role of different ligands for reactivity of these complexes M. Chemoresistance in rat ovarian tumours. Eur. J. Cancer, 27:62-67, 1991. with DNA. This knowledge in combination with data of in vivo 15. Zeller, W. J., FrÃ¼hauf,S., Chen, G.. Eisenbrand, G., and Lijinsky, W. Biological activity of hydroxylated chloroethylnitrosoureas. Cancer Res., 49: studies on their anticancer activity could be used in the design 3267-3270. 1989. of new metal complexes with higher activity and less toxicity. 16. Micetich, K. C.. Barnes. D., and Erickson, L. C. A comparative study of the cytotoxicity and DNA-damaging effects of a'i-(diammino) (1,1-cyclobutane- dicarboxylato)-platinum(II)andm-diammincdichloroplatinum(Il)on LI210 ACKNOWLEDGMENTS cells. Cancer Res., 45: 4043-4047. 1985. 17. Laurent, G., Erickson, L. C., Sharkey. N. A., and Kohn. K. W. DNA cross- linking and cytotoxicity induced by m-diamminedichloroplatinum(II) in We wish to thank Dr. B. K. Keppler, Institute of Inorganic Chemis human normal and tumor cell lines. Cancer Res., 41: 3347-3351. 1981. try, University of Heidelberg, for kindly supplying the new platinum, 18. Litters!, C. L. Plasma pharmacokinetics. urinary excretion, and tissue distri titanium, and ruthenium metal complexes. We are indebted to A. Keller bution of platinum following i.v. administration of cyclobutanedicarboxyla- and J. Engel for their skillful technical assistance. toplatinum-II and c/s-platinum to rabbits. In: M. P. Hacker, E. B. Douple. and I. H. Krakoff(eds.). Platinum Complexes in Cancer Chemotherapy, pp. 71-81. Boston: Martinus Nijhoff, 1984. 19. Cleare, M. J., and Hoeschele, J. D. Studies on the antitumor activity of group REFERENCES Vili transition metal complexes. Part I. Platinum complexes. Bioinorg. Chem., 2: 187-210, 1973. 1. Plooy, A. C. M., van Dijk, M.. Berends, F.. and Lohman, P. H. M. Formation 20. KÃ¶pf.H., and KÃ¶pf-Maier,P. Tumor inhibition by metallocene dihalides of and repair of DNA interstrand cross-links in relation to cytotoxicity and early transition metals, chemical and biological aspects. In: S. J. 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S. Frühauf and W. J. Zeller

Cancer Res 1991;51:2943-2948.

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