Platinum(II) complexes containing iminoethers: a trans platinum antitumour agent

ELSEVIER

Chemico-Biological Interactions 98 (1995) 251-266

Platinum(I1) complexes containing iminoethers: a bans platinum antitumour agent Mauro Coluccia*a, Angela Boccarellia, Maria A. Mariggiba, Nicola Cardellicchiob, Paola Caputoc, Francesco P. Intini’, Giovanni NatileC ‘Diportimento

di Scienze Biomediche e Oncologia Umano, Sez. Potologia Generole e Oncologia Sperimentale,

Policlinico,

bCNR, Isiituto ‘Dipartimento

Piazza Giulio Cesore II.

Sperimentale

Tolassogrojico,

Formaco-Chimico,

I-70124

Bari. Italy

Toronto, Italy

Universitti di Bari, Bari, Italy

Received 27 March 1995; revision received 21 June 1995; accepted 3 July 1995

Abstract The biological activity of cis and truns complexes of formula [PtCl,( HN=C(OMe)Me )J has been investigated. The iminoether ligands can have either E or Z configuration about the C=N double bond, therefore EE, EZ and ZZ isomers are obtainable. Substitution of iminoether with EE configuration for amine leads to unexpectedly high antitumour activity for the complex with tram geometry which turns out to be more active than the cis congener in the P388 leukaemia system. The same frans-EE complex shows an activity comparable to that of cisplatin in reducing the primary tumour mass and lung metastases in mice bearing Lewis lung carcinoma, thus representing a tram platinum complex active on both limphoproliferative and solid metastasizing murine tumours. Also the cytotoxicity, the inhibition of DNA synthesis and the mutagenic activity, which are greater for the cis- with respect to the fruns-isomer in the amine complexes, are instead greater for the trans- than for the cisisomer in the case of iminoether compounds. Binding to calf thymus DNA is slower for iminoether complexes than it is for amine complexes, however after 24 h reaction time the level of binding is similar for both types of complexes. Tram-EE, like trans-DDP, does not give the DNA conformational alterations (terbium fluorescence) typical of antitumour-active cisAbbreviations:

cis-DDP,

cisplatin,

cis-[PQ(NH,),];

trans-DDP,

frans-[PtCl&NH,),];

(DM)b,

number of bound platinum atoms/nucleotide; (D/N)f, platinum complex/nucleotide formal concentration ratio; cis- and trons-EE, cis- and trans-[PQ( (E)HN=C(OMe)Me) *I; ID,, and ID,, concentrations inhibiting 50% and 90% cell growth, respectively; PBS, phosphate buffered saline. * Corresponding author, Tel.: +39 80 278412. 0009-2797/95/$09.50

0

1995 Elsevier Science Ireland

SSDI 0009-2797(95)03650-B

Ltd. AI1 rights reserved

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platinum compounds, but, under strictly analogous experimental conditions, reduced DNA interstrand cross-linking ability (heat denaturation/renaturation spect to either trans-DDP or cis-EE and cis-DDP. The data in hand point platinum antitumour complex with a mechanism of action different from that classical analogues. Keywords: Platinum; Iminoether; binding

Tram geometry; Antitumour

shows a greatly assay) with reto a new tram of cis-DDP and

activity; Mechanism; DNA

1. Introduction For many years, cis-[PtC12(NH3)2J has been successfully used as a chemotherapeutic agent against testicular, ovarian, cervical, bladder, head and neck tumours. Unfortunately, cis-DDP has minor activity against common malignancies, such as colon and breast cancers, and its efficacy is limited by a variety of adverse effects

[1,4. The search for new platinum derivatives on the basis of the structure-activity relationships valid for cis-DDP [3,4] has been rather disappointing, some success having been achieved only with carboplatin which is devoid of the major side effects of cisDDP but shows a broadly similar spectrum of activity [5]. The antitumour activity of this class of drugs is believed to stem from the ability to damage cellular DNA, thus exerting a cytotoxic effect. The most common DNA adducts of cis-DDP are intrastrand cross-links at d(pGG) (65%) and d(pAG) (20%) sites; moreover, up to 8% of the platinum bound can form interstrand cross-links between guanines in the complementary strands [6-lo]. Recently, new active platinum complexes with structural features that violate the ‘classical’ structure-activity relationships have been described. Non-neutral compounds of the form [PtCi(NH&(Am)]’ (Am = pyridine, pyrimidine, purine, piperidine or aniline ligand) have been reported to be active against murine and human tumour systems [ 11,121. Also complexes with tram geometry and general formula trans-[PtC12(L)(L’)] (L = L’ = pyridine or thiazole; L = quinoline and L’ = NH, or substituted sulfoxides) [ 13- 151 and complexes with bridging diamines [ ( trans-PtC1(NHj)z ) 2( HzN(CH&NH* j] 2+ have shown considerable cytotoxic effects in murine and human tumour cell lines [ 16,171. In vitro and in vivo antitumour activity has also been reported for the platinum(IV) complex trans-[PtC12(0H)s(NH~)INH,(C,H,I)]I V81. We have investigated the effect of substitution of iminoethers for amines in cisand trans-DDP. Iminoethers, like amines, are potential N-donor ligands and the nitrogen atom carries a hydrogen atom suitable for hydrogen-bond formation (Fig. 1). Therefore, the platinum-iminoether complexes cis- and tram- [ PtCl2 ( HN=C(OMe)Me12 are expected to show rather strong analogies with platinum-amine complexes. In addition to the cis and tram geometry, platinum-iminoether complexes can have either E or Z configuration about the C=N double bond of the iminoether ligands, and this brings in another feature to be considered for structure-activity relationships.

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MBC/oMe

MBC/oMe H ‘L / ,‘!q c / pt\&H

253

2S1-266

II C

C

\

/%H Pt

“b/

II

‘Cl II

C

Me/ 'OMe cis-EE

Me,

MekcNMe

,..OMe

NLH cLptJ’

r::

ANNH

“, C f

“h/ ‘Cl II

’ t\N,H II Me0 64e

Me0 /‘be trans-EZ

cis-EZ

MeO,

MeO,

,Me

,Me I7

c C \ C I

P/%H \N/H I/ Med

c\

Me

C Me’

cis-zz

Fig. 1, Schematic drawing of the structural

‘OMe

tra ns-ZZ

formulae

of platinum-iminoether

complexes.

A preliminary investigation of the biological activity of the platinum-iminoether complexes had shown that the complex with trans geometry was endowed with significant in vivo antileukaemic activity [19]. In this study we have extended the antitumour investigation of tram-EE, and undertaken a mechanistic study in order to address the biological properties of the new compounds. In particular, some biological effects (inhibition of DNA synthesis and mutagenicity) and interaction with DNA (binding affinity, secondary structure modification, interstrand cross-link formation) have been studied in comparison with those of cis- and trans-DDP. The results indicate that the antitumour-active tram iminoether complex behaves differently from cisplatin and classical cisplatin analogues.

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2. Materials and methods 2.1. Chemicals All reagents were purchased from Sigma Chemical Co., except for terbium chloride (Aldrich), [methyl-3H]thymidine (Amersham), RPM1 1640 and fetal bovine serum (Bio-Wittaker). 2.2. Preparation

of the complexes

The compounds cis- and truns-[PtCl*( HN=C(OMe)Mej2] having either E or Z configurations at the iminoether ligands (2 and E configurations correspond to having the platinum and the O-Me groups mutually cis and trans with respect to the C=N double bond, respectively) were prepared as already described [20]. 2.3. Antitumour

activity

The P388 leukaemia and the Lewis lung carcinoma lines were obtained as frozen stock from the National Cancer Institute (Bethesda, MD) and maintained in DBA/2J and C57BL/6 female mice, respectively (Charles River, Italy). For experimental purposes, adult female B6D2Fl mice weighing 18-20 g were used. The antileukaemic effects were determined using the life extension assay. Briefly, B6D2Fl mice were inoculated i.p. with lo6 P388 cells on day zero. Platinum complexes, freshly dissolved in water, were administered i.p. on days l-7 (0.1 ml/10 g body weight). The antitumour activity was expressed as (T/C) x 100, with T the mean survival time of treated mice and C that of untreated control ones. A dose in which the T/C% value was < 85% or the body weight of mice decreased by more than 4 g was considered as a toxic dose. For the evaluation of effects on Lewis lung carcinoma-bearing mice, B6D2Fl mice were inoculated S.C. in the axillary region with lo6 single viable tumour cells obtained from donors similarly inoculated 2 weeks before. The platinum complexes were administered at equitoxic dosages, corresponding to LDo.ss [21], by daily i.p. injections (0.1 ml/l0 g body weight) for 14 consecutive days, starting 24 h after tumour implantation. The effects of truns-EE and cis-DDP on primary tumour growth, spontaneous metastases and survival time were evaluated according to previously described methods [22]. Experimental data were subjected to statistical analysis with the Student-Newmann-Keuls test. 2.4. Biological studies Inhibition of DNA synthesis. P388 cells in exponential growth phase (5.5 x lo5 cells/ml in RPM1 1640 medium supplemented with 10% fetal bovine serum and 100 &ml kanamycin) were treated with the platinum complex (0.1 ml of drug solution per 0.9 ml of cell suspension) and incubated at 37°C under gentle agitation in a 5% COz humilied incubator. In the dose-response experiment the incubation time was fixed at 1 h and the platinum concentration varied from O-60 PM. In the timeresponse experiment, P388 cells were exposed for 1 h to the IDss concentration of cis-DDP (11.2 PM), truns-DDP (80 PM), cis-EE (48 PM) and truns-EE (7.4 PM). At the end of treatment, the cells were washed twice with PBS, resuspended in fresh culture medium and further incubated for a time between 0 and 24 h. 1 &I of [methyl-

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255

3H]thymidine was added to the samples 20 min before the end of the incubation time, and the DNA synthesis was measured according to the Freshney protocol [23]. Inhibition of cell growth and DNA synthesis and DNA interstrand cross-links formation for drug-treated P388 leukaemia ceils. The inhibition of cell proliferation, the inhibition of DNA synthesis and the formation of DNA interstrand cross-links were measured simultaneously for drug treated P388 cells. P388 cells in exponential growth phase (5.0 x lo5 cells/ml in RPM1 1640 medium supplemented with 10% fetal calf serum and 100 pg/ml kanamycin) were treated with the platinum complex (concentrations ranging from O-60 PM) at 37°C in a 5% CO2 humilied incubator. After 5 h exposure, the cells were washed 3 times with ice-cold PBS and resuspended in fresh culture medium. The percentage of interstrand cross-linked DNA was evaluated immediately after exposure by the ethidium bromide fluorescence technique, as described by Morgan and Pulleyblank [24] and Brent [25]. DNA was extracted (Genomix kit for DNA extraction, Talent, Italy) from samples containing 25 x lo6 cells and resuspended in 2 mM Tris-HCl, pH 7.3, its yield and purity were estimated by measuring the absorbance of the resulting solutions at 260 and 280 nm. Three ml of a solution containing ethidium bromide (0.5 pg/ml in 0.4 mM EDTA, 20 mM K2HP04, pH 11.8) were added to 0.2 ml (20 pg) aliquots of DNA extracted from control and treated cells. The fluorescence was measured before and after heating at 90°C for 10 min (PerkinElmer LS-5B spectrofluorimeter; excitation wavelength, 525 nm; emission wavelength, 580 nm). The percentage of cross-linked DNA (%ICL) was determined by the formula: %ICL = (I; - f,)l( 1-f,) x 100, where ft and j, = fluorescence after denaturation divided by fluorescence before denaturation of treated v,) and control (f,) samples. The inhibition of DNA synthesis was measured in parallel, as changes in thymidine incorporation after pulse-labelling for the last 20 min of incubation time, following the procedure quoted above. The inhibition of cell growth was evaluated after 48 h of post-incubation in drugfree medium by the trypan blue exclusion test. Mutagenic assay. The mutagenic activity of the platinum complexes was evaluated according to the protocol of Maron and Ames [26] on TA 100 and TA 98 S. typhimurium strains without metabolic activation. 2.5. Interaction with DNA Platinum-DNA binding. The quantitation of platinum-DNA binding was performed at 37°C by reaction of calf thymus DNA (6 x lo-’ mol nucleotides) with the platinum complex at a (D/N), of 0.08 in a total volume of 2.2 ml of 2 mM Tris-HCl, pH 7.4. At fixed time intervals, 150 ~1 aliquots of the solution were withdrawn and centrifuged through Sephadex G-50 columns in order to remove unbound platinum. The DNA concentration was measured by UV spectroscopy, and the platinum content by flameless atomic absorption spectroscopy on a Perkin Elmer 560 instrument, at 267 nm. Terbium fluorescence enhancement assay. Calf thymus DNA (6 x lo-’ mol nucleotides) was allowed to react for 6 h at 37°C with the platinum complex at

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(D/N)r ranging from 0.02-0.16 in a total volume of 220 ~1 of 2 mM Tris-HCl, pH 7.3. At the end of incubation time, 3 ml of a solution of terbium chloride (100 PM TbCI, .6H20 in 20 mM Tris-HCl, pH 7.3) were added to the samples, and these were equilibrated for 30 min at room temperature in the dark. The fluorescence intensity was measured on a Perkin-Elmer LS-SB spectrofluorimeter (excitation wavelength, 290 nm; emission wavelength, 544 nm; entrance and exit slit widths, 10 nm). DNA interstrand cross-links formation on calf thymus DNA. Calf thymus DNA (1.2 x 10T6mol nucleotides) was reacted with the platinum complex at (D/N), of 2 x 1O-4in a total volume of 4.4 ml of 2 mM Tris-HCI at 37°C. At fixed time intervals 220 ~1 aliquots of the solution were withdrawn and the DNA interstrand crosslinks evaluated as described above. 3. Results 3.1. Synthesis of the complexes Platinum complexes with iminoethers, [PtCl, ( HN=C(OMe)Me ) 2], are prepared by treatment of platinum nitriles with methanol in basic medium [27,28]. The geometry of the metal centre does not change during the alcoholysis reaction. The iminoether ligands can have either E or Z configurations, therefore 3 cis and 3 trans isomers can be formed. The configuration could be determined by ‘H-NMR spectroscopy [27]. A downfield shift is suffered by signals of the protons close to platinum: the O-bonded Me protons in the 2 configuration, the C-bonded Me protons in the E configuration. Compounds having EZ and EE configurations are more

Table I In vitro and in vivo antileukaemic Complex

effects (P388 system) of platinum

In vitro”

In vivob

ID,, W4

Dose (mgikg, qd l-7)

cis-DDP

2

0.6

cis-EE

7.5

4

cis-EE tram-EE

2.2

iransEE

% T/Cc

8

211 I50 toxicd

8

I71

I2

complexes

196

YZells in exponential growth phase are exposed for I h to different concentrations of platinum complexes. Following a 48 h post-incubation time in drug-free medium. the cell number is measured (trypan blue exclusion test) and proliferation inhibition is calculated as percentage of control (data from [l9]). bP388 leukaemia cells (106/mouse) were implanted i.p. on day 0 in B6D2FI female mice weighing 18 g (6 animals/group, 8 controls). The treatment with platinum complexes freshly dissolved in water was performed i.p. on days 1-7. ‘%T/C = mean survival time dBody weight loss > 4 g.

(X

100) of treated

animals

vs. controls.

M. Coluccia et al. / Chemico-Biological Inieractions 98 (199s) 251-266

257

Table 2 Comparison of the antitumour activity of trans-E/Z and cis-DDP in mice bearing Lewis lung carcinomaa Complex

Daily dose

Primary tumourb (mg)

Lung metastasesC (number)

Lung metastasesC (mg)

Survival time (days)

148 +z 34

30 f I

(mg/kg) Vehicle

-

941 * 110

31 zt6

trans-EE

4

530 f 72d

15 f Id

65 zt 6d

cis-DDP

0.6

450 * 52d

10 l 2d

44

l

Id

33 f

I

35 f 2

‘Lewis lung carcinoma cells (106/mouse) were implanted S.C.into B6D2FI female mice on day 0 (16 animals/group). The i.p. treatment was performed on days l- 14 with equitoxic dosages (LD,,,,) of platinum complexes freshly dissolved in water. Each value is the mean f SE. in groups of 16 animals (primary tumour weight) and 8 animals (lung metastases and survival time). bMeasured on day 14 from tumour implantation. ‘Measured on day 21 from tumour implantation. d!’ < 0.05 vs. controls, Student-Newman-Keuls test.

soluble in organic solvents and can be obtained in a pure form by chromatography on silica gel. Compounds having ZZ configuration are much less soluble in common solvents and were obtained as solid residues after extraction of the product mixture with methanol and dichloromethane. The purity of all compounds was estimated to be >96% by ‘H-NMR. Compounds having EZ configuration were always obtained in very small yields. Moreover the solubility of tram-ZZ was not sufficient for biological testing.

120

0”

‘2 100. b

s 8 c

.-

80.

E

0

‘E 60. $ ,= 40P E 6 20-

0

15

30 Complex concentration

45

60

(uM)

Fig. 2. DNA synthesis of P388 cells incubated for 1 h with various concentrations of cis-DDP (A), IransDDP (m, cis-EE (A) and fransEE (II). The incorporation of [methyl-‘HJthymidine was measured after pulse-labelling for the last 20 min of incubation. Each point represents the mean f SD. from 3 independent experiments.

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3.2. Antitumour activity The in vitro and in vivo antileukaemic (P388 system) effects of platinum iminoether complexes are reported in Table 1. Cis- and tram-EE show an in vitro growth inhibitory capability, expressed as 50% growth inhibitory concentration, of the same order as that of cisplatin. On the contrary, in vivo, tram-EE is less toxic and more active than cis-EE and shows an activity comparable to that of cisplatin in increasing the life span of treated animals. The effects of administration of tram-EE to mice bearing subcutaneous Lewis lung carcinoma are reported in Table 2. A significant inhibition of primary tumour growth and of lung metastases is observed in comparison with untreated controls. The overall efficacy of tram-EE appears to be slightly lower than that obtained with equitoxic dosages of cis-DDP, but the difference is not statisticahy significant. 3.3. Biological studies The DNA synthesis, as [methyl-3H]thymidine incorporation, of P388 cells exposed for 1 h to various concentrations of platinum complex is reported in Fig. 2. The [3H]thymidine incorporation decreases as the concentration of platinum complex increases and, at equimolar concentration, the DNA synthesis decreases in the order, cis-EE > trans-DDP > cis-DDP > tram-EE. The DNA synthesis, as a function of time of post-incubation, of P388 cells exposed for 1 h to IDs0 concentration of platinum complex is reported in Fig. 3. A relevant inhibition of thymidine incorporation is observed in the case of the trans

6

12 Post-incubation

18

24

time (hours)

of cis-DDP (A), Fig. 3. Delayed DNA synthesis of P388 cells after I h exposure to IDw concentrations rrans-DDP (m, cis-EE (A) and tram-EE (0). The incorporation of [methyl-‘Hlthymidine was measured after pulse-labelling for the last 20 min of post-incubation in drug-free medium. Each point represents the mean * SD. from 3 independent experiments.

M. Coluccia et al. / Chemico- Biological Inieractions 98 (I 995) 251-246

259

complexes after a short time of post-incubation, while in the case of cis-DDP and cis-EE a comparable effect is observed only after a longer post-incubation time. Rapid inhibition of DNA synthesis has already been reported for truns-DDP and related trans complexes [ 141. Interactions with replenishable cellular components different from DNA cannot be excluded. The DNA interstrand cross-links formation was investigated in parallel to the effects on DNA synthesis and cell growth of P388 cells exposed to different concentrations of platinum complex for 5 h (Table 3). For cis-DDP, trans-DDP and cis-EE complexes the DNA interstrand cross-links formation is proportional to the drug concentration and correlates with the inhibition of DNA synthesis and of cell proliferation In contrast, trans-EE inhibits the DNA synthesis and cell proliferation but does not form detectable interstrand cross-links on cellular DNA. The mutagenic activity of the platinum-iminoether complexes on the TA 100 strain of S. typhimurium is reported in Fig. 4. Truns-EE is definitely more mutagenic than cis-EE while the opposite trend is observed for truns- and cis-DDP [29]. Both cis- and truns-EE, and cis- and truns-DDP, are inactive towards the TA 98 strain which undergoes mutation via a frame-shift mechanism (data not shown).

Table 3 In vitro effects of platinum complexes eration of P388 leukaemia cell? Complex

Concentration (PM)

cis-DDP

cis-EE

tram-DDP

tram-EE

on DNA synthesis,

% Inhibition of DNA synthesis

interstrand

cross-link

% ICL formation

formation

% Inhibition proliferation

29 zt 4 40 f I2

4.6 f 0.9 7.1 f I.1

83 f 0.7 91 f 0.6

I5

54 f I2

16.2 zt 1.4

97 f 0.6

I5 30

15 f 2 21 f 4

ND 2.1

0.2

78 f 4 86 f 2

60

43 f 9

7.6 f 0.5

92 f 2

15 30

41 f 6 67 f 9

3.9 l 0.2 9 f 0.9

76 zt I.5 91 l 3

60

89 f 5

17.4 f I.1

98 f

55 f 3 86 f 1

ND ND

80 zt 5 98 f 2

96 + I

ND

3.75 1.5

3.75 1.5 I5

l

lOO*

and cell prolif-

of cell

1

I

aP388 cells (500 OOO/ml) were incubated for 5 h with platinum complexes in RPM1 1640 medium. At the end of incubation time, the DNA was extracted and the % interstrand cross-link formation was evaluated on 20 pg aliquots of DNA from control and treated cells by the ethidium bromide fluorescence assay. The inhibition of DNA synthesis was measured in parallel as changes in [methyl-‘Hlthymidine incorporation after pulse-labelling for the last 20 min of incubation. The inhibition of cell proliferation was evaluated after 48 h post-incubation in drug-free medium (trypan blue exclusion assay). Data are expressed as mean f SD. ND, not detectable.

of 3 separate

experiments.

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500

400 a,

I

,a g 300 s! P ~200

0 ;i

100

A

0 0

25

50

75

100

125

Complex concentration

150

175

200

@g/plate)

Fig. 4. Mutagenic activity of tram-EE (q and cis-EE (A) towards TA 100 strain of Salmonella typhimurium. Revertant colonies are expressed as mean value of 3 different experiments. The spontaneous revertants (116 * 15)have been subtracted.

J

0

4

6

12

16

20

24

Reaction time (hours) Fig. 5. Kinetics of binding of cis-DDP (A), cis-.EE (A), trans-DDP (m and tram-EE (a) to calf thymus DNA [(D/N)r = 0.08)] in 2 mM Tris-HCI. pH 7.4, 37OC. The amount of platinum bound/nucleotide, (DM)t,, was measured by atomic absorption spectroscopy.

hi.

Coluccia

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et al. / Chemico-Biological

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I

/

I

I

1

0

4

8

12

16

261

Drug/nucleotide formal ratio (x100)

Fig. 6. Change in terbium fluorescence rrans-DDP (H), cis-EE (A) and tram-EE was set at unity. Each point represents bars are seen they are smaller than the

produced on calf thymus DNA after reaction with cis-DDP (A). (n) at various (D/N), values. Fluorescence of untreated control the mean f SD. of 3 independent experiments. Where no S.D. size of symbols.

3.4. Interaction with DNA The ratio of platinum bound per nucleotide of calf thymus DNA as a function of time is reported in Fig. 5. The rates of binding of both cis-EE and trans-EE appear to be slower than those of cis- and trans-DDP. However, after 24 h, all complexes have reached approximately the same level of binding. The terbium fluorescence enhancement of calf thymus DNA incubated with

6

12

16

24

Reaction time (hours) Fig. 7. Kinetics of inter-strand cross-link formation in calf thymus DNA treated for the indicated time with cis-DDP (A), trans-DDP (m, cis-EE (A) and tram-EE (0) at a (D/N),.= 2 x 10V4. Each point represents the mean f S.D. of 3 independent experiments,

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various concentrations of the platinum-iminoether and platinum-diamine complexes is shown in Fig. 6. Tram-EE and trans-DDP produce no change in terbium fluorescence, cis-DDP causes the largest enhancement, while cis-EE exhibits an intermediate behaviour. Our results agree with previous reports on cis- and trans-DDP [30,3 1] and indicate that only cis complexes alter the DNA structure in such a way to produce terbium fluorescence enhancement. The interstrand cross-linking ability of the platinum complexes on calf thymus DNA is reported in Fig. 7. Substitution of iminoethers for amines reduces the interstrand cross-linking ability. The effect is dramatic for tram-EE which, even after 24 h, has a very small DNA interstrand cross-linking efficacy. In contrast, transDDP shows an interstrand cross-linking efficacy similar to that of cis-DDP. 4. Discussion In a preliminary account on the biological activities of platinum-iminoether complexes the isomer trans-[PtC12 ((E’)HN=C(OMe)Me] Xl (trans-EE) was shown to be endowed with greater antileukaemic activity than the cis congeners, thus violating the paradigm on therapeutic selectivity of platinum complexes with cis geometry [19]. We have extended the investigation on anticancer properties of platinum iminoether complexes and found that tram-EE shows an activity comparable to that of cisplatin in the P388 leukaemia system and exerts antitumoural effects also on Lewis lung carcinoma, thus representing a trans platinum complex active in vivo on both limphoproliferative and solid metastasizing murine turnours. Furthermore trans-EE is active against a cisplatin-resistant subline of P388 leukaemia [19] and does not show cross-resistance with cisplatin in several human cancer cell lines (M. Coluccia, manuscript in preparation), suggesting that the mechanism of action of trans-platinum iminoether complexes might be different from that of cis-isomers. There is general agreement that cisplatin and related platinum amine chloride complexes manifest their activity through covalent interaction of their hydrolysis products with cellular DNA. The DNA binding properties of cisplatin have been widely investigated and compared to those of trans-DDP in an effort to explain the differences of biological activity between the two isomers. Therefore, the aims of this study were also to investigate the relevance of DNA as a biological target of platinum iminoether complexes and their DNA interaction properties. The experiments on DNA synthesis inhibition and mutagenic activity indicate that platinum iminoether complexes, likewise platinum amine complexes, interact with cellular DNA. interestingly, the DNA synthesis inhibition and the mutagenic activity of tram-EE are markedly greater than those of cis-EE. while an opposite behaviour has been reported for amine complexes. Ongoing studies on the different cytotoxic potency of cis- and trans-EE towards wild-type and repair-deficient cells further indicate that platinum iminoether compounds damage cellular DNA. Replication mapping experiments showed that trans-EE blocks DNA polymerase at guanine residues in 3 ‘AGN (N = G or C) sites and thus exhibits a sequence selectivity not only different from that of cisplatin and cis-EE but also different from that

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of truns-DDP 1191.The DNA interaction properties of platinum iminoether complexes have been further investigated for binding affinity, conformational alterations and interstrand cross-links formation. As observed for cis- and trans-DDP, also for platinum iminoether complexes the hydrolysis appears to be a prerequisite for their binding to DNA. In fact, treatment of plasmid pBR 322 DNA with cis- and trans-EE under extra-cellular chloride concentration does not inhibit the activity of BumHI restriction enzyme; in contrast, analogous treatment under lower chloride concentration inhibits the restriction enzyme (data not shown). Both the cis and tram isomers of the iminoether complexes react slower with DNA than cis and truns isomers of the amine species, but they reach approximately the same level of binding after 24 h reaction time. The iminoether ligands are more sterically demanding than NH3 and this could account for the slower reactions with nucleobases in DNA duplexes. The results obtained with the fluorescent terbium probe indicate that, independently from the type of ligand (amine or iminoether), cis complexes disrupt the duplex secondary structure more than the trans complexes. Trans-EE, similarly to trans-DDP, does not induce the local conformational alterations that are believed to depend on intrastrand cross-links between adjacent purines (i.e. the main lesion of platinum complexes with cis geometry). More detailed structural studies in progress support this conclusion. It is well known that both cis- and trans-DDP produce interstrand cross-links when reacting with DNA and, more recently, the binding sites and the conformational changes have been identified [32,33]. Interstrand cross-links are among the critical molecular lesions able to inactivate the DNA as a template for replication; these lesions correlate with the cytotoxic activity of cis-DDP [34,35] and characterize the binding mode of other classes of cytotoxic trans platinum complexes [ 15,181.The ability of platinum-iminoether complexes to form DNA interstrand cross-links was evaluated either on the DNA extracted from treated cells or on purified calf thymus DNA. The results showed that under strictly analogous experimental conditions truns-EE has a greatly reduced ability to form DNA interstrand cross-links with respect to either truns-DDP or cis-EE and cis-DDP. However, before drawing a definite conclusion it is necessary to check if trans-EE does not form DNA cross-links which are thermally unstable and that is why such cross-links are not detected. It has been demonstrated that the interstrand cross-links induced by some non metal-based anticancer drugs such as acridines [36], anthracyclines [37] and nitrogen mustards [38,39] may be destroyed either in severe alkaline conditions (alkaline elution method) or at high temperature (heat denaturationhenaturation assay) [39-411. This behaviour could also apply to the iminoether compounds if one of the drug-DNA interactions does not involve directly the metal atom but is mediated by the iminoether ligand. For instance metathesis of the alkoxide group of the iminoether ligand by the aminic group of a nucleobase could create a cross-link unstable at high pH or high temperature. Such a type of interaction with DNA would place the platinum-iminoether complexes at the junction between cis-DDP and non-metallic cytotoxic agents which also are known to

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react bifunctionally with DNA [40-421. This could represent the novelty of this new class of drugs. In conclusion, the trans iminoether platinum complex with in vivo anticancer efftcacy broadens the spectrum of the antitumour platinum complexes with unusual structure and has relevant mechanistic implications. Besides making a tram platinum complex an effective antitumour agent, the substitution of iminoethers for amines causes a reversal of the dependence on cis and trans geometry of several biological activities such as inhibition of DNA synthesis, mutagenicity and cytotoxicity. As far as the DNA interaction properties are concerned, the cis-EE complex behaves basically like cis-DDP, in contrast the tram-EE species behaves differently from trans-DDP for sequence specificity and interstrand cross-link formation. More detailed studies on the nature of DNA adducts will allow a better understanding of the mechanistic properties of truns-platinum iminoether complexes. Acknowledgements

This work was supported by the contributions of CNR (A.C.R.O. Project), MURST (40%) and EC (contract Cl l-CT92-0016 and COST D1/02/92). References [I]

R. Canetta, M. Rozenweig, R.E. Wittes and L.P. Schacter. Platinum coordination complexes in cancer chemotherapy: an historical perspective, in: K. Kimura, K. Yamada, SK. Carter and D.P. Griswold Jr. (Eds.), Cancer Chemotherapy: Challenges for the Future, 5, Excerpta Medica, Tokyo, 1990, pp. 318-323. [2] M.J. McKeage, J.D. Higgins III and L.R. Kelland, Platinum and other metal coordination com-

pounds in cancer chemotherapy, Br. J. Cancer, 64 (1991) 788-792. [3] M.J. Cleare and J.D. Hoeschele, Antitumor activity of group VIII transition metal complexes. I. Platinum(H) complexes, Bioinorg. Chem., 2 (1973) 187-210. [4] M.J. Cleare, P.C. Hydes, D.R. Hepburn and B.W. Malerbi, Antitumor platinum complexes: structure-activity relationships, in: S. Prestayko. T. Crooke and SK. Carter (Eds.), Cisplatin: Current Status and Development, Academic Press Inc., New York, 1980, pp. 149-170. [5] E. Eisenhauer, K. Swerton, J. Sturgeon, S. Fine, S. O’Reilly and R. Canetta, Carboplatin therapy for recurrent ovarian carcinoma: National Cancer Institute of Canada experience and a review of the literature, in: P. Bunn, R. Canetta. R. Ozols and M. Rozenweig (Eds.), Carboplatin: Current Perspectives and Future Directions, W.B. Saunders, Philadelphia, 1990. pp. 133-140. [6] A.M.J. Fichtinger-Schepman, J.L. van der Veer, J.H.J. den Hartog, P.H.M. Lohman and J. Reedijk, Adducts of the antitumor drug ris-diamminedichloroplatinum(l1) with DNA: formation, identification and quantitation, Biochemistry, 24 (1985) 707-713. [7] A. Eastman, The formation, isolation, and characterization of DNA adducts produced by anticancer platinum complexes, Pharmacol. Ther.. 34 (1987) 155-166. [8] C.A. Lepre and S.J. Lippard, Interaction of platinum antitumor compounds with DNA, in: F. Eckstein and D.M.J. Lilley (Eds.), Nucleic Acids and Molecular Biology, 4, Springer-Verlag. Berlin, 1990, pp. 9-38. [9] J.C. Jones, W. Zeen, E. Reed, R.J. Parker, A. Sancar and V.A. Bohr, Gene-specific formation and repair of cisplatin intra-strand adducts and inter-strand cross-links in Chinese hamster ovary cells, J. Biol. Chem., 266 (1991) 7101-7107. [IO] M.A. Lemaire, A. Schwartz, A.R. Rahmouni and M. Leng. Inter-strand cross-links are preferentially formed at the d(GC) sites in the reaction between cis-diamminedichloroplatinum(I1) and DNA. Proc. Natl. Acad. Sci. USA, 88 (1991) 1982-1985

M. Coluccia

et al. /Chemico-Biological

Interactions

98 (1995)

251-266

265

[I l] L.S. Hollis, A.R. Amundsen and E.W. Stem, Chemical and biological properties of a new series of cis-diammineplatinum(I1) antitumor agents containing three nitrogen donors: cis-[Pt(NHj)z(N-donor)Cl]+, J. Med. Chem., 32 (1989) 128-136. [12] L.S. Hollis, W.I. Sundquist, J.N. Burstyn, W.J. Heiger-Bernays, S.F. Bellon, K.J. Ahmed, A.R. Amundsen, E.W. Stem and S.J. Lippard, Mechanistic studies of a novel class of trisubstituted platinum(I1) antitumor agents, Cancer Res., 51 (1991) 1866-1875. [I9 M. Van Beusichem and N. Farrell, Activation of the tram geometry in platinum antitumor complexes. Synthesis, characterization and biological activity of complexes with the planar ligands pyridine, N-methylimidazole, thiazole and quinoline. Crystal and molecular structure of trans-dichlorobis(thiazole)platinum(II), Inorg. Chem., 31 (1992) 634-639. [14] N. Farrell, L.R. Kelland, J.D. Roberts and M. Van Beusichem, Activation of the frans geometry in platinum antitumor complexes: a survey of the cytotoxicity of tram complexes containing planar ligands in murine L1210 and human tumor panels and studies on their mechanism of action, Cancer Res., 52 (1992) 5065-5072. [15] Y. Zou, B. Van Houten and N. Farrell, Ligand effects in platinum binding to DNA. A comparison of DNA binding properties for cis- and frans-[PtClz(amine)z] (amine = NH3, pyridine), Biochemistry, 32 (1993) 9632-9638. [16] N. Farrell, Y. Qu and M.P. Hacker, Cytotoxicity and antitumor activity of bis(platinum) complexes. A novel class of platinum complexes active in cell lines resistant of both cisplatin and I ,2diaminocyclohexane complexes, 1. Med. Chem., 33 (1990) 2179-2183. [17] Y. Qu and N. Farrell, Interaction of bis(platinum) complexes with the mononucleotide 5’guanosine monophosphate. Effect of diammine linker and nature of the bis(platinum) complex on product formation, J. Am. Chem. Sot., 113 (1991) 4851-4857. [18] L.R. Kelland, C.F.J. Barnard, K.J. Mellish, M. Jones, P.M. Goddard, M. Valenti, A. Bryant, B.A. Murrer and K.R. Harrap, A novel rrans-platinum coordination complex possessing in vitro and in vivo antitumor activity, Cancer Res., 54 (1994) 5618-5622. [19] M. Coluccia, A. Nassi, F. Loseto, A. Boccarelli, M.A. Mariggib, D. Giordano, F.P. Intini, P. Caputo and G. Natile, A trans platinum complex showing higher antitumor activity than the cis congeners, J. Med. Chem., 36 (1993) 510-512. [20] R. Cini, P.A. Caputo, F.P. Intini and G. Natile, Mechanistic and stereochemical investigation of iminoethers formed by alcoholysis of coordinated nitriles: X-ray crystal structure of cis- and trans[bis(l-imino-l-methoxyethane)dichloroplatinum(II)], Inorg. Chem., 34 (1995) 1130- 1137. [21] J. Litchfield and F.A. Wilcoxon, A simplified method of evaluating dose-effects experiments, J. Pharmacol. Exp. Ther., 96 (1949) 99-l 13. [22] M. Coluccia, F.P. Fanizzi, G. Giannini, D. Giordano, F.P. Intini, G. Lacidogna, F. Loseto, M.A. Mariggib, A. Nassi and G. Natile, Synthesis, mutagenicity, binding to pBR 322 DNA and antitumour activity of platinum(I1) complexes with ethambutol, Anticancer Res., I1 (1991) 281-288. [23] R.I. Freshney, in: Culture of Animal Cells, A Manual of Basic Technique, A.R. Liss Inc., New York, 1987, pp. 236-237. [24] A.R. Morgan and D.E. Pulleybank, Native and denatured DNA, cross-linked and palindromic DNA and circular covalently-closed DNA analysed by a sensitive fluorometric procedure, B&hem. Biophys. Res. Commun., 61 (1974) 396-403. [25] T.O. Brent, Suppression of cross-link formation in chloroethyl-nitrosourea-treated DNA by an activity in extracts of human leukemic limphoblasts, Cancer Res., 44 (1984) 1887-1892. [26) D.M. Maron and B.N. Ames, Revised methods for the salmonella mutagenicity test, Mutat. Res.. 113 (1983) 173-215. [27] F.P. Fanizzi, F.P. Intini and G. Natile, Nucleophylic attack of methanol on bis(bcnzonitrile)dichloroplatinum: formation of mono- and bis-imido ester derivatives, J. Chem. Sot. Dalton Trans., (1989) 947-951. (28) J.M. Casas, M.H. Chisholm, M.V. Sicilia and W.E. Streib, Imino ether complexes of platinum: cis[PtCl&HN=C(OR)Me),] and [PtCl,(HN=C(OR)Me)& where R = Me, Et and Pr’. Preparation, characterization and X-ray structure for [PtClz(HN=C(OPr’)Me)l] 2, Polyedron. 10 (1991) 1573-1578. [29] M. Coluccia, M. Correale, F.P. Fanizzi, D. Giordano, L. Maresca, M.A. Mariggib, G. Natile and

266

M. Coluccia er al. / Chemico-Biological lnieracrions 98 I 1995) 251-266

M. Tamaro, Mutagenic activity of some platinum complexes: chemical properties and biological activity, Toxicol. Environ. Chem., 8 (1984) l-8. L.F. Pearlman and H. Simpkins, Effect of platinum antitumor [301 M. Arquilla, L.M. Thompson, agents on DNA and RNA investigated by terbium fluorescence. Cancer Res., 43 ( 1983) 12 II- I2 16. of fluorescence of terbium ion-DNA complexes. 1311 Z. Balcarova and V. Brabec, Reinterpretation Biophys. Chem., 33 (1989) 55-61. induced in DNA by cisI321 M. Sip, A. Schwartz, F. Vovelle, M. Ptak and M. Leng, Distortions platinum inter-strand adducts, Biochemistry, 31 (1992) 2508-2513. are I331 V. Brabec and M. Leng, DNA inter-strand cross-links of trans-diamminedichloroplatinum(I1) preferentially formed between guanine and complementary cytosine residues, Proc. Natl. Acad. Sci. USA, 90 (1993) 5345-5349. 1341 G. Laurent, L.C. Erickson, N.A. Sharkey and K.W. Kohn, DNA cross-linking and cytotoxicity induced by ris-diamminedichloroplatinum(l1) in human normal and tumor cell lines, Cancer Res.. 41 (1981) 3347-3351. interI351 J.J. Roberts, R.J. Knox, M.F. Pera, F. Friedlos and D.A. Lydall, The role of platinum-DNA actions in the cellular toxicity and antitumor effects of platinum coordination compounds, in: M. Nicolini (Ed.), Platinum and Other Metal Coordination Compounds in Cancer Chemotherapy, Martinus Nijoff Publishing, Boston, 1988, pp. 16-31. lI361 J. Konopa, J.W. Pawlak and K. Pawlak. The mode of action of cytotoxic and antitumor nitroacridines. III. In vivo interstrand cross-linking of DNA of mammalian or bacterial cells by lnitroacridines, Chem.-Biol. Interact., 43 (1983) 175- 197. in tumor I371 A. Skladanowski and J. Konopa, Interstrand DNA crosslinking induced by anthracyclines cells, B&hem. Pharmacol., 47 (1994) 2269-2278. alkylations and inter-strand crosslinks 1381 K. Kohn and C.L. Spears, Stabilization of nitrogen-mustard in DNA by alkali, B&him. Biophys. Acta, 145 (1967) 734-741. I391 D.C. Gruenert and J.E. Cleaver, Sensitivity of mitomycin C and nitrogen mustard crosslinks to extreme alkaline conditions, B&hem. Biophys. Res. Commun., 123 (1984) 549-554. Adriamycin and Daunomycin induce inter-strand cross-links in HeLa S3 cells, I401 J. Konopa, B&hem. Biophys. Res. Commun., 110 (1983) 819-826. in 1411 P.G. Parson, Dependence on treatment time of melphalan resistance and DNA cross-linking human melanoma cell lines, Cancer Res.. 44 (1984) 2773-2778. I421 E.P. Geiduschek, ‘Reversible’ DNA, Proc. Natl. Acad. Sci. USA. 47 (1961) 950-955.