Tumor-inhibiting

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J Cancer Res Clin Oncol (1990) 116:434-438 ,.~,,,,,.~a~176

Research

. n d

99 9

@ Springer-Verlag 1990

Tumor-inhibiting

[1,2-bis(fluorophenyl)ethylenediamine]platinum(II) complexes

V. Synthesis and evaluation

of enantiomeric [ 1,2-bis-(4-fluorophenyl)ethylenediaminel dichloroplatinum(II) complexes *

Hans-Dieter vom Orde 1, Herta Reile 1, Richard Miiller 1, Ronald Gust 1, Giinther Bernhardt 1, Thilo Sprufl 1, Helmut Sch6nenberger 1, Thomas Burgemeister 2, and Albrecht Mannschreck 2

Institut ffir Pharmazie, Lehrstuhl Pharmazeutische Chemie II, Sonderforschungsbereich 234 (SFB 234), Universitfit Regensburg, Universit/itsstraBe 31, D-8400 Regensburg, Federal Republic of Germany

2 Institut fiir Organische Chemie (SFB 234), UniversiQit Regensburg, UniversitfitsstraBe 31, D-8400 Regensburg, Federal Republic of Germany

Received 18 June 1990/Accepted 25 June 1990

Summary. The enantiomeric [1,2-bis(4-fiuorophenyl)eth- ylenediamine]dichloroplatinum(II) complexes were syn- thesized and tested on the hormone-sensitive h u m a n M C F 7 breast cancer cell line and on the P388 leukemia of the mouse. They showed a strong and comparable ac- tivity on both t u m o r models.

Key words: (R,R)- and (S,S)-[1,2-bis(4-fluorophenyl)eth- ylenediamine]dichloroplatinum(II) Synthesis - M C F 7 breast cancer cell line P388 mutine leukemia

Introduction

R,S- and R,R/ S,S-configurated diaqua[1,2-bis(4-fluoro- phenyl)ethylenediamine]platinum(II) sulfates and ni- trates (1-4) display a strong growth inhibition o f the hor- mone-dependent M X T - M 3 . 2 m a m m a r y carcinoma o f the mouse (Reile et al. 1990 b). Their effect is independent o f the ligand configuration, R,S (meso) or R,R/S,S (race- mate), and identical with that o f cisplatin. In contrast to cisplatin, however, the diaqua [1,2-bis(4-fluorophenyl)- ethylenediamine]platinum(II) sulfates and nitrates cause a strong decrease in uterine weight o f the adult but not o f the juvenile mouse. This p h e n o m e n o n is thought to be a consequence o f an interference in the steroid bio- synthesis. Therefore we assume that [1,2-bis(4-fluorophe- nyl)ethylenediamine]platinum(II) complexes exert their mammary-tumor-inhibiting properties by a decrease o f the estrogen and progesterone level as well as by an inter- ference with D N A replication in analogy to the mode o f action o f cisplatin. In fact, c o m p o u n d 2, with R,R/S,S configuration, inhibits the [3H]thymidine incorporation into hormone-independent human MDA-MB231 breast cancer cells to the same extent as cisplatin (Reile et al.

* Dedicated to Professor Dr. D. Schm/ihl on the occasion of his 65th birthday

Abbreviations. R,R/S,S, racemate; R,S, meso Offprint requests to: H. Sch6nenberger

1990b). C o m p o u n d 1, with R,S-configuration, is some- what less active in this experiment. The same results were seen with the analogous dichloroplatinum(II) com- plexes (Reile et al. 1990b). Platinum complexes that do not only act on the hormone-dependent but also on the hornaone-independent m a m m a r y carcinoma (i.e. es- trogen-receptor-positive and -negative, respectively) are of great therapeutic interest, since they delay or perhaps prevent the development o f resistance, which originates from estrogen-receptor-negative m a m m a r y carcinoma clones.

The aim o f this work was to show whether the effect o f one enantiomer o f [1,2-bis(4-fluorophenyl)ethylenedi- amine]dichloroplatinum(II) is superior to that of its racemic mixture (compound 8). In the following we will describe the resolution of c o m p o u n d 8 into its enan- tiomers 9 and 10, the determination o f the absolute con- figuration by circular dichroism (CD) spectroscopy and the testing on the hormone-sensitive human M C F 7 breast cancer cell line and on the P388 leukemia of the mouse.

F F F F

H C - - C H - H C - - C H

/ \ / \

H2N NH z H 2 N NH2

\ /

8a.,ga,10a cI/Pt~.cI

Compound Configuration

8a, 8

^R.R/S.5

9a, 9

^R.R

10a, 10 S,S

8, 9, 10

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Table 1. 1 H - N M R data of enantiomeric [1,2-bis(4-fluorophenyl)ethylenediamine]dichloroplatinum(II) complexes and of their ligands

Compound Configuration R Leaving Counter

group (L) ion

R R 1 R,S 4~F OH 2 SO 4

~ 2 R,R/S,S 4 - F O H 2 8 0 4

3 R,S 4 - F O H 2 (NO3) 2

4 R,R/S,S 4 - F O H 2 (NO3) 2

5 R,R/S,S 3-OH C1 -

HC / CH \ 6 R,R/S,S 4-OH C1 -

H2 N NH 2 7 R,S 4 - F C1 -

~ p t /

8 R,R/S,S ZbF Cl -

9 R,R 4 - F C1 -

L /

~ L

10 S,S

4-F

C1 -

Compound

5

(ppm) (tetramethylsilane, internal)

Aromatic H CH (benzylic) NH

9a a 6.90-6.99 (m, 4 H) 4.01 (s, 2 H) 7.13-7.20 (m, 4 H)

lOa" 6.89-6.99 (m, 4 H) 4,01 (s, 2 H) 7.13-7.19 (m, 4 H)

9a. 2HCL b 7.01-7.61 (m, 8 H) 5.2 (s, 2 H) lOa. 2HC1 b 6.98 7.56 (m, 8 H) 5.2 (s, 2 H)

9 c 7.03-7.10 (m, 4 H) 5.2 (m, br, 2 H) 7.75-7.80 (m, 4 H)

10 c 7.03-7.10 (m, 4 H) 5.2 (m, br, 2 H) 7.75 7.81 (m, 4 H)

1.6 (s, br, 4 H) 1.6 (s, br, 4 H )

6.0 (m, br, 2 H) 6.4%6.52 (m, 2 H) 5.926.03 (m, 2 H) 5.49-6.53 (m, 2 H) a CDC13, 250 MHz

b CD3OD, 60 MHz

c dv_dimethylformamide ' 250 MHz

M a t e r i a l s a n d m e t h o d s Chem&try

The synthesis of (• (8a)

has been described in one of our previous publications (Miiller et al.

1989). Since it turned out that neither ethylenediamine ligands nor dichloroplatinum(II) complexes were appreciably resolved by HPLC on triacetylcellulose, compound 8 a was resolved in MeOH by fractional crystallization of its diastereomeric salts with optically active tartaric acid (method A). This procedure leads to complete enantiomeric purity of the diamines [(+)9 a and ( - ) 1 0 ai. For ana- lytical characterization, the oily bases were transformed into their solid dihydrochlorides ( + ) 9 a . 2 H C 1 and ( - ) 1 0 a . 2 H C 1 (method B). The enantiomeric [1,2-bis(4-fluorophenyl)ethylenediamine]

platinum(II) complexes [(+)9 and ( - ) 1 0 ] were synthesized by reacting K2PtC14 with ( + ) 9 a . 2 H C 1 and ( - ) I 0 a . 2 H C 1 , respectively, in aqueous solution (method C). 1H-NMR data and elemental analyses of these substances are listed in Tables 1 and 2.

The absotute configuration of the enantiomeric [1,2-bis(4- fluorophenyl)ethylenediamine]dichloroplatinum(II) complexes (9

Table 2. Enantiomeric [1,2-bis(4-fluorophenyl)ethylenediamine]

dichloroplatinum(II) complexes, 9 and 10 and their ligands 9a. 2HC1 and lOa - 2HCI: elemental analyses

Com- C (%) H (%) N (%)

pound

Calcd. Found Calcd. Found Calcd. Found

9a. 2HC1 52,4 52.4 5.02 4.91 8.7 8.5

lOa. 2HC1 52.4 4.87 8.5

9 32.7 33.2 2.75 2.83 5.5 5.3

10 33.0 2.90 5.2

and 10) was determined by comparison of their CD spectra with those of (+)(R,R)- and (-)(S,S)-dichloro[l,2-diphenylethylenediamine] platinum(II), the structural assignment ofwhich derives from the known absolute configuration of (+)-l,2-diphenylethylenediamine (R,R) (Meric and Vigneron 1974). In analogy to the 3- and 4-hydroxy-substituted ( _+ )-dichloro[1,2-diphenylethylenediamine]

platinum(II) complexes 5 (Jennerwein et al. 1989a) and 6 (Wappes et al. 1984), the dextrorotatory enantiomer of the (•

fluorophenyl)ethylenediamine]dichloroplatinum(II) (8) has the R,R

F F

~ ~

^cHN

/ \

H2N NH2 3.12 2.95

+

F3Cs /OH (+) I

H',,IIC

3.12

(si _ _

6.3 equivalents 2.96

Fig. 1 . 1 H - N M R benzyl signals of ligands in CDC13 at 250 MHz in the presence of 6.3 equivalents of an optically active auxiliary and o f a small amount of D20. Top: ( + ) 8 a shows singlets for both enantiomers. Center: ( + ) 9 a shows one singlet only, i.e. an enantiomeric purity of approx. 100%. Bottom: ( - ) ! 0 a shows another singlet, exclusively (i.e. purity approx. 100%).

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(compound 9) and the levorotatory enantiomer S,S configuration (compound 10).

( +_ )- 1,2-Bis (4-fluorophenyl) ethylenediamine [8 ai. The synthesis of 8a has been reported by Mfiller et al. (1989).

( + )- and ( - ) -1,2-Bis (4-fluorophenyl) ethylenediamine [ ( + ) 9 a and ( - ) l O a ] , method A. Compound 8a (7.49g, 30.17 mmol), dissolved in 35 ml MeOH, was added to the solution of L(+)-tartaric acid (13.58 g, 90.51 mmol) in 85 ml MeOH and boiled for 10 min. The solution was allowed to cool slowly. The tartrate of ( - ) 1 0 a crystallized at room temperature and was recrystallized from MeOH several times. The free base was obtained by treatment with 5% NaOH, extraction with CH2C1 z, washing with H 2 0 and drying over MgSO4. The product was a colorless oil, yield 28%, [ o E ] 2 1 6 : - - 1 1 0 , c = 1.0, MeOH, enantiomeric purity approximately 100%. From the mother liquors of the precipitates the diamine, which contains an excess of ( + ) 9 a , was isolated as free base and purified in the saine manner by crystallization of the D ( - )-tartrate.

The diamine was a colorless oil, yield 34%, [oE]sz416 = + 109, c = 1.0, MeOH, enantiomeric purity: approximately 100%. The enantiomeric purity was determined by 1H-NMR spectroscopy (250 MHz) by means of the formation of diastereomeric complexes with (S)-I- (9-anthryl)-2,2,2-trifluoroethanol. The molar ratio of this alcohol to compound ( + ) 9 a and compound ( - ) 1 0 a , respectively, was 6.3 [10.24mg ( + ) 9 a or ( - ) 1 0 a , 71.87mg (S)-l-(9-anthryl)-2,2,2-trifluoroethanol and two drops DzO in 0.5 ml CDCl~]. The enantiomeric purity was established by the integral ratio of the two singlets for the benzylic protons. The absorption band of compound ( - ) 1 0 a is shifted to higher field (6 = 2.96 ppm) than that of ( + ) 9 a (6 = 3.12 ppm) (Fig. 1).

( + )-l,2-Bis(4-fluorophenyl)ethylenediaminedihydrochloride [(+)9a.2HC1], method B. To compound ( + ) 9 a , dissolved in a small amount of MeOH, ethereal HC1 was added dropwise under ice cooling. After addition of Et20 to tarnish, the mixture was allowed to stand in the refrigerator. The precipitate was sucked off. Color- less crystals, re.p. 241.0 241.5 ~ C (decomp.), yield 50%, [oE]2] 6 = + 49, c = 1.0, MeOH.

MHz) spectrometer and 1H-NMR spectra of the platinum complexes with a Bruker P F T - N M R spectrometer WM 250 at 250 MHz. Elemental analyses were carried out by the Microlabora- tory of the University of Regensburg. The polarimeter was a Perkin- Elmer 241 MC (Perkin-Elmer, Uberlingen, FRG).

Biological methods

Hormone-sensitive human MCF7 breast cancer cell line. The MCF7 cell line was obtained from the pleural effusion of a patient with dis- seminated mammary carcinoma (Soule et al. 1973) and was obtained from the American Type Culture Collection, Rockville, USA. Cell line banking and quality control were performed according to the "seed stock concept" reviewed by Hay (1988). The cells were maintained in Eagle's minimum essential medium (Sigma) containing NaHCO3 (2 g/l), gentamicin (50 mg/l), 10% fetal calf serum (Gibco) in 75-cm 2 flasks at 37 ~ C in a H20-saturated atmosphere of 95% air and 5% CO2. The cells were serially passaged weekly following trypsinization using trypsin/EDTA (Boehringer). For chemosensitivity testing the cells (in passage 155) were plated in 96- well microplates (100 pl/well) at a deusity of about 19 cells/micro- scopic field (Leitz Diavert, 320 • ) and were allowed to attach. After 73.5 h, the medium was removed by suction and replaced with fresh medium (200 ~d/well) containing the drug (drugs were added as a 1000-fold stock solution in dimethylformamide or pure solvent. On every plate the rows 5 and 6 (n = 16) acted as controls, whereas two vertical rows (n = 16) per drug concentration and time point were used. After varying times of incubation the cells were fixed with glu- taraldehyde and stored under phosphate-buffered saline at 4 ~ C. All plates were stained with crystal violet simultaneously. The pro- cessing procedure and data anatysis were performed as described by Reile et al. (1990a). Drug effects were calculated as corrected TIC values according to: TIC ... : (T-- Co)/(C-- Co)" 1001%], where T is the absorbance of treated cells, C that of the controls and Co the absorbancc at the time ( t = 0 ) when drug was added. The calculated experimental crrors for TIC ... (according to the Gaussian formula) amount to about 10% after prolonged incubation.

( - )-l,2-Bis (4-fluorophenyl) ethylenediaminedihydrochloride [ ( - ) l O a . 2 H C l ] . This was obtained as colorless crystals, re.p.

240.5-241.5"C (decomp.), yield 78%, [oE]546-- --46, C= 1.0, 2 1 _ MeOH.

[( ++ )-l,2-Bis(4-fluorophenyl)ethylenediamine] dichloroplatinum(II) (8), method C. Synthesis has been described by Miiller et al. (1989).

[ ( + )- 1,2-Bis (4-fluorophenyl) ethylenediamine] dichloroplatinum (II) (( + ) 9). Compound ( + ) 9 was obtained as a yellow powder, yield 86%, [c~]~26 = + 148, c=0.5, dimethylformamide.

[ ( - )-l,2-Bis (4-fluorophenyl) ethylenediamine] dichloroplatinum (Il) ( ( - ) 10). Compound ( ( - ) 1 0 ) was a yellow powder, yield 88%,

2 2 =

[oE]87 -- 144, e=0.5, dimethylformamide.

P388 Leukemia. The P388 leukemia (Dawe and Potter 1957; Geran et al. 1972) was maintained by routine passage in female DBA/2 mice (Charles River Wiga, Sulzfetd, FRG). For determination of antitumor activity, female CDF1 mice (18-22 g, Zentralinstitut ffir Versuchstiere Hannover, FRG), were inoculated i.p. with 106 leukemia cells in 0.1 ml phosphate-buffered saline (day 0). The animals were randomly assigned to groups of six (ten animals to the solvent control) and the complexes were administered i.p. on days 1-9 as a solution or suspension in polyethylenegtycol-400/1.8% NaC1 in H 2 0 (1 : 1). The antitumor activity was evaluated as median survival time (days) compared to the untreated control.

Results and discussion

C D spectra

The spectra were obtained with a JASCO J-40 A spectropolarimeter (time constant 4 s; scan speed 50 nm/min) and recored in Me2SO at room temperature in 0.2 cm quartz cells. The concentrations were 0.7 m M f o r (+)9, and 0.8 m M f o r ( - ) 1 0 , 2 m M for (S,S)-dichloro- [1,2-diphenylethylenediamine]platinum(II) and 4 m M for (R,R)-di- chloro[1,2-diphenylethylenediamine]platinum(II).

General procedures

Melting points (uncorrected) were recorded in a Btichi 510 melting- point apparatus, 1H-NMR spectra with a Varian EM 360 L (60

T h e s t r o n g effect o f t h e d i a s t e r e o m e r i c [ 1 , 2 - b i s ( 4 - f l u o r o - p h e n y l ) e t h y l e n e d i a m i n e ] p l a t i n u m ( I I ) c o m p l e x e s f o u n d o n t h e h o r m o n e - d e p e n d e n t M X T m a m m a r y c a r c i n o m a o f t h e m o u s e , w a s r e e v a l u a t e d i n t h e e x p e r i m e n t o n t h e h o r m o n e - s e n s i t i v e h u m a n M C F 7 b r e a s t c a n c e r cell line b y m e a n s o f t h e d i c h l o r o p l a t i n u m ( I I ) d e r i v a t i v e s 7 - 1 0 ( F i g . 2).

I n this test we s t u d i e d t h e g r o w t h k i n e t i c s i n t h e p r e s - e n c e o f i n c r e a s i n g d r u g c o n c e n t r a t i o n s (0.5 g M , 1 I-tM, a n d 5 p M ) b y s t a i n i n g w i t h c r y s t a l violet. A m i c r o c o m - p u t e r t e c h n i q u e , w h i c h a l l o w s a l a r g e n u m b e r o f s p e c t r o - p h o t o m e t r i c m e a s u r e m e n t s n e c e s s a r y f o r t h e c o n s t r u c - t i o n o f t h e g r o w t h c u r v e s , w a s u s e d . I n o r d e r to p r o v i d e a m a x i m u m o f i n f o r m a t i o n , t h e d a t a a r e p r e s e n t e d as cor-

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120

80

z~O

0 0

8

(R,R/S,S)

I I I I

100 2 0 0

120

80

7 (R,5)

~0 ™ ~

0 . . . _D_-~~..~.~ _ _

0 100 200

120

(..)

8o

oe

L L O L 0

0

9

(R,R)

¤244244244

. . . . -D-- O---O---'D--

I I I I

0 100 200

120

1o (s.s)

LO z~ ""zx~z~~'szx

~ 0 ~ 0 ~ 0 0

0 . . . ~ - ~ - ~~-D=~ 5 -- -

l I I I

0 100 2 0 0

120

cDDP

~o ~i~i--~~

^&o

o

. . . --~-- --=- .. . .

I I I E

o lOO 200

I n e u b o t i o n time (h)

Fig. 2. Effect of compounds 7-10 and of cisplatin on MCF7 breast cancer cell line; plot of corrected TIC values versus time of drug ex- posure, zx, 0.5 laM," o, 1 gM; n, 5 gM

rected TIC values versus time of drug exposure. A gradual decrease o f TIC ... values with time indicates an inhibition o f cell growth (i.e. a slowing d o w n or a cessa- tion ofcell proliferation = cytostatic drug action). An in- crease of TIC ... values with time o f drug exposure represents p r i m a r y drug resistance or recovery of the cells f r o m drug-induced damage, which m a y result in full reproductive integrity o f the cells (see also Mfiller et al.

1990). TIC ... values < 0 indicate cytocidal drug action.

437 On the M C F 7 breast cancer cell line, the R, S-config- urated complex (7) shows a similar effect to cisplatin in a concentration o f 1 p M and 5 pM. At the lowest concen- tration (0.5 g M ) the cells are weakly inhibited initially but recover after a b o u t 150 h o f drug exposure. This be- havior can be explained by a development o f resistance o f the cell line against low concentrations o f 7. In the case o f the m o r e active complex 8, with the R,R/S,S configu- ration, a clearcut dose-dependent inhibition is observed, producing a cytocidal effect in the highest concentration (5 gM). Surprisingly, neither o f the enantiomers (9 and 10) was m o r e active t h a n the racemate (8).

On the P388 leukemia o f the mouse the enantio- meric [1,2-bis(4-fluorophenyl)ethylenediamine]dichloro- platinum(II) complexes (9 and 10) showed a strong but not dose-dependent inhibition o f t u m o r growth, which p r o b a b l y results f r o m their low water solubility (Table 3, c o m p a r e also Reile et al. 1990 c). In accordance with the results on the M C F 7 breast cancer cell line, differences in activity between the two enantiomers (9 a n d 10) were n o t detectable. F u r t h e r on, the antileukemic effect o f 9 and 10 is similar to that o f their racemate 8.

Differences in the a n t i t u m o r activity o f R,R- a n d S,S- configurated [1,2-diphenylethylenediamine]platinum (II) complexes due to the chirality of D N A were postulated for the first time by Gulotti and coworkers (1982, 1984), but could not be p r o v e d so far in experiments on the P388 leukemia of the mouse. In contrast to these results Noji and coworkers (1984) reported on a different activity o f enantiomeric [diaqua]l,2-diphenylethylenediamine]

platinum(II) sulfates and nitrates in the L1210 mouse leukemia experiment. However, the significance o f these data is questionable, since the order o f activity changed depending on the nature of the counter ion. In the m o r e meaningful in vitro experiment on the h u m a n M D A - MB231 breast cancer cell line we observed equal effects with b o t h enantiomers of dichloro[1,2-diphenylethyl- enediamine]platinum(II), but m a r k e d differences be- tween diastereomers (Wappes et al. 1984 b). The complex with the R,R/S,S configuration was m o r e active t h a n its R,S counterpart.

However, small differences we observed with enantio- meric 3- and 4-hydroxy-substituted dichloro[1,2-di- phenylethylenediamine]platinum(II) complexes (compare formulae of c o m p o u n d s 5 and 6) (Wappes et al. 1984a;

Table 3. Antitumor effect of R,R- and S,S-configurated [1,2-bis(4-fluorophenyl)ethytenediamine]dichloroplatinum(II), compounds 9 and 10, on the P388 leukemia of the CDF I mouse

Compound Total dose" Change ofmean body weight (g) Median survival TIC

(pmol/kg) days (range) (%)

d5-dl d9-dl d16 dl Control

9 10

0.3 4.7 - 9.0 (9 11) 100

160 -0.9 -2.3 4.0 19.0 (17-22) 211

120 -1.3 -1.4 19.5 (5oE0) 217

60 0.2 -0.7 - 17.5 (17-20) 194

160 -1.1 -1.2 3.9 18.5 (16-19) 206

120 -0.3 -0.7 - 17.5 (16-20) 194

60 -0.7 -0.3 - 16.5 (15-19) 183

" The compounds were administered intraperitoneally as polyethylcne-glycol-400/l.8% NaC1 solution on days 1-9

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J e n n e r w e i n et al. 1989 b). T h e i n f l u e n c e o f steric f a c t o r s o n the a n t i t u m o r a c t i v i t y o f p l a t i n u m c o m p l e x e s is t h o r - o u g h l y d i s c u s s e d in v o n A n g e r e r et al. (1989).

T h o u g h the e n a n t i o m e r i c [ 1 , 2 - b i s ( 4 - f l u o r o p h e n y l ) e t h y l e n e d i a m i n e ] d i c h l o r o p l a t i n u m ( I I ) c o m p l e x e s 9 a n d 10 s h o w n o d i f f e r e n c e in a c t i v i t y o n the M C F 7 b r e a s t c a n c e r cetl line, it is c o n c e i v a b l e t h a t u n d e r in v i v o c o n d i - t i o n s the b i o l o g i c a l effects o f t h e e n a n t i o m e r s 9 a n d 10 a r e d i f f e r e n t , since the d i s c u s s e d e s t r o g e n - a n d p r o g e s - t e r o n e - l o w e r i n g effects o f these d r u g s ( c o m p a r e M / i l l e r et al. 1989) a l s o i n f l u e n c e the h o r m o n e - d e p e n d e n t m a m m a r y c a r c i n o m a . T h e steric f a c t o r s a r e s u p p o s e d to be m o r e i m p o r t a n t f o r the a c t i v i t y o f the e n a n t i o m e r i c [ 1 , 2 - b i s ( 4 - f l u o r o p h e n y l ) e t h y l e n e d i a m i n e ] d i c h l o r o - p l a t i n u m ( I I ) c o m p l e x e s o n t h e s t e r o i d b i o s y n t h e s i s t h a n o n the D N A r e p l i c a t i o n .

W h e t h e r e n a n t i o m e r i c [ 1 , 2 - b i s ( 4 - f l u o r o p h e n y l ) e t h y l - e n e d i a m i n e ] d i c h l o r o p l a t i n u m ( I I ) c o m p l e x e s h a v e differ- ing effects o n the h o r m o n e - d e p e n d e n t m a m m a r y c a r c i - n o m a in v i v o will be s t u d i e d o n the e s t r o g e n - r e c e p t o r - p o s i t i v e M X T m a m m a r y c a r c i n o m a o f t h e m o u s e .

Acknowledgements.

The technical assistance of E. Aichinger, D.

Krisam, P. Pistor and P. Richthammer is gratefully acknowledged.

Thanks are also due to the Deutsche Forschungsgemeinschaft (SFB 234), the Matthias Lackas-Stiftung ffir Krebsforschung and the Fonds der Chemischen Industrie for financial support.

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