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Reduction of the Estrogenic Side Effects of the Mammary Tumor-Inhibiting Drug [12-Bis26-dichloro-4-hydroxyphenyl-ethylenediamine]dichloroplatinumII by Variation of Ring Substituents.

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Estrogenic Side Effects
Reduction of the Estrogenic Side Effects of the Mammary
Tumor-Inhibiting Drug [1,2-Bis(2,6-dichloro-4-hydrox~henyl)ethylenediamine]dichloroplatinum(II) by Variation of Ring
Substituents+)
Ronald Gust*, Johann Karl, Michael Faderl, and Helmut Schbnenberger
Institut fiir Pharmazie. Lehrstuhl PharmazeutischeChemie I1 der Universitit Regensburg, Universititsstr. 31, D-93040 Regensburg, Germany
Received October 11,1994; revised form received December 23. 1994
Vennindemng der estmgenen Nebenwirkungen der brustkrebshemmenden Vehindung [ l P B i s ( 2 6 ~ ~ o r 4 - h y d r o x y p h e n y l ) e t h y amin]clichloroplatin(II)durch Veriinderung der Ringsubstituentem
[ 1,2-Bis(4-methoxy /4-hydroxyphenyl)ethylenediamine]dic~or~latinu~
[ 1,2-Bis(4-methoxy/4-hydroxyhenyl)ethylendiamin]dichloroplatin(II)
(II) complexes with CI-, CH3-, or OCHrsubstituents in the orrho-positions
Komplexe mit CI-, CH3-, oder OCfi-Substituenten in den ortho-Positionen
of the aromatic rings (meso-1-PtClz,~ , ~ - l - P t C lmso-2-PtCl2,
z,
~ , ~ - 2 P t C l 2 , der aromatischen Ringe (meso-1-PtClz, ~ , ~ - l - P t C lmeso-l-PtCh,
z.
D,L-2me.so-1PtClz. meso-QPtCIz, meso-5-PtClz)were tested on the MDA-MB
PtClz,~ o - J - P t C l zmeso-QPtClz.
,
meso-5-PtClz) wurden an der MDA-MI3
231 breast cancer cell line, the lymphocytic leukemia P388,and theestrogen
231 Brustkrehszellinie. der lymphozytischen LeuEmie p388 und dem
receptor-positive and -negative MXT mammary carcinoma of the mouse
Estrogenrezeptor-positiven und -negativen MXT Mammakaninom der
(MXT,ER(+)-MC, MXT,ER(-)-MC). The comparison of the effects of
Maus (MXT,ER(+>MC, MXT.ER(-)-MC) gepriift. Vergleicht m a n die
Wirkung der 4-OCH3-substituierten Komplexe (mesel-PtClz. D,L-~-P~C~Z,
meso-3-PtCIz)
methoxy-substitutedcomplexes (meso-l-PtCIz. D,L-~-P~CIZ
D,L-~with those of the respective hydroxy-substitutedones (meso-%PtClz,D , L - ~ - meso-3-PtClz) mit der der jeweiligen 4-OH-Derivate (mesc~ZPtClz,
PtClz, mesd-PtClz) shows that a reduction of estrogenic effects as well as PtCIz, mese4-PtClz), so findet man eine Vemngerung der estrogenen
a total loss of the mammary tumor-inhibitingactivity takes place on methyWirkung und einen totalen Verlust der mammaturnorhemmendenWirkung
durch Methylierung der COH-Gruppe. Der Austausch der 2.6-stiindigen
lation of the 4-OH group. The exchange of the 2,bstandingchlorine atoms
Chlorsubstituenten in meso-ZPtClz durch CHs-Gmppen ftihrt zu einem
by methyl groups in meso-2-PtClz led to the non-estrogenic, but on the
Komplex (meso-4-PtClz) ohne estrogene Eigenschaften. der aber hochaktiv
MXT,ER(+)-MC highly effective derivative meso-4-PtClz which proved to
am MXT.ER(+>MC ist und sich gleichzeitig als zytotoxisch an ER(->Tube also cytotoxic on ER(-)-tumors such as MXT,ER(->MC, and the P 388
moren wie dem MXT.ER(-)-MC und der P 388 LeuErnie envies.
leukemia.
Cisplatin is currently one of the most widely used chemotherapeutics
against malignant tumors of the testis, ovary, and bladder as well as the head
and neck I). However, its high toxicity and its inactivity against important
cancer diseaseslike breast (MC) and prostate (PC) cancerlimit its application
in the oncological therapy. An approach to overcome these disadvantages
was the combinationof cisplatin with estrogen receptor @R) affinic carriers
derived from estrogens or antiestrogensw). One of the most interesting
compounds of this kind, [meso-1.2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine]dichloroplatinum(II) (meso-2-PtClz,formula see scheme I), is
derived from the synthetic estrogen hexestrol '). It is supposed that its carrier
ligand directs the cytotoxic dichloroplatinum(I1) moiety to ER-positive
(ER(+)) MC and PC cells thus achieving selective damage of the latter. The
high activity of meso-ZPtClz and its water-soluble sulfatoplatinum(I1) derivative meso-2-PtS04 on ER-positive rodent tumors (DMBA-induced MC,
MXT,ER(+)-MC, and R3327 Dunning K)and the fact that much higher Pt
levels were found in the DMBA-induced MC as well as in the uterine tissue
of the test animals than in their skeletal muscle confirm the validity of this
concept3*').
The mode of action of meso-2-PtClz was studied in detail. Indeed, on the
ER-positive MCF7 breast cancer cell line strong estrogenic effects but only
weak cpotoxic activity were found@.Meso-ZptClz drastically reduces the
number of Ez binding sites (ER processing) and increases the level of
progesterone receptor (progesterone receptor biosynthesis). In this model
meso-2-PtClz competes also with 17P-estradiol(Ez) for ER binding sites in
a competitive manner, but only at a relatively high concentration (RBA =
0.3%)
Surprisingly, meso-ZPtClz is much more estrogenic than its ER affinity
suggests. In a recent publication we have discussed the uselessness of the
common competitivebinding assay. in which 13H]E2is applied as a competitor, and we have proposed a binding site for meso-ZPtClz differentfrom that
for E2". Furthermore, we showed in these studies that in [mso-l,Z-bis(hy+) Dedicated
to Prof. Dr.F. Eden, Miinchen, on the occasion of his 70th
birthday.
Arch. Phann. (We&m)
328,457463 (1995)
droxyphenyl)ethylenediamine]platinm(Il) complexes lipophilic substituents in the 2,dposition of both phenyl rings, an 0-0distance between the
hydroxy group of about 8 A, additionalN-standing alkyl residues as well as
a rigid 5-membered chelate ring (especially in the N-alkyl derivatives)
promote the estrogenic activity, though they did not change the ER
affinity7-'0).
The goal of this study was both the reduction of the estrogenic side effects of meso-2-PtCl2, which are thought to be
responsible for cardiovascular disorders' and the preservation of the ER affinity and of the cytotoxicity. To this end we
methylated the OH groups in the parent compound meso-2PtC12 and/or exchanged also its ring standing chlorine atoms
by methyl or methoxy groups. The pharmacological properties of the new compoundswere estimated and compared with
those of the hydroxy series, which have already been described3).
Results
Chemistry
The 4-hydroxy-substituted platinum complex series has
been described in ref3). The methoxy-substituted ligands
,
and meso-5 were synthesized accordmeso-1, D , L - ~mey-3,
ing to Karl el al. . To obtain the related dichloro[l,Zdiphenylethylenediamine]platinum(II) complexes meso-lPtC12, D,L-l-PtClz, rneso-3-PtCl2, and rneso-S-PtCl2, the respective ligands were reacted with K2PtCl4 in a 50% ten-butanoVH2O mixture at 40 "C and pH 4.5 to 5.5 (Scheme 1).
For structural characterizationof the dichloroplatinum(I1) complexes 'HNMR spectroscopy was used. Upon coordinatioii to platinum the proton
signals of the 1,2-diphenylethylenediamineligands are charaderistically
0 VCH Valagsgesellschaft mbH, D-69451Weinheim 1995
0365-6233/95/0505-0457 $5.00 + 2 9 3
Gust, Karl, Faded, and Schonenberger
458
/
“2N
N.O-1
t o maw-5
DL-1 to DL-2
mew-l-PtCl2 to meso-6-PtC12
D.L-WtC12 to D.L-2-PtClg
I
compd.
X
Y
meso-1-PtClp
CI
ocH3
D.L-l-PtC12
CI
OCH3
meso-2-PtClp
CI
OH
O.L-2-PtC12
CI
OH
meso-3-PtCl2
c h
OCH3
OCH3
OCH3
meso-4-PtCb
meso-5-PtCb
I
Scheme 1
changed. Due to the blocked rotation around the N-C axis and the neighborhood of asymmetric benzylic C-atoms the N-standing H-atom show a
diastereomericsplittingwith separated signals of the axially and equatorially
arranged protons which are broadened by coupling with benzylicprotons and
I9’F’t (Table 1).
Comparison of the data listed in Table 1 indicates that
protection of the hydroxy groups by C H 3 only marginally
influences the proton shifts and splitting. Therefore, for the
respective OH- and OCHysubstituted complexes the same
spatial structure can be assumed. Coordination of rac. 1,2diphenylethylenediamines (D,L- 1 and D,L-2) to platinum results in a 5-membered chelate ring which exists predominantly in a conformation with both aromatic rings in the
equatorial position (6conformation7)).
An interconversion of the chelate ring into the energetically
unfavored conformation with axially arranged rings (h-conformation) can be excluded 7J2.13)
On the other hand, the 6 and the h-conformers of the mesocompounds meso-l-PtCl2 to meso-5-PtClz (Scheme 2) are
energetically equivalent and should exist in equilibrium. Investigations with the diiodo derivative of meso-4-PtCl2 have
shown that this is true at room temp. or physiological temp.
(about 40 “C).Only cooling to about 200 K slows down this
dynamic Gh-interconversion. At 193 K the benzylic protons
are diastereotopically split in an AB-pattern (6 = 4.73 and
5.04, JH-H= 6.6Hz). Furthermore, the rotation of the aromatic
rings is frozen, since four singlets stemming from the ortho-
Table 1: ‘H-NMR-Data of Ring-Substituted [1,2-Diphenylethylenediamine]dichloroplatinum~I)
Complexes
Y
compd.
a
X
Y
arom. H
NH
CHbenzylic
OWOCH3
OCH3
7.01 (br,ZH)
7.04 (br.2H)
5.37 (br,ZH)
6.75 (br,2H)
5.37 (br,2H)
3.86 (s,~H.OCH~)
OCH3
6.96 (d?J=2SHz,2H) 5.58 (br.2H)
7.19 (d:J=2.5Hz,2H) 6.90 (br,ZH)
4.99 (br.2H)
3.86 (s.~H,OCH~)
OH
6.82 (br,2H)
6.85 (br,2H)
5.30 (br,2H)
6.73 (br,2H)
5.30 (br,2H)
10.86 (br,2H,OH)
OH
6.78 (d:J=2.5Hz,2H)
6.98 (d:J=ZSHz,2H)
5.50 (br,2H)
6.88 (br,ZH)
4.92 (br.2H)
10.57 (br,2H,OH)
OCH3
6.53 (s,~H)
4.90 (br,ZH)
6.41 (br.2H)
4.90 (br2H)
3.74 (s,~H,OCH~) 2.03 (br.6H)
2.74 (brdH)
OH
6.41 (s.4H)
4.83 (br,2H)
6.35 (br,2H)
4.83 (br.2H)
9.43 (br.ZH,OH)
OCH3
6.11 (s.4H)
5.07 (br,2H)
5.91 (br,2H)
4.86 (br,ZH)
3.70 (s,12H,OCH3)
3.77 (s,~H,OCH~)
250 MHz in pprn, solvent: DMF-d7, TMS as internal standard
Data from Ref. 3,
AK-CH~
1.97 (brdH)
2.67 (br,6H)
Estrogenic Side Effects
459
Ar
HI
Ar
Ar
A-conformation
J
6-conformation
Meme 2
methyl groups appear in the spectrum. Temp.-dependent
'H-NMR measurements yield a coalescence temp. for the
benzylic protons of 225 K and of 243 K for the CH3 signals
(first coalescence) indicating an activation enthalpy of AG*
= 45 kl/mol for the interconversion of the 5-membered
chelate ring (to be published in detail). The second coalescence of the Ar-CH3 (freerotation of the aromaticrings) takes
place at 344 K (AG*= 68 Id/mol).
The existence of the two separate signals for the rneta-standing aromatic protons in the spectra of rneso-1-PtCl2 and
rneso-2-PtC12 reveals interconversion of the 5-membered
chelate ring and restricted rotation of the aromatic rings
(coalescence temp. of the Ar-H of meso-2-PtClz = 313 K). A
hindered &A-conversion would entail 4 doublets for the
Ar-H, as was found for rneso-4-PtI2 at 193 K.
Biology
The binding affinity to the ER of ligands and platinum(I1)
complexes can readily be measured by a competitive binding
assay using the dextran-coated charcoal technique. Th affinities were obtained relative to the tracer compound [4HI-
Table 2 Estrogenic Activity of Ring-Substituted 1,2-Diphenylethylenediaminesand of their DichIoroplatinumQI)Complexes
estrogenic activity EA[%]
at a dose of [nmoI]
compd.
a
b
X
Y
RBA a
10
4.001
74
<0.001
29
0.45
0.30
<0.001
llOd
<o.m
91
5
3
<0.001
1
<0.001
4
40
200
100
135
82
100
54
140
59
'
'
82'
114'
0
17'
2
0
loo0
'
'
'
md
3
54
13
'
0
RF3A = ratio of molar concentrationsof 17pestradiol (E2) and inhibitor required to decrease the amount of bound t3H]Ez by 509b x 100
EA = [(ET-EV)/(EE-EV)]x 100;ET = effect of test compound, Ev = effect of vehicle, EE= effect of estrone standard, effect = uterus dry weight
(mg)/body weight (s) x 100; the U-test according to Wilcaxon, Mam. and Whimey was used.
dose per animal and day, administered as solution in olive oil.
'
d .
significantto solvent treated control (p < 0.001).
dto (p < 0.05).
data from Ref. 3,
Gust, Karl, Faderl, and Schonenberga
460
Table 3: Antituma Effect of Ring-Substituted [1,2-Diphenylethylenedi~mineldichloroplatinum~I)
Complexes against Hormone-Independent MDA-MB 231 B r w t Cancer Cells
cell growth
[3H]thymidineincorporation
cow.
meSo-l-PtC12
D,L-I-F%CIZ
ntesO-zPtclz
D.L-2-PtaZ
MO-3-PtclZ
n&?S*4-FJtCl2
me~0-5-PtC12
a
TIC,%
at 5x104M EDso a [MI
49
43
100
95
43
28
96
4.4 x lo4
3.6 x lod
> I x 10-~
> IX 10-~
3.6 x lod
2.1 x lod
> I x 10-~
TIC%
at 5~10% ED% a [MI
49
43
90
94
49
34
98
3.9 x lod
4.0 x lod
>I
>I x 10-~
4.6 x lo4
2.1 x lo4
>I x
EDm = the effective dose, which decreases tumor growth by 50%;mean of 2 tests.
data from Ref. 3,
estradiol and are conveniently expressed on a % scale where enediamine]platinum(II) complexes as determined on the
MDA-MB 231 cell line.
the binding of estradiol is 100%.
Meso-l-PtCl2 and D,L-I-PtC12 were equipotent, inhibiting
Only the 2,6-C12,4-OH-substitutedligand meso-2 (RBA =
0.45%) and its dichloroplatinum(I1) complex meso-2-PtC12 cell growth and [3Hlthymidine incorporation to the same
(RBA = 0.30%)showed a weakER-affinity 3, (Table 2). Both extent (Table 3). The ED50 amounted to about 4 x lo4 M,
protection of the hydroxy group with CH3 groups (rneso-1, comparable to the activity of the 2,6-dimethyl-substituted
meso-l-PtClz) and exchange of the ortho-chlorine substi- complexes meso-J-PtC12 and meS0-4-PtC12. The 2,4,6-trituents by CH3 (meso-4, meso-4-PtClz) or OCH3 (meso-5- methoxy-substituted complex meso-S-PtCl;?was quite inactive.
PtCIz) led to a complete loss in binding affinity.
These results differ strongly from those found in vivo on the
Simultaneously, Omethylation reduced the estrogenic
properties of the ligands. Meso-1 showed the hormonal pro- P388 leukemia implanted in mice. In the case of meso-2-PtClz
file of a “true” estrogen (Table 2) but reached the maximum and meso-4-PtC12, methylation of the hydroxy groups led to
effect of estrone at a 10 fold higher dose than meso-2 in the complete inactivation. Among the methoxy-substituted deimmature mouse uterine weight test. Coordination of meso-1 rivatives only meso-5-PtClz prolonged the survival time (TIC
to platinum reduced the growth stimulating effects once = 133%).On the hormone-insensitiveMXT,ER(-)-MC of the
again. The dose activity relationship of rneso-l-PtCl2 resem- mouse, too, the methoxy-substituted complexes were inacbles that of an “impeded” estrogen. Since meso-1 as well as tive. Even at the highest dose of 20 p o l k g no significant
meso-l-PtC12 show no affinity to the estrogen receptor, the reduction of the tumor growth was found.
On the hormone-sensitive variant of this tumor
estrogenic activity may be the result of metabolic activation,
(MXT,ER(+)-MC),
the excellent activities of meso-2-PtC12
e.g. cleavage of the OMe group to give the receptor affinic
(T/C
=
5%
at
a
dose
of 1.5 pmolkg) and meso-l-PtCl;! (T/C
OH derivatives meso-2 and meso-2-PtClz. In order to avoid
=
9%
at
a
dose
of
20
p o l k g ) were abolished by O-methythis, we tried to bypass the metabolism of meso-1 by using
lation.
At
a
dose
of
20
wmolkg meso-J-PtC1~did not show
topical application to the target organ, the uterus. For this
purpose we used a transvaginaYUanscervical pulse admini- antitumor effects (T/C = 95%), and meso-J-PtC12even stimustration as described by Chwulisz et ~ 1 . ’ to
~ )place the test lated the tumor growth to TIC = 149%.The latter effect seems
compounds into the uterine lumen of juvenile SD-rats. In this to be caused by the OCH3 groups, since OCH3 trisubstitution
test the methoxy-substitutedcompound meso-1 showed com- (meso-5-PtClz)enhanced the tumor growth to a T/C = 244%.
In this test none of the OCH3 derivatives showed any estroparable estrogenic activity (EA = 90%; 100 nmol/anigenic properties on adult BDF1-mice.
mal/day), as was found in the uterine weight test on NMRI
mice (EA = 135%. 100 nmoYanimal/day),thus excluding its
Discussion
metabolic activation.
A drastic decrease of the endocrine activity of meso-2 and
As expected, the kind of 2,6-standing ring substituents
meso-2-PtClz was brought about b the change of the diamine strongly influenced the estrogenic potency of the complex
configuration from RS to RRISS 3 7as well as by exchange of
type [meso-1,2-bis(4-hydroxyphenyl)ethylenediamine]dithe ortho-chlorine substituents in the aromatic rings by CH3 chloroplatinum(I1). For example, the exchange of the four
or OCH3. Only the free 2,6-dimethyl-substituted ligand C1-atoms in meso-2-PtClz by space-requiringmethyl or methmeso-4 slightly stimulated the growth of the uterus at the very oxy groups led to a total loss of hormonal activity (Table 2).
high dose of lo00 nmol/animaYday (EA=54%). The com- Corresponding with this the 2,6-methyl-substitutedcomplex,
plexes meso-l-PtCl;! and meso-S-PtC12 as well as ~ , ~ - l - P t C 1 2meS04PtClz, showed even at the highest dosage of lo00
and D,L-2-PtClz were totally inactive.
nmol/animal/day no estrogenic effect in the mouse uterine
On the other hand the exchange of the OH groups by the weight test. The same is true for the 2,6-dimethoxy-substiOCH3 groups enhanced the in v i m cytotoxicity of the dia- tuted complex meso-S-PtClZ. Furthermore, we stated a markstereomeric [ l ,2-bis(2,6-dichloro-4-hydroxyphenyl)ethyl- ed decrease but not a loss in the estrogenic potency, if the
Arch Pharm (Weinheim)328 457-443 (1995)
46 1
Estrogenic Side Effects
Table 4 Effect of Ring-Substituted [1.2-Diphenylethylenediamine]~~l~~l~inum~I)
Complexes on the Growth of Hormone-Dependent(ER(+)) and
the Hormone-Independent (ER(-)) MXT Mammary Carcinoma of the BDFI-Mouse
yQxay
x
ffi-
H
d
J2
''
tP'
aa
MXT,ER(+)
dose a
cod.
X
Y
[ w o ~ k g[rnsflisl
l
a
a
a
OCH3
OCH3
OH
20.0
20.0
1.5
4.5
13.5
20.0
a
OH
CH3
CH3
OCH3
OCH3
OH
OCH3
4.5
20.0
20.0
20.0
20.0
2.0
a
'
13.5
13.5
1.0
3.0
9.0
13.0
3.0
13.0
12.2
11.3
13.2
899
15
med. tumor
weight [mgl
T/C
%
MXT,ER(-)
uterotr. T/C
effectb" 9%
850
1248
23
95
39
95
139
5
19
8
-
82.3
116.4
135.2
193.8
-
849
195
-
76.9
108
1342
40'
2195
100
3
149
9
244
88.1
95
71.1
80
86.6
%
-
-
96.4
-
64.8
100
114
105
93
138
160
229
-
75
288
49
med. tumor
area [mm21
TIC
96
286
126
99
330
102
241
260
203
252
74
90
63
88
363
100
15
the compounds were administered 3 times a week S.C. as a solution in olive oil.
determined at the end of the &week therapy.
uterotrophic effect = [uterus dry weight ( m g ) M y weight (s)] x 100
determined at the end of the 2-week therapy
med. tumor weight of control group: 488 mg;uterotr. effect of control group: 84.5
med. tumor weight of control group: 446
uterotr. effect of control group: 71.2
med. tumor area of control group: 325 mm
data from Ref. 3,
9;
C1-atoms in rneso-2-PtClz were exchanged by the less bulky
F-atoms"). It is worth mentioning that in spite of hormonal
inactivity rneso4-Ptc12 strongly inhibits the hormone-sensitive MXT,ER(+)-MC of the mouse (T/C = 9%, Table 4).
However, the lack of estrogenic effects need not necessarily
mean the absence of affinity to the ER. Corresponding with
the drug targeting concept described in the introduction of
this publication, the marked inhibitory effect of rneso-4-PtC12
on the ER-positive MC could also be caused by the combination of ER-affinity and cytotoxicity. While the proof of binding of rneso-4-PtC12 to the ER is still missing, we have
demonstrated cytotoxic effects in tests on the ER-negative
human MDA-MB 231 breast cancer cell line(Tab1e 3) and
also on the P388 leukemia of the mouse. These effects were
more pronounced in meso-4-PtC1;! than in the parent compound meso-2-PtC12. The transformation of rneso-QPtC12
into its 0-methyl ether, rnesu-3-PtCl2, led to a loss of activity
against the ER-positive MXT,ER(+)-MC of the mouse,
though the latter compound still possesses cytotoxic properties (c$ Table 3). In this context it is recalled that O-methylation of the highly active complex rneso-2-PtCl2 also
resulted in a compound (rneso-1-PtC12)which proved to be
inactive in the test on the hormone-sensitive MXT,ER(+)MC of the mouse. This is all the more surprising, since this
structural modification led to an increase in the cytotoxic
potency (compare rneso-l-PtC12 with rneso-2-PtCl2 in Table
3). As a rule, 0-methylation of estrogens strongly reduces
their estrogenic properties, and is accompanied by a diminu-
Arch. Phann (Weinheim)328,457463(1995)
tion of their ER-affinities. This is also true for transformation
of rneso-2-PtCl2 into rnesu-1-PtC12. For the same estrogenic
effect in the mouse uterine weight test the corresponding
0-methyl ether (rneso-1-PtCl2) must be ten times higher
dosed than the hydroxy compound rneso-2-RCl2 (Table 2).
In addition to this we have proved that the estrogenic
potency of rnesu-1-PtClzcannotbe due to metabolic transformation to the hydroxy derivative (rnesu-2-PtCl2), since its
transcervical (i.e. topical) administration led to effects comparable with those seen after subcutaneous injection. This
experiment, too, confirms the assumption that rneso-1-PtC12
does not exert its estrogeniceffect by liberation of the diamine
ligand, which itself is equipotent under identical conditions
(prodrug mechanism). Numerous studies with rneso-2-PtC12
support the idea of a direct estrogen agonistic effect of this
type of complex.
The experimentswith meso-2-PtClz (and also with rneso-4PtC12) indicate that 0-methylation is not a useful means of
diminishing the estrogenic side effects, since this derivation
was accompaniedby loss of activity on the hormone-sensitive
MC. However, our second concept, the use of less hydrophobic ring substituents, was successful. It led to a compound
(rr~es0-4-PtC12)which was indeed free from estrogenic side
effects as well as strongly active on the hormone-sensitive
MC. We also obtained similar data by exchange of one of the
four C1-atoms in rneso-2-PtClz by H (estrogenic effect: 1/10
of rneso-2-PtCl2; effect on the MXT,ER(+): T/C= 22% at
15 pmolkg)'2). The experimental studies with rneS0-4-PtC12
462
can also explain the negative results which we achieved with
the 2,4,6-trimethoxy-substitutedcompound meso-5-PtClz.
Our studies on meso-4-PtC12lead to the following conclusions concerning its mode of action:
1. The low cytotoxic potency found in several ER(-) in vitroand in vivo-tumor models contradicts an unspecific cisplatin-like mechanism of action.
2. The unexpected high activity of meso-4-PtC12 on the
MXT,ER(+)-MC of the mouse suggests a more specific,
presumably ER-mediated cytotoxic effect.
To find out whether the specific activity of the cytotoxic complex meso4-PtClzonthe hormone-sensitiveMCiscausedby anER-mediatedtransport
into the tumor cell, further studies, especially on the interaction between
complex and ER, are necessary.
The investigations described in this paper indicate that the
introduction of various substituents in the 2,6-position of
[rneso-l,2-bis(4-hydroxyphenyl)ethylenediamine]dichloroplatinum(II) allows the development of derivatives with a
specific activity on the hormone-sensitive breast cancer and
with only marginal estrogenic side effects.
The search for further types of compounds endowed with
the discussed activity is ongoing.
The technical assistanceof L Schneider, D. G&$ried, S. Paulus, P. Pistor,
and A. Rob1 is gratefully acknowledged. Thanks are also due to the Deutsche
Forschungsgemeinschaft (SFB 234). the Matthias Lackas-Stiftung fiir
Krebsforschung, and the Fonds der Chemischen Industrie for financial
~Pport.
Gust, Karl, Faderl, and Schonenberger
MDA-MB 231 human breast cancer cell line
The MDA-MB 231 cell line'@was kindly provided by Dr.M. E. Lippman,
NCI, Bethesda, USA. Cells were grown in a humidified incubator in 5% COz
at 37 "C. McCoy 5a supplemented with gentamycin (40pg/ml), 10% NCS.
and NaHC03 (I 1g/5 1) was used as culture medium The cells were harvested
with trypin/EDTA, diluted with 5% NCS-containing medium, and syringed
gently toprevent clumping. Approximately 2 x lo4cells in 2 ml were plated
in duplicate in dwell dishes (Costar). 2 Days later the medium was changed
and the platinum complexes were added as freshly prepared 1000-fold
concentrated solutions in DMF. After incubation for 2 days, which covered
a triple duplication time, the cells were labeled with 1 pCi [3H]thymidine/well for 2h. Cells were washed with ice-cold PBS and harvested with
PBSEDTA buffer. After centrifugation, the cell pellet was resuspended in
1 ml of PBS and divided into two 0.5 ml aliquots. One part was counted in
a ZI Coulter Counter, the other was sonicated. Afta addition of 4 ml 10%
trichloroaceticacid, the acid-insoluble fraction was collected on a 0.45 pn
Filter (Metricel Gelman) and counted after addition of 10 ml scintillation
liquid.
Estrogen receptor binding assay
The applied method has been described by Hartmann et a1."). The relative
binding affinity (RBA) of the test compounds was determined by the displacement of 17P-f'Hlestradiol. At 4 OC the test compounds were shaken
with calf uterine cytosol and 17b[3H]estradiol for 16 h. To stop incubation.
dextran-coated charcoal was added, and after centrifugationthe radioactivity
of 200 pl supernatant aliquot was counted. On a semilog plot the % of bound
labeled steroid vs. concentration of the competitor was plotted. Six concentrations of each compound were chosen to get a linear graph. From this plot
these molar concentrations of unlabeled estradiol and of the competitors,
respectively. were determined which reduce the binding of the radioligand
by 50 %.
Assay for estrogenic activity
Experimental Part
General procedures
'H-NMR spectra of the platinum complexes: Bruker PFT-NMR spectrometer WM 250 at 250 MHz- Elemental analyses: Mkroanalytisches
Laboratorium University of Regensburg.
Estrogenic effects were determined by stimulation of the uterine growth
as describedbySchneideretal.'*).On threeconsecutive daysthecompounds,
dissolved in polyethylene glycol 4oo/HzO (0.9% NaCI), 1:1(0.1 d m u s e ) .
were daily administered S.C. to female, immature NMRI mice (age: 20 days
at test beginning: body weight: 10-12 g; 6 mice/group). The uteri were
excised 24 h after the last injection, fixed with Bouin's solution, dried and
weighed.
Assay for estrogenic activity by intrauterine adminktration in rats
Chemical methods
Juvenile, female SDrats, 30 days old. were randomized in groups of 11
animals. The test compounds were dissolved in olive oil and applied daily
for three days. The untreated control received olive oil only, the positive
control estrone.24h after the last applicationthe rats were killedand weighed.
The uteri were removed and fixed in Bouin's solution for 4h. After removal
of connective tissue the uteri were washed with ethanol dried for 24h at
[D,~ - 1 , 2 - B ~ 2 , 6 - d i c h l o r o - 4 - m e t h a r y p h e n y l ) e t h y l f f l e ~ ~ ] ~ c h l o r o105
- "C, and weighed.
phtinUm(II)(D,L-I-PtCI2)
'Ihe following method is representative of the syntheses of all dichlore
platinum(I1) complexes. The C,H,N values found for all compounds analyzed were within kO.408 of the calculated values.
KzPta (415 mg, 1 mmol) was dissolved in 20 ml of water and added to
a solution of D , L - ~(676 mg, 1 mmol) in 50% tefl-butanoVHfl. The mixture
was stirred in the dark at pH 5 and 40 "C. The solution was adjusted to pH 5
several times by 0.1 N NaOH. After 24h the yellow precipitate was sucked
off,washed with 0.1 N HCI and water, and dried over PzOs (75% pale yellow
powder).
Biological mtW
[3H]thymidine was obtained from New England Nuclear, Dreieich, Germany, newborn calf serum (NCS) from BiochromBerlin, McCoy 5a medium
and trypsin (O.OS%)/EDTA (0.02%) solution from Boehringer, Mannheim,
Germany, and scintillation liquid Quickszint 212 from Zinsser, Frankfurt,
Germany. The phosphate-buffered saline (PBS) contained NaCl(8 g). KCI
(0.2 g), Na2HP04 x 2HzO (lg), NaHzP04 x HzO (0.15g), and KHzP04
(0.2 g) in 1 1of HzO. To prepare PBSEDTA buffer 0.02% EDTA was added
to PBS.
Procedure for transvagidtranscervical application
The rats were anesthetized with ether and fixed in a supine pasition, head
down. The vagina was distended by deeply inserting a speculum. Using a
shadow-free lamp for illumination the vaginal orifice of the cervix was
centered. With gentle pressure, taking care not to injure the uterus, a blunt,
stainless steel tube with a curved tip was then successivelyintroduced in both
uterine horns according to the positioning of the curvature. The needle was
connected with flexible tubing to a 1 ml gas-tight Hamilton syringe. With a
multiple dispenser unit a total volume of 100 pl test solution per uterine horn
was delivered in 20 pl portions.
P388 kukemia of the CD2FI -mouse
This tumor was propagated in female DBAL? mice, To test the antitumor
activity female CDFi-mice (age: 6-8 weeks; body weight: 17-21 g) were
injected i.p. with 1 x lo6 leukemiacells in 0.1 ml PBS buffer (day 0). On day
1,5,and 9 the animals were randomly distributed into g r o u p of 6 and treated
Arch Pharm (Weinheim)328,457463(1995)
463
Estrogenic Side Effects
i.p. with solutions of the test compounds. Cisplatin was used as positive
control. The median survival time compared with the control (treated with
solvent only) shows the antitumor activity.
Home-&pendent, tmnsplantable MXT-M 3.2 mammary tumor of the
BDA-mouse
The applied method was described by Schneider et al. I*). In female
BDFi-mice (age: 8 weeks at test beginning: body weight about 20 g: Charles
River Wiga, Germany) the tumor was transplanted S.C.in pieces of about
2 mm3 (one piecelmouse). The mice were then randomly assigned to test
groups of 9 animals each. On the first day after transplantationthe treatment
with test compounds, dissolved in polyethyleneglycol 4OO/HzO (0.9%NaCI)
1:1, was started. Three times a week (Monday, Wednesday, Friday)
0.1 ml/mouse was injected S.C. for 6weeks. On day 43 the animals were killed
by cervical dislocation and weighed. The tumors were removed and after
washing in 0.9% NaCl solution dabbed dry and weighed. The median tumor
weight was then calculated. To assess the estrogenic or antiestrogenicside
effects the uteri were excised, fixed, dried,and weighed.
Home-independent, transplantable M n ,ER(-) mammary tumor of the
BD A-mouse
4 N. Knebel C.-D. Schiller, M. R. Schneider, H. Schonenberger. E. von
Angerer, Eur. J. Cancer Clin. Oncol. 1989.25.293-299.
5 M. R. Schneider, C.-D. Schiller, A. H u m Th. Sprup, H. Schonenberger, W. Amselgruber, F. Sinowatz, Prostate 1989,15, 135-148.
6 A.Otto. M.Faderl. H. Schonenberger,Cancer Rex. 1991.51.3217-3223.
7 R. Gust, H. Schonenberger, U. Klement, K.-J. Range, Arch. Phunn
(Weinheim)1993,326,967-976,
8 R. Gust, H. Schonenberger,Arch. P h n n (Weinheim) 1993,326,405413.
9 R. Gust, K.Niebler, H. SchonenbergerJ. Med.Chem, accepted.
10 R. Gust, H. SchonenbergerArch. Phann (Weinheim) 1994,327,763769.
11 V. T. De Vita Jr, S. Helman, S. A. Rosenberg, Cancer, Principles rmd
Pmctice of Oncology, 3rd edition, J. B. Lippincott Company, Philadelphia, 1989.
12 R. Gust, T. Burgemeister, A. Mannschreck, H. Schonenberger,J. Med
Chem 1990,33,2535-2544.
The MXT,ER(-) tumor was maintained by routine passage in ovariectomized BDFi-mice. The tumor was transplanted in pieces of about 2 m3 13 S.Yano. T. Tukada, S. Yoshikawa. Inorg, Chem 1978,17,252&2526.
(one piecehouse) in intact female BDF1-mice (age: 8 weeks at test begin14 K. Chwalisz, A. Hughes. Ch. Zou,Acfu Endocriml. 1983,103,131-137.
ning: body weight: 20 g: Charles River Wiga, Germany: 9 animalsfgroup)
and then the animals were treated with the solutions of the compounds as
15 R. Gust, unpublished results.
described in the preceding paragraph. but only for 14 days. On day 15 the
tumor area (the produd of two perpendicular diameters: one across the
16 M. E. Lippmann. M. E. Monaco, C. Bolan, Cancer Res. 1977. 37,
longest side) was measured.
1901-1907.
References
1 P.J. Loehrer, H. Einhorn. Ann. lnt. Med 1984,100,704-713.
17 R. Hartmann. G. Kranzfelder, E. von Angerer, H. Schonenberger,J. Med
Chem 1980,23,841-848.
2 H. Brunner, G. Sped, Momtsh. Chem 1993,124.83-102.
18 M. Schneider. H. Ball, H. Schonenberger, J. Med. Chem 1985,28,
1880-1885.
3 J. Karl, R. Gust, Th. SpruB, M. Schneider, H. Schonenberger,J. Eagel
K.-H.Wrobel, F. Lux, S. Trebert-Haeberlin,J. Med Chem 1988,31,
71-83.
Arch Pham (Weinheim)32845743(1995)
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estrogen, ethylenediamine, variation, inhibition, reduction, substituents, ring, hydroxyphenyl, bis26, effect, drug, side, mammary, dichloro, tumors, dichloroplatinumii
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