- Email: [email protected]
Guide-lines in the design of new antiestrogens and cytotoxic-linked estrogens for the treatment of breast cancer
J. steroid Biochem. Vol. 19. No.1. pp. 75-85. 1983
0022-4731/83 $3.00+0.00 Copyright © 1983 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
GUIDE-LINES IN THE DESIGN OF NEW ANTIESTROGENS AND CYTOTOXIC-LINKED ESTROGENS FOR THE TREATMENT OF BREAST CANCER G. LECLERCQ, N. DEVLEESCHOUWER and J. C. HEUSON Laboratoire de Cancerologie Mammaire, Service de Medecine, Institut J. Bordet. !Ooo Brussels, Belgium SUMMARY
Antiestrogens (Tamoxifen) are used for the treatment of breast cancer. However these compounds are also weak estrogens that may stimulate tumor growth. Cytotoxic-linked estrogens (Estradiol Mustard, Estracyt) are devoid of major therapeutic activity. This led to search for new antiestrogens devoid of estrogenicity and for active cytotoxic-estrogens. Hydroxylation of C-4 of triphenylethylene antiestrogens (tamoxifen, CI628 and U 23,469) largely increases their binding affinity for the estrogen receptor (ER). Hydroxylation also increases the in vitro antitumor activity of the drugs as shown by their higher ability to inhibit the growth of the ER-positive cell line MCF-7. Triphenylethylene antiestrogens contain an aminoethoxy side chain which appears essential for their physiological activity. Removal of the chain of tamoxifen suppresses its antiestrogenicity and antitumor activity. The grafting of side chains on a weak estrogen of the gem-diphenylethylene category produces "symmetrical" antiestrogens devoid of estrogenic activity. This observation raises the question of the role played by the third phenyl ring of the triphenylethylenes since the trans-isomers of the latter display antiestrogenicity and the cis-isomers estrogenicity. Comparison of the binding affinity for ER and antitumor activity of di- and triphenylethylene antiestrogens suggests that this third phenyl ring increases the interaction with ER of the 4-phenolic group of the drugs and/or their aminoethoxy side chain. An analogue of this chain is without any biological activity suggesting that the di-(tri)phenylalkene ntructure is required for promoting the interaction of the chain with ER. New chemical structures yielding antiestrogens with antitumor activity are also reviewed. New cytotoxic estrogens designed for producing lethal damage of DNA show a low binding affinity for ER. Moreover, there is no evidence suggesting specific antitumor activity. Such activity may be more easily obtained with estrogens bearing reagents for proteins rather than DNA. The biological properties of a 2-mesylate derivative of estrone irreversibly interacting with ER supports the concept. On MCF-7 cells, the drug displays a strong antitumor activity which can only be suppressed by high, equimolar, concentrations of estradiol. It is devoid of cytotoxic activity on the ER-negative cell line Evsa-T suggesting that ER is involved in its action.
INTRODUCTION
This stresses the need for new antiestrogens devoid of estrogenic activity. On the other hand, estrogen-linked cytotoxic agents such as Estradiol Mustard and Estracyt have been produced (Fig. 1). The rationale for using such compounds relied on the principle that they may bind to ER and thereby concentrate their cytotoxic agent (nitrogen mustard) in the mammary tumor cells. Unfortunately these drugs are devoid of major therapeutic activity in the treatment of advanced breast cancer [6--9]. The absence of a significant binding affinity of these compounds for ER as well as their metabolism with production offree estradiol might be responsible for their lack of antitumor activity [10, 11]. These disappointing results led to a reappraisal of the design of cytotoxic-estrogens [12]. It is our purpose to review the more recent investigations devoted to the production of new antiestrogens and cytotoxic-estrogens and define guide-lines for the design of new drugs of potential therapeutic activity.
Specificity of action is a problem of major importance in the development of new antitumor drugs. In breast cancer, the presence of estrogen receptor (ER) could well form the basis for an advance in this respect. One may legitimately hope that receptor-mediated chemotherapy could enhance therapeutic efficacy while decreasing the toxic side effects. In fact this hope has already found support in the clinical use of non steroidal antiestrogens [IJ such as nafoxidine, clomiphene or tamoxifen (Fig. 1). In advanced breast cancer, these compounds give a remission rate close to 30% which is similar to those reported with other endocrine treatments [2]. The lack of major side effect of tamoxifen prompted its use as the standard antiestrogen for the treatment of the disease [3]. Moreover, in primary breast cancer, it has been shown to act as an active additive to adjuvant chemotherapy [4]. However, this drug as all other antiestrogens [IJ displays weak estrogenic activity which may stimulate mammary tumor growth [5]. s.•.
19-I(A)-G
75
76
G.
LECLERCQ
et al.
ANTIESTROGENS
CH30 ~
I~ Natoxidint
Clomiftnt
(U-II,IOOA)
Tamoxiftn ( ICI-46,474)
CYTOTOXIC - ESTROGENS
Estradiol Mustard
Estracyt
Fig. 1. Chemical formulae of antiestrogens and cytotoxic-estrogens tested in clinical trials. On tamoxifen the star indicates the position of the hydroxyl function in the hydroxylated form of the drug (position 4).
MODELS FOR EVALUATING THE POTENTIAL THERAPEUTIC ACTIVITY OF THE DRUGS
Several animal mammary tumor models are useful for evaluating the in vivo antitumor activity of antiestrogens and cytotoxic-estrogens (review; [13]). Among them, the DMBA-induced rat mammary carcinoma has been the most widely used [14]. In our laboratory, we recently introduced a new hormonesensitive transplantable model, the MXT-mouse mammary carcinoma developed by Watson et al.[15], which seems more appropriate dor drug screening. This ER-positive tumor shows a marked sensivity to ovarian and pituitary hormones: its growth is slowed down by castration [15,16], CB-154 [17] and tamoxifen [16]. In vivo screening always requires large amounts of drugs which could not always be easily produced. This led to the development of in vitro tests on ERpositive breast cancer cell lines such as the MCF-7 [18]. In our laboratory, this line as well as the ER-negative line Evsa-T (control) is routinely used for the selection of new compounds of potential therapeutic activity. The following experimental protocol has been designed. MCF-7 and Evsa-T cells are grown in Falcon plastic flasks (75 cm 2 ) containing Earle's minimal essential medium (MEM) supplemented with 0.6 mg L-glutamine/ml, 40 J-lg gentamycin/ml, 100 U penicillin/ml, 100 J-lg streptomycin/ml and 10% fetal calf serum. At confluency, cells are removed by trypsinisation (trypsin 0.05%, EDTA 0.025%) and suspended (50-200 x 103 cells/ml) in the growth medium supplemented with charcoal stripped fetal calf serum (0.5%
charcoal, 0.005% dextran in 1.5 ml medium/ml serum; overnight incubation at 4°C). Cells are then plated in 35 mm Petri dishes containing this medium and cultured at 37°C in a humidified 95% air 5% CO 2 atmosphere. After 24h, drugs (solvent:ethanol at the final concentration of 0.1 %) are added to the culture dishes. Forty-eight hours later the medium is replaced by fresh medium containing the drugs. The cultures are then pursued for an additional n-h period before harvest. At this time, the cells are washed twice with 2 ml of Earle's base before being suspended in 1.5 rnl trypsin-EDTA. Total DNA of collected cells are precipated in 0.5 N perchloric acid and evaluated by the diphenylamine method. Experiments are always performed in quadruplicate.
ANTIESTROGENS
All strong antiestrogens synthetised within the last three decades are of the triphenylethylene category [1] (Fig. 1). Recently it has been shown that other chemical structures produce antiestrogenic and mammary antitumor activities. Thus, Bouton and Raynaud demonstrated that steroidal estrogens fastly dissociating from ER may have antiestrogenic properties [19]. Such a compound (RU 16,117; Fig. 2) was found to produce regression of DMBA-induced rat mammary carcinomas '[20]. On the other hand, Schonenberger and his group [21-27] showed that chemical modifications of the synthetic estrogens diethylstilbestrol, hexestrol and indenestrolled to drugs with antiestrogenic activity. Roughly, displacement of the phenolic groups from the para to the meta position pro-
77
Antiestrogens and cytotoxic-linked estrogens OAvNJ
OH H30"
~
.·eaCH ~. BOUTON AND
CI 628
J. P. RA YNAUD
(ROUSSEL (0)
Incr~as~
I~
HO
~
~
20
~
RU-16\l7
10
HO~ {DOH OJ~·
c +1
III
-OH
- OCH 3
j II~
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~[n-OH
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HO
I
_10""
~ ~ (II vs135)
OH OH O/"L./
z
c H.
ScHONUaUGER
(REGENS_G,6E_>
U 23,469
Cll ~
CH3 ~ CH3
Of
J.T.
PENTO
Control
.8 _7 _6
e\
Fig. 2. Chemicals formulae of new antiestrogens producing mammary tumor growth.
duces a strong decrease in estrogenicity with a gain in antiestrogenicity [21-24]. Similarly, structural modifications of the diphenylethane (ethene) skeleton of these estrogens produce strong antiestrogenicity [25-27]. Such an observation was also reported by Pento et al.[28, 29]. Notably, all these modifications led to drugs able to inhibit the growth of the DMBAinduced rat mammary carcinomas (examples III Fig. 2). Recent studies have shown that hydroxylation of one phenyl ring of tamoxifen (position 4; Fig. 1) led to a drug with very high binding affinity for ER (Relative Binding Affinity; RBA ~ 1(0) and strong antiestrogenicity [30-33]. These properties largely increased the in vitro antitumor activity of the drug as shown by its higher cytotoxicity on the MCF-7 cells [34, 35]. This effect of hydroxylation in position 4 might be common to all antiestrogens of the triphenylethylene category since we have found that the replacement of the methoxy group by a hydroxyl group of the two antiestrogens CI 628 and U 23,469 [36]* also produced a significant increase of growth inhibition (Fig. 3). Noteably, the difference was more marked for U 23,469 which is consistent with the increase in binding affinity for ER produced by the hydroxylation of these drugs (CI 628 = - IO-fold; U 23,469 = - 300-fold) [36].
* Compounds provided by Dr 1. A. Katzenellenbogen, Urbana, Illinois.
.8 .7 .6
log Molarity
(OKL_ (ITY. 010
Fig. 3. Growth inhibition of MCF-7 cells by C1628, U 23,469 and their hydroxylated derivatives. Variance analysis [64] shows that CI628 and derivative are active (P < 0.(01) at all concentrations (Newman-Keuls test); the derivative is more effective at 10- 7 M (P < 0.(01). U 23,469 is not active while its derivative is active at 10- 7 and 10- 6 M (P < 0.(01). Symbols: 0 control without any drug; ~ experimental drugs.
I Alone I
Cl VI
+1 «l
z
Cl Ol
~:J 0 JI I
Trans
1+5xIO-7M NAFI
20
::1..
10
[1. JlI~
Controls
.7.6
log Molarity
Fig. 4. Estrogenicity of a tamoxifen derivative devoid of aminoethoxy side chain. The compound is in the trans configuration [65]. Variance analysis shows that the compound does not significantly modulate growth of MCF-7 cells. At 10- 6 M, it significantly suppresses (interaction P < 0.01) the inhibition (P < 0.01) of 5 x 10- 7 M nafoxidine. The upper pannel refers to the drug alone; the lower to the drug with nafoxidine. Symbols: 0 and ~ as in Fig. 3; • nafoxidine alone; I§I nafoxidine with experimental drug.
78
G.
LECLERCQ
et al.
I Alonll I 30
II I ~ .1
20
10
1+5xI0-7M NAFJ 30 0 I/)
+1 20 4 Z 0
10
01 ~
1+10_8M 30
Ezi
~~I
20
10
Controls
_8 _7 _6 log Molarity
Fig. 5. Antiestrogenicity of a gem-diphenylethylene bearing an aminoethoxy side chain. Variance analysis shows that the diphenolic compound does not significantly modulate growth. At 10- 6 M it significantly suppresses (interaction P < 0.001) the inhibition of 5 x 10- 7 M nafoxidine. The two derivatives with an aminoethoxy side chain (A and B) significantly reduce growth (P < 0.01) at 10- 6 M (NewmanKeuls test). At 10- 8 and 10- 7 M they are devoid of activity on the inhibition of 5 x 10- 7 M nafoxidine. Estradiol at 10- 8 M significantly suppresses (interaction P < 0.01) their inhibition. The upper pannel refers to the drugs alone; the intermediate to the drugs with nafoxidine; the lower to the drugs with estradiol. Symbols: 0, ~, • and S as in Fig. 4; ~ estradiol alone; ~ experimental drug with estradiol.
In vivo hydroxytamoxifen was found to be a less effective inhibitor than tamoxifen in the DMBAinduced rat mammary tumor model [37]. The reason would be the shorter biological half-life of this polar drug. The hydroxyl function being of major importance in the in vitro antitumor activity of the triphenylethylene antiestrogens, search for its in vivo stabilization seems of prominent interest. Antiestrogens of the triphenylethylene category contain an aminoethoxy side chain which seems essential for their physiological activity: without such * Compound provided by Dr A. B. Foster, Chester Beatty Institute, Sutton, Surrey, U.K. t Compound provided by Dr I. Niculescu-Duvaz, Oncologic Institute Bucharest. Romania.
a chain the compounds lose their antiestrogenic properties and become potent estrogens [I, 38]. Substitution of the side chain of tamoxifen by a methoxy group* suppresses its in vitro ability to inhibit the growth of MCF-7 cells (Fig. 4). The resulting molecule behaves as a weak estrogen in view of its ability to partly suppress at high concentration the inhibition of nafoxidine (the minimal effective concentration is 10- 6 M while that of estradiol is about 100 times lower; Fig. 6, Ref [18]). In this context, it should be stressed that the grafting of an aminoethoxy side chain on a weak estrogen of the gem-diphenylethylene categoryt [39] produces compounds able to inhibit the growth of the MCF-7 cells (Fig. 5). This inhibitory effect is similar to that found with antiestrogens of the triphenylethylene category since it could be sup-
79
Antieslrogens and cytotoxic-linked estrogens
Cis
Trans
20 10
o VI
+'
o
Z
o 0'
"-
\+10- 8 ""
£21
7 1+5XIO· M NAF
Fig. 7. Role of the third phenyl ring of the triphenyiethylenes in their antiestrogenic activity. The arrows indicate two possible influences: (1) an increase of interaction of the phenol with the receptor; (2) an increase of interaction of the aminoethoxy side chain with the receptor.
I
~:] [J ]~ I ~: ~ 1 Control~
.7
.b
Control~
log Molarity
.7.G log Molarity
Fig. 6. Antiestrogenicity of lrlllls-tamoxifen (ICI-46.474) and estrogenicity of cis-lamoxifen (lCl-47.699). Variance analysis shows that lnms-tamamoxifen significantly reduces growth of MCF-7 cells (P < 0.01) at 10- 7 and 10- 6 M (Newman-Keuls test); estradiol at 10- 8 M significantly suppresses (interaction P < 0.01) its inhibition. cisTamoxifen does not significantly modulate growth; at JO- 6 M it suppresses (interaction P = 0.1) the inhibition (P < 0.01) of 5 x 10- 7 M nafoxidine. The upper pannel refers to the drugs alone; the lower to the drugs with estradiol or nafoxidine. Symbols as in Fig. 5.
pressed by estradiol. On the other hand, these compounds do not suppress the inhibition of nafoxidine indicating that they are devoid of estrogenicity. That the grafting of an aminoethoxy side chain on a "symmetrical" diphenylethylene produces antiestrogenicity is of interest in view of the fact that triphenylethylene antiestrogens are always in the trans configuration [I, 40--42]. When they are in the cis configuration, they usually display estrogenic activity [1,40-42]. This geometrical isomerism also influences the MCF-7 cell growth: the trailS isomer§
* Compounds provided by Dr J. S. Patterson. lCI. Macclesfield, Cheshire, U.K.
of tamoxifen shares the classical antiestrogenic activity (inhibitory action suppressed by estradiol) while the cis isomer* behaves as a weak estrogen (no inhibitory action; partial suppression of nafoxidine inhibition) (fig. 6). The analogy in biological activity between the gemdiphenylethylene antiestrogens and the classical triphenylethylene antiestrogens raises the question of the role played by the third phenyl of the latter (Fig. 7). Assuming that the phenol of the gem-diphenylethy(enes corresponds to the phenol of the classical antiestrogens, hydroxytamoxifen, CI 628 M and U 23,469 M (position 4), it appears that the third phenyl of the latter largely increases the binding affinity for ER as well as the antitumor activity (Table 1). This observation highly suggests that this third phenyl in the rrans configuration increases the interaction of the 4-phenolic group with the receptor [13, 43] and thereby enhances antiestrogenecity. This third phenyl may also favor the interaction of the side chain with a specific site on ER which would essentially mediate antiestrogenicity [44]. In this regard, it should be stressed that such a specific interaction has never been demonstrated. If it exists, it seems that it would involve a hydrogen bound interaction in view of the fact that residues able to such binding only produce antiestrogenicity (-N-{Alkh; -CHOH-CH 2 0H; - N->O; Ref.[ I.35.36,45]).
Table I. Hydroxylated antiestrogens. Relative binding affinity and inhibition ability of MCF-7 cells
Compound
RBA (E 2 = 1(0)
Diphenylethylene A Diphenylethylene B U 23,469 M Cl628 M OH-tamoxifen
0.1 0.2
30 ;:;: 100 ;:;:100
Concentration producing 50'~" growth inhibition
10-1> M 10- 6 M 10- 7 M -10- H M _1O- M M
Reference
This This [36] [36]
work work This work This work
[30--35]
G.
80
LECLERCQ
et at.
,....,
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.c:: u os
0
:r:
E
~
Ill::
os
+1
~
I+OH.Tam
~ 10
5
Controls
_6 _5 log Molarity
Fig. 8. Absence of influence of an analogue of the aminoethoxy side chain (1-[2(p-bromophenoxy)ethyl]pyrrolidine) on the growth of MCF-7 cells. The upper pannel refers to the drug alone; the lower to the drug with 10 - 8 and 10 -, M hydroxytamoxifen. Symbols 0, lSI as in Fig. 3; • S experimental drug with hydroxytamoxifen; hydroxytamoxifen.
The hypothesis of a specific site on ER for the side chain of the antiestrogens was investigated by using a free analogue (1-[2(p-bromophenoxy)ethyl]pyrrolidine; Aldrich Co.). In the competitive test of the binding of eH]-estradiol to ER, this compound does not share any competitive activity indicating a total absence of affinity for the estradiol binding site. On MCF-7 growth, it does not display any antitumor activity, nor does it suppress the inhibition by hydroxytamoxifen even at a lOoo-fold higher concentration (Fig. 8). These results indicate that, if a specific interaction does exist between the side chain and ER, the di-(tri-)phenylalkene structure of the antiestrogens is required for promoting the interaction.
'-
o
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'':.:.c
CQ.
'en = 0;:: Q.,
I:
=~ ~ 0 os ...
Q.,t;
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DJ
DJ
os
DJ
CYTOTOXIC ESTROGENS
Estradiol Mustard and Estracyt (Fig. I) are devoid of significant binding affinity for ER [lO, 11]. In both compounds the 3-phenolic group of estradiol is substituted by the cytotoxic agent. In view of the major importance of this group in the binding of the estrogen to ER [13,43], it has been proposed that this substitution was responsible for the lack of affinity of these drugs for the receptor. Drugs with free phenol were therefore synthetized [13, 46-52]. Table 2 provides the formulae, binding affinities and biological properties of some alkylating derivatives recently produced (three of these drugs being in a methoxylated form, their binding was not assessed). Although
0. :l
...o 1>0 1>0
.S ~
>. ~
< "
'f i
/
Y
o :r:,..
:r:Q. u,..
u
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0
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I
NO
-
-0
'"
CHzCHzCl
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CHzCHzCI
'"
/
CHzCHzCI
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'"
/
CHzCHzCI
CHzCHzCl
O-CHzCHz-N/
---eH zCH zCH z-N
-
--0
J
-CHzNHC-N---eHz-CHzCl
0
II
-NHC-N---eH z-CH zC1
I
NO
J
---eHzNH---e-N-CHz-CHzCl
I
NO
6dehydro E z (3MeO, J7P OAc)
Ez (3MeO, 17P OAc)
Ez (3MeO)
Ez
Ez
Ez
6P
7P
171X
171X
17P
17tX
-
-
-
0.2
0.4
2
Non estrogenic Non antiestrogenic
Non estrogenic Non antiestrogenic
Weakly estrogenic
Growth inhibition of rat mammary tumor 13762 (specificity?)
Growth inhibition of rat mammary tumor (specificity'!)
ICI 140, 175 [52]
ICI 140,496 [52]
Iel 141,857 [52]
Lam [49, 50]
Chavis [51]
Lam [49, 50]
00
'"
(JQ
0 n> ::0
'" ::t
n> 0n>
"..
5'
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82
G.
LECLERCQ
almost all these compounds bind significantly to ER, the substitution always produced a marked reduction of binding affinity (the methoxylated drugs did not display potent estrogenic or antiestrogenic activities suggesting a weak affinity). This observation is consistent with the known fact that substitutions usually reduce the interaction of the steroid with the receptor [13, 53]. Substitutions of non steroid estrogens may be more fruitful. However, in such cases, important spatial modifications of the estrogen moeity would normally occur most likely resulting in a reduction of binding affinity [54, 55]. In the design of cytotoxic estrogens a fundamental question is the concentration of cytotoxic agent required for lethal damage to the DNA. It is implicitly assumed that the tumor ER levels are sufficient to concentrate the required amounts of cytotoxicestrogens. However, this assumption has never been demonstrated and deserves more substantial analysis. The chemical reactivity of the cytotoxic agent has not been sufficiently taken into account either. Strong alkylating derivatives may logically react with all the tissues they reach, especially the liver, which contain ER and enzymes of the estrogen metabolism. Such undesirable alkylation might compromise the whole concept of specific tumoral killing [52]. On the other hand, systematic studies of alkylating drugs have shown that significant antitumor activity may only occur with drugs bearing at least two alkylating residues [56]. The presence of only one aziridine .in the aziridine derivative of estradiol described in Table 2 (ORG 5895) may explain its total lack of antitumor activity on the MXT mouse mammary tumor and the P388 leukemia [57]. Since multiple substitutions brought to an estrogen usually produce a marked reduction of its binding affinity to ER, its seems likely that monoalkylating residues (i.e. aziridines and epoxydes) should not be selected in future syntheses. Finally, it is unknown whether the antitumor properties of the cytotoxic agents are maintained when the latter are linked to an estrogen interacting with ER. Drugs able to dissociate from the receptor in the cell nucleus might be desirable. However, knowledge on the stability of the nuclear ER-estrogen complex is still too scarce to propose suitable chemical structures. These considerations suggest that specific therapeutic activity may be more easily obtained with cytotoxic agents interacting with ER rather than with DNA. Efforts have been made in this direction [58-62J yielding compounds that irreversibly [63J interact with the receptor. These compounds may be strong estrogens or strong antiestrogens as shown by the behavior of a IIp-chloromethyl derivative of estradiol [47, 59J and a 2-mesylate derivative of estrone (62). Thus, in MCF-7 cells the chloromethyl
* Compound provided by Dr F. J. Zeelen, Organon, Oss, The Netherlands. t Compound provided by Dr R. L. Morgan, Louisiana State University, New Orleans, Louisiana.
et al.
ORG 4333
I
Alone
20
HO~
10
o
+1
1+ 10_6M
~
o
01
NAF
I
20
:I...
10
_II _10 _9 _6 log Molarity
Control~
Fig. 9. Estrogenicity of IIp-chloromethylestradiol (ORG 4333) on the growth of the MCF-7 cells. Variance analysis shows thai the compound does not significantly modulate growth. At 10- 9 and 10- 8 M, it significantly suppressess (interaction P < 0.01) the inhibition (P < 0.01) of 10- 6 M nafoxidine. The upper panne! refers to the drug alone; the lower to the drug with nafoxidine. Symbols as in Fig. 4. derivative (ORG 4333, Fig. 9)* suppresses the growth inhibition of nafoxidine in the same range of concentrations as estradiol. In contrast, the mesylate derivativet (Fig. 10) inhibits growth at similar concentration 0""
Z
S02 CH 3
0 II
°bSD
HO
~I ~
15
10
o oil
+1 10
5
o
Cl
5
:l..
Control
_8 _7 _6 log Molarity
Control
_8 _7 _6 log Molarity
Fig. 10. Influence of a 2-mesylate derivative of estrone on the growth of MCF-7 and Evsa-T cells. Variance analysis shows thai the compound significantly reduces growth of MCF-7 cells (P < 0.01) at all concentrations (NewmanKeuls test). The compound is not active on Evsa-T cells. Symbols as in Fig. 3.
Antiestrogens and cytotoxic-linked estrogens
IE2 alone I 15
10
5 o Vl
J
+1 ~ Ol
1,....~-2-+-IO-_-::6-M-M-e-s-E-d 15
:J...
10
5
~~ _8 _7
Controls
_6
log Molarity
Fig. II. Suppression by estradiol of the growth inhibition of a 2-mesylate derivate of estrone on the growth of MCF-7 cells. Estradiol at 10- 6 M significantly suppresses (interaction P < 0.0 I) the inhibition of 10 - 6 M of the drug. The upper panne! refers to the estradiol alone; the lower to the experimental drug with estradiol. Symbols as in Fig. 5.
to the strong hydroxylated antiestrogens hydroxytamoxifen, and CI 628 M. That no growth inhibition occurs in Evsa-T cells (ER-) in the presence of Ihe drug indicates that it has no major general cytotoxicity. Remarkably, the inhibition in the MCF-7 cells is suppressed by estradiol but only at high, equimolecular, concentrations (Fig. 11). This contrasts with the behavior of conventional antiestrogens such as tamoxifen which are counteracted by low levels of estradiol [18]. Finally, hydroxylated and methoxylated analogues of estrone (2-0H, 2-0CH 3 ) do not inhibit growth suggesting that the cytotoxicity of the mesylate derivative was associated with its alkylating residue. These remarkable properties highly suggest that estrogens interacting irreversibly with ER may provide a new class of "suicide inhibitors" of prominent therapeutic interest. CONCLUSIONS
The studies reported here reveal a considerable effort in the production of new antiestrogens and cytotoxic estrogens for the treatment of breast cancer. Guide-lines for the design of new drugs emerge from these investigations. In the case of triphenylethylene antiestrogens a close parallelism was found between the known endocrinological properties of the drugs and their antitumoral activity. This observation highly suggests that a large part of the literature on these compounds is relevant in the design of new antineoplastic drugs.
83
In this regard. it seems that the mere triphenylethylene structure should be abandoned in view of its metabolic instability which might yield both trans and cis metabolites. A more rigid structure that mimics the A and B rings of estradiol (cf. nafoxidine) would be investigated. On the other hand it seems that metabolic studies [32, 35, 42J might be extremely helpful in the identification of active metabolites which may be produced at a later stagr (or production of stable analogues). In view of the prominent function of the aminoethoxy side chain of the antiestrogens, effort should be made to increase interaction of former with ER by introducing it into reactive groups [61]. Finally, the production of several gem-diphenylethylene derivatives bearing an aminoethoxy side chain could be proposed. The data on the potential antitumor activity of cytotoxic-estrogens are still too scarce to ascertain an important therapeutic role for such drugs. However, evidence is given here suggesting that suicide inhibitors that irreversibly interact with ER may be useful. Effort should therefore be made to identify on estrogens and antiestrogens suitable positions for grafting reagents for proteins yielding such very strong antiestrogens. Acknowledgements-We wish to thank Drs A. B. Foster, J. A. Katzenellenbogen. L. R. Morgan, I. Niculescu-Duvaz, J. S. Patterson and F. J. Zeelen for the gift of drugs. This work was supported by a grant from lCl-Belgium (Dr M. A. Lassance) and by a grant from the Fonds Cancerologique de la Caisse d'Epargne et de Retraite (Belgium).
REFERENCES
1. Sutherland R. L. and Jordan Y. C: Non-steroidal antiestrogens. Molecular Pharmacology and Antitumor Acticity. Academic Press, Sydney (1981). 2. Legha S. S. and Carter S. K.: Antiestrogens in the treatment of breast cancer. Cancer Treat. Rev. 3 (1976) 205--216. 3. Mouridsen H., Palshof T., Patterson J. and Battersby L.: Tamoxifen in advanced breast cancer. Cancer Treat. Rell. 5 (1978) 131-141. 4. Fisher B.. Redmond C, Brown A., Wolmark N., Wittliff 1. Fisher E. R.. Plotkin D., Bowman D., Sachs S., Wolter.l.. Frelick R., Desser R., Licalzi N., Geggie P., Campbell T.. Elias G., Prager D., Koontz P., Yolk H., Dimilrov N.. Gardner B.• Lerner H., Shibata H. and other NSABP Investigators: Treatment of primary breast cancer with chemotherapy and tamoxifen. New Engl. J. Merl. 305 (1981) 1-6. 5. Legault-Poisson S., Jolivet J.. Poisson R.. Beretta-Piccoli M. and Band P. R.: Tamoxifen-induced tumor stimulation and withdrawal response. Cancer Treat. Rep. 63 (1979) 1839-1841. 6. Schapira D.. Hall T. Benett 1. M.. Lavin P., Colsky J., Perlia C. Brodovsky H. and SChnider B.: A phase II study of oestradiol mustard (NSC-112259) by Eastern Cooperative Oncology Group. Cancer Clin. Trials 1 (1978) 5-8. 7. EORTC Breast Cancer Cooperative Group: Essai clinique du phenol (2-chloroethyl) carbamate d'oestradiol dans Ie cancer mammaire en phase avancee. Eur. J. Cancer 5 (1969) 1-4. 8. Alexander N. c.. Hannock A. K., Masood M. B., Peet
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c.:
1,l,2,2-tetraalkyl-l,2-diphenylethanes. J. med. Chem.23 (1980) 841-848. 26. Hartmann R. W., Buchborn H., Kranzfelder G., Schonenberger H. and Bogden A.: Potential antiestrogens. Synthesis and evaluation of mammary tumor inhibiting activity of 1,2-dialkyl-I,2-bis(3' hydroxyphenyl) ethanes. J. med Chem. 24 (1981) 1192-1197. 27. von Angerer E., Egginger G., Krantzfelder G., Bernhauer H. and Schonenberger H.: N,N'-Dialkyl-l,2bis(hydroxyphenyl)ethylenediamines and N,N' -dialkyl-4,5-bis(4-hydroxyphenyl)imidazolidines: syntheses and evaluation of their mammary tumor inhibiting activity. J. med. Chem. 25 (1982) 832-837. 28. Pento 1. T., Magarian R. A., Wright R. 1., King M. M. and Benjamin E. 1.: Non steroidal estrogens and antiestrogens: biological activity of cyclopropyl analogs of stilbene and stilbenediol. J. Pharm. Sci. 70 (1981) 399-403. 29. Pento 1. T., Magarian R. A. and King M. M.: A comparison of the efficacy for antitumor activity of the non-steroidal antiestrogens analog II and tamoxifen in 7,12-dimethylbenz[lX]anthracene-induced rat mammary tumors. Cancer Lett. 15 (1982) 261-269. 30. Jordan V. c., Collins M. M., Rowsby L. and Prestwich G.: A new monohydroxylated metabolite of tamoxifen with potent antiestrogenic activity. J. Endocr. 75 (1977) 305-316. 31. Nicholson R. I., Synes 1. S., Daniel C. P. and Griffiths K.: The binding of tamoxifen to oestrogen receptor proteins under equilibrium and non equilibrium conditions. Eur. J. Cancer 15 (1979) 317-329. 32. Borgna J. L. and Rochefort H.: High affinity binding to the estrogen receptor of [3H] 4-hydroxytamoxifen, an active antiestrogen metabolite. Mol. cell. Endocr. 20 (1980) 71-85. 33. Wakeling A. E. and Slater S. R.: Estrogen receptor binding and biological activity of tamoxifen and its metabolites. Cancer Treat. Rep. 64 (1980) 741-744. 34. Coezy E., Borgna 1. L. and Rochefort H.: Tamoxifen and metabolites in MCF-7 cells: correlation between binding to estrogen receptors and inhibition of cell growth. Cancer Res. 42 (1982) 317-323. 35. Bates D. 1., Foster A. B., Griggs L. 1., Jarman M., Leclercq G. and Devleeschouwer N.: Metabolism of tamoxifen by isolated rat hepatocytes: antiestrogenic activity of tamoxifen N-oxide. Biochem. Pharmac. 31 (1982) 2823-2827. 36. Hayes J. R., Rorke E. A., Robertson D. W., Katzenellenbogen B. S. and Katzenellenbogen 1. A.: Biological potency and uterine estrogen receptor interaction of the metabolites of the antiestrogens CI 628 and U 23,469. Endocrinology 108 (1981) 164-172. 37. Jordan V. C. and Allen K. E.: Evaluation the antitumor activity of the non-steroidal antiestrogen monohydroxytamoxifen in the DMBA-induced rat mammary carinorna model. Eur. J. Cancer 16 (1980) 239-253. 38. Lednicer D., Lyster S. C. and Duncan G. W.: Mammalian anti-fertility agents VI Basic 3,4-dihydronaphtalenes and 1,2,3,4-tetrahydro-I-naphthols. J. med. Chem. 10 (1967) 78-84. 39. Devleeschouwer N., Leclercq G. and Heuson 1. Antagonism of cyclofenil and nafoxidine in the growth of the human breast cancer cell line MCF-7. IRCS Med. Sci. 8 (1980) 849. 40. Harper M. 1. K. and Walpole A. L. Contrasting endocrine activity of cis and trans isomers in a serie of substituted triphenylethylenes. Nature 212 (1966) 87. 41. Jordan V. c., Hadelmann B. and Allen K. E. Geometric isomers of substituted triphenylethylenes and antiestrogen action. Endocrinology 108 (1981) 1353-1361. 42. Robertson D. W., Katzenellenbogen 1. A., Long D. 1.,
of
c.:
Antiestrogens and cytotoxic-linked estrogens Rorke E. A. and Katzenellenbogen B. S.: Tamoxifen antiestrogens. A comparison of the activity, pharmacokinetics and metabolic activation of the cis and trans isomers of tamoxifen. J. steroid Biochem. 16 (1982)
1-13. 43. Duax W. L., Griffin J. F., Rohrer D. C and Weeks C M.: Steroid agonists and antagonists: molecular conformation, receptor binding and activity. In Hormone Antagonists (Edited by M. K. Argawal). Walter de Gruyter, Berlin (1982) pp. 3-24. 44. Durani S., Agarwal A. K., Saxena R., Setty B. S., Gupta R. C, Kole P. L., Ray S. and Anand N.: Seco-oestradiols and some non-steroidal oestrogens: structure correlates of oestrogenic action. J. steroid Biochem. 11 (1979) 67-77. 45. Robertson D. W., Katzenellenbogen J. A., Hayes J. R. and Katzenellenbogen B. S.: Antiestrogen basicityactivity relationships: a comparison of the estrogen receptor binding and antiuterotrophic potencies of several analogues of (z)-l ,2-diphenyl-l-[4-[2-(dimethylamino)ethoxy]phenylJ-l-butene(Tamoxifen, Novadex) having altered basicity. J. med. Chern. 25 (1982) 167-171. 46. Schoneman K. H., Van Vliet N. P. and Zeelen F. J.: Potential antitumor agents: II fl-( (-aziridinylmethyl)derivatives of oestradiol and oestrone. Eur. J. med. Chern. 15 (1980) 333-335. 47. Leclercq G., Devleeschouwer N., Legros N. and Heuson J. C: In vitro screening for cytotoxic estrogens of potential therapeutic activity. [n Cytotoxic Estrogens in Hormone Receptive Tumors (Edited by J. Raus, H. Martens and G. Leclercq). Academic Press, London (1980) pp. 165-181. 48. Hamacher H.: Potential antineoplastics. 4th communication: N-mustard derivatives of estrone. ArzneimitteJForsch. (Drug Res.) 29 (1979) 463-466. 49. Lam H. Y. P., Begleiter A. and Goldenberg G. J.: Synthesis of steroidal nitrosoureas with antitumor activity. J. med. Chem. 22 (1979) 200-202. 50. Lam H. Y. P.: In Cytoxic Estrogens in Hormone Receptive Tumors (Edited by J. Raus, H. Martens and G. Leclercq). Academic Press, London (1980) pp. 118-120. 51. Chavis C., de Goucy C, Borgna J. L. and Imbach J. L.: New steroidal nitrosoureas. Steroids 39 (1982) 129-147. 52. Jordan V. C, Fenuik (Rowsby) L., Allen K. E., Cotton R. C, Ridchardson D., Walpole A. L. and Bowler J.: Structural derivatives of tamoxifen and oestradiol 3-methyl ether as potential alkylating antioestrogens. Eur. J. Cancer 17 (1981) 193-200. 53. Zeelen F. J. and Bergink E. W.: Structure-activity relationships of steroid estrogens. In Cytotoxic Estrogens in Hormone Receptive Tumors (Edited by J. Raus, H. Martens and G. Leclercq). Academic Press, London (1980) pp. 39-47.
85
54. Kilbourn M. R.. Arduengo A. 1., Park J. T. and Katzenellenbogen J. A.: Conformational analysis of non steroidal estrogens. The effect of conformer population on the binding affinity of meso and dl-hexestrol to the estrogen receptor. Mol. Pharmac. 19 (1981) 388-398. 55. Landvatter S. W. and Katzenellenbogen 1. A.: Stereochemical considerations in the binding of non steroidal' estrogens to the estrogen receptor. Mol. Pharmac. 20 (1981) 43-51. 56. Connors T. A. Mechanism of action of 2-chloro-ethylamine derivatives, sulfur mustards, epoxides and aziridines. In Antineoplastic and Immunosuppressive Agents II (Edited by A. G. Sartorelli and D. G. Johns). Springer. Berlin (1975) pp. 18-34. 57. Devleeschouwer N., Leclercq G.. Danguy A. and Atassi G.: Absence of antitumor activity of ORG 5895. a lIfl-aziridimylmethyl derivative of estradiol on the MXT mammary tumor and the P388 leukemia. Cancer Chemother. Pharmac. 8 (1982) 315-316. 58. Katzenellenbogen 1. A., McGorrin R. 1., Tatee T., Kempton R. 1., Carlson K. E. and Kinder D. H.: Chemically reactive estrogens: synthesis and estrogen receptor interaction of hexesterol ether derivatives and 4-substituted deoxyhexestrol derivatives bearing alkylating functions. J. med. Chern. 24 (1981) 435-450. 59. Ratajczak T., Sheppard P. N., Capon R. J. and Hahnel R.: The synthesis and study of some potential affinity labeling reagents for estrogen receptors. Steroids 38 (1981) 537-555. 60. Vilkas M., Epztein R., Sainton J. and Leclercq G.: Synthesis and properties of some new 3,3'-substituted diethylstilbestrol derivatives. Eur. J. med. Chern. 17 (1982) 191-192. 61. Robertson D. W., Wei L. L. Hayes 1. R.. Carlson K. E., Katzenellenbogen J. A. and Katzenellenbogen B. S.: Tamoxifen aziridines: effective inactivators of the estrogen receptor. Endocrinology 109(1981) 1298-1300. 62. De Vleeschouwer N., Leclercq G .. Heuson J. C and Morgan L. R.: A new mesylate derivative of estrone with potential activity for the treatment of hormone dependent tumor. 1983. 63. Katzenellenbogen 1. A., Johnson H. J. Jr. and Karlson K. E.: Studies on uterine cytoplasmic estrogen binding protein. Thermal stability and ligand dissociation rate. An assay for empty and filled sites by exchange. Biochemistry 12 (1973) 4073-4099. 64. Hicks R. H.: Fundamental Concepts in the Design of Experiments. Holt, Rinehart & Winston. New York (1973) p. 30. 65. Sohar P.. Abraham G., Schneider G., Horvath 1. CiIlU Fuggerth E.: Synthesis and structure determination of geometric isomers of l-aryl-2-ethyl-I-2-diphenylethylenes by H-NMR spectroscopy. Acta chim. Acad. Sci. Hung. 100 (1979) 69-74.
0022-4731/83 $3.00+0.00 Copyright © 1983 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
GUIDE-LINES IN THE DESIGN OF NEW ANTIESTROGENS AND CYTOTOXIC-LINKED ESTROGENS FOR THE TREATMENT OF BREAST CANCER G. LECLERCQ, N. DEVLEESCHOUWER and J. C. HEUSON Laboratoire de Cancerologie Mammaire, Service de Medecine, Institut J. Bordet. !Ooo Brussels, Belgium SUMMARY
Antiestrogens (Tamoxifen) are used for the treatment of breast cancer. However these compounds are also weak estrogens that may stimulate tumor growth. Cytotoxic-linked estrogens (Estradiol Mustard, Estracyt) are devoid of major therapeutic activity. This led to search for new antiestrogens devoid of estrogenicity and for active cytotoxic-estrogens. Hydroxylation of C-4 of triphenylethylene antiestrogens (tamoxifen, CI628 and U 23,469) largely increases their binding affinity for the estrogen receptor (ER). Hydroxylation also increases the in vitro antitumor activity of the drugs as shown by their higher ability to inhibit the growth of the ER-positive cell line MCF-7. Triphenylethylene antiestrogens contain an aminoethoxy side chain which appears essential for their physiological activity. Removal of the chain of tamoxifen suppresses its antiestrogenicity and antitumor activity. The grafting of side chains on a weak estrogen of the gem-diphenylethylene category produces "symmetrical" antiestrogens devoid of estrogenic activity. This observation raises the question of the role played by the third phenyl ring of the triphenylethylenes since the trans-isomers of the latter display antiestrogenicity and the cis-isomers estrogenicity. Comparison of the binding affinity for ER and antitumor activity of di- and triphenylethylene antiestrogens suggests that this third phenyl ring increases the interaction with ER of the 4-phenolic group of the drugs and/or their aminoethoxy side chain. An analogue of this chain is without any biological activity suggesting that the di-(tri)phenylalkene ntructure is required for promoting the interaction of the chain with ER. New chemical structures yielding antiestrogens with antitumor activity are also reviewed. New cytotoxic estrogens designed for producing lethal damage of DNA show a low binding affinity for ER. Moreover, there is no evidence suggesting specific antitumor activity. Such activity may be more easily obtained with estrogens bearing reagents for proteins rather than DNA. The biological properties of a 2-mesylate derivative of estrone irreversibly interacting with ER supports the concept. On MCF-7 cells, the drug displays a strong antitumor activity which can only be suppressed by high, equimolar, concentrations of estradiol. It is devoid of cytotoxic activity on the ER-negative cell line Evsa-T suggesting that ER is involved in its action.
INTRODUCTION
This stresses the need for new antiestrogens devoid of estrogenic activity. On the other hand, estrogen-linked cytotoxic agents such as Estradiol Mustard and Estracyt have been produced (Fig. 1). The rationale for using such compounds relied on the principle that they may bind to ER and thereby concentrate their cytotoxic agent (nitrogen mustard) in the mammary tumor cells. Unfortunately these drugs are devoid of major therapeutic activity in the treatment of advanced breast cancer [6--9]. The absence of a significant binding affinity of these compounds for ER as well as their metabolism with production offree estradiol might be responsible for their lack of antitumor activity [10, 11]. These disappointing results led to a reappraisal of the design of cytotoxic-estrogens [12]. It is our purpose to review the more recent investigations devoted to the production of new antiestrogens and cytotoxic-estrogens and define guide-lines for the design of new drugs of potential therapeutic activity.
Specificity of action is a problem of major importance in the development of new antitumor drugs. In breast cancer, the presence of estrogen receptor (ER) could well form the basis for an advance in this respect. One may legitimately hope that receptor-mediated chemotherapy could enhance therapeutic efficacy while decreasing the toxic side effects. In fact this hope has already found support in the clinical use of non steroidal antiestrogens [IJ such as nafoxidine, clomiphene or tamoxifen (Fig. 1). In advanced breast cancer, these compounds give a remission rate close to 30% which is similar to those reported with other endocrine treatments [2]. The lack of major side effect of tamoxifen prompted its use as the standard antiestrogen for the treatment of the disease [3]. Moreover, in primary breast cancer, it has been shown to act as an active additive to adjuvant chemotherapy [4]. However, this drug as all other antiestrogens [IJ displays weak estrogenic activity which may stimulate mammary tumor growth [5]. s.•.
19-I(A)-G
75
76
G.
LECLERCQ
et al.
ANTIESTROGENS
CH30 ~
I~ Natoxidint
Clomiftnt
(U-II,IOOA)
Tamoxiftn ( ICI-46,474)
CYTOTOXIC - ESTROGENS
Estradiol Mustard
Estracyt
Fig. 1. Chemical formulae of antiestrogens and cytotoxic-estrogens tested in clinical trials. On tamoxifen the star indicates the position of the hydroxyl function in the hydroxylated form of the drug (position 4).
MODELS FOR EVALUATING THE POTENTIAL THERAPEUTIC ACTIVITY OF THE DRUGS
Several animal mammary tumor models are useful for evaluating the in vivo antitumor activity of antiestrogens and cytotoxic-estrogens (review; [13]). Among them, the DMBA-induced rat mammary carcinoma has been the most widely used [14]. In our laboratory, we recently introduced a new hormonesensitive transplantable model, the MXT-mouse mammary carcinoma developed by Watson et al.[15], which seems more appropriate dor drug screening. This ER-positive tumor shows a marked sensivity to ovarian and pituitary hormones: its growth is slowed down by castration [15,16], CB-154 [17] and tamoxifen [16]. In vivo screening always requires large amounts of drugs which could not always be easily produced. This led to the development of in vitro tests on ERpositive breast cancer cell lines such as the MCF-7 [18]. In our laboratory, this line as well as the ER-negative line Evsa-T (control) is routinely used for the selection of new compounds of potential therapeutic activity. The following experimental protocol has been designed. MCF-7 and Evsa-T cells are grown in Falcon plastic flasks (75 cm 2 ) containing Earle's minimal essential medium (MEM) supplemented with 0.6 mg L-glutamine/ml, 40 J-lg gentamycin/ml, 100 U penicillin/ml, 100 J-lg streptomycin/ml and 10% fetal calf serum. At confluency, cells are removed by trypsinisation (trypsin 0.05%, EDTA 0.025%) and suspended (50-200 x 103 cells/ml) in the growth medium supplemented with charcoal stripped fetal calf serum (0.5%
charcoal, 0.005% dextran in 1.5 ml medium/ml serum; overnight incubation at 4°C). Cells are then plated in 35 mm Petri dishes containing this medium and cultured at 37°C in a humidified 95% air 5% CO 2 atmosphere. After 24h, drugs (solvent:ethanol at the final concentration of 0.1 %) are added to the culture dishes. Forty-eight hours later the medium is replaced by fresh medium containing the drugs. The cultures are then pursued for an additional n-h period before harvest. At this time, the cells are washed twice with 2 ml of Earle's base before being suspended in 1.5 rnl trypsin-EDTA. Total DNA of collected cells are precipated in 0.5 N perchloric acid and evaluated by the diphenylamine method. Experiments are always performed in quadruplicate.
ANTIESTROGENS
All strong antiestrogens synthetised within the last three decades are of the triphenylethylene category [1] (Fig. 1). Recently it has been shown that other chemical structures produce antiestrogenic and mammary antitumor activities. Thus, Bouton and Raynaud demonstrated that steroidal estrogens fastly dissociating from ER may have antiestrogenic properties [19]. Such a compound (RU 16,117; Fig. 2) was found to produce regression of DMBA-induced rat mammary carcinomas '[20]. On the other hand, Schonenberger and his group [21-27] showed that chemical modifications of the synthetic estrogens diethylstilbestrol, hexestrol and indenestrolled to drugs with antiestrogenic activity. Roughly, displacement of the phenolic groups from the para to the meta position pro-
77
Antiestrogens and cytotoxic-linked estrogens OAvNJ
OH H30"
~
.·eaCH ~. BOUTON AND
CI 628
J. P. RA YNAUD
(ROUSSEL (0)
Incr~as~
I~
HO
~
~
20
~
RU-16\l7
10
HO~ {DOH OJ~·
c +1
III
-OH
- OCH 3
j II~
«l
~[n-OH
HO~~
HO
I
_10""
~ ~ (II vs135)
OH OH O/"L./
z
c H.
ScHONUaUGER
(REGENS_G,6E_>
U 23,469
Cll ~
CH3 ~ CH3
Of
J.T.
PENTO
Control
.8 _7 _6
e\
Fig. 2. Chemicals formulae of new antiestrogens producing mammary tumor growth.
duces a strong decrease in estrogenicity with a gain in antiestrogenicity [21-24]. Similarly, structural modifications of the diphenylethane (ethene) skeleton of these estrogens produce strong antiestrogenicity [25-27]. Such an observation was also reported by Pento et al.[28, 29]. Notably, all these modifications led to drugs able to inhibit the growth of the DMBAinduced rat mammary carcinomas (examples III Fig. 2). Recent studies have shown that hydroxylation of one phenyl ring of tamoxifen (position 4; Fig. 1) led to a drug with very high binding affinity for ER (Relative Binding Affinity; RBA ~ 1(0) and strong antiestrogenicity [30-33]. These properties largely increased the in vitro antitumor activity of the drug as shown by its higher cytotoxicity on the MCF-7 cells [34, 35]. This effect of hydroxylation in position 4 might be common to all antiestrogens of the triphenylethylene category since we have found that the replacement of the methoxy group by a hydroxyl group of the two antiestrogens CI 628 and U 23,469 [36]* also produced a significant increase of growth inhibition (Fig. 3). Noteably, the difference was more marked for U 23,469 which is consistent with the increase in binding affinity for ER produced by the hydroxylation of these drugs (CI 628 = - IO-fold; U 23,469 = - 300-fold) [36].
* Compounds provided by Dr 1. A. Katzenellenbogen, Urbana, Illinois.
.8 .7 .6
log Molarity
(OKL_ (ITY. 010
Fig. 3. Growth inhibition of MCF-7 cells by C1628, U 23,469 and their hydroxylated derivatives. Variance analysis [64] shows that CI628 and derivative are active (P < 0.(01) at all concentrations (Newman-Keuls test); the derivative is more effective at 10- 7 M (P < 0.(01). U 23,469 is not active while its derivative is active at 10- 7 and 10- 6 M (P < 0.(01). Symbols: 0 control without any drug; ~ experimental drugs.
I Alone I
Cl VI
+1 «l
z
Cl Ol
~:J 0 JI I
Trans
1+5xIO-7M NAFI
20
::1..
10
[1. JlI~
Controls
.7.6
log Molarity
Fig. 4. Estrogenicity of a tamoxifen derivative devoid of aminoethoxy side chain. The compound is in the trans configuration [65]. Variance analysis shows that the compound does not significantly modulate growth of MCF-7 cells. At 10- 6 M, it significantly suppresses (interaction P < 0.01) the inhibition (P < 0.01) of 5 x 10- 7 M nafoxidine. The upper pannel refers to the drug alone; the lower to the drug with nafoxidine. Symbols: 0 and ~ as in Fig. 3; • nafoxidine alone; I§I nafoxidine with experimental drug.
78
G.
LECLERCQ
et al.
I Alonll I 30
II I ~ .1
20
10
1+5xI0-7M NAFJ 30 0 I/)
+1 20 4 Z 0
10
01 ~
1+10_8M 30
Ezi
~~I
20
10
Controls
_8 _7 _6 log Molarity
Fig. 5. Antiestrogenicity of a gem-diphenylethylene bearing an aminoethoxy side chain. Variance analysis shows that the diphenolic compound does not significantly modulate growth. At 10- 6 M it significantly suppresses (interaction P < 0.001) the inhibition of 5 x 10- 7 M nafoxidine. The two derivatives with an aminoethoxy side chain (A and B) significantly reduce growth (P < 0.01) at 10- 6 M (NewmanKeuls test). At 10- 8 and 10- 7 M they are devoid of activity on the inhibition of 5 x 10- 7 M nafoxidine. Estradiol at 10- 8 M significantly suppresses (interaction P < 0.01) their inhibition. The upper pannel refers to the drugs alone; the intermediate to the drugs with nafoxidine; the lower to the drugs with estradiol. Symbols: 0, ~, • and S as in Fig. 4; ~ estradiol alone; ~ experimental drug with estradiol.
In vivo hydroxytamoxifen was found to be a less effective inhibitor than tamoxifen in the DMBAinduced rat mammary tumor model [37]. The reason would be the shorter biological half-life of this polar drug. The hydroxyl function being of major importance in the in vitro antitumor activity of the triphenylethylene antiestrogens, search for its in vivo stabilization seems of prominent interest. Antiestrogens of the triphenylethylene category contain an aminoethoxy side chain which seems essential for their physiological activity: without such * Compound provided by Dr A. B. Foster, Chester Beatty Institute, Sutton, Surrey, U.K. t Compound provided by Dr I. Niculescu-Duvaz, Oncologic Institute Bucharest. Romania.
a chain the compounds lose their antiestrogenic properties and become potent estrogens [I, 38]. Substitution of the side chain of tamoxifen by a methoxy group* suppresses its in vitro ability to inhibit the growth of MCF-7 cells (Fig. 4). The resulting molecule behaves as a weak estrogen in view of its ability to partly suppress at high concentration the inhibition of nafoxidine (the minimal effective concentration is 10- 6 M while that of estradiol is about 100 times lower; Fig. 6, Ref [18]). In this context, it should be stressed that the grafting of an aminoethoxy side chain on a weak estrogen of the gem-diphenylethylene categoryt [39] produces compounds able to inhibit the growth of the MCF-7 cells (Fig. 5). This inhibitory effect is similar to that found with antiestrogens of the triphenylethylene category since it could be sup-
79
Antieslrogens and cytotoxic-linked estrogens
Cis
Trans
20 10
o VI
+'
o
Z
o 0'
"-
\+10- 8 ""
£21
7 1+5XIO· M NAF
Fig. 7. Role of the third phenyl ring of the triphenyiethylenes in their antiestrogenic activity. The arrows indicate two possible influences: (1) an increase of interaction of the phenol with the receptor; (2) an increase of interaction of the aminoethoxy side chain with the receptor.
I
~:] [J ]~ I ~: ~ 1 Control~
.7
.b
Control~
log Molarity
.7.G log Molarity
Fig. 6. Antiestrogenicity of lrlllls-tamoxifen (ICI-46.474) and estrogenicity of cis-lamoxifen (lCl-47.699). Variance analysis shows that lnms-tamamoxifen significantly reduces growth of MCF-7 cells (P < 0.01) at 10- 7 and 10- 6 M (Newman-Keuls test); estradiol at 10- 8 M significantly suppresses (interaction P < 0.01) its inhibition. cisTamoxifen does not significantly modulate growth; at JO- 6 M it suppresses (interaction P = 0.1) the inhibition (P < 0.01) of 5 x 10- 7 M nafoxidine. The upper pannel refers to the drugs alone; the lower to the drugs with estradiol or nafoxidine. Symbols as in Fig. 5.
pressed by estradiol. On the other hand, these compounds do not suppress the inhibition of nafoxidine indicating that they are devoid of estrogenicity. That the grafting of an aminoethoxy side chain on a "symmetrical" diphenylethylene produces antiestrogenicity is of interest in view of the fact that triphenylethylene antiestrogens are always in the trans configuration [I, 40--42]. When they are in the cis configuration, they usually display estrogenic activity [1,40-42]. This geometrical isomerism also influences the MCF-7 cell growth: the trailS isomer§
* Compounds provided by Dr J. S. Patterson. lCI. Macclesfield, Cheshire, U.K.
of tamoxifen shares the classical antiestrogenic activity (inhibitory action suppressed by estradiol) while the cis isomer* behaves as a weak estrogen (no inhibitory action; partial suppression of nafoxidine inhibition) (fig. 6). The analogy in biological activity between the gemdiphenylethylene antiestrogens and the classical triphenylethylene antiestrogens raises the question of the role played by the third phenyl of the latter (Fig. 7). Assuming that the phenol of the gem-diphenylethy(enes corresponds to the phenol of the classical antiestrogens, hydroxytamoxifen, CI 628 M and U 23,469 M (position 4), it appears that the third phenyl of the latter largely increases the binding affinity for ER as well as the antitumor activity (Table 1). This observation highly suggests that this third phenyl in the rrans configuration increases the interaction of the 4-phenolic group with the receptor [13, 43] and thereby enhances antiestrogenecity. This third phenyl may also favor the interaction of the side chain with a specific site on ER which would essentially mediate antiestrogenicity [44]. In this regard, it should be stressed that such a specific interaction has never been demonstrated. If it exists, it seems that it would involve a hydrogen bound interaction in view of the fact that residues able to such binding only produce antiestrogenicity (-N-{Alkh; -CHOH-CH 2 0H; - N->O; Ref.[ I.35.36,45]).
Table I. Hydroxylated antiestrogens. Relative binding affinity and inhibition ability of MCF-7 cells
Compound
RBA (E 2 = 1(0)
Diphenylethylene A Diphenylethylene B U 23,469 M Cl628 M OH-tamoxifen
0.1 0.2
30 ;:;: 100 ;:;:100
Concentration producing 50'~" growth inhibition
10-1> M 10- 6 M 10- 7 M -10- H M _1O- M M
Reference
This This [36] [36]
work work This work This work
[30--35]
G.
80
LECLERCQ
et at.
,....,
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.c:: u os
0
:r:
E
~
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os
+1
~
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~ 10
5
Controls
_6 _5 log Molarity
Fig. 8. Absence of influence of an analogue of the aminoethoxy side chain (1-[2(p-bromophenoxy)ethyl]pyrrolidine) on the growth of MCF-7 cells. The upper pannel refers to the drug alone; the lower to the drug with 10 - 8 and 10 -, M hydroxytamoxifen. Symbols 0, lSI as in Fig. 3; • S experimental drug with hydroxytamoxifen; hydroxytamoxifen.
The hypothesis of a specific site on ER for the side chain of the antiestrogens was investigated by using a free analogue (1-[2(p-bromophenoxy)ethyl]pyrrolidine; Aldrich Co.). In the competitive test of the binding of eH]-estradiol to ER, this compound does not share any competitive activity indicating a total absence of affinity for the estradiol binding site. On MCF-7 growth, it does not display any antitumor activity, nor does it suppress the inhibition by hydroxytamoxifen even at a lOoo-fold higher concentration (Fig. 8). These results indicate that, if a specific interaction does exist between the side chain and ER, the di-(tri-)phenylalkene structure of the antiestrogens is required for promoting the interaction.
'-
o
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'':.:.c
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'en = 0;:: Q.,
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=~ ~ 0 os ...
Q.,t;
'"
,.. DJ "0
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DJ
os
DJ
CYTOTOXIC ESTROGENS
Estradiol Mustard and Estracyt (Fig. I) are devoid of significant binding affinity for ER [lO, 11]. In both compounds the 3-phenolic group of estradiol is substituted by the cytotoxic agent. In view of the major importance of this group in the binding of the estrogen to ER [13,43], it has been proposed that this substitution was responsible for the lack of affinity of these drugs for the receptor. Drugs with free phenol were therefore synthetized [13, 46-52]. Table 2 provides the formulae, binding affinities and biological properties of some alkylating derivatives recently produced (three of these drugs being in a methoxylated form, their binding was not assessed). Although
0. :l
...o 1>0 1>0
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/
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---eH zCH zCH z-N
-
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-CHzNHC-N---eHz-CHzCl
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6dehydro E z (3MeO, J7P OAc)
Ez (3MeO, 17P OAc)
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Ez
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7P
171X
171X
17P
17tX
-
-
-
0.2
0.4
2
Non estrogenic Non antiestrogenic
Non estrogenic Non antiestrogenic
Weakly estrogenic
Growth inhibition of rat mammary tumor 13762 (specificity?)
Growth inhibition of rat mammary tumor (specificity'!)
ICI 140, 175 [52]
ICI 140,496 [52]
Iel 141,857 [52]
Lam [49, 50]
Chavis [51]
Lam [49, 50]
00
'"
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82
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LECLERCQ
almost all these compounds bind significantly to ER, the substitution always produced a marked reduction of binding affinity (the methoxylated drugs did not display potent estrogenic or antiestrogenic activities suggesting a weak affinity). This observation is consistent with the known fact that substitutions usually reduce the interaction of the steroid with the receptor [13, 53]. Substitutions of non steroid estrogens may be more fruitful. However, in such cases, important spatial modifications of the estrogen moeity would normally occur most likely resulting in a reduction of binding affinity [54, 55]. In the design of cytotoxic estrogens a fundamental question is the concentration of cytotoxic agent required for lethal damage to the DNA. It is implicitly assumed that the tumor ER levels are sufficient to concentrate the required amounts of cytotoxicestrogens. However, this assumption has never been demonstrated and deserves more substantial analysis. The chemical reactivity of the cytotoxic agent has not been sufficiently taken into account either. Strong alkylating derivatives may logically react with all the tissues they reach, especially the liver, which contain ER and enzymes of the estrogen metabolism. Such undesirable alkylation might compromise the whole concept of specific tumoral killing [52]. On the other hand, systematic studies of alkylating drugs have shown that significant antitumor activity may only occur with drugs bearing at least two alkylating residues [56]. The presence of only one aziridine .in the aziridine derivative of estradiol described in Table 2 (ORG 5895) may explain its total lack of antitumor activity on the MXT mouse mammary tumor and the P388 leukemia [57]. Since multiple substitutions brought to an estrogen usually produce a marked reduction of its binding affinity to ER, its seems likely that monoalkylating residues (i.e. aziridines and epoxydes) should not be selected in future syntheses. Finally, it is unknown whether the antitumor properties of the cytotoxic agents are maintained when the latter are linked to an estrogen interacting with ER. Drugs able to dissociate from the receptor in the cell nucleus might be desirable. However, knowledge on the stability of the nuclear ER-estrogen complex is still too scarce to propose suitable chemical structures. These considerations suggest that specific therapeutic activity may be more easily obtained with cytotoxic agents interacting with ER rather than with DNA. Efforts have been made in this direction [58-62J yielding compounds that irreversibly [63J interact with the receptor. These compounds may be strong estrogens or strong antiestrogens as shown by the behavior of a IIp-chloromethyl derivative of estradiol [47, 59J and a 2-mesylate derivative of estrone (62). Thus, in MCF-7 cells the chloromethyl
* Compound provided by Dr F. J. Zeelen, Organon, Oss, The Netherlands. t Compound provided by Dr R. L. Morgan, Louisiana State University, New Orleans, Louisiana.
et al.
ORG 4333
I
Alone
20
HO~
10
o
+1
1+ 10_6M
~
o
01
NAF
I
20
:I...
10
_II _10 _9 _6 log Molarity
Control~
Fig. 9. Estrogenicity of IIp-chloromethylestradiol (ORG 4333) on the growth of the MCF-7 cells. Variance analysis shows thai the compound does not significantly modulate growth. At 10- 9 and 10- 8 M, it significantly suppressess (interaction P < 0.01) the inhibition (P < 0.01) of 10- 6 M nafoxidine. The upper panne! refers to the drug alone; the lower to the drug with nafoxidine. Symbols as in Fig. 4. derivative (ORG 4333, Fig. 9)* suppresses the growth inhibition of nafoxidine in the same range of concentrations as estradiol. In contrast, the mesylate derivativet (Fig. 10) inhibits growth at similar concentration 0""
Z
S02 CH 3
0 II
°bSD
HO
~I ~
15
10
o oil
+1 10
5
o
Cl
5
:l..
Control
_8 _7 _6 log Molarity
Control
_8 _7 _6 log Molarity
Fig. 10. Influence of a 2-mesylate derivative of estrone on the growth of MCF-7 and Evsa-T cells. Variance analysis shows thai the compound significantly reduces growth of MCF-7 cells (P < 0.01) at all concentrations (NewmanKeuls test). The compound is not active on Evsa-T cells. Symbols as in Fig. 3.
Antiestrogens and cytotoxic-linked estrogens
IE2 alone I 15
10
5 o Vl
J
+1 ~ Ol
1,....~-2-+-IO-_-::6-M-M-e-s-E-d 15
:J...
10
5
~~ _8 _7
Controls
_6
log Molarity
Fig. II. Suppression by estradiol of the growth inhibition of a 2-mesylate derivate of estrone on the growth of MCF-7 cells. Estradiol at 10- 6 M significantly suppresses (interaction P < 0.0 I) the inhibition of 10 - 6 M of the drug. The upper panne! refers to the estradiol alone; the lower to the experimental drug with estradiol. Symbols as in Fig. 5.
to the strong hydroxylated antiestrogens hydroxytamoxifen, and CI 628 M. That no growth inhibition occurs in Evsa-T cells (ER-) in the presence of Ihe drug indicates that it has no major general cytotoxicity. Remarkably, the inhibition in the MCF-7 cells is suppressed by estradiol but only at high, equimolecular, concentrations (Fig. 11). This contrasts with the behavior of conventional antiestrogens such as tamoxifen which are counteracted by low levels of estradiol [18]. Finally, hydroxylated and methoxylated analogues of estrone (2-0H, 2-0CH 3 ) do not inhibit growth suggesting that the cytotoxicity of the mesylate derivative was associated with its alkylating residue. These remarkable properties highly suggest that estrogens interacting irreversibly with ER may provide a new class of "suicide inhibitors" of prominent therapeutic interest. CONCLUSIONS
The studies reported here reveal a considerable effort in the production of new antiestrogens and cytotoxic estrogens for the treatment of breast cancer. Guide-lines for the design of new drugs emerge from these investigations. In the case of triphenylethylene antiestrogens a close parallelism was found between the known endocrinological properties of the drugs and their antitumoral activity. This observation highly suggests that a large part of the literature on these compounds is relevant in the design of new antineoplastic drugs.
83
In this regard. it seems that the mere triphenylethylene structure should be abandoned in view of its metabolic instability which might yield both trans and cis metabolites. A more rigid structure that mimics the A and B rings of estradiol (cf. nafoxidine) would be investigated. On the other hand it seems that metabolic studies [32, 35, 42J might be extremely helpful in the identification of active metabolites which may be produced at a later stagr (or production of stable analogues). In view of the prominent function of the aminoethoxy side chain of the antiestrogens, effort should be made to increase interaction of former with ER by introducing it into reactive groups [61]. Finally, the production of several gem-diphenylethylene derivatives bearing an aminoethoxy side chain could be proposed. The data on the potential antitumor activity of cytotoxic-estrogens are still too scarce to ascertain an important therapeutic role for such drugs. However, evidence is given here suggesting that suicide inhibitors that irreversibly interact with ER may be useful. Effort should therefore be made to identify on estrogens and antiestrogens suitable positions for grafting reagents for proteins yielding such very strong antiestrogens. Acknowledgements-We wish to thank Drs A. B. Foster, J. A. Katzenellenbogen. L. R. Morgan, I. Niculescu-Duvaz, J. S. Patterson and F. J. Zeelen for the gift of drugs. This work was supported by a grant from lCl-Belgium (Dr M. A. Lassance) and by a grant from the Fonds Cancerologique de la Caisse d'Epargne et de Retraite (Belgium).
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of
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