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cervix in daughters of women receiving DES during
pregnancy.5 The good pharmacokinetic profile and high
binding affinity for the estrogen receptor (ER) make
DES attractive as the starting point for the development
of antiestrogens for clinical use. The main question we
wanted to answer in this study was whether it is possible
to convert this potent estrogen into an estrogen antago-
nist devoid of any agonist activity. To achieve this goal
we replaced one of the ethyl groups by long alkyl chains
that incorporate functional groups. Previous studies
have shown that sulfur functions in a distance of 10 car-
bon atoms from the core of the molecule are appropriate
for this purpose.2
All of the stilbenes were obtained as E/Z mixtures with
the E-stereoisomer as the dominant product. HPLC
studies revealed a ratio of E-4 to Z-4 of approximately
85:15. When the stereoisomers had been separated by
HPLC they rapidly isomerized to give the original ratio
of isomers. Thus, no attempt was made to study the ste-
reoisomers separately.
The first step in the biological characterization of the
new stilbene derivatives was the determination of the
binding affinities for the ER. Calf uterine cytosol was
used as receptor source as described previously.6 The
introduction of long side chains reduced the affinity con-
siderably (Table 1). A similar observation was made
with steroids, for example, fulvestrant.7 The data
showed that the affinity is mainly dependent on the type
of side chain used. The most favorable conditions are
provided by the bifunctional side chains as demon-
strated by derivatives 9 and 10. Their values are close
to those found for fulvestrant and the indole derivative
ZK 164,015.
The synthesis of the new DES derivatives started from
the corresponding desoxyanisoins 1 (Scheme 1). The
first side chain was introduced by deprotonation and
subsequent reaction with ethyl bromide or the bromo
alkane with the respective sulfur function to give the ke-
tones 2. The second substituent was introduced by a
Grignard reaction, which led to the formation of a dou-
ble bond with orientation towards the side chain (3).
Since the acidity of the sulfone prevented its direct use
as the Grignard reagent, the thio ether function had to
be oxidized with m-CPBA after the Grignard reaction
to give 3h. Cleavage of the methoxy groups in 3 led to
mixtures of the stereoisomeric phenols 4 and 5 with a
preference for the stilbene structure 4. For the prepara-
tion of DES derivatives 9 and 10 with side chains con-
taining an additional methylamino group the synthesis
had to be modified (Scheme 2): First the desoxyanisoin
1b was reacted with ethyl 6-bromohexanoate to give 6,
followed by the Grignard reaction with EtMgBr to af-
ford the ester 7, which was then reacted with the appro-
priate amine to give 8. Deprotection with BBr3 resulted
in double bond migration from a styrene-like to stilbene-
like position. In the last step, the phenolic stilbenes 9
were reduced with LiAlH4 to yield the amines 10.
Estrogenic and antiestrogenic activities were determined
in vitro6 using ER+ human MCF-7/2a cells8 stably
transfected with a luciferase reporter gene under the
control of an ERE. Since pure antiestrogens should be
devoid of any agonist action by definition, all new stil-
bene derivatives were evaluated for agonism. At a con-
centration of 10À6 M they exhibited values for
luciferase activity below that of control cells grown in
steroid depleted medium (Fig. 2). Luciferase activities
below baseline are characteristic for pure antiestrogens
and indicate the blockade of ligand-independent activa-
tion of the ER, responsible for the basal luciferase activ-
ity in control cells.8
Antiestrogenic activity was determined in a similar
assay by simultaneous treatment of the cells with
R2
R2
OMe
OMe
OMe
b
a
R1
R1
R1
O
O
Me
1a: R1 = 3-OMe
1b: R1 = 4-OMe
2 R2 = Me, (CH2)9SC5H11
,
3
(CH2)9SO2C5H11
OMe
Me
OMe
Me
OMe
Me
d
c
O
(CH2)9SO2C5H11
MeO
(CH2)9SC5H11
MeO
R2
MeO
3h
3c
2b
R2
OH
OH
e
R1
+
3
R1
R3
5a-h R1 = OH
Scheme 1. Reagents and conditions: (a) 1. NaH, DMF, 0°C; 2. R2CH2Br, rt, 2h, 62–86%; (b) EtMgBr, Et2O, reflux, 2h, 54–76%; (c)
11C5S(CH2)10MgBr, Et2O, reflux, 2h, 33%; (d) m-CPBA, CHCl3, rt, 2h, 76%; (e) BBr3, CH2Cl2, À5°C–rt, 7h, 52–95%.
R3
4a-h R1 = OH
H