Novel estradiol derivatives labeled with Ru, W, and Co complexes. Influence on hormone-receptor affinity of several organometallic groups at the 17α position

Article abstract of DOI:10.1002/1521-3765(20021115)8:22<5241::AID-CHEM5241>3.0.CO;2-M

In order to elucidate the extent to which recognition of the estrogen receptor is influenced by addition of an organometallic substituent at the 17α position, modification of 17β-estradiol at this position was carried out by using the organometallic groups -C≡C-(η⁵-C₅H₄)RuCp, CH₂-(η⁵-C₅H₄)RuCp, -C≡C-(η⁵-C₅H₄)- W(CO)₃(Me), -(C≡CCHO)Co₂(CO)₆, and -(C≡CCH₂OH)Co₂(CO)₆. The relative binding affinity (RBA) values for estradiol receptor alpha showed that recognition was good (RBA between 20 and 13.5%) when the organometallic moiety was attached at the end of a rigid alkyne spacer. However, the affinity of the modified hormone for the receptor was severely reduced. (RBA = 1 %) for a substituent such as -CH₂-(η⁵-C₅H₄)- RuCp, in which the spacer is reduced to a single flexible sp³ carbon atom, allowing the organometallic moiety greater freedom of movement around the attachment point. The RBA values found were in agreement with results obtained from a molecular-modeling study in which 5, an organometallic hormone with a rigid spacer, or 7, a molecule with a flexible spacer, was inserted into the cavity of the recently characterized Ligand-Binding Domain of estrogen receptor alpha.

Full text of DOI:10.1002/1521-3765(20021115)8:22<5241::AID-CHEM5241>3.0.CO;2-M

FULL PAPER
Novel Estradiol Derivatives Labeled with Ru, W, and Co Complexes.
Influence on Hormone-Receptor Affinity of Several Organometallic Groups
at the 17a Position
Siden Top,*[a] Hassane El Hafa,[a] Anne Vessie¡res,[a] Michel Huche¬,[a]
Jacqueline Vaissermann,[b] and Ge¬rard Jaouen*[a]
Abstract: In order to elucidate the
extent to which recognition of the
estrogen receptor is influenced by
addition of an organometallic substi-
tuent at the 17a position, modifica-
tion of 17b-estradiol at this position
was carried out by using the organo-
recognition was good (RBA between 20
and 13.5%)when the organometallic
moiety was attached at the end of a rigid
alkyne spacer. However, the affinity of
the modified hormone for the receptor
was severely reduced (RBA ˆ 1%)for a
substituent such as -CH2-(h5-C5H4)-
RuCp, in which the spacer is reduced
to a single flexible sp3 carbon atom,
allowing the organometallic moiety
greater freedom of movement around
the attachment point. The RBA values
found were in agreement with results
obtained from a molecular-modeling
study in which 5, an organometallic
hormone with a rigid spacer, or 7, a
molecule with a flexible spacer, was
inserted into the cavity of the recently
characterized Ligand-Binding Domain
of estrogen receptor alpha.
5
ꢀ
metallic groups -C C-(h -C5H4)RuCp,
5
CH2-(h5-C5H4)RuCp, -C C-(h -C5H4)-
ꢀ
ꢀ
W(CO)3(Me), -(C CCHO)Co2(CO)6,
Keywords: bioorganometallic chem-
istry ¥ cobalt ¥ molecular modeling
¥ ruthenium ¥ steroids ¥ tungsten
ꢀ
and -(C CCH2OH)Co2(CO)6. The rela-
tive binding affinity (RBA)values for
estradiol receptor alpha showed that
Introduction
Bioorganometallic chemistry is a strongly emerging field,
whose promise was underlined by a recent special issue of the
Journal of Organometallic Chemistry devoted to the subject.[1]
At the basis of bioorganometallic chemistry is the addition of
an organometallic functional group onto a vector or a
biological target–a biomolecule, a protein, DNA–in order
to modify its properties.[2] Over the past few years this
approach has given rise to new types of radiopharmaceu-
ticals,[3] various cytotoxic entities,[4] innovative analytical con-
cepts,[5] and novel bioconversions.[6] The principle of attaching
an organometallic functional group to a tamoxifen-type
antiestrogen delivery system may well lead to new therapeutic
approaches, since such molecules, as for example hydroxyfer-
rocifen, are simultaneously active on both hormone-depen-
dent (MCF7)and hormone-independent (MDAMB231)
breast-cancer cell lines.[7]
Over the last few years we have been studying the natural
estrogen, estradiol, and searching for organometallic modifi-
cations to its skeletal structure that would retain good affinity
for its specific receptor.[8] In preference to the chemically
more difficult modification at position 11,[9] we chose to attach
an alkyne-type rigid spacer at position 17a of the steroid.[10]
Molecules such as (17a-ethynylcyrhetrene)estradiol in fact
prove to be very well recognized by estrogen receptor a.[10] It
[a] Dr. S. Top, Prof. Dr. G. Jaouen, Dr. H. El Hafa,
¡
¬
Dr. A. Vessieres, Dr. M. Huche
¬
Laboratoire de Chimie Organometallique
¬
Ecole Nationale Superieure de Chimie de Paris, UMR C.N.R.S. 7576
11, rue Pierre et Marie Curie, 75231 Paris Cedex 05 (France)
Fax : (‡33)1-43-26-00-61
ꢀ
remains to be seen whether this (estradiol) (17 a)-C C-OM
E-mail: siden@ext.jussieu.fr, jaouen@ext.jussieu.fr
combination (OM ˆ organometallic)can be generalized syn-
thetically to organometallic systems other than rhenium and
ferrocene,[11] whether these also show good binding affinities
for the receptor, and, if so, why this is the case. We thus
[b] Dr. J. Vaissermann
¬
¬
Laboratoire de Chimie Inorganique et Materiaux Moleculaires
¬
¬
Universite Pierre et Marie Curie, Unite C.N.R.S. 7071
75252 Paris Cedex 05 (France)
Chem. Eur. J. 2002, 8, No. 22
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5241

G. Jaouen, S. Top et al.
FULL PAPER
prepared a number of acetyl-
ene estradiols modified at po-
sition 17a with new complexes
of Ru, W, and Co, evaluated
their relative binding affinity
for estrogen receptor a, and
used molecular-modeling tech-
niques to analyze the factors
involved in the recognition of
the modified hormone by the
receptor. The potential appli-
cations of these organometallic
moieties are related to new
cytotoxic effects,[4e] new radio-
pharmaceuticals,[12] and heavy
metals for X-ray structural de-
termination.[13]
COMe
H C C
1) POCl3, DMF
2) H2O
NaOH
dioxane
Ru
Ru
1
2
Li C C
O
OH
C
C
Ru
Ru
THF, -78¡C to RT
tBuMe2SiO
tBuMe2SiO
4
3
THF
nBu4NF
OH
C
Results and Discussion
C
Synthesis of ruthenium com-
plexes derived from estradiol:
Scheme 1 shows the synthesis
of 17a-(ruthenocenylethynyl)-
estradiol (5). This synthesis
involves the use of ethynylru-
Ru
HO
5
Scheme 1. Synthesis of 17a-(ruthenocenylethynyl)estra-1,3,5(10)-trien-3,17b-diol (5).
thenocene (2)obtained from acetylruthenocene ( 1)following
the method reported by Rausch et al.[14]
O
OH
Li
CH2
CH2
Ru
Ethynylruthenocenyl lithium is obtained by addition of
butyllithium to 2. The organolithium thus formed is then
allowed to react with the protected estrone 3. The protected
hormone complex 4 is formed initially in 55% yield. This
complex is converted into 17a-(ruthenocenylethynyl)-estra-
diol 5 simply by stirring 4 with the deprotection agent,
nBu4NF.
Ru
THF, -78¡C
HO
HO
6
7
Scheme 2. Synthesis of 17a-(ruthenocenylmethyl)estra-1,3,5(10)-trien-
3,17b-diol (7).
In order to be able to com-
O
pare the results with those for a
complex containing a shorter
O
O
MeO
O
HO
and less rigid spacer, 17a-
(ruthenocenylmethyl)-estradiol
(7)was prepared from 17 b-
spiro-oxiranyl estradiol (6)
NaH, CH3I
THF, HMPA
PhCH2O
9
PhCH2O
(Scheme 2). Lithium rutheno-
cenyl, obtained by addition of
8
1) THF, H2, Pd(C)
2) methanol, HCl
nBuLi to ruthenocene, was al-
lowed to react with compound
OH
O
Li C C
MeO
C C
MeO
6 at À788C. Complex 7 was
Ru
obtained in 65% yield.
Ru
Attachment of an organic
group in the 11b position often
affects recognition.[10] The syn-
thesis of 17a-(ruthenocenyl-
ethynyl)-11b-methoxyestradiol
THF, -78¡C
HO
HO
10
11
Scheme 3. Synthesis of 17a-(ruthenocenylethynyl)-11b-methoxyestra-1,3,5(10)-trien-3,17b-diol (11).
(11)(Scheme 3)utilizes dipro-
the benzyl and the glycoxy ethylene by hydrogenation and by
action of HCl gives 11b-methoxy estrone 10. This is finally
allowed to react with ethynylruthenocenyl lithium to give 11,
with a 60% yield for this final step.
tected 11b-hydroxy-estrone 8. Compound 8, obtained by the
method described by Zeicher and Quivy,[15] is allowed to react
with NaH and then with an excess of MeI to give, in the first
instance, the methoxy derivative 9. Successive deprotection of
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Chem. Eur. J. 2002, 8, No. 22

Organometallic Estradiol Derivatives
5241 5249
tributylstannyl acetylene to
give 13.[16] Lithiation of 13 fol-
lowed by addition of protected
estrone 3 gave the complex 14
in 60% yield.
HC≡C-SnBu3
DMF, Pd(MeCN)2Cl2
I
H
C
C
W(CO)3Me
12
W(CO)3Me
13
Synthesis of estradiol deriva-
tives of cobalt complexes: The
Co complex 17 was prepared
from estrone in three steps
O
1) Li C C
OH
C
C
W(CO)3Me
THF, -78¡C to RT
2) nBu4NF
W(CO)3Me
(Scheme 5).
Addition
of
HO
tBuMe2SiO
ꢀ
LiC CCH(OEt)2 to estrone
yielded 15. Stirring 15 with
Co2(CO)8 yielded the complex
16. Deprotection of 16 with
HCOOH gave the final product
17. This compound is unusual in
that an aldehyde function re-
mains at the end of the chain;
this offers further applicability.
The Co complex 19 was pre-
14
3
Scheme 4. Synthesis of tungsten complex 14.
OH
O
C
C CH(OEt)2
LiC≡C-CH(OEt)2
THF, -78¡C
HO
15
HO
pared in
a
similar way
(Scheme 6). We found that the
estrone
ꢀ
addition of LiC CCH2OLi,
Co2(CO)8
THF
generated from propargylic al-
cohol, to estrone produced 18 in
low yield. An acceptable yield
of 18 (47%)was obtained by
OH
OH
C
C
CHO
C
C
CH(OEt)2
Co2(CO)6
Co2(CO)6
ꢀ
adding LiC CCH2OLi to pro-
HCOOH
CH2Cl2
tected estrone 3, followed by
deprotection with nBu4NF. Stir-
ring 18 with Co2(CO)8 yielded
the complex 19.
HO
HO
17
16
Scheme 5. Synthesis of cobalt complex 17.
The addition reaction of an
organolithium to estrone to
give compounds 5, 11, 14, 15,
and 18 is theoretically a diaster-
eogenic reaction. However due
to the particular structure of
estrone, and especially to the
presence of the methyl group in
position 13b, this reaction is
stereospecific and yields almost
exclusively the 17a isomer.
This behavior is well known in
this series.[10, 17] NMR studies
show the presence of only one
diastereoisomer; this is con-
sistent with the X-ray crys-
tallographic analysis of com-
pound 5.
OH
O
C
C CH2OH
1) LiC≡C-CH2OLi
THF, -78¡C
2) nBu4NF
HO
18
tBuMe2SiO
3
Co2(CO)8
THF
OH
C
C
CH2OH
Co2(CO)6
HO
19
Scheme 6. Synthesis of cobalt complex 19.
Synthesis of estradiol derivatives of tungsten complexes:
Scheme 4 shows the synthetic route for the tungsten complex
14. (Iodocyclopentadienyl)(methyl)tungsten tricarbonyl, 12,
was first prepared by addition of an excess of iodine to lithium
(cyclopentadienyl)(methyl)tungsten tricarbonyl. Compound
12 was then allowed to undergo a Stille coupling reaction with
X-ray crystal structure of 5: Determination of the structure of
the modified hormone is very useful in the later analysis of
receptor binding. Compound 5 gave crystals suitable for an
X-ray crystallographic-structural determination, and the data
are given in Table 1. The ORTEP diagram of 5 is shown in
Figure 1.
Chem. Eur. J. 2002, 8, No. 22
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G. Jaouen, S. Top et al.
FULL PAPER
Table 1. Summary of crystallographic data for 5.
Formula
Mw
C30H32O2Ru
525.6
a [ä]
7.587(2)
b [ä]
c [ä]
V [ä3]
Z
16.540(3)
19.344(6)
2427(1)
4
crystal system
space group
orthorhombic
P212121
6.56
m [cmÀ1
]
1 [gcmÀ3
]
1.44
diffractometer
radiation
scan type
Enraf-Nonius CAD4
MoKa (l ˆ 0.71069 ä)
w/2q
scan range [8]
q limits [8]
0.8‡0.345 tgq
Figure 1. X-ray structure of 5.
1 24
T
RT
Octants collected
No of data collected
No of unique data collected
No of unique data used for refinement
R ˆ S j jFo jÀj Fc j j/S j Fo j
0.8, 0.18, 0.22
2212
with those of 20[10] and 21[19] determined previously by us
(Figure 2). In the case of 21, in which the organometallic
moiety (h5-C5H4)RuCp is attached without a spacer in
position 17a, the ruthenocenyl group is twisted towards the
opposing face of the steroidal skeleton, probably owing to the
steric effect of the neighboring D ring. In compound 5, on the
other hand, the existence of the ethynyl spacer eliminates this
problem. The ruthenocenyl group can turn freely and prefers
a position almost directly below the D ring, probably for
reasons of compactness. In contrast, there is a great structural
similarity between 5 and 20. As in the case of 20, the ethynyl
linkage is not perfectly linear, and shows a slight deformation.
In fact the C(17)-C(19)-C(20) angle proves to be 177.08 (20)
and the C(19)-C(20)-C(21) angle is 174.78 (20).
2188
959 (Fo)2 > 3s(Fo)2
0.0539
0.0624
Rw ˆ {Sw(jFo jÀj Fc j )2/SwFo2}1/2
[a]
absorption correction
extinction parameter
goodness of fit (s)1.17
No. of variables
DIFABS (min ˆ 0.87, max ˆ 1)
none
149
D1min [eäÀ3
D1max [eäÀ3
]
]
À 0.47
0.62
[a] w ˆ w'[1-((j j Fo jÀj Fc j j)/6s(Fo))2]2 with w' ˆ 1/S1ArTr(X)with three coeffi-
cients 3.79, À0.810, and 2.74 for a Chebyshev Series, for which X is Fc/Fc(max).
The structure of 5 shows that the steroid×s skeletal arrange-
ment is not noticeably different from that of 17b-estradiol.[18]
Structural analysis confirmed the position of the ruthenocenyl
group at 17a. It is interesting to compare the structure of 5
Biochemical studies: At this point it is important to establish
the affinity of the modified hormones for the estradiol
Figure 2. X-ray structures of 20 and 21.
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Organometallic Estradiol Derivatives
5241 5249
receptor (ER). Measurement of Relative Binding Affinities
(RBA)was performed by using the previously published
method[8d] based on competition between the modified
hormone and tritium-labeled 17b-estradiol. The values ob-
tained are summarized in Table 2. By definition, the RBA
value of 17b-estradiol is 100%.
firmed for compound 18, in which the organic group CH2OH
is attached to the terminal carbon. An RBA of 19% was found
in this case. On the other hand, when steric hindrance is
introduced close to the D ring by complexation of the triple
bond by a Co2(CO)6 group, as in compound 19, the RBA is
again reduced, to 3.3%. This matches the result for 17
(RBA ˆ 2.9%).
We also measured the lipophilic value (logPo/w)of these
complexes, since this value is an indication of the facility with
which compounds can cross the cellular membrane. The
values found for representative complexes are listed in
Table 3. As observed previously,[10, 19] it can be seen that all
Table 2. Relative binding affinity (RBA)of the compounds for the
estrogen receptor. R2 ˆ H, except for 11 (R2 ˆ MeO); R3 ˆ OH, except
for 4 (R3 ˆ tBuMe2SiO).
Table 3. Partition coefficients (logPo/w)of some 17 a-estradiol deriva-
tives.[a]
Compound
logPo/w
Compound
R1
RBA
[08C, 3 h]
estradiol
5
14
22
3.3
5.3
4.9
5.3[b]
17b-estradiol
4
H
100
0
1
5
ꢀ
R ˆ C C-(h -C5H4)RuCp
R3 ˆ tBuMe2SiO
5
[a] Octanol/water partition coefficient (logPo/w)were determined by the
HPLC method as described in ref. [10]. [b] Value from ref. [10].
ꢀ
5
7
11
14
C C-(h -C5H4)RuCp
13.5
1.0
19.5
CH2-(h5-C5H4)RuCp
1
5
2
ꢀ
R ˆ C C-(h -C5H4)RuCp, R ˆ MeO
5
ꢀ
C C-(h -C5H4)W(CO)3(Me)17
the organometallic hormones are more lipophilic than estra-
diol. There is no noticeable variation between the three
metals Ru, W, and Re. These values appear to be a good
compromise for a potential pharmaceutical product. They are
high enough to allow the product to enter the cell, and, being
under 6, they are also not too high–higher values could cause
them to be retained preferentially by fatty tissues in vivo, thus
making it more difficult for them to reach their target tissues.
17
18
19
2.9
19
ꢀ
C CCH2OH
3.3
5
16[a]
0.8[a]
ꢀ
22
23
C C-(h -C5H4)Re(CO)3
CH2-(h5-C5H4)Re(CO)3
[a] Value from ref. [10].
It is known that the hydroxyl in position 3 forms a hydrogen
bond with residues Glu353 and Arg394 of the receptor, and
that elimination of this phenol function lowers recognition
considerably. This observation is confirmed again here by the
affinity of 4, in which the hydroxyl in position 3 has been
exchanged for a silylated ether. This compound elicits no
recognition by the receptor. In 5, the regeneration of the
hydroxyl in position 3 increased the RBA to 13.5%. This value
can be compared to that of the tungsten compound 14, which
differs from 4 only in the organometallic group. The affinity of
14 for the receptor is slightly higher (17%)but closely
comparable to that of 5. The presence of a methoxy in the 11b
position, as in compound 11, increases the RBA from 13.5%
for 5 to 19.5% for 11. Conversely, replacing the rigid ethynyl
spacer by methylene in 7 strongly decreases recognition,
giving an RBAvalue of only 1%. This result confirms those we
obtained with the rhenium complexes 22 and 23.[10] These
complexes gave RBA values of 16% and 0.8%, respectively.
In light of these results it can be seen that a neutral
organometallic moiety attached to the terminal carbon of
the 17a-ethynyl group of estradiol is well tolerated by the
receptor. Within certain limits, the particular metal group
used (Ru, W, Re)does not seem to be an important factor,
since the RBA values of the compounds all fall within a
narrow range (13.5 20%). This observation was also con-
Molecular modeling: The X-ray crystallographic-structural
determination of the Ligand Binding Domain (LBD)of
estrogen receptor a with estrogenic and antiestrogenic ligands
attached at the binding site has recently become available.[20]
Since the crystal structure of the organometallic bioligand 5
has been obtained here, we were able to perform molecular
modeling studies to attempt to visualize the differences in
receptor binding between 5 and 7, and thus perhaps account
for their great difference in affinity.
The choice of the human ER (hERa)site, initially occupied
by hydroxytamoxifen,[20b] was dictated by the fact that it is the
only cavity described as being large enough to allow
calculation of the correlations. Since deformation of the
corresponding protein only impacts the 11b position of
estradiol, this has little effect on the amino acid residues of
the protein chain at 17a of estradiol, and it is the latter that is
the subject of this study.
Mac Spartan Pro software was used for the molecular
modeling.[21] Only the amino acids forming the wall of the
cavity were retained. Hydroxytamoxifen was removed and
replaced successively by the bioligands under study. The
position of the bioligand was energetically optimized, with all
the heavy atoms (i.e., all except hydrogen)in the cavity
immobilized. Then the side chain of the amino acid His524
was released. This was justified in view of the fact that this
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5245

G. Jaouen, S. Top et al.
FULL PAPER
part of the cavity has been shown to be flexible.[22] Energy
minimization was then carried out, with all the heavy atoms
immobilized except for those of the mediator and of the side
chain of His524, by using the Merck molecular force field
(MMFF).[21] This established the ideal position for the
bioligand. We then established the affinity of the hormone
for its cavity by semiempirical PM3 quantum-mechanical
methods. This involved calculation of the overall bioligand-
cavity energy as well as the individual bioligand and cavity
energies, the last two conserving the conformation they had in
the complete molecule. This allows calculation of the DrH8
enthalpy variation of the reaction:
bioligand ‡ protein binding site ! supramolecular complex
For 17a-ruthenocenylethynylestradiol 5, the enthalpy varia-
tion is 1.6 kcalmolÀ1, a low endothermic value that favors
association. The molecular model obtained with the Molview
program[23] (Figure 3)shows that there is a constricted area in
the cavity at the ethynyl level, but the narrow structure of the
alkyne is a good fit and allows the organometallic complex to
pass comfortably through the bottleneck.
Figure 4. 17a-ruthenocenylmethylestradiol (7)in the hER a cavity. The
hormone 7 is represented as a space-filling model and the amino acids as
rods. The amino acid His524 is clearly visible on the right of the cavity. Note
the steric hindrance between the -CH2(h5-C5H4)RuCp group and the
receptor residues neighboring His524.
Figure 5. 17a-ethynyl-estradiol in the hERa cavity. The hormone is
represented as a space-filling model and the amino acid as rods. The
amino acid His524 is clearly visible on the right hand of the cavity. It can be
seen here that the ethynyl group at the 17a position does not present any
steric constraint at this level, near to the narrow hydrophobic channel.
Figure 3. 17a-ruthenocenylethynylestradiol (5)in the hER a cavity. The
bioligand 5 is represented as a space-filling model and the amino acids as
rods. The His524 amino acid is clearly visible on the right of the cavity. The
ethynylruthenocenyl group is also bordered in its lower side by two
hydrophobic amino acid residues Met343 and Met421. A shrinkage, which
is well adapted to accommodate the rigid ethynyl group, can be clearly seen
in front of the 17a-position of 5; this allows the ruthenocenyl group to avoid
steric constraints inside the cavity. This model must be similar for related
structures.
The software used does not permit calculation of the free
enthalpy variation, DrG8, or of the entropic variation DrS8.
However, the two DrH8 energy values found for 5 and 7 are so
different that it is possible to conclude that the difference in
affinities correlates well with the RBAvalues, and thus to have
confidence in the observed experimental results. In addition,
several authors have stressed the importance of the hydrogen
bond between the carboxylate function of Glu353 and the
phenol group at position 3 of estradiol.[25] According to our
modeling studies, this length for compounds 5 and 7 is,
respectively, 2.72 and 2.82 ä. This lengthening also reflects
the steric hindrance at the other end of the organometallic
steroid. For ethynyl estradiol, which exhibits no steric
constraint, this distance is 1.77 ä.
Conversely, for 17a-ruthenocenylmethylestradiol, 7, the
enthalpy variation 12.3 kcalmolÀ1, an endothermic value that
is unfavorable for association. The molecular model (Fig-
ure 4)shows that the bottleneck mentioned earlier is a bad fit
for the methylene, which is not linear. This is aggravated by
the closeness of the bulky ruthenocenyl group, which steri-
cally hinders this part of the receptor.
Finally, Figure 5 shows 17a-ethynylestradiol in the cavity. It
can be seen that there are minimal steric constraints, as
suggested by the RBA value of 107%.[24]
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Organometallic Estradiol Derivatives
5241 5249
Ethynylruthenocene 2: POCl3 (0.918 g; 6 mmol)was dissolved in DMF
(4 mL)at 0 8C. A solution of acetyl ruthenocene (0.546 g, 2 mmol)in DMF
(8 mL)was added dropwise to this solution. The mixture was stirred at 0 8C
for 15 min and then at room temperature for 2 h. The resulting orange
solution was poured into a MeCOONa solution (20%, 100 mL). After 1 h
of stirring, the solution had turned yellow. The product was extracted with
dichloromethane. After drying over magnesium sulfate, the solution was
filtered and concentrated with a rotary evaporator. The crude product
obtained was dissolved in dioxane (40 mL). The solution was heated at
reflux, then NaOH solution (0.5n, 50 mL)was added. The reflux was
maintained for 20 min. After hydrolysis with ice water, neutralization with
Conclusion
In summary, the affinity measurements suggest three trends:
a)If the organometallic, or an organic group, is attached at
ꢀ
the end of a rigid -C C- ethynyl chain at the 17a position
of the steroid, recognition of estrogen receptor a is good
for any type of organometallic group, in this case neutral or
lipophilic.
3
b)If the spacer is shortened to a single flexible sp carbon
1
³10 HCl solution, ether extraction, and solvent removal, the residue was
atom, creating greater mobility for the organometallic
entity at 17a and a greater space-filling volume, affinity is
reduced.
chromatographed on silica gel plates with dichloromethane/pentane (1:6)
as eluent. Ethynyl ruthenocene was finally isolated as a yellow solid
(0.100 g, 20% yield). 1H NMR (200 MHz, CDCl3) d ˆ 4.87 and 4.56 (t, t,
2H; 2H; C5H4), 4.61 (s, 5H; C5H5), 2.66 (s, 1H; CH).
c)If the bulky organometallic moiety is moved towards the D
ring of the steroid, even on a rigid spacer, affinity is again
decreased.
17a-(Ruthenocenylethynyl)estra-1,3,5(10)-trien-3,17b-diol (5): nBuLi
(0.50 mL of a 1.6m solution in hexane, 0.8 mmol)was added to a solution
of ethynylruthenocene (0.200 g, 0.78 mmol)in THF (5 mL)cooled to
À788C. After the mixture had been stirring for 1 h, a solution of 3-tert-
butyldimethylsiloxyestrone 3 (0.200 g, 0.78 mmol)in THF (3 mL)was
slowly added over 30 min. The stirring was continued for 3 h during which
time the temperature was allowed to rise slowly to room temperature.
After hydrolysis with ice water, ether extraction, and solvent removal, the
residue was chromatographed on silica gel plates with ether/pentane (1:2)
as eluent. The first fraction to elute was unreacted ethynylruthenocene
(0.010 g). The second fraction yielded 17a-(ruthenocenylethynyl)estra-
1,3,5(10)-trien-3-tert-butyldimethylsiloxy-17b-ol (0.170 g, 55% yield). The
latter was dissolved in THF (5 mL), and a solution of nBu4NF (2 mL 1m)in
THF was added. After 10 min of stirring and a work-up, the crude product
obtained was purified by TLC chromatography with diethyl ether/pentane
(1:1)as eluent. Finally, 17 a-(ruthenocenylethynyl)estra-1,3,5(10)-trien-
3,17b-diol was isolated in 60% yield. M.p. 2308C; 1H NMR (250 MHz,
CD3COCD3): d ˆ 7.94 (s, 1H; OH), 7.13 (d, J ˆ 8.3 Hz, 1H; H(1)), 6.59 (dd,
J ˆ 8.3 and 2.6 Hz, 1H; H(2)), 6.52 (d, J ˆ 2.6 Hz, 1H; H(4)), 4.80 (t, J ˆ
1.7 Hz, 2H; C5H4), 4.54 (t, J ˆ 1.7 Hz, 2H; C5H4), 4.57 (s, 5H; Cp), 2.78 (m,
2H; H(6)), 0.89 (s, 3H; Me-13); 13C NMR (62.89 MHz, CD3COCD3) d ˆ
155.9 (C(3)), 138.4 (C(5)), 131.9 (C(10)), 127.1 (C(1)), 115.9 (C(4)), 113.6
The results observed are in agreement with the molecular-
modeling studies performed with 5 and 7. On the His524 side,
facing a possible substituent at position 17a, the estrogen
receptor possesses a narrow region that can be turned to
advantage, since it accommodates rigid, narrow substituents
such as an alkyne group. In addition, if a bulky organometallic
moiety is attached to the end of this rigid spacer, the affinity
remains acceptable since, in this case, the organometallic
group lies outside the zone of steric constraint with this part of
the protein. This general pattern should hold true for a whole
series of molecules with this type of structure. To our
knowledge, this is the first molecular-level explanation for
this kind of behavior; it may prove useful in the future in
evaluating factors that influence recognition when designing
customized estrogen vectors for well-defined targets, for
example organometallic radiopharmaceutical products at-
tached to bioligands of this type.[3d, 2f]
ꢀ
(C(2)), 90.8 (C5H4), 80.0 ( C), 82.5 (C(17)), 74.2 74.1, 71.2, 71.1 (C5H4),
ꢀ
72.1 (Cp), 69.7 (C , 50.4 (C(14)), 48.5 (C(13)), 44.9 (C(9)), 40.6 (C(8)),
39.8 33.8 (C(12), C(16)), 30.3 (C(6)), 28.3 27.4 (C(7), C(11)), 23.5
(C(15)), 13.4 (Me-13); MS (70 eV, EI): m/z: 526 (100)[ M ], 508 (21)
‡
‡
[M À H2O], 298 (26), 270 (78), 256 (56): elemental analysis calcd (%) for
C30H32O2Ru: C 68.55, H 6.13; found: C 68.43, H 6.06.
Experimental Section
17a-Ruthenocenylmethylestra-1,3,5(10)-trien-3,17b-diol (7): Ruthenocene
(0.181 g, 0.78 mmol)in THF (5 mL)was cooled to À788C and treated with
nBuLi (0.50 mL of 1.6m solution in hexane, 0.80 mmol). After stirring for
General procedures: All reactions were performed under a dry argon
atmosphere by using standard Schlenk techniques. Re2(CO)10 was pur-
chased from Strem Co., other reagents and solvents were obtained from
Aldrich Chemical Co and Janssen Chemical Co. Solvents were purified by
conventional distillation techniques under argon. IR spectra were recorded
on a Bomem Michelson 100 spectrophotometer. 1H NMR and 13C NMR
spectra were recorded on Bruker AM-250 and AM-200 spectrometers.
Mass spectra were obtained by the ™Service de Spectrometrie de Masse∫ of
the E.N.S.C.P., Paris, and C.N.R.S., Vernaison, France. 11b-Chloromethyl-
1 h,
a solution of spiro-17b-oxiranylestra-1,3,5(10)-trien-3-ol, (0.100 g,
0.35 mmol)in THF (2 mL)was slowly added to the organolithium solution
maintained at À788C. The stirring was continued for 2 h, during which time
the temperature was allowed to rise slowly to room temperature. After
hydrolysis with ice water, ether extraction, and solvent removal, the residue
was chromatographed on silica gel plates with diethyl ether/pentane (4:6)
as eluent to give 17a-ruthenocenylmethylestra-1,3,5(10)-trien-3,17b-diol
(0.117 g, 65% yield. M.p. 1308C, Rf ˆ 0.22; 1H NMR (250 MHz, CDCl3)
d ˆ 7.15 (d, J ˆ 8.4 Hz, 1H; H(1)), 6.63 (dd, J ˆ 8.4 and 2.5 Hz, 1H; H(2)),
6.56 (d, J ˆ 2.5 Hz, 1H; H(4)), 4.65 (m, 2H; C5H4), 4.55 (s, 5H; Cp) 4.52 (m,
2H; C5H4), 2.81 (m, 2H; H(6)), 2.53 and 2.47 (d,d, J ˆ 14.0 Hz, 1H, 1H;
CH2), 0.94 (s, 3H; Me-13); 13C NMR (62.89 MHz, CDCl3) d ˆ 153.5 (C(3)),
138.4 (C(5)), 132.8 (C(10)), 126.6 (C(1)), 115.3 (C(4)), 112.7 (C(2)), 87.6
(Cip of C5H4), 82.6 (C(17)), 73.4 72.7, 70.4, 70.0 (C5H4), 71.1 (5C, Cp), 49.4
(C(14)), 46.5 (C(13)), 43.9 (C(9)), 39.6 (C(8)), 36.6 34.7 31.7 (C(12),
CH2C5H4, C(16)), 29.8 (C(6)), 27.5 26.4 (C(7), C(11)), 23.6 (C(15)), 14.5
estrone and 17b-oxiranylestra-1,3,5(10)-trien-3-ol were provided by Medg-
enix S.A. (h5-C5H4I)Re(CO)3, (h5-C5H4I)Mn(CO)3, (h -C5H4C CH)-
Re(CO)3 and (h -C5H4C CH)Mn(CO)3 were prepared according to the
5
ꢀ
5
ꢀ
literature method.[16]
Acetylruthenocene 1: Acetyl ruthenocene was prepared following the
procedure described by Rausch et al.[14] Ruthenocene (0.600 g, 2.59 mmol)
was dissolved in acetic anhydride (1 mL)and 1,2-dichloroethane (4 mL).
Phosphoric acid (0.2 mL, 85%)was added to this solution. The mixture was
heated at reflux for 15 min. The solution was then hydrolyzed with ice
water, and the product was extracted with dichloromethane. After drying
over magnesium sulfate, the solution was filtered and concentrated with a
rotary evaporator. The crude product was purified by TLC chromatog-
raphy with dichloromethane as eluent to yield acetyl ruthenocene as a
yellow solid (0.400 g, 56% yield). M.p. 1188C; 1H NMR (200 MHz, CDCl3)
d ˆ 5.10 and 4.78 (t, t, 2H; 2H; C5H4), 4.59 (s, 5H; C5H5), 1.58 (s, 3H; Me);
elemental analysis calcd (%)for C 12H12ORu: C 52.74, H 4.43; found: C
52.56, H 4.55. Unreacted ruthenocene (100 mg)was also isolated.
‡
(Me-13); MS (70 eV, EI): m/z: 516 (21)[ M ], 245 (100), 167 (17).
Synthesis of 9: In a flask equipped with a dropping funnel, compound 8
(1.500 g, 3.57 mmol)was dissolved in THF (50 mL)and HMPA (3.7 mL). A
suspension of NaH (0.70 g, 17.8 mmol)in THF (10 mL)was added
dropwise to the solution. After 2 h of stirring at room temperature, an
excess of MeI (15 mL)was added to the mixture. The mixture was then
heated at reflux for 12 h. The solvent was evaporated, and the crude
product was dissolved in dichloromethane. The solution obtained was
Chem. Eur. J. 2002, 8, No. 22
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0947-6539/02/0822-5247 $ 20.00+.50/0
5247

G. Jaouen, S. Top et al.
FULL PAPER
washed with water, dried with MgSO4, and evaporated. The yellow oil
obtained was chromatographed on silica gel plates with diethyl ether/
pentane (1:3)as eluent. Finally, 9 was obtained as a white solid (1.06 g, 71%
yield). M.p. 1208C; 1H NMR (250 MHz, CDCl3) d ˆ 7.2 (d, J ˆ 8.5 Hz, 1H;
H(1)), 6.8 (dd, J ˆ 8.5 and 2.6 Hz, 1H; H(2)), 6.77 (d, J ˆ 2.6 Hz, 1H; H(4)),
5.04 (s, 2H; PhCH2), 4.2 (m, 1H; H(11)), 3.31 (s, 3H; OCH3), 3.89 (m, 4H;
O(CH2)O), 1.10 (s, 3H; Me-18); MS (70 eV, EI): m/z: 434 (3)[ M ], 372 (1),
287 (1); elemental analysis calcd (%) for C 28H34O4Ru: C 77.39, H 7.89;
found: C 76.54, H 8.10.
solvent removal, the residue was chromatographed on silica gel plates with
diethyl ether/pentane (2:3)as eluent. Compound 15 was isolated as a white
solid (0.491 g, 62% yield). M.p. 808C; 1H NMR (250 MHz, CDCl3) d ˆ 7.09
(d, J ˆ 8.5 Hz, 1H; H(1)), 6.65 (dd, J ˆ 8.5 and 2.7 Hz, 1H; H(2)), 6.57 (d,
J ˆ 2.7 Hz, 1H; H(4)), 5.92 (s, 1H; OH-3), 5.39 (s, 1H; CH(OEt)2), 3.86
3.59 (m, 4H; OCH2CH3), 2.77 (m, 2H; H(6)), 1.26 (t, J ˆ 7.1 Hz, 6H;
OCH2CH3), 0.86 (s, 3H; Me-13); 13C NMR (62.89 MHz, CD3CN) d ˆ 153.6,
(C(3)), 138.0 (C(5)), 132.3, (C(10), 126.4 (C(1)), 115.2 (C(4)), 112.7 (C(2)),
91.5 (C(21)), 89.1 or 81.4 (C(19) or C(20)), 79.9 (C(17)), 61.1 (OCH2CH3),
49.7 (C(14)), 47.3 (C(13)), 43.3 (C(9)), 39.3 (C(8)), 38.9 (C(16)), 32.9
(C(12)), 29.6 (C(6)), 27.1 (C(7)), 26.3 (C(11)), 22.8 (C(15)), 15.0 (C(18)),
‡
Synthesis of 10: Compound 9 (0.500 g, 1.15 mmol)was dissolved in THF
(18 mL). Pd/C (10%, 0.25 g) was added, and the flask was filled with
hydrogen at atmospheric pressure. The mixture was stirred overnight. After
filtration and solvent removal, the crude compound 10 obtained was
dissolved in methanol (40 mL), and concentrated aqueous HCl (9 mL)
solution was added. The mixture was heated at reflux for 1 h. The solution
was concentrated, and then ethyl acetate (100 mL)was added. The solution
was washed with water, dried over MgSO4, and evaporated. The crude
product obtained was recrystallized from dichloromethane/heptane to give
‡
‡
12.7 (OCH2CH3); MS (70 eV, EI): m/z: 398 (67)[ M ], 354 (100)[ M
EtOH]; elemental analysis calcd (%)for C 25H34O4: C 75.34, H 8.60; found:
C 75.27, H 7.52.
À
ꢀ
17a-[(C CH(OEt)2)(Co2(CO)6)] estradiol (16): A solution of 15 (0.395 g,
1 mmol)in diethyl ether (5 mL)was added to the solution of Co 2(CO)8
(0.384 g, 1.12 mmol)in diethyl ether (5 mL). After 2 h of stirring at room
temperature, the solvent was evaporated. The crude product was chroma-
tographed on silica gel column with diethyl ether/pentane (2:3)as an
eluent. Compound 16 was isolated as a red solid (0.507 g, 74% yield).
Decomp. 1708C; 1H NMR (250 MHz, CDCl3) d ˆ 7.15 (d, 1H; H(1)), 6.62
(dd, 1H; H(2)), 6.57 (d, 1H; H(4)), 5.54 (s, 1H; CH(OEt)2), 4.67 (s, 1H;
OH-3), 3.86 3.59 (m, 4H; OCH2CH3), 2.83 (m, 2H; H(6)), 1.32 and 1.31 (t,
t, 6H; OCH2CH3), 1.05 (s, 3H; Me-13); 13C NMR (62.89 MHz, CDCl3) d ˆ
199.5 (Co2(CO)6), 153.4 (C(3)), 138.2 (C(5)), 132.6 (C(10)), 126.3 (C(1)),
115.2 (C(4)), 112.6 (C(2)), 103.7 (C(21)), 103.2 and 96.1 (C(19) and C(20)),
86.1 (C(17)), 65.8 (OCH2CH3), 49.9 (C(14)), 48.5 (C(13)), 42.8 (C(9)), 40.0
(C(8)), 39.5 (C(16)), 32.4 (C(12)), 29.5 (C(6)), 27.4 (C(7)), 26.2 (C(11)), 23.4
(C(15)), 15.5 (C(18)), 15.2 (OCH2CH3); IR (KBr) nÄCO ˆ 2093, 2055,
1
10 as white crystals (0.32 g, 92% yield). M.p. 2608C; H NMR (250 MHz,
CDCl3) d ˆ 7.00 (d, J ˆ 8.5 Hz, 1H; H(1)), 6.60 (dd, J ˆ 8.5 and 2.6 Hz, 1H;
H(2)), 6.57 (d, J ˆ 2.6 Hz, 1H; H(4)), 4.20 (m, 1H; H(11)), 3.32 (s, 3H;
‡
OCH3), 1.11 (s, 3H; Me-18); MS (70 eV, EI) m/z: 300 (13)[ M ], 241 (8),
197 (8), 170 (21), 157 (31), 146 (76); elemental analysis calcd (%) for
C19H24O3: C 75.97, H 8.05; found: C 75.01, H 8.26.
Synthesis of 11: The synthetic procedure is similar to that for 17a-
(ruthenocenylethynyl)estra-1,3,5(10)-trien-3,17b-diol. Compound 11 was
1
obtained in 60% yield. M.p. 1608C; H NMR (250 MHz, CDCl3) d ˆ 7.03
(d, J ˆ 8.6 Hz, 1H; H(1)), 6.64 (dd, J ˆ 8.6 and 2.7 Hz, 1H; H(2)), 6.54 (d,
J ˆ 2.7 Hz, 1H; H(4)), 4.80 (t, J ˆ 1.5 Hz, 2H; C5H4), 4.53 (t, J ˆ 1.5 Hz,
2H; C5H4), 4.55 (s, 5H; Cp), 3.31 (s, 3H; CH3O), 2.80 (m, 2H; H(6)), 1.08
(s, 3H; Me-13); 13C NMR (62.89 MHz, CDCl3) d ˆ 153.26 (C(3)), 138.9
(C5), 129.0 (C(10), 126.9 C(1)), 115.7 (C(4)), 113.7 (C(2)), 88.8 (C5H4), 83.6
‡
2030 cmÀ1; MS (ElectroSpray): m/z: 707 (92)[ M ‡Na]; elemental analysis
calcd (%)for C 31H34O10Co2: C 54.39, H 5.01; found: C 54.57, H 5.13.
ꢀ
17a-[(C CHO)(Co2(CO)6)]estradiol 17: Compound 16 (0.300 g,
0.44 mmol)was dissolved in dichloromethane (5 mL. ) Formic acid
(0.312 g, 6.78 mmol)was added, and the mixture was stirred at room
temperature for 2 h. After hydrolysis with water, extraction with dichloro-
methane, and solvent removal, 17 (0.300 g)was isolated. Crystallization
from diethyl ether/pentane gave red crystals. M.p. 1608C; 1H NMR
(250 MHz, CDCl3) d ˆ 10.37, (s, 1H; CHO), 7.13 (d, 1H; H(1)), 6.62 (dd,
1H; H(2)), 6.57 (d, J ˆ 2.7 Hz, 1H; H(4)), 4.70 (s, 1H; OH-3), 2.83 (m, 2H;
H(6)), 0.89 (s, 3H; Me-13); 13C NMR (62.89 MHz, CDCl3) d ˆ 198.1
(Co2(CO)6), 191.4 (CHO), 153.4, (C(3)), 138.0 (C(5)), 132.2, (C(10)), 126.4
(C(1)), 115.2 (C(4)), 112.7 (C(2)), 105.1 and 87.6 (C(19) and C(20)), 86.3
(C(17)), 50.1 (C(14)), 48.7 (C(13)), 42.8 (C(9)), 42.2 (C(8)), 39.6 (C(16)),
32.5 (C(12)), 29.4 (C(6)), 27.4 (C(7)), 26.0 (C(11)), 23.3 (C(15)), 15.4
(C(18)); IR (KBr) nÄCO ˆ 2101, 2064, 2034 cmÀ1; MS (ElectroSpray): m/z:
ꢀ
ꢀ
(C ), 80.5 (C(17)), 76.8 (C(11), 73.8 73.7, 70.7 (3C of C5H4), 68.1 (C ),
71.72 (Cp), 56.4 (OCH3), 50.6 (C(14)), 49.2 (C(9)), 47.7 C(13)), 39.0 (C(8)),
34.7 (C(16)), 33.0 (C(12)), 29.7 (C(6)), 27.5 (C(7)), 22.8 (C(15)), 14.0 (Me-
‡
‡
13); MS (70 eV, EI) m/z: 556 (100)[ M ], 538 (27)[ M À H2O], 298 (19),
256 (25).
(Iodocyclopentadienyl)(methyl)tungsten tricarbonyl 12: CpW(CO)3Me
(0.522 g, 1.5 mmol)was dissolved in THF (12 mL.) The solution was
cooled to À608C, and nBuLi (2 mL, 1.3m, 2.6 mmol)was added. The
mixture was stirred at À608C for 1 h. Iodine (0.635 g 2.5 mmol)was then
added in one portion. The stirring was maintained for 1 h, during which
time the temperature was allowed to rise slowly to room temperature. The
solvent was evaporated, and the crude product obtained was chromato-
graphed on silica gel plates with diethyl ether/pentane (1:10)as eluent.
(Iodocyclopentantadienyl)(methyl)tungsten tricarbonyl was isolated as a
yellow oil (0.300 g 42% yield). 1H NMR (200 MHz, CDCl3) d ˆ 5.53 (t, J ˆ
2.3 Hz, 2H; C5H4), 5.30 (t, J ˆ 2.3 Hz, 2H; C5H4), 0.54 (s, 3H; Me); IR
‡
633 (76)[ M ‡Na].
[26]
ꢀ
17a-(C CH2OH) estradiol 18: nBuLi (2.80 mL of a 2.5m solution in
ꢀ
hexane, 7 mmol)was added to a solution of HC CH2OH (0.336 g, 6 mmol)
(CH2Cl2) nÄCO ˆ 2016, 1922 cmÀ1
.
in THF (25 mL)cooled to À608C. After the mixture had been stirred for
30 min, the cooling bath was removed for 10 min, and then the solution was
cooled again to À788C. A solution of protected estrone 3 (0.384 g, 1 mmol)
in THF (10 mL)was slowly added over 1 h. The stirring was continued
overnight, during which time the temperature was allowed to rise slowly to
room temperature. A solution of nBu4NF (1m, 1 mmol)in THF (1 mL)was
then added, and the stirring was maintained for 10 min. After hydrolysis
with ice water, neutralization with 10% HCl solution, extraction with
dichloromethane, and solvent removal, the residue was chromatographed
on silica gel column with diethyl ether as eluent. Compound 18 was isolated
as a colorless solid (0.152 g, 47%). M.p. 2288C; 1H NMR (200 MHz,
[D6]acetone) d ˆ 8.01 (s, 1H; OH), 7.09 (d, J ˆ 8.4 Hz, 1H; H(1)), 6.58 (dd,
J ˆ 8.4 and 2.7 Hz, 1H; H(2)), 6.51 (d, J ˆ 2.7 Hz, 1H; H(4)), 4.23 (s, 2H;
CH2OH), 2.77 (m, 2H; H(6)), 0.87 (s, 3H; Me-13); MS (70 eV, EI): m/z: 326
5
ꢀ
Synthesis of HC C(h -C5H4)W(CO)3Me (13): The procedure was similar
5
ꢀ
to that for ethynyl ruthenocene. HC C(h -C5H4)W(CO)3Me was obtained
in 60% yield. 1H NMR (200 MHz, CDCl3) d ˆ 5.55 (t, J ˆ 2.3 Hz, 2H;
C5H4), 5.30 (t, J ˆ 2.3 Hz, 2H; C5H4), 2.96 (s, 1H; CH), 0.54 (s, 3H; Me); IR
(CH2Cl2) nÄCO ˆ 2017, 1922 cmÀ1
.
Synthesis of tungsten complex 14: The procedure was similar to that for
17a-(ruthenocenylethynyl)estra-1,3,5(10)-trien-3,17b-diol. Complex 14
1
was obtained in 60% yield. M.p. 2128C. H NMR (250 MHz, CDCl3) d ˆ
7.19 (d, J ˆ 8.4 Hz, 1H; H(1)), 6.61 (dd, J ˆ 8.4 and 2.1 Hz, 1H; H(2)), 6.55
(d, J ˆ 2.1 Hz, 1H; H(4)), 5.53 (t, 2H; C5H4), 5.31 (t, 2H; C5H4), 2.82 (m,
2H; H(6)), 0.91 (s, 3H; Me-13), 0.56 (s, 3H; Me-W); IR (CH2Cl2) nÄCO
ˆ
‡
‡
2017, 1922 cmÀ1; MS (70 eV, EI): m/z: 642 (33)[ M ], 558 (69)[ M À 3CO];
elemental analysis calcd (%)for C 29H30O5W: C 54.22, H 4.71; found: C
54.03, H 4.91.
‡
(12)[ M ], 308 (6), 293 (10), 270 (6); elemental analysis calcd (%) for
C21H26O3.H2O: C 73.22, H 8.19; found: C 73.00, H 8.39.
ꢀ
ꢀ
17a-[C CH(OEt)2] estradiol (15): nBuLi (2.50 mL of a 2.5m solution in
17a-[(C CH2OH)(Co2(CO)6)] estradiol 19: Compound 18 (0.152 g,
ꢀ
hexane, 6 mmol)was added to a solution of H-C CH(OEt)2 (0.769 g,
0.47 mmol)was dissolved in THF (3 mL,) and Co
2(CO)8 (0.161 g,
6 mmol)in THF (30 mL)cooled at À708C. After the mixture had been
stirred for 1.5 h, a solution of estrone (0.540 g, 2 mmol)in THF (25 mL)was
slowly added over 2 h. The stirring was continued overnight, during which
time the temperature was allowed to rise slowly to room temperature.
After hydrolysis with ice water, extraction with dichloromethane, and
0.47 mmol)was added to the solution. The mixture turned red after it
had been stirred for 20 min. The solution was then filtered over a 1 cm-
thick silica gel pad. After evaporation of solvent, the crude product was
obtained as a red oil (0.266 g). After crystallization in diethyl ether/
pentane, 19 was isolated as red crystals (0.065 g, 23%). Decomp. 1708C;
5248
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0947-6539/02/0822-5248 $ 20.00+.50/0
Chem. Eur. J. 2002, 8, No. 22

Organometallic Estradiol Derivatives
5241 5249
1H NMR (200 MHz, [D6]acetone) d ˆ 7.95 (s, 1H; OH), 7.09 (d, J ˆ 8.4 Hz,
1H; H(1)), 6.58 (dd, J ˆ 8.4 and 2.8 Hz, 1H; H(2)), 6.51 (d, J ˆ 2.8 Hz, 1H;
H(4)), 4.97 (m, 2H; CH2OH), 2.83 (m, 2H; H(6)), 1.08 (s, 3H; Me-13); MS
(ElectroSpray): m/z: 635 (100)[ M ‡Na].
¡
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¡
‡
¡
Determination of the relative binding affinity (RBA) of the complexes for
the estrogen receptor alpha: Sheep-uterine cytosol prepared as previously
described[8d] was used as the source of ERa. Aliquots (200 mL)were
incubated for 3 h at 08C with 2 Â 10À9 m of [6,7-3H]-estradiol (2 Â 10À9 m,
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¡
¡
¡
¡
¬
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