7alpha- and 17alpha-substituted estrogens containing tridentate tricarbonyl rhenium/technetium complexes: synthesis of estrogen receptor imaging agents and evaluation using microPET with technetium-94m.

Article abstract of DOI:10.1016/j.bmc.2003.09.004

To develop technetium and rhenium-labeled imaging agents for estrogen receptor (ER) positive breast tumors, we have prepared tridentate metal tricarbonyl chelates substituted at the 7alpha- and 17alpha-positions of estradiol. Some of the Re(CO)(3) conjuga

Full text of DOI:10.1016/j.bmc.2003.09.004

Bioorganic & Medicinal Chemistry 11 (2003) 4977–4989
7ꢀ- and 17ꢀ-Substituted Estrogens Containing Tridentate
Tricarbonyl Rhenium/Technetium Complexes: Synthesis of
Estrogen Receptor Imaging Agents and Evaluation Using
MicroPET with Technetium-94m
Leonard G. Luyt,a Heather M. Bigott,b Michael J. Welchb
and John A. Katzenellenbogena,*
aDepartment of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, IL 61801, USA
bMallinckrodt Institute of Radiology, Washington University School of Medicine, 510 S. Kingshighway, Campus Box 8225,
St. Louis, MO 63110, USA
Received 10 March 2003; revised 14 August 2003; accepted 3 September 2003
Abstract—To develop technetium and rhenium-labeled imaging agents for estrogen receptor (ER) positive breast tumors, we have
prepared tridentate metal tricarbonyl chelates substituted at the 7a- and 17a-positions of estradiol. Some of the Re(CO)3 conjugates
have high binding for the ER in vitro. The in vivo biodistribution of the highest affinity of these novel metal tricarbonyl conjugates,
prepared as the 94mTc labeled analogue, was evaluated by tissue dissection and microPET imaging. Although target tissue-selective
uptake was not apparent, it is notable that microPET imaging identified the stomach as a major site of activity deposition, a site
that might have been missed by standard tissue distribution studies.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
the clinical effectiveness of hormone therapy with
tamoxifen.15,16
Selecting the most effective therapy for women with
breast cancer requires early detection and accurate sta-
ging of the disease. This includes determining the estro-
gen receptor (ER) status of the tumor, because hormone
therapy with antiestrogens is beneficial only in tumors
that are ER positive (ER+).1 Imaging of the ER in vivo
using an ER binding radiopharmaceutical has the
potential for determining the ER status of all tumor
sites simultaneously and non-invasively. Previously, we
and others have labeled ER ligands with fluorine-18
and other cyclotron-produced short-lived radionuclides
to develop such in vivo imaging agents.2ꢀ14 In parti-
cular, we have used 16a-[18F]fluoro-estradiol (FES) to
image primary and metastatic breast tumors, and we
have shown that these images have value in predicting
Short-lived, cyclotron-produced radionuclides, how-
ever, are expensive and are not widely available, and
this limits the utility of ER imaging agents such as FES
for routine clinical studies. By contrast, technetium-99m
is the most commonly used radioisotope in nuclear
medicine, because of its favorable nuclear emission
characteristics and its wide availability at low cost from
a convenient generator system. A number of attempts
have been made to conjugate technetium (or its non-
radioactive congener rhenium) with estradiol, the nat-
ural ligand for the ER, to create an agent suitable for
imaging ER in vivo that would be more widely available
than FES.17ꢀ26 Many of these estradiol complexes
showed excellent in vitro binding to the ER; however,
none of them gave favorable tissue distribution in vivo.
Recently, organometallic technetium and rhenium com-
plexes in low oxidation states have been described and have
been applied in receptor-targeted radiopharmaceutical
*Corresponding author. Tel.:+1-217-333-6310; fax:+1-217-333-7325;
e-mail: jkatzene@uiuc.edu
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.09.004

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L. G. Luyt et al. / Bioorg. Med. Chem. 11 (2003) 4977–4989
development.24,25 Advances in the preparation of the
technetium-99m tricarbonyl core from generator-pro-
duced pertechnetate has provided a method of radi-
olabelling using a convenient aqueous-based kit.27
The organometallic precursor formed is fac-
Results and Discussion
Tridentate chelation units to form rhenium(I) and techne-
tium(I) tricarbonyl complexes
Two novel chelation systems were prepared to investi-
gate estrogens containing rhenium or technetium tri-
carbonyl with a tridentate chelation core. The first
chelate utilizes iminodiacetic acid and a thioether, the
synthesis of which is described in Scheme 1. This chel-
ator, prepared via an alternate synthetic route, has been
reported in the literature, but it was used to form
Tc(V)–oxo complexes, not as a tridentate chelator for
M(I) tricarbonyl complexes (M=Tc or Re).42 Similar
chelators utilizing iminodiacetic acid as part of a che-
lator system were reported by Schibli et al., but they did
not include a thioether as part of the chelation unit.33
Thioether ligands are believed to be strong coordinating
ligands for the metal(I) carbonyl center, due to their p-
accepting properties.43ꢀ47
[
99mTc(OH2)3(CO)3]+, a stable, water-soluble precursor
that can be readily coordinated to a variety of ligand
systems via substitution of the coordinated water mol-
ecules.28,29 Similarly, the organometallic aqua species
fac-[Re(OH2)3(CO)3]+ can be readily formed,30,31 and it
may hold promise for application using the therapeutic
radionuclide 188Re.32
We are interested in exploring the use of tridentate
chelators, conjugated to estradiol, that would enable us
to form Tc(I) or Re(I) tricarbonyl complexes as estra-
diol–radiometal complexes. When appropriately
designed, these estradiol complexes would be stable and
neutral, and they would have a substantially different
lipophilicity than previously reported estradiol com-
plexes containing the Tc(I) or Re(I) tricarbonyl moiety.
In addition, it has been reported in other systems that
such tridentate complexes have better pharmacokinetic
characteristics than bidentate complexes; the latter suffer
from interaction with cellular nucleophiles through their
labile third coordination site.33 As is typically the case,
in these exploratory studies we have used rhenium as a
congener for technetium to facilitate the complete char-
acterization and safe handling of the estrogen deriva-
tives, and the measurement of their binding affinity to
the ERs.
The synthesis starts with the reaction of iminodiacetic
acid with ethyl chloromethyl sulfide under basic condi-
tions, giving material, isolated first as a barium salt, and
then liberated with sulfuric acid to furnish the ami-
nothioether diacid 1 in 46% yield. This material is ready
for conjugation through amide coupling of one of the
carboxylic acid groups to an amino group on the bio-
molecule. To confirm the metal chelating ability of
compound 1, it was complexed with the rhenium tri-
carbonyl cation under aqueous conditions with one
equivalent of base, resulting in the formation of
rhenium(I) tricarbonyl complex 2 in 88% yield.
The generator-produced technetium-99m isotope has
nuclear characteristics (t1/21=2=6 h, Eg=140.5 keV,
Ig=87.2%) that make it an ideal isotope for single
photon emission computed tomography (SPECT).
Technetium-94m has the same chemistry as 99mTc, but has
nuclear characteristics (t1/2=52 min, E+bmax=2.44 MeV,
Ib+=72%) suitable for imaging using positron emis-
sion tomography (PET), with which more quantitative
measurements of radiopharmaceutical uptake, biodis-
tribution and clearance can be made. Such studies have
been performed by imaging small numbers of animals at
multiple time points on a microPET scanner (a small
animal PET imaging device),34 rather than by tradi-
tional, time-consuming tissue dissection biodistribution
studies.35ꢀ41
The second chelation system utilizes a carboxylic acid
and two thioethers, which upon chelation with rhenium
or technetium would give a neutral, and very small
metal complex. This system can provide a complex that
is achiral, which is a substantial advantage, because it
avoids formation of diastereomers when the complex is
attached to a chiral biomolecule. Formation of such
diastereomers has introduced unnecessary stereo-
chemical complications in metal-labeled receptor-binding
radiopharmaceuticals.48ꢀ50
The dithioether chelator was prepared as outlined in
Scheme 2. Glyoxalic acid was reacted with two equiva-
lents of ethanethiol to yield the thioketal acid 3 in 93%
There are two components to the investigations pre-
sented in this report: The first component involves the
development of four estradiol derivatives containing the
rhenium tricarbonyl moiety tethered through two novel
tridentate chelates, one having a typical amide linkage
between the biomolecule (estradiol) and the metal com-
plex, and the other being unique in having an all carbon
linkage, potentially representing a more stable biomol-
ecule-chelator junction. The second component of this
study involves the labeling of the highest affinity estro-
gen–technetium conjugate with technetium-94m. This
has enabled us to perform both in vivo tissue distribu-
tion and microPET imaging studies in the rat, thus
expediting the evaluation of novel technetium-labeled
radiopharmaceuticals.
Scheme 1. Reagents and conditions (yield): (a) NaOH,
ClCH2CH2SCH2CH3, H2O, EtOH; (b) BaCl2, H2O; (c) H2SO4, H2O
(46% three steps); (d) Re(CO)+3 , H2O (88%).

L. G. Luyt et al. / Bioorg. Med. Chem. 11 (2003) 4977–4989
4979
estradiol (estradiol=100%). While the receptor binding
assays indicate that these compounds have reasonably
high ER binding affinity, the behavior of these bio-
conjugates in vivo has proved to be disappointing, with
little or no ER-specific target tissue uptake being evi-
dent. Although the reasons were not fully clear, we
ascribed the poor behavior of these compounds in vivo
to be the result of their large size and in some cases their
very high lipophilicity. Bidentate Tc(I)/Re(I) tricarbonyl
estradiol systems have also been previously repor-
ted,19,53 but they also proved to be poorly behaved in
vivo. In this case, the poor behavior is ascribed to ligand
exchange at the open coordination site, as was men-
tioned earlier. We hoped that by using a tridentate tri-
carbonyl metal complex to label an ER ligand, we might
obtain more effective ER imaging agents in vivo, ones
that would be labeled through a small, stable, neutral,
and moderately lipophilic metal-containing moiety.
Scheme 2. Reagents and conditions (yield): (a) EtSH, TsOH, benzene
(93%); (b) nBuLi, THF; (c) BnBr, THF (68%); (d) Re(CO)+3 , H2O
(79%).
yield. To explore the use of this chelation system for
conjugation to biomolecules, a benzyl group was placed
adjacent to the carboxylic acid. This was accomplished
by forming the anion of 3 followed by addition of ben-
zyl bromide, yielding alkylated product 4 in 68% yield.
While a previously described procedure suggests that a
mixed cation pair (e.g., sodium–lithium) is required for
successful bismetallation of 3,51 we found that the use of
two equivalents of n-butyl lithium gave satisfactory
results when benzyl bromide is the alkylation substrate.
Chelation with the rhenium tricarbonyl cation under
aqueous conditions yielded the complex 5 in 79% yield.
This system has the unique ability to provide a rhenium
or technetium labeled biomolecule in which the linker is
an aliphatic chain, without a more typical amide or ester
linkage to the biomolecule. This may prove to be a more
stable linkage for in vivo applications.
Two 7a-substituted estradiol derivatives have been pre-
pared using a common precursor, a protected 7a-(6-
hydroxyhexan-1-yl)estradiol 6, previously described by
Skaddan et al., which was prepared from estradiol via a
6-keto-estradiol intermediate.19,54,55 Conversion of the
alcohol 6 to the 6-aminohexyl 7 proceeded following the
literature procedure (Scheme 3), with initial Mitsunobu
conditions to produce a phthalimide, and then hydra-
zinolysis to give the amine 7. Carbodimide coupling to
chelator 1 provided conjugate 8 in 62% yield, with
concomitant deprotection of the phenolic hydroxy
group. Complete deprotection was carried out with HF,
providing amine 9 quantitatively . The rhenium(I) tri-
carbonyl complex 10a was then prepared by chelation of
9 with aqueous Re(CO)+3 and one equivalent of base.
The neutral rhenium estrogen conjugate was purified by
column chromatography, yielding the metal complex
10a in 48% yield.
Design and synthesis of 7ꢀ-substituted estradiol derivatives
Previous work from our laboratory and elsewhere has
shown that bulky substituents are well tolerated at the
7a-position of estradiol, without a significant decrease
in ER binding (Fig. 1).19,52 In particular, having a hex-
amethylene spacer between a metal chelator and estra-
diol results in estrogens having relative binding affinities
in vitro that are from 5% to as high as 43% that of
The second 7a-substituted estradiol derivative was
prepared as outlined in Scheme 4. The alcohol 6 was
converted to methanesulfonate 11, as previously
detailed.19 The anion of ethyl bis(ethylthio)acetate
was prepared using potassium t-butoxide in a manner
similar to that described by Lerner et al.56 This
Figure 1. Representative examples of 7a-substituted estradiols with their corresponding relative binding affinity (RBA, estradiol=100%).

4980
L. G. Luyt et al. / Bioorg. Med. Chem. 11 (2003) 4977–4989
Scheme 3. Reagents and conditions (yield): (a) PPh3, DEAD, phthalimide, THF;19 (b) NH2NH2, DME, EtOH;19 (c) 1, EDC, DMAP, DMF,
CH2Cl2 (40%); (d) 40% HF, THF, CH3CN (quant); (e) Re(CO)+3 , H2O (48%); (f) 94mTc(CO)3+, H2O.
Scheme 4. Reagents and conditions (yield): (a) MsCl, NEt3, THF (quant); (b) (CH3CH2S)2CHCO2Et, KOtBu, THF; (c) 40% HF, THF, CH3CN
(30% two steps); (d) NaOH, H2O, MeOH (57%); (e) Re(CO)+3 , H2O (44%).
Design and synthesis of 17ꢀ-ethynyl substituted estradiol
derivatives
carbanion then displaced the methanesulfonate, pro-
viding the conjugated estradiol as a mixture of di-TBS
protected and mono-deprotected material. Attempts to
prepare 12 via the alkylation of the anion of 6-keto-
estradiol with 8-iodo-2,2-bis(ethanthiol)-octanoic acid
ethyl ester, gave a low yield. Complete alcohol deprotec-
tion was carried out using HF, providing the estradiol
conjugate 12 in moderate yield, followed by base saponi-
fication to provide 13. Rhenium complexation provided
the neutral 14, which was purified by flash chromato-
graphy prior to being tested for ER binding affinity.
High affinity ligands for the ER can be designed by
placing a compact, electron-rich substituent, connected
through a rigid linker to estradiol at the 17a-position.52,57
This concept was extended to Tc/Re estradiol conjugates,
examples of which are indicated in Fig. 2.21,24ꢀ26,53
Attaching the unique bisthioether chelator 3 to estradiol
through a rigid alkyne was accomplished by preparing

L. G. Luyt et al. / Bioorg. Med. Chem. 11 (2003) 4977–4989
4981
Figure 2. Representative examples of 17a-substituted estradiols with their corresponding relative binding affinity (RBA, estradiol=100%).
Estrogen receptor binding affinity determination
Table 1 presents the binding affinity of the estrogens
developed in this study. Binding affinities were deter-
mined by competitive radiometric binding assays with
[3H]estradiol, using cytosol preparations of lamb uterus
or full-length human ERa and ERb, as previously
described.58,59 Binding affinities are expressed relative to
that of the standard, estradiol, as relative binding affin-
ity (RBA) values, where the affinity of estradiol is
100%.
The binding affinity of the two 7a-substituted estrogen
rhenium tricarbonyl tridentate complexes, 10a and 14, is
very good, being in the nanomolar range. These affin-
ities are very comparable to previously reported 7a-
substituted estradiol derivatives, which indicates again
the ability of the ER to tolerate a variety of bulky sub-
stituents at this position.19 The binding affinity of these
rhenium-labeled estrogens showed no preference for
either of the two ER subtypes, ERa or ERb. The RBA
values for the rhenium-chelated 17a-ethynyl substituted
estradiol derivatives 21a and 21b are much poorer than
for the 7a-substituted estradiols, and chain extension
from n=1 to n=3 results in a lowering of the RBA.
Overall, the 7a-substituted estrogen rhenium conjugates
proved to be much better ER ligands than the 17a-sub-
stituted ones.
Scheme 5. Reagents and conditions (yield): (a) KOtBu, THF, pro-
pargyl bromide or 15 (16a 58%, 16b 57%); (b) NaOH, H2O, MeOH
(90%); (c) Re(CO)+3 , H2O (49%).
alkynyl–chelator analogues as shown in Scheme 5. We
started with commercially available 2,2-bisthioether
ethyl acetate and reacted the corresponding anion
with propargyl bromide or with 5-iodopentyne to
yield the propargylic dithioketal acetate ester 16. To
test the chelation ability of this alkynyl system, the
ester 16b was converted to the acid 17b in excellent
yield. The rhenium(I) tricarbonyl complex 18b was
then prepared by reaction of the chelate system 17b
with triaquatricarbonylrhenium(I) in an aqueous
solution.
Radiolabelling with technetium-94m, in vivo biodistribu-
tion by tissue dissection and microPET imaging in rats
These two alkynyl compounds provide an excellent
means of preparing 17a-substituted estradiol, with a
built in rigid linker (Scheme 6). The lithium anions of
alkynes 16a and 16b were reacted with TBS-protected
estrone to provide the conjugated estradiols 19a and
19b. Saponification with 3 M NaOH solution gave the
carboxylic acid 20a/20b with concomitant deprotection
Compound 10a, which overall had the highest affinity
for ER in vitro, was chosen for preliminary in vivo bio-
logical assessment. In this manner, we sought to test
whether using a neutral, tridentate M(CO)3 chelation
unit would result in improved uptake in ER-rich tissues
of an animal model. The radiolabelling was carried out
with both 99mTc (data not included) and 94mTc. Tech-
netium-94m is a positron emitter, which allows for
imaging using small-animal PET as well as standard
of
tricarbonylrhenium(I) in aqueous medium provided the
the
phenol.
Chelation
using
triaqua-
rhenium(I) complexes 21a and 21b.

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L. G. Luyt et al. / Bioorg. Med. Chem. 11 (2003) 4977–4989
Scheme 6. Reagents and conditions (yield): (a) 16a or 16b, nBuLi, THF (19a 53%, 19b 46%); (b) NaOH, H2O, MeOH (20a 65%, 20b 78%); (c)
Re(CO)+3 , H2O (21a 29%, 21b 68%).
Table 1. Relative binding affinities (RBA)a of rhenium(I) tricarbonyl
estrogens
was found in the liver, though these values are within
the ranges reported for estradiol compounds labeled
with 18F.2,4ꢀ7
Ligand
RBA (%)
Cytosol
ERa
ERb
MicroPET images of 10b in a mature female Sprague–
Dawley rat, at four times after injection, are shown in
Figure 3. Although activity was evident in large organs,
in these images, we were unable to resolve the smaller
organs, including the target tissues (uterus and ovaries).
This is due to the long positron range distribution of
94mTc, which results in a strong contrast reduction
of the images. Because 94mTc has a transaxial resolution
of 4.3 mm,63 it is necessary to perform tissue bio-
distribution studies in conjunction to the microPET
imaging studies. Work is underway on developing
reconstruction algorithms that may account for these
characteristics and therefore improve image quality of
94mTc-labeled radiopharmaceuticals.
Estradiol
10a
14
21a
21b
100
100
27ꢂ3
100
38ꢂ2
18ꢂ1
1.9ꢂ0.4
0.25ꢂ0.04
0.14ꢂ0.01
36ꢂ9
27ꢂ7
0.26ꢂ0.07
0.22ꢂ0.04
0.14ꢂ0.03
0.072ꢂ0.006
aDetermined by
a competitive radiometric binding assay with
[3H]estradiol, using cytosol preparations of lamb uterus or full-length
human ERa and ERb; see Experimental for details. Values are repor-
ted as the meanꢂrange (n=2) or SD (n>2). Under these conditions,
the Kd for estradiol is 0.2 nM for ERa and 0.5 nM for ERb.
tissue dissection biodistribution analysis. This radio-
nuclide was produced by proton irradiation (approxi-
mately 13 MeV) of an enriched molybdenum target
according to the 94Mo(p,n)94mTc nuclear reaction.60ꢀ62
The 94mTc was separated by sublimation as previously
described,61 and 94mTcOꢀ4 was isolated using a normal
phase Sep-Pak1 cartridge. We were able to prepare
compound 10b, the technetium-94m-labelꢀed analogue of
compound 10a, by first reducing 94mTcO4 with sodium
borohydride in the presence of CO(g) to give the triaqua
cation 94mTc(CO)+3 , and then adding 9 and heating at
80 ꢁC for 20min. This material ( 10b) was shown to be
radiochemically homogeneous and free from chemical
impurities after purification by reverse-phase HPLC,
and it was used in both tissue distribution and micro-
PET imaging studies of mature female Sprague–Dawley
rats.
In the microPET images (Fig. 3), high liver uptake was
noted at early times, followed by substantial uptake in
the gastrointestinal system. Most striking, however, was
the very high stomach activity levels evident in these
images. While it is not surprising to find high intestinal
activity following injection of lipophilic radio-
pharmaceuticals that undergo elimination by a hepato-
biliary route, the high activity levels in the stomach were
unexpected. One should note that this site of activity
deposition, clearly apparent on the microPET images,
would likely have been missed entirely if the distribution
studies had relied solely on tissue dissection experi-
ments, which do not routinely include the stomach as a
potential site of uptake. This highlights the utility of using
microPET imaging paired with the positron-emitting
radionuclide technetium-94m to expedite the evaluation
of novel technetium-labeled radiopharmaceuticals.
Table 2 presents the uptake of 10b in selected tissues of
mature (180–200 g) female Sprague–Dawley rats at 1 h
post-injection, at 1 h with co-injected estradiol to block
the ER, and at 2 h. Uptake in ER-rich, target tissues
(uterus and ovaries) was quite low, yet uterus/muscle and
ovary/muscle ratios were reasonably high. However, the
fact that target tissue uptake and target tissue to muscle
ratios did not show a significant reduction when the
receptors were blocked by co-injection of a large excess
of estradiol indicates that this uptake is not mediated by
binding to ER. Of the tissues assayed, highest uptake
At this point, we do not know the mechanism by which
activity becomes deposited in the stomach after injec-
tion of 10b, nor its chemical form. We believe, however,
it is likely that at the site of acidic gastric secretions, the
chelate donor heteroatoms become protonated and the
complex decomposes, releasing the free 94mTc(CO)3
cation that is then deposited in the surrounding tissue
from which it is cleared more slowly than the original

L. G. Luyt et al. / Bioorg. Med. Chem. 11 (2003) 4977–4989
4983
the metal center is buried more centrally in the core of
the ligand structure—may prove to be a more successful
approach for developing ER imaging agents.64 Never-
theless, it is notable that by using microPET to image
the biodistribution of the 94mTc labeled estrogen, we
were able to identify a major site of activity deposition,
the stomach, that would have been missed by tissue
distribution studies as they are routinely performed.
Table 2. Biodistribution of 10b in female Sprague–Dawley ratsa
Tissue
1 h
1 h block
2 h
Blood
Lung
Liver
Spleen
Kidney
Heart
Uterus
Ovaries
Muscle
Uterus/blood
Uterus/muscle
Ovaries/blood
Ovaries/muscle
0.84ꢂ0.07
1.55ꢂ0.15
3.55ꢂ0.21
1.04ꢂ0.04
1.76ꢂ0.16
0.69ꢂ0.05
0.17ꢂ0.05
0.65ꢂ0.14
0.06ꢂ0.00
0.20ꢂ0.06
2.64ꢂ0.86
0.77ꢂ0.13
10.02ꢂ1.56
0.56ꢂ0.29
1.07ꢂ0.77
2.24ꢂ1.54
0.71ꢂ0.51
1.08ꢂ0.71
0.48ꢂ0.35
0.15ꢂ0.11
0.58ꢂ0.42
0.05ꢂ0.03
0.23ꢂ0.10
2.90ꢂ0.71
0.91ꢂ0.37
11.69ꢂ3.23
0.48ꢂ0.02
1.38ꢂ0.18
3.54ꢂ0.25
0.97ꢂ0.09
1.61ꢂ0.26
0.67ꢂ0.07
0.20ꢂ0.05
0.58ꢂ0.08
0.06ꢂ0.01
0.41ꢂ0.09
3.10ꢂ0.41
1.19ꢂ0.14
9.14ꢂ0.45
Experimental
Reagents and solvents were purchased from Acros,
Aldrich, Fisher, Strem, or TCI. Estrone was purchased
from Steraloids (Newport, RI, USA). Methylene chlo-
ride and tetrahydrofuran were dried by a Solvent
Delivery System (neutral alumina columns) fabricated
by J. C. Meyer, based on a design published by Pagborn
et al.65 Methods for the production and the purification
of 94mTc by sublimation have been previously repor-
ted.61 The Re(I) aqua ion, [Re(H2O)3(CO)3]+, was pre-
pared by refluxing Re(CO)5Cl in water over-night, with
the Re concentration being determined by ICP analysis.
All air-sensitive reactions were carried out under a
nitrogen or argon atmosphere, using flame or oven-
dried glassware. Reaction progress was monitored by
analytical thin-layer chromatography on silica gel med-
ium. Flash chromatography was performed using
Woelm silica gel (0.040–0.063 mm) packing. Hexane for
chromatography was distilled prior to use.
a% Injected dose per gram; n=3 for each time point; 1 h block indi-
cates co-administration of 15 mg estradiol.
1H and 13C NMR spectra were obtained using a Unity
400, Unity 500, or Unity INOVA 500NB Varian FT-
NMR spectrometer. Chemical shifts (d) are reported in
parts per million downfield from internal tetra-
methylsilane and referenced from solvent resonances.
NMR coupling constants are reported as absolute
values in Hertz. Low resolution electron impact (EI)
mass spectra and high resolution EI mass spectra
(HRMS) were obtained on Micromass 70-VSE, or
Micromass 70-SE-4F spectrometers. Both low and high
resolution fast atom bombardment (FAB) mass spectra
were obtained on Micromass ZAB-SE and 70-SE-4F
spectrometers, respectively. Melting points were deter-
mined on a Thomas Hoover melting point apparatus
and are uncorrected.
Figure 3. Coronal microPET image slices of a female Sprague–Dawley
rat at 5 (a), 30(b), 60(c), and 120(d) min post-injection of compound
10b (87 mCi). Note: the entire rat was in the imaging plane in panel (a),
whereas in panels (b)–(d), the rat was imaged from the neck down.
complex 10b. If this proves to be the case, then our
observation of high stomach activity suggests that this
tissue should be routinely examined when the biodis-
tribution of radiolabeled metal chelates is being
determined by the commonly used tissue dissection
method.
N-(2-Ethanethioethyl)iminodiacetic acid (1). Iminodiace-
tic acid (0.61 g, 4.58 mmol) was dissolved in 10 mL
water and 30mL ethanol, with 4.6 mL of a 1.0M
NaOH solution. To this stirred solution was added an
additional 4.6 mL 1.0M NaOH solution and 540 mL
ethyl chloroethyl sulfide (4.6 mmol) portionwise over 10
min. The flask was then heated at 70 ꢁC, with the
reagents remaining dissolved. After 12 h, an additional
4.6 mL 1.0M NaOH solution was added, and heating
was continued for 4 h. The reaction was cooled to rt; the
solvents were removed by a rotary evaporator, and 20
mL water was added with the slurry being heated to
80 ꢁC. To this was added barium chloride dihydrate
(1.12 g, 4.58 mmol) in 3 mL hot water. The mixtꢁure was
heated at 80 ꢁC for 30min, and then cooled to 0 C. The
resulting white precipitate was removed by filtration
Conclusions
Novel tridentate Re(CO)3 chelates substituted at the 7a-
and 17a- positions of estradiol gave estrogen conjugates
having good in vitro binding for the ER. Compounds
14, 21a, and 22a, in particular, are unique in their con-
jugation through an aliphatic chain, with the absence of
an amide or ester linkage that might be susceptible to
hydrolysis. Despite favorable ER binding of these
Re(CO)3 conjugates in vitro, the highest affinity of these
novel metal tricarbonyl conjugates, prepared as the
94mTc labeled analogue, did not show target tissue-
directed biodistribution in vivo. An integrated approach
to the labeling of estrogens with technetium—in which

4984
L. G. Luyt et al. / Bioorg. Med. Chem. 11 (2003) 4977–4989
and dried under vacuum overnight, yielding 0.90 g of
the barium salt. A slurry was made with the barium salt
in water, to which was added 140 mL concd sulfuric
acid, and the mixture was heated at 80 ꢁC for 1 h. The
solution was hot filtered, and the product was recovered
graphy (silica, gradient: 0–2% MeOH/CH2Cl2) yielding
1
46 mg (79%) of 5. H NMR (CDCl3): d 1.30(t, 6H,
J=7.5), 2.61 (dq, 2H, J=7.5, 11.6), 2.79 (dq, 2H, J=7.5,
11.6), 3.47 (s, 2H), 7.33–7.46 (5H). 13C NMR (CDCl3): d
12.20, 32.02, 46.54, 80.89, 128.17, 128.79, 131.04, 133.12,
191.53, 194.28, 197.99. MS (FAB, m.b.): m/z 541
(M[187Re]++H, 100%), 539 (M[185Re]++H, 46%);
HRMS calcd C16H18O5S1287Re, 541.0153, found 541.0153.
1
by evaporation of the filtrate yielding 0.46 g (46%). H
NMR (D2O): d 1.09 (t, 3H, J=7.3 Hz), 2.49 (q, 2H,
J=7.3 Hz), 2.82 (t, 2H, J=7.1 Hz), 3.40(t, 2H, J=7.1
Hz), 3.89 (s, 4H). 13C NMR (D2O): d 13.76, 24.92,
24.92, 54.41, 55.97, 169.45. MS (FAB): m/z 244 (100%,
M++H+Na), 222 (86%, M++H); HRMS calcd
C8H16NO4S, 222.0800, found 222.0799.
7ꢀ-(6-(N-(acetylimino-N0-acetic acid-N0-ethylthioethyl)-
amino)hexan-1-yl)-estra-1,3,5(10)-triene-3,17ꢁ-diol (8).
7a-(6-Aminohexan-1-yl)-3,17a-bis(t-butyldimethylsilanyl-
oxy)-estra-1,3,5(10)-trienediol (7, 30.9 mg, 0.052 mmol),19
1 (12.5 mg, 0.057 mmol), EDC (10.9 mg, 0.057 mmol),
and dimethylaminopyridine (3 mg, 0.02 mmol) were
combined in a flask with 4 mL DMF and 2 mL CH2Cl2.
The mixture was stirred at rt for 2 days, at which time
the reaction was deemed complete by tlc (20% MeOH/
80% CHCl3). After solvent removal under vacuum, the
solids were dissolved in chloroform and separated by
flash chromatography (silica, gradient: 10–30% MeOH/
CHCl3), yielding 22 mg of crude product (62%). This
was partitioned between ethyl acetate and 0.01 M HCl,
with two subsequent ethyl acetate extractions. The
combined organics were combined, washed with brine,
dried with Na2SO4, evaporated under vacuum to give a
clear colorless glassy solid (14 mg, 40%). 1H NMR
(CDCl3): d 0.02 (s, 3H), 0.03 (s, 3H), 0.74 (s, 3H), 0.89
(s, 9H), 1.16–1.96 (21H), 2.25 (m, 2H), 2.52 (q, 2H,
J=7.6 Hz), 2.62–2.86 (5H), 2.99 (m, 2H), 3.22 (m, 2H),
3.50(m, 4H), 3.65 (t, 2H, J=8.1 Hz), 6.58 (m, 1H), 6.63
(m, 1H), 7.11 (d, 1H, J=8.3 Hz), 7.71 (m, 1H, NH). 13C
NMR (CDCl3): d ꢀ4.51, ꢀ4.83, 11.37, 14.66, 18.07,
22.75, 25.15, 25.83, 25.97, 26.52, 27.31, 27.65, 28.94,
29.21, 30.90, 33.32, 34.69, 37.34, 38.36, 39.46, 42.04,
43.69, 46.08, 54.16, 55.35, 57.56, 81.83, 113.05, 116.23,
127.03, 131.51, 136.98, 153.87, 164.51, 172.35. MS
(FAB): m/z 690(M ++H, 78%); HRMS calcd
C38H65N2O5SiS, 689.4383, found, 689.4383.
N-(2-Ethanethioethyl)iminodiacetic acid rhenium(I) tri-
carbonyl (2). The iminodiacetic acid 1 (12 mg, 0.05
mmol) was dissolved in 0.5 mL H2O and heated to
70 ꢁC. Re(CO)+3 in an aqueous solution (0.55 mL,
0.098 M) was added, and the reaction was stirred at 70 ꢁC
for 30min with a white ppt forming. The product was
filtered, washed with H2O and dried under vacuum to
yield 23 mg (88%). MS (FAB): m/z 492 (M[187Re]++H,
100%), 490 (M[185Re]++H, 57%); HRMS calcd
C11H15NO7S187Re, 492.0127, found 492.0125.
2,2-Bis(ethylthio)acetic acid (3). This compound was
prepared as previously described.51 1H NMR (CDCl3):
d 1.27 (t, 6H, J=7.5 Hz), 2.74 (m, 4H,), 4.35 (s, 1H),
11.40(br s, 1H). 13C NMR (CDCl3): d 14.01, 25.19,
49.74, 175.87. MS (EI): m/z 180(M +, 27%); HRMS
calcd C6H12O2S2, 180.0279, found 180.0279.
2,2-Bis(ethanethio)-3-phenylpropanoic acid (4). A dry
argon filled flask was charged with the thioketal acetic
acid 3 (132 mg, 0.74 mmol) and 8 mL THF. The vessel
was cooled to ꢀ23 ꢁC, and then 0.81 mL of 2.0 M n-
butyl lithium solution (1.62 mmol) was slowly added,
producing a precipitate that soon re-dissolved. The
solution was maintained at ꢀ23 ꢁC for 1 h, and was then
cooled to ꢀ78 ꢁC, at which point benzyl bromide (87
mL, 0.74 mmol) was added. The reaction was kept at
ꢀ78 ꢁC for 50min, and then warmed to rt and stirred
overnight. The reaction was quenched with water, acid-
ified with 1 M HCl, and extracted with ethyl acetate
(3ꢃ). The combined organics were washed with brine,
dried over Na2SO4, and evaporated to dryness in vacuo.
Purification by flash chromatography (silica, gradient:
0–10% MeOH/CH2Cl2) yielded 4 (136 mg, 68%) as a clear
colorless oil. 1H NMR (CDCl3): d 1.25 (t, 6H, J=7.5 Hz),
2.70(m, 4H), 3.35 (s, 2H), 7.26–7.33 (5H). 13C NMR
(CDCl3): d 13.34, 24.53, 42.49, 65.17, 127.30, 128.05,
130.59, 135.23, 176.21. MS (EI): m/z 270(M +, 8%);
HRMS calcd C13H18O2S2, 270.0748, found 270.0743.
7ꢀ-(6-(N-(Acetylimino-N0-acetic acid-N0-ethylthioethyl)-
amino)hexan-1-yl)-estra-1,3,5(10)-triene-3,17ꢁ-diol (9).
A flask containing 8 (12.4 mg, 0.017 mmol) was charged
with 1 mL THF and 0.5 mL acetonitrile. The solution
was heated to 50 ꢁC, 0.1 mL 40% HF was added, and
the reaction was stirred at 50 ꢁC for 20min. The reac-
tion was made neutral with satd NaHCO3 and extracted
into methylene chloride (3ꢃ). Removal of the solvent
1
yielded 11 mg (quant) of 9. H NMR (CD3OD): d 0.77
(s, 3H), 1.25 (t, 3H, J=7.3 Hz), 1.28–1.54 (14H), 1.60
(m, 2H), 1.73 (m, 1H), 1.91 (m, 1H), 2.03 (m, 1H), 2.29
(m, 2H), 2.60(q, 2H, J=7.3 Hz), 2.68 (d, 1H, J=16.6
Hz), 2.81 (dd, 1H, J=4.9, 16.6 Hz), 2.89 (t, 2H, J=7.6
Hz), 3.22 (t, 2H, J=7.1 Hz), 3.52 (t, 2H, J=7.6 Hz),
3.66 (t, 2H, J=8.7 Hz), 4.11 (s, 2H), 4.23 (s, 2H), 6.45
(d, 1H, J=2.4 Hz), 6.54 (dd, 1H, J=2.4, 8.5 Hz), 7.07
(d, 1H, J=8.5 Hz). MS (FAB): m/z 575 (M++H,
30%); HRMS calcd C32H51N2O5S, 575.3519, found
575.3517.
2,2-Bis(ethanethio)-3-phenylpropanoic acid rhenium(I)
tricarbonyl (5). The chelator 4 (29 mg, 0.11 mmol) was
dissolved in 1 mL water, 0.5 mL MeOH, and 120 mL
1 M NaOH solution. The solution was heated to 60 ꢁC,
and then 1.1 mL of a 0.098 M Re(CO)+3 aqueous solu-
tion was added and a ppt formed. After 20min, the
reaction was cooled to rt and the solvents were removed
by rotary evaporation. The product was dissolved in
CHCl3, filtered, and the filtrate was evaporated to dry-
ness. Purification was carried out by flash chromato-
7ꢀ-(6-(N-(Acetylimino-N0-acetic acid-N0-ethylthioethyl)-
amino)hexan-1-yl)-estra-1,3,5(10)-triene-3,17ꢁ-diol rhe-
nium(I) tricarbonyl (10a). The estradiol analogue 9 (8.1

L. G. Luyt et al. / Bioorg. Med. Chem. 11 (2003) 4977–4989
4985
mg, 0.014 mmol) was dissolved in 1.5 mL MeOH, 1.5
mL H2O, and 30 mL of 1.0M KOH. The solution was
heated to 70 ꢁC, and 139 mL of 0.098 M Re(CO)+3 aqu-
eous cation in solution was added, with a cloudy sus-
pension immediately forming. After 45 min, the mixture
was cooled to 0 ꢁC, 2 mL of H2O was added, the solid
was removed by filtration, washed with H2O, and dried
under vacuum. Purification was carried out by flash
chromatography (silica, 10% MeOH/90% CHCl3) to
give 10a as a white solid (5.5 mg, 48%). 1H NMR
(CD3OD): d 0.77 (s, 3H), 1.20–1.53 (18H), 1.60 (m, 2H),
1.74 (m, 1H), 1.91 (m, 1H), 2.03 (m, 1H), 2.29 (m, 2H),
2.66 (m, 2H), 2.78–2.95 (m, 2H), 3.17 (m, 4H), 3.34 (m,
1H), 3.66 (t, 1H, J=8.5 Hz), 3.80(m, 1H), 3.96–4.32 (m,
4H), 6.45 (d, 1H, J=2.4 Hz), 6.53 (dd, 1H, J=2.6, 8.3
Hz), 7.07 (d, 1H, J=8.3 Hz). MS (FAB): m/z 845
(M[187Re]++H, 3%), 843 (M[185Re]++H, 2%); HRMS
calcd C35H50N2O8S187Re, 845.2846, found, 845.2845.
1H NMR (CDCl3): d 0.03 (s, 3H), 0.04 (s, 3H), 0.20 (s,
6H), 0.75 (s, 3H), 0.90 (s, 9H), 0.98 (s, 9H), 1.18–1.75
(18H), 1.84 (m, 1H), 1.94 (m, 1H), 2.27 (m, 2H), 2.68 (d,
1H, J=16.7 Hz), 2.85 (dd, 1H, J=4.9, 16.3 Hz), 2.99 (s,
3H), 3.67 (t, 1H, J=8.4 Hz), 4.20(t, 2H, J=6.5 Hz),
6.54 (d, 1H, J=2.6 Hz), 6.62 (dd, 1H, J=2.6, 8.6 Hz),
7.12 (d, 1H, J=8.6 Hz). 13C NMR (CDCl3): dꢀ4.84,
ꢀ4.52, ꢀ4.43, 11.33, 18.06, 22.75, 25.46, 25.65, 25.82,
27.24, 27.98, 29.08, 29.38, 30.88, 33.24, 34.53, 37.30,
38.22, 41.88, 43.67, 46.04, 70.10, 81.79, 117.18, 120.79,
126.69, 132.51, 136.70, 153.22.
7ꢀ-[7-Carboxyethyl-7,7-bis(ethanethio)-heptan-1-yl]-estra-
1,3,5(10)-triene-3,17ꢁ-diol (12). A flame-dried, nitrogen
filled flask was charged with ethyl bis(ethanethio)acetate
(41 mg, 0.19 mmol) and 5 mL THF. The solution was
cooled to 0 ꢁC, and then 194 mL of 1.0M potassium t-
butoxide solution (THF) was added and the reaction
was stirred at 0 ꢁC for 30min. The mesylate 11 (120mg,
0.18 mmol) was added in 2 mL THF, the mixture was
stirred at 0 ꢁC for 2 h, and then at rt overnight. The
reaction was quenched with satd NH4Cl, the organics
were extracted into CHCl3 (3ꢃ), dried over MgSO4, and
evaporated to dryness. Flash chromatography (silica,
gradient: 10–20% ethyl acetate/hexanes) provided the
desired product as a mixture of mono-protected and di-
protected alcohols. Complete deprotection was carried
out by dissolving the mixture in 2 mL acetonitrile and
0.2 mL THF, heating to 60 ꢁC, and adding 0.3 mL of
40% HF. After 15 min, the reaction was quenched with
sat. NaHCO3, extracted into CH2Cl2 (3ꢃ), dried over
Na2SO4, and evaporated in vacuo to give the crude 12.
Purification was carried out by flash chromatography
(silica, 30% ethyl acetate/70% hexanes) to give 12 (30
mg, 30% over two steps) as a clear colorless glassy solid.
1H NMR (CDCl3): d 0.78 (s, 3H), 1.18–1.64 (26H), 1.72
(m, 1H), 1.89 (m, 2H), 2.13 (m, 1H), 2.29 (m, 2H), 2.51
(m, 1H), 2.60(m, 4H), 2.70(m, 1H), 2.85 (m, 1H), 3.76
(t, 1H, J=8.5 Hz), 4.22 (q, 2H, J=7.1 Hz), 5.05 (br s,
1H), 6.55 (d, 1H, J=2.4 Hz), 6.63 (dd, 1H, J=2.7, 8.4
Hz), 7.14 (d, 1H, J=8.4 Hz). 13C NMR (CDCl3): d
11.04, 13.58, 14.09, 22.59, 23.64, 25.24, 25.49, 27.19,
28.03, 29.49, 29.54, 29.68, 30.46, 33.10, 34.50, 36.22,
36.80, 37.98, 41.88, 43.33, 46.37, 62.01, 65.44, 82.02,
112.82, 116.13, 127.05, 131.79, 137.07, 153.43, 170.82 .
MS (EI) m/z 562 (M+, 32%); HRMS calcd
C32H50O4S2, 562.3151, found 562.3153.
7ꢀ-(6-(N-(Acetylimino-N0-acetic acid-N0-ethylthioethyl)-
amino)hexan-1-yl)-estra-1,3,5(10)-triene-3,17ꢁ-diol tech-
netium-94m(I) tricarbonyl (10b). A pressure vessel was
charged with sodium borohydride (5 mg, 0.13 mmol),
sodium carbonate (5 mg, 0.05 mmol), and a stir bar.
The vessel was flushꢀed with CO (g) for 5 min, and then
14.6 mCi of 94mTcO4 in 0.4 mL saline was added while
under CO flow. The pressure vessel was sealed and
heated at 80 ꢁC for 30min in an aluminum block,
behind a safety shield. The vessel was then cooled to
0 ꢁC, opened, and 0.3 mL of a buffer (2 parts 1 M HCl, 1
part 1 M Na2HPO4) was added. After brief mixing, the
ligand 9 (1 mg, 0.002 mmol), dissolved in 0.1 mL
methanol, was added. The pressure vessel was sealed
and heated at 80 ꢁC for 20min (see safety precautions
above), followed by cooling to 0 ꢁC. The reaction solu-
tion was passed through a syringe filter (0.2 mm) and
rinsed with 0.5 mL H2O. The product was eluted from
the filter with 0.5 mL CH3CN to yield 1.74 mCi (27%
decay corrected radiochemical yield) of crude product.
Purification was carried out by reversed-phase C18
HPLC, using an eluent composition of 65% CH3CN/
35% H2O. The fractions containing the product (as
determined by prior HPLC analysis of 10a) were com-
bined, extracted with ether (2ꢃ), dried over Na2SO4, and
gently evaporated to dryness yielding 303 mCi of 10b (8%
decay corrected radiochemical yield). The product was
dissolved in 20 mL ethanol, and then diluted with 180 mL
saline containing 0.1% Tween-80, and the injectable
solution was divided into syringes in the required dosage.
7ꢀ-[7-Carboxy-7,7-bis(ethanethio)-heptan-1-yl]-estra-
1,3,5(10)-triene-3,17ꢁ-diol (13). The ester 12 (26 mg,
0.046 mmol) was dissolved in 5 mL methanol and 3 mL
3 M NaOH solution. The mixture was heated at reflux
for 3 h, cooled to rt, and the methanol was removed by
rotary evaporation. The solution was then diluted with
water, acidified with 3 M HCl, and then extracted with
CH2Cl2 (3ꢃ). The combined organics were washed with
brine, dried over Na2SO4, and evaporated to dryness in
vacuo. Purification by flash chromatography (silica,
ethyl acetate then 20% MeOH/80% CHCl3) yielded the
3,17ꢁ-Bis(t-butyldimethylsilanyloxy)-7ꢀ-[6-(methanesul-
fonyloxy)-hexan-1-yl]-estra-1,3,5(10)-triene (11). 3,17b-
Bis(t-butyldimethylsilanyloxy)-7a-[6-hydroxy-hexan-1-
yl]-estra-1,3,5(10)-triene (6, 106 mg, 0.18 mmol) was
dissolved in 15 mL THF and 49 mL triethylamine (0.35
mmol). The solution was cooled to 0 ꢁC, and then
methanesulfonyl chloride (27 mL, 0.35 mmol) was
added, and the reaction was stirred overnight with
warming to rt. The mixture was partitioned between
satd NaHCO3 and CHCl3 (3ꢃ), the organics were com-
bined and evaporated in vacuo. Purification by flash
chromatography (silica, 10% ethyl acetate/90% hexane)
provided 11 (121 mg, 0.18 mmol) in quantitative yield.
1
pure 13 (14 mg, 57%). H NMR (CDCl3): d 0.76 (s,
3H), 1.15–1.60(22H), 1.70–1.82 (m, 4H), 1.94 (m, 1H),
2.03 (m, 1H), 2.27 (m, 2H), 2.57 (m, 4H), 2.67 (m, 1H),
2.80(m, 1H), 3.66 (t, 1H, J=8.6 Hz), 6.45 (d, 1H,

4986
L. G. Luyt et al. / Bioorg. Med. Chem. 11 (2003) 4977–4989
J=2.4 Hz), 6.53 (dd, 1H, J=2.5, 8.6 Hz), 7.06 (d, 1H,
J=8.6 Hz). 13C NMR (CDCl3): d 11.72, 14.13, 18.35,
23.66, 24.70, 26.60, 28.62, 29.17, 30.68, 30.75, 31.07,
34.63, 35.75, 38.06, 38.24, 39.61, 43.67, 44.52, 47.78,
58.32, 82.57, 113.95, 116.96, 127.90, 131.81, 137.61,
156.01, 174.00. MS (FAB) m/z 535 (M++H, 26%);
HRMS calcd C30H46O4S2, 534.2838, found 534.2840.
2,2-Bis(ethanethio)-hept-6-ynoic acid ethyl ester (16b). A
flame-dried flask was charged with 2,2-bis(ethanethio)-
acetate (1.02 g, 4.90 mmol) and 10 mL THF, and then
cooled to 0 ꢁC. A potassium t-butoxide solution (5.4 mL
1.0M in THF) was slowly added to the flask and stirred
for 30min at 0 ꢁC. 5-Iodopentyne (601 mL, 4.90mmol)
was then added and the mixture was stirred for 2 h at
0 ꢁC, and then warmed to rt and stirred overnight. The
reaction was quenched with satd NH4Cl, extracted with
diethyl ether (3ꢃ), the combined organics were washed
with brine, dried with Na2SO4, evaporated and dried
under vacuum. The crude material was purified by flash
chromatography (silica, 10% ethyl acetate/90% hex-
anes) and was then placed under vacuum at 130 ꢁC (0.3
mm Hg) to remove residual 2,2-bisthioetherethyl ace-
tate, yielding 0.76 g of 16b as a clear colorless oil (57%).
1H NMR (CDCl3): d 1.22 (t, 6H, J=7.5 Hz), 1.30(t,
3H, J=7.1 Hz), 1.70(m, 2H), 1.96 (t, 1H, J=2.6 Hz),
2.05 (m, 2H), 2.23 (dt, 2H, J=2.6, 6.9 Hz), 2.63 (dq,
4H, J=1.5, 7.5 Hz), 4.23 (q, 2H, J=7.1 Hz). 13C NMR
(CDCl3): d 13.52, 14.08, 18.19, 23.62, 24.22, 35.19,
62.08, 64.79, 68.86, 83.65, 170.46. MS (EI): m/z 274
(M+, 1%), 246 (M+ꢀEt, 5%); HRMS calcd
C13H22O2S2, 274.1061, found, 274.1059.
7ꢀ-[7-carboxy-7,7-bis(ethanethio)-heptan-1-yl]-estra-
1,3,5(10)-triene-3,17ꢁ-diol rhenium(I) tricarbonyl (14).
The estradiol analogue 13 (12 mg, 0.022 mmol) was
dissolved in 0.5 mL MeOH, 1 mL H2O, and 23 mL 1 M
NaOH solution. The mixture was heated to 60 ꢁC at
which point 202 mL of a 0.122 M Re(CO)+3 aqueous
solution was added, with the solution soon turning tur-
bid. After 10 min tlc (silica, 20% MeOH/80% CHCl3)
indicated the complete disappearance of the starting 13
and the formation of a new, less polar product. The
solvents were then removed in vacuo, and the product
purified by flash chromatography (silica, gradient 5–
10% MeOH/CHCl3) to give 8 mg (44%) of 14. 1H
NMR (CDCl3): d 0.78 (s, 3H), 1.20–1.73 (24H), 1.93 (m,
3H), 2.13 (m, 1H), 2.30(m, 2H), 2.71 (m, 1H), 2.79 to
3.00 (5H), 3.73 (m, 1H), 6.56 (d, 1H, J=2.8 Hz), 6.63
(dd, 1H, J=2.6, 8.6 Hz), 7.15 (d, 1H, J=8.6 Hz). MS
(FAB) m/z 805 (M[187Re]++H, 54%), 803
(M[185Re]++H, 37%); HRMS calcd C33H46O7S2,
805.2243, found, 805.2241.
2,2-Bis(ethanethio)-hept-6-ynoic acid (17b). The ester
16b (155 mg, 0.57 mmol) was dissolved in 6 mL MeOH,
to which 3 mL 3 M NaOH solution was added. The
mixture was heated at 40 ꢁC for 6 h, and then was
cooled to rt and diluted with H2O. Unreacted ester was
removed by ether extraction, followed by acidification,
and the product was extracted with ether (3ꢃ), washed
with brine, dried over MgSO4. The solvent was removed
by rotary evaporation, and then dried under vacuum to
give 17b as a clear colorless oil (125 mg, 90%). 1H NMR
(CDCl3): d 1.24 (t, 6H, J=7.5 Hz), 1.77 (m, 2H), 1.97 (t,
1H, J=2.7 Hz), 2.06 (m, 2H), 2.24 (dt, 2H, J=2.7, 6.8
Hz), 2.67 (dq, 4H, J=1.9, 7.5 Hz). 13C NMR (CDCl3):
d 13.34, 18.17, 23.80, 24.09, 34.81, 64.08, 68.99, 83.51,
176.50. MS (EI): m/z 247 (M+, 10%); HRMS calcd
C11H18O2S2, 246.0748, found 246.0747.
5-Iodopentyne (15). 5-Chloropentyne (1.12 g, 10.9
mmol), sodium iodide (8.2 g, 55 mmol), and acetone
were heated at reflux overnight. The mixture was cooled
to rt, filtered, and the filtrate was evaporated to dryness.
The crude material was partitioned between water and
CH2Cl2 (3ꢃ), dried over Na2SO4, and the solvent was
removed by rotary evaporation, with final drying under
a steady stream of nitrogen to yield 1.9 g (90%) of 15 as
1
a clear and colorless oil. H NMR (CDCl3): d 1.99 (t,
1H, J=2.7 Hz), 2.00 (quintet, 2H, J=6.8 Hz), 2.34 (dt,
2H, J=2.7, 6.8 Hz), 3.31 (t, 2H, J=6.8 Hz). 13C NMR
(CDCl3): d 5.06, 19.39, 31.75, 69.45, 82.24.
2,2-Bis-(ethanethio)-pent-4-ynoic acid ethyl ester (16a).
A flame-dried flask was charged with 2,2-bis-(ethan-
ethio)acetate (1.00 g, 4.80 mmol) and 6 mL THF, and
then cooled to 0 ꢁC. Slowly added 4.8 mL 1.0M
potassium t-butoxide solution (in THF) to the flask
and warmed the solution to room temperature. After
30min, propargyl bromide (80% solution, 535 mL, 4.80
mmol) was added, and the mixture was stirred for 5 h.
The reaction was quenched with satd NH4Cl, extracted
with diethyl ether (3ꢃ), and the combined organics
were washed with brine, dried with Na2SO4, evapo-
rated and dried under vacuum. The crude material was
purified by flash chromatography (silica, 10% ethyl ace-
tate/90% heaxanes) to yield 0.69 g of 16a as a clear col-
orless oil (58%). 1H NMR (CDCl3): d 1.24 (t, 6H, J=7.5
Hz), 1.32 (t, 3H, J=7.2 Hz), 2.12 (t, 1H, J=2.6 Hz), 2.70
(m, 4H), 2.93 (d, 2H, J=2.6 Hz), 4.27 (q, 2H, J=7.2 Hz).
13C NMR (CDCl3): d 13.54, 14.01, 24.33, 28.38, 62.41,
62.71, 71.48, 78.85, 169.44 . MS (EI): m/z 246 (M+,
100%); HRMS calcd C11H18O2S2, 246.0748, found,
246.0751.
2,2-Bis(ethanethio)-hept-6-ynoic acid rhenium(I) tricar-
bonyl (18b). The acid 17b (31 mg, 0.13 mmol) was dis-
solved in 0.5 mL MeOH, 1 mL H2O, and 126 mL 1.0M
NaOH solution. The mixture was heated to 60 ꢁC, and
then 1.24 mL of a 0.122 M Re(CO)+3 aqueous solution
was added. After 15 min tlc (silica, 5% MeOH/95%
CHCl3) indicated the absence of s.m., and the reaction
was cooled to 0 ꢁC. The beige ppt. was removed by fil-
tration to give 65 mg of crude product. Purification by
flash chromatography (silica, gradient: 0–2% MeOH/
CH2Cl2) yielded 32 mg of 18b (49%). 1H NMR
(CDCl3): d 1.39 (t, 6H, J=7.5 Hz), 1.82 (m, 2H), 2.01 (t,
1H, J=2.8 Hz), 2.13 (m, 2H), 2.35 (dt, 2H, J=2.6, 6.6
Hz), 2.85 (m, 2H), 3.00 (m, 2H).
17ꢀ-(40,40-Bis(ethanethio)-40-ethoxycarbonyl-butyn-10-yl)-
3-tbutyldimethylsilanyloxy-estra-1,3,5(10)-triene-17ꢁ-ol
(19a). A flame-dried flask was charged with alkyne 16a
(290mg, 1.18 mmol) and 20mL THF, and then was
cooled to ꢀ78 ꢁC. A 1.2-mL portion of 1.0M n-butyl
lithium was slowly added via syringe and the reaction

L. G. Luyt et al. / Bioorg. Med. Chem. 11 (2003) 4977–4989
4987
was stirred for 30min. A portion of 3- tbutyldimethylsi-
lanyloxy-estrone (377 mg, 0.98 mmol) dissolved in 5 mL
THF was slowly added, and the reaction was main-
tained at ꢀ78 ꢁC for 8 h, and then warmed to room
temperature overnight. The reaction was quenched with
satd NH4Cl, the organics were extracted with CHCl3
(3ꢃ), the combined organics were washed with brine,
dried over Na2SO4, evaporated and dried under vacuum.
Purification was carried out by flash chromatography
(silica, gradient 10% ethyl acetate/90% hexanes to 20%
ethyl acetate/80% hexanes) yielding 329 mg (53%) of a
(CDCl3): d 0.83 (s, 3H), 1.19 (t, 3H, J=7.5 Hz), 1.20(t,
3H, J=7.5 Hz), 1.30–1.40 (4H), 1.65 (m, 1H), 1.72 (m,
1H), 1.85 (m, 1H), 1.90–1.97 (m, 2H), 2.12 (m, 1H), 2.22
(m, 2H), 2.32 (m, 1H), 2.64–2.78 (6H), 2.93/3.01 (AB,
2H, J=18.7 Hz), 6.45 (d, 1H, J=2.6 Hz), 6.52 (dd, 1H,
J=2.6, 8.4 Hz), 7.06 (d, 1H, J=8.4 Hz). 13C NMR
(CDCl3): d 13.41, 14.12, 23.72, 25.29, 25.36, 27.84,
28.67, 30.17, 30.77, 33.88, 39.83, 41.23, 44.88, 48.63,
50.16, 67.62, 80.65, 82.62, 87.61, 113.73, 116.04, 127.26,
132.73, 138.84, 155.87, 176.46. MS (EI): m/z 488 (M+,
1%), 487 (M-H, 1%), 470(M ꢀH2O, 4%); HRMS calcd
C27H35O4S2, 487.1977, found 487.1975.
1
clear, pale yellow oil. H NMR (CDCl3): d 0.19 (s, 6H),
0.85 (s, 3H), 0.98 (s, 9H), 1.23 (t, 6H, J=7.5 Hz), 1.31 (t,
3H, J=7.3 Hz), 1.32–1.50(4H), 1.63–2.01 (6H), 2.22–
2.33 (3H), 2.68 (m, 4H), 2.79 (m, 2H), 2.96 (d, 2H, J=3.0
Hz), 4.24 (q, 2H, J=7.1 Hz), 6.55 (d, 1H, J=2.6 Hz),
6.61 (dd, 1H, J=2.6, 8.4 Hz), 7.12 (d, 1H, J=8.6 Hz).
13C NMR (CDCl3): d ꢀ4.45, 12.65, 13.66, 13.69, 14.08,
18.11, 22.69, 24.20, 24.24, 25.66, 26.38, 27.26, 28.51,
29.60, 32.47, 38.88, 39.35, 43.41, 47.33, 49.00, 62.29,
17ꢀ-(60,60-Bis(ethanethio)-60-carboxy-hexyn-10-yl)-estra-
1,3,5(10)-triene-3,17ꢁ-diol (20b). A flask with attached
condenser was charged with 19b (210mg, 0.32 mmol),
12 mL MeOH, and 12 mL 3 M NaOH aqueous solu-
tion. The mixture was heated at reflux overnight, cooled
to room temperature, and the methanol was evaporated
in vacuo. The aqueous solution was made slightly acidic
with the addition of 3 M HCl. The organics were
extracted with CHCl3 (4ꢃ), the combined organics were
washed with brine, dried over Na2SO4, evaporated and
dried under vacuum. Purification was carried out by
flash chromatography (silica, gradient: 0–20% MeOH/
63.66, 79.95, 80.87, 87.06, 117.09, 119.88, 126.12,
+
133.17, 137.85, 153.24, 169.59 . MS (EI): m/z 630(M
,
73%); HRMS calcd C35H54O4SiS2, 630.3233, found,
630.3232.
17ꢀ-(60,60-Bis(ethanethio)-60-ethoxycarbonyl-hexyn-10-yl)-
3-tbutyldimethylsilanyloxy-estra-1,3,5(10)-triene-17ꢁ-ol
(19b). A flame-dried flask was charged with alkyne 16b
(290mg, 1.06 mmol) and 10mL THF, and then was
cooled to ꢀ78 ꢁC. A 1.1 mL portion of 1.0M n-butyl
lithium was slowly added via syringe and the reaction
was stirred for 30min. A portion of 3- tbutyldimethyl-
silanyloxy-estrone (333 mg, 0.87 mmol) dissolved in 5
mL THF was slowly added, and the reaction was
maintained at ꢀ78 ꢁC for 6 h, and then warmed to room
temperature overnight. Workup and purification were
carried out as described for 19a, yielding 264 mg (46%)
CHCl3) to give a clear, colorless oil (129 mg, 78%). H
1
NMR (CD3OD): d 0.84 (s, 3H), 1.14 (dt, 6H, J=2.8, 7.5
Hz), 1.36–1.41 (4H), 1.67–1.79 (5H), 1.85 (m, 1H), 1.95
(m, 2H), 2.06 (m, 2H), 2.17–2.26 (2H), 2.30 (t, 2H,
J=6.5), 2.34 (m, 1H), 2.59 (m, 4H), 2.75 (m, 2H), 6.46
(d, 1H, J=2.6), 6.53 (dd, 1H, J=2.6, 8.4), 7.07 (d, 1H,
J=8.4). 13C NMR (CD3OD): d 13.47, 13.93, 19.32,
23.73, 24.62, 24.70, 25.74, 27.75, 28.56, 30.78, 34.24,
37.17, 39.96, 41.14, 45.05, 48.43, 50.84, 67.97, 80.56,
85.73, 85.86, 113.68, 116.00, 127.31, 132.57, 138.82,
155.85, 175.55.
of a clear, colorless oil. H NMR (CDCl3): d 0.18 (s,
17ꢀ-(40,40-Bis(ethanethio)-40-carboxy-butyn-10-yl)-estra-
1,3,5(10)-triene-3,17ꢁ-diol rhenium(I) tricarbonyl (21a).
Compound 20a (27 mg, 0.054 mmol) was dissolved in 1
mL MeOH, 1 mL water, and 100 mL of satd NaHCO3.
The solution was heated to 70 ꢁC and 337 mL of a
0.178 M Re(CO)+3 aqueous solution was added. After
90min the solution was cooled and evaporated to dry-
ness under vacuum. The crude material was purified by
flash chromatography (silica, gradient: 0–20% MeOH/
CHCl3) to give 12 mg (29%) of 21a. 1H NMR
(CD3OD): d 0.88 (s, 3H), 1.12–1.49 (10H), 1.76–2.07
(6H), 2.16–2.38 (3H), 2.64–2.81 (8H), 6.48 (d, 1H,
J=2.4 Hz), 6.54 (dd, 1H, J=2.4, 8.1 Hz), 7.09 (d, 1H,
J=8.1 Hz). MS (FAB): m/z 759 (M[187Re]++H, 2.9%),
757 (M[185Re]++H, 2.2%), 741 (M[187Re]+ꢀOH,
2.7%), 739 (M[185Re]+ꢀOH, 1.7%). HRMS calcd
C30H35O7S2Re, 759.1460, found 759.1457.
1
6H), 0.87 (s, 3H), 0.97 (s, 9H), 1.20 (t, 6H, J=7.5 Hz),
1.29 (t, 3H, J=7.1 Hz), 1.32 to 1.51 (4H), 1.64–1.90
(7H), 2.01 (m, 1H), 2.08 (m, 2H), 2.20–2.36 (5H), 2.63
(m, 4H), 2.80(m, 2H), 4.22 (q, 2H, J=7.1 Hz), 6.55 (d,
1H, J=2.6 Hz), 6.61 (dd, 1H, J=2.6, 8.4 Hz), 7.12 (d,
1H, J=8.4 Hz). 13C NMR (CDCl3): d ꢀ4.46, 12.78,
13.49, 14.08, 14.09, 18.11, 18.63, 22.76, 23.63, 23.70,
24.49, 25.65, 26.33, 27.18, 29.62, 32.93, 35.54, 39.05,
39.33, 43.59, 47.14, 49.53, 62.08, 64.63, 80.00, 84.53,
85.33, 117.11, 119.88, 126.14, 133.01, 137.82, 153.22,
170.42 . MS (EI): m/z (M+, 1%); HRMS calcd
C37H58O4SiS2, 658.3546, found, 658.3548.
17ꢀ-(40,40-Bis(ethanethio)-40-carboxy-butyn-10-yl)-estra-
1,3.5(10)-triene-3, 17ꢁ-diol (20a). A flask with attached
condenser was charged with 19a (253 mg, 0.40 mmol),
12 mL MeOH, and 12 mL 3 M NaOH aqueous solu-
tion. The mixture was heated at reflux for 5 h, cooled to
room temperature, and made slightly acidic with the
addition of 1 M HCl. The organics were extracted with
CHCl3 (3ꢃ), the combined organics were washed with
brine, dried over Na2SO4, evaporated and dried under
vacuum. Purification was carried out by flash chroma-
tography (silica, gradient: 0–30% MeOH/CHCl3) to
give a clear, colorless oil (128 mg, 65%). 1H NMR
17ꢀ-(60,60-Bis(ethanethio)-60-carboxy-hexyn-10-yl)-estra-
1,3,5(10)-triene-3,17ꢁ-diol rhenium(I) tricarbonyl (21b).
Compound 20b (26 mg, 0.051 mmol) was dissolved in 2
mL methanol, 2 mL water, and 52 mL of 1 M NaOH.
The solution was heated to 70 ꢁC and 500 mL of a
0.122 M Re(CO)+3 aqueous solution was added. After
15 min the solution was cooled and evaporated to dry-
ness under vacuum. The crude material was purified by

4988
L. G. Luyt et al. / Bioorg. Med. Chem. 11 (2003) 4977–4989
flash chromatography (silica, gradient: 5–20% MeOH/
CHCl3) to give 27 mg (68%) of 21b as a as a clear color-
less oil. H NMR (CD3OD): d 0.85 (s, 3H), 1.10–1.46
(10H), 1.69–2.02 (8H), 2.14–2.46 (7H), 2.56–2.84 (4H),
3.11 (m, 1H), 3.30(m, 1H), 6.47 (m, 1H), 6.53 (m, 1H),
7.08 (m, 1H). MS (FAB): m/z 787 (M[187Re]++H, 2%),
769 (M[187Re]+ꢀOH, 12%), 767 (M[185Re]+ꢀOH, 6%).
HRMS calcd (for M[187Re]+ꢀOH) C32H38O6S1287Re,
769.1667, found, 769.1671.
NMR spectra were obtained in the Varian Oxford
Instrument Center for Excellence in NMR Laboratory.
Funding for this instrumentation was provided in part
from the W. M. Keck Foundation, the National Insti-
tutes of Health (PHS 1 S10 RR104444-01), and the
National Science Foundation (NSF CHE 96-10502).
Mass spectra were obtained on instruments supported
by grants from the National Institute of General Medi-
cal Sciences (GM 27029), the National Institutes of
Health (RR 01575), and the National Science Founda-
tion (PCM 8121494). We also thank Kathryn Carlson
for the binding affinity measurements.
1
Estrogen receptor binding affinity
Relative binding affinities were determined by competi-
tive radiometric binding assays using [3H]estradiol, fol-
lowing previously published procedures.58,59 The
relative binding affinities (RBA) are expressed as a per-
centage, with estradiol being set to 100%. The receptor
sources were lamb uterine cytosol, purified full-length
human ERa, or purified full-length human ERb (Pan-
Vera Inc.). The RBA values are reported as the average
of two or more determinationsꢂthe range (n=2) or
standard deviation (n>2).
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PET imaging
Positron emission tomography imaging was performed
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We are grateful for support of this research through
grants from the National Institute of Health [PHS 5R37
CA25836 (to J.A.K.) and PHS 5P01HL13851 (to
M.J.W.)] and the Department of Energy [DE FG02
86ER60401 (to J.A.K.) and DE FG02 84ER60218 (to
M.J.W.)]. Technetium-94m was provided by Washing-
ton University Medical School and partially funded
through an NCI grant R24 CA86307. MicroPET ima-
ging was supported by an NIH/NCI SAIRP grant (1
R24 CA83060). We would also like to thank the Small
Animal Imaging Core of the Alvin J. Siteman Cancer
Center at Washington University and Barnes-Jewish
Hospital in St. Louis, MO, USA for additional support
of the microPET imaging. The Core is supported by an
NCI Cancer Center Support Grant # 1 P30CA91842.

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