Synthesis and estrogen receptor affinity of a 4-hydroxytamoxifen-labeled ligand for diagnostic imaging

Article abstract of DOI:10.1016/S0968-0896(02)00329-2

A 10-step synthesis of a novel 4-hydroxytamoxifen-DTPA ligand (HOTam-DTPA) is reported. Tamoxifen and its primary metabolite 4-hydroxytamoxifen are common estrogen receptor ligands. Consequently, tamoxifen has found utility as the targeting component of various diagnostic agents for selective imaging of estrogen receptor-rich tissue, specifically breast cancer. An L-aspartic acid-derived DTPA analogue was attached to the ethyl side chain of 4-hydroxy-tamoxifen using N,N′-dimethylethylenediamine as a hydrophilic linker. A competitve estrogen receptor binding assay using [³H]-17β-estradiol was performed to determine the effect of the ethyl side chain modification on estrogen receptor affinity. The results show that while the relative affinity of HOTam-DTPA for the estrogen receptor is ～10-fold lower than that of tamoxifen, it still remains a potent ligand at relatively low concentrations.

Full text of DOI:10.1016/S0968-0896(02)00329-2

Bioorganic & Medicinal Chemistry 10 (2002) 4075–4082
Synthesis and Estrogen Receptor Affinity of a
4-Hydroxytamoxifen-Labeled Ligand for Diagnostic Imaging
Matthew R. Lashley,a Edmund J. Niedzinski,a Jane M. Rogers,b
Michael S. Denisonb and Michael H. Nantza,*
aDepartment of Chemistry, University of California, Davis, CA 95616, USA
bDepartment of Environmental Toxicology, University of California, Davis, CA 95616, USA
Received 26 December 2001; accepted 24 June 2002
Abstract—A 10-step synthesis of a novel 4-hydroxytamoxifen-DTPA ligand (HOTam-DTPA) is reported. Tamoxifen and its primary
metabolite 4-hydroxytamoxifen are common estrogen receptor ligands. Consequently, tamoxifen has found utility as the targeting
component of various diagnostic agents for selective imaging of estrogen receptor-rich tissue, specifically breast cancer. An l-aspartic
acid-derived DTPA analogue was attached to the ethyl side chain of 4-hydroxy-tamoxifen using N,N0-dimethylethylenediamine as a
hydrophilic linker. A competitve estrogen receptor binding assay using [3H]-17b-estradiol was performed to determine the effect of the
ethyl side chain modification on estrogen receptor affinity. The results show that while the relative affinity of HOTam-DTPA for the
estrogen receptor is ꢀ10-fold lower than that of tamoxifen, it still remains a potent ligand at relatively low concentrations.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
efforts to elucidate the mechanism of action,9 the asso-
ciated metabolic pathways,10 and structure–activity
relationships11 have led to an understanding of ER-ligand
interactions. Tamoxifen is reported to bind the ER
ligand binding site with a relative affinity of between
Breast cancer is the leading form of cancer in women
today, affecting as many as one in eight women.1
Depending on the type of breast cancer, one therapy is
to administer an anti-estrogen to compete with estradiol
(Fig. 1) for binding the estrogen receptor (ER).2 The ER
is a 66 kDa protein member of the nuclear hormone
receptor family of transcription factors3 that exists in
two forms, ERa and ERb.4 The ER is over-expressed in
cancerous cells relative to healthy tissues and high ER
concentrations are associated with decreased overall
breast cancer survival.5,6 In addition, changes in the
relative levels of expression of each form of ER are
reported to occur in cancer cells, with decreases in ERb
expression common in mammary and other tumors.7
The abundance of ER in forms of breast cancer suggests
that this protein is a candidate for preferential targeting
of cancer cells, an important consideration for any form
of chemotherapy. Indeed, anti-estrogen therapy affects
principally tissues rich in ER.2 Tamoxifen (Fig. 1), the
prototypical anti-estrogen, has been employed over the
course of three decades for the treatment of ER-positive
tumors.8 Since FDA approval for tamoxifen in 1978,
*Corresponding author. Tel.: +1-530-752-6357; fax: +1-530-752-
8995; e-mail: mhnantz@ucdavis.edu
Figure 1. Structures and approximate relative binding values of com-
mon estrogen receptor ligands.4,12,13
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00329-2

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M. R. Lashley et al. / Bioorg. Med. Chem. 10 (2002) 4075–4082
20–200-fold lower than that of 17b-estradiol, while
4-hydroxytamoxifen, the primary metabolite of tam-
oxifen, is significantly more potent, with reported affi-
nities ranging between 1.8-fold more potent to 10-fold less
potent than 17b-estradiol.4,12,13 Although the two ER
forms appear to differ somewhat in their tissue distribution
and in their specificity and relative affinity for ligands,
tamoxifen binds both subtypes with similar affinity.4
that sex hormone-binding globulin (SHBG), a serum
glycoprotein that specifically binds testosterone and
17b-estradiol,21 may have contributed to the uptake of
these tamoxifen-based agents exists since SHBG also
binds membranes of estrogen-dependent tissues such as
breast cancer.22 However, tamoxifen-based agents gen-
erally have failed to provide sufficient contrast between
tissues rich in ER and surrounding tissues to warrant
their diagnostic use in humans. To underscore this
problem, Podoloff et al. suggested that clearance of the
111In-chelate of 4 from tissues surrounding ER-rich
tumors might be improved if the hydrophilicity of the
agent were increased.18
An emerging area of research that seeks to exploit the
tamoxifen affinity for the ER is the use of tamoxifen as
a targeting element of diagnostic and imaging agents.14
Since the initial observation by Bloomer et al. that an
125I-labeled tamoxifen analogue was cytotoxic to var-
ious cells,15 several isotopically enriched, halogenated
analogues of tamoxifen have been synthesized and tes-
ted for their ability to localize in ER-rich tissue (e.g., 1–3,
Fig. 2).16,17 For a similar purpose, tamoxifen also has
been fitted with moieties for chelation of radionuclides
and magnetic resonance imaging (MRI) elements (e.g.,
4–6).18ꢁ20 Positron emission tomography (PET) and
single photon emission computed tomography (SPECT)
studies in animals using analogues 2 and 3 confirmed
the central hypothesis underlying the design of tamoxifen-
based imaging agents, namely that uptake of the agents
occurred via an ER-mediated process.17 The possibility
With these considerations in mind, we set out to design
and synthesize the first 4-hydroxytamoxifen (HOTam)
analogue containing a hydrophilic link to diethylene-
triaminepentaacetic acid (DTPA), a common23 ligand
for chelation of radionuclides and MRI-elements. The
synthesis and ER-binding measurements of HOTam-
DTPA are described here. Our rationale for using
4-hydroxytamoxifen as the targeting element is two-
fold: (i) the greater ER-binding affinity of 4-hydroxy-
tamoxifen relative to tamoxifen is expected to improve
uptake and retention of HOTam-derived agents by
ER-rich tissue, while (ii) the 4-hydroxyl group, com-
bined with a hydrophilic ethylenediamine linker,24 is
expected to improve clearance from tissue that expresses
lower levels of ER. Thus, the increased hydrophilicity
and ER-binding affinity of this new anti-estrogen-
labeled DTPA are designed to improve retention of the
agent in ER-rich tissue.
Of greatest concern was whether an appended DTPA
would significantly affect interaction of the HOTam-
DTPA conjugate with the ER. It is reasonable to expect
that factors such as linker composition and length, and
site of attachment could alter the ER-binding affinity of
the conjugate. The knowledge that the A-ring of
tamoxifen cannot be modified, including the A-ring
b-(dimethylamino)ethoxy substituent,25 without altering
the pharmacological activity helped guide our selection
for a site of ‘chelate arm’ attachment onto HOTam.26
Furthermore, the recent crystallographic findings of
Brzozowski et al.27 and Shiau et al.28 indicate that
tamoxifen and HOTam bind the ER in a manner that
places the B- and C-rings firmly within a binding
pocket. In agreement with this model for ER-binding, in
vitro and in vivo studies of 5, a substituted C-ring ana-
logue, indicated very limited ER-binding.20 In contrast
to the binding requirements for the aryl rings, the ethyl
side chain of tamoxifen protrudes out of the ER binding
pocket and thus appears to be a suitable position for
functionalization. This assumption is supported by the
results of an [3H]-17b-estradiol competition binding
assay that indicated the ethyl side chain derivative 4
bound ER.18 Although no other ER-binding data for
ethyl side chain derivatives is available, we felt that
functionalization of hydroxytamoxifen at this site is the
most attractive option for attachment of DTPA.
A final consideration in our design of HOTam-DTPA
involves the mode of DTPA attachment. Recent studies
Figure 2. Tamoxifen radionuclides and chelates for imaging.
Tr=CPh3.

M. R. Lashley et al. / Bioorg. Med. Chem. 10 (2002) 4075–4082
4077
We modified the procedure for assembly of the tamoxi-
fen core first described by Olier-Reuchat et al.34 by
reacting benzophenone 13 with the 1,6-dianion gener-
ated from 5-phenyl-1-pentanol (14) (Scheme 2). This
unique example of 1,2-addition by a 1,6-dianion gave
adduct 15 as a mixture of diastereomers in good yield.
However, all attempts to selectively dehydrate the ter-
tiary benzylic alcohol of 15 resulted in the formation of
an undesired cyclized product.35 We circumvented this
problem by silylating the primary alcohol and then
effecting dehydration. Treatment of silyl ether 16 with
thionyl chloride in pyridine gave alkene 17 as a separ-
able 1:1 mixture of E and Z isomers in quantitative
yield. The two isomers are differentiated by the 1H
NMR chemical shifts of the respective methylene
groups adjacent to the A-ring side chain oxygen.36
Although the isomeric forms of tamoxifen and its
metabolites have different therapeutic effects, both iso-
mers bind the ER, which, for imaging and diagnostic
purposes, obviates the need for isomer separation.12a
Furthermore, the observation that the E and Z isomers
of hydroxytamoxifen readily isomerize in vivo also sug-
gests that 17 may be elaborated as a mixture of iso-
mers.37 We therefore used 17 as obtained and pursued
conversion of its silyl ether into a suitable leaving group
for nucleophilic attachment of the ethylenediamine linker.
Figure 3. Hydroxytamoxifen-linked chelate.
point toward maintaining the full complement of
DTPA-carboxylates for maximum stability of the metal-
ligand complex.29 Several investigators have synthesized
functionalized DTPA derivatives so that covalent
attachment of the chelate need not occur through one of
the five DTPA carboxylic acid groups.30 With this in
mind, we selected a hydrophilic L-aspartic acid-derived
DTPA which can be conjugated without consuming any
of the DTPA-carboxylates.31 This feature and the
aforementioned considerations led to the design of
HOTam-DTPA, the target chelate represented by
structure 7 (Fig. 3).
Results and Discussion
Synthesis
We envisioned a convergent synthesis of 7 wherein the
alkene would result from dehydration of a benzylic
alcohol. Fragment coupling to form the benzylic alcohol
suggested the synthesis of an A,B-ring benzophenone.
Thus, we prepared benzophenone derivative 13 (Scheme 1)
from methoxymethyl protected p-anisaldehyde (9)32 and
aryl bromide 11.33 Treatment of 9 with the Grignard
reagent derived from 11 gave the corresponding alcohol,
12, and subsequent MnO2 oxidation provided benzo-
phenone 13 in near quantitative yield.
Scheme 2. (a) (i) t-BuOK (1 equiv), pentane, rt, 5 min, (ii) s-BuLi
(2 equiv), TMEDA (2 equiv), 0 ꢂC–rt, 1 h, (iii) 13 (0.17 equiv), THF,
0 ꢂC–rt, 4 h, 80%; (b) TBS-Cl, imidazole, DMAP (cat.), CH2Cl2, rt,
12 h, 79%; (c) SOCl2, pyridine, ꢁ10 ꢂC, 4.5 h, 99%; (d) TBAF, THF,
rt, 6 h, 98%; (e) CBr4 (1.2 equiv), PPh3 (1.2 equiv), CH2Cl2, 0 ꢂC–rt,
1 h; (f) MeNHCH2CH2NHMe (10 equiv), CH3CN, rt, 12 h, 73% two
steps.
Scheme 1. (a) NaH (1.1 equiv), MOMCl (1 equiv), DMF, 0 ꢂC–rt;
.
4.5 h, 74%; (b) K2CO3 (4 equiv), Me2NCH2CH2Cl HCl (2 equiv),
DMF, rt, 24 h, 80%; (c) (i) Mg, THF, reflux 2 h, (ii) 9, THF, 0 ꢂC–rt,
3.5 h, 97%; (d) MnO2 (20 equiv), pentane, rt, 18 h, 98%.

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M. R. Lashley et al. / Bioorg. Med. Chem. 10 (2002) 4075–4082
Figure 4. Competition by diethylstilbestrol, tamoxifen and chelate 22
for [3H]-17b-estradiol binding to the ER. Calf uterine cytosol was
incubated with 3 nM [3H]-17b-estradiol in the absence or presence of
increasing concentrations of diethylstilbestrol (DES), tamoxifen
(TAM) or HOTam–DTPA (22) for 2 h at 4 ꢂC. Specific binding and
competitive displacement of [3H]-17b-estradiol was determined using
the dextran-coated charcoal binding assay as described in the material
and methods. Values are expressed as the meanꢃSD of incubations
performed in triplicate.
Scheme 3. (a) DCC, HOBT, 20, CHCl3, 64%; (b) TFA, Anisole
(30 equiv), CH2Cl2, ꢁ10 ꢂC – rt, 12 h.
Silyl ether deprotection using n-Bu4NF gave alcohol 18
in near quantitative yield. The alcohol was converted to
its tosylate derivative using standard conditions, but
this tosylate was prone to decomposition. Attempts to
directly convert silyl ether 17 to the corresponding bro-
mide (PPh3, Br2) also failed.38 We were gratified, how-
ever, when application of a literature method39 for
alcohol to bromide conversion transformed 18 to the
corresponding bromide. Not surprisingly, the bromide
was also unstable and reacted within hours at room
temperature. Thus, immediately after its formation, the
crude bromide was passed through a short SiO2 column
and then immediately treated with an acetonitrile solu-
tion of N,N0-dimethylethylenediamine. In this way, we
obtained amine 19 from alcohol 18 in a two-step 73%
yield.
competitive ligand binding analysis. Equilibrium bind-
ing of [3H]-17b-estradiol to the ER in the presence of
increasing concentrations of a competitor provides a
means of estimating the relative binding affinity of the
competitor to the ER. Comparison of the resulting
competitive binding curves (Fig. 4) revealed that the
relative binding affinity of HOTam-DPTA (22) was
ꢀ10-fold lower than that of tamoxifen and ꢀ200 fold
lower than that of the most potent ER ligand diethyl-
stilbestrol. While these data suggest that ligand 22 binds
ER with greater affinity than other tamoxifen ligands
for which ER-binding data is available, the in vivo suf-
ficiency of its metal chelates in diagnostic studies needs
to be determined experimentally. Considering that the
ER content in estrogen-responsive cells is roughly
100,000 ERs per cell,42 it is unlikely that the corre-
sponding gadolinium chelate of 22 would afford the
detection sensitivity needed to image ER positive
tumors using MRI methods. However, since signal
detection in scintigraphy is approximately 2–3 orders of
magnitude more sensitive than in MRI, the corre-
sponding indium and technetium chelates of 22 may be
used for this purpose. Work is in progress to evaluate
the in vivo diagnostic capability of chelate 7 (M=111In).
At this stage in the synthesis, we opted to attach the
DTPA-chelate to the ethylenediamine moiety of 19. To
reduce DTPA chelation of undesired metals during pre-
paration and handling, we attached the t-butyl-protected
DTPA analogue 20 (Scheme 3).31 HOBT-mediated DCC
coupling of 19 and 20 gave hydroxytamoxifen analogue
21. Purification of 21 was achieved using normal silica
gel chromatography. The MOM group and all t-butyl
esters were cleaved by treatment of 21 with TFA and
anisole40 to liberate HOTam-DTPA chelate 22. The
average concentration of free chelating groups in a
given solution of 22, determined using the 57Co-method
developed by Meares et al.41 indicated that 73% of the
theoretical titer of 22 was available for chelation.
Conclusion
To our knowledge, no reported imaging agents have
been prepared using 4-hydroxy-tamoxifen as the target-
ing element. The investigations reported here describe a
method for attaching DTPA to the ethyl side chain of
HOTam and the influence of this structural modification
on ER-binding. Although modification of the ethyl side
chain with a hydrophilic EDA–DTPA substituent does
With HOTam-DTPA fully assembled, we next eval-
uated the effect of the ethyl side chain modifications on
the ability of 22 to bind to the ER by performing a

M. R. Lashley et al. / Bioorg. Med. Chem. 10 (2002) 4075–4082
4079
decrease its affinity for the ER, the ability of the con-
jugate to effectively compete with [3H]-17b-estradiol for
binding to the ER at relatively low concentrations
demonstrates that it still remains a relatively potent
ligand. In fact, the relative binding affinity of HOTam–
DTPA is still greater than that of numerous ER ligands
that have been shown to produce effects in cells and
animals in vivo.4,12b
temperature was added MnO2 (55.77 g, 642 mmol). The
suspension was stirred at room temperature for 18 h and
then filtered by passing through a bed of Celite. The
retentate was washed with pentane (200 mL) and then
diethyl ether (200 mL). The combined solvents were
removed in vacuo to give 13 (10.35 g, 98%) as a reddish
oil that was used without further purification; Rf=0.26
(CH2Cl2/MeOH, 9:1); IR 2944, 1648, 1603, 1507 cmꢁ1
;
1H NMR d 7.69–7.73 (m, 4H), 7.03 (d, J=8.8 Hz, 2H),
6.91 (d, J=8.2 Hz, 2H), 5.18 (s, 2H), 4.08 (t, J=5.9 Hz,
2H), 3.43 (s, 3H), 2.71 (t, J=5.9 Hz, 2H), 2.29 (s, 6H);
13C NMR d 194.5, 162.3, 160.5, 132.4, 132.2, 131.8,
130.7, 115.6, 114.2, 94.2, 66.3, 58.3, 56.4, 46.0; MS (EI)
m/z (rel intens) 330 (MH+ 100), 259 (50); HRMS (EI)
calcd for C19H23NO4 329.1627, found 330.1692 (MH+).
Experimental procedures
All reactions were carried out under an atmosphere of
nitrogen. CH2Cl2 was distilled from calcium hydride
immediately prior to use. THF and Et2O were distilled
from sodium benzophenone ketyl immediately prior to
use. Column chromatography was carried out using
230–400 mesh silica gel, slurry packed in glass columns,
eluting with the solvents indicated. Yields were calcu-
lated for material judged to be homogeneous by TLC
and NMR. TLC was performed on kieselgel 60 F254
[4-(2-Dimethylamino-ethoxy)-phenyl]-(4-methoxymethoxy-
phenyl)-2-phenyl-hexane-1,6-diol (15). To a stirred sus-
pension of t-BuOK (4.03 g, 36.0 mmol) in pentane
(36 mL) at room temperature was added 5-phenyl-
1-pentanol (14) (6.06 mL, 36.0 mmol). The mixture was
cooled to 0 ꢂC and s-BuLi (55.3 mL of a 1.3 M solution,
71.9 mmol) was added dropwise. To the resultant red
suspension was added TMEDA (10.9 mL, 72.0 mmol)
followed by warming to room temperature. After stir-
ring 1 h, the dark red solution was recooled to 0 ꢂC and
an ice cold solution of the ketone 13 (1.98 g, 6.00 mmol)
in dry THF (20 mL) was added dropwise via cannula.
After the addition was complete, the mixture was stirred
for 2 h at room temperature and then quenched by
pouring over saturated aqueous NaHCO3 (50 mL). The
aqueous layer was extracted three times with diethy-
lether (25 mL). The combined organics were washed
successively with water and brine, then dried (Na2SO4).
The solvents were evaporated and the residue was pur-
ified by flash chromotography, eluting with a gradient
(CH2Cl2 to CH2Cl2/MeOH, 19:1), to obtain 15 (2.55 g,
80%) as a 1:1 mixture of diastereomers; Rf=0.35
(CH2Cl2/MeOH, 4:1); IR 3400, 2942, 1607, 1508,
1235 cmꢁ1; 1H NMR d 7.33–7.37 (m, 4H), 6.92–7.05 (m,
16H), 6.80 (d, J=6.8 Hz, 2H), 6.66 (d, J=6.8 Hz, 2H),
6.55 (d, J=9.0 Hz, 2H), 5.09 (s, 2H), 4.97 (s, 2H), 3.99
(t, J=5.9 Hz, 2H), 3.87 (t, J=5.9 Hz, 2H), 3.55 (s, 1H),
3.52 (s, 1H), 3.40 (s, 3H), 3.38 (m, 4H), 3.31 (s, 3H), 2.66
(t, J=5.9 Hz, 2H), 2.57 (t, J=5.9 Hz, 2H), 2.40 (bs, 2H),
2.26 (s, 6H), 2.23 (bs, 2H), 2.21 (s, 6H), 1.6–1.8 (m, 4H),
1.3–1.5 (m, 4H), 1.0–1.2 (m, 4H); 13C NMR d 157.3,
156.9, 155.8, 155.3, 140.1, 139.9, 139.4, 138.8, 138.1,
130.05, 130.03, 127.7, 127.4, 126.9, 126.8, 126.3, 115.6,
115.1, 113.9, 113.4, 94.39, 94.30, 80.3, 65.7, 65.5, 62.6,
58.2, 58.1, 56.0, 55.8, 54.4, 45.8, 45.7, 32.6, 30.08, 30.00,
24.1; MS (EI) m/z (rel intens) 494 (MH+, 100), 332 (45),
58, (40); HRMS (EI) calcd for C30H39NO5 493.2828,
found 494.2895 (MH+).
1
plates. H NMR (300 MHz) and 13C NMR (75 MHz)
were recorded in CDCl3. High resolution mass spectro-
metry was performed by Mass Spectrometry Service
Lab, UC Riverside and combustion analyses were per-
formed by Midwest Microlab, Indianapolis, IN. Infra-
red (IR) data were obtained on neat samples.
[4-(2-Dimethylamino-ethoxy)-phenyl]-(4-methoxymethoxy-
phenyl)-methanol (12). A three necked flask fitted with a
reflux condenser and a pressure equalizing addition
funnel was flame dried and charged with magnesium
turnings (1.34 g, 55.2 mmol) and THF (80.0 mL). The
suspension was heated to 60 ꢂC and a mixture of 11
(11.2 g, 46.0 mmol) and 1,2-dibromoethane (0.60 mL,
7.00 mmol) in dry THF (10 mL) was added dropwise via
the addition funnel. The resulting suspension was
heated at the reflux temperature until the metal was
consumed and then cooled to room temperature. The
reaction solution was transferred via cannula into an
empty, flame-dried flask charged with argon and then
cooled to 0 ꢂC. To the cooled solution was added a chilled
solution of 9 (5.35 g, 32.2mmol) in THF (15 mL) via can-
nula over 90min. After addition, the mixture was stirred
for 1 h at 0 ꢂC, and then warmed to room temperature and
stirred for 1 h. The reaction was quenched by slowly
pouring into saturated aqueous NaHCO3 (100mL). The
aqueous layer was extracted with diethyl ether (3ꢄ50mL),
the combined organic fraction was washed with water
(100mL), brine (100mL) and dried (Na2SO4). The sol-
vents were removed in vacuo to give 12 (10.4 g, 97%) as a
light yellow oil; Rf=0.15 (CH2Cl2/MeOH, 9:1); IR 3360,
1
2780, 1609, 1508cmꢁ1; H NMR d 7.29–7.33 (m, 4H),
7.02 (d, J=6.4Hz, 2H), 6.90 (d, J=8.8 Hz, 2H), 5.73 (s,
1H), 5.18 (s, 2H) 4.03 (t, J=5.9 Hz, 2H), 3.89 (br. s, 1H),
3.50 (s, 3H), 2.73 (t, J=5.9Hz, 2H), 2.31 (s, 6H); 13C
NMR d 157.6, 156.1, 138.0, 136.8, 127.5, 127.4, 94.1, 74.7,
65.4, 57.7, 55.7, 45.4; MS (EI) m/z (rel intens) 332 (MH+,
26), 58 (100), 72 (10); HRMS (EI) calcd for C19H25NO4
331.1764, found 332.1850 (MH+).
6-(tert-Butyl-dimethyl-silanyloxy)-1-[4-(2-dimethylamino-
ethoxy)-phenyl]-(4-methoxymethoxy-phenyl)-2-phenyl-
hexan-1-ol (16). To a stirred solution of diol 15 (4.01 g,
8.13 mmol) in CH2Cl2 (30 mL) at room temperature was
added imidazole (1.13 g, 16.7 mmol) and DMAP
(50.0 mg, 0.40 mmol). After 5 min TBSCl (1.29 g,
8.54 mmol) was added and the reaction mixture was
stirred overnight at room temperature. The reaction
[4-(2-Dimethylamino-ethoxy)-phenyl]-(4-methoxymethoxy-
phenyl)-methanone (13). To a solution of carbinol 12
(10.63 g, 32.07 mmol) in pentane (640 mL) at room

4080
M. R. Lashley et al. / Bioorg. Med. Chem. 10 (2002) 4075–4082
mixture was poured over saturated aqueous NaHCO3
(30 mL), the organic layer was dried over anhydrous
Na2SO4, filtered and concentrated in vacuo. The crude
oil was purified by flash chromotography, eluting with a
gradient (CH2Cl2–CH2Cl2/MeOH, 9:1, v/v), to give 16
(3.89 g, 79%) as a pale yellow oil; Rf=0.33 (CH2Cl2/
MeOH, 9:1); IR 2950, 1607, 1507, 1257 cmꢁ1; 1H NMR
d 7.42 (m, 4H), 6.99–7.16 (m 16H), 6.88 (d, J=5.9 Hz,
2H), 6.75 (d, J=5.9 Hz, 2H), 6.63 (d, J=5.9 Hz, 2H)
5.17 (s, 2H), 5.05 (s, 2H), 4.07 (t, J=5.9 Hz, 2H), 3.93
(t, J=5.9 Hz, 2H), 3.62 (s, 1H), 3.59 (s, 1H), 3.48 (s,
3H), 3.40–3.47 (m, 4H), 3.39 (s, 3H), 2.74 (t, J=5.9 Hz,
2H), 2.65 (t, J=5.9 Hz, 2H), 2.34 (s, 6H), 2.32 (bs, 2H),
2.29 (s, 6H), 1.69–1.83 (m, 4H), 1.33–1.49 (m, 4H), 1.1–
1.15 (m, 4H); 0.88 (s, 18H), 0.00 (s, 12H); 13C NMR d
158.6, 157.3, 156.8, 155.7, 155.2, 140.2, 140.0, 139.5, 138.9,
138.2, 130.0, 129.9, 127.5, 127.3, 126.9, 126.8, 126.1, 120.6,
115.5, 115.0, 114.4, 113.8, 113.4, 94.3, 94.2, 80.2, 65.7,
65.69, 65.60, 62.8, 58.19, 58.13, 55.8, 55.7, 54.3, 53.3, 45.8,
45.7, 32.6, 30.0, 29.9, 25.8, 23.9, 18.1, ꢁ5.4; MS (EI) m/z
(rel intens) 608 (MH+ 100), 330 (65), 259 (40); HRMS (EI)
calcd for C36H53NO5Si 607.3693, found 608.3788 (MH+).
column chromotography, eluting with a gradient
(CH2Cl2–CH2Cl2/MeOH, 19:1), to afford 18 (1.12 g,
98%) as a pale yellow oil; IR 3393, 2932, 1601, 1498,
1236 cmꢁ1 1H NMR d 7.08–7.15 (m, 14H), 7.01 (d,
;
J=0.03 Hz, 2H), 6.89 (d, J=0.03 Hz, 2H), 6.75–6.87 (m,
4H), 6.65 (d, J=0.03 Hz, 2H), 6.52 (d, J=0.03 Hz, 2H),
5.18 (s, 2H), 5.03 (s, 2H), 4.07 (t, J=5.9 Hz, 2H), 3.91 (t,
J=5.9 Hz, 2H), 3.50 (s, 3H), 3.46–3.48 (m, 4H), 3.39 (s,
3H), 2.74 (t, J=5.9 Hz, 2H), 2.63 (t, J=5.9 Hz, 2H),
2.43–2.47 (m, 4H), 2.33 (s, 6H), 2.27 (s, 6H), 1.65 (bs,
2H), 1.44–1.49 (m, 4H), 1.33–139 (m, 4H); 13C NMR d
157.4, 156.6, 155.8, 155.0, 142.5, 139.5, 139.4, 138.2,
137.2, 136.7, 136.0, 135.5, 131.74, 131.71, 130.47,
130.44, 129.4, 127.8, 125.9, 125.8, 115.7, 114.9, 114.0,
113.2, 94.4, 94.2, 65.6, 65.4, 62.3, 58.1, 58.0, 55.9, 55.8,
45.7, 45.6, 35.5, 35.4, 32.6; MS (EI) m/z (rel intens) 476
(MH+, 100), 215 (10); HRMS (EI) calcd for
C30H37NO4 475.2723, found (MH+) 476.2786.
N-[6-[4-(2-Dimethylamino-ethoxy)-phenyl]-6-(4-methoxy-
methoxy-phenyl)-5-phenyl-hex-5enyl]-N,N0-dimethyl-eth-
ane-1,2-diamine (19). To a solution of 18 (1.01 g,
2.13 mmol) and CBr4 (0.85 g, 2.55 mmol) in CH2Cl2
(5 mL) at 0 ꢂC was added dropwise a solution of PPh3
(0.67 g, 2.55 mmol) in CH2Cl2 (5 mL) over 30 min. The
reaction mixture was warmed to room temperature and
stirred 30 min whereupon the solvent was removed by
rotary evaporation. The residue was dissolved in a
minimal quantity of ethyl acetate and passed through a
short column of silica gel, eluting with CH2Cl2/MeOH
(9:1, v/v). The fractions were concentrated to afford the
corresponding bromide which was immediately dis-
solved in acetonitrile (10 mL) and treated at room tem-
perature with N,N0-dimethylethylenediamine (2.3 mL,
21 mmol). After stirring 12 h, the reaction mixture was
poured into water (50 mL) and the resultant mixture
was extracted using CH2Cl2 (3ꢄ50 mL). The combined
organic layer was washed with water, brine, and then
dried (Na2SO4). The solvent was removed and the resi-
due purified by column chromotography, eluting with a
gradient (CH2Cl2/MeOH/TEA, 19/1/0.1 to 4/1/0.1) to
give 19 (0.85 g, 73%) as a viscous yellow oil; Rf=0.12
(2-{4-[6-(tert-Butyl-dimethyl-silanyloxy)-1-[4-(2-dimethyl-
amino-ethoxy)-phenyl]-(4-methoxymethoxy-phenyl)-2-
phenyl-hex-1-enyl]-phenoxy}-ethyl)-dimethyl-amine (17).
To a solution of carbinol 16 (108 mg, 0.18 mmol) in dry
pyridine (0.4 mL) at ꢁ10 ꢂC was added SOCl2
(0.023 mL, 0.32 mmol) via syringe, and the reaction was
stirred at this temperature 4.5 h. The reaction mixture
was then poured into water and extracted with diethyl
ether. The organic layer was dried (Na2SO4) and then
concentrated in vacuo to give the crude product. The
crude, orange oil was purified by column chromoto-
graphy, eluting with a gradient (CH2Cl2 to CH2Cl2/
MeOH, 9:1), to afford 17 (104mg, 99%) as a 1:1 mixture
of diastereomers; Rf=0.27 (CH2Cl2/MeOH, 9:1); IR 2951,
1
1609, 1507, 1240 cmꢁ1; H NMR d 7.07–7.14 (m, 14H),
6.97 (d, J=11.7 Hz, 2H), 6.86 (d, J=11.1 Hz, 2H), 6.74–
6.77 (m, 4H), 6.63 (d, J=11.7Hz, 2H), 6.52 (d, J=11.7 Hz,
2H), 5.18 (s, 2H), 5.04 (s, 2H), 4.13 (t, J=5.9 Hz, 2H), 3.97
(t, J=5.9 Hz, 2H), 3.51 (s, 3H), 3.47 (m, 4H), 3.40 (s, 3H),
2.84 (t, J=5.9 Hz, 2H), 2.75 (t, J=5.9Hz, 2H), 2.42 (s,
6H), 2.38 (m, 4H), 2.36 (s, 6H), 1.28–1.40 (m, 8H), 0.84 (s,
18H), ꢁ0.02 (s, 12H); 13C NMR d 157.4, 156.6, 155.8,
255.0, 142.6, 139.8, 139.7, 138.0, 137.4, 136.8, 136.1, 135.6,
131.7, 130.5, 129.5, 127.7, 125.8, 115.6, 114.9, 113.9, 113.2,
94.4, 94.3, 65.7, 65.5, 62.9, 58.3, 58.2, 56.0, 55.9, 45.88,
45.84, 35.6, 32.9, 25.9, 25.1, 18.2, ꢁ5.3; MS (EI) m/z (rel
intens) 590 (MH+ 100); HRMS (EI) calcd for
C36H51NO4Si 589.3587, found 590.3652 (MH+).
(CH2Cl2/MeOH, 4:1); IR 2941, 1604, 1501, 1236 cmꢁ1
;
1H NMR d 7.16–7.07 (m, 14H), 6.9 (d, J=6.6 Hz, 2H),
6.68 (d, J=8.4 Hz, 2H), 6.76–6.73 (m, 4H), 6.66 (d,
J=6.6, 2H), 6.54 (d, J=8.6, 2H), 5.18 (s, 2H), 5.03 (s,
2H), 4.07 (t, J=5.9 Hz, 2H), 3.90 (t, J=5.9 Hz, 2H),
3.50 (s, 3H), 3.39 (s, 3H), 2.73 (t, J=5.9 Hz, 2H), 2.62 (t,
J=5.9 Hz, 2H), 2.61–2.56 (m, 2H), 2.45–2.41 (m, 10H),
2.38 (s, 6H), 2.34 (s, 6H), 2.27 (s, 6H), 2.20–2.16 (m,
6H), 2.11 (s, 6H), 1.34–1.28 (m, 8H); 13C NMR d 157.4,
156.6, 155.8, 155.0, 142.6, 139.7, 139.6, 138.1, 137.3,
136.8, 136.1, 135.6, 131.8, 131.7, 130.5, 130.4, 129.5,
127.7, 125.8 (2), 115.7, 114.9, 114.0, 113.6, 94.4, 94.3,
65.8, 65.5, 58.3, 58.2, 57.8, 56.7, 56.0, 55.9, 49.2, 45.8 (2),
42.1, 36.3, 35.8, 35.7, 27.3, 26.7; MS (EI) m/z (rel intens)
546 (MH+, 100), 501 (55), 147 (45); HRMS (EI) calcd
for C34H47N3O3 545.3617, found (MH+) 546.3714.
6-[4-(2-Dimethylamino-ethoxy)-phenyl]-6-(4-methoxy-
methoxy-phenyl)-5-phenyl-hex-5en-1-ol (18). To a solu-
tion of silyl ether 17 (1.43 g, 2.4 mmol) in anhydrous
THF (10 mL) at 0 ꢂC was added TBAF (4.8 mL of a
1.0 M solution in THF, 4.8 mmol). The reaction was
allowed to room temperature and stirred for 6 h. The
reaction mixture was poured over water (10 mL) and
extracted several times with diethyl ether. The combined
organic layer was washed with water (30 mL), brine
(30 mL), and then dried (Na2SO4). The solvents were
concentrated in vacuo, and the residue was purified by
tert-Butyl(2S)-3-(N-{2-[((5Z)-6-{4-[2-(dimethylamino)-
ethoxy]phenyl}-5,6-diphenylhex-5-enyl)methylaino]ethyl}-
N-methylcarbamoyl)-2-{bis[2-(bis{[(tert-butyl)oxycarbonyl]
methyl}amino)ethyl]amino}propanoate (21). To a solution

M. R. Lashley et al. / Bioorg. Med. Chem. 10 (2002) 4075–4082
4081
of 20 (0.877 g, 1.20 mmol) and amine 19 (0.658 g,
1.20 mmol) in CHCl3 (8.0 mL) at 0 ꢂC were added DCC
(0.272 g, 1.318 mmol) and HOBT (0.238 g, 1.56 mmol).
The reaction was warmed to room temperature and
stirred overnight. The reaction mixture was poured into
water (20 mL) and extracted several times with CHCl3.
The combined extracts were dried (Na2SO4), and the
solvent was removed in vacuo. The crude yellowish oil
was purified using column chromotography using a
gradient elution (CH2Cl2/MeOH/TEA (19/1/0.1, v/v to
CH2Cl2/MeOH:TEA, 4/1/0.1) to give 21 (0.953 g, 64%)
as a viscous yellow oil; Rf=0.05 (CH2Cl2/MeOH, 4:1),
(v/v) glycerol and 20 mM sodium molybdate). Diluted
cytosol (0.5 mL) was incubated with 3 nM [3H]
17b-estradiol (33.5 Ci/mmol, NEN Life Sciences, Bos-
ton, MA) in the absence or presence of the indicated
concentration of each competitor, for 2 h at 4 ꢂC. Die-
thylstilbestrol and tamoxifen were obtained from Sigma
Chemical Company (St. Louis). Incubations were then
mixed with a dextran-coated charcoal pellet that had
been prepared by centrifuging 0.5 mL of a DCC solu-
tion (0.5 mg charcoal (Norit A, Fisher Scientific)/
0.05 mg dextran (average MW 127,000)/mL water) at
3500 rpm for 15 min and removing the supernatant. The
sample/DCC mixture was incubated for 15 min at 4 ꢂC,
followed by centrifugation at 3500 rpm for 15 min and
the supernatant collected. Radioactivity in an aliquot of
the supernatant (300 mL) was then determined by liquid
scintillation counting. Specific binding of [3H] estradiol
to the ER was computed by subtracting the amount of
[3H] estradiol bound in the presence of competitor (die-
thylstilbestrol, tamoxifen or 22) from the amount of
total [3H] estradiol bound in the absence of excess com-
petitor. Specific binding values were expressed as fmoles
[3H] estradiol specific binding/mg protein.
1
IR 2974, 1724, 1642, 1153 cmꢁ1; H NMR d 7.08–7.14
(m, 8H), 6.99 (d, J=8.7 Hz, 2H), 6.74 (d, J=8.1 Hz,
2H), 6.54 (8.7 Hz, 2H), 5.18 (s, 2H), 3.94 (t, J=5.4 Hz,
2H) 3.80 (m, 1H), 3.50 (s, 3H), 3.422 (s, 8H) 2.98 (s,
2H), 2.82–2.85 (m, 2H), 2.69–2.72 (m, 10H), 2.37–2.41
(m, 5H), 2.32 (s, 8H), 2.17–2.23 (m, 4H), 1.80 (m, 4H)
1.44 (s, 45H); 13C NMR d 171.3,171.2, 170.6, 156.1,
155.9, 143.6, 142.3, 139.1, 137.0, 135.8, 131.8, 130.4,
129.4, 127.9, 127.5, 126.0, 124.5, 123.5, 118.7, 115.8,
114.9, 113.2. 110.6, 94.4, 64.6, 61.2, 57.5, 56.8, 56.1,
56.0, 53.7, 51.2, 45.0, 40.8, 35.8, 35.4, 33.9, 29.6, 28.19,
28.14, 28.0; MS (EI) m/z (rel intens) 1282 (MNa+, 100),
560 (25), 825 (15); HRMS (EI) calcd for C70H110N6O14
1258.8080, found (MNa+) 1281.7943. Anal. calcd for
C70H110N6O14: C, 66.74; H, 8.80; N, 6.67. Found C,
66.27; H, 8.61; N, 6.66.
Acknowledgements
We thank the University of California Cancer Research
Coordinating Committee and National Institute of
Envirnonmental Health Sciences (ESO4699) for support
of this research. MRL is grateful for support from the
SEGRF program of the Lawrence Livermore National
Laboratory. The departmental spectrometers, Varian
Invoa 400 and Mercury 300 MHz, were funded by the
National Science Foundation (CHE-9808183).
(2S)-3-(N-{2-[((5Z)-6-{4-[2-(dimethylamino)ethoxy]phenyl}-
6-(4-hydroxyphenyl)-5-phenylhex-5-enyl)methylamino]-
ethyl}-N-methylcarbamoyl)-2-(bis{2-[bis(carboxymethyl)
amino]ethyl}amino)propanoic acid (22). Prior to
removal of the MOM and tert-butyl protecting groups,
all glassware was soaked in a 6M HCl bath overnight.
The glassware was then rinsed with nano pure water
and dried. To a solution of 21 (0.060 g, 0.047 mmol) in
CH2Cl2 (0.5 mL) at ꢁ10 ꢂC was added anisole
(0.155 mL, 1.43 mmol). After 5 min, TFA (0.11 mL,
1.43 mmol) was added dropwise. The reaction was stir-
red for 2 h at ꢁ10 ꢂC and then was warmed to room
temperature. The anisole and the TFA were removed by
reduced pressure distillation. The residue was dissolved
in CH2Cl2 (0.5 mL) and again treated with TFA
(0.11 mL, 1.43 mmol) followed by stirring at room tem-
perature overnight. The solvent and excess TFA were
removed by bulb-to-bulb distillation, and the residue
was dissolved in a 19:1 mixture of CH2Cl2/MeOH
(5.5 mL) to obtain a stock solution of 22. The titer of 22
in the stock solution was established using the method
of analysis developed by Meares.41 Measurement of
bound (7, M=57Co) versus unbound (22) material was
performed using Selecto Scientific Silica Gel 60, F-254
TLC plates eluting with normal saline. The results of
the 57Co assay counted using a LKB Wallac 1282 uni-
versal gamma counter determined the effective con-
centration of 22 equaled 6.25 mM (73%).
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