Click synthesis of estradiol-cyclodextrin conjugates as cell compartment selective estrogens

Article abstract of DOI:10.1016/j.bmc.2009.11.046

Cyclodextrin (CD) is a well known drug carrier and excipient for enhancing aqueous solubility. CDs themselves are anticipated to have low membrane permeability because of relatively high hydrophilicity and molecular weight. CD derivatization with 17-beta estradiol (E₂) was explored extensively using a number of different click chemistries and the cell membrane permeability of synthetic CD-E₂ conjugate was explored by cell reporter assays and confocal fluorescence microscopy. In simile with reported dendrimer-E₂ conjugates, CD-E₂ was found to be a stable, extranuclear receptor selective estrogen that penetrated into the cytoplasm.

Full text of DOI:10.1016/j.bmc.2009.11.046

Bioorganic & Medicinal Chemistry 18 (2010) 809–821
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Click synthesis of estradiol–cyclodextrin conjugates as cell compartment
selective estrogens
Hye-Yeong Kim a, Johann Sohn a, Gihani T. Wijewickrama a, Praneeth Edirisinghe a, Teshome Gherezghiher a,
Madhubani Hemachandra a, Pei-Yi Lu a, R. Esala Chandrasena a, Mary Ellen Molloy b, Debra A. Tonetti b,
Gregory R. J. Thatcher a,
*
a Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 S. Wood Street, Chicago, IL 60612, United States
b Department of Biopharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, 833 S. Wood Street, Chicago, IL 60612, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclodextrin (CD) is a well known drug carrier and excipient for enhancing aqueous solubility. CDs them-
selves are anticipated to have low membrane permeability because of relatively high hydrophilicity and
molecular weight. CD derivatization with 17-beta estradiol (E2) was explored extensively using a number
of different click chemistries and the cell membrane permeability of synthetic CD–E2 conjugate was
explored by cell reporter assays and confocal fluorescence microscopy. In simile with reported dendri-
mer–E2 conjugates, CD–E2 was found to be a stable, extranuclear receptor selective estrogen that pene-
trated into the cytoplasm.
Received 19 September 2009
Revised 16 November 2009
Accepted 21 November 2009
Available online 27 November 2009
Keywords:
Cyclodextrin
Estrogen
Ó 2009 Elsevier Ltd. All rights reserved.
Bioconjugate
Membrane
Nuclear receptor
1. Introduction
in part on the assumed membrane impermeability of CD conju-
gates, based upon the natural hydrophilicity of CD conjugates
b-Cyclodextrins (CDs) are torus-shaped saccharides consisting
of a shallow interior cavity, a primary face of 7 hydroxyls and a sec-
ondary face of 14 hydroxyl groups. CDs and modified CDs have
many interesting biological properties due to their unique struc-
ture, which can be altered most readily by chemical modification
of the primary face.1,2 The hydrophobicity of the CD cavity has
dominated thinking in terms of CD host–guest chemistry via
formation of non-covalent inclusion complexes and native and
commercially available CD derivatives, the latter generally hetero-
geneous compounds, have been used as efficient drug carriers.3
Alternatively, the CD torus can be used as a scaffold to attach drug
moieties in order to enhance their effectiveness by facilitating drug
delivery or specificity; for example, CD–epoxysuccinyl peptide
conjugates were reported as inhibitors of membrane-associated
cathepsin B, known to be secreted in several tumor cell lines.4
The synthesized CD–peptide conjugates were full inhibitors of
extranuclear cysteine proteases and were reported not to be mem-
brane-permeant. Other examples of CD conjugates as specific li-
gands of cell-surface receptors include opioid peptides,5 gastrin
peptides,6 and mono- and oligosaccharides.7 These approaches rely
and a molecular weight (>1500) above the perceived limits of fac-
ile, passive membrane transport.
The development of chemically and biologically stable estro-
gen–macromolecule conjugates has been a challenge of increasing
importance given the interest in differentiating non-classical li-
gand-activated estrogen receptor (ER) actions versus the classical
ER function as a ligand-activated nuclear receptor. Estrogens are
able to elicit rapid responses that are thought to be mediated by
extranuclear receptors that may be the classic ER, splice variants,
or G-protein coupled receptors.8–10 The non-classical, extranuclear
action of E2 elicit activity via cytosolic kinase cascades, which may
have genomic effects via epigenetic actions or transcription factors
other than ER.11,12 The classical, genomic actions of ER as a nuclear
receptor have been differentiated from membrane-associated ER
activity by use of commercially available protein conjugates such
as E2–BSA12–14 and E2–peroxidase.15 These conjugates have been
widely used, but as remarked by Katzenellenbogen, they have crit-
ical limitations such as leakage of free E2 either non-covalently
bound or from biological degradation of the conjugate in cell cul-
tures.8,13,16 In order to overcome these problems and develop effi-
cient E2–macromolecule conjugates, Katzenellenbogen and co-
workers utilized a polyamidoamine (PAMAM) dendrimer as core
architecture instead of a protein, leading to an E2-conjugate selec-
tive for extranuclear ER.
* Corresponding author. Tel.: +1 312 355 5282; fax: +1 312 916 7107.
E-mail address: thatcher@uic.edu (G.R.J. Thatcher).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.046

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H.-Y. Kim et al. / Bioorg. Med. Chem. 18 (2010) 809–821
Click chemistry is commonly used today as a synonym for the
of (EE2)6ACD and (EE2)5ACD. Chromatographic and spectroscopic
examination revealed no evidence for an inclusion complex be-
tween 7EACD and either EE2 or the reaction by-product, 5, compat-
ible with the extensive washing and chromatography employed in
synthesis, and the known propensity of ACD to former weak inclu-
sion complexes.18 7EACD is comparable to the heterogeneous E2–
dendrimer conjugate described by Katzenellenbogen.
The lack of a homogeneous product from the imine synthetic
route (Scheme 1) was surprising given that the condensation of
2-pyridinecarboxaldehyde with 3 had been previously demon-
strated in our lab to proceed to completion, yielding the persubsti-
tuted ACD (7) after reduction in situ (Table 1, entry 2).26 To
examine the limitations of this synthetic methodology for persub-
stituted ACD derivatives, other approaches were attempted. Con-
Huisgen cycloaddition, although originally defined by Sharpless
as encompassing simple synthetic steps giving quantitative con-
versions.17 Quantitative procedures for formation of CD-conjugates
avoid the complex chromatography required to remove by-prod-
ucts of incomplete derivatization. Herein, novel estradiol–CD con-
jugates were prepared using click chemistry and the estrogenic
activity was assayed in order to test the ability of CD derivatives
to provide selective probes of estrogenic activity.
2. Results and discussion
2.1. Synthesis of ACD conjugates
densation of
3 with 4-benzyloxybenzaldehyde yielded the
A CD-conjugate persubstituted at the C6 position of the primary
face was the initial target. Aminocyclodextrins (ACDs) are a specific
subset of persubstituted CDs, which may be readily prepared by di-
rect amination of per-6-bromo-per-6-deoxy-CD or per-6-iodo-per-
6-deoxy-CD ACDs possess an annulus of amino groups in place of
the ether oxygens of native CD and a corona of pendant side
chains; at neutral pH the presence of the positively charged annu-
lus diminishes the ability of the ACD cavity to include hydrophobic
small molecules.18 The ACD scaffold provides ACD derivatives with
demonstrated, inherent biological activity.19 Synthesis from the
more reactive per-6-iodo-per-6-deoxy-CD was revealed by careful
chromatography to yield an intramolecular cross-linked impu-
rity,20 whereas synthesis via the less reactive per-6-bromo-per-6-
deoxy-CD is accomplished in quantitative steps requiring no chro-
matography (Scheme 1).21,22 However, this ‘click’ strategy is lim-
ited to reactions with amines in the liquid phase or with high
solubility in DMF,21 an alternative click methodology is imine for-
mation from reaction of aldehyde with per-6-amino-per-6-deoxy-
b-CD (3), easily obtained from the per-azido derivative 2, and sub-
sequent reduction, ultimately leading to the desired CD–E2 conju-
gate, 7EACD (Scheme 1).
desired product 8 (Table 1, entry 3). The possibility that complete
reaction of 3 with 4 was hindered by the steric bulk of the ethynyl
estradiol group was explored by using a longer linker arm between
estradiol and aldehyde groups. However, neither condensation of
10 (Scheme 2) nor the synthon 9 with ACD (3) gave the desired
imine products (Table 1, entries 5 and 6), indicating the preference
in imine condensation for the more reactive arylaldehydes. Other
alternatives were explored, including reaction via the known b-
CD derivative, 15 (Scheme 2; Table 1, entry 7).27 however, under
all reactions conditions tested, the persubstituted product was
the major, but not the sole product observed. The imine condensa-
tion reaction of 19 with 4 produced the monosubstituted E2-conju-
gate 1EACD (20; Table 1, entry 8), and a similar product (21) was
obtained by the simple nucleophilic displacement of the tosyl
group in mono-6-tosyl-b-CD using EE2 derivative 12 (Table 1, entry
9). Both E2-conjugates 20 and 21 contained small amounts of
unmodified b-CD carried through the synthesis as shown by NMR
spectra and MALDI-TOF mass spectroscopic analysis.
2.2. Synthesis of ‘qCD’ conjugates
EE2 is a biologically active ER ligand of synthetic utility because
of the reactivity of the terminal acetylene group.23 Elaboration of
EE2 is readily achieved using palladium catalyzed Sonogashira
cross coupling with halide reagents,24 allowing attachment of a
benzaldehyde moiety for ACD conjugation. Subsequent reduction
of benzaldehyde 4 gave the benzyl alcohol 5 that was used as a
control compound. The previously reported heterogeneous dendri-
mer E2–PAMAM conjugate was obtained by PAMAM condensation
with 4 in MeOH at room temperature.8,25 Similarly, condensation
of ACD (3) with 4 was attempted, followed by in situ NaBH4 reduc-
tion (Scheme 1; Table 1 entry 1). However, reaction did not go to
completion, giving a heterogeneous mixture that, from mass spec
and NMR examination, contained (EE2)7ACD as major product in
addition to smaller amounts of (EE2)6ACD and (EE2)5ACD (Fig. 1).
Optimization of reaction conditions failed to yield a homogeneous
product, however, semi-preparative RP-HPLC did yield conjugate
7EACD (6) that consists of (EE2)7ACD (>90%) with small amounts
Cycloaddition click chemistry is currently used extensively for
bioconjugation because of the efficient conversion and good yields
in aqueous media.28–30 Given the ease with which mono- and per-
substituted azido-CD are prepared, this synthetic methodology
was explored for E2–CD conjugates. The presence of seven triazole
groups in the annulus of such cyclodextrin conjugates is likely to
confer inherently different physicochemical properties to CD and
ACD conjugates, hence we coin the click-derived conjugates as
qCD derivatives.
A variety of click reactions were explored based upon literature
precedent (Table 2),31,32 monitoring conjugation products by MAL-
DI-TOF spectroscopy. In situ [1,3]-cycloaddition ligation was
unsuccessful from 1 (Table 2, entry 10), but was successful from
mono-6-tosyl-b-CD giving the desired product 22 in 50% conver-
sion along with unreacted b-CD contaminant 17 (Table 2, entry
11).33–35 The reaction mixture of 2 with seven equivalent of EE2
Scheme 1. Modifications of b-cyclodextrin.

Table 1
Per-6-amino-b-CD (ACD) derivatives and conjugates
Entry
CD
Ligand
Reaction condition
Product
1a
10–70 equiv
60–70 °C, 10 d
2b
8 equiv
8 equiv
rt, 5 h
rt, 7 d
3c
4c
8 equiv
8 equiv
60–70 °C, 18 h
60–70 °C, 18 h
5d
S.M recovered
S.M recovered
6d
10 equiv
21 equiv
60–70 °C, 10 d
60–70 °C, 10 d
7d
8e
1–3 equiv
21 equiv
60–70 °C, 3 d
60–70 °C, 7 d
9f
a
b
c
d
e
f
(1) DMF, 60–70 °C, 10 d, (2) NaBH4, MeOH, 2 h.
(1) rt, 5 h, (2) NaBH4, MeOH, 2 h.
(1) DMF, rt, 7 d, (2) NaBH4, MeOH, 2 h.
(1) DMF, 60–70 °C, 18 h, (2) NaBH4, MeOH, 2 h.
(1) DMF, 60–70 °C, 3 d, (2) NaBH4, MeOH, 2 h.
DMF, 60–70 °C, 7 d.

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H.-Y. Kim et al. / Bioorg. Med. Chem. 18 (2010) 809–821
Figure 1. (I) (A–D) HPLC–UV chromatograms of crude and purified 7EACD product compared to synthetic monomer standards; (E) MALDI-TOF mass spectrum of purified
7EACD. (II) (A) 13C NMR (75 MHz, DMSO-d6) spectrum of 7EqCD (d ppm) 154.8, 153.6, 137.1, 130.4, 125.9, 124.4, 114.9, 112.6, 101.5, 82.0, 81.0, 72.4, 71.8, 69.5, 49.5, 47.6,
46.8, 43.1, 37.1, 32.6, 30.7, 29.2, 27.2, 26.1, 23.4, 14.3); (B) MALDI-TOF spectrum of 7EqCD; (C) HPLC–UV chromatogram of 7EqCD, detection at 280 nm, m/z = 1261.7 MH33þ).
azido-b-CD under the same reaction conditions gave 1EqCD (24)
in 68% isolated yield (Table 2, entry 13). In all procedures, exten-
sive washing was carried out to remove residual Cu ions. As a con-
trol compound for bioassay, a triazole modified estradiol was
synthesized (qE2, 25): methyl azide was prepared for in situ click
ligation to EE2, resulting in the desired product 25 obtained in
60% yield after chromatography (Table 2, entry 14).
In order to estimate the membrane permeability of 7EqCD, rho-
damine was coupled to the secondary face of 7EqCD (26; Table 2,
entry 15). Non-covalent inclusion of rhodamine would be difficult
to rule out in the rhodamine–7EqCD conjugates, therefore the tri-
azole-CD derivative (26) was prepared and the rhodamine–qCD
conjugate synthesized for comparison (Table 2, entries 16 and
17). Both compounds 23 and 26 were coupled with 1 mol equiv
of fluorophore, either as the free carboxylic acid (rhodamine-19-
perchlorate, yielding conjugates 23R0 and 26R0, respectively) or
as the succinimidyl ester (NHS–rhodamine, yielding products
23R and 26R, respectively). In each case, the presence of the mon-
Scheme 2. Synthesis of EE2 derivatives and synthons.
osubstituted rhodamine conjugate was demonstrated by MALDI-
TOF spectroscopy.
in the presence of CuI and DIEA changed color from green to brown
coincident with the loss of the TLC spot for unreacted EE2. Reaction
time was extended for one day beyond this indicator point, yield-
ing quantitative conversion to 7EqCD (23) as a powder in 58%
isolated yield after washing (Table 2, entry 12; Fig. 1). The mono-
2.3. Summary of synthetic strategies
Elaboration of the ACD scaffold via imine formation with aryl
aldehydes is efficient when the aldehyde can be used as a co-sol-

H.-Y. Kim et al. / Bioorg. Med. Chem. 18 (2010) 809–821
813
Table 2
Click chemistry routes to qCD derivatization
Entry CD
Ligand
Reaction condition
Product
CuSO4–5H2O, NaN3
sodium ascorbate
10g
EE2 recovered
CuSO4–5H2O, NaN3
sodium ascorbate
11h
12i
7 equiv
1 equiv
CuI, DIEA
CuI, DIEA
13i
14j
Dimethylsulfate, NaN3
CuSO4–5H2O,
sodium ascorbate
15k
NHS–rhodamine
Rhodamine–perchlorate
16i
CuI, DIEA
17k
NHS–rhodamine
Rhodamine–perchlorate
g
DMF/H2O (3:1), 60 °C, 4 d.
DMF/H2O (3:1), 60 °C, 4 d.
DMF/MeOH, rt, 1 d.
t-BuOH/H2O (3:1), rt, 1 d.
h
i
j
k
DMAP/DMF, EDC–HCl/0 °C.
vent or in high concentration, however other aldehydes do not give
quantitative reaction. Monitoring of reactions by MALDI-TOF is
useful to identify qualitatively the extent of multivalent substitu-
tion of CD conjugates, however, the intensity of signals in MAL-
DI-TOF is not directly related to relative quantities of each
multivalent ACD conjugate. The problematic ability of mass spec
to provide quantitation has been previously described for CD deriv-
atives.36 Furthermore, only perfacially substituted CD derivatives
give simple NMR spectra; incompletely substituted b-CD deriva-
tives have 42 chemically and spectroscopically non-equivalent glu-

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H.-Y. Kim et al. / Bioorg. Med. Chem. 18 (2010) 809–821
cose carbons. From HPLC–UV chromatography, 7EACD was esti-
mated predominantly to contain the desired persubstituted prod-
uct, (EE2)7ACD, however the MALDI-TOF spectrum indicated the
presence of both (EE2)6ACD and (EE2)5ACD isomers (Fig. 1). Cyclo-
addition click chemistry provided a more efficient route to perfa-
cial modification, yielding qCD conjugates perfacially derivatized
with EE2 on the primary face (Figs. 1 and 2). Four biocompatible
E2–CD conjugates were obtained for bioassay: 7EACD, 1EACD,
7EqCD, and 1EqCD.
Rapid signaling via non-classical estrogen-activated pathways
has been recognized for some time in the CNS and cardiovascular
systems.39,40 However, the quantification of non-classical estro-
genic activity is not trivial, in part because different molecular
identities and cellular localization have been proposed for these
receptors.9 The debate on ligand-activated G-protein-coupled ER
(GPR-ER, or GPR-30) has focused on the plasma membrane and
endoplasmic reticulum,41 whereas ER itself has been proposed
to be active in the plasma membrane, cytoplasm, or mitochon-
dria.42 Rapid assays of MAPK pathway activation and intracellular
Ca levels have been used for extranuclear ER activation, however,
several pathways may elicit such responses. The classical activity
of ER as a nuclear receptor can be assayed by E2-responsive gene
reporter assays such as the alkaline phosphatase assay presented
above, however, it has been shown that of E2-responsive genes, a
full 25% are induced by E2–dendrimer conjugates.43 Recently To-
2.4. Estrogenic activity of CD conjugates
The ERa-positive human endometrial Ishikawa cell line is rou-
tinely used to quantify estrogenic and antiestrogenic activity of test
compounds.37 In this cell line, the induction of alkaline phosphatase
is an ER-mediated estrogenic response, allowing measurement of
EC50 values for estrogenic and IC50 for antiestrogenic potency by en-
zyme assay. The measured EC50 values for 1EACD, 1EqCD, 7EACD,
netti and co-workers reported that T47D/PKCa cells (human
breast cancer cells stably transfected with protein kinase C) were
stimulated by E2-BSA, whereas the parent cell line (T47D/neo)
was unresponsive.44 Therefore, the activity of 7EqCD was assayed
in both T47D cell lines (Fig. 4). The classical ER-dependent po-
tency of 7EqCD in T47D/neo cells was low: EC50 for 7EqCD was
and 7EqCD were 4.0 lM, 4.0 lM, 300 nM, and 200 nM, respectively;
both polyvalent conjugates showed higher potency than monova-
lent derivatives ( Fig. 3). There was no significant antiestrogenic
activity or cytotoxicity observed from the test compounds at the
concentrations studied (data not shown). EE2 derivatives 5 and 12
and ACD (3) represent control compounds for EACD: ACD was
inactive, whereas 5 and 12 were 200-fold more potent than 7EACD
(Fig. 3). Similarly, qE2 and CD derivative 2 represent controls for
EqCD; 2 was inactive, whereas qE2 was a full estrogen (Fig. 3). All
non-conjugated estrogen control compounds were substantially
less potent than EE2 itself, for which EC50 was measured as 28 pM
in Ishikawa cells. Katzenellenbogen has referred to potency for
E2–dendrimer conjugates where observed potency is divided by
the average number of E2 moieties in the conjugate.8 Applying this
>100 nM in T47D/neo cells. However, the response of T47D/PKC
a
cells to 7EqCD was similar to that of E2-BSA; 7EqCD (10 nM) was
a full estrogen, with the EC50 estimated as 2 nM. Taken together,
the data shown in Figure 4 suggest that 7EqCD at 2–10 nM may
be useful as an extranuclear ER selective ligand and warrants fur-
ther study.
2.5. Membrane permeability and mechanism of action
Rhodamine was conjugated to 7EqCD (23R, 23R0) using two
different coupling agents, and a control qCD (26R, 26R0) was con-
jugated in the same way to provide comparison. Both fluorescent
E2-conjugates retained estrogenic activity at higher concentra-
tions as shown by measured EC50 values in Ishikawa cells of
measure, the potency for: 5, 1EACD, and 7EACD were 27 nM, 4
and 2 M, respectively; and for qE2, 1EqCD, and 7EqCD were
27 nM, 4 M, and 1.4 M, respectively.
In the standard radioligand displacement assay for measuring
relative binding affinity to recombinant human ER , qE2 was ob-
served to be a weak ER ligand (IC50 = 0.4 0.1 M). In this assay,
lM,
l
l
l
a
1.5 0.47 lM and 2.6 0.63 l
M for 23R and 23R0, respectively.
a
l
ER positive MCF-7 mammary cancer cells were incubated with
either 23R, 23R0, or the control compound (26R) for 24 h at which
time nuclei and conjugate were localized by confocal fluorescence
microscopy (Fig. 5). Both 7EqCD conjugates were observed in the
cytoplasm, but not the nucleus of MCF-7 cells, whereas no
ER binding of EACD and EqCD derivatives was not observed within
solubility limits, however, other compounds with submicromolar
estrogenic potency in cell culture have shown low or negligible
affinity in the radioligand displacement assay.38
Figure 2. Structure of 7EqCD and one representative conformer optimized at the AM1level in Spartan 04 (Wavefunction Inc., CA) showing top and side views with small
cavity (yellow), annulus of triazole groups (cyan), and large flexible corona of pendant estradiol groups visible.

H.-Y. Kim et al. / Bioorg. Med. Chem. 18 (2010) 809–821
815
N3
7
100
75
50
25
0
OH
OH
HO
OH
NH2
HO
-9
-8
-7
-6
-5
Log [Compound], M
Figure 3. Estrogenic activity determined measuring alkaline phosphatase activity induced in ER(+) Ishikawa cells. Left: concentration–response for cells treated with 1EACD
(closed triangles), 1EqCD (open triangles), 7EACD (closed circles), 7EqCD (open circles): 100% estrogenic activity corresponds to E2 (1 nM). Data show mean and s.d. from
triplicate experiments. Right: table of potency for all compounds for which concentration–response was measured in Ishikawa cells.
E2 1 nM
The estradiol conjugates best described to date are E2–BSA, E2–
peroxidase, and E2–dendrimers; at higher concentrations
(P10 nM for protein conjugates and P100 nM for dendrimer conju-
7EqCD
E2-BSA 1 nM
gates) all three demonstrate classical estrogenic actions reasoned to
result from leaching of non-covalently bound estrogen (E2 or EE2) or
degradation to release estrogens. There is no reason to expect that
inclusion of EE2 would be an important factor for either 7EACD or
7EqCD, because EE2 has low reported affinity for b-CD (Kd = 8
mM),45,46 and a more polar CD annulus is known to reduce the affin-
ity for hydrophobic aromatic guests further.18,21 The presence of EE2
in the 7EqCD product was tested in the same Ishikawa cell cultures
used for assessment of classical estrogenic activity. For comparison,
the presence of E2 was measured in incubations of E2–BSA in Ishik-
awa cell culture. After four days, the cell supernatant was collected
and assayed for EE2 and E2 using LC–MS/MS quantitation, against
standard curves, of estrogens derivatized with dansyl chloride
(Fig. 6). In the 7EqCD incubation, EE2 was not observed above
detection limits, but E2 was observed in incubations of E2–BSA in cell
cultures as anticipated from literature reports.
100
50
0
1
10 100
1
10 100
7EqCD, nM
7EqCD, nM
T47D/neo
T47D/PKC
Figure 4. Activation of ERE and induction of luciferase activity in T47D/neo and
T47D/PKC cell lines by 7EqCD compared to E2–BSA. In all experiments, relative
luciferase activity was corrected for transfection efficiency and normalized to
vehicle (0%) and E2 (1 nM; 100%). Data show mean and s.d. from triplicate
measurements on two separate cell passages.
a
7EACD bears comparison to the heterogeneous E2–dendrimer
conjugate prepared by Katzenellenbogen andco-workers, sinceboth
bioconjugates are prepared from a similar EE2 synthon via an imine
intermediate, with the same spacer unit linking macromolecular
amine with EE2, and both are heterogeneous.8 In MCF-7 cells, the
evidence for cell membrane permeation was obtained for the con-
trol compound.
Figure 5. Localization of (A) 26R, (B) 23R, and (C) 23R0 in ER positive MCF-7 cells by confocal microscopy. Drugs (100 nM) were incubated with cells for 24 h. Clockwise from
bottom-left: Ex/Em: 544/576 nm rhodamine-conjugates; Ex/Em: 345 nm/420 nm nuclear marker; differential interference contrast image; overlay of rhodamine and nuclear
dyes showing cytoplasmic localization of rhodamine conjugates for 23R and 23R0. Data shown are representative of several images of different cells and planes.

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H.-Y. Kim et al. / Bioorg. Med. Chem. 18 (2010) 809–821
Figure 6. LC–MS/MS analysis of free estrogen in cell culture supernatant after four days incubation of E2–BSA or 7EqCD. Positive ion electrospray selected reaction monitoring
(SRM) chromatograms of estrogens as dansyl derivatives: (A) EE2 (70 nM) incubated in Ishikawa cell culture (peak intensity 3.0 ꢂ 105); (B) 7EqCD (10 nM) incubated with
Ishikawa cells (maximum y-axis intensity 1.4 ꢂ 103) ꢁ EE2 peak was not observed above detection limits (<50 pM); SRM conditions EE2–dansylꢃ[MH+] m/z 530?171 collision
energy 53 eV; (C) E2 (70 nM) incubated in Ishikawa cell culture (peak intensity 5.5 ꢂ 105); (D) E2–BSA (1
l
g/mL) incubated with Ishikawa cells (peak intensity 1.1 ꢂ 104)
showing approximately 1 nM based upon standard curves; SRM conditions E2–dansyl [MH+] m/z 506?171 collision energy 57 eV.
E2–dendrimer induced E2-responsive genes, but E2 itself was 3–4 or-
ders of magnitude more potent than the conjugate (E2–dendrimer;
EC50 ꢀ 10ꢁ9–10ꢁ8 M).8 These and other data were compatible with
permeation of the E2–dendrimer conjugate into the cytoplasm, but
not into the nucleus. Recently, the combination of E2–BSA (mem-
brane selective estrogen) and E2–dendrimer (extranuclear selective
estrogen) conjugates has been used to distinguish membrane and
extranuclear inputs into E2-responsive gene induction.43
cell nucleus, suggesting that this E2–CD conjugate would provide
a selective extranuclear estrogen comparable to E2–dendrimer
conjugates. Furthermore, at concentrations giving negligible classi-
cal ER activity (1–10 nM), 7EqCD was shown to deliver activity in
cell culture equivalent to E2, via a mechanism attributed in the lit-
erature to extranuclear ER. Thus, 7EqCD bears comparison to E2–
dendrimer conjugates as a biological probe to explore extranuclear
ER mechanisms; 7EqCD is the first homogeneous probe of this
type.
3. Conclusions
4. Experimental
The classical mechanism of estrogen action is defined by: (i)
binding of an estrogenic ligand to ER in the cytoplasm; (ii) ER
dimerization and translocation to the nucleus; (iii) complexation
with the ERE domain of DNA; (iv) recruitment of coregulator pro-
teins; (v) transcription and expression of E2-responsive gene prod-
ucts. Non-classical mechanisms utilize extranuclear receptors that
are activated by estrogenic ligands, often via rapid signaling cas-
cades, however these may also induce ER-dependent, genomic
activity.43 It is recognized that selective ligands are required to
untangle the role of nuclear and extranuclear receptors and the
cross-talk between classical and non-classical pathways. b-CD
derivatives play an important role in drug delivery, and less fre-
quently have been used as molecular scaffolds for biomimet-
ics;47–49 the expectation that these large, hydrophilic molecules
seldom penetrate cellular membranes supports the exploration of
E2–CD conjugates as cell compartment selective estrogens.
Several click chemistry approaches were attempted towards a
b-CD persubstituted at the primary face with estradiol via a stable
linker. Reactions using the amino-CD (ACD) scaffold proved insuf-
ficiently generalized; selected examples giving quantitative per-
substitution, but others such as the ACD–estradiol conjugate,
7EACD, not providing an homogeneous persubstituted product.
Conjugation via [1,3]-cycloaddition proved a suitable alternative
method for preparation of a homogeneous, persubstituted E2–CD,
namely 7EqCD. The four E2–CD conjugates studied in cell culture
were not cytotoxic below solubility limits and all manifested estro-
genic activity in cell culture. 7EqCD as an homogeneous E2–CD
conjugate was preferred for further study. Labeling with rhoda-
mine allowed localization of 7EqCD in the cytoplasm, but not the
4.1. Synthesis of compounds
4.1.1. Materials and general methods
All chemicals and solvents were obtained from Sigma–Aldrich
and Fisher unless otherwise stated and purified prior to use accord-
ing to known procedures when necessary: 17b-ethynylestradiol
(Steraloids, Newport, RI), NHS–rhodamine (PIERCE, Rockford, IL),
and all other reagents were used as received. DMF and Et3N were
distilled from CaH2. b-Cyclodextrin and all CD derivatives were
dried under vacuum in a drying pistol over P2O5 with refluxing ace-
tone prior to use. Moisture sensitive reactions were performed un-
der nitrogen using oven dried glassware. Analytical thin layer
chromatography (TLC) was carried out on 60 Å silica gel-flexible
baked plates (Whatman), and compounds were visualized by UV
or by staining with I2, KMnO4, or phosphomolybdic acid solutions.
Flash column chromatography was performed using silica gel (32–
63 lm, Selecto Scientific).
4.1.2. Instrumentation
NMR spectra were recorded on Bruker Ultrashield (400 MHz) or
Advance (300 MHz) spectrometers using CDCl3, DMSO-d6, or
CD3CN at 300 K. Variable temperature 1H NMR at 323 K was per-
formed on 7EqCD in order to improve chemical splitting resolution.
Chemical shifts of 1H NMR spectra were reported using TMS
(0 ppm) as a reference, and 13C NMR spectra were referenced to
internal deuterated NMR solvents. All 13C NMR spectra of CD deriv-
atives were obtained from overnight runs at optimized condition
(D1; 3–5 s, TD; 32 K, SI; 265 K). Analytical HPLC (C8 column,

H.-Y. Kim et al. / Bioorg. Med. Chem. 18 (2010) 809–821
817
5
l
m, 4.6 ꢂ 150 mm, Agilent Technologies) was carried out with
Perkin–Elmer (Fremont, CA) equipment, and semi-preparative re-
versed-phase C8 column (5
idue was purified by silica gel column chromatography (EtOAc/Hx,
2:1) giving a white solid (423 mg, 90% yield). 1H NMR (400 MHz,
DMSO-d6) dppm 9.00 (s, 1H), 7.37 (d, J = 8 Hz, 2H), 7.30 (s,
J = 7.6 Hz 2H), 7.07 (d, J = 8.4 Hz, 1H), 6.51 (d, J = 8.4 Hz, 1H), 6.44
(s, 1H), 5.44 (s, 1H), 5.25 (s, 1H), 4.50 (s, 2H), 2.71 (s, 2H), 2.32
(d, J = 11.6 Hz, 1H), 2.24–1.69 (m, 9H), 1.37–1.19 (m, 5H),
0.86 (m, 1H), 0.81 (s, 3H). 13C NMR (100 MHz, DMSO-d6) dppm
155.0, 142.7, 137.2, 131.0, 130.3, 126.5, 126.1, 121.1, 115.0,
112.7, 94.5, 84.1, 78.6, 62.5, 49.4, 47.2, 43.4, 39.0, 33.0, 29.3,
27.1, 26.3, 22.7, 13.0. APCI-MS: calcd for C27H30O3 402.53, found
[MꢁH]ꢁ 401.50.
l
m, 10 ꢂ 250 mm, VYDAC) was used
for purification on the same equipment. Matrix-assisted laser
desorption ionization time-of-flight (MALDI-TOF) mass spectra
were obtained on a Voyager (Applied Biosystems Co. Ltd) using
DHB as a matrix. Mass spectra for monomer precursors were re-
corded on an Agilent 1100 series LC/MSD ion trap instrument,
using APCI as ionization method.
4.1.3. Per-6-azido-6-deoxy-b-cyclodextrin (2)50
Per-6-iodo-b-cyclodextrin (1; 4.5 g, 2.36 mmol) obtained by lit-
erature procedures was dissolved in DMF (51 mL), and sodium
azide (1.32 g, 20.3 mmol) was added. The suspension was stirred
under N2 atmosphere at 70 °C for 20 h. The resulting yellow solu-
tion was concentrated under reduced pressure to half volume,
and was poured into 90% EtOH (280 mL) to precipitate the final
product, a fine white powder that was filtered and was washed
with H2O and dried in a P2O5 drying pistol for one day (2.8 g,
90% yield). 1H NMR (300 MHz, DMSO-d6) dppm 5.91 (br d,
J = 6.7 Hz, 7H), 5.76 (s, 7H), 4.92 (s, 7H), 3. 80–3.70 (m, 14H),
3.61–3.56 (m, 14H), 3.40–3.33 (m, 14H). 13C NMR (75 MHz,
DMSO-d6) dppm 102.05, 83.20, 72.60, 72.00, 70.33, 51.33. MALDI-
TOF: calcd C42H63O28N21 [M]+ 1310.08, found [M+Na]+ 1333.91.
4.1.7. Per-6-(estradiol-17-ethynylbenzylamino)-6-deoxy-b-
cyclodextrin (6)
Per-6-amino-b-cyclodextrin (50 mg, 0.044 mmol) and EE2–Ph–
CHO 4 (176 mg, 3.08 mmol) were dissolved in DMF (1 mL), and the
reaction mixture was refluxed at room temperature or 65 °C for 4–
10 d under N2 atmosphere. The resulting brown imine suspension
was reduced in situ by freshly prepared 0.1 M NaBH4 solution
(2.1 mL) at 0 °C and allowed to stir for 2 h. The warmed reaction mix-
ture was poured into acetone (25 mL) to precipitate the desired
product. The beige solid was repeatedly washed and filtered until
no corresponding alcohol 5 from EE2–Ph–CHO reduction was de-
tected on TLC. The resulted precipitates were collected and dried
yielding a beige powder (51 mg, 30% yield). MALDI-TOF: m/z calcd
for C231H273N7O42 3818.9, C204H245N7O40 3434.7, C177H217N7O38
3050.5; found [M+Na]+ 3843.46, 3458.81, 3073.22.
4.1.4. Per-6-amino-6-deoxy-b-cyclodextrin (3)
Per-6-azido-b-cyclodextrin 2 (1.53 g, 1.17 mmol) was dissolved
in DMF (30.8 mL), and Ph3P (4.85 g, 18.5 mmol) was added. The
evolution of N2 was observed by the formation of bubbles in the
reaction vessel. After 1 h, the evolution of N2 ceased, 35% aqueous
NH3 solution (6.0 mL) was added dropwise into the solution. After
the addition of aqueous NH3 was completed, the reaction mixture
turned to a white suspension that was stirred at room temperature
for 18 h. The resulting mixture was condensed under vacuum at
60 °C to one fourth of volume, and then EtOH (77 mL) was added
to precipitate the product. The white powder was washed with
EtOH (77 mL, three times) and dried for one day (1 g, 82% yield).
For NMR spectroscopy, the free amino cyclodextrin was converted
to amine salt by addition of diluted HCl until the pH had reached 6.
1H NMR (300 MHz, DMSO-d6) dppm 5.21 (d, J = 2.4 Hz, 7H), 4.25
(ddd, 7H), 4.03 (t, J = 2.7 Hz, 7H), 3. 72 (dd, J = 2.4, 7.5 Hz, 7H),
3.63 (t, J = 6.9 Hz, 7H), 3.49 (dd, J = 2.1, 9.9 Hz, 7H), 3.33–3.28
(dd, J = 5.7 Hz, 7H), 13C NMR (75 MHz, DMSO-d6) dppm 100.94,
81.69, 71.67, 71.15, 67.29, 39.72. MALDI-TOF: calcd C42H77N7O28
[M]+ 1128.10, found [M+Na]+ 1151.96.
4.1.8. Per-6-(pyridine-2-ylmethylamino)-6-deoxy-b-
cyclodextrin (7)
2-Pyridinecarboxaldehyde (33.5
lL, 0.35 mmol) was syringed
into a suspension of 3 (50 mg, 0.044 mmol) in DMF (100
lL). The
yellow reaction solution was stirred at room temperature for 5
hr. The resulting imine was reduced in situ by freshly prepared
0.1 M NaBH4 solution (2.58 mL) at 0 °C and allowed to stir for
2 h. The warmed reaction mixture was poured into Et2O (50 mL)
to precipitate product. After washing and filtration, the white pre-
cipitate was collected (60 mg, 77% yield). For NMR assay, the HBr
salt was prepared after acidification with 5% HBr to bring pH to
4.0. After water was evaporated, the beige powder was taken for
NMR spectroscopy. 13C NMR (75 MHz, HBr salt in D2O) dppm
148.1, 138.5, 124.3, 123.6, 100.9, 81.8, 71.7, 71.2, 67.3, 50.8, 48.4.
MALDI-TOF: calcd for C84H112N14O28 1765.89, found [M+Na]+ mass
1788.31.
4.1.9. Per-6-(4-(benzyloxy)benzylamino)-6-deoxy-b-
cyclodextrin (8)
4-Benzyloxybenzaldehyde (312 mg, 1.47 mmol) was added to a
suspension of 3 (40 mg, 0.035 mmol) in DMF (580 lL) and MeOH
4.1.5. 17a-[(4-Formylphenyl)]ethynyl-estra-1,3,5-(10)-triene-
3,17b-diol, EE2–Ph–CHO (4)
Compound 4 was synthesized by literature procedures.25 After
silica gel column chromatography (EtOAc/Hx, 1:2) and vacuum
drying, a fine beige powder was obtained (945 mg, 70% yield). 1H
NMR (300 MHz, DMSO-d6) dppm 10.00 (s, 1H), 8.99 (s, 1H), 7.89
(d, J = 8.4 Hz, 2H), 7.62 (d, J = 8.1, 7.2 Hz, 2H), 6.51 (dd, J = 8.4,
2.4 Hz, 1H), 6.43 (d, J = 2.4 Hz, 1H), 5.59 (s, 1H), 2.70–1.23 (m,
14H), 0.82 (s, 3H). 13C NMR (75 MHz, CDCl3) dppm 192.5, 154.9,
137.2, 135.2, 131.9, 130.2, 129.7, 128.9, 126.1, 114.9, 112.8, 99.2,
83.6, 78.8, 49.5, 47.4, 43.4, 33.0, 29.2, 27.0, 26.2, 22.6, 12.9. APCI-
MS: calcd for C27H28O3 400.52, found [MꢁH]ꢁ 399.70.
(1.17 mL). The reaction solution was stirred at room temperature
for seven days. The resulting imine was reduced in situ with freshly
prepared 0.1 M NaBH4 solution (2.1 mL) stirring at 0 °C for 2 h. The
warmed reaction mixture was poured into acetone (25 mL) to pre-
cipitate the product. After repetitive washing followed by filtration,
a white precipitate was collected and dried (71 mg, 81% yield).
MALDI-TOF: calcd for C140H161N7O35 2501.84, found [M+Na]+
2523.94.
4.1.10. 4-(4-Iodophenyl) butanal (9)
Compound 9 was prepared by adaptation of literature proce-
dures.51,52 To a mixture of 1,4-diiodobenzene (5 g, 0.015 mol),
palladium(II) acetate (68 mg, 0.304 mmol), sodium hydrogen car-
bonate (3.2 g, 0.038 mol), and tetra-N-butyl ammonium chloride
(4.2 g, 0.015 mol) purged with nitrogen gas was added DMF until
dissolution. 3-Butene-1-ol (2 mL, 0.023 mol) was added via syr-
inge, and the mixture stirred for 2 d at 40 °C. During the reaction,
4.1.6. 17a-[(4-Hydroxymethylphenyl)]ethynyl-estra-1,3,5-(10)-
triene-3,17b-diol (5)
EE2–Ph–CHO 4 (470 mg, 1.17 mmol) was dissolved in MeOH
(4 mL) and DMF (1.3 mL). Under N2 atmosphere NaBH4 (110 mg,
2.91 mmol) was quickly added. The dark brown reaction mixture
was stirred at room temp for 3 h. After removal of solvent, the res-

818
H.-Y. Kim et al. / Bioorg. Med. Chem. 18 (2010) 809–821
the mixture turned from orange to dark brown in color. After cool-
ing acetone (20 mL) was added, and the resulting precipitates were
removed by filtration. This procedure was repeated until no more
precipitate was produced, and the filtrates then concentrated. Puri-
fication by silica gel chromatography (EtOAc/Hx, 1:7) afforded a
light-yellow oil (1.08 g, yield 26%; 80:20 to branched aldehyde).
1H NMR (300 MHz, CDCl3) dppm 9.72 (s, 1H), 7.57 (d, J = 8.4 Hz,
2H), 6.90 (d, J = 8.1 Hz, 2H), 2.56 (t, J = 7.8, 7.2 Hz, 2H), 2.41 (td,
J = 7.2, 1.2 Hz, 2H), 1.89 (p, J = 7.2, 7.5 Hz, 2H). 13C NMR (75 MHz,
CDCl3) dppm 202.0, 141.0, 137.5, 130.6, 101.78, 81.24, 72.43,
69.74, 68.93, 59.61, 21.23.
4.1.15. Per-6-(phthalimidoethylthio)-6-deoxy-b-cyclodextrin (14)
Compound 14 was synthesized according to literature proce-
dures27,56 as
a
white solid (112 mg, 17% yield). 1H NMR
(400 MHz, DMSO-d6) dppm 7.71 (s, 4H), 5.84 (d, J = 23 Hz, 2 Hz,
2H), 4.86 (s, 1H), 3. 94 (br s, 1H), 3.81 (m, 2H), 3.59 (m, 1H),
3.42–3.33 (m, overlaps with DOH), 3.04 (d, J = 11.6 Hz, 1H), 2.91–
2.78 (m, 3H). 13C NMR (100 MHz, DMSO-d6) dppm 167.4, 134.2,
131.3, 122.9, 102.1, 84.2, 72.5, 72.2, 71.2, 36.9, 33.0, 30.8. MALDI-
TOF: calcd for C112H119N7O42S7 2459.66, found [M+Na]+ 2482.62.
4.1.16. Per-6-(2-aminoethylthio)-6-deoxy-b-cyclodextrin (15)
Compound 15 was prepared by literature procedures27 as a
white powder (50 mg, 81% yield). 1H NMR (400 MHz, DMSO-d6)
dppm 5.45 (d, J = 24, 4 Hz, 2H), 4.51 (br s, 1H), 3.40 (br s, 1H), 3.
19 (t, J = 8.4 Hz, 8.8 Hz, 1H), 3.02–2.48 (m, overlaps with DOH),
2.09 (s, 1H). 13C NMR (100 MHz, DMSO-d6) dppm 102.1, 84.5, 72.6,
72.2, 71.4, 38.6, 32.8, 29.7. MALDI-TOF: calcd for C56H105N7O28S7
1548.94, found [M]+ 1548.90.
4.1.11. 17a-[(4-Butanalphenyl)]ethynyl-estra-1,3,5 (10)-triene-
3,17b-diol (10)
Compound 10 was prepared by literature procedures25 as a
light-yellow powder (900 mg, 60% yield). 1H NMR (400 MHz,
CD3CN) dppm 9.75 (s, 1H), 7.45 (d, J = 8 Hz, 2H), 7.25 (d, J = 8 Hz,
2H), 7.20 (d, J = 8 Hz, 2H), 6.91 (s, 1H), 6.69 (d, J = 8 Hz, 1H), 6.62
(s, 1H), 3.64 (s, 1H), 2.85 (m, 2H), 2.69 (t, J = 8 Hz, 2H), 2.48–2.46
(m, 4H), 1.96–1.86 (m, 6H), 1.49–1.34 (m, 4H), 0.98 (s, 3H). 13C
NMR (100 MHz, DMSO-d6) dppm 203.2, 154.9, 141.8, 137.1, 131.2,
130.3, 128.6, 126.1, 120.5, 114.9, 112.7, 94.5, 84.0, 78.6, 49.4,
47.3, 43.4, 42.3, 40.4, 39.0, 34.2, 32.9, 29.2, 27.0, 26.3, 23.2, 12.9.
APCI-MS: calcd C30H32O3 442.25, found [MꢁH]ꢁ 442.10.
4.1.17. Per-6-(estradiol-17-(4-((2-(propylthio)ethylamino)-
methyl)phenyl)ethynyl)-6-deoxy-b-cyclodextrin (16)
Compounds 15 (40 mg, 0.0258 mmol) and 4 (310 mg, 0.774
mmol) were dissolved in DMF, and stirred at 60–70 °C for 10 days.
2 mL of aqueous NaOH (1 N in MeOH) and stirred for a further
2.5 h. After cooling and concentration, the residue was precipitated
in acetone (25 mL) giving a beige powder that was repeatedly
washed and filtered until no trace of 4 was detected by TLC. The pre-
cipitates were dried to give a light-yellow solid product containing
16 as the major product in a heterogeneous mixture. MALDI-TOF:
calcd for C245H301N7O42S7 4240.49, found [M+H2O+H]+ 4259.84.
4.1.12. Per-6-(4-iodobenzylamino)-6-deoxy-b-cyclodextrin (11)
ACD 3 (50 mg, 0.044 mmol) and 4-iodobenzaldehyde (85.1 mg,
0.352 mmol) were dissolved in DMF (0.1 mL), and the reaction
mixture was stirred at 60–70 °C for 18 h under N2 atmosphere.
The resulted brown imine solution was reduced in situ by freshly
prepared 0.1 M NaBH4 solution (2.1 mL) at 0 °C and allowed to stir
for 2 h. The warmed reaction mixture was poured into acetone
(25 mL) to precipitate the product as beige powder that was
repeatedly washed and filtered until no corresponding alcohol
from 4-iodobenzaldehyde was detected by TLC. The precipitates
were collected, and overnight drying gave a light-yellow powder
that by MALDI-TOF analysis gave signals corresponding to a mix-
ture of 1–7-fold substitution.
4.1.18. Mono-6-(p-tolylsulfonyl)-6-deoxy-b-cyclodextrin (17)
Compound 17 was prepared by adaptation of literature proce-
dures57 as a white powder was obtained (1 g, 10% yield). 1H NMR
(300 MHz, DMSO-d6) dppm 7.76 (d, J = 7.6 Hz, 2H), 7.44 (d,
J = 7.2 Hz, 2H), 5.71 (br s, 14H), 4.84 (s, 4H), 4.77 (s, 3H), 4.33 (d,
J = 10.4 Hz, 3H), 4.20 (m, 3H), 3.66–3.51 (m, overlaps with HOD),
3.38–3.22 (m, overlaps with HOD), 2.09 (s, 3H), 13C NMR
(100 MHz, DMSO-d6) dppm 144.9, 132.7, 129.9, 127.6, 102.0 (m),
81.5 (m), 73.1–72.8, 60.0, 21.3. MALDI-TOF: calcd C49H76O37
S
4.1.13. 17a-(4-Aminophenyl)ethynyl-estra-1,3,5 (10)-triene-
[M]+ 1289.19, found [M+Na]+ 1313.43.
3,17b-diol (12)53–55
A solution of Pd(OAc)2 (80.7 mg, 0.169 mmol) and P(Ph)3
(90.4 g, 0.338 mmol) in TEA (20 mL) was stirred under N2 for
10 min. CuI (65.7 mg, 0.338 mmol) and 4-iodoaniline (748 mg,
3.376 mmol) were added, and after 5 min, 17b-ethynylestradiol
(1 g, 3.376 mmol) was transferred. The dark brown mixture was
stirred at room temperature for 5 h. TEA was evaporated, and the
residue was purified by silica gel column chromatography
(CH2Cl2/MeOH, 20:1). After drying under vacuum, yellow powder
was obtained (1.10 g, 84% yield). 1H NMR (400 MHz, CD3CN) dppm
8.98 (s, 1H), 7.06 (s, 2H), 6.50 (d, J = 7.2 Hz, 2H), 6.43 (s, 1H), 5.39
(s, 2H), 5.25 (s, 1H), 3.33 (s, 1H), 2.70 (s, 2H), 2.31 (d, J = 9.6 Hz,
1H), 2.15–1.68 (m, 9H), 1.31 (m, 4H), 0.79 (s, 3H). 13C NMR
(100 MHz, CD3CN) dppm 154.9, 148.8, 137.1, 132.3, 130.3, 126.1,
114.9, 113.6, 112.7, 109.1, 91.4, 85.2, 78.6, 49.2, 47.1, 43.4, 32.9,
29.2, 27.0, 26.3, 22.6, 12.9. APCI-MS: calcd For C26H29NO2 387.2,
found [MꢁH]ꢁ 386.6.
4.1.19. Mono-6-azido-6-deoxy-b-cyclodextrin (18)57
Compound 17 (350 mg, 0.271 mmol) and NaN3 (189 mg,
2.90 mmol) were suspended in H2O (3.53 mL), which was heated
and to a clear solution with stirring at 80 °C for 15 h. The reaction
mixture was cooled, poured into acetone (60 mL) to precipitate the
product as a white powder that was dried in a drying pistol at 56 °C
under vacuum. A pure white solid was obtained (492.8 mg, 90%
yield). 1H NMR (400 MHz, DMSO-d6) dppm 5.73 (br s, 14 H), 4.84
(m, 7 H), 3.75–3.55 (m, 28 H), 3.41–3.28 (m, overlaps with HOD),
13C NMR (100 MHz, DMSO-d6) dppm 102.0 (m), 83.0, 81.6 (m),
73.0 (m), 72.4, 72.2, 72.1, 70.2, 60.2 (m), 51.1. MALDI-TOF: calcd
C42H69N3O34 [M]+ 1160.00, found [M+Na]+ 1183.74.
4.1.20. Mono-6-amino-6-deoxy-b-cyclodextrin hydrochloride
(19)57
Compound 18 (300 mg, 0.259 mmol) and PPh3 (116 mg,
0.442 mmol) were dissolved in DMF (5.6 mL) to which 30% NH4OH
(2.7 mL) was added by syringe. The reaction mixture was stirred at
room temperature for 18 h, and poured into acetone (50 mL)
yielding after successive washes and drying a white powder
(200.0 mg, 68% yield). 1H NMR (400 MHz, DMSO-d6) dppm 5.15 (br
s, 7 H), 4.06–4.00 (m, 21 H), 3.73–3.63 (m, 10 H). 13C NMR
(100 MHz, DMSO-d6) dppm 101.8, 101.5, 82.8, 81.0, 80.8, 73.1,
4.1.14. (2-Phthalimidoethyl) isothiouronium hydrobromide (13)
Compound 13 was synthesized according to literature proce-
dures.54,55 as a white solid (5.1, 79% yield). 1H NMR (400 MHz,
DMSO-d6) dppm 9.14 (s, 4H), 7.91 (m, 4H), 3.39 (t, J = 5.2 Hz,
6.4 Hz, 2H), 3.55 (t, J = 5.2 Hz, 6.4 Hz, 2H). 13C NMR (100 MHz,
DMSO-d6) dppm 169.2, 167.6, 134.7, 131.4, 123.2, 36.0, 29.3. APCI-
MS: calcd for C11H12BrN3O2S 328.98, found [MꢁHBr]+ 249.80.

H.-Y. Kim et al. / Bioorg. Med. Chem. 18 (2010) 809–821
819
73.0, 72.1, 71.8, 71.7, 60.2, 41.2. MALDI-TOF: calcd C42H71NO34
4.1.25. Mono-6-(17-estradiol-17-(1H-1,2,3-triazol-4-yl))-6-
deoxy-b-cyclodextrin (24)
[M]+ 1134.01, found [M+Na]+ 1157.57.
Mono-6-azido-b-cyclodextrin 2 (100 mg, 0.086 mmol) and 17b-
ethynylestradiol (25.5 mg, 0.086 mmol) were dissolved in 50% of
MeOH and DMF (2.15 mL). CuI (82.0 mg, 0.43 mmol) was added,
and the reaction mixture formed brown suspension. DIEA
4.1.21. Mono-6-(17-estradiolphenylmethylamino)-6-deoxy-b-
cyclodextrin (20)
Compounds 19 (50 mg, 0.044 mmol) and
4
(370.6 mg,
0.926 mmol) were dissolved in DMF (1 mL), and the reaction mix-
ture was refluxed at 65 °C for 3 d under N2. The resulting brown
imine suspension was reduced in situ by the addition of a freshly
prepared 0.1 M NaBH4 solution (3 mL) at 0 °C with stirring for
2 h. The warmed reaction mixture was precipitated into acetone
(25 mL) and the precipitate repeatedly washed and filtered until
no presence of 5 was detected by TLC. After drying a white powder
was obtained (30 mg, 45% yield). MALDI-TOF: calcd for C69H99NO36
1518.53, found [M+Na]+ 1541.97.
(75.0 lL) was added via syringe into the reaction mixture. The
green suspension was stirred at room temperature for one day un-
til there was no EE2 detected on TLC. The solvent was evaporated
under vacuum, and the residue was dissolved with 6 mL of aque-
ous NH4OH (30%). The deep blue colored solution was precipitated
into cold Ac2O (50 mL). This washing process was repeated three
times for the complete removal of copper ions. After centrifugation
at 4 °C (4000 rpm, 20 min), fine beige powder was obtained, 1EqCD
(80 mg, 64% yield). MALDI-TOF: calcd for C62H93N3O36 1456.42,
found [M+Na]+ 1479.20.
4.1.22. Mono-6-(estradiol-17-ethynylbenzylamino)-6-deoxy-b-
cyclodextrin (21)
4.1.26. 17a-(17-1,2,3-Triazolmethyl)ethynyl-estra-1,3,5 (10)-
Compounds 17 (100 mg, 0.078 mmol) and 12 (630 mg,
1.63 mmol) were dissolved in DMF (0.5 mL), and the reaction mix-
ture was refluxed at 60–70 °C for 7 d under N2. The solvent was
evaporated under vacuum, and the dark brown residue was precip-
itated in acetone (50 mL). The beige powder was repeatedly
washed and filtered until no trace of compound 12 was detected
by TLC. The precipitates were collected, and overnight drying gave
triene-3,17b-diol (25)
This procedure was modified from the literature.31 Dimethylsul-
fate (90 mL, 0.676 mmol) was added to the solution of 17b-ethyny-
lestradiol (100 mg, 0.338 mmol) and sodium azide (46 mg,
0.676 mmol) in t-BuOH/H2O (0.61/0.2 mL) at 0 °C. CuSO4 (4.22 mg,
5 mol%) in 200
l
L
of water and sodium ascorbate (6.7 mg,
10 mol%) in 200
lL of water were added stepwisely. This reaction
a
light-yellow solid (51 mg, 43%). MALDI-TOF: calcd for
suspension was gradually warmed up to room temperature and stir-
red for 1 d. It was worked up by addition of water and extracted with
EtOAc. The combined organic layers were washed with brine, and
the solvent was removed. The resulting mixture was evaporated un-
der pressure, and the residue was purified by silica gel column chro-
matography (EtOAc/Hx, 1:5). After drying under vacuum, white
powder was obtained (71.7 mg, 60% yield). 1H NMR (400 MHz,
DMSO-d6) dppm 8.94 (s, 1H), 7.79 (s, 1H), 6.95 (d, J = 8 Hz, 1H), 6.46
(d, J = 8.4 Hz, 1H), 6.40 (s, 1H), 5.04 (s, 1H), 4.01 (s, 3H), 2.68 (m,
2H), 2.50 (m, 1H), 2.10 (d, J = 12 Hz, 1H), 1.88–1.70 (m, 5H), 1.42–
1.26 (m, 6H), 0.95 (s, 3H), 0.65 (t, J = 12 Hz, 11.6 Hz, 1H). 13C NMR
(100 MHz, DMSO-d6) dppm 154.8, 154.2, 137.1, 130.4, 126.0, 123.5,
114.8, 112.6, 81.1, 47.5, 46.6, 43.1, 37.1, 36.0, 32.6, 29.2, 27.2, 26.1,
23.5, 14.3.
C69H99NO36 1518.53, found [M+Na]+ 1541.60.
4.1.23. Mono-6-(estradiol-17-(1H-1,2,3-triazol-4-yl))-6-deoxy-
b-cyclodextrin (22)
Compound 17 (100 mg, 0.078 mmol), 17b-ethynylestradiol
(23.1 mg, 0.078 mmol), and sodium azide (6.6 mg, 0.10 mmol)
were dissolved in DMF and H2O (3:1, 0.9 mL). A solution of
CuSO4–5H2O (400 mg, 1.60 mmol) and sodium ascorbate
(360 mg, 1.82 mmol) was prepared separately in the same DMF/
H2O solvent system (2 mL) and added via syringe. The brown sus-
pension was stirred at 60–70 °C for four days. The solvent was
evaporated, and the residue was precipitated in H2O (50 mL). This
washing process was repeated three times for complete removal of
residual copper ions. After centrifugation at 4 °C (4000 rpm,
20 min), a fine beige powder was obtained (60 mg, 53%). MALDI-
TOF: calcd for C62H93N3O36 1456.42, found [M+Na]+ 1479.44.
4.1.27. Per-6-(4-hydroxymethyl-1,2,3-triazolmethyl)-6-deoxy-
b-cyclodextrin (26)
Azide 2 (50 mg, 0.038 mmol) and CuI (36.0 mg, 0.19 mmol) were
dissolved in 50% of MeOH and DMF (1.0 mL) to which propagyl alco-
4.1.24. Per-6-(estradiol-17-(1H-1,2,3-triazol-4-yl))-6-deoxy-b-
cyclodextrin (23)
hol (15.8 lL, 0.27 mmol) and DIEA (33.0 lL, 0.19 mmol) were added
Compound
0.532 mmol) were dissolved in 50% of MeOH and DMF (1.9 mL).
CuI (72.4 mg, 0.38 mmol) was added, and DIEA (66.2 L) added
2
(100 mg, 0.076 mmol) and EE2 (157.6 mg,
by syringe, and the mixture then stirred at room temperature for 1 d.
Theresultinggreensuspensionwasclarifiedusing2.0 mLofaqueous
30%NH4OH, andChelex100sodiumformwasaddedtothedeepblue
solution to complex copper ions. The solution was allowed to stir
vigorously overnight, and the yellow supernatant obtained was con-
centrated to give a beige powder (11 mg, 15% yield). 1H NMR
(400 MHz, DMSO-d6) dppm 7.80 (s, 1H), 6.0 (br s, 4H), 5.1 (m, 6H),
4.6–4.3 (m, 11H), 4.0 (m, 3H), 3.7 (t, J = 8.8, 8.4 Hz, 4H), 2.9 (br s,
11H), 2.1 (s, 6H). 13C NMR (100 MHz, DMSO-d6) dppm 147.6, 124.1,
101.5, 82.5, 72.5, 71.8, 69.7, 54.6, 49.3, MALDI-TOF: m/z calcd for
C63H91N21O35 1701.6, found [M+Mg]+ 1726.0.
l
by syringe to the reaction mixture. The green suspension was stir-
red at room temperature for 1 d until there was no EE2 detected by
TLC. The solvent was evaporated under vacuum, and the residue
was dissolved with 6 mL of aqueous NH4OH (30%). The deep blue
solution was precipitated into cold acetone (50 mL). This washing
process was repeated three times for removal of residual copper.
After addition of H2O and centrifugation at 4 °C three times
(4000 rpm, 20 min), a fine beige powder was obtained (150 mg,
58%). 1H NMR (300 MHz, DMSO-d6, 323 K) dppm 8.61 (br s, 1H),
7.78 (s, 1H), 6.88 (d, J = 8.1 Hz, 1H), 6.46 (s, 1H), 5.61 (s, 3H), 5.12
(s, 1H), 4.66 (s, 2H), 4.60 (s, 2H), 4.14 (m, 1 H), 3.74 (t, J = 8.7 Hz,
2H), 3.27–3.15 (m, overlaps with DOH), 2.653 (s, 3H), 2.20–1.26
(18H), 0.903 (s, 3H), 0.69 (m, 1H). 13C NMR (75 MHz, DMSO-d6,
300 K) dppm 154.8, 153.6, 137.1, 130.4, 125.9, 124.4, 114.9, 112.6,
101.5, 82.0, 81.0, 72.4, 71.8, 69.5, 49.5, 47.6, 46.8, 43.1, 37.1,
32.6, 30.7, 29.2, 27.2, 26.1, 23.4, 14.3. MALDI-TOF: m/z calcd for
C182H231N21O42 3383.7, found [M+H2O]+ 3402.90. HRMS: m/z calcd
for C182H233N21O42 [MH2]2+ 1692.3366; found 1692.3302.
4.1.28. General rhodamine conjugation procedure
Standard synthetic procedures were used. The appropriate qCD
derivative (50 mg), the appropriate rhodamine coupling reagent
(1 equiv of NHS–rhodamine for 23R, 26R and rhodamine-19-per-
chlorate for 23R0, 26R0) and DMAP (1 equiv) were dissolved in
DMF (1 mL) under N2. The reaction mixture was cooled to 0 °C
and EDC–HCl salt (1 equiv) was quickly added and the solution al-
lowed to warm, before stirring overnight at room temperature in
the dark. The resulting dark red suspension was precipitated in

820
H.-Y. Kim et al. / Bioorg. Med. Chem. 18 (2010) 809–821
acetone (15 mL) giving a pink powder after filtration, which was
washed twice with acetone followed by centrifugation at 4 °C for
1 h (10,000 rpm). The collected solids were lyophilized overnight
to give a pink solid and kept at ꢁ20 °C in the dark.
added to determine EC50 values, and the cells in a total volume
of 200 L media/well were incubated at 37 °C for four days. For
l
the determination of antiestrogenic activity, 2 ꢂ 10ꢁ8 M estradiol
was added to the media. The enzyme activity was measured by
reading the release of p-nitrophenyl phosphate at 405 nm every
15 s with a 10 s shake between readings for 16 readings using a
Power Wave 200 microplate scanning spectrophotometer (Bio-
Tek Instruments, Winooski, VT). For estrogenic determination,
the percent induction as compared with the estradiol control was
calculated. For antiestrogenic determination, the percent induction
was calculated compared to the background induction. The sulfo-
rhodamine (SRB) assay was used as a measure of cytotoxicity in
parallel with alkaline phosphatase (ALP) induction assays using
ꢀ1000 cells per well in a 96-well plate (5 ꢂ 106 cells/mL). Cells
were treated with the same samples used in the ALP induction as-
say. The plates were read using the endpoint mode at 515 nm. Cal-
culation of the percent cytotoxicity is essential to rule out cell
death as a causal factor in reduced induction and apparent anties-
trogenic activity.
4.1.29. Rhodamine coupled 7EqCD (23R)
Yield: 35 mg, 61%. MALDI-TOF: calcd for C210H259N23O46 3841.43,
found [M+Na]+ 3864.41.
4.1.30. Rhodamine coupled 7EqCD (23R0)
Yield: 36 mg, 63%. MALDI-TOF: calcd C209H259N23O43 3781.42,
found [M+Na]+ 3804.41.
4.1.31. Rhodamine coupled per-6-(4-hydroxymethyl-1,2,3-
triazolmethyl)-6-deoxy-b-cyclodextrin (26R)
Yield: 30 mg, 47%. MALDI-TOF: calcd for C91H119N23O38 2143.05,
found [M+K]+ 2181.75.
4.1.32. Rhodamine coupled per-6-(4-hydroxymethyl-1,2,3-
triazolmethyl)-6-deoxy-b-cyclodextrin (26R0)
Yield: 30 mg, 49%. MALDI-TOF: m/z calcd for C90H119N23O36
2099.04, found [M+K]+ 2138.15.
4.2.4. ERa competitive binding assay
Competitive displacement of [3H] estradiol (E2) radioligand
(Amersham Biosciences, Piscataway, NJ) from full-length recombi-
4.2. Bioassay procedures
nant human (h) ER
sayed as described.59 Briefly, the reaction mixture consisted of
L of compound in DMSO, 5 L of hER (0.5 pmol) in ER binding
buffer [consisting of 10 mM Tris–HCl (pH 7.5), 10% glycerol, 2 mM
a (PanVera/Invitrogen, Carlsbad, CA) was as-
4.2.1. Cell culture
5
l
l
a
The Ishikawa cell line was provided by Dr. R. B. Hochberg (Yale
University, New Haven, CT) and the cells were grown in Dulbecco’s
Modified Eagle medium (DMEM/F12) containing 1% sodium pyru-
vate, 1% non-essential amino acids (NEAA), 1% glutamax-1, 0.05%
insulin, and 10% heat-inactivated FBS. All cultures were maintained
at 37 °C with 5% CO2:95% air. For phenol red-free complete med-
ium, estrogen-stripped FBS was used. Human breast cancer cell
line T47D:A18 is a hormone-responsive clone: the stable transfec-
tant cell lines T47D:A18/neo and T47D:A18/PKCa58 were main-
tained in RPMI 1640 (phenol red) supplemented with 10% fetal
dithiothrietol, 1 mg/mL BSA], 5 lL of ‘Hot Mix’ [400 nM, prepared
fresh using 95 Ci/mmol [3H] estradiol, diluted in 1:1 ethanol/ER
binding buffer; obtained from NEN Life Science Products (Boston,
MA)] and 85 lL of ER binding buffer. The incubation was carried
out with test compounds at room temperature for 2 h. Radioactiv-
ity was counted using a Beckman LS 5801 liquid scintillation coun-
ter (Schamburg, IL). The IC50 was calculated from a concentration–
response data obtained in triplicate experiments.
bovine serum-containing G418 (500
transfection, T47D:A18/neo and T47D:A18/PKC
tained for three days in phenol red-free, E2-depleted RPMI 1640
supplemented with G418. After stripping, cells were transiently
transfected by electroporation as described previously.58
l
g/mL). Before transient
4.2.5. Fluorescence confocal microscopy
a
cells were main-
MCF-7 cells were grown (106 cells/mL) on each of eight wells on a
sterile Nunc™ chambered cover glass and incubated for 48 h at 37 °C
with 5% CO2 in phenol-free medium supplemented with 10%
stripped fetal bovine serum medium. Cells were incubated with
rhodamine conjugates (100 nM) for 24 h at 37 °C, 5% CO2. Cells were
4.2.2. Measurement of ERE activation
rinsed twice with PBS to remove the unincorporated dye and 0.2 lg/
T47D cells were transiently transfected by electroporation as de-
scribed previously.58 Briefly, 8 ꢂ 106 cells were harvested and
resuspended in 0.5 mL serum-free, phenol red-free RPMI 1640.
ERE-tk-Luc plasmid containing the luciferase reporter gene con-
mL Hoechst stain was added to the cells to detect nuclear staining.
Imaging was performed with a Zeiss LSM 510 laser-scanning
confocal microscope with the detector gain adjusted to eliminate
the background autofluorescence. The fluorescence signal from
rhodamine conjugate was monitored with a 544 nm argon/krypton
laser and a 576 nm band pass filter. Hoechst nuclear staining signal
was monitored with a 345 nm UV laser and 420 nm band pass filter.
A ꢂ 63 (1.2 numerical aperture) water immersion objective was
used for all experiments. Images were analyzed using the analysis
tool provided in the Zeiss biophysical software package.
trolled by the ERE (5 lg) and b-galactosidase (b-gal; 1 lg) express-
ing plasmid pCMVb were added to the cell suspension and
incubated and processed. On the following day, medium containing
E2 (10ꢁ9 mol/L), 7EqCD or qE2 (10ꢁ9–10ꢁ7 mol/L), or vehicle (etha-
nol) was added. Luciferase activities were measured using Lucifer-
ase Reporter Gene Assay System from Applied Biosystems. b-gal
signals were measured with Galacto-Light Plus assay systems
(Applied Biosystems). After 18 h incubation, cells were washed
with ice-cold PBS and lysed in the lysis buffer provided. The cell
lysates were cleared by centrifugation and luciferase activity and
b-gal signals were determined after adding corresponding sub-
strates and read by a Monolight 2010 luminometer (Analytical
Luminescence Laboratory). The results were normalized relative
to pRL-TK activity, to account for transfection efficiency, by dividing
the sum of the luciferase activity by the sum of the b-gal signals.
Acknowledgments
This research was supported by the National Institutes of Health
(CA 102590) and DOD contract W81XWH-07-1-0445. Dr. Zhiqiang
Wang and Dr. Kuanqiang Gao are acknowledged for technical
assistance.
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