Design, synthesis, and initial biological evaluation of a steroidal anti-estrogen - Doxorubicin bioconjugate for targeting estrogen receptor-positive breast cancer cells

Article abstract of DOI:10.1021/bc200645n

As part of our program to develop breast cancer specific therapeutic agents, we have synthesized a conjugate agent that is a conjugate of the steroidal anti-estrogen and the potent cytotoxin doxorubicin. In this effort, we employed a modular assembly appr

Full text of DOI:10.1021/bc200645n

Article
pubs.acs.org/bc
Design, Synthesis, and Initial Biological Evaluation of a Steroidal
Anti-Estrogen−Doxorubicin Bioconjugate for Targeting Estrogen
Receptor-Positive Breast Cancer Cells
Kinh-Luan Dao,† Rupa R. Sawant,‡ J. Adam Hendricks, Victoria Ronga,† Vladimir P. Torchilin,‡
and Robert N. Hanson*,†
‡
†Department of Chemistry and Chemical Biology Center for Pharmaceutical Biotechnology and Nanomedicine 360 Huntington
Avenue, Northeastern University, Boston, Massachusetts 02115-5000, United States
S
* Supporting Information
ABSTRACT: As part of our program to develop breast cancer
specific therapeutic agents, we have synthesized a conjugate
agent that is a conjugate of the steroidal anti-estrogen and the
potent cytotoxin doxorubicin. In this effort, we employed a
modular assembly approach to prepare a novel 11β-substituted
steroidal anti-estrogen functionalized with an azido-tetra-
ethylene glycol moiety, which could be coupled to a complementary doxorubicin benzoyl hydrazone functionalized with a
propargyl tetraethylene glycol moiety. Huisgen [3 + 2] cycloaddition chemistry gave the final hybrid that was evaluated for
selective uptake and cytotoxicity in ER(+)-MCF-7 and ER(−)-MDA-MB-231 breast cancer cell lines. The results demonstrated
that the presence of the anti-estrogenic component in the hybrid compound was critical for selectivity and cytotoxicity in ER(+)-
MCF-7 human breast cancer cells as the hybrid was ∼70-fold more potent than doxorubicin in inhibition of cell proliferation and
promoting cell death.
variants that would enhance or optimize the activity of the
product.
INTRODUCTION
■
Breast cancer is the most common cancer diagnosis among
women, with the majority of cases linked with the hormone-
responsive form of the disease.1 Because of the well-established
association of estradiol with estrogen receptors (ER), endocrine
therapy using anti-estrogens, such as tamoxifen (TAM) and
Faslodex, is the typical regimen for the treatment of hormone-
responsive breast cancer.2−5 Unfortunately, prolonged treat-
ment of breast cancer patients with anti-estrogens frequently
leads within 2−5 years to the emergence of recurrent disease
that no longer responds to endocrine therapy. More aggressive
and nonselective interventions are required that produce
significant side-effects and morbidity to the patient population.
Therefore, there is a continued need to develop therapeutic
agents that are more effective from the very beginning and/or
that do not develop resistance.
One approach to develop such agents involves combining
two drugs into a single entity as a conjugate that can interact
with two relevant components of the disease process. The
general criteria and challenges associated with such an approach
have been the subject of several reviews.6−12 In addition to
fundamental concerns related to choice of targeting strategies
(e.g., receptor vs antibody), of single target versus independ-
ent/related targets, and of pathway interactions between the
biological targets, one also needs to address the issues related to
the chemical synthesis of such an agent. In particular, the two
components need to be joined in a fashion that does not
compromise the activity of either, and ultimately, the synthetic
approach needs to be sufficiently robust to tolerate structural
In the field of hormone-responsive breast cancer, this
approach typically involved linking a potent estrogen receptor
targeting agent to a second component, such as an
antimetabolite, intercalating agent, antimitotic, alkylating
agent, or metal chelating group.13−28 In the case of estrogen
receptor-targeted hybrids, these efforts have been almost
invariably unsuccessful. To a significant extent, the lack of
success can be traced to an over-reliance on chemical
transformations of readily available estrogens or easily modified
sites on those estrogens to prepare the target compounds.
While the attachment of functional groups at the 17β-,17α-,
16α-, and 7α-positions of estradiol (Figure 1) is readily
achieved through simple transformations of estradiol or its
derivatives, the analysis of the crystal structures of agonist and
antagonist−estrogen receptor−ligand binding domain (ER-
LBD) complexes suggests that such modifications seriously
impair receptor binding.29 Several examples of recent 17β-,
17α-, and 16α -substituted estradiol hybrids with specific
therapeutic R groups have shown low ER binding affinity.30−35
While the introduction of substituents at the 7α-position of
estradiol (such as those found in the anti-estrogen Faslodex) is
synthetically more challenging, the resultant products retain
significant ER binding capacity and modest ER-based
Received: December 2, 2011
Revised: February 15, 2012
Published: March 11, 2012
© 2012 American Chemical Society
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Figure 1. (1) 17-β substituted estradiol;28 (2) 17-α substituted estradiol;27,31−33 (3) 16-α substituted estradiol;30,34,35 and (4) 7-α substituted
estradiol.36−40
Figure 2. Prodrug approaches in the development of doxorubicin conjugates.
Figure 3. Tamoxifen−doxorubicin conjugate.50,51
selectivity.36−40 X-ray crystal structures of complexes of
similarly 7α-substituted ligands with ERα-LBD indicated that
the steroidal scaffold was rotated around the 3−17 axis, and
there was a disorder associated with helix-12, suggesting that
the significant steric interactions were involved.41
groups, including breast cancer selective agents, have been
described.47−49 For example, a nonsteroidal anti-estrogen, such
as tamoxifen (Figure 3), has been used to target doxorubicin to
ER(+)-breast cancer; however, it is associated with significant
problems. The parent compound, tamoxifen, has low ER
affinity and exhibits substantial non-ER binding capacity.
Although the hydroxylated metabolite, 4-hydroxytamoxifen,
has higher ER affinity, this compound exists as a mixture of E/
Z-isomers and is chemically less stable.5,50 Nevertheless,
preliminary studies suggested that an enhanced and selective
cytotoxicity in breast cancer cells may be achieved using a
better targeting group. The conjugation of amino-sugar
component doxorubicin to the amino terminus of hydroxyl
tamoxifen using a releasable linker led to an increase of potency
(antiproliferative activity) in a variety of breast cancer cell lines.
The investigators suggested that the targeting in this case was
due to a combination of ER and anti-estrogen binding site
(AEBS) effects.47,48
The second component of the bioconjugate involves the
therapeutic agent. To address the need for more effective drugs
for estrogen-responsive breast cancer, we have chosen the
clinically effective anthracycline doxorubicin. While highly
effective as a cytotoxic agent, its use is compromised by dose-
limiting cardiotoxic side effects.42−44 Strategies to improve its
clinical utility have focused on pro-drug approaches to reduce
side effects and on targeted drug delivery to improve its
efficacy. Some of the most promising doxorubicin derivatives
involve hydrazone formation through the ketone or amide
conjugation on the carbohydrate amino-group (Figure
2).42,45,46 In both approaches, intracellular processes, such as
pH-dependent hydrolysis or enzymatic cleavage of the amide
bond lead to free doxorubicin that generates the observed
therapeutic response. While such conjugation strategies may
reduce cardiotoxic effects, selective or enhanced delivery of the
agent to the tumor is not improved.42
Much of the (anti-)estrogen−doxorubicin conjugate research
has focused on the targeting and therapeutic groups; however,
the linking moiety is also important. For the hybrid to be
effective in vitro or in vivo, the linker must be long enough to
permit the ER-binding component to interact with the target
protein while maintaining a stable bond with the doxorubicin.
Likewise, the interaction with doxorubicin must be stable in the
Enhanced tumor delivery of doxorubicin requires the
incorporation of an appropriate targeting agent. Several
examples of doxorubicin derivatives bearing tumor-selective
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Figure 4. Strategy for conjugate AE-Dox synthesis.
Scheme 1. Synthesis of Anti-Estrogen−Doxorubicin (AE-Dox) Conjugate 15
extracellular environment while permitting facile dissociation
within the target breast cancer cells. The linker also needs to
have physicochemical properties that do not compromise its
formulation or biological compatibility. Therefore, our strategy
in this study considered all three components of hybrid drug
designthe estrogen targeting component, the doxorubicin
drug delivery component, and the linker componentthat
would tether the targeting, readily release mechanism, and
chemotherapeutic units.
In our design of the anti-estrogen−doxorubicin conjugate, we
have used a strategy involving modular assembly, i.e., each
component can be developed independently and ultimately
incorporated in a modular fashion. We selected an analogue of
steroidal anti-estrogen similar to RU39411, as our targeting
component. Not only is the parent compound a pure
antagonist, it possesses significantly higher ER binding affinity
compared to most nonsteroidal anti-estrogens such as
tamoxifen and raloxifene, and it is less lipophilic.51,52 The
elimination of the triarylethylene pharmacophore would also
reduce interactions with the anti-estrogen binding sites not
associated with the estrogen receptor. The analysis of its
binding in comparison to tamoxifen-ERα-LBD complexes
suggested that the ligation of linker groups via the tertiary
amine should retain ER affinity, as the linker would be external
to the ligand-binding pocket. The modified anti-estrogen 9 is
chemically accessible via the multistep synthesis from the
steroidal intermediate deltenone (as shown in the Experimental
section), a process with which we have significant experi-
ence.53−56 The preparation of doxorubicin hydrazones is well-
described as is their intracellular release under acidic
conditions;45,46,57−62 however, we needed to develop a specific
linker derivative to form the desired hydrazone. A key aspect of
our approach involves the use of half-linkers that would be
ligated in the final step to form the final conjugate. Each part
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would consist of a heterobifunctional tetraethylene glycol, in
which one functional group consists of a “click” partner and the
other functionality would interact with the targeting (anti-
estrogen) or chemotherapeutic (doxorubicin) moiety. Our
ultimate approach is shown in Figure 4, in which we take each
component, attach it to the appropriate half-linker, and finally
ligate them to form the intact bioconjugate. We hypothesize
that the resultant bioconjugate should retain antiproliferative
effects comparable to doxorubicin in all cancer cell lines, but
demonstrate selective antiproliferative effects in membrane ER-
expressing cancer cells. We suggest that endosomal uptake of
our AE-Dox hybrid is selectively mediated through membrane
ER.63 Once within the acidic cytoplasm compartment,
hydrolysis of the hydrazone-Dox linkage releases the free
doxorubicin to generate the antiproliferative response. As our
results illustrate, the final compound achieves these target
properties and demonstrates selectivity toward ER(+)-breast
cancer cells and promotes enhanced cytotoxicity against those
cells compared to the unmodified parent components.
liquor was purified via flash chromatography to afford 2.0 g (5.8
mmol, 18.2%) of the β-isomer, and an additional 600 mg (1.8
mmol, 5.7%) of 2α-isomer. Overall 3,3-ethylenedioxy-5(10)-α-
epoxy-estr-9(11)-ene-17-one (5.8 g, 17.5 mmol, 56% yield) and
3,3-ethylenedioxy-5(10)-β-epoxy-estr-9(11)-ene-17-one (2.0 g,
5.8 mmol, 18% yield) were obtained in a final ratio of 3:1 in
favor of the α-isomer and a total yield of 74%. For 2α-isomer:
1H NMR (CDCl3, 400 MHz): δ 0.88 (3H, s), 1.32−1.12 (1H,
s), 2.52−2.44 (2H, m), 3.98−3.88 (4H, m), 6.06 (1H, s). 13C
NMR (CDCl3, 100 MHz): δ 14.89, 22.05, 22.33, 25.23, 28.19,
31.73, 33.76, 36.05, 37.23, 40.40, 46.13, 46.81, 60.18, 61.73,
64.24, 64.45, 107.04, 125.80, 136.81, 221.37; mp 154 °C. For β-
1
isomer: H NMR (400 MHz, CHCl3): δ 0.87 (s, 3H), 1.32−
1.12 (m, 1H), 2.52−2.44 (m, 2H), 3.98−3.88 (m, 4H), 5.87(s,
1H); C20H26O4, TOF-MS: m/z 330.18 (calcd); 353.42 [M
+Na]+ (found).
11β-(4-Hydroxy-phenyl)-estra-4,9-diene-3,17-dione 3. 3,3-
Ethylenedioxy-5(10)-α-epoxy-estr-9-ene-17-one 2α (2.014 g,
6.1 mmol) was dissolved in anhydrous THF (15 mL) under an
argon atmosphere. Copper(I) iodide (0.160 g, 0.840 mmol)
was added to the solution at −10 °C and stirred for 15 min.
Freshly prepared Grignard reagent, (4-((trimethylsilyl)oxy)-
phenyl)magnesium bromide, was added dropwise in 5.0 mL
aliquots. The reaction was gradually warmed to ambient
temperature, and stirring was continued for 16 h. The reaction
was quenched by the addition of ammonium chloride (0.8 g, 15
mmol) in 35 mL of water and 35 mL of EtOAc at 10 °C. The
organic layer was washed with water (2 × 35 mL). The organic
solvent was removed under reduced pressure, and the resulting
residue was dissolved in a mixture of acetic acid (14 mL) and
water (6 mL). The resultant mixture was warmed at 50−60 °C
for 1.5 h, after which it was diluted with ethyl acetate (20 mL).
The solution was neutralized by the addition of saturated
aqueous sodium bicarbonate. The organic layer was separated,
washed with brine solution, dried over magnesium sulfate, and
evaporated to dryness to give a crude, yellow oil. Purification
using silica gel column chromatography (70:30 hexane/ethyl
acetate) afforded the product 3 (2.00 g, 76%) as a yellow solid:
1H NMR (CDCl3, 400 MHz): δ 0.53 (3H, s), 4.38 (1H, d, J =
6.9), 5.78 (1H, s), 6.71 (2H, d), 6.97 (2H, d). 13C NMR
(CDCl3, 100 MHz): δ 14.17, 21.76, 25.99, 27.06, 30.68, 35.02,
36.87, 38.14, 38.34, 39.71, 47.53, 50.77, 115.53, 122.82, 128.30,
129.83, 135.52, 145.62, 155.59, 155.99, 197.54, 217.44;
C24H26O3, TOF-MS: m/z 363.19 (calcd); 384.99 [M+Na]+
(found); m.p. 248 °C.
EXPERIMENTAL SECTION
■
General Methods. All solvents and reagents involved in the
synthesis were reagent grade, purchased from either Sigma-
Aldrich or Fisher Scientific, and used without further
purification. Thin-layer chromatography (TLC) was done on
polyester sheets precoated with silica gel matrix 60 F254
obtained from Sigma-Aldrich. Separations were performed
using automated flash chromatography (Argonaut FlaskMaster)
or packed column chromatography with Sorbent Technologies
silica gel particle size 32−63 μm and 60 Å pore size. Liquid
chromatography−mass spectroscopy (LC-MS) was performed
using Alliance HT -LCT Premier 2489, Waters instrument
equipped with time-of-flight (TOF) MS module. High-
performance liquid chromatography (HPLC) trace analysis
was performed using a Waters HPLC system, equipped with a
Waters 2695 binary pump, a Waters 2998 fluorescence
photodiode array detector, and an XBridge C18 column (3.5
μm, 4.6 × 75 mm). 1H and 13C NMR spectra were recorded on
400 or 500 MHz Varian FT-NMR spectrometers. Chemical
shifts (δ) are reported in parts per million (ppm) by reference
to proton resonances resulting from incomplete deuteration of
the NMR solvent. The concentrations of test compounds were
determined spectrophotometrically with a diode array UV mini
1240, Shimadzu spectrophotometer. Ultracentrifugation of cell
lysates was accomplished with Sorvall RT 6000B Refrigerated
Centrifuge. All tissue culture materials were obtained from
Gibco Life Technologies (Grand Island, NY) unless otherwise
stated. MCF-7 and MDA-MB-231 cells were obtained from
American Type Culture Collection (Rockville, MD).
11β-[4-(2-Dimethylamino-ethoxy)-phenyl]-estra-4,9-
diene-3,17-dione 4. To a solution of 3 (250 mg, 0.69 mmol)
and cesium carbonate (1.1 g, 3.45 mmol) in 10 mL acetone was
added 2-N,N-dimethyl chloroethyl amine hydrochloride (223
mg, 2.07 mmol). The reaction mixture was heated at reflux for
16 h. The solvent was removed under reduced pressure, and
the residue was extracted with ethyl acetate. The organic layer
was washed with water, brine solution, dried over magnesium
sulfate, and evaporated under reduced pressure to give a crude
oil. Purification using flash chromatography yielded the product
Synthesis. 3,3-Ethylenedioxy-5(10)-α-epoxy-ester-9(11)-
ene 2α. To a solution of estra-5(10),9(11)-diene-3,17-dione 3-
ethylenedioxy ketal 1 (10.0 g, 31.8 mmol), hexafluoroacetone
(0.46 mL, 3.6 mmol), and pyridine (0.23 mL, 2.86 mmol) in 10
mL of dichloromethane was added hydrogen peroxide (50%,
2.28 mL, 74 mmol) at 0 °C. After 18 h stirring at ambient
temperature, the reaction was terminated by the addition of 4 g
of sodium thiosulfate in 100 mL of water and extracted with
dichloromethane (3 × 100 mL). The organic layer was dried
over magnesium sulfate, filtered, and the solvent removed
under reduced pressure. The resulting colorless solid was
triturated with diethyl ether (35 mL). The precipitate was
collected by filtration and rinsed with diethyl ether (25 mL) to
yield 5.23 g (15.8 mmol) 49.8% of the 2α-isomer. The mother
1
4 (287 mg, 96%) as light yellow oil. H NMR (CDCl3, 400
MHz): δ 0.56 (3H, s), 1.22−1.41 (2H, m), 1.48−1.72 (2H, m),
1.91 (2H, dd, J = 13.92, 6.60 Hz), 1.99−2.23 (6H, m), 2.28−
2.52 (6H, m), 2.59−2.67 (2H, m), 2.67−2.89 (2H, m), 4.03
(1H, t, J = 5.86 Hz), 4.38 (2H, d, J = 7.33 Hz), 5.79 (1H, s),
6.84 (2H, d, J = 8.79 Hz), 7.08 (2H, d, J = 8.06 Hz). 13C NMR
(CDCl3, 100 MHz): δ 15.6, 22.1, 26.1, 27.0, 29.6, 31.1, 32.0,
35.6, 37.0, 38.0, 39.8, 46.1, 47.9, 50.9, 54.0, 58.5, 66.1, 114.9,
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123.6, 127.9, 128.0, 128.2, 130.2, 136.1, 145.2, 156.2, 157.2,
199.6; C28H35NO3, TOF-MS: m/z 433.26 (calcd); 457.99 [M
+Na]+ (found).
35.5, 38.3, 40.2, 44.8, 47.8, 48.3, 48.4, 52.4, 57.4, 64.7, 64.9,
82.5, 114.0, 116.4, 128.6, 130.4, 131.6, 138.2, 139.1, 158.6,
155.7; C27H35NO3, TOF-MS: m/z 422.26 (calcd); 443.99 [M
+Na]+ (found).
3-Acetoxy-11β-[4-(2-dimethylamino-ethoxy)-phenyl]-
estra-1,3,5(10)-triene-17-one 5. To a solution of 4 (200 mg,
0.42 mmol) in dichloromethane (10 mL) was added acetic
anhydride (47 mg, 0.46 mmol) and acetyl bromide (142 mg,
1.15 mmol) at room temperature. The reaction solution was
stirred for 16 h, after which the product was extracted with
ethyl acetate and washed with water. The organic layer was
separated, dried over magnesium sulfate, and evaporated under
reduced pressure to give a yellow crude oil. Column
chromatography afforded the desired product 5 (207 mg,
95%). 1H NMR (CDCl3, 400 MHz): δ 0.45 (3H, s), 1.25 (2H,
s), 1.47−1.73 (2H, m), 1.84−2.02 (2H, m), 2.03−2.20 (6H,
m), 2.22−2.29 (6H, m), 2.35 (3H, d, J = 10.26 Hz), 2.41−2.64
(2H, m), 2.87−3.03 (2H, m), 3.07 (1H, br. s.), 4.00−4.22 (2H,
m), 6.59−6.76 (3H, m), 6.86 (1H, d, J = 2.20 Hz), 6.96 (2H, d,
J = 8.79 Hz). 13C NMR (CDCl3, 100 MHz): δ 15.4, 21.6, 22.3,
23.1, 27.3, 30.2, 35.1, 35.5, 38.3, 40.2, 44.8, 47.8, 48.3, 48.4,
52.4, 57.4, 64.7, 64.9, 114.0, 119.4, 121.9, 128.6, 130.9, 135.6,
135.7, 137.7, 148.3, 155.7, 169.9, 176.1; C30H37NO4, TOF-MS:
m/z 475.27 (calcd); 498.26 [M+Na]+ (found).
11β-[4-(2-Dimethylamino-ethoxy)-phenyl]-estra-1,3,5(10)-
triene-3,17β-diol 6. To a solution of 5 (200 mg, 0.42 mmol) in
methanol (5 mL) was added sodium borohydride (24 mg, 0.63
mmol) and the reaction mixture was stirred at room
temperature. After 1 h, 10 N sodium hydroxide (0.025 mL,
0.25 mmol) was added and the reaction continued for 16 h.
The reaction solution was poured into an ice-cold mixture of
ethyl acetate (20 mL) and water (20 mL), after which the
organic layer was separated, washed sequentially with water and
brine, and dried over magnesium sulfate. The solvent was
removed under reduced pressure to give a yellow crude oil.
Silica column chromatography afforded the product 6 (176 mg,
96%). 1H NMR (CDCl3, 400 MHz): δ 0.36 (3H, s), 1.17−1.29
(2H, m), 1.29−1.44 (2H, m), 1.70 (2H, d, J = 8.79 Hz), 1.78
(2H, dd, J = 13.18, 5.86 Hz), 1.86−1.99 (2H, m), 2.03−2.14
(2H, m), 2.27−2.38 (6H, m), 2.51 (3H, d, J = 12.21 Hz), 2.61−
2.67 (2H, m), 2.69−2.85 (2H, m), 3.68 (1H, t, J = 8.06 Hz),
3.89−4.04 (2H, m), 6.37 (1H, dd, J = 8.30, 2.44 Hz), 6.44−
6.58 (3H, m), 6.76 (1H, d, J = 8.79 Hz), 6.95 (2H, d, J = 8.30
Hz). 13C NMR (CDCl3, 100 MHz): δ 15.4, 21.6, 22.3, 23.1,
27.3, 30.2, 35.1, 35.5, 38.3, 40.2, 44.8, 47.8, 48.3, 48.4, 52.4,
57.4, 64.7, 64.9, 82.5, 114.0, 116.4, 128.6, 130.4, 131.6, 138.2,
139.1, 158.6, 155.7; C28H37NO3, TOF-MS: m/z 435.26 (calcd);
437.99 [M+Na]+ (found).
2-{2-[2-(2-Azido-ethoxy)-ethoxy]-ethoxy}-ethyl Tosylate 8.
To a solution of tetraethylene glycol di-para-toluene sulfonate
(2 g, 4 mmol) in ethanol (25 mL) was added sodium azide
(0.28 g, 4.3 mmol). The resulting solution was heated at 80 °C
overnight. The reaction mixture was poured into ice water (125
mL), and the product was extracted with ethyl acetate. The
combined organic extracts were washed sequentially with water
and brine solution, and dried over magnesium sulfate. The
crude material was purified using silica gel chromatography to
yield the product as clear oil 8 (1.2 g, 79%). 1H NMR (CDCl3,
400 MHz): δ 1.21 (2H, td, J = 6.96, 2.93 Hz), 2.45 (3H, s),
3.40 (2H, m), 3.52−3.75 (10H, m), 3.93 (2H, m), 7.37 (2H,
d), 7.79 (2H, d); C15H23N3O7S, TOF-MS: m/z 389.42 (calcd);
412.56 [M+Na]+ (found).
11β-(4-{2-[(2-{2-[2-(2-Azido-ethoxy)-ethoxy]-ethoxy}-
ethyl)-methyl-amino]-ethoxy}-phenyl)-estra-1,3,5(10)-triene-
3,17β-diol 9. To a solution of 7 (8 mg, 0.205 mmol) and
potassium carbonate (43 mg, 0.31 mmol) in acetonitrile (10
mL) was added dropwise at ambient temperature under an
inert atmosphere a solution of 8 (69 mg, 0.16 mmol) in
acetonitrile. The solution was heated at reflux for 16 h. The
reaction solvent was evaporated under reduced pressure, and
the resulting residue was extracted with ethyl acetate. The
solvent was evaporated under reduced pressure and the crude
material was purified using silica gel column chromatography to
yield a light yellow oil 9 (77 mg, 60%). 1H NMR (CDCl3, 400
MHz): δ 0.31 (3H, s), 1.18−1.45 (2H, m), 1.66−1.81 (2H, m),
1.99−2.20 (2H, m), 2.33−2.41 (4H, m), 2.51 (1 H, d, J = 12.46
Hz), 2.68 (2H, t, J = 5.86 Hz), 2.77−2.90 (2H, m), 3.34−3.42
(2H, m), 3.45−3.71 (14H, m), 3.81 (2H, t, J = 4.76 Hz), 3.92−
4.00 (4H, m), 4.00−4.07 (3H, m), 6.49 (1H, dd, J = 8.43, 2.56
Hz), 6.56−6.67 (2H, m), 6.86 (2H, d, J = 8.79 Hz), 6.94 (2H,
d, J = 8.79 Hz). 13C NMR (CDCl3, 100 MHz): δ 13.0, 17.5,
23.4, 28.2, 30.5, 30.7, 35.6, 38.4, 43.6, 43.8, 45.8, 47.6, 50.9,
52.1, 56.8, 57.4, 67.4, 69.6, 70.0, 70.3, 70.6, 70.9, 71.0, 76.9,
82.8, 112.6, 113.7, 114.8, 127.7, 130.7, 131.1, 136.0, 137.7,
155.9, 156.3; C35H50N4O6, TOF-MS: m/z 622.37 (calcd);
657.99 [M+Na]+ (found).
2-{2-[2-(2-Prop-2-ynyloxy-ethoxy)-ethoxy]-ethoxy}-etha-
nol 10. To a solution of tetraethylene glycol (2.00 g, 10.3
mmol) in tetrahydrofuran (10.0 mL) was slowly added sodium
hydride (60% in paraffin, 580 mg, 15 mmol) at −20 °C. To the
reaction vessel was added dropwise at −20 °C a solution of
propargyl bromide (3.0 g, 22 mmol) in 5.0 mL tetrahydrofuran.
The reaction mixture was stirred at −20 °C for 30 min, allowed
to warm to ambient temperature, and then stirred for an
additional 24 h. The reaction was partitioned between ethyl
acetate (20 mL) and water (20 mL), after which the organic
layer was washed with water and brine. The organic layer was
dried over magnesium sulfate, filtered, and the solvent removed
via rotary evaporation. The crude product was purified using
silica gel column chromatography to yield 1.55 g (65%) of the
product 10 as oil. 1H NMR (CDCl3, 400 MHz): δ 1.26 (1H, t, J
= 7.1 Hz), 2.08−2.09 (1H, m), 2.17 (1H, s), 2.44 (2H, t, J = 2.4
Hz), 3.65−3.77 (14H, m), 4.21 (2H, d, J = 2.4 Hz). 13C NMR
(CDCl3, 100 MHz): δ 58.3, 61.5, 69.0, 70.3, 70.3, 70.5, 70.5,
70.6, 72.7, 75.1, 79.7; C11H20O5, TOF-MS: m/z 233.42 (calcd);
256.34 [M+Na]+ (found).
11β-[4-(2-methylamino-ethoxy)-phenyl]-estra-1,3,5(10)-
triene-3,17β-diol 7. To a solution of 6 (100 mg, 0.23 mmol) in
anhydrous dichloromethane (10 mL) was added α-chloroethyl
chloroformate (53 μL, 0.48 mmol). The reaction mixture was
stirred at 0 °C for 30 min, and then heated at reflux for 24 h.
The solvent was removed by rotary evaporation, methanol (3
mL) was added, and the reaction solution was heated at reflux
for 3 h. The solvent was removed by rotary evaporation to give
the crude product as clear oil. Purification using silica gel
chromatography gave the product 7 (82 mg, 85%) as yellow oil.
1H NMR (CDCl3, 400 MHz) δ 0.34 (3H, s), 1.20−1.45 (2H,
m), 1.63−1.87 (2H, m), 1.90−2.18 (2H, m), 2.32 (1H, s), 2.51
(4H, m), 2.72−2.97 (4H, m), 3.69 (1H, t, J = 8.43 Hz), 3.88−
4.03 (2H, m), 6.39 (1H, dd, J = 8.43, 2.56 Hz), 6.47−6.61 (3H,
m), 6.79 (1H, d, J = 8.06 Hz), 6.97 (2H, d, J = 8.06 Hz). 13C
NMR (CDCl3, 100 MHz): δ 15.4, 21.6, 22.3, 23.1, 27.3, 30.2,
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2-(2-(2-(2-(Prop-2-ynnyloxy)-ethoxy)-ethoxy)-ethoxy)-
ethanol Tosylate 11. To a solution of propargyl tetraethylene
glycol 10 (1.0 g, 4.31 mmol) in CH2Cl2 (15.0 mL) was added
triethylamine (1.2 mL, 8.6 mmol) and p-toluenesulfonyl
chloride (0.99 g, 5.2 mmol). The reaction mixture was stirred
at ambient temperature for 16 h. The solvent was under
reduced pressure to give a crude product as dark oil. Separation
using silica gel column chromatography gave the product 11
(1.53 g, 92%) as a yellow oil. 1H NMR (CDCl3, 400 MHz): δ.
2.45 (3H, s), 2.49 (1H, J = 2.2 Hz), 3.59−3.64 (14H, m),
3.65−3.72 (2H, m), 4.13−4.17 (2 H, m), 4.18 (2H, J = 2.2 Hz),
7.36 (2H, J = 8.1 Hz), 7.79 (2H, d, J = 8.1 Hz). 13C NMR
(CDCl3, 100 MHz): δ 21.9, 21.9, 58.6, 68.9, 69.3, 69.5, 70.6,
70.7, 70.7, 70.8, 70.9, 74.8, 74.8, 79.9, 128.2, 130.1, 133.2,
145.0; C18H26O7S, TOF-MS: m/z 402.13 (calcd); 422.99 [M
+Na]+ (found).
Methyl 4-(2-(2-(2-(2-(Prop-2-ynyloxy)-ethoxy)-ethoxy)-
ethoxy)-ethoxybenzoate 12. To a solution of 11 (1.0 g, 2.6
mmol) in dichloromethane (10 mL) was added dropwise a
mixture of cesium carbonate (126 mg, 388 mmol) and methyl
4-hydroxybenzoate (590 mg, 3.9 mmol) in 5 mL of
dichloromethane. The reaction was heated at reflux for 16 h.
The reaction mixture was filtered and then concentrated under
rotary evaporation. The residue was purified using silica gel
column chromatography to afford the product 12 (1.1 g, 70%)
as clear oil. 1H NMR (CDCl3, 400 MHz): δ 2.05 (1H, s), 2.44
(3H, t, J = 2.56 Hz), 3.62−3.75 (12H, m), 3.82−3.92 (2H, m),
4.09−4.22 (4H, m), 6.93 (2H, d, J = 8.79 Hz), 7.98 (2H, d, J =
8.79 Hz). 13C NMR (CDCl3, 100 MHz): δ 22.6, 52.1, 52.3,
58.6, 67.8, 69.3, 69.7,70.4, 70.6, 70.8, 71.1, 74.8, 79.9, 114.4,
122.9, 131.7, 162.8, 167.0; C19H26O7, TOF-MS: m/z 366.16
(calcd); 389.10 [M+Na]+ (found).
38.3, 48.1, 51.2, 61.0, 67.6, 69.4, 70.1, 70.3, 70.4, 72.4, 114.2,
117.3, 122.4, 125.8, 131.4, 140, 153.2, 155.4, 163.1, 167.4,
187.2; C45H53N3O15, TOF-MS: m/z 875.35 (calcd); 898.39 [M
+Na]+ (found).
Anti-Estrogen−Doxorubicin Conjugate 15. To a solution
of 9 (10 mg, 0.015 mmol) in 500 μL of tert-butanol/water
(1:1) was added a solution of 14 (13.1 mg, 0.015 mmol) in 500
μL of tert-butanol/water (1:1). The reaction was stirred at
room temperature for 30 min, followed by the addition of
copper(II) sulfate pentahydrate (3.75 μL, 0.15 μmol) and
(+)-sodium L-ascorbate (14.8 μL, 0.75 μmol). The reaction
mixture was warmed to 40 °C, stirred for 24 h, and then
partitioned between water (10 mL) and dichloromethane (10
mL). The organic layer was washed with water (2 × 10 mL).
The aqueous layers were combined; sodium chloride was added
and then back-extracted with dichloromethane (10 mL). The
organic fractions were combined, dried over magnesium sulfate,
filtered, and concentrated leaving a dark red residue. The
product was isolated using column chromatography (85:15
dichloromethane/methanol) to yield the product 15 (12.7 mg,
55%) as a red solid. TLC (Si2O, 80:20 dichloromethane/
1
methanol) Rf = 0.25. H NMR (CH3OH-d, 500 MHz): δ =
7.93 (d, J = 7.2, 2H), 7.89 (s, 1H), 7.85 (s, 1H), 7.76 (s, 1H),
7.61−7.67 (m, 3H), 7.56 (s, 1H), 7.49 (s, 1H), 7.25 (d, J = 7.8
Hz, 2H), 7.03 (d, J = 8.8 Hz, 2H), 6.99 (d, J = 8.8 Hz, 2H),
6.90 (s, 1H), 6.83 (s, 2H), 6.75−6.79 (m, 2H), 4.36 (t, J = 7.3
Hz, 1H), 4.27 (m, 2H), 4.16−4.20 (m, 1H), 4.03 (d, J = 5.9 Hz,
1H), 3.86−3.90 (m, 2H), 3.71−3.75 (m, 3H), 3.63−3.71 (m,
28H), 3.55−3.61 (m, 2H), 3.50−3.54 (m, 1H), 3.39−3.44 (m,
1H), 2.39 (s, 2H), 2.27 (s, 3H), 1.93 (s, 1H), 1.64−1.74 (m,
2H), 1.41−1.50 (m, 6H), 1.33−1.41 (m, 2H), 1.30 (s, 2H),
1.28 (br. s., 1H), 1.26 (m, 3H), 1.15 (m, 3H), 1.00 ppm (m,
3H). C45H53N3O15, TOF-MS: m/z 1277.59 (calcd); 1299.91
[M+Na]+ (found).
High-Performance Liquid Chromatography (HPLC)
Trace Analysis. The compounds were analyzed with Waters
HPLC system, equipped with a Waters 2695 binary pump, a
Waters 2998 fluorescence photodiode array detector, and a
XBridge C18 column (3.5 μm, 4.6 × 75 mm). HPLC-grade
acetonitrile/water/trifluoroacetic acid (50/50/0.1%, v/v) was
used as the mobile phase at 25 °C with a flow rate of 1.0 mL
min−1. Fluorescence detector was set at 254 nm for excitation
and 570 nm for emission and linked to Empower III software
for data analysis.
4-(2-{2-[2-(2-Prop-2-ynyloxy-ethoxy)-ethoxy]-ethoxy}-
ethoxy)-benzoic Acid Hydrazide 13. To a solution of 12 (200
mg, 0.55 mmol) in ethanol (10 mL) was added hydrazine
hydrate (44 mg, 1.4 mmol). The solution was heated at reflux
for 10 h. The solvent was removed by rotary evaporation to
give a crude material that was purified by using amino column
chromatography. The product 13 was isolated (144 mg, 72%)
1
as a pale, yellow oil. H NMR (CDCl3, 400 MHz): δ d 1.81
(2H, s), 2.16 (1H, s), 2.21−2.35 (2H, m), 3.66−3.81 (14H, m),
4.2 (2H, m), 6.96 (2H, d), 7.78 (2H, d), 7.82 (1H, s). 13C
NMR (CDCl3, 100 MHz): δ 67.7, 69.7, 70.1, 70.7, 71.0, 73.2,
114.5, 114.6, 125.2, 126.0, 129.0, 129.5, 144.0, 161.8, 161.9,
168.5, 169.8, 173.6; C18H26N2O6, TOF-MS: m/z 371.17
(calcd); 371.3 [M]+ (found).
Cell Cultures. The ER(+)MCF7 (human breast adenocar-
cinoma) and ER(−)MBA-MD-231 (human breast adenocarci-
noma) cell lines were maintained in Dulbecco’s Modified Eagle
Medium (DMEM) at 37 °C, 5% CO2. DMEM were
supplemented with 10% fetal bovine serum (FBS), 50 U/mL
penicillin, and 50 μg/mL streptomycin.
4-(2-(2-(2-(2-(Prop-2-ynyloxy)ethoxy)ethoxy)ethoxy)-
ethoxybenzohydrazone−Doxorubicin Conjugate 14. To a
solution of 12 (14 mg, 0.04 mmol) in ethanol (5 mL) was
added doxorubicin hydrochloride (20 mg, 0.004 mmol) and
trifluoroacetic acid (64.5 mg, 0.6 mmol). The reaction mixture
was stirred at 20 °C for 24 h, concentrated to approximately 1.0
mL, and triturated with ether to yield a red precipitate. The red
precipitate was collected by filtration, washed with ether, and
Cytotoxicity Assays. Cells were plated at a 5 × 103 cells
per well density in 96-well plates (Corning Inc., Corning, NY,
USA). The stock solutions of free drug or drug-conjugates were
prepared in DMSO and diluted in complete media before
adding to cells. After 24 h, the medium was replaced with
medium containing free drug or drug-conjugates. After 24 h
incubation, each well was washed twice with complete media,
and cell survival was measured using the Cell Titer-Blue Cell
Viability Assay method. The conversion of Resaruzin to
Resorufin by viable cells results in the fluorescence excitation
at 550 nm. The fluorescence produced is proportional to the
number of viable cells. The emitted fluorescence was measured
at 590 nm (the measurement of the cytotoxicity) using a
1
dried under vacuum to afford the product (28.5 mg, 85%). H
NMR (CDCl3, 400 MHz): δ 1.29 (3H, d, J = 6.6 Hz), 1.81−
1.94 (3H, m), 1.97−2.10 (2H, m), 2.11−2.22 (1H, m), 2.30−
2.47 (1H, m), 2.89−3.03 (1H, m), 3.06−3.15 (1H, m), 3.22−
3.37 (12H, m), 3.52−3.74 (3H, m), 3.78−3.89 (1H, m), 4.04
(3H, s), 4.14−4.24 (2H, m), 4.26−4.33 (1H, m), 4.71 (2H, d, J
= 2.2 Hz), 5.03−5.17 (1H, m), 5.39−5.52 (2H, m), 6.97−7.13
(2H, m), 7.51−7.62 (1H, m), 7.74−7.89 (3H, m), 7.94−8.00
(1H, m). 13C NMR (CDCl3, 100 MHz): δ 11.6, 20.5, 28.6,
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1
Figure 5. (A) Partial stacking H NMR of AE-Dox 15; (B) HPLC of AE-Dox 15 and its components.
Labsystems Multiskan MCC/340 microplate reader (Labsys-
tems and Life Sciences International, UK).
transformed to the requisite components in high yields and
purity. In particular, the conversion of the readily available
tetra(oligo)ethylene glycols to heterobifunctional half-linkers 8
and 13 is notable because of the possibility of subsequent
variations that one can generate. If necessary, one can control
the length of the spacer groups as well as the modes of ligation
to address concerns not apparent at the outset of the study. In
this example, the use of small tetraethylene glycol moieties
facilitated the individual reactions, purifications, and subsequent
ligations. The conjugation the propargylated-tetraethylene
glycol benzohydrazide 13 to doxorubicin to form the
alkynylated intermediate 14, proceeded with good yield, and
remained stable under neutral and basic conditions. The
preparation of the steroidal anti-estrogen 9 was readily achieved
from the key starting material 1 and transformed via N-
demethylation into the reactive intermediate 7. The subsequent
alkylation with the azido-tetraethylene glycol tosylate 8 gave the
stable component 9. The conversion into the final product 15
was achieved with good yield using the classical “click”
conditions.
For estradiol competition assay, the cells were pretreated
with 17β-estradiol (ES) (50 μM) for 1 h, followed by addition
of free drug or drug-conjugates in complete media with 50 μM
of ES. After 24 h incubation, cell viability was analyzed as
described above.
Flow Cytometry. The ER(+)MCF-7 and ER(−)MDA-MB-
231 cells were grown in 12-well tissue culture plates until 70−
80% confluency. The medium was removed from the wells;
cells were washed with complete media and incubated with or
without 50 μM ES for 1 h. After incubation, the cells were
washed twice with complete media, and exposed to 0.1 μM of
Dox, AE-Dox in complete media with or without 50 μM of ES.
After 1 h, cells were washed, trypsinized, and finally
resuspended in 800 μL of 4% paraformaldeyhde in phosphate
buffered saline (PBS) pH 7.4. The cell-associated fluorescence
was quantified by Becton Dickinson FACScan (Becton
Dickinson, San Jose, CA) at the emission wavelength of 580
nm (channel FL-2).The data analysis was performed using
CellQuest software (Becton Dickinson). A total of 10 000 events
were acquired for each sample. Data shown were derived from
three separate experiments.
Fluorescence Microscopy. The ER(+) MCF-7 cells were
seeded on a coverslip in six-well tissue culture plates at a
concentration of 1 × 105 cells per well. After 24 h, the cells
were washed twice with complete media and then incubated
with 0.1 μM Dox or AE-Dox 15. After 1 h incubation, medium
was removed, and cells were washed twice with sterile PBS
followed by fixation of the cells with 4% paraformaldehyde (15
min at room temperature). Hoechst 33342 (1 μg/mL) was
added to the cells for 15 min, and cells were washed twice with
sterile PBS. Cells were observed immediately on a Nikon
Eclipse E400 fluorescence microscope equipped with appro-
priate filters for Rhodamine and Hoechst detection. For
estradiol competition assay, the cells were pretreated with
17β-estradiol (ES) (50 μM) for 1 h, followed by addition of 0.1
μM of AE-Dox in complete media with 50 μM of ES.
To verify the ligation of the AE-Dox conjugate 15 from its
two intermediates, the azido anti-estrogen 9 and the
1
alkynylated Dox 14, we performed stacking H NMR analysis,
FT-IR, and HPLC. AE-Dox formation was indicated by the
signature triazole proton, shown at approximately 8.7 ppm in
1
the H NMR spectrum, and the presence of the characteristic
aromatic signature protons for both Dox and AE components
(Figure 5A). The FTIR spectrum supported the ligation of the
two intermediates by the disappearance of the azide stretch
approximately at 2100 cm−1 from compound 9, and the alkyne
stretch at 2050 cm−1 (SI) from compound 14. In addition, the
HPLC data (Figure 5B) showed the elution of a single peak for
15 that was distinct from the azide 15 or the alkyne 14.
Cell Studies. The preliminary evaluation of the parent
compounds (Dox and RU39411), linker modified components
14 and 9, and the final AE-Dox hybrid 15 was determined using
ER(+)-MCF-7 and ER(−)-MDA-MB-231 breast cancer cell
lines. ER(+)-MCF-7 is a human breast adenocarcinoma cell line
that overexpresses ER and is an excellent in vitro assay system
to demonstrate anti-estrogenic effects,64−66 while ER(−)-
MDA-MB-231 cells are insensitive to antihormonal interven-
tions.67 Both cell lines are responsive to doxorubicin, such that
structural modifications affecting the activity should be readily
apparent. Under the conditions of the bioassay, neither cell line
RESULTS AND DISCUSSION
■
Synthesis. The convergent modular approach proved to be
a successful strategy for assembling the target anti-estrogen
doxorubicin conjugate AE-Dox 15. The starting materials were
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metabolizes doxorubicin or the anti-estrogen to a significant
degree, so that any activity observed would be due primarily to
the parent drug and not potential metabolites.50,68
represented here were evaluated in a duplicate of a triplicate
count. Standard deviation was measured from the mean of six
wells for each compound.
Initial cell-based assays evaluated whether the presence of the
linker groups would affect the individual components as
inhibitors of cell proliferation (cytotoxicity). The steroidal
anti-estrogen and its azido-linker modified derivative (N3-AE 9)
had no significant cytotoxic effects on ER(+)-MCF-7 cells at
concentrations below 1 μM and only modest effects at 10 μM
(see SI). In the same cell line, doxorubicin and its alkynyl
hydrazone derivative 14 displayed statistically similar IC50
values (Table 1) and in vitro cytotoxicity profiles (results also
The subsequent study was undertaken both to support the
ER-related effect and to identify the nature of that effect. Cell
uptake/targeting of the hybrid AE-Dox 15 with ER(+)-MCF-7
and ER(−)-MDA-MB-231 cells was evaluated using fluores-
cence activated cell sorting (FACS), in both presence and
absence of ES, as shown in Figure 7. The AE-Dox 15
demonstrated enhanced cell binding only to ER(+)-MCF-7
cells in the absence of ES. In presence of ES, cell binding of AE-
Dox 15 was similar to that of Dox alone. Also, the ER(−)-
MDA-MB-231cells did not show any significant change in cell
binding of Dox and AE-Dox 15, and as expected, the presence
or absence of ES had no effect on cell binding of the drugs. The
results in Figure 7 illustrated a marked targeting effect imparted
by the presence of the steroidal anti-estrogen component in the
hybrid agent 15. These effects were consistent with the
interaction selective for the membrane ER.
Table 1. Inhibition Concentration, IC50 (nM), of Various
Dox Compounds
compound
Dox
MCF-7
MDA-MB-231
MCF-7 (+ES)
602 20
597 20
89
86
90
7
5
8
585 30
594 20
Dox-Linker 14
AE-Dox 15
11
6
589
3
Since FACS studies do not distinguish among membrane,
cytoplasmic, or nuclear localization of the fluorescent group, we
undertook cellular studies using fluorescence microscopy. The
results shown in Figure 8 clearly support enhanced AE-Dox 15
targeting to the ER-positive breast cancer cells. The cellular
localization of the fluorescence from AE-Dox 15 and Dox was
evaluated via ER(+)-MCF-7 cells in the presence (+) and
absence (−) of ES. The low level of fluorescence for cells
treated with Dox alone (panel B) is associated primarily with
the nucleus since the drug is known as DNA binding agent and
accumulates in nucleus.69,70 AE-Dox 15 (panel C) shows a
significantly enhanced uptake within the ER(+)-MCF-7 cells,
compared to Dox alone (panel B), suggesting that the AE
component of the conjugate has substantially facilitated uptake
by cells. Moreover, the fluorescence appeared to be associated
with nucleus, suggesting Dox should have been hydrolyzed
from the targeting group coupled with the translocation of free
Dox. The incubation of the cells with AE-Dox 15 and ES (panel
D) decreased the uptake of the AE-Dox (panel B) confirming
that the uptake is ER-mediated. This study supported the
observation for the initial cytotoxicity results in which AE-Dox
15 would generate a higher intracellular concentration of Dox
and therefore more rapid cell death.
in SI). Therefore, the introduction of the tetraethylene glycol
(TEG) linker onto the amino terminus of the steroidal anti-
estrogen and the TEG hydrazone linker onto the ketone of
doxorubicin had no observable effect on the cytotoxic
properties of the parent compounds. However, the AE-Dox
conjugate 15 showed a significant enhancement of cytotoxicity
(approximately 70-fold) in ER(+)-MCF-7 cells compared to
the other formulations (Figure 6A). The IC50 for the AE-Dox
conjugate 15 was 0.011 μM compared to 0.602 μM and 0.597
μM for the free Dox and the linker-Dox 14, respectively. In the
ER(−)-MDA-MB-231 cell line (Figure 6B), the presence of the
anti-estrogenic component had no significant effect. The
cytotoxicity curves for all three compounds in this cell line
were essentially superimposable, with IC50 values in a narrow
range (0.125−0.080 μM).
Then, we determined whether the effect of the anti-
estrogenic component in the hybrid AE-Dox 15 could be
reversed by the addition of estradiol (ES). Cytotoxicity of Dox
and the hybrid AE-Dox 15 were analyzed in ER(+)MCF-7 cells
in the presence or absence of ES. The results (Table 1) show
that the enhanced cytotoxicity of the AE-Dox hybrid was
completely abolished by the addition of ES, whereas ES has
virtually no effect on either Dox itself, or the linker-Dox 14.
Therefore, it appeared that the effect was ER-dependent and
not a nonspecific process.
CONCLUSION
■
In this study, we have demonstrated that we can prepare the
anti-estrogen−doxorubicin conjugate 15 efficiently and in high
yield using our modular assembly approach. Because the
The IC50 was estimated by using GraphPad 3 parameters
curve fitting for 24 h drug exposure data points. All values are
Figure 6. Cytotoxicity of compounds 11β-AE 9 (black), Dox-linker 14-red, Dox (orange), and AE-Dox 15 (purple), in (A) ER(+)MCF-7 and (B)
ER(−)MDA-MB-231 cell lines.
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Figure 7. FACS analysis of ER(+)MCF-7 and ER(−)MDA-MB-231 cells. (A) Histogram analysis of cells treated with 0.1 μM of Dox and AE-Dox
15 with and without ES; (purple) cells only, (green) Dox treated cells, and (red) AE-Dox treated cells. (B) The percentage of Dox-positive cells in
ER(+)MCF-7 and ER(−)MDA-MB-231cells. *P < 0.01 (Dox vs AE-DOX (-ES)); §P < 0.01 (AE-Dox (-ES) vs AE-Dox(+ES).
Figure 8. Fluorescence microscopy images ER(+)MCF-7 cells treated for 1 h with 0.1 μM of (B) Dox; (C) AE-Dox 15; (D) AE-Dox 15 after
pretreatment with 50 μM estradiol; and (A) untreated cell. Hoechst fluorescent (upper panel). Red fluorescence (lower panel).
components can be prepared independently and conjugated
using simple chemistry, potential modifications of the conjugate
properties are relatively easy. Initial cytotoxicity experiments
demonstrated that the AE-Dox conjugate 15 was 70-fold more
potent than Dox alone in ER(+)-MCF-7 cells, but equipotent
compared to Dox in ER(−)-MDA-MB-231 cells. The enhanced
cytotoxic effect in MCF-7 cells was reversed by preincubation
with ES, suggesting an ER-mediated process. Subsequent FACS
studies on both cell lines in the presence or absence of ES
supported this hypothesis. Additional studies using fluorescence
microscopy in MCF-7 cells suggested that the uptake
proceeded via a membrane ER-mediated effect leading to an
enhanced cellular accumulation of Dox. Within the cell, the pH-
sensitive hydrazone release mechanism incorporated into the
conjugate leads ultimately to the elevated levels of free Dox in
nucleus. Studies to further characterize the individual steps of
the process are in progress.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra for compounds 2−15 are provided.
Infrared spectra for 15, and cytotoxicity data for 9 and 14 are
provided. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: r.hanson@neu.edu.
Notes
The authors declare no competing financial interest.
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Article
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ACKNOWLEDGMENTS
■
We acknowledge the financial support from the Northeastern
University and the Department of Defense- Congressional
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[K.-L.D.], Department of Energy (DE-SC0001781)[RNH],
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