Bis-arylidene oxindoles as anti-breast-cancer agents acting via the estrogen receptor

Article abstract of DOI:10.1002/cmdc.201400003

We report a new family of bis-arylidene oxindole derivatives that show highly selective estrogen receptor (ER)-mediated anticancer activity at low-nanomolar concentrations in ER-positive (ER+) breast cancer cells. In terms of cell growth inhibition, IC₅₀ values for these compounds in ER+ breast cancer cells are two to three orders of magnitude lower than in ER-negative (ER-) breast cancer cells and non-cancer cells. In comparison with known bis-arylidene drugs, these compounds are at least three orders of magnitude more toxic than tamoxifen and 1.5-4-fold more toxic than 4-hydroxytamoxifen in ER+ MCF-7 cancer cells. These oxindoles inhibit ER transactivation, and their anticancer activities are inhibited in ER-depleted MCF-7 cells. Some of these nonsteroidal molecules also exhibit essential properties of selective ER down-regulation. From the development of two series of bis-arylidene oxindole-based compounds, we report a new series of anticancer agents for estrogen-responsive breast cancer. A trip to the ER: We developed oxindole derivatives that exhibit anticancer activity at low-nanomolar IC ₅₀ concentrations (30-70 nM) against ER-positive breast cancer cells. Results of siRNA studies reveal that the anticancer effects of these molecules are mediated by the estrogen receptor (ER). These compounds also inhibit ER-mediated transactivation and exhibit selective ER modulating and down-regulating properties.

Full text of DOI:10.1002/cmdc.201400003

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DOI: 10.1002/cmdc.201400003
Bis-arylidene Oxindoles as Anti-Breast-Cancer Agents
Acting via the Estrogen Receptor
Abhishek Pal,[c] Anirban Ganguly,[a] Avijit Ghosh,[b] Md Yousuf,[b] Bhowmira Rathore,[a]
Rajkumar Banerjee,*[a] and Susanta Adhikari*[b]
We report a new family of bis-arylidene oxindole derivatives
that show highly selective estrogen receptor (ER)-mediated an-
ticancer activity at low-nanomolar concentrations in ER-posi-
tive (ER+) breast cancer cells. In terms of cell growth inhibi-
tion, IC50 values for these compounds in ER+ breast cancer
cells are two to three orders of magnitude lower than in ER-
negative (ERÀ) breast cancer cells and non-cancer cells. In
comparison with known bis-arylidene drugs, these compounds
are at least three orders of magnitude more toxic than tamoxi-
fen and 1.5–4-fold more toxic than 4-hydroxytamoxifen in ER+
MCF-7 cancer cells. These oxindoles inhibit ER transactivation,
and their anticancer activities are inhibited in ER-depleted
MCF-7 cells. Some of these nonsteroidal molecules also exhibit
essential properties of selective ER down-regulation. From the
development of two series of bis-arylidene oxindole-based
compounds, we report a new series of anticancer agents for
estrogen-responsive breast cancer.
influencing the pathological processes of hormone-dependent
breast, endometrial, prostate, ovarian, and thyroid cancers.[7,8]
This makes the ER one of the principal targets for cancer thera-
py. We have shown that ES can be conveniently used as
a ligand for targeting bioactive cargoes to ER-positive (ER+)
breast cancer cells; if appropriately modified, ES can also be
used for the targeted delivery of kinase inhibitors for treating
breast cancer.[9] Estrogens, both natural and synthetic, have
various levels of agonistic or antagonistic character in different
target tissues.[10,11] For example, ES stimulates responses in the
uterus, breast, bone, and liver, whereas the nonsteroidal estro-
gen reloxifene blocks estrogen action in uterus and breast, but
has agonistic activity in bone and liver. There are several ER an-
tagonists, aromatase inhibitors, and selective ER modulators
(SERMs) that alone, or in combination, provide several clinical
options for breast cancer therapy with limited efficacy.[12–16]
Small polyphenolic molecules such as stilbenes, flavonoids,
pro-anthocyanidins, and their derivatives found throughout
the vegetable world, are the most acknowledged antioxidants
working against free radicals associated with diseases related
to certain cancers and cardiac, ocular, and degenerative prob-
lems.[17] They also act as protein function modulators. For ex-
ample, resveratrol is an endocrine modulator, whereas a struc-
turally similar stilbene, trans-diethylstilbesterol, acts as a power-
ful estrogen via ERa and ERb. Similarly, the synthetic diphenol-
ethylene, 1,1-bis(4’-hydroxyphenyl)-2-phenylbut-1-ene (1c),
shows estrogenic effects.[17] Accordingly, in our design, we
sought to modify the biological properties of organic polyphe-
nols, originally inefficacious at therapeutic levels, through co-
valent addition of an oxindole group to the organic skeleton.
In this work we show that by grafting the oxindole moiety to
organic diphenol, the estrogenic properties can be profoundly
modified, in fact, reversed. Oxindole was chosen as the key
molecular fragment in the present design because it is a privi-
leged heterocyclic motif that is known to possess a wide range
of biological activities and medicinal applications.[18,19] Earlier
we reported a new synthesis of enantiopure 3,3-disubstituted
oxindoles directed toward the total synthesis of the complex
antitumor pyrrolidinoindoline alkaloid, laptosin D, by starting
from isatin.[20] In our quest to develop more potent anticancer
molecules with potential anti-estrogenic properties, we incor-
porated an oxindole unit into the bisphenolic or bis-arylidene
moieties. These have the base skeleton of the active metabo-
lite, 4-hydroxytamoxifen (1b), bearing the crucial hydroxy and
dimethylamino groups, which are known to maintain excellent
ER recognition (Scheme 1). We also intended for the designed
Estrogen and its receptor are classically involved in a majority
of cancers of gynecological origin.[1] The estrogen receptor
(ER), a nuclear hormone receptor, is expressed in estrogen-re-
sponsive organs such as ovary, uterus, and mammary glands.
Estrogen and estrogen-bound ER possess both genomic and
non-genomic functions.[2–6] Because the functional expression
of the ER is confined to the initial stages of neoplastic transfor-
mation, the design of compounds that interfere with ER func-
tion is expected to be an effective strategy in preventing these
types of cancer.
17b-Estradiol (ES), the chief endogenous steroid hormone
ligand of the ER, primarily regulates a broad range of physio-
logical processes such as growth, differentiation, and physiolo-
gy of reproductive processes. Estrogen-bound ER classically
acts as a transcription factor, regulating cell proliferation and
[a] A. Ganguly,+ B. Rathore, Dr. R. Banerjee
Biomaterials Group, Division of Lipid Science & Technology
CSIR-Indian Institute of Chemical Technology, Hyderabad 500607 (India)
E-mail: rkbanerjee@yahoo.com
banerjee@iict.res.in
[b] A. Ghosh, M. Yousuf, Dr. S. Adhikari
Department of Chemistry, University of Calcutta
92, A.P.C. Road, Kolkata 700 009 (India)
E-mail: adhikarisusanta@yahoo.com
[c] A. Pal+
Department of Organic Chemistry, IACS, Jadavpur, Kolkata 700032 (India)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201400003.
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The synthetic procedure is il-
lustrated in Scheme 1B. It ap-
peared that a McMurry coupling
reaction[25] would be most effec-
tive for preparing the desired
alkene derivatives. Compounds 2
and 2a were obtained as a mix-
ture of Z and E isomers via
a
cross-coupling reaction be-
tween isatin (4) and 4,4’-dihy-
droxybenzophenone derivatives
6 and 6a, respectively. Com-
pounds 6 and 6a were obtained
by mono-alkylation of 4,4’-dihy-
droxybenzophenone (5) with re-
spective N,N-dimethylaminoalkyl
chlorides in the presence of
ethanolic sodium ethoxide. Com-
pound 2 and 2a were present as
respective mixtures of Z and E
isomers, and could not be sepa-
rated by flash column chroma-
tography or fractional crystalliza-
tion. This can be explained from
the consequence of the interac-
tion between the central double
bond and the hydroxy group of
the phenol, leading to intercon-
version between Z and E iso-
mers, as observed in other in-
stances in the stilbene series.[23a]
Similarly, compounds 3 and 3a
were also obtained as Z/E iso-
Scheme 1. Synthesis of bis-arylidineoxindoles. Reagents and conditions: a) Zn, TiCl4, THF, reflux, 3 h; b) 1. NaH, THF,
RT, 1 h, 2. PMB-Br, DMF, RT, 5 h, 78%.
compounds to have higher selectivity for cancer cells with
minimal toxicological liability.
meric mixtures from the subsequent coupling between 7 and
6/6a.
Initially, we undertook the synthesis of 2 and 2a with the re-
spective basic N,N-dimethylaminoalkoxy side chain in tamoxi-
fen-based molecules (1a,b), as it is readily available. It is well
documented that increasing the number of methylene units in
the alkylamino side chain of various nonsteroidal anti-estro-
gens such as tamoxifen,[21] nafoxidine, idoxifene,[22] and organo-
metallic hormones[23] allows maintenance of antagonistic po-
tency and antiproliferative effects toward breast-cancer cell
lines. To verify the significance of the oxindole nucleus in de-
termining antagonistic potency with increased cytotoxic effects
in MCF-7 breast cancer cells, we also synthesized N-4-methoxy-
benzylated analogues 3 and 3a by incorporation of hydropho-
bic substituents such as 4-methoxybenzyl at the oxindole ni-
trogen atom, based on computational modeling and published
precedent.[24] While assuming a possible binding of ligand to
ER, we preliminarily studied the interaction by molecular mod-
eling. We found that among the various N-alkylating groups
on the oxindole nitrogen, “4-methoxybenzyl group” with an
acceptor atom “O” has an additional stabilizing electrostatic in-
teraction with the neighboring Ile424 residue of human ERa
(data not shown).
The compounds were first evaluated for their cytotoxic ef-
fects in ER+ MCF-7 breast cancer cells. The molecules with
promising cytotoxicity were further tested against ER-negative
(ERÀ) MDA-MB-231 cells and non-cancer cells, CHO and
HEK293 (Table 1). Compounds 2/2a and 3/3a showed anti-
proliferative effects against ER+ breast cancer cells only. Com-
pounds 2/2a and 3 were 1.5–4-fold more potent than the
most active tamoxifen metabolite 4-hydroxytamoxifen (1b)
and 1000-fold more potent than tamoxifen (1a), whereas 3a
exhibited activity similar to that of 1b in MCF-7 cells. The intro-
duction of an oxindole group to the diphenol moiety classical-
ly associated with tamoxifen, with or without para-methoxy-
benzyl (PMB) protection, did not change its selective toxicity
against ER+ cancer cells. This ER selectivity was also unaffect-
ed by the change in side chain carbon number, which differed
from tamoxifen derivatives. It appears that the oxindole
moiety imparted a strong influence on ERa recognition. Inci-
dentally, compound 3a, with PMB protection and a two-
carbon side chain, showed a 2–4-fold lower anticancer effect
than 2, 2a, and 3 in ER+ breast cancer cells. It seems that the
ER-targeting ability is slightly more tolerated and stabilized in
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compounds 2/2a and 3/3a, as described above. Notably, IC50
values for 13b,c are 4–5-fold greater than those for 2 and 3 in
ERÀ MDA-MB-231 cells. This indicates that as far as selectivity
toward ER-associated breast cancer cells is concerned, com-
pounds 13b,c are possibly even more potent than 2/2a and
3/3a.
Table 1. Growth inhibition elicited by test compounds against various
cell lines.
IC50 [mm]
Compound
MCF-7[a]
>35
MDA-MB-231[b]
CHO[a] HEK293[a]
1a
1b
ICI182780
2
2a
3
3a
8
ND
>35
>35
ND
ND
>35
ND
0.096Æ0.004
0.023Æ0.003
0.032Æ0.004
0.037Æ0.004
0.033Æ0.006
0.112Æ0.006
0.602Æ0.033
>35
The most potent molecules, 2, 3, 13b, and 13c, and one of
the potent parent oxindoles, 8, were further tested to deter-
mine if their anticancer effects could be antagonized by the ER
endogenous ligand, 17b-estradiol (ES). This was done to cor-
roborate the ER-specificity of these molecules. Figure 1 shows
that the anticancer effect of all the compounds tested against
MCF-7 were significantly inhibited (p<0.001) if cells were pre-
treated and co-treated with 10 mm ES. The results indicate that
ES, the most potent natural ER ligand possessing the highest
ER binding affinity, could restrict and antagonize the anticancer
effect of oxindoles. This could be due to possible restriction of
oxindole binding to the ES binding site in the ER. These data
were corroborated independently when we observed similar
inhibition of cytotoxicity with MCF-7 cells pre-treated with the
ER antagonist/anti-estrogen ICI182780 (fulvestrant) for 4 h fol-
lowed by prolonged treatment with our new oxindoles (Sup-
porting Information figure S1). This ER antagonist also has
binding affinity for the ES binding site of the ER, and hence in-
dicates further for a possible ER-binding capacity of these mol-
ecules. However, it is clear that whether or not these oxindoles
ND
5.764Æ0.005
>35
>35
>35
>35
>35
ND
>35
>35
>35
>35
>35
ND
>35
5.896Æ0.005
>35
>35
8a
8b
8c
9
13
13a
13b
13c
>35
ND
>35
>35
>35
>35
ND
ND
ND
ND
ND
ND
ND
>35
ND
ND
ND
ND
ND
ND
>35
>35
0.672Æ0.005
ND
0.069Æ0.021
0.055Æ0.031
26.324Æ0.005
20.251Æ0.005
>35
>35
[a] Values are the meanÆSD for n=3 replicates performed in triplicate
on different days. [b] Values are the meanÆSD for n=2 replicates per-
formed in duplicate on different days.
PMB-protected, three-carbon side chain oxindoles than in two-
carbon side chain oxindoles.
Encouraged by the above result, we proceeded to examine
the biological efficacy of diphenols (without an alkyl-
amino-terminated side chain) as shown in Scheme 2.
The desired molecule 8 was obtained in 56% yield
(Scheme 2A) by an analogous McMurry coupling of
isatin (4) and 4,4’-dihydroxybenzophenone (5). We
also prepared the 5-halo derivatives (F, Cl, I), as pre-
cursors are commercially available. The PMB-protect-
ed iodoisatin derivative 10 was subjected to McMurry
coupling to yield 11, which in turn provided access
to
a number of C5-substituted aryl derivatives
through Suzuki–Miyaura coupling using the appropri-
ate commercially available boronic acids (Scheme 2B)
under microwave irradiation.[26]
The cytotoxicities of these new compounds 8, 8a–
c, 9, 13, and 13a–c were again tested. Moreover,
they were also tested against MDA-MB-231 (ERÀ)
and non-cancer cell lines, CHO and HEK293. The se-
lective anticancer effects against ER+ breast cancer
cells exhibited by these compounds are clearly evi-
dent in Table 1. The parent molecule 8, in compari-
son with its halogenated analogues (8a–c) showed
the highest anticancer effect against MCF-7 cells. Al-
though the PMB-protected derivative 9 did not ex-
hibit anticancer activity, compounds 13a–c, which
are all PMB protected, exhibited significant and selec-
tive anticancer effects against MCF-7 cells. However,
among these PMB-protected molecules, the C5-sub-
stituted para-methoxyphenyl analogue 13 surprising-
ly showed no activity. That the PMB group per se is
not detrimental to biological activity was independ-
Scheme 2. Synthesis of bis-phenol analogues. Reagents and conditions: a) Zn, TiCl4, THF,
reflux, 3 h; b) 1. NaH, THF, RT, 1 h, 2. PMB-Br, DMF, RT, 5 h, 72%; c) K2CO3, Pd(PPh3)4, micro-
ently proven by similar, low-level IC50 values between wave, 1208C, 30 min.
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Figure 1. ER-mediated anticancer activity of oxindoles: Viability of MCF-7
cells treated with indicated test compounds (0.1 mm) in the presence (&)
and absence (&) of the endogenous ER ligand, 17b-estradiol (*p<0.001).
have ER binding affinity, the anticancer activities of these com-
pounds are clearly mediated by the ER.
Figure 2. A) Trans-repression of ERE-promoted protein expression by oxin-
doles: Relative light units (RLU) of luciferase expression per unit of b-galac-
tosidase (b-gal) expression obtained from MCF-7 cell lysates, preconditioned
in either hormone-free (i.e., charcoal stripped, CS) serum or normal serum,
then pre-transfected with ERE–luciferase and b-gal plasmids and respectively
kept untreated (UT) or treated with 17b-estradiol (ES) and indicated test
compounds (*p<0.001). B) RT-PCR analyses of the BRCA-1 gene: The ERE-
regulated BRCA-1 gene in MCF-7 cells was analyzed following treatment
with the indicated compounds (UT: untreated control; 18S: internal control).
The ratios of increased or decreased BRCA-1 expression levels with respect
to 18S are indicated below.
Toward elucidating any potential anti-estrogenic effects that
may be linked to the anticancer effects of these oxindoles,
MCF-7 cells were treated with the potent compounds 2, 3, 8,
and 13b,c. Two more compounds were incorporated into this
study: compound 1a is a well-known ER antagonist that binds
to the ER and inhibits any further estrogen-responsive gene
transactivation; 1b acts as an anti-estrogen and binds to the
ER, repressing estrogen-responsive genes. Following the bind-
ing of an estrogenic or anti-estrogenic compound, the ER di-
merizes and acts on the estrogen responsive element (ERE)
promoter region in chromosomal DNA to regulate the tran-
scription and expression of various genes (ERE genes).[8,10] To
quantitatively assess the effect of lead oxindoles on the expres-
sion of ERE-promoted genes, we pre-transfected MCF-7 cells
with an ERE–luciferase plasmid and a CMV–b-gal plasmid. It is
known that only the estrogenic/anti-estrogenic molecules in-
fluence the expression of the ERE-promoted reporter luciferase
gene, whereas b-gal expression should remain independent of
any such treatment. Following plasmid transfection, treatment
with test compounds was performed. Figure 2A shows that
under normal serum conditions, in which all the endogenous
hormones in cell culture serum are present, the known antago-
nist 1a, anti-estrogen 1b, and test compounds 2, 3, 8, and
13c clearly down-regulated luciferase expression under ERE
promoter control. Notably, under hormone-free conditions
(charcoal-stripped serum), ES indeed induced the up-regulation
of luciferase expression with respect to untreated control (UT),
thereby indicating that the luciferase expression in these ERE–
luciferase transfected cells was truly estrogen sensitive. Gener-
ally, upon binding with a pro-estrogenic molecule such as nat-
ural estrogen ES, the ER favorably up-regulates expression of
the pro-proliferative, ERE-promoted genes. On the other hand,
anti-estrogens, following ER binding, tend to down-regulate
ERE genes, whereas ER antagonists, upon ER binding, oppose
any further up-regulation of these genes. Our data show that
the tested oxindoles behave more like anti-estrogens, the ERE-
trans-repression activities of which remain undeterred even in
the presence of natural hormonal conditions.
The repression of ERE gene translation was not an isolated
effect. We observed similar effects on ERE gene transcription.
For these, we chose to monitor the gene transcription (mRNA
expression) levels of two ERE-promoted genes, BRCA-1 and Bcl-
2, by RT-PCR techniques in the presence of these compounds.
The 18S gene was selected as a homing control gene whose
mRNA expression should remain undeterred by any treatment.
MCF-7 cells were treated with key molecules 2, 8, and 13c,
which showed ER-mediated anticancer activity in MCF-7 cells.
Other control treatment groups such as pro-estrogenic ES and
anti-estrogenic 1b were also kept along with untreated control
(UT). Following treatment, cells were isolated for their respec-
tive RNAs, which were converted into cDNA by reverse tran-
scriptase. This was followed by PCR using individual primers
for BRCA-1 and 18S for their respective amplification. The re-
spective DNAs were separated by gel electrophoresis for deter-
mining their respective levels of expression (Figure 2B and
Supporting Information figure S2). The trend in the mRNA ex-
pression of these ERE genes corroborated this luciferase pro-
tein expression pattern, indicating that the compounds do
indeed exhibit a down-regulating effect on ERE-promoted
genes at both transcriptional and translational levels, as partic-
ularly exhibited by known anti-estrogens.
To demonstrate that the induced cytotoxicity of these newly
designed lead molecules are indeed mediated by the ER, we
performed a silencing RNA (siRNA)-based ER knock-down
study. The hypothesis is that if compound-induced cytotoxicity
in MCF-7 (ER+) cells is significantly inhibited following ER de-
pletion, then the given compound induces ER-mediated cyto-
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We also performed molecular docking analyses with the
complex between human the ERa ligand binding site and 1b
as the template. Preliminarily, the analysis showed that all the
synthesized molecules are not only potent in binding to the
ER ligand binding site, but also maintain a trend in the docking
score among the respective series, such as 2/3 or 8 or 13 oxin-
dole groups, roughly equivalent to their trend in cellular toxici-
ty (data not shown). Detailed modeling studies may further
help elucidate the tentative ER binding capability, if any, of
these oxindoles.
Figure 3. Reversal of cytotoxicity in ERa-depleted MCF-7 cells: Percent viabil-
ity of either siRNA-untreated (UT, &), control siRNA (&), or ERa siRNA-pre-
treated (&) MCF-7 cells under 36 h continuous treatment with lead com-
pounds 2, 2a, 3, 3a, 8, 13b, and 13c, as well as known anti-estrogen 1b
(*p<0.001).
In summary, we report herein the development of highly
potent, ER-selective, anti-breast-cancer oxindole-conjugated bi-
sphenols. Our data show that the ER-targeted anticancer activi-
ties of arylidenes are not compromised by changing or replac-
ing basic alkylaminoethoxy side chains present in highly anti-
estrogenic 4-hydroxytamoxifen (1b). The uncompromised ER-
targeting ability and anti-estrogenic or reverse agonistic prop-
erties are equivocally strengthened by the introduction of ox-
indole moieties in arylidenes. Serendipitously, N-4-methoxy-
benzyl protection of oxindoles rather reinforced ER-mediated
anti-breast-cancer activity. Some of these compounds down-
regulate the ER and hence behave as SERDs. The in vivo evalu-
ation in breast cancer models for the most active compound is
currently in progress. Toward further structure–activity relation-
ship studies, the syntheses of additional oxindole derivatives
are underway to expand our knowledge in optimizing the
pharmacophore with improved anti-breast-cancer potential.
toxicity in ER+ cells. Toward understanding this, MCF-7 cells
were pre-treated with either ERa siRNA or with control (non-
targeted) siRNA. Cells were then treated with lead oxindoles.
Figure 3 shows that the most potent oxindoles 2, 2a, 3, 3a, 8,
and 13b,c exhibited maximum toxicity (i.e., minimum viability)
in siRNA-untreated or control-siRNA-treated MCF-7 cells. How-
ever, there were significant, if not complete reversal of cytotox-
icities (or in other words, exhibition of 100% viability) mediat-
ed by these molecules in ERa-depleted MCF-7 cells. ERa deple-
tion was evident from its protein level following ERa-targeted
siRNA treatment (Supporting Information figure S3). The 100%
reversal of toxicity in ERa-depleted cells indicates the possibili-
ty that the cellular toxicities of these oxindoles are predomi-
nantly mediated via ERa and not via ERb.
Experimental Section
Further to this, we sought to find the ERa expression status
in MCF-7 cells treated with lead molecules 2, 2a, 3, 13b, and
13c compared with levels obtained by treating cells with
known selective ER down-regulators (SERDs), 1b and
ICI182780. SERDs are functionally different from selective ER
modulators (SERMs). Unlike SERMs, SERDs degrade ER levels,
while exhibiting anti-estrogenic properties, regulation of ERE
genes, etc.[27] Figure 4 indicates that under the same subclass,
compounds 2, 2a, and 3 behave as SERDs in MCF-7 cells, in
which ERa levels are clearly degraded relative to those in un-
treated cells. Interestingly, among the other subclass, 13c de-
grades ERa, but 13b maintains ERa levels. So, despite exhibit-
ing similar cytotoxicities, these molecules under the same
broad class, individually exhibit either SERM- or SERD-type
properties. Further experiments are certainly warranted to as-
certain the specific and important roles of these oxindoles.
General
Chemicals and solvents were purchased from commercial suppliers
and used as received. 1H and 13C NMR spectra were recorded on
Bruker Avance III HD (300 MHz) or Avance III 500 (500 MHz) spec-
trometers. Chemical shifts (d) are reported in parts per million
(ppm), and the residual solvent peak was used as an internal refer-
1
ence: H (CHCl3 d=7.26 ppm), 13C (CHCl3 d=77.16 ppm), or tetra-
methylsilane (TMS d=0.00 ppm) was used as a reference. Multiplic-
ity is indicated as follows: s (singlet), d (doublet), t (triplet), q (quar-
tet), m (multiplet), dd (doublet of doublets), brs (broad singlet),
appt (apparent triplet). Coupling constants are reported in Hertz
(Hz). High-resolution MS data were obtained on a Micromass/Q-ToF
microspectrometer. Melting points were determined on a Thomas
Hoover capillary melting point apparatus. IR spectra were mea-
sured on Thermo Scientific Nicolet 380 instrument. A CEM Discover
(MN-542470) microwave was used for Suzuki–Miyaura cross-cou-
pling reactions. Merck pre-coated TLC plates (Merck 60 F254) were
used for thin-layer chromatography, and compounds were visual-
ized with UV light at l 254 nm. Further visualization was achieved
by staining with iodine. Flash chromatography separations were
performed on SRL 230–400 mesh silica gel.
Starting materials
Compounds 4, 4a, 4b, and 4c were purchased from Avra Synthe-
sis Pvt. Ltd. (India), compound 5 was brought from Sigma (India),
and compounds 12, 12a, 12b, and 12c were purchased from
Frontier Scientific (India). Compounds 6 and 6a were prepared ac-
cording to published methods.[23a]
Figure 4. Western blot analysis of cell lysates for ERa content: Differential
expression of ERa along with the housekeeping control gene b-actin in cell
lysates of MCF-7 kept untreated (UT) or treated with various test compounds
as indicated (ICI=ICI182780).
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Representative procedure for McMurry coupling
Keywords: anticancer agents · estrogen response element ·
McMurry coupling · oxindoles · receptors · transactivation
To a suspension of Zn0 dust (314 mg, 4.8 mmol) in THF (10 mL),
TiCl4 (0.26 mL, 2.4 mmol) was added at À108C. The cooling bath
was removed, and the mixture was held at reflux for 1 h. After
cooling to room temperature, anhydrous pyridine (0.38 mL,
4.8 mmol) was added, and the mixture was stirred for 5 min. A so-
lution of the appropriate oxindole (4/4a/4b/4c/7; 0.8 mmol) and
4,4’-dihydroxybenzophenone (5) (171 mg, 0.8 mmol) in THF (4 mL)
was added, and the mixture was held at reflux for 2 h (monitored
by TLC). After cooling to room temperature, the mixture was hy-
drolyzed with 10 mL of 8% aqueous K2CO3. The reaction mixture
was extracted with several 10-mL portions of EtOAc. The organic
phase was washed with H2O (2ꢁ20 mL) and brine (20 mL), dried
over Na2SO4, and evaporated to dryness. The crude product was
purified by flash chromatography on silica gel (230–400 mesh)
with petroleum ether/EtOAc (3:2) as eluent to obtain the first un-
reacted oxindole moiety [Rf ꢀ0.7; ~10–15%] followed by the prod-
uct (8/8a/8b/8c/9) [Rf ꢀ0.4; ~32–63%] and finally the homo-cou-
pled product derived from the corresponding benzophenone
moiety [Rf ꢀ0.35; ~15–20%]. It was observed that going from 8 to
8c, the product yield decreases due to additional formation of
homo-coupled product from the oxindole motif.
[1] C. K. Osborne, Breast Cancer Res. Treat. 1998, 51, 227–238.
[2] P. Thomas, Y. Pang, E. J. Filardo, J. Dong, Endocrinology 2005, 146, 624–
632.
[3] M. Razandi, A. Pedram, G. L. Greene, E. R. Levin, Mol. Endocrinol. 1999,
13, 307–319.
[4] Z. Chen, I. S. Yuhanna, Z. Galcheva-Gargova, R. H. Karas, M. E. Mendel-
sohn, P. W. Shaul, J. Clin. Invest. 1999, 103, 401–406.
[5] R. X. D. Song, R. A. McPherson, L. Adam, Y. Bao, M. Shupnik, R. Kumar,
R. J. Santen, Mol. Endocrinol. 2002, 16, 116–127.
[6] P. Monje, R. Boland, Mol. Cell. Endocrinol. 1999, 147, 75–84.
[7] P. Ascenzi, A. Bocedi, M. Marino, Mol. Aspects Med. 2006, 27, 299–402.
[8] S. T. Pearce, V. C. Jordan, Crit. Rev. Oncol. Hematol. 2004, 50, 3–22.
[9] a) B. S. Reddy, R. Banerjee, Angew. Chem. Int. Ed. 2005, 44, 6723–6727;
Angew. Chem. 2005, 117, 6881–6885; b) S. Sinha, S. Roy, B. S. Reddy, K.
Pal, G. Sudhakar, S. Iyer, S. Dutta, E. Wang, P. K. Vohra, K. R. Roy, P. Red-
danna, D. Mukhopadhyay, R. Banerjee, Mol. Cancer Res. 2011, 9, 364–
374.
[10] D. P. McDonnell, Trends Endocrinol. Metab. 1999, 10, 301–311.
[11] D. P. McDonnell, J. Soc. Gynecol. Invest. 2000, 7, S10–S15.
[12] L. W. Wattenberg, Cancer Res. 1985, 45, 1–8.
[13] A. Fugh-Berman, S. Epstein, Lancet 1992, 340, 1143–1145.
[14] J. F. Robertson, Cancer Treat. Rev. 2004, 30, 695–706.
[15] K. L. Jones, A. U. Buzdar, Endocr.-Relat. Cancer 2004, 11, 391–406.
[16] I. C. Henderson, Semin. Oncol. 2004, 31, 31–34.
Representative procedure for Suzuki–Miyaura coupling
[17] A. Vessiꢂres, S. Top, P. Pigeon, E. Hillard, L. Boubeker, D. Spera, G.
Jaouen, J. Med. Chem. 2005, 48, 3937–3940, and references cited there-
in.
[18] N. V. Hanhan, Y. C. Tang, N. T. Tran, A. K. Franz, Org. Lett. 2012, 14, 2218–
2221, and references cited therein.
[19] C. R. Reddy, E. Jithender, G. Krishna, G. V. Reddy, B. Jagadeesh, Org.
Biomol. Chem. 2011, 9, 3940–3947, and references cited therein.
[20] S. Adhikari, S. Caille, M. Hanbauer, V. X. Ngo, L. E. Overman, Org. Lett.
2005, 7, 2795–2797.
[21] D. W. Robertson, J. A. Katzenellenbogen, J. R. Hayes, B. S. Katzenellenbo-
gen, J. Med. Chem. 1982, 25, 167–171.
[22] L. Jin, N. Legros, G. Leclercq, I. R. Hardcastle, M. Jarman, Cancer Chemo-
ther. Pharmacol. 1998, 41, 339–342.
[23] a) S. Top, A. Vessiꢂres, G. Leclercq, J. Quivy, J. Tang, J. Vaissermann, M.
Huchꢃ, G. Jaouꢃn, Chem. Eur. J. 2003, 9, 5223–5236; b) E. Hillard, A. Ves-
sieres, L. Thouin, G. Jaouen, C. Amatore, Angew. Chem. Int. Ed. 2006, 45,
285–290; Angew. Chem. 2006, 118, 291–296.
A septum-sealed microwave tube charged with 11 (100.0 mg,
0.174 mmol), appropriate arylboronic acid (12/12a/12b/12c;
0.261 mmol), Pd(PPh3)4 (4.0 mg, 0.0035 mmol), and K2CO3 (72.0 mg,
0.52 mmol) in dioxane (1.0 mL) was irradiated in a microwave
cavity (160 W, 1208C, 30 min). The reaction mixture was quenched
with dilute AcOH and extracted with EtOAc. The combined organic
extracts were dried over anhydrous Na2SO4, then filtered off, and
the solvent was evaporated under reduced pressure. The crude
product was purified by flash chromatography on silica gel (230–
400 mesh) with petroleum ether/EtOAc (3:2) as eluent to give first
unreacted starting material [Rf ꢀ0.45; ~20–25%] followed by the
cross-coupled products (13/13a/13b/13c) [Rf ꢀ0.4; ~50–59%].
Acknowledgements
[24] K. L. Vine, J. M. Locke, M. Ranson, S. G. Pyne, J. B. Bremner, J. Med.
Chem. 2007, 50, 5109–5117.
We acknowledge Prof. Amitabha Sarkar (IACS, Kolkata) for his
generous suggestions and support. We also thank Priyanka
Sharma (IICT, Hyderabad) for technical assistance in cell culture
experiments. A.P. and M.Y. acknowledge fellowship support from
CSIR, New Delhi. A. Ghosh is thankful to UGC for a fellowship.
R.B. acknowledges the DST (project no. SR/S1/OC-64/2008) and
IICT (in-house project OLP0003). S.A. acknowledges the DST (proj-
ect no. SR/S1/OC-101/2012).
[25] J. E. McMurry, Acc. Chem. Res. 1983, 16, 405–411.
[26] A. K. Sharma, K. Gowdahalli, J. Krzeminski, S. Amin, J. Org. Chem. 2007,
72, 8987–8989.
[27] a) J. Hoffmann, R. Bohlmann, N. Heinrich, H. Hofmeister, J. Kroll, H.
Kꢄnzer, R. B. Lichtner, Y. Nishino, K. Parczyk, G. Sauer, H. Gieschen, H.-F.
Ubrich, M. R. Schneider, J. Natl. Cancer Inst. 2004, 96, 210–218; b) B. M.
Wittmann, A. Sherk, D. P. McDonnell, Cancer Res. 2007, 67, 9549–9560.
Received: January 1, 2014
Published online on January 31, 2014
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 727 – 732 732