Bismuth nitrate-induced novel nitration of estradiol: An entry to new anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2014.06.010

Direct nitration of estradiol was carried out using metal nitrates on solid surfaces under mild condition, and a combination of bismuth nitrate pentahydrate impregnated KSF clay was found to be the best reagent to synthesize 2- and 4-nitroestradiol effectively. Furthermore, various basic side chains were introduced, through O-linker at C-3, to these nitroestradiols. The ability of these derivatives to cause cytotoxicity in Estrogen Receptor (ER)-positive and ER-negative breast cancer cell lines, as well as cancer cell lines of other origins, was examined. Qualitative structure activity relationship (SAR) has also been studied. We found that a basic side chain containing either a piperidine or morpholine ring, when conjugated to 2-nitroestradiol, was particularly effective at causing cytotoxicity in each of the cancer cell lines examined. Surprisingly, this effective cytotoxicity was even seen in ER-negative breast cancer cells.

Full text of DOI:10.1016/j.ejmech.2014.06.010

Accepted Manuscript
Bismuth nitrate-induced novel nitration of estradiol: An entry to new anticancer agents
Debasish Bandyopadhyay , Gildardo Rivera , Jorge L. Sanchez , Jesse Rivera , Jose
C. Granados , Adrian M. Guerrero , Fang-Mei Chang , Robert K. Dearth , John D.
Short , Bimal K. Banik
PII:
S0223-5234(14)00529-7
10.1016/j.ejmech.2014.06.010
EJMECH 7053
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 April 2014
Revised Date: 1 June 2014
Accepted Date: 7 June 2014
Please cite this article as: D. Bandyopadhyay, G. Rivera, J.L. Sanchez, J. Rivera, J.C. Granados, A.M.
Guerrero, F.-M. Chang, R.K. Dearth , J.D. Short, B.K. Banik, Bismuth nitrate-induced novel nitration
of estradiol: An entry to new anticancer agents, European Journal of Medicinal Chemistry (2014), doi:
10.1016/j.ejmech.2014.06.010.
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ACCEPTED MANUSCRIPT
Highlights
Bismuth nitrate-induced novel nitration of estradiol: An entry to new anticancer agents
Debasish Bandyopadhyay, Gildardo Rivera, Jorge L. Sanchez, Jesse Rivera, Jose C. Granados, Adrian M. Guerrero,
Fang-Mei Chang, Robert K. Dearth, John D. Short, and Bimal K. Banik
ꢀ
ꢀ
ꢀ
An ecofriendly direct procedure for the nitration of estradiol is developed.
Carbon linkers on the oxygen at C-3 with basic terminal moieties are introduced.
Compounds 3 and 4 are cytotoxic for HepG2, Hepa1-6, Hep3B, HeLa and HT-29 cells.
ꢀ
ꢀ
Compound 3 is more potent than 4-HT for both ER-positive and ER-negative cells.
Compound 3 circumvented P-gp mediated drug resistance in ovarian cancer cells.

ACCEPTED MANUSCRIPT
Bismuth nitrate-induced novel nitration of estradiol: An entry to
new anticancer agents
1
2†
1,4†
3
Debasish Bandyopadhyay , Gildardo Rivera , Jorge L. Sanchez , Jesse Rivera , Jose C.
4
1,4
4
3
Granados , Adrian M. Guerrero , Fang-Mei Chang , Robert K. Dearth , John D.
4
,5
1
Short *, and Bimal K. Banik *
1
Department of Chemistry, The University of Texas-Pan American, 1201 West University Drive,
Edinburg, Texas 78539, USA
2
Centro de Biotecnologia Genomica, Instituto Politecnico Nacional, Blvd. del Maestro, s/n, esq.
Elias Piña, Col.Narciso Mendoza, 88710, Reynosa, Mexico
3
Department of Biology, The University of Texas-Pan American, 1201 West University Drive,
Edinburg, Texas 78539, USA
4
University of Texas Health Science Center at San Antonio-Regional Academic Health Center,
Medical Research Division, 1214 West Schunior Street, Edinburg, TX 78541, USA
5
Department of Pharmacology, University of Texas Health Science Center at San Antonio,
7
703 Floyd Curl Drive, San Antonio, TX 78229, USA
†
Contributed equally
*
Corresponding authors: Ph: +1(956)6658741, Fax: +1(956)3845006, E-mail:
banyopad@utpa.edu or shortj@uthscsa.edu

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Abstract
Direct nitration of estradiol was carried out using metal nitrates on solid surfaces under mild
condition, and a combination of bismuth nitrate pentahydrate impregnated KSF clay was found
to be the best reagent to synthesize 2- and 4-nitroestradiol effectively. Furthermore, various basic
side chains were introduced, through O-linker at C-3, to these nitroestradiols. The ability of
these derivatives to cause cytotoxicity in Estrogen Receptor (ER)-positive and ER-negative
breast cancer cell lines, as well as cancer cell lines of other origins, was examined. Qualitative
structure activity relationship (SAR) has also been studied. We found that a basic side chain
containing either a piperidine or morpholine ring, when conjugated to 2-nitroestradiol, was
particularly effective at causing cytotoxicity in each of the cancer cell lines examined.
Surprisingly, this effective cytotoxicity was even seen in ER-negative breast cancer cells.
Keywords
Nitration, Bismuth nitrate, Estradiol, Anticancer, Apoptosis, Solid-support, Estrogen receptor

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Article Outline
1
2
. Introduction
. Results
2
2
.1 Synthesis of novel nitroestradiol compounds
.2 Selected nitroestradiol compounds decrease viability & block estradiol effects in breast cancer
cell lines
2
2
2
.3 Compound 3 circumvents p-glycoprotein-mediated drug resistance
.4 Selected nitroestradiol compounds decrease viability of multiple mammalian cell lines
.5 Estradiol compounds potentially cause cytotoxicity via induction of apoptosis
3
4
. Discussion & Conclusions
. Experimental Section
4
4
4
4
4
4
4
.1 Materials
.2 Synthesis of the compounds 1-6
.3 Spectroscopic analyses of compounds (1-6)
.4 Mammalian cell culture
.5 Mammalian cell viability assays
.6 Estradiol-induced cell proliferation
.7 Membrane blebbing
Acknowledgements
References & Notes

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1
. Introduction
According to World Health Organization, cancer was the leading cause of death worldwide in
2
008, accounting for 7.6 million deaths (around 13% of all deaths) [1]. Furthermore, cancer-
1
related mortalities are projected to continue to rise to over 13.1 million by 2030. Therefore,
there is a continuous need to develop new anticancer agents to reduce both cancer incidence and
cancer-related mortalities.
Breast cancer is the most commonly occurring type of cancer globally [2], and estrogens and
Estrogen Receptors (ERs), including ERα and ERβ, play an important role in numerous cancers,
including breast, ovarian, uterine, prostate, and colon cancers [3,4]. In fact, ERs are key targets
for the treatment of breast cancer [5], and the selective estrogen receptor modulator (SERM),
Tamoxifen (Tam), is an ERα antagonist that has been used to treat breast cancer since the 1970s
[6]. However, many breast cancers become resistant to Tam therapy, and Tam therapy leads to
an increased risk of other tumor types; therefore, recent studies have focused on identifying
novel SERMs that are tissue selective, on targeting other mechanisms of ER function, and on
identifying novel compounds that target ERβ instead of ERα [5].
In addition to modulating SERMs, medicinal chemistry has focused on inhibiting the
synthesis of estrogens by inhibiting the activities of Aromatase or 17-β-hydroxysteroid
dehydrogenase or by modulating sulfotransferase enzymes (e.g., steroid sulfotransferase or
steroid sulfatase) that affect the sulfation of estrone and/or estradiol [7]. In this respect,
nitroestrogens have potential application in cancer chemotherapy. For example, 2-nitroestradiol
was reported to be a more effective inhibitor of a steroid alcohol sulfotransferase when compared
with 4-nitroestradiol [8], whereas 4-substituted estrogen derivatives have been reported to inhibit
the steroid sulfatase enzyme more effectively than 2-substituted estrogen derivates [9].
We have previously reported that the introduction of small carbon chains with a basic
terminal moiety or heterocyclic scaffold to polyaromatic systems can induce significant
anticancer activity to the resultant molecules [10‒22]. In the present study we have developed (i)
a direct procedure for the nitration of estradiol and (ii) introduced carbon linkers on the oxygen
at C-3 with basic terminal moieties to the nitroestradiol derivatives. The resultant compounds
have been screened against a small panel of cancer cell lines, including ER-positive and ER-
negative breast cancer cells, as well as ovarian cancer cells and colon cancer cells. We have also
analyzed specified cellular and molecular mechanisms by which certain nitroestradiol derivatives

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affect these cancer cell lines. Some of these novel nitroestradiol compounds were more potent
cytotoxic agents than 4-hydroxytamoxifen (4-HT) when used to treat breast cancer cell lines, and
these compounds were also potent cytotoxic agents against ER-negative breast cancer cells.
Furthermore, one of these novel nitroestradiol compounds circumvented P-glycoprotein-
mediated drug resistance in ovarian cancer cells.
2
. Results
2
.1. Synthesis of novel nitroestradiol compounds
Organo-bismuth chemistry is considered an emerging field in synthetic organic chemistry
research, and we have previously demonstrated the selective activity of trivalent bismuth nitrate
pentahydrate in a number of examples. These experiments resulted in various methods that
include nitration of aromatic systems [23‒25], Michael reaction [26], protection of carbonyl
compounds [27], deprotection of oximes and hydrazones [28], Paal-Knorr synthesis of pyrroles
[29], hydrolysis of amide [30], electrophilic substitution of indoles [31,32], synthesis of α-
aminophosphonates [33], Biginelli condensation[34], and selective synthesis of 1,4-
dihydropyridines [35]. To determine the best route for the direct nitration of β-estradiol, different
metal nitrates were analyzed in combination with various solid supports (Scheme 1). We
observed that a mixture of 4- and 2-nitroestradiol was obtained when bismuth nitrate
pentahydrate was used as nitrating agent on solid supports, such as florisil, silica gel, molecular
sieves 4Å, montmorillonite KSF clay and alumina in two solvents (benzene and
dichloromethane) under refluxing condition using Dean-Stark water separator (Table 1, entries
1
,2, 4-8). 2-Nitroestradiol was isolated as the major product (40-50% yield) using the same
nitrating agent when molecular sieve was used as solid surface and the reflux was carried out in
benzene using Dean-Stark water separator (Table 1, entry 3) or when KSF clay (Table 1, entry 9)
or alumina (Table 1, entry 10) was used as solid surface in dichloromethane using Dean-Stark
water separator. No reaction was observed when β-estradiol was mixed with solid supports
impregnated with bismuth nitrate (Table 1, entries 11-15). Mixture of the nitro derivatives was
obtained under microwave irradiation in solvent free condition (Table 1, entries 16-20).
Moderate to excellent yield of both the regioisomers was obtained under reflux in benzene
(Table 1, entries 21-25). Taken together, these data indicate that nitration of β-estradiol depends
strongly on the nature of the solid support and metal nitrate, with a high to excellent yield of

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nitroestradiols (70-95%) being obtained when bismuth nitrate is used as nitrating agent
impregnated with KSF clay (Table 1).
Based on this observation, a comparative study was performed using other metal nitrates,
including ammonium cerium (IV) nitrate (CAN), zinc nitrate, calcium nitrate, lanthanum nitrate,
sodium nitrate and cupric nitrate, in combination with KSF clay. We found that CAN, lanthanum
nitrate and sodium nitrate did not yield nitroestradiols of any kind when used as a metal nitrate in
combination with KSF clay (Table 1, entries 26, 29, and 30). Additionally, zinc nitrate and
calcium nitrate formed 2-nitroestradiol regioselectively and cupric nitrate formed a mixture of
both the isomers in a ratio 1:1 with an overall 60% yield (Table 1, entries 27-28 and 31
respectively).
Upon obtaining 2- or 4-nitroestradiol, we coupled carbon chains with different terminal basic
moieties to the phenolic hydroxy group of the nitroestradiol derivatives. This was accomplished
by refluxing hydrochloride salt of the corresponding chloride compound in the presence of
potassium carbonate in acetone (Scheme 2), and the final products (1-4) were purified by column
chromatography. These six novel nitroestradiol derivatives (1-6), including 2- and 4-
nitroestradiol, were then analyzed as potential anticancer agents in vitro.
2
.2. Select Nitroestradiol Compounds decrease viability & block estradiol effects in breast
cancer cell lines
5
Since ERs are a major target for anticancer agents and potential anticancer agents , including
tamoxifen and estradiol derivatives, we initially analyzed the ability of our novel estradiol
derivatives to reduce viability of breast cancer cell lines, including ER-positive (both early and
late-stage MCF-7 cells and MD-MBA-453 cells) and ER-negative cells (AU565). Although
compounds 1, 5, and 6 did not effectively reduce viability of any of the breast cancer cell lines
examined, three compounds (3, 4, and 5) were capable of reducing viability of breast cancer cell
lines-compounds 3 and 4 were the most potent cytotoxic agents, reducing viability with an
estimated IC50 value of less than 10 µM (Table 2). In fact, the estradiol derivatives (3 and 4), as
well as a known ER-targeting drug, 4-hydroxytamoxifen (4-HT), reduced the viability of ER-
positive and ER-negative cells with similar efficacy, and the toxicity exhibited by 3 was slightly
better when compared to that 4-HT, with and IC50 value of approximately 2 µM in breast cancer
cells (Table 2). These data suggest that selective novel nitroestradiol compounds effectively

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cause cytotoxicity in breast cancer cells and that this cytotoxicity might occur in an ER-
independent manner.
To further examine whether our novel estradiol derivatives could affect breast cancer cell
lines in an ER-dependent manner, we compared the ability of compound 3 and 4-HT to inhibit
estradiol-induced cell proliferation of MCF-7 cells when used at sub-lethal doses. After three
days or five days of estrogen treatment, the number of Estradiol-treated MCF-7 cells, but not
AU565 cells, was increased 2.5- to 3-fold when compared to cells grown in serum free media
(
Figure 1A and data not shown). This increase in MCF-7 cell number after 3 days of estradiol
exposure was significantly blocked by addition of 4-HT in a dose dependent manner; however,
compound 3 had no detectable effect on the increase in cell number in response to estradiol
exposure for three days (Figure 1A). In contrast, both 4-HT and 3 significantly inhibited
increased cell numbers in response to estradiol exposure for five days (Figure 1B), although 4-
HT exhibited a greater inhibition when compared to 3. Taken together, these data indicate that
compound 3 functions, at least in part, as an antagonist of estradiol, albeit less effectively than 4-
HT.
2
.3. Compound 3 circumvents p-glycoprotein-mediated drug resistance in ovarian carcinoma
cells
We also analyzed the ability compound 3 to reduce viability of an ovarian cancer cell line,
SKOV3. Similar to the breast cancer cell lines, we found that 3 was a slightly more effective
cytotoxic agent against SKOV3 cells when compared with 4-HT, although both 3 and 4-HT were
much less potent cytotoxic agents when compared with the microtubule stabilizing agent,
Paclitaxel (Figure 2).
Next, we analyzed the ability compound 3 to reduce viability of a retrovirally transduced
SKOV3 cell line overexpressing P-glycoprotein, SKOV3-MDR1-M6/6. These SKOV3-MDR1-
M6/6 cells have previously been shown to be resistant to cytotoxicity induced by anticancer
drugs, including daunorubicin and paclitaxel, when compared with SKOV3 cells [36,37].
Similarly, we found that SKOV3-MDR1-M6/6 cells were resistant to cytotoxicity in response to
paclitaxel treatment when compared with SKOV3 cells, since the IC50 value for paclitaxel was
greater than 1000-fold higher in SKOV3-MDR1-M6/6 cells when compared with SKOV3 cells
(
Figure 2A). In addition, paclitaxel was incapable of killing over 45% of SKOV3-MDR1-M6/6

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cells, even when used at dosages up to 20 µM (Figure 2A and data not shown). In contrast, no
resistance or only a modest level of resistance was observed for 4-HT or compound 3, which had
IC values equal to or only 2.2-fold higher in SKOV3-MDR1-M6/6 when compared to SKOV3
5
0
cells, respectively (Figure 2B and 2C). These data indicate that compound 3, as well as 4-HT,
bypasses the P-glycoprotein-mediated resistance of SKOV3-MDR1-M6/6 cells.
2
.4. Select Nitroestradiol Compounds decrease viability of multiple mammalian cell lines
Since our novel nitroestradiol derivatives killed both ER-positive and ER-negative breast
cancer cell lines with similar efficacy and compound 3 overcame drug resistance in ovarian
cancer cells, we decided to test the effects of these novel compounds on the viability of a small
panel of human and mouse cancer cell lines that originated from tissues other than reproductive
tissues. These included a colon cancer cell line (HT-29), liver cancer cell lines (HepG2, Hepa1-6,
and Hep3B), and a cervical cancer cell line (HeLa). Similar to the cancer cell lines from
reproductive tissue, 4-nitroestradiol (6) was the least effective in reducing cell viability in this
small panel of cell lines as the majority of cell lines had an estimated IC value greater than 50
5
0
ꢀ
M when treated with this compound (Table 3). Also similar to cancer cell lines from
reproductive tissues, compounds 2, 3, and 4 were the most potent cytotoxic compounds when
used to treat colon, liver or cervical cancer cells, with an estimated IC value of 8 µM or lower
5
0
(
Table 3).
To determine when compounds 2, 3, and 4 were causing cytotoxicity, HepG2 cells were
treated with each compound at a concentration of twice the calculated IC50 value for 24-, 48-, or
2-hour time-points. Both 2 and 4 caused cytotoxicity between 24 and 48 hours, reducing
7
viability greater than 80% after 48 hours (Figure 3); however, 3 caused a steady reduction in
viability, reducing viability greater than 80% only after 72 hours (Figure 3). Treatment of cells
with compound 3 only reduced viability approximately 30% after 24 hours and 55% after a 48-
hour treatment (Figure 3).
2
.5. Estradiol compounds potentially cause cytotoxicity via induction of apoptosis
To determine the mechanism by which our novel nitroestradiol derivatives were killing cells,
we analyzed the effects of compounds 3 or 4 on plasma membrane blebbing, which is a known
indicator of apoptosis, in HeLa cells over the course of 24 hours. Both 3 and 4 caused membrane

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blebbing on the surface of cells, whereas non-treated cells or vehicle-treated cells did not exhibit
blebbing on the cell surface (Figure 4). This blebbing started after 12 hours of treatment and
continued throughout the 24-hour period in which cells were observed, and similar results were
observed in HepG2 cells (data not shown). These data suggest that the ability of these novel
nitroestradiol derivatives to reduce cell viability occurs through an apoptosis-dependent
mechanism.
3
. Discussion and Conclusions
Conventional nitration of aromatic compounds involves nitric acid-sulfuric acid treatment or
nitronium tetrafluoborate, which typically requires a mixture of concentrated or fuming nitric
acid with sulfuric acids leading to excessive acid waste streams and added expense. Furthermore,
nitration of estrogens has been reported using nitric acid [38,39] or peroxidase/H O /NO [40],
2
2
2
and either of these procedures includes the use of hazardous chemicals and utilizes a difficult
work-up procedure with low yield. In this study, a total of 31 different reaction conditions were
used involving several metal nitrates in combination with various solid supports and techniques
to develop a novel, eco-friendly procedure for the nitration of estradiol. Of the 31 reaction
conditions tested, we found that bismuth nitrate, when used as a nitrating agent impregnated with
KSF clay, could efficiently generate either 2- or 4-nitroestradiol.
We also conjugated three different side chains containing basic moieties to either 2- or 4-
nitroestradiol, and we examined the potential anti-cancer effects of these novel agents. 2-
nitroestradiol was a more effective cytotoxic agent when compared with 4-nitroestradiol (Tables
2
and 3). These data are similar to a previous study showing that either 2-hydroxyestradiol or 2-
methoxyestradiol inhibited cell proliferation and caused cellular apoptosis, whereas 4-
hydroxyestradiol or 4-methoxyestradiol had no effect on cell proliferation or apoptosis [41].
Although 2-nitroestradiol wasn’t as potent at causing cytotoxicity as previously reported 2-
methoxyestradiol [42], the addition a side chain containing either a morpholine or piperidine ring
(3 or 4, respectively) improved the cytotoxic potency dramatically, making the cytotoxicity of
these compounds comparable to 2-methoxyestradiol and, in some cases, better than 4-HT in
breast cancer cells as well as cisplatin in colon cancer cells and other cell types [42] (Table 2).
Interestingly, the addition of an ethylamine group to 4-nitroestradiol improved its cytotoxicity

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beyond that of 2-nitroestradiol with the same side chain (Tables 2 and 3). Thus, it would be
interesting to conjugate side chains containing either the morpholine or piperidine ring onto 4-
nitroestradiol to determine whether the potency could be improved even further.
Our results also indicate that the novel nitroestradiol derivatives 3 and 4, along with 4-HT,
can inhibit estradiol-induced proliferation of ER-positive breast cancer cells, but that these
estradiol derivatives can affect toxicity of ER-positive and ER-negative breast cancer cell lines in
a similar manner (Table 2). This data supports previous results showing that Tam could inhibit
growth of ER-negative AU565 cells [43]. Taken together, these data suggest that, like 4-HT, our
novel nitroestradiol derivatives can affect cells by regulation of Estrogen Receptor, but
regulation of ER is not likely to be the mechanism by which these agents cause cytotoxicity. This
notion is plausible, since Tam has been shown to induce oxidative stress and apoptosis in Jurkat
cells, which lack ER expression [44], and another group showed that Tam induced apoptosis via
2
+
Ca influx in ER-negative HepG2 cells [45]. In addition, Tam, when used at pharmacological
concentrations (above 5 µM), was reported to induce cytochrome c release from mitochondria
resulting in acute cell death in both ER-positive (MCF-7) and ER-negative (MDA-MB-231)
breast cancer cells [46]. Thus, it is possible that our novel nitroestradiol derivatives are causing
toxicity through one or several of these mechanisms. In fact, our data indicates that 3 and 4 cause
plasma membrane blebbing (Figure 4), suggesting that these nitroestradiol derivatives, like Tam,
are causing apoptosis. One possible ER-independent mechanism by which our novel
nitroestradiol derivatives might cause cytotoxicity is by integrating into the plasma membrane of
cells, since it is well known that sterols function, at least in part, in this manner [47]. Future
studies will focus on elucidating the cellular and molecular mechanisms by which novel estradiol
derivatives cause cytotoxicity in cancer cells and in determining the role, if any, that ER plays in
cytotoxicity in response to these and other novel estradiol derivatives.
In addition to killing ER-positive and ER-negative breast cancer cell lines, our nitroestradiol
compounds, 3 and 4, also caused cytotoxicity in ovarian and colon cancer cells, as well as
cervical and hepatic cancer cell lines. Interestingly, compound 3 was also capable of
circumventing the drug resistance in ovarian cancer cells overexpressing P-glycoprotein (Figure
2
), although SKOV3-MDR1-M6/6 cells did display a subtle resistance to 3 when compared with
SKOV3 cells. This result is similar to a previous study showing that SKOV3-MDR1-M6/6 cells
exhibited a mild resistance to 2-methoxyestradiol and a robust resistance to paclitaxel [36]. P-

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glycoprotein (Pgp) is one of nine Multidrug Resistant Proteins (MRPs), and MRPs are expressed
in various tissues of the body and capable of transporting numerous physiological substrates or
anticancer drugs out of cells [48].
In conclusion, an ecofriendly direct procedure for the nitration of estradiol is reported using
KSF clay impregnated bismuth nitrate pentahydrate as nitrating agent. A total of 31 different
conditions have been tested to figure out the best condition. In the following step, carbon linkers
with diversely substituted basic terminal moieties have been introduced on the oxygen at C-3.
The biological evaluation showed that compounds 3 and 4 are cytotoxic for HepG2, Hepa1-6,
Hep3B, HeLa and HT-29 cancer cell lines. Interestingly, the compound 3 is more potent than 4-
HT (4-hydroxytamoxifen, the well-known anticancer drug) for both ER-positive and ER-
negative cells and this compound also circumvented P-gp mediated drug resistance in ovarian
cancer cells. Qualitative structure activity relationship (SAR) has also been studied. Thus, it
would be interesting to assess whether the nitroestradiol derivatives described here, or other
novel nitroestradiol derivatives, circumvented the multidrug resistance mediated by MDR1 and
other MDR proteins in vitro and in vivo. If so, then nitroestradiol derivatives could become
chemotherapeutic drugs with clinical usefulness.
4
. Experimental Section
4
.1. Materials
Dimethylsulfoxide (DMSO; Sigma Aldrich Corp., St. Louis, MO), phosphate-buffered saline
(PBS), Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen, Carlsbad, CA), Fetal
Bovine Serum (FBS; Invitrogen), McCoy’s media (Invitrogen) were purchased. All other
chemicals were purchased from Sigma-Aldrich Corporation (analytical grade). Throughout the
project solvents were purchased from Fisher-Scientific. Deionized water was used for the
preparation of all aqueous solutions.
Melting points were determined in a Fisher Scientific electrochemical Mel-Temp* manual
melting point apparatus (Model 1001) equipped with a 300°C thermometer. FT-IR spectra were
1
registered on a Bruker IFS 55 Equinox FTIR spectrophotometer as KBr discs. H-NMR (600
1
3
MHz) and C-NMR (150 MHz) spectra were obtained at room temperature with Bruker
TM
superconducting Ultrashield Plus 600 MHz NMR spectrometer with central field 14.09 Tesla,
3
coil inductance 89.1 Henry and magnetic energy 1127.2 kJ using CDCl as solvent. Elemental

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analyses (C, H, N) were conducted using the Perkin-Elmer 2400 series II elemental analyzer,
their results were found to be in good agreement (± 0.2%) with the calculated values for C, H, N.
4
.2. Synthesis of the compounds 1-6
4
.2a Nitration of estradiol: A representative procedure (Entry 24, Table 1) for the nitration of
estradiol is as follow: Estradiol (1 mmol) and montmorillonite KSF (500 mg) were added to a
suspension of bismuth nitrate pentahydrate (1 equiv.) in anhydrous benzene (30 mL). The
mixture was refluxed for 2 hours and the reaction was monitored by TLC. After completion of
the reaction, the mixture was filtered through vacuum and the solid was washed thrice (3x10 mL)
by dichloromethane and washed with saturated solution of sodium bicarbonate, brine and water
(10 mL each) successively. The organic layer was dried over anhydrous sodium sulfate and
filtered through cotton. It was then concentrated to afford the crude mixture. The pure products 5
and 6 were isolated by chromatographic column (hexane/ethyl acetate over silica gel.
4
.2b Introduction of carbon linker with basic terminal moiety: A representative procedure
leading to the synthesis of compound (2) is as follow: 4-Nitroestradiol (1 mmol), N,N-dimethyl-
-chloroethylamine and potassium carbonate (1 equiv.) were mixed in anhydrous acetone (5 mL)
2
and the mixture was refluxed for 4-6 hours (monitored by TLC). After completion of the mixture
was filtered through vacuum and the solid was washed thrice (3x10 mL) by dichloromethane.
The solvent was removed by rotavapor and 20 mL of dichloromethane was added to the crude
mixture. The organic layer was washed with saturated solution of sodium bicarbonate, brine and
water (10 mL each) successively. It was then dried over anhydrous sodium sulfate and filtered
through cotton. The organic layer was concentrated through rotavapor again to afford the crude
mixture. The pure product (2) was isolated by chromatographic column (hexane/ethyl acetate
over silica gel (65%).
4
.3. Spectroscopic analyses of compounds (1-6)
The physical and spectral data are of the compounds are given below:
4
.3a. 3-(2-(Dimethylamino)ethoxy)-13-methyl-2-nitro-7,8,9,11,12,13,14,15,16,17-decahydro-
H-cyclopenta[a]phenanthren-17-ol (1).
6

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Brick red solid (71%); mp 142°C; IR (KBr) 3416, 2916, 1611, 1564, 1517, 1466, 1345, 1263,
-
1 1
1
1
2
091, 899, 759 cm ; H NMR (600 MHz, d -DMSO) δ 0.66 (s, 3H, H-18), 1.28 (m, 7H, H-7, H-
6
4, 15,16), 1.59 (m, 1H, H-8), 1.85 (m, 3H, H-9 & H-11), 2.11 (m, 1H, H-17), 2.20 (s, 6H, H-3′),
.62 (t, J = 5.70 Hz, 2H, H-12), 2.84 (m, 2H, H-2′), 3.52 (t, J = 8.64 Hz, 2H, H-6), 4.17 (m, 2H,
1
3
H-1′), 4.50 (broad s, 1H, OH), 7.05 (s, 1H, H-4), 7.69 (s, 1H, H-1); C NMR (150 MHz, d -
6
DMSO) δ 11.13 (C-18), 22.66 (C-15), 25.67 (C-11), 26.24 (C-7), 29.22 (C-6), 29.84 (C-16),
3
6
5
8
6.29 (C-12), 37.88 (C-8), 42.70 (C-9), 42.91 (C-13), 45.55 (C-14,), 49.38 (2C-3′), 57.31 (C-2′),
7.82 (C-1′), 79.90 (C-17), 114.92 (C-4), 121.73 (C-1), 132.79 (C-10), 137.24 (C-2), 144.36 (C-
), 149.21 (C-3). Anal. Calcd for C22
.17; N, 7.16.
32 2 4
H N O : C, 68.01; H, 8.30; N, 7.21. Found: C, 67.86; H,
4
.3b. 3-(2-(Dimethylamino)ethoxy)-13-methyl-4-nitro-7,8,9,11,12,13,14,15,16,17-decahydro-
H-cyclopenta[a]phenanthren-17-ol (2).
6
Reddish yellow solid (65%); mp 133°C; IR (KBr) 3244, 2947, 1618, 1571, 1529, 1462, 1372,
-
1 1
1
1
290, 1232, 1079, 1017, 814, 790, 669 cm ; H NMR (600 MHz, d -DMSO) δ 0.67 (s, 3H, H-
6
8), 1.26 (m, 6H, H-7,12,16), 1.57 (m, 1H, , H-14), 1.89 (m, 3H, H-8, H-11), 2.10 (m, 3H, H-9,
H-15), 2.18 (s, 6H, H-3′), 2.31 (m, 2H, H-6b, H-17), 2.62 (m, 2H, H-2′), 3.52 (distorted t, J =
8
7
2
4
2
.40 Hz, 1H, H-6a), 4.15 (m, 2H, H-1′), 4.51 (broad s, 1H, OH), 7.13 (d, J = 8.82 Hz, 1H, H-2),
1
3
6
.44 (d, J = 8.82 Hz, 1H, H-1); C NMR (150 MHz, d -DMSO) δ 11.13 (C-18), 22.63 (C-15),
3.41 (C-11), 25.52 (C-7), 25.88 (C-6), 29.82 (C-16), 36.36 (C-12), 37.55 (C-8), 42.67 (C-9),
3.22 (C-13), 45.38 (C-14), 49.21 (2C-3′), 57.15 (C-2′), 67.55 (C-1′), 79.92 (C-17), 111.51 (C-
), 128.03 (C-5), 131.51 (C-1), 133.68 (C-10), 141.20 (C-4), 146.97 (C-3). Anal. Calcd for
C H N O : C, 68.01; H, 8.30; N, 7.21. Found: C, 67.91; H, 8.13; N, 7.27.
22
32
2
4
4
.3c. 13-Methyl-3-(2-morpholinoethoxy)-2-nitro-7,8,9,11,12,13,14,15,16,17-decahydro-6H-
cyclopenta[a]phenanthren-17-ol (3).
Deep yellow solid (74%); mp 104 °C; IR (KBr) 3240, 2926, 1616, 1541, 1476, 1419, 1245,
-
1 1
1
7
3
2
179, 1048, 911, 833, 747 cm ; H NMR (600 MHz, d -DMSO) δ 0.66 (s, 3H, H-18), 1.26 (m,
6
H, H-7,12,14,16), 1.47 (distorted t, J = 5.58, 11.04 Hz, 2H, H-15), 1.58 (m, 1H, H-8), 1.84 (m,
H, H-9,11), 2.13 (m, 2H, H-6), 2.34 (m, 4H, H-2″,6″), 2.58 (m, 4H, H-3″,5″), 2.87 (m, 2H, H-
′), 3.53 (ddd, J = 3.78, 5.82, 9.84 Hz, 1H, H-17), 4.18 (dddd, J = 5.82, 8.94, 13.26, 19.14 Hz,

ACCEPTED MANUSCRIPT
1
3
2
H, H-1′), 4.54 (d, J = 4.50 Hz, 1H, OH), 7.07 (s, 1H, H-4), 7.69 (s, 1H, H-1); C NMR (150
MHz, d -DMSO) δ 11.15 (C-18), 22.64 (C-15), 23.91 (C-11), 25.91 (C-7), 26.10 (C-6), 29.85
6
(
C-16), 36.27 (C-12), 38.05 (C-8), 42.66 (C-9), 42.72 (C-13), 46.09 (C-14), 49.30 (C-2′), 49.36
C-2″,6″), 53.53 (C-3″,5″), 66.17 (C-1′), 79.96 (C-17), 117.00 (C-4), 123.05 (C-1), 133.12 (C-
(
1
0), 135.28 (C-2), 149.59 (C-5), 157.54 (C-3). Anal. Calcd for C H N O : C, 66.95; H, 7.96;
24 34 2 5
N, 6.51. Found: C, 67.06; H, 8.00; N, 6.43.
4
.3d. 13-Methyl-2-nitro-3-(2-(piperidin-1-yl)ethoxy)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-
cyclopenta[a]phenanthren-17-ol (4).
Brownish yellow solid (62%); mp 146°C; IR (KBr) 3448, 2991, 1617, 1516, 1458, 1350, 1275,
-
1 1
1
3
115, 1068, 944 cm ; H NMR (600 MHz, d
6
-DMSO) δ 0.66 (s, 3H, H-18), 1.23 (m, 10H, H-
″,5″,7,12,16), 1.46 (m, 3H, H-14,15), 1.58 (m, 1H, H-8), 2.11 (m, 3H, H-9,11), 2.25 (distorted
d, 2H, H-4″), 2.42 (m, 4H, H-2″,6″), 2.65 (t, J = 5.82 Hz, 2H, H-6), 2.85 (m, 2H, H-2′), 3.52 (t, J
8.10 Hz, 1H, H-17), 4.18 (dddd, J = 5.76, 8.64, 12.96, 18.78 Hz, 2H, H-1′), 4.52 (broad s, 1H,
=
1
3
OH), 7.07 (s, 1H, H-4), 7.69 (s, 1H, H-1); C NMR (150 MHz, d -DMSO) δ 11.13 (C-18), 22.66
6
(C-15), 23.81 (C-11), 25.56 (C-3″,5″), 25.69 (C-7), 26.24 (C-4″), 29.21 (C-6), 29.82 (C-16),
3
2
1
6
6.28 (C-12), 37.90 (C-8), 42.71 (C-9), 42.92 (C-13), 49.37 (C-14), 54.28 (C- 2′), 56.97 (C-
″,6″), 67.69 (C-1′), 79.91 (C-17), 115.17 (C-4), 121.69 (C-1), 132.84 (C-10), 137.31 (C-2),
44.35 (C-5), 149.26 (C-3). Anal. Calcd for C25
9.91; H, 8.39; N, 6.43.
36 2 4
H N O : C, 70.06; H, 8.47; N, 6.54. Found: C,
4
.3e. 13-Methyl-2-nitro-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-
,17-diol (5).
3
Bright yellow solid (75%, Entry 9, Table 1); mp 162°C; IR (KBr) 3363, 2929, 2360, 1631, 1577,
-
1 1
1
3
1
525, 1480, 1432, 1312, 1266, 1052, 762, 656 cm ; H NMR (600 MHz, d -DMSO) δ 0.66 (s,
6
H, H-18), 1.11 (m, 1H, H-14), 1.20 (m, 3H, H-7,15b), 1.26 (m, 2H, H-12), 1.35 (m, 1H, H-16b),
.57 (m, 1H, H-8), 1.79 (m, 1H, H-15a), 1.87 (m, 2H, H-11), 2.09 (m, 1H, H-16a), 2.24 (m, 1H,
H-9), 2.78 (m, 2H, H-6), 3.52 (t, J = 8.16 Hz, 1H, H-17), 4.51 (s, 1H, 17-OH), 6.81 (s, 1H, H-4),
1
3
7
6
.74 (s, 1H, H-1), 10.52 (s, 1H, 3-OH); C NMR (150 MHz, d -DMSO) δ 11.11 (C-18), 22.67
(
C-15), 25.69 (C-11), 26.21 (C-7), 28.88 (C-6) , 29.82 (C-16), 36.25 (C-12), 37.87 (C-8), 42.68
C-9), 42.81 (C-13), 49.42 (C-14), 79.91 (C-17), 118.45 (C-4), 121.41 (C-1), 132.18 (C-2),
(

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1
4
33.97 (C-10), 146.06 (C-5), 150.22 (C-3). Anal. Calcd for C18
.41. Found: C, 67.99; H, 7.21; N, 4.35.
4
H23NO : C, 68.12; H, 7.30; N,
4
3
1
1
1
.3f. 13-Methyl-4-nitro-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-
,17-diol (6). Brownish yellow solid (48%, Entry 24, Table 1); mp 224°C; IR (KBr) 3184, 2925,
-1 1
6
570, 1527, 1497, 1348, 1294, 1046, 794 cm ; H NMR (600 MHz, d -DMSO) δ 0.66 (s, 3H, H-
8), 1.15 (m, 1H, H-14), 1.26 (m, 8H, H-7,8,12,15, 16b), 1.86 (m, 2H, H-11), 2.09 (m, 1H, H-
6a), 2.25 (m, 1H, H-9), 2.62 (m, 2H, H-6), 3.52 (m, 1H, H-17), 4.50 (broad s, 1H, 17-OH), 6.85
1
3
(
d, J = 8.70 Hz, 1H, H-2), 7.28 (d, J = 8.70 Hz, 1H, H-1), 10.49 (s, 1H, 3-OH); C NMR (150
MHz, d -DMSO) δ 11.23 (C-18), 22.76 (C-15), 26.06 (C-11), 26.93 (C-7), 29.13 (C-6), 29.89
6
(
C-16), 36.58 (C-12), 38.68 (C-8), 42.78 (C-9), 43.51 (C-13), 49.52 (C-14), 80.04 (C-17), 114.89
C-2), 125.96 (C-5), 128.28 (C-1), 130.40 (C-10), 137.08 (C-4), 154.86 (C-3). Anal. Calcd for
(
C H NO : C, 68.12; H, 7.30; N, 4.41. Found: C, 67.96; H, 7.26; N, 4.32.
18
23
4
4
.4. Mammalian cell culture
HepG2, Hepa1-6, Hep3B, MCF-7 (less than 100 passages), MCF-7L (over 500 passages), MDA-
MB-453 and HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)
(
Invitrogen, Carlsbad, CA) containing 10% Fetal Bovine Serum (FBS, Invitrogen), and HT-29
cells were cultured in McCoy’s media (Invitrogen) containing 10% FBS. SKOV3, and SKOV3-
MDR1-M6/6 cells were grown in Basal Medium Eagle (BME) media containing 10% FBS. All
cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA)
except MCF-7L, MDA-MB-453, SKOV3, and SKOV3-MDR1-M6/6. MCF-7L and MDA-
MB453 cells were a provided by Dr. R.K. Dearth, while SKOV3 and SKOV3-MDR1-M6/6 cells
o
were provided by Dr. Susan Mooberry (UTHSCSA). All cells were incubated at 37 C with 5%
CO2.
4
.5. Mammalian cell viability assays
o
Cells were plated (5,000 cells/well) onto a 96-well dish and incubated overnight at 37 C. The
following day, cells were treated with increasing dosages (0.1 ꢀM to 100 ꢀM) of each estradiol
derivative, which had been dissolved in DMSO. The DMSO concentration of treatments was
limited to 0.25%, and cells were treated with DMSO alone (0.25%) or 10 ꢀM cisplatin as

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negative and positive controls for cytotoxicity, respectively. For breast cancer cell lines, estradiol
derivatives were compared with 4-hydroxytamoxifen (4-HT). After 48 hours, cells were fixed
and cell viability was analyzed using the Sulforhodamine B colorimetric assay as described
previously [49]. Absorbance of SRB was measured utilizing a SpextraMaxM5 plate reader and
absorbance values were normalized to non-treated cells. Normalized cell viabilities, with
increasing drug doses, were plotted on a 4-parameter logistical curve, and the IC50 of each
compound in each cell line was calculated using SigmaPlot software (Systat Software, Inc.).
Each compound was used in two independent cell viability assays. The mean IC , with the
5
0
corresponding standard deviation, of the two independent treatments was then calculated. For
time-courses, viability was assessed after treating HepG2 cells (5,000cells/well) with compounds
at twice the IC50 concentrations for 24, 48, and 72 hrs. After treatment, cells were fixed and cell
viability was analyzed using SRB assay as described above.
4
.6. Estradiol-induced cell proliferation
MCF-7 cells were plated onto 12-well dishes at a density of 1.0x10 cells/well. The following
5
day, cells were placed into phenol red-free, serum-free Improved MEM (IMEM: Mediatech,
Manasses, VA) containing 10-mM HEPES, 1-µg/mL apotransferrin, and 1-µg/mL fibronectin for
2
4 hours. Cells were then treated with 10-nM 17β-Estradiol (E ) in the absence or presence of 4-
2
HT or 3 (compounds were used at either 250 nM or 500 nM, respectively). Media and treatments
were changed and repeated, respectively, every 48 hours, and cells were counted on Day 3 and
Day 5 of treatments using a cellometer Vision automated cell counter (Nexcelom Bioscience,
2
Lawrence, MA). Both E and 4-HT were dissolved in ethanol (EtOH), and 3 was dissolved using
DMSO. Cells were treated with the vehicle for 4-HT (EtOH) or the vehicle for 3 (DMSO) in
combination with 10nM E and neither vehicle affected E -induced cell proliferation. Statistical
2
2
analyses were performed using SigmaPlot software (Systat Software, Inc.), and P values were
calculated using a Mann-Whitney U test.
4
.7. Membrane Blebbing
To detect membrane blebbing, HeLa cells were plated onto 6-well dishes at a density of 1.5-
5
2
.0x10 cells/well. Cells were then treated with the compound 3 (16 µM) or 4 (10 µM) and
visualized every 3 hours using an Olympus CKX31 culture microscope (Olympus Corp., Tokyo,
Japan). Images were captured using a Nikon Coolpix E995 eyepiece camera (Nikon, Japan).

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Acknowledgements
The authors would like to thank Drs. Susan Mooberry and April Risinger (UTHSCSA) for
providing ovarian cancer cell lines and Drs. Joanne Rampersad and David Ammons for
providing the Nikon eyepiece camera. We gratefully acknowledge the funding support from
Kleberg Foundation of Texas.
Conflict of Interest
The authors declare no competing financial interest.

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Figure Legends
Scheme 1. Reaction whereby
β-estradiol is nitrated with metal nitrate on a solid surface.
Scheme 2. Reactions and end products in which different side chains with basic terminal moieties were
added to nitroestradiols.
Figure 1. The effect of compound 3 on estradiol-induced cell proliferation in MCF-7 cells. MCF-7 cells
were treated with 17- Estradiol in the absence or presence of sub-lethal doses of 4-HT or compound 3
(
dosages are indicated) for 3 days (A) or 5 days (B) as described in Materials and Methods. After
treatment, cells were counted and compared statistically using the Mann Whitney U test. A representative
experiment is shown, and each data point represents the mean ± standard deviation value of four
a
replicates. Statistically significant (p<0.05) when compared with cells grown in serum free media.
b
Statistically significant (p<0.05) when compared with vehicle-treated cells.
Figure 2. The effect of Paclitaxel, 4-HT, or compound 3 on viability of ovarian cancer cells that
overexpress P-glycoprotein. SKOV3 cells (left panels) or SKOV3-MDR1-M6/6 cells (right panels) were
treated with increasing concentrations of Paclitaxel (A), 4-HT (B), or compound 3 (C) for 48 hrs, and
then cell viability was analyzed using SRB assay. The dose curve for each compound in each cell line was
plotted, and the calculated IC value for each compound is indicated in the top right corner of each graph.
5
0
A representative experiment is shown (n
≥ 2).
Figure 3. The effect of 2, 3, or 4 on HepG2 cell viability over the course of time. HepG2 cells were
treated with compounds 2, 3, and 4 at 2X IC concentrations for 24, 48, or 72 hrs, and then cell viability
50
was analyzed using SRB assay. A representative experiment of two independent experiments performed
in duplicates is shown. Each data point represents the mean ± standard deviation value.
Figure 4. The effects of 3 or 4 on cell morphology. HeLa cells were treated with the indicated compounds
(
3: 16
 M; 4: 10  M) for 24 hours. After treatment, cells were observed using bright-filed microscopy
and photographed every 3 hours. Arrows indicate cells exhibiting membrane blebbing.

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Scheme 1 – Bandyopadhyay et al.

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Scheme 2 – Bandyopadhyay et al.

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Figure 1 – Bandyopadhyay et al.

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Figure 2 – Bandyopadhyay et al.