Synthesis and biological evaluation of selective aromatase expression regulators in breast cancer cells

Article abstract of DOI:10.1021/jm061133j

Aromatase converts androgens to estrogens and is a particularly attractive target in the treatment of estrogen receptor positive breast cancer. The enzyme is encoded by the CYP19 gene, which is expressed in a tissue-specific manner. Prostaglandin E_2<

Full text of DOI:10.1021/jm061133j

J. Med. Chem. 2007, 50, 1635-1644
1635
Synthesis and Biological Evaluation of Selective Aromatase Expression Regulators in Breast
Cancer Cells
Bin Su,† Serena Landini,† Danyetta D. Davis,‡ and Robert W. Brueggemeier*,†
DiVision of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, and Ohio State Biochemistry Program, The Ohio State UniVersity,
500 W. 12th AVenue, Columbus, Ohio 43210
ReceiVed September 28, 2006
Aromatase converts androgens to estrogens and is a particularly attractive target in the treatment of estrogen
receptor positive breast cancer. The enzyme is encoded by the CYP19 gene, which is expressed in a tissue-
specific manner. Prostaglandin E2 (PGE2), the major product of cyclooxygenase-2 (COX-2), stimulates
aromatase gene expression via protein kinase A and C signaling pathways. Our previous study demonstrated
that COX-2 selective inhibitor nimesulide decreased aromatase activity from the transcriptional level in
breast cancer cells. In this manuscript, the synthesis and biological evaluation of a series of nimesulide
analogues as potential selective aromatase expression regulators are described. Several novel sulfonanilide
compounds demonstrate IC50 values from 0.33 to 2.68 µM in suppressing aromatase enzyme activity in
SK-BR-3 breast cancer cells and are 10- to 80-fold more active than nimesulide. Also, the sulfonanilide
compounds selectively decrease aromatase gene expression in breast cancer cells, without exhibiting cytotoxic
or apoptotic effects at low micromole concentrations.
1. Introduction
Aromatase inhibitors, such as anastrozole, letrozole, and ex-
emestane, significantly decrease plasma estrogen levels, are
emerging as alternatives to tamoxifen due to their clinical
efficacy, and have favorable safety profiles for the treatment of
hormone-dependent breast cancer in postmenopausal women.7-9
However, since the AIs inhibit aromatase enzyme in a global
fashion, a major long-term side effect of the AIs is the reduction
of the bone density which can lead to osteoporosis.10
In the United States, breast cancer is the most common cancer
diagnosed in women and is the second leading cause of death
in women after lung cancer. It is estimated that 212 920 new
cases of invasive breast cancer will be detected among women
in the United States in the 2006 and 40 970 women will die
from this disease in the same year.1 Approximately 60% of
premenopausal and 75% of postmenopausal breast cancer
patients have estrogen-dependent carcinoma.2 Hormone-de-
pendent breast tumors require estrogens for their growth and
are characterized by high expression of estrogen receptors
(ERs).3 As a result, efforts to block estrogen action by interfering
with the binding to its receptor or by decreasing estrogen
production are used as strategies to treat hormone-dependent
breast cancer. The first approach involves antiestrogen molecules
that can compete with estrogen for binding to the ERs. The
second method utilizes aromatase inhibitors (AIs) that inhibit
the enzyme catalyzing the final rate-limiting step of the estrogen
biosynthesis.4
Significant research has focused on developing antiestrogens
that can selectively inhibit the estrogen effects in breast tissue
without antagonizing the physiological roles of estrogens in other
tissues. Agents that exhibit such tissue-specific antiestrogenic/
estrogenic activities have been termed selective estrogen receptor
modulators (SERMs). Tamoxifen is the most widely used SERM
in hormone-dependent breast cancer therapy and has made a
substantial contribution to the reduced mortality rate in many
developed countries since 1990.5 Although tamoxifen is still
considered the “gold standard” for endocrine therapy in
hormone-dependent breast cancer, its use is associated with
tumor resistance and increased risk of endometrial cancer.6
To reduce the risk of the long-term side effects, a new
pharmacological approach in the treatment of estrogen-depend-
ent postmenopausal breast cancer is the use of tissue-specific
inhibitors of aromatase. This concept is based on the tissue-
specific regulation of aromatase expression. Ten different
promoter regions have been identified upstream of the coding
region of the aromatase gene (CYP19).11 The employment of
alternative promoters results in tissue-specific regulation of the
CYP19 expression. Each promoter is regulated by distinct
hormones and second messenger pathways. In postmenopausal
women, estrogens are produced by adipose tissue, including
breast tissue and skin.12,13 In these tissues aromatase expression
is directed by promoter I.4 that is regulated by the synergistic
actions of glucocorticoids and class I cytokines such as
interleukin (IL)-6, IL-11, and tumor necrosis factor alpha
(TNFR).13 In breast adenocarcinoma, aromatase expression and
activity increase significantly compared to normal breast tissue.14
Breast tumors produce factors that stimulate aromatase expres-
sion locally.15 This stimulation is associated with switching of
the aromatase gene promoter from promoter I.4 to c-AMP-
dependent promoter I.3 and promoter II in breast cancer and
surrounding adipose stromal cells.16,17 Current evidence suggests
that tumor-derived factors, including the cyclooxygenase (COX)
product prostaglandin E2 (PGE2), can mediate the induction of
aromatase expression via promoter II by stimulating adenylate
cyclase in adipose stromal cells.16-18 This biochemical mech-
anism may explain epidemiological observations of the benefi-
cial effect of nonsteroidal anti-inflammatory drugs (NSAIDs)
on breast cancer.
* To whom correspondence should be addressed. Telephone: 614-292-
5231, Facsimile: 614-292-3113, E-mail: Brueggemeier.1@osu.edu.
† Division of Medicinal Chemistry and Pharmacognosy.
‡ Ohio State Biochemistry Program.
a Abbreviations: PGE2, Prostaglandin E2; COX-2, cyclooxygenase-2;
NSAIDs, nonsteroidal anti-inflammatory drugs; ER, estrogen receptors; AIs,
aromatase inhibitors; SERMs, selective estrogen modulators; TNFR, tumor
necrosis factor alpha; cAMP, adenosine 3′,5′-cyclic monophosphate.
Previously, we demonstrated that COX-2 inhibitors, N-(2-
phenoxy-4-nitrophenyl)methanesulfonamide (nimesulide),19 and
10.1021/jm061133j CCC: $37.00 © 2007 American Chemical Society
Published on Web 02/22/2007

1636 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Su et al.
Figure 1. Chemical structures of COX-2 inhibitors nimesulide, N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide, and the target compounds
Scheme 1. Synthesis of Compounds 1b-4c
N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide (NS-
398),20 suppress aromatase activity in breast cancer cells by
suppressing aromatase transcription,21 and these two agents share
very similar chemical structures (Figure 1). Interestingly,
derivatives of N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfon-
amide suppresses aromatase activity at similar levels with the
compound itself, whereas the N-methyl and the 2-methyl
derivatives of N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfon-
amide exhibit no COX-2 inhibition.22 In the present manuscript,
we described the synthesis of novel sulfonanilide compounds
based on nimesulide (Figure 1) and the biological effects of
these new agents as selective aromatase expression regulators.
starting material 2-amino-5-nitrophenol was commercially avail-
able. In Scheme 1, 2-amino-5-nitrophenol was refluxed with
K2CO3 and benzyl bromide to obtain compounds 1a.23 Sodium
hydride and methanesulfonyl chloride were added to compound
1a in dry dimethylformamide (DMF) at room temperature, and
the reaction mixture was stirred at room temperature overnight
to obtain the N,N-bimethanesulfonamido (1b). Dealkylation
reaction of compound 1b was performed with BF3‚OEt2-Me2S
in dichloromethane yielding hydroxy compound 1c. In addition,
1b can be hydrolyzed with 10% NaOH solution to generate 1e
as a monomethanesulfonamido compound. Compound 1e was
treated with K2CO3 and substituted benzyl chloride/bromide in
acetone or CH3CN at room temperature or refluxed to obtain
2a-4a. Hydrolysis of 2a-4a generated 2b-4b. Methylation
of 1e and 2b-4b gave compounds 1f, 2c-4c, respectively. In
Scheme 2, 2-amino-5-nitrophenol was treated with K2CO3 and
substituted benzyl chloride/bromide in DMF to obtain com-
2. Chemistry
The synthesis of the target compounds was carried out in
Scheme 1 and Scheme 2, in which R represents substituted
benzyl moieties and X represents chloride or bromide. The

Synthesis of SelectiVe Aromatase Expression Regulators
Scheme 2. Synthesis of Compounds 5a-14c
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1637
(which produce aromatase expression from promoter I.1 medi-
ated by protein kinase C). Comparisons of bioactivity between
these two cell lines enable us to differentiate compounds that
can only suppress aromatase activity in breast cancer cells and
not suppress aromatase activity in placental cells. Those
compounds with differential effects on aromatase activity were
further investigated for their potential to suppress CYP19
transcription in breast cancer cells.
The cellular aromatase assays for these compounds were
performed in the choriocarcinoma placental JAR cell line and
the breast cancer SK-BR-3 cell line according to the modified
method of the procedure previously reported by our labora-
tory.21,22 These two cell lines, the SK-BR-3 and JAR cells, were
used because of their high cellular aromatase enzyme activity
and their different regulation of CYP19 expression.21 Other
human breast cancer cell lines express lower levels of aromatase
activity. If the compounds decrease aromatase activity ef-
fectively in JAR cells, they may decrease aromatase transcription
from promoter I.1 by interfering with protein kinase C pathway.
If the compounds decrease aromatase activity effectively in SK-
BR-3 breast cancer cells, these compounds may decrease
aromatase expression from promoter II and I.3 mediated by
cAMP. If the compounds decrease aromatase activity in both
cells lines, then several alternative mechanisms for these
compounds may be involved: (1) they may directly inhibit
aromatase enzyme, (2) they may increase aromatase degradation,
or (3) they may decrease aromatase mRNA stability. In addition,
the compounds were tested for their general cell cytotoxicity
and the ability to induce apoptosis. Compounds only targeting
aromatase expression in breast cancer cells and not affecting
other cell functions will be the ideal final target compounds.
pounds 5a-14a. Sodium hydride and methanesulfonyl chloride
were added and resulted in a mixture of N,N-bimethanesulfon-
amido and N-methanesulfonamido compounds. The mixture was
hydrolyzed with 10% NaOH solution to generate monomethane-
sulfonamido compounds 5b-14b, respectively. Methylation of
5b-14b gave compounds 5c-14c respectively.22 The structures
of all the synthesized compounds were confirmed by 1H NMR,
13C NMR, and HRMS, and the composition of key compounds
in the biological studies was also confirmed by elemental
analysis.
4. Results and Discussion
3. Biological Evaluation
The ability of the synthesized compounds to suppress
aromatase activity was performed in JAR cells (Figure 2).
Unexpectedly, most of the compounds tested exhibited aro-
matase suppression activity at 15 µM concentration. The results
indicate that the compounds may be involved in multiple
pathways to decrease aromatase activity. Most of the compounds
significantly decrease aromatase activity in SK-BR-3 cells as
well at 2.5 µM (Figure 3). Among all the compounds tested,
only the ones that do not affect aromatase activity in JAR cells
but decrease aromatase activity in SK-BR-3 breast cancer cells
might be selective aromatase expression regulators. In Figure
2, compounds 1f, 2c, 3c, 4c, 5b, 5c, 7b, 8c, 11b, 11c, 13b,
13c, 14b, and 14c at 15µM decrease aromatase activity less
than 50% in JAR cells. In Figure 3, compounds 1e, 1f, 2b, 2c,
3b, 3c, 4b, 4c, 5c, 6c, 7c, 8c, 9b, 9c, 10c, 12b, 12c, and 13b
decrease aromatase activity greater than 50% at 2.5µM in SK-
BR-3 cells. Compounds 1f, 2c, 3c, 4c, 5c, and 8c may be
potential selective aromatase expression regulators because they
do not decrease aromatase activity very effectively in JAR cells
at 15 µM but significantly suppress aromatase activity in SK-
BR-3 breast cancer cells at 2.5 µM.
To further investigate the six compounds, dose-response
studies on aromatase activity were performed in SK-BR-3 cells.
All six compounds exhibited dose-response of suppression
aromatase activity (Figure 4), and the corresponding IC50 values
are listed in Table 1. The IC50 of nimesulide is listed as well.
The results of the cellular aromatase assay exhibited that
extending one carbon at the 2-position of nimesulide results in
a significant increase in suppression of aromatase in breast
cancer cells compared with nimesulide. Compound 4c, which
is the most bulky compound, showed the best IC50 value with
Aromatase in the endoplasmic reticulum of cells catalyzes
the biosynthesis of estrogen. The regulation of aromatase is
complex and different in various tissues, and several tissue-
specific promoter regions have been identified upstream from
the CYP19 gene. These tissue-specific promoters include
promoter PI.1, PI.3, PI.4, PI.6, PI.7, and PII. Promoter PI.1 is
the major promoter used in placental tissues, and the PII and
I.3 promoters are used in the ovary and in breast cancer tissues.
Due to the unique organization of tissue-specific promoters,
various promoters employ different signaling pathways and
different transcription factors. This use of tissue-specific
promoters allows for the development of possible selective
aromatase expression regulators.4 Promoter I.1 (mainly used in
placenta) is regulated through a protein kinase C-mediated
mechanism.14,24 Studies from several research groups indicated
that promoters II and I.3, which are adenosine 3′,5′-cyclic
monophosphate (cAMP)-regulated promoters, are the major
promoters driving aromatase expression in breast cancer and
surrounding adipose stromal cells. In addition, alternate exon
PII and 1.3 were also found to be the major exons in aromatase
transcripts in four breast cancer cell lines (MCF-7, T-47D, SK-
BR-3, and MDA-MB-231 cells).15 Therefore, the major pro-
moter used in breast tumors and the four breast cancer cells
(i.e., cAMP-stimulated promoters I.3 and II) is different from
that placental tissue (i.e., protein kinase C-mediated promoter
I.1).
In the current research, evaluations of the synthetic com-
pounds were performed in both SK-BR-3 breast cancer cells
(which produce aromatase expression from promoter II and I.3
mediated by cAMP) and JAR choriocarcinoma placental cells

1638 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Su et al.
Figure 2. Aromatase activity in JAR cells treated with novel sulfonanilide. JAR cells were treated with indicated compounds at 15 µM, and
aromatase activity was measured as described in the Experimental Section. The results were normalized against a control treatment with vehicle,
and the value of 100% is equal to 4.0 pmol/h/106 cells. Each data bar represents the mean results of three independent determinations.
Figure 3. Aromatase activity in SK-BR-3 cells treated with novel
sulfonanilide. SK-BR-3 cells were treated with indicated compounds
Figure 5. Real-time RT-PCR analysis of CYP19 mRNA expression
at 2.5 µM, and aromatase activity was measured as described in the
in SK-BR-3 cells treated by novel sulfonanilide. Cells were treated for
Experimental Section. The results were normalized against a control
24 h with the indicated agents at 5µM, and total RNA was isolated.
treatment with vehicle, and the value of 100% is equal to 0.03 pmol/
Results are expressed as means of CYP19 (normalized to 18S rRNA)
h/106 cells. Each data bar represents the mean results of three
( SEM., *P < 0.05 vs control by unpaired t test (n ) 9).
independent determinations.
expression. This result suggests that 12b decreases aromatase
activity through a posttranscriptional mechanism. In addition,
some researchers found that aromatase was also regulated via
phosphorylation processes.25,26 Possible mechanisms by which
an agent only decreases aromatase enzyme activity without
suppression of its gene expression are direct enzyme inhibition,
increased enzyme degradation, or alterations in posttranslational
modifications.
Fortunately, all six compounds, 1f, 2c, 3c, 4c, 5c, and 8c,
did not cause significant cell cytotoxicity in SK-BR-3 breast
cancer cells at higher concentrations (10 µM) than the effective
dose for suppression of aromatase expression (Figure 6).
However, some of them induce some cytotoxicity at higher
concentrations (30 µM). The compounds do not produce
Figure 4. Dose-response suppression of aromatase activity in SK-
BR-3 cells by novel sulfonanilide. SK-BR-3 cells were treated with 2c
apoptotic effects in SK-BR-3 cells at 5 µM, which is effective
dose for suppressing aromatase expression (Figure 7). These
two results show that, at low micromolar concentrations, the
six compounds did not cause any cytotoxicity and apoptotic
effects.
(9), 1f (2), 8c (1), 4c ((), 5c (b), and 3c (0) and aromatase activity
was measured as described in the Experimental Section. The results
were normalized against a control treatment with vehicle, with the value
of 100% equal to 0.03 pmol/h/106 cells. Each data point represents the
mean results of three independent determinations, and the data were
statistically analyzed by a nonlinear regression analysis method.
In summary, the biological results indicated that several agents
selectively decreased aromatase activity and enzyme gene
expression at low micromolar concentrations in SK-BR-3 breast
cancer cells. These compounds were 10- to 80-fold more active
than nimesulide and did not affect the aromatase activity in
choriocarcinoma placental JAR cells. Furthermore, this sup-
pression of aromatase activity occurs at the transcriptional level.
Several potential lead compounds were developed in this study
as selective aromatase expression regulators by structural
modifications of the COX-2 inhibitor nimesulide. Because of
the limited amount of effective compounds, it is difficult to draw
an 80-fold increase. Furthermore, real time PCR demonstrated
that compounds 1f, 2c, 3c, 4c, and 8c at 5 µM significantly
decreased CYP19 gene expression in SK-BR-3 cells (Figure 5).
This suggests that the suppression of aromatase enzyme activity
occurs at the transcriptional level. Compound 12b was chosen
as a negative control because it inhibited aromatase activity in
both JAR cells and SK-BR-3 cells. Real time RT-PCR assay
of CYP19 demonstrated that compound 12b might only decrease
aromatase enzyme activity but not affect the CYP19 gene

Synthesis of SelectiVe Aromatase Expression Regulators
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1639
Table 1. Suppression of Aromatase Activity in SK-BR-3 Breast Cancer Cells
compd
chemical description
N-(2-phenoxy-4-nitrophenyl)methanesulfonamide
IC50 (µM)a
27.0 ( 4.70b
0.81 ( 0.29
0.49 ( 0.14
2.68 ( 0.91
0.33 ( 0.15
2.33 ( 0.66
1.78 ( 0.63
nimesulide
1f
N-methyl-N-(2-benzyloxy-4-nitrophenyl) methanesulfonamide
2c
3c
4c
5c
8c
N-methyl-N-[2-(4′-nitrobenzyloxy)-4-nitrophenyl]methanesulfonamide
N-methyl-N-[2-(â-naphthylmethoxy) 4-nitrophenyl]methanesulfonamide
N-methyl-N-[2-(2′-phenylbenzyloxy)-4-nitrophenyl]methanesulfonamide
N-methyl-N-[2-(4′-methylbenzyloxy)-4-nitrophenyl]methanesulfonamide
N-methyl-N-[2-(4′-fluorobenzyloxy)-4-nitrophenyl]methanesulfonamide
a IC50 values were calculated by a nonlinear regression analysis (GraphPad Prism). Each dose-response curve contained six concentrations, each in
triplicate. b See ref 21.
the NMR spectra were recorded on a Bruker DPX 250 and DRX
400 MHz in either DMSO-d6 or CDCl3. Chemical shifts (δ) for 1H
NMR spectra are reported in parts per million to residual solvent
protons. Chemical shifts (δ) for 13C NMR spectra are reported in
parts per million relative to residual solvent carbons.
2-Benzyloxy-4-nitroaniline (1a). K2CO3 (9.0 g, 65.1 mmol) and
benzyl bromide (5.7 g, 33.1 mmol) were successively added to a
solution of 2-amino-5-nitrophenol (5.0 g, 32.5 mmol) in acetone
(250 mL), and the mixture was refluxed for 8 h. After being cooled,
the inorganic precipitate was filtered through a Celite pad, and the
filtrate was concentrated in Vacuo. The resulting solid was
recrystallized from AcOEt to afford 1a (7.2 g, 91%) as yellow
1
needles: mp 148-149 °C; H NMR (400 MHz, CDCl3) δ 7.84
(1H, dd, J ) 8.7, 2.4 Hz), 7.80 (1H, d, J ) 2.4 Hz), 7.38 (5H, m),
6.69 (1H, d, J ) 8.7 Hz), 4.73 (2H, br); 13C NMR (100 MHz,
CDCl3) δ 145.20, 136.07, 129.19, 128.98, 128.33, 119.72, 112.86,
107.67, 71.32.
Figure 6. Cell cytotoxicity in SK-BR-3 cells treated with novel
sulfonanilide. SK-BR-3 cells were treated with indicated compounds
at different concentrations, and cell viability was measured by MTT
assay as described in the Experimental Section.
N,N-(2-Benzyloxy-4-nitrophenyl)dimethanesulfonamide (1b).
NaH (95%, 3.03 g, 120 mmol) was added to a solution of 1a (7.33
g, 30 mmol) in anhydrous DMF (80 mL) at room temperature. After
the mixture was stirred at the same temperature for 30 min, MsCl
(10.31 g, 90 mmol) was added slowly and the stirring was continued
overnight at room temperature. H2O (150 mL) and saturated Na2-
CO3 (20 mL) were added to the mixture, and the precipitated solid
was collected by filtration and washed with H2O and a cold ether/
hexane mixture to afford 1b as yellow powder (11.62 g, 97%):
mp 188-189 °C; 1H NMR (400 MHz, DMSO-d6) δ8.04 (1H, d, J
) 1.7 Hz), 7.89 (2H, m), 7.53 (2H, d, J ) 7.7 Hz), 7.38 (3H, m),
5.41 (2H, s), 3.46 (6H, s); 13C NMR (100 MHz, DMSO-d6) δ
157.42, 150.16, 136.25, 134.69, 129.40, 129.38, 129.17, 128.57,
116.70, 109.42, 71.80, 44.59.
N,N-(2-Hydroxy-4-nitrophenyl)dimethanesulfonamide (1c).
To a stirred suspension of 1b (800.7 mg, 2 mmol) in Me2S (15
mL) and CH2Cl2 (15 mL) was slowly added BF3‚OEt2 (5.07 mL,
40 mmol) at room temperature. The resulting yellow solution was
vigorously stirred at room-temperature overnight. After being cooled
to 0 °C, the reaction mixture was quenched with water and
concentrated under reduced pressure. The insoluble product was
collected by filtration and washed with H2O and CHCl3 to give a
Figure 7. Cell apoptosis in SK-BR-3 cells treated with novel
sulfonanilide. SK-BR-3 cells were treated with indicated compounds
at different concentrations, and apoptotic cells were measured as
described in the Experimental Section.
extensive structure-activity relationships for the compounds.
However, these initial findings on the sulfonanilide analogues
will enable further lead optimization for novel drug discovery
and examination of structural requirements for the selective
regulation of aromatase expression. Our current research also
focuses on discerning the specific molecular target of the
compounds.
1
pale yellow solid (0.554 g, 89%): mp 249-253 °C (decomp); H
NMR (400 MHz, DMSO-d6) δ 11.65 (1H, s), 7.69 (3H, m), 3.56
(6H, s); 13C NMR (100 MHz, DMSO-d6) δ 157.29, 149.75, 134.54,
128.32, 114.83, 111.82, 44.74.
N-(2-Benzyloxy-4-nitrophenyl)methanesulfonamide (1e). Com-
pound 1c (0.4 g, 1 mmol) was added to a 3 N NaOH aq solution
and was stirred at 80-90 °C overnight. After cooling, it was
neutralized with 5 N HCl until pH 1-2. The precipitated solid was
collected and washed with H2O and cold ether to provide the desired
product, and then it was recrystallized from ethyl acetate/hexane
5. Experimental Section
1
5.1. Chemistry. Chemicals were commercially available and
used as received without further purification unless otherwise noted.
Moisture-sensitive reactions were carried out under a dry argon
atmosphere in flame-dried glassware. Solvents were distilled before
use under argon. Thin-layer chromatography was performed on
precoated silica gel F254 plates (Whatman). Silica gel column
chromatography was performed using silica gel 60A (Merck, 230-
400 mesh). High-resolution electrospray ionization mass spectra
were obtained on the Micromass QTOF Electrospray mass spec-
trometer at The Ohio State Chemical Instrumentation Center. All
to afford a pale yellow solid (0.30 g, 92%): mp 150-152 °C; H
NMR (400 MHz, CDCl3) δ 7.94 (1H, dd, J ) 8.9, 2.3 Hz), 7.91
(1H, d, J ) 2.3 Hz), 7.68 (1H, d, J ) 8.9 Hz), 7.43 (5H, m), 7.26
(1H, br), 5.23 (2H, s), 3.09 (3H, s); 13C NMR (100 MHz, CDCl3)
δ 147.12, 144.14, 134.74, 133.40, 129.63, 129.53, 128.65, 118.30,
116.96, 107.90, 72.30, 40.65; HRMS calculated for C14H14N2NaO5S
(M + Na)+ 345.0521, found 345.0531.
N-Methyl-N-(2-benzyloxy-4-nitrophenyl)methanesulfon-
amide (1f). Compound 1e (0.16 g, 0.5 mmol) was dissolved in 3
mL dry DMF, and NaH powder (15.2 mg 95%, 0.6 mmol, 1.2

1640 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Su et al.
equiv) was added. The mixture was stirred at room temperature
for 10 min, iodomethane (0.6 mmol, 1.2 equiv) was added, and
the stirring was maintained for 2 h at room temperature. Then the
mixture was taken up with 7 mL of water and 2 mL of Na2CO3 aq
solution. The precipitated solid was collected by filtration and
washed with water and cold ether to afford the desired product,
and then it was recrystallized from ethyl acetate/hexane to afford
a pale yellow solid (0.16 g, 96%): mp 138-140 °C; 1H NMR (400
MHz, CDCl3) δ 7.95 (1H, d, J ) 2.4 Hz), 7.89 (1H, dd, J ) 8.6,
2.5 Hz), 7.56 (1H, d, J ) 8.6 Hz), 7.43 (5H, m), 5.23 (2H, s), 3.28
(3H, s), 2.83 (3H, s); 13C NMR (100 MHz, CDCl3) δ 155.68,
148.41, 135.77, 134.97, 133.10, 129.52, 129.46, 128.60, 116.98,
108.48, 71.98, 38.91, 37.92; HRMS calculated for C15H16N2NaO5S
(M + Na)+ 359.0678, found 359.0663. Anal. (C15H16N2O5S) C,
H, N.
128.91, 128.67, 128.51, 127.40, 127.23, 127.06, 126.72, 119.89,
118.77, 109.17, 71.30, 41.54; HRMS calculated for C18H16N2NaO5S
(M + Na)+ 395.0678, found 395.0685.
N-[2-(2′-Phenylbenzyloxy)-4-nitrophenyl]methanesulfona-
mide (4b). It is stirred in 3 N NaOH aq solution at 85-90 °C
1
overnight. Yellow solid, 94%: mp 116-119 °C; H NMR (400
MHz, DMSO-d6) δ 7.65 (2H, dd, J ) 9.1, 3.0 Hz), 7.51 (2H, d, J
) 7.0 Hz), 7.31 (7H, m), 7.19 (1H, d, J ) 9.2 Hz), 4.93 (2H, s),
2.73 (3H, s); 13C NMR (100 MHz, DMSO-d6) δ 149.33, 142.49,
140.87, 135.02, 130.63, 130.52, 130.02, 129.10, 129.00, 128.33,
128.21, 120.32, 116.59, 109.09, 69.14, 41.27; HRMS calculated
for C20H18N2NaO5S (M + Na)+ 421.0834, found 421.0840.
N-Methyl-N-[2-(4′-nitrobenzyloxy)-4-nitrophenyl]methane-
sulfonamide (2c). Compound 2b (0.11 g, 0.3 mmol) was dissolved
in 4 mL of dry DMF, and K2CO3 powder (0.083 g, 0.6 mmol) was
added. The mixture was stirred at room temperature for 10 min,
iodomethane (0.085 g, 0.6 mmol) was added, and then it was stirred
at 45-50 °C overnight. Then the mixture was taken up with 7 mL
of water and 2 mL of Na2CO3 aq solution. The precipitated solid
was collected by filtration and washed with water and cold ether
to afford a white solid, 108 mg (94%): mp 209-211 °C; 1H NMR
(400 MHz, DMSO-d6) δ 8.29 (2H, d, J ) 8.7 Hz), 8.00 (1H, d, J
) 2.3 Hz), 7.89(1H, dd, J ) 8.7, 2.4 Hz), 7.80 (2H, d, J ) 8.6
Hz), 7.64 (1H, d, J ) 8.7 Hz), 5.53 (2H, s), 3.22 (3H, s), 3.05 (3H,
s); 13C NMR (100 MHz, DMSO-d6) δ 155.92, 148.23, 148.12,
144.44, 136.65, 132.64, 129.43, 124.65, 117.12, 109.49, 70.33,
39.17, 38.12. HRMS calculated for C15H15N3NaO7S (M + Na)+
404.0528, found 404.0501. Anal. (C15H15N3O7S) C, H, N.
General Procedure for the Preparation of 3c-14c. Methane-
sulfonamide compound (0.5 mmol) was dissolved in 3 mL of dry
DMF, and NaH powder (15.2 mg 95%, 0.6 mmol, 1.2 equiv) was
added. The mixture was stirred at room temperature for 10 min,
iodomethane (0.6 mmol, 1.2 equiv) was added, and stirring was
maintained for 2 h at room temperature. Then the mixture was taken
up with 7 mL of water and 2 mL of Na2CO3 aq solution. The
precipitated solid was collected by filtration, washed with water
and cold ether to afford the desired product, and then recrystallized
from ethyl acetate/hexane.
General Procedure for the Preparation of 2a-4a. K2CO3 (0.69
g, 5 mmol) and aryl halide (5 mmol, 1.0 equiv) were successively
added to a solution of compound 1c (0.77 g, 5 mmol) in CH3CN
or acetone, and the mixture was stirred at room temperature or
refluxed from 3 h to overnight. After the mixture was cooled, 20
mL of H2O and 5 mL of saturated aqueous Na2CO3 were added
and the precipitated solid was collected by filtration and washed
with H2O and cold ethyl ether to afford the desired compounds.
N,N-[2-(4′-Nitrobenzyloxy)-4-nitrophenyl]dimethanesulfona-
mide (2a). It is stirred and refluxed in acetone for 2 h. White
1
powder, 89%: mp 265-269 °C (decomp); H NMR (400 MHz,
DMSO-d6) δ 8.28 (2H, d, J ) 8.8 Hz), 8.05 (1H, d, J ) 2.3 Hz),
7.93 (1H, dd, J ) 8.6, 2.3 Hz), 7.89 (2H, d, J ) 8.6 Hz), 7.80 (2H,
d, J ) 8.8 Hz), 5.59 (2H, s), 3.50 (6H, s); 13C NMR (100 MHz,
DMSO-d6) δ 157.10, 150.20, 148.13, 144.10, 135.00, 129.29,
129.21, 124.55, 117.06, 109.48, 70.58, 44.61.
N,N-[2-(â-Naphthylmethoxy)-4-nitrophenyl]dimethanesulfona-
mide (3a). It is stirred in CH3CN at room temperature overnight.
White powder, 98%: mp 184-185 °C; 1H NMR (400 MHz, CDCl3)
δ 8.05 (1H, d, J ) 2.4 Hz), 8.04 (1H, s), 7.95 (2H, dd, J ) 8.5, 2.3
Hz), 7.88 (2H, m), 7.64 (1H, dd, J ) 8.5, 1.7 Hz), 7.52 (3H, m),
5.44 (2H, s), 3.37 (6H, s); 13C NMR (100 MHz, CDCl3) δ 157.35,
150.21, 133.77, 133.57, 133.47, 132.00, 129.24, 128.55, 128.22,
127.69, 127.11, 127.02, 125.74, 116.88, 109.14, 72.52, 44.29.
N,N-[2-(2′-Phenylbenzyloxy)-4-nitrophenyl]dimethanesulfona-
mide (4a). It is stirred in CH3CN at room temperature overnight.
White powder, 91%: mp 182-184 °C; 1H NMR (400 MHz, CDCl3)
δ 7.88 (1H, dd, J ) 8.6, 2.4 Hz), 7.68 (2H, m), 7.40 (9H, m), 5.19
(2H, s), 3.38 (6H, s); 13C NMR (100 MHz, CDCl3) δ 157.12,
150.10, 142.79, 140.10, 133.15, 131.84, 130.81, 130.20, 129.59,
129.49, 129.08, 129.03, 128.33, 128.22, 116.68, 109.15, 70.31,
44.24.
N-Methyl-N-[2-(â-naphthylmethoxy)-4-nitrophenyl]methane-
1
sulfonamide (3c). Pale yellow solid, 96%: mp 119-121 °C; H
NMR (400 MHz, CDCl3) δ 8.02 (1H, d, J ) 2.4 Hz), 7.96 (1H, s),
7.89 (4H, m), 7.55 (4H, m), 5.39 (2H, s), 3.30 (3H, s), 2.84 (3H,
s); 13C NMR (100 MHz, CDCl3) δ 155.73, 148.43, 135.84, 133.79,
133.58, 133.06, 132.30, 129.44, 128.45, 128.27, 128.08, 127.29,
127.23, 125.80, 117.03, 108.52, 72.16, 38.98, 37.96. HRMS
calculated for C19H18N2NaO5S (M + Na)+ 409.0834, found
409.0825. Anal. (C19H18N2O5S) C, H, N.
General Procedure for the Preparation of 2b-4b. Compound
2a-4a was added to a 3 N NaOH aq solution or CH3CN/K2CO3
suspension and was stirred at 80-90 °C from 24 to 48 h. After
cooling, H2O and saturated Na2CO3 were added, the mixture was
stirred for 30 min, and then the insoluble solid was filtered off.
The mother liquid was neutralized with 5N HCl until pH 1-2. The
precipitated solid was collected and washed with H2O and cold
ether to provide the desired product.
N-Methyl-N-[2-(2′-phenylbenzyloxy)-4-nitrophenyl]methane-
sulfonamide (4c). Pale yellow solid, 82%: mp 128-129 °C; H
1
NMR (400 MHz, CDCl3) δ 7.84 (1H, dd, J ) 8.6, 2.4 Hz), 7.66
(1H, d, J ) 2.4 Hz), 7.37 (10H, m), 5.19 (2H, s), 3.24 (3H, s),
2.83 (3H, s); 13C NMR (100 MHz, CDCl3) δ 155.51, 148.34,
142.74, 140.20, 132.81, 132.31, 131.05, 129.88, 129.55, 129.41,
129.04, 128.38, 128.24, 116.88, 108.59, 69.76, 38.94, 38.00. HRMS
calculated for C21H20N2NaO5S (M + Na)+ 435.0991, found
435.0996. Anal. (C21H20N2O5S) C, H, N.
N-[2-(4′-Nitrobenzyloxy)-4-nitrophenyl]methanesulfon-
amide (2b). It is stirred and refluxed in CH3CN for 48 h. Yellow
General Procedure for the Preparation of 5a-14a. K2CO3
(0.69 g, 5 mmol) and aryl halide (5 mmol, 1.0 equiv) were
successively added to a solution of 2-amino-5-nitrophenol (0.77 g,
5 mmol) in DMF or acetone (10 mL), and the mixture was stirred
at room temperature or 75-80 °C from 3 h to overnight. After the
mixture was cooled, 20 mL of H2O and 5 mL of saturated aqueous
Na2CO3 were added to the mixture and the precipitated solid was
collected by filtration and washed with H2O and cold ethyl ether.
If the product precipitated as an oil, the aqueous phase was extracted
with CH2Cl2. The organic solution was washed with saturated
aqueous Na2CO3 solution and H2O, dried over anhydrous MgSO4,
and concentrated. The residue was chromatographed on silica gel
[AcOEt-hexane (1:5)] to afford desired compounds.
1
powder, 74%: mp 168-169 °C; H NMR (400 MHz, CDCl3) δ
8.32 (2H, d, J ) 8.7 Hz), 7.98 (1H, dd, J ) 8.9, 2.3 Hz), 7.86 (1H,
d, J ) 2.3 Hz), 7.71 (1H, d, J ) 8.9 Hz), 7.63 (1H, d, J ) 8.8 Hz),
5.35 (2H, s), 3.16 (3H, s); 13C NMR (100 MHz, CDCl3) 148.73,
146.40, 143.96, 141.70, 133.38, 128.97, 124.77, 118.88, 116.90,
107.70, 70.74, 40.95; HRMS calculated for C14H13N3NaO7S (M +
Na)+ 390.0372, found 390.0364.
N-[2-(â-Naphthylmethoxy)-4-nitrophenyl]methanesulfona-
mide (3b). It is stirred in 3 N NaOH aq solution at 85-90 °C
overnight. Pale yellow solid, 95%: mp 239-243 °C (decomp); 1H
NMR (400 MHz, DMSO-d6) δ 8.07 (1H, s), 7.92 (3H, m), 7.83
(1H, s), 7.80 (1H, d, J ) 2.5 Hz), 7.68 (1H, dd, J ) 8.4, 1.5 Hz),
7.53 (2H, m), 7.45 (1H, J ) 8.4 Hz), 5.44 (2H, s), 3.01 (3H, s);
13C NMR (100 MHz, DMSO-d6) δ 149.72, 135.00, 133.60, 133.48,
2-(4′-Methylbenzyloxy)-4-nitroaniline (5a). 4-Methylbenzyl
chloride was used, and it was stirred in DMF at 75-80 °C for 3 h.

Synthesis of SelectiVe Aromatase Expression Regulators
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1641
Yellow solid, 85%: mp 149-150 °C; 1H NMR (400 MHz, CDCl3)
δ 7.83 (1H, dd, J ) 8.7, 2.3 Hz), 7.79 (1H, d, J ) 2.3 Hz), 7.35
(2H, d, J ) 8.0 Hz), 7.24 (2H, d, J ) 7.8 Hz), 6.66 (1H, d, J ) 8.7
Hz), 5.12 (2H, s), 4.61(2H, s), 2.41 (3H, s); 13C NMR (100 MHz,
CDCl3) δ 144.97, 143.93, 138.97, 138.87, 133.11, 129.86, 128.49,
119.75, 112.31, 107.63, 71.23, 21.67.
129.57, 129.32, 127.41, 127.35, 126.88, 126.27, 124.81, 120.78,
112.06, 108.25, 69.17.
2-(3′,6′-Dimethylbenzyloxy)-4-nitroaniline (14a). 3,6-Dimeth-
ylbenzyl chloride was used, and it was stirred in DMF at 75-80
1
°C for 3h. Yellow solid, 93%: mp 160-161 °C; H NMR (400
MHz, DMSO-d6) δ 7.75 (1H, dd, J ) 8.7, 2.2 Hz), 7.73 (1H, d, J
) 2.3 Hz), 7.32 (1H, s), 7.11 (1H, d, J ) 7.7 Hz), 7.07 (1H, d, J
) 7.7 Hz), 6.70 (1H, dd, J ) 8.7, 1.0 Hz), 6.39 (2H, br), 5.15(2H,
s), 2.30 (3H, s), 2.29 (3H, s); 13C NMR (100 MHz, DMSO-d6) δ
147.05, 144.37, 136.36, 135.57, 135.00, 134.41, 130.94, 130.14,
129.54, 120.68, 112.00, 107.83, 69.45, 21.44, 18.93.
2-(4′-Methoxylbenzyloxy)-4-nitroaniline (6a). 4-Methoxylben-
zyl chloride was used, and it was stirred in DMF at 75-80 °C for
1
3 h. Yellow solid, 89%: mp 131-132 °C; H NMR (400 MHz,
CDCl3) δ 7.83 (1H, dd, J ) 8.7, 2.3 Hz), 7.79 (1H, d, J ) 2.3 Hz),
7.38 (2H, d, J ) 8.7 Hz), 6.95 (2H, d, J ) 8.7 Hz), 6.65 (1H, d,
J ) 8.7 Hz), 5.09 (2H, s), 4.60(2H, s), 3.85 (3H, s); 13C NMR
(100 MHz, CDCl3) δ 160.25, 144.96, 143.94, 138.96, 130.14,
128.17, 119.74, 114.54, 112.30, 107.65, 71.10, 55.75.
General Procedure for the Preparation of Compounds 5b-
14b. NaH (95% powder, 0.265 g, 10.5 mmol, 3.5 equiv) was added
to a solution of aryl-substituted 2-amino-5-nitrophenol (3.0 mmol)
in anhydrous DMF (8 mL) at room temperature. After the mixture
was stirred at the same temperature for 30 min, MsCl (1.031 g, 9.0
mmol, 3 equiv) was added and the stirring was continued overnight
at room temperature. H2O was added to the mixture, and then it
was neutralized with 5 N HCl until pH 1-2. The intermediate
precipitated as a yellow solid. It was collected by filtration, washed
with H2O, and then used without further purification in the next
reaction. The intermediate was added to a 3 N NaOH aq solution
and was stirred at 80-90 °C overnight. After cooling, it was
neutralized with 5 N HCl until pH 1-2. The precipitated solid was
collected and washed with H2O and cold ether to provide the desired
product, and then it was recrystallized from ethyl acetate/hexane.
N-[2-(4′-Methylbenzyloxy)-4-nitrophenyl]methanesulfona-
2-(4′-Isopropylbenzyloxy)-4-nitroaniline (7a). 4-Isopropylben-
zyl chloride was used, and it was stirred in DMF at 75-80 °C for
1
3 h. Yellow solid, 91%: mp 99-101 °C; H NMR (400 MHz,
CDCl3) δ 7.84 (1H, dd, J ) 8.7, 2.4 Hz), 7.80 (1H, d, J ) 2.4 Hz),
7.39 (2H, d, J ) 8.1 Hz), 7.30 (2H, d, J ) 8.0 Hz), 6.66 (1H, d,
J ) 8.7 Hz), 5.13 (2H, s), 4.60(2H, s), 2.95 (1H, m), 1.29 (6H, d,
J ) 6.9 Hz); 13C NMR (100 MHz, CDCl3) δ 149.84, 145.04,
143.88, 133.48, 128.57, 127.25, 119.74, 112.30, 107.54, 71.23,
34.34, 24.38.
2-(4′-Fluorobenzyloxy)-4-nitroaniline (8a). 4-Fluorobenzyl chlo-
ride was used, and it was stirred in DMF at room temperature
1
overnight. Yellow solid, 98%: mp 125-127 °C; H NMR (400
MHz, CDCl3) δ 7.84 (1H, dd, J ) 8.7, 2.3 Hz), 7.78 (1H, d, J )
2.3 Hz), 7.43 (2H, m), 7.08 (2H, m), 6.73 (1H, d, J ) 8.7 Hz),
5.15 (2H, s), 5.05 (2H, br); 13C NMR (100 MHz, CDCl3) δ 164.43,
161.97, 145.28, 131.83, 130.32, 130.24, 119.70, 116.26, 116.04,
113.38, 107.69, 70.66.
1
mide (5b). Pale yellow solid, 87%: mp 151-152 °C; H NMR
(400 MHz, CDCl3) δ 7.93 (1H, dd, J ) 8.9, 2.4 Hz), 7.91 (1H, d,
J ) 2.3 Hz), 7.67 (1H, d, J ) 8.8 Hz), 7.32 (2H, d, J ) 8.0 Hz),
7.25 (2H, d, J ) 7.9 Hz), 5.19 (2H, s), 3.08 (3H, s), 2.41 (3H, s);
13C NMR (100 MHz, CDCl3) δ 147.20, 144.15, 139.64, 133.42,
131.73, 130.17, 128.77, 118.19, 116.96, 107.91, 72.25, 40.59, 21.68;
HRMS calculated for C15H16N2NaO5S (M + Na)+ 359.0678, found
359.0670.
2-(4′-Chlorobenzyloxy)-4-nitroaniline (9a). 4-Chlorobenzyl
chloride was used, and it was stirred in DMF at room temperature
1
overnight. Yellow solid, 90%: mp 128-130 °C; H NMR (400
MHz, CDCl3) δ 7.84 (1H, dd, J ) 8.7, 2.3 Hz), 7.75 (1H, d, J )
2.3 Hz), 7.41 (4H, s), 6.68 (1H, d, J ) 8.7 Hz), 5.13 (2H, s), 4.61
(2H, s); 13C NMR (100 MHz, CDCl3) δ 144.64, 143.84, 138.95,
134.86, 134.60, 129.64, 129.41, 119.97, 112.50, 107.64, 70.49.
N-[2-(4′-Methoxylbenzyloxy)-4-nitrophenyl]methanesulfona-
1
mide (6b). Pale yellow solid, 86%: mp 150-152 °C; H NMR
(400 MHz, CDCl3) δ 7.93 (1H, dd, J ) 8.8, 2.4 Hz), 7.91 (1H, d,
J ) 2.2 Hz), 7.67 (1H, d, J ) 8.8 Hz), 7.36 (2H, d, J ) 8.6 Hz),
6.97 (2H, d, J ) 8.6 Hz), 5.16 (2H, s), 3.86 (3H, s), 3.08 (3H, s);
13C NMR (100 MHz, CDCl3) δ 160.72, 147.19, 144.13, 133.42,
130.50, 126.73, 118.16, 116.91, 114.87, 107.90, 72.12, 55.80, 40.60;
HRMS calculated for C15H16N2NaO6S (M + Na)+ 375.0627, found
375.0625.
2-(4′-Bromobenzyloxy)-4-nitroaniline (10a). 4-Bromobenzyl
bromide was used, and it was stirred in DMF at room temperature
1
overnight. Yellow solid, 66%: mp 131-133 °C; H NMR (400
MHz, CDCl3) δ 7.83 (1H, dd, J ) 8.7, 2.3 Hz), 7.75 (1H, d, J )
2.3 Hz), 7.55 (2H, d, J ) 8.4 Hz), 7.33 (2H, d, J ) 8.5 Hz), 6.67
(1H, d, J ) 8.7 Hz), 5.12 (2H, s), 4.62 (2H, s); 13C NMR (100
MHz, CDCl3) δ 144.62, 143.84, 138.94, 135.12, 132.36, 129.92,
129.09, 122.99, 119.98, 112.51, 107.63, 70.51.
N-[2-(4′-Isopropylbenzyloxy)-4-nitrophenyl]methanesulfona-
1
mide (7b). Pale yellow solid, 86%: mp 171-172 °C; H NMR
(400 MHz, CDCl3) δ 7.95 (1H, dd, J ) 8.8, 2.5 Hz), 7.92 (1H, d,
J ) 2.4 Hz), 7.68 (1H, d, J ) 8.8 Hz), 7.35 (2H, d, J ) 8.2 Hz),
7.31 (2H, d, J ) 8.3 Hz), 7.25 (1H, br), 5.18 (2H, s), 3.08 (3H, s),
2.96 (1H, m), 1.30 (6H, d, J ) 6.9 Hz); 13C NMR (100 MHz,
CDCl3) δ 150.63, 147.25, 144.18, 133.39, 132.02, 128.97, 127.60,
118.20, 116.92, 107.84, 72.30, 40.58, 34.39, 24.33; HRMS
calculated for C17H20N2NaO5S (M + Na)+ 387.0991, found
387.0981.
2-(4′-Phenylbenzyloxy)-4-nitroaniline (11a). 4-Phenylbenzyl
chloride was used, and it was stirred in DMF at room temperature
for 48 h. Yellow solid, 82%: mp 151-153 °C; 1H NMR (400 MHz,
CDCl3) δ 7.84 (1H, dd, J ) 8.7, 2.3 Hz), 7.82 (1H, d, J ) 2.3 Hz),
7.61 (4H, m), 7.54 (2H, d, J ) 8.4 Hz), 7.46 (2H, m), 7.40 (1H,
m), 6.74 (1H, d, J ) 8.6 Hz), 5.21 (2H, s), 5.01 (2H, br); 13C NMR
(100 MHz, CDCl3) δ 145.33, 141.94, 140.88, 139.56, 134.99,
129.28, 128.84, 128.53, 128.01, 127.90, 119.69, 113.11, 107.70,
71.09.
N-[2-(4′-Fluorobenzyloxy)-4-nitrophenyl]methanesulfona-
1
mide (8b). Yellow solid, 83%: mp 162-164 °C; H NMR (400
2-Phenylethoxy-4-nitroaniline (12a). Phenylethyl bromide was
MHz, CDCl3) δ 7.95 (1H, dd, J ) 8.9, 2.4 Hz), 7.89 (1H, d, J )
2.4 Hz), 7.68 (1H, d, J ) 8.9 Hz), 7.41 (2H, m), 7.25 (1H, br),
7.13 (2H, m), 5.19 (2H, s), 3.11 (3H, s); 13C NMR (100 MHz,
CDCl3) δ 164.79, 162.32, 146.93, 144.06, 133.38, 130.76, 130.68,
130.60, 130.56, 118.38, 116.86, 116.67, 116.46, 107.76, 71.54,
40.71; HRMS calculated for C14H13FN2NaO5S (M + Na)+ 363.0427,
found 363.0417.
used, and it was refluxed in DMF for 48 h. Yellow solid, 28%:
1
mp 83-85 °C; H NMR (400 MHz, CDCl3) δ 7.81 (1H, dd, J )
8.7, 2.4 Hz), 7.68 (1H, d, J ) 2.4 Hz), 7.29 (5H, m), 6.62 (1H, d,
J ) 8.7 Hz), 4.48 (2H, br), 4.31 (2H, dd, J ) 6.8, 6.8 Hz), 3.17
(2H, dd, J ) 6.7, 6.7 Hz); 13C NMR (100 MHz, CDCl3) δ 144.92,
143.82, 139.04, 138.12, 129.30, 129.08, 127.18, 119.65, 112.30,
107.39, 69.74, 35.96.
N-[2-(4′-Chlorobenzyloxy)-4-nitrophenyl]methanesulfona-
1
2-(R-Naphthylmethoxy)-4-nitroaniline (13a). R-Naphthyl-
mide (9b). Pale yellow solid, 90%: mp 171-173 °C; H NMR
methyl chloride was used, and it was stirred in DMF at room
(400 MHz, CDCl3) δ 7.94 (1H, dd, J ) 8.9, 2.4 Hz), 7.87 (1H, d,
J ) 2.3 Hz), 7.68 (1H, d, J ) 8.9 Hz), 7.43(2H, d, J ) 8.5 Hz),
7.37(2H, d, J ) 8.5 Hz), 5.20 (2H, s), 3.11 (3H, s); 13C NMR (100
MHz, CDCl3) δ 146.85, 144.04, 135.62, 133.38, 133.19, 130.01,
129.76, 118.46, 116.89, 107.77, 71.43, 40.74; HRMS calculated
for C14H13ClN2NaO5S (M + Na)+ 379.0131, found 379.0136.
1
temperature overnight. Yellow solid, 98%: mp 197-199 °C; H
NMR (400 MHz, DMSO-d6) δ 8.19 (1H, d, J ) 8.0 Hz), 7.94 (2H,
m), 7.90 (1H, d, J ) 2.4 Hz), 7.76 (2H, m), 7.52 (3H, m), 6.70
(1H, d, J ) 8.9 Hz), 6.39 (2H, br), 5.70 (2H, s); 13C NMR (100
MHz, DMSO-d6) δ 147.08, 144.24, 136.39, 134.15, 132.85, 131.91,

1642 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
N-[2-(4′-Bromobenzyloxy)-4-nitrophenyl]methanesulfona-
Su et al.
116.87, 108.48, 71.87, 38.89, 37.93, 34.32, 24.32; HRMS calculated
for C18H22N2NaO5S (M + Na)+ 401.1147, found 401.1143.
N-Methyl-N-[2-(4′-fluorobenzyloxy)-4-nitrophenyl]methane-
1
mide (10b). Pale yellow solid, 82%: mp 183-185 °C; H NMR
(400 MHz, CDCl3) δ 7.94 (1H, dd, J ) 8.9, 2.4 Hz), 7.87 (1H, d,
J ) 2.3 Hz), 7.68 (1H, d, J ) 8.9 Hz), 7.59 (2H, d, J ) 8.4 Hz),
7.31(2H, d, J ) 8.4 Hz), 5.18 (2H, s), 3.11 (3H, s); 13C NMR (100
MHz, CDCl3) δ 146.81, 144.04, 133.69, 133.38, 132.73, 130.25,
123.78, 118.48, 116.89, 107.78, 71.47, 40.75; HRMS calculated
for C14H13BrN2NaO5S (M + Na)+ 422.9626, found 422.9636.
1
sulfonamide (8c). Pale yellow solid, 92%: mp 168-169 °C; H
NMR (400 MHz, CDCl3) δ 7.94 (1H, d, J ) 2.4 Hz), 7.90 (1H,
dd, J ) 8.4, 2.4 Hz), 7.55 (1H, d, J ) 8.6 Hz), 7.44 (2H, m), 7.13
(2H, m), 5.20 (2H, s), 3.27 (3H, s), 2.84 (3H, s); 13C NMR (100
MHz, CDCl3) δ 164.67, 162.20, 155.57, 148.36, 135.86, 132.87,
130.89, 130.86, 130.63, 130.55, 117.10, 116.62, 116.41, 108.46,
71.24, 38.97, 37.93; HRMS calculated for C15H15FN2NaO5S (M
+ Na)+ 377.0583, found 377.0586. Anal. (C15H15FN2O5S) C,H,N.
N-Methyl-N-[2-(4′-chlorobenzyloxy)-4-nitrophenyl]methane-
N-[2-(4′-Phenylbenzyloxy)-4-nitrophenyl]methanesulfona-
1
mide (11b). Yellow solid, 94%: mp 253-255 °C; H NMR (400
MHz, DMSO-d6) δ 9.68 (1H, br), 7.87 (1H, s), 7.84 (1H, d, J )
2.5 Hz), 7.64 (6H, m), 7.46 (3H, m), 7.36 (1H, m), 5.35 (2H, s),
3.08 (3H, s); 13C NMR (100 MHz, DMSO-d6) δ 149.70, 140.76,
140.61, 136.25, 129.82, 129.43, 128.41, 127.61, 127.56, 118.32,
108.80, 70.89, 41.53; HRMS calculated for C20H18N2NaO5S (M +
Na)+ 421.0834, found 421.0815.
1
sulfonamide (9c). Pale yellow solid, 93%: mp 168-170 °C; H
NMR (400 MHz, CDCl3) δ 7.90 (2H, m), 7.55 (1H, m), 7.40 (4H,
m), 5.20 (2H, s), 3.27 (3H, s), 2.86 (3H, s); 13C NMR (100 MHz,
CDCl3) δ 155.54, 148.36, 135.85, 135.49, 133.48, 132.87, 129.91,
129.72, 117.17, 108.48, 71.15, 39.03, 37.95; HRMS calculated for
C15H15ClN2NaO5S (M + Na)+ 393.0288, found 393.0268.
N-Methyl-N-[2-(4′-bromobenzyloxy)-4-nitrophenyl]methane-
sulfonamide (10c). Pale yellow solid, 90%: mp 151-152 °C; 1H
NMR (400 MHz, CDCl3) δ 7.90 (2H, m), 7.55 (3H, m), 7.34 (2H,
d, J ) 8.3 Hz), 5.19 (2H, s), 3.28 (3H, s), 2.86 (3H, s); 13C NMR
(100 MHz, CDCl3) δ 155.52, 148.36, 135.86, 133.99, 132.87,
132.68, 130.15, 123.63, 117.19, 108.48, 71.18, 39.05, 37.96; HRMS
calculated for C15H15BrN2NaO5S (M + Na)+ 436.9783, found
436.9791.
N-(2-Phenylethoxy 4-nitrophenyl)methanesulfonamide (12b).
Yellow solid, 77%: mp 120-121 °C; 1H NMR (400 MHz, CDCl3)
δ 7.90 (1H, dd, J ) 8.9, 2.5 Hz), 7.78 (1H, d, J ) 2.4 Hz), 7.63
(1H, d, J ) 8.9 Hz), 7.38 (2H, m), 7.29(3H, m), 7.05 (1H, br),
4.38 (2H, dd, J ) 6.6, 6.6 Hz), 3.19 (2H, dd, J ) 6.6, 6.6 Hz),
2.89 (3H, s); 13C NMR (100 MHz, CDCl3) δ 147.40, 144.33,
137.55, 133.23, 129.43, 129.21, 127.52, 118.10, 117.42, 107.43,
70.54, 40.27, 35.76; HRMS calculated for C15H16N2NaO5S (M +
Na)+ 359.0678, found 359.0668. Anal. (C15H16N2O5S) C, H, N.
N-[2-(R-Naphthylmethoxy) 4-nitrophenyl]methanesulfona-
N-Methyl-N-[2-(4′-phenylbenzyloxy)-4-nitrophenyl]methane-
sulfonamide (11c). Pale yellow solid, 94%: mp 152-153 °C; 1H
NMR (400 MHz, CDCl3) δ 7.98 (1H, d, J ) 2.3 Hz), 7.90 (1H,
dd, J ) 8.6, 2.3 Hz), 7.68 (2H, d, J ) 8.1 Hz), 7.63 (2H, dd, J )
7.8, 1.4 Hz),7.48 (5H, m), 7.41 (1H, d, J ) 7.4 Hz), 5.28 (2H, s),
3.31 (3H, s), 2.88 (3H, s); 13C NMR (100 MHz, CDCl3) δ 155.70,
148.42, 142.42, 140.56, 135.80, 133.87, 133.08, 129.35, 129.05,
128.20, 128.12, 127.52, 117.02, 108.53, 71.71, 39.00, 37.97; HRMS
calculated for C21H20N2NaO5S (M + Na)+ 435.0991, found
435.0985.
1
mide (13b). Pale yellow solid, 83%: mp 193-195 °C; H NMR
(400 MHz, CDCl3) δ 8.09 (1H, d, J ) 2.4 Hz), 7.96 (4H, m), 7.69
(1H, d, J ) 8.9 Hz), 7.58 (3H, m), 7.53(1H, d, J ) 8.1 Hz), 7.14
(1H, br), 5.66 (2H, s), 2.90 (3H, s); 13C NMR (100 MHz, CDCl3)
δ 147.24, 144.24, 134.39, 133.62, 132.03, 130.81, 130.23, 129.58,
128.62, 127.58, 126.83, 125.71, 123.37, 118.44, 117.33, 108.20,
71.07, 40.35; HRMS calculated for C18H16N2NaO5S (M + Na)+
395.0678, found 395.0686.
N-[2-(3′,6′-Dimethylbenzyloxy)-4-nitrophenyl]methanesulfona-
1
mide (14b). Pale yellow solid, 93%: mp 178-179 °C; H NMR
N-Methyl-N-(2-phenylethoxy 4-nitrophenyl)methanesulfona-
mide (12c). Yellow solid, 93%: mp 126-128 °C; H NMR (400
(400 MHz, DMSO-d6) δ 7.95 (1H, d, J ) 2.5 Hz), 7.88 (1H, dd,
J ) 8.9, 2.5 Hz), 7.55 (1H, d, J ) 8.9 Hz), 7.32 (1H, s), 7.14 (1H,
d, J ) 7.7 Hz), 7.08 (1H, d, J ) 7.6 Hz), 5.25 (2H, s), 3.10 (3H,
s), 2.32 (3H, s), 2.28 (3H, s); 13C NMR (100 MHz, DMSO-d6) δ
150.09, 144.19, 135.59, 134.80, 134.47, 131.01, 130.73, 129.85,
121.28, 117.81, 108.61, 70.30, 41.61, 21.45, 18.97; HRMS
calculated for C16H18N2NaO5S (M + Na)+ 373.0834, found
373.0829.
1
MHz, CDCl3) δ 7.85 (2H, m), 7.52 (1H, m), 7.35 (2H, m), 7.30
(2H, m), 4.45 (2H, dd, J ) 6.5, 6.5 Hz), 3.21 (2H, dd, J ) 6.4, 6.4
Hz), 3.13 (3H, s), 2.63 (3H, s); 13C NMR (100 MHz, CDCl3) δ
155.73, 148.45, 137.51, 135.20, 133.53, 129.32, 129.08, 127.55,
116.73, 108.00, 69.83, 38.34, 37.60, 35.76; HRMS calculated for
C16H18N2NaO5S (M + Na)+ 373.0834, found 373.0829.
N-Methyl-N-[2-(R-naphthylmethoxy) 4-nitrophenyl]methane-
sulfonamide (13c). Pale yellow solid, 94%: mp 163-165 °C; 1H
NMR (400 MHz, CDCl3) δ 8.12 (1H, d, J ) 2.4 Hz), 8.01 (1H,
dd, J ) 6.2, 3.5 Hz), 7.92 (3H, m), 7.51 (5H, m), 5.65 (2H, s),
3.13 (3H, s), 2.55 (3H, s); 13C NMR (100 MHz, CDCl3) δ 155.79,
148.46, 135.89, 134.28, 133.22, 131.88, 130.70, 130.57, 129.46,
128.38, 127.32, 126.84, 125.77, 123.74, 117.07, 108.37, 71.37,
38.62, 37.96; HRMS calculated for C19H18N2NaO5S (M + Na)+
409.0834, found 409.0842.
N-Methyl-N-[2-(3′,6′-dimethylbenzyloxy)-4-nitrophenyl]-
methanesulfonamide (14c). Yellow solid, 87%: mp 137-139 °C;
1H NMR (400 MHz, DMSO-d6) δ 8.05 (1H, d, J ) 2.5 Hz), 7.86
(1H, dd, J ) 8.6, 2.5 Hz), 7.61 (1H, d, J ) 8.6 Hz), 7.30 (1H, s),
7.14 (1H, d, J ) 7.7 Hz), 7.11 (1H, d, J ) 7.6 Hz), 5.28 (2H, s),
3.15 (3H, s), 2.94 (3H, s), 2.32 (3H, s), 2.28 (3H, s); 13C NMR
(100 MHz, DMSO-d6) δ 156.34, 148.19, 136.80, 135.72, 134.52,
134.36, 132.01, 131.06, 130.56, 129.97, 116.75, 109.33, 70.17,
39.04, 38.07, 21.44, 18.84; HRMS calculated for C17H20N2NaO5S
(M + Na)+ 387.0991, found 387.0983.
5.2. Biological Study. 5.2.1. Cell Culture. JAR and SK-BR-3
cells were obtained from ATCC (Rockville, MD). SK-BR-3 cells
were maintained in phenol red-free custom media (MEM, Earle’s
salts, 1.5x amino acids, 2x nonessential amino acids, L-glutamine,
1.5x vitamins, Gibco BRL) supplemented with 10% fetal bovine
serum (FBS), 2 mM L-glutamine and 20 mg/L gentamycin. JAR
cells were maintained in RPMI 1640 medium with 2 mM L-
glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L
Preparation of 5c-14c with General Procedures: N-Methyl-
N-[2-(4′-methylbenzyloxy)-4-nitrophenyl]methanesulfonamide
1
(5c). Yellow solid, 87%: mp 96-98 °C; H NMR (400 MHz,
CDCl3) δ 7.95 (1H, d, J ) 2.4 Hz), 7.88 (1H, dd, J ) 8.6, 2.4 Hz),
7.55 (1H, d, J ) 8.6 Hz), 7.33 (2H, d, J ) 8.0 Hz), 7.25 (2H, d,
J ) 7.8 Hz), 5.18 (2H, s), 3.27 (3H, s), 2.83 (3H, s), 2.41 (3H, s);
13C NMR (100 MHz, CDCl3) δ 155.72, 148.42, 139.48, 135.73,
133.14, 131.93, 130.10, 128.73, 116.86, 108.46, 71.89, 38.90, 37.90,
21.69; HRMS calculated for C16H18N2NaO5S (M + Na)+ 373.0834,
found 373.0825. Anal. (C16H18N2O5S) C, H, N.
N-Methyl-N-[2-(4′-methoxylbenzyloxy)-4-nitrophenyl]methane-
1
sulfonamide (6c). Pale yellow solid, 92%: mp 108-109 °C; H
NMR (400 MHz, CDCl3) δ 7.95 (1H, d, J ) 2.4 Hz), 7.87 (1H,
dd, J ) 8.6, 2.5 Hz), 7.54 (1H, d, J ) 8.6 Hz), 7.37 (2H, d, J )
8.6 Hz), 6.96 (2H, d, J ) 8.7 Hz), 5.15 (2H, s), 3.86 (3H, s), 3.25
(3H, s), 2.81(3H, s); 13C NMR (100 MHz, CDCl3) δ 160.58, 155.70,
148.39, 135.78, 133.05, 130.42, 126.95, 116.83, 114.76, 108.44,
71.73, 55.75, 38.88, 37.88; HRMS calculated for C16H18N2NaO6S
(M + Na)+ 389.0783, found 389.0774.
N-Methyl-N-[2-(4′- isopropylbenzyloxy)-4-nitrophenyl]methane-
1
sulfonamide (7c). Yellow solid, 85%: mp 83-85 °C; H NMR
(400 MHz, CDCl3) δ 7.96 (1H, d, J ) 2.4 Hz), 7.88 (1H, dd, J )
8.6, 2.4 Hz), 7.56 (1H, d, J ) 8.6 Hz), 7.37 (2H, d, J ) 8.1 Hz),
7.30 (2H, d, J ) 8.1 Hz), 5.19 (2H, s), 3.28 (3H, s), 2.95 (1H, m),
2.83 (3H, s), 1.29 (6H, d, J ) 6.9 Hz); 13C NMR (100 MHz, CDCl3)
δ 155.74, 150.40, 148.43, 135.72, 133.16, 132.28, 128.70, 127.49,

Synthesis of SelectiVe Aromatase Expression Regulators
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1643
glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate, 90%/FBS,
10%. Fetal bovine serum was heat-inactivated for 30 min in a 56
°C water bath before use. Cell cultures were grown at 37 °C, in a
humidified atmosphere of 5% CO2 in a Hereaus CO2 incubator.
For all experiments, cells were plated in six-well plates and grown
to subconfluency. Before treatment, the media was changed to a
defined one containing DMEM/F12 media (Sigma) with 1.0 mg/
mL human albumin (OSU Hospital Pharmacy), 5.0 mg/L human
transferrin, and 5.0 mg/L bovine insulin.
5.2.2. Tritiated Water-Release Assay in JAR and SK-BR-3
Cell Lines. Measurement of aromatase enzyme activity was based
on the tritium water release assay. Cells in six-well plates were
treated with 0.1% DMSO (control) and inhibitors at the indicated
concentrations. After 24 h, the cells were incubated 3 h (SK-BR-3
cells) or 1 h (JAR cells) with fresh media along with 100 nM [1â-
3H]-androst-4-ene-3,17-dione (1 µCi). Subsequently, the reaction
mixture was removed, and proteins were precipitated using 10%
trichloroacetic acid at 42 °C for 20 min. After a brief centrifugation,
the media was extracted three times with an equal amount of
chloroform to remove remaining substrate and further treated with
dextran-treated charcoal. After centrifugation, a 250-µL aliquot
containing the product was counted in 5 mL of liquid scintillation
mixture. Results were corrected for blanks and for the cell contents
of culture flasks, and results were expressed as picomoles of 3H2O
formed per hour incubation time per million live cells (pmol/h/106
cells). To determine the amount of cells in each flask, the cells
were lysed and analyzed using the diphenylamine DNA assay
adapted to a 96-well plate. IC50 sigmoidal dose-response data were
analyzed with Microsoft Excel and the Graphpad Prism (Version
3.0) program.
5.2.3. Diphenylamine DNA Assay. To determine the amount
of viable cells in each flask, the cells were lysed with 0.5 N NaOH
aqueous solution and analyzed using the diphenylamine DNA assay
adapted to a 96-well plate. DNA standards (0-30 µg) were prepared
using double-stranded DNA reconstituted in PBS and added in
triplicate directly to the wells. A uniform cell suspension was
prepared from the six-well plate in 300 µL of 0.5 N NaOH aqueous
solution, and 60 µL of the unknown samples was added in triplicate
to separate wells. A solution of 0.16% acetaldehyde in water was
prepared and mixed at a 1:5 ratio with perchloric acid (20% vol/
vol). This solution (60 µL) was added to each well along with 100
µL of a 4% diphenylamine solution in glacial acetic acid. The plates
were incubated at 37 °C for 24 h. After centrifugation, 100 µL
supernatant of each well was transferred to a new 96-well plate,
and the OD595 was measured using a microplater reader. The DNA
concentration was determined by extrapolation to the standard curve,
and the amount of cells/well was calculated using the equation:
CYP19 total gene, the PCR reaction mixture consisted of Taqman
Universal PCR Master Mix (Applied Biosystems), 600 nM CYP19
primer (sense: 5′-TGT CTC TTT GTT CTT CAT GCT ATT TCT
C-3′; antisense: 5′-TCA CCA ATA ACA GTC TGG ATT TCC-
3′); 250 nM Taqman probe (6FAM 5′-TGC AAA GCA CCC TAA
TGT TGA AGA GGC AAT-3′TAMRA)(Invitrogen), and 2.0 µL
of each cDNA sample in a final volume of 20 µL. For the 18S
house-keeping total gene, the PCR reaction mixture consisted of
Taqman Universal PCR Master Mix (Applied Biosystems), 500 nM
18S primer (sense: 5′-CAG TTC ATA CAG CGG AAC ACT G-3′;
antisense: 5′-TTT GCT GGA GAA CAG GGC TG-3′), 50 nM
Taqman probe (6FAM 5′-TGC TGG CAC CAG ACT TGC CCT
C-3′TAMRA) (Invitrogen), and 2.0 µL of each cDNA sample in a
final volume of 20 µL. The Taqman probes for aromatase and 18S
were designed to anneal to a specific sequence of the aromatase
and 18S gene correspondingly between the forward and the reverse
primers. Cycling conditions were 50 °C for 2 min and 95 °C for
10 min, followed by 50 cycles at 95 °C for 15 s and 60 °C for 1
min.
5.2.7. Cell Viability Analysis. The effect of nimesulides
derivatives on SK-BR-3 cell viability was assessed by using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide
assay in six replicates. Cells were grown in custom media in 96-
well, flat-bottomed plates for 24 h and were exposed to various
concentrations of nimesulide derivatives dissolved in DMSO (final
concentration 0.1%) in defined media for different time intervals.
Controls received DMSO vehicle at a concentration equal to that
in drug-treated cells. The medium was removed and replaced by
200 µL of 0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
2H-tetrazolium bromide in fresh media, and cells were incubated
in the CO2 incubator at 37 °C for 2 h. Supernatants were removed
from the wells, and the reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-2H-tetrazolium bromide dye was solubilized in 200 µL/
well DMSO. Absorbance at 570 nm was determined on a plate
reader.
5.2.8. Analysis of Apoptosis. Apoptosis was determined by
selective denaturation of DNA in apoptotic cells by formamide and
detection of denatured DNA with a monoclonal antibody to single-
stranded DNA using an ELISA kit (CHEMICON, Temecula, CA).
Cells were plated in a 96-well flat bottom plate from 0.5 × 104 to
1 × 104 cells/well in custom media. Cells were allowed to adhere
to wells overnight. Following incubation, compounds were made
up in defined media, and a 5 µM screen was performed in each
cell line with respective compounds in triplicate for 24 h. Following
treatment, the plate was centrifuged at 200g for 5 min and media
was removed followed by the addition of 200 µL of fixative. The
plate was incubated for 30 min at 37 °C, at which point the fixative
was removed and the plate dried for 1-2 h at room temperature.
Fifty microliters of formamide was added to each well following a
brief incubation at room temperature for 10 min. The DNA in
apoptotic cells was denatured by heating the plate for 10 min and
then briefly cooling the plate for 5 min at 4 °C following removal
of formamide. The plate was rinsed three times with 200 µL of
PBS following 1 h incubation at 37 °C with 200 µL of 3% blocking
agent. After removal of the blocking agent, 100 µL of antibody
mixture was added to each well for 30 min at room temperature.
The plate was washed three times with 1X wash buffer using 250
µL of wash buffer/well followed by the addition of 200 µL of ABTS
solution added to each well for a 15-60 min incubation. The
reaction was stopped by the addition of 100 µL of a stop solution
added to each well, and absorbance was measured at 405 nm on a
SpectroMax 340 UV PlateReader.
1 cell ≈ 7 pg DNA.
5.2.4. RNA Extraction. Total RNA was isolated using the
TRIzol reagent according to the manufacturer’s protocol. Total RNA
pellets were dissolved in Dnase,RNase-free water and quantitated
using a spectrophotometer. The quality of RNA samples was
determined by electrophoresis through agarose gels and staining
with ethidium bromide; the 18S and 28S rRNA bands were
visualized under ultraviolet light.
5.2.5. cDNA Synthesis. Isolated total RNA (2 µg) was treated
with DNase I Amplification grade, according to the recommended
protocol to eliminate any DNA before reverse transcription. Treated
total RNA was denatured at 65 °C for 5 min in the presence of 2.5
ng/µL random hexamers and 0.5 mM dNTP mix. The samples were
snap-cooled on ice and centrifuged briefly. Complementary DNA
(cDNA) was synthesized using Superscript II reverse transcriptase
according to the recommended protocol. Briefly, the reactions were
conducted in the presence of 1X First-Strand Buffer and 20 mM
DTT at 42 °C for 50 min and consequently inactivated at 70 °C
for 15 min. The cDNA generated was used as a template in real-
time PCR reactions.
Acknowledgment. This work was supported by the National
Institutes of Health (NIH) Grant R01 CA73698 (R.W.B.), and
The Ohio State University Comprehensive Cancer Center Breast
Cancer Research Fund.
Supporting Information Available: Elemental analysis for
compounds 1f, 2c, 3c, 4c, 5c, 8c, and 12b. This material is available
free of charge via the Internet at http://pubs.acs.org.
5.2.6. Real-Time PCR. Real-time PCR was performed using
the Opticon 2 system from MJ Research (Waltham, MA). For the

1644 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
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