Novel sulfonanilide analogues suppress aromatase expression and activity in breast cancer cells independent of COX-2 inhibition

Article abstract of DOI:10.1021/jm051126f

Aromatase is a particularly attractive target in the treatment of estrogen receptor positive breast cancer. Aromatase levels in breast cancer cells are enhanced by prostaglandins and reduced by COX inhibitors. The synthesis and biological evaluation of a

Full text of DOI:10.1021/jm051126f

J. Med. Chem. 2006, 49, 1413-1419
1413
Novel Sulfonanilide Analogues Suppress Aromatase Expression and Activity in Breast Cancer
Cells Independent of COX-2 Inhibition
Bin Su, Edgar S. Diaz-Cruz, Serena Landini, and Robert W. Brueggemeier*
DiVision of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State UniVersity, 500 W. 12th AVenue,
Columbus, Ohio 43210
ReceiVed NoVember 8, 2005
Aromatase is a particularly attractive target in the treatment of estrogen receptor positive breast cancer.
Aromatase levels in breast cancer cells are enhanced by prostaglandins and reduced by COX inhibitors.
The synthesis and biological evaluation of a novel series of sulfonanilide analogues derived from the COX-2
selective inhibitor NS-398 are described. The compounds suppress aromatase enzyme activity in SK-BR-3
breast cancer cells in a dose- and time-dependent manner. The effect of these compounds on COX-2 inhibition
is investigated in breast cancer cells as well. Structure-activity analysis does not find a correlation between
aromatase suppression and COX-2 inhibition. Microsomal aromatase inhibition studies rule out the possibility
of direct enzyme inhibition. Real-time PCR analysis demonstrates that the sulfonanilide analogues decrease
aromatase gene transcription in SK-BR-3 cells. These studies suggest that the novel sulfonanilide compounds
suppress aromatase activity and transcription in SK-BR-3 breast cancer cells independent of COX-2 inhibition.
1. Introduction
Approximately one-third of all breast cancer patients and two-
thirds of postmenopausal breast cancer patients have estrogen-
dependent (ER+) breast cancer, which contains estrogen
receptors and requires estrogen for tumor growth.1 Approaches
for hormonal therapy of ER+ breast cancer are either blocking
the mechanism of action of estrogens by selective estrogen
receptor modulators (SERMs) or inhibiting estrogen biosynthesis
by aromatase inhibitors (AIs).2 For more than 30 years, the
antiestrogen tamoxifen has been the mainstay of hormonal
therapy in postmenopausal women with ER+ breast cancer.
Figure 1. Compound design.
However, drug resistance and increased uterine and endometrial
cancer limit its clinical application.3,4 Recent reports demonstrate
the transcriptional level.14 Therefore, COX selective inhibitors
that the third generation of aromatase inhibitors are more
could serve as the first generation of selective aromatase
effective than tamoxifen in postmenopausal breast cancer
expression regulators (SAERs).
patients.5 However, these compounds inhibit aromatase activity
throughout the body and could adversely impact sites where
estrogen is required for normal function, such as bone and brain.6
As a consequence, other approaches are being investigated to
develop agents that can decrease aromatase activity in a tissue
selective manner.
Research in our laboratory demonstrates that the COX-2
selective inhibitor NS-398 is significantly stronger in suppression
of aromatase activity than other COX-2 inhibitors in breast
cancer cells.14 This manuscript describes the synthesis of a small
library of novel sulfonanilide analogues and their biological
activity on aromatase and COX-2 in breast cancer cells.
In humans, cytochrome P450 aromatase is encoded by the
CYP19 gene, which contains nine coding exons (exons II to
X). The expression of this gene is regulated in a tissue-specific
manner by the alternative use of eight promoters, each one being
associated with a specific 5′ untranslated exon I.7 Furthermore,
due to the unique organization of tissue-specific promoters, the
various promoters employ different signaling pathways and
different transcription factors.8-12 In breast cancer tissue,
aromatase expression switches from promoter I.4 to promoter
I.3 and II. Thus drugs that target promoter I.3 and II-driven
expression of aromatase would be useful as chemotherapeutic
agents for the treatment of ER+ breast cancer. 7 Prostaglandin
2. Results and Discussion
2.1. Drug Design. In our previous study, different COX-2
inhibitors with similar IC50 values (concentration for 50%
inhibition) for COX-2 inhibition differ significantly in their
ability to suppress aromatase activity.14 This observation sug-
gests differences in the mechanisms by which these COX
inhibitors modulate aromatase expression in SK-BR-3 cells. It
is noteworthy that the effect of aromatase suppression by the
COX-2 selective inhibitor NS-398 was greater than other COX-2
inhibitors, even though NS-398 has weak COX-2 inhibitory
activity. To determine whether the modulation of aromatase
expression by NS-398 required the inhibition of COX-2 enzyme
activity, we designed and synthesized NS-398 analogues with
no COX-2 inhibitory activity. Introduction of a methyl group
at the N atom of the sulfonamide group to the COX-2 inhibitor
nimesulide resulted in no COX-2 inhibitory activity (Figure
1).15,16 This structural modification was utilized in our drug
E2 (PGE2) is a major regulator driving aromatase expressing
13
from promoter I.3 and II in breast cancer.
Recently, we
demonstrated that NSAIDs, COX-1, and COX-2 inhibitors can
suppress aromatase activity in SK-BR-3 breast cancer cells in
* To whom correspondence should be addressed.Phone: 614-292-5231.
Fax: 614-292-3113. E-mail: Brueggemeier.1@osu.edu.
10.1021/jm051126f CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/02/2006

1414 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Scheme 1. Synthesis of Sulfonanilide Analogues
Su et al.
design. The nitrate group at the 4 position of NS-398 was
retained and modifications of the sulfonamide and of the 2
position alkyl group were made to generate the new compounds.
corresponding unsubstituted compounds with the exception of
compounds 23 and 24. One possible explanation is the pKa value
of the reagents.
Compounds without N-methyl group have low pKa (3-4) and
are deprotonated very easily in the cell culture media, with the
ionization process illustrated in Figure 3. The negative charge
may reduce the ability of the drug to penetrate the cell
membrane. For compounds 23 and 24, the phenomenon is
reversed. They are less effective than their corresponding
unsubstituted compounds 14 and 15, respectively. The possible
reason is the large side chain on the four compounds, which
imparts low water solubility due to their hydrophobic effect.
However, compounds 14 and 15 are easily deprotonated in cell
culture media; the resulting negative charge may increase the
solubility of the drugs to enhance their activity. The best three
pairs of compounds, NS-398 and 17, 12 and 21, 13 and 22,
have a medium-sized side chain, which may bind more
effectively to the target molecule. Compounds 13 and 22 with
more flexible side chains are the best compounds in this library
for suppression of aromatase activity.
2.2. Synthesis. All the compounds were synthesized accord-
ing to a general procedure described in Scheme 1, in which R
represents alkyl structures and X represents halogen atom.15-17
The starting material 2-amino-5-nitrophenol is commercially
available and was treated with K2CO3 and alkyl halide in DMF
at room temperature or refluxed to obtain compounds 1-8.
Powder sodium hydride is added to compounds 1-8 in dry
dimethylformamide (DMF) at room temperature. After the
evolution of hydrogen ended, methanesulfonyl chloride was
added and the reaction mixture was stirred at room-temperature
overnight. After workup, the resulting mixture of N,N-
bimethanesulfonamido and N-methanesulfonamido was hydro-
lyzed with 10% NaOH solution to generate NS-398 and
compounds 9-16 as monomethanesulfonamido compounds.
Methylation of NS-398 and compounds 9-16 yielded com-
pounds 17-25, respectively. All the synthesized compounds
1
tested in the following biological study are confirmed by H
NMR and HRMS. Key compounds are also confirmed by
elemental analysis.
2.3. Biological Evaluation. 2.3.1. Aromatase Activity Assay
in SK-BR-3 Breast Cancer Cell Line. To investigate whether
these compounds decrease aromatase activity in breast cancer
cells, we first performed a 25 µM bioassay in SK-BR-3 breast
cancer cells (data not show). All the compounds tested, with
the exception of 16 and 25, suppressed aromatase activity by
almost 80-90%. At 1 µM, most compounds still significantly
decrease aromatase activity (Figure 2). In an effort to discrimi-
nate among compounds in this library, dose-response studies
of the active compounds were performed, and the resulting IC50
values of the compounds are listed in Table 1. Our results
suggest that the length of the group on position 2 of the
compounds is important for the suppression of aromatase
activity. Compounds containing a methoxy (16 and 25) or an
isopropyloxy (10 and 19), which are relatively short, have low
ability to suppress aromatase activity. Extremely long chain
substituents (15, 24, 14, and 23) have reduced activity as well,
which may also be due to the poor solubility of the compounds.
All the N-methyl compounds exhibited better activity than their
Figure 2. Suppression of aromatase activity in SK-BR-3 breast cancer
cells. SK-BR-3 cells were treated with indicated compounds (1 µM).
Aromatase activity was measured as described in the Experimental
Section. Values are expressed as picomoles 3H2O formed per hour
incubation time per million cells. The results were normalized against
a control treatment with vehicle. The value of 100% is equal to 0.03
pmol/h/106 cells. Each data bar represents the mean results of three
independent determinations. *P < 0.05 vs control by unpaired t test.

Sulfonanilide Analogues Decrease Aromatase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1415
Table 1. Suppression of Aromatase Activity in SK-BR-3 Breast Cancer
Cellsa
Figure 4. Time-course of aromatase suppression by NS-398 (2 µM,
9), compound 17 (2 µM, 2), letrozole (10 nM, 1), and cycloheximide
(10 µM, [) in SK-BR-3 cells. (A) Cells were treated with the indicated
agents for different period of times, and then aromatase activity was
subsequently determined during a 3 h assay as described. The results
were normalized against a corresponding control treatment with vehicle.
(B) Cells were pretreated with the indicated agents for 24 h, and then
the reagents were removed. The cells were also extensively washed
three times with PBS, followed by incubation in fresh media without
any agents for different periods of time. Aromatase activity was
subsequently determined during a 3 h assay as described. The results
were normalized against a control treatment with vehicle and washed
as the same process. The value of 100% is equal to 0.03 pmol/h/106
cells.
a IC50 values were calculated by a nonlinear regression analysis (Graph-
Pad Prism). Each dose-response curve contained five concentrations, each
in triplicate.
Figure 3. Ionization of the compounds in cell culture media.
2.3.2. Time-Course Studies for Suppression of Aromatase
Activity in SK-BR-3 Cells. Time-course studies in SK-BR-3
cells demonstrated a prolonged reversible suppression of aro-
matase by NS-398 (2 µM) and compound 17 (2 µM) in a time-
dependent manner. Cycloheximide (10 µM), which blocks de
novo protein synthesis, showed similar results, whereas the
aromatase inhibitor letrozole (10 nM) showed an acute inhibition
(Figure 4A). In a separate time-course study, the suppression
of aromatase activity of the four agents were shown to be
reversible (Figure 4B). After removal of the drugs, aromatase
activity returned in a time-dependent manner. However, in
letrozole treatment, aromatase activity returned and resulted in
a 150% increase after removing the drug. This is consistent with
the finding that aromatase inhibitors can stabilize aromatase
enzyme and thus reduce enzyme degradation.18 In brief, time-
course studies demonstrated a prolonged, reversible suppression
of aromatase by NS-398 and compound 17 in SK-BR-3 cells.
Figure 5. Microsomal aromatase inhibitory activities of NS-398 and
analogues. The results were normalized against a control treatment with
vehicle. Each data bar represents the mean results of three independent
determinations.
assay. The result rules out the possibility that the compounds
can work as aromatase inhibitors in the cell study.
2.3.3. Aromatase Activity Assay in Human Placental
Microsomes. Aromatase inhibition is performed with human
placental microsomes using methods previously reported by our
laboratory.19 Enzyme inhibition studies demonstrated that all
the compounds cannot significantly inhibit aromatase activity
at 5 µM (Figure 5), which is higher than the IC50 from the cell
2.3.4. Level of PGE2 Production in MDA-MB-231 Cell
Line. The production of PGE2 was measured in cells treated
with NS-398 and the novel sulfonanilide derivatives. MDA-
MB-231 cell line was chosen because of its high cyclooxygenase
activity. Cells were treated for 24 h with the indicated
concentration (25 µM) of the agents. NS-398, compound 12

1416 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Su et al.
levels. This suggests that the compounds interfere with pathways
affecting aromatase expression in breast cancer cells that do
not involve prostaglandins and COX enzyme activities.
3. Conclusion
The selective COX-2 inhibitor, NS-398, proved to be a good
lead compound for decreasing aromatase activity in breast cancer
cells by suppression of CYP19 mRNA at the transcriptional
level. The potent regulatory activity of this compound suggests
that COX-2 independent mechanisms may be involved in its
mechanism of action. The present study reports a convenient
synthetic approach for preparation of novel sulfonanilide
compounds. NS-398 and the sulfonanilide analogues suppress
aromatase activity in a dose- and time-dependent manner in SK-
BR-3 breast cancer cells. Human placenta microsomal assay
demonstrates that the compounds do not directly inhibit the
aromatase enzyme reaction at concentration above the IC50 from
cell study. In the COX-2 inhibition study, NS-398 and com-
pound 12 and 13 showed COX-2 inhibitory activity, but their
corresponding N-methyl compounds (17, 21, and 22, respec-
tively) and compounds 11 and 20 did not have any COX-2
inhibitory activity. This suggests that COX-2 inhibitory activity
is not necessary for the suppression of aromatase activity.
Furthermore, real time PCR demonstrated that NS-398 and
derivatives decreased CYP19 gene expression. These results
suggest that the novel sulfonanilide compounds suppressed
aromatase activity and transcription in SK-BR-3 cells indepen-
dent of COX-2 inhibition. The expression of aromatase is very
complex, and researchers also found that some orphan/nuclear
receptors such as ERRR-1, EAR-2, COUP-TFI, and RARγ are
involved in the regulation of aromatase expression.20,21 In
addition, MAPK pathway is involved to regulate aromatase
expression as well.22 It is still too early to speculate which
pathway(s) the compounds are targeting for the regulation of
aromatase activity and transcription. The specific molecular
target of these compounds in breast cancer cells is still under
investigation. Future chemistry studies will focus on developing
expanded libraries for detailed SAR studies and future drug
development.
Figure 6. Effect of NS-398 derivatives on PGE2 production of MDA-
MB-231 cells. Cells were treated for 24 h with the indicated agents at
25 µM. Results are expressed as means of the concentration of PGE2
produced per microgram protein ( SEM., *P < 0.05 vs control by
unpaired t test (n ) 6).
Figure 7. Real-time RT-PCR analysis of CYP19 mRNA expression
in SK-BR-3 cells. Cells were treated for 24 h with the indicated agents
at 25µM, and total RNA was isolated. Results are expressed as means
of CYP19 (normalized to 18S rRNA) ( SEM., *P < 0.05 vs control
by unpaired t test (n ) 9).
and 13 resulted in a significantly decrease in PGE2 production.
Compounds 17, 20, 21, and 22 did not show any inhibitory
activity (Figure 6). This is consistent with our design approach
that the introduction of a methyl group in to the N atom of the
sulfonamide group results in analogues that cannot be depro-
tonated and thus loses COX-2 inhibitory activity. In addition,
compound 11 did not show any COX-2 inhibitory activity, and
compound 11 has one carbon longer side chain comparing with
NS-398. This result suggests that the size of the side chain is
very important for the COX-2 inhibitory activity and that this
extension affects the binding of the compound with COX-2 and
results in no COX-2 inhibitory activity.
2.3.5. CYP19 mRNA Expression by Real-Time PCR.
Analysis of total CYP19 mRNA transcripts was performed using
real-time PCR in order to determine whether the decrease in
aromatase activity by NS-398 in SK-BR-3 cells was due to a
down-regulation of aromatase expression at transcriptional level.
SK-BR-3 cells were treated with NS-398, compound 17, 13,
and 22 for 24 h at concentrations at 25 µM. Total RNA was
extracted at 24 h, and CYP19 transcript levels were compared
to control (vehicle) treatment. All four compounds significantly
decreased CYP19 gene expression in SK-BR-3 cells relative to
the control (Figure 7). No effect on the expression level of the
housekeeping 18S rRNA was observed with any of the
compounds. Compounds 17 and 22, which do not show COX-2
inhibitory activity, decrease aromatase expression at similar
4. Experimental Section
4.1. 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
the NMR spectra were recorded on a Bruker DPX 250 and 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.
A. General Procedure for the Preparation of 1-8. The
halohydrocarbon (6 mmol, 1.2 equiv) and K2CO3 (0.69 g, 5 mmol)
were successively added to a solution of 2-amino-5-nitrophenol
(0.77 g, 5 mmol) in DMF (10 mL), and the mixture was refluxed
from 2 h to 7 days. After being cooled, 20 mL of H2O and 5 of
mL saturated aqueous Na2CO3 was added to the mixture, and 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 chro-
matographed on silica gel [AcOEt-hexane (1:5)] to afford desired
compounds.
2-Cyclohexyloxy-4-nitroaniline (1). Cyclohexyl iodide was used
and it was refluxed for 7 days. Yellow oil, 6.5%: 1H NMR (400

Sulfonanilide Analogues Decrease Aromatase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1417
MHz, CDCl3) δ 7.76 (1H, dd, J ) 8.7, 2.3 Hz), 7.66 (1H, d, J )
2.1 Hz), 6.64 (1H, d, J ) 8.8 Hz), 4.66 (2H,br), 4.34 (1H, m), 2.00
(2H, m), 1.78 (2H, m), 1.56 (3H, m), 1.38 (3H, m).
3.13 (s, 3H), 1.89 (m, 2H), 1.08 (t, J ) 7.4, 7.4 Hz, 3H); HRMS
calculated for C10H14N2NaO5S (M + Na)+ 297.0521, found
297.0533.
N-(2-Isopropyloxy-4-nitrophenyl)methanesulfonamide (10).
Yellow solid, 81.8%: mp 128-131 °C; 1H NMR (400 MHz,
CDCl3) δ 7.90 (dd, J ) 2.3, 9.0 Hz, 1H), 7.79 (d, J ) 2.3 Hz, 1H),
7.66 (d, J ) 9.0 Hz, 1H), 7.26 (br, 1H), 4.77 (m, 1H), 3.12 (s,
3H), 1.45 (d, J ) 6.1 Hz, 6H); HRMS calculated for C10H14N2-
NaO5S (M + Na)+ 297.0521, found 297.0501.
2-Propyloxy-4-nitroaniline (2). 1-Iodopropane was used and it
was refluxed for 2 h. Yellow solid, 82.8%: mp 59-61 °C; 1H NMR
(400 MHz, CDCl3) δ 7.81 (1H, dd, J ) 8.7, 2.3 Hz), 7.67 (1H, d,
J ) 2.1 Hz), 6.65 (1H, d, J ) 8.7 Hz), 4.58 (2H,br), 4.05 (2H, dd,
J ) 6.5, 6.5 Hz), 1.87 (2H, m), 1.08 (3H, dd, J ) 7.5, 7.5 Hz).
2-Isopropyloxy-4-nitroaniline (3). 2-Iodopropane was used and
it was refluxed for 24 h. Yellow oil, 69.3%: 1H NMR (400 MHz,
CDCl3) δ 7.73 (1H, dd, J ) 8.7, 2.4 Hz), 7.62 (1H, d, J ) 2.3 Hz),
6.63 (1H, d, J ) 8.8 Hz), 4.77 (2H, br), 4.59 (1H, m), 1.34 (6H, d,
J ) 6.0 Hz).
N-(2-Methylcyclohexyloxy-4-nitrophenyl)methanesulfon-
1
amide (11). Yellow powder, 80.5%: mp 138-142 °C; H NMR
(250 MHz, DMSO-d6) δ 7.64 (dd, J ) 2.6, 9.1 Hz, 1H), 7.41 (d,
J ) 2.7 Hz, 1H), 7.13 (d, J ) 9.2 Hz, 1H), 3.72 (d, J ) 6.4 Hz
2H), 2.71 (s, 3H), 1.69 (m, 6H), 0.99 (m, 5H); HRMS calculated
for C14H20N2NaO5S (M + Na)+ 351.0991, found 351.1017. Anal.
Calcd for C14H20N2O5S: C, 51.21; H, 6.14; N, 8.53. Found: C,
51.08; H, 6.10; N, 8.34.
2-Methylcyclohexyloxy-4-nitroaniline (4). Bromoethyl cyclo-
hexane was used and it was refluxed for 6 h. Yellow solid, 87.5%:
1
mp 54-56 °C; H NMR (400 MHz, CDCl3) δ 7.79 (1H, dd, J )
8.7, 1.9 Hz), 7.64 (1H, d, J ) 1.9 Hz), 6.64 (1H, d, J ) 8.7 Hz),
4.62 (2H,br), 3.86 (2H, d, J ) 6.1 Hz), 1.78 (6H, m), 1.08 (5H,
m).
N-(2-Cyclopentyloxy-4-nitrophenyl)methanesulfonamide (12).
Yellow solid 88.5%: mp 139-140 °C; 1H NMR (400 MHz, CDCl3)
δ 7.90 (dd, J ) 2.3, 8.9 Hz, 1H), 7.79 (d, J ) 2.3 Hz, 1H), 7.65(d,
J ) 8.9 Hz, 1H), 7.19(br, 1H), 4.95(m, 1H), 3.12 (s, 3H), 2.06 (m,
2H), 1.59 (m, 6H); HRMS calculated for C12H16N2NaO5S (M +
Na)+ 323.0678, found 323.0673. Anal. Calcd for C12H16N2O5S: C,
47.99; H, 5.37; N, 9.33. Found: C, 47.76; H, 5.45; N, 9.14.
N-(2-(1-Ethyl-propyloxy-4-nitrophenyl))methanesulfon-
amide (13). Yellow solid, 81.6%: mp 100-102 °C; 1H NMR (400
MHz, CDCl3) δ 7.89 (dd, J ) 2.4, 9.0 Hz, 1H), 7.78 (d, J ) 2.3
Hz, 1H), 7.67 (d, J ) 8.9 Hz, 1H), 4.38 (m, 1H), 3.12 (s, 3H),
1.75 (m, 4H), 0.98 (t, J ) 7.4, 7.4 Hz, 6H); HRMS calculated for
C12H18N2NaO5S (M + Na)+ 325.0834, found 325.0823. Anal. Calcd
for C12H18N2O5S: C, 47.67; H, 6.00; N, 9.27. Found: C, 47.78;
H, 6.11; N, 9.22.
2-Cyclopentyloxy-4-nitroaniline (5). Cyclopentyl iodide was
1
used and it was refluxed for 3 days. Red oil, 47%; H NMR (400
MHz, CDCl3) δ 7.78 (1H, dd, J ) 8.7, 2.3 Hz), 7.66 (1H, d, J )
2.3 Hz), 6.63 (1H, d, J ) 8.6 Hz), 4.87 (1H, m), 4.56 (2H,br),
1.68-2.02 (8H, m).
2-(1-Ethyl-propyloxy)-4-nitroaniline (6). 3-Bromopentane was
used and it was refluxed for 5 days. Yellow solid, 51.7%: mp 62-
1
63 °C; H NMR (400 MHz, CDCl3) δ 7.79 (1H, dd, J ) 8.7, 2.3
Hz), 7.67 (1H, d, J ) 2.2 Hz), 6.65 (1H, d, J ) 8.7 Hz), 4.58
(2H,br), 4.29 (1H, m), 1.72 (4H, m), 0.98(6H, dd, J ) 7.4, 7.4
Hz).
2-Nonyloxy-4-nitroaniline (7). 1-Iodononane was used and it
1
was refluxed for 24 h. Yellow solid, 86.2%: mp 74-75 °C; H
N-(2-Nonyloxy-4-nitrophenyl)methanesulfonamide (14). Yel-
NMR (400 MHz, CDCl3) δ 7.80 (1H, dd, J ) 8.7, 2.4 Hz), 7.67
(1H, d, J ) 2.3 Hz), 6.64 (1H, d, J ) 8.7 Hz), 4.57 (2H,br), 4.07
(2H, dd, J ) 6.6, 6.6 Hz), 1.84 (2H, m), 1.31(14H, m), 0.89 (3H,
dd, J ) 6.7, 6.7 Hz).
1
low solid, 94.9%: mp 70-71 °C; H NMR (400 MHz, CDCl3) δ
7.92 (dd, J ) 2.3, 8.9 Hz, 1H), 7.79 (d, J ) 2.4 Hz, 1H), 7.66 (d,
J ) 8.9 Hz, 1H), 7.24 (br, 1H), 4.14 (t, J ) 6.7, 6.7 Hz, 2H), 3.12
(s, 3H), 1.86 (m, 2H), 1.31 (m, 12H), 0.89 (t, J ) 6.4, 6.4 Hz,
3H); HRMS calculated for C16H26N2NaO5S (M + Na)+ 381.1460,
found 381.1482.
2-Hexyloxy-4-nitroaniline (8). 1-Iodohexane was used and it
1
was refluxed for 6 h. Yellow solid, 72.9%: mp 101-104 °C; H
NMR (400 MHz, CDCl3) δ 7.81 (1H, dd, J ) 8.7, 2.4 Hz), 7.67
(1H, d, J ) 2.3 Hz), 6.64 (1H, d, J ) 8.7 Hz), 4.58 (2H,br), 4.08
(2H, dd, J ) 6.5, 6.5 Hz), 1.85 (2H, m), 1.48 (2H, m), 1.35 (4H,
m), 0.92 (3H, dd, J ) 6.9, 6.9 Hz).
N-(2-Hexyloxy-4-nitrophenyl)methanesulfonamide (15). Pale
yellow solid, 86.8%: mp 74-76 °C; 1H NMR (400 MHz, CDCl3)
δ 7.92 (dd, J ) 2.5, 8.6 Hz, 1H), 7.78 (d, J ) 2.5 Hz, 1H), 7.65
(d, J ) 8.7 Hz, 1H), 7.23 (br, 1H), 4.14 (t, J ) 6.5, 6.5 Hz, 2H),
3.12 (s, 3H), 1.85 (m, 2H), 1.38 (m, 6H), 0.93 (t, J ) 6.8, 6.8 Hz,
3H); HRMS calculated for C13H20N2NaO5S (M + Na)+ 339.0991,
found 339.0986.
B. General Procedure for the Preparation of NS-398 and
Compounds 9-16. NaH (95% powder, 0.265 g, 10.5 mmol, 3.5
equiv) was added to a solution of alkyl instituted 2-amino-5-
nitrophenol (3.0 mmol) in anhydrous DMF (8 mL) at room
temperature. After being stirred at the same temperature for 30 min,
MsCl (1.031 g, 9.0 mmol, 3 equiv) was added to the mixture, 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 and washed with H2O, which was
used to the next reaction without further purification.
The intermediate was added to a 3 N NaOH aq solution and
was stirred at 80-90 °C overnight. After being cooled, 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 recrystalized from ethyl acetate/
hexane.
N-(2-Cyclohexyloxy-4-nitrophenyl)methanesulfonamide (NS-
398). Pale yellow powder, 59.5%: mp 124-126 °C; 1H NMR (250
MHz, CDCl3) δ 7.89 (dd, J ) 2.3, 8.9 Hz, 1H), 7.79 (d, J ) 2.4
Hz, 1H), 7.66(d, J ) 8.9 Hz, 1H), 7.24(br, 1H), 4.44(m, 1H), 3.12
(s, 3H), 2.06 (m, 2H), 1.84 (m, 2H), 1.41 (m, 6H). Anal. Calcd for
C13H18N2O5S: C, 49.67; H, 5.77; N, 8.91. Found: C, 49.75; H,
5.77; N, 8.80.
N-(2-Propyloxy-4-nitrophenyl)methanesulfonamide (9). Yel-
low powder, 67.7%: mp 117-119 °C; 1H NMR (250 MHz, CDCl3)
δ 7.92 (dd, J ) 2.4, 8.9 Hz, 1H), 7.79 (d, J ) 2.4 Hz, 1H), 7.66
(d, J ) 8.9 Hz, 1H), 7.25 (br, 1H), 4.12 (t, J ) 6.6, 6.6 Hz 2H),
N-(2-Methoxy-4-nitrophenyl)methanesulfonamide (16). Yel-
low solid, 81.1%: mp 128-130 °C; 1H NMR (250 MHz, DMSO-
d6) δ 7.83 (dd, J ) 2.5, 8.9 Hz, 1H), 7.72 (d, J ) 2.5 Hz, 1H),
7.46 (d, J ) 8.9 Hz, 1H), 3.90 (s 3H), 3.06 (s, 3H); HRMS
calculated for C8H10N2NaO5S (M + Na)+ 269.0208, found 269.0223.
C. General Procedure for the Preparation of 17-25. The
methanesulfonamide 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, and iodomethane (0.6 mmol, 1.2 eq) was added; the stirring
was kept 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
recrystalized from ethyl acetate/hexane. If oil precipitated, it was
extracted by using CH2Cl2. The organic phase was washed with
water and Na2CO3 aq solution, dried over anhydrous MgSO4, and
concentrated. The residue was chromatographed on silica gel
[AcOEt-hexane (1:5)] to afford the product.
N-Methyl-N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfon-
amide (17). White solid, 89.8%: mp 129-132 °C; 1H NMR (400
MHz, CDCl3) δ 7.82 (dd, J ) 2.4, 8.6 Hz, 1H), 7.80 (d, J ) 2.2
Hz, 1H), 7.55 (d, J ) 8.6 Hz, 1H), 4.49 (m, 1H), 3.30 (s, 3H),
2.99 (s, 3H), 2.11 (m, 2H), 1.85 (m, 2H), 1.50 (m, 6H); HRMS
calculated for C14H20N2NaO5S (M + Na)+ 351.0991, found

1418 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Su et al.
351.0970. Anal. Calcd for C14H20N2O5S: C, 51.21; H, 6.14; N,
8.53. Found: C, 51.23; H, 6.16; N, 8.41.
M potassium phosphate buffer (pH 7.0) with 5% propylene glycol.
All samples contained a NADPH regenerating system consisting
of 2.85 mM glucose-6-phosphate, 1.8 mM NADP+, and 1.5 units
of glucose-6-phosphate dehydrogenase (Sigma, St. Louis, MO).
Samples contained 100 nM androst-4-ene-3,17-dione (400 000-
450 000 dpm). Reactions were initiated with the addition of 50 µg
microsomal protein. The total incubation volume was 2.0 mL.
Incubations were allowed to proceed for 15 min in a shaking water
bath at 37 °C. Reactions were quenched by the addition of 2.0 mL
of chloroform. Samples were then vortexed and centrifuged for 5
min, and the aqueous layer was removed. The aqueous layer was
subsequently extracted twice in the same manner with 2.0 mL of
chloroform. A 0.5 mL aliquot of the final aqueous layer was
combined with 5 mL of 3a70B scintillation cocktail (Research
Products International Corp., Mt. Prospect, IL) and the amount of
radioactivity determined. Each sample was run in triplicate, and
background values were determined with microsomal protein
inactivated by boiling. Samples containing 50 µM (()-aminoglu-
tethimide (Sigma, St. Louis, MO) were used a positive control.
The data were analyzed with the Graphpad Prism (Version 3.0)
program.
N-Methyl-N-(2-propyloxy-4-nitrophenyl)methanesulfon-
1
amide (18). Pale yellow solid, 82.9%: mp 66-69 °C; H NMR
(400 MHz, CDCl3) δ 7.86 (dd, J ) 2.5, 8.6 Hz, 1H), 7.83 (d, J )
2.5 Hz, 1H), 7.55 (d, J ) 8.5 Hz, 1H), 4.12 (t, J ) 6.6, 6.6 Hz,
2H), 3.32 (s, 3H), 2.99 (s,3H), 1.92 (m, 2H), 1.12 (t, J ) 7.5, 7.5
Hz, 3H); HRMS calculated for C11H16N2NaO5S (M + Na)+
311.0678, found 311.0658.
N-Methyl-N-(2-isopropyloxy-4-nitrophenyl)methanesulfon-
amide (19). Yellow solid, 89.5%: mp 99-100 °C; 1H NMR (400
MHz, CDCl3) δ 7.83 (dd, J ) 2.5, 8.5 Hz, 1H), 7.81 (d, J ) 2.5
Hz, 1H), 7.55 (d, J ) 8.6 Hz, 1H), 4.80 (m, 1H), 3.30 (s, 3H),
2.99 (s,3H), 1.47 (d, J ) 6.1 Hz, 6H); HRMS calculated for
C11H16N2NaO5S (M + Na)+ 311.0678, found 311.0661.
N-Methyl-N-(2-methylcyclohexyloxy-4-nitrophenyl)methane-
1
sulfonamide (20). Yellow powder, 86.7%: mp 105-107 °C; H
NMR (400 MHz, CDCl3) δ 7.86 (dd, J ) 2.5, 8.6 Hz, 1H), 7.83
(d, J ) 2.4 Hz, 1H), 7.56 (d, J ) 8.6 Hz, 1H), 3.96 (d, J ) 6.0 Hz
2H), 3.33 (s, 3H), 2.98 (s, 3H), 1.76 (m, 6H), 1.13(m, 5H); HRMS
calculated for C15H22N2NaO5S (M + Na)+ 365.1147, found
365.1169. Anal. Calcd for C15H22N2O5S: C, 52.62; H, 6.48; N,
8.18. Found: C, 52.62; H, 6.55; N, 8.11.
4.2.3. Aromatase Tritiated Water-Release Assay in SK-BR-3
Cell Lines. SK-BR-3 cells were obtained from ATCC (Rockville,
MD). Cell cultures were maintained in phenol red-free custom
media (MEM, Earle’s salts, 1.5× amino acids, 2× nonessential
amino acids, L-glutamine, 1.5× vitamins, Gibco BRL) supplemented
with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 20
mg/L gentamycin. Measurement of aromatase enzyme activity was
based on the tritium water release assay.24 Cells in 100 mm Petri
dish were treated with 0.1% DMSO (control), and inhibitors at the
indicated concentrations. After 24 h, the media was changed and
the cells were incubated with 100 nM [1â-3H]-androst-4-ene-
3,17-dione (2 Ci) for 3 h. 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
extract unused substrate and further 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
N-Methyl-N-(2-cyclopentyloxy-4-nitrophenyl)methanesulfon-
amide (21). Yellow solid 92.9%: mp 102-104 °C; 1H NMR (400
MHz, CDCl3) δ 7.90 (dd, J ) 2.3, 8.5 Hz, 1H), 7.82 (d, J ) 2.5
Hz, 1H), 7.54 (d, J ) 8.5 Hz, 1H), 4.98 (m, 1H), 3.29 (s, 3H),
2.97 (s, 3H), 2.08 (m, 2H), 1.78 (m, 6H); HRMS calculated for
C13H18N2NaO5S (M + Na)+ 337.0834, found 337.0824. Anal. Calcd
for C13H18N2O5S: C, 49.67; H, 5.77; N, 8.91. Found: C, 49.87;
H, 5.93; N, 8.78.
N-Methyl-N-(2-(1-ethyl-propyloxy-4-nitrophenyl))methane-
sulfonamide (22). Yellow solid, 89.5%: mp 89-90 °C; 1H NMR
(400 MHz, CDCl3) δ 7.82 (dd, J ) 2.5, 8.6 Hz, 1H), 7.80 (d, J )
2.4 Hz, 1H), 7.55 (d, J ) 8.6 Hz, 1H), 4.42 (m, 1H), 3.31 (s, 3H),
2.98 (s, 3H), 1.77 (m, 4H), 1.02 (t, J ) 7.4, 7.4 Hz, 6H); HRMS
calculated for C13H20N2NaO5S (M + Na)+ 339.0991, found
339.0967. Anal. Calcd for C13H20N2O5S: C, 49.35; H, 6.37; N,
8.85. Found: C, 49.55; H, 6.43; N, 8.74.
N-Methyl-N-(2-nonyloxy-4-nitrophenyl)methanesulfon-
3
1
expressed as picomoles of H2O formed per hour incubation time
amide (23). Yellow solid, 98.8%: mp 83-84 °C; H NMR (400
per million live cells (pmol/h/106 cells). To determine the amount
of cells in each flask, the cells were trypsinized and analyzed using
the diphenylamine DNA assay adapted to a 96-well plate.25 IC50
sigmoidal dose-response data were analyzed with Microsoft Excel
and the Graphpad Prism (Version 3.0) program.
MHz, CDCl3) δ 7.85 (dd, J ) 2.5, 8.6 Hz, 1H), 7.82 (d, J ) 2.5
Hz, 1H), 7.55 (d, J ) 8.6 Hz, 1H), 4.14 (t, J ) 6.6, 6.6 Hz, 2H),
3.31 (s, 3H), 2.99 (s, 3H), 1.88 (m, 2H), 1.50 (m, 2H), 1.31 (m,
10H), 0.89 (t, J ) 6.6, 6.6 Hz, 3H); HRMS calculated for C17H28N2-
NaO5S (M + Na)+ 395.1617, found 395.1612.
N-Methyl-N-(2-hexyloxy-4-nitrophenyl)methanesulfon-
4.2.4. Enzyme Immunoassay of PGE2 in MDA-MB-231 Cells.
MDA-MB-231 cells were obtained from ATCC (Rockville, MD).
Cell cultures were maintained in phenol red-free custom media
(MEM, Earle’s salts, 1.5× amino acids, 2× nonessential amino
acids, L-glutamine, 1.5× vitamins, Gibco BRL) supplemented with
10% fetal bovine serum (FBS), 2 mM L-glutamine and 20 mg/L
gentamycin. To study PGE2 synthesis in cell culture media,
experiments were performed in 6015 mm Petri dishes.14 An aliquot
of MDA-MB-231 cells (15 × 104 cells) were added to each dish
and then incubated overnight to allow the cells to adhere to the
dish. After that, cells were serum starved in defined media for 24
h. This step was followed by replacement of media with fresh media
containing either vehicle (DMSO) or the indicated concentration
of agents. After 24 h incubation at 37 °C the media were collected
and the amount of PGE2 was determined by ELISA (Cayman
Chemical) according to the protocol provided by the manufacturer.
PGE2 concentration was normalized to total protein. Total proteins
were extracted from adhered cells by 30 min treatment with 0.5 M
NaOH at room temperature and shaking. Protein concentrations in
these extracts were determined using a protein assay method (Bio-
Rad Laboratories, Inc., Hercules, CA).
1
amide (24). Yellow oil, 99.3%: mp 44-46 °C; H NMR (400
MHz, CDCl3) δ 7.84 (dd, J ) 2.5, 8.6 Hz, 1H), 7.82 (d, J ) 2.5
Hz, 1H), 7.54 (d, J ) 8.7 Hz, 1H), 4.15 (t, J ) 6.6, 6.6 Hz, 2H),
3.31 (s, 3H), 2.98 (s, 3H), 1.88 (m, 2H), 1.52 (m, 2H), 1.37 (m,
4H), 0.92 (t, J ) 7.0, 7.0 Hz, 3H); HRMS calculated for C14H22N2-
NaO5S (M + Na)+ 353.1147, found 353.1178.
N-Methyl-N-(2-methoxy-4-nitrophenyl)methanesulfon-
1
amide (25). Pale yellow solid, 89%: mp 125-126 °C; H NMR
(400 MHz, DMSO-d6) δ 7.87 (d, J ) 2.1 Hz, 1H),7.84 (dd, J )
2.1, 8.5 Hz, 1H), 7.56 (d, J ) 8.5 Hz, 1H), 3.98 (s, 3H), 3.18 (s,
3H), 3.09 (s, 3H); HRMS calculated for C9H12N2NaO5S (M + Na)+
283.0365, found 283.0373.
4.2. Biological Study. 4.2.1. Preparation of Human Placental
Microsomes. Human term placentas were processed immediately
after delivery from The Ohio State University Hospitals at 4 °C.
The placenta was washed with normal saline, and connective and
vascular tissue was removed. Microsomes were prepared from the
remaining tissue using the method previously described.23 Microso-
mal suspensions were stored at -80 °C until required.
4.2.2. Inhibition Study. Inhibition of human placental aromatase
was determined by monitoring the amount of 3H2O released as the
enzyme converts [1â-3H]androst-4-ene-3,17-dione to estrone. All
the compounds were tested at 5 µM for their potential aromatase
inhibitory activity. Aromatase activity assays were carried in 0.1
4.2.5. RNA Extraction. Total RNA was isolated using the
TRIzol reagent according to the manufacturer’s protocol. Total RNA
pellets were dissolved in DNase- and RNase-free water and
quantitated using a spectrophotometer. The quality of RNA samples

Sulfonanilide Analogues Decrease Aromatase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1419
(8) Chen, S. Aromatase and breast cancer. Frontiers Biosci. 1998, 3,
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was determined by electrophoresis through agarose gels and staining
with ethidium bromide; the 18S and 28S rRNA bands were
visualized under ultraviolet light.
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and its exon I usage in human breast tumors. Detection of aromatase
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4.2.6. 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.
4.2.7. Real-Time PCR. Real-time PCR was performed using
the Opticon 2 system from MJ Research (Waltham, MA). For the
CYP19 total gene the PCR reaction mixture consisted of Taqman
Universal PCR Master Mix (Applied Biosystems), 600 nM of
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),
18S rRNA (Applied Biosystems, Foster City, CA), and 2.0 µL of
each RT sample in a final volume of 20 µL. The Taqman probe
was designed to anneal to a specific sequence of the aromatase
gene 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.
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stability. Endocr.-Rel. Cancer 1999, 6, 211-218.
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and characterization of azole isoflavone inhibitors of aromatase.
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regulation of aromatase expression in human breast tissue. J. Steroid
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Acknowledgment. This work was supported by the National
Institutes of Health (NIH) Grant R01 CA73698 (R.W.B.), the
NIH Chemistry and Biology Interface Training Program Grant
T32 GM08512 (E.S.D.-C.), and The Ohio State University
Comprehensive Cancer Center Breast Cancer Research Fund.
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