Novel sulfonanilide analogs decrease aromatase activity in breast cancer cells: Synthesis, biological evaluation, and ligand-based pharmacophore identification

Article abstract of DOI:10.1021/jm701107h

Aromatase converts androgens to estrogens and is a particularly attractive target in the treatment of estrogen receptor positive breast cancer. Previously, the COX-2 selective inhibitor nimesulide and analogs decreased aromatase expression and enzyme acti

Full text of DOI:10.1021/jm701107h

1126
J. Med. Chem. 2008, 51, 1126–1135
Novel Sulfonanilide Analogs Decrease Aromatase Activity in Breast Cancer Cells: Synthesis,
Biological Evaluation, and Ligand-Based Pharmacophore Identification
Bin Su,† Ran Tian,‡ Michael V. Darby,† and Robert W. Brueggemeier*,†
DiVision of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State UniVersity, 500 West 12th AVenue,
Columbus, Ohio 43210, and State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking UniVersity,
XueYuan Road 38, Beijing, China 100083
ReceiVed September 6, 2007
Aromatase converts androgens to estrogens and is a particularly attractive target in the treatment of estrogen
receptor positive breast cancer. Previously, the COX-2 selective inhibitor nimesulide and analogs decreased
aromatase expression and enzyme activity independent of COX-2 inhibition. In this manuscript, a
combinatorial approach was used to generate diversely substituted novel sulfonanilides by parallel synthesis.
Their pharmacological evaluation as agents for suppression of aromatase activity in SK-BR-3 breast cancer
cells was extensively explored. A ligand-based pharmacophore model was elaborated for selective aromatase
modulation (SAM) using the Catalyst HipHop algorithms. The best qualitative model consisted of four
features: one aromatic ring, two hydrogen bond acceptors, and one hydrophobic function. Several lead
compounds have also been tested in aromatase transfected MCF-7 cells, and they significantly suppressed
cellular aromatase activity. The results suggest that both genomic and nongenomic mechanisms of these
compounds are involved within the aromatase suppression effect.
tamoxifen as adjuvant therapy experience tumor relapse and die
from their disease. These findings strongly suggest primary or
acquired resistance to tamoxifen occurs in breast cancer patients
and affect the efficacy of this treatment.7–10
1. Introduction
Breast cancer is the second leading cause of cancer death in
women in the United States. About 178480 women in the United
States will be found to have invasive breast cancer in 2007.
About 40460 women will die from the disease this year.
Approximately over 2 million women living in the United States
have been treated for breast cancer.1 Two-thirds of breast cancers
are hormone-dependent, contain estrogen receptors (ERsa), and
require estrogen for tumor growth. These patients are, therefore,
suitable candidates for hormonal therapy, which targets blocking
estrogen stimulation of breast cancer cells. This can be achieved
by different approaches.2,3 In postmenopausal women, who are
characterized by the absence of ovarian production of estrogen,
ovarian suppression is not required. Two strategies that ame-
liorate the effects of estrogen in promoting breast cancer are
currently being compared in clinical trials of postmenopausal
patients with ER-positive breast cancer. Antiestrogens (e.g.,
tamoxifen and fulvestrant) block binding of estrogen to its
receptor, whereas aromatase inhibitors (e.g., letrozole, anastro-
zole, and exemestane) block estrogen biosynthesis. AIs are now
proving to be more effective and increase survival.4–6
Significant improvements in the efficacy of endocrine therapy
for ER-positive breast cancer have resulted from the introduction
of AIs, which induce estrogen deprivation in postmenopausal
women.7 The “third generation AIs” are potent and highly
selective in their inhibition of the cytochrome P450 enzyme,
aromatase. As a consequence, they efficiently block conversion
of androgens (testosterone and androstenedione) to estrogens
(estradiol and estrone, respectively) in peripheral tissues and
breast tumors.11 Of the three aromatase inhibitors (AIs) currently
approved for breast cancer treatment, letrozole and anastrozole
are nonsteroidal triazole compounds, whereas exemestane is a
steroidal analog of androstenedione. In recent years, AIs have
become first-line hormonal therapy for ER-positive patients with
advanced breast cancer. 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. As a consequence, other approaches are being
investigated to develop agents that can decrease aromatase
activity selectively.
Nonsteroidal anti-inflammatory drugs (NSAIDs) are beneficial
in breast cancer treatment.12 Recently, it has been shown that
the COX-2 selective inhibitor, celecoxib, has strong chemopre-
ventive activity against mammary carcinoma in rats.13 In
addition to COX inhibition, these small molecules can also target
other molecular pathways. For example, celecoxib can block
phosphoinositide 3-kinase (PI3K)/phosphoinositide-dependent
kinase (PDK)/Akt pathway to induce apoptosis in prostate cancer
cells.14,15
Tamoxifen has been the mainstay of hormonal therapy in both
early and advanced breast cancer patients for approximately
three decades. Almost all patients with ER-positive tumors in
Western countries have been treated with this drug either as
adjuvant treatment following surgery or as first-line treatment
for advanced disease. However, approximately 50% of patients
with advanced disease do not respond to first-line treatment with
tamoxifen. Furthermore, almost all patients with metastatic
disease and approximately 40% of the patients that receive
The COX-2 inhibitor nimesulide suppressed the development
of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-
induced mammary gland carcinogenesis in rats.16,17 Research
in our laboratory demonstrated that nimesulide also suppressed
aromatase activity and expression in several breast cancer cell
lines.18 Two small libraries of nimesulide analogs have been
* To whom correspondence should be addressed. Telephone: 614-292-
5231. Fax: 614-292-3113. E-mail: brueggemeier.1@osu.edu.
†
The Ohio State University.
Peking University.
‡
a Abbreviations: PGE2, prostaglandin E2; COX-2, cyclooxygenase-2;
NSAIDs, nonsteroidal anti-inflammatory drugs; ER, estrogen receptors; AIs,
aromatase inhibitors; SERMs, selective estrogen modulators.
10.1021/jm701107h CCC: $40.75
2008 American Chemical Society
Published on Web 02/14/2008

Sulfonanilides Suppress Aromatase in Breast Cancer
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1127
Table 1. Suppression of Aromatase Activity in SK-BR-3 Breast Cancer
Cells by Compounds with Modifications at the A-Position with Alkyl
Groups
Figure 1. Structural modifications of nimesulide.
synthesized in our laboratory. Their pharmacological effect on
aromatase has been extensively studied.19,20 In this manuscript,
a combinatorial chemistry approach was used to generate
diversely substituted nimesulide derivatives by parallel synthesis.
Their pharmacological evaluation as agents for suppression
aromatase activity in breast cancer cells was also further
explored. A ligand-based pharmacophore model was identified
by a qualitative approach, that is, Catalyst HipHop algorithms,
based on the nimesulide derivatives library. The best qualitative
model consisted of four features: one aromatic ring, two
hydrogen bond acceptors, and one hydrophobic function. Several
lead compounds also significantly decreased aromatase activity
in aromatase transfected MCF-7 cells. The results suggest that
both genomic and nongenomic mechanisms of these compounds
are involved within the aromatase suppression effect.
2. Results and Discussion
2.1. Compound Design. In this study, 81 novel sulfonanil-
ides are described and are based on nimesulide structure as a
platform (Figure 1). We systematically altered the structure of
nimesulide using the combinatorial strategies to modify the four
moieties depicted in Figure 1. The A position aromatic ring of
nimesulide was modified to either alkyl or substituted aryl
groups to generate compounds 1–46, as reported previously.19,20
Alkyl or substituted benzyl groups at the B position were
introduced to produce compounds 47-60. Next, the C position
was modified with benzamide, substituted benzamide, or
substituted benzenesulfonamide to produce compounds 61-72.
Last, the D position nitro group was reduced to the amine moiety
and substituted benzamide groups were then introduced to
generate compounds 73-81. This parallel combinatory strategy
can easily be extended to produce hundreds of new analogs,
which is well suited for future lead modification and drug
development.
2.1. Pharmacological Evaluation of Known Nimesulide
Analogs. We tested some of the nimesulide derivatives in SK-
BR-3 breast cancer cells of their IC50 for aromatase suppression
(compounds in Table 1 and part of the compounds in Table 2)
and also their COX-2 inhibition in breast cancer cells.19 The
results suggest that selective aromatase suppression is indepen-
dent of the COX-2 inhibition. The functionality at the B-position
is very critical for the COX-2 inhibitory activity. The compounds
inhibit COX-2 only when the N-H is available in the ionized
form. Introduction of any group in the B-position eliminates
this ionization process and produces compounds with no COX-2
inhibitory activity.21 However, this ionization is not important
for aromatase suppression. On the contrary, blocking this
position results in better agents for suppression of aromatase
expression and activity.19
a From ref 19.
bulky substituted benzyl group to the A-position is better for
the aromatase suppression (Tables 1 and 2). Compounds 1, 2,
5, 6, 9, 10, 25, and 26 decreased aromatase activity in SK-
BR-3 cells, with IC50 around 0.5 µM. At the B-position, methyl
is slightly better than hydrogen in most of the compounds.
However, methyl and hydrogen are insufficient to fully char-
acterize this position, and additional substituted compounds at
the B-position were synthesized and discussed next.
2.2. Parallel Synthesis of Diversely Substituted Sulfon-
anilides. Modifications at the A-position have been extensively
explored previously.19,20 In the current study, we mainly focus
on modifications of the B-, C-, and D-position of nimesulide.
The B-position modifications are described in Scheme 1. The
starting material 2-amino-5-nitrophenol was refluxed with
K2CO3 and benzyl bromide to obtain compound 1a. 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). Compound 1b
was hydrolyzed with 10% NaOH solution to generate compound
19 as a monomethanesulfonamido compound. Compound 19
was treated with K2CO3 and substituted benzyl chloride/bromide,
alkyl bromide, or iodine in DMF at room temperature or refluxed
to obtain compounds 47–60, respectively.
Modifications at the C-position are described in Scheme 2.
Compound 1a was treated with different acyl chlorides and
K2CO3 to generate the carboxamides 61–70. For aryl sulfona-
mide compounds, sodium hydride and aryl sulfonyl chloride
were added to compound 1a in dry dimethylformamide (DMF)
at room temperature, and the reaction mixture was stirred
overnight to obtain compounds 71 and 72.
For the purpose of building detailed structure–activity rela-
tionships to facilitate the future drug development, all the
compounds synthesized previously were investigated here for
their IC50 in suppression of aromatase in SK-BR-3 breast cancer
cells. The results suggest that introducing short bulky alkyl or

1128 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Su et al.
Table 2. Suppression of Aromatase Activity in SK-BR-3 Breast Cancer
Cells by Compounds with Modifications at the A-Position with Aryl
Groups
Modifications at the D-position are described in Scheme 3.
First, the D-position nitro was reduced to amine to obtain
compound 73 (Scheme 1), then 73 was treated with different
acyl chlorides and K2CO3 to generate the carboxamides 74–81,
respectively.
This parallel combinatory strategy can produce numerous new
analogs to facilitate lead optimization and future drug develop-
ment. The structure of all the synthesized compounds in the
1
following biological study were determined by H NMR and
HRMS and the purity is confirmed by HPLC.
2.3. Biological Evaluation of the Sulfonanilides Sub-
stituted at the B-, C-, and D-Position. The diversely substituted
sulfonanilides were evaluated for their ability to suppress
aromatase activity in SK-BR-3 breast cancer cells. The results
demonstrate that the B-position alkyl- or aryl-substituted
compounds lost their biological activity in suppression of
aromatase. Compounds 47–60 only slightly decreased SK-BR-3
cellular aromatase activity at 50 µM (data not shown).
The majority of the C-position benzamide-substituted deriva-
tives did not decrease aromatase activity (Table 3). However,
compound 63, which has an acetyl group at the C-position,
showed an IC50 of 1.18 µM in suppression of aromatase.
Compounds 71 and 72, which have different phenol sulfonamide
groups at the C-position, showed an IC50 of 2.17 µM and 17.37
µM, respectively. This suggests that an electron-withdrawing
group is better for the aromatase suppression with phenol
sulfonamide analogs. Overall, the main factor that controls the
suppression of aromatase activity at the C-position is steric
effect. Smaller methyl sulfonamide or acetyl groups are the best
fit and bulky groups will decrease the pharmacological activity.
Unexpectedly, introduction of different benzamide groups to
the D-position significantly increased the suppression of aro-
matase activity (Table 4). Compound 73, which has an amine
group at the D-position, showed similar activity as compound
20, which has a nitro group at the D-position. Introducing an
acetyl group to the D-amine (compound 74, IC50 16.67 µM)
significantly decreased the biological activity. On the other hand,
other various benzamide-substituted derivatives significantly
suppressed cellular aromatase activity, with IC50 from 0.12 µM
to 1.77 µM (Table 4). Compound 81, which has a 4-phenol
benzamide group at the D-position, is the most active analog
in this library, with an IC50 of 0.12 µM. Compound 76, which
has a cyclohexaneacetamide group at the D-position, exhibited
an IC50 of 0.25 µM. These results suggest that bulky hydro-
phobic groups at the D-position are better for the suppression
of aromatase in SK-BR-3 breast cancer cells.
2.4. Ligand-Based Pharmacophore Identification. The
range of inhibitory activity (∼2 log units) was insufficient to
allow us to generate a meaningful activity-based (predictive)
pharmacophore model using Catalyst/Hypogen technology. We
employed the Catalyst/HipHop approach to evaluate the com-
mon feature required for suppression of cellular aromatase
activity. Thus, a training set consisting of 10 sulfonanilide
derivatives (compounds 2, 8, 20, 22, 40, 42, 63, 75, 76, and
81), having structural diversity and exhibiting the greatest
suppression of aromatase activity, was submitted for pharma-
cophore model generation based on common chemical features.
The resulting pharmacophore model contained four chemical
features: one hydrophobe (blue), two hydrogen bond acceptors
(green), and one ring aromatic (yellow) (Figure 2). Compound
41 was mapped onto the model. The hydrophobe maps to the
A-position of phenol ring with a two-carbon chain and a
hydrogen bond acceptors map to the C-position sulfonamide
b From ref 20.

Sulfonanilides Suppress Aromatase in Breast Cancer
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1129
Scheme 1. Modifications of the B-Position
Scheme 2. Modifications of the C-Position
and the D-position nitro group. The aromatic ring maps with
the platform center phenol ring.
2.5. Aromatase Assays in Aromatase Transfected MCF-
7 Cells. Our previous study demonstrated that sulfonanilide
analogs decrease cellular aromatase activity and transcription.
However, the concentrations for the suppression of aromatase
activity are much lower than the concentrations for the sup-
pression of CYP19 gene expression.19 This suggests that these
compounds might also suppress cellular aromatase activity with
From the biological activity and the structure of the respective
compounds, the D-position as a hydrophobic area enhances the
pharmacological activity. The synthesis of more diverse com-
pounds at the D-position will be necessary to further optimize
the model.

1130 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Scheme 3. Modifications of the D-Position
Su et al.
Table 3. Suppression of Aromatase Activity in SK-BR-3 Breast Cancer
Cells by Compounds with Modifications at the C-Position
Table 4. Suppression of Aromatase Activity in SK-BR-3 Breast Cancer
Cells by Compounds with Modifications at the D-Position
tion is controlled by tetracycline, which eliminates the possibility
that the compounds decrease CYP19 gene expression.22 Thus,
any suppression of aromatase activity by these compounds in
the transfected cells would be the result of nongenomic action.
Compounds 9, 10, 25, 26, 35, 38, 41, 45, 76, 79, and 81,
which have IC50 below 0.5 µM for suppression of aromatase
activity in SK-BR-3 cell, were tested in aromatase transfected
MCF-7 cells at 1 µM. The results demonstrate that some of the
sulfonanilide analogs significantly suppressed cellular aromatase
activity (Figure 3). This suggests that these compounds suppress
cellular aromatase activity via a nongenomic mechanism in this
transfected cell line.
3. Conclusion
a nongenomic mechanism. Because the compounds do not
inhibit aromatase enzyme directly, post-transcriptional modifica-
tion may be a primary mechanism. Aromatase transfected
MCF-7 cells were used to investigate the possible post-
transcriptional mechanisms. In these cells, aromatase transcrip-
In summary, the biological results indicated that novel
sulfonanilides significantly decrease aromatase activity at low
micromolar to submicromolar concentrations in SK-BR-3 breast
cancer cells. These compounds were more active than the lead

Sulfonanilides Suppress Aromatase in Breast Cancer
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1131
nongenomic mechanisms are also involved for this suppression.
Because these compounds also suppress CYP19 gene expression
in SK-BR-3 breast cancer cells,19,20 both genomic and nonge-
nomic mechanisms are involved in the suppression of cellular
aromatase activity. Our current research focuses on discerning
the specific molecular target(s) of these compounds. In addition,
the pharmacophore identification of the sulfonanilide analogs
will enable further lead optimization for novel drug discovery
and examination of the structural requirements for the suppres-
sion of cellular aromatase.
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 electro-
spray mass spectrometer at The Ohio State Chemical Instru-
mentation Center. All the NMR spectra were recorded on either
a Bruker DPX 250 or a DRX 400 MHz spectrometer in either
1
DMSO-d6 or CDCl3. Chemical shifts (δ) for H NMR spectra
are reported in parts per million to residual solvent protons.
For the HPLC analysis, a 1.00 mg/mL stock solution of each
standard was prepared in either methanol or acetonitrile. HPLC
analysis was performed on a HP1100 system (Hewlett-Packard,
Palo Alto, CA), which consists of a vacuum degasser, binary
pump, autosampler, column compartment, and a UV detector.
Reverse-phase HPLC was carried out on a C18 column (3.0 ×
150 mm, 5µm) from Beckman (Beckman Instruments, Fullenton,
CA) at room temperature, with a flow rate of 0.5 mL/min. Two
mobile phases (mobile phase A: 35% water, 65% acetonitrile;
mobile phase B: 25% water, 75% methanol) were employed to
run 35 min. An injection volume of 5–15 µL was used. The
UV detector was set up at 254 and 330 nm.
Figure 2. (a) Pharmacophore generated using Catalyst methodology
that describes the suppression of aromatase activity of the sulfonanilide
derivatives. Pharmacophore features are color-coded (yellow, aromatic
ring; blue, hydrophobic group; green, hydrogen bond acceptor). (b)
Compound 41 mapped onto the pharmacophore.
Compounds 1a, 1b, 19, and 20 were prepared as described
by Su et al.20
General Procedure for the Preparation of B-Position-
Substituted Nimesulide Analogs 47–60 from Compound
19. K2CO3 (0.69 g, 5 mmol) and aryl or alkyl halide (1 mmol,
1.0 equiv) were successively added to a solution of compound
19 (1.2 mmol, 1.2 equiv) in dry DMF and the mixture was
stirred at room temperature or 80 °C from several hours to
overnight. After being cooled, 5 mL of H2O and 1 mL of satd
aqueous Na2CO3 was added to the mixture and it was stirred at
room temperature overnight. The precipitated solid was collected
by filtration and washed with H2O and cold ethyl ether/hexane
to afford desired compounds.
Figure 3. Aromatase activity in MCF-7 transfected aromatase cell
treated with sulfonanilide derivatives. Cells were treated with indicated
compounds at 1 µM and aromatase activity was measured as described
in the Experimental Section. The results were normalized against a
control treatment with vehicle. Each data bar represents the mean results
of three independent determinations. *P < 0.05, **P < 0.005.
N-(2-Benzyloxy-4-nitro-phenyl)-N-isopropyl-methane-
sulfonamide (47). Isopropyl iodine was used and it was stirred
in DMF at 80 °C overnight. Pale yellow solid, yield 28%: H
nimesulide. Structure–activity analysis demonstrated that im-
proved pharmacological activity of the novel sulfonanilides is
achieved with substitutions at the A-position as short bulky
hydrophobic groups, at the B-position as a hydrogen or methyl
group, at the C-position as methyl sulfonamide, and at the
D-position as substituted benzamide groups. Ligand-based
pharmacophore model identification based on the sulfonanilide
library reveals that the best qualitative model consisted of four
features: one aromatic ring, two hydrogen bond acceptors, and
one hydrophobic function.
1
NMR (250 MHz, DMSO-d6) δ 8.02 (1H, d, J ) 2.5 Hz), 7.90
(1H, dd, J ) 8.6, 2.6 Hz), 7.56 (3H, m), 7.44 (3H, m), 5.32
(2H, s), 4.24 (1H, m), 2.99 (3H, s), 1.21 (3H, d, J ) 5.7 Hz),
1.10 (3H, d, J ) 5.6 Hz); HRMS calcd for C17H20N2NaO5S (M
+ Na)+, 387.0991; found, 387.0990.
N-(2-Benzyloxy-4-nitro-phenyl)-N-propyl-methanesulfon-
amide (48). Propyl iodine was used and it was stirred in DMF
at 80 °C overnight. Pale yellow solid, yield 53%: 1H NMR (250
MHz, DMSO-d6) δ 8.03 (1H, d, J ) 2.5 Hz), 7.90 (1H, dd, J
) 8.6, 2.5 Hz), 7.61 (6H, m), 5.36 (2H, s), 3.58 (2H, dd, J )
Several lead compounds also suppressed aromatase activity
in aromatase transfected MCF-7 cells, in which aromatase
transcription is controlled by tetracycline. This suggests that

1132 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Su et al.
6.9, 6.9 Hz), 2.95 (3H, s), 1.39 (2H, m), 0.86 (3H, dd, J ) 7.3,
7.3 Hz); HRMS calcd for C17H20N2NaO5S (M + Na)+,
387.0991; found, 387.0991.
m), 1.16 (6H, d, J ) 6.9 Hz); HRMS calcd for C24H26N2NaO5S
(M + Na)+, 477.1460; found, 477.1461.
N-(2-Benzyloxy-4-nitro-phenyl)-N-(2,5-dimethyl-benzyl)-
methanesulfonamide (57). 2,5-Dimethyl-benzyl chloride was
used and it was stirred in DMF at 80 °C overnight. Pale yellow
solid, yield 75%: 1H NMR (250 MHz, DMSO-d6) δ 7.95 (1H,
d, J ) 2.5 Hz), 7.76 (1H, dd, J ) 2.6, 8.6 Hz), 7.59 (2H, d, J
) 6.7 Hz), 7.50 (4H, m), 6.96 (3H, m), 5.36 (2H, s), 4.79 (2H,
s), 3.04 (3H, s), 2.16 (3H, s), 2.13 (3H, s); HRMS calcd for
C23H24N2NaO5S (M + Na)+, 463.1304; found, 463.1307.
N-(2-Benzyloxy-4-nitro-phenyl)-N-cyclohexylmethyl-meth-
anesulfonamide (58). Cyclohexylmethyl bromide was used and
it was stirred in DMF at 80 °C for four days. Pale yellow solid,
yield 52%: 1H NMR (250 MHz, DMSO-d6) δ 8.03 (1H, d, J )
2.5 Hz), 7.90 (1H, dd, J ) 2.5, 8.6 Hz), 7.62 (2H, d, J ) 8.6
Hz), 7.54 (5H, m), 5.36 (2H, s), 3.44 (2H, d, J ) 7.1 Hz), 2.93
(3H, s), 1.71 (5H, m), 1.09 (6H, m); HRMS calcd for
C21H26N2NaO5S (M + Na)+, 441.1460; found, 441.1461.
N-(2-Benzyloxy-4-nitro-phenyl)-N-hexyl-methanesulfon-
amide (49). Hexyl iodine was used and it was stirred in DMF
1
at 80 °C overnight. Yellow solid, yield 66%: H NMR (250
MHz, DMSO-d6) δ 8.03 (1H, d, J ) 2.5 Hz), 7.90 (1H, dd, J
) 8.6, 2.5 Hz), 7.60 (6H, m), 5.36 (2H, s), 3.60 (2H, dd, J )
6.5, 6.5 Hz), 2.95 (3H, s), 1.32 (8H, m), 0.84 (3H, dd, J ) 7.0,
7.0 Hz); HRMS calcd for C20H26N2NaO5S (M + Na)+,
429.1460; found, 429.1457.
N-Benzyl-N-(2-benzyloxy-4-nitro-phenyl)-methanesulfon-
amide (50). Benzyl bromide was used and it was stirred in DMF
at room temperature overnight. Pale yellow solid, yield 88%:
1H NMR (250 MHz, DMSO-d6) δ 7.97 (1H, d, J ) 2.4 Hz),
7.76 (1H, dd, J ) 8.6, 2.3 Hz), 7.58 (6H, m), 7.25 (5H, s), 5.36
(2H, s), 4.81 (2H, s), 3.06 (3H, s); HRMS calcd for
C21H20N2NaO5S (M + Na)+, 435.0991; found, 435.0989.
N-(2-Benzyloxy-4-nitro-phenyl)-N-(4-methoxy-benzyl)-
methanesulfonamide (51). 4-Methoxy-benzyl chloride was
used and it was stirred in DMF at 80 °C overnight. Pale yellow
solid, yield 97%: 1H NMR (250 MHz, DMSO-d6) δ 7.96 (1H,
d, J ) 2.1 Hz), 7.77 (1H, dd, J ) 8.6, 2.2 Hz), 7.57 (6H, m),
7.15 (2H, d, J ) 8.5 Hz), 6.82 (2H, d, J ) 8.5 Hz), 5.35 (2H,
s), 4.73 (2H, s), 3.69 (3H, s), 3.04 (3H, s); HRMS calcd for
C22H22N2NaO6S (M + Na)+, 465.1096; found, 465.1099.
N-(2-Benzyloxy-4-nitro-phenyl)-N-biphenyl-4-ylmethyl-
methanesulfonamide (59). Biphenyl-4-ylmethyl chloride was
used and it was stirred in DMF at 80 °C overnight. Yellow
solid, yield 71%: 1H NMR (250 MHz, DMSO-d6) δ 7.99 (1H,
d, J ) 2.5 Hz), 7.64 (17H, m), 5.37 (2H, s), 4.86 (2H, s), 3.08
(3H, s); HRMS calcd for C27H24N2NaO5S (M + Na)+,
511.1304; found, 511.1308.
N-(2-Benzyloxy-4-nitro-phenyl)-N-naphthalen-2-ylmethyl-
methanesulfonamide (60). Naphthalen-2-ylmethyl bromide was
used and it was stirred in DMF at room temperature overnight.
Pale yellow solid, yield 72%: 1H NMR (250 MHz, DMSO-d6)
δ 7.95 (1H, d, J ) 2.5 Hz), 7.86 (5H, m), 7.59 (9H, m), 5.38
(2H, s), 4.98 (2H, s), 3.11 (3H, s); HRMS calcd for
C25H22N2NaO5S (M + Na)+, 485.1147; found, 485.1148.
General Procedure for the Preparation of C-Position-
Substituted Nimesulide Analog Compounds 61–70 from
Compounds 1a. K2CO3 (0.69 g, 5 mmol) and substituted acyl
chloride (1.2 mmol, 1.2 equiv) were successively added to a
solution of compound 1a (1.0 mmol, 1.0 equiv) in dry
1,4-dioxane, and the mixture was stirred at room temperature
or 80 °C from 3 h to overnight. After being cooled, 10 mL of
H2O and 3 mL of satd aqueous Na2CO3 was added to the
mixture and it was stirred at room temperature overnight. The
precipitated solid was collected by filtration and washed with
H2O and cold ethyl ether/hexane to afford the desired compounds.
N-(2-Benzyloxy-4-nitro-phenyl)-N-(4-methyl-benzyl)-meth-
anesulfonamide (52). 4-Methyl-benzyl chloride was used and
it was stirred in DMF at 80 °C overnight. Pale yellow solid,
1
yield 75%: H NMR (250 MHz, DMSO-d6) δ 7.95 (1H, dd, J
) 1.8, 1.8 Hz), 7.75 (1H, m), 7.56 (6H, m), 7.12 (2H, d, J )
7.8), 7.07 (2H, d, J ) 7.5 Hz), 5.35 (2H, s), 4.74 (2H, s), 3.04
(3H, s), 2.22 (3H, s); HRMS calcd for C22H22N2NaO5S (M +
Na)+, 449.1147; found, 449.1147.
N-(2-Benzyloxy-4-nitro-phenyl)-N-(4-fluoro-benzyl)-meth-
anesulfonamide (53). 4-Fluoro-benzyl chloride was used and
it was stirred in DMF at 80 °C overnight. White solid, yield
1
72%: H NMR (250 MHz, DMSO-d6) δ 7.97 (1H, d, J ) 2.5
Hz), 7.78 (1H, dd, J ) 2.5, 8.6 Hz), 7.58 (6H, m), 7.30 (2H,
m), 7.12 (2H, m), 5.36 (2H, s), 4.79 (2H, s), 3.06 (3H, s); HRMS
calcd for C21H19FN2NaO5S (M + Na)+, 453.0896; found,
453.0898.
N-(2-Benzyloxy-4-nitro-phenyl)-N-(4-chloro-benzyl)-meth-
anesulfonamide (54). 4-Chloro-benzyl chloride was used and
it was stirred in DMF at 80 °C overnight. Pale yellow solid,
yield 75%: 1H NMR (250 MHz, DMSO-d6) δ 7.97 (1H, d, J )
2.5 Hz), 7.79 (1H, dd, J ) 2.5, 8.6 Hz), 7.57 (10H, m), 5.36
(2H, s), 4.79 (2H, s), 3.07 (3H, s); HRMS calcd for
C21H19ClN2NaO5S (M + Na)+, 469.0601; found, 469.0599.
N-(2-Benzyloxy-4-nitro-phenyl)-benzamide (61). Benzoyl
chloride was used and it was stirred at room temperature
overnight. White solid, yield 96%: 1H NMR (250 MHz, DMSO-
d6) δ 9.77 (1H, s), 8.30 (1H, d, J ) 9.6 Hz), 7.99 (4H, m), 7.65
(5H, m), 7.39 (3H, m), 5.41 (2H, s); HRMS calcd for
C20H16N2NaO4 (M + Na)+, 371.1008; found, 371.1009.
N-(2-Benzyloxy-4-nitro-phenyl)-N-(4-bromo-benzyl)-meth-
anesulfonamide (55). 4-Bromo-benzyl bromide was used and
it was stirred in DMF at room temperature overnight. White
solid, yield 92%: 1H NMR (250 MHz, DMSO-d6) δ 7.97 (1H,
d, J ) 2.5 Hz), 7.75 (1H, dd, J ) 2.6, 8.6 Hz), 7.57 (8H, m),
7.22 (2H, d, J ) 8.3 Hz), 5.36 (2H, s), 4.78 (2H, s), 3.06 (3H,
s); HRMS calcd for C21H19BrN2NaO5S (M + Na)+, 513.0096;
found, 513.0095.
Cyclohexanecarboxylic Acid (2-Benzyloxy-4-nitro-phe-
nyl)-amide (62). Cyclohexanecarbonyl chloride was used and
it was stirred at room temperature overnight. White solid, yield
99%: 1H NMR (250 MHz, DMSO-d6) δ 9.39 (1H, s), 8.32 (1H,
dd, J ) 5.3, 9.6 Hz), 7.88 (2H, m), 7.56 (2H, d, J ) 7.8 Hz),
7.42 (3H, m), 5.41 (2H, s), 2.64 (1H, m), 1.86 (5H, m), 1.43
(5H, m); HRMS calcd for C20H22N2NaO4 (M + Na)+, 377.1477;
found, 377.1477.
N-(2-Benzyloxy-4-nitro-phenyl)-N-(4-isopropyl-benzyl)-
methanesulfonamide (56). 4-Isopropyl-benzyl chloride was
used and it was stirred in DMF at 80 °C overnight. Pale yellow
solid, yield 91%: 1H NMR (250 MHz, DMSO-d6) δ 7.98 (1H,
d, J ) 2.5 Hz), 7.79 (1H, dd, J ) 2.5, 8.6 Hz), 7.57 (6H, m),
7.14 (4H, s), 5.35 (2H, s), 4.77 (2H, s), 3.04 (3H, s), 2.85 (1H,
N-(2-Benzyloxy-4-nitro-phenyl)-acetamide (63). Acetyl chlo-
ride was used and it was stirred at room temperature overnight.
White solid, yield 89%: 1H NMR (250 MHz, DMSO-d6) δ 9.59
(1H, s), 8.36 (1H, d, J ) 9.6 Hz), 7.89 (2H, m), 7.57 (2H, dd,
J ) 1.6, 6.6 Hz), 7.45 (3H, m), 5.41 (2H, s), 2.21 (3H, s); HRMS
calcd for C15H14N2NaO4 (M + Na)+, 309.0851; found, 309.0850.

Sulfonanilides Suppress Aromatase in Breast Cancer
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1133
N-(2-Benzyloxy-4-nitro-phenyl)-4-nitro-benzamide(64).4-Ni-
tro-benzoyl chloride was used and it was stirred at room
temperature overnight. Pale yellow solid, yield 94%: 1H NMR
(250 MHz, DMSO-d6) δ 10.26 (1H, s), 8.42 (1H, d, J ) 8.9
Hz), 8.20 (3H, m), 7.99 (2H, d, J ) 7.3 Hz), 7.58 (2H, d, J )
7.2 Hz), 7.45 (3H, m), 5.41 (2H, s); HRMS calcd for
C20H15N3NaO6 (M + Na)+, 416.0859; found, 416.0859.
N-(2-Benzyloxy-4-nitro-phenyl)-3-nitro-benzamide(65).3-Ni-
tro-benzoyl chloride was used and it was stirred at room
temperature overnight. Pale yellow solid, yield 91%: 1H NMR
(250 MHz, DMSO-d6) δ 10.31 (1H, s), 8.77 (1H, d, J ) 2.0
Hz), 8.47 (2H, dd, J ) 1.0, 8.8 Hz), 8.20 (1H, d, J ) 8.6 Hz),
8.00 (3H, m), 7.60 (2H, d, J ) 7.7 Hz), 7.42 (3H, m), 5.41
(2H, s); HRMS calcd for C20H15N3NaO6 (M + Na)+, 416.0859;
found, 416.0856.
collected by filtration and washed with H2O and a cold ether/
hexane mixture to afford the desired compounds.
N-(2-Benzyloxy-4-nitro-phenyl)-4-nitro-benzenesulfon-
amide (71). 4-Nitro-benzene sulfonyl chloride was used. Pale
1
yellow solid, yield 60%: H NMR (400 MHz, DMSO-d6) δ
10.70 (1H, s), 8.24 (2H, d, J ) 8.9 Hz), 7.98 (2H, d, J ) 8.7
Hz), 7.87 (1H, dd, J ) 2.4, 8.7 Hz), 7.81 (1H, d, J ) 2.3 Hz),
7.55 (1H, d, J ) 8.8 Hz), 7.34 (5H, m), 5.11 (2H, s); HRMS
calcd for C19H15N3NaO7S (M + Na)+, 452.0528; found,
452.0531.
N-(2-Benzyloxy-4-nitro-phenyl)-4-methyl-benzenesulfon-
amide (72). 4-Methyl-benzene sulfonyl chloride was used. Pale
1
yellow solid, yield 84%: H NMR (400 MHz, DMSO-d6) δ
10.28 (1H, s), 7.81 (4H, m), 7.52 (1H, d, J ) 8.8 Hz), 7.39
(7H, m), 5.21 (2H, s), 2.34 (3H, s); HRMS calcd for
C20H18N2NaO5S (M + Na)+, 421.0834; found, 421.0836.
N-(4-Amino-2-benzyloxy-phenyl)-N-methyl-methanesulfon-
amide (73). A mixture of ferric chloride (4 mmol, 4 equiv) and
nitrobenzene compound 20 (1 mmol, 1 equiv) were added to a
solvent mixture of dimethyl formamide and water (6:1, 7 mL).
It was stirred for 30 min, and then zinc dust (10 mmol, 10 equiv)
was added slowly. After completion of the reaction (10 min,
monitored by TLC), the reaction mixture was filtered, bypassing
the celite pad. The filtrate was diluted with water and basified
by adding satd aqueous Na2CO3. The precipitated solid was
collected by filtration, dried, and then dissolved in acetone. After
filtration of the insoluble residues, the desired compound was
recovered by distillation of the acetone under reduce pressure.
Pale yellow solid, yield 82%: 1H NMR (250 MHz, DMSO-d6)
δ 7.50 (5H, m), 6.91 (1H, d, J ) 8.3 Hz), 6.36 (1H, d, J ) 2.2
Hz), 6.14 (1H, dd, J ) 2.3, 8.3 Hz), 5.29 (2H, s), 5.07 (2H, s),
3.07 (3H, s), 2.79 (3H, s); HRMS calcd for C15H18N2NaO3S
(M + Na)+, 329.0936; found, 329.0936.
N-(2-Benzyloxy-4-nitro-phenyl)-4-chloro-3-nitro-benzam-
ide (66). 4-Chloro-3-nitro-benzoyl chloride was used and it was
stirred at room temperature overnight. White solid, yield 83%:
1H NMR (250 MHz, DMSO-d6) δ 10.31 (1H, s), 8.61 (1H, d,
J ) 2.1 Hz), 8.26 (1H, dd, J ) 2.1, 8.4 Hz), 8.16 (1H, dd, J )
1.1, 9.1 Hz), 8.02 (3H, m), 7.58 (2H, d, J ) 7.8 Hz), 7.44 (3H,
m), 5.41 (2H, s); HRMS calcd for C20H14ClN3NaO6 (M + Na)+,
450.0469; found, 450.0468.
N-(2-Benzyloxy-4-nitro-phenyl)-4-chloro-3-chloro-benzam-
ide (67). 3,4-Dichloro-benzoyl chloride was used and it was
stirred at room temperature overnight. White solid, yield 92%:
1H NMR (250 MHz, DMSO-d6) δ 10.07 (1H, s), 8.17 (2H, dd,
J ) 1.9, 9.0 Hz), 7.97 (4H, m), 7.59 (2H, d, J ) 7.0 Hz), 7.44
(3H, m), 5.39 (2H, s); HRMS calcd for C20H14Cl2N2NaO4 (M
+ Na)+, 439.0228; found, 439.0225.
N-(2-Benzyloxy-4-nitro-phenyl)-4-cyano-benzamide (68).
4-Cyano-benzoyl chloride was used and it was stirred at room
1
temperature overnight. White solid, yield 84%: H NMR (400
General Procedure for the Preparation of D-Position-
Substituted Nimesulide Analogs 74–81 from Compound 73.
K2CO3 (0.69 g, 5 mmol) and substituted acyl chloride (1.2
mmol, 1.2 equiv) were successively added to a solution of
compound 73 (1.0 mmol, 1.0 equiv) in dry 1,4-dioxane, and
the mixture was stirred at room temperature or 80 °C from 3 h
to overnight. After being cooled, 10 mL of H2O and 3 mL of
satd aqueous Na2CO3 was added to the mixture and it was stirred
at room temperature overnight. The precipitated solid was
collected by filtration and washed with H2O and cold ethyl ether/
hexane to afford desired compounds.
N-[3-Benzyloxy-4-(methanesulfonyl-methyl-amino)-phe-
nyl]-acetamide (74). Acetyl chloride was used and it was stirred
at room temperature overnight. White solid, yield 80%: 1H NMR
(250 MHz, DMSO-d6) δ 10.09 (1H, s), 7.56 (6H, m), 7.23 (2H,
m), 5.13 (2H, s), 3.12 (3H, s), 2.87 (3H, s), 2.05 (3H, s); HRMS
calcd for C17H20N2NaO4S (M + Na)+, 371.1041; found,
371.1044.
MHz, DMSO-d6) δ 10.14 (1H, s), 8.18 (2H, d, J ) 9.0 Hz),
8.11 (4H, m), 7.97 (2H, m), 7.56 (2H, d, J ) 4.6 Hz), 7.43
(3H, m), 5.40 (2H, s); HRMS calcd for C21H15N3NaO4 (M +
Na)+, 396.0960; found, 396.0958.
Naphthalene-2-carboxylic Acid (2-Benzyloxy-4-nitro-
phenyl)-amide (69). Naphthalene-2-carbonyl chloride was used
and it was stirred at room temperature for two days. White solid,
yield 91%: 1H NMR (400 MHz, DMSO-d6) δ 9.97 (1H, s), 8.57
(1H, s), 8.33 (1H, dd, J ) 0.9, 5.7 Hz), 8.10 (6H, m), 7.69
(4H, m), 7.45 (3H, m), 5.43 (2H, s); HRMS calcd for
C24H18N2NaO4 (M + Na)+, 421.1164; found, 421.1165.
Biphenyl-4-carboxylic Acid (2-Benzyloxy-4-nitro-phenyl)-
amide (70). Biphenyl-4-carbonyl chloride was used and it was
stirred at 80 °C for three days. Pale yellow solid, yield 57%:
1H NMR (400 MHz, DMSO-d6) δ 9.84 (1H, s), 8.31 (1H, d, J
) 9.3 Hz), 8.06 (2H, d, J ) 8.5 Hz), 7.98 (2H, dd, J ) 2.0, 7.1
Hz), 7.88 (2H, d, J ) 8.4 Hz), 7.79 (2H, dd, J ) 1.3, 8.5 Hz),
7.61 (2H, d, J ) 7.5 Hz), 7.54 (2H, dd, J ) 7.3, 7.3 Hz), 7.46
(3H, m), 7.37 (1H, m), 5.42 (2H, s); HRMS calcd for
C26H20N2NaO4 (M + Na)+, 447.1321; found, 447.1322.
General Procedure for the Preparation of C-Position-
Substituted Nimesulide Analogs, Compounds 71 and 72
from Compounds 1a. NaH (95%, 1 mmol) was added to a
solution of 1a (0.4 mmol) in anhydrous DMF (3 mL) at room
temperature. After being stirred at the same temperature for 30
min, substituted benzene sulfonyl chloride (0.4 mmol) was added
to the mixture slowly and the stirring was continued for 30 min
at room temperature. H2O (5 mL) was added, and the solution
was acidified by adding 2 N HCl until pH ) 2–3. It was stirred
at room temperature overnight. The precipitated solid was
N-[3-Benzyloxy-4-(methanesulfonyl-methyl-amino)-phe-
nyl]-benzamide (75). Benzoyl chloride was used and it was
stirred at room temperature overnight. Pale yellow solid, yield
1
93%: H NMR (250 MHz, DMSO-d6) δ 10.39 (1H, s), 7.98
(2H, d, J ) 6.8 Hz), 7.79 (1H, s), 7.63 (10H, m), 5.18 (2H, s),
3.15 (3H, s), 2.89 (3H, s); HRMS calcd for C22H22N2NaO4S
(M + Na)+, 433.1198; found, 433.1197.
Cyclohexanecarboxylic Acid [3-Benzyloxy-4-(methane-
sulfonyl-methyl-amino)-phenyl]-amide (76). Cyclohexanecar-
bonyl chloride was used and it was stirred at room temperature
1
overnight. Pale yellow solid, yield 90%: H NMR (250 MHz,
DMSO-d6) δ 9.96 (1H, s), 7.65 (1H, s), 7.53 (5H, m), 7.22 (2H,
m), 5.13 (2H, s), 3.11 (3H, s), 2.86 (3H, s), 2.37 (1H, m), 1.77

1134 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Su et al.
(5H, m), 1.48 (5H, m); HRMS calcd for C22H28N2NaO4S (M
+ Na)+, 439.1667; found, 439.1667.
cancer cells, five concentrations are used and each in triplicate.
After a 24 h treatment, the cells were incubated 3 h with fresh
media along with 100 nM [1ꢀ-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 contain-
ing 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.
N-[3-Benzyloxy-4-(methanesulfonyl-methyl-amino)-phe-
nyl]-3,4-dichloro-benzamide (77). 3,4-Dichloro benzoyl chlo-
ride was used and it was stirred at room temperature overnight.
White solid, yield 87%: 1H NMR (250 MHz, DMSO-d6) δ 10.25
(1H, s), 8.23 (1H, s), 7.94 (2H, m), 7.74 (1H, s), 7.55 (7H, m),
5.18 (2H, s), 3.15 (3H, s), 2.90 (3H, s); HRMS calcd for
C22H20Cl2N2NaO4S (M + Na)+, 501.0419; found, 501.0420.
N-[3-Benzyloxy-4-(methanesulfonyl-methyl-amino)-phe-
nyl]-4-chloro-3-nitro-benzamide (78). 4-Chloro-3-nitro ben-
zoyl chloride was used and it was stirred at room temperature
overnight. White solid, yield 98%: 1H NMR (250 MHz, DMSO-
d6) δ 10.67 (1H, s), 8.65 (1H, s), 8.29 (1H, d, J ) 8.5 Hz),
8.02 (1H, d, J ) 8.5 Hz), 7.73 (1H, s), 7.46 (7H, m), 5.19 (2H,
s), 3.15 (3H, s), 2.90 (3H, s); HRMS calcd for C22H20
ClN3NaO6S (M + Na)+, 512.0659; found, 512.0658.
N-[3-Benzyloxy-4-(methanesulfonyl-methyl-amino)-phe-
nyl]-4-cyano-benzamide (79). 4-Cyano benzoyl chloride was
used and it was stirred at room temperature overnight. White
solid, yield 97%: 1H NMR (400 MHz, DMSO-d6) δ 10.59 (1H,
s), 8.12 (2H, d, J ) 8.3 Hz), 8.06 (2H, d, J ) 8.3 Hz), 7.76
(1H, d, J ) 1.8 Hz), 7.54 (2H, d, J ) 7.2 Hz), 7.45 (5H, m),
5.18 (2H, s), 3.14 (3H, s), 2.89 (3H, s); HRMS calcd for
C23H21N3NaO4S (M + Na)+, 458.1150; found, 458.1151.
Naphthalene-2-carboxylic Acid [3-Benzyloxy-4-(methane-
sulfonyl-methyl-amino)-phenyl]-amide (80). Naphthalene-2-
carbonyl chloride was used and it was stirred at room temper-
4.2.3. Diphenylamine DNA Assay. To determine the amount
of viable cells in each well, the cells were lysed with 0.5 N
NaOH aqueous solution and analyzed in triplicate 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 directly to the wells. A uniform
cell suspension was prepared from the six-well-plate in 300 µL
0.5 N NaOH aqueous solution, and 60 µL of the unknown
samples were added 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 of the supernatant of
each well was transferred to a new 96-well plate, and the OD595
was measured using a microplater reader. The DNA concentra-
tion was determined by extrapolation to the standard curve.
1
ature overnight. Pale yellow solid, yield 96%: H NMR (250
MHz, DMSO-d6) δ 10.58 (1H, s), 8.60 (1H, s), 8.13 (4H, m),
7.84 (1H, s), 7.67 (9H, m), 5.21 (2H, s), 3.16 (3H, s), 2.91 (3H,
s); HRMS calcd for C26H24N2NaO4S (M + Na)+, 483.1354;
found, 483.1355.
Biphenyl-4-carboxylic Acid [3-Benzyloxy-4-(methanesulfo-
nyl-methyl-amino)-phenyl]-amide (81). Biphenyl-4-carbonyl
chloride was used and it was stirred at room temperature
4.2.4. Computational Methods. All molecular modeling
studies were performed using Catalyst 4.11 (Accelrys Inc., San
Diego, CA) installed on Silicon Graphics O2 work station
equipped with a 300 MHz MIPS R5000 processor (128 MB
RAM) running the Irix 6.5 operating system.
1
overnight. Pale yellow solid, yield 81%: H NMR (400 MHz,
DMSO-d6) δ 10.42 (1H, s), 8.09 (1H, d, J ) 8.2 Hz), 7.88 (5H,
m), 7.56 (10H, m), 5.19 (2H, s), 3.15 (3H, s), 2.90 (3H, s);
HRMS calcd for C28H26N2NaO4S (M + Na)+, 509.1511; found,
509.1513.
All structures were built and minimized within the Catalyst
software package, and conformational analysis for each molecule
was implemented using the Poling algorithm. The settings in
conformer generation were 250 as the maximum number of
conformers, best quality generation type, and an energy range
of 20 kcal/mol beyond the calculated potential energy minimum.
All other parameters used were kept at their default settings.
4.2. Biological Study. 4.2.1. Cell Culture. SK-BR-3 cells
were obtained from ATCC (Rockville, MD). MCF-7 transfected
with aromatase was kindly provided by Dr. Yue Wei (University
of Virginia). SK-BR-3 cells 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. MCF-7 transfected with
aromatase cells were maintained in DMEM/F12 supplemented
with 5% fetal bovine serum (FBS) and 20 mg/L gentamycin.
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 transferin, and 5.0 mg/L bovine insulin.
The range of inhibitory activity was not broad enough (4–5
orders of magnitude are required) for us to generate a meaningful
activity-based (predictive) pharmacophore model using Catalyst/
Hypogen technology. We employed the Catalyst/HipHop ap-
proach to evaluate the common feature required for suppression
of cellular aromatase. Therefore, 10 nimesulide derivatives that
have the highest aromatase suppression activity (compounds 2,
8, 20, 22, 40, 42, 63, 75, 76, and 81) and also structure diversity
were submitted for pharmacophore model generation based on
common chemical features.
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. We thank Dr. Yue Wei (University of
Virginia) for generously providing the aromatase transfected
MCF-7 cells.
4.2.2. Tritiated Water-Release Assay in Breast Cancer
Cells. 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. For the IC50 of aromatase suppression in breast

Sulfonanilides Suppress Aromatase in Breast Cancer
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1135
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