Synthesis, biological evaluation, and docking study of indole aryl sulfonamides as aromatase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.111815

In order to identify new aromatase enzyme inhibitors, thirty aryl sulfonamide derivatives containing an indole nucleus have been synthesized. The enzyme inhibition assay showed that four compounds inhibit aromatase in the sub-micromolar range. Loading concentrations of these four compounds were afterwards tested for cell viability and cytotoxicity on MCF7 human breast cancer cells, revealing a time- and dose-dependent decrease of active metabolizing cells over the time of the culture (0–72 h), starting from a concentration of 100 μM. Likewise LDH released raised up to 40% at early time of exposures (24 h). Finally, the docking study showed that the best active compounds efficiently bound in the active site of the aromatase; high values of HBD and low levels of HBA are the principal requirement evidenced by the QSAR model.

Full text of DOI:10.1016/j.ejmech.2019.111815

Journal Pre-proof
Synthesis, biological evaluation, and docking study of indole aryl sulfonamides as
aromatase inhibitors
Marialuigia Fantacuzzi, Barbara De Filippis, Marialucia Gallorini, Alessandra
Ammazzalorso, Letizia Giampietro, Cristina Maccallini, Zeineb Aturki, Enrica Donati,
Reham S. Ibrahim, Eman Shawky, Amelia Cataldi, Rosa Amoroso
PII:
S0223-5234(19)30967-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.111815
EJMECH 111815
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 July 2019
Revised Date: 23 October 2019
Accepted Date: 24 October 2019
Please cite this article as: M. Fantacuzzi, B. De Filippis, M. Gallorini, A. Ammazzalorso, L. Giampietro,
C. Maccallini, Z. Aturki, E. Donati, R.S. Ibrahim, E. Shawky, A. Cataldi, R. Amoroso, Synthesis,
biological evaluation, and docking study of indole aryl sulfonamides as aromatase inhibitors, European
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111815.
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Synthesis, Biological Evaluation, and Docking Study of Indole Aryl Sulfonamides as Aromatase
Inhibitors
Marialuigia Fantacuzzia*, Barbara De Filippisa, Marialucia Gallorinib, Alessandra Ammazzalorsoa, Letizia
Giampietroa, Cristina Maccallinia, Zeineb Aturkic, Enrica Donatic, Reham S. Ibrahimd, Eman Shawkyd,
Amelia Cataldib, Rosa Amorosoa
SYN20028567
1-30

Synthesis, Biological Evaluation, and Docking Study of Indole Aryl Sulfonamides as Aromatase
Inhibitors
Marialuigia Fantacuzzia*, Barbara De Filippisa, Marialucia Gallorinib, Alessandra Ammazzalorsoa, Letizia
Giampietroa, Cristina Maccallinia, Zeineb Aturkic, Enrica Donatic, Reham S. Ibrahimd, Eman Shawkyd,
Amelia Cataldib, Rosa Amorosoa
b
aUnit of Medicinal Chemistry, Department of Pharmacy, “G. d’Annunzio” University, Chieti, Italy; Unit of
c
Anatomy, Department of Pharmacy, “G. d’Annunzio” University, Chieti, Italy; Institute for Biological
Systems (ISB), Italian National Research Council, Monterotondo (Rome), Italy; dPharmacognosy
Department, Faculty of Pharmacy, Alexandria University, Egypt
*Corresponding author
E-mail: marialuigia.fantacuzzi@unich.it
Department of Pharmacy, “G. d’Annunzio” University, Chieti, Italy; Tel: ++39-0871-355-4684, Fax: ++39-
0871-355-4911;
Abstract
In order to identify new aromatase enzyme inhibitors, thirty aryl sulfonamide derivatives containing an
indole nucleus have been synthesized. The enzyme inhibition assay showed that four compounds inhibit
aromatase in the sub-micromolar range. Loading concentrations of these four compounds were afterwards
tested for cell viability and cytotoxicity on MCF7 human breast cancer cells, revealing a time- and dose-
dependent decrease of active metabolizing cells over the time of the culture (0-72 h), starting from a
concentration of 100 µM. Likewise LDH released raised up to 40 % at early time of exposures (24 hours).
Finally, the docking study showed that the best active compounds efficiently bound in the active site of the
aromatase; high values of HBD and low levels of HBA are the principal requirement evidenced by the
QSAR model.
Keywords
aromatase, breast cancer, aromatase inhibitors, sulfonamide, indole, docking, cytochromes P450
1

1.
Introduction
Cytochrome P450 (CYP) enzymes are crucial for biological homeostasis. These enzymes possess an iron-
porphyrin group (Fe2+-containing heme) at the active site [1,2]. Aromatase is a CYP enzyme, a
monooxygenase coded by the gene CYP19 located in chromosome 15q21, responsible for the conversion of
androgens (C19) to estrogens (C18) by demethylation and aromatization of the steroidal A-ring [3-5]. The
natural substrate of aromatase is the androstenedione (Figure 1) [6]. The physiological functions of
estrogens range from glucose and lipid homeostasis, brain function, follicular growth, bone mineralization,
epiphyseal closure, to coordination of the ovulatory process [7,8]. Perturbation in aromatase level can initiate
a number of diseases in both genders, even though gender-specific expression of this enzyme is evident.
Hyper level of aromatase could be found in gastric, hepatic, lung, and pituitary cancer, short stature, and
obesity in both gender; in female it could be found in breast cancer, breast hypertrophy, endometriosis,
polycystic ovary syndrome and ovarian cancer, while prostate cancer, gynecomastia and male hypogonadism
are the diseases caused by the excess of aromatase in males [9,10].
Breast cancer is the most predominant female cancer and estrogens play a central role in its development and
progression. In post-menopausal women, an abnormally elevated expression of aromatase is found in breast
cancer tissues that sustains the growth and progression of the tumor because of the endogenous production of
estrogens [11]. The use of antiestrogens and aromatase inhibitors (AIs) is the first line of treatments or the
adjuvant hormonal therapy after surgery for post-menopausal estrogen-dependent breast cancer [12-14].
AIs suppress the aromatase activity interacting with the substrate-binding site of the aromatase, and, based
on their structure, were usually divided in steroidal (exemestane, Figure 1) and non-steroidal (letrozole and
anastrozole, Figure 1). The steroidal AIs, structurally related to the natural substrate androstenedione, bind
to the catalytic site of aromatase and attach irreversibly the active site provoking an irreversible inhibition of
the enzyme, while the non-steroidal inhibitors coordinate to the heme iron of aromatase in a reversible
manner. Nowadays the third generation of AIs, including extremely potent and specific inhibitors, is
clinically used with strong effect and well tolerated. Limitations to third AIs generation are side effects (bone
loss, joint pain and cardiac events), and the prolonged therapy of five years that could develop acquired
resistance [15-21]. For these reason, restrain the side effects and enhance the clinical efficacy is the goal of
different medicinal chemistry research groups that synthesized novel steroidal AIs, non-steroidal AIs, or
investigate the effect of natural product, such as resveratrol [22-27].
Another recent approach to reduce side effects and preventing the development of acquired resistance after
prolonged treatments of ER+ breast cancer with AIs, is the use of molecules directed to allosteric sites of
aromatase. In this manner a modulation of the estrogen biosynthesis could be realized because of the non-
complete inhibition of estrogen biosynthesis, and not competitive binding to the active site [28].
2

Figure 1. Chemical structures of androstenedione and steroidal and non-steroidal third generation of AIs.
Our research group is involved in the discovery of non-steroidal sulfonamide-containing AIs [29]. The goal
of the present work is the synthesis, the biological evaluation, and the docking study of new chemical entities
that could overpass the limitations of the third generation of AIs. Based on the research made by Caporuscio
et al. [30], the imidazolyl methyl piperidine sulfonamide SYN20028567 (Figure 2) was chosen as lead
compound. The lead possesses an in vitro aromatase IC50 of 9.4 nM and the docking study revealed the
interaction between the azole ring and the heme iron, the H-bond of the oxygen of the sulfonamide moiety
with Ser478, and interactions between the piperidyl moiety and the hydrophobic pocket (Ile133, Phe134,
Phe221, Trp224, Ile305, Ala306).
Figure 2. Chemical structure of the lead compound SYN20028567.
Starting from this considerations, in the current study a library of thirty non-steroidal compounds (1-30,
Figure 3) containing an aryl sulfonamide group linked to the indole moiety was developed. In our purpose of
inhibiting the active site of CYP19A1, the sulfonamide group of the lead was kept constant for the H-
bonding, but the nitrogen was not included in a cycle, the aryl moiety of the new compounds was un-
substituted or substituted with different groups to interact with the hydrophobic pocket, and the imidazole
ring was substituted by an indole to interact with the heme iron or gave van der Waals contacts, whereas
indole is one of the privileged scaffold in medicinal chemistry [31-33].
3

The effect on the catalytic activity (percentage of inhibition and IC50) of the human aromatase was measured
by in vitro kinetic experiments by a fluorescence-based assay using letrozole as reference compound.
Moreover, the cell viability and citotoxicity were valued using MTT assay and LDH release assay over the
MCF7 breast cancer cell line. Further, the interactions between the best active compounds and the active site
of the enzyme were analyzed through docking study using the human placental aromatase (3EQM) in
complex with androstenedione.
cmp
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30
Figure 3. Chemical structure of newly synthesized arylsulfonamides 1-30.
2.
Results
2.1 Chemistry
The synthesis of compounds 1-30 was realized by the reaction of the appropriate commercial indolamine and
the commercial arylsulfonamide in the presence of trimethylamine in dry dichloromethane keeping the
reaction at 0 °C for 2 hours and then at room temperature for 18-22 hours. The purification by Liquid
Chromatography gave the purified compounds 1-30. The reaction scheme is presented in Scheme 1.
4

R
R
1
2
R
R
1
2
O
O
O
O
H N
2
+
R
S
3
R
S
3
N
N
n
H
Cl
H
n
N
H
Scheme 1. General synthesis of compounds 1-30.
Reagents and conditions: NEt3, dry CH2Cl2, N2, 0°C for 2h, t.a. 18h-22h.
2.2
Aromatase Inhibition Studies
The in vitro anti-aromatase activity of the compounds 1-30 was valued using a commercial fluorimetric
assay kit (Aromatase-CYP19A Inhibitor Screening kit, BioVIsion) with letrozole (IC50 = 1.9 nM) as the
reference drug, based on methods already described [34]. First of all, the newly synthesized compounds 1-30
were tested at 1 µM to understand the percentage of aromatase inhibition referred to letrozole 1 µM (100%
aromatase inhibition) and in absence of inhibitor (0% aromatase inhibition). The percentage of inhibition is
reported in Figure 4 only for compounds showing values superior than 10%.
150
129.3
109.7
100
100
50
0
83.2
75.2
43.5
33.4
30.6
20.7
19.7
18.6
16.4
15.3
12.8
0
Figure 4. Percentage of aromatase enzyme inhibition of newly synthesized compounds. Concentration of
compounds 1-30 and letrozole (LTR) 1 µM. Experiments were repeated in triplicate.
Compounds 1, 4, 11, 13-15, 24, 26-27 mildly inhibit the enzyme, showing percentage of inhibition in the
range of 12.8%-43.5%. Compounds 3, 7, 22, and 23, tested at 1 µM, strongly inhibit the aromatase, showing
values of 83.2%, 129.3%, 75.2%, and 109.7%, respectively.
Compounds 3, 7, 22, and 23, that showed a percentage of inhibition of aromatase enzyme superior than 50%,
were chosen to evaluate the IC50, considering five concentrations in the range 0-100 µM. The IC50 values are
reported in Table 1.
5

Table 1. The IC50 values of the aromatase-active compounds.
IC50 (µM)a
Compound
3
0.49 ± 0.03
7
0.16 ± 0.01
0.75 ± 0.05
22
23
0.20 ± 0.01
aThe value represents mean of triplicate determinations ±RSD
The IC50 values of compounds were in the sub-micromolar range (0.16-0.75 µΜ). The best values were
shown by compounds 7 and 23 (0.16 and 0.20 µM, respectively).
2.3
Viability and Cytotoxicity Assay
The cell viability was assessed by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)
assay on human breast cancer cell line (MCF7) in the concentration range of 0-250 µM for 24, 48 and 72 h
only for the best active compounds 3, 7, 22 and 23. The results are shown in Figure 5.
Figure 5. Effect of compounds 3, 7, 22, and 23 at the concentration range of 0-250 µM on the viability of
MCF7 breast cancer cell line at 24, 48 and 72 h.
There is a general decrease of cell metabolic activity with respect to the DMSO control, which is assessed
around 45% after 24 hours of exposure. The metabolic activity of MCF7 cells is reduced up to 36.63% and
38.62% by 250 µM of compound 3 and 7. At 48 hours, there is a fall in the percentage of active metabolizing
cells, mainly for compounds 3 and 7 at 100 and 250 μM (20.78% and 22.08% for compound 3; 19.53% and
20.08% for compound 7). After 72 hours of exposure, there is a clear dose-dependent decrease of cell
viability for compounds 3 and 7. Conversely, compound 22 is more effective at 100 µM as well as
compound 23.
6

To assess the cytotoxicity of compounds 3, 7, 22 and 23, the LDH (lactate dehydrogenase) assay was used,
measuring the LDH released from human breast cancer cell line (MCF7) in the concentration range of 0-250
µM for 24 h. Results are shown in Figure 6.
Figure 6. Effect of compounds 3, 7, 22, and 23 at the concentration range of 0-250 µM at 24h on the
cytotoxicity (LDH release) over MCF7 breast cancer cell line.
In general, the percentage of LDH released is assessed around 35% for all the inhibitors without displaying
dose-dependent effects. These data can be related to the decreased cell metabolic activity measured by the
MTT assay and to the exiguous number of cells found at 24 hours of exposure.
2.4
Molecular docking study
2.4.1 Validation of the docking procedure
Human placental aromatase enzyme (3EQM) in complex with the natural substrate androstenedione (AD)
was chosen as target protein for molecular docking based on its highly resolved crystal structure at 2.9 Å.
For validation of the docking process, the co-crystallized ligand (AD) was re-docked into the prepared
aromatase protein structure binding pocket. RMSD value of 0.3 was obtained indicating a successful docking
procedure. Therefore, the adopted docking strategy was implemented for analyzing the binding interactions
between indole-sulfonamide derivatives and aromatase enzyme.
2.4.2 Molecular docking of indole-sulphonamide derivatives
The top four indole-sulfonamide derivatives (compounds 3, 7, 22, and 23) possessing the highest in vitro
aromatase inhibitory activity (IC50 values of 0.49, 0.16, 0.75 and 0.20 µM, respectively) were subjected to
docking study. The two-dimensional and three-dimensional interaction diagrams (Figure 7 and Figure 8)
revealed that these potent indole-sulfonamides were bound to aromatase enzyme via most of the previously
described binding residues of AD, the native substrate [35]. These amino acid residues include Phe134,
Trp224, Val370, Val373 and Met374 in addition to the cofactor heme group (HEM) which plays an essential
7

role in binding [36]. This fact confirmed that the binding poses of the investigated compounds (3, 7, 22, and
23) were successfully docked in the correct binding site in the enzyme cleft.
Compound 22 (Figure 7.a and Figure 8.a) interacted via hydrogen bonding with Met374 by its sulfonyl
oxygen atom, in addition to π- π stacking interaction with Trp224 and hydrophobic interactions with Ile 133,
Phe134, Phe221, Ala306, Val370, Leu 372, Val373 and Leu477, that led to a high binding affinity to the
enzyme. Compound 3 (Figure 7.b and Figure 8.b) formed π- π interaction with the cofactor (HEM) group
and Trp224 amino acid residue by the indole nucleus. Moreover, many hydrophobic interactions with
Phe134, Val370, Val373 and Met374 that enhanced its affinity. The binding modalities of compound 7
(Figure 7.c and Figure 8.c) and compound 23 (Figure 7.d and Figure 8.d) were hydrogen-bonding between
the sulfonyl oxygen atom and Thr310 and hydrophobic interactions with Ile133, Phe134, Val370, Leu372,
Val373, Met374 and Leu477. In addition, compound 7 exhibited cation-π interactions between Arg115 and
the indole nucleus.
a.
b.
d.
c.
Figure 7. 3D interaction diagrams representing the docked conformation of compound 22 (a), compound 3
(b) compound 7 (c) and compound 23 (d) inside the human placental aromatase. Interactions between
binding residues and the ligands are represented by dashed lines and arrows.
8

a.
b.
c.
d.
Figure 8. 2D interaction diagrams representing the docked conformation of compound 22 (a) compound 3
(b) compound 7 (c) and compound 23 (d) inside the human placental aromatase. Interactions between
binding residues and the ligands are represented by dashed lines and arrows.
2.4.3 Physicochemical Descriptors and Multiple linear regression QSAR analysis
Ten physicochemical properties were used as molecular descriptors to build the Multiple Linear Regression
(MLR) model through which the aromatase inhibitory activity of the dataset compounds were predicted. The
following physicochemical descriptors were calculated: octanol-water partition coefficient (aLogP),
hydrogen bond acceptors (HBA), hydrogen bond donors (HBD), number of rotatable bonds (RB), and heavy
atom count (HAC), ring count (RC), polar surface area (PSA), electrotopological state (E-state), molar
refractivity (MR), and molecular polarizability (Polar). A QSAR model was successfully constructed using
MLR algorithm, equation (1).
PIC50=-0.092aLogP-0.42HBA+0.41HBD+0.36RB-0.82HAC+0.45RC-0.06PSA+0.22E-state+0.37Polar-0.032MR
9

2.4.4 Kernel-Based Partial Least Squares
A Canvas KPLS model coupling direct kernel-based partial least squares regression and Canvas fingerprints
was applied to obtain predictive and interpretable 2D QSAR models.
Canvas fingerprints allows for the direct mapping of each bit in the model back to the atoms in the molecule
where the model can readily be deconstructed into atomic contributions that can be visualized in terms of
favorable and unfavorable structural characteristics on the molecule.
Four molecular fingerprint types (dendritic, linear, molprint2D, and radial) were used as molecular
descriptors to generate the 2D QSAR models. These descriptors were correlated with the aromatase
inhibitory activity of the dataset compounds using KPLS regression.
3.
Discussion
Breast cancer is the prevalent cancer in the female sex in the world. Although the incidence of women
affected by this cancer rises annually, survival increases due to screening programs that progressively cover
a larger number of people, and the progress of therapeutic approaches. Surely an early diagnosis increases
survival, but the use of more specific drugs for cancer cells should be evidenced.
Over 70% of breast cancer is estrogen dependent cancer, and the inhibition or modulation of estrogen
biosynthesis is fundamental. Although the SERMs have less clinical efficacy and greater adverse effects, in
clinical practice they are preferred to the AIs that instead show a greater development of resistance due to the
long period of use.
With the aim of identifying new AIs with higher efficacy and lower adverse effects thirty compounds (1-30)
with an indole-sulfonamide structure were synthesized, starting from the structure of the lead SYN
20028567. As shown in Figure 3, the series can be divided into four groups depending on whether the
nitrogen atom of the sulfonamide function is directly linked to the C6 of the indole (1-6), directly linked to
C5 (7-12), linked by a methylene to C5 (13-21) or linked by a chain of 2 carbon atoms to C3 (22-30).
Considering the aryl moiety of 1-30, it can be an unsubstituted benzyl ring, an unsubstituted, mono- or di-
substituted phenyl ring, or a 5-chlorothiophene.
A first screening of potentially active inhibitors of aromatase was performed with an enzymatic assay at the
concentration of 1 µM. Considering the four groups of compounds, in the series 1-6, arylsulfonamide
substituted in 6 position of the indole, and in the series 7-12, arylsulfonamide substituted in 5 position of the
indole, only compound 3 and 7 possess a good inhibitor activity at 1 µM. In compound 3 the benzene ring,
containing a para-methyl substituent, is directly bound to the sulfur atom while in compound 7 the
unsubstituted benzene ring is linked to the sulfur by a methylene group. The series 13-21 that contain the
sulfonamide in the 5 position linked by a methyl group did not affect the activity of aromatase; while in the
last series 22-30, only compounds 22 and 23 containing an unsubstituted benzene ring were good inhibitors.
Analyzing the structure of compounds, a Structure Activity Relationship (SAR) can be delineated. The
presence of one or two substituents on the benzene ring or the substitution with a thiophene is deleterious for
the aromatase inhibition. Only the presence of a methyl in para position of the benzene could be tolerated
10

(compounds 3, 15 and 24, 83.2 %, 33.4 % and 43.5 %, respectively). The presence of a methylene bridge
between the sulfur atom and the unsubstituted benzene ring was positive for compounds 7 and 22 (129.3 and
75.2%). Finally, compound 23, unsubstituted on the aryl moiety and with a linker of 2 carbon atoms bound
to the C3 of the indole, showed a percentage of inhibition equal to 109.7%
Based on the data obtained with the first screening, compounds 3, 7, 22-23, possessing a percentage of
aromatase inhibition superior than 50%, were tested at different concentrations (0-100 µM) to calculate the
value of IC50. The best active inhibitors possess an IC50 in the sub-micromolar range, 0.16-0.75 µM.
Cell viability (MTT assay) and cytotoxicity (LDH release assay) were also performed on human breast
cancer cell line MCF-7. Cell metabolic activity shows a time and dose-dependent decrease with loading
concentrations of the four compounds 3, 7, 22-23. After exposure to the lower dose administered (10 µM) at
48 h, cell metabolic activity is assessed at around 40-45% whereas for compounds 3 and 7 this trend is even
more enhanced at higher doses reaching values of metabolic activity around 20%. Moreover, cytotoxicity test
(LDH release assay) shows that there is a secretion of the enzyme assesses at 40% already after 24 hours of
exposure, being doubled with respect to the amount of LDH released with DMSO alone.
Finally, the most active compounds were subjected to a docking study, demonstrating that they interact with
aromatase enzyme via the binding residues of the natural substrate, androstenedione. Considering the
interaction summarized in Table 2 and the IC50 values, it can be supposed that H-bond between the
sulfonamide function and Thr310 for compounds 7 and 23 (IC50 0.16 µM and 0.20 µM, respectively) allows
better interaction in the active site. These two compounds interact with the same hydrophobic pocket, but
only compound 7 showed an additional cation-π interaction. Compound 3 and 22 made hydrophobic
interactions with the hydrophobic pocket; compound 3 made double π-π stacking interactions between the
indole and Trp224 and HEM, while 22 made a single π-π stacking interaction between the indole and Trp224
and hydrogen bonding with Met374.
Table 2. Type of interaction between the best active compounds and aromatase.
Cmp
type of interaction
π-π stacking
hydrophobic
H-bond
Trp224, HEM
3
Phe134, Val370, Val373, Met374
Thr 310
Arg115
cation-π
7
Ile133, Phe134, Val370, Leu372,
Val373, Met374, Leu477
hydrophobic
H-bond
Met374
22
23
Ile 133, Phe134, Phe221, Ala306,
Val370, Leu 372, Val373, Leu477
hydrophobic
H-bond
Thr310
Ile133, Phe134, Val370, Leu372,
Val373, Met374, Leu477
hydrophobic
11

A QSAR model was successfully constructed using multiple linear regression (MLR) algorithm (eq (1)). The
predictive performance of the constructed QSAR was acceptable as designated by the R2 (0.831) for training
set, and Q2 (0.762) for test set while the RMSE value was 0.023 indicating acceptable predictive errors of
the models. The QSAR model indicated that the descriptors octanol-water partition coefficient (aLogP),
hydrogen bond donors (HBD), number of rotatable bonds (RB), ring count (RC), electrotopological state (E-
state) and molecular polarizability (Polar) are the key properties overriding the aromatase inhibition of the
compounds as indicated by their high regression coefficient values (equation (1)). These descriptors are
strongly related to bioavailability and permeation of cell membranes, and therefore, the ability of a
compound to reach its molecular target. The QSAR model demonstrated that high values of hydrogen bond
donors (HBD) and low value of hydrogen bond acceptors (HBA) are required for the compounds to be
active.
For the training set, models generated with dendritic and linear fingerprints exhibited the best correlation
coefficients (R2 = 0.84). Dendritic model also showed the best prediction ability for the test set (Q2 = 0.79)
with RMSE value of 0.09. A plot of the experimental and predicted pIC50 values produced by the dendritic
model is shown in Figure 9. Considering these results, this model was selected to investigate how the
structure of the dataset compounds correlate with the aromatase inhibitory activity of the compounds.
Figure 9. Plots of experimental versus predicted pIC50 values of aromatase inhibitory activities generated by
(A) Multiple linear regression QSAR and (B) Kernel-Based Partial Least Squares QSAR model (compounds
are represented by red circles in training set and as blue circles in test set).
The generation of atomic contribution maps from KPLS models allows for the assessment of favorable and
unfavorable structural characteristics to reveal the most relevant structural features for the aromatase
inhibitory. Figure 10 shows the atomic contribution maps of the four compounds which showed
considerable aromatase inhibitory activity, positive contributions are depicted in red while negative
contributions are depicted in blue, white for neutral contributions, the color intensity shows the magnitude of
the effect.
12

Figure 10. Contribution maps based on the dendrite Kernel-Based Partial Least Squares QSAR model for
the active compounds in the dataset; A) compound 22; B) compound 3; C) compound 7; D) compound 23.
Positive contributions are depicted in red while negative contributions are depicted in blue, white for neutral
contributions, the color intensity shows the magnitude of the effect.
In general, the benzene ring and the sulphonamide moiety contributed positively to the activity. In
compounds 7 and 22, the aliphatic hydrocarbon chain linking the sulphonamide moiety to the indole ring
contributed favorably to the activity while the methylene group linking the benzene ring to the sulphur of the
sulphonamide in compound 22 possessed no contribution to the activity in contrast to the methylene group in
compound 7 which showed positive influence on the activity. In compound 3, the para methyl group in the
aromatic ring had a favorable contribution to the activity.
4.
Materials and Methods
4.1 Chemistry
All chemical reagents for synthesis were purchased from Sigma-Aldrich Co., and were used without further
purification. Chemical reactions were monitored by thin-layer chromatography (TLC) on F254 silica gel 60
TLC plates. Flash chromatography was performed on silica gel 60 (Merck). Melting points were determined
1
13
in open capillary tubes on a Buchi apparatus and were uncorrected. H and C Nuclear magnetic resonance
spectra were monitored on a Varian instrument 300 MHz spectrometer using tetramethylsilane as an internal
reference, and chemical shifts (δ) are reported in ppm. Elemental analyses were carried out using a Perkin-
Elmer 240 B microanalyzer and were found to be within ±0.4% of the theoretical values for C, H, and N for
all compounds. The purity of all compounds was over 98%. Table 3 lists the physical and analytical data of
the new prepared compounds.
13

Table 3. Physical and analytical data of all new synthesized compounds.
Calculated (found)%
Cmp
1
M. formula
MW
color
m.p. (°C)
C
H
N
C15H14N2O2S
286.35
light green
113.5-135.4
62.92 (61.40)
4.93 (4.78)
9.78 (9.56)
2
C14H12N2O2S
C15H14N2O2S
C14H11N3O4S
C15H13N3O4S
C15H10ClN3O2S
C15H14N2O2S
C14H12N2O2S
C15H14N2O2S
C14H11N3O4S
C15H13N3O4S
C15H10ClN3O2S
C16H16N2O2S
C15H14N2O2S
C16H16N2O2S
C15H13N3O4S
C16H15N3O4S
C16H12ClN3O2S
C16H13N3O2S
C17H18N2O4S
C13H11ClN2O2S2
C17H18N2O2S
C16H16N2O2S
C17H18N2O2S
C16H15N3O4S
C17H17N3O4S
C17H14ClN3O2S
C17H15N3O2S
C18H19N2O4S
C14H13ClN2O2S2
272.32
286.35
317.32
331.35
331.78
286.35
272.33
286.35
317.32
331.35
331.78
300.38
286.35
300.38
331.35
345.37
345.80
311.36
346.40
326.82
314.40
300.38
314.40
345.37
359.40
359.83
325.38
360.43
340.85
white
brown
190.7-191.4
159.4-161.8
197.3-197.7
210.1-211.3
158.8-161.7
165.8-167.1
162.8-163.4
158.7-161.3
174.3-174.9
173.5-174.8
197.6-198.8
n.d.
61.75 (60.84)
62.92 (62.01)
52.99 (51.94)
54.37 (53.60)
54.30 (53.59)
62.92 (61.83)
61.75 (60.29)
62.92 (61.95)
52.99 (51.74)
54.37 (53.22)
54.30 (52.98)
63.98 (61.96)
62.92 (60.89)
63.98 (62.21)
54.37 (53.01)
55.64 (53.94)
55.57 (53.79)
62.75 (61.49)
58.94 (57.58)
47.78 (46.32)
64.94 (63.21)
63.98 (62.55)
64.94 (63.45)
55.64 (54.29)
56.81 (55.20)
56.74 (55.93)
61.72 (60.37)
59.98 (58.21)
49.33 (48.26)
4.44 (4.37)
4.93 (4.82)
3.49 (3.43)
3.95 (3.89)
3.04 (3.01)
4.93 (4.86)
4.44 (4.36)
4.93 (4.79)
3.49 (3.38)
3.95 (3.86)
3.04 (2.99)
5.37 (5.22)
4.93 (4.75)
5.37 (5.23)
3.95 (3.83)
4.38 (4.27)
3.50 (3.45)
4.65 (4.57)
5.24 (5.11)
3.39 (3.31)
5.77 (5.68)
5.37 (5.31)
5.77 (5.71)
4.38 (4.26)
4.77 (4.63)
3.92 (3.89)
4.21 (4.12)
5.59 (5.48)
3.84 (3.73)
10.29 (10.03)
9.78 (9.61)
3
4
orange
orange
brown
13.24 (12.47)
12.68 (12.32)
12.67 (12.33)
9.78 (9.67)
5
6
7
pink
8
light pink
orange
yellow
10.29 (10.01)
9.78 (9.68)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
13.24 (12.97)
12.68 (12.43)
12.67 (12.46)
9.33 (9.14)
yellow
salmon
light orange
white
167.6-168.0
161.7-162.6
131.6-133.3
149.0-151.1
188.6-190.0
159.2-160.5
110.8-111.7
109.8-110.7
141.3-142.2
87.7-88.6
9.78 (9.62)
white-pink
yellow
9.33 (9.10)
12.68 (12.64)
12.17 (11.79)
12.15 (11.84)
12.91 (12.76)
8.09 (7.94)
yellow
orange
white
light yellow
orange
white
8.57 (8.40)
8.91 (8.78)
light yellow
beige
9.33 (9.14)
112.4-114.1
100.6-102.8
145.3-146.6
173.6-174.9
209.3-210.2
131.7-132.9
111.1-112.0
8.91 (8.84)
orange
light yellow
light yellow
light pink
light yellow
beige
12.17 (11.88)
11.69 (11.41)
11.68 (11.44)
13.50 (13.23)
7.77 (7.64)
8.22 (8.04)
4.1.1 General procedure for synthesis of compounds 1-30
To a stirred solution of the appropriate indolamine (1.50 mmol), and Et3N (4.50 mmol) in dry CH2Cl2 (3
mL/mmol) at 0 °C was added dropwise a solution of benzylsulfonamide (2.25 mmol) under nitrogen. The
mixture was reacted at 0°C for 2 h, and for 18-22 h at room temperature. The residue was poured into water
(20 mL) and extracted with CH2Cl2 (3 x 20 mL). The combined organic layers were dried (Na2SO4), filtered
and concentrated under reduced pressure. Purification by column chromatography on silica gel
(CH2Cl2/MeOH, 98:2) gave compounds 1-30.
14

N-(1H-indol-6-yl)-1-phenyl methanesulfonamide (1)
yield 21.1%, 1H NMR (CD3OD) δ 4.31 (s, 2H, CH2), 6.41 (d, H, Harom), 6.87 (dd, H, Harom), 7.22 (m, H,
Harom), 7.30 (m, 5H, Harom), 7.39 (s, H, Harom), 7.49 (d, H, Harom); 13C NMR (CD3OD) δ 55.86, 100.89,
103.67, 113.71, 120.31, 124.66, 127.99, 128.08, 129.54, 130.75, 131.68.
N-(1H-indol-5-yl)benzenesulfonamide (2)
1
yield 61.2%, H NMR (DMSO) δ 6.30 (m, H, Harom), 6.78 (dd, H, Harom), 7.20 (m, 2H, Harom), 7.26 (t,
2H, Harom), 7.51 (m, 3H, Harom), 7.65 (m, 2H, Harom); 13C NMR (DMSO) δ 101.52, 112.00, 114.32,
117.58, 126.69, 127.15, 128.05, 129.30, 129.41, 132.88, 134,06, 140.18.
N-(1H-indol-6-yl)-4-methyl benzenesulfonamide (3)
yield 57.4%, 1H NMR (CD3OD) δ 2.31 (s, 3H, CH3), 6.34 (d, H, Harom), 6.64 (dd, H, Harom), 7.18 (m, 4H,
13
Harom), 7.32 (d, H, Harom), 7.54 (s, H, Harom), 7.56 (s, H, Harom); C NMR (CD3OD) δ 19.97, 100.80,
105.64, 115.13, 119.86, 124.70, 125.93, 126.92, 128.92, 131.02, 136.13, 136.64, 143.21.
N-(1H-indol-6-yl)-3-nitrobenzenesulfonamide (4)
yield 48.5%, 1H NMR (CD3OD) δ 6.35 (d, H, Harom), 6.64 (dd, H, Harom), 7.17 (d, H, Harom), 7.20 (m, H,
Harom), 7.35 (d, H, Harom), 7.65 (t, H, Harom), 7.96 (m, H, Harom), 8.36 (m, H, Harom), 8.53 (t, H,
Harom); 13C NMR (CD3OD) δ 89.62, 104.81, 106.81, 109.89, 118.99, 124.11, 125.65, 129.01, 130.52,
134.01, 134.13, 136.45, 145.52.
N-(1H-indol-6-yl)-4-methyl-3-nitrobenzenesulfonamide (5)
1
yield 48.3%, H NMR (CD3OD) δ 2.55 (s, 3H, CH3), 6.35 (s, H, Harom), 6.65 (d, H, Harom), 7.18 (m, 2H,
Harom), 7.36 (d, H, Harom), 7.47 (s, H, Harom), 7.73 (d, H, Harom), 8.27 (m, H, Harom); 13C NMR
(CD3OD) δ 18.81, 100.85, 105.84, 115.00, 120.13, 122.98, 125.00, 126.27, 130.41, 130.69, 133.22, 136.12,
137.83, 138.89.
2-chloro-4-cyano-N-(1H-indol-6-yl)benzenesulfonamide (6)
yield 50.8%, 1H NMR (CD3OD) δ 6.32 (d, H, Harom), 6.76(dd, H, Harom), 7.16 (d, H, Harom), 7.22 (m, H,
Harom), 7.34 (d, H, Harom), 7.67 (dd, H, Harom), 7.99 (d, H, Harom), 8.04 (m, H, Harom); 13C NMR
(CD3OD) δ 100.83, 105.58, 110.00, 114.82, 115.87, 117.11, 120.12, 120.74, 125.03, 130.43, 132.41, 134.58,
155.11, 165.49.
N-(1H-indol-5-yl)-1-phenylmethanesulfonamide (7)
yield 47.1%, 1H NMR (CD3OD) δ 4.28 (s, 2H, CH2), 6.43 (d, H, Harom), 7.02 (dd, H, Harom), 7.29 (m, 7H,
Harom), 7.47 (d, H, Harom); 13C NMR (CDCl3) δ 55.87, 101.06, 111.24, 111.30, 113.49, 116.82, 125.55,
125.71, 127.96, 128.09, 128.38, 128.41, 129.16, 129.57, 130.75.
N-(1H-indol-6-yl)benzenesulfonamide (8)
1
yield 57.4%, H NMR (DMSO) δ 6.28 (s, H, Harom), 6.71 (dd, H, Harom), 7.13 (s, H, Harom), 7.22 (t, H,
Harom), 7.32 (d, H, Harom), 7.51 (m, 4H, Harom), 7.68 (dd, H, Harom); 13C NMR (CDCl3) δ 101.34,
104.95, 114.73, 120.62, 125.38, 125.91, 127.10, 129.48, 131.65, 133.01, 136.23, 140.13.
15

N-(1H-indol-5-yl)-4-methyl benzenesulfonamide (9)
1
yield 55.0%, H NMR (CD3OD) δ 2.39 (s, 3H, CH3), 4.07 (s, 2H, CH2), 6.33 (d, H, Harom), 6.93 (dd, H,
Harom), 7.24 (d, 2H, Harom), 7.29 (m, 3H, Harom), 7.68 (m, 2H, Harom); 13C NMR (CD3OD) δ 20.51,
47.63, 101.41, 111.19, 119.83, 121.69, 125.24, 127.01, 127.85, 129.30, 129.47, 135.72, 142.76.
N-(1H-indol-5-yl)-3-nitrobenzenesulfonamide (10)
yield 45.7%, 1H NMR (CD3OD) δ 6.32 (d, H, Harom), 6.79 (dd, H, Harom), 7.22 (m, 3H, Harom), 7.65 (t, H,
Harom), 7.92 (m, H, Harom), 8.35 (m, H, Harom), 8.48 (t, H, Harom); 13C NMR (CD3OD) δ 101.05, 108.17,
111.11, 115.48, 117.98, 121.75, 125.58, 126.51, 127.83, 130.00, 132.56, 141.53, 156.01.
N-(1H-indol-5-yl)-4-methyl-3-nitrobenzenesulfonamide (11)
yield 47.6%, 1H NMR (CD3OD) δ 2.54 (s, 3H, CH3), 6.33 (d, H, Harom), 6.80 (dd, H, Harom), 7.22 (m, 3H,
Harom), 7.44 (d, H, Harom), 7.69 (dd, H, Harom), 8.22 (s, H, Harom); 13C NMR (CD3OD) δ 18.81, 101.05,
111.10, 115.39, 117.95, 123.00, 125.55, 127.94, 128.19, 130.73, 133.19, 134.58, 137.75, 138.85, 148.89.
2-chloro-4-cyano-N-(1H-indol-5-yl)benzenesulfonamide (12)
yield 38.8%, 1H NMR (CD3OD) δ 6.32 (d, H, Harom), 6.79 (dd, H, Harom), 7.22 (m, 3H, Harom), 7.65 (t, H,
Harom), 7.92 (m, H, Harom), 8.35 (m, H, Harom), 8.48 (t, H, Harom); 13C NMR (CD3OD) δ 101.05, 108.17,
111.11, 115.48, 117.98, 121.75, 125.58, 126.51, 127.83, 130.00, 132.56, 141.53, 156.01.
N-((1H-indol-5-yl)methyl)-1-phenylmethanesulfonamide (13)
1
yield 40.2%, H NMR (CDCl3) δ 4.20 (s, 2H, CH2), 4.25 (d, 2H, CH2), 6.53 (m, H, Harom), 7.11 (dd, H,
Harom), 7.22 (m, 4H, Harom), 7.34 (m, 3H, Harom), 7.52 (s, H, Harom); 13C NMR (CDCl3) δ 48.32, 59.26,
102.65, 104.45, 111.53, 120.59, 122.40, 125.134, 129.99, 128.70, 128.77, 129.28, 130.69, 135.42.
N-((1H-indol-5-yl)methyl)benzenesulfonamide (14)
1
yield 56.7%, H NMR (CD3OD) δ 4.10 (s, 2H, CH2), 6.34 (d, H, Harom), 6.93 (dd, H, Harom), 7.18 (d, H,
Harom), 7.25 (d, H, Harom), 7.34 (s, H, Harom), 7.52 (m, 3H, Harom), 7.82 (m, 2H, Harom); 13C NMR
(CD3OD) δ 100.88, 110.73, 119.51, 121.24, 124.63, 126.60, 126.61, 127.21, 127.96, 128.64, 131.98, 140.76.
N-((1H-indol-5-yl)methyl)-4-methylbenzenesulfonamide (15)
1
yield 62.6%, H NMR (CDCl3) δ 2.44 (s, 3H, CH3), 4.19 (d, 2H, CH2), 6.47 (t, H, Harom), 7.02 (d, H,
Harom), 7.21 (t, H, Harom), 7.32(d, 2H, Harom), 7.41 (s, 2H, Harom), 7.79 (d, 2H, Harom); 13C NMR
(CDCl3) δ 21.58, 47.94, 102.60, 111.37, 120.41, 122.27, 124.94, 127.26, 129.73, 136.85, 143.08.
N-((1H-indol-5-yl)methyl)-3-nitrobenzenesulfonamide (16)
1
yield 46.4%, H NMR (CD3OD) δ 4.23 (s, 2H, CH2), 6.24 (d, H, Harom), 6.84 (dd, H, Harom), 7.12 (d, H,
Harom), 7.14 (d, H, Harom), 7.22 (s, H, Harom), 7.51 (t, H, Harom), 8.00 (m, H, Harom), 8.11 (m, H,
Harom), 8.31 (t, H, Harom); 13C NMR (Acetone-D6) δ 47.77, 101.34, 111.16, 120.15, 121.76, 124.83,
125.38, 126.28, 127.04, 130.49, 132.59, 135.43, 143.49, 147.55.
N-((1H-indol-5-yl)methyl)-4-methyl-3-nitrobenzenesulfonamide (17)
1
yield 47.7%, H NMR (CD3OD) δ 2.43 (s, 3H, CH3), 4.22 (s, 2H, CH2), 6.24 (d, H, Harom), 6.86 (dd, H,
Harom), 7.15 (m, 3H, Harom), 7.28 (d, H, Harom), 7.73 (dd, H, Harom), 8.08 (d, H, Harom),; 13C NMR
16

(CD3OD) δ 18.78, 45.73, 100.77, 110.64, 119.81, 121.38, 122.80, 124.78, 126.37, 127.80, 130.28, 132.96,
135.29, 137.06, 140.82.
N-((1H-indol-5-yl)methyl)-2-chloro-4-cyanobenzenesulfonamide (18)
1
yield 44.0%, H NMR (CD3OD) δ 4.27 (s, 2H, CH2), 6.26 (d, H, Harom), 6.86 (dd, H, Harom), 7.06 (d, H,
Harom), 7.18 (m, 2H, Harom), 7.34 (dd, H, Harom), 7.52 (d, H, Harom), 7.76 (d, H, Harom); 13C NMR
(CD3OD) δ 101.34, 111.16, 120.15, 121.76, 124.83, 125.38, 126.28, 127.04, 130.49, 132.59, 135.43, 143.49,
147.55.
N-((1H-indol-5-yl)methyl)-4-cyanobenzenesulfonamide (19)
1
yield 70.6%, H NMR (DMSO) δ 4.06 (s, 2H, CH2), 6.31 (s, H, Harom), 6.90 (d, H, Harom), 7.23 (d, H,
Harom), 7.29 (s, 2H, Harom), 7.92 (m, 4H, Harom); 13C NMR (DMSO) δ 47.45, 101.34, 111, 62, 114.91,
118.24, 119.95, 121.69, 126.19, 127.56, 127.64, 127.82, 133.59, 135.59, 145.62.
N-((1H-indol-5-yl)methyl)-2,4-dimethoxybenzenesulfonamide (20)
1
yield 77.2%, H NMR (CDCl3) δ 3.72 (s, 3H, CH3), 3.84 (s, 3H, CH3), 4.11 (d, 2H, CH2), 6.36 (d, H,
Harom), 6.44 (m, H, Harom), 6.54 (dd, H, Harom), 6.96 (dd, H, Harom), 7.20 (t, H, Harom), 7.26 (s, H,
Harom), 7.30 (d, H, Harom), 7.89 (d, H, Harom); 13C NMR (CDCl3) δ 48.41, 55.69, 56.02, 99.24, 102.52,
104.34, 111.10, 119.63, 120.37, 122.30, 124.84, 127.71, 127.85, 132.23, 135.23, 157.49, 164.68.
N-((1H-indol-5-yl)methyl)-5-chlorothiophene-2-sulfonamide (21)
1
yield 58.5%, H NMR (CD3OD) δ 4.21 (s, 2H, CH2), 6.37 (d, H, Harom), 6.93 (d, H, Harom), 6.97 (dd, H,
Harom), 7.20 (d, H, Harom), 7.29 (m, 2H, Harom), 7.38 (m, H, Harom); 13C NMR (CDCl3) δ 48.23, 102.66,
111. 50, 120.51, 122.21, 125.09, 126.69, 126.83, 127.99, 131.70, 135.45, 138.97.
N-(2-(1H-indol-5-yl)ethyl)-1-phenylmethanesulfonamide (22)
1
yield 50.1%, H NMR (CDCl3) δ 2.97 (t, 2H, CH2), 3.31 (q, 2H, CH2), 4.16 (s, 2H, CH2), 6.98 (m, H,
Harom), 7.15 (m, H, Harom), 7.23 (m, 6H, Harom), 7.37 (s, H, Harom), 7.55 (s, H, Harom); 13C NMR
(CDCl3) δ 26.18, 43.53, 58.33, 111.42, 111.53, 118.54, 119.70, 122.45, 122.71, 126.84, 128.61, 128.75,
129.24, 130.47, 136.49.
N-(2-(1H-indol-3-yl)ethyl)benzenesulfonamide (23)
1
yield 75.3%, H NMR (CDCl3) δ 2.94 (t, 2H, CH2), 3.29 (q, 2H, CH2), 6.97 (d, H, Harom), 7.06 (t, H,
Harom), 7.20 (m, H, Harom), 7.35 (m, H, Harom), 7.44 (m, 2H, Harom), 7.53 (m, 2H, Harom), 7.76 (m, 2H,
Harom); 13C NMR (CDCl3) δ 25.54, 43.07, 111.31, 111.53, 118.48, 119.63, 122.36, 122.59, 126.84, 126.95,
129.04, 132.56, 136.42, 139.81.
N-(2-(1H-indol-3-yl)ethyl)-4-methylbenzenesulfonamide (24)
1
yield 88.2%, H NMR (CDCl3) δ 2.40 (s, 3H, CH3), 2.93 (t, 2H, CH2), 3.27 (q, 2H, CH2), 6.97 (d, H,
Harom), 7.06 (t, H, Harom), 7.20 (m, 3H, Harom), 7.40 (s, 2H, Harom), 7.61 (m, H, Harom), 7.64 (m, H,
13
Harom); C NMR (CDCl3) δ 21.53, 25.48, 43.00, 111.31, 111.54, 118.51, 119.55, 122.30, 122.62, 126.81,
127.01, 129.65, 136.36, 136.68, 143.34.
N-(2-(1H-indol-3-yl)ethyl)-3-nitrobenzenesulfonamide (25)
17

1
yield 78.1%, H NMR (CDCl3) δ 2.95 (t, 2H, CH2), 3.36 (q, 2H, CH2), 6.98 (m, 2H, Harom), 7.15 (m, H,
Harom), 7.30 (m, H, Harom), 7.48 (t, H, Harom), 7.96 (m, H, Harom), 8.03 (s, H, Harom), 8.22 (m, H,
Harom), 8.42 (m, H, Harom); 13C NMR (CDCl3) δ 25.42, 42.98, 111.04, 111.38, 118.21, 119.67, 121.89,
122.46, 122.77, 126.49, 126.78, 129.94, 132.34, 136.33, 141.57, 147.85.
N-(2-(1H-indol-3-yl)ethyl)-4-methyl-3-nitrobenzenesulfonamide (26)
1
yield 80.6%, H NMR (CDCl3) δ 2.60 (s, 3H, CH3), 2.96 (t, 2H, CH2), 3.35 (q, 2H, CH2), 7.01 (m, 2H,
Harom), 7.16 (m, H, Harom), 7.30 (m, 2H, Harom), 7.72 (dd, H, Harom), 8.03 (m, H, Harom), 8.22 (m, H,
Harom); 13C NMR (CDCl3) δ 20.71, 25.41, 42.93,111.10, 111.36, 118.28, 119.63, 122.39, 122.72, 123.29,
126.51, 130.60, 133.49, 136.36, 138.16, 138.96.
N-(2-(1H-indol-3-yl)ethyl)-2-chloro-4-cyanobenzenesulfonamide (27)
1
yield 71.4%, H NMR (CD3OD) δ 2.85 (t, 2H, CH2), 3.42 (t, 2H, CH2), 6.89 (m, H, Harom), 7.03 (m, H,
Harom), 7.18 (m, H, Harom), 7.23 (m, H, Harom), 7.33 (m, H, Harom), 7.51 (m, H, Harom), 7.73 (m, H,
Harom); 13C NMR (CD3OD) δ 25.19, 43.13, 110.41, 111.04, 115.83, 116.26, 117.63, 118.16, 120.86, 123.11,
129.83, 129.89, 131.47, 133.91, 141.95.
N-(2-(1H-indol-3-yl)ethyl)-4-cyanobenzenesulfonamide (28)
1
yield 85.6%, H NMR (DMSO) δ 2.77 (t, 2H, CH2), 3.05 (t, 2H, CH2), 6.91 (t, H, Harom), 7.02 (m, H,
Harom), 7.07 (s, H, Harom), 7.27 (d, H, Harom), 7.33 (d, H, Harom), 7.85 (m, 2H, Harom), 7.92 (m, 2H,
Harom); 13C NMR (DMSO) δ 25.75,43.69, 111.04, 111.76, 115.07, 118.19, 118.35, 118.68, 121.32, 123.32,
127.27, 127.45, 133.54, 136.41, 145.05.
N-(2-(1H-indol-3-yl)ethyl)-2,4-dimethoxybenzenesulfonamide (29)
yield 62.2%, 1H NMR (CDCl3) δ 2.97 (t, 2H, CH2), 3.10 (s, 3H, CH3), 3.15 (t, 2H, CH2), 3.82 (s, 3H, CH3),
6.17 (d, H, Harom), 6.52 (dd, H, Harom), 7.03 (m, H, Harom), 7.17 (dd, H, Harom), 7.35 (t, 2H, Harom),
7.80 (d, H, Harom), 8.19 (m, H, Harom); 13C NMR (CDCl3) δ 24.93, 42.99, 55.12, 55.66, 98.87, 104.25,
111.33, 111.53, 118.33, 118.56, 119.49, 122.25, 123.03, 126.80, 132.14, 136.61, 157.25, 164.66.
N-(2-(1H-indol-3-yl)ethyl)-5-chlorothiophene-2-sulfonamide (30)
1
yield 59.9%, H NMR (CDCl3) δ 3.00 (t, 2H, CH2), 3.38 (q, 2H, CH2), 6.81 (d, H, Harom), 7.02 (t, H,
Harom), 7.10 (t, H, Harom), 7.20 (m, H, Harom), 7.24 (m, H, Harom), 7.38 (d, H, Harom), 7.46 (d, H,
Harom); 13C NMR (CDCl3) δ 25.39, 43.24, 103.97, 104.96, 111.37, 118.42, 119.75, 122.40, 122.51, 122.62,
126.57, 131.39, 138.64.
4.2 Biological Assays
4.2.1 Aromatase activity inhibition assay and calculation of the IC50
The aromatase inhibitory activity of the new compounds was determined using a commercial fluorimetric
assay (Aromatase-CYP19A Inhibitor Screening kit, BioVision, Milpitas, CA, USA). The assay utilizes a
fluorogenic aromatase substrate that is converted into a highly fluorescent metabolite detected in the visible
range (Ex/Em = 488/527 nm), ensuring a high signal-to-background ratio with little interference by
autofluorescence. Briefly, each inhibitor and the provided letrozole were diluted in acetonitrile (ACN) from a
18

stock solution of 1 mM to a final concentration of 5 µM (5X solution) and kept on ice. In parallel, the
provided recombinant human aromatase stock (2X) was reconstituted with 1 ml of Aromatase Assay Buffer,
mixed thoroughly by vortexing and transferred to a 15 ml conical tube. After having brought the volume up
to 2450 µl with Aromatase Assay Buffer and having added 50 µl of the NADPH Generating System (100X)
for a final total volume of 2.5 ml, 50 µl of this solution were afterwards pipetted into each well. Then 20 µl
of test inhibitors and letrozole 5 µM were added. No inhibitor control (only assay bugger) and solvent
control (0.5% of ACN in assay buffer) as well as a background control (containing no fluorogenic Aromatase
Substrate) were added to the experiment. The plate was incubated for 10 minutes at 37° C to allow test
ligands to interact with aromatase. During the incubation, Aromatase Substrate/NADP+ mixture (3X) was
prepared by adding 6 µl of 1 mM Aromatase Substrate stock solution and 50 µl of 10 mM β-NADP+ Stock
(100X) to 1444 µl of Aromatase Assay Buffer for a total volume of 1.5 ml. The reaction was started by
adding 30 µl of the Aromatase Substrate/NADP+ (3X) mixture to each well (aside from the background
control) reaching a final reaction volume of 100 µl/well. The fluorescence at Ex/Em = 488/527 nm in kinetic
mode for 60 min was measured immediately (within 1 min) by means of a fluorometer equipped with a 488
nm filter (GloMax® - Multi Detection System, Promega, Madison, WI, USA).
To determine the IC50 values for the best tested inhibitors, 5X test compound solutions were prepared in a
range of concentrations (0.05-500 µM in ACN) in order to generate a multi-point dose-response curve (0.01-
100 µM). The assay was performed as for the inhibitory activity in the same experimental conditions.
Dose-response curves were fitted with GraphPad Prism 5.0 [37].
4.2.2 Cell viability assay (MTT)
Human breast cancer MCF7 (HTB-22 ™) cells were purchased from ATCC and maintained in Dulbecco’s
modified Eagle’s medium (DMEM) high glucose (EuroClone, Milan, Italy) supplemented with 10% of fetal
bovine serum (FBS) and 1% of penicillin/streptomycin (Gibco - Thermofisher Scientific, MD, USA).
Cell viability of MCF7 human breast cancer cells was assessed by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-
Diphenyltetrazolium Bromide) test (Sigma Aldrich, Milan, Italy). Cells were seeded (1x104/well) in a 96-
well tissue culture-treated plate (Falcon®, Corning Incorporated, NY, USA) and let them adhere for 24 h.
Next, medium was removed and the cell monolayer was incubated in the presence of loading concentrations
of compounds 3, 7, 22 and 23 (0-250 µM) for 24, 48 and 72 h. After the exposure time, cells were incubated
with 100 µl/well of MTT (1mg/ml) 1:10 with fresh growth medium for 4 h at 37°C and 5% CO2. Finally, the
MTT solution was removed and replaced with 100 µl/well of DMSO. Cells were incubated for additional 20
minutes at 37°C and 5% CO2 and gently swirled for 10 minutes at room temperature. The optical density was
measured at 540 nm by means of a spectrophotometer (Multiskan GO, Thermo Scientific, Monza, Italy). The
results were expressed as the percentage of DMSO treated cells (final concentration of DMSO < 0.2%) and
each experiment was performed three times in triplicate (n=9).
4.2.3 Cytotoxicity test (lactate dehydrogenase release)
19

MCF7 cells were seeded and stimulated as previously described for the MTT test. After the exposure time,
cell supernatants were collected, centrifuged at 450xg for 4 minutes and stored on ice. In order to quantify
the cytotoxicity of loading concentrations of compounds 3, 7, 22 and 23 (0-250 µM), the CytoTox 96® Non-
Radioactive Cytotoxicity Assay (Promega Corporation, WI, USA) was performed. The CytoTox 96® Assay
quantitatively measures lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released upon cell
lysis. Released LDH in culture supernatants is measured with a 30-minute coupled enzymatic assay, which
results in the conversion of a tetrazolium salt (iodonitrotetrazolium violet; INT) into a red formazan product.
The absorbance signal was measured at 490 and 690 nm with a spectrophotometer (Multiskan GO, Thermo
Scientific, Monza, Italy). The results were expressed according to the formula: %LDH releasedꢀ=ꢀ[(A–B)/(C–
B)] ×100, with Aꢀ=ꢀLDH activity measured at 490 nm, Bꢀ=ꢀLDH activity measured at 690 nm (background)
and Cꢀ=ꢀLDH activity of the positive control (cell lysate).
4.3
Docking Study Assay
All prediction and processes were performed on an Intel i7-core workstation with 2.5+GHz, 8 GB of RAM,
and a high-end RADEON graphical processing unit. All programs run on the Windows 10 Pro platform.
Structure-based virtual screening (SBVS) was performed using Schrodinger Maestro 9.1 software package
(LLC, New York, NY).
4.3.1 Ligands preparation for docking study
The structures of compounds 3, 7, 22 and 23 (having IC50 values of 0.49, 0.16, 0.75 and 0.20 µM,
respectively) were drawn using ChemDraw® Professional software 17.1. (PerkinElmer’s). The structures
were saved as (.Mol) files.
Ligands were optimized and energetically minimised through OPLS 3 force field algorithm embedded in the
LigPrep module of Schrödinger suite. The ionization states of the ligands were predicted at pH 7 +/- 2 and
Epik tool was selected to generate tautomers. Ligands were then desalted and specified chiralities were
retained. Structures of unidentified stereochemistry are automatically subjected to all possible
conformational changes for selection of best pose. The optimized structures were saved as maestro format
(.mae) to retain 3D conformers.
4.3.2 Preparation of protein structure
X-ray crystal structures of human placental aromatase complexed with natural ligand, androstenedione (AD)
(PDB ID: 3EQM) was imported (as .pdb file) from the RCSB Protein Data Bank (PDB)
(http://www.rcsb.org/). Preparation of 3EQM structure was performed using the PrepWiz module
implemented in Schrodinger suite. The protein was first preprocessed where bond orders were assigned. All
the water molecules beyond 5 Å from the active site were deleted. Iron states of heme group at PH 7±3 were
generated and the state with lowest penalty was selected. Hydrogen bonds were assigned using PROPKA
20

function at PH 7 finally, energy was minimized using OPLS 3 force field. For grid generation; a box
enclosing the centroid of co-crystallized ligand (AD) was set as the grid.
4.3.3 Molecular docking
The prepared ligands were flexibily docked using GLIDE docking with extra precision (XP) mode. Maestro
interface was implemented to investigate the 2D and 3D interactions between the docked ligands and the
target such as hydrogen bonding, ion-pairing and hydrophobic interactions.
The co-crystallized ligand (AD) was redocked to the aromatase binding cavity for validation of the docking
process. Root Mean Square Deviation (RMSD) was used for evaluation of redocked results.
4.3.4 Physicochemical Descriptors and Multiple linear regression QSAR analysis
The physicochemical properties of the compounds were calculated using Canvas 2.9 (Schrodinger LLC, New
York, NY, USA). The following descriptors were calculated: MW, aLogP, hydrogen bond acceptor
(
HBA), hydrogen bond donors (HBD)
electrotopological state (E-state) molar refractivity (MR)
Atom Coun HAC), and ring count (RC). The calculated physicochemical descriptors and pIC50 values for
,
number of rotatable bonds (RB)
, polar surface area (PSA),
,
,
molecular polarizability (Polar), Heavy
(
the training set were used to construct MLR models using Canvas (Schrodinger LLC, New York, NY, USA).
The physicochemical data of the new prepared compounds are listed in Table 4.
Table 4. Physicochemical data of all new synthesized compounds.
Cmp
1
M. formula
MW
aLogP HBA HBD RB HAC RC PSA E-state
MR
Polar
C15H14N2O2S
C14H12N2O2S
C15H14N2O2S
C14H11N3O4S
C14H10N3O4S-
C15H13N3O4S
C15H12N3O4S-
C15H10ClN3O2S
C15H9ClN3O2S-
C15H14N2O2S
C14H12N2O2S
C14H11N2O2S-
C15H14N2O2S
C14H11N3O4S
C14H10N3O4S-
C15H13N3O4S
C15H12N3O4S-
C15H10ClN3O2S
C15H9ClN3O2S-
C16H16N2O2S
C15H14N2O2S
286.3489
272.3223
286.3489
317.3198
316.3119
331.3464
330.3385
331.7768
330.7689
286.3489
272.3223
271.3143
286.3489
317.3198
316.3119
331.3464
330.3385
331.7768
330.7689
300.3754
286.3489
2.1023
2.0954
2.5816
1.9898
2.6047
2.476
2
2
2
2
2
2
2
3
3
2
2
2
2
2
2
2
2
3
3
2
2
2
2
2
2
1
2
1
2
1
2
2
1
2
2
1
2
1
2
1
2
2
4
3
3
4
4
4
4
3
3
4
3
3
3
4
4
4
4
3
3
5
4
20
19
20
22
22
23
23
22
22
20
19
19
20
22
22
23
23
22
22
21
20
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
70.34
70.34
70.34
113.48
101.45
113.48
101.45
94.13
82.1
48.085
46.585
48.252
62.252
59.752
63.919
61.419
58.53
78.1035
73.2685
78.3097
80.5932
77.2932
85.6344
82.3344
83.811
38.855
37.02
2
3
38.855
38.566
38.179
40.401
40.014
40.842
40.455
38.855
37.02
4
4(-)
5
5(-)
6
3.0909
2.6387
3.2536
2.1023
2.0954
2.7103
2.5816
1.9898
2.6047
2.476
6(-)
7
56.03
80.511
70.34
70.34
58.31
70.34
113.48
101.45
113.48
101.45
94.13
82.1
48.085
46.585
44.085
48.252
62.252
59.752
63.919
61.419
58.53
78.1035
73.2685
69.9685
78.3097
80.5932
77.2932
85.6344
82.3344
83.811
8
8(-)
9
36.633
38.855
38.566
38.179
40.401
40.014
40.842
40.455
40.69
10
10(-)
11
11(-)
12
12(-)
13
14
3.0909
2.6387
3.2536
2.1092
2.1023
56.03
80.511
70.34
70.34
49.585
48.085
82.9385
78.1035
38.855
21

15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
C16H16N2O2S
C15H13N3O4S
C16H15N3O4S
C16H12ClN3O2S
C16H13N3O2S
C17H18N2O4S
C13H11ClN2O2S2
C17H18N2O2S
C16H16N2O2S
C17H18N2O2S
C16H15N3O4S
C17H17N3O4S
C17H14ClN3O2S
C17H15N3O2S
C18H19N2O4S
C14H13ClN2O2S2
300.3754
331.3464
345.373
345.8034
311.3583
346.4008
326.8216
314.402
300.3754
314.402
345.373
359.3996
359.83
2.5885
1.9967
2.4829
2.6456
1.9812
2.0695
2.8178
2.4304
2.4235
2.9097
2.3179
2.8041
2.9668
2.3024
2.3907
3.139
2
2
2
3
3
4
2
2
2
2
2
2
3
3
4
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
4
5
5
4
4
6
4
6
5
5
6
6
5
6
7
5
21
23
24
23
22
24
20
22
21
22
24
25
24
23
25
21
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
70.34
113.48
113.48
94.13
94.13
88.8
49.752
63.752
65.419
60.03
83.1447
85.4282
90.4694
88.646
40.69
40.401
42.236
42.677
40.707
43.799
39.259
42.525
40.69
56.252
58.419
49.696
51.085
49.585
51.252
65.252
66.919
61.53
83.8412
91.0299
81.3704
87.6931
82.8581
87.8993
90.1828
95.224
98.58
70.34
70.34
70.34
113.48
113.48
94.13
94.13
88.8
42.525
42.236
44.071
44.512
42.542
45.634
41.094
93.4006
88.5958
95.7845
86.125
325.3849
360.4274
340.8482
57.752
59.919
51.196
98.58
4.3.5 Molecular Fingerprints and 2D Contribution Maps
Canvas molecular fingerprints (Schrodinger LLC, New York, NY, USA) was used for construction of KPLS
models. Four types of fingerprints were explored: linear, dendritic, radial, and molprint2D [38, 39]. Dendritic
KPLS model, showed the best results and was thus used to generate the 2D contribution maps in which red,
and blue indicated positive and negative contributions, respectively.
5.
Conclusions
The pharmacological treatment of early- and advanced-stage breast cancer in post-menopausal women
makes use of aromatase inhibitors. Although AIs are currently in clinical use, the development of new
molecules with more clinical efficacy and minimal side effects is challenging.
A series of thirty aryl sulfonamide indole derivatives structurally related to SYN20028567 was synthesized
and valued for their ability to inhibit aromatase enzyme (CYP19A1). The synthesis of the compounds was a
one-step chemical process starting from commercially available products. The evaluation of the ability to
inhibit the aromatase enzyme revealed that four compounds (3, 7, 22, and 23) showed IC50 values in the
range of sub-micromolar (0.49, 0.16, 0.75 and 0.20 µM, respectively). The cell viability (MTT) and
cytotoxicity (LDH release) assays on MCF7 human breast cancer cells demonstrated a time- and dose-
dependent decrease of active metabolizing cells over the time of the culture (0-72 h), starting from a
concentration of 100 µM. The docking study revealed that all the best active compounds interact with the key
residues in the active site of the aromatase. The fundamental characteristics that emerged from this series of
compounds are the presence of an indole nucleus capable of interacting with the heme of the aromatase
enzyme, the presence of a sulfonamide linker for hydrogen bonding and the presence of an unsubstituted
benzene or replaced with a para-methyl group that interact with a hydrophobic pocket. In particular, the
22

QSAR model demonstrated that high values of hydrogen bond donors (HBD) and low value of hydrogen
bond acceptors (HBA) are required for the compounds to be active.
Thus, novel aryl sulfonamide derivatives have emerged as aromatase inhibitors and may be used as potential
lead for the development of more potent AIs.
Author Information
Corresponding Author
Fantacuzzi
Marialuigia:
phone,
+39-0871-3554684;
fax,
+39-0871-3554911;
Email,
marialuigia.fantacuzzi@unich.it
ORCID
Fantacuzzi Marialuigia: 0000-0003-2401-7547
Funding
We gratefully acknowledge the financial support from University ‘G. d’Annunzio’ of Chieti local grants.
Conflicts of Interest
The authors declare no conflicts of interest.
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26

Highlights:
•
•
Aromatase inhibitors are used in postmenopausal women for hormone-responsive breast cancer.
The use of aromatase inhibitors, usually divided in steroidal and non-steroidal, increased drastically
over the years.
•
•
New chemical entities that could overpass the limitations of the third generation of aromatase
inhibitors are required.
Four novel indole aryl sulfonamides were identified as aromatase inhibitors.

The authors declare no conflicts of interest.