Synthesis, molecular docking, and QSAR study of sulfonamide-based indoles as aromatase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.10.057

Thirty four of indoles bearing sulfonamides (11–44) were synthesized and evaluated for their anti-aromatase activities. Interestingly, all indole derivatives inhibited the aromatase with IC₅₀ range of 0.7–15.3 μM. Indoles (27–36) exerted higher aromatase inhibitory activity than that of ketoconazole. The phenoxy analogs 28 and 34 with methoxy group were shown to be the most potent compounds with sub-micromolar IC₅₀ values (i.e., 0.7 and 0.8 μM, respectively) without affecting to the normal cell line. Molecular docking demonstrated that the indoles 28, 30 and 34 could occupy the same binding site on the aromatase pocket and share several binding residues with those of the natural substrate (androstenedione), which suggested the competitive binding could be the mode of inhibition of the compounds. The most potent analog 28 could mimic H-bond interactions of the natural androstenedione with MET374 and ASP309 residues on the aromatase. QSAR model also revealed that the para-phenoxy indole (28) affords the higher value of electronegativity descriptor MATS6e as well as the higher inhibitory activity compared with that of the ortho-phenoxy compound (34). The study highlighted a series of promising indoles to be potentially developed as novel aromatase inhibitors for therapeutics.

Full text of DOI:10.1016/j.ejmech.2017.10.057

Accepted Manuscript
Synthesis, molecular docking, and QSAR study of sulfonamide-based indoles as
aromatase inhibitors
Ratchanok Pingaew, Prasit Mandi, Veda Prachayasittikul, Supaluk Prachayasittikul,
Somsak Ruchirawat, Virapong Prachayasittikul
PII:
S0223-5234(17)30859-0
10.1016/j.ejmech.2017.10.057
EJMECH 9849
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 June 2017
Revised Date: 27 August 2017
Accepted Date: 19 October 2017
Please cite this article as: R. Pingaew, P. Mandi, V. Prachayasittikul, S. Prachayasittikul, S.
Ruchirawat, V. Prachayasittikul, Synthesis, molecular docking, and QSAR study of sulfonamide-
based indoles as aromatase inhibitors, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.10.057.
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ACCEPTED MANUSCRIPT
Compound 28: anti-aromatase activity
IC50 0.7 µM without affecting to the normal cell line
Compound 34: anti-aromatase activity
IC50 0.8 µM without affecting to the normal cell line

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Synthesis, molecular docking, and QSAR study of sulfonamide-
based indoles as aromatase inhibitors
Ratchanok Pingaew a,*, Prasit Mandi b, Veda Prachayasittikul c,*, Supaluk
Prachayasittikul c, Somsak Ruchirawat d,e,f, Virapong Prachayasittikul g
aDepartment of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok 10110,
Thailand
bDepartment of Community Medical Technology, Faculty of Medical Technology, Mahidol
University, Bangkok 10700, Thailand
cCenter of Data Mining and Biomedical Informatics, Faculty of Medical Technology,
Mahidol University, Bangkok 10700, Thailand
dChulabhorn Research Institute, Bangkok 10210,Thailand
eProgram in Chemical Biology, Chulabhorn Graduate Institute, Bangkok 10210,Thailand
fCenter of Excellence on Environmental Health and Toxicology (EHT), CHE, Ministry
of Education, Thailand
gDepartment of Clinical Microbiology and Applied Technology, Faculty of Medical
Technology, Mahidol University, Bangkok 10700, Thailand
*Corresponding authors:
E-mail: ratchanok@swu.ac.th (R. Pingaew)
Tel: 662-649-5000 ext 18253; Fax: 662-260-0128
E-mail: veda.pra@mahidol.ac.th (V. Prachayasittikul)
Tel: 662-441-4376; Fax: 662-441-4380

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Abstract
Thirty four of indoles bearing sulfonamides (11-44) were synthesized and evaluated
for their anti-aromatase activities. Interestingly, all indole derivatives inhibited the aromatase
with IC50 range of 0.7-15.3 µM. Indoles (27-36) exerted higher aromatase inhibitory activity
than that of ketoconazole. The phenoxy analogs 28 and 34 with methoxy group were shown
to be the most potent compounds with sub-micromolar IC50 values (i.e., 0.7 and 0.8 µM,
respectively) without affecting to the normal cell line. Molecular docking demonstrated that
the indoles 28, 30 and 34 could occupy at the same binding site on the aromatase pocket and
share several binding residues with those of the natural substrate (androstenedione), which
suggested the competitive binding could be the mode of inhibition of the compounds. The
most potent analog 28 could mimic H-bond interactions of the natural androstenedione with
MET374 and ASP309 residues on the aromatase. QSAR model also revealed that the para-
phenoxy indole (28) affords the higher value of electronegativity descriptor MATS6e as well
as the higher inhibitory activity compared with that of the ortho-phenoxy compound (34).
The study highlighted a series of promising indoles to be potentially developed as novel
aromatase inhibitors for therapeutics.
Keywords: Indole; Sulfonamide; Anti-aromatse activity; Molecular docking; QSAR

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1. Introduction
Aromatase, a cytochrome P450 enzyme complex present in breast tissues, plays a
significant role in the biosynthesis of estrogen from androgen [1-3]. Inhibition of the
aromatase is a crucial strategy to prevent the growth stimulation effect of estrogens in breast
cancer tissues in postmenopausal women [4,5]. So, aromatase inhibitors (AIs) have been
developed and used as anti-breast cancer agents. AIs can be catagorized by their mechanisms
of action into two types, steroidal and nonsteroidal aromatase inhibitors. The former type of
agents are androstenedione analogs which may bind to the active site of the aromatase via
irreversible process either by a mechanism-based or by a competitive manner via covalent
interactions. The latter AIs interact reversibly to the active site of the aromatase through
noncovalent interaction [3,6,7]. Di- and triarylmethanes are core structures regularly found in
the most potent nonsteroidal AIs such as letrozole (1) and anastrozole (2) bearing triazole
ring as well as liarozole (3) containing imidazole and benzimidazole rings [1]. Generally, the
important structural feature of the nonsteroidal AIs is represented by a heterocyclic nitrogen
atom, which interacts with the heme iron of the aromatase enzyme while the rest of the
molecule interacts with amino acid residues of the active site [1,8,9].
The indole scaffold, defined as a privileged structure, is one of the most important
heterocyclic systems for pharmaceutical applications [10]. A simple naturally occurring
indole i.e., indole-3-carbinol (4) is a constituent of cruciferous vegetables of the genus
Brassica. 3,3'-Diindolylmethane (5) is a major product of acid condensation of indole 4
formed in the stomach. Indoles 4 and 5 have been proven to be chemoproventive agents.
They were developed for preventing, inhibiting and reversing the progession of many cancer
cells, especially, estrogen-dependent reproductive cancers [11,12]. Licznerska et al. have
reported that the indoles 4 and 5 could inhibit the aromatase expression in estrogen-
dependent MCF7 breast cancer cell line [13]. In addition, many indoles containg-molecules
have been reported to display potent aromatase inhibitory activity [14-16]. Furthermore,
sulfonamide scaffold has also been found in AIs [17-20] in which such group could form
hydrogen bond interaction with the target protein [17-19]. However, due to the expansion of
resistance to AIs and the side effects related to currently utilized compounds, the need for
improved AIs persists [21,22].
Computational approaches are considered as effective facilitating tools in drug design
and discovery [23]. Molecular docking is a computational method to find possible binding

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modes of the compound against its biological target, and has been successfully used to
investigate binding modes of many classes of aromatase inhibitors [17-19]. Quantitative
structure-activity relationship (QSAR) is a method that correlates chemical structure of the
compound with its biological activity [24-26]. QSAR has been widely used to provide useful
information for guiding the design and discovery of many classes of drugs, including
aromatase inhibitors [7].
Therefore, the rational design of target compounds is based on the hybrid of attractive
phenotypic bis-indole (5, Fig. 1) and sulfonamide moieties, in which the indole core might
anticipate in hydrophobic interaction, and the functionality of sulfonamide group may involve
in H-bonding with the site of action. Structural feature of both indole and sulfonamide
moieties was modified using many substitution patterns and substituents. In this study,
various hybrids of indole-sulfonamide derivatives were synthesized (Scheme 1) and
evaluated for their aromatase inhibitory activities. Molecular docking and QSAR modeling
were also performed to provide insights into the important interaction of compounds and
target site as well as useful structure-activity relationship (SAR) information.
2. Results and discussion
2.1 Chemistry
The synthesis of bis- and tris-indoles containing sulfonamides (11-13, 28-29, 31-38
and 41-44) has been previously reported by our group [27,28]. In the same manner,
alkylations of N-sulfonyltryptamines 8, derived from the condensation of tryptamine (6) and
N-benzenesulfonyl chlorides 7, with various benzaldehyde derivatives 9 catalyzed by 20 mol
% H4O40SiW12 aq in acetonitrile yielded a series of novel indole-sulfonamide derivatives (14-
27, 30 and 39-40) in moderate to good yields (40–68%) (Scheme 1).
Structures of the novel indoles were confirmed based on their HRMS and NMR
spectra. 1H NMR spectra showed a characteristic signal of methine proton as a singlet at δ in
the range of 5–7 ppm indicating that the alkylation products (14-27, 30 and 39-40) were
formed. In addition, the spectra displayed a typical symmetrical signal of two N-arylsulfonyl
tryptamine parts (IndCH2CH2NHSO2Ph) in which showed eight methylene protons as
multiplet at δ in the range of 2-3 ppm and two NH protons as a singlet at δ in the range of 10-
11 ppm.

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2.2 Aromatase inhibitory activity / Structure-activity relationship
Thirty four indoles bearing sulfonamide (11-44) were evaluated for their aromatase
inhibitory activities using ketoconazole (IC50 = 2.6 µM) and letrozole (IC50 = 1.9 nM) as the
reference drugs summarized in Table 1. The investigated indoles (11-40, Table 1) constitute
bis-indole sulfonamide moieties and different R groups (phenyl and substituted phenyl) on
the methine carbon. The benzene sulfonamide parts were substituted by X groups (CH3,
OCH3, F, Cl, Br, CF3, NO2). Tris-indoles (41-44) have X groups as OCH3, NO2 and Cl.
SAR of the compounds has been discussed. Results showed that all indoles (11-44)
exhibited aromatase inhibitory activities with IC50 in the range of 0.7-15.3 µM. Bis-indoles
(11-13) with unsubstituted benzene (R = C6H5) showed IC50 = 3.5-11.9 µM in which
compound 13 with X = 4-OCH3 group displayed 3-fold higher potency than compounds 11
and 12 with substituents (X) = 4-CH3 and 4-NO2. Bis-indoles containing electron
withdrawing substituents, R = 4-nitrophenyl (14-16), R = 4-bromophenyl (20-21) and R = 4-
trifluorophenyl (25-27), exerted comparable activity with IC50 = 2.3-4.0 µM. Bis-indoles (17-
19) with 4-cyanophenyl (R) showed IC50 = 5.5-15.3 µM in which the effect of electron
withdrawing groups (X) increased the aromatase inhibitory potency in the following trend: Cl
> F > CF3. Bis-indoles with 4-fluorophenyl (R) provided compounds (22-24) with IC50 = 4.4-
7.6 µM in which the effect of X moiety increased the aromatase inhibitory potency in the
following trend: CF3 > Cl > Br. The inhibition of aromatase was significantly improved when
the R group was para (p)-OHC6H4 as seen in compound 28 (X = 4-OCH3), 29 (X = 4-Cl) and
30 (X = 4-CF3). These p-phenoxy compounds (28-30, R = p-OHC6H4) exhibited better
activity compared with the derivatives having the same X moiety but different R groups i.e.,
28 > 13 (R = C6H5) with X = 4-OCH3, 29 > 26 (R = 4-CF3C6H4), 17 (R = 4-CNC6H4), 20 (R
= 4-BrC6H4), 23 (R = 4-FC6H4) with X = 4-Cl, and 30 > 16 (R = 4-NO2C6H4), 19 (R = 4-
CNC6H4), 21 (R = 4-BrC6H4), 24 (R = 4-FC6H4), 27 (R = 4-CF3C6H4) with X = 4-CF3. When
hydroxyl group was moved to the ortho (o)-position of the phenyl group (R), compounds 32-
37 were obtained with IC50 = 0.8-4.6 µM in which the effect of X substituent increased the
aromatase inhibitory potency in the following trend: 4-OCH3 > 3-NO2 > 4-NO2 > 4-CH3 ~ 4-
Cl ~ 2-NO2. Comparable inhibitory potency of p- and o-OHC6H4 (R) was noted for 28 (IC50
= 0.7 µM) vs 34 (IC50 = 0.8 µM), X = 4-OCH3, and 31 (IC50 = 2.3 µM) vs 36 (IC50 = 1.7
µM), X = 3-NO2. However, 3-fold reduction of the activity was noted for o-OHC6H4

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compound 35 (IC50 = 4.2 µM) compared with p-OHC6H4 compound 29, X = 4-Cl. The best
result was seen in derivatives with electron donating substituent (X = 4-OCH3) of both p- and
o-phenoxy (R) compounds (28 and 34), but it was not found in the compound without OH
group on the phenyl ring (R) like derivative 13. Obviously, phenoxy compounds (28 and 34)
displayed 5-fold higher activity compared with compound 13 (IC50 = 3.5 µM). Introduction
of dimethoxy groups at o-position of phenoxy group of compound 28 led to compound 38
(IC50 = 2.5 µM) with decreasing 3.6-fold of inhibition potency compared with the compound
28. This could be due to the dimethoxy groups that hindered the interaction of p-OH group
with its site of action. A steric bulky tricyclic ring (R = fluorene ring) reduced activity of the
compound (39, X = 4-Cl, IC50 = 8.8 µM) compared with other compounds having phenyl
rings (R) and X = 4-Cl as noted for compound (39 < 17, 20, 23, 26, 29, 35, IC50 = 5.5, 2.7,
5.4, 3.2, 1.4, 4.2 µM, respectively). A steric replacement of fluorene ring with N-ethyl-3-
carbazole group led to compound 40 with 2-fold higher potency compared with compound
39. Tris-indoles 41-44 also showed anti-aromatase activiy with IC50 = 2.8-11.9 µM.
These indoles (11-44) were also tested against the noncancerous (Vero) cell line obtained
from African green monkey kidney (Table 1). Most analogs were non-cytotoxic toward
normal cell, except for compounds 20, 21, 24, 26, 29, 30 and 32. The most active compounds
28 and 34 displayed high safety index with SI values of > 93 and > 81, respectively.
2.3. Molecular docking
Molecular docking was performed to investigate binding modalities of the synthesized
indole derivatives, and the target protein, aromatase. Co-crystallized androstenedione (ASD)
was re-docked to the binding site of aromatase protein structure to validate the docking
protocol. The re-docking showed that ASD could re-docked in the binding cavity of the
enzyme providing RMSD value of 0.714. This suggested that the docking protocol is
acceptable for the analysis of binding modalities of the investigated compounds as
demonstrated by the RMSD value within a range of less than 2.000. The re-docking position
of ASD to aromatase is shown in Fig. 2A.
The top three indole derivatives (28, 30 and 34) possessing the greatest aromatase
inhibitory activity were selected for the docking study. Results suggested that the most potent
indole analogs possibly bind to aromatase via the same binding sites of the co-crystallized
ASD, the natural substrate of aromatase (Fig. 2B). The protein-ligand interaction was

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analyzed and displayed as two dimensional interaction diagrams (Fig. 3). Considering the
ASD binding that was described in the literature [29]
(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3469775), the key interaction was suggested
to be the hydrogen bonding with MET374 as well as the hydrophobic interactions with the
pockets of PHE134, TRP224, VAL370 and VAL373. Moreover, the cofactor heme (HEM)
group also plays a role in the binding with ASD via hydrophobic interaction (Fig. 3D).
Interestingly, the binding poses of the investigated compounds (28, 30, and 34) suggested that
all of them can bind to the same binding site on the aromatase enzyme, and share several
binding residues with the natural substrate ASD (Fig. 3A-3C). All of the selected compounds
can form hydrogen bonds with MET374 in which p-OCH3 (X) of compounds 28 and 34 are
responsible for H-bonding whereas compound 30 used p-CF3 (X) for H-bond interaction.
This is similar to that was observed in the docking pose of ASD (Fig. 3D). Furthermore, such
compounds form hydrophobic interactions with the aromatase via the same residues that the
ASD formed with the protein. For example, compound 28 and compound 34 interact with the
hydrophobic VAL370 and VAL373 (Fig. 3A and 3C) as well as compound 30 interacts with
PHE134 residue (Fig. 3B) as noted for the ASD. In addition, indole ring of the investigated
compounds (28 and 34) interacted with the HEM via Pi-Pi bond while benzene ring of
sulfonamide 30 formed hydrophobic interaction with the HEM. This is similar to that was
found in the docking pose of ASD (Fig. 3A-3D). Of note, these investigated compounds
share common characteristics of binding with the aromatase. Particularly, the interaction of
compounds 28, 34 (X = p-OCH3) and 30 (X = p-CF3) with ALA306 used the indole ring that
linked to the sulfonamide moiety, which formed H-bonding with the MET374 of the enzyme.
This suggested that the ALA306 residue may be essential for the aromatase binding of the
indole derivatives. Moreover, the investigated compounds possess various aromatic ring
substructures which could enable them to bind tightly with the binding cavity via the Pi-based
interactions (i.e., Pi-Pi, Pi-Amide, Pi-Sigma, and Pi-charge interactions), Fig. 3A-3C. It
should be noted that p-phenoxy (R) group of the most potent bis-indole compound 28
affected its molecular shape in such a way that 4-OCH3 (X) group on the sulfonamide moiety,
and NH of the indole ring participate in H-bond forming with the MET374 backbone amide
and with ASP 309 side chain, respectively. Additionally, weak hydrophobic interaction of
benzene ring (part of sulfonamide) with ARG115 was noted. These interactions fit the
requirement of compound to mimic the H-bonding of C-17 and C-3 keto functions of the

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natural ASD [30,31]. Another indolesulfonamide moiety of compound 28 formed H-bonds
with ARG192 and SER478 via sulfonyl (O atom) and NH of the sulfonamide, respectively. In
addition, two indole rings of compound 28 formed Pi-Sigma interaction with the same
THR310. Notably, p-phenoxy (R) of the most potent inhibitor 28 showed some distinctive
characteristic such as H-bonding of the OH group with LEU372 that was not observed for
other compounds. The binding pose of compound 28 enabled it to form hydrogen bonding
network with six amino acid residues within the binding pocket of the aromatase enzyme
including ARG115, ARG192, ASP309, LEU372, MET374, and SER478. This characteristic
might provide the higher inhibitory potency of the compound 28 than those of compounds 30
and 34, which formed three and two hydrogen bonds with the enzyme, respectively, as well
as those of the others in this class.
The docking results provided insights into possible binding modalities and also
suggested the inhibitory mechanism of the investigated indole derivatives. The possible
mechanism of inhibition was more likely to be the competitive inhibition, which prevents the
binding of aromatase with its natural substrate ASD.
2.4. QSAR analysis
Chemical structures of the investigated compounds (11-44) together with their
experimental aromatase inhibitory activities (pIC50 values) were used as data set for QSAR
analysis. All compounds were drawn, geometrically optimized, and calculated to obtain a set
of 13 quantum chemical and 3,244 molecular descriptors. The feature selection was
performed to obtain a set of 4 informative descriptors i.e., MATS6e, Mor28u, E2m, and
nArOH. Definitions and descriptors of the investigated compounds are provided in Table 2
and Table 3, respectively. The QSAR model was successfully constructed using multiple
linear regression (MLR) algorithm, equation (1).
pIC50 = 0.6048(MATS6e) - 0.3918(Mor28u) + 1.1045(E2m) + 0.3656 (nArOH) – 1.3587
(1)
Experimental and predicted activities (pIC50 values) of the compounds are shown in
Table 4 and Figure 4. The constructed QSAR model provides acceptable predictive
performance as indicated by Rtr = 0.8121 and RMSEtr = 0.1887 for training set, and Rcv =
0.7312 and RMSEcv = 0.2228 for leave-one-out cross validation set. The QSAR model
indicated that the descriptors representing mass (E2m) and electronegativity (MATS6e) are
two main properties governing aromatase inhibition of the investigated indoles as shown by

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their high regression coefficient values of 1.1045 and 0.6048, respectively (equation 1). The
QSAR model demonstrated that high values of E2m, MATS6e, and nArOH, but low value of
Mor28u are required for potent activity of the compounds. Notably, the nArOH descriptor
representing the presence of aromatic hydroxyl group in the QSAR equation is coincided
with the SAR findings from the experimental results, which demonstrated that the
compounds bearing hydroxyphenyl (R) moiety mostly exhibited more potent activity than
those substituted by other R groups (i.e., R = phenyl, halophenyl, cyanophenyl, Table 1).
Additionally, it was observed that the para-hydroxyl group substituted on the benzene ring
provided more potent activity than the ortho-substitution (i.e., the most potent compound 28
(p-OH) exhibited higher activity than the second most potent compound 34 (o-OH), Table 1).
Considering the descriptor values of these two compounds, the positions of hydroxyl group
on the phenyl ring may mainly affect the electronegativity of the compounds as shown by
MATS6e values (28 = 0.225 and 34 = 0.173, Table 3) indicating the higher MATS6e value
affords the higher activity. Physicochemical properties such as electronegativity, dipole
moment, and van der Waals volume as well as molecular size and shape, steric factor, and
hydrophobicity are crucial factors governing the aromatase inhibitory activity [7]. Different
types of compounds require different factors in exerting the bioactivity. The QSAR study of
indole derivatives such as 3-azolylmethylindoles with the lower polar volume afforded the
better aromatase inhibitory activity [7].
The molecular docking results also revealed that the hydroxyl group substituted at the
para-position of compound 28 may be essential for better activity as the H-bonding
interaction with the residue LEU372 of the aromatase enzyme in which such interaction can
be only observed for this compound (Fig. 3A). The substitution (X group) on both of benzene
sulfonyl rings also may influence activity of the compounds as one of them (X = OCH3, Fig.
3A) could mimic H-bonding with the residue MET374 on the enzyme similar to that of the
natural substrate, androstenedione (Fig. 3D).
3. Conclusions
A series of indole-based sulfonamides (11-44) have been synthesized using the
alkylation reaction. Their aromatase inhibitory activities, molecular docking, and QSAR
were studied. It was observed that all indoles exerted aromatase inhibitory activity (IC50
=
0.8-15.3 M). Particularly, the bis-indoles 28 and 34 were shown to be the most potent
µ

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inhibitors (IC50 of 0.7 and 0.8
µM, respectively) without affecting the normal cell line.
Molecular docking results revealed that the top three ranked indoles possessing the most
potent aromatase inhibitory activities (compounds 28, 30 and 34) could occupy at the same
binding site of the natural substrate ASD on the aromatase pocket, and share the same
binding residues with those of the ASD (i.e. MET374, VAL370, VAL373, and PHE134).
These suggested that the investigated indole derivatives may elicit their aromatase inhibitory
activities via acting as competitive inhibitors. The QSAR study demonstrated MATS6e,
Mor28u, E2m, and nArOH as a set of influential descriptors. The most potent inhibitors with
para- and ortho-phenoxy groups (R) had high value of electronegativity (MATS6e) in which
the value of para-OH compound (28) was greater than that of ortho-OH compound (34). The
QSAR results are well correlated with the molecular docking that the most potent compound
(28) could tightly bind the cavity of the aromatase enzyme to mimic H-bonding of C-17 and
C-3 keto functions (via MET374 and ASP309, respectively) as well as to mimic hydrophobic
interactions of the natural substrate. In summary, the study highlighted a series of indole
derivatives as potential compounds to be further developed as novel aromatase inhibitors for
therapeutic applications.
4. Experimental section
4.1 Chemistry
Column chromatography was carried out using silica gel 60 (70–230 mesh ASTM).
Analytical thin-layer chromatography (TLC) was performed using silica gel 60 F254
aluminum sheets. 1H- and 13C- NMR spectra were recorded on a Bruker AVANCE 300 NMR
1
spectrometer (operating at 300 MHz for H and 75 MHz for 13C). The following standard
abbreviations were used for signal multiplicities: singlet (s), doublet (d), triplet (t), quartet
(q), multiplet (m), and broad (br). Mass spectra were recorded on a Bruker Daltonics
(microTOF). IR spectra were obtained using a universal attenuated total reflectance attached
on a Perkin–Elmer Spectrum One spectrometer. Melting points were determined using a
Griffin melting point apparatus and were uncorrected.
Synthesis of indole-sulfonamides (11-13, 28, 29, 31-38 and 41-44) has been reported
in the literatures [27,28].
4.2 General procedure for the synthesis of N-(2-(1H-indol-3-yl)ethyl)-benzenesulfonamides
(8)

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A solution of tryptamine 6 (10 mmol) in dichloromethane (50 mL) was added in
dropwise manner to a stirred mixture of appropriate benzenesulfonyl chloride 7 (10 mmol)
and sodium carbonate (14 mmol) in dichloromethane (20 mL). The reaction mixture was
stirred at room temperature overnight, and distilled water (20 mL) was added. The organic
phase was separated and the aqueous phase was extracted with dichloromethane (2×30 mL).
The organic extracts were combined and washed with water (30 mL). The organic layer was
dried over anhydrous sodium sulfate, filtered and evaporated to dryness under reduced
pressure. The crude product was further purified by column chromatography on silica gel
using 1:9 acetone/hexane to obtain the pure product.
4.2.1 4-Bromo-N-(2-(1H-indol-3-yl)ethyl)-benzenesulfonamide (8a)
White solid. 88% yield; mp 129-130 oC; IR (UATR) cm-1: 3399, 3260, 1575, 1317,
1155. 1H NMR (300 MHz, DMSO-d6) δ 2.79 (t, J=7.5 Hz, 2H, ArCH2), 3.03 (t, J=7.5 Hz, 2H,
CH2NH), 6.95 (t, J=7.5 Hz, 1H, C5-ArH), 7.05 (t, J=7.5 Hz, 1H, C6-ArH), 7.11 (s, 1H, C2-
ArH), 7.32 (d, J=8.0 Hz, 1H, C7-ArH), 7.37 (d, J=8.0 Hz, 1H, C4-ArH), 7.65 (d, J=8.6 Hz,
2H, C3′-ArH and C5′-ArH), 7.77 (d, J=8.6 Hz, 2H, C2′-ArH and C6′-ArH C4-ArH), 7.85 (s,
13
1H, NHSO2), 10.80 (s, 1H, NH). C NMR (75 MHz, DMSO-d6) δ 25.8, 43.9, 111.3, 111.9,
118.4, 118.8, 121.4, 123.4, 126.5, 127.4, 128.9, 132.7, 136.6, 140.4. TOF-MS m/z: 400.9939
(Calcd for C16H15BrN2NaO2S: 400.9930).
4.2.2 4-Fluoro-N-(2-(1H-indol-3-yl)ethyl)-benzenesulfonamide (8b)
Brown solid. 86% yield; mp 119-120 oC; IR (UATR) cm-1: 3401, 3264, 1592, 1494,
1317, 1153. 1H NMR (300 MHz, DMSO-d6) δ 2.78 (t, J=7.5 Hz, 2H, ArCH2), 3.01 (t, J=7.5
Hz, 2H, CH2NH), 6.94 (t, J=7.1 Hz, 1H, C5-ArH), 7.03 (t, J=7.1 Hz, 1H, C6-ArH), 7.10 (s,
1H, C2-ArH), 7.28-7.42 (m, 4H, C4-ArH, C7-ArH, C3′-ArH and C5′-ArH), 7.78-7.88 (m,
3H, C2′-ArH, C6′-ArH and NHSO2), 10.81 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6) δ
25.8, 43.9, 111.3, 111.9, 116.7 (d, 2JCF=23), 118.4, 118.7, 121.4, 123.4, 127.4, 129.9 (d,
3JCF=9), 136.6, 137.5, 164.5 (d, 1JCF=249). TOF-MS m/z: 341.0729 (Calcd for
C16H15FN2NaO2S: 341.0731).
4.2.3 4-Trifluoromethyl-N-(2-(1H-indol-3-yl)ethyl)-benzenesulfonamide (8c)
White solid. 84% yield; mp 145-146 oC; IR (UATR) cm-1: 3414, 3254, 1320, 1124. 1H
NMR (300 MHz, DMSO-d6) δ 2.80 (t, J=7.3 Hz, 2H, ArCH2), 3.07 (t, J=7.3 Hz, 2H,
CH2NH), 6.94 (t, J=7.4 Hz, 1H, C5-ArH), 7.04 (t, J=7.4 Hz, 1H, C6-ArH), 7.12 (s, 1H, C2-

12
ACCEPTED MANUSCRIPT
ArH), 7.31 (d, J=8.0 Hz, 1H, C7-ArH), 7.36 (d, J=7.8 Hz, 1H, C4-ArH), 7.88-8.08 (m, 5H,
C2′-ArH, C3′-ArH, C5′-ArH, C6′-ArH and NHSO2), 10.80 (s, 1H, NH). 13C NMR (75 MHz,
DMSO-d6) δ 25.9, 43.9, 111.2, 111.9, 118.4, 118.7, 121.4, 123.5, 124.0 (q, 1JCF=271), 126.7,
2
126.8, 127.4, 127.8, 132.5 (q, JCF=32), 136.6, 145.0. TOF-MS m/z: 391.0700 (Calcd for
C17H15F3N2NaO2S: 391.0699).
4.3 General procedure for the synthesis of indole-sulfonamides (11-13, 28-29, 31-38 and 41-
44)
A mixture of N-sulfonyltryptamine (8) (0.5 mmol), benzaldehyde derivative (9) (0.3
mmol), and H4O40SiW12 aq (20 mol%) in acetonitrile (5 mL) was stirred at room temperature
for 2-6 h. After the complete conversion, as indicated by TLC, the reaction mixture was
concentrated in a vacuum and purified by silica gel column chromatography using 1:9
acetone/hexane to obtain the corresponding pure indole-sulfonamide products.
4.3.1 N,N'-((2,2'-((4-nitrophenyl)methylene)bis(1H-indole-3,2-diyl))bis(ethane-2,1-
diyl))bis(4-bromobenzenesulfonamide) (14)
Yellow solid. 68% yield; mp 119-120 °C; IR (UATR) cm-1: 3378, 1596, 1458, 1345,
1
1156. H NMR (300 MHz, dmso-d6) δ 2.65-2.88 (m, 8H, 4×CH2), 6.09 (s, 1H, CH), 6.99 (t,
J=7.3 Hz, 2H, 2xC5-ArH), 7.07 (t, J=7.3 Hz, 2H, 2xC6-ArH), 7.29 (d, J=7.8 Hz, 2H, 2xC7-
ArH), 7.30 (d, J=8.8 Hz, 2H, C2′′-ArH and C6′′-ArH), 7.38 (d, J=7.6 Hz, 2H, 2xC4-ArH),
7.54 (d, J=8.5 Hz,, 4H, 2×C3′-ArH and 2×C5′-ArH), 7.63 (d, J=8.5 Hz,, 4H, 2×C2′-ArH and
2×C6′-ArH), 7.75 (br s, 2H, 2×NHSO2), 8.15 (d, J=8.7 Hz, 2H, C3′′-ArH and C5′′-ArH,
10.47 (s, 2H, 2×NH). 13C NMR (75 MHz, dmso-d6) δ 25.1, 39.7 (superimposed with dmso-d6
peaks), 43.7, 109.4, 111.9, 118.4, 119.3, 121.6, 124.1, 126.5, 128.1, 128.8, 130.0, 132.6,
134.2, 136.3, 140.0, 146.8, 149.4. TOF-MS m/z: 912.0158 (Calcd for C39H33Br2N5NaO6S2:
912.0131).
4.3.2 N,N'-((2,2'-((4-nitrophenyl)methylene)bis(1H-indole-3,2-diyl))bis(ethane-2,1-
diyl))bis(4-fluorobenzenesulfonamide) (15)
Yellow solid. 63% yield; mp 111-112 °C; IR (UATR) cm-1: 3377, 1592, 1458, 1346,
1
1152. H NMR (300 MHz, dmso-d6) δ 2.63-2.84 (m, 8H, 4×CH2), 6.08 (s, 1H, CH), 6.97 (t,
J=7.2 Hz, 2H, 2xC5-ArH), 7.05 (t, J=7.2 Hz, 2H, 2xC6-ArH), 7.20-7.33 (m, 8H, 2xC7-ArH,
C2′′-ArH, C6′′-ArH, 2×C3′-ArH and 2×C5′-ArH), 7.37 (d, J=7.7 Hz, 2H, 2xC4-ArH), 7.62-
7.70 (m, 6H, 2×C2′-ArH, 2×C6′-ArH and 2×NHSO2), 8.12 (d, J=8.7 Hz, 2H, C3′′-ArH and

13
ACCEPTED MANUSCRIPT
C5′′-ArH, 10.45 (s, 2H, 2×NH). 13C NMR (75 MHz, dmso-d6) δ 24.6, 39.7 (superimposed
2
with dmso-d6 peaks), 43.2, 109.0, 111.4, 116.1 (d, JCF=32), 117.9, 118.8, 121.1, 123.6,
3
127.6, 129.3 (d, JCF=9), 129.5, 133.7, 135.9, 136.6, 146.3, 148.9, 163.9 (d, 1JCF=249). TOF-
MS m/z: 792.1710 (Calcd for C39H33F2N5NaO6S2: 792.1733).
4.3.3 N,N'-((2,2'-((4-nitrophenyl)methylene)bis(1H-indole-3,2-diyl))bis(ethane-2,1-
diyl))bis(4-trifluoromethylbenzenesulfonamide) (16)
Yellow solid. 55% yield; mp 121-122 °C; IR (UATR) cm-1: 3386, 1607, 1458, 1322,
1
1162. H NMR (300 MHz, dmso-d6) δ 2.65-2.85 (m, 8H, 4×CH2), 6.08 (s, 1H, CH), 6.98 (t,
J=7.5 Hz, 2H, 2xC5-ArH), 7.04 (t, J=7.5 Hz, 2H, 2xC6-ArH), 7.26 (d, J=8.0 Hz, 2H, C2′′-
ArH and C6′′-ArH), 7.28 (d, J=7.4 Hz, 2H, 2xC7-ArH), 7.34 (d, J=7.7 Hz, 2H, 2xC4-ArH),
7.73 (d, J=8.7 Hz, 4H, 2×C3′-ArH and 2×C5′-ArH), 7.77 (d, J=8.7 Hz,, 4H, 2×C2′-ArH and
2×C6′-ArH), 7.90 (br s, 2H, 2×NHSO2), 8.11 (d, J=8.6 Hz, 2H, C3′′-ArH and C5′′-ArH)
10.44 (s, 2H, 2×NH). 13C NMR (75 MHz, dmso-d6) δ 25.1, 39.7 (superimposed with dmso-d6
1
peaks), 43.7, 109.3, 111.9, 118.4, 119.3, 121.7, 123.9 (q, JCF=271), 124.0, 126.6, 126.7,
2
127.7, 128.1, 130.0, 132.5 (q, JCF=32), 134.2, 136.3, 144.6, 146.8, 149.3. TOF-MS m/z:
892.1646 (Calcd for C41H33F6N5NaO6S2: 892.1669).
4.3.4 N,N'-((2,2'-((4-cyanophenyl)methylene)bis(1H-indole-3,2-diyl))bis(ethane-2,1-
diyl))bis(4-chlorobenzenesulfonamide) (17)
Light brown solid. 49% yield; mp 153-155 °C; IR (UATR) cm-1: 3368, 2230, 1586,
1
1457, 1324, 1158. H NMR (300 MHz, dmso-d6) δ 2.65-2.85 (m, 8H, 4×CH2), 6.01 (s, 1H,
CH), 6.97 (t, J=7.3 Hz, 2H, 2xC5-ArH), 7.04 (t, J=7.3 Hz, 2H, 2xC6-ArH), 7.20 (d, J=8.3 Hz,
2H, C2′′-ArH and C6′′-ArH), 7.27 (d, J=7.9 Hz, 2H, 2xC7-ArH), 7.35 (d, J=7.7 Hz, 2H,
2xC4-ArH), 7.48 (d, J=8.5 Hz,, 4H, 2×C3′-ArH and 2×C5′-ArH), 7.60 (d, J=8.5 Hz,, 4H,
2×C2′-ArH and 2×C6′-ArH), 7.73 (d, J=8.2 Hz, 2H, C3′′-ArH and C5′′-ArH), 7.74 (br s, 2H,
13
2×NHSO2), 10.44 (s, 2H, 2×NH). C NMR (75 MHz, dmso-d6) δ 25.0, 39.7 (superimposed
with dmso-d6 peaks), 43.8, 109.4, 110.1, 111.9, 118.4, 119.2, 119.3, 121.6, 128.0, 128.8,
129.6, 129.7, 132.8, 134.2, 136.3, 137.6, 139.6, 147.3. TOF-MS m/z: 804.1217 (Calcd for
C34H37Cl2N3NaO10S2: 804.1190).
4.3.5 N,N'-((2,2'-((4-cyanophenyl)methylene)bis(1H-indole-3,2-diyl))bis(ethane-2,1-
diyl))bis(4-fluorobenzenesulfonamide) (18)

14
ACCEPTED MANUSCRIPT
Light brown solid. 44% yield; mp 104-105 °C; IR (UATR) cm-1: 3369, 2229, 1592,
1
1457, 1324, 1151. H NMR (300 MHz, dmso-d6) δ 2.65-2.80 (m, 8H, 4×CH2), 6.02 (s, 1H,
CH), 6.97 (t, J=7.3 Hz, 2H, 2xC5-ArH), 7.04 (t, J=7.3 Hz, 2H, 2xC6-ArH), 7.18-7.30 (m, 8H,
2xC7-ArH, 2×C3′-ArH, 2×C5′-ArH, C2′′-ArH and C6′′-ArH), 7.36 (d, J=7.8 Hz, 2xC4-ArH),
7.62-7.75 (m, 8H, 2×C2′-ArH, 2×C6′-ArH, C3′′-ArH, C5′′-ArH and 2×NHSO2), 10.42 (s,
13
2H, 2×NH). C NMR (75 MHz, dmso-d6) δ 25.0, 39.7 (superimposed with dmso-d6 peaks),
43.8, 109.4, 110.1, 111.9, 116.6 (d, 2JCF=22), 118.4, 119.2, 119.3, 121.6, 128.1, 129.7, 129.8
3
1
(d, JCF=9), 132.8, 134.2, 136.3, 137.1, 147.3, 164.4 (d, JCF=250). TOF-MS m/z: 772.1814
(Calcd for C40H33F2N5NaO4S2: 772.1834).
4.3.6 N,N'-((2,2'-((4-cyanophenyl)methylene)bis(1H-indole-3,2-diyl))bis(ethane-2,1-
diyl))bis(4-trifluoromethylbenzenesulfonamide) (19)
Light brown solid. 44% yield; mp 139-140 °C; IR (UATR) cm-1: 3323, 2231, 1608,
1
1458, 1321, 1161. H NMR (300 MHz, dmso-d6) δ 2.65-2.85 (m, 8H, 4×CH2), 6.03 (s, 1H,
CH), 6.95 (t, J=7.2 Hz, 2H, 2xC5-ArH), 7.03 (t, J=7.2 Hz, 2H, 2xC6-ArH), 7.20 (d, J=8.3 Hz,
2H, C2′′-ArH and C6′′-ArH), 7.26 (d, J=7.9 Hz, 2H, 2xC7-ArH), 7.34 (d, J=7.8 Hz, 2H,
2xC4-ArH), 7.72 (d, J=8.6 Hz, 2H, C3′′-ArH and C5′′-ArH), 7.76 (d, J=9.6 Hz,, 4H, 2×C3′-
ArH and 2×C5′-ArH), 7.80 (d, J=9.6 Hz,, 4H, 2×C2′-ArH and 2×C6′-ArH), 7.94 (br s, 2H,
13
2×NHSO2), 10.46 (s, 2H, 2×NH). C NMR (75 MHz, dmso-d6) δ 25.1, 39.7 (superimposed
1
with dmso-d6 peaks), 43.8, 109.3, 110.1, 111.9, 118.4, 119.3, 121.6, 123.9 (q, JCF=273),
126.6, 126.7, 127.7, 128.0, 129.7, 132.5 (q, 2JCF=32), 132.8, 134.3, 136.3, 144.7, 147.3. TOF-
MS m/z: 872.1761 (Calcd for C42H33F6N5NaO4S2: 872.1770).
4.3.7 N,N'-((2,2'-((4-bromophenyl)methylene)bis(1H-indole-3,2-diyl))bis(ethane-2,1-
diyl))bis(4-chlorobenzenesulfonamide) (20)
Light brown solid. 50% yield; mp 131-132 °C; IR (UATR) cm-1: 3379, 1587, 1458,
1
1323, 1157. H NMR (300 MHz, dmso-d6) δ 2.65-2.85 (m, 8H, 4×CH2), 5.88 (s, 1H, CH),
6.92-7.07 (m, 6H, 2xC5-ArH, 2xC6-ArH, C2′′-ArH and C6′′-ArH), 7.27 (d, J=7.9 Hz, 2H,
2xC7-ArH), 7.34 (d, J=7.6 Hz, 2H, 2xC4-ArH), 7.42-7.51 (m, 6H, 2×C3′-ArH, 2×C5′-ArH,
C3′′-ArH and C5′′-ArH), 7.60 (d, J=8.4 Hz,, 4H, 2×C2′-ArH and 2×C6′-ArH), 7.72 (br s, 2H,
13
2×NHSO2), 10.38 (s, 2H, 2×NH). C NMR (75 MHz, dmso-d6) δ 25.0, 39.7 (superimposed
with dmso-d6 peaks), 43.8, 109.1, 111.8, 118.3, 119.2, 120.4, 121.5, 128.1, 128.8, 129.6,

15
ACCEPTED MANUSCRIPT
130.8, 131.8, 134.8, 136.2, 137.6, 139.6, 141.0. TOF-MS m/z: 857.0391 (Calcd for
C39H33BrCl2N4NaO4S2: 857.0396)
4.3.8 N,N'-((2,2'-((4-bromophenyl)methylene)bis(1H-indole-3,2-diyl))bis(ethane-2,1-
diyl))bis(4-trifluoromethylbenzenesulfonamide) (21)
Brown solid. 58% yield; mp 112-113 °C; IR (UATR) cm-1: 3385, 1609, 1458, 1322,
1
1163. H NMR (300 MHz, dmso-d6) δ 2.65-2.85 (m, 8H, 4×CH2), 5.91 (s, 1H, CH), 6.90-
7.05 (m, 6H, 2xC5-ArH, 2xC6-ArH, C2′′-ArH and C6′′-ArH), 7.26 (d, J=7.9 Hz, 2H, 2xC7-
ArH), 7.33 (d, J=7.5 Hz, 2H, 2xC4-ArH), 7.45 (d, J=8.4 Hz, 2H, C3′′-ArH and C5′′-ArH),
7.78 (s, 8H, 2×C2′-ArH, 2×C3′-ArH, 2×C5′-ArH and 2×C6′-ArH), 7.93 (br s, 2H,
13
2×NHSO2), 10.41 (s, 2H, 2×NH). C NMR (75 MHz, dmso-d6) δ 25.1, 39.7 (superimposed
1
with dmso-d6 peaks), 43.8, 109.0, 111.8, 118.3, 119.1, 120.4, 121.5, 123.9 (q, JCF=271),
126.7, 127.7, 127.8, 128.1, 130.9, 131.8, 132.5 (q, 2JCF=32), 134.9, 136.2, 141.0, 144.7. TOF-
MS m/z: 925.0918 (Calcd for C41H33BrF6N4NaO4S2: 925.0923).
4.3.9 N,N'-((2,2'-((4-fluorophenyl)methylene)bis(1H-indole-3,2-diyl))bis(ethane-2,1-
diyl))bis(4-bromobenzenesulfonamide) (22)
Brown solid. 55% yield; mp 120-122 °C; IR (UATR) cm-1: 3382, 1575, 1458, 1324,
1
1158. H NMR (300 MHz, dmso-d6) δ 2.60-2.85 (m, 8H, 4×CH2), 5.87 (s, 1H, CH), 6.92-
7.10 (m, 8H, 2xC5-ArH, 2xC6-ArH, C2′′-ArH , C3′′-ArH , C5′′-ArH and C6′′-ArH), 7.27 (d,
J=7.9 Hz, 2H, 2xC7-ArH), 7.33 (d, J=7.6 Hz, 2H, 2xC4-ArH), 7.52 (d, J=8.6 Hz,, 4H, 2×C3′-
ArH and 2×C5′-ArH), 7.62 (d, J=8.4 Hz,, 4H, 2×C2′-ArH and 2×C6′-ArH), 7.74 (br s, 2H,
13
2×NHSO2), 10.40 (s, 2H, 2×NH). C NMR (75 MHz, dmso-d6) δ 25.0, 39.7 (superimposed
2
with dmso-d6 peaks), 43.8, 108.9, 111.8, 115.6 (d, JCF=21), 118.3, 119.1, 121.4, 126.5,
1
128.1, 128.9, 130.4 (d, 3JCF=8), 132.6, 135.2, 136.2, 137.6, 140.0, 161.5 (d, JCF=241). TOF-
MS m/z: 885.0190 (Calcd for C39H33Br2FN4NaO4S2: 885.0186).
4.3.10 N,N'-((2,2'-((4-fluorophenyl)methylene)bis(1H-indole-3,2-diyl))bis(ethane-2,1-
diyl))bis(4-chlorobenzenesulfonamide) (23)
Brown solid. 51% yield; mp 109-110 °C; IR (UATR) cm-1: 3381, 1587, 1458, 1324,
1
1157. H NMR (300 MHz, dmso-d6) δ 2.60-2.80 (m, 8H, 4×CH2), 5.88 (s, 1H, CH), 6.92-
7.10 (m, 8H, 2xC5-ArH, 2xC6-ArH, C2′′-ArH , C3′′-ArH , C5′′-ArH and C6′′-ArH), 7.27 (d,
J=7.9 Hz, 2H, 2xC7-ArH), 7.34 (d, J=7.6 Hz, 2H, 2xC4-ArH), 7.47 (d, J=8.6 Hz,, 4H, 2×C3′-
ArH and 2×C5′-ArH), 7.60 (d, J=8.4 Hz,, 4H, 2×C2′-ArH and 2×C6′-ArH), 7.74 (br s, 2H,

16
ACCEPTED MANUSCRIPT
13
2×NHSO2), 10.40 (s, 2H, 2×NH). C NMR (75 MHz, dmso-d6) δ 24.6, 39.7 (superimposed
2
with dmso-d6 peaks), 43.4, 108.5, 111.4, 115.2 (d, JCF=21), 117.9, 118.8, 121.1, 127.7,
1
128.4, 129.2, 130.0 (d, 3JCF=8), 134.8, 135.8, 137.2, 137.3, 139.2, 161.1 (d, JCF=242). TOF-
MS m/z: 797.1196 (Calcd for C39H33Cl2FN4NaO4S2: 797.1197).
4.3.11 N,N'-((2,2'-((4-fluorophenyl)methylene)bis(1H-indole-3,2-diyl))bis(ethane-2,1-
diyl))bis(4-trifluoromethylbenzenesulfonamide) (24)
Light yellow solid. 43% yield; mp 111-112 °C; IR (UATR) cm-1: 3384, 1607, 1458,
1
1323, 1162. H NMR (300 MHz, dmso-d6) δ 2.65-2.85 (m, 8H, 4×CH2), 5.90 (s, 1H, CH),
6.94 (t, J=7.3 Hz, 2H, 2xC5-ArH), 6.98-7.08 (m, 6H, 2xC6-ArH, C2′′-ArH, C3′′-ArH, C5′′-
ArH and C6′′-ArH), 7.26 (d, J=8.0 Hz, 2H, 2xC7-ArH), 7.32 (d, J=7.6 Hz, 2H, 2xC4-ArH),
7.76 (d, J=9.0 Hz,, 4H, 2×C3′-ArH and 2×C5′-ArH), 7.79 (d, J=9.0 Hz,, 4H, 2×C2′-ArH and
2×C6′-ArH), 7.92 (br s, 2H, 2×NHSO2), 10.41 (s, 2H, 2×NH). 13C NMR (75 MHz, dmso-d6)
2
δ 25.1, 39.7 (superimposed with dmso-d6 peaks), 43.8, 108.8, 111.8, 115.6 (d, JCF=21),
118.2, 119.1, 121.4, 123.9 (q, 1JCF=272), 126.6, 126.7, 127.7, 128.1, 130.4 (d, 3JCF=8), 132.4
1
(q, 2JCF=32), 135.2, 136.2, 137.7, 144.7, 161.5 (d, JCF=242). TOF-MS m/z: 865.1732 (Calcd
for C41H33F7N4NaO4S2: 865.1724).
4.3.12 N,N'-((2,2'-((4-trifluoromethylphenyl)methylene)bis(1H-indole-3,2-diyl))bis(ethane-
2,1-diyl))bis(4-bromobenzenesulfonamide) (25)
Brown solid. 49% yield; mp 102-103 °C; IR (UATR) cm-1: 3380, 1575, 1458, 1323,
1
1159. H NMR (300 MHz, dmso-d6) δ 2.70-2.90 (m, 8H, 4×CH2), 6.03 (s, 1H, CH), 6.98 (t,
J=7.4 Hz, 2H, 2xC5-ArH), 7.06 (t, J=7.4 Hz, 2H, 2xC6-ArH), 7.24 (d, J=8.0 Hz, 2H, C2′′-
ArH and C6′′-ArH), 7.30 (d, J=7.9 Hz, 2H, 2xC7-ArH), 7.37 (d, J=7.6 Hz, 2H, 2xC4-ArH),
7.55 (d, J=8.5 Hz,, 4H, 2×C3′-ArH and 2×C5′-ArH), 7.63 (d, J=8.5 Hz,, 4H, 2×C2′-ArH and
2×C6′-ArH), 7.64 (d, J=7.8 Hz, 2H, C3′′-ArH and C5′′-ArH, 7.76 (br s, 2H, 2×NHSO2),
10.46 (s, 2H, 2×NH). 13C NMR (75 MHz, dmso-d6) δ 25.0, 39.7 (superimposed with dmso-d6
1
peaks), 43.8, 109.4, 111.8, 118.4, 119.2, 121.6, 124.7 (q, JCF=264), 125.8, 127.9 (q,
2JCF=32), 128.0, 128.9, 129.4, 132.6, 134.5, 136.3, 140.0, 146.3. TOF-MS m/z: 935.0131
(Calcd for C40H33Br2F3N4NaO4S2: 935.0154).
4.3.13 N,N'-((2,2'-((4-trifluoromethylphenyl)methylene)bis(1H-indole-3,2-diyl))bis(ethane-
2,1-diyl))bis(4-chlorobenzenesulfonamide) (26)

17
ACCEPTED MANUSCRIPT
Brown solid. 40% yield; mp 119-120 °C; IR (UATR) cm-1: 3380, 1587, 1458, 1323,
1158. 1H NMR (300 MHz, dmso-d6) δ 2.70-2.85 (m, 8H, 4×CH2), 6.01 (s, 1H, CH), 6.97 (t,
J=7.4 Hz, 2H, 2xC5-ArH), 7.05 (t, J=7.4 Hz, 2H, 2xC6-ArH), 7.23 (d, J=8.1 Hz, 2H, C2′′-
ArH and C6′′-ArH), 7.28 (d, J=7.8 Hz, 2H, 2xC7-ArH), 7.36 (d, J=7.8 Hz, 2H, 2xC4-ArH),
7.47 (d, J=8.5 Hz,, 4H, 2×C3′-ArH and 2×C5′-ArH), 7.61 (d, J=8.5 Hz,, 4H, 2×C2′-ArH and
2×C6′-ArH), 7.62 (d, J=7.9 Hz, 2H, C3′′-ArH and C5′′-ArH), 7.75 (br s, 2H, 2×NHSO2),
10.46 (s, 2H, 2×NH). 13C NMR (75 MHz, dmso-d6) δ 25.0, 39.7 (superimposed with dmso-d6
peaks), 43.9, 109.4, 111.8, 118.4, 119.2, 121.6, 124.8 (q, 1JCF=270), 125.8, 127.9 (q,
2JCF=32), 128.0, 128.8, 129.4, 129.6, 134.5, 136.3, 137.6, 139.6, 146.3. TOF-MS m/z:
847.1171 (Calcd for C40H33Cl2F3N4NaO4S2: 847.1165).
4.3.14 N,N'-((2,2'-((4-trifluoromethylphenyl)methylene)bis(1H-indole-3,2-diyl))bis(ethane-
2,1-diyl))bis(4-trifluoromethylbenzenesulfonamide) (27)
Yellow solid. 50% yield; mp 102-103 °C; IR (UATR) cm-1: 3384, 1618, 1458, 1323,
1162. 1H NMR (300 MHz, dmso-d6) δ 2.65-2.90 (m, 8H, 4×CH2), 6.03 (s, 1H, CH), 6.95 (t,
J=7.4 Hz, 2H, 2xC5-ArH), 7.03 (t, J=7.5 Hz, 2H, 2xC6-ArH), 7.22 (d, J=8.1 Hz, 2H, C2′′-
ArH and C6′′-ArH), 7.27 (d, J=8.0 Hz, 2H, 2xC7-ArH), 7.35 (d, J=7.7 Hz, 2H, 2xC4-ArH),
7.61 (d, J=8.1 Hz, 2H, C3′′-ArH and C5′′-ArH), 7.74 (d, J=8.6 Hz,, 4H, 2×C3′-ArH and
2×C5′-ArH), 7.79 (d, J=8.6 Hz,, 4H, 2×C2′-ArH and 2×C6′-ArH), 7.92 (br s, 2H, 2×NHSO2),
10.44 (s, 2H, 2×NH). 13C NMR (75 MHz, dmso-d6) δ 25.1, 39.7 (superimposed with dmso-d6
peaks), 43.9, 109.3, 111.8, 118.4, 119.2, 121.6, 123.9 (q, 1JCF=278), 125.7, 126.6, 127.7,
128.0, 129.4, 132.5 (q, 2JCF=32), 134.5, 136.3, 144.7, 146.3. TOF-MS m/z: 915.1706 (Calcd
for C42H33F9N4NaO4S2: 915.1692).
4.3.15 N,N'-((2,2'-((4-hydroxyphenyl)methylene)bis(1H-indole-3,2-diyl))bis(ethane-2,1-
diyl))bis(4-trifluoromethylbenzenesulfonamide) (30)
Red solid. 45% yield; mp 141-142 °C; IR (UATR) cm-1: 3417, 3266, 1611, 1457,
1322, 1138. 1H NMR (300 MHz, dmso-d6) δ 2.65-2.85 (m, 8H, 4×CH2), 5.78 (s, 1H, CH),
6.65 (d, J=8.4 Hz, 2H, C3′′-ArH and C5′′-ArH), 6.81 (d, J=8.4 Hz, 2H, C2′′-ArH and C6′′-
ArH), 6.92 (t, J=7.4 Hz, 2H, 2xC5-ArH), 7.00 (t, J=7.4 Hz, 2H, 2xC6-ArH), 7.26 (d, J=7.7
Hz, 2H, 2xC7-ArH), 7.29 (d, J=7.7 Hz, 2H, 2xC4-ArH), 7.76 (d, J=9.0 Hz, 4H, 2×C3′-ArH
and 2×C5′-ArH), 7.79 (d, J=9.0 Hz,, 4H, 2×C2′-ArH and 2×C6′-ArH), 7.90 (br s, 2H,
2×NHSO2), 9.37 (br s, 1H, OH), 10.34 (s, 2H, 2×NH). 13C NMR (75 MHz, dmso-d6) δ 25.1,

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39.7 (superimposed with dmso-d6 peaks), 43.9, 108.5, 111.7, 115.7, 118.1, 119.0, 121.2,
123.9 (q, 1JCF=271), 126.6, 126.7, 127.7, 128.1, 129.5, 131.6, 132.5 (q, 2JCF=32), 136.0,
136.1, 144.7, 156.6. TOF-MS m/z: 863.1803 (Calcd for C41H34F6N4NaO5S2: 863.1767).
4.3.16 N,N'-((2,2'-((9H-fluoren-2-yl)methylene)bis(1H-indole-3,2-diyl))bis(ethane-2,1-
diyl))bis(4-chlorobenzenesulfonamide) (39)
Brown solid. 44% yield; mp 156-158 °C; IR (UATR) cm-1: 3322, 1586, 1457, 1324,
1157. 1H NMR (300 MHz, dmso-d6) δ 2.65-2.85 (m, 8H, 4×CH2), 3.82 (s, 2H, CH2), 5.98 (s,
1H, CH), 6.92-7.10 (m, 5H, ArH), 7.25-7.43 (m, 11H, ArH), 7.54-7.60 (m, 5H, ArH), 7.75
(br s, 2H, 2×NHSO2), 7.77 (t, J=8.0 Hz,, 1H, ArH), 7.88 (d, J=7.5 Hz,, 1H, ArH), 10.45 (s,
2H, 2×NH). 13C NMR (75 MHz, dmso-d6) δ 24.6, 36.4, 39.7 (superimposed with dmso-d6
peaks), 43.3, 108.3, 111.3, 117.7, 118.6, 119.9, 120.8, 124.8, 125.1, 126.6, 126.7, 126.8,
127.7, 128.2, 129.0, 135.1, 135.7, 137.0, 139.2, 139.8, 139.9, 140.8, 143.1, 143.3. TOF-MS
m/z: 867.1610 (Calcd for C46H38Cl2N4NaO4S2: 867.1604).
4.3.17 N,N'-((2,2'-((9-ethyl-9H-carbazol-3-yl)methylene)bis(1H-indole-3,2-diyl))bis(ethane-
2,1-diyl))bis(4-chlorobenzenesulfonamide) (40)
Dark green solid. 40% yield; mp 148-150 °C; IR (UATR) cm-1: 3327, 1586, 1458,
1326, 1157. 1H NMR (300 MHz, dmso-d6) δ 1.30 (t, J=6.9 Hz, 3H, CH2CH3), 2.65-2.85 (m,
8H, 4×CH2), 4.42 (q, J=7.0 Hz, 3H, CH2CH3), 6.09 (s, 1H, CH), 6.96 (t, J=7.0 Hz, 2H, 2xC5-
ArH), 7.02 (t, J=7.0 Hz, 2H, 2xC6-ArH), 7.11 (d, J=7.4 Hz, 1H, ArH), 7.16 (d, J=8.4 Hz, 1H,
ArH) 7.25-7.62 (m, 15H, ArH), 7.73 (br s, 2H, 2×NHSO2), 7.89 (s, 1H, ArH), 7.97 (d, J=7.7
Hz, 1H, ArH), 10.40 (s, 2H, 2×NH). 13C NMR (75 MHz, dmso-d6) δ 14.2, 25.2, 39.2, 39.7
(superimposed with dmso-d6 peaks), 43.9, 108.5, 109.5, 111.8, 118.2, 119.0, 120.2, 120.9,
121.2, 122.5, 122.7, 126.2, 126.5, 128.3, 128.7, 129.5, 132.0, 136.2, 136.3, 137.5, 139.0,
139.7, 140.4. TOF-MS m/z: 896.1868 (Calcd for C47H41Cl2N5NaO4S2: 896.1869).
4.4 Aromatase inhibition assay
Aromatase inhibitory effect was evaluated using the modified method described by
Stressor et al. [32]. The assay was carried out according to the Gentest kit using CYP19
enzyme and O-benzyl fluorescein benzyl ester (DBF) as a fluorometric substrate. The DBF
substrate was dealkylated by the aromatase and then hydrolyzed to give the fluorescein
product.

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In brief, 100 µL of cofactor, containing 78.4 µL of 50 mM phosphate buffer (pH 7.4);
20 µL of 20x NADPH-generating system (26 mM NADP+, 66 mM glucose-6-phosphate, and
66 mM MgCl2); and 1.6 µL of 100 U/mL glucose-6-phosphate dehydrogenase, were added
o
into a 96-well black plate and preincubated in water bath (37 C) for 10 min. The reaction
was initiated by adding 100 µL of enzyme/substrate mixture containing 77.3 µL of 50 mM
phosphate buffer (pH 7.4); 12.5 µL of 16 pmol/mL CYP19; 0.2 µL of 0.2 mM DBF, and 10
µL of tested sample or 10% DMSO as a negative control or ketoconazole/letrozole as a
positive control. Fluorescence signal was recorded using an excitation wavelength of 490 nm
and emission wavelength of 530 nm with cutoff 515 nm. Percentage (%) of inhibition was
calculated using equation (2). Samples with % inhibition greater than 50 were further
determined to obtain their IC50 values.
(2)
4.5 Cytotoxicity assay: primate cell line (Vero)
The cytotoxicity was investigated using the Green Fluorescent Protein (GFP)
detection method [33]. The GFP-expressing Vero cell line was generated in-house by stably
transfecting the African green monkey kidney cell line (Vero, ATCC CCL-81), with pEGFP-
N1 plasmid (Clontech). The assay was carried out by adding 45 µL of cell suspension (3.3 ×
104 cells/mL) to each well of 384-well plates containing 5 µL of test compounds diluted in
0.5% DMSO, and then incubated for 4 days at 37 oC incubator with 5% CO2. Fluorescence
signals were measured using SpectraMax M5 microplate reader (Molecular Devices, USA)
with excitation and emission wavelengths of 485 and 535 nm, respectively. IC50 values were
determined from dose-response curves, using 6 concentrations of 3-fold serially diluted
samples, by the SOFTMax Pro software (Molecular device). Ellipticine and 0.5% DMSO
were used as a positive and a negative controls, respectively. In this study, the cell line was
maintained in a minimal essential medium supplemented with 10% heat-inactivated fetal
bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 1.5 g/L sodium bicarbonate and
0.8 mg/mL geneticin, at 37 °C in a humidified incubator with 5% CO2.
4.6 Molecular Docking

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Molecular docking of the indole derivatives was performed using crystal structure of
human placental aromatase protein co-crystalized with, natural substrate, ASD (PDBID:
3S79) which was retrieved from RSCB protein data bank (http://www.rcsb.org/). The target
protein structures were prepared by adding essential hydrogen atoms and repairing missing
side chains using the WHAT IF web server version 10.1 [34]. Non-polar hydrogen atoms
were merged, Gasteiger atomic charges were assigned, and atom type of the protein
structures were specified using AutoDock Tools version 1.5.6. [35,36]. The top three indole
derivatives that provide the aromatase inhibitory activity were selected to investigate the
binding modality. All selected ligands were constructed using Marvin Sketch version 6.1.4
[37] and were geometrically optimized by Gaussian 09 [38] using Becke’s three-parameter
hybrid method with the Lee-Yang-Parr correlation functional (B3LYP) together with the 6-
31g(d) basis set. Follow by the structural optimization, all ligands were prepared for docking
by merging non-polar hydrogen atoms, defining rotatable bonds and assigning partial atomic
charge using AutoDock Tools version 1.5.6 [35,36]. The grid box size of 25 × 25 × 25 A°
was generated and allocated to center of binding cavity using x, y and z coordinates of 83.13,
49.73, and 46.20 for intend searching modality. AutoDock Vina, as a part of the PyRx 0.8
software [39], was used to perform the molecular docking simulation. The co-crystallized
ASD were re-docked to the aromatase protein to validate docking protocol. Re-docked results
were evaluated by calculation of root mean standard deviation (RMSD) using Chimera
software [40]. Docking poses of the investigated compounds were visualized using PyMOL
[41], and two-dimensional ligand-protein interaction diagrams were generated using
Discovery Studio Visualization version 16.1.0.15350 [42].
4.7. QSAR analysis
4.7.1. Data set
Conceptually, the investigated sulfonamide-based indoles (11-44) and their
experimental aromatase inhibitory activities (IC50 values) were used as data set for QSAR
analysis. Chemical structures of the compounds (11-44) were drawn and calculated to obtain
descriptor values. IC50 values were converted to pIC50 values by taking the negative
logarithm to the base of 10 (-log IC50) to obtain the distribution of data points.
4.7.2. Molecular structure optimization and descriptor calculation

21
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Chemical structures of 34 investigated compounds (11-44) were drawn by the Marvin
Sketch version 6.1.4 [37] and initially geometrically optimized using Gaussian 09 [38] at the
semi-empirical Austin Model 1 (AM1) level followed by density functional theory (DFT)
calculation using the Becke’s three-parameter hybrid method with the Lee-Yang-Parr
correlation functional (B3LYP) together with the 6-31g(d) level. The geometrical
optimization was performed to obtain the low-energy conformers of the compounds and the
output files that were consequently used for the extraction of quantum chemical descriptors
and calculation of molecular descriptors. A set of quantum chemical descriptors comprising
the mean absolute atomic charge (Qm), total energy (Etotal), total dipole moment (µ), highest
occupied molecular orbital energy (EHOMO), lowest unoccupied molecular orbital energy
(ELUMO), energy difference of HOMO and LUMO (HOMO-LUMOGap), electron affinify
(EA), ionization potential (IP), Mulliken electronegativity (χ), hardness (η), softness (S),
electrophilic index (ωi) and electrophilicity (ω) were extracted using an in-house developed
script. A set of 3,224 molecular descriptors were calculated from the output files of optimized
molecular structures using Dragon software (version 5.5) [43]. The molecular descriptors
obtained from Dragon are comprised of 22 classes as follows: Constitutional descriptors,
Topological descriptors, Walk and path counts, Connectivity indices, Information indices, 2D
autocorrelation, Edge adjacency indices, Burden eigenvalues, Topological charge indices,
Eigenvalue-based indices, Randic molecular profiles, Geometrical descriptors, RDF
descriptors, 3D-MoRSE descriptors, WHIM descriptors, GETAWAY descriptors, Functional
group counts, Atom-centred fragments, Charge descriptors, Molecular properties, 2D binary
fingerprints and 2D frequency fingerprints.
4.7.3. Feature selection
Feature selection was performed to select informative descriptors from a large set of
descriptors obtained from calculation. Initially, a subset of descriptors was selected by
Waikato Environment for Knowledge Analysis (WEKA) version 3.4.5 [44] using attribute
evaluator CfsSubsetEval with BestFirst search method. The CfsSubsetEval selected a subset
of informative descriptors by considering the predictive ability of each descriptor along with
the degree of redundancy among them [44]. Values of the initially selected descriptors along
with bioactivity of the compounds (pIC50) then were used as input data set for further

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descriptor selection by stepwise multiple linear regression (MLR) using SPSS 18.0 (SPSS
Inc., USA) to obtain the final set of informative descriptors for multivariate analysis.
4.7.4. Multivariate analysis using multiple linear regression
Multivariate analysis was performed using the data set comprised of selected
descriptors and pIC50 values to construct QSAR model using multiple linear regression
(MLR) by WEKA version 3.4.5 [44]. The QSAR models were constructed according to the
following equation:
Y = B
0
+
B
nXn
(3)
∑
where Y is the pIC50 values of compounds, B0 is the intercept and Bn are the regression
coefficient of descriptors Xn.
The data set was divided into (i) training set and (ii) testing set by means of leave-
one-out cross validation (LOO-CV). One sample was removed from the whole data set (N) to
be used as the testing set, while the remaining samples (N-1) were used as the training set.
The same sampling process was continued until every sample in the data set was used as the
testing set to predict Y variable.
4.7.5. Evaluating the performance of QSAR models
Two statistical parameters such as correlation coefficient (R) and root mean square
error (RMSE) were used to assess predictive performance of the constructed QSAR model.
The R parameter represented predictive performance and the RMSE represented predictive
error of the model.
Acknowledgments
This project is financially supported by Srinakharinwirot University (grant no.
055/2560). Great supports from the office of the Higher Education Commission and Mahidol
University under the National Research Universities Initiative are appreciated. We are also
indebted to Chulabhorn Research Institute for recording mass spectra and bioactivity testing.
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Figure legends
Fig. 1. Representative aromatase inhibitors
Scheme 1. Synthesis of indole-sulfonamide derivatives (11-44)
Fig. 2. A: The re-docking pose of androstenedione to aromatase. The original position is
shown in red and the re-docked position is shown in yellow. B: Docking pose of compounds
28, 30 and 34. Tested compounds are shown in green and androstenedione is shown in red.
Heme group is shown in orange for A and B.
Fig. 3. Two dimensional interactions scheme of compounds 28 (A), 30 (B), 34 (C) and
androstenedione (D) to aromatase.

27
ACCEPTED MANUSCRIPT
Fig. 4. Plots of experimental versus predicted pIC50 values of aromatase inhibitory activities
generated by QSAR models (training set: compounds are represented by closed circle and
regression line is shown by solid line, leave-one-out validated testing set: compounds are
represented by open triangle and regression line is shown by dash line).

ACCEPTED MANUSCRIPT
Table 1 Anti-aromatase and cytotoxic activities (IC50, µM) of compounds (11 - 44).
Compound
R
X
Inhibitory
activity
cytotoxic activity Selectivity
(Vero cell line)a
index (SI)b
(aromatase)
11.9 ± 1.2
4-CH3
4-NO2
Non-cytotoxic
Non-cytotoxic
>5.86
>5.73
11
12
11.2 ± 1.7
3.5 ± 1.3
2.7 ± 0.1
3.5 ± 1.0
4.0 ± 1.1
5.5± 0.9
9.8 ± 0.8
15.3± 1.7
4-OCH3
4-Br
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
>19.07
>20.77
>18.56
>14.37
>11.61
>6.80
13
14
15
16
17
18
19
4-F
4-CF3
4-Cl
4-F
4-CF3
>3.85
4-Cl
2.7 ± 0.3
3.4 ± 1.6
7.6 ± 2.0
44.86
16.61
7.30
20
21
22
4-CF3
4-Br
24.81
Non-cytotoxic
>7.61
4-Cl
5.4 ± 1.2
Non-cytotoxic
>11.94
23
4-CF3
4-Br
4.4± 0.1
3.8 ± 0.4
3.2 ± 0.1
2.3 ± 0.1
15.06
3.42
24
25
26
27
Non-cytotoxic
16.18
>14.39
5.06
4-Cl
4-CF3
Non-cytotoxic
>24.35
4-OCH3
0.7 ± 0.0
Non-cytotoxic
>93.39
28
4-Cl
1.4 ± 0.1
1.3 ± 0.0
2.3 ± 0.4
4.1 ± 0.1
2.8 ± 0.1
14.98
10.70
10.25
>27.35
3.60
29
30
31
32
33
4-CF3
3-NO2
4-CH3
4-NO2
13.33
Non-cytotoxic
14.76
Non-cytotoxic
>22.46
4-OCH3
4-Cl
0.8 ± 0.1
4.2 ± 1.3
Non-cytotoxic
Non-cytotoxic
>81.71
>15.39
34
35