Design, synthesis and biological evaluation of novel naphthoquinone-4-aminobenzensulfonamide/carboxamide derivatives as proteasome inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112890

A series of novel 4-aminobenzensulfonamide/carboxamide derivatives bearing naphthoquinone pharmacophore were designed, sythesized and evaluated for their proteasome inhibitory and antiproliferative activities against human breast cancer cell line (MCF-7). The structures of the synthesized compounds were confirmed by spectral and elemental analyses. The proteasome inhibitory activity studies were carried out using cell-based assay. The antiproteasomal activity results revealed that most of the compounds exhibited inhibitory activity with different percentages against the caspase-like (C-L, β1 subunit), trypsin-like (T-L, β2 subunit) and chymotrypsin-like (ChT-L, β5 subunit) activities of proteasome. Among the tested compounds, compound 14 bearing 5-chloro-2-pyridyl ring on the nitrogen atom of sulfonamide group is the most active compound in the series and displayed higher inhibition with IC₅₀ values of 9.90 ± 0.61, 44.83 ± 4.23 and 22.27 ± 0.15 μM against ChT-L, C-L and T-L activities of proteasome compared to the lead compound PI-083 (IC₅₀ = 12.47 ± 0.21, 53.12 ± 2.56 and 26.37 ± 0.5 μM), respectively. The antiproliferative activity was also determined by MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay in vitro. According to the antiproliferative activity results, all of the compounds exhibited cell growth inhibitory activity in a range of IC₅₀ = 1.72 ± 0.14–20.8 ± 0.5 μM and compounds 13 and 28 were found to be the most active compounds with IC₅₀ values of 1.79 ± 0.21 and 1.72 ± 0.14 μM, respectively. Furthermore, molecular modeling studies were carried out for the compounds 13, 14 and 28 to investigate the ligand-enzyme binding interactions.

Full text of DOI:10.1016/j.ejmech.2020.112890

Journal Pre-proof
Design, Synthesis and Biological Evaluation of Novel Naphthoquinone-4-
aminobenzensulfonamide/carboxamide Derivatives as Proteasome Inhibitors
Sirin Uysal, Zeynep Soyer, Merve Saylam, Ayse H. Tarikogullari, Sinem Yilmaz,
Petek Ballar Kirmizibayrak
PII:
S0223-5234(20)30862-X
DOI:
https://doi.org/10.1016/j.ejmech.2020.112890
EJMECH 112890
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 July 2020
Revised Date: 23 September 2020
Accepted Date: 24 September 2020
Please cite this article as: S. Uysal, Z. Soyer, M. Saylam, A.H. Tarikogullari, S. Yilmaz, P.B.
Kirmizibayrak, Design, Synthesis and Biological Evaluation of Novel Naphthoquinone-4-
aminobenzensulfonamide/carboxamide Derivatives as Proteasome Inhibitors, European Journal of
Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112890.
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Graphical Abstract
O
O
Cl
O
N
H
N
H
S
N
Cl
O
compound 14
IC50= 9.90±0.61 µM ( 5)

Design, Synthesis and Biological Evaluation of Novel Naphthoquinone-4-aminobenzensulfonamide/carboxamide
Derivatives as Proteasome Inhibitors
Sirin UYSAL1, Zeynep SOYER1*, Merve SAYLAM2, Ayse H. TARIKOGULLARI1, Sinem YILMAZ3,4, Petek
BALLAR KIRMIZIBAYRAK5
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ege University, İzmir, Turkey.
2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, İzmir Katip Celebi University, İzmir, Turkey.
3Department of Biotechnology, Institute of Science, Ege University, İzmir, Turkey.
4Department of Bioengineering, Faculty of Engineering, University of Alaaddin Keykubat, Antalya, Turkey.
5Department of Biochemistry, Faculty of Pharmacy, Ege University, İzmir, Turkey.
* Corresponding Author
Zeynep SOYER
Department of Pharmaceutical Chemistry
Faculty of Pharmacy, Ege University
35100 Bornova, İzmir-Turkey
Phone: + 90-232-3114078
Telefax: + 90-232-3115258
e-mail: zeynep.soyer@ege.edu.tr

Abstract
A series of novel 4-aminobenzensulfonamide/carboxamide derivatives bearing naphthoquinone pharmacophore were
designed, sythesized and evaluated for their proteasome inhibitory and antiproliferative activities against human breast
cancer cell line (MCF-7). The structures of the synthesized compounds were confirmed by spectral and elemental
analyses. The proteasome inhibitory activity studies were carried out using cell-based assay. The antiproteasomal
activity results revealed that most of the compounds exhibited inhibitory activity with different percentages against the
caspase-like (C-L, β1 subunit), trypsin-like (T-L, β2 subunit) and chymotrypsin-like (ChT-L, β5 subunit) activities of
proteasome. Among the tested compounds, compound 14 bearing 5-chloro-2-pyridyl ring on the nitrogen atom of
sulfonamide group is the most active compound in the series and displayed higher inhibition with IC50 values of
9.90±0.61, 44.83±4.23 and 22.27±0.15 µM against ChT-L, C-L and T-L activities of proteasome compared to the lead
compound PI-083 (IC50=12.47±0.21, 53.12±2.56 and 26.37±0.5 µM), respectively. The antiproliferative activity was
also determined by MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay in vitro.
According to the antiproliferative activity results, all of the compounds exhibited cell growth inhibitory activity in a
range of IC50= 1.72±0.14-20.8±0.5 µM and compounds 13 and 28 were found to be the most active compounds with
IC50 values of 1.79±0.21 and 1.72±0.14 µM, respectively. Furthermore, molecular modeling studies were carried out
for the compounds 13, 14 and 28 to investigate the ligand-enzyme binding interactions.
Keywords: proteasome inhibitor; antiproliferative activity; naphthoquinone; sulfonamide/carboxamide; molecular
docking; synthesis.

1.
Introduction
The ubiquitin-proteasome system (UPS) is the main proteolytic pathway that plays a critical role in protein
homeostasis in eukaryotic cells.1-3 This system is involved in several fundamental cellular processes such as cell cycle
regulation, DNA repair, apoptosis, immune and inflammatory responses.4 26S proteasome, the vital component of the
UPS, is responsible for the ATP-dependent degredation of poly-ubiquitinated proteins and is composed of two 19S
regulatory particles and one multicatalytic proteinase complex which is known as the 20S proteasome core particle.5-7
The 20S proteasome possesses multiple catalytic activities, including caspase-like (C-L, β1 subunit), trypsin-like (T-L,
β2 subunit) and chymotripsin-like (ChT-L, β5 subunit) activities. These β catalytic subunits have slightly different
substrate specificity but they all perform a common mechanism of proteolysis by nucleophilic attack through a
hydrophilic-γ-hydroxyl group of the N-terminal threonine.8,9 Increased levels of proteasome and subsequent protein
breakdown have been implicated in many disorders such as cancer, inflammation, neurodegenerative and immune
diseases.10,11 Proliferation and apoptosis pathways are tightly regulated in a cell by the UPS and alterations of this
system may result in cellular transformation or other important pathologies especially cancer.12 The high rate of
protein production in cancer cells and the need for proteasome function in these cells for keeping up the cancer cell
survival besides proliferation are making them more sensitive to proteasome inhibition than normal cells.7,8 Therefore,
the development of proteasome inhibitors has emerged as an attractive target for the treatment of cancer and other
disorders.13-16
Bortezomib, Ixazomib and Carfilzomib have been approved by the U.S. Food and Drug Administration (FDA) for
the treatment of multiple myeloma (MM) (Figure 1).17 All of the approved inhibitors carry a peptide backbone
containing an electrophilic warhead and they covalently bound to the N-terminal threonine residue in the catalytic
subunits of the proteasome. Despite the remarkable success of these inhibitors in the clinic, intrinsic defects such as
chemical instability, poor membrane permeability and acquired drug resistance are the major problems.18 In addition,
these inhibitors have also been unsuccessful in the treatment of solid cancers.19 The peptide backbone and the
electrophilic reactive groups of these agents are thought to be the major cause of side effects, acquired drug resistance
and unsatisfactory pharmacokinetic profiles.20 Therefore, much efforts have been dedicated for the development of
non-covalent and non-peptidic proteasome inhibitors with different scaffolds which may limit some of these intrinsic
pharmacokinetic drawbacks and may translate into a broader clinical profile in recent years.21-23 As a result of these
studies, several classes of proteasome inhibitors bearing distinct pharmacophoric groups have been developed.24-26 In
2009, Kazi et al. discovered PI-083 compound, a novel non-peptide proteasome inhibitor, that possesses a unique
naphthoquinone skeleton by screening compounds from the NCI (National Cancer Institute) chemical libraries and is
proved to have a broader antitumor activity and is more selective against cancer cells compared to Bortezomib in vitro
and in vivo.27 Furthermore, studies regarding the non-peptide PI-083 and its analogues suggest the potential interaction
of the γ-hydroxyl group of the catalytic threonine residue with the 2-chloronaphthoquinone unit. PI-083 and its
naphthoquinone scaffold have been acknowledged as promising structural phenotypes for the development of new
proteasome inhibitors.28
According to the existing literature evaluation on proteasome inhibitors, PI-083 was selected as the lead compound
and a series of novel 4-aminobenzensulfonamide/carboxamide derivatives bearing naphthoquinone pharmacophoric
group have been designed by molecular modification method. As illustrated in Figure 2, the design of the compounds
was constructed based on the suggested hypothetical structure as a result of docking and the structure-activity
relationship studies on PI-083 by Xu et al.29 Accordingly, pharmacophoric unit chloronaphthoquinone was linked to
hydrogen bond acceptor sulfonamide or carboxamide group by phenylamine linker. Then, different
carbocyclic/heterocyclic rings bearing substituents with distinct electronic and steric properties were substituted to
amide nitrogen. Herein, the synthesis, antiproliferative and proteasome inhibitory activities of the designed
compounds were reported. Also molecular modeling studies were carried out for the active compounds 13, 14 and 28
to investigate the ligand enzyme binding interactions.
2.
Results and Discussion
2.1.
Chemistry
In this study, novel thirty-three naphthoquinone-4-aminobenzensulfonamide/carboxamide derivatives have been
synthesized to evaluate their antiproliferative and proteasome inhibitory activities against MCF-7 cell line. PI-083 lead
compound was also synthesized in order to compare the proteasome inhibitory activity.
The synthesis of the target compounds was illustrated in Schemes 1 and 2. As shown in Scheme 1, the sulfonamide
derivatives were synthesized in three steps according to the literature.28,30,31 First, acetanilide and chlorosulfonic acid
were reacted to yield 4-acetamidobenzensulfonyl chloride. After this compound was reacted with appropriate amines
by the nucleophilic substitution (SN2) reaction, 4-amino-N-substitutedbenzenesulfonamide intermediates were obtained
by the deacetylation reaction. Finally, 4-amino-N-substitutedbenzenesulfonamides were treated with 2,3-dichloro-1,4-
naphthoquinone to give the title compounds.
The carboxamide derivatives were prepared in two-step synthesis described in Scheme 2. Initially, 2,3-dichloro-1,4-
naphthoquinone was coupled with 4-aminobenzoic acid to obtain 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)amino)benzoic acid. After this carboxylic acid derivative was converted to acyl halide by using SOCl2 reagent, it

was reacted with corresponding amines to effort the carboxamide products (compounds 18, 20-25, 27, 32 and 33).32,33
For the synthesis of compounds 19, 26, 28-31, carboxylic intermediate 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-
2-yl)amino)benzoic acid was converted to the ester functional group in the presence of NMM and IBCF, before it was
treated with appropriate amines.34
1
The structures of the target compounds were confirmed by spectral (IR, H NMR, 13C NMR and Mass) and
1
elemental analyses. According H and 13C NMR spectra, the proton and carbon resonance signals of aromatic and
aliphatic groups were observed at the expected regions. The mass spectra of the title compounds were verified by ESI
or APCI spectra where the m/z values of molecular ion peaks were in complete agreement with the calculated
molecular weight for each compound. The purity levels of compounds were determined by elemental analysis (C, H, N
and S) and the results were within ± 0.4% of the calculated values. The compounds 2, 3, 7, 11, 12, 15-17 and 21-25
had water of crystallization (see Experimental section).
Regarding the literature survey, only compound 14 was reported previously. However, except for predicted
antimalarial activity using constructed QSAR models in silico, its synthesis and detailed spectral data have not been
described.35 The synthesis and spectral data of the title compounds were reported for the first time in this study. The
details of synthesis protocol and structural characterization are reported in the Experimental section.
2.2.
Biological activity
2.2.1. Cellular proliferation inhibition assay
Considering the fundamental role of proteasome in cell proliferation, we investigated the antiproliferative activity
of all the compounds on MCF-7 human breast cancer cell line by MTT method compared to the reference drug
doxorubicin. The activity results are listed in Table 1. The results revealed that all compounds exhibited cell growth
inhibitory activity at different ratios with the IC50 values varying from 1.72±0.14 to 20.8±0.5 µM. The most active
compound in sulfonamide series (1-17) is compound 13 bearing 5-methyl-2-pyridyl ring on the nitrogen atom of
sulfonamide group with an IC50 value of 1.79±0.21 µM. In terms of carboxamide derivatives, the most active
compound is compound 28 that bearing 4-methyl-2-pyridyl ring on the nitrogen atom of carboxamide group with an
IC50 value of 1.72 ±0.14 µM. These two most active compounds in both series showed slightly better antiproliferative
activity compared to the lead compound PI-083 with an IC50 value of 1.96±0.14 µM. The rest of carboxamide
derivatives did not produce any better activity than the lead compound PI-083, whereas most of the compounds in
sulfonamide series exhibited antiproliferative activities comparable or superior to the lead compound PI-083.
Replacing the sulfonamide group with a carboxamide group decreased the antiproliferative activity. In general,
sulfonamides had better antiproliferative activity profile than carboxamides except for compounds 19, 23, 24, 27-29,
31 and 32. In terms of compound 17 where nitrogen atom of sulfonamide group is converted into member of an
aliphatic cyclic ring provided a favorable effect on the activity. In addition, introduction of methyl substituent into the
2-pyridyl ring on the nitrogen atom of amide group afforded the most active compounds in both series. These results
indicated that the structure of the group attached on the amide group has considerable effect on the antiproliferative
activity.
2.2.2. Cell-based proteasome inhibition assay
The proteasome inhibitory activities of the synthesized compounds were evaluated for the three activities (ChT-L,
C-L and T-L) of proteasome by cell-based assay using specific fluorogenic substrates (Suc-LLVY-AMC for the ChT-
L, Z-LLE-AMC for the C-L and Boc-LRR-AMC for the T-L) on MCF-7 human breast cancer cell line. MG132 was
used as a positive control. Inhibition potency of lead compound PI-083 against ChT-L, C-L and T-L activities of
proteasome was also determined for the comparison of biological activity. In initial screening, the percentage of ChT-
L, C-L and T-L proteasome inhibitory activities of the compounds were measured and on the basis of these results a
dose-response IC50 values were obtained for compounds that displayed higher or comparable inhibition percentage
than the lead compound PI-083 at 10 µM. The antiproteasomal activity results of the compounds are reported in Tables
2 and 3.
Generally, most of the compounds possessed inhibitory activity with varying ratios against the ChT-L, C-L and T-L
activities of proteasome. Among the tested compounds, only one sulfonamide (compound 13) and one carboxamide
derivative (compound 23) did not display any inhibitory activity against C-L activity of proteasome, while seven
carboxamide derivatives (compounds 19, 25, 27-31) have been found inactive against T-L activity of proteasome.
Therefore, it can be concluded that sulfonamide derivatives possessed slightly better inhibitory activity than
carboxamide derivatives against all three proteasomal activities.
Under the set of the compounds studied, compound 14 bearing 5-chloro-2-pyridyl ring on the nitrogen atom of
sulfonamide group is the most active compound with 42.31%, 28.89% and 35.02% inhibition values against all three
ChT-L, C-L and T-L activities of proteasome, respectively. Additionally, this compound exhibited higher inhibitory
potency than lead compound PI-083 bearing 2-pyridyl ring on the nitrogen atom of sulfonamide group having 36.76%,
18.81% and 29.59% inhibition values against ChT-L, C-L and T-L activities of proteasome, respectively. The
introduction of chlorine atom into the 5-position of 2-pyridyl ring resulted in an increase on the inhibition of all three
proteasomal acitivities and produced the most active compound in the series, whereas the replacement of chlorine at
the 5-position on 2-pyridyl ring with methyl led to a decrease or loss in activity (compound 13). The positional change

of this methyl group from the 5-position to 4 or 3 positions on 2-pyridyl ring did not cause a significant improvement
on activity. On the other hand, the conversion of sulfonamide group of the most active compound (compound 14) to
carboxamide counterpart (compound 30) led to a significant decrease in ChT-L and C-L inhibitory activities and loss
of T-L inhibitory activity. These findings suggested that the characterization of substituent on the 2-pyridyl ring and
amide is important for the inhibitory activity potency.
N-phenylcarboxamide derivative, compound 21, bearing 4-nitrophenyl ring on the amide nitrogen is the second
most active compound in the series with 35.34%, 22.77% and 30.52% inhibition values against ChT-L, C-L and T-L
activities of proteasome, respectively and exhibited nearly similar potency with the lead compound PI-083. The
introduction of an electron-withdrawing nitro group on 4-position of N-phenyl ring dramatically enhanced the
inhibition potency compared to non-substituted N-phenylcarboxamide derivative, compound 18. Overall, in the N-
phenylcarboxamide derivatives, the presence of a substituent on the N-phenyl ring appeared to be benefical for the
potency when non-substituted derivative 18 is compared to other compounds with different substituents on the N-
phenyl ring, except for compounds 19 and 23 which exhibit low or no inhibition against C-L and T-L activities.
On the other hand, 3-chloro and 4-nitro substitutions on the N-phenyl ring in the N-phenylsulfonamide derivatives
yielded the most active compounds and rest of the substituents studied, namely 2-ethyl, 2-isopropyl, 3,5-dimethoxy, 2-
nitro, 3-nitro and 4-bromo, produced compounds with nearly equal activity (see Table 2). Interestingly, same activity
trend was observed in the N-phenylcarboxamide derivatives. 3-Chloro and 4-nitro substitution patterns on the N-
phenyl ring seems to give superior results than other substitutions in each series.
Concerning heterocyclic substitution on amide nitrogen in both series, the inhibitory effect of non-substituted or
substituted heterocyclic ring depended on its substitution nature and pattern. In general, the sulfonamide derivatives
(compounds 11-16) exhibited varying inhibition percentage against the three proteasome subunits, whereas
heterocyclic ring substituted carboxamide derivatives did not follow similar trend except for compounds 26, 32 and 33.
These carboxamide derivatives displayed higher ChT-L inhibitory activity than C-L inhibitory activitiy and most of
them did not show any inhibitory activity against T-L proteasomal activity. Therefore, it can be speculated that the
carboxamide derivatives are selective against ChT-L activity of proteasome.
According to the literature survey, a compound that possesses high antiproliferative activity is expected to have
good antiproteasomal activity. However, 5-methyl-2-pyridyl substituted sulfonamide derivative (compound 13) and 4-
methyl-2-pyridyl substituted carboxamide derivative (compound 28) exhibited high antiproliferative activity, whilst
they possessed weak proteasomal inhibitory activity. The discrepancy between antiproliferative activity and cell-based
proteasome inhibitory activity can be attributed to the fact that compounds exhibit cytotoxicity through a molecular
mechanism other than proteasome inhibition.
In continuation of our work, IC50 values were determined for the most active compounds (compounds 14 and 21)
which played higher or comparable inhibition percentage than the lead compound PI-083 against all three activities of
proteasome. The results suggested that compound 14 showed higher inhibitory activity with IC50 values of 9.90±0.61,
44.83±4.23 and 22.27±0.15 µM against ChT-L, C-L and T-L activities of proteasome, respectively than the lead
compound PI-083 with IC50 values of 12.47±0.21, 53.12±2.56 and 26.37±0.5 µM.
Since compound 14 showed the lowest IC50 value on the activity of ChT-L among tested proteasome catalytic
subunits and PI-083 has been previously determined as a chymotrypsin-like reversible proteasome inhibitor27, we next
determined the expression level of 20S proteasome subunit β5 which is related to ChT-L activity using MCF-7 cells
treated with compound 14, PI-083 or solvent control (DMSO) at IC50 concentrations for 12 hours. Our immunoblotting
results showed that neither compound 14 nor PI-083 caused significant change on the expression levels of β5 subunit
suggesting the inhibition of ChT-L activity by compound 14 does not depend on the expression level of target protein
(Figure 3A). On the other hand, the evaluation of K48-linked polyubiquitinated protein levels clearly revealed that
treatment of cells with compound 14 at IC50 concentration caused significantly more accumulation of
polyubiquitinated proteins compared to treatment of cells with PI-083 at IC50 concentration (Figure 3B). To sum, our
data suggested that compound 14 potently inhibits ChT-L catalytic activity without affecting 20S proteasome subunit
β5 protein expression.
Under the set of the compounds studied, the most active compound in the series had N-heterocyclic ring substituted
sulfonamide structure, whereas the second most active compound possessed N-phenyl ring substituted carboxamide
structure. Acccording to the contribution of substitution on amide nitrogen in both series, 5-chloro substitution of 2-
pyridyl ring and 4-nitro substitution of phenyl ring seemed to yield superior compounds.
2.3.
Docking Studies
Docking is a very effective method to propose the binding interactions between the ligand and the receptor. In this
study, we have chosen the most active compound (compound 14) against β1, β2 and β5 subunits of human 20S
proteasome for molecular modeling studies in order to propose the non-covalent binding interactions between
compound 14 and the active sites. Also docking studies were carried out for compounds 13 and 28 which possessed
the highest antiproliferative but low antiproteasomal activity to suggest the reason of discrepancy between the two
activities in terms of binding interactions.

The proposed binding pose of compound 14 in complex with β1 subunit has three important H-bonds (Figure 4A).
Two of these H-bonds are located between the carbonyl groups of naphthoquinone ring and Ala49 and Gly23 amino
acid residues. These H-bonds demonstrate that the core structure of the ligand is stabilized by the two key amino acids
of the active site. Ala49 draws attentions with the H-bonds that forms between the important inhibitors like PI-083 and
Bortezomib.28,36 The third H-bond of compound 14 is observed between the NH of the sulfonamide group of the side
chain and Asp51 amino acid.
On the other hand, the proposed binding pose of compound 14 in complex with β2 subunit has four H-bonds: two
H-bonds between the carbonyl groups of naphthoquinone ring and Ala20 and Gly47 amino acid residues, one H- bond
between the NH group adjacent to the naphthoquinone ring and Thr21 amino acid and one H-bond between the NH of
the sulfonamide group of the side chain and Asp166 amino acid (Figure 4B). Three of these H-bonds are formed in the
active site and two H-bonds formed are remarkable as they were formed with Thr21 and Gly47 key amino acids. These
two amino acids take place in forming binding interactions with some important proteasome inhibitors in molecular
modeling studies.27
Binding interactions of compound 14 and the β5 subunit of proteasome are different from the other proposed
binding poses. Naphthoquinone ring makes a strong π-π interaction with Tyr169 which is heading out of the pocket,
NH group located next to the ring forms a H-bond with Ser130 and also SO group of the sulfonamide on the side chain
stabilizes with Gly47, one of the key amino acids (Figure 4C).
Docking studies of compound 13 and compound 28 in complex with β1, β2 and β5 subunits of human 20S
proteasome reveal different binding interactions and different orientations in the active sites than compound 14 (Figure
5). In β1 subunit's active site, compounds 13 and 28 show similar orientation but settled in a different geometry than
compound 14. Compound 13 has one hydrogen bond formed between SO group and Ala50 amino acid residue.
Compound 28 also has one hydrogen bond formed between NH of carboxamide and Thr22 (Figures 5A, 6 and 7).
Similar case can be observed in β2 subunit's active site for compounds 13 and 28. They both settled in a different
geometry than compound 14 but relatively similar orientation in each other (Figure 5B). Compound 13 has two
hydrogen bonds which are located between SO group and Thr1 and pyridine-N and Thr21. Compound 28 formed only
one hydrogen bond between the carbonyl group of naphthoquinone ring and Gly128 amino acid residue (Figures 8 and
9).
In case of the proposed binding modes of compound 13 and 28 in the active site of β5 subunit, they both have
different orientations in the active gorge than compound 14 and also in themselves (Figure 5C). Compound 13 has
three hydrogen bonds which of two are formed between Thr1 and NH and SO of sulfonamide. The third bond was
formed between the carbonyl group of naphthoquinone ring andAla49. Compound 28 formed two hydrogen bonds
with Thr 21 and the carbonyl group of naphthoquinone ring and NH group next to the naphthoquinone ring (Figures 10
and 11). See supplementary data for figures (5-11) of the proposed binding poses of the compounds 13 and 28.
In the light of these proposed binding poses and the binding interactions, it can be observed that compound 14
forms important bonds with naphthoquinone pharmacophoric group and the side chains while compound 13 and
compound 28 settles in different orientations and forms their binding interactions mostly with the side chains or linker
parts. It can be speculated that the proteasome inhibitory activity difference may be explained by the geometry of the
compounds in the active sites and the difference of binding interactions.
2.3.1. Molecular Dynamics Simulations
Molecular dynamics (MD) simulations are an effective technique usually used to investigate the binding
interactions between the ligand and the active site, dynamically. The purpose of this technique is to simulate the body
enviroment and test if the ligand is stable in the active site and the binding interactions of the proposed binding pose
are conserved or not in a time period.
With this aim, free molecular dynamics simulations was performed in order to test the stability of β1, β2 and β5
subunits of human 20S proteasome, compound 14 in complex with β1, β2 and β5 subunits, individually and see if the
binding interactions of compound 14 with the active sites were conserved.
The MD of the apo form of β1 subunit demonstrated that the subunit was stable individually with RMSD value of
2.28 Ǻ throughout the 30 ns simulation (Figures 12 and 15). However, the MD simulation of compound 14 in the
active site of β1 subunit was stable in the active site but formed different bonds than the proposed binding interactions.
It can be speculated that even though the naphthoquinone ring possessed two H-bonds, it was unable to reach Thr1
amino acid which located in the deepest part of the gorge and very important for the inhibitor activity (Figure 12).
MD simulation of compound 14 in complex with β2 subunit was performed for 30 ns, also. The RMSD value
obtained from the MD of the apo form of β2 subunit was 11.36 Ǻ. When the simulation images were examined in
detail, it is observed that the active site was stable during the simulation but the random coil part of the subunit which
is located between Arg187 and Glu220 lead to the high RMSD value (Figures 13 and 16). MD simulation of
compound 14 in the active site of β2 subunit demonstrated that the ligand was stable in the active site but the binding
interactions were not conserved. In the beginning of the simulation the H-bond formed with Gly47 was broken and

when the ligand reached the stable position, Thr21 and Asp166 bonds were conserved but the naphthoquinone ring
was located with a 90o angle compared to its initial pose due to the loss of the H-bond with Gly47 (Figure 13).
RMSD value of the MD simulation obtained from the apo form of β5 subunit had an avarage of 1.16 Ǻ which
showed that the enzyme was stable throughout the simulation (Figures 14 and 17). On the other hand, the graphics of
compound 14 in complex with β5 subunit indicated that the π-π interaction was lost in the beginning of the simulation
and the ligand was leaned towards the deep of the gorge and located between Ala20, Ala22 and Val31 amino acids
near the end of the simulation. See supplementary data for figures (12-17) of RMSD plot and ribbon model of the
compounds.
3.
Conclusion
In this study, novel thirty-three 4-aminobenzensulfonamide/carboxamide derivatives bearing naphthoquinone
pharmacophoric group were synthesized and evaluated for their antiproliferative and proteasome inhibitory activities
against MCF-7 cell line in vitro. The antiproteasomal activity results revealed that most of the compounds exhibited
inhibitory activity with varying percentage against the chymotrypsin-like (ChT-L, β5 subunit), caspase-like (C-L, β1
subunit) and trypsin-like (T-L, β2 subunit) activities of proteasome. Among the screened compounds, compound 14
bearing 5-chloro-2-pyridyl ring on the nitrogen atom of sulfonamide group is the most active compound in the series
and displayed higher inhibition with IC50 values of 9.90±0.61, 44.83±4.23 and 22.27±0.15 µM against ChT-L, C-L and
T-L activities of proteasome, respectively compared to the lead compound PI-083 (IC50=12.47±0.21, 53.12±2.56 and
26.37±0.5 µM). According to the antiproliferative activity results, all compounds displayed different ratios of cell
growth inhibitory activity with the IC50 values varying from 1.72±0.14 to 20.8±0.5 µM against MCF-7 cell line. In
particular, compounds 13 and 28 were found to be the most active compounds with IC50 values of 1.79±0.21 and
1.72±0.14 µM, respectively. Those results suggested that these derivatives can be starting compounds to obtain new
potent proteasome inhibitors.
4.
Experimental
4.1. Chemistry
The chemical reagents and solvents in this study were purchased from common commercial suppliers and were
used without further purification. Reactions were checked by thin-layer chromatography (TLC) on precoated silica gel
plates (Kieselgel 60, F254, E. Merck, Germany). Column chromatography was performed using silica gel (200-300
mesh). All yields were unoptimized and generally represented. Melting points were determined on a Stuart SMP30
(Staffordshire, ST15 OSA, United Kingdom) melting point apparatus and are not corrected. IR spectra of the
compounds were recorded on a Perkin Elmer 100 FT-IR (ATR) spectrophotometer (Perkin Elmer Inc., MA) and the
representative absorption bands were reported. NMR spectra were recorded on a Varian AS 400 Mercury Plus NMR
1
(Varian Inc., Palo Alto, CA, USA) at 400 MHz for H and 101 MHz for 13C using DMSO-d6 as a solvent. The
1
chemical shifts (δ) of H and 13C NMR were reported in parts per million (ppm) relative to internal tetramethylsilane
(TMS), and coupling constants (J) were reported in hertz (Hz). Splitting patterns were indicated as follows: s (singlet);
bs (broad singlet); d (doublet); dd (doublet-doublet); dt (doublet-triplet); t (triplet); td (triplet-dublet); tt (triplet-triplet)
and m (multiplet). Molecular masses were determined with atomic pressure chemical ionization (APCI-MS) and
electron spray ionization mass spectra (ESI-MS) on a Thermo MSQ Plus LC/MS instrument (Thermoscientific Inc.,
San Jose, CA). Elemental analyses (C, H, N and S) were performed by Leco TruSpec Micro (Leco, St. Joseph, MI) and
are within ±0.4% of the theoretical values.
4.1.1. General procedure for the synthesis of sulfonamide derivatives (1-17)
4.1.1.1. Synthesis of 4-acetamidobenzenesulfonyl chloride
The chlorosulfonic acid (0.08 mol, 5.2 ml) was slowly added to the N-phenylacetamide (0.015 mol, 2 g) and stirred
o
at 60 C for 30 minutes. After cooling to room temperature, the mixture was poured into the ice water, then
precipitated 4-acetamidobenzenesulfonyl chloride was filtered and washed with water. The compound was obtained
without further purification. Yield 66%, mp: 145 oC.
4.1.1.2. Synthesis of 4-amino-N-substitutedbenzenesulfonamide intermediates
4-Acetamidobenzenesulfonyl chloride (0.016 mol, 3.72 g) was added in portion to a solution of appropriate amine
o
(0.02 mol) in pyridine (0.129 mol, 10 ml) at 0 C, then stirred for 1 h. The reaction mixture was poured into the ice
water, after stirring another 3 h at room temperature. The resulting solid was filtered and dried followed by dissolved
in 10% NaOH aqueous solution. Impurities were removed by filtration. The filtrate was acidified with HCl (5 M) to
pH 3-4. The precipitate formed in reaction flask was filtered and dried. After precititate was dissolved in 20 ml of
NaOH (5 M) solution, 12 ml of methanol was added and heated at 70 oC for 3h. Then, 2 M HCl solution was added to
the reaction mixture until the pH was 6. The crude product formed by acid addition was recrystallized from ethanol/
water.
4.1.1.3. Synthesis of sulfonamide derivative final compounds (1-17)
2,3-Dichloro-1,4-naphthoquinone (0.03 mol, 0.6 g) and appropriate 4-amino-N-substitutedbenzenesulfonamide
o
(0.03 mol) were suspended in 15 ml of absolute ethanol and refluxed at 115 C for 3 days. The resultant precipitate

colored with orange/red was filtered and washed with ethanol. The crude product was rinsed with EtOAc (5 ml), DCM
(5–10 ml), methanol (5–10 ml) and the mixture of DCM: aceton (1:1; 5 ml) to remove the impurities, respectively and
recrystallized from ethanol.
4.1.1.4. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(2-ethylphenyl) benzenesulfonamide (1)
o
Yield 13%; mp 258 C; IR υmaks (FT/ATR): 3245 (NH), 3227 (NH), 3020 (CH-arom), 2971 (CH-aliph), 1673
1
(C=O), 1591 (C=C), 1329 (SO2-asym), 1158 (SO2-sym) cm-1; H NMR (400 MHz, DMSO-d6): δ 9.52 (1H, bs,
SO2NH), 9.43 (1H, bs, NH-Phenyl), 8.03 (2H, dd, J=7.6; 1.2 Hz, Naphthoquinone H-5 and H-8), 7.86 (1H, td, J=7.6;
1.2 Hz, Naphthoquinone H-6 or H-7), 7.81 (1H, td, J=7.6; 1.2 Hz, Naphthoquinone H-6 or H-7), 7.52 (2H, d, J=8.4
Hz, Benzene H-2 and H-6), 7.17 (2H, d, J=8.4 Hz, Benzene H-3 and H-5), 7.19-7.11 (2H, m, Phenyl H-4 and H-6),
7.04 (1H, td, J=7.6; 1.3 Hz, Phenyl H-5), 6.88 (1H, dd, J=8.0; 1.2 Hz, Phenyl H-3), 2.46 (2H, q, J=7.6 Hz, CH2), 0.98
(3H, t, J=7.6 Hz, CH3) ppm; 13C NMR (101 MHz, DMSO-d6): δ 180.4, 177.4, 143.5, 143.1, 140.8, 135.2, 134.9, 134.6,
134.0, 132.2, 130.9, 129.3, 127.2, 127.2, 127.0, 126.7, 126.5, 122.8, 118.8, 23.5, 14.8 ppm; MS (APCI) m/z: 467
(M+H+), 469 (M+H+2+); Anal. Calcd. for C24H21ClN2O4S (468.95): C, 61.73; H, 4.10; N, 6.10; S, 6.87%, Found: C,
61.74; H, 4.18; N, 5.73; S, 7.10%.
4.1.1.5. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(2-isopropylphenyl)benzenesulfonamide(2)
o
Yield 43%; mp 186 C; IR υmaks (FT/ATR): 3238 (NH), 3085 (CH-arom), 2968 (CH-aliph), 1672 (C=O), 1590
(C=C), 1327 (SO2-asym), 1155 (SO2-sym) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 9.47 (1H, bs, NH-Phenyl), 8.03
(2H, d, J=7.2 Hz, Naphthoquinone H-5 and H-8), 7.88-7.79 (2H, m, Naphthoquinone H-6 and H-7), 7.53 (2H, d, J=8.0
Hz, Benzene H-2 and H-6), 7.26 (1H, d, J=7.6 Hz, Phenyl H-6), 7.16 (2H, d, J=8.4 Hz, Benzene H-3 and H-5), 7.19-
7.15 (1H, m, Phenyl H-4), 7.02 (1H, t, J=7.6 Hz, Phenyl H-5), 6.82 (1H, d, J=7.6 Hz, Phenyl H-3), 3.29-3.23 (1H, m,
CH(CH3)2), 0.98 (6H, d, J=6.8 Hz, 2xCH3) ppm; 13C NMR (101 MHz, DMSO-d6): 180.3, 177.4, 146.3, 143.4, 143.1,
135.2, 135.1, 134.9, 134.0, 132.2, 131.4, 130.9, 127.4, 127.0, 126.7, 126.2, 122.7, 119.0, 27.1, 24.1 ppm; MS (APCI)
m/z: 479 (M+H+), 481 (M+H+2+); Anal. Calcd. for C25H21ClN2O4S. 0.2 H2O (484.57): C, 61.97; H, 4.45; N, 5.78; S,
6.62%, Found: C, 61.58; H, 4.07; N, 5.49; S, 6.71%.
4.1.1.6. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(3,5-dimethoxyphenyl)benzenesulfonamide(3)
o
Yield 49%; mp 254 C; IR υmaks (FT/ATR): 3231 (NH), 2981 (CH-aliph), 1674 (C=O), 1606 (C=C), 1591 (C=C),
1
1340 (SO2-asym), 1290 (=C-O-C), 1161 (SO2-sym) cm-1; H NMR (400 MHz, DMSO-d6): δ 10.11 (1H, s, SO2NH),
9.47 (1H, s, NH-Phenyl), 8.03-8.00 (2H, m, Naphthoquinone H-5 and H-8), 7.86-7.77(2H, m, Naphthoquinone H-6
and H-7), 7.67 (2H, d, J=8.4 Hz, Benzene H-2 and H-6), 7.18 (2H, d, J=8.4 Hz, Benzene H-3 and H-5), 6.26 (2H, s,
Phenyl H-2 and H-6), 6.15 (1H, t, J=2.2 Hz, Phenyl H-4), 3.63 (6H, s, 2xOCH3) ppm; 13C NMR (101 MHz, DMSO-
d6): δ 180.2, 177.4, 161.1, 143.8, 143.0, 140.1, 135.1, 133.9, 133.6, 132.1, 130.9, 127.4, 127.0, 126.6, 122.7, 119.4,
98.6, 95.8, 56.5, 55.5 ppm; MS (APCI) m/z: 497 (M-H-), 499 (M-H+2-); Anal. Calcd. for C24H19ClN2O6S. 3.41 H2O
(560.37): C, 51.44; H, 4.64; N, 5.00; S, 5.72%, Found: C, 51.00; H, 4.37; N, 5.15; S, 6.11%.
4.1.1.7. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(2-nitrophenyl)benzenesulfonamide(4)
Yield 23%; mp 244 oC; IR υmaks (FT/ATR): 3308 (NH), 3276 (NH), 3100 (CH-arom), 2925 (CH-aliph), 2854 (CH-
aliph), 1672 (C=O), 1647 (C=O), 1591 (C=C), 1567 (C=C), 1514 (NO2-asym), 1350 (SO2-asym), 1328 (NO2-sym),
1
1160 (SO2-sym) cm-1; H NMR (400 MHz, DMSO-d6): δ 10.12 (1H, s, SO2NH), 9.52 (1H, s, NH-Phenyl), 8.03 (2H,
dd, J=7.6; 1.5 Hz, Naphthoquinone H-5 and H-8), 7.92 (1H, dd, J=8.0; 1.5 Hz, Phenyl H-5), 7.86 (1H, td, J=7.6, 1.6
Hz, Naphthoquinone H-6 or H-7), 7.81 (1H, td, J= 7.6; 1.6 Hz, Naphthoquinone H-6 or H-7), 7.63-7.59 (1H, m,
Phenyl H-3 or H-4), 7.60 (2H, d, J=8.6 Hz, Benzene H-2 and H-6), 7.36 (1H, td, J=7.1; 1.2 Hz, Phenyl H-3 or H-4),
7.28 (1H, dd, J=8.2; 1.3 Hz, Phenyl H-2), 7.16 (2H, d, J=8.8 Hz, Benzene H-3 and H-5) ppm; 13C NMR (101 MHz,
DMSO-d6): δ 180.3, 177.5, 144.2, 143.4, 142.9, 135.2, 134.6, 134.0, 133.0, 132.1, 130.9, 130.9, 127.5, 127.0, 126.7,
126.7, 126.0, 125.9, 122.6, 119.7 ppm; MS (ESI) m/z: 482 (M-H-), 484 (M-H+2-); Anal. Calcd. for C23H19ClN3O6S.
0.9 C2H6O (525.34): C, 54.41; H, 3.72; N, 8.00; S, 6.10%, Found: C, 54.60; H, 4.08; N, 8.38; S, 6.57%.
4.1.1.8. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(3-nitrophenyl)benzenesulfonamide(5)
o
Yield 59%, mp 268 C; IR υmaks (FT/ATR): 3260 (NH), 3257 (NH), 3100 (CH-arom), 1677 (C=O), 1590 (C=C),
1527 (NO2-asym), 1350 (NO2-sym), 1331 (SO2-asym), 1158 (SO2-sym) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 10.76
(1H, s, SO2NH), 9.50 (1H, s, NH-Phenyl), 8.02 (2H, dd, J=8.0; 1.6 Hz, Naphthoquinone H-5 and H-8), 7.90-7.78 (4H,
m, Naphthoquinone H-6 and H-7, Phenyl H-2 and H-4), 7.67 (2H, d, J=8.8 Hz, Benzene H-2 and H-6), 7.55-7.50
(2H, m, Phenyl H-5 and H-6), 7.16 (2H, d, J=8.8 Hz, Benzene H-3 and H-5) ppm; 13C NMR (101 MHz, DMSO-d6): δ
180.2, 177.4, 176.3, 148.6, 144.2, 142.9, 142.9, 139.7, 135.1, 135.1, 134.0, 132.7, 132.1, 131.4, 131.2, 130.9, 127.5,
127.5, 127.0, 126.6, 126.1, 122.7, 119.8, 118.9, 114.1 ppm; MS (APCI) m/z: 484 (M+H+), 486 (M+H+2+); Anal.
Calcd. for C22H14ClN3O6S (483.88): C, 54.61; H, 2.92; N, 8.68; S, 6.63%, Found: C, 54.59; H, 3.16; N, 8.29; S, 6.86%.
4.1.1.9. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(4-nitrophenyl)benzenesulfonamide(6)
o
Yield 21%; mp 271 C; IR υmaks (FT/ATR): 3300 (NH), 3252 (NH), 2990 (CH-arom), 1676 (C=O), 1592 (C=C),
1508 (NO2-asym), 1332 (SO2-asym), 1155 (SO2-sym) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 11.10 (1H, bs,
SO2NH), 9.49 (1H, bs, NH-Phenyl), 8.12 (2H, d, J=9.1 Hz, Phenyl H-3 and H-5), 8.02 (2H, dd, J=8.0; 1.6 Hz,
Naphthoquinone H-5 and H-8), 7.86 (1H, td, J=7.4; 1.5 Hz, Naphthoquinone H-6 or H-7), 7.81 (1H, td, J=7.4; 1.5 Hz,
Naphthoquinone H-6 or H-7), 7.74 (2H, d, J=8.8 Hz, Benzene H-2 and H-6), 7.30 (2H, d, J=9.1 Hz, Phenyl H-2 and
H-6), 7.18 (2H, d, J=8.8 Hz, Benzene H-3 and H-5) ppm; 13C NMR (101 MHz, DMSO-d6): δ 180.2, 177.4, 144.8,

144.5, 142.9, 142.9, 135.1, 134.0, 132.8, 132.1, 131.0, 127.5, 127.0, 126.7, 125.7, 122.5, 120.2, 118.5 ppm; MS
(APCI) m/z: 484 (M+H+), 486 (M+H+2+); Anal. Calcd. for C22H14ClN3O6S (483.88): C, 54.61; H, 2.92; N, 8.68; S,
6.63%, Found: C, 54.52; H, 3.11; N, 8.54; S, 6.48%.
4.1.1.10. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(2-chlorophenyl)benzenesulfonamide(7)
o
Yield 8%; mp 254 C; IR υmaks (FT/ATR): 3262 (NH), 3047 (CH-arom), 1671 (C=O), 1591 (C=C), 1337 (SO2-
1
asym), 1166 (SO2-sym) cm-1; H NMR (400 MHz, DMSO-d6): δ 9.80 (1H, bs, SO2NH), 9.51 (1H, bs, NH-Phenyl),
8.03 (2H, dd, J=7.6; 1.2 Hz, Naphthoquinone H-5 and H-8), 7.88-7.79 (2H, m, Naphthoquinone H-6 and H-7), 7.56
(2H, d, J=7.6 Hz, Benzene H-2 and H-6), 7.37 (1H, d, J=7.6 Hz, Phenyl H-3), 7.26-7.25 (2H, m, Phenyl H-5 and H-6),
7.16-7.14 (3H, m, Phenyl H-4, Benzene H-3 and H-5) ppm; 13C NMR (101 MHz, DMSO-d6): δ 180.3, 177.5, 143.7,
143.0, 135.2, 134.5, 134.1, 134.0, 132.2, 130.9, 130.3, 129.6, 128.1, 128.0, 128.0, 127.3, 127.0, 126.7, 122.7, 119.0
ppm; MS (APCI) m/z: 473 (M+H+), 475 (M+H+2+), 477 (M+H+4+); Anal. Calcd. for C22H14Cl2N2O4S. 0.3 H2O
(478.73): C, 55.19; H, 3.07; N, 5.85; S, 6.70%, C, 55.56; H, 3.25; N, 5.51; S, 6.70%.
4.1.1.11. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(3-chlorophenyl)benzenesulfonamide(8)
o
Yield 9%; mp 266 C; IR υmaks (FT/ATR): 3254 (NH), 3085 (CH-arom), 1677 (C=O), 1592 (C=C), 1329 (SO2-
1
asym), 1156 (SO2-sym) cm-1; H NMR (400 MHz, DMSO-d6): δ 10.38 (1H, bs, SO2NH), 9.48 (1H, bs, NH-Phenyl),
8.02 (2H, dd, J=7.6; 1.6 Hz, Naphthoquinone H-5 and H-8), 7.87-7.80 (2H, m, Naphthoquinone H-6 and H-7), 7.63
(2H, d, J=8.0 Hz, Benzene H-2 and H-6), 7.24 (1H, t, J=8.4 Hz, Phenyl H-5), 7.16 (2H, d, J=8.0 Hz, Benzene H-3 and
H-5), 7.07-7.04 (3H, m, Phenyl H-2, H-4 and H-6) ppm; 13C NMR (101 MHz, DMSO-d6): δ 180.3, 177.4, 144.0,
143.0, 139.9, 135.2, 134.0, 133.8, 133.1, 132.1, 131.3, 130.9, 127.4, 127.0, 126.7, 124.2, 122.7, 119.8, 119.5, 118.8
ppm; MS (APCI) m/z: 473 (M+H+), 475 (M+H+2+), 477 (M+H+4+); Anal. Calcd. for C22H14Cl2N2O4S (473.32): C,
55.82; H, 2.98; N, 5.92; S, 6.7%, Found: C, 55.70; H, 3.23; N, 5.57; S, 6.41%.
4.1.1.12. N-(4-bromophenyl)-4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)benzenesulfonamide(9)
o
Yield 59%; mp 276 C; IR υmaks (FT/ATR): 3296 (NH), 3229 (NH), 3085 (CH-arom), 1670 (C=O), 1588 (C=C),
1341 (SO2-asym), 1152 (SO2-sym) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 10.29 (1H, s, SO2NH), 9.49 (1H, s, NH-
Phenyl), 8.03 (1H, dd, J=7.6; 1.6 Hz, Naphthoquinone H-5 or H-8), 8.02 (1H, dd, J=7.6; 1.6 Hz, Naphthoquinone H-5
or H-8), 7.86 (1H, td, J=7.4; 1.5 Hz, Naphthoquinone H-6 or H-7), 7.81 (1H, td, J=7.4; 1.5 Hz, Naphthoquinone H-6
or H-7), 7.61 (2H, d, J=8.8 Hz, Benzene H-2 and H-6), 7.40 (2H, d, J=8.8 Hz, Phenyl H-3 and H-5), 7.15 (2H, d, J=8.4
Hz, Benzene H-3 and H-5), 7.02 (2H, d, J=8.8 Hz, Phenyl H-2 and H-6) ppm; 13C NMR (101 MHz, DMSO-d6): 180.2,
177.4, 143.9, 143.0, 137.7, 135.1, 134.0, 133.3, 132.4, 132.1, 130.9, 127.4, 127.0, 126.6, 122.7, 122.5, 119.5, 116.6
ppm; MS (APCI) m/z: 517 (M+H+), 519 (M+H+2+), 521 (M+H+4+); Anal. Calcd. for C22H14BrClN2O4S. 0.1 C2H6O
(522.38): C, 51.04; H, 2.82; N, 5.36; S, 6.14%, Found: C, 50.79; H, 2.86; N, 4.99; S, 5.74%.
4.1.1.13. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(naphthalen-1-yl)benzenesulfonamide(10)
Yield 47%; mp 291oC; IR υmaks (FT/ATR): 3278 (NH), 3225 (NH), 2990 (CH-arom), 1670 (C=O), 1588 (C=C),
1574 (C=C), 1341 (SO2-asym), 1151 (SO2-sym) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 10.08 (1H, bs, SO2NH), 9.46
(1H, bs, NH- Phenyl), 8.02 (2H, dd, J=7.6; 1.2 Hz, Naphthoquinone H-5 and H-8), 7.99 (1H, d, J=8.0 Hz, Naphthalene
H-4), 7.86 (1H, td, J=7.4; 1.5 Hz, Naphthoquinone H-6 or H-7), 7.80 (1H, td, J=7.4; 1.5 Hz, Naphthoquinone H-6 or
H-7), 7.76 (2H, d, J=8.0 Hz, Naphthalene H-5 and H-8), 7.53 (2H, d, J=8.4 Hz, Benzene H-2 and H-6), 7.43 (2H, td,
J=8.0; 1.2 Hz; Naphthalene H-6 and H-7), 7.39 (1H, t, J=7.6 Hz, Naphthalene H-3), 7.17 (1H, d, J=6.8 Hz,
Naphthalene H-2), 7.09 (2H, d, J=8.4 Hz, Benzene H-3 and H-5) ppm; 13C NMR (101 MHz, DMSO-d6): δ 180.3,
177.4, 143.4, 143.0, 135.2, 134.4, 134.3, 133.9, 132.9, 132.2, 130.8, 129.9, 128.4, 127.3, 127.2, 127.0, 126.6, 126.6,
126.4, 125.8, 124.0, 123.6, 122.7, 118.8 ppm; MS (APCI) m/z: 489 (M+H+), 491 (M+H+2+); Anal. Calcd. for
C26H17ClN2O4S (488.94): C, 63.87; H, 3.50; N, 5.73; S, 6.56%, Found: C, 63.74; H, 3.66; N, 5.37; S, 6.39%.
4.1.1.14. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(3-methylpyridin-2-yl)benzenesulfonamide(11)
Yield 54%; mp 284oC; IR υmaks (FT/ATR): 3329 (NH), 3259 (NH), 2972 (CH-arom), 2884 (CH-aliph), 1671 (C=O),
1607 (C=C), 1592 (C=C), 1329 (SO2-asym), 1163 (SO2-sym) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 9.44 (1H, bs,
NH- Phenyl), 8.03 (1H, dd, J=7.6; 1.2 Hz, Naphthoquinone H-5 or H-8), 8.02 (1H, dd, J=7.6; 1.2 Hz, Naphthoquinone
H-5 or H-8), 7.86 (1H, td, J=7.4; 1.5 Hz, Naphthoquinone H-6 or H-7), 7.83-7.78 (1H, m, Naphthoquinone H-6 or H-
7), 7.79 (2H, d, J=8.0 Hz, Benzene H-2 and H-6), 7.61-7.59 (2H, m, Pyridine H-4 and H-6), 7.17 (2H, d, J=8.0 Hz,
Benzene H-3 and H-5), 6.83-6.75 (1H, m, Pyridine H-5), 2.11 (3H, s, CH3) ppm; 13C NMR (101 MHz, DMSO-d6):
180.3, 177.3, 143.2, 142.8, 140.5, 135.2, 133.9, 132.2, 130.9, 127.5, 127.0, 126.9, 126.6, 122.6, 122.2, 118.5, 17.7
ppm; MS (APCI) m/z: 454 (M+H+), 456 (M+H+2+); Anal. Calcd. for C22H16ClN3O4S. 0.55 H2O (463.81): C, 56.97; H,
3.72; N, 9.06; S, 6.91%, Found: C, 56.53; H, 3.68; N, 8.70; S, 7.38%.
4.1.1.15. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(4-methylpyridin-2-yl)benzenesulfonamide(12)
o
Yield 51%; mp 283 C; IR υmaks (FT/ATR): 3270 (NH), 3047 (CH-arom), 2917 (CH-aliph), 1681 (C=O), 1595
1
(C=C), 1332 (SO2-asym), 1145 (SO2-sym) cm-1; H NMR (400 MHz, DMSO-d6): δ 9.46 (1H, bs, NH-Phenyl), 8.02
(1H, dd, J=7.6; 1.2 Hz, Naphthoquinone H-5 or H-8), 8.01 (1H, dd, J=7.6; 1.2 Hz, Naphthoquinone H-5 or H-8), 7.85
(1H, td, J=7.4; 1.5 Hz, Naphthoquinone H-6 or H-7), 7.82-7.78 (2H, m, Naphthoquinone H-6 or H-7, Pyridine H-6),
7.74 (2H, d, J=8.4 Hz, Benzene H-2 and H-6), 7.16 (2H, d, J=8.8 Hz, Benzene H-3 and H-5), 6.97 (1H, s, Pyridine H-
3), 6.66 (1H, d, J=4.8 Hz, Pyridine H-5), 2.22 (3H, s, CH3) ppm; 13C NMR (101 MHz, DMSO-d6): δ 180.3, 177.3,
153.9, 143.2, 142.8, 141.5, 137.2, 135.2, 133.9, 132.2, 130.9, 127.0, 126.6, 122.8, 118.4, 114.4, 21.6 ppm; MS (APCI)

m/z: 454 (M+H+), 456 (M+H+2+); Anal. Calcd. for C22H16ClN3O4S. 0.6 H2O (464.71): C, 56.86; H, 3.73; N, 9.04; S,
6.90%, Found: C, 56.46; H, 3.78; N, 8.61; S, 6.50%.
4.1.1.16. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(5-methylpyridin-2-yl)benzenesulfonamide(13)
o
Yield 48%; mp 223 C; IR υmaks (FT/ATR): 3268 (NH), 2985 (CH-arom), 2907 (CH-aliph), 1679 (C=O), 1592
1
(C=C), 1365 (SO2-asym), 1133 (SO2-sym) cm-1; H NMR (400 MHz, DMSO-d6): δ 9.46 (1H, bs, NH-Phenyl), 8.02
(1H, dd J=7.6; 1.2 Hz, Naphthoquinone H-5 or H-8), 8.01 (1H, dd, J=7.6; 1.2, Naphthoquinone H-5 or H-8), 7.86 (1H,
td, J=7.4; 1.5 Hz, Naphthoquinone H-6 or H-7), 7.88-7.86 (1H, m, Pyridine H-6), 7.82 (1H, td, J=7.4; 1.5 Hz,
Naphthoquinone H-6 or H-7), 7.72 (2H, d, J=8.8 Hz, Benzene H-2 and H-6), 7.53 (1H, dd, J=8.8; 2.4 Hz, Pyridine H-
4), 7.15 (2H, d, J=8.8 Hz, Benzene H-3 and H-5), 7.06 (1H, d, J=8.8 Hz, Pyridine H-3), 2.12 (3H, s, CH3) ppm; 13
C
NMR (101 MHz, DMSO-d6): δ 180.3, 177.4, 151.1, 143.2, 143.1, 141.1, 136.0, 135.1, 133.9, 132.1, 130.9, 127.5,
127.3, 127.0, 126.6, 126.2, 122.6, 118.9, 113.6, 17.3 ppm; MS (APCI) m/z: 454 (M+H+), 456 (M+H+2+); Anal. Calcd.
for C22H16ClN3O4S (453.90): C, 58.21; H, 3.55; N, 9.26; S, 7.06%, Found: C, 57.99; H, 3.59; N, 9.21; S, 7.31%.
4.1.1.17. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(5-chloropyridin-2-yl)benzenesulfonamide(14)
o
Yield 53%; mp 230 C; IR υmaks (FT/ATR): 3336 (NH), 3228 (NH), 3039 (CH-arom), 1667 (C=O), 1588 (C=C),
1335 (SO2-asym), 1159 (SO2-sym) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 11.14 (1H, bs, SO2NH), 9.51 (1H, bs, NH-
Phenyl), 8.20 (1H, d, J=2.4 Hz, Pyridine H-6), 8.03 (1H, dd, J=7.6; 1.2 Hz, Naphthoquinone H-5 or H-8), 8.02 (1H,
dd, J=7.6; 1.2 Hz, Naphthoquinone H-5 or H-8), 7.86 (1H, td, J=7.4; 1.5 Hz, Naphthoquinone H-6 or H-7), 7.83-7.76
(2H, m, Naphthoquinone H-6 or H-7, Pyridine H-4), 7.77 (2H, d, J=8.8 Hz, Benzene H-2 and H-6), 7.17 (2H, d, J=8.8
Hz, Benzene H-3 and H-5), 7.08 (1H, d, J=8.8 Hz, Pyridine H-3) ppm; 13C NMR (101 MHz, DMSO-d6): δ 180.2,
177.4, 150.6, 146.4, 144.0, 143.0, 138.7, 135.1, 134.1, 134.0, 132.1, 130.9, 127.8, 127.0, 126.7, 125.6, 125.5, 122.4,
119.6, 113.8 ppm; MS (APCI) m/z: 474 (M+H+), 476 (M+H+2+), 478 (M+H+4+); Anal. Calcd. for C21H13Cl2N3O4S.
0.5 C2H6O. 0.4 CH2Cl2 (535.92): C, 50.65; H, 3.27; N, 7.84; S, 5.98%, Found: C, 50.84; H, 2.91; N, 7.94; S, 5.80%.
4.1.1.18. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(2-chloropyridin-3-yl)benzenesulfonamide (15)
o
Yield 51%; mp 282 C; IR υmaks (FT/ATR): 3256 (NH), 3085 (CH-arom), 1670 (C=O), 1591 (C=C), 1331 (SO2-
1
asym), 1163 (SO2-sym) cm-1; H NMR (400 MHz, DMSO-d6): δ 9.53 (1H, bs, NH-Phenyl), 8.20 (1H, dd, J=4.4; 1.6
Hz, Pyridine H-6), 8.03 (2H, dd, J=7.8; 1.6 Hz, Naphthoquinone H-5 and H-8), 7.86 (1H, td, J=7.6; 1.2 Hz,
Naphthoquinone H-6 or H-7), 7.81 (1H, td, J=7.6; 1.2 Hz, Naphthoquinone H-6 or H-7), 7.72 (1H, dd, J=8.0; 1.6 Hz,
Pyridine H-4), 7.58 (2H, d, J=8.0 Hz, Benzene H-2 and H-6), 7.4 (1H, dd, J=8.0; 4.8 Hz, Pyridine H-5), 7.17 (2H, d,
J=8.0 Hz, Benzene H-3 and H-5) ppm; 13C NMR (101 MHz, DMSO-d6): δ 180.3, 177.5, 147.2, 146.2, 144.0, 143.0,
136.1, 135.2, 135.1, 134.0, 132.2, 131.3, 130.9, 127.5, 127.3, 127.0, 126.7, 124.1, 122.7, 122.1, 119.4 ppm; MS
(APCI) m/z: 474 (M+H+), 476 (M+H+2+), 478 (M+H+4+); Anal. Calcd. for C21H13ClN3O4S. 2 H2O (510.34): C, 49.42;
H, 3.36; N, 8.23; S, 6.28%, Found: C, 49.47; H, 2.94; N, 7.80; S, 6.24%.
4.1.1.19. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(4-methylthiazol-2-yl)benzenesulfonamide(16)
o
Yield 49%; mp 271 C; IR υmaks (FT/ATR): 3328 (NH), 3224 (NH), 3085 (CH-arom), 1677 (C=O), 1571 (C=C),
1386 (SO2-asym), 1140 (SO2-sym) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 12.56 (1H, bs, SO2NH), 9.45 (1H, s, NH-
Phenyl), 8.02 (2H, dd, J= 7.2; 1.2, Naphthoquinone H-5 and H-8), 7.86 (1H, td, J= 7.4; 1.5 Hz, Naphthoquinone H-6
or H-7), 7.81 (1H, td, J=7.4; 1.5 Hz, Naphthoquinone H-6 or H-7), 7.67 (2H, d, J= 8.8 Hz, Benzene H-2 and H-6),
7.17 (2H, d, J= 8.8 Hz, Benzene H-3 and H-5), 6.36 (1H, s, Thiazole H-4), 2.06 (3H, s, CH3) ppm; 13C NMR (101
MHz, DMSO-d6): δ 180.3, 177.3, 169.0, 143.1, 142.9, 137.3, 135.2, 134.1, 133.9, 132.2, 130.9, 127.0, 126.6, 126.4,
122.8, 118.5, 102.6, 13.7 ppm; MS (APCI) m/z: 460 (M+H+), 462 (M+H+2+); Anal. Calcd. for C20H14ClN3O4S2. 0.5
H2O (468.93): C, 51.23; H, 3.22; N, 8.96; S, 13.68%, Found: C, 51.20; H, 3.49; N, 8.56; S, 13.40%.
4.1.1.20. 2-chloro-3-((4-(morpholinosulfonyl)phenyl)amino)naphthalene-1,4-dione(17)
o
Yield 55%; mp 230 C; IR υmaks (FT/ATR): 3281 (NH), 3085 (CH-arom), 2990 (CH-aliph), 1682 (C=O), 1607
(C=C), 1344 (SO2-asym), 1159 (SO2-sym), 1134 (C-O-C) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 9.57 (1H, s, NH-
Phenyl), 8.04 (2H, dd, J=7.6; 1.2 Hz, Naphthoquinone H-5 and H-8), 7.86 (1H, td, J=7.4; 1.5 Hz, Naphthoquinone H-6
or H-7), 7.82 (1H, td, J=7.4; 1.5 Hz, Naphthoquinone H-6 or H-7), 7.62 (2H, d, J=8.0 Hz, Benzene H-2 and H-6), 7.28
(2H, d, J=8.0 Hz, Benzene H-3 and H-5), 3.63.3.60 (4H, m, Morpholine-H), 2.86-2.83 (4H, m, Morpholine-H) ppm;
13C NMR (101 MHz, DMSO-d6): δ 194.4, 180.2, 177.4, 144.4, 143.0, 135.1, 134.0, 132.1, 131.0, 128.5, 128.3, 127.0,
126.7, 122.6, 119.9, 65.7, 46.4 ppm; MS (APCI) m/z: 433 (M+H+), 435 (M+H+2+); Anal. Calcd. for C20H17ClN2O5S.
0.9 H2O (449.09): C, 53.49; H, 3.87; N, 6.11; S, 7.00%, Found: C, 53.14; H, 3.96; N, 5.82; S, 7.02%.
4.1.1.21. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(pyridin-2-yl)benzenesulfonamide (PI-083)
Yield 44%; mp 272 oC; 3221 (NH), 3085 (CH-arom), 1683 (C=O), 1605 (C=C), 1352 (SO2-asym), 1135 (SO2-sym)
1
cm-1; H NMR (400 MHz, DMSO-d6): δ 9.44 (1H, bs, NH-Phenyl), 8.04-8.00 (3H, m, Naphthoquinone H-5 and H-8,
Pyridine H-6), 7.85 (1H, td, J=7.4; 1.5 Hz, Naphthoquinone H-6 or H-7), 7.80 (1H, td, J=7.4; 1.5 Hz, Naphthoquinone
H-6 or H-7), 7.75 (2H, d, J=8.0 Hz, Benzene H-2 and H-6), 7.72-7.69 (1H, m, Pyridine H-5), 7.16 (2H, d, J=8.0 Hz,
Benzene H-3 and H-5), 7.17-7.13 (1H, m, Pyridine H-3), 6.86 (1H, t, J=6.4 Hz, Pyridine H-4) ppm; 13C NMR (101
MHz, DMSO-d6): δ 180.3, 177.4, 153.4, 143.2, 143.1, 140.5, 136.3, 135.2, 133.9, 132.1, 130.9, 127.5, 127.2, 127.0,
126.6, 122.7, 118.8, 116.2, 114.1 ppm; MS (APCI) m/z: 440 (M+H+), 442 (M+H+2+); Anal. Calcd. for
C21H14ClN3O4S. 0.3 H2O (445.27): C, 56.64; H, 3.30; N, 9.44; S, 7.20%, Found: C, 56.27; H, 3.43; N, 9.30; S, 6.88%.

4.1.2. General procedure for the synthesis of carboxamide derivatives (18-33)
4.1.2.1. Synthesis of 4-[(3-chloro-1,4-dioxo-1,4-dihydronaphthalene-2-yl)amino]benzoic acid
2,3-Dichloro-1,4-naphthoquinone (0.002 mol, 0.5 g) and 4-aminobenzoic acid (0.002 mol, 0.3 g) were refluxed in
the presence of anhydrous K2CO3 (0.0007 mol, 0.1 g) in 20 ml of ethanol for 6 h. The reaction mixture was filtered off
and the solid was washed with ethanol and water. The crude product was recrystallized from ethanol. Yield 33%, mp:
307 oC.
4.1.2.2. Synthesis of carboxamide derivative final compounds (18, 20-25, 27, 32 and 33)
The previously synthesized 4-[(3-chloro-1,4-dioxo-1,4-dihydronaphthtalen-2-yl)amino]benzoic acid was refluxed in
SOCl2 for 3 h. Then, excess SOCl2 was removed from the reaction mixture by rotary evoporation, the residue was
dissolved in 15 ml of dichloromethane (DCM) and stirred under argon atmosphere. Appropriate amine was added to
reaction mixture in an ice bath, and it was allowed to stir for 3 h, gradually warming up to room temperature. The
resulting precipitate was filtered off and washed with DCM. The crude product was purified by recrystallization from
ethanol or methanol. Compound 32 and 33 were purified via coloumn chromatography utilizing a 4:4:1 hexane:
chloroform/dichloromethane: methanol.
4.1.2.3. Synthesis of carboxamide derivative final compounds (19, 26, 28-31)
To a stirring solution of 4-[(3-chloro-1,4-dioxo-1,4-dihydronaphthtalen-2-yl)amino]benzoic acid (0.00053 mol)
suspended in 10 ml of tetrahydrofuran (THF) cooled to 0 oC, N-methylmorpholine (NMM) (0.00107 mol) and isobutyl
chloroformate (IBCF) (0.00114 mol) were added under argon atmosphere and stirred for 4h at 0 oC. Then, appropriate
amine (0.00053 mol) was added and the reaction mixture was refluxed up to 36-72 h. After completion of the reaction
was monitered by TLC, the precipitated NMM.HCl was removed by filtration. The organic phase was evaporated
under reduced pressure, the obtained solid was purified via column chromatography utilizing a 4:4:1 hexane:
chloroform/dichloromethane:methanol.
4.1.2.4. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-phenylbenzamide(18)
o
Yield 40%; mp 304 C; IR υmaks (FT/ATR): 3239 (NH), 3066 (CH-arom), 1673 (C=O), 1644 (Amide I), 1595
1
(C=C), 1569 (Amide II), 1288 (=C-N) cm-1; H NMR (400 MHz, DMSO-d6): δ 10.12 (1H, s, CONH), 9.46 (1H, s,
NH-Phenyl), 8.04 (2H, dt, J=8.0; 1.6 Hz, Naphthoquinone H-5 and H-8), 7.90 (2H, d, J=8.0 Hz, Benzene H-2 and H-
6), 7.86-7.79 (2H, m, Naphthoquinone H-6 and H-7), 7.75 (2H, d, J=8.0 Hz, Phenyl H-2 and H-6), 7.32 (2H, t, J=8.0
Hz, Phenyl H-3 and H-5), 7.20 (2H, d, J=8.0 Hz, Benzene H-3 and H-5), 7.07 (1H, t, J=7.6 Hz, Phenyl H-4) ppm; 13C
NMR (101 MHz, DMSO-d6): δ 180.6, 177.3, 165.6, 156.5, 143.2, 139.7, 138.9, 135.2, 133.8, 132.3, 130.9, 130.1,
129.0, 128.1, 127.0, 126.6, 123.9, 122.7, 120.8 ppm; MS (APCI) m/z: 403 (M+H+), 405 (M+H+2+); Anal. Calcd. for
C23H15ClN2O.0.5 C2H6O (425.87): C, 68.02; H, 3.99; N, 6.84%, Found: C, 67.75; H, 4.39; N, 6.88%.
4.1.2.5. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(2-isopropylphenyl)benzamide(19)
Yield 17%; mp 245 oC; IR υmaks (FT/ATR): 3213 (NH), 2959 (CH-arom), 2925 (CH-aliph), 2854 (CH-aliph), 1676
1
(C=O), 1638 (Amide I), 1596 (C=C), 1568 (Amide II), 1285 (=C-N) cm-1; H NMR (400 MHz, DMSO-d6): δ 9.82
(1H, s, CONH), 9.47 (1H, s, NH-Phenyl), 8.08-8.05 (2H, m, Naphthoquinone H-5 and H-8), 7.93 (2H, d, J=8.1 Hz,
Benzene H-2 and H-6), 7.89 (1H, td, J=7.5; 1.5 Hz, Naphthoquinone H-6 or H-7), 7.83 (1H, td, J=7.5; 1.3 Hz,
Naphthoquinone H-6 or H-7), 7.35 (1H, d, J=7.5 Phenyl H-6), 7.28-7.23 (1H, m, Phenyl), 7.22 (2H, d, J=7.3 Hz,
Benzene H-3 and H-5), 7.22-7.18 (2H, m, Phenyl H), 3.18 (1H, m, CH), 1.14 (6H, d, J=6.7 Hz, 2xCH3) ppm; 13C
NMR (101 MHz, DMSO-d6): δ 180.5, 177.3, 165.9, 145.3, 143.2, 142.5, 135.6, 135.2, 133.8, 132.3, 130.8, 129.7,
128.6, 128.0, 127.3, 127.0, 126.6, 126.2, 126.0, 122.8, 117.5, 28.0, 23.6 ppm; MS (ESI) m/z: 445 (M+H+), 447
(M+H+2+); Anal. Calcd. for C26H21ClN2O3.0.1 CHCl3 (456.85): C, 68.62; H, 4.66; N, 6.13%, Found: C, 68.32; H,
4.58; N, 9.60%.
4.1.2.6. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(m-tolyl)benzamide(20)
o
Yield 46%; mp 261 C; IR υmaks (FT/ATR): 3227 (NH), 3066 (CH-arom), 2971 (CH-aliph), 1674 (C=O), 1641
(Amide I), 1593 (C=C), 1567 (Amide II), 1288 (=C-N) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 10.08 (1H, s, CONH),
9.45 (1H, s, NH-Phenyl), 8.04 (2H, dd, J=8.0; 1.2 Hz, Naphthoquinone H-5 and H-8), 7.90 (2H, d, J=8.0 Hz, Benzene
H-2 and H-6), 7.87-7.79 (2H, m, Naphthoquinone H-6 and H-7), 7.61 (1H, s, Phenyl H-2), 7.56 (1H, d, J=8.0 Hz,
Phenyl H-6), 7.21-7.17 (1H, m, Phenyl H-5), 7.19 (2H, d, J=8.0 Hz, Benzene H-3 and H-5), 6.89 (1H, d, J=8.0 Hz,
Phenyl H-4), 2.28 (3H, s, CH3) ppm; 13C NMR (101 MHz, DMSO-d6): δ 180.4, 177.3, 165.3, 143.2, 142.5, 139.8,
138.1, 135.2, 133.9, 132.4, 130.8, 129.9, 128.8, 128.8, 126.6, 124.6, 123.9, 122.7, 121.4, 120.6, 117.6, 21.7 ppm; MS
(APCI) m/z: 417 (M+H+), 419 (M+H+2+); Anal. Calcd. for C24H17ClN2O3.1.1 C2H6O (467.54): C, 67.31; H, 5.09; N,
5.99%, Found: C, 67.07; H, 5.49; N, 6.33%.
4.1.2.7. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(4-nitrophenyl)benzamide(21)
o
Yield 13%; mp >300 C; IR υmaks (FT/ATR): 3206 (NH), 3028 (CH-arom), 1673 (C=O), 1645 (Amide I), 1594
1
(C=C), 1568 (Amide II), 1539 (NO2-asym), 1331 (NO2-sym), 1290 (=C-N) cm-1; H NMR (400 MHz, DMSO-d6): δ
10.69 (1H, s, CONH), 9.50 (1H, s, NH-Phenyl), 8.25 (2H, d, J=8.2 Hz, Phenyl H-3 and H-5), 8.05 (2H, d, J=8.4 Hz,
Phenyl H-2 and H-6), 8.08-8.02 (2H, m, Naphthoquinone H-5 and H-8), 7.92 (2H, d, J=8.4 Hz, Benzene H-2 and H-6),
7.89-7.80 (2H, m, Naphthoquinone H-6 and H-7), 7.22 (2H, d, J=8.4 Hz, Benzene H-3 and H-5) ppm; 13C NMR (101
MHz, DMSO-d6): δ 180.4, 177.4, 166.1, 146.1, 143.3, 143.2, 142.8, 135.2, 133.9, 132.2, 130.9, 129.1, 128.5, 127.0,

126.6, 125.2, 122.6, 120.2, 118.3 ppm; MS (APCI) m/z: 446 (M-H-), 448 (M-H+2-); Anal. Calcd. for
C23H14ClN3O5.0.5 H2O (456.84): C, 60.00; H, 3.37; N, 9.02%, Found: C, 60.39; H, 3.46; N, 8.62%.
4.1.2.8. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(3-chlorophenyl)benzamide(22)
o
Yield 28%; mp 267 C; IR υmaks (FT/ATR): 3230 (NH), 3028 (CH-arom), 1672 (C=O), 1644 (Amide I), 1592
1
(C=C), 1568 (Amide II), 1285 (=C-N) cm-1; H NMR (400 MHz, DMSO-d6): δ 10.36 (1H, s, CONH), 9.47 (1H, s,
NH-Phenyl), 8.03 (2H, dd, J=7.2; 1.2 Hz, Naphthoquinone H-5 and H-8), 7.97 (1H, d, J=1.6 Hz, Phenyl H-6), 7.91
(2H, d, J=7.2 Hz, Benzen H-2 and H-6), 7.88-7.79 (2H, m, Naphthoquinone H-6 and H-7), 7.72 (1H, d, J=8.0, Phenyl
H-2), 7.35 (1H, td, J=8.0; 1.2 Hz, Phenyl H-5), 7.20 (2H, d, J=7.2 Hz, Benzene H-3 and H-5), 7.13 (1H, td, J=8.0; 1.2
Hz, Phenyl H-4) ppm; 13C NMR (101 MHz, DMSO-d6): δ 180.4, 177.3, 165.6, 143.2, 142.9, 141.3, 135.2, 133.9,
133.3, 132.2, 130.9, 130.7, 129.8, 129.5, 128.3, 127.0, 126.6, 123.6, 122.7, 122.5, 120.2, 119.1, 117.9 ppm; MS
(APCI) m/z: 437 (M+H+), 439 (M+H+2+), 441 (M+H+4+); Anal. Calcd. for C23H14Cl2N2O3.0.35 H2O (479.63): C,
62.28; H, 3.34; N, 6.32%, Found: C, 62.41; H, 3.14; N, 6.29%.
4.1.2.9. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(4-chlorophenyl)benzamide(23)
o
Yield 26%; mp 275 C; IR υmaks (FT/ATR): 3222 (NH), 3047 (CH-arom), 1674 (C=O), 1640 (Amide I), 1595
1
(C=C), 1568 (Amide II), 1286 (=C-N) cm-1; H NMR (400 MHz, DMSO-d6): δ 10.31 (1H, s, CONH), 9.46 (1H, s,
NH-Phenyl), 8.04 (2H, dt, J=7.6; 1.6 Hz, Naphthoquinone H-5 and H-8), 7.91 (2H, d, J=8.8 Hz, Benzene H-2 and H-
6), 7.88-7.83 (2H, m, Naphthoquinone H-6 and H-7), 7.82 (2H, d, J=9.2, Phenyl H-2 and H-6), 7.38 (2H, d, J=8.8 Hz,
Phenyl H-3 and H-5), 7.20 (2H, d, J=8.4 Hz, Benzene H-3 and H-5) ppm; 13C NMR (101 MHz, DMSO-d6): δ 180.4,
177.3, 165.5, 143.2, 142.7, 138.7, 135.2, 133.9, 132.2, 130.9, 129.7, 128.9, 128.2, 127.5, 127.0, 126.6, 122.7, 122.3,
117.8 ppm; MS (APCI) m/z: 437 (M+H+), 439 (M+H+2+), 441 (M+H+4+); Anal. Calcd. for C23H14Cl2N2O3.0.5
C2H6O.0.5 H2O (505.37): C, 61.42; H, 3.87; N, 5.97%, Found: C, 61.66; H, 4.07; N, 5.85%.
4.1.2.10. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(naphthalen-1-yl)benzamide(24)
o
Yield 42%; mp 264 C; IR υmaks (FT/ATR): 3227 (NH), 3047 (CH-arom), 1674 (C=O), 1640 (Amide I), 1597
1
(C=C), 1569 (Amide II), 1287 (=C-N) cm-1; H NMR (400 MHz, DMSO-d6): δ 10.33 (1H, s, CONH), 9.49 (1H, s,
NH-Phenyl), 8.07-7.95 (5H, m, Naphthoquinone H-5 and H-8, Naphthalene-H), 7.91-7.81 (3H, m, Naphthoquinone
H-6 and H-7, Naphthalene-H), 7.80-7.71 (1H, m, Naphthalene-H), 7.60-7.52 (3H, m, Benzene H-2 and H-6,
Naphthalene-H), 7.45-7.36 (1H, m, Naphthalene-H), 7.24 (2H, d, J=8.4 Hz, Benzene H-3 ve H-5) ppm; 13C NMR (101
MHz, DMSO-d6): δ 180.5, 177.4, 166.1, 143.3, 142.7, 135.2, 134.4, 134.2, 133.9, 132.3, 130.9, 129.7, 129.6, 128.7,
128.3, 127.2, 127.0, 126.7, 126.6, 126.5, 126.4, 126.2, 126.0, 124.4, 122.8, 122.4, 117.7 ppm; MS (APCI) m/z: 451
(M-H-), 453 (M-H+2-); Anal. Calcd. for C27H17ClN2O3.0.6 H2O (463.70): C, 69.94; H, 3.96; N, 6.04%, Found: C,
69.75; H, 4.14; N, 6.29%.
4.1.2.11. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(pyridin-3-yl)benzamide(25)
o
Yield 35%; mp 197 C; IR υmaks (FT/ATR): 3213 (NH), 3100 (CH-arom), 2960 (CH-arom), 1675 (C=O), 1648 (
Amide I), 1599 (C=C), 1572 (Amide II), 1294 (=C-N) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 10.36 (1H, s, CONH),
9.52 (1H, bs, NH-Phenyl), 8.92 (1H, d, J=2.4 Hz, Pyridine H-2), 8.29 (1H, dd, J=4.4; 1.4 Hz, Pyridine H-6), 8.18 (1H,
dt, J=8.4; 2.0 Hz, Pyridine H-4), 8.04 (2H, dt, J=7.6; 1.6 Hz, Naphthoquinone H-5 and H-8), 7.91 (2H, d, J=8.8 Hz,
Benzene H-2 and H-6), 7.87 (1H, td, J=7.6; 1.6 Hz, Naphthoquinone H-6 or H-7), 7.82 (1H, td, J=7.6; 1.6 Hz,
Naphthoquinone H-6 and H-7), 7.38 (1H, dd, J=8.4; 4.8 Hz, Pyridine H-5), 7.22 (2H, d, J=8.8 Hz, Benzene H-3 and
H-5) ppm; 13C NMR (101 MHz, DMSO-d6): δ 180.4, 177.3, 165.8, 144.8, 143.3, 143.0, 142.4, 136.4, 135.2, 133.9,
132.3, 130.9, 129.8, 129.4, 128.2, 127.7, 127.0, 126.6, 123.9, 122.7, 122.6, 117.9 ppm; MS (APCI) m/z: 404 (M+H+),
406 (M+H+2+); Anal. Calcd. for C22H14ClN3O3.0.3 H2O (409.23): C, 64.57; H, 3.60; N, 10.27%, Found: C, 64.38; H,
3.76; N, 10.28%.
4.1.2.12. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(pyridin-4-yl)benzamide(26)
o
Yield 17%; mp 233 C (decomp.); IR υmaks (FT/ATR): 3223 (NH), 3000 (CH-arom), 1673 (C=O), 1639 (Amide I),
1593 (C=C), 1565 (Amide II), 1287 (=C-N) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 10.49 (1H, s, CONH), 10.07 (1H,
bs, NH- Phenyl), 8.47-8.41 (1H, m, Pyridine-H), 8.05-8.00 (2H, m, Pyridine-H), 7.89 (2H, d, J=6.9 Hz, Benzene H-2
and H-6), 7.90-7.81 (3H, m, Naphthoquinone H-6 and H-7, Pyridine-H), 7.77 (2H, d, J=7.2 Hz, Naphthoquinone H-5
and H-8), 7.16 (2H, d, J=6.9 Hz, Benzene H-3 and H-5) ppm; 13C NMR (101 MHz, DMSO-d6): δ 180.6, 166.3, 150.7,
147.3, 146.7, 146.6, 135.1, 133.6, 132.6, 131.0, 128.5, 127.0, 126.5, 122.4, 114.5, 114.4 ppm; MS (ESI) m/z: 404
(M+H+), 406 (M+H+2+); Anal. Calcd. for C22H14ClN3O3.0.78 CHCl3. 0.46 C6H12 (535.64): C, 57.27; H, 3.82; N,
7.84%, Found: C, 57.68; H, 3.85; N, 8.13%.
4.1.2.13. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(3-methylpyridin-2-yl)benzamide(27)
o
Yield 7%; mp 257 C; IR υmaks (FT/ATR): 3228 (NH), 3090 (CH-arom), 2920 (CH-aliph), 1674 (C=O), 1644 (
Amide I), 1592 (C=C), 1570 (Amide II), 1283 (=C-N) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 10.40 (1H, s, CONH),
9.46 (1H, bs, NH-Phenyl), 8.29 (1H, d, J=5.1 Hz, Pyridine-H), 8.04 (2H, d, J=7.6 Hz, Naphthoquinone H-5 and H-8),
7.92 (2H, d, J= 8.7 Hz, Benzene H-2 and H-6), 7.86 (1H, td, J=7.6; 1.6 Hz, Naphthoquinone H-6 or H-7), 7.81 (1H, td,
J=7.4; 1.6 Hz, Naphthoquinone H-6 or H-7), 7.70 (1H, dd, J=8.1; 1.6 Hz, Pyridine-H), 7.23 (1H, dd, J=7.5; 4.6 Hz,
Pyridine-H), 7.18 (2H, d, J= 8.6 Hz, Benzene H-3 and H-5), 2.18 (1H, s, CH3) ppm; 13C NMR (101 MHz, DMSO-d6):
δ 180.5, 177.4, 165.3, 150.9, 146.3, 143.3, 142.8, 139.7, 135.2, 133.9, 132.3, 130.9, 130.2, 129.0, 128.4, 127.0, 126.6,

122.7, 122.5, 117.9, 18.1 ppm; MS (ESI) m/z: 418 (M+H+), 420 (M+H+2+); Anal. Calcd. for C23H16ClN3O3 (417.85):
C, 66.11; H, 3.86; N, 10.06%, Found: C, 65.75; H, 3.76; N, 9.67%.
4.1.2.14. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(4-methylpyridin-2-yl)benzamide(28)
o
Yield 2%; mp 259 C; IR υmaks (FT/ATR): 3217 (NH), 3045 (CH-arom), 2922 (CH-aliph), 2852 (CH-aliph), 1673
1
(C=O), 1636 (Amide I), 1595 (C=C), 1561 (Amide II), 1286 (=C-N) cm-1; H NMR (400 MHz, DMSO-d6): δ 10.52
(1H, s, CONH), 9.46 (1H, bs, NH-Phenyl), 8.21 (1H, d, J=5.1, Pyridine-H), 8.10-8.01 (3H, m, Naphthoquinone H-5
and H-8, Pyridine H), 7.97 (2H, d, J= 9.8 Hz, Benzene H-2 and H-6), 7.91-7.80 (2H, m, Naphthoquinone H-6 and H-
7), 7.17 (2H, d, J=8.3 Hz, Benzene H-3 and H-5), 7.01-6.96 (1H, m, Pyridine-H), 2.34 (1H, s, CH3) ppm; 13C NMR
(101 MHz, DMSO-d6): δ 180.5, 177.4, 165.6, 152.8, 149.1, 148.0, 143.2, 143.0, 135.2, 133.9, 132.3, 130.9, 129.1,
128.5, 127.0, 126.6, 122.5, 121.1, 118.1, 115.6, 21.4 ppm; MS (ESI) m/z: 418 (M+H+), 420 (M+H+2+); Anal. Calcd.
for C23H16ClN3O3. 0.07 CHCl3. 0.28 C6H12 (449.77): C, 66.09; H, 4.35; N, 9.34%, Found: C, 66.53; H, 3.91; N, 9.31%.
4.1.2.15. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(5-methylpyridin-2-yl)benzamide(29)
o
Yield 8%; mp 241 C; IR υmaks (FT/ATR): 3213 (NH), 3100 (CH-arom), 2996 (CH-aliph), 1674 (C=O), 1648
(Amide I), 1594 (C=C), 1568 (Amide II), 1289 (=C-N) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 10.53 (1H, s, CONH),
9.45 (1H, bs, NH-Phenyl), 8.20-8.19 (1H, m, Pyridine-H), 8.07-8.03 (3H, m, Naphthoquinone-H, Pyridine-H), 7.98-
7.96 (2H, m, Benzene H-2 and H-6), 7.89-7.85 (1H, m, Naphthoquinone H-6 or H-7), 7.84-7.79 (1H, m,
Naphthoquinone H-6 or H-7), 7.64 (1H, dd, J=8.5; 2.4 Hz, Pyridine H-4), 7.17 (2H, d, J=8.6 Hz, Benzene H-3 and H-
5), 2.27 (1H, s, CH3) ppm; 13C NMR (101 MHz, DMSO-d6): δ 180.4, 177.4, 165.5, 150.5, 148.0, 142.9, 138.8, 135.2,
133.9, 132.3, 130.9, 129.1, 128.4, 126.6, 122.5, 118.0, 114.7, 40.6, 17.8 ppm; MS (ESI) m/z: 418 (M+H+), 420
(M+H+2+); Anal. Calcd. for C23H16ClN3O3. 0.27 C6H12. 0.4 CHCl3 (488.32): C, 61.54; H, 4.05; N, 8.61%, Found: C,
61.98; H, 3.34; N, 8.54%.
4.1.2.16. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(5-chloropyridin-2-yl)benzamide(30)
o
Yield 3%; mp 283 C (decomp); IR υmaks (FT/ATR): 3388 (NH), 3304 (NH), 3070 (CH-arom) 1676 (C=O), 1665
(Amide I), 1598 (C=C), 1572 (Amide II), 1289 (=C-N) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 10.83 (1H, s, CONH),
9.50 (1H, bs, NH-Phenyl), 8.42-8.41 (1H, m, Pyridine-H), 8.21 (1H, dd, J=8.8; 1.2 Hz, Pyridine H-3), 8.04 (2H, dd,
J=7.6; 1.6 Hz, Naphthoquinone H-5 and H-8), 8.01-7.92 (1H, m, Pyridine-H), 7.96 (2H, d, J=7.2 Hz, Benzene H-2 and
H-6), 7.87 (1H, tt, J=7.6, 1.5 Hz, Naphthoquinone H-6 or H-7), 7.82 (1H, tt, J=7.6, 1.5 Hz, Naphthoquinone H-6 or H-
7), 7.18 (2H, d, J=7.2 Hz, Benzene H-3 and H-5) ppm; 13C NMR (101 MHz, DMSO-d6): δ 180.5, 165.9, 151.4, 146.7,
138.2, 135.2, 133.9, 130.9, 128.6, 127.0, 126.6, 125.8, 122.4, 119.0, 116.3 ppm; MS (ESI) m/z: 436 (M-H-), 438 (M-
H+2-); Anal. Calcd. for C22H13Cl2N3O3.1.34 C6H12 (551.04): C, 65.48; H, 5.32; N, 7.63%, Found: C, 65.92; H, 4.95 N,
9.38%.
4.1.2.17. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(thiazol-2-yl)benzamide(31)
Yield 2%; mp 246 oC; IR υmaks (FT/ATR): 3221 (NH), 2960 (CH-arom), 1676 (C=O), 1636 (Amide I), 1596 (C=C),
1
1560 (Amide II), 1289 (=C-N) cm-1; H NMR (400 MHz, DMSO-d6): δ 12.46 (1H, bs, CONH), 9.48 (1H, s, NH-
Phenyl), 8.07-8.02 (2H, m, Naphthoquinone H-5 and H-8), 8.03 (2H, d, J=8.4 Hz, Benzene H-2 and H-6), 7.88 (1H, td,
J=7.6; 1.6 Hz, Naphthoquinone H-6 or H-7), 7.82 (1H, td, J=7.6; 1.6 Hz, Naphthoquinone H-6 or H-7), 7.53 (1H, d,
J=3.6 Hz, Thiazole H-3), 7.24 (1H, d, J=3.6 Hz, Thiazole H-4), 7.19 (2H, d, J= 8.4 Hz, Benzene H-3 and H-5) ppm;
13C NMR (101 MHz, DMSO-d6): δ 180.4, 177.4, 176.4, 143.6, 143.1, 142.9, 135.2, 135.1, 133.9, 132.3, 131.4, 130.9,
128.7, 127.5, 127.0, 126.7, 122.4, 118.8, 114.1, 113.6 ppm; MS (ESI) m/z: 408 (M-H-), 410 (M-H+2-); Anal. Calcd.
for C20H12ClN3O3S.0.64 CHCl3.1.12 C6H12 (580.50): C, 56.61; H, 4.53; N, 7.24; S, 5.52%, Found: C, 56.97; H, 4.11;
N, 6.80; S, 5.42%.
4.1.2.18. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(4-methylthiazol-2-yl)benzamide(32)
Yield 16%; mp 204 oC; IR υmaks (FT/ATR): 3365 (NH), 3269 (NH), 3096 (CH-arom), 3032 (CH-arom), 2918 (CH-
1
aliph), 1665 (C=O), 1646 (Amide I), 1599 (C=C), 1574 (Amide II), 1241 (=C-N) cm-1; H NMR (400 MHz, DMSO-
d6): δ 9.48 (1H, bs, NH-Phenyl), 8.06-8.01 (4H, m, Naphthoquinone H-5 and H-8, Benzene H-2 and H-6), 7.87 (1H,
td, J=7.4; 1.6 Hz, Naphthoquinone H-6 or H-7), 7.82 (1H, td, J=7.5; 1.5 Hz, Naphthoquinone H-6 or H-7), 7.18 (2H, d,
J=8.6 Hz, Benzene H-3 and H-5), 6.78 (1H, s, Thiazole H-5), 2.29 (3H, s, CH3) ppm; 13C NMR (101 MHz, DMSO-d6):
δ 180.4, 177.4, 143.5, 143.1, 135.2, 133.9, 132.2, 130.9, 128.6, 127.0, 126.6, 122.4, 118.7, 108.4, 17.3 ppm; MS (ESI)
m/z: 422 (M-H-), 424 (M-H+2-); Anal. Calcd. for C21H14ClN3O3S. 0.15CHCl3. 0.2CH3OH (448.18): C, 57.22; H, 3.36;
N, 9.38; S, 7.15%, Found: C, 57.65; H, 2.44; N, 8.09; S, 6.70%.
4.1.2.19. 4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(5-methylisoxazol-3-yl)benzamide(33)
o
Yield 5%, mp 159 C; IR υmaks (FT/ATR): 3284 (NH), 3215 (NH), 3081 (CH-arom), 2972 (CH-aliph), 2852 (CH-
1
aliph), 1692 (C=O), 1668 (Amide I), 1597 (C=C), 1575 (Amide II), 1280 (=C-N) cm-1; H NMR (400 MHz, DMSO-
d6): δ 9.49 (1H, bs, NH-Phenyl), 8.04 (2H, dt J=7.6; 1.6 Hz, Naphthoquinone H-5 and H-8), 7.90-7.84 (3H, m,
Naphthoquinone H-6 or H-7, Isoxazole-H), 7.86 (2H, d, J=8.8 Hz, Benzene H-2 and H-6), 7.82 (1H, td, J=7.6; 1.6 Hz,
Naphthoquinone H-6 or H-7), 7.16 (2H, d, J=8.8 Hz, Benzene H-3 and H-5), 3.81 (3H, s, CH3) ppm; 13C NMR (101
MHz, DMSO-d6): δ 180.4, 177.5, 166.4, 144.2, 143.1, 135.2, 134.0, 132.2, 130.9, 129.7, 127.0, 126.7, 124.4, 122.5,
119.0, 52.4 ppm; MS (APCI) m/z: 407 (M+), 409 (M+2+); Anal. Calcd. for C21H17Cl2N2O4.0.9 C2H6O (449.27): C,
60.95; H, 4.35; N, 9.35%, Found: C, 61.38; H, 4.76; N, 9.28%.

4.2.
Biological evaluation
4.2.1. Cell culture
Human breast cancer cell line (MCF-7) was obtained from American Type Culture Collection (ATCC) and
maintained as exponentially growing monolayers by culturing according to the supplier's instructions.
4.2.2. Cell proliferation inhibition assay
The antiproliferative activity of the synthesized compounds was determined on MCF-7 cells. Cells were seeded in
96-well plates at a density of 6000 cells/well. After 24 h incubation, cells were treated for 48 h with desired
concentration of compounds according to their solubility ranges. Cell viability was determined by MTT (3-(4,5-
Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) (Sigma Aldrich, UK). After 48 h incubation of
compounds, the mixture of MTT and medium (1:9) was replaced with old media in each well and the plates were
incubated for 4 h at 37°C. 150 µL of DMSO was added to dissolve violet formazan crystals and the amount of
formazan product was measured absorbance at wavelength 570 nm with a microplate reader a (Varioscan, Thermo
Fisher Scientific, US). DMSO (dimethyl sulfoxide, VWR, US) was used a solvent control or a negative control. All
tests were performed in triplicate. Cytotoxic effects of the compounds were determined as IC50 which refers to the
concentration of the drug to inhibit 50% of cell viability and calculated by Graph Pad Prism 5 (San Diego, CA, US).
4.2.3. Cell-based proteasome inhibition assay
To determine proteasome inhibitory activity of compounds in protein extracts, MCF-7 cells were harvested with a
lysis buffer of 8.56 g sucrose, 0.6 g HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 0.2 g MgCl2, 0.037
g EDTA (ethylenediaminetetraacetic acid) prepared in 100 ml (before lysis, DTT (dithiothreitol) was added freshly to
the lysis buffer at a final concentration of 1 mM). The supernatant was used to measure the activity of proteasome
subunit activity. Reaction mixture included 225 mM Tris buffer (pH 7.8) containing 7.5 mM MgOAc, 7.5 mM MgCl2,
45 mM KCl, and 1 mM DTT.37 The protein extracts were incubated with reaction mixture for 10 min. Then, potential
proteasome inhibitors with determined doses according to the cell viability assay were added and incubated for 1h at
37 °C. While the fluorogenic peptide Suc-Leu-Leu-Val-Tyr-AMC (Enzo Life Sciences; US) was used as a substrate at
a concentration of 200 µM to measure chymotrypsin-like activity of the proteasome, Ac-Gly-Pro-Leu-Asp-AMC (100
µM) (Enzo Life Sciences; US) and Z-Leu-Leu-Glu-AMC (40 µM) (Enzo Life Sciences; US) flourogenic peptides were
used to determine caspase-like and trypsin-like activity of the proteasome, respectively. After the incubation, the
fluorogenic substrates were added with desired compounds for 1 h at 37 °C. Methyl coumarin (MCA) liberation was
measured with a fluorescence reader at 360 nm excitation/460 nm emission (Varioscan, Thermo Fisher Scientific, US).
While DMSO was used as a solvent control, MG-132, known proteasome inhibitor, was used as a positive control.
The selected and potential proteasome inhibitors were treated to MCF-7 cells for 12 h to determine IC50 value at
proteasome subunits activity. Then, proteasome subunit activities were determined as mentioned above. The results
were normalized via total protein level performed by BCA (Bicinchonic acid) assay (Thermo Fisher Scientific, US).
4.2.4. Immunoblotting assay
MCF-7 cells were treated with IC50 values of compound 14 and PI-083 for 12 hours. Cells then lysed with RIPA
buffer (50mM Tris-HCl, pH 8.0, 1% NP-40, 0.1% SDS, 150mM NaCl, 0.1% Triton X-100, 5mM EDTA) with
protease inhibitors (Roche, Switzerland) in order to prepare whole cell lysates. Protein concentrations were determined
by bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, US). 40 µg total cellular proteins were used for
immunoblotting studies. Samples were denatured in 4x Laemmli buffer at 95 °C for 5 min and were separated by
handcast SDS-PAGE electrophoresis and transferred to PVDF membranes (EMD Milipore, Thermo Fisher Scientific,
US). After blocking the membranes with PBS–0.1% Tween-20 with 5% non-fat dry milk, first primary antibodies
namely anti-20S β5 subunit (Enzo, BML-PW8895-, US), anti-K48 linkage Specific Polyubiquitin (CST, 8081, US)
and anti-actin (Sigma-Aldrich-A5316, UK) and then goat anti-rabbit and anti-mouse HRP-Conjugated secondary
antibodies (Thermo Fisher Scientific, US) were used for determination of target proteins. Protein signals were detected
using Clarity ECL substrate solution (BIORAD, US) by Fusion-FX7 (Vilber Lourmat, Thermo Fisher Scientific, US).
A well known proteasome inhibitor MG-132 was used at 10 µM concentration.
4.3.
Molecular modeling studies
4.3.1. Docking studies
4.3.1.1. Compounds 13, 14 and 28 in β1, β2 and β5 subunits of human 20S proteasome
In order to prepare the ligand and the subunits of the protein for docking studies, compounds 13, 14 and 28 were
built, protonated and energy minimized by using MMFF94x in MOE2014.09. On the other hand, the crystal structure
of human 20S proteasome in complex with bortezomib (PDB code 5LF3) resolved at 2.1 Ǻ were downloaded from
Protein Data Bank. After the ligand and the water molecules were removed, β1, β2 and β5 subunits were saved
individually. Then, each subunit was protonated via protonate 3D protocol and subjected to energy minimization using
AMBER99 force field in MOE2014.09.38

Docking simulations of compounds 13, 14 and 28 in complex with β1, β2 and β5 subunits were carried out with
GOLD 5.2.1 program with default settings.39,40 The binding site was defined around the backbone carbon of Gly47
with a sphere of 22 Å in each active site of β1, β2 and β5 subunits. Goldscore and Chemscore standard precision were
selected as the scoring function and 50 confirmations were allowed. Proposed binding poses of the ligand-subunit
complexes were selected by visual inspection using GOLD 5.2.1 program.
4.3.2. Molecular dynamics simulations
Molecular dynamics simulations were carried out for the apo forms of the β1, β2 and β5 subunits of human 20S
proteasome and the selected poses of compound 14 in complex with β1, β2 and β5 subunits of human 20S proteasome,
individually. AMBER 14 program was used for all the preparation steps of the apo forms of enzymes and the protein-
ligand complexes and AMBER 12 program was used for the simulation part.41
After the atom types and the partial charges of compound 14 with selected geometries were calculated by AM1-
BCC charge model of antechamber module, protein subunits and protein subunit-ligand complexes were prepared for
energy minimisation and MD simulations by using the xLeap module of AmberTools14.41,42 In order to generate the
model, compound 14 and the enzyme subunits were parameterized with Gaff and AMBERff99SB force field using
parmchk module, respectively.43,44 While compound 14-β1 subunit and compound 14-β5 subunit complexes were
neutralized by 7K+ and 3Cl- counterions, respectively; compound 14-β2 subunit was already neutral in its current form.
The complexes were solvated by water molecules for each complex, respectively in octahedral box using TIP3P
leaving at least 10 Å between the solute atoms and the border of the box.45 The solvated and neutralized complexes
were energy minimized by Sander module of AMBER. Then, the systems were heated from 50 to 300K with
positional restrains (force constant: 10 kcal/mol/ Å) for 0.3 ns for compound 14-β1 subunit and compound 14-β2
subunit; 0.1 ns for compound 14-β5 subunit allowing the solvent and the counterions to move freely. 9 Å cut off for the
short-range nonbonded interactions was used combined with the particle mesh Ewald option.46 In order to constrain
bond vibrations involving the hydrogen atoms, Settle algorithm was used.47 Then the positional restrains were reduced
to allow unrestrained MD simulations for all atoms and were reached the equilibration state.
Apo forms of the three protein subunits and compound 14-β1 subunit, compound 14-β2 subunit and compound 14-
β5 subunit complexes were subjected to free MD simulations for 30 ns for compound 14-β1 subunit, compound 14-β2
subunit complexes and 10 ns for compound 14-β5 subunit complex. VMD was used for the visualization of the
trajectories and XMGRACE software was used for preparing the plots.48,49 (See Supplementary Data)
Declaration of Competing Interest
The authors declare that they have no conflict of interest.
Acknowledgments
This study was supported by grants from The Scientific and Technological Research Council of Turkey
(TÜBİTAK, Project Number:116S300) and Ege University (Project Number: 14ECZ042). The authors thank also to
the Pharmaceutical Sciences Research Centre (FABAL) at Ege University, Faculty of Pharmacy for spectral and
elemental analyses of the compounds.
Appendix A. Supplementary data
Supplementary data to this article can be found online at…
References
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Tables 1, 2 and 3
Table 1. In vitro antiproliferative activity of final compounds against MCF-7 cell line.
Compound No
IC50(µM)a
Compound No
IC50(µM) a
1
2
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
2.43±0.11
5.38±1.16
2.20±0.14
3.99±0.35
2.17±0.19
2.84±0.13
2.56±0.37
2.10±0.31
3.37±0.48
3.32±0.45
1.91±0.13
2.11±0.15
1.79±0.21
4.43±0.31
4.65±0.63
4.23±0.59
1.84±0.18
1.96±0.14
15.4 ±5.25
2.61±0.2
3
14.43±3.29
16.15±2.53
16.57±2.71
4.52±0.78
4.80±0.11
6.04±0.26
20.80±0.5
4.32±0.4
4
5
6
7
8
9
10
11
12
13
14
15
16
17
PI-083
1.72±0.14
4.16±0.4
6.19±0.1
3.84±0.27
2.44±0.19
10.15±0.12
Doxorubicin
0.93±0.06
aThe IC50 values are shown as an average of three independent determinations.

Table 2. 20S proteasome β5, β1 and β2 subunits inhibition of final compounds
Compound No
Inhibition % (10 µM)a
ChT-L(β5) activity
17.36
20.01
19.98
17.31
23.51
26.03
3.25
C-L (β1) activity
13.88
11.5
T-L(β2) activity
21.08
17.12
22.66
21.27
19.38
22.70
6.29
1
2
3
12.01
14.25
7.44
4
5
6
14.13
1.21
7
8
28.83
22.17
18.44
14.76
11.70
10.06
42.31
22.41
24.82
10.79
4.03
14.20
10.02
15.40
9.01
24.54
23.81
21.16
12.96
7.26
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
PI-083
MG132
3.35
n.i.
11.56
35.02
22.08
20.96
2.76
28.89
11.17
15.44
16.17
10.72
9.94
3.19
17.24
14.61
35.34
21.64
15.85
16.92
16.28
8.96
n.i.
15.06
22.77
11.71
n.i.
3.39
30.52
27.55
1.55
16.71
1.20
4.41
n.i.
7.87
2.43
11.41
16.65
22.01
19.40
19.65
22.29
18.89
36.76
9.52
n.i.
8.37
n.i.
7.42
n.i.
8.55
n.i.
3.59
n.i.
7.76
7.11
8.36
6.55
18.81
29.59
38.23
18.39
51.42
aThe values reported are the average of three independent determinations; n.i.: no inhibition

Table 3. IC50 values of compounds 14, 21 and PI-083 for antiproteasomal activity
Compound No
IC50 (µM)a
ChT-L(β5) activity
9.90±0.61
C-L (β1) activity
44.83±4.23
>50
T-L(β2) activity
22.27±0.15
>50
14
21
33.14±0.29
PI-083
12.47±0.21
53.12±2.56
26.37±0.5
aThe values reported are the mean±SEM of three independent experiments.

Figures 1-4
Figure 1. Chemical structures of FDA-approved proteasome inhibitors
O
O
O
O
O
O
Cl
Cl
Cl
NH
NH
NH
linker
phenyl
O
sulfonamide/
carboxamide
S
H-bond
acceptor
O
HN
N
carbocyclic/
heterocyclic
ring
tailing
(b)
(a)
(c)
Figure 2. (a) Structure of PI-083 lead compound (b) General schematic structure of proteasome inhibitors bearing 1,4-naphthoquinone
pharmacophoric unit (c) General structure of the designed compounds

Figure 3. Evaluation of the expression level of 20S proteasome subunit β5 which is related to ChT-L activity (A) and accumulation of K-48
linked polyubiquitinated proteins (B). MCF-7 cells were treated with compound 14 and PI-083 at their IC50 concentrations for 12 h (A, B). 20S
proteasome subunit β5 protein level and K48-linked polyubiquitinated proteins were detected by immunoblotting with the antibody against β5 and
K48 linkage Specific Polyubiquitin, respectively. β-actin was used as loading control. MG-132, well known proteasome inhibitor was used at 10
μM as positive control. The experiments were repeated three times independently; with one representative result shown.
(A)
(B)
(C)
Figure 4. Proposed binding poses of compound 14 in the active gorge of (A) β1 subunit (B) β2 subunit and (C) β5subunit of human 20S
proteasome. (Compound 14 is colored as pink sticks while the involved residues are named using three letters code and colored as dark gray
sticks)

Schemes 1 and 2
Scheme 1. Synthesis of compounds 1-17
Scheme 2. Synthesis of compounds 18-33

Highlights
•
•
A series of novel 4-aminobenzensulfonamide/carboxamide derivatives were explored as proteasome inhibitors
Compound 14 inhibited ChT-L (IC50 =9.90 µM), C-L (IC50 =44.83 µM) and T-L (IC50 = 22.27 µM) activities of
proteasome.
•
•
All of the compounds exhibited good antiproliferative activity in a range of IC50= 1.72- 20.8 µM.
The binding mechanism of compound 14 was carried out with docking studies.