Novel 1,4-naphthoquinone-based sulfonamides: Synthesis, QSAR, anticancer and antimalarial studies

Article abstract of DOI:10.1016/j.ejmech.2015.09.001

A novel series of 1,4-naphthoquinones (33-44) tethered by open and closed chain sulfonamide moieties were designed, synthesized and evaluated for their cytotoxic and antimalarial activities. All quinone-sulfonamide derivatives displayed a broad spectrum o

Full text of DOI:10.1016/j.ejmech.2015.09.001

Accepted Manuscript
Novel 1,4-naphthoquinone-based sulfonamides: synthesis, QSAR, anticancer and
antimalarial studies
Ratchanok Pingaew, Veda Prachayasittikul, Apilak Worachartcheewan, Chanin
Nantasenamat, Supaluk Prachayasittikul, Somsak Ruchirawat, Virapong
Prachayasittikul
PII:
S0223-5234(15)30245-2
10.1016/j.ejmech.2015.09.001
EJMECH 8098
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 June 2015
Revised Date: 11 August 2015
Accepted Date: 2 September 2015
Please cite this article as: R. Pingaew, V. Prachayasittikul, A. Worachartcheewan, C. Nantasenamat,
S. Prachayasittikul, S. Ruchirawat, V. Prachayasittikul, Novel 1,4-naphthoquinone-based sulfonamides:
synthesis, QSAR, anticancer and antimalarial studies, European Journal of Medicinal Chemistry (2015),
doi: 10.1016/j.ejmech.2015.09.001.
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ACCEPTED MANUSCRIPT

1
ACCEPTED MANUSCRIPT
Novel 1,4-naphthoquinone-based sulfonamides: synthesis, QSAR,
anticancer and antimalarial studies
a,
b,c
c,d
Ratchanok Pingaew *, Veda Prachayasittikul , Apilak Worachartcheewan ,
b,c
c
e,f
Chanin Nantasenamat , Supaluk Prachayasittikul , Somsak Ruchirawat ,
b,
Virapong Prachayasittikul *
a
Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok 10110,
Thailand
b
Department of Clinical Microbiology and Applied Technology, Faculty of Medical
Technology, Mahidol University, Bangkok 10700, Thailand
c
Center of Data Mining and Biomedical Informatics, Faculty of Medical Technology,
Mahidol University, Bangkok 10700, Thailand
d
Department of Clinical Chemistry, Faculty of Medical Technology, Mahidol University,
Bangkok 10700, Thailand
e
Laboratory of Medicinal Chemistry, Chulabhorn Research Institute, and Program in
Chemical Biology, Chulabhorn Graduate Institute, Bangkok 10210,Thailand
f
Center of Excellence on Environmental Health and Toxicology, Commission on Higher
Education (CHE), Ministry of Education, Thailand
*
Corresponding authors:
E-mail: ratchanok@swu.ac.th (R. Pingaew)
Tel: 662-649-5000 ext 18253; Fax: 662-260-0128
E-mail: virapong.pra@mahidol.ac.th (V. Prachayasittikul)
Tel: 662-441-4376; Fax: 662-441-4380

2
ACCEPTED MANUSCRIPT
Abstract
A novel series of 1,4-naphthoquinones (33-44) tethered by opened and closed chain
sulfonamide moieties were designed, synthesized and evaluated for their cytotoxic and
antimalarial activities. All quinone-sulfonamide derivatives displayed a broad spectrum of
cytotoxic activities against all of the tested cancer cell lines including HuCCA-1, HepG2,
A549 and MOLT-3. Most quinones (33-36 and 38-43) exerted higher anticancer activity
against HepG2 cell than that of the etoposide. The opened chain analogs 36 and 42 were
shown to be the most potent compounds. Notably, the restricted sulfonamide analog 38 with
6
,7-dimethoxy groups exhibited the most potent antimalarial activity (IC50 = 2.8 µM).
Quantitative structure-activity relationships (QSAR) study was performed to reveal important
chemical features governing the biological activities. Five constructed QSAR models
provided acceptable predictive performance (R 0.5647 - 0.9317 and RMSE 0.1231 -
cv
cv
0
.2825). An additional set of structurally modified compounds were generated in silico (34a-
4d, 36a-36k, 40a-40d and 42a-42k) in which their activities were predicted using the
3
constructed QSAR models. A comprehensive discussion of the structure-activity relationships
was made and a set of promising compounds (i.e., 33, 36, 38, 42, 36d, 36f, 42e, 42g and 42f)
were suggested for further development as anticancer and antimalarial agents.
Keywords: Naphthoquinone; Sulfonamide; QSAR; Anticancer activity; Antimalarial
activity

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1
. Introduction
Quinones are widely distributed in nature [1,2], and are represented in many clinically
used drugs (e.g. doxorubicin, daunorubicin, bleomycins, mitomycin-C) [3]. Apparently, 1,4-
naphthoquinones have been found to possess a diverse range of biological activities including
anticancer and antimalarial activities [4-6]. For example, calothrixins A and B isolated from
cyanobacterial Calothrix exhibited potent anticancer and antimalarial activities [7].
Biological properties of quinones have been related by a wide range of molecular targets thus
rendering them to be promising lead molecules for various therapeutic applications. Two
carbonyl groups of quinone have been well-documented to act as electrophile and electron
acceptor, therefore, alkylation and reactive oxygen species (ROS) production have been
stimulated on the two carbonyl sites (1,4-C), respectively [8-10]. In addition, cytotoxic
quinone derivatives were proven to be topoisomerase I and II inhibitors [11,12]. Furthermore,
1
,4-naphthoquinone derivatives were previously proposed to display antimalarial activity by
involving a cascade of redox reactions in the parasite [13,14].
Aminonaphthoquinone, an important class of the quinone family, has been proven to
be interesting scaffold for pharmacological applications [15-21]. For instance, 5-hydroxy-3-
amino-2-aceto-1,4-naphthoquinone (1) and 2-amino-3-chloro-1,4-naphthoquinone (2), as
shown in Fig. 1, exhibited significant cytotoxic [15] and antimalarial [16] activities
respectively. The extended studies to structural variations of the naphthoquinones revealed
that the presence of amino or chloro substituents on the 1,4-naphthoquinone would contribute
to their redox potentials [17-21]. Enhancing of biological potency was observed when amino
group was inserted at 2- and/or 3- positions [17]. Phenylaminonaphthoquinones showed
superior anticancer activity than that of alkylamino counterparts, and the incorporation of
acetyl group on the phenylamino part led to improvement of their cytotoxic potencies [17].
Furthermore, insertion of sulfonamide moiety to phenylamino-1,4-naphthoquinone affording
PI-083 (3, Fig. 1) that was capable of inhibition of cell proliferation, and inducing apoptosis
with selective for malignant over normal cells [22].
To find novel lead candidates as promising anticancer and antimalarial agents based
on the attractive phenotypes, thus, target 1,4-naphthoquinone-sulfonamide derivatives (Fig.
2
) were rationally designed as follows:
(i) use 2-chloro-3-anilino-1,4-naphthoquinone as a core structure;
(ii) vary the position of sulfonyl group on aniline ring (3- or 4-position); and

4
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(iii) vary the type of N-substituent of sulfonamide (opened chain alkylphenyl group, opened
chain alkylpyridyl group or restricted isoquinoline ring).
A variety of expected 1,4-naphthoquinone derivatives were synthesized and evaluated
for their in vitro antiproliferative activity against four cancer cells and normal cell line as well
as antimalarial activity toward Plasmodium faciparum. To provide insights into the effect of
sulfonamide substituents on the quinone ring in their biological activities, quantitative
structure-activity relationships (QSAR) of the synthesized and structurally modified
compounds were investigated.
2
. Results and discussion
2
.1 Chemistry
The preparation of aminobenzenesulfonamides 20-31 as starting materials for the
synthesis of phenylaminonaphthoquinone is illustrated in Scheme 1. Initially, condensation of
various primary amines (4-7) with either 3- or 4-nitrobenzenesulfonyl chloride yielded the
corresponding phenethyl sulfonamides (8-15). Consequently, N-sulfonyl-1,2,3,4-
tetrahydroisoquinolines (16-19) were readily obtained using the Pictet-Spengler reaction by
treatment of nitrobenzenesulfonamides (12-15) with paraformaldehyde in refluxing formic
acid. Reduction of the nitro derivatives (8-11) was carried out by catalytic hydrogenation
using palladium on charcoal whereas the reduction of nitro analogs (12-19) was performed
using stannous chloride to give the corresponding aminobenzenesulfonamides (20-31). With
the amino derivatives (20-31) on hands, a series of desired naphthoquinones (33-44) were
then synthesized by nucleophilic substitution of 2,3-dichloro-1,4-naphthoquinone 32 with the
appropriate aminobenzenesulfonamide derivatives 20-31 in refluxing ethanol as shown in
Scheme 2. However, reaction of 2,3-dichloro-1,4-naphthoquinone 32 with amines in the
presenc of bases (KOH or Et N) and surfactant (LD or SDS) in aqueous medium have been
3
recently reported.[23] The obtained compounds (33-44; Fig. 3) are categorized as para-
isomer (amino and sulfonyl groups at 1,4-position of benzene ring, 33-38) and meta-isomer
(amino and sulfonyl groups at 1,3-position of benzene ring, 39-44). N-substituents of these
sulfonamides are opened chain alkylpyridines (33, 36, 39 and 42), alkylphenyls (33-34, 40-
4
1) and closed chain or restricted isoquinolines 37-38 and 43-44).
Structures of the desired naphthoquinone 33-44 were confirmed based on their HRMS
1
and NMR spectra. H NMR spectra showed the presence of NH proton as a singlet at δ

5
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around 9.5-9.6 ppm indicating that the nucleophilic displacement of the phenylamino
1
3
derivative occurred at a chloro group of 2,3-dichloro-1,4-naphthoquinone. In addition, C
NMR spectra displayed a characteristic signal of two carbonyl groups (C=O) at chemical
shift in the range of 177-181 ppm.
2
2
.2 Biological activities
.2.1 Cytotoxic activity
In assessing the cytotoxicity, the series of 1,4-naphthoquinones (33-44) were examined
against four human cancer cell lines including HuCCA-1 (cholangiocarcinoma), HepG2
hepatocellular carcinoma), A549 (lung carcinoma) and MOLT-3 (lymphoblastic leukemia)
cell lines as summarized in Table 1.
,3-Dichloro-1,4-naphthoquinone 32 was reported to exert cytotoxic activity against
HuCCA-1, HepG2, A549 and MOLT-3 cells with IC values 19.20, 65.19, 45.37 and 13.04
(
2
5
0
µM, respectively [16]. Distinctively, replacement of the chloro atom of compound 32 with
various aminobenzenesulfonamide moieties afforded the compounds with improved their
inhibitory potencies. It was observed that all the tested naphthoquinone derivatives displayed
a broad spectrum of cytotoxic activities, in which their inhibition potencies depended on the
type of cancer cells.
Most naphthoquinones exhibited cytotoxic activities with comparable IC values lesser
5
0
than 10 µM against HuCCA-1 cell line, except for the restricted isoquinolines 37 (IC =
5
0
5
7.77 µM) and 44 (IC = 26.42 µM). Apparently, the opened chain pyridine analog 42 was
50
shown to be the most potent compound with IC 3.66 µM; however the meta-compound 42
5
0
displayed 9-folds lower potency than the control drug doxorubicin. Additionally, the
compound 42 was also shown to be the most potent compound against A549 cell line (IC =
5
0
2
.3 µM). The effects of chemical structures, meta- and para- isomers, and substituents
towards bioactivities of the compounds are discussed (section 2.3).
Interestingly, most 1,4-naphthoquinones (para-analogs, 33-36 and meta-analogs, 38-43)
showed higher anticancer activity against HepG2 cell than that of the etoposide. Among these
compounds, the analog 36 was shown to be the most potent compound (IC = 3.30 µM).
5
0
Reducing the alkyl chain length of pyridine analog 33 led to 3-folds and 5-folds improved
inhibition potencies as seen in the analog 36 against HepG2 and A-549 cells, respectively.
However those results could not be observed in meta-analogs (39 vs 42). Obviously, in

6
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MOLT-3 cell line, most compounds exerted comparable cytotoxic activities with IC lesser
5
0
than 5 µM, in which the pyridine analogs (33 and 36) have shown to be the most potent
compounds with identical IC value of 1.37 µM.
5
0
It was observed that naphthoquinones bearing opened chain sulfonamides exhibited
higher cytotoxicity (all tested cells) than the analogs containing cyclic sulfonamide as seen
for 34 vs 37, 35 vs 38 (except for MOLT-3 cell) and 41 vs 44 (except for MOLT-3 cell). At
this point, it seemed reasonable that NH group of the sulfonamide can act as a hydrogen bond
donor which might play a crucial role for cytotoxic activity.
These naphthoquinones (33-44) were also tested against the noncancerous (Vero) cell
line obtained from African green monkey kidney (Table 1). Comparison of the IC values
5
0
measured in cancer cells versus that obtained in normal cells, the isoquinolines (37, 38, 43
and 44) had no selectivity. Most of the meta derivatives seem to be more cytotoxic to normal
cell than that of the corresponding para-analogs. Notably, the opened chain compounds 33
and 34 were found to be the most promising compounds showing a broad spectrum cytotoxic
activity with the best safety index. Both compounds displayed cytotoxicities against the Vero
cell line with IC values of 99.89 and >107.08 µM, respectively.
5
0
2
.2.2 Antimalarial activity
Antimalarial activity of the quinone-sulfonamides (33-44) was evaluated against P.
falciparum (K1, multidrug resistant strain) as shown in Table 1. The synthesized quinones
with opened chain sulfonamides (35, 40 and 41) and restricted-ring sulfonamides (37-38 and
4
3-44) showed significant antimalarial activity with IC values in the range of 2.80-14.74
50
µM. Dimethoxy group insertion on the benzene ring in para analogs 34 and 37 created the
corresponding compounds 35 and 38 with enhanced antimalarial activity; however the
reduced activity was observed for meta analogs (40 vs 41 and 43 vs 44). Replacement of
alkyl benzene with alkyl pyridine in para analogs (34 vs 33, 36) could not improve the
antimalarial potency while the reduced activity was observed for meta analogs (40 vs 39, 41).
Improving of the potency was observed when the opened chain of para analogs 34 and 35
was cyclized to the respective restricted analogs 37 and 38 in which the analog 38 was shown
to be the most potent compound (IC50 = 2.80 µM). These may be attributed to the
lipophilicity of dimethoxyphenyl and the restricted appropriate tetrahydroisoquinoline

7
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sulfonamide which may be required for enhancing the absorption of the compounds into the
cells.
2
.3. QSAR analysis
Chemical structures of the tested 1,4-naphthoquinones 33-44 (Fig. 3) along with their
experimental activity (pIC ) were used for performing QSAR study. The chemical structures
50
were geometrically optimized and their physicochemical descriptors were calculated using
Gaussian 09 [24] and Dragon software (version 5.5) [25] to obtain 13 quantum chemical and
3
224 molecular descriptors, respectively. Correlation-based feature selection was employed
to initially select a set of correlated descriptors for further selection process. Pearson’s
correlation coefficient (r) between descriptor values and bioactivities were calculated and
descriptors having |r| ≥ 0.5 was retained and further subjected to stepwise multiple linear
regression (MLR) as implemented in SPSS statistics 18.0 or attribute elevator CfsSubsetEval
as implemented in the Waikato Environment for Knowledge Analysis (WEKA) version 3.4.5
[
26] to obtain a sets of 9 important descriptors in which their definitions and their calculated
descriptor values are shown in Table 2 and Supplementary Table S1, respectively. The
multivariate analysis was performed by (WEKA) version 3.4.5 [26] using multiple linear
regression (MLR) algorithm. Five QSAR models were constructed and their predictive
performance are summarized in Table 3.
The constructed models provided correlation coefficient (R ) and root mean square
cv
error (RMSE ) values in range of 0.5647 – 0.9317 and 0.1231 – 0.2825, respectively. The
cv
A549 model provided the best performance as represented by its highest R and its lowest
cv
RMSEcv values. The experimental and predicted activities (pIC50 values) of the tested
compounds 33-44 against four cancer cell lines and P. falciparum are presented in
Supplementary Table S2 and Fig. 7.
HuCCA-1 cell line:
pIC50 = 34.1344
(
X1A
)
+ 23.1368
(
Gm
)
+ 9.4788
(
G2u
)
− 21.1565
Eq. 1
The QSAR model revealed that connectivity (X1A) and symmetry (Gm and G2u) of
the compound influenced anticancer activity against HuCCA-1 cell line, Eq. 1. The
connectivity indices descriptor, X1A, was noted as the most influential descriptor as

8
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represented by its highest regression coefficient. The molecular connectivity indices represent
the molecular area which is accessible from outside molecule and are successfully used for
describing physicochemical properties of the molecules and their intermolecular interactions
[
27]. Positive regression coefficients in the QSAR equation indicated that high values of
X1A, Gm and G2u are required for potent activity against HuCCA-1 cell line.
HepG2 cell line:
pIC50 = −0.097
(
RDF105m
)
− 0.2179
Eq. 2
According to QSAR equation, a mass-weighted RDF descriptor (RDF105m) was
noted as an influential descriptor for anticancer activity against HepG2 cell line, Eq. 2.The
negative regression coefficient indicated low value of RDF105m is required for potent
activity against HepG2 cell line.
A549 cell line:
pIC50 = −0.0949
(
RDF105m
)
+ 0.6903
(
Mor12u
)
+ 0.366
Eq. 3
The RDF105m descriptor was also found to influence anticancer activity against
A549 cell line (Eq. 3). In addition, a 3D-MoRSE descriptor, Mor12u, was noted as the most
important descriptor as shown by its high regression coefficient (Eq. 3). The negative and
positive regression coefficient values of RDF105m an Mor12u indicated the low RDF105m
value together with the high Mor12u value are essential for the compound to exhibit potent
activity against A549 cell line.
MOLT-3 cell line:
pIC50 =10.9192
(
G2u
)
+ 0.0185
(
G(N..Cl)
)
− 2.5605
Eq. 4
In addition to the G2u descriptor, a geometrical distances between nitrogen (N) atom
and chlorine (Cl) atom, represented by G(N..Cl) descriptor, was found to influence anticancer
activity against MOLT-3 cell line. Positive regression coefficients presented in the QSAR
equation (Eq. 4) indicated that high values of both descriptors are required for high pIC50
value.
Antimalarial:

9
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pIC50 = −1.6078
(
Mor22v
)
+ 0.1457
(
B05[N − O]
)
+ 1.2647
(
Mor 24u
)
−1.4305 Eq. 5
3
D-MoRSE descriptors (i.e., Mor22v and Mor24u) and the presence/absence of N-O
at topological distance 5 were noted as important descriptors governing antimalarial activity.
A van der Waals volume descriptor (Mor22v) was found to be the most influential descriptor
as indicated by its highest regression coefficient in the QSAR model, Eq. 5. The QSAR
equation revealed that low value of Mor22v along with high values of Mor24u and B05[N-O]
are essential for potent activity.
2
.4. Prediction of structurally modified compounds using the constructed QSAR models
The predicted pIC values of structurally modified compounds (34a-34d, 36a-36k,
5
0
4
0a-40d, and 42a-42k, Figs. 4-6) were computed using the QSAR equations in which
important descriptor values were assigned as independent (X) variables to calculated
activities (dependent variable, Y). Predicted activities of structurally modified compounds are
summarized in Supplementary Table S3.
2
.5. Understanding structure-activity relationships
Understanding structure-activity relationships (SAR) is useful for the search of
potential compounds to be further developed [28,29]. QSAR modelling has been successfully
employed for understanding SAR of various classes of compounds and bioactivities [17, 30-
3
6]. In this study, a comprehensive analysis of relationships between identified important
descriptor values and pIC value of the individual compound in the QSAR equations was
50
performed to gain insights into the SAR. The analyzed results of tested compounds (33-44,
Fig. 3) and structurally modified compounds (34a-34d, 36a-36k, 40a-40d, and 42a-42k,
Figs. 4-6) are summarized in Tables S4–S6. The SAR analysis revealed that closed/opened
chain structure, para-/meta- position of amino and sulfonyl groups on the benzene ring,
length of the carbon chain linker as well as type of substituted ring and certain moieties may
affect activities of the compounds.
In overview, contrast effects of structural variations were observed between para-
(33–38) and meta- (39-44) series of the tested compounds (Supplementary Table S4). In
particular, N-atom of sulfonamide group substituted with benzene ring provided compound
with more potent anticancer activity than the pyridine ring for the para-compounds (34 > 33)

1
0
ACCEPTED MANUSCRIPT
except for MOLT-3 cell while the pyridine substitution was found to be more effective for
meta-compounds (39 > 40). The addition of dimethoxy (diOMe) moieties was noted to
improve activity of the para-compounds against malarial (38 > 37) and cancer cell lines (35 >
3
4 and 38 > 37), except for HuCCA-1 and A549 cell lines (34 > 35). For the meta-
compounds, diOMe groups were observed to ameliorate (41 > 40) and aggravate (43 > 44)
anticancer activities against all cancer cell lines (except for MOLT-3), and antimalarial
activities (40 > 41).
The structural modifications can cause an alteration, either increase or decrease, of
descriptor values thereby influence activities of the compounds. Herein, QSAR equations
along with descriptor values were used to reveal the relationships between these interactive
factors. The effects of chemical structure on descriptor values are summarized in
Supplementary Table S7. Detailed discussion of each cell line/activity is provided in
Supplementary data. In brief, the closed/opened chain structure influenced anticancer activity
against A549 and HepG2 cell lines, whereas the para-/meta-structure dominated the activity
towards HuCCA-1 cell line. Position of aminopyridine and aminoquinoline as well as length
of alkyl chain linker were shown to be crucial factors for anticancer activity against MOLT-3
cell line. Particular moieties i.e., diOMe, CF , and pyrrole ring were noted for their roles in
3
antimalarial activity.
A series of potential compounds exhibiting the most potent experimental and
predicted activities are summarized in Fig. 8. Two isomeric pyridine derivatives with 1C
length linker, compounds 36 and 42, were found to be the most potent anticancer agents. The
para-compound 36 was the most active compound against HepG2 and MOLT-3 cell lines,
whereas the meta-compound 42 was noted for HuCCA-1 and A549 cell lines. Various types
of substituted rings on sulfonamide group were seen for the most potent modified compounds
against each cell lines or P. falciparum. It should not be overlooked that the potent anticancer
activity against A549 and HepG2 cell lines were observed from a set of 5-menbered ring
derivatives (thiophene 42e and furan 42f, respectively). Particularly, the positive pIC values
5
0
of 42e were predicted against A549 cell line. The chloro group was suggested to be a crucial
moiety governing the potent anticancer activity against MOLT-3 cell line. Likewise, the
diOMe substituents (38) were noted for antimalarial activity. The results suggested that meta-
and para- isomers, types of substituted ring, length of alkyl chain linker and particular

1
1
ACCEPTED MANUSCRIPT
moieties of the compounds are essential factors influencing anticancer activities against
distinct cell line, and antimalarial activity.
3
. Conclusions
A novel series of quinones (33-44) have been achieved by nucleophilic displacement
of 2,3-dichloro-1,4-naphthoquinone 32 with appropriate aminobenzenesulfonamide
derivatives 20-31. Most of the naphthoquinones bearing opened chain sulfonamides (33-36,
3
9 and 41-42) exhibited higher anticancer activity than the cyclic sulfonamides, in which the
analog 34 displayed the best safety index. Apparently, the opened chain analogs 36 (para-
isomer) and 42 (meta-isomer) were shown to be the most potent compounds against HepG2
and MOLT-3; and HuCCA-1 and A549 cells, respectively. The para-isomer of restricted
sulfonamide with diOMe groups (38) exerted the most potent antimalarial activity. The
QSAR analysis revealed a set of important descriptors which influence the activity of the
compound against particular cell lines. The predictions of structurally modified compounds
indicated that certain chemical features (i.e, meta-/para- and opened/closed chain structures)
and particular substituents (i.e., OMe, Cl and length of alkyl linker) are essential for potent
activities. Finally, a set of promising compounds against cancer and malarial cell lines were
highlighted for guiding the design, synthesis and development of naphthoquinone-based
anticancer and antimalarial agents.
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
1
13
aluminum sheets. H- and C- NMR spectra were recorded on a Bruker AVANCE 300 NMR
1
13
spectrometer (operating at 300 MHz for H and 75 MHz for C). 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 or on a FT-IR Spectrum BX (Perkin Elmer)
in KBr. Melting points were determined using a Griffin melting point apparatus and were
uncorrected.

1
2
ACCEPTED MANUSCRIPT
4
.2 General Procedure for the synthesis of benzenesulfonamides (8-15)
A solution of the corresponding amine (10 mmol) in dichloromethane (50 mL) was
added dropwise to a stirred mixture of benzenesulfonyl chloride (10 mmol) and sodium
carbonate (Na CO , 14 mmol) in dichloromethane (20 mL). The reaction mixture was stirred
2
3
at room temperature overnight, and added distilled water (20 mL). 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 (anh. Na SO ), filtered and evaporated to dryness under
2
4
reduced pressure. The crude product was further purified either by recrystalization or column
chromatography.
1
H NMR of 4-nitro-N-(pyridin-2-ylmethyl)benzenesulfonamide (9) [37], 4-nitro-N-
phenethylbenzenesulfonamide
nitrobenzenesulfonamide (13) [38], 3-nitro-N-phenethylbenzenesulfonamide (14) [39] and N-
3,4-dimethoxyphenethyl)-3-nitrobenzenesulfonamide (15) [39] were consistent with that
reported in the literature.
.2.1 4-nitro-N-(2-(pyridin-2-yl)ethyl)benzenesulfonamide (8)
From 2-pyridylethylamine 4 and 4-nitrobenzenesulfonyl chloride. Pale yellow solid. Yield:
(12)
[38],
N-(3,4-dimethoxyphenethyl)-4-
(
4
o
-1
1
8
8%. mp 147-148 C. R 0.33 (30% acetone:hexane). IR (UATR) cm : 3280. H NMR (300
f
MHz, CDCl ) δ 2.98 (t, J = 5.8 Hz, 2H, PyCH ), 3.44 (t, J = 5.8 Hz, 2H, CH NH), 6.76 (br s,
3
2
2
1
H, NH), 7.10 (d, J = 7.7 Hz, 1H, PyH), 7.18 (dd, J = 7.1, 5.1 Hz, 1H, PyH), 7.61 (dt, J =
.7, 1.7 Hz, 1H, PyH), 8.05 (d, J = 7.6 Hz, 2H, ArH), 8.33 (d, J = 7.6 Hz, 2H, ArH), 8.47 (d,
7
1
3
J = 4.4 Hz, 1H, PyH ). C NMR (75 MHz, CDCl ): δ 35.6, 42.3, 122.0, 123.5, 124.3 (2),
3
+
1
28.2 (2), 136.9, 146.3, 149.0, 149.9, 158.8. HRMS-TOF: m/z [M+H] 308.0701 (Calcd for
C H N O S: 308.0700).
13
14
3
4
4
.2.2 3-nitro-N-(2-(pyridin-2-yl)ethyl)benzenesulfonamide (10)
From 2-pyridylethylamine 4 and 3-nitrobenzenesulfonyl chloride. Light brown solid. Yield:
o
-1
1
8
0%. mp 119-120 C. R 0.38 (30% acetone:hexane). IR (KBr) cm : 3451. H NMR (300
f
MHz, CDCl ) δ 2.99 (t, J = 6.1 Hz, 2H, PyCH ), 3.44 (br q, 2H, CH NH), 6.79 (br s, 1H,
3
2
2
NH), 7.09 (d, J = 7.7 Hz, 1H, PyH), 7.16 (dd, J = 6.9, 5.0 Hz, 1H, PyH), 7.61 (dt, J = 7.7, 1.8
Hz, 1H, PyH), 7.72 (t, J = 8.0 Hz, 1H, ArH), 8.20 (dt, J = 87.7, 1.5 Hz, 1H, ArH), 8.40 (d, J
1
3
=
8.0 Hz, 1H, ArH), 8.48 (d, J = 4.9 Hz, 1H, PyH ), 8.69 (t, J = 1.9 Hz, 1H, ArH). C NMR

1
3
ACCEPTED MANUSCRIPT
(
75 MHz, CDCl ): δ 35.6, 42.3, 122.0, 122.2, 123.5, 126.8, 130.3, 132.6, 136.9, 142.6, 148.3,
3
+
1
49.0, 158.7. HRMS-TOF: m/z [M+Na] 330.0516 (Calcd for C H N NaO S: 330.0519).
1
3
13
3
4
4
.2.3 3-nitro-N-(pyridin-2-ylmethyl)benzenesulfonamide (11)
From picolylamine 7 and 3-nitrobenzenesulfonyl chloride. Pale yellow solid. Yield: 81%. mp
o
-1
1
1
30-131 C. R 0.38 (30% acetone:hexane). IR (UATR) cm : 3285. H NMR (300 MHz,
f
CDCl ) δ 4.33 (d, J = 1.8 Hz, 2H, PyCH ), 6.32 (br s, 1H, NH), 7.07-7.17 (m, 2H, PyH),
3
2
7
.53-7.67 (m, 2H, ArH and PyH), 8.15 (d, J = 7.8 Hz, 1H, ArH), 8.31 (d, J = 7.8 Hz, 1H,
1
3
ArH), 8.39 (d, J = 4.4 Hz, 1H, PyH ), 8.61 (s, 1H, ArH). C NMR (75 MHz, CDCl ): δ 47.3,
3
1
22.0, 122.4, 122.9, 126.8, 130.2, 132.6, 136.9, 142.3, 148.2, 149.1, 153.9 HRMS-TOF: m/z
+
[
M+Na] 316.0353 (Calcd for C H N NaO S: 316.0363).
1
2
11
3
4
4
.3 General procedure for the synthesis of 1,2,3,4-tetrahydro-2-((nitrophenyl)sulfonyl)-
isoquinolines (16-19)
A mixture of nitrobenzenesulfonamide (0.67 mmol) and paraformaldehyde (0.72
mmol) in formic acid (15 mL) was refluxed for 2 h, and then allowed to cool to room
temperature. The reaction mixture was added to 30 mL of water, and the product was
extracted with CH Cl (2 × 30 mL). Combined extracts were washed with saturated aqueous
2
2
NaHCO , dried (anh. Na SO ) and evaporated to dryness under reduced pressure. The crude
3
2
4
product was recrystallized from methanol.
1
H NMR of 2-((4-nitrophenyl)sulfonyl)-1,2,3,4-tetrahydroisoquinoline (16) [40], 6,7-
dimethoxy-2-((4-nitrophenyl)sulfonyl)-1,2,3,4-tetrahydroisoquinoline (17) [41], 2-((3-
nitrophenyl)sulfonyl)-1,2,3,4-tetrahydroisoquinoline (18) [42] and 6,7-dimethoxy-2-((3-
nitrophenyl)sulfonyl)-1,2,3,4-tetrahydroisoquinoline (19) [39] were consistent with that
reported in the literature.
4
.4 General procedure for the synthesis of aminobenzenesulfonamides (20-23)
A mixture of nitrobenzenesulfonamide (4 mmol) and 10% Pd-C (0.02 g) in absolute ethanol
20 mL) was stirred at room temperature under a hydrogen atmosphere until completion of
(
the reaction as monitored by TLC. The catalyst was removed by filtration and the filtrate was
concentrated under reduced pressure. The residue was purified using silica gel column
chromatography.
4
.4.1 4-amino-N-(2-(pyridin-2-yl)ethyl)benzenesulfonamide (20)

1
4
ACCEPTED MANUSCRIPT
From 4-nitro-N-(2-(pyridin-2-yl)ethyl)benzenesulfonamide (8). Yellow solid. Yield: 43%. mp
o
-1
1
1
32 C (d). R 0.15 (30% acetone:hexane). IR (KBr) cm : 3418, 3335, 3214. H NMR (300
f
MHz, DMSO-d ) δ 2.79 (t, J = 7.7 Hz, 2H, PyCH ), 2.99 (t, J = 7.7 Hz, 2H, CH NH), 5.91 (s,
6
2
2
2
H, NH ), 6.58 (d, J = 8.6 Hz, 2H, ArH), 7.15-7.22 (m, 3H, NHSO and PyH), 7.39 (d, J =
2 2
1
3
8
.6 Hz, 2H, ArH), 7.66 (t, J = 7.7 Hz, 1H, PyH), 8.43 (d, J = 3.9 Hz, 1H, PyH ). C NMR
(
75 MHz, DMSO-d ): δ 37.7, 42.8, 113.2 (2), 122.0, 123.7, 125.7, 128.9 (2), 137.0, 149.5,
6
+
1
52.9, 159.0. HRMS-TOF: m/z [M+H] 278.0973 (Calcd for C H N O S: 278.0963).
1
3
16
3
2
4
.4.2 4-amino-N-(pyridin-2-ylmethyl)benzenesulfonamide (21)
From 4-nitro-N-(pyridin-2-ylmethyl)benzenesulfonamide (9). Yellow solid. Yield: 52%. mp
o
-1
1
1
42-143 C. R 0.16 (30% acetone:hexane). IR (UATR) cm : 3468, 3374, 3243. H NMR
f
(
300 MHz, DMSO-d ) δ 3.97 (d, J = 6.4 Hz, 2H, PyCH ), 5.93 (s, 2H, NH ), 6.60 (d, J = 8.7
6 2 2
Hz, 2H, ArH), 7.24 (dd, J = 6.5, 4.8 Hz, 1H, PyH ), 7.37 (d, J = 7.9 Hz, 1H, PyH ), 7.43 (d, J
1
3
=
8.7 Hz, 2H, ArH), 7.69-7.77 (m, 2H, NHSO and PyH), 8.44 (d, J = 4.8 Hz, 1H, PyH ). C
2
NMR (75 MHz, DMSO-d ): δ 48.4, 113.1 (2), 122.0, 122.7, 125.8, 129.0 (2), 137.1, 149.1,
6
+
1
53.0, 158.1. HRMS-TOF: m/z [M+Na] 286.0633 (Calcd for C H N NaO S: 286.0621).
1
2
13
3
2
4
.4.3 3-amino-N-(2-(pyridin-2-yl)ethyl)benzenesulfonamide (22)
From 3-nitro-N-(2-(pyridin-2-yl)ethyl)benzenesulfonamide (10). Yellow solid. Yield: 48%.
o
-1
1
mp 133-134 C. R 0.28 (30% acetone:hexane). IR (UATR) cm : 3458, 3374, 3246. H NMR
f
(
300 MHz, DMSO-d ) δ 2.83 (t, J = 7.7 Hz, 2H, PyCH ), 3.08 (t, 2H, CH NH), 5.56 (s, 2H,
6 2 2
NH ), 6.74 (d, J = 8.0 Hz, 1H, ArH), 6.86 (d, J = 7.7 Hz, 1H, ArH), 6.97 (s, 1H, ArH),
2
7
.14-7.24 (m, 3H, ArH and PyH), 7.50 (br s, 1H, NHSO ), 7.67 (t, J = 7.6 Hz, 1H, PyH), 8.45
2
1
3
(
d, J = 4.4 Hz, 1H, PyH ). C NMR (75 MHz, DMSO-d ): δ 37.8, 42.8, 111.6, 113.6, 117.6,
6
+
1
22.1, 123.7, 130.0, 137.0, 141.2, 149.5, 149.8, 158.8. HRMS-TOF: m/z [M+Na] 300.0781
(
Calcd for C H N NaO S: 300.0777).
13 15 3 2
4
.4.4 3-amino-N-(pyridin-2-ylmethyl)benzenesulfonamide (23)
From 3-nitro-N-(pyridin-2-ylmethyl)benzenesulfonamide (11). Yellow solid. Yield: 55%. mp
o
-1
1
1
10-111 C. R 0.29 (30% acetone:hexane). IR (KBr) cm : 3456, 3363, 3236. H NMR (300
f
MHz, DMSO-d ) δ 4.03 (d, J = 6.3 Hz, 2H, PyCH ), 5.56 (s, 2H, NH ), 6.75 (d, J = 8.0 Hz,
6
2
2
1
H, ArH), 6.90 (d, J = 7.7 Hz, 1H, ArH), 7.00 (s, 1H, ArH), 7.18 (t, J = 7.8 Hz, 1H, ArH),
.25 (dd, J = 7.5, 4.8 Hz, 1H, PyH), 7.38 (d, J = 7.7 Hz, 1H, PyH), 7.75 (t, J = 7.7 Hz, 1H,
7
1
3
PyH), 8.05 (t, J = 6.2 Hz, 1H, NHSO ), 8.44 (d, J = 4.7 Hz, 1H, PyH ). C NMR (75 MHz,
2

1
5
ACCEPTED MANUSCRIPT
DMSO-d ): δ 48.4, 111.6, 113.7, 117.7, 121.9, 122.8, 130.0, 137.2, 141.3, 149.1, 149.8,
6
+
1
57.9. HRMS-TOF: m/z [M+Na] 286.0632 (Calcd for C H N NaO S: 286.0621).
1
2
13
3
2
4
.5 General procedure for the synthesis of aminobenzenesulfonamides (24-31)
A mixture of nitrobenzenesulfonamide (4 mmol) and SnCl ⋅2H O (20 mmol) in absolute
2
2
ethanol (20 mL) was stirred under reflux until completion of the reaction as monitored by
TLC until completion of the reaction as monitored by TLC, then concentrated under reduced
pressure. Water (20 mL) was added and extracted with EtOAc (3 × 20 mL). The organic
extracts were combined and washed with water (20 mL) and brine (20 mL). The organic
layer was dried over anh. Na SO , filtered and concentrated. The crude product was purified
2
4
using silica gel column chromatography.
1
H NMR of 4-amino-N-phenethylbenzenesulfonamide (24) [43], 4-amino-N-(3,4-
dimethoxyphenethyl)benzenesulfonamide
phenethylbenzenesulfonamide (26)
yl)sulfonyl)aniline (28) [45],
yl)sulfonyl)aniline (29) [45] and 3-((3,4-dihydroisoquinolin-2(1H)-yl)sulfonyl)aniline (30)
42] and 3-((6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)sulfonyl)aniline (31) [39] were
consistent with that reported in the literature.
.5.1 3-amino-N-(3,4-dimethoxyphenethyl)benzenesulfonamide (27)
From N-(3,4-dimethoxyphenethyl)-3-nitrobenzenesulfonamide (15). Yellow solid. Yield:
(25)
[44],
4-((6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-
[43],
3-amino-N-
4-((3,4-dihydroisoquinolin-2(1H)-
[
4
o
-1
1
8
0%. mp 89-90 C. R 0.30 (30% acetone:hexane). IR (UATR) cm : 3467, 3372, 3259. H
f
NMR (300 MHz, CDCl ) δ 2.69 (t, J = 6.8 Hz, 2H, ArCH ), 3.17 (q, J = 6.8 Hz, 2H,
3
2
CH NH), 3.80, 3.83 (2s, 6H, 2 × OCH ), 4.40 (t, J = 6.0 Hz, 1H, NH), 6.56 (d, J = 1.8 Hz,
2
3
1
8
=
H, ArH), 6.61 (dd, J = 8.1, 1.8 Hz, 1H, ArH), 6.75 (d, J = 8.1 Hz, 1H, ArH), 6.80 (dd, J =
.0, 1.6 Hz, 1H, ArH), 7.02 (t, J = 1.8 Hz, 1H, ArH), 7.10 (d, J = 7.7 Hz, 1H, ArH), 7.22 (t, J
13
7.9 Hz, 1H, ArH),. C NMR (75 MHz, DMSO-d ) δ 35.3, 44.4, 55.9 (2), 111.4, 111.8,
6
1
12.7, 116.6, 118.8, 120.8, 130.0, 130.1, 140.5, 147.2, 147.9, 149.1. HRMS-TOF: m/z [M +
+
Na] 359.1027 (Calcd for C H N NaO S: 359.1036).
16
20
2
4
4
.6 General procedure for the synthesis of naphthoquinones (33-44)
A mixture of the appropriate amine (5 mmol) and 2,3-dichloronaphthoquinone 32 (5 mmol)
in absolute ethanol (20 mL) was stirred under reflux until completion of the reaction as
monitored by TLC, and then concentrated under reduced pressure. Water (20 mL) was added

1
6
ACCEPTED MANUSCRIPT
and extracted with EtOAc (3 × 20 mL). The organic layer was dried over anh. Na SO ,
2
4
filtered and concentrated under reduced pressure. The crude product was purified using silica
gel column chromatography to afford the pure product.
4
.6.1
4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(2-(pyridin-2-
yl)ethyl)benzenesulfonamide (33)
From 4-amino-N-(2-(pyridin-2-yl)ethyl)benzenesulfonamide (20). Red solid. Yield: 20%. mp
o
-1
1
2
02-203 C. R 0.21 (30% acetone:hexane). IR (UATR) cm : 3254, 1672, 1593. H NMR
f
(
300 MHz, DMSO-d ) δ 2.82 (t, J = 7.4 Hz, 2H, PyCH ), 3.10 (q, J = 6.9 Hz, 2H, CH NH),
6 2 2
7
.17-7.25 (m, 4H, ArH and PyH ), 7.59-7.70 (m, 4H, ArH , PyH and NHSO ), 7.82 (dt, J =
2 2 2 2
7
.4, 1.3 Hz, 1H, NTQH ), 7.85 (dt, J = 7.4, 1.3 Hz, 1H, NTQH ), 8.04 (d, J = 7.3 Hz, 2H,
13
NTQH), 8.44 (d, J = 4.5 Hz, 1H, PyH), 9.53 (s, 1H, NH ). C NMR (75 MHz, DMSO-d ): δ
6
3
1
7.7, 42.8, 118.9, 122.1, 122.9 (2), 123.7, 126.7, 127.1, 127.2 (2), 131.0, 132.2, 134.0, 134.9,
+
35.2, 136.9, 143.2, 143.4, 149.5, 158.8, 177.4, 180.4. HRMS-TOF: m/z [M+H] 468.0777
(
Calcd for C H ClN O S: 468.0779).
23 19 3 4
4
.6.2
4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-
phenethylbenzenesulfonamide (34)
o
From 4-amino-N-phenethylbenzenesulfonamide (24). Red solid. Yield: 45%. mp 181-182 C.
-1
1
R 0.31 (30% acetone:hexane). IR (KBr) cm : 3275, 1674, 1591. H NMR (300 MHz,
f
DMSO-d ) δ 2.66 (t, J = 7.8 Hz, 2H, ArCH ), 2.96 (q, J = 6.3 Hz, 2H, CH NH), 7.15 (d, J =
6
2
2
8
.6 Hz, 2H, ArH), 7.17-7.30 (m, 5H, ArH), 7.63 (t, J = 5.8 Hz, 1H, NHSO ), 7.69 (d, J = 8.6
2
Hz, 2H, ArH), 7.83 (t, J = 7.4 Hz, 1H, NTQH ), 7.88 (t, J = 7.4 Hz, 1H, NTQH ), 8.05 (d, J =
1
3
7
1
1
.4 Hz, 2H, NTQH), 9.56 (s, 1H, NH ). C NMR (75 MHz, DMSO-d ): δ 35.7, 44.6, 118.9,
6
22.9 (2), 126.7 (2), 127.1, 127.2 (2), 128.8 (2), 129.1 (2), 130.9, 132.2, 134.0, 134.9, 135.2,
+
39.2, 143.2, 143.3, 177.5, 180.4. HRMS-TOF: m/z [M+H] 467.0821 (Calcd for
C H ClN O S: 467.0827).
24
20
2
4
4
.6.3
4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(3,4-
dimethoxyphenethyl)benzenesulfonamide (35)
From 4-amino-N-(3,4-dimethoxyphenethyl)benzenesulfonamide (25). Red solid. Yield: 55%.
o
-1
1
mp 191-192 C. R 0.21 (30% acetone:hexane). IR (UATR) cm : 3261, 1671, 1591. H NMR
f
(
300 MHz, DMSO-d ) δ 2.57 (t, J = 7.8 Hz, 2H, ArCH ), 2.94 (q, J = 6.3 Hz, 2H, CH NH),
6 2 2
3
.68, 3.70 (2s, 6H, 2 × OCH ), 6.63 (dd, J = 8.2, 1.8 Hz, 1H, ArH), 6.72 (d, J = 1.8 Hz, 1H,
3
ArH), 6.81 (d, J = 8.2 Hz, 1H, ArH), 7.22 (d, J = 8.6 Hz, 2H, ArH), 7.57 (t, J = 5.7 Hz, 1H,

1
7
ACCEPTED MANUSCRIPT
NHSO ), 7.67 (d, J = 8.6 Hz, 2H, ArH), 7.82 (t, J = 7.6 Hz, 1H, NTQH ), 7.87 (t, J = 7.6 Hz,
2
1
3
1
H, NTQH ), 8.04 (d, J = 7.6 Hz, 2H, NTQH), 9.53 (s, 1H, NH ). C NMR (75 MHz,
DMSO-d ): δ 34.8, 44.2, 55.4, 55.5, 112.0, 112.6, 118.2, 120.4, 122.4 (2), 126.2, 126.5, 126.7
6
(2), 130.5, 131.1, 131.8, 133.4, 134.5, 134.7, 142.8, 143.1, 147.3, 148.6, 176.8, 179.9.
+
HRMS-TOF: m/z [M+H] 527.1036 (Calcd for C H ClN O S: 527.1038).
2
6
24
2
6
4
.6.4
4-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(pyridin-2-
ylmethyl)benzenesulfonamide (36)
From 4-amino-N-(pyridin-2-ylmethyl)benzenesulfonamide (21). Red-brown solid. Yield:
o
-1
1
3
1%. mp 225-226 C. R 0.38 (30% acetone:hexane). IR (UATR) cm : 3261, 1675, 1591. H
f
NMR (300 MHz, DMSO-d ) δ 4.10 (d, J = 6.2 Hz, 2H, PyCH ), 7.17-7.28 (m, 3H, ArH and
6
2
2
PyH), 7.36 (d, J = 7.8 Hz, 1H, PyH), 7.65-7.77 (m, 3H, ArH and PyH), 7.84 (t, J = 7.4 Hz,
2
1
H, NTQH ), 7.89 (t, J = 7.4 Hz, 1H, NTQH ), 8.06 (d, J = 7.4 Hz, 2H, NTQH), 8.19 (t, J =
13
6
.2 Hz, 1H, NHSO ), 8.44 (d, J = 4.3 Hz, 1H, PyH), 9.56 (s, 1H, NH ). C NMR (75 MHz,
2
DMSO-d ): δ 48.4, 119.0, 122.0, 122.8 (2), 122.9, 126.7, 127.1, 127.2 (2), 131.0, 132.2,
6
1
34.0, 135.0, 135.2, 137.2, 143.1, 143.3, 149.2, 157.7, 177.5, 180.4. HRMS-TOF: m/z
+
[
M+Na] 476.0448 (Calcd for C H ClN NaO S: 476.0442).
2
2
16
3
4
4
1
.6.5 2-chloro-3-((4-((3,4-dihydroisoquinolin-2(1H)-yl)sulfonyl)phenyl)amino)naphthalene-
,4-dione (37)
From 4-((3,4-dihydroisoquinolin-2(1H)-yl)sulfonyl)aniline (28). Red solid. Yield: 38%. mp
o
-1
1
1
79-180 C. R 0.36 (30% acetone:hexane). IR (KBr) cm : 3275, 1674, 1591. H NMR (300
f
MHz, DMSO-d ) δ 2.84 (t, J = 7.8 Hz, 2H, C4-IQH), 3.31 (t, J = 6.3 Hz, 2H, C3-IqH), 4.21
6
(s, 2H, C1-IQH), 7.07-7.18 (m, 4H, IQH), 7.25 (d, J = 8.7 Hz, 2H, ArH), 7.72 (d, J = 8.7
Hz, 2H, ArH), 7.83 (t, J = 7.4 Hz, 1H, NTQH ), 7.88 (t, J = 7.4 Hz, 1H, NTQH ), 8.05 (d, J =
1
3
7
1
1
.4 Hz, 2H, NTQH), 9.59 (s, 1H, NH ). C NMR (75 MHz, DMSO-d ): δ 28.3, 44.0, 47.7,
6
19.7, 122.7 (2), 126.6, 126.7, 126.9, 127.0, 127.1, 128.1 (2), 129.1, 130.0, 130.1, 131.0,
+
32.1, 133.5, 134.0, 135.2, 143.0, 144.1, 177.5, 180.3. HRMS-TOF: m/z [M+H] 479.0829
(
Calcd for C H ClN O S: 479.0827).
25 20 2 4
4
.6.6
2-chloro-3-((4-((6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)sulfonyl)phenyl)amino)naphthalene-1,4-dione (38)
From 4-((6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)sulfonyl)aniline (29). Red solid.
o
-1
Yield: 41%. mp 161-162 C. R 0.29 (30% acetone:hexane). IR (KBr) cm : 3313, 1671,
f
1
1
594. H NMR (300 MHz, DMSO-d ) δ 2.75 (br t, J = 5.1 Hz, 2H, C4-IQH), 3.31 (t, J = 5.2
6

1
8
ACCEPTED MANUSCRIPT
Hz, 2H, C3-IqH), 3.68 (s, 6H, 2 × OCH ), 4.11 (s, 2H, C1-IQH), 6.66, 6.76 (2s, 2H, IQH),
3
7
.25 (d, J = 8.6 Hz, 2H, ArH), 7.70 (d, J = 8.6 Hz, 2H, ArH), 7.83 (t, J = 7.3 Hz, 1H,
NTQH ), 7.88 (t, J = 7.3 Hz, 1H, NTQH ), 8.05 (d, J = 7.3 Hz, 2H, NTQH), 9.59 (s, 1H,
1
3
NH ). C NMR (75 MHz, DMSO-d ): δ 27.8, 44.2, 47.5, 55.9 (2), 110.2, 112.2, 119.6, 122.7
6
(2), 123.7, 125.2, 126.7, 127.1, 128.1 (2), 130.1, 131.0, 132.2, 134.0, 135.2, 143.1, 144.1,
+
1
47.8, 148.0, 177.5, 180.3. HRMS-TOF: m/z [M+Na] 561.0859 (Calcd for
C H ClN NaO S: 561.0863).
27
23
2
6
4
.6.7
3-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(2-(pyridin-2-
yl)ethyl)benzenesulfonamide (39)
From 3-amino-N-(2-(pyridin-2-yl)ethyl)benzenesulfonamide (22). Red solid. Yield: 22%.
o
-1
1
mp 172-173 C. R 0.21 (30% acetone:hexane). IR (KBr) cm : 3291, 1668, 1588. H NMR
f
(
300 MHz, DMSO-d ) δ 2.85 (t, J = 7.4 Hz, 2H, PyCH ), 2.96 (q, J = 6.9 Hz, 2H, CH NH),
6 2 2
7
1
7
7
.18-7.24 (m, 2H, PyH), 7.34-7.40 (m, 1H, ArH), 7.47-7.55 (m, 3H, ArH), 7.68 (dt, J = 7.7,
.7 Hz, 1H, PyH), 7.75 (t, J = 5.7 Hz, 1H, NHSO ), 7.81 (dt, J = 7.4, 1.1 Hz, 1H, NTQH ),
2
.88 (dt, J = 7.4, 1.1 Hz, 1H, NTQH ), 8.02 (dd, J = 7.4, 1.1 Hz, 1H, NTQH), 8.05 (dd, J =
13
.4, 1.1 Hz, 1H, NTQH), 8.45 (d, J = 4.5 Hz, 1H, PyH), 9.52 (s, 1H, NH ), C NMR (75
MHz, DMSO-d ): δ 37.8, 42.8, 116.8, 121.6, 122.1, 122.2, 123.7, 126.6, 127.0, 127.3, 129.3,
6
1
30.9, 132.3, 133.8, 135.2, 136.9, 140.3, 140.7, 143.5, 149.5, 158.7, 177.3, 180.3. HRMS-
+
TOF: m/z [M+H] 468.0768 (Calcd for C H ClN O S: 468.0779).
2
3
19
3
4
4
.6.8
phenethylbenzenesulfonamide (40)
From 3-amino-N-phenethylbenzenesulfonamide (26). Red solid. Yield: 41%. mp 161-462 C.
3-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-
o
-
1
1
R 0.36 (30% acetone:hexane). IR (UATR) cm : 3289, 1673, 1592. H NMR (300 MHz,
f
DMSO-d ) δ 2.66 (t, J = 7.8 Hz, 2H, ArCH ), 2.96 (q, J = 6.3 Hz, 2H, CH NH), 7.13-7.31
6
2
2
(m, 5H, ArH), 7.34-7.39 (m, 1H, ArH), 7.48-7.55 (m, 3H, ArH), 7.75 (t, J = 5.7 Hz, 1H,
NHSO ), 7.81 (t, J = 7.5 Hz, 1H, NTQH ), 7.88 (t, J = 7.5 Hz, 1H, NTQH ), 8.01 (d, J = 7.5
2
1
3
Hz, 1H, NTQH), 8.05 (d, J = 7.5 Hz, 1H, NTQH), 9.52 (s, 1H, NH ). C NMR (75 MHz,
DMSO-d ): δ 35.7, 44.5, 116.8, 121.6, 122.2, 126.6, 126.7, 127.0, 127.3, 128.8 (2), 129.1 (2),
6
1
29.4, 130.9, 132.3, 133.9, 135.2, 139.1, 140.3, 140.7, 143.5, 177.3, 180.3. HRMS-TOF: m/z
+
[
M+Na] 489.0652 (Calcd for C H ClN NaO S: 489.0646).
2
4
19
2
4
4
.6.9
3-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(3,4-
dimethoxyphenethyl)benzenesulfonamide (41)

1
9
ACCEPTED MANUSCRIPT
From 3-amino-N-(3,4-dimethoxyphenethyl)benzenesulfonamide (27). Red solid. Yield: 49%.
o
-1
1
mp 163-164 C. R 0.33 (30% acetone:hexane). IR (UATR) cm : 3291, 1674, 1592. H NMR
f
(
300 MHz, DMSO-d ) δ 2.60 (t, J = 5.7 Hz, 2H, ArCH ), 2.95 (q, J = 5.7 Hz, 2H, CH NH),
6 2 2
3
.69, 3.70 (2s, 6H, 2 × OCH ), 6.64 (dd, J = 8.2, 1.8 Hz, 1H, ArH), 6.74 (d, J = 1.8 Hz, 1H,
3
ArH), 6.82 (d, J = 8.2 Hz, 1H, ArH), 7.33-7.38 (m, 1H, ArH), 7.46-7.55 (m, 3H, ArH), 7.69
t, J = 5.7 Hz, 1H, NHSO ), 7.79 (t, J = 7.6 Hz, 1H, NTQH ), 7.87 (t, J = 7.6 Hz, 1H,
(
2
NTQH ), 7.99 (d, J = 7.6 Hz, 1H, NTQH), 8.04 (d, J = 7.6 Hz, 1H, NTQH), 9.49 (s, 1H,
1
3
NH ). C NMR (75 MHz, DMSO-d ): δ 35.4, 44.7, 55.9, 56.0, 112.4, 113.1, 121.0 (2), 121.6,
6
1
1
5
4
22.2, 126.6, 127.0, 127.3, 129.3, 130.9, 131.6, 132.3, 133.8, 135.2, 140.4, 140.8, 143.6,
+
47.8, 149.1, 177.3, 180.3. HRMS-TOF: m/z [M+H] 527.1053 (Calcd for C H ClN O S:
26
24
2
6
27.1038).
.6.10
3-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(pyridin-2-
ylmethyl)benzenesulfonamide (42)
From 3-amino-N-(pyridin-2-ylmethyl)benzenesulfonamide (23). Red-brown solid. Yield:
o
-1
1
2
8%. mp 180-181 C. R 0.30 (30% acetone:hexane). IR (UATR) cm : 3209, 1680, 1593. H
f
NMR (300 MHz, DMSO-d ) δ 4.11 (d, J = 7.4 Hz, 2H, PyCH ), 7.21-7.28 (m, 1H, PyH),
6
2
7
.31-7.40 (m, 2H, ArH and PyH), 7.45-7.58 (m, 3H, ArH), 7.74 (t, J = 7.7 Hz, 1H, PyH),
.82 (t, J = 7.3 Hz, 1H, NTQH ), 7.88 (t, J = 7.3 Hz, 1H, NTQH ), 8.05 (d, J = 7.3 Hz, 2H,
7
NTQH), 8.29 (t, J = 6.3 Hz, 1H, NHSO ), 8.44 (d, J = 4.7 Hz, 1H, PyH), 9.50 (s, 1H, NH ).
2
1
3
C NMR (75 MHz, DMSO-d ): δ 48.4, 116.8, 121.6, 122.0, 122.1, 122.9, 126.6, 127.1,
6
1
1
4
1
27.3, 129.3, 130.9, 132.3, 133.9, 135.2, 137.2, 140.2, 140.8, 143.5, 149.2, 157.7, 177.3,
+
80.4. HRMS-TOF: m/z [M+Na] 476.0443 (Calcd for C H ClN NaO S: 476.0442).
2
2
16
3
4
.6.11 2-chloro-3-((3-((3,4-dihydroisoquinolin-2(1H)-yl)sulfonyl)phenyl)amino)naphthalene-
,4-dione (43)
From 3-((3,4-dihydroisoquinolin-2(1H)-yl)sulfonyl)aniline (30). Red solid. Yield: 32%. mp
o
-1
1
2
24-225 C. R 0.51 (30% acetone:hexane). IR (KBr) cm : 3286, 1671, 1591. H NMR (300
f
MHz, DMSO-d ) δ 2.87 (t, J = 7.8 Hz, 2H, C4-IQH), 3.29 (t, J = 6.3 Hz, 2H, C3-IqH), 4.22
6
(s, 2H, C1-IQH), 7.15-7.29 (m, 4H, IQH), 7.39-7.44 (m, 1H, ArH), 7.51-7.60 (m, 3H, ArH),
7
.82 (dt, J = 7.3, 1.3 Hz, 1H, NTQH ), 7.88 (dt, J = 7.3, 1.3 Hz, 1H, NTQH ), 8.02 (dd, J =
1
3
7
.3, 1.3 Hz, 1H, NTQH), 8.06 (dd, J = 7.3, 1.3 Hz, 1H, NTQH), 9.52 (s, 1H, NH ). C NMR
(
75 MHz, DMSO-d ): δ 28.5, 44.1, 47.7, 117.0, 122.3, 123.0, 126.6, 126.8, 127.0, 127.2,
6

2
0
ACCEPTED MANUSCRIPT
1
1
4
27.5, 128.1, 129.2, 129.5, 131.0, 132.0, 132.3, 133.5, 133.9, 135.2, 136.3, 140.7, 143.6,
+
77.4, 180.3. HRMS-TOF: m/z [M+H] 479.0842 (Calcd for C H ClN O S: 479.0827).
2
5
20
2
4
.6.12
2-chloro-3-((3-((6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)sulfonyl)phenyl)amino)naphthalene-1,4-dione (44)
From 3-((6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)sulfonyl)aniline (31). Red solid.
o
-1
Yield: 39%. mp 199-200 C. R 0.38 (30% acetone:hexane). IR (UATR) cm : 3297, 1672,
f
1
1
2
592. H NMR (300 MHz, DMSO-d ) δ 2.77 (t, J = 7.8 Hz, 2H, C4-IQH), 3.26 (t, J = 6.3 Hz,
6
H, C3-IqH), 3.69 (s, 6H, 2 × OCH ), 4.12 (s, 2H, C1-IQH), 6.69, 6.72 (2s, 2H, C5-IQH and
3
C8-IQH), 7.38-7.44 (m, 1H, ArH), 7.48-7.60 (m, 3H, ArH), 7.82 (dt, J = 7.4, 1.4 Hz, 1H,
NTQH ), 7.88 (dt, J = 7.4, 1.4 Hz, 1H, NTQH ), 8.02 (dd, J = 7.4, 1.4 Hz, 1H, NTQH), 8.05
1
3
(
dd, J = 7.4, 1.4 Hz, 1H, NTQH), 9.52 (s, 1H, NH ). C NMR (75 MHz, DMSO-d ): δ 28.1,
6
4
4.2, 47.4, 55.9 (2), 110.2, 112.3, 116.9, 122.3, 123.1, 123.6, 125.2, 126.6, 127.0, 128.2,
1
29.5, 130.9, 132.3, 133.9, 135.2, 136.2, 140.6, 143.6, 147.8, 148.0, 177.4, 180.3. HRMS-
+
TOF: m/z [M+Na] 561.0862 (Calcd for C H ClN NaO S: 561.0858).
2
7
23
2
6
4
.7 Cytotoxic assay: cancer cell lines
The cells suspended in the corresponding culture medium were inoculated in 96-well
microtiter plates (Corning Inc., NY, USA) at a density of 10,000-20,000 cells per well, and
o
incubated for 24 h at 37 C in a humidified atmosphere with 95% air and 5% CO . An equal
2
volume of additional medium containing either the serial dilutions of the test compounds,
positive control (etoposide and/or doxorubicin), or negative control (DMSO) was added to
the desired final concentrations, and the microtiter plates were further incubated for an
additional 48 h. The number of surviving cells in each well was determined using MTT assay
[
46,47] (for adherent cells: HuCCA-1, HepG2, and A549 cells) and XTT assay [48] (for
suspended cells: MOLT-3 cells). The IC50 value is defined as the drug (or compound)
concentration that inhibits cell growth by 50% (relative to negative control).
4
.8 Antimalarial assay: Radioisotope techniques
P.falciparum (K1, multidrug resistant strain) was cultivated in vitro conditions,
according to Trager & Jensen (1976) [49], in RPMI 1640 medium containing 20 mM HEPES
(
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 32 mM NaHCO and 10% heat
3
o
activated human serum with 3% erythrocytes, in humidified 37 C incubator with 3% CO .
2

2
1
ACCEPTED MANUSCRIPT
The culture was passaged with fresh mixture of erythrocytes and medium every day to
maintain cell growth. Quantitative assessment of antimalarial activity in vitro was determined
by microculture radioisotope techniques based upon the methods described by Desjardins et
al. (1979) [50]. Briefly, a mixture of 200 µL of 1.5% erythrocytes with 1% parasitemia at the
early ring stage was pre-exposed to 25 µL of the medium containing a test sample dissolved
3
in 1% DMSO (0.1% final concentration) for 24 h. Subsequently, 25 µL of [ H] hypoxanthine
(Amersham, USA) in culture medium (0.5 µCi) was added to each well and the plates were
incubated for an additional 24 h. Levels of incorporated radioactive labeled hypoxanthine,
indicating parasite growth, were determined using the Top Count microplate scintillation
counter (Packard, USA). The percentage of parasite growth was calculated using the signal
count per minute of treated (CPMT) and untreated conditions (CPMU) as shown by the
following equation;
%
parasite growth = CPMT/CPMU x 100
Eq. 6
4
.9 Cytotoxicity assay: primate cell line (Vero)
The cytotoxicity assay was performed using the Green Fluorescent Protein (GFP)
detection method [51]. 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 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% CO . The assay was carried out by adding 45 µL of cell
2
4
suspension at 3.3 × 10 cells/mL to each well of 384-well plates containing 5 µL of test
o
compounds previously diluted in 0.5% DMSO, and then incubated for 4 days at 37 C
incubator with 5% CO . Fluorescence signals were measured using SpectraMax M5
2
microplate reader (Molecular Devices, USA) in the bottom reading mode with excitation and
emission wavelengths of 485 and 535 nm, respectively. Fluorescence signal at day 4 was
subtracted from background fluorescence at day 0. IC50 values were derived 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.

2
2
ACCEPTED MANUSCRIPT
4
.10 QSAR analysis
Conceptually, the chemical structures of the tested compounds (33-44, Fig. 3)
together with their experimental activities (IC50 values) were used as data sets for
construction of five QSAR models. The additional set of 30 structurally modified compounds
(34a-34d, 36a-36k, 40a-40d, and 42a-42k, Figs. 4-6) was virtually constructed, and their
activities were predicted using the constructed QSAR models.
4
.10.1 Data set
Chemical structures of the tested compounds (33-44) along with their IC values
50
were used as data sets for QSAR analysis. Regarding anticancer activity against 4 cancer cell
lines and antimalarial activity of the investigated compounds, data sets were arranged as 5
data sets in which QSAR models were separately developed. To obtain distribution of data
points, IC values of the tested compounds 33-44 were converted to pIC values by taking
5
0
50
the negative logarithm to the base of 10 (-log IC ). Experimentally inactive compounds were
5
0
excluded from the data set.
4
.10.2 Molecular structure optimization and descriptor calculation
Chemical structures of 12 tested (33-44, Fig. 3) and 30 structurally modified (34a-
4d, 36a-36k, 40a-40d, and 42a-42k, Figs. 4-6) compounds were drawn using the
3
GaussView software [52] and were subjected to geometrical optimization to obtain the low-
energy conformers. Initially, all structures were geometrically optimized using Gaussian 09
[
24] at the semi-empirical Austin Model 1 (AM1) level. Subsequently, density functional
theory (DFT) calculation was performed using the Becke’s three-parameter hybrid method
with the Lee-Yang-Parr correlation functional (B3LYP) together with the LANL2DZ ECP
basis set. The optimized structures were consequently used as an input for extracting
descriptor values. A set of 13 quantum chemical descriptors i.e., mean absolute atomic charge
(
Q ), total energy (Etotal), total dipole moment (µ), highest occupied molecular orbital energy
m
(EHOMO), lowest unoccupied molecular orbital energy (ELUMO), energy difference of HOMO
and LUMO (HOMO-LUMO ), electron affinity (EA), ionization potential (IP), Mulliken
Gap
electronegativity (χ), hardness (η), softness (S), electrophilic index (ω ) and electrophilicity
i
(
ω) were extracted using an in-house developed script. The optimized structures were further
used for calculation of an additional set of 3,224 molecular descriptors using Dragon software
version 5.5) [25]. Dragon descriptors comprised of 22 classes including Constitutional
(

2
3
ACCEPTED MANUSCRIPT
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
.10.3 Descriptor selection
Feature selection was performed to select a set of informative descriptors from the
whole set of descriptors obtained from the calculation. Correlation-based feature selection
was employed for initial selection. The pair-correlation of each descriptor value and
bioactivity (pIC ) was calculated and the Pearson’s correlation coefficient (r) value of 0.5
5
were used as the cut-off value in which descriptors with |r| < 0.5 were considered as low
correlated descriptors and were excluded while descriptors with |r| ≥ 0.5 were used for further
selection using stepwise multiple linear regression (MLR) as implemented in the SPSS
statistics 18.0 software or subjected to the attribute evaluator CfsSubsetEval using the
BestFirst search method as implemented in Waikato Environment for Knowledge Analysis
(WEKA) version 3.4.5 [26]. Finally, a set of important descriptors were obtained for
multivariate analysis.
4
.10.4 Multivariate analysis
Multivariate analysis was performed by Waikato Environment for Knowledge
Analysis (WEKA) version 3.4.5 [26] using multiple linear regression (MLR) algorithm where
the selected descriptors and pIC values were assigned as independent variables (X) and
5
0
dependent variable (Y), respectively. The MLR models were constructed according to the
following equation.
Y = B +
∑Bn Xn
Eq. 7
0
where Y is the pIC50 values of compounds, B is the intercept and B are the regression
0
n
coefficient of descriptors Xn.
4
.10.5 Data sampling
Leave-one-out cross validation (LOO-CV) was used for splitting the data set into a
training set and a testing set. Principally, one sample was left out as the testing set while the

2
4
ACCEPTED MANUSCRIPT
remaining N-1 samples were used as the training set. This procedure was continued until
every sample in the data set was used as the testing set to predict Y variable (activity).
4
.10.6 Evaluating the performance of QSAR models
The performance of constructed QSAR models was evaluated using two statistical
parameters. The correlation coefficient (R) indicated the predictive performance, and the root
mean square error (RMSE) represented predictive error of the models.
4
.10.7 Prediction of structurally modified compounds using the constructed QSAR models
The important descriptor values of the structurally modified compounds (34a-34d,
6a-36k, 40a-40d, and 42a-42k, Figs. 4-6) were obtained from the calculation as described
3
previously. The QSAR equations were used to predict activities of these modified compounds
in which important descriptor values of each modified compound were assigned in the
equation for computing its predicted activity (predicted pIC value).
5
0
Acknowledgments
This project was financially supported from Srinakharinwirot University under the
Government Budget. R.P. is grateful for the Thailand Research Fund (MRG5680001). Great
supports from the Office of 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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