New chalcone-sulfonamide hybrids exhibiting anticancer and antituberculosis activity

Article abstract of DOI:10.1016/j.ejmech.2019.05.013

New sulfonamides 5/6 derived from 4-methoxyacetophenone 1 were synthesized by N-sulfonation reaction of ammonia (3)and aminopyrimidinone (4)with its sulfonyl chloride derivative 2. Sulfonamides 5 and 6 were used as precursors of two new series of chalcones 8a-f and 9a-f, which were obtained through Claisen-Schmidt condensation with aromatic aldehydes 7a-f. Compounds 5/6, 8a-d, 8f, 9a-d, and 9f were screened by the US National Cancer Institute (NCI)at 10 μM against sixty different human cancer cell lines (one-dose trial). Chalcones 8b and 9b satisfied the pre-determined threshold inhibition criteria and were selected for screening at five different concentrations (100, 10, 1.0, 0.1, and 0.01 μM). Compound 8b exhibited remarkable GI₅₀ values ranging from 0.57 to 12.4 μM, with cytotoxic effects being observed in almost all cases, especially against the cell lines K-562 of Leukemia and LOX IMVI of Melanoma with GI₅₀ = 0.57 and 1.28 μM, respectively. Moreover, all compounds were screened against Mycobacterium tuberculosis H37Rv, chalcones 8a-c and 9a-c were the most active showing MIC values between 14 and 42 μM, and interestingly they were devoid of antibacterial activity against Mycobacterium smegmatis and Staphylococcus aureus. These antituberculosis hits showed however low selectivity, being equally inhibitory to M. tuberculosis and mammalian T3T cells. The chalcone-sulfonamide hybrids 8a-f and 9a-f resulted to be appealing cytotoxic agents with significant antituberculosis activity.

Full text of DOI:10.1016/j.ejmech.2019.05.013

Accepted Manuscript
New chalcone-sulfonamide hybrids exhibiting anticancer and antituberculosis activity
Lina Fernanda Castaño, Viviana Cuartas, Anthony Bernal, Alberto Insuasty, Juan
Guzman, Oscar Vidal, Vivian Rubio, Gloria Puerto, Pavol Lukáč, Vladimir Vimberg,
Gabriela Balíková-Novtoná, Luca Vannucci, Jiri Janata, Jairo Quiroga, Rodrigo
Abonia, Manuel Nogueras, Justo Cobo, Braulio Insuasty
PII:
S0223-5234(19)30422-2
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.013
EJMECH 11323
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 November 2018
Revised Date: 7 February 2019
Accepted Date: 6 May 2019
Please cite this article as: L.F. Castaño, V. Cuartas, A. Bernal, A. Insuasty, J. Guzman, O. Vidal, V.
Rubio, G. Puerto, P. Lukáč, V. Vimberg, G. Balíková-Novtoná, L. Vannucci, J. Janata, J. Quiroga,
R. Abonia, M. Nogueras, J. Cobo, B. Insuasty, New chalcone-sulfonamide hybrids exhibiting
anticancer and antituberculosis activity, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.05.013.
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GRAPHICAL ABSTRACT
New chalcone-sulfonamide hybrids exhibiting anticancer and antituberculosis activity
Lina Fernanda Castaño a, Viviana Cuartas a,b, Anthony Bernal c, Alberto Insuasty c, Juan Guzman c,d
,
e
f
f
g
Oscar Vidal , Vivian Rubio , Gloria Puerto , Pavol Lukáč
d, Vladimir Vimberg , Gabriela
g
d
d
Balíková-Novtoná , Luca Vannucci , Jiri Janata , Jairo Quiroga a,b, Rodrigo Abonia a,b, Manuel
Nogueras h, Justo Cobo h and Braulio Insuasty a,b*
a Heterocyclic Compounds Research Group, Department of Chemistry, Universidad del Valle, Cali,
A.A. 25360, Colombia
b Centre for Bioinformatics and Photonics-CIBioFI, Universidad del Valle, Cali A.A. 25360,
Colombia
c Department of Chemistry and Biology, Basic Sciences Division, Universidad del Norte,
Barranquilla, 081007, Colombia
d Institute of Microbiology, Czech Academy of Sciences, Videnska 1083, 142 20 Prague, Czech
Republic
e Department of Medicine, Health Sciences Division, Universidad del Norte, Barranquilla, 081007,
Colombia
f Mycobacteria Laboratory, National Health Institute, Bogotá, 111321, Colombia
g Institute of Microbiology, Czech Academy of Sciences, BIOCEV, Pr
Vestec, Czech Republic
ůmyslová 595, 252 50
h Department of Inorganic and Organic Chemistry, Universidad de Jaén, Jaén, 23071, Spain
* braulio.insuasty@correounivalle.edu.co; Tel: +(57)–2–3392440

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New chalcone-sulfonamide hybrids exhibiting anticancer and antituberculosis activity
Corresponding Author: Braulio Insuasty, Ph.D.
Corresponding Author's Institution: Universidad del Valle
First Author: Lina Fernanda Castaño, B.Sc.
Order of Authors: Lina Fernanda Castaño, B.Sc.; Viviana Cuartas, B.Sc.; Anthony Bernal, M.Sc.;
Alberto Insuasty, Ph.D.; Juan Guzman, Ph.D.; Oscar Vidal, Ph.D.; Vivian Rubio, M.Sc.; Gloria
Puerto, Ph.D.; Pavol Lukáč, RNDr.; Vladimir Vimberg, Ph.D.; Gabriela Balíková-Novtoná, Ph.D.;
Luca Vannucci, Ph.D.; Jiri Janata, Ph.D.; Jairo Quiroga, Ph.D.; Rodrigo Abonia, Ph.D.; Manuel
Nogueras, Ph.D.; Justo Cobo, Ph.D.; Braulio Insuasty, Ph.D.
Abstract
New sulfonamides 5/6 derived from 4-methoxyacetophenone 1 were synthesized by N-sulfonation
reaction of ammonia (3) and aminopyrimidinone (4) with its sulfonyl chloride derivative 2.
Sulfonamides 5 and 6 were used as precursors of two new series of chalcones 8a-f and 9a-f, which
were obtained through Claisen-Schmidt condensation with aromatic aldehydes 7a-f. Compounds
5/6, 8a-d, 8f, 9a-d, and 9f were screened by the US National Cancer Institute (NCI) at 10 µM
against sixty different human cancer cell lines (one-dose trial). Chalcones 8b and 9b satisfied the
pre-determined threshold inhibition criteria and were selected for screening at five different
concentrations (100, 10, 1.0, 0.1, and 0.01
ranging from 0.57-12.4 M, with cytotoxic effects being observed in almost all cases, especially
against the cell lines K-562 of Leukemia and LOX IMVI of Melanoma with GI50 = 0.57 and 1.28
µM). Compound 8b exhibited remarkable GI50 values
µ

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µ
M, respectively. Moreover, all compounds were screened against Mycobacterium tuberculosis
H37Rv, chalcones 8a-c and 9a-c were the most active showing MIC values between 14 and 42 µM,
and interestingly they were devoid of antibacterial activity against Mycobacterium smegmatis and
Staphylococcus aureus. These antituberculosis hits showed however low selectivity, being equally
inhibitory to M. tuberculosis and mammalian T3T cells. The chalcone-sulfonamide hybrids 8a-f and
9a-f resulted to be appealing cytotoxic agents with significant antituberculosis activity.
Keywords: Antibacterial, anticancer, antituberculosis, chalcones, sulfonamides.
Highlights
•
•
New chalcone-sulfonamide hybrids were synthesized from 4-methoxyacetophenone.
The 4-chloro-substituted compounds 8b and 9b showed important anticancer activity with
GI50 0.57-40.1 ꢀM.
•
Hybrids 8b and 9b showed significant growth inhibition (MIC ≤ 20 µM) of M. tuberculosis
H37Rv.
*Corresponding author: Dr. Braulio Insuasty, Facultad de Ciencias Naturales y Exactas,
Departamento de Química, Universidad del Valle, Santiago de Cali, Colombia.
E–mail: braulio.insuasty@correounivalle.edu.co
FAX: +57–2–3392440

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1 Introduction
It is well known that cancer and tuberculosis (TB) are major global health challenges. Cancer is the
second cause of human mortality (below cardiovascular disease) and the number of cases is
currently increasing with an estimation of 22 million new cases by 2030 [1]. Cancer diagnosis and
treatment are a great challenge to physicians [2], and the development of new cancer therapeutic
entities is still a major focus of medicinal chemistry efforts worldwide. TB is currently the leading
cause of death from a single infectious agent. The Mycobacterium tuberculosis bacilli are able to
remain latent in one-third of human population [3], and there are multidrug-resistant bacteria
isolates circulating in all the continents [4]. Early phases of drug discovery for TB are quite
important in medicinal chemistry research, and it is not clear if there are enough lead compounds
entering the pipeline to assure novel therapeutics in the following decade.
Sulfonamides are important scaffolds for the development of biologically relevant molecules
because they display a wide variety of pharmacological properties including anti-inflammatory [5],
antimicrobial [6–8], anticancer [9,10], antiviral [11], diuretic [12] and hypoglycemic [13]. The
target versatility of sulfonamides allows the properties tuning, for example by assembling hybrids
with other relevant pharmacophores [14]. The hybrids by combination of the sulfonamide moiety
with heterocyclic rings [15] or chalcones have shown anticancer activity [16,17]. Sulfonamides,
such as sulfamethoxazole, have widely been used in antibacterial chemotherapy because they
interfere with folic acid biosynthesis [18]. Recent studies have also employed the sulfonamide
group in the development of antituberculosis agents [19,20]. Their biochemical advantages are the
coordination to metal ions in diverse metalloenzymes by the S=O and NH2 polar groups, the
establishment of particular hydrogen bonds reproducing the tetrahedral transition states of many
enzymatic reactions and the possibility of forming lipophilic interactions via the aryl groups
[10,21]. These chemical characteristics grant sulfonamides their ability to inhibit a large number of

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metalloenzymes including carbonic anhydrases [9] and peptidases [22], among others. We report
here the synthesis of new chalcone-sulfonamide hybrids derived from 4-methoxyacetophenone and
the study of their anticancer and antituberculosis activities.
2 Results and Discussion
2.1 Chemistry
The synthesis was started with the preparation of the intermediate benzenesulfonyl chloride 2, wich
was carried out by the chlorosulfonation reaction of compound 1, using chlorosulfonic acid and
thionyl chloride (Scheme 1). The use of an equimolar amount of the chlorinating agent resulted in a
low yield (20%). In order to enhance the conversion, an excess 2:1 of the chlorinating agent was
employed reaching 93% yield after 26 h. Compound 2 showed to be stable and it was possible to
1
confirm its structure by spectroscopic techniques such as FTIR, H NMR, 13C NMR and mass
spectrometry. The IR spectrum of chloride 2 shows absorption bands at 3108, 2871, 1684, 1254 and
1167 cm-1 corresponding to stretching vibration of C-H Ar, C-H, C=O, C-O-C and S=O bonds,
respectively. The 1H NMR spectrum showed two downfield signals at 8.52 ppm as a doublet with J
= 2.0 Hz and 8.31 ppm as a double doublet with J = 8.8 and 2.0 Hz corresponding to Ho and Hp
protons, respectively. At 7.21 ppm a doublet with J = 8.8 Hz was assigned to the signal of Hm. The
13C NMR spectrum showed the expected nine signals for carbons in compound 2. The mass
spectrum revealed characteristic isotopic profile for the molecular peak m/z 248/250 for the
presence of one chlorine atom in the molecule.

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Compound
5
R
Reaction time (h) Yield (%)
-
H
4
82
(3)
2
81
6
(4)
Scheme 1. General procedure for the synthesis of sulfonamides 5/6. Reagents and conditions: i) HOSO2Cl,
SOCl2 at 0 °C, 1 and then rt, ii) EtOH, ammonia, rt (for 5) and TEA, R-NH2, rt (for 6).
The synthesis of the sulfonamides intermediates (5/6) was performed through N-sulfonation
reactions of compound 2 with ammonia 3 and aminopyrimidinone 4 (Scheme 1). Compound 5 was
synthetized using a mixture 1:1 ammonia:ethanol in 82% yield. However, several synthetic
conditions were tested ranging diverse solvents and bases for the synthesis of sulphonamide 6
(Table 1). In low polar solvents (e.g. dichlorometane or chloroform) the reactions were unfavored.
In contrast, the use of a protic solvent such as ethanol increased the yield and reduced the reaction
time even at room temperature. Finally, the reaction was optimized by adding triethylamine as acid
scavenger, to afford the desired product 6 in 81% yield.
Table 1. Screening conditions for the synthesis of sulfonamide 6.
Solvent
Base
Concentration
Entry
Temperature Time (h)
Yield (%)
(1.5 mL) (3 drops)
(w/v %)
---
1
2
3
4
5
DCM
CHCl3
EtOH
EtOH
EtOH
Et3N
Et3N
rt
rt
170
170
2
Traces
---
Traces
Et3N
---
rt
81
Complex mixture
30
Et3N
---
reflux
rt
0.25
2.5
NaOH
50

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The structures of compounds 5/6 were confirmed by spectroscopic techniques. The most relevant
features in the IR spectrum of sulfonamide 5 correspond to the presence of two stretching vibration
bands at 3329 and 3208 cm-1 belonging to the N-H bond in a primary amino group, confirming the
1
substitution. In the H NMR spectrum, the product 5 showed similar chemical shift values for
aromatic protons to those of compound 2 but slightly shifted upfield, additionally, at 7.25 ppm is
observed a singlet integrating for two protons, corresponding to the sulfonamidic amino group. The
structure of sulfonamide 5 was also confirmed from single-crystal X-ray data, Figure 1 (a).
(a)
(b)
Figure 1. Molecular structure of the compounds 5 (a) and 8c (b) determined from single-crystal X-ray data,
showing the atom-labelling scheme.
Sulfonamides 5/6 reacted with several substituted aromatic aldehydes 7a-f through a Claisen-
Schmidt condensation to obtain two series of chalcone-sulfonamide hybrids 8a-f and 9a-f. In all
cases, ethanol was used as solvent and an aliquot of aqueous NaOH solution as the base (Table 2).
Chalcones derived from sulfonamide 6 (i.e. 9a-f, Figure 3) turned out to be more sensitive to base
concentration and some by-products were observed under the same reaction conditions used for
preparation of chalcones derived from sulfonamide 5 (i.e. 8a-f, Figure 2) as shown in Entry 1. For
this reason, Entry 2 was considered as the best reaction conditions to provide products 9a-g. By
using the methodologies of Entry 1 and 2, products 8a-g and 9a-g were initially obtained in their

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sodium salt forms, respectively. Therefore, a subsequent treatment with aqueous HCl solution was
necessary.
Table 2. Screening conditions for the synthesis of chalcone-sulfonamide hybrid 9b as model reaction.
Solvent
Base
Entry
Concentration (w/v %) Temperature Time (h)
Yield (%)
(1.5 mL) (2 drops)
1
2
3
EtOH
EtOH
EtOH
NaOH
NaOH
NaOH
50
30
30
rt
rt
6
2.5
3
Complex mixture
56
reflux
Complex mixture
Regarding to the NMR spectra, the twelve compounds synthetized (8a-f and 9a-f) showed similar
chemical shifts, although in compounds 9a-f the presence of the corresponding signals for the
pyrimidine moiety are also present, and therefore we discuss only the spectroscopic data for 8b as
1
the representative of this series. The H NMR spectrum revealed the C=C double bond formation
due to the presence of a multiplet between 7.98 and 7.92 ppm integrating for three protons assigned
to Hα and Ho' protons, a doublet at 7.74 ppm with J = 15.5 Hz corresponding to Hβ and a doublet for
two hydrogens at 7.53 ppm with J = 7.6 Hz assigned to Hm'. The coupling constant for Hβ disclosed
the E configuration on the enone moiety. All proton signals from the aryl ring bound to sulfonamide
13
were observed, including those corresponding to the amino group. In the C NMR spectrum was
observed the two signals for
ꢁ and ꢂ carbon atoms at 122.2 and 142.6 ppm, respectively. The
structure of hybrid 8c was confirmed by single-crystal X-ray data. The ORTEP representation is
shown in Figure 1 (b).

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Figure 2. Chalcone-sulfonamide hybrids derived from sulfonamide 5.
Figure 3. Chalcone-sulfonamide hybrids derived from compound 6.
2.2 Anticancer activity
A preliminary screening of the structures of compounds 5/6, 8a-f and 9a-f was carried out by
COMPARE algorithm, all of them excepting chalcones 8e and 9e were selected by the
Developmental Therapeutics Program (DTP) at National Cancer Institute (NCI) for single

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concentration trials. The assay involves a primary in vitro evaluation of submitted compounds
against 60 human cancer cell lines at 10 µM concentration. The results for each compound are
reported as a mean value of the growth percent (GP) of the treated cells (Figure 4a). Table 3
summarizes the results obtained for compounds 5/6, 8a-d, 8f, 9a-d and 9f.
Table 3. Range and mean growth percent of NCI human cancer cell lines treated with selected compounds at
one dose (10 ꢀM).
Growth percent of most
sensitive cell line (%)
Compound
Mean growth
Most sensitive cell lines
5
6
98.28
99.84
68.07
HL-60(TB) (Leukemia)
M14 (Melanoma)
74.74
94.85
K-562 (Leukemia)
7.90
8a
RPMI-8226 (Leukemia)
K-562 (Leukemia)
-31.74*
3.05
HOP-92 (Non-Small Cell Lung Cancer)
HCT-116 (Colon Cancer)
M14 (Melanoma)
-26.57*
-78.33*
-57.32*
-30.67*
-55.63*
3.04
44.42
8b
786-0 (Renal Cancer)
PC-3 (Prostate Cancer)
K-562 (Leukemia)
56.09
78.45
8c
SR (Leukemia)
28.10
8d
K-562 (Leukemia)
38.78
HCT-116 (Colon Cancer)
K-562 (Leukemia)
19.22
82.97
80.00
8f
44.59
HCT-116 (Colon Cancer)
K-562 (Leukemia)
35.18
9a
35.63
SF-539 (CNS Cancer)
K-562 (Leukemia)
-5.49*
52.87
84.26
3.84
9b
9c
U251(CNS Cancer)
-4.20*
HCT-116 (Colon Cancer)
10.78

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K-562 (Leukemia)
HCT-116 (Colon Cancer)
K-562 (Leukemia)
38.79
24.01
54.26
18.36
53.79
90.59
89.03
9d
9f
HCT-116 (Colon Cancer)
K-562 (Leukemia)
*
Negative value means lethality. This percentage indicates there is no net growth of tumor cells over the
course of the experiment, instead, compound causes the death of the respective cancer cell.
Sulfonamides 5/6 exhibited low activity against the cancer cell lines analyzed. Nevertheless, their
α,β-unsaturated derivatives showed a significant increase in the antitumor activity. In general, both
series of chalcones displayed significant activity against Leukemia panel, on cancer cell lines K-562
(GP = 3.04-54.26 %). Specifically, the compounds 8a, 8b, 8c and 9b at 10 µM showed the strongest
effect, decreasing the viability of this cell line to less than 10% (Figure 4a). The K-562 cell line are
known to be positive for the BCR-ABL fusion gene, as well as to show similarity to primary
granulocytes and erythrocytes [23]. K-562 cells contain mutations on the genes cyclin-dependent
kinase (CDKN2A) and tumor protein (TP53) [24], which induce a cell phenotype with an active
proliferative capacity driven by inhibition of apoptosis. As apoptosis is an important mechanism
providing targets for antitumor drugs, that has been shown to inhibit K-562 cells grow [25-28], we
hypothesised, that these chalcones activate apoptosis causing the effect that we see in table 3. All
compounds also showed activity against Colon Cancer in sub-panel HCT-116 (GP = -78.33-
44.62%) and CNS Cancer on cell line U251 (GP = -4.20-35.40%). Compounds 8b and 9b were the
most active of both series against the majority of cancer cell lines, especially HCT-116 (Colon
Cancer) for 8b and SF-539 (CNS Cancer) for 9b with lethality values of 78.33% and 5.49%,
respectively. Both compounds satisfied the pre-determined threshold inhibition criteria of NCI,
therefore they were selected for a Five-Dose screening, involving the evaluation against 60 cancer
cell lines at five different concentrations (100, 10, 1.0, 0.1, and 0.01
µM) to determine GI50 (growth
inhibitory activity) and LC50 (cytotoxic activity) values. Compounds 8b and 9b exhibited potent

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anticancer activity against several cancer cell lines with GI50 values between 0.57-12.4 µM and
1.56-40.1
especially in K-562 and SR cell lines with GI50 values of 0.57 and 0.86
displayed important activity against HCT-116 (Colon Cancer) with GI50 = 1.36
(Melanoma) with GI50 = 1.28 M and MCF7 (Breast Cancer) with GI50 = 1.30
µ
M, respectively (Table 4). Chalcone 8b showed the best results against Leukemia panel,
M. This compound also
M, LOX IMVI
M. Cytotoxic
µ
µ
µ
µ
activity of 8b was also remarkable, most relevant results were obtained against HCT-116 (Colon
Cancer), SF-539 (CNS Cancer) and LOX IMVI (Melanoma) with LC50 values of 5.36, 6.82 and
5.47
(Colon Cancer) with LC50 = 5.55
(Breast Cancer) LC50 = 6.17 M. Ras proteins are essential components of signalling networks that
µ
M (Table 4). Compound 9b demonstrated to be a good cytotoxic agent against HCT-116
µ
M, U251 (CNS Cancer) with LC50 = 6.16 M and BT-549
µ
µ
control cell proliferation [29], and the HCT-116 cells have a mutation in codon 13 of the KRAS
proto-oncogene [30]. Studies in HCT-116 cells have shown that a number of anticancer drugs target
the cell cycle molecular pathways [31], and thus, we hypothesize that compound 8b could be
targeting similar biochemical mechanisms. CDKN2A mutation in HCT-116, modifies the cell cycle
cellular control, and as such, many CDK inhibitors have been shown anticancer effect in these cells.
For example, roscovitine, purvalanol, and 9-nitropaullone inhibit CDC2, CDK2 and CDK5 [32].
Similar to the K-562 Leukemia cells, the SF-539 CNS cancer cells also contained mutations on the
TP53 gene, which encodes for a tumor suppressor protein that in case of DNA damage, either
activates DNA repair or directs the cell to apoptosis. We propose that the synthetic chalcone-
sulfonamide hybrids could either inhibit CDKs and thus arrest cell cycle, or induce apoptosis or
both, on the cells that lack a functional TP53 protein, as there is strong evidence of apoptosis
induction by different types of chalcones [33-35]. As summary, both 4-chloro-substituted chalcones
8b and 9b showed strong cytotoxic effects against several cancer cell lines, achieving LC50 values
in the low micromolar range (Figure 4b). Both 8b and 9b constitute hit structures for the
development of further anticancer agents.

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Table 4. Cytotoxic activity of compounds 8b and 9b in Five-Dose test expressed as GI50 and LC50 of cancer
cell lines.a
Compounds
Panel/Cell line
8b
9b
b
c
GI50
LC50
GI50
LC50
Leukemia
CCRF-CEM
HL-60(TB)
K-562
3.34
3.08
0.57
2.93
2.61
0.86
>100
>100
>100
>100
>100
>100
2.66
3.20
2.77
3.71
2.17
3.57
>100
>100
>100
>100
>100
>100
MOLT-4
RPMI-8226
SR
Non-Small Cell Lung Cancer
30.8
A549/ATCC
EKVX
2.69
4.56
2.51
2.14
3.66
2.54
2.04
2.58
1.93
2.57
3.94
1.81
2.59
2.34
2.53
2.38
2.49
1.88
30.0
80.3
8.70
32.6
52.9
40.4
28.0
29.1
8.83
50.5
31.2
21.4
95.7
40.9
28.8
30.0
12.1
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
2.03
1.78
1.36
2.14
3.26
1.73
1.76
17.4
7.48
5.36
59.3
84.0
8.26
8.45
1.76
1.92
1.56
3.24
2.93
2.09
1.58
6.68
6.50
5.55
83.9
45.3
9.76
28.3
KM12
SW-620

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CNS Cancer
SF-268
SF-295
SF-539
SNB-19
U251
3.13
3.04
1.60
3.10
1.96
38.3
2.69
2.72
2.10
2.52
1.58
36.1
34.7
15.1
32.2
6.16
31.9
6.82
33.4
9.33
Melanoma
5.47
LOX IMVI
MALME-3M
M14
1.28
5.89
2.13
4.34
12.4
3.24
5.69
12.2
4.88
1.63
3.02
2.00
2.95
12.8
2.10
2.72
8.27
2.33
6.34
41.8
15.7
48.0
61.1
17.0
32.0
50.1
27.4
54.8
21.2
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
41.6
53.2
37.2
42.7
50.8
44.8
Ovarian Cancer
48.1
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
2.67
2.04
3.47
3.08
3.55
3.64
5.96
3.25
1.94
4.50
2.83
3.07
40.1
3.65
80.2
8.00
43.6
38.2
37.1
>100
36.6
9.70
40.4
44.9
32.6
>100
43.1
Renal Cancer
7.82
786-0
A498
1.74
10.4
1.94
2.72
1.98
2.72
2.47
1.89
1.95
10.6
3.23
3.83
1.60
3.45
2.98
1.89
8.74
47.6
36.5
79.5
6.67
73.4
32.3
12.9
47.1
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
8.75
32.5
7.96
31.5
27.5
22.0

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Prostate Cancer
PC-3
2.24
3.42
17.0
2.53
3.58
32.8
33.9
DU-145
36.3
Breast Cancer
>100
MCF7
MDA-MB-231/ATCC
HS 578T
1.30
3.48
3.28
2.29
3.20
1.97
3.42
3.56
1.63
2.45
>100
40.3
>100
6.17
38.8
40.1
>100
BT-549
27.3
T-47D
59.0
a
b
Data obtained from NCI’s in vitro disease–oriented human cancer cell lines screen in µM. GI50 was the
drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in
control cells during the drug incubation, determined at five concentration levels (100, 10, 1.0, 0.1, and 0.01
µM). c LC50 is a parameter of cytotoxicity that reflects the concentration needed to kill 50% of the cells.
2.3 Antibacterial activity and mammalian cytotoxicity
Among the synthesized compounds, the chalcone-sulfonamide hybrids 8a, 8b and 9b were the most
potent growth inhibitors of Mycobacterium tuberculosis H37Rv, with minimum inhibitory
concentration (MIC) values of 16, 14 and 20
µ
M respectively (Table 5). The compounds 8c, 9a and
M. The structure-activity
9c also showed significant activity with MIC values between 30 and 42
µ
analysis revealed that the absence of substitution (8a and 9a), the 4-chloro- (8b and 9b) or 4-
methyl-substitution (8c and 9c) on ring B, consistently increased the anti-TB effect. However, the
4-methoxy- (8d and 9d), 3,4,5-trimethoxy- (8e and 9e) and 3,4-methylenedioxy-substitution (8f and
9f) eliminated the antituberculosis effect. Sulfonamides 5 and 6 without the chalcone moiety, were
also devoid of any anti-TB activity (MIC >130
µM). All the evaluated compounds 5/6, 8a-f and 9a-
f, showed little inhibitory effect against Mycobacterium smegmatis mc2155 and Staphylococcus
aureus strains including drug-resistant clinical isolates, with MIC values >175 ꢀM and >57 ꢀM,

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respectively. This result suggested antibacterial specificity against M. tuberculosis, and opens the
door to explore in detail their mechanism of action. However, evaluation of mammalian
cytotoxicity on normal (non-cancer) mouse fibroblasts showed that most of the synthetic
compounds were highly citotoxic (Table 5) with LC50 values <20
exception of the sulfonamides 5 and 6 and the hybrids 8d and 8f with respective LC50 values >2.7
mM, >1.6 mM, and 96 M and 132 M (Figure 4c). The chalcone-sulphonamide hybrids 8e and 9e
were highly cytotoxic to fibroblasts with LC50 values lower around 2 M. Surprisingly, the tri-
methoxylated chalcone-sulphonamide hybrids 8e and 9e were highly cytotoxic to fibroblasts with
LC50 values around 2 M and were devoid of anti-tuberculosis activity (Figure 4d), suggesting a
µM for most of them, with the
µ
µ
µ
µ
mammalian-specific mechanism of action. The selectivity index (SI) is calculated as the ratio
between LC50 and MIC values against M. tuberculosis. In order to progress to further in vitro and in
vivo anti-TB assays, an experimental compound must be more selective to kill mycobacteria than
mammalian cells and the SI value should be higher than 10 [36]. None of the synthetic compounds
showed SI higher than 1, indicating that the compounds were equally toxic to M. tuberculosis and
mammalian cells.
Table 5. Anti-TB and cytotoxic activities of the synthesized compounds 5/6, 8a-f and 9a-f.
Minimum inhibitory
concentration against
Cytotoxicity LC50 on
3T3 fibroblast cells (µM)
Selectivity index
ꢅꢆꢇꢈ
(ꢃꢄ =
)
Compound
ꢉꢄꢆꢊꢋꢌꢍꢎ
M. tuberculosis
H37Rv (µM)
>218
>130
16
>2.7 mM
>1.6 mM
7.7 ±1.5
4.1 ± 1.8
4.3 ± 0.2
96 ± 37
nd
nd
5
6
0.48
0.29
0.14
nd
8a
8b
8c
8d
8e
14
30
>144
>123
2.4 ± 0.7
nd

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8f
>138
42
132 ± 58
17 ± 3
nd
0.40
0.65
0.18
nd
9a
20
13 ± 5
9b
41
7.2 ± 0.8
6.5 ± 4.6
2.1 ± 1.1
7.1 ± 1.1
nd
9c
>100
>89
>97
0.36
nd
9d
nd
9e
9f
nd
nd
Isoniazid
Cycloheximide
< 2.2
nd
Figure 4. Anti-cancer and anti-tuberculosis activities of the synthetized compounds. a. Average viability of
the cancer cell lines and the leukemia K256 line, evaluated at a single concentration (10 µM) of the
compounds. b. Cytotoxicity of the most active 8b and 9b compounds on selected cell lines. c. Dose-response

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3T3 fibroblast inhibitory plot of the synthetized compounds and cycloheximide (CH). d. Comparison of
growth inhibition of M. tuberculosis H37Rv and cytotoxicity on 3T3 fibroblasts.
3 Conclusions
New sulfonamides 5/6 derived from the starting 4-methoxyacetophenone were synthesized by N-
sulfonation reactions of ammonia 3 and aminopyrimidinone 4 with sulfonyl chloride 2. Two series
of chalcones 8a-f and 9a-f were obtained through Claisen-Schmidt condensation of sulfonamides 5
and 6 with aromatic aldehydes 7a-f in basic media, with short reaction times and good yields.
Compounds 5/6, 8a-d, 8f, 9a-d and 9f were selected by NCI for single concentration assay.
Sulfonamides 5/6 showed low anticancer activity against the cancer cell lines evaluated, however,
their chalcone hybrids exhibited significant activity against Leukemia, Colon and CNS cancer cells.
The 4-chloro-substituted compounds 8b and 9b were the most active and passed to five-dose
screen. In this trial, compound 8b demonstrated remarkable growth inhibition and cytotoxic values,
and thus constitute a promising hit in the development of anticancer agents. The hybrids 8b and 9b
also showed potent growth inhibition (MIC
≤ 20 µM) of M. tuberculosis H37Rv, with no activity
against rapid-growing bacteria M. smegmatis or S. aureus. The evaluation of cytotoxicity against
3T3 mouse fibroblasts showed low selectivity, and thus these agents were considered equally toxic
to M. tuberculosis and mammalian cells. The chalcone-sulfonamide hybrids are appealing
cytotoxic agents with antituberculosis activity. They may be further optimized to target distinctively
cancer or mycobacterial cells.
4 Experimental
4.1 General

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All chemicals and solvents were purchased from Sigma-Aldrich unless stated otherwise. Melting
points were measured using a Stuart SMP10 melting point device and are uncorrected. ATR-FTIR
1
13
spectra were recorded on a Shimadzu IRAffinity–1. The H and C NMR spectra were run on a
BRUKER DPX 400 spectrometer operating at 400 and 100 MHz respectively, using DMSO–d6 and
CDCl3 as solvents and TMS as internal standard. The mass spectra were recorded on a
SHIMADZU–GCMS–QP2010 spectrometer operating at 70 eV. Elemental analyses were
performed on a Thermo Finnigan Flash EA1112 CHN elemental analyzer and the values are within
±0.4% of the theoretical values. The single cristal x-ray data were collected in a Diffractometer
Bruker D8 Venture at “Centro de Instrumentación Científico y Técnico”, (CICT) in “Universidad
de Jaén” (UJA). Thin layer chromatography (TLC) was performed on a 0.2 mm pre–coated
aluminum plates of silica gel (Merck 60 F254) and spots visualized with ultraviolet irradiation.
4.2 Chemistry
4.2.1 General procedure for the synthesis of benzenesulfonyl chloride 2
A mixture of chlorosulfonic acid (60 mmol) and thionyl chloride (20 mmol) was stirred in an ice
bath for 30 min. Subsequently, 4-methoxyacetophenone 1 (10 mmol) was added portionwise and
the reaction mixture was stirred at room temperature for 26 h. Then, it was quenched in ice-water
and the precipitate obtained was filtered and washed with water affording compound 2. The product
did not require further purification.

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5-Acetyl-2-methoxybenzenesulfonyl chloride (2)
Orange solid; 93 % yield; m.p. 104-105 °C. FTIR (ATR)
(C-O-C), 1167 (S=O). 1H NMR (400 MHz, CDCl3)
ν
(cm-1): 3108 (C-H), 1684 (C=O), 1254
δ
ppm 8.52 (d, J = 2.0 Hz, 1H, Ho), 8.31 (dd, J
= 8.8, 2.0 Hz, 1H, Hp), 7.21 (d, J = 8.8 Hz, 1H, Hm), 4.14 (s, J = 7.8 Hz, 3H, O-CH3), 2.61 (s, 3H,
CH3). 13C NMR (101 MHz, CDCl3)
δ
ppm 194.7 (C), 160.6 (C), 137.1 (CH), 131.8 (C), 130.7 (CH),
129.7 (C), 113.4 (CH), 57.3 (CH3), 26.5 (CH3). MS (EI, m/z (%)): 248/250 (M+/M+2+, 22/8), 233
(100), 119 (22), 69 (23), 43 (43). Anal. calcd. for C9H9ClO4S: C, 43.47; H, 3.65; S, 12.89. Found: C,
43.50; H, 3.75; S, 13.00.
4.2.2 General procedure for the synthesis of sulfonamide 5
A mixture of chloride 2 (0.31 mmol) and ammonia 3 (1 mL) in ethanol (1 mL) was stirred at room
temperature and the progress was monitored by TLC. After completion, the solid formed was
filtered and washed with ethanol and water, compound 5 did not required further purification.
5-Acetyl-2-methoxybenzenesulfonamide (5)
Pale pink solid; 82 % yield; m.p. 195-198 °C. FTIR (ATR)
ν
(cm-1): 3329 and 3208 (N-H), 3098 (C-
H), 1657 (C=O), 1159 (S=O). 1H NMR (400 MHz, DMSO-d6)
δ
ppm 8.29 (d, J = 2.3 Hz, 1H, Ho),
8.19 (dd, J = 8.7, 2.3 Hz, 1H, Hp), 7.32 (d, J = 8.7 Hz, 1H, Hm), 7.24 (s, 2H, NH2), 4.00 (s, 3H, O-
CH3), 2.56 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6)
δ
ppm 195.7 (C), 159.6 (C), 134.4 (CH),
131.4 (C), 128.8 (C), 127.7 (CH), 112.6 (CH), 56.7 (CH3), 26.4 (CH3). MS (EI, m/z (%)): 229 (M+,
22), 214 (100), 119 (25), 76 (57), 43 (87). Anal. calcd. for C9H11NO4S: C, 47.15; H, 4.84; N, 6.11;
S, 13.98. Found: C, 47.09; H, 4.93; N, 6.15; S, 14.01. Crystal data for 5 were deposited at CCDC

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with reference CCDC 1865855: Chemical formula C9H11NO4S, Mr 229.25, Triclinic, P-1, 100 K,
cell dimensions a, b, c (Å) 8.1560 (7), 8.4673 (7), 8.5234 (7) Å
α, β, γ (º) 80.690 (3), 74.603 (3),
61.443 (3). V (Å3) 498.03 (7), Z = 2, F(000)= 240, Dx (Mg m-3) = 1.53, Mo K
α, µ (mm-1)= 0.32,
pale pink crystal, size (mm) = 0.23 × 0.14 × 0.1. Data collection: Diffractometer Bruker D8 Venture
(APEX 3), Monochromator multilayer mirror, CCD rotation images, thick slices scans, Mo
φ
& θ
INCOATEC high brilliance microfocus sealed tube (λ= 0.71073 Å), multiscan absorption correction
(SADABS-2016/2), Tmin, Tmax 0.717, 0.746. No. of measured, independent and observed [I >
2
σ
(I)] reflections 25127, 2277, 2108, Rint= 0.028,
θ
values (°):
θmax = 27.5, θmin = 2.5; Range h =
-10
→10, k = -10
→
10, l = -11
→
11, Refinement on F2:R[F2 > 2
σ
(F2)]= 0.026, wR(F2)= 0.072,
S=1.104. No. of reflections 2277, No. of parameters 138, No. of restraints 0. Weighting scheme: w
= 1/σ2(Fo2) + (0.0324P)2 + 0.3110P where P = (Fo2 + 2Fc2)/3. ( ) < 0.002, ∆ρmax, ∆ρmin (e Å-3)
∆/σ
0.32, −0.5.
4.2.3 General procedure for the synthesis of sulfonamide 6
To a mixture of chloride 2 (0.31 mmol) and aminopyrimidinone 4 (0.26 mmol) in ethanol (1.5 mL),
TEA (0.15 mL) was added. The reaction mixture was stirred at room temperature and the progress
was monitored by TLC. After completion, a further amount of TEA was added until pH = 7 and the
solid thus formed was filtered and washed with ethanol to provide compound 6.
5-Acetyl-N-(4-amino-2-(methylthio)-6-oxo-1,6-dihydropyrimidin-5-yl)-2
methoxybenzenesulfonamide (6)
Pale pink solid; 81 % yield; m.p. 235-238 °C. FTIR (ATR)
ν
(cm-1): 3470 and 3343 (N-H), 3109 (C-
1
H), 1676 and 1643 (C=O), 1151 (S=O). H NMR (400 MHz, DMSO-d6)
δ
ppm 11.68 (s, 1H, NH-

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CO), 8.39 (s, 1H, NH-C5), 8.17 – 8.11 (m, 2H, Ho and Hp), 7.25 (d, J = 8.4 Hz, 1H, Hm), 6.39 (s, 2H,
NH2-C6), 3.99 (s, 3H, OCH3), 2.52 (s, 3H, CH3), 2.41 (s, 3H, SCH3). 13C NMR (101 MHz, DMSO-
d6) δ ppm 195.7 (C), 161.3 (C), 161.0 (C), 159.8 (C), 159.8 (C), 134.9 (C), 129.5 (CH), 128.9 (CH),
128.1 (C), 112.7 (CH), 92.6 (C), 56.8 (CH3), 26.4 (CH3), 12.8 (CH3). MS (EI, m/z (%)): 384 (M+,
32), 199 (38), 171 (100), 135 (100), 70 (61). Anal. calcd. for C14H16N4O5S2: C, 43.74; H, 4.20; N,
14.57; S, 16.68. Found: C, 43.65; H, 4.15; N, 14.60; S, 16.45.
4.2.4 General procedure for the synthesis of chalcone-hybrids 8a-f
To a mixture of sulfonamide 5 (0.20 mmol) and the corresponding aldehyde 7a-f (0.24 mmol) in
ethanol (1.5 mL), it was added 2 drops of 50% (w/v) NaOH solution. The reaction mixture was
stirred at room temperature for 2-3.7 h. The sodium salt formed was filtered and washed with
ethanol. Then, it was poured into water and neutralized by adding 10% (w/v) HCl aqueous solution.
The precipitate was filtered and washed with water to afford the corresponding compounds 8a-f.
5-Cinnamoyl-2-methoxybenzenesulfonamide (8a)
Beige solid; 57 % yield; m.p. 191-193 °C. FTIR (ATR)
ν
(cm-1): 3352 and 3260 (N-H), 2985 (C-H),
1
1656 (C=O), 1593 (C=C), 1155 (S=O). H NMR (400 MHz, DMSO-d6)
δ
ppm 8.48 (d, J = 8.7, 1.9
Hz, 1H, Hp), 8.45 (s, J = 1.9 Hz, 1H, Ho), 7.97–7.87 (m, 3H, Hα and Ho’), 7.76 (d, J = 15.6 Hz, 1H,
Hβ), 7.50–7.44 (m, 3H, Hm’ and Hp’), 7.37 (d, J = 8.7 Hz, 1H, Hm), 7.28 (s, 2H, NH2), 4.03 (s, 3H,
OCH3). 13C NMR (101 MHz, DMSO-d6)
δ ppm 187.0 (C), 159.7 (C), 144.1 (CH), 134.8 (CH),
134.6 (C), 131.8 (C), 130.7 (CH), 129.4 (C), 129.0 (CH), 129.0 (CH), 128.2 (CH), 121.6 (CH),
112.7 (CH), 56.8 (CH3). MS (EI, m/z (%)): 317 (M+, 14), 316 (23), 81 (45), 69 (100), 55 (42), 41
(42). Anal. calcd. for C16H15NO4S: C, 60.55; H, 4.76; N, 4.41; S, 10.10. Found: C, 60.50; H, 4.80;
N, 4.52; S, 10.23.

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(E)-5-(3-(4-Chlorophenyl)acryloyl)-2-methoxybenzenesulfonamide (8b)
White solid; 65 % yield; m.p. 230-232 °C. FTIR (ATR)
ν
(cm-1): 3364 and 3219 (N-H), 3090 (C-H),
ppm 8.58–8.33 (m, 2H,
1
1655 (C=O), 1591 (C=C), 1155 (S=O). H NMR (400 MHz, DMSO-d6)
δ
Ho and Hp), 7.98-7.92 (m, 3H, Hα and Ho’), 7.74 (d, J = 15.5 Hz, 1H, Hβ), 7.53 (d, J = 7.6 Hz, 2H,
13
Hm’), 7.36 (d, J = 8.4 Hz, 1H, Hm), 7.28 (s, 2H, NH2), 4.03 (s, 3H, OCH3). C NMR (101 MHz,
DMSO-d6)
δ ppm 186.8 (C), 159.7 (C), 142.6 (CH), 135.1 (C), 134.8 (CH), 133.6 (C), 131.8 (C),
130.6 (CH), 129.3 (C), 128.9 (CH), 128.2 (CH), 122.2 (CH), 112.3 (CH), 56.8 (CH3). MS (EI, m/z
(%)): 351/353 (M+/M+2+, 100/41), 316 (49), 271 (63), 214 (36), 165 (19). Anal. calcd. for
C16H14ClNO4S: C, 54.63; H, 4.01; N, 3.98; S, 9.11. Found: C, 54.59; H, 3.99; N, 4.02; S, 9.04.
(E)-2-Methoxy-5-(3-(p-tolyl)acryloyl)benzenesulfonamide (8c)
Beige solid; 82 % yield; m.p. 205-207 °C. FTIR (ATR)
1653 (C=O), 1595 (C=C), 1153 (S=O). 1H NMR (400 MHz, DMSO-d6)
ν
(cm-1): 3381 and 3265 (N-H), 2955 (C-H),
δ
ppm 8.47 (dd, J = 8.6, 2.2
Hz, 1H, Hp), 8.44 (d, J = 2.2 Hz, 1H, Ho), 7.87 (d, J = 15.6 Hz, 1H, Hα), 7.78 (d, J = 8.0 Hz, 2H,
Ho’), 7.73 (d, J = 15.6 Hz, 1H, Hβ), 7.35 (d, J = 8.6 Hz, 1H, Hm), 7.29-7.27 (m, 4H, Hm’ and NH2),
13
4.03 (s, 3H, OCH3), 2.35 (s, 3H, CH3). C NMR (101 MHz, DMSO-d6)
δ ppm 186.8 (C), 159.6
(C), 144.1 (CH), 140.8 (C), 134.6 (CH), 131.9 (C), 131.7 (C), 129.5 (C), 129.5 (CH), 128.9 (CH),
128.1 (CH), 120.4 (CH), 112.6 (CH), 56.7 (CH3), 21.1 (CH3). MS (EI, m/z (%)): 331 (M+, 36), 330
(43), 316 (100), 251 (14), 214 (8). Anal. calcd. for C17H17NO4S: C, 61.62; H, 5.17; N, 4.23; S, 9.67.
Found: C, 61.60; H, 5.22; N, 4.23; S, 9.46. Crystal data for 8c were deposited at CCDC with
reference CCDC 1865863: Chemical formula C17H17NO4S, Mr 331.38, Orthorhombic, P212121,
100 K, cell dimensions a, b, c (Å) 10.4960 (8), 12.2051 (8), 12.5300 (8),
α,
β,
γ
(º) 90, 90, 90. V
(Å3) 1605.15 (19), Z = 4, F(000)= 696.0, Dx (Mg m-3) = 1.371, Mo K
α, µ (mm-1)= 0.221, colorless
block crystal, size (mm) = 0.26 × 0.23 × 0.1. Data collection: Diffractometer Bruker D8 Venture
(APEX 3), Monochromator multilayer mirror, CCD rotation images, thick slices scans, Mo
INCOATEC high brilliance microfocus sealed tube (λ= 0.71073 Å), multiscan absorption
φ
& θ

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correction (SADABS-2016/2), Tmin, Tmax 0.657, 0.746. No. of measured, independent and
observed [I > 2
σ
(I)] reflections 24961, 3698, 3213, Rint= 0.059,
16, Refinement on F2:R[F2 > 2
wR(F2)= 0.071, S=1.073. No. of reflections 3698, No. of parameters 210, No. of restraints 0.
θ
values (°):
θ
max = 27.5,
θmin =
2.3; Range h = -13
→
13, k = -15
→
15, l = -16
→
σ
(F2)]= 0.035,
Weighting scheme: w = 1/σ2(Fo2) + (0. 0358 P)2 + 0.2642 P where P = (Fo2 + 2Fc2)/3. (
0.003, ∆ρmax, ∆ρmin (e Å-3) 0.18, −0.38. Flack parameter x -0.01(3).
∆/σ) <
(E)-2-Methoxy-5-(3-(4-methoxyphenyl)acryloyl)benzenesulfonamide (8d)
Pale yellow solid; 83 % yield; m.p. 214-216 °C. FTIR (ATR)
(C-H), 1655 (C=O), 1597 (C=C), 1153 (S=O). 1H NMR (400 MHz, DMSO-d6)
ν
(cm-1): 3375 and 3265 (N-H), 3073
δ
ppm 8.47-8.43 (m,
2H, Ho and Hp), 7.85 (d, J = 8.8 Hz, 2H, Ho’), 7.80 (d, J = 15.5 Hz, 1H, Hα), 7.73 (d, J = 15.5 Hz,
1H, Hβ), 7.35 (d, J = 8.6 Hz, 1H, Hm), 7.27 (s, 2H, NH2), 7.03 (d, J = 8.8 Hz, 2H, Hm’), 4.02 (s, 3H,
OCH3), 3.82 (s, 3H, p’-OCH3). 13C NMR (101 MHz, DMSO-d6)
δ ppm 186.7 (C), 161.4 (C), 159.5
(C), 144.1 (CH), 134.6 (CH), 131.7 (C), 130.9 (CH), 129.6 (C), 128.1 (CH), 127.3 (C), 119.0 (CH),
114.4 (CH), 112.6 (CH), 56.7 (CH3), 55.4 (CH3). MS (EI, m/z (%)): 347 (M+, 100), 316 (23), 267
(18), 161 (25), 133 (18). Anal. calcd. for C17H17NO5S: C, 58.78; H, 4.93; N, 4.03; S, 9.23. Found:
C, 58.88; H, 4.90; N, 3.98; S, 8.96.
(E)-2-Methoxy-5-(3-(3,4,5-trimethoxyphenyl)acryloyl)benzenesulfonamide (8e)
Pale yellow solid. 75 % yield; m.p. 204-208 °C. FTIR (ATR)
(C-H), 1659 (C=O), 1595 (C=C), 1150 (S=O). 1H NMR (400 MHz, DMSO-d6)
ν
(cm-1): 3366 and 3260 (N-H), 3086
δ
ppm 8.49 (dd, J =
8.7, 2.2 Hz, 1H, Hp), 8.43 (d, J = 2.2 Hz, 1H, Ho), 7.87 (d, J = 15.5 Hz, 1H, Hα), 7.71 (d, J = 15.5
Hz, 1H, Hβ), 7.36 (d, J = 8.7 Hz, 1H, Hm), 7.27 (s, 2H, NH2), 7.23 (s, 2H, Ho’), 4.03 (s, 3H, OCH3),
13
3.87 (s, 6H, m’-OCH3), 3.72 (s, 3H, p’-OCH3). C NMR (101 MHz, DMSO-d6)
δ ppm 186.9 (C),
159.6 (C), 153.1 (C), 144.6 (CH), 139.9 (C), 134.8 (CH), 131.8 (C), 130.2 (C), 129.5 (C), 128.2
(CH), 120.8 (CH), 112.5 (CH), 106.7 (CH), 60.2 (CH3), 56.8 (CH3), 56.2 (CH3). MS (EI, m/z (%)):

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407 (M+, 100), 376 (48), 364 (11), 214 (14). Anal. calcd. for C19H21NO7S: C, 56.01; H, 5.20; N,
3.44; S, 7.87. Found: C, 56.00; H, 5.23; N, 3.51; S, 7.79.
(E)-5-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-2-methoxybenzenesulfonamide (8f)
Yellow solid. 84 % yield; m.p. 272-275 °C. FTIR (ATR)
ν
(cm-1): 3289 and 3182 (N-H), 3113 (C-
1
H), 1641 (C=O), 1595 (C=C), 1250 (C-O-C), 1157 (S=O). H NMR (400 MHz, DMSO-d6)
δ ppm
8.48 (d, J = 8.7 Hz, 1H, Hp), 8.44 (s, 1H, Ho), 7.81 (d, J = 15.4 Hz, 1H, Hα), 7.69 (d, J = 15.4 Hz,
1H, Hβ), 7.66 (s, 1H, Ho’’), 7.36-7.32 (m, 2H, Ho’ and Hm’), 7.26 (s, 2H, NH2), 6.99 (d, J = 8.7 Hz,
13
1H, Hm), 6.11 (s, 2H, OCH2O), 4.02 (s, 3H, OCH3). C NMR (101 MHz, DMSO-d6)
δ ppm 186.7
(C), 159.5 (C), 149.6 (C), 148.1 (C), 144.1 (CH), 134.6 (CH), 131.7 (C), 129.6 (C), 129.2 (C),
128.1 (CH), 126.0 (CH), 119.4 (CH), 112.5 (CH), 108.6 (CH), 107.0 (CH), 101.7 (CH2), 56.7
(CH3). MS (EI, m/z (%)): 361 (M+, 76), 236 (38), 83 (60), 57 (100), 43 (76). Anal. calcd. for
C17H15NO6S: C, 56.50; H, 4.18; N, 3.88; S, 8.87. Found: C, 56.45; H, 4.18; N, 3.91; S, 8.65.
4.2.5 General procedure for the synthesis of chalcone-hybrids 9a-f
To a mixture of sulfonamide 6 (0.20 mmol) and the corresponding aldehyde 7a-f (0.24 mmol) in
ethanol (1.5 mL), it was added 2 drops of 30% (w/v) NaOH solution. The reaction mixture was
stirred at room temperature until the reaction was finished by TLC. Then, 10% (w/v) HCl was
added to reach neutral pH (7.0) and the precipitate was filtered and washed with water and ethanol.
The purification of compounds 9a, 9b, 9e and 9f was carried out by recrystallization from ethanol,
while compounds 9c and 9d were purified by CC using silica gel as the stationary phase and a
mixture AcOEt: EtOH (15:1) as mobile phase.

ACCEPTED MANUSCRIPT
N-(4-Amino-2-(methylthio)-6-oxo-1,6-dihydropyrimidin-5-yl)-5-cinnamoyl-2-
methoxybenzenesulfonamide (9a)
Beige solid; 61 % yield; m.p. 239-240 °C. FTIR (ATR)
ν
(cm-1): 3474, 3362 and 3298 (N-H), 3057
1
(C-H), 1661 and 1645 (C=O), 1616 (C=C), 1148 (S=O). H NMR (400 MHz, DMSO-d6)
δ ppm
11.73 (s, 1H, NH-CO), 8.49 – 8.39 (m, 2H, Hp and NH-C5), 8.34 (d, J = 2.3 Hz, 1H, Ho), 7.97 –
7.83 (m, 3H, Hα and Ho’), 7.72 (d, J = 15.6 Hz, 1H, Hβ), 7.51 – 7.42 (m, 3H, Hm’ and Hp’), 7.30 (d, J
13
= 8.8 Hz, 1H, Hm), 6.41 (s, 2H, NH2-C6), 4.03 (s, 3H, OCH3), 2.41 (s, 3H, SCH3). C NMR (101
MHz, DMSO-d6)
δ ppm 186.9 (C), 161.4 (C), 161.1 (C), 144.0 (CH), 135.1 (CH), 134.7 (C), 130.6
(CH), 129.8 (C), 129.5 (CH), 128.9 (CH, CH), 128.7 (C), 127.7 (C), 123.8 (C), 121.6 (CH), 112.8
(CH), 92.6 (C), 56.9 (CH3), 12.8 (CH3). MS (EI, m/z (%)): 472 (M+, 0.6), 238 (100), 135 (79), 86
(56), 77 (70). Anal. calcd. for C21H20N4O5S2: C, 53.38; H, 4.27; N, 11.86; S, 13.57. Found: C,
53.40; H, 4.27; N, 11.89; S, 13.36.
(E)-N-(4-Amino-2-(methylthio)-6-oxo-1,6-dihydropyrimidin-5-yl)-5-(3-(4-chlorophenyl)acryloyl)-
2-methoxybenzenesulfonamide (9b)
Pale yellow solid; 56 % yield; m.p. 242-244 °C. FTIR (ATR)
(C-H), 1643 (C=O), 1595 (C=C), 1155 (S=O). 1H NMR (400 MHz, DMSO-d6)
ν
(cm-1): 3478 and 3364 (N-H), 3053
δ
ppm 11.73 (s, 1H,
NH-CO), 8.43 (dd, J = 8.6, 2.2 Hz, 1H, Hp), 8.35 (d, J = 2.2 Hz, 1H, Ho), 7.97-7.92 (m, 3H, Hα and
Hm’), 7.71 (d, J = 15.6 Hz, 1H, Hβ), 7.52 (d, J = 8.5 Hz, 2H, Ho’), 7.30 (d, J = 8.6 Hz, 1H, Hm), 6.42
13
(s, 2H, NH2-C6), 4.03 (s, 3H, OCH3), 2.41 (s, 3H, SCH3). C NMR (101 MHz, DMSO-d6)
δ ppm
186.7 (C), 161.5 (C), 159.3 (C), 158.2 (C), 155.5 (C), 142.4 (CH), 135.1 (CH), 135.0 (C), 133.6
(C), 130.6 (CH), 129.8 (C), 129.5 (CH), 128.9 (CH), 128.6 (C), 122.3 (CH), 112.7 (CH), 92.5 (C),
56.9 (CH3), 12.7 (CH3). MS (EI, m/z (%)): 507 (M+, 2), 236 (28), 83 (68), 57 (100), 43 (89). Anal.
calcd. for C21H19ClN4O5S2: C, 49.75; H, 3.78; N, 11.05; S, 12.65. Found: C, 49.82; H, 3.70; N,
10.99; S, 12.43.

ACCEPTED MANUSCRIPT
(E)-N-(4-Amino-2-(methylthio)-6-oxo-1,6-dihydropyrimidin-5-yl)-2-methoxy-5-(3-(p-
tolyl)acryloyl)benzenesulfonamide (9c)
White solid; 62 % yield; m.p. 237-239 °C. FTIR (ATR)
ν
(cm-1): 3478 and 3362 (N-H), 2851 (C-H),
ppm 11.74 (s, 1H, NH-
1
1649 (C=O), 1597 (C=C), 1153 (S=O). H NMR (400 MHz, DMSO-d6)
δ
CO), 8.45 – 8.37 (m, 2H, Hp and NH-C5), 8.32 (d, J = 2.2 Hz, 1H, Ho), 7.85 (d, J = 15.5 Hz, 1H,
Hα), 7.77 (d, J = 8.1 Hz, 2H, Ho’), 7.69 (d, J = 15.5 Hz, 1H, Hβ), 7.30-7.26 (m, 3H, Hm and Hm’),
13
6.40 (s, 2H, NH2-C6), 4.02 (s, 3H, OCH3), 2.41 (s, 3H, SCH3), 2.35 (s, 3H, CH3). C NMR (101
MHz, DMSO-d6)
δ ppm 187.0 (C), 161.4 (C), 161.2 (C), 144.2 (CH), 140.9 (C), 136.0 (C), 135.2
(CH), 132.0 (C), 129.8 (C), 129.7 (CH), 129.5 (CH), 129.3 (CH), 129.0 (C), 128.9 (C), 120.6 (CH),
112.8 (CH), 92.7 (C), 56.9 (CH3), 21.2 (CH3), 12.8 (CH3). MS (EI, m/z (%)): 486 (M+, 0.5), 252
(85), 237 (100), 135 (72), 43 (59). Anal. calcd. for C22H22N4O5S2: C, 54.31; H, 4.56; N, 11.52; S,
13.18. Found: C, 54.29; H, 4.55; N, 11.61; S, 12.98.
(E)-N-(4-Amino-2-(methylthio)-6-oxo-1,6-dihydropyrimidin-5-yl)-2-methoxy-5-(3-(4-
methoxyphenyl)acryloyl)benzenesulfonamide (9d)
Pale yellow solid; 60 % yield; m.p. 230-232 °C. FTIR (ATR)
(C-H), 1645 (C=O), 1591 (C=C), 1153 (S=O). 1H NMR (400 MHz, DMSO-d6)
ν
(cm-1): 3462 and 3335 (N-H), 3055
δ
ppm 11.73 (s, 1H,
NH-CO), 8.47 – 8.37 (m, 2H, Hp and NH-C5), 8.32 (d, J = 2.0 Hz, 1H, Ho), 7.84 (d, J = 8.6 Hz, 2H,
Ho’), 7.77 (d, J = 15.5 Hz, 1H, Hα), 7.69 (d, J = 15.5 Hz, 1H, Hβ), 7.29 (d, J = 8.8 Hz, 1H, Hm), 7.01
(d, J = 8.6 Hz, 2H, Hm’), 6.41 (s, 2H, NH2-C6), 4.02 (s, 3H, OCH3), 3.82 (s, 3H, p’-OCH3), 2.41 (s,
3H, SCH3). 13C NMR (101 MHz, DMSO-d6)
δ ppm 187.5 (C), 161.9 (C), 161.6 (C), 161.5 (C),
160.3 (C), 144.6 (CH), 136.4 (C), 135.5 (CH), 131.2 (CH), 129.8 (CH), 129.3 (C), 127.6 (C), 119.3
(CH), 114.9 (CH), 113.2 (CH), 93.0 (C), 63.6 (C), 57.2 (CH3), 55.8 (CH3), 13.2 (CH3). MS (EI, m/z
(%)): 268 (80), 135 (50), 71 (63), 57 (99), 43 (100). Anal. calcd. for C22H22N4O6S2: C, 52.58; H,
4.41; N, 11.15; S, 12.76. Found: C, 52.65; H, 4.41; N, 11.04; S, 12.90.

ACCEPTED MANUSCRIPT
(E)-N-(4-Amino-2-(methylthio)-6-oxo-1,6-dihydropyrimidin-5-yl)-2-methoxy-5-(3-(3,4,5-
trimethoxyphenyl))acryloyl)benzenesulfonamide (9e)
Yellow solid; 71 % yield; m.p. 241-243 °C. FTIR (ATR)
ν
(cm-1): 3472, 3362 and 3310 (N-H), 2947
1
(C-H), 1653 (C=O), 1603 (C=C), 1273 (C-O-C), 1128 (S=O). H NMR (400 MHz, DMSO-d6)
δ
ppm 11.73 (s, 1H, NH-CO), 8.40 (d, J = 31.3 Hz, 3H, Hp, Ho and NH-C5), 7.89 (d, J = 15.2 Hz, 1H,
Hα), 7.69 (d, J = 15.2 Hz, 1H, Hβ), 7.30 (d, J = 7.9 Hz, 1H, Hm), 7.23 (s, 2H, Ho’), 6.41 (s, 2H, NH2-
13
C6), 4.03 (s, 3H, OCH3), 3.86 (s, 6H, m’-OCH3), 3.72 (s, 3H, p’-OCH3), 2.42 (s, 3H, SCH3). C
NMR (101 MHz, DMSO-d6)
δ ppm 187.3 (C), 161.6 (C), 161.4 (C), 153.4 (C), 144.8 (CH), 140.1
(C), 136.3 (C), 135.6 (CH), 130.5 (C), 130.0 (C), 129.7 (CH), 129.1 (C), 127.4 (C), 121.1 (CH),
113.0 (CH), 106.8 (CH), 92.9 (C), 60.5 (CH3), 57.2 (CH3), 56.5 (CH3), 13.1 (CH3). MS (EI, m/z
(%)): 563 (M+, 1), 368 (10), 83 (75), 57 (100), 43 (82). Anal. calcd. for C24H26N4O8S2: C, 51.24; H,
4.66; N, 9.96; S, 11.40. Found: C, 51.11; H, 4.72; N, 9.98; S, 11.53.
(E)-N-(4-Amino-2-(methylthio)-6-oxo-1,6-dihydropyrimidin-5-yl)-5-(3-(benzo[d][1,3]dioxol-5)-
yl)acryloyl)-2-methoxybenzenesulfonamide (9f)
Yellow solid; 65 % yield; m.p. 198-200 °C. FTIR (ATR)
ν
(cm-1): 3607, 3520, 3456 and 3308 (N-
1
H), 3152 (C-H), 1665 and 1635 (C=O), 1595 (C=C), 1227 (C-O-C), 1157 (S=O). H NMR (400
MHz, DMSO-d6) δ ppm 11.73 (s, 1H, NH-CO), 8.43 (dd, J = 8.8, 2.3 Hz, 1H, Hp), 8.40 (s, 1H, NH-
C5), 8.32 (d, J = 2.3 Hz, 1H, Ho), 7.80 (d, J = 15.5 Hz, 1H, Hα), 7.68-7.64 (m, 2H, Hβ and Ho’’), 7.33
(dd, J = 8.0, 1.5 Hz, 1H, Ho’), 7.28 (d, J = 8.8 Hz, 1H, Hm), 6.99 (d, J = 8.0 Hz, 1H, Hm’), 6.40 (s,
13
2H, NH2-C6), 6.10 (s, 2H, OCH2O), 4.02 (s, 3H, OCH3), 2.41 (s, 3H, SCH3). C NMR (101 MHz,
DMSO-d6) δ ppm 186.6 (C), 161.6 (C), 161.3 (C), 159.8 (C), 149.6 (C), 149.0 (C), 148.4 (C), 148.1
(C), 144.0 (CH), 135.9 (CH), 129.4 (CH), 129.2 (C), 128.9 (C), 126.0 (CH), 119.5 (CH), 112.6
(CH), 108.5 (CH), 107.1 (CH), 101.6 (CH2), 92.6 (C), 56.8 (CH3), 12.7 (CH3). MS (EI, m/z (%)):