Synthesis, structural characterization, and cytotoxic evaluation of chalcone derivatives

Article abstract of DOI:10.1007/s00044-019-02434-1

Chalcones containing amino or acetamide groups on ring A and electron donating/withdrawing groups on ring B have been shown to have great cytotoxic potential against human cancer cell lines. In this work, a series of twenty chalcones, including nine 1-(4′-aminophenyl)-3-(substituted aryl)-2-propen-1-ones (1–9), nine 1-(4′-acetamidophenyl)-3-(substituted aryl)-2-propen-1-ones (1a–9a), and two 1-(3′-methoxy-4′-hydroxyphenyl)-3-(substituted aryl)-2-propen-1-ones (10, 11), were synthesized and submitted for initial biological screening using HCT-116 cells. Among the evaluated compounds, chalcone 6a showed strong and selective activity against HCT-116 cells (IC₅₀ = 2.37 ± 0.73 μM). The preliminary structure–activity relationship analysis indicated that the cytotoxic effect of these compounds might be attributed to the combined effect of two electron withdrawing groups: the nitro group (NO₂) at the meta-position of ring B and the acetyl group at the para-position of ring A. Moreover, chalcone 6a was able to induce G2/M cell cycle arrest and apoptosis at a concentration of 10 μM after 24 h of incubation. These data reinforce that compound 6a could be a promising lead compound for the future exploration of selective anti-colon carcinoma cancer agents.

Full text of DOI:10.1007/s00044-019-02434-1

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02434-1
ORIGINAL RESEARCH
Synthesis, structural characterization, and cytotoxic evaluation
of chalcone derivatives
Paulo N. Bandeira1 Telma L. G. Lemos2 Hélcio S. Santos1,3 Mylena C. S. de Carvalho4 Daniel P. Pinheiro4
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Manoel O. de Moraes Filho4 Cláudia Pessoa4,5 Francisco W. A. Barros-Nepomuceno6 Tigressa H. S. Rodrigues1
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Paulo R. V. Ribeiro7 Herbert S. Magalhães2 Alexandre M. R. Teixeira
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Received: 16 March 2019 / Accepted: 28 August 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Chalcones containing amino or acetamide groups on ring A and electron donating/withdrawing groups on ring B have been
shown to have great cytotoxic potential against human cancer cell lines. In this work, a series of twenty chalcones, including
nine 1-(4′-aminophenyl)-3-(substituted aryl)-2-propen-1-ones (1–9), nine 1-(4′-acetamidophenyl)-3-(substituted aryl)-2-
propen-1-ones (1a–9a), and two 1-(3′-methoxy-4′-hydroxyphenyl)-3-(substituted aryl)-2-propen-1-ones (10, 11), were
synthesized and submitted for initial biological screening using HCT-116 cells. Among the evaluated compounds, chalcone
6a showed strong and selective activity against HCT-116 cells (IC50 = 2.37 0.73 µM). The preliminary structure–activity
relationship analysis indicated that the cytotoxic effect of these compounds might be attributed to the combined effect of two
electron withdrawing groups: the nitro group (NO2) at the meta-position of ring B and the acetyl group at the para-position
of ring A. Moreover, chalcone 6a was able to induce G2/M cell cycle arrest and apoptosis at a concentration of 10 µM after
24 h of incubation. These data reinforce that compound 6a could be a promising lead compound for the future exploration of
selective anti-colon carcinoma cancer agents.
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Keywords Synthesis NMR Infrared Chalcone 4′-acetamidochalcones Cytotoxic activity
Introduction
Chalcones are open chain flavonoids that are characterized
by two aromatic rings joined by a three-carbon α,
β-unsaturated carbonyl system (Fig. 1). These compounds
can be isolated from natural sources due to their widespread
distribution in fruits, vegetables, and tea or they can be
synthesized by chemical processes (Abbas et al. 2014; Di
Carlo et al. 1999).
Over the last several years, chalcones have instigated the
interest of chemical and pharmacological researchers due to
their simple chemical structure and varied biological
activities. The chalcone biological activity spectrum
includes antinociceptive, anti-inflammatory (Padaratz et al.
2009; Corrêa et al. 2001; De Campos-Buzzi et al. 2007;
Nowakowska 2007; de Campos-Buzzi et al. 2006), anti-
tumor (Zingales and Moore 2016; Cabrera et al. 2007),
antibacterial, antifungal (Karaman et al. 2010), antil-
eishmanial (Boeck et al. 2006), and antioxidant activity
(Prasad et al. 2013). This wide range of activity is mainly
due to the numerous substitution possibilities on the chal-
cone aromatic rings. Moreover, the synthesis of chalcones
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02434-1) contains supplementary
material, which is available to authorized users.
* Alexandre M. R. Teixeira
amrteixeira@gmail.com
1
Curso de Química, Universidade Estadual Vale do Acaraú,
Sobral, CE, Brazil
2
Departamento de Química Orgânica e Inorgânica, Universidade
Federal do Ceará, Fortaleza, CE, Brazil
3
Departamento de Química Biológica, Universidade Regional do
Cariri, Crato, CE, Brazil
4
Núcleo de Pesquisa e Desenvolvimento de Medicamentos,
Universidade Federal do Ceará, Fortaleza, CE, Brazil
5
Fundação Oswaldo Cruz, FIOCRUZ-CE, Eusebio, CE, Brazil
6
Instituto de Ciências da Saúde, Universidade da Integração
Internacional da Lusofonia Afro-Brasileira, Acarape, CE, Brazil
7
Laboratório Multiusuário de Química de Produtos Naturais,
Embrapa Agroindústria Tropical, Fortaleza, CE, Brazil

Medicinal Chemistry Research
based on the Claisen–Schmidt condensation reaction allows
a vast number of compounds to be obtained, providing the
desired structural variety (Ducki et al. 1998).
This study describes the synthesis and characterization of
twenty chalcones, including nine 4′-aminochalcones, nine
4′-acetamidechalcones, and two 3′-hydroxy-4′-methox-
ychalcones. Figure 2 shows the molecular structures of
these twenty chalcones. Among these compounds, a new
chalcone (11) is being reported for the first time. After the
initial cytotoxic screening for all synthesized compounds,
we explored the cytotoxic effect of chalcone 6a against a
panel of the tumor and nontumor cell lines. In addition, we
evaluated the induction of apoptosis and cell cycle pro-
gression in HCT-116 cells incubated with chalcone 6a for
24 h.
There is a continuous scientific effort that has been
expended on the design and development of anticancer
chalcones that has resulted in many novels and chemically
diverse chalcones with therapeutic potential against differ-
ent types of cancer (Karthikeyan et al. 2015). In this con-
text, chalcones are able to induce cell cycle arrest and
apoptosis in various human cancer cell lines. These abilities
can be attributed, in part, by tubulin polymerization inhi-
bition and/or the capacity to bind at the DNA minor groove
of neoplastic cells. The antimetastatic potential and anti-
angiogenic actions are related to flavokawain B, a chalcone
isolated from the root extracts of kava-kava plants (Linda-
mulage et al. 2017; Hassan et al. 2017; Shankaraiah et al.
2017; Rossette et al. 2017).
Materials and methods
Synthesis of the chalcones
Recent studies have shown the cytotoxic anticancer
potential of chalcones that contain an amino or acetamide
group on ring A (Santos et al. 2017; Jardim et al. 2015;
Tristão et al. 2012.). Chalcones with hydroxyl, methoxy,
and halogen groups on the B ring have also presented
effective cytotoxic potential against human cancer cell lines
(Karthikeyan et al. 2015). Based on this information,
chalcones containing amino or acetamide groups on ring A
and electron donating/withdrawing groups on ring B were
synthesized, and their cytotoxicity was evaluated in the
present work.
The description of the procedure of the synthesis of the
twenty chalcones are shown in Fig. 3. The 4′-ami-
nochalcones (1–9) and 3′-methoxy-4′-hydroxy chalcones
(10, 11) were synthesized by a Claisen–Schmidt con-
densation reaction in basic medium (Bhat et al. 2005). An
ethanol solution of 4-aminoacetophenone (2 mmol) and 3-
methoxy-4-hydroxyacetophenone (2 mmol) were added to a
solution of benzaldehyde and the derivatives (2 mmol),
followed by the addition of ten drops of 50% w/v aq. NaOH
with stirring for 48 h. The solid that formed was filtered
under reduced pressure, washed with cold water, and ana-
lyzed by TLC, giving yields of 25.65–69.85%. The 4′-
acetamidechalcones (1a–9a) were prepared by the acetyla-
tion reaction of the 4′-aminochalcones (2 mmol) with acetic
anhydride (2 mmol) in a buffered medium (5 mL) at pH =
5.0 with AcOH/AcONa (de Campos-Buzzi et al. 2007). The
reaction mixture was refluxed for 1 h. The product was
filtered under vacuum and washed with cold water, giving
yields of 22.27–88.92%.
The structural data of the twenty chalcones which were
synthesized in this work are given below.
Fig. 1 Fundamental structure of a chalcone
Fig. 2 Molecular structures of
the synthesized chalcones

Medicinal Chemistry Research
Fig. 3 Description of the
procedure of the synthesis of the
chalcones (1–11) and (1a–9a).
a NaOHaq. 50% w /v, ethanol, r.
t., 48 h. b (CH3CO)2, (AcOH
/AcONa), r.t. *Novel chalcone
(2E)-1-(4′-aminophenyl) -3-(phenyl)-prop-2-en-1-one
(1)
(CD3OD, 300 MHz): δ = 7.76 (2H, dd, J = 8.8, 2.1 Hz, H-
2, H-6), 7.14 (2H, t, J = 8,8 Hz, H-3, H-5), 7.91 (2H, d, J =
6.93 Hz H-2′,H-6′), 6.73 (2H, d, J = 8.8. Hz, H-3′, H-5′),
7.67 (1H, d, J = 12.1 Hz, H-α), 7.74 (1H, d, J = 14.2 Hz,
H-β). 13C-NMR (CD3OD, 75 MHz): δ = 132.6 (C, C-1),
132.6 (CH, C-2, C-6), 116.1 (CH, C-3,C-5), 163.8 (C, C-4),
128.1 (C, C-1′), 131.8 (CH, C-2′, C-6′), 114.9 (CH, C-3′,C-
5′), 155.0 (C, C-4′), 123.2 (CH, C-α), 142.9 (CH, C-β),
190.1 (C, COBz); EIMS m/z (M+. 241), calcd for
C15H12NOF/241.
Yellow amorphous powder (25.65 %), m.p. 109.3–109.9 °C;
1
FT-IR (KBr) νmax 3522, 3434, 1623, 1578, 1554 cm−1; H-
NMR (CD3OD, 300 MHz): δ = 7.40–7.42 (3H, m, H-3,H-5,
H-4), 7.93 (2H, d, J = 8.7 Hz H-2′, H-6′), 6.73 (2H, d, J =
8.7 Hz, H-3′, H-5′), 7.69–7.72 (4H, m, H-2/H-6, H-α, H-β).
13C-NMR (CD3OD, 75 MHz): δ = 136.8 (C, C-1), 129.6
(CH, C-2, C-6), 130.1(CH, C-3, C-5), 131.4 (CH, C-4),
128.2 (C, C-1′), 132.6 (CH, C-2′, C-6′), 115.1 (CH, C-3′, C-
5′), 154.8 (C, C-4′), 123.3 (CH, C-α), 144.4 (CH, C-β),
190.3 (C, COBz); EIMS m/z (M+. 223), calcd for
C15H13NO/223.
(2E)-1-(4′-aminophenyl)-3-(4-methoxyphenyl)-prop-
2-en-1-one (3)
Yellow amorphous powder (38%), m.p.148–148,5oC; FT-
(2E)-1-(4′-aminophenyl)-3-(4-fluophenyl)-prop-2-en-
1-one (2)
IR (KBr) νmax 1628, 1570, 1560 1555, 1480, 1240 cm−1
;
1H-NMR (CDCl3, 300 MHz): δ = 7.92 (2H, d, J = 8.61 Hz,
H-2,H-6), 6.70 (2H, d, J = 8.58 Hz, H-3,H-5), 7.59 (2H, d,
J = 8.64 Hz, H-2′,H-6′), 6.93 (2H, d, J = 8.67 Hz,H-3′,H-
5′), 7.42 (1H, d, J = 15.57 Hz, H-α), 7,76 (1H, d, J =
Yellow amorphous powder (28%), m.p. 161.5–162.5 9 °C;
1
FT-IR (KBr) νmax 3600, 1660, 1590, 1570 cm−1; H-NMR

Medicinal Chemistry Research
15.39 Hz, H-β), 3.89 (3H, s, OCH3). 13C-NMR (CDCl3,
75 MHz): δ = 128.3 (C, C-1), 131.1(CH, C-2,C-6), 114.2
(CH, C-3,C-5), 161.5 (C, C-4), 127.8 (C, C-1′), 130.2 (CH,
C-2′,C-6′), 114.5 (CH, C-3′,C-5′), 151.1 (C, C-4′), 120.1
(CH, C-α), 143.2 (CH, C-β), 188.4 (C, COBz), 55.8 (CH3,
OCH3); EIMS m/z (M+. 253), calcd for C16H15NO2/253.
(CH, C-5), 135.2 (CH, C-6), 127.6 (C, C-1′), 132.3 (CH, C-
2′, C-6′), 114.2 (CH, C-3′,C-5′), 154.7 (C, C-4′), 123.9
(CH, C-α), 140.0 (CH, C-β), 186.9 (C, COBz); EIMS m/z
(M+. 268), calcd for C15H12N2O3/268.
(2E)-1-(4′-aminophenyl) -3-(4-dimethyaminophenyl)
-prop-2-en-1-one (7)
(2E)-1-(4′-aminophenyl)-3-(4-ethoxyphenyl)-prop-2-
en-1-one (4)
Orange amorphous powder (35.66%), m.p. 169–170 °C;
FT-IR (KBr) νmax 3500, 3460, 1610, 1570, 1350, 1160,
1
Yellow amorphous powder (33.89%), m.p. 140–140,8 °C;
FT-IR (KBr) νmax 1634, 1600, 1588, 1575, 1480, 1167 cm
980 cm−1; H-NMR (CD3OD, 300 MHz): δ = 7.57 (2H, d,
J = 8.85 Hz, H-2, H-6), 6.69 (2H, d, J = 8.76 Hz, H-3,H-5),
7.89 (2H, d, J = 8.73 Hz, H-2′,H-6′), 6.76 (2H, d, J =
8.88 Hz, H-3′,H-5′), 7.46 (1H, d, J = 15.39 Hz, H-α), 7.68
(1H, d, J = 15.39 Hz, H-β)), 3.02 (6H, s, 2CH3). 13C-NMR
(CD3OD, 75 MHz): δ = 124.7 (C, C-1), 131.5 (CH, C-2, C-
6), 113.3 (CH, C-3, C-5), 153.8 (C, C-4), 40.4 (C, N(CH3)
2), 128.4 (C, C-1′), 132.3 (CH, C-2′, C-6′), 114.6 (CH, C-
3′,C-5′), 155.2 (C, C-4′), 117.6 (CH, C-α), 145.8 (CH, C-
β), 190.8 (C, COBz); EIMS m/z (M+. 266), calcd for
C17H18N2O/266.
1
−1; H-NMR (CD3OD, 300 MHz): δ = 7.90 (2H, d, J =
8.70 Hz, H-2,H-6), 6.69 (2H, d, J = 8.73 Hz, H-3,H-5), 7.63
(2H, d, J = 7.98 Hz, H-2′,H-6′), 6.94 (2H, d, J = 8.70 Hz,
H-3′,H-5′), 7.58 (1H, d, J = 15.57 Hz, H-α), 7.65 (1H, d,
J = 15.06 Hz, H-β), 4,08 (2H, q, J = 6.96, OCH2CH3), 1.39
(3H, t, J = 6.99 Hz, OCH2CH3). 13C-NMR (CD3OD,
75 MHz): δ = 129.3 (C, C-1), 132.5 (CH, C-2, C-6), 114.6
(CH, C-3, C-5), 155.5 (C, C-4), 127.9 (C, C-1′), 131.4 (CH,
C-2′,C-6′), 116.1 (CH, C-3′,C-5′), 162.6 (C, C-4′), 120.7
(CH, C-α), 144.4 (CH, C-β), 199.3 (C, COBz), 64.8 (CH2,
OCH2CH3), 15.2 (CH3, OCH2CH3); EIMS m/z (M+. 267),
calcd for: C17H17NO2/267.
(2E)-1-(4′-aminophenyl) -3- (furan-2-yl-prop-2-en-1-
one (8)
(2E)-1-(4′-aminophenyl)-3-(4-clorophenyl)-prop-2-
en-1-one (5)
Orange amorphous powder (69.85%), m.p. 118–118,3 °C;
FT-IR (KBr) νmax 3447, 3434, 1640, 1584, 1545,
1173 cm−1; 1H-NMR (CD3OD, 300 MHz): δ = 6.78 (1H, d,
J = 3.36 Hz, H-3), 6.55 (1H, m, H-4); 7.62 (1H, s, H-5),
7,85 (2H, d, J = 8.73 Hz, H-2′, H-6′), 6.68 (2H, d, J =
8.73 Hz, H-3′,H-5′), 7.50–7.62 (2H, s broad, H-α, H-β).
13C-NMR (CD3OD, 75 MHz): δ = 153.4 (C, C-2), 113.7
(CH, C-3), 116.5 (CH, C-4), 146.3 (CH, C-5), 127.6 (C, C-
1′), 132.4 (CH, C-2′, C-6′), 114.6 (CH, C-3′,C-5′), 155.5
(C, C-4′), 130.5 (CH, C-α), 120.5 (CH, C-β), 189.7 (C,
COBz); EIMS m/z (M+. 213), calcd for C13H11NO2/213.
Yellow amorphous powder (53.59%), m.p. 162,9–163,3 °C;
FT-IR (KBr) νmax 3555, 3350, 1621, 1570, 1550,
1490 cm−1; 1H-NMR (CD3OD, 300 MHz): δ = 7.91 (2H, d,
J = 8.73 Hz, H-2,H-6), 7.69 (2H, d, J = 8.79, H-3,H-5),
7.42 (2H, d, J = 8.46 Hz, H-2′,H-6′), 6.68 (2H, d, J =
8.76 Hz, H-3′, H-5′), 7.70 (1H, d, J = 14.28 Hz, H-α), 7.74
(1H, d, J = 15.60 Hz, H-β). 13C-NMR (CD3OD, 75 MHz):
δ = 135.6 (C, C-1), 132.7 (CH, C-2, C-6), 130.9 (CH, C-3,
C-5), 137.5 (C, C-4), 127.6 (C, C-1′), 130.8 (CH, C-2′’,C-
6′’), 114.6 (CH, C-3′,C-5′), 155.8 (C, C-4′), 124.3 (CH, C-
α), 142.9 (CH, C-β), 189.9 (C, COBz); EIMS m/z (M+.
257.5), calcd for: C15H12NOCl/257.5.
(2E,4E)-1-(4-aminophenyl)-5-phenylpenta-2,4-dien-
1-one (9)
Orange amorphous powder (34.00%), m.p. 151,8–152 °C;
FT-IR (KBr) νmax 3458, 3376, 1635, 1611, 1576 1564 cm−1
1H-NMR (CD3OD, 300 MHz): δ = 7.26–7.35 (5H, m, Ar),
7.84 (2H, d, J = 8.79 Hz, H-2′,H-6′), 6.67 (2H, d, J =
8.76 Hz, H-3′,H-5′), 7.00–7.25 (3H, m, H-α, H-7, H-8),
7.38 (1H, ddd, J = 15.96, 8.43, 1.65 Hz, H-β). 13C-NMR
(CD3OD, 75 MHz): δ = 138.0 (C, C-1), 128.7 (CH, C-2, C-
6), 130.0 (CH, C-3, C-5), 128.4 (C, C-4), 126.8 (CH, C-7),
142.6 (CH, C-8), 127.7 (C, C-1′), 132.5 (CH, C-2′,C-6′),
114.6 (CH, C-3′,C-5′), 155.6 (C, C-4′), 130.5 (CH, C-α),
144.6 (CH, C-β), 190.3 (C, COBz); EIMS m/z (M+. 253),
calcd for C17H15NO/253.
(2E)-1-(4′-aminophenyl) -3- (3-nitrophenyl) prop-2-
en-1-one (6)
Orange amorphous powder (69.85%), m.p. 208–208,7 °C;
1
FT-IR (KBr) νmax 3400, 1640, 1600, 980 cm−1; H-NMR
(CD3COCD3, 300 MHz): δ = 8.26 (1H, d, J = 1.95 Hz, H-
2), 7.98–8.26 (2H, m, H-4,H-5), 7.75 (2H, d, J = 7.89 Hz,
H-2′,H-6′), 6.74 (2H, d, J = 8.67 Hz, H-3′,H-5′), 7.72 (1H,
d, J = 15.96 Hz, H-α)), 7.80 (1H, d, J = 17.34 Hz, H-β).
13C-NMR (CD3COCD3, 75 MHz): δ = 138.6 (C, C-1),
124.9 (CH, C-2), 149.9 (C, C-3), 126.3 (CH, C-4), 132.2

Medicinal Chemistry Research
1
N-(4′[(2E)-3-(phenyl)-1-(phenyl) prop-2-en-1-one])
980 cm−1; H-NMR (CDCl3, 300 MHz): δ = 7.57 (2H, d,
acetamide (1a)
J = 8.50 Hz, H-2,H-6), 6.91 (2H, d, J = 8.45 Hz, H-3,H-5),
8.00 (2H, d, J = 8.40, H-2′H-6′), 7.66 (2H, d, J = 7.75 Hz,
H-3′,H-5′), 7.39 (1H, d, J = 15.55 Hz, H-α), 7.78 (1H, d,
J = 15.60 Hz, H-β), 4.08 (2H, q, J = 6.85 Hz, OCH2CH3),
1.43 (3H, t, J = 6.90 Hz, OCH2CH3), 2.21 (3H, s, COCH3).
13C-NMR (CDCl3, 75 MHz): δ = 127.6 (C, C-1), 130.0
(CH, C-2, C-6), 115.1 (CH, C-3, C-5), 161.3 (C, C-4),
134.2 (C, C-1′), 130.5 (CH, C-2′, 6′), 119.2 (CH, C-3′,5′),
142.3 (C, C-4′), 63.9 (CH2, OCH2CH3), 14.9 (CH3,
OCH2CH3), 25.0 (CH3, COCH3), 119.5 (CH, C-α), 144.8
(CH, C-β), 189.4 (C, COBz), 168.9 (C, N–C=O); EIMS m/
z (M+. 309), calcd for C19H19NO3/309.
Yellow amorphous powder (88.92%), m.p. 158–158.5 °C;
FT-IR (KBr) νmax 3330, 1658, 1600, 1310, 980 cm−1
;
1H-NMR (CD3OD, 300 MHz): δ = 7.40–7,72 (5H, m, Ar),
8.06 (2H, d, J = 8.79 Hz, H-2′, H-6′), 7.71–7.75 (2H, s
broad, H-α, H-β), 2.14 (3H, s, COCH3). 13C-NMR
(CD3OD, 75 MHz): δ = 136.5 (C, C-1), 129.8 (CH, C-2,
C-6), 131.8 (CH, C-3, C-5), 131.1 (C, C-4), 134.6 (C, C-1′),
130.2 (CH, C-2′,C-6′), 123.0 (CH, C-3′,C-5′), 144.9 (C, C-
4′), 24.2 (CH3, COCH3), 120.4 (CH, C-α), 145.9 (CH, C-β),
191.0 (C, COBz), 172.1 (C, N–C=O); EIMS m/z (M+. 265),
calcd for C17H15NO2 /265.
N-(4′[(2E)-3-(4-clorophenyl-1-(phenyl) prop-2-en-1-one])
acetamide (5a)
N-(4′[(2E)-3-(4-flurophenyl) -1-(phenyl) prop-2-en-1-one])
acetamide (2a)
Yellow amorphous powder (54.02%), m.p. 198.5–199 °C;
1
Yellow amorphous powder (88.21%), m.p. 178.4–179 °C;
FT-IR (KBr) νmax 3270, 1650, 1600, 1310, 980 cm−1; H-
FT-IR (KBr) νmax 3430, 1650, 1615, 1500, 1300, 980 cm−1
;
NMR (CD3OD, 300 MHz): δ = 7.42 (2H, d, J = 8.49 Hz,
H-2,H-6), 7.90 (2H, d, J = 8.76 Hz, H-3,H-5), 8.00 (2H, d,
J = 8.79 Hz, H-2′,H-6′), 6,69 (2H, d, J = 8,76 Hz, H-3′,H-
5′), 7.40 (1H, d, J = 17.04 Hz, H-α,), 8.71 (1H, d, J =
14.88 Hz, H-β), 2.15 (3H, s, COCH3). 13C-NMR (CD3OD,
75 MHz): δ = 134.5 (C, C-1), 130.4 (CH, C-2, C-6), 131.0
(CH, C-3, C-5), 137.5 (C, C-4), 135.3 (C, C-1′), 132.7 (CH,
C-2′,6′), 120.4 (CH, C-3′5′), 142.6 (C, C-4′), 130.3 (CH, C-
α), 145.0 (CH, C-β), 190.0 (C, COBz), 172.1 (C, N–C=O),
24.2 (CH3, COCH3); EIMS m/z (M+. 283.5), calcd for
C17H14NOCl/283.5.
1H-NMR (CD3OD, 300 MHz): δ = 7.76 (2H, m, H-2,H-6),
7.16 (2H, t, J = 8.70 Hz, H-3,H-5)), 8.07 (2H, d, J =
8.67 Hz, H-2’,H-6’), 7.73 (2H, m, H-3′,H-5′), 7,72–7.81
(2H, s broad, H-α, H-β), 2.16 (3H, s, COCH3). 13C-NMR
(CD3OD, 75 MHz): δ = 133.1 (C, C-1), 131.1 (CH, C-2, C-
6), 116.9 (CH, C-3, C-5), 164.0 (C, C-4), 132.1 (C, C-1′),
131.1 (CH, C-2′,C-6′), 122.9 (CH, C-3′,C-5′), 144.9 (C, C-
4′), 120.4 (CH, C-α), 144.5 (CH, C-β), 190.8 (C, COBz),
172.1 (C, N–C=O); EIMS m/z (M+. 283), calcd for
C17H14NO2F/283.
N-(4′[(2E)-3-(4-methoxyphenyl) -1-(phenyl) prop-2-en-1-
one]) acetamide (3a)
N- (4′- [(2E) -3- (3-nitrophenyl) -1- (phenyl) prop-2-en-1-
one]) acetamide (6a)
Yellow amorphous powder (49.40%), m.p. 202.5–202.8 °C;
Orange amorphous powder (51.29%), m.p. 198–198,3 °C;
FT-IR (KBr), νmax 3330, 1610, 1660, 1580, 1340,
FT-IR (KBr) νmax 3260, 1640, 1600, 1310, 1160, 970 cm−1
;
1H-NMR (CD3OD, 300 MHz): δ = 7.71 (2H, m, H-2,H-6),
7.00 (2H, d, J = 8.70 Hz,H-3,H-5), 8.00 (2H, d, J =
8.70 Hz, H-2′,H-6′)), 7.74 (2H, d, J = 8.55 Hz, H-3′,H-5′),
7,72 (1H, d, J = 15.63 Hz, H-α), 7.76 (1H, d, J = 16.47 Hz,
H-β), 3.85 (3H, s, OCH3), 2.16 (3H, s, COCH3). 13C-NMR
(CD3OD, 75 MHz): δ = 129.1 (C, C-1), 131.4 (CH, C-2,
C-6), 115.7 (CH, C-3,C-5), 163.6 (C, C-4), 132.6 (C, C-1′),
131.8 (CH, C-2′,C-6′), 120.4 (CH, C-3′,C-5′), 146.2 (C,
C-4′), 120.4 (CH, C-α), 144.7 (CH, C-β), 191.1 (C, COBz),
172.1 (C, N–C=O), 56.1 (C, OCH3); EIMS m/z (M+. 295),
calcd for C18H17NO3/295.
970 cm−1; H-NMR (CD3SOCD3, 300 MHz): δ = 8.17–7.75
1
(2H, m, H-4, H-5), 8,17 (1H, d, J = 10.53 Hz, H-6), 7.96
(2H, d, J = 7.41 Hz, H-2′,H-6′), 6.63 (2H, d, J = 7.32 Hz, H-
3′,H-5′), 8.00 (1H, d, J = 17.04, H-α), 8.71 (1H, d, 14.88 Hz,
H-β), 2.09 (3H, s, COCH3). NMR de 13C (CD3SOCD3,
75 MHz): δ = 134.9 (C, C-1), 122.8 (CH, C-2), 148.4 (C, C-
3), 122.6 (CH, C-4), 130.1 (C, C-5), 134.7 (CH, C-6), 137.1
(C, C-1′), 131.4 (CH, C-2′, C-6′), 112.9 (CH, C-3′,5′), 144.0
(C, C-4′), 122.9 (CH, C-α), 144.0 (C, C-β), 187.4 (C, COBz),
169.2 (C, N–C=O), 24.2 (CH3, COCH3); HRESIMS, m/z:
310.1445(C19H19NO3) [M+H]+ (calcd. 310.1443).
N- (4′- [(2E) -3- (4-ethoxyphenyl) -1- (phenyl) prop-2-en-1-
one]) acetamide (4a)
N-(4′[(2E)-3-(4-dimetilaminophenyl) -1-(phenyl) prop-2-en-
1-one]) acetamide (7a)
Yellow amorphous powder (38.02%), m.p. 134–135 °C;
FT-IR (KBr) νmax 3310, 1650, 1670, 1600, 1310, 1155,
Yellow amorphous powder (60.40%), m.p. 207–207.5 °C;
FT-IR (KBr) νmax 3410, 1680, 1650, 1580, 1380, 1030 cm

Medicinal Chemistry Research
−1
;
1H-NMR (CDCl3, 300 MHz): δ = 7.66 (2H, d, J =
1H-NMR (CD3COCD3, 300 MHz): δ = 7.50 (2H, d, J =
7,50 Hz, H-2,H-6), 7.70 (2H, d, J = 8.73 Hz, H-3, H-5),
7.76 (1H, d, J = 1.92 Hz, H-2′), 6.70 (1H, d, J = 8.22 Hz,
H-5′), 7.74 (1H, dd, J = 8.85, 2.01 Hz, H-6′), 7.73 (1H, d,
J = 17.90 Hz, H-α), 7.91 (1H, d, J = 15.48 Hz, C-β), 3.90
(3H, s, OCH3). 13C-NMR (CD3COCD3, 75 MHz): δ =
135.3 (C, C-1), 129.9 (CH, C-2,C-6), 130.9 (CH, C-3, C-5),
136.9 (C, C-4), 136.2 (C, C-1‘), 112.6 (CH, C-2′), 148.7 (C,
C-3′), 152.7 (C, C-4′), 115.6 (CH, C-5′), 124.6 (CH, C-6′),
123.7 (CH, C-α), 142.2 (CH, C-β), 56.5 (C, OCH3), 187.9
(C, COBz); EIMS m/z (M+. 288.5), calcd for C16H13NO3Cl
/288.5.
8,20 Hz, H-2,H-6)), 7.53 (2H, d, J = 8.65 Hz H-3,H-5),
8.00 (2H, d, J = 8.30 Hz, H-2′,H-6′), 6.71 (2H, d broad, H-
3′/H-5′), 7.34 (1H, d, J = 15.45 Hz, H-α), 7.78 (1H, d, J =
15.45 Hz, H-β), 3.04 (6H, s, 2CH3), 2.21 (3H, s, COCH3).
13C-NMR (CDCl3, 75 MHz): δ = 134.6 (C, C-1), 130.1
(CH, C-2, C-6), 112.3 (CH, C-3,C-5), 152.0 (C, C-4), 40.5
(C, N(CH3)2), 133.0 (C, C-1′), 130.6 (CH, C-2′,C-6′), 116.9
(CH, C-3′,C-5′), 145.7 (C, C-4′), 24.9 (CH3, COCH3),
119.2 (CH, C-α), 142.0 (CH, C-β), 189.5 (C, COBz), 169.0
(C, N–C=O), 40.5(C, N(CH3)2), 24.9(CH3, COCH3); EIMS
m/z (M+. 308), calcd for C19H20N2O2/308.
N-(4′[(2E)-3- (furan-2-il) -1-(phenyl) prop-2-en-1-one])
acetamide (8a)
(2E) -1- (3′-methoxy-4′-hydroxyphenyl) -3- (3-
nitrophenyl) prop-2-en-1-one (11)
Orange amorphous powder (76.17%), m.p. 122–122.4 °C;
FT-IR (KBr) νmax 3470, 1690, 1640, 1600, 1375, 1005 cm
−1; 1H-NMR (CDCl3, 300 MHz): δ = 6.51 (1H, s broad, H-
3), 6.70 (1H, d, J = 3,09 Hz, H-4), 7.46 (1H, s, H-5), 8.00
(2H, d, J = 8,55 Hz, H-2′, H-6′), 7.66 (2H, d, J = 8,31 Hz,
H-3′, H-5′), 7.33 (1H, d, J = 15.33 Hz, H-α), 7.51 (1H, d,
J = 17.58 Hz, H-β), 2.21 (3H, s, COCH3). 13C-NMR
(CDCl3, 75 MHz): δ = 151.9 (C, C-2), 112.7 (CH, C-3),
116.2 (CH, C-4), 145.1 (CH, C-5), 133.9 (C, C-1′), 130.0
(CH, C-2′, C-6′), 119.3 (CH, C-3′, C-5′), 24.8 (CH3,
COCH3), 119.4 (CH, C-α), 130.7 (CH, C-β), 188.8 (C,
COBz), 169.0 (C, N–C=O), 24.9 (CH3, COCH3); EIMS m/
z (M+. 239), calcd for C15H13NO2/239.
Yellow amorphous powder (46.33%), m.p. 179.4–180 °C;
1
FT-IR (KBr) νmax 3425, 1621, 1575, 1350, 990 cm−1; H-
NMR (CD3COCD3, 300 MHz): δ = 6.96–8.26 (5H, m, Ar),
8.65 (1H, s, H-2′), 7.73 (1H, m, H-5′), 8.23 (1H, d, J =
6.36 Hz, H-6′), 7.82 (1H, d, J = 15.35 Hz, H-α), 7.87 (1H,
d, J = 15.35 Hz, H-β), 3.90 (3H, s, OCH3). 13C-NMR
(CD3COCD3, 75 MHz): δ = 131.1 (C, C-1), 124.9 (CH,
C-2), 152.9 (C, C-3), 125.1 (CH, C-4), 131.2 (CH, C-5),
135.4 (CH, C-6), 138.3 (C, C-1′), 124.9 (CH, C-2′), 148.8
(C, C-3′), 149.9 (C, C-4′), 115.6 (CH, C-5′), 125.8 (CH,
C-6′), 123.5 (CH, C-α), 141.1 (CH, C-β), 56.5 (C, OCH3),
187.8 (C, COBz); HRESIMS m/z: 300.0867 (C16H13NO3)
[M+H]+ (calcd. 300.0872).
N-(4′[(2E)-5-[phenylpenta-2,4-dien) -1-(phenyl) prop-2-en-1-
one]) acetamide (9a)
NMR, GC–MS, and IR measurements
1
The chemical reagents were from Sigma-Aldrich. H and
Orange amorphous powder (22.27%), m.p. 176.5–177 °C;
FT-IR (KBr) νmax 3300, 1670, 1650, 1600, 1300,
13C NMR spectra were obtained using a Bruker Spectro-
meter, either model Avance DPX-300 or model Avance
DRX-500 operating at frequencies of 300 or 500 MHz for
hydrogen, and 75 or 125 MHz for carbon, respectively. The
spectra were measured in the solvents CD3OD,
CD3COCD3, and CDCl3, and chemical shifts are reported as
δ values in parts per million (ppm) relative to tetra-
methylsilane (δ 0.00) as the internal standard. The mass
spectra were obtained with a Shimadzu QP201 GC-MS (gas
chromatography coupled to mass spectrometry) using an
RTX-5MS capillary column (30.0 m × 0.25 mm × 0.30 mm)
for compounds within the literature record, and UPLC-
QTOF-MS was performed with an ACQUITY UPLC BEH
column (150 × 2.1 mm, 1.7 µm; Waters Co.) on a Waters
Acquity UPLC system. The column temperature was set at
40 °C. The binary gradient elution system consisted of 0.1%
formic acid in water (A) and 0.1 % formic acid in acet-
onitrile (B), with a linear gradient from 2 to 95% B
(0–15 min), with a flow rate of 0.4 mL.min−1 for the new
compound (11). The high mass spectra of the chalcone 11
1000 cm−1 1H-NMR (CDCl3, 300 MHz): δ = 7.33–7.40
;
(5H, m, Ar), 7.98 (2H, d, J = 8,64 Hz, H-2′,H-6′), 7.01 (2H,
d, J = 7.71 Hz, H-3′,H-5′), 7.19 (1H, d, J = 14.79 Hz, H-α),
7.51 (1H, dd, J = 17.40, 8.76 Hz, H-β), 7.02–7.07 (2H,
m, H-7,H-8), 2.23 (3H, s, COCH3). 13C-NMR (CDCl3,
75 MHz): δ = 136.4 (C, C-1), 127.5 (CH, C-2, C-6), 129.1
(CH, C-3,C-5), 129.4 (C, C-4), 134.1 (C, C-1′), 130.0 (CH,
C-2′, C-6′), 119.2 (CH, C-3′, C-5′), 142.1 (C, C-4′), 24.9
(CH3, COCH3), 127.2 (CH, C- α), 144.8 (CH, C-β), 125.4
(CH, C-7), 142.0 (CH, C-8), 189.2 (C, COBz), 168.8 (C,
N–C=O), 24.9 (CH3, COCH3); EIMS m/z (M+. 291), calcd
for C19H17NO2/291.
(2E)-1-(3′-methoxi-4′-hidroxiphenyl)-3-(4-
clorophenyl)-prop-2-en-1-one (10)
Yellow amorphous powder (20.89%), m.p. 141–141.3 °C;
FT-IR (KBr) νmax 3390, 1640, 1590, 1290, 1180, 970 cm−1
;

Medicinal Chemistry Research
was obtained with LC–MS on an Acquity UPLC system
coupled with quadrupole/TOF mass analyzers (Waters)
equipped with an ESI source operated in the negative
ionization mode (UPLC-ESI-qTOF). The chromatographic
separations were performed using a Waters Acquity UPLC
BEH column (150.0 × 2.1 mm × 1.7 μm) at 40 °C. Water
and acetonitrile were used for the mobile phase, both with
0.1% formic acid. The gradient ranged from 95 to 2% water
in 15 min at a flow rate of 0.4 mL/min and an injection
volume of 5.0 μL. The desolvation gas was N2 at 350 °C at a
flow rate of 350 L/h and a source temperature of 120 °C.
The capillary voltage was set to 2.600 V. The collision
energies/cone voltages were set to 6 eV/15 V (low) and
30–50 eV/30 V (high) to achieve sufficient fragmentation.
The MS spectra were acquired in negative ionization mode
between 100 and 1180 Da in MSE tandem mode. The
samples were prepared by filtering 1 mL of the chalcones
directly through a 0.22 µm PTFE syringe filter (Simple
Pure, USA). Infrared spectra were determined on a Perkin
Elmer FT/IR 1000 spectrophotometer and reported as the
wavenumber (cm−1). The melting point was determined
with the MQAPF-302 apparatus (microchemistry) with a
heating rate of 3.0 °C/min.
595 nm (DTX 880 Multimode Detector, Beckman Coulter,
Inc. Fullerton, California, USA).
To carry out a structure-activity relationship analysis, all
compounds were tested against HCT-116 cells at different
concentrations (0.04–20 µM) to determine the half-maximal
inhibitory concentration (IC50) after 72 h of incubation. In
addition, the IC50 value of the most active compound was
determined for four other human tumor cell lines (PC-3,
HL-60, K-562, and KASUMI-1) and one nontumor cell line
(L-929) after 72 h of incubation. Doxorubicin
(0.07–8.6 μM; Sigma Chemical Co. St. Louis, MO, USA)
was used as a positive control.
Study of the mechanism of cytotoxicity
In this stage, the study aimed to describe the chalcone 6a-
induced cytotoxicity against HCT116 cells (chosen based
on the preliminary cytotoxicity screening). For this purpose,
the cells (0.7 × 105 cells/well) were incubated with the
chalcone 6a for 24 h at three different concentrations (2.5, 5
and 10 μM; chosen based on the IC50 values obtained by the
MTT assay), and the experiments below were performed
(Pereira et al. 2016). The control group received the same
amount of vehicle whose concentration was kept constant
(below 0.5%). Cell treatments were carried out in triplicate.
Doxorubicin (2 µM; Sigma Chemical Co. St. Louis, MO,
USA) was used as a positive control due to its known
cytotoxic effects, as well as its ability to induce cell cycle
arrest in the G2/M phase. Stock solutions of the compounds
tested were prepared in DMSO and diluted in culture
medium to obtain the desired concentrations.
Cell culture
The human cancer cell lines (HCT-116, colon carcinoma; PC-
3, prostate adenocarcinoma; HL-60, promyelocytic leukemia;
K-562, chronic myeloid leukemia; and KASUMI-1, acute
myeloid leukemia) were obtained from the National Cancer
Institute, Bethesda, MD, USA. The L-929 (murine fibroblasts)
nontumor cell line was purchased from ATCC® and deposited
in the Rio de Janeiro cell bank. The cell lines were maintained
in RPMI-1640 medium or DMEM (Gibco) supplemented with
10% fetal bovine serum, 2 mM glutamine, 100 U/mL peni-
cillin, and 100 µg/mL streptomycin at 37 °C in a humidified
atmosphere with 5% CO2.
Morphological analysis
The morphological features of HCT116 cells were assessed
using light microscopy (Olympus, Tokyo, Japan). Cells
were harvested, transferred to Cytospin slides, and stained
using a quick panoptic kit (Laborlin, Brazil). Cells were
fixed with methanol and counterstained with 0.1% xan-
thanes and 0.1% thiazines before the analysis.
Determination of cytotoxic activity
The cytotoxicity of the compounds was determined using the
MTT assay (Mosmann 1983). Briefly, cells were seeded in 96-
well plates (0.7 × 104 cells/well for adherent cells and 0.3 ×
105 cells/well for suspended cells). The compounds dissolved
in DMSO were added to each well using high-throughput
screening with a Biomek 3000 (Beckman Coulter, Inc. Full-
erton, California, USA) and incubated for 3 days (72 h). The
control group received the same amount of vehicle, and the
final concentration of DMSO in the culture media was con-
stant (below 0.5%). After incubation, the supernatant was
replaced by fresh medium containing MTT (0.5 mg.mL−1).
Three hours later, the MTT formazan product was dissolved in
150 µL of DMSO, and the absorbance was measured at
Apoptosis assay
Apoptosis was examined by Annexin V/7-AAD staining
followed by flow cytometry. Briefly, HCT116 cells were
collected (both floating and attached cells) and stained using
an Annexin V-PE/7-AAD apoptosis detection kit. Annexin
V binds to phosphatidylserine (PS) in cells undergoing
apoptosis due to the translocation of PS from the inner
leaflet to the outer leaflet of the cytoplasmic membrane. 7-
AAD is a fluorescent intercalator of DNA impermeant to the
cell, indicating membrane integrity. Double staining is used to
distinguish between viable, early apoptotic cells, and necrotic

Medicinal Chemistry Research
or late apoptotic cells. The resulting fluorescence (Annexin V-
PE at 583 nm and 7-AAD at 680 nm) of all samples was
analyzed with BD FACSVerse™. 7-AAD-positive and
annexin V-negative cells were considered necrotic. Annexin
V-positive cells (both 7-AAD positive and negative cells)
were considered apoptotic cells. 7-AAD-negative and
Annexin V-negative cells were considered viable.
those chalcones registered in the literature, and 1D and 2D
(COSY, HSQC and HMBC) NMR for the new chalcone
(11), electron ionization mass spectrometry, high-resolution
electrospray ionization mass spectrometry (HRESIMS), and
1
Fourier-transform infrared spectroscopy (FT-IR). The H
(500 MHz) and 13C (125 MHz) NMR spectra in CDCl3
(d, ppm, J/Hz) are presented in the Supplementary Material
in Figs S1–S44. The mass spectra of these chalcones are
shown in Figs S45–S64 and their infrared spectra are given
in Figs S65–S84. The values of the coupling constants
between Hα and Hβ (J = 12.10–17.98 Hz) confirm that for
this reaction, the products generated were only E-isomers.
Determination of cell viability and membrane
integrity
Cell viability and cell membrane integrity analyses were per-
formed with propidium iodide (5 µg/mL, Sigma Aldrich Co.,
St. Louis, MO, USA) exclusion using flow cytometry.
HCT116 cells were plated in 24-well plates and treated with
PJOV56. After cell harvesting, the treated and untreated cells
were incubated with propidium iodide in the dark for 5 min.
Then, the fluorescence was measured by flow cytometry with
BD FACSVerse™ (BD Biosciences, San Jose, CA).
Cytotoxic activity against tumor and nontumor cells
The MTT assay was used to determine the cytotoxicity of
the synthesized chalcones. This test is extremely useful for
both determining cell viability studies and defining the
anticancer potential of new compounds (Kepp et al. 2011).
Table 1 displays the IC50 values of the investigated com-
pounds whose data were used for the structure–activity
relationship analysis.
DNA fragmentation and cell cycle analysis
The DNA content of HCT116 cells was assessed by pro-
pidium iodide DNA staining followed by flow cytometry
analysis. Briefly, cells were treated with PJOV56, harvested
and incubated in the dark with a solution containing 5 µg.
mL−1 propidium iodide, 5 µg.mL−1 RNase, 0.1 % sodium
citrate, and 0.1 % Triton X-100 at room temperature for
40 min. The DNA fragmentation and cell cycle profile were
obtained by cell counting and fluorescence measurements
using a BD FACSVerse™ flow cytometer (BD Biosciences,
San Jose, CA). Data were analyzed by ModFit LT software
(Verity Software House, Inc., Topsham, ME).
Out of 20 compounds tested, only 1a (IC50 = 4.96
0.03 µM), 2a (IC50 = 7.05 0.02 µM), 5a (IC50 = 5.75
0.44 µM), and 6a (IC50 = 3.56 0.03 µM) showed a strong
cytotoxic effect (IC50 < 10 µM) against HCT-116 cells,
revealing that small modifications to the chalcone chemical
structure were able to alter the biological activity. Careful
analysis of the structures suggests that the cytotoxicity can
be attributed to the combined effects of the two electron-
withdrawing groups in the structure of 6a (an amide group
on ring A and a nitro group on ring B). For the amide group
contribution, compound 1a (IC50 = 4.96 0.03 µM) pre-
sented higher cytotoxic activity when compared to 1 (IC50
Statistical analysis
> 20 µM). Regarding the nitro group, compounds 6 (IC50 =
13.96 0.03 µM) and 6a (IC50 = 3.56 0.03 µM) presented
greater activity when compared with 1 and 1a, respectively.
Moreover, the presence of an extended conjugated system
significantly reduced the cytotoxic effect. Compound 9a
(IC50 > 20 µM) showed no cytotoxic effect when compared
to compound 1a (IC50 = 4.96 0.03 µM), and this effect
was influenced by the presence of an additional double
bond in the conjugated system. Another important structural
feature that reduced the cytotoxic activity was a high elec-
tronic density in ring B (IC50 > 20 µM) for compounds 3,
3a, 4, 4a, 7 (% RCV = 4.25 2.60 %), 7a (% RCV = 0.00
%), 8 (% RCV = 9.15 3.01 %), and 8a (% RCV = 9.45
1.37 %). These data are corroborated by a review of the
anticancer activity of chalcones that states that the cytotoxic
properties of chalcones against cancer cell lines are mainly
influenced by the substituents on the two aryl rings of the
chalcone molecule simultaneously (Karthikeyan et al.
2015). Recent studies (Nazir et al. 2013; Guilherme et al.
Data are presented as the mean standard error of the mean
(SEM) from three independent experiments performed in
triplicate as calculated by Prism 8.0 (GraphPad/Intuitive
Software for Science, San Diego, CA). The IC50 values of
the compounds were obtained from the nonlinear regression
of the cytotoxicity curves. Statistical analyses for multiple
comparisons were performed using one-way ANOVA fol-
lowed by Turkey’s Test. A value of p < 0.05 was considered
statistically significant.
Results and discussion
Structural characterization
The structures of the twenty synthetic chalcones were elu-
cidated by spectroscopic methods, including 1D NMR for

Medicinal Chemistry Research
Table 1 Cytotoxicity of the chalcones 1–11 and 1a–9a at different
concentrations (0.02–20 µM) against HCT-116 cells (human colon
carcinoma) after 72 h of incubation using MTT assay
2015) have also demonstrated the high cytotoxic effects of
nitro- and amide-containing chalcones against various types
of human cancer cell lines, including colon cancer. More-
over, Guo et al. (2018) showed that a synthetic compound
bearing the strongly electron-withdrawing NO2 group dis-
played the highest anticancer activity (7.3 1.2 μM) against
the A549 cell line (lung cancer) compared with the other
synthesized compounds. All of these findings reinforce the
synthetic proposal of the present work. For these reasons,
chalcone 6a was selected for further IC50 value determina-
tion against four human tumor cell lines (PC-3, HL-60, K-
562, and KASUMI-1) and nontumor murine fibroblasts (L-
929 cells). Table 2 shows the cytotoxicity of chalcone 6a at
different concentrations (0.02–20 µM) against six cell lines
after 72 h of incubation using MTT assay. Against the
tumor cells, the IC50 values of 6a ranged from 5.65 to
10.52 µM (strong cytotoxic activity). Against L-929 cells,
compound 6a was not active (IC50 > 20 µM). This suggests
that there are specific characteristics of the tumor cells that
are being affected by compound 6a at the tested con-
centrations. In fact, cancer has six biological capabilities,
including sustaining proliferative signaling, evading growth
suppressors, resisting cell death, enabling replicative
immortality, inducing angiogenesis, and activating invasion
and metastasis (Hanahan and Weinberg 2011). In this
context, Karthikeyan et al. (2015) reported the cytotoxic
activity of chalcones through multiple mechanisms,
including cell cycle disruption, angiogenesis inhibition,
tubulin polymerization inhibition, apoptosis induction,
blockade of the NF-κB signaling pathway, and inhibition of
cell cycle regulatory kinases.
Chalcone Chemical groupb
HCT-116
O
R2
R
R1
R
R
R
IC50 S.E.M.
(µM)a
1
2
1
NH2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Ph
>20
1a
2
–NHCOCH3
NH2
Ph
4.96 0.03
15.16 0.06
7.05 0.02
>20
p-F-Ph
p-F-Ph
p-OCH3-Ph
p-OCH3-Ph
2a
3
–NHCOCH3
NH2
3a
4
–NHCOCH3
NH2
>20
p-OCH2CH3-Ph >20
p-OCH2CH3-Ph >20
4a
5
–NHCOCH3
NH2
p-Cl-Ph
>20
5a
6
–NHCOCH3
NH2
p-Cl-Ph
5.75 0.44
13.96 0.03
3.56 0.03
>20
m-NO2-Ph
m-NO2-Ph
p-N(CH3)2-Ph
p-N(CH3)2-Ph
Furan-2-il
Furan-2-il
6a
7
–NHCOCH3
NH2
7a
8
–NHCOCH3
NH2
>20
>20
8a
9
–NHCOCH3
NH2
>20
Phenylpenta-4-
en
>20
9a
–NHCOCH3
H
Phenylpenta-4-
en
>20
Mechanism of cytotoxicity against human colon
cancer cells
10
11
OH
OH
OCH3 p-Cl-Ph
>20
>20
OCH3 m-NO2-Ph
aData are presented as half maximal inhibitory concentration (IC50 in
µM) Standard Error of the Mean (S.E.M.) obtained from three
independent experiments performed in triplicate
bSpecific chemical groups of each compound bonded to
fundamental core
Based on the initial data indicating the in vitro cytotoxic
potential of the chalcone 6a, we decided to investigate the
pattern of cell death induced by this chalcone in HCT-116
cells at three concentrations (2.5, 5, and 10 µM) after 24 h of
incubation. First, microscopic analysis showed that
Table 2 Cytotoxicity of
chalcone 6a at different
Chalcone
Cell line
concentrations (0.02–20 µM)
against six cell lines after 72 h of
incubation using MTT assay
HCT-116
IC50 S.E.M. (µM)a
PC-3
HL-60
K-562
KASUMI-1
L-929
6a
Doxb
3.56 0.03
0.11 0.03
9.33 0.05
0.44 0.10
5.65 0.41
7.49 1.35
0.46 0.01
10.52 0.10
0.13 0.007
>20
0.02 0.002
0.66 0.17
aData are presented as half maximal inhibitory concentration (IC50 in µM) Standard Error of the Mean (S.E.
M.) for tumor cells (HCT-116, human colon carcinoma; PC-3, human prostate adenocarcinoma; HL-60,
human promyelocytic leukemia; K-562, human chronic myeloid leukemia; and KASUMI-1, human acute
myeloid leukemia), and nontumor cells (L-929, murine fibroblast), obtained from at least two independent
experiments performed in triplicate
bDoxorubicin (Dox, 0.07–8.6 μM) was used as positive control of the assay

Medicinal Chemistry Research
Fig. 4 Effect of chalcone 6a on the morphology of HCT-116 cells
determined by light microscopy (400×) using panoptic kit after 24 h of
incubation. Cells untreated (a) or treated with chalcone 6a (2.5 µM, c;
5 µM, d; and 10 µM, e) were fixed with methanol and counter-stained
with 0.1% xanthanes and 0.1% thiazines before the analysis. Black
arrows show apoptotic cells (nuclear fragmentation, chromatin con-
densation, apoptotic bodies, and cell shrinkage)
chalcone 6a was able to induce morphological changes in
HCT-116 cells that were characteristic of apoptosis (nuclear
fragmentation, chromatin condensation, apoptotic bodies,
and cell shrinkage), and these changes were more evident at
the concentrations of 5 and 10 µM (Fig. 4d, e) (Kroemer
et al. 2008).
Chalcone 6a-induced apoptosis was confirmed by the
externalization of phosphatidylserine accompanied by
maintenance of membrane integrity and a reduction in the
number of cells as determined by flow cytometry (Figs 5
and 6). At 10 µM, chalcone 6a induced a significant
increase in apoptotic cells (9.60 1.68 %) when compared
with the negative control (1.55 0.18 %). At this con-
centration, we also observed 90.29 0.62 % membrane
integrity (no difference when compared with the negative
control value of 95.10 0.55 %) and 4.21 0.19 × 105 cells/
mL (statistically significant when compared with the
negative control, 7.34 0.35 × 105 cells/mL) after 24 h of
incubation. Corroborating these results, Das and Manna
(2016) highlighted the participation of apoptosis in the
anticancer activity of chalcones. Specifically, against human
colon cancer, apoptosis induction might be correlated with a
cell cycle arrest in the G2/M phase, involving disruption of
the microtubular network and/or DNA damage. The mitotic
spindle network is critical for the process of cell division,
whereas disruption leads to apoptosis (Bond et al. 2018).
In this work, we confirmed this effect for chalcone 6a
against HCT-116 cells (Fig. 7). Similar to the positive control

Medicinal Chemistry Research
Fig. 5 Effect of chalcone 6a on the PS externalization of HCT-116
cells determined by flow cytometry using Annexin V-PE/7-AAD after
24 h of incubation. Data are presented as mean S.E.M. from three
independent experiments performed in triplicate. *p < 0.05 compared
with negative control (c) by One-way ANOVA followed by the Tur-
key’s Test. Ten thousand events were analyzed in each experiment,
and menadione (M; 20 µM) was used as positive control
doxorubicin (2 µM; %G2/M = 79.78 1.35 %), chalcone 6a
induced significant G2/M arrest (59.87 5.15 %) in HCT-
116 cells when compared with the negative control (treated
with only vehicle; %G2/M = 36.70 2.47 %). This means
that compound 6a may interfere with the process of cellular
division that is detectable by cell cycle checkpoints (Das and
Manna 2016). Consequently, the cells are triggered to cell
death by apoptosis, as we can observe in the data shown
above (morphological and flow cytometry data). It is worth
mentioning that the chalcone 6a was not able to induce DNA
at the tested concentrations and incubation time (data not
shown). It seems that DNA fragmentation is not involved
with the chalcone 6a-induced cell cycle arrest in HCT-116
cells, at least not as a starting point. This evidence reinforces
the hypothesis of mitotic spindle network interference that
shall be investigated further. In fact, Martel-Frachet et al.
(2015) identified a new chalcone derivative that is capable of
inducing prometaphase arrest and subsequent apoptosis of
bladder cancer cells that acts as a microtubule inhibitor.
Finally, it is important to note that additional tests are
necessary to evaluate the specific mechanism of action
involved in the selective cytotoxicity of chalcone 6a against
HCT-116 cells.
Fig. 6 Effect of chalcone 6a on cell membrane integrity (a) and cell
number (b) of HCT-116 cells determined by flow cytometry using
propidium iodate (PI) after 24 h incubation. Data are presented as
mean S.E.M. from three independent experiments performed in tri-
plicate. *p < 0.05 compared with negative control (c) by One-way
ANOVA followed by the Turkey’s Test. Ten thousand events were
analyzed in each experiment, and doxorubicin (d; 2 µM) was used as
positive control
Conclusions
Fig. 7 Effect of chalcone 6a on the DNA content of HCT-116 cells
determined by flow cytometry using propidium iodate (PI) after 24 h of
incubation. Data are presented as mean S.E.M. from three indepen-
dent experiments performed in triplicate. *p < 0.05 compared with
negative control (c) by ANOVA followed by Turkey’s Test. Ten
thousand events were analyzed in each experiment, and doxorubicin
(d; 2 µM) was used as positive control
A series of twenty E-isomer chalcones were synthetized by
the Claisen–Schmidt condensation reaction; nine 4′-ami-
nochalcones, nine 4′-acetamidechalcones, and two 3′-
methoxy-4′-hydroxychalcones. Regarding cytotoxic activ-
ity, this work demonstrated that small changes in the

Medicinal Chemistry Research
structure of the chalcone are capable of improving the
in vitro effects against human cancer cells. In this context,
chalcone N-(4′-[(E)-3-(3-nitrophenyl)-1-(phenyl)prop-2-en-
1-one]) acetamide (6a) containing an amide group on ring A
and a nitro group on ring B showed a strong and selective
cytotoxic effect against HCT-116 cells and was able to
induce cell death by apoptosis related to G2/M cell cycle
arrest. These data provide us with the prospect of con-
ducting studies on the specific mechanism of action of
chalcone 6a using human cancer cell-based assays.
De Campos-Buzzi F, Padaratz P, Meira VA, Corrêa R, Nunes JR,
Cechinel-Filho V (2007) 4′-acetamidochalcone derivatives as
potential antinociceptive agents. Molecules 12:896–906
de Campos-Buzzi F, Pereira de Campos J, Pozza Tonini P, Corrêa R,
Augusto Yunes R, Boeck P, Cechinel-Filho V (2006) Anti-
nociceptive effects of synthetic chalcones obtained from xan-
thoxyline. Arch Pharm 339:361–365
Das M, Manna K (2016) Chalcone scaffold in anticancer arma-
mentarium: a molecular insight. J Toxicol 6:7651047
Di Carlo G, Mascolo N, Izzo AA, Capasso F (1999) Flavonoids: old
and new aspects of a class of natural therapeutic drugs. Life Sci
65:337–353
Ducki S, Forrest R, Hadfield JA, Kendall A, Lawrence NJ, McGown
AT, Rennison D (1998) Potent antimitotic and cell growth inhi-
bitory properties of substituted chalcones. Bioorg Med Chem Lett
8:1051–1056
Guilherme AM, Jardim T, Guimaraes T, Maria do Carmo FR, Pinto
BC, Cavalcanti KMF, Pessoa C, Gatto CC, Nair DK, Nam-
boothiri INN, Junior ENS (2015) Naphthoquinone-based chal-
cone hybrids and derivatives: synthesis and potent activity against
cancer cell lines. Med Chem Comm 6:120–130
Acknowledgements The authors thank the National Cancer Institute
(Bethesda, MD, USA) for donating of all human tumor cell lines used
in this study. CENAUREMN—Northeastern Center for the Applica-
tion and Use of Nuclear Magnetic Resonance and EMBRAPA
AGROINDÚSTRIA TROPICAL-Multiuser Laboratory of Natural
Product Chemistry by obtaining the spectral data. Teixeira AMR also
acknowledges financial support from the PQ/CNPq (Grant#: 3 05719/
2018-1).
Guo L, Zhang H, Tian M, Tian Z, Xu Y, Yang Y, Peng H, Liu P, Liu Z
(2018) Electronic effects on reactivity and anticancer activity by
half-sandwich N,N-chelated iridium (III) complexes. New J
Chem 42:1–22
Compliance with ethical standards
Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next
generation. Cell 144:646–674
Hassan GS, El-Messery SM, Abbas A (2017) Synthesis and anticancer
activity of new thiazolo[3,2-a]pyrimidines: DNA binding and
molecular modeling study. Bioorg Chem 74:41–52
Jardim GAM, Guimarães TT, Pinto MCFR, Cavalcanti BC, Farias
KM, Pessoa C, Gatto CC, Nair DK, Namboothir INN, Júnior
ENS (2015) Naphthoquinone-based chalcone hubrids and deri-
vatives: synthesis and potent activity against cancer cell lines.
Med Chem Comm 6:120–130
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Karaman İ, Gezegen H, Gürdere MB, Dingil A, Ceylan M (2010)
Screening of biological activities of a series of chalcone deriva-
tives against human pathogenic microorganisms. Chem Biodivers
7:400–408
Karthikeyan CN, Moorthy SH, Ramasamy N, Vanam S, Manivannan
U, Karunagaran ED, Trivedi P (2015) Advances in chalcones
with anticancer activities. Recent Pat Anticancer Drug Discov
10:97–115
References
Abbas A, Naseer MM, Hasan A, Hadda TB (2014) Synthesis and
cytotoxicity studies of 4-Alkoxychalcones as new antitumor
agents. J Mater Environ Sci 5:281–292
Bhat BA, Dhar KL, Puri SC, Saxena AK, Shanmugavel M, Qazi GN
(2005) Synthesis and biological evaluation of chalcones and their
derived pyrazoles as potential cytotoxic agents. Bioorg Med
Chem Lett 15:3177–3180
Boeck P, Bandeira Falcão CA, Leal PC, Yunes RA, Filho VC, Torres-
Santos EC, Rossi-Bergmann B (2006) Synthesis of chalcone
analogues with increased antileishmanial activity. Bioorg Med
Chem 14:1538–1545
Bond MJ, Bleiler M, Harrison LE, Scocchera EW, Nakanishi M, G-
Dayanan N, Keshipeddy S, Rosenberg DW, Wright DL, Giar-
dina C (2018) Spindle assembly disruption and cancer cell
apoptosis with a CLTC-binding compound. Mol Cancer Res
16:1361–1372
Cabrera M, Simoens M, Falchi G, Lavaggi ML, Piro OE, Castellano
EE, Vidal A, Azqueta A, Monge A, de Ceráin AL, Sagrera G,
Seoane G, Cerecetto H, González M (2007) Synthetic chalcones,
flavanones, and flavones as antitumoral agents: biological eva-
luation and structure–activity relationships. Bioorg Med Chem
15:3356–3367
Corrêa R, Pereira MAS, Buffon D, dos Santos L, Filho VC, Santos
ARS, Nunes RJ(2001) Antinociceptive properties of chalcones.
Structure-activity relationships. Arch Pharm 334:332–334
Kepp O, Galluzzi L, Lipinski M, Yuan J, Kroemer G (2011) Cell death
assays for drug discovery. Nat Rev Drug Discov 10:221–237
Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES,
Baehrecke EH, Blagosklonny MV, El-Deiry WS, Golstein P,
Green DR, Hengartner M, Knight RA, Kumar S, Lipton SA,
Malorni W, Nuñez G, Peter ME, Tschopp J, Yuan J, Piacentini
M, Zhivotovsky B, Melino G (2008) Classification of cell death:
recommendations of the Nomenclature Committee on Cell Death
2009. Cell Death Differ 16:3–11
Lindamulage IK, Vu H-Y, Karthikeyan C, Knockleby J, Lee Y-F,
Trivedi P, Lee H (2017) Novel quinolone chalcones targeting
colchicine-binding pocket kill multidrug-resistant cancer cells by
inhibiting tubulin activity and MRP1 function. Sci Rep 7:1–5
Martel-Frachet V, Keramidas M, Nurisso A, DeBonis S, Rome C, Coll
JL, Ahcène B, Skoufias DA, Ronot X (2015) IPP51, a chalcone
acting as a microtubule inhibitor with in vivo antitumor activity
against bladder carcinoma. Oncotarget 6:14669–14686
Mosmann T (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. J
Immunol Methods 65:55–63

Medicinal Chemistry Research
Nazir S, Ansari FL, Hussain T, Mazhar K, Muazzam AG, Qasmi ZU,
Makhmoor T, Noureen H, Mirza B (2013) Brine shrimp lethality
assay ‘an effective prescreen’: microwave-assisted synthesis,
BSL toxicity and 3DQSAR studies-based designing, docking and
antitumor evaluation of potent chalcones. Pharm Biol
51:1091–1093
Rossette MC, Moraes DC, Sacramento EK, Romano-Silva MA, Car-
valho JL, Gomes DA, Caldas H, Friedman E, Bastos-Rodrigues
L, De Marco L (2017) The in vitro and in vivo antiangiogenic
effects of Flavokawain B. Phytother Res 31:1607–1613
Santos MB, Pinhanelli VC, Garcia MAR, Silva G, Baek SJ, França
SC, Fachim AL, Marins M, Regasini LO (2017) Antiproliferative
and pro-apoptotic activities of 2- and 4-aminochalcones against
tumor canine cells. Eur J Med Chem 138:884–889
Shankaraiah N, Nekkanti S, Brahma UR, Praveen Kumar N, Desh-
pande N, Prasanna D, Senwar KR, Jaya Lakshmi U (2017)
Synthesis of different heterocycles-linked chalcone conjugates as
cytotoxic agents and tubulin polymerization inhibitors. Bioorg
Med Chem 25:4805–4816
Nowakowska
Z (2007) A review of anti-infective and anti-
inflammatory chalcones. Eur J Med Chem 42:125–137
Padaratz P, Fracasso M, De Campos-Buzzi F, Corrêa R, Niero R,
Monache FD, Cechinel-Filho V (2009) Antinociceptive activity
of a new benzofuranone derived from a chalcone. Basic Clin
Pharm Toxicol 105:257–261
Pereira UA, Moreira TA, Barbosa LCA, Maltha CRA, Bomfim IS,
Maranhão SS, Moraes MO, Pessoa C, Barros-Nepomuceno FWA
(2016) Rubrolide analogues and their derived lactams as potential
anticancer agents. Med Chem Comm 7:345–352
Zingales SK, Moore ME (2016) Design and Synthesis of (2-(furanyl)
vinyl)-1-tetralone chalcones as anticancer agents. Der Pharma
Chem 8:40–47
Prasad YR, Rani VJ, Rao AS (2013) In vitro antioxidant activity and
scavenging effects of some synthesized 4’-aminochalcones.
Asian J Chem 25:52–58
Tristão TC, Campos-Buzzi F, Cruz RCB, Filho VC, Cruz AB (2012)
Antimicrobial and citotoxicity potential of acetamido, amino and
nitrochalcones. Arzneimittelforschung 62:590–594