Synthesis and biological evaluation of novel aminochalcones as potential anticancer and antimicrobial agents

Article abstract of DOI:10.3390/molecules24224129

A series of 18 aminochalcone derivatives were obtained in yields of 21.5-88.6% by applying the classical Claisen-Schmidt reaction. Compounds 4-9, 14 and 16-18 with 4-ethyl, 4-carboxy-, 4-benzyloxy- and 4-benzyloxy-3-methoxy groups were novel, not previously described in the scientific literature. To determine the biological properties of the synthesized compounds, anticancer and antimicrobial activity assays were performed. Antiproliferative potential was evaluated on four different human colon cancer cell lines-HT-29, LS180, LoVo and LoVo/DX-using the SRB assay and compared with green monkey kidney fibroblasts COS7. Anticancer activity was described as the IC₅₀ value. The best results were observed for 2′-aminochalcone (1), 3′-aminochalcone (2) and 4′-aminochalcone (3) (IC₅₀ = 1.43-1.98 μg·mL^-1) against the HT-29 cell line and for amino-nitrochalcones 10-12 (IC₅₀ = 2.77-3.42 μg·mL^-1) against the LoVo and LoVo/DX cell lines. Moreover, the antimicrobial activity of all derivatives was evaluated on two strains of bacteria: Escherichia coli ATCC10536 and Staphylococcus aureus DSM799, the yeast strain Candida albicans DSM1386 and three strains of fungi: Alternaria alternata CBS1526, Fusarium linii KB-F1 and Aspergillus Niger DSM1957. In the case of E. coli ATCC10536 almost all derivatives hindered the bacterial growth (ΔOD = 0). Furthermore, the best results were observed in the presence of 4′-aminochalcone (3), that completely limited the growth of all tested strains at the concentration range of 0.25-0.5 mg·mL^-1. The strongest bacteriostatic activity was exhibited by novel 3′-amino-4-benzyloxychalcone (14), that prevented the growth of E. coli ATCC10536 with MIC = 0.0625 mg·mL^-1

Full text of DOI:10.3390/molecules24224129

molecules
Article
Synthesis and Biological Evaluation of Novel
Aminochalcones as Potential Anticancer and
Antimicrobial Agents
3
Joanna Kozłowska 1,
Mirosław Anioł 1
*
, Bartłomiej Potaniec 1,2 , Dagmara Baczyn´ska , Barbara Zarowska 4 and
˙
1
Department of Chemistry, Wrocław University of Environmental and Life Sciences,
Norwida 25, 50-375 Wrocław, Poland; b.potaniec@gmail.com (B.P.); miroslaw.aniol@upwr.edu.pl (M.A.)
ŁUKASIEWICZ Research Network - PORT Polish Center for Technology Development, Stabłowicka 147,
54-066 Wrocław, Poland
Department of Molecular and Cellular Biology, Faculty of Pharmacy with Division of Laboratory Diagnostics,
Wroclaw Medical University, Borowska 211A, 50-556 Wrocław, Poland; dagmara.baczynska@umed.wroc.pl
Department of Biotechnology and Food Microbiology, Wrocław University of Environmental
and Life Sciences, Chełmon´skiego 37, 51-630 Wrocław, Poland; barbara.zarowska@upwr.edu.pl
Correspondence: joannakozlowska3@gmail.com
2
3
4
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Nuno Vale
Received: 9 October 2019; Accepted: 13 November 2019; Published: 15 November 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: A series of 18 aminochalcone derivatives were obtained in yields of 21.5–88.6% by
applying the classical Claisen-Schmidt reaction. Compounds 14 and 16 18 with 4-ethyl,
4–
9
,
–
4-carboxy-, 4-benzyloxy- and 4-benzyloxy-3-methoxy groups were novel, not previously described
in the scientific literature. To determine the biological properties of the synthesized compounds,
anticancer and antimicrobial activity assays were performed. Antiproliferative potential was
evaluated on four different human colon cancer cell lines—HT-29, LS180, LoVo and LoVo/DX
—using the SRB assay and compared with green monkey kidney fibroblasts COS7. Anticancer
activity was described as the IC50 value. The best results were observed for 20-aminochalcone (
30-aminochalcone ( ) and 40-aminochalcone ( mL−1) against the HT-29 cell
) (IC50 = 1.43–1.98
line and for amino-nitrochalcones 10 12 (IC50 = 2.77–3.42
mL−1) against the LoVo and LoVo/DX
1),
2
3
µ
g
·
–
µg
·
cell lines. Moreover, the antimicrobial activity of all derivatives was evaluated on two strains of
bacteria: Escherichia coli ATCC10536 and Staphylococcus aureus DSM799, the yeast strain Candida albicans
DSM1386 and three strains of fungi: Alternaria alternata CBS1526, Fusarium linii KB-F1 and Aspergillus
niger DSM1957. In the case of E. coli ATCC10536 almost all derivatives hindered the bacterial growth
(
∆
OD = 0). Furthermore, the best results were observed in the presence of 40-aminochalcone (
completely limited the growth of all tested strains at the concentration range of 0.25–0.5 mg
3
·
), that
mL−1
.
The strongest bacteriostatic activity was exhibited by novel 30-amino-4-benzyloxychalcone (14), that
prevented the growth of E. coli ATCC10536 with MIC = 0.0625 mg·mL−1
.
Keywords: aminochalcone; anticancer activity; antimicrobial activity
1. Introduction
Chalcones (1,3-diphenyl-2-propene-1-ones) belong to a large group of bioactive flavonoids.
They are known as secondary metabolites of plants, which protect them from damage caused by
microorganisms, insects and animals. The chemical structure of these compounds is based on the
presence of two aromatic rings—A and B—connected with the three carbon chain containing an
α
,
β
-unsaturated bond and a carbonyl group [
1
]. Chalcones maintain an unflagging interest among
], antifungal [ ],
scientists due to their broad spectrum of biological activities, including antibacterial [
2
3
Molecules 2019, 24, 4129; doi:10.3390/molecules24224129
www.mdpi.com/journal/molecules

Molecules 2019, 24, 4129
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anticancer [
4–
6], antioxidant [7] and anti-inflammatory properties [
8]. Their biocapability is correlated
with the different electron-donor and electron-acceptor groups attached to the aromatic rings [9].
The literature describes chalcones containing a single amino group as promising agents with
antitumor [10], antimicrobial [11,12] and antioxidant activity [13]. The DPPH assay performed on
20-amino- and 40-aminochalcones proved their ability to scavenge free radicals. The mentioned activity
was satisfactory and comparable to that of ascorbic acid, a reference compound known for its high
antioxidant properties [14,15]. Additionally, the presence of less than five hydrogen donors and no
more than 10 hydrogen bond acceptors in the chalcones’ chemical structure, molecular weight lower
than 500 Da and the octanol-water partition coefficient (logP) not greater than 5, is consistent with
Lipinski’s rule of five and indicates the possible applicability of aminochalcones as drug candidates [16].
Molecular modeling techniques confirm the potential of monosubstituted chalcones and their usefulness
as anti-inflammatory agents. Interesting results were obtained for 20-aminochalcones, which docking
score was on a similar level to that of the widely used drug ibuprofen [17]. Moreover, 40-aminochalcone
(3
), 40-amino-4-fluorochalcone and 40-amino-4-methyl- chalcone exhibited strong inhibitory activity
against myeloperoxidase (MPO)—an enzyme, involved in numerous inflammatory diseases. The
IC50 values of these aminochalcones against MPO were comparable to that of 5-fluorotryptamine—a
potent inhibitor of this enzyme [18]. Furthermore, the position of the amino group attached to the
aromatic ring has a significant influence on the biological properties of the compounds. Trein et al.
tested the activity of 20-aminochalcone (
1 2 3) against the
), 30-aminochalcone ( ) and 40-aminochalcone (
parasitic protozoan Trichomonas vaginalis, which contributes to serious reproduction disorders, cervical
cancer and a predisposition to prostate cancer. The scientists observed the best results for compound
compared to a popular antibiotic—metronidazole [19].
2,
Besides, 20-aminochalcones substituted at the C-40, C-50, C-2, C-3 and C-4 positions have
demonstrated cytotoxic activity against epidermoid carcinoma of the nasopharynx (KB), breast cancer
(MCF-7), lung carcinoma (A-549) and ovarian cancer (1A9) cell lines [20]. Also, 20-aminochalcone
(1
) was described as the most potent inductor of apoptosis in TRAIL-resistant human colon cancer
(HT-29) cells [ ]. Santos et al. reported that chalcones with hydrophobic substituents on ring B such
4
as 40-amino-4-fluorochalcone, 40-amino-4-chlorochalcone and 20-amino-4-methylchalcone were the
strongest inhibitors of canine malignant histiocytic cell line (DH82) proliferation with IC50 values of
34.4, 31.4 and 38.2 mM, respectively. The authors proved that the mechanism of action is based on
apoptosis rather than necrosis compared to doxorubicin [21]. Moreover, 40-amino-4-fluorochalcone
and 40-amino-2-chlorochalcone were also described as promising agents against human gastric cancer
cell line (SGC7901) with MIC values of 7.44
µM and 11.15
µM, respectively. It is noteworthy that
their cytotoxic effects were higher than that of the reference—5-fluorouracil (MIC = 29.19
µM) [12].
The anticancer activity of 40-amino-1-naphthyl- chalcone and 40-amino-4-methyl-1-naphthylchalcone
described by Seba et al. proved the ability of these derivatives to inhibit the migration and invasion of
osteosarcoma cells, especially in p53-expressing cells (U2OS) [22]. Moreover, Santos et al. showed
that aminochalcones containing a fluorine atom at the C-2 position and a pyridyl group at the C-3
position induced apoptosis due to upregulated p53 protein expression in the breast cancer cell lines
MCF-7 (ER) and MDA-MB-231 (TNBC) [23]. The studies of Pati et al. proved that incorporation of an
amino moiety into the ring B of the methoxychalcones may increase their cytotoxicity against a murine
melanoma cell line (B16) with the effect depending on the location of the substituent [10].
The search for novel antibacterial and antifungal candidates, especially of natural origin,
maintains an unabated interest in the scientific community. Lin et al. described the high potential of
30-amino-2-iodochalcone to inhibit the growth of Mycobacterium tuberculosis, a Gram-positive pathogenic
bacterium which is an tuberculosis agent [24]. Moreover, 40-aminochalcones, especially those with
methoxy substituents attached to the ring B, exhibited antimicrobial activity against Escherichia
coli, Staphylococcus aureus, Candida albicans and Trichophyton mentagrophytes [11,25]. Furthermore,
the presence of a -NO2 group attached to the C-4 position enhances by 2–3 times the antifungal
activity against Microsporum canis, Trichophyton rubrum and Epidermophyton floccosum in comparison

Molecules 2019, 24, 4129
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to unsubstituted chalcone [26]. Additionally, 40-aminochalcones with fluorine, chlorine or bromine
atoms at the C-2, C-3 and C-4 positions in the B ring inhibited the growth of Bacillus pumilis, Bacillus
subtilis and Proteus vulgaris. Likewise, strong fungistatic activity of these derivatives was observed
against Aspergillus niger, C. albicans and Rhizopus oryzae. Both antibacterial and antifungal properties of
aminochalcones were comparable with the appropriate standards—amikacin and fluconazole [27].
In this paper, we describe the chemical synthesis of a library of 18 aminochalcones, including
10 novel derivatives, which have not been previously described in scientific reports. The next step
was the evaluation of the biological properties of all synthesized compounds. Cytotoxicity tests were
performed on four types of human colon cancer cell line—HT-29, LS180, LoVo and LoVo/DX and
normal green monkey fibroblast line COS7. Antitumor activity was expressed as the IC50 value and
compared with standard compounds—cisplatin and doxorubicin. Additionally, the antimicrobial
activity was determined against two strains of bacteria Escherichia coli ATCC10536 and Staphylococcus
aureus DSM799, the strain of yeast Candida albicans DSM1386 and three strains of fungi—Alternaria
alternata CBS1526, Fusarium linii KB-F1 and Aspergillus niger DSM1957 and expressed as microbial
growth curves. For compounds which completely hindered the growth of microorganisms (
the minimal inhibitory concentrations (MIC) values were evaluated.
∆OD = 0),
2. Results and Discussion
Aminochalcones were obtained as the result of base-catalyzed condensation reactions
of 20-aminoacetophenone, 30-aminoacetophenone and 40-aminoacetophenone with appropriate
benzaldehydes containing 4-ethyl, 4-nitro, 4-carboxy, 4-benzyloxy and 4-benzyloxy-3-methoxy groups.
Compounds 1–6 and 10–18 were obtained according to modified protocol described by Amir et al. [28].
Reactions were performed on a magnetic stirrer using a mixture of methanol and 1,4-dioxane 1:1 (v:v)
as solvent in the presence of sodium hydroxide (Scheme 1). After complete conversion of the substrates,
the reaction mixtures were poured into ice water and the precipitated crystals were collected and
purified using liquid column chromatography.
Scheme 1. Synthesis of aminochalcones 1–18.
In the case of amino-carboxy derivatives 7–9, the reactions were performed in mixture of ethanol
and water 2.5:1 (v/v) in the ice-bath at the time of addition of 4-formylbenzoic acid. After the
completion of the reaction, 1 M hydrochloric acid was added until the product crystals precipitated [29].
The reaction progress and the purity of synthesized compounds were analyzed using thin layer
chromatography (TLC) and high performance liquid chromatography (HPLC). As a result of our
research, the 18 aminochalcones were obtained with the yields of 21.5–88.6%. Compounds 4–9, 14 and
16–18 have never been synthesized earlier, according to scientific reports.

Molecules 2019, 24, 4129
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The structures of all compounds were determined by 1H and 13C nuclear magnetic resonance
(NMR), high resolution mass spectrometry (HRMS) and Fourier-transform infrared spectroscopy
(FTIR). Analysis of the 1H-NMR spectra confirmed the presence of the
α
,
β-unsaturated bonds in each
derivative. Characteristic signals from H-α and H-β appeared as doublets with coupling constants
J = 15.5–15.7 Hz. Furthermore, a peak in the 185.39–191.69 ppm range in the 13C-NMR spectra indicated
the presence of the carbonyl group. Moreover, two signals in the 120.83–127.58 ppm and 138.63–144.50
ppm regions were assigned to C-α and C-β, respectively.
Lipin´ski’s rule of five (known also as Pfizer’s rule of five) allows one to predict if a compound has
chemical and physical properties that would likely make it an orally active drug. In relation to this
principle, we calculated the molecular weight (MW), partition coefficient (logP), number of hydrogen
bond acceptors (nON) and number of hydrogen bond donors (nOHNH) of all synthesized derivatives
(Table 1). According to the rule of five, all aminochalcones 1–18 were characterized by parameters
consistent with Lipin´ski’s rule of five, which suggest that they could be taken into consideration as
potential medicines.
Table 1. Aminochalcones 1–18 and their Lipin´ski’s rule of five parameters.
Substituent
on Ring A
Substituent on
Ring B
No.
MW
logP
nON
nOHNH
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
20-NH2
30-NH2
40-NH2
20-NH2
30-NH2
40-NH2
20-NH2
30-NH2
40-NH2
20-NH2
30-NH2
40-NH2
20-NH2
30-NH2
40-NH2
20-NH2
30-NH2
40-NH2
4-H
4-H
4-H
4-CH2CH3
4-CH2CH3
4-CH2CH3
4-COOH
4-COOH
4-COOH
4-NO2
4-NO2
4-NO2
4-OCH2C6H5
4-OCH2C6H5
4-OCH2C6H5
3-OMe-4-OCH2C6H5
3-OMe-4-OCH2C6H5
3-OMe-4-OCH2C6H5
223.28
223.28
223.28
251.33
251.33
251.33
267.28
267.28
267.28
268.27
268.27
268.27
329.40
329.40
329.40
359.43
359.43
359.43
3.25
2.86
2.89
4.16
3.78
3.80
3.16
2.77
2.80
3.21
2.82
2.85
4.90
4.51
4.54
4.49
4.11
4.13
2
2
2
2
2
2
4
4
4
5
5
5
3
3
3
4
4
4
2
2
2
2
2
2
3
3
3
2
2
2
2
2
2
2
2
2
MW: Molecular Weight; logP: logarithm of octanol-water partition coefficient; nON: number of hydrogen bond
acceptors; nOHNH: number of hydrogen bond donors.
The next step of our research was the evaluation of the biological properties of all synthesized
aminochalcones. It is worth mentioning that there is no correlation between anticancer and antimicrobial
activities. However, as a result of our work, we present the biological potential of the synthesized
aminochalcones 1–18 concerning their antiproliferative and also antimicrobial activity.
Anticancer activity was evaluated on four different human colon cancer cell lines: HT-29, LS180,
LoVo and LoVo/DX. Due to problems with the mortality of cells isolated from healthy tissues, which
were characterized by a low proliferative potential and poor survivability, green monkey fibroblasts
COS7 were selected as a normal cell line to determine the toxicity of aminochalcones. The cytotoxic effect
of the compounds was investigated using the SRB assay and indicated as IC50 values in comparison
with two reference drugs—cisplatin (19) and doxorubicin (20) [30]. Santos et al.’s research on the
molecular mechanism of death of a canine malignant histiocytic cell line (DH82) proved that the action
of 40-amino-4-fluorochalcone, 40-amino-4-chlorochalcone and 20-amino-4- methylchalcone is based

Molecules 2019, 24, 4129
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on apoptosis [21]. Furthermore, Mai et al. described 20-aminochalcone (
1
) as the most potent and
selective agent against TRAIL-resistant breast (MCF-7), ovarian (Caov-3), lung (A549), liver (HepG2),
colorectal (HT-29), nasopharyngeal (CNE-1), erythromyeloblastoid (K-562) and T-lymphoblastoid
(CEM-SS) cancer cells. The IC50 for compound
as 4.39 M (0.98
mL−1). The authors also determined the molecular mechanism of action of
20-aminochalcone (
) against the HT-29 cell line, which, like in the case of the DH82 cell line, is based
on apoptosis [ ) was the strongest inhibitor of HT-29
]. In our investigation, 20-aminochalcone (
cell line proliferation (IC50 = 1.43
mL−1) (Table 2), exhibiting almost 12 times higher activity than
1 when tested on the HT-29 cell line was calculated
µ
µ
g
·
1
4
1
µg
·
the reference compound cisplatin (19, IC50 = 16.73
µg
mL−1) and four times lower in comparison to
·
doxorubicin (20, IC50 = 0.33 µg·mL−1). Furthermore, it was cytotoxic against the COS7 cell line with
an IC50 value of 2.64
of cisplatin.
µg
mL−1, which indicated its poor selectivity against cancer cells, similar to that
·
Table 2. Anticancer activity of aminochalcones 1–18 (IC50, [µg·mL−1]).
No.
Aminochalcone
HT-29
LS180
LoVo
LoVo/DX
COS7
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
20-Aminochalcone
1.43 ± 0.16
1.60 ± 0.01
1.98 ± 0.18
3.98 ± 0.03
3.43 ± 0.05
3.61 ± 0.21
4.26 ± 0.13
12.32 ± 0.32
2.06 ± 0.18
2.13 ± 0.18
4.68 ± 0.09
5.10 ± 0.65
4.16 ± 0.12
5.20 ± 0.30
7.86 ± 0.35
9.22 ± 0.72
1.56 ± 0.04
1.88 ± 0.12
2.84 ± 0.09
4.77 ± 0.37
1.79 ± 0.10
4.26 ± 0.19
7.68 ± 0.34
1.43 ± 0.02
1.97 ± 0.30
2.26 ± 0.05
4.78 ± 0.19
2.35 ± 0.08
3.68 ± 0.30
11.08 ± 0.44
2.64 ± 0.47
3.25 ± 0.66
3.85 ± 0.28
9.57 ± 0.52
4.42 ± 0.27
7.16 ± 0.34
7.78 ± 0.75
30-Aminochalcone
40-Aminochalcone
20-Amino-4-ethylchalcone
30-Amino-4-ethylchalcone
40-Amino-4-ethylchalcone
20-Amino-4-carboxychalcone
30-Amino-4-carboxychalcone
40-Amino-4-carboxychalcone
20-Amino-4-nitrochalcone
30-Amino-4-nitrochalcone
40-Amino-4-nitrochalcone
20-Amino-4-benzyloxychalcone
30-Amino-4-benzyloxychalcone
40-Amino-4-benzyloxychalcone
31.50 ± 0.13 27.44 ± 0.11 15.93 ± 1.04
32.42 ± 0.99 18.37 ± 0.19 73.54 ± 0.98 66.18 ± 1.32 32.00 ± 8.11
3.24 ± 0.27
2.77 ± 0.49
3.42 ± 0.54
ND
2.00 ± 0.35
2.89 ± 0.53
4.64 ± 0.54
ND
6.51 ± 0.62
8.23 ± 0.49
6.64 ± 0.29
3.76 ± 0.38
4.59 ± 0.29
1.49 ± 0.13
0.05 ± 0.02
1.11 ± 0.07
1.34 ± 0.10
1.80 ± 0.10
ND
0.96 ± 0.12
1.36 ± 0.06
2.00 ± 0.12
ND
5.19 ± 0.41
6.66 ± 0.48
4.59 ± 0.21
1.77 ± 0.09
ND
1.79 ± 0.14
2.30 ± 0.45
3.10 ± 0.50
ND
7.61 ± 0.43
13.39 ± 2.78
9.02 ± 0.17
3.45 ± 0.22
ND
7.54 ± 0.18
ND
7.83 ± 0.14
7.64 ± 0.53
5.77 ± 0.38
1.71 ± 0.13
3.54 ± 0.30
2.09 ± 0.12
0.73 ± 0.18
20-Amino-4-benzyloxy-3-methoxychalcone 4.85 ± 0.08
30-Amino-4-benzyloxy-3-methoxychalcone 2.90 ± 0.27
40-Amino-4-benzyloxy-3-methoxychalcone 5.01 ± 0.49
Cisplatin
Doxorubicin
16.73 ± 0.58
0.33 ± 0.02
2.03 ± 0.17
3.86 ± 0.62
5.32 ± 1.44
3.55 ± 1.15
ND—not detected in the tested concentration range.
Another scientific reports described compound
cancer cell line T47D with an IC50 value of 5.28
proved that 40-amino-2-chlorochalcone demonstrated the strongest anticancer activity on the HT-29
cell line with an IC50 value of 26.25 ) exhibited the
M [12]. In our research, 40-aminochalcone (
highest anticancer activity on the HT-29 cell line (IC50 = 1.98
mL−1) among the 40-aminochalcones
12 15 ) against all tested human
18. Similar activity was observed for 30-aminochalcone (
colon cancer cell lines (IC50 values in range of 1.60–2.13
mL−1). Moreover, compounds with ethyl,
3
·
as the most active against the human breast
µg
mL−1
[
31]. Furthermore, Wu et al.’s studies
µ
3
µg
·
3
,
6,
9,
,
,
2
µg
·
nitro and benzyloxy groups attached at the C-4 position and 4-benzyloxy-3-methoxy moieties on
ring B also represented a group of inhibitors stronger than cisplatin against the HT-29 cell line. In
the case of amino-carboxy derivatives
7
–
9
, the location of the amino group had a significant impact
) >
) > 40-amino-4-carboxychalcone (
8 9). A similar trend was observed on
on the IC50 value. The activity decreased in the following order: 20-amino-4-carboxychalcone (
7
30-amino-4-carboxychalcone (
other tested cell lines.
In the case of 20-amino-4-nitrochalcone (10), the scientific literature reports it as a weaker
antitumor agent in comparison to 20-aminochalcone (
1) against HT-29 [4]. In our research a similar
effect was observed. Incorporation of the electron-withdrawing -NO2 group at the C-4 position
resulted in about two times less cytotoxic effect on the HT-29 cell line. However, in the case of the
LS180, LoVo and LoVo/DX cell lines, the activities of all investigated amino-nitrochalcones were

Molecules 2019, 24, 4129
6 of 18
similar to that of compounds
1
–
3
and comparable to cisplatin (19). Unfortunately, they were also
toxic against monkey fibroblasts COS7 with IC50 values in range of 1.79–3.10
20-amino-4-benzyloxychalcone (13) was it not possible to determine the antitumor properties in the
tested concentration range for all tested cell lines.
µg
mL−1. Only for
·
Antimicrobial activity assays were performed in two stages. First, screening procedure was carried
out on two strains of bacteria—E. coli ATCC10536 and S. aureus DSM799, strain of yeast C. albicans
DSM1386 and three strains of fungi—A. alternata CBS1526, F. linii KB-F1 and A. niger DSM1957. Chosen
method allowed to present the microbial growth curves and calculate the duration of the lag-phase
and increase of optical density (
∆OD) in the presence of 18 aminochalcones at the concentration of 0.1%
(w/v) (Table 3). In all cases of OD = 0, minimal inhibitory concentration (MIC) values were evaluated
∆
(Table 4) and compared with reference substances: oxytetracycline (against bacteria), cycloheximide
(against yeast) and nystatin (against fungi).
Table 3. Antimicrobial activity of aminochalcones 1–18.
Strain
E. coli
S. aureus
C. albicans
A. alternata
F. linii
A. niger
Lag-phase [h]
∆OD
Lag-phase [h]
∆OD
Lag-phase [h]
∆OD
Lag-phase [h]
∆OD
Lag-phase [h]
∆OD
Lag-phase [h]
∆OD
Lag-phase [h]
∆OD
Lag-phase [h]
∆OD
Lag-phase [h]
∆OD
Lag-phase [h]
∆OD
Lag-phase [h]
∆OD
Lag-phase [h]
∆OD
Lag-phase [h]
∆OD
Lag-phase [h]
∆OD
Lag-phase [h]
∆OD
Lag-phase [h]
∆OD
4.0
1.61
10.0
0.12
-
0.0
-
0.0
-
0.0
-
0.0
3.5
0.16
-
0.0
-
0.0
-
0.0
-
0.0
-
0.0
-
0.0
-
0.0
-
0.0
-
0.0
-
0.0
-
2.5
1.67
-
0.0
-
3.0
1.58
24.5
0.14
-
16.5
1.86
-
0.0
-
0.0
-
0.0
26.5
0.40
12.0
0.39
-
0.0
-
0.0
26.0
1.14
12.5
1.12
0.5
0.20
42.0
0.55
13.0
0.87
13.0
0.92
12.0
0.63
19.5
1.04
8.5
13.5
1.95
18.5
0.73
30.0
0.23
-
0.0
7.5
1.04
8.0
0.80
9.0
0.40
26.0
0.37
13.0
0.45
13.5
0.99
-
0.0
-
0.0
-
11.0
2.14
-
0.0
1.5
0.34
-
0.0
30.0
0.60
25.5
0.36
36.5
0.41
-
Control
1
2
0.0
-
0.0
-
3
0.0
32.5
0.84
29.5
0.98
28.0
0.87
-
0.0
6.0
0.98
9.5
0.61
9.5
0.44
-
0.0
23.0
0.28
8.5
0.97
-
0.0
-
0.0
12.0
0.13
-
0.0
4.5
1.42
16.0
0.37
8.5
0.66
9.0
0.55
4.5
0.68
4
5
6
7
0.0
-
0.0
39.0
1.11
35.0
0.51
23.0
0.79
39.5
0.83
-
8
0.0
37.0
0.35
11.5
0.15
-
0.0
-
0.0
-
0.0
33.5
0.82
8.0
0.22
-
0.0
33.5
0.36
33.5
0.26
-
9
10
11
12
13
14
15
16
17
18
0.0
0.0
16.0
0.32
6.0
0.87
11.5
0.50
-
21.5
0.78
20.5
0.98
25.0
0.57
40.5
0.41
-
Lag-phase [h]
∆OD
Lag-phase [h]
∆OD
Lag-phase [h]
∆OD
Lag-phase [h]
∆OD
Lag-phase [h]
∆OD
Lag-phase [h]
∆OD
0.64
-
0.0
21.5
0.22
0.0
11.0
0.24
6.5
0.0
-
0.0
-
0.0
28.5
0.65
0.41
Oxytetracycline
Cycloheximide
Nystatin
NA
NA
NA
NA
0.0
0.0
-
0.0
NA
NA
NA
NA
NA
NA
NA
-
0.0
10.0
0.13
4.5
0.74
NA
OD—Optical Density (measured for λ = 560 nm); “-“—not determined in view of ∆OD = 0; NA—not applicable.

Molecules 2019, 24, 4129
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Table 4. Minimal inhibitory concentrations [mg·mL−1] for aminochalcones 1–18.
Strain
Compound
E. coli
S. aureus
C. albicans
A. alternata
F. linii
A. niger
1
2
3
-
0.25
0.5
0.25
0.25
0.25
-
0.5
0.5
1.0
0.5
0.5
-
-
0.125
-
0.5
0.25
4
0.5
-
-
-
-
-
5
6
1.0
-
-
-
-
-
-
1.0
-
-
-
-
7
8
9
10
11
12
13
14
0.125
0.125
0.125
0.25
1.0
0.25
0.25
0.0625
0.125
0.5
0.125
0.125
-
0.125
-
-
0.5
0.25
-
0.5
-
-
-
-
-
0.5
-
-
-
-
-
-
-
-
-
-
-
0.5
0.5
1.0
-
-
-
0.125
-
-
-
1.0
-
-
-
-
-
0.25
0.25
0.25
-
-
0.5
-
15
16
17
-
1.0
-
0.5
-
-
-
1.0
-
0.125
0.5
18
-
Oxytetracycline
Cycloheximide
Nystatin
0.5
NA
NA
0.125
NA
NA
NA
0.5
NA
NA
NA
1.0
NA
NA
0.5
NA
NA
0.25
“-“—not determined; NA—not applicable.
Prasad et al. described the antimicrobial potential of 40-aminochalcones with halogen atoms
and methoxy groups attached at various positions of ring B. These compounds effectively inhibited
the growth of B. subtilis, B. pumilus, S. aureus, E. coli and P. vulgaris [27]. In our investigations,
almost all aminochalcones (compounds
2
–
5
,
7
–
18) prevented the growth of E. coli ATCC10536 at
) as the strongest inhibitor of
the tested concentration. Suwito et al. described 40-aminochalcone (
3
E. coli ATCC25923 growth, comparable with two reference standards—sulfamerazine and sulfadiazine.
Furthermore, this derivative was also active against S. aureus ATCC25922 and C. albicans ATCC1023
and described by the authors as the best wide spectrum antimicrobial agent candidate [31]. In our
research, compound
3 was two times stronger against Gram-positive bacteria S. aureus DSM799 than
Gram-negative E. coli ATCC10536, confirming the known phenomenon of higher susceptibility of
Gram-negative strains to flavonoids [32]. Moreover, the activity of 40-aminochalcone (
3) against all
tested microorganisms was expressed with MIC values of 0.25–0.5 mg·mL−1 (Table 4).
In the case of S. aureus DSM799 complete growth inhibition was observed in the presence of
20-, 30-, 40-aminochalcones
13) and 4-benzyloxy-3-methoxy (16) moieties (
20-amino-4-carboxychalcone ( ) and 30-amino-4-carboxychalcone (
as the reference substance oxytetracycline, with a MIC value of 0.125 mg
aminochalcones ( 10 11 and 13) prevented the growth of C. albicans DSM1386. Additionally,
compound showed four times higher activity in comparison to cycloheximide and was the
best inhibitor of growth of this strain with a MIC value of 0.125 mg
mL−1 Only 30-amino-4-
1
–
3
, and aminochalcones with carboxy (
OD = 0). Among them, the new compounds
) exhibited just as strong activity
mL−1. Moreover, six
7–8), nitro (11–12), benzyloxy
(
∆
7
8
·
2,
3,
7
,
,
7
·
.
benzyloxychalcone (14) did not affect to the growth of C. albicans DSM1386, but in contrast limited
the growth of S. aureus DSM799, A. alternata CBS1526, F. linii KB-F1 and A. niger DSM1957 by
half. Moreover, compound 14 prevented the growth of E. coli ATCC10536 with an MIC value of
0.0625 mg
mL−1, which was the lowest concentration among all obtained results. This proved that
·
30-amino-4-benzyloxychalcone (14) could be a selective antibacterial agent against this bacterial strain,
eight times more effective than the reference substance—oxytetracycline.

Molecules 2019, 24, 4129
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In the case of A. alternata CBS1526, the strongest inhibition was observed for 20-, 30-,
40-aminochalcones , 40-amino-4-ethylchalcone ( ), 20-amino-4-carboxychalcone ( ) and 30-amino-
1
–3
6
7
4-benzyloxy-3-methoxychalcone (17). The lowest MIC values were determined for compounds
and 7 (MIC = 0.5 mg·mL−1), which were two times lower than for nystatin.
2, 3
Zhang et al.
investigated the antifungal activities of chalcones with 20-substituents,
including 20-aminochalcones, against the dermatophyte Trichophyton rubrum. Among them,
20-amino-3-nitro- chalcone and 20-amino-4-nitrochalcone (10) showed the highest activity with
MIC80 values of 0.5
µg
mL−1, which were only two times weaker than the standard—fluconazole
·
(
MIC80 = 0.25 µg·mL−1) [33]. In our studies, 40-aminochalcone (
3
), amino-nitrochalcones 10
–12 and
20-amino-4- benzyloxy-3-methoxychalcone (16) prevented the growth of F. linii KB-F1 (
∆
OD = 0).
Moreover, among all tested aminochalcones 10
–12 with -NO2 moieties attached at the C-4 position of
ring B, the best results were obtained in the presence of 20-amino-4-nitrochalcone (10) and 30-amino-4-
nitrochalcone (11), with MIC values of 0.5 mg
mL−1 for both derivatives. However, the minimal
inhibitory concentration of 40-amino-4-nitrochalcone (12) was two times higher in comparison to
compounds 10 and 11. These results proved that the position of the -NH2 group attached at ring A has
·
a significant impact on the biological activity of aminochalcones. Also amino-carboxychalcones 7–9
hindered the growth of F. linii KB-F1 by 2–4 fold in comparison to the untreated control. Interestingly,
the double extended lag-phase was observed only for 20-amino-4-carboxychalcone (7).
In the case of A. niger DSM1957, five compounds (
inhibition. Their activity was stronger than that of the reference substance (
20-aminochalcone ( ) and 20-amino-4-carboxychalcone (
) had the higher activity, with MIC values
of 0.125 mg
mL−1, which indicated they were two times more effective than nystatin. Incubations
1
,
3,
7
,
12 and 17) caused a complete growth
∆
OD = 0.74). Among them,
1
7
·
with other aminochalcones resulted in elongation of the lag-phase by up to 40 h. Simultaneously, in
comparison to the control culture, even a 4-fold decrease of optical density value was observed.
3. Materials and Methods
3.1. Chemicals
Reagents for synthesis: 20-aminoacetophenone, 30-aminoacetophenone, 40-aminoacetophenone,
benzaldehyde, 4-ethylbenzaldehyde, 4-formylbenzoic acid, 4-nitrobenzaldehyde, 4-(benzyloxy)-
benzaldehyde, 4-(benzyloxy)-3-methoxy-benzaldehyde and sodium hydroxide were purchased from
Sigma Aldrich (St. Louis, MO, USA).
3.2. Analysis
The progress of reactions was followed by thin layer chromatography (TLC) on silica gel-coated
aluminum sheets with a fluorescent indicator (DC-Alufolien, Kieselgel 60 F254; Merck, Darmstadt,
Germany). The TLC plates were sprayed with a solution of 1% Ce(SO4)2 and 2% H3[P(Mo3O10)4]
in 5% H2SO4 and heated to visualize synthesized compounds. The crude products were purified
by liquid column chromatography filled with silica gel (Kieselgel 60, 230–400 mesh, Merck). The
purity of the compounds was analysed by high performance liquid chromatography (HPLC) on a
Waters 2690 system (Milford, MA, USA) equipped a with Waters 996 Photodiode Array Detector. The
analysed samples were dissolved in methanol and were separated on a reverse-phase C-18 column
(Phenomenex, Torrance, CA, USA, Kinetex 5u XB-C18 100A, 250 mm
×
4.6 mm), thermostated at 28 ◦C.
The analysed samples were kept at 12 ◦C. The mobile phase consisted of two eluents: A—1% HCOOH
in H2O and B—1% HCOOH in MeCN. Elution gradient was started from 55% of eluent A to 45% of
eluent B over 15 min and a flow rate was amounted 1.5 mL·min−1
.
The structures of the obtained products were determined by 1H and 13C nuclear magnetic resonance
(NMR). Compounds for NMR analysis were dissolved in deuterated solvents: acetone-d6 or dimethyl
sulfoxide-d6. The spectra were recorded on a an AvanceTM600 MHz spectrometer (Bruker, Billerica,
MA, USA) (Supplementary Materials). The high-resolution ESI-MS spectra were measured on a Bruker

Molecules 2019, 24, 4129
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ESI-Q-TOF Maxis Impact Mass Spectrometer. The direct infusion ESI-MS parameters were: the mass
spectrometer was operated in positive ion mode with the potential between the spray needle and the
orifice of 3.5 kV, a nebulizer pressure of 0.4 bar, and a drying gas flow rate of 3.0 L
·
min−1 at 200 ◦C. The
sample flow rate was 3.0
µL
·
min−1. Ionization mass spectra were collected in a range of m/z 50–1250.
Melting points (uncorrected) were determined on a Boetius apparatus (Jena, Germany). Infrared spectra
were determined using a Tensor 27 FTIR spectrometer (Bruker) with ATR accessory with diamond
crystal in the wavelength range 400–4000 cm−1 (Supplementary Materials). Partition coefficient (logP)
was calculated by using the JSME Molecular Editor, ©Molinspiration Cheminformatics software
(Novartis Institutes for BioMedical Research Inc. and Bruno Bienfait).
3.3. Synthesis of Aminochalcones 1–8
3.3.1. Method I (Compounds 1–6, 10–18)
In a round bottomed flask 0.24 g (6.00 mmol) of sodium hydroxide was dissolved in 10 mL
methanol and 10 mL dioxane. After that, an appropriate aminoacetophenone (3.67 mmol) and the
corresponding benzaldehyde (3.67 mmol) were added. Reactions were performed in room temperature
on a magnetic stirrer until complete conversion of the substrates. After that, the reaction mixtures
were poured into ice water. Precipitated crystals were collected and purified by liquid column
chromatography on silica gel using mixtures of hexane-acetone, hexane-ethyl acetate or hexane-ethyl
acetate-methylene chloride as eluents. Compounds
4 and 6 were synthesized at 2.5 times smaller scale.
3.3.2. Method II (Compounds 7–9)
To 0.24 g (6.00 mmol) of sodium hydroxide dissolved in water (25 mL) and ethanol (10 mL),
4-formylbenzoic acid (3.67 mmol) was added. The resulting mixture was stirred in the ice-bath for
15 min and after that the corresponding aminoacetophenone (3.67 mmol) was added. The reaction was
continued on a magnetic stirrer in room temperature until complete conversion of the substrates. After
that, 1 M hydrochloric acid was added dropwise to the reaction mixtures until pH 6–7 was reached.
Precipitated crystals were filtered on a Büchner funnel, washed with distilled water and dried under
vacuum. Crude products
chloroform-methanol as eluent.
Spectroscopic data of all synthesized aminochalcones is described below:
8 and 9 were purified by liquid column chromatography using a mixture of
20-Aminochalcone ( ), yield 71.4% (0.59 g), yellow solid, m.p. 68–69 ◦C, lit. 70–71 ◦C [34]. FTIR-ATR
1
(cm−1): 3377.97, 3325.85, 3282.72, 1644.58, 1613.85, 1574.34, 1540.18, 1495.37, 1481.22, 1445.84, 1339.54,
1300.52, 1286.16, 1261.81, 1205.41, 1153.70, 1081.03, 1030.55, 1010.82, 997.22, 974.44, 854.19, 773.64,
736.01, 689.67, 660.81, 573.21, 555.57, 525.29, 509.08, 479.08, 427.87; 1H-NMR (600 MHz, acetone-d6)
δ
(ppm): 8.06 (dd, J = 8.2, 1.5 Hz, 1H, H-60), 7.93 (d, J = 15.5 Hz, 1H, H-
7.72 (d, J = 15.5 Hz, 1H, H-
), 7.47–7.39 (m, 3H, H-3, H-4, H-5), 7.30–7.26 (m, 1H, H-40), 7.13 (s, 2H,
-NH2), 6.85 (dd, J = 8.4, 1.2 Hz, 1H, H-30), 6.67–6.61 (m, 1H, H-50); 13C-NMR (150 MHz, acetone-d6)
(ppm): 191.61 (C=O), 153.05 (C-20), 142.98 (C- ), 136.38 (C-1), 134.99 (C-40), 132.01 (C-60), 130.80 (C-4),
129.72 (C-3, C-5), 129.23 (C-2, C-6), 124.08 (C-
), 119.15 (C-10), 117.90 (C-30), 115.71 (C-50); HR ESI-MS
m/z calculated for C15H14NO [M + H]+ 224.1070, found [M + H]+ 224.1069.
α), 7.82–7.79 (m, 2H, H-2, H-6),
β
δ
β
α
30-Aminochalcone ( ), yield 53.4% (0.44 g), yellow solid, m.p. 121–124 ◦C, lit. 115–118 ◦C [35]. FTIR-ATR
2
(cm−1): 3467.58, 3365.18, 2923.54, 1655.93, 1619.34, 1585.37, 1489.68, 1454.03, 1331.41, 1306.82, 1288.12,
1183.40, 1101.75, 1074.94, 1041.05, 992.90, 977.83, 918.50, 887.18, 874.56, 860.34, 758.17, 719.43, 688.09,
677.35, 563.60, 511.04, 488.38; 1H-NMR (600 MHz, acetone-d6)
7.76 (d, J = 15.7 Hz, 1H, H- ), 7.73 (d, J = 15.7 Hz, 1H, H-
(m, 1H, H-20), 7.38 (ddd, J = 7.6, 1.7, 1.0 Hz, 1H, H-60), 7.26–7.20 (m, 1H, H-50), 6.94 (ddd, J = 7.9, 2.4,
1.0 Hz, 1H, H-40), 4.91 (s, 2H, -NH2); 13C-NMR (150 MHz, acetone-d6)
(ppm): 190.25 (C=O), 149.81
(C-30), 144.11 (C- ), 140.03 (C-10), 136.15 (C-1), 131.13 (C-4), 130.04 (C-50), 129.79 (C-3, C-5), 129.34 (C-2,
δ
(ppm): 7.83–7.78 (m, 2H, H-2, H-6),
β
α), 7.49–7.42 (m, 3H, H-3, H-4, H-5), 7.43–7.38
δ
β

Molecules 2019, 24, 4129
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C-6), 123.34 (C-
[M + H]+ 224.1070, found [M + H]+ 224.1072.
α
), 119.55 (C-40), 117.84 (C-60), 114.46 (C-20); HR ESI-MS m/z calculated for C15H14NO
40-Aminochalcone ( ), yield 65.5% (0.54 g), yellow solid, m.p. 92–94 ◦C, lit. 90–92 ◦C [25]. FTIR-ATR
3
(cm−1): 3328.68, 3218.16, 1625.81, 1602.80, 1575.64, 1541.66, 1516.96, 1494.00, 1446.84, 1339.18, 1302.12,
1286.15, 1227.37, 1172.18, 1134.22, 1030.61, 1018.78, 997.56, 971.61, 829.45, 766.37, 733.12, 694.83, 673.12,
638.13, 619.11, 559.57, 521.60, 501.64, 479.66, 421.47; 1H-NMR (600 MHz, acetone-d6)
δ
(ppm): 8.01–7.94
), 7.80–7.77 (m, 2H, H-2, H-6), 7.71 (d, J = 15.6 Hz, 1H,
), 7.47–7.37 (m, 3H, H-3, H-4, H-5), 6.80–6.72 (m, 2H, H-30, H-50), 5.57 (s, 2H, -NH2); 13C-NMR
(150 MHz, acetone-d6)
(ppm): 187.03 (C=O), 154.30 (C-40), 142.52 (C- ), 136.51 (C-1), 131.79 (C-20,
C-60), 130.67 (C-4), 129.69 (C-3, C-5), 129.14 (C-2, C-6), 127.76 (C-10), 123.14 (C- ), 114.01 (C-30, C-50);
HR ESI-MS m/z calculated for C15H14NO [M + H]+ 224.1070, found [M + H]+ 224.1074.
(m, 2H, H-20, H-60), 7.84 (d, J = 15.6 Hz, 1H, H-
H-
α
β
δ
β
α
20-Amino-4-ethylchalcone ( ), yield 72.8% (0.27 g), yellow solid, m.p. 34–36 ◦C. FTIR-ATR (cm−1): 3484.35,
4
3334.87, 2962.18, 2927.8, 2868.20, 1636.56, 1611.41, 1563.57, 1533.53, 1510.28, 1478.79, 1441.51, 1418.43,
1348.67, 1332.47, 1287.79, 1261.38, 1206.76, 1181.12, 1156.39, 1061.64, 1000.90, 990.92, 972.77, 867.49,
841.81, 826.64, 772.55, 744.57, 682.33, 655.83, 565.38, 526.32, 501.57; 1H-NMR (600 MHz, acetone-d6)
δ
(ppm): 8.05 (dd, J = 8.2, 1.3 Hz, 1H, H-60), 7.88 (d, J = 15.5 Hz, 1H, H-
H-
), 7.32–7.24 (m, 3H, H-3, H-5, H-40), 7.11 (s, 2H, -N 2), 6.84 (dd, J = 8.3, 1.3 Hz, 1H, H-30), 6.66–6.60
(m, 1H, H-50), 2.67 (q, J = 7.6 Hz, 2H, -CH2-CH3), 1.23 (t, J = 7.6 Hz, 3H, -CH3); 13C-NMR (150 MHz,
acetone-d6)
(ppm): 191.68 (C=O), 153.00 (C-20), 147.49 (C-4), 143.10 (C- ), 134.89 (C-40), 133.91 (C-1),
131.94 (C-60), 129.39 (C-2, C-6), 129.24 (C-3, C-5), 123.08 (C- ), 119.26 (C-10), 117.89 (C-30), 115.70 (C-50),
H3); HR ESI-MS m/z calculated for C17H18NO [M + H]+ 252.1383, found
α), 7.73–7.69 (m, 3H, H-2, H-6,
β
H
δ
β
α
29.32 (-CH2-CH3), 15.84 (-C
[M + H]+ 252.1388.
30-Amino-4-ethylchalcone ( ), yield 47.9% (0.45 g), yellow solid, m.p. 69–71 ◦C. FTIR-ATR (cm−1): 3447.32,
5
3358.78, 2964.59, 2929.94, 1660.30, 1646.31, 1620.74, 1578.10, 1508.68, 1488.31, 1455.59, 1416.74, 1329.17,
1289.15, 1204.55, 1178.87, 1043.10, 992.38, 976.26, 951.87, 921.46, 869.62, 822.57, 776.37, 742.39, 717.24,
675.97, 653.54, 551.26, 534.59, 489.12, 423.57; 1H-NMR (600 MHz, acetone-d6)
H-2, H-6, H-α, H-β
δ
(ppm): 7.75–7.68 (m, 4H,
), 7.43–7.39 (m, 1H, H-20), 7.40–7.34 (m, 1H, H-60), 7.32–7.27 (m, 2H, H-3, H-5), 7.23
(t, J = 7.8 Hz, 1H, H-50), 6.96–6.91 (m, 1H, H-40), 4.91 (s, 2H, -NH2), 2.67 (q, J = 7.6 Hz, 2H, -CH2-CH3),
1.22 (t, J = 7.6 Hz, 3H, -CH3); 13C-NMR (150 MHz, acetone-d6) (ppm): 190.27 (C=O), 149.75 (C-30),
147.85 (C-4), 144.22 (C-
), 140.14 (C-10), 133.64 (C-1), 130.00 (C-50), 129.47 (C-2, C-6), 129.28 (C-3, C-5),
122.34 (C- H2-CH3), 15.80 (- H3); HR ESI-MS m/z
), 119.46 (C-40), 117.80 (C-60), 114.45 (C-20), 29.32 (-
calculated for C17H18NO [M + H]+ 252.1383, found [M + H]+ 252.1391.
δ
β
α
C
C
40-Amino-4-ethylchalcone ( ), yield 70.8% (0.26 g), yellow solid, m.p. 125–125 ◦C. FTIR-ATR (cm−1):
6
3465.95, 3334.06, 3217.87, 2961.76, 2928.20, 1628.78, 1596.43, 1579.38, 1556.72, 1509.02, 1457.23, 1437.83,
1417.82, 1336.26, 1303.40, 1288.36, 1224.09, 1171.45, 1119.89, 1018.06, 992.87, 814.32, 752.79, 731.09,
680.00, 636.54, 608.50, 550.12, 497.23, 419.01; 1H-NMR (600 MHz, acetone-d6)
δ
(ppm): 8.00–7.94 (m, 2H,
), 7.32–7.25 (m, 2H, H-3, H-5),
6.79–6.71 (m, 2H, H-30, H-50), 5.55 (s, 2H, -NH2), 2.67 (q, J = 7.6 Hz, 2H, -CH2-CH3), 1.22 (t, J = 7.6 Hz
3H, -C
3); 13C-NMR (150 MHz, acetone-d6) (ppm): 187.08 (C=O), 154.21 (C-40), 147.32 (C-4), 142.60
(C- ),
), 134.04 (C-1), 131.72 (C-20, C-60), 129.29 (C-2, C-6), 129.21 (C-3, C-5), 127.89 (C-10), 122.16 (C-
114.01 (C-30, C-50), 29.31 (- H3); HR ESI-MS m/z calculated for C17H18NO [M + H]+
H2-CH3), 15.84 (-
252.1383, found [M + H]+ 252.1391.
H-20, H-60), 7.79 (d, J = 15.5 Hz, 1H, H-
α), 7.72–7.67 (m, 3H, H-2, H-6, H-β
,
H
δ
β
α
C
C
20-Amino-4-carboxychalcone ( ), yield 70.0% (0.69 g), yellow solid, m.p. 188–191 ◦C. FTIR-ATR (cm−1):
7
2980.74, 2883.94, 1688.08, 1649.77, 1610.72, 1576.61, 1540.98, 1508.41, 1483.29, 1422.81, 1321.34, 1287.94,
1263.36, 1211.48, 1159.09, 1126.15, 1112.59, 1013.62, 980.03, 964.25, 930.50, 852.74, 782.57, 751.37, 700.21,
664.52, 624.92, 548.47, 524.65, 485.60, 444.08, 419.91; 1H-NMR (600 MHz, DMSO-d6)
δ
(ppm): 13.10
), 8.01–7.94 (m, 4H,
), 7.44 (s, 2H, -NH2), 7.32–7.26 (m, 1H, H-40), 6.81
(s, 1H, -COO
H-2, H-3, H-5, H-6), 7.66 (d, J = 15.5 Hz, 1H, H-
H α
), 8.10 (dd, J = 8.3, 1.5 Hz, 1H, H-60), 8.06 (d, J = 15.5 Hz, 1H, H-
β

Molecules 2019, 24, 4129
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(
dd, J = 8.4, 1.2 Hz, 1H, H-30), 6.62–6.56 (m, 1H, H-50); 13C-NMR (150 MHz, DMSO-d6)
(C=O), 166.93 (-
OOH), 152.20 (C-20), 140.49 (C-
(C-60), 129.71 (C-3, C-5), 128.63 (C-2, C-6), 125.65 (C-
ESI-MS m/z calculated for C16H14NO3 [M + H]+ 268.0968, found [M + H]+ 268.0975.
δ
(ppm): 190.27
C
β
), 139.22 (C-1), 134.51 (C-40), 131.65 (C-4), 131.53
α
), 117.32 (C-10), 116.94 (C-30), 114.50 (C-50); HR
◦
30-Amino-4-carboxychalcone (
8), yield 27.2% (0.27 g), yellow solid, m.p. decomposition > 200 C.
FTIR-ATR (cm−1): 2922.41, 1676.92, 1656.20, 1591.50, 1566.88, 1541.38, 1508.17, 1489.29, 1455.63, 1419.46,
1324.25, 1295.40, 1181.20, 1039.90, 992.10, 962.87, 880.80, 851.20, 799.93, 773.12, 757.12, 728.41, 692.02,
1
676.85, 620.45, 551.66, 503.42, 486.51, 456.88, 419.46; H-NMR (600 MHz, DMSO-d6)
δ
(ppm): 7.99
), 7.72 (d, J = 15.7
), 7.37–7.32 (m, 1H, H-60), 7.28 (t, J = 2.0 Hz, 1H, H-20), 7.21 (t, J = 7.8 Hz, 1H, H-50), 6.86
(ddd, J = 8.0, 2.4, 0.9 Hz, 1H, H-40), 5.49 (s, 2H, -N 2); 13C-NMR (150 MHz, DMSO-d6)
(ppm): 189.54
(C=O), 167.29 (-
OOH), 149.21 (C-30), 142.03 (C- ), 138.47 (C-10), 138.23 (C-1), 133.15 (C-4), 129.74 (C-3,
C-5), 129.26 (C-50), 128.65 (C-2, C-6), 124.38 (C- ), 118.84 (C-40), 116.50 (C-60), 112.99 (C-20); HR ESI-MS
m/z calculated for C16H14NO3 [M + H]+ 268.0968, found [M + H]+ 268.0971.
(
d, J = 8.4 Hz, 2H, H-3, H-5), 7.96–7.92 (m, 2H, H-2, H-6), 7.88 (d, J = 15.7 Hz, 1H, H-
α
Hz, 1H, H-
β
H
δ
C
β
α
◦
40-Amino-4-carboxychalcone (
9), yield 78.8% (0.78 g), yellow solid, m.p. decomposition > 200 C.
FTIR-ATR (cm−1): 1652.41, 1636.15, 1606.86, 1582.74, 1558.26, 1541.17, 1522.22, 1508.32, 1417.78, 1397.72,
1362.63, 1339.42, 1317.64, 1284.60, 1226.77, 1175.68, 1077.07, 1024.67, 973.97, 827.35, 779.52, 699.74,
1
664.81, 636.17, 594.70, 539.31, 498.02, 443.93, 418.55; H-NMR (600 MHz, DMSO-d6)
δ
(ppm): 8.00
), 7.87 (d, J = 8.0 Hz, 2H, H-2, H-6), 7.63
), 6.64 (d, J = 8.5 Hz, 2H, H-30, H-50), 6.26 (s, 2H, -NH2); 13C-NMR (150 MHz,
(ppm): 185.74 (C=O), 154.11 (- OOH), 140.60 (C-
), 137.71 (C-40), 131.23 (C-1, C-20, C-60),
), 112.78 (C-30, C-50); HR ESI-MS m/z
(
d, J = 8.0 Hz, 2H, H-3, H-5), 7.97–7.90 (m, 3H, H-20, H-60, H-
(d, J = 15.5 Hz, 1H, H-
DMSO-d6) β
α
β
δ
C
129.77 (C-10, C-3, C-5), 128.10 (C-2, C-6), 125.17 (C-4), 123.70 (C-
α
calculated for C16H14NO3 [M + H]+ 268.0968, found [M + H]+ 268.0972.
20-Amino-4-nitrochalcone (10), yield 77.9% (0.77 g), orange solid, m.p. 114–117 ◦C. FTIR-ATR (cm−1):
3458.66, 3336.15, 1646.30, 1615.73, 1577.18, 1542.17, 1509.30, 1480.24, 1447.16, 1338.57, 1282.27, 1260.70,
1205.19, 1167.95, 1108.82, 1005.11, 985.97, 852.37, 771.81, 740.52, 676.50, 660.28, 633.89, 563.69, 538.21,
524.51, 481.80, 440.76; 1H-NMR (600 MHz, DMSO-d6)
(m, 4H, H-2, H-6, H-60, H-
), 7.69 (d, J = 15.5 Hz, 1H, H-
6.82 (dd, J = 8.5, 1.2 Hz, 1H, H-30), 6.63–6.57 (m, 1H, H-50); 13C-NMR (150 MHz, DMSO-d6)
189.98 (C=O), 152.30 (C-20), 147.73 (C-4), 141.64 (C-1), 139.07 (C- ), 134.65 (C-40), 131.60 (C-60), 129.56
δ
(ppm): 8.28–8.23 (m, 2H, H-3, H-5), 8.17–8.09
), 7.48 (s, 2H, -N
2), 7.33–7.27 (m, 1H, H-40),
(ppm):
α
β
H
δ
β
(C-2, C-6), 127.58 (C-α
), 123.88 (C-3, C-5), 117.16 (C-10), 116.94 (C-30), 114.47 (C-50); HR ESI-MS m/z
calculated for C15H13N2O3 [M + H]+ 269.0921, found [M + H]+ 269.0926.
30-Amino-4-nitrochalcone (11), yield 55.1% (0.55 g), orange solid, m.p. 166–168 ◦C. FTIR-ATR (cm−1):
3425.98, 3353.4, 1658.08, 1578.12, 1541.87, 1508.46, 1488.46, 1455.75, 1412.60, 1332.24, 1316.38, 1289.71,
1186.59, 1106.99, 1076.88, 1040.63, 979.55, 968.98, 943.39, 920.05, 881.78, 869.10, 835.22, 789.52, 767.13,
749.70, 715.47, 678.11, 620.66, 533.29, 476.30, 451.08, 420.64; 1H-NMR (600 MHz, DMSO-d6)
8.30–8.25 (m, 2H, H-3, H-5), 8.15–8.11 (m, 2H, H-2, H-6), 7.99 (d, J = 15.7 Hz, 1H, H-
1H, H-
), 7.38 (ddd, J = 7.6, 1.7, 0.9 Hz, 1H, H-60), 7.30–7.26 (m, 1H, H-20), 7.22 (t, J = 7.8 Hz, 1H, H-50),
6.87 (ddd, J = 8.0, 2.4, 0.9 Hz, 1H, H-40), 5.40 (s, 2H, -NH2); 13C-NMR (150 MHz, DMSO-d6)
(ppm):
189.40 (C=O), 149.24 (C-30), 147.99 (C-4), 141.30 (C-1), 140.44 (C- ), 137.98 (C-10), 129.71 (C-2, C-6),
δ
(ppm):
α
), 7.76 (d, J = 15.7 Hz
,
β
δ
β
129.26 (C-50), 126.49 (C- ), 123.94 (C-3, C-5), 119.04 (C-40), 116.65 (C-60), 112.89 (C-20); HR ESI-MS m/z
α
calculated for C15H13N2O3 [M + H]+ 269.0921, found [M + H]+ 269.0927.
40-Amino-4-nitrochalcone (12), yield 71.0% (0.70 g), orange solid, m.p. 205–207 ◦C, lit. 206–207 ◦C [36].
FTIR-ATR (cm−1): 3467.21, 3377.82, 1638.35, 1601.18, 1581.43, 1560.34, 1508.85, 1442.14, 1413.84, 1344.40,
1316.83, 1289.13, 1233.98, 1180.20, 1135.16, 1110.55, 1017.59, 1003.98, 987.79, 967.72, 853.58, 829.10, 780.21,
758.82, 734.39, 697.42, 666.20, 634.07, 599.08, 541.89, 479.30, 412.79; 1H-NMR (600 MHz, DMSO-d6)
δ
(ppm): 8.27–8.23 (m, 2H, H-3, H-5), 8.14–8.10 (m, 2H, H-2, H-6), 8.06 (d, J = 15.6 Hz, 1H, H-
α), 7.98–7.93
(m, 2H, H-20, H-60), 7.69 (d, J = 15.6 Hz, 1H, H- ), 6.67–6.61 (m, 2H, H-30, H-50), 6.25 (s, 2H, -NH2);
β

Molecules 2019, 24, 4129
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13C-NMR (150 MHz, DMSO-d6)
δ
(ppm): 185.39 (C=O), 154.25 (C-40), 147.68 (C-4), 141.81 (C-1), 138.63
(C-β α
), 131.42 (C-20, C-60), 129.46 (C-2, C-6), 126.67 (C- ), 124.99 (C-10), 123.89 (C-3, C-5), 112.77 (C-30,
C-50); HR ESI-MS m/z calculated for C15H13N2O3 [M + H]+ 269.0921, found [M + H]+ 269.0929.
20-Amino-4-benzyloxychalcone (13), yield 88.6% (1.08 g), yellow solid, m.p. 89–91 ◦C. FTIR-ATR (cm−1):
1646.91, 1612.83, 1570.88, 1541.84, 1508.20, 1483.15, 1454.26, 1419.95, 1379.69, 1350.66, 1337.33, 1288.39,
1265.74, 1243.87, 1207.27, 1174.49, 1155.63, 1115.90, 1079.11, 1006.35, 993.30, 916.28, 861.16, 848.14,
833.92, 809.90, 765.23, 735.03, 694.17, 679.03, 658.83, 637.27, 609.25, 515.69, 419.10; 1H-NMR (600 MHz,
acetone-d6)
H-2, H-6), 7.70 (d, J = 15.4 Hz, 1H, H-
7.37–7.30 (m, 1H, H-4”), 7.30–7.23 (m, 1H, H-40), 7.21–6.90 (m, 4H, H-3, H-5, -N
Hz, 1H, H-30), 6.67–6.58 (m, 1H, H-50), 5.19 (s, 2H, -O-CH2-); 13C-NMR (150 MHz, acetone-d6)
191.66 (C=O), 161.42 (C-4), 152.93 (C-20), 142.89 (C- ), 137.98 (C-1”), 134.77 (C-40), 131.88 (C-60), 130.99
), 119.39
H2-); HR ESI-MS m/z calculated for
δ
(ppm): 8.05 (dd, J = 8.1, 1.5 Hz, 1H, H-60), 7.80 (d, J = 15.4 Hz, 1H, H-
), 7.53–7.45 (m, 2H, H-2”, H-6”), 7.43–7.37 (m, 2H, H-3”, H-5”),
2), 6.83 (dd, J = 8.4, 1.1
(ppm):
α), 7.78–7.74 (m, 2H,
β
H
δ
β
(C-2, C-6), 129.31 (C-3”, C-5”), 129.21 (C-1), 128.73 (C-4”), 128.47 (C-2”, C-6”), 121.72 (C-
α
(C-10), 117.87 (C-30), 116.05 (C-3, C-5), 115.68 (C-50), 70.56 (-O-
C
C22H20NO2 [M + H]+ 330.1489, found [M + H]+ 330.1501.
◦
30-Amino-4-benzyloxychalcone (14), yield 71.2% (0.87 g), yellow solid, m.p. 115–120 C. FTIR-ATR
(cm−1): 3450.90, 3359.11, 2970.58, 1739.03, 1655.55, 1628.42, 1588.24, 1571.05, 1509.14, 1490.46, 1455.41,
1421.22, 1380.61, 1339.93, 1311.31, 1288.24, 1252.87, 1205.51, 1170.44, 1079.94, 1038.87, 1013.49, 986.95,
919.05, 873.84, 837.55, 805.06, 785.36, 763.25, 744.73, 734.80, 700.21, 677.19, 636.81, 610.87, 519.12, 433.36;
1H-NMR (600 MHz, acetone-d6)
7.62 (d, J = 15.6 Hz, 1H, H-
δ (ppm): 7.78–7.73 (m, 2H, H-2, H-6), 7.72 (d, J = 15.6 Hz, 1H, H-β),
α
), 7.52–7.46 (m, 2H, H-2”, H-6”), 7.44–7.38 (m, 3H, H-20, H-3”, H-5”), 7.37
(ddd, J = 7.6, 1.7, 1.0 Hz, 1H, H-60), 7.35–7.32 (m, 1H, H-4”), 7.22 (t, J = 7.7 Hz, 1H, H-50), 7.12–7.06
(m, 2H, H-3, H-5), 6.93 (ddd, J = 7.9, 2.4, 1.0 Hz, 1H, H-40), 5.19 (s, 2H, -O-CH2-), 4.89 (s, 2H, -NH2);
13C-NMR (150 MHz, acetone-d6)
δ β), 140.30
(ppm): 190.17 (C=O), 161.64 (C-4), 149.71 (C-30), 143.99 (C-
(C-10), 137.92 (C-1”), 131.11 (C-2, C-6), 129.97 (C-50), 129.32 (C-3”, C-5”), 128.94 (C-1), 128.75 (C-4”),
128.48 (C-2”,C-6”), 121.00 (C-α
), 119.34 (C-40), 117.76 (C-60), 116.10 (C-3, C-5), 114.44 (C-20), 70.57
(-O-CH2-); HR ESI-MS m/z calculated for C22H20NO2 [M + H]+ 330.1489, found [M + H]+ 330.1494.
40-Amino-4-benzyloxychalcone (15), yield 21.5% (0.26 g), yellow solid, m.p. 128–131 ◦C. FTIR-ATR (cm−1):
3471.33, 3375.40, 3336.84, 1646.24, 1620.54, 1597.14, 1583.39, 1570.48, 1557.08, 1508.12, 1453.58, 1438.61,
1421.51, 1382.48, 1348.70, 1313.09, 1290.93, 1257.82, 1220.24, 1170.16, 1133.07, 1080.19, 1025.84, 989.25,
818.93, 734.80, 696.30, 680.81, 637.97, 625.64, 597.14, 540.67, 517.95, 494.69, 412.69; 1H-NMR (600 MHz,
acetone-d6)
1H, H- ), 7.68 (d, J = 15.5 Hz, 1H, H-
7.37–7.30 (m, 1H, H-4”), 7.11–7.05 (m, 2H, H-3, H-5), 6.77–6.71 (m, 2H, H-30, H-50), 5.52 (s, 2H, -NH2),
5.18 (s, 2H, -O-CH2-); 13C-NMR (150 MHz, acetone-d6)
(ppm): 187.03 (C=O), 161.31 (C-4), 154.09
(C-40), 142.36 (C- ), 138.01 (C-1”), 131.65 (C-20, C-60), 130.86 (C-2, C-6), 129.35 (C-1), 129.31 (C-3”, C-5”),
δ
(ppm): 7.98–7.94 (m, 2H, H-20, H-60), 7.77–7.73 (m, 2H, H-2, H-6), 7.72 (d, J = 15.5 Hz,
), 7.52–7.47 (m, 2H, H-2”, H-6”), 7.43–7.37 (m, 2H, H-3”, H-5”),
α
β
δ
β
128.73 (C-4”), 128.47 (C-2”, C-6”), 128.02 (C-10), 120.83 (C- ), 116.03 (C-3, C-5), 113.99 (C-30, C-50), 70.55
α
(-O-CH2-); HR ESI-MS m/z calculated for C22H20NO2 [M + H]+ 330.1489, found [M + H]+ 330.1494.
20-Amino-4-benzyloxy-3-methoxychalcone (16), yield 73.4% (0.98 g), yellow solid, m.p. 94–96 ◦C. FTIR-ATR
(cm−1): 1748.36, 1643.61, 1610.51, 1573.59, 1541.47, 1507.85, 1475.96, 1449.99, 1438.81, 1418.98, 1396.47,
1380.93, 1305.43, 1255.22, 1229.51, 1203.47, 1154.88, 1143.52, 1076.24, 1033.12, 1023.47, 1003.5, 967.44,
904.14, 868.01, 846.46, 835.22, 800.7, 764.94, 732.05, 693.46, 660.11, 630.13, 615.46, 597.6, 552.26, 519.17,
501.56, 466.17, 419.96; 1H-NMR (600 MHz, acetone-d6)
δ
(ppm): 8.02 (dd, J = 8.2, 1.5 Hz, 1H, H-60), 7.82
β), 7.53–7.48 (m, 3H, H-2, H-2”, H-6”), 7.43–7.36
(d, J = 15.4 Hz, 1H, H- ), 7.69 (d, J = 15.4 Hz, 1H, H-
α
(m, 2H, H-3”, H-5”), 7.36–7.31 (m, 1H, H-4”), 7.30 (ddd, J = 8.2, 2.0, 0.6 Hz, 1H, H-6), 7.29–7.23 (m, 1H,
H-40), 7.13–7.05 (m, 3H, H-5, -NH2), 6.83 (dd, J = 8.3, 1.2 Hz, 1H, H-30), 6.64–6.58 (m, 1H, H-50), 5.18
(s, 2H, -O-CH2-), 3.91 (s, 3H, -O-CH3); 13C-NMR (150 MHz, acetone-d6)
δ 191.69 (C=O), 152.92 (C-4),
151.34 (C-3), 150.96 (C-20), 143.39 (C- ), 138.13 (C-1”), 134.75 (C-40), 131.88 (C-60), 129.67 (C-1), 129.25
β

Molecules 2019, 24, 4129
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(C-3”, C-5”), 128.68 (C-4”), 128.51 (C-2”, C-6”), 123.71 (C-6), 121.85 (C-
α
), 119.40 (C-10), 117.86 (C-30),
115.64 (C-50), 114.36 (C-5), 111.86 (C-2), 71.19 (-O-
C
H2-), 56.28 (-O-CH3); HR ESI-MS m/z calculated for
C23H22NO3 [M + H]+ 360.1594, found [M + H]+ 360.1601.
30-Amino-4-benzyloxy-3-methoxychalcone (17), yield 56.4% (0.75 g), yellow solid, m.p. 38 ◦C. FTIR-ATR
(cm−1): 3446.89, 3359.02, 3031.48, 2935.80, 1738.77, 1652.27, 1622.82, 1570.76, 1505.91, 1453.29, 1419.23,
1379.82, 1311.17, 1250.32, 1186.02, 1161.21, 1134.98, 1078.16, 1023.51, 991.84, 914.30, 843.29, 787.07,
734.08, 695.78, 679.72, 663.21, 626.50, 586.66, 550.42, 468.48, 425.64; 1H-NMR (600 MHz, acetone-d6)
δ
(ppm): 7.70 (d, J = 15.6 Hz, 1H, H-
7.49 (d, J = 2.1 Hz, 1H, H-2), 7.43–7.36 (m, 3H, H-20, H-3”, H-5”), 7.36–7.31 (m, 2H, H-60, H-4”), 7.30
ddd, J = 8.2, 2.1, 0.5 Hz, 1H, H-6), 7.21 (t, J = 7.8 Hz, 1H, H-50), 7.09 (d, J = 8.3 Hz, 1H, H-5), 6.92 (ddd,
J = 8.0, 2.4, 1.0 Hz, 1H, H-40), 5.18 (s, 2H, -O-C 3); 13C-NMR
2-), 4.89 (s, 2H, -NH2), 3.91 (s, 3H, -O-C
(150 MHz, acetone-d6) ),
(ppm): 190.26 (C=O), 151.57 (C-4), 150.96 (C-3), 149.70 (C-30), 144.50 (C-
140.34 (C-10), 138.07 (C-1”), 129.94 (C-50), 129.39 (C-1), 129.26 (C-3”, C-5”), 128.70 (C-4”), 128.52 (C-2”,
C-6”), 123.84 (C-6), 121.19 (C-
), 119.34 (C-40), 117.78 (C-60), 114.40 (C-20), 114.35 (C-5), 111.95 (C-2),
71.18 (-O- H2-), 56.29 (-O-
H3); HR ESI-MS m/z calculated for C23H22NO3 [M + H]+ 360.1594, found
[M + H]+ 360.1606.
β), 7.64 (d, J = 15.6 Hz, 1H, H-α), 7.52–7.49 (m, 2H, H-2”, H-6”),
(
H
H
δ
β
α
C
C
◦
40-Amino-4-benzyloxy-3-methoxychalcone (18), yield 51.5% (0.69 g), yellow solid, m.p. 181–184 C.
FTIR-ATR (cm−1): 3377.26, 2970.42, 1738.59, 1649.85, 1625.86, 1581.76, 1559.96, 1513.66, 1456.72, 1440.93,
1415.56, 1375.66, 1345.90, 1316.69, 1295.71, 1257.10, 1234.23, 1215.19, 1176.88, 1131.86, 1018.94, 1003.29,
975.16, 922.53, 872.90, 855.89, 829.71, 812.35, 794.53, 747.84, 730.35, 698.60, 614.60, 588.78, 557.84, 504.01,
459.73; 1H-NMR (600 MHz, acetone-d6)
δ
(ppm): 7.96–7.92 (m, 2H, H-20, H-60), 7.73 (d, J = 15.5 Hz,
1H, H- ), 7.65 (d, J = 15.5 Hz, 1H, H- ), 7.53–7.48 (m, 2H, H-2”, H-6”), 7.49 (d, J = 2.0 Hz, 1H, H-2),
α
β
7.42–7.37 (m, 2H, H-3”, H-5”), 7.36–7.31 (m, 1H, H-4”), 7.30–7.27 (m, 1H, H-6), 7.09 (d, J = 8.3 Hz, 1H,
H-5), 6.76–6.70 (m, 2H, H-30, H-50), 5.51 (s, 2H, -NH2), 5.19 (s, 2H, -O-CH2-), 3.91 (s, 3H, -O-CH3);
13C-NMR (150 MHz, acetone-d6)
(C-
C-6”), 128.04 (C-1), 123.52 (C-6), 120.98 (C-
(-O- H2-), 56.29 (-O-
H3); HR ESI-MS m/z calculated for C23H22NO3 [M + H]+ 360.1594, found [M +
H]+ 360.1605.
δ
(ppm): 187.04 (C=O), 154.10 (C-40), 151.23 (C-4), 150.99 (C-3), 142.84
β
), 138.20 (C-1”), 131.65 (C-20, C-60), 129.85 (C-10), 129.27 (C-3”, C-5”), 128.69 (C-4”), 128.53 (C-2”,
), 114.44 (C-5), 113.97 (C-30, C-50), 111.84 (C-2), 71.22
α
C
C
3.4. Anticancer Activity
Anticancer activity was evaluated using the sulforhodamine B assay (SRB) on four different
human colon cancer cell lines: HT-29, LS180, LoVo and LoVo/DX and green monkey kidney fibroblasts
COS7 (ATCC, Manassas, VA, USA). Cell lines HT-29 and COS7 were cultured in
(IIET PAS, Wrocław, Poland), LS180 in OptiMEM medium (IIET PAS), LoVo and LoVo/DX in mixture
of -MEM and OptiMEM medium (1:1 (v:v)). Each culture medium was supplemented with 10% fetal
bovine serum, 2 mM glutamine, 100
α-MEM medium
α
µ
g
·
mL−1 penicillin, 100
µ
g
·
mL−1 streptomycin and 0.25
µg
mL−1
·
amphotericin (all supplements purchased from Gibco (Paisley, UK). Cells were incubated at 37 ◦C, 5%
CO2 in HeraCELL 150i incubator (Thermo Fisher Scientific, Waltham, MA, USA).
For the SRB assay, cells were seeded onto 96-well plates (Sarstedt, Nümbrecht, Germany) at a
density of 5
removed and replaced with fresh medium, supplemented with aminochalcone derivatives in the
concentration ranging from 1
mL−1 to 100 mL−1. All stock solutions were prepared in DMSO at
a concentration of 10 mg
mL−1. Cells were incubated for 48 h and then the SRB assay was performed
·
103/well and cultured overnight as described above. Then, the growth medium was gently
µg
·
µg
·
·
as described previously [30]. Each experiment was performed in quadruplicate. The final results were
reported as the IC50 values calculated for three independent experiments. Cisplatin (Acros Organics,
Waltham, MA, USA) and doxorubicin (Fisher BioReagents, Waltham, MA, USA) were used as positive
controls of the test.

Molecules 2019, 24, 4129
14 of 18
3.5. Antimicrobial Activity
3.5.1. Microbial Growth Curve Calculation
The antimicrobial screening tests were performed on two strains of bacteria: E. coli ATCC10536
and S. aureus DSM799, strain of yeast C. albicans DSM1386 and three strains of fungi: F. linii KB-F1,
A. alternata CBS1526 and A. niger DSM1957. All microorganisms were obtained from the collection
of the Faculty of Biotechnology and Food Microbiology, Wrocław University of Environmental and
Life Sciences. The bacteria were cultured in nutrient broth (Biocorp, Warsaw, Poland), yeast and fungi
in YM medium (3 g yeast extract, 3 g malt extract, 5 g bacteriological peptone and 10 g of glucose
dissolved in 1 L of distilled water). The tests were carried out in 100-well microtiter plates. To each
well 280
dissolved in dimethyl sulfoxide (3% (w/v)) were added. The final concentration of the tested compound
18) was 0.1% (w/v). Each experiment were performed in 3 replications. The optical density of the
µL of culture medium, 10 µL of microorganism suspension and 10 µL of aminochalcone (1–18)
(1–
cell suspension was measured on Bioscreen C (Automated Growth Curve Analysis System Lab System,
Vantaa, Finland) at 560 nm automatically, at regular intervals of 30 min for 2–3 days. Cell cultures were
maintained at 28 ◦C on a continuous shaker. To prepare the growth curves for each strain, the mean
values of the absorbance in a function of time were used. The resulting antimicrobial activity was
expressed as the increase of optical density (∆OD) and was compared to that of the control cultures in
the medium supplemented with dimethyl sulfoxide and also with reference substances: oxytetracycline
(against bacteria), cycloheximide (against yeast) and nystatin (against fungi) (Sigma Aldrich).
3.5.2. Minimal Inhibitory Concentration (MIC) Evaluation
In all cases of complete growth inhibition in the screening tests (∆OD = 0), the minimal inhibitory
concentration (MIC) values were evaluated using the broth method. Sterile 96-well microtiter plates
(Sarstedt) with nutrient broth (for bacteria) (Biocorp) or YM medium (for yeast and filamentous
fungi) were inoculated with a standardized cell suspension of bacteria and yeast or fungal spores
of 105 cell mL−1, and supplemented with aminochalcones dissolved in DMSO. Final concentration
·
of tested compounds in the incubation mixtures ranged from 0.1% to 0.00625% and was achieved
with the series of two-fold dilutions. All analysis were conducted in triplicates. The plates were
incubated at 30 ◦C (bacteria and yeast) or 25 ◦C (filamentous fungi) for 48–72 h. The results were
measured with the Spark® Multimode Microplate Reader (Tecan, Männedorf, Switzerland) at a 560 nm
wavelength. The minimal inhibitory concentration was defined as the lowest concentration of a tested
compound that completely restricted the growth of the microorganism. In order to verify the obtained
results, microbial cultures subjected to each concentration of aminochalcone were inoculated onto
nutrient agar or YM plates and incubated as described above. The lack of growth on agar plate was a
confirmation of the MIC of the tested compound. The resulting MIC values were compared with the
reference substances: oxytetracycline (against bacteria), cycloheximide (against yeast) and nystatin
(against fungi) (Sigma Aldrich).
4. Conclusions
In this paper we describe the synthesis of a library of 18 aminochalcones with different
electron-withdrawing and electron-donating substituents at the C-4 position. Among them,
10 compounds were novel, never previously described in the scientific literature.
We evaluated the anticancer and antimicrobial activity of all synthesized derivatives. The best
results were observed for compounds
activity expressed as the IC50 value below 2
12 times stronger in comparison to the reference substance–cisplatin (19) (IC50 = 16.73
Similar effects were observed for aminochalcones with nitro groups attached to the C-4 position
(compounds 10 12) against the LoVo and LoVo/DX cell lines (IC50 < 2
mL−1). This proves that
the presence of a strong electron withdrawing group -NO2 group in the para position as well as the
1–
3
against the HT-29 human colon cancer cell line with
mL−1. This inhibition of proliferation was almost
mL−1).
µg
·
µg
·
–
µg
·

Molecules 2019, 24, 4129
15 of 18
aminochalcones without moieties attached to ring B significantly enhances the biological properties.
However, these derivatives are characterized by a high toxicity against green monkey fibroblasts
COS7. Based on the available knowledge concerning the mechanism of action of 20-aminochalcone
(1), we could assume that aminochalcones induce apoptosis rather than necrosis in colon cancer
cell lines. However, to confirm this hypothesis with certainty, further research into the molecular
mechanism of action is required and it will be taken into account in further studies. In the case of
antimicrobial activity, complete inhibition of the growth of all tested microorganisms under 0.1%
(w/v) concentration was observed for 40-aminochalcone (
derivatives (compounds
observed for novel 30-amino-4-benzyloxychalcone (14) with an MIC value of 0.0625 mg
presence of an amino group in the meta position and the additional aromatic ring in compound 14, which
increases the hydrophobicity of the molecule, may facilitate the penetration into the microorganisms’
cells. It is worth mentioning that the obtained aminochalcones fulfill all Lipinski’s rules, which allows
us to qualify them as potential drug candidates. Moreover, strong inhibition of proliferation against
different human colon cancer cell lines and prevention of the microbial growth proved their potential
applicability as anticancer and also antimicrobial agents.
3
). Furthermore, sixteen of the synthesized
18) prevented the growth of E. coli ATCC10536 and the best results were
mL−1. The
2–5, 7–
·
Supplementary Materials: The following are available online. Figure S1: 1H-NMR (600 MHz, acetone-d6)
spectrum of 20-aminochalcone ( ), Figure S2: 13C-NMR (150 MHz, acetone-d6) spectrum of 20-aminochalcone
1
(
1
), Figure S3: FTIR-ATR spectrum of 20-aminochalcone (
spectrum of 30-aminochalcone ( ), Figure S5: 13C-NMR (150 MHz, acetone-d6) spectrum of 30-aminochalcone
), Figure S6: FTIR-ATR spectrum of 30-aminochalcone ( ), Figure S7: 1H-NMR (600 MHz, acetone-d6)
spectrum of 40-aminochalcone ( ), Figure S8: 13C-NMR (150 MHz, acetone-d6) spectrum of 40-aminochalcone
), Figure S9: FTIR-ATR spectrum of 40-aminochalcone ( ), Figure S10: 1H-NMR (600 MHz, acetone-d6)
spectrum of 20-amino-4-ethylchalcone ( ), Figure S11: 13C-NMR (150 MHz, acetone-d6) spectrum of
20-amino-4-ethylchalcone ( ), Figure S12: FTIR-ATR spectrum of 20-amino-4-ethylchalcone (
), Figure S13:
1H-NMR (600 MHz, acetone-d6) spectrum of 30-amino-4-ethylchalcone ( ), Figure S14: 13C-NMR (150 MHz,
acetone-d6) spectrum of 30-amino-4-ethylchalcone ( ), Figure S15: FTIR-ATR spectrum of 30-amino-4-ethylchalcone
), Figure S16: 1H-NMR (600 MHz, acetone-d6) spectrum of 40-amino-4-ethylchalcone (
), Figure S17:
13C-NMR (150 MHz, acetone-d6) spectrum of 40-amino-4-ethylchalcone (
), Figure S18: FTIR-ATR spectrum of
40-amino-4-ethylchalcone ( ), Figure S19: 1H-NMR (600 MHz, DMSO-d6) spectrum of 20-amino-4-carboxychalcone
), Figure S20: 13C-NMR (150 MHz, DMSO-d6) spectrum of 20-amino-4-carboxychalcone (
), Figure S21:
FTIR-ATR spectrum of 20-amino-4-carboxychalcone ( ), Figure S22: 1H-NMR (600 MHz, DMSO-d6)
spectrum of 30-amino-4-carboxychalcone ( ), Figure S23: 13C-NMR (150 MHz, DMSO-d6) spectrum
of 30-amino-4-carboxychalcone ( ), Figure S24: FTIR-ATR spectrum of 30-amino-4-carboxychalcone (
),
Figure S25: 1H-NMR (600 MHz, DMSO-d6) spectrum of 40-amino-4-carboxychalcone (
), Figure S26:
13C-NMR (150 MHz, DMSO-d6) spectrum of 40-amino-4-carboxychalcone (
), Figure S27: FTIR-ATR
spectrum of 40-amino-4-carboxychalcone ( ), Figure S28: 1H-NMR (600 MHz, DMSO-d6) spectrum of
20-amino-4-nitrochalcone (10), Figure S29: 13C-NMR (150 MHz, DMSO-d6) spectrum of 20-amino-4-nitrochalcone
1
), Figure S4: 1H-NMR (600 MHz, acetone-d6)
2
(
2
2
3
(
3
3
4
4
4
5
5
(
5
6
6
6
(
7
7
7
8
8
8
9
9
9
(
10), Figure S30: FTIR-ATR spectrum of 20-amino-4-nitrochalcone (10), Figure S31: 1H-NMR (600 MHz,
DMSO-d6) spectrum of 30-amino-4-nitrochalcone (11), Figure S32: 13C-NMR (150 MHz, DMSO-d6)
spectrum of 30-amino-4-nitrochalcone (11), Figure S33: FTIR-ATR spectrum of 30-amino-4-nitrochalcone
(
11), Figure S34: 1H-NMR (600 MHz, DMSO-d6) spectrum of 40-amino-4-nitrochalcone (12), Figure S35:
13C-NMR (150 MHz, DMSO-d6) spectrum of 40-amino-4-nitrochalcone (12), Figure S36: FTIR-ATR
spectrum of 40-amino-4-nitrochalcone (12), Figure S37: 1H-NMR (600 MHz, acetone-d6) spectrum
of 20-amino-4-benzyloxychalcone (13), Figure S38: 13C-NMR (150 MHz, acetone-d6) spectrum of
20-amino-4-benzyloxychalcone (13), Figure S39: FTIR-ATR spectrum of 20-amino-4-benzyloxychalcone
(
13), Figure S40: 1H-NMR (600 MHz, acetone-d6) spectrum of 30-amino-4-benzyloxychalcone (14),
Figure S41: 13C-NMR (150 MHz, acetone-d6) spectrum of 30-amino-4-benzyloxychalcone (14), Figure S42:
FTIR-ATR spectrum of 30-amino-4-benzyloxychalcone (14), Figure S43: 1H-NMR (600 MHz, acetone-d6)
spectrum of 40-amino-4-benzyloxychalcone (15), Figure S44: 13C-NMR (150 MHz, acetone-d6) spectrum
of 40-amino-4-benzyloxychalcone (15), Figure S45: FTIR-ATR spectrum of 40-amino-4-benzyloxychalcone
(
(
(
15), Figure S46: 1H-NMR (600 MHz, acetone-d6) spectrum of 20-amino-4-benzyloxy-3-methoxychalcone
16), Figure S47: 13C-NMR (150 MHz, acetone-d6) spectrum of 20-amino-4-benzyloxy-3-methoxychalcone
16), Figure S48: FTIR-ATR spectrum of 20-amino-4-benzyloxy-3-methoxychalcone (16), Figure S49:
1H-NMR (600 MHz, acetone-d6) spectrum of 30-amino-4-benzyloxy-3-methoxychalcone (17), Figure S50:

Molecules 2019, 24, 4129
16 of 18
13C-NMR (150 MHz, acetone-d6) spectrum of 30-amino-4-benzyloxy-3-methoxychalcone (17), Figure S51:
FTIR-ATR spectrum of 30-amino-4-benzyloxy-3-methoxychalcone (17), Figure S52: 1H-NMR (600 MHz,
acetone-d6) spectrum of 40-amino-4-benzyloxy-3-methoxychalcone (18), Figure S53: 13C-NMR (150 MHz,
acetone-d6) spectrum of 40-amino-4-benzyloxy-3-methoxychalcone (18), Figure S54: FTIR-ATR spectrum of
40-amino-4-benzyloxy-3-methoxychalcone (18).
˙
Author Contributions: Conceptualization, J.K.; Formal analysis, J.K., B.P., D.B. and B.Z.; Investigation, J.K., B.P.,
˙
˙
D.B. and B.Z.; Methodology, J.K., B.P., D.B. and B.Z.; Project administration, J.K.; Supervision, M.A.; Visualization,
˙
J.K.; Writing–original draft, J.K.; Writing–review & editing, B.P., D.B., B.Z. and M.A.
Funding: This research was funded by the National Science Centre, Poland, Grant No. 2016/23/N/NZ7/01856.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Gomes, M.N.; Muratov, E.N.; Pereira, M.; Peixoto, J.C.; Rosseto, L.P.; Cravo, P.V.L.; Andrade, C.H.; Neves, B.J.
Chalcone derivatives: Promising starting points for drug design. Molecules 2017, 22, 1210. [CrossRef]
[PubMed]
2.
Moreira Os
ório, T.; Delle Monache, F.; Domeneghini Chiaradia, L.; Mascarello, A.; Regina Stumpf, T.; Roberto
Zanetti, C.; Bardini Silveira, D.; Regina Monte Barardi, C.; De Fatima Albino Smânia, E.; Viancelli, A.; et al.
Antibacterial activity of chalcones, hydrazones and oxadiazoles against methicillin-resistant Staphylococcus
aureus. Bioorganic Med. Chem. Lett. 2012, 22, 225–230. [CrossRef] [PubMed]
0
˙
3.
4.
Kozłowska, J.; Potaniec, B.; Zarowska, B.; Anioł, M. Microbial transformations of 4 -methylchalcones as an
efficient method of obtaining novel alcohol and dihydrochalcone derivatives with antimicrobial activity.
RSC Adv. 2018, 8, 30379–30386. [CrossRef]
Mai, C.W.; Yaeghoobi, M.; Abd-Rahman, N.; Kang, Y.B.; Pichika, M.R. Chalcones with electron-withdrawing
and electron-donating substituents: Anticancer activity against TRAIL resistant cancer cells, structure-activity
relationship analysis and regulation of apoptotic proteins. Eur. J. Med. Chem. 2014, 77, 378–387. [CrossRef]
Kar Mahapatra, D.; Kumar Bharti, S.; Asati, V. Anti-cancer chalcones: Structural and molecular target
perspectives. Eur. J. Med. Chem. 2015, 98, 69–114. [CrossRef]
Rybka, M.; Mercader, A.G.; Castro, E.A. Predictive QSAR study of chalcone derivatives cytotoxicity activity
against HT-29 human colon adenocarcinoma cell lines. Chemom. Intell. Lab. Syst. 2014, 132, 18–29. [CrossRef]
Chen, J.J.; Cheng, M.J.; Shu, C.W.; Sung, P.J.; Lim, Y.P.; Cheng, L.Y.; Wang, S.L.; Chen, L.C. A New Chalcone
and Antioxidant Constituents of Glycyrrhiza glabra. Chem. Nat. Compd. 2017, 53, 632–634. [CrossRef]
Vijaya Bhaskar Reddy, M.; Hung, H.Y.; Kuo, P.C.; Huang, G.J.; Chan, Y.Y.; Huang, S.C.; Wu, S.J.;
Morris-Natschke, S.L.; Lee, K.H.; Wu, T.S. Synthesis and biological evaluation of chalcone, dihydrochalcone,
and 1,3-diarylpropane analogs as anti-inflammatory agents. Bioorganic Med. Chem. Lett. 2017, 27, 1547–1550.
[CrossRef]
5.
6.
7.
8.
9.
Singh, P.; Anand, A.; Kumar, V. Recent developments in biological activities of chalcones: A mini review.
Eur. J. Med. Chem. 2014, 85, 758–777. [CrossRef]
10. Pati, H.N.; Holt, H.L.; LeBlanc, R.; Dickson, J.; Stewart, M.; Brown, T.; Lee, M. Synthesis and cytotoxic
properties of nitro-and aminochalcones. Med. Chem. Res. 2005, 14, 19–25. [CrossRef]
11. Tristão, T.C.; Campos-Buzzi, F.; Corrêa, R.; Cruz, R.C.B.; Cechinel Filho, V.; Bella Cruz, A. Antimicrobial and
Cytotoxicity Potential of Acetamido, Amino and Nitrochalcones. Arzneimittelforschung 2012, 62, 590–594.
[CrossRef] [PubMed]
12. Wu, J.; Wang, C.; Cai, Y.; Peng, J.; Liang, D.; Zhao, Y.; Yang, S.; Li, X.; Wu, X.; Liang, G. Synthesis and crystal
structure of chalcones as well as on cytotoxicity and antibacterial properties. Med. Chem. Res. 2012, 21,
444–452. [CrossRef]
13. Sulpizio, C.; Roller, A.; Giester, G.; Rompel, A. Synthesis, structure, and antioxidant activity of methoxy- and
hydroxyl-substituted 20-aminochalcones. Mon. Fur Chem. 2016, 147, 1747–1757. [CrossRef] [PubMed]
14. Prasad, Y.R.; Rani, V.J.; Rao, A.S. In vitro Antioxidant Activity and Scavenging Effects of Some Synthesized
40-Aminochalcones. Asian J. Chem. 2013, 25, 52–58. [CrossRef]
15. Ruanwas, P.; Chantrapromma, S.; Fun, H.K. Synthesis, Characterization, Antioxidant, and Antibacterial
Activities of 2-Aminochalcones and Crystal Structure of (2E)-1-(2-aminophenyl)-3-(4-ethoxyphenyl)-2-propen-
1-one. Mol. Cryst. Liq. Cryst. 2015, 609, 126–139. [CrossRef]

Molecules 2019, 24, 4129
17 of 18
16. Iqbal, H.; Prabhakar, V.; Sangith, A.; Chandrika, B.; Balasubramanian, R. Synthesis, anti-inflammatory and
antioxidant activity of ring-A-monosubstituted chalcone derivatives. Med. Chem. Res. 2014, 23, 4383–4394.
[CrossRef]
17. Balasubramanian, R.; Gopal, R.V. Design and in silico Analysis of Ring-A Monosubstituted Chalcones as
Potential Anti-Inflammatory Agents. Bull. Pharm. Res. 2012, 2, 70–77. [CrossRef]
18. Zeraik, M.; Ximenes, V.; Regasini, L.; Dutra, L.A.; Silva, D.; Fonseca, L.; Coelho, D.; Machado, S.; Bolzani, V.
40-Aminochalcones as novel inhibitor of the chlorinating activity of myeloperoxidase. Curr. Med. Chem.
2012, 19, 5405–5413. [CrossRef]
19. Trein, M.R.; Rodrigues e Oliveira, L.; Rigo, G.V.; Garcia, M.A.R.; Petro-Silveira, B.; da Silva Trentin, D.;
Macedo, A.J.; Regasini, L.O.; Tasca, T. Anti-Trichomonas vaginalis activity of chalcone and amino-analogues.
Parasitol. Res. 2019, 118, 607–615. [CrossRef]
20. Xia, Y.; Yang, Z.; Xia, P.; Bastow, K.F.; Nakanishi, Y.; Lee, K. Antitumor Agents. Part 202: Novel 20-Amino
Chalcones: Design, Synthesis and Biological Evaluation. Bioorganic Med. Chem. Lett. 2000, 10, 699–701.
[CrossRef]
21. Santos, M.B.; Pinhanelli, V.C.; Garcia, M.A.R.; Silva, G.; Baek, S.J.; França, S.C.; Fachin, A.L.; Marins, M.;
Regasini, L.O. Antiproliferative and pro-apoptotic activities of 20-and 40-aminochalcones against tumor
canine cells. Eur. J. Med. Chem. 2017, 138, 884–889. [CrossRef] [PubMed]
22. Seba, V.; Silva, G.; Bastos dos Santos, M.; Baek, S.J.; França, S.D.C.; Fachin, A.L.; Regasini, L.O.; Marins, M.
Chalcone Derivatives 40-Amino-1-Naphthyl-Chalcone (D14) and 40-Amino-4-Methyl-1-Naphthyl-Chalcone
Suppress Migration and Invasion of Osteosarcoma Cells Mediated by p53 Regulating EMT-Related Genes.
Int. J. Mol. Sci. 2018, 19, 2838. [CrossRef] [PubMed]
23. Bastos dos Santos, M.; Anselmo, D.B.; Gisleine de Oliveira, J.; Jardim-perassi, B.V.; Monteiro, D.A.; Silva, G.;
Gomes, E.; Fachin, A.L.; Marins, M.; Pires de Campos Zuccari, D.A.; et al. Antiproliferative activity and
p53 upregulation effects of chalcones on human breast cancer cells. J. Enzym. Inhib. Med. Chem. 2019, 34,
1093–1099. [CrossRef] [PubMed]
24. Lin, Y.M.; Zhou, Y.; Flavin, M.T.; Zhou, L.M.; Nie, W.; Chen, F.C. Chalcones and flavonoids as anti-tuberculosis
agents. Bioorganic Med. Chem. 2002, 10, 2795–2802. [CrossRef]
25. Suwito, H.; Jumina; Mustofa; Pudjiastuti, P.; Fanani, M.Z.; Kimata-Ariga, Y.; Katahira, R.; Kawakami, T.;
Fujiwara, T.; Hase, T.; et al. Design and synthesis of chalcone derivatives as inhibitors of the
ferredoxin—Ferredoxin-NADP+ reductase interaction of Plasmodium falciparum: Pursuing new antimalarial
agents. Molecules 2014, 19, 21473–21488. [CrossRef]
26.
López, S.N.; Castelli, M.V.; Zacchino, S.A.; Domínguez, J.N.; Lobo, G.; Charris-Charris, J.; Cortes, J.C.G.;
Ribas, J.C.; Devia, C.; Rodriguez, A.M.; et al. In vitro antifungal evaluation and structure–activity relationships
of a new series of chalcone derivatives and synthetic analogues, with inhibitory properties against polymers
of the fungal cell wall. Bioorganic Med. Chem. 2001, 9, 1999–2013. [CrossRef]
27. Prasad, Y.R.; Rao, A.S.; Rambabu, R. Synthesis of Some 40-Amino Chalcones and their Antiinflammatory and
Antimicrobial Activity. Asian J. Chem. 2009, 21, 907–914.
28. Amir, M.; Kumar, H.; Khan, S.A. Synthesis and pharmacological evaluation of pyrazoline derivatives as new
anti-inflammatory and analgesic agents. Bioorganic Med. Chem. Lett. 2008, 18, 918–922. [CrossRef]
29. Dimmock, J.R.; Jha, A.; Zello, G.A.; Quail, J.W.; Oloo, E.O.; Nienaber, K.H.; Kowalczyk, E.S.;
Allen, T.M.; Santos, C.L.; De Clercq, E.; et al. Cytotoxic N-[4-(3-aryl-3-oxo-1-propenyl)phenylcarbonyl]-3,5-
bis(phenylmethylene)-4-piperidones and related compounds. Eur. J. Med. Chem. 2002, 37, 961–972.
[CrossRef]
30. Kozłowska, J.; Grela, E.; Baczyn´ska, D.; Grabowiecka, A.; Anioł, M. Novel O-alkyl Derivatives of Naringenin
and Their Oximes with Antimicrobial and Anticancer Activity. Molecules 2019, 24, 679. [CrossRef]
31. Suwito, H.; Nyoman, N.; Puspaningsih, T. Anticancer and antimicrobial activity of methoxy amino chalcone
derivatives. Der Pharma Chem. 2015, 7, 89–94.
32. Cushnie, T.P.T.; Lamb, A.J. Antimicrobial activity of flavonoids. Int. J. Antimicrob. Agents 2005, 26, 343–356.
[CrossRef] [PubMed]
33. Zhang, K.-X.; Wang, W.; Zhao, F.; Meng, Q.-G.; Jin, Y.-S. Synthesis of Substituted Chalcones and Assessment
of their Antifungal Activity Against Trichophyton rubrum. Chem. Nat. Compd. 2018, 54, 158–160. [CrossRef]

Molecules 2019, 24, 4129
18 of 18
34. Castaing, M.; Wason, S.L.; Estepa, B.; Hooper, J.F.; Willis, M.C. 2-aminobenzaldehydes as versatile substrates
for rhodium-catalyzed alkyne hydroacylation: Application to dihydroquinolone synthesis. Angew. Chem.
Int. Ed. 2013, 52, 13280–13283. [CrossRef] [PubMed]
35. Karaman, I.; Gezegen, H.; Gürdere, M.B.; Dingil, A.; Ceylan, M. Screening of Biological Activities of a Series
of Chalcone Derivatives against Human Pathogenic Microorganisms. Chem. Biodivers. 2010, 7, 400–408.
[CrossRef] [PubMed]
36. Dimmock, J.R.; Jha, A.; Zello, G.A.; Allen, T.M.; Santos, C.L.; Balzarini, J.; De Clercq, E.; Manavathu, E.K.;
Stables, J.P. Cytotoxic 40-aminochalcones and related compounds. Pharmazie 2003, 58, 227–232. [CrossRef]
[PubMed]
Sample Availability: Samples of the compounds are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).