The cytotoxic properties of some tricyclic 1,3-dithiolium flavonoids

Article abstract of DOI:10.3390/molecules24132459

Background: Due to the emergence of multidrug resistant microorganisms, new classes of antibiotics are needed. In this paper, we present the cytotoxic effects of five tricyclic flavonoids, one of which was previously identified as a potent antimicrobial a

Full text of DOI:10.3390/molecules24132459

molecules
Article
The Cytotoxic Properties of Some Tricyclic
1,3-Dithiolium Flavonoids
Laura G. Sarbu 1, Sergiu Shova 2, Dragos Peptanariu 2 , Isabela A. Sandu 2, Lucian M. Birsa 1,
*
and Lucian G. Bahrin 1,2,
*
1
Alexandru Ioan Cuza University of Iasi, Department of Chemistry, 11 Carol I Blvd., 700506 Iasi, Romania
Petru Poni Institute of Macromolecular Chemistry, Intelcenter. 41A Grigore Ghica Vodă Alley,
700487 Iasi, Romania
2
*
Correspondence: lbirsa@uaic.ro (L.M.B.); bahrin.lucian@icmpp (L.G.B.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 17 June 2019; Accepted: 3 July 2019; Published: 4 July 2019
Abstract: Background: Due to the emergence of multidrug resistant microorganisms, new classes of
antibiotics are needed. In this paper, we present the cytotoxic effects of five tricyclic flavonoids, one of
which was previously identified as a potent antimicrobial agent. Methods: All five derivatives were
tested against human HOS and MCF7 cancer cell lines using a wound scratch assay. The cytotoxic
properties of previously reported flavonoid 4a were also evaluated using the standard MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner
salt) and live/dead assays, using NHDF, HOS and MCF7 cell lines. Results: All five derivatives were
found to inhibit to some degree the proliferation of cancer cells. 4a was also found to be less toxic
towards regular versus cancerous human cells. Moreover, the minimum bactericidal concentration
of 4a against Staphylococcus aureus was found to be non-toxic for any of the tested human cell lines.
Conclusions: Derivative 4a has the potential of being used as a therapeutic agent against certain
microorganisms. Further structure optimization is required for use against tumors.
Keywords: flavonoids; dithiolium; cytotoxicity
1. Introduction
Since the discovery of penicillin in 1928 and until present day, antibiotics have been used extensively,
for both medical and agricultural purposes. This ultimately led to the emergence of multidrug resistant
microorganisms, as pathogens acquired resistance to multiple classes of antibiotics [
to develop new drugs is now greater than ever [ ]. Moreover, it is desirable that said drugs belong to
novel classes of antibiotics, to which microorganisms have not had time to adapt to [ ]. In this respect,
1]. As such, the need
2
3
flavonoids are good candidates when looking for new antimicrobial agents. As natural polyphenols,
flavonoids are widely distributed in the plant kingdom and present a variety of biological activities,
such as antiviral [
4], antifungal [5], anti-inflammatory [6], antitumoral [7], cardio-protective [8],
neuro-protective [9] and antibacterial [10] properties.
With respect to their antibacterial properties, the most studied flavonoids are the naturally occurring
ones. However, it was shown that semi-synthetic flavonoids, obtained by modifying natural ones, can lead
to better antimicrobial agents [11,12]. Moreover, several synthetic flavonoids were also found to display
potent antimicrobial properties, a subject which was recently reviewed by our group [13].
Although strong antimicrobial properties are desired, they are not enough when looking for new
antibiotics. Thus, any new drug must also be well tolerated by the human body, inflicting as little
damage as possible to human cells. Therefore, once the antimicrobial capabilities of a new agent have
been established, cytotoxicity studies are needed.
Molecules 2019, 24, 2459; doi:10.3390/molecules24132459
www.mdpi.com/journal/molecules

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Our continued interest in synthetic flavonoids has prompted us to synthesize and evaluated
the cytotoxic effect of five tricyclic flavonoids, whose backbone is known to induce antimicrobial
properties [14–18]. Out of the five, three are reported here for the first time.
2. Results and Discussion
2.1. Chemistry
The synthetic route used to obtain 1,3-dithiolium flavonoids 4a
–
e
is described in Scheme 1 and
follows the protocol used for the model compound 4a [19].
O
O
S
N
S
H
N
N
N
O
O
O
R1
R2
S
R1
R2
S
EtOH
+
reflux, 2h
OH
H
R3
R3
Cl
Cl
2
1a-e
3a-e
i. AcOH:H2SO4=1:3 (v/v)
80 °C, 30 min;
ii. NaBF4 (aq)
N
S
BF4
S
R1
R2
O
R3
4a-e
Cl
c
1, 3, 4
a
b
d
e
1
R
Br
Me
H
Br
H
Br
M
I
2
R
H
H
e
3
R
H
Br
Me
Br
I
Scheme 1. The synthesis of tricyclic flavonoids 4a–e.
Methylated flavonoids 4b
–d were thought as an extension to our previous structure-activity
studies [16 17], which found that the optimal structure when it comes to antibacterial properties
,
contains either a Br or I substituent on the benzopyran core and a Cl or Br substituent on the side-ring,
while unsubstituted derivatives at the benzopyran ring and 2-aryl moiety displayed the lowest
antibacterial acitivity.
The first step of the synthesis sees the formation of the benzopyran core, by reacting previously
reported phenacyl dithiocarbamates 1a–e with aminal 2, leading to dithiocarbamic flavanones 3a–e.
This kind of reaction, used by our group in the past [20], runs smoothly in refluxing ethanol, over a period
of 2 h. The reaction product, which precipitates upon cooling, is filtered and recrystallised from ethanol.
1
The formation of flavanones 3a
–
e
is confirmed by NMR and mass spectrometry. H NMR analysis
indicates the disaprearance of the phenolic protons of 1a
–
e, present around 11.00–12.00 ppm. In turn,
two new doublets appear between 5.00–7.00 ppm, corresponding to the H-2 and H-3 protons of the
newly formed benzopyran core. Because these two protons can be located either on the same side

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or on opposite sides of the plane of the molecule, two stereomers, namely syn-
3
and anti-
3
can be
obtained. For all new dithiocarbamic flavonoids 3b–e the syn/anti ratio was always identified to be 4:6.
This was established on the basis of coupling constants between the H-2 and H-3 protons. The anti
isomers always displayed a coupling constant between 9.5–10.7 Hz and the syn isomers around 2.8 Hz.
13C NMR analysis confirms the presence of the C-2 carbon atom, found around 80.0 ppm, while the
C-3 carbon atom can be found around 60.0 ppm.
The next step, the closure of the 1,3-dithiolium ring, was performed by heating flavanones 3a–e
in a mixture of acetic acid and sulfuric acid, followed by treatment with an aqueous solution of
sodium tetrafluoroborate. The desired products, obtained as colorless precipitates, are filtered and
1
recrystallised from ethanol. H NMR analysis indicates the disappearance of the H-3 signal and a shift
of the H-2 signal to about 6.80 ppm, while the 13C NMR spectra show the disappearance of the carbonyl
and thiocarbonyl signals from around 190.0 ppm. Instead, a new signal belonging to the positive
carbon atom of the 1,3-dithiolium ring can be found around 185.0 ppm.
2.2. X-Ray Crystallography
The structure of tricyclic flavonoid 4b has been unambiguously proved by single crystal X-ray
diffraction study [21]. It was confirmed that compound 4b crystalizes in the P21/n space group of
-
monoclinic system. Its ionic crystal structure consists of flavonoid cations and BF4 anions in 1:1 ratio.
No co-crystallized solvent has been found in the crystal. The result of X-ray diffraction investigation is
shown in Figure 1, while the bond lengths and angles are summarized in Table 1. It is to note, that the
chlorophenyl fragment, as well as one of the sulphur atoms were found to be disordered into two
resolvable positions with components ratio refined at 0.7:0.3.
Figure 1. X-ray molecular structure of flavonoid 4b cation with atom labeling scheme and thermal
ellipsoids at 50% probability. Only the major components of disordered fragments are shown [21].
Table 1. Bond distances (Å) and angles (◦) for compound 4b.
Br1–C4
S1–C8
S1–C10
O1–C5
O1–C15
O1–C15X
1.895(5)
1.730(5)
1.719(5)
1.376(6)
1.443(7)
1.443(10)
C8–C9
1.351(10)
1.738(6)
1.501(11)
1.510(8)
1.723(4)
1.529(9)
C10–S2
C11–C12
C13–C14
Cl1–C19
C9–C15

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Table 1. Cont.
N1–C10
N1–C11
N1–C13
C1–C2
C2–C3
C2–C7
C3–C4
C4–C5
1.301(6)
C15–C16
C17–C16
C17–C18
C16–C21
C21–C20
C20–C19
C19–C18
F1–B1
1.509(7)
1.39
1.39
1.39
1.39
1.478(8)
1.485(7)
1.518(8)
1.384(8)
1.388(7)
1.380(8)
1.377(7)
1.410(7)
1.375(8)
1.457(7)
95.0(2)
118.2(4)
120.3(5)
121.4(5)
118.3(4)
122.3(5)
117.8(5)
119.9(6)
121.1(5)
118.7(4)
119.9(4)
121.4(5)
120.9(5)
121.2(4)
117.9(5)
115.5(5)
120.2(5)
124.2(5)
121.6(5)
121.5(4)
117.9(4)
120.4(5)
116.4(3)
1.39
1.39
1.356(9)
1.304(9)
1.369(8)
1.348(8)
122.0(4)
121.6(4)
112.1(7)
112.1(5)
107.2(6)
111.2(5)
112.0(6)
C5–C6
C6–C7
C6–C8
F2–B1
F3–B1
F4–B1
C10–S1–C8
C5–O1–C15
C10–N1–C11
C10–N1–C13
C11–N1–C13
C3–C2–C1
C3–C2–C7
C7–C2–C1
C4–C3–C2
C3–C4–Br1
C5–C4–Br1
C5–C4–C3
O1–C5–C4
O1–C5–C6
C4–C5–C6
C5–C6–C8
C7–C6–C5
C7–C6–C8
C6–C7–C2
C6–C8–S1
C9–C8–S1
C9–C8–C6
S1–C10–S2
N1–C10–S1
N1–C10–S2
N1–C11–C12
N1–C13–C14
O1–C15–C9
O1–C15–C16
C16–C15–C9
C16–C17–C18 120
C17–C16–C15 119.5(4)
C17–C16–C21 120
C21–C16–C15 120.5(4)
C20–C21–C16 120
C21–C20–C19 120
C20–C19–Cl1
C18–C19–Cl1
C18–C19–C20 120
C19–C18–C17 120
F1–B1–F3
F2–B1–F1
F2–B1–F3
F2–B1–F4
F4–B1–F1
F4–B1–F3
120.7(3)
119.2(3)
107.5(7)
104.3(8)
109.7(6)
111.9(7)
107.1(7)
115.6(6)
The bond length C10–N1 of 1.311(2) Å corresponds to a double bond and thus confirms the formal
positive charge at the nitrogen atom, while C3–C4 is of 1.342(2) Å indicating a double bond character.
The C–S bond distances in the five-membered ring vary in the range between 1.727(1)–1.746(1) Å.
The crystal structure of compound 4b essentially results from the parallel packing of the discrete
two-dimensional supramolecular layers (Figure 2), where the cationic and anionic components are
interacting through C-H—F hydrogen bonding. The further analysis of the 2D architecture shows
the presence of
π–π stacking between bromophenyl rings of two centrosymmetrically related cations,
which is evidenced by centroid-to-centroid distance of 3.700(8) Å.

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Figure 2. Partial view of the crystal packing viewed along a axis. H-bonds are drawn in dashed black,
while centroid-to-centroid contacts are in dashed orange lines.
2.3. Wound Scratch Assay
Wound scratch assay is commonly used to evaluate migration and proliferation of tumor cells
in response to a treatment. We wanted to know if our compounds have any antitumor activity, therefore
their effect was investigated on two neoplastic cell lines: HOS-osteosarcoma and MCF7-a breast cancer
cell line (Figure 3; S1 in Supplementary Material).
Figure 3. Wound scratch assay. Following scratch, cells were treated with 5
for 4 h. Graphs represent ratios between treated samples and control percentages of the initial open
area which remained uncovered by cells after 24 h. The results are presented as a mean value the
µg/mL compound solutions
±
standard error of the mean (S.E.M.), n = 3–8. * p < 0.05, ** p < 0.01, and *** p < 0.001 treated vs. control.
(a) Results on HOS cell line. (b) Results on MCF7 cell line.
The test consists of producing a wound on cell culture and tracking the evolution of the open
area created while cells were under the influence of drugs to be tested. All compounds demonstrated
inhibition on cell proliferation and migration on both cell lines; however the most effective ones proved
to be 4a, 4b and 4c. Upon treatment of HOS cells with these three compounds, the wound was between

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5.6 to 6.6 times slower to heal than control, while in the case of MCF7 cells the inhibition of wound
healing after treatment with 4a, 4b and 4c was around 0.3.
The inhibition induced by 4d and 4e relative to control on HOS cells was about 2.4 and 3.3 fold
change respectively, while on MCF7 cells they were 0.24 and 0.20 respectively.
Encouraged by these results, we decided to further investigate the cytotoxic effects of flavonoids of
type
4
on normal and cancerous human cells. Considering that the antibacterial properties of 4a were
g/mL; E. coli—MIC = 3.9 µg/mL,
found by us to be very promising (S. aureus—MIC and MBC = 0.24
µ
MBC = 7.8 µg/mL) [15], we decided to focus on it as a model compound.
2.4. MTS Assay
A metabolic test CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) which
measures the mitochondrial reductase activity was used to investigate the influence of 4a on cells.
Cells with unaffected mitochondrial function could reduce the MTS reagent to a soluble and colorful
formazan and as a result, the mitochondrial reductase activity is used as an indirect indicator for
cell viability.
After 48 h of treatment, 4a (Figure 4) demonstrates greater cytotoxic activity on HOS versus NHDF
cells at concentrations between 1.7–8
Likewise, 4a is killing MCF7 cells more efficiently than NHDF cells in the interval 3.3–6.4 µg/mL.
In terms of magnitude of effect the interval 4.1–5.1 g/mL appears to be the most beneficial. Thus,
the relative viability at 4.1 g/mL 4a for NHDF, HOS and MCF7 cells is 62%, 49% and 39% respectively,
while at 5.1 g/mL it is 62%, 49% and 31% respectively. At concentrations lower than 1.7 µg/mL for
HOS and 3.3 g/mL for MCF7, 4a does not kill these tumoral cells better than fibroblasts, and at
µg/mL, statistically significant in the interval 2.6–4.1 µg/mL.
µ
µ
µ
µ
concentrations lower than 1.0 µg/mL all tested cells are virtually unaffected.
Figure 4. MTS assay, graphical representation of relative viability compared to untreated cells. NHDF,
HOS and MCF7 cells were treated for 48 h with various concentrations of 4a. The results are presented as
a mean value
±
the standard error of the mean (S.E.M.), n = 13–15. * p < 0.05, ** p < 0.01, and *** p < 0.001
MCF7 vs. NHDF; , p < 0.05, ,, p < 0.01, and ,,, p < 0.001 HOS vs. NHDF by Student’s t-test.
These results suggest that 4a could potentially be used as an antitumor agent, although the rather
high toxicity exhibited against normal cells is a drawback. Addressing this issue will require further
research, in order to identify the optimal structure of compounds of type 4 that would decrease their
effect on normal cells, while still retaining the ability to act against cancerous cells.
In a previous study, we also documented the antibacterial properties of 4a
[
15]. It was then
found that the minimum inhibitory concentration, as well as minimum bactericidal concentration
against S. aureus is 0.24 g/mL. At this concentration, the tested human fibroblasts are virtually
µ
unaffected, suggesting that tricyclic flavonoids similar to 4a could be used as antimicrobial drugs
against gram-positive bacteria.

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2.5. Live/Dead Staining
MTS assay is a metabolic test which evaluates cell viability in an indirect manner. To get more
insight over antitumor activity and cytotoxicity, a live/dead staining was performed, which offers more
direct information.
The inhibition of proliferation and migration of malignant cells while maintaining a lower cytotoxic
effect on healthy cells is a desirable feature of antitumor drugs. As could be seen in Figure 5, compound
4a at a concentration of 5 µg/mL is visibly affecting the viability and proliferation of HOS and MCF7
cells (48 h control—untreated cells vs. 48 h treatment—with 4a). These effects are noticeable for NHDF
cells as well but to a lower extent, compared to the two cancerous cell lines.
Figure 5. Live/dead staining. Cells were treated with 5 µg/mL compound 4a for 48 h. Live cells are
stained in green while dead cells, in red. Scale bar represents 200 µm.
These results confirm those recorded using the MTS assay, namely that 4a affects the HOS and
MCF7 cell lines to a greater extent than the NHDF cell line.
3. Experimental
3.1. Chemistry
Melting points were obtained on a KSPI melting-point meter and are uncorrected. Melting points
of flavonoids 3b–d concern the mixture of isomers. IR spectra were recorded on a Bruker Tensor 27
instrument. NMR spectra were recorded on a Bruker 500 MHz spectrometer. Chemical shifts are
reported in ppm downfield from TMS. Mass spectra were recorded on a Thermo Scientific ISQ LT
instrument. All reagents were commercially available and used without further purification.
3.1.1. General Procedure for 8-bromo-2-(4-chlorophenyl)-6-methyl-4-oxochroman-3-yl
N,N-diethyldithiocarbamate (3b)
To a solution of 1-(3-bromo-2-hydroxy-5-methylphenyl)-1-oxoethan-2-yl-N,N-diethyldithiocarbamate
(1b) [22] (0.75 g, 2 mmol) in EtOH (30 mL) aminal 2 (0.59 g, 2 mmol) was added and the reaction mixture was
heated under reflux for 2 h. After cooling the solid material was filtered off and purified by recrystallization
from ethanol to give 3b (0.82 g, 83%) as colorless crystals. M.p. 149–150 ◦C. IR (ATR, cm−1) 2889, 1692, 1492,
1
1271, 1239, 803, 649, 548. H NMR (DMSO-d6, selected data for the major isomer)
δ
7.81 (d, J = 1.4 Hz, 1H),
7.62 (d, J = 1.4 Hz, 1H), 7.50 (m, 4H), 6.17 (d, J = 10.4 Hz, 1H), 5.85 (d, J = 10.4 Hz, 1H), 3.83 (m, 4H), 2.31 (s,
3H), 1.10 (m, 6H). 13C NMR (DMSO-d6, selected data for the major isomer)
δ 189.8, 187.1, 154.8, 140.7, 135.0,
134.0, 133.3, 130.1, 128.8, 128.5, 126.8, 111.2, 80.0, 57.7, 50.1, 47.6, 20.1, 12.7, 11.5. MS (EI) m/z: 497.1 (M+, 7%)
for C21H2179BrClNO2S2.

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3.1.2. 6-Bromo-2-(4-chlorophenyl)-8-methyl-4-oxochroman-3-yl N,N-diethyldithiocarbamate (3c)
The compound was synthesized from 1-(5-bromo-2-hydroxy-3-methylphenyl)-1-oxoethan-2-yl-
N,N-diethyldithiocarbamate (1c) [23] following the general procedure. Colorless crystals (0.46 g, 80%).
1
M.p. 178–179 ◦C. IR (ATR, cm−1) 2877, 1696, 1490, 1419, 1260, 1200, 811, 652, 539. H NMR (DMSO-d6,
selected data for the major isomer)
δ 7.70 (m, 2H), 7.50 (m, 4H), 6.13 (d, J = 10.7 Hz, 1H), 5.85 (d,
J = 10.4 Hz, 1H), 3.82 (m, 4H), 2.19 (s, 3H), 1.10 (m, 6H). 13C NMR (DMSO-d6, selected data for the
major isomer) δ 189.9, 186.9, 157.9, 139.6, 135.1, 133.3, 130.8, 130.1, 128.7, 126.8, 121.2, 113.7, 79.5, 57.7,
51.0, 47.6, 15.5, 12.9, 11.5. MS (EI) m/z: 496.0 (M+, 4%) for C21H2179BrClNO2S2.
3.1.3. 6,8-Dibromo-2-(4-chlorophenyl)-7-methyl-4-oxochroman-3-yl N,N-diethyldithiocarbamate (3d
)
The compound was synthesized from 1-(3,5-dibromo-2-hydroxy-4-methylphenyl)-1-oxoethan-2-
yl-N,N-diethyldithiocarbamate (1d) [24] following the general procedure. Colorless crystals (0.83 g,
1
72%). M.p. 164–165 ◦C. IR (ATR, cm−1) 2888, 1697, 1490, 1417, 1337, 1199, 1011, 802, 642, 528. H NMR
(DMSO-d6, selected data for the major isomer)
δ 7.94 (m, 2H), 7.52 (m, 4H), 6.22 (d, J = 9.5 Hz, 1H), 5.89
(d, J = 9.5 Hz, 1H), 3.80 (m, 4H), 2.58 (s, 3H), 1.08 (m, 6H). 13C NMR (DMSO-d6, selected data for the
major isomer) δ 190.7, 186.0, 158.3, 145.7, 135.5, 134.2, 130.3, 128.8, 128.7, 121.1, 117.1, 115.2, 82.5, 57.3,
50.7, 47.7, 24.9, 12.7, 11.4. MS (EI) m/z: 574.9 (M+, 10%) for C21H2079Br2ClNO2S2.
3.1.4. 2-(4-Chlorophenyl)-6,8-diiodo-4-oxochroman-3-yl N,N-diethyldithiocarbamate (3e)
The compound was synthesized from 1-(3,5-diiodo-2-hydroxyphenyl)-1-oxoethan-2-yl-N,N-
diethyldithiocarbamate (1e) [25] following the general procedure. Colorless crystals (0.92 g, 70%).
1
M.p. 160–161 ◦C. IR (ATR, cm−1) 3078, 1685, 1570, 1491, 1422, 1261, 1200, 1085, 972, 819, 655, 513. H
NMR (DMSO-d6, selected data for the major isomer)
δ 8.39 (m, 1H), 8.03 (m, 1H), 7.51 (m, 4H), 6.20 (d,
J = 10.7 Hz, 1H), 5.85 (d, J = 10.7 Hz, 1H), 3.81 (m, 4H), 1.08 (m, 6H). 13C NMR (DMSO-d6, selected
data for the major isomer) δ 189.6, 186.3, 159.1, 152.2, 135.6, 134.6, 134.2, 133.5, 130.2, 128.7, 80.2, 57.2,
50.7, 47.7, 12.8, 11.5. MS (EI) m/z: 656.9 (M+, 26%) for C20H18ClI2NO2S2.
3.1.5. General Procedure for 2-N,N-diethylamino-6-bromo-4-(4-chlorophenyl)-8-methyl-4H-1,3-dithiol
[4,5-c]chromen-2-ylium tetrafluoroborate (4b)
To a mixture of sulfuric acid (0.5 mL) and acetic acid (1.5 mL), flavanone 3b (0.5 g, 1 mmol) was
added and the resulting solution was heated to 80 ◦C for 20 min. The reaction mixture was then left to
cool to room temperature and a solution of sodium tetrafluoroborate (250 mg) in water (10 mL) was
added dropwise, with vigorous stirring. The resulting precipitate was then filtered, washed thoroughly
with water and recrystallized from ethanol, yielding the desired tetrafluoroborate 4b in the form of
colorless crystals (0.5 g, 88%). M.p. 232–233 ◦C. IR (ATR, cm−1) 1538, 1441, 1235, 1041, 774, 734, 460,
1
451. H NMR (DMSO-d6)
δ
7.52 (m, 4H), 7.48 (d, J = 1 Hz, 1H), 7.31 (d, J = 1 Hz,) 6.92 (s, 1H), 3.94 (m,
185.1, 145.7,
4H), 2.27 (s, 3H), 1.41 (t, J = 7.1 Hz, 3H), 1.34 (t, J = 7.1 Hz, 3H). 13C NMR (DMSO-d6)
δ
135.9, 135.6, 135.0, 134.6, 129.7, 129.6, 128.3, 127.9, 125.1, 117.9, 110.8, 75.1, 54.7, 54.6, 20.1, 10.8, 10.6. MS
(EI) m/z: 480.1 (M+-BF4, 7%) for C21H2079BrClNOS2]+.
3.1.6. 2-N,N-Diethylamino-8-bromo-4-(4-chlorophenyl)-6-methyl-4H-1,3-dithiol[4,5-c]chromen-2-ylium
tetrafluoroborate (4c)
Colorless crystals (0.48 g, 84%). M.p. 225–226 ◦C. IR (ATR, cm−1) 1549, 1454, 1210, 1036, 789, 741,
1
467, 448. H NMR (DMSO-d6)
δ 7.54 (m, 6H), 6.87 (s, 1H), 3.90 (m, 4H), 2.21 (s, 3H), 1.40 (t, J = 7.2 Hz,
3H), 1.33 (t, J = 7.2 Hz, 3H). 13C NMR (DMSO-d6)
δ 185.1, 148.4, 136.2, 135.7, 135.0, 129.7, 129.6, 129.5,
128.2, 127.8, 125.0, 118.3, 114.3, 74.7, 54.7, 54.6, 15.6, 10.8, 10.6. MS (EI) m/z: 480.1 (M+-BF4, 11%) for
C21H2079BrClNOS2]+.

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3.1.7. 2-N,N-Diethylamino-6,8-dibromo-4-(4-chlorophenyl)-7-methyl-4H-1,3-dithiol[4,5-c]chromen-2-ylium
tetrafluoroborate (4d)
Colorless crystals (0.51 g, 79%). M.p. 241–242 ◦C. IR (ATR, cm−1)1561, 1450, 1228, 1154, 1041, 717,
1
489. H NMR (DMSO-d6)
δ 7.81 (s, 1H), 7.51 (m, 4H), 6.99 (s, 1H), 3.92 (m, 4H), 2.19 (s, 3H), 1.41 (t,
J = 7.2 Hz, 3H), 1.34 (t, J = 7.2 Hz, 3H). 13C NMR (DMSO-d6)
δ 185.1, 147.5, 140.7, 135.7, 135.2, 129.8,
129.6, 128.3, 127.2, 127.1, 117.4, 117.1, 114.8, 75.5, 54.8, 54.6, 24.3, 10.8, 10.6. MS (EI) m/z: 557.9 (M+-BF4,
8%) for C21H1979Br2ClNOS2]+.
3.1.8. 2-N,N-Diethylamino-4-(4-chlorophenyl)-6,8-diiodo-4H-1,3-dithiol[4,5-c]chromen-2-ylium
tetrafluoroborate (4e)
Colorless crystals (0.54 g, 75%). M.p. 240–241 ◦C. IR (ATR, cm−1) 1596, 1544, 1427, 1224, 1076, 990,
1
809, 701, 620. H NMR (DMSO-d6)
δ
8.13 (d, J = 1.9 Hz, 1H), 7.83 (d, J = 1.9 Hz, 1H), 7.55 (m, 4H), 7.00
184.5, 149.8,
(s, 1H), 3.92 (m, 4H), 1.43 (t, J = 6.9 Hz, 3H), 1.33 (t, J = 6.9 Hz, 3H). 13C NMR (DMSO-d6)
δ
147.3, 135.1, 134.6, 132.6, 129.3, 129.1, 128.3, 126.6, 118.4, 88.1, 87.3, 74.9, 54.2, 54.1, 10.3, 10.0. MS (EI)
m/z: 639.8 (M+-BF4, 14%) for C20H17ClI2NOS2]+.
3.2. X-Ray Structure Determination
X-ray diffraction data were collected with an Oxford-Diffraction XCALIBUR E CCD diffractometer
equipped with graphite-monochromated MoK radiation. A single crystal was positioned at 40 mm
from the detector, and 2552 frames were measured each at 15 and 100 s over a 1◦ scan. The unit
cell determination and data integration were carried out using the CrysAlis package of Oxford
Diffraction [26]. The structures were solved by direct methods using Olex2 [27] software with
the SHELXS structure solution program and refined by full-matrix least-squares on F2 with
SHELXL-2015 [28]. Hydrogen atoms attached to carbon were placed in fixed, idealized positions and
refined as rigidly bonded to the corresponding non-hydrogen atoms. Whenever necessary, restraints
were imposed on geometry and displacement parameters of disordered fragments. CCDC-1919638
contains the supplementary crystallographic data for this contribution. These data can be obtained free
of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or deposit@ccdc.ca.ac.uk).
Crystal Data for Compound 4b
C21H20BrClF4NOS2 (Mr = 568.67 g mol-1), monoclinic, a = 8.9673(6) Å, b = 14.7904(8) Å,
·
c = 17.5523(9), β
= 92.335(5)◦, V = 2326.0(2) Å3, T = 180 K, space group P21/c, Z = 4, 10,919 coll.
refl., 4114 indep. (Rint = 0.0608), Gof = 1.027, R1 = 0.0661, wR(F2) = 0.1447. CCDC–1919638.
4. Materials and Methods
4.1. General Information
Human osteosarcoma (HOS) and MCF7 cell lines were purchased from CLS Cell Lines Service
GmbH (Eppelheim, Germany) and Normal Human Dermal Fibroblasts (NHDF) cell line from PromoCell,
(Heidelberg, Germany); Eagle’s Minimal Essential Medium alpha (aMEM), Dulbecco’s Modified
Eagle Medium (DMEM) without phenol red, 1% Penicillin–Streptomycin–Amphotericin B mixture
(10K/10K/25 µg in 100 mL) and Trypsin–Versene (EDTA) mixture from Lonza, Verviers, Begium;
fetal bovine serum (FBS) from Biochrom GmbH, Germany, CellTiter 96® Aqueous One Solution Cell
Proliferation Assay from Promega (Madison, WI, USA); Tryple from Gibco, (Langley, VA, USA);
LIVE/DEAD Viability/Cytotoxicity Kit and phosphate buffered saline (PBS) from Invitrogen, (Eugene,
OR, USA), 35-mm Cell Imaging Dishes from Eppendorf (Hamburg, Germany).
Absorbance at 490 nm from MTS assay was measured with an iMark plate reader (Biorad,
Hercules, CA, USA) and images for live/dead staining and wound scratch assays were recorded with
a Leica DMI 3000B inverted microscope (Wetzlar, Germany).

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4.2. Cell Culture
All cell lines were cultured in alpha-MEM medium supplemented with 10% FBS and 1%
◦
penicillin–streptomycin–amphotericin B mixture under 5% CO2 humidified atmosphere at 37 C.
For passaging cells, Trypsin–Versene was used for HOS and MCF7 cells while Tryple was used for
NHDF cells.
Stock solutions in DMSO were prepared for each compound to be tested and working solutions
were prepared by diluting the stock solutions 100 times with cell culture medium. Negative control
cells were treated with cell culture medium containing 1% DMSO.
4.3. Wound Scratch Assay
A stock solution of 500 µg/mL in DMSO was prepared for each compound and working solutions
of 5 µg/mL were prepared by diluting the stock solution with cell culture medium.
For wound scratch assay, cells were seeded in 24-well plates at a density of 24
×
104 cells/well
for HOS cell line and 12
scratching the cell culture in the form of a cross in the center of the well with a pipette tip. Wells were
washed with PBS and cells were treated with the compounds to be tested, using 5 g/mL solutions
×
104 cells/well for MCF7 cell line. After 48 h a wound was performed by
µ
in cell culture medium except control wells which were treated with 1% DMSO in cell culture medium.
Pictures with cell cultures were acquired under microscope and these were denoted as time 0 in the
experiment. After 4 h, the compounds solutions were removed and replaced with fresh culture medium.
The following day, cell cultures were photographed again marking the 24 h time point in the experiment.
Pictures were analyzed using TScratch version 1.0 software developed by the Koumoutsakos group
(CSE Lab), at ETH Zürich [29]. First, open wound area is calculated as a percentage of open image
area at 24 h compared to time 0 of the experiment: (open wound area 24 h) = 100 x (open image area
24 h)/(open image area 0 h). Finally a graph is made representing the ratio between open wound areas
of treated samples and control.
4.4. MTS Assay
For MTS assay, cells were plated on 96-well plates at densities of 10
for HOS, MCF7 and NHDF cell lines respectively in 100 L/well aMEM medium and incubated for 24 h.
The next day the media were replaced with 4a solutions in serial dilutions and the plates were incubated
for another 48 h. The media containing the test compound were then replaced with 100 L/well DMEM
×
103, 7
×
103 and 5
×
103 cells/well
µ
µ
without phenol red and 20 µL/well MTS reagent and the plates were returned to the incubator for another
3 h. Following the last incubation, the plates were removed from the incubator and absorbance at 490 nm
was recorded with a plate reader.
The relative cell viability is expressed as percentage of the viability of control (cells treated only
with cell culture medium and 1% DMSO). Cumulated replicates from three independent experiments
were used in the analysis.
4.5. Live/Dead Staining
A stock solution of 500
µg/mL in DMSO was prepared for 4a and working solution of 5 µg/mL
were prepared by diluting stock with cell culture medium.
Cells were seeded on 35-mm Cell Imaging Dishes with glass bottoms at a density of 24
×
104,
104 cells/dish for HOS, MCF7 and NHDF cell lines respectively. The following day
g/mL solution of 4a and incubated for 24 h. Finally, the 4a treatment
L 50:50 mixture of LIVE/DEAD staining solution with DMEM without
12 × 104 and 5
×
they were treated with 1 mL of 5
µ
solution was replaced with 500
µ
phenol red. Images were acquired after 15 min of staining and processed with ImageJ Fiji distribution
version 1.52.

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4.6. Data Analysis
Data analysis was performed with GraphPad Prism software version 7.00 for Windows (GraphPad
Software, San Diego, CA). Results are presented as means standard error of the mean (S.E.M.).
±
To determine statistical significance between two groups parametric unpaired t-test with Welch’s
correction was applied. Where groups failed Shapiro–Wilk normality test, nonparametric unpaired
Mann–Whitney test was used instead. The difference was considered significant when p < 0.05.
5. Conclusions
Four new tricyclic flavonoids have been synthesized and characterized. These, along a previously
studied flavonoid, were tested against HOS and MCF7 human cancer lines using a wound scratch
assay. The results showed that all five compounds inhibit cell proliferation, with derivatives 4a–c being
the most active. Further cytotoxicity tests suggested that flavonoid 4a is less toxic for normal human
cells when compared to cancerous ones, although more research is required to optimize this property.
When referring to the antibacterial properties of 4a, the previously reported minimum inhibitory
concentration against S. aureus of 0.24
µg/mL is well under the concentration of 1 µg/mL, at which the
tested compound begins to be too toxic for the NHDF cell line.
Supplementary Materials: The following are available online http://www.mdpi.com/1420-3049/24/13/2459/s1,
Figure S1: Wound scratch assay.
Author Contributions: All the authors have equal contributions for conceptualization, methodology, investigation
and writing.
Funding: This research received no external funding.
Acknowledgments: CNCS—UEFISCDI support within PNCDI III is acknowledged by LGS and LGB for
project numbers PN-III-Pl-l.1-PD-2016-0962 and PN-III-Pl-l.1-PD-2016-1117, respectively. Romanian Ministry of
Research and Innovation support within the Program 1—Development of the national RD system, Subprogram
1.2—Institutional Performance—RDI excellence funding projects, Contract no. 34PFE/19.10.2018 is acknowledged
by LGS and MLB. DP acknowledges the support provided by H2020 WIDESPREAD 2-2014: ERA Chairs Project
no 667387: SupraChem Lab Laboratory of Supramolecular Chemistry for Adaptive Delivery Systems ERA
Chair initiative and by the Romanian Ministry of Research and Innovation, CNCS-UEFISCDI, project number
PN-III-P1-1.1-TE-2016-1180, within PNCDI III. MLB is indebted to the Alexander von Humboldt Foundation for
a stay in Braunschweig.
Conflicts of Interest: The authors declare no conflict of interest.
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©
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/).