Synthesis and anticancer activity of novel 9-O-substituted berberine derivatives

Article abstract of DOI:10.3390/ijms20092169

Berberine is a bioactive isoquinoline alkaloid derived from many plants. Although berberine has been shown to inhibit growth and induce apoptosis of several tumor cell lines, its poor absorption and moderate activity hamper its full therapeutic potential. Here, we describe the synthesis of a series of 9-O-substituted berberine derivatives with improved antiproliferative and apoptosis-inducing activities. An analysis of novel berberine derivatives by EPR spectroscopy confirmed their similar photosensitivity and analogous behavior upon UVA irradiation as berberine, supporting their potential to generate ROS. Improved antitumor activity of novel berberine derivatives was revealed by MTT assay, by flow cytometry and by detection of apoptotic DNA fragmentation and caspase-3 activation, respectively. We showed that novel berberine derivatives are potent inhibitors of growth of HeLa and HL-60 tumor cell lines with IC50 values ranging from 0.7 to 16.7 μM for HL-60 cells and 36 to >200 μM for HeLa cells after 48 h treatment. Further cell cycle analysis showed that the observed inhibition of growth of HL-60 cells treated with berberine derivatives was due to arresting these cells in the G₂/M and S phases. Most strikingly, we found that berberine derivative 3 (9-(3-bromopropoxy)-10-methoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquino[3,2-a] isoquinolin-7-ylium bromide) possesses 30-fold superior antiproliferative activity with an IC50 value of 0.7 μM and 6-fold higher apoptosis-inducing activity in HL-60 leukemia cells compared to berberine. Therefore, further studies are merited of the antitumor activity in leukemia cells of this berberine derivative.

Full text of DOI:10.3390/ijms20092169

ASryticnle thesis and Anticancer Activity of Novel 9-O-
Substituted Berberine Derivatives
1
2
3
1
Viktor Milata , Alexandra Svedova , Zuzana Barbierikova , Eva Holubkova , Ingrid Cipakova
4
4
4
5
2
3,
, Dana Cholujova , Jana Jakubikova , Miroslav Panik , Sona Jantova , Vlasta Brezova * and
4,
Lubos Cipak *
1
Institute of Organic Chemistry, Catalysis and Petrochemistry, Faculty of Chemical and Food Technology,
Slovak University of Technology, Radlinskeho 9, 812 37 Bratislava, Slovakia; viktor.milata@stuba.sk (V.M.);
evaholubicka@gmail.com (E.H.)
Institute of Biochemistry and Microbiology, Faculty of Chemical and Food Technology, Slovak University
2
of Technology, Radlinskeho 9, 812 37 Bratislava, Slovakia; s.svedova@gmail.com (A.S.);
sona.jantova@stuba.sk (S.J.);
Institute of Physical Chemistry and Chemical Physics, Faculty of Chemical and Food Technology, Slovak
3
University of Technology in Bratislava, Radlinskeho 9, 812 37 Bratislava, Slovakia;
zuzana.barbierikova@stuba.sk
Cancer Research Institute, Biomedical Research Center, University Science Park for Biomedicine, Slovak
4
Academy of Sciences, Dubravska cesta 9, 845 05 Bratislava, Slovakia; ingrid.cipakova@savba.sk (I.C.);
dana.cholujova@savba.sk (D.C.); jana.jakubikova@savba.sk (J.J.)
Institute of Management, Slovak University of Technology, 812 33 Bratislava, Slovakia;
5
miroslav.panik@stuba.sk
* Correspondence: vlasta.brezova@stuba.sk (V.B.); lubos.cipak@savba.sk (L.C.)
Received: 11 April 2019; Accepted: 30 April 2019; Published: 1 May 2019
Abstract: Berberine is a bioactive isoquinoline alkaloid derived from many plants. Although
berberine has been shown to inhibit growth and induce apoptosis of several tumor cell lines, its
poor absorption and moderate activity hamper its full therapeutic potential. Here, we describe the
synthesis of a series of 9-O-substituted berberine derivatives with improved antiproliferative and
apoptosis-inducing activities. An analysis of novel berberine derivatives by EPR spectroscopy
confirmed their similar photosensitivity and analogous behavior upon UVA irradiation as
berberine, supporting their potential to generate ROS. Improved antitumor activity of novel
berberine derivatives was revealed by MTT assay, by flow cytometry and by detection of apoptotic
DNA fragmentation and caspase-3 activation, respectively. We showed that novel berberine
derivatives are potent inhibitors of growth of HeLa and HL-60 tumor cell lines with IC50 values
ranging from 0.7 to 16.7 µM for HL-60 cells and 36 to >200 µM for HeLa cells after 48 h treatment.
Further cell cycle analysis showed that the observed inhibition of growth of HL-60 cells treated with
berberine derivatives was due to arresting these cells in the G2/M and S phases. Most strikingly, we
found that berberine derivative 3 (9-(3-bromopropoxy)-10-methoxy-5,6-dihydro-[1,3]dioxolo[4,5-
g]isoquino[3,2-a] isoquinolin-7-ylium bromide) possesses 30-fold superior antiproliferative activity
with an IC50 value of 0.7 µM and 6-fold higher apoptosis-inducing activity in HL-60 leukemia cells
compared to berberine. Therefore, further studies are merited of the antitumor activity in leukemia
cells of this berberine derivative.
Keywords: berberine; berberine derivatives; EPR spectroscopy; antitumor activity; cell cycle arrest;
apoptosis
1. Introduction
Int. J. Mol. Sci. 2019, 20, 2169; doi:10.3390/ijms20092169
www.mdpi.com/journal/ijms

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Berberine is a well-known bioactive isoquinoline alkaloid present in a wide variety of medicinal
plants such as Berberis aristata, B. aquifolium, B. vulgaris, Hydrastis canadensis, Coptis chinensis and many
others [1–3]. It has been shown to be active in several diseases such as diabetes, diarrhea, hormonal
disorder, coronary heart disease, obesity or hyperlipidemia [2]. Berberine has also shown promising
antiprotozoal, antifungal and antimicrobial activities with a range of bacteria [4–6]. Furthermore,
recent studies have shown that berberine exerts potent anticancer activity towards a variety of cancer
cell types, such as colorectal, leukemia, prostate, lung, glioma, esophageal, and ovarian [7–13].
Detailed studies have shown that berberine can induce apoptosis of tumor cells through its
modulation of activity of several pro-apoptotic and anti-apoptotic genes. For example, it alters the
Bcl-2/Bax ratio and decreases the mitochondrial membrane potential of selected tumor cells [14]. In
addition, it might induce apoptosis through the mitochondrial/caspase pathway, activate caspase-3,
and caspase-8, and release the cytochrome c [3,8]. It has been also shown that berberine has a variety
of effects on the cell cycle and its lower concentrations arrest tumor cells in the G1 phase [15,16]. In
contrast, higher concentrations of berberine induce the arrest of tumor cells in the G2/M phase [17].
Interestingly, it has been reported that berberine might also induce apoptosis of tumor cells by a
mechanism that involves the production of a reactive oxygen species (ROS) [18–21]. Most
importantly, several reports have demonstrated that berberine could be combined with radiotherapy
or chemotherapy drugs to neutralize their toxicity and enhance their therapeutic activities, thereby
improving therapeutic outcomes [22–28].
Several studies have shown that berberine selectively inhibits tumor cell growth and induces
apoptosis while eliciting less cytotoxic effects in normal cells [29,30]. Its poor absorption and
moderate activity, however, hamper its full antitumor therapeutic potential [31]. As a result, many
laboratories have focused their efforts towards the design and synthesis of novel berberine
derivatives with improved biological activities, which has led to the synthesis of numerous berberine
derivatives with various modifications at the C8, C9, C10, C12 and C13 positions of the berberine
structure [32–38]. These studies have shown that modifications of berberine at C8 or C13 positions
mainly increase its antimicrobial activity, which is closely related to the length of the substituent
chains. On the other hand, the modifications to the C9 position of berberine usually increased its
antitumor activities [39].
Over the past years, several novel 9-O-substituted berberine derivatives, including the 9-O-
pyrazole alkyl group [40], 9-O-chlorohexyl group [41], or 9-O-alkyl- and 9-O-terpenyl group [35]
modified berberine derivatives with improved aqueous solubility and enhanced absorption have
been synthesized and tested for their anticancer activity. These studies have shown berberine
derivatives to be more potent antitumor agents with improved apoptosis-inducing activity than that
of berberine. Additionally, it has been shown that the substitution of berberine on C9 with lipophilic
group substitutes, such as ibuprofen and naproxen [42], or cinnamic acid [36], significantly increased
the anti-inflammatory and enhanced the hypoglycemic activity of berberine, respectively. These
studies point out that modification of berberine on the C9 position can significantly improve its
biological activities. Several recent reviews have summarized and outlined in more detail the most
important findings on the relationship between various structural modifications of berberine and its
improved pharmacological activities [38,43,44].
Here, we report the synthesis and evaluation of biological activity of four novel 9-O-substituted
berberine derivatives. To improve the pharmacological properties and increase the therapeutic
efficiency of berberine, we synthesized its 9-O-substituted derivatives bearing 2-chloro- and 2-bromo-
acetoxy substituents (derivate 1 and 2), 3-bromopropoxy substituent (derivate 3) and ciprofloxacin
(derivate 4) (Figure 1, Scheme 1-5). Ciprofloxacin, as a quinolone antibiotic, was selected for the
synthesis of berberine derivative 4 as it was reported to have high intrinsic lipophilicity [45] and
various biological activity, including the activity against multidrug resistant bacteria Klebsiella
pneumoniae [46,47].
We demonstrate that novel berberine derivatives maintain similar photosensitivity and
analogous behavior upon UVA irradiation to that of berberine, supporting their potential to induce
apoptosis through generation of the reactive oxygen species (ROS). We show that novel berberine

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derivatives are more potent antiproliferative and apoptosis-inducing agents compared to berberine.
Moreover, these derivatives arrest HL-60 leukemia cells in the G2/M and S phases, with the most
promising derivative 3 showing superior apoptosis-inducing activity.
Figure 1. Chemical structures of berberine, ciprofloxacin and newly synthesized berberine
derivatives.
2. Results and Discussion
Berberine molecule offers a variety of possible modifications at positions C2, C3, C8, C9, C10,
+
C12, C13 or C=N [32–38,48]. Based on previous reports that derivatization of berberine at the C9
position usually increases its antitumor activities [39], we prepared a set of novel 9-O-substituted
berberine derivatives. Following the established procedures of heating the berberine to 190 °C for at
least 15 min under vacuum [35] or under microwave irradiation [49,50], we performed selective
demethylation of the berberine on its methoxy group at position C9 to obtain berberrubine (Scheme
1, see section Materials and Methods).
Berberrubine was then treated with 1,3-dibromopropane (DBP) to prepare the berberine
derivative 3 (Scheme 3). No base was used here to avoid cyclisation to nitrogen. Various conditions
for the synthesis of berberine derivative 3 were tested and obtained yields were summarized in Table
1.
Table 1. Reaction conditions for the preparation of berberine derivative 3 (1 equivalent of substrate,
no base used).
Reaction DBP (Equivalents) Solvent Temperature (°C) Time (h)
Yield (%)
R1
R2
R3
R4
3
CH3CN
CH3CN
DMF
60
60
80
80
2
2
2
2
48
55
59
74
10
10
58
DMF
As presented, we found that the high excess of alkylating agent DBP was the most important
factor in increasing the yield of the reaction. Similar to the alkylation of berberrubine by DBP, the
excess of the haloacetylhalogenides was limiting factor for derivatization of berberrubine to
haloacetylesters 1 and 2 (Scheme 2). It should be noted that, under these conditions, the separation of
berberine derivatives from reactions was simple because the products were soluble in polar aprotic
solvents, thus distinguishing them from the berberrubine. Concerning the synthesis of the berberine-
ciprofloxacin conjugate (referred as berberine derivative 4), the first step of its synthesis was the
derivatization of ciprofloxacin to its acylated form (Scheme 4) [51,52]. Prepared carbonyl chloride of
ciprofloxacin was further mixed with berberrubine and the reaction was microwave irradiated to
produce the berberine derivative 4 in 31% yield (Scheme 5). Importantly, no berberine derivative 4
was obtained without microwave irradiation. It is widely accepted that microwave irradiation is an
unconventional energy source with great potential in synthetic organic chemistry [53]. In this respect,

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microwave irradiation has been applied with success to synthesize berberine derivatives under mild
reaction conditions and with short reaction times [49,50,54].
1
13
The synthetic routes and characterization of novel berberine derivatives 1–4 by H NMR and C
NMR spectroscopy are described in detail in the Materials and Methods section (Schemes 2–5).
It was previously shown that berberine, like many other bioactive molecules, exhibits sensitivity
towards light and its photochemical behavior is strongly affected by the solvent and the presence of
molecular oxygen [55]. As a photosensitive agent, berberine was previously studied, and the
photoinduced generation of ROS, via both electron and energy transfer mechanisms (Type I and II),
was evidenced by EPR spectroscopy and direct detection of singlet oxygen phosphorescence
[20,56,57]. The investigation of the cytotoxic/phototoxic effect of berberine on murine fibroblast non-
cancer NIH-3T3 and Ehrlich ascites carcinoma (EAC) cells showed a statistically significant degree of
UVA induced increase of the cytotoxic effect only in cell line EAC. The cell cycle profile analysis
revealed that the DNA damage triggered by berberine in combination with UVA irradiation led to a
significant blockage of cells in the S and G2/M phases with no sub-G0 cell fraction detected [20].
Consequently, to provide a more complex view on the newly prepared berberine derivatives, their
ability to produce ROS upon UVA irradiation (λmax = 365 nm) was tested using the in situ EPR spin
trapping technique.
The UV/vis absorption spectra of the prepared derivatives were recorded to follow the effect of
the structural changes on the absorption maxima (Figure 2). We found that berberine derivative 3
exhibits an analogous spectrum to that of berberine with the characteristic absorption maxima at 348
nm and 421 nm (Figure 2B), while berberine derivatives 1 and 2 reveal a slight bathochromic shift of
these bands (355 and 464 nm) together with the presence of other bands in the 300–600 nm region
(Figure 2A). The absorption bands reaching higher wavelength up to 600 nm reflect the extended
conjugation in berberine derivatives 1 and 2 due to the presence of 1-chloro-acetyl or 1-bromo-acetyl
substituents. A combination of berberine with ciprofloxacin (berberine derivative 4) led to a slight
bathochromic shift of the absorption maxima (325 and 337 nm) compared to the ciprofloxacin (321
and 334 nm) and the appearance of broad shoulders at higher wavelength (370 and 490 nm),
expanding the absorption more towards the visible region (Figure 2C). As novel berberine derivatives
reveal a suitable overlap of their absorption spectra and the emission of UVA light, a source with a
maximum at 365 nm was applied in further photochemical experiments.
2.5
2.0
1.5
1.0
0.5
0.0
1.0
0.8
0.6
0.4
0.2
0.0
2.5
2.0
1.5
1.0
0.5
0.0
1.0
0.8
0.6
0.4
0.2
0.0
1
BBR-Clac
BBR-Brac
2
Berberine
Ber rine
3
be
BBR-Bralk
X = Cl, Br
200
300
400
500
600
700
200
300
400
500
600
700
Wavelength, nm
Wavelength, nm
(A)
(B)
2.5
1.0
Ciprofloxacin
Ciprofloxacin
BBR-Ciprofloxacin
4
2.0
1.5
1.0
0.5
0.0
0.8
0.6
0.4
0.2
0.0
200
300
400
500
600
700
Wavelength, nm
(C)
Figure 2. Electronic absorption spectra of (A) berberine derivatives 1 and 2, (B) berberine and
berberine derivative 3, (C) ciprofloxacin and berberine derivative 4, all in the mixed solvent
DMSO/water (1:100, v:v; concentration 0.07 mM). Each graph contains the relative spectral
distribution of the UVA LED source (λmax = 365 nm) applied in the EPR spin trapping experiments.

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The ROS generation initiated by UVA photoexcitation of berberine derivatives was investigated
using indirect techniques of EPR spectroscopy. The application of spin trapping agent 5,5-dimethyl-
1-pyrroline N-oxide (DMPO) enabled us to follow the formation of reactive radical intermediates
upon in situ photoexcitation of berberine derivatives as the corresponding spin-adducts. The EPR
spin trapping experiments confirmed that berberine derivatives preserved the photosensitivity of the
parent berberine molecule, since they exhibited the photoinduced formation of DMPO spin-adducts
upon UVA irradiation, reaching the highest EPR signal intensity for derivative 3 (Figure 3).
333
334
335
336
337
338
339
333
334
335
336
337
338
339
Magnetic field, mT
Magnetic field, mT
(A)
(B)
Figure 3. Experimental (–) and simulated (–) EPR spectra obtained after 13 min UVA exposure of the
DMSO solution of (A) berberine and (B) berberine derivative 3 in the presence of DMPO spin trap
under air. (Initial concentrations: c0(berberine) = c0(berberine derivative 3) = 0.001 M, c0(DMPO) = 0.042
M, λmax = 365 nm, irradiance 13 mW·cm−2).
The dominant twelve-line signal corresponding to berberine derivative 3 in the spectrum was
•
−
β
γ
assigned to the DMPO-O2 (spin-Hamiltonian parameters, aN = 1.279 mT, aH = 1.025 mT; aH = 0.137
mT; g = 2.0059) based on the simulation analysis. This evidences the effective electron transfer from
the photoexcited berberine derivative 3 to the molecular oxygen generating the superoxide radical
anion stabilized in the aprotic DMSO solvent. Further spin-adduct recognizable in the spectrum
•
β
γ
belongs to the spin-adduct DMPO-OCH3 (aN = 1.303 mT, aH = 0.842 mT; aH = 0.147 mT; g = 2.0059)
•
•−
originating from the OCH3 produced by the interaction of O2 with DMSO. Even though the
absorption of the berberine derivatives is shifted towards the visible region, only a negligible
concentration of DMPO spin-adducts upon the visible-light irradiation ( > 420 nm) was found.
The EPR spectra monitored during the continuous irradiation of the berberine and its derivatives
1–4 in a mixed water/DMSO (2:3, v:v) solvent containing DMPO are more complex and represent
superposition of signals attributable to four individual DMPO spin-adducts, as shown in the
berberine derivative 2 in Figure 4A. Based on the spin-Hamiltonian parameters elucidated from the
simulation analysis, the signals were assigned to the spin-adducts DMPO-O2 /O2H (aN = 1.340 mT,
aH = 1.069 mT; aH = 0.131 mT; g = 2.0059) and DMPO-OH (aN = 1.433 mT, aH = 1.303 mT; aH = 0.071
mT; g = 2.0057) [58] and to the two spin-adducts of the carbon-centered radicals with slightly different

−

β
γ
β
γ


β
parameters: DMPO-CR1 (aN = 1.551 mT, aH = 2.197 mT; g = 2.0056) and DMPO-CR2 (aN = 1.506 mT,
aH = 2.121 mT; g = 2.0057). The last two spin-adducts were produced via interaction of
β
photogenerated ROS with DMSO or berberine molecules. Moreover, a low-intensity single-line signal
(g ~ 2.0043, asterisk in Figure 4A) with unresolved hyperfine structure was found upon UVA
photoexcitation of the berberine derivative 2 in water/DMSO (2:3, v:v) solution under given
experimental conditions, which was most probably produced by the electron transfer reactions of the
photoexcited berberine derivative 2, and its g-value is compatible with the oxygen-centered radicals
[59]. The generation of this radical species was observed also upon continuous UVA irradiation of
the aqueous solution of berberine derivative 2 in the presence of DMPO spin trap, along with the

β
low-intensity four-line signal of DMPO-OH spin-adduct (aN = 1.506 mT, aH = 1.478 mT; g = 2.0057)
(Figure 4B).

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*
*
333
334
335
336
337
338
339
333
334
335
336
337
338
339
Magnetic field, mT
Magnetic field, mT
(A)
(B)
Figure 4. Experimental (–) and simulated (–) EPR spectra obtained after 13 min UVA exposure of
berberine derivative 2 in the presence of DMPO spin trap under air in: (A) water/DMSO (2:3, v:v) and
(B) water. (Initial concentrations: c0(berberine derivative 2) = 0.8 mM, c0(DMPO) = 0.042 M, λmax = 365
nm, irradiance 13 mW·cm−2).
Upon the continuous UVA irradiation of berberine derivatives 1–4 in the presence of sterically-
hindered amine 4-oxo-2,2,6,6-tetramethylpiperidine (TMPO) in DMSO, we only monitored the
generation of low-intensity EPR signal of 4-oxo-2,2,6,6-tetramethylpiperidine N-oxyl (Tempone)
produced via TMPO oxidation by singlet oxygen. The generated nitroxide radical Tempone is most
•
likely transformed to the diamagnetic products by the interaction of >NO moiety with other radical
intermediates [60], effectively formed upon the UVA photoexcitation of berberine derivatives 1–4.
Overall, the EPR spin trapping experiments demonstrated analogous behaviour of novel
berberine derivatives upon UVA irradiation involving effective ROS generation to that of berberine.
It will be interesting to examine these berberine derivatives for their dose-dependent accumulation
of intracellular ROS to find out if generated ROS are involved in their cytotoxicity.
The antiproliferative activity of berberine derivatives was carried out by the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and rated in half-maximal
inhibitory concentration, which is a measure of the effectiveness of an agent in inhibiting the growth
and viability of cells [61]. The antiproliferative activity of berberine derivatives in tumor HeLa and
HL-60 cell lines is summarized in Table 2.
Table 2. IC50 values (µM) for antiproliferative activity of berberine, ciprofloxacin and berberine
derivatives 1–4 determined by MTT assay.
Cell Line Time (h) Berberine Ciprofloxacin
1
2
3
4
24
48
24
48
˃200
˃200
175 ± 8.7
˃200
˃200
115 ± 7.4
˃200
95 ± 4.8
58 ± 2.6
36 ± 1.9
˃200
˃200
HeLa
126 ± 7.8
43.7 ± 1.1
27.1 ± 2.6
23.4 ± 2.6 14.6 ± 1.7 2.7 ± 0.9 19.3 ± 1.3
16.7 ± 0.8 11.2 ± 1.6 0.7 ± 0.2 15.7 ± 1.9
HL-60
142 ± 5.7
We observed an increase in antiproliferative activity of berberine derivatives 1–4 against tested
tumor cell lines compared to berberine or ciprofloxacin. Cytotoxic activity was observed in berberine
derivatives 1, 2 and 4 with IC50 values ranging from 95.0 to ˃200 µM for HeLa cells and from 11.2 to
23.4 µM for HL-60 cells. The lowest antiproliferative activity was found for berberine derivative 4
with IC50 values ranging from 15.7 to ˃200 µM. Most strikingly, the highest antiproliferative activity
towards tested tumor cell lines was observed in berberine derivative 3 with IC50 values of 58.0 and
36.0 for HeLa cells and 3.7 and 0.7 µM for HL-60 cells treated for 24 and 48 h, respectively. This
finding is in agreement with the previous study showing that 9-O-pyrazole alkyl substituted
berberine derivatives have increased anticancer activity compared to berberine, with IC50 values
ranging from 48.8 to 100.4 µM for HeLa cells after 24 h treatment [40].

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The effect of berberine derivatives towards tumor HeLa and HL-60 cells was further assessed by
analyzing their impact on the cellular morphology of cells treated for 24 and 48 h (Figure 5). As
shown, untreated HeLa cells had normal epithelial morphology. In contrast, in some tumor cells,
exposure to 10 µM concentrations of berberine or its derivatives 1, 2 and 3 induced the formation of
cell membrane blebbing after 24 and 48 h of treatment. However, HeLa cells treated with 10µM
concentrations of ciprofloxacin and berberine derivative 4 did not show any morphological changes.
On the other hand, compared to untreated HL-60 cells, which have the rounded morphology
characteristic of leukemia cells, the treatment of HL-60 cells with 10 µM concentrations of berberine,
ciprofloxacin and berberine derivatives 1–4 resulted in profound formation of cell membrane blebs.
Figure 5. Assessment of changes in cellular morphology of HeLa and HL-60 cells treated with
berberine, ciprofloxacin, and berberine derivatives (10 µM) for the time indicated. Magnification of
microscope: ×600.
As cell membrane blebbing, observed in sub-population of berberine derivative-treated HeLa
and HL-60 cells, is one of the defining features of apoptosis [62,63], next, we analyzed the potential
of novel berberine derivatives to induce apoptosis. We compared their ability to induce apoptosis by
employing DNA laddering and caspase-3 activation assays. As presented, after incubation of HL-60
cells with 10 µM concentrations of berberine derivatives for 48 h, only berberine derivative 3 treated
HL-60 cells were positive for apoptotic DNA fragmentation (Figure 6A) and had active caspase-3
(Figure 6B). Comparing the intensities of apoptotic DNA fragmentation and activation of caspase-3
in HL-60 cells treated with 30 µM berberine and 10 µM berberine derivative 3, berberine derivative 3
clearly exhibited 30-fold superiority to that of berberine.

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Figure 6. (A) Detection of apoptotic DNA fragmentation in HL-60 cells treated with indicated
concentrations of berberine, ciprofloxacin and berberine derivatives for 48 h. (B) Activation of caspase-
3 expressed as the mean absorbance at 405 nm (the relative amount of the pNA released from the DEVD-
pNA). Each point represents a mean ± S.D. from 3 independent experiments. * p < 0.05, ** p < 0.01.
To further extend our cytotoxicity studies, we analyzed the effect of novel berberine derivatives
on the cell cycle of HL-60 leukemia cells. We found that in contrast to berberine, which at 30 µM
concentrations arrests HL-60 cells in the G0/G1 phase, the novel berberine derivatives 1 and 2 (30 µM)
and 3 (10 µM) arrest HL-60 cells at the G2/M phase with a proportional decrease of cells in the G0/G1
and S phases, while berberine derivative 4 induces G2/M and S phases-arrest (Figure 7).
Figure 7. Analysis of cell cycle distribution of HL-60 cells treated with indicated concentrations of
berberine, ciprofloxacin and berberine derivatives 1–4 for 24 h (A) and 48 h (B).

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This finding indicates that novel berberine derivatives are more active molecules than berberine and
ciprofloxacin, which in the same concentration have no effect or induce G0/G1 cell cycle arrest of HL-60
leukemia cells [15–17]. The observed G2/M cell cycle arrest of HL-60 cells treated with berberine
derivatives 1–4 supports their higher antiproliferative activity. Interestingly, berberine derivative 3
induced a significant increase in the number of cells defined as the sub-G0 cell population (1.8% in
untreated vs. 21.9% in berberine derivative 3 treated cells, Figure 7B), confirming its apoptotis-inducing
potential.
In conclusion, we have synthesized a series of four novel 9-O-substituted berberine derivatives
and evaluated their antiproliferative activity in human HeLa and HL-60 tumor cell lines. We found
these derivatives to be more active antitumor agents than their parental molecules, berberine and
ciprofloxacin. Most importantly, we showed that berberine derivative 3 (9-(3-bromopropoxy)-10-
methoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquino[3,2-a]isoquinolin-7-ylium bromide) possesses the
highest cytotoxic activity and ability to induce apoptosis of HL-60 leukemia cells. Therefore, this
compound could become a promising candidate for anticancer therapy, and has been selected for the
follow-up studies to reveal the molecular mechanisms of its antitumor action.
3. Materials and Methods
3.1. Experimental Methods
3.1.1. Chemistry
General
®
All reagents and solvents were purchased from Sigma-Aldrich (Darmstadt, Germany), Alfa-
®
®
®
Aesar (Ward Hill, MA, USA), Fluka (Buchs, Switzerland) and Mikrochem (Pezinok, Slovakia).
Solvents (acetonitrile, DMF, dioxane, THF) were purified and/or dried using standard laboratory
methods and stored over molecular sieves (4Å ). Column chromatography was performed using
®
silicagel Nomasil—40–63 μm (VWR , Randor, PA, USA) and displayed eluent. Reaction progress was
monitored by thin layer chromatography on Silufol or Alufol plates (Merck , Darmstadt, Germany)
®
with a UV indicator for λ = 254 nm.
Melting points (m. p.) of the prepared compounds were established on a Kofler block using
®
digital thermometer TD 121 (VWR , Randor, PA, USA).
Spectrometers INOVA 300 (300 MHz, 75 MHz, 282 MHz; Varian Inc., Palo Alto, CA, USA), VXR-
300, Bruker (300 MHz, 75 MHz, Karlsruhe, Germany), and VNMRS 600 (600 MHz, 151 MHz, 564
1
13
19
MHz; Varian Inc., Palo Alto, CA, USA) were used for measurements of H, C and F NMR spectra,
at their frequencies at RT. Chemical shifts in (δ)–[ppm] (parts per million) were referenced to residual
signal of the solvent. Coupling constants (J) were given in [Hz] with multiplicity: s (singlet), d
(doublet), dd (doublet of the doublet), t (triplet), q (quartet), q (quintet) and m (multiplet).
®
Spectrometer Nicolet NEXUS 470 FT-IR (Thermo Fisher Scientific , Madison, WI, USA) was used
-1
to obtain ATR IR absorption bands (ν) in [cm ]. High resolution mass spectra (HR-MS) were recorded
®
with spectrometer LTQ Orbitrap XL (Thermo Fisher Scientific , Madison, WI, USA). Microwave
irradiation was realized in a CEM Discover microwave reactor (CEM Corporation, Matthews, NC,
USA).
Selective Demethylation of Berberine on its C9 Position
Berberine (2.0 g, 5.38 mmol) was placed into a round-bottomed flask equipped with a cross head
magnetic stirrer. The flask was inserted into an oil bath preheated to 200 °C and attached to a vacuum
pump. The berberine was then intensively stirred at 195 °C for 40 min (Scheme 1). After that, the obtained
berberrubine was crystallized using a mixture of ethanol:methanol (1:1) by refluxing for 10 min. The
obtained crystals of berberrubine were filtered using a Büchner funnel, washed with ethanol, and vacuum
dried in a water bath at 40 °C.

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Berberine
Berberrubine
Scheme 1. Synthesis of berberrubine. Reaction conditions: (i) 195 °C (temperature of oil bath), 40 min.
The yield of berberrubine was 1.25 g (62.5%, red-brownish powder, m. p.: 275–279 °C).
1
3
H NMR: (CD3OD) δ 9.28 (s, 1H, H-8), 8.70 (s, 1H, H-13), 7.54 (t, 1H, JHH=8.04 Hz, H-12), 7.49 (s,
3
3
1H, H-1), 6.91 (d, 1H, JHH=7.60, H-11), 6.85 (s, 1H, H-4), 6.04 (s, 2H, H-2’), 4.60 (t, 2H, JHH=5.32, H-6),
3
3.89 (s, 3H, H-10´), 3.12 (t, 2H, JHH=5.46, H-5)
13
C NMR: (CD3OD) δ 149.5 (C-13´), 149.4 (C-9), 148.0 (C-10), 145.9 (C-8), 134.2 (C-2), 132.2 (C-3),
129.2 (C-12´), 122.6 (C-8´), 121.4 (C-4´), 120.1 (C-13´´), 118.3 (C-12), 107.7 (C-11), 107.1 (C-13), 104.4 (C-
4),101.8 (C-1), 56.9 (C-2´), 55.2 (C-10´), 54.1 (C-6), 27.5 (C-5)
Synthesis of 9-(2-Chloro- or Bromo-acetoxy)-10-metoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquino[3,2-
a] isoquinoline-7-ylium Chloride or Bromide
Berberrubine (0.7 g, 1.96 mmol) was dissolved in dry CH3CN and stirred at 40 °C for 15 min.
After that, haloacetylhalides chloracetyl chloride (0.29 mL, 2.55 mmol) or bromacetyl bromide (0.22
mL, 2.55 mmol) were dropped into the stirred reaction mixture, followed by stirring for additional 2
h. After cooling the mixture to RT, the products were filtered off, washed with dry CH3CN and left
to dry in a vacuum.
Berberrubine
1 (X = Cl); 2 (X = Br)
Scheme 2. Synthesis of berberine derivative 1 and derivative 2. Reagents and conditions: (i) CH3CN,
40 °C, 15 min, C2H2Cl2O/C2H2Br2O, 2 h.
The yield of berberine derivative 1 was 0.42 g (49%, yellow powder, m. p. > 300 °C).
1
3
H NMR: (CD3OD) δ 10.03 (s, 1H, H-8), 8.72 (s, 1H, H-13), 8.54 (d, 1H, JHH=8.05, H-11), 8.19 (d,
3
3
1H, JHH=8.61, H-12), 7.27 (s, 1H, H-1), 7.16 (s, 1H, H-4), 6.34 (s, 2H, H-2´), 5.12 (t, 2H, JHH=5.55 Hz, H-
3
6), 4.19 (s, 2H, H-9´´), 3.78 (s, 3H, H-10´), 3.14 (t, 2H, JHH=5.34 Hz, H-5)
13
C NMR: (CD3OD) δ 159.8 (C-9´), 152.6 (C-10), 147.9 (C-3), 147.1 (C-2), 146.3 (C-8), 140.1 (C-9),
135.5 (C-13´), 132.2 (C-12´), 131.9 (C-4´), 124.4 (C-11), 121.8 (C-12), 120.1 (C-8´), 119.1 (C-13´´), 118.2
(C-13), 106.7 (C-4), 105.1 (C-1), 100.3 (C-2´), 61.1 (C-10´), 53.8 (C-6), 44.7 (C-9´´) 21.77 (C-5)
The yield of berberine derivative 2 was 0.38 g (38%, yellow-orange powder, m. p. > 300 °C).
1
3
H NMR: (CD3OD) δ 9.83 (s, 1H, H-8), 8.71 (s, 1H, H-13), 8.64 (d, 1H, JHH=8.15, H-11), 7.87 (d,
3
3
1H, JHH=8.25, H-12), 7.23 (s, 1H, H-1), 7.14 (s, 1H, H-4), 6.72 (s, 2H, H-2´), 5.13 (t, 2H, JHH=5.74 Hz, H-
3
6), 4.22 (s, 2H, H-9´´), 3.92 (s, 3H, H-10´), 3.03 (t, 2H, JHH=5.21 Hz, H-5)
13
C NMR: (CD3OD) δ 161.3 (C-9´), 149.2 (C-10), 148.1 (C-3), 145.7(C-2), 143.1 (C-8), 142.6 (C-9),
138.4 (C-13´), 134.8 (C-12´), 131.6 (C-4´), 129.1 (C-11), 123.3 (C-12), 122.7 (C-8´), 120.1 (C-13´´), 117.8
(C-13), 109.2 (C-4), 107.4 (C-1), 100.9 (C-2´), 63.1 (C-10´), 52.5 (C-6), 45.9 (C-9´´) 23.8 (C-5)
Synthesis of 9-(3-Bromopropoxy)-10-methoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquino[3,2-a]
isoquinolin-7-ylium Bromide
Berberrubine (0.5 g, 1.40 mmol) was dissolved in dry DMF (10 mL) at 80 °C and stirred for 15
min. Then, 1,3-dibromopropane (8.2 mL, 80.75 mmol) was added dropwise and the reaction was
stirred for 2 h. After cooling to RT, the diethyl ether (20 mL) was added. Separated precipitate was

Int. J. Mol. Sci. 2019, 20, 2169
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filtered off using Büchner funnel. Obtained berberine derivative 3 was further purified using column
chromatography (eluent CHCl3:MeOH—10:1).
Berberrubine
3
Scheme 3. Synthesis of berberine derivative 3. Reagents and conditions: (i) C3H6Br2, DMF, 80 °C, 2 h.
The yield of berberine derivative 3 was 0.54 g (74%, yellow powder, m. p. > 300 °C).
1
3
H NMR: (CD3OD) δ 9.79 (s, 1H, H-8), 8.94 (s, 1H, H-13), 8.20 (d, 1H, JHH=, H-11), 8.00 (d, 1H,
3
3
JHH=, H-12), 7.79 (s, 1H, H-1), 7.09 (s, 1H, H-4), 6.17 (s, 2H, H-2´), 4.94 (t, 2H, JHH=5.98 Hz, H-6), 4.42
(t, 2H, JHH=9.28 Hz, H-9´), 4.06 (s, 3H, H-10´), 3.94 (t, 2H, H-9´´´), 3.21 (t, 2H, JHH=5.58 Hz, H-5), 2.33
3
3
(p, 2H, H-9´´)
13
C NMR: (CD3OD) δ 150.2 (C-10), 149.8 (C-3), 147.6 (C-2), 145.2 (C-8), 142.4 (C-9), 137.5 (C-13´),
133.0 (C-12´), 130.7 (C-4´), 126.7 (C-11), 123.5 (C-12), 121.4 (C-8´), 120.4 (C-13´´), 120.2 (C-13), 108.4 (C-
4), 105.4 (C-1), 102.0 (C-2´), 71.2 (C-9´), 57.1 (C-10´), 55.2 (C-6), 42.1 (C-9´´´), 32.6 (C-9´´), 26.3 (C-5)
Synthesis of 1-Cyclopropyl-6-fluoro-4-oxo-7-piperazin-1-yl-1,4-dihydroquinoline-3-carbonyl
Chloride
A round bottomed flask (equipped with Dimroth condenser and calcium chloride tube)
containing thionyl chloride (0.85 mL, 11.7 mmol) was cooled in ice bath and the apparatus was filled
with argon. After cooling off the flask content, ciprofloxacin (0.1 g, 0.3 mmol) was added. The colour
of the suspension turned orange. The sample was stirred for 2 h. During stirring, the temperature
was allowed to reach RT. Then dry CH2Cl2 (20 mL) was slowly added and the suspension was
allowed to exsiccate until dry using a rotary vacuum evaporator. The procedure was repeated 3 times
to remove the excess of HCl and thionyl chloride. As the result, the target carbonyl chloride derived
from ciprofloxacin 5 was obtained [42].
Ciprofloxacin
5
Scheme 4. Synthesis of chlorinated ciprofloxacin 5. Reagents and conditions: (i) SOCl2, RT, 2 h.
The yield of chlorinated ciprofloxacin 5 was 0.085 g (81 %, red powder, m. p. 246–247 °C).
1
3
4
H NMR: (TFA-d) δ 9.63 (s, 1H, H-2), 9.47 (d, 1H, JHF=12.92 Hz, H-5), 8.13 (d, 1H, JHF=5.36 Hz,
3
H-8), 3.93-4.49 (m, 5H, H-10, H-14, H-1´), 3.13 (t, 4H, JHH=4.71 Hz, H-11, H-13), 2.43 (s, 1H, H-12),
1.47-1.85 (m, 4H, H-1´, H-1´´)
13
C NMR: (TFA-d) δ 178.2 (C-4), 162.3 (C-3´), 155.4 (C-2), 149.9 (C-6), 149.5 (C-7), 148.1 (C-8´),
117.2 (C-3), 115.1 (C-4´), 112.8 (C-5), 107.6 (C-8), 52.1, 52.3 (C-10, C-14), 44.6, 44.9 (C-11, C-13), 32.6 (C-
1´), 12.6, 12.8 (C-1´´, C-1´´´)
19
F NMR: (TFA-d) δ −77.95 (F-6)
Synthesis of 9-(1-Cyclopropyl-6-fluoro-4-oxo-7-piperazin-1-yl-1,4-dihydroquinoline-3-
carbonyloxy)-10-methoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquino[3,2-a]isoquinolin-7-ylium
Chloride
During continuous stirring, berberrubine (0.071 g, 0.19 mmol) and NaH (0.03 g, 1.14 mmol) and
after 15 min chlorinated ciprofloxacin 5 (0.07 g, 0.19 mmol) were slowly added to dry dioxane (5 mL).
The reaction mixture was then irradiated for 10 min using 300 W power at a temperature of 100 °C.

Int. J. Mol. Sci. 2019, 20, 2169
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After cooling, the mixture was exsiccated until dry and further purified using column
chromatography on silicagel using MeOH:CHCl3 (1:10) as an eluent.
5
4
Scheme 5. Synthesis of berberine derivative 4. Reagents and conditions: (i) berberrubine, NaH,
dioxane, 300 W, 100 °C, 10 min.
The yield of berberrubine-ciprofloxacin conjugate, also referred to as berberine derivative 4 was
0.039 g (31 %, dark brown powder, m. p. > 300 °C).
1
3
H NMR: (TFA-d) δ 9.43 (s, 1H, H-8), 9.31 (s, 1H, H-2C), 8.98 (d, 1H, JHF=13.83 Hz, C-5C), 8.47
3
3
(d, 1H, JHF=5.15 Hz, H-8C), 8.27 (d, 1H, JHH=8.73 Hz, H-11), 7.98-8. 12 (m, 3H, H-1, H-4, H-12), 6.63
(s, 2H, H-2´), 5.28 (t, 2H, JHH=6.02 Hz H-6), 4.25 (s, 3H, H-10´), 3.25-4.18 (m, 11H, H-5, H-10C, H-11C,
3
H-13C, H-14C, H-1´C), 2.98 (s, 1H, H-12C), 1.53-1.72 (m, 4H, H-1´´C, H-1´´´C)
13
C NMR: (TFA-d) δ 176.1 (C-4C), 167.5 (C-3´C), 159.7 (C-2C), 152.6 (C-10), 151.7 (C-6C), 150.3
(C-7C), 148.1 (C-3), 146.9 (C-2), 144.9 (C-8), 143.5, 144.1 (C-9, C- 8´), 140.0 (C-13´), 135.1 (C-12´), 131.7
(C-4´), 123.8, (C-11), 123.1 (C-12), 119.3, 120.7, 121.9 (C-8´, C-13´´, C-3C), 118.5 (C-13), 115.4 (C-8C),
106.3, 106.9, 108.2 (C-1, C-4, C-8C), 100.1 (C-2´), 62.1 (C-10´), 53.8, 54.6, 55.1 (C-6, C-10C, C-14C), 48.7,
49.3 (C-11,C-13), 38.7 (C-1´C), 31.4 (C-5), 14.7, 14.9 (C-1´´, C-1´´´)
19
F NMR: (TFA-d) δ −75.71 (F6)
+
HRMS (ESI): calculated for C36H32ClFN4O6 (M+H ): 671.1994; found: 670.9106.
3.1.2. Electron Paramagnetic Resonance (EPR) Spectroscopy Studies of Berberine Derivatives
The UV/visible spectra of berberine, ciprofloxacin and newly synthesized berberine derivatives
dissolved in the mixed solvent DMSO/water (1:100, v:v) were recorded using a UV-3600 UV-vis-NIR
spectrophotometer (Shimadzu, Kyoto, Japan) with a 1-cm square quartz cell.
The formation of paramagnetic intermediates of the studied berberine derivatives upon their
UVA irradiation was monitored in situ using the EPR spin trapping technique. The photoinduced
production of singlet oxygen during the excitation of berberine derivatives was followed by the
oxidation of a sterically-hindered amine 4-oxo-2,2,6,6-tetramethylpiperidine (TMPO). The solution of
the berberine derivatives was mixed either with the spin trapping agent 5,5-dimethyl-1-pyrroline N-
oxide (DMPO) or TMPO solution, and immediately after a careful air or argon saturation using a
slight gas stream, the prepared sample was transferred to a small quartz flat cell (WG 808-Q, optical
cell length 0.04 cm; Wilmad-LabGlass, Vineland, NJ, USA) optimized for the TE102 cavity (Bruker,
Rheinstetten, Germany) of the X-band EPR spectrometer (EMX Plus, Bruker, Rheinstetten, Germany).
The photoexcitation of the samples took place directly in the EPR resonator at 295 K and the EPR
spectra were recorded in situ during the continuous irradiation or after a defined exposure. A UV
LED monochromatic radiator (λmax = 365 nm; Bluepoint LED, Hönle UV Technology,
–2
Gräfelfing/München, Germany) reaching the irradiance value of 13 mW·cm within the EPR cavity,
determined using a UVX radiometer (UVP, Upland, CA, USA) or the visible-light source (KL
1600LED (T = 5600 K; Schott)) were used as the irradiation sources. The g-values were determined
using a built-in nuclear magnetic resonance teslameter (ER 036TM, Bruker) and an integrated
frequency counter. The EPR spectra were analyzed and simulated using Bruker software WinEPR
and EasySpin toolbox working under Matlab software (MathWorks, Natick, MA, USA) [64].
3.1.3. Evaluation of Antiproliferative Activities of Berberine Derivatives
Cell Cultures and Determination of Cell Viability and Morphology

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HL-60 (human promyelocytic leukemia) and HeLa (human cervix epithelioid carcinoma) cells
were grown in a humidified atmosphere containing 5% CO2 and 95% air at 37 °C. HeLa cells were
maintained in a DMEM medium and HL-60 cells were maintained in RPMI 1640 medium, both
supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 50 U/mL penicillin, and 50
µg/mL streptomycin. Stock solutions of berberine and its derivatives were prepared in DMSO, except
for a stock solution of ciprofloxacin, which was prepared in 0.1 M HCl. In all experiments, the final
concentration of DMSO was kept ≤ 0.1% (v/v) and 0.1% DMSO alone was used as vehicle control. Cell
viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
4
4
(MTT) assay [61]. HL-60 cells (2 × 10 cells/well) or HeLa cells (4 × 10 cells/well) were seeded in 96-
well plates. Cells were treated with various concentrations of berberine, ciprofloxacin and berberine
derivatives (0–100 µM) for 24 and 48 h. At the end of the treatment, MTT solution (final concentration
1 mg/mL) was added to each well and the plates were incubated for another 4 h. The precipitated
formazan was dissolved in DMSO and its concentration was measured at 595 nm using a microplate
reader (xMark, BioRad, Tokyo, Japan). Based on the curve fitting using nonlinear regression (Origin
7.0, Microcal), the median inhibitory concentrations IC50 values (the concentration resulting in 50%
inhibition of the cell proliferation that was recorded in control experiments) were determined
separately for each experiment. The values were calculated from three independent experiments.
Data are presented as a mean ± SD (n=3).
The morphology of cells treated with tested drugs was evaluated using light microscopy (Zeiss
Jenalumar, Jena, Germany) [65].
Detection of Induction of Apoptosis by DNA Laddering Assay
6
Cells (2 × 10 ) treated with various concentrations of tested derivatives were washed with ice-
cold PBS containing 10 mM EDTA. Pellets of cells were resuspended in 250 µL of lysis buffer (10 mM
Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM EDTA, 5% Triton-X, 0.25% SDS) supplemented with 0.4
mg/mL DNase-free RNase A, incubated at 65 °C for 15 min, cooled to RT for 10 min, and incubated
at 37 °C for 90 min. After that, Proteinase K was added to a final concentration of 0.2 mg/mL and
samples were incubated at 50 °C for an additional 1 h. The DNA was extracted with 200 µL of
phenol:chloroform:isoamyl alcohol (25:24:1) for 1 min and centrifuged at 14,500 g for 5 min. The DNA
was precipitated from the aqueous phase with 3 volumes of chilled EtOH containing 0.3 M sodium
acetate. The DNA pellets were washed with 70% EtOH, dried, and dissolved in 100 µL of TE buffer.
The DNA was separated on 1.5% agarose gel and visualized under UV illumination after being
stained with GelRed Nucleic Acid Gel Stain (254 nm, Ultra-Lum Electronic UV Transilluminator,
Paramount, CA, USA) [66].
Detection of Apoptosis by Caspase-3 Assay
TM
Activation of caspase-3 was measured using a colorimetric caspase-3 assay kit (CaspACE
Assay System, Promega, Madison, WI, USA). Briefly, exponentially growing HL-60 cells were treated
6
with tested derivatives (10–30 µM) for 48 h. Cells (5 × 10 ) were pelleted by centrifugation (1200 × g,
3 min at 4 °C) and resuspended in 50 μL of lysis buffer (Promega, Madison, WI, USA). The lysed cell
mixture was then incubated on ice for 15 min before centrifugation (13,000 × g, 5 min at 4 °C). 42 μL
of reaction buffer supplemented with 10 mM DTT were mixed with 20 µL of cell lysates. The substrate
DEVD-pNA (2 µL) was added and the samples were incubated for 4 h at 37 °C. The release of p-
nitroaniline from specific caspase-3 substrate was measured at 405 nm using a microplate reader
(xMark, BioRad, Tokyo, Japan).
Determination of Cell Cycle Distribution
6
Cells (0.5 × 10 ) treated with berberine derivatives (0–30 µM) for 24 and 48 h were washed with
PBS and fixed in ice-cold 70% ethanol. Next, the cells were washed twice with PBS and resuspended
in 0.1% Triton-X in PBS containing 50 µg/mL RNase A and incubated for 30 min at 37 °C. Afterwards,
DNA was stained using PI (50 µg/mL) for 15 min at 4 °C. The cell cycle distribution was analyzed

Int. J. Mol. Sci. 2019, 20, 2169
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using a FACS Canto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). A minimum of
10,000 cells per sample was analyzed at a flow rate of 200 cells/s [67].
Author Contributions: Data curation, J.J., M.P. and L.C.; Funding acquisition, V.M., I.C., D.C., V.B. and L.C.;
Investigation, V.M., A.S., Z.B., E.H., I.C., D.C., S.J., V.B. and L.C.; Project administration, V.M. and L.C.;
Supervision, V.M., V.B. and L.C.; Visualization, S.J.; Writing—original draft, V.M., Z.B., I.C., S.J., V.B. and L.C.
Writing—review & editing, V.M., V.B. and L.C.
Funding: This work was supported by the by People Programme (Marie Curie Actions) European Union’s
Seventh Framework Programme (REA Grant Agreement No. 609427, SASPRO Project No. 0032/01/02), by the
VEGA Grants 2/0004/10, 2/0076/17, 2/0026/18 and 2/0039/19 and by the Slovak Research and Development
Agency under the contract No. APVV-15-0053, No. APVV-16-0120 and No. APVV-17-0513. V.B. thanks the
Ministry of Education, Science, Research and Sport of the Slovak Republic for funding within the scheme
“Excellent research teams”.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
Abbreviations
DBP
DMF
DMPO
DMSO
DTT
MTT
PBS
1,3-Dibromopropane
Dimethylformamide
5,5-Dimethyl-1-pyrroline N-oxide
Dimethyl Sulfoxide
Dithiothreitol
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Phosphate buffer saline
Propidium iodide
PI
ROS
RT
Reactive Oxygen Species
Room temperature
Tempone 4-Oxo-2,2,6,6-tetramethylpiperidine N-oxyl
THF
Tetrahydrofuran
TMPO
4-Oxo-2,2,6,6-tetramethylpiperidine
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