Substituted piperazines as novel potential radioprotective agents

Article abstract of DOI:10.3390/molecules25030532

The increasing risk of radiation exposure underlines the need for novel radioprotective agents. Hence, a series of novel 1-(2-hydroxyethyl)piperazine derivatives were designed and synthesized. Some of the compounds protected human cells against radiation-

Full text of DOI:10.3390/molecules25030532

molecules
Article
Substituted Piperazines as Novel Potential
Radioprotective Agents
Alzbeta Filipova 1, Jan Marek 2, Radim Havelek 3, Jaroslav Pejchal 4, Marcela Jelicova 1,
Jana Cizkova 1, Martina Majorosova 3, Lubica Muckova 4 , Tomas Kucera 4, Lukas Prchal 2,
Miroslav Psotka 2, Natalie Zivna 2, Darja Koutova 3, Zuzana Sinkorova 1, Martina Rezacova 3
and Ales Tichy 1,
*
1
Department of Radiobiology, Faculty of Military Health Sciences, University of Defence in Brno, Trebesska
1575, 500 01 Hradec Kralove, Czech Republic; alzbeta.filipova@unob.cz (A.F.);
Marcela.Jelicova@unob.cz (M.J.); Jana.Cizkova@unob.cz (J.C.); Zuzana.Sinkorova@unob.cz (Z.S.)
Biomedical Research Center, University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec Kralove,
Czech Republic; marekjjja@gmail.com (J.M.); lukas.prchal@fnhk.cz (L.P.); miroslav.psotka@fnhk.cz (M.P.);
natalie.zivna@natur.cuni.cz (N.Z.)
Department of Medical Biochemistry, Faculty of Medicine in Hradec Kralove, Charles University, Simkova
870, 500 03 Hradec Kralove, Czech Republic; havelekr@lfhk.cuni.cz (R.H.); SeifrtovaM@lfhk.cuni.cz (M.M.);
koutova.darja@lfhk.cuni.cz (D.K.); rezacovaM@lfhk.cuni.cz (M.R.)
Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences, University of Defence
in Brno, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic; jaroslav.pejchal@unob.cz (J.P.);
Lubica.Muckova@unob.cz (L.M.); Tomas.Kucera@unob.cz (T.K.)
2
3
4
*
Correspondence: ales.tichy@unob.cz; Tel.: +420973253216
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 3 December 2019; Accepted: 20 January 2020; Published: 25 January 2020
Abstract: The increasing risk of radiation exposure underlines the need for novel radioprotective
agents. Hence, a series of novel 1-(2-hydroxyethyl)piperazine derivatives were designed and
synthesized. Some of the compounds protected human cells against radiation-induced apoptosis and
exhibited low cytotoxicity. Compared to the previous series of piperazine derivatives, compound
8
exhibited a radioprotective effect on cell survival in vitro and low toxicity in vivo. It also enhanced
the survival of mice 30 days after whole-body irradiation (although this increase was not statistically
significant). Taken together, our in vitro and in vivo data indicate that some of our compounds are
valuable for further research as potential radioprotectors.
Keywords: piperazine; radiation-protective agents; cytotoxicity; maximum tolerated dose; synthesis
de novo
1. Introduction
Ionizing radiation (IR) was discovered in the 19th century; since then, its use has had a dramatic
impact on medicine, space travel, research, and the energy industry. Unfortunately, the catastrophic
effects of IR have also been exploited by the development of nuclear weapons; moreover, a number
of accidents have occurred at nuclear power reactors resulting in ecological disasters and loss of
lives [1–3].
Exposure to IR is associated with cell death, genetic mutations, and carcinogenesis [4]. It induces
DNA damage in the form of double-strand DNA breaks, which is considered the underlying mechanism
of the resulting cell death: apoptosis.
Apoptosis is a very complex process, which is tightly controlled in mammalian cells [5].
Its multi-level regulation has been the subject of medical research for a long time because some
pathologies (such as cancer and autoimmune and neurodegenerative disease) are closely associated
Molecules 2020, 25, 532; doi:10.3390/molecules25030532
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Molecules 2020, 25, 532
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with the dysregulation of programmed cell death. Above that, individual regulatory systems provide
suitable targets for novel compounds with desirable pharmacological potential [ ]. The testing
6–9
of thousands of synthetic and natural compounds has consequently led to the discovery of novel
radioprotectants or mitigators. Unfortunately, these compounds can often be insoluble or cytotoxic,
with low tolerance, or with very low circulating half-life. As a result, their clinical application is rather
limited [10,11].
The increasing risk of radiation exposure underlines the need for novel radioprotective
agents [3,12,13]
.
However, there is very limited current literature concerned with small
molecules as potential radioprotective agents. Based on previous studies, we have selected the
1-(2-hydroxyethyl)piperazine moiety (main part of compounds in Scheme 1, compound 1 in Scheme
S4 in Supplementary Materials) as the essential component of the molecule [10
our structures are already mentioned in the literature, but none has been reported in the context of
radioprotection. Namely, compound had already been prepared as a leading structure for a novel
group of cholinesterase inhibitors [15]. Compound was prepared afterwards as a novel structure
according to the procedure for preparation of these inhibitors. Compound was searched for in the
PubChem and ZINC database [16], but no synthesis or biological data were found. The symmetrical
molecule consisting of two aniline residues had been prepared by Dains et al. in 1922 [17]. None of
,14]. Notably, some of
2
3
4
9
the other compounds had ever been prepared or tested before (5, 6, 7, 8, and 10).
Thus, the aim of this work was to investigate a group of 1-(2-hydroxyethyl)piperazine derivatives
as potential affordable radioprotective agents. We focused on various modifications of the part of the
molecule attached to the basic 1-(2-hydroxyethyl)piperazine moiety. We characterized the prepared
compounds using nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS),
and evaluated their cytotoxicity and radioprotective properties in vitro and in vivo using human cell
lines and an animal experimental model.
2. Results
2.1. Chemistry
All the compounds developed in this study are outlined in Scheme 1. Compound
synthesized as previously described; the results (yields, analytical data) are comparable to those formerly
reported [15]. Compound was prepared by the reaction of with Hunig s base and 2-bromoethanol,
following also previously reported protocol (Scheme S1 in Supplementary Materials) [15]. The synthetic
route for compound is a two-step reaction. The first step is the reaction of 4-chloro-3-methylphenol
4a) with an excess of epibromohydrin under reflux for 2 h in the presence of piperidine, according to
2 was
3
2
´
4
(
the process published in [14] (Scheme S2 in Supplementary Materials). This solvent-free reaction led to
the preparation of intermediate 4b in 70% yield (Scheme S2 in Supplementary Materials). 4b was used
for a further step without any purification. Accordingly, 1-(2-hydroxyethyl)piperazine was alkylated
by 4b, affording 4 in 79% yield (Scheme S2 in Supplementary Materials).
Compounds
preparation of intermediates 5b
appropriate aryl amines 5a 6a, and 7a, respectively, with the 1-bromo-3-chloropropane in acetonitrile
under microwave conditions, all with rather poor yield around 30% (Scheme S3 in Supplementary
Materials). 5b 6b, and 7b were used for the following step without any purification. The next step
involved the alkylation of 1-(2-hydroxyethyl)piperazine ( ) by the intermediates 5b 6b, and 7b, resulting
in compounds , and , respectively, in 60–70% yields (Scheme S3 in Supplementary Materials).
The procedure for the preparation of compound was the reaction of 1-(2-hydroxyethyl)piperazine
) with epibromohydrin in ethanol (Scheme S4 in Supplementary Materials). The yield of this
one-step reaction was 27% due to the formation of side products. Symmetric molecule was prepared
5,
6, and
7
were synthesized similarly via a two-step reaction. The first step was the
,
6b, and 7b (Scheme S3 in Supplementary Materials) by treatment
,
,
1
,
5,
6
7
8
(1
9
using solvent-free reaction under microwave conditions in 25% yield (Scheme S5 in Supplementary
Materials). Compound 10 was synthesized via a two-step reaction. The first step was the preparation

Molecules 2020, 25, 532
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of intermediate 10b (Scheme S6 in Supplementary Materials). This intermediate was isolated during
the procedure of preparation of as the second expected product in a yield of 45%. The next step
involved the alkylation of 1-(2-hydroxyethyl)piperazine ( ) by the intermediate (10b), resulting in
9
1
compound 10 in 90% yield (Scheme S6 in Supplementary Materials).
Scheme 1. Synthesized compounds as bases with potential radioprotective properties.
Finally, the compounds (2–10) (Table 1) were converted to their salts by treatment with hydrochloric
acid in methanol. All the final compounds were characterized by their 1H- and 13C-NMR spectra and
high-resolution mass spectrometry (HRMS). All analytical measurements confirmed the structure and
purity of over 95% of the final compounds as determined by LC-MS analysis. All the spectral data of
the compounds are provided in the Experimental section.
Table 1. Structures, yields, and ClogP of prepared compounds.
Compound
Yield (%)
Melting Point (◦C)
*ClogP
2
3
4
5
6
7
8
9
10
78
75
79
68
69
61
27
25
90
172–173
157–158
189–190
185–186
181–182
182–183
oil
2.255
1.948
1.478
0.682
1.671
0.524
−2.107
2.090
−0.009
oil
154–155
* ClogP was calculated with MarvinSketch (version 14.9.8.0) software.
2.2. Molecular Docking with Anti-Apoptotic Protein Bcl-2
The docking calculations with ligand anti-apoptotic protein Bcl-2 (B-cell lymphoma 2) were
performed for all of the investigated compounds. All of them showed hydrophobic interaction of the
aromatic part (tetrahydroacridine, naphtyl, or phenyl moiety) with the hydrophilic pocket of the Bcl-2
protein (Leu134, Phe101, Tyr105, Phe109, Met112, Phe150 and Val130), except for compound 8. The
quarternary hydrogen of all compounds interacted with Asp108, except for compound 9.

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A hydrogen bond of uncharged piperazine nitrogen with Arg143 was observed frequently. Some
of the compounds showed a hydrogen bond of aniline nitrogen with Asp108. Other interactions could
not be judged as group characteristics but rather individual features.
Interaction of compounds 4, 8, and 10 with Bcl-2 are shown in Figure 1.
Figure 1. Interaction of selected compounds with Bcl-2 protein. (A) Interaction of compound 4 with
Bcl-2: ionic interaction of protonated piperacilin nitrogen with Asp108, hydrogen bond of hydroxyl
oxygen with Arg143, hydrophobic interaction of chlormethylphenyl part with the pocket Leu134,
Phe101, Tyr105, Phe109, Met112, Phe150, and Val130. (
interaction of protonated piperacilin nitrogen with Asp108, hydrogen bonds Arg143 with hydroxyl
oxygen. ( ) Interaction of compound 10 with Bcl-2: hydrogen bonds of aniline group and of hydroxyl
B) Interaction of compound 8 with Bcl-2: ionic
C
group with Asp108, and of piperacilin nitrogen and hydroxyl oxigen with Arg143, hydrophobic
interaction of phenyl part with the pocket Leu134, Phe101, Tyr105, Phe109, Met112, Phe150, and Val130.
2.3. In Vitro Cytotoxicity of Newly Synthesized Compounds 2, 3, 4, 5, 6, 7, 8, 9, and 10
In this study, the cytotoxic effects of nine new compounds (
10 human cell lines (nine cancer and one non-cancer cell line). The cell lines were treated with each
compound at concentrations of 10 M and 100 M for 48 h. The proliferation capacity of the treated
2, 3, 4, 5, 6, 7, 8, 9, 10) were tested on
µ
µ
cells was determined by WST-1 test and compared against the proliferation rate of the untreated control
cells (100%).
These experiments revealed that compound
tested, as indicated by the percentage of viable cells which was less than 44%, especially in MOLT-4
cells. Accordingly, compound was not tested at 100 M. The remaining compounds had no evident
cytotoxic effect on any of the tested cell lines.
Further experiments at 100 M revealed a significantly lower number of living cells when
compound was used. The cytotoxic effect of this compound was detected in all the tested cell lines,
decreasing cell viability by 70% and more, especially in MOLT-4 and MRC-5 cells. However, the
2 (10 µM) was cytotoxic across all the 10 cell lines
2
µ
µ
3
remaining compounds (4, 5, 6, 7, 8, 9, and 10) had no significant cytotoxic effects on the studied cell
lines (Table 2).

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Table 2. Cytotoxic effect of novel compounds in vitro. Values represent cell viability after treatment
with 10 M ( ) and 100 M ( ) of the tested compounds. The results are expressed as relative
viability (%) when compared to an untreated control (100%). Compound was not tested at 100 M.
DOX—doxorubicin (1 M). Each value represents the mean of three independent experiments SD.
µ
A
µ
B
2
µ
µ
±
Values from the intervals 0–25% (red color), 26–50% (orange color) and 50–75% (yellow color) are
highlighted with different colors.
(A)
Cell line
2
3
4
5
6
7
8
9
10
DOX
Jurkat
MOLT-4
A549
31 ± 14
95 ± 9
81 ± 13
94 ± 15
85 ± 11
78 ± 6
105 ± 5
88 ± 6
100 ± 4
100 ± 2
97 ± 7
98 ± 2
106 ± 2
105 ± 4
99 ± 6
97 ± 8
98 ± 4
93 ± 9
86 ± 7
91 ± 2
86 ± 16
93 ± 5
100 ± 4
101 ± 6
96 ± 4
98 ± 7
101 ± 1
90 ± 7
103 ± 7
86 ± 4
92 ± 11
74 ± 3
108 ± 6
102 ± 8
94 ± 6
91 ± 4
(B)
100 ± 5
93 ± 3
95 ± 5
96 ± 3
99 ± 10
94 ± 3
96 ± 8
105 ± 6
102 ± 7
100 ± 3
103 ± 5
106 ± 8
97 ± 5
104 ± 5
103 ± 8
107 ± 11
102 ± 5
93 ± 12
90 ± 3
108 ± 7
112 ± 6
105 ± 9
104 ± 11
95 ± 7
2 ± 0
0 ± 0
1 ± 0
27 ± 3
28 ± 6
39 ± 2
44 ± 9
25 ± 1
15 ± 1
28 ± 2
23 ± 2
42
55
±
±
11
18
HT-29
101 ± 1
98 ± 9
PANC-1
A2780
HeLa
MCF-7
SAOS-2
MRC-5
78 ± 7
84 ± 7
104 ± 4
103 ± 4
101 ± 5
102 ± 6
95 ± 7
103 ± 6
110 ± 7
98 ± 2
8 ± 4
104 ± 13
84 ± 9
99 ± 9
76
±
12
100 ± 4
106 ± 2
102 ± 8
32 ± 3
22 ± 4
22 ± 2
80 ± 4
97 ± 6
84 ± 2
102 ± 3
2 - not
tested
Cell line
3
4
5
6
7
8
9
10
DOX
Jurkat
MOLT-4
A549
x
x
x
x
x
x
x
x
x
x
12 ± 5
2 ± 0
89 ± 1
92 ± 6
99 ± 0
83 ± 4
111 ± 24
89 ± 1
137 ± 5
90 ± 3
88 ± 4
93 ± 5
98 ± 3
104 ± 7
104 ± 8
109 ± 2
115 ± 7
105 ± 8
116 ± 10
106 ± 1
111 ± 6
102 ± 8
93 ± 2
112 ± 4
93 ± 3
97 ± 4
111 ± 5
95 ± 4
104 ± 7
94 ± 9
111 ± 4
94 ± 2
96 ± 0
99 ± 3
96 ± 13
81 ± 6
79 ± 11
71 ± 4
95 ± 4
100 ± 4
105 ± 2
113 ± 2
111 ± 6
96 ± 2
2 ± 0
1 ± 0
13 ± 2
14 ± 5
30 ± 4
20 ± 5
11 ± 3
10 ± 2
10 ± 2
9 ± 6
115 ± 0
118 ± 2
116 ± 3
91 ± 2
111 ± 11
154 ± 11
117 ± 11
76 ± 10
100 ± 18
96 ± 7
98 ± 13
148 ± 2
118 ± 5
64 ± 5
42
55
±
±
11
18
HT-29
PANC-1
A2780
HeLa
MCF-7
SAOS-2
MRC-5
78 ± 7
8 ± 4
132 ± 3
104 ± 6
109 ± 2
104 ± 3
90 ± 14
98 ± 11
90 ± 4
121 ± 3
96 ± 6
76
±
12
32 ± 3
22 ± 4
22 ± 2
87 ± 7
102 ± 2
102 ± 8
119 ± 16
120 ± 16
2.4. In Vitro Toxicity Determination of New Inhibitors
In further experiments, MTT (3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
assay was employed to evaluate the toxicological indexes of the newly synthesized compounds,
including IC50 (half-maximal inhibitory concentration) and MTC (maximum tolerated concentration).
Since the assay is applicable only for adherent cells, the A-549 cell line was selected for these evaluations.
In A549 cells, the lowest IC50 was found for compound
compounds increased in the following order from to
2
(0.04
±
0.002 mM). The tolerance of other
2
3,
9,
6
,
4
,
5
,
7
,
10, and , with corresponding IC50
8
values being 3.5-, 6.0-, 11.3-, 16.0-, 33.8-, 63.8-, 126.5-, and 625-fold higher than that for compound
(Table 3).
2
Table 3. Inhibitory concentration (IC50) and maximum tolerated concentration (MTC) in A549 cells.
Compounds
IC50 ± SEM (mM)
0.04 ± 0.002
0.14 ± 0.01
0.64 ± 0.03
1.35 ± 0.12
0.45 ± 0.01
2.55 ± 0.21
> 25
MTC (mM)
0.002
0.01
2
3
4
0.05
5
0.05
6
0.02
7
0.39
8
6.25
9
0.24 ± 0.01
5.06 ± 0.23
0.09
10
1.56

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2.5. Pre-Treatment with Compounds 4, 5, 6, 7, 8, and 10 Reduced Radiation-Induced Apoptosis In Vitro
Phosphatidylserine, a major component of the inner leaflet of the cytoplasmic membrane bilayers,
is frequently used as an indicator of cell death. The externalization of this phospholipid can be detected
through Annexin V, which binds to phosphatidylserine in the presence of calcium. Cell viability was
quantified by Annexin V-Alexa Fluor® 488/propidium iodide (PI) staining and flow cytometry analysis
to determine whether the tested compounds could prevent radiation-induced cell death in MOLT-4
cells. The percentage of viable cells was 92% for non-irradiated control (0.1% DMSO vehicle) and
40% for untreated irradiated cells (1 Gy) (Figure 2A). A 60-min pre-treatment with the compounds at
100
(compound
(Figure 2B). Compound
µ
M significantly increased cell viability after irradiation (1 Gy), with the following values: 61%
4
), 69% (compound ), 64% (compound ), 57% (compound ), and 51% (compound 10)
6
7
8
9
exhibited no significant radioprotective effect in vitro. Although compound
5
did not show statistically significant radioprotective effect (59%), it was further evaluated in vivo
due to its low cytotoxicity.
B
A
Figure 2. The viability of MOLT-4 cells after exposed to ionizing radiation (IR) alone or in
combination with inhibitors at 100 M. Viability of MOLT-4 cells was determined as the percentage
of Annexin-V–Alexa Fluor® 488 and propidium iodide (PI)-negative cells by flow cytometry 24 h
following irradiation with a dose of 1 Gy. ( ) Representative flow cytometry histograms depicting the
percentage of viable MOLT-4 cells are shown. ( ) The bar graph represents the percentage of viable
µ
A
B
MOLT-4 cells detected by flow cytometry. Results are shown as the mean
*—significantly different from irradiation alone (t-test, * p < 0.05).
±
SD from four experiments.
2.6. The Compounds Administered to Mice at MTD Caused No Pathologies
Mild to moderate signs of intoxication could be observed once the MTD (maximum tolerated
dose) was reached. In vivo, MTD is described as the highest dose of a given compound that can be
administered before showing signs of toxicity (based on the same dose regimen or series) and whose
excess would be expected to be fatal, yielding unreliable results [18]. Therefore, a staged approach
(including core and intermediate dose levels) has become a suitable strategy in this regard.
The symptoms described in Tables S1 (in Supplementary Materials) and S2 (in Supplementary
Materials) diminished spontaneously within 2 h. After observing these symptoms, MTD thresholds
were set at 100, 200, 100, 200, 2000, and 650 mg/kg for compounds 4, 5, 6, 7, 8, and 10, respectively.
Animals receiving MTD were further examined by necropsy 48 h after administration. Plasma
(reflecting biochemical changes in blood), intestine with mesentery (site of administration), and liver
and kidney (elimination organs) were collected to evaluate the toxic potential of the tested compounds.
For evaluation of the impact of the compounds on organ function, we performed blood biochemistry

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test 48 h after administration. The test focused on seven important indicators: glucose and amylase for
normal function of pancreas, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and
alkaline phosphatase (ALP) for the normal function of liver, and urea and creatinine for the normal
function of kidneys. According to necropsy, blood biochemistry and histopathology analyses, the
tested compounds caused no pathologies, were found harmless, and were suitable for in vivo survival
tests (Table S3 in Supplementary Materials). The only histopathological change observed was limited
focal necrosis in one male administered with compound
4 (one small focus), one female administered
with compound 10 (five small foci), and one control male (two small foci). According to Thoolen et
al. [19], focal necrosis can be occasionally seen in untreated rodents and therefore is not pathognomonic
of hepatotoxicity, which is consistent with our findings.
2.7. Pre-Treatment with the Compounds Increased Survival of Whole-Body Irradiated Mice
To estimate the radioprotective effect on whole-body irradiated mice, compounds
4 (50 mg/kg),
5
(100 mg/kg), (50 mg/kg), (100 mg/kg), (1000 mg/kg), and 10 (325 mg/kg) were injected
6
7
8
(intraperitoneally, i.p.) 5 min before receiving 7.15 Gy of gamma radiation. These compounds were
selected based on the cytotoxicity screening in vitro, and applied at a dose corresponding to 50% of
the MTD in vivo. Notably, the tested compounds did not affect the survival of non-irradiated mice.
The follow-up period was 30 days.
The lethal effect of IR could be observed after 10 days in the non-treated group, and only four mice
remained alive after the 30-day period of the study. Mice pre-treated with the compounds retained
longer (or equal) survival than the non-treated control with the exception of the group pre-treated with
compound
7, which showed an earlier lethal effect at 9 days post-irradiation. In addition, compounds 5
and caused a decline in survival at the same time as the non-treated control (10 days post-irradiation).
6
In contrast, the groups pre-treated with
12 post-irradiation.
4, 8, and 10 had a higher survival rate, with a drop after day
At the end of experiment (day 30 post-irradiation), the survival values were as follows: control
(40%), (50%), (30%), (20%), (0%), (70%), and 10 (30%). In summary, although compounds and
10 led to a higher survival rate in the short-term, it was compound that conferred a radioprotective
4
5
6
7
8
4
8
effect and showed the highest survival values after 30 days of irradiation; however, these increases
were not found to be statistically significant (Figure 3).
Figure 3. Kaplan–Meier survival curves of irradiated mice pre-treated intraperitoneally (i.p.) with
saline buffer (7.15 Gy) and with compounds
4, 5, 6, 7, 8, and 10. Each group (n = 10) was observed until
termination of the experiment (30 days after irradiation).

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3. Discussion
In recent years, medical radiation uses have increased significantly together with the risk of
radiation/nuclear events. Current pre-clinically tested radioprotectors for radiotherapy involve immune
modulators, nutraceuticals (isoflavonoids), free-radical scavengers, corticosteroids, or recombinant
cytokines. Several promising radioprotective agents have been synthesized de novo.
Ex-RAD(®), which is also known as the sodium salt of 4-carboxystyryl-4-chlorobenzylsulfone, was
reported as a small molecule with radioprotective properties based on targeting p53 and its downstream
regulators [20]. Ex-RAD prevented radiation-induced apoptosis and increased the survival rate of
whole-body irradiated mice [21]. Tang et al. prepared a group of novel benzyl naphthyl sulfoxide
derivatives from Ex-RAD. Interestingly, one of the new derivatives exhibited a remarkable increase
(100%) in mice survival after irradiation [22].
Another attempt to develop radioprotective agents was made by Hosseinimehr, who synthesized
a group of 2-imino-3-[(chromone-2-yl)carbonyl] thiazolidines. Some of these compounds protected
mice from whole-body gamma irradiation with high statistical significance [23].
Obviously, numerous papers were published regarding novel agents for enhanced radiation
protection in the last decade; notwithstanding, the only radioprotector approved by the FDA is
amifostine [24].
Amifostine was reported to possess a high dose-reducing factor and to prevent radiation-induced
injury repeatedly [25,26]. However, FDA has approved it for limited clinical indications only and
not for non-clinical applications. Despite the efforts that have been put into increasing amifostine
effectiveness, the issues with its toxicity and side effects have not been resolved. Thus, further studies
are needed to improve the drug design and safety of future radioprotectors.
When searching for appropriate drug targets in radioprotection, many researchers turned their
attention toward the regulation of apoptotic machinery, as both acute and chronic radiotoxicity has
been associated with cell death. IR induces apoptosis through a complex DNA damage pathway.
While this effect is desirable during the radiotherapy of cancer cells, it leads to radiotoxicity in normal
healthy tissues.
There has been growing evidence that a complex of pro-apoptotic and anti-apoptotic proteins (e.g.,
PUMA-Bcl-2, PUMA-Mcl-1) play the crucial role in apoptosis regulation, and their selective inhibition
represents an exciting therapeutic opportunity [27].
Consistently, according to our previous observations, we identified PUMA together with its
interaction partners as the key molecule of radiation-induced cell death signaling, and thus a promising
target in medical radioprotection, but also in the therapy of neurodegenerative and cardiovascular
diseases [28].
Previously, we reported a group of 1-(4-(2-hydroxyethyl)piperazin-1-yl)-3-phenoxypropan-2-ol
derivatives with a possible mechanism of radioprotective effect based on interference with Bcl-2
family protein–protein interaction [14]. The goal of this work was to describe the second generation of
piperazine derivates, as we anticipate a similar mechanism of action with an improved pharmacological
profile. Although the molecular explanation of the potential radioprotective effect is a subject of
parallel study (currently undergoing), the molecular docking data indicate possible interaction with
the hydrophilic pocket of anti-apoptotic protein Bcl-2.
In the presented study, 10 new compounds were synthesized based on 1-(2-hydroxyethyl)
piperazine derivatives. Compound 2 has already been published as a cholinesterase inhibitor with
intended application for the treatment of Alzheimer’s disease [15]. Since it contained a piperazine
structural moiety linked through an aminoethyl group to an aromatic moiety, we presumed its inhibition
of other protein–protein interactions. Unfortunately, the compound was discarded in baseline screening
due to high cytotoxicity.
Therefore, the substance was modified by substituting the piperazine core with a hydroxyethyl
group. By substituting the secondary amino group of the piperazine of compound
2, we presumed
that there would be a reduction in the cytotoxic effect. This phenomenon was confirmed during the

Molecules 2020, 25, 532
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baseline screening but at a higher concentration (100
µM). Compound
3
was eventually found to be
excessively toxic to the cell lines and excluded from further testing.
Compound showed no radioprotective effect in vitro. A possible explanation could be that the
compound does not contain a basic piperidine moiety, which seems to be essential for this property.
Such phenomenon has already been published by our group [14]. Compound could not be tested
9
9
in vivo due to the low solubility. Compared to the other compounds, the low solubility was due to the
presence of two aromatic moieties that also significantly affect the ClogP value (Table 1). All other
substances (4, 5, 6, 7, 8, and 10) have been tested in vitro and in vivo based on cytotoxic screening and
physicochemical properties.
For structurally similar substances
and cytotoxic effect on various cell lines. Compound
5
,
6
,
7
, and 10, there is an evident correlation between ClogP
, substituted with a naphthalene moiety (with the
6
highest ClogP and hence MTC), appears to be the most promising radioprotector of all the compounds
evaluated in vitro. On the other hand, no significant effect was observed under in vivo conditions.
The effect is comparable to substances 5 and 7. According to the results for compounds 5 and 10, the
presence or absence of a hydroxyl group on the linker plays no significant role concerning the potential
radioprotective effect.
Substance
4 is structurally comparable to those prepared recently by our group [14]. In addition,
a halogen atom (Cl) was added to the structure, which could influence other binding interactions.
In vitro and in vivo tests indicate that the compound (despite its relatively higher toxicity) possesses a
radioprotective effect in vivo.
The structurally different compound
8 contains no aromatic moiety. This compound was
synthesized by linking two 1-(2-hydroxyethyl)piperazine moieties with an alkyl linker. The compound
is minimally non-toxic and excellently soluble in media. After in vivo evaluation, it appeared to be the
most effective compound in this group in terms of radioprotection. In addition, according to the safety
profile, it might be considered an appropriate compound for further studies.
Regarding the structure–activity relationship, we observed that acridine moiety increased the
cytotoxicity of the compounds
was achieved by substitution of the secondary amine at the 4-position of piperazine nitrogen, whereby
a tertiary amine was formed (compound ). The radioprotective properties are reduced in compounds
2 and 3 and therefore were omitted. Interestingly, lower cytotoxicity
3
with the methoxy group. Furthermore, the 1-(2-hydroxyethyl)piperazine moiety seems to be crucial,
as the compounds lacking this structure possess increased cytotoxicity and insolubility (compound
9
). The longest survival was found after the treatment with compound 8. Moreover, it is lacking
aromatic moieties and is entirely non-toxic and well soluble. Thus, it might be the most promising
radioprotective compound that is suitable for further development.
4. Experimental Section
4.1. Synthesis and Analysis
Analytical grade reagents were purchased from Sigma-Aldrich, Fluka and Merck (Darmstadt,
Germany). The solvents were purchased from Penta Chemicals (Prague, Czech Republic). Reactions
were monitored by thin layer chromatography (TLC) using a precoated silica gel 60 F254 TLC aluminium
sheet. Column chromatography was performed with silica gel 0.063–0.200 mm. Melting points were
determined on a microheating stage PHMK 05 (VEB Kombinat Nagema, Radebeul, Germany) and are
uncorrected. All compounds were fully characterized by NMR spectra and HRMS. NMR spectra were
recorded on a Varian VNMR S500 (operating at 500 MHz for 1H and 126 MHz for 13C; Varian Comp.,
Palo Alto, CA, USA). The chemical shifts (δ) are given in ppm and related to tetramethylsilane (TMS)
as an internal standard. Coupling constants (J) are reported in Hz. Splitting patterns are designated as
s, singlet; d, doublet; t, triplet; dd, doublet of doublets; and m, multiplet. Mass spectra were recorded
using a combination of liquid chromatography and mass spectrometry: high-resolution mass spectra
(HRMS) and sample purities were obtained by high-performance liquid chromatography (HPLC)

Molecules 2020, 25, 532
10 of 16
with UV and the mass spectrometry (MS) gradient method. The system used in this study was a
Dionex Ultimate 3000 UHPLC: RS Pump, RS Column Compartment, RS Autosampler, Diode Array
Detector, Chromeleon software (version 6.80 SR13 build 3967, Thermo Fisher Scientific, Germering,
Germany) with a Q Exactive Plus Orbitrap mass spectrometer with Thermo Xcalibur software (version
3.1.66.10, Thermo Fisher Scientific, Bremen, Germany). Detection was performed by mass spectrometry
in positive mode. Settings of the heated electrospray source were: spray voltage 3.5 kV, capillary
temperature 220 ◦C, sheath gas 55 arbitrary units, auxiliary gas 15 arbitrary units, spare gas 3 arbitrary
units, probe heater temperature 220 ◦C, max spray current 100
µA, S-lens RF Level 50. A C18 column
(Kinetex EVO C18, 3
A was ultrapure water of ASTM I type (resistance 18.2 M
×
150 mm, 2.6
µ
m, Phenomenex, Japan) was used in this study. Mobile phase
◦
Ω
.cm at 25 C) prepared by a Barnstead
Smart2Pure 3 UV/UF apparatus (Thermo Fisher Scientific, Bremen, Germany) with 0.1% (v/v) formic
acid (LC-MS grade, Sigma Aldrich, Steinheim, Germany); mobile phase B was acetonitrile (MS grade,
Honeywell-Sigma Aldrich, Steinheim, Germany) with 0.1% (v/v) of formic acid. The flow was constant
at 0.4 mL/min and began with 1 min of isocratic flow of 10% B, and then 3 min of gradient to 100% B,
followed by 1 min of constant 100% B. Then, the composition reverted to 10% B and was equilibrated
for 2.5 min. Samples were dissolved in methanol (LC-MS grade, Fluka-Sigma Aldrich, Steinheim,
Germany) at a concentration of 1 mg/mL, and sample injection was 1 µl. Purity was determined from
UV spectra measured at a wavelength of 254 nm. HRMS was determined by the total ion current
spectra from the mass spectrometer. Clog P was calculated with MarvinSketch software (version
14.9.8.0, ChemAxon, Budapest, Hungary).
7-Methoxy-N-(2-(piperazin-1-yl)ethyl)-1,2,3,4-tetrahydroacridin-9-amine trihydrochloride (
The synthetic procedure has already been published elsewhere [15]. The product was obtained as a
yellow solid (0,49 g, 78%), mp = 172–173 ◦C; 1H-NMR (methanol-d4):
7.72 (d, 1H, J = 9.2 Hz), 7.55
(d, 1H, J = 2.7 Hz), 7.40 (dd, 1H, J = 9.2, 2.7 Hz), 3.96 (s, 3H), 3.86 (m, 2H), 3.78 (m, 2H), 2.98 (m, 6H),
2).
δ
2.78 (m, 2H), 2.59 (m, 4H), 1.96 (m, 4H); 13C-NMR (126 MHz, cd3od):
δ 158.1, 154.9, 151.6, 132.8, 125.6,
123.7, 120.4, 115.7, 104.2, 58.7, 52.9, 45.9, 45.2, 41.4, 31.5, 25.5, 22.8, 22.1; HRMS: m/z 341.2330 [M + H]+
(calculated for [C20H29N4O]+ 341.2336).
7-Methoxy-N-{2-[4-(2-hydroxyethyl)piperazin-1-yl]ethyl}-1,2,3,4-tetrahydroacridin-9-amine
trihydrochloride (3). Hunig’s base (0.45 g, 3.6 mmol) and 2-bromoethanol (1.8 mmol) were added to
intermediate (0.2 g, 0.6 mmol) dissolved in anhydrous CH2Cl2. The reaction mixture was refluxed
2
under nitrogen overnight, followed by the evaporation of volatile solvents under reduced pressure.
The crude product was purified by column chromatography eluting with EtOAc/MeOH/NH3 (25%
aq.) (40:1:0.2
methanol and treated with hydrochloric acid to give
◦C; 1H-NMR (500 MHz, methanol-d4)
7.79 (d, 1H, J = 9.2 Hz), 7.76 (d, 1H, J = 2.7 Hz), 7.54 (dd, 1H, J
= 9.2, 2.7 Hz), 4.35 (t, J = 6.2 Hz, 2H), 4.04 (s, 3H), 3.98–3.93 (m, 2H), 3.47–3.41 (m, 2H), 3.09 (t, J = 6.0
Hz, 2H), 2.91 (t, J = 5.7 Hz, 2H), 1.98 (m, 12H), 1.43–1.36 (m, 2H); 13C-NMR (126 MHz, methanol-d4):
→
15:1:0.2) to obtain a yellow oily residue. The resulting free base was dissolved in
3
as a yellow solid (0.17 g, 75%), m.p. = 157–158
δ
δ
158.2, 154.7, 152.6, 133.1, 125.5, 123.7, 120.4, 115.7, 104.2, 61.5, 58.7, 52.9, 45.9, 45.2, 41.4, 35.2 31.5, 25.5,
22.6, 22.2; HRMS: m/z 385.2590 [M + H]+ (calculated for [C22H33N4O2]+ 385.2598).
1-(4-chloro-3-methylphenoxy)-3-(4-(2-hydroxyethyl)piperazin-1-yl)propan-2-ol dihydrochloride
(4). 1-(2-hydroxyethyl)piperazine 1 (0.2 g, 1.5 mmol) was dissolved in anhydrous acetonitrile and K2CO3
(0,4 g. 3.0 mmol) with appropriate intermediate 4b (0.2g, 1 mmol) was added. The reaction mixture was
stirred under nitrogen for 4 h. The mixture was filtered and the filtrate was concentrated under reduced
pressure. The residue was purified by column chromatography eluting with EtOAc/MeOH/NH3 (25%
aq) (6:2:0.2) to obtain a colorless oily residue. The resulting free base was dissolved in methanol and
treated with hydrochloric acid to give
(500 MHz, methanol-d4) 7.23 (d, J = 8.7 Hz, 1H), 6.90 (d, J = 3.0 Hz, 1H), 6.78 (dd, J = 8.8, 3.0 Hz,
1H), 4.20–4.12 (m, 1H), 4.02–3.92 (m, 2H), 3.82–3.79 (m, 2H), 3.14–3.00 (m, 6H), 2.96–2.89 (m, 6H),
2.82–2.70 (m, 2H), 2.33 (s, 3H); 13C-NMR (126 MHz, methanol-d4)
158.97, 138.08, 130.63, 126.93, 118.17,
4
as white solid. (0.27 g, 79%) m.p. = 189–190 ◦C; 1H-NMR
δ
δ

Molecules 2020, 25, 532
11 of 16
114.53, 71.82, 67.85, 61.03, 60.09, 57.91, 53.23, 52.65, 20.28; HRMS: m/z 329.1620 [M + H]+ (calculated for
[C16H26ClN2O3]+ 329.1621).
General procedure for the synthesis of 2-(4-(3-(arylamino)propyl)piperazin-1-yl)ethanol
trihydrochloride derivatives (5–7). 1-Bromo-3-chloropropane (1 mmol) was dissolved in MeCN
(anhydrous, 5 mL) and KI (potassium iodide, 0.1 mmol) was added. After 5 min of stirring, the
solution of appropriate arylamine (aniline 5a, naphtyl-1-amine 6a or p-anisidine 7a) (3 mmol) in MeCN
(anhydrous, 10 mL) was added. The final mixture was stirred under microwave conditions (110 ◦C,
15 min). The mixture was filtered and the filtrate was concentrated under reduced pressure. The
residue was purified by column chromatography eluting with petroleum ether/EtOAc (20:1) to obtain
intermediates 5b, 6b, or 7b as a yellow (6b violet) oily residue in a yield around 30%.
1-(2-hydroxyethyl)piperazine
(3.0 mmol) and the appropriate intermediate (5b
1
(1.5 mmol) was dissolved in anhydrous MeCN (20 mL) and K2CO3
6b or 7b) were added. The reaction mixture was
,
stirred under nitrogen for 4 h. The mixture was filtered, and the filtrate was concentrated under reduced
pressure. The residue was purified by flash chromatography eluting with petroleum ether/EtOAc
(10:1) to obtain yellow or white residues in 61–69% yields. The resulting free bases were dissolved in
methanol and treated with hydrochloric acid to give 5–7 as yellow or white solids.
2-(4-(3-(phenylamino)propyl)piperazin-1-yl)ethanol trihydrochloride (
m.p. = 185–186 ◦C; 1H-NMR (500 MHz, chloroform-d)
7.21–7.16 (m, 2H), 6.73–6.67 (m, 1H), 6.64–6.59
(m, 2H), 3.66–3.62 (m, 2H), 3.20 (t, J = 6.4 Hz, 2H), 2.61–2.57 (m, 2H), 2.69–2.36 (m, 8H), 2.51 (t, J = 6.7
Hz, 2H), 1.82 (p, J = 6.5 Hz, 2H); 13C-NMR (126 MHz, chloroform-d)
148.7, 129.2, 117.0, 112.6, 59.2,
57.7, 57.2, 53.2, 52.9, 43.4, 25.8; HRMS: m/z 264.2065 [M + H]+ (calculated for [C15H26N3O]+ 264.2070).
2-(4-(3-(naphthalen-1-ylamino)propyl)piperazin-1-yl)ethanol trihydrochloride ( ). White solid
(0.22 g, 69%) m.p. = 181–182 ◦C; 1H-NMR (500 MHz, chloroform-d)
8.00–7.96 (m, 1H), 7.79 (dd, J =
5). White solid (0.18 g, 68%)
δ
δ
6
δ
7.9, 1.5 Hz, 1H), 7.48–7.38 (m, 2H), 7.38–7.33 (m, 1H), 7.21 (d, J = 8.1 Hz, 1H), 6.56 (d, J = 7.5 Hz, 1H),
3.68 (t, J = 5.4 Hz, 2H), 3.41–3.35 (m, 2H), 2.83–2.50 (m, 12H), 2.00 (p, J = 6.1 Hz, 2H); 13C-NMR (126
MHz, chloroform-d)
53.6, 53.0, 44.8, 24.9; HRMS: m/z 314.2283 [M + H]+ (calculated for [C19H28N3O]+ 314.2227).
2-(4-(3-((4-methoxyphenyl)amino)propyl)piperazin-1-yl)ethanol trihydrochloride ( ). Yellow
solid (0.18 g, 61%) m.p. = 182–183 ◦C; 1H-NMR (500 MHz, chloroform-d)
6.89–6.85 (m, 2H), 6.85–6.81
(m, 2H), 3.85 (s, 3H), 3.71–3.67 (m, 2H), 3.15 (t, J = 6.4 Hz, 2H), 2.61–2.55 (m, 2H), 2.692.16 (m, 8H), 2.52
δ 144.5, 134.5, 128.8, 127.0, 125.8, 124.4, 123.7, 121.1, 116.8, 103.8, 59.7, 58.5, 57.9,
7
δ
(t, J = 6.7 Hz, 2H), 1.88 (p, J = 6.5 Hz, 2H); 13C-NMR (126 MHz, chloroform-d)
δ 149.1, 130.2, 118.0, 112.4,
59.3, 57.8, 57.2, 53.2, 52.9, 49.2, 43.5, 23.9; HRMS: m/z 294.2172 [M + H]+ (calculated for [C16H28N3O2]+
294.2176).
1.3-bis [4-(2-hydroxyethyl)piperazin-1-yl)propan-2-ol tetrahydrochloride (8). 1-(2-hydroxyethyl)
piperazine (1 mmol) dissolved in anhydrous MeOH (0.9 mL) was treated with 1,3-dibromo-2-propanol
1
(1 mmol) at room temperature and the mixture was stirred for 48 h. Silica gel was added to the
mixture and the solvent was evaporated. The residue was purified by column chromatography using
CH2Cl2/MeOH/NH3 (25% aq.) (5:1:0.2) as the eluent to obtain colorless oily residue. The resulting free
base was dissolved in methanol and treated with hydrochloric acid to give
27%) 1H-NMR (500MHz; DMSO-d6)
3.73 (m; 1H), 3.47 (t; 4H; J = 6.3 Hz), 2.41 (m; 16H), 2.36 (t; 4H; J
= 6.3 Hz), 2.30 (m; 2H), 2.20 (m; 2H); 13C-NMR (126 MHz; DMSO-d6)
65.1, 62.8, 60.2, 58.4, 53.3, 53.1;
HRMS: m/z 317.2543 [M + H]+ (calculated for [C15H33N4O3]+ 317.2542).
1,3-bis(phenylamino)propan-2-ol dihydrochloride ( ). Epibromohydrin (1 mmol) was slowly
8 as a colorless oil. (85 mg,
δ
δ
9
added with stirring to aniline (1 mmol) at 0 ◦C, and the final mixture was stirred for 1 h at 50 ◦C using
a microwave reactor. Then, the reaction mixture was diluted with MeOH, silica gel was added, and the
mixture was adsorbed onto silica gel using a rotary vacuum evaporator. The product was purified by
column chromatography using CH2Cl2/MeOH/NH3 (25% aq.) (300:1:0.1) as eluent to obtain a colorless
oily residue. The resulting free base was dissolved in methanol and treated with hydrochloric acid to
give
9 δ 7.05 (m; 4H), 6.59 (m; 4H), 6.51
as a colorless oil. (61 mg, 25%); 1H-NMR (500MHz; DMSO-d6)

Molecules 2020, 25, 532
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(m; 2H), 3.83 (m; 1H), 3.49 (m; 2H), 3.16 (m; 2H); 13C-NMR (126 MHz; DMSO-d6) δ 148.4, 128.8, 115.9,
112.2, 68.3, 47.2; HRMS: m/z 243.1488 [M + H]+ (calculated for [C15H19N2O]+ 243.1492).
1-(phenylamino]-3-[4-(2-hydroxyethyl)piperazin-1-yl)propan-2-ol
trihydrochloride
(10).
Epibromohydrin (1 mmol) was slowly added with stirring to aniline 5a (1 mmol) at 0 ◦C, and the
final mixture was stirred for 1 h at 50 ◦C using a microwave reactor. Then, the reaction mixture was
diluted with MeOH, silica gel was added, and the mixture was adsorbed on to silica gel using a rotary
vacuum evaporator. The product was purified by column chromatography using CH2Cl2/MeOH/NH3
(25% aq.) (300:1:0.1) as eluent to obtain intermediate 10b as a colorless oily residue in a yield of 45%.
The intermediate (1-Bromo-3-(phenylamino)propan-2-ol 10b (1 mmol) was dissolved in anhydrous
MeCN (4.5 mL) and K2CO3 (1.5 mmol) was added at 0 ◦C and stirred for 15 min. Then, a solution
of 1-(2-hydroxyethyl)piperazine 1 (2 mmol) in anhydrous MeCN (2.5 mL) was added dropwise at 0
◦C, and the final mixture was stirred overnight from 0 ◦C to room temperature. The solid residue
was removed by filtration under reduced pressure, and the filtrate was evaporated. The mixture was
purified by column chromatography using CH2Cl2/MeOH/NH3 (25% aq.) (5:1:0.2) as eluent to yield
251 mg of product as a colorless oil. The resulting free base was dissolved in methanol and treated
with hydrochloric acid to give 10 as a white solid. (0.25 g, 90%) m.p. = 154–155 ◦C; 1H-NMR (500MHz;
DMSO-d6) δ 7.05 (m; 2H), 6.58 (m; 2H), 6.51 (m; 1H), 5.44 (m; 1H), 3.48 (m; 2H), 3.11 (m; 1H), 2.92 (m;
1H), 2.40 (m; 8H), 2.35 (m; 2H), 2.29 (m; 2H); 13C-NMR (126 MHz; DMSO-d6)
δ 149.9, 128.8, 115.5, 112.0,
66.2, 62.6, 60.3, 58.5, 53.5, 53.3, 48.6; HRMS: m/z 280.2016 [M + H]+ (calculated for [C15H26N3O2]+
280.2008).
4.2. Molecular Docking
The structure of receptor was gained from the Protein Data Bank, PDB ID 4LXD (Bcl_2-Navitoclax
Analog complex) [29]. The structure has a resolution of 1.9 Å and it does not have any Ramachandran’s
outliers. Therefore, it was found to be suitable for molecular docking. The structure was prepared by
the DockPrep function of UCSF Chimera (version 1.4) and converted to pdbqt-files by AutodockTools
(v. 1.5.6) [30
,
31]. Three-dimensional structures of ligands were minimized by Avogadro (v. 1.1.0) and
33]. The docking calculations were done
converted to a pdbqt-file format by Open Babel (v. 2.3.1) [32
,
by Autodock Vina (v. 1.1.2) with the exhaustiveness of 8 [34]. Calculation was repeated 10 times
for each ligand, and the best-scored result was selected for manual inspection. The visualization of
enzyme–ligand interactions was prepared using Pymol (v. 1.7.4.5) [The PyMOL Molecular Graphics
System, Version 1.7.4.5, Schrödinger, LLC, Mannheim, Germany].
4.3. Cell Culture and Treatment with Novel Compounds
The cell lines Jurkat, MOLT-4, A2780, A549, HT-29, PANC-1, HeLa, MCF-7, SAOS-2, and MRC-5
(all purchased from Sigma Aldrich, Czech Republic) were included in this study. All cell lines were
maintained according to the provider’s guidelines, and plated and treated in 96-well plates (500 to 30
×
103 cells per well). The tested compounds
2, 3, 4, 5, 6, 7, 8, 9, and 10 were dissolved in 0.1% DMSO
and kept in 10 mM stock solutions. Before use, the stock solution was diluted in culture media at ratios
of 1:100 and 1:1000. The studied cell lines were treated with the compounds 2, 3, 4, 5, 6, 7, 8, 9, and
10 at concentrations of 10 and 100 µM for 48 h. Control cells were tested in culture media with and
without DMSO (0.1%). The cells were also treated with 1 µM doxorubicin as a cytotoxicity control.
4.4. Cytotoxicity of Novel Compounds In Vitro
The WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium
salt) test (Roche, Mannheim, Germany) was used according to the manufacturer’s protocol to determine
cell viability in all 10 cell lines 48 h after treatment with the tested compounds. The cells were analyzed in
a Tecan Infinite M200 spectrometer (Tecan Group, Männedorf, Switzerland) at 440 nm.
IC50 and MTC were determined through MTT assays. The MTT assay was performed according
to [35]. In brief, the A549 cell line was plated in 96-well plates (100
µL, 9
×
103 cells per well)

Molecules 2020, 25, 532
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and incubated overnight. The tested compounds were prepared in phosphate buffer saline (PBS;
Sigma–Aldrich) and serially diluted in DMEM (Dulbecco’s Modified Eagle Medium). An untreated
control was included as a viability standard in each plate. After 24 h of treatment, the viability of A549
cells was measured at 570 nm. All experiments were performed in triplicate. The GraphPad Prism
statistics software v5.04 (GraphPad Software Inc., San Diego, CA, USA) was used to calculate the IC50
values for parameter nonlinear regression. Values are expressed as mean ± SEM.
4.5. Evaluation of Radioprotection In Vitro
Fresh 50 mM stock solutions of the compounds were prepared in DMSO (Sigma-Aldrich, St.
Louis, MO, USA) before use in the study. For the radioprotection experiments, MOLT-4 cells were
M solutions of the novel compounds and irradiated after 60 min with a 60Co
treated with 100 µ γ-ray
source (Chisotron Chirana, Prague, Czech Republic). The non-irradiated and irradiated control cells
were treated with DMSO vehicle only (0.1%) and handled in parallel with the experimental cells.
Non-irradiated control cells were handled in the same manner. Cell viability was determined by
flow cytometry using an Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis kit (Life Technologies,
Grand Island, NY, USA) according to the manufacturer’s instructions. The Alexa Fluor® 488 Annexin
V/Dead Cell Apoptosis kit employs the property of Alexa Fluor® 488 conjugated to Annexin V to bind
to phosphatidylserine in the presence of Ca2+, and the property of propidium iodide (PI) to enter
cells with damaged cell membranes and bind to DNA. Measurement was performed using a CyAn
(Beckman Coulter, Miami, FL, USA) flow cytometer. Listmode data was analyzed with the Kaluza
Analysis v1.3 software (Beckman Coulter, Miami, FL, USA).
4.6. Safe Use Evaluation In Vivo
Both male and female BALB/c mice (Velaz s r.o., Prague, Czech Republic) with an average weight
of 21
housed in environmentally controlled breeding units (Faculty of Military Health Sciences vivarium;
temperature 21
1 ◦C, 12/12 h light/dark cycle) with access to food (Cerea corp., Pardubice, Czech
±
0.8 g and 21
±
0.8 g, respectively, were selected for the safe use evaluation in vivo. The mice were
±
Republic) and water ad libitum. The acclimatization period of the mice was at least 10 days before each
experiment. All experimental procedures and protocols were reviewed and approved by the Ethics
Committee of the Faculty of Military Health Sciences in Hradec Kralove, University of Defence in
Brno. Compounds 2, 3, 4, 5, 6, 7, 8, 9, and 10 were dissolved in physiological saline solution (B. Braun
Melsungen AG, Melsungen, Germany) and injected intraperitoneally (i.p.) into the corresponding
mouse groups, which were composed of two males and two females. Several doses were administered
to determine the maximum tolerated dose (MTD), which was assessed by close observation for signs
of toxicity within the first two hours after administration of the compounds, and then regularly over
the following 48 h. The observed symptoms were registered according to the laboratory Animal
Science Association UK guidelines (for a detailed description, see [36]. After 48 h, the surviving
animals were euthanized with CO2 and evaluated by basic macroscopic necropsy. Venous blood
was collected into heparinized tubes (Scanlab Systems, Prague, Czech Republic) and the plasma was
separated in a U-320R centrifuge (Boeco, Hamburg, Germany) to further evaluate the toxic potential of
the tested compounds. The concentrations of glucose, urea, and creatinine and the levels of alanine
aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and amylase
were evaluated in a qualified biochemical laboratory of the University Hospital Hradec Kralove
(Hradec Kralove, Czech Republic). Additionally, intestine, liver, and kidney samples were collected for
histopathologic examination. The samples were fixed in 10% neutral buffered formalin, dehydrated
through increasing concentrations of ethanol and xylene, and transferred into liquid paraffin (all from
Bamed, Ceske Budejovice, Czech Republic) using a Leica TP1020 tissue processor (Leica, Wetzlar,
Germany). The paraffin-embedded samples were cut into 5 µm thick sections (Microtome model
SM2000 R, Leica, Wetzlar, Germany), re-hydrated (xylene and decreasing ethanol concentrations),
stained with hematoxylin and eosin (both from Merck, Darmstadt, Germany), dehydrated once again,

Molecules 2020, 25, 532
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and mounted in DPX (Dibutylphthalate Polystyrene Xylene) media (Merck). The histopathological
analysis was performed on a BX-51 microscope (Olympus, Tokyo, Japan).
4.7. Animals and Gamma Radiation
Adult female BALB/c mice (21
±
0.8 g) were used for all experiments (Velaz a.s., Prague, Czech
Rep.). All the performed methods were in accordance with NIRS Guidelines for the Care and Use of
Laboratory Animals and approved by the Committee for animal rights of the Ministry of Defence of
ˇ
the Czech Republic no. CJ MO 255172/2019-684800. The mice, arranged in groups of 10 animals each,
received a dose of 7.15 Gy from a 60Co gamma source (Chisotron, Chirana, Czech Republic) at a dose
rate of 1.04 Gy/min. Non-irradiated and non-treated control groups were included. Their survival and
health status were checked on a daily basis for 30 days.
4.8. Preparation of the Novel Compound Solutions and Evaluation of Radioprotection In Vivo
Compounds
4 (50 mg/kg), 5 (100 mg/kg), 6 (50 mg/kg), 7 (100 mg/kg), 8 (1000 mg/kg), and 10 (325
mg/kg) were selected and administered i.p. 5 min before irradiation. The doses were prepared from
100 mg/mL stock before each experiment.
4.9. Statistical Analysis
The charts were made with the GraphPad Prism 6 biostatistics software (GraphPad Software, La
Jolla, CA, USA). The evaluated groups were analyzed by Student’s t-test or one-way ANOVA, followed
by a post hoc Tukey test (p < 0.05).
5. Conclusions
In summary, all the compounds were tested in vitro, revealing that compounds
M) were highly cytotoxic, while compound was not cytotoxic, but was insufficiently soluble
for necessary concentrations, making it inappropriate for further tests in vivo. After determining the
MTD in vivo of the remaining compounds ( , and 10), we used only 50% of the calculated
MTD and assessed the radioprotective effect through the long-term survival of the irradiated mice.
Our results show that compounds and 10 were able to prolong survival but failed to prevent
fatal radiation-induced injury in the long term. On the other hand, pre-treatment with compound led
to an increased survival rate in the irradiated animals. Although this increase was not statistically
significant, we propose compound as a valuable candidate for further research due to its low toxicity
2 (10 µM) and 3
(100
µ
9
4, 5, 6, 7, 8
4
8
8
and appropriate yield during synthesis.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/25/3/532/s1,
Schemes S1–S6: Synthetic pathway of novel compounds, Table S1: Symptoms induced by MTD of novel
compounds in male mice, Table S2: Symptoms induced by MTD of novel compounds in female mice, Table S3:
Biochemical parameters evaluated 48 h after administering MTD of novel compounds in male (m) and female (f)
BALB/c mice.
Author Contributions: Conceptualization, Z.S., M.R. and A.T.; Formal analysis, A.F., J.M., R.H., M.J., M.M., T.K.,
M.P., N.Z., Z.S. and M.R.; Funding acquisition, J.M., J.P., Z.S., M.R. and A.T.; Investigation, J.P., J.C., L.M., D.K. and
Z.S.; Methodology, L.P. and Z.S.; Supervision, A.T.; Writing—original draft, A.F., J.M., J.P. and A.T.; Writing—review
and editing, M.R. and A.T. All authors have read and agree to the published version of the manuscript.
Funding: This work was supported by grant project No. 17-13541S of the Czech Science Foundation.
Acknowledgments: The authors thank Dr. Ian McColl for proof-reading the manuscript.
Conflicts of Interest: The authors declare no conflicts of interest.
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Sample Availability: Samples of the compounds 2, 3, 4, 5, 6, 7, 8, 9 and 10 are available from the authors.
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