A novel class of small molecule inhibitors with radioprotective properties

Article abstract of DOI:10.1016/j.ejmech.2019.111606

The goal of this study was to develop novel radioprotective agents targeting the intrinsic apoptotic pathway and thus decreasing the radiation-induced damage. For that purpose, we designed, synthesized and analyzed ten new compounds based on the 1-(4-(2-hydroxyethyl)piperazin-1-yl)-3-phenoxypropan-2-ol leading structure. The cytotoxicity of the newly synthesized substances was tested in vitro on cell lines derived from different progenitor cells by WST-1 proliferation assay. MTT test was utilized to assess half-maximal inhibitory concentrations and maximum tolerated concentrations of novel compounds in A-549 cells. Screening for radioprotective properties was performed using flow-cytometry in MOLT-4 cells exposed to ⁶⁰Co ionizing gamma radiation. Selected candidates underwent in vivo testing in C57Bl/6 J mice having a positive impact on their immunological status. In summary, we report here promising compounds with radioprotective effect in vivo.

Full text of DOI:10.1016/j.ejmech.2019.111606

European Journal of Medicinal Chemistry xxx (xxxx) xxx
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
A novel class of small molecule inhibitors with radioprotective
properties
Jan Marek a, Ales Tichy b, Radim Havelek d, Martina Seifrtova d, Alzbeta Filipova b,
Lenka Andrejsova b, Tomas Kucera c, Lukas Prchal a, Lubica Muckova c, Martina Rezacova d,
,
*
Zuzana Sinkorova b, Jaroslav Pejchal c
a Biomedical Research Center, University Hospital Hradec Kralove, Sokolska 581, 500 05, Hradec Kralove, Czech Republic
b Department of Radiobiology and Military Pharmacy, Faculty of Military Health Sciences, University of Defence in Brno, Trebesska 1575, 500 01, Hradec
Kralove, Czech Republic
c Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences, University of Defence in Brno, Trebesska 1575, 500 01, Hradec
Kralove, Czech Republic
d Department of Medical Biochemistry, Faculty of Medicine in Hradec Kralove, Charles University, Simkova 870, 500 03, Hradec Kralove, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
The goal of this study was to develop novel radioprotective agents targeting the intrinsic apoptotic
pathway and thus decreasing the radiation-induced damage. For that purpose, we designed, synthesized
and analyzed ten new compounds based on the 1-(4-(2-hydroxyethyl)piperazin-1-yl)-3-
phenoxypropan-2-ol leading structure. The cytotoxicity of the newly synthesized substances was
tested in vitro on cell lines derived from different progenitor cells by WST-1 proliferation assay. MTT test
was utilized to assess half-maximal inhibitory concentrations and maximum tolerated concentrations of
novel compounds in A-549 cells. Screening for radioprotective properties was performed using flow-
cytometry in MOLT-4 cells exposed to 60Co ionizing gamma radiation. Selected candidates underwent
in vivo testing in C57Bl/6 J mice having a positive impact on their immunological status. In summary, we
report here promising compounds with radioprotective effect in vivo.
Received 27 June 2019
Received in revised form
1 August 2019
Accepted 7 August 2019
Available online xxx
Keywords:
1-(2-hydroxyethyl)piperazine derivative
Synthesis
Radioprotection
Ionizing radiation
In vitro
© 2019 Elsevier Masson SAS. All rights reserved.
Mice
1. Introduction
mechanisms (enhancement of DNA repair, free-radical scavenging,
cell synchronization, modulation of growth factors and cytokines,
The increasing risk of acute large-scale radiological/nuclear ex-
posures of the population underlines the need to improve efficient
radioprotective tools that could be used for radiation protection
purposes in public situations such as a nuclear accident, an act of
radiologic terrorism, or military conflict [1]. In addition, radio-
therapy is still a key modality for treatment of cancer [2]. Unfor-
tunately, it is associated with a number of negative side effects such
as the death of bone marrow haematopoietic stem cells or enter-
ocytes, due to the DNA damage, induced signaling pathways, and
inflammatory responses and associated generation of reactive
oxidative species (ROS), eventually leading to cell death or senes-
cence of cells in normal healthy tissue [3,4].
modulation of redox sensitive genes, inhibition of apoptosis,
interaction and chelation of the radionuclides, or via gene therapy
or stem cell therapy [5]. When searching for an appropriate target,
our effort was based on the fact that ionizing radiation (IR) is able to
trigger programmed cell death in mammalian cells. While this ef-
fect is desirable during radiotherapy of cancer cells, it leads to
radiotoxicity in normal healthy tissues.
Two major signaling pathways of apoptosis can be recognized in
normal and tumor cells. Extrinsic pathway occurs after ligation of
cytokines or factors, such as Tumor necrosis factor alpha (TNF-
alpha) or Fas ligand (FasL), to their specific cytoplasmic membrane
receptors. In contrast, the intrinsic one is activated by a wide range
of stimuli, including growth factor deprivation, oxidants, DNA-
damaging agents, or microtubule targeting drugs [6]. We previ-
ously identified p53-upregulated mediator of apoptosis (PUMA) as
the key molecule of IR-induced cell-death signaling and thus a
A radioprotective effect can be achieved through various
* Corresponding author.
E-mail address: jaroslav.pejchal@unob.cz (J. Pejchal).
https://doi.org/10.1016/j.ejmech.2019.111606
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.
Please cite this article as: J. Marek et al., A novel class of small molecule inhibitors with radioprotective properties, European Journal of
Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2019.111606

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J. Marek et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
Abbreviations
DMEM
DMSO
DOX
GP
Dulbecco's modified Eagle's medium
dimethyl sulfoxide
doxorubicin
growth percent
HPLC
HRMS
i.p.
high performance liquid chromatography
high resolution mass spectrometry
intraperitoneally
IC50
IR
half maximal inhibitory concentration
ionizing radiation
m. p.
MS
melting point
mass spectrometry
MTC
NMR
PBS
maximum tolerated concentration
nuclear magnetic resonance
phosphate buffer saline
Fig. 1. Docking pose of the ligand 3e in the cavity of Bcl-2 protein.
PI
SEM
TLC
propidium iodide
standard error of the mean
thin layer chromatography
group with Asp108 and Arg143, of the methoxy-group with Arg143,
of the ether oxygen with Arg143, of the distal hydroxymethyl group
with Arg126, and of the protonated nitrogen with Met112. The
docking pose was compared with the pose of a bavitoclax analog
co-crystallized with Bcl-2. Both of them have an aromatic ring
placed in the lipophilic cavity bordered by Phe101 and Phe109.
Surprisingly, the piperazine motif of 3e is located differently from
the bavitoclax analog. Therefore, the protonated nitrogen of
piperazine can interact with Glu133.
promising target in radioprotection, but also in the therapy of
neurodegenerative and cardiovascular diseases [7]. PUMA is often
associated with IR-induced apoptosis and it can be upregulated in
both p53-dependent and p53-independent manner [8e12].
In this paper, we present the basic characteristics of novel
compounds - derivatives of 1-(4-(2-hydroxyethyl)piperazin-1-yl)-
3-phenoxypropan-2-ol that was screened and selected as one of
the leading structures by Mustata et al., in 2011 [13]. Cytotoxicity
screening was performed on a panel of human cell lines. In vitro
experiments were carried out in order to assess basic parameters
such as half-maximal inhibitory concentration (IC50), maximum
tolerated concentration (MTC), and inhibition of IR-induced
apoptosis in selected cell lines. Finally, the most promising com-
pounds were subjected to in vivo tests on whole-body irradiated
mice in order to evaluate their radioprotective potential.
The docking study indicates that our compounds might interfere
with anti-apoptotic and pro-apoptotic protein interaction with
possible implications for cell-death signaling.
2.3. Synthesis and analysis
The general synthetic procedure for the novel 1-(4-(2-
hydroxyethyl)piperazin-1-yl)-3-phenoxypropan-2-ol like com-
pounds is displayed in Scheme 1. The first step is reaction of the
appropriate aryl alcohol (1a-j) with an excess of epibromohydrin
under reflux for 2 h in the presence of piperazine (i), according to
the process published by Kreighbaum et al. [20]. These solvent-free
reactions led to the preparation of intermediates 2a-j. Purification
by column chromatography gave yields of around 80%. The purity of
intermediates 2a-j was relatively low (around 60e70%), but suffi-
cient for the second step of the reaction. The next step involved the
alkylation of 1-(2-hydroxyethyl)piperazine by the differently-
substituted intermediates 2a-j, resulting in compounds 3a-j. The
reactions were refluxed with potassium carbonate in acetonitrile
for 4 h (ii). The crude products were purified by column chroma-
tography, with yields ranging from 57 to 81% (Table 1) and sufficient
purity (ꢀ95%). Finally, the compounds (3a-j) were converted into
their more soluble hydrogen chloride salts by treatment with hy-
drochloric acid in methanol. All of the 1-(4-(2-hydroxyethyl)
piperazin-1-yl)-3-phenoxypropan-2-ol derivatives were addition-
ally characterized by the 1H and 13C nuclear magnetic resonance
(NMR) spectra and high resolution mass spectrometry (HRMS). All
analytical measurements confirmed the structure and high purity
of the prepared compounds. The detailed analyses of all com-
pounds are provided in the Supporting information.
2. Results and discussion
2.1. Compounds
Several published structures are already mentioned in the
literature. The patent ES411826 describes the preparation of com-
pounds 3b, 3c, 3f, 3j, and 3i without specification of the preparation
process or usage [14]. Some compounds (3a, 3b, 3c, 3 g, and 3 h) are
mentioned in ZINC database, again without the preparation process
and biological activities [15e18]. A similar strategy of preparation
process of aryloxy derivatives (preparation of 2a-j) was found in the
literature [19e22]. These compounds were published as antiviral
agents, adrenoceptor antagonists or platelet aggregation inhibitors.
Compounds 3b and 3c had already been published by Mustata et al.,
in 2011 as PUMA inhibitors [13]. Nevertheless, the preparation
process and in vivo activity are not mentioned here. The 3e
analogue was not found elsewhere.
2.2. In silico study
2.4. In vitro cytotoxicity
The selected compounds underwent a docking study with anti-
apoptotic Bcl-2 protein involving its interaction site with pro-
apoptotic PUMA, which negatively regulates Bcl-2 protein and
triggers apoptosis [23].
An example is illustrated in Fig. 1, depicting the docking pose of
the ligand 3e. It shows hydrogen bond interactions of the hydroxyl
The cytotoxicity of the 10 new inhibitors at concentrations of 10
and 100
lines, including 9 human cancerous cell lines and 1 non-cancerous
cell line. Doxorubicin at a concentration of 1 M was used as a
positive control. The cell lines were exposed to these compounds
mM was determined on a screening panel of 10 human cell
m
Please cite this article as: J. Marek et al., A novel class of small molecule inhibitors with radioprotective properties, European Journal of
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J. Marek et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
3
Scheme 1. Synthetic pathway of 1-(4-(2-hydroxyethyl)piperazin-1-yl)-3-phenoxypropan-2-ol like derivatives (3a-j). Reagents and conditions: (i) Epibromohydrin, 135 ꢁC, 2 h, 1
drop of piperazine; (ii) 1-(2-hydroxyethyl)piperazine, acetonitrile, reflux, 4 h.
Table 1
2.5. Half maximal inhibitory concentration (IC50) and maximum
tolerated concentration (MTC)
Structures, yields and C log P of prepared compounds.
Compound
R
Yield (%)
C log P
In addition to WST-1 proliferation assay, MTT test was carried
out to provide information on toxicological indexes of the new
compounds, including IC50 and MTC. MTT assay is a sensitive
colorimetric method assessing metabolic activity of cells. The
method is suitable for adherent cell lines. Therefore, A-549 cell line
was randomly selected for the test.
In A549 cells, the lowest toxicity was detected in compound 3i
(IC50 ¼ 9.27 1.18 mM) using MTT assay. The toxicity of potential
radioprotective compounds increased from 3i towards 3g, 3d, 3e,
3j, 3c, 3h, 3a, 3f, and 3b with IC50 values being 1.2-, 1.6-, 2.1-, 2.7-,
3.8-, 8.3-, 22.1-, 25.8-, and 29.9 lower than in 3i treated cells
(Table 3). In relation to the structure, it is evident that the tolera-
bility of cells is lower for compounds with larger substituents (3a,
3b) or additional alkyl substituents on the phenyl ring (3f). The data
indicates that increased lipophilicity is associated with cytotoxicity
[24]. The correlation of ClogP value with toxicity status of the
prepared compounds is obvious.
3a
3b
3c
3d
3e
3f
3g
3h
3i
1-naphthyl
2-naphthyl
4-methyl-1-phenyl
Phenyl
4-nitro-2-methoxy-1-phenyl
2-isopropyl-5-methyl-1-phenyl
2,6-dimethoxy-1-phenyl
4-allyl-2-methoxy-1-phenyl
2-methoxy-1-phenyl
2-methyl-1-phenyl
65
57
74
77
81
72
59
64
71
75
1,35
1,35
0,874
0,361
0,143
2119
0,046
1302
0,203
0,874
3j
for 48 h. Cell proliferation was then determined by WST-1 prolif-
eration assay and related to the proliferation of untreated control
cells (100%). The results have shown that the inhibitors had no
cytotoxic effect at 10
cells varying from 87% to 112%. At a concentration of 100
compound 3a decreased the viability of HT-29 and MCF-7 cells to
53% and 64%, respectively. Similarly, compounds 3f and 3h showed
a mild cytotoxic effect in HT-29 and MOLT-4 with the percentage of
viable cells being 64% and 65%, respectively. The other compounds
mM in all 10 cell lines, the percentage of viable
m
M,
2.6. Protective effect on irradiated cell lines
showed no signs of cytotoxicity at 100 mM. The data of all screened
The radioprotective effect of the ten compounds was evaluated
by flow cytometric detection of Annexin V/propidium iodide pos-
itive cells. To avoid interference of the detachment procedure with
the binding of Annexin V to the cytoplasmic membrane, non-
adherent MOLT-4 lymphoblastic leukemia cells were selected for
the testing.
Novel potential radioprotectives improved the viability of
MOLT-4 cells in vitro. As per Fig. 3A, the Annexin V/propidium io-
dide (PI) staining clearly demonstrates the decline in the percent-
age of viable cells 24 h after gamma irradiation by a dose of 1 Gy
compounds for each cell line are presented in Tables 2a and 2b.
To express the overall inhibitory activity of each compound, the
growth percent value (GP) was calculated. GP represents the mean
viability of 10 cell lines treated with the same concentration. The
compounds at 10
mM concentrations showed no cytotoxic effect. At
this concentration, GP values were 96%e103%. At the higher 100
mM
concentration, the GP values ranged from 86% to 102% (Fig. 2). The
basic screening at a given concentration showed no significant
cytotoxic effect in any tested compound.
Table 2a
Cytotoxic effect of inhibitors on 10 human cell lines. Values represent cell viability after treatment with the compounds at doses of 10
mM. The results are expressed as
percentage of viability of untreated control cells (100%). DOX e doxorubicin (1
0e25%, 26e50% and 50e75% are highlighted with different colors.
mM). Each value is a mean of three independent experiments SD. Values from the intervals
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J. Marek et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
Table 2b
Cytotoxic effect of inhibitors on 10 human cell lines. Values represent cell viability after treatment with the compounds at doses of 100 mM. The results are expressed as
percentage of viability of untreated control cells (100%). DOX e doxorubicin (1
0e25%, 26e50% and 50e75% are highlighted with different colors.
m
M). Each value is a mean of three independent experiments SD. Values from the intervals
stand-alone (40%) as compared to the untreated cells (92%).
Conversely, pretreatment with compounds 3a-3j at 100 M 1 h
possibly other anti-apoptotic Bcl-2 members and inhibits its anti-
m
apoptotic action. If confirmed, such substance could be of poten-
tial value as an anticancer agent [28]. Other tested substances,
including 3e, 3 g, 3 h, and 3i, increased survival from 0% to 40%, 20%,
30%, and 30%, respectively; however, this increase was not statis-
tically significant.
before gamma irradiation exerted protective effects against
ionizing radiation-induced death of MOLT-4 cells. In the 24 h in-
terval after the exposure to IR, the amount of viable cells increased
up to 65%, 59%, 52%, and 62% after being pretreated with inhibitors
3b, 3e, 3h, and 3j, respectively, when compared to only IR-treated
cells (Fig. 3B).
2.7.3. Impact of the compounds on the immunological status of
irradiated mice
2.7. Radioprotective effect on mice
Fig. 5 shows that the i.p. application of compound 3j signifi-
cantly decreased the absolute number of lymphocytes in non-
irradiated animals (0 Gy) 7 and 12 days after the treatment,
whereas substance 3h showed a similar effect in the 12 day inter-
val. Fig. 6 demonstrates that the immunotoxicity of 3j was directed
selectively towards B-lymphocytes.
In contrast, a significantly increased trend in the absolute
number of lymphocytes in irradiated mice was observed in groups
treated with the compounds 3e (day 12, day 30) and 3i (day 30;
Fig. 5). Substance 3e showed the most pronounced radioprotective
effect, maintaining a significant B cell population throughout the
experiment and supporting T and NK cell populations in the later
time intervals (Fig. 6).
Compounds for this stage of study were selected based on
toxicological evaluation and physico-chemical properties. Com-
pounds 3b, 3e, 3h, and 3j significantly improved the viability of
irradiated MOLT-4 cells in vitro; however, compound 3b induced
undesired effects when applied to healthy mice and possessed the
lowest solubility. Thus, it was excluded from further testing. Com-
pounds 3g and 3i showed the lowest toxicity/highest MTC when
applied to A549 cells and were therefore included in the in vivo
radioprotective study.
2.7.1. Safety of selected compounds in vivo
The experimental mice were injected (i.p.) with 100 mg/kg of
tested substances 3e, 3g, 3h, 3i, and 3j.
The selected compound administered at this dose appeared to
be safe, with no deaths or adverse effects recorded within the 30-
day interval. Weight gain was similar to the control group (Table 4).
3. Conclusion
Despite the current large-scale screening of thousands of syn-
thetic or natural compounds with the goal of finding an appropriate
radioprotective agent, only a few of them find application in clinical
practice. As an example, antioxidants scavenging ROS, such as
amifostine, alpha-tocopherol (vitamin E), curcumin, or metformin,
have shown very promising results [29,30]. Other agents, including
angiotensin converting enzyme inhibitors, statins or immunosup-
pressive agents, have been demonstrated to target inflammatory
pathways [29]. However, there is a concern that such therapies,
given concomitantly with radiotherapy, could reduce tumor control
rates.
With a deeper molecular understanding of pathways involved in
response to ionizing radiation, new targeted therapies could be
developed with the advantage of greater specificity towards diverse
molecular pathways activated in various distinct cell types (e.g.
tumor vs. nonmalignant). Multitude of pathways is involved in cell
death following irradiation e apoptosis induction by intrinsic
2.7.2. Treatment with potential radioprotectives prolongs mice
survival after gamma irradiation
To assess radioprotective properties of selected substances, the
animals were injected (i.p.) with 100 mg/kg of 3e, 3g, 3h, 3i, and 3j
5 min before receiving an IR dose of 8.5 Gy. This dose can be
considered as supra-lethal for the C57BL/6 J mouse strain with LD50
ranging from 7.76 to 8.05 Gy [25e27]. The radioprotection effect of
these compounds was assessed for 30 days after irradiation.
Survival curves are presented in Fig. 4, median survival values
are summarized in Table 5, and weight gain of surviving animals is
shown in Table 6. The substances did not affect animal survival with
one exception. Substance 3j significantly decreased mouse survival
from 12 to 7 days. The mechanism underlying this effect remains
unknown. We may only hypothesize that 3j interacts with Bcl-2 or
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Fig. 2. Viability of tested cell lines Growth percent value was calculated for each compound at 10
(expressed as percent of control) of all the 10 cell lines. Control: untreated cells, DOX e doxorubicin (1
m
M (A) and 100
M).
mM (B) concentration. The values represent the average viability
m
pathway, but also chronic inflammation and oxidative stress
[31,32], as well as DNA repair suppression. All of these could be a
target of possible radioprotective small molecular inhibitors. Our
efforts were driven by the hypothesis that small molecule in-
hibitors targeting the apoptosis pathway (particularly those inter-
fering with Bcl-2 proteins interaction) should exhibit
radioprotective properties. Supported by initial docking study data,
we designed a group of potentially radioprotective compounds.
These might be applied as inhibitors of pro-apoptotic and anti-
apoptotic protein-protein interactions with obvious impact on
radiation-induced cell death signaling. This might have great im-
plications not only for radioprotection but also for other areas such
as the therapy of neurodegenerative and cardiovascular diseases
that rely on the apoptotic pathway [7].
For this study, our team built a unique pipeline consisting of in
silico study, in-house synthesis, physicochemical analysis, in vitro
cytotoxicity and in vivo toxicity testing followed by radioprotective
effect evaluation. As an outcome of this approach we report the
synthesis and biological characterization of a novel series of 1-(4-
(2-hydroxyethyl)piperazin-1-yl)-3-phenoxypropan-2-ol
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Table 3
Table 4
Toxicity of new inhibitors in A549 cells measured using MTT assay.
Average weight of mice before (day 0) and after (day 30) the treatment with selected
compounds (100 mg/kg) SD.
Compounds
IC50 SEM (mM)
MTC (mM)
Control (g) 3e (g)
26.3 2.4
3 g (g)
3 h (g)
3i (g)
3j (g)
3a
3b
3c
3d
3e
3f
3g
3h
3i
0.42 0.04
0.31 0.02
2.43 0.16
5.78 0.30
4.45 0.05
0.36 0.01
7.81 0.48
1.12 0.10
9.27 0.68
3.40 0.05
0.07
0.02
0.29
0.59
0.78
0.16
1.56
0.20
1.56
0.39
Day 0
27.8 2.3 27.0 0.9 25.7 2.5 27.9 4.4 26.2 2.0
28.5 2.9 27.6 2.1 27.0 2.3 28.5 4.6 27.2 2.7
Day 30 27.0 3.2
103 12
%
103 10 102
8
105
9
102 16 104 10
%: procentage change since day 0 (100%).
3j
MTC: maximum tolerated concentration, i.e. the highest concentration showing no
significant difference in comparison with non-treated control (t-test).
Fig. 4. Survival curves of irradiated mice and irradiated mice injected (i.p.) with
selected compounds 3e, 3g, 3h, 3i, and 3j for 30 days.
Table 5
Median survival values of irradiated mice treated (i.p.) with selected compounds 3e,
3g, 3h, 3i, and 3j for 30 days.
Group
Median survival in days (95% confidence interval)
saline þ 8.5 Gy
3e þ 8.5 Gy
3 g þ 8.5 Gy
3 h þ 8.5 Gy
3i þ 8.5 Gy
12 (9.9e14.1)
11 (6.9e15.1)
12 (10.5e13.6)
12 (5.8e18.2)
13 (8.4e17.6)
7 (6.4e7.6)a
3j þ 8.5 Gy
a
p ꢂ 0.05 when compared with saline-treated mice.
Table 6
Average weight of mice before (day 0) and after (day 30) the treatment with selected
compounds (100 mg/kg) and combined with gamma radiation (8.5 Gy) SD.
Control (g) 3e (g)
3 g (g)
3 h (g)
3i (g)
3j (g)
Day 0
Day 30
%
25.4 2.6
26.1 1.9 27.3 1.6 25.1 2.5 26.8 2.1 27.3 1.4
y
26.5 2.4 27.7 0.9 27.3 1.2 27.6 1.9
101 109 103 102
ǂ
ǂ
y
3
5
7
9
%: procentual change since day 0 (100%).
y Mice lost (death) before the analysis could be performed.
ǂ Analysis was not performed e only one mouse left.
well-soluble substances. Beyond that, the presented data show that
our compounds had no cytotoxic effect on 10 human cell lines
Fig. 3. The viability of MOLT-4 cells after the exposure of ionizing radiation stand-
alone or in combination with novel compounds at 100 mM concentrationViability
(even at 100 mM). Subsequently, we measured the maximum
of MOLT-4 cells was determined as the percentage of Annexin-V and propidium iodide
(PI)-double-negative cells by flow cytometry 24 h following irradiation by a dose of
1 Gy. (A) Representative flow cytometry histograms depicting the percentage of viable
MOLT-4 cells are shown. (B) The bar graph represents the percentage of viable MOLT-
4 cells detected by flow cytometry as Annexin V negative/PI negative cells. Results are
shown as the mean SD from four experiments.
tolerable concentration for all compounds.
Furthermore, we evaluated the protective effect on irradiated
MOLT4 cells. Finally, we selected the most suitable compounds
based on their toxicological and physicochemical profile. We have
performed in vivo study on whole-body irradiated mice. Impor-
tantly, we observed an interesting structural relationship between
the methoxy group in position 2 on the aromatic ring and an
improved radioprotective effect of our compounds. That said,
analogue 3e was determined as the most effective radioprotective
compound. The data from in vitro experiments related to radio-
protection were comparable with other compounds, but the in vivo
radioprotective effect together with MTC indicate that analogue 3e
derivatives as compounds with radioprotective properties.
We modified the aryl moiety of our leading structure to prepare
an entire group of compounds. The influence of substituents has
been compared with the important parameter of solubility, which
is often limiting for further biological tests. We managed to prepare
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Fig. 5. Absolute lymphocyte counts in peripheral blood after the application of 3e, 3g, 3h, 3i, and 3j (100 mg/kg) in non-irradiated and radiated mice at (A) 7, (B) 12 and (C) 30 days
post-treatment. Inhibitors were applied i.p. 5 min before irradiation by a single dose of 8.5 Gy. Values represent the mean SEM. *p < 0.05, **p < 0.01, ***p < 0.001. y Mice lost
(death) before the analysis could be performed. ǂ Analysis was not performed e only one mouse left.
Fig. 6. Effect of radioprotectives (3e, 3g, 3h, 3i, and 3j; 100 mg/kg) on the proportion of B- and T-lymphocytes and NK cells in non-irradiated and irradiated mice at (A) 7, (B) 12, and
(C) 30 days post-radiation. Inhibitors were applied i.p. 5 min before irradiation by a single dose of 8.5 Gy. Values represent the mean SEM. *p < 0.05, **p < 0.01, ***p < 0.001. y Mice
lost (death) before the analysis could be performed. ǂ Analysis was not performed e only one mouse left.
possesses the highest potential as the radioprotective therapeutic
utility.
superoxide) contribute to cell death [33]. Synergic combination of
small molecule inhibitor targeting intrinsic apoptotic pathway
together with an anti-inflammatory drug could be an effective
strategy for radioprotection in mass casualty scenarios.
Further research is ongoing in order to reveal a mechanistic
explanation of the radioprotective effect, but as our in silico data
indicate, the compounds are likely to interfere with Bcl-2 protein-
protein interaction, interfering with apoptosis induction. The actual
mechanistic explanation will be necessary to design patient-
tailored radioprotection in cancers with dysfunctional intrinsic
apoptotic pathways. It should be also noted that in radiosensitive
organs (such as bone marrow) immune system activation and
signaling mechanism (involving FasL, TNF-alpha, nitric oxide, and
4. Experimental
4.1. In silico study
The 3D structure of Bcl-2 was gained from RCSB Protein Data
Bank e PDB ID 4LXD (Bcl_2-Navitoclax Analog complex) [23]. The
Please cite this article as: J. Marek et al., A novel class of small molecule inhibitors with radioprotective properties, European Journal of
Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2019.111606

8
J. Marek et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
structure was inspected and found as suitable for molecular dock-
ing e it's resolution is 1,9 Å and it has no outliers according to
Ramachandran's plot [34] The receptor structure was prepared by
the DockPrep function of UCSF Chimera (version 1.4) [35] and
converted to pdbqt-files by AutodockTools (v. 1.5.6) [36]. Three-
dimensional structures of ligands were built by Open Babel (v.
2.3.1) [37], minimized by Avogadro (v 1.1.0) [38], and converted to
pdbqt-file format by AutodockTools [36]. The docking calculations
were done by Autodock Vina (v 1.1.2) with an exhaustiveness of 8
[39]. Calculation was done 15 times for each ligand and receptor,
and the best-scored result was selected for manual inspection. The
visualization of enzymeeligand interactions was prepared using
Pymol (v. 1.7.4.5) [The PyMOL Molecular Graphics System, Version
4.2.1. General procedure for synthesis of 1-(4-(2-hydroxyethyl)
piperazin-1-yl)-3-phenoxypropan-2-ol derivatives (3a-3j)
1-(2-hydroxyethyl)piperazine (0.2 g, 1.5 mmol) was dissolved in
anhydrous acetonitrile and K2CO3 (0.4 g, 3.0 mmol) with appro-
priate intermediate (2a-2j) were added. The reaction mixture was
stirred under a nitrogen atmosphere for 4 h. The mixture was
filtered and the filtrate concentrated under reduced pressure. The
residue was purified by flash chromatography eluting with EtOAc/
MeOH/NH3 (25% aq) (6:2:0.2) to obtain yellow or white product in
57e81% yields.
4.2.1.1. 1-(4-(2-hydroxyethyl)piperazin-1-yl)-3-(naphthalen-1-
yloxy)propan-2-ol (3a). Yellow oil (0,43 g, 86%); 1H NMR (500 MHz,
€
1.7.4.5, Schrodinger, LLC, Mannheim, Germany].
Methanol-d4)
d
8.31 (dd, J ¼ 7.6, 1.9 Hz, 1H), 7.83e7.75 (m, 1H),
7.51e7.40 (m, 3H), 7.40e7.33 (m, 1H), 6.91 (dd, J ¼ 7.6, 1.0 Hz, 1H),
4.27 (m, 1H), 4.21e4.09 (m, 2H), 3.69 (t, J ¼ 6.0 Hz, 2H), 2.74e2.52
4.2. Synthesis and analysis
(m,14H); 13C NMR (126 MHz, Methanol-d4)
d 155.87,136.04,128.45,
Analytical grade reagents were purchased from Sigma-Aldrich,
Fluka and Merck (Darmstadt, Germany). The solvents were pur-
chased from Penta Chemicals (Prague, Czech Republic). Reactions
were monitored by thin layer chromatography (TLC) using precoated
silica gel 60 F254 TLC aluminium sheet. Column chromatography was
performed with silica gel 0.063e0.200 mm. Melting points (m. p.)
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 Varian VNMR S500 (operating at 500 MHz for H1
and 125 MHz for C13; Varian Comp., Palo Alto, USA). The chemical
127.37, 126.99, 126.07, 123.02, 121.41, 105.98, 72.13, 68.45, 62.36,
61.25, 59.77, 54.43, 54.35; HRMS: m/z 331.20111 [MþH]þ (calcu-
lated m/z 331.20162 for [C19H27N2O3]þ).
4.2.1.2. 1-(4-(2-hydroxyethyl)piperazin-1-yl)-3-(naphthalen-2-
yloxy)propan-2-ol (3b). Yellow oil (0,42 g, 85%); 1H NMR (500 MHz,
Methanol-d4) d 7.78e7.71 (m, 3H), 7.45e7.38 (m, 1H), 7.34e7.28 (m,
1H), 7.24 (d, J ¼ 2.5 Hz, 1H), 7.18 (dd, J ¼ 9.0, 2.5 Hz, 1H), 4.22e4.15
(m, 1H), 4.12 (dd, J ¼ 9.8, 4.1 Hz, 1H), 4.05 (dd, J ¼ 9.8, 5.9 Hz, 1H),
3.69 (t, J ¼ 5.9 Hz, 2H), 2.75e2.47 (m, 12H); 13C NMR (126 MHz,
Methanol-d4) d 158.21, 136.10, 130.54, 130.36, 128.57, 127.82, 127.33,
shifts (
d
) are given in ppm, related to tetramethylsilane (TMS) as
124.65, 119.80, 107.78, 71.96, 68.33, 62.17, 61.25, 59.77, 54.41, 54.33;
HRMS: m/z 331.20102 [MþH]þ (calculated m/z 331.20162 for
[C19H27N2O3]þ).
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) with UV and
mass spectrometry (MS) gradient method. The system used in this
study was Dionex Ultimate 3000 UHPLC: RS Pump, RS Column
Compartment, RS Autosampler, Diode Array Detector, Chromeleon
(version 6.80 SR13 build 3967) software (Thermo Fisher Scientific,
Germering, Germany) with Q Exactive Plus Orbitrap mass spec-
trometer with Thermo Xcalibur (version 3.1.66.10.) software
(Thermo Fisher Scientific, Bremen, Germany). Detection was per-
formed 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 tempera-
4.2.1.3. 1-(4-(2-hydroxyethyl)piperazin-1-yl)-3-(4-methylphenoxy)
propan-2-ol (3c). White solid, m. p. 201e203 ꢁC (0,30 g, 69%); 1H
NMR (500 MHz, Methanol-d4)
d 7.11e7.03 (m, 2H), 6.89e6.78 (m,
2H), 4.14e4.05 (m, 1H), 3.95 (dd, J ¼ 9.8, 4.3 Hz, 1H), 3.89 (dd,
J ¼ 9.8, 5.9 Hz, 1H), 3.69 (t, J ¼ 5.9 Hz, 2H), 2.70e2.46 (m, 12H), 2.26
(s, 3H); 13C NMR (126 MHz, Methanol-d4)
d 158.22, 131.12, 130.82,
115.46, 71.98, 68.38, 62.18, 61.25, 59.78, 54.40, 54.32, 20.52; HRMS:
m/z 295.20117 [MþH]þ (calculated m/z 295.20162 for
[C16H27N2O3]þ).
4.2.1.4. 1-(4-(2-hydroxyethyl)piperazin-1-yl)-3-phenoxypropan-2-ol
(3d). Yellow oil (0,28 g, 67%); 1H NMR (500 MHz, DMSO‑d6)
d
7.31e7.22 (m, 3H), 6.94e6.90 (m, 2H), 6.67 (m, 1H), 3.98e3.89 (m,
2H), 3.87e3.79 (m, 1H), 3.46 (t, J ¼ 6.4 Hz, 2H), 2.48e2.28 (m, 12H);
13C NMR (126 MHz, DMSO‑d6)
158.90, 129.62, 120.59, 114.65,
ture: 220 ꢁC, Max spray current: 100
column (Kinetex EVO C18, 3 ꢃ 150 mm, 2.6
was used in this study. Mobile phase A was ultrapure water of ASTM I
type (resistance 18.2 M
cm at 25 ꢁC) prepared by Barnstead
mA, S-lens RF Level: 50. C18
d
mm, Phenomenex, Japan)
71.13, 66.66, 61.28, 58.67, 53.71, 53.47, 22.68; HRMS: m/z 281.18527
[MþH]þ (calculated m/z 281.18597 for [C15H25N2O3]þ).
U
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. The method started
with 1 min of isocratic flow of 10% B, and then the gradient of B rose
to 100% B in 3 min and remained constant at 100% B for 1 min. The
composition then went back to 10% B and equilibrated for 2.5 min.
Samples were dissolved in methanol (LC-MS grade, Fluka-Sigma
Aldrich, Steinheim, Germany) at concentration 1 mg/ml and sam-
4.2.1.5. 1-(4-(2-hydroxyethyl)piperazin-1-yl)-3-(2-methoxy-4-
nitrophenoxy)propan-2-ol (3e). Yellow solid, m. p. 184e186 ꢁC
(0,47 g, 88%);1H NMR (500 MHz, DMSO‑d6)
d
7.87 (dd, J ¼ 9.0,
2.7 Hz, 1H), 7.73 (d, J ¼ 2.7 Hz, 1H), 7.19 (d, J ¼ 9.0 Hz, 1H), 4.12 (dd,
J ¼ 9.8, 3.0 Hz, 1H), 4.05e3.94 (m, 2H), 3.88 (s, 3H), 3.46 (t,
J ¼ 6.3 Hz, 2H), 2.48e2.31 (m, 12H); 13C NMR (126 MHz, DMSO‑d6)
d
154.44, 148.90, 140.75, 117.82, 112.01, 106.72, 72.61, 66.46, 61.14,
58.66, 56.20, 53.69, 53.45, 22.68; HRMS: m/z 356.18094 [MþH]þ
(calculated m/z 356.18161 for [C16H26N3O6]þ).
ple injection was 1
m
l. Purity was determined from UV spectra
4.2.1.6. 1-(4-(2-hydroxyethyl)piperazin-1-yl)-3-(2-isopropyl-5-
measured at wavelength 254 nm. HRMS was determined by total ion
current spectra from the mass spectrometer.
Clog P was calculated with MarvinSketch (version 14.9.8.0)
software.
methylphenoxy)propan-2-ol (3f). Yellow oil (0,36 g, 71%); 1H NMR
(500 MHz, DMSO‑d6)
1H), 6.68 (dd, J ¼ 7.6, 1.5 Hz, 1H), 3.97e3.88 (m, 2H), 3.88e3.81 (m,
d
7.02 (d, J ¼ 7.6 Hz, 1H), 6.72 (d, J ¼ 1.5 Hz,
1H), 3.46 (t, J ¼ 6.3 Hz, 2H), 3.27e3.17 (m, 1H), 2.52e2.31 (m, 12H),
Please cite this article as: J. Marek et al., A novel class of small molecule inhibitors with radioprotective properties, European Journal of
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J. Marek et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
9
2.24 (s, 3H), 1.13 (dd, J ¼ 6.9, 1.3 Hz, 6H); 13C NMR (126 MHz,
culture medium to reach final concentrations of 10 and 100
Cells were exposed to 10 and 100
for 48 h. Cells were also exposed to doxorubicin at a concentration
of 1 M. The maximal concentration of DMSO in cultivation media
m
M.
DMSO‑d6)
d
155.82, 135.95, 133.36, 125.68, 121.02, 112.60, 70.97,
mM newly synthesized inhibitors
66.74, 61.44, 60.50, 58.67, 53.78, 53.47, 26.23, 22.85, 22.83, 21.13;
HRMS: m/z 337.24792 [MþH]þ (calculated m/z 337.24857 for
[C19H33N2O3]þ).
m
was 0.1%.
4.2.1.7. 1-(2,6-dimethoxyphenoxy)-3-(4-(2-hydroxyethyl)piperazin-
4.3.2. Cell proliferation assay and growth percent calculation
The WST-1 (Roche, Mannheim, Germany) reagent was used to
determine the cytotoxic effect of the tested compounds. At the end
of the cultivation period, WST-1 test was performed according to
the manufacturer's protocol. The absorbance was measured using a
1-yl)propan-2-ol (3g). Yellow oil (0,41 g, 81%);1H NMR (500 MHz,
Methanol-d4)
d
7.02 (t, J ¼ 8.4 Hz, 1H), 6.67 (d, J ¼ 8.4 Hz, 2H),
4.08e3.99 (m, 1H), 3.95 (dd, J ¼ 10.1, 4.9 Hz, 1H), 3.86 (dd, J ¼ 10.1,
6.2 Hz, 1H), 3.84 (s, 6H), 3.69 (t, J ¼ 6.0 Hz, 2H), 2.70e2.60 (m, 10H),
2.55 (t, J ¼ 6.0 Hz, 2H), 2.51 (dd, J ¼ 13.1, 7.9 Hz, 1H); 13C NMR
€
Tecan Infinite M200 spectrometer (Tecan Group, Mannedorf,
(126 MHz, Methanol-d4)
d 154.70, 138.39, 125.16, 106.63, 77.24,
Switzerland). Each value is the mean of three independent exper-
iments and represents the percentage of proliferation of control,
non-treated cells (100%). The GP value was calculated for each in-
hibitor tested. GP represents the mean of viability decrease as a
percentage of all the 10 cell lines treated with the same inhibitor.
68.94, 62.01, 61.24, 59.76, 56.57, 54.36, 54.23; HRMS: m/z 341.20691
[MþH]þ (calculated m/z 341.20710 for [C17H29N2O5]þ).
4.2.1.8. 1-(4-Allyl-2-methoxyphenoxy)-3-(4-(2-hydroxyethyl)piper-
azin-1-yl)propan-2-ol (3h). Yellow oil (0,42 g, 79%); 1H NMR
(500 MHz, Methanol-d4)
d
6.89 (d, J ¼ 8.1 Hz, 1H), 6.81 (d, J ¼ 2.1 Hz,
1H), 6.72 (dd, J ¼ 8.2, 2.0 Hz, 1H), 6.02e5.90 (m, 1H), 5.10e5.00 (m,
2H), 4.17e4.08 (m,1H), 3.98 (dd, J ¼ 9.9, 4.4 Hz,1H), 3.91 (dd, J ¼ 9.9,
6.0 Hz, 1H), 3.83 (s, 3H), 3.70 (t, J ¼ 6.0 Hz, 2H), 3.33 (d, J ¼ 1.5 Hz,
1H), 2.74e2.54 (m, 13H); 13C NMR (126 MHz, Methanol-d4)
4.4. Half maximal inhibitory concentration (IC50) and maximum
tolerated concentration (MTC)
For the cytotoxicity assays, A-549 cells were cultivated in Dul-
becco's modified Eagle's medium (DMEM, Biosera, Nuaille, France)
supplemented with 10% fetal bovine serum (Biosera) and 1%
penicillin-streptomycin antibiotic solution (SigmaeAldrich). The
cells were maintained in a humidified atmosphere of 5% CO2 at
37 ꢁC.
d
150.87, 148.05, 139.12, 135.06, 121.96, 115.75, 115.54, 113.96, 73.61,
68.38, 61.87, 61.05, 59.46, 56.49, 54.10, 54.08, 40.73; HRMS: m/z
351.22748 (calculated m/z 351.22783 for
[MþH]þ
[C19H31N2O4]þ).
4.2.1.9. 1-(4-(2-hydroxyethyl)piperazin-1-yl)-3-(2-methoxyphenoxy)
Cell viability was evaluated by colorimetric viability assay ac-
cording to Muckova et al. (2018). Briefly, A-549 cells were plated
propan-2-ol (3i). White solid, m. p. 192e194 ꢁC (0,34 g, 74%); 1H
NMR (500 MHz, Methanol-d4)
d
7.01e6.88 (m, 4H), 4.18 (m, 1H),
into 96-well plates in a volume of 100
m
L and density of 9 ꢃ 103 cells
4.01 (dd, J ¼ 9.8, 4.5 Hz, 1H), 3.96 (dd, J ¼ 9.8, 5.8 Hz, 1H), 3.85 (d,
per well. Cells were left to attach overnight. The working solutions
of tested compounds were prepared in phosphate buffer saline
(PBS; SigmaeAldrich) and serially diluted in DMEM. For determi-
nation of 100% viability, we measured the untreated control in each
plate. After 24 h incubation, the cell viability was determined using
colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (SigmaeAldrich) reduction assay. The exper-
iment was carried out in triplicate and repeated three independent
times.
J ¼ 5.0 Hz, 3H), 3.76 (t, J ¼ 5.6 Hz, 2H), 3.03e2.67 (m, 10H), 1.94 (d,
J ¼ 0.6 Hz, 3H); 13C NMR (126 MHz, Methanol-d4)
d 150.96, 149.67,
122.91, 122.26, 115.42, 113.49, 73.18, 68.06, 61.33, 60.43, 58.52,
56.49, 53.43, 53.22, 22.08; HRMS: m/z 311.19586 [MþH]þ (calcu-
lated m/z 311.19653 for [C16H27N2O4]þ).
4.2.1.10. 1-(4-(2-hydroxyethyl)piperazin-1-yl)-3-(2-methylphenoxy)
propan-2-ol (3j). Yellow oil (0,30 g, 67%); 1H NMR (500 MHz,
Methanol-d4)
d
7.17e7.09 (m, 2H), 6.89 (dd, J ¼ 8.1, 1.0 Hz, 1H),
6.87e6.81 (m, 1H), 4.23 (s, 1H), 4.01 (d, J ¼ 5.0 Hz, 2H), 3.87e3.81
(m, 2H), 3.23e3.19 (m, 8H), 3.12e3.09 (m, 3H), 2.93e2.89 (m, 3H),
4.4.1. Statistical analysis
The IC50 values were calculated using four parametric nonlinear
regressions by GraphPad Prism statistics software (version 5.04,
GraphPad Software Inc., San Diego, CA, USA) from the logarithmic
concentration-response curve. The values were expressed as a
mean standard error of the mean (SEM).
2.23 (s, 3H); 13C NMR (126 MHz, Methanol-d4)
d 158.15, 131.61,
127.95, 127.75, 121.80, 112.32, 71.40, 60.92, 59.67, 52.78, 49.51,
49.34, 49.17, 49.00, 48.83, 48.66, 48.49, 22.08, 16.37; HRMS: m/z
295.20099
[C16H27N2O3]þ).
[MþH]þ
(calculated
m/z
295.20162
for
4.3. In vitro cytotoxicity
4.5. In vitro radioprotection studies
4.3.1. Cell cultivation and treatment
4.5.1. Cell treatment and gamma irradiation
The selected 10 human cell lines e Jurkat (acute T-cell leuke-
mia), MOLT-4 (acute lymphoblastic leukemia), A2780 (ovarian
carcinoma), A549 (lung carcinoma), HT-29 (colorectal adenocarci-
noma), PANC-1 (pancreas epithelioid carcinoma), HeLa (cervix
adenocarcinoma), MCF-7 (breast adenocarcinoma), SAOS-2 (oste-
osarcoma), and MRC-5 (lung fibroblast) e were purchased from
Sigma Aldrich and cultivated according to the provider's culture
method guidelines. At the start of experiments, each cell line was
Fresh stock solutions of PUMA inhibitors in concentrations of
50 mM were dissolved in dimethyl sulfoxide - DMSO (Sigma-
Aldrich, St. Louis, MO, USA). Stock solutions were freshly prepared
before use in the study. For the radioprotection experiments, MOLT-
4 cells were treated with inhibitors at 100 mM, making sure the
concentration of DMSO was <0.1% to avoid toxic effects on the cells.
Sixty minutes after inhibitor addition, MOLT-4 cells were irradiated
at room temperature using a60Co
g-ray source (Chisotron Chirana,
seeded
at
previously
established
optimal
density
Prague, Czech Republic). Non-irradiated control and irradiated-
alone cells were treated with a DMSO vehicle only (0.1%; Control)
and handled in parallel with the test cells. Non-irradiated control
cells were handled in the same way as described for inhibitor-
treated cells, except that irradiation was omitted.
(500e30 ꢃ 103 cells per well) in a 96-well plate and the cells were
allowed to settle overnight. The derivatives to be tested were dis-
solved in DMSO to stock solutions (10 mM). For the experiments,
the stock solution was diluted with the appropriate complete
Please cite this article as: J. Marek et al., A novel class of small molecule inhibitors with radioprotective properties, European Journal of
Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2019.111606

10
J. Marek et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
4.5.2. Analysis of viability
cytometer CyAn ADP (Beckman Coulter, San Jose, CA, US).
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 in-
structions. 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 Ca2þ, and the
property of PI to enter cells with damaged cell membranes and to
bind to DNA. Measurement was performed immediately using a
CyAn (Beckman Coulter, Miami, FL, USA) flow cytometer. Listmode
data were analysed using Kaluza Analysis 1.3 software (Beckman
Coulter, Miami, FL, USA).
Author contributions
The manuscript was written through contributions of all au-
thors. All authors have given approval to thefinal version of
themanuscript.
Declarations of interest
None.
Acknowledgements
4.5.3. Statistical analysis
The descriptive statistics of the results were calculated, and the
charts made in either Microsoft Office Excel 2010 (Microsoft, Red-
mond, WA, USA) or GraphPad Prism 5 biostatistics (GraphPad
Software, La Jolla, CA, USA). In this study, all of the values were
expressed as arithmetic means with SD of triplicates, unless
otherwise noted. The significant differences between the groups
were analyzed using the Student's t-test and a P value < 0.05 was
considered significant.
This work was supported by grant project No. 17-13541S of the
Czech Science Foundation.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.111606.
References
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Experimental C57Bl/6 mice (27 3 g) were obtained from Velaz
a.s. (Prague, Czech Republic). All used methods were in accordance
to EU Directive 2010/63/EU for animal experiments and NIRS
Guidelines for the Care and Use of Laboratory Animals and
approved by the Committee for animal rights of the Ministry of
Defence of Czech Republic. The mice were organized in groups of 10
animals each and received a dose of 8.5 G from a60Co gamma source
(Chisotron, Chirana, Czech Republic) at a dose rate of 1.3 Gy/min;
non-irradiated and non-treated control groups were also included.
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Please cite this article as: J. Marek et al., A novel class of small molecule inhibitors with radioprotective properties, European Journal of
Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2019.111606