5-(N-trifluoromethylcarboxy)aminouracil as a potential dna radiosensitizer and its radiochemical conversion into n-uracil-5-yloxamic acid

Article abstract of DOI:10.3390/ijms21176352

Hypoxia—a hallmark of solid tumors—dramatically impairs radiotherapy, one of the most common anticancer modalities. The adverse effect of the low-oxygen state can be eliminated by the concomitant use of a hypoxic cell radiosensitizer. In the present paper, we show that 5-(N-trifluoromethylcarboxy) aminouracil (CF₃CONHU) can be considered as an effective radiosensitizer of DNA damage, working under hypoxia. The title compound was synthesized in the reaction of 5-aminouracil and trifluoroacetic anhydride in trifluoroacetic acid. Then, an aqueous and deoxygenated solution of the HPLC purified compound containing tert-butanol as a hydroxyl radical scavenger was irradiated with X-rays. Radiodegradation in a 26.67 ± 0.31% yield resulted in only one major product—N-uracil-5-yloxamic acid. The mechanism that is possibly responsible for the formation of the observed radioproduct has been elucidated with the use of DFT calculations. The cytotoxic test against the PC3 prostate cancer cell line and HDFa human dermal fibroblasts confirmed the low cytotoxicity of CF₃CONHU. Finally, a clonogenic assay and flow cytometric analysis of histone H2A.X phosphorylation proved the radiosensitization in vitro.

Full text of DOI:10.3390/ijms21176352

International Journal of
Molecular Sciences
Article
5-(N-Trifluoromethylcarboxy)aminouracil as a
Potential DNA Radiosensitizer and Its Radiochemical
Conversion into N-Uracil-5-yloxamic Acid
Paulina Spisz, Witold Kozak , Lidia Chomicz-Man´ka, Samanta Makurat , Karina Falkiewicz,
Artur Sikorski , Anna Czaja, Janusz Rak and Magdalena Zdrowowicz *
Department of Physical Chemistry, Faculty of Chemistry, University of Gdan´sk, Wita Stwosza 63,
80-308 Gdan´sk, Poland; paulina.rewers@phdstud.ug.edu.pl (P.S.); davelombardo@wp.pl (W.K.);
lidia.chomicz-manka@ug.edu.pl (L.C.-M.); samanta.makurat@ug.edu.pl (S.M.); karina.falki@gmail.com (K.F.);
artur.sikorski@ug.edu.pl (A.S.); czaja.ania@yahoo.com (A.C.); janusz.rak@ug.edu.pl (J.R.)
* Correspondence: magdalena.zdrowowicz@ug.edu.pl
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 21 July 2020; Accepted: 28 August 2020; Published: 1 September 2020
Abstract: Hypoxia—a hallmark of solid tumors—dramatically impairs radiotherapy, one of
the most common anticancer modalities. The adverse effect of the low-oxygen state can be
eliminated by the concomitant use of a hypoxic cell radiosensitizer. In the present paper, we show
that 5-(N-trifluoromethylcarboxy) aminouracil (CF3CONHU) can be considered as an effective
radiosensitizer of DNA damage, working under hypoxia. The title compound was synthesized in
the reaction of 5-aminouracil and trifluoroacetic anhydride in trifluoroacetic acid. Then, an aqueous
and deoxygenated solution of the HPLC purified compound containing tert-butanol as a hydroxyl
radical scavenger was irradiated with X-rays. Radiodegradation in a 26.67
±
0.31% yield resulted in
only one major product—N-uracil-5-yloxamic acid. The mechanism that is possibly responsible for
the formation of the observed radioproduct has been elucidated with the use of DFT calculations.
The cytotoxic test against the PC3 prostate cancer cell line and HDFa human dermal fibroblasts
confirmed the low cytotoxicity of CF3CONHU. Finally, a clonogenic assay and flow cytometric
analysis of histone H2A.X phosphorylation proved the radiosensitization in vitro.
Keywords: radiosensitizer; uracil derivatives; modified nucleobases; electron-induced degradation;
DFT calculations
1. Introduction
Tumor hypoxia, defined as a low level of oxygenation, causes resistance to radiotherapy, which is
one of the treatments of choice in many types of cancer [
1]. In a hypoxic environment, cancer cells
are two to three times more resistant to ionizing radiation (IR) than normoxic ones [
2]. The oxygen
effect is explained by the oxygen fixation hypothesis, which postulates that radical damage produced
in DNA by the direct or indirect action of radiation can be repaired under hypoxia or “fixed” by
molecular oxygen even at a very small concentration [3,4]. One of the ways to overcome this important
impediment to effective cancer therapy is introducing radiosensitizers. Radiosensitizers are agents
that sensitize tumor cells to IR. A “true” radiosensitizer meets the stricter criteria of being (i) relatively
nontoxic and (ii) selective for tumors. These species may be grouped into three categories based on
their structures [
nanoparticles, are promising potential radiosensitizers because of their ability to absorb, scatter
and emit a radiation energy, satisfying chemical stability, high biocompatibility and low toxicity [ ].
Second, macromolecules such as miRNAs, proteins, peptides and oligonucleotides are also capable of
regulating radiosensitivity and can be combined with biological therapy as well as drug delivery [ ].
5]. First, nanomaterials such as nanometallic materials, and especially gold-based
5
5
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www.mdpi.com/journal/ijms

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Finally, a number of small molecules have been explored to develop radiosensitizers. The latter group
includes oxygen mimetics, repair process inhibitors, apoptosis activators, suppressors of radioprotective
substances or substrates for the biosynthesis of modified DNA—modified nucleosides [5].
Another classification of radiosensitizers takes into account only two groups of sensitizers that
can be really used in clinical practice, because of their selectivity: (i) hypoxic-cell radiosensitizers
and (ii) halogenated pyrimidines. The first group of radiosensitizers enhances the radiation effects
only in hypoxic cells (characteristic of tumor tissue) but not in normoxic ones. For this reason,
the selectivity toward cancer is due to the difference in oxygenation levels between cancer and normal
cells. The selectivity of halogenated pyrimidines results from rapid growth and uncontrolled division
of cancer cells, which means a higher degree of incorporation of pyrimidine analogs into the DNA of
cancer cells, compared to healthy ones [6].
Uracil derivatives possess exceptional properties, making them especially good agents for the
radiation-induced cell killing [7]. Some of them are able to substitute native nucleobases in DNA
without affecting its structure and biological functions, and consequently they are incorporated into
the DNA of tumor cells during their non-controlled proliferation. This results in a much larger
extent of DNA labeling in cancer cells. Additionally, if nucleosides are appropriately modified,
their radiosensitizing properties may occur only under hypoxia.
Such a modification should rely on the introduction of suitable substituents in nucleobases,
which increases nucleosides’ sensitivity to degradation induced by solvated electrons [
These electrons are one of the major products of water radiolysis under hypoxic conditions [
(in normoxic conditions solvated electrons can be scavenged by oxygen) and remain non-reactive to
native DNA under biological conditions [ 10]. Thus, a modified, radiosensitizing nucleobase
7].
8]
9,
has usually an electrophilic substituent and undergoes an efficient dissociative electron
attachment (DEA) that leaves behind a nucleoside radical, which, in secondary reactions,
is able to produce DNA damage leading to cancer cell death [7]. One of the best known
radiosensitizers of this type are the halo derivatives of uracil. In the past, it was shown
that cells undergoing DNA synthesis are unable to distinguish between thymidine and
5-halogenated 2’-deoxyuridines, such as 5-bromo-2’-deoxyuridine (BrdU). Therefore, such analogues
are incorporated into newly synthesized DNA [11
prone to dissociative electron attachment leading to a reactive uracil-5-yl radical via the unstable
anion [13 15]. The promising characteristics of the electron-induced degradation of halo uracils
have been demonstrated many times [16 20]. Recently, several new C5-pyrimidine analogues
have been proposed in the context of efficient electron-induced degradation [21 22]. Among others,
,12]. BrdU is a highly electron-affinic molecule,
–
–
,
one could name: 5-thiocyanato-2’-deoxyuridine [23], 5-selenocyanato-2’-deoxyuridine [24], 5-
trifluoromethanesulfonyl-2’-deoxyuridine [25] and 5-iodo-4-thio-2’-deoxyuridine [26].
In the present paper, we demonstrate that 5-(N-trifluoromethylcarboxy) aminouracil (CF3CONHU)
can be considered as an effective radiosensitizer. Indeed, the theoretical profile of dissociative
electron attachment to CF3CONHU, concerning the release of the fluoride anion, suggests that
electron-induced damage to the molecule is kinetically and thermodynamically accessible. A steady
state radiolysis of the deoxygenated solution of CF3CONHU (obtained in the reaction of 5-aminouracil
and trifluoroacetic anhydride in trifluoroacetic acid) containing an •OH radical scavenger has been
performed. This experiment, coupled with high performance liquid chromatography and mass
spectrometry, allowed us to identify the product of the electron-induced degradation of CF3CONHU
and determine the efficiency of the process. The mechanism responsible for the formation of the
observed radioproduct has been proposed with use of quantum chemical calculations. In view of the
radiodamage observed in the radiolytic studies, we also performed a cytotoxicity test and clonogenic
assay and found out that the treatment of PC3 prostate cancer cells with the studied compound brings
about no significant decrease in cell viability, although it results in a reduction of cell survival after
exposure to IR. The results of cytometric studies on histone H2A.X phosphorylation suggest that

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the radiosensitivity of CF3CONHU-treated PC3 cells is related to (at least in part) the formation of
double-strand breaks (DSBs).
2. Results and Discussion
2.1. Design and Synthesis of 5-(N-Trifluoromethylcarboxy)aminouracil
The uracil molecule can be transformed into a potential radiosensitizer by introduction of an
appropriate substituent which increases the electron affinity of the system [7]. 5-Substituted uracils
are by far the most common derivatives of uracil, due to the susceptibility of this position to
chemical transformation. Besides halogenation reactions that lead to the C5–X bond (X = F, Cl, Br or
I), other substituents can be relatively easily introduced into this nucleobase [27]. For instance,
the amination reaction of 5-bromouracil may serve to obtain 5-aminouracil (NH2U). However,
as indicated by the computational adiabatic electron affinity (AEA), the vulnerability of 5-aminouracil
to accept the excess electron is lower than that of uracil itself (AEA = 2.04 for NH2U and 2.09 eV
for U calculated at the M06-2X/6-31++G (d,p) level). Yet the amino group can be relatively easily
converted into a more electrophilic one. Namely, introducing a highly electronegative trifluoroacetyl
group onto the -NH2 substituent should improve the electron affinity of NH2U. Indeed, the AEA of
5-(N-trifluoromethylcarboxy) aminouracil calculated at the M06-2X/6-31++G (d,p) level amounts to
2.41 eV, which is only 0.02 eV lower than that of 5-bromouracil (AEA = 2.43 eV at the same level of
theory), a well-known radiosensitizer.
It is worth emphasizing that an increased electron affinity itself is not a sufficient condition
for a molecule to be an efficient radiosensitizer. Besides the ability to accept an excess electron,
which is necessary to trap a solvated electron, a potential radiosensitizer has to be prone to electron
attachment-induced dissociation. In Figure 1, the energetic profile (in the free energy scale) for the
possible DEA process in CF3CONHU, obtained at the DFT level, is depicted. We considered the
cleavage of particular bonds—C5–N, N–C, C–CF3 and C–F—in the substituent.
Figure 1. Energetics of dissociative electron attachment (DEA) processes in CF3CONHU, calculated
at the M06-2X/6-31++G (d,p) level. The arrow associated with three values indicates the process that
actually occurs in the studied system.
As indicated by the values gathered in Figure 1, only the release of the fluoride anion, induced by
electron attachment, is possible. The thermodynamic barriers for the dissociation of the remaining
bonds—C5–N, C–N and C–CF3—span the range between 24.5 and 34.6 kcal mol−1, which is far too
high to be competitive with the release of F−. Undoubtedly, the latter process is associated with a

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small activation barrier and a slightly positive thermodynamic stimulus of 5.6 kcal mol−1. It is worth
underlining that at the B3LYP level this driving force amounts to
4.0 kcal mol−1, which on the one
−
hand reflects the semi-empirical nature of the DFT approach and on the other hand suggests that the
release of fluoride anions from the primary radical anion can be spontaneous.
In view of the promising results described above, we decided to obtain CF3CONHU. The synthesis
of the above-mentioned compound relies on the acylation reaction, according to Figure 2. The title
compound has been obtained via the reaction of 5-aminouracil with trifluoroacetic anhydride (TFAA)
in trifluoroacetic acid (TFA) [28].
Figure 2. Synthesis of 5-(N-trifluoromethylcarboxy) aminouracil.
2.2. Crystallography
Single-crystal X-ray diffraction measurements show that CF3CONHU crystallized in the monoclinic
C2/c space group with one molecule in the asymmetric unit (Figure 3 and Table S1 in the Supplementary
Materials). In the crystal of a title compound, the molecules are linked through N1–H1···O7 and
N3–H3···O8 hydrogen bonds to create sheets of asymmetric ribbons (Figure 4 and Table S2 in
the Supplementary Materials), previously observed in a crystal of form II of 5-fluorouracil [29].
The neighboring, anti-parallel ribbons are connected via N9–H9···O8, C6–H6···O11 hydrogen bonds
and F15···F15 contact (d (F···F) = 2.81 Å) to form a 3D framework.
Figure 3. Molecular structure of 5-(N-trifluoromethylcarboxy) aminouracil, showing the atom-labeling
scheme (displacement ellipsoids are drawn at the 25% probability level, and H-atoms are shown as
small spheres of arbitrary radius).

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Figure 4. Crystal packing of 5-(N-trifluoromethylcarboxy) aminouracil viewed along the b-axis
(hydrogen bonds are represented by dashed lines).
2.3. Radiolysis
Two of the most important properties which a DEA type radiosensitizer should possess are (i) high
electron affinity of the nucleobase and (ii) the ease of dissociation of the radical anion, formed as
a result of electron attachment, leading to reactive radicals that in secondary steps may produce
strand breaks [7]. Therefore, the steady state radiolysis has been performed for an aqueous solution
of CF3CONHU. Thus, the aqueous solution of 5-(N-trifluoromethylcarboxy) aminouracil has been
irradiated at pH = 7 with a dose of 140 Gy in the presence of phosphate buffer and tert-butanol (as •OH
radical scavenger). Prior to IR exposure, samples were deoxygenated by Ar saturation in order to
avoid the scavenging of electrons by oxygen molecules. In the end, the HPLC and LC-MS analyses of
the irradiated and non-irradiated samples were performed.
Figure 5 shows that only one main radioproduct has been produced. Based on the MS and
MS/MS spectra, we found that the product of the electron-induced degradation of CF3CONHU is
N-uracil-5-yloxamic acid (for MS and MS/MS spectra with ion identities, see the Supplementary
Materials Figures S3–S6). This product has been generated due to the DEA process, which, in the first
step, leads to a fluoride anion detachment and the generation of a •CF2CONHU radical (for the detailed
mechanism, see Figure 6). The yield of CF3CONHU degradation, 26.67
comparing the areas of its HPLC signals before and after IR exposure. It is worth noting that the decay of
BrdU—a well-known radiosensitizer—is equal to 19.31 0.86% under the same experimental conditions.
±
0.31%, has been determined by
±
Altogether, the above-mentioned results suggest that 5-(N-trifluoromethylcarboxy) aminouracil should
exhibit radiosensitizing properties.
2.4. A Possible Mechanism of Electron-Induced Degradation of CF3CONHU
Without doubt, the C–F bond is one of the strongest single bonds involving carbon [30]. However,
one can find examples of reactions of trifluoromethylated aromatic compounds resulting in an expulsion
of fluoride anions. One of them concerns the hydrolysis of hydroxybenzotrifluorides and fluorinated
uracil derivatives showing the impact of a deprotonation at the N1 and N3 positions on the labilization
of one of the fluoride atoms within the CF3 group [31]. The other work shows various examples of

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reactions of trifluoromethylated compounds with different nucleophilic agents [32]. Not only hard
nucleophiles can be responsible for the CF3 group decomposition. Trifluorothymidine treated with a
phosphate buffer in an aqueous solution affords 5-carboxy-2’-deoxyuridine in a very good yield [33].
Moreover, trifluoroacetates undergo hydrolysis in mild basic conditions [34] or by sodium borohydride
treatment [35].
In light of the aforementioned facts, one could expect the formation of either a deacetylation
reaction product or hypothetical 2-oxazolidinone (see Figure S7) in the course of CF3CONHU radiolysis.
Instead, N-uracil-5-yloxamic acid is generated (see Figure 5). The overnight conditioning of samples
containing CF3CONHU in a basic aqueous solution, phosphate buffer or pure water confirmed that
hydrolysis is not involved in the studied process and the formation of N-uracil-5-yloxamic acid occurs
exclusively through the DEA process. To the best of our knowledge, there are no literature reports
regarding the obtainment of oxalic acid derivatives starting from N-substituted trifluoroacetamides.
Thus, one may treat the above-described protocol as a general approach that enables various oxalic
acid analogues to be synthesized radiolytically.
Figure 5. HPLC traces for 5-(N-trifluoromethylcarboxy) aminouracil radiolysis. Samples before (black)
and after irradiation with a dose of 140 Gy (red). Chemical structures, as indicated by the LC-MS/MS
analyses, are assigned to particular HPLC peaks.

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Figure 6. Gibbs free energy profile for the degradation of an anion radical of 5-(N-trifluoromethylcarboxy) aminouracil. Thermodynamic (∆G) and kinetic (∆G*)
barriers in kcal mol−1
.

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In Figure 6 we present a computational proposal for the mechanism of the formation of N-oxamic
acid derivative under the action of a solvated electron. In the first step, a solvated electron attaches to
CF3CONHU and initiates the release of the fluoride anion from the primary radical anion (A
→
B,
see Figure 6). The obtained difluoromethylene radical (B) reacts with water, giving, after hydrogen
fluoride detachment, a fluorohydroxy intermediate (D). However, the aforementioned reaction with a
water molecule turned out to be kinetically forbidden, as the activation barrier for water attachment
was estimated to be as high as 37 kcal mol−1. Indeed, if the elemental reaction with such activation
barrier is the bottleneck step, the reaction completion time (99% of substrate converted into product)
amounts to ca. 30 million years in 298 K. To make sure that such a high kinetic barrier is not an artifact
of the employed computational method, we recalculated it with the use of a popular B3LYP [36
and the MPWB1K [39] functional, dedicated to kinetic calculations (in both cases using the 6-31++G
(d,p) basis set and the polarization continuum model (PCM) model of water), and with the G2MP2 [40
–38]
]
model of chemical accuracy, obtaining
∆GB3LYP* = 31.5, ∆GMPWB1K* = 39.3 and ∆GG2MP2* = 35.5
kcal mol−1. On the one hand this confirms the estimate obtained at the M06-2X/6-31++G (d,p) level
and, on the other hand, indicates that the reaction of B with water is unlikely. Therefore, we proposed
a hydroxyl anion rather than a water molecule as a nucleophile in the elemental reaction involving
B (B
→
C, Figure 6), although the concentration of the former was only 10−7 M (pH = 7), while that
of water amounted to 55.5 M under the experimental conditions. It turned out that B reacts with the
hydroxyl anion without a kinetic barrier. More precisely, the optimization of the B···−OH complex,
even for a distance between B and −OH as large as 8 Å, converged spontaneously to intermediate C
(Figure 6), and the thermodynamic stimulus associated with the formation of C was highly favorable
(
∆
G =
−
41.9 kcal mol−1). The subsequent elimination of fluoride anion from difluorohydroxy
G* = 10.7 and
G = 5.6 kcal mol−1),
anion radical C proceeded with relatively low barriers (
∆
∆
giving fluorohydroxy radical D. In the next step, a water molecule assisted in a concerted elimination
of the last fluoride atom (in the hydrogen fluoride form) to produce radical E with an acceptable kinetic
barrier (
of intermediate E (with low activation barrier
G =
19.2 kcal mol−1) and spontaneously transferred one of hydrogen atoms to the neighboring
∆
G* = 24.6 kcal mol−1; D
→
E, Figure 6). Then, a water molecule attached to the radical center
∆
G* = 10.3 and favorable thermodynamic stimulus
∆
−
carbonyl oxygen, producing intermediate radical F (Figure 6). Such radical can be stabilized via the
detachment of hydrogen atom by any encountered radical. For instance, if a tert-butanol radical
•
(tert-butanol radicals (tert-But•) are formed in the amount equivalent to that of the OH radicals
during the reaction between the hydroxyl radicals (from water radiolysis) and tert-butanol used as an
•OH radical scavenger [
8
]) occurs in the proximity of F, it spontaneously (barrier-free) detaches the
hydrogen atom from F, giving closed shell products: a tert-butanol molecule and the experimentally
observed N-uracil-5-yloxamic acid (F
G, Figure 6). The complex of a radical F and tert-But• is
→
so reactive that its geometry optimization, even for a distance of 10 Å (between radical centers of
F and tert-But• molecules), leads barrier-free to the product complex. Due to the above-mentioned
fact, the thermodynamic stimulus for the final step,
∆
Gsep
=
67.2 kcal mol−1, was estimated for the
−
separated reagents (F + tert-But• → G + tert-butanol).
However, one could still argue that despite the fact that reacti−on B
→
C is barrier-less when a
hydroxyl anion is a nucleophile, a much smaller concentration of OH compared to that of water
makes the rate of this reaction insufficient for the whole process to be completed in a reasonable time.
The numerical integration of the set of appropriate kinetic equations (see Figure 6) revealed that the
formation of N-uracil-5-yloxamic acid was completed in ca. 7 h (see Scheme S1, Equation (S1), Table S4
in the Supplementary Materials and Figure 7) which, at least intuitively, seems to be reasonable. It is
worth noticing that the DFT model does not possess chemical accuracy, and the activation barrier
of a bottleneck step (D
→
F, Figure 6) lowered by only 1 kcal mol−1 led to ca. 0.6 h for the reaction
completion time, which additionally shows that the proposed mechanism may indeed be operative.

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Figure 7. Kinetic curves for particular species involved in the radiolysis of a CF3CONHU solution
(R = NHU). The vertical broken line indicates the moment at which the X-ray source was turned off.
The system of kinetic equations (Equation S1) has been integrated for 3 × 104 s.
2.5. Cytotoxicity
A good radiosensitizer should be characterized by low cytotoxicity. In order to determine the
cytotoxicity of CF3CONHU toward PC3 (prostate cancer cells) and HDFa (normal fibroblasts) lines,
the WST-1 assay has been performed. This test is based on the conversion of a tetrazolium salt to a
chromatic formazan in the presence of intracellular mitochondrial dehydrogenase. The amount of dye
generated by the enzyme is directly proportional to the amount of living cells [41].
CF3CONHU has been tested at seven concentrations: 0 (for control), 10−4, 10−5, 10−6, 10−7, 10−8
and 10−9 M; and two incubation time variants: 24 and 48 h (Figure 8). The data show a statistically
significant reduction of viability in two cases. When it comes to the PC3 line, it is observed at the
10−4 M concentration in a 48 h time variant (the reduction of viability to 90.79
of the incubation time of PC3 cells with the tested uracil derivative to 72 and 96 h results in a
similar effect—the obtained results (see Figure S9 in the Supplementary Materials) show that the
tested derivative causes a statistically significant reduction in cell viability compared to the control
±
1.3%). The extension
(up to 86.5
±
1.6% for 72 h incubation and up to 87.3
±
1.7% for 96 h incubation) only for the
highest concentration tested (10−4 M). The curves of proliferation are presented in the Supplementary
Materials (Figure S10). In the case of the HDFa line, the statistically significant reduction of viability to
92.53
±
1.4% is observed also at 10−4 M, in the 48 h time variant. The presented data show that the
cytotoxicity of the studied compound is very low for both lines and, furthermore, no meaningful
differences have been observed between both lines.

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Figure 8.
5-(N-trifluoromethylcarboxy)aminouracil in a range of concentrations from 0 to 10−4 M. Results are
shown as mean SD of three independent experiments performed in triplicate. * a statistically
The viability of PC3 and HDFa cell lines after 24 and 48 h treatment with
±
significant difference is present in the treated culture when compared with control (untreated culture).
2.6. Clonogenic Assay
The radiosensitivity of PC3 cells treated with the studied compound was determined by clonogenic
assays. The effect of CF3CONHU on PC3 cells’ survival, when not combined with irradiation, was not
observed. Figure 9 shows the change in cell survival with doses of 0.5, 1, 2 and 4 Gy in the presence
of the tested derivative, compared to the survival of the control. The comparison of curves clearly
shows that the presence of CF3CONHU enhances the radiosensitivity of the cells. The pre-treatment
with 100
from 86.2
reduced from 69.5
pre-treatment caused a significant reduction in the proliferation of PC3 cells (from 43.4
32.5 4.6% and from 11.5 3.2% to 4.2 1.3%, for 2 Gy and 4 Gy, respectively). The dose enhancement
factor (ID50( treatment)/ID50(+treatment)) [42], where ID50 means the radiation dose causing 50%
growth inhibition) representing the radiosensitizing effect was equal to 1.52. Furthermore, based on
these survival curves, the parameters for cellular radiosensitivity, such as (coefficient for linear killing)
and (coefficient for quadratic killing) values, were calculated by a fitting using the linear–quadratic
model (SF = exp( 1*( D + D2), where SF is the survival fraction and D is the dose). Average
and values were 0.29 0.015 and 0.064 0.007, respectively, for the non-treated PC3 cells and
0.591 0.092 and 0.027
0.014, respectively, for PC3 cells treated with 10−4 M. In general, the incubation
µ
M of the studied analogue reduced the survival of the PC3 cells irradiated with 0.5 Gy
2.2% to 68.8 7.1%. With a somewhat higher dose (1 Gy), the surviving fraction was
4.1% to 56.6 1.9% after incubation. Also at higher doses of ionizing radiation,
2.8% to
±
±
±
±
±
±
±
±
−
α
β
−
α
β
α
β
±
±
±
±
of cancer cells with CF3CONHU causes an enhancement of radiation-induced cancer cell killing,
which suggests that the studied compound can be considered as a promising radiosensitizer.

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Figure 9. Effect of 100
irradiation on the survival of PC3 cells. Data obtained with the clonogenic assay. Experiments
were performed at least three times, and the results are expressed as mean standard deviation.
µM 5-(N-trifluoromethylcarboxy) aminouracil treatment combined with
±
The average plating efficiencies for the controls with and without pretreatment are equal to 40.66%
(0 µM), and 42.09% (100 µM)
2.7. Histone H2A.X Phosphorylation
Double-strand breaks generation is one of the most desirable type of DNA damage in the context
of radiosensitization. Phosphorylation of histone H2A.X is the biomarker of such a damage. The flow
cytometric assay was performed for human prostate cancer cells treaded with CF3CONHU at a
concentration of 10−4 M and/or irradiated with a dose of 0 or 2 Gy in order to check the relationship
between the formation of DNA double-strand breaks and the radiosensitivity of pre-treated PC3 cells.
Our studies show that treatment with CF3CONHU results in a significant increase in the population of
γ
H2A.X positive cells after irradiation with the dose of 2 Gy (Table 1 and Figure S8 in the Supplementary
Materials). The exposure of treated cells results in an enhancement of the H2A.X level from
16.32 0.12% to 37.48 1.72%. This result suggests that the radiosensitivity of CF3CONHU-treated
PC3 cells is related to (at least in part) the formation of DSBs.
γ
±
±
Table 1. Fractions of
deviation (SD) of two independent flow cytometry experiments.
γ
H2A.X positive cells 1 h after irradiation. Results are shown as mean
±
standard
% of γH2AX Positive Cells
Control (Non-Treated)
Dose [Gy]
CF3CONHU (10−4 M, 48 h)
0 Gy
2 Gy
6.91 ± 0.17
7.00 ± 0.55
16.32 ± 0.12
37.48 ± 1.72
3. Materials and Methods
3.1. Materials
5-Aminouracil, TFAA, TFA, streptomycin, penicillin, WST-1 and phosphate buffer were purchased
from Sigma-Aldrich (Saint Louis, MO, USA). HDFa, F-12K, DMEM and fetal bovine serum (FBS) were
obtained from Gibco (Life Technologies Limited, Paisley, Scots, UK). Thin-layer chromatography was
performed on silica gel plates, 60G, F254 (Sigma-Aldrich, Saint Louis, MO, USA). The NMR spectra
were recorded on a Bruker AVANCE III, 500 MHz spectrometer (Bruker, Billerica, MA, USA). Chemical
shifts are reported in ppm relative to the residual signals of DMSO-d6 (2.49 ppm for 1H, 39.5 ppm for
13C). The radiolysis was carried out in a Cellrad X-ray cabinet (Faxitron X-ray Corporation, Tucsconcity,
AZ, USA). HPLC analyses were performed on a Dionex UltiMate 3000 system with a diode array
detector (Dionex Corporation, Sunnyvale, CA, USA). The LC-MS analyses were performed using a
mass spectrometer TripleTOF 5600+ (ABSciex, Darmstadt, Germany) with UHPLC system Nexera
X2 (Shimadzu, Canby, OR, USA). The absorbance was measured with an EnSpire microplate reader

Int. J. Mol. Sci. 2020, 21, 6352
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(PerkinElmer, Waltham, MA, USA). The colony size was assessed by using an inverted fluorescence
microscope (IX73, Olympus, Tokyo, Japan).
3.2. Synthesis of 5-(N-trifluoromethylcarboxy)aminouracil
To a stirred solution of 5-aminouracil (100 mg, 0.79 mmol) in TFA (1 mL), TFAA (0.122 mL,
0.86 mmol) was added. The mixture was stirred for 24 h. After that time it was evaporated to dryness,
and the crude product was purified with HPLC. The analytes were separated on a Synergy Polar-RP
(Phenomenex) reverse-phase column. The isocratic elution with 5% phase B was used (mobile phase A:
0.1% formic acid and B: 80% ACN). The resulting product was obtained as a white solid (127 mg) in a
72.3% yield.
1H NMR (Bruker AVANCE III, 500 MHz, DMSO),
7.79 (s, 1H, CHAr); 13C NMR (125 MHz, DMSO),
δ
: 11.40 (brs, 1H, NH), 10.79 (brs, 1H, NH),
δ
: 161.0, [156.68, 156.39, 156.10, 155.81 (q)], 150.8,
138.6, [119.68, 117.39, 115.09, 112.80 (q)], 109.5. HRMS (TripleTOF 5600+, SCIEX), m/z: [M–H]− calcd
for C6H4F3N3O3 222.0131, found 222.0081; UV spectrum (water), λmax: 270 nm.
CAS Registry number: 31385-11-2.
3.3. Crystallography
XRD Measurements and Refinements. A good-quality single-crystal of CF3CONHU was selected
for the X-ray diffraction experiments at T = 295 K (Table S1 in the Supplementary Materials). It was
mounted with an epoxy glue at the tip of glass capillaries. Diffraction data were collected on an
Oxford Diffraction Gemini R ULTRA Ruby CCD diffractometer with MoKα (λ = 0.71073 Å) radiation.
The lattice parameters were obtained by least-square fit to the optimized setting angles of the reflections
collected by means of CrysAlis CCD [43]. Data were reduced using CrysAlis RED software41 and
applying multi-scan absorption corrections (empirical absorption correction using spherical harmonics,
implemented in the SCALE3 ABSPACK scaling algorithm). The structural resolution procedure
was carried out using the SHELX package [44]. The structure was solved with direct methods that
carried out refinements by full-matrix least-squares on F2 using the SHELXL-2017/1 program [44].
H-Atom bound to aromatic C-atom was placed geometrically and refined using a riding model with
C–H = 0.93 Å and Uiso(H) = 1.2Ueq(C). All H-atoms bound to N-atoms were placed geometrically
and refined freely with Uiso(H) = 1.2Ueq(N). All interactions were calculated using the PLATON
program [45]. The ORTEPII, [46] PLUTO-78 [47] and Mercury [48] programs were used to prepare the
molecular graphics.
Full crystallographic details of the title compound have been deposited in the Cambridge
Crystallographic Data Center (deposition no. CCDC 2016475), and they may be obtained from:
ccdc.cam.ac.uk, e-mail: deposit@ccdc.cam.ac.uk, or The Director, CCDC, 12 Union Road, Cambridge,
CB2 1EZ, UK.
3.4. Radiolysis
An aqueous solution of the studied compound at a concentration of 10−4 M was prepared.
The mixture contained t-BuOH (0.03 M) and phosphate buffer (10−2 M, pH = 7.0). In order to
deoxygenate the solution, argon was purged for ca. 3 min. All samples were subsequently IR-exposed
with a dose of 140 Gy. The studied samples were prepared in triplicate, while the non-irradiated ones
were used as controls.
3.5. HPLC and LC-MS
HPLC analysis. All CF3CONHU samples were analyzed with reversed-phase HPLC. For the
separation of analytes, a C18 column (Wakopak Handy ODS, 4.6
×
150 mm, 5 µm in particle size
and 100 Å in pore size) and an isocratic elution with 0.1% HCOOH flow rate 1 mL min−1 were used.
The HPLC analyses were performed on a Dionex UltiMate 3000 System with a Diode Array Detector
set at 260 nm.

Int. J. Mol. Sci. 2020, 21, 6352
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LC-MS analysis. In order to identify the irradiation products, a Nexera X2 ultra high performance
liquid chromatography (UHPLC) system coupled with a tandem mass spectrometer Triple TOF
5600+ (SCIEX) with a double electrospray interface operating in a negative mode has been
used. Chromatographic separation has been performed on a C18 column (Kinetex Phenomenex,
2.1
×
150 mm2, 2.6 m in particle size and 100 Å in pore size) at 25 ◦C, with an isocratic elution with
µ
0.2% formic acid flow (0.3 mL min−1). The effluent was directed to waste for 1 min after each injection
during analysis. MS parameters were as follows: the nebulizer gas (N2) pressure 30 psi, the spray
voltage −4.5 kV, the flow rate 11 L min−1 and the source temperature 300 ◦C.
3.6. Calculations
A mechanism of a radiodegradation of CF3CONHU was studied computationally at the density
functional theory level, using the M06-2X functional [49] and a 6-31++G (d,p) basis set [50,51].
The aqueous reaction environment was mimicked with the use of a polarization continuum model
(PCM) [52]. All the geometries were fully optimized without any geometrical constraints, and the
analysis of harmonic frequencies demonstrated that all of them were geometrically stable (all force
constants were positive) or first-order transition states (all but one force constant positive). The Gibbs
free energies of particular elementary reactions (∆Gs) were estimated as ∆Es (electronic energy change)
between the product and substrate complexes corrected for zero-point vibration terms, thermal
contributions to energy, the pV term and the entropy term. These terms were calculated in the rigid
rotor-harmonic-oscillator approximation for T = 298 K and p = 1 atm [53]. For the last step, where the
substrate complex is highly unstable and spontaneously transforms into products,
as the difference of the sum of Gibbs free energies of products and the sum of Gibbs free energies of
substrates (each reagent optimized separately). Activation barriers, denoted as G*, were calculated as
∆Gsep was calculated
∆
the difference between the Gibbs free energies of transition state (TS) and the corresponding substrate
complex. Each TS geometry was confirmed with the intrinsic reaction coordinate (IRC) procedure [54].
The adiabatic electron affinity, AEAG, was defined as the difference in Gibbs free energies of the
neutral and anion radical at their corresponding fully relaxed geometries.
All calculations have been carried out with the Gaussian 16 package [55].
3.7. Cytotoxicity
In order to establish the level of cytotoxicity of the tested derivative, a WST-1 assay was performed.
Normal human dermal fibroblasts (HDFa) were grown in a DMEM medium (high concentration of
glucose and sodium puryvate), with antibiotics (streptomycin and penicillin) at a concentration of
100 U mL−1 and 10% FBS. The human prostate cancer cell line (PC3) was grown in a F-12K medium
with 10% FBS and antibiotics (streptomycin and penicillin) at the same concentration. HDFa and PC3
cells were seeded into a 96-well plate in a density of 4
×
103 per well and incubated in 37 ◦C and 5%
CO2 overnight. Subsequently, the medium was replaced with the fresh one and the cells were treated
with 5-(N-trifluoromethylcarboxy)aminouracil at 7 tested concentrations: 0 (for the control), 10−4
10−5, 10−6, 10−7, 10−8 and 10−9 M. Next, treated cells were incubated (in the same conditions) with the
studied compound for 24 and 48 h. After this time, the aqueous solution of WST-1 salt was added in
,
an amount of 10 µL to each well and again incubated for further 4 h. The absorbance was measured at
440 nm. The vitality of control was taken as 100%. Received results were analyzed with the GraphPad
Prism software. The statistical evaluation of the treated samples and untreated control was calculated
using the one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test.
The data were obtained from three independent experiments and each treatment condition assayed in
triplicate. The differences were considered significant at p < 0.05.
3.8. Clonogenic Assay
PC3 cells (human prostate cancer cells obtained from Cell Line Service, Eppelheim, Germany)
were treated with CF3CONHU in a concentration of 10−4 M and plated on 60 mm dishes in a density

Int. J. Mol. Sci. 2020, 21, 6352
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of 106 cells per dish. After 48 h incubation (RPMI medium supplemented with 10% fetal bovine serum
and with antibiotics (streptomycin and penicillin) at a concentration of 100 U mL−1, 37 ◦C, 5% CO2)
the cells were exposed to 0.5, 1, 2 and 4 Gy, with the use of a Cellrad X-ray cabinet (Faxitron X-ray
Corporation, 1.27 Gy min−1, 130 kV, 5.0 mA). After 6 h (the assay was performed in the variant with
delayed plating after treatment to allow for repair processes [56]), the cells were trypsinized and
plated on 100 mm dishes in a density of 800 cells per dish. The cell culture and irradiations have been
performed under normoxic conditions. After 13 days, the formed colonies were fixed with 6.0% (v/v)
glutaraldehyde and 0.5% crystal violet. The stained colonies were counted manually, and the colony
size was evaluated using an inverted fluorescence microscope (Olympus, IX73, Tokyo, Japan). The data
were analyzed, and survival curves were plotted following the linear-quadratic equation using the
GraphPad Prism Software (San Diego, CA, USA). Plating efficiencies and survival fractions are shown
in Table S3 in the Supplementary Materials.
3.9. Histone H2A.X Phosphorylation
To determine the level of DSBs caused by IR, the analysis of histone H2A.X phosphorylation,
which is the marker of such a damage, was performed. PC3 cells were seeded at a density of 0.2
×
106
per plate and incubated for 24 h (37 ◦C, 5% CO2). Next, the cells were treated with CF3CONHU at a
concentration of 10−4 M and again incubated under the same conditions for 48 h. Upon this time, the
plates with cells were irradiated with 0 and 2 Gy doses (1.27 Gy min−1, 130.0 kV, 5.0 mA). The incubation
was extended for another hour (this time was optimized in previous experiments). Subsequently,
the PC3 cells were dissociated with 1 Accutase solution and fixed, permeabilized and, in the last
×
step, stained with anti-phospho-Histone H2A.X, FITC conjugate (the fixation, permeabilization and
staining were performed according to the manufacturer’s protocol, FlowCellectTM Histone H2A.X
Phosphorylation Assay Kit, Merck, Hayward, CA, USA). The prepared cells were cytometrically
analyzed (Guava easyCyteTM 12, Merck, Kenilworth, NJ, USA). Nontreated cultures were the controls.
4. Conclusions
In this paper, we demonstrate that stationary radiolysis leads to the efficient radiochemical
transformation of CF3CONHU to N-uracil-5-yloxamic acid and that CF3CONHU sensitizes prostate
cancer cells (PC3 line) to X-rays.
The title compound belongs to the group of modified nucleobases, which undergoes DEA induced
by solvated electrons. Synthesized and HPLC-purified CF3CONHU was dissolved in water and
irradiated with a dose of 140 Gy of ionizing radiation, and the product analysis was carried out by
mass spectrometry. It turned out that the attachment of a solvated electron to CF3CONHU leads
to the generation of N-uracil-5-yloxamic acid. The decay of CF3CONHU (26.67
±
0.31%), induced
by ionizing radiation, was compared to the decomposition of BrdU (19.31
experimental conditions.
±
0.86%) under the same
The mechanism of degradation of the studied reaction has been proposed using quantum chemical
calculations carried out at the M06-2X/6-31++G (d,p) level. We demonstrated that the formation of
N-uracil-5-yloxamic acid from CF3CONHU is allowed due to a barrier-less attack of a hydroxyl anion
on the intermediate [CF2CONHU]• radical. The rate constants for elementary reactions calculated
using the M06-2X/6-31++G (d,p) activation barriers lead to a reaction completion time of the order of
hours, which remains in accordance with the experiment.
A very low in vitro cytotoxicity of CF3CONHU was evaluated by the WST-1 assay. Finally,
a clonogenic assay showed that the enhancement factor representing the radiosensitizing effect is equal
to 1.52, and the cytometric analysis of histone H2A.X phosphorylation showed that the radiosensitizing
effect of CF3CONHU is associated, at least in part, with the formation of double-strand breaks. Thus,
one can reach the conclusion that CF3CONHU can act as a DNA radiosensitizer.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/17/
6352/s1.

Int. J. Mol. Sci. 2020, 21, 6352
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Author Contributions: Conceptualization, J.R., L.C.-M., S.M. and M.Z.; investigation, P.S., W.K.; L.C.-M., S.M.,
K.F., A.S., A.C., J.R., M.Z.; writing original draft preparation, L.C.-M., P.S., W.K., A.S., J.R., M.Z.; writing review
and editing, P.S., J.R., M.Z.; supervision, J.R., M.Z.; funding acquisition, J.R. All authors have read and agreed to
the published version of the manuscript.
Funding: This research was funded by the Polish National Science Center (NCN) under the grant no.
UMO-2014/14/A/ST4/00405 (J.R.).
Acknowledgments: Calculations have been carried out in the Wroclaw Center for Networking and
Supercomputing (wcss.wroc.pl), grant no. 209, and at a local cluster.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
Abbreviations
CF3CONHU
IR
5-(N-trifluoromethylcarboxy) aminouracil
Ionizing Radiation
DEA
AEA
Dissociative Electron Attachment
Adiabatic Electron Affinity
Polarization Continuum Model
Prostate Cancer Cell Line
Human Dermal Fibroblasts
5-bromo-2’-deoxyuridine
Fetal Bovine Serum
PCM
PC3
HDFa
BrdU
FBS
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