Inhibition of tyrosyl-DNA phosphodiesterase 1 by lipophilic pyrimidine nucleosides

Article abstract of DOI:10.3390/molecules25163694

Inhibition of DNA repair enzymes tyrosyl-DNA phosphodiesterase 1 and poly(ADP-ribose) polymerases 1 and 2 in the presence of pyrimidine nucleoside derivatives was studied here. New effective Tdp1 inhibitors were found in a series of nucleoside derivatives possessing 2′,3′,5′-tri-O-benzoyl-d-ribofuranose and 5-substituted uracil moieties and have half-maximal inhibitory concentrations (IC50) in the lower micromolar and submicromolar range. 2′,3′,5′-Tri-Obenzoyl- 5-iodouridinemanifested the strongest inhibitory effect on Tdp1 (IC50 = 0.6 μM). Adecrease in the number of benzoic acid residues led to a marked decline in the inhibitory activity, and pyrimidine nucleosides lacking lipophilic groups (uridine, 5-fluorouridine, 5-chlorouridine, 5-bromouridine, 5-iodouridine, and ribothymidine) did not cause noticeable inhibition of Tdp1 (IC50 > 50 μM). No PARP1/2 inhibitors were found among the studied compounds (residual activity in the presence of 1mMsubstances was 50-100%). Several O-benzoylated uridine and cytidine derivatives strengthened the action of topotecan on HeLa cervical cancer cells.

Full text of DOI:10.3390/molecules25163694

molecules
Article
Inhibition of Tyrosyl-DNA Phosphodiesterase 1 by
Lipophilic Pyrimidine Nucleosides
Alexandra L. Zakharenko 1,†, Mikhail S. Drenichev 2,† , Nadezhda S. Dyrkheeva 1,†
,
Georgy A. Ivanov 2, Vladimir E. Oslovsky 2 , Ekaterina S. Ilina 1, Irina A. Chernyshova 1,
Olga I. Lavrik 1,3,
*
and Sergey N. Mikhailov 2,
*
1
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of
Sciences, 8 Lavrentiev Ave., 630090 Novosibirsk, Russia; a.zakharenko73@gmail.com (A.L.Z.);
elpida80@mail.ru (N.S.D.); katya.plekhanova@gmail.com (E.S.I.); chernyshova0305@gmail.com (I.A.C.)
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilova Str., 119991 Moscow,
Russia; mdrenichev@mail.ru (M.S.D.); georgyivanovk423@gmail.com (G.A.I.);
vladimiroslovsky@gmail.com (V.E.O.)
Department of Natural Sciences, Novosibirsk State University, 2 Pirogova Str., 630090 Novosibirsk, Russia
Correspondence: lavrik@niboch.nsc.ru (O.I.L.); smikh@eimb.ru (S.N.M.);
Tel.: +7(383)363-5195 (O.I.L.); +8(499)135-9733 (S.N.M.)
2
3
*
†
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Katherine Seley-Radtke and Luigi A. Agrofoglio
Received: 19 May 2020; Accepted: 9 August 2020; Published: 13 August 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Inhibition of DNA repair enzymes tyrosyl-DNA phosphodiesterase 1 and poly(ADP-ribose)
polymerases 1 and 2 in the presence of pyrimidine nucleoside derivatives was studied here.
New effective Tdp1 inhibitors were found in a series of nucleoside derivatives possessing
20,30,50-tri-O-benzoyl-d-ribofuranose and 5-substituted uracil moieties and have half-maximal
inhibitory concentrations (IC50) in the lower micromolar and submicromolar range. 20,30,50-Tri-O-
benzoyl-5-iodouridine manifested the strongest inhibitory effect on Tdp1 (IC50 = 0.6 µM). A decrease in
the number of benzoic acid residues led to a marked decline in the inhibitory activity, and pyrimidine
nucleosides lacking lipophilic groups (uridine, 5-fluorouridine, 5-chlorouridine, 5-bromouridine,
5-iodouridine, and ribothymidine) did not cause noticeable inhibition of Tdp1 (IC50 > 50 µM).
No PARP1/2 inhibitors were found among the studied compounds (residual activity in the presence of
1 mM substances was 50–100%). Several O-benzoylated uridine and cytidine derivatives strengthened
the action of topotecan on HeLa cervical cancer cells.
Keywords: nucleosides; DNA repair; tyrosyl-DNA phosphodiesterase; Tdp1 inhibition; topotecan
1. Introduction
DNA repair systems are resistant to the action of chemotherapeutic drugs and ionizing radiation,
and therefore the beneficial effect of antitumor therapy depends on the effectiveness of DNA repair
systems in tumor cells. Accordingly, targeted inhibition of DNA repair enzymes can increase the
effectiveness of chemotherapeutic drugs used in clinical practice and may enable reducing their dose,
which in turn can decrease the overall toxicity of treatment [
1–8]. A number of enzymes and protein
factors are involved in DNA repair [ 15]. Poly(ADP-ribose)polymerase 1 (PARP1, EC 2.4.2.30)—a key
9
–
regulator of DNA repair mechanisms—is the most studied pharmacological target for the creation of
targeted drugs [16]. Four PARP1 inhibitors (olaparib, rucaparib, niraparib, and talazoparib) are used
in clinical practice for the treatment of ovarian cancer.
Among promising targets is the enzyme tyrosyl-DNA phosphodiesterase 1 (Tdp1), which is
considered an important target for the antitumor therapy based on topoisomerase 1 (Top1) inhibitors [17].
Molecules 2020, 25, 3694; doi:10.3390/molecules25163694
www.mdpi.com/journal/molecules

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Tdp1 plays a key role in the removal of Top1–DNA adducts stabilized by Top1 inhibitors such as
camptothecin and its clinically relevant derivatives. The catalytic action of Tdp1 is the hydrolysis of
the phosphotyrosyl bond between the 30 end of DNA and a tyrosine residue of Top1 in a stabilized
Top1–DNA complex, with hydrophobic interactions playing an important part in the binding of Tdp1
to the Top1–DNA complex [18]. The literature describes Tdp1 inhibitors of various structures based
on different classes of natural and synthetic compounds [3,4]; these inhibitors have half-maximal
inhibitory concentrations (IC50) in the range 0.015–10.000 M. Recently, a novel group of Tdp1 inhibitors
µ
effective in the submicromolar range of concentrations was found among disaccharide nucleosides
with lipophilic groups [19]. The current work represents a continuation of our earlier studies on the
inhibition of DNA repair enzymes by nucleoside compounds [19–24].
2. Results
2.1. DNA Repair Enzyme Inhibition
To study the inhibition of the DNA repair enzyme by nucleoside derivatives, a number of
5-substituted derivatives of 20,30,50-tri-O-benzoyluridine were synthesized according to the procedures
elaborated earlier by Vorbrüggen and coworkers [25] and by Prasad and coworkers [26] (Scheme S1,
Supplementary Material).
Previously, we designed a real-time oligonucleotide biosensor based on the capability of Tdp1
to remove fluorophore quenchers from the 30 end of DNA [27]. The single-stranded substrate was
a 16-mer oligonucleotide containing both a 50-FAM fluorophore donor and a quenching 30-BHQ
moiety. When the quencher is removed by Tdp1, FAM fluorescence flares up and can be detected by a
fluorimeter. Fluorescence intensity diminishes in the presence of an inhibitor; curves of the obtained
Tdp1 activity versus inhibitor concentration can be used to calculate IC50 values. The results of the Tdp1
assay for nucleoside derivatives
related compounds obtained by previously developed methods [25
modification either in a carbohydrate or heterocyclic moiety (Table 1).
1
and
2
are shown in Table 1. We also investigated some structurally
,
26,28]; these compounds feature a
Table 1. Inhibition of Tdp-1 by nucleoside derivatives.
Cmpd
1a
Structure
LogP 1
IC50 µM
HeLa CC50 µM
ND 2
−2.28
>50
1b
1f
−2.64
−2.24
>50
>50
ND
ND

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Table 1. Cont.
Cmpd
Structure
LogP 1
IC50 µM
HeLa CC50 µM
1c
1d
1e
2a
2b
2f
−1.97
−1.44
−1.39
5.07
>50
ND
>50
ND
ND
>50
6.3 ± 0.4
8.5 ± 1.4
3.6 ± 1.1
1.5 ± 0.9
ND
5.27
>100
>100
>100
5.73
2c
5.90

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Table 1. Cont.
Cmpd
Structure
LogP 1
IC50 µM
0.6 ± 0.9
HeLa CC50 µM
2d
6.06
>100
2e
2g
3a
4a
5.47
5.27
0.08
2.58
2.7 ± 0.6
3.4 ± 0.2
>100
>100
>100
ND
23 ± 6
>100
5
6
5.07
5.28
18 ± 1
ND
6.0 ± 0.7
>100

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Table 1. Cont.
Cmpd
Structure
LogP 1
IC50 µM
HeLa CC50 µM
2.91 ±
7
5.55
>100
0.01
8
9
4.69
5.62
4.3 ± 0.7
4.2 ± 0.3
>100
ND
1 The values of the partition coefficient of the compounds between the 1-octanol-water phases (logP) were calculated
2
using the Instant J. Chem. (ChemAxon®). ND—not determined.
In addition to the effects on Tdp1, we tested the effects of the lipophilic nucleosides on DNA
break sensors poly(ADP-ribose)polymerases 1 and 2 (PARP1 and PARP2), in a test system based on
tritium-labeled NAD+ [20]. None of the tested nucleosides caused significant inhibition of either
PARP1 or PARP2 (residual activity in the presence of 1 mM substances was 50–100%, data not shown).
2.2. Cytotoxicity and Sensitization of Tumor Cells to the Effect of Topotecan
2.2.1. Cytotoxicity
We evaluated the cytotoxicity of compounds that showed a Tdp1-inhibitory activity as well as
their impact on the cytotoxic effect of topotecan. First, we assessed the toxicity of the compounds
to HeLa cells (cervical cancer cells). Analysis of the intrinsic cytotoxicity of the compounds was
examined in the EZ4U Cell Proliferation and Cytotoxicity Assay (Biomedica, Austria). The tested
compounds were added to the medium (the volume of added reagents was 1/100 of the total volume
of the culture medium, and the amount of DMSO (dimethyl sulfoxide) was 1% of the final volume),
and the cell culture was monitored for 3 days. Control cells were grown in the presence of 1% DMSO.
Intrinsic cytotoxicity of the compounds was either absent or negligible at concentrations up to 100
µM (>80% live cells) with the exception of compounds 2b, 2c, and 7, in the presence of which, at a
concentration of 100 µM, 30–45% of the cells died (Table 1 and Figure 1).
2.2.2. Tumor Cell Sensitization to Topotecan
To study the influence of the inhibitors on the cytotoxic effect of topotecan, the latter from the
manufacturer ACTAVIS GROUP PTC ehf. was used. To select a nontoxic but effective concentration
of the nucleoside compounds, we investigated their cytotoxicity in the presence of 2 µM topotecan
(concentration causing the death of half the HeLa cells). Three independent cytotoxicity assays were
performed with each inhibitor in combination with topotecan. For the cells treated with a nucleoside
derivative alone, values in the control wells treated with 1% DMSO were set to 100% viability. For the
cells treated with a combination of the drugs, the viability of cells treated only with topotecan was set to
100%. Topotecan significantly (p = 0.05, Mann–Whitney U test) enhanced the cytotoxicity of compounds

Molecules 2020, 25, 3694
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2g
,
7, and
8
(Figure 2a). For compound
5
, the increase in cytotoxicity in the presence of topotecan was
significant only at the maximum concentration, 100 µM. For compound 6, the increase in cytotoxicity
in the presence of topotecan (Figure 2a, cyan bars) was statistically insignificant. The cytotoxicity of
the remaining compounds in the presence of topotecan did not change significantly (Figure 2b).
120
100
5
2d
6
80
7
2f
60
2e
2g
2c
40
2b
2a
8
20
0
0
20
40
60
80
100
Tdp1 inhibitors concentration, M
Figure 1. Dose-dependent action of the lipophilic pyrimidine nucleosides on HeLa cell viability
measured in the EZ4U assay.
(a)
(b)
Figure 2. Dose-dependent action of the lipophilic nucleosides in combination with topotecan (Tpc)
on HeLa cell viability in the EZ4U assay. The unshaded histogram bars denote cell viability in the
presence of a single Tdp1 inhibitor. The hatched histogram bars indicate cell viability in the presence
of a combination of a Tdp1 inhibitor with 2
topotecan. The colors indicate: orange–compound
) Inactive compounds. The colors indicate: cyan—compound 2f, yellow—2e, gray—2d, magenta—2a,
µM topotecan. (
a) Compounds showing synergy with
7
, magenta—2g, cyan—
6
, gray— , and yellow—5.
8
(b
orange—2b, and green—2c. Each of the four bars corresponds to an inhibitor concentration (from left
to right) of 1, 5, 20, and 100 µM.

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For the three compounds 2g
concentration, 20
,
7
, and
8
that had a synergistic effect with topotecan, a nontoxic
µ
M, which exerted a noticeable synergistic effect, was chosen for further experiments
on HeLa cells at various doses of topotecan (Figure 3).
140
120
100
80
60
40
20
0
Tpc
Tpc + 7
Tpc + 2g
Tpc + 8
Tpc + 5
0
2
4
6
8
10
Topotecan, M
Figure 3. Dose-dependent action of topotecan (Tpc) in combination with one of the nucleoside
derivatives on HeLa cell viability in the EZ4U assay.
For
5
, a low cytotoxic concentration, 50
µ
M, was chosen because at a concentration of 20
µ
M,
had a weak sensitizing
M; Figure 3, the orange
no synergy with topotecan was observed (Figure 2a, yellow bars). Compound
effect on topotecan-treated cells at high concentrations (a significant effect at 10
5
µ
graph compared to the black one). Compounds 2g, 7, and 8 doubled the cytotoxicity of topotecan.
3. Discussion
It has been shown earlier that disaccharide nucleosides with lipophilic groups inhibit Tdp1 in a
submicromolar range of concentrations and are only weakly toxic to cancerous and healthy cells [19].
On the other hand, disaccharide nucleosides tend to inhibit PARP1 [20,22], and this activity may reduce
their selectivity toward Tdp1 in the cell. Therefore, we focused on nucleoside inhibitors of Tdp1,
which are structurally modified previously described disaccharide inhibitors of Tdp1. New efficient
Tdp1 inhibitors were found in a series of ribofuranose nucleoside derivatives and have IC50 values in the
low micromolar and submicromolar range (Table 1). It was demonstrated here that for the manifestation
of an inhibitory effect, the presence of the 2,3,5-tri-O-benzoyl-β-d-ribofuranose (2,3,5-tri-O-Bz-β-d-Rib)
residue is necessary. Pyrimidine nucleosides (uridine, 5-fluorouridine, 5-chlorouridine, 5-bromouridine,
5-iodouridine, and ribothymidine) without lipophilic groups did not inhibit Tdp1 (IC50 > 50 µM).
According to the calculations of the coefficient of distribution (logP) between the 1-octanol-water
phases, the introduction of benzoyl groups (Bz) into pyrimidine nucleosides led to an increase in
logP and therefore could improve their penetration of cellular and nuclear membranes (Table 1).
The magnitude of Tdp1 inhibition by the modified uridine nucleosides linearly correlated with the
increase in logP (Figure 4 and Supplementary Materials S18).
The presence of the bulky 2,3,5-tri-O-Bz-
activity (IC50 = 6.3
M for 20,30,50-tri-O-benzoyluridine 2a and 0.6
5-iodouridine 2d) in comparison with nucleoside derivatives lacking benzoyl groups (Table 1).
1-O-acetyl-2,3,5-tri-O-Bz- -d-Rib ( ) exerted a Tdp1-inhibitory activity similar to that of O-benzoylated
cytidine ( ) derivatives and a higher Tdp1-inhibitory activity than that of O-benzoylated uridine (2a),
5-fluorouridine (2b), and pyrimidone ( and ) derivatives (Table 1). Most of these compounds were
found to be characterized by lower logP than that of ribofuranose derivative (Table 1, logP = 5.62).
O-benzoylated uridine derivatives with logP comparable to or higher than 5.62 manifested a higher
β
-d-Rib residue significantly enhanced the inhibitory
µ
µ
M for 20,30,50-tri-O-benzoyl-
β
9
8
5
6
9

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Tdp1-inhibitory activity in comparison with a ribofuranose derivative (
9). A decrease in the number of
benzoic acid residues caused a marked decline in the inhibitory activity (Figure 5, IC50 = 23
20,30-di-O-benzoyluridine 4a and IC50 > 100 µM for 50-O-benzoyluridine 3a).
µM for
7
6.06
5.90
5.73
6
5.62
5.55
5.47
5.28
5.27
5.27
5
4
3
2
1
0
5.07
y = -0.1479x + 6.0978
R² = 0.9503
2.58
0
5
10
15
20
25
IC50 10-6M
Figure 4. Correlation diagram between Tdp-1 inhibition IC50 and logP of nucleoside derivatives for
compounds 2a–2g, 4a, 6–7 and 9 with exclusion of the data for compounds 5, 8.
Figure 5. Structure-activity relationship between Tdp-1 inhibition and the quantity of benzoyl groups
in nucleoside ribofuranose moiety.

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Thus, it can be claimed that lipophilicity and the number of benzoyl groups in the modified
nucleoside compounds strongly influence Tdp1 inhibition.
It has been previously reported that disaccharide nucleosides [20
,22] and nucleoside derivatives
with modified nitrogen bases [29 30] inhibit PARP1. Therefore, in addition to the effect on Tdp1,
,
a possible effect of the lipophilic nucleosides on DNA break sensors PARP1 and PARP2 was tested.
Our compounds had virtually no impact on the activity of either enzyme (residual activity in the
presence of each 1 mM substance was 50–100%), and therefore may be employed for the development
of selective nucleoside inhibitors of Tdp1.
Several papers have been published in the past decade on the applicability of Tdp1 inhibitors for
the sensitization of tumors to the action of anticancer topoisomerase 1 inhibitors (e.g., topotecan) see
reviews [3,4,17]. Therefore, on HeLa cells, we also studied the effect of the synthesized Tdp1 inhibitors
in combination with topotecan, which is the Top1 poison used in clinical practice. Given that Tdp1
inhibitors are supposed to be used in therapeutic cocktails, it is important that they do not exert their own
toxicity and do not enhance the existing adverse effects of cancer therapy. According to the experiments
on the viability of HeLa cells in the presence of the synthesized pyrimidine lipophilic nucleosides,
the intrinsic cytotoxicity of the compounds was either absent or negligible at concentrations up to
100
was then studied. Four nucleoside compounds (2g
M topotecan solution (Figure 2a), while the cytotoxicity of topotecan in the presence of the other
compounds did not significantly change (Figure 2b). It was shown that compounds (containing a
6-methoxy-2-oxopyrimidine residue), 2g (containing a 6-methyluracil residue), and cytosine derivative
at a nontoxic concentration, 20 M, have a sensitizing effect on topotecan-treated cells, thereby doubling
topotecan’s activity toward HeLa cells (Figure 3). These results suggest that nucleoside compounds
containing a 2,3,5-tri-O-Bz- -d-Rib group and a uracil base modified at positions 4 or 6 can penetrate
µM (Figure 1). The enhanced cytotoxicity of topotecan in the presence of the synthesized compounds
,
5,
7
, and ) each enhanced the cytotoxicity of a
8
2
µ
7
8
µ
β
into a cell nucleus and accumulate in it.
Tdp1 was discovered because of its ability to hydrolyze the stalled covalent 30-phospho-tyrosyl
that represents the chemical linkage between the active-site tyrosine of DNA topoisomerase I (Topo1)
and DNA [31
such as camptothecin-stabilized Topo1–DNA adducts, and modified nucleotides, including oxidized
nucleotides, apurinic/apyrimidinic sites, and chain-terminating nucleoside analogs reviewed in [17
and described in [18 34]. Because many substrates of Tdp1 arise when anticancer and antiretroviral
–33]. The list of substrates of Tdp1 has since grown and consists of protein–DNA adducts,
]
,
drugs are used, this enzyme is becoming an important therapeutic target. In addition, a natural
catalytic-site Tdp1 mutant increases the stability of a Tdp1–DNA reaction intermediate and forms the
molecular basis for an autosomal recessive neurodegenerative disease called spinocerebellar ataxia
with axonal neuropathy (SCAN1).
A number of studies confirmed the hypothesis that Tdp1 is responsible for the resistance
of some types of cancer to these drugs [35]: Tdp1-deficient human cells are hypersensitive to
camptothecin [36–38]. Furthermore, recent data indicate that suppression of Tdp1 expression by
minocycline enhances the antimetastatic effect of irinotecan and increases the life span of experimental
animals [39]. Conversely, in cells with increased Tdp1 expression, camptothecin and etoposide cause
less DNA damage [40,41]. Moreover, the response to irinotecan therapy is lower in tumors of the
intestine with overexpression of Tdp1 [42]. Thus, it is expected that a combination of anticancer drugs
and Tdp1 inhibitors can significantly increase the effectiveness of chemotherapy.
4. Materials and Methods
4.1. General
The solvents and materials were reagent grade and were used without additional purification.
Column chromatography was performed on silica gel (Kieselgel 60, 0.040–0.063 mm, Merck, Darmstadt,
Germany. Thin-layer chromatography was carried out on Alugram SIL G/UV254 (Macherey-Nagel,

Molecules 2020, 25, 3694
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Düren, Germany) with UV visualization. Melting points were determined by means of Electrothermal
1
Melting Point Apparatus IA6301 and are uncorrected. H and 13C (with complete proton decoupling)
NMR spectra were recorded on a Bruker AMX 400 NMR instrument (Bruker, Billerica, MA, USA).
1H-NMR-spectra were acquired at 400 MHz, and 13C-NMR spectra at 100 MHz. Chemical shifts in
parts per million (ppm) were measured relative to the residual solvent signals as internal standards
(CDCl3, 1H: 7.26 ppm, 13C: 77.1 ppm; DMSO-d6, 1H: 2.50 ppm, and 13C: 39.5 ppm). Spin–spin coupling
constants (J) are given in Hertz (Hz). The following compounds were prepared according to the methods
reported earlier: compounds 2a
pyrimidine-2-one, 2-oxo-4-methoxypyrimidine (
–
e
[
26], 2f
[
43], compound 2g 44], and derivatives of pyrimidine-4-one,
[
5–7) [25,44,
45], and of 20,30,50-tri-O-benzoylcytidine
(8) [26]. The compounds’ coefficients of partition between the octanol–water phases (logP) were
calculated in the Instant J. Chem. (ChemAxon®) software (Budapest, Hungary).
4.2. Real-Time Detection of Tdp1 Activity
This procedure was reported in our previous work [27]. The approach consists of fluorescence
intensity measurement in a reaction of quencher removal from a fluorophore quencher–coupled DNA
oligonucleotide catalyzed by Tdp1 in the presence of an inhibitor (the control samples contained 1% of
DMSO). The reaction mixtures (200
µL) contained Tdp1 buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl,
and 7 mM -mercaptoethanol), 50 nM biosensor, an inhibitor being tested, and purified Tdp1 (1.5 nM).
β
The biosensor was a single-stranded oligonucleotide (50-[FAM] AAC GTC AGGGTC TTC C [BHQ]-30)
containing a fluorophore at the 50 end (5,6-FAM) and Black Hole Quencher 1 (BHQ) at the 30 end and
was synthesized in the Laboratory of Biomedical Chemistry at the Institute of Chemical Biology and
Fundamental Medicine (Novosibirsk, Russia).
◦
The reactions were incubated at a constant temperature of 26 C on a POLARstar OPTIMA
fluorimeter (BMG LABTECH, GmbH, Ortenberg, Germany) to measure fluorescence every 55 s
(ex. 485/em. 520 nm) during the linear phase (here, data from minute 0 to minute 8). The values of IC50
were determined using a six-point concentration response curve in three independent experiments
and were calculated using MARS Data Analysis 2.0 (BMG LABTECH).
4.3. Cell Culture Assays
Intrinsic toxicity of the compounds to human cell line HeLa (cervical cancer) was examined by
means of the EZ4U Cell Proliferation and Cytotoxicity Assay (Biomedica, Wien, Austria), according to
the manufacturer’s protocols. The cells were grown in Iscove’s modified Dulbecco’s medium (IMDM)
with 40 µg/mL gentamicin, 50 IU/mL penicillin, 50 µg/mL streptomycin (MP Biomedicals), and 10% of
fetal bovine serum (Biolot) in a 5% CO2 atmosphere. After formation of a 30–50%-confluent monolayer,
one of the tested compounds was added to the medium (the volume of the added reagents was 1/100 of
the total volume of the culture medium, and the amount of DMSO was 1% of the final volume), and the
cell culture was monitored for 3 days. Control cells were grown in the presence of 1% DMSO. To assess
the influence of the inhibitors on the cytotoxic effect of topotecan, the latter from the manufacturer
ACTAVIS GROUP PTC ehf. (Bucharest, Romania) was used. First of all, 50% cytotoxic concentrations
of topotecan and of each inhibitor were determined to attain a defined single-agent effect. Second,
three independent tests were performed with each inhibitor in combination with topotecan.
5. Conclusions
We studied the inhibition of DNA repair enzymes (PARP1, PARP2, and Tdp1) by lipophilic
pyrimidine nucleosides. New effective Tdp1 inhibitors were found in a series of lipophilic pyrimidine
nucleosides and have IC50 in the lower micromolar and submicromolar range. The 2,3,5-tri-O-Bz-β-d-Rib
moiety appears to be the pharmacophore that is essential for Tdp1 inhibition by each synthesized
compound. It was shown that the lipophilic pyrimidine nucleosides inhibiting Tdp1 in vitro have only
low cytotoxicity. Some derivatives strengthen the action of topotecan on HeLa cells and thus may be

Molecules 2020, 25, 3694
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considered promising for further optimization of the structure and the development of compounds
sensitizing cancer cells to topotecan.
6. Patents
The application for RU patent №2019141448 was registered 13.12.2019.
Supplementary Materials: The following are available online, Figure S1: 1H-NMR-spectrum (400 MHz)
of 20,30,50-tri-O-benzoyluridine in DMSO-d6 at 298 K, Figure S2: 13C-NMR-spectrum (400 MHz)
of 20,30,50-tri-O-benzoyluridine in DMSO-d6 at 298 K, Figure S3: 1H-NMR-spectrum (400 MHz) of
5-fluoro-20,30,50-tri-O-benzoyluridine in DMSO-d6 at 298 K, Figure S4: 13C-NMR-spectrum (100 MHz) of
5-fluoro-20,30,50-tri-O-benzoyluridine in DMSO-d6 at 298 K, Figure S5: 1H-NMR-spectrum (400 MHz) of
5-chloro-20,30,50-tri-O-benzoyluridine in DMSO-d6 at 298 K, Figure S6: 13C-NMR-spectrum (100 MHz) of
5-chloro-20,30,50-tri-O-benzoyluridine in DMSO-d6 at 298 K, Figure S7: 1H-NMR-spectrum (400 MHz) of
5-bromo-20,30,50-tri-O-benzoyluridine in DMSO-d6 at 298 K, Figure S8: 13C-NMR-spectrum (100 MHz)
of 5-bromo-20,30,50-tri-O-benzoyluridine in DMSO-d6 at 298 K, Figure S9: 1H-NMR-spectrum (400 MHz)
of 5-iodo-20,30,50-tri-O-benzoyluridine in DMSO-d6 at 298 K, Figure S10: 13C-NMR-spectrum (100 MHz)
of 5-iodo-20,30,50-tri-O-benzoyluridine in CDCl3 at 298 K, Figure S11: 1H-NMR-spectrum (400 MHz)
of 20,30,50-tri-O-benzoylribotimidine in DMSO-d6 at 298 K, Figure S12: 13C-NMR-spectrum (100 MHz)
of 20,30,50-tri-O-benzoylribotimidine in DMSO-d6 at 298 K, Figure S13: 1H-NMR-spectrum (400 MHz)
of 20,30,50-tri-O-benzoylcytidine in DMSO-d6 at 298 K, Figure S14: 13C-NMR-spectrum (100 MHz) of
20,30,50-tri-O-benzoylcytidine in DMSO-d6 at 298 K, Table S1: Inhibition of Tdp-1 by nucleoside derivatives,
Figure S15: Dose-dependent action of the lipophilic pyrimidine nucleosides on HeLa cell viability measured in the
EZ4U assay, Figure S16: Dose-dependent action of the lipophilic nucleosides in combination with topotecan (Tpc)
on HeLa cell viability in the EZ4U assay, Figure S17: Dose-dependent action of topotecan (Tpc) in combination
with one of the nucleoside derivatives on HeLa cell viability in the EZ4U assay, Figure S18: Correlation diagram
between Tdp-1 inhibition IC50 and logP of nucleoside derivatives for compounds 2a–2g, 4a, 5–9, Figure S19:
Structure-activity relationship between Tdp-1 inhibition and the quantity of benzoyl groups in nucleoside
ribofuranose moiety.
Author Contributions: Conceptualization, S.N.M. and O.I.L.; methodology, A.L.Z., N.S.D. and M.S.D.;
investigation, A.L.Z., M.S.D., N.S.D., G.A.I., E.S.I., I.A.C.; data analysis, A.L.Z., M.S.D., N.S.D., V.E.O.;
writing—original draft preparation, A.L.Z., M.S.D., V.E.O.; writing—review and editing, S.N.M. and O.I.L.;
supervision, S.N.M.; project administration, S.N.M.; funding acquisition, S.N.M. All authors have read and agreed
to the published version of the manuscript.
Funding: This research was funded by RUSSIAN FOUNDATION FOR BASIC RESEARCH (RFBR),
grant number 18-29-09037.
Acknowledgments: We thank Juliana Raikova, a student of Moscow Institute of Fine Chemical Technology
(MITHT), for assistance in chemical synthesis of nucleoside compounds.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
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Multidisciplinary Digital Publishing Institute
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Sample Availability: Samples of the compounds 1–9 are available from the authors.
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