New 5-Hydroxycoumarin-Based Tyrosyl-DNA Phosphodiesterase I Inhibitors Sensitize Tumor Cell Line to Topotecan

Tyrosyl-DNA-phosphodiesterase 1 (TDP1) is an important enzyme in the DNA repair system. The ability of the enzyme to repair DNA damage induced by a topoisomerase 1 poison such as the anticancer drug topotecan makes TDP1 a promising target for complex antitumor therapy. In this work, a set of new 5-hydroxycoumarin derivatives containing monoterpene moieties was synthesized. It was shown that most of the conjugates synthesized demonstrated high inhibitory properties against TDP1 with an IC50 in low micromolar or nanomolar ranges. Geraniol derivative 33a was the most potent inhibitor with IC50 130 nM. Docking the ligands to TDP1 predicted a good fit with the catalytic pocket blocking access to it. The conjugates used in non-toxic concentration increased cytotoxicity of topotecan against HeLa cancer cell line but not against conditionally normal HEK 293A cells. Thus, a new structural series of TDP1 inhibitors, which are able to sensitize cancer cells to the topotecan cytotoxic effect has been discovered.


Introduction
Tyrosyl-DNA phosphodiesterase 1 (TDP1) is an important target in cancer therapy because it repairs damaged 3 ends of DNA caused by various drugs used commonly in cancer chemotherapy [1]. These damages include the covalent adduct of topoisomerase 1 (Top1) and DNA, so-called Top1cc (Top1/DNA cleavage complex), which is formed during the normal catalytic action of Top1 and is stabilized by the antitumor drugs topotecan and irinotecan [2]. Such a "stuck" complex leads to the formation of double-strand breaks and cell death. TDP1 eliminates Top1cc, thereby interfering with the action of clinically used drugs and being one of the causes of resistance to them [3,4].
Thus, TDP1 inhibitors could increase the efficacy of therapy with Top1 inhibitors such as topotecan and irinotecan and/or reduce the dose, and hence toxicity, of the latter. Indeed, there is a large amount of experimental evidence for this: cells or animals deficient in TDP1 are more sensitive to Top1 inhibitors [5][6][7][8][9]. Conversely, increased expression of TDP1 protects tumor cells from these drugs [10][11][12][13][14]. In addition, our team demonstrated the effectiveness of TDP1 inhibitors as sensitizers of the action of topotecan against a number of mouse tumors in vivo. These compounds belong to usnic acid derivatives (Figure 1,  1 and 2) [15][16][17] and nucleosides (Figure 1, 3) [18]. Nevertheless, no TDP1 inhibitors are in clinical trials now, making the search for new inhibitors of this enzyme important. Thus, several new structural types of TDP1 inhibitors were developed recently [19][20][21]. A number of good TDP1 inhibitors were developed using natural compounds [22] including monoterpenes. For example, compounds 4-6 ( Figure 1) were synthesized by reactions of monoterpenes with aldehydes [23,24]. In contrast, inhibitors 7-10 ( Figure 1) contain monoterpene fragments without significant modification of their core [25][26][27][28].
Previously, we have shown that 7-hydroxycoumarin derivatives with monoterpene moieties 11 and 12 ( Figure 2) inhibit TDP1 in the submicromolar concentration range and sensitize the action of camptothecin (a Top1 inhibitor of natural origin) [29] and topotecan on the Krebs-2 ascites in vivo tumor model [30]. Of note here is that many coumarins demonstrated diverse biological activity [31][32][33]. Although it is known that some coumarins are toxic and can induce hepatotoxicity, for example [34], usually they are safe enough for pharmacological application [35]. Based on molecular modeling, it was suggested that analogues of these compounds containing an oxyterpene residue at position 5 of coumarin and having an aryl fragment at position 3 of the model structure 13 (Figure 2) could have high inhibitory activity. In order to verify this assumption experimentally, novel monoterpene esters of 5-hydroxycoumarins containing aromatic substituents in the 3-position of the pyran ring were synthesized. Substituted 3-arylylcoumarins has various pharmacological applications and could be considered as a privileged scaffold in medicinal chemistry [36]. It has been shown that these compounds are indeed capable of effectively inhibiting TDP1, as well as sensitizing tumor cell lines to the action of topotecan.

Chemistry
Target compounds were synthesized by condensation of substituted phenylacetic acids with 2,6-dihydroxyacetophenone, followed by removal of the acetyl group and subsequent reaction with monoterpene bromides.

Biology
To determine the inhibitory properties of the new compounds against TDP1 we used a previously designed [41] real-time oligonucleotide biosensor with 5(6)-carboxyfluorescein (FAM) at the 5 end and fluorophore quencher BHQ1 (Black Hole Quencher-1) at the 3 -end.
As can be seen from Table 1, all geraniol derivatives 33a-38a demonstrated good inhibitory activity against TDP1 with IC 50 values in low micromolar or submicromolar ranges. The most active compound with IC 50 130 nM contains a fluorine substituent in the 4-position of the pendant aromatic ring. Methoxy substituted derivatives were slightly less active than their halogen containing counterparts. The introduction of a nitro group did not lead to significant changes in inhibitory activity.
Compound 33b containing a fully saturated monoterpenoid fragment was twofold less active than its analogue 33a. It is interesting that in this case the 4-methoxy substituted compound also demonstrated high inhibitory activity.
Among the myrtenol-coumarin conjugates, the most active were with the derivative bearing 4-bromo (34c) and 2-methoxy (37c) substituents on the pendant aryl ring. Elongation of the linker when passing from conjugate 37c to conjugate 37d led to a decrease in inhibitory activity. Compound 33b containing a fully saturated monoterpenoid fragment was twofold less active than its analogue 33a. It is interesting that in this case the 4-methoxy substituted compound also demonstrated high inhibitory activity.
Among the myrtenol-coumarin conjugates, the most active were with the derivative bearing 4-bromo (34c) and 2-methoxy (37c) substituents on the pendant aryl ring. Elongation of the linker when passing from conjugate 37c to conjugate 37d led to a decrease in inhibitory activity.
Thus, monoterpene esters of 5-hydroxycoumarins containing aromatic substituents in the 3-position of the pyran ring indeed demonstrated good inhibitory activity against TDP1 as we proposed at the start of our work, with compound 33a being the most active.
It is interesting that 5-hydroxycoumarin derivatives bearing an acyclic monoterpene fragment were generally more active than their counterparts containing a bicyclic monoterpene substituent, whilst earlier studies [30] demonstrated that 7-hydroxycoumarin conjugates with both types of monoterpene residue had comparable inhibitory activity. Taking into account that most active 7-hydroxycoumarin derivatives have IC50 not less than 0.4 μM [29,30], the new coumarin-monoterpene conjugates reported here are the most potent TDP1 coumarin-based inhibitors found so far.
The cytotoxicity of the compounds was studied against the cell lines HeLa (cervical carcinoma) and HEK293A (human embryonic kidney). Since TDP1 inhibitors are planned to be used in combination with other chemotherapy drugs that have significant side effects, the toxicity of these TDP1 inhibitors should be minimal. Overall, the compounds exhibited moderate to mild toxicity (see Table 2 and Figure S1 in Supplementary), the semi-toxic concentration CC50 was 60 μM or more for both tested cell lines. Compounds 34a and 37a turned out to be the most toxic for HeLa cells (CC50 values 32 and 18 μM, respectively). For cells of non-cancerous origin, HEK293A, compound 37a was also the most toxic (16 μM), while 33b and 38c were somewhat more toxic than the rest of the compounds (40 and 32 μM, respectively). The most effective inhibitor of TDP1 33a turned out to be one of the least toxic compounds (turquoise plot in Figure S1 in the Supplementary Information).  Compound 33b containing a fully saturated monoterpenoid fragment was twofold less active than its analogue 33a. It is interesting that in this case the 4-methoxy substituted compound also demonstrated high inhibitory activity.
Among the myrtenol-coumarin conjugates, the most active were with the derivative bearing 4-bromo (34c) and 2-methoxy (37c) substituents on the pendant aryl ring. Elongation of the linker when passing from conjugate 37c to conjugate 37d led to a decrease in inhibitory activity.
Thus, monoterpene esters of 5-hydroxycoumarins containing aromatic substituents in the 3-position of the pyran ring indeed demonstrated good inhibitory activity against TDP1 as we proposed at the start of our work, with compound 33a being the most active.
It is interesting that 5-hydroxycoumarin derivatives bearing an acyclic monoterpene fragment were generally more active than their counterparts containing a bicyclic monoterpene substituent, whilst earlier studies [30] demonstrated that 7-hydroxycoumarin conjugates with both types of monoterpene residue had comparable inhibitory activity. Taking into account that most active 7-hydroxycoumarin derivatives have IC50 not less than 0.4 μM [29,30], the new coumarin-monoterpene conjugates reported here are the most potent TDP1 coumarin-based inhibitors found so far.
The cytotoxicity of the compounds was studied against the cell lines HeLa (cervical carcinoma) and HEK293A (human embryonic kidney). Since TDP1 inhibitors are planned to be used in combination with other chemotherapy drugs that have significant side effects, the toxicity of these TDP1 inhibitors should be minimal. Overall, the compounds exhibited moderate to mild toxicity (see Table 2 and Figure S1 in Supplementary), the semi-toxic concentration CC50 was 60 μM or more for both tested cell lines. Compounds 34a and 37a turned out to be the most toxic for HeLa cells (CC50 values 32 and 18 μM, respectively). For cells of non-cancerous origin, HEK293A, compound 37a was also the most toxic (16 μM), while 33b and 38c were somewhat more toxic than the rest of the compounds (40 and 32 μM, respectively). The most effective inhibitor of TDP1 33a turned out to be one of the least toxic compounds (turquoise plot in Figure S1 in the Supplementary Information).  Compound 33b containing a fully saturated monoterpenoid fragment was twofold less active than its analogue 33a. It is interesting that in this case the 4-methoxy substituted compound also demonstrated high inhibitory activity.
Among the myrtenol-coumarin conjugates, the most active were with the derivative bearing 4-bromo (34c) and 2-methoxy (37c) substituents on the pendant aryl ring. Elongation of the linker when passing from conjugate 37c to conjugate 37d led to a decrease in inhibitory activity.
Thus, monoterpene esters of 5-hydroxycoumarins containing aromatic substituents in the 3-position of the pyran ring indeed demonstrated good inhibitory activity against TDP1 as we proposed at the start of our work, with compound 33a being the most active.
It is interesting that 5-hydroxycoumarin derivatives bearing an acyclic monoterpene fragment were generally more active than their counterparts containing a bicyclic monoterpene substituent, whilst earlier studies [30] demonstrated that 7-hydroxycoumarin conjugates with both types of monoterpene residue had comparable inhibitory activity. Taking into account that most active 7-hydroxycoumarin derivatives have IC50 not less than 0.4 μM [29,30], the new coumarin-monoterpene conjugates reported here are the most potent TDP1 coumarin-based inhibitors found so far.
The cytotoxicity of the compounds was studied against the cell lines HeLa (cervical carcinoma) and HEK293A (human embryonic kidney). Since TDP1 inhibitors are planned to be used in combination with other chemotherapy drugs that have significant side effects, the toxicity of these TDP1 inhibitors should be minimal. Overall, the compounds exhibited moderate to mild toxicity (see Table 2 and Figure S1 in Supplementary), the semi-toxic concentration CC50 was 60 μM or more for both tested cell lines. Compounds 34a and 37a turned out to be the most toxic for HeLa cells (CC50 values 32 and 18 μM, respectively). For cells of non-cancerous origin, HEK293A, compound 37a was also the most toxic (16 μM), while 33b and 38c were somewhat more toxic than the rest of the compounds (40 and 32 μM, respectively). The most effective inhibitor of TDP1 33a turned out to be one of the least toxic compounds (turquoise plot in Figure S1 in the Supplementary Information).  Compound 33b containing a fully saturated monoterpenoid fragment was twofold less active than its analogue 33a. It is interesting that in this case the 4-methoxy substituted compound also demonstrated high inhibitory activity.
Among the myrtenol-coumarin conjugates, the most active were with the derivative bearing 4-bromo (34c) and 2-methoxy (37c) substituents on the pendant aryl ring. Elongation of the linker when passing from conjugate 37c to conjugate 37d led to a decrease in inhibitory activity.
Thus, monoterpene esters of 5-hydroxycoumarins containing aromatic substituents in the 3-position of the pyran ring indeed demonstrated good inhibitory activity against TDP1 as we proposed at the start of our work, with compound 33a being the most active.
It is interesting that 5-hydroxycoumarin derivatives bearing an acyclic monoterpene fragment were generally more active than their counterparts containing a bicyclic monoterpene substituent, whilst earlier studies [30] demonstrated that 7-hydroxycoumarin conjugates with both types of monoterpene residue had comparable inhibitory activity. Taking into account that most active 7-hydroxycoumarin derivatives have IC50 not less than 0.4 μM [29,30], the new coumarin-monoterpene conjugates reported here are the most potent TDP1 coumarin-based inhibitors found so far.
The cytotoxicity of the compounds was studied against the cell lines HeLa (cervical carcinoma) and HEK293A (human embryonic kidney). Since TDP1 inhibitors are planned to be used in combination with other chemotherapy drugs that have significant side effects, the toxicity of these TDP1 inhibitors should be minimal. Overall, the compounds exhibited moderate to mild toxicity (see Table 2 and Figure S1 in Supplementary), the semi-toxic concentration CC50 was 60 μM or more for both tested cell lines. Compounds 34a and 37a turned out to be the most toxic for HeLa cells (CC50 values 32 and 18 μM, respectively). For cells of non-cancerous origin, HEK293A, compound 37a was also the most toxic (16 μM), while 33b and 38c were somewhat more toxic than the rest of the compounds (40 and 32 μM, respectively). The most effective inhibitor of TDP1 33a turned out to be one of the least toxic compounds (turquoise plot in Figure S1 in the Supplementary Information).
Thus, monoterpene esters of 5-hydroxycoumarins containing aromatic substituents in the 3-position of the pyran ring indeed demonstrated good inhibitory activity against TDP1 as we proposed at the start of our work, with compound 33a being the most active.
It is interesting that 5-hydroxycoumarin derivatives bearing an acyclic monoterpene fragment were generally more active than their counterparts containing a bicyclic monoterpene substituent, whilst earlier studies [30] demonstrated that 7-hydroxycoumarin conjugates with both types of monoterpene residue had comparable inhibitory activity. Taking into account that most active 7-hydroxycoumarin derivatives have IC 50 not less than 0.4 µM [29,30], the new coumarin-monoterpene conjugates reported here are the most potent TDP1 coumarin-based inhibitors found so far.
The cytotoxicity of the compounds was studied against the cell lines HeLa (cervical carcinoma) and HEK293A (human embryonic kidney). Since TDP1 inhibitors are planned to be used in combination with other chemotherapy drugs that have significant side effects, the toxicity of these TDP1 inhibitors should be minimal. Overall, the compounds exhibited moderate to mild toxicity (see Table 2 and Figure S1 in Supplementary), the semi-toxic concentration CC 50 was 60 µM or more for both tested cell lines. Compounds 34a and 37a turned out to be the most toxic for HeLa cells (CC 50 values 32 and 18 µM, respectively). For cells of non-cancerous origin, HEK293A, compound 37a was also the most toxic (16 µM), while 33b and 38c were somewhat more toxic than the rest of the compounds (40 and 32 µM, respectively). The most effective inhibitor of TDP1 33a turned out to be one of the least toxic compounds (turquoise plot in Figure S1 in the Supplementary Information).
To study the effect of our compounds on the cytotoxic effect of topotecan, we chose a low-toxic concentration of TDP1 inhibitor of 10 µM. With respect to HeLa cervical cancer cells, many compounds increased the cytotoxicity of topotecan (Figure 3a), with 33a being the most effective sensitizer (Table 2).
With respect to cells of non-cancerous origin HEK293A, no pronounced sensitizing effect of 5-hydroxycoumarins was found (Table 2, Figure 3b). Moreover, some compounds (e.g., 38a, 34c, 37c) protect cells from the cytotoxic effects of topotecan. Previously, we also observed the absence of sensitization of the cytotoxic effect of topotecan by TDP1 inhibitors of a different structure on HEK293A cells [26].  Thus, the most active TDP1 inhibitor demonstrating the promising sensitization effect, compound 33a, significantly increased cytotoxicity of topotecan against HeLa cancer cell line but not against conditionally normal HEK293A cells.
When the predicted binding poses of the compounds were analyzed no dominant configurations were seen. However, the ligands overlapped with the co-crystallized ligand and were predicted to sit in the catalytic pocket with the catalytic His263 and His493 amino acid residue except for 35c. The configuration predicted by ChemPLP, of 33a is shown in Figure 4. The aliphatic chain moiety overlaps with the co-crystallized ligand whereas the coumarin and the para fluorophenyl rings are sitting in a groove. The methylene terpene carbon from the oxygen coming off the 5 position of the coumarin forms a weak H-bond with the hydroxyl side chain of the Ser400 amino acid residue. In addition, Ser403 forms one weak hydrogen bond from its side chain methylene with the carbonyl group in the coumarin ring system as well as hydrogen-π ring system interaction between the backbone's amino group and the ester containing ring (see Figure 4B). Structural activity relations and modelling studies of usnic acid derivatives suggest an allosteric binding site close to the catalytic pocket as shown in Figure 4A [43]. This potential allosteric site was also suggested by molecular dynamics simulation work [51]. In this study, none of the ligands were predicted to reach this potential pharmacophore, which may explain their relatively modest efficacy.

Chemical Space
The mainstream molecular descriptors water-octanol partition coefficient (log P), molecular weight (MW, g mol −1 ), hydrogen bond donors (HD), hydrogen bond acceptors (HA), rotatable bonds (RB) and polar surface area (PSA, Å 2 ) are given in Table S2. The values of the descriptors span different definitions of chemical space: HD are in lead-like chemical space; PSA is both in lead-and drug-like space; and in drug-like space only are RB, MW and HA. Log P is in Known Drug Space (KDS) and beyond (see definitions for regions in chemical space see ref. [52] and in Table S3).
To find the balance of the physicochemical properties reflected in the molecular descriptors described above the Known Drug Indexes (KDIs) for the ligands were derived. The KDIs are derived from drugs in clinical use, i.e., a weighted index from a normalized (to a maximum of 1) statistical distribution was fitted to a Gaussian function for each descriptor. Both the summation of the indexes (KDI 2a -see Equation (1)) and multiplication (KDI 2b -see Equation (2)) methods were used [40]. All the numerical values are given in Table S2. All product yields are given for pure compounds purified by recrystallization or isolated by column chromatography (SiO 2 ; 60-200 µ; Macherey-Nagel, Düren, Germany). All of the target compounds have a purity of no less than 95% (GC-MS data).

Synthesis of coumarins 26-31.
Syntheses were carried out from 2,6-dihydroxyacetophenone and appropriate phenylacetic acids (14)(15)(16)(17)(18)(19) in accordance with article [37]. A mixture of the appropriate phenylacetic acid (18.6 mmol) and 2,6-dihydroxyacetophenone (19.5 mmol) in the presence of triethylamine (56.0 mmol) in acetic anhydride (15 mL) was refluxed for 3 h. After the completion of the reaction, water was added and the mixture was extracted with dichloromethane. The organic phase was separated, washed with saturated sodium bicarbonate, brine and water, dried over anhydrous Na 2 SO 4 , filtered, and evaporated to give the crude product. The products were purified by recrystallization from methanol and dichloromethane. A mixture of the appropriate 5-acetyloxy-coumarin 20-25 (4.5 mmol) and hydrazine monohydrate (22.5 mmol) in methanol (40 mL) was stirred at 40 • C for 2 h. After the completion of the reaction, water was added and the mixture was then extracted with ethyl acetate. The organic phase was separated, washed with brine and water, dried over anhydrous Na 2 SO 4 , filtered, and evaporated to give the crude product. The products were triturated with methanol.

Synthesis of bromides 32a-d.
Bromide 32a was synthesized from geraniol via the reaction with PBr 3 [30]. PBr 3 (8.9 mmol) was added to cooled (0-5 • C) solution of geraniol (26.7 mmol) in dry ether (30 mL) and the reaction mixture was stirred for 2 h at r.t. Saturated aqueous NaHCO 3 was added and the product was extracted with ether. The extracts were washed with brine, dried with Na 2 SO 4 and evaporated.
Other used bromides 32b,c were synthesized as described above. Compounds 32a-c (the yields 91%, 65%, and 55%, respectively) were sufficiently pure and used for the next step without purification.
Triphenylphosphine (2.0 equiv, 6.1 g, 23 mmol) was dissolved in dry DCM (23 mL) under argon. To this solution cooled by ice-water bath, N-bromosuccinimide (NBS) (2.0 equiv, 4.2 g, 23 mmol) was added in small portions over 5 min. Later the resulting mixture was stirred at r.t. for 30 min. Pyridine (1 mL) was added and then (-)-nopol (1.0 equiv, 2.0 mL, 12 mmol) was added dropwise over 10 min. The reaction mixture was stirred overnight at r.t. The mixture was diluted with hexane (40 mL) and filtered through a silica gel plug, diluted with EtOAc-hexane (1:1, 40 mL) and filtered through the silica gel plug. The residue was concentrated in vacuo and purified by chromatography on SiO 2 (hexane) to obtain bromide 32d (2.3 g, 70% yield).   detection of TDP1 enzyme activity was carried out as described [53]. The recombinant TDP1 was purified as described [54], using plasmid pET 16B-TDP1, kindly provided by Dr. K.W. Caldecott (University of Sussex, Brighton, UK). To the reaction mixture (200 µL) containing 50 nM oligonucleotide in buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, and 7 mM β-mercaptoethanol), varied concentrations of the tested compounds and purified TDP1 in a final concentration of 1.5 nM were added.

Synthesis of compounds 33-38.
Fluorescence intensity was measured (Ex485/Em520 nm) using POLARstar OPTIMA fluorimeter (BMG LABTECH, GmbH, Ortenberg, Germany) every 1 min for 7 min. The half maximal inhibitory concentrations (IC 50 ) were determined using a six-point concentration response curve and calculated using MARS Data Analysis 2.0 (BMG LABTECH). At least three independent experiments were carried out to obtain the IC 50 values.

Cytotoxicity Assays
The cytotoxicity of the compounds to HeLa (human cervical cancer) and HEK293A (human embryonic kidney) cell lines was determined using standard colorimetric MTTtest [55]. Cells were seeded in 96-well plates (5000 cells per well) and cultured in DMEM medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), penicillin (100 units/mL), and streptomycin (100 µg/mL) at 37 • C and 5% CO 2 in a humid atmosphere. Control wells contained 1% DMSO. At 30-50% confluence, the tested compounds were added to the medium. To determine the cytotoxicity of Tdp1 inhibitors, the cells were allowed to attach for 24 h and were treated with compounds with concentrations ranging from 1 to 100 µM for 72 h at 37 • C. All measurements were repeated three times.
To study the effect of the compounds on the cytotoxicity of topotecan, an aqueous solution of topotecan was used at concentrations from 0.2 to 2 µM for HeLa cells and from 0.05 to 0.5 µM for HEK293A cells against the background of 10 µM of the compounds. Cells treated with compounds alone without topotecan were used as controls.

Computer Aided Drug Design
The compounds were modelled using the TDP1 crystal structure (PDB ID: 6W7K, resolution 1.70 Å) [42], obtained from the Protein Data Bank (PDB) [56,57]. The GOLD (v2020.2.0) software was used to prepare the crystal structures for docking, i.e., the hydrogen atoms were added, water molecules deleted and the co-crystallized ligand identified: 4-[(2-phenylimidazo[1,2-a]pyridin-3-yl)amino]benzene-1,2-dicarboxylic acid (TG7). The Scigress version FQ 3.4.4 program [58] was used to build the compounds and the MM3 [59][60][61] force field was applied to identify the global minimum using the CONFLEX method [62] followed by energy minimization. The docking center for the TDP1 catalytic pocket was defined as the position of the co-crystallized ligand TG7 with 10 Å radius. Fifty docking runs were allowed for each ligand with default search efficiency (100%). The basic amino acids lysine and arginine were defined as protonated. Furthermore, aspartic and glutamic acids were assumed to be deprotonated. The GoldScore(GS) [44] and ChemScore(CS) [45,46] ChemPLP(Piecewise Linear Potential) [47] and ASP(Astex Statistical Potential) [48] scoring functions were implemented to predict the binding modes and relative binding energies of the ligands using the GOLD v2020.2.0 software suite.
The QikProp 6.2 [63] software package was used to calculate the molecular descriptors for all the ligands. QikProp was benchmarked for the calculated descriptors [64]. The KD were derived from the descriptors as described by Eurtivong and Reynisson [52]. For application in Excel, columns for each property were created and the following equa-

Conclusions
Compounds combining the 5-hydroxycoumarin and monoterpenoid moieties were synthesized for the first time by the condensation of the coumarin derivatives containing an aryl fragment at position 3 with acyclic and bicyclic monoterpenoid bromides. A study of TDP1 inhibitory activities of the compounds showed that the most potent inhibitors are 5-hydroxycoumarin derivatives bearing an acyclic monoterpene fragment, with IC 50 up to 130 nM. Such activity makes these compounds the most potent coumarin-based TDP1 inhibitors found so far. These inhibitors generally demonstrated low or no cytotoxicity against cancer HeLa and conditionally normal HEK 293A cell lines. Importantly, the most active TDP1 inhibitor demonstrated a significant synergistic effect with anticancer drug topotecan against the HeLa cancer cell line but not against HEK293A cells. The ability of new inhibitors to occupy the catalytic pocket of TDP1 was confirmed by molecular modeling studies. Based on chemical space analysis, it is assumed that the inhibitors could have good biocompatibility. Thus, we developed a new structural type of potent TDP1 inhibitors which are promising for further pharmacological studies as adjuvant therapy against cancer in combination with Top 1 poisons, such as topotecan.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author.