Novel Coumarin–Nucleobase Hybrids with Potential Anticancer Activity: Synthesis, In Vitro Cell-Based Evaluation, and Molecular Docking

A new series of compounds planned by molecular hybridization of the nucleobases uracil and thymine, or the xanthine theobromine, with coumarins, and linked through 1,2,3-triazole heterocycles were evaluated for their in vitro anticancer activity against the human tumor cell lines: colon carcinoma (HCT116), laryngeal tumor cells (Hep-2), and lung carcinoma cells (A549). The hybrid compound 9a exhibited better activity in the series, showing an IC50 of 24.19 ± 1.39 μM against the HCT116 cells, with a selectivity index (SI) of 6, when compared to the cytotoxicity against the non-tumor cell line HaCat. The in silico search for pharmacological targets was achieved through molecular docking studies on all active compounds, which suggested that the synthesized compounds possess a high affinity to the Topoisomerase 1–DNA complex, supporting their antitumor activity. The in silico toxicity prediction studies suggest that the compounds present a low risk of causing theoretical mutagenic and tumorigenic effects. These findings indicate that molecular hybridization from natural derivative molecules is an interesting approach to seek new antitumor candidates.


Introduction
One of the key challenges in medicinal chemistry is the development of more effective, selective.and safer compounds for treating known and new pathologies [1].Even though there are almost infinite possibilities of novel chemical structures wanted for their bioactive properties' evaluation, the molecular hybridization of known bioactive entities can be used as an alternative approach to optimize the process.Molecular hybridization involves the rational design and association of two or more pharmacophores into a single molecule, aiming at new molecular entities that allow multiple biological activities, modified selectivity profiles, different or dual modes of action, and reduced undesired side effects [2][3][4][5][6][7][8].
Although molecular hybridization aims to combine the pharmacophoric properties of the distinct "parent" molecules as multi-targeted agents, it is relevant to consider that the new molecular structure of the hybrid differs from the molecular structure of the "parent" molecules.Thus, the comparison of using the combined "parent" pharmacophores as a drug cocktail administration is limited.Such disadvantages include the difficulty of establishing the ratio of activities at different targets or obtaining a clear interpretation of the molecular and cellular basis of the hybrid activity.In addition, a more complex synthesis can also be considered a disadvantage along with the inevitable increase in molecular mass and lipophilicity [6,7].
The 2H-chromen-2-one core is considered a privileged structure due to its rigid and conjugated structure.The aromatic ring allows a series of hydrophobic, π-π, CH-π, and cation-π interactions, and the two oxygen atoms in the lactone ring can also hydrogenbond with amino acid residues in different classes of enzymes and receptors [21].The relevant pharmacological profile of the H-chromen-2-one nucleus is illustrated by its presence in the main backbone of approved drugs such as warfarin (anticoagulant), carbochromen (vasodilator), and novobiocin (antibiotic) [22].Regarding the molecular hybridization strategy, coumarins have also been employed along with diverse bioactive compounds [4,[22][23][24].
Pyrimidine and its derivatives are also a significant and widespread class of nitrogencontaining heterocycles that are an integral part of DNA and RNA building blocks.They play an essential role in the biological process and, consequently, have considerable chemical and pharmacological importance.5-fluorouracil (5-FU) is an example of a pyrimidine derivative that has been used in cancer treatment.5-FU, such as other pyrimidine derivatives, acts as an antagonist in the biosynthetic pathways of pyrimidine nucleobases [25], competing for the same binding sites of naturally occurring compounds [26].
Coumarin and uracil derivatives conjugated to 1,2,3-triazoles have been reported in the literature and evaluated as possible anticancer agents.For instance, coumarin-chalcone hybrids linked by the 1,2,3-triazole ring were synthesized and evaluated as anticancer and antimalarial agents [32].Example A (Figure 1) showed antiproliferative activity against human acute T lymphoblastic leukemia cell lines (MOLT-3) with an IC 50 of 0.53 µM.
The 2H-chromen-2-one core is considered a privileged structure due to its rigid and conjugated structure.The aromatic ring allows a series of hydrophobic, π-π, CH-π, and cation-π interactions, and the two oxygen atoms in the lactone ring can also hydrogenbond with amino acid residues in different classes of enzymes and receptors [21].The relevant pharmacological profile of the H-chromen-2-one nucleus is illustrated by its presence in the main backbone of approved drugs such as warfarin (anticoagulant), carbochromen (vasodilator), and novobiocin (antibiotic) [22].Regarding the molecular hybridization strategy, coumarins have also been employed along with diverse bioactive compounds [4,[22][23][24].
Pyrimidine and its derivatives are also a significant and widespread class of nitrogencontaining heterocycles that are an integral part of DNA and RNA building blocks.They play an essential role in the biological process and, consequently, have considerable chemical and pharmacological importance.5-fluorouracil (5-FU) is an example of a pyrimidine derivative that has been used in cancer treatment.5-FU, such as other pyrimidine derivatives, acts as an antagonist in the biosynthetic pathways of pyrimidine nucleobases [25], competing for the same binding sites of naturally occurring compounds [26].
Coumarin and uracil derivatives conjugated to 1,2,3-triazoles have been reported in the literature and evaluated as possible anticancer agents.For instance, coumarinchalcone hybrids linked by the 1,2,3-triazole ring were synthesized and evaluated as anticancer and antimalarial agents [32].Example A (Figure 1) showed antiproliferative activity against human acute T lymphoblastic leukemia cell lines (MOLT-3) with an IC50 of 0.53 µM.
An example of a pyrimidine derivative employed in the molecular hybridization strategy is the uracil-isatin conjugates hybridized via the 1,2,3-triazole bridge [33].Among the series prepared in this study, compound B (Figure 1) showed considerable selectivity for the human prostate cancer cell line (DU145) with an IC50 of 13.9 µM.An example of a pyrimidine derivative employed in the molecular hybridization strategy is the uracil-isatin conjugates hybridized via the 1,2,3-triazole bridge [33].Among the series prepared in this study, compound B (Figure 1) showed considerable selectivity for the human prostate cancer cell line (DU145) with an IC 50 of 13.9 µM.
Considering the pharmacological relevance of the coumarin and pyrimidine derivatives, in this work, we explored their molecular hybridization linked by a triazole unit aiming for a non-cleavable conjugated pharmacophore.Thus, we investigate the potential anticancer activities of the novel coumarin-nucleobase hybrids against three human cell lines, colon carcinoma (HCT116), laryngeal tumor (Hep-2), and lung carcinoma (A549), and one non-tumor cell line, namely HaCat (human keratinocyte), using the colorimetric MTT assay.

Chemistry
The synthetic protocol to obtain the target hybrid compounds started with the Pechmann condensation of the phenols phloroglucinol (1a) and 1-naphthol (1b) with the β-ketoester ethyl 4-chloroacetoacetate (2) to achieve the 4-chloromethylcoumarins 3a and 3b with 86 and 70% yield, respectively.This procedure was carried out in the absence of solvent and using sulfamic acid as a catalyst [34].Then, the azide group was installed by the nucleophilic displacement of the chlorine atom [35], affording the 4-(azidomethyl)coumarins 4a and 4b with 89 and 86% yields, respectively, as shown in Figure 2.
Considering the pharmacological relevance of the coumarin and pyrimidine derivatives, in this work, we explored their molecular hybridization linked by a triazole unit aiming for a non-cleavable conjugated pharmacophore.
Thus, we investigate the potential anticancer activities of the novel coumarinnucleobase hybrids against three human cell lines, colon carcinoma (HCT116), laryngeal tumor (Hep-2), and lung carcinoma (A549), and one non-tumor cell line, namely HaCat (human keratinocyte), using the colorimetric MTT assay.

Chemistry
The synthetic protocol to obtain the target hybrid compounds started with the Pechmann condensation of the phenols phloroglucinol (1a) and 1-naphthol (1b) with the β-ketoester ethyl 4-chloroacetoacetate (2) to achieve the 4-chloromethylcoumarins 3a and 3b with 86 and 70% yield, respectively.This procedure was carried out in the absence of solvent and using sulfamic acid as a catalyst [34].Then, the azide group was installed by the nucleophilic displacement of the chlorine atom [35], affording the 4-(azidomethyl)coumarins 4a and 4b with 89 and 86% yields, respectively, as shown in Figure 2. Next, we prepare the portions constituted by the N1-propargylated uracil (6a), N1-propargylated thymine (6b), or N1,N2-dipropargylated uracil (6c).Initially, we envisioned the monopropargylation reaction of the nucleobases at the N1 position, retaining the hydrogen at the N3 position free due to its importance in biological activity.The preservation of the hydrogen at the N3 position is intended to maintain the same hydrogen-bond pairing scheme presented by uracil.In addition, the propargylation at the N1 position is necessary to introduce lipophilic groups or to bind other groups to the nucleobase skeleton [26].
Thus, to selectively prepare the N1-propargylated nucleobases 6a,b, the bis(trimethylsilyl)acetamide (BSA) was employed as a base, using acetonitrile as a solvent, at 45 °C for 72 h [36].This methodology allows us to obtain the desired compounds as unique products with a 62% and 76% yield for 6a and 6b, respectively (Figure 3, Scheme A).Afterward, the N1,N2-dipropargylated uracil 6c was synthesized in order to obtain the hybrid compounds bis-(coumaronyl-triazolyl)uracil, which is based on similar molecular structures that present anticancer activity [33].In this case, the dipropargylation reaction was carried out using the simple base K2CO3 in anhydrous DMF at room temperature for 24 h.The N1,N2-dipropargylated uracil 6c was obtained with an 86% yield (Figure 3, Scheme A).Next, we prepare the portions constituted by the N1-propargylated uracil (6a), N1-propargylated thymine (6b), or N1,N2-dipropargylated uracil (6c).Initially, we envisioned the monopropargylation reaction of the nucleobases at the N1 position, retaining the hydrogen at the N3 position free due to its importance in biological activity.The preservation of the hydrogen at the N3 position is intended to maintain the same hydrogen-bond pairing scheme presented by uracil.In addition, the propargylation at the N1 position is necessary to introduce lipophilic groups or to bind other groups to the nucleobase skeleton [26].
Thus, to selectively prepare the N1-propargylated nucleobases 6a,b, the bis(trimethylsilyl)acetamide (BSA) was employed as a base, using acetonitrile as a solvent, at 45 • C for 72 h [36].This methodology allows us to obtain the desired compounds as unique products with a 62% and 76% yield for 6a and 6b, respectively (Figure 3, Scheme A).Afterward, the N1,N2-dipropargylated uracil 6c was synthesized in order to obtain the hybrid compounds bis-(coumaronyl-triazolyl)uracil, which is based on similar molecular structures that present anticancer activity [33].In this case, the dipropargylation reaction was carried out using the simple base K 2 CO 3 in anhydrous DMF at room temperature for 24 h.The N1,N2-dipropargylated uracil 6c was obtained with an 86% yield (Figure 3, Scheme A).
In addition to uracil and thymine, we also decided to investigate the influence of the theobromine (5c, 3,7-dimethylxanthine) moiety in the activity.The propargylated theobromine (6d) was prepared using K 2 CO 3 as a base, in anhydrous DMF, at 40 • C [33].However, a longer reaction time of 48 h was necessary to achieve only 50% yield (Figure 3, Scheme B).
Theobromine is an alkaloid of the family methylxanthines.It is found mainly in cocoa products and has a diuretic action [37].In addition, theobromine derivatives demonstrated potential antitumor activities through multiple mechanisms and could reverse resistance to multiple drugs.In view of this potential, theobromine derivatives can also lead to potent antitumor agents that are selective and have low toxicity [38,39].
In addition to uracil and thymine, we also decided to investigate the influence of the theobromine (5c, 3,7-dimethylxanthine) moiety in the activity.The propargylated theobromine (6d) was prepared using K2CO3 as a base, in anhydrous DMF, at 40 °C [33].However, a longer reaction time of 48 h was necessary to achieve only 50% yield (Figure 3, Scheme B).Theobromine is an alkaloid of the family methylxanthines.It is found mainly in cocoa products and has a diuretic action [37].In addition, theobromine derivatives demonstrated potential antitumor activities through multiple mechanisms and could reverse resistance to multiple drugs.In view of this potential, theobromine derivatives can also lead to potent antitumor agents that are selective and have low toxicity [38,39].
The propargylated nucleobases 6a-d and the 4-(azidomethyl)coumarins 4a,b were hybridized through a 1,2,3-triazole ring by a Cu(I)-catalyzed [3+2]-cycloaddition reaction, as outlined in Figure 4.The click reaction was successfully employed for all intermediates, using the simple copper(II) sulfate and sodium ascorbate system to generate the copper(I) catalyst.All the reactions were carried out in ethanol-water (10:1), at 30 °C, for 24 h.
After the reaction completion, the target compounds were simply purified by pouring the reaction contents into an ice-water mixture.The precipitated products were filtered and presented a high purity degree, as observed by 1 H NMR. However, when impurities were noticed, they were removed by a short pad of silica gel.The target compounds 7a,b, 8a,b, 9a,b, and 10a,b were prepared from good to excellent yield, as shown in Figure 4.  4. The click reaction was successfully employed for all intermediates, using the simple copper(II) sulfate and sodium ascorbate system to generate the copper(I) catalyst.All the reactions were carried out in ethanol-water (10:1), at 30 • C, for 24 h.
After the reaction completion, the target compounds were simply purified by pouring the reaction contents into an ice-water mixture.The precipitated products were filtered and presented a high purity degree, as observed by 1 H NMR. However, when impurities were noticed, they were removed by a short pad of silica gel.The target compounds 7a,b, 8a,b, 9a,b, and 10a,b were prepared from good to excellent yield, as shown in Figure 4.
All compounds were characterized by 1 H and 13 C NMR, HRMS, and melting point.The NMR and HRMS spectra for the final compounds are reported in the Supplementary Materials.All compounds were characterized by 1 H and 13 C NMR, HRMS, and melting point.The NMR and HRMS spectra for the final compounds are reported in the Supplementary Materials.

Inclusion Complex
Before the cytotoxic evaluation, it was noticed that the target compounds showed low solubility in the cell medium, even the N1-(cumaronyltriazolyl)uracils 7a,b and 8a,b, with the free hydrogen at the N3 position.In order to enhance the solubility, we proposed the use of non-toxic (2-hydroxylpropyl)-β-cyclodextrin (HP-β-CD) as a complexing agent.The HP-β-CD is suitable for cell cultures and has been used to enhance the solubility of non-polar compounds, such as vitamins and hormone derivatives [40].Hydrophobic molecules are incorporated into the cavity by water molecule displacement, thus favoring the solubility in the medium.During the cytotoxic experiment, the reverse process occurs due to low concentration, releasing the active compound.Thus, the complex compounds were prepared by vigorous mixing of a solution of the targets in ethanol with an equimolar amount of HP-β-CD in water.After 15 h of vigorous mixing, a clean solution was noticed, in which, in turn, the solvent was removed under a vacuum, and the residual water was removed by the lyophilization method to obtain a solid.The complex compounds were characterized by 1 H NMR, and besides the presence of HP-β-CD signals, no significant changes in the chemical shift of the signals of the target compounds were noticed (Figure S26).

Biological Activities Cytotoxicity Assay
The potential antitumor activity of eight new hybrids was evaluated through colorimetric microculture MTT assay against three tumor cell lines: human colon carcinoma cells (HCT116), human laryngeal tumor cells (Hep-2), and human lung carcinoma cells (A549).The hybridized compounds were also assayed against the non-tumor cell line HaCat (human keratinocyte) in order to obtain an indication of their selective cytotoxicity.Also, the commercial anticancer drug doxorubicin was employed as a positive drug standard to compare the cytotoxicity activities.Each experiment was carried out in triplicate on three different days.The selectivity index (SI) was calculated as the ratio between the IC 50 (concentration that reduces cell viability by 50%) of HaCat (non-tumor line) and the IC 50 of the tumor lines.The results are outlined in Table 1.The mono N1-(coumaronyltriazolyl)uracils 7a,b and 8a,b did not show activity against the tumor cell lines, with the exception of compound 8b, which showed poor cytotoxicity only against HCT116 cells with an IC 50 of 87.58 ± 1.94 µM.Compound 9a, resulting from the molecular hybridization of the uracil and two coumarins derived from phloroglucinol, exhibits the highest cytotoxicity against HCT116 cells with an IC 50 of 24.19 ± 1.39 µM for a 48 h experiment (Table 1, entry 1).Interestingly, compound 9a did not exhibit appreciable cytotoxicity against the non-tumor cells HaCat, showing a good selectivity index (SI) of 6.0.No cytotoxicity effect was observed for compound 9a against A549 cells in 24 and 48 h treatments.For Hep-2 cells, an IC 50 of 51.55 ± 1.71 µM was noticed for 24 h of exposure.
Compound 9b, which is an analog to 9a, but with the coumarin moiety derived from 1-naphthol, presented an IC 50 for HCT116 cells of 54.50 ± 1.74 and 59.17 ± 1.77 µM for 24 and 48 h treatments.It was the only compound in the series that showed activity against A549 cells, with an IC 50 of 51.03 ± 1.71 µM for 24 h of exposure.Besides the moderated cytotoxicity, the selectivity index when compared to HaCat cells was 63.No appreciative cytotoxicity was observed for Hep-2 cells.
Compounds 10a,b, both derived from the nucleobase theobromine, showed activity against HCT116 cells.However, compound 10b showed higher potency with an IC 50 40.98± 1.61 µM for 24 h of exposure with a selectivity index of 6. Compounds with SI ≥ 10 can be considered selective [41].Compound 10a showed an IC 50 of 78.26 ± 1.89 in 24 h of exposure for HCT116 cells, and an IC 50 of 56.55 ± 1.75 µM for Hep-2 cells, with a selectivity index of 4 and 5, respectively.None of the tested compounds showed cytotoxicity against the non-tumor cell line (HaCat), even at the highest evaluated concentration of 100 µM at all exposure times tested.Also, the reference compound doxorubicin was used as a positive drug standard to compare the cytotoxicity activities.Besides being used as an effective antitumor agent, doxorubicin exhibits well-documented toxic effects, including tissue ulceration, necrosis, and cardiac toxicity [42].For the HCT116 cell line, compound 9a showed a moderated activity (approximately 20-fold) when compared to doxorubicin (IC 50 of 1.23 ± 0.05 µM).

In Silico Studies
The Osiris Property Explorer ® (Idorsia Pharmaceuticals Ltd., Allschwil, Switzerland) [43] software was used to calculate the mutagenic, tumorigenic, irritant, and toxicant reproductive system effects of compounds 9a,b and 10a,b, and therefore, to predict the toxicity risks.The calculations are based on the functional group similarity between the query molecules within the compounds present in the software database.The results were compared with the standard H 2 O 2 and are outlined in Table 2.
The evaluation showed that compounds 9a and 9b showed a low risk of causing theoretical mutagenic and also tumorigenic effects.No risk of irritant effect was determined.Only compound 9a showed a medium risk of causing an effect on the reproductive system.All compounds showed lower predicted toxic effects in comparison with H 2 O 2 .These results suggest that the compounds present a low potential to cause theoretical toxicity risks and, thus, can be submitted to drug design development.
In order to understand the cytotoxicity presented by the compounds in the MTT assay, we decided to employ computational techniques to suggest the most likely biological target.The chemical similarity ensemble algorithm (SEA) [44] was employed based on the hypothesis of the identification of candidate target proteins to interact with the shared scaffolds of uracil, coumarin, and triazole, such as (1-((1-((5,7-dihydroxy-2-oxo-2H-chromen-4-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)-3-methylpyrimidine-2,4(1H,3H)-dione), from the structures of 9a, 9b, 10a, and 10b.The results indicated the potential of several protein targets, in which the main target model is related to the Topoisomerase 1-DNA complex (Topo 1-DNA complex), which showed a p-value of 3.83 × 10 −31 and a maximum Tanimoto coefficient (max-TC) of 0.29.All predicted results are shown in Table 3. Topoisomerase inhibitors have been shown to be potent antineoplastic agents.The explanation for this potential is due to the mechanism of action of topoisomerase-targeted drugs; the higher the cellular concentration of topoisomerases, the more lethal these drugs become.Tumor cells grow faster and generally express higher concentrations of topoisomerase than normal cells.Therefore, drugs generate more DNA breaks and are more toxic to tumor cells [45].
Compounds containing triazole, coumarin, or uracil nuclei in their structures have shown antitumor activity attributed to topoisomerase inhibitory activity [46][47][48], which could indicate the importance of these cores in the construction of new molecules aimed at this biological target.Thus, according to the obtained results, the Topo 1-DNA complex was selected as the target protein in further docking studies.

Molecular Modeling and Docking Studies
For the docking analyses, the lowest-energy molecular conformers for the most active compounds 9a,b and 10a,b were generated by Spartan'08 modeling software (version 1.2.0,Wavefunction Inc., Irvine, CA, USA).Further, the docking studies were performed to identify the individual pose that presents lower energy that selectively binds to the Topo 1-DNA complex active site.The molecular docking analysis was accomplished by using the iGEMDOCK software (version 2.1) [49] and applying the generic evolutionary method (GA).The empirical scoring function was measured using the total sum of the energies of van der Waal forces (VDW), hydrogen bond (H-bond), and electrostatic interactions occurring between the compounds and the target protein, as shown in Table 4. Compound 9a complexed with the Topo 1-DNA complex was the ligand with the lowest energy in the series, with a total binding energy of −118.5 kcal mol −1 .Ligands 9b and 10a show similar total binding energy values, which are −113.3and −113.7 kcal mol -1 , respectively.However, for ligand 9b, the contribution of van der Waal energy was larger than that of ligand 10a.In turn, the contribution of H-bond energy for ligand 10a was larger than for ligand 9b.Ligand 10b showed the lowest total binding energy in the series, with a value of −102.7 kcal mol −1 .
Considering the interaction with the amino acid residues in the active site of the Topo 1-DNA complex, the docking studies reveal that the main interactions occur with the residues ARG 364, ARG 488, LYS 532, ASP 533, ILE 535, HIS 632, THR 718, LEU 721, ASN 722, TYR 723 (O-phospho-L-tyrosine), and LYS 571, as illustrated in Figure 5A.The measured energies are represented in Table 5.The residues ARG 364, LYS 532, ASP 533, ASN 722, and TYR 723 are the same as those observed in docking studies performed by Laco (2011) [50], who suggest the same active site of the Topo 1-DNA complex herein studied.
detected as the main residue evolved in this ligand-receptor binding (with Z-score 1.94 and WPharma 1.00).
Van der Waals interactions are the main type between the ligands and the residues in the binding site of the Topo 1-DNA complex.Some relevant interactions to compound poses are observed in LYS 532, HIS 632, and LEU 721.The main molecular regions of interaction between 9a and the Topo 1-DNA complex in the in silico model are shown in Figure 5B.The carbonyl group from the coumarin moiety showed relevant interaction with HIS 632, while the uracil moiety interacted with ARG 364 and THR 718.One of the triazole rings showed interaction with ARG 364 from the Topo 1-DNA complex.Thus, based on the results from the docking studies, the most active compounds are potential ligands and fit as candidates as Topo 1-DNA complex inhibitors.In the post-screening analysis using Residues Consensus Analysis, HIS 632 was detected as the main residue evolved in this ligand-receptor binding (with Z-score 1.94 and WPharma 1.00).
Van der Waals interactions are the main type between the ligands and the residues in the binding site of the Topo 1-DNA complex.Some relevant interactions to compound poses are observed in LYS 532, HIS 632, and LEU 721.The main molecular regions of interaction between 9a and the Topo 1-DNA complex in the in silico model are shown in Figure 5B.The carbonyl group from the coumarin moiety showed relevant interaction with HIS 632, while the uracil moiety interacted with ARG 364 and THR 718.One of the triazole rings showed interaction with ARG 364 from the Topo 1-DNA complex.Thus, based on the results from the docking studies, the most active compounds are potential ligands and fit as candidates as Topo 1-DNA inhibitors.

Materials and Instrumentation
All reagents and solvents used were purchased from commercial suppliers (Sigma-Aldrich ® , São Paulo, Brazil) and used without further purification.The NMR experiments were performed on a Fourier 300 FT-NMR spectrometer (Bruker Daltonics, Bremen, German; 7.05 Tesla, 300 MHz for the 1 H nucleus and 75 MHz for the 13 C nucleus).The chemical shifts (δ) values are expressed in part per million (ppm) and are relative to DMSO-d6 (2.50 ppm for 1 H NMR spectra and 39.52 ppm for 13 C NMR spectra).The coupling constants are reported in Hz.The spectra were acquired at a temperature of 293 K, using 5 mm quartz tubes.For the NMR data acquisition and processing, the TopSpin™ software (version 4.0.7 Bruker BioSpin, Rheinstetten, Germany) was used.The high-resolution electrospray ionization mass spectrometry (ESI-QTOF) analyses were performed on a micrOTOF-Q II instrument (Bruker Daltonics, Billerica, MA, USA) in a positive mode under the following conditions: capillary and cone voltages were set to +3500 V and +40 V, respectively, with a de-solvation temperature of 200 • C. The samples were solubilized in HPLC-grade methanol containing 0.1% formic acid and injected into the ESI source by means of a syringe pump at a flow rate of 5.0 µL min 1.Melting points were recorded on a capillary melting point apparatus (Fisatom, model 431, São Paulo, Brazil), with a measurement range from 50 • C to 350 • C, and they are uncorrected.

The General Procedure for the Preparation of the 4-(Chloromethyl)-coumarins 3a,b
Phenol (1a or 1b) (5 mmol), ethyl 4-chloroacetoacetate (7.5 mmol), and sulfamic acid (10 mol%, 0.0485 g) were added to a 30 mL glass vial and tightly sealed with a Teflon cap.The reaction mixture was heated at 100 • C, for 20 min (1a) or 6 h (1b).After, the reaction mixture was cooled to room temperature and dissolved in 25 mL of hot ethanol, filtered, and poured into an ice-water mixture (100 mL).The precipitate that formed was filtered and recrystallized from ethanol.White solid (970 mg, 86%); mp 231-233 • C (lit.[34], 240-242  In a 50 mL round-bottomed flask equipped with a magnetic stirrer, 4-(chloromethyl)coumarins 3a,b (2 mmol) were taken in 4 mL of acetone.After, a solution of sodium azide (2.4 mmol) in 0.6 mL of water was added dropwise with a continuous stirring, which was kept for an additional 10 h at 30 • C. The reaction mixture was poured into an ice-water mixture.The precipitate that formed was filtered and recrystallized from ethanol [35].White solid (414 mg, 89%); mp 215-217 • C (lit.[52]  In a 50 mL round-bottomed flask, equipped with a magnetic stirrer, uracil (5a, 0.560 g, 5 mmol) or thymine (5b, 0.631 g, 5 mmol) was suspended in dry acetonitrile (15 mL), N,O-bis-(trimethylsilyl)acetamide (BSA, 3.06 mL, 12.5 mmol) was added, and the mixture was stirred until a clear solution was obtained.Subsequently, propargyl bromide (80 wt.% in toluene, 0.615 mL, 6.9 mmol) was added dropwise and the reaction mixture was heated at 45 • C for 72 h.The acetonitrile was evaporated under a vacuum, and the residue was treated with a saturated aqueous NH 4 Cl solution (15 mL) and extracted with CH 2 Cl 2 .The organic phase was dried with anhydrous Na 2 SO 4 and concentrated under a vacuum.The crude product was purified by recrystallization from CH 2 Cl 2 /hexane (1:2 v/v).

Synthesis of 1,3-Di
In a 50 mL round-bottomed flask, equipped with a magnetic stirrer, a suspension of potassium carbonate (4 mmol, 0.552 g) in anhydrous DMF (10 mL) and uracil (1 mmol, 0.112 g) was added.The mixture was stirred for 1 h at room temperature.Afterward, propargyl bromide (80 wt.% in toluene, 0.212 mL, 2.4 mmol) was added, and the reaction mixture was kept under stirring for 24 h at room temperature.After the reaction completion, the mixture was treated with brine (20 mL) and extracted with ethyl acetate (3 × 20 mL).The organic phase was dried with anhydrous Na 2 SO 4 and concentrated under reduced pressure.The product 6c was purified by chromatography column using hexane/ethyl acetate (65:35) and obtained as a white solid (161 mg, 86%); mp 98-100 • C (lit.[54] 102-104 the remaining water was removed by lyophilization to give a water-soluble compound of the HP-β-CD complex in a powder [56].The stock solutions of the compounds were prepared by dissolving the complexed compounds in 200 µL of DMSO, followed by dissolving in 1800 µL of Dulbecco's Modified Eagle Medium (DMEM).The stock solution and dilutions were prepared prior to the cytotoxicity assays.

Cells Cultures
The human colon carcinoma (HCT116), human laryngeal tumor (Hep-2), and human lung carcinoma (A549) cell lines, and the non-tumor human keratinocyte cell line (HaCat) were purchased from Rio de Janeiro Cell Bank.The cells were maintained in DMEM supplemented with The, Gibco BRL; Life Technologies, Carlsbad, CA, USA) and 1% penicillin-streptomycin in a humidified 5% CO 2 atmosphere at 37 • C.

Cell Viability MTT Assay
Cytotoxicity was assayed through the colorimetric microculture MTT assay.Cell suspensions were plated in 96-well plates in triplicate at an initial density of 7 × 10 4 cells mL −1 and incubated at 37 • C for 24 h.After the incubation, the cells were treated with various concentrations of the evaluated compounds (7a,b, 8a,b, 9a,b, or 10a,b) and incubated for 24 and 48 h.After the treatment, the medium was removed, and an MTT solution (0.4 mg mL −1 ) was added to each well and further incubated for 2 h at 37 • C.Then, DMSO (100 µL) was added for the solubilization of the formazan crystals, and the absorbance intensities were measured in a microplate reader (SpectraMax M2e, Molecular Devices, Sunnyvale, CA, USA) at 570 nm.The percentage of viable cells was calculated in relation to the control to determine the cytotoxic concentration that reduces 50% of the cell viability (IC 50 ).

Molecular Modeling and Docking Studies
All molecular modeling studies were performed with more active compounds 9a,b and 10a,b, aiming to find information that may support the understanding of their biological activity.Geometry optimization and conformational analysis were calculated using Spartan'08 ® modeling software (V.1.2.0,Wavefunction Inc., Irvine, CL, USA) based on the DFT-B3LYP/6-311G* method, set in the gas phase.The geometry of compounds was optimized, followed by submitting them to systematic conformational analysis with a torsion angle increment of 30 • in the range 0-360 • .The lowest energy conformer for the chemical structure was saved in the mol2 file before being used in docking studies.The structure of the human Topo 1-DNA complex encoded by PDB ID: 1K4S [57] was downloaded from the Protein Data Bank (PDB, http://www.rcsb.org/pdb,accessed on 12 August 2020), before performing the docking studies.The cavity of interaction from the Topo 1-DNA model was used in this study.The protein structure was prepared by removing the water molecules and adding polar hydrogens using Autodock Tools (version 1.5.6)[58].Docking studies were performed using iGemdock software (version 2.1) [49], in which the individual binding poses of compounds were assessed and submitted to dock in the protein.Docking calculations were performed in a drug screening Docking Accuracy Setting with generic evolutionary method (GA) parameters set for population size, generation, and the number of solutions as 200, 70, and 8, respectively, ligand entry energy option active, and Gemdock score function of hydrophobic and electrostatic (1:1 preference).iGemdock software was used to propose the pharmacological pose interactions between the biological receptor and the compound studied.

Conclusions
In conclusion, we described the synthesis of eight novel compounds that originated from the molecular hybridization of the nucleobases uracil, thymine, or theobromine, with coumarins linked through triazole rings.Among the synthesized compounds, the hybrid compound 9a, composed of uracil and the coumarin derivative phloroglucinol, showed antitumor activity against colon carcinoma (HCT116) with the lowest value of IC 50 of 24.19 ± 1.39 µM and a selectivity index of 6.0.The findings of these in vitro results were supported by in silico experiments that, according to the SEA algorithm, indicate that the synthesized compounds show affinity to the Topo 1-DNA complex.Also, molecular docking studies demonstrate that the target compounds 9a,b and 10a,b are potential ligands for the Topo 1-DNA complex and possibly act as its inhibitor.Thus, these findings support the potential antitumor activity revealed by these compounds since the Topo 1-DNA complex has been identified as a potential biological target for several anticancer drugs.This protein complex is more expressed in tumor cells than in normal cells.Lastly, in silico toxicity prediction studies have shown that the target compounds possess a low risk of causing theoretical mutagenic and tumorigenic effects compared to H 2 O 2 .Thus, based on the low cytotoxicity shown by compounds 9a,b and 10a,b, they can be considered lead scaffolds for further studies.

Figure 3 .
Figure 3. Synthetic methodologies employed to prepare the N1-propargylated uracil 6a, the N1-propargylated thymine 6b, the N1,N2-dipropargylated uracil 6c, and the propargylated theobromine 6d.The propargylated nucleobases 6a-d and the 4-(azidomethyl)coumarins 4a,b were hybridized through a 1,2,3-triazole ring by a Cu(I)-catalyzed [3+2]-cycloaddition reaction, as outlined in Figure4.The click reaction was successfully employed for all intermediates, using the simple copper(II) sulfate and sodium ascorbate system to generate the copper(I) catalyst.All the reactions were carried out in ethanol-water (10:1), at 30 • C, for 24 h.After the reaction completion, the target compounds were simply purified by pouring the reaction contents into an ice-water mixture.The precipitated products were filtered and presented a high purity degree, as observed by 1 H NMR. However, when impurities were noticed, they were removed by a short pad of silica gel.The target compounds 7a,b, 8a,b, 9a,b, and 10a,b were prepared from good to excellent yield, as shown in Figure4.All compounds were characterized by 1 H and 13 C NMR, HRMS, and melting point.The NMR and HRMS spectra for the final compounds are reported in the Supplementary Materials.

Figure 5 .Table 5 . 0 a
Figure 5. Predicted binding of most active in vitro compounds 9a,b and 10a,b poses (A) and 9a pose (B) in active site of Topo 1-DNA complex (PDB code: 1K4S).Graphic visualization obtained using UCSF Chimera (v.1.10.1)[51].Table5.Van der Waals (VDW) and H-bonding (H-bond) pharmacological interactions between ligands 9a,b, 10a,b, and the amino acid residues in the binding site of the Topo 1-DNA complex applying the Residues Consensus Analysis a .

Table 1 .
Compounds assessed in this study and their cytotoxicity against three different cell lines via the colorimetric MTT assay.

IC 50 (µM ± SD) a (Selectivity Index)
HaCat: human keratinocyte.c IC 50 for the positive control was not measured for the A459 cell line due to a lack of activity of the target compounds.

Table 2 .
Prediction of theoretical toxicity of 9a,b and 10a,b in comparison with H 2 O 2 a .

Table 3 .
Main biological target candidates identified by SEA predictions.

9a,b and 10a,b derivatives
on the Topo 1-DNA complex a .
a All energy values are given in kcal mol −1 .b Total binding energy = VDW + H-Bond + electrostatic interactions.VDW: van der Waal forces; H-bond: hydrogen bond.