New Hybrid Scaffolds Based on 5-FU/Curcumin: Synthesis, Cytotoxic, Antiproliferative and Pro-Apoptotic Effect

A series of 5-FU-Curcumin hybrids were synthesized, and their structures were elucidated by spectroscopic analysis. The synthesized hybrid compounds were evaluated in different colorectal cancer cell lines (SW480 and SW620) and in non-malignant cells (HaCaT and CHO-K1), to determine their chemopreventive potential. Hybrids 6a and 6d presented the best IC50 value against the SW480 cell line with results of 17.37 ± 1.16 µM and 2.43 ± 0.33 µM, respectively. Similarly, compounds 6d and 6e presented IC50 results of 7.51 ± 1.47 µM and 14.52 ± 1.31 µM, respectively, against the SW620 cell line. These compounds were more cytotoxic and selective than curcumin alone, the reference drug 5-fluorouracil (5-FU), and the equimolar mixture of curcumin and 5-FU. In addition, hybrids 6a and 6d (in SW480) and compounds 6d and 6e (in SW620) induced cell cycle arrest in S-phase, and, compounds 6d and 6e caused a significant increase in the sub-G0/G1 phase population in both cell lines. Hybrid 6e was also observed to induce apoptosis of SW620 cells with a respective increase in executioner caspases 3 and 7. Taken together, these results suggest that the hybrids could actively act on a colorectal cancer model, making them a privileged scaffold that could be used in future research.


Introduction
Colorectal cancer (CRC) is the second-deadliest and most widely diagnosed cancer in the world, accounting for 10% of all cancers. This pathology presents a great geographical distribution, and the patterns are very similar among men and women, being the third most common cancer in men and the second in women. Among the risk factors that contribute to CRC development, it is possible to include alcohol consumption, smoking, physical inactivity, obesity, diets high in fat and red meat, and inadequate intake of dietary fiber, vegetables, and fruits. Due to the widespread occurrence of the risk factors and the increase in the statistics, extensive research is ongoing to develop new pharmaceutical agents with chemopreventive potential against CRC [1]. 5-fluorouracil (5-FU) is one of the major anticancer agents used clinically for the treatment of several kinds of cancer, including CRC. However, this compound not only exhibits a short half-life and low selectivity but also various toxic side effects [2]. For these reasons, it has been modified in different ways to obtain conjugates and hybrid molecules, which may improve its therapeutic index and reduce the side effects [3]. Curcumin is one of the bioactive components of Curcuma longa Linn. Several studies with this compound have revealed important functions in cancer control [4], including antiproliferative activity against different cancer cell lines, inhibition at different stages of cancer cell progression [5], and its effect on a variety of growth factor receptors and cell adhesion molecules involved in tumor growth, metastasis, apoptosis, and multidrug resistance [6][7][8]. Considering the background described above and the urgent need to develop new therapeutic alternatives for the treatment of colorectal cancer, we synthesized several hybrids based on 5-FU and curcumin via the click reaction as a key step in the synthesis ( Figure 2). Besides, we evaluated their biological activity using colon adenocarcinoma cells (SW480) and their metastatic derivative (SW620) to determine the chemopreventive potential against this type of cancer. The chemopreventive potential of hybrids based on curcumin and resveratrol was evaluated against SW480 human colon adenocarcinoma cells, its metastatic derivative, and the SW620 cell line. Hybrid G displayed the best cytotoxic activity in both cell lines, with IC 50 values of 29.18 µM for SW480 and 20.44 µM for SW620 [18]. Curcumin-Pharmaceutics 2023, 15, 1221 3 of 25 chromone hybrid H was evaluated for its cytotoxic activity against human liver cancer cells (SMMC-7721), human gastric cancer cells (SGC-7901, MGC-803), and human glioma cells (U87). This compound displayed IC 50 values of 1.08, 0.56, 3.18, and 3.93 µM over SGC-7901, MGC-803, SMMC-7721, and U87, respectively. Moreover, this hybrid showed that it could induce apoptosis in gastric cancer cells by arresting the cell cycle, breaking mitochondrial function, and inhibiting TrxR activity [19]. A hybrid molecule based on curcumin and thalidomide I was tested on human multiple myeloma MM1S, RPMI8226, and U266 cells. The biological results showed that this compound exhibited significantly improved lethal effects towards all three human MM cell models (IC 50 = 4.55, 6.44, and 3.96 µM against RPMI8226, MM1S, and U266 cells, respectively). Furthermore, mechanistic studies in U266 cells demonstrated that compound I can induce the production of reactive oxygen species (ROS) and cause G1/S arrest, thus leading to apoptosis and cell death [20]. Hybrid J, which is based on curcumin and coumarin linked by a triazole ring, showed significant cytotoxic potential against THP-1, COLO-205, and HCT-116 cell lines with IC 50 values of 0.82, 4.68, and 2.21 µM, respectively. This compound was found to significantly inhibit tubulin polymerization (IC 50 = 0.82 µM in THP-1 tumor cells) [21] (Figure 1).
Considering the background described above and the urgent need to develop new therapeutic alternatives for the treatment of colorectal cancer, we synthesized several hybrids based on 5-FU and curcumin via the click reaction as a key step in the synthesis ( Figure 2). Besides, we evaluated their biological activity using colon adenocarcinoma cells (SW480) and their metastatic derivative (SW620) to determine the chemopreventive potential against this type of cancer. Considering the background described above and the urgent need to devel therapeutic alternatives for the treatment of colorectal cancer, we synthesized sev brids based on 5-FU and curcumin via the click reaction as a key step in the sy ( Figure 2). Besides, we evaluated their biological activity using colon adenocar cells (SW480) and their metastatic derivative (SW620) to determine the chemopre potential against this type of cancer.

General Remarks
Microwave reactions were carried out in a CEM Discover microwave reactor i vessels (monowave, maximum power 300 W, temperature control by an IR sens

General Remarks
Microwave reactions were carried out in a CEM Discover microwave reactor in sealed vessels (monowave, maximum power 300 W, temperature control by an IR sensor, and fixed temperature). 1 H and 13 C NMR spectra were recorded on a Varian instrument operating at 300 and 75 MHz, respectively. The signals of the deuterated solvent (CDCl 3 or DMSO-D6) were used as references. Chemical shifts (δ) are expressed in ppm with the solvent peak as a reference and TMS as an internal standard; coupling constants (J) are given in Hertz (Hz). HRMS was obtained using a Bruker Impact II UHR-Q-TOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) in positive mode. Silica gel 60 (0.063-0.200 mesh, Merck, Whitehouse Station, NJ, USA) was used for column chromatography, and precoated These compounds were obtained as described below. A mixture of 4-hydroxyacetophenone (1 mmol), K 2 CO 3 (1.5 mmol), and DMF (10 mL) was placed in a flat-bottomed flask of 25 mL equipped with a magnetic stirring bar and stirred for a period of 30 min. 1,ωdibromoalkane (1.2 mmol) was added to the reaction, and the mixture was sonicated for 1 h at 25 • C. After this time, water was added, and the mixture was transferred to a separation funnel and extracted with ethyl acetate. Anhydrous sodium sulfate was used to dry the organic phase. The liquid phase was concentrated under reduced pressure on a rotatory evaporator, and the residue was purified by flash chromatography on silica gel using a mixture of hexanes/ethyl acetate of different ratios as eluent. Bromoalkyl derivatives were obtained in yields ranging from 73-84%.  (3 mmol), and DMF (5 mL) were placed in a 10 mL flat-bottomed flask equipped with a magnetic stirring bar. Then, the mixture was heated to 100 • C at 200 W for a period of 15 min under microwave irradiation. Then, water was added, and the mixture was transferred to a separation funnel and extracted with ethyl acetate. Anhydrous sodium sulfate was used to dry the organic phase. The liquid phase was concentrated under reduced pressure on a rotatory evaporator, and the residue was purified by flash chromatography on silica gel using a mixture of hexanes/ethyl acetate of different ratios as the eluent. Alkylazide derivatives were obtained in yields ranging from 85-96%.  Acetic acid (5 mmol) was added to pyrrolidine (5 mmol) cooled in a bath of ice and water for 10 min, and then the mixture was warmed to 30 • C. Then, compounds 3a-h (1 mmol) and vanillin (1 mmol) were added. The mixture was stirred at 30 • C for 2 h. Then, water was added, and the mixture was transferred to a separation funnel and extracted with ethyl acetate. Anhydrous sodium sulfate was used to dry the organic phase. The liquid phase was concentrated under reduced pressure on a rotatory evaporator, and the residue was purified by flash chromatography on silica gel using a mixture of hexanes/ethyl acetate of different ratios as the eluent. Alkylazide derivatives were obtained in yields ranging from 68-82%.  13 13 (5) 5-fluorouracil (1 mmol), HMDS (2.0 mmol), KI (0.5 mmol), and ammonium sulfate (0.5 mmol) were placed in a 25 mL flat-bottomed flask. The mixture was refluxed at 120 • C for 3 h. Then, the temperature was lowered to 90 • C, and 5 mL of acetonitrile and propargyl bromide (2.0 mmol) was added; the mixture was left to reflux at 90 • C for 12 h. The reaction was then concentrated under reduced pressure on a rotatory evaporator, and the residue was purified by flash chromatography on silica gel using a mixture of hexanes/ethyl acetate of different ratios as the eluent to obtain compounds 5 in 70% yield.
Yield In a 10 mL flat-bottomed flask, were placed propargyl-5-FU (5) (1 mmol), chalconealkylazides (4a-h) (1 mmol) DMF (5 mL), and the mixture was sonicated for 5 min to 40 • C. After this time, was added a mixture of ascorbic acid (0.5 mmol), copper acetate (0.5 mmol), DMF (1 mL), and water (1 mL) and the reaction mixture and sonicated for 1 h to 40 • C. Then, 10% HCl was added and extracted with ethyl acetate. The organic phase was dried on anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was subjected to crystallization (MeOH:H 2 O, 1:1 ratio). Finally, the solid obtained was purified by preparative chromatography on silica gel to obtain compounds 6a-h (88-96% yield).     , supplemented with 10% equine serum previously inactivated at 56 • C, 1% penicillin/streptomycin, and 1% non-essential amino acids, and incubated at 37 • C in an atmosphere with 5% CO 2 . To carry out the experiments, the serum was diluted to 3% and additionally supplemented with 10 mg/mL insulin, 5 mg/mL transferrin, and 5 ng/mL selenium. Cells were used for experimentation when they reached 80-90% confluence, using a 1X trypsin enzymatic disintegration method. To avoid contamination by Mycoplasma spp., cell cultures were periodically analyzed by polymerase chain reaction (PCR) [22].

Cell Viability
Cell viability was evaluated with a sulforhodamine B (SRB) assay. This technique provides an index of protein contents in living and adherent cells, whose optical density can be related to cell concentration. Initially, 20,000 cells were seeded per well in 96-well culture plates. An initial adherence period of 24 h was performed at 37 • C, 5% CO 2 , and after this, the medium was replaced by another that contained the hybrid compounds in different concentrations. Then, the cells were left incubating for 24 and 48 h under the same conditions previously described. After the respective incubation times, the cells were fixed with trichloroacetic acid (50% v/v) for 1 h at 4 • C and stained with 0.4% (p/v) of SRB. Subsequently, washings with 1% acetic acid were carried out to eliminate possible nonspecific bindings, and after solubilization with 10 mM Tris-base, the absorbance was read at 492 nm in a microplate reader (Mindray MR-96A) [23]. All the experiments were carried out in triplicate.

Antiproliferative Activity
For this experiment, the same colorimetric method (SRB) from the evaluation of cell viability was used, although with some modifications. Initially, 2500 cells per well were seeded in 96-well culture plates, and a 24 h adherence period was performed at 37 • C in a 5% CO 2 atmosphere. Afterwards, the cell lines were subjected to treatment with increasing concentrations (ranges dependent on the IC 50 values) of the hybrid compounds that exhibited greater activity in the initial screening. For this assay, incubation periods of 0, 2, 4, 6, and 8 days were used, and the fixation, staining, and reading conditions were the same as those previously described for the cytotoxicity assay [23].

Cell Cycle Analysis
The method used for the recognition of cell cycle phases (G0/G1, S, G2/M) was based on staining with a fluorescent dye (propidium iodide-PI), as a direct relationship between fluorescence intensity and DNA content can be established. For this method, 250,000 cells/well were initially seeded in 6-well culture plates with an adherence period of 24 h at 37 • C in a 5% CO 2 atmosphere. After this time, the cells were treated with the respective IC 50 value of the hybrid compound and vehicle control for 48 h. After treatment, cells were obtained by mechanical disaggregation, and the cell pellet was centrifuged and washed with a versene buffer. The cell pellet was fixed with 1.8 mL of cold 70% ethanol and kept at 4 • C overnight. Subsequently, two washes with a versene buffer were performed, and the pellet was resuspended in 300 µL of PBS containing 0.25 mg/mL of RNAsa and 0.1 mg/mL of PI. After a 30 min period of dark incubation, the fluorescence of 10,000 cells was analyzed using a FACS Canto II flow cytometer. The PI signal was analyzed with excitation at 488 nm using a sapphire laser, and fluorescence was detected at 610 nm. The data analysis was performed with FlowJo 10.8.1 software [24].

Measurement of Mitochondrial Membrane Potential (∆Ψm)
The fluorescent dyes PI and DIOC6 (3,3'-dihexyloxacarbocyanine iodide) were used to observe the depolarization of the mitochondrial membrane as well as possible cell membrane damage. For this method, 250,000 cells/well were initially seeded in 6-well culture plates during a 24 h adherence period at 37 • C in a 5% CO 2 atmosphere. After this time, the cells were treated with the respective IC 50 of the hybrid compound and vehicle control for 48 h. After treatment, cells were obtained by mechanical disaggregation, stained with DiOC6 and PI, and incubated at room temperature for 30 min in the dark. Ten thousand events were analyzed by flow cytometry with excitation at 488 nm and emission detection with green (530/15 nm) and red (610/20 nm) filters [24].

Cell Death Induction by 5-FU/Curcumin Hybrids
For this method, 250,000 cells/well were initially seeded in 6-well culture plates during a 24 h adherence period at 37 • C in a 5% CO 2 atmosphere. After this time, the cells were treated with the respective IC 50 of the hybrid compound and vehicle control for 48 h. After treatment, cells were obtained by mechanical disaggregation, resuspended in a solution with Annexin-V-FLUOS and PI, and incubated for 20 min in the dark before flow cytometry analysis [25].

Determination of Apoptotic Biomarkers
To provide an approach to the possible molecular mechanisms through which the hybrid compounds induce an effect on the cell lines, different proteins related to the process of death by apoptosis were determined. For this method, 1,000,000 cells were initially seeded per Petri dish, with an adherence period of 24 h at 37 • C in a 5% CO 2 atmosphere. After this time, the cells were treated with the respective IC 50 of the hybrid compounds for 48 h. Later, the consumed medium was discarded, and after the addition of the versene buffer, the cells were obtained by mechanical disaggregation. The cells were then lysed with a cell lysis buffer and centrifuged to obtain the supernatant, which was used to determine the biomarkers of interest by enzyme-linked immunosorbent assays. These ELISA kits were provided by Elabscience Biotechnology Co., Houston, TX, USA [26].

Statistical Analysis
The results were analyzed with GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA). The experiments were performed in triplicate, and the data were presented as the mean ± standard error (SE). Results were analyzed with one-way ANOVA, and statistical differences were identified using Tukey's or Dunnett's test. In all cases, a p < 0.05 was considered significant.

Chemistry
The synthesis of hybrids began with the Williamson reaction between acetophenone (1) and 1, ω-dibromoalkanes (ω = 3, 4, 5, 8, 9, and 12) to afford the bromo-alkylacetophenones 2a-h with 73-84% yields [27,28]. Compounds 2a-h were treated with sodium azide, leading to the obtention of the acetophenone-alkylazides 3a-h in 85-96% yields [29,30]. The above compounds were subjected to an aldol condensation reaction with vanillin, giving the chalcones 4a-h in 68-82% [31]. Besides, the reaction of 5-FU with propargyl bromide led to propargyl-5-FU (5) with a 70% yield [32]. Finally, the click reaction between chalcone-alkylazides 4a-h and compound 5 led to the obtention of hybrids 6a-h in 88-96% yields [30,33] (Scheme 1). The parental compounds curcumin and 5-fluorouracil (5-FU, the reference drug) were used as controls. All the compounds, with the exception of the 6g and 6h hybrids, showed an improvement in their cytotoxic activity against the SW480 and SW620 tumor cell lines 48 h after the start of the treatments, compared to the results obtained at 24 h (Tables 1 and 2). The range of IC 50 values was found between 2.43 and 29.71 µM in SW480 cells and between 7.51 and 45.10 µM in SW620 cells. These values were lower than those found for the starting compound curcumin and the reference drug 5-FU. Additionally, the hybrid compounds except for 6f, 6g, and 6h presented better IC 50 values in the SW480 cell line regarding curcumin and the equimolar mixture. The same effect could be seen in SW620 cells with the hybrid compounds 6d and 6e. During the treatments, the cells were observed under an optical microscope. As shown in Figure 3, it was identified that cells presented an altered morphology with changes in size and shape, in addition to an evident decrease in cell population compared to the control. The severity of these changes increased as a function of time and dose.  The increase in cytotoxic activity over time was also reflected in some of the obtained on the non-tumor cell lines HaCaT and CHO-K1, a situation that negativ fects the selectivity indices (SI) by generating a decrease in them. Only compounds 6d in SW480 cells and compounds 6d and 6e in SW620 cells increased their SI ov post-treatment; moreover, these same compounds presented the lowest IC50 comp the starting compounds and the equimolar mixture.
Similar results regarding the same starting compounds, curcumin and 5-FU, a in combination with other pharmacophores, have been reported. Zhang et al., 20 The increase in cytotoxic activity over time was also reflected in some of the results obtained on the non-tumor cell lines HaCaT and CHO-K1, a situation that negatively affects the selectivity indices (SI) by generating a decrease in them. Only compounds 6a and 6d in SW480 cells and compounds 6d and 6e in SW620 cells increased their SI over 48 h post-treatment; moreover, these same compounds presented the lowest IC 50 compared to the starting compounds and the equimolar mixture.   Regarding the structure-activity relationship (SAR), one of the objectives of the biological evaluation was to recognize the importance of the length of the aliphatic chain that links the chalcone analog of curcumin with the triazole, which in turn binds to 5-FU. After analyzing the results of the IC 50 value, it was possible to evidence an improvement in the biological activity of the hybrid compounds when the chain length of the linker increased until it reached a length of 3 carbons. At this point, the most representative IC 50 value of the entire screening was obtained (2.43 ± 0.33 µM in SW480 and 7.51 ± 1.47 µM in SW620). This allowed us to affirm that when the hybrid compound had a specific chain length of 3 carbons, the interaction with one or more molecular targets in the cell reached its maximum point of activity, and as a result, the highest levels of specific cytotoxicity were generated. Therefore, when observing the results obtained with the hybrids having chain lengths of 4, 6, 7, and 10 carbons, it was evident how the biological activity of these cell lines decreased (Figure 4).    In accordance with the cytotoxicity and high SI, compounds 6a, 6d, and 6e were chosen to continue with the other biological assays.

Antiproliferative Effect of 5-FU/Curcumin Hybrids on SW480 and SW620 Cells
According to the antiproliferative activity of compounds 6a and 6d (against the SW480 cell line) and 6d and 6e (against the SW620 cell line), it was possible to identify that the hybrid compounds showed a decrease in the percentages of cell viability with a dependence on concentration and time (Table 3). Thus, compound 6a in the SW480 cell line showed significant differences in the inhibition percentages from day 2 at a concentration of 5 µM compared to the control and maintained a trend of decreasing viability until day 8 at all concentrations evaluated. A similar situation was presented with hybrid 6d in this same cell line, where the main differences in inhibition began to appear from day 2 with concentrations higher than 2.5 µM ( Figure 5). . Antiproliferative activity of hybrids 6d and 6e in SW620 cells. Data are presented as the mean ± SE of three independent experiments. (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). Vehicle control was assumed to be 100% viable.  . Antiproliferative activity of hybrids 6d and 6e in SW620 cells. Data are presented as the mean ± SE of three independent experiments. (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). Vehicle control was assumed to be 100% viable.
Regarding the results obtained against the SW620 cell line with hybrids 6d and 6e, it was possible to state that from concentrations of 5 µM and 20 µM on day 2, a significant decrease in cell viability began to be observed, reaching inhibition percentages higher than 30%. Similar results were obtained and published by our research group from hybrids derived from 5-FU and genistein linked with a triazole [17], demonstrating the antiproliferative activity of compounds 4a and 4g on SW480 and SW620 cell lines with a significant inhibitory effect over time at low concentrations (6.25 µM). Similarly, Goldhahn et al., 2015 [37] evaluated the antiproliferative potential of the curcumin analog HP102 on Jurkat T cells, finding an enhanced effect with respect to the other analogs and curcumin.

5-FU/Curcumin Hybrids Induce Cell Cycle Arrest on SW480 and SW620 Cells
Cell lines SW480 and SW620 were treated with the previously obtained IC 50 value, and their effect on cell cycle distribution was evaluated. The hybrid compound 6a showed in cell line SW480 a significant increase in the cell population in the S phase of the cycle with a slight decrease in the population in the G0/G1 phase, but no significant changes in the G2/M and sub-G0/G1 phases. This could suggest an arrest in the cell cycle and the possible activity of this compound as a cytostatic agent. A different situation was presented with the hybrid 6d in the SW480 cell line, which generated significant changes in all phases of the cycle at the expense of an increase in the sub-G0/G1 population with a corresponding decrease in the cell population in the G0/G1, S, and G2/M phases, suggesting that the cells suffered death after 48 h of treatment ( Figure 6). The same situation was presented with this same hybrid compound and with compound 6e when evaluated in the SW620 cell line, where both showed that more than 40% of the cell population was in the sub-G0/G1 phase (Figure 7). Similar findings were reported by Zhang et al., 2019 [34], with the hybrid compound 14 derived from the drugs 5-FU and Oxaliplatin. They reported its cytostatic activity by presenting cell cycle S-phase arrest against HCT-116 cells post-treatment. Similarly, Focaccetti et al., 2015 [38] confirmed the activity of 5-FU on the S phase of the cell cycle by inhibiting DNA synthesis due to a restriction in the availability of the enzyme thymidylate [38]. Other studies performed with the starting compound curcumin showed similar results to ours. Rahim et al. 2021 [39] reported that the DMCH analog of curcumin increased by more than 70% the sub-G0/G1 population in the cell cycle after treatment against HT29 colorectal adenocarcinoma cells and the metastatic SW620. The results of our study allowed us to demonstrate that the hybrid compounds 6a, 6d, and 6e affect the cell cycle machinery, exerting their cytotoxic and cytostatic effects against SW480 and SW620 cells.
curcumin showed similar results to ours. Rahim et al. 2021 [39] reported that the DMCH analog of curcumin increased by more than 70% the sub-G0/G1 population in the cell cycle after treatment against HT29 colorectal adenocarcinoma cells and the metastatic SW620. The results of our study allowed us to demonstrate that the hybrid compounds 6a, 6d, and 6e affect the cell cycle machinery, exerting their cytotoxic and cytostatic effects against SW480 and SW620 cells.

Changes in Mitochondrial Membrane Potential (ΔΨm) Induced by 5-FU/Curcumin Hybrids
Changes in the mitochondrial membrane potential are an indicator to assess the loss of mitochondrial integrity, which would reflect the onset of a proapoptotic signal [40]. According to the results obtained and illustrated in Figure 8, hybrid compounds 6a and 6d in the treatment against SW480 cells and hybrids 6d and 6e in the treatment against Figure 7. Cell cycle distribution after treatment with hybrids 6d and 6e in SW620 cells and representation of distribution percentages in bar chart form. All experiments were performed in duplicate and showed similar results. p-values lower than 0.05 were considered statistically significant (** p < 0.01; *** p < 0.001; **** p < 0.0001).

Changes in Mitochondrial Membrane Potential (∆Ψm) Induced by 5-FU/Curcumin Hybrids
Changes in the mitochondrial membrane potential are an indicator to assess the loss of mitochondrial integrity, which would reflect the onset of a proapoptotic signal [40]. According to the results obtained and illustrated in Figure 8, hybrid compounds 6a and 6d in the treatment against SW480 cells and hybrids 6d and 6e in the treatment against SW620 cells do not induce significant changes in the mitochondrial membrane potential; this was evidenced by not finding a cell population located in the lower left quadrant (low Dioc6). Additionally, in the treatments with the hybrid compounds 6a and 6d in SW480 cells, an increase in the PI+ population (upper quadrant) of 15% and 23.7%, respectively, was observed, as well as compound 6d versus SW620 cells that concentrated most of their cells in this same quadrant (76.1%), which is indicative of dormant cells that lose the polarization of the plasma membrane, with damage to the membrane, or correspond to dead cells. concentration-dependent treatment with the hybrid compound 7d. In contrast, we reported in our research group with hybrids 4a and 4g, derived from 5-FU and genistein, that no significant changes in the ΔΨm were induced, supporting the hypothesis related to a possible cytostatic-like activity on SW480 and SW620 cells [17].

Cell Death Induction by 5-FU/Curcumin Hybrids
In the early stages of apoptosis, changes occur at the cell surface. One of these changes is the translocation of phosphatidylserine from the inner side of the plasma membrane to the outer layer, exposing it to the cell surface. Annexin V is a phospholipid-binding protein with a high affinity for phosphatidylserine, therefore, cells in early apoptosis show unique staining for Annexin V/FITC. After 48 h of exposure to the treatments with the hybrid compounds, it was observed that hybrid 6a did not induce significant changes in the integrity of the plasma membrane of the SW480 cell line. These results were similar to those observed for the control, preserving the higher percentage of cells in the Q4 quadrant of viable cells. On the other hand, the hybrid compound 6d against this same cell line induced a significant displacement of almost half of the cell population to the Q3 quadrant (50.2%), possibly implying a mechanism of cell death by apoptosis (early apoptotic cells), where the exposure of phosphatidylserine in the plasma membrane was evidenced with- These results support the previous hypothesis in the analysis of the cell cycle distribution with hybrid 6a, where a possible cytostatic effect was observed, while the other hybrids showed cytotoxic activity on the cell lines, possibly exerting a mitochondria-independent cell death mechanism. Other studies performed with curcumin-derived hybrid compounds reported a decrease in mitochondrial membrane potential in human cancer cells. Quan et al., 2018 [19] reported a significant decrease in mitochondrial membrane potential due to ROS accumulation in gastric cancer cells following time-and concentrationdependent treatment with the hybrid compound 7d. In contrast, we reported in our research group with hybrids 4a and 4g, derived from 5-FU and genistein, that no significant changes in the ∆Ψm were induced, supporting the hypothesis related to a possible cytostatic-like activity on SW480 and SW620 cells [17].

Cell Death Induction by 5-FU/Curcumin Hybrids
In the early stages of apoptosis, changes occur at the cell surface. One of these changes is the translocation of phosphatidylserine from the inner side of the plasma membrane to the outer layer, exposing it to the cell surface. Annexin V is a phospholipid-binding protein with a high affinity for phosphatidylserine, therefore, cells in early apoptosis show unique staining for Annexin V/FITC. After 48 h of exposure to the treatments with the hybrid compounds, it was observed that hybrid 6a did not induce significant changes in the integrity of the plasma membrane of the SW480 cell line. These results were similar to those observed for the control, preserving the higher percentage of cells in the Q4 quadrant of viable cells. On the other hand, the hybrid compound 6d against this same cell line induced a significant displacement of almost half of the cell population to the Q3 quadrant (50.2%), possibly implying a mechanism of cell death by apoptosis (early apoptotic cells), where the exposure of phosphatidylserine in the plasma membrane was evidenced without PI entering the cell ( Figure 9A). This same compound 6d against the SW620 cell line induced plasma membrane disintegration, as evidenced by the significative displacement of the cell population through the upper quadrant with positive staining for PI and Annexin V-FITC. Finally, treatment with compound 6e concentrated most of its cell population (84.7%) in the Q4 quadrant of viable cells (negative for Annexin V-FITC and negative for PI) ( Figure 9B). Other authors reported that the anticancer effect of curcumin on colorectal cancer cells is associated with the activation of the apoptosis pathway, involving multiple molecular targets [41]. Weng et al., 2015 [42] reported the treatment of SGC-7901 gastric cancer cells with curcumin-derived compounds, where the 5-and 28-hybrids significantly and dose-dependently increased cell apoptosis compared to negative control cells. Similarly, Ndreshkjana et al., 2019 [15] reported a significant increase in the fraction of apoptotic cells in 5-FU and thymoquinone-derived hybrid (SARB) treatments against HCT-116 cells.

Determination of Apoptotic Biomarkers
To complement the findings of the previous experiments, we proceeded to evaluate some biomarkers of importance in the process of cell death by apoptosis. The levels of caspase 3, caspase 7, caspase 8, and the tumor suppressor protein p53 were calculated after treatment with the hybrid compounds 6a, 6d, and 6e in the SW480 and SW620 cell lines. Significantly increased levels of apoptotic cell death executioner caspases 3 and 7 were detected after treatment with hybrid compound 6e in the SW620 cell line, which could be indicative of a possible mechanism of apoptotic cell death independent of the p53 status; however, further investigations are needed to confirm this hypothesis ( Figure 10) [41] demonstrated that curcumin induces cell death by apoptosis in a p53 status-independent manner in colon cancer cells, highlighting its therapeutic potential in cancer treatment when there is resistance to chemotherapy due to defects in p53 expression or function. We also evaluated the same biomarkers in SW480 cells; however, we did not observe significant changes regarding the control. 1 1 Figure 9. Apoptosis of SW480 (A) and SW620 (B) cells following treatment with the hybrid compounds 6a, 6d, and 6e. The histograms represent one of at least two different experiments. Q1 + Q2: Cells that lost membrane integrity (late apoptotic cells, dead cells, necroptotic cells, secondary necrotic cells, others); Q3: Early apoptotic cells; Q4: Viable cells. Data are presented as the mean of these two populations ± SE of three independent experiments. p-values lower than 0.05 were considered statistically significant (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).
Regarding the other results, no significant differences were found in the levels of biomarkers after treatments in cells with hybrid compounds 6a and 6d; therefore, it may be inferred that hybrid compound 6a presents a cytostatic rather than a cytotoxic effect and that compound 6d induces a caspase-independent cell death process.

Conclusions
This study allowed us to demonstrate for the first time the chemopreventive potential of the hybrids synthesized from the natural compound curcumin and the reference anticancer drug 5-fluorouracil. The hybrid compounds exhibited cytotoxic activity against colon adenocarcinoma cells SW480 and its metastatic derivative SW620, with the most active compounds 6a, 6d, and 6e having IC50 values ranging from 2.43-29.71 µM and 7.51-45.10 µM, respectively, for each cell line 48 h post-treatment. Furthermore, these compounds were selective when evaluated in the non-tumor cell lines HaCaT and CHO-K1, with better results than the starting compounds curcumin and 5-FU and their equimolar mixture. Under the conditions evaluated, we were able to demonstrate the antiproliferative effect of the hybrids and identify in compound 6a its probable mechanism of cytostatic action against SW480 cells by analyzing the cell cycle effect and its S-phase arrest. Likewise, we were able to conclude that compound 6d induced SW480 and SW620 cell death by a mitochondria-and caspases-independent death mechanism. Finally, compound 6e induced significant upregulation of executioner caspases 3 and 7, suggesting a possible cell death by apoptosis; however, further experimental testing is required to clearly determine the death mechanisms associated with the evaluated hybrids.
All these findings highlight the chemopreventive potential of natural compounds in the molecular hybridization strategy, especially those inspired by curcumin in combination with different scaffolds such as the drug 5-FU, pointing out the importance of using them in further research, contributing to the design of new molecules with great potential and better selectivity compared to conventional chemotherapy.

Conclusions
This study allowed us to demonstrate for the first time the chemopreventive potential of the hybrids synthesized from the natural compound curcumin and the reference anticancer drug 5-fluorouracil. The hybrid compounds exhibited cytotoxic activity against colon adenocarcinoma cells SW480 and its metastatic derivative SW620, with the most active compounds 6a, 6d, and 6e having IC 50 values ranging from 2.43-29.71 µM and 7.51-45.10 µM, respectively, for each cell line 48 h post-treatment. Furthermore, these compounds were selective when evaluated in the non-tumor cell lines HaCaT and CHO-K1, with better results than the starting compounds curcumin and 5-FU and their equimolar mixture. Under the conditions evaluated, we were able to demonstrate the antiproliferative effect of the hybrids and identify in compound 6a its probable mechanism of cytostatic action against SW480 cells by analyzing the cell cycle effect and its S-phase arrest. Likewise, we were able to conclude that compound 6d induced SW480 and SW620 cell death by a mitochondria-and caspases-independent death mechanism. Finally, compound 6e induced significant upregulation of executioner caspases 3 and 7, suggesting a possible cell death by apoptosis; however, further experimental testing is required to clearly determine the death mechanisms associated with the evaluated hybrids.
All these findings highlight the chemopreventive potential of natural compounds in the molecular hybridization strategy, especially those inspired by curcumin in combination with different scaffolds such as the drug 5-FU, pointing out the importance of using them in further research, contributing to the design of new molecules with great potential and better selectivity compared to conventional chemotherapy.