In silico and in vitro evaluation of drug-like properties and anticancer potential of novel 5-fluorouracil derivatives

Abstract

The development of more effective and safer anticancer agents remains a critical focus in cancer therapy. While biologics are gaining attention, small molecules continue to play a significant role due to their versatility and cost-effectiveness. This study investigates the potential of synthesized 5-fluorouracil derivatives to improve its drug-like properties, as well as anticancer activity in human lung cancer cells. Among the synthesized compounds, two (2c and 3a) demonstrated greater cytotoxicity and selectivity toward A549 lung cancer cells compared to 5-FU. These derivatives did not induce oxidative stress but instead affected antioxidant enzyme activity, suggesting interference with cellular metabolism. Additionally, these derivatives shifted cells toward hypodiploidy, suggesting the presence of apoptotic cells, while 5-FU significantly increased A549 cell ploidy, a trait linked to cancer progression and treatment resistance. In silico predictions revealed improved pharmacokinetic properties for the 2c derivative and a low probability of hepatotoxicity or neurotoxicity compared to 5-FU. These findings suggest that 2c and 3a derivatives warrant further research as potentially safer and more effective alternatives to 5-FU.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-23237-y.

Introduction

The search for more effective and safer anticancer agents remains a critical focus in cancer therapeutics, with the need to enhance the efficacy of existing treatments while minimizing their toxicity. While biologic therapies and immunotherapies are increasingly gaining attention in cancer treatment for their ability to specifically target cancer cells and modulate the immune system, small molecules continue to hold a prominent position in cancer research due to their ability to target a wide variety of cellular processes, their potential for development into oral medications with favorable pharmacokinetic properties, ease of synthesis, lower production costs, and other advantages. Patient access to drugs is a pressing issue, especially when comparing biologics to small molecules. On average, a daily dose of a biologic is 22 times more expensive than that of a small molecule¹. The generation of new small molecules is often more effective when starting with a known scaffold that has an established mechanism of action. This approach can significantly enhance the likelihood of success in drug development by optimizing efficacy, safety, and pharmacokinetic properties².

The 5-fluorouracil (5-FU) molecule is one of the earliest chemotherapy drugs developed globally and continues to be used to this day³. 5-FU is an antimetabolite drug that is widely used to treat different malignant tumors, including breast, skin, esophageal, pancreatic, stomach, as well as colorectal cancer and head and neck cancers^4,5. 5-FU’s cytotoxic mechanism involves the inhibition of thymidylate biosynthesis or the misincorporation of fluorinated nucleotides into newly synthesized DNA or RNA⁶. As shown by numerous studies summarized in a number of reviews, 5-FU affects such important cellular functions as the cell cycle, pro-oxidant and antioxidant systems, apoptosis and autophagy, causes epigenetic modifications and metabolic changes, etc^7–10. Over the past four decades, various fluoropyrimidines, such as capecitabine, tegafur, and a different class like cytarabine, have been developed to address 5-FU’s limitations, including its low bioavailability, short half-life, rapid metabolism, and the development of drug resistance following chemotherapy, all of which reduce its therapeutic efficacy^5,8.

Lung cancer is one of the most common cancers and has one of the highest mortality rates in both men and women. Patients with lung cancer are mainly treated with chemotherapy. However, certain patients do not respond to this therapy or may respond well initially but gradually show signs of a relapse. This results in increased drug doses, which may cause adverse drug reactions or the development of drug resistance¹¹. Thus, developing new drugs for the treatment of lung cancer is crucial. Although 5-FU is widely used to treat cancers, consequent drug resistance seriously limits its clinical use in lung cancer treatment¹².

This study aimed to synthesize 5-FU derivatives and evaluate potential enhancements in their drug-like properties using in silico modeling and their anticancer efficacy through in vitro assays in lung cancer cells. From the drug-likeliness perspective we aimed to synthesize 5-FU derivatives to prolong systemic exposure by increasing plasma-protein binding (PPB), thereby offsetting 5-FU’s short half-life; increase oral absorption (HIA) and passive permeability (Caco-2), addressing its low oral bioavailability; reduce off-target liabilities (hepato-, neuro-, and nephrotoxicity; hERG liability; and mutagenic/genotoxic/carcinogenic potential relevant to secondary cancer risk, without compromising a drug-like physicochemical profile. To assess anticancer potential relative to 5-FU, cytotoxicity, oxidative stress modulation, activities of antioxidant enzymes such as superoxide dismutase and catalase, and alterations in cellular ploidy were studied.

Results and discussion

Chemistry

The target N-mono- and N-bis-substituted pyrimidine-2,4(1 H,3 H)-diones were synthesized by the interaction of pyrimidines 1a-c according to the scheme:

1a-c: R = H (a), F (b), Br (c). 2a-e, 3a-e: R, R¹ = H, 2-MeO-5-CHO (a), F, 3-NO₂−4-MeO (b), Br, 2-MeO-5-CHO (c), H, 3-NO₂−4-MeO (d), F, 2-MeO-5-Cl (e). 4a, b: R, R¹, R² = H, 2-MeO-5-CHO, CH₂(4-FC₆H₄) (a), F, 3-NO₂−4-MeO, CH₂C CH (b).

Heating of pyrimidines 1a–c with the corresponding benzyl chlorides in DMSO in the presence of K₂CO₃ at 70 °C for 2 h afforded the target pyrimidines as mixtures of N¹-monosubstituted derivatives 2a–e and N¹,N³-bis-substituted derivatives 3a–e, which were separated by treatment with 15% aqueous NaOH (see Experimental part). Previously, the position of the N¹-(2,4-dimethyl)benzyl and N¹-(2,5-dimethyl)benzyl groups in uracil and 5-fluorouracil derivatives was confirmed based on NOESY spectra, which revealed a sterically close mutual arrangement of the CH₂ groups and the 6-H atom in the ring of monosubstituted pyrimidines¹³. N¹-Mono- and N¹.N³-bisubstituted uracil derivatives 2a, 3a¹⁴, 5-fluorouracil derivatives 2b, 3b^15,16 and 5-bromouracil derivatives 2c, 3c¹⁴ have been described previously. The resulting N¹-derivatives of uracil and 5-fluorouracil were then alkylated under the above-described conditions with 4-fluoro-1-chloromethylbenzene¹⁷ and 3-bromoprop-1-yne, respectively, to afford disubstituted uracil derivative 4a and 5-fluorouracil derivative 4b.

The 5-FU molecule is analogous to the uracil nucleotide, though with a fluorine atom at the hydrogen site at the C-5 position. The replacement of the hydrogen atom by fluoride causes significant changes in the reactivity of the pyridine ring. Although the size of the fluorine atom is similar to that of hydrogen, fluorine is an electron-withdrawing atom, which lowers the pKa of 5-FU compared to natural pyrimidines¹⁸. In acidic environments, such as those found in tumors, drugs with a lower pKa tend to perform better. From this perspective, electron-withdrawing groups like nitro (NO₂), formyl (CHO), and fluoro-substituted phenyl groups (4-FC₆H₄) as well as chlorine and to a lesser extent bromine, may further lower the pKa of molecules compared to 5-FU. In contrast, the methoxy group (MeO) is electron-donating, which could increase the pKa. However, this electron-donating effect may also influence the metabolism of 5-FU derivatives. Methoxy groups often enhance metabolic stability by reducing the molecule’s reactivity, making it less susceptible to rapid metabolism by enzymes like cytochrome P450. This could lead to a longer half-life and more controlled drug release, improving therapeutic effectiveness and reducing dosing frequency. Methoxy groups can also slightly increase a molecule’s lipophilicity by introducing hydrophobic character through their methyl group. It potentially can enhance cell membrane permeability. Compared to 5-F, a highly polar molecule with low affinity for cell membranes and limited bioavailability¹⁹, this can be an advantage. The vinyl group (CH2C ≡ CH) supposed to have minimal impact on the pKa due to its lack of significant electron effects, but it can increase lipophilicity, which may enhance cell membrane penetration. Additionally, the vinyl group is reactive and can undergo various chemical reactions, including nucleophilic and electrophilic additions. This increased reactivity could be advantageous for designing prodrugs. Based on general drug-like criteria, the most balanced physicochemical properties among they 2a-e group were observed for compound 2e, which demonstrated the optimal combination of molecular weight (MW), topological polar surface area (TPSA), logP, and rigidity (Fig. 1). Compounds 2c and 2d also exhibited favorable properties and were selected for further experiments. From the 3a-e group, only compound 3a was selected, as the others exhibited excessively high TPSA, flexibility, or logP values, while 3a’s properties remained within an acceptable range for drug-likeness. Compounds 4a and 4b were selected for further experiments due to their acceptable drug-like properties and complex functionalities, making them valuable for detailed analysis.

Fig. 1.

Heatmap of the physicochemical properties. The color bar represents the score for physicochemical properties, where 0 (light red) indicates values within the acceptable range for potential pharmaceuticals, and 1 (dark red) indicates values outside the acceptable range.

Evaluation of in silico drug-like and toxicity properties predictions

The ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) properties of the 5-FU derivatives were predicted using the Toxometris-ADMET-Suite application. The complete list of predictions is available in Supplementary information Table S2. The drug-like properties of 5-FU derivatives were evaluated based on selected predictions of ADMET endpoints, including solubility (logS), human intestinal absorption (HIA), Caco-2 permeability (Caco2), and plasma protein binding (PPB). These parameters are essential to evaluate the pharmacokinetic profile of 5-FU derivatives as potential drugs²⁰. The prediction score was calculated by assessing how the predicted values aligned with the acceptable range established for potential pharmaceuticals (Fig. 2a). The predicted logS values for the derivatives ranged from − 3.6 to −4.6, indicating low aqueous solubility for the compounds. However, these logS values fall within the typical range for most commercially available drugs, which have logS values between − 5 and 1²¹. Thus, the scoring function categorized these compounds as having moderate to good potential; however, they were later dissolved in DMSO due to their low solubility in media and/or water, as predicted. The predicted logS value for 5-FU was − 1.1, which corresponds to the experimental data of approximately − 1.03²². This suggests that the modifications made to its derivatives have led to a reduction in solubility for these compounds. Predictions for HIA and Caco-2 permeability offer information on a compound’s ability to cross intestinal barriers, which is essential for evaluating its potential oral bioavailability²³. While the Caco-2 permeability predictions, which are based on in vitro cellular assay data, indicated strong permeability and thus good bioavailability for all derivatives, the HIA model, based on human absorption data, predicted moderate bioavailability for derivative 2c, and low bioavailability for the remaining compounds. It is important to note that 5-FU is typically administered intravenously due to its low oral bioavailability, though conclusive studies are limited²⁴. Interestingly, in vitro Caco-2 permeability studies have demonstrated a high absorption rate for 5-FU²⁵, suggesting potential limitations of this assay in predicting in vivo absorption. In this case, greater emphasis should be placed on HIA model predictions. Rather than focusing solely on absolute values, a comparative analysis of the probability scores suggests that derivative 2c may offer higher bioavailability potential than 5-FU. The 5-FU is known for its rapid degradation, with reported in vivo half-life times of only 10 to 20 min following administration. This short half-life demands continuous intravenous infusion to maintain therapeutic levels of 5-FU. In the case of new 5-FU derivatives, the PPB has been predicted to understand the potential half-life of the molecules. In contrast to 5-FU the derivatives were predicted to have good plasma protein binding properties, indicating a potential for an extended half-life and sustained therapeutic effects (Fig. 2a). It is particularly noteworthy that the predicted value for 5-FU closely matches its clinical data (as reported in DrugBank DB00544, Supplementary Table S1), which provides confidence in the reliability of the predictions for newly synthesized derivatives.

Fig. 2.

Prediction scores for selected in silico drug-like and toxicity properties (Toxometris-ADMET-Suite), estimated based on in silico prediction values for synthesized 5-FU derivatives. A lower score indicates a higher likelihood that the compounds fall within the desirable ranges established for pharmaceuticals.

A high risk of hepatotoxicity and neurotoxicity has been predicted for 5-FU, which corresponds to reported data²⁶ (Fig. 2b). All 5-FU derivatives demonstrated a lower risk of causing hepatotoxicity and an equal or reduced risk of neurotoxicity compared to 5-FU. Notably, derivative 2c showed no predicted risk of either hepatotoxicity or neurotoxicity. A low risk of nephrotoxicity and hERG-related cardiotoxicity has been predicted for 5-FU. Although 5-FU is known to cause both nephrotoxicity and cardiotoxicity, the nephrotoxicity arises primarily from its metabolites rather than from 5-FU itself²⁷. Additionally, the cardiotoxicity associated with 5-FU is not hERG-related, as studies have shown that the effects of 5-FU do not correlate with hERG expression levels in cells²⁸. For 5-FU derivatives, a high risk of nephrotoxicity was predicted only for 4b, while the other derivatives exhibited moderate to low risk. Furthermore, all derivatives demonstrated a very low risk of hERG-related cardiotoxicity.

Although anticancer drugs target existing cancers, some can increase the risk of secondary cancers due to DNA reactivity²⁹. 5-FU, as an antimetabolite, carries a relatively lower risk of secondary cancers compared to DNA-reactive agents like alkylating agents. Nonetheless, secondary cancer risk remains a crucial consideration for potential pharmaceuticals intended for prolonged therapy³⁰. For synthesized 5-FU derivatives mutagenicity, genotoxicity, and carcinogenicity endpoints were predicted, and overall risk scores were calculated to compare compounds (Fig. 2c). The predicted and experimental values for 5-FU showed absolute concordance^31–33, which strengthens the reliability of other predictions. The cumulative risk score for carcinogenicity prediction, which considers all carcinogenicity-associated endpoints, was higher for the 2e and 4b derivatives compared to 5-FU. All other derivatives showed lower risk scores, with 2c and 2d having the lowest scores.

Overall, guided by drug-likeness criteria, the 5-FU derivatives were designed to enhance oral absorption and systemic exposure while reducing off-target toxicities. In silico ADMET predictions indicated strong Caco-2 permeability across all derivatives and a higher HIA probability for compound 2c relative to 5-FU. Plasma protein binding was consistently increased compared with 5-FU, suggesting an extended half-life. Predicted hepatotoxicity and neurotoxicity were reduced for the series, with compound 2c showing no risk for either endpoint; hERG liability was uniformly very low, and nephrotoxicity was predicted as moderate-to-low for most derivatives (elevated only for 4b). For carcinogenicity-associated endpoints, 2c and 2 d were predicted to be lower risk than 5-FU, whereas 2e and 4b showed higher risk. A concomitant drawback was decreased aqueous solubility compared with 5-FU (logS − 3.6 to − 4.6 vs. − 1.1), although values remained within typical drug space and were experimentally managed by dissolution in DMSO.

Cytotoxicity of novel derivatives of 5-FU

The cytotoxicity of 5-FU and its derivatives was evaluated in A549 (human lung adenocarcinoma) and MRC-5 (human normal lung fibroblast) cell lines. The A549 cell line was selected as a well-characterized model of non-small cell lung cancer (NSCLC), the most common form of lung cancer^34,35. It is also the most widely used human NSCLC cell line in both basic research and drug discovery, owing to its chemotherapy resistance^36,37, active metabolic profile³⁸, and expression of key targets involved in metabolism, apoptosis, and stress responses. These properties make it particularly suitable for evaluating the mechanisms and selective toxicity of 5-fluorouracil derivatives. The MRC-5 cell line served as a comparative model, commonly used as a negative control in lung cancer^39,40. Three concentrations were used to assess the cytotoxicity of the compounds (25, 50 and 100 µM), with the highest concentration determined by solubility limitations in DMSO. A final highest DMSO concentration of 0.4% was chosen, as it had no significant or minimal impact (~ 10%) on cell viability. Results are presented in Fig. 3. All treatments were compared to their corresponding vehicle controls (0.1% DMSO for 25 µM, 0.2% DMSO for 50 µM, and 0.4% DMSO for 100 µM) and to the corresponding 5-FU treatments. A statistically significant dose-dependent decrease in cell viability was observed with 5-FU, reaching up to 70% reduction at the highest concentration (100 µM) (Fig. 3a). Although literature reports on the IC50 of 5-FU for the A549 cell line vary widely, ranging from 51 µM⁴¹ to 11 mM⁴², likely due to differences in cell concentrations, interlaboratory variability, and other factors, our findings are consistent with at least two studies, where approximately 30–40% inhibition was observed at 5-FU concentrations between 100 and 190^43,44. The 5-FU derivatives demonstrated a dose-dependent reduction in A549 cell viability across all tested compounds, with the exception of 4a. While most compounds exhibited either non-cytotoxic (e.g., 4a) or low cytotoxic activity (2c, 2d, 4b), with inhibition ranging from 10% to 20%, compounds 2c and 3a significantly reduced cancer cell viability by up to 60%, a greater cytotoxic effect than that of 5-FU at the same concentration (100 µM) (Fig. 3a).

Fig. 3.

Cytotoxicity of the synthesized 5-FU derivatives (25, 50, and 100 µM) after 24 h of treatment (MTT assay) in A549 and MRC5 cells. Data are presented as mean ± SE of three independent experiments. *p < 0.05 compared to corresponding DMSO (vehicle control), ^#p < 0.05 compared to corresponding 5-FU.

No signs of cytotoxicity were observed for 5-FU at any of the tested concentrations in the MRC5 cell line, with cell viability remaining approximately 90% compared to the vehicle control (Fig. 3b). This type of selectivity, in which 5-FU exhibits cytotoxicity against the A549 cancer cell line while being non-cytotoxic to the normal MRC5 cell line, has been demonstrated earlier⁴⁵. All 5-FU derivatives exhibited a non-cytotoxic profile in MRC5 cells, demonstrating a dose-dependent tendency to promote cell proliferation or enhance cell metabolic activity. Since the MTT assay does not directly measure cell number but instead assesses the proportion of metabolically active cells based on mitochondrial enzyme activity, it is challenging to determine whether the observed increase reflects enhanced cell proliferation or an upregulation of metabolic activity within the same population of cells. Despite this ambiguity, both increased cell proliferation and heightened metabolic activity can be interpreted as compensatory responses of normal cells to potential antimetabolites. This is particularly relevant given that the most pronounced effect was observed with compound 3a, which also exhibited the highest cytotoxicity against cancer cells (Fig. 3a). Cancer cells are known to undergo significant metabolic reprogramming, with many relying predominantly on glycolysis for both energy production and biosynthesis (the Warburg effect)⁴⁶. In contrast, normal cells generally follow glycolysis with oxidative phosphorylation in the mitochondria, making them less dependent on glycolysis for proliferation and metabolic activity⁴⁷. This fundamental metabolic distinction likely underlies the observed differential responses between cancerous and normal cells.

So, based on these results, 5-FU derivatives 2c and 3a showed higher cytotoxicity against A549 lung cancer cells than 5-FU, while maintaining low toxicity in normal MRC-5 cells. This selective activity suggests that these derivatives may offer more effective and safer anticancer options compared to 5-FU.

Induction of oxidative stress and antioxidant response by 5-FU derivatives

In addition to its DNA-targeting effects, 5-FU induces oxidative stress, characterized by elevated levels of reactive oxygen species (ROS)⁴⁸. Cancer cells usually respond to this oxidative stress by enhancing their antioxidant defenses, such as upregulating enzymes like superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase, as well as increasing intracellular levels of glutathione⁴⁹. However, when these defenses are insufficient to counteract the high ROS levels, the cells may undergo cell death^9,48. In this study, oxidative stress induced by a 5-FU derivative was assessed using two-photon microscopy to analyze carboxy-DCFDA-stained A549 cells. This method is based on the ability of carboxy-DCFDA, a cell-permeable fluorescent probe, to detect ROS by being oxidized within the living cell, resulting in fluorescence emission that can be quantitatively measured⁵⁰. Two-photon was chosen to confine excitation to the focal plane and use near-infrared light, thereby reducing photobleaching and phototoxicity, minimizing out-of-focus background/autofluorescence, and improving signal-to-noise in intact cell monolayers. These features help preserve the cellular redox state during imaging and yield more reliable quantitative measurements of carboxy-DCF fluorescence⁵¹. The dysregulation of SOD and catalase enzymes was studied to evaluate the cellular antioxidant response, which may provide insights into the mechanisms by which 5-FU derivatives influence oxidative stress in cancer cells.

A significant reduction in intracellular ROS levels was observed in cells treated with DMSO, the vehicle control, compared to the negative control (cells incubated with growth media alone) (Fig. 4). This reduction is likely due to well-known free radical scavenging properties of DMSO⁵². A threefold increase in ROS levels was observed following 5-FU treatment of cells compared to the vehicle control. These results are comparable to literature data obtained from the same cell line⁴³. The 5-FU derivatives 2c, 2e, 3a, 4a were found to demonstrate equal or even lower levels of ROS compared to the vehicle control. It is worth noting that derivatives 2c and 3a, as shown above, exhibited cytotoxic activity, potentially through a decrease in metabolic activity, as indicated by the MTT assay. Therefore, it can be assumed that oxidative stress is not involved in the cytotoxic activity of these derivatives. Instead, low metabolic state could suppress biosynthetic processes that typically generate ROS. A drastic increase in ROS generation was observed with the 2d derivative, showing approximately a 13-fold increase compared to the vehicle control and a 4.4-fold increase compared to 5-FU. The 4b derivative induces a 3-fold increase in ROS levels compared to the vehicle control, which was similar to the levels observed with 5-FU.

Fig. 4.

Two-photon fluorescence intensity of carboxy-DCFDA in A549 cells measured after 24 h of treatment with 5-FU derivatives at a concentration of 100 µM. Data are presented as mean ± SE of three independent experiments. NC- negative control (cells incubated with growth media), PC - positive control (cells incubated with 0.1% H₂O₂). **p < 0.01 compared to DMSO (vehicle control), ^##p < 0.01 compared to 5-FU, †† < 0.01compared to negative control, ‡ ‡ < 0.01compared to positive control.

Enzymatic and non-enzymatic antioxidant systems are involved in the formation of an adaptive response to oxidative stress. Two key enzymes that protect cells from the action of ROS include SOD and CAT^53,54. SOD is a metal containing homodimer that catalyses the removal of superoxides, producing hydrogen peroxides. CAT is a heme-containing enzyme, which catalyses the conversion of hydrogen peroxide to oxygen and water and thus acts as a synergist antioxidant agent with SOD. Therefore, simultaneously with the evaluation of the intensity of oxidative stress induced by studied compounds, the response of the antioxidant enzymes SOD and CAT, was assessed. Data (Fig. 5) indicate that DMSO has no effect on the activity of these enzymes. The 5-FU significantly reduced the activity of both enzymes, leading to complete inactivation of SOD. All tested 5-FU derivatives, with the exception of 2d, caused a reduction in SOD activity ranging from 20% to 60%, with the most pronounced inactivation observed for derivatives 2c and 4b. These two compounds also significantly reduced CAT activity, with 2c causing a notable 40% decrease. A statistically significant increase in CAT activity was observed with the 2e and 2d derivatives, while the 3a, 4a compounds did not affect CAT activity.

Fig. 5.

Effect of 5-FU derivatives at a concentration of 100 µM on SOD and CAT activity in A549 cells after 24 h of treatment. Activity (%) is expressed relative to the vehicle control (DMSO).

It should be noted that, compared to SOD, CAT exhibits significant resistance to the effects of the studied derivatives, which is likely due to the subunit structure of SOD, highly sensitive to microenvironmental conditions. When these conditions change (such as temperature, pH, redox potential, chemical composition) the dissociation of the molecule into separate subunits happens, followed by aggregation and the formation of labile complexes, which is accompanied by a change in the biological activity of SOD⁵⁵. 5-FU and its derivatives could inhibit SOD and CAT both indirectly, by inducing excessive ROS⁹ and consequently causing oxidative modification of enzyme proteins⁵⁶, as well as directly through direct interaction with enzyme cofactor metals and/or essential amino acids in their active sites⁵⁷. The potential advantage of the latter pathway is indicated by the absence of ROS induction in the case of 2c, 2e, 3a, 4a (Fig. 4a) which cause a decrease in SOD activity, and the significant increase in ROS concentration in the case of 2d (Fig. 4a), which, however, does not affect enzyme activity (Fig. 5). Moreover, considering the mechanism of the therapeutic action of fluoropyrimidines, based on their interaction with nucleoside metabolism⁶, the studied compounds may also cause disruptions in the expression and synthesis of SOD and CAT, leading to a reduction in their activity.

So, of the tested derivatives, only 2 d significantly increased ROS levels in A549 cells, which may explain its modest cytotoxicity. In contrast, 2c and 3a demonstrated cytotoxicity without inducing oxidative stress, suggesting alternative mechanisms of action. Most derivatives, including 2c and 3a, led to a reduction in SOD activity, while CAT activity decreased only with 2c, suggesting a potential disruption in antioxidant enzyme function. These findings suggest that the cytotoxicity of 2c and 3a could be linked to interference with cellular metabolic pathways, rather than oxidative stress induction.

Effect of 5-FU derivatives on cellular ploidy

Polyploidy, where cells have extra sets of chromosomes, is common in various cancers⁵⁸ and often leads to aneuploidy, with recent studies linking oncogenes to this⁵⁹. The development of abnormal ploidy in cancer cells may result from multiple mechanisms, including chromosomal instability, and failure of cell cycle checkpoints⁶⁰, cytokinesis failure and DNA repair defects⁶¹. These changes contribute to cancer progression by increasing genetic diversity and allowing cancer cells to adapt to environmental pressures, including therapeutic interventions. Polyploidy can be triggered by genotoxic stresses such as chemotherapy, radiation, hypoxia, oxidative stress or environmental factors⁶². Cancer cells treated with certain chemotherapy drugs often show increased polyploidy, with about 30% of primary tumors and 56% of metastatic tumors undergoing whole-genome duplication⁶³. Combination treatment with 5-FU and sulforaphane resulted in the formation of multiple nuclei and subsequent polyploidy in triple-negative breast cancer cells⁶⁴. Conversely, in colorectal cancer cells, monotherapy with 5-FU did not induce polyploidization⁶⁵, while repeated administration of 5-FU did⁶⁶. The depolyploidization of cancer cells may lead to increased aggressiveness and contribute to the development of resistance and tumor recurrence; therefore, contemporary anti-cancer therapies should be designed with attention to this process⁶².

In this study, the impact of 5-FU derivatives on A549 cells ploidy at a concentration of 100 µM was investigated. The data indicate that the average DNA amount in ploidy units (“c”) of the control A549 cells is 2.6 ± 0.7 (Fig. 6j), characterizing them as hypotriploid cells, which is consistent with the cytogenetic characteristics published by ATCC (American Type Culture Collection) for this cell line. The DMSO (vehicle control) resulted in a slight yet statistically significant reduction in the average DNA amount in ploidy units (“c”) of these cells (2.4 ± 0.7 compared to 2.6 ± 0.7) (Fig. 6j). 5-FU induced the highest statistically significant level of DNA amount, represented in “с” units (3.3 ± 0.8), followed by compounds 2d (2.9 ± 0.6) and 4a (2.7 ± 0.1), which exhibited slightly lower values compared to 5-FU. It can be suggested that the lack of cytotoxicity in 4a and the slight cytotoxicity of 2d, despite its dramatic effect on OS (Fig. 4a), may be associated with the increase in DNA amounts displayed in ploidy levels they induce, which, in turn, triggers adaptive cell resistance⁵⁹. All other derivatives, including 2c, 2e, 3a, and 4b had no effect on the ploidy of the cells.

Fig. 6.

Distribution frequency of cells by ploidy (the average DNA amount in ploidy units “c”) levels in A549 cells after 24-h treatment with 5-FU derivatives at a concentration of 100 µM (a-i). (j) Data are presented as mean ± SE of three independent experiments. **p < 0.01 compared to DMSO, ##p < 0.01 compared to 5-FU.DNA content was expressed on a “c” scale, in which 1c is the haploid amount of nuclear DNA occurring in normal diploid populations in G0/G1.

Figure 6 (a-i) presents the distribution frequency of cells by ploidy levels, enabling a more precise interpretation of the data. 5-FU, which induced the highest increase in the average ploidy level of the cells (Fig. 6j), caused a rightward shift in the distribution of cells by ploidy, resulting in an increase in the proportion of cells with 4c ploidy and higher to 26% compared to 10% in the DMSO. This finding supports the previously discussed risk that 5-FU treatment can induce polyploidy in solid tumors. Polyploid cells can survive genotoxic stress, such as chemotherapy, and later undergo depolyploidization, giving rise to aneuploid progeny. These progeny are genetically diverse, which increases the chance that some subclones will have enhanced adaptability, drug tolerance, and proliferative advantages, thereby contributing to adaptive resistance and tumor recurrence. It was found that the 2c derivative, while exhibiting an average ploidy level comparable to that of the DMSO control, still caused a leftward shift in the distribution of cells by ploidy, indicating an increase in hypodiploid cells, which constituted 24% of the total pool compared to approximately 6% in the DMSO and 16% in the original (non-treated) cancer cells (Fig. 6d, b). This suggests the activation of apoptosis in the studied cells, as the presence of a hypodiploid (sub-G1) population is widely accepted as a marker of apoptosis, reflecting chromatin fragmentation and DNA loss⁶⁷ and aligns with the significant cytotoxic effect of this compound (Fig. 3a). However, complementary assays (e.g., Annexin V/PI staining, caspase-3/7 activity) will be necessary to further confirm the apoptotic pathways engaged by these derivatives. A similar pattern was observed with the 3a derivative. In the case of the 4a compound, there was an emergence of hypertetraplod cells which account for 12% compared to 4% in the DMSO. The distribution pattern of cells by ploidy treated by 2e and 4b derivatives closely mirrors that of the DMSO. Conversely, the 4d compound leads to an increase in the proportion of hyperdiploid cells at the expense of diploid cells. In this case, as indicated above, polyploidy apparently induces adaptive resistance of cells at elevated oxidative stress (Fig. 4), which ensures low cytotoxicity of 2d (Fig. 3a). It is worth noting here that polyploidy can offer both benefits and drawbacks to cancer cells. On one hand, polyploid cells may exhibit greater resistance to apoptosis and enhanced adaptability to environmental stresses. On the other hand, polyploidy can trigger cell death pathways, including apoptosis induced by mitotic catastrophe, as well as stress associated with the maintenance of polyploidy, high metabolic demands, and increased genomic and chromosomal instability. The dual effects of polyploidy on cancer cells may stem from varying levels of polyploidy, leading to distinct outcomes⁵⁹.

So, the study reveals that 5-FU, along with derivatives 2 d and 4a, significantly increased A549 cell ploidy, which is generally associated with cancer progression and treatment resistance. Conversely, derivatives 2c and 3a caused a shift toward hypodiploid cells, suggesting the presence of apoptotic cells, which is a favorable characteristic for potential cancer therapeutics.

In summary, among all synthesized 5-FU derivatives, 2c and 3a demonstrated higher cytotoxicity against A549 lung cancer cells than 5-FU, while exhibiting low toxicity in normal MRC-5 cells. The cytotoxic activity of 2c and 3a was independent of oxidative stress induction. Instead, these derivatives disrupted antioxidant enzyme activity, particularly 2c, which affected both SOD and CAT, suggesting possible interference with cellular metabolic pathways. Additionally, while 5-FU, increased ploidy in A549 cells, often linked to cancer resistance and progression, 2c and 3a shifted cells toward hypodiploidy, a marker of apoptosis, supporting their potential as promising anticancer candidates. In terms of pharmacokinetics 2c was predicted to have enhanced human intestinal permeability and plasma protein binding properties, suggesting it may offer improved bioavailability and an extended half-life compared to 5-FU. The 2c derivative also showed no predicted risks for hepatotoxicity, neurotoxicity, or carcinogenicity, further enhancing its safety profile compared to 5-FU. These results suggest that 3a, and especially 2c, may offer more effective anticancer options than 5-FU.

Conclusions

The present study reports the synthesis of novel N1-mono- and N1, N3-bisarylmethyl derivatives of uracil incorporating combinations of electron-donating and electron-withdrawing groups, as well as derivatives bearing diverse substituents at the N1 and N3 positions of the pyrimidine ring. These modifications were explored to determine whether they confer structural advantages over 5-FU with respect to drug-likeness and anticancer activity in vitro, and to prioritize leads for further study. Two compounds emerged as leads: 2c, an N1-monoarylmethyl 5-bromouracil derivative (R = Br) bearing a 2-methoxy-5-formylbenzyl substituent at N1 position; and 3a, an N1,N3-bis(arylmethyl) uracil derivative (R = H) carrying 2-methoxy-5-formylbenzyl groups at both N1 and N3 positions. Both 2c and 3a showed enhanced cytotoxicity and selectivity toward A549 lung cancer cells while sparing normal MRC-5 fibroblasts. Cytotoxicity was independent of ROS induction and, unlike 5-FU, not accompanied by polyploidization. In silico ADMET profiling for the series indicated improved developability in respect to higher plasma-protein binding and oral-absorption potential with reduced predicted risks of hepatotoxicity, neurotoxicity, and carcinogenicity—particularly for 2c. These results suggest that 2c and 3a may represent safer, more effective alternatives to 5-FU. Translation to clinical application requires addressing several challenges, including validation of pharmacokinetics and toxicity in animal models, overcoming solubility limitations through optimized formulation, and demonstrating efficacy within the tumor microenvironment and immune context. Further preclinical studies are warranted to confirm these advantages and to evaluate their therapeutic potential in vivo.

Materials and methods

Synthesis

Solvents were purified by distillation, crystalline starting compounds were used directly without additional purification. IR spectra were recorded on a Nicolet Avatar 330, NMR spectra ¹H and ¹³C – on a Varian «Mercury-300VX» device with a frequency of 300.80 and 75.46 MHz, respectively, in DMSO-d₆ and DMSO-d₆–CCl₄ solutions (1:3), internal standard – TMS. Mass spectra were recorded on a «WatersTM XevoTM G3 QTOF MS ES+». Elemental analysis was performed on an automatic elemental analyzer (Euro EA 3000 (Evrovetktor, Italy). TLC was carried out on «Silufol UV-254» plates, development – UV irradiation. (The obtained spectra are shown in the supplementary Fig S1-S24).

General method for the preparation of 2 d,3d and 2e,3e compounds

A mixture of 0.01 mol of uracil 1a or 5-fluorouracil 1b, 0.01 mol of substituted benzyl chloride and 1.38 g (0.01 mol) of dry K₂CO₃ in 15 ml of dry DMSO is heated for 3 h at 70 °C, poured into 40 ml of 1% NaOH solution and left for 3 h at room temperature. The filtered precipitate of bis-substituted derivatives 3d, 3e is repeatedly washed on the filter with 10 ml of 1% NaOH solution, water, and dried. The mother liquor is acidified with HCl, left in the cold, the formed precipitate of monosubstituted derivatives 2d, 2e is filtered off and dried.

Compounds 2d, 3d were obtained by the reaction of pyrimidine-2,4(1H,3H)-dione (1a) with 4-(chloromethyl)−1-methoxy-2-nitrobenzene⁶⁸, compounds 2e, 3e – by the reaction of 5-fluoropyrimidine-2,4(1H,3H)-dione (1b) with 4-chloro-2-(chloromethyl)−1-methoxybenzene⁶⁹.

1-(4-Methoxy-3-nitrobenzyl)pyrimidine-2,4(1H,3H)-dione (2d).

Yield 0.4 g (43.9%), mp: 170–171 °C, TLC (toluene: ethylacetate, 1:1 v/v): R_f = 0.65; ¹H NMR (300 MHz, DMSO/CCl₄,1/3): δ 3.95 (s, 3 H, OCH₃), 4.85 (s, 2H, CH₂), 5.48 (dd, 1H, J = 7.9, 2.2 Hz, H⁵pyrimidine), 7.23 (d, 1H, J = 8.7 Hz, H⁵C₆H₃), 7.61 (dd, 1H, J = 8.7, 2.2 Hz, H⁶C₆H₃), 7.73 (d, 1H, J = 7.9 Hz, H⁶ pyrimidine), 7.91 (d, 1H, J = 2.2 Hz, H²C₆H₃), 11.11 (broad, 1H, NH); ¹³C NMR (75 MHz, DMSO/CCl₄,1/3): δ 162.9, 151.6, 150.6, 144.2, 138.9, 133.9, 128.7, 124.8, 113.8, 101.5, 56.0 (CH₃), 48.8 (CH₂); IR (Nujol): 1685, 1621 cm-¹; HRMS (m/z): [M]⁺ calcd. for C₁₂H₁₁N₃O₅, 278.0777; found, 278.0789; analysis (calcd., found for C₁₂H₁₁N₃O₅): C (51.99, 51.97), H (4.00, 3.98), N (15.16, 15.15), O (28.85, 28.88).

1,3-Bis(4-methoxy-3-nitrobenzyl)pyrimidine-2,4(1H,3H)-dione (3d).

Yield 0.9 g (47.4%), mp: 250–251 °C, TLC (toluene: ethylacetate, 1:1 v/v): R_f = 0.68; ¹H NMR (300 MHz, DMSO/CCl₄,1/3): δ 3.94 (s, 3 H, OCH₃), 3.95 (s, 3 H, OCH₃), 4.93 (s, 2 H, NCH₂), 4.97 (s, 2 H, NCH₂), 5.68 (d, 1H, J = 7.9, 2.2 Hz, H⁵ pyrimidine), 7.17 (d, 1H, J = 8.7 Hz, C₆H₃), 7.22 (d, 1H, J = 8.7 Hz, C₆H₃), 7.63 (dd, 1H, J = 8.7, 2.2 Hz, C₆H₃), 7.66 (dd, 1H, J = 8.7, 2.2 Hz, C₆H₃), 7.81 (d, 1H, J = 2.2 Hz, C₆H₃), 7.87 (d, 1H, J = 7.9 Hz, H⁶ pyrimidine), 7.94 (d, 1H, J = 2.2 Hz, C₆H₃); ¹³C NMR (75 MHz, DMSO/CCl₄,1/3): δ 161.4, 151.7, 151.3, 150.9, 143.3 (NCH), 138.9, 138.8, 134.3 (CH), 134.0 (CH), 129.0, 128.3, 125.1 (CH), 124.9 (CH), 113.7 (CH), 113.3 (CH), 100.7 (= CH), 56.0 (OCH₃), 55.9 (OCH₃), 50.1 (NCH₂), 42.0 (NCH₂); IR (Nujol): 1698, 1649, 1624 cm-¹; HRMS (m/z): [M]⁺ calcd. for C₂₀H₁₈N₄O₈, 443.1203; found, 443.1203; analysis (calcd., found for C₂₀H₁₈N₄O₈): C (54.30, 54.33), H (4.10, 4.08), N (12.67, 12.66), O (28.93, 28.91).

1-(5-Chloro-2-methoxybenzyl)−5-fluoropyrimidine-2,4(1H,3H)-dione (2e).

Yield 0.4 g (43.9%), mp: 200–202 °C, TLC (toluene: ethylacetate, 1:1 v/v): R_f = 0.62; ¹H NMR (300 MHz, DMSO): δ 3.82 (s, 3H, OCH₃), 4.74 (s, 2 H, NCH₂), 7.05 (d, 1H, J = 8.7 Hz, H³ C₆H₃), 7.17 (d, 1H, J = 2.6 Hz, H⁶ C₆H₃), 7.34 (d, 1H, J = 8.7, 2.6 Hz, H⁴ C₆H₃), 8.04 (d, 1H, J_{H, F} = 6.7 Hz, H⁶ pyrimidine), 11.78 (s, 1H, NH); ¹³C NMR (75 MHz, DMSO): δ 46.8 (CH₂), 55.8 (CH₂), 112.6 (CH, C³C₆H₃), 124.0, 125.9, 128.1 (CH), 128.6 (CH), 130.4 (d, J_{C, F} = 33.6 Hz, C⁶pyrimidine), 139.5 (d, J_{C, F} = 230.0 Hz, C⁵pyrimidine), 149.5, 155.7, 157.5 (d, J_{C, F} = 25.7 Hz, C⁴pyrimidine); IR (Nujol): 3151 cm-¹ (NH), 1745, 1710 cm-¹ (CO),1650 cm-¹ (C = C–C = N); HRMS (m/z): [MH]⁺ calcd. for C₁₂H₁₀ClFN₂O₃, 285.0442; found, 285.0450; analysis (calcd., found for C₁₂H₁₀ClFN₂O₃): C (50.63, 50.64), H (3.54, 3.55), Cl (12.45, 12.43), F (6.67, 6.68), N (9.84, 9.82), O (16.86, 16.85).

1,3-Bis(5-chloro-2-methoxybenzyl)−5-fluoropyrimidine-2,4(1H,3H)-dione (3e).

Yield 1.2 g (42.9%); m.p.129–130 °C, TLC (toluene: ethylacetate, 1:1 v/v): R_f = 0.60; ¹H NMR (300 MHz, DMSO): 3.80 (s, 3H, OCH₃), 3.82 (s, 3H, OCH₃), 4.85 (s, 2 H, N¹CH₂), 4.92 (s, 2 H, N³CH₂), 6.84 (d, 1H, J = 2.6 Hz, H⁶ N³C₆H₃), 7.02 (d, 1H, J = 2.6 Hz, H³ N³C₆H₃), 7.06 (d, 1H, J = 8.8 Hz, H³ N¹C₆H₃), 7.22 (d, 1H, J = 2.6 Hz, H⁶ N¹C₆H₃), 7.27 (dd, 1H, J = 8.7, 2.6 Hz, H⁴ N³C₆H₃), 7.34 (dd, 1H, J = 8.8, 2.6 Hz, H⁴ N¹C₆H₃), 8.23 (d, 1H, J_{H, F} = 6.4 Hz, H⁶ pyrimidine); ¹³C NMR (75 MHz, DMSO): δ 48.0 (2•CH₂), 55.8 (OCH₃), 55.9 (OCH₃), 112.4 (CH), 112.6 (CH), 124.07, 124.10, 125.4 (CH), 125.7, 126.1, 127.6 (CH), 128.2 (CH),128.7 (CH), 129.4 (d, J_{C, F} = 33.6 Hz, C⁶pyrimidine), 139.1 (d, J_{C, F} = 227.0 Hz, C⁵pyrimidine), 149.5, 155.3, 155.7, 156.8 (d, J_{C, F} = 25.7 Hz, C⁴pyrimidine); IR (Nujol): 1701 cm-¹ (CO), 1650 cm-¹ (C = C–C = N); HRMS (m/z): [MH]⁺ calcd. for C₂₀H₁₇Cl₂FN₂O₄) 439.0628; found, 439.0639; analysis (calcd., found for C₂₀H₁₇Cl₂FN₂O₄): C (54.69, 54.70), H (3.90, 3.92), Cl (16.14, 16.12), F (4.33, 4.34), N (6.38, 6.36), O (14.57, 14.58).

Pyrimidines 4a, b.

A mixture of 0.01 mol of pyrimidines 2a or 2b, 0.01 mol of the corresponding halide and 1.38 g of 0.01 mol K₂CO₃ in 10 ml of DMSO was heated for 3 h at 80 °C, poured into 50 ml of water, the precipitated product was filtered off and recrystallized 80% AcOH.

3-((3-(4-Fluorobenzyl)−2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-4-methoxybenzaldehyde (4a).

Prepared by alkylation of pyrimidine 2a with 1-(chloromethyl)−4-fluorobenzene. Yield 2.8 g (76.1%), mp: 160–162 °C TLC (toluene: ethylacetate, 1:1 v/v): R_f = 0.60; ¹H NMR (300 MHz, DMSO): 3.95 (s, 3H, OCH₃), 4.91 (s, 2 H, CH₂), 4.96 (s, 2 H, CH₂), 5.64 (d, 1H, J = 7.9 Hz, H⁵pyrimidine), 6.93–7.01 (m, 2 H, H^3,5, C₆H₄), 7.15 (d, 1H, J = 8.5 Hz, H³, C₆H₃), 7.36–7.43 (m, 2 H, H^2,6, C₆H₄),), 7.61 (d, 1H, J = 7.9 Hz, H⁶pyrimidine), 7.75 (d, 1H, J = 2.1 Hz, H⁶, C₆H₃), 7.85 (dd, 1H, J = 8.5, 2.1 Hz, H⁴, C₆H₃), 9.84 (s, 1H, CHO); ¹³C NMR (75 MHz, DMSO): δ 42.4 (CH₂), 47.4 (CH₂), 55.6 (CH₃), 100.1 (C⁵pyrimidine), 110.6 (C³C₆H₃), 114.4 (d, J_{C, F} = 21.3 Hz, C^3,5, C₆H₄), 124.3, 129.1, 130.2 (d, J_{C, F} = 8.1 Hz, C^2,6, C₆H₄), 130.8 (C⁴C₆H₃), 131.7 (C⁶C₆H₃), 132.7 (d, J_{C, F} = 2.9 Hz, C₆H₄), 143.5 (C⁶pyrimidine), 150.7, 161.3 (d, J_{C, F} = 244.4 Hz, C₆H₄), 161.5 (CO), 161.6 (CO), 189.3 (CHO); IR (Nujol): 1702, 1679, 1649 cm-¹ (CO), 1604 cm-¹ (C = C–C = N); HRMS (m/z): [MH]⁺ calcd. for C₂₀H₁₇FN₂O₄, 369.1251; found, 369.1260; analysis (calcd., found for C₂₀H₁₇FN₂O₄): C (65.21, 65.22), H (4.65, 4.67), F (5.16, 5.14), N (7.60, 6.58), O (17.37, 17.38).

5-Fluoro-1-(4-methoxy-3-nitrobenzyl)−3-(prop-2-yn-1-yl)pyrimidine-2,4(1H,3H)-dione (4b)

Prepared by alkylation of pyrimidine 2b with 3-bromoprop-1-yne. Yield 2.6 g (78.1%), mp: 130–131 °C TLC (toluene: ethylacetate, 1:1 v/v): R_f = 0.70; ¹H NMR (300 MHz, DMSO-d₆–CCl₄ (1:3): 2.61 (t, 1H, J = 2.5 Hz, CH), 3.96 (s, OCH₃), 4.57 (d, J = 2.1 Hz, 2H, CH₂), 4.92 (s, 2H, NCH₂), 7.25 (d, J = 8.7 Hz, 1H, H⁵arom.), 7.70 (dd, J = 8.7, 2.2 Hz, 1H, H⁶arom.), 7.99 (d, J = 2.2 Hz, 1H, H²arom.), 8.33 (d, J = 6.2 Hz, 1H, H⁶pyrimidine); ¹³C NMR (75 MHz, DMSO-d₆–CCl₄ (1:3): δ 30.1 (CH₂), 50.3 (NCH₂), 56.1 (OCH₃), 71.9 (CH), 77.5 (C),113.8, 125.2, 127.8, 128.1 (d, J_{C, F} = 33.6 Hz), 134.2, 139.01, 139.04 (d, J_{C, F} = 231.4 Hz), 148.7, 151.7, 155.2 J_{C, F} = 26.1 Hz); IR (Nujol): 3261 cm-¹ (CH), 1715, 1682 cm-¹ (CO), 1627 cm-¹ (C = C–C = N); HRMS (m/z): [MH]⁺ calcd. for C₁₅H₁₂FN₃O₅, 334.0839; found, 334.0848; analysis (calcd., found for C₁₅H₁₂FN₃O₅): C (54.06, 54.07), H (3.63, 3.64), F (5.70, 5.68), N (12.61, 12.63), O (24.00, 24.01).

Cell culture

Human fetal lung fibroblasts MRC5 (CCL-171™, ATCC) and human lung cancer epithelial cells A549 (CCL-185™, ATCC) were obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in Eagle’s modified Dulbecco’s medium (DMEM, Gibco), supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin-streptomycin (P/S, Gibco), and 1% L-glutamine (Sigma-Aldrich) at 37 °C in a humidified atmosphere with 5% CO₂. Growth medium was refreshed every 3 days, and cells were subcultured upon reaching 80–90% confluency by trypsinization with a 0.5% Trypsin-EDTA solution (Gibco).

MTT cytotoxicity assay

A549 and MRC5 cells were seeded at a density of 2 × 10⁴ cells per well in 96-well plates in 200 µl of growth medium containing 10% FBS, and cultured overnight at 37 °C with 5% CO₂. The test compounds, dissolved in dimethyl sulfoxide ((DMSO) Carl Roth), were added at final concentrations of 25 µM, 50 µM, and 100 µM and incubated for 24 h at 37 °C with 5% CO2. Control groups included untreated cells (negative control with culture media only) and cells treated with DMSO at final concentrations of 0.1%, 0.2%, and 0.4% (vehicle controls). After incubation with the test compounds, 40 µl of MTT (5 mg/ml) (3-[(4,5)-dimethylthiazol-2-yl]−2,5-diphenyl tetrazolium bromide ((MTT) Sigma -Aldrich) was added to each well, and the cells were incubated for an additional 3 h at 37 °C with 5% CO₂. The formazan crystals formed by viable cells were dissolved in 150 µl DMSO, and the optical density was measured at 570 nm (BioTek’s plate reader EPOCH/2). Cell viability was calculated relative to the control group (vehicle control).

Imaging of oxidative stress by two-photon microscopy

The intracellular ROS was determined using carboxy-DCFDA staining assay⁵⁰. A549 cells were seeded in a 35 mm Petri dish on a coverslip at a density of 2 × 10⁴ cells and incubated for 24 h. The test compounds were added at a final concentration of 100 µM and incubated for 24 h at 37 °C with 5% CO₂. Control groups included untreated cells (negative control with culture media only), cells treated with DMSO at final concentrations of 0.4% (vehicle controls), and cells treated with 0.1% H₂O₂ 10 min (positive control). After incubation, the samples were rinsed with PBS and stained. For staining, carboxy-DCFDA (5(6)-carboxy-2’,7’-dichlorofluorescein diacetate, Sigma-Aldrich Chemic GmbH, Germany), a ROS-sensitive, membrane-permeable fluorescent dye, was added to each sample at a final concentration of 10 µM in PBS, followed by incubation at 37 °C with 5% CO₂ for 30 min in the dark. After incubation, the cells were washed with PBS (Sigma-Aldrich) and immediately prepared for two-photon microscopy imaging. All measurements were made at room temperature (20–22 °C). Two-photon imaging was performed using a diode-pumped Yb: KGW ultrafast oscillator (“t-pulse”, Amplitude Systems, France) available at the AREAL facility (CANDLE SRI, Armenia) attached to a two-photon laser scanning upright microscope (MOM- Movable Objective Microscope, Sutter Instruments, USA) with 20× water immersion objective and numerical aperture of 1.0 and 2.0 mm working distance. Two-channel system with green filter was used providing 70 nm of full width at half maximum, 525 nm of maximum transmission and 92% of average transmission. A photomultiplier with 185–900 nm bandwidth (R6357; Hamamatsu Photonics Deutschland GmbH, Herrsching, Germany) was used to detect the carboxy-DCFDA fluorescence. A final power of 300 mW was maintained at the sample. Images were obtained by x, y galvanometric scanner in standard (512 × 512 pixels; 3.05 fps frame rate) modes on 12 bits photomultiplier with a pixel clock of 1000 ns.

Image processing

The images were processed using ImageJ software (version 1.50; https://imagej.net/ij/download.html). The image fluorescence intensity was calculated as a sum of intensities of all the cells from the ROI. The following formula was used to calculate the corrected total cell fluorescence (CTCF)⁷⁰.

Cytometric quantification of the DNA-staining

A549 cells were seeded in 6-well plates at a density of 3 × 10⁵ cells/well for 24 h. The test compounds were then added at the concentration of 100µM and incubated for 24 h at 37 °C with 5% CO₂. Controls After incubation the cells were fixed and stained with Feulgen-Naphthol Yellow⁷¹. Briefly, DNA hydrolyses were performed in 5 N HCl, 60 min at 22 °C. After being rinsed in sulfite and distilled water, the samples were then put directly into a solution of 0.1% Naphthol Yellow S in 1% acetic acid (pH 2.8) for 30 min, thereafter were de-stained with 1% acetic acid 3 times for 0.5 min, then samples were dehydrated 3 times with tert-butanol, treated with xylol for 5 min. In order to measure DNA content (in conventional units) by image scanning cytometry, computer-equipped microscope-cytometer SMP 05 (OPTON) was used at 575 nm wavelength. Quantity of DNA first was defined by image cytophotometry in conventional units (C.U.)^72,73. Cytometric quantification of DNA staining, measured as the Integrated Optical Density (IOD) of the nucleus in human cells, was used as a measure of DNA content. The DNA content of unstimulated human lymphocytes was used as a diploid standard for measurements. For the quantification of DNA IOD, values were evaluated by comparison with those from cells with the known DNA content. DNA content was expressed on a “c” scale, in which 1c is the haploid amount of nuclear DNA that occurred in normal (non-pathologic) diploid populations in the G0/G1 phase. In DNA measurements, a nucleus was identified as aneuploid if it deviates from 2c, 4c, 8c, or 16c by more than 10%⁷⁴.

Preparation of cell lysate for SOD and catalase activity studies

A549 cells were seeded in 6-well plates at a density of 3 × 10⁵ cells/well for 24 h. The test compounds were then added at the concentration of 100 µM and incubated for 24 h at 37 °C with 5% CO₂. DMSO at a final concentration of 0.4% was used as the vehicle control. After incubation cells were trypsinized, washed with PBS and the cell pellet was resuspended with 0.1 mL of cold lysis buffer (10 mM Tris, pH 7.5, 150mM NaCl, 0.1 mM EDTA + 1% Triton − 100) for 30 min. After centrifugation the supernatant was collected.

Determination of SOD activity

Superoxide dismutase (SOD) activity in cell lysates was measured using the epinephrine (adrenaline) autoxidation spectrophotometric assay, in which SOD inhibits the superoxide-driven conversion of epinephrine to adrenochrome, as described previously^75,76,77. A 10 µL of a 0.1% adrenaline solution (pharmaceutical grade) was added to 200 µL of carbonate buffer (0.2 M, pH 10.65), mixed quickly, and the optical density was recorded at a wavelength of 340 nm after 3 min (control sample). For the experimental samples, 10 µL of cell lysate was added to 200 µL of buffer, followed by 10 µL of 0.1% adrenaline. After 3 min, the increase in optical density was measured. The superoxide dismutase (SOD) activity in cell lysates was determined based on the degree of inhibition of adrenaline autoxidation. The inhibition percentage was calculated using the equation:

where ΔD_experiment and ΔD_control represent the optical densities (or autoxidation rates) in the presence and absence of cell lysate, respectively. Results were expressed as units per milliliter of protein (U/mL). One SOD unit was defined as the amount of enzyme required to achieve 50% inhibition of adrenaline autoxidation^76,77.

Determination of CAT activity

Catalase activity was determined in cell lysates using the ammonium molybdate colorimetric assay, in which residual H₂O₂ reacts with ammonium molybdate to yield a peroxomolybdate complex quantified spectrophotometrically⁷⁸. CAT activity was determined by mixing 50 µL of the sample (cell lysate) with 50 µL of substrate (0.06% H₂O₂) for 30 min. Following incubation, 100 µL of 4% ammonium molybdate was added, and absorbance was measured at 420 nm. Catalase activity (U/mL) was calculated using a standard curve⁷⁹.

Computational studies

The Toxometris.ai platform (https://portal.toxometris.ai) was used to calculate physicochemical descriptors and predict the ADMET properties and drug-likeness of the compounds. The Toxometris-ADMET-Suite uses an ensemble approach for endpoint predictions, combining the outputs of three statistical models, each based on unique molecular representation methods. Detailed information about these models, documented in QSAR Model Reporting Format (QMRF) files in compliance with OECD (Q)SAR validation principles⁸⁰, can be accessed at https://portal.toxometris.ai. The SMILES representations of compounds were generated and subsequently converted to canonical SMILES using RDKit: Open-Source Cheminformatics Software (Version 2020.09.1; https://www.rdkit.org/)⁸¹.

Predictions are provided as risk scores assigned to each compound, allowing compounds to be ranked from the most to the least promising for pharmaceutical suitability. These scores are derived from a scoring function that integrates the preferred ranges of predicted properties along with the reliability of those predictions. The overall risk score is calculated as a weighted average of individual scores across various predicted endpoints, offering a comprehensive evaluation of potential ADMET risks. Scores range from 0 to 1, with lower values indicating a higher likelihood that a compound meets the desired criteria for pharmaceutical applications.

Statistical analysis

All experiments were performed in triplicates, with six replicates for MTT assay. Data are presented as the mean ± standard error (SE) of three independent experiments. Statistical analysis was conducted using GraphPad Prism version 8.0 (Boston, Massachusetts USA). To evaluate differences between treatments, the non-parametric Mann-Whitney U test was applied. The level of p < 0.05 was considered as statistically significant.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

K.H., L.N., and N.B. contributed to methodology, formal analysis, validation, data curation, investigation, and writing the original draft. E.A., M.M., M.S., S.T. contributed to data acquisition, image analysis. V.A. performed the studies related to oxidative stress. Z.K., H.A. were responsible for cellular ploidy studies. A.H. synthesized the compounds and led the chemistry section. G.T., N.B. supervised the study design and conceptualization. L.N., N.B. performed review and editing. N.B. was responsible for funding acquisition.

Data availability

All data generated or analyzed during this study are included in this manuscript (and its Supplementary Information file). Any other datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.