Introduction

Colorectal cancer (CRC) is considered one of the main cancers, being the third most diagnosed malignancy and the fourth leading cause of cancer death worldwide [1]. 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 one in women [2]. Current treatments for CRC are effective, however, they all cause high-grade toxicity, which often results in dose limitations or cessation of the anticancer therapy [3,4,5]. Due to the widespread occurrence of the risk factors and the increase in the statistics, extensive research is ongoing to develop new potential chemopreventive agents against colorectal cancer.

Dihydropyrimidinone (DHPM) is characterized by its multi-functionalized scaffold that exhibits diverse biological activities [6,7,8,9,10] and especially anticancer activity [11]. Monastrol (Fig. 1), a DHPM, causes mitotic arrest at the G2/M phase by blocking bipolar mitotic spindle in mammalian cells due to inhibition of motility of mitotic kinesin Eg5, leading to cell apoptosis [12, 13]. On the other hand, the neurohormone melatonin (Fig. 1) is generated in several organs, mostly in the pineal gland, but also in the retina and gastrointestinal system. Numerous physiological activities, including the regulation of the immunological and endogenous antioxidant systems, are mediated by this molecule. Several in vitro and in vivo experimental models of tumors have shown that melatonin has an antiproliferative effect, suggesting that it may be an oncostatic drug in several cancer types [14]. It has been demonstrated to specifically impede the proliferation of prostate cancer cells that are both androgen-dependent and independent, through various mechanisms [15, 16].

Fig. 1
figure 1

Chemical structure of monastrol, melatonin, and hybrids molecules from these compounds

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Furthermore, molecular hybridization is a useful tool that has emerged in medicinal chemistry for the design of new drug prototypes. This strategy is based on the chemistry union of two pharmacophoric moieties of different bioactive substances to produce a new compound with distant physic-chemistry and pharmacokinetic properties. The subject of this strategy is to obtain compounds with improved affinity and efficacy, modified selectivity profile, and reduced undesired side effects [17, 18].

In this way, different authors have synthesized and evaluated different hybrids. For instance, Sashidhara and colleagues (2013) designed several coumarin-monastrol hybrids and evaluated their anti-breast cancer response. Insight this study, hybrid A showed good activity against MCF-7, T47D, and MDA-MB-231 cell lines. Besides, this compound induced apoptosis in both primary and metastatic cell lines (Fig. 1) [19]. In addition, another research showed that compound B, a monastrol-curcumin hybrid, showed maximum activity and it was found to be sensitive on different cancer cell lines as breast cancer MDA-MB-231 and HS 578T with growth percent (GP) values of 55.45 and 73.39, respectively; Prostate Cancer PC-3 (GP = 58.50); central nervous system cancer SNB-75 (GP = 59.60); leukemia RPMI-8226 (GP = 60.07), MOLT-4 (GP = 64.11) and CCRF-CEM (GP = 72.84) (Fig. 1) [20]. On the other hand, the tryptamine-salicylic acid hybrid C, exhibited a cytotoxic effect against A549 and MGC-803 causing cell-cycle arrest in the G2/M phase and apoptosis of MGC-803 cells in a concentration-dependent manner (Fig. 1) [21]. Finally, the melatonin-arylboronate hybrid D, exhibited strong toxic effects in cervical cancer cells (HeLa), without causing significant toxicity to normal NCTC-2544 cells. Besides, the cytotoxicity was accompanied by depletion of cellular glutathione (GSH) and increased ROS levels (Fig. 1) [22].

Considering all these facts and the urgent need for searching new therapeutic alternatives to treat colorectal cancer, this research was focused on the design and synthesis of a series of monastrol-melatonin hybrids (Fig. 2) together with their further biological evaluation in colorectal adenocarcinoma cells (SW480) and nonmalignant human colon mucosal epithelial cell line (NCM460) to determine the chemopreventive potential of these new molecules against this type of lethal pathology.

Fig. 2
figure 2

Design of monastrol-melatonin hybrids as anticancer agents

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Results and discussion

Chemistry

As illustrated in Scheme 1, the synthesis of monastrol-melatonin hybrids uses the Biginelli multicomponent reaction method to generate the monastrol intermediates 2a–k. In this way, different aldehydes (1A) were condensed with ethyl acetoacetate (1B) and thiourea (1C), using citric acid as a catalyst. The reaction was carried out under solventless conditions [23] and obtained yields between 60–99%. These compounds have already been reported [12, 24,25,26,27,28,29].

Scheme 1
scheme 1

Synthesis of target hybrids

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On the other hand, the reaction of 5-methoxytryptamine (3) with chloroacetyl chloride (4) led to amide (5) with 60% yield [30]. Finally, nucleophilic substitution between 2a–k and the amide 5 led to the formation of hybrids 6a–k in 40–80% yields [24] (Table 1).

Table 1 Structure and yield of title monastrol-melatonin hybrids 6a–k
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Using a combination of 1H-NMR, 13C-NMR, and HRMS-ESI (m/z) spectra analysis, the structures of all compounds were set. The molecular weights of the compounds were shown by the typical [M + H]+ peaks in the HRMS-ESI (m/z) spectra. J-constants and typical δ-values were used to allocate the signals to specific H or C-atoms. The 1H-NMR spectra of hybrids dissolved in CDCl3 showed between 5.40-5.50 ppm, a signal corresponding to CH-Ar. The CH3-C=C- and S-CH2 appear around 2.28 and 2.84 ppm, respectively. 13C-NMR spectra of the hybrids showed around 170 and 167 ppm a signal corresponding to the carbonyl groups of the amide and ester, respectively. The indole ring exhibited a signal around 122.96 ppm. Finally, the signals corresponding to NH-CH2 and S-CH2 were observed around 39.80 and 33.48 ppm, respectively.

Biological assays

The cytotoxic activity of the monastrol-melatonin hybrids 6a–k was tested against a human adenocarcinoma cell line (SW480) and nonmalignant human colon mucosal epithelial cells through the SRB assay. This research included different controls such as monastrol, melatonin, and the physical mixture of both to check the effectiveness of molecular hybridization; in addition, we included the vehicle control to establish the baseline, and the control with the reference drug (5-fluorouracil, 5-FU). The results are presented as inhibitory concentration 50 (IC50), which indicates the concentration of the compound needed the reduce the cell viability by 50%, besides, we also report the selectivity index (SI). According to the results shown in Table 2, after 48 h of treatment, all synthesized compounds 6a–k exert good cytotoxic activity against the SW480 cell line with IC50 values in the range of 2.2 ± 1.08 to 28.9 ± 1.14 µM, highlighting the importance of molecular hybridization since neither the parent compounds nor their physical mixture exhibited activity under the conditions evaluated (IC50 > 40 µM). In addition, we also observed that compounds 6a, 6c, 6i, and 6j significantly increased the selectivity over time becoming slightly more selective than the reference drug (5-FU) after 96 h of treatment, with selectivity indices of 2.1, 2.4, 3.1, and 2.1, respectively, which probably indicates that these compounds are likely to become less toxic over time against the nonmalignant cell line NCM460 without affecting the activity in SW480 cell. This effect of time was proved through two-way ANOVA, with a p-value of 0.0047.

Table 2 Effect of monastrol-melatonin hybrids on growth inhibition (IC50) of colorectal adenocarcinoma cell line (SW480) and nonmalignant human colon mucosal epithelial cell line (NCM460)
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In addition, as shown in Fig. 3 we observed that these four compounds decrease cell viability of SW480 cells in a concentration and time dependent manner. These results were also evidenced by an optical microscope. Thereon, in Fig. 4 we illustrate those changes that were clearly visible in malignant cells, altering cell number, shape, size, adhesion, and inducing granularity, while nonmalignant cell lines preserve healthy shape after the treatment with different concentrations (5, 10, and 20 µM, close to the IC50 value at the different points of evaluation) of the hybrid molecules. Different authors have also evaluated other monastrol or melatonin derivatives using in vitro models. Mervat and colleagues published in 2020 several monastrol derivatives with potential activity against a variety of cancers including the colon, finding activity through the inhibition of the Eg5 kinesin enzyme which probably leads to mitotic cell arrest at the G2/M phase, and subsequent cell death [31]. In addition, we previously published some chalcone-melatonin hybrids with remarkable cytotoxic and antiproliferative activity against SW480 cells [32]. Moreover, SanJuan et al. (2023) published for the first time the potent biological activity of Agomelatine, a naphthalene analog of melatonin, through cell-cycle arrest and caspase-dependent apoptotic death of colon cancer cells [33]. These studies support our findings about the antiproliferative effect exerted by our monastrol-melatonin hybrids on these colon cancer cell lines, highlighting the importance of realizing further studies on this matter.

Fig. 3
figure 3

Effect of the most active monastrol/melatonin hybrids (6a, 6c, 6i, and 6j) on cell proliferation of SW480 cells 48 h post treatment with different concentrations of compounds (0.6–40 µM). Data are presented as the mean ± SE of at least three independent experiments. Vehicle control (DMSO 0.5%) was assumed as 100% of viability

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Fig. 4
figure 4

Representative images of SW480 and NCM460 cells 48 h after treatment with different concentrations of the most selective hybrid molecule (6i) and the vehicle control (magnification: 40×)

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Structure–activity Relationship (SAR)

According to the SAR analysis illustrated in Fig. 5, the hybrid 6a without any substitution displayed moderate selectivity, even though it did not show the lowest IC50 value. In addition, we could observe that substitution at position 3 with an electron-donating group (-OCH3) seems to be a key feature in the cytotoxic activity and selectivity as evidenced in the response induced by compounds 6c, 6i, and 6j. The IC50 values in these three molecules were even lower regarding 6a. The importance of this position and methoxy group was also evidenced by the results of compound 6b, where the presence of an electron-withdrawing group (-Cl) in position 3 of the aromatic ring decreased selectivity. In addition, we could observe that the mono substitution at position 4 was not very relevant for the selectivity since compounds 6d substituted with an electron-withdrawing group (-F)) and 6e with an electron-donating group (-N(CH3)2) although displayed low IC50 values after 96 h of treatment (6.6 and 7.2 µM, respectively), they did not improve the selectivity over time. Besides, contrasting the results observed with disubstituted compounds 6g (with substitution pattern 2,4-OCH3) which decreased the selectivity, with hybrid 6i (3,4-OCH3 substitution), which displayed an important improvement in selectivity over time, we propose that the substitution in position 4 imparts an improvement in the activity when it cooperates with another substituent on the aromatic ring, being stronger the synergia between positions 3 and 4 because it has a positive effect in selectivity, while in positions 2 and 4 the selectivity decreases. Similar results were observed regarding position 2 with compounds 6f (substitution pattern 2,3-OCH3) and 6h (substitution pattern 2,5-OCH3) since these compounds displayed the lowest activity against malignant cells with the highest IC50 values after 96 h of treatment (18.9 and 14.7 µM, respectively), besides, they displayed poor selectivity. We also appreciate that the synergistic effect observed with the above-mentioned patterns is lost when we have a trisubstituted compound since 6k showed low cytotoxicity and selectivity, so it seems that the synergic effect on positions 3 and 4 are lost when the compound has three substitutions on the aromatic ring rather than having only two.

Fig. 5
figure 5

Schematic representation of SAR analysis of the monastrol-melatonin hybrids 6a–k

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Theoretical drug-likeness and toxicological evaluation for promising hybrids 6a, 6c, 6i, and 6j

Characteristics such as drug-likeness, pharmacokinetic, and physicochemical properties can be used as efficient filters in the development of new anticancer candidates. Preclinical and clinical trials for a lead molecule could be encouraged by early biopharmaceutical profiling forecasts that increase the likelihood of success in drug discovery settings and result in a significant reduction of time and cost. In this work, pharmacokinetic parameters were computed by using the SwissADME web tool [34]. As previously discussed, among all the synthesized compounds, 6a (-H), 6c (3-OMe), 6i (3,4-OMe), and 6j (3,5-OMe) showed great ability to induce cytotoxicity in human colorectal cancer cells (SW480) with good selectivity (IS > 2), therefore, in the course of this section we focused our study only on these hybrids. Thereby, eleven crucial biopharmaceutical parameters were calculated for the selected hybrids and then compared against those major approved drugs (Table 3).

Table 3 Biopharmaceutic score for the most promising conjugates 6a, 6c, 6i, and 6j
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Our findings suggest suitable pharmacokinetics indices for hybrids compared to major marketed drugs, showing their therapeutic potential as orally administrable drug candidates for further pre-clinical experiments. In particular, the degree of lipophilicity (calculated as logPo/w) was predicted to be around 3.03, fitting well within the ideal range for lipid-based formulations (−2.0 to 6.0) [35]. In addition to this crucial permeability parameter, we also calculated the PSA, which similar to logP well correlates to the passive permeation of drugs through the biological cell membranes [36]. Notably, hybrids showed optimal PSA values of 130.11 and 148.57 Å2, respectively, which together with the logPo/w value would suggest good oral absorption and bioavailability. Besides, we also investigated both the fraction of carbon atoms that are sp3 hybridized (Fsp3) and the aromatic ring count (#ArRNG) which are two key new drug-likeness parameters relating to the possibility of liability or movement of a molecule through a biological barrier [37,38,39,40,41]. Major commercially available drugs have Fsp3 < 0.5. In this context, optimal 0.30 and 0.34 of Fsp3 values were found for hybrids, respectively. Further, the number of aromatic and heteroaromatic rings (#ArRNG) was counted. Current evidence has shown that molecules containing ≤3 aromatic rings (∼96% of marketed drugs meet this criterion) would have a better chance during drug development [40]. In this respect, the top-three hybrids bearing three aromatic rings would have an optimal developability profile. On the other hand, the in silico passive transmembrane permeation calculated for the title compounds using Caco-2 cell monolayers or MDCK cells as models was also considered. Both models are often recommended as a simplified in vitro model of intestinal absorption after oral administration in drug discovery [41,42,43,44]. Thus, promising compounds would possess a permeability range from 467 to 307 nm/s, respectively, across the intestinal Caco-2 cell monolayers model, and an apparent permeability (Papp) between 524 and 578 nm/s across a monolayer of MDCK cells. These in silico permeability results mean that these hybrids would be suitable to test in further studies for oral dosing. Another approach that predicted the ability of the drug candidates to bind blood plasma proteins was computed for 6a, 6c, 6i, and 6j. The most crucial factor influencing the distribution and transportation of anticancer formulations in the systemic circulation and a crucial factor in the initial stages of drug discovery is binding to human serum albumin (expressed as logKHSA) [45, 46]. According to the predictive model, compounds with positive values are expected to have a higher affinity for binding HSA, whereas compounds with negative values may exhibit a lower affinity for binding HSA. The calculated binding affinity to HSA for the top-three hybrids resulted to be positive numbers of 0.957 (for 6a), 0.950 (for 6c), 0.903 (for 6i), and 0.896 (for 6j), which fit well within the recommended range for potential oral drugs candidates (–1.5 to 1.5).

In addition to pharmacokinetic studies, ten toxicological endpoints have also been estimated for the lead-hybrids 6a, 6c, 6i, and 6j by employing several open-source chemoinformatic servers such as OSIRIS, TEST, ProTox-II, Pred-hERG, pkCSM, SwissADME, ToxTree, and ADMET-SAR (Table 4). These parameters are closely associated with adverse effects in the progress of a lead molecule, and their early estimation through artificial intelligence tools offer an attractive and rapid low-cost approach toward the design of safer pharmaceutical lead candidates [47, 48]. Regarding their in silico toxicological diagnosis, promising 6a, 6c, 6i, and 6j would have no apparent warnings, precautions and adverse events as mutagenic, tumorogenic, irritant, hepato/nephro/neuro/cardio/immunotoxic, any reproductive toxic effect, any structural alert for covalent DNA binding, and any conspicuous oral toxicity. As well, no alerts for pan-assay promiscuity (PAINS) were found for hit-hybrids.

Table 4 Toxicological profile for the most potent conjugates 6a, 6c, 6i, and 6j
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Conclusions

Eleven monastrol-melatonin hybrids were synthesized and their structures were confirmed by means of 1H-NMR, 13C-NMR, DEPT-135 spectroscopy, and ESI-MS. The biological activity of these compounds was tested against a colorectal cancer cell line SW480 and a normal human colon mucosal epithelial cell line NCM460. The most active and selective compound 6i showed cytotoxicity against SW480 cells, with an IC50 value of 5.2 ± 1.22 µM, and the IC50 value against NCM460 was 16.4 ± 1.07 µM after 96 h of treatment, with a SI of 3.1, being much more active than parental compounds (melatonin and monastrol) and slightly more selective than the reference drug 5FU. The increase in the activity and selectivity was supposed to be attributed to the presence of electron-donating groups like methoxy at 3 and 4 positions. In addition, theoretical drug-likeness and toxicological studies would suggest that the promising hybrids 6a, 6c, 6i, and 6j exhibit optimal biopharmaceutical indices to be considered in further pre-clinical testing. Taken altogether, hybrids merging monastrol/MLT of the present study could serve as a promising scaffold in the future for the development of more effective anti-colorectal cancer agents. However, further theoretical, and experimental assays are needed to further validate these preliminary findings.

Experimental methods and materials

Chemistry

1H NMR and 13C NMR spectra were measured in CDCl3 solutions on a Varian instrument operating at 300 and 75 MHz, respectively, at 25 °C and referenced to tetramethylsilane (TMS). Chemical shifts are expressed in terms of parts per million (ppm, δ) in relation to TMS. High-resolution mass spectra of every hybrid were obtained using electrospray ionization mass spectrometry (ESI-MS). In the W-mode, a QTOF Premier apparatus (Waters, Manchester, UK) featuring an orthogonal Z-spray-electrospray interface was employed. Using a Stuart SMP10 Digital Melting Point apparatus, the melting point (uncorrected) was ascertained. Commercial sources provided all common reagents and solvents, which were utilized without additional purification. Analytical thin-layer chromatography (TLC) was used to track the progress of the chemical reactions on precoated silica gel 60 F254 (Merck), and UV light (λ = 254 nm) was used to detect the spots. On Merck 230–400 mesh silica gel, column chromatography separations were carried out under standard pressure (Merck).

General procedure for the synthesis of monastrol derivatives (2a–k)

a mixture of aldehyde 1A (1.4 mmol), ethyl acetoacetate 1B (2,1 mmol), thiourea 1C (2.4 mmol) and citric acid (0.8 mmol) was taken into a round bottom flask and the reaction mixture heated at 80 °C for 5 h. Then the reaction mixture was dissolved in ethyl acetate and it was transferred to a separatory funnel and washed twice with water, the organic layer was dried with Na2SO4 which, after filtration, was concentrated on a rotatory evaporator, and the residue was purified by column chromatography over silica gel using a mixture of hexane/ethyl acetate of different ratios to obtain the respective monastrol derivative 2a–k.

Synthesis of 2-chloro-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)acetamide (5)

5-methoxytryptamine (3) (1,6 mmol) was dissolved in dry THF (7 mL) in a reaction flask equipped with a magnetic stirrer and then the system was sealed and saturated with argon. This solution was stirred in an ice-cold water bath for 15 min, then at this temperature, DIPEA (1,7 mmol) and 2-chloroacetyl chloride 4 (1,7 mmol) were added to the reaction flask using a syringe. The reaction mixture was stirred in the same water bath for 2 more hours. After completion of the reaction, the mixture is vacuum dried, and then it is re-dissolved in ethyl acetate, transferred to a separatory funnel, and washed twice initially with water and then with an HCl 10% solution. The organic layer was dried with Na2SO4 and then it was vacuum dried to acquire a crude material which was purified by silica gel chromatography column using a solution of hexane/ethyl acetate of different polarities to acquire amide 5 with a yield of 60%.

Synthesis of monastrol-melatonin hybrids 6a–k

A mixture of monastrol derivatives (2a–k) (0,2 mmol), amide 5 (0,2 mmol), Et3N (0,6 mmol), KI (0,3 mmol), and absolute EtOH (8 mL) was refluxed for 20 h. After reaction completion, the mixture was vacuum dried, and then it was re-dissolved in ethyl acetate, transferred to a separatory funnel, and washed twice with water. The organic layer was dried with Na2SO4 and then it was vacuum dried to acquire a crude material which was purified by a silica gel preparative plate using a solution of hexane/ethyl acetate in a proportion of 4/7 as eluent to yield hybrids 6a–k between 40–80%.

Ethyl-2-((2-((2-(5-methoxy-1H-indol-3-yl)ethyl)amino)-2-oxoethyl)thio)-4-methyl-6-phenyl-1,6-dihydropyrimidine-5-carboxylate (6a)

Pale yellow solid; yield: 40%, mp: 116–118 °C; 1H NMR (300 MHz, CDCl3) δ 7.39–7.19 (m, 6H), 7.04 (d, J = 2.5 Hz, 1H), 6.95 (d, J = 2.5 Hz, 1H), 6.87 (dd, J = 8.8, 2.5 Hz, 1H), 5.53 (s, 1H, CH-Ar), 4.16 (dd, J = 7.1, 3.1 Hz, 2H, CH3-CH2-O), 3.90 (s, 3H), 3.52 (d, J = 3.7 Hz, 2H), 3.48 (s, 2H), 2.80 (s, 2H, S-CH2), 2.31 (s, 3H), 1.24 (t, J = 7.1 Hz, 3H, CH3-CH2-O). 13C NMR (75 MHz, CDCl3) δ 170.07(NH-C = O), 166.81 (O-C=O), 153.88 (Ar-O), 144.28 (Ar-CH), 131.59 (Ar-NH), 128.63 (Ar), 127.76 (Ar), 127.59 (Ar), 126.84 (Ar), 123.01 (C=CH-NH), 112.56 (CH=C-CH2), 112.17 (Ar), 111.96(C–C=O), 100.72 (Ar), 60.14 (CH2-O), 56.06(CH-Ar), 39.76 (NH-CH2), 33.48 (S-CH2), 24.94, 14.25. HRMS: m/z 507.2100, calcd. for C27H30N4O4S, [M]+ 507.2096.

Ethyl 6-(3-chlorophenyl)-2-((2-((2-(5-methoxy-1H-indol-3-yl)ethyl)amino)-2-oxoethyl)thio)-4-methyl-1,6-dihydropyrimidine-5-carboxylate (6b)

Pale yellow solid; yield: 80%, mp: 57–59 °C; 1H NMR (300 MHz, MeOD) δ 7.04–6.98 (m, 4H), 6.91 (d, J = 7.6 Hz, 1H), 6.82 (d, J = 2.4 Hz, 1H), 6.78 (s, 1H), 6.55 (dd, J = 8.7, 2.4 Hz, 1H), 5.13 (s, 1H, CH-Ar), 3.92–3.81 (m, 2H, CH3-CH2-O), 3.62 (s, 3H), 3.40 (d, J = 14.4 Hz, 2H), 3.33–3.18 (m, 2H), 3.11 (s, 2H, S-CH2), 2.08 (s, 3H), 0.96 (t, J = 7.1 Hz, 3H, CH3-CH2-O). 13C NMR (75 MHz, MeOD) δ 170.34 (NH-C=O), 166.81 (O-C=O), 153.53 (C-S), 133.76 (Ar-NH), 131.98 (Ar), 129.67 (Ar-Cl), 127.63 (Ar), 126.69 (Ar), 125.04 (C=CH-NH), 122.80 (Ar), 111.54 (CH=C-CH2), 111.40 (Ar), 111.25 (Ar), 99.81(C–C=O), 59.57 (CH2-O), 54.95 (CH-Ar), 39.90(NH-CH2), 32.79 (S-CH2), 24.61, 13.12. HRMS: m/z 542.1716, calcd. for C27H29ClN4O4S, [M]+ 542.1711.

Ethyl 2-((2-((2-(5-methoxy-1H-indol-3-yl)ethyl)amino)-2-oxoethyl)thio)-6-(3-methoxyphenyl)-4-methyl-1,6-dihydropyrimidine-5-carboxylate (6c)

Pale yellow solid; yield: 42%, mp: 67–69 °C; 1H NMR (300 MHz, CDCl3) δ 7.23 (m, 3H), 7.04 (d, J = 2.4 Hz, 1H), 6.94 (d, J = 2.4 Hz, 1H), 6.88 (s, 1H), 6.85 (s, 1H), 6.83–6.76 (m, 1H), 5.52 (s, 1H, CH-Ar), 4.25–4.07 (m, 2H, CH3-CH2-O), 3.90 (s, 3H), 3.78 (s, 3H), 3.50 (d, J = 5.1 Hz, 4H), 2.81 (s, 2H, S-CH2), 2.30 (s, 3H), 1.25 (t, J = 7.1 Hz, 3H, CH3-CH2-O). 13C NMR (75 MHz, CDCl3) δ 170.00(NH-C=O), 166.78(O-C=O), 159.76 (C-S), 153.88 (Ar-O), 131.58 (Ar-CH), 129.67 (Ar-NH), 127.77 (Ar), 122.96 (C=CH-NH), 119.20 (Ar), 113.13 (Ar), 112.58 (Ar), 112.30 (CH=C-CH2), 112.17 (Ar), 111.95 (C–C=O), 100.66 (Ar), 60.16, 56.03, 55.24, 39.6. HRMS: m/z 537.2264, calcd. for C28H32N4O5S, [M]+ 537.2258.

Ethyl 6-(4-fluorophenyl)-2-((2-((2-(5-methoxy-1H-indol-3-yl)ethyl)amino)-2-oxoethyl)thio)-4-methyl-1,6-dihydropyrimidine-5-carboxylate (6d)

Off-white solid; yield: 40%, mp: 89–91 °C; 1H NMR (300 MHz, CDCl3) δ 7.32–7.24 (m, 2H), 7.23–7.14 (m, 2H), 7.06 (d, J = 2.4 Hz, 1H), 6.96 (d, J = 8.7 Hz, 2H), 6.89 (dd, J = 8.7, 2.4 Hz, 1H), 5.47 (s, 1H, CH-Ar), 4.16 (dd, J = 7.1, 4.6 Hz, 2H, CH3-CH2-O), 3.91 (s, 3H), 3.51 (m, 4H), 2.83 (s, 2H, S-CH2), 2.32 (s, 3H), 1.24 (t, J = 7.1 Hz, 3H, CH3-CH2-O). 13C NMR (75 MHz, CDCl3) δ 169.95 (NH-C=O), 166.73 (O-C=O), 153.95 (Ar-O), 131.60 (Ar-CH), 128.55 (Ar-NH), 128.44 (Ar), 127.79 (Ar), 122.96 (C=CH-NH), 115.49 (Ar), 115.20 (CH=C-CH2), 112.61 (Ar), 112.27 (Ar), 111.97 (C–C=O), 100.74(Ar), 60.16 (CH2-O), 56.07 (CH-Ar), 39.87 (NH-CH2), 33.52 (S-CH2), 24.97, 14.26. HRMS: m/z 525.2001, calcd. for C27H29FN4O4S, [M]+ 525.1995.

Ethyl 6-(4-(dimethylamino)phenyl)-2-((2-((2-(5-methoxy-1H-indol-3-yl)ethyl)amino)-2-oxoethyl)thio)-4-methyl-1,6-dihydropyrimidine-5-carboxylate (6e)

Pale yellow solid; yield: 51%, mp: 92–94 °C; 1H NMR (300 MHz, CDCl3) δ 7.25 (d, J = 8.8 Hz, 1H), 7.13 (d, J = 8.8 Hz, 2H), 7.05 (d, J = 2.5 Hz, 1H), 6.94 (d, J = 2.5 Hz, 1H), 6.87 (dd, J = 8.8, 2.5 Hz, 1H), 6.63 (d, J = 8.8 Hz, 1H), 5.42 (s, 1H, CH-Ar), 4.15 (q, J = 7.1 Hz, 2H, CH3-CH2-O), 3.90 (s, 3H), 3.53 (s, 4H), 2.88 (s, 6H), 2.81 (s, 2H, S-CH2), 2.26 (s, 3H), 1.26 (t, J = 7.1 Hz, 3H, CH3-CH2-O). 13C NMR (75 MHz, CDCl3) δ 170.19(NH-C=O), 167.11(O-C=O), 153.85 (Ar-O), 150.03 (Ar-N(CH3)2), 132.60 (Ar-CH), 131.58 (Ar-NH), 127.77 (Ar), 127.65 (2Ar), 122.96 (C=CH-NH), 112.64 (CH=C-CH2), 112.46 (Ar), 112.13 (Ar), 111.95 (C–C=O), 100.61 (Ar), 60.02 (CH2-O), 56.01 (CH-Ar), 40.52 (NH(CH3)2), 39.70 (NH-CH2), 33.51 (S-CH2), 24.97, 14.32. HRMS: m/z 550.2477, calcd. for C29H35N5O4S, [M]+ 550.2476.

Ethyl 6-(2,3-dimethoxyphenyl)-2-((2-((2-(5-methoxy-1H-indol-3-yl)ethyl)amino)-2-oxoethyl)thio)-4-methyl-1,6-dihydropyrimidine-5-carboxylate (6f)

Pale yellow solid; yield: 41%, mp: 101–102 °C; 1H NMR (600 MHz, CDCl3) δ 7.22 (d, J = 8.9 Hz, 1H), 7.04 (d, J = 2.4 Hz, 1H), 6.99–6.95 (m, 2H), 6.85–6.80 (m, 2H), 6.72 (dd, J = 7.7, 1.5 Hz, 1H), 5.81 (s, 1H, CH-Ar), 4.09 (q, J = 7.1 Hz, 2H, CH3-CH2-O), 3.90 (s, 3H), 3.88 (s, 3H), 3.82 (s, 3H), 3.53–3.42 (m, 4H), 2.82 (s, 2H, S-CH2), 2.31 (s, 3H), 1.15 (t, J = 7.1 Hz, 3H, CH3-CH2-O). 13C NMR (75 MHz, DMSO) δ 170.01(NH-C=O), 160.72(O-C=O), 153.41 (Ar-O), 145.94 (Ar-O), 136.50 (Ar-NH), 134.73 (Ar-CH), 133.17 (Ar), 131.40 (Ar), 127.56 (C=CH-NH), 123.35 (Ar), 112.11 (CH=C-CH2), 110.66 (Ar), 108.32 (Ar), 106.88 (Ar), 100.23 (C–C=O), 99.29 (Ar), 57.41 (CH3-O), 55.78, 55.55 (CH-Ar), 42.07, 22.69. HRMS: m/z 567.2319, calcd. for C29H34N4O6S, [M]+ 567.2314.

Ethyl 6-(2,4-dimethoxyphenyl)-2-((2-((2-(5-methoxy-1H-indol-3-yl)ethyl)amino)-2-oxoethyl)thio)-4-methyl-1,6-dihydropyrimidine-5-carboxylate (6g)

Pale yellow solid; yield: 54%, mp: 90–92 °C; 1H NMR (300 MHz, CDCl3) δ 7.26 (d, J = 8.8 Hz, 1H), 7.06 (d, J = 2.4 Hz, 1H), 7.03–6.94 (m, 2H), 6.88 (dd, J = 8.8, 2.4 Hz, 1H), 6.49 (d, J = 2.4 Hz, 1H), 6.40 (dd, J = 8.4, 2.4 Hz, 1H), 5.79 (s, 1H, CH-Ar), 4.12 (q, J = 7.1 Hz, 2H, CH3-CH2-O), 3.91 (s, 3H), 3.85 (s, 3H), 3.77 (s, 3H), 3.58–3.40 (m, 4H), 2.86 (s, 2H, S-CH2), 2.28 (s, 3H), 1.19 (t, J = 7.1 Hz, 3H, CH3-CH2-O). 13C NMR (75 MHz, CDCl3) δ 169.87 (NH-C=O), 166.89 (O-C=O), 160.53 (Ar-O), 157.60 (C-S), 153.93 (Ar-O), 131.58 (Ar-NH), 127.78 (Ar), 122.94 (C=CH-NH), 112.72 (CH=C-CH2), 112.18 (Ar), 111.92 (Ar), 104.04 (C–C=O), 100.59 (Ar), 98.86 (Ar), 59.95 (CH2-O), 55.99 (2CH3-O), 55.57 (CH3-O), 55.35 (CH-Ar), 39.79 (NH-CH2), 33.64 (S-CH2), 25.08, 14.24. HRMS: m/z 567.2349, calcd. for C29H34N4O6S, [M]+ 567.2344.

Ethyl 6-(2,5-dimethoxyphenyl)-2-((2-((2-(5-methoxy-1H-indol-3-yl)ethyl)amino)-2-oxoethyl)thio)-4-methyl-1,6-dihydropyrimidine-5-carboxylate (6h)

Pale yellow solid; yield: 46%, mp: 97–99 °C; 1H NMR (300 MHz, CDCl3) δ 7.25 (d, J = 8.7 Hz, 1H), 7.06 (d, J = 2.5 Hz, 1H), 6.99 (d, J = 8.7 Hz, 2H), 6.87 (dd, J = 8.7, 2.5 Hz, 1H), 6.48 (d, J = 2.4 Hz, 1H), 6.39 (dd, J = 8.7, 2.4 Hz, 1H), 5.79 (s, 1H, CH-Ar), 4.12 (q, J = 7.1 Hz, 2H, CH3-CH2-O), 3.90 (s, 3H), 3.85 (s, 3H), 3.76 (s, 3H), 3.57–3.38 (m, 4H), 2.85 (s, 2H, S-CH2), 2.28 (s, 3H), 1.19 (t, J = 7.1 Hz, 3H, CH3-CH2-O). 13C NMR (75 MHz, CDCl3) δ 169.98(NH-C=O), 166.94(O-C=O), 160.49 (C-S), 157.59 (CH-CH3), 153.90 (Ar-O), 131.59 (Ar-NH), 127.76 (Ar-CH), 122.98 (C=CH-NH), 112.66 (Ar), 112.14 (CH=C-CH2), 111.94 (C–C=O), 104.05 (Ar), 100.57 (Ar), 98.85 (Ar), 59.95 (CH2-O), 55.99 (3CH3-O), 55.57 (CH3-O), 55.36 (CH-Ar), 39.80 (NH-CH2), 33.61 (S-CH2), 25.06, 14.24. HRMS: m/z 567.2342, calcd. for C29H34N4O6S, [M]+ 567.2337.

Ethyl 6-(3,4-dimethoxyphenyl)-2-((2-((2-(5-methoxy-1H-indol-3-yl)ethyl)amino)-2-oxoethyl)thio)-4-methyl-1,6-dihydropyrimidine-5-carboxylate (6i)

Pale yellow solid; yield: 47%, mp: 104–106 °C; 1H NMR (300 MHz, CDCl3) δ 7.25 (d, J = 8.8 Hz, 1H), 7.05 (d, J = 2.4 Hz, 1H), 6.96 (d, J = 2.4 Hz, 1H), 6.88 (d, J = 2.4 Hz, 1H), 6.85 (d, J = 2.1 Hz, 1H), 6.78 (d, J = 2.1 Hz, 1H), 6.76 (s, 2H), 5.49 (s, 1H, CH-Ar), 4.17 (dd, J = 7.1, 1.8 Hz, 2H, CH3-CH2-O), 3.89 (s, 3H), 3.86 (s, 3H), 3.81 (s, 3H), 3.62–3.41 (m, 4H), 2.81 (s, 2H, S-CH2), 2.32 (s, 3H), 1.26 (t, J = 7.1 Hz, 3H, CH3-CH2-O). 13C NMR (75 MHz, CDCl3) δ 170.01 (NH-C=O), 166.85 (O-C=O), 153.89 (Ar-O), 148.90 (Ar-O), 148.47 (Ar-O), 137.02 (Ar-CH), 131.58 (Ar-NH), 127.75 (Ar), 123.03 (C=CH-NH), 118.87 (Ar), 112.48 (CH=C-CH2), 112.13 (Ar), 111.96 (Ar), 111.18 (Ar), 110.40 (C–C=O), 100.65 (Ar), 60.17 (CH2-O), 55.93 (CH-Ar), 39.80 (NH-CH2), 33.48 (S-CH2), 24.96, 14.33. HRMS: m/z 567.2318, calcd. for C29H34N4O6S, [M]+ 567.2314.

Ethyl 6-(3,5-dimethoxyphenyl)-2-((2-((2-(5-methoxy-1H-indol-3-yl)ethyl)amino)-2-oxoethyl)thio)-4-methyl-1,6-dihydropyrimidine-5-carboxylate (6j)

Pale yellow solid; yield: 44%, mp: 104–106 °C; 1H NMR (300 MHz, CDCl3) δ 7.25 (d, J = 8.8 Hz, 1H), 7.05 (d, J = 2.4 Hz, 1H), 6.94 (d, J = 2.4 Hz, 1H), 6.87 (dd, J = 8.8, 2.4 Hz, 1H), 6.47 (d, J = 2.2 Hz, 2H), 6.37 (t, J = 2.2 Hz, 1H), 5.48 (s, 1H, CH-Ar), 4.18 (dd, J = 7.1, 2.0 Hz, 2H, CH3-CH2-O), 3.90 (s, 3H), 3.76 (s, 6H), 3.51 (s, 4H), 2.81 (s, 2H, S-CH2), 2.27 (s, 3H), 1.27 (t, J = 7.1 Hz, 3H, CH3-CH2-O). 13C NMR (75 MHz, CDCl3) δ 170.02 (NH-C=O), 166.85 (O-C=O), 160.92 (2Ar-O), 153.87 (Ar-O), 131.58 (Ar-CH), 127.77 (Ar-NH), 122.94 (C=CH-NH), 112.56 (CH=C-CH2), 112.16 (Ar), 111.94 (Ar), 105.21 (2Ar-O), 100.61 (C–C=O), 98.80 (Ar), 60.15 (CH2-O), 56.00 (CH-Ar), 55.31 (3CH3-O), 39.64 (NH-CH2), 33.58 (S-CH2), 24.94, 14.33. HRMS: m/z 567.2313, calcd. for C29H34N4O6S, [M]+ 567.2308.

Ethyl 2-((2-((2-(5-methoxy-1H-indol-3-yl)ethyl)amino)-2-oxoethyl)thio)-4-methyl-6-(3,4,5-trimethoxyphenyl)-1,6-dihydropyrimidine-5-carboxylate (6k)

Pale yellow solid; yield: 42%, mp: 87–89 °C; 1H NMR (300 MHz, CDCl3) δ 7.31 (s, 1H), 7.26 (d, J = 8.7 Hz, 1H), 7.05 (d, J = 2.5 Hz, 2H), 6.97 (s, 1H), 6.88 (dd, J = 8.7, 2.5 Hz, 1H), 6.51 (s, 1H), 5.49 (s, 1H, CH-Ar), 4.27–4.12 (m, 2H, CH3-CH2-O), 3.90 (s, 2H), 3.84 (s, 2H), 3.54 (m, 4H), 2.35 (s, 2H, S-CH2), 1.84 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H, CH3-CH2-O). 13C NMR (75 MHz, CDCl3) δ 169.83 (NH-C=O), 167.00 (O-C=O), 153.91 (CH-CH3), 153.24 (3Ar-O), 131.59 (Ar-NH), 127.77 (Ar), 122.96 (C=CH-NH), 112.52 (CH=C-CH2), 112.20(Ar), 111.95(Ar), 104.02 (C–C=O), 100.62(Ar), 60.81, 60.18, 56.16, 56.02, 39.82, 33.62, 25.02, 14.39. HRMS: m/z 597.2432, calcd. for C30H36N4O7S, [M]+ 597.2426.

In vitro biological assays

Cell lines and culture medium

Biological activity was investigated using nonmalignant cells (NCM460) and human colorectal adenocarcinoma cells (SW480), obtained from the European Collection of Authenticated Cell Cultures (ECACC, UK). Dulbecco’s Modified Eagle Medium (DMEM) was used to maintain the cells, supplemented with 10% horse serum previously inactivated at 56 °C, non-essential amino acids (1%), and antibiotics (penicillin/streptomycin, 1%) (Gibco Invitrogen, Carlsbad, CA, USA). For all the experiments, the horse serum was reduced to 3%, and the culture media was additionally supplemented with ITS-defined medium (10 mg/mL insulin, 5 mg/mL transferrin, and 5 ng/mL selenium) (Gibco, Invitrogen, Carlsbad, CA, USA) [49, 50].

Cell viability

The colorimetric test with Sulforhodamine B (SRB) was used to evaluate cell viability. Cell density was adjusted to 6500 cells/well in 96-well tissue culture plates and cells were first incubated for 24 h. After this, cell lines were daily treated with increasing concentrations (0.6 to 40 µM) of the different compounds, from 24 to 96 h. In addition to the hybrid molecules, in this study, we included different controls. The first one was the vehicle used to solubilize the compounds, DMSO (0.5%), to establish the baseline of the experiments. In addition, we used the lead compounds (monastrol, melatonin, and the equimolar mixture of both), and the reference drug (5-FU). The optimum conditions were 37 °C and 5% CO2. Following the conclusion of every treatment, culture plates were kept at 4 °C for an hour in order to fix the cells. Cold trichloroacetic acid (MERCK, 50% v/v) was then added. After that, 0.4% (w/v) SRB (Sigma-Aldrich, St. Louis, MO, USA) was used to stain the cell protein content. To get rid of any unbound SRB, acetic acid (1%) was used as a wash. Protein-bound SRB was allowed to air dry before being solubilized in 10 mM Tris-base and then the absorbance at 492 nm was measured using a Mindray MR-96A microplate reader [51, 52]. Every experiment was run through at least three times.

Statistical analysis

Each experiment had a minimum of three repeats according to the experimental design. The data was presented as mean ± standard error, or SE. Tukey’s test was run after the two-way ANOVA statistical analysis. Significant p-values were those less than 0.05. Data were analyzed with GraphPad Prism version 8.0.1 for (GraphPad Software, San Diego, CA, USA).

Theoretical pharmacokinetics and toxicological studies

For promising 6a, 6c, 6i, and 6j, twelve biopharmaceutics indices together were computed by using the open-source platform SwissADME [34]. Importantly, these parameters govern oral exposure, as well as permeability, absorption, motion, and action of potential oncological drug candidates. Besides, we explored the toxicological profile for hybrids using eight in silico freeware platforms such as OSIRIS [53], T.E.S.T Version 5.1.2 [54], ProTox-II [55], Pred-Herg [56], pkCSM [57], SwissADME [34], ToxTree [58] and ADMET-SAR [59].