Design, synthesis and evaluation of fluorescent dihydropyridine–dihydropyrimidinone hybrids as inducers of cell-cycle arrest in a prostate cancer cell line via Aurora kinase interactions

Abstract

Fluorescent dihydropyridine–dihydropyrimidinone (DHP–DHPM) hybrids were easily synthesized through the combination of Hantzsch and Biginelli multicomponent reactions followed by a copper-catalyzed azide–alkyne cycloaddition reaction (CuAAC, click chemistry) protocol. Nine hybrids showed promising antitumor activity for the PC3 prostate cancer cell line, notably compounds 9d and 9g. Both hybrids exhibited high selectivity for tumor cells, with significant selectivity indices (SI), particularly 9g (SI >68.8). Selectivity was qualitatively observed by the internalization of the fluorescent hybrids through high-resolution confocal laser scanning microscopy (CLSM). In silico investigations and western blotting analysis showed a selective inhibition of the isoform C of Aurora kinase by hybrid 9d. A mechanism of action including cell cycle arrest at the G₀/G₁ phase, inhibition of cell migration and invasion, and modulation of key signaling pathways such as MAPK, AKT, and mTOR are discussed.

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1. Introduction

1,4-Dihydropyridines (DHPs), also known as Hantzsch esters, are heterocyclic nitrogen-containing compounds which have great importance due to their associated pharmacological properties, mainly as calcium channel blockers¹ responsible for the regulation of blood pressure in human hypertension.²

Several 1,4-dihydropyridines such as nifedipine, nicardipine or amlodipine are commercially available as anti-hypertensive drugs. Nifedipine(I), trade name Adalat®, was the first marketed DHP drug approved in 1975 by Bayer AG.³ DHP exhibits a variety of bioactivities such as antioxidant,^4a neuroprotective (cerebrocrast II),^4b antidiabetic,^4c antimicrobial,^4d leishmanicidal^4e or anticancer agents⁵ (Fig. 1). The intrinsic fluorescence of DHP⁶ was explored as a molecular probe to detect chemical species⁷ or simply as a flashlight to “illuminate” the trajectory and position of the molecule inside the cell (Fig. 1).⁸

Fig. 1 Examples of bioactive 1,4-dihydropyridines reported in the literature.

3,4-Dihydropyrimidin-2-one (DHPM), also known as Biginelli adduct, has a pyrimidine pharmacophoric nucleus which presents a plethora of different bioactivities as discussed in recent reviews.⁹ Among them, dihydropyrimidin-2-thiones displaying diverse structural variability can show potent anticancer activity against UACC-62 (melanoma),^10a A549 (lung),^10b HepaRG (hepatic),^10c or L-5178-y (lymphoma),^10d a biological profile which has attracted our interest (Fig. 2). Monastrol, a dihydropyrimidin-2-thione, is known as an Eg5 kinesin inhibitor¹¹ as well as an inhibitor of tyrosinase, cytochrome P450, estrogen and progesterone receptors that could be overexpressed in tumor cells, promoting cell survival and proliferation.¹²

Fig. 2 Examples of the structural diversity of anticancer dihydropyrimidin-2-thiones.

Until a few years ago, the “magic bullet” paradigm was the main strategy for the development of new drugs. However, monofunctional drugs have several limitations that make it difficult to treat multifactorial diseases.¹³ The use of drug cocktails for multifactorial diseases is not always effective due to extensive drug interactions and multiple side effects that often interfere with a patient's adherence to treatment.

For complex diseases, most drugs remain largely ineffective, and the rigid concept of “one target, one drug” is gradually being abandoned.¹⁴ To overcome the limited success of monofunctional drugs, the concept of polypharmacology began to be developed and applied.¹⁵ In this context, the molecular hybridization strategy creates a single molecular entity with multifunctional activity aiming to avoid the limitations of the “magic bullet” paradigm.¹⁶

This methodology involves the covalent linkage of two or more pharmacophores through an appropriate spacer to generate a single molecular entity, commonly referred to as a hybrid.¹⁷ The principal rationale underlying hybrid drug design is the possibility of mitigating the inherent limitations associated with monotherapy or conventional combination chemotherapy.¹⁸ These limitations include the emergence of drug resistance, suboptimal pharmacokinetic profiles, and solubility issues of the individual constituents. Of particular significance is the potential for enhanced selectivity: one moiety of the hybrid may function as a tumor-targeting ligand, while the other confers the therapeutic effect.¹⁹

Furthermore, hybrids may exhibit synergistic pharmacological activity, thereby surpassing the efficacy of their parent compounds.²⁰ The hybrids may incorporate both pharmacophores in their active forms, or alternatively, one or both moieties may be introduced in a prodrug.²¹

Previous reports from our laboratory demonstrated the success of this strategy of developing dihydropyrimidinone-based hybrid compounds such as perillyl–DHPM,^22a fatty acid–DHPM,^22b arylbenzothiazole–DHPM,^22c piplartine–DHPM^22d and chalcone–DHPM¹⁷ (Fig. 3).

Fig. 3 Dihydropyrimidinone-based hybrids with anticancer activity.

Motivated by the growing interest in designing novel hybrid compounds based on DHPM scaffolds, we visualized the strategy of molecular hybridization involving different pharmacophores such as DHP and DHPM, aiming to enhance bioactivity.²³ In this work, a series of nine molecular hybrids combining dihydropyridines and dihydropyrimidinones (DHP–DHPM) was synthesized and various physical and biological properties were evaluated, including cytotoxicity against breast and prostate cancer cell lines, fluorescence cell imaging, effects on cell migration and invasion, inhibition of protein kinases, and molecular docking studies.

The synthesis of these hybrids represents an opportunity to develop new fluorescent hybrid small molecules with anticancer activity.

2. Results and discussion

2.1. Synthesis of hybrid compounds

The construction of molecules with complex architecture has imposed significant challenges in organic and medicinal chemistry, typically demanding multi-step sequences, expensive reagents, and tedious purification protocols. In recent years, the strategic integration of multicomponent reactions (MCRs)²⁴ and click chemistry²⁵ has transformed the synthetic approaches of structurally diverse pharmacophores, heterocycles, and drug-like scaffolds, offering a streamlined pathway to intricate structures with high efficiency, selectivity, and atom economy.²⁶ This integrated methodology exemplifies how classic reactions, when paired with modern click chemistry, can revolutionize the drug discovery and development process from simplicity to a cornerstone of modern organic synthetic chemistry.²⁷

Thus, the copper-catalyzed azide–alkyne cycloaddition reaction (CuAAC, click chemistry)²⁸ was chosen as the protocol to connect the two pharmacophores. Hence, it was necessary firstly to prepare the azide and the alkyne components. Propargyloxy–dihydropyridines and azido–dihydropyrimidinones were established as the azide and the alkyne components for the CuAAC reaction (Scheme 1).

Scheme 1 General scheme to synthesize the DHP–DHPM hybrids.

This protocol involves the use of CuSO₄ as a pre-catalyst and sodium ascorbate as the reducing agent to generate the active catalytic Cu(I) species. This method is particularly advantageous once a non-hydrolyzable triazole linker is formed. In addition, the triazole moiety can exhibit bioisosterism related to the amide bond that can be important for the biological properties.²⁹

2.1.1. Synthesis of propargyloxy–dihydropyridines (alkyne component). One of the main methods to obtain 1,4-dihydropyridines is the classical multicomponent Hantzsch 1,4-dihydropyridine synthesis, discovered in 1881.³⁰ The multicomponent synthesis of propargyloxy–dihydropyridines was performed by a modification of a previous report using (NH₄)₂CO₃ as the ammonia component and CeCl₃·7H₂O as the Lewis acid catalyst, which obviates the use of dry solvents.³¹ The reaction was carried out by mixing benzaldehyde derivatives 1a–1c, ethyl acetoacetate (2a) or dimedone (2b), (NH₄)₂CO₃ (3), and CeCl₃·7H₂O (20 mol%) in iPrOH to afford the propargyloxy–DHPs 4a–4e (Scheme 2). The propargyloxy–benzaldehydes 1a–1e were prepared through conventional O-alkylation of the corresponding hydroxybenzaldehydes.^22a

Scheme 2 Multicomponent synthesis of propargyloxy–dihydropyridines 4a–4e.

The propargyloxy–DHPs 4a–4e were obtained in good yields after chromatographic purification. The results are shown in Table 1.

Table 1 Synthesis of dihydropyrimidinones 4a–4e

Entry	1	R^1	R^2	2	DHP 4	Rend. (%)
1	1a	–OCH2C≡CH	H	2a	4a	75
2	1b	–OCH2C≡CH	OMe	2a	4b	74
3	1c	H	–OCH2C≡CH	2a	4c	79
4	1a	–OCH2C≡CH	H	2b	4d	80
5	1b	H	–OCH2C≡CH	2b	4e	75

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The ¹H NMR spectra of compounds 4a–4e showed a characteristic singlet signal in the range of 4.95–5.09 ppm assigned to the benzylic hydrogen. On the other hand, the benzylic carbon in 4a–4e appeared between 38.7 and 40.7 ppm in the ¹³C NMR spectra. The observed HRMS data were in accordance with the theoretically calculated molecular mass for all compounds.

2.1.2. Synthesis of azido–dihydropyrimidinones (azide component). The azido–dihydropyrimidinones were prepared in two steps. The first one involved the preparation of 6-chloro-DHPMs 7a and 7b through the classical multicomponent Biginelli reaction starting from benzaldehydes 1d and 1e, ethyl 4-chloroacetoacetate (5), and urea (6).³² Compounds 7a and 7b were obtained in 82% and 80% yield, respectively. Next, 7a and 7b were converted to the azido DHPMs 8a and 8b in excellent yields (88% and 91%, respectively) by reaction with NaN₃ (Scheme 3).³³ The ¹H NMR spectra of compounds 6-chloro-DHPMs 7a and 7b and 6-azido-DHPMs 8a and 8b showed diastereotopic methylene hydrogens at C-6 as two doublets, with vicinal coupling constants around 16 Hz for all compounds. The spectral data and melting points of the compounds were compatible with the previously reported data in the literature (see Experimental section).

Scheme 3 Synthesis of 6-azidomethyl-dihydropyrimidinones 8a and 8b.

2.1.3. Synthesis of dihydropyridine–dihydropyrimidinone hybrid compounds. Once the azide and alkyne moieties were installed in the respective pharmacophores, the CuAAC reaction protocol was applied for coupling between propargyloxy–dihydropyridines 4a–4e and the 6-azido–dihydropyrimidinones 8a and 8b. The reaction was performed at room temperature and was monitored by TLC until the complete consumption of the reagents (Scheme 4).

Scheme 4 Synthesis of hybrid compounds 9a–9ivia the CuAAC reaction.

The hybrid compounds 9a–9i were obtained in reasonable to good yields after purification through silica gel column chromatography. The results are presented in Table 2.

Table 2 Synthesis of hybrid compounds 9a–9i

Entry	Compound	Structure	Yield (%)
1	9a		70
2	9b		78
3	9c		74
4	9d		73
5	9e		51
6	9f		64
7	9g		85
8	9h		53
9	9i		79

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2.2. Hybrid molecules reduce the viability of prostate tumor cell lines

The in vitro cytotoxic activity of the novel DHP–DHPM hybrid compounds 9a–9i was evaluated against PC3 human prostate adenocarcinoma cells and PNT2 human non-tumorigenic prostate cells using the CCK-8 assay after 48 h of treatment. For comparison, we also assessed the cytotoxicity of monastrol (the most studied DHPM compound) and the commercial anticancer drug cisplatin, both employed as positive controls. The results are expressed as the half-maximum inhibitory concentration (IC₅₀) values and are summarized in Table 3 as well as the corresponding selectivity index (SI) of each compound.

Table 3 In vitro cytotoxic activities and selectivity indices (SI) of DHP–DHPM hybrids 9a–9i

Entry	Compound	IC50 (μM) ± SD^a		SI^b
		PC3	PNT2
a The half-maximum inhibitory concentration (IC50) value for each compound was calculated from two independent experiments performed in triplicate per plate. b SI: selectivity index defined as the ratio between the IC50 value for the normal cell line (PNT2) and the IC50 value for the cancer cell line (PC3); SI prostate = IC50 (PNT2)/IC50 (PC3).
1	9a	24.0 ± 0.7	529.5 ± 90.3	22.1
2	9b	22.7 ± 1.8	534.3 ± 4.4	23.6
3	9c	12.4 ± 3.8	61.4 ± 5.9	4.9
4	9d	6.4 ± 0.0	50.1 ± 2.0	7.8
5	9e	46.7 ± 12.4	64.9 ± 7.1	1.4
6	9f	56.4 ± 6.6	>1500	>26.6
7	9g	21.8 ± 2.5	>1500	>68.8
8	9h	52.3 ± 9.6	>1500	>28.7
9	9i	44.1 ± 0.8	212.1 ± 34.2	4.8
10	Cisplatin	7.5 ± 0.5	6.6 ± 0.5	0.9
12	Monastrol	96.1 ± 22.2	159.3 ± 27.9	1.7

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According to the data shown in Table 3, after 48 h of treatment the synthesized hybrid 9d exhibited the highest cytotoxic activity toward PC3 cells, with an IC₅₀ value of 6.4 ± 0.0 μM. Notably, 9d displayed an IC₅₀ value significantly lower than that of the positive control monastrol (96.1 ± 22.2 μM), a well-known potent and cell-permeant inhibitor of mitosis due to its capacity to bind specifically to the kinesin Eg5 motor protein.³⁴ Furthermore, 9d also presented cytotoxicity of the same magnitude as the reference anticancer drug cisplatin (7.5 ± 0.5 μM). The largest IC₅₀ values were observed for hybrids 9e (46.7 ± 12.4 μM), 9f (56.4 ± 6.6 μM), 9h (52.3 ± 9.6 μM) and 9i (44.1 ± 0.8 μM). Thus, the cytotoxic profile of 9d is in line with the recommendations of the NIH screening program which states that the IC₅₀ values of candidate molecules should be less than 10 μM.³⁵

Additionally, among the novel DHP–DHPM hybrids, 9d displayed the highest cytotoxicity (IC₅₀ = 13.8 ± 3.9 μM) against MCF-7 human breast cancer cells, which is comparable to the potency of cisplatin (IC₅₀ = 14.8 ± 4.5 μM). Hybrid 9d also exhibited significantly higher cytotoxic activity than monastrol (IC₅₀ = 115.1 ± 16.8 μM, Table S1 in the SI).

Besides potency, another key factor in the search for new anticancer drugs is their selectivity against cancer cells. Thus, as a first approximation of the safety profile of the DHP–DHPM hybrids, we determined their cytotoxic activity in PNT2 normal prostate cells. The selectivity index (SI) was calculated for each compound by dividing the IC₅₀ value observed for the noncancer cell line by the IC₅₀ value obtained for the corresponding cancer cell line. The results in Table 3 showed that in general, hybrids exhibited weaker cytotoxicity towards PNT2 cells when compared to PC3 ones, indicating selectivity towards these prostate cancer cells. Only 9e (SI = 1.4) demonstrated low selectivity for PC3 cancer cells but superior to cisplatin (SI = 0.9) and close to monastrol (SI = 1.7). The other synthesized hybrids showed SI values higher than 4.8, with notably high indices for 9f (SI >26.6), 9h (SI >28.7), 9d (SI = 7.8), and especially 9g (SI >68.8).

Thus, compound 9d was selected as the lead compound due to its highest potency among the tested hybrids (IC₅₀ = 6.4 μM). Compound 9g, although less potent (IC₅₀ = 21.8 μM), was also prioritized because of its markedly higher selectivity index (SI >68.8).

To clarify the differences in cytotoxic potency observed among the hybrids, a structure–activity relationship (SAR) analysis was performed. This evaluation considered three main aspects: the structural features of the DHP moiety, the specific positions on its aromatic ring where hybridization occurred, and the type of substituents present on the aromatic ring of the DHPM moiety. Structurally, the DHP fragment differs between the two series: in hybrids 9a–9e it is a monocyclic ring, whereas in hybrids 9f–9i it adopts a linear tricyclic structure. Despite the limited number of compounds, a general trend was observed: monocyclic DHP derivatives are typically more potent than tricyclic ones. Within the monocyclic series (9a–9e), the introduction of methoxy substituents in the aromatic ring of the DHPM moiety enhanced potency. For example, 9a (IC₅₀ = 24.0 μM) was less active than 9c (IC₅₀ = 12.4 μM). Importantly, the hybridization site on the DHP aromatic ring also played a key role: 9c, hybridized through the 4-position, showed an IC₅₀ of 12.4 μM, while its isomer 9d, hybridized through the 3-position, was 2-fold more potent (IC₅₀ = 6.4 μM). A similar trend was found in the tricyclic series (9f–9i). 9f (IC₅₀ = 56.4 μM, hybridized at the 4-position) was less active than its isomer 9g (IC₅₀ = 21.8 μM, hybridized at the 3-position). Likewise, 9h (IC₅₀ = 52.3 μM) was slightly less potent than 9i (IC₅₀ = 44.1 μM). However, unlike the monocyclic series, the presence of methoxy groups did not consistently improve potency. For instance, 9h (methoxylated, IC₅₀ = 52.3 μM) showed no advantage over 9f (non-methoxylated, IC₅₀ = 56.4 μM), and 9g (non-methoxylated, IC₅₀ = 21.8 μM) was more active than its methoxylated analogue 9i (IC₅₀ = 44.1 μM).

In summary, these results suggest that the nature of the DHP scaffold strongly influences the activity of these hybrids. For monocyclic derivatives (9a–9e), both hybridization position and methoxy substitution are critical for potency. In contrast, for tricyclic derivatives (9f–9i), the hybridization position appears to be the dominant factor, while methoxy substitution has little to no beneficial effect.

2.3. Hybrids 9d and 9g are selective for tumor cell lines and potential molecular fluorescent flashlights

The selectivity and/or internalization of the fluorescent hybrid compounds 9d and 9g were assessed by high-resolution confocal laser scanning microscopy (CLSM) due to their intrinsic captured fluorescence. In these experiments, the cell lines PC3 (tumor) and PNT2 (non-tumor) were incubated with the fluorescent hybrids for 8 h and/or 24 h under physiological conditions (37 °C). The treatments were carried out using concentrations corresponding to 50% of the IC₅₀ values presented in Table 3, i.e. 10.9 μM for 9g and 3.2 μM for 9d. Afterwards, cellular, nuclear and F-actin filaments were stained with DRAQ5 (blue channel) and Alexa Fluor 555 Phalloidin (green channel), respectively.

A fast internalization of 9d was observed as soon as 8 h of exposure, while no significant differences were observed after 8 h and 24 h (Fig. 4). As revealed in the merged CLSM image, this compound homogeneously accumulated inside the cell, including in the nucleus.

Fig. 4 (A) Representative CLSM images of PC3 cells incubated for 8 h and 24 h at 37 °C with hybrid 9d. The subcellular localization of nuclei (DRAQ5 stained, blue channel), F-actin cytoskeleton (Alexa Fluor 555 Phalloidin, green channel), and 9d (red channel) is presented. (B) Quantitative analysis of the mean fluorescence intensity of 9d internalized into PC3 cells (calculated using Fiji ImageJ software). Data are shown as mean ± SD (n = 2). Statistical significance was calculated using one-way ANOVA with a Tukey multiple comparison test (ns indicates statistically not significant).

According to Fig. 5, a red fluorescence is observed inside PC3 cells, indicating that hybrid 9g also quickly internalized into these prostate cancer cells after 8 h. Furthermore, 9g interestingly exhibited a very selective profile as it did not internalize into the PNT2 cell line after 8 h, in accordance with its selectivity index of more than 68.8 observed in the cytotoxicity assay (see Table 3).

Fig. 5 (A) Representative CLSM images of PC3 and PNT2 cells incubated for 8 h at 37 °C with hybrid 9g. The subcellular localization of nuclei (DRAQ5 stained, blue channel), F-actin cytoskeleton (Alexa Fluor 555 Phalloidin, green channel), and 9g (red channel) is presented. (B) Quantitative analysis of mean fluorescence intensity of 9g internalized into PC3 and PNT2 cells (calculated using Fiji ImageJ software). Data are shown as mean ± SD (n = 2). Statistical significance was calculated using one-way ANOVA with a Tukey multiple comparison test (***p < 0.001).

These results confirm the efficient and selective cellular uptake of hybrids 9d and 9g by PC3 cells, highlighting their potential not only as antineoplastic fluorescent compounds and confirming their internalization into the cell.

2.4. Hybrids 9d and 9g lead to cell cycle arrest in the G₀/G₁ phase and reduce the migration and invasiveness

The hybrid molecules did not cause any changes in the cell death patterns analyzed after 24 h of exposure to the PC3 tumor cell line. We detected a slight increase in tumor cells in the late death phase when treated with 9g (3.8%) and 9d (7.7%) but no significant difference compared to the control population (DMSO, 5.5%, Fig. 6A). On the other hand, significant changes were observed in cell cycle populations after treatment with hybrids 9d and 9g. There was a significant reduction in cells in the S phase compared to the negative (DMSO) and positive (cisplatin) controls (p = 0.01). Additionally, hybrid 9d promoted significant cell cycle arrest, with an increase in the G₀/G₁ population (p = 0.05) (Fig. 6B). The migration and invasion capacity of the PC3 tumor cell line was assessed after exposure to the hybrid molecules 9d and 9g for 24 h. A significant reduction in cell migration was identified for both molecules as well as for the positive control (cisplatin) (Fig. 6C and D).

Fig. 6 Evaluation of cell death, cell cycle, migration, and invasion. (A) Flow cytometry analysis of the PC3 cell population exposed to hybrid molecules (at IC₅₀ concentrations) for 24 h, showing early and late apoptotic phases. (B) Flow cytometry analysis of cell cycle distribution (G₀/G₁, S, and G₂/M phases) in PC3 cells exposed to hybrid molecules (at IC₅₀ concentrations) and cisplatin for 24 h. (C) Photomicrographs (40× magnification) illustrating PC3 cell migration after 24 h of treatment with hybrid molecules and cisplatin. (D) Bar graphs depicting the number of migrated cells. (E) Photomicrographs (40× magnification) illustrating PC3 cell invasion after 24 h of treatment with hybrid molecules and cisplatin. (F) Bar graphs depicting the number of invaded cells. DMSO was used as a negative control. **p < 0.05 and ***p < 0.001.

Similarly, the hybrid molecules significantly reduced the invasion of PC3 tumor cells compared to the control (DMSO), with hybrid 9d showing a greater reduction in invasion than the other treatments (p = 0.05) (Fig. 6E and F). In summary, hybrid 9d was able to promote G₀/G₁ cell cycle arrest in PC3 cancer cells and a significant reduction in invasion and migration rates. Similarly, a novel molecule designed from new dihydropyridine and pyridine analogues was able to arrest the cell cycle at the G₁ phase by inhibiting CDK4/6 in MCF7 cells.³⁶

2.5. Hybrids 9d and 9g modulate the MAP kinase pathways

After a 24 h treatment with the hybrid molecules 9d and 9g, the protein content of the PC3 cell line was analyzed using protein arrays. Treatment with the above hybrids positively regulated the phosphorylation of AKT1, AKT2 and AKT3 isoforms and generally increased the phosphorylation of the pan-AKT form, indicating an activation of this pathway (Fig. 7A and B). Among the p38 isoforms evaluated, hybrid 9d was able to reduce the phosphorylation of p38δ and p38γ proteins. In addition, both molecules significantly reduced the phosphorylation of MSK2, RSK1 and mTOR (Fig. 7A and B). In summary, hybrid molecules 9d and 9g were able to modulate intracellular pathways responsible for proliferation such as AKT, MSK2 and mTOR.

Fig. 7 Influence of hybrid molecules on proliferative signaling pathways. (A) Nitrocellulose membrane arrays containing 26 different capture antibodies in duplicate, exposed to protein extracts treated with 9d, 9g, and DMSO for 24 h. Red squares indicate duplicate antibodies with differential expressions between groups. (B) Bar graph showing the densitometric analysis of the captured dots.

Both hybrid molecules modulated targets within the MAP kinase-mediated signaling pathway. Specifically, molecule 9d reduced the expression of MSK2, ERK1, p53, RSK1, and mTOR, while molecule 9g decreased the protein levels of AKT1, CREB, HSP27, RSK1, and mTOR. These proteins are frequently dysregulated in tumors and play critical roles in regulating gene transcription, protein synthesis, cell proliferation, immune cell differentiation, and tumor metabolism.³⁷ Although both molecules demonstrated similar effects, they were able to inhibit MSK2 and mTOR, two key proteins involved in cell cycle progression. Notably, the inhibition of mTOR and MSK2, a kinase involved in the phosphorylation of H3S10, may be linked to the activity of the Aurora kinase family, which also phosphorylates H3S10/S28 at active promoters, further implicating these molecules in cell cycle regulation.³⁸ One series of dihydropyrimidinone derivatives has demonstrated potent enzyme inhibition against mTOR, showing greater efficacy compared to rapamycin.³⁹

2.6. Hybrids 9d and 9g interact with Aurora kinase C

In the molecular docking assays with Aurora kinase C (AURKC), the pose of tozasertib (VX-680), a commercially available pan-Aurora kinase inhibitor⁴⁰ displaying the lowest binding energy showed a binding score of −9.7 kcal mol⁻¹. The hybrid compounds 9d and 9g demonstrated similar binding scores (−8.4 and −10.4 kcal mol⁻¹, respectively) in the top-ranked poses at the AURKC binding pocket with the major interaction being a π–π stacking between the aromatic rings of the compounds and the Phe54 residue.

Regarding the analysis of specific intermolecular interactions, tozasertib interacts by π–π stacking with the phenyl ring of Phe54 (magenta dashed lines in Fig. 8) and π–π T-shaped interactions between the five-membered ring and the same residue. In addition, this compound also forms hydrogen bonds (H-bonds, green dashed lines in Fig. 8) with Glu121 and Ala123. Compounds 9d and 9g displayed π–π stacking interactions with Phe54 similar to tozasertib, with 9g also interacting with Leu173 through a π–σ interaction (purple dashed lines in Fig. 8), potentially increasing the ligand affinity within the binding pocket. Hybrid 9d displays H-bonds with Tyr122, and with Ser852 and Gly848 residues of the inner centromere protein (INCENP) chain. Given the hydrophobic nature of the binding pockets of Aurora kinases,⁴¹ hydrophobic interactions such as π–alkyl (light pink circles in Fig. 8) were the most prevalent.

Fig. 8 3D and 2D representations of the molecular interactions between Aurora kinase C (AURKC) and three different compounds. (A) Tozasertib, (B) 9g, and (C) 9d. The top panel shows the 3D visualization of the binding pocket of AURKC with the respective ligands, where key residues involved in interactions are labeled. The specific interactions, including conventional H-bonds (green dashed lines), pi–pi stacking interactions (pink dashed lines) and pi–sigma interactions (purple dashed lines) are highlighted between the ligands and residues within the binding site. The bottom panel displays the 2D interaction diagrams for each compound, depicting the array of interactions between the ligands and the key amino acids in the AURKC binding pocket. Hydrogen bonds, pi interactions, and van der Waals forces are represented with distinct colors as on the legend: van der Waals (light green), conventional hydrogen bonds (green), pi–sigma (purple), pi–pi stacking (pink), and pi–alkyl (light pink). Figure generated with PyMOL and BIOVIA Discovery Studio.

The Glu121, Tyr122, and Ala123 residues were notable for exhibiting a relevant number of H-bonds with these residues in the FTMap results (Fig. 9D). Most of these amino acids in the binding pocket are conserved between the structures of AURKC and AURKA.⁴² This conservation could explain why the hybrid compounds also induced dephosphorylation of AURKC in the western blot assays.⁴³ However, despite this binding site similarity, the proteins of the Aurora kinase family have distinct roles in the cell cycle and are associated with different types of tumors. Therefore, it is crucial to search for new molecules that can interact selectively with these proteins, unlike tozasertib, which acts as a nonspecific pan-Aurora kinase inhibitor.⁴⁴

Fig. 9 3D representations showing the interaction of AURKC's active site in complex with the compounds. (A) Tozasertib, (B) 9g, and (C) 9d. The key residues of the AURKC binding pocket are labeled and represented as white sticks; the yellow sticks are clusters of probes identified by the FTMap software, indicating potential binding hot spots. (D) The graph represents the percentage of hydrogen bonds formed by various residues in the AURKC binding site. The blue bars correspond to the frequency of hydrogen bond formation across different amino acids, with the red arrows highlighting the residues Glu121, Tyr122, and Ala123, suggesting a significant contribution to the binding interactions of these ligands. Figure generated with PyMOL and FTMap server. (E) Western blot analysis performed after 24 h of exposure to hybrid molecules 9d and 9g to detect inhibition of Aurora kinase isoforms (A, B, and C), the cell cycle regulator p21, and DNA damage by PARP cleavage status.

Also, AURKA is regulated by androgen in prostate cancer cells that highly express androgen receptors (AR).⁴⁵ These findings further underscore the role of AURK in the malignant transformation of prostate cancer cells. The DHP–DHMP hybrids can also inhibit the MKK family of kinases, RSK1 and mTOR in protein array experiments.

To better understand the compound poses and interactions identified in the molecular docking analysis, a probe-guided assay was performed with FTMap. The results indicated that clusters of probes (yellow molecules in Fig. 9A–C) were formed within the binding pocket region of AURKC, predominantly occupied by hydrophobic molecules such as ethane. These clusters also exhibited a prevalence of molecules with aromatic rings, like benzaldehyde, and hydrogen bond acceptors such as dimethyl ether, which might form hydrogen bonds with specific residues such as Glu121, Tyr122, and Ala123 (red arrows in Fig. 9D). Additionally, some of these probes are likely to form interactions with Phe54 and Leu173 through π–π stacking and π–σ interactions, respectively. Overall, the results suggest the relevance of these residues to establish interactions with the DHP–DHMP hybrids, reinforcing their importance for the design of new inhibitors for AURKC.

As observed in in silico analyses, the inhibition of Aurora kinase isoforms was evaluated by western blotting. The PC3 cell line was exposed to hybrid molecules 9d and 9g as well as to the potent Aurora kinase inhibitor tozasertib for 24 h. While the inhibitor tozasertib significantly reduced the phosphorylation of all isoforms (A, B and C) at a concentration of 100 μM, after 24 h of treatment with hybrid 9d a significant reduction in the AURKC isoform (35 kDa) was observed following exposure at the initial concentration of 10 μM, with complete reduction at the maximum concentration of 100 μM. This effect was not observed for 9g (Fig. 9E). Additionally, a significant increase in p21 (21 kDa) and the cleaved PARP isoforms (116 and 89 kDa) was detected after treatment with 9d. Analysis of target proteins (Aurora kinase) and cell cycle regulators indicated that 9d exhibited greater inhibitory efficiency compared to 9g and exclusive action on the AURKC isoform.

Certain 2,4-diaminopyrimidine derivatives exhibit selectivity for the Aurora kinase family, particularly Aurora kinase A, which was partially inhibited at concentrations of 50 nM in the HeLa cell line. However, these compounds were not evaluated for activity against the AURKC isoform.³⁷

To validate the docking protocol, as mentioned in section 4.2.4 (In silico approach), we performed a redocking of the co-crystallized Aurora kinase inhibitor VX-680 (tozasertib) in complex with AURKC (PDB ID 6GR9). The receptor and ligand were prepared as described in the section In silico approach: receptor processing in AutoDockTools with addition of polar hydrogens and Kollman charges; ligand preparation with Gasteiger charges and full torsional flexibility. The grid box was centered on the co-crystallized ligand and defined as 24 Å × 24 Å × 24 Å. Docking runs were executed with AutoDock Vina version 1.1.2, with exhaustiveness set to 20. Under these conditions, the highest scoring pose reproduced the crystallographic binding mode, yielding an RMSD of 0.357 Å relative to the VX-680 coordinates in the X-ray structure. This agreement supports the validation of the used docking protocol (see Fig. S88 in the SI).

Thus, a possible mechanism for the effect on Aurora kinase, as indicated by cell cycle analysis and protein profiling in PC3 cells treated with compounds 9d and 9g, was validated through Western blot analysis. Molecular docking studies further supported these results, showing that 9d and 9g had binding affinities similar to known inhibitors such as tozasertib.

2.7. Hybrids 9d and 9g induce cell death and decrease viability in a 3D tumor model

The 3D model is considered to provide information usually missing in monolayer conditions, such as quiescence, ECM-mediated signaling pathways and hypoxia.⁴⁶ Additionally, drug penetration as well as the gradient of nutrients and oxygen are also somewhat limited in spheroids, closely mimicking the restricted diffusion observed in solid tumors.

To validate the results obtained in the 2D cell model, tests were carried out using 3D models to obtain the cytotoxicity profile of the 9d and 9g molecules. The PC3 and androgen-sensitive human prostate adenocarcinoma (LNCAP) tumor cells were grown in 3D models and treated for 72 h with the two hybrid compounds above. Both 9d and 9g significantly reduced the perimeter of the spheroids when compared to the control in the PC3 cell line (Fig. 10A and B). The inhibitor tozasertib also showed a significant reduction in the length of the spheroids, which was more evident in the PC3 cells (Fig. 10A and B).

Fig. 10 Cytotoxicity of the hybrid molecules 9g and 9d in three-dimensional cell models. (A) Representative micrographs of tumor spheroids derived from PC3 and LNCaP cell lines treated with hybrid molecules 9g and 9d and tozasertib for 72 h, captured at 40× magnification. (B and C) Bar graphs depicting the perimeter measurements of 30 spheroids following 72 h of treatment with hybrid molecules 9g and 9d and tozasertib. (D and E) Bar graphs showing the cell viability quantified in 5 spheroids after 72 h of treatment. (F) Representative micrographs of spheroids after 72 h of treatment, stained with propidium iodide and imaged at 40× magnification. (G and H) Bar graphs illustrating the relative fluorescence intensity (red) measured in 5 spheroids, reflecting propidium iodide incorporation.

The spheroids of the LNCaP cell line showed a greater reduction in perimeter when treated with the 9g molecule compared to the other treatments (Fig. 10A and C). The spheroids from the PC3 cell line showed a significant reduction in viability when treated with the hybrid molecules and tozasertib (Fig. 10D). A similar result was detected in the spheroids of the LNCAP cell line, with a significant reduction in spheroid viability, which was even more pronounced when treated with the inhibitor tozasertib (Fig. 10F).

The intensity of fluorescence captured by propidium iodide (PI) impregnation corroborated these findings, indicating an increase in intensity in the treatments with the hybrid molecules when compared to the control in both PC3 and LNCAP cell lines (Fig. 10E–G). However, for the LNCAP tumor cell line, PI impregnation was significantly higher in the treatment with the inhibitor tozasertib compared to the treatments with the hybrid molecules (Fig. 10E and G). Tests to detect PI-stained foci revealed a significant increase following treatment with the hybrid molecule 9d in the PC3 cell line, which was identified as the most sensitive to the compounds.

3. Conclusion

This study demonstrated the potential of dihydropyridine–dihydropyrimidinone hybrid molecules, particularly compounds 9d and 9g, as promising anti-tumor agents for prostate cancer treatment. The easy synthesis of hybrid molecules was achieved through sequential multicomponent reactions and copper-catalyzed azide–alkyne cycloaddition (CuAAC), yielding satisfactory results for a chemically diverse structure. Cytotoxicity assays revealed that compound 9d exhibited the highest antitumor activity against the PC3 cell line with comparable potency to cisplatin. Furthermore, both hybrids demonstrated high selectivity for tumor cells, with significant selectivity indices (SI), particularly for 9g (SI >68.8), also observed through the selectivity and/or internalization of the fluorescent hybrid compounds 9d and 9g, assessed by high-resolution laser scanning confocal microscopy.

Hybrids 9d and 9g showed promising mechanisms of action, including cell cycle arrest at the G₀/G₁ phase, inhibition of cell migration and invasion, and modulation of key signaling pathways such as MAPK, AKT, and mTOR. Molecular docking studies revealed their potential to interact with Aurora kinase C, with binding affinities comparable to that of known inhibitors like tozasertib. Additionally, the hybrids induced significant cell death and reduced viability in 3D tumor models, further validating their efficacy in mimicking solid tumor conditions.

These findings highlight the role of 9d and 9g not only as potent antitumoral agents but also as potential fluorescent flashlights due to their intrinsic fluorescence and selective cellular uptake.

Finaly, the DHP–DHPM hybrids, especially 9d and 9g, represent a promising class of compounds for prostate cancer therapy, combining potent antitumor activity with high selectivity. Further studies are needed to explore the in vivo efficacy and potential clinical applications.

4. Experimental

4.1. Chemistry

4.1.1. General procedure for the preparation of propargyloxy–DHPs 4a–4e. A 50 mL round-bottom flask equipped with a reflux condenser was charged with oxy-propargylated aldehyde (1.0 mmol), 1,3-dicarbonyl compound (2.2 mmol), (NH₄)₂CO₃ (3 mmol), CeCl₃·7H₂O (20 mol%) and isopropanol (2.5 mL) in this order. The reaction mixture was refluxed for 24 h under magnetic stirring. After the consumption of starting materials, monitored by TLC, the solvent was evaporated under reduced pressure and the product was purified by chromatography on a silica gel column, using a gradient of the hexane/ethyl acetate mixture as an eluent.
4.1.1.1. Diethyl 2,6-dimethyl-4-(4-(prop-2-yn-1-yloxy)phenyl)-1,4-dihydropy ridine-3,5-dicarboxylate (4a)⁴⁷. Yield: 75%; pale yellow solid; M.P. 151–152 °C; ¹H NMR (300 MHz, CDCl₃): δ 7.21 (d, J = 8.8 Hz, 2H), 6.82 (d, J = 8.8 Hz, 2H), 5.67 (brs, 1H), 4.95 (s, 1H), 4.64 (d, J = 2.4 Hz, 2H), 4.17–4.02 (m, 4H), 2.50 (t, J = 2.4 Hz, 1H), 2.32 (s, 6H), 1.23 (t, J = 7.3 Hz, 6H); ¹³C NMR (75 MHz, CDCl₃): δ 167.6; 155.9; 143.6; 141.2; 129.0; 114.1; 104.3; 78.9; 75.2; 59.7; 55.8; 38.8; 19.5; 14.2. HRMS (ESI⁺) calcd for C₂₂H₂₅NNaO₅ [M + Na]⁺: 406.1625, found: 406.1633.
4.1.1.2. Diethyl 4-(3-methoxy-4-(prop-2-yn-1-yloxy)phenyl)-2,6-dimethyl-1,4- dihydropyridine-3,5-dicarboxylate (4b)⁴². Yield: 74%; pale beige solid; M.P. 136–137 °C; ¹H NMR (400 MHz, CDCl₃): δ 6.90 (d, J = 2.0 Hz, 1H), 6.88 (d, J = 8.3 Hz, 1H), 6.79 (dd, J = 8.3 and 2.0 Hz, 1H), 5.67 (brs, 1H), 4.96 (s, 1H), 4.70 (d, J = 2.3 Hz, 2H), 4.15–4.07 (m, 4H), 3.83 (s, 3H), 2.48 (t, J = 2.4 Hz, 1H), 2.34 (s, 6H), 1.24 (t, J = 7.0 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃): δ 167.7; 148.7; 145.1; 143.8; 142.1; 119.7; 113.8; 112.2; 104.0; 78.9; 75.5; 59.7; 56.7; 55.7; 39.1; 19.5; 14.3. HRMS (ESI⁺) calcd for C₂₃H₂₇NNaO₆ [M + Na]⁺: 436.1731, found: 436.1734.
4.1.1.3. Diethyl 2,6-dimethyl-4-(3-(prop-2-yn-1-yloxy)phenyl)-1,4-dihydropy- ridine-3,5-dicarboxylate (4c). Yield: 80%; pale yellow solid; M.P. 139–141 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.14 (t, J = 7.9 Hz, 1H), 6.95 (dt, J = 7.6 Hz e 1.2 Hz, 1H), 6.92–6.91 (m,1H), 6.75 (ddd, J = 8.2 Hz, 2.6 H, 1.0 Hz, 1H), 5.73 (brs, 1H), 5.00 (s, 1H), 4.64 (d, J = 2.5 Hz, 2H), 4.14–4.06 (m, 4H), 2.51 (t, J = 2.4 Hz, 1 H), 2.33 (s, 6H), 1.23 (t, J = 2.1 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃): δ 167.5; 157.4; 149.4; 144.0; 128.6; 121.5; 115.0; 111.8; 103.9; 78.8; 75.2; 59.7; 55.7; 39.5; 19.6; 14.3. HRMS (ESI⁺) calcd for C₂₂H₂₅NNaO₅ [M + Na]⁺: 406.1625, found: 406.1621.
4.1.1.4. 3,3,6,6-Tetramethyl-9-(4-(prop-2-yn-1-yloxy)phenyl)-3,4,6,7,9,10-hexahydroacridine-1,8 (2H,5H)-dione (4d). Yield: 79%; pale beige solid; M.P. 213–214 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.66 (brs, 1H); 7.28–7.25 (m, 2H); 6.81–6.79 (m, 2H); 5.05 (s, 1H); 4.56 (d, J = 2.5 Hz, 2H); 2.46 (t, J = 2.4 Hz, 1H); 2.32–2.13 (m, 8H); 1.07 (s, 6H); 0.96 (s, 6H); ¹³C NMR (100 MHz, CDCl₃): δ 195.9; 155.9; 148.7; 140.0; 128.9; 114.2; 113.4; 78.8; 75.3; 55.8; 50.9; 40.8; 32.6; 29.5; 27.2. HRMS (ESI⁺) calcd for C₂₆H₂₉NNaO₃ [M + Na]⁺: 426.2040, found: 426.2037.
4.1.1.5. 3,3,6,6-Tetramethyl-9-(3-(prop-2-yn-1-yloxy)phenyl)-3,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-dione (4e). Yield: 75%; pale yellow solid; M.P. >199 °C, dec.; ¹H NMR (400 MHz, CDCl₃): δ 7.21 (brs, 1H), 7.12 (t, J = 7.8 Hz, 1H), 7.01 (d, J = 7.8 Hz, 1H), 6.94–6.92 (m, 1H), 6.70 (ddd, J = 8.1, 2.6, 0.9 Hz, 1H), 5.09 (s, 1H), 4.61 (d, J = 2.5 Hz, 2H), 2.49 (t, J = 2.4 Hz, 1H), 2.35–2.15 (m, 8H), 1.08 (s, 6H), 0.98 (s, 6H); ¹³C NMR (100 MHz, CDCl₃): δ 196.0; 157.6; 150.1; 148.0; 129.0; 121.5; 115.0; 112.9; 111.9; 78.8; 75.5; 55.9; 50.6; 40.6; 32.6; 29.4; 27.2. HRMS (ESI⁺) calcd for C₂₆H₂₉NNaO₃ [M + Na]⁺: 426.2040, found: 426.2042.

4.1.2. General procedure for the preparation of 6-chloro–DHPMs 7a and 7b. In a 100 mL round-bottom flask equipped with a reflux condenser, the aryl aldehyde (5 mmol), ethyl 4-chloroacetoacetate (7.5 mmol), urea (7.5 mmol) and glacial acetic acid (12.5 mL) were added in this order. The reaction mixture was heated at 50 °C for 48 h under magnetic stirring. The reaction mixture was cooled in an ice bath and slowly poured into distilled water (100 mL), promoting the precipitation of a white solid. The solid was filtered in a Büchner funnel under reduced pressure, washed with water, saturated NaHCO₃ solution and water. Finally, the resulting solid was dried under vacuum.
4.1.2.1. Ethyl 6-(chloromethyl)-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (7a). Yield: 82%; pale beige solid; M.P. 198–200 °C; ¹H-NMR (400 MHz, DMSO-d₆): δ 9.50 (brs,1H), 7.86 (brs, 1H), 7.36–7.32 (m, 2H), 7.28–7.25 (m, 3H), 5.19 (d, J = 3.3 Hz, 1H), 4.78 (d, J = 10.6 Hz, 1H), 4.60 (d, J = 10.6 Hz, 1H), 4.04 (q, J = 7.1 Hz, 2H), 1.11 (t, J = 7.1 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 164.2; 152.1; 146.0; 144.0; 128.5; 127.6; 126.3; 101.8; 60.0; 53.9; 39.2; 13.9. IR-ATR (ν_max, cm⁻¹): 3344, 3080, 2929, 1687, 1646, 1514, 1459, 1434, 1224, 1135, 1020, 803, 611. HRMS (ESI⁺) calcd for C₁₄H₁₆ClN₂O₃ [M + H]⁺: 295.0844; found: 295.0840.
4.1.2.2. Ethyl 6-(chloromethyl)-2-oxo-4-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (7b). Yield: 80%; dark-yellow solid; M.P. 243–244 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 9.50 (d, J = 1.6 Hz, 1H), 7.82 (m, 1 H), 6.55 (s, 2H), 5.14 (d, J = 3.3 Hz, 1H), 4.80 (d, J = 10.6 Hz, 1H), 4.63 (d, J = 10.6 Hz, 1H), 4.07 (q, J = 7.1 Hz, 2H), 4.72 (s, 6H), 3.63 (s, 3H), 1.13 (t, J = 7.1 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 164.7, 153.3, 152.5, 146.8, 139.9, 137.3, 103.9, 101.8, 60.5, 60.4, 56.2, 54.2, 39.8, 14.4. IR-ATR (ν_max, cm⁻¹): 3315, 3106, 2957, 1687, 1643, 1594, 1454, 1305, 1223, 1132, 1098, 997, 781, 680, 639. HRMS (ESI⁺) calcd for C₁₇H₂₁ClN₂O₆ [M+ Na]⁺: 407.0980; found: 407.0986.

4.1.3. General procedure for the preparation of 6-azido–DHPMs 8a and 8b. In a 100 mL round-bottom flask equipped with a reflux condenser, chloro-dihydropyrimidinone (3 mmol), NaN₃ (3.6 mmol), acetone (7.5 mL) and distilled water (3 mL) were added in this order. The reaction mixture was maintained at 60 °C for approximately 4 h under magnetic stirring. The reaction was monitored by TLC until the consumption of starting materials. After the reaction completion, the solvent was evaporated under reduced pressure, and the solid formed was filtered in a Büchner funnel. Finally, the resulting solid product was dried under vacuum.
4.1.3.1. Ethyl 6-(azidomethyl)-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (8a). Yield: 88%; yellow solid; M.P. 110–112 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 9.47 (brs, 1H), 7.88 (brs, 1H), 7.36–7.32 (m, 2H), 7.28–7.24 (m, 3H), 5.21 (d, J = 3.3 Hz, 1H), 4.47 (d, J = 12.8 Hz, 1H), 4.35 (d, J = 12.9 Hz, 1H), 4.03 (q, J = 7.1 Hz, 2H), 1.11 (t, J = 7.1 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 164.6, 151.9, 144.4, 144.1, 128.5, 127.6, 126.3, 102.3, 60.0, 54.1, 47.9, 13.9. IR-ATR (ν_max, cm⁻¹): 3361, 3116, 2975, 2103, 1690, 1649, 1444, 1224, 1094, 696. HRMS (ESI⁺) calcd for C₁₄H₁₆N₅O₃ [M + H]⁺ 302.1248, found: 302.1244.
4.1.3.2. Ethyl 6-(azidomethyl)-2-oxo-4-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (8b). Yield: 91%; white solid; M.P. 175–177 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 9.46 (d, J = 1.4 Hz, 1H), 7.84 (m, 1H), 6.57 (s, 2H), 5.19 (d, J = 3.2 Hz, 1H), 4.49 (d, J = 12.8 Hz, 1H), 4.31 (d, J = 12.8 Hz, 1H), 4.12–4.01 (m, 2H), 3.73 (s, 6H), 3.63 (s, 3H), 1.13 (t, J = 7.1 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 164.7, 152.8 (2×), 151.9, 144.5, 139.6, 136.9, 103.5, 102.0, 60.0, 55.8, 54.1, 48.0, 14.0. IR-ATR (ν_max, cm⁻¹): 3320, 3203, 3104, 2950, 2094, 1680, 1632, 1593, 1454, 1312, 1218, 1128, 1094, 989, 775. HRMS (ESI⁺) calcd for C₁₇H₂₂N₅O₆ [M + H]⁺: 392.1565, found: 392.1558.

4.1.4. General procedure for the synthesis of dihydropyridine–dihydropyrimidinone hybrids 9a–9i. In a 25 ml round-bottom flask were added (in this order) the DHPM–azide compound (0.2 mmol), the propargyloxy–DHP compound (0.2 mmol), dichloromethane (2 mL), water (2 mL), CuSO₄·5H₂O (10 mol%) and sodium ascorbate (20 mol%). The reaction mixture was stirred at room temperature until the complete consumption of reagents, monitored by TLC. After the reaction was complete, a 0.1 M solution of EDTA (4 mL) was added to the crude mixture under stirring and the biphasic solution was extracted with CH₂Cl₂ (3 × 2 mL). The organic phases were combined and washed with saturated NaCl solution. After separation, the organic phase was dried over anhydrous MgSO₄, simply filtered and the solvent removed under reduced pressure. The crude product was purified by chromatography on a silica gel column using a gradient of a hexane/ethyl acetate mixture as an eluent.
4.1.4.1. Diethyl 4-(4-((1-((5-(ethoxycarbonyl)-2-oxo-6-phenyl-1,2,3,6-tetrahydropyrimidin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (9a + diastereoisomer). Yield: 70%; yellow solid; M.P. 122–123 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.25 (brs, 1H), 7.88 (s, 1H), 7.30–7.23 (m, 5H), 7.20 (d, J = 8.8 Hz, 2H), 6.81 (d, J = 8.8 Hz, 2H), 6.02 (brs,1H), 5.90 (brs, 1H), 5.85 (d, J = 14.7 Hz, 1H), 5.63 (d, J =14.7 Hz, 1H), 5.41 (d, J = 2.5 Hz, 1H), 5.13 (s, 2H), 4.93 (s, 1H), 4.14–4.04 (m, 6H), 2.32 (s, 3H), 2.31 (s, 3H), 1.22 (t, J = 7.3 Hz, 6H), 1.16 (t, J = 7.1 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 164.8; 159.7; 152.8; 152.0; 143.6; 142.6; 141.2; 129.3; 129.0; 128.9; 128.9; 128.4; 126.6; 126.6; 124.2; 115.0; 114.0; 104.4; 104.3; 104.1; 62.7; 61.9; 61.0; 59.7; 55.7; 47.9; 38.7; 20.6; 19.5; 18.6; 14.3; 14.0; 13.7. HRMS (ESI⁺) calcd for C₃₆H₄₀N₆O₈ [M + Na]⁺: 7072908; found: 707.2800.
4.1.4.2. Diethyl 4-(4-((1-((5-(ethoxycarbonyl)-2-oxo-6-phenyl-1,2,3,6-tetrahydropyrimidin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)-3-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate) (9b + diatereisomer). Yield: 78%; pale yellow solid; M.P. 122–124 °C: ¹H NMR (400 MHz, CDCl₃): δ 8.13 (brs, 1H), 7.88 (s, 1H), 7.28–7.21 (m, 5H), 6.90 (d, J = 2.0 Hz, 1H), 6.86 (d, J = 8.3 Hz, 1H), 8.76 (dd, J = 8.3 e 2.0 Hz, 1H), 5.93 (brs, 1H), 5.89 (brs, 1H), 5.84 (d, J = 14.9 Hz, 1H), 5.59 (d, J = 14.9 Hz, 1H), 5.39 (d, J = 2.8 Hz, 1H), 5.19 (s, 2H), 4.95 (s, 1H), 4.16–4.04 (m, 6H), 3.78 (s, 3H), 2.32 (s, 3H), 2.31 (s, 3H) 1.24 (t, J = 7.2 Hz, 6H), 1.15 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 167.8; 167.7; 164.7; 152.2; 148.6; 145.9; 144.8; 144.0; 143.9; 142.7; 141.8; 141.3; 128.8; 128.2; 126.6; 124.2; 119.8; 113.8; 112.2; 104.2; 103.9; 63.1; 60.8; 59.7; 55.7; 47.7; 38.9; 19.4; 14.3; 13.9. HRMS (ESI⁺) calcd for C₃₇H₄₂N₆O₉ [M + Na]⁺: 737.3013; found: 737.2905.
4.1.4.3. Diethyl 4-(4-((1-((5-(ethoxycarbonyl)-2-oxo-6-(3,4,5-trimethoxyphenyl)-1,2,3,6-tetrahydropyrimidin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (9c + diastereoisomer). Yield: 74%; pale yellow solid; M.P. 125–127 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.05 (brs, 1H), 7.88 (s, 1H), 7.20 (d, J = 8.8 Hz, 2H), 6.81 (d, J = 8.6 Hz, 2H), 6.43 (s, 2H), 5.92 (d, J = 14.6 Hz, 1H), 5.90 (brs, 1H), 5.77 (brs, 1H), 5.56 (d, J = 14.6 Hz, 1H), 5.37 (d, J = 2.3 Hz, 1H), 5.11 (s, 2H), 4.94 (s, 1H), 4.16–4.05 (m, 6H), 3.81 (s, 3H), 3.75 (s, 6H), 2.33 (s, 3H), 2.32 (s, 3H), 1.23 (t, J = 7.0 Hz, 6H), 1.18 (t, J = 7.0 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 167.7; 164.7; 156.5; 153.5; 152.1; 143.8; 141.5; 141.0; 138.3; 137.8; 129.0; 123.9; 113.9; 104.1; 103.4; 61.8; 61.0; 60.8; 59.7; 56.1; 55.9; 48.05; 38.7; 19.5; 14.3; 14.1; 13.7. HRMS (ESI⁺) calcd for C₃₉H₄₆N₆O₁₁ [M + Na]⁺: 797.3225; found: 797.3117.
4.1.4.4. Diethyl 4-(3-((1-((5-(ethoxycarbonyl)-2-oxo-6-(3,4,5-trimethoxyphenyl)-1,2,3,6-tetrahydropyrimidin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (9d + diastereoisomer). Yield: 73%; pale yellow solid; M.P. 119–121 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.32 (s, 1H), 7.94 (s, 1H), 7.13 (t, J = 7.8 Hz, 1H), 6.97 (d, J = 7.8 Hz, 1H), 6.86 (brs, 1H), 6.74 (dd, J = 8.0 e 2.2 Hz, 1H), 6.52 (brs, 1H), 6.40 (s, 2H), 6.08 (d, J = 14.3 Hz, 1H), 5.99 (s, 1H), 5.43 (d, J = 14.3 Hz, 1H), 5.37 (d, J = 2.5 Hz, 1H), 5.12 (d, J = 11.8 Hz, 1H), 5.08 (d, J = 11.8 Hz, 1H), 4.96 (s, 1H), 4.17–4.05 (m, 6H), 3.82 (s, 3H), 3.73 (m, 6H), 2.34 (s, 3H), 2.25 (s, 3H), 1.25–1.17 (m, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 167.7; 164.7; 158.3; 153.5; 153.4; 150.0; 144.6; 144.5; 138.6; 137.5; 128.9; 128.5; 126.6; 123.9; 121.5; 114.7; 111.2; 104.2; 103.7; 103.6; 103.5; 103.3; 61.0; 60.8; 59.7; 59.6; 56.2; 56.1; 39.4; 19.2; 19.1; 14.3; 14.1; 13.6. HRMS (ESI⁺) calcd for C₃₉H₄₆N₆O₁₁ [M + Na]⁺: 797.3225; found: 797.3117.
4.1.4.5. Diethyl 4-(4-((1-((5-(ethoxycarbonyl)-2-oxo-6-(3,4,5-trimethoxyphenyl)-1,2,3,6-tetrahydropyrimidin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)-3-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (9e). Yield: 51%; yellow solid; M.P. 115–117 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.25 (brs, 1H), 7.93 (s, 1H), 6.90 (d, J = 1.8 Hz, 1H), 6.85 (d, J = 8.3 Hz, 1H), 6.74 (dd, J = 8.1 e 1.8 Hz, 1H), 6.43 (s, 2H), 5.95 (brs, 1H), 5.90 (brs, 1H), 5.85 (d, J = 14.6 Hz, 1H), 5.60 (d, J = 14.6 Hz, 1H), 5.34 (d, J = 1.5 Hz, 1H), 5.17 (s, 2H), 4.95 (s, 1H), 4.17–4.05 (m, 6H), 3.81 (s, 3H), 3.79 (s, 3H), 3.74 (s, 6H), 2.33 (s, 3H), 2.31 (s, 3H), 1.24 (t, J = 7.0 Hz, 6H), 1.17 (t, J = 7.0 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 167.7; 164.7; 153.5; 152.0; 148.6; 145.9; 144.8; 144.0; 143.9; 141.9; 141.4; 138.4; 137.7; 124.4; 119.8; 113.8; 112.1; 104.1; 103.9; 103.4; 63.1; 60.8; 59.7; 56.1; 55.7; 47.9; 38.9; 19.5; 14.3; 14.1; 13.7. HRMS (ESI⁺) calcd for C₄₀H₄₈N₆O₁₂ [M + Na]⁺: 8273330; found: 827.3222.
4.1.4.6. Ethyl 2-oxo-4-phenyl-6-((4-((4-(3,3,6,6-tetramethyl-1,8-dioxo-1,2,3,4, 5,6,7,8,9,10-decahydroacridin-9-yl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (9f). Yield: 64%; beige solid; M.P. 178–180 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 9.59 (brs, 1H), 9.26 (brs, 1H), 8.15 (s, 1H), 7.89 (brs, 1H), 7.34–7.26 (m, 5H), 7.06 (d, J = 8.8 Hz, 2H), 6.82 (d, J = 8.8 Hz, 2H), 5.67 (d, J = 13.7 Hz, 1H), 5.45 (d, J = 13.7 Hz, 1H), 5.20 (d, J = 3.4 Hz, 1H), 5.04 (s, 2H), 4.76 (s, 1H), 3.99 (q, J = 7.1 Hz, 2H), 2.44 (d, J = 17.0 Hz, 2H), 2.32 (d, J = 17.0 Hz, 2H), 2.16 (d, J = 16,1 Hz, 2H), 1.98 (d, J = 16.1 Hz, 2H), 1.04 (t, J = 7.1 Hz, 3H), 1.00 (s, 6H), 0.87 (s, 6H); ¹³C NMR (100 MHz, DMSO-d₆): δ 194.4; 164.5; 156.0; 151.7; 149.1; 143.9; 143.1; 142.6; 139.9; 128.5; 127.6; 126.4; 124.9; 120.4; 113.6; 111.6; 102.9; 60.8; 60.1; 54.1; 50.3; 47.9; 32.2; 31.9; 29.1; 26.5; 13.8. HRMS (ESI⁺) calcd for C₄₀H₄₄N₆O₆ [M + Na]⁺: 7273322; found: 727.3215.
4.1.4.7. Ethyl 2-oxo-4-phenyl-6-((4-((3-(3,3,6,6-tetramethyl-1,8-dioxo-1,2,3,4, 5,6,7,8,9,10-decahydroacridin-9-yl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (9g). Yield: 85%; yellow solid; M.P. 169–171 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 9.60 (d, J = 1.7 Hz, 1H), 9.31 (s, 1H), 8.19 (s, 1H), 7.90 (brs, 1H), 7.36–7.26 (m,5H), 7.11–7.07 (m,1H), 6.78–6.76 (m, 3H), 5.69 (d, J = 13.9 Hz, 1H), 5.47 (d, J = 13.9 Hz, 1H), 5.23 (d, J = 3.2 Hz, 1H), 5.04 (s, 2H), 4.80 (s, 1H), 4.01 (q, J = 7.1 Hz, 2H), 2.44 (d, J = 17.1 Hz, 2H), 2.35 (d, J = 17.1 Hz, 2H), 2.17 (d, J = 16.1 Hz, 2H), 2.01 (d, J = 16.0 Hz, 2H), 1.06 (t, J = 7.1 Hz, 3H), 1.01 (s, 6H), 0.88 (s, 6H); ¹³C NMR (100 MHz, DMSO-d₆): δ 194.5; 164.5; 157.6; 151.8; 149.5; 148.7; 143.9; 143.1; 142.5; 128.6; 128.5; 127.7; 126.5; 125.0; 120.4; 114.7; 111.3; 111.0; 103.0; 60.8; 60.1; 54.2; 50.3; 48.0; 32.8; 32.2; 29.0; 26.6; 13.9. HRMS (ESI⁺) calcd for C₄₀H₄₄N₆O₆ [M + Na]⁺: 7273322; found: 727.3215.
4.1.4.8. Ethyl 2-oxo-6-((4-((4-(3,3,6,6-tetramethyl-1,8-dioxo-1,2,3,4,5,6,7,8, 9,10-decahydroacridin-9-yl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-4-(3,4,5-trimethoxy-phenyl)1,2,3,4-tetrahydropyrimidine-5-carboxylate (9h). Yield: 53%; yellow solid; M.P. 175-177 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 9.60 (brs, 1H), 9.27 (brs, 1H), 8.21 (s, 1H), 7.84 (brs, 1H), 7.06 (d, J = 8.3 Hz, 2H), 6.82 (d, J = 8.8 Hz, 2H), 6.55 (s, 2H), 5.82 (d, J = 14.2 Hz, 1H), 5.33 (d, J = 14.2 Hz, 1H), 5.17 (d, J = 3.4 Hz, 1H), 5.04 (s, 2H), 4.75 (s, 1H), 4.00 (q, J = 7.3 Hz, 2H), 3.70 (s, 6H), 3.61 (s, 3H), 2.44 (d, J = 17.1 Hz, 2H), 2.31 (d, J = 16.6 Hz, 2H), 2.16 (d, J = 16.1 Hz, 2H), 1.98 (d, J = 16.1 Hz, 2H), 1.05 (t, J = 7.1 Hz, 3H), 1.00 (s, 6H), 0.86 (s, 6H); ¹³C NMR (100 MHz, DMSO-d₆): δ 194.5; 164.5; 156.0; 152.9; 151.7; 149.1; 143.5; 142.6; 139.9; 139.5; 136.8; 128.6; 125.2; 113.6; 111.7; 103.5; 102.2; 60.8; 60.0; 59.9; 55.8; 54.2; 50.3; 48.2; 32.2; 31.9; 29.1; 26.6; 13.9. HRMS (ESI⁺) calcd for C₄₃H₅₀N₆O₉ [M + Na]⁺: 817.3639; found: 817.3531.
4.1.4.9. Ethyl 2-oxo-6-((4-((3-(3,3,6,6-tetramethyl-1,8-dioxo-1,2,3,4,5,6,7,8,9,10-decahydroacridin-9-yl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-4-(3,4,5-trimethoxy-phenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (9i). Yield: 79%; yellow solid; M.P. 163–164 °C; ¹H NMR (400 MHz, CDCl₃): δ 9.16 (brs, 1H), 8.05 (brs, 1H), 7.95 (s, 1H), 7.09 (t, J = 7.8 Hz, 1H), 6.96 (brs, 1H), 6.91 (d, J = 7,5 Hz, 1H), 6.66 (dd, J = 8.1 e 1.0 Hz, 1H), 6.48 (s, 2H), 6.42 (s, 1H), 6.01 (d, J =14.1 Hz, 1H), 5.43 (d, J = 14.1 Hz, 1H), 5.38 (d, J = 2.3 Hz, 1H), 5.07 (s, 2H), 5.01 (s, 1H), 4.16–4.08 (m, 2H), 3.81 (s, 3H), 3.75 (s, 6H), 2.30–2.09 (m, 8H), 1.17 (t, J = 7.0 Hz, 3H), 1.02 (s, 3H), 0.99 (s, 3H), 0.91 (s, 3H), 0.85 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 196.1; 164.7; 157.7; 153.5; 152.6; 149.6; 148.2; 144.0; 141.7; 138.7; 137.4; 129; 1; 120.1; 115.1; 113.0; 112.4; 104.2; 103.4; 100.0; 61.4; 60.9; 60.7; 56.2; 50.6; 48.0; 40.6; 33.6; 32.5; 29.4; 26.9; 14.1. HRMS (ESI⁺) calcd for C₄₃H₅₀N₆O₉ [M + Na]⁺: 8173639; found: 817.3531.

4.2. Biology

4.2.1. Cell lines and reagents. Prostate cancer cell lines PC3, LNCAP and non-tumorigenic cell line PNT2 used in this study were obtained from the Cell Bank of the Barretos Cancer Hospital. Breast cancer cells MCF-7 were purchased from Rio de Janeiro Cell Bank. Cell lines were maintained in RPMI 1640 complete medium containing 10% fetal bovine serum (FBS), 2 mM glutamine, and 1% penicillin/streptomycin. Cell culture reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell lines were incubated in a humidified atmosphere of 5% CO₂ at 37 °C. To avoid misidentification and/or cross-contamination, cell lines were authenticated by STR analysis.⁴⁸ Cultures pellets were regularly tested for mycoplasma contamination using a MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, Walkersville, MD, USA).

4.2.2. Pharmacological agents. Cisplatin (Cat. No. S8830), tozasertib anti-pan-Aurora kinase (Cat. No. S1048), and inhibitors were purchased from Selleck Chemicals (Houston, TX, USA). All drugs were diluted in DMSO at 10 mM and stored at −20 °C for future use. In all experiments, DMSO was used as a control vehicle at a final concentration of 1% (v/v).

4.2.3. Cellular viability assay. Cell viability was evaluated 48 h post-drug treatment using the colorimetric CCK-8 assay following the manufacturer's protocol. This assay is based on the amount of WST-8 (colorless compound) converted to the water-soluble WST-8 formazan (orange compound) due to the action of the dehydrogenases in viable cells. For assessing the compounds' cytotoxicity, 7.5 × 10³ cells per well were plated in 96-well plates with complete RPMI 1640 medium and allowed to adhere overnight at 37 °C in a humidified atmosphere with 5% CO₂. Cells were then exposed to increasing concentrations of hybrid compounds (0.1–1000 μM), and absorbance (450 nm) was measured using a multiwell plate reader (FlashScan 530 Analytik Jena). The results were normalized to DMSO control values. The cytotoxicity of cisplatin (0.8–833 μM), used as a positive control, was evaluated in the same manner. IC₅₀ values, expressed as the mean ± SD of at least two independent experiments carried out in triplicate, were determined using GraphPad Prism software (version 9.0) by fitting a nonlinear regression curve.

4.2.4. In silico approach. After the insights on in vitro assays, a computer-aided approach was used to perform in silico protein–ligand interaction analysis to investigate the binding mode and the potential of the compounds to interact with the target. First, the atomic coordinates of Aurora kinase C (AURKC) in complex with inner centromere protein (INCENP) were obtained from the Protein Data Bank (PDB ID 6GR9, resolution 2.25 Å) (Berman, 2000). This PDB structure was chosen based on its resolution and the presence of the co-crystallized ligand tozasertib (VX-680). The AURKC structure was submitted to FTMap, a computational server for mapping and identification of binding hot spots in macromolecules based on scans of 16 small organic molecule probes over the macromolecule surface.⁴⁹

Docking simulations were performed using AutoDock Vina 1.2.0 with Vina's default scoring function (a non-directional hydrogen bond and a hydrophobic term; a conformational entropy penalty; and steric terms: two attractive Gaussians and repulsion term), as originally described by Trott and Olson⁵⁰ and in the Vina 1.2.0 paper⁵¹ (Eberhardt et al., 2021). In our molecular docking protocol, the receptor (Aurora kinase) was treated as rigid, while the ligands were considered flexible, allowing for free rotation of a chosen set of covalent bonds. The search exhaustiveness parameter was adjusted to ensure coverage of the accessible conformational space⁵² and the search space was defined based on the coordinates of the crystallographic ligand.

AutoDock Tools v1.5.7 was used to prepare receptor and ligand structures in PDBQT file format. Protein structure preparation steps involved deleting water molecules, repairing missing atoms, and adding polar hydrogens and Kollman charges. The compounds were prepared by setting the rotatable bonds and adding hydrogens and Gasteiger charges. For molecular docking, the grid was centralized on the geometric center of the co-crystallized ligand tozasertib with a size of 24 Å in each direction.⁵³ AutoDock Vina v1.1.2 software was used to accomplish the redocking of tozasertib, which had a calculated RMSD of 0.357 Å between the top-ranked pose and the co-crystallized ligand of 6GR9 PDB complex. Docking assays of 9g and 9d were also performed, both with an exhaustive parameter of 20.⁵⁰ Finally, the molecular docking results were evaluated in BIOVIA Discovery Studio Visualizer v24.1.0 and PyMOL software v3.0.4.⁵⁴

4.2.5. Confocal analysis. For immunofluorescence, PC3 and PNT2 cells were seeded at a density of 2.5 × 10⁵ cells per glass cover slide inside the wells of 6-well plates containing 3 mL of complete RPMI 1640 medium and allowed to adhere overnight at 37 °C in a humidified atmosphere with 5% CO₂. Cells were then treated with vehicle [0.5% (v/v) DMSO], hybrid 9d (3.2 μM), and hybrid 9g (10.9 μM), where the concentrations correspond to 50% of the IC₅₀ values of the samples. After treatment for 8 h and 24 h, the cells were gently washed with 3× PBS, fixed with 3.7% paraformaldehyde in PBS for 20 min at room temperature, washed again with 1× PBS, and permeabilized with Triton X-100 (0.2% in PBS) for 5 min. Following washing with PBS, cells were blocked in 10% fetal bovine serum albumin (FBS) and diluted in PBS for 10 min at room temperature. The cells were then incubated with DRAQ5 diluted in PBS (1 : 1000) to stain the nuclei and with Alexa Fluor 555 Phalloidin diluted in PBS (1 : 300) for labelling the F-actin following instructions provided by the manufacturer. Finally, the slides containing the cells were rinsed again with 3× PBS and mounted with one drop of the antifading solution ProLong Gold (Invitrogen). High-resolution confocal laser scanning microscopy (CLSM) images were captured using an Upright LSM780-NLO Zeiss microscope (Carl Zeiss AG, Germany) using an EC Plan-Neofluar 40×/1.3 oil objective. Images for hybrid compounds 9d and 9g were collected using a 405 nm laser line for excitation and a 420–445 nm emission filter. Alexa Fluor 555 Phalloidin was excited at 555 nm and detected at 568–639 nm, whereas a 633 nm and 661–759 nm HeNe laser were used to excite and detect DRAQ5, respectively. Before imaging analysis, raw.czi files were automatically processed using Zen Black 2.3 software. All images were collected in the same day using identical parameters, and they were analyzed with ImageJ software for the evaluation of red fluorescence.

4.2.6. Cellular migration, invasion and adhesion assays. Cell migration was assessed using inserts without Matrigel, and invasion was detected using the Invasion Chamber Kit (BD Biosciences, USA) according to the manufacturer's instructions. A total of 1.0 × 10⁶ cells were seeded into 24-well Transwell inserts in serum-free RPMI. RPMI with 10% FBS was used as a chemoattractant. After 48 h, the insert membranes were fixed with iced methanol and stained with hematoxylin/eosin. Photomicrographs of the membranes were taken using a microscope at 40× magnification, and cell counts were performed using ImageJ software. The results were expressed as the mean percentage relative to DMSO control (considered 100% invasion). For both assays, the cell lines were fixed with 10% trichloroacetic acid (TCA), stained with 0.5% crystal violet solution, and dissolved in 10% acetic acid solution for absorbance detection at 590 nm using a Varioskan microplate reader (Thermo Scientific, Finland). The absorbance values were plotted using GraphPad Prism software (Version 9.0). All experiments were conducted in triplicate and repeated at least three times.

4.2.7. Cell cycle and death analysis. To assess cell death by apoptosis, 3 × 10⁵ cells of the PC3 cell line were seeded in 6-well plates and treated for 48 h with hybrid compounds selected in the previous steps at concentrations equivalent to their IC₅₀ values. Cells were then stained with annexin V (apoptosis) and propidium iodide (PI – cell viability) using the annexin V-FITC Apoptosis Detection Kit I (BD Biosciences) following the manufacturer's instructions. To evaluate interference with the cell cycle, the same selected compounds were applied to the cell lines at their respective IC₅₀ concentrations. Cells were then incubated with PI using the Cycle Test Plus kit (BD Biosciences) according to the manufacturer's instructions. Data acquisition was performed on a BD Accuri C6 flow cytometer (BD Biosciences) and analyzed using the BD Accuri C6 software (BD Biosciences).

4.2.8. Western blot and reverse phase protein arrays (RPPAs). Protein lysates of the PC3 cell line were used to perform western blot analysis. In summary, cells were rinsed in DPBS and then lysed in lysis buffer following our group protocol.⁵⁵ Cells were incubated overnight with primary antibodies: pAKT-Ser473 (#4060), AKT (pan) (#4691), P21 (#3063), pan-pAUK (mAb #2914) and β-actin (#3700). The primary antibodies were purchased from Cell Signaling and all were diluted in 5% BSA solution at 1 : 1000. Membranes were incubated with anti-rabbit (#7074) or anti-mouse (#7076) secondary antibody at 1 : 5000 dilution. Chemiluminescent signals were detected by ECL in an automatic ImageQuant mini LAS4000 system (GE Healthcare). Protein array Human Phospho-Mitogen-activated (ARY002B; R&D Systems, MN) was conducted according to the manufacturers' instructions. A total of 1000 μg mL⁻¹ protein lysate was incubated in the array membrane, and immune dot signal was detected by ECL in the automatic ImageQuant mini LAS4000 system (GE Healthcare). Densitometric analysis of western blotting and RPPA were carried in ImageJ software. All the experiments were performed in triplicate.

4.2.9. Viability of spheroids (3D model). For the construction of the spheroid model, PC3 and LNCaP cell lines were cultured under ideal conditions in culture flasks and incubated at 37 °C, 5% CO₂, and 90% humidity until they reached confluence. A total of 5.0 × 10⁶ cells were seeded in 12-well plates previously covered with agarose + DMEM solution (1 : 1 v/v). After the agarose solidifies, a 3D stamp-like device is used to generate spheroids following the manufacturer's instructions,⁵⁶ incubated for 72 h to cohesive spheroid formation, and the baseline photomicrography was recorded. Spheroids were treated with 5 times IC₅₀ values calculated previously in a 2D model for the hybrid compound and tozasertib for 72 h and finally photographed. The spheroid diameter (n = 50) from each group was obtained using ImageJ software and the experiments were conducted in biological and experimental triplicates. Viability of spheroids was measured using CellTiter Glo 3D assay (G9681) according to the manufacturer's instructions. The luminescence signal of spheroids was obtained at 590 nm using a Varioskan microplate reader (Thermo Scientific, Finland). Additionally, after treatment the spheroids were incubated with propidium iodide (PI) solution (1 mg mL⁻¹) for 15 min and then photographed with a fluorescence microscope using a Texas Red filter (559-34 Ex, 630-69 Em). Fluorescence intensity was calculated using ImageJ software. All the experiments were performed in triplicate.

4.2.10. Statistical analysis. Both the 2D and 3D cellular models were performed in biological and experimental triplicates. For simple comparisons between the different studied conditions, the Student's t-test was used, and differences between groups were tested using one-way analysis of variance (ANOVA) followed by the Bonferroni test and a p-value <0.05 was considered statistically significant. All statistical analyses were conducted using GraphPad Prism software v.9.

Author contributions

Vanessa P. de Souza: synthesis, conceptualization. Izabela N. F. Gomes: writing – original draft, validation. Samuel J. Santos: synthesis, conceptualization. Carolyne B. Braga: writing – original draft, methodology, validation. Aryel J. A. Bezerra: writing – original draft, validation. Eric A. Philot: writing – original draft, validation. Cíntia R. N. Ramos: writing – original draft, validation. Simone Q. Pantaleão: writing – original draft, validation. Luciane S. da Silva: writing – original draft, validation. Ronaldo A. Pilli: conceptualization, writing – original draft, funding acquisition. Rui M. Reis: writing – original draft, funding acquisition, conceptualization. Renato J. S. Oliveira: writing – original draft, synthesis, supervision, validation, resources, funding acquisition, conceptualization. Dennis Russowsky: writing – original draft; synthesis, validation, supervision, resources, funding acquisition, conceptualization.

Conflicts of interest

The authors declare no conflict of interest.

Data availability

The data supporting this article have been included as part of the supplementary information (SI).

Supplementary information is available. See DOI: https://doi.org/10.1039/D5MD00635J.

Acknowledgements

The authors acknowledge FAPERGS (Grant 19/2551-0001767-7 – D. R.), FAPESP (Grants 2013/07607-8; 2019/13104-5 to R. A. P. and 2023/02032-9 to R. J. S. O.) and a fellowship (2017/06146-8 to C. B. B.), CNPq (Grants 310438/2020-9 to D. R. and 2020/306747 to R. A. P.) and CAPES for fellowships (V. P. S., S. J. S. and C. B. B.). We would like to give credit to the Vecteezy website which offers free images that were used as part of the background composition for the Table of Contents (https://pt.vecteezy.com/vetor-gratis/gesto).

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