Synthesis and Antiproliferative Activity of Novel Imipridone–Ferrocene Hybrids with Triazole and Alkyne Linkers

Imipridones, including ONC201, ONC206 and ONC212 (which are emblematic members of this class of compounds developed by Oncoceutics) constitute a novel class of anticancer agents, with promising results in clinical trials. With the aim of increasing the ROS (reactive oxygen species) responsivity of the synthesized molecules, a set of novel ferrocene–imipridone hybrids were designed and synthesized. Our strategy was motivated by the documented interplay between the imipridone-triggered activation of TRAIL (the tumor necrosis factor-related apoptosis-inducing ligand) and mitochondrial ClpP (Caseinolytic protease P) and the ROS-mediated effect of ferrocene-containing compounds. In order to obtain novel hybrids with multitarget characters, the ferrocene moiety was tethered to the imipridone scaffold through ethynylene and 1,2,3-triazolyl linkers by using Sonogashira coupling of Cu(I)- and Ru(II)-catalyzed azide–alkyne cycloadditions. The biological activities of the new hybrids were examined by using in vitro cell viability assays on four malignant cell lines (PANC-1, A2058, EBC-1 and Fadu), along with colony formation assays on the most resistant PANC-1 cell line. Several hybrids caused a significantly greater drop in the cell viability compared to ONC201, and two of them completely overcame the resistance, with IC50 values comparable to those produced by ONC201. The two most potent hybrids, but not ONC201, induced apoptosis/necrosis in PANC-1 and A2058 cells after 24 h of treatment.


Introduction
ONC201, the first-in-class member of imipridones, which are a novel family of anticancer agents, is currently in Phase I/II clinical trials for solid and hematologic cancers of the CNS (central nervous system). Other imipridones, such as ONC206 and ONC212, were also identified as highly potent anticancer agents ( Figure 1) that displayed enhanced in vitro and in vivo efficacy compared to ONC201 [1].

Introduction
ONC201, the first-in-class member of imipridones, which are a novel family of anticancer agents, is currently in Phase I/II clinical trials for solid and hematologic cancers of the CNS (central nervous system). Other imipridones, such as ONC206 and ONC212, were also identified as highly potent anticancer agents ( Figure 1) that displayed enhanced in vitro and in vivo efficacy compared to ONC201 [1].

ONC201 ONC206 ONC212
Previously, our research group synthesized a series of such imipridones, in which the skeletal N-benzyl substituents were replaced by the ferrocenylalkyl groups. The hybrids were found to exhibit much more pronounced long-term cytotoxic effects against the A-2058 melanoma cell line than ONC201 and ONC212, which is possibly due to the contribution of the ROS-implicating apoptotic pathways that are activated by the ferrocenyl moieties. Furthermore, these ferrocenylalkyl-substituted imipridones also displayed a marked efficacy against the COLO-205 and EBC-1 cell lines [25]. At this stage, it must be pointed out that convincing preclinical evidence has been disclosed on the interplay between the TRAIL and redox signaling pathways that is implicated in cancer [26]. Prompted by these precedents and by the anticipated synergistic effects of ferrocene-driven oxidative stress and imipridone-induced ClpP and TRAIL activation [27], we undertook to construct and evaluate a small library of novel and potentially proapoptotic organometallic imipridone hybrids, which contain ferrocene fragments that are tethered by triazole and/or alkyne linkers at different positions of the N-benzyl groups on the terminal regions of the heterocyclic skeleton.
We hypothesized that the dual promotion of ROS formation in the mitochondria by the ferrocene moiety and the imipridone scaffold in the designed hybrids leads to a more efficient decrease in the cancer cell viability and, more importantly, can eliminate the resistance that is often experienced to imipridone treatment.

Synthesis of the Reference and Hybrid Imipridones
In the designed compounds, the ferrocenyl moiety is connected to the terminal aromatic rings (Figure 1) of the imipridone scaffold via a 1,2,3-triazole or an ethynylene linker.
The targeted imipridones were synthesized by using a well-established pathway that is described in our previous work [25], as shown in Scheme 1. The commercially available primary amines (1a-e) were reacted with an excess amount of methyl acrylate in methanol at room temperature to obtain intermediates (2a-e), which were then cyclized by sodium hydride, which afforded the N-substituted piperidone carboxylates (3a-e). Primary amine building blocks (1e-g) were also reacted with activated methyltioimidazoline (5) to obtain cyclic guanidines (6e-g), which were then cyclized with piperidone carboxylates (3a-e) under standard conditions to access the imipridones, 7ag, 7be, 7bf, 7ce, 7cf, 7df, 7ef and 7eg. The derivatives carrying a Boc-protected amino group on either of the aromatic rings (7be, 7ce, 7ef and 7eg) were used as precursors to obtain the appropriate azido derivative (7bh, 7ch, 7hf and 7hg) by deprotection, diazotation and sequence-terminating azide introduction.
It should be noted that Compound 7hf-which carries 4-N3 and 4-I substituen the aromatic rings-participated exclusively in azide-alkyne cycloadditions, witho ing accompanied by undesired side reactions. The iodoaryl group remained intac thus, it could be used for the Sonogashira coupling in the following step.
It should be noted that Compound 7hf-which carries 4-N 3 and 4-I substituents on the aromatic rings-participated exclusively in azide-alkyne cycloadditions, without being accompanied by undesired side reactions. The iodoaryl group remained intact and, thus, it could be used for the Sonogashira coupling in the following step.
To examine the effect of the ferrocenyl group, reference compounds were also synthesized. In these molecules, the ferrocenyl group connected to the triazole-ring (in 12a and 17c) was replaced with the phenyl group (13, 17d) or was left unsubstituted (14, 17e). These derivatives were prepared by using phenylacetylene or trimethylsilylacetylene in copper-catalyzed azide-alkyne cycloaddition protocols. The removal of the trimethylsilylprotecting group was carried out by tetra-n-butylammonium fluoride (TBAF) in THF.

Cell Viability Screenings of the Imipridone Analogues
Cell viability screenings were performed on PANC-1 (pancreatic ductal adenocarcinoma), A2058 (melanoma), EBC-1 (lung squamous carcinoma) and Fadu (hypopharynx squamous carcinoma) human cancer cell lines at three concentrations (2.8, 8.3 and 25 µM) to determine the structure-activity relationship and to narrow down the range of compounds for further investigations. The results of the viability screenings are shown in Table 1. Table 1. Cell viability screening on PANC-1, A2058, EBC-1 and Fadu cell lines. (Data represent the mean ± SD; n = 4). Cell viability data below 10% and in between 10 and 20% are presented in bold and in italics, respectively.

Cell Viability Screenings of the Imipridone Analogues
Cell viability screenings were performed on PANC-1 (pancreatic ductal adenocarcinoma), A2058 (melanoma), EBC-1 (lung squamous carcinoma) and Fadu (hypopharynx squamous carcinoma) human cancer cell lines at three concentrations (2.8, 8.3 and 25 µM) to determine the structure-activity relationship and to narrow down the range of compounds for further investigations. The results of the viability screenings are shown in Table 1.

Cell Viability Screenings of the Imipridone Analogues
Cell viability screenings were performed on PANC-1 (pancreatic ductal adenocarcinoma), A2058 (melanoma), EBC-1 (lung squamous carcinoma) and Fadu (hypopharynx squamous carcinoma) human cancer cell lines at three concentrations (2.8, 8.3 and 25 µM) to determine the structure-activity relationship and to narrow down the range of compounds for further investigations. The results of the viability screenings are shown in Table 1.

Structure-Activity Relationships
It can be clearly seen that the cells treated with the reference compound, ONC201, had a consistent reduction in viability at all three concentrations; however, depending on the cell line, a significant portion survived the treatment (47-63% for PANC-1 and A2058, 31-37% for EBC-1 and 16-25% for Fadu). The structure of Compound 18 is the most closely related to that of ONC201 in terms of the steric bulk, even though it contains electronwithdrawing groups on the aromatic rings. Nevertheless, the cell viability results of 18 indicate no significant difference between 18 and ONC201 at these concentrations.
However, upon treatment with compounds 17b-d at a concentration of 25 µM, the viability of the investigated cell lines dropped down to 1-3%. All of the three models, with such remarkable efficiency, are 4-(4-(3-aminoprop-1-yn-1-yl)benzyl)-substituted imipridones that carry a triazole ring at the para position on the benzyl group at the N-7 position of the angular tricyclic scaffold. A comparison of the performance of the ferrocene-containing hybrids, 17b and 17c, indicates that the 1,4-disubstitution of the triazole ring is more favorable than the alternative 1,5 substitution pattern. Since 17d contains a phenyl instead of a ferrocenyl group on the 1,4-disubstituted triazole ring, the comparison of the results that were obtained at an imipridone concentration of 25.0 µM would apparently suggest that it is not the ferrocenyl moiety that is prominently responsible for the low viability of the cells; however, at a concentration of 8.3 µM, the organometallic counterpart, 17c, performed better on all four cell lines compared to 17d.
Compound 17e induced only a small decrease in the cell viability at the highest concentration, and little-to-no decrease at lower concentrations, which proves that the substitution of the triazole ring is crucial for achieving low cell viability.
From the aspect of structure-activity relationships (SAR), it is of importance that Compounds 11, 12a,b, 13 and 16d, which are also decorated with triazole-based substituents on the para position of the N-7-benzyl group, do not display effects that are similar to those that are produced by 17b-d, which proves that the para-aminopropynyl-substituted benzyl group on the N-4 skeletal atom is also necessary for the production of significant

Structure-Activity Relationships
It can be clearly seen that the cells treated with the reference compound, ONC201, had a consistent reduction in viability at all three concentrations; however, depending on the cell line, a significant portion survived the treatment (47-63% for PANC-1 and A2058, 31-37% for EBC-1 and 16-25% for Fadu). The structure of Compound 18 is the most closely related to that of ONC201 in terms of the steric bulk, even though it contains electronwithdrawing groups on the aromatic rings. Nevertheless, the cell viability results of 18 indicate no significant difference between 18 and ONC201 at these concentrations.
However, upon treatment with compounds 17b-d at a concentration of 25 µM, the viability of the investigated cell lines dropped down to 1-3%. All of the three models, with such remarkable efficiency, are 4-(4-(3-aminoprop-1-yn-1-yl)benzyl)-substituted imipridones that carry a triazole ring at the para position on the benzyl group at the N-7 position of the angular tricyclic scaffold. A comparison of the performance of the ferrocene-containing hybrids, 17b and 17c, indicates that the 1,4-disubstitution of the triazole ring is more favorable than the alternative 1,5 substitution pattern. Since 17d contains a phenyl instead of a ferrocenyl group on the 1,4-disubstituted triazole ring, the comparison of the results that were obtained at an imipridone concentration of 25.0 µM would apparently suggest that it is not the ferrocenyl moiety that is prominently responsible for the low viability of the cells; however, at a concentration of 8.3 µM, the organometallic counterpart, 17c, performed better on all four cell lines compared to 17d.
Compound 17e induced only a small decrease in the cell viability at the highest concentration, and little-to-no decrease at lower concentrations, which proves that the substitution of the triazole ring is crucial for achieving low cell viability.
From the aspect of structure-activity relationships (SAR), it is of importance that Compounds 11, 12a,b, 13 and 16d, which are also decorated with triazole-based substituents on the para position of the N-7-benzyl group, do not display effects that are similar to those that are produced by 17b-d, which proves that the para-aminopropynyl-substituted

Structure-Activity Relationships
It can be clearly seen that the cells treated with the reference compound, ONC201, had a consistent reduction in viability at all three concentrations; however, depending on the cell line, a significant portion survived the treatment (47-63% for PANC-1 and A2058, 31-37% for EBC-1 and 16-25% for Fadu). The structure of Compound 18 is the most closely related to that of ONC201 in terms of the steric bulk, even though it contains electronwithdrawing groups on the aromatic rings. Nevertheless, the cell viability results of 18 indicate no significant difference between 18 and ONC201 at these concentrations.
However, upon treatment with compounds 17b-d at a concentration of 25 µM, the viability of the investigated cell lines dropped down to 1-3%. All of the three models, with such remarkable efficiency, are 4-(4-(3-aminoprop-1-yn-1-yl)benzyl)-substituted imipridones that carry a triazole ring at the para position on the benzyl group at the N-7 position of the angular tricyclic scaffold. A comparison of the performance of the ferrocene-containing hybrids, 17b and 17c, indicates that the 1,4-disubstitution of the triazole ring is more favorable than the alternative 1,5 substitution pattern. Since 17d contains a phenyl instead of a ferrocenyl group on the 1,4-disubstituted triazole ring, the comparison of the results that were obtained at an imipridone concentration of 25.0 µM would apparently suggest that it is not the ferrocenyl moiety that is prominently responsible for the low viability of the cells; however, at a concentration of 8.3 µM, the organometallic counterpart, 17c, performed better on all four cell lines compared to 17d.
Compound 17e induced only a small decrease in the cell viability at the highest concentration, and little-to-no decrease at lower concentrations, which proves that the substitution of the triazole ring is crucial for achieving low cell viability.
From the aspect of structure-activity relationships (SAR), it is of importance that Compounds 11, 12a,b, 13 and 16d, which are also decorated with triazole-based substituents on the para position of the N-7-benzyl group, do not display effects that are similar to those that are produced by 17b-d, which proves that the para-aminopropynyl-substituted

Structure-Activity Relationships
It can be clearly seen that the cells treated with the reference compound, ONC201, had a consistent reduction in viability at all three concentrations; however, depending on the cell line, a significant portion survived the treatment (47-63% for PANC-1 and A2058, 31-37% for EBC-1 and 16-25% for Fadu). The structure of Compound 18 is the most closely related to that of ONC201 in terms of the steric bulk, even though it contains electron-withdrawing groups on the aromatic rings. Nevertheless, the cell viability results of 18 indicate no significant difference between 18 and ONC201 at these concentrations.
However, upon treatment with compounds 17b-d at a concentration of 25 µM, the viability of the investigated cell lines dropped down to 1-3%. All of the three models, with such remarkable efficiency, are 4-(4-(3-aminoprop-1-yn-1-yl)benzyl)-substituted imipridones that carry a triazole ring at the para position on the benzyl group at the N-7 position of the angular tricyclic scaffold. A comparison of the performance of the ferrocene-containing hybrids, 17b and 17c, indicates that the 1,4-disubstitution of the triazole ring is more favorable than the alternative 1,5 substitution pattern. Since 17d contains a phenyl instead of a ferrocenyl group on the 1,4-disubstituted triazole ring, the comparison of the results that were obtained at an imipridone concentration of 25.0 µM would apparently suggest that it is not the ferrocenyl moiety that is prominently responsible for the low viability of the cells; however, at a concentration of 8.3 µM, the organometallic counterpart, 17c, performed better on all four cell lines compared to 17d. Compound 17e induced only a small decrease in the cell viability at the highest concentration, and little-to-no decrease at lower concentrations, which proves that the substitution of the triazole ring is crucial for achieving low cell viability.
From the aspect of structure-activity relationships (SAR), it is of importance that Compounds 11, 12a,b, 13 and 16d, which are also decorated with triazole-based substituents on the para position of the N-7-benzyl group, do not display effects that are similar to those that are produced by 17b-d, which proves that the para-aminopropynyl-substituted benzyl group on the N-4 skeletal atom is also necessary for the production of significant antiproliferative activity. On the other hand, the viability assays also indicate that, at least in combination with the triazolylbenzyl substituents on the N-7 skeletal position, the introduction of the 2-methyl-, 4-iodo-or 4-ferrocenylethynyl group in the benzyl group, depending on the N-4 skeletal atom, is not beneficial for lowering the cell viability. It must be pointed out here that, although, in the case of the isomer pair, 17b/17c, the 1,4-disubtitution on the triazole ring was found favorable over the 1,5-disubstitution pattern. Among the triazole derivatives, 11, 12a,b, 13 and 16d, the experiments on the A2058 and EBC-1 cell lines identified 11, a 4-(4-iodobenzyl)-containing imipridone, as the most effective model, with the 4-(5-ferrocenyl-1H-1,2,3-triazol-1-yl)benzyl group attached to the N-7 position instead of its isomer, 12a, which carries the 4-(4-ferrocenyl-1H-1,2,3-triazol-1-yl)benzyl group on the N-7 skeletal atom.
Demonstrating a further SAR, compounds 9a,b, which are equipped with the 1,4 disubstituted triazolyl-substituted benzyl group at Position 4, caused only a little-to-no decrease in the cell viability, whereas 10a,b, which contain the 1,5-disubstituted triazole ring on the benzyl group that is attached to the same (N-4) position, induced a more pronounced decrease in the viability of the investigated cell lines. In fact, 10a was able to affect a substantial drop in the viability of the EBC-1 cells, down to the level (3%) at 25 µM, which was achieved by 17b-d at the same concentration. (Pointing to the cell selectivity, the treatment with 10a also led to the relatively low viability of the other cell lines at this concentration, but not as low as was achieved by 17b-d).
The compounds with a ferrocenylethynyl group at the para position on the benzyl group that is attached to the N-4 atom (16a-c) showed moderate-to-no decrease and, interestingly, on PANC-1, even showed an increase in the cell viability. The increment in the viability might be ascribed to the fact that pancreatic cancer cells naturally have an elevated ROS level, and a small elevation in the ROS concentration can even be beneficial in attenuating their proliferation [16]. This tendency has also been revealed for other cell lines [17] that are associated with less pronounced increments in their viability.
As mentioned above, Compound 18 exhibited very similar effects to those of ONC201. Interestingly, 17a, which also carries the 3-cyanobenzyl group on N-7 and the 4-(3-aminoprop-1-yn-1-yl)benzyl group, an extended alkyne residue on N-4 gave rise to an appreciable drop in the viability of all of the studied cells to markedly lower levels, relative to those induced by 18 and ONC201. This means that, cooperating with the aryltriazolyl moiety that pends on the benzyl group at the N-7 position, the presence of the aminopropynyl group on the benzyl group at the opposite N-4 position is necessary to attain very low-level or nearly complete loss of viability. Thus, the aforementioned SAR could also imply that: (i) Compounds 17b-d act on a molecular target that is different from those identified for ONC201 and other emblematic imipridones; and (ii) The arytriazolyl and aminopropynyl groups, which are appropriately located on the two terminals of the imipridone framework, are necessary for cooperatively creating effective interactions with target(s) undisclosed so far. It is of note that the abovementioned substituent patterns seem incompatible with the known imipridone targets, with special regard to ClpP. This hypothesis is also supported by the fact that the PANC-1 (pancreas) and A2058 (melanoma) cells were less susceptible to ONC201 treatment than the Fadu (hypopharynx) or EBC-1 (lung) cancer cells; however, for the treatment with Compounds 17b-d, the sensitivity of the four cell lines showed much less significant differences. Furthermore, during treatment with these compounds, especially in the case of the PANC-1 cell line, the cell viability dropped more rapidly with the same increment in concentration, which also points to the involvement of different biological target(s).
Since Compounds 10a and 17b-d can be considered as the most effective hybrids among the tested ones, these aryltriazolyl derivatives were selected for the dose-response curve determination and for the colony formation assays. It must be noted here that 10a, which is a fluorinated compound, was identified as one the most potent antiproliferative agents in the recent series of our imipridone hybrids, which also include other monoand difluoro-substituted derivatives (9a,b, 10b and 16a,b). Besides the conjugation with ferrocene-containing fragments, the introduction of fluoro substituents was considered to be an attractive strategy to attenuate the antiproliferative activity of the targeted hybrids. In terms of decreasing the efficient dose, we identified highly active imipridones, which were fluorinated on the meta position(s) of the benzyl group that was attached to the N-7 skeletal position, that feature IC 50 values in the low nanomolar range on different human malignant cell lines [31]. On the other hand, the improved drug-like properties of the fluorinated compounds can be ascribed to the significant influence of fluorine on the polarity, acidity, conformation, membrane permeability and, thus, on the pharmacokinetics of the potential drug candidates [32]. Moreover, in the context of the possible prospects of our research on ferrocene-containing hybrids, it must be mentioned here that fluorination has also been successfully explored in labeling studies that were aimed at improving the bioanalytical sensitivity in drug uptake studies on organometallic anticancer agents [33].

Dose-Response Curves of Selected Imipridone Hybrids
As detailed above, the most effective compounds, 10a and 17b-d, were selected for the determination of their dose-response curves and IC 50 values on previously examined cancer cell lines (PANC-1, A2058, EBC-1 or Fadu) by an MTT cell viability assay. ONC201 was applied as a reference compound. The dose-response curves are shown in Figure 2. The IC 50 values are presented in Table 2.  10a, 17b, 17c and 17d) and reference compound, ONC201, on different cancer cell lines based on MTT cell viability assay. Curves were fitted by GraphPad Prism 8 software using nonlinear regression (variable slope; 95% CI; n = 4).  10a, 17b, 17c and 17d) and reference compound, ONC201, after 72 h of treatment, based on MTT assay. IC50 values were determined by GraphPad Prism 8 software (nonlinear regression; variable slope; best-fit values; n = 4).
Treated Cells 10a 17b 17c 17d ONC201 Figure 2. Dose-response curves of selected imipridone hybrids (10a, 17b, 17c and 17d) and reference compound, ONC201, on different cancer cell lines based on MTT cell viability assay. Curves were fitted by GraphPad Prism 8 software using nonlinear regression (variable slope; 95% CI; n = 4). Although the novel imipridone hybrids (10a and 17b-d) resulted in higher IC 50 values than the reference imipridone, ONC201, the dose-response curves reveal that each of the tested cancer cell lines contained a significant amount of viable cells after 72 h of ONC201 treatment. On the contrary, the novel imipridone hybrids, 17b, 17c and 17d, were able to reduce the cell viability to near zero on every tested cancer cell line.

Colony Formation Assay
The cell viability screening and the dose-response curves revealed that PANC-1 and A2058 exhibit the least susceptibility to ONC201 treatment. Accordingly, previously published data [3] confirm that PANC-1 can be categorized as a cell line that is resistant to ONC201 (and ONC212) imipridone derivatives. On the basis of these findings, the PANC-1 cell line was selected for the colony formation assay, and the cells were treated with compounds at a 10 µM concentration for 24 and 72 h, followed by 5 and 3 days of postincubation periods.
The results of the colony formation assay are presented in Figure 3, and they clearly demonstrate that, even after 72 h of exposure time with ONC201 (Figure 3b), there were still viable PANC-1 cells that were able to form colonies. Treatment with Compound 10a led to a similar result. However, in the cases of Compounds 17c and 17d, the PANC-1 cells were completely eradicated, even after 24 h of exposure time (Figure 3a). In accordance with the IC 50 data that were gained by the MTT test, 10 µM proved to be insufficient for 17b to eradicate PANC-1 cells. On the basis of these results, 17c and 17d proved to be the most potent imipridone derivatives, and they bear the capacity to overcome the resistance to ONC201 treatment.
Pharmaceuticals 2022, 15, 468 11 still viable PANC-1 cells that were able to form colonies. Treatment with Compound led to a similar result. However, in the cases of Compounds 17c and 17d, the PANC-1 were completely eradicated, even after 24 h of exposure time (Figure 3a). In accord with the IC50 data that were gained by the MTT test, 10 µM proved to be insufficien 17b to eradicate PANC-1 cells. On the basis of these results, 17c and 17d proved to be most potent imipridone derivatives, and they bear the capacity to overcome the resist to ONC201 treatment.

Cytotoxicity Assay with the Most Potent Hybrids on Cancer Cell Lines and Nontumorou Fibroblast Cells
The imipridone hybrids, 17c and 17d, proved to be the most potent antiprolifera agents in the dose-response and colony formation assays. However, it is critical, from aspect of their therapeutic potential, to assess their toxicity in nontumorous cells. To end, concentration-dependent viability studies were performed with these two c pounds on the four investigated tumor cell lines and on primary fibroblast cells by u

Cytotoxicity Assay with the Most Potent Hybrids on Cancer Cell Lines and Nontumorous Fibroblast Cells
The imipridone hybrids, 17c and 17d, proved to be the most potent antiproliferative agents in the dose-response and colony formation assays. However, it is critical, from the aspect of their therapeutic potential, to assess their toxicity in nontumorous cells. To this end, concentration-dependent viability studies were performed with these two compounds on the four investigated tumor cell lines and on primary fibroblast cells by using CellTiter, which is a method that is more sophisticated than MTT analysis. The obtained doseresponse curves (Figure 4) unambiguously reveal that the ferrocenyltriazole analogue, 17c, can be considered to offer substantial therapeutic windows, as it led to the practically complete eradication of all the investigated tumor cells at a 10 µM concentration, while it displayed no toxicity on the fibroblast cells at the same concentration. The IC 50 values that were obtained from these of experiments (Table 3) further support our view about the promising therapeutic potential of 17c. The dose-response curves and the IC 50 data that were obtained from the experiments with the phenyltriazolyl counterpart, 17d, which were carried out under the same conditions, are indicative of significantly narrower therapeutic windows with regard to this compound. Nevertheless, at a 10 µM concentration, 17d also completely eradicates PANC-1 cells, without showing toxicity on the fibroblast cells.

Apoptosis and Necrosis Quantitation Assay
In order to gain preliminary information about the mechanism of action of the most potent imipridone hybrids, 17c and 17d, we performed an apoptosis-necrosis detection assay on PANC-1 and A2058 cells by using ONC201 as the structurally related reference, paclitaxel as the positive control and untreated cells as the negative control ( Figure 5). The experiments demonstrated a marked apoptosis-inducing effect of both the selected novel imipridone hybrids on both cell lines, while no apoptotic/necrotic signal was observed after 24 h of treatment with ONC201. These findings indicate differences in the kinetics, and possibly the targets, of the proapoptotic/pronecrotic effects of 17c and 17d, and those identified for ONC201.

Apoptosis and Necrosis Quantitation Assay
In order to gain preliminary information about the mechanism of action of the most potent imipridone hybrids, 17c and 17d, we performed an apoptosis-necrosis detection assay on PANC-1 and A2058 cells by using ONC201 as the structurally related reference, paclitaxel as the positive control and untreated cells as the negative control ( Figure 5). The experiments demonstrated a marked apoptosis-inducing effect of both the selected novel imipridone hybrids on both cell lines, while no apoptotic/necrotic signal was observed after 24 h of treatment with ONC201. These findings indicate differences in the kinetics, and possibly the targets, of the proapoptotic/pronecrotic effects of 17c and 17d, and those identified for ONC201.
In order to gain preliminary information about the mechanism of action of the most potent imipridone hybrids, 17c and 17d, we performed an apoptosis-necrosis detection assay on PANC-1 and A2058 cells by using ONC201 as the structurally related reference, paclitaxel as the positive control and untreated cells as the negative control ( Figure 5). The experiments demonstrated a marked apoptosis-inducing effect of both the selected novel imipridone hybrids on both cell lines, while no apoptotic/necrotic signal was observed after 24 h of treatment with ONC201. These findings indicate differences in the kinetics, and possibly the targets, of the proapoptotic/pronecrotic effects of 17c and 17d, and those identified for ONC201. Figure 5. Apoptosis-necrosis detection assay on PANC-1 and A2058 cancer cells by confocal laser scanning microscopy. Cells were treated with ONC201, 17c and 17d for 24 h at 10 µM. Paclitaxel was used as a positive control at 1 µM, and untreated cells as a negative control. Annexin V labeled Figure 5. Apoptosis-necrosis detection assay on PANC-1 and A2058 cancer cells by confocal laser scanning microscopy. Cells were treated with ONC201, 17c and 17d for 24 h at 10 µM. Paclitaxel was used as a positive control at 1 µM, and untreated cells as a negative control. Annexin V labeled with CF488A stains apoptotic cells with green fluorescence by binding to phosphatidylserine exposed on the cell surface. Ethidium Homodimer III is a nucleic acid probe that is impermeant to live cells and early apoptotic cells, but that stains necrotic cells and late apoptotic cells with red fluorescence.

Conclusions
Drug resistance that develops in the highly adaptive cancer cells is a major factor that hampers the clinical efficacy of anticancer chemotherapy. The prosurvival mechanisms that are upregulated in some tumor cells were also found to limit the anticancer activity of ONC201, which is the first-in-class imipridone. To address the problem that is associated with the resistance of cancer cells to imipridone treatment, we synthesized a set of novel ferrocene-containing hybrids with potential ROS-generating capabilities to attenuate the antiproliferative activity exerted by the imipridone core. This strategy of fragment-based design is in line with the concept on the hybrid compounds that emerged as prominent multitargeted anticancer agents that contain the inducers of two or more cytotoxic mechanisms [34].
The initial screenings revealed that four newly synthesized imipridone hybrids (10a, 17b, 17c and 17d) were significantly more effective at decreasing the cell viability compared to ONC201, on all four examined cell lines (PANC-1, A2058, EBC-1 and Fadu). Although the IC 50 values of these compounds were found to be higher than those of ONC201, the colony formation assays performed on the most treatment-resistant PANC-1 cell line [3] proved that two of the emergent hybrids (17c and 17d) eliminated the resistance, and no viable cells could be observed after treatments at 10 µM concentrations.
The SAR discussed above strongly suggest that-in a cooperative manner-a 4/5-aryl-1H-1,2,3-triazol-1-yl residue (aryl = phenyl, ferrocenyl or others not investigated here) and the protruding basic 3-aminopropynyl substituent placed at the para positions of the benzyl substituents that pend on the distant imipridone skeletal positions, 7 and 4, respectively, are able to overcome the resistance of cancer cells, for which the assays are evaluated in this research.
However, the dose-response curves show that the cell viability drops very rapidly with the change in the concentration of the presented hybrids, so that even the two most potent compounds should be applied at relatively high concentrations within a very narrow window to evade cell resistance, which might limit their applicability. On the other hand, by highlighting the beneficial properties of 17c and 17d, it was also demonstrated that, in shorter treatments (24 h), these compounds are far more efficient in apoptosis/necrosis induction in PANC-1 and A2058 cells than ONC201, which caused no detectable apoptotic/necrotic signal after an identical period of treatment. Finally, in comparative cytotoxicity studies on PANC-1 and A2058 cells and on nontumorous primary fibroblasts, the organometallic derivative, 17c, emerged as the most potent anticancer agent in the presented research. Without exerting an observable toxicity on the fibroblasts at 10 µM, this lead compound was capable of selectively eradicating the tumor cells at the aforementioned concentration. The results of all of the comparative experiments suggest that the advantageous properties of the two most potent compounds, with particular regard to overcoming resistance, might probably be associated with their multitarget character, which warrants the activation of at least a dual mechanism of action.
In summary, despite the fact that the IC 50 values that were produced by our most potent hybrids are 5-10 times higher than those of ONC201 on the same cell lines, the overall results are very promising with regard to 17c and 17d as potential anticancer drug candidates. We believe this work is worth continuing by fine-tuning the structures of the aforementioned most potent hybrids with the variation and reasonable modification of the substitution pattern on the imipridone core, and by exploring the molecular fragments introduced so far and their structurally related versions. The cytotoxicity of 17c,d, and their modified versions, are to be assessed on a significantly extended platform of cell lines to evaluate the effects of diverse functionalizations on the aromatic rings and on the amino group, as well as further versions of the hybridization on the triazole ring with other pharmacophores.

Data Availability Statement:
The data generated and analyzed during our research are not available in any public database or repository but will be shared by the corresponding author upon reasonable request.