Synthesis and Antiproliferative Effect of New Alkyne-Tethered Vindoline Hybrids Containing Pharmacophoric Fragments

In the frame of our diversity-oriented research on multitarget small molecule anticancer agents, utilizing convergent synthetic sequences terminated by Sonogashira coupling reactions, a preliminary selection of representative alkyne-tethered vindoline hybrids was synthesized. The novel hybrids with additional pharmacophoric fragments of well-documented anticancer agents, including FDA-approved tyrosine-kinase inhibitors (imatinib and erlotinib) or ferrocene or chalcone units, were evaluated for their antiproliferative activity on malignant cell lines MDA-MB-231 (triple negative breast cancer), A2780 (ovarian cancer), HeLa (human cervical cancer), and SH-SY5Y (neuroblastoma) as well as on human embryonal lung fibroblast cell line MRC-5, which served as a reference non-malignant cell line for the assessment of the therapeutic window of the tested hybrids. The biological assays identified a trimethoxyphenyl-containing chalcone-vindoline hybrid (36) as a promising lead compound exhibiting submicromolar activity on A2780 cells with a marked therapeutic window.


Introduction
Cancer is one of the most severe health problems.Even though the associated survival rate has improved, different types of tumorous diseases are still among the leading causes of mortality, with low survival rates [1,2].Although in clinical practice, chemotherapy is generally considered one of the most widely used tools for cancer treatment, often in combination with other therapies, such as surgery, radiation, or hormone therapy, the efficacy of most anticancer chemotherapies is decreased by several therapy-limiting factors, including multidrug resistance (MDR) [3,4] and adverse effects.Consequently, developing more potent novel drugs possessing improved activity, selectivity, and enhanced potency to overcome MDR is continuously the focus of research.One of the most appealing new strategies for developing improved chemotherapy is the fragment-based design and synthesis of hybrid compounds by coupling a reasonable selection of pharmacophoric fragments [5][6][7].Such hybrid drugs capable of interacting with more than one cellular molecular target can be considered highly potent anticancer agents with enhanced efficiency as they trigger cell death by multiple mechanisms, thus having a real potential to overcome the typical disadvantages of single anticancer agents, including resistance and adverse effects.For an expansion of novel potent therapeutic agents, the implication of compounds of natural origin and their chemically modified versions also seems an attractive strategy.In this regard, several representatives from alkaloid families are of pronounced interest [8][9][10][11].The representative tubulin inhibitor bis-indole alkaloid Vinblastine (1: Figure 1) can also be considered a hybrid composed of the coupled monomers catharanthine (1a) and vindoline (1b).This alkaloid is one of the most widely used therapeutic agents for treating cancers, e.g., breast cancer [12], acute lymphocytic leukemia [13], and testicular germ-cell carcinomas [14] either as a single agent or in combination with other drugs, exerting its activity by inhibiting tubulin polymerization [15].
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 2 of 21 with more than one cellular molecular target can be considered highly potent anticancer agents with enhanced efficiency as they trigger cell death by multiple mechanisms, thus having a real potential to overcome the typical disadvantages of single anticancer agents, including resistance and adverse effects.For an expansion of novel potent therapeutic agents, the implication of compounds of natural origin and their chemically modified versions also seems an attractive strategy.In this regard, several representatives from alkaloid families are of pronounced interest [8][9][10][11].The representative tubulin inhibitor bis-indole alkaloid Vinblastine (1: Figure 1) can also be considered a hybrid composed of the coupled monomers catharanthine (1a) and vindoline (1b).This alkaloid is one of the most widely used therapeutic agents for treating cancers, e.g., breast cancer [12], acute lymphocytic leukemia [13], and testicular germ-cell carcinomas [14] either as a single agent or in combination with other drugs, exerting its activity by inhibiting tubulin polymerization [15].Vinca alkaloids, including vinblastine, can be isolated from Catharanthus roseus.The monomers occur in much greater quantity in the plant than the dimers; however, these single fragments do not display perceptible anticancer effects [16,17].To address this problem, various vinca alkaloids have been synthesized as hybrids, such as amino acidand steroid-containing hybrids, and with other pharmacophore moieties [18][19][20].In this context, pursuing our diversity-oriented research on the development of novel small molecule anticancer agents with enhanced anticancer potency, we aimed at constructing a preliminary selection of novel alkyne-tethered vindoline-based hybrids in which position 10 is coupled to fragments of well-documented pharmacophore residues replacing catharanthine (1a), which is present in vinblastine 1. Accordingly, we planned to introduce the FDA-approved tyrosine-kinase inhibitor anticancer agents imatinib (2) [21] and erlotinib (3) [22] as well as the ferrocene-and chalcone-containing moieties 4 and 5 (Figure 2) in the targeted vindoline hybrids.Representative pharmacophores, or their fragments, which were selected for introduction in the targeted vindoline hybrids: FDA-approved kinase inhibitors (imatinib 2 and erlotinib 3), ferrocene (4), and chalcone (5).
Besides its commercial availability and enhanced stability compared to catharanthine, the choice of vindoline as an essential fragment in our targeted hybrids can be justified by the results published over recent years pointing to the possibility of Vinca alkaloids, including vinblastine, can be isolated from Catharanthus roseus.The monomers occur in much greater quantity in the plant than the dimers; however, these single fragments do not display perceptible anticancer effects [16,17].To address this problem, various vinca alkaloids have been synthesized as hybrids, such as amino acid-and steroid-containing hybrids, and with other pharmacophore moieties [18][19][20].In this context, pursuing our diversity-oriented research on the development of novel small molecule anticancer agents with enhanced anticancer potency, we aimed at constructing a preliminary selection of novel alkyne-tethered vindoline-based hybrids in which position 10 is coupled to fragments of well-documented pharmacophore residues replacing catharanthine (1a), which is present in vinblastine 1. Accordingly, we planned to introduce the FDA-approved tyrosine-kinase inhibitor anticancer agents imatinib (2) [21] and erlotinib (3) [22] as well as the ferrocene-and chalcone-containing moieties 4 and 5 (Figure 2) in the targeted vindoline hybrids.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 2 of 21 with more than one cellular molecular target can be considered highly potent anticancer agents with enhanced efficiency as they trigger cell death by multiple mechanisms, thus having a real potential to overcome the typical disadvantages of single anticancer agents, including resistance and adverse effects.For an expansion of novel potent therapeutic agents, the implication of compounds of natural origin and their chemically modified versions also seems an attractive strategy.In this regard, several representatives from alkaloid families are of pronounced interest [8][9][10][11].The representative tubulin inhibitor bis-indole alkaloid Vinblastine (1: Figure 1) can also be considered a hybrid composed of the coupled monomers catharanthine (1a) and vindoline (1b).This alkaloid is one of the most widely used therapeutic agents for treating cancers, e.g., breast cancer [12], acute lymphocytic leukemia [13], and testicular germ-cell carcinomas [14] either as a single agent or in combination with other drugs, exerting its activity by inhibiting tubulin polymerization [15].Vinca alkaloids, including vinblastine, can be isolated from Catharanthus roseus.The monomers occur in much greater quantity in the plant than the dimers; however, these single fragments do not display perceptible anticancer effects [16,17].To address this problem, various vinca alkaloids have been synthesized as hybrids, such as amino acidand steroid-containing hybrids, and with other pharmacophore moieties [18][19][20].In this context, pursuing our diversity-oriented research on the development of novel small molecule anticancer agents with enhanced anticancer potency, we aimed at constructing a preliminary selection of novel alkyne-tethered vindoline-based hybrids in which position 10 is coupled to fragments of well-documented pharmacophore residues replacing catharanthine (1a), which is present in vinblastine 1. Accordingly, we planned to introduce the FDA-approved tyrosine-kinase inhibitor anticancer agents imatinib (2) [21] and erlotinib (3) [22] as well as the ferrocene-and chalcone-containing moieties 4 and 5 (Figure 2) in the targeted vindoline hybrids.Besides its commercial availability and enhanced stability compared to catharanthine, the choice of vindoline as an essential fragment in our targeted hybrids can be justified by the results published over recent years pointing to the possibility of Besides its commercial availability and enhanced stability compared to catharanthine, the choice of vindoline as an essential fragment in our targeted hybrids can be justified by the results published over recent years pointing to the possibility of types of structural engineering that can produce real anticancer agents incorporating this alkaloid conjugated to suitable pharmacophores including amino acids [23], steroids [18], and N-heterocycles [19].
The introduction of ferrocene-containing fragments can be justified by the following findings.In 1984, Köpf-Maier et al. reported the anticancer properties of ferrocene salts [24].Later, more studies reported ferrocene derivatives exhibiting antiproliferative effects on several cancer cell lines and low toxicity against non-transformed cells.It is of importance that due to its stability, super-aromaticity, elevated membrane-penetrating ability, and implication in substituent-dependent, and thus fine-tunable, ROS-generating single electron transfer (SET) events, organoferrocene fragments became the most widespread structural motifs in the emerging group of organometallics displaying anticancer activity triggered by versatile mechanisms of action [25][26][27][28][29][30][31][32][33][34][35].It has also been demonstrated that replacing the aromatic nucleus of certain organic compounds for a ferrocene unit can lead to products with antiproliferative activity that is absent or less manifested in the parent molecule [36][37][38][39].
Chalcones are also privileged scaffolds embedded in a plethora of highly potent anticancer drug candidates inducing cancer cell death by versatile mechanisms of action, including cell cycle arrest in the subG1, S, and G2/M phases, inhibition of tubulin polymerization, enzyme dynamics [40][41][42][43][44][45], and signal transductions initiated by nuclear factor κB [46].On the other hand, it is also of pronounced importance that various chalcone-containing scaffolds feature marked potency even in overcoming drug resistance, as they were found to exhibit in vitro and in vivo effects on both drug-susceptible and drug-resistant cancers by targeting the aromatase enzyme (CIP19A1), breast cancer resistance protein (BCRP), vascular endothelial growth factor (VEGF), and ATP binding cassette subfamily G member 2 (ABCG2) [47,48].Since indole is an integrated structural motif in vindoline, chalconeindol hybrids are particularly worth highlighting as further examples with demonstrated antiproliferative activity against A549, MCF-7, HepG2, paclitaxel-resistant HCT-8/T, and vincristine-resistant HCT-8/V cell lines [49].In this regard, Yan et al. reported the low nanomolar activity of chalcone-indol hybrids detected, again, with A549, MCF-7, and HCT-8 cancer cells [50].

Results and Discussion
The targeted alkyne-tethered hybrids were synthesized via two straightforward convergent synthetic pathways both terminated by Sonogashira reactions involving the readily available 10-iodovindoline [51] and the primarily prepared propargylated pharmacophoric moieties, or the 10-iodovindoline-derived silyl-protected 10-ethynylvindoline and iodinated chalcones as alternative coupling partners.Besides their synthetic aspects, the introduction of an acetylenic linker into anticancer drug candidates can also be beneficial in terms of their bioactivity, as justified by characteristic literature examples reporting on alkyne derivatives identified as potent antitumor agents of natural and synthetic origins [52][53][54][55][56][57][58].A representative alkyne-coupled pyrrolo [2,3-d]pyrimidine, BIIB028, displaying therapeutic activity with excellent drug-like properties and an acceptable safety profile in the treatment of breast cancer, melanoma, gastrointestinal cancer, lymphoma, and myeloma [59][60][61], further supports the view about the benefits of adding carbon-carbon triple bonds as rigid spacers into potential anticancer agents.

Synthesis of Propargylated Imatinib Fragments
The propargylated imatinib fragments were synthesized utilizing the feasibility of the introduction of propargyl groups into the intermediates 9, 14, and 18, synthesized by the reaction sequences developed by Liu et al. [62], as shown in Scheme 1. Accordingly, 3-acetylpyridine (6) was reacted with DMF-DMA to obtain the intermediate enaminone 7, which was cyclized with guanidine-nitrate to obtain aminopyrimidine 8, the diazotization followed by chlorination of which formed chloropyrimidine 9.This electrophilic intermediate was reacted with propargylamine to obtain compound 10, an alkynefunctionalized imatinib moiety suitable for Sonogashira coupling.Using another pathway, the pyrimidine-forming ring closure of enaminone 7 was performed with N-(2-methyl-5- 7, which was cyclized with guanidine-nitrate to obtain aminopyrimidine 8, the diazotization followed by chlorination of which formed chloropyrimidine 9.This electrophilic intermediate was reacted with propargylamine to obtain compound 10, an alkyne-functionalized imatinib moiety suitable for Sonogashira coupling.Using another pathway, the pyrimidine-forming ring closure of enaminone 7 was performed with N-(2-methyl-5-nitrophenyl)guanidine nitrate 12, previously generated from nitroaniline 11.The catalytic hydrogenation of the resulting nitroaryl derivative 13 gave aniline 14 as a nucleophilic key intermediate, the N-alkylation of which with propargyl bromide conducted under standard conditions led to the formation of the next imatinib-based terminal alkyne 15, featuring a more extended structural motif compared to the biaryl-type intermediate 10.Finally, the reaction pathway starting with the double chlorination of 4-(hydroxymethyl)benzoic acid (16 → 17) followed by N-acylation (14 + 17 → 18) and the sequential N-propargylation of the benzyl chloride-type intermediate 18 created 19, the most complex propargylated fragment comprising the majority of the structural motifs of imatinib.Scheme 1. Synthesis of propargylated imatinib fragments suitable for Sonogashira reactions with 10-iodovindoline.

Sonogashira Coupling Reactions Terminating the Synthetic Pathways to the Targeted Alkyne-Tethered Vindoline Hybrids
The first group of the hybrids containing the fragments of FDA-approved anticancer agents 21-24 was synthesized by coupling 10-iodovindoline 20 with propargyalated imatinib fragments (10,15,19) and intact erlotinib (3), as outlined in Scheme 2. The reactions were conducted for 24 h at room temperature in DMF using CuI(20%)/PdCl2(PPh3)2(10%) and N,N-diisopropylethylamine (DIPEA) (3 eq.) as a catalyst system and base, respectively (Scheme 2).Under the same conditions, ferrocene-containing hybrids 28 and 29 were obtained when the commercially available ethynylferrocene 25 and N-propargyl ferrocene carboxamide 27, respectively, were used as alkyne components in the coupling reactions.The carboxamide 27 was obtained by the well-established acylation of propargylamine with N-ferrocenoylimidazole 26 [63].

Sonogashira Coupling Reactions Terminating the Synthetic Pathways to the Targeted Alkyne-Tethered Vindoline Hybrids
The first group of the hybrids containing the fragments of FDA-approved anticancer agents 21-24 was synthesized by coupling 10-iodovindoline 20 with propargyalated imatinib fragments (10,15,19) and intact erlotinib (3), as outlined in Scheme 2. The reactions were conducted for 24 h at room temperature in DMF using CuI(20%)/PdCl 2 (PPh 3 ) 2 (10%) and N,N-diisopropylethylamine (DIPEA) (3 eq.) as a catalyst system and base, respectively (Scheme 2).Under the same conditions, ferrocene-containing hybrids 28 and 29 were obtained when the commercially available ethynylferrocene 25 and N-propargyl ferrocene carboxamide 27, respectively, were used as alkyne components in the coupling reactions.The carboxamide 27 was obtained by the well-established acylation of propargylamine with N-ferrocenoylimidazole 26 [63].
In the first step of the synthetic route to creating the representative chalcone-containing hybrids, 10-iodovindoline 20 was coupled with trimethylsilylacetylene under the same Sonogashira reaction conditions to create protected alkyne 30.In a one-pot procedure, without isolation and purification, the unstable 30 was subjected to TBAF-mediated desilylation followed by Sonogashira coupling of the resulting non-isolated 10-ethynylvindoline with iodinated chalcones 34 and 35 to obtain hybrids 36 and 37, respectively (Scheme 2).Iodochalcones 34 and 35 were previously prepared by Claisen-Schmidt condensation of 4-iodobenzaldehyde 31 with 3,4,5-trimethoxyacetophenone (32) and acetylferrocene (33), respectively (Scheme 2).In the first step of the synthetic route to creating the representative chalcone-containing hybrids, 10-iodovindoline 20 was coupled with trimethylsilylacetylene under the same Sonogashira reaction conditions to create protected alkyne 30.In a one-pot procedure, without isolation and purification, the unstable 30 was subjected to TBAF-mediated desilylation followed by Sonogashira coupling of the resulting non-isolated 10-ethynylvindoline with iodinated chalcones 34 and 35 to obtain hybrids 36 and 37, respectively (Scheme 2).Iodochalcones 34 and 35 were previously prepared by Claisen-Schmidt condensation of 4-iodobenzaldehyde 31 with 3,4,5-trimethoxyacetophenone (32) and acetylferrocene (33), respectively (Scheme 2).
The reactions of 34 and 35 with phenylacetylene 38 led to the formation of chalcones 36a and 37a (Scheme 3), serving as reference models in the biological assays.The reactions of 34 and 35 with phenylacetylene 38 led to the formation of chalcones 36a and 37a (Scheme 3), serving as reference models in the biological assays.Scheme 3. Synthesis of reference chalcones containing phenyl group in place of vindoline moiety.

In Vitro Antiproliferative Evaluation of the Novel Vindoline Hybrids and Reference Compounds
The antiproliferative effect of the novel vindoline-containing hybrids was initially tested on MRC-5 cells (non-cancerous human embryonal lung fibroblasts) to obtain results concerning the cytotoxicity of the tested molecules.The anticancer properties of the substances were characterized utilizing MDA-MB-231 (breast adenocarcinoma), HeLa (human cervical cancer), A2780 (ovarian cancer), and SH-SY5Y (neuroblastoma) cells.Two concentrations, 10 and 30 µM, were applied at the initial screening.In the case of the Scheme 3. Synthesis of reference chalcones containing phenyl group in place of vindoline moiety.

In Vitro Antiproliferative Evaluation of the Novel Vindoline Hybrids and Reference Compounds
The antiproliferative effect of the novel vindoline-containing hybrids was initially tested on MRC-5 cells (non-cancerous human embryonal lung fibroblasts) to obtain results concerning the cytotoxicity of the tested molecules.The anticancer properties of the substances were characterized utilizing MDA-MB-231 (breast adenocarcinoma), HeLa (human cervical cancer), A2780 (ovarian cancer), and SH-SY5Y (neuroblastoma) cells.Two concentrations, 10 and 30 µM, were applied at the initial screening.In the case of the most potent analogs, i.e., when the cell growth inhibition was higher than 50% at 10 µM on any cancer cell line, the assay was repeated with a set of dilutions to determine the IC 50 values.The results of the growth inhibition screening and the IC 50 values of the most potent compounds are listed in Tables 1 and 2, respectively.The percentage of cell growth inhibition caused by vindoline (1b) and the newly synthesized hybrids (21-24, 28, 29, 36, 37, 36a, and 37a) are listed in Table 1.It can be seen that vindoline extended with pharmacophore units shows some cytotoxic effects.Of the hybrids containing imatinib fragments (21-23), component 22 showed cell division inhibition above 85% with three cell lines (MDA-MB-231, A2780, SH-SY5Y) at a concentration of 30 µM.The ferrocene-containing compounds (28 and 29) did not elicit substantial cell division inhibitory effects at a concentration of 30 µM.
Trimethoxyphenyl derivative 36 was identified as the most potent antiproliferative agent (IC 50 = 0.6-2.55µM), especially against the A2780 cell line.A comparison of the effects of 36 and its simplified analog 36a led to the conclusion that the contribution of the vindoline residue to the antiproliferative effect on malignant cells is 3-5-fold more substantial than that of the phenyl group (Table 2).These most promising compounds were comparable to the reference agent cisplatin.Though the IC 50 values measured with the malignant and MRC-5 cells show comparable therapeutic windows for 36 and 36a, the latter seems less toxic against non-cancerous cells.However, it must be emphasized that based on the ratio of the IC 50 values (MRC-5/A2780 = 4.25 and 3.35 for 36 and 36a, respectively), A2780 ovarian cancer cells are particularly susceptible to these novel agents.The exact identification of the potential therapeutic target requires further investigation, including in vivo experiments; however, at this stage of our research in which we are collecting data for establishing SAR followed by performing mechanistic studies and designing more potent drug candidates, it can only be assumed that hybrid 36 exerts its effect by dual-targeting the vinca and colchicine sites of tubulin heterodimers, as supported by the following facts.In the vinblastine molecule, the vindoline domain was found to bind to the vinca site [65], and the trimethoxyphenyl group is a typical fragment present in a large number of tubuline-targeting anticancer agents, including a variety of chalcones and combretastatin analogs, such as the FDA-approved drug fosbretabulin, all of which bind to the colchicine site [66].The concept of simultaneously targeting two binding sites of microtubules was also exploited by Passarella et al., who synthesized hybrids comprising vinca alkaloids and other documented polymerization inhibitors [67].The hybrids containing vindoline tethered at position 17 to thiocolchicine via diacyl spacers of different lengths demonstrated the significant inhibition of tubulin polymerization and antiproliferative activity on A549 lung cancer cells [67].Finally, our view about the possible molecular targets of 36 is in good accordance with the significantly reduced effect produced by 37 in which the trimethoxyphenyl group is replaced by three-dimensional ferrocene.However, the IC 50 values produced by 36a and 37a indicate that the same structural modification caused a smaller decrease in the effect of phenylacetylene-derived hybrids than in the vindoline-containing analogs.Finally, it must be pointed out that our lead compound 36 proved to be superior to cisplatin with each investigated cell line in terms of both the antiproliferative effect and selectivity, as indicated by the data listed in Table 2.

Materials and Methods
All chemicals were obtained from commercially available sources (Merck, Budapest, Hungary; Fluorochem, Headfield, UK; Molar Chemicals, Halásztelek, Hungary; VWR, Debrecen, Hungary) and used without further purifications.Equipment from Merck Kieselgel (230-400 mesh, 60 Å) was used for flash column chromatography.Melting points (uncorrected) were determined with a Büchi M-560.The 1 H-and 13 C-NMR spectra were recorded in DMSO-d 6 solution in 5 mm tubes at room temperature on a Bruker DRX-500 spectrometer (Bruker Biospin, Karlsruhe, Baden Württemberg, Germany) at 500 ( 1 H) and 125 ( 13 C) MHz, with the deuterium signal of the solvent as the lock and TMS as internal standard ( 1 H and 13 C).The 2D-HSQC-, HMBC-, and NOESY spectra, which support the exact assignments of 1 H-and 13 C NMR signals, were measured by using the standard Bruker pulse programs.Exact mass measurements were performed on a high-resolution Waters ACQUITY RDa Detector (Waters Corp., Wilmslow, UK) equipped with an electrospray ionization source using on-line UHPLC coupling.UHPLC separation was performed on a Waters ACQUITY UPLC H-Class PLUS system using a Waters Acquity UPLC BEH C18 column (2.1 × 150 mm, 1.7 µm).Samples were dissolved in MeOH/Water 5:95 v/v, and 5-5 µL sample solutions were injected.Linear gradient elution (0 min 5% B, 1.0 min 5% B, 7.0 min 80% B, 7.1 min 100% B, 8.0 min 100% B, 8.1 min 5% B, 12.0 min 5% B) with eluent A (0.1% formic acid in water, v/v) and eluent B (0.1% formic acid in Methanol, v/v) was used at a flow rate of 0.200 mL/min at 45 • C column temperature.High-resolution mass spectra were acquired in the m/z 50-2000 range in the positive ionization mode.Leucine enkephalin peptide was used for single lock mass calibration correction.
For each compound characterized in this study, the numbering of atoms used for the assignment of 1 H-and 13 C-NMR signals do not correspond to the IUPAC rules reflected in the given systematic names.Imatinib fragments (7-9, 12-14, 17, and 18) were synthesized by reported procedures [62].N-Ferrocenoylimidazole (26) was prepared using the method reported by Imrie et al. [63].
Copies of the NMR spectra of the novel propargylated pharmacophore fragments (S2-S5), the novel iodinated chalcones (S6-S7) and the novel alkyne-tethered vindoline hybrids (S8-S16) along with the copies of the HRMS spectra of the novel alkyne-tethered vindoline hybrids (S17-S20) are included in the Supplementary Materials.18) (2 mmol) was dissolved in MeCN (20 mL), then propargylamine (1.27 mL, 1.10 g, 20 mmol) was added dropwise to the solution.The obtained mixture was stirred at reflux temperature for 12 h and concentrated in vacuo.The residue was purified by column chromatography on silica using solvent mixture DCM:MeOH (15:1) as eluent, followed by crystallization with Et 2 O to obtain the pure product (10,19).(10) standard Bruker pulse programs.Exact mass measurements were performed on a high-resolution Waters ACQUITY RDa Detector (Waters Corp., Wilmslow, UK) equipped with an electrospray ionization source using on-line UHPLC coupling.UHPLC separation was performed on a Waters ACQUITY UPLC H-Class PLUS system using a Waters Acquity UPLC BEH C18 column (2.1 × 150 mm, 1.7 µm).Samples were dissolved in MeOH/Water 5:95 v/v, and 5-5 µL sample solutions were injected.Linear gradient elution (0 min 5% B, 1.0 min 5% B, 7.0 min 80% B, 7.1 min 100% B, 8.0 min 100% B, 8.1 min 5% B, 12.0 min 5% B) with eluent A (0.1% formic acid in water, v/v) and eluent B (0.1% formic acid in Methanol, v/v) was used at a flow rate of 0.200 mL/min at 45 °C column temperature.High-resolution mass spectra were acquired in the m/z 50-2000 range in the positive ionization mode.Leucine enkephalin peptide was used for single lock mass calibration correction.
For each compound characterized in this study, the numbering of atoms used for the assignment of 1 H-and 13 C-NMR signals do not correspond to the IUPAC rules reflected in the given systematic names.Imatinib fragments (7-9, 12-14, 17, and 18) were synthesized by reported procedures [62].N-Ferrocenoylimidazole (26) was prepared using the method reported by Imrie et al. [63].
Copies of the NMR spectra of the novel propargylated pharmacophore fragments (S2-S5), the novel iodinated chalcones (S6-S7) and the novel alkyne-tethered vindoline hybrids (S8-S16) along with the copies of the HRMS spectra of the novel alkyne-tethered vindoline hybrids (S17-S20) are included in the Supplementary Materials.

Synthesis of N-Propargylferrocenecarboxamide (27)
Propargylamine (0.32 mL, 0.275 g, 5 mmol), ferrocenoylimidazolide 26 (1.704 g; 6 mmol, 1.2 eq.), and DMAP (0.184 g; 6 mmol, 1.2 eq.) were dissolved in freshly distilled pyridine (15 mL).This reaction mixture was purged with argon and stirred for 12 h at room temperature, then poured onto crushed ice.The resulting suspension was extracted with DCM (5 × 20 mL).The combined organic layers were washed with brine solution, dried over anhydrous Na 2 SO 4 , and evaporated to dryness on a rotary evaporator.The dark solid residue was purified by column chromatography on silica using solvent mixture DCM:MeOH (20:1) as eluent, followed by sequential crystallization with water and Et 2 O to obtain the pure product as a light orange solid.Yield: 1.00 g (75%).
room temperature, then poured onto crushed ice.The resulting suspension was extracted with DCM (5 × 20 mL).The combined organic layers were washed with brine solution, dried over anhydrous Na2SO4, and evaporated to dryness on a rotary evaporator.The dark solid residue was purified by column chromatography on silica using solvent mixture DCM:MeOH (20:1) as eluent, followed by sequential crystallization with water and Et2O to obtain the pure product as a light orange solid.Yield: 1.00 g (75%).

Synthesis of Iodochalcone Intermediates 34 and 35
The corresponding methyl ketone 32 or 33 (1 mmol) and 4-iodobenzaldehyde 31 (232 mg, 1 mmol) were dissolved in EtOH (3 mL).To this solution, 2% NaOH/H2O (2 mL) was added, and the resulting mixture was stirred for 12 h at room temperature under argon atmosphere.The precipitated crystals were filtered out and first purified by column chromatography on silica using solvent mixture DCM:MeOH (40:1) as eluent and crystallized from EtOH to obtain the pure product.

Synthesis of Iodochalcone Intermediates 34 and 35
The corresponding methyl ketone 32 or 33 (1 mmol) and 4-iodobenzaldehyde 31 (232 mg, 1 mmol) were dissolved in EtOH (3 mL).To this solution, 2% NaOH/H 2 O (2 mL) was added, and the resulting mixture was stirred for 12 h at room temperature under argon atmosphere.The precipitated crystals were filtered out and first purified by column chromatography on silica using solvent mixture DCM:MeOH (40:1) as eluent and crystallized from EtOH to obtain the pure product.room temperature, then poured onto crushed ice.The resulting suspension was extracted with DCM (5 × 20 mL).The combined organic layers were washed with brine solution, dried over anhydrous Na2SO4, and evaporated to dryness on a rotary evaporator.The dark solid residue was purified by column chromatography on silica using solvent mixture DCM:MeOH (20:1) as eluent, followed by sequential crystallization with water and Et2O to obtain the pure product as a light orange solid.Yield: 1.00 g (75%).

Determination of Antiproliferative Activities
The antiproliferative properties of a selected set of the prepared vindoline analogs

Determination of Antiproliferative Activities
The antiproliferative properties of a selected set of the prepared vindoline analogs were determined by the standard MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay using MDA-MB-231, HeLa, A2780, and SH-SY5Y cell lines isolated from human breast, cervical, ovarian cancer, and neuroblastoma, respectively [68].Non-cancerous human fibroblast MRC-5 cells were additionally used to characterize the cancer selectivity of the active tested agents.Cells were obtained from the European Collection of Cell Cultures (Salisbury, UK) and maintained in Eagle's minimal essential medium (EMEM) supplemented with 10% fetal calf serum, 1% non-essential amino acids, and 1% antibioticantimycotic at 37 • C in a humidified atmosphere with 5% CO 2 .All media and supplements were purchased from Capricorn Scientific Ltd. (Ebsdorfergrund, Germany).Cells were seeded onto 96-well plates (5000/well), and after overnight incubation, the tested substances were added at two concentrations (10 or 30 µM).After incubation for 72 h under cell-culturing conditions, the MTT solution was added (20 µL of 5 mg/mL per well), and the medium was removed after 4 h.The generated formazan crystals were solubilized in 100 µM dimethylsulfoxide, and the absorbance was measured at 545 nm using a microplate reader (BMG Labtech, Ortenberg, Germany).Background-corrected values were used for further calculations.In the case of the test substances exhibiting higher than 50% growth inhibition at 10 µM on any cancer cell lines, the assays were repeated using a set of dilutions to obtain IC 50 values.In the same cases, the compounds were tested against MRC-5 fibroblasts to characterize their cancer selectivity.Two independent experiments were performed with five parallel wells.Cisplatin (Ebewe Pharma GmbH, Unterach, Austria) was included as an additional reference agent.The IC 50 values were calculated by fitting sigmoid concentration-response curves using GraphPad Prism 10.0 software (GraphPad Software, San Diego, CA, USA).

Conclusions
This contribution presents feasible synthetic pathways for the synthesis of the first representatives of alkyne-tethered vindoline-based hybrids as potential anticancer agents.The antiproliferative assays identified a trimethoxyphenyl-containing chalcone-vindoline hybrid (36) as a highly efficient and selective lead compound featuring a wide therapeutic window determined by its submicromolar activity against A2780 cells and a substantially decreased activity against MRC-5 fibroblast cells.Consequently, ovarian cancer might be considered a prioritized target of treatment with 36, which merits more extended investigation to disclose its cellular targets and mechanism of action, paving the way for developing rationally designed follow-up molecules with enhanced potency in clinical applications.

nitrophenyl)guanidine nitrate 12 ,
previously generated from nitroaniline 11.The catalytic hydrogenation of the resulting nitroaryl derivative 13 gave aniline 14 as a nucleophilic key intermediate, the N-alkylation of which with propargyl bromide conducted under standard conditions led to the formation of the next imatinib-based terminal alkyne 15, featuring a more extended structural motif compared to the biaryl-type intermediate 10.Finally, the reaction pathway starting with the double chlorination of 4-(hydroxymethyl)benzoic acid (16 → 17) followed by N-acylation (14 + 17 → 18) and the sequential N-propargylation of the benzyl chloride-type intermediate 18 created 19, the most complex propargylated fragment comprising the majority of the structural motifs of imatinib.

a
Selectivity indices (IC 50 on MRC-5/IC 50 on the cancer cell).b Reference drug.Values are from reference[64] except for unpublished IC 50 measured for SH-SY5Y.

Author Contributions:
Conceptualization, A.C. and L.H.; methodology, E.F. and P.K.; validation, G.S. and I.Z.; formal analysis, G.S.; investigation, E.F., B.A.T., R.M., D.P. and A.C.; resources, L.H., I.Z.and A.C.; data curation, E.F.; writing-original draft preparation, E.F.; writing-review and editing, I.Z.and A.C.; visualization, A.C.; supervision, I.Z.; project administration, E.F.; funding acquisition, L.H., I.Z.and A.C.All authors have read and agreed to the published version of the manuscript.Funding: This work was funded by the Hungarian Scientific Research Fund [OTKA K_129037], ELTE Thematic Excellence Programme Synth+, supported by the Hungarian Ministry for Innovation and Technology and by the Ministry of Innovation and Technology of Hungary from the National Research, Development and Innovation Fund [TKP2021-EGA-32].This work has also been connected to project no.RRF-2.3.1-21-2022-00015 with support provided by the European Union (Széchenyi Plan Plus, National Laboratory Program, PharmaLab).Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.

Table 1 .
Initial screening of the antiproliferative effect of the alkyne-tethered vindoline hybrids and reference compounds, including vindoline 1b, measured using the investigated cells.

Table 2 .
IC 50 values obtained for the most active compounds identified in the initial viability tests.