Novel Piperazine Derivatives of Vindoline as Anticancer Agents

A series of novel vindoline–piperazine conjugates were synthesized by coupling 6 N-substituted piperazine pharmacophores at positions 10 and 17 of Vinca alkaloid monomer vindoline through different types of linkers. The in vitro antiproliferative activity of the 17 new conjugates was investigated on 60 human tumor cell lines (NCI60). Nine compounds presented significant antiproliferative effects. The most potent derivatives showed low micromolar growth inhibition (GI50) values against most of the cell lines. Among them, conjugates containing [4-(trifluoromethyl)benzyl]piperazine (23) and 1-bis(4-fluorophenyl)methyl piperazine (25) in position 17 of vindoline were outstanding. The first one was the most effective on the breast cancer MDA-MB-468 cell line (GI50 = 1.00 μM), while the second one was the most effective on the non-small cell lung cancer cell line HOP-92 (GI50 = 1.35 μM). The CellTiter-Glo Luminescent Cell Viability Assay was performed with conjugates 20, 23, and 25 on non-tumor Chinese hamster ovary (CHO) cells to determine the selectivity of the conjugates for cancer cells. These compounds exhibited promising selectivity with estimated half-maximal inhibitory concentration (IC50) values of 2.54 μM, 10.8 μM, and 6.64 μM, respectively. The obtained results may have an impact on the design of novel vindoline-based anticancer compounds.

Piperazine (2), as a privileged structure, is found in many drugs [14] and natural compounds [15].It is especially used in pharmaceutical research due to its excellent physicochemical characteristics; it provides beneficial pharmacodynamic and pharmacokinetic effects (e.g., solubility, bioavailability, etc.) to the molecule to which it has been coupled [16,17].
In summary, the aim of this work was to combine a Vinca alkaloid with one of the Based on these results and the literature data, we continued our project, during which we coupled various N-substituted piperazines to position 17 of vindoline (1) via two types of linkers.A more flexible linker was formed using 4-bromobutyric acid, while a slightly more rigid linker was formed with succinic anhydride.To study the structure-activity relationships, the piperazine derivatives were also attached to position 10 of vindoline (1); in this case, the linker was built with chloroacetyl chloride.The piperazine pharmacophores planned for coupling were selected based on the work of ˙Ibiş et al. [16].In this project, piperazine-oxazole hybrids were produced, which demonstrated remarkable cytotoxicity on all examined cell lines with IC 50 values in the range of 0.09-11.7 µM.We chose the following six cheap and easily available piperazine derivatives: 1-methylpiperazine (5), 1-(4-trifluoromethylphenyl)piperazine ( 6), 1-[4-(trifluoromethyl)benzyl]piperazine (7), 1-(4-fluorobenzyl)piperazine ( 8), 1-bis(4-fluorophenyl)methyl piperazine (9), and 1-(2furoyl)piperazine (10) (Figure 2).
In summary, the aim of this work was to combine a Vinca alkaloid with one of the most frequent pharmacophore molecules, which is used in a wide range of fields of biological action.It was presumed that even the ineffective vindoline could possess important antitumor activity when connected with piperazines.Moreover, we had another goal, namely, the investigation of the cytotoxic activity of the new compounds not only on cancerous cells but also on non-tumor cells to determine the selectivity.biological action.It was presumed that even the ineffective vindoline could possess important antitumor activity when connected with piperazines.Moreover, we had another goal, namely, the investigation of the cytotoxic activity of the new compounds not only on cancerous cells but also on non-tumor cells to determine the selectivity.

Preparation of the Linker-Containing Vindoline Derivatives
The substitution possibilities for forming linkers on the vindoline skeleton are primarily positions 10 and 17 (Scheme 1).The linkers were built according to known procedures.
The synthesis of 10-chloroacetamidovindoline (12) was previously presented by us [26] through the N-acylation reaction of 10-aminovindoline (11) with chloroacetyl chloride.Although a three-step synthesis of 10-aminovindoline (11) was also described by our research group, in this project, a simpler and shorter synthetic procedure was applied [26].The nitrosation of vindoline (1) with sodium nitrite in an acidic medium and the following reduction by sodium borohydride resulted in the desired amino derivative (11) in a better overall yield (92%) than the previously described process [27].

Chemistry 2.1.1. Preparation of the Linker-Containing Vindoline Derivatives
The substitution possibilities for forming linkers on the vindoline skeleton are primarily positions 10 and 17 (Scheme 1).The linkers were built according to known procedures.12) was reacted with the corresponding piperazine derivative (5-10) (Figure 2) in acetonitrile solution in the presence of potassium carbonate (Scheme 2) in N-alkylation reactions, and this resulted in the 16-21 vindoline-piperazine conjugates in medium to excellent yields.The synthesis of 10-chloroacetamidovindoline (12) was previously presented by us [26] through the N-acylation reaction of 10-aminovindoline (11) with chloroacetyl chloride.Although a three-step synthesis of 10-aminovindoline (11) was also described by our research group, in this project, a simpler and shorter synthetic procedure was applied [26].The nitrosation of vindoline (1) with sodium nitrite in an acidic medium and the following reduction by sodium borohydride resulted in the desired amino derivative (11) in a better overall yield (92%) than the previously described process [27].

Coupling of the Linker-Containing Vindoline Derivatives With Piperazines
10-Chloroacetamidovindoline ( 12) was reacted with the corresponding piperazine derivative (5-10) (Figure 2) in acetonitrile solution in the presence of potassium carbonate (Scheme 2) in N-alkylation reactions, and this resulted in the 16-21 vindoline-piperazine conjugates in medium to excellent yields.
Similarly, compounds 3 and 22-26 were prepared from the 17-O-4-bromobutanoyl derivative ( 14) with the given piperazines (5-10) (Scheme 3) using two methods, namely, with triethyl amine in dichloromethane solution (Method A) and in acetonitrile in the presence of potassium carbonate (Method B).Method B was used in cases where conversion was low with Method A. As mentioned, compound 3 had already been synthesized before by us [25]; however, now it was obtained with a slightly better yield.The latter was also necessary because we planned to subject it to a more extensive biological screening (NCI60) than before.Similarly, compounds 3 and 22-26 were prepared from the 17-O-4-bromobutanoyl derivative (14) with the given piperazines (5-10) (Scheme 3) using two methods, namely, with triethyl amine in dichloromethane solution (Method A) and in acetonitrile in the presence of potassium carbonate (Method B).Method B was used in cases where conversion was low with Method A. As mentioned, compound 3 had already been synthesized before by us [25]; however, now it was obtained with a slightly better yield.The latter was also necessary because we planned to subject it to a more extensive biological screening (NCI60) than before.
with triethyl amine in dichloromethane solution (Method A) and in acetonitrile in the presence of potassium carbonate (Method B).Method B was used in cases where conversion was low with Method A. As mentioned, compound 3 had already been synthesized before by us [25]; however, now it was obtained with a slightly better yield.The latter was also necessary because we planned to subject it to a more extensive biological screening (NCI60) than before.Thus, since the same six piperazine derivatives (5-10) were coupled to different sites of vindoline, these piperazine conjugates through various types of linkers make a preliminary study of the structure-activity relationship in connection with the anticancer activities possible.Thus, since the same six piperazine derivatives (5-10) were coupled to different sites of vindoline, these piperazine conjugates through various types of linkers make a preliminary study of the structure-activity relationship in connection with the anticancer activities possible.
The screening results are given in the Supplementary Materials (Tables S1-S3), where the biological activities were determined for the 10 −5 M concentration.The percentages of growths show the amount of living cancer cells compared to a reference.The negative numbers indicate a significant decrease in the cell number.As expected, a notable antiproliferative effect was not shown by vindoline (1), the linker-containing vindoline derivatives (14 and 15), and their precursors (11 and 13), except 10-chloroacetamidovindoline (12), which exerted moderate cytostatic activity.
The experimental data obtained for compounds 16-21 containing the piperazine side chain coupled at position 10 of vindoline (1) are presented in Table S1.One of them, derivative 17, in which the piperazine nitrogen atom contains a 4-trifluoromethylphenyl substituent, proved to be highly effective in several types of cancer.In the case of colon cancer, the growth percent rate was found to be −84.40% on the KM12 cell line.For CNS cancer, more than −80% reduction was obtained on SF-539 and SNB-75 brain tumor cell lines.For melanoma, 17 was very effective on almost all cell lines, and the two most outstanding values were −98.17% (on SK-MEL-5) and −95.37% (on LOX-IMVI).In the case of breast cancer for the cell line MDA-MB-231/ATCC, a −86.10% growth rate was obtained.The 1-bis(4-fluorophenyl)methyl piperazine-containing compound (20) showed moderate antiproliferative activity on some colon, CNS, and melanoma cell lines.
Data of compounds 27-32 coupled with vindoline (1) in position 17 through an amide bond are shown in Table S3.The activity of compound 28 containing the 4trifluoromethylphenyl substituent is also noteworthy in this context.Similar to 1-[4-(trifluoromethyl)benzyl]piperazine-containing derivative 29, compound 28 is highly effective and rather selective in the cases of colon cancer (COLO-205, −90.33%) and melanoma (SK-MEL-5, −92.46%).The 1-bis(4-fluorophenyl)methyl piperazine-containing compound 31 should also be mentioned; it proved to be effective on several types of cancer, particularly on the leukemia MOLT-4 cell line (−98.81%).
Since compounds 17, 20, 22-25, 28, 29, and 31 showed significant antiproliferative effects on several cancer cell lines during the one-dose test, they were subjected to a fivedose screening.The 50% growth inhibition (GI 50 ) and their mean values are given in Table 1.Among them, the ([4-(trifluoromethyl)benzyl]piperazine-containing derivative 23 and the (1-bis(4-fluorophenyl)methyl piperazine-containing compound 25 were the most potent agents.The latter two derivatives resulted in less selectivity but outstanding cytotoxic activity with GI 50 < 2 µM on almost all cell lines.The most significant activity was shown by compound 23 on the MDA-MB-468 cell line of breast cancer (GI 50 = 1.00 µM), while compound 25 had the most significant activity on the HOP-92 cell line of non-small cell lung cancer (GI 50 = 1.35 µM).In addition, compounds 22, 28, and 31 exhibited mean GI 50 values below 4 µM.It should also be emphasized that compound 24 had a GI 50 value of 1.00 µM on the renal cancer RXF 393 cell line and that derivative 28 had a GI 50 value of 1.17 µM on the leukemia MOLT-4 cell line.

Effect of Selected Conjugates on Cell Viability of Non-Tumor Chinese Hamster Ovary (CHO) Cell Lines
Three promising conjugates (20,23,25) were selected for testing on the non-tumor CHO cell line in the CellTiter-Glo Luminescent Cell Viability Assay (Promega Corporation, Madison, WI, USA) to reveal their selectivity for cancer cells.Treatment of CHO cells for 48 h with the compounds in the 10 −7 -10 −5 M concentration range resulted in a concentrationdependent decrease in the luminescent signal proportional to the amount of ATP produced by living cells as an indicator of cell viability.Piperazine conjugate treatment did not affect CHO cell viability in 10 −7 and 10 −6 M concentrations, while treatment in 10 −5 M concentrations resulted in significantly decreased cell viability with values of 1.25 ± 0.77%, 52.76 ± 7.25%, and 33.45 ± 19.62% for compounds 20, 23, and 25, respectively (Figure 3).Based on these data, IC50 values of compounds 20, 23, and 25 can be roughly estimated to be 2.54 μM, 10.8 μM, and 6.64 μM, respectively; these results show the promising selectivity of the compounds on tumor cells, as a significant inhibitory effect on non-tumor cell viability is exerted at remarkably higher concentrations compared to the investigated tumor cell lines.

General
All chemicals were purchased from Sigma-Aldrich (Budapest, Hungary) and were used as received.Melting points were measured on a VEB Analytik Dresden PHMK-77/1328 apparatus (Dresden, Germany) and were uncorrected.IR spectra were recorded on Zeiss IR 75 and 80 instruments (Thornwood, NY, USA).NMR measurements were performed on a Bruker Avance III HDX 400 MHz NMR spectrometer equipped with a 31 P-15 N{1H- 19   Based on these data, IC 50 values of compounds 20, 23, and 25 can be roughly estimated to be 2.54 µM, 10.8 µM, and 6.64 µM, respectively; these results show the promising selectivity of the compounds on tumor cells, as a significant inhibitory effect on non-tumor cell viability is exerted at remarkably higher concentrations compared to the investigated tumor cell lines.

General
All chemicals were purchased from Sigma-Aldrich (Budapest, Hungary) and were used as received.Melting points were measured on a VEB Analytik Dresden PHMK-77/1328 apparatus (Dresden, Germany) and were uncorrected.IR spectra were recorded on Zeiss IR 75 and 80 instruments (Thornwood, NY, USA).NMR measurements were performed on a Bruker Avance III HDX 400 MHz NMR spectrometer equipped with a 31 P-15 N{1H-19 F} 5 mm CryoProbe Prodigy BBO probe, a Bruker Avance III HDX 500 MHz NMR spectrometer equipped with a 1 H{ 13 C/ 15 N} 5 mm TCI CryoProbe, a Varian VNMRS 600 MHz NMR System NMR spectrometer, and a Bruker Avance III HDX 800 MHz NMR spectrometer equipped with a 1 H-19 F{ 13 C/ 15 N} 5 mm TCI CryoProbe (Bruker Corporation, Billerica, MA, USA). 1 H And 13 C chemical shifts are given on the delta scale as parts per million (ppm) relative to tetramethyl silane.One-dimensional 1 H, and 13 C spectra and two-dimensional 1 H-1 H COSY, 1 H-1 H NOESY, 1 H-13 C HSQC, and 1 H- 13 C HMBC spectra were acquired using pulse sequences included in the standard spectrometer software package (Bruker TopSpin 3.5, Bruker Corporation).NMR spectra were processed with Bruker TopSpin 3.5 pl 6 (Bruker Corporation, Billerica, MA, USA) and ACD/Spectrus Processor version 2017.1.3(Advanced Chemistry Development, Inc., Toronto, ON, Canada).ESI-HRMS and MS-MS analyses were performed on a Thermo Velos Pro Orbitrap Elite (Thermo Fisher Scientific, Bremen, Germany) system.The ionization method was ESI and operated in positive ion mode.The protonated molecular ion peaks were fragmented by CID (collisioninduced dissociation) at a normalized collision energy of 35-65%.For the CID experiment, helium was used as the collision gas.The samples were dissolved in methanol.EI-HRMS analyses were performed on a Thermo Q Exactive GC Orbitrap (Thermo Fisher Scientific, Bremen, Germany) system.The ionization method was EI and operated in positive ion mode.The electron energy was 70 eV, and the source temperature was set at 250 • C. Data acquisition and analysis were accomplished with Xcalibur software version 4.0 (Thermo Fisher Scientific).The reactions were followed by analytical thin layer chromatography (TLC) on DC-Alufolien Kieselgel 60 F 254 (Merck, Budapest, Hungary) plates.Preparative TLC analyses were performed on silica gel 60 PF 254+366 (Merck) glass plates.Column chromatography was carried out using Silica gel 60 (0.040-0.063 mm) (Merck).

NCI60 Screening
A detailed description of the NCI screening procedures [30][31][32][33][34], including the onedose and five-dose tests, can be found in the Supplementary Materials, on the website of NCI [35], and in our previous work [26].

CellTiter-Glo Luminescent Cell Viability Assay on Non-Tumor CHO Cells
Compounds 20, 23, and 25 were dissolved in DMSO in 10 mM stock solutions and stored frozen until use.CHO cells were cultured in complete Dulbecco's Modified Eagle's Medium (DMEM, low glucose (1 g/L)) supplemented with 10% fetal bovine serum, 1% Gibco GlutaMAX-I (100×) solution, 1% Gibco MEM non-essential amino acid solution (100×), and 0.1% penicillin-streptomycin mixture.Cells were grown in T-25 size cell culture flasks under standard cell culture conditions (37 • C, 5% CO 2 ) and passaged at 70-80% confluency.The assay was performed according to the manufacturer's protocol, as described previously [36][37][38].Briefly, CHO cells were seeded to opaque 96-well culture plates at a density of 5000 cells/100 µL complete DMEM/well.The side rows and columns of the plate were filled with sterile phosphate-buffer saline to avoid an edge effect.Following overnight incubation (37 • C, 5% CO 2 ), the culture medium was aspirated from the cells and replaced with increasing concentrations of drug solutions (10 −5 , 10 −6 , and 10 −7 M) diluted from the stock solution in sterile complete DMEM (100 µL/well).Untreated cells served as a control, and complete DMEM served as a luminescent background control.Drug-treated cells were incubated for 48 h (37 • C, 5% CO 2 ), and then equilibrated to room temperature in 30 min.At the end of the incubation period, 100 µL of room temperature CellTiter-Glo reagent was added to each well, and the plates were placed on an orbital shaker for 2 min and incubated for additional 10 min at room temperature without shaking.The luminescent signal was measured using an EnSpire AlphaLisa microplate reader (Perkin Elmer, Inc., Waltham, MA, USA).The normalized luminescent values of the treated cells were compared to those of the untreated control.The statistical analysis and calculation of IC 50 values were performed in GraphPad Prism 8.0.1 (GraphPad, La Jolla, CA, USA).Estimated IC 50 values were calculated by using non-linear regression by fitting a sigmoidal dose-response curve to the data points.

Conclusions
Monomer Vinca alkaloid vindoline, which does not show any anticancer effect by itself, was coupled via different positions and linkers with N-substituted piperazine derivatives.The latter were chosen because the piperazine skeleton is a well-known pharmacophore, widely used in pharmaceutical research and the field of medicine for different indications.The substituents on the nitrogen atom of piperazines were alkyl, aryl, aralkyl, and heterocyclic groups.The products were prepared using simple, three-step synthetic routes.Among the compounds synthesized, several derivatives presented significant and excellent antiproliferative activity during the in vitro NCI-60 cell line screening, especially the derivatives with N-[4-(trifluoromethyl)benzyl] and N-bis(4-fluorophenyl)methyl substituents on the piperazine ring.Compound 23 was identified as the most potent antitumor candidate, exhibiting a growth inhibition (GI 50 ) value of 1.00 µM on the breast cancer MDA-MB-46 cell line.In addition, several other conjugates showed low micromolar GI 50 values against most of the examined cell lines.It is important to emphasize that a particularly valuable result is the selectivity that the most effective compounds showed on non-tumor cells with compound 23 having a half-maximal effective concentration (EC 50 ) of 10.8 µM.The results obtained in this study are promising for further development; in particular, with the involvement of other piperazines, a more complete SAR could be elaborated.For example, an exciting continuation of the work could be the synthesis and biological evaluation of the N-bis [4-(trifluoromethyl)phenyl]methyl analog by hybridizing the piperazine units of the two most effective derivatives (23 and 25).Furthermore, the elucidation of the mechanism of action of these types of molecules would also be an interesting scope of study.Finally, we would like to highlight that this study may have a significant impact on the design of new Vinca alkaloid-based antitumor agents.

14 2. 2 . 2 .
Int. J. Mol.Sci.2024, 25, 7929 11 of Effect of Selected Conjugates on Cell Viability of Non-Tumor Chinese Hamster Ovary (CHO) Cell Lines Three promising conjugates (20, 23, 25) were selected for testing on the non-tumor CHO cell line in the CellTiter-Glo Luminescent Cell Viability Assay (Promega Corporation, Madison, WI, USA) to reveal their selectivity for cancer cells.Treatment of CHO cells for 48 h with the compounds in the 10 −7 -10 −5 M concentration range resulted in a concentration-dependent decrease in the luminescent signal proportional to the amount of ATP produced by living cells as an indicator of cell viability.Piperazine conjugate treatment did not affect CHO cell viability in 10 −7 and 10 −6 M concentrations, while treatment in 10 −5 M concentrations resulted in significantly decreased cell viability with values of 1.25 ± 0.77 %, 52.76 ± 7.25 %, and 33.45 ± 19.62 % for compounds 20, 23, and 25, respectively (Figure 3).

25 *Figure 3 .
Figure 3.Effect of compounds 20, 23, and 25 on the viability of native CHO cells.The percentage values of viable cells after 48 h treatment are presented as mean ± SD of three independent experiments in technical triplicates (n = 9), One-way ANOVA, Dunnett's post hoc test; * p < 0.0001 vs. control).

Figure 3 .
Figure 3.Effect of compounds 20, 23, and 25 on the viability of native CHO cells.The percentage values of viable cells after 48 h treatment are presented as mean ± SD of three independent experiments in technical triplicates (n = 9), One-way ANOVA, Dunnett's post hoc test; * p < 0.0001 vs. control).