Data for the cytotoxicity, self-assembling properties and synthesis of 4-pyridinium-1,4-dihydropyridines

In this data file the synthetic procedures for preparation of the original 4-pyridinium-1,4-dihydropyridines (4-Py-1,4-DHP) and their parent compounds – dialkyl 2,6-dimethyl-4-(3-pyridyl)-1,4-dihydropyridine-3,5-dicarboxylates were described. In total, 5 unpublished compounds were obtained and characterised. All the structures of original compounds were confirmed by Nuclear Magnetic Resonance (NMR, including 1H NMR and 13C NMR) and low resolution mass spectra (MS) data. Additionally, the cytotoxic properties of four 4-Py-1,4-DHPs were evaluated on 3 cell lines – normal NIH3T3 (mouse embryonic fibroblast), cancerous HT-1080 (human lung fibrosarcoma) and MH-22A (mouse hepatoma) and self-assembling properties were studied and characterisation of formed nanoparticles were performed using dynamic light scattering technique. In this article provided data are directly related to the previously published research articles – “Novel cationic amphiphilic 1,4-dihydropyridine derivatives for DNA delivery” [1] where compound 5 was tested as gene delivery agent without full physico-chemical characterisation and “Synthesis and studies of calcium channel blocking and antioxidant activities of novel 4-pyridinium and/or N-propargyl substituted 1,4-dihydropyridine derivatives” [2] where synthesis and physico-chemical characterisation as well as calcium channel blocking and antioxidant activities were described for compound 6. Synthesis of other compounds – parent 1,4-DHPs 1 and 2, and 4-Py-1,4-DHPs 3–5, their characterisation, estimation of cytotoxicity and self-assembling properties for all 4-Py-1,4-DHPs 3–6 are reported herein for the first time. Information provided in this data file can be used in medicinal chemistry by other scientists to estimate structure-activity relationships for the analysis and construction of various cationic 1,4-dihydropyridine derivatives and related heterocycles.

tested as gene delivery agent without full physico-chemical characterisation and "Synthesis and studies of calcium channel blocking and antioxidant activities of novel 4-pyridinium and/or N-propargyl substituted 1,4-dihydropyridine derivatives" [2] where synthesis and physico-chemical characterisation as well as calcium channel blocking and antioxidant activities were described for compound 6. Synthesis of other compounds -parent 1,4-DHPs 1 and 2, and 4-Py-1,4-DHPs 3-5, their characterisation, estimation of cytotoxicity and selfassembling properties for all 4-Py-1,4-DHPs 3-6 are reported herein for the first time. Information provided in this data file can be used in medicinal chemistry by other scientists to estimate structure-activity relationships for the analysis and construction of various cationic 1,4-dihydropyridine derivatives and related heterocycles.

Value of the Data
• The data contain the synthetic procedures for preparation of dialkyl 2,6-dimethyl-4-(3pyridyl)-1,4-dihydropyridine-3,5-dicarboxylates with following quaternisation of pyridine moiety giving 4-pyridinium-1,4-dihydropyridines which may serve as valuable guidance for other organic chemists. • The data provide characterisation of physico-chemical properties of original compoundsparent 4-pyridine-1,4-DHPs and quaternised pyridine moiety containing 1,4-DHPs which have not been reported before. • Moreover, the described synthetic procedures and obtained spectral data will be useful for preparation and structure elucidation of representatives in related heterocycles. • Besides the estimated cytotoxicity and basal toxicity data of quaternised pyridine moiety containing 1,4-DHPs may be used for studies and definitions of structure-activity relationships. • Additionally, self-assembling properties of quaternised pyridine moiety containing 1,4-DHPs were estimated and obtained nanoparticles characterised by dynamic light scattering measurements. These data may be respected for the design and development of delivery systems.

Data Description
As a part of our research topic towards the development of novel pyridinium moieties containing biologically active compounds, we continue studies on original single-charged cationic lipid-like compounds on the 1,4-DHP core. Synthetic procedure and the structures of 1,4-DHP derivatives with quaternised pyridine moiety at the position 4 were depicted in Fig. 1 . According to the literature data 4-phenyl-, 4-pyridyl-and 4-unsubstituted 1,4-DHPs is performed via the classical well-known synthetic procedures and target compounds can be obtained in good yields (78-80%) [3][4] . Also in this case, the parent 4-pyridyl-1,4-DHPs 1 and 2 as intermediates for preparation of target cationic moiety containing 1,4-DHPs were synthesised with high yields, 71% and 75% respectively, via typical Hantzsch method performing the one-pot cyclocondensation of the corresponding acetoacetate, 3-pyridinecarboxaldehyde and ammonium acetate in ethanol under refluxing.
The purities of the studied compounds were at least 97% according to high-performance liquid chromatography (HPLC) data.
Previously it was shown that related compound -4-(N-dodecyl)pyridinium-1,4-DHPs demonstrated the ability to block brain calcium channels and improve memory by enhancing the GABAergic processes [8] . The evaluation of cytotoxicity for all 4-Py-1,4-DHPs 3-6 in vitro was assessed using the MTT {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolinium bromide} and CV {tris(4-(dimethylamino)phenyl)methylium chloride} assays on two monolayer tumour cell lines, namely HT-1080 (human fibrosarcoma) and MH-22A (mouse hepatoma) and also the compound influence on "normal" mouse fibroblasts (NIH3T3) was estimated for the studies of structure-activity relationships. Using an alternative in vitro method it is possible to estimate possible toxicity of new compounds and selected compounds for further study that vastly reducing the number of animal experiments. The results are presented in Table 1 .
IC 50 is a quantitative measure of the compound concentration (μg/ml), at which 50% of the cells die. CV is a triarylmethane dye that can bind to ribose type molecules such as DNA in nuclei. CV staining can be used to quantify the total DNA of the remaining population and thus is used to determine the number of live cells based on the concentration of the dye which remains after staining. MTT is a standard colorimetric assay used to measure a cellular proliferation. Yellow MTT is reduced to purple formazan in the mitochondria of living cells.
Dynamic light scattering (DLS) technique was used for evaluation of self-assembling properties of 4-pyridinium-1,4-DHP derivatives 3-6 and characterisation of obtained nanoparticles,  including determination of average diameter (D av ), zeta-potential (Zeta-pot.) and polydispersity index (PDI). Samples were prepared by the thin-film hydration method. The results are obtained for freshly prepared samples after 24 h storage and their values are presented in Table 2 .
It was demonstrated that 4-pyridinium-1,4-DHPs 5 and 6 formed relatively homogenous nanoparticles, their PDI values are around 0.340 and average particle diameter around 90 and 600 nm, respectively. While other 4-pyridinium-1,4-DHPs 3 and 4 formed very heterogeneous samples with PDI value 1, which means a broad particle size distribution and will not be discussed further here, because these compounds are not perspective for further studies.
For the CV assay, cells were stained with 0.05% crystal violet (Sigma-Aldrich) in 30% methanol for 20 min at room temperature. After incubation the staining solution was removed, the cells were washed for 4 times with water. For dye solubilisation 200 μL a solution of 0.1 M citrate buffer, pH 4.2 with 50% ethanol; 1:1 v/v was added. The absorbence of the solution was measured using an Infinite M10 0 0 Tecan microplate spectrophotometer at a wavelength of 570 nm [11] .

Basal cytotoxicity test
The Neutral Red Uptake (NRU) Assay was performed according to the standard protocol of Stokes [12] modified by NICEATM-ECVAM validation study [13] . The NRU cytotoxicity assay procedure based on the ability of viable cells to incorporate and bind neutral red, a supravital dye.
Balb/c NIH3T3 (Mouse Swiss Albino embryo fibroblast, ATCC® CRL-1658 TM ) cells (90 0 0 cells/well) were placed into 96-well plates for 24 h in Dulbecco's modified Eagle's (DMEM) medium containing 5% fetal bovine serum. Then exposed to the test compound over a range of eight concentrations (10 0 0, 316, 10 0, 31, 10, 3, 1 μg/mL) for 24 h. Untreated cells were used as a control. After 24 h, the medium was removed from all plates. Then, 150 μL of neutral red solution was added (0.05 mg/mL NR in DMEM 24 h pre-incubated at 37 °C and then filtered before use through 0.22 μm syringe filter). Plates were incubated for 3 h and then cells were washed three times with PBS. The dye within viable cells was released by extraction with a mixture of acetic acid, ethanol and water (1:50:49). The absorbence of neutral red was measured using a spectrophotometer multiplate reader (TECAN, Infinite M10 0 0) at 540 nm. The optical density (OD) was calculated using the formula: OD (treated cells) × 100/OD (control cells). The IC 50 values were calculated using the program Graph Pad Prism® 3.0.

Self-assembling properties by dynamic light scattering measurements
Compound samples for characterisation with dynamic light scattering (DLS) were prepared by thin-film hydration method in an aqueous solution at a concentration of 0.05 mM. A certain amount of compound was weighted in a round-bottom flask and dissolved in chloroform; then, the organic solvent was removed in vacuo , and the residue was dried in high vacuo for 2 h. Deionised water was added and samples were prepared by sonication using a bath-type sonicator (Cole Parmer Ultrasonic Cleaner 8891CPX). Samples were sonicated for 60 min at 50 °C.
The DLS measurements of the nanoparticles in an aqueous solution were carried out on a Zetasizer Nano ZSP (Malvern Panalytical Ltd.) instrument with Malvern Instruments Ltd. Software 7.12, using the following specifications -medium: water; refractive index: 1.33; viscosity: 0.8872 cP; temperature: 25 °C; dielectric constant: 78.5; nanoparticles: liposomes; refractive index of materials: 1.60; detection angle: 173 °; wavelength: 633 nm. Data were analysed using the multimodal number distribution software that was included with the instrument.

Statistical analysis
Results are expressed as mean ± standard deviation (SD). All of the biological experiments were performed six times and self-assembling properties by dynamic light scattering measurements three times.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.
Authors are thankful to Dr. N.Makarova for elaboration of the preparation method for acetoacetate 2.

Supplementary Materials
Supplementary material associated with this article can be found in the online version at doi: 10.1016/j.dib.2020.106545 .