Amino- and polyaminophthalazin-1(2H)-ones: synthesis, coordination properties, and biological activity

Amino- and polyaminophthalazinones were synthesized by the palladium‐catalyzed amination (alkyl- and arylamines, polyamines) of 4-bromophthalazinones in good yields. The coordinating properties of selected aminophthalazinones towards Cu(II) ions were investigated and the participation of the nitrogen atoms in the complexation of the metal ion was shown. A biological screening of the potential cytotoxicity of selected synthesized compounds on HT-29 and PC-3 cell lines, as well as on the L-929 cell line, proved that some amino derivatives of phthalazinone show interesting anticancer activities. The detailed synthesis, spectroscopic data, and biological assays are reported.

The sample was introduced into the ESIMS source by continuous infusion by means of the instrument syringe pump at a rate of 10 μL min −1 . The ESI source was operated at 5.00 kV and the capillary heater was set to 350 °C and the cone voltage within the range 50-150 V. Scanning was performed from m/z = 200 to 1000. Nitrogen (N2) was used as the drying gas and the nebulizer gas. For fragmentation experiments, massselected monoisotopic molecular ions were isolated in the ion trap and collisionally activated using Helium damping gas. All sample solutions were prepared in methanol by Baker.

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The analytical thin layer chromatography tests (TLC) were carried out on Sigma-Aldrich (Supelco) silica gel plates (Kieselgel 60 F254, layer thickness 0.2 mm) and the spots were visualized using a UV lamp. The flash column chromatography purifications were performed on Fluka silica gel (Silica gel 60, 0.040-0.063 mm).
All reactions with organopalladium compounds were performed under an argon atmosphere using standard Schlenk techniques. Toluene and 1,4-dioxane were distilled from sodium benzophenone ketyl prior to use. Commercially available reagents: 2-formylbenzoic acid, hydrazine monohydrate, methyl iodide (MeI), isopropyl Sigma-Aldrich and used without further purification.
Compounds 1, 2, 3a were obtained as described previously and their characterization data were in agreement with already reported analysis [1].
Method A: Analogous as described in [1].

Synthesis of 4-amino-and 4-polyamino-phthalazinones 5, 6
General procedure: The reaction was carried out under an argon atmosphere in an oven-dried sealable

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Cells were maintained in a humidified incubator at 37 °C in a 5% CO2 atmosphere and were regularly screened for mycoplasma contamination. Exponential growth of the cells was controlled by their routine passaging at 90% confluence three times a week using 0.025% trypsin/EDTA.

Cytotoxicity analysis -MTT assay:
The viability of the cells is associated with their mitochondrial integrity and capacity.
For that reason, in the MTT microplate assay with 3-(4,5-dimethylthiazol-2-yl)-2,3diphenyltetrazolium bromide, first described by Mosmann [5], only viable cells with intact metabolism have the ability to incorporate the yellow, water-soluble tetrazolium salt which is subsequently reduced by the mitochondrial enzyme succinate dehydrogenase to the violet-blue formazan compound. After dissolving the formazan crystals in an organic solvent, e.g., dimethyl sulfoxide (DMSO), the absorbance of the formazan solution can be measured spectrophotometrically at 570 nm [6].
In our research, the MTT assay was used to describe the effect of the tested compounds on cytotoxicity, proliferation and growth of HT-29, PC-3 and L-929 cells.
Before treatments with the tested compounds the suspension of 8×10 3 cells in 100 µL medium was transferred to each well of a standard 96-well microplate. Subsequently, the plates were incubated for 24 h (5% CO2; 37 °C) to ensure cell growth.
Afterwards, the examined substances at 10 different concentrations were added in 2-

Experimental
Methanolic solutions of the ligands 5i or 6d or 7 and CuCl2·2H2O (c = 10 −3 mol/dm 3 each) were freshly prepared prior to analysis. Next, the solutions were diluted with methanol up to 10 −4 mol/dm 3 . The solutions prepared this way were then mixed in 1:1 (v/v, ligand / copper salt) at ambient temperature and analyzed by ESIMS. In case of ligand 6d absence of complex was observed, due to that the sample was further mixed for 24 hours.   S43 Figure S39: 13 C NMR spectrum of 5h. Figure S40: FTIR spectrum of 5h.