Cu(II), Mn(II) and Zn(II) complexes of hydrazones with quaternary ammonium moiety: synthesis, experimental and theoretical characterization and cytotoxic activity

In this paper Cu(II), Mn(II) and Zn(II) complexes with N , N , N -trimethyl-2-oxo-2-(2-(1-(thiazol-2-yl)ethylidene)hydrazinyl)ethan-1-aminium chloride ( HL 1 Cl) were synthesized and characterized by single-crystal X-ray diffraction, IR spectroscopy, elemental analysis and DFT calculations. In all three complexes ligand ( L 1 ) is coordinated in deprotonated formally neutral zwitter-ionic form via NNO donor set atoms. Cu(II) and Zn (II) form mononuclear penta-coordinated complexes [Cu L 1 (N 3 )(CH 3 OH)]BF 4 and [Zn L 1 (N 3 ) 2 ], respectively, while with Mn(II) a binuclear [Mn 2 L 1 2 ( μ -1,1 -N 3 ) 2 (N 3 ) 2 ] ‧ 2CH 3 OH complex, with unusual distorted trigonal-prismatic geometry around metal centers, has been obtained. Antimicrobial activity was tested against a panel of Gram-negative and Gram-positive bacteria, two yeasts and one fungal strain. The binuclear Mn(II) complex showed antifungal activity of similar intensity as amphotericin B. Based on the results of the brine shrimp test and DPPH radical scavenging activity, the most active, Cu(II) and Mn(II) complexes, were selected for evaluation of cytotoxic activity against five malignant cancer cell lines (HeLa, A375, MCF7, PC-3 and A549) and one normal cell line HaCaT. Both complexes showed a significant activity. It should be pointed out that the activity of Mn(II) complex against breast cancer MCF7 cell is only slightly weaker than that of cisplatin, but with selectivity to the tumor cell line in comparison to normal HaCaT cells, which is non-existent in case of cisplatin.

Spectroscopy IR spectra. The IR spectroscopy data confirm that the HL 1 Cl ligand (Fig. S4) is coordinated in a deprotonated form, since the ν(N-H) band at 2955 cm -1 is absent in the spectrum of all complexes 31 . The presence of a medium, sharp peak at 3050 cm -1 in the spectrum of 1 (Fig. S5), and a medium, broad peak at 3388 cm -1 in the spectrum of 2 (Fig. S6) Table S1. The Cu(II) ion has fivefold coordination with in-plane coordinated L 1 through NNO-set of donor atoms and one nitrogen atom (N5) of the azide ligand, while the apical position is occupied by an oxygen atom (O2) from methanol. In general, the distortion in the five-coordinated systems is described by an index of trigonallity  = (−)/60, where  is the greatest basal angle and  is the second greatest angle 32 (Table   S4 and Fig. S1). The counter anion (BF 4 -) mediates in joining the H-bonded dimers into the layers parallel with the (202) lattice plane through intermolecular C-HF hydrogen bonds (Table S4 and Fig. S1a) and assists in connecting the neighboring layers by means of C-HF hydrogen bonds (Table S4 and Fig. S1b). The Cu1Cu1 a (a = 1−x, 1−y, −z) separation of 4.9983 (7)  However, in the crystal structure of 1, the shortest CuCu b (b = −x, 1−y, −z) separation of 3.384 Å has been observed between the Cu(II) ions belonging to the neighboring (202) layers (Fig. S1b).
Crystal structure of complex 2. The crystal structure of 2 displays a centrosymmetric binuclear complex with the asymmetric unit comprising one Mn(II) center, one ligand L 1 , two azide anions (one bridging and one terminal) and one solvent (methanol) molecule. The molecular structure of [Mn 2 L 1 2 (μ-1,1 -N 3 ) 2 (N 3 ) 2 ] with atom numbering scheme is shown in Fig. 2. Selected bond distances and valence angles are given in Table S2. The Mn(II) ion is hexacoordinated with three donor atoms N1, N2 and O1 of ligand L 1 , two nitrogen atoms (N5 and N5 c were c is 1−x, 1−y, 1−z) from bridging azide anions, and one nitrogen atom (N8) from terminal azide anion. The polyhedron around the Mn(II) ion is described as a distorted trigonal prism (TPR-6) with the twist angle  of 14.19 (mean value) being calculated applying the method 1 reported in Ref. 36 (Table S6 and Fig. S3).    Table 1). The results reveal that thermodynamically most favorable  Table 1) and formation of six-coordinate complexes (reactions 11 and 12, Table 1). The calculations disclose that [ZnL 1 (N 3 ) 2 ] complex (complex 3) is thermodynamically preferred in DMSO solution.  Reaction

Antimicrobial activity
The antibacterial activity of the synthesized complexes, their precursors HL 1 Cl, NaN 3 and appropriate salts was evaluated against a panel of five Gram-positive and five Gram-negative bacteria. The MIC data are given in Table 2. All three complexes showed antibacterial activity against all tested bacterial strains. For complexes 1 and 2, precursor compounds either do not have or show low antibacterial activity. The most active complex 2 is also the only binuclear complex in the series. Its activity towards P. aeruginosa is over twice lower than the activity of chloramphenicol, while against P. hauseri, the activity was four times lower than the control compound. Complex 1 displayed the best activity towards E. coli strain and very weak selectivity towards Gram-negative bacteria. The lowest antibacterial activity was obtained for complex 3. In some cases, its activity was lower than that of the parent salt. A comparison of antimicrobial activity of binuclear azido bridged complexes of Mn(II) (complex 2) and Ni(II) with the same ligand system 42 , showed that complex 2 has higher antimicrobial activity. Even with this slightly lower activity, binuclear Ni(II) complex in most cases has higher activity than of mononuclear Cu(II) and Zn(II) complexes. Bearing in mind these two facts, the reason for higher antibacterial activity in the case of bimetallic Mn(II) complex can be explained by the existence of two metal centers.
The antifungal activity of the tested compounds is given in Table 3. All tested complexes showed a very good activity towards A. braziliensis and S. cerevisiae, and the strongest activity against these strains was displayed by binuclear complex 2. Its activity (MIC 0.09 mM) is comparable to the control compound amphotericin B. Against C. albicans, all three complexes showed moderate activity.

Assessment of toxicity and radical scavenging activity
The obtained results of the assessment of the toxicity compounds against freshly hatched nauplii Artemia salina as well as radical scavenging activity are given in Table 4. All synthesized complexes manifested moderate toxicity, compound 2 exhibiting the highest one. A possible interpretation of this result could be based on its good antibacterial activity. Since the nauplii live in symbiosis with some bacterial strains, it would be reasonable to assume that complex 2 exhibits its toxicity in this way.
Radical scavenging activity was determined by the DPPH test. Complex 1 showed the best activity. This is in line with the structure of the complexes. Namely, the central ion of complex 1 is redox-active Cu 2+ . The radical scavenging activity of complex 1 is comparable to that of ascorbic acid.

Cytotoxic activities of Cu(II) and Mn(II)complexes
Examination of cytotoxic activities of Cu(II) complex (1), Mn(II) complex (2), Zn(II) complex (3) and their precursor compounds against human cancer cell lines and normal keratinocyte cell line is presented in Table 5. All three complexes showed concentration-dependent cytotoxic effects on

X-ray crystallography
Crystal data and refinement parameters of compounds 1-3 are listed in Table 6. Single crystal Xray diffraction data were collected at room temperature on an Agilent SuperNova dual-source diffractometer with an Atlas detector equipped with mirror-monochromated Mo-K α radiation (λ = 0.71073 Å). Data processing was performed with CrysAlis PRO 42 . The structures were solved with Olex software 43 using SHELXT 44 or SHELXS 45 and refined by a full-matrix least-squares based on F 2 using SHELXL 46 where n is the number of reflections and p is the total number of parameters refined.

Computational Details
All DFT calculations were done with the ADF program package (version 2017) 49

Brine shrimp assay
About 20 g of commercially purchased lyophilized eggs of Artemia salina was added to 0.5 L of tap water, and air was passed through the suspension by a pump under illumination for 48 h. All tested compounds were dissolved in DMSO and various amounts (0.01-1 mg) were added to 950 μL of artificial seawater with freshly hatched nauplii. After 24 h illumination at room temperature, the number of dead and surviving nauplii were counted and statistically analyzed. LC 50 was defined as a concentration of compounds that caused the death of 50% of the nauplii. All samples were done in triplicate.

DPPH radical scavenging activity
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was determined by the method of Blois 79 . Commercially available free radical DPPH was dissolved in methanol at concentration of 6.58×10 -5 M, while tested compounds were dissolved in DMSO. Into a 96-well microplate, 50 μL solutions of the tested compounds at concentrations range 10 to 0.02 mg/mL were loaded (50 μL DMSO in the control), and 100 μL of DPPH solution were added. After incubation for 30 min at room temperature in the dark, the absorbance was measured at 517 nm.
All the measurements were performed in triplicate and the scavenging activity of the tested derivatives was calculated as: Scavenging activity (%) = 100 × (A control -(A sample -A 0 )) / A control where A control and A sample refer to the absorbance of DPPH in control solution and sample, respectively, while A 0 refers to the absorbance of the solutions of compounds, because of their color.
The IC 50 was defined as the antioxidant concentration necessary to decrease the amount of the initial DPPH radical by 50 % and was calculated from the plotted graph of scavenging activities against the concentrations of the tested compounds. Ascorbic acid was employed as the positive control (concentrations from 50 to 500 μg mL -1 ).

Determination of cytotoxic activity
The cytotoxic activity of newly synthesized Cu(II) and Mn(II) complexes and their precursor compounds was examined on five human cancer cell lines: cervical adenocarcinoma HeLa, melanoma A375, lung carcinoma A549, prostate adenocarcinoma PC-3, breast adenocarcinoma

Conflicts of interest
There are no conflicts to declare.