Copper(II) Complexes of 5–Fluoro–Salicylaldehyde: Synthesis, Characterization, Antioxidant Properties, Interaction with DNA and Serum Albumins

The synthesis, characterization and biological profile (antioxidant capacity, interaction with calf-thymus DNA and serum albumins) of five neutral copper(II) complexes of 5–fluoro–salicylaldehyde in the absence or presence of the N,N’–donor co–ligands 2,2′–bipyridylamine, 2,9–dimethyl–1,10–phenanthroline, 1,10–phenanthroline and 2,2′–bipyridine are presented herein. The compounds were characterized by physicochemical and spectroscopic techniques. The crystal structures of four complexes were determined by single-crystal X-ray crystallography. The ability of the complexes to scavenge 1,1–diphenyl–picrylhydrazyl and 2,2′–azinobis(3–ethylbenzothiazoline–6–sulfonic acid) radicals and to reduce H2O2 was investigated in order to evaluate their antioxidant activity. The interaction of the compounds with calf-thymus DNA possibly takes place via intercalation as suggested by UV–vis spectroscopy and DNA–viscosity titration studies and via competitive studies with ethidium bromide. The affinity of the complexes with bovine and human serum albumins was examined by fluorescence emission spectroscopy revealing the tight and reversible binding of the complexes with the albumins.


Introduction
Nowadays, there is no doubt that we need new drugs to counter viral pandemics, like Covid-19, super-resistant bacteria and drug-resistance for diseases like cancer [1]. After the FDA approved the medicinal administration of cisplatin in 1978, the scientific community has shown great interest in the study of bioactive metal compounds. In the literature, the main interest is focused on the study of compounds with precious and non-endogenous metals, such as platinum, gold and ruthenium. This approach has some disadvantages like the high cost at the production of the drugs and the severe side-effects of their use. A suggestion to overcome these problems is to try using less expensive endogenous metals like copper [1].

Synthesis and Characterization
All complexes were prepared in high yields in methanolic solutions. Complex 1 was prepared from the reaction of Cu(NO 3 ) 2 ·3H 2 O with deprotonated 5-fluoro-salicylaldehyde Molecules 2022, 27, 8929 3 of 18 in a 1:2 ratio. The reaction of methanolic solutions of Cu(II) salts with deprotonated 5fluoro-salicylaldehyde in the presence of the a-diimines bipyam, neoc, phen or bipy in a 1:1:1 ratio led to the formation of complexes 2-5, respectively. Evidence of the coordination mode of the ligands in the complexes has also arisen from the interpretation of their IR and UV-vis spectra. The crystal structures of complexes 1-4 were further verified by single-crystal X-ray diffraction analysis.
All complexes are soluble in DMF and DMSO, but insoluble in most organic solvents and H 2 O. Molar conductivity measurements have shown that complexes 1-5 are nonelectrolytes in DMSO solution, since the values of the Λ M of the complexes in 1 mM DMSO solution were found in the range 8-12 mho·cm 2 ·mol −1 [27].
The coordination of the ligands to the copper(II) ion may be confirmed by IR spectroscopy. More specifically, the broad band at 3227 cm −1 and the sharp one at 1381 cm −1 , originating from the stretching and the bending vibration, respectively, of the O-H group of free 5-F-saloH, did not appear in the IR spectra of all complexes ( Figure S1), confirming the successful deprotonation of the phenolate group. In addition, the shift of the band at 1271 cm −1 assigned to v(C ar -O hydroxo ) in the spectra of complexes may indicate the binding via the phenolato oxygen to Cu(II). The coordination of the aldehydo oxygen can be confirmed by the shift of the band at 1663 cm −1 to lower wavenumbers. These features reveal the bidentate coordination of the 5-F-salo − ligands to Cu(II) ion. The coexistence and the coordination of the N,N'-donors bipyam, neoc, phen and bipy may be detected by the bands at 755 cm −1 , 732 cm −1 , 722 cm −1 and 767 cm −1 , respectively, which may be attributed to the out-of-plane vibration ρ(C ar -H) that is characteristic for each co-ligand [28]. For complexes 4 and 5, the coordination of the NO 3 − ligand is denoted by the presence of two characteristic vibrations at 1315 cm −1 and 1422-1428 cm −1 which are attributed to the symmetric (v s ) and the asymmetric (v a ) stretching vibration, respectively. The magnitude of the splitting parameter ∆ (∆ = v a − v s ) is~110 cm −1 and is typical of monodentate coordination (M-O-NO 2 ) of nitrato ligands [29]. The suggestions from the IR spectroscopy are in good agreement with the structures determined by X-ray crystallography.
The UV-vis spectra of the complexes were recorded as nujol mull (corresponding to the solid state) and in DMSO ( Figure S2) or buffer solutions used in biological experiments (150 mM NaCl and 15 mM trisodium citrate at pH values regulated in the range 6-8 by HCl solution). The spectra in nujol and DMSO did not show any appreciable differences, suggesting that the complexes keep their structure in solution [17]. In the visible region, one band appeared with λ max in the range 625-750 nm which is typical for geometries expected for tetra-and penta-coordinated copper(II) complexes [30,31].

Description of the Structures of Complexes 2-4
The structures of complexes 2-4 present similarities and differences and will be discussed together. The molecular structures of the complexes are illustrated in Figure 3 and selected bond lengths and bond angles are summarized in Table 3. Complexes 2 and 3 crystallized in a monoclinic system and P2 1 /n space group and complex 4 crystallized in a triclinic system and P-1 space group. The structures of complexes 2-4 present similarities and differences and will be discussed together. The molecular structures of the complexes are illustrated in Figure 3 and selected bond lengths and bond angles are summarized in Table 3. Complexes 2 and 3 crystallized in a monoclinic system and P21/n space group and complex 4 crystallized in a triclinic system and P-1 space group.  Complexes 2-4 are all neutral mononuclear Cu(II) complexes, having a deprotonated bidentate chelating 5-F-salo − ligand coordinated to Cu(II) ion via its two oxygen atoms, a bidentate α-diimine (bipyam, neoc or phen) ligand coordinated via its two nitrogen atoms and a chlorido (in complexes 2 and 3) or nitrato ligand (in complex 4) completing the coordination sphere. A distorted square pyramidal geometry around the five-coordinated Cu(I) ions in complexes 2-4 may be derived via the values of 0.11-0.27 (Table 3) for the trigonality index τ5 [37], and the values of 0.79-0.92 (Table 3) for the tetragonality T 5 [38].  (Table 3) for the trigonality index τ 5 [37], and the values of 0.79-0.92 (Table 3) for the tetragonality T 5 [38]. The arrangement of the ligand atoms around Cu1 is not similar for all complexes: in complexes 2 and 4, O1, O2, N1 and N2 form the basal plane and Cl1 and O3 (nitrato) , respectively, are lying in the apical position, while in complex 3, O1, O2, N1 and Cl1 atoms constitute the vertices of the base and N2 is on the apex.

Proposed Structure for Complex 5
According to the findings of the IR and UV-vis spectroscopic data, elemental analysis and molar conductivity measurements, and after the comparison with the crystal structures of complexes 2-4 and with those of similar mixed-ligand copper(II) complexes found in the literature [17,39,40], we suggest that complex 5 is a mononuclear and neutral complex presenting distorted square pyramidal geometry around the penta-coordinated copper(II) ion. The 5-F-salo − ligand is expected to bind in a bidentate manner to Cu(II) through the carbonyl and phenolato oxygen atoms, bipy is coordinated to Cu(II) ion through its nitrogen atoms while an oxygen atom of the monodentate nitrato ligand completes the coordination sphere.

Study of the Antioxidant Activity
Generally, antioxidants found mainly in food are rich in organic compounds (phenolic, hydroxyphenolic and hydroxycinnamic acids, flavones and flavonoids, etc.). The carboxylic groups in these acids or a near hydroxyl group and an oxo group for flavonoids and flavones enable them to coordinate to metal ions through their oxygen atoms leading to the formation of stable complexes. The combination of the redox properties of metal ions with such ligands is an interesting method to develop antioxidant compounds [41].
For the above reasons, the antioxidant ability of 5-F-saloH and Cu(II) complexes 1-5 has been evaluated via their scavenging activity towards DPPH and ABTS radicals, as well as the ability to reduce H 2 O 2 , and in comparison with that of well-known antioxidant agents such as nordihydroguaiaretic acid (NDGA), butylated hydroxytoluene (BHT), 6hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox) and L-ascorbic acid (these are the most commonly used standard reference antioxidant agents [42][43][44]). The results are summarized in Table 4. The DPPH-radical assay was developed in the 1950s [45] and this method has been used to assess the antioxidant capacity of several metal complexes [41]. The DPPH-scavenging ability of compounds has often been related to their ability to prevent ageing, cancer and inflammation [46]. The ability of a compound to scavenge the cationic ABTS radicals (ABTS +• ) has been considered a measure of its total antioxidant activity [46]. Further, hydrogen peroxide has the ability to penetrate biological membranes and, although it is not very reactive itself, it can sometimes be toxic since it may give rise to hydroxyl radicals in cells. For this reason, the removal of H 2 O 2 is very important for the protection of living systems [47]. When a compound is incubated with H 2 O 2 using a peroxidase assay system, the loss of H 2 O 2 can be measured [48]. Complexes 1-5 presented a low ability to scavenge DPPH and were found significantly less active than the reference compounds NDGA and BHT. The DPPH-scavenging ability of most complexes was found similar when incubated for 30 and for 60 min, so time did not seem to improve their action, except for complex 3 which presented enhanced DPPHscavenging activity over time. Almost all complexes 1-5 can scavenge ABTS radicals more effectively than 5-F-saloH, but they are significantly less active than the reference compound trolox. Complex 1 was proved to be a much more active ABTS-scavenger (ABTS = 78.89 ± 0.18%) than the other complexes. Most complexes presented higher ability to reduce H 2 O 2 than the reference compound L-ascorbic acid with complex 2 being the most active compound (H 2 O 2 % = 99.69 ± 0.29%). On average, complexes 1-5 presented similar or lower antioxidant activity when compared to other metal complexes with substituted salicylaldehydes as ligands [19][20][21][22].

Interaction of the Complexes with CT DNA
The interaction of the complexes with CT DNA was studied by UV-vis spectroscopy, viscosity measurements and via competitive studies with ethidium bromide. UV-vis spectroscopy may be considered a preliminary method for the study of the complexes with CT DNA, while viscosity measurements and competitive studies with EB were used to give more insight about the mode of interaction of the complexes with CT DNA, as metal complexes may interact by more than one way with DNA. In covalent binding, DNA-base nitrogen may be coordinated to metal ions after displacing at least one labile ligand of the complex. In the case of non-covalent interactions, the metal complexes interact with DNA via weak interactions: (i) π-π stacking interactions of the complexes between DNA base pairs (resulting in intercalation), (ii) Coulomb forces leading to electrostatic interaction outside of the helix, and (iii) van der Waals forces (hydrogen bonding, hydrophobic interactions) upon groove-binding [49].
Initially, the UV-vis spectra of complexes 1-5 (2.5 × 10 −5 -1 × 10 −4 M) were recorded in the presence of incremental amounts of CT DNA (Figure 4), and the changes of the λ max of the bands observed in the spectra of the complexes were monitored as a means to study the interaction between complexes and CT DNA [50] and to calculate the corresponding DNAbinding constants (K b ). As observed, in the UV-vis spectra of the complexes, at least two bands were observed: band I in the range 314-339 nm and band II in the region 380-428 nm. Upon addition of the CT DNA solution, band I exhibited a significant hypochromism, and band II a rather intense hyperchromism which was mainly accompanied by a red-shift (Table 5). These features indicate the interaction of the complexes with CT DNA [51], but may not provide sufficient information to reveal the possible interaction mode. Therefore, for the elucidation of the CT DNA interaction mode, further experiments involving DNAviscosity measurements and competitive studies with EB were performed. similar or lower antioxidant activity when compared to other metal complexes with sub-stituted salicylaldehydes as ligands [19][20][21][22].

Interaction of the Complexes with CT DNA
The interaction of the complexes with CT DNA was studied by UV-vis spectroscopy, viscosity measurements and via competitive studies with ethidium bromide. UV-vis spectroscopy may be considered a preliminary method for the study of the complexes with CT DNA, while viscosity measurements and competitive studies with EB were used to give more insight about the mode of interaction of the complexes with CT DNA, as metal complexes may interact by more than one way with DNA. In covalent binding, DNA-base nitrogen may be coordinated to metal ions after displacing at least one labile ligand of the complex. In the case of non-covalent interactions, the metal complexes interact with DNA via weak interactions: (i) π-π stacking interactions of the complexes between DNA base pairs (resulting in intercalation), (ii) Coulomb forces leading to electrostatic interaction outside of the helix, and (iii) van der Waals forces (hydrogen bonding, hydrophobic interactions) upon groove-binding [49].
Initially, the UV-vis spectra of complexes 1-5 (2.5 × 10 −5 -1 × 10 −4 M) were recorded in the presence of incremental amounts of CT DNA (Figure 4), and the changes of the λmax of the bands observed in the spectra of the complexes were monitored as a means to study the interaction between complexes and CT DNA [50] and to calculate the corresponding DNA-binding constants (Kb). As observed, in the UV-vis spectra of the complexes, at least two bands were observed: band I in the range 314-339 nm and band II in the region 380-428 nm. Upon addition of the CT DNA solution, band I exhibited a significant hypochromism, and band II a rather intense hyperchromism which was mainly accompanied by a red-shift (Table 5). These features indicate the interaction of the complexes with CT DNA [51], but may not provide sufficient information to reveal the possible interaction mode. Therefore, for the elucidation of the CT DNA interaction mode, further experiments involving DNA-viscosity measurements and competitive studies with EB were performed.     (Table 5) were relatively high (in the order of 10 5 -10 6 M −1 ), with complex 1 showing the highest K b constant (=2.37 (±0.07) × 10 6 M −1 ) among them and, in most cases, they are higher than the K b value of the typical intercalator EB (=1.23 (±0.07) × 10 5 M −1 ) [53]. The K b values of the compounds under study are lying in the range found for analogous metal complexes of X-saloH [17][18][19][20][21][22][23][24][25][26].
The viscosity of DNA is related to the length changes occurring when interacting with a compound [54]. For this study, the viscosity of a CT DNA solution (0.1 mM) was monitored in the presence of increasing amounts (up to r = [compound]/[DNA] = 0.36) of the compounds at room temperature. All complexes 1-5 induced an increase in the relative DNA viscosity ( Figure 5), which was higher in the case of complex 5. This increase is considered evidence of an intercalative binding mode to DNA, since the DNA viscosity increases because of an increase in the separation distances between DNA bases in order to provide the necessary space for the accommodation of the intercalating compound [54]. EB is a well-known indicator of DNA intercalation, since its insertion in-between adjacent DNA base pairs may lead to the development of effective π-π stacking interactions. A solution containing the EB-DNA adduct presents an intense fluorescence emission band at 592 nm when excited at λex = 540 nm [55]. The addition of a compound intercalating to DNA equally or more tightly than EB into this solution may induce changes to the emission band which are monitored, in order to gain insight into its competition with EB for the DNA intercalation site. The compounds under study do not present any fluorescence emission bands at RT in solution or in the presence of CT DNA or EB under the same experimental conditions; so, any changes observed in the fluorescence emission spectra of the EB-DNA solution, when the compounds are added, are useful to examine the EB-displacing ability of the complexes,  EB is a well-known indicator of DNA intercalation, since its insertion in-between adjacent DNA base pairs may lead to the development of effective π-π stacking interactions. A solution containing the EB-DNA adduct presents an intense fluorescence emission band at 592 nm when excited at λ ex = 540 nm [55]. The addition of a compound intercalating to DNA equally or more tightly than EB into this solution may induce changes to the emission band which are monitored, in order to gain insight into its competition with EB for the DNA intercalation site. The compounds under study do not present any fluorescence emission bands at RT in solution or in the presence of CT DNA or EB under the same experimental conditions; so, any changes observed in the fluorescence emission spectra of the EB-DNA solution, when the compounds are added, are useful to examine the EB-displacing ability of the complexes, as indirect evidence of their intercalating ability [55,56].
The fluorescence emission spectra of pretreated EB-DNA ([EB] = 20 µM, [DNA] = 26 µM) were recorded in the presence of increasing amounts of the complexes (representatively shown for complex 1 in Figure 6A). The addition of the complexes resulted in a significant decrease in the intensity of fluorescence emission band of the DNA-EB compound at 592 nm ( Figure 6B), with complex 5 inducing the highest quenching ( Table 6). The complexes present significant ability to displace EB from the EB-DNA adduct, as it can be deducted from the observed quenching, and it can be indirectly suggested that the complexes interact with CT DNA via intercalation [56]. EB is a well-known indicator of DNA intercalation, since its insertion in-between adjacent DNA base pairs may lead to the development of effective π-π stacking interactions. A solution containing the EB-DNA adduct presents an intense fluorescence emission band at 592 nm when excited at λex = 540 nm [55]. The addition of a compound intercalating to DNA equally or more tightly than EB into this solution may induce changes to the emission band which are monitored, in order to gain insight into its competition with EB for the DNA intercalation site. The compounds under study do not present any fluorescence emission bands at RT in solution or in the presence of CT DNA or EB under the same experimental conditions; so, any changes observed in the fluorescence emission spectra of the EB-DNA solution, when the compounds are added, are useful to examine the EB-displacing ability of the complexes, as indirect evidence of their intercalating ability [55,56].
The fluorescence emission spectra of pretreated EB-DNA ([EB] = 20 µ M, [DNA] = 26 µ M) were recorded in the presence of increasing amounts of the complexes (representatively shown for complex 1 in Figure 6A). The addition of the complexes resulted in a significant decrease in the intensity of fluorescence emission band of the DNA-EB compound at 592 nm ( Figure 6B), with complex 5 inducing the highest quenching ( Table 6). The complexes present significant ability to displace EB from the EB-DNA adduct, as it can be deducted from the observed quenching, and it can be indirectly suggested that the complexes interact with CT DNA via intercalation [56].  The Stern-Volmer (K SV ) constants (Table 6) of the complexes were calculated with the Stern-Volmer equation (Equation (S2)) and Stern-Volmer plots. The K SV values are relatively high (of the 10 −4 -10 −5 M −1 magnitude), indicating a tight binding to CT DNA. Among the complexes, complex 1 exhibits the highest K SV constant (=1.56 (±0.02) × 10 5 M −1 ). The EB-DNA quenching constants (k q ) of the compounds (Table 6) were calculated with Equation (S3) (considering τ o = 23 ns as the EB-DNA fluorescence lifetime [57]); the k q values are higher than 10 10 M −1 s −1 [56], proposing the existence of a static quenching mechanism [21], which may confirm the interaction with the fluorophore and the displacement of EB.

Study of the Interaction with Serum Albumins
Serum albumins are among the important proteins of the circulatory system. Their main role is to carry drugs and other bioactive small molecules through the bloodstream [58,59]. BSA and HSA are structurally homologous albumins, having two and one tryptophan residues, respectively [60]. The tryptophan residues of both albumins are responsible for the intense fluorescence emission band with λ em,max = 342 nm for BSA and 350 nm for HSA, respectively, when their solutions are excited at 295 nm [55]. The solutions of the complexes exhibited a maximum emission in the region 395-415 nm under the same experimental conditions and the SA-fluorescence emission spectra were corrected before the calculation processing. The inner-filter effect was calculated with Equation (S4) [61] and it was found too low to affect the measurements.
When the compounds were added to a solution of the albumins (3 µM), a significant quenching of the BSA (λ em = 342 nm) and HSA (λ em = 350 nm) fluorescence emission bands was observed ( Figure 7) which was more pronounced in the case of BSA (Table 7 and Figure 8). The appearance of a second emission with band λ max,em in the region 395-415 nm was attributed to the compound and, in many cases, resulted in the existence of an isoemissive point at~385-390 nm (Figure 7). The observed quenching may be ascribed to changes in the tryptophan environment of SA resulting from possible denaturation of their secondary structure, induced by the binding of the complexes to the albumins [62].  The SA-quenching constants (k q ) for complexes 1-5 (Table 7) (calculated from the corresponding Stern-Volmer plots with the Stern-Volmer quenching equation (Equation (S2) and (S3)) are much higher than 10 10 M −1 s −1 , indicating the existence of a static quenching mechanism [56] which may indirectly verify the interaction of the compounds with the albumins. The k q constants of complexes 1-5 are similar to those reported for similar Pd(II) and other metal complexes with substituted salicylaldehydes as ligands [12,13,[20][21][22][23][24][25][26][27].
The SA-binding constants (K) of the complexes (calculated from the corresponding Scatchard plots with the Scatchard equation (Equation (S5)) ( Table 7) are relatively high suggesting a tight interaction of the compounds with the albumins in order to be transported towards their potential biological targets. Furthermore, the K values are significantly lower than the value of 10 15 M −1 (which is the binding constant with avidin and it is considered as the limit between reversible and irreversible interactions), suggesting a rather reversible interaction of the compounds with the albumins and revealing their ability to get released when they approach their desired destinations [63]. Table 7. The quenching of the SA-fluorescence (∆I/Io, %), the albumin-quenching (k q , in M −1 s −1 ) and albumin-binding (K, in M −1 ) constants for 5-F-saloH and complexes 1-5.  The SA-quenching constants (kq) for complexes 1-5 (Table 7) (calculated from the corresponding Stern-Volmer plots with the Stern-Volmer quenching equation (Equation (S2) and (S3)) are much higher than 10 10 M −1 s −1 , indicating the existence of a static quenching mechanism [56] which may indirectly verify the interaction of the compounds with the albumins. The kq constants of complexes 1-5 are similar to those reported for similar Pd(II) and other metal complexes with substituted salicylaldehydes as ligands [12,13,[20][21][22][23][24][25][26][27].
The SA-binding constants (K) of the complexes (calculated from the corresponding Scatchard plots with the Scatchard equation (Equation (S5)) ( Table 7) are relatively high suggesting a tight interaction of the compounds with the albumins in order to be transported towards their potential biological targets. Furthermore, the K values are significantly lower than the value of 10 15 M −1 (which is the binding constant with avidin and it is considered as the limit between reversible and irreversible interactions), suggesting a ra-

Materials-Instrumentation-Physical Measurements
All chemicals and solvents were reagent grade and were used as purchased from commercial sources: 5-F-saloH, Cu(NO 3 ) 2 ·3H 2 O, CuCl 2 ·2H 2 O, CH 3 ONa, trisodium citrate, NaCl, BSA, HSA, CT DNA, EB, ABTS, K 2 S 2 O 8 , NaH 2 PO 4 , NDGA and BHT were purchased from Sigma-Aldrich Co; trolox from J&K; DPPH from TCI; L-ascorbic acid and all solvents from Chemlab. Infrared (IR) spectra (400-4000 cm −1 ) were recorded on a Nicolet FT-IR 6700 spectrometer with samples prepared as KBr pellets (abbreviations used: s = strong, sm = strong-to-medium, and m = medium). UV-visible (UV-vis) spectra were recorded as nujol mulls and in DMSO solutions at concentrations in the range 2 × 10 −5 -5 × 10 −3 M on a Hitachi U-2001 dual-beam spectrophotometer. C, H and N elemental analyses were performed on a PerkinElmer 240B elemental microanalyzer. Molecular conductivity measurements of 1 mM DMSO solutions of the complexes were carried out with a Crison Basic 30 conductometer. Fluorescence spectra were recorded in solution on a Hitachi F-7000 fluorescence spectrophotometer. Viscosity experiments were carried out using an ALPHA L Fungilab rotational viscometer equipped with an 18 mL LCP spindle and the measurements were performed at 100 rpm.
DNA stock solution was prepared by dilution of CT DNA with buffer (containing 150 mM NaCl and 15 mM trisodium citrate at pH 7.0) and kept at 4 • C for no longer than a week. The stock solution of CT DNA gave a ratio of UV absorbance at 260 and 280 nm (A 260 /A 280 ) of 1.88, indicating that the DNA was sufficiently free of protein contamination [64]. The DNA concentration per nucleotide was determined by the UV absorbance at 260 nm after 1:20 dilution using ε = 6600 M −1 cm −1 [65].

X-ray Crystal Structure Determination
Single-crystals of complexes 1-4 suitable for crystal structure analysis were obtained by slow evaporation of their mother liquids at RT. They were mounted at room temperature on a Bruker Kappa APEX2 diffractometer equipped with a triumph monochromator using Mo Kα (λ = 0.71073 Å, source operating at 50 kV and 30 mA) radiation. Unit cell dimensions were determined and refined by using the angular settings of at least 200 high intensity reflections (>10σ(I)) in the range 11 < 2 θ < 36 • . Intensity data were recorded using ϕ and ω-scans. All crystals presented no decay during the data collection. The frames collected for each crystal were integrated with the Bruker SAINT Software package [67], using a narrow frame algorithm. Data were corrected for absorption using the numerical method (SADABS) based on crystal dimensions [68]. The structure was solved using the SUPERFLIP package [69], incorporated in Crystals. Data refinement (full-matrix leastsquares methods on F 2 ) and all subsequent calculations were carried out using the Crystals version 14.61 build 6236 program package [70]. All non-hydrogen non-disordered atoms were refined anisotropically. For the disordered atoms, their occupation factors under fixed (an)isotropic displacement parameters were first detected. For 3, the methanol solvent molecule is disordered over two positions with site occupation factors of 0.5 each. The same holds for the non-coordinating nitrate oxygen atoms in complex 4. The disordered atom positions in 3 were refined isotropically, but anisotropically in the case of 4.
Hydrogen atoms riding on non-disordered parent atoms were located from difference Fourier maps and refined at idealized positions riding on the parent atoms with isotropic displacement parameters Uiso(H) = 1.2 Ueq(C) or 1.5 Ueq(methyl, -NH and -OH hydrogens) and at distances C-H 0.95 Å, N-H 0.83 Å and O-H 0.82 Å. All methyl, amine and OH hydrogen atoms were allowed to rotate but not to tip. Hydrogen atoms riding on disordered oxygen atoms of methanol solvent molecules were positioned geometrically to fulfill hydrogen bonding demands. The rest of the methyl hydrogen atoms were positioned geometrically to their parent atoms. Illustrations with 50% ellipsoids probability were drawn by CAMERON [71]. Crystallographic data for complexes 1-4 are presented in Table 1.

Study of the Biological Profile of the Compounds
The biological activity of the compounds (interaction with CT DNA and albumins, antioxidant activity) was evaluated in vitro after the compounds were dissolved in DMSO (1 mM), due to their low solubility in water. The studies were conducted in the presence of aqueous buffer solutions, where mixing of each solution never exceeded 5% DMSO (v/v) in the final solution. Control experiments were undertaken to assess the effect of DMSO on the data. Minimum or no changes were observed in the spectra of the SAs or CT DNA and appropriate corrections were performed, when needed.
All the protocols and relevant equations regarding the in vitro study of the biological activity (antioxidant activity, interaction with CT DNA, HSA and BSA) of the compounds can be found in the Supporting Information File (Sections S1-S3).

Conclusions
A total of five novel Cu(II) complexes of 5-fluoro-salicylaldehyde were synthesized and characterized by diverse techniques. The crystal structures of complexes 1-4 were determined by single crystal X-ray diffraction analysis, with complex 1 presenting square planar geometry and complexes 2-4 distorted square pyramidal arrangement of the ligands around Cu(II). 5-fluoro-salicylaldehyde ligands are bound in a bidentate manner to the Cu(II) ion in all complexes, via the carbonyl and phenolato oxygen atoms.
Complexes 1-5 presented a low ability to scavenge DPPH radicals, moderate ability (with the exception of complex 2) to reduce H 2 O 2 , and had significantly high scavenging activity toward ABTS radicals, which was close to that of the reference compound trolox. The interaction of the compounds with CT DNA probably takes place via intercalation, as deduced by UV-vis spectroscopic, viscosity measurements and EB displacement studies, leading to a rather tight DNA binding. Furthermore, the complexes have the ability to interact strongly and reversibly with serum albumins, as well as to get released upon reaching their biotarget(s).
The herein reported results concerning the antioxidant capacity and interaction of the complexes with biomacromolecules are interesting and may lead to more specific biological studies, which could reveal pathways for further biological applications of these types of compounds.
Author Contributions: Z.P.: synthesis, characterization, interaction with DNA and albumins, manuscript preparation; E.D.: synthesis, characterization, interaction with DNA and albumins, manuscript preparation; A.Z.: antioxidant activity studies, interaction with DNA and albumins, manuscript preparation; A.G.H.: X-ray structural determination, manuscript preparation; G.P.: supervisor of Z.P., E.D. and A.Z., manuscript preparation and editing, corresponding author, supervisor of the project. All authors have read and agreed to the published version of the manuscript.