Solvent effects on the crystallization and structure of ternary copper(ii) coordination compounds with l-threonine and 1,10-phenanthroline

Solution-based and solid-state reactions of copper(ii) compounds, 1,10-phenanthroline and l-threonine were investigated. Eight new ternary coordination compounds were obtained: [Cu(l-Thr)(H2O)(phen)]2SO4∙10H2O (1a∙10H2O), [Cu(l-Thr)(H2O) (phen)]2SO4∙4.3H2O (1a∙4.3H2O), {[Cu(μ-l-Thr)(phen)]2SO4∙3.5H2O}n (1b∙3.5H2O), [Cu(l-Thr)(H2O)(phen)][Cu(l-Thr)(CH3OH)(phen)]SO4∙2H2O∙CH3OH (1c∙2H2O∙CH3OH), [Cu(l-Thr)(H2O)(phen)][Cu(l-Thr)(CH3OH)(phen)]SO4∙4H2O (1c∙4H2O), [Cu(l-Thr)(CH3OH)(phen)]2SO4∙H2O (1d∙H2O), [Cu(l-Thr)(H2O)(phen)][Cu(l-Thr)(CH3OH)(phen)][Cu(SO4)(l-Thr)(phen)]HSO4∙H2O∙3CH3OH (1e·H2O·3CH3OH), [Cu(l-Thr)(H2O)(phen)][Cu(l-Thr)(phen)(py)]SO4 (1f) (phen = 1,10-phenanthroline, l-Thr = l-threoninate, py = pyridine). X-ray crystal structure analysis of the prepared ternary coordination compounds revealed extensive hydrogen bonding and π-interactions that link complex species, anions and solvent molecules into 3D architectures. The water/methanol solvent molecules are found in pockets and/or channels in seven solvates. ESR spectra of different types of compounds were also investigated. In all measured compounds the unpaired electron of the copper(II) ion is located in the dx2-y2 orbital which is in agreement with elongated square-pyramidal geometry. Compound 1a∙10H2O showed substantial cytotoxic activity toward human hepatocellular carcinoma (HepG2) and acute monocytic leukaemia (THP-1) cell lines.

(1f) (phen ¼ 1,10-phenanthroline, L-Thr ¼ L-threoninate, py ¼ pyridine). X-ray crystal structure analysis of the prepared ternary coordination compounds revealed extensive hydrogen bonding and π-interactions that link complex species, anions and solvent molecules into 3D architectures. The water/methanol solvent molecules are found in pockets and/or channels in seven solvates. ESR spectra of different types of compounds were also investigated. In all measured compounds the unpaired electron of the copper(II) ion is located in the d x2-y2 orbital which is in agreement with elongated square-pyramidal geometry. Compound 1a10H 2 O showed substantial cytotoxic activity toward human hepatocellular carcinoma (HepG2) and acute monocytic leukaemia (THP-1) cell lines.

Introduction
Investigation of ternary coordination compounds of essential metals with amino acids and heterocyclic bases have been in focus for many years. Previous studies of such coordination compounds can be divided into three main study directions: biological activity, crystal structures and porosity. Most studies of these systems include copper as an essential metal. Copper is important for the function of several enzymes and proteins (cytochrome oxidase, superoxide dismutase, ascorbate oxidase, tyrosinase). Its deficiency or overload is associated with Menkes and Wilson disease, respectively [1].
Also, an aminocarboxylate as a ligand with various binding possibilities contributes to other properties such as plasticity of the coordination sphere, prevention of oxidation and biocompatibility of the metal ion environment. Some of these complexes can induce cell apoptosis in vitro and also exhibit excellent in vivo antitumor activity [13].
Since amino acids possess different side chains, their coordination compounds can create structures of higher dimensionalities (1D, 2D and 3D) through different non-covalent interactions (hydrogen bonds, π-stacking). In most of the ternary copper coordination compounds with amino acids and 1,10-phenanthroline the copper(II) atom is either fiveor six-coordinated [14]. Depending on the amino acid and the presence of a heterocyclic base, different architectures can be formed and, among others, porous structures into which can be incorporated guest molecules such as solvents to produce different solvatomorphs [5,15]. In CSD there are 98 data sets of ternary copper coordination compounds involving aminoacidates and 1,10-phenanthroline. These 98 data sets being published in 80 different articles show the lack of systematic exploration of the crystal structures and structure-property relationship of such copper(II) ternary coordination compounds. Only in two research articles different solvates of these ternary coordination compounds were investigatedmonohydrate and unhydrated alaninato complexes [16] and six serinato solvates involving water and/or methanol [5]. Both described alaninato compounds are coordination polymers, where carboxylic group acts as bridge between two copper(II) atoms. In serinato compounds, all six compounds contain monomeric complex cations of general formula [Cu(ser) (L)(phen)] þ (ser ¼ L-serinato; L ¼ H 2 O or CH 3 OH; phen ¼ 1, 10-phenanthroline). Three of those compounds contain only water as solvent, two of them contain only methanol, while one compound contains mixture of water and methanol molecules (coordinated and crystallization molecules).
In the early papers published in the 1980s coordination of the copper atom was the main concern of researchers [17,18,19], while in later papers intermolecular interactions were explored [20,21]. In the recent years, as mentioned earlier, such ternary coordination compounds are mostly investigated for their antiproliferative activity [3,13,22,23]. In contrast to solid-state, these complexes are well explored in solution.
As a part of our ongoing research on ternary copper(II) coordination compounds with amino acids and heterocyclic bases [5,29,30,31], in this work we explored the versatility of intermolecular interactions in copper ternary coordination compounds with L-threonine and 1,10-phenanthroline for the purpose of crystal engineering and investigation of structure-property relationships, such as magnetic property (ESR spectroscopy) or biological activity. We have performed the reactions of copper (II) compounds with L-threonine and 1,10-phenanthroline by solution methods and solid-state reactions. These two ligands were chosen having in mind a polar ligand and a nonpolar ligand, and their ability to form different types of intermolecular contacts. The effects of solvent (water, methanol, pyridine (py) or their binary mixtures) on crystallization and crystal structures was investigated.

Materials and methods
Copper(II) sulfate pentahydrate (Gram-Mol), anhydrous copper (II) sulfate (Acros), 1,10-phenanthroline monohydrate (Merck), L-threonine (Fischer Scientific), methanol (Alkaloid), DMEM/F12 medium (Sigma), FBS (Sigma), DMSO (Sigma) and staurosporine (Sigma) were obtained from commercial sources and used without further purification. Copper(II) hydroxide was prepared by the method of Agte [32,33]. Copper(II) sulfate trihydrate was prepared by heating copper(II) sulfate pentahydrate to 100 C and copper(II) sulfate monohydrate by heating copper(II) sulfate pentahydrate to 150 C. Anhydrous 1,10-phenanthroline was prepared by heating 1,10-phenanthroline monohydrate to 130 C. Attenuated total reflectance infrared (ATR-IR) spectra were measured on a Thermo Scientific ™ Nicolet ™ iS50 FTIR Spectrometer equipped with an ATR module in the spectral range 4000-400 cm À1 with a resolution of 4 cm À1 . Powder X-ray diffraction (PXRD) was performed on a Malvern Panalytical Aeris diffractometer in a Bragg-Brentano geometry with CuK α radiation (λ ¼ 1.54184 Å) at room temperature. The samples were contained on a silicon holder and the diffractograms were measured in 2θ range 5-40 with a step size of 0.022 and 15.0 s of measurement time per step. PXRD data were analyzed using HighScore Plus program [34]. Mechanochemical syntheses were performed on a Retch MM200 grinder operating at frequency of 25 Hz for 15 min. Teflon jars (volume 14 mL) and stainless steel balls (diameter 8 mm) were used for grinding.  Copper(II) sulphate monohydrate (46.8 mg, 0.25 mmol), copper(II) hydroxide (24.9 mg, 0.25 mmol), anhydrous 1,10-phenanthroline (90.9 mg, 0.5 mmol), and L-threonine (62.3 mg, 0.5 mmol) were dissolved in methanol (10 mL). The solution was heated at 80 C for 45 min, filtered and left to evaporate. Eventually, dark blue prisms (1c4H 2 O) and/or needles (1e⋅H 2 O⋅3CH 3 OH) formed after a few weeks. Crystals of 1e⋅H 2 O⋅3CH 3 OH are highly unstable outside solution and decompose in few seconds. Crystals of 1c4H 2 O were suitable for single-crystal X-ray diffraction, while crystals of 1e⋅H 2 O⋅3CH 3 OH diffracted poorly and were analysed by synchrotron irradiation.

Mechanochemical syntheses
Copper(II) sulphate pentahydrate (62.5 mg, 0.25 mmol), copper(II) hydroxide (24.4 mg, 0.25 mmol), anhydrous 1,10-phenanthroline (90.2 mg, 0.5 mmol) and L-threonine (59.5 mg, 0.5 mmol) were mixed in a Teflon jar (V ¼ 14 mL) and 24 μL of water was added (η ¼ 0.1 μL mg À1 ) [36]. Grinding was performed using one steel ball (8 mm) at a vibration frequency 25 Hz for 15 min. The product was analysed by powder X-ray diffraction. Powder diffraction pattern was consistent with powder pattern calculated from crystal structure of 1a⋅10H 2 O (Fig. S1 in Supplementary information). Another PXRD experiments were performed on the same sample after 2 and 5 min and it was confirmed that the decomposition of compound began (Fig. S1 in Supplementary information).

Synthesis of [Cu
Anhydrous copper(II) sulphate (39.9 mg, 0.5 mmol), copper(II) hydroxide (24.7 mg, 0.25 mmol), anhydrous 1,10-phenanthroline (90.1 mg, 0.5 mmol) and L-threonine (59.5 mg, 0.5 mmol) were mixed in a Teflon jar (V ¼ 14 mL) and 43 μL of methanol was added (η ¼ 0.2 μL mg À1 ). Grinding was perfomed using one steel ball (8 mm) at vibration frequency 25 Hz for 15 min. The product was analysed by powder X-ray diffraction. Most diffraction maxima were consistent with the powder pattern calculated from the crystal structure of 1d⋅H 2 O, but a small amount of unknown phase was also detected (Fig. S1 in Supplementary information).

Single-crystal X-ray diffraction
Single-crystal X-ray diffraction data of 1a10H 2 O, 1b3.5H 2 O, 1c2H 2 OCH 3 OH, 1c4H 2 O and 1f were measured on a Rigaku XtaLAB Synergy diffractometer using CuK α radiation (λ ¼ 1.54184 Å) with HyPix6000HE detector at 170 (1b3.5H 2 O, 1c2H 2 OCH 3 OH, 1c4H 2 O and 1f) or 180 K (1a10H 2 O). Diffraction data of 1dH 2 O were collected on an Oxford Diffraction Xcallibur3 CCD diffractometer using MoK α radiation (λ ¼ 0.71073 Å) at room temperature. X-ray diffraction data of 1a4.3H 2 O and 1e⋅H 2 O⋅3CH 3 OH were collected at Elettra Sincrotrone Trieste using synchrotron radiation (λ ¼ 0.70000 Å) at XRD1 beamline equipped with Dectris Pilatus 2M detector at 100 K. Data reduction for all data sets was performed using the CrysAlis software package [37]. All crystal structures except 1f were solved by SHELXS [38] and 1f was solved by SHELXT [39] program. Full-matrix least-squares refinements based on F 2 against all reflections were carried out by a SHELXL-2017/1 program [40]. Structures were visualized using the Mercury program [41]. Geometrical parameters were calculated with PLATON [42]. All nonhydrogen atoms were refined anisotropically. Most of the hydrogen atoms were refined at calculated positions. Hydrogen atoms belonging to the water or methanol molecules and hydrogensulfate ions were found in the Fourier difference maps and restrained to positions according to the idealized geometry of the respective group. In 1a4.3H 2 O hydrogen atoms belonging to water molecules were constrained to positions found in the Fourier difference map according to the idealized geometry. One of the symmetrically independent L-threoninate residue in 1a4.3H 2 O is disordered over two sites with occupancies of 0.52 and 0.48. In the part with the higher occupancy factor one water molecule is also present (with the same occupancy factor), while in the other part there is no additional water molecule, so the average number of crystallization water molecules per formula unit is 4.3. In 1b3.5H 2 O one of the symmetrically independent sulfate ion is disordered over two positions around one of the S-O axis, so that three oxygen atoms are split in two parts with individual occupancy factors 0.67 and 0.33. After visualization of the crystal structure of 1a10H 2 O it was noticed that two unusually large voids are present in the structure (volume of each void %20 Å 3 , calculated with PLATON), however, there is no significant residual electron density inside voids.

ESR spectroscopy
The ESR measurements were performed on a Bruker Elexsys 580 FT/ CW spectrometer from the room down to the liquid nitrogen temperature. The microwave frequency was around 9.7 GHz with the magnetic field modulation amplitude of 0.5 mT and modulation frequency of 100 kHz. The investigated compounds 1a⋅10H 2 O and 1b3.5H 2 O were unstable outside of solution so the samples were kept in respective mother liquors till measurements.

In vitro cytotoxic activity
Cytotoxicity experiments were performed at the School of Medicine, University of Zagreb. Since all prepared compounds contain similar complex species which are biologically active, we chose 1a⋅10H 2 O as the representative compound. All prepared compounds are highly soluble in water and moderately soluble in methanol and DMSO. The in vivo cytotoxicity of the 1a10H 2 O was assessed on the hepatocellular carcinoma (HepG2, ATCC HBÀ8065) and human acute monocytic leukaemia (THP-1, ATCC TIBÀ202) cell lines. CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (MTS) was used. It is a tetrazolium-based cell viability assay that measures the metabolic capacity of cells in culture [43]. Serial dilutions of the tested compound in 96-well microtiter plates were prepared in 100-0.05 μM concentration range in an appropriate cell medium (50 μL). Cells were added to plates in an appropriate number per well (50 μL). Control wells consisted of media only (blank) or cells with 1% DMSO added (control). Plates were incubated overnight at 37 C in a 5% CO 2 atmosphere. For determining inhibition of cellular proliferation or inducement of cytotoxic effect CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, G3580) was used. 10 μL of MTS reagent was dispensed per well. Plates were incubated for 2 h at 37 C in a 5% CO 2 atmosphere and the absorbance was recorded at 490 nm using a 96-well Spectramax i3x plate reader. Results were analyzed in GraphPad Prism software.

Synthesis and crystallization
Overview of the solution-based synthetic procedures of all eight coordination compounds is given in Scheme 1. All monomeric compounds except 1e⋅H 2 O⋅3CH 3 OH contain two complex cations, [Cu(L-Thr)(L)(phen)] þ (L ¼ H 2 O, CH 3 OH and/or py), and a sulfate counterion while 1e⋅H 2 O⋅3CH 3 OH contains three complex units, [Cu(L-Thr)(H 2 O)(phen)] þ , [Cu(L-Thr)(CH 3 OH)(phen)] þ and [Cu(SO 4 ) (L-Thr)(phen)] À and a hydrogen sulfate counterion. Compound 1b⋅3.5H 2 O contains polymeric chains {[Cu(μ-L-Thr)(phen)] þ } n and sulfate counter ions. As seen in Scheme 1, many synthetic conditions yielded two or three different products. If fully hydrated reactants (copper(II) sulfate pentahydrate, 1,10-phenanthroline monohydrate) in pure water or high ratio of water is used, 1a⋅10H 2 O crystallizes in most cases, and in some cases 1a⋅4.3H 2 O or their mixture. If fully hydrated reactants and pure methanol as solvent are used three different products crystallized: 1a⋅10H 2 O, 1c⋅2H 2 O⋅CH 3 OH and/or 1d⋅H 2 O. If less hydrated reactants (anhydrous or monohydrate of copper(II) sulfate, anhydrous 1,10-phenanthroline) and pure methanol were used 1c⋅4H 2 O, 1d⋅H 2 O and/or 1e⋅H 2 O⋅3CH 3 OH crystallized from the solution. If we look at the formula units of 1c⋅4H 2 O, 1c⋅2H 2 O⋅CH 3 OH, 1d⋅H 2 O and 1e⋅H 2 O⋅3CH 3 OH it is seen that all these compounds contain both water and methanol molecules (coordinated and/or as crystallization molecules) so it is possible that at high methanol to water ratio, there is small difference in their stability and there is a possibility for any of them to crystallize. Small variances of conditions, such as room temperature, evaporation rate or size and shape of the crystallization dish, may influence the final crystal product. 1b3.5H 2 O crystallized from the water/pyridine mixture (v/v ¼ 1:9). The same product crystallizes from the methanol/pyridine solution (v/v ¼ 7:3), but in a mixture with 1d⋅H 2 O. It seems pyridine has an effect as a tailor-made additive on the formation of polymeric 1b3.5H 2 O. At higher pyridine to methanol ratio 1f crystallizes but with an extremely low yield, due to low solubility of reactants. Although in some of the synthetic conditions there is a very low water content, water is present in all products (coordinated and/or as crystallization molecules). The ability of water molecules to act as donors and acceptors of hydrogen bonds may be crucial for the stability of crystal structures in these ternary coordination compounds.
Similar to the solution-based syntheses, small differences in water content influenced the final product of mechanochemical syntheses so we tried to use different hydrates of copper(II) sulfate and 1,10-phenanthroline. Three compounds were obtained from mechanochemical syntheses: 1a⋅10H 2 O, 1c⋅2H 2 O⋅CH 3 OH and 1d⋅H 2 O. Compound 1a⋅10H 2 O was obtained when copper(II) sulphate pentahydrate, copper(II) hydroxide, anhydrous 1,10-phenanthroline and small amount of water were used for LAG synthesis. The same product, but in a mixture with 1c⋅2H 2 O⋅CH 3 OH, was obtained when water was replaced by a small amount of methanol. If copper(II) sulfate pentahydrate was replaced by copper(II) sulfate trihydrate and small amount of methanol was used for LAG synthesis, pure 1c⋅2H 2 O⋅CH 3 OH was obtained. When anhydrous reactants (copper(II) sulfate and 1,10-phenanthroline) and small amount of methanol were used 1d⋅H 2 O was obtained, but a small amount of an unknown phase was also present.

Infrared spectroscopy
IR (ATR) spectra were measured for 1a10H 2 O, 1b3.5H 2 O, 1c2H 2 OCH 3 OH and 1f. IR spectra of all analysed compounds are very similar to each other due to similarity in the structure and coordination of the ligands. A broad band in the range 3600-2900 cm À1 (3550-2900 cm À1 for 1f) corresponding to O-H stretching suggests extensive hydrogen bonding in the crystal structures. Since there are two or more symmetrically independent L-threoninate ligands in all compounds, several bands corresponding to asymmetric stretching of the carboxylate groups are overlapped. In all cases, at least two bands can be Scheme 1. Solution-based syntheses of all eight copper(II) coordination compounds.
distinguished one at higher wavenumbers in the range 1658-1623 cm À1 (uncoordinated or bridging oxygen atom) and one at lower wavenumbers in the range 1610-1600 cm À1 (coordinated oxygen atom).

Crystal structures
All compounds crystallize in noncentrosymmetric space groups in either monoclinic or triclinic crystal system. Crystallographic data are given in Tables S1 and S2 in Supplementary information.

Coordination of copper(II) ion
All compounds are composed of complex species of three different types (type 1, type 2 and/or type 3), considering a molecule or ion coordinated to copper(II) ion in the apical position (Figure 1), with sulfate or hydrogensulfate counterions (Fig. S2 and S3 in Supplementary information).
In complex cations of type 1, solvent molecule (water, methanol or pyridine) is coordinated in the apical position, forming complex cations [Cu(L-Thr)(L)(phen)] þ (L ¼ H 2 O, CH 3 OH or py). All compounds except 1b⋅3.5H 2 O contain type 1 complex cations. We have obtained two different hydrates with two cations of type 1 with apically coordinated water molecule, 1a10H 2 O and 1a4.3H 2 O; one hydrate with two cations of type 1 with apically coordinated methanol molecule, 1dH 2 O; two different solvates with one cation of type 1 with apically coordinated water and one with a methanol molecule, 1c2H 2 OCH 3 OH and 1c4H 2 O; as well as compound 1f with one cation of type 1 with apically coordinated water and one with coordinated pyridine molecule. Type 2 complex anion has coordinated sulfate anion in the apical position forming complex anion [Cu(SO 4 )(L-Thr)(phen)] À . Only compound 1e⋅H 2 O⋅3CH 3 OH contains type 2 complex anion together with two complex cations of type 1 (one cation with an apically coordinated water and one with a methanol molecule) ( Figure 2). Complex unit of type 3 is quite different since it contains carboxylate of adjacent complex cation coordinated to copper(II) ion in the apical position forming a polymeric chain {[Cu(μ-L-Thr)(phen)] þ } n . 1b⋅3.5H 2 O is the only compound containing a type 3 complex unit.
All eight ternary coordination compounds contain copper(II) squarepyramidal pentacoordinated complex species, with elongated apical Cu-X bond (X ¼ O or N), due to the Jahn-Teller effect. In all types of the investigated compounds, 1,10-phenanthroline and L-threoninate are    Table S3 in Supplementary information). The apical Cu-N pyridine bond (2.175 (9) Å) in 1f is shorter than the apical Cu-O bonds (Table S3 in Supplementary information).

Non-covalent interactions and structural isomerism
All structures contain numerous potential hydrogen bond donors and acceptors, such as amino-, hydroxyl-and carboxylate groups of L-threoninate, solvent molecules, sulfate or hydrogensulfate ions, so most of the compounds contain complex hydrogen-bonded frameworks. Some hydrogen bond motifs are conserved in all eight compounds. At least one Figure 3. Right-hand rule for defining a) "up", ↑ and b) "down", ↓ optical isomers in ternary copper(II) coordination compounds. Rotation is chosen from heavier towards lighter atoms in L-threoninate, and the straight arrow shows: "up", ↑, or "down", ↓, direction. amino-and one hydroxyl group of L-threoninate ligand in each compound are involved in N-H⋅⋅⋅O sulfate and O-H⋅⋅⋅O sulfate hydrogen bonds, which act as a bridge between two adjacent complex species (in 1eH 2 O3CH 3 OH one N-H⋅⋅⋅O sulfate and O-H⋅⋅⋅O sulfate hydrogen bond is intramolecular) (Table S4 and S5 in Supplementary information).
In 1eH 2 O3CH 3 OH a rare hydrogen sulfate⋅⋅⋅sulfate hydrogen bond is found (Figure 2). In a CSD search in which close contacts of hydrogen sulfate and sulfate anions were defined as an intermolecular contact with H⋯O distance shorter than the sum of van der Waals radii, 298 data sets were found. In another search, the same query was defined but limited to the structures containing any transitional metal. 51 data sets were found, which is only 1.5% of all data sets found in CSD containing any transitional metal and sulfate ions. In 1eH 2 O3CH 3 OH the sulfate ion is coordinated to the copper atom in one of the symmetrically independent complex species and acts as an acceptor for the hydrogen bond with the hydrogen sulfate ion  longer with a distance of 1.568 (6) Å, which is in accordance with literature data [45,46]. Due to asymmetrically coordinated copper in the basal plane, two optical isomers may be distinguished based on the position of the apically coordinated atom. "Up", ↑, and "down", ↓, positions of apically coordinated atom are defined using the right-hand rule shown in Figure 3.
All compounds contain both ↑ and ↓ isomers, however, since all structures are noncentrosymmetric, not all structures contain mixtures of those isomers. In 1a10H 2 O, 1a4.3H 2 O, 1b3.5H 2 O, and 1dH 2 O only one type of the apical ligand is present, and all those structures contain mixtures of ↑ and ↓ isomers. In 1c2H 2 OCH 3 OH, 1c4H 2 O, 1eH 2 O3CH 3 OH, and 1f, all complex cations with apically coordinated water are ↓ isomers, while complexes with apically coordinated methanol, pyridine and sulfate ions are all ↑ isomers. In 1b⋅3.5H 2 O polymeric chains are interlocked by π-interactions forming supramolecular 2D sheets (Figure 4b.). In 1c⋅H 2 O⋅2CH 3 OH and 1f ↑ and ↓ complex cations form sandwich-like dimers which are alternating in a zig-zag pattern (Figure 4c). Finally, 1eH 2 O3CH 3 OH contains three different complex species, having apically coordinated water or methanol molecules or sulfate ions. In this compound 1D pillars are formed by stacking all three complex species alternating in order ↑ MeOH ↑ SO4 ↓ H2O (Figure 4d).

Crystallization solvent molecules
Specific structure of investigated copper(II) ternary complex species, with half of the complex being hydrophobic, and the other half hydrophilic, has a consequence that hydrophobic phenanthroline ligands are oriented towards the inner side of π-stacked 1D pillars, while the hydrophilic parts of the complex species are oriented towards the outside of 1D pillars. This feature of the complex species is probably the reason why so many different solvates may be formed from very similar synthetic conditions. Between the pillars, crystallization solvent molecules form either 1D infinite chains (1a10H 2 O), pockets and 1D chains (1a4.3H 2 O, 1c⋅4H 2 O) or pockets (1b3.5H 2 O, 1cH 2 O2CH 3 OH, 1dH 2 O, 1eH 2 O3CH 3 OH), as shown in Table 1 and Figure 5. It seems that not only the type and number of crystallization solvent molecules but also the type of coordinated species in the apical position has an influence on the formation of 1D chains. In 1a10H 2 O crystallization water molecules occupy 25% of the unit cell volume, and this is the only compound having only 1D chains. Influence of the apically coordinated molecule on the formation of pockets or channels of solvent molecules is also observed in the crystal structure of 1f. In this structure, one of the symmetrically independent complex cations has coordinated pyridine, while the other has a coordinated water molecule. Coordinated pyridine does not have a free acceptor or strong hydrogen bond donor atoms, and the structure does not contain crystallization solvent molecules.

ESR spectroscopy
Three polycrystalline ternary copper(II) complexes 1a10H 2 O, 1b3.5H 2 O and 1dH 2 O were investigated by X-band electron spin resonance (ESR) spectroscopy. The representative spectra, obtained at room temperature (297 K) and liquid nitrogen temperature (78 K), are shown in Figure 6.
The spectral simulations were performed by EasySpin software [47] using the following form of the spin-Hamiltonian for copper (II) ions [48,49]: In Eq. (1) μ B is Bohr magneton, g is the g-tensor, B is the magnetic field, S and I are the electron and nuclear spin operator, respectively while hyperfine tensor A describes interaction between electron and nuclear spins. For all simulations, the second term in Eq. (1) was omitted because hyperfine interaction was not experimentally observed.
The spectra were simulated using the same values of g-tensor at both temperatures, allowing linewidth of assumed Lorentzian lineshape to change with temperature. The spin-Hamiltonian values, obtained from the simulations, are given in Table 2. Small variations in the local geometry of the Cu(II) coordination can cause distribution of g x , g y and g zvalues around some average values [50]. This effect described by g strain parameters is also considered in the simulations with the values presented in Table 2. The obtained g-values g x % g y < g z are expected for the elongated octahedral, square pyramidal or square planar copper geometry [51], and are in agreement with structurally determined distorted square pyramidal geometries in these compounds. Although all investigated complexes contain more than one copper(II) atom per unit cell, only one ESR line is observed in the powder spectra due to similar geometries and g-parameters. The distances between two nearest copper atoms are 6.584 Å and 6.389 Å, for 1a10H 2 O and 1dH 2 O, respectively, while 1b3.5H 2 O is an one-dimensional zig-zag polymer with the distance between two nearest copper atoms of 5.487 Å. Therefore, the Cu-Cu distances are large enough to observe significant exchange interactions in these compounds. 1b3.5H 2 O is unstable outside of solution and a sample was poorly grinded into powder, resulting in observation of additional crystalline peaks in the powder spectrum.

Cytotoxicity
The results of the cell viability assay with IC 50 values are presented in Table 3. 1a10H 2 O showed substantial cytotoxic activity toward human hepatocellular carcinoma (HepG2) and acute monocytic leukaemia (THP-1) cell lines. Moreover, it exhibited somewhat lowered cytotoxicity against THP-1 cells. Similar ternary copper complexes, investigated by different research groups, showed that mode of their action against tumor cells was DNA cleavage via reactive oxygen species (ROS) generation, mitochondrical toxicity or direct interaction with DNA after their administration to tumor cells [13,52,53,54,55]. Quantitative structure-activity relationship (QSAR) studies on similar ternary copper compounds indicated that the presence of phenanthroline was necessary to preserve the antiproliferative activity and that the nature of the O,N coligand had a poor influence on biological activity [56]. We believe that the square pyramidal structure of compound 1a10H 2 O with planar phenathroline ligand, L-Thr coligand and apically coordinated water molecule provides an optimal geometry for interaction with DNA strands through π-stacking interaction (phenathroline with DNA bases) and hydrogen bonds (L-Thr and apically coordinated water molecule with DNA bases) [3,57]. However, comprehensive research on mode of action of 1a10H2O is needed to compare antitumor activities on different cell lines.

Conclusions
Eight new ternary copper(II) coordination compounds with 1,10-phenanthroline and L-threonine were obtained by solution-based syntheses (1a10H 2 O, 1a4.3H 2 O, 1b3.5H 2 O, 1c2H 2 OCH 3 OH, 1c4H 2 O, 1dH 2 O, 1e⋅H 2 O⋅3CH 3 OH, 1f) and three of them also by mechanochemical syntheses (1a10H 2 O, 1c2H 2 OCH 3 OH and 1dH 2 O). We have found that different synthetic approach can control porous and nonporous structures in a controlled manner. It was shown that small differences in water content in synthetic mixtures (and possibly atmospheric conditions) have a high influence on the final product in both solution-based and mechanochemical syntheses, so in some cases two or three different products were obtained. All compounds consist of three different types of complex unit (named type 1, type2 and type 3). In all complex units the copper(II) ion is pentacoordinated by a L-threoninate and a phenanthroline ligand in the equatorial plane, and apically by a solvent molecule (either a water, methanol or pyridine molecule) in the type 1 complex cation, by sulphate in the type 2 complex anion and by carboxylate of a neighbouring complex cation in the type 3 complex unit. Two types of diastereomers are found for the complex unit regarding the position of the apically coordinated species in relation to the chiral Lthreoninato ligand (named up and down). All compounds except 1f are different solvates with solvent molecules situated either in the endless 1D channels and/or pockets. In all crystal structures of the solvates, the complex units, sulfate or hydrogensulfate anions and solvent molecules are linked by hydrogen bonds and π-interactions into 3D supramolecular frameworks. Compounds described in this study form some predictive intermolecular interactions such as π-interactions or certain hydrogen bond motifs (N-H⋅⋅⋅O sulfate and O-H⋅⋅⋅O sulfate ). Knowledge of intermolecular interactions might help in the design of new compounds for potential drug design and/or functional material in the separation industry. ESR analysis of the different types of compounds showed agreement of the obtained g-values of the copper unpaired electron with the elongated square-pyramidal geometries of copper ions. Compound 1a10H 2 O showed substantial cytotoxic activity toward human HepG2 and THP-1 cell lines. Found biologycal activity provides an incentive to further study in different tumor cells.

Associated content
Supporting Information. The supporting material contains crystallographic data, ORTEP drawings of crystal structures, selected hydrogen bond distances and angles, powder X-ray diffraction patterns.

Author contribution statement
Darko Vu sak, Jurica Jurec: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.
Katarina Le zai c: Performed the experiments; Analyzed and interpreted the data.
Dijana Zili c, Biserka Prugove cki: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Funding statement
This work was supported by the Croatian Government and the European Union through the European Regional Development Fund-Competitiveness and Cohesion Operational Programme (Grant KK.01.1.1.02.0016), and The Croatian Science Foundation (grant no IP-2018-01-3168).

Data availability statement
Data included in article/supplementary material/referenced in article.

Declaration of interests statement
The authors declare no conflict of interest.

Additional information
Supplementary content related to this article has been published online at https://doi.org/10.1016/j.heliyon.2022.e09556.