Synthesis, Structure and Cytotoxic Properties of Copper(II) Complexes of 2-Iminocoumarins Bearing a 1,3,5-Triazine or Benzoxazole/Benzothiazole Moiety

A series of copper(II) complexes of 2-imino-2H-chromen-3-yl-1,3,5-triazines 2a-h, 3-(benzoxazol-2-yl)-2H-chromen-2-imines 4a-b, and 3-(benzothiazol-2-yl)-2H-chromen-2-imines 6a-c were obtained by reacting of appropriate 2-iminocoumarin ligands L1a-h, L3a-b, and L5a-c with 3-fold molar excess of copper(II) chloride. The structure of these compounds was confirmed by IR spectroscopy, elemental analysis, and single-crystal X-ray diffraction data (2f, 2g, 2h, and 6c). All the synthesized complexes were screened for their activity against five human cancer cell lines: DAN-G, A-427, LCLC-103H, SISO, and RT-4 by using a crystal violet microtiter plate assay and relationships between structure and in vitro cytotoxic activity are discussed. The coordination of 2-iminocoumarins with copper(II) ions resulted in complexes 2a-h, 4a-b, and 6a-c with significant inhibitory properties toward tested tumor cell lines with IC50 values ranging from 0.04 μM to 15.66 μM. In comparison to the free ligands L1a-h, L3a-b, and L5a-c, the newly prepared Cu(II) complexes often displayed increased activity. In the series of copper(II) complexes of 2-imino-2H-chromen-3-yl-1,3,5-triazines 2a-h the most potent compound 2g contained a 4-phenylpiperazine moiety at position 6 of the 1,3,5-triazine ring and an electron-donating diethylamino group at position 7′ of the 2-iminocoumarin scaffold. Among the Cu(II) complexes of 3-(benzoxazol-2-yl)-2H-chromen-2-imines 4a-b and 3-(benzothiazol-2-yl)-2H-chromen-2-imines 6a-c the most active was benzoxazole-2-iminocoumarin 4b that also possessed a diethylamino group at position 7′ of the 2-iminocoumarin moiety. Moreover, compound 4b was found to be the most prominent agent and displayed the higher potency than cisplatin against tested cell lines.


Introduction
Cancer-a multifactorial disease-constitutes the second leading cause of worldwide mortality, being a major public health issue. Despite the fact that there are several various approaches in cancer treatment, chemotherapy still remains one of the most important strategies [1]. Among clinically approved antineoplastic drugs cisplatin and its analogues have a leading and well-established position in the treatment of cancer. Unfortunately, their use is limited by toxicity and severe side effects [2]. In addition to high toxicity, another serious problem is acquired resistance of cancer cells to platinum drugs [3,4]. Therefore, there is a need for novel transition metal-complexes as a drug-candidates with less undesirable effects. Copper(II) complexes of 2-imino-2H-chromen-3-yl-1,3,5-triazine derivatives 2a-h were prepared by reaction of copper(II) chloride dihydrate with previously described ligands L1a-h [65]. The reactions were carried out at 20-22 °C in solvents such as Copper(II) complexes of 2-imino-2H-chromen-3-yl-1,3,5-triazine derivatives 2a-h were prepared by reaction of copper(II) chloride dihydrate with previously described ligands L1a-h [65]. The reactions were carried out at 20-22 • C in solvents such as dimethylformamide, ethanol, or methanol, containing 1-2% of water (Scheme 1). Dimethylformamide turned out to be the most convenient due to better solubility of 2-iminocoumarin ligands. Copper(II) complexes of 2-iminocoumarin are formed by self-assembly of a mixture of copper(II), chloride, and appropriate ligand containing nitrogen donor atoms of 2iminocoumarin skeleton and the 1,3,5-triazine ring. Formally, we are dealing here with a β-diimine system (β-NN) and due to the conjugation of π electrons of both heterocyclic rings, we may consider two possible conformations of copper(II) complex (structures C and D, as shown in Figure 2. Thus, the reaction of 2-iminocoumarins A and B may lead to complexes in which the central copper atom is chelated by a bidentate neutral ligand forming a six-membered ring -Cu-N5-C4-C3′-C2′-N (structure C) or -Cu-N3-C6-C3′-C2′-N (structure D). Therefore, to obtain better insight into the structure of the ligands used for complexation with copper salt, the relative stability of conformers A and B for ligand L1a (Figure 2) containing piperidine moiety was determined using the Spartan computer program (version '14 V 1.1.4.'). The obtained data revealed that conformer A was calculated to be slightly lower in energy than conformer B (ΔE = 1.36 kcal/mol). However, based on their calculated dipole moments, the conformer B (μ = 2.85 debye) would be predicted to predominate over the conformer A (μ = 1.98 debye) in a polar solvent used for the reaction-ethanol or dimethylformamide ( Figure 3). It should be noted, that more stable conformer A upon complexation gives rise the formation of mononuclear copper(II) complex (structure C, Figure 2), where central metal atom is chelated by neutral 2iminocoumarin-1,3,5-triazine ligand with formation of six-membered ring involving two nitrogen atoms-at position 5 of heterocyclic ring and imine moiety (C2=NH), respectively. Dark brown, orange, violet, or green crystals of copper(II) complexes were obtained upon slow evaporation of the solvent over 1-4 days. It should be noted that this process in some cases resulted in the isolation of a significant amount of coumarin as a result of the hydrolysis of the imine moiety. For this reason, dimethylformamide was replaced with lower boiling ethanol (compounds: 2b, 2c, and 2f) or methanol (compounds: 2e, 2g, and 2h).
The best yields (26-27%) were achieved with ligands L1b and L1g containing morpholine or phenylpiperazine rings (R = morpholine, phenylpiperazine) and an electrondonating substituent-diethylamino group (R 1 = H, R 2 = (C 2 H 5 ) 2 N). It was found that the best results were obtained using appropriate ligand and copper(II) chloride in a molar ratio of 1:3. Stoichiometric amounts, as well as 1:2 ratio of ligand, copper(II), and copper salt, led to the products constituting a mixture of the 2-iminocoumarin and coumarin complexes.
In the IR spectra of copper(II) complexes of 2-imino-2H-chromen-3-yl-1,3,5-triazine derivatives 2a-h a strong band in a rage of 3110 to 3488 cm −1 is assigned to the N-H stretching vibrations of the −C=N-H group and the primary amine group (NH2) of the 1,3,5-triazine ring. On the other hand, a broad absorption observed at 3209-3392 cm −1 in the IR spectra of the free ligands L1a-h [65], has disappeared in the IR spectra of complexes 2a-h. In the discussed spectra of complexes 2a-h a strong absorption attributable to the −C=N-of the −C=N-H group occurs in the range of 1643-1654 cm −1 and is shifted toward higher or lower wavenumbers in comparison with the spectra of the free ligands L1a-h (1649-1672 cm −1 ) [65]. In turn, spectroscopic characterization of the copper(II) complexes of 3-(benzoxazol-2-yl)-2H-chromen-2-imines 4a-b and 3-(benzothiazol-2-yl)-2H-chromen-2-imines 6a-c shows that the N-H of the −C=N-H group vibrational stretching modes appear in the 3119 to 3292 cm −1 and 3178 to 3338 cm −1 regions, and are shifted toward lower or higher wavenumbers in comparison with the IR spectra of the free ligands L3ab and L5a-c (3290-3279 cm −1 and 3213-3230 cm −1 , respectively) [66]. The vibration modes of ν(C=N) of the −C=N-H group appear in the ranges of 1635-1654 cm −1 and 1636-1664 cm −1 . In general, these frequencies shift toward lower frequencies in comparison with those of the free ligands L3a-b and L5a-c (1655-1663 cm −1 and 1662-1664 cm −1 , respectively) [66].
Molecules 2022, 27, x FOR PEER REVIEW 7 o resonance (NMR) spectra cannot be registered. Thus, the great emphasis was placed obtaining the single crystals suitable for X-ray diffraction studies to confirm structure synthesized copper(II) complex compounds. In all studied compounds, the organic ligands L act as bidentate N,N-chel forming neutral mononuclear [LCuCl2] or binuclear [(LCuCl)2(μ-Cl)2] complexes w CuCl2. In complexes 2f, 2g, and 2h, the ligand on binding to the metal ion adopts th conformation. In mononuclear complexes 2h, 2g, and 6c, the Cu (II) atom tetracoordinated with the coordination environment intermediate between tetrahe and square planar as indicated by the analysis with the SHAPE program (version 2.1 the dinuclear complex, 2f, the pentacoordinated metal center is in a slightly disto tetragonal pyramidal environment. In this compound, one of the Cl ligands is acting bridging mode binding asymmetrically the metal centers in the Cu2Cl2 coordination [Cu-Cl distances are 2.3330(6) and 2.6959(7) Å]. The bond formed by the metal center w the primary imine group N atom of the 2-iminocoumarin fragment, which is in the ra 1.930(2)-1.957(2) Å, is always shorter than the bond to the secondary imine group N a in the chelate ring (1.994(2)-2.053(2) Å).
In 2f, 2h, and 2g primary NH2 amino groups are involved in intramolecular N-H hydrogen bonds and intermolecular N-H···N or N-H···Cl interactions, whereas the im =N-H group does not take part in any hydrogen bonding. In the case of 6c, where primary NH2 group is missing, the imino C=N-H proton takes part in N-H···Cl interac assembling the coordination molecules into hydrogen-bonded centrosymmetric dim where the distance between the two-metal center is 3.9916(5) Å.
Some additional geometrical details can be found in the Supplementary Materia
It should be noted that the presence of copper(II) ions, that can act as Lewis acid in solution, could facilitate the hydrolysis of the 2-iminocoumarin ligands under experimental conditions. On the other hand, due to the presence of an unpaired electron resulting in paramagnetic properties of copper(II) complexes, the nuclear magnetic resonance (NMR) spectra cannot be registered. Thus, the great emphasis was placed on obtaining the single crystals suitable for X-ray diffraction studies to confirm structures of synthesized copper(II) complex compounds.
In all studied compounds, the organic ligands L act as bidentate N,N-chelates forming neutral mononuclear [LCuCl 2 ] or binuclear [(LCuCl) 2 (µ-Cl) 2 ] complexes with CuCl 2 . In complexes 2f, 2g, and 2h, the ligand on binding to the metal ion adopts the B conformation. In mononuclear complexes 2h, 2g, and 6c, the Cu (II) atom is tetracoordinated with the coordination environment intermediate between tetrahedral and square planar as indicated by the analysis with the SHAPE program (version 2.1). In the dinuclear complex, 2f, the pentacoordinated metal center is in a slightly distorted tetragonal pyramidal environment. In this compound, one of the Cl ligands is acting in a bridging mode binding asymmetrically the metal centers in the Cu 2 Cl 2 coordination ring [Cu-Cl distances are 2.3330(6) and 2.6959(7) Å]. The bond formed by the metal center with the primary imine group N atom of the 2-iminocoumarin fragment, which is in the range 1.930(2)-1.957(2) Å, is always shorter than the bond to the secondary imine group N atom in the chelate ring (1.994(2)-2.053(2) Å).
In 2f, 2h, and 2g primary NH 2 amino groups are involved in intramolecular N-H···Cl hydrogen bonds and intermolecular N-H···N or N-H···Cl interactions, whereas the imino =N-H group does not take part in any hydrogen bonding. In the case of 6c, where the primary NH 2 group is missing, the imino C=N-H proton takes part in N-H···Cl interaction assembling the coordination molecules into hydrogen-bonded centrosymmetric dimer, where the distance between the two-metal center is 3.9916(5) Å.
Some additional geometrical details can be found in the Supplementary Materials.

3-(benzoxazol-2-yl)-2H-chromen-2-imines 4a-b, and 3-(benzothiazol-2-yl)-2H-chromen-2-imines 6a-c in Aqueous Buffer Solution
The chemical stability of copper(II) complexes 2a-h, 4a-b, and 6a-c in phosphatebuffered saline solution (PBS, Dulbecco's buffer, pH 7.4) at 37 • C was investigated using UV-Vis spectroscopy. The Figure 8 shows the time-dependent changes in the UV-Vis spectra of the selected complexes 2b and 2d over 6 h when they were incubated at 37 • C in PBS. In general, all copper(II) complexes tested proved to be relatively stable in the PBS solution, as exemplified by the complexes 2b and 2d, since no new spectra with the formation of isosbestic points were observed. The complex 2d ( Figure 8A) showed no noticeable timedependent changes, whereas complex 2b ( Figure 8B) displayed a decrease in the intensity of the initial spectrum, but no change in the shape of the spectrum.
In regards to the cytotoxicity studies, it should be mentioned that the bond length of the halide bridge (Cu1-Cl2) of copper(II) complex 2f is 2.696 Å (Figure 9). Compared to the Cu-Cl bond lengths observed in the mononuclear complexes 2g-h (2.24-2.33 Å), the dimer 2f under physiological conditions can easily dissociate to form a mononuclear copper(II) complex with the structure analogous to the discussed complexes 2a-e and 2g-h. Thus, it can be assumed that in the solution, the tested complex 2f possesses a similar structure to that of the complexes 2a-e and 2g-h.   In regards to the cytotoxicity studies, it should be mentioned that the bond length of the halide bridge (Cu 1 -Cl 2 ) of copper(II) complex 2f is 2.696 Å (Figure 9). Compared to the Cu-Cl bond lengths observed in the mononuclear complexes 2g-h (2.24-2.33 Å), the dimer 2f under physiological conditions can easily dissociate to form a mononuclear copper(II) complex with the structure analogous to the discussed complexes 2a-e and 2g-h. Thus, it can be assumed that in the solution, the tested complex 2f possesses a similar structure to that of the complexes 2a-e and 2g-h.

General Information
The melting points were determined with a Boëtius apparatus and are uncorrected. The infrared spectra were recorded on a Nicolet 380 FT-IR spectrophotometer (Thermo Fisher Scientific Inc., Waltham, Massachusetts, MA, USA). The elemental analyses of carbon, hydrogen, nitrogen, and sulfur determined for compounds were within ± 0.4% of the theoretical values.
UV-Vis spectra were recorded on Carl Zeiss Technology Spekol 1200 (Analytik Jena AG, Jena, Germany) in a 1.0 cm cuvette maintained at 37 °C by a thermostatically controlled cuvette holder.
The above pattern was also seen in the series of 4-phenylpiperazine-containing complexes 2f-h. Thus, replacement of the 7 -diethylamino group (R 2 = (C 2 H 5 ) 2 N) for an electron-withdrawing 6 -Cl substituent (R 1 = Cl) in the 2-iminocoumarin scaffold led to compound with similar activity (compare 2g with 2h: IC 50 = 1.21-1.66 µM vs. 1.97-3.28 µM). It should be pointed out that the lack of the substituents R 1 and R 2 yielded less active analogue 2f (IC 50 = 6.29-8.84 µM) (Table 1, Figure 10), but this compound still retained pronounced growth inhibitory properties toward the tested tumor cell lines. The results obtained seem to indicate that electronic and steric effects brought about the R 1 and R 2 substituents and may affect the biological activity of these compounds.

General Information
The melting points were determined with a Boëtius apparatus and are uncorrected. The infrared spectra were recorded on a Nicolet 380 FT-IR spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The elemental analyses of carbon, hydrogen, nitrogen, and sulfur determined for compounds were within ± 0.4% of the theoretical values.
UV-Vis spectra were recorded on Carl Zeiss Technology Spekol 1200 (Analytik Jena AG, Jena, Germany) in a 1.0 cm cuvette maintained at 37 • C by a thermostatically controlled cuvette holder.
Diffraction experiments were carried out at room temperature with an Oxford Diffraction SuperNova diffractometer (Agilent Technologies Inc., Santa Clara, CA, USA) using Cu Kα radiation. Diffraction data were processed with CrysAlisPro software [68]. The structures were solved with the program SHELXT [69] and refined by full-matrix least-squares method on F 2 with SHELXL-2018/3 [70] within the Olex2 software [71]. C-H hydrogen atoms were placed in calculated positions and refined as riding on their carriers. In 2f, 2h and 6c hydrogen atoms from the N-H groups were freely refined, whereas those in 2g were placed in the geometrically predicted positions and refined as riding on their carriers.
Molecular modeling studies were performed at ab initio level using the density functional method (B3LYP) with the 6-31G* basis set as implemented into SPARTAN program version '14 V 1.1.4' [72]. diluted by 50% in DMF or DMSO to give the feed solutions, which were diluted 500-fold into the culture medium. The controls received DMF or DMSO at a final concentration of 0.1%. Each concentration was tested in eight wells, with each well receiving 100 µL of the medium containing the substance. The concentration ranges were chosen to bracket the expected IC 50 values as best as possible. Cells were then incubated for 96 h, after which time the medium was removed and replaced with 1% glutaraldehyde/phosphate-buffered saline for 30 min. The glutaraldehyde solution was discarded, and the plates washed with water before staining cells for 30 min with an aqueous solution of 0.02% Crystal Violet. After washing out excess Crystal Violet from the plates, optical density (OD) was measured at λ = 570 nm by use of an Anthos 2010 plate reader (Salzburg, Austria). Corrected T/C values were calculated according to the Equation (1)

Conclusions
In this paper we have described the synthesis, structure, and cytotoxic activity of two series of novel Cu(II) complexes of 2-iminocoumarins bearing a 1,3,5-triazine or benzoxazole/benzothiazole moiety. The coordination of 2-iminocoumarin ligands with copper(II) results in metal complexes with significant growth inhibitory properties toward tested human tumor cell lines with IC 50 values ranging from 0.04 to 15.66 µM. Furthermore, it was observed that newly prepared Cu(II) complexes often possess higher activity than the related ligands. As expected, in the series of Cu(II) complexes of 2-imino-2H-chromen-3-yl-1,3,5-triazine derivatives, the highest potency was shown for compound 2g bearing 4-phenylpiperazine moiety at position 6 of the 1,3,5-triazine ring and an electron-donating diethylamino group at position 7' of the 2-iminocoumarin scaffold. On the other hand, the SAR studies of this series indicate that 2-iminocoumarin scaffold is open for variation such that halogen, diethylamino, and methyl substituents are allowed. In turn, in the category of Cu(II) complexes of benzoxazole/benzothiazole-2H-chromen-2-imines the best antiproliferative activity was found for 7'-diethylamino-substituted benzoxazole derivative 4b. Additionally, compound 4b was found to be the most promising agent with higher potency than cisplatin against the LCLC-103H, SISO, and RT-4 cell lines. Based on the results obtained, one may conclude that the most active compounds in either triazine or benzazole series possess a diethylamino group in the 2-iminocoumarin skeleton as evidenced by compounds 2g and 4b, suggesting that this group is somehow involved in the mechanism of action. In this context, it is worth noting that coumarin-based agents [65,[73][74][75][76] display the intercalative mode of binding properties with DNA and are well-known DNA minor groove binders. Thus, the presence of diethylamino group in the structure of designed compounds may increase the efficiency in the intercalative binding giving extra non-covalent force between the substituent and DNA grooves. Therefore, additional work is needed to elucidate their mechanism of action.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules27217155/s1, checkCIF/PLATON reports for compounds 2h, 2f, 2g and 6c. Crystallographic data (CCDC 2191576-2191579) associated with this article are available online at: www.ccdc.cam.ac.uk/conts/retrieving.html (accessed on 1 September 2022). Acknowledgments: The X-ray structure analysis was carried out at The Structural Research Laboratory, Faculty of Chemistry, Adam Mickiewicz University, Poznań, Poland. Anna Makowska thanks the European Union student exchange program Erasmus for partial financial support of a research stay in Greifswald.