Transition Metal Complexes with Flufenamic Acid for Pharmaceutical Applications—A Novel Three-Centered Coordination Polymer of Mn(II) Flufenamate

Five complexes of Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) with non-steroidal anti-inflammatory drug, flufenamic acid were synthesized: (1) [Mn3(fluf)6EtOH)(H2O)]·3EtOH; (2) [Co(fluf)2(EtOH)(H2O)]·H2O; (3) [Ni(fluf)2(EtOH)(H2O)]·H2O; (4) [Cu(fluf)2·H2O]; (5) [Zn(fluf)2·H2O]. All complexes were characterized by elemental analysis (EA), flame atomic absorption spectrometry (FAAS), Fourier-transform infrared spectroscopy (FTIR), and thermogravimetric analysis (TGA). The crystal structure of 1 was determined by the single crystal X-ray diffraction technique. It crystallizes in the triclinic space group P1¯ with three independent Mn(II) cations, six coordinated flufenamato ligands augmented with water and ethanol molecules in the inner coordination sphere. In this crystal, manganese atoms are multiplied by symmetry and form infinite, polymeric chains which extend along the [001] dimension. The Hirshfeld Surface analysis revealed changes in interaction assemblies around all metal centers. The antioxidant and antimicrobial activities were established for all complexes and free ligand for comparison. All compounds exhibit good or moderate bioactivity against Gram-positive bacteria and yeasts.


Introduction
Coordination polymers (CPs) have attracted attention of numerous scientists, and material engineers well over 30 years [1]. The oldest known CP is the Prussian blue (Fe 4 [Fe(CN) 6 ]3H 2 O) which, since the pioneering work of Keggin and Miles [2], initiated remarkable development in the field [3,4]. Following the IUPAC recommendation [5], CP is an array of constitution repeating units linked by coordinate-covalent bonding. Therefore, a one-dimensional coordination polymer contains metal ions or their clusters connected by coordinating organic ligands into an infinitive chain [6]. Progress in the efficient synthesis and design of CPs [7,8] prompted diverse applications in technologies crucial for civilization development. The gas and energy storage [9,10], intelligent drug delivery [11], production of smart catalysts and recent, remarkable advancements in tunable catalysis are among the most important of these applications [12,13].
In this contribution, we report synthesis, spectroscopic characterization and thermal behavior of Mn, Co, Ni, Cu and Zn complexes with HFluf 1-5, respectively. Antioxidant and antimicrobial activities of all investigated compounds augmented with those of free flufenamic acid were also determined. Moreover, the structure of 1 was solved and unequivocally refined by X-ray crystallography. Scheme 1. Structural scheme of flufenamic acid 6.

Results and Discussion
Compounds 1-5 are air stable at room temperature. They are highly soluble in polar organic solvents like methanol, ethanol DMF and DMSO. Their properties were thoroughly investigated by the elemental analysis (EA), flame atomic absorption spectrometry (FAAS), Fourier-transform infrared spectroscopy (FTIR) and thermogravimetric (TGA) methods augmented by antimicrobial and antioxidant studies, Table 1. Crystal structure of 1 was determined by the single crystal X-ray diffraction technique, Table 2 In this contribution, we report synthesis, spectroscopic characterization and thermal behavior of Mn, Co, Ni, Cu and Zn complexes with HFluf 1-5, respectively. Antioxidant and antimicrobial activities of all investigated compounds augmented with those of free flufenamic acid were also determined. Moreover, the structure of 1 was solved and unequivocally refined by X-ray crystallography.

Results and Discussion
Compounds 1-5 are air stable at room temperature. They are highly soluble in polar organic solvents like methanol, ethanol DMF and DMSO. Their properties were thoroughly investigated by the elemental analysis (EA), flame atomic absorption spectrometry (FAAS), Fourier-transform infrared spectroscopy (FTIR) and thermogravimetric (TGA) methods augmented by antimicrobial and antioxidant studies, Table 1. Crystal structure of 1 was determined by the single crystal X-ray diffraction technique, Table 2.  Complex 1 crystallizes in the centrosymmetric, triclinic space group P1 with asymmetric unit containing three independent Mn(II) cations, six coordinating flufenamato ligands augmented with water and ethanol molecules in the inner coordination sphere. Additionally, three disordered ethanol molecules are placed in voids out of the manganese coordination spheres (Figure 1a). All metal ions are six coordinated and exhibit twisted octahedral geometry. Deprotonated HFluf ligands (Fluf) link manganese cations by complexation with carboxylate groups. Two of them adopt a bidentate (µ 2 :η 1 :η 1 ) binding mode while the remaining four ligands are tridentate (µ 2 :η 2 :η 1 ). In particular, the central Mn1 ion is surrounded by six single oxygen O1A, O1B, O1C, O1D, O1E, O2F atoms from six diverse symmetrically independent carboxylate groups. Oxygens O1B and O1E occupy axial positions while O1A, O1C, O1D, O2F are equatorial. Parent oxygens of Fluf residues are also involved in coordination with the Mn2 and Mn3 wing atoms. Namely, O2B, O2D, O2F coordinate Mn2 while O2A, O1C, O2E are associated with Mn3. Oxygens O1F and O2C are exclusively connected to Mn2 and Mn3. Furthermore, coordination spheres of the latter ions are completed by O1G water and O1H ethanol molecules, respectively. Atoms O1C, O2B, O2E and O2F are bridging between neighboring Mn1-Mn2, Mn2-Mn2[1-x, 1-y, -z], Mn3-Mn3[1-x, 1-y, 1-z] and Mn1-Mn3 metal centers, respectively. In the crystal, those coordination bonds are multiplied by symmetry and form infinite, polymeric ribbons which expand along the [001] dimension (Figure 1b- Crystal is stabilized by an extensive network of inter-and intramolecular hydrogen bonds. Especially, strong intramolecular interactions are formed between amine groups (N1A, N1B, N1C, N1D, N1E and N1F) and the oxygen (O1A, O1B, O2C, O1D, O1E and O1F) atoms of carboxylate group of flufenamato ligands, Table 3. Hydrogen of the coordinated water molecule participates in the hydrogen bond O1G-H1G … O1I with the hydroxyl group of the neighboring ethanol molecule which is also involved in O1I-H1I … O1KA bonding with the consecutive ethanol molecule. The hydrogen H1KA of the latter species forms O1KA-H1KA … O1L bond with the third disordered ethanol molecule which is located in the void. Those interactions are replicated by the crystal symmetry and form chain (13) [39,40] which extends along the [010] dimension. The O1K and O1L ethanols are associated through the O1L-H1L … O1KA hydrogen bond which is transformed by the inversion center to form ring additionally stabilized by the chain O1KA-H1KA … O1L interaction, (Figure 1c).  Table 3. Hydrogen bond geometry in 1 (Å,°).

Hirshfeld Surface Analysis of the Mn 2+ Centers
The structural investigations of 1 have been augmented by the Hirshfeld Surface (HS) analysis (University of Western Australia, Crawley, Australia) of symmetrically independent metal centers Mn1, Mn2 and Mn3. This approach has been successfully applied for metal complexes by Pinto et al. [41] and Abendrot et al. [42]. As shown in the Figure 2, HS of the central Mn1 is diverse than those generated for wing atoms Mn2 and Mn3. Their shapes reflect the differences in coordination of all Mn ions. Symmetry codes: ( a ) 1-x, 1-y, -z.

Hirshfeld Surface Analysis of the Mn 2+ Centers
The structural investigations of 1 have been augmented by the Hirshfeld Surface (HS) analysis (University of Western Australia, Crawley, Australia) of symmetrically independent metal centers Mn1, Mn2 and Mn3. This approach has been successfully applied for metal complexes by Pinto et al. [41] and Abendrot et al. [42]. As shown in the Figure 2, HS of the central Mn1 is diverse than those generated for wing atoms Mn2 and Mn3. Their shapes reflect the differences in coordination of all Mn ions.

Near Infrared Spectra of 1-6
The infrared spectra of 1-6 were collected within the range 4000 and 400 cm −1 and are shown in the  Figures S1-S6). The parameter ∆ν(CO2) = νas -νs is slightly smaller for all complexes than that for sodium fenamate (∆ν(CO2) = 192 cm -1 ) [14] and, according to the well-recognized Nakamoto [43] criteria, the coordination mode of carboxylate groups is the bidentate chelating.  Figure 5 shows the TG/DTG/DTA curves of 1-5, indicating that their decompositions are multistage processes. Compounds 2-5 start to decompose through the dehydration process at the 70-170 °C temperature range. It is associated with the loss of a sole water molecule as in 2, 3, 4

Near Infrared Spectra of 1-6
The infrared spectra of 1-6 were collected within the range 4000 and 400 cm −1 and are shown in the Figure 4. All complexes exhibit a broad absorption band 3300-3600 cm −1 which is assigned to the ν(OH) stretching vibrations of either water or ethanol molecules. Another appearance in this region were bands from ν(NH) stretching vibrations of flufenamato ligands.

Near Infrared Spectra of 1-6
The infrared spectra of 1-6 were collected within the range 4000 and 400 cm −1 and are shown in the Figure 4. All complexes exhibit a broad absorption band 3300-3600 cm -1 which is assigned to the ν(OH) stretching vibrations of either water or ethanol molecules. Another appearance in this region were bands from ν(NH) stretching vibrations of flufenamato ligands.
The characteristic bands at 1651 and 1255 cm -1 are attributed to the respective ν(C=O) and ν(C-O) stretching vibrations of the carboxylic group in the free Hfluf. In 1-5, they are shifted towards ranges 1586-1580 and 1399-1391 cm -1 which correspond to νas(COO − ) and νs(COO − ) stretching vibrations, respectively. Both are affected by ligands coordination to metal ions. Additionally, in the IR spectra of 1-5 there exist 460-490 cm -1 bands associated with metal-oxygen vibrations ( Figures S1-S6). The parameter ∆ν(CO2) = νas -νs is slightly smaller for all complexes than that for sodium fenamate (∆ν(CO2) = 192 cm -1 ) [14] and, according to the well-recognized Nakamoto [43] criteria, the coordination mode of carboxylate groups is the bidentate chelating.  Figure 5 shows the TG/DTG/DTA curves of 1-5, indicating that their decompositions are multistage processes. Compounds 2-5 start to decompose through the dehydration process at the 70-170 °C temperature range. It is associated with the loss of a sole water molecule as in 2, 3, 4 (Figures S1-S6). The parameter ∆ν(CO 2 ) = ν as -ν s is slightly smaller for all complexes than that for sodium fenamate (∆ν(CO 2 ) = 192 cm −1 ) [14] and, according to the well-recognized Nakamoto [43] criteria, the coordination mode of carboxylate groups is the bidentate chelating. Figure 5 shows the TG/DTG/DTA curves of 1-5, indicating that their decompositions are multistage processes. Compounds 2-5 start to decompose through the dehydration process at the 70-170 • C temperature range. It is associated with the loss of a sole water molecule as in 2, 3, 4 and 5 (mass loss exp. 2.12%, 2.72%, 3.70%, 2.30% and calc. 2.57%, 2.57%, 2.81%, 2.80%, respectively). The corresponding exo-effects were identified on respective DTA curves. Complex 1 begins to decompose by elimination of water together with all ethanol molecules (exp. 10.57% and calc. 9.88%) at 120-220 • C. It is accompanied by the endo-and egzo-thermic effects on the DTA curve. The following step in 3 (250-400 • C) corresponds to elimination one water and a single ethanol molecules (exp. 8.66% and calc. 9.15%). In 2, it is additionally combined (200-400 • C) with the release of a half-flufenamate ligand (exp. 28.27% and calc. 29.12%). The characteristic exo-and endothermic effects related to the latter transformations are clearly visible on DTA curves. The second step of 1 and 4 decomposition (220-600 and 150-550 • C) is assigned to a ligand destruction in a single process (exp. 78.30%, calc. 78.82% and exp. 83.56%, calc. 84.80%), respectively. On the contrary, a two-step flufenamato elimination (250-600 • C)

Antioxidant and Antimicrobial Activities
The antibacterial, antifungal and antioxidant activities have been determined for all complexes 1-5 and additionally for the free Hfluf 6 which was taken as a reference. The in vitro antimicrobial activity has been evaluated against several bacterial and fungal reference strains (Table 4).

Antioxidant and Antimicrobial Activities
The antibacterial, antifungal and antioxidant activities have been determined for all complexes 1-5 and additionally for the free Hfluf 6 which was taken as a reference. The in vitro antimicrobial activity has been evaluated against several bacterial and fungal reference strains (Table 4). Vancomycin (Van), ciprofloxacin (Cip) and nystatin (Nys) were used as the standard drugs. In this study, minimum inhibitory concentrations (MIC s) < 1000 mg/L were considered as noteworthy [44]. No bioactivity of tested compounds was observed for Gram-negative reference strains (MIC > 1000 mg/L) except for P. mirabilis strain which had inhibited growth in concentration of 250 mg/L for all tested compounds. From those findings, it was revealed that good bioactivity of tested compounds (MIC 26-125 mg/L) against most of Gram-positive reference bacteria was comparable to that of free ligand and was not affected by the coordinated metal ions. All compounds showed moderate activity (MIC 126-500 mg/L) against S. mutans and E. faecalis strains. Either free ligand or complexes demonstrated comparable, moderate antifungal activity against Candida spp. reference strains.
The DPPH radical scavenging activities (RSA) are low with the exception of 4 which exhibits noticeable RSA (60.72%).

Preparation of Complexes and Crystallization
Compounds 1-5 were synthesized by the following procedure. Flufenamic acid (2 mmol) was dissolved in 30 mL of a freshly prepared aqueous-ethanol solution (v/v 1:2) of NaOH (0.02 mol·L −1 ). Then, sodium flufenamate was heated up to 60 • C and subsequently added to an aqueous-ethanol solution (v/v 1:2) of respective metal chlorides (1.0 mol in 15 mL) heated to the same temperature. Reaction mixtures were carried out at 50 • C for 3 h. After a few days, the polycrystalline powders of 2-5 were isolated by filtration, washed with a hot water, and dried in air. Green single crystals of 1 suitable for X-ray structure analysis were obtained from mother liquor after one week. Numerous attempts to obtain single crystals of 2-5 were unsuccessful.
The chemical compositions of 1-5 were determined by the elemental analysis and flame atomic absorption spectrometry (FAAS) (Complexes (20 mg) were mineralized by the Anton Paar Multiwave 3000 closed microwave system (Anton Paar GmbH, Graz, Austria); the mixture of concentrated HNO 3 and HCl (6:1, v/v) was applied. Metals concentration were measured by the HR-CS FAAS with the ContrAA 300 Analytik Jena spectrometer (Analytik Jena, Jena, Germany) plus spectrometer. Hydrogen, carbon and nitrogen contents were measured with the Vario EL III Elemental Analyzer (Elementar Analysensysteme GmbH, Langenselold, Germany).
Thermal stability and decomposition pathways of 1-5 were studied by thermogravimetric techniques. All complexes were measured with the Netzsch TG 209 apparatus (Netzsch, Selb, Germany). Samples (10 mg) were heated up to 1000 • C, at a heating rate 10 • C min −1 in air atmosphere.
X-ray data were collected at 100 K on the XtaLAB Synergy, Dualflex, Pilatus 300K diffractometer apparatus (Rigaku Corporation, Tokyo, Japan) equipped with the PhotonJet microfocus X-ray tube apparatus (Rigaku Corporation, Tokyo, Japan). Data reduction was performed with CrysAlisPro (Agilent Technologies UK Ltd., Yarnton, England) [45]. The structure was solved by Intrinsic Phasing as implemented in ShelXT [46] and further refined on F 2 in ShelXL [47] by full-matrix least-squares minimalization. Non-hydrogen atoms were refined anisotropically. Four of six trifluoromethyl groups are rotationally disordered. Sites of disordered hydrogen atoms were identified as peaks on difference Fourier map and divided into groups using PART instruction. Their occupancy ratios were determined by the refinement of respective free variables. Geometrical parameters of disordered -CF 3 groups were constrained to geometrically and spatially reasonable values using DFIX, DANG and SAME procedures. Moreover, atomic displacement ellipsoids of disordered fluorine atoms were restrained using SIMU, DELU and RIGU procedures. Anisotropic displacement parameters of fluorine atoms in molecule A were constrained in pairs by the EADP procedure. Hydrogen atoms were placed in calculated positions and refined riding on their parent atoms. The complex 1 crystallizes with three ethanol molecules placed in voids out of the Mn coordination sphere. The residual electron density map suggested substantial ethanol deficiency in the crystal. Freely refined occupancies of all three ethanol molecules add up to approximately 2.5 molecules per asymmetric unit. To validate that non-stoichometric number, the structure was subjected to the PLATON/SQUEEZE [48] procedure. The total potential solvent accessible void volume was ca. 577.7 Å 3 , the electron count per unit cell was 118 which corresponds to 59 electrons per asymmetric unit. Bearing in mind that ethanol molecule has 26 electrons, the latter is equivalent to 2.3 ethanol species per asymmetric unit. This number is approximately equal to the sum of refined ethanol occupancies.
Molecular plots and packing diagrams were drawn using Mercury [49]. Molecular geometry parameters were computed with PLATON [50].
Hirshfeld Surfaces of metallic centers Mn1, Mn2 and Mn3 were generated using the CrystalExplorer 17.5 program (University of Western Australia, Crawley, Australia). Molecular geometries were the same as in crystal structures. Only major components of crystallographic disorders were considered. On the generated HSs of Mn 2+ centers, the normalized contact distances (d norm ) were mapped. The explored d norm function is defined as the sum of the distances from the HS to the nearest atom interior and exterior to the surface (d i and d e , respectively). Distances (d i and d e ) were calculated and plotted as scattergrams, namely Fingerprint Plots (FPs) [51][52][53][54]. A quantitative decomposition analysis of atom to surface contacts was calculated as a percentage of the points in the Hirshfeld Surface with d i and d e for specific atom pairs.
The CIF file for 1 is available from the Cambridge Crystallographic Data Centre (CCDC) (deposition numbers CCDC: 2019658).
The antibacterial and antifungal activities were screened for 1-6 via the micro-dilution broth method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (www.eucast.org). Minimal inhibitory concentrations (MIC) of the tested compounds were evaluated for the panel of the reference microorganisms from the American Type Culture Collection (ATCC), including Gram-negative bacteria ( The antibacterial activities of 1-6 and their radical scavenging activity (RSA) were measured by method as described by us in detail [28].

Conclusions
In this contribution we synthesized and thoroughly investigated metal [Mn(II), Co(II), Ni(II), Cu(II) and Zn(II)] complexes with flufenamic acid. The structure of [Mn 3 (fluf) 6 (H 2 O)(EtOH)]·3EtOH was determined by X-ray crystallography. Asymmetric unit of that crystal contains three independent Mn(II) cations, six coordinated flufenamato ligands augmented with water and ethanol molecules in the inner coordination sphere. Additionally, three disordered ethanol molecules are placed in voids. Flufenamato ligands are bound to manganese ions by carboxylato groups which adopt distinct binding modes. Manganese atoms form infinite, polymeric 1-D chains which extend along the [001] dimension. Their Hirshfeld Surface analysis highlighted reduction of Mn . . . O contacts contribution within coordination spheres prompted by replacement of carboxylate ligands with water and ethanol as in Mn2 and Mn3, respectively. This study firmly showed that geometrical properties mapped on the HS of the metal centers in crystals may be a useful tool to identify subtle effects in the metal coordination spheres.
The investigated compounds exhibited moderate to good activities against Gram-positive bacteria and yeasts. Moreover, the Cu(II) complex is a good antioxidant scavenger as determined in the experiment with the DPPH free radical.