Quasi-Isostructural Co(II) and Ni(II) Complexes with Mefenamato Ligand: Synthesis, Characterization, and Biological Activity

Three metal complexes of mefenamato ligand 1 were synthesized: [Co2(mef)4(EtOH)2(H2O)4]: 2; [Co(mef)2(MeOH)4]∙2MeOH: 3; and [Ni(mef)2(MeOH)4]∙2MeOH: 4. Their compositions and properties were investigated by elemental analysis (EA), flame atomic absorption spectrometry (FAAS), Fourier-transform infrared spectroscopy (FTIR), and thermogravimetric analysis (TGA). Crystal structures were determined by the single crystal X-ray diffraction technique. Additionally, their antioxidant and antimicrobial activity were established, thus proving good/moderate bioactivity against Gram-positive bacteria and yeasts. In the crystal structure of 2, an apical water molecule is shared between two adjacent cobalt(II) ions, resulting in the formation of a polymeric chain extending along the [100] direction. Meanwhile, structures 3 and 4 have strong intermolecular hydrogen bonds with diverse topologies that yield unique quasi-isostructural arrangements. The packing topology is reflected by the Hirshfeld surface analysis of intermolecular contacts.


Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most popular pharmaceuticals that have been used against inflammation, pain, and fever over the years [1]. Their therapeutic effect is mainly based on their ability to inhibit the conversion of the arachidonic acid to prostaglandins [2]. Unfortunately, NSAIDs exhibit acute side effects and may cause dyspepsia, nausea, abdominal pain, constipation, headache, dizziness, and rash [2,3]. Additionally, several NSAIDs can be effectively applied in treatments of colon, lung, and breast cancers [4,5] due to their profound chemopreventive and chemosuppressive effects [6][7][8].
The structural similarity of crystals [9] plays a vital role in understanding the pharmaceutical activity of solid-state drugs. It is often described by the term isostructurality [10] and is profoundly associated with intermolecular interactions and often prompted by symmetry restrictions. A given packing arrangement may tolerate tiny molecular geometry adjustments while keeping the crystals almost isostructural [11,12]. However, certain crystals of coordination compounds which diverse metal ions are characterized by the same chemical constituents may not be classified as entirely isostructural due to subtle differences in their interaction arrangement [13]. That quasi-isostructurality was observed in systems 3 and 4.

Results and Discussion
Compounds 2, 3, and 4 are air stable at room temperature. They are highly soluble in polar organic solvents like DMF, DMSO, methanol, and ethanol. Their properties were thoroughly investigated by the FAAS, EA, FTIR, and TGA methods augmented by antimicrobial and antioxidant studies; see Table 1. The crystal structures of 2-4 were determined by the single crystal X-ray diffraction technique; see Table 2. The crystals of 3 and 4 are isomorphic. The analytical data were in good agreement with crystal structures of all complexes.  The molecular structure of complex 2 is presented in Figure 1. It crystallizes in the triclinic space group P1 with two symmetrically independent cobalt ions, namely Co1 and Co2, placed in a special positions on inversion centers. Therefore, the single asymmetric unit contains half Co1 and half Co2 ions, two mefenamato moieties, two coordinated waters, and a single coordinated ethanol molecule. Both Co(II) ions are six coordinated, adopt octahedral environments, and share a single apical water O5. This arrangement propagates along the [100] direction, thus leading to a 1-D polymeric structure with a Co1-O5-Co2 repetitive unit ( Figure S1). Similar supramolecular motifs were recently identified and published by us [2].
The presented crystal is stabilized by an extensive network of inter-and intramolecular hydrogen bonds (Table 3). In particular, a strong intramolecular interaction is formed between a sole hydrogen at the coordinating water molecule O6 and the carboxylate O1 of a mefenamato moiety. The hydrogen of the ethanol O7 molecule participates in a hydrogen bond with the carboxylate O3. Moreover, the O5 water molecules are hydrogen donors to bifurcated carboxylate acceptors O2 and O4, and the latter oxygen atoms additionally attract hydrogens of neighboring amine groups N1 and N2A, respectively. The resulting chain motif may be described by the Etter's symbol C 2 4 (9) [41,42]. Hydrogen atoms are omitted for clarity. Displacement ellipsoids are drawn at the 50% probability level. Only the asymmetric unit is labeled, and the major component of disordered atoms are display for clarity. Table 3. Hydrogen bond geometry in 2 (Å,°). The centrosymmetric, triclinic crystals of 3 and 4 ( Figure 2) are quasi-isostructural [43]. Their asymmetric units contain one-half metal ions, a single mefenamato ligand, two coordinated methanol species, and an additional methanol molecule in the outer coordination sphere. The inner coordination spheres of both complexes are quite similar. Octahedral metal ions are placed on inversion centers. They are surrounded by two equatorial mefenamato carboxylate oxygen atoms augmented by two equatorial and two apical methanol molecules. Similarly to 2, the 2-((2,3-dimethylphenyl)amino)benzoate moieties in 3 and 4 adopt twisted conformations with dihedral angles between phenyl rings equal 74.60(3) and 74.38(18)°, respectively. Apical Co-O3A (2.1220(15) Å) and Ni-O3B (2.113(3) Å) bonds involving methanol species are longer than relevant equatorial bonds (Co-O4A and Ni-O4B; 2.0803(13) and 2.051(2) Å, respectively). The equatorial coordination bonds involving the carboxylate O1 atoms of the mefenamato moieties are slightly shorter (2.0556(8) and 2.0207(8) Å for 3 and 4, respectively). A structurally similar complex (Mn(mef)2(MeOH)4) was recently reported by Feng et al. [32]. Following the larger atomic radius of manganese, all bonds within the Mn coordination sphere of this compound are longer than the respective bonds observed in 3 and 4.

D-H … A D-H H … A D … A D-H … A
The quasi-isostructurality of 3 and 4 is driven by the diverse topologies of the strong hydrogen bonds in both crystals; see Tables 4 and 5, respectively. The superposition of both structures is presented in Figure 3. In 3, the carboxylate O2 of the mefanamato moiety is involved in hydrogen bonding with the O4 equatorial methanol molecule. The O5 methanol of the outer coordination  The centrosymmetric, triclinic crystals of 3 and 4 ( Figure 2) are quasi-isostructural [43]. Their asymmetric units contain one-half metal ions, a single mefenamato ligand, two coordinated methanol species, and an additional methanol molecule in the outer coordination sphere. The inner coordination spheres of both complexes are quite similar. Octahedral metal ions are placed on inversion centers. They are surrounded by two equatorial mefenamato carboxylate oxygen atoms augmented by two equatorial and two apical methanol molecules. Similarly to 2, the 2-((2,3dimethylphenyl)amino)benzoate moieties in 3 and 4 adopt twisted conformations with dihedral angles between phenyl rings equal 74.60(3) and 74.38(18) • , respectively. Apical Co-O3A (2.1220(15) Å) and Ni-O3B (2.113(3) Å) bonds involving methanol species are longer than relevant equatorial bonds (Co-O4A and Ni-O4B; 2.0803(13) and 2.051(2) Å, respectively). The equatorial coordination bonds involving the carboxylate O1 atoms of the mefenamato moieties are slightly shorter (2.0556(8) and 2.0207(8) Å for 3 and 4, respectively). A structurally similar complex (Mn(mef) 2 (MeOH) 4 ) was recently reported by Feng et al. [32]. Following the larger atomic radius of manganese, all bonds within the Mn coordination sphere of this compound are longer than the respective bonds observed in 3 and 4.
sphere is connected to the axial O3. A diverse situation is observed in 4, where carboxylate O2 interacts with axial O3, while equatorial O4 is involved in H bonding with O5. The latter is a hydrogen donor for contact with carboxylate O2 in either 3 or 4. The resulting chain may be characterized by motif 7 ( Figure S2). The conformation of mefanamato moieties in all investigated complexes 2-4 is stabilized by the intramolecular interactions of the amine nitrogen atoms with carbonyl groups.   Table 4. Hydrogen bond geometry in 3 (Å,°). Symmetry codes: ( a )-1+x, y, z;. Table 5. Hydrogen bond geometry in 4 (Å,°). (7) 2.12 (7) 2.732 (6) 131 (8) Symmetry codes: ( a ) 1-x, 1 -y, 1-z; ( b ) -1+x, y, z; The quasi-isostructurality of 3 and 4 is driven by the diverse topologies of the strong hydrogen bonds in both crystals; see Tables 4 and 5, respectively. The superposition of both structures is presented in Figure 3. In 3, the carboxylate O2 of the mefanamato moiety is involved in hydrogen bonding with the O4 equatorial methanol molecule. The O5 methanol of the outer coordination sphere is connected to the axial O3. A diverse situation is observed in 4, where carboxylate O2 interacts with axial O3, while equatorial O4 is involved in H bonding with O5. The latter is a hydrogen donor for contact with carboxylate O2 in either 3 or 4. The resulting chain may be characterized by motif C 3 3 (7) ( Figure S2). The conformation of mefanamato moieties in all investigated complexes 2-4 is stabilized by the intramolecular interactions of the amine nitrogen atoms with carbonyl groups.   Figure S2). The conformation of mefanamato moieties in all investigated complexes 2-4 is stabilized by the intramolecular interactions of the amine nitrogen atoms with carbonyl groups.    (11) 134(5) Figure 3. Superposition of structures 3 (red) and 4 (green). Hydrogen atoms and methanol O5 molecules in the outer coordination sphere are omitted for clarity.

Crystal Packing Analysis of 2-4
The molecular shapes of structures 2-4 were visualized as Hirshfeld surfaces (HSs) (Figure 4). In 2, the mer unit of infinite polymeric chain was selected for the HS visualization. Relevant fingerprint plots (FPs) derived from the HSs were generated for each structure to characterize the propensity of these species to form particular intermolecular contacts. Crystal packing arrangements in all structures may be defined by three types of contacts, namely H . The molecular shapes of structures 2-4 were visualized as Hirshfeld surfaces (HSs) (Figure 4). In 2, the mer unit of infinite polymeric chain was selected for the HS visualization. Relevant fingerprint plots (FPs) derived from the HSs were generated for each structure to characterize the propensity of these species to form particular intermolecular contacts. Crystal packing arrangements in all structures may be defined by three types of contacts, namely H … H, C … H, and O … H. Additionally, the fingerprint analysis revealed the limited presence of N … H contacts, as indicated by their contributions below 1%. Due to the polymeric character of 2, the H … H contacts contribution of 63.7% is notably lower than that in 3 and 4. The latter approach 70.2% and 70.4%, respectively. The FPs of all systems feature symmetrical wings that visualize C … H contacts and follow the C-H … π and O-H … π interactions. The contribution of C … H contacts in the crystal packing is visibly higher for polymeric structure 2 (26.2%) than those in 3 and 4 (21.9% and 22.4%, respectively). Similarly, the O … H contacts contributions over all structures range from 7.0% to 8.2%. These contacts are depicted as sharp spikes for 2, while for 3 and 4, those spikes are slightly bold. The latter presumable follow the methanol molecule disorder in the crystal structures of 3 and 4. Interestingly, a difference in the intermolecular hydrogen bond topology is also apparent in the O … H contact contribution. Structure 3, where axial O3 is a hydrogen donor for the outer O5 methanol, is characterized by an O … H contacts contribution of 7.0%. The equatorial position of the relevant hydrogen bond donor in 4 results in an increase of the O … H contacts' contribution to 7.7%.
The fingerprint analysis showed that supramolecular systems of 2-4 are defined by a relatively similar set of contacts. The most distinguished is a polymeric system in 2. Nonetheless, the  The fingerprint analysis showed that supramolecular systems of 2-4 are defined by a relatively similar set of contacts. The most distinguished is a polymeric system in 2. Nonetheless, the quantitative analysis of intermolecular contacts helped to expose the subtle differences in supramolecular assemblies, as observed in the quasi-isostructural systems of 3 and 4.

Infrared Spectra of 2-4
All complexes exhibit sharp bands in the range of 3290-3305 cm −1 and weak broad absorption bands in the region of 2900-3200 cm −1 associated with the ν(NH) and ν(OH) stretching vibrations, respectively ( Figure S3). Additionally, bands at 3610-3630 cm −1 for 3 and 4 are assigned to the ν(OH) stretching vibration of methanol molecules. The characteristic bands originated from carboxylate group appear in the range of 1350-1700 cm −1 . In particular, sharp absorption peaks appearing at 1610.3, 1604.5, and 1604.5 cm −1 correspond to ν as (COO -) and peaks at 1380.8, 1382.8, and 1384.3 cm −1 are assigned to ν s (COO -) stretching vibrations of carboxylate group for 2, 3, and 4, respectively. The parameter ∆ν=ν as (COO -) -ν s (COO -) is larger than that for sodium mefenamate [40], and according to Nakamoto [44], the criteria the coordination mode of carboxylate groups may be described as monodentate.

Thermal Analysis of 2-4
The TG/DTG/DTA curves of 2, 3, and 4 are summarized in Figure 5. The decomposition pathways are slightly different for each complex. Compound 2 starts to decompose through the concurrent elimination of the ethanol molecule together with two water molecules (mass loss exp. 12.70% and calc. 13.23%) proceeding at the temperature range of 80-250 • C. It is accompanied by endo-and exo-thermic effects on the DTA curve. The next step represents the mass loss (exp. 12.70% and calc. 13.23%) associated with the destruction of two mefenamate ligands at the temperature range of 250-480 • C. The latter leads to the final product Co 3 O 4 (exp. 12.93% and calc. 12.57%). The corresponding two endo-thermic and one exo-thermic effects were also identified and are shown on the DTA curve.

Antioxidant and Antimicrobial Activities
The antibacterial, antifungal, and antioxidant activities were determined for complexes 2-4 and, additionally, 5-10 published by us [40]. The above three activities were also analyzed, for the mefenamic acid 1 taken as a reference.
The investigated complexes exhibited visible RSAs (i.e., higher than that of 1), with those of 5, 4, 2, and 8 being the highest. The final antioxidant activities are in the order of 5 > 4 > 2 > 8 > 3 > 7 > 9 > 10 > 6 ( Figure 6). Similar results were reported by Altun and Suözer [45], who pointed out that the electron withdrawing effect of the metal(II) ion facilitates the release of hydrogen, which further reduces the DPPH radical. The thermal decomposition of 3 and 4 begins with the release of methanol molecules. In 3, it proceeds in one step, while two distinguished steps were identified for 4. The weight loss of the former (exp. 26.04% and calc. 26.27%) at 60-260 • C is consistent with the elimination of six methanol molecules altogether. In 4, two methanol molecules are lost at the temperature range of 100-180 • C (exp. 9.87% and calc. 8.76%), while further four molecules are released at 180-430 • C (exp. 18.13% and calc. 17.52%). The DTA curves exhibit characteristic exo-effects related to those steps. In 4, both symmetry-related O5 methanols from the outer coordination sphere form weaker hydrogen bonds with the carbonyl O2 than the relevant molecules in 3 (Tables 4 and 5, respectively). Presumably, the former facilitates a two-step methanol elimination with premature O5 release, as observed in 4.
Further heating leads to the destruction of both mefenamato moieties in a single step.

Antioxidant and Antimicrobial Activities
The antibacterial, antifungal, and antioxidant activities were determined for complexes 2-4 and, additionally, 5-10 published by us [40]. The above three activities were also analyzed, for the mefenamic acid 1 taken as a reference.
The investigated complexes exhibited visible RSAs (i.e., higher than that of 1), with those of 5, 4, 2, and 8 being the highest. The final antioxidant activities are in the order of 5 > 4 > 2 > 8 > 3 > 7 > 9 > 10 > 6 ( Figure 6). Similar results were reported by Altun and Suözer [45], who pointed out that the electron withdrawing effect of the metal(II) ion facilitates the release of hydrogen, which further reduces the DPPH radical.    The results of the antibacterial and antifungal activities are presented in Table 6. Vancomycin (Van) and nystatin (Nys) were used as the standard drugs. Notably, the tested derivatives showed no bioactivity against Gram-negative bacteria (E. coli ATCC 25922, S. Typhimurium ATCC 14028, K. pneumoniae ATCC 13883, P. aeruginosa ATCC 9027, and P. mirabilis activity of the tested compounds [46]. The antibacterial efficiency of the tested derivatives was in the order of 10 > 3 > 2 > 6 > 4 > 9 > 7 > 5 > 8 > 1. The antifungal bioactivity (C. albicans ATCC 2091, C. glabrata ATCC 90030, and C. parapsilosis ATCC 22019) of the tested compounds was mild or moderate, with that of 10 being the highest.

Preparation of Complexes and Crystallization
Compounds 2, 3, and 4 were synthesized by the dissolution of mefenamic acid (1 mmol) in 50 mL of a freshly precipitated aqueous-ethanol solution (1:1) of NaOH (0.02 mol·L −1 ). Further, sodium mefenamate was heated up to 60 • C and slowly added to an aqueous solution of metal chlorides (0.5 mol in 25 mL). The reaction mixture was kept at 60 • C for 2 hours. After several days, the polycrystalline powders were isolated by filtration, washed with a hot water, and dried in air [40].
Single crystals of the 2, 3, and 4 complexes suitable for X-ray structure analysis were obtained at room temperature by slow evaporation from the aqueous/ethanol (1:2 v/v) solution for 2 and from a pure methanol for 3 and 4. Good quality crystals appeared after two weeks.

Materials and Methods
Mefenamic acid was purchased from Sigma-Aldrich and used without further purification. Methanol, ethanol, and DPPH (2,2-diphenyl-1-picrylhydrazyl) were purchased from Lab-Scan, and other chemicals were from POCh-Gliwice, Poland. All reagents were chemically pure.
The chemical compositions of 2-4 were determined by the elemental analysis (EA) and flame atomic absorption spectrometry (FAAS). Initially, samples (20 mg) were mineralized by the Anton Paar Multiwave 3000 closed microwave system; a mixture of concentrated HNO 3 and HCl (6:1, v/v) was applied. Metals concentration were measured by the FAAS with the GBC Scientific Equipment 932 Plus spectrometer. Hydrogen, carbon, and nitrogen contents were measured with the Vario EL III Elemental Analyzer.
The infrared spectra of 2-4 were recorded with the Thermo Scientific Nicolet 6700 FTIR spectrometer equipped with liquid nitrogen-cooled MCT (mercury cadmium telluride-HgCdTe) detector. Samples were prepared as KBr pellets and measured over the range of 4000-400 cm −1 .
The thermal stability and decomposition pathways of 2-4 were studied by thermogravimetric techniques. All complexes were measured with a Netzsch TG 209 apparatus. Samples (10 mg) were heated (in ceramic crucibles) up to 1000 • C at a heating rate 10 • C min −1 in an air atmosphere.
The single-crystal X-ray diffraction experiments of 2-4 were performed on a Rigaku XtaLAB Synergy Dualflex Pilatus 300K diffractometer. Measurements were conducted using PhotonJet microfocus X-ray source CuKα (λ = 1.54184 Å) for 2, while MoKα (λ = 0.71073 Å) radiation was used for 3 and 4. Crystals were kept at 100.0(1) K during data collection. The data were integrated using the CrysAlisPro software [47]. All structures were solved using the intrinsic phasing method in ShelXT [48] and refined by the full matrix least squares minimization on F 2 with the ShelXL [49] refinement package. The structure of 2 was refined as a two-component twin with a twin scale factor of 0.458 (1). The structures of 2-4 were notably affected by static disorder, with the mefenamato fragments being disordered over two positions. Relevant occupancy factors were refined to 0.52 (3) . In all structures, the sums of occupancies of relevant sites were set equal to 1 and refined using free variables. The PART instruction was applied to exclude bonding between equivalent disordered atoms. The anisotropic displacement parameters of neighboring disordered atoms were restrained using the SIMU and RIGU procedures in ShelXL. In 2 and 4, a few disordered atoms were additionally fixed with the EADP instruction. The geometries of disordered fragments were restrained using the DFIX and SADI commands, while O-H and N-H hydrogen atoms were located in difference Fourier maps, C-H hydrogens were generated geometrically using the HFIX command, and a riding model was applied for the refinement. Molecular plots and packing diagrams were drawn using Mercury [50]. Geometry parameters were computed with PLATON [51].
The CIF file for 2-4 is available from the Cambridge Crystallographic Data Centre (CCDC) (deposition numbers CCDC: 1989350, 1989353, and 1989354, respectively). Hirshfeld surfaces (HSs) were generated using the CrystalExplorer17 program. Molecular geometries were the same as in crystal structures. For molecular fragments where crystallographic disorder was identified, the major components were only considered. The distances from the HS to the nearest atom interior and exterior to the surface (di and de, respectively) were calculated and plotted as scattergrams [52]. A quantitative decomposition analysis of atom-to-surface contacts was calculated as a percentage of the points in the Hirshfeld surface with di and de for specific atom pairs.
The radical scavenging activities (RSAs) of 1-10 were evaluated by measuring scavenging ability of the DPPH free radical. The solutions were prepared as following: 0.5 mL methanolic solutions of the complexes (0.04, 0.06, 0.08, and 0.1 mol/L) were mixed with the DPPH methanolic solution (60 µM and 0.5 mL) in the dark. The samples were incubated at 25 • C for 30 min in the dark to reach equilibrium before the measurement. The radical scavenging ability of compounds was calculated using the equation: I% = (1 − A s /A 0 ) × 100, where A 0 is the absorbance of the sample at 0 min and A S is the absorbance of the sample at 30 min. The RSA experiment was repeated in triplicate. The average values were noted.
The antibacterial and antifungal activities were screened for 1-10 via the micro-dilution broth method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) using a Mueller-Hinton broth and a Mueller-Hinton broth with 5% lysed sheep blood for the growth of non-fastidious and fastidious bacteria, respectively, or RPMI with MOPS for the growth of fungi. The complexes dissolved in DMSO were first diluted to the concentration of 1000 mg/L in an appropriate broth medium recommended for bacteria or yeasts. Then, using the same media, serial two-fold dilutions were made in order to obtain final concentrations of the tested derivatives ranged from 1.95 to 1000 mg/L. The sterile 96-well polystyrene microtitrate plates (Nunc, Denmark) were prepared by dispensing 200 µL of the appropriate dilution of the tested derivatives in broth medium per well. The inocula were prepared with fresh microbial cultures in sterile 0.85% NaCl to match the turbidity of 0.5 McFarland standard, and 2 µL were added to wells to obtain a final density of 1.5 × 10 6 CFU/mL for bacteria and 5 × 10 4 CFU/mL for yeasts; CFU-colony forming units. After incubation (bacterial strains-35 • C for 24 h; yeast strains-30 • C for 48 h), the MICs were visually assessed as the lowest concentration of the extracts showing the complete growth inhibition of the reference microbial strains. An appropriate DMSO control (at a final concentration of 10%), a positive control (containing inoculum without the tested derivatives), and a negative control (containing the tested derivatives without inoculum) were included on each microplate. Minimal bactericidal concentration (MBC) or minimal fungicidal concentration (MFC) was determined by subculturing 5 µL of the microbial culture from each well that showed growth inhibition, from the last positive one and from the growth control onto the recommended agar plates. The plates were incubated at 35 • C for 24 h, and the MBC/MFC was defined as the lowest concentration of the extracts without the growth of microorganisms. Each experiment was repeated in triplicate. The highest MIC value was noted [53].

Conclusions
In summary, three metal complexes of cobalt and nickel with mefenamato ligand were characterized by X-ray structure analysis combined with elemental and thermal analyses that were further augmented by spectroscopic and biological activity studies. The structure of 3 and 4 are stabilized by strong intermolecular hydrogen bonds of diverse topologies, thus yielding quasi-isostructural arrangements. Their crystal packing formation is reflected by the Hirshfeld surface analysis of intermolecular contacts. The latter shows subtle differences in the O . . . H contacts contributions, which are closely related to diverse hydrogen bonds topologies and thermal decomposition patterns, as observed in 3 and 4. In particular, the higher contribution of O . . . H contacts prompts a single step of methanol molecule elimination. On the contrary, a two-stage elimination can be observed in 4.

Conflicts of Interest:
The authors declare that they have no conflicts of interest.