Syntheses and Structural Investigations of Penta-Coordinated Co(II) Complexes with Bis-Pyrazolo-S-Triazine Pincer Ligands, and Evaluation of Their Antimicrobial and Antioxidant Activities

Two penta-coordinated [Co(MorphBPT)Cl2]; 1 and [Co(PipBPT)Cl2]; 2 complexes with the bis-pyrazolyl-s-triazine pincer ligands MorphBPT and PipBPT were synthesized and characterized. Both MorphBPT and PipBPT act as NNN-tridentate pincer chelates coordinating the Co(II) center with one short Co-N(s-triazine) and two longer Co-N(pyrazole) bonds. The coordination number of Co(II) is five in both complexes, and the geometry around Co(II) ion is a distorted square pyramidal in 1, while 2 shows more distortion. In both complexes, the packing is dominated by Cl…H, C-H…π, and Cl…C (anion-π stacking) interactions in addition to O…H interactions, which are found only in 1. The UV-Vis spectral band at 564 nm was assigned to metal–ligand charge transfer transitions based on TD-DFT calculations. Complexes 1 and 2 showed higher antimicrobial activity compared to the respective free ligand MorphBPT and PipBPT, which were not active. MIC values indicated that 2 had better activity against S. aureus, B. subtilis, and P. vulgaris than 1. DPPH free radical scavenging assay revealed that all the studied compounds showed weak to moderate antioxidant activity where the nature of the substituent at the s-triazine core has a significant impact on the antioxidant activity.


Introduction
Triazine is a prototypal molecule that has, together with its derivatives, a wide commercial uses, for example, in resins, dyes, herbicides, or as sulfide removal agents [1,2]. These compounds are a well-suited model system in molecular imprinting [3]. s-Triazines are widely used within the pharmaceutical, textile, plastic, and rubber industries, and as pesticides, dyestuffs, optical bleaches, explosives, and surface-active agents [4][5][6]. s-Triazine and its compounds are also used as subunits in the formation of supramolecular structures because they possess good optical and electronic properties and are able to form three strong hydrogen bonds with the host molecule [7].
The packing of complex molecules in 1 is controlled by O1…H11 and Cl1…H6 contacts shown in Figure 3 (upper part). The donor (D)-acceptor (A) distances are 3.503(3) and 3.550(2) Å for O1…H11 and Cl1…H6 hydrogen bond contacts, respectively ( Table 3). Figure 3 (lower part).  The packing of complex molecules in 1 is controlled by O1 . . . H11 and Cl1 . . . H6 contacts shown in Figure 3    The [Co( Pip BPT)Cl2] complex (2) crystallized in the less symmetric triclinic crystal system and P-1 space group with Z = 4 and two molecular units as an asymmetric formula. In both units, the Co(II) is penta-coordinated with CoN3Cl2 coordination sphere comprising one Pip BPT ligand chelating the Co(II) ion in a pincer fashion augmented with two Co-Cl bonds at almost equal distances ( Figure 4). Generally, the bond distances and angles of the two asymmetric formulas are very similar (   The [Co( Pip BPT)Cl 2 ] complex (2) crystallized in the less symmetric triclinic crystal system and P-1 space group with Z = 4 and two molecular units as an asymmetric formula. In both units, the Co(II) is penta-coordinated with CoN 3 Cl 2 coordination sphere comprising one Pip BPT ligand chelating the Co(II) ion in a pincer fashion augmented with two Co-Cl bonds at almost equal distances ( Figure 4). Generally, the bond distances and angles of the two asymmetric formulas are very similar (  The packing of complex molecules in 2 is controlled by Cl2…H6A, Cl2…H8A, and Cl2…H13A contacts shown in Figure 5 (upper part). The donor (D)-acceptor (A) distances are 3.524(2), 3.768(2) and 3.611(2) Å, respectively ( Table 3). The hydrogen-bonding network in which the complex units are interconnected via C-H…Cl interactions as shown in Figure 5 (lower part).

Hirshfeld Topology Analyses
In order to further explore the different intermolecular contacts in the solid-state structure of the studied complexes, we employed Hirshfeld calculations (Figures S1-S3; Supplementary Data). Quantitative analysis results of all possible interactions are presented in Figure 6.

Hirshfeld Topology Analyses
In order to further explore the different intermolecular contacts in the solid-state structure of the studied complexes, we employed Hirshfeld calculations (Figures S1-S3; Supplementary Data). Quantitative analysis results of all possible interactions are presented in Figure 6.   (Figures 7 and 8). The shortest intermolecular contacts are listed in Table 4. In complex 1, the packing is controlled by O…H and Cl…H hydrogen bonds as well as C-H…π and Cl…C (anion-π stacking) interactions. The latter belongs to the interaction between the coordinated chloride anion Cl2 and the C1 atom from the electron-deficient s-triazine moiety. In complex 2, the packing is also dominated by short Cl…H, C-H…π and Cl…C (anion-π stacking) interactions. It is noted that the Cl2…C1 (3.297 Å) in complex 1 is significantly shorter than the Cl2A…C3 (3.407 Å) and Cl2A…C2 (3.412 Å) interactions in complex 2. The H…C(π-system) are in the range of 2.642-2.751 and 2.722-2.727 Å in complexes 1 and 2, respectively. Also, the C…C/C…N contacts having longer distances than the vdWs radii sum of the interacting elements indicated weak π-π interactions.    (Figures 7 and 8). The shortest intermolecular contacts are listed in Table 4. In complex 1, the packing is controlled by O…H and Cl…H hydrogen bonds as well as C-H…π and Cl…C (anion-π stacking) interactions. The latter belongs to the interaction between the coordinated chloride anion Cl2 and the C1 atom from the electron-deficient s-triazine moiety. In complex 2, the packing is also dominated by short Cl…H, C-H…π and Cl…C (anion-π stacking) interactions. It is noted that the Cl2…C1 (3.297 Å) in complex 1 is significantly shorter than the Cl2A…C3 (3.407 Å) and Cl2A…C2 (3.412 Å) interactions in complex 2. The H…C(π-system) are in the range of 2.642-2.751 and 2.722-2.727 Å in complexes 1 and 2, respectively. Also, the C…C/C…N contacts having longer distances than the vdWs radii sum of the interacting elements indicated weak π-π interactions.

The FTIR spectra of [Co( Morph BPT)Cl 2 ] (1) and [Co( Pip BPT)Cl 2 ]
(2) showed some variations compared to the free ligands. The free Morph BPT and Pip BPT showed the C=N stretching modes at 1609 and 1603 cm −1 , respectively. The corresponding values in complexes 1 and 2 showed significant shifts toward higher wavenumbers of 1633 and 1636 cm −1 , respectively, due to the coordination of the Co(II) with the pincer ligand. Additionally, the ν C = C modes in the free ligands were observed at 1529 and 1512 cm −1 for Morph BPT and Pip BPT, respectively. The ν C = C modes are also significantly shifted to higher wave numbers of 1589 and 1597 cm −1 in [Co( Morph BPT)Cl 2 ] (1) and [Co( Pip BPT)Cl 2 ] (2), respectively. The presentation of the calculated vibrational spectra of complex 2 compared with the experimental FTIR spectra is shown in Figure 9. The results indicated two sharp bands at 1670.7 and 1558.1 cm −1 with relatively high intensity corresponding to the mixed C=N and C=C stretching vibrations. A comprehensive comparison of the experimental and calculated vibrational characteristics for complex 2 is provided in Table

Electronic Spectra
The electronic spectra of 4 × 10 −3 M solution of complex 2 were recorded in ethanol as solvent. The experimentally observed UV-Vis spectra along with the simulated electronic spectra calculated using the TD-DFT method for complex 2 are shown in Figure 10. The recorded electronic spectra showed a broad spectral band at 564 nm, which was calculated at 589.9 nm.

Electronic Spectra
The electronic spectra of 4 × 10 −3 M solution of complex 2 were recorded in ethanol as solvent. The experimentally observed UV-Vis spectra along with the simulated electronic spectra calculated using the TD-DFT method for complex 2 are shown in Figure 10. The recorded electronic spectra showed a broad spectral band at 564 nm, which was calculated at 589.9 nm.

Electronic Spectra
The electronic spectra of 4 × 10 −3 M solution of complex 2 were recorded in ethanol as solvent. The experimentally observed UV-Vis spectra along with the simulated electronic spectra calculated using the TD-DFT method for complex 2 are shown in Figure 10. The recorded electronic spectra showed a broad spectral band at 564 nm, which was calculated at 589.9 nm. In order to assign the origin of this electronic spectral band, the calculated excited and ground states included in this spectral band are shown in Figure 11. The band observed in the visible region could be assigned to electronic transitions from HOMO, HOMO-1, HOMO-9, and HOMO-10 as ground states to LUMO+4 as an excited state where all are βtype orbitals. This electronic transition could be described as mainly metal-ligand ( Pip BPT) charge transfer-based transition. In order to assign the origin of this electronic spectral band, the calculated excited and ground states included in this spectral band are shown in Figure 11. The band observed in the visible region could be assigned to electronic transitions from HOMO, HOMO-1, HOMO-9, and HOMO-10 as ground states to LUMO+4 as an excited state where all are β-type orbitals. This electronic transition could be described as mainly metalligand ( Pip BPT) charge transfer-based transition.

Antimicrobial Activity
The biological activity of the free ligands ( Morph BPT and Pip BPT), as well as the Co(II) complexes [Co( Morph BPT)Cl2] (1) and [Co( Pip BPT)Cl2] (2), were evaluated against S. aureus and B. subtilis as Gram-positive bacteria, E. coli and P. vulgaris as Gram-negative bacteria and two fungi (A. fumigatus and C. albicans). Minimum inhibition zone diameters were determined for the studied compounds (10 mg/mL) and the results are listed in Table 5.

Antimicrobial Activity
The biological activity of the free ligands ( Morph BPT and Pip BPT), as well as the Co(II) complexes [Co( Morph BPT)Cl 2 ] (1) and [Co( Pip BPT)Cl 2 ] (2), were evaluated against S. aureus and B. subtilis as Gram-positive bacteria, E. coli and P. vulgaris as Gram-negative bacteria and two fungi (A. fumigatus and C. albicans). Minimum inhibition zone diameters were determined for the studied compounds (10 mg/mL) and the results are listed in Table 5. The results shown in Table 5 indicated that the free ligands have no antimicrobial activity against all the studied microbes at the applied concentration (10 mg/mL) except Pip BPT, which is active only against the Gram-positive bacteria B. subtilis (13 mm). In contrast, the Co(II) complexes showed interesting antibacterial activities. Complex 1 is active against the two tested Gram-positive bacteria (S. aureus (20 mm) and B. subtilis (24 mm)) and one Gram-negative bacteria (E. coli (16 mm)). On the other hand, complex 2 showed significant antibacterial activities against all the studied bacteria strains with inhibition zone diameters ranging from 15 mm (E. coli) to 30 mm (P. vulgaris). An additional observation that could be concluded from these results; complex 2 has better antibacterial activity against P. vulgaris (30 mm) and very close antibacterial activities against B. subtilis (26 mm) compared to control (gentamycin: 27 mm). Both complexes showed no antifungal activity against the two tested fungi at the experimental conditions. The results indicated that the synthesized Co(II) complexes are promising antibacterial agents rather than antifungal agents.
Moreover, the minimum inhibitory concentrations (MIC) in µg/mL were determined and the results are depicted in Table 6. The results are in accord with our observations. The MIC values are the lowest for complex 2 against B. subtilis, P. vulgaris, and S. aureus indicated potent activities against these microbes. It is also more potent (complex 2; 39 µg/mL) than Pip BPT against B. subtilis (87 µg/mL). Complex 1 has lower potency against the studied bacteria with higher MIC values ranging from 156-625 µg/mL.

Antioxidant Activity
The DPPH free radical scavenging assay enabled us to determine the antioxidant activity of the studied complexes compared to the free ligands. The detailed results are tabulated in Tables S2-S5 (Supplementary Data) and summarized graphically in Figure 12.
Although the results showed that the studied systems have weak to moderate antioxidant activity, especially for the free ligands and complex 2, but the most significant conclusion is that complex 1 has improved antioxidant activity compared to the free ligand Morph BPT while the antioxidant activity of Pip BPT and its [Co( Pip BPT)Cl 2 ]; 2 are comparable indicating that varying the substituent at the s-triazine core of the functional ligand have a significant impact on the antioxidant activity of this class of Co(II) complexes.

Materials and Methods
Chemicals were purchased from Sigma-Aldrich Company (Chemie GmbH, 82024 Taufkirchen, Germany). The CHN analyses were determined using a Perkin-Elmer 2400 instrument (PerkinElmer, Inc., 940 Winter Street, Waltham, MA, USA). Cobalt content was determined using Shimadzu atomic absorption spectrophotometer (AA-7000 series, Shimadzu, Ltd., Kyoto, Japan). An Alpha Bruker spectrophotometer (Billerica, MA, USA) was used to measure the FTIR spectra in KBr pellets ( Figures S4 and S5, Supplementary Data). The FTIR spectra were recorded in the range of 4000-400 cm −1 at a spectral resolution of 2 cm −1 and with 40 scans. The UV-Vis electronic spectra were recorded in ethanol using Pg instruments T80+ spectrophotometer (Alma Park, Wibtoft, UK). The melting points were ascertained in open capillary tubes using a Gallenkamp melting point apparatus (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) and were uncorrected.
A 10 mL methanolic solution of 0.5 mmol of the functional s-triazine chelate was added to 10 mL aqueous solution of the CoCl2 (64.9 mg, 0.5 mmol). Purple color crystals of the titled complexes were obtained after five days. Yield

Materials and Methods
Chemicals were purchased from Sigma-Aldrich Company (Chemie GmbH, 82024 Taufkirchen, Germany). The CHN analyses were determined using a Perkin-Elmer 2400 instrument (PerkinElmer, Inc., 940 Winter Street, Waltham, MA, USA). Cobalt content was determined using Shimadzu atomic absorption spectrophotometer (AA-7000 series, Shimadzu, Ltd., Kyoto, Japan). An Alpha Bruker spectrophotometer (Billerica, MA, USA) was used to measure the FTIR spectra in KBr pellets ( Figures S4 and S5, Supplementary Data). The FTIR spectra were recorded in the range of 4000-400 cm −1 at a spectral resolution of 2 cm −1 and with 40 scans. The UV-Vis electronic spectra were recorded in ethanol using Pg instruments T80+ spectrophotometer (Alma Park, Wibtoft, UK). The melting points were ascertained in open capillary tubes using a Gallenkamp melting point apparatus (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) and were uncorrected.
A 10 mL methanolic solution of 0.5 mmol of the functional s-triazine chelate was added to 10 mL aqueous solution of the CoCl 2 (64.9 mg, 0.5 mmol). Purple color crystals of the titled complexes were obtained after five days.

Antioxidant Activity
The antioxidant activity of complexes 1 and 2 was determined at the Regional Center for Mycology and Biotechnology (RCMB) at the Al-Azhar University by the DPPH free radical scavenging assay in triplicate and average values were considered [63,64]. More details regarding the bio-experiments are found in (Method S3 Supplementary Data).

DFT Calculations
The structure of complex 2 was optimized in the gas phase using the B3LYP method employing 6-31G(d,p) for nonmetal atoms and LANL2DZ for Co [65] with the aid of Gaussian 09 software [66]. All frequency results are positive and no imaginary frequency indicating real minima. The gas-phase optimized structure was used as the input for simulating the structure in ethanol as solvent followed by TD-DFT calculations in the same solvent in order to simulate and assign the experimentally observed UV-Vis spectra [67,68].

Conclusions
Two penta-coordinated Co(II) complexes with bis-pyrazolo-s-triazine pincer ligands bearing morpholino ( Morph BPT) and piperidino ( Pip BPT) substituents were synthesized and their structure aspects were analyzed using single-crystal X-ray diffraction and Hirshfeld analysis. The mononuclear [Co( Morph BPT)Cl 2 ]; 1 and [Co( Pip BPT)Cl 2 ]; 2 pincer complexes have a similar coordination environment comprising a tridentate functional ligand and two coordinated chloride ions. Complex 2 has higher potency against all the studied bacteria (except E. coli) than complex 1. In addition, the antioxidant activity of complex 1 is higher than Morph BPT, while both 2 and Pip BPT have comparable results. These outcomes shed light on the importance of the nature of the substituent on the s-triazine ring of the coordinated functional ligand on the antioxidant activity. The design of s-triazine ligands carrying different substituents could improve the antioxidant activity, which is one of our future perspectives.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.