Synthesis, Structure and Biological Evaluations of Zn(II) Pincer Complexes Based on S-Triazine Type Chelator

2,4-bis (3,5-dimethyl-1H-pyrazol-1-yl)-6-methoxy-1,3,5-triazine (BPMT) pincer ligand was used to synthesize the new [Zn(BPMT)(NCS)2] (1) and [Zn(BPMT)(Br)2] (2) complexes by a reaction with Zn(NO3)2·6H2O in the presence of either KSCN or KBr, respectively. The structure of complex 1 has been exclusively confirmed using single crystal X-ray diffraction. In this neutral heteroleptic complex, the BPMT is a pincer chelate coordinating the Zn(II) ion via three interactions with the two pyrazole moieties and the s-triazine core. Hence, BPMT is a tridentate NNN-chelate. The coordination environment of Zn(II) is completed by two strong interactions with two terminal SCN− ions via the N-atom. Hence, the Zn(II) is penta-coordinated with a distorted square pyramidal coordination geometry. Hirshfeld analysis indicated the predominance of H…H, H…C and N…H intermolecular interactions. Additionally, the S…H, S…C and S…N contacts are the most significant. The free ligand has no or weak antimicrobial, antioxidant and anticancer activities while the studied Zn(II) complexes showed interesting biological activity. Complex 1 has excellent antibacterial activity against B. subtilis (2.4 μg/mL) and P. vulgaris (4.8 μg/mL) compared to Gentamycin (4.8 μg/mL). Additionally, complex 1 (78.09 ± 4.23 µg/mL) has better antioxidant activity than 2 (365.60 ± 20.89 µg/mL). In addition, complex 1 (43.86 ± 3.12 µg/mL) and 2 (30.23 ± 1.26 µg/mL) have 8 and 12 times the anticancer activity of the free BPMT ligand (372.79 ± 13.64 µg/mL).


Introduction
Triazines are known lead compounds which have a diverse range of pharmaceutical applications [1]. These heterocyclic compounds have interesting biological activities as antimicrobial [2], anticancer [3,4], anti-HIV [5,6], and anti-inflammatory activities [1]. Among the different types of triazines, 1,3,5-triazine (s-triazine) is a highly symmetrical molecule. In this regard, the self-assembly of such highly symmetrical ligands with metal salts is a simple way to build interesting supramolecular architectures for different applications, such as gas storage, catalysis, magnetism, nonlinear optics, and luminescence [7][8][9][10]. s-Triazine pincer ligands are an interesting class of polydentate ligands as they form stable complexes with different metal ions [11]. The bis-pyrazolyl-s-triazine ligand shown in Figure 1 attracted our attention due to its simple route of preparation and its interesting coordination chemistry with different metal ions [12][13][14][15].  [11,12].

Synthesis and Characterizations
The reaction of an aqueous solution of Zn(NO3)2.6H2O with an ethanolic solution of the pincer ligand BPMT in presence of either KSCN or KBr afforded two new Zn(II) complexes. Due to the relatively weak coordinating power of the nitrate ion compared with the SCN − and Br − anions, the corresponding [Zn(BPMT)(NCS)2] (1) and [Zn(BPMT)(Br)2] (2) complexes were obtained, respectively. The structures of the two complexes were characterized using FTIR spectra and elemental analysis. The FTIR spectra of both complexes showed the vibrational fundamentals of the ligand BPMT with some observable shifts which confirm the coordination with the Zn(II) ion. The υC=N and υC=C modes were detected at 1593 and 1555 cm −1 , respectively, in the case of the ligand BPMT. The υC=N mode was found to shift to higher wavenumbers of 1626 and 1622 cm −1 in complexes 1 and 2, respectively. On the other hand, the υC=C modes were detected at 1582-1540 cm −1 and 1555, respectively. A strong intense band was detected at 2082 cm −1 confirming the presence of the SCN − in the structure of complex 1. In addition, the molecular and supramolecular structures of 1 are exclusively determined using a single crystal X-ray structure. Additionally, the structure of complex 2 is confirmed using 1 H NMR spectra where two singlets that appeared at  2.37 and 2.69 ppm are related to the two methyls of the pyrazole ring at positions 3 and 5, respectively, and a singlet at  4.13 ppm is related to the methoxy group, which are at almost the same position as in the ligand BPMT; the CH of pyrazole for complex 2 showed some shift (6.52 ppm) compared to the free ligand (6.05 ppm).

Synthesis and Characterizations
The reaction of an aqueous solution of Zn(NO 3  (2) complexes were obtained, respectively. The structures of the two complexes were characterized using FTIR spectra and elemental analysis. The FTIR spectra of both complexes showed the vibrational fundamentals of the ligand BPMT with some observable shifts which confirm the coordination with the Zn(II) ion. The υ C=N and υ C=C modes were detected at 1593 and 1555 cm −1 , respectively, in the case of the ligand BPMT. The υ C=N mode was found to shift to higher wavenumbers of 1626 and 1622 cm −1 in complexes 1 and 2, respectively. On the other hand, the υ C=C modes were detected at 1582-1540 cm −1 and 1555, respectively. A strong intense band was detected at 2082 cm −1 confirming the presence of the SCN − in the structure of complex 1. In addition, the molecular and supramolecular structures of 1 are exclusively determined using a single crystal X-ray structure. Additionally, the structure of complex 2 is confirmed using 1 H NMR spectra where two singlets that appeared at δ 2.37 and 2.69 ppm are related to the two methyls of the pyrazole ring at positions 3 and 5, respectively, and a singlet at δ 4.13 ppm is related to the methoxy group, which are at almost the same position as in the ligand BPMT; the CH of pyrazole for complex 2 showed some shift (6.52 ppm) compared to the free ligand (6.05 ppm).  (Table S1). The unit cell volume is 2190.6 (12) Å 3 and Z = 2. The complex crystallized in the P1c1 space group and two [Zn(BPMT)(NCS) 2 ] complex units as an asymmetric formula.  (Table S1). The unit cell volume is 2190.6 (12) Å 3 and Z = 2. The complex crystallized in the P1c1 space group and two [Zn(BPMT)(NCS)2] complex units as an asymmetric formula. The structure of the [Zn(BPMT)(NCS)2] complex showed a penta-coordinated Zn(II) with one BPMT ligand as a tridentate NNN-pincer chelate and two terminally coordinated thiocyanate anions ( Figure 2). In both molecules found in the asymmetric formula, the bonds Zn1-N7 (2.055 (7) Å ) and Zn2-N14 (2.061 (6) Å ) with the s-triazine core are shorter than the corresponding Zn-N (pyrazole) bonds. In the molecule with lower atom numbering, the Zn1-N9 and Zn1-N3 bonds are found to be 2.191 (7) Å and 2.223 (7) Å , respectively, while in the other unit, the Zn2-N12 and Zn2-N18 are found to be 2.202 (7) and 2.234 (7) Å , respectively. In this complex, the pincer ligand forms two five membered chelate rings with small N-Zn-N bite angles of 72.8 (3)° (N7-Zn1-N9) and 72.9 (3)° (N7-Zn1-N9) in one unit. In the other unit, the bite angles N14-Zn2-N18 and N14-Zn2-N12 are 72.5 (3) and 73.0 (3)°, respectively. The penta-coordination environment of the Zn(II) is completed by two short interactions with two isothiocyanate groups (NSC − ) via the nitrogen atom while the S-atom sets were freely uncoordinated. Hence, the thiocyanate ion acts as an N-terminal ligand. The Zn-N distances with the thiocyanate anions are found to be the shortest among the rest of the Zn-N distances. The Zn1-N1 and Zn1-N2 bond distances are 1.932 (8) Å and 1.938 (9) Å while the corresponding Zn2-N11 and Zn2-N10 bond distances are 1.918 (8) Å and 1.935 (7) Å , respectively. The N1-Zn1-N2 and N10-Zn2-N11 angles are found to be 117.3 (4) and 116.6 (3)°, respectively. In this regard, the ZnN5 coordination environment [25] is considered highly distorted compared to any of the two ideal geometries of the five coordinated systems; the trigonal bipyramidal and square pyramidal configurations. The τ5 parameter ((β − α)/60) was used as a measure for the extent of distortion in the five coordinated metal ion systems. The β and α values are the largest angles in the coordination sphere. Using the results listed in Table 1, the τ5 values were calculated to be 0.355 and 0.278 for Zn1 and Zn2, respectively. Hence, the coordination geometry is more likely to be a distorted square pyramidal for both Zn(II) sites.  The structure of the [Zn(BPMT)(NCS) 2 ] complex showed a penta-coordinated Zn(II) with one BPMT ligand as a tridentate NNN-pincer chelate and two terminally coordinated thiocyanate anions ( Figure 2). In both molecules found in the asymmetric formula, the bonds Zn1-N7 (2.055 (7) Å) and Zn2-N14 (2.061 (6) Å) with the s-triazine core are shorter than the corresponding Zn-N (pyrazole) bonds. In the molecule with lower atom numbering, the Zn1-N9 and Zn1-N3 bonds are found to be 2.191 (7) Å and 2.223 (7) Å, respectively, while in the other unit, the Zn2-N12 and Zn2-N18 are found to be 2.202 (7) and 2.234 (7) Å, respectively. In this complex, the pincer ligand forms two five membered chelate rings with small N-Zn-N bite angles of 72.8 (3) • (N7-Zn1-N9) and 72.9 (3) • (N7-Zn1-N9) in one unit. In the other unit, the bite angles N14-Zn2-N18 and N14-Zn2-N12 are 72.5 (3) and 73.0 (3) • , respectively. The penta-coordination environment of the Zn(II) is completed by two short interactions with two isothiocyanate groups (NSC − ) via the nitrogen atom while the S-atom sets were freely uncoordinated. Hence, the thiocyanate ion acts as an N-terminal ligand. The Zn-N distances with the thiocyanate anions are found to be the shortest among the rest of the Zn-N distances. The Zn1-N1 and Zn1-N2 bond distances are 1.932 (8) Å and 1.938 (9) Å while the corresponding Zn2-N11 and Zn2-N10 bond distances are 1.918 (8) Å and 1.935 (7) Å, respectively. The N1-Zn1-N2 and N10-Zn2-N11 angles are found to be 117.3 (4) and 116.6 (3) • , respectively. In this regard, the ZnN5 coordination environment [25] is considered highly distorted compared to any of the two ideal geometries of the five coordinated systems; the trigonal bipyramidal and square pyramidal configurations. The τ 5 parameter ((β − α)/60) was used as a measure for the extent of distortion in the five coordinated metal ion systems. The β and α values are the largest angles in the coordination sphere. Using the results listed in Table 1, the τ 5 values were calculated to be 0.355 and 0.278 for Zn1 and Zn2, respectively. Hence, the coordination geometry is more likely to be a distorted square pyramidal for both Zn(II) sites.
The supramolecular structure of [Zn(BPMT)(NCS) 2 ] complex is controlled by the S . . . H, S . . . N and S . . . C contacts shown in Figure 3 while the corresponding contact distances are listed in Table 2. The weak S . . . H interactions occurred alternatively between the two molecular units found in the asymmetric formula leading to the 1D chain shown in Figure 4A. The hydrogen-acceptor distances of the S2 . . . H23B and S1 . . . H30 interactions are 2.915 and 3.377 Å, respectively. Additionally, there are some intermolecular interactions between the free S-atom of the coordinated thiocyanate ion from one unit with the carbon and nitrogen atoms of the s-triazine core from another complex unit. The most important C . . . S and S . . . N contacts are given in Table 2. The resulting packing scheme is shown in Figure 4B.
The supramolecular structure of [Zn(BPMT)(NCS)2] complex is controlled by t S…H, S…N and S…C contacts shown in Figure 3 while the corresponding contact d tances are listed in Table 2. The weak S…H interactions occurred alternatively betwe the two molecular units found in the asymmetric formula leading to the 1D chain show in Figure 4A. The hydrogen-acceptor distances of the S2…H23B and S1…H30 inter tions are 2.915 and 3.377 Å , respectively. Additionally, there are some intermolecular teractions between the free S-atom of the coordinated thiocyanate ion from one unit w the carbon and nitrogen atoms of the s-triazine core from another complex unit. The m important C…S and S…N contacts are given in Table 2. The resulting packing scheme shown in Figure 4B.      Figure 4B for better clarity.

Hirshfeld Analysis
Hirshfeld analysis is an important tool for decomposing all intermolecular interactions in the structure of crystalline materials. For this task, the Crystal Explorer 17.5 program [26] was used to perform the Hirshfeld analysis. The Hirshfeld surfaces of the two complex molecules in the asymmetric unit are shown in Figure 5. Curvedness and shape index maps could give enough evidence of the presence or absence of π-π stacking interactions. The absence of a green flat area in the former and the absence of red/blue triangles in the latter confirm the absence of any π-π stacking interactions in the studied system. All hydrogen atoms were omitted from Figure 4B for better clarity.

Hirshfeld Analysis
Hirshfeld analysis is an important tool for decomposing all intermolecular interactions in the structure of crystalline materials. For this task, the Crystal Explorer 17.5 program [26] was used to perform the Hirshfeld analysis. The Hirshfeld surfaces of the two complex molecules in the asymmetric unit are shown in Figure 5. Curvedness and shape index maps could give enough evidence of the presence or absence of π-π stacking interactions. The absence of a green flat area in the former and the absence of red/blue triangles in the latter confirm the absence of any π-π stacking interactions in the studied system. On the other hand, there are many red spots in the dnorm map corresponding to the S…H, S…C and S…N intermolecular interactions. The percentages of these contacts in the Zn1 unit are 20.2, 3.5 and 5.8%, respectively while the respective values in the Zn2 unit are 18.8, 4.3 and 4.6%. All these intermolecular contacts are significant and appeared as spikes in the fingerprint plots shown in Figure 6. Additionally, there is large number of weak intermolecular contacts contributing to the molecular packing of this complex (Figure 7). These contacts appeared in the dnorm map as blue or white regions. Hence, these contacts have either longer or equal interaction distances than the sum of the vdWs radii of the two interacting atoms. Among these interactions, the H…H, H…C and N…H are the most dominant. Their percentages were found to be 30.3, 18.2 and 12.9% in the Zn1 unit, respectively. In the case of the Zn2 unit, the respective values are 29.7, 20.6 and 14.4%.  Additionally, there is large number of weak intermolecular contacts contributing to the molecular packing of this complex (Figure 7). These contacts appeared in the dnorm map as blue or white regions. Hence, these contacts have either longer or equal interaction distances than the sum of the vdWs radii of the two interacting atoms. Among these interactions, the H…H, H…C and N…H are the most dominant. Their percentages were found to be 30.3, 18.2 and 12.9% in the Zn1 unit, respectively. In the case of the Zn2 unit, the respective values are 29.7, 20.6 and 14.4%.

Biological Studies 2.4.1. Antimicrobial Activity
The biological activity of the free BPMT, complexes 1 and 2 against different microbes including bacteria and fungi were determined by detecting the sizes of inhibition zones at 10 mg/mL of the investigated compounds. All results are listed in Table 3  Additionally, the minimum inhibitory concentrations (MIC) in µg/mL were determined. Complex 1 showed interestingly low MIC values against B. subtilis (2.4 µg/mL) and P. vulgaris (4.8 µg/mL) compared to Gentamycin (4.8 µg/mL). Additionally, complex 2 has relatively low MIC values against the same microbes (9.7 and 39.1 µg/mL, respectively (Table 4).

Antioxidant and Anticancer Activities
The DPPH method was used to detect the antioxidant activity of the studied compounds ( Figure 8). The free BPMT ligand exhibited no antioxidant activity. In contrast, complexes 1 (IC 50 = 78.09 ± 4.23 µg/mL) and 2 (IC 50 = 365.60 ± 20.89 µg/mL) showed antioxidant activity which is found to depend on the coordinating anion. It is obvious that the thiocyanate complex (1) has better antioxidant activity than the bromo complex (2). For the reference ascorbic acid, the IC 50 value was determined to be 12.3 ± 0.51 µg/mL. Hence, complex 1 has better antioxidant activity than complex 2. These results indicated that the antioxidant activity of the studied Zn(II) complexes is sensitive to the nature of the coordinating anion.

Antioxidant and Anticancer Activities
The DPPH method was used to detect the antioxidant activity of the studied compounds (Figure 8). The free BPMT ligand exhibited no antioxidant activity. In contrast, complexes 1 (IC50 = 78.09 ± 4.23 µ g/mL) and 2 (IC50 = 365.60 ± 20.89 µ g/mL) showed antioxidant activity which is found to depend on the coordinating anion. It is obvious that the thiocyanate complex (1) has better antioxidant activity than the bromo complex (2). For the reference ascorbic acid, the IC50 value was determined to be 12.3 ± 0.51 µ g/mL. Hence, complex 1 has better antioxidant activity than complex 2. These results indicated that the antioxidant activity of the studied Zn(II) complexes is sensitive to the nature of the coordinating anion.  Tables S2-S4. In addition, in vitro cytotoxic activity of complexes 1 and 2 against the lung carcinoma A-549 cell line was examined and compared with the free BPMT ligand (Figure 9). Both Zn(II) complexes have promising cytotoxic activity against the lung carcinoma A-549 cell line, with enhanced anticancer activity compared to the free BPMT ligand. For complexes 1 and 2, the IC50 values are 43.86 ± 3.12 and 30.23 ± 1.26 µ g/mL, respectively, while the corresponding value for the free BPMT ligand is 372.79 ± 13.64 µ g/mL. The studied Zn(II) complexes have 8 and 12 times the anticancer activity of the free BPMT ligand, respectively. Hence, complex 2 has the best anticancer activity against the lung carcinoma A-549 cell line. For cis-platin as control, the IC50 value is 7.53  0.69 µ g/mL indicating moderate anticancer activity for both Zn(II) complexes against the lung carcinoma A-549 cell line. In addition, in vitro cytotoxic activity of complexes 1 and 2 against the lung carcinoma A-549 cell line was examined and compared with the free BPMT ligand (Figure 9). Both Zn(II) complexes have promising cytotoxic activity against the lung carcinoma A-549 cell line, with enhanced anticancer activity compared to the free BPMT ligand. For complexes 1 and 2, the IC 50 values are 43.86 ± 3.12 and 30.23 ± 1.26 µg/mL, respectively, while the corresponding value for the free BPMT ligand is 372.79 ± 13.64 µg/mL. The studied Zn(II) complexes have 8 and 12 times the anticancer activity of the free BPMT ligand, respectively. Hence, complex 2 has the best anticancer activity against the lung carcinoma A-549 cell line. For cis-platin as control, the IC 50 value is 7.53 ± 0.69 µg/mL indicating moderate anticancer activity for both Zn(II) complexes against the lung carcinoma A-549 cell line.

Synthesis of Zn(II) Complexes
The synthesis of the [Zn(BPMT)X2] complexes (X = SCN − ; 1 and X = Br − ; 2) was performed by mixing 10 mL ethanolic solution of BPMT (29.9 mg, 0.1 mmol) with Zn(NO3)2.6H2O (29.8 mg, 0.1 mmol) in 5 mL distilled water followed by the addition of 1 mL saturated KSCN (for 1) or KBr (for 2) aqueous solution. The resulting solutions were left for slow evaporation at room temperature, and colorless crystals of the target compounds were obtained after two weeks. Only for complex 1, the quality of single crystals was found suitable for the single crystal X-ray structure measurement.

Biological Studies
The bio-activities of the free BPMT and the two Zn(II) complexes as antimicrobial, anticancer and antioxidant agents were investigated. The experimental details are described in Methods S2-S4 (Supplementary Materials).

Synthesis of Zn(II) Complexes
The synthesis of the [Zn(BPMT)X 2 ] complexes (X = SCN − ; 1 and X = Br − ; 2) was performed by mixing 10 mL ethanolic solution of BPMT (29.9 mg, 0.1 mmol) with Zn(NO 3 ) 2 ·6H 2 O (29.8 mg, 0.1 mmol) in 5 mL distilled water followed by the addition of 1 mL saturated KSCN (for 1) or KBr (for 2) aqueous solution. The resulting solutions were left for slow evaporation at room temperature, and colorless crystals of the target compounds were obtained after two weeks. Only for complex 1, the quality of single crystals was found suitable for the single crystal X-ray structure measurement.

Biological Studies
The bio-activities of the free BPMT and the two Zn(II) complexes as antimicrobial, anticancer and antioxidant agents were investigated. The experimental details are described in Methods S2-S4 (Supplementary Materials).

Conclusions
Synthesis and biological evaluations of two new penta-coordinated Zn(II) complexes with an s-triazine pincer type ligand were presented. Mixing an aqueous solution of Zn(NO 3 ) 2 .6H 2 O and ethanolic solution of the BPMT pincer ligand in presence of either KSCN or KBr afforded [Zn(BPMT)(NSC) 2 ] (1) and [Zn(BPMT)(Br) 2 ] (2) complexes at a good yield. The structure of complex 1 is confirmed by X-ray single crystal structure where the Zn(II) ion is coordinated with one tridentate BPMT ligand and two isothiocyanate ions. Its supramolecular structure aspects were analyzed using Hirshfeld analysis. The complexes are compared with the results of the BPMT ligand. The latter has no or very weak biological activity while the studied Zn(II) complexes are biologically active. It is found that; complex 1 has better antibacterial and antioxidant activities than complex 2. In contrast, complex 2 (30.23 ± 1.26 µg/mL) has better anticancer activity than complex 1 (43.86 ± 3.12 µg/mL) against the lung carcinoma A-549 cell line.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27113625/s1. Chemicals and instrumentations; X-ray structure measurement details; Figure S1: FTIR spectra of BPMT; Figure S2: FTIR spectra of 1; Figure  S3: FTIR spectra of 2; Figure S4: 1 H and 13 C NMR spectra of the ligand (BPMT); Figure S5: 1 H NMR spectra of the complex 2. Chemical shifts are reported in parts per million (ppm); Scheme S1: Synthesis of the ligand (BPMT); Method S1: Synthesis of BPMT; Method S2: Antimicrobial studies; Method S3: DPPH Radical Scavenging Activity; Method S4: Evaluation of Cytotoxic activity; Table S1: Crystal data and refinement details of 1; Table S2: Evaluation of antioxidant activity of 1; Table S3: Evaluation of antioxidant activity of 2; Table S4: Evaluation of antioxidant activity of BPMT; Table S5: Evaluation of cytotoxicity of 1 against A-549 cell line; Table S6: Evaluation of cytotoxicity of 2 against A-549 cell line; Table S7