Synthesis, Structure and In Vitro Anticancer Activity of Pd(II) Complex of Pyrazolyl- s -Triazine Ligand; A New Example of Metal-Mediated Hydrolysis of s -Triazine Pincer Ligand

: The square planar complex [Pd(PT)Cl(H 2 O)]*H 2 O ( HPT : 6-(3,5-dimethyl-1 H -pyrazol-1-yl)-1,3,5-triazine-2,4(1 H ,3 H )-dione) was obtained by the reaction of 2-methoxy-4,6- bis (3,5-dimethyl-1 H -pyrazol-1-yl)-1,3,5-triazine ( MBPT ) pincer ligand with PdCl 2 in a molar ratio (1:1) under thermal conditions and using acetone as a solvent. The reaction proceeded via C-N cleavage of one C-N moiety that connects the pyrazole and s -triazine combined with the hydrolysis of the O-CH 3 group. The reaction of the chloride salt of its higher congener (PtCl 2 ) gave [Pt(3,5-dimethyl-1 H -pyrazole) 2 Cl 2 ]. The crystal structure of [Pd(PT)Cl(H 2 O)]*H 2 O complex is stabilized by inter- and intra-molecular hydrogen bonding interactions. Hirshfeld analysis revealed that the H...H (34.6%), O...H (23.6%), and Cl...H (7.8%) interactions are the major contacts in the crystal. The charges at Pd, H 2 O, Cl and PT are changed to 0.4995, 0.2216, − 0.4294 and − 0.2917 instead of +2, 0, − 1 and − 1, respectively, using the MPW1PW91 method. [Pd(PT)Cl(H 2 O)]*H 2 O complex has almost equal activities against MDA-MB-231 and MCF-7 cell lines with IC 50 of 38.3 µ g/mL.


Materials and Methods
Solvents and reagents were bought from Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany. The C, H, and N analyses were determined using Perkin-Elmer 2400 elemental analyzer.

Synthesis of MBPT Ligand
The ligand MBPT was prepared following the reported method [30,31]. The spectral data agreed with the reported one (see in Supplementary Material, Figure S1). Following the same procedures, the reaction of PtCl2 with the same ligand afforded the [Pt(3,5-dimethyl-1H-pyrazole)2Cl2] complex also indicating the hydrolysis of MBPT. The crystals were isolated and the solid-state structure was established using X-ray diffraction of a single crystal, and it is found to agree with the previously reported structure by Khripun et al. [32].

Materials and Methods
Solvents and reagents were bought from Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany. The C, H, and N analyses were determined using Perkin-Elmer 2400 elemental analyzer. The ligand MBPT was prepared following the reported method [30,31]. The spectral data agreed with the reported one (see in Supplementary Material, Figure S1). Following the same procedures, the reaction of PtCl 2 with the same ligand afforded the [Pt(3,5-dimethyl-1H-pyrazole) 2 Cl 2 ] complex also indicating the hydrolysis of MBPT. The crystals were isolated and the solid-state structure was established using X-ray diffraction of a single crystal, and it is found to agree with the previously reported structure by Khripun et al. [32].

Crystal Structure Determination
The crystal of [Pd(PT)Cl(H 2 O)]*H 2 O was immersed in cryo-oil, mounted in a loop, and measured at a temperature of 170 K. The X-ray diffraction data was collected on a Bruker Kappa Apex II diffractometer using MoKα radiation. The Denzo-Scalepack [33] software package was used for cell refinement and data reduction. A numerical absorption correction (SADABS [34]) was applied to the intensities before structure solution. The structure was solved by the intrinsic phasing method using the SHELXT [35] software. Structural refinement was carried out using SHELXL [36] software. The H 2 O and NH hydrogen atoms were located from the difference Fourier map and refined isotropically. Other hydrogen atoms were positioned geometrically and constrained to ride on their parent atoms, with C-H = 0.95-0.98 Å and U iso = 1.2-1.5 U eq (parent atom). The crystallographic details are summarized in Table 1.

Computational Details
Gaussian 09 program [38] was used for DFT calculations. MPW1PW91 and ωB97XD methods [39,40] combined with cc-PVTZ and cc-PVTZ-PP [41-43] as basis sets for nonmetal atoms and Pd, respectively, were used for natural charge populations [44] at the X-ray structure coordinates of the studied Pd(II) complex.

In Vitro Anti-Cancer Activity
In vitro anti-cancer activities against two breast adenocarcinoma (MDA-MB-231 and MCF-7) cell lines were tested (see in Supplementary Material, Method S1).

Cell Culture Conditions
Breast cancer cell lines MDA-MB-231 and MCF-7 were obtained from the German Type Cell Culture Collection (DSMZ, Germany). Cells were maintained in high glu-

MTT Assay
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out according to the previous report by Abutaha et al. [45]. Briefly, trypsin was added to MDA-MB-231, and MCF-7 cells, and then cells were counted and seeded at 5 × 10 4 cells/well in the 24-well plate for 24 h. The following day, cells were incubated with different concentrations of the compounds for 48 h with 5% CO 2 at 37 • C. After that, 100 µL of MTT (5 mg/mL) (Thermo, USA) was added to each well and left for 2 h. Next, the supernatant was discarded, and 1000 µL of methanol was added, and the formazan was quantified at 595 nm using a microplate reader. Triplicates were used to calculate the cell viability percentage and the IC 50 values using OriginPro 8.5 software.

Results and Discussion
is coordinated by a chloride anion, one water molecule as monodentate ligands and two nitrogen atoms from the chelating ligand PT −1 as a mononegative bidentate NN-chelate. The Pd(II) exhibiting slightly distorted square planar coordination geometry (Table 2, Figure 2). The structure of the metal complex is supported by one intramolecular H-bond between the coordinated water molecule (H1A) and the oxygen (O2), forming the organic chelate (PT −1 ), leading to the six-membered ring ( Figure 3). The asymmetric unit also contains water of crystallization. The NH 2 -group and the oxygen O3 are involved in a pair of hydrogen bonds binding the metal complex with the adjacent molecule at the equivalent position of −x + 1, y, −z + 1.5. The aqua ligand is also hydrogen bonded to the water of crystallization ( Figure 3). The water of crystallization forms additional hydrogen bonds with chloride ligand as well as with O6 of the neighboring metal complexes. Additional weak CH···O type H-bonds (C6-H6···O4 and C8-H8C···O4) are supporting the overall packing of the molecules. The hydrogen bonds are summarized in Table 3 and Figure 3. Packing of the Pd(II) complex units stacked along the crystallographic b-axis is shown in Figure 4.

Hirshfeld Analysis of Molecular Packing
Hirshfeld surfaces mapped over d norm , shape index (SI) and curvedness for the studied complex are shown in Figure S2 (see in Supplementary Material). Quantitative analysis of molecular packing is given in Figure 5.

Hirshfeld Analysis of Molecular Packing
Hirshfeld surfaces mapped over dnorm, shape index (SI) and curvedness for the studied complex are shown in Figure S2 (see in Supplementary Material). Quantitative analysis of molecular packing is given in Figure 5 ..Cl contact distance of 2.415 Å and one sharp spike in the fingerprint plot indicating that the chloride ion inside the surface acting as hydrogen bond acceptor. In addition, every two complex units forming a dimer via two equivalent C1...N5 contacts (3.344 Å) along the crystallographic b-direction indicating weak π-π stacking interactions, which is further indicated by the presence of blue/red triangle in the shape index map.

Natural Population Analysis
The divalent Pd ion is coordinated with two negatively charged ligand groups, which are Cl − and PT − . These isolated ions have a net charge of −1 e. As a result of the interactions between the Pd(II) ion as Lewis acid and these ligand groups as Lewis base, there are some electrons that are transferred from the ligand groups to Pd(II) ion (Table 4). Two DFT methods (MPW1PW91 and ωB97XD) employing natural charge population analysis [46] which has low sensitivity to the basis set variations, were used for this task. The chloride ion transferred 0.571-0.527 e to the Pd(II) while the anionic organic ligand (PT − ) as a bidentate chelate transferred a large amount (0.708-0.701 e) of its negative charge to the metal center while the coordinated water molecule has a net charge of 0.2216-0.2172 e. As a result, the water molecule as ligand transferred about 0.2 e to Pd(II). The net charge of Pd was decreased to 0.500-0.527 e. Since the charges transferred are not associated with a physical observable [47], one could conclude that there are some charges that are transferred from the ligand groups to the Pd(II) central metal ion, which confirm the coordination between the Pd(II) and ligand groups.

Natural Population Analysis
The divalent Pd ion is coordinated with two negatively charged ligand groups, which are Cl − and PT − . These isolated ions have a net charge of −1 e. As a result of the interactions between the Pd(II) ion as Lewis acid and these ligand groups as Lewis base, there are some electrons that are transferred from the ligand groups to Pd(II) ion (Table 4). Two DFT methods (MPW1PW91 and ωB97XD) employing natural charge population analysis [46] which has low sensitivity to the basis set variations, were used for this task. The chloride ion transferred 0.571-0.527 e to the Pd(II) while the anionic organic ligand (PT − ) as a bidentate chelate transferred a large amount (0.708-0.701 e) of its negative charge to the metal center while the coordinated water molecule has a net charge of 0.2216-0.2172 e. As a result, the water molecule as ligand transferred about 0.2 e to Pd(II). The net charge of Pd was decreased to 0.500-0.527 e. Since the charges transferred are not associated with a physical observable [47], one could conclude that there are some charges that are transferred from the ligand groups to the Pd(II) central metal ion, which confirm the coordination between the Pd(II) and ligand groups. Another interesting feature that could be discussed is the HOMO and LUMO patterns of the studied system (Figure 7). The energies of these frontier molecular orbitals were calculated to be -7.146 and -2.358 eV, respectively, and the HOMO-LUMO transition required an energy of 4.789 eV using the MPW1PW91 method. As can be seen from Figure 7, the HOMO is mainly localized over the Pd(II), which has a major contribution from the dz 2 orbital, while the LUMO is distributed over the metal and organic ligand skeleton, suggesting d-d transition mixed with metal-ligand charge transfer transitions.  Another interesting feature that could be discussed is the HOMO and LUMO patterns of the studied system (Figure 7). The energies of these frontier molecular orbitals were calculated to be -7.146 and -2.358 eV, respectively, and the HOMO-LUMO transition required an energy of 4.789 eV using the MPW1PW91 method. As can be seen from Figure 7, the HOMO is mainly localized over the Pd(II), which has a major contribution from the dz 2 orbital, while the LUMO is distributed over the metal and organic ligand skeleton, suggesting d-d transition mixed with metal-ligand charge transfer transitions.

In Vitro Anti-Cancer Activity
Presently, palladium-based drugs are among the most studied drugs in oncology and are attractive substitute metal-based drugs because of considerable similarities to platinum agents regarding structure and coordination chemistry [48]. , and many more [49]. In addition, Pd(II) complexes were reported to have more activity against cancer cell lines with less side effects compared to cisplatin [15,17]. The studied [Pd(PT)Cl(H2O)]*H2O complex showed a cell growth reduction against both of the tested breast cell lines compared with the control that was inactive at all the tested concentra-

In Vitro Anti-Cancer Activity
Presently, palladium-based drugs are among the most studied drugs in oncology and are attractive substitute metal-based drugs because of considerable similarities to platinum agents regarding structure and coordination chemistry [48]. Palladium-based drugs are known to be active against a wide range of cancer cells with different IC 50 2 ), and many more [49]. In addition, Pd(II) complexes were reported to have more activity against cancer cell lines with less side effects compared to cisplatin [15,17]. The studied [Pd(PT)Cl(H 2 O)]*H 2 O complex showed a cell growth reduction against both of the tested breast cell lines compared with the control that was inactive at all the tested concentrations. In addition, the complex showed good activity against MDA-MB-231 and MCF-7 cells with same IC 50 of 38.3 µg/mL for both cells (Figure 8). This data is somewhat better than the reported data for cisplatin and the