Synthesis, Structural Studies, and Anticancer Properties of [CuBr(PPh 3 ) 2 (4,6-Dimethyl-2-Thiopyrimidine-

: CuBr(PPh 3 ) 2 (4,6-dimethylpyrimidine-2-thione)(Cu-L)wassynthesizedbystirringCuBr(PPh 3 ) 3 and 4,6-dimethylpyrimidine-2-thione in dichloromethane. The crystal structure of Cu-L was obtained, and indicated that the complex adopts a distorted tetrahedral structure with several intramolecular hydrogen bonds. Moreover, a centrosymmetric dimer is formed by the intermolecular hydrogen bonding of the bromine acceptor created by symmetry operation 1 − x, 1 − y, 1 − z to the methyl group (D 3 = C42) of the pyrimidine–thione ligand. HSA-binding of Cu-L and its ligand were evaluated, revealing that Cu-L binds to HSA differently than its ligand. The HSA-bindings were modeled by molecular docking, which suggested that Cu-L binds to the II A domain while L binds between the I B and II A domains. Anticancer activities toward OVCAR-3 and HeLa cell lines were tested and indicated the signiﬁcance of the copper center in enhancing the cytotoxic effect; negligible toxicities for L and Cu-L were observed towards a non-cancer cell line. The current study highlights the potential of copper(I)-phosphine complexes containing thione ligands as therapeutic agents. ideal tetrahedral angle (109.4 The largest deviation of 125 is observed for the P–Cu–P angle, which is more than 6 ◦ wider the P–Cu–P angle (119 ◦ ) = 3.879(1) Å is slightly longer than the intramolecular hydrogen bridges. This type of structural feature was also observed in the crystal structure of [CuBr(PPh 3 ) 2 (MTUC)] [14]. The intra- and intermolecular hydrogen bonding distances are compiled in Table S7 of the SI. No π – π stacking was observed in the crystal structure of the title complex.


Synthesis and Characterization
Equimolar amounts of CuBr(PPh 3 ) 3 (0.198 g, 0.214 mmol) and 4,6-dimethylpyrimidien-2-thione ligand (0.030 g, 0.214 mmol) were stirred under nitrogen atmosphere in the appropriate amount of dichloromethane (DCM). The colorless solution turned yellow in a few minutes and was left under stirring for 2 h. The reaction solution was then reduced in volume and 50 mL petroleum spirit (40-60 • C) was added, leading to the precipitation of the product. The product was collected by filtration, washed with diethylether, and dried, affording the complex as a yellow powder (0.149 g, 86%). 31

Single-Crystal X-ray Diffraction Study
Single crystals of the [CuBr(PPh 3 ) 2 (C 6 H 8 N 2 S)] complex were grown by vapor diffusion of hexane into a saturated solution of the compound in dichloromethane at 5 • C. A suitable single crystal was selected and mounted on a Bruker D8 Venture single crystal X-ray diffractometer equipped with Photon II detector (Billerica, MA, USA). The data was collected at 120 K with Mo Kα (λ = 0.71073 Å) radiation using the Bruker APEX III software package. The crystal structure was solved by the SHELXT [36] structure solution program using Intrinsic Phasing method and refined by least squares method with the SHELXL [37] program implemented in Olex2 [38]. All the non-hydrogen atomic positions were refined anisotropically. The H-atoms were treated as riding, and U iso (H) values were set at 1.5U eq (C) for methyl and 1.2U eq (C) for aromatic groups. Table 1 summarizes the crystal structure refinement details of the [CuBr(C 18 H 15 P) 2 (C 6 H 8 N 2 S)].

HSA Binding Studies
Human serum albumin (HSA, Product No.: A9511) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. The stock solution of HSA was prepared by using the Tris-HCl/NaCl aqueous buffer system (pH = 7.4). The concentration of HSA (1.2 × 10 −5 mol dm −3 ) was measured spectroscopically according to the reported procedure [39]. The solution was titrated with different concentrations of the complex and the ligand in DMSO (the amount of DMSO was maintained at ca. 20% v/v). An incubation time of 5 min was used to achieve a homogeneous mixing of both the HSA and the compounds. The changes in the absorption spectra of the solutions were monitored at 295 K.

Molecular Docking
The crystal structure of the human serum albumin receptor was downloaded from the protein data bank (PDB: 1H9Z) (http://www.rcsb.org/pdb/home/home.do accessed on 10 June 2020) [40]. The ligand and its copper complex were sketched in ChemBioOffice Ultra, vers ion 13. All docking studies used the MOE program. HSA (1H9Z) was prepared for the docking study by removing all water and cofactor molecules from the downloaded protein. Then, all invalid charges and broken bonds were corrected, and all hydrogen atoms were added. The parameters and charges were assigned with the MMFF94x force field. After alpha-site spheres were identified using the site finder module of MOE, the two complexes were docked to the same active site of the downloaded compound using the DOCK module of MOE [41,42]. The docking scores in the MOE software were obtained using the London dG scoring function. The highest ten docking scores were used to compare between the two complexes and the co-crystallized reference compound; the values were optimized using two independent refinement methods. The docking results were validated following the reported method [43,44]. The method (the pose selection method) involves re-docking the co-crystallized ligand into the receptor's active site. If the program is able to identify the best pose under a preselected Root Mean Square Deviation (RMSD) value from the known conformation (regularly 1.5 or 2 Å, depending on ligand size), the method is considered "validated". In the current study, pose selection and docking score for Rwarfarin (the co-crystallized compound with 1H9Z) were determined; the docking result of the same compound reached a 1.09 Å resolution of the co-crystal structure.

Anticancer Studies
The cells were supplied by the Egyptian Holding Company for Biological Products and Vaccines (VACSERA) and then kept in the tissue culture unit. The growth of the cells was effected in RPMI-1640 medium, supplemented with 10% heat inactivated FBS, 50 units/mL of penicillin, and 50 mg/mL of streptomycin, and maintained in a humidified atmosphere with 5% carbon dioxide [45,46]. The cells were maintained as monolayer cultures by serial sub-culturing, obtaining cell culture reagents from Lonza (Basel, Switzerland). The antitumor activities of the complexes were assessed against OVCAR-3 (ovarian cancer) and HOPE-62 (small cell lung cancer) cell lines.
The sulforhodamine B (SRB) assay method was applied to determine the cytotoxicity, as described in the literature [47]. Exponentially-growing cells were collected using 0.25% Trypsin-EDTA and seeded in 96-well plates at 1000-2000 cells/well in RBMI-1640 supplemented medium. The cells were kept in the medium for 24 h and then they were incubated for 3 days with various concentrations of the copper complexes. Following 3 days of treatment, the cells were fixed with 10% trichloroethanoic acid for 1 h at 4 • C. Wells were stained for 10 min at room temperature with 0.4% SRBC dissolved in 1% acetic acid. The plates were air dried for 24 h and the dye was dissolved in Tris-HCl for 5 min with shaking at 1600 rpm. The optical density (OD) of each well was assessed spectrophotometrically at 564 nm with an ELISA microplate reader (ChroMate-4300, Westport, CT, USA). The minimum inhibitory concentration (IC 50 ) values were calculated from a Boltzmann sigmoidal concentration response curve using the nonlinear regression fitting models (Graph Pad, Prism Version 9).

Synthesis and Characterization
The synthesis of the complex was achieved by stirring equimolar amounts of CuBr(PPh 3 ) 3 and 4,6-dimethylpyrimidine-2-thione in DCM. One phosphine ligand is substituted with the thione, and the driving force of this substitution is the high steric hindrance of the triphenylphosphine as indicated by comparing the X-ray single crystallographic data of the reactant and the product. 31 P NMR spectroscopy, elemental analysis, and mass spectrometry were utilized to characterize the compound. In the 31 P NMR spectra, the compound shows a sharp singlet signal around −1.5 ppm. The mass spectrum of the compound afforded the peak of [M-Br] + ion. Elemental analysis provided data that are in agreement with the proposed formula. Further confirmation of the structure is provided by the X-ray diffraction crystallographic study.

Crystal Structure Study
The title compound crystallizes in a monoclinic crystal system with P2 1 /c symmetry forming a monomeric complex with four formula units per unit cell (Z = 1). Fractional atomic coordinates, atomic displacement parameters, site occupancy factors, bond lengths, bond angles, and torsion angles are shown in Tables S1-S6 of the Supplementary Materials. Figure 1 shows the molecular structure of [CuBr(PPh 3 ) 2 (C 6 H 8 N 2 S)]. The geometrical feature of the molecule resembles the parent [CuBr(PPh 3 ) 3 ] complex and other similar Cu(I) halide complexes with CuXSP 2 cores [14,28,[48][49][50]. The heavily distorted tetrahedral coordination of the copper compound is realized by the S-atom of the -thione, the P-atom of phosphine, and bromo ligands. The bond angles of the CuBrSP 2 tetrahedron varied between 98 • and 125 • , which notably deviates from the ideal tetrahedral angle (109.4 • ). The largest deviation of 125 • is observed for the P-Cu-P angle, which is more than 6 • wider than the P-Cu-P angle (119 plates were air dried for 24 h and the dye was dissolved in Tris-HCl for 5 min with shaking at 1600 rpm. The optical density (OD) of each well was assessed spectrophotometrically at 564 nm with an ELISA microplate reader (ChroMate-4300, Westport, CT, USA). Th minimum inhibitory concentration (IC50) values were calculated from a Boltzmann sig moidal concentration response curve using the nonlinear regression fitting models (Graph Pad, Prism Version 9).

Synthesis and Characterization
The synthesis of the complex was achieved by stirring equimolar amounts o CuBr(PPh3)3 and 4,6-dimethylpyrimidine-2-thione in DCM. One phosphine ligand is sub stituted with the thione, and the driving force of this substitution is the high steric hin drance of the triphenylphosphine as indicated by comparing the X-ray single crystallo graphic data of the reactant and the product. 31 P NMR spectroscopy, elemental analysis and mass spectrometry were utilized to characterize the compound. In the 31 P NMR spec tra, the compound shows a sharp singlet signal around −1.5 ppm. The mass spectrum o the compound afforded the peak of [M-Br] + ion. Elemental analysis provided data that ar in agreement with the proposed formula. Further confirmation of the structure is pro vided by the X-ray diffraction crystallographic study.

Crystal Structure Study
The title compound crystallizes in a monoclinic crystal system with P21/c symmetry forming a monomeric complex with four formula units per unit cell (Z′ = 1). Fractiona atomic coordinates, atomic displacement parameters, site occupancy factors, bond lengths, bond angles, and torsion angles are shown in Tables S1-S6 of the Supplementary Materials. Figure 1 shows the molecular structure of [CuBr(PPh3)2(C6H8N2S)]. The geomet rical feature of the molecule resembles the parent [CuBr(PPh3)3] complex and other simila Cu(I) halide complexes with CuXSP2 cores [14,28,[48][49][50]. The heavily distorted tetrahedra coordination of the copper compound is realized by the S-atom of the -thione, the P-atom of phosphine, and bromo ligands. The bond angles of the CuBrSP2 tetrahedron varied be tween 98° and 125°, which notably deviates from the ideal tetrahedral angle (109.4°). Th largest deviation of 125° is observed for the P-Cu-P angle, which is more than 6° wide than the P-Cu-P angle (119°) found in [CuBr(PPh3)3].  This type of tetrahedral distortion is very common in similar complexes [11,28,51] and is mainly attributed to the valence-shell electron-pair repulsion and/or steric imposition of the bulky triphenyl-phosphine group. The distortion of the tetrahedral coordination around an atom can be judged by the so called τ 4 parameter (τ 4 = [360−(α + β)]/141, where α and β are the largest angles around the central atom), whose value should be equal to one for exact tetrahedral and zero for square planar coordination. For the title compound, τ 4 = 0.93 is slightly higher than the τ 4 = 0.90 of the [CuBr(PPh 3 ) 3 ] complex, which means that the overall tetrahedral character around the Cu-atom was not substantially altered by the substitution of the PPh 3 group by the -thione ligand. In general, all the interatomic distances are comparable to the corresponding distances reported in identical Cu-complexes [14,28,[48][49][50]52]. In the title compound, the Cu-Br distance (2.5412(2) Å) is slightly longer, while the Cu-P average distance of 2.275 Å became shorter compared to the respective distances  (1) Å is slightly longer than the intramolecular hydrogen bridges. This type of structural feature was also observed in the crystal structure of [CuBr(PPh 3 ) 2 (MTUC)] [14]. The intra-and intermolecular hydrogen bonding distances are compiled in Table S7 of the SI. No π-π stacking was observed in the crystal structure of the title complex.
This type of tetrahedral distortion is very common in similar complexes [11,28,51 and is mainly attributed to the valence-shell electron-pair repulsion and/or steric imposi tion of the bulky triphenyl-phosphine group. The distortion of the tetrahedral coordina tion around an atom can be judged by the so called τ4 parameter (τ4 = [360−(α + β)]/141 where α and β are the largest angles around the central atom), whose value should b equal to one for exact tetrahedral and zero for square planar coordination. For the titl compound, τ4 = 0.93 is slightly higher than the τ4 = 0.90 of the [CuBr(PPh3)3] complex which means that the overall tetrahedral character around the Cu-atom was not substan tially altered by the substitution of the PPh3 group by the -thione ligand. In general, a the interatomic distances are comparable to the corresponding distances reported in iden tical Cu-complexes [14,28,[48][49][50]52]. In the title compound, the Cu-Br distance (2.5412(2 Å) is slightly longer, while the Cu-P average distance of 2.275 Å became shorter compared to the respective distances The intermolecular hydrogen bonding distance of d(D3···A) = 3.879(1) Å is slightly longe than the intramolecular hydrogen bridges. This type of structural feature was also ob served in the crystal structure of [CuBr(PPh3)2(MTUC)] [14]. The intra-and intermolecula hydrogen bonding distances are compiled in Table S7 of the SI. No π-π stacking was ob served in the crystal structure of the title complex.

Protein Binding Study
Drug-protein (serum albumin) interactions have been identified as crucial for thei delivery [53][54][55][56]. Human serum albumin is widely used as a model system for protei folding, aggregation, and drug delivery and it is one of the most abundant proteins pre sent in the blood plasma, comprising about 55% of the blood plasma protein [57-59

Protein Binding Study
Drug-protein (serum albumin) interactions have been identified as crucial for their delivery [53][54][55][56]. Human serum albumin is widely used as a model system for protein folding, aggregation, and drug delivery and it is one of the most abundant proteins present in the blood plasma, comprising about 55% of the blood plasma protein [57][58][59]. Structural characteristics of HSA have been well investigated. The HSA structure has nearly 580 amino acid residues in one chain, which presumes a solid equilateral triangular shape with sides of ca. 80 Å and depth of ca. 30 Å [60,61]. The structure of HSA (secondary and tertiary) is comprised of three α-helical domains I, II, and III. Each of these domains consists of two subdomains stabilized by disulfide bonds [60,61]. Subdomain IIA (site I) is of significance in binding with hydrophobic materials, while subdomain IIIA (site II) is involved in a combination of hydrophobic, hydrogen bonding, and electrostatic interactions with the guest molecules [62][63][64]. Exploring protein-drug binding by UV-vis absorption is a simple but effective technique. The absorption spectrum of HSA features a strong absorption band at 240 nm related to the protein backbone (α-helix structure), and a weak absorption band at 278 nm assigned to the absorption of aromatic amino acids (Trp, Tyr and Phe) [65]. The absorption spectra of HSA was collected in the presence of increasing amounts of the ligand and its copper(I) complex. As illustrated in Figure 3, the two compounds behave differently with the protein. The band intensity of HSA at 240 nm increased significantly with a slight red-shift upon increasing concentrations of the ligand, while the intensity of the band at 278 nm increased with a blue-shift in the absorption maxima. The presence of pronounced changes around 240 nm suggests conformational changes in the α-helix structure of the protein, while changes around 278 nm indicates a strong impact on the microenvironment of the HSA aromatic amino acids by the binding of the ligand (Figure 3). However, the addition of Cu-L into the protein causes weak decreases in the intensities of the absorption band at 240, suggesting that the complex interacts differently from its ligand. The complex slightly affects the α-helix structure of the protein, but insignificantly changes the microenvironment of the HSA aromatic amino acids.
Structural characteristics of HSA have been well investigated. The HSA structure has nearly 580 amino acid residues in one chain, which presumes a solid equilateral triangular shape with sides of ca. 80 Å and depth of ca. 30 Å [60,61]. The structure of HSA (secondary and tertiary) is comprised of three α-helical domains I, II, and III. Each of these domains consists of two subdomains stabilized by disulfide bonds [60,61]. Subdomain IIA (site I) is of significance in binding with hydrophobic materials, while subdomain IIIA (site II) is involved in a combination of hydrophobic, hydrogen bonding, and electrostatic interactions with the guest molecules [62][63][64]. Exploring protein-drug binding by UV-vis absorption is a simple but effective technique. The absorption spectrum of HSA features a strong absorption band at 240 nm related to the protein backbone (α-helix structure), and a weak absorption band at 278 nm assigned to the absorption of aromatic amino acids (Trp, Tyr and Phe) [65]. The absorption spectra of HSA was collected in the presence of increasing amounts of the ligand and its copper(I) complex. As illustrated in Figure 3, the two compounds behave differently with the protein. The band intensity of HSA at 240 nm increased significantly with a slight red-shift upon increasing concentrations of the ligand, while the intensity of the band at 278 nm increased with a blue-shift in the absorption maxima. The presence of pronounced changes around 240 nm suggests conformational changes in the α-helix structure of the protein, while changes around 278 nm indicates a strong impact on the microenvironment of the HSA aromatic amino acids by the binding of the ligand (Figure 3). However, the addition of Cu-L into the protein causes weak decreases in the intensities of the absorption band at 240, suggesting that the complex interacts differently from its ligand. The complex slightly affects the α-helix structure of the protein, but insignificantly changes the microenvironment of the HSA aromatic amino acids.

Molecular Docking
Molecular docking is a powerful tool for computer-assisted drug design with the aim to predict a drug's key binding modes with the 3D structure of the biomolecular target. Docking can be utilized to justify experimental results and extract structural-property relationships, which are significant for further optimization [66]. The docking study was conducted to gain insight into the binding sites and the potential interactions of the compounds in the protein microenvironment [67]. The lowest five binding energies of the two forms of the ligand (thiole and thione) and its copper complex were obtained. For the ligand, the docking scores suggest that the thione form has a slightly better docking score than the thiole form. Hence, the thione form will be discussed in detail. The docking results suggest that the thione ligand binds in the pocket between subdomain I B and subdomain II A, forming hydrogen bonding with Gln 196, Lys 199, and Cys 200 (Figure 4) [68]. This explains the strong impact induced by the ligand on the spectrum of the protein (at 240 nm) since cysteine units are responsible for the α-helix structure. The presence of the ligand in this area may allow for hydrophobic interactions with tryptophan (214), phenylalanine (149, 156, 157, 165, 206, and 211), and tyrosine (148, 150, and 161) amino acids available in this area. In contrast, Cu-L has a better binding score toward HSA than that

Molecular Docking
Molecular docking is a powerful tool for computer-assisted drug design with the aim to predict a drug's key binding modes with the 3D structure of the biomolecular target. Docking can be utilized to justify experimental results and extract structural-property relationships, which are significant for further optimization [66]. The docking study was conducted to gain insight into the binding sites and the potential interactions of the compounds in the protein microenvironment [67]. The lowest five binding energies of the two forms of the ligand (thiole and thione) and its copper complex were obtained. For the ligand, the docking scores suggest that the thione form has a slightly better docking score than the thiole form. Hence, the thione form will be discussed in detail. The docking results suggest that the thione ligand binds in the pocket between subdomain I B and subdomain II A, forming hydrogen bonding with Gln 196, Lys 199, and Cys 200 (Figure 4) [68]. This explains the strong impact induced by the ligand on the spectrum of the protein (at 240 nm) since cysteine units are responsible for the α-helix structure. The presence of the ligand in this area may allow for hydrophobic interactions with tryptophan (214), phenylalanine (149, 156, 157, 165, 206, and 211), and tyrosine (148, 150, and 161) amino acids available in this area. In contrast, Cu-L has a better binding score toward HSA than that calculated for the ligand ( Table 2). The data indicate that Cu-L has a strong preference to bind exclusively to subdomain II A (Figure 4). The complex forms some H-bonding interactions with Lys 199 and Arg 222; there is proximity for Cu-L with any cysteine unit, which explains the small impact of the molecule on the α-helix structure. However, the binding site of Cu-L is surrounded by several tryptophan, phenylalanine, and tyrosine amino acids, which may allow for weak electrostatic, hydrophobic, and/or van der Waals interactions with Cu-L. The dramatic difference in the binding sites for L and Cu-L rationalize the spectral changes induced by the molecule-protein binding.

Anticancer Activities
The anticancer properties of L and Cu-L were assessed against ovarian carcinoma (OVCAR-3) and cervical cancer (HeLa) cell lines (see Table 3), as well as rhesus monkey normal kidney epithelial cells (LLC-MK2). The obtained IC50 values against the cancer cell lines for 4,6-dimethylpyrimidine-2-thione ranged from 19.37 to 21.35 µM; coordinating the thione to the CuBr(PPh3)2 moiety enhances the anticancer activities, reducing IC50 val-

Anticancer Activities
The anticancer properties of L and Cu-L were assessed against ovarian carcinoma (OVCAR-3) and cervical cancer (HeLa) cell lines (see Table 3), as well as rhesus monkey normal kidney epithelial cells (LLC-MK2). The obtained IC 50 values against the cancer cell lines for 4,6-dimethylpyrimidine-2-thione ranged from 19.37 to 21.35 µM; coordinating the thione to the CuBr(PPh 3 ) 2 moiety enhances the anticancer activities, reducing IC 50 values by nearly 30%. The minimum inhibitory concentrations for Cu-L against the two cancer cell lines are within a narrow range from that of cisplatin, which does not allow conclusive comparison. L and Cu-L were also examined against the LLC-MK2 cell line as a model to evaluate cytotoxicity for normal cells. The compounds show good selectivity toward cancerous cells, as can be represented by the "selectivity index". The selectivity index was previously used to measure selective cytotoxic activity [69]. The selectivity index (SI) can be calculated from the ratio of IC 50 of the compound on LLC-MK2 to the IC 50 of the compound on a cancer cell line. The selectivity index for L was in the range of 25.6 to 28.2 while it was in the range 43.0 to 49.8 for Cu-L, highlighting the higher selectivity for the copper(I) complex.

Conclusions
In the current work, a complex with the formula CuBr(PPh 3 ) 2 (4,6-dimethylpyrimidine-2-thione) (Cu-L) was synthesized by stirring CuBr(PPh 3 ) 3 and 4,6-dimethylpyrimidine-2thione in dichloromethane. The crystal structure of Cu-L was obtained, and indicated that the complex adopts a distorted tetrahedral structure. Compared to the parent complex, the Cu-Br distance is slightly longer, while Cu-P bond lengths are shorter. Several intramolecular hydrogen bonds are established, while a centrosymmetric dimer is formed by the intermolecular hydrogen bonding of the bromine acceptor created by symmetry operation 1−x, 1−y, 1−z to the methyl group (D 3 = C42) of the pyrimidine-thione ligand. The binding of Cu-L and its ligand against HSA was conducted by following the changes in the UV-vis absorption of the protein upon the addition of the compounds; Cu-L binds to HSA differently from its ligand. The ligand seems to affect the α-helix structure while Cu-L has an insignificant impact on the α-helix structure of the HSA protein. The Cu-L binds to the II A domain while L binds between the I B and II A domains, as suggested by docking modelling. Anticancer activities against OVCAR-3 and HeLa cell lines highlighted the significance of the copper center in enhancing the cytotoxic effect. Moreover, Cu-L has more selectivity toward cancer cell lines than the thione ligand. Overall, the current results highlight that complexes of the type CuXP 2 S can be explored for accessing new therapeutic agents.