Synthesis, Characterization, and In Vitro Cytotoxicity of Platinum(II) Complexes Bearing Chiral Tetradentate Salicylaldimine Ligands

A series of platinum(II) complexes with chiral Schiﬀ base ligands derived from various salicylaldehydes with ( R,R ′ )- and ( S,S ′ )-cyclohexanediamine were synthesized and characterized by ESI-MS, IR, and NMR. Obtained spectra with typical signals were in agreement with suggested molecular formulae of the complexes. Their photophysical properties were studied by UV-visible and emission spectroscopies. The UV-Vis showed the typical band with low energy at visible range 400–500nm for MLCT, and this band can emit the luminescent band with emission maximum wavelengths at 529–595nm. The in vitro cytotoxicity of obtained platinum(II) complexes was screened for KB and MCF-7 human cancer cell lines. The results showed that ( S )-enantiomers were more active than ( R )-enantiomers and the diﬀerent positions of methoxy group in salicyl ring gave diﬀerent cytotoxicities. with some salicylaldehydes. Characterization of


Introduction
Schiff bases are considered as a very important class of organic compounds which have been applied in many biological aspects such as antimicrobial, antifungal, and antitumor activity [1]. eir coordination with transition metals generated a great deal of relatively stable transition metal complexes which have showed interesting properties used in chemical catalysis [2][3][4], analysis [5][6][7], and advanced materials [8][9][10].
ese coordination compounds have also exhibited wonderful biological applications including antimicrobial, antifungal, anti-inflammatory, and anticancer activity [11][12][13][14][15]. e medicinal properties of metal Schiff base complexes depend on the nature of metal ions and ligands. One of the life's biochemical features is chirality. Sometimes, chirality plays a decisive role in the area of pharmaceuticals, agrochemicals, flavors, and fragrances. In most cases, all the enantiomers of a chiral compound are considered as different "chemicals" and each stereoisomer is tested separately for its drug action [16]. So, chiral metal complexes have attracted the attention of many research groups. Some chiral Schiff base complexes of Ni(II), Cu(II), and Zn(II) and their DNA binding, antioxidant, and antibacterial activities were studied, and the results showed that S-enantiomers of the complexes were more efficient for DNA interaction, antioxidant, and antibacterial activities than their R-enantiomers [17,18]. Chiral Mn(IV) complexes with Schiff base ligands were synthesized, and the interaction of the chiral Mn(IV) complexes with CT-DNA was studied, and the results showed that the complexes with (S)-ligands exhibited more efficient CT-DNA interaction than the complexes with (R)-ligands [19]. Recently, there were some studies on bioactivity of tetradentate salicylaldimine platinum(II) complexes [20][21][22][23]; however, the research number of chiral Schiff base platinum(II) complexes in this field is quite limited. In this work, we continue to study on the synthesis of a series of platinum(II) complexes with chiral tetradentate salicylaldimine ligands obtained by the reaction of (R,R′)-cyclohexanediamine and (S,S′)-cyclohexanediamine with some salicylaldehydes. Characterization of obtained complexes was studied by ESI-MS, FT-IR, NMR, and CD spectroscopies, and their photophysical properties were studied by UV-Vis and luminescent spectra. eir in vitro antitumor activities are also evaluated against KB and MCF-7 human cancer cell lines.

Materials and Methods
Analytical reagent grade chemicals were used as received from commercial companies without further purification. All solvents were purified by following the appropriate purification procedures.
Mass spectra (m/z) were recorded on Agilent 6310 Ion Trap spectrometer. Infrared spectra (cm −1 ) were obtained from KBr pellets using a Perkin Elmer Spectrum Two spectrophotometer. 1 H-NMR and 13 C-NMR spectra were analyzed using a Bruker Advance 500 MHz NMR spectrometer with TMS as internal standard, and chemical shifts (δ) were recorded in ppm. Optical rotations were recorded on Atago POLAX-2L polarimeter at the wavelength of measuring light 589 nm for studied ligands. CD spectra (235-400 nm) were measured on a Chirascan CD spectrometer for obtained chiral salen platinum(II) complexes.

Preparation of Chiral Schiff Base Ligands
Chiral Schiff base ligands were prepared similarly according to the known procedure from the condensation of (R,R′)-or (S,S′)-cyclohexanediamine with the relative salicylaldehydes [24]. Pure (R,R)-1,2-diaminonium cyclohexane mono-L-(+)-tartrate or (S,S)-1,2-diaminonium cyclohexane mono-D-(+)-tartrate (2.05 g, 7.6 mmol) and Na 2 CO 3 (1.65 g, 15.5 mmol) with added methylene chloride (20 mL) and distilled water (15 mL) in a 100 ml flask were stirred for 1 h. After the mixture turned to clear solutions, relative salicylaldehyde (15.4 mmol) was added to organic layer and the new mixture was put under ultrasound for 1 h and then separated to dryness. e organic layer was dried over anhydrous Na 2 SO 4 , and the solvent was removed under reduced pressure to obtain a yellowish product.

Photophysical Property of Chiral Salen Platinum(II) Complexes
e UV-visible absorption spectra of the complexes were measured in DCM (dichloromethane) solutions (2 × 10 −5 M) on Perkin Elmer Lambda UV-35 spectrophotometer. e emission spectra of the solutions were recorded on Horiba Fluorolog spectrofluorometer at room temperature. e obtained photophysical data are shown in Table 1.

In Vitro Cytotoxicity Assay
Human cancer cells KB and MCF-7 were cultured in DMEM with 10% fetal bovine serum, 100 µg/mL streptomycin, 100 units/mL penicillin, and 2 mmol/L L-glutamine at 37oC in humidified atmosphere with 5% CO 2 and 95% air. Cancer cells were cultivated in 96 well plates for 24 h followed by treating with different concentrations of complexes in DMSO for 24 h more. en, testing cells were exposed to 10 µL of freshly prepared MTT (5 mM) solution and incubated for 2 h at 37oC in the atmosphere of 5% CO 2 . e formazan crystals obtained during MTT incubation were dissolved in 100 µL of DMSO. e absorbance was recorded at 540 nm on Genios TECAN spectrophotometer. e experiments were carried out in triplicates for every concentration of the complexes. e percent viable cells were plotted as a function of concentration to determine the IC 50 values presented in Table 2.

Results and Discussion
7.1. Synthesis and Characterization. LH 2 ligands were formed by the condensation of (R,R′)-cyclohexanediamine and (S,S′)-cyclohexanediamine with salicylaldehydes (Figure 1), and then the precipitates formed were collected by filtration and washed by cold ethanol in high yields (>90%). All obtained chiral ligands (Table 3)  e signals at 99.61-133.24 ppm could belong to carbon signals of the salicylidene moiety without substitution. e chemical shifts for carbons with substituted groups moved the higher field compared to the carbon signals without substituted groups, carbons of C−OMe at Pt(II) complexes in the list (Table 3) were obtained in moderate yields (67-73%) by the reaction of K 2 PtCl 4 with each ligand in DMSO. e complexes were characterized by ESI-MS, IR, and NMR. e results received by ESI-MS were suitable to the suggested formulae (Figure 1). e IR spectra of obtained complexes showed the typical signals at 1601-1627 cm It was similar to IR spectra, and there were no big different signals on 1 H and 13 C-NMR spectra of (R)-and (S)enantiomers of ligands and platinum(II) complexes except opposite rotations of polarized light. e CD spectra of these couples of enantiomeric Pt(II) complexes in methanol or DCM (4R, 4S and 6R, 6S) were measured at room temperature from 235 to 400 nm. e couples of chiral enantiomers emitted the equal intense peaks with the opposite orientation ( Figure 2), respectively, which demonstrated the existence of the chiral enantiomers.

Photophysical Property of Chiral Salen Platinum(II)
Complexes.
e photophysical data are shown in Table 1. When dissolved in DCM, all complexes afforded the yellow   solution and exhibited UV-Vis absorption and emission spectra. ere were three main absorption bands as usual on UV-Vis spectra, two intense absorption bands at 288-323 nm and 331-360 nm are probably attributed to intraligand absorption, and the visible band at 398-450 nm may belong to the MLCT (metal-to-ligand charge transfer) state due to Pt(5d) ⟶ π * transition (Figure 3).
ere were no meaningful difference in λ max(abs) between enantiomeric Pt(II) complexes in (R) and (S) configuration. e luminescence spectra from the corresponding excited state possessed an emission maximum at 529-595 nm. e emission maximum wavelengths of complexes' (S)-enantiomers were similar to (R)-enantiomers (Table 1). e Pt(II) complexes with substituted groups at aromatic ring C5position show emission peaks at 566-595 nm, red-shifted

In Vitro Cytotoxicity Assay.
In order to estimate possible effect of ligand enantiomers and substituted groups to the platinum(II) complexes' antitumor activity, the in vitro cytotoxicity of the studied Pt(II) complexes was validated against two human cancer cell lines of KB and MCF-7. e cytotoxicity of ellipticine, a standard anticancer compound, was also evaluated in the same condition for comparison. e IC 50 values (the concentration that inhibited in 50% cellular proliferation) of the obtained complexes are presented in Table 2.
It was noted that all complexes exhibited the antitumor effect with low IC 50 value (<50 µM) for KB and MCF-7 human cancer cell lines. In particular, complexes 1S, 5S, 6R, and 6S had good anticancer activity for both KB and MCF-7 human cancer cell lines. Enantiomers in (S) configuration gave more effect for the Pt(II) complexes' cytotoxicity than ones in (R) configuration (Table 2). e obtained results were similar to the previous results of chiral Ru(II) and Mn(III) salen complexes because the DNA binding affinity of chiral salen complexes (S)-would be greater than (R)- [25,26]. e complexes 3-6 with different methoxy group positions on salicyl ring had different activities. e introduction of methoxy groups to salicylidene moiety afforded the complex 6S with the best antitumor activity obviously for KB and MCF-7 human cancer cells. e complex 6S had the anticancer activity for MCF-7 human cancer cells with IC 50 of 5.72 µM which was quite close to IC 50 of the standard compound ellipticine (2.07 µM).

Conclusions
In this research, a series of platinum(II) complexes with chiral tetradentate salen ligands were synthesized from salicylaldehydes and (R,R′)-cyclohenxanediamine and (S,S′)-cyclohenxanediamine and characterized by IR, ESI-MS, NMR, and CD spectra. Photophysical properties showed their typical absorption and luminescence in UVvisible region. e luminescent spectra exhibited the effect of substituted groups to their luminescent wavelengths obviously; however, there was no big difference between enantiomeric complexes in (R) and (S) configuration. Complex 5 with the substituted MeO at C5-position of salicylidene moiety gave farthest red-shift luminescence (at 595 nm). e in vitro antitumor activity of the studied complexes have been evaluated against KB and MCF-7 human cancer cells by MTT assay, and the results showed all complexes had activity against tested human cancer cell lines. e antitumor activity of enantiomers in (S) configuration was more favourable than enantiomers in (R) configuration. e complexes 6S were the most effective agents to KB and MCF-7 human cancer cells, respectively.

Data Availability
All data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.  Journal of Chemistry