Synthesis and Biological Activities of Luminescent 5,6-Membered Bis(Metallacyclic) Platinum(II) Complexes

Four couples of 5,6-membered bis(metallacyclic) Pt(II) complexes with acetylide and isocyanide auxiliary ligands have been prepared and characterized. The structures of (−)-2 and (−)-3 are confirmed by single-crystal X-ray diffraction, showing a distorted square-planar coordination environment around the Pt(II) nucleus. Both solutions and solid samples of all complexes are emissive at RT. Acetylide-coordinated Pt(II) complexes have a lower energy emission than those isocyanide-coordinated ones. The emission spectra of N^N′*C-coordinated Pt(II) derivatives show a lower energy emission maximum relative to N^C*N′-coordinated complexes with the same auxiliary ligand. Moreover, the difference between cyclometalated N^N′*C and N^C*N′ ligands exerts a more remarkable effect on the emission than the auxiliary ligands acetylide and isocyanide. Cytotoxicity and cell imaging of luminescent 5,6-membered bis(metallacyclic) Pt(II) complexes have been evaluated.

Although the square geometry is improved by forming a 5,6-membered bis(metallacycle), introducing a 5,6-membered bis(metallacycle) would increase molecular flexibility, which is adverse to the emission efficiency.The rectified geometry may alone seem not to result in a large d orbital splitting enough to eliminate thermally accessible nonradiative d-d transition, and NˆC*N -and NˆN *C-coordinated Pt(II) chloride complexes may be weakly emissive [7,8].Acetylide and isocyanide ligands induce a much stronger ligand field than the chloride, which could efficiently raise the nonradiative d−d transition to higher energy, leading to a big jump in emission efficiency in cyclometalated NˆCˆN -and NˆN ˆC-coordinated Pt(II) complexes [15,16].Therefore, introducing strong donors such as acetylide and isocyanide may be a suitable alternative.The emissions of NˆC*N -and NˆN *C-coordinated Pt(II) phenylacetylene and isocyanide derivatives are highly intensive, with Φ em exceeding 40% [9].The phosphorescent phenylacetylene derivatives could be utilized as triplet photosensitizers for triplet-triplet annihilation upconversion [10].
In previous work, we reported a series of pinene-fused NˆCˆN -, NˆN ˆC-, NˆC*N -, and NˆN *C-coordinated Pt(II) complexes and investigated their solvent-, mechano-, vapor-, and thermo-induced color, luminescent, and chiroptical switching behaviors [17][18][19].Pinenecontaining NˆC*N -and NˆN *C-coordinated Pt(II) complexes displayed significant AIE properties in the THF-water solution system by restricting intramolecular vibration and rotation in the poor solvent.A reversible mechanochromic luminescence was found for the NˆN *C-coordinated Pt(II) chloride complex, and its potential utility in an anti-counterfeiting application was attempted [19].As an extension, acetylide and isocyanide ligands are introduced in pinene-containing NˆC*N -and NˆN *C-coordinated Pt(II) complexes (Scheme 1).Four pairs of 5,6-membered bis(metallacyclic) Pt(II) complexes were characterized by NMR, HRMS, and elemental analysis, and the structures of (−)-2 and (−)-3 were unambiguously confirmed by single-crystal X-ray diffraction.We studied all obtained complexes' photophysical and luminescent properties and explored their cytotoxic properties and applications in cell imaging.
Molecules 2023, 28, x FOR PEER REVIEW 2 of 18 accessible nonradiative d−d transition, and N^C*N′-and N^N′*C-coordinated Pt(II) chloride complexes may be weakly emissive [7,8].Acetylide and isocyanide ligands induce a much stronger ligand field than the chloride, which could efficiently raise the nonradiative d−d transition to higher energy, leading to a big jump in emission efficiency in cyclometalated N^C^N′-and N^N′^C-coordinated Pt(II) complexes [15,16].Therefore, introducing strong donors such as acetylide and isocyanide may be a suitable alternative.The emissions of N^C*N′-and N^N′*C-coordinated Pt(II) phenylacetylene and isocyanide derivatives are highly intensive, with Φem exceeding 40% [9].The phosphorescent phenylacetylene derivatives could be utilized as triplet photosensitizers for triplet-triplet annihilation upconversion [10].
In previous work, we reported a series of pinene-fused N^C^N′-, N^N′^C-, N^C*N′-, and N^N′*C-coordinated Pt(II) complexes and investigated their solvent-, mechano-, vapor-, and thermo-induced color, luminescent, and chiroptical switching behaviors [17][18][19].Pinene-containing N^C*N′-and N^N′*C-coordinated Pt(II) complexes displayed significant AIE properties in the THF-water solution system by restricting intramolecular vibration and rotation in the poor solvent.A reversible mechanochromic luminescence was found for the N^N′*C-coordinated Pt(II) chloride complex, and its potential utility in an anti-counterfeiting application was attempted [19].As an extension, acetylide and isocyanide ligands are introduced in pinene-containing N^C*N′-and N^N′*C-coordinated Pt(II) complexes (Scheme 1).Four pairs of 5,6-membered bis(metallacyclic) Pt(II) complexes were characterized by NMR, HRMS, and elemental analysis, and the structures of (−)-2 and (−)-3 were unambiguously confirmed by single-crystal X-ray diffraction.We studied all obtained complexes' photophysical and luminescent properties and explored their cytotoxic properties and applications in cell imaging.

Spectroscopic Properties
The solution spectroscopic properties of all the complexes are shown in   3).Both complexes (−)-1 and (−)-3 display a vibronically structured emission with λmax at 493 and 511 nm and shoulders at 528 and 541 nm, respectively (Figure 3 and Table 2).In contrast, a nearly structureless The CH 2 Cl 2 solutions of NˆC*N -coordinated Pt(II) derivatives (−)-1 and (−)-3 emit in the green-yellow region at RT, while the solutions of NˆN *C-coordinated Pt(II) complexes (−)-2 and (−)-4 emit orange light (Figure 3).Both complexes (−)-1 and (−)-3 display a vibronically structured emission with λ max at 493 and 511 nm and shoulders at 528 and 541 nm, respectively (Figure 3 and Table 2).In contrast, a nearly structureless band in the orange region with lower energy emission peaks at 578 and 595 nm is perceived for complexes (−)-2 and (−)-4, respectively.Acetylide-coordinated Pt(II) complexes have a lower energy emission in CH 2 Cl 2 solution than those isocyanide-coordinated ones (∆λ = ca.20 nm) (Figure 3 and Table 2), which is consistent with their absorption spectra.In addition to this, the emission spectra of NˆN *C-coordinated Pt(II) derivatives show a lower energy emission maximum relative to NˆC*N -coordinated complexes with the same auxiliary ligand (∆λ = ca.80 nm) (Figure 3 and Table 2).The difference between cyclometalated NˆN *C and NˆC*N ligands exerts a more remarkable effect on the emission than the auxiliary ligands acetylide and isocyanide.The emission dependences on concentration and solvent are insignificant (Figures S16-S23).When changing from toluene to methanol, the solvatochromic emission shift is almost less than 10 nm, and the emission profiles stay the same with increasing concentration.All the complexes provide a more structured and narrower emission band in frozen glass at 77 K than in a fluid at RT (Figure S28).The complexes ((−)-1 and (−)-3) with NˆC*N skeleton show a negligible rigidochromic effect with the value of emission maximum shift 5-8 nm.In contrast, the rigidochromic shift of derivatives ((−)-2 and (−)-4) with NˆN *C parent ligand is evident with a 22-34 nm difference between the emission maximum in the fluid and the rigid glass.According to the reported investigations on 5,6-membered bis(metallacyclic) Pt(II) complexes, not only the cyclometalated parent ligand (NˆC*N and NˆN *C) and the auxiliary ligand (isocyanide and acetylide) could affect the emission of complexes but also the conjugation degree of the 5,6-membered bis(metallacycle) through the amino N atom exerts a considerable influence on the emission [8,19].The emissive state of all complexes can be assigned to a ligand-centered (LC) triplet transition ( 3 π,π) with some CT transitions [7][8][9]19].The low energy emissions (578 and 595 nm) have more CT character than the high-energy ones (493 and 511 nm) [8,19].For complexes (−)-2 and (−)-4, the geometric change to maximize conjugation of the 5,6-membered bis(metallacycle) through the amino N atom is unfavorable in the rigid matrix.Hence, the emission state has more LC 3 π,π characteristic in the rigid glass at 77 K, showing a high energy emission.
The solids of all complexes are emissive from the green-yellow to orange regions at RT (Figure 4 and Table 2).The emission spectra of NˆC*N -coordinated complexes are more structured and show a higher energy emission maximum than the ones of NˆN *Ccoordinated derivatives.At 77 K, the emission profiles become highly structured and narrow.The solids show higher emission quantum efficiencies (Φ em ) than the solutions (Table 2).The NˆN *C-coordinated derivatives with isocyanide and acetylide could not matrix.Hence, the emission state has more LC 3 π,π characteristic in the rigid glass at 77 K, showing a high energy emission.
The solids of all complexes are emissive from the green-yellow to orange regions at RT (Figure 4 and Table 2).The emission spectra of N^C*N′-coordinated complexes are more structured and show a higher energy emission maximum than the ones of N^N′*C-coordinated derivatives.At 77 K, the emission profiles become highly structured and narrow.The solids show higher emission quantum efficiencies (Φem) than the solutions (Table 2).The N^N′*C-coordinated derivatives with isocyanide and acetylide could not form aggregates under external stress like chloride precursors, and the mechanochromic luminescence phenomenon is not realized for (−)-2 and (−)-4 [19].

Theoretical Investigation
Time-dependent density functional theory (TD-DFT) calculations have been performed to explore the origin of the transitions [27].As revealed in Figure 5 and Table S3, the nature of S 0 →S 1 transition of all complexes mainly derive from HOMO (the highest occupied molecular orbital)→LUMO (the lowest unoccupied molecular orbital) with the overwhelming contribution of over 90%.Also, the S 0 →S 1 transition of complex (−)-1 involves some composition of HOMO-1→LUMO (7.0%).Based on molecular orbital (MO) patterns and orbital composition analysis (Figure 5 and Table S3), the HOMO of complexes (−)-1 and (−)-2 is mainly delocalized on Ring B, Ring C, and their bridging atom amino N with a total contribution of over 80%.Their LUMO concentrates on phenyl-pyridine or dipyridine moiety (Ring A and Ring B).In addition, the central Pt atom in (−)-1 and (−)-2 contributes little to HOMO and LUMO.
For acetylide-coordinated Pt(II) complexes (−)-3 and (−)-4, the HOMO primarily comes from Pt nucleus and phenylacetylene with a total contribution of over 60%, and the fragments Ring B and Ring C also contribute some to the HOMO (Figure 5 and Table S3).Similar to the LUMO distribution of (−)-1 and (−)-2, the phenyl-pyridine or dipyridine moiety (Ring A and Ring B) gives a dominant contribution (>75%) to the LUMO of (−)-3 and (−)-4.Therefore, a mixture of 1 ILCT (NˆC*N or NˆN *C), 1 MLCT (from Pt(II) atom to NˆC*N or NˆN *C) and 1 LLCT (from phenyl isocyanide or phenylacetylene to NˆC*N or NˆN *C) transitions should be responsible for the lowest energy absorption band in the UV-vis spectra of all complexes.Furthermore, more 1 MLCT and 1 LLCT components are involved in acetylide-coordinated Pt(II) complexes (−)-3 and (−)-4, showing a calculated longer absorption wavelength than (−)-1 and (−)-2 with the same cyclometalated ligand (Table S3), which accords with the ones observed in the experiment (Figure 3).
To gain an insightful understanding of emission, natural transition orbital (NTO) analysis has been accomplished to examine the S 0 →T 1 excitation based on optimized T 1 geometries (Figure 6 and Table S4) [19,28].The hole (H) orbital of (−)-1 is mainly resident in the phenyl-pyridine or dipyridine moiety (Ring A and Ring B), and the one of (−)-2 is spread over the whole NˆN *C ligand (amino N atom, Ring A, Ring B, and Ring C).For complexes (−)-3 and (−)-4, the central Pt atom holds a considerable distribution (18.16% and 11.07%) of the H orbital in addition to the contribution of the NˆC*N or NˆN *C parent.All complexes' particle (P) orbitals are mainly distributed on the phenyl-pyridine or dipyridine moiety (Ring A and Ring B), with a predominant contribution of over 85%.Hence, the luminescence of (−)-1 and (−)-2 can be mainly assigned as a ligand-centered (LC) triplet state ( 3 π,π*) with minor CT character.In contrast, the phosphorescence of (−)-3 and (−)-4 originates from a mixture of 3  To gain an insightful understanding of emission, natural transition orbital (NTO) analysis has been accomplished to examine the S0→T1 excitation based on optimized T1 geometries (Figure 6 and Table S4) [19,28]

Cytotoxicity
All the prepared 5,6-membered bis(metallacyclic) Pt(II) complexes, including chloride precursors, were evaluated for their cytotoxicity against human cancer cell lines K562, SGC-7901, BEL-7402, A549, and HeLa with cisplatin as the positive control [29,30].From Table 3, the obtained half-inhibitory concentration (IC50) values range from 0.47 to Figure 6.Natural transition orbital patterns for S 0 -T 1 excitation for all complexes on the basis of their optimized T 1 geometries.

Cell Imaging
Given the intriguing luminescence properties of 5,6-membered bis(metallacyclic) Pt(II) complexes, we tentatively explore their applications in cell imaging.The cultured HeLa cells were inoculated in glass-bottom cell culture dishes.After overnight culture, the cells were stained with 10 µmol/L phosphorescent Pt complexes in a DMSO/DMEM mixture (5/95) for 15 min.After the removal of extracellular fluorescent dyes through washing with PBS buffer, the cell samples were imaged using confocal laser scanning fluorescence microscopy in blue, green, and red channels [38,39].As shown in Figure 7, the chloride precursor Pt(NˆC*N )Cl can efficiently permeate cells, showing bright blue luminescence and bright green emission, and the image in the red channel is not satisfactory enough.

General Methods
All reagents were purchased from commercial suppliers and used as received.Highresolution ESI (HR-ESI) mass spectrometry spectra were acquired on Thermo Scientific (Waltham, MA, USA) Q Exactive Mass, Thermo Scientific Q Exactive Focus, and Aglient (Santa Clara, CA, USA) 7250 & JEOL-JMS-T100LP AccuTOF (Tokyo, Japan) Spectrometer.The 1 H and 13 C NMR spectra were obtained on Bruker (Mannheim, Germany) DRX-400 spectrometer.Coupling constants are provided in hertz.UV-vis spectra were measured on a UV-3600 spectrophotometer.Photoluminescence (PL) spectra were measured by a Hitachi (Tokyo, Japan) F-4600 PL spectrophotometer (λ ex = 420 nm).Emission quantum yields (λ ex = 420 nm) and lifetimes were measured on a HORIBA JY (Kyoto, Japan) system.The circular dichroism (CD) spectra in CH 2 Cl 2 solution were recorded on a Jasco (Tokyo, Japan) J-810 spectropolarimeter at a scan rate of 100 nm•min −1 and 1 nm resolution at room temperature (using 10 mm quartz cell for the concentration of 5 × 10 −5 mol•L −1 , bandwidth = 1 nm, response = 1 s, accumulations = 3).Images of cells were obtained using a Nikon (Tokyo, Japan) A1 confocal laser scanning microscope.

Preparation of Chloride Precursors
NˆC*N -and NˆN *C-coordinated Pt(II) chloride precursors were prepared according to our reported literature [19].

Preparation of (−)-1
An equivalent of 2,6-dimethylphenyl isocyanide (18.3 mg, 0.14 mmol) dissolved in dichloromethane was added dropwise into a vigorously stirred dichloromethane solution of Pt((−)-NˆC*N )Cl (100 mg, 0.14 mmol) pre-covered by an aqueous solution of excess AgOTf.After stirring at RT for 1 h, the dichloromethane solution was separated, and the aqueous phase was extracted with dichloromethane (20 mL × 3).The organic phase was washed with brine water and then dried over anhydrous Na 2 SO 4 .After removing the solvent in vacuo, the final product was obtained as green-yellow powder.Yield: 80%. 1

Preparation of (−)-3
A methanol solution of phenylacetylene (20.5 mg, 0.2 mmol) and sodium hydroxide (8 mg, 0.2 mmol) was stirred for 30 min at RT.Then, the chloride precursor Pt((−)-NˆC*N )Cl (97 mg, 0.15 mmol) was added to the above solution, reacting for a further 24 h.The solvent was removed in vacuo, and the solid was washed with methanol several times (20 mL × 3).The final product, a yellow powder, was obtained after recrystallization in a mixed methanol/chloromethane solution.Yield: 85%. 1

Single-Crystal X-ray Structure Determination
Single-crystal X-ray diffraction measurements were performed on a Bruker SMART APEX CCD and Rigaku (Tokyo, Japan) XtaLAB Synergy R. Intensities were collected with graphite monochromatized Mo Kα radiation (λ = 0.71073 Å) operating at 50 kV and 30 mA using ω/2θ scan mode.The data reduction was performed with the Bruker SAINT package [42].Absorption corrections were performed using the SADABS program [43].The structures were solved by direct methods and refined on F 2 by full-matrix least-squares using SHELXL-2018/3 (Sheldrick, 2018) with anisotropic displacement parameters for all non-hydrogen atoms in the two structures.Hydrogen atoms bonded to carbon atoms were placed in calculated positions and refined as riding mode, with C−H = 0.93 Å (methane) or 0.96 Å (methyl) and Uiso(H) = 1.2 Ueq (C methane ) or Uiso(H) = 1.5 Ueq (C methyl ).All computations were carried out using the SHELXL-2018/3 program package [44].CCDC numbers 2279411-2279412 contain the supplementary crystallographic data for this paper.These data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures/ (accessed on 5 July 2023) or by e-mailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44(0)1223-336033.

Calculation Methods
The crystal structures of (−)-2 and (−)-3 were used as starting geometries, and calculations were performed with the Gaussian 09 program [45].Geometry optimizations of ground states were simulated with density functional theory (DFT) at the hybrid functional PBE1PBE-D3/LANL2DZ (Pt) and PBE1PBE-D3/6-31g(d,p) (H, C, N) levels using CH 2 Cl 2 as solvent.The solvent effect is based on the polarizable continuum model (PCM).The optimized structures were used to calculate the lowest singlet electronic transition using the time-dependent density functional theory (TDDFT) method.The geometry of the first triplet state (T 1 ) was optimized, and the analysis of the natural transition orbital (NTO) was carried out for the excitation of S 0 →T 1 [28,46].Mulliken population analysis (MPA) was utilized to obtain the electron density distribution of each atom in the specific molecular orbital of the Pt(II) complexes using the Multiwfn program [47].

Cytotoxicity
The cytotoxic activities of obtained 5,6-membered bis(metallacyclic) Pt(II) complexes were assessed against K562 (human leukemia cell line), SGC-7901 (human gastric carcinoma cells line), BEL-7402 (human hepatocellular carcinoma cell line), A549 (human non-small cell lung cancer cell line), and HeLa (human cervical cancer cell line) by MTT assay [29,30].Briefly, the logarithmic phase cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 mg/mL streptomycin under conditions of 37 • C, 5% CO 2 , and 90% humidity.These human tumor cell lines with a density of 5 × 10 4 unit/mL were seeded onto 96-well plates and then, after 24 h of incubation, treated with different concentrations of the sample dissolved in DMSO, respectively, while cisplatin was used as the positive control and DMSO was used as the negative control.After 72 h of incubation, MTT was dissolved at 5 mg/mL in PBS and used essentially as previously described.Finally, the inhibition rates were calculated using OD mean values measured by the MK3 Microtiter plate reader at 490 nm, and the IC 50 value expressed as the mean standard deviation was determined using the Bliss method.

Conclusions
In summary, we have synthesized four groups of enantiomeric NˆC*N -and NˆN *Ccoordinated Pt(II) complexes featuring a fused 5,6-membered bis(metallacycle).Their structures have been determined by NMR, HRMS, and single-crystal X-ray diffraction.Distorted square-planar coordination of the Pt(II) nucleus is observed for both isocyanideand acetylide-containing derivatives.The solution of all complexes is emissive in the greenyellow or orange regions, and acetylide-coordinated Pt(II) complexes show a lower energy emission than those isocyanide-coordinated ones with the same cyclometalated ligand (∆λ = ca.20 nm in the solution).Furthermore, the difference between cyclometalated NˆN *C and NˆC*N ligands induces a more significant effect (∆λ = ca.80 nm in the solution) on the emission.The influence trend is also observed for the solid-state emission.The emissive state of all complexes can be attributed to a ligand-centered (LC) triplet transition ( 3 π,π) with some CT transitions.Platinum(II) complex (−)-2 coordinated with isocyanide displays high cytotoxicity against the above five human cancer lines K562, SGC-7901, BEL-7402, A549, and HeLa.The existence of NˆN *C 5,6-membered bis(metallacycle) may be important for high cytotoxicity.All the complexes can efficiently permeate cells, mainly distributing in cell membranes and showing a clear cell outline.This research provides a reference for developing biologically active 5,6-membered bis(metallacyclic) Pt(II) complexes.

18 Figure 5 .
Figure 5. Molecular orbital (MO) patterns of all complexes on the basis of their optimized S0 geometries.
. The hole (H) orbital of (−)-1 is mainly resident in the phenyl-pyridine or dipyridine moiety (Ring A and Ring B), and the one of (−)-2 is spread over the whole N^N′*C ligand (amino N atom, Ring A, Ring B, and Ring C).For complexes (−)-3 and (−)-4, the central Pt atom holds a considerable distribution (18.16% and 11.07%) of the H orbital in addition to the contribution of the N^C*N′ or N^N′*C parent.All complexes' particle (P) orbitals are mainly distributed on the phenyl-pyridine or dipyridine moiety (Ring A and Ring B), with a predominant contribution of over 85%.Hence, the luminescence of (-)-1 and (−)-2 can be mainly assigned as a ligand-centered (LC) triplet state ( 3 π,π*) with minor CT character.In contrast, the phosphorescence of (-)-3 and (−)-4 originates from a mixture of 3 π,π* and 3 MLCT (from Pt(II) atom to N^C*N′ or N^N′*C) emissive states.

Figure 5 . 18 Figure 6 .
Figure 5. Molecular orbital (MO) patterns of all complexes on the basis of their optimized S 0 geometries.Molecules 2023, 28, x FOR PEER REVIEW 10 of 18

Table 2 .
Luminescent data in different states at RT and 77 K.