Design of a Tris-Heteroleptic Ru(II) Complex with Red-Light Excitation and Remarkably Improved Photobiological Activity

Article

Design of a Tris-Heteroleptic Ru(II) Complex with Red-Light

Excitation and Remarkably Improved Photobiological Activity

Shuang Li, Jian Zhao,* Xinyi Wang, Gang Xu, Shaohua Gou,* and Qiang Zhao*

Cite This: https://dx.doi.org/10.1021/acs.inorgchem.0c01860

Read Online

ACCESS

Metrics & More

Article Recommendations

sı Supporting Information

ABSTRACT: Ru(II)-polypyridyl complexes are of increasing interest in photodynamic therapy (PDT) due to their easily tunable

photophysical and photochemical properties. However, short-wavelength absorption of Ru(II)-polypyridyl complexes has limited

their penetration depth in PDT. Herein, the series of Ru(II)-polypyridyl complexes 1−4 was designed by replacing one bipyridine in

[

Ru(bpy) ]Cl with Schiﬀ bases (iminopyridine or iminoquinoline analogues) to achieve red-shifted absorption of Ru(II)-

3 2

polypyridyl photosensitizers. To further shift the absorption to longer wavelength and improve the photobiological activity of

Ru(II)-polypyridyl complexes, the three tris-heteroleptic Ru(II) complexes 5−7 with benzo[i]dipyrido[3,2-a:2′,3′-c]phenazine

(

dppn) as a ligand were designed to achieve long-lived intraligand ( IL) excited states. Cytotoxicity data against A549 and HepG2

cells revealed that complex 7 showed extraordinarily high cytotoxicity under 650 nm irradiation, resulting in IC values of 56 and 63

nM with exceptionally large phototoxicity index (PI) values of 763 and 613, respectively. Thus, the resulting complex 7 with

considerable red-light photocytotoxicity and high PI values shows a promising potential for therapeutic applications, which

represents a new scaﬀold of Ru(II)-polypyridyl photosensitizers for PDT in the “therapeutic window”. This study delivers a rational

strategy for the design of tris-heteroleptic Ru(II) complexes as promising photosensitizers for cancer therapy.

7−31

INTRODUCTION

tissue treatment.

absorption band (generally a metal to ligand charge transfer

MLCT) transition) of most Ru(II) complexes is located in

However, the longest-wavelength

■

The successful application of platinum-based anticancer drugs

in the clinic has stimulated increasing interest in discovering

new metallodrugs, in which ruthenium complexes have been

(

the blue region (<500 nm), which has limited their

phototherapeutic applications for deep-tissue diseases.

Consequently, exploring novel Ru(II) complexes with one-

photon red-light excitation is highly desired for PDT.

Schiﬀ bases are useful for chelating ligands to metal ions,

resulting in complexes with diﬀerent physical and chemical

properties. Thus, metal complexes of Schiﬀ bases have been

extensively designed and investigated for biomedical applica-

tions, such as antitumor, anti-inﬂammatory, antibacterial, and

32−34

−6

thought to be promising alternatives.

To date, two

ruthenium(III) complexes (NAMI-A, KP1019 and its sodium

salt KP1339) have been evaluated in clinical trials for cancer

chemotherapy. To further improve the therapeutic eﬃcacy,

Ru(II)-polypyridyl complexes with unique photophysical and

photochemical properties have been designed as photo-

dynamic therapy (PDT) agents.

−20

A well-known example

is TLD1433, which successfully completed phase Ib clinical

trials in 2018.

Received: June 23, 2020

In PDT, the photosensitizer (PS) used is generally nontoxic

in the dark but can be activated to produce cytotoxic reactive

oxygen species (ROS) by light of a suitable wavelength.

22−26

As red and near-infrared lights enable maximum tissue

penetration with minimum damage, PS activated in the

“therapeutic window” of 600−850 nm is suitable for deep-

XXXX American Chemical Society

Inorg. Chem. XXXX, XXX, XXX−XXX

Figure 1. Ru(II)-polypyridyl complexes studied in this work.

Article

Figure 2. Frontier molecular orbital diagrams and energy proﬁles for the HOMOs and LUMOs of [Ru(bpy)₃]²⁺and complexes 1−7.

Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Figure 3. UV−vis absorption spectra of complexes 1−7 and [Ru(bpy) ]Cl in methanol. The insets show expansions of the absorption above 460

nm.

antifungal activities. Recently, our group reported that

dinuclear Ru(II)-arene complexes containing Schiﬀ base

ligands (iminopyridine or iminoquinoline) exhibited unique

RESULTS AND DISCUSSION

■

Synthesis and Photophysical Properties. Complexes

−7 were synthesized and characterized as described in the

Figures S1 −S21 and Scheme S1in the Supporting Information.

The UV−vis absorption spectra of these complexes are

presented in Figure 3. These complexes exhibit intense

absorptions in the ultraviolet region, which are typical

absorption bands of Ru(II)-polypyridyl complexes due to the

ππ* transitions of the ligands. Notably, the longest absorption

maxima of complexes 1−7 are located above 470 nm, which

are red-shifted in comparison to that of [Ru(bpy) ]Cl at 450

biological characteristics. Moreover, replacing pyridine

ligands with imine ligands in Ru(II) complexes can result in

a red shift of the MLCT absorption band.

intend to introduce iminopyridine and iminoquinoline ligands

to Ru(II)-polypyridyl complexes with improved photophysical

and biological properties for PDT. Herein we utilized

38,39

Therefore, we

[

Ru(bpy) ]Cl as a model complex and replaced its one

3 2

bipyridine with imine ligands to obtain complexes 1−4 (Figure

Prior to undertaking the experiments, we performed DFT

density functional theory) calculations to investigate the

nm. Moreover, complexes 3, 4, and 7 with iminoquinoline

ligands showed longer wavelength absorption in the visible

region in comparison to the corresponding complexes 1, 2, and

6 with iminopyridine ligands due to the highly delocalized π-

system of the iminoquinoline ligand. In addition, a close

examination of the UV−vis absorption spectra of complexes 4

and 7 revealed that the absorption tails of both complexes

extend over 650 nm, indicating that they have the potential to

be excited by red light. In particular, complex 7 displayed weak

but clearly observable absorption bands above 700 nm (700

(

HOMO/LUMO energy levels of complexes 1−4. As shown in

Figure 2, the HOMO−LUMO energy gaps of complexes 1−4

are smaller than that of [Ru(bpy) ] , which may lead to red

shifts of the MLCT bands of complexes 1−4, providing a

theoretical foundation for the following study. In addition,

complexes 2 and 4 possess donor (dimethylamino)−acceptor

pyridine or quinoline) structures, which have relatively lower

(

−

−1

nm, ε = 200 M cm ). It was reported that Ru(II) complexes

band gap energies in comparison with complexes 1 and 3,

respectively. However, according to the energy gap law, red-

light-absorbing metal complexes often exhibit a short excited-

state lifetime that may compromise the therapeutic eﬃciency

with long-lived IL excited states are highly sensitive to red-

light (630 nm) excitation, which could be excited at

wavelengths even where their molar extinction coeﬃcients

−

−1 21,46

are very low (ε < 100 M cm ).

The luminescence

of these complexes. Actually, the features of the triplet

excited states of metal-based PSs can be tuned for diﬀerent

application purposes such as phototherapy, catalysis, and

electroluminescence. Subtle ligand modiﬁcations on metal-

based PSs can lead to diﬀerent photophysical and photo-

chemical properties and result in signiﬁcant changes in the

spectra of complexes 1 −7 and [Ru(bpy) ]Cl were studied and

are presented in Figure S22 . All of the complexes showed near-

infrared emission spectra between 700 and 900 nm in

methanol, which are red-shifted in comparison to the emission

of [Ru(bpy) ]Cl , which might be attributed to CN bond

distortion in the excited state. It is noted that the emission

intensity of complexes 1−7 is so low that the quantum yields

are below 1% in deaerated methanol solutions. In addition, the

anticancer eﬃcacy. McFarland has demonstrated that Ru(II)

complexes with long-lived intraligand ( IL) excited states are

extremely sensitive to O and can result in considerable

emission of complexes 1−7 can be quenched by O ,

demonstrating that the luminescence can be attributed to

phosphorescence from the triplet excited state.

Theoretical Calculations. DFT calculations were under-

taken to gain insight into the electronic transitions of these

Ru(II) complexes, and the fully optimized geometries can be

found in Table S1 in the Supporting Information. The selected

1,43−46

photocytotoxicity.

Moreover, the IL excited states of

Ru(II)-polypyridyl complexes can be achieved by π-expansive

ligands, such as benzo[i]dipyrido[3,2-a:2′,3′-c]phenazine

7−49

(

dppn),

which can greatly improve the photocytotoxicity

of the resulting Ru(II) complexes, as reported by Turro and

co-workers. Thus, in order to improve the photobiological

activity of Ru(II)-imine complexes, the three tris-heteroleptic

Ru(II) complexes 5−7 were further designed to achieve long-

vertical transition energies between the ground state (S ) and

the singlet excited states (S ) are presented in Table 1. For

lived IL excited states with dppn as a ligand. It was anticipated

complexes 2, 4, 6, and 7, it is found that the molecular

HOMOs are mostly localized on the donor group

(dimethylamino), while the LUMOs (LUMO+1 for complexes

6 and 7) are more localized on the acceptor core (pyridine or

that complex 7 with a remarkable red-shifted absorption could

exhibit signiﬁcantly high photocytotoxicity in the PDT

therapeutic window.

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

Table 1. Selected Calculated Singlet Excited-State Transitions for Complexes 1−7 and [Ru(bpy) ]Cl₂

quinoline). Therefore, the absorption bands (S −S ) of

acceptor) or LLCT (Schiﬀ base ligands to bipyridine/dppn)

along with MLCT transitions, while complexes 1, 3, and

complexes 2, 4, 6, and 7 are composed of ILCT (donor to

Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Figure 4. Absorption spectra of ABDA (50 μM) in the presence of 1 (a), 2 (b), 3 (c), 4 (d), 5 (e), 6 (f), 7 (g), and [Ru(bpy) ]Cl (h) at

concentrations of 10 μM upon 465 nm irradiation.

Table 2. Photobiological Activity of Complexes 1−7 and [Ru(bpy) ]Cl toward A549 and HepG2 Cancer Cells

A549

HepG2

IC , μM

(dark)

compound

(dark)

(465 nm)

(650 nm)

(465 nm)

(650 nm)

64.3 ± 2.9

51.6 ± 3.1

52.5 ± 2.6

49.6 ± 3.7

51.8 ± 3.3

48.7 ± 2.8

42.7 ± 3.5

54.4 ± 4.7

24.9 ± 3.4

29.5 ± 2.6

22.6 ± 1.3

2.6

1.7

2.3

3.1

582

667

43.8 ± 3.1

35.5 ± 3.5

30.4 ± 1.7

1.5

1.7

2.6

214

327

763

59.4 ± 1.9

48.5 ± 2.4

55.7 ± 4.6

45.9 ± 2.7

46.4 ± 3.0

40.4 ± 2.5

38.6 ± 2.1

53.2 ± 4.2

23.6 ± 1.7

25.8 ± 2.3

34.1 ± 3.1

2.5

1.9

1.6

4.0

713

897

965

40.3 ± 3.1

37.1 ± 2.2

33.6 ± 2.5

1.5

1.3

1.7

2.7

201

306

613

16.2 ± 1.1

18.8 ± 1.2

11.5 ± 1.0

17.3 ± 1.4

0.089 ± 0.008

0.073 ± 0.011

0.038 ± 0.003

8.5 ± 0.6

0.242 ± 0.006

0.149 ± 0.005

0.056 ± 0.004

53.1 ± 1.6

0.065 ± 0.008

0.045 ± 0.006

0.040 ± 0.003

7.5 ± 0.4

0.231 ± 0.012

0.132 ± 0.007

0.063 ± 0.004

49.1 ± 1.8

1124

[

Ru(bpy) ]

6.4

1.0

7.1

1.1

PI = dark IC₅₀value/light IC₅₀value.

[

Ru(bpy) ] are composed of MLCT transitions (Table 1 and

4 and 7 can lead to an obvious decrease in ABDA absorbance,

Figures S23 −S30 ). In addition, the calculated energy level of

the S state of [Ru(bpy) ] is 2.72 eV (456 nm), which is

higher than those of complexes 1−7, in accordance with the

experimental results that the absorption tails of complexes 1−7

terminate at longer wavelength in comparison with [Ru-

indicating that they are capable of producing O under 650

nm light irradiation. Strikingly, complexes 5 and 6 with low

molar absorption coeﬃcients at 650 nm can cause severe

reduction in ABDA, which is probably due to their highly

photosensitizing IL excited states. In addition, little decrease

in ABDA absorbance was observed for complexes 1−3 and

[Ru(bpy) ]Cl due to their negligible molar absorption

(

bpy) ] .

O₂Generation. The singlet oxygen ( O ) generation

ability was evaluated for complexes 1−7 together with

coeﬃcients in the red-light region. This study indicates that

complexes 4−7 could be excited by red light, which provided

the conditions for these complexes to realize PDT.

[

Ru(bpy) ]Cl as a control by using 9,10-anthracenediyl-

bis(methylene)dimalonic acid (ABDA) as a probe, which can

react with O and convert to a steady-state endoperoxide

In Vitro Photocytotoxicity. The in vitro photocytotox-

icity of complexes 1−7 was evaluated against A549 and HepG2

cancer cells by using an MTT assay, together with [Ru-

(bpy) ]Cl for comparison (Table 2 and Figure S32 ). Upon

product, thereby leading to a decrease in absorption intensity

at around 378 nm. As shown in Figure 4, the absorbance of

ABDA decreased dramatically in the presence of complexes 5−

−

and [Ru(bpy) ]Cl upon 465 nm irradiation (1.26 J cm ),

blue-light irradiation (465 nm), the photocytotoxicity enhance-

whereas a slight decrease in ABDA absorbance was observed

ment of complexes 1−4 and [Ru(bpy) ]Cl toward the tested

for complexes 1−4. By using [Ru(bpy) ]Cl as a reference (Φ

cell lines is modest with PI (phototoxicity index) values

ranging from 1.6 to 7.1 (Table 2). Unexpectedly, complexes

5−7 exhibited greatly enhanced cytotoxicity against A549 and

HepG2 cancer cells with blue-light irradiation, resulting in IC

0.41 in water), the O quantum yields of complexes 1−7

were determined to be 0.025, 0.036, 0.061, 0.046, 0.27, 0.33,

and 0.36, respectively, revealing that complexes 5−7 and

[

Ru(bpy) ]Cl are capable of producing O more eﬃciently

values ranging from 38 and 89 nM with exceptionally large PI

values from 582 to 1124, respectively, which are 2 orders of

magnitude larger than those of complexes 1−4. This may be

than complexes 1−4 under 465 nm irradiation.

Considering the red-light absorption ability of complexes 1−

, we further investigated their O generation with irradiation

attributed to the highly photosensitizing IL excited state of

−

at 650 nm (19.2 J cm ) (Figure S31). As expected, complexes

complexes 5−7. Importantly, upon red-light irradiation (650

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

respectively (Figure 5 and Figure S33 ). In addition, complex

Article

nm), considerable cytotoxicity was also observed for complexes

−7 against A549 and HepG2 cells with PI values ranging

from 201 to 763. In contrast, complexes 1−4 and [Ru(bpy) ]-

Cl produced no discernible photodynamic activities (PI =

.0−2.7) upon irradiation with red light. When these results

are taken together, complex 7 with the most considerable red-

light photocytotoxicity as well as the highest PI value within

these complexes shows a promising potential for therapeutic

applications.

To further conﬁrm the considerable photocytotoxicity of

complex 7, a calcein AM and propidium iodide (PI) costaining

assay was carried out to label the living and dead cells as

indicators by staining the cytoplasm with the green

ﬂuorescence generated by the enzymatic hydrolysis of calcein

AM and the nucleus with the red ﬂuorescence of PI,

Figure 6. Confocal ﬂuorescence images of ROS generation in A549

cells incubated with complexes 4 and 7 and [Ru(bpy) ]Cl upon 465

and 650 nm irradiation.

Cellular Accumulation. To investigate the possible

mechanism of the considerable photocytotoxicity of complex

on A549 cancer cells, the intracellular content of Ru was

detected using ICP-MS (inductively coupled plasma mass

spectrometry) together with complex 4 and [Ru(bpy) ]Cl for

comparison. As shown in Figure S34 , there was no obvious

diﬀerence of the intracellular Ru contents for complex 4 (91.7

8.3 ng/10 cells) and 7 (98 ± 7.2 ng/10 cells). Thus, it is

rational to conclude that the cellular accumulation is not the

main reason for the markedly improved photocytotoxicity of

complex 7.

DNA Photocleavage. Ru(II)-polypyridyl complexes are

known to induce DNA photocleavage, which may be

responsible for the observed photocytotoxicity to some extent.

Thus, the DNA photocleavage ability of complex 7 was

investigated under blue (465 nm)- and red-light (650 nm)

irradiation by agarose gel electrophoresis together with

complex 4 and [Ru(bpy) ]Cl for comparison. As shown in

Figure 5. Confocal ﬂuorescence images of calcein AM (green, live

cells)/propidium iodide (PI; red, dead cells) costained A549 cells

after treatment with complexes 4, 7, and [Ru(bpy) ]Cl at

concentrations of 1 μM: (a, left) without irradiation, (b, right)

irradiation at 650 nm.

Figure 7 A, complex 4 did not show any observable DNA

and [Ru(bpy) ]Cl were also studied for comparison. As

3 2

shown in Figure 5a, no signiﬁcant cell death was observed

without irradiation for A549 cells after treatment with

complexes 4, 7 and [Ru(bpy) ]Cl . Once irradiation was

conducted under 465 or 650 nm, cells treated by complex 7

were eﬀectively killed as revealed by the intense red

ﬂuorescence, whereas complex 4 or [Ru(bpy) ]Cl treated

cells were negligibly damaged, demonstrating the robust PDT

ability of complex 7 again.

Intracellular ROS Production. The intracellular ROS

generation in A549 cells after treated with complexes 4, 7, and

[

Ru(bpy) ]Cl was evaluated using a 2,7-dichlorodihydro-

3 2

ﬂuorescein diacetate (DCFH-DA) staining method. DCFH-

DA is a nonﬂuorescent cell-permeable indicator for ROS,

which can be converted to the highly ﬂuorescent DCF upon

intracellular oxidation by ROS. As shown in Figure 6, no ROS

production was observed in A549 cells without light

irradiation. However, after they were exposed to 465 nm

light irradiation, the compound-treated A549 cells showed

obvious green ﬂuorescence, demonstrating the successful

production of ROS. Notably, in the presence of 650 nm

illumination, ROS production was detected after the cells were

treated with complexes 4 and 7. In contrast, negligible ROS

signals were observed in [Ru(bpy) ]Cl -treated cells, matching

Figure 7. (A) Gel electrophoretic mobility pattern of pBR322 plasmid

DNA incubated with various concentrations of Ru(II) complexes

under blue and/or red light irradiation. Lanes 1−9 (0, 1, 5, 20, 40, 80,

60, 320, and 640 μM) + DNA: (a) 4 + 465 nm; (b) 7 + 465 nm; (c)

[Ru(bpy) ]Cl + 465 nm; (d) 4 + 650 nm; (e) 7 + 650 nm; (f)

3 2

[Ru(bpy) ]Cl + 650 nm. (B) Stereoview of the molecular docking of

well with the extracellular O generation results.

complex 7 with DNA duplex (PDB ID: 4JD8).

Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Figure 8. Flow cytometry analysis for apoptosis of A549 cancer cells in the presence of (a) blue light and (b) red light.

cleavage at the tested concentrations under blue light

irradiation. In contrast, complex 7 exhibited considerable

DNA photocleavage activity, which can completely convert

supercoiled DNA form (form I) to nicked circular form (form

II) at an extremely low concentration of 5 μM (drug to

nucleotide ratio: 0.032). Moreover, the plasmid DNA gradually

disappeared with increasing concentrations of complex 7,

indicating that it can inhibit the intercalation of EtBr in

ascribed to its strong DNA binding ability as well as high O

generation eﬃciency.

Cell Death Study. The potential of complexes 4 and 7 and

[Ru(bpy) ]Cl to induce cell death was determined with blue

465 nm)- and red-light (650 nm) irradiation by using an

Annexin V-FITC/propidium iodide (PI) assay. A549 cancer

cells were treated with the Ru(II) complexes at concentrations

of their IC values. As shown in Figure 8, all Ru(II) complexes

can induce obvious incidences of early- to late-stage apoptosis

in A549 cancer cells in comparison with untreated cells

(

plasmid DNA at high concentrations. In addition, [Ru(bpy) ]-

Cl displayed much weaker DNA photocleavage activity in

(

control) under blue- and red-light irradiation with apoptotic

comparison to complex 7 under the experimental conditions.

For the red-light irradiation, a concentration-dependent DNA

cleavage pattern was also observed for complex 7, while

complex 4 and [Ru(bpy) ]Cl did not show any obvious

rates of ∼50%. This result was in accordance with the results of

the MTT assay. Overall, this study indicates that the two

Ru(II) complexes produced cancer cell death through an

apoptotic pathway under light exposure.

cleavage, which is indicative of the eﬃcient DNA photo-

cleavage ability of complex 7 upon red-light irradiation. When

these results are taken together, the considerable DNA

photocleavage activity of complex 7 may be one of the causes

for its high photocytotoxicity.

CONCLUSION

■

[

In summary, complexes 1−7 were designed as analogues to

Ru(bpy) ]Cl with the aim to red-shift the MLCT

absorption of Ru(II)-polypyridyl complexes into the PDT

The binding of the PS to DNA is a key step for DNA

window (600−850 nm). DFT calculations indicated that the

photocleavage. It has been reported that dppz-containing

Ru(II) complexes (dppz = dipyrido[3,2-a:2′,3′-c]phenazine)

can intercalate between DNA base pairs and serve as DNA

HOMO−LUMO energy gaps of complexes 1−7 are much

smaller than that of [Ru(bpy) ] , which is responsible for the

red shifting of the MLCT absorption band of these

molecular light switches. The dppn ligand in complex 7 is a

derivative of dppz, which may have the potential to intercalate

into DNA due to the excellent planar conjugated structure of

complexes. Cytotoxicity data against A549 and HepG2 cells

revealed that complex 7 showed extraordinarily high

^c^y^t^o^t^o^xⁱ^cⁱ^t^y^uⁿ^d^e^r⁶⁵⁰ⁿ^mⁱ^r^r^a^dⁱ^a^tⁱ^oⁿ^,^r^e^s^u^l^tⁱⁿ^gⁱⁿ^I^C50 values

of 56 and 63 nM with exceptionally large phototoxicity index

4,55

dppn.

Thus, a molecular docking study was carried out on

a DNA duplex structure (PDB ID: 4JD8) to elucidate the

(

PI) values of 763 and 613, respectively. Thus, the resulting

DNA binding mode of complex 7 using the AutoDock 4.2

complex 7 with considerable red-light photocytotoxicity and

high PI values shows a promising potential for therapeutic

applications, which represents a new scaﬀold of Ru(II)-

polypyridyl photosensitizers for PDT in the “therapeutic

window”. This study delivers a rational strategy for the design

of tris-heteroleptic Ru(II) complexes as promising photo-

sensitizers for cancer therapy.

6,57

package.

behavior similar to that of dppz-containing Ru(II) complexes

Figure 7B), and the binding energy was calculated to be

10.78 kcal/mol, indicating that complex 7 can eﬀectively

Obviously, complex 7 showed an intercalation

(

−

bind to the DNA. When these results are taken together, the

considerable DNA photocleavage activity of complex 7 may be

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

Complex 4. Yield: 0.48 g (63.5%), reddish brown powder. Anal.

EXPERIMENTAL SECTION

■

Calcd for C H Cl N Ru: C, 60.08; H, 4.38; N, 12.91. Found: C,

Materials and Measurements. All analytical grade chemicals

and solvents were used without further puriﬁcation. cis-[Ru(bpy)-

(

0.19; H, 4.35; N, 12.94. ESI mass spectrum data: m/z 344.5973

[M/2 − Cl] ). H NMR (600 MHz, DMSO-d ): δ 2.81 (s, 6H),

(

dppn)Cl ] was prepared according to previous literature methods.

.30−6.32 (d, 2H, J = 8.9 Hz), 6.50−6.51 (d, 2H, J = 8.9 Hz), 7.15−

.16 (d, 1H, J = 8.9 Hz), 7.30−7.32 (t, 1H, J = 6.6 Hz), 7.34−7.36

H and C NMR spectra were recorded on a Bruker Avance III-HD

00 MHz spectrometer. Elemental analysis of C, H, and N used a

Vario MICRO CHNOS elemental analyzer (Elementar). UV−vis

absorption and luminescence spectra were measured on a Shimadzu

UV2600 instrument and a FluoroMax-4 ﬂuorometer, respectively.

Mass spectrometry was performed using an Agilent 6224 ESI/TOF

MS instrument. Cell accumulation was conducted on a PerkinElmer

NexION 1000G ICP mass spectrometer.

m, 1H), 7.52−7.53 (d, 1H, J = 5.4 Hz), 7.58−7.60 (t, 1H, J = 5.4

Hz), 7.62−7.65 (q, 2H, J = 6.4 Hz), 7.67−7.70 (t, 1H, J = 7.5 Hz),

.80−7.81 (t, 1H, J = 5.3 Hz), 7.95−7.97 (t, 1H, J = 7.6 Hz), 7.98−

.99 (d, 1H, J = 5.4 Hz), 8.15−8.19 (m, 2H), 8.24−8.27 (t, 2H, J =

7.8 Hz), 8.54−8.55 (d, 1H, J = 5.4 Hz), 8.56−8.60 (d-d, 2H, J = 8.5

Hz, J = 12.4 Hz), 8.79−8.82 (m, 2H), 8.90−8.91 (d, 1H, J = 8.2 Hz),

8.99−9.01 (d, 1H, J = 8.2 Hz), 9.67 (s, 1H). C NMR (150 MHz,

Preparation of 1. Complex 1 was prepared according to previous

DMSO-d ): δ 40.34, 111.68, 122.65, 123.96, 124.33, 124.66, 125.08,

literature. Yield: 0.57 g (71.3%), yellowish brown powder. Anal.

Calcd for C H Cl N Ru: C, 57.66; H, 3.93; N, 12.61. Found: C,

125.41, 125.52, 127.88, 128.29, 128.62, 128.64, 129.60, 129.75,

130.38, 132.18, 138.39, 138.55, 138.89, 139.10, 139.23, 139.30,

149.73, 150.43, 151.39, 151.58, 152.42, 153.43, 156.77, 156.85,

157.05, 157.21, 159.49, 169.01.

(

7.71; H, 3.91; N, 12.64. ESI mass spectrum data: m/z 298.0647

[M/2 − Cl] ). H NMR (600 MHz, DMSO-d ): δ 6.63 (d, J = 7.6

Hz, 2H), 7.07 (t, J = 7.8 Hz, 2H), 7.16 (t, J = 7.4 Hz, 1H), 7.35 (t, J =

.6 Hz, 1H), 7.53 (t, J = 6.2 Hz, 1H), 7.61−7.65 (m, 2H), 7.67−7.73

m, 3H), 7.75 (d, J = 5.4 Hz, 1H), 7.85 (d, J = 5.5 Hz, 1H), 7.90 (m,

H), 8.26−8.28 (m, 4H), 8.44 (d, J = 8.1 Hz, 1H), 8.58 (d, J = 7.7

Complex 5. Yield: 0.28 g (33.2%), reddish brown powder. Anal.

(

Calcd for C44

H30Cl N Ru: C, 62.71; H, 3.59; N, 13.30. Found: C,

2 8

62.73; H, 3.53; N, 13.31. ESI mass spectrum data: m/z 386.0775

([M/2 − Cl] ). H NMR (600 MHz, DMSO-d ): δ H NMR (600

Hz, 1H), 8.64 (d, J = 8.2 Hz, 1H), 8.69 (d, J = 5.3 Hz, 1H), 8.94−

MHz, DMSO): δ 6.75 (d, J = 7.6 Hz, 2H), 7.11 (t, J = 7.8 Hz, 2H),

7.25−7.16 (m, 2H), 7.53−7.48 (m, 1H), 7.77−7.73 (m, 4H), 7.83 (d,

J = 5.2 Hz, 1H), 7.89−7.85 (m, 1H), 7.91 (d, J = 5.6 Hz, 1H), 8.02

.00(m, 2H), 9.42 (s, 1H). C NMR (150 MHz, DMSO-d ): δ

21.60, 124.06, 124.27, 125.15, 125.23, 127.79, 128.29, 128.55,

28.58, 128.70. 129.33, 129.55, 131.25, 138.02, 138.17, 138.60,

38.68, 138.77, 148.98, 151.64, 151.73, 152.05, 152.18, 153.43,

56.54, 156.76, 156.82, 156.93, 157.31, 169.88.

General Procedure for the Synthesis of Complexes 2−7. A

methanol solution (30 mL) of aniline (for the synthesis of complexes

(

dd, J = 8.1, 5.4 Hz, 1H), 8.25−8.15 (m, 3H), 8.29 (td, J = 8.1, 1.2

Hz, 1H), 8.50−8.41 (m, 3H), 8.62 (d, J = 7.8 Hz, 1H), 8.71 (d, J =

.3 Hz, 1H), 9.19 (dd, J = 5.2, 0.9 Hz, 1H), 9.23 (d, J = 8.7 Hz, 2H),

.49 (s, 1H), 9.59 (dd, J = 8.1, 1.0 Hz, 1H), 9.66 (dd, J = 8.1, 1.0 Hz,

H). C NMR (150 MHz, DMSO-d ): δ 121.78, 123.98, 124.33,

27.60, 128.28, 128.45, 128.62, 128.74, 129.10, 129.39, 131.13,

31.22, 133.82, 133.95, 135.08, 138.06, 138.29, 138.37, 138.96,

41.61, 141.64, 148.99, 151.21, 151.32, 152.44, 152.51, 152.80,

54.00, 155.63, 156.55, 157.20, 169.93.

and 5) or N,N-dimethyl-1,4-phenylenediamine (for the synthesis of

complexes 2, 4, 6, and 7) (1.2 mmol) and the corresponding

aldehydes (1.2 mmol) was heated at reﬂux for 12 h to obtain a

yellowish brown solution. The solution was used without puriﬁcation.

Then cis-[Ru(bpy) Cl ] or cis-[Ru(bpy)(dppn)Cl ] (1.0 mmol) was

Complex 6. Yield: 0.35 g (39.5%), reddish brown powder. Anal.

Calcd for C H Cl N Ru: C, 63.41; H, 4.17; N, 13.91. Found: C,

added, and the resulting mixture was stirred under reﬂux for 12 h,

during which time the mixture turned dark brown. The solvent was

then removed by evaporation under reduced pressure. The crude

product was puriﬁed using dichloromethane/methanol (20/1, v/v)

through preparative column chromatography (basic Al O ) to aﬀord

(

3.38; H, 4.19; N, 13.94. ESI mass spectrum data: m/z 407.5984

[M/2 − Cl] ). H NMR (600 MHz, DMSO-d ): δ 2.35 (s, 6H),

.01 (d, J = 9.1 Hz, 2H), 6.43 (d, J = 9.0 Hz, 2H), 7.47 (t, J = 6.4 Hz,

H), 7.67−7.62 (m, 1H), 7.72−7.67 (m, 2H), 7.80−7.76 (m, 1H),

.87 (t, J = 6.2 Hz, 2H), 7.95 (d, J = 5.4 Hz, 1H), 8.14−8.13 (m, 1H),

.16 (dd, J = 8.0, 5.3 Hz, 1H), 8.21−8.18 (m, 2H), 8.26−8.22 (m,

H), 8.32 (dd, J = 11.7, 4.4 Hz, 1H), 8.38−8.35 (m, 2H), 8.59 (d, J =

.8 Hz, 1H), 8.69 (d, J = 5.2 Hz, 1H), 9.04 (d, J = 11.1 Hz, 2H),

the product.

Complex 2. Yield: 0.58 g (68.11%), yellowish brown powder. Anal.

Calcd for C H Cl N Ru: C, 57.55; H, 4.40; N, 13.82. Found: C,

(

7.58; H, 4.39; N, 13.86. ESI mass spectrum data: m/z 319.5857

.10−9.06 (m, 2H), 9.33−9.31 (m, 1H), 9.35 (s, 1H), 9.54 (dd, J =

[M/2 − Cl] ). H NMR (600 MHz, DMSO-d ): δ 2.81 (s, 6H),

.0, 1.1 Hz, 1H). C NMR (150 MHz, DMSO-d ): δ 40.55, 111.59,

.31 (d, J = 9.0 Hz, 2H), 6.59 (d, J = 8.6 Hz, 2H), 7.41 (t, J = 6.6 Hz,

H), 7.52−7.58 (m, 2H), 7.64 (d, J = 5.0 Hz, 1H), 7.73−7.65 (m,

H), 7.73−7.77 (m, 2H), 7.96−8.02 (m, 1H), 8.24−8.15 (m, 3H),

.25 (m, 1H), 8.52−8.63 (m, 3H), 8.74 (t, J = 9.3 Hz, 1H), 9.02 (m,

22.82, 128.29, 128.44, 129.06, 130.09, 130.15, 135.02, 138.09,

38.21, 138.73, 140.84, 150.08, 150.90, 151.11, 156.90, 157.09,

57.98, 166.10.

Complex 7. Yield: 0.22 g (24.0%), reddish brown powder. Anal.

H), 9.29−9.34 (m, 1H). C NMR (150 MHz, DMSO-d ): δ 48.99,

Calcd for C H Cl N Ru: C, 64.17; H, 3.99; N, 13.47. Found: C,

11.66, 122.95, 124.33, 128.52, 137.97, 138.53, 138.67, 150.56,

51.47, 151.70, 151.73, 151.81, 153.31, 156.75, 156.88, 157.02,

57.82, 165.42.

(

4.22; H, 4.02; N, 13.49. ESI mass spectrum data: m/z 432.6187

[M/2 − Cl] ). H NMR (600 MHz, DMSO-d ): δ 2.33 (s, 6H),

.93−5.94 (d, 2H, J = 8.9 Hz), 6.26−6.27 (d, 2H, J = 8.8 Hz), 7.28−

.29 (d, 1H, J = 8.9 Hz), 7.40−7.43 (t, 1H, J = 7.8 Hz), 7.51−7.53 (t,

Complex 3. Yield: 0.45 g (52.33%), reddish brown powder. Anal.

Calcd for C H Cl N Ru: C, 60.34; H, 3.94; N, 11.73. Found: C,

1H, J = 6.6 Hz), 7.68−7.70 (d, 1H, J = 6.5 Hz), 7.73−7.75 (t, 1H, J =

(

0.40; H, 3.92; N, 11.75. ESI mass spectrum data: m/z 323.0726

.5 Hz), 7.78−7.81 (m, 3H), 7.98−7.99 (t, 1H, J = 5.0 Hz), 8.07−

8.10 (d-d, 1H, J = 5.4 Hz, J = 8.1 Hz), 8.19−8.27 (m, 4H), 8.35−8.36

d, 1H, J = 5.0 Hz), 8.46−8.47 (m, 2H), 8.61−8.62 (d, 1H, J = 8.4

[M/2 − Cl] ). H NMR (600 MHz, DMSO-d ): δ 6.59 (d, J = 7.3

Hz, 2H), 7.08 (t, J = 7.2 Hz, 2H), 7.16 (t, J = 7.0 Hz, 1H), 7.24 (d, J =

(

.9 Hz, 2H), 7.39 (t, J = 7.5 Hz, 1H), 7.49 (d, J = 4.8 Hz, 1H), 7.61−

.67 (m, 3H), 7.73 (t, J = 7.2 Hz, 1H), 7.88 (d, J = 12.7 Hz, 2H), 8.00

d, J = 4.8 Hz, 1H), 8.15 (t, J = 7.6 Hz, 1H), 8.22 (d, J = 7.9 Hz, 1H),

Hz), 8.65−8.66 (d, 1H, J = 5.4 Hz), 8.86−8.88 (d, 1H, J = 8.4 Hz),

.94−8.95 (d, 1H, J = 8.2 Hz), 9.02−9.04 (d, 1H, J = 8.2 Hz), 9.21 (s,

H), 9.24 (s, 1H), 9.36−9.37 (d, 1H, J = 8.0 Hz), 9.64−9.65 (d, 1H, J

.24−8.30 (m, 2H), 8.48 (d, J = 7.8 Hz, 1H), 8.65 (d, J = 7.5 Hz,

= 8.0 Hz), 9.70 (s, 1H). C NMR (150 MHz, DMSO-d ): δ 63.28,

H), 8.77 (d, J = 7.8 Hz, 1H), 8.84−8.93 (m, 2H), 8.99 (d, J = 7.9

111.46, 122.37, 124.18, 125.10, 125.40, 125.58, 128.44, 128.53,

128.59, 128.74, 129.15, 129.89, 130.05, 130.44, 132.33, 133.73,

134.15, 135.15, 135.17, 138.24, 138.31, 138.33, 138.73, 139.18,

139.56, 140.83, 141.01, 149.87, 149.96, 151.21, 151.32, 152.55,

152.81, 154.11, 156.00, 156.81, 157.05, 159.68, 169.52.

Hz, 1H), 9.83 (s, 1H). C NMR (150 MHz, DMSO-d ): δ 121.37,

24.15, 124.71, 125.05, 125.41, 125.05, 125.41, 125.97, 127.93,

28.34, 128.41, 128.71, 128.80, 129.26, 129.99, 130.04, 130.42,

32.39, 138.42, 138.66, 139.14, 139.21, 139.55, 149.21, 149.66,

51.29, 151.81, 152.33, 153.78, 156.68, 156.84, 157.08, 158.99,

72.53.

DFT Calculations. All calculations were performed using the

Gaussian 09 suite of programs. Full geometry optimizations were

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

The Supporting Information is available free of charge at

Article

−2

carried out for complexes 1−7 and [Ru(bpy) ]Cl by using the

red light (650 ± 10 nm, 72.0 J cm ). After 24 h of incubation, the

mixtures (5 μL) with loading buﬀer (1 μL) were submitted to

electrophoresis in agarose gel in TA buﬀer at 100 V for 90 min.

Agarose gels were then dyed with ethidium bromide (0.5 mg/L) for

20 min. Bands were imaged by using a Molecular Imager (Bio-Rad,

USA) under UV light.

B3LYP density functional with the LanL2DZ basis set and an eﬀective

core functional used for the ruthenium atom, while the 6-31G(d,p)

59,60

basis set was used for the other atoms.

The time-dependent

density functional theory (TD-DFT) calculations were performed at

the same level to predict the singlet electronic transitions and the

UV−visible spectra.

Molecular Docking. A molecular docking simulation was carried

Generation. The ¹O₂generation of complexes 1−7 was

out using AutoDock 4.2. The crystal structure of the DNA duplex

evaluated through monitoring the absorption spectral change at 378

was obtained from the Protein Data Bank (PDB ID: 4JD8). The

docking procedure was conducted using a Lamarckian genetic

algorithm for 200 docking runs. Visualization results were performed

by PyMOL.

nm of ABDA, and [Ru(bpy) ]Cl (Φ = 0.41 in water) was used as a

standard in water. The experiment was conducted for complexes 1−7

(10 μM) in DMSO/water (1/99, v/v) containing ABDA (50 μM).

The absorption spectra were recorded every 30 s under 465 ± 10 nm

LED irradiation (1.26 J cm ) or every 4 min with red-light

Apoptosis Analysis by Flow Cytometry. Complexes 4, 7, and

−

[Ru(bpy) ]Cl with concentrations of IC50 values were added to the

3 2

irradiation (650 ± 10 nm, xenon lamp with a band-pass ﬁlter, 19.2 J

A549 cells. After incubation for 4 h, cells were irradiated with 465 ±

−

−2

cm ). The Φ values were calculated with the equation Φ (PS) =

10 nm LED irradiation (25.2 J cm ) or red light (650 ± 10 nm,

−

Φ (Std)S × F_S_t_d/(S × F ), where PS designates the complexes

144.0 J cm ). Then the cells were incubated in the dark for a further

24 h and collected by centrifugation (5 min, 25 °C, 2000 rpm).

Afterward, the A549 cells were dyed by Annexin V-FITC/PI and

analyzed with a ﬂow cytometer.

std

−7 and Std designates [Ru(bpy) ]Cl ; S is the decomposition rate of

ABDA at 378 nm, and F is the correction factor of absorption, which

−

is given by F = 1 − 10

complexes 1−7 and [Ru(bpy) ]Cl at 465 nm).

(OD denotes the optical density of

Cytotoxicity Assay of PDT. The photocytotoxicity of complexes

−7 and [Ru(bpy) ]Cl against A549 and HepG2 cells was

ASSOCIATED CONTENT

sı Supporting Information

https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c01860.

■

determined by means of an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide) assay. Cells (10 per well) were seeded

in 96-well plates and allowed to adhere for 24 h. After that, complexes

−7 and [Ru(bpy) ]Cl were dissolved with DMF and diluted with

H and C NMR and ESI mass spectra of complexes 1−

the medium to the required concentrations (the ﬁnal concentration of

DMF was less than 0.4%). After being incubated in the dark for 4 h,

cells were irradiated with 465 ± 10 nm nm LED irradiation (25.2 J

cm ) or red light (650 ± 10 nm, 144.0 J cm ), and then the cells

were incubated in the dark for a further 48 h. After that, the cells were

stained with MTT (5 mg/mL) for another 5 h. The inhibition of cell

growth was detected using an enzyme-labeling instrument. The IC₅₀

values were calculated by SPSS software.

[

, frontier molecular orbitals of complexes 1−7 and

Ru(bpy) ]Cl , production of O by complexes 1−7

−

−2

and [Ru(bpy) ]Cl upon 650 nm irradiation and dose-

3 2

dependent cell viability curves and synthetic route of 1−

7, confocal ﬂuorescence images of calcein AM and

propidium iodide (PI) costained A549 cells, cell

accumulation of complexes 4, 7, and [Ru(bpy) ]Cl on

A549 cells, and Cartesian coordinates of all optimized

structures of complexes 1−7 and [Ru(bpy)₃] (PDF)

Cellular Accumulation. A549 cells with good viability were

transferred into 6-well plates and cultured overnight at 37 °C. Then,

complexes 4, 7, and [Ru(bpy) ]Cl were added with a concentration

of 20 μM and incubated with the cells for 12 h. Then the supernatants

were removed, and the cells were washed three times with ice-cold

AUTHOR INFORMATION

Corresponding Authors

■

PBS. The cells were then digested with HNO (65%), and the Ru

contents were measured by ICP-MS.

Jian Zhao − Jiangsu Province Hi-Tech Key Laboratory for

Biomedical Research and Pharmaceutical Research Center,

School of Chemistry and Chemical Engineering, Southeast

University, Nanjing 211189, People’s Republic of China; Key

Laboratory for Organic Electronics and Information Displays,

Institute of Advanced Materials (IAM), Jiangsu National

Synergetic Innovation Center for Advanced Materials (SICAM),

Nanjing University of Posts and Telecommunications (NUPT),

Nanjing 210023, People’s Republic of China; orcid.org/

Calcein AM and Propidium Iodide (PI) Costaining. For the

calcein AM and propidium iodide (PI) costaining assay, A549 cells

(

10 per well) were seeded and cultured in confocal dishes overnight

at 37 °C. Then complexes 4, 7, and [Ru(bpy) ]Cl were added to the

cells with a ﬁnal concentration of 1 μM. After 4 h of incubation, the

−2

cells were exposed to LED light (465 ± 10 nm, 25.2 J cm ) or red

−

light (650 ± 10 nm, 144.0 J cm ). Thereafter, the cells were stained

with Calcein AM/PI Double Stain Kit according to the instruction

manual. Fluorescence images of the stained cells were then taken

using a confocal microscope.

000-0002-9365-7727 ; Email: zhaojianzhaokuan@163.com

Intracellular ROS Production. The ROS generation in A549

Shaohua Gou − Jiangsu Province Hi-Tech Key Laboratory for

Biomedical Research and Pharmaceutical Research Center,

School of Chemistry and Chemical Engineering, Southeast

University, Nanjing 211189, People ’s Republic of China;

orcid.org/0000-0003-0284-5480; Email: sgou@seu.edu.cn

Qiang Zhao − Key Laboratory for Organic Electronics and

Information Displays, Institute of Advanced Materials (IAM),

Jiangsu National Synergetic Innovation Center for Advanced

Materials (SICAM), Nanjing University of Posts and

Telecommunications (NUPT), Nanjing 210023, People ’s

Republic of China; orcid.org/0000-0002-3788-4757;

Email: iamqzhao@njupt.edu.cn

cells was measured by DCFH-DA staining. A549 cells were seeded in

a 6-well plate at a density of 2 × 10 cells/well and cultured for 12 h at

37 °C. Then, the tested complexes were added with a ﬁnal

concentration of 30 μM. After 4 h of incubation, DCFH-DA was

added and the cells were incubated for another 30 min. Thereafter,

the cells were washed with fresh medium three times followed by

−

irradiation with 465 ± 10 nm LED light (25.2 J cm ) or red light

−

(

650 ± 10 nm, 144.0 J cm ). The photos were captured using a

confocal microscope.

Gel Electrophoresis Study. DNA photocleavage activities of

complexes 4, 7, and [Ru(bpy) ]Cl were evaluated by agarose gel

electrophoresis. The tested complexes were ﬁrst dissolved in DMF

10 mM) and then diluted to the desired concentrations with Tris-

H PO (50 mM, pH 7.2) buﬀer. The tested complexes (5 μL; lanes

(

Authors

−9 0, 1, 5, 20, 40, 80, 160, 320, and 640 μM) and the ﬁnal

concentration of pBR322 plasmid DNA (50 ng/μL, 5 μL) were mixed

together and irradiated with blue light (465 ± 10 nm, 12.5 J cm ) or

Shuang Li − Jiangsu Province Hi-Tech Key Laboratory for

Biomedical Research and Pharmaceutical Research Center,

−

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

(12) Jakubaszek, M.; Goud, B.; Ferrari, S.; Gasser, G. Mechanisms of

Article

School of Chemistry and Chemical Engineering, Southeast

University, Nanjing 211189, People’s Republic of China

Xinyi Wang − Jiangsu Province Hi-Tech Key Laboratory for

Biomedical Research and Pharmaceutical Research Center,

School of Chemistry and Chemical Engineering, Southeast

University, Nanjing 211189, People’s Republic of China

Gang Xu − Jiangsu Province Hi-Tech Key Laboratory for

Biomedical Research and Pharmaceutical Research Center,

School of Chemistry and Chemical Engineering, Southeast

University, Nanjing 211189, People’s Republic of China

action of Ru(II) polypyridyl complexes in living cells upon light

irradiation. Chem. Commun. 2018, 54, 13040 −13059.

(13) Heinemann, F.; Karges, J.; Gasser, G. Critical overview of the

use of Ru(II) polypyridyl complexes as photosensitizers in one-

photon and two-photon photodynamic therapy. Acc. Chem. Res. 2017,

(

0, 2727−2736.

14) Huang, H.; Yu, B.; Zhang, P.; Huang, J.; Chen, Y.; Gasser, G.;

Ji, L.; Chao, H. Highly charged ruthenium(II) polypyridyl complexes

as lysosome-localized photosensitizers for two-photon photodynamic

therapy. Angew. Chem., Int. Ed. 2015, 54, 14049−14052.

(15) Zeng, L.; Kuang, S.; Li, G.; Jin, C.; Ji, L.; Chao, H. A GSH-

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.inorgchem.0c01860

activatable ruthenium(II)-azo photosensitizer for two-photon photo-

dynamic therapy. Chem. Commun. 2017, 53, 1977−1980.

(16) Tian, N.; Sun, W.; Guo, X.; Lu, J.; Li, C.; Hou, Y.; Wang, X.;

Notes

Zhou, Q. Mitochondria targeted and NADH triggered photodynamic

activity of chloromethyl modified Ru(II) complexes under hypoxic

conditions. Chem. Commun. 2019, 55, 2676−2679.

The authors declare no competing ﬁnancial interest.

(17) Zheng, Y.; Zhang, D. Y.; Zhang, H.; Cao, J. J.; Tan, C. P.; Ji, L.

ACKNOWLEDGMENTS

■

N.; Mao, Z. W. Photodamaging of mitochondrial DNA to overcome

We are grateful to the National Natural Science Foundation of

China (Grants 21601034 and 21571033) and Jiangsu Province

Natural Science Foundation (Grant BK20160664) for ﬁnancial

aid for this work. The Fundamental Research Funds for the

Central Universities (Projects 2242019K40142) are also

appreciated.

cisplatin resistance by a Ru -Pt bimetallic complex. Chem. - Eur. J.

018, 24, 18971−18980.

(18) He, L.; Liao, S. Y.; Tan, C. P.; Ye, R. R.; Xu, Y. W.; Zhao, M.; Ji,

L. N.; Mao, Z. W. Ruthenium-arene-β -carboline complexes as potent

inhibitors of cyclin-dependent kinase 1: synthesis, characterization

and anticancer mechanism studies. Chem. - Eur. J. 2013, 19, 12152−

12160.

19) Ye, R. R.; Ke, Z. F.; Tan, C. P.; He, L.; Ji, L. N.; Mao, Z. W.

REFERENCES

■

Histone-deacetylase-targeted fluorescent ruthenium(II) polypyridyl

complexes as potent anticancer agents. Chem. - Eur. J. 2013, 19,

10160−10169.

(

1) Zeng, L.; Gupta, P.; Chen, Y.; Wang, E.; Ji, L.; Chao, H.; Chen,

Z.-S. The development of anticancer ruthenium(II) complexes: from

single molecule compounds to nanomaterials. Chem. Soc. Rev. 2017,

(20) Zamora, A.; Denning, C. A.; Heidary, D. K.; Wachter, E.;

Nease, L. A.; Ruiz, J.; Glazer, E. C. Ruthenium-containing P450

inhibitors for dual enzyme inhibition and DNA damage. Dalton.

Trans. 2017, 46, 2165−2173.

2) Liu, J.; Zhang, C.; Rees, T. W.; Ke, L.; Ji, L.; Chao, H.

6, 5771−5804.

Harnessing ruthenium(II) as photodynamic agents: Encouraging

advances in cancer therapy. Coord. Chem. Rev. 2018 , 363 , 17 −28.

3) Notaro, A.; Gasser, G. Monomeric and dimeric coordinatively

saturated and substitutionally inert Ru(II) polypyridyl complexes as

anticancer drug candidates. Chem. Soc. Rev. 2017, 46, 7317−7337.

4) Poynton, F. E.; Bright, S. A.; Blasco, S.; Williams, D. C.; Kelly, J.

(

21) Monro, S.; Colon, K. L.; Yin, H.; Roque, J., III; Konda, P.;

Gujar, S.; Thummel, R. P.; Lilge, L.; Cameron, C. G.; McFarland, S.

A. Transition metal complexes and photodynamic therapy from a

tumor-centered approach: Challenges, opportunities, and highlights

from the development of TLD1433. Chem. Rev. 2019, 119, 797−828.

(

M.; Gunnlaugsson, T. The development of ruthenium(II) polypyridyl

complexes and conjugates for in vitro cellular and in vivo applications.

Chem. Soc. Rev. 2017, 46, 7706−7756.

(

22) Wachter, E.; Heidary, D. K.; Howerton, B. S.; Parkin, S.;

Glazer, E. C. Light-activated ruthenium complexes photobind DNA

and are cytotoxic in the photodynamic therapy window. Chem.

Commun. 2012, 48, 9649−9651.

5) Liu, J.; Lai, H.; Xiong, Z.; Chen, B.; Chen, T. Functionalization

and cancer-targeting design of ruthenium complexes for precise

cancer therapy. Chem. Commun. 2019, 55, 9904−9914.

6) Farrer, N. J.; Salassa, L.; Sadler, P. J. Photoactivated

chemotherapy (PACT): the potential of excited-state d-block metals

in medicine. Dalton. Trans. 2009, 0, 10690−10701.

7) Trondl, R.; Heffeter, P.; Kowol, C. R.; Jakupec, M. A.; Berger,

(

23) Jiang, J.; Qian, Y.; Xu, Z.; Lv, Z.; Tao, P.; Xie, M.; Liu, S.;

Huang, W.; Zhao, Q. Enhancing singlet oxygen generation in

semiconducting polymer nanoparticles through fluorescence reso-

nance energy transfer for tumor treatment. Chem. Sci. 2019, 10,

24) Whittemore, T. J.; White, T. A.; Turro, C. New ligand design

085−5094.

(

W.; Keppler, B. K. NKP-1339, the first ruthenium-based anticancer

provides delocalization and promotes strong absorption throughout

the visible region in a Ru(II) complex. J. Am. Chem. Soc. 2018, 140,

drug on the edge to clinical application. Chem. Sci. 2014, 5, 2925−

(

932.

(

29−234.

8) Conti, L.; Bencini, A.; Ferrante, C.; Gellini, C.; Paoli, P.; Parri,

25) Sun, W.; Thiramanas, R.; Slep, L. D.; Zeng, X.; Mailander, V.;

M.; Pietraperzia, G.; Valtancoli, B.; Giorgi, C. Highly charged

ruthenium(II) polypyridyl complexes as effective photosensitizer in

photodynamic therapy. Chem. - Eur. J. 2019, 25, 10606 −10615.

Wu, S. Photoactivation of anticancer Ru complexes in deep tissue:

How deep can we go? Chem. - Eur. J. 2017, 23, 10832−10837.

(

26) Foxon, S. P.; Alamiry, M. A.; Walker, M. G.; Meijer, A. J.;

9) Shum, J.; Leung, P. K. K.; Lo, K. K. W. Luminescent

Sazanovich, I. V.; Weinstein, J. A.; Thomas, J. A. Photophysical

properties and singlet oxygen production by ruthenium (II)

complexes of benzo[i]dipyrido [3,2-a:2 ′,3-c] phenazine: spectro-

scopic and TD-DFT study. J. Phys. Chem. A 2009, 113, 12754−12762.

ruthenium(II) polypyridinecomplexes for a wide variety of bio-

molecular and cellular applications. Inorg. Chem. 2019, 58, 2231−

10) Li, A.; Turro, C.; Kodanko, J. J. Ru(II) polypyridyl complexes

247.

(

27) Zhao, J.; Liu, N.; Sun, S.; Gou, S.; Wang, X.; Wang, Z.; Li, X.;

derived from tetradentate ancillary ligands for effective photocaging.

Acc. Chem. Res. 2018, 51, 1415−1421.

11) Lv, Z.; Wei, H.; Li, Q.; Su, X.; Liu, S.; Zhang, K. Y.; Lv, W.;

Zhang, W. Light-activated ruthenium (II)-bicalutamide prodrugs for

prostate cancer. J. Inorg. Biochem. 2019, 196, 110684.

(

Zhao, Q.; Li, X.; Huang, W. Achieving efficient photodynamic therapy

under both normoxia and hypoxia using cyclometalated Ru(II)

photosensitizer through type I photochemical process. Chem. Sci.

(28) Sgambellone, M. A.; David, A.; Garner, R. N.; Dunbar, K. R.;

Turro, C. Cellular toxicity induced by the photorelease of a caged

bioactive molecule: design of a potential dual-action Ru (II) complex.

J. Am. Chem. Soc. 2013, 135, 11274−11282.

2018, 9, 502−512.

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

29) Zhang, Y.; Zhou, Q.; Tian, N.; Li, C.; Wang, X. Ru(II)-

Predictive strength of photophysical measurements for in vitro

photobiological activity in a Series of Ru(II) polypyridyl complexes

derived from π -extended ligands. Inorg. Chem. 2019, 58, 3156−3166.

(45) Sainuddin, T.; McCain, J.; Pinto, M.; Yin, H.; Gibson, J.; Hetu,

M.; McFarland, S. A. Organometallic Ru(II) photosensitizers derived

from π -expansive cyclometalating ligands: surprising theranostic PDT

effects. Inorg. Chem. 2016, 55, 83−95.

Complex-based DNA photocleaver having intense absorption in the

phototherapeutic window. Inorg. Chem. 2017, 56, 1865−1873.

(

Felgentrager, A.; Maisch, T.; Spiccia, L.; Gasser, G. Synthesis,

characterization, and biological evaluation of new Ru(II) polypyridyl

photosensitizers for photodynamic therapy. J. Med. Chem. 2014, 57,

30) Frei, A.; Rubbiani, R.; Tubafard, S.; Blacque, O.; Anstaett, P.;

31) Karges, J.; Blacque, O.; Goldner, P.; Chao, H.; Gasser, G.

280−7292.

Towards long wavelength absorbing photodynamic therapy photo-

(46) Yin, H.; Stephenson, M.; Gibson, J.; Sampson, E.; Shi, G.;

Sainuddin, T.; Monro, S.; McFarland, S. A. In vitro multiwavelength

PDT with IL states: Teaching old molecules new tricks. Inorg. Chem.

sensitizers via the extension of a [Ru(bipy)₃ ] core. Eur. J. Inorg.

Chem. 2019, 2019, 3704−3712.

32) Bahreman, A.; Cuello-Garibo, J. A.; Bonnet, S. Yellow-light

2014, 53, 4548−4559.

(47) Rohrabaugh, T. N.; Collins, K. A.; Xue, C.; White, J. K.;

Kodanko, J. J.; Turro, C. New Ru(II) complex for dual photo-

sensitization of a ligand photosubstitution reaction in a ruthenium

polypyridyl complex covalently bound to a rhodamine dye. Dalton.

Trans. 2014, 43, 4494−4505.

chemotherapy: release of cathepsin K inhibitor and O production.

Dalton. Trans. 2018, 47, 11851−11858.

(48) Sun, Y.; Joyce, L. E.; Dickson, N. M.; Turro, C. Efficient DNA

(

33) Zhou, Q. X.; Lei, W. H.; Li, C.; Hou, Y. J.; Wang, X. S.; Zhang,

photocleavage by [Ru(bpy) (dppn)] with visible light. Chem.

B. W. DNA photocleavage in anaerobic conditions by a Ru(II)

polypyridyl complex with long wavelength MLCT absorption. New J.

Chem. 2010, 34, 137−140.

Commun. 2010, 46, 2426−2428.

(49) Zhao, J.; Zhang, X.; Fang, L.; Gao, C.; Xu, C.; Gou, S.

Iridium(III) Complex-Derived Polymeric Micelles with Low Dark

Toxicity and Strong NIR Excitation for Phototherapy and Chemo-

therapy. Small 2020, 16, 2000363.

(34) Zhou, Q. X.; Lei, W. H.; Chen, J. R.; Li, C.; Hou, Y. J.; Wang,

X. S.; Zhang, B. W. A new heteroleptic ruthenium(II) polypyridyl

complex with long-wavelength absorption and high singlet-oxygen

quantum yield. Chem. - Eur. J. 2010, 16, 3157−3165.

(50) Albani, B. A.; Pena, B.; Leed, N. A.; De Paula, N. A.; Pavani, C.;

Baptista, M. S.; Dunbar, K. R.; Turro, C. Marked improvement in

photoinduced cell death by a new tris-heteroleptic complex with dual

action: singlet oxygen sensitization and ligand dissociation. J. Am.

Chem. Soc. 2014, 136, 17095−17101.

35) Mahmoud, W. H.; Mohamed, G. G.; El-Sayed, O. Y.

Coordination compounds of some transition metal ions with new

Schiff base ligand derived from dibenzoyl methane. Structural

characterization, thermal behavior, molecular structure, antimicrobial,

anticancer activity and molecular docking studies. Appl. Organomet.

Chem. 2018, 32, No. e4051.

(51) Feng, Z.; Tao, P.; Zou, L.; Gao, P.; Liu, Y.; Liu, X.; Wang, H.;

Liu, S.; Dong, Q.; Li, J.; Xu, B.; Huang, W.; Wong, W. Y.; Zhao, Q.

Hyperbranched phosphorescent conjugated polymer dots with

iridium(III) complex as the core for hypoxia imaging and photo-

dynamic therapy. ACS Appl. Mater. Interfaces 2017, 9, 28319−28330.

(52) Nomula, R.; Wu, X.; Zhao, J.; Munirathnam, N. R.

Photodynamic effect of light-harvesting, long-lived triplet excited

state Ruthenium (II)-polyimine-coumarin complexes: DNA binding,

photocleavage and anticancer studies. Mater. Sci. Eng., C 2017, 79,

710−719.

(53) Hartshorn, R. M.; Barton, J. K. Novel dipyridophenazine

complexes of ruthenium(II): exploring luminescent reporters of DNA.

J. Am. Chem. Soc. 1992, 114, 5919−5925.

(54) Sun, Y.; Joyce, L. E.; Dickson, N. M.; Turro, C. DNA

photocleavage by an osmium (II) complex in the PDT window. Chem.

Commun. 2010, 46, 6759−6761.

(55) Chen, X.; Gao, F.; Yang, W. Y.; Zhou, Z. X.; Lin, J. Q.; Ji, L. N.

Structure activity relationship of polypyridyl ruthenium(II) complexes

as DNA intercalators, DNA photocleavage reagents, and DNA

topoisomerase and RNA polymerase inhibitors. Chem. Biodiversity

2013, 10, 367−384.

(56) Hall, J. P.; Cook, D.; Morte, S. R.; McIntyre, P.; Buchner, K.;

Beer, H.; Cardin, D. J.; Brazier, J. A.; Winter, G.; Kelly, J. M.; Cardin,

C. J. X-ray crystal structure of rac-[Ru(phen)₂ dppz] with

(36) El-Sonbati, A. Z.; Mahmoud, W. H.; Mohamed, G. G.; Diab, M.

A.; Morgan, S. M.; Abbas, S. Y. Synthesis, characterization of Schiff

base metal complexesand their biological investigation. Appl. Organo-

met. Chem. 2019, 32, No. e5048.

(37) Zhao, J.; Li, S.; Wang, X.; Xu, G.; Gou, S. Dinuclear

organoruthenium complexes exhibiting antiproliferative activity

through DNA damage and a reactive-oxygen-species-mediated

endoplasmic reticulum stress pathway. Inorg. Chem. 2019, 58,

(

208−2217.

38) Hotze, A. C.; Faiz, J. A.; Mourtzis, N.; Pascu, G. I.; Webber, P.

R.; Clarkson, G. J.; Yannakopoulou, K.; Pikramenou, Z.; Hannon, M.

J. Far-red luminescent ruthenium pyridylimine complexes; building

blocks for multinuclear arrays. Dalton. Trans. 2006, 24, 3025−3034.

(

39) Bozic-Weber, B.; Constable, E. C.; Housecroft, C. E.;

Neuburger, M.; Price, J. R. Sticky complexes: Carboxylic acid-

functionalized N-phenylpyridin-2-ylmethanimine ligands as anchoring

domains for copper and ruthenium dye-sensitized solar cells. Dalton.

Trans. 2010, 39, 3585−3594.

(40) Juris, A.; Balzani, V.; Barigelletti, F.; Campagna, S.; Belser, P. L.;

von Zelewsky, A. V. Ru (II) polypyridine complexes: photophysics,

photochemistry, eletrochemistry, and chemiluminescence. Coord.

Chem. Rev. 1988, 84, 85−277 1998.

d(ATGCAT) shows enantiomer orientations and water ordering. J.

(41) Higgins, S. L.; Brewer, K. J. Designing red-light-activated

Am. Chem. Soc. 2013, 135, 12652−12659.

multifunctional agents for the photodynamic therapy. Angew. Chem.,

Int. Ed. 2012, 51, 11420−1422.

(57) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew,

R. K.; Goodsell, D. S.; Olson, A. J. AutoDock4 and AutoDockTools4:

Automated docking with selective receptor flexibility. J. Comput.

Chem. 2009, 30, 2785−2791.

(42) Karges, J.; Heinemann, F.; Jakubaszek, M.; Maschietto, F.;

Subecz, C.; Dotou, M.; Vinck, R.; Blacque, O.; Tharaud, M.; Goud,

B.; Vinuelas Zah ı nos, E.; Spingler, B.; Ciofini, I.; Gasser, G. Rationally

Designed Long-Wavelength Absorbing Ru(II) Polypyridyl Complexes

as Photosensitizers for Photodynamic Therapy. J. Am. Chem. Soc.

(58) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;

Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci,

B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H.

P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;

Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.;

Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.,

Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M. J.;

Heyd, J.; Brothers, E. N.; Kudin, K. N.; Staroverov, V. N.; Kobayashi,

R.; Normand, J.; Raghavachari, K.; Rendell, A. P.; Burant, J. C.;

Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, N. J.; Klene,

M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.;

Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.;

(

020, 142, 6578−6587.

43) Lincoln, R.; Kohler, L.; Monro, S.; Yin, H.; Stephenson, M.;

Zong, R.; Chouai, A.; Dorsey, C.; Hennigar, R.; Thummel, R. P.;

McFarland, S. A. Exploitation of long-lived IL excited states for

metal-organic photodynamic therapy: verification in a metastatic

melanoma model. J. Am. Chem. Soc. 2013, 135, 17161−17175.

(

44) Reichardt, C.; Monro, S.; Sobotta, F. H.; Colon, K. L.;

Sainuddin, T.; Stephenson, M.; Sampson, E.; John Roque, J., III; Yin,

H.; Brendel, J. C.; Cameron, C. G.; McFarland, S.; Dietzek, B.

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

(59) Wadt, W. R.; Hay, P. J. Ab initio effective core potentials for

Article

Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.;

Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.;

Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.;

Cioslowski, J.; Fox, D. J. Gaussian 09, E01; Gaussian, Inc.:

Wallingford, CT, 2009.

molecular calculations. Potentials for the transition metal atoms Sc to

Hg. J. Chem. Phys. 1985 , 82 , 284 −298.

(60) Becke, A. D. Density-functional exchange-energy approxima-

tion with correct asymptotic behavior. Phys. Rev. A: At., Mol., Opt.

Phys. 1988, 38, 3098−3100.