Arene ruthenium(II) complexes with chalcone, aminoantipyrine and aminopyrimidine based ligands: Synthesis, structure and preliminary evaluation of anti-leukemia activity

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C. Raja, S. Srinivasan, S. Kar Mahapatra, D. Moon, S. P. Anthony and V. Madhu, RSC Adv., 2016, DOI:

10.1039/C6RA18504E.

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Arene Ruthenium (II) Complexes with Chalcone, Aminoantipyrine

and Aminopyrimidine Based Ligands: Synthesis, Structure and

Preliminary Evaluation of Anti-leukemia Activity

Received 00th January 20xx,

Accepted 00th January 20xx

Jayaraman Pitchaimani,^aMamilla R. Charan Raja,^bSrinivasan Sujatha,^bSantanu Kar Mahapatra,^b*

Dohyun Moon,^c*Savarimuthu Philip Anthony^d*and Vedichi Madhu^a*

DOI: 10.1039/x0xx00000x

www.rsc.org/

A series of arene ruthenium(II) (Ru) complexes with N-monodentate (AAP) and N,O- and N,N-bidentate chelating ligands

(AAPS, ADABS, AAPPA and P2P) have been synthesized and evaluated for preliminary antileukemia activity. Single crystal

X-ray studies of AAP-Ru, ADABS-Ru and P2P-Ru indicates that Ru(II) adopted typical “three-leg piano-stool” geometry with

pseudo-tetrahedral (AAP-Ru) and quasi-octahedral arrangement (ADABS-Ru and P2P-Ru). Supramolecular interactions (C–

H···Cl, N–H···Cl and π-π) in the crystal lattice of AAP-Ru and ADABS-Ru resulted in dimeric structure whereas P2P-Ru

showed supramolecular 1D chain. Importantly, preliminary in-vitro anticancer activities for Ru-arene complexes have been

evaluated against K562 (Human chronic myeloid leukemia cell line) by means of MTT assay that showed strong anti-

leukemic activity for AAPS-Ru (IC₅₀= 12.46 ± 0.47 μM) and P2P-Ru (IC₅₀= 11.8 ± 0.49 μM). The mechanistic studies

indicated reactive oxygen species (ROS) mediated selective leukemic cells death without affecting the normal cells by

AAPS-Ru. The initiation of apoptosis by AAPS-Ru treatment, particularly TNF-α induced cell death, confirmed by pre-

treatment with selective TNF-α inhibitor.

activity, multiple stable oxidation states under physiological

conditions, light-activated reactions with DNA, high delivery to

cancer cells, low side effects, high cytotoxicity toward cancer

and lower toxicity toward healthy cells.^5,2fThus, different types

of ruthenium (II) arene complexes have been investigated as a

potential anti-cancer drug candidate.⁶Sadler et al prepared

cationic arene ruthenium complex and reported very good

anticancer activity both in vitro and in vivo.⁷

Introduction

In recent years, there has been a growing interest in the

development of transition metal based drugs for application of

chemotherapeutic agents. In this perspective, platinum-based

anti-cancer drugs have primarily been used in the clinic as

chemotherapeutic agents.¹However, extensive side effects

and cancer cell resistance, renal toxicity and neurotoxicity of

platinum based drugs have often restricted their application

potential.²These limitation of cisplatin drugs have lead to

intensive research on non-platinum based metal complex for

anticancer activity. In particular, organometallic complexes of

iron, ruthenium and osmium showed promising activity.³Of

that, arene ruthenium(II) complexes are highly promising

materials and considered as potential alternatives to platinum

chemotherapeutic agents.⁴In particular, ruthenium complexes

exhibited admiring properties like acceptable antitumor

Scheme 1 Structure of ruthenium (II) and ruthenium (III) complexes exhibited strong

anticancer activity.

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Ru(II)-arene chloroquine showed noticeable anticancer were purchased from GIBCO, Invitrogen. RPMI 1640, MTT, was

activity against various tumor cell lines.⁸Curcumin based purchased from Himedia, India. TNF- α and IL-10 ELISA sets

arene-Ru(II) complex displayed high in vitro activity against were purchased from R&D systems. All other chemicals were

breast MCF7 and ovarian A2780 cell lines.⁹The incorporation from Merck-Millipore, SRL and Himedia were of the highest

of carborane and ferrocene with Ru(II) arene complexes purity grade available. All other reagents and solvents were

significantly increases apoptosis in human lung cancer HCC827 obtained from commercial suppliers (Aldrich, AlfaAesar, and

cell line.¹⁰The intensive research effort of ruthenium TCI Chemicals) and used as received; solvents were purified

complexes resulted in developing two strong anticancer active from appropriate drying agents when necessary. All the

ruthenium(III) compounds, namely KP1019 and NAMI-A. It is reactions were carried out under nitrogen atmosphere. The

noteworthy that, arene-Ru(II) complexes RAPTA-C (developed compounds, 4-(2-hydroxybenzylideneamino)-2,3-dimethyl-1-

by Dyson and coworkers) and RM175 (developed by Sadler and phe nyl-1,2-dihydropyrazol-5-one (AAPS)¹⁹, 1-(4-aminophenyl)-

coworkers) displayed strong anticancer activity in vivo also 3-(4-(dimethylamino)phenyl) prop -2-en-1-one (ADAB)²⁰and 4-

(Scheme 1).^11-15Further, the prototype compound RAPTA-C (pyridine-2-yl) pyrimidin-2-amine(P2P)²¹were synthesized as

reduces the growth of primary tumors in preclinical models for previously described in the literature, are also presented in the

ovarian and colorectal carcinomas as well as it has shown Supporting Information. The complex (ɳ6-p-cymene)(2,2-

moderate effects on solid tumour metastases.¹¹The ligand bipyridine)Chloro ruthenium(II) chloride (BPY-Ru) was

scaffolds plays important role on the arene ruthenium synthesized based on reported in literature.²²Absorption and

complexes in controlling the anticancer activities such as fluorescence spectra were recorded using Perking Elmer

improving water solubility and cell permeability. For example, Lambda 1050 and Jasco fluorescence spectrometer-FP-8200

incorporation of bioactive ligand scaffolds, such as flavonoids, instruments. FT-IR spectra were measured using a Shimadzu

isoflavonoids,

naphthoquinones,

staurosporine

or IRAffinity-1S spectrophotometer with KBr pellets.

thiosemicarbazone to arene Ru(II) complexes enhanced

anticancer activities.¹⁶Pyrimidine analogues have also been

Synthesis and characterization data

evaluated as viable anti-tumour drugs.¹⁷The aminoantipyrine Synthesis of Ru(ɳ⁶-p-cymene)Cl₂(4-amino antipyrine) (AAP-

derivatives exhibited wide range of biological activities such as, Ru). [(η⁶-p-cymene)RuCl₂]₂(50 mg, 0.08 mmol) was added to a

antimicrobial, fungicidal anti-inflammatory properties.¹⁸In this solution of 4-aminoantipyrine (33 mg, 0.16 mmol) in degassed

manuscript, we report the synthesis of Ru-arene complex with dichloromethane (5 mL) under N₂atmosphere. The mixture

antipyrine, chalcone and pyrimidine based mono and was allowed to stir at room temperature for 3 h. The

bidendate ligands as new chemotherapeutic agents and completion of the reaction was monitored by TLC. After

preliminary screening for anti-cancer activities against completion of the reaction, the solvent was removed under

leukaemia cell. The structure of Ru-arene complexes (AAP-Ru, vacuum and the solid residue was washed with n-

AAPPA-Ru, AAPS-Ru, ADABS-Ru, P2P-Ru and BPY-Ru) have hexane/diethyl ether and dried under vacuum that afforded

been confirmed by spectroscopy (1H NMR, 13C NMR FT-IR and red solid. The reaction mixture was filtered, and ether was

mass spectroscopy) and X-ray diffraction methods. Further allowed to diffuse into the filtrate for a week. Orange crystals

anti-cancer activities of Ru-arene complexes have been of AAP-Ru formed which were filtered off, washed with diethyl

evaluated against K562 (Human chronic myeloid leukaemia cell ether, and dried in air at room temperature. The crystals were

line) by means of MTT assay in comparison with 5-fluorouracil suitable for single crystal X-ray crystallography. Yield: 0.115 g

(5-FU) as a control anticancer drug. Interestingly, AAPS-Ru, (92%). ¹H NMR (400 MHz,CDCl₃): δ 9.26 (s, 2H), 7.55 – 7.42 (m,

antipyrine based N,O-bidendate ligand coordinated complex 2H), 7.41 – 7.28 (m, 1H), 7.21 (d, J = 7.4 Hz, 1H), 5.93 (t, J = 5.8

and pyrimidine based positively charged P2P-Ru complex Hz, 1H), 5.72 (dd, J = 34.3, 5.4 Hz, 1H), 5.37 (qd, J = 18.8, 13.0

exhibited strong anticancer activities compared to other Hz, 3H), 4.56 – 3.22 (m, 2H), 3.18 – 2.97 (m, 3H), 2.94 – 2.67

complexes. AAPS-Ru anticancer activity has also been tested (m, 1H), 2.43 – 2.32 (m, 3H), 2.25 – 2.03 (m, 3H), 1.46 – 1.05

with human peripheral blood mononuclear cells (PBMC) in (m, 6H). ¹³C NMR (400 MHz,CDCl₃): δ 168.13, 161.07, 146.48,

vitro to assess its effect on normal healthy cell. The detailed 145.33, 134.39, 131.46,129.96, 129.74, 127.49, 126.25, 124.07,

mechanistic studies suggest that AAPS-Ru facilitated ROS 116.42, 113.44, 103.19, 100.68, 95.81, 82.44, 81.20, 79.57,

mediated leukemic cell death selectively without affecting the 36.41, 34.46, 31.01, 30.79, 22.74, 22.77, 21.97, 18.74.

normal cells while control drug kills the normal cell also.

12.20,11.28. IR (KBr ): ν = 3178(m), 29549(m), 1672(s), 1566(s),

1494(s), 1296(s), 1130(s), 760(s),698(s), 576(m), 497(m).

Synthesis of 2,3-dimethyl-1-phenyl-4-(pyridine-2-ylmethyl

Experimental

eneamino)-1,2-dihydropyrazol-5-one

(AAPPA).

4-

2-

aminoantipyrine (200 mg, 0.98 mmol) and

Materials and methods

pyridinecarboxalaldehyde (105 mg, 0.98 mmol) were refluxed

in ethanol (15 mL) for 5h. The solvent was evaporated and

dried under vacuum. The yellow solid product was obtained.

Histopaque 1077, acridine orange, ethidium bromide (EtBr),

2,7-dichlorofluorescein diacetate (DCFH2-DA), pentoxifylline

(POF), N-acetyl-L-cysteine (NAC), 5-fluorouracil, cell culture

grade DMSO were procured from Sigma (St. Louis, MO, USA).

Fetal bovine serum (FBS), penicillin, streptomycin, gentamicin

1

Yield: 0.256g (89%). H NMR (400 MHz, CDCl₃): δ 9.76 (d, J =

21.4 Hz, 1H), 8.69 (d, J = 4.7 Hz, 1H), 7.99 (d, J = 7.9 Hz, 1H),

7.74 (td, J = 7.8, 1.5 Hz, 1H), 7.52 – 7.45 (m, 2H), 7.40 (d, J = 7.5

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Hz, 2H), 7.33 (t, J = 7.3 Hz, 1H), 7.27 (t, J = 3.3 Hz, 1H), 3.19 (s, NMR (400 MHz, CDCl₃): δ 11.61 (s, 1H), 6.93 (s, 1H), 6.69 (d, J =

3H), 2.52 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 160.37 (s), 8.4 Hz, 2H), 6.64 (s, 2H), 6.39 (s, 2H), 6.37 (s, 2H), 6.15 (s, 2H),

156.59 (s), 156.17 (s), 152.46 (s), 149.71 (s), 136.28 (s), 134.65 6.01 (s, 2H), 5.85 (s, 2H), 1.66 (s, 6H).¹³C NMR (100 MHz,

(s), 129.26 (s), 129.03 (s), 127.15 (s), 124.79 (s), 123.94 (s), CDCl₃): δ 163.89 (s), 161.18 (s), 151.96 (s), 145.98 (s), 145.23

122.84 (s), 121.60 (s), 118.11 (s), 76.80 (s), 35.58 (s), 10.25 (s). (s), 133.76 (s), 132.68 (s), 130.86 (s), 130.45 (d, J = 15.7 Hz),

IR (KBr ): ν = 3041(m), 1645(s), 1566(s), 1457(m), 1414(m), 129.80 (d, J = 13.6 Hz), 121.30 (s), 121.01 (s), 119.31 (s), 117.39

1305(s), 1134(m), 764(s), 567(m).

Synthesis of

(ɳ⁶-p-cymene)(2,3-dimethyl-1-phenyl-4- (KBr ): ν = 3420(w), 2900(m), 1645(s), 1558(s), 1540(s), 1367(s),

(pyridine-2-ylmethyleneamino)-1,2-dihydropyrazol-5- 1224(m), 1157(s), 806(s), 7549(m), 518(m). Yield: 0.240g

(s), 116.85 (s), 116.47 (s), 111.85 (s), 111.55 (s), 40.17 (s). IR

one)chlororuthenium(II) chloride (AAPPA-Ru). Ligand AAP-PA (87%). ESI-MS (CHCl₃) m/z calcd for C₂₄H₂₂N₂O₂: 370.44 found:

(50 mg, 0.17 mmol) was suspended in dichloromethane (6 mL), 371.17.

followed by [Ru(p-cymene)Cl₂]₂(52 mg, 0.08 mmol) was Synthesis of (ɳ⁶-p-cymene)(1-(4-((Z)-2-hydroxybenzylidene

added. The reaction mixture was stirred for 3 h at room amino)phenyl)-3-(4-(dimethylamino)

phenyl)prop-2-en-1-

temperature. After completion of reaction, the solvent was one)chlororuthenium(II) (ADABS-Ru). Ligand ADABS (59 mg,

removed in vacuum under reduced pressure and the solid 0.13 mmol) was suspended in dichloromethane (10 mL) and

residue was washed with n-hexane/ether and dried under triethyamine (23 mg, 0.16 mmol) was added. The solution was

1

vacuum, obtained as a red solid. Yield: 0.093 g (95%). H NMR stirred at room temperature for 15 min and [Ru(p-

(400 MHz,CDCl₃): δ 9.73 (d, J = 4.6 Hz, 1H), 8.97 (s, 1H), 8.24 (d, cymene)Cl₂]₂(50 mg, 0.065 mmol) was added The reaction

J = 7.6 Hz, 1H), 7.98 (t, J = 7.5 Hz, 1H), 7.84 – 7.67 (m, 1H), 7.52 mixture was stirred for 3 h at room temperature. After

(d, J = 7.3 Hz, 2H), 7.48 (d, J = 7.5 Hz, 2H), 7.41 (t, J = 7.2 Hz, completion of reaction, the solvent was removed under

1H), 6.15 (d, J = 5.7 Hz, 1H), 6.06 (dd, J = 8.6, 6.3 Hz, 2H), 5.87 vacuum and the solid residue was washed with n-

(d, J = 5.6 Hz, 1H), 3.38 (s, 3H), 2.69 (d, J = 9.1 Hz, 3H), 2.57 (s, hexane/ether and dried under vacuum, obtained as a red solid.

1H), 2.18 (s, 3H), 1.02 (dd, J = 13.8, 6.8 Hz, 6H). IR (KBr ): ν = Yield: 0.093 g (91%) The reaction mixture was filtered, and

3412(w), 2964(m), 1645(s), 1491(m), 1309(m), 1236(m), ether was allowed to diffuse into the filtrate for a week. Red

1029(m), 773(s), 698(m), 589(m).

Synthesis of (ɳ⁶-p-cymene)(4-(2-hydroxybenzylidene amino)- off, washed with diethyl ether, and dried in air at room

2,3-dimethyl-1-phenyl-1,2-dihydropyrazol-5-one) temperature. The crystals were suitable for single crystal X-ray

crystals of compound ADABS-Ru formed which were filtered

1

chlororuthenium(II) (AAPS-Ru). Ligand AAPS (50 mg, 0.16 crystallography. H NMR (400 MHz, CDCl₃): δ 8.36 (s, 1H), 8.11

mmol) was suspended in dichloromethane (6 mL) and (dd, J = 23.7, 8.5 Hz, 2H), 7.99 – 7.70 (m, 4H), 7.70 – 7.52 (m,

triethyamine (23 mg, 0.16 mmol) and [Ru(p-cymene)Cl₂]₂(50 2H), 7.41 (dd, J = 15.4, 6.1 Hz, 1H), 7.28 (d, J = 3.1 Hz, 1H), 7.01

mg, 0.08 mmol) were added. The reaction mixture was stirred (dd, J = 12.7, 8.3 Hz, 1H), 6.84 – 6.66 (m, 3H), 6.46 (t, J = 6.8 Hz,

for 3 h at room temperature. After completion of reaction, the 1H), 5.66 – 4.87 (m, 4H), 4.26 (d, J = 5.6 Hz, 1H), 3.11 – 3.07 (m,

solvent was removed under vacuum and the solid residue was 6H), 2.66 (dt, J = 13.8, 7.1 Hz, 1H), 2.19 (dd, J = 13.1, 6.3 Hz,

washed with n-hexane/ether and dried under vacuum, 3H), 1.39 (t, J = 7.3 Hz, 6H). IR (KBr ): ν = 3445(m), 2677(w),

obtained as a red solid. Yield: 0.089 g (93%). ¹H NMR (400 1589(s) 1524(s), 1439(s), 1332(s), 1224(m), 1165(m), 1031(m),

MHz, CDCl₃): δ 7.64 (s, 1H), 7.50 (t, J = 7.7 Hz, 2H), 7.42 (d, J = 812(w), 661(w), 416(w).

7.8 Hz, 2H), 7.33 (t, J = 7.4 Hz, 1H), 7.16 (t, J = 7.4 Hz, 1H), 7.02 Synthesis of (ɳ⁶-p-cymene)(4-(pyridine-2-yl)pyrimidin-2-

– 6.78 (m, 2H), 6.36 (t, J = 7.3 Hz, 1H), 5.64 (dd, J = 20.7, 5.9 Hz, amine)chlororuthenium(II) tetrafluoroborate (P2P-Ru). 0.1

2H), 5.47 – 5.17 (m, 2H), 4.89 (d, J = 5.8 Hz, 1H), 3.10 (s,s 3H), g,0.58 mmol of 4-(pyridine-2-yl)pyrimidin-2-amine was

2.73 (dt, J = 13.8, 6.9 Hz, 1H), 2.50 – 2.40 (m, 3H), 2.11 – 1.97 suspended in 15ml methanol and 0.178 g, 0.29 mmol of

(m, 3H), 1.32 (d, J = 7.3 Hz, 6H). ¹³C NMR (400 MHz,CDCl₃): δ dichloro(p-cymene)Ru(II)dimer and followed by the addition of

169.44, 166.00, 161.48, 149.51, 135.78, 134.84, 132.00, 0.032 g, 0.29 mmol of NaBF₄allow to stir this reaction at RT for

130.44, 129.61, 129.40, 127.40, 124.77, 124.28, 122.95, 3h. The resulting solution was evaporated in vacuum under

118.51, 114.29, 102.28, 95.77, 85.27, 83.27, 83.84, 81.49, reduced pressure. Yield: 0.240 g (96%) the complex was

79.81, 45.99, 36.25, 30.57, 22.34, 21.90, 18.29, 11.90, 8.76. IR crystallized by slow diffusion of ether into methanol; dark red

1

(KBr ): ν = 3437(w), 2935(m), 2677(m), 2494(m), 1660(m), crystals were formed after 2 days. H NMR (300 MHz, D₂O): δ

1602(s), 1525(s), 1441(s), 1323(m), 1286(m), 1145(s), 1031(m), 9.23 (d, J = 5.5 Hz, 1H), 8.39 (d, J = 5.0 Hz, 1H), 8.14 (d, J = 8.1

760(s).

Synthesis of 1-(4-((Z)-2-hydroxybenzylideneamino)phenyl) -3- 5.0 Hz, 1H), 6.10 (d, J = 6.1 Hz, 1H), 5.80 (s, 2H), 5.68 (d, J = 6.1

(4-(dimethylamino) phenyl)prop-2-en-1-one (ADABS). Hz, 1H), 4.70 (s, 2H). 2.10 (d, J = 11.6 Hz, 3H), 0.76 (dd, J = 27.6,

Hz, 1H), 8.01 (t, J = 7.8 Hz, 1H), 7.78 – 7.54 (m, 1H), 7.39 (d, J =

Compound ADABS was prepared from the reaction of chalcone 6.9 Hz, 6H). IR (KBr): ν = 3383(w), 3240(m), 3136(m), 1606(s),

derivative, 1-(4-(Z)-2-hydroxybenzylideneamino) phenyl)prop- 1550(s), 1481(s), 1352(m), 1222(m), 1120(m), 1028, 779, 540,

2-en-1-one (200 mg, 0.74 mmol) and salicylaldehyde (90 mg, 462.

0.74 mmol) were refluxed in methanol (25 mL) for 6h. Then,

Biological Studies

the reaction mixture was cooled to room temperature; the

resulting precipitate was filtered off and dried under vacuum.

1

The product was obtained as a yellow crystalline product. H

Drug preparation

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All the ruthenium complexes, 5-FU, and others inhibitors were measured after derivatization of GSH with 2-vinyl pyridine

dissolved in cell culture grade DMSO to prepare the stock followed by using Ellman’s reagent according to the previously

solutions which were freshly diluted in RMPI 1640 to prepare described method by Kar Mahapatra et al.²⁵Protein was

the working concentration. All the experiments were done in determined according to Lowry et al., 1951 using bovine serum

triplicate and repeated at least two times.

albumin as standard.²⁶

K562 cell culture and PBMC isolation

TNF-α and IL-10 release assay by ELISA

K562 cells, obtained from National centre for cell sciences K562 cells and PBMCs were treated with AAPS-Ru (12.5 μM)

(NCCS), Pune, India, were cultured in RMPI-1640 media for 24 hrs. TNF-α and IL-10 release by K562 cells and PBMC

supplementated with 10% fetal bovine serum (Gibco) and 1X were determined from cell free supernatants of culture media

pen-strep antibiotics in T25 cell culture flask with regular (200 μl) with an enzyme-linked immunosorbent assay (ELISA)

passage as described earlier.²³The confluent culture of K562 (R&D systems) according to the manufacturer's instructions.

cells were used in all experiments. PBMCs were isolated from

Apoptotic morphology by AO/EtBr staining

heparinised blood of healthy donor with prior consent. 1:1

diluted blood samples with PBS were layered with Histopaque Apoptotic morphology was detected by K562 cells and PBMC

1077 (Sigma) followed by centrifuged at 1500 rpm for 30 min. were treatment followed by AO/EtBr staining for 24 h. After

The buffy coat was collected carefully washed three times with the treatment with AAPS-Ru (12.5 μM), K562cells and PBMCs

PBS and resuspended in complete culture media and used in were collected, washed with cold PBS, and then stained with a

different experiments.^{23, 24}

mixture of AO (100 μg/mL) and Et–Br (100 μg/mL) at room

temperature in dark for 5 min. After that cells were washed

with PBS, and thin single mono layer films were prepared and

Cell cytotoxicity

MTT

(3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium observed under fluorescence microscope (NIKON, Japan).

bromide) assays were performed to estimate cell cytotoxicity

of K562 cells and PBMCs in the presence or absence of

Statistical analysis

ruthenium complexes (0 – 100 μM ) at different time point (0 Each of the above assays was performed in triplicate repeated

– 72 hrs) using 2 × 10⁴cells/well as initial cell number in a 96 at least two times. The data were expressed as mean ±

well microplate. The absorbance of MTT formazan product standard deviation. Comparisons between the means of

was measured using a microplate reader (Biotek, Synergy H1, control and treated group were made by two-way ANOVA test

India) at 570 nm. Cell viability was calculated as a relative (using a statistical package, Origin 6.1, Northampton, MA

percentage to untreated cells.

01060 USA) with multiple comparison t-tests.

Inhibitors assay

K562 cells were pre-treated with 60 μM necrostatin-1

(inhibitor of necrosis), 2 mM pentoxifylline (a potent TNF-α

inhibitor), 30 μM SP600125 (JNK inhibitor), 10 mM NAC and

then treated with AAPS-Ru (12.5 μM) for 24 h. After the

treatment schedule, the cells were washed with culture

medium and cell viability was determined by MTT assay as

described earlier.

Intracellular measurement of reactive oxygen species (ROS)

ROS generation was measured using DCFH₂-DA as described

previously²³using H₂O₂(100 mM) as positive control.

Fluorescence images were also taken by phase contrast Scheme 2 Structure of the arene ruthenium (II) complexes

microscope (NIKON, Japan). All the experiments were done in studied for their anticancer activity.

triplicate and repeated at least two times.

Biochemical parameters

Results and Discussion

Lactic dehydrogenase (LDH) releases assay was done using a

Synthesis and structural characterization

spectrophotometric LDH assay kit (Reckon Diagnostics Pvt. Ltd,

The arene-ruthenium(II) complexes were synthesized using

India) to evaluate cell membrane integrity. Malondialdehyde

biologically active N-monodentate and bidentate N,N-, N,O-

(MDA), the extent of lipid peroxidation, was estimated as the

chelating ligand scaffolds (Scheme 2). Bidendate N,O- and N,N-

concentration of thiobarbituric acid reactive product according

chelating Schiff base ligands, AAPS and AAPPA, ADABS have

to the previously describe by Kar Mahapatra et al.²⁵Reduced

been synthesized by reacting 4-aminoantipyrine and amino-

glutathione estimation in cell lysate was performed by

chalcone (1-(4-(Z)-2-hydroxybenzylideneamino) phenyl)prop-2-

Ellman’s reagent according to the previously described method

en-1-one with relevant aldehydes (salicylaldehyde and

by Kar Mahapatra et al.²⁵The oxidized glutathione level was

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pyridine-2-carbaldehyde)

in

refluxing

alcohol Cl^–anion. The details are depicted in supporting information

(ethanol/methanol) (Scheme S1, S5 and S6). The reaction of (Fig. S24-S27). The mass spectral data’s are suggested that, the

dimeric ruthenium precursor [Ru(η⁶-

-cymene)(μ-Cl)Cl]₂with chloride ion, which is attached to arene Ru(II) complexes is

different ligands AAP, AAPPA, AAPS, ADABS and P2P) produced more labile.

p

corresponding arene Ru(II) complexes AAP-Ru, AAPPA-Ru,

The absorption spectra of ligands, 4-Aminoantipyrine

2,3-dimethyl-1-phenyl-4-(pyridine-2-

4-(2-

amino)-2,3-dimethyl-1-phenyl-1,2-

(AAPS), and 1-(4-((Z)-2-

AAPS-Ru, ADABS-Ru and P2P-Ru respectively (Scheme S7-S11). (AAP),

¹H and ¹³C NMR spectra of arene Ru(II) complexes recorded in ylmethyleneamino)-1,2-dihydropyrazol-5-one(AAPPA),

CDCl₃clearly indicate the coordination of ligand with Ru(II) hydroxy

(Fig. S1-S14). The ¹H NMR spectra of arene Ru(II) complexes dihydropyrazol-5-one

benzylidene

showed proton signals in the range of δ = 5.37−6.15 ppm due hydroxybenzylideneamino)phenyl)-3-(4-(dimethylamino)

to p-cymene ring protons. The proton chemical shifts in the phenyl)prop-2-en-1-one (ADAB-S) and their complexes AAP-

range between δ = 0.76−1.39 ppm and 2.10-2.32 correspond Ru, AAPPA-Ru, AAPS-Ru, ADABS-Ru and P2P-Ru have been

to isopropyl and methyl protons, respectively. AAP-Ru, AAPPA- recorded in CH₂Cl₂(Fig. 1). The absorption spectral data are

Ru, AAPS-Ru, and ADABS-Ru showed proton signals in the summarized in Table 1. The electronic absorption spectra of

range of 6.36-7.52 ppm corresponding to benzene moiety. The free ligand showed peaks in the range between 254–350 nm

ligands AAPS and ADABS showed ¹H NMR signals of -OH proton that can be assigned to intra-ligand n-π* and π-π*

at 9.80 ppm and 11.61 ppm respectively, but the phenolic transitions.²⁷The absorption bands in the ranges of 350-400

proton resonance has been absent in their corresponding nm can be assigned to inter-ligand-charge transfer (ILCT)

metal complexes AAPS-Ru and ADABS-Ru which confirmed the transition. Besides, the absorption spectra of the complexes

coordination of hydroxyl group with ruthenium metal centre. exhibited low energy bands at 340-425 nm corresponding to

Besides, in complex P2P-Ru, the protons of the pyrimidine and the metal to-ligand charge transfer [MLCT= dπ(Ru) → π*(L)].

pyridine core shifted to downfield between 7.38-9.24 ppm and Further to ascertain the coordination and structure of arene

NH₂protons shifted up-field to 4.70 ppm compared to free P2P Ru complexes, single crystals were attempted to grow for all

ligand. The ¹H NMR spectra for arene Ru(II) complexes showed compounds. However, only AAP-Ru, ADABS-Ru and P2P-Ru

downfield shift compared to their corresponding free ligands, produced quality single crystals by slow diffusion of diethyl

which confirmed the coordination of AAP, AAPPA, AAPS, ether into CH₂Cl₂solution of reaction mixture.

ADABS and P2P ligands to Ru(II) metal centres. The ¹³C NMR

The complex AAP-Ru crystallized in the monoclinic space

spectrum of the arene Ru(II) complexes AAP-Ru, AAPPA-Ru, group C2/c whereas ADABS-Ru and P2P-Ru crystallized in

AAPS-Ru, and ADABS-Ru showed chemical shifts in the range triclinic P-1 space group (Table 2). A thermal ellipsoidal plot of

of 160-161 ppm corresponding to -C=O carbon atom. Further, AAP-Ru, ADABS-Ru and P2P-Ru compounds are shown in Fig. 2.

the complexes showed carbon atom signals between 161-169 The selected bond lengths and bond angles of compound AAP-

ppm owing to the presence of -C=N feature. The aromatic Ru are presented in supporting information. The molecular

carbons are resonated between 108-156 ppm. FT-IR spectra structure of AAP-Ru, ADABS-Ru and P2P-Ru indicates that

also confirmed the characteristic stretching frequencies and Ru(II) adopted a typical ‘three leg piano-stool’ conformation

functional groups present in the free ligands as well as (three coordination sites of a quasi-octahedral array and three

complexes (Fig. S15-S23). For instance, compound AAP-Ru other donor atoms). In AAP-Ru, two chlorido ligands and NH₂

showed ν(NH₂) stretching vibration bands at 3178 cm^-1and group of 4-aminoantipyrine ligand (N-monodentate with

2955 cm^-1that are at lower wavenumbers in comparison to primary terminal amine groups -NH2) act as three legs. In

the free ligand of AAP bands 3431 cm^-13325 cm^-1, thus ADABS-Ru, chloride ligand and bidendate hydroxyl oxygen (O1)

authenticates the coordination of AAP with Ru metal centre.

Compound AAPPA exhibited ν(C=N) stretching vibration band Table

1 Absorption data for ligands and arene Ru(II)

at 1566 cm^-1, upon coordination the ν(C=N) stretching band complexes.

shifted to lower wave number considerably. In compound

AAPS, the ν(C=N) band appears at 1593 cm^-1, that is shifted to

Free

λ_max(nm)

Arene Ru(II) λ_max(nm)

Ligands

(ε, M⁻¹cm⁻¹)

Complexes

(ε, M⁻¹cm⁻¹)

1525 cm^-1after coordination with Ru centre. Similarly, the IR

spectrum of compound ADABS-Ru showed typical ν(C=N) band

at 1523 cm^-1, which is considerably at lower wave-number

compared to free ADABS (1540 cm^-1). Complex, P2P-Ru

exhibited typical ν(C=N) bands at 1607 cm^-1and after

coordination it is shifted to 1550 cm^-1. Further, Mass analyses

of the complexes have also revealed the presence of molecular

species. Mass spectral analysis of AAP-Ru is showed molecular

ion peak at 474.08, which was assigned to the m/z of the AAP-

Ru molecule with loss of a Cl^–anion. The AAPPA-Ru is showed

main peak at 563.11, which is based on the m/z of the cationic

AAPPA-Ru molecule. In case of AAPS-Ru, the molecular ion

peak at m/z 542.31 is due to AAPS-Ru molecule with loss of a

AAP

AAP-Ru

287 (7500)

425 (1400)

255(8200)

285(11000)

AAPPA

AAPS

254 (1,40000)

341 (170000)

AAPPA-Ru

AAPS-Ru

286 (14800)

418 (7500)

350 (21000)

290 (10700)

348(9600)

417(3000)

256 (34000)

289 (22500)

428 (41200)

255 (12200)

332(8300)

367(4600)

400(2100)

ADABS

P2P

264 (18500)

316 (24500)

424 (45000)

273(49700)

327(50000)

ADABS-Ru

P2P-Ru

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(c)

Fig. 2 Thermal ellipsoid plot at the 50% probability level of the arene Ruthenium

(II) Complexes, AAP-Ru (a); ADABS-Ru(b); and P2P-Ru (c). Hydrogen atoms have

been omitted for clarity.

and imine nitrogen (N1) atoms of chalcone based Schiff-base

ligand act as the legs. Whereas in P2P-Ru, chloride ligand and

two nitrogens, N1 and N2 from 2-Amino-4--(pyridin-2-

yl)pyrimidine bidendate, act as the legs. Ru(II) is π-bonded to

the

p

-cymene ring in ⁶ɳ- coordination mode in all three

compound. The selected bond lengths and bond angles of

AAP-Ru, ADABS-Ru and P2P-Ru are presented in Table S1-S3.

Ru(II) in AAP-Ru adopted a pseudo-tetrahedral coordination

geometry. The bond distance between Ru metal and the

centroid of the p-cymene is 1.6554(4) Å. The bond lengths of

Ru(1)-Cl(1) and Ru(1)-Cl(2), and Ru(1)-N(1) are found to be

2.4143(7) Å, 2.4047(7) Å and 2.1723(18) Å respectively. The

coordination sphere and coordination bond lengths matched

with reported arene-Ru(II) complexes.²⁸The bond angles

between Cl(1)–Ru–Cl(2), N(1)–Ru–Cl(1) and N(1)–Ru–Cl(2) are

87.32(1), 82.96(5) and 81.77(5) respectively. The bond

parameters around the Ru-center are remarkably similar to

RAPTA-C.²⁹The average Ru–C bond distance is 2.180(2) Å for

Fig. 1 Absorption spectra of ligands and arene Ru(II) complexes in CH₂Cl₂(1x10^-5M) at

298 K (a) Uncoordinated free ligands (b) Arene ruthenium(II) complexes.

(a)

AAP-Ru. Notably, the two longest Ru–C bonds (Ru(1)-C(13)

2.195(2) and Ru(1)-C(16) 2.199(2); see Table S1) are trans with

respect to Cl atoms as a result of the strong trans influence of

these atoms. The monomeric complex AAP-Ru showed N-H···Cl

hydrogen bonding interactions and produced supramolecular

dimeric structure (Fig. 3). The relevant N−H···Cl hydrogen

bonding distances are 2.622(10)Å (H(1B)···Cl(2)] and

2.700(35)Å[(H(1A)···Cl(1)] and the N–H···Cl hydrogen bond

angles are 157.13(1) [(N(1)–H1B···Cl(2)] and 125.01(1) [(N(1)–

H1A···Cl(1)]. The distance between Ru to Ru is 5.940(67) Å,

which is similar to the previously studied arene ruthenium(II)

complexes.³⁰

(b)

In ADABS-Ru and P2P-Ru, Ru(II) adopted a quasi-octahedral

arrangement. Further structural analysis of ADABS-Ru reveals

that the Ru(1)-Cl(1) distance is 2.4409(6) Å is slightly longer

than reported in literature,³¹this might be due to the C−H···Cl

intermolecular hydrogen bonding interaction. However, Ru(1)-

O(1) and Ru(1)-N(1) bond lengths are 2.0668(12) Å and

2.0801(14)

Å

are comparable to those of reported

ruthenium(II)-arene complexes. The bond distance observed

between the Ru metal and centroid of the p-cymene is

1.6603(5) Å.²⁷Interestingly, ADABS-Ru molecule interact with

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lattice of P2P-Ru resulted in the formation of infinite 1-D chain

(Fig. 5).

Table 2 Crystal data and structure refinement for compounds.

Compound

Empirical

formula

Formula

weight

AAP-Ru

ADABS-Ru

P2P-Ru

RuC₂₁H₂₇Cl₂N₃O

C₃₄H₃₅ClN₂O₂RuC₁₉H₂₂N₄ClBF₄

Ru

509.42

640.16

529.73

T [K]

100(2)

0.610

100(2)

0.610

100(2)

0.610

λ [Å]

Crystal system

Space group

a, Å

monoclinic

C2/c

triclinic

P-1

12.175(2)

11.563(2)

30.760(6)

90

9.0960(18)

11.993(2)

14.283(3)

74.20(3)

75.55(3)

89.72(3)

1448.5(6)

2

9.3290(19)

10.369(2)

11.557(2)

94.36(3)

96.55(3)

112.79(3)

1015.0(4)

2

b, Å

c, Å

α , deg

β, deg

97.18(3)

90

Fig. 4 Supramolecular Dimer of ADABS-Ru. The ruthenium complex(ADABS-Ru)

dimer linking through C_Cym–H···Cl hydrogen bond. Hydrogen atoms that are not

involved in H-bonding are omitted for clarity. Selected hydrogen bond distance:

Cl1–H14 = 2.875 Å. Color code: N, blue; O, red; C,gray; Cl, green; Ru, purple.

γ, deg

V (Å³)

4296.4(15)

8

Z

d_calc(mg/cm³)

µ (mm^-1)

R[I>2σ(I)]

1.575

1.468

1.733

0.657

0.444

0.632

R₁= 0.0326

wR₂=0.0859

R₁= 0.0378

wR₂=0.0875

1.295 /-2.106

R₁= 0.0272

wR₂=0.0272

R₁= 0.0314

wR₂=0.0762

1.316 /-

1.427

R₁= 0.0336

wR₂= 0.0893

R₁= 0.0398

wR₂= 0.0916

0.646/-2.064

R (all data)

Largest diff.

Peak/hole [e

Å^–3

]

Fig. 5 Supramolecular 1D chain formed by the combination C−H···Cl and N−H···Cl

hydrogen bonding and π-π stacking interaction in the crystal structure of [(η⁶-p-

cymene)-RuCl(P2P)]BF₄(P2P-Ru). The intermolecular hydrogen bonds are shown

as dashed lines. π-π interactions: aromatic Interatomic distances,

C(15)−C(15)=3.431(42) Å; C(14)−C(16)=3.774(42) Å; C(15)−C(16)= 3.319(35) Å.

In Vitro anti-leukaemia activities

The in vitro anticancer activities of Ru-arene complexes were

screened against K562, a chronic myeloid leukemia cell line, to

screen the best complex for further studies (Table 3).

Interestingly, Ru-arene complex based N, O-bidendate Schiff

base derived from AAP (AAPS-Ru) and a positively charged

bipyridine complex (P2P-Ru) exhibited strong inhibition of cells

growth. The IC₅₀values of anti-leukemic activity for AAPS-Ru

and P2P-Ru were found to be 12.46 ± 0.47 μM and 11.8± 0.49

μM, respectively (Table 3). It is noted that Ru-arene complex

with simple AAP (AAP-Ru) and bipyridine (BPY-Ru) did not

show any inhibition of leukemia cells growth even though the

later complex is positively charged. In contrast to Ru-arene

Fig. 3 Supramolecular interactions in the crystal structure of AAP-Ru. Dimers of

the 4-Aminoantipyrine-Ru(II) complex (AAP-Ru) linking through N-H·····Cl

hydrogen bonds. Hydrogen atoms that are not involved in H-bonding are omitted

for clarity. Selected hydrogen bond distance: Cl1–H14 = 2.875 Å.

each other through C−H···Cl (Cl(1)···H(14), 2.875(26) Å)

hydrogen-bonding and π‒π stacking interaction (3.659(74) Å)

(Fig. 4). The combination of both intermolecular hydrogen

bonding and π−π interactions leads to dimeric structure of

complex. Ru(1)-Cl(1) distance is 2.4040(8) Å and the average

Ru-N distances (Ru(1)-N(1), 2.082(2) Å; Ru(1)-N(2), 2.112(2) Å)

are comparable to those of ruthenium(II)-arene complexes

reported in literature.³⁰The bond angles for N(1)-Ru(1)-Cl(1)

and N(2)-Ru(1)-Cl(1) are 87.60(6)°, and 87.18(6)° respectively.

Further hydrogen bonding (both C−H···Cl and N−H···Cl

hydrogen bonding) and π-π stacking interactions in the crystal

complex with AAPS ligand, complex prepared from N,O

-

bidendate Schiff base ligand of chalcone derivative (ADABS)

showed only weak activity against leukemia cells. Similarly, Ru-

arene complexes of N,N-bidendate Schiff base ligand (AAPPA)

also exhibited weak activity. These structure-activity

comparison indicate that N,O-bidendate ligand with

hydrophilic functionality and N, N’-bidendate ligand with

hydrophilic functionality and ionic nature such as positive

charge could be suitable for antileukemic activities. Although

ADABS-Ru complex has N,O-bidendate chelating unit, the

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increased conjugation via phenyl groups are expected to

increase the hydrophobic character. The positively charged Ru

complexes have already shown to exhibit good anticancer

activities since the positive charge facilitate strong interaction

with negatively charged membrane that helps in the

infiltration in both cancer and normal healthy cells to kill

them.^7,9It is noted that positively charged P2P-Ru showed

strong inhibition of both cancer as well as healthy cells, while ,

neutral AAPS-Rs exhibited inhibition of cancer cell growth

alone (Table 3).

Table 3 IC₅₀value (μM) of ruthenium complexes against K562 and human PBMC.

Values not sharing a common letter (a–f) are significantly different (p<0.05).

Fig. 7. AAPS-Ru induced alteration in nuclear morphology of K562 cells and PBMCs by

acridine orange/ethidium bromide (AO/EB) staining. Data represented hare as best

photographs obtained from all experimental sets.

Compound

IC50

against IC50

against

PBMC

K562 (μM)

human

(μM)

(mean ± SEM)

>100

The decreased cell viability of AAPS-Ru was correlated with

induction of apoptosis by studying morphology changes of

K562 cells using fluorescence microscopy. The untreated and

AAPS-Ru treated K562 cells and PBMCs were stained with

acridine orange/ethidium bromide (AO/EB) staining. Viable

cells will uptake only AO and emit green fluorescence where

apoptotic cell will uptake AO/EtBr both and emit green with

red/orange fluorescence. In this study, the presences of

apoptosis associated nuclear morphological changes were

inferred as follows: orange–red color significantly increased in

AAPS-Ru treated K562 cells but not in PBMC, indicating the

early and late apoptotic K562 cells. The absence of apoptotic

cells in PBMC were confirmed by only green fluorescence since

it uptakes only AO (Fig. 7). It is noted that 5-FU control drug

treated K562 and human PBMC cells clearly show orange-red

color and suggest apoptosis in both infected as well as normal

cells. To study the mechanism of action, we have performed

AAPS-Ru induced ROS generation experiments since ROS

generation is the early events in drug-induced killing of K562

cells.³²AAPS-Ru (12.5 µM) induced ROS generation in K562

cells were measured after 60 min exposure. The results

strongly indicate that AAPS-Ru selectively and significantly

BPY-Ru

P2P-Ru

65.83± 2.76 ^a

11.8 ± 0.49 ^b

87.93 ± 4.22 ^c

59.81 ± 2.53 ^{a, d}

12.46 ± 0.47 ^{b, e}

53.25 ± 2.59 ^{d, f}

28.73 ± 1.49

92.54 ± 4.72

84.68 ± 3.43

>100

AAP-Ru

AAPPA-Ru

AAPS-Ru

ADABS-Ru

71.95 ± 3.02

Fig. 6 (a) Concentrations and (b) time dependent K562 cell viability decreased due to

AAPS-Ru exposure. Data represented here as mean ± SEM from all the experimental

values. *p<0.05, **p<0.01, and ***p<0.001 compared with respective untreated

control cells.

(p<0.01) provoked ROS generation in K562 cells (Fig. 8a).

Moreover, AAPS-Ru was more efficient than control 5-FU in

ROS generation in K562 cells. Fluorescence microscopy images

further supported ROS generation in K562 cells and PBMC (Fig.

8b). To confirm the critical role of AAPS-Ru-induced ROS

generation in K562 cells killing, we have also performed

experiments with pre-treatment of N-acetyl-l-cysteine (NAC), a

selective ROS inhibitor in wide range of cells.³³It clearly

indicates that, inhibition of ROS generation by NAC interfered

with AAPS-Ru promoted decreased cell viability in K562 cells

Hence, we have chosen the neutral AAPS-Ru arene complex

for further detailed anti-leukemia activity studies in

comparison with 5-FU as a control drug to elucidate the

possible mechanism of action. Cell viability study on K562 and

human PBMC cells were performed after AAPS-Ru treatment

to evaluate the cell killing ability of the Ru-arene complex drug

by exposing different concentration (0.78 µM to 100 µM) after

24 h of time period (Fig. 6a). It was observed that AAPS-Ru

exhibited cytotoxicity on K562 cells from 0.78 µM onwards and

100 µM concentrations decreased the cell viability by 84.5 %.

Interestingly, cell viability of normal cells (PBMC) was

decreased by 38 % only when exposing 100 µM of AAPS-Ru.

The time dependent studies of cell viability with AAPS-Ru drug

suggest that exposing 12.5 µM can reduce the cell viability by

65 % after 72 h (Fig. 6b). So, further studies were performed

using this concentration with 24 h incubation time.

significantly (p<0.05). Hence, it could be assumed that, AAPS-

Ru-induced ROS generations were the critical events in K562

cells death. Most of the standard antileukemic drugs induce

apoptosis passing through the ROS generation inside the

leukemic cells that lead to oxidative damages of the lipid,

protein and DNA molecule.³²Hence, AAPS-Ru induces ROS

generation inside the K562 cells might damage the cellular

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were significantly (p<0.01) decreased in K562 cells when treated

with AAPS-Ru (Fig. 9d,e). Moreover, the GSH/GSSG ratios were

drastically decreased in K562 cells (Fig. 9f), indicating the cellular

damage in K562 cells and corroborating with ROS generations, LDH

releases and MDA levels in AAPS-Ru treated K562 cells. Tumor

necrosis factor α (TNF-α) is well known to induce apoptosis/cell

death in K562 cells.³⁶The TNF- α release in cell free supernatant

from AAPS-Ru treated K562 cells and PBMC was measured using

ELISA method. The results indicate strong TNF-α release associated

with AAPS-Ru treatment that might induce the K562 cell killing but

not in PBMC (Fig. 9a).

building blocks and finally destroy the cells via apoptosis

process.

Fig. 8 Induction of ROS generation is critical for the AAPS-Ru induced K562 cells killing.

(a) ROS generations by K562 and PBMC were measured by spectrofluorometric method

using DCFH₂DA probe and bar represented here as arbitrary unit (a.u.) of fluorescence

intensity of DCF. (b) Photographs of ROS generation in K562 and PBMC were taken

using fluorescence microscope (Nikon) and represented hare as best photographs

obtained from duplicate experimental sets obtaining similar kind of results. (c) Cell

viability determination in presence of 10 mM NAC (potent ROS inhibitor). Data

represented hare as mean ± SEM from all the experimental values. *p<0.05, **p<0.01,

and ***p<0.001 compared with respective untreated control cells.

Fig. 9 AAPS-Ru induced changes in (a) LDH release, (b) lipid peroxidation, (d) GSH level,

(e) GSSG level, (f) GSH/GSSG ratio in K562 cells. (c) Effect of necrostatin-1 pretreatment

in cell viability of AAPS-Ru treated K562 cells. All the experiments were done in

duplicate and repeated at least three times. Data represented hare as mean ± SEM

from all the experimental values. *p<0.05, **p<0.01, and ***p<0.001 compared with

respective untreated control cells.

LDH release, loss of cellular membrane integrity, lipid peroxidations

are the major events in ROS-induced cells death.³⁴The LDH release

and lipid peroxidation has also been evaluated in AAPS-Ru treated

K562 cells and PBMC. The results showed that LDH release and

MDA levels (indicator of lipid peroxidation) were significantly

(p<0.01) increased in AAPS-Ru treated K562 cells (Fig. 9a, b). This

increased LDH leakages in cell culture medium of AAPS-Ru treated

K562 cells were due to swift cell-membrane damage that directed

to cell death and provided evidences that the membrane leakages

were occurred as a consequence of the apoptosis.³⁵It is well known

that, ROS generation induced cellular damage as well as LDH

release either through apoptosis or necrosis process. Necrostatin-1

(selective inhibitor of necrosis) pre-treatment could not affect the

decreased cell viability in AAPS-Ru treated K562 cells (Fig. 9c).

Hence, it may be assumed that AAPS-Ru induced ROS generation

activated the programmed cell death events (Apoptosis) in K562

cells. ROS mediated apoptosis is solely depended upon the cellular

antioxidant and pro-oxidant status. Reduced glutathione (GSH) is a

major oxidant and oxidized glutathione (GSSG) is a major pro-

oxidant in cellular level, and their ratio determines the cellular

damage intensity.^23,25Hence, we have measured their levels and

their ratio in AAPS-Ru treated K562 cells. The results suggest that,

GSH levels were significantly (p<0.01) decreased and GSSG levels

Fig. 10. (a, b) AAPS-Ru increased TNF-α and decreased IL-10 to kill the K562 cells. (c)

Pentoxifylline (POF) and JNK inhibitor (SP600125) increased the cell viability of AAPS-Ru

treated K562 cells. Data represented hare as mean ± SEM from all the experimental

values. *p<0.05, and **p<0.01 compared with respective untreated control cells.

To provide evidence of critical role of TNF-α, we have used

pentoxifylline (POF; irreversible inhibitor of TNF-α). Pre-

treatment with POF almost totally reversed back the affected

cell viability during AAPS-Ru treatment (Fig. 10a). These result

strongly evidenced that, AAPS-Ru selectively induced TNF-α

potentiated for the cancer cell death without much affecting

healthy cells, PBMC. It is well established that cancer cells

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secret more interleukin 10 (IL-10) for their survival through gratitude. VM is also grateful to UGC, Government of India for

opposing the role of TNF-α and inducing immune suppression. UGC research award-2014-16 (No: 30-11/2015 (SA-II). SKM is

Therefore, we have also studied the level of IL-10 in cell acknowledging DST-FIST programme, Govt. of India

culture supernatant of K562 cells with or without treatment of (SR/FST/ETI-331/2013) and SERB, Govt. of India (SB/YS/LS-

AAPS-Ru (Fig. 10b). AAPS-Ru exposure to K562 cells decreased 285/2013) for instrumental and financial support.

the IL-10 production significantly (p<0.01) that might be

responsible for inducing TNF-α mediated cell death. c-Jun-N-

terminal kinase (JNK) is a member of the mitogen-activated

protein (MAP) kinase family. Several reports indicated the vital

role of JNK in induction of apoptosis through critical regulation

of TNF- α and IL-10. ^{28, 33}AAPS-Ru-treated K562 cells death that

was pre-treated with JNK inhibitor (SP600125) has also

provided evidence of involving JNK signalling molecule in the

apoptosis (Fig. 10c).

References

1

(a) E. Alessio, Bioinorganic Medicinal Chemistry; Wiley-VCH:

Weinheim, Germany, 2011. (b) A. L. Noffke, A. Habtemarian,

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5219−5246. (c) S. Komeda, A. Casini, Curr. Top. Med. Chem.,

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Schellens, J. H. Beijnen, G. J. Sava, Inorg. Biochem., 2012,

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Conclusions

2

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In conclusion, we have synthesized and characterized new

arene-Ru complexes with N-donor monodentate (AAP) and

N,O- and N,N-bidentate chelating ligands (AAPS, ADABS,

AAPPA and P2P) and screened for antileukemic activities. Ru(II)

adopts typical “three-leg piano-stool” geometry with pseudo-

tetrahedral (AAP-Ru) and quasi-octahedral arrangement

(ADABS-Ru and P2P-Ru) in the coordination complex. Further,

supramolecular interactions (C–H···Cl, N–H···Cl and π-π)

produced dimeric structure in AAP-Ru and ADABS-Ru crystal

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3

evaluated for in-vitro anticancer activities against K562 cells,

human chronic myeloid leukemia cell line, by using MTT assay.

The results indicate strong anti-leukemic activity for neutral

A.

Jakupec, V. B. Arion, B. K. Keppler,

AAPS-Ru (IC₅₀= 12.46 ± 0.47 μM) and cationic P2P-Ru (IC₅₀

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Importantly, the neutral AAPS-Ru kills selectively chronic

leukemic cells without killing healthy PBMC cells. Thus neutral

complex arene-Ru complex would be more suitable for

chemotherapeutic agents. The detailed mechanistic studies

indicated selective ROS generation in K562 by AAPS-Ru that

mediated cells death via apoptosis without affecting the

normal cells. Further, the initiation of apoptosis by AAPS-Ru

4

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treatment, particularly TNF-α induced cell death, confirmed by

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interaction studies of Ru complexes with DNA and protein are

under progress by using UV-Vis and fluorescence techniques.

5

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,

Ethics statement: Human Blood sample from Healthy

volunteers, collected by Medical personal, was used to isolate

the PBMC. Prior written consent was taken from the

volunteers. All the experiments with PBMC was carried out in

strict guideline of Institutional Biosafety committee (IBSC) with

prior approval (Number: IBSC/21/2014, Dated: 08.09.2014).

6

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Acknowledgements

We are thankful to SERB, DST, Government of India (DST Fast

Track scheme no. SB/FT/CS-182/2011) is acknowledged with

10 | J. Name., 2012, 00, 1-3

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Arene ruthenium(II) complexes with chalcone, aminoantipyrine and aminopyrimidine based ligands: Synthesis, structure and preliminary evaluation of anti-leukemia activity

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