Synthesis, characterization, DNA binding and cytotoxicity of fluoro-dipyrrin based arene ruthenium(II) complexes

Article abstract of DOI:10.1016/j.ica.2016.03.003

Synthesis of four new arene ruthenium(II) complexes [(η⁶-C₁₀H₁₄)RuCl(MFPdpm)] (1); [(η⁶-C₁₂H₁₈)Ru-Cl(MFPdpm)] (2); [(η⁶-C₁₀H₁₄)RuCl(PFPdpm)] (3) and [(η

Full text of DOI:10.1016/j.ica.2016.03.003

Accepted Manuscript
Synthesis, characterization, DNA binding and cytotoxicity of fluoro-dipyrrin
based arene ruthenium (II) complexes
Rajendra Prasad Paitandi, Roop Shikha Singh, Sujay Mukhopadhyay, Gunjan
Sharma, Biplob Koch, Pratap Vishnoi, Daya Shankar Pandey
PII:
S0020-1693(16)30095-0
http://dx.doi.org/10.1016/j.ica.2016.03.003
ICA 16938
DOI:
Reference:
Inorganica Chimica Acta
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Revised Date:
Accepted Date:
23 January 2016
2 March 2016
3 March 2016
Please cite this article as: R.P. Paitandi, R.S. Singh, S. Mukhopadhyay, G. Sharma, B. Koch, P. Vishnoi, D.S. Pandey,
Synthesis, characterization, DNA binding and cytotoxicity of fluoro-dipyrrin based arene ruthenium (II) complexes,
Inorganica Chimica Acta (2016), doi: http://dx.doi.org/10.1016/j.ica.2016.03.003
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Synthesis, characterization,DNA binding and cytotoxicity of fluoro-dipyrrin
based arene ruthenium(II) complexes
Rajendra Prasad Paitandia, Roop Shikha Singha, Sujay Mukhopadhyaya,Gunjan Sharmab, Biplob
Kochb*,Pratap Vishnoic,Daya Shankar Pandeya*
aDepartment of Chemistry, Institute of Science, Banaras Hindu University, Varanasi – 221 005
(U.P.) India.
bDepartment of Zoology, Institute of Science, Banaras Hindu University, Varanasi – 221 005
(U.P.) India
cDepartment of Chemistry, Indian Institute of Technology, Bombay, Powai, Mumbai 400076,
India.
Abstract
Synthesis of four new areneruthenium(II) complexes [(6-C10H14)RuCl(MFPdpm)] (1); [(6-
C12H18)Ru-Cl(MFPdpm)] (2); [(6-C10H14)RuCl(PFPdpm)] (3) and [(6-C12H18)RuCl(PFPdpm)]
(4)containingdipyrrin ligands5-(4-fluoro)phenyldipyrromethene (MFPdpm) and 5-(penta-
fluoro)phenyldipyrromethene (PFPdpm) have beendescribed. The ligands and complexes have
been thoroughly characterized by elemental analyses,spectroscopic studies (ESI-MS, IR, 1H, 13C
NMR, UVvis.) and structure of the representative complex 4 determined by X-ray single crystal
analyses. DNA binding activities of 14 havebeen investigated byUVvis and fluorescence
spectroscopy and their binding through the minor groove of DNA has been established by
molecular docking studies. The complexes 14 exhibit significant cytotoxicity toward human
lung cancer cell line (A549). Cytotoxicity, morphological changes, and apoptosis studies have
been evaluated through MTT assay, Hoechst 33342/PI staining, and cell cycle analysis by
fluorescenceactivated cell sorting (FACS). In vitro antitumor activity and cytotoxicity of the
complexes lie in the order 4 >3>2 >1.
*Corresponding author. E-mail: dspbhu@bhu.ac.in; Tel.: + 91 542 6702480; Fax: + 91 542
2368174.
1

1. Introduction:
Since successful application of cis-platin and its derivatives in the treatment of a broad
spectrum of cancer numerous platinum [13] and non-platinum based metal complexes have
fascinated many research groups [412]. Although,cis-platin is one of the most active anticancer
drugs, its usage is limited due to high toxicity, drug selectivity, intrinsic and acquired resistance.
To overcome the disadvantagesof platinum based drugs,attempts are being made to develop
alternative metal-based antitumor agents [414].In this context, ruthenium based complexes have
drawn special attention andamong these KP1019 and NAMI-A have shown great promise owing
to their therapeutic potential, low toxicity, and activity against primary tumors and metastases
[1518]. Meanwhile, intercalators have been target of many studies because intercalation distorts
the helical structure of DNA and causes inhibition of replication enzymes [1924]. Thus in the
burgeoning area of DNA intercalation studies, ruthenium based intercalators have been
imperative due to their specific redox and photophysical properties [1921].Further,antitumor
activity of many Ru(II)-arene complexes has been related to their enhanced DNA binding
affinity, through covalent and/or non-covalent interactions [2224].
In addition, dipyrrins present conjugated π-systems consisting of two pyrrolic units andform a
variety of metal complexes which find applications in diverse areas such as dye-sensitized solar
cells, biological stains/probes, light harvester and anticancer agents [2531]. Recently we have
synthesized some stable neutral homo/heteroleptic Ru(II), Rh(III), and Ir(III) dipyrrinato
complexes exhibiting enhanced anticancer activity[2931]. To explore possible modes of
intercalation we have supplemented the dipyrrin core with various substituents ranging from
thioethers, ferrocenyl to 2-methoxypyridyl etc. [2931].Further, to enable covalent interaction
with biological targets, modern day drugs are usually equipped with a halide leaving group
particularly, fluorine as it allows simultaneous modulation of the electronic, lipophilic and steric
parameters [32 33]. In this directionconsidering wide range of biological functions numerous
fluorinated organic molecules have been developed.
Furthermore, fluorinated anti-cancer agents have attracted huge interest towards development
of new therapies for cancer treatment [3435].Therefore,in view of the advancements made
toward development of potential bioorganometallic compoundswe wished to develop some arene
ruthenium (II) complexes based on fluorinated dipyrrin core which could be potential anticancer
2

agents.Through this contribution we presentfour half-sandwich complexes of the type [Ru(η6-
arene)-(L)Cl], where arene is p-cymene (1, 3) and hexamethylbenzene (2, 4) and L is 4-
fluorophenyldipyrrins (MFPdpmH) and 1,2,3,4,5-penta-fluorophenyldipyrrins(PFPdpmH)and
their anticancer activity (Scheme 1). Also, we describe herein anticancer activity of the
synthesized complexes and structure-activity relationship with different fluorodipyrrinato ligands
and arene moiety.
.
Scheme 1.Synthesis of complexes 14
2.Experimental Section
2.1.Reagents.
Reagent grade chemicals were used throughout.Solvents were dried and distilled
following literature procedures and reactions carried out under nitrogen atmosphere [36]. Metal
chlorides (RuCl3·xH2O), agarose,-terpinene, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ), 4-(fluoro)benzaldehyde, pentafluorobenzaldehyde,and pyrrole were procured from
Sigma Aldrich Chemical Co.and used without further purifications. 3-(4, 5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT), fetal bovine serum (FBS) and RPMI-1640, were
purchased from Hi-Mediaand ethidium bromide (EB) from Loba Chemie, while acridine orange
(AO) from Sisco Research Laboratory (SRL), Mumbai, India. Triton X-100 and other chemicals
of analytical grades were purchased from Lobachemie Pvt. Ltd., India and RNAse was obtained
from GeNie, Merck, India. Propidium iodide was purchased from Calbiochem, USA. Lung
3

cancer cell line (A549) was obtained from National centre for cell science (NCCS) Pune. Calf
thymus (CT DNA) was purchased from Bangalore Genei, India and cis-platin 50 mg/50 ml was
obtained from Cipla with the name cis-platin injection BP Cytoplatin-50 Aqueous (Each 50 ml
vial contains: cis-platin IP 50 mg, sodium chloride IP 0.9% w/v and water for injection IP to 50
ml).The precursor complexes [{(6-arene)Ru(-Cl)Cl}2] (6-arene =C10H14, C12H18) [3738]and
ligands 5-(4-fluoro) phenyldipyrromethane (MFPdpmH), 5-(pentafluoro)phenyldipyrro-methane
(PFPdpmH)wereprepared and purifiedbyliteratureprocedures [39].
2.2. General Methods.
Elemental analyses for C, H and N were performed on a Euro-E 3000 Elemental
Analyzer.Infrared and electronic absorption spectra were acquired on a PerkinElmer Spectrum
Version 10.03.05 FT-IR and Shimadzu UV-1601 spectrophotometer respectively. 1H (300 MHz)
13
and C (75.45 MHz) NMR spectra were obtained on a JEOL AL300 FT-NMR spectrometer
using tetramethylsilane (TMS) as an internal reference. Fluorescence spectra were obtained on
PerkinElmer LS-55 spectrofluorimeter equipped with a xenon lamp in a 10.0 mm quartz cell
with excitation and emission slit widths of 10 and 5 nm, respectively. Fluorescence microscopic
images were taken on EVOS FL cell imaging system and electrospray ionization mass
spectrometric data were obtained on a micrOTF-Q IImass spectrometer.
2.3. Synthesis
Synthesis of [(6-C10H14)RuCl(MFPdpm)] (1)
DDQ (0.114 g, 0.50 mmol) dissolved in benzene (20 ml) was added dropwise with
stirring to an ice cooled solution ofMFPdpmH (0.120 g, 0.50 mmol) in CH2Cl2 (50 mL)and
reaction mixture stirred for an additional 1 h. After completion of the reaction (monitored by
TLC) solvent was removed under reduced pressure. Resulting residue was dissolved in
CH2Cl2/MeOH (70 mL; 1:1 v/v) and treated successively withtriethylamine (1.0 mL) and
dimeric complex [{(6-C10H14)Ru(-Cl)Cl}2] (0.153 g, 0.25 mmol). The reaction mixture
wasfurther stirred for ~4 h at room temperature. The solvent was removed under reduced
pressure and resulting residue was purified bycolumn chromatography (SiO2, CH2Cl2 with 2%
MeOH) to afford complex 1 as a red solid. Yield: 50% (0.254 g). Anal. Calc for C25H24N2FClRu:
1
C, 60.77; H, 5.41; N, 12.89. Found: C, 60.71; H, 5.36; N, 12.84%. H NMR (CDCl3, δ ppm):
1.08 (d, 6H, J = 6.9 Hz, CH(CH3)2), 2.17 (s, 3H, CH3), 2.43 (m, 1H, CH(CH3)2), 5.27 (s,
4

4H,C6H4), 6.47 (d, 2H, J = 4.2 Hz, pyrrole), 6.55 (d, 2H, J = 3.6 Hz, pyrrole), 7.0 (t, 2H, J = 8.7
Hz,phenyl), 7.35 (t, 2H, J = 8.7 Hz,phenyl), 8.01 (s, 2H, pyrrole).13C NMR (CDCl3, δ ppm):
22.06 (CH(CH3)2), 29.0 (CH3), 30.6 (CH(CH3)2), 53.1 (C- meso, dipyrrin), 84.5, 84.8, 108.2,
118.9,130.5, 155.4, 180.5. ESI-MS. (Calcd, found, m/z) 473.0967, 473.1000 [MCl]+. IR (KBr
pellets, cm-1): 1027, 1249, 1345, 1376, 1466, 1550.UVvis. (c, 10 M; EtOH:H2O, 1:1, v: v; pH
~7.3; λmax nm, ε M-1 cm-1): 491 (2.8 ×104), 430 (1.2 ×104), 315 (0.7 ×104), 221 (2.4 ×104)
Synthesis of [(6-C12H18)RuCl(MFPdpm)] (2)
This complex was prepared following the above procedure for 1 except that [{(6-
C12H18)Ru(-Cl)Cl}2] (0.167 g, 0.25 mmol) was used in place of [{(6-C10H14)Ru(-Cl)Cl}2]
(0.153 g, 0.25 mmol). Yield: 56% (0.230 g). Anal. Calc for C27H28N2FClRu: requires: C, 62.71;
H, 5.57; N, 10.75. Found: C, 62.67; H, 5.48; N, 10.80%.1H NMR (CDCl3, δ ppm): 1.9 (s, 18H,
CH3), 6.48 (d, 2H, J = 3.9 Hz, pyrrole), 6.64 (d, 2H, J = 4.2 Hz, pyrrole), 7.0 (t, 2H, J = 8.4
Hz,phenyl), 7.35 (t, 2H, J = 8.4 Hz,phenyl), 8.01 (s, 2H, pyrrole). 13C NMR (CDCl3, δ ppm):
29.6 (CH3,), 55.4 (C- meso, dipyrrin), 114.4, 114.6, 116.9, 119.4, (dipyrrin) 132.3, 132.9, 143.9,
148.7, 154.1 (Phenyl ring). ESI-MS. (Calcd, found, m/z) 501.12, 501.10[MCl]+. IR (KBr
pellets, cm-1): 1028, 1250, 1345, 1375, 1429, 1551. UVvis. (c, 10 M; EtOH:H2O, 1:1, v: v; pH
~7.3; λmax nm, ε M-1 cm-1): 438 (1.5 ×104), 322 (1.1 ×104), 261 (1.0 ×104).
Synthesis of [(6-C10H14)RuCl(PFPdpm)] (3)
It was prepared following the above procedure for 1 except thatPFPdpmH (0.156 g, 0.5
mmol) was used in place of MFPdpmH (0.120 g, 0.50 mmol) Yield: 48% (0.292 g). Anal. Calc
for C27H24N2F5ClRu, requires: C, 53.34; H, 3.98; N, 4.61. Found C, 53.28; H, 3.94; N, 4.57%.1H
NMR (CDCl3, δ ppm): 1.09 (d, 6H, J = 6.6 CH(CH3)2), 2.14 (s, 3H,CH3), 2.55 (m,
1H,CH(CH3)2), 5.25 (d, 2H, J = 5.7Hz, C6H4), 5.33 (d, 2H, J = 5.7Hz, C6H4) ,6.51 (s, 4H,
pyrrole),8.02 (s, 2H, pyrrole).13C NMR (CDCl3, δ ppm): 30.8 (C- meso, dipyrrin),
30.5CH(CH3)2),21.9(CH3) , 18.2 CH(CH3)2, 99.7, 103.4, 119.6, 134.2, 156.35, 156.36
(dipyrrin).ESI-MS. (Calcd, found, m/z) 573.0903, 573.0865 [MCl]+. IR (KBr pellets, cm-1):
987, 1026, 1248, 1343, 1377, 1496, 1518, 1567. UVvis (c, 10 M; EtOH:H2O, 1:1, v: v; pH
~7.3; λmax nm, ε M-1 cm-1): 499 (0.77 ×104), 441 (0.47 ×104), 303 (0.24 ×104), 221 (0.97 ×104).
Synthesis of [(6-C12H18)RuCl(PFPdpm)] (4)
5

It was prepared following the above procedure for 2 except that PFPdpmH (0.156 g, 0.5
mmol) was used in place of MFPdpmH (0.120 g, 0.50 mmol) Yield: 52% (0.237 g). Anal. Calc
for C27H24N2F5ClRu: requires: C, 53.34; H, 3.98; N,4.61. Found: C, 53.29; H, 3.93; N, 4.55%.
1H NMR (CDCl3, δ ppm): 1.91 (s, 18H, CH3), 6.45 (d, 2H, J = 4.2 Hz,pyrrole), 6.53 (d, 2H, J =
13
4.2 Hz pyrrole), 7.75 (s, 2H, pyrrole), C NMR (CDCl3, δ ppm): 15.0 (CH3,), 56.9 (C- meso,
dipyrrin), 93.8, 119.2, 130.2, 134.5, 155.7. ESI-MS. (Calcd, found, m/z) 545.0590, 545.0550 [M-
Cl]+. IR (KBr pellets, cm-1): 988, 1042, 1114, 1441, 1499, 1521, 1594. UVvis (c, 10 M;
EtOH:H2O, 1:1, v:v; pH ~7.3; λmax nm, ε M-1 cm-1): 513 (1.40 ×104), 453 (1.49 ×104), 306 (0.46
×104), 223 (1.71 ×104)
2.4. X-ray Structure Determination.
Crystals suitable for X-ray single crystal analyses for 4 were obtained by slow diffusion
of hexane over a dichloromethane solution of the complex. X-ray data for4were collected on a
Rigaku Saturn 724+ CCD diffractometer with a fine-focus 1.75 kW sealed tube Mo Kα (λ =
0.71075 Å) X-ray source.Structures were solved by direct methods (SHELXS 97) and refined by
full-matrix least squares on F2 (SHELX 97)[40].Disordered solvent molecules were removed by
SQUEEZE command in PLATON [4142]. All the non-H atoms were treated anisotropically.
The H-atoms attached to aromatic carbon were included as fixed contribution and geometrically
calculated and refined using SHELX riding model. Intermolecular interactions and stacking
distances were analysed by using computer program PLATON.
2.5. DNA Binding Studies.
UVvistitration studies have been performedby gradual additions of increasing
concentrations of CT DNA (Na-phosphate buffer solution, pH 7.2) to a solution of the complexes
14[fixed concentration,10 μM, ethanol/water (EtOH/H2O), 1:1, v/v].Absorbance of the stock
solution of CT DNA at 260 and 280 nm gave a ratio of 1.9, suggesting the DNA to beadequately
free of proteins.Concentration of DNA was determined using the molar extinction coefficient of
6600 M1cm1 at 260 nm. An analogous method has been used for fluorescence studies also.
Ethidium bromide (EB) displacement experiments were executed by addition of 14to an
aqueous solution of EB-DNA and simultaneous monitoring of the changes influorescence
intensity of emission maxima at 602 nm (ex, 525nm).
6

2.6. Partition Coefficient Determination.
Lipophilicity of the complexes under study was evaluatedby“Shake flask” method in
octanol/water phase partitions as described elsewhere [29].
2.7. Molecular Docking.
Molecular docking studies on 14 have been performed using HEX 6.1 software and Q-site
finder which is an interactive molecular graphics program for interaction and docking
calculations and to classify possible binding site of the biomolecules [43].The DFT calculations
were carried out using Gaussian 09 program, B3LYP method [44]. Geometry of the complexes
14were optimized with standard 631G** basis sets for C, H, N, O, and Cl and LANL2DZ for
Ru with effective core pseudo potential for metal[4546]. The coordinates of metal complexes
were taken from its optimized structure as a .mol file and transformed to PDB format using
CHIMERA 1.5.1 software.Crystal structure of B-DNA (PDB ID: 1BNA) was recovered from the
protein data bank (http://www.rcsb.org./pdb). Visualization of the docked structures has been
performed by Discovery Studio 3.5 software. The by default parameters were used for docking
calculations with correlation type shape only, FFT mode at 3D level, grid dimension was 6 with
receptor range 180 and ligand range 180 with twist range 360 and distance range 40.
2.8. Cytotoxicity and proliferation assay by MTT Assay.
The MTT assay is a quantitative, sensitive and reliable colorimetric assay generally used
for assessing viability and proliferation of the cells. In this assay mitochondrial dehydrogenase
enzymes of live cells convert the yellow water soluble substrate, 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetra-zolium bromide (MTT) into a purple formazan crystal product which is
insoluble in water or medium[47].
MTT of working concentration of 5 mg/ml was prepared in 1X phosphate buffered saline
(PBS) and 10µL added into each well of 96 well cell culture plates where 10,000 cells were
seeded in each well except the blank with different concentrations of 14and incubated for 24 h
at 37 °C and in 5% CO2 atmosphere. After adding MTT the cells were again incubated for 2 h at
37 °C. Mitochondrial dehydrogenase of the viable cells cleave tetrazolium ring of the substrate
(MTT) and produce purple formazan crystal which is insoluble in medium, after that 100µL of
DMSO was added to dissolve the formazan crystals and incubated for 30 minutes at 37 °C and
absorbance was measured spectrophotometrically in ELISA plate reader at 570 nm. All the
7

experiments were carried out in three to five replicates and IC50 value for each compound
estimated.
2.9. Analysis of cell/nucleus morphology with Hoechst 33342/PI staining.
Cell death is discriminated into two main forms, apoptosis and necrosis. In contrast to
necrosis, apoptosis is a programmed and regulated pathway of cell death [48]. Morphological
hallmarks of apoptosis in the nucleus are chromatin condensation and nuclear fragmentation[49].
This condensation and fragmentation can be followed byfluorescence microscopy after staining
with DNA binding fluorescence dyes.Hoechst and PI double staining provide a fast and suitable
assay for nucleus morphology and apoptosis. Hoechst binds in the minor groove of double
stranded DNA preferably AT rich region while PI intercalates between the bases without any
sequence preference. PI is normally used to identify dead cells as it is membrane impermeable
and gets excluded from viable cells while Hoechst dye is permeable to the cell membrane and
binds to DNA in live or fixed cells.
In the present study cells were seeded in 6 well plates and kept for 24 h at 37 °C in a
humidified atmosphere of 5% CO2. After that cells were treated with different concentrations
(15, 20 and 25µM) of complex 4 for 24 h. Subsequently, cells were washed with PBS and stained
with Hoechst (10µg/mL) and propidium iodide (10µg/mL) solution. These were then washed
with PBS and images were taken in inverted fluorescent microscope in red and blue channel.
2.10. FACS analysis with PI staining.
Besides other typical features apoptotic cells are characterized by DNA fragmentation
and consequently loss of nuclear DNA content. Use of a fluorochrome such as PI(propidium
iodide) that is capable of binding and labeling DNA makes it possible to get a rapid and precise
evaluation of cellular DNA content by flow cytometric analysis and subsequent identification of
hypodiploid cells[50].
The DNA content during cell cycle was evaluatedby flow cytometry. 1 × 105 A549 lung
cancer cells were seeded in 6 well plates and kept at 37 °C in a humidified atmosphere of 5%
CO2. After that these were treated with three different concentrations (15, 20 and 25 µM) of
complex 4 for 24 h in DMEM medium with 10% FBS. After 24 h the treated cells were washed,
trypsinised and fixed in ice cold 70% ethanol and incubated for overnight at 20oC. Then the
cells were washed twice in PBS, 500 µL of staining solution [460 µL of 0.1% (v/v) Triton x-100
8

solution, 20 µL of PI (1 mg/mL) solution and 20 µL of RNase (10 mg/mL in MQ water)] was
added in each sample and incubated for 40 minutes at 37oC in the dark and samples evaluated by
a flow cytometer.
3. Results and Discussion
3.1. Synthesis and Characterization
Ligands5-(4-fluoro)phenyldipyrromethane (MFPdpmH) and 5-(pentafluoro)phenyldipyrro-
methane (PFPdpmH)werepreparedin reasonably good yield (7075%) by one-pot solvent free
reaction of the respective aldehydes (4-fluorobenzaldehyde/ pentafluorobenzaldehyde) with an
excess of pyrrole in presence of catalytic amounts of trifluoroacetic acid (TFA) [39]. The
complexes 14,viz. [(6-C10H14)RuCl(MFPdpm)], 1; [(6-C12H18)RuCl(MFPdpm)],2; [(6-
C10H14)RuCl-(PFPdpm)], 3 and [(6-C12H18)RuCl(PFPdpm)],4 were synthesized by reaction of
chloro bridged dimeric arene ruthenium precursors [{(6-arene)Ru(-Cl)Cl}2] (6-arene =
C10H14,C12H18) with MFPdpm/PFPdpm obtained in-situby oxidation of respective dipyrro-
methanes MFPdpmH-/PFPdpmH with DDQ in CH2Cl2/CH3OH (1:1, v/v) in presence of
triethylamine at room temperature.Simple scheme showing synthesis of the complexes isdepicted
in Scheme 1.
The complexes under investigation are non-hygroscopic, orange-red crystalline solids,
soluble in common organic solvents like methanol, ethanol, acetone, dichloromethane,
chloroform, dimethylformamide, dimethylsulfoxide, and partially soluble in diethyl ether,
petroleum ether, hexane and stable in air as well as in solution. All the complexes have been
1
characterized satisfactorily by elementalanalyses and spectral studies [ESI-MS, IR, H, 13C
NMR, UVvis]. IR spectra of 14 displayed diagnostic bands due to pyrrolic ring(C=Npyr)
vibrations at 1590, 1; 1591, 2; 1521, 3; 1520, 4 cm1 which slightly shifted towards lower
frequency than the free ligand (1596 cm1,MFPdpmH, 1559 cm1,PFPdpmH) indicating
coordination of the ligand to metal center [2931].
3.2. NMR Spectral Studies
The NMR spectra of dipyrromethanes and complexes 14have been acquired in CDCl3and
1
resulting data is summarized in the experimental section. H NMR spectra of MFPdpmH-
/PFPdpmHexhibited
peakscorresponding
tomeso(5.44,
MFPdpmH;
5.90ppm,
PFPdpmH),pyrrolic [5.88, 6.15, 6.67, and 7.90 (br, NH), MFPdpmH; 6.02, 6.16,6.72, 8.15 ppm
9

(br, NH), PFPdpmH] and aromatic protons (6.98, 7.17, MFPdpmH) [39].1H NMR spectra of
14displayed loss of meso-subsituted (–CH) and NH protonssuggesting oxidation of the
ligandwhile pyrrolic and phenyl ring protons resonated at their usual positionwith
asmalldownfield shift (Figure S1S4, SI). The change in peak position and integrated intensity
clearly indicated coordination of the ligand with metal centerand thereby formation of dipyrrin
complexes[29-31].Further, arene protons in 14displayed an up-field shift relative to respective
1
precursor complexes[2931]. In the H NMR spectra of 1 and 3the protons due to arene ring
exhibited a little upfield shift [1.08 (CH(CH3)2), 2.17 (CH3), 2.43(CH(CH3)2), 5.27 (C6H4),
(merged peak) 1; 1.09 (CH(CH3)2), 3; 2.14 (CH3), 2.55 (CH(CH3)2), 5.25 ppm (merged peak)
C6H4), 3]relative to precursor complexes. Shift in the position of MFPdpmH/PFPdpmH and
arene protonsfurther suggested complexation of the ligands with metal center and formation of
respective complexes. 13C NMR spectral data of 1-4 further supported formation of these
complexes and their proposed formulations and is consistent with earlier reports[2931].
3.3. Mass spectral studies.
The composition and stability of complexes has been studied by ESIMS spectral studies. In
their positive ESI mass spectra 14 showedmajor peaks due to cationic fragment [(arene)-
Ru(dpm)]+ generated by loss of the Cl (m/z found 473.10; 1, 501.102;545.053; 573.08, 4) from
the respective complexes. The results clearly suggested that chloro (Cl) group is labile and
possibly replaced by targeted biomolecules (Figure S5S8, SI) [51].
3.4. Crystal Structure.
Crystalstructure of 4 has been determined by X-ray single crystal data. It crystallizes in
monoclinic system with P21/nspace group. Details about the data collection, solution and
refinement have been given in the experimental section and (Table 1). Pertinent view along with
partial atom numbering scheme is shown in (Fig. 1) and important bond length and angles are
summarized in (Table 2). The Ru(II) center in this complex adopted typical piano stool geometry
with two positions occupied by pyrrolic nitrogen (N1, N2) from PFPdpm, the chloro (Cl1) group
and arenering bonded in -manner[2931].The ruthenium to pyrrolic nitrogen bond distances
are identical [RuN1, 2.07 Å; RuN2, 2.08 Å], while RuCl bond distance (RuCl1) is 2.40 Å,
these are normal and comparable to other closely related complexes[2931].The bond angles
NRuN and NRuCl about metal center are close to 90° [N1RuN2, 83.54°; N1RuCl1,
10

87.18°; N2RuCl1, 87.18°] (Table 2). The arene ring is symmetrically bonded to the metal ion
with an average RuC bond distance of 2.16 Å [range, 2.1352.175 Å]. The carbon atoms in
arene ring are planar and distance between ruthenium and centroid of the hexamethylbenzene
ring is 1.69 Å, (4) which is normal and consistent with other arene ruthenium complexes
[2931].
3.5. Electronic absorption spectroscopy.
UVvis absorption spectra of 14 were recorded in EtOH: H2O, c, 10 M; 1:1, v/v; pH
~7.3(Fig. 2). The absorption spectra exhibited three major bands, an intense low energy band at ~
500 nm [489, 1; 491, 2; 494, 3; 513 nm 4] corresponding to π-π* charge transfer from conjugated
dipyrrin core, and low intensity bands at ~ 440 nm [427, 1; 432, 2; 438, 3; 457 nm 4] assignable
to metal to ligand charge transfer (MLCT) transitions. Third one appeared in UV region at ~ 305
nm [319, 1; 322, 2; 304, 3;339 nm 4] and has been ascribed to dipyrrin based intra-ligand ππ*
transitions [2931].
3.6. Electronic absorption titration.
Electronic absorption spectroscopy is an effective tool to examine the binding mode of
complexes with DNA by monitoring changes in absorption intensity and position of the bands.
The absorption spectra have been recorded with a fixed concentration of 14 (10 M) with
increasing concentrations of CT DNA (00 M) and resulting spectra is depicted in (Fig. S9).
In case of complex 1 the band centered at 427 nm exhibited hypochromism (10%) with a small
red shift of 4 nm and the weak band centered at 268 nm showed a hyperchromism (85%) with a
red shift of 12 nm.Absorption spectraof2 and 3displayed similar changes under identical
conditions(Fig S9). On the other hand, gradual addition of CT DNA to a solution of 4led to
hypochromism (8 and 6%) for the bands centered at 513 and 457 nm with small red shift of 2
nm. Further, appearance of a new band at 478 nm with apparent isosbestic points at 494 and 467
nm suggested existence of more than two species(Fig. 3). In addition, intra-ligand charge transfer
transition centered at 281 nm displayed hyperchromism (70%) with blue shift of 3 nm and
generation of a new shoulder at 336 nm with a clear isosbestic point at 390 nm. Observed
spectral changes clearly revealed that complex 4 can interact with DNA through intercalation as
well as groove binding via electrostatic interactions leading to DNA stabilization [20, 52].To
11

quantitatively compare DNA binding affinities intrinsic binding constant Kbhas been calculated
using the equation [53].
………….. (Eq. 1)
where [DNA] is the concentration of DNA in base-pairs, εa is apparent extinction coefficient
calculated as Aobs/[complex], εf corresponds to extinction coefficient of the complex in its free
form and εb refers to extinction coefficient of the complex in bound form. Each set of data upon
fitting in the above equation gives a straight line with a slope of 1/ (εb− εf) and a y-intercept of
1/Kb (εb− εf) and Kbwas determined from the ratio of slope to intercept. The values of Kb varies
in the order 4 (6 .5 × 104) >3 (5.5 × 104) >2 (2.2 × 104) >1(1.8 × 104).
3.7. Competitive binding between EB and complexes for CT DNA
Ethidium bromide (EB = 3,8-diamino-5-ethyl-6-phenyl-phenanthridiniumbromide) is a
fluorescence dye having a planar structure that binds DNA in an intercalation mode. To affirm
binding of 14withDNA,competitive studieshave been performedusing DNA bound ethidium
bromide (EB) by successive addition of 0–50 μM of complexes.Intercalation of the complexes
with DNA causes decrease in binding sites of DNA available for EB, which in turn leads to a
decrease in fluorescence intensity of the EB–DNA system [5455]. The fluorescence emission
intensity of DNA–EB system in presence of the compounds under investigation causes an
appreciable lowering in fluorescence intensity upon increasing their amounts (by51.6%,1;
55.7%, 2, 67.2%, 3 and 72%, 4;(Fig. 4 and S10). From the data we conclude that 14 are capable
of displacing EBfrom EB-DNA complex and strongly interact with DNA binding sites.The
quenching parameters for 14 have been calculated using SternVolmer equation-
………………………………… (Eq. 2)
where I0 and I represent emission intensities of EB-DNA in absence and presence of 14
respectively, Kq is the quenching constant and [Q] is concentration of 14(Fig. S11).
The Kq values for 14 were found to be 1.22 × 104 M−1, 1.33 × 104 M−1, 1.66 × 104 M−1,
and 2.00 × 104 M−1, respectively. Furthermore, apparent binding constant (Kapp) values for the
complexes have been deduced using following equation:
……………………………. (Eq. 3)
12

where, [complex] is the value of decrease in the fluorescence intensity of EB at 50%, KEB (1.0
× 107 M-1) is binding constant of EB with CT DNA and [EB] presents the concentration of EB
(10 M). The apparent binding constants for 14 came out to be 3.2 × 104 M−1, 3.4 × 104 M−1,
6.4 × 104 M−1and 8.2 × 104 M−1, respectively and are comparable to earlier reports [20,
2931].The above results suggest that 4 intercalated rather strongly than the other complexes
whichis in good agreement with the UVvis titration studies.
3.8. Partition coefficient determination.
Lipophilicity is an important physiological parameter in determining penetration behavior of
drugs[5657]. It has been estimated in terms of partition coefficient (log P) based on the amount
of 14 dispersed in biphasic system (noctanol/water) using the equation:
……………….. (Eq. 4)
The calculated log P values for14 are 1.24, 1.26, 1.42 and 1.45,respectively(Table S1).
Thelipophilicity of 4 and 3 are higher than 1 and 2which may be attributed to presence of greater
number of fluoro groups. The cytotoxicity increases with an increase in lipophilicity, therefore
cytotoxicity of 4 and 3 should be higher than 1 and2, which has further been validated by MTT
assay.
3.9. Density functional theory calculations.
Geometry optimization for all the complexes under study has been carried out at DFT level.
The rutheniummetal centre has been described by LANL2DZ basis set while non-metal atoms
using 6-31G**. The starting geometries were taken from single crystal X-ray data for4 and
subjected to optimization employing Gaussview 5.0 and resulting coordinates have been
energetically optimized. Calculated geometrical parameters such as bond lengths, bond angles
and bond energies are consistent with single crystal X-ray data (Tables S2). Contour plots for
some selected molecular orbitals of 1–4 were generated using Gauss view 5.0 and are shown in
(Fig. 5). DFT calculations on4 reproduced familiar “three-leg piano-stool” structures confirming
the optimized structures for13(Fig 6 and S12).
From the DFT calculation it is clear that electron density of highest occupied molecular
orbital (HOMO) is localized primarily on Ru atom, dipyrrin, and Cl− ion whereas lowest
13

unoccupied molecular orbital (LUMO) mainly on dipyrrin and to some extent on phenyl unit.
The calculated HOMO energies of the complexes are in the order of 1 (−5.22) <2(−5.11)
>3(−5.35) <4 (−5.22 eV) and those for LUMO exhibited the following trend: 1(−2.01) ~2(−2.01)
>3(−2.25) ~4(−2.25 eV). On the basis of DFT calculations and frontier molecular orbital theory,
it is well established that DNA molecule acts as an electron donor and intercalated complex as
electron acceptor [30, 5859].In this regardenergy and population of LUMO of1–4 could be
considered as key factors for affecting DNA binding. As the number of fluoro substituent
increases from 12 to 34 the LUMO energy of the complexes decreases which favours transfer
of electron cloud from the HOMO of DNA towards LUMO of the complexes, indicating stronger
interactions [6064].The difference between the HOMO–LUMO in 1 (−), 2 (−), 3
(−), and 4 (−2.97eV) are almost identical. At the basis of LUMO energy, the order of
interaction of the complexesis4 ~ 3 > 2 ~1. The results arein good agreement with the
experimental data and also number of important factors such as lipophilicity, H-bonding, steric
bulk, rotational motion etc.which can also affect their biological activity [59,61].
3.10. Molecular docking investigation on the interaction of DNA with 14
Molecular docking has been established as an effective tool for rational drug designing
through recognition of the drug–DNA interactions. The mechanism of action of reactants is
ascertained by placing a small molecule into binding site of the target specific region of DNA,
mainly in a noncovalent fashion, however a covalent bond may also be formed between the
reactants. Binding mode varies depending upon structural properties of the reactants and
molecular shape is believed to be the most important factor governing it. Earlier reports suggest
that DNA–intercalator complex is stabilized via van der Waals forces, hydrogen bonding,
hydrophobic charge transfer and electrostatic complementarity[31, 65].To predict preferred
binding site along with orientation of the ligands inside the DNA minor groovedocking
studiesusing 14 were performed with DNA duplex of the sequence d(CG-CGAATTCGCG)2
dodecamer (PDB ID: 1BNA) [31]. Resulting model shows that compounds 14recognise DNA
in minor groove situated in the GC rich region via electrostatic interaction. The planarity of
dipyrrin core is comfortable for strong ππ stacking interactions which fits inside the DNA
strands via van der Waals interaction and hydrophobic contacts (Fig. 7 and S13S14, SI)
[31,65].Moreover, fluorine atoms of 4 may be engaged in hydrogenbonding interactions with
14

DNA nucleobases available in the minor grooves in GC rich region (Fig. S15). The docked
structures for 14 with 1B-DNA exhibited relative binding affinity (248.14, 1; 249.34, 2;
256.68, 3;263.57, 4;eV) indicating 4 have better binding capability relative to other complexes
under investigation. These results are in good agreement with the experimentalobservations.
3.11. Determination of IC50 value.
The cytotoxicity of ligands and complexes has been studied by MTT colorimetric assay
against lung cancer cell line (A549). All the complexes induced cytotoxic response in a
concentration dependent manner. On the basis of this assay, 4 exhibited the highest toxicity or
lowest IC50 value followed by complex 3, whereas 2 and 1 displayed poor toxicity and anti-
proliferative activity against A549 cells. However, ligands did not show any toxicity up to
100µM concentration. The IC50 values are 20, 4; 6575, 3 and >100 µM2and1.On the basis of
cytotoxicity it can be arranged as 4>3>2 > 1(Fig.8) (Table S3). It is clearfrom the IC50 values
that complex 4 is most efficient cytotoxic drug in comparison to others. Thus we have attempted
further studies like changes in nuclear morphology, cell cycle analysis with the complex 4 only.
3.12. Change in nuclear morphology of cells.
Nuclear fragmentation is one of the prime features of apoptotic cell death.To investigate
the cell death by apoptosis and changes in nuclear morphology, DNA binding dyes Hoechst and
PI were used to investigate nucleus morphology of the control and treated cells.Hoechst is
permeable to cell membrane and typically used to stain the nucleus of live/dead cells. Hoechst
binds to the minor groove of double strand DNA preferably at AT rich regions and stains nucleus
in blue colour.PI is usually used to identify the dead cells and it is membrane impermeable and
gets excluded from viable cells. PI binds to DNA by intercalating between the bases without any
sequence preference and gives red colour at respective wavelength.
In control cells, almost uniform level of blue fluorescent nuclei were seen which indicate
that all cells are live and healthy. In treated cells, dead ones could be clearly visualized due to
toxicity of the drugs and cell membrane disruption.At increasing concentrations of 4 early
apoptotic cells with deep blue fluorescence (indicated with yellow circle), late apoptotic cells
with fragmented nuclei (indicated with blue circle) and necrotic dead cells with red fluorescence
(indicated with red circle) were clearly observed. At IC50 value (20 µM) of complex 4
15

fragmented nuclei were clearly observed and it indicates that at this concentration above
complex induced apoptosis very efficiently in A549 lung cancer cells (Fig.9).
3.13. Cell cycle analysis.
Cell death and cell cycle delay on the basis of DNA content, fluorescence activated cell
sorting (FACS) was done with PI staining (Fig. 10). Cells were treated at three different
concentrations (15, 20 and 25µM) of complex 4. According to FACS histogram statistics,
percentage of sub-G1 (apoptotic cell death) cells significantly increased from 2.67% to 32.8% in
treated group as compared to control. Whereas at IC50 value, significant increase in G2/M phase
of the cell cycle 18% to 25.57% were observed. On the other hand, in G1 phase cells were
decreased from 71.67% to 38% while S phase was almost constant in control and treated group.
Histogram statistics of FACS clearly indicated that complex 4 potentially induced apoptosis
(increase in sub G1) as well as cell cycle delay (increase in G2/M) in A549.
4. Conclusions
In conclusion through this work a new set of organometallic complexes have been synthesized
and thoroughly characterized by various physicochemical techniques. The DNA binding affinity
of the complexes has been followed by spectrophotometric methods. The results supported
interaction of the complexes with CT DNA through intercalation. Further, molecular docking
studies suggest that these complexes bind with minor groove of DNA. Moreover, all the
complexes (14) exhibited significant cytotoxicity toward A549 cell line with best performance
from 4, showing lowest IC50 value, prominent blebbing at low concentration (c, 20 M).
Acknowledgements
Authors gratefully acknowledge financial support from the Department of Science and
Technology (DST), New Delhi, India for providing financial assistance through the scheme
[SR/S1/IC-25/2011] and also to CSIR, New Delhi, India for the award of a senior Research
Fellowship to RPP (No. 09/013(0514)/2013-EMR-I). We are also thankful to the Head,
Departments of Chemistry and Zoology,Institute of Science, Banaras Hindu University, Varanasi
(U.P.) India, for extending laboratory facilities.We are also thankful to Prof. R. Murugavel (IIT
16

Bombay) for the access to his Single Crystal X-ray Diffraction Facility established through a
DAE-SRC Outstanding Investigator Award.
Supporting Information
13
1H, C NMR, ESI-MS, UVvis titration curves, fluorescence spectra, and Tables S1S3 are
provided.CCDC No. 1448655(4) contains the supplementary crystallographic data for this paper.
It can be had free of charge via https://summary.ccdc.cam.ac. uk/structure-summary-form (or
from the CCDC, 12 Union Road, Cambridge CB2 1EZ, U.K.; Fax: +44−1223−336033; Email:
deposit@ccdc.cam.ac.uk).
17

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Table 1. Crystal data and structure refinement parameters for 4
Empirical formula
C27H24ClF5N2Ru
Monoclinic
P21/n
Crystal system
Space group
a (Å)
7.3790(18)
14.783(4)
23.820(6)
90.00
b (Å)
c (Å)
α (deg)
β (deg)
92.972(4)
90.00
(deg)
V (Å3), Z
2594.9(11), 4
0.71075
λ (Å)
Colour and habit
T (K)
Red, needle
150(2)
reflns collected
refins/restraint/params
Dcalcd (Mg m-3)
µ (mm-1)
19435
4562/0/325
1.647
0.762
GOF on F2
final R indices I>2σ(I)
1.149
R1 = 0.0486
wR2 = 0.0971
R1 = 0.0522
wR2 = 0.0990
R indices (all data)
22

Table 2 Selected bond lengths (Å) and angles (˚) for 4
Bond
4
Bond
4
length (Å)
RuN1
Angle (˚)
N2Ru1N1
2.0628
2.0715
2.4134
1.6738
2.182
83.87
87.24
RuN2
RuCl1
RuCg
RuCav
N2Ru1Cl1
N1Ru1Cl1
CgRuCl1
CgRuN1
CgRuN2
88.72
126.87
127.71
128.28
Cg= metal centroid bond distance, Cav = average metal-carbon bond distance.
23

Fig. 1. ORTEP views of 4 at 30% thermal ellipsoid probability (Solvent molecule and H-atoms
have been omitted for clarity).
Fig. 2. Electronic spectra of ligand and complexes 14 in ((EtOH:H2O, c, 10 M; 1:1, v/v; pH
~7.3).
24

Fig. 3.Absorption titration spectra of 4 (EtOH:H2O, c, 10 M; 1:1, v/v; pH ~7.3) in the absence
(red line) and presence (other lines) of CT DNA (120 M) at room temperature. Arrow shows
the changes in absorption intensity upon increasing the CT DNA concentration.
Fig. 4. Emission spectra of EB (dotted black line) EB bound to CT DNA (Red solid line) and in
presence (other lines) of 4 (withincreasing amounts 050 μM). [EB] = 10 μM, [CT DNA] = 10
μM.
25

COM.
HOMO
LUMO
1
2
3
4
Fig.5. Frontier MOs contour plots of complex 14.
26

Fig. 6.DFT optimised structure of complex 4
27

Fig. 7. Molecular docked model of complex 4 with DNA (PDB ID: 1BNA).
Fig. 8.Cell viability and antiproliferation profile of complex 14 against A549 lung cancer cells
after drug treatment for 24 h by MTT assay.
28

Fig. 9. Images of control and drug treated A549 lung cancer cells with Hoechst and PI staining
after 24 h of incubation with three different concentrations of complex 4. Early apoptotic cells
indicated with yellow circle, late apoptotic cells with fragmented nucleus indicated with blue
circle and necrotic dead cells with red fluorescence indicated with red circle.
29