Half Sandwich Rhodium(III) and Iridium(III) Complexes as Cytotoxic and Metallonuclease Agents

Article abstract of DOI:10.1007/s12010-018-2835-y

Half sandwich complexes of the type [(η5-C₅Me₅)M(L^1–3)Cl]Cl.2H₂O were synthesized using [{(η⁵-C₅Me₅)M(μ-Cl)Cl}₂], where M = Rh(III)/Ir(III) and L^1–3 = pyrim

Full text of DOI:10.1007/s12010-018-2835-y

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https://doi.org/10.1007/s12010-018-2835-y
Half Sandwich Rhodium(III) and Iridium(III) Complexes
as Cytotoxic and Metallonuclease Agents
Pankajkumar A. Vekariya1 & Parag S. Karia1
Bhupesh S. Bhatt1 & Mohan N. Patel1
&
Received: 21 April 2018 /Accepted: 2 July 2018
#
Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract Half sandwich complexes of the type [(η5-C5Me5)M(L1–3)Cl]Cl.2H2O were syn-
thesized using [{(η5-C5Me5)M(μ-Cl)Cl}2], where M = Rh(III)/Ir(III) and L1–3 = pyrimidine-
based ligands. The complexes were characterized by spectral analysis. DNA interaction studies
by absorption titration and hydrodynamic measurement and suggest intercalative mode of
binding of complexes with CT-DNA. The molecular docking study also supports intercalation
of the complexes between the stacks of nucleotide base pairs. The gel electrophoresis assay
demonstrated the ability of the complexes to interact and cleave plasmid DNA. Minimum
inhibitory concentrations (MIC) of the complexes were investigated by the microdilution broth
method. The cytotoxic properties of the metal complexes were evaluated using brine shrimp
lethality bioassay.
.
.
.
Keywords Half sandwich compounds Rh(III)/Ir(III) metal complexes NMR Molecular
docking
Introduction
The discovery of chemical nuclease activity of transition metal complexes and exploring their
application in antineoplastic medication, molecular biology, and bioengineering have become
hotspots in recent years [1, 2]. Transition metal complexes offer enormous scope for the design
of anticancer candidates due to the large diversity of structure, bonding modes, and the wide
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12010-018-
2835-y) contains supplementary material, which is available to authorized users.
* Mohan N. Patel
jeenen@gmail.com
Bhupesh S. Bhatt
bhupeshbhatt31@gmail.com
1
Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar, Gujarat 388120, India

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range of ligand substitution rates [3]. The anticancer activity of most of the transition metal
complexes is mainly due to the formation of metal-DNA adducts which interfere DNA
replication and results in cell death [4].
After discovery as an antitumor agent, cisplatin represents today one of the most successful
metal-based drugs in chemotherapy. The undesirable side effects and easily acquired drug
resistance of platinum-based drugs stimulated tremendous research efforts in the field of
medicinal inorganic chemistry [5, 6]. It is expected that transition metal-based anticancer
drugs with mechanisms of action different from those of the platinum-based drugs would
demonstrate diminished side effects, less tendency to acquire drug resistance and the broader
spectrum of anticancer activities [7, 8]. Recently transition metal complexes received signif-
icant attention for their anticancer activity [7, 9–19].
Pyrimidine-based scaffolds have been explored for diverse biological applications in a
recent decade [20–22], but the literature lacks more information about their DNA interaction
and anticancer activities. So, exploring the application of pyrimidine-based metal complexes
could bring a major breakthrough in medicinal inorganic chemistry, and within this context,
herein, we report synthesis, characterization, and biological activities of half sandwich Rh(III)
and Ir(III) complexes with pyrimidine-based neutral bidentate ligand.
Experimental Section
Materials and Reagents
All the chemicals were of analytical grade. Solvents were dried and distilled prior to their use
by following standard procedure [23]. Dinuclear dichloro complexes [{(η5-C5Me5)M(μ-
Cl)Cl}2] (M = Rh/Ir) were prepared (Supply. 1) according to the literature method [24–26].
RhCl3.2H2O, IrCl3.2H2O, CT-DNA, bromophenol blue, acetic acid, and ethylenediaminetet-
raacetic acid disodium salt (EDTA) were purchased from S. D. Fine-Chem Limited, Mumbai,
India. Agarose, Luria broth, ethidium bromide, and Tris(hydroxymethyl)methylamine (Tris-
HCl) were purchased from Hi-media Laboratories Pvt. Ltd., India. Culture of pUC19 (MTCC
47), two Gram(+ve) i.e., Staphylococcus aureus (S. aureus) (MTCC–3160), Bacillus subtilis
(B. subtilis) (MTCC–7193) and three Gram(−ve) i.e., Serratia marcescens (S. marcescens)
(MTCC–7103), Pseudomonas aeruginosa (P. aeruginosa) (MTCC–1688), and Escherichia
coli (E. coli) (MTCC–433) bacterial species were purchased from the Institute of Microbial
Technology (Chandigarh, India).
Physical Measurements
Euro Vector EA3000 elemental analyzer (240) was used to analyze carbon, hydrogen, and
nitrogen content of the compounds. Thermogravimetric analysis and differential scanning
calorimetric study were performed with a model 5000/2960 SDTA, TA instrument (USA).
1
The H NMR spectra were recorded on a Bruker Avance (400 MHz). Infrared spectra were
recorded on an FT-IR Shimadzu spectrophotometer as KBr pellets in the range 4000 to
400 cm−1. In the range 800 to 200 nm, the absorption spectra of the complexes were recorded
on UV-160A UV–vis spectrophotometer, Shimadzu, Japan and 10-mm path length quartz cell.
The magnetic moments were measured by Gouy’s method using mercury
tetrathiocyanatocobaltate(II) as the calibrant (χg = 16.44 × 10−6 cgs units at 20 °C). The

Appl Biochem Biotechnol
diamagnetic correction was made using Pascal’s constant. Photo quantization of the gel after
electrophoresis was carried out on AlphaDigiDocTM RT, Version V.4.0.0 PC–Image software.
Synthesis
The reaction of 2-acetyl pyridine with different substituted aldehydes in 10% aq. NaOH and
30 ml methanol was carried out to yield corresponding chalcones. To a solution of guanidine
hydrochloride (1.5 equiv.) in 50 mL of absolute ethanol, potassium t-butoxide (1.1 equiv.) was
added. The reaction mixture was refluxed for 15 min and then the solution of chalcone (1.0
equiv.) in methanol was added to it and further refluxed for 6 h to afford the corresponding
pyrimidine derivatives, i.e., ligands Ln (Supply. 2). The progress of the reaction was measured
by TLC (40% Ethyl acetate/Hexane). The solvent was removed from the reaction mixture
under reduced pressure. Water was added and the aqueous phase was extracted with chloro-
form (3 × 20 mL). The organic phases were dried over anhydrous Na2SO4, filtered, and
concentrated. The crude product was purified by crystallization from ethanol or sometimes
by column chromatography (2% Methanol in chloroform) to afford the pure compounds. All
the synthesized compounds were well characterized by spectroscopic methods such as LCMS,
NMR, FT-IR, and elemental analysis.
4-(4-Fluorophenyl)-6-(pyridin-2-yl)pyrimidin-2-amine (L1)
Mp: 131–133 °C. Yield: 58%. FT-IR (KBr, 4000–400 cm−1): 3340 ν(NH2); 3059, 3003 ν(C–
H)ar; 1537, 1477 ν(C = C); 1361 ν(C = N); 1230 ν(C–F); 1361 (pyridine skeleton band); 840
1
δ(C–H). H NMR (CDCl3, 400 MHz) δ/ppm: 8.775–8.703 (4H, m, phenyl), 8.581 (1H, dd,
J = 2.0, 8.4 Hz, pyridyl), 8.145 (1H, s, pyrimidyl), 7.917 (1H, dd, J = 8.0, 8.8 Hz, pyridyl),
7.259 (1H, dd, J = 7.6 Hz, pyridyl), 7.076 (1H, dd, J = 2.8, 8.4 Hz, pyridyl), 6.645 (2H, s,
amine). 13C NMR (CDCl3, 100 MHz) δ/ppm: 163.61 (C), 158.93 (C), 155.76 (C), 154.56 (C),
153.60 (C), 148.27 (CH), 139.45 (CH), 132.58 (CH), 130.23 (C), 123.43 (CH), 121.46 (CH),
113.64 (CH), 104.34 (CH). Anal. Calcd. for C15H11FN4: C, 67.66; H, 4.16; N, 21.04. Found:
C, 67.64; H 4.15; N 21.06.
4-(4-Chlorophenyl)-6-(pyridin-2-yl)pyrimidin-2-amine (L2)
Mp: 154–156 °C. Yield: 69%. FT-IR (KBr, 4000–400 cm−1): 3325 ν(NH2); 3055, 3001 ν(C–
H)ar; 1535, 1473 ν(C = C); 1392 ν(C = N); 1091 ν(C–Cl); 1357 (pyridine skeleton band); 829
δ(C–H). 1H NMR (CDCl3, 400 MHz) δ/ppm: 8.824 (2H, dd, J = 2.4, 8.8 Hz, phenyl), 8.702 (2H,
dd, J = 2.4, 8.8 Hz, phenyl), 8.512 (1H, dd, J = 2.4, 7.2 Hz, pyridyl), 8.378 (1H, s, pyrimidyl),
8.092 (1H, dd, J = 8.0 Hz, pyridyl), 7.774 (1H, dd, J = 7.6 Hz, pyridyl), 7.340 (1H, dd, J = 2.4,
8 Hz, pyridyl), 6.639 (2H, s, amine). 13C NMR (CDCl3, 100 MHz) δ/ppm: 160.52 (C), 155.37
(C), 154.64 (C), 153.43 (C), 149.77 (CH), 142.68 (C), 138.31 (CH), 132.24 (C), 127.82 (CH),
124.65 (CH), 123.15 (CH), 121.47 (CH), 104.55 (CH). Anal. Calcd. for C15H11ClN4: C, 63.72;
H, 3.92; N, 19.82. Found: C, 63.70; H, 3.95; N, 19.80.
4-(4-Bromophenyl)-6-(pyridin-2-yl)pyrimidin-2-amine (L3)
Mp: 140–142 °C. Yield: 67%. FT-IR (KBr, 4000–400 Cm−1): 3325 ν(NH2); 3055, 3005 ν(C–
H)Ar; 1539, 1485 ν(C = C); 1361 ν(C = N); 1157, 825 (p–Substituted Aromatic Ring); 1009

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1
ν(C–Br); 786 ν(C–H). H NMR (CDCl3, 400 MHz) δ/Ppm: 8.752 (2H, Dd, J = 2.4, 7.6 Hz,
Phenyl), 8.701 (1H, S, Pyrimidyl), 8.510 (2H, Dd, J = 2.4, 8 Hz, Phenyl), 8.364 (1H, Dd, J =
2.4, 6.8 Hz, Pyridyl), 7.994 (1H, Dd, J = 8 Hz, Pyridyl), 7.623 (1H, Dd, J = 7.2 Hz, Pyridyl),
7.323 (1H, dd, J = 2.4, 8 Hz, Pyridyl), 6.651 (2H, S, Amine). 13C NMR (CDCl3, 100 MHz) δ/
Ppm: 160.71 (C), 158.12 (C), 155.73 (C), 153.83 (C), 150.12 (CH), 137.37 (CH), 133.19
(CH), 131.05 (CH), 130.03 (C), 124.79 (CH), 123.87 (CH), 123.11 (C), 105.24 (CH). Anal.
Calcd. For C15H11BrN4: C, 55.06; H, 3.39; N, 17.12. Found: C, 55.08; H, 3.32; N, 17.15.
Synthesis of Complexes
To a solution of the dinuclear dichloro complex [{(η5-C5Me5)M(μ-Cl)Cl}2] (0.20 mmol) in
CH2Cl2 (10 mL), an excess of the solid ligand (L1-L3) was added. The resulting mixture was
stirred at room temperature for 16 h. Then the solution was filtered through Celite in order to
remove solid particles, and the solvent was removed under reduced pressure. The residue was
dissolved in methanol (5 mL), and the product was precipitated by addition of diethyl ether
(30 mL), isolated by filtration, and dried in vacuo to obtain the corresponding complexes
(Scheme 1). The complexes are soluble in methanol, ethanol, acetone, dichloromethane,
chloroform, acetonitrile, dimethylformamide, and dimethyl sulphoxide but insoluble in petro-
leum ether and diethyl ether. The metal complexes were characterized by various analytical
and spectroscopic methods. The 1H-NMR, 13C-NMR, and IR spectra of metal complexes were
provided in the supplementary material (Supply. 3).
[(ƞ5-C5Me5)Rh(L1)Cl]Cl·2H2O (1)
Mp: 221–223 °C. Yield: 89%. FT-IR (KBr, 4000–340 cm−1): 347 ν(Rh–Cl); 779 δ(C–H);
1026, 848 (p–substituted aromatic ring); 1381, 1234 (pyridine skeleton band); 1481 ν(C=N);
1589 ν(C=Npyridyl); 1658 ν(C=C); 3070 ν(C–H)ar; 3441 ν(N-H). 1H NMR (DMSO,
400 MHz) δ/ppm: 8.801–8.723 (4H, m, phenyl), 8.624 (1H, dd, J = 2.0, 8.4 Hz, pyridyl),
8.423 (1H, s, pyrimidyl), 8.020–7.978 (1H, ddpoor resolved, pyridyl), 7.417–7.378 (1H,
ddpoor resolved, pyridyl), 7.235 (1H, dd, J = 2.8, 8.4 Hz, pyridyl), 6.678 (2H, s, amine),
1.597 (15H, s, Cp). 13C NMR (DMSO, 100 MHz) δ/ppm: 169.92 (C), 163.76 (d, J = 275 Hz,
C), 162.08 (C), 158.12 (C), 149.16 (CH), 135.00 (CH), 134.68 (CH), 130.80 (C), 128.81
(CH), 128.48 (CH), 126.30 (C), 114.94 (CH), 112.19 (CH), 94.74 (Cp), 8.36 (Cp). Anal.
Calcd. for C25H30Cl2FN4O2Rh: C, 49.12; H, 4.95; N, 9.16; Rh, 16.83. Found: C, 49.56; H,
4.65; N, 9.35; Rh, 16.95.
Scheme 1 Reaction scheme for the synthesis of complexes 1–6

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[(ƞ5-C5Me5)Rh(L2)Cl]Cl·2H2O (2)
Mp: 203–205 °C. Yield: 95%. FT-IR (KBr, 4000–340 cm−1): 378 ν(Rh–Cl); 779 δ(C–H);
1087, 840 (p–substituted aromatic ring); 1381, 1242 (pyridine skeleton band); 1473 ν(C=N);
1589 ν(C=Npyridyl); 1692 ν(C=C); 3070 ν(C–H)ar; 3443 ν(N-H). 1H NMR (DMSO,
400 MHz) δ/ppm: 8.860–8.742 (4H, m, phenyl) 8.590 (1H, dd, J = 2.4, 7.2 Hz, pyridyl),
8.456 (1H, s, pyrimidyl), 8.167–8.126 (1H, ddpoor resolved, pyridyl), 7.851–7.814 (1H,
ddpoor resolved, pyridyl), 7.422 (1H, dd, J = 2.4, 8 Hz, pyridyl), 6.675 (2H, s, amine),
1.596 (15H, s, Cp). 13C NMR (DMSO, 100 MHz) δ/ppm: 170.02 (C), 162.80 (C), 157.88
(C), 149.36 (CH), 142.65 (C), 134.94 (CH), 133.86 (CH), 131.85 (C), 129.34 CH), 124.51
(C), 123.33 (CH), 119.08 (CH), 115.88 (CH), 94.58 (Cp), 8.63 (Cp). Anal. Calcd. for
C25H30Cl3N4O2Rh: C, 47.83; H, 4.82; N, 8.92; Rh, 16.39. Found: C, 47.55; H, 4.62; N,
8.75; Rh, 16.58.
[(ƞ5-C5Me5)Rh(L3)Cl]Cl·2H2O (3)
Mp: 227–229 °C. Yield: 90%. FT–IR (KBr, cm–1): 357 ν(Rh–Cl); 786 δ(C–H); 1010, 835
(p–substituted aromatic ring); 1357, 1288 (pyridine skeleton band); 1481 ν(C=N); 1543,
ν(C=Npyridyl); 1674 ν(C=C); 3062 ν(C–H)ar; 3434 ν(N-H). 1H NMR (DMSO, 400 MHz)
δ/ppm: 8.812–8.764 (4H, m, phenyl), 8.576 (1H, dd, J = 2.4, 7.2 Hz, pyridyl), 8.432 (1H, s,
pyrimidyl), 8.207–8.167 (1H, ddpoor resolved, pyridyl), 7.731–7.695 (1H, ddpoor resolved,
pyridyl), 7.432 (1H, dd, J = 2.4, 8 Hz, pyridyl), 6.677 (2H, s, amine), 1.610 (15H, s, Cp). 13C
NMR (DMSO, 100 MHz) δ/ppm: 169.66 (C), 163.14 (C), 160.31 (C), 151.34 (CH), 135.16
(CH), 133.24 (CH), 130.14 (C), 129.69 (CH), 129.05 (CH), 127.44 (CH), 125.55 (C), 122.70
(C), 115.18 (CH), 94.08 (Cp), 8.69 (Cp). Anal. Calcd. for C25H30BrCl2N4O2Rh: C, 44.67; H,
4.50; N, 8.33; Rh, 15.31. Found: C, 44.13; H, 4.22; N, 8.19; Rh, 15.61.
[(ƞ5-C5Me5)Ir(L1)Cl]Cl·2H2O (4)
Mp: 242–244 °C. Yield: 96%. FT-IR (KBr, 4000–340 cm−1): 354 ν(Ir–Cl); 779 δ(C–H);
1034, 848 (p–substituted aromatic ring); 1381, 1234 (pyridine skeleton band); 1481 ν(C=N);
1540 ν(C=Npyridyl); 1597 ν(C=C); 3070 ν(C–H)ar; 3427 ν(N-H). 1H NMR (DMSO,
400 MHz) δ/ppm: 8.808–8.734 (4H, m, phenyl), 8.692 (1H, dd, J = 2.0, 8.0 Hz, pyridyl),
8.486 (1H, s, pyrimidyl), 8.194–8.153 (1H, ddpoor resolved, pyridyl), 7.496–7.456 (1H,
ddpoor resolved, pyridyl), 7.327 (1H, dd, J = 2.8, 8.4 Hz, pyridyl), 6.685 (2H, s, amine),
1.621 (15H, s, Cp). 13C NMR (DMSO, 100 MHz) δ/ppm: 170.80 (C), 164.81 (C), 162.77 (C),
158.58 (C), 150.20 (CH), 136.17 (CH), 134.97 (CH), 132.12 (C), 128.62 (CH), 127.47 (CH),
126.51 (C), 116.59 (CH), 113.89 (CH), 95.69 (Cp), 9.11 (Cp). Anal. Calcd. for
C25H30Cl2FIrN4O2: C, 42.86; H, 4.32; Ir, 27.43; N, 8.00. Found: C, 42.17; H, 4.52; Ir,
27.46; N, 8.15.
[(ƞ5-C5Me5)Ir(L2)Cl]Cl·2H2O (5)
Mp: 213–215 °C. Yield: 95%. FT-IR (KBr, 4000–340 cm−1): 351 ν(Ir –Cl); 779 δ(C–H);
1033, 840 (p–substituted aromatic ring); 1381, 1242 (pyridine skeleton band); 1481 ν(C=N);
1533 ν(C=Npyridyl); 1597 ν(C=C); 3062 ν(C–H)ar; 3421 ν(N-H). 1H NMR (DMSO,
400 MHz) δ/ppm: 8.877–8.757 (4H, m, phenyl), 8.596 (1H, dd, J = 2.4, 7.2 Hz, pyridyl),
8.481 (1H, s, pyrimidyl), 8.241–8.200 (1H, ddpoor resolved, pyridyl), 7.925–7.887 (1H,
ddpoor resolved, pyridyl), 7.437 (1H, dd, J = 2.4, 8.0 Hz, pyridyl), 6.681 (2H, s, amine),
1.631 (15H, s, Cp). 13C NMR (DMSO, 100 MHz) δ/ppm: 173.09 (C), 163.83 (C), 158.04 (C),
150.24 (CH), 143.19 (C), 135.66 (CH), 134.24 (CH), 132.44 (C), 130.09 CH), 125.25 (C),
124.25 (CH), 119.62 (CH), 116.09 (CH), 95.32 (Cp), 9.79 (Cp). Anal. Calcd. for
C25H30Cl3IrN4O2: C, 41.87; H, 4.22; Ir, 26.80; N, 7.81. Found: C, 41.77; H, 4.17; Ir, 26.74;
N, 7.75.

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[(ƞ5-C5Me5)Ir(L3)Cl]Cl·2H2O (6)
Mp: 219–221 °C. Yield: 92%. FT-IR (KBr, 4000–340 cm–1): 362 ν(Ir–Cl); 771 δ(C–H);
1030, 833 (p–substituted aromatic ring); 1381, 1242 (pyridine skeleton band); 1481 ν(C=N);
1
1589 ν(C=Npyridyl); 1627 ν(C=C); 3078 ν(C–H)ar; 3417 ν(N-H). H NMR (DMSO,
400 MHz) δ/ppm: 8.837–8.798 (4H, m, phenyl), 8.589 (1H, dd, J = 2.8, 7.6 Hz, pyridyl),
8.485 (1H, s, pyrimidyl), 8.254–8.214 (1H, ddpoor resolved, pyridyl), 7.797–7.758 (1H, ddpoor
resolved, pyridyl), 7.482 (1H, dd, J = 2.0, 8.0 Hz, pyridyl), 6.682 (2H, s, amine), 1.652 (15H, s,
Cp). 13C NMR (DMSO, 100 MHz) δ/ppm: 170.37 (C), 164.79 (C), 161.57 (C), 152.01 (CH),
135.42 (CH), 134.20 (CH), 131.51 (C), 130.34 (CH), 129.49 (CH), 128.73 (CH), 126.54 (C),
123.24 (C), 116.16 (CH), 95.29 (Cp), 9.27 (Cp). Anal. Calcd. for C25H30BrCl2IrN4O2: C, 39.43;
H, 3.97; Ir, 25.24; N, 7.36. Found: C, 39.34; H, 3.88; Ir, 25.63; N, 7.30.
Antibacterial Activity
To understand quantitatively the magnitude of the antibacterial activity, minimum inhibitory
concentrations (MIC) of ligands and metal complexes were investigated according to the
literature [27, 28].
DNA Binding Studies
UV-Visible Absorption
The binding constants of the metal complexes to CT-DNA determined by optical titrations at
room temperature were measured with 5 μM metal complex, and the CT-DNA concentration
was varied from 0 to 100 μM (5 mM Tris/HCl, pH 7.5) as reported in the literature [29–31].
Viscosity Measurement
Viscosity measurement was performed in the thermostatic viscosity bath maintained at 37
0.1 °C using Ubbelohde viscometer by measuring the flow time of DNA in Tris-HCl buffer,
pH 7.2 as per the reported procedure [32–34].
Molecular Docking Study
The rigid molecular docking studies on complexes 1–6 have been performed using HEX 8.0
software. The crystal structure of the B-DNA dodecamer d(CGCGAATTCGCG)2 was
downloaded from the Protein Data Bank (PDB ID: 1BNA) (http://www.rcsb.org/pdb). The
default parameters were used for docking calculation with correlation type shape only, FFT
mode at 3D level, grid dimension of 6 with receptor range 180, and ligand range 180 with twist
range 360 and distance range 40 [35, 36].
Chemical Nuclease Activity
For gel electrophoresis, 15 μL reaction mixture containing plasmid DNA in TE buffer (10 mM
Tris, 1 mM EDTA, pH 8.0) and 200 μM complex were incubated for 24 h at 37 °C; followed by
addition of 5 μL loading buffer (0.25% bromophenol blue, 40% sucrose, 0.25% xylene cyanole,
and 200 mM EDTA) for reaction quenching. The gel was stained with 0.5 μg/mL EtBr and was

Appl Biochem Biotechnol
photographed on a UV illuminator. The percentage of each form of DNA was quantified using
AlphaDigiDoc™ RT. Version V.4.0.0 PC–Image software. The degree of DNA cleavage activity
was expressed in terms of the percent of cleavage of the SC-DNA according to the following Eq. 1:
ꢀ
ꢁ
ð%of SC−DNAÞcontrol−ð%of SC−DNAÞsample
ð%of SC−DNAÞcontrol
%DNA cleavage activity ¼
 100
Brine Shrimp Lethality Bioassay
The cytotoxicity of complexes was studied on brine shrimp, Artemia cysts, according to the
method reported by Meyer et al. [37] for the series of complex concentrations, in vials
containing 1450 μL sea salt solution + 1000 μL sea salt containing ten nauplii + 50 μL
complex in 2% (V/V) DMSO. After 24 h of incubation, live and dead larvae were counted
and LC50 was determined for each complex.
Result and Discussion
Analytical and Spectral Characterization
The molar conductivities of half sandwich Rh/Ir complexes in DMSO (10−3 M) at room
temperature are observed in the range 81–96 Ω−1 cm2 mol−1, indicate the 1:1 electrolytic nature
of the complexes [38]. The magnetic moment values of synthesized complexes are found to be
zero, which indicates the absence of unpaired electron and diamagnetic nature of complexes. The
TGA curve of complexes shows weight loss up to 120 °C, indicates the presence of two water
molecules in the compounds as the water of crystallization [39]. The weight loss occurs from 320
to 600 °C corresponds to the loss of ligands and leaving behind the metal residue (Supply. 4).
The IR spectra of half sandwich complexes exhibit intense bands due to υ(C = Npyridyl) at
1533–1589 cm−1, in conjunction with other bands associated with dipyridyl moieties. The IR
spectral data support the coordination of the dipyridyl ligand and formation of the respective
complexes. The band in complexes at 3417–3443 cm−1 is attributed to the υ(N-H) stretching
vibration. The band around 3070 cm−1 are assigned to the υ(C-H)ar stretching of the ligands.
1H-NMR and 13C-NMR spectra of complexes indicate that the coordination of ligand results in
shifting of all peaks of heterocyclic ring in the downfield region. This suggests that coordina-
tion occurs through N atom of pyrimidine and pyridine ring. The pentamethylcyclopentadienyl
ligand gives rise to a characteristic singlet for all the methyl protons.
Antibacterial Activity
The in vitro antimicrobial activities of the complexes were screened against three Gram(−ve)
bacteria, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and two Gram(+
ve) bacteria, Bacillus subtilis and Serratia marcescens. The minimum inhibitory concentration
values of complexes indicate that all the complexes are active against all the five different
microorganisms compared to ligand and metal salt alone (Supply 5). The higher antimicrobial
activity of the metal complexes (MIC = 180–369 μM) compared to the metal salt (MIC >
1000 μM) can be the result of chelate effects, nuclearity of the metal center in the complexes,
nature of the ligands, and ion neutralizing the complexes [40].

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DNA Binding Studies
UV-Visible Absorption
Electronic absorption spectroscopy is one of the most widely used techniques to follow the
interaction of metal complexes with DNA. There are mainly two binding modes in the drug–
DNA interaction, i.e., covalent binding and non-covalent binding mode (intercalative, elec-
trostatic, and groove binding). Absorption titration study was performed to investigate the
binding mode and intrinsic equilibrium binding constant (Kb) for the complexes with CT-DNA
by monitoring the change in absorbance at various concentrations of DNA. DNA binding
constants (Kb) was calculated from the Eq. 1 using the extinction coefficient of the compounds,
the free complex concentration, and the ratio of bound complex per mole of DNA [41].
½DNAŠ
½DNAŠ
1
À
Á
À
Á
À
Á
¼
þ
ð1Þ
εa−εf
εb−εf
Kb εb−εf
where, [DNA] = concentration of DNA in base pairs, εa = extinction coefficient observed for the
MLCT absorption band at the given DNA concentration, εf = the extinction coefficient of the
complex in solution, and εb = the extinction coefficient of the complex when fully bound to
DNA. The plot of [DNA]/(εa − εf) vs [DNA] gave a slope 1/ (εa − εf) and intercept 1/Kb(εa − εf),
respectively. The intrinsic binding constant (Kb) is the ratio of the slope to the intercept [42].
The pronounced decreases in absorbance with bathochromic shifts of ~ 2 nm for the MLCT
maxima at a wavelength of around ~ 295 nm (Fig. 1) indicate that complex intercalates in
between the base pairs of DNA, the π* orbital of the intercalated ligand of the complex couples
with π orbital of the base pairs of DNA, thus decreasing the π-π* transition energy and
resulting in bathochromism. The Kb values of the complexes 1–6 are 9.48 × 105, 7.17 × 105,
4.66 × 105, 7.50 × 105, 5.85 × 105, and 3.55 × 105 M−1, respectively. The plot of [DNA]/
(εa − εf) vs. [DNA] of complexes 1–6 are shown in supplementary 6. These values are
comparable to the classical intercalator ethidium bromide (7.16 × 105 M−1) [43], and higher
than some metal complexes reported in literature [44–47].
Viscosity Measurement
The binding behavior was further confirmed by viscosity measurements. A classical
intercalative mode causes an increase in the DNA viscosity due to increasing of overall length
of DNA [48]. The relative viscosity of the DNA solutions increased progressively with an
increase in the concentrations of all the complexes (Supply 7), which suggests intercalation of
the complexes between the stacks of DNA base pairs and thus this study supports the UV-Vis.
absorption data. The intercalation of complexes into the DNA lengthens the DNA base stacks,
which results in an increase in the viscosity of the DNA solution.
Molecular Docking Study
DNA-binding is the critical step for the study of metal-based drugs, illuminating the mechanisms
involved in site-specific DNA recognition and designing new types of pharmaceutical molecules.
The binding behavior of the synthesized compounds toward DNA was explored both

Appl Biochem Biotechnol
Fig. 1 Absorption spectra of
complex 1 (5 μM) at 295 nm with
increasing amount of CT DNA (0–
60 μM) in Tris–HCl buffer (pH
7.2), with an incubation period of
10 min at room temperature. The
arrow shows absorbance change
with increasing DNA concentra-
tions Inset: plot of [DNA]/ (εa − εf)
vs. [DNA]
theoretically and experimentally with the aid of different procedures and techniques. The
theoretical evaluation includes the computational study of the interaction between compounds
and DNA duplex with sequence d(ACCGACGTCGGT)2. Docking was performed to prelimi-
narily predict the binding affinity and binding site with the sterically acceptable conformations.
Lower binding free energy implies a better binding interaction between the receptor (DNA) and
Bthe ligand^ molecules. The minimum energy docked pose clearly show minor groove with a
rich G-C region as the first interaction site of metal complexes as shown in Fig. 2, followed by the
intercalation of complexes in between the stacks of DNA base pairs as suggested by UV-Vis
absorption study and viscosity measurement. The interaction is the result of van der Waals forces
of attraction and hydrophobic contacts, and the binding energy or Docking scores obtained for
the interaction of metal complexes 1–6 with DNA duplex sequence are − 322.09, − 323.86, −
323.86, − 321.90, − 324.64, and − 324.64 kJ mol−1, respectively. The docked structure for
complexes 1–6 are shown in supplementary 8. The negative values of the binding free energy
of the docked complexes suggest reasonably binding of metal complexes with DNA.
Chemical Nuclease Activity
Gel electrophoresis experiment provides valuable information of plasmid DNA hydrolytic or
oxidative cleavage reaction by compounds [49]. The cleaved DNA molecule migrates in the
gel as a function of their mass, charge, and shape, with supercoiled DNA migrating faster than
an open circular molecule of the same mass and charge [50]. The plasmid cleavage influenced

Appl Biochem Biotechnol
Fig. 2 Molecular docked model of complex 1 located within the hydrophobic pocket of DNA (PDB ID: 1BNA)
by metal salt and complexes is shown in Fig. 3, and data of plasmid cleavage is presented in
Table 1. Here, all the complexes show the efficient cleavage ability (Supply 9). The similar
DNA cleavage activity was shown by half sandwich Rh(III) and Ir(III) complexes in the
literature [51]. The different DNA-cleavage efficiency of the complexes, metal salt, and drugs
is due to the difference in binding affinity of the complexes to DNA and the structural
dissimilarities of ligands [52]. This can be verified by the fact that complexes having larger
binding constant value as evaluated by UV-Vis absorption study, are better nuclease agents.
Also fluorine-containing ligands are better metallonuclease agents than ligands containing less
electronegative Cl and bulkier Br atoms.
Brine Shrimp Lethality Bioassay
Brine shrimp lethality bioassay is a recent development in the assay procedure of bioactive
compound, which indicates cytotoxicity as well as a wide range of pharmacological activities
(such as anticancer, antiviral, insecticidal, and pesticidal) of the compounds. From the data
(Table 1), the variation in lethality results may be due to the difference in the amount and kind
of cytotoxic substances and substituents. The complexes show good cytotoxic activity against
brine shrimps but this investigation is a primary one, and further tests are required to
investigate its actual mechanism of cytotoxicity and its probable effects on cancer cell line
Fig. 3 Photogenic view of cleavage of pUC19 DNA with metal salts and a series of complexes 1–6 using 1%
agarose gel containing 0.5 μM EB. All reactions were incubated in TE buffer (pH 8) at a final volume of 15 μL
for 3 h at 37 °C

Appl Biochem Biotechnol
Table 1 Biological screening of
the complexes
Complex
% Cleavage
LC50 (μg mL−1
)
1
2
3
4
5
6
85.99
84.84
83.18
85.48
83.31
81.40
5.75
6.45
7.58
5.88
6.91
7.76
and higher animal model. It suggests that the complexes can be used as potent cytotoxic agents
with the hope of adding an arsenal of weapons used against the fatal disease cancer.
Conclusions
Half sandwich metal complexes of pyrimidine-2-amine derivatives coordinated to Rh(III) and
Ir(III) have been synthesized and characterized by various analytical and spectral techniques.
NMR and FT-IR spectra suggest coordination of metal with N atom of pyridine and pyrimidine
ring of ligands. Antimicrobial screening of the complexes shows that complexes are more
potent antimicrobial agent than ligand and metal salt against all the microbes used for the
study. Absorption titration, viscosity measurement and docking studies suggest that all com-
plexes intercalate in between the stacks of DNA base pair. Such type of interaction is also
supported by docking study. Gel electrophoresis of plasmid DNA in the presence of complexes
evidenced metal complexes as potent chemical nucleases agents. From the above studies,
complex 1 emerges as the most potent metallonuclease agent among all complexes, which can
be attributed to its highest binding affinity with DNA. The highest nuclease activity and
binding constant value of complex 1 may be attributed to its least bulky nature and having
more electronegative F atom as a substituent ligand. The highest potency may be attributed to
Brine shrimp lethality bioassay which proposes metal complexes as potent cytotoxic agents.
Acknowledgements We are thankful to the Head, Department of Chemistry, Sardar Patel University, India for
making it convenient to work in a laboratory.
Funding Information This study was financially supported by the University Grant Commission for providing
under the scheme BUGC-CAS^ and BSR One Time Grant.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of interest.
References
1. Gubendran, A., Kesavan, M. P., Ayyanaar, S., Mitu, L., Athappan, P., & Rajesh, J. (2017). Non-enolisable
Knoevenagel condensate appended Schiff bases-metal (II) complexes: Spectral characteristics, DNA-
binding and nuclease activities. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy,
181, 39–46.

Appl Biochem Biotechnol
2. Liu, J.-B., Yang, C., Ko, C.-N., Vellaisamy, K., Yang, B., Lee, M.-Y., Leung, C.-H., & Ma, D.-L. (2017). A
long lifetime iridium(III) complex as a sensitive luminescent probe for bisulfite detection in living zebrafish.
Sensors and Actuators B: Chemical, 243, 971–976.
3. Gasser, G., Ott, I., & Metzler-Nolte, N. (2011). Organometallic Anticancer Compounds. Journal of
Medicinal Chemistry, 54(1), 3–25.
4. Johnstone, T. C., Suntharalingam, K., & Lippard, S. J. (2016). The next generation of platinum drugs:
targeted Pt(II) agents, nanoparticle delivery, and Pt(IV) prodrugs. Chemical Reviews, 116(5), 3436–3486.
5. Muhammad, N., Sadia, N., Zhu, C., Luo, C., Guo, Z., & Wang, X. (2017). Biotin-tagged platinum(iv)
complexes as targeted cytostatic agents against breast cancer cells. Chemical Communications, 53(72),
9971–9974.
6. Xu, G., Zhao, J., Gou, S., & Pang, J. (2015). Antitumor platinum(II) complexes of N-cyclobutyl-1R,2R-
diaminocyclohexane with dicarboxylates as leaving groups. Bioorganic & Medicinal Chemistry Letters,
25(2), 221–224.
7. Leonidova, A., & Gasser, G. (2014). Underestimated potential of organometallic rhenium complexes as
anticancer agents. ACS Chemical Biology, 9(10), 2180–2193.
8. Liu, Z., & Sadler, P. J. (2014). Organoiridium complexes: anticancer agents and catalysts. Accounts of
Chemical Research, 47(4), 1174–1185.
9. Tian, M., Li, J., Zhang, S., Guo, L., He, X., Kong, D., Zhang, H., & Liu, Z. (2017). Half-sandwich
ruthenium(ii) complexes containing N^N-chelated imino-pyridyl ligands that are selectively toxic to cancer
cells. Chemical Communications, 53(95), 12810–12813.
10. Hegde, D., Dodamani, S., Kumbar, V., Jalalpure, S., & Gudasi, K. B. (2017). Synthesis, crystal structure,
DNA interaction and anticancer evaluation of pyruvic acid derived hydrazone and its transition metal
complexes. Applied Organometallic Chemistry, 31(12).
11. Wang, X. L., Zheng, K., Wang, L. Y., Li, Y. T., Wu, Z. Y., & Yan, C. W. (2016). Synthesis and structure of a
new ternary monocopper(II) complex containing mixed ligands of 2,2′-diamino-4,4′-bithiazole and picrate:
in vitroanticancer activity, molecular docking and reactivity towards DNA. Applied Organometallic
Chemistry, 30(9), 730–739.
12. Nagaj, J., Kołkowska, P., Bykowska, A., Komarnicka, U. K., Kyzioł, A., & Jeżowska-Bojczuk, M. (2015).
Interaction of methotrexate, an anticancer agent, with copper(II) ions: coordination pattern, DNA-cleaving
properties and cytotoxic studies. Medicinal Chemistry Research, 24(1), 115–123.
13. Ahmadi, F., Saberkari, M., Abiri, R., Motlagh, H. M., & Saberkari, H. (2013). In vitro evaluation of Zn–
Norfloxacin complex as a potent cytotoxic and antibacterial agent, proposed model for DNA binding.
Applied Biochemistry and Biotechnology, 170(4), 988–1009.
14. Heydari, M., Moghadam, M. E., Tarlani, A., & Farhangian, H. (2017). DNA as a target for anticancer Phen-
Imidazole Pd(II) complexes. Applied Biochemistry and Biotechnology, 182(1), 110–127.
15. Vellaisamy, K., Li, G., Ko, C.-N., Zhong, H.-J., Fatima, S., Kwan, H.-Y., Wong, C.-Y., Kwong, W.-J., Tan,
W., & Leung, C.-H. (2018). Cell imaging of dopamine receptor using agonist labeling iridium(iii) complex.
Chemical Science, 9(5), 1119–1125.
16. Wang, W., Vellaisamy, K., Li, G., Wu, C., Ko, C.-N., Leung, C.-H., & Ma, D.-L. (2017). Development of a
long-lived luminescence probe for visualizing β-galactosidase in ovarian carcinoma cells. Analytical
Chemistry, 89(21), 11679–11684.
17. Liu, L.-J., Wang, W., Huang, S.-Y., Hong, Y., Li, G., Lin, S., Tian, J., Cai, Z., Wang, H.-M. D., & Ma, D.-L.
(2017). Inhibition of the Ras/Raf interaction and repression of renal cancer xenografts in vivo by an
enantiomeric iridium(iii) metal-based compound. Chemical Science, 8(7), 4756–4763.
18. Pouryasin, Z., Yousefi, R., Nabavizadeh, S. M., Rashidi, M., Hamidizadeh, P., Alavianmehr, M.-M., &
Moosavi-Movahedi, A. A. (2014). Anticancer and DNA binding activities of platinum (IV) complexes;
importance of leaving group departure rate. Applied Biochemistry and Biotechnology, 172(5), 2604–2617.
19. Yousefi, R., Aghevlian, S., Mokhtari, F., Samouei, H., Rashidi, M., Nabavizadeh, S. M., Tavaf, Z.,
Pouryasin, Z., Niazi, A., & Faghihi, R. (2012). The anticancer activity and HSA binding Properties of
the structurally related platinum (II) complexes. Applied Biochemistry and Biotechnology, 167(4), 861–872.
20. Saddik, A. A., El-Dean, K., Adel, M., El-Sokary, G. H., Hassan, K. M., Abbady, M. S., Ismail, I. A., &
Saber, S. H. (2017). Synthesis and cytotoxicity of some Thieno[2,3-d]pyrimidine derivatives. Journal of the
Chinese Chemical Society, 64(1), 87–93.
21. Gorle, S., Maddila, S., Chokkakula, S., Lavanya, P., Singh, M., & Jonnalagadda, S. B. (2016). Synthesis,
biological activity of pyrimidine linked with morpholinophenyl derivatives. Journal of Heterocyclic
Chemistry, 53(6), 1852–1858.
22. Mohana, K. N., Kumar, B. N. P., & Mallesha, L. (2013). Synthesis and biological activity of some
pyrimidine derivatives. Drug Invention Today, 5(3), 216–222.
23. Furniss, B. S. H., J, A., Smith, P. W. G., & Tatchell, A. R. V. (2004). Vogel’s textbook of practical organic
chemistry (5th ed.). London: ELBS and Longman.

Appl Biochem Biotechnol
24. White, C., Yates, A., Maitlis, P., & Heinekey, D. (2007). (η5-Pentamethylcyclopentadienyl)Rhodium and
Iridium Compounds Inorganic Syntheses, 29, 228–234.
25. Ball, R., Graham, W., Heinekey, D., Hoyano, J., McMaster, A., Mattson, B., & Michel, S. (1990). Synthesis
and structure of dicarbonylbis(.eta.-pentamethylcyclopentadienyl)diiridium. Inorganic Chemistry, 29(10),
2023–2025.
26. Kang, J. W., Moseley, K., & Maitlis, P. M. (1969). Pentamethylcyclopentadienylrhodium and -iridium
halides. I. Synthesis and properties. Journal of the American Chemical Society, 91(22), 5970–5977.
27. Fernandes, C., Horn Jr., A., Vieira-da-Motta, O., de Assis, V. M., Rocha, M. R., Mathias, L. d. S., Bull, É.
S., Bortoluzzi, A. J., Guimarães, E. V., Almeida, J. C. A., & Russell, D. H. (2010). Synthesis, character-
ization and antibacterial activity of FeIII, CoII, CuII and ZnII complexes probed by transmission electron
microscopy. Journal of Inorganic Biochemistry, 104(11), 1214–1223.
28. Patel, M., Chhasatia, M., & Bhatt, B. (2011). In vitro bacteriostatic and DNA interaction studies of drug-
based mixed-ligand complexes of cobalt(II). Medicinal Chemistry Research, 20(2), 220–230.
29. Reichmann, M. E., Rice, S. A., Thomas, C. A., & Doty, P. (1954). A further examination of the molecular
weight and size of desoxypentose nucleic acid. Journal of the American Chemical Society, 76(11), 3047–
3053.
30. Patel, M. N., Bhatt, B. S., & Dosi, P. A. (2012). Thermal, spectral, and thermodynamic studies for
evaluation of calf thymus DNA interaction activity of some copper(II) complexes. Journal of Thermal
Analysis and Calorimetry, 107(1), 55–64.
31. Shahabadi, N., Kashanian, S., Shalmashi, K., & Roshanfekr, H. (2009). DNA Interaction with PtCl2(LL)
(LL = Chelating Diamine Ligand: N,N-Dimethyltrimethylendiamine) Complex. Applied Biochemistry and
Biotechnology, 158(1), 1–10.
32. Bi, S., Zhao, T., Wang, Y., Zhou, H., Pang, B., & Gu, T. (2015). Spectrochimica Acta A, 150, 921–927.
33. Patel, M. N., Dosi, P. A., & Bhatt, B. S. (2012). Synthesis, characterization and biological activities of
ciprofloxacin drug based metal complexes. Acta Chimica Slovenica, 59, 622-631
34. Patel, M. N., Bhatt, B. S., & Dosi, P. A. (2012). Topoisomerase inhibition, nucleolytic and electrolytic
contribution on DNA binding activity exerted by biological active analogue of coordination compounds.
Applied Biochemistry and Biotechnology, 166(8), 1949–1968.
35. Arthi, P., Shobana, S., Srinivasan, P., Mitu, L., & Kalilur Rahiman, A. (2015). Synthesis, characterization,
biological evaluation and docking studies of macrocyclic binuclear manganese(II) complexes containing 3,
5-dinitrobenzoyl pendant arms. Spectrochimica Acta A, 143, 49–58.
36. Mehta, J. V., Gajera, S. B., & Patel, M. N. (2016). Design, synthesis and biological evaluation of pyrazoline
nucleus based homoleptic Ru(iii) compounds. Medicinal Chemistry Communications, 7(7), 1367–1380.
37. Meyer, B. N., Ferrigni, N. R., Putnam, J. E., Jacobsen, L. B., Nichols, D. E., & McLaughlin, J. L. (1982).
Brine shrimp: a convenient general bioassay for active plant constituents. Planta Medica, 45(05), 31–34.
38. Rani, S., Kumar, S., & Chandra, S. (2011). Synthesis, structural, spectral, thermal and antimicrobial studies
of palladium(II), platinum(II), ruthenium(III) and iridium(III) complexes derived from N,N,N,N-tetradentate
macrocyclic ligand. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 78(5), 1507–
1514.
39. Bellis, E., Hajba, L., Kovacs, B., Sandor, K., Kollar, L., & Kokotos, G. (2006). Three generations of α,γ-
diaminobutyric acid modified poly(propyleneimine) dendrimers and their cisplatin-type platinum com-
plexes. Journal of Biochemical and Biophysical Methods, 69(1-2), 151–161.
40. Dendrinou-Samara, C., Psomas, G., Raptopoulou, C. P., & Kessissoglou, D. P. (2001). Copper(II) com-
plexes with phenoxyalkanoic acids and nitrogen donor heterocyclic ligands: structure and bioactivity.
Journal of Inorganic Biochemistry, 83(1), 7–16.
41. Swavey, S., DeBeer, M., & Li, K. (2015). Photoinduced interactions of supramolecular Ruthenium(II)
complexes with plasmid DNA: synthesis and spectroscopic, electrochemical, and DNA photocleavage
studies. Inorganic Chemistry, 54(7), 3139–3147.
42. Asatkar, A. K., Tripathi, M., Panda, S., Pande, R., & Zade, S. S. (2017). Cu(I) complexes of
bis(methyl)(thia/selena) salen ligands: synthesis, characterization, redox behavior and DNA binding studies.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 171, 18–24.
43. Singh, R., Jadeja, R. N., Thounaojam, M. C., Patel, T., Devkar, R. V., & Chakraborty, D. (2012). Synthesis,
DNA binding and antiproliferative activity of ternary copper complexes of moxifloxacin and gatifloxacin
against lung cancer cells. Inorganic Chemistry Communications, 23, 78–84.
44. Shahabadi, N., Hadidi, S., & Taherpour, A. A. (2014). Synthesis, characterization, and DNA binding studies
of a new Pt(II) complex containing the drug Levetiracetam: combining experimental and computational
methods. Applied Biochemistry and Biotechnology, 172(5), 2436–2454.
45. Dehkhodaei, M., Sahihi, M., Rudbari, H. A., Gharaghani, S., Azadbakht, R., Taheri, S., & Kajani, A. A.
(2017). Studies of DNA- and HSA-binding properties of new nano-scale green synthesized Ni (II) complex

Appl Biochem Biotechnol
as anticancer agent using spectroscopic methods, viscosity measurement, molecular docking, MD simula-
tion and QM/MM. Journal of Molecular Liquids, 248, 24–35.
46. Lo, K. K. W., Chung, C. K., & Zhu, N. (2006). Nucleic acid intercalators and avidin probes derived from
luminescent cyclometalated iridium(III)-dipyridoquinoxaline and -dipyridophenazine complexes.
Chemistry-A European Journal, 12(5), 1500–1512.
47. Lo, K. K.-W., & Tsang, K. H.-K. (2004). Bifunctional luminescent Rhenium(I) complexes containing an
extended planar diimine ligand and a biotin moiety. Organometallics, 23(12), 3062–3070.
48. Shoair, A., El-Shobaky, A., & Azab, E. (2015). Synthesis, characterization, DNA binding and catalytic
applications of Ru(III) complexes. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy,
151, 322–334.
49. Rabindra, G., & Mahyoub, M. (2010). London AiT & First5 Committee: the voice of London General
Practice Trainees within the Royal College of General Practitioners (RCGP). London Journal of Primary
Care, 3(1), 58–58.
50. Dhanaraj, C. J., & Johnson, J. (2016). Transition metal complexes of a novel quinoxaline-based tridentate
ONO donor ligand: synthesis, spectral characterization, thermal,in vitropharmacological and molecular
modeling studies. Applied Organometallic Chemistry, 30(10), 860–871.
51. Gras, M., Therrien, B., Süss-Fink, G., Casini, A., Edafe, F., & Dyson, P. J. (2010). Anticancer activity of
new organo-ruthenium, rhodium and iridium complexes containing the 2-(pyridine-2-yl)thiazole N,N-
chelating ligand. Journal of Organometallic Chemistry, 695(8), 1119–1125.
52. Liu, Y.-J., Guan, X.-Y., Wei, X.-Y., He, L.-X., Mei, W.-J., & Yao, J.-H. (2007). Ruthenium(II) complexes
containing 2,9-dimethyl-1,10-phenanthroline and 4,4′-dimethyl-2,2′-bipyridine as ancillary ligands: synthe-
sis, characterization and DNA-binding. Transition Metal Chemistry, 33, 289–294.