Antimicrobial selectivity of ruthenium, rhodium, and iridium half sandwich complexes containing phenyl hydrazone Schiff base ligands towards B. thuringiensis and P. aeruginosa bacteria

Article abstract of DOI:10.1016/j.ica.2018.09.067

A series of new hydrazone mononuclear complexes of the type [(arene)MLCl]PF₆ (M = Ru, Rh, Ir) have been synthesized in this study. All these complexes were fully characterized with the help of FT-IR, UV–Vis, mass and NMR spectroscopy. The molec

Full text of DOI:10.1016/j.ica.2018.09.067

Accepted Manuscript
Research paper
Antimicrobial selectivity of ruthenium, rhodium, and iridium half sandwich
complexes containing phenyl hydrazone Schiff base ligands towards B. thurin-
giensis and P. aeruginosa bacteria
Agreeda Lapasam, Lincoln Dkhar, Nidhi Joshi, Krishna Mohan Poluri, Mohan
Rao Kollipara
PII:
S0020-1693(18)30733-3
https://doi.org/10.1016/j.ica.2018.09.067
ICA 18525
DOI:
Reference:
Inorganica Chimica Acta
To appear in:
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Revised Date:
Accepted Date:
11 May 2018
10 August 2018
20 September 2018
Please cite this article as: A. Lapasam, L. Dkhar, N. Joshi, K.M. Poluri, M. Rao Kollipara, Antimicrobial selectivity
of ruthenium, rhodium, and iridium half sandwich complexes containing phenyl hydrazone Schiff base ligands
towards B. thuringiensis and P. aeruginosa bacteria, Inorganica Chimica Acta (2018), doi: https://doi.org/10.1016/
j.ica.2018.09.067
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Antimicrobial selectivity of ruthenium, rhodium, and iridium half sandwich complexes
containing phenyl hydrazone Schiff base ligands towards B. thuringiensis and P. aeruginosa
bacteria
Agreeda Lapasama, Lincoln Dkhara, Nidhi Joshib, Krishna Mohan Polurib,
Mohan Rao Kolliparaa*
aCentre for Advanced Studies in Chemistry, North-Eastern Hill University, Shillong-793 022,
India.
bDepartment of Biotechnology and Center for Nanotechnology, Indian Institute of Technology
Roorkee, Roorkee-247667, Uttarakhand, India.
E-mail: mohanrao59@gmail.com; kmrao@nehu.ac.in
1

Abstract
A series of new hydrazone mononuclear complexes of the type [(arene)MLCl]PF6 (M =
Ru, Rh, Ir) have been synthesized in this study. All these complexes were fully characterized
with the help of FT-IR, UV-Vis, mass and NMR spectroscopy. The molecular structures of
representative complexes (1, 2, 7 and 8) were established by single crystal X-ray diffraction
study. The molecular structures of the complexes revealed typical piano stool geometry around
the metal center in which the ligand acts as NNʹ donor chelating ligand. In the case of complexes
(1-3), the ligand coordinates to the metal by using both the pyridine nitrogen atoms forming a six
membered metallocycle whereas in complexes (4-9) one hydrazone nitrogen atom and one
pyridine nitrogen atom coordinates to the metal ion forming a five membered metallocycle.
These compounds were evaluated for their in vitro antibacterial activity by agar well diffusion
method against two Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, and two
Gram-positive bacteria Staphylococcus aureus, Bacillus thuriengiensis. Results show that all the
complexes inhibit the growth of bacteria.
Keywords: ruthenium, rhodium, iridium, hydrazone, antibacterial.
2

Introduction
Antimicrobials are one of the most significant weapons in fighting bacterial infections [1-
2]. Antibacterial substances therefore are of great importance and necessity in treating infectious
diseases caused by pathogenic bacteria [3-8]. However, the wide use of antibiotics caused
pathogenic bacteria to grow increasingly resistant to commercially available antimicrobial
agents, resulting in poor treatment efficacy and significant economic losses [9-11]. The
increasing cases of microbial resistance pose a major concern to the scientific community and
have become a threat for human life worldwide [12]. To overcome these problems, the
development of new and safe antimicrobial agents with better effectiveness is required day by
day. Therefore, the search for antimicrobials is a never-ending task. Now a day a number of
hydrazone derivatives have been developed and evaluated for their antibacterial and antifungal
activity [13]. Hydrazone derivatives also draw considerable attention due to their extensive
applications in pharmaceutical and biological activities like anticancer [14], anti-inflammatory
[15], antimalarial [16], antitubercular [17] activities. In previous studies, metallo elements such
as, copper, cobalt, nickel, zinc and ruthenium, have been investigated because of their small size,
comparatively high nuclear charge and consequently great ability to form coordination
compounds [18-21]. But among the transition metal complexes, ruthenium-based complexes have
been widely studied and displayed significant biological activity. This can be due to their ability to
strongly bind nucleic acids and proteins, ligand exchange kinetics similar to those of their platinum
counterparts, the prevalence of two main oxidation states (II and III) and the iron-mimicking property
when bound to biological molecules [22-28]. In addition, both the commonly accessible oxidation
states of ruthenium are octahedral and relatively inert and moreover the synthetic chemistry is very
well established [27]. Over the last decade their therapeutic potential as anticancer and antimicrobial
3

agents have been demonstrated that may have advantages, such as minimal side effects and
immunity to drug resistance [29-32].
Nevertheless, Cp*Rh and Cp*Ir complexes have also been considered as alternatives to
ruthenium based drugs mainly because of their water solubility and inert facial co-ligand Cp*
which allows half-sandwich complexes to be easily tailored for catalytic or biological
applications. This has led to a growing interest in the chemistry of pentamethylcyclopentadienyl
complexes of the type [Cp*M(L)Cl]2+ (M = Rh/Ir, L a chelating ligand). These complexes have
also been employed as catalysts for various organic reactions namely C-H activation, oxidation
of alcohols, reduction of ketones and water oxidation. Rhodium(III) and iridium(III) complexes
with nitrogen containing ligands have also been used as antibacterial as well as antitumor agents
[33].
In our present work we report the synthesis of ruthenium, rhodium and iridium half-
sandwich hydrazone complexes and their antibacterial activity have also been studied. Ligands
used in this study are shown in Chart-1.
2. Experimental Section
2.1. Physical methods and materials
The reagents were of commercial quality and used without further purification. Di(2-
pyridyl) ketone, α-phellandrene, pentamethylcyclopentadiene, 2- benzoyl pyridine and phenyl
hydrazine were purchased from Sigma-Aldrich. 2,4 Di-Nitro phenyl hydrazine was obtained
from Alfa Aesar. The syntheses of all the metal complexes were performed without using any
inert atmosphere. All solvents used for syntheses were dried and distilled prior to use according
to standard procedures [34]. Starting compounds [(p-cymene)RuCl2]2 and [Cp*MCl2]2 (M =
Rh/Ir) were prepared according to literature methods [35-36]. Infrared (IR) spectra (400-4000
4

cm-1) were recorded on a Perkin-Elmer 983 spectrophotometer with compounds being dispersed
as KBr discs. 1H NMR spectra were recorded on a Bruker Avance II 400 MHz instrument using
CDCl3 and DMSO-d6 as solvents chemical shifts were referenced to TMS. Mass spectra were
obtained from Waters ZQ 4000 mass spectrometer by ESI method using acetonitrile as solvent.
Absorption spectra were recorded on a Perkin-Elmer Lambda 25 UV/Vis spectrophotometer in
the range of 200-600 nm at room temperature in acetonitrile. Elemental analyses of the
complexes were performed on a Perkin-Elmer 2400 CHN/S analyzer.
2.2. Structure determination by X-ray crystallography
Crystals suitable for X-ray analyses for complexes (1, 2, 7 and 8) were obtained by slow
diffusion of the solutions of the compounds in dichloromethane with a fourfold excess of hexane
and allowing them to stand undisturbed for three days. The single crystals of complexes were
attached to a glass fiber and placed into the Oxford Diffraction Xcalibur Eos Gemini
diffractometer. Single crystal X-ray diffraction data for the complexes were collected on an
Oxford Diffraction Xcalibur Eos Gemini diffractometer using graphite monochromated Mo-Kα
radiation (λ = 0.71073 Å). The strategy for the data collection was evaluated using the
CrysAlisPro CCD software. Crystal data were collected by standard ‘‘phi–omega scan’’
techniques and were scaled and reduced using CrysAlisPro RED software. The structures were
solved by direct methods using SHELXS-97 and refined by full-matrix least squares with
SHELXL-97 refining on F2 [37-38]. The metal atoms in the complex were located from the E-
maps and non-hydrogen atoms were refined anisotropically. The hydrogen atoms bound to the
carbon were placed in geometrically constrained positions and refined with isotropic temperature
factors, generally 1.2 Ueq of their parent atoms. The crystallographic and structure refinement
parameters for the complexes are listed in Table 1 and selected bond lengths and bond angles are
5

presented in Table 2. Figures 1-3 were drawn with ORTEP3 program whereas Figures S14-S17
was drawn using MERCURY 3.6 program [39].
2.3. In vitro antimicrobial assay
The effect of synthesized complexes on the broad spectrum of bacterial strains was
assayed by agar well diffusion method. In the present study, two-gram positive (Staphylococcus
aureus; Bacillus thuringiensis) and two-gram negative (Escherichia coli, Pseudomonas
aeruginosa) bacterial strains have been used. The agar nutrient broth media was prepared,
sterilized at 121°C for 15 min. The chosen bacterial strains were inoculated in nutrient broth and
incubated for overnight. Petriplates containing 30 ml of fresh Muller Hinton (MH) agar medium
were seeded with 24 hour grown culture of bacterial strains. Wells of 5 mm diameter were cut
and 100 µl of the each complex were added. The plates were then incubated at 37°C for 24
hours. The antibacterial activity was evaluated by measuring the diameter of the inhibition zone
formed around the well. Each experiment was performed in triplicate. For well diffusion assay 1
mg mL-1 concentration of complexes were used. Dimethylsulphoxide (DMSO) was used as a
negative control and the antibiotic gentamycin was applied as positive control drug.
2.3. Fluorescence studies on DNA interaction
Salmon milt DNA (SM-DNA) was used for DNA binding experiments. SM-DNA was
dissolved in 5 mM Tris–HCl and 50 mM NaCl buffer of pH 7.4. The concentration of
DNA/nucleotide was evaluated by measuring UV-absorption at the wavelength of 260 nm. The
fluorescence titration experiments of complexes were performed at a fixed concentration of
ligand L1, complex 1 and complex 2 metal complexes (20µM) with increasing concentration of
DNA (5-40 µM). The emission spectra of compounds were recorded at 390-600 nm by exciting
ligand L1, complex 1 and complex 2 at 375 nm, 380 nm and 365 nm respectively. All
6

fluorescence titration experiments were recorded at room temperature and the samples were
allowed to incubate for 15 min to attain thermodynamic equilibrium before spectral recording.
2.4. General procedure for preparation of ligands.
The ligands (L1-L3) were synthesized by modified literature methods [40-41] by mixing
equimolar amounts of di(2-pyridyl)ketone or 2-benzoylpyridine with phenyl hydrazine in
methanol and stirred for 30 minutes. To this solution three drops of acetic acid was added and the
reaction mixture was kept under reflux for 6 h. After cooling to room temperature the resulting
solids were filtered off, washed with ethanol and ether and dried in vacuum.
2.5. General procedure for synthesis of cationic complexes (1-3)
A mixture of starting metal precursor (0.1 mmol) and ligand di(2-pyridyl)ketone 2,4-
dinitrophenylhydrazone (L1) (0.2 mmol) and 2.5 equivalents of NH4PF6 were dissolved in dry
methanol (15 ml) and stirred at room temperature for 8 hours (Scheme-1). The solvent was
evaporated to dryness. The residue was then dissolved in dichloromethane and filtered through a
bed of celite to remove excess NH4Cl. The filtrate was concentrated to 1 ml and on addition of
excess hexane yellow solid precipitates out. The precipitate was further washed with diethyl
ether and dried in vacuum.
2.5.1. [(p-cymene)RuL1Cl] PF6 (1)
Yield: 87%; IR (KBr, cm-1): 3313 ν(N-H), 3160 ν(C-H),1614 ν(C=N), 1505, 1328 ν(N-O) 841 ν(P-F)
1
558 ν(P-F) (bending); H NMR (400 MHz) [(CD3)2SO] (ppm): 11.79 (s, 1H, NH), 9.31 (d,
(stretching)
2H, J = 8Hz), 7.97-8.14 (m, 4H), 7.64 ( t, 1H, J = 8Hz), 7.58 (d, 2H, J = 8Hz), 7.45 (d, 2H, J =
12Hz), 6.14 (d, 2H, J = 4Hz), 5.93 (d, 2H, J = 4Hz), 2.92-2.99 (sept, 1H), 2.28 (s, 3H), 1.22 (d,
6H, J = 8Hz); ESI-MS (m/z): 635.06 [M-PF6]+; UV-Vis {Acetonitrile, λmax, nm (ε/10-4 M-1 cm-
7

1)}: 230 (5.65), 365 (4.69). Anal. Calc. for C27H26ClN6O4RuPF6 (780.02): C, 41.57; H, 3.36; N,
10.77. Found: C, 41.68; H, 3.33; N, 10.91%.
2.5.2. [Cp*RhL1Cl]PF6 (2)
Yield: 83%; IR (KBr, cm-1): 3293 ν(N-H), 3109 ν(C-H),1618 ν(C=N), 1504,1342 ν(N-O) 843 ν(P-F)
(stretching) 558 ν(P-F) (bending); 1H NMR (400 MHz) [(CD3)2SO] (ppm) : 11.75 (s, 1H), 9.08 (d, 1H, J
= 8Hz), 8.46 (t, 1H, J = 8Hz), 8.17-8.39 (m, 5H), 8.01 ( t, 1H, J = 8Hz), 7.91 (t, 1H, J = 8Hz),
7.82 (t, 1H, J = 8Hz), 1.43 (s, 15H); ESI-MS (m/z): 637.01 [M-PF6]+; UV-Vis {Acetonitrile,
λmax, nm (ε/10-4 M-1 cm-1)}: 265 (2.58), 365 (3.72). Anal. Calc. for C27H27ClN6O4RhPF6
(782.86): C, 41.42; H, 3.48; N, 10.73. Found: C, 41.55; H, 3.50; N, 10.87%.
2.5.3. [Cp*IrL1Cl]PF6 (3)
Yield: 76%; IR (KBr, cm-1): 3295 ν(N-H), 3115 ν(C-H), 1617 ν(C=N), 1503,1331 ν(N-O), 843 ν(P-F)
(stretching) 558 ν(P-F) (bending); 1H NMR: (400 MHz) [(CD3)2SO] (ppm): 11.75 (s, 1H), 9.05 (d,
1H, J = 4Hz), 8.79 (d, 1H, J = 8Hz), 8.43 (t, 1H, J = 8Hz), 8.16-8.38 (m,5H), 7.97 (t, 1H, J =
8Hz), 7.89 (t, 1H, J = 8Hz), 7.78 (t, 1H, J = 8Hz), 1.39 (s, 15H); ESI-MS (m/z): 727.10 [M-
PF6]+; UV-Vis {Acetonitrile, λmax, nm (ε/10-4 M-1 cm-1)}: 260 (2.20), 350 (2.97). Anal. Calc. for
C27H27ClN6O4IrPF6 (872.18): C, 37.18; H, 3.12; N, 9.64. Found: C, 37.29; H, 3.07; N, 9.77%.
2.6. General procedure for synthesis of cationic complexes (4-6)
A mixture of starting metal precursor (0.1 mmol) and ligand 2-benzoylpyridine 2,4-
dinitrophenylhydrazone (L2) (0.2 mmol) were dissolved in dry methanol (15 ml) and stirred at
room temperature for 8 hours (Scheme-2). A yellow colored compound precipitated out from the
reaction mixture. The precipitate was filtered, washed with cold methanol and diethyl ether and
dried in vacuum.
2.6.1. [(p-cymene)RuL2Cl]Cl (4)
8

1
Yield: 81%; 3306 ν(N-H), 3117 ν(C-H), 1621 ν(C=N), 1513, 1329 ν(N-O); H NMR (400 MHz)
[(CD3)2SO] (ppm): 8.96 (d, 3H, J = 4Hz), 8.89 (s, 1H), 8.42 (d, 2H, J = 12Hz), 8.19 (d, 2H, J =
8Hz), 8.06 (t, 2H, J = 8Hz), 7.44 (d, 3H, J = 8Hz), 5.80 (d, 2H, J = 8Hz), 5.76 (d, 2H, J = 8Hz),
2.80 ( sept, 1H), 2.06 (s, 3H), 1.16 (d, 6H, J = 4Hz); ESI-MS (m/z): 634.52 [M-Cl]+; UV- Vis
{Acetonitrile, λmax, nm (ε/10-4 M-1 cm-1)}: 270 (2.94), 390 (2.53), 525 (1.28). Anal. Calc. for
C28H27Cl2N5O4Ru- (669.52): C, 50.23; H, 4.06; N, 10.46. Found: C, 50.38; H, 4.21; N, 10.54%.
2.6.2. [Cp*Rh L2Cl ]Cl (5)
Yield: 76%; IR (KBr, cm-1): 3303 ν(N-H), 3107 ν(C-H),1621 ν(C=N),1516,1350 ν(N-O) ; 1H NMR (400
MHz, CDCl3) (ppm): 11.20 (s, 1H), 9.03 (d, 1H, J = 4Hz), 8.32 (d, 1H, J = 8Hz), 8.17 (d,1H, J =
12Hz), 7.61-7.66 (m, 4H), 7.32-7.41 (m, 5H), 1.56 (s, 15H); ESI-MS (m/z):636.15 [M-Cl]+;
UV- Vis {Acetonitrile, λmax, nm (ε/10-4 M-1 cm-1)}: 265 (1.85), 375 (1.35).
2.6.3. [Cp*IrL2Cl]Cl (6)
Yield: 89%; IR (KBr, cm-1): 3305 ν(N-H), 3113 ν(C-H), 1623 ν(C=N) 1508,1341 ν(N-O); 1H NMR (400
MHz, CDCl3) (ppm) : 11.41 (s, 1H), 9.46 (s, 1H), 9.10 (d, 1H, J = 4 Hz), 8.33 (t, 1H, J = 12Hz),
8.26 (d, 1H, J = 8Hz), 7.84 (t, 2H, J = 8Hz), 7.65 (t, 2H, J = 8Hz), 7.50 (d, 2H, J = 4Hz), 7.44 (d,
1H, J = 8Hz), 7.41 (d, 1H, J = 8Hz); ESI-MS (m/z):741.71 [M-Cl]+; UV- Vis {Acetonitrile, λmax
,
-
nm (ε/10-4 M-1 cm-1)}: 235 (2.47), 375 (1.08). Anal. Calc. for C28H28Cl2IrN5O4 (689.04): C,
44.15; H, 3.71; N, 9.19. Found: C, 44.57; H, 3.68; N, 9.32%.
2.7. General procedure for synthesis of cationic complexes (7-9)
A mixture of starting metal precursor (0.1 mmol) and ligand 2 benzoyl pyridine phenyl
hydrazine (L3) (0.2 mmol) and 2.5 equivalents of NH4PF6 were dissolved in dry methanol (15
ml) and stirred at room temperature for 8 hours (Scheme-3). The solvent was evaporated to
dryness and the residue was dissolved in dichloromethane and filtered to remove excess NH4Cl.
9

The filtrate was then concentrated to 1 ml and addition of excess hexane resulted in the
precipitation of the compound. The precipitate was further washed with diethyl ether and dried in
vacuum.
2.7.1. [(p-cymene)RuL3Cl]PF6 (7)
Yield: 72%; IR (KBr, cm-1): 3298 ν(N-H), 3152 ν(C-H),1619ν(C=N), 844 ν(P-F) (stretching) 560 ν(P-F)
1
(bending); H NMR (400 MHz, CDCl3) (ppm): 9.29 (d, 1H, J = 4Hz), 8.30 (s,1H), 8.86 (t, 1H, J =
8Hz), 7.74 (t, 1H, J = 4Hz), 7.63 (t, 2H, J = 8Hz), 7.55 (d, 4H, J = 8Hz), 7.45 (t, 2H, J = 8Hz),
7.15-7.22 (m, 3H), 5.74 (d, 1H, J = 8Hz), 5.68 (d, 1H, J = 8Hz), 5.46 (d, 1H, J = 8Hz), 4.71 (d,
1H, J = 4Hz), 2.71-2.78 (sept, 1H), 2.04 (s, 6H), 1.16 (d, 3H, J = 4Hz), 1.08 (d, 3H, J = 8Hz) ,
ESI-MS (m/z): 544.20 [M-PF6]+; UV-Vis {Acetonitrile, λmax, nm (ε/10-4 M-1 cm-1)}: 245 (2.49),
390 (1.25).
2.7.2. [Cp*RhL3Cl]PF6 (8)
Yield: 64%; IR (KBr, cm-1): 3295 ν(N-H), 3114 ν(C-H),1621 ν(C=N) , 845 ν(P-F) (stretching) 560 ν(P-F)
(bending); 1H NMR (400 MHz, CDCl3) (ppm): 8.72 (d, 1H, J=8Hz), 8.31 (s, 1H), 7.93 (t, 1H), 7.80
(d, 2H, J = 8Hz), 7.71 (t, 1H, J = 8Hz), 7.63 (t, 1H, J = 8Hz), 7.52 (d, 2H , J = 8Hz), 7.47 (d, 2H,
J = 8Hz), 7.37 (t, 2H, J = 8Hz), 7.24 (d, 1H, J = 8Hz), 7.13 (t, 2H, J = 4Hz), 1.52 (s, 15H); ESI-
MS (m/z):546.23 [M-PF6]+; UV-Vis {Acetonitrile, λmax, nm (ε/10-4 M-1 cm-1)}: 260 (2.52), 355
(3.87). Anal. Calc. for C28H30ClN3RhPF6 (691.88): C, 48.61; H, 4.37; N, 6.07. Found: C, 48.73;
H, 4.52; N, 6.19%.
2.7.3. [Cp*IrL3Cl]PF6 (9)
Yield: 73%; IR (KBr,cm-1):3300 ν(N-H), 3118 ν(C-H), 1622 ν(C=N), 841 ν(PF6) (stretching) 561
1
ν(PF6) (bending); H NMR (400 MHz, CDCl3) (ppm): 9.29 (s, 1H), 8.75 (d, 1H, J = 4Hz), 7.98
10

(t, 1H, J = 8Hz), 7.73-7.80 (m, 9H), 1.52 (s, 15H); ESI-MS (m/z): 636.15 [M-PF6]+; UV- Vis
{Acetonitrile, λmax, nm (ε/10-4 M-1 cm-1)}: 245 (2.81), 265 (2.42), 320 (2.03), 420 (3.98).
3.
RESULT AND DISCUSSION
3.1. Synthesis of complexes
The mononuclear complexes (1-9) were synthesized by the reaction of precursor
complexes with the ligands L1-L3 in methanol as presented in schemes1-3. Complexes 1-3 and
7-9 were isolated as cationic complexes with PF6 as counter ion whereas complexes 4-6 were
isolated as cationic complexes with chloride as counter ion. All these metal complexes were
obtained in good yield and are non hygroscopic. The complexes were obtained as yellow solids
and were found to be soluble in common organic solvents like dichloromethane, acetone and
acetonitrile but insoluble in petroleum ether, hexane and diethyl ether. The synthesized
complexes were characterized by various spectroscopic techniques and the molecular structures
of some of the complexes were established by single crystal X-ray analysis.
3.2. Spectral studies of complexes (1-9)
The preliminary confirmation of the formation of complexes 1-9 can be justified to some
extent from their IR spectra. Complexes (1-9) exhibit characteristic bands around 3300 cm-1,
1619 cm-1 which correspond to the stretching frequencies of N-H and C=N respectively. A
strong band is also observed in the IR spectra of the complexes (1-6) in the region 1503–1516
cm-1, which correspond to the asymmetric stretching vibration νN-O while the symmetric
stretching vibration is observed in the region 1328–1350 cm-1. These bands appear in the lower
frequency regions compared to the free ligands L1-L3 where the ν(N-H), ν(C=N) are observed at
3400, 1638 cm-1 respectively suggesting the coordination of the ligands to the metal centers. In
11

addition, these complexes show a characteristic stretching and bending frequencies for P-F
counter anions around 844 cm-1 and 550 cm-1 respectively.
1
To reveal the coordination behavior of the ligands to metal in complexes the H NMR
analyses of the ligand and complexes were recorded in deuterated solvent at room temperature.
The 1H NMR spectra of complexes (1-9) display one singlet in the range 8.31-11.75 ppm for the
NH proton which shift to the down field region with respect to the free ligand indicating the co-
ordination of the ligand to the metal center. It may be mentioned that in few complexes the NH
signal is not observed due to solvent interaction. The protons signals of the pyridine ring are
observed as doublets in the downfield region around of 9.06-8.79 ppm indicating the co-
ordination of the pyridyl nitrogen to the metal center which arises due to the donation of lone
pairs of electron from the nitrogen atom to the metal center. In all the complexes the aromatic
proton signals associated with the ligands are observed in the range 8.47–7.04 ppm. Complexes
(1), (4) and (7) display a singlet and septet in the range 2.04-2.99 ppm corresponding to the
methyl group and methine protons of the isopropyl group of the p-cymene ligand. The signal for
the methyl protons of the isopropyl group is observed as one doublet in the range 1.07-1.21 ppm
for complexes (1) and (4) and two doublets for complex (7) instead of one doublet as observed in
the metal precursor. In addition, a singlet at 1.39-1.61 ppm is observed in complexes 2, 3, 5, 6, 8
and 9 corresponding to the methyl protons of the pentamethylcyclopentadienyl ring.
The mass spectra of all the complexes display the loss of counter ion which is quite
common on the case of half sandwich cationic complexes. Complexes (1-3 and 7-9) display their
molecular ion peaks at m/z: 635.06, 637.01, 727.10, 544.20, 546.23, and 636.15 respectively
which corresponds to the [M-PF6]+ ion, whereas complexes (4-6) display their molecular ion
peaks at m/z: 634.52, 636.15, and 741.71 which corresponds to the [M-Cl]+ ion. The peaks
12

corresponding to the loss of the ligands as well as the arene ring are not observed which indicates
the strong metal to ligand and metal to arene bond. The mass spectral values strongly justify the
composition and formulation of these complexes.
The UV–visible spectra of complexes (1-9) were recorded 20 μM acetonitrile solution in
the range 200-600 nm. All the ligands and complexes showed two bands around 260 and 375
nm. The higher energy band around 245-260 nm is attributed to n→π* and π→π* transitions
whereas the lower energy absorption band in the region 350-390 nm are ascribed to metal to
ligand charge transfer (MLCT) (Figure S13).
3.3. Single crystal X-ray structure determination of complexes:
The molecular structures of complexes (1, 2, 7 and 8) were established by single crystal
X-ray analysis. Because of low theta value the crystal structures of complexes (3, 4 and 6) are
presented here to only confirm the structures and compositions of the molecules. The crystals of
the complexes suitable for X-ray diffraction study were obtained by solvent diffusion method by
diffusing hexane into a saturated solution of the complexes in dichloromethane. Suitable single
crystals were attached to a glass fiber and transferred into the Oxford Diffraction Xcalibur Eos
Gemini diffractometer. The ORTEP representations of the complexes at 50% probability along
with the crystallographic numbering schemes are depicted in figures (1-3). Crystal data
parameters along with selected bond lengths and bond angles are given in the Tables 1 and 2
respectively. Complexes (1), (2), and (7) crystallized in triclinic crystal system with space group
1 whereas complex (8) crystallized in monoclinic with P121/n1 space group. The molecular
structures of all these complexes display typical three legged “piano stool” geometry around the
metal center which is common for ruthenium, rhodium and iridium half-sandwich compounds
[42, 43]. The metal center is coordinated to the p-cymene and pentamethylcyclopentadienyl in a
13

η6/η5 manner, which occupies one face of the octahedron, two nitrogen donor atoms from
chelating ligand in a bidentate N∩N fashion and one chloride [44]. The X-ray structure of the
complexes reveals the different coordination modes of the ligands to the metal ion. In complexes
(1-3) the ligand coordinates to the metal center through both the pyridine nitrogen atoms forming
a six membered chelate ring whereas in the case of complexes (4-9) the ligand coordinates to the
metal center through one hydrazone nitrogen and one pyridine nitrogen atom forming a five
membered chelate ring as evidenced from the molecular structure.
The metal-centroid bond distances in ruthenium complexes (1) and (7){ 1.68 Å} are
found to shorter than the metal-centroid bond distances in rhodium complexes (2) and (8){ 1.728
Å} as indicated in Table 2. The Ru–N and Ru–Cl bond distances in complex (1) and complex (7)
are found to be 2.07-2.09 and 2.38-2.39 Å respectively which are consistent with other
related system. Likewise, the Rh–N and Rh–Cl bond distances in complex (2) and (8) are found
to be in the range 2.09-2.12 and 2.39- 2.41Å respectively which are also in good agreement with
the reported complexes [45]. The bond angle values N1-M-N2 and N-M-Cl in these complexes
are very to close 90° which is comparable to the pseudo-octahedral geometry about the metal
center.
Furthermore, the crystal structure of complex (1) display two different types of
intermolecular hydrogen bonding; the first interaction is C-H∙∙∙O between the nitro - O and H-
atom from methyl group of p-cymene moiety (2.625 Å), the second C-H∙∙∙O between the nitro -
O and methine hydrogen of p-cymene ligand (2.407 Å) (Figure S14). Similarly the crystal
structure of complex (2) exhibits three different types of intermolecular interactions viz. C-H∙∙∙O
(2.674 Å) between the nitro - O and H-atom of pyridyl ring, C-H∙∙∙Cl (2.616 Å) interaction
between chloride atom and aromatic H atom from pyridyl ring and π∙∙∙π interaction (4.008Å)
14

between the two ring of phenylhydrazone group (Figure S15). Complex (7) displays π∙∙∙π (4.655
Å) interaction between the two rings of p-cymene moiety as presented in figure S16. The crystal
packing of complex (8) exhibit a π∙∙∙π (4.175 Å) interaction between two pyridine ring and one
C-H∙∙∙π (3.714 Å) interaction of one C-H and one pyridine ring (Figure S17).
Figure 1. ORTEP rendered views of complex (1) and complex (2) with 50% probability.
Hydrogen atoms except N-H and counter ion (PF6) are omitted for clarity.
15

Figure 2. ORTEP rendered views of complex (3), complex (4) and complex (6) with 50%
probability. Hydrogen atoms except N-H and counter ion Cl- are omitted for clarity. Because of
low theta value the crystal structure of these complexes are presented here to only confirm the
structure and composition of molecule.
16

Figure 3. ORTEP rendered views of complex (7) and complex (8) with 50% probability.
Hydrogen atoms except N-H and counter ion (PF6) are omitted for clarity.
3.4. In vitro antimicrobial assay
The starting metal precursor complexes, synthesized ligands and complexes were tested
for their activity against two Gram positive bacteria S. aureus and B. thuringiensis and two Gram
negative bacteria such as E. coli and P. aeruginosa taking gentamycin as a positive control. The
starting precursor of [(p-cymene)RuCl2]2 were found to be inactive as reported in the previous
literature [46]. We have carried out the antimicrobial activity of [Cp*RhCl2]2 and [Cp*IrCl2]2 but
none of them show any significant activity. This indicates that the antimicrobial activity is solely
attributed to the ligands and the metal-ligand complexes. The effectiveness of an antibacterial
agent in sensitivity testing is based on the size of the diameter of zones of inhibition against the
tested organism. The histograms of the resultant zone of inhibition are presented in Figure 4 and
Figure 5. The diameter of the zone is measured to the nearest millimeter, and the results are
17

summarized in Table 3. For Gram negative bacteria, it was found that all the complexes show
maximum antibacterial selectivity against P. aeruginosa. However, none of the complexes show
significant antimicrobial activity against E.coli bacteria.
Figure 4. Histogram of the zone of inhibition (mm) of the ligands (1-3) in comparison with
gentamycin. Where “*” indicates that compound exhibited no zone of inhibition. All the
complexes expressed standard deviation of error with in the ± 2 and ±1.
18

Figure 5. Histogram of the zone of inhibition (mm) of the complexes (1-9) in comparison with
gentamycin. Where “*” indicates that compound exhibited no zone of inhibition. All the
complexes expressed standard deviation of error with in the ± 2 and ±1.
Similarly, in the case of Gram positive bacteria it was found that all the complexes show
antibacterial selectivity against B. thuringiensis with significant activity. However, these
complexes did not show any noticeable activity for S.aureus. Among the ligands, L2 are more
effective in inhibiting the specific bacteria, L3 showed moderate activity whereas L1 did not
show any activity for all the selected bacteria. As evident from the results all p-cymene
ruthenium complexes showed good activity against P. aeruginosa and B. thuringiensis compared
to Cp*Rh and Cp*Ir complexes. However, complexes have not superseded the activity of the
standard gentamycin. These observations indicate that all the complexes are exclusive to
particular strains of both gram-positive and gram negative bacteria evidencing them as strong
selective antibacterial agents.
DNA binding studies of platinum group metal complexes
The biological activity of platinum group metal complexes such as half-sandwich
ruthenium, rhodium and iridium metal complexes has been partially attributed to their interaction
with DNA molecules [47]. In order to elucidate any such kind of interaction is present between
the newly synthesized complexes with DNA; we have performed titration experiments with
fluorescent metal complexes (Ligand 1, Complex 1 and complex 2) using SM-DNA as a
biomolecule. The emission spectra of these complexes on increasing amounts of SM-DNA do
not show a significant change in the spectral intensity or peak shift even up to a ratio of 1:2
(Figure. S18). Hence, these titration experiments clearly evidenced that none of the chosen
platinum group metal complexes containing phenyl hydrazone Schiff base ligands does not bind
19

to DNA, which is in contrast to Cisplatin and other known platinum group complexes that form
DNA adducts [48].
4. Conclusion
In summary, we have successfully synthesized a series of nine mononuclear complexes
of p-cymene ruthenium, Cp* rhodium and Cp* iridium with phenyl hydrazone derived ligands
L1, L2 and L3. The complexes were obtained in good yields. All the complexes were
characterized by various spectroscopic studies and X-ray analysis. Spectral studies strongly
support the formation of the compounds. The X-ray structure of the complexes reveals the
different coordination modes of the ligands to the metal ion. In complexes (1-3) the ligand
coordinates to the metal center through both the pyridine nitrogen atoms forming a six membered
chelate ring whereas in the case of complexes (4-9) the ligand coordinates to the metal center
through one hydrazone nitrogen and one pyridine nitrogen atom forming a five membered
chelate ring as evidenced from the molecular structure. All the nine complexes investigated in
the present study exhibited interesting selectivity in the antibacterial studies performed against
four pathogenic bacteria viz., S. aureus, E. coli, B. thuringiensis and P. aeruginosa with zone of
inhibition in the range of 13–21 mm. All the complexes exhibit selectivity for P. aeruginosa and
B. thuringiensis by showing better activity and it did not show any activity against the bacteria
E.coli and S.aureus.
Acknowledgements
Agreeda Lapasam thanks UGC, New Delhi, India for providing financial assistance in the
form of university fellowship (UGC-Non-Net). I thank DST-PURSE SCXRD, NEHU-SAIF,
Shillong, India for providing Single crystal X-ray analysis and other spectral studies.
20

Appendix A. Supplementary data
CCDC 1842504 (1), 1842505 (2), 1842506 (7) and 1842507 (8) contains the supplementary
crystallographic data for this paper. These data can be obtained free of charge via
www.ccdc.cam.ac.uk/data_request/cif, by e-mailing data_request@ccdc.cam.ac.uk, or by
contacting The Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ,
UK; Fax: +44 1223 336033.
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24

Chart 1: Ligands used in the present study
Scheme 1: Schematic representation of the synthesis of complexes (1-3).
25

Scheme 2: Schematic representation of the synthesis of complexes (4- 6).
Scheme 3: Schematic representation of the synthesis of complexes (7- 9).
26

Table 1. Crystal data and structure refinement details of complexes.
Complexes
1
C27 H26 Cl N6 O4 RuPF6
780.03
293 (2)
0.71073
2
7
8
C28 H30 Cl N3 Rh PF6
691.88
293.0 (2)
0.71073
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
C27 H27 Cl F6 N6 O4 P Rh C28 H29 Cl N3 Ru PF6
782.87
293(2)
0.71073
Triclinic
689.03
293.0 (2)
0.71073
Triclinic
Triclinic
'monoclinic'
'P 1 21/n 1'
' 1'
' 1'
' 1'
a (Å)/α (°)
b (Å)/β (°)
c (Å)/γ (°)
8.2067(3)/84.430(4)
13.1749(6)/87.712(3)
14.4250(7)/80.885(3)
1532.22(12)
8.9243(5)/92.281(3)
12.6858(5)/107.099(4)
14.7599(6)/97.993(4)
1575.86(13)
10.4195(5)/104.857(5)
10.8269(5)/94.486(5)
14.1435(10)/102.316(4)
1491.71(15)
11.3169(6)/90
8.6057(5)/96.606(4)
30.0275(15)/90
2905.0(3)
3
Volume (Å )
Z
2
2
1
4
-
3
1.691
1.650
1.534
1.582
Density (calc) (Mg/m )
Absorption coefficient (µ) 0.732
0.757
0.729
0.797
-1)
(mm
F(000)
784
788
696
1400
3
0.25 x 0.21 x 0.19
3.7890 to 25.0470
0.23 x 0.18 x 0.15
4.1600 to 26.3690
0.39 x 0.25 x0.23
4.0220 to 26.1770
0.25 x 0.16 x 0.12
3.8840 to 27.0480
Crystal size (mm )
Theta range for data
collection
Index ranges
-10<=h<=10,
-17<=k<=12,
-19<=l<=17
10665
-10<=h<=11,
-13<=k<=17,
-20<=l<=20
11090
-11<=h<=13,
-14<=k<=14,
-19<=l<12
10287
-14<=h<=15,
-10<=k<=11,
-40<=l<18
12195
Reflections collected
Independent reflections
6879 [R(int)=0.0393]
99.15
7137 [R(int)=0.0376]
99.41
4789 [R(int)=0.0340]
99.36
5318 [R(int)=0.0347]
99.04
Completeness to theta =
25.00°
Absorption correction
Semi-empirical from
equivalents
Semi-empirical from
equivalents
Semi-empirical from
equivalents
Semi-empirical from
equivalents
Refinement method
Full-matrix least-squares Full-matrix least-squares
Full-matrix least-squares Full-matrix least-
2
2
on F2
2
on F
on F
squares on F
6617/0/361
1.116
Data/restraints/parameters
6879/0/415
1.056
7137/0/415
1.036
6711/0/361
1.023
2
Goodness-of-fit on F
Final R indices
[I>2sigma(I)]
R1 = 0.0617, wR2 =
0.1265
R1 = 0.0598, wR2 =
0.1408
R1=0.0629, wR2=
0.1566
R1=0.0651, wR2 =
0.1351
R indices (all data)
R1 = 0.0873, wR2 =
0.1406
R1 = 0.0865, wR2 =
0.1591
R1=0.0895,
wR2=0.1786
R1=0.0864, wR2 =
0.1460
Largest diff. peak and hole 0.713 and -0.458
-3
0.924 and -0.747
0.983 and -0.704
0.678 and -0.695
(e.Å
)
CCDC No.
1842504
1842505
1842506
1842507
Structures were refined on F0 : wR2 = [Σ[w(F0 - Fc2)2] / Σw(F0 ) ] , where w-1
=
2
2
2 2 1/2
2
2
[Σ(F0 )+(aP)2+bP] and P = [max(F0 , 0)+2Fc2]/3
27

Table 2: Selected bond lengths (Å) and bond angles (°) of complexes
Complexes
1
2
7
8
Bond distances (Å)
M(1)-CNT
Bond distances (Å)
M(1)-CNT
1.689
1.784
1.686
1.787
M(1)-N(1)
2.099(4)
2.093(4)
2.096(3) M(1)-N(1)
2.11 9(4) M(1)-N(2)
2.074(4)
2.098(3)
2.097(4)
2.120(4)
M(1)-N(2)
M(1)-Cl(1)
2.394(1)
1.344(6)
2.399(1) M(1)-Cl(1)
2.384(2)
1.367(5)
2.412(1)
1.390(6)
N(3)-N(4)
1.344(7)
N(2)-N(3)
Bond Angles (°)
N(1)-M(1)-N(2)
N(1)-M(1)-Cl(1)
N(2)-M(1)-Cl(1)
Bond Angles (°)
N(1)-M(1)-N(2)
N(1)-M(1)-Cl(1)
N(2)-M(1)-Cl(1)
84.8(2)
85.9(1)
86.9(1)
86.2(1)
87.6(1)
87.1(1)
76.3(2)
82.4(1)
91.6(1)
76.0(1)
83.3(1)
91.4(1)
28

Table 3: Resultant zone of inhibition
Sl. No. Compounds
Bacterial Strains
Gram positive
Gram negative
E.coli
P. aeruginosa S.aureus
B. thuringiensis
+ve control Gentamycin
23±2
23±2
-
24±2
24±2
-
1
Ligand 1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
Ligand 2
19±2
15±1
21±2
18±2
17±1
20±2
15±1
16±2
18±2
17±1
19±1
21±1
14±2
17±1
19±2
17±2
20±1
13±1
16±1
21±2
18±1
19±1
3
Ligand 3
4
Complex 1
Complex 2
Complex 3
Complex 4
Complex 5
Complex 6
Complex 7
Complex 8
Complex 9
5
6
7
8
9
10
11
12
29