Biological assessment of substituted quinoline based heteroleptic organometallic compounds

Article abstract of DOI:10.1039/c6md00251j

A series of substituted quinoline derivatives were synthesized via the Friedl?nder condensation reaction, using substituted 2-aminobenzophenone with 2-acetyl pyridine or 2-acetyl thiophene in the presence of sodium methoxide. The series of substituted qui

Full text of DOI:10.1039/c6md00251j

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PAPER
Biological assessment of substituted quinoline based heteroleptic
organometallic compounds†
Jugal V. Mehta,a Sanjay B. Gajera,a Dilip B. Raval,b Vasudev R. Thakkarb and Mohan N. Patel*a
A series of substituted quinoline derivatives were synthesized via the Friedlӓnder condensation reaction using substituted
2-aminobenzophenon with a 2-acetyl pyridine or 2-acetyl thiophene in presence of sodium methoxide. A series of
substituted quinoline based organometallic ruthenium complexes of the type [(Cp*)Ru(Ln)Cl]• Cl (where, Cp*=
pentamethylcyclopentadienyl ring, Ln=L1-7= quinoline ligands) were synthesized and characterized by elemental analysis,
electronic spectra, conductance measurements, thermogravimetric analysis (TGA), Fourier transform infrared (FT-IR) and
mass spectrometry. By analogy with structure to a piano stool these are known as piano stool complexes, in which metal
centre being coordinated by pentamethylcyclopentadienyl ring, chlorido ligand and chelating quinoline ligand. All
compounds were studied for their in vitro antimicrobial activity against five unlike bacterial strain. All compounds were
investigated for their interaction with Herring Sperm (HS) DNA, developing absorption titration (Kb=0.34‒6.25×105 L mol-1)
and viscosity measurements. The studies conclude the classical intercalative mode of DNA binding. The DNA-binding
property of all the compounds were also examined theoretically using molecular docking study and it prove intercalation
binding mode between compounds and nucleotide base pairs of HS DNA. The capability of the compounds to cleave
pUC19 DNA was examined by chemical nuclease activity. The results indicate that the ruthenium complexes effectively
promote the cleavage of plasmid DNA than that of respective quinoline ligands. All compounds were evaluated for
cytotoxicity activity against S. pombe cells at a cellular level and exhibit enhanced activity against S. pombe cells compared
to the quinoline ligands. All synthesized compounds were also evaluated for their in vitro antimalarial activity [50%
Inhibition concentration (IC50) = 0.55‒1.84 mg/L] against Plasmodium falciparum strain as well as in vitro cytotoxic activity
[50% Lethal concentration (LC50) = 5.64–119.67 mg/L] against brine shrimp (Artemia cysts) eggs.
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Industrial, biological, and synthetic significance places this
scaffold in a significant position. Therefore, significant effort
continues to be directed toward the development of new
1. Introduction:
In view of the environmental, practical and economic issues,
the development of a clean procedure for the preparation of
heterocyclic compounds is a major task in modern heterocyclic
chemistry. Heterocyclic ring containing compounds have
concerned significant interest owing to their immense
biological activities in the field of bioorganic and biomedical
compounds, pharmaceuticals and others. Nitrogen-containing
heterocycles are a very important group of organic compounds
because of their wide application in medicine, agriculture, and
technology. Among these, quinoline derivatives are of
significant synthetic interest due to their diverse range of
biological activities. In the recent time, quinoline nucleus has
gathered an immense attention among chemists as well as
biologists as it is one of the key building elements for many
quinolines. In particular, there is much current interest in the
quinoline ring system especially in the area of medicinal
chemistry, and moreover it is a ubiquitous sub-structure found
in many biologically active natural products.2-4 In this regard,
quinolines have occupied a unique position in the design and
synthesis of novel biologically active compounds since they are
often used as anti-inflammatory, anti-asthmatic, anti-
tuberculosis, anti-bacterial, anti-hypertensive, anti-tumor and
anti-malarial agents.5
The complexes as drug work in one of the two ways: some use
a process called redox chemistry to take electrons from the
bonds holding the target molecules together. Others use
hydrolysis, meaning that they breakdown the target’s chemical
waterproofing, so that the water that is naturally present in a
cell react with the target. The field of inorganic chemistry in
medicine may usefully be divided into two main categories:
firstly, metal-based drugs where central metal ion is usually
the key feature of the mechanism and secondly, ligands as
drugs which target metal ions in some form, whether free or
protein-bound. Metals can play an important role in modifying
the pharmacological properties of known drugs after
naturally occurring compounds. Compounds containing
a
quinoline framework have been found applications as
pharmaceuticals and being general synthetic building blocks.1
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coordinating to a metal. Chelation causes drastic change in intercalation of extended aromatic groups and specific
biological properties of ligands as well as metal moiety and in hydrogen bonding depending on the particular type of ligands
many cases it causes synergistic effect of metal ion and ligand used. In this regard, substituted quinoline ligands were
both. For example, complexation of nonsteroidal anti- synthesized and combined with the ruthenium(III)-
inflammatory drugs to copper overcomes some of the gastric pentamethylcyclopentadienyl ring moiety to enhance their
side effects of these drugs.6 Metal based drugs play an DNA-binding properties, so as to achieve different biological
important part in the history of medicinal chemistry. A variety functions and to maximize their effectiveness as therapeutic
of transition metal complexes with different metal centres and agents. Here, we used pyridine/thiophene substituted
ligands of diverse structures were synthesized and studied for quinoline ligands. Because pyridine is found to have a large
their biological activity. Because of the range of coordination number of biological activities such as antiviral, anticancer,
geometries, metal complexes provide more stereochemical antimicrobial, antidiabetic, antitubercular etc. Pyridine is also
variability than is possible in organic molecules and often found in the form of vitamin B3 i.e pyridoxine.18 In medicinal
introduce new elements of chirality which may be important chemistry, thiophene derivatives have been very well known
for biological molecule recognition and interaction. The metal for their therapeutic applications. Many thiophene derivatives
complexes can also be extremely positively-charged: since have been developed as chemotherapeutic agents and are
many biological structures, such as several types of widely used. Thiophene nucleus is one of the most important
phospholipids, DNA and RNA, and some regions of proteins – heterocycles
exhibiting
remarkable
pharmacological
are negatively charged, for electrostatic reasons the positive activities.19 Also we used halogen substituted quinoline
charge of metal complexes could aid the binding with ligands because halogens are more electronegative atoms
intracellular targets.
which improves its biological activities.
Among the transition metal complexes or coordination This article mainly focuses on exploring the trend in DNA-
compounds, ruthenium-based complexes have been widely binding affinities of synthesized compounds and important
studied and some have displayed significant biological activity. differences in some related properties. Understanding the
This can be due to their ability to strongly bind nucleic acids features that contribute to recognition of DNA by metal based
and proteins, ligand exchange kinetics similar to those of their drugs or small molecules is crucial for the enhancement of
platinum counterparts. Ruthenium based organometallic drugs targeted at DNA. In present study, a series of substituted
compounds are progressively gaining in importance as quinoline derivatives were synthesized via the Friedlӓnder
promising aspirants for the design of novel and more effective condensation as well as a series of piano-stool ruthenium
metal-based drugs.7-11 Also various metal-containing coordination compounds were synthesized and well
compounds, ruthenium compounds are considered stable characterized. All synthesized compounds were evaluated for
under both highly acidic and alkaline conditions.12 Applications their biological applications. In assessment of these
of ruthenium compounds have a long history. This includes inspections and in persistence of the research work on
antimicrobial activity, anti-cancer activity, DNA binding, and bioactive heterocycles, it was anticipated to plan and viewing
DNA cleavage. It is well known that, deoxyribonucleic acid them for biological applications.
(DNA) is an important target in the organism for some metal
based drugs or reagents. These reagents can interact with DNA
2. Materials and methods
Details of materials, reagents and physical measurements are
thereby changing the replication of DNA and inhibiting the
growth of the tumor cells.13 Therefore, considerable attentions
have focused on the design and synthesis of new complexes,
especially transition metal complexes, which have enhanced
selectivity and novel modes of DNA interaction, such as non-
covalent interactions that mimic the mode of interaction of
proteins with DNA.14 It has been reported that the metal
complexes can interact with DNA non-covalently in the mode
shown in supplementary material 1.
2.1. General method for synthesis of quinoline ligands (3a-3g)
Detail process for the synthesis andof quinoline ligands is
shown in supplementary material 1. The proposed reaction for
1
the synthesis of ligands (3a-3g) is shown in scheme 1. H NMR
and 13C NMR spectra of ligands are shown in supplementary
material 2 and 3, respectively. Detail characterisation of
ligands are shown in supplementary material 1. The possible
mechanism for 4-phenyl-2-(pyridin-2-yl) quinoline (3a) via
Friedlӓnder condensation reaction is proposed in scheme 2.
2.2. General synthesis of the complexes
16
of intercalation, groove binding and electrostatic effect.15,
The effectiveness mainly depends on the mode and affinity of
the binding between the complexes and DNA.17 The insertion
of biologically-active ligands into metal coordination
compounds deals much scope for the design of novel drugs
with improved and targeted activity. Studies on such
complexes indicate that new mechanisms of action are
favorable when combining the bioactivity of the ligand with
the properties inherent to the metal, leading to the possibility
of overcoming current drug resistance pathways.
An organometallic half-sandwich Ru(III) metal complexes (5a-
5g) of the general formula [(Cp*)Ru(Ln)Cl]•Cl were synthesized
by the reactions of [{(Cp*)Ru(µ-Cl)Cl} with the respective
ligands (3a-3g) in a 1:2 molar ratio of dichloromethane and
methanol, respectively. Detail synthesis and characterisation
of complexes are shown in supplementary material 1.
The antitumor activity of many organometallic ruthenium
complexes is generally related to their enhanced DNA binding
affinity, which involves covalent coordination or simultaneous
2.3 Biological applications of synthesized compounds
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All experimental part of biological studies are included in
supplementary material 1.
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Scheme 1. Synthesis of quinoline derivatives (3a‒3g) and its complexation with ruthenium metal ion.
Scheme 2. The possible mechanism of formation 4-phenyl-2-(pyridin-2-yl) quinoline via Friedlӓnder condensation reaction.
The room temperature magnetic moments show that the
organometallic Ru(III) complexes are paramagnetic (2.15–2.23
BM), which corresponds to the +3 oxidation state of
ruthenium. The μeff values indicate a single unpaired (S = ½)
3.
Results and discussion
3.1 Magnetic moments, electronic spectra analysis and
conductance measurements
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electron and are consistent with low-spin t2g5 configuration for decomposition of the Cp* and quinoline ligand (71%), leaving
ruthenium(III) ion in all complexes.20
behind metal oxide as residue.
The electronic spectra of synthesized complexes are recorded 3.4. Biological applications of synthesized compounds
5
using DMSO as a reference. The ground state of Ru(III) (t2g
2
3.4.1 Cellular level bioassay using S. pombe cells
configuration) is T2g.21 In most of the Ru(III) complexes, the
electronic spectra show only charge transfer bands.22 All Ru(III)
Concentration(mg/L)
Compounds
3a
2
4
6
8
10
complexes exhibit three dissimilar bands, one band ranging
from 358‒370 nm, which is attributed to metal to ligand
charge transfer (MLCT) transition. A high energy two bands,
ranging from approximately 345‒351 nm and 285‒292 nm in
the ultraviolet region are attributed to the LMCT transition (n‒
π*) and intra-ligand charge transfer (π‒π*) transitions for the
quinoline ligands, respectively. The pattern of the electronic
spectra of all the organometallic Ru(III) complexes indicate the
existence of piano-stool environment around the
ruthenium(III) metal ion.23, 24
% Viability
69±2
77±2
75±2
72±2
70±1
82±3
80±2
79±2
65±2
63±1
60±1
59±1
73±1
69±1
68±1
74±2
72±2
70±2
69±2
79±2
77±2
76±5
63±2
61±1
57±1
56±1
70±2
67±1
66±1
67±2
66±2
63±1
61±1
73±3
72±2
68±2
56±1
53±1
50±1
49±1
62±2
60±1
58±1
62±2
61±1
59±1
57±1
70±2
68±2
65±1
48±1
46±1
45±1
44±1
52±1
51±1
50±1
3b
68±2
66±2
65±2
76±2
75±2
72±2
60±1
58±1
55±1
53±1
65±2
63±1
62±1
96±3
97±3
3c
3d
3e
3f
3g
The molar conductance values for the synthesized low spin
Ru(III) complexes were determined in methanol and are found
in the range of 39–67 cm2 Ω-1 mol-1, indicating electrolytic
nature and presence of one counter ion outside the
coordination sphere of Ru(III) complexes. So, we conclude that
all synthesized organometallic Ru(III) complexes are ionic in
nature.
5a
5b
5c
5d
5e
5f
5g
3.2 LC–MS analysis
The LC–MS spectrum and probable mass fragmentation
pattern of complex (5a) are shown in supplementary material
4 and 5, respectively. Mass spectrum of the complex (5a)
shows molecular ion peak [M−Cl]+ and [M−Cl+2]+ at 554 m/z
and 556 m/z, respectively due to presence of one chlorine
atom. The peak at 519 m/z is due to loss of one chlorine atom.
The peak at 384 m/z is due to loss of
pentamethylcyclopentadienyl ring. The peak at 237 m/z is due
to loss of quinoline ligand. The peak at 282 m/z corresponds to
quinoline moiety. The peak at 135 m/z corresponds to
pentamethylcyclopentadienyl ring.
DMSO
Untreated cells
Table 1: Effect of compounds on viability of S. Pombe cells at different concentrations
with error uncertainty in the value ±5%
3.3 Thermo gravimetric (TG) studies
Thermo gravimetric analysis determines the weight changes of
a sample, as a function of temperature. Weight changes as a
function of temperature indicate decomposition of material.
The temperature, at which no weight loss takes place, reveals
the stability of the material. Another important piece of
information that can be obtained by TGA is the amount of
weight lost by heating the sample to a given temperature. This
information helps the chemist to determine the composition
of a compound and to follow the reactions involved in its
decomposition.
The characteristic thermo gravimetric plot (mass loss % to
temperature in °C) of complex (5a) shows three distinct mass
losses.25 1st mass loss occurs between 40–130 °C, 2nd between
180–240 °C, and last between 260–650 °C. Mass loss occurring
during 1st decomposition step corresponds to lose of one
chlorine atom (6%), whereas mass loss during 2nd step relates
to decomposition of other chlorine atom which bind by one
covalent bond to metal center (6%), and 3rd step relates to
Figure 1. Effect of compounds on S. pombe cells. Dead cells are seen dark whereas live
cells are seen transparent.
The cellular level study of free quinoline ligands (3a-3g) and
synthesized organometallic Ru(III) complexes (5a-5g) was
tested against S. pombe cells in terms of the percentage
viability. Table 1 shows that all the complexes are active
against S. pombe cells than those of respective quinoline
ligands. S. pombe cells has become an important tool to study
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cell biology due to its eukaryotic and fairly big size respective ligands. The degree of lethality is found to be
characteristics. Cell death caused by toxicity of the chemically directly proportional to the concentration of the compounds.
synthesized compounds could be easily observed by vital The tested complexes have strong cytotoxic activity but this
staining which is shown in figure 1. The toxicity is found to vary examination is a primary one and further tests are required to
with the type of substituent present and concentrations of the investigate its actual mechanism of cytotoxicity and its
synthesized compounds. General observation is that the probable effects on higher animal model and on cancer cell
cytotoxicity is increases with concentration of compounds line. It recommends that the complexes can be used as potent
increases. After the treatment, many of the S. pombe cells are cytotoxic agents with the hope of adding arsenal of weapons
killed due to toxic nature of the compounds. Complex (5d) is used against the fatal disease cancer.
the most active amongst all the compounds. All the complexes
show comparable study to [RuIII(L1-7)3]•(PF6)3 compounds
reported in published literature.11
3.4.3 In vitro antimicrobial study
A relative study of in vitro antimicrobial screening values of the
quinoline ligands and their complexes indicates that the metal
3.4.2 In vitro brine shrimp lethality bioassay (BSLB)
The brine shrimp lethality bioassay is considered as a useful microorganisms when incorporated to the ligands. The
method for preliminary calculation of toxicity of compounds minimum concentrations of compounds that induced
complexes show superior activity against five unlike
a
and development in the assay procedure of bioactive complete growth inhibition will be recorded as MIC value. An
compound, which indicates cytotoxicity as well as a wide range satisfactory reason for this increase in antibacterial activity
of pharmacological activities of the compounds. All the may be considered in the light of Overtone’s concept29 and
synthesized compounds were studied for their cytotoxicity chelation theory.30 According to Overtone’s concept of cell
using the protocol of Meyer et al.26 The method is inexpensive, permeability, the lipid membrane that surrounds cell favours
rapid, reliable and economical. Results for the lethality are the passage of only lipid soluble materials, so that
noted in terms of deaths of larvae. The mortality rate of brine liposolubility is an important factor which controls
shrimp nauplii is found to increase with increasing the bacteriostatic activity. On chelation, the polarity of the Ru(III)
concentration of compounds. A plot of the log of sample’s ion will be lowered to a greater extent due to the overlap of
concentration versus percentage of mortality showed a linear the ligand orbital and partial sharing of the positive charge of
correlation. From the graph, the LC50 values of the compounds the Ru(III) ion with donor groups. Further, it increases the
are calculated, and they are found in the range of 5.64–119.67 delocalization of π-electrons over whole chelate ring and
mg/L (supplementary material 6). All the complexes show enhances lipophilicity of the complexes. This increased
superior
cytotoxicity
than
[RuIII(OFL)(PPh3)2Cl2],27 lipophilicity enhances the penetration of the complexes into
[RuIII(PFL)(PPh3)2Cl2],27 [RuIII(L4-6)PPh3)Cl3]28 and comparable to lipid membranes and blocks the metal binding sites in bacterial
[RuIII(L1-7)3]•(PF6)3 compounds reported in published enzymes. The complexes also disturb the respiratory processes
literature.11
of the cell and thus block the synthesis of proteins, which
restricts further growth of the organism.
Figure 2. Plot of LC50 values of different compounds in mg/L using Brine shrimp. Error
bars represent standard deviation of three replicates.
Figure 3. Effect of different concentration of free ligands and synthesized complexes on
five unlike microorganisms. Error bars represent standard deviation of three replicates.
From the data recorded, complex (5d) is the most potent
amongst all the compounds. Figure
2 shows that the
From figure 3, we observed that the antimicrobial screening of
all complexes against different microorganisms better than
synthesized complexes are good cytotoxic agent than that of
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that of respective ligands (supplementary material 7). The DNA. An absorption spectrum of complexes with HS DNA was
results indicated that a lower concentration of complex (5d) recorded for a constant concentration of complexes (20 µmol
was required to inhibit the bacterial growth and kill the L-1) with varying concentration of DNA (100 µmol L-1) to obtain
bacterial strain, leading to a higher efficiency in antimicrobial different DNA/complex mixing ratio is shown in figure 4.
activities. Complex (5d) is the most active amongst all the Complexes binding to DNA through intercalation usually result
complexes due to presence of fluorine atom and pyridine ring in hypochromism and bathochromism called red shift 35. When
substituents in quinoline ligand. Presence of more the complex intercalates with base pairs of DNA, the π* orbital
electronegative environment in complex (5d) improves its of the intercalated ligand of the complex couples with π orbital
biological property. All the complexes show lower MIC value of the base pairs of DNA, thus decreasing the π–π* transition
against all bacterial species than [RuIII(OFL)(PPh3)2Cl2]27 and energy and resulting in the bathochromism.36 The absorption
[RuIII(SPF)(PPh3)2Cl2]27 but higher than [RuIII(L1–6)PPh3)Cl3]28 and spectral changes were monitored around 270−292 nm for the
11
comparable to [RuIII(L1-7)3]•(PF6)3 compounds reported in investigation of DNA binding mode and strength. As the DNA
published literature. All the complexes show better activity concentration is increased, transition bands of complexes
against S. aureus
,
E. coli and P. aeruginosa than those of (5a−5g) exhibit hypochromicity [hypochromicity, H% = [(Afree
[Ru(4,7-Me2phen)3]Cl2 and lower than − Abound) / Afree] × 100%] of about 20.23−23.14%, and
[Ru(phen)3]Cl2,
[Ru(3,4,7,8-Me4phen)3]Cl2 reported by Fangfei Li et al 31
.
and bathochromicity of 2−4 nm. The complex (5d) has highest
comparable activity against S. aureus than those of reported percentage hypochromicity (23.14%). These spectral features
by P.-L. Lam et al 32
.
may recommend a mode of binding that covers a stacking
interaction between the aromatic chromophore and the DNA
base pairs.
3.4.4 DNA interactions
DNA can be regarded as a double stranded chromonic, side-
chain polymer, with a core of stacked bases resembling a
chromonic column, It is the π–π interactions between the
bases that holds the stack together and the sugar/phosphate
chains are wound round the column loosely enough to allow
intercalation of a guest molecule. In order to accommodate
the foreign molecule, the chain must unwind, far enough to
allow two bases to move apart and make room for the
intercalating molecule to enter the stack. A large number of
soluble, multi-ring aromatic molecules are able to intercalate
in this way. These include dyes, the fluorescent marker,
ethidium bromide, many antibiotics, cancer-causing agents
and anticancer drugs 33. Since DNA is the key target for many
metal-based drugs, and DNA-binding is the critical step for the
study of effective metal-based drugs, which is of importance in
illuminating the mechanisms involved in the site specific
recognition of DNA, and designing new types of
pharmaceutical molecules, the binding behaviors of the
synthesized compounds toward DNA are explored both
theoretically and experimentally with the aid of different
procedures and techniques.
Figure 4. Absorption spectral changes on addition of HS DNA to the solution of complex
(5d) after incubating it for 10 minutes at room temperature in phosphate buffer
(Na2HPO4/NaH2PO4, pH=7.2). Inset: plot of [DNA]/Ƴꢀꢁ Ǝ ꢀ!Ʒ vs. [DNA]. Error bars
represent standard deviation of three replicates. (Arrow shows the change in
absorption with increase in concentration of DNA).
(I) By absorption spectral analysis
The binding ability of the complexes with HS DNA can be
characterized by measuring their effects on electronic
absorption spectra. It is one of the most useful techniques for
the study of binding mode and binding strength of the
compounds with DNA. Ruthenium metal complexes as well as
most drugs tend to interact with DNA non-covalently through
three selective modes: (I) a groove-bound fashion stabilized by
a mixture of hydrophobic, electrostatic and hydrogen-bonding
interactions; (II) an intercalative association with planar,
heteroaromatic moieties between the DNA base pairs; and (III)
electrostatic binding.34 The intrinsic DNA binding constants i.e.
Kb of the complexes to HS DNA were quantitatively
determined by observing the change in the absorption
intensity of the spectral bands by subsequent addition of HS
The Kb values of all compounds are found in the range
0.34×105 ‒ 6.25×105 L mol-1 (supplementary material 6). It is
lower than Kb value of classical intercalators ethidium bromide
(7.16×107 L mol-1), [Ru(PEF)Cl2(H2O)2]•5H2O (5.00×107 L mol-
1 37
)
and [RuIIICl2(PPh3)2L1] (1.2±0.14 ×106 L mol−1)38 but higher
than [RuIII(cipro)3]•4H2O (2.5±0.9 ×104
[RuIII(L1)(H2O)2]Cl (8.72×104 mol−1),40 [RuIII(L2)(H2O)2]Cl
(6.35×104
mol−1),38 [RuIIICl2(AsPh3)2L2] (1.6±0.11 ×104
L
mol−1),39
L
L
mol−1),40 [RuIIICl2(PPh3)2L2] (3.9±0.12 ×104
L
L
mol−1),38
[RuCl(AsPh3)L2] (3.6×104 L mol−1),41 [RuCl(AsPh3)L3] (3.2×104
L
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mol−1)41 and comparable with [RuIIICl2(AsPh3)2L1] (5.6±0.09 > 5a > 5g > 5f > 5e > quinoline ligands (supplementary material
×105 L mol−1),38 [RuCl(AsPh3)L1] (4.7×105 L mol−1) 41
From the 8). This behavior is similar to that of ruthenium complexes
Kb value and red shift, it is clear that the complexes bind to the reported by Ihtshamul Haq et al42 and Caipihg. Tan et al 43, 44
.
.
DNA by intercalation mode and complex (5d) has the highest The viscosity results may reveal the tendency of each ligand to
binding ability (figure 5) than the other complexes and ligands intercalate into DNA base pairs. The increase in DNA viscosity
due to an existence of fluorine atom. An existence of fluorine observed in the complexes suggests a classical intercalative
atoms act as chemical isosteres for the oxygen atoms in the mode.
heterocyclic base of thymidine, as a result it shows better DNA
interaction activity than other. The binding mode is further
confirmed by hydrodynamic volume (i.e. viscosity)
measurement.
Figure 6. Effect on relative viscosity of HS DNA under the influence of increasing
amounts of complexes at 27 (±0.1) °C in phosphate buffer at pH=7.2. Error bars
represent standard deviation of three replicates.
Figure 5. Plot of Kb values of synthesized compounds in L mol-1. Error bars represent
standard deviation of three replicates.
(III) By molecular docking with DNA sequence d(ACCGACGTCGGT)2
(II) By hydrodynamic volume or viscosity measurement
Spectroscopic study are essential, but not satisfactory to
support a binding mode. The binding mode or nature of the
interaction of all the compounds with HS DNA was further
confirmed by viscosity measurements. A partial and non-
classical ligand intercalation causes a bend in the DNA helix,
reducing its effective length and thereby its viscosity. Whereas
in classical intercalation, the DNA helix lengthens as base pairs
are separated to accommodate the bound ligand, leading to
increase in DNA viscosity. Therefore viscosity measurement is
regarded as the least ambiguous and the most critical means
Figure 7. Molecular docking of the complex 5a (ball and stick) with the DNA duplex
for studying the binding mode of metal complexes with DNA in
(VDW spheres) of sequence d(ACCGACGTCGGT)2. The complex is docked in to the DNA
solution and provides stronger arguments for intercalative
binding mode.25 The effects of synthesized complexes on the
showing intercalation between the DNA base pairs.
viscosity of HS DNA are shown in figure 6.
Molecular docking studies of the compounds with the DNA
As illustrated in this figure, on increasing the amount of the
duplex were performed to predict the chosen binding site
complexes the relative viscosity of HS DNA increases steadily,
along with the preferred orientation of compound inside the
which confirms that the organometallic Ru(III) complexes are
DNA helix which is shown in figure 7. To understand the
bound to HS DNA by intercalation. This phenomenon may be
preferred orientation of sterically acceptable compound with
explained by the insertion of the compounds between the
the DNA sequence at the theoretical level, molecular docking
DNA base pairs, leading to an increase in the separation of
study was performed. By placing a small molecule into the
base pairs at intercalation sites and thus an increase in overall
binding site of the target specific area of DNA, this study has
DNA length. It is clear from the figure that all these complexes
played important roles in the mechanistic study as well as
show an increase in the relative viscosity of HS DNA. The
understanding the metal based drug-DNA interactions for
increase in the degree of viscosity of all compounds depends
rational drug discovery and design. The study shown that the
on their affinity to DNA, with an order as follows: 5d > 5c > 5b
compounds under investigation interact with DNA via an
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intercalation mode involving outside edge stacking interaction supercoiled (SC) form, also known as covalently coiled coil
with oxygen atom of the phosphate backbone. From the DNA, here designated as Form I. Single strand cleavage results
resultant docked models, it is clear that the compounds fit well in so called nicked or open circular (OC) form of DNA
into the intercalative mode of the targeted DNA and A−T rich (designated as Form II), whereas the double-strand cleavage
region stabilized by van der Waal’s interaction and results in linear (LC) form of DNA (designated as Form III). The
46
hydrophobic contacts.45, The resulting binding energies of migration rate during agarose gel electrophoresis depends on
docked complexes (5a−5g) are found to be −274.94, −295.61, both size (base pairs) and conformation, with smaller or
−300.01, −327.01, −310.34, −319.77 and −325.71 kJ mol−1, condensed DNA migrating faster than larger or unfolded DNA.
respectively. Also the binding energies of docked compounds Form I has a tightly packed conformation and therefore
(3a−3g) are found to be −232.08, −244.75, −242.87, −252.56, migrates faster through agarose gels than linear DNA
−229.80, −246.00 and −250.55 kJ mol−1, respectively. All the (intermediate migration) or open circle DNA (slowest
complexes show comparable result to [RuIII(L1-7)3]•(PF6)3 migration).
compounds reported in published literature.11 Therefore,
molecular docking together with absorption spectral and
viscosity studies indicate that the compound interacts with the
DNA through the classical intercalation mode mainly inside the
DNA helix.
(IV)By agarose gel electrophoresis: photo quantization technique
There has been considerable interest in DNA nucleolytic
cleavage reactions that are activated by complexes. Cleavage
study on plasmid DNA induced by synthesized compounds can
be monitored by agarose gel electrophoresis using pUC19
DNA. Agarose gel electrophoresis is a convenient method to
assess cleavage of DNA by metal based drugs to assess the
factors affecting the nucleolytic efficiency of a compound and
to compare the nucleolytic property of different compounds.
The DNA cleavage can occur by hydrolytic and oxidative
pathways, in which hydrolytic DNA cleavage involves cleavage
Figure 9. Plot of DNA cleavage data by agarose gel electrophoresis of different
of phosphodiester bond to generate fragments which can be
compounds using pUC19 DNA. Error bars represent standard deviation of three
subsequently relegated; started in a modest way of converting
replicates.
supercoil (SC) form of DNA to the open-circular (OC) form and
last in linear (L) form, is now being used for identifying the
Figure 8 shows the electrophoretic separation of pUC19 DNA
percentage of cleavage as a function of concentration of
when reacted with compounds under aerobic conditions. This
nuclease. Oxidative DNA cleavage involves either oxidation of
clearly show that the relative binding efficacy of compounds to
the deoxyribose moiety by abstraction of sugar hydrogen or
DNA is much higher than the binding efficacy of ruthenium salt
oxidation of nucleobases.29
and free quinoline ligands (figure 9 and supplementary
material 10). The difference in DNA-cleavage efficiency of
complexes was due to the difference in binding affinity of
complexes to DNA. The similar behavior of Ru(III) complexes
with plasmid DNA was shown by reported compounds of type
[RuIII(PFL)(PPh3)2Cl2],27
[RuIII(OFL)(PPh3)2Cl2],27
[RuIII(SFL)(PPh3)2Cl2],27 mer-[RuIIICl3(CH3CN)(dpq)]43 and mer-
[RuIIICl3(dmso)(N−N)].44
3.4.5 In vitro antimalarial screening
Malaria cause vast medical, financial, and emotional problem
in the world. Plasmodium falciparum is the parasite
responsible for most malaria cases up to 80%, which often
proves harmless. Malaria is one of the most prevalent
infectious diseases worldwide and it represents a major global
health issue for which new effective chemotherapies are
urgently needed. As part of this search for novel drugs against
malaria, we report encouraging results for compounds
resulting from the modification of quinoline moiety by
coordination to ruthenium metal centres; the new compounds
Figure 8. Photogenic view of cleavage of pUC19 DNA (300 µg/cm3) with series of
compounds using 1% agarose gel containing 0.5 µg/cm3 EtBr. All reactions were
incubated in TE buffer (pH 8) at a final volume of 15 mm3 for 24 h at 37 °C.
The principle of this method is that molecules migrate in the
gel as a function of their mass, charge and shape, with
supercoiled DNA migrating faster than open circular molecules
of the same mass and charge. The native DNA remains in the
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MedChemComm
are highly active against a chloroquine resistant strain of coordination sphere which accomplish all complexes are ionic
Plasmodium falciparum. The results of the pharmacological in nature. All the complexes show strong in vitro cytotoxic, in
screening are expressed as the drug concentration resulting in vitro antimalarial as well as cellular level bioassay compared to
50% inhibition (IC50) of parasite growth. The mean values of free ligands. The antimicrobial activity of the all compounds
IC50 in mg/L for compounds are given in supplementary has been tested on five dissimilar microorganisms and the data
material 6 (0.55‒1.84 mg/L).
show an improved biological activity of all complexes in
Figure 10 shows that all complexes exhibit good antimalarial relation to the free ligands. Hypochromism and
activity than that of respective ligands. It is thus clear that the bathochromism (red sift) of band in absorption spectral
combination of ruthenium metal and quinoline ligand in a titration and increase in relative viscosity of DNA recommend
single molecule does produce an improvement of the activity that all complexes bind with DNA via classical intercalative
against resistant strains of the parasite, demonstrating the mode.
validity of our concept in the search for novel antimalarial Ligand (3d) is the most active amongst all the ligands due to
drugs capable of overcoming resistance. The IC50 values of all presence of fluorine atom and pyridine ring substituents in
III
11
Ru(III) complexes are comparable to [Ru (L1-7)3]•(PF6)3,
quinoline ligand. Presence of more electronegative
[Ru(A)2(B)]Cl2 (10 mg/L),47 [Cu(terpyridyl)Cl]Cl (0.52 µmol L-1) environment in ligand (3d) improves its biological property.
and Fe(terpyridyl)Cl3 (0.63 µmol L-1).48
Therefore, complex (5d) of ligand (3d) is more potent than
other complexes. Presence of fluorine atoms act as not only
chemical isosteres for the oxygen atoms in the heterocyclic
base of thymidine, but shows strong affinity of binding due to
strong van der Waals force of interaction between fluorine and
hydrogen atom compared to other substituents as a result it
shows superior antimicrobial and DNA interaction activity than
other compounds. Molecular docking studies of the
synthesized compounds with the DNA duplex were performed
to predict the chosen binding site, which suggests intercalation
between compounds and DNA base pairs. The DNA cleavage
study of pUC19 shows that all complexes have high cleavage
ability than metal salt and ligands. Presence of more
electronegative environment in complex (5d) improves its
biological property. Hence, we concluded that pyridine
substituted quinoline is more potent than thiophene
substituted quinoline because pyridine contain nitrogen atom
is more electronegative than sulphur atom. The preliminary
studies encourage for carrying out further in vivo experiments.
The results are of importance towards further designing and
developing ruthenium based complexes and systematic
assessment of biological activity for their potential applications
as therapeutic agents.
Figure 10. Plot of IC50 values of different compounds in mg/L using Plasmodium
falciparum strain. Error bars represent standard deviation of three replicates.
4. Final Remarks
A series of substituted quinoline nucleus based half-
sandwich organo-ruthenium complexes were synthesized and
well characterized., in search of new metal based drugs Acknowledgements
looking promising as potent cytotoxic, DNA binding, DNA
The authors thankful to the Head, Department of
cleavage, antimicrobial as well as antimalarial agents. This
synthetic approach were carried out with an aim to study their
biological activity and allows the incorporation of potent
bioactive nuclei in a single skeleton through an easy way.
Likewise arene ring, pentamethylcyclopentadienyl ring
derivatives occupy three coordination sites at the octahedral
metal centre and provide to the metal a lipophilic protecting
face. On the other hand, the remaining three coordination
sites allow the introduction of ligands with hydrophilic
character. By analogy with their structure to a piano stool
these are known as piano stool complexes, with the metal
centre being coordinated by pentamethylcyclopentadienyl
ring, a chlorido ligand and a chelating quinoline ligand. The
molar conductivities values of all organometallic Ru(III)
complexes are indicates, one counter ion present outside the
Chemistry, Sardar Patel University, Vallabh Vidyanagar,
Gujarat, India, for providing the laboratory facilities, Dhanji P.
Rajani, microcare laboratory, Surat for in vitro antimalarial
activity, IIT SAIF, Bombay for EPR analysis, U.G.C., New Delhi
for providing financial assistance of UGC BSR grant No.
C/2013/BSR/Chemistry/59 and “BSR UGC One Time Grant”,
vide UGC letter No.F.19-119/2014(BSR).
Declaration of interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of the paper.
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28 P. S. Karia, P. A. Vekariya, A. P. Patidar, R. R. Patel and M. N.
Patel, Monatsh. Chem., 2016, DOI: 10.1007/s00706-016-
1708-8, 1-12.
29 J. V. Mehta, S. B. Gajera and M. N. Patel, Spectrochim. Acta,
Part A, 2015, 136, Part C, 1881-1892.
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Graphical abstract and synopsis
“Spectral changes accompanying addition of HS DNA to solution of quinoline nucleus based piano
stool coordination compounds indicate intercalative mode of binding of the compound between
DNA base pairs.”