Oxadiazole based Os(IV) compounds as potential DNA intercalator and cytotoxic agents

Article abstract of DOI:10.1016/j.inoche.2020.108070

Os(IV) compounds and ligands have been synthesized and well characterized. DNA cleavage tendency of compounds has been evaluated using gel electrophoresis and binding behavior has been evaluated by viscosity measurements, absorption titration, fluorescenc

Full text of DOI:10.1016/j.inoche.2020.108070

Journal Pre-proofs
Short communication
Oxadiazole based Os(IV) compounds as potential DNA intercalator and cyto-
toxic agents
Bharat H. Pursuwani, Bhupesh S. Bhatt, Foram U. Vaidya, Chandramani
Pathak, Mohan N. Patel
PII:
S1387-7003(20)30660-2
DOI:
https://doi.org/10.1016/j.inoche.2020.108070
INOCHE 108070
Reference:
To appear in:
Inorganic Chemistry Communications
Received Date:
Revised Date:
Accepted Date:
27 April 2020
25 June 2020
28 June 2020
Please cite this article as: B.H. Pursuwani, B.S. Bhatt, F.U. Vaidya, C. Pathak, M.N. Patel, Oxadiazole based
Os(IV) compounds as potential DNA intercalator and cytotoxic agents, Inorganic Chemistry Communications
(2020), doi: https://doi.org/10.1016/j.inoche.2020.108070
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Oxadiazole based Os(IV) compounds as potential DNA intercalator and cytotoxic agents
Bharat H. Pursuwani, Bhupesh S. Bhatt, Foram U. Vaidya, Chandramani Pathak and
Mohan N. Patel*
Department of Chemistry, Sardar Patel University,
Vallabh Vidyanagar–388 120, Gujarat, India.
Corresponding author. Tel.: +91 2692 226856
E-mail: jeenen@gmail.com
Abstract
Os(IV) compounds and ligands have been synthesized and well characterized. DNA
cleavage tendency of compounds has been evaluated using gel electrophoresis and binding
behavior has been evaluated by viscosity measurements, absorption titration, fluorescence, and
docking studies. All the compounds show the intercalation mode of binding. The binding constant
of complexes falls at about 1.8-7.6×104 M-1. Bacteriostatic activity of compounds has been
evaluated on a series of gram+ve and gram-ve bacteria in terms of their MIC values. Also, the
compounds have been screened for in vivo and in vitro cytotoxicity assays on S. Pombe cell’s DNA
and HCT-116 cell line. LC50 of ligands and Os(IV) complexes are in the range of 7.92-19.91 and
4.00- 11.74 μg/mL respectively.
Keywords: Os(IV), DNA cleavage, Bacteriostatic, S.Pombe, HCT-116.
1. Introduction
Nitrogen and oxygen atoms bearing fused heterocycles have been proved successful in
keeping footprint in the world of medicinal chemistry [1-3]. Oxadiazoles have been proved as
successful in bringing a variety of novel drugs.[4] Iso-oxazoles have been explored widely in
medicinal chemistry [5, 6]. Iso-oxazoles have been widely used in marketed products such as
leflunomide [7], sulfafurazole [8], isooxaflutole, [9] and hymexazol [10] (Supplementary material
1). Oxadiazoles are analogues heterocycles of iso-oxazoles, oxadiazoles moieties fused with
phenyl ring exhibit a wide variety of biological activities [11-13]. They have an impactful
biological role, efficacy, and pharmacokinetics importance [14]. Various drugs with this parent
1

moiety have been proved successful, such as sulfamethoxazole, which has been proved as
antibiotic and proved to have a wide broad spectrum against bacteria [15]. Risperidone has been
effective in the treatment of schizophrenia[16]. These have been a class of highly stable electron
donating species. Synthesis of oxadiazoles has been widely explored in synthetic organic
chemistry [17]. 1,3,4-Oxadiazoles have been proved as effective tuberculosis analogues [18].
Oxadiazole has been a similar class of compounds. Geometry and conformational environment of
molecules are important to adhere interaction mechanism of the molecule with DNA [19, 20].
Synthesis and biological applications of oxadiazole heterocycles have been widely explored [21,
22]. Oxadiazole nucleus based ruthenium(II) complexes have been synthesized and reported in the
literature [23]. Oxadiazole heterocycles have also proved to be anticonvulsant and antidepressant
agents [24]. These class of heterocyclic ligands readily coordinate with 5d transition metal ions
[25]. Metal complexes have been popular amongst inorganic chemists to explore a wide plethora
of aspects in biological applications [26, 27]. Osmium metal ion based complexes are quite inert
in comparison to ruthenium which provides stability to molecule [28]. Osmium metal ion based
complexes have been proved successful in resisting the growth of tumor cells, which highlights its
potentiality in its anti-cancer behavior [29, 30]. They have been also proved successful in photo-
cleavage of macromolecules [31]. Osmium complexes with tetrazolo quinoline moiety have been
reported to have good biological efficacy and DNA interaction mode [29]. Osmium compounds
have been most investigated as non-platinum metallodrug undergoing clinical screenings [28].
These have proved to have more selectivity towards diseased cells [32]. FY026 has been proved
to be a successful osmium class of drug over HCT-116 cell line and has greater tolerability
compared to ruthenium class of drugs which are of pharmacokinetic importance [33]. Osmium
complex with azopyridine nucleus has been proved to be effective at sub-micromolar
concentration over wide variety of cell lines and are competing to ruthenium complexes and cis-
platin analogues [34].
2. Experimental
General method for synthesis of ligands, metal salt, and complexes
Ligand L1 i.e. 1-(2-(4-chlorophenyl)-5-phenyl-1,3,4-oxadiazol-3(2H)-yl)ethan-1-one have
been synthesized by reacting 4-chloro benzaldehyde (2 g, 14 mmol) with (1.93 g, 14 mmol)
benzhydrazide to form Schiff base in presence of potassium hydroxide (0.39 g, 7 mmol). The
Schiff base formed (1 g, 3 mmol) has been further refluxed with acetic anhydride (0.36 mL, 3
2

mmol) to form ligand L1. (NH4)2OsBr6 has been synthesized by refluxing OsO4 (25 mg dissolved
in 25 mL millipore water) with hydrobromic acid (9 mL) at 80 °C for the period of 2h. The resulting
solution has been mixed with ammonium bromide (0.36 g) at room temperature with constant
stirring. The solution has been kept in a deep freezer at 2-4 °C till the salt formation [35, 36].
Complex C1 i.e. tetrabromido(1-(2-(4-chlorophenyl)-5-phenyl-1,3,4-oxadiazol-3(2H)-yl)ethan-1-
one)osmium(IV) has been synthesized using (NH4)2OsBr6 (60 mg, 8 mmol) and ligand L1 (25 mg,
8 mmol).
3

Scheme 1. Synthesis of compounds.
4

3. Results and discussion
3.1. Characterization
Ligands L1-L8 have been synthesized by benzhydrazide as reactant. In 1H-NMR, aromatic
proton region of ligand L1 falls in the range of 7.30-8.48 δ ppm. Aliphatic methine proton is
justified at a signal of 6.789 δ ppm. Methyl protons show its presence at 2.59 δ ppm. Ligands L9-
L14 have been synthesized by 4-hydroxy benzhydrazide as reactant. In ligand L9, aromatic proton
region is justified at 7.51-7.94 δ ppm. Aliphatic methine proton shows its presence at 6.77 δ ppm.
Methyl protons and hydroxy proton show its presence at 1.97 and 12.03 δ ppm respectively. In
ligand L1, there are 5 quaternary carbon signals observed. Carbonyl carbon falls at 163.2 δ ppm.
Whereas carbons labeled C2, C1’, C1’’ and C4’’ show its presence at 145.9, 135.2, 134.7, and
124.1 respectively. Aromatic methine carbons are well justified at 133.9, 130.1, 129.3, 129.0 and
123.5 assigned to (C2’, C3’), C4’, (C5’, C6’), (C2’’, C3’’) and (C5’’, C6’’) respectively. Aliphatic
carbon C5 shows its presence at 83.53 δ ppm whereas, methyl functionality as primary carbon
signal falls at 25.15 δ ppm. In ligand L9, there are 6 quaternary carbon signals observed. Carbonyl
carbon falls at 163.7 δ ppm. Whereas carbons labeled C2, C1’, C4’, C1’’ and C4’’ show its
presence at 146.8, 137.4, 137.3, 132.3 and 131.3 respectively. Aromatic methine carbons are well
justified at 133.7, 131.4, 128.9 and 128.1 assigned to (C2’, C3’), (C5’, C6’), (C2’’, C3’’) and
(C5’’, C6’’) respectively. Aliphatic carbon C5 shows its presence at 83.22 δ ppm whereas, methyl
functionality as primary carbon signal falls at 25.40 δ ppm. A detailed experimental section has
1
been given in supplementary material 2. H-NMR and 13C-APT spectra of all the ligands is given
in supplementary materials 3 and 4. ICP-OES data justifies metal percentage present in complexes
relative to their theoretical values.
LC-MS spectrum of ligand L1 shows a high intense base peak at 258 m/z whereas mass
peak is obtained at 300 m/z. Complex C1 has been characterized using ESI-MS spectrometry.
Molecular ion peak of complex is observed at 810.59 m/z with [M+], [M+2], [M+4], [M+6] and
[M+8] peaks. In ligand L1, the conjugated methine group shows its presence at 3032 cm-1, which
shifts slightly to high frequency 3044 cm-1 in case of its respective Os(IV) complex C1. The
presence of C-N, C-H, C=C, and C=O bonds in ligands and complexes are justified at 1551, 1366,
1651 and 1605 cm-1 to 3044, 1560, 1370, 1655, and 1610 cm-1 respectively. Further, N-Os bond
is justified at 486 cm-1 which move towards higher frequency in N-Os-Br bond at 671 cm-1 due to
5

electron withdrawing behavior of inductive bromine. In complex C9, bands are relatively shifted
compare to ligand L9. The ν(=C-H), ν(C-N), ν(C-H), ν(C=C), ν(C=O) bands are observed at 3083,
1589, 1402, 1652, 1625 cm-1, respectively. Additionally, ν(N-Os) band is observed at 447 cm-1,
whereas ν(N-Os-Br) linkage is justified its presence at 671 cm-1. LC-MS spectra of ligands and
ESI-MS spectrum with fragmentation pattern of the complex are given in supplementary materials
5 and 6. IR spectra and CHNS elemental analysis data are given in supplementary materials 7 and
8 respectively.
Magnetic property exhibited by Os(IV) complexes has been measured using Guoy’s
balance. All the Osmium(IV) complexes bear 2.70-2.80 B.M. with low spin electronic
0
configuration t2g4 eg proving paramagnetic behavior containing d2sp3 hybridization and possess
octahedral geometry [37]. Conductance measurements have been carried out using DMSO
solubilized test compounds which shows non-electrolytic behavior. The electronic spectral study
concludes that ligands show the transition in the range of 222-284 nm, whereas their respective
Os(IV) complexes show bands in the range of 222-296 nm. In complex C1, bands at 284, 340, and
458 nm are assigned to n-π*, π-π*, and d-d transitions, respectively. This adds support to electronic
spectral study in justifying octahedral behavior and paramagnetic behavior exhibited by the Os(IV)
compounds.
3.2. Biological activities
3.2.1. DNA binding and cleavage activity
Compounds have interacted in the presence and absence of HS-DNA solution taking the
ethidium bromide as reference. Flow time taken by Os(IV) complexes has been relatively higher
compared to their respective ligands. Plots in figure 1 show an increase in viscosity of compounds
with an increase in concentration of compounds. Comparing ligands amongst series, it can be
concluded that ligands L9-L14 took more flow time compared to ligands L1-L8 due to the presence
of electron releasing hydroxy functionality which enhances interaction and π character of the ring
while interacting with HS-DNA solution in thermostatic viscometry bath. All the ligands and
Os(IV) complexes show partial intercalation mode of binding [38].
6

Fig. 1. The plot of effect on relative viscosity of HS- DNA under the influence of an increasing
amount of L1-L14 and complexes C1-C14 at 27±0.1 oC in phosphate buffer (pH 7.2)
Os(IV) complexes and ligands have been interacted with B-DNA receptor to evaluate
minimum energy acquired by molecules in the best possible flexible conformational structure
allowed to run in docking processing software Hex 6.0. Docked images of the interaction of all the
compounds are presented in supplementary material 9. There has been an π-π stacking interactions
observed between compound and phosphate backbone when compounds interacted with
d(CGCCGAATTCGCCG)2 sequence. Compounds intercalate between A-T rich region of DNA.
This adds additional proof to titrimetric data and viscometric measurement in showing effective
binding by compounds while interacted with DNA. Docking energy of ligands and Os(IV)
complexes are given in supplementary material 10.
Upon absorption titrimetric analysis in the absence and varying concentration of DNA
solution, a decrease in absorbance has been observed resulting in a slight hypochromism shift.
Comparing Os(IV) complexes and their ligands, it is observed that intrinsic binding constant and
relative shift in percentage hypochromism is greater in complexes than their respective ligands
because of bonding of metal with four bromine atoms which allow dominating π-π stacking
interaction between π character of the ring with the lone pairs of nitrogen atom bases of the
macromolecule. The interaction is steady and a relatively small shift in absorbance is observed
accounting to justify the intercalation mode of binding. Intrinsic binding constant and percentage
hypochromism of compounds have been calculated and are given in supplementary material 11.
7

Figure 2 shows absorption titration curve of complex C1. The binding constants of Os(IV)
complexes are higher than that of copper(II) complexes [39].
Fig. 2. Absorption spectral changes on addition of HS-DNA to the solution of complex I (after
incubating it for 10 min. at room temperature in phosphate buffer. Graph: plot of [DNA]/(εa–εf)
vs. [DNA]. (Arrow shows the change in absorption with an increase in the concentration of DNA)
amount of complexes and EtBr at 27±0.1 oC in phosphate buffer (pH 7.2)
Os(IV) complexes do not show fluorescence at room temperature because of the limitations
of quenching ethidium bromide probe in emission spectra. EB-DNA emission band is observed at
470 nm, which is subjected to excitation at 610 nm. Os(IV) complexes displace EB from EB-DNA
adduct in various concentration range, thereby decrease in fluorescence intensity have been
observed. The constants have been evaluated using Stern Volmer and Scatchard plots graphically
with an accurate regression coefficient (r=3.50). The binding constant, quenching constant and
binding sites are presented in supplementary material 12. The plots are represented in figure 3. All
the complexes show better binding except C7, C9, and C10 than reported platinum(II) complexes.
Amongst, benzhydrazide derived ligands and complexes C1-C8, complex C7 has electron releasing
methoxy functionality which lowers the tendency of binding. While amongst 4-hydroxy
benzhydrazide derived ligands and complexes C9-C14, complex C9 has electron releasing hydroxy
and withdrawing chlorine at para positions of reactants counter and nullify such effect as a result
only hyperconjugation forces are active in heterocyclic environment. Whereas, complex C10 has
8

electron releasing hydroxy functionality along with the hyperconjugation effect of the methyl
group in the ligand environment. Hence, they dominate in showing effective binding strength.
Fig. 3. Fluorescence emission of EB bound to HS-DNA in presence of complex C1. Plots: I0/I vs.
[Quencher] and plot of log[I0-I] vs. log[Q] for the titration of HS-DNA EB system with Os(IV)
complex C1 in 1M phosphate buffer (pH 7.2) medium.
Gel electrophoresis experiment has been carried out on S. Pombe cell’s DNA. Os(IV)
complexes have been proved impactful in degrading DNA when allowed to run over 1% agarose
gel in casting apparatus. There have been no smearing observed over untreated DNA samples
which conclude the tendency of Os(IV) complexes to show degradation phenomenon of the
macromolecule. Relative tendency of complexes to show effective smearing is in order of
C3>C5>C4>C1>C6>C8>C2>C7>C13>C9>C11>C12>C10>C14. Complexes C1-C8 show better
photolytic smearing compare to C9-C14 since the former has electron releasing functionality which
pushes the electron cloud of nucleus thereby resisting effective smearing. The presence of nitro
functionality readily withdraws electron cloud towards itself ruptures DNA hence there is intense
smearing observed in the case of complex C3. Comparing complexes C1 and C4, complex C4
dominates in showing cleavage property since electron withdrawing chlorine present at ortho
position and in former its present at the para position which is closer to nucleus. The same
9

phenomenon is observed in complexes C13 and C9. Photolytic cleavage image photographed by
spectrophotometer and intensity visualized by alphadigidoc software is given in figure 4.
Fig. 4. Photogenic view of cleavage of S. Pombe cell’s DNA (1 μgL-1) with a series of compounds
using 1% agarose gel containing 0.5 (μgL-1) EtBr.
3.1.2. Bacteriostatic activity
Lipids in bacteria play a key role in developing growth and providing an immune response
to its walls. Therefore, moieties with functionalities that resist lipid growth or suppress response
are equally important to resist the growth of bacteria. Comparing series of ligands, ligands L1-L8
show better MIC compare to ligands L9-L14 because ligands L9-L14 have hydroxy functionality
which pushes electron towards the nucleus thereby having less impact on resisting cell wall
synthesis of bacteria. On the other hand, ligands L1-L8, there is hyperconjugation of bonds taking
part in the absence of any electron withdrawing or releasing functionality which is comparatively
more impactful in resisting bacterial growth. Os(IV) complexes have high electron withdrawing
bromine atoms able to rupture cell walls of bacteria in resisting the growth of bacteria hence show
superior MIC compared to their respective ligands as shown in figure 5. in terms of µM
concentration. Ligands and Os(IV) complexes show MIC in range of 142.50-180 and 72.50-110
μg/mL, respectively.
10

Fig. 5. MIC values of synthesized compounds in μM.
3.1.3. Cytotoxicity
Compounds have been stained over S.Pombe DNA’s cell culture to evaluate their cytotoxic
count. With a relative increase in the concentration of DMSO solubilized compounds, relative
percentage cell viability increase. Cell death has been observed under a digital microscope. It is
concluded that ligands L1-L8 dominate in percentage cell viability compared to ligands L9-L14, the
reason is due to the presence of electron pushing functionality on phenyl ring which has less effect
in resisting the development of cell growth cycle. Further, their respective Os(IV) complexes also
play analogues behavior in potency unlike ligands but they further proved to have more percentage
cell viability compared to ligands L1-L14. Complexes C1-C8 show better cytotoxic effect than
complexes C9-C14 because of having different classes of ligands. Supplementary material 13
shows the relative percentage cell viability of Os(IV) complexes and their respective ligands. The
successive order of percentage cell viability is C1-C8 > C9- C14> L1-L8> L9-L14. Cytotoxicity of
Os(IV) complexes have been better than iridium(III) and palladium(II) complexes [40, 41].
Further, cytotoxicity has been evaluated using brine shrimp lethality assay. With the help
of the plot, LC50 values have been calculated. LC50 of ligands L1-L14 fall in the range of 4.00-11.74
μg/mL whereas, osmium(IV) complexes show LC50 in the range of 7.92-19.91 μg/mL. The bar
graph in figure 6 shows relative LC50 of all the synthesized compounds. The reason behind the
dominance of Os(IV) complexes is resistance for fertility of brine shrimps, thereby controlling the
11

pathogenic population by delocalizing the cell growth cycle with the help of nature of groups
around the molecule. In ligands, such impactful groups absentia does not play a vital role in
resisting growth but heterocyclic π character rich environment and substituents have an optimum
effect on resisting brine shrimp growth. Complex C4 has good cytotoxicity since it possesses nitro
functionality at the meta position. Further, complex C5 has fluorine in the ring which shows
comparable but less cytotoxicity compare to complex C4. Comparing C1 and C4, it is observed that
both compounds possess chlorine in rings. But one with chlorine at ortho position dominates in
cytotoxicity. On widely comparing compounds C1-C8 and C9-C14, former ones have better
cytotoxicity effect since C9-C10 has electron releasing functionality which relatively reduces effect.
Fig. 6. LC50 of ligands and Os(IV) complexes.
IC50 values of synthesized compounds have been calculated using HCT-116 lung cancer
cell line. IC50 values of compounds C1-C14 fall at about 46-1000 μg/mL given in plots mentioned
in supplementary material 14. Amongst the complexes C1-C8, complex C4 is the most effective
compound. Whereas, amongst the complexes C9-C14 having ligands with 4-hydroxy
benzhydrazide, complex C13 shows most effective compound, which are dominating in showing
antiproliferative activity compared to some of the reported platinum(II) complexes [42]. The
reason is the presence of electronegative chlorine atom in respective compounds which dominant
in showing cytotoxicity compare to other isostructural compounds with different substituents.
12

4. Conclusion
In this work, we conclude that compounds show intercalation mode of DNA binding with
HS-DNA as observed in absorption titration and docking measurements. The binding constants of
complexes fall at about 1.8-7.6×104 M-1. Oxadiazole based Os(IV) complexes show better binding
compare to reported copper(II) complexes. Whereas compounds C7, C9, and C10 dominate
compare to reported platinum ion-based complexes in binding revealed by fluorescence quenching
study. LC50 values of ligands L1-L14 fall in the range of 4.00-11.74 μg/mL whereas, osmium(IV)
complexes show LC50 values in the range of 7.92-19.91 μg/mL. The minimum inhibitory
concentration of Os(IV) dominates over their respective ligands. Ligands and Os(IV) complexes
show MIC in range of 142.50-180 and 72.50-110 μM, respectively. Relative tendency of
complexes
to
show
effective
smearing
is
in
order
of
C3>C5>C4>C1>C6>C8>C2>C7>C13>C9>C11>C12>C10>C14. The successive order of percentage cell
viability is C1-C8 > C9- C14> L1-L8> L9-L14. Cytotoxicity of Os(IV) complexes have been better
than iridium(III) complexes. Amongst the complexes C1-C8 having co-ligand benzhydrazide,
complex C4 has the most effective IC50. Whereas, amongst the complexes C9-C14 having co-ligand
ligands 4-hydroxy benzhydrazide, complex C13 shows the most effective IC50 value, which are
dominating in showing antiproliferative activity compared to some of reported platinum(II)
complexes.
Acknowledgements
The authors are thankful to the Head, Department of Chemistry, Sardar Patel University, Vallabh
Vidyanagar, Gujarat, India, for providing the laboratory facilities, U. G. C., New Delhi for
providing financial assistance of UGC BSR grant No. C/2015/BSR/Chemistry/1579. Authors are
also thankful to UGC-CPEPA and UGC-CAS programmes.
1
Supporting information: Materials and reagents, Physical measurements, Experimental, H-
NMR, 13C-NMR, LC-MS, ESI-MS, ICP-OES, Electronic spectrum, IR spectra, CHNS elemental
analysis, docking images, biological activities data.
13

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Highlights
 Os(IV) compounds show good binding strength and intercalation mode of binding with
HS-DNA.
 Os(IV) compounds show effective impact on displacing intercalator ethidium bromine in
fluorescence quenching study..
 Os(IV) compounds show good cytotoxicity over different sets of assays viz. percentage
cell viability on S. Pombe cell and BSLA.
 Os(IV) compounds show good antiproliferative activity against human cancer cell line
HCT-116.
17