Design, synthesis and biological evaluation of pyrazoline nucleus based homoleptic Ru(III) compounds

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Design, synthesis and biological evaluation of pyrazoline nucleus

based homoleptic Ru(III) compounds^†

Jugal V. Mehta, Sanjay B. Gajera, and Mohan N. Patel*

A

series of tri-substituted pyrazoline nucleus based homoleptic Ru(III) complexes of type [Ru(L^1-7)₂]•(PF₆)₃(L^1-7

=

heterocyclic pyrazoline derivatives) have been synthesized and characterized by elemental analysis, electronic spectra,

conductance measurements, thermo gravimetric analysis (TGA), electron paramagnetic resonance (EPR), fourier transform

infrared (FT-IR) and mass spectrometry (MS). An octahedral geometry around ruthenium has been assigned in all

complexes using electronic spectral analysis and EPR measurements. All compounds have been evaluated for cytotoxicity

activity against S. pombe cells at a cellular level. A comparative study of cellular level cytotoxicity values of all compounds

indicates that the metal complexes show better activity against S. pombe cells compared to the free heterocyclic

pyrazoline ligand. All complexes are good in vitro cytotoxic agents and LC₅₀values are in range of 5.57–8.01 mg/L. All

compounds have been screened for their in vitro antimicrobial study against five unlike microorganisms. All compounds

have been inspected for their interaction with Herring Sperm (HS) DNA expending absorption titration (K_b=0.37‒5.02×10⁵L

mol^-1) and viscosity measurement study, suggest the classical intercalative mode of DNA binding. Moreover, DNA-binding

property of all compounds have been examined theoretically using molecular docking study and suggests intercalation

binding mode between complex and nucleotide base pairs of DNA. The cleavage study on pUC19 DNA has been checked

by agarose gel electrophoresis. All newly synthesized compounds have been also evaluated for their in vitro antimalarial

study against Plasmodium falciparum strain (IC₅₀=0.46‒1.50 mg/L).

Received 00th January 20xx,

Accepted 00th January 20xx

DOI: 10.1039/x0xx00000x

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unsaturated carbonyl) with different hydrazine derivatives

under the basic conditions.

In coordination chemistry, ligands are most often electron

1. Introduction:

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. Amongst these, pyrazoline is a

nitrogen containing a five membered heterocyclic molecule

and plays major role in heterocyclic chemistry, a fact well

understood through its extensive usage as a synthon and

pharmacophore. Pyrazoline are an important class of

heterocyclic compounds containing two nitrogen atoms in the

donating organic compounds that bind with metal ions, thus

modifying the physical and chemical properties. Ligands can be

introduced into a system to limit the adverse effects of metal

ion in excess. Upon chelation of ligands to the metal ion some

drastic changes occurs which include increase in lipophilicity,

stabilizing specific oxidation states, and contributing to

substitution inertness. Properly designed ligands for metal-

based medicinal agents can play an integral role in achieving

five membered ring. These heterocyclic compounds widely the potential toxicity of a metallodrug, which can be used for

occur in nature in the form of vitamins, alkaloids, pigments

and as constituents of plant and animal cell. Pyrazoline

derivatives are the electron rich nitrogen heterocycles and

considerable attention has been focused on the pyrazoline and

substituted pyrazoline due to their diverse biological

diagnosis and therapy. 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. Among the transition metal complexes,

ruthenium-based complexes have been widely studied and

some have 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. In addition, both the commonly

applications. They displayed

a

wide spectrum of

pharmacological activities such as anticancer,¹antibacterial,²

antiviral,³

analgesic,⁴

anti-inammatory,⁵

antimalarial,

antituberculosis and antifungal activities.⁶These compounds

are generally prepared from the reactions of chalcones (α, β-

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accessible oxidation states of ruthenium complexes are

octahedral and relatively inert. Octahedral transition metal

complexes that contain one or more intercalating ligands have

drawn considerable interest due to their potential uses as

nucleic acid probes and as therapeutic agents.^7-10Also various

metal-containing compounds, ruthenium compounds are

considered stable under both highly acidic and alkaline

conditions.¹¹Applications of ruthenium compounds have a

long history. This includes antimicrobial activity, anti-cancer

activity, DNA binding, and DNA cleavage.

chlorobenzaldehyde,

p-bromobenzaldehyde,

m-chlorobenzaldehyde,

p-

methoxybenzaldehyde,

m-

bromobenzaldehyde, m-fluorobenzaldehyde, benzhydrazide,

potassium tert-butoxide (PTB), KPF_6,HS DNA and

ethylenediaminetetraacetic acid disodium salt (edta) were

purchased from Sigma Aldrich Chemical Co. (India). Agarose,

Luria Broth (LB), ethidium bromide (EtBr), tris-acetyl-edta

(TAE), bromophenol blue and xylene cyanol FF were purchased

from Himedia (India). S. pombe Var. Paul Linder 3360 was

obtained from IMTECH, Chandigarh.

2.2 Physical measurements

Metal based drugs play an important part in the history of

medicinal chemistry. A variety of transition metal complexes

with different metal centres and ligands of diverse structures

were synthesized and studied for their biological activity. The

metal complexes can also be extremely positively-charged:

since many biological structures – such as several types of

phospholipids, DNA and RNA, and some regions of proteins –

are negatively charged, for electrostatic reasons the positive

charge of metal complexes could aid the binding with

intracellular targets.

The ¹H NMR and ¹³C NMR were recorded with a Bruker Avance

(400 MHz). IR spectra were recorded on a FT–IR Shimadzu

spectrophotometer with sample prepared as KBr pellets in the

range 4000–400 cm^-1. C, H, and N elemental analysis were

performed thermofinnigan at CHN Lab, Panjab University,

Chandigarh. X-band electron paramagnetic resonance (EPR)

spectra were recorded with a Varian E-line Century Series

Spectrometer equipped with a dual cavity and operating at X-

band (~9.5 GHz) with 100 kHz modulation frequency at room

temperature. TCNE was used as field marker. Melting points

(°C, uncorrected) were determined in open capillaries on

ThermoCal₁₀melting point apparatus (Analab Scientific Pvt.

Ltd, India). Precoated silica gel plates (silica gel 0.25 mm, 60 G

F 254; Merck, Germany) were used for TLC. The LC-MS spectra

were recorded using make waters model micromass quattro

micro API mass spectrometer. The electronic spectra were

It is well known that, DNA is central target in the organism for

some metal based drugs or reagents. These reagents can

interact with DNA thereby changing the replication of DNA and

inhibiting the growth of the tumor cells.¹²Therefore,

considerable attentions have focused on the design and

synthesis of new 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.¹³It has been reported that

the metal complexes can interact with DNA non-covalently in

the mode of intercalation, groove binding and electrostatic

effect^{14, 15}and the effectiveness mainly depends on the mode

and affinity of the binding between the complexes and DNA.¹⁶

There is also a group of metal complexes containing existing

antibacterial as ligands: compared to their parent organic

antibacterial, the metalloantibacterial normally show

enhanced antimicrobial activity, especially against drug-

resistant bacterial strains.¹⁷

recorded on

Shimadzu, Kyoto (Japan). The magnetic moments were

measured by Gouy’s method using mercury

a

UV–160A UV–Vis spectrophotometer,

tetrathiocyanatocobaltate(II) as the calibrant (χ_g= 16.44 × 10^–6

cgs units at 20°C), Citizen balance. Antibacterial study was

carried out by means of laminar air flow cabinet Toshiba, Delhi

(India). The thermogram of complexes was recorded with a

Mettler Toledo TGA/DSC

1 thermogravimetric analyser.

Conductance measurement was carried out using conductivity

meter.

2.3 Synthesis of the ligands

In present study, a series of heterocyclic substituted pyrazoline

based homoleptic Ru(III) compounds have been synthesized

and well characterized. All synthesized compounds were

evaluated for their biological applications. This article mainly

focuses on exploring the trend in DNA-binding affinities of

synthesized compounds and important differences in some

related properties. Understanding the features that contribute

to recognition of DNA by small molecules or metal complexes

is essential for the improvement of drugs targeted at DNA. In

assessment of these inspections and in persistence of the

research work on bioactive heterocycles, it was anticipated to

plan and viewing them for biological applications.

2.3.1 General method for synthesis of enones (chalcones) (3a-

3g)

To the solution of 2-acetyl thiophene (1) (10 mmol, 1.17 mL)

in 10 mL of methanol, freshly prepared methanolic KOH

solution (20 mmol, 1.12 g, 25 mL) was added and stirred for

15 min. To this appropriate aldehyde (2a–g) (10 mmol) was

added and the reaction mixture was stirred at room

temperature for 24 h. The reaction mixture was cooled on an

ice bath and neutralized with dilute hydrochloric acid. The

precipitate appeared was separated by filtration and washed

three times with 60 mL distilled water to give the crude

product. The obtained product was recrystallized from

methanol. The purity of the products was checked on TLC by

using mixture of ethyl acetate and hexane as mobile phase.

2.3.2 General method for synthesis of 1, 3, 5-trisubstituted

2. Materials and methods

2.1 Materials and reagents

All the chemicals and solvents were of reagent grade and used

as purchased; double distilled water was used throughout the

studies. RuCl₃•3H₂O was purchased from Chemport (Mumbai,

India). 2-Acetyl thiophene, p-fluorobenzaldehyde, p-

pyrazoline (5a-5g)

To the solution of the appropriate enones (3a– 3g) (6 mmol) in

10 mL of methanol, benzhydrazide (4) (6 mmol, 0.82 g) and

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2.3.2.3 Synthesis of [5-(4-chlorophenyl)-3-(thiophen-2-yl)-4, 5-

dihydro-1H-pyrazol-1-yl]-phenyl methanone (5c)

freshly prepared methanolic potassium tert-butoxide (3 mmol,

0.34 g) solution were added and the reaction mixture was

refluxed for 5–6 h. Conversion was monitored in every 60 min

interval on precoated silica TLC plates by using mixture of ethyl

acetate and hexane as mobile phase. The excess of solvent was

removed under reduced pressure and the reaction mixture

was cooled on an ice bath. The products precipitated out at

low temperature were washed five times with 60 mL distilled

water, reconstituted in minimum amount of methanol and

dried under reduced pressure. The proposed reaction for the

synthesis of ligands 5a-5g is shown in scheme 1. ¹H NMR

spectra and ¹³C NMR spectra of ligands are shown in

supplementary material 1 and 2, respectively.

Prepared by above method from 3-(4-chlorophenyl)-1-

(thiophen-2-yl)-prop-2-en-1-one (3c) (6 mmol, 1.49 g) and

benzhydrazide (6 mmol, 0.82 g) after 5–6 h reflux; yield: 80%;

white amorphous solid; mp: 166°C; mol. wt. 366.86 g/mol;

anal. calc. for: C₂₀H₁₅N₂OClS, calc. (found) (%): C, 65.48

(65.41); H, 4.12 (4.05); N, 6.81 (6.75); MS (m/z): 367 [M+], 369

[M+2]; ¹H NMR (400 MHz, CDCl₃-d₁) δ/ppm: 3.199 (1H, dd, J =

5.2 Hz, 17.6 Hz, 4-H_a), 3.830 (1H, dd, J = 11.6 Hz, 17.6 Hz, 4-H_b),

5.834 (1H, dd, J = 4.8 Hz, 11.6 Hz, 5-H), 7.065-6.965 (3H, m,

3′′,4′′,6′′ -H), 7.091 (1H, dd, J = 3.6 Hz, 4.8 Hz, 3′-H), 7.542-

7.143 (6H, m, 2′′,2′′′,3′′′,4′′′,5′′′,6′′′-H), 8.054 (2H, d, J =7.2 Hz,

2′-H & 4′-H); ¹³C NMR (100 MHz, CDCl₃-d₁) δ/ppm: 166.05

(C=O), 150.11 (C₃), 140.14 (C_1′′), 134.80 (C_1′′′), 133.81 (C_4′′),

2.3.2.1 Synthesis of [5-(4-fluorophenyl)-3-(thiophen-2-yl)-4, 5-

dihydro-1H-pyrazol-1-yl]-phenyl methanone (5a)

Prepared by above method from 3-(4-fluorophenyl)-1- 133.59 (C_1′), 131.18 (C_4′′′), 130.23 (C_{3′′′, 5′′′}), 129.02 (C_{2′′, 6′′}),

(thiophen-2-yl)-prop-2-en-1-one (3a) (6 mmol, 1.39 g) and 128.88 (C_{2′′′, 6′′′}), 127.69 (C_{3′′, 5′′}), 127.18 (C_2′), 125.98 (C_3′),

benzhydrazide (6 mmol, 0.82 g) after 5–6 h reflux; yield: 77%; 124.54 (C₄′), 60.90 (C₅), 42.09 (C₄); [Signals observed=16: Ar-

white amorphous solid; mp: 147°C; mol. wt. 350.41 g/mol; C=4, Ar-CH=8, pyrazole-C=1, pyrazole-CH=1, pyrazole-CH₂=1,

anal. calc. for: C₂₀H₁₅N₂OFS, calc. (found) (%): C, 68.55 (68.50); CO=1]; IR (KBr, 4000–400 cm^–1): 3067, ν(C–H)_{ar stretching}; 1634,

H, 4.31 (4.22); N, 7.99 (7.90); MS (m/z): 351 [M+]; ¹H NMR ν(C=O); 1524, ν(C=N); 1321, ν(C=C); 1180, ν(C–N); 1084, ν(C–

(400 MHz, CDCl₃-d₁) δ/ppm: 3.192 (1H, dd, J = 4.8 Hz, 17.2 Hz, Cl); 1011, 919, (p–substituted aromatic ring); 780, ν(C–H)_ar

4-H_a), 3.822 (1H, dd, J = 11.6 Hz, 17.2 Hz, 4-H_b), 5.795 (1H, dd, J

= 5.2 Hz, 11.6 Hz, 5-H), 7.093 (1H, dd, J = 3.6 Hz, 5.2 Hz, 3′-H),

7.542-7.219 (9H, m, 2′′,3′′,5′′,6′′,2′′′,3′′′,4′′′,5′′′,6′′′-H), 8.054

(2H, d, J =7.2 Hz, 2′-H & 4′-H); ¹³C NMR (100 MHz, CDCl₃-d₁)

δ/ppm: 166.06 (C=O), 163.47 (C_4′′), 150.15 (C₃), 137.48 (C_1′′′),

137.44 (C_1′′), 134.87 (C_1′), 133.89 (C_4′′′), 131.13 (C_{3′′′, 5′′′}), 130.22

(C_{2′′, 6′′}), 128.98 (C_{2′′′, 6′′′}), 128.86 (C_2′), 127.69 (C_3′), 127.67 (C₄′),

115.98 (C_{3′′, 5′′}), 60.82 (C₅), 42.19 (C₄); [Signals observed=16: Ar-

C=4, Ar-CH=8, pyrazole-C=1, pyrazole-CH=1, pyrazole-CH₂=1,

CO =1]; IR (KBr, 4000–400 cm^–1): 3067, ν(C–H)_{ar stretching}; 1631,

ν(C=O); 1505, ν(C=N); 1324, ν(C=C); 1220, ν(C–F); 1152, ν(C–N);

.

bending

2.3.2.4 Synthesis of [5-(3-chlorophenyl)-3-(thiophen-2-yl)-4, 5-

dihydro-1H-pyrazol-1-yl]-phenyl methanone (5d)

Prepared by above method from 3-(3-chlorophenyl)-1-

(thiophen-2-yl)-prop-2-en-1-one (3d) (6 mmol, 1.49 g) and

benzhydrazide (6 mmol, 0.82 g) after 5–6 h reflux; yield: 81%;

white amorphous solid; mp: 150°C; mol. wt. 366.86 g/mol;

anal. calc. for: C₂₀H₁₅N₂OClS, calc. (found) (%): C, 65.48

(65.41); H, 4.12 (4.07); N, 7.64 (7.60); MS (m/z): 367 [M+], 369

[M+2]; ¹H NMR (400 MHz, CDCl₃-d₁) δ/ppm: 3.192 (1H, dd, J =

4.8 Hz, 17.2 Hz, 4-H_a), 3.825 (1H, dd, J = 11.6 Hz, 17.2 Hz, 4-H_b),

5.806 (1H, dd, J = 5.2 Hz, 12.0 Hz, 5-H), 7.093 (1H, dd, J = 3.6

1017, 922, (p–substituted aromatic ring); 786, ν(C–H)_{ar bending}

.

2.3.2.2 Synthesis of [5-(3-fluorophenyl)-3-(thiophen-2-yl)-4, 5-

Hz,

5.2

Hz,

3′-H),

7.543-7.239

(9H,

m,

dihydro-1H-pyrazol-1-yl]-phenyl methanone (5b)

2′′,3′′,4′′,6′′,2′′′,3′′′,4′′′,5′′′,6′′′-H), 8.057 (2H, d, J =7.2 Hz, 2′-H &

Prepared by above method from 3-(3-fluorophenyl)-1- 4′-H); ¹³C NMR (100 MHz, CDCl₃-d₁) δ/ppm: 166.11 (C=O),

(thiophen-2-yl)-prop-2-en-1-one (3b) (6 mmol, 1.39 g) and 150.13 (C₃), 144.13 (C_1′′), 144.06 (C_1′′′), 134.77 (C_1′), 133.79

benzhydrazide (6 mmol, 0.82 g) after 5–6 h reflux; yield: 76%; (C_3′′), 131.13 (C_4′′′), 130.67 (C_2′′), 130.59 (C_4′′), 129.18 (C_5′′),

white amorphous solid; mp: 155°C; mol. wt. 350.41 g/mol; 127.37 (C_{3′′′, 5′′′}), 121.38 (C_{2′′′, 6′′′}), 114.89 (C_6′′), 114.68 (C_2′),

anal. calc. for: C₂₀H₁₅N₂OFS, calc. (found) (%): C, 68.55 (68.48); 112.85 (C_3′), 112.63 (C₄′), 60.98 (C₅), 42.12 (C₄); [Signals

H, 4.31 (4.25); N, 7.99 (7.90); MS (m/z): 351 [M+]; ¹H NMR observed=18: Ar-C=4, Ar-CH=10, pyrazole-C=1, pyrazole-CH=1,

(400 MHz, CDCl₃-d₁) δ/ppm: 3.199 (1H, dd, J = 5.2 Hz, 17.6 Hz, pyrazole-CH₂=1, CO=1]; IR (KBr, 4000–400 cm^–1): 3081, ν(C–

4-H_a), 3.830 (1H, dd, J = 11.6 Hz, 17.6 Hz, 4-H_b), 5.834 (1H, dd, J H)_ar; 1631, ν(C=O); 1524, ν(C=N); 1327, ν(C=C); 1076, ν(C–Cl);

= 4.8 Hz, 11.6 Hz, 5-H), 7.065-6.965 (3H, m, 3′′,4′′,6′′ -H), 7.091 1200, ν(C–N); 922, (m–substituted aromatic ring); 787, ν(C–H)_ar

(1H, dd, J = 3.6 Hz, 4.8 Hz, 3′-H), 7.542-7.143 (6H, m,

2′′,2′′′,3′′′,4′′′,5′′′,6′′′-H), 8.054 (2H, d, J =7.2 Hz, 2′-H & 4′-H); ¹³C

NMR (100 MHz, CDCl₃-d₁) δ/ppm: 166.10 (C=O), 150.11 (C₃),

143.60 (C_1′′), 134.87 (C_1′′′), 134.73 (C_1′), 133.75 (C_3′′), 131.21

(C_4′′′), 130.32 (C_2′′), 130.26 (C_4′′), 129.06 (C_5′′), 128.94 (C_6′′),

128.05 (C_{3′′′, 5′′′}), 127.70 (C_{2′′′, 6′′′}), 125.91 (C_2′), 124.90 (C_3′),

123.97 (C₄′), 60.98 (C₅), 42.11 (C₄); [Signals observed=18: Ar-

C=4, Ar-CH=10, pyrazole-C=1, pyrazole-CH=1, pyrazole-CH₂=1,

CO=1]; IR (KBr, 4000–400 cm^–1): 3066, ν(C–H)_{ar stretching}; 1635,

ν(C=O); 1523, ν(C=N); 1323, ν(C=C); 1246, ν(C–F); 1129, ν(C–N);

.

bending

2.3.2.5 Synthesis of [5-(4-bromophenyl)-3-(thiophen-2-yl)-4, 5-

dihydro-1H-pyrazol-1-yl]-phenyl methanone (5e)

Prepared by above method from 3-(4-bromophenyl)-1-

(thiophen-2-yl)-prop-2-en-1-one (3e) (6 mmol, 1.76 g) and

benzhydrazide (6 mmol, 0.82 g) after 5–6 h reflux; yield: 72%;

white amorphous solid; mp: 152°C; mol. wt. 411.31 g/mol;

anal. calc. for: C₂₀H₁₅N₂OBrS, calc. (found) (%): C, 58.40

(58.33); H, 3.68 (3.61); N, 6.8 (6.75); MS (m/z): 411 [M+], 413

[M+2]; ¹H NMR (400 MHz, CDCl₃-

°d₁) δ/ppm: 3.174 (1H, dd, J

887, (m–substituted aromatic ring); 787, ν(C–H)_{ar bending}

.

= 5.2 Hz, 17.6 Hz, 4-H_a), 3.818 (1H, dd, J = 12.0 Hz, 17.6 Hz, 4-

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H_b), 5.791 (1H, dd, J = 5.2 Hz, 12.0 Hz, 5-H), 7.089 (1H, dd, J = ν(C–O–C)_sy; 1029 ν(C–O–C)_asy

4.0 Hz, 5.2 Hz, 3′-H), 7.535-7.231 (9H, m, aromatic ring); 787 ν(C–H)_{ar bending}

;

1076, 919 (p–substituted

.

2.3.3 General synthesis of the complexes

2′′,3′′,5′′,6′′,2′′′,3′′′,4′′′,5′′′,6′′′-H), 8.041 (2H, d, J =7.2 Hz, 2′-H &

4′-H); ¹³C NMR (100 MHz, CDCl₃-d₁) δ/ppm: 166.05 (C=O),

150.12 (C₃), 140.66 (C_1′′), 134.77 (C_1′′′), 133.79 (C_4′′), 132.12

(C_4′′′), 131.19 (C_{3′′, 5′′}), 130.23 (C_{3′′′, 5′′′}), 129.04 (C_{2′′, 6′′}), 128.90

(C_{2′′′, 6′′′}), 127.70 (C_2′), 127.69 (C_3′), 127.55 (C₄′), 121.69 (C_1′),

60.97 (C₅), 42.04 (C₄); [Signals observed=16: Ar-C=4, Ar-CH=8,

pyrazole-C=1, pyrazole-CH=1, pyrazole-CH₂=1, CO=1]; IR (KBr,

4000–400 cm^–1): 3067, ν(C–H)_{ar stretching}; 1637, ν(C=O); 1527,

ν(C=N); 1324, ν(C=C); 1068, ν(C–Br); 1139, ν(C–N); 1008, 919,

The homoleptic Ru(III) metal complexes (6a-6g) of the general

formula [RuL₂](PF₆)₃were synthesized by the reactions of

RuCl₃•3H₂O with the respective ligands (5a-5g) in a 1:2 molar

ratio in methanol.

2.3.3.1 Synthesis of Ru^IIIcomplex (6a)

A methanolic suspension of the precursor RuCl₃•3H₂O (0.130

g, 0.5 mmol) was refluxed for 10 min. Then a solution of ligand

5a (0.350 g, 1.0 mmol in 50 mL methanol), was added and the

reaction was refluxed overnight yielding a red-brown solution.

The reaction was filtered to remove the residual undissolved

material. Then, a saturated solution of excess KPF₆was added

(p–substituted aromatic ring); 784, ν(C–H)_{ar bending}

.

2.3.2.6 Synthesis of [5-(-bromophenyl)-3-(thiophen-2-yl)-4, 5-

dihydro-1H-pyrazol-1-yl]-phenyl methanone (5f)

Prepared by above method from 3-(3-bromophenyl)-1- drop wise to the reaction mixture to far-reaching precipitation

(thiophen-2-yl)-prop-2-en-1-one (3f) (6 mmol, 1.76 g) and and the mixture was kept at 4°C overnight. The brown

benzhydrazide (6 mmol, 0.82 g) after 5–6 h reflux; yield: 74%; precipitate was filtered off, washed with water as well as

white amorphous solid; mp: 142°C; mol. wt. 411.31 g/mol; diethyl ether and dried in vacuo at 35°C. The proposed

anal. calc. for: C₂₀H₁₅N₂OBrS, calc. (found) (%): C, 58.40 reaction for the synthesis of complexes (6a-6g) is shown in

(58.36); H, 3.68 (3.62); N, 6.81 (6.78); MS (m/z): 411 [M+], 413 scheme 1. Yield: 72%; mp: ≥ 300°C; µ_eff: 1.91 BM; mol. wt.

[M+2]; ¹H NMR (400 MHz, CDCl₃-d₁) δ/ppm: 3.214 (1H, dd, J = 1236.78 g/mol; anal. calc. for C₄₀H₃₀F₂₀N₄O₂P₃S₂Ru, calc.

4.8 Hz, 17.6 Hz, 4-H_a), 3.928 (1H, dd, J = 12.0 Hz, 18.0 Hz, 4-H_b), (found) (%): C, 38.85 (38.80); H, 2.44 (2.41); N, 4.53 (4.48); Ru,

5.772 (1H, dd, J = 4.8 Hz, 11.6 Hz, 5-H), 7.155 (1H, dd, J = 3.6 8.17 (8.12); IR (KBr, 4000–400 cm^–1): 3074, ν(C–H)_ar

;

stretching

Hz,

4.8

Hz,

3′-H),

7.735-7.273

(9H,

m, 1628, ν(C=O); 1523, ν(C=N); 1324, ν(C=C); 1232, ν(C–F); 1161,

2′′,3′′,4′′,6′′,2′′′,3′′′,4′′′,5′′′,6′′′-H), 7.836 (2H, d, J =7.2 Hz, 2′-H & ν(C–N); 1015, 923 (p–substituted aromatic ring); 841, ν(PF₆);

4′-H); ¹³C NMR (100 MHz, CDCl₃-d₁) δ/ppm: 166.85 (C=O), 791, ν(C–H)_{ar bending}conductance: 188 cm²Ω^-1mol^-1; UV-Vis: λ

;

159.13 (C_4′′), 150.52 (C₃), 135.12 (C_1′′′), 133.85 (C_1′′), 130.97 (nm) (ε, L mol^-1cm^-1): 572 (830), 310 (16320), 240 (21721).

2.3.3.2 Synthesis of Ru^IIIcomplex (6b)

(C_4′′′), 130.23 (C_{3′′′, 5′′′}), 128.79 (C_{2′′′, 6′′′}), 128.72 (C_{2′′, 6′′}), 127.64

(C_2′), 127.59 (C_3′), 127.07 (C₄′), 124.54 (C_1′), 114.34 (C_{3′′, 5′′}),

60.96 (C₅), 55.30 (-OCH₃), 42.21 (C₄); [Signals observed=18: Ar-

C=4, Ar-CH=10, pyrazole-C=1, pyrazole-CH=1, pyrazole-CH₂=1,

CO=1]; IR (KBr, 4000–400 cm^–1): 3074, ν(C–H)_{ar stretching}; 1633,

ν(C=O); 1527, ν(C=N); 1324, ν(C=C); 1068, ν(C–Br); 1139, ν(C–

It was synthesized using ligand 5b (0.350 g, 1.0 mmol). Yield:

74%; mp: ≥ 300°C; µ_eff1.82 BM; mol. wt. 1236.78 g/mol;

:

anal. calc. for C₄₀H₃₀F₂₀N₄O₂P₃S₂Ru, calc. (found) (%): C, 38.85

(38.79); H, 2.44 (2.38); N, 4.53 (4.48); Ru, 8.17 (8.11); IR (KBr,

4000–400 cm^–1): 3081, ν(C–H)_{ar stretching}; 1632, ν(C=O); 1531,

ν(C=N); 1324, ν(C=C); 1247, ν(C–F); 1130, ν(C–N); 879, (m–

N); 823, (m–substituted aromatic ring); 784, ν(C–H)_{ar bending}

.

2.3.2.7 Synthesis of [5-(4-methoxyphenyl)-3-(thiophen-2-yl)-4, 5-

dihydro-1H-pyrazol-1-yl]-phenyl methanone (5g)

substituted aromatic ring); 845, ν(PF₆); 787, ν(C–H)_ar

;

bending

conductance: 182 cm²Ω^-1mol^-1; UV-Vis: λ (nm) (ε, L mol^-1cm^-

¹): 585 (733), 313 (16840), 243 (22330).

Prepared by above method from 3-(4-methoxyphenyl)-1-

2.3.3.3 Synthesis of Ru^IIIcomplex (6c)

(thiophen-2-yl)-prop-2-en-1-one (3g) (6 mmol, 1.46 g) and

benzhydrazide (6 mmol, 0.82 g) after 5–6 h reflux; yield: 73%; It was synthesized using ligand 5c (0.366 g, 1.0 mmol). Yield:

White amorphous solid; mp: 156°C; mol. wt. 362.44 g/mol; 76%; mp: ≥ 300°C; µ_eff1.84 BM; mol. wt. 1269.69 g/mol; anal.

:

anal. calc. for: C₂₁H₁₈N₂O₂S, calc. (found) (%): C, 69.59 (69.55); calc. for C₄₀H₃₀F₁₈Cl₂N₄O₂P₃S₂Ru, calc. (found) (%): C, 37.84

H, 5.01 (4.97); N, 7.73 (7.68); MS (m/z): 363 [M+]; ¹H NMR (37.76); H, 2.38 (2.32); N, 4.41 (4.33); Ru, 7.96 (7.88); IR (KBr,

(400 MHz, CDCl₃-d₁) δ/ppm: 3.213 (1H, dd, J = 4.8 Hz, 17.6 Hz, 4000–400 cm^–1): 3088, ν(C–H)_{ar stretching}; 1630, ν(C=O); 1534,

4-H_a), 3.799 (1H, dd, J = 11.6 Hz, 17.2 Hz, 4-H_b), 3.800 (3H, s, ν(C=N); 1324, ν(C=C); 1093, ν(C–Cl); 1182, ν(C–N); 1015, 923

4′′-OCH₃), 5.802 (1H, dd, J = 4.8 Hz, 11.6 Hz, 5-H), 6.931-6.873 (p–substituted aromatic ring); 844, ν(PF₆); 787, ν(C–H)_{ar bending}

;

(3H, m, 3′′,4′′,6′′ -H), 7.089 (1H, dd, J = 3.6 Hz, 4.8 Hz, 3′-H), conductance: 186 cm²Ω^-1mol^-1; UV-Vis: λ (nm) (ε, L mol^-1cm^-

7.543-7.234 (6H, m, 2′′, 2′′′,3′′′,4′′′,5′′′,6′′′-H), 8.029 (2H, d, J ¹): 588 (778), 317 (17744), 225 (24825).

2.3.3.4 Synthesis of Ru^IIIcomplex (6d)

=7.2 Hz, 2′-H & 4′-H); ¹³C NMR (100 MHz, CDCl₃-d₁) δ/ppm:

166.25 (C=O), 159.06 (C_4′′), 154.76 (C₃), 140.72 (C_4′), 134.53

(C_1′′′), 134.19 (C_1′′), 130.82 (C_4′′′), 130.16 (C_{3′, 5′}), 129.43 (C_{3′′′, 5′′′}),

128.69 (C_1′), 127.59 (C_{2′′′, 6′′′}), 127.09 (C_{2′, 6′}), 126.75 (C_{2′′, 6′′}),

114.30 (C_{3′′,5′′}), 60.68 (C₅), 55.30 (-OCH₃), 41.58 (C₄), 21.52 (-

CH₃); [Signals observed=17: Ar-C=4, Ar-CH=8, pyrazole-C=1,

pyrazole-CH=1, pyrazole-CH₂=1, -CO =1, -OCH₃=1]; IR (KBr,

It was synthesized using ligand 5d (0.366 g, 1.0 mmol). Yield:

74%; mp: ≥ 300°C; µ_eff: 1.80 BM; mol. wt. 1269.69 g/mol; anal.

calc. for C₄₀H₃₀F₁₈Cl₂N₄O₂P₃S₂Ru, calc. (found) (%): C, 37.84

(37.75); H, 2.38 (2.31); N, 4.41 (4.35); Ru, 7.96 (7.90); IR (KBr,

4000–400 cm^–1): 3081, ν(C–H)_{ar stretching}; 1630, ν(C=O); 1523,

ν(C=N); 1328, ν(C=C); 1079, ν(C–Cl); 1200, ν(C–N); 923, (m–

4000–400 cm^–1): 3076, ν(C–H)_{ar stretching}; 2931 ν(C–H)_al

substituted aromatic ring); 844, ν(PF₆); 787, ν(C–H)_ar

;

stretching;

bending

conductance: 175 cm²Ω^-1mol^-1; UV-Vis: λ (nm) (ε, L mol^-1cm^-

¹): 593 (840), 333 (18322), 235 (23830).

1631 ν(C=O); 1516 ν(C=N); 1321 ν(C=C); 1182 ν(C–N); 1249

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2.3.3.5 Synthesis of Ru^IIIcomplex (6e)

ν(C=N); 1325, ν(C=C); 1068, ν(C–Br); 1139, ν(C–N); 1012, 919

(p–substituted aromatic ring); 844, ν(PF₆); 784, ν(C–H)_{ar bending}

;

It was synthesized using ligand 5e (0.411 g, 1.0 mmol). Yield:

conductance: 177 cm²Ω^-1mol^-1; UV-Vis: λ (nm) (ε, L mol^-1cm^-

¹): 590 (630), 330 (17485), 232 (23540).

80%; mp: ≥ 300°C; µ_eff: 1.78 BM; mol. wt. 1358.59 g/mol; anal.

calc. for C₄₀H₃₀F₁₈Br₂N₄O₂P₃S₂Ru, calc. (found) (%): C, 35.36

(35.30); H, 2.23 (2.17); N, 4.12 (4.08); Ru, 7.44 (7.38); IR (KBr,

4000–400 cm^–1): 3074, ν(C–H)_{ar stretching}; 1630, ν(C=O); 1527,

Scheme 1. Synthesis of pyrazoline ligands (5a‒5g) and its complexation with ruthenium metal ion.

2.3.3.6 Synthesis of Ru^IIIcomplex (6f)

incubated at 30°C on shaker at 150 rpm till the exponential

growth of S. pombe obtained (24 to 30 h). Then the cell culture

was treated with the different concentrations (2, 4, 6, 8, 10

mg/L) of synthesized complexes, free ligands and also with

dimethylsulphoxide (DMSO) as a control and further allowed

to grow for 16-18 h. Next day, by centrifugation at 10,000 rpm

10 min; treated cells were collected and dissolved in 500 µL of

PBS. The 80 µL of yeast culture dissolved in PBS and 20 µL of

0.4 % trypan blue prepared in PBS were mixed and cells were

observed in a compound microscope (40X). The dye could

enter the dead cell only so they appeared blue whereas live

cells resisted the entry of dye. The number of dead cells and

number of live cells were counted in one field. Cell counting

was repeated in two more of the microscopic fields and

average percentage of cells died due to synthesized

compounds were calculated.

It was synthesized using ligand 5f (0.411 g, 1.0 mmol). Yield:

79%; mp: ≥ 300°C; µ_eff1.90 BM; mol. wt. 1358.59 g/mol; anal.

:

calc. for C₄₀H₃₀F₁₈Br₂N₄O₂P₃S₂Ru, calc. (found) (%): C, 35.36

(35.30); H, 2.23 (2.20); N, 4.12 (4.06); Ru, 7.44 (7.35); IR (KBr,

4000–400 cm^–1): 3074, ν(C–H)_{ar stretching}; 1630, ν(C=O); 1523,

ν(C=N); 1324, ν(C=C); 1068, ν(C–Br); 1140, ν(C–N); 823, (m–

substituted aromatic ring); 848, ν(PF₆); 784, ν(C–H)_ar

;

bending

conductance: 190 cm²Ω^-1mol^-1; UV-Vis: λ (nm) (ε, L mol^-1cm^-

¹): 596 (648), 335 (16745), 244 (26825).

2.3.3.7 Synthesis of Ru^IIIcomplex (6g)

It was synthesized using ligand 5g (0.362 g, 1.0 mmol). Yield:

71%; mp: ≥ 300°C; µ_eff: 1.91 BM; mol. wt. 1260.85 g/mol; anal.

calc. for C₄₂H₃₆F₁₈N₄O₄P₃S₂Ru, calc. (found) (%): C, 40.01

(39.95); H, 2.88 (2.82); N, 4.44 (4.40); Ru, 8.02 (7.99); IR (KBr,

4000–400 cm^–1): 3074, ν(C–H)_{ar stretching}; 1629, ν(C=O); 1513,

2.4.2 In vitro cytotoxic study using brine shrimp lethality

ν(C=N); 1321, ν(C=C); 1250, ν(C–O–O)_sy; 1030, ν(C–O–O)_asy

1183, ν(C–N); 1079, 920, (p–substituted aromatic ring); 844,

ν(PF₆); 784, ν(C–H)_ar

;

bioassay (BSLB)

;

conductance: 194 cm²Ω^-1mol^-1; Brine shrimp (Artemia cysts) eggs were hatched in a shallow

UV-Vis: λ (nm) (ε, L mol^-1cm^-1): 582 (640), 330 (17628), 227 rectangular plastic dish (22×32 cm), filled with artificial

bending

(22833).

seawater, which was prepared with commercial salt mixture

and double distilled water. An unequal partition was made in

2.4 Biological applications of synthesized compounds

the plastic dish with the help of

a perforated device.

2.4.1 Cellular level bioassay using S. pombe cells

Approximately 50 mg of eggs were sprinkled into the large

compartment and was opened to ordinary light. After two

days, nauplii were collected by a pipette from the lighted side.

Cellular level bioassay was done using S. pombe cells, which

were grown in liquid yeast extract media in 150 mL Erlenmeyer

flask containing 50 mL of yeast extract media. Flask was

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The detail experimental process is reported in newly published level, grid dimension of 6 with receptor range 180 and ligand

range 180 with twist range 360 and distance range 40.²²

literatures.^{18, 19}

2.4.3 In-vitro antimicrobial screening

(IV)By gel electrophoresis

The in-vitro antimicrobial activities of free ligands, and Gel electrophoresis of pUC19 DNA was carried out in TAE

ruthenium complexes were tested against two gram positive; buffer (0.04 mol L^-1Tris–Acetate, pH 8, 0.001 mol L^-1edta). 15

Staphylococcus aureus, Bacillus subtilis and three gram µL reaction mixture containing 300 µg/mL plasmid DNA in TE

negative; Serratia marcescens, Escherichia coli, Pseudomonas buffer (10 mmol L^-1Tris, 1 mmol L^-1edta, pH 8.0) and 200 µmol

aeruginosa, microorganisms. The MIC value was determined L^-1complex solution. Reactions were allowed to proceed for 3

by broth dilution technique.²⁰The detail experimental process h at 37°C in dark and reactions were satiated by addition of 5

is reported in newly published literatures.^{18, 19}

2.4.4 DNA interactions

µL loading buffer (0.25% bromophenol blue, 40% sucrose,

0.25% xylene cyanol and 200 mmol L^-1edta). The aliquots were

loaded directly on to 1% agarose gel and electrophoresed at 50

V in 1× TAE buffer. Gel was stained with 0.5 mg/L ethidium

bromide and was photographed on a UV illuminator. After

electrophoresis, the proportion of DNA in each fraction was

estimated quantitatively from the intensity of the bands using

AlphaDigiDoc™ RT. Version V.4.0.0 PC–Image software. The

degree of DNA cleavage activity was expressed in terms of the

percentage of conversion of the SC-DNA to OC-DNA and L-DNA

according equation reported in published literatures.^{18, 19, 23}

2.4.5 In vitro antimalarial study

(I) By absorption spectral analysis

DNA-mediated hypochromic and bathochromic shifts under

the influence of the ruthenium complexes were measured

with the help of UV–Visible absorbance spectra. UV–Visible

spectral titration of Ru(III) complexes (in DMSO) with HS DNA

in phosphate buffer was carried out to inspect the binding

mode for the complexes. The concentration of HS DNA was

determined by measuring absorbance at 260 nm and using

12858 L mol^‒1cm^‒1as the molar extinction coefficient value.²¹

In experiment, fixed amount of DNA solution (100 µL) in

phosphate buffer was added to sample cell holding in definite

concentration of complex solution (20 µmol L^‒1) and reference

cell to nullify the effect of HS DNA, and allowed to incubate for

10 min prior to the spectra being recorded. DMSO was also

added into the reference cell as a control to nullify the effect

of DMSO. The intrinsic binding constant, K_b, was determined

by equation reported in published literatures.^{18, 19}

All the synthesized compounds were screened for their

antimalarial activity against the Plasmodium falciparum strain.

The P. falciparum strain was acquired from Shree R. B Shah

Mahavir Superspeciality hospital, Surat, Gujarat, India, and was

used in in vitro tests. The P. falciparum strains were cultivated

by a modified method described by Trager and Jensen.²⁴The

detail experimental process is reported in published

literatures.^{18, 19}

(II) By viscosity measurement

An Ubbelohde viscometer maintained at

a

constant

temperature of 27±0.1°C in a thermostatic jacket, was used to

measure the flow time of DNA in phosphate buffer

(Na₂HPO₄/NaH₂PO₄, pH 7.2) with accuracy of 0.01 second and

precision of 0.1 second. DNA samples, approximately 200 base

pairs in average length, were prepared by sonicating in order

to minimize complexities arising from DNA flexibility. Flow

Results and discussion

3.1 Magnetic moments, electronic spectra analysis and

conductance measurements

The room temperature magnetic moments show that the

homoleptic Ru(III) complexes are paramagnetic(1.78–1.91

BM), which corresponds to the +3 oxidation state of

ruthenium. The μ_effvalues indicate a single unpaired (S = ½)

electron and are consistent with low-spin t_2g⁵configuration for

ruthenium(III) ion in an octahedral environment.²⁵

time for buffer alone was measured and was termed as ꢀ_ꢁ. The

detail experimental process is reported in published

literatures.^{18, 19}

(III) By molecular docking with DNA sequence d(ACCGACGTCGGT)₂

The rigid molecular docking study has been performed using

HEX 8.0 software to determine the orientation of the Ru(III)

complexes binding to DNA. Docking was performed and the

most stable configuration was chosen as input for

investigation. The coordinates of metal complexes were taken

from their optimized structure as a .mol file and were

converted to .pdb format using CHIMERA 1.5.1 software. HS-

DNA used in the experimental work was too large for current

computational resources to dock, therefore, the structure of

the DNA of sequence d(ACCGACGTCGGT)₂(PDB id: 423D, a

familiar sequence used in oligodeoxynucleotide study)

obtained from the Protein Data Bank (www.rcsb.org/pdb). All

calculations were carried out on an Intel CORE i5, 2.5 GHz

based machine running MS Windows 8 64bit as the operating

system. The by default parameters were used for the docking

calculation with correlation type shape only, FFT mode at 3D

2

The ground state of Ru(III) (t_2g⁵configuration) is T_2g.²⁶In most

of the Ru(III) complexes, the electronic spectra show only

charge transfer bands.²⁷The electronic spectra of homoleptic

Ru(III) complexes show three bands in 225–596 nm region. The

band in the 572–596 nm region have been assigned to the d–d

transition. Other bands in the region 310–335 nm and 225–

244 nm have been assigned to the charge transfer transitions

designated as π–π* and n–π* transitions, respectively of non-

bonding electrons present on nitrogen atom of the

coordinated pyrazoline ligand in Ru(III) complexes. The shape

of the electronic spectra of all the Ru(III) complexes indicates

the existence of an octahedral environment around the

ruthenium(III) metal ion.²⁸

The molar conductance values for the synthesized low spin

Ru(III) complexes were determined in methanol and values are

found in the range of 175–194 cm²Ω^-1mol^-1, indicating

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electrolytic nature and presence of three counter ion (PF6^–) all the synthesized complexes exhibit a characteristic of an

outside the coordination sphere of Ru(III) complexes. So we axially symmetric system with g_⊥around 1.983–1.989, g_||

around 2.269–2.306 and g_avg= 2.078‒2.089 computed from

the formula g_avg= (1/3) (2g_⊥+ g_||) (Table 1). Overall the

location of lines and nature of the EPR spectra of the Ru(III)

complexes are characteristic of low spin octahedral

complexes.^30-32

g_||

Complexes

g_⊥

g_average

2.088

2.085

2.088

2.089

2.084

2.078

2.087

2.306

2.291

2.288

2.302

2.287

2.269

2.285

1.989

1.983

1.989

1.983

1.989

6a

6b

6c

6d

6e

6f

Table 1 EPR spectral data of synthesized complexes

Concentration(mg/L)

2

4

6

% Viability

71

8

10

6g

Compounds

conclude that all synthesized homoleptic Ru(III) complexes are

ionic in nature.

5a

5b

80

81

82

75

78

77

67

69

68

63

64

65

72

5c

5d

5e

5f

71

73

75

77

53

54

55

56

59

62

3.2 LC–MS spectrum analysis

83

84

85

60

61

62

64

68

72

78

79

81

57

58

61

63

67

70

72

75

50

51

52

53

55

60

67

68

70

72

45

47

48

49

50

52

55

The LC–MS spectrum and probable mass fragmentation

pattern of complex (6a) are shown in supplementary material

3 and 4, respectively. Mass spectrum of the complex (6a)

shows molecular ion peak [M−(PF₆)₃]⁺at 802 m/z. The peaks at

451 m/z is due to pyrazole ligands attached with ruthenium

metal ion. The peak at 350 m/z corresponds to pyrazole

moiety. The peaks at 245 m/z and 105 m/z are due to

fragmentation of pyrazole ligand.

5g

6a

6b

6c

6d

6e

6f

3.3 Thermogravimetric (TG) studies

Thermogravimetric analysis determines the weight changes of

a

sample, as

a

function of temperature or time.

6g

Thermogravimetric analysis is concerned with the change in

weight of a material as its temperature changes. This indicates

the temperature at which the material loses weight and the

loss indicates decomposition of the sample. 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.

DMSO

96

97

Untreated cells

3.5.Biological applications of synthesized compounds

3.5.1 Cellular level bioassay using S. pombe cells

The cellular level study of free pyrazoline (5a-5g) and

synthesized Ru(III) complexes (6a-6g) has been tested against

S.

pombe cells in terms of the percentage viability. Table 2 shows

that all the complexes are active against S. pombe cells than

those of respective pyrazoline ligands. S. pombe cells has

become an important tool to study cell biology due to its

eukaryotic and fairly big size characteristics. Cell death caused

by toxicity of the chemically synthesized compounds could be

easily observed by vital staining which is shown in figure 1.

TGA was carried out at a heating rate of 10°C per minute in the

range of 20–800°C under nitrogen atmosphere. The

characteristic thermo gravimetric gram in which mass loss % to

temperature in °C of complex (6a) shows two distinct mass

losses (supplementary material 5). The thermo gram of

complex (6a) shows no weight loss up to the temperature of

~200°C which infers the absence of lattice and coordinated

water. The mass loss (about 35%) during first step between

200 to 350°C corresponds to decomposition of three PF6⁻ion

from complex (6a).²⁹The second step (about 56%) corresponds

to decomposition of two tridentate ligand, in which six

coordinate covalent bonds are involved and leaving behind the

metal oxide as residue.

3.4 EPR analysis

The solid state X-band EPR spectrum of all complexes acquired

in the polycrystalline state at room temp using reference

material [2, 2-diphenyl-1-picrylhydrazyl (DPPH), g = 2.0023]

showed spectrum, exhibiting both parallel (g_||) and

perpendicular (g_⊥) g values. The low spin d⁵configuration is a

good examination of molecular structure and bonding since

the experimental ‘g’ values are very sensitive to small changes

in structure and to metal ligand covalency. The EPR spectra of

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[Ru^III(OFL)(PPh₃)₂Cl₂],³⁵

[Ru^III(PFL)(PPh₃)₂Cl₂],³⁵

[Ru^III(L^4-

Table 2: Effect of compounds on viability of S. Pombe cells at different concentrations

⁶)PPh₃)Cl₃]³⁶and comparable to [Ru^III(L^1-7)₃]•(PF₆)₃

compounds reported in published literature. The degree of

lethality was found to be directly proportional to the

concentration of the compounds. The mortality rate of nauplii

was found to increase with increasing the concentration of

compounds. The tested complexes have strong cytotoxic

activity but this examination is a primary one and further tests

are required to investigate its actual mechanism of cytotoxicity

and its probable effects on higher animal model and on cancer

cell line. It recommends that the complexes can be used as

potent cytotoxic agents with the hope of adding arsenal of

weapons used against the fatal disease cancer.

33

with error uncertainty in the value ±5%

Figure 1. Effect of compounds on S. pombe cells. Dead cells are seen dark whereas live

cells are seen transparent.

The toxicity was found to vary with the type of substituent

present and concentrations of the synthesized compounds.

General observation is that as concentration of compounds

increased the cytotoxicity was also increased. After 17 hours of

the treatment, many of the S. pombe cells were killed due to

toxic nature of the compounds. Complex (6a) is the most

active amongst all the compounds. All the complexes show

comparable study to [Ru^III(L^1-7)₃]•(PF₆)₃compounds reported in

published literature.³³

Figure 2. Plot of LC₅₀values of different compounds in mg/L using Brine shrimp. Error

bars represent standard deviation of three replicates.

3.6.2 In vitro cytotoxic study using brine shrimp lethality bioassay

(BSLB)

3.6.3 In-vitro antimicrobial screening

The BSLB is considered as a useful method for preliminary

calculation of toxicity of compounds and development in the

assay procedure of bioactive compound, which indicates

cytotoxicity as well as a wide range of pharmacological

activities

A relative study of in vitro antimicrobial screening values of the

pyrazoline ligands and their complexes indicates that the metal

complexes show superior activity against five unlike

microorganisms when incorporated to the pyrazoline ligand.

The minimum concentrations of compounds that induced a

complete growth inhibition are recorded as MICs value.

Satisfactory reason for this increase in antibacterial activity

may be considered in the light of Overtone’s concept¹⁹and

chelation theory.³⁷According to Overtone’s concept of cell

permeability, the lipid membrane that surrounds cell favours

the passage of only lipid soluble materials, so that

liposolubility is an important factor which controls

bacteriostatic activity. On chelation, the polarity of the Ru(III)

ion will be lowered to a greater extent due to the overlap of

the ligand orbital and partial sharing of the positive charge of

the Ru(III) ion with donor groups. Further, it increases the

delocalization of π-electrons over whole chelate ring and

enhances lipophilicity of the complexes. This lipophilicity

enhances the penetration of the complexes into lipid

membranes and blocks the metal binding sites in bacterial

enzymes. The complexes also disturb the respiratory processes

of the compounds. All the synthesized compounds were

studied for their cytotoxicity using the protocol of Meyer et

al ³⁴

The method is inexpensive, rapid, reliable and

.

economical.

Results for the lethality were noted in terms of deaths of

larvae. The mortality rate of brine shrimp nauplii was found to

increase with increasing the concentration of compounds. A

plot of the log of sample’s concentration versus percentage of

mortality showed a linear correlation. From the graph, the

LC50 values of the compounds were calculated, and they were

found in the range of 5.568 to 119.12 mg/L (supplementary

material 7). From the data recorded, complex (6a) is the most

potent amongst all the compounds. Figure 2 shows that the

synthesized complexes are good cytotoxic agent than that of

respective ligands. The order of potency of compounds is 6a >

6b > 6c > 6f > 6d > 6e > 6g > pyrazoline ligands. All the

complexes

show

superior

cytotoxicity

than

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of the cell and thus block the synthesis of proteins, which three selective modes: (I) a groove-bound fashion stabilized by

restricts further growth of the organism.

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.³⁹The intrinsic DNA binding constants i.e.

K_bof 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

DNA. An absorption spectrum of complexes with HS DNA was

recorded for a constant concentration of complexes (20 µmol

L^-1) with varying concentration of DNA (100 µmol L^-1) to obtain

different DNA/complex mixing ratio is shown in figure 4.

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 figure 3, we observed that the antimicrobial activity of all

complexes against different microorganisms are better than

that of respective ligands. The results concerning MIC values of

ligands and their complexes are represented in supplementary

material 8. The results indicate that a lower concentration of

complex (6a) is required to inhibit the bacterial growth and kill

the bacterial strain, leading to

a higher efficiency in

Figure 4. Absorption spectral changes on addition of HS DNA to the solution of complex

(6a) after incubating it for 10 minutes at room temperature in phosphate buffer

(Na₂HPO₄/NaH₂PO₄, pH=7.2). Inset: plot of [DNA]/ꢂꢃ_ꢄꢅ ꢃ_ꢆꢇ vs. [DNA]. Error bars

antimicrobial activities. Complex (6a) is the most active

amongst all the complexes due to presence of fluorine atom at

para position. Presence of more electronegative environment represent standard deviation of three replicates. (Arrow shows the change in

absorption with increase in concentration of DNA)

in complex (6a) expands its biological property. All the

complexes show lower MIC value against all bacterial species

than [Ru^III(OFL)(PPh₃)₂Cl₂]³⁵and [Ru^III(SPF)(PPh₃)₂Cl₂]³⁵but

Complexes binding to DNA through intercalation usually result

in hypochromism and bathochromism called red shift.⁴⁰When

the complex intercalates with base pairs of DNA, the π* orbital

of the intercalated ligand of the complex couples with π orbital

of the base pairs of DNA, thus decreasing the π–π* transition

energy and resulting in the bathochromism.⁴¹The absorption

spectral changes were monitored around 310−340 nm for the

investigation of DNA binding mode and strength. As the DNA

concentration is increased, transition bands of complexes

higher than [Ru^III(L^1–6)PPh₃)Cl₃]³⁶and comparable to [Ru^III(L^1-

33

⁷)₃]•(PF₆)₃compounds reported in published literature. All

the complexes show better activity against S. aureus, E. coli

and P. aeruginosa than those of [Ru(phen)₃]Cl₂, [Ru(4,7-

Me₂phen)₃]Cl₂and lower than [Ru(3,4,7,8-Me₄phen)₃]Cl₂

reported by Fangfei Li et al ³⁸

.

3.6.4 DNA interactions

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.

(6a−6g) exhibit hypochromicity [hypochromicity, H% = [(A_free

A_boundA_free100%] of about 17.94−23.36%, and

−

)

/

] ×

bathochromicity of 2−4 nm. The complex (6a) has highest

percentage hypochromicity (23.36%). These spectral features

may recommend a mode of binding that covers a stacking

interaction between the aromatic chromophore and the DNA

base pairs.

In order to elucidate the binding strength of the compounds,

the DNA-binding constants K_bwere determined by monitoring

the changes of absorbance in the transition band with increasing

(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

concentration of HS DNA. The K_bvalues of all compounds were

found in the range 0.36×10⁵‒ 5.02×10⁵L mol⁻¹(supplementary

material 7). It is lower than K_bvalue of classical intercalators

ethidium bromide (7.16×10⁵

L

mol⁻¹), [Ru(PEF)Cl₂(H₂O)₂]•5H₂O

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(5.00×10⁷L mol⁻¹),⁴²and [Ru^IIICl₂(PPh₃)₂L¹] (1.2±0.14 ×10⁶L mol⁻¹

MedChemComm

43

)

DNA by intercalation. This phenomenon may be explained by

the insertion of the compounds between the DNA base pairs,

but higher than [Ru^III(cipro)₃]•4H₂O (2.5±0.9 ×10⁴

L

mol⁻¹),⁴⁴

[Ru^III(L¹)(H₂O)₂]Cl (8.72×10⁴L mol⁻¹),⁴⁵[Ru^III(L²)(H₂O)₂]Cl (6.35×10⁴L leading to an increase in the separation of base pairs at

mol⁻¹),⁴⁵

[Ru^IIICl₂(PPh₃)₂L²]

(3.9±0.12

×10⁴

L

mol⁻¹),⁴³

intercalation sites and thus an increase in overall DNA length.

[Ru^IIICl₂(AsPh₃)₂L²] (1.6±0.11 ×10⁴

(3.6×10⁴

comparable with [Ru^IIICl₂(AsPh₃)₂L¹] (5.6±0.09 ×10⁵

L

mol⁻¹),⁴³[RuCl(AsPh₃)L²]

It is clear from the figure that all these complexes show an

increase in the relative viscosity of HS DNA. The increase in the

degree of viscosity of all compounds depends on their affinity

to DNA, with an order as follows: 6a > 6b > 6c > 6d > 6e > 6g >

6f > ligands (supplementary material 9). This behavior is similar

to that of ruthenium complexes mer-[Ru^IIICl₃(dmso)(bpy)],

mer-[Ru^IIICl₃(dmso)(bpy)], mer-[Ru^IIICl₃(dmso)(dpq)], mer-

[Ru^IIICl₃(dmso)(dppz)] and mer-[Ru^IIICl₃(CH₃CN)(dpq)] reported

46

L

mol⁻¹),⁴⁶[RuCl(AsPh₃)L³] (3.2×10⁴

L

mol⁻¹

)

and

L

mol⁻¹),⁴³

[RuCl(AsPh₃)L¹] (4.7×10⁵L mol⁻¹).⁴⁶From the K_bvalue and red shift,

it is clear that the complexes bind to the DNA by intercalation mode

and complex (6a) has the highest binding ability (figure 5) than the

other complexes and ligands due to an existence of fluorine atom.

An existence of fluorine atoms act as not only chemical isosteres for

the oxygen atoms in the heterocyclic base of thymidine, but also

participate in “strong” F-H bonding as a result it shows better

antibacterial and DNA interaction activity than other. The binding

mode is further confirmed by hydrodynamic volume (i.e. viscosity)

measurement.

by Caipihg. Tan et al ^47,48

The viscosity results may reveal the

.

tendency of each ligand to intercalate into DNA base pairs. The

increase in DNA viscosity observed in the complexes suggests a

classical intercalative mode.

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 K_bvalues of synthesized complexes in L mol⁻¹using HS DNA. Error bars

(III) By Molecular docking with DNA sequence d(ACCGACGTCGGT)₂

To understand the preferred orientation of sterically acceptable

compound with the DNA sequence at the theoretical level,

represent standard deviation of three replicates.

(II) By viscosity measurement

molecular docking study was performed. By placing

a small

The binding mode or nature of the interaction of all the

compounds was further confirmed by viscosity measurements.

Spectroscopic study are essential, but not satisfactory to

support a binding mode. 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

for studying the binding mode of metal complexes with DNA in

solution and provides stronger arguments for intercalative

binding mode.¹⁸The effects of synthesized complexes on the

viscosity of HS DNA are shown in figure 6.

molecule into the binding site of the target specific area of DNA,

this study has played important roles in the mechanistic study as

well as understanding the metal based drug-DNA interactions for

rational drug discovery and design. Molecular docking studies of the

compounds with the DNA duplex were performed to predict the

chosen binding site along with the preferred orientation of

compound inside the DNA helix which is shown in figure 7. The

study shown that the compounds under investigation interact with

DNA via an intercalation mode involving outside edge stacking

interaction with oxygen atom of the phosphate backbone. From the

resultant docked models, it is clear that the compounds fit well into

the intercalative mode of the targeted DNA and A−T rich region

stabilized by van der Waal’s interaction and hydrophobic

contacts.^{49, 50}The resulting binding energies of docked complexes

(6a−6g) were found to be −308.19, −294.45, −313.24, −297.67,

−313.24, −297.67 and −319.74 kJ mol⁻¹, respectively. Also the

As illustrated in this figure, on increasing the amount of the

complexes the relative viscosity of HS DNA increases steadily,

which confirms that the Ru(III) complexes are bound to HS

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binding energies of docked compounds 5a−5g were found to be

−252.46, −257.49, −257.50, −252.07, −256.68, −256.70 and −241.56

kJ mol⁻¹, respectively. All the complexes show comparable result to

[Ru^III(L^1-7)₃]•(PF₆)₃compounds reported in published literature.³³

Therefore, molecular docking together with absorption spectral and

viscosity studies indicate that the complex interacts with the DNA

through the classical intercalation mode mainly inside the DNA

helix.

Figure 8. Photogenic view of cleavage of pUC19 DNA (300 µg/cm³) with series of

compounds using 1% agarose gel containing 0.5 µg/cm³EtBr. All reactions were

incubated in TE buffer (pH 8) at a final volume of 15 mm³for 24 h at 37 °C.

Figure 8 clarifies the cleavage of pUC19 DNA induced by the

compounds under aerobic conditions. This clearly show that

the relative binding efficacy of complexes to DNA is much

higher than the binding efficacy of ruthenium salt and free

pyrazoline ligands (figure 9 and supplementary material 11).

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

Figure 7. Molecular docking of the complexes (6a) (ball and stick) with the DNA duplex

(VDW spheres) of sequence d(ACCGACGTCGGT)2. The complex is docked in to the DNA

showing intercalation between the DNA base pairs.

was

shown

by

reported

compounds

of

type

[Ru^III(PFL)(PPh₃)₂Cl₂],³⁵

[Ru^III(OFL)(PPh₃)₂Cl₂],³⁵

[Ru^III(SFL)(PPh₃)₂Cl₂],³⁵mer-[Ru^IIICl₃(CH₃CN)(dpq)]⁴⁷and mer-

[Ru^IIICl₃(dmso)(N−N)].⁴⁸

(IV)By gel electrophoresis

The cleavage study on plasmid DNA induced by synthesized

compounds can be monitored by agarose gel electrophoresis.

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

supercoiled (SC) form, also known as covalently coiled coil

DNA, here designated as Form I. Single strand cleavage results

in so called nicked or open circular (OC) form of DNA

(designated as Form II), whereas the double-strand cleavage

results in linear (LC) form of DNA (designated as Form III). The

migration rate during agarose gel electrophoresis depends on

both size (base pairs) and conformation, with smaller or

condensed DNA migrating faster than larger or unfolded DNA.

Form I has a tightly packed conformation and therefore

migrates faster through agarose gels than linear DNA

(intermediate migration) or open circle DNA (slowest

migration).

Figure 9. Plot of DNA cleavage data by agarose gel electrophoresis of compounds using

pUC19 DNA. Error bars represent standard deviation of three replicates.

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 of

phosphodiester bond to generate fragments which can be

subsequently relegated; started in a modest way of converting

supercoil (SC) form of DNA to the open-circular (OC) form and

last in linear (L) form, is now being used for identifying the

percentage of cleavage as a function of concentration of

nuclease. Oxidative DNA cleavage involves either oxidation of

the deoxyribose moiety by abstraction of sugar hydrogen or

oxidation of nucleobases.¹⁹

3.6.5 In vitro antimalarial screening

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.

Plasmodium falciparum is the parasite responsible for most

malaria cases up to 80%, which often proves harmless. Malaria

cause vast medical, financial, and emotional problem in the

world. As part of this search for novel drugs against malaria,

we report encouraging results for compounds resulting from

the modification of pyrazoline moiety by coordination to

ruthenium metal centres; the new compounds are highly

active against a chloroquine resistant strain of Plasmodium

falciparum

.

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MedChemComm

All newly synthesized Ru(III) complexes clearly shows the compared to free ligands. The antimicrobial activity of the all

advantage of ruthenium complexation and the requirement of compounds has been tested on five different microorganisms

six coordinated geometry around ruthenium as some of the and the data show an improved biological activity of all

key structural features for designing such metal-based complexes in relation to the free ligands. Hypochromism and

antimalarial agent. The results of the pharmacological bathochromism (red sift) of band in absorption spectral

screening are expressed as the drug concentration resulting in titration and increase in relative viscosity of DNA recommend

50% inhibition (IC₅₀) of parasite growth. The mean values of that all complexes bind with DNA via classical intercalative

IC₅₀in µg/mL for compounds are given in supplementary mode. Complex (6a) is bound more strongly than the other

material

7

(0.46‒1.50 mg/L). Figure 10 shows that all compounds due to presence of fluorine atom at 4^thposition of

complexes exhibit good antimalarial activity than respective aromatic ring. Presence of fluorine atoms act as not only

ligands. It is thus clear that the combination of ruthenium chemical isosteres for the oxygen atoms in the heterocyclic

metal and pyrazoline ligand in a

base of thymidine, but shows strong affinity of binding due to

single molecule does produce an improvement of the activity strong van der Waals force of interaction between fluorine and

against resistant strains of the parasite, demonstrating the hydrogen atom compared to other substituents as a result it

validity of our concept in the search for novel antimalarial shows superior antimicrobial and DNA interaction activity than

drugs capable of overcoming resistance. The IC₅₀values of all other compounds. Molecular docking studies of the complexes

III

33

Ru(III) complexes are comparable to [Ru (L^1-7)₃]•(PF₆)_3,

with the DNA duplex were performed to predict the chosen

[Ru(A)₂(B)]Cl₂(10 mg/L),⁵¹[Cu(terpyridyl)Cl]Cl (0.52 µmol L^-1) binding site which suggests intercalation between complex

and Fe(terpyridyl)Cl₃(0.63 µmol L^-1).⁵²

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 (6a) improves its biological property.

The ruthenium complexes have limited potential as

antimicrobial agents but found as good antimalarial agents.

However the cytotoxicity values indicate that complexes are

good cytotoxic agents compared to its antibacterial potency.

As the antibacterial activity and cytotoxic activity depends on

different mode of action and species being studied are

different. The preliminary studies encourage for carrying out

further in vivo experiments.

Acknowledgements

The authors thankful to the Head, Department of

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 University Grant Commission

under the scheme of “BSR UGC One Time Grant”, vide UGC

letter No.F.19-119/2014 (BSR).

Figure 10. Plot of IC₅₀values of different compounds in mg/L using Plasmodium

falciparum organism. Error bars represent standard deviation of three replicates.

4. Conclusions

A series of tri-substituted pyrazoline nucleus based homoleptic

Ru(III) compounds have been synthesized and well

characterized, in search of new metal based drugs looking

promising as potent cytotoxic, antimicrobial, antimalarial, DNA

cleavage as well as DNA binding 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. Data of magnetic

moment behavior, electronic spectra and EPR measurement

points towards the d⁵system with an octahedral environment

around the metal ion. The molar conductivities values of all

Ru(III) complexes are indicates, three PF₆counter ion present

outside the coordination sphere which accomplish all

complexes are ionic in nature. The Ru(III) complexes have

more potential than the free ligands as potential new

therapeutic agents. All the complexes show strong in vitro

cytotoxic, in vitro antimalarial as well as cellular level bioassay

Declaration of interest

The authors report no conflicts of interest. The authors alone

are responsible for the content and writing of the paper.

References

1

S. Fletcher, E. P. Keaney, C. G. Cummings, M. A.

Blaskovich, M. A. Hast, M. P. Glenn, S.-Y. Chang, C. J.

Bucher, R. J. Floyd, W. P. Katt, M. H. Gelb, W. C. Van

Voorhis, L. S. Beese, S. M. Sebti and A. D. Hamilton,

Journal of Medicinal Chemistry, 2010, 53, 6867-6888.

12 | MedChemComm., 2016, 00, 1-3

Please do not adjust margins

MedChemComm

Page 13 of 15

MedChemComm

View Article Online

DOI: 10.1039/C6MD00149A

PAPER

2

3

4

A. Tanitame, Y. Oyamada, K. Ofuji, M. Fujimoto, N. 21

Iwai, Y. Hiyama, K. Suzuki, H. Ito, H. Terauchi, M.

Kawasaki, K. Nagai, M. Wachi and J.-i. Yamagishi,

W. Zhong, J.-S. Yu and Y. Liang, Spectrochimica Acta

Part A: Molecular and Biomolecular Spectroscopy,

2003, 59, 1281-1288.

Journal of Medicinal Chemistry, 2004, 47, 3693-3696.

G. Ouyang, X.-J. Cai, Z. Chen, B.-A. Song, P. S. Bhadury,

S. Yang, L.-H. Jin, W. Xue, D.-Y. Hu and S. Zeng, Journal

22

M.-L. Liu, M. Jiang, K. Zheng, Y.-T. Li, Z.-Y. Wu and C.-W.

Yan, Journal of Coordination Chemistry, 2014, 67, 630-

648.

of Agricultural and Food Chemistry, 2008, 56, 10160- 23

10167.

J. Yang, R. N. S. Wong and M. S. Yang, Chemico-

Biological Interactions, 2000, 125, 221-232.

W. Trager and J. Jensen, Science, 1976, 193, 673-675.

M. M. T. Khan, D. Srinivas, R. I. Kureshy and N. H. Khan,

Polyhedron, 1991, 10, 2559-2565.

A. Hall, A. Billinton, S. H. Brown, N. M. Clayton, A. 24

Chowdhury, G. M. P. Giblin, P. Goldsmith, T. G. 25

Hayhow, D. N. Hurst, I. R. Kilford, A. Naylor, B.

Passingham and L. Winyard, Bioorganic & Medicinal 26

Chemistry Letters, 2008, 18, 3392-3399.

C. J. Ballhausen, Introduction to Ligand Field Theory,

McGarw Hill, New York, 1962.

5

6

B. P. Bandgar, S. S. Gawande, R. G. Bodade, N. M. 27

A. B. P. Lever, Inorganic Electronic Spectroscopy,

Elsevier, New York, 2 edn., 1984.

Gawande and C. N. Khobragade, Bioorganic

Medicinal Chemistry, 2009, 17, 8168-8173.

&

28

R. Samanta, B. Mondal, P. Munshi and G. Kumar Lahiri,

Journal of the Chemical Society, Dalton Transactions,

2001, DOI: 10.1039/B100163I, 1827-1833.

G. Sathyaraj, T. Weyhermüller and B. Nair, J Chem

Crystallogr, 2011, 41, 353-358.

S. C. Karad, V. B. Purohit, D. K. Raval, P. N. Kalaria, J. R.

Avalani, P. Thakor and V. R. Thakkar, RSC Advances,

2015, 5, 16000-16009.

29

30

31

7

8

9

N. W. Luedtke, J. S. Hwang, E. Nava, D. Gut, M. Kol and

Y. Tor, Nucleic Acids Research, 2003, 31, 5732-5740.

C. Metcalfe and J. A. Thomas, Chemical Society

Reviews, 2003, 32, 215-224.

M. M. T. Khan, D. Srinivas, R. I. Kureshy and N. H. Khan,

Inorganic Chemistry, 1990, 29, 2320-2326.

R. Ramesh and S. Maheswaran, Journal of Inorganic

Biochemistry, 2003, 96, 457-462.

B. M. Zeglis, V. C. Pierre and J. K. Barton, Chemical

Communications, 2007, DOI: 10.1039/B710949K, 4565- 32

4579.

O. K. Medhi and U. Agarwala, Inorganic Chemistry,

1980, 19, 1381-1384.

10

11

F. R. Keene, J. A. Smith and J. G. Collins, Coordination 33

Chemistry Reviews, 2009, 253, 2021-2035.

J. V. Mehta, S. B. Gajera, P. Thakor, V. R. Thakkar and

M. N. Patel, RSC Advances, 2015, 5, 85350-85362.

B. N. Meyer, N. R. Ferrigni, J. E. Putnam, L. B. Jacobsen,

D. E. Nichols and J. L. McLaughlin, Planta Med, 1982,

45, 31-34.

A. Bolhuis, L. Hand, J. E. Marshall, A. D. Richards, A. 34

Rodger and J. Aldrich-Wright, European Journal of

Pharmaceutical Sciences, 2011, 42, 313-317.

12

13

Y.-B. Zeng, N. Yang, W.-S. Liu and N. Tang, Journal of 35

Inorganic Biochemistry, 2003, 97, 258-264.

M. Patel, H. Joshi and C. Patel, J Chem Sci, 2014, 126,

739-749.

I. Meistermann, V. Moreno, M. J. Prieto, E. Moldrheim, 36

E. Sletten, S. Khalid, P. M. Rodger, J. C. Peberdy, C. J.

Isaac, A. Rodger and M. J. Hannon, Proceedings of the

P. S. Karia, P. A. Vekariya, A. P. Patidar, R. R. Patel and

M. N. Patel, Monatshefte für Chemie

- Chemical

Monthly, 2016, DOI: 10.1007/s00706-016-1708-8, 1-12.

B. G. Tweedy, Phytopathalogy, 1964, 55, 910.

F. Li, Y. Mulyana, M. Feterl, J. M. Warner, J. G. Collins

and F. R. Keene, Dalton Transactions, 2011, 40, 5032-

5038.

National Academy of Sciences, 2002, 99, 5069-5074.

37

14

15

C.-Y. Zhou, J. Zhao, Y.-B. Wu, C.-X. Yin and Y. Pin, 38

Journal of Inorganic Biochemistry, 2007, 101, 10-18.

F. Gao, H. Chao, F. Zhou, Y.-X. Yuan, B. Peng and L.-N.

Ji, Journal of Inorganic Biochemistry, 2006, 100, 1487- 39

1494.

C. V. Kumar and E. H. Asuncion, Journal of the

American Chemical Society, 1993, 115, 8547-8553.

J. R. J. Sorenson, Journal of Medicinal Chemistry, 1984,

27, 1747-1749.

16

17

18

19

B. A. Chabner and T. G. Roberts, Nat Rev Cancer, 2005, 40

5, 65-72.

L.-J. Ming, Medicinal Research Reviews, 2003, 23, 697- 41

762.

R. Indumathy, S. Radhika, M. Kanthimathi, T.

Weyhermuller and B. Unni Nair, Journal of Inorganic

Biochemistry, 2007, 101, 434-443.

J. V. Mehta, S. B. Gajera, D. D. Patel and M. N. Patel,

Applied Organometallic Chemistry, 2015, 29, 357-367.

42

H. Muslu, A. Golcu, M. Tumer and M. Ozsoz, Journal of

Coordination Chemistry, 2011, 64, 3393-3407.

K. Sampath, S. Sathiyaraj, G. Raja and C.

Jayabalakrishnan, Journal of Molecular Structure, 2013,

1046, 82-91.

J. V. Mehta, S. B. Gajera and M. N. Patel,

Spectrochimica Acta Part A: Molecular and 43

Biomolecular Spectroscopy, 2015, 136, Part C, 1881-

1892.

20

D. Sinha, A. K. Tiwari, S. Singh, G. Shukla, P. Mishra, H. 44

Chandra and A. K. Mishra, European Journal of

Medicinal Chemistry, 2008, 43, 160-165.

M. K. Tanimoto, K. Dias, S. Dovidauskas and S.

Nikolaou, Journal of Coordination Chemistry, 2012, 65,

1504-1517.

MedChemComm., 2016, 00, 1-3 | 13

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Page 14 of 15

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DOI: 10.1039/C6MD00149A

PAPER

MedChemComm

45

A. A. Abdel Aziz and H. A. Elbadawy, Spectrochimica

Acta Part A: Molecular and Biomolecular Spectroscopy,

2014, 124, 404-415.

46

G. Prakash, R. Manikandan, P. Viswanathamurthi, K.

Velmurugan and R. Nandhakumar, Journal of

Photochemistry and Photobiology B: Biology, 2014,

138, 63-74.

47

48

49

C. Tan, J. Liu, H. Li, W. Zheng, S. Shi, L. Chen and L. Ji,

Journal of Inorganic Biochemistry, 2008, 102, 347-358.

C. Tan, J. Liu, L. Chen, S. Shi and L. Ji, Journal of

Inorganic Biochemistry, 2008, 102, 1644-1653.

E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch,

D. M. Greenblatt, E. C. Meng and T. E. Ferrin, Journal of

Computational Chemistry, 2004, 25, 1605-1612.

S. Tabassum, M. Zaki, M. Afzal and F. Arjmand, Dalton

Transactions, 2013, 42, 10029-10041.

50

51

S. S. Anchuri, S. Thota, R. N. Bongoni, R. Yerra, R. N.

Reddy and S. Dhulipala, Journal of the Chinese Chemical

Society, 2013, 60, 153-159.

52

J. Paraskevopoulosa, P. Smithc, H. Hopped, D. Choprae,

T. Govenderb, H. Krugera and G. Maguirea, S. Afr. J.

Chem., 2013, 66, 80-85.

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Graphical abstract and synopsis

Biological applications of homoleptic Ru(III) compounds towards molecular docking, DNA-

binding and DNA cleavage.

Article Doi

Design, synthesis and biological evaluation of pyrazoline nucleus based homoleptic Ru(III) compounds

DOI: 10.1039/c6md00149a

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Article abstract of DOI:10.1039/c6md00149a

Full text of DOI:10.1039/c6md00149a

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