Stabilization of the ruthenium (II) and -(III) centres by chelating N-donor ligands: Synthesis, characterization, biomolecular affinities and computational studies

DOI: 10.1016/j.molstruc.2021.130986

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Journal of Molecular Structure (2021)

Update date:2022-08-03

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Authors:

Booysen, Irvin Noel

Maikoo, Sanam

Ramasami, Ponnadurai

Rhyman, Lydia

Xulu, Bheki

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Article abstract of DOI:10.1016/j.molstruc.2021.130986

Anticancer activities of ruthenium Schiff base metal complexes have been closely correlated to their steric factors and physicochemical properties. The core objectives of our research study were to synthesize and characterize new ruthenium Schiff base com

Full text of DOI:10.1016/j.molstruc.2021.130986

Journal of Molecular Structure 1244 (2021) 130986

Contents lists available at ScienceDirect

Journal of Molecular Structure

journal homepage: www.elsevier.com/locate/molstr

Stabilization of the ruthenium (II) and -(III) centres by chelating

N-donor ligands: Synthesis, characterization, biomolecular aﬃnities

and computational studies

Sanam Maikoo^a, Irvin Noel Booysen^a,∗, Bheki Xulu^a, Lydia Rhyman^b,c

Ponnadurai Ramasami^b,c

^aSchool of Chemistry and Physics, University of KwaZulu-Natal, Pietermaritzburg, South Africa

^bComputational Chemistry Group, Department of Chemistry, Faculty of Science, University of Mauritius, Réduit 80837, Mauritius

^cCentre of Natural Product, Department of Chemical Sciences, University of Johannesburg, Doornfontein, Johannesburg 2028, South Africa

a r t i c l e i n f o

a b s t r a c t

Article history:

Anticancer activities of ruthenium Schiff base metal complexes have been closely correlated to

their steric factors and physicochemical properties. The core objectives of our research study were

to synthesize and characterize new ruthenium Schiff base compounds derived from cinnamalde-

hyde, cuminaldehyde or 4-aminoantipyrine. In addition, the stereo-electronic features of the afore-

mentioned metal compounds and two selected previously reported ruthenium Schiff base com-

pounds were related to their antioxidant and biomolecular interaction capabilities. Therefore, we

demonstrated the formation of novel mononuclear ruthenium(III) compounds with chelating N-

donor ligands: fac-[RuCl₃(PPh₃)(ap)] (1) (ap = 4-aminoantipyrine), trans-P-[Ru(PPh₃)₂(cinap)₂](PF₆) (2)

Received 20 February 2021

Revised 21 June 2021

Accepted 25 June 2021

Available online 30 June 2021

Keywords:

Ruthenium

Spectral characterization and crystal

structures

(cinap

1,5-dimethyl-2-phenyl-4-{[3-phenylprop-2-en-1-ylidene]amino}-1,2-dihydro-3H-pyrazol-3-one)

N’-(4-isopropylbenzylidene)benzohydrazide).

Computational studies

DNA/BSA interaction studies

Radical scavenging

and cis-Cl, trans-P-[RuCl₂(PPh₃)₂(cumbh)] (3) (cumbh

Structural conﬁrmations were conducted primarily by spectroscopic techniques while the single crys-

tal X-ray analysis revealed the distorted octahedrons of the respective metal compounds. Voltammetry

experiments of 1–3 illustrated one-electron quasi-reversible redox waves which are attributed to metal

oxidation state interconversions. CT-DNA binding aﬃnities and modes of the novel metal complexes 1–

3 as well as the formerly published ruthenium Schiff base compounds, trans-P, cis-Cl-[Ru(pch)Cl₂(PPh₃)₂]

(4) (pch = 4-((pyridine-2yl-imino)methylene)-chromone) and cis-[RuCl₂(bpap)(PPh₃)] (5) (bpap = 2,6-bis-

((antipyrine-imino)methylene)pyridine) were experimentally and computationally investigated. The para-

magnetism of 1–4 and the proton-donor abilities of all the metal compounds promoted signiﬁcantly

higher NO and DPPH radical scavenging activities than the natural antioxidant, vitamin C. Experimental

electronic BSA titrations revealed that the metal compounds are ideal binders which preferentially bind

to site IIA. The X-ray and spectroscopic data were supported by the computed data which was simulated

using the density functional theory method.

1. Introduction

compounds than platinum chemotherapeutic drugs. In addition,

non-sterically hindered ruthenium anticancer drugs can be re-

Innovative metal-based anticancer drugs are essential to evolve

their current chemotherapeutic eﬃcacies from the current lack of

speciﬁcity and prevalent cancer resistant development of estab-

lished platinum-based drugs. In particular, ruthenium shares sim-

ilar electronic and redox properties to its physiologically-relevant

group congener, iron and these commonalities have accounted to

lower toxicity to non-cancerous cells of the ruthenium-anticancer

garded as pro-drugs as their substitution kinetics towards biolog-

ical nucleophiles and bimolecular modes of interactions mimics

that of cisplatin [1].

However, the next generation of ruthenium anticancer drugs

should have tailored biodistribution patterns and hence current

design strategies entail the inclusion of bio-active moieties into

multidentate ligands, that can facilitate bioavailability, target-

speciﬁcity and cytotoxicity. This design strategy is illustrated in the

diamagnetic arene ruthenium(II) compound, Ru(ɳ⁵-Cp)(PPh₃)(2,2’-

bipy-4,4’-R)]⁺(R = dibiotin ester) which contains biotinylated

∗

Corresponding author.

E-mail address: Booyseni@ukzn.ac.za (I.N. Booysen).

https://doi.org/10.1016/j.molstruc.2021.130986

S. Maikoo, I.N. Booysen, B. Xulu et al.

Journal of Molecular Structure 1244 (2021) 130986

groups anchored via a bipyridine chelator [2]. The aforementioned

metal complex salt showed markedly higher anticancer activities

than cisplatin in selected breast cancer cell lines. The use of Schiff

bases not only allow the stabilization of ruthenium in its com-

mon oxidation states +II or +III but also inclusion of various

bio-vectors within different Schiff base architectures [3]. In our

present research study, we have utilized chromone-, antipyrine-

encompassing imines and cinnamaldehyde, cuminaldehyde-derived

Schiff bases as potential biomarkers for cancerous cells.

antioxidant abilities and biological interactive studies with DNA

and BSA of the above mentioned compounds, as well as two

previously synthesised compounds containing the chromone and

4-aminoantipyrine moieties (trans-P, cis-Cl-[Ru^III(pch)Cl₂(PPh₃)₂]

(4) (pch

cis-[Ru^IICl₂(bpap)(PPh₃)] (5) (bpap

4-((pyridine-2ylimino)methylene)-chromone) and

2,6-bis-((antipyrine-

imino)methylene)pyridine)) were investigated [18]. In addition,

we used density functional theory method to complement the

experimental studies.

The secondary metabolite, chromone has been incorporated

as a pharmacophore in various medicinal drugs while those de-

rived from the essential oils, cinnamaldehyde and cuminaldehyde

portrays a wide variety of their inherent antioxidant and an-

timicrobial activities as well as new bio-activities [4,5]. Particu-

larly, Schiff bases of the essential oils when coordinated to M(II)

centers afforded metallo-drugs, [Ni(tcum)₂] (Htcum = cuminalde-

hyde thiosemicarbazone) and [Cu(tcin)(H₂O)Cl] (Htcin = trans-

cinnamaldehyde thiosemicarbazone), which exhibited proliferation

against the U937 human cell lines [6]. Organic derivatives of an-

tipyrine have also displayed a broad range of anticancer activi-

ties which has been improved for ruthenium complexes containing

antipyrine analogous [7]. Ruthenium Schiff base complexes with

chromone and antipyrine groups have also shown considerable

biological activities, namely [Ru(ɳ⁶-p-cymene)-(chromone)Cl] and

[RuCl(CO)(PPh₃)L] (HL = 4-(2-(2-hydroxyphenyl)ethylideneamino)-

1,2-dihydro-2,3-dimethyl-1-phenylpyrazol-5-one) produced good

cytotoxic effects when screen against the human cervical cancer

cell line, HeLa [8,9].

2. Experimental

2.1. Materials and methods

The metal precursor, trans-[RuCl₂(PPh₃)₃] as well as the organic

precursors, 4-aminoantipyrine, cinnamaldehyde, cuminaldehyde,

benzohydrazide and ammonium hexaﬂuorophosphate were all ac-

quired from Sigma-Aldrich. High purity ascorbic acid, Sodium ni-

troprusside, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Griess reagent,

phosphate buffered saline tablets (PBS), calf thymus (CT)-DNA,

bovine serum albumin (BSA), Ibuprofen, Warfarin and electrochem-

ical analysis grade and tetrabutylammonium hexaﬂuorophosphate

were also procured from Sigma Aldrich. Organic solvents were pur-

chased from Merck SA and used without further puriﬁcation. The

Schiff bases, cumap and cinap, as well as the ruthenium(II) metal

compounds 4 and 5 were prepared according to literature methods

[18,19].

¹H NMR spectra were run in the solvent d⁶-DMSO by using

a Bruker Advance 400 MHz spectrometer equipped with an au-

tosampler. A Bruker EMX-Plus X-band spectrometer operated at

9.83 GHz was used for EPR measurements at room temperature.

Operation parameters were as follows: microwave power of 2 mW,

centre magnetic ﬁeld of 330 mT, sweep width of 200 mT, modu-

lation frequency of 100 kHz and modulation amplitude of 0.6 mT.

Electronic spectra were run on a Perkin-Elmer Lambda 25 whereas

solid-state infrared spectra were collected on a Perkin-Elmer Spec-

trum 100. Melting point ranges were attained with a Stuart SMP3

melting point device. Redox properties of the respective ruthenium

compounds were explored using a Metrohm Autolab potentiostat

combined with a three electrode system: a glassy carbon work-

ing electrode (GCWE), a pseudo Ag|AgCl reference electrode and

an auxiliary Pt counter electrode. Electrochemical grade tetrabuty-

lammonium hexaﬂuorophosphate (0.1 M) was utilized as a sup-

porting electrolyte to the 2 mM dichloromethane solutions of the

metal compounds. Fluorescence experiments were conducted using

a 1 cm quartz emission cell and a Perkin Elmer LS-45 ﬂuorescence

spectrometer with a xenon lamp source.

Probing the interactions between metal complexes and various

biomolecules are essential in elucidating plausible mechanisms

of anticancer activity [10–12]. In particular, Schiff base ruthe-

nium compounds have illustrated distinctive binding to DNA

nucleic acids and protein residues in enzymes associated cancer

pathogenesis and their uptake by human serum album is critical

for effective physiological distribution [13–15]. Imino ruthenium

compounds can also infer antioxidant activities by scavenging re-

active oxygen species (ROS) which have been correlated with DNA

mutation [15,16]. A typical example includes the octahedral ruthe-

nium(III) species: [RuCl(AsPh₃)L¹] (H₂L¹= bis-(salicylaldehyde)-

S-methylisothiosemicarbazone) and [RuCl(AsPh₃)L²] (H₂L²= bis-

(5-chloro-salicylaldehyde)-S-methylisothiosemicarbazone)

which

could intercalate between the DNA base pairs despite the presence

of the bulky AsPh₃co-ligand within their respective coordination

spheres [1 7]. Furthermore, the individual unpaired d-electrons of

the paramagnetic metal complexes rendered superior radical neu-

tralizing capabilities over vitamin C and butylated hydroxytoluene.

In addition, the ruthenium compounds could form adducts with

BSA where quenching rate (K_q) constants ranging in the vicinity of

10⁵M⁻¹were attained.

In this research study, three novel ruthenium(III) com-

pounds were isolated from equimolar reactions of the

metal precursor, trans-[RuCl₂(PPh₃)₃] precursor with each

of the Schiff bases, cumap (1,5-dimethyl-2-phenyl-4-[4-

(propan-2-yl)benzylidene]amino-1,2-dihydro-3H-pyrazol-3-

2.2. Synthesis of N’-(4-isopropylbenzylidene)benzohydrazide (cumbh)

The compound cumbh was formed from the condensation re-

action between benzohydrazide (0.250 g, 1.84 mmol) and cumi-

naldehyde (0.272 g, 1.84 mmol) in the presence of three drops

of glacial acetic acid, see Scheme 1. The reaction mixture was

heated until reﬂux in ethanol (30 cm³) for 5 h. A light-yellow so-

lution was attained which was cooled to room temperature and

then ﬁltered. Excess cuminaldehyde was removed by washing with

petroleum ether to afford a cream precipitate. Yield: 61 %; m.p:

192.1–196.0 ˚C. IR (ν_max/cm⁻¹): ʋ(N-H)_amide3236; ʋ(C=O)_ketonic

1647; ʋ(C=N)_imine1551. ¹H NMR (295 K/ d⁶- CD₆SO/ ppm, see

Fig. S1): 8.45 (s, 1H, H10); 7.92 (d, 2H, H6, H8); 7.69–7.51 (m, 6H,

N2H, H13, H14, H15, H16, H17); 7.36 (d, 2H, H5, H9); 2.95 (p, 1H,

H2); 1.244 (d, 6H, H1, H1’, H1”, H3, H3’, H3”). UV-Vis (DCM, ε, M⁻¹

cm⁻¹): 229 nm (10100); 306 nm (17540).

one)

cinap

(1,5-dimethyl-2-phenyl-4-[3-phenylprop-2-en-

and N’-(4-

1-ylidene]amino-1,2-dihydro-3H-pyrazol-3-one)

isopropylbenzylidene)benzohydrazide (cumbh). These coordination

reactions resulted in the formation of the paramagnetic metal

compounds fac-[RuCl₃(PPh₃)(ap)] (1), trans-P-[Ru(PPh₃)₂(cinap)₂]

(2) and cis-Cl, trans-P-[RuCl₂(PPh₃)₂(cumbh)] (3). Structural elu-

cidations were established using single X-Ray diffraction and

supplemented with spectroscopic characterization. Interestingly

for 1, the solid state structure showed that cumap underwent

hydrolysis and consequently, only the ap moiety coordinated to

the trans-[RuCl₂(PPh₃)₃] unit, as opposed to 2 and 3 where the

Schiff bases remained intact upon coordination. Furthermore, the

S. Maikoo, I.N. Booysen, B. Xulu et al.

Journal of Molecular Structure 1244 (2021) 130986

742 (vs). UV-Vis (DCM, ε, M⁻¹cm⁻¹): 260 nm (sh, 18360); 265

nm (sh, 17870); 271 nm (sh, 14330); 325 nm (5420); 389 nm (sh,

3260); 624 nm (1200). Anal. Found: C, 60.479; H, 4.150; N, 2.727.

Anal. Calcd (with 1 molecule of DCM): C, 59.98; H, 4.47; N, 2.59.

2.6. X-Ray diffraction

Crystallographic data for the mononuclear compounds was col-

lected on a Bruker Apex Duo furnished with an Oxford Instru-

ments Cryojet. The diffractometer was operated at a low temper-

ature of 100(2) K and an Incoatec microsource was tuned at 30 W

power. Their crystal and structure reﬁnement are summarized in

Table 1 while the experimental geometrical parameters are shown

in Tables 2–4. An X-ray beam was generated with MoKα radiation

source with a wavelength of 0.71073 A and radiation exposures

were at a common crystal-to-detector distance of approximately

50 mm. In addition, data collection was achieved via omega and

phi scans at 0.50 frame widths, using APEX2 [20]. Data reduc-

tion were implemented with the aid of the SAINT program [20],

whereby scan speed scaling, standard Lorentz and polarization cor-

rection factors and also outlier rejection were applied. A semi-

empirical multi-scan absorption correction algorithm, SADABS was

used for data correction [21]. Direct methods, WinGX [22] and

SHELX-2016 [23] were applied to the solid-state structures. The x,

y, z coordinates of the non-hydrogen atoms were established using

the difference density map and anisotropically reﬁned with SHELX-

2016 [23]. Furthermore, three-dimensional orientations of the hy-

drogens were incorporated as idealized contributors in the least

squares process and their positions were computed using a stan-

Scheme 1. The condensation reaction between benzohydrazide and cuminaldehyde.

2.3. Synthesis of [RuCl₃(PPh₃)(ap)] (1)

A 1:1 molar reaction between cumap (0.0348 g, 0.104 mmol)

and trans-[RuCl₂(PPh₃)₃] (0.100 g, 0.104 mmol) was carried out

at elevated temperature until reﬂux for 5 hours in toluene (30

cm³). The dark red solution was cooled to room temperature, ﬁl-

tered at stored at STP conditions. After several days, rectangular-

shaped, red crystals were formed in the mother liquor which were

dard riding model with C-H_methylenedistances of 0.99 A, U_iso= 1.2

U_eq, C-H_methyldistances of 0.98 A, U_iso= 1.5 U_eq, C-H_aromaticdis-

tances of 0.93 A and U_iso= 1.2 U_eq

2.7. Computational studies

appropriate for X-ray analysis. Yield: 46 %. m.p: 271.6–274 C.

Density functional theory (DFT) method was used to perform all

computations. Full geometry optimisations using the X-ray struc-

tures of complexes 1-3 were carried out using the B3LYP [24,25]

functional in the gas phase with the LANL2DZ (Los Alamos Na-

tional Laboratory 2 double-ζ) [26–28] basis set on all atoms. The

LANL2DZ effective core potential (ECP) basis set on the ruthenium

atom, the 6-31G(2d,p) basis for chlorine atom and the 6–31G(d,p)

basis for carbon, nitrogen, phosphorus and oxygen atoms and 6-

31G basis for hydrogen atom were also used and this basis set

combination is denoted as BS1. In addition, the def2-tzvp basis set

for ruthenium atom and cc-pVDZ basis set for all other atoms were

also employed and this is denoted as BS2. The selected combina-

tion of B3LYP and basis sets provide reliable results as previously

justiﬁed in the literature [29,30]. Frequency computation of each

of the optimised geometry was also performed to ensure that the

complex was a true local minimum.

IR (ν_max/cm⁻¹): ʋ(N-H) 3050 (m); ʋ(C=O) 1565 (s); ʋ(Ru-[PPh₃])

692 (vs). UV-Vis (DCM, ε, M⁻¹cm⁻¹): 290 nm (20540); 339 nm

(18310); 461 nm (3360). Anal. Found: C, 60.653; H, 4.065; N, 5.225.

Anal. Calcd. (with 2 molecules of toluene): C, 60.32; H, 5.06; N,

4.91.

2.4. Synthesis of trans-P-[Ru(PPh₃)₂(cinap)₂](PF₆) (2)

A mixture of cinap (0.0331 g, 0.104 mmol), trans-[RuCl₂(PPh₃)₃]

(0.100 g, 0.104 mmol) and ammonium hexaﬂuorophosphate

(17.00 mg, 0.104 mmol) in methanol (20 cm³) was heated until re-

ﬂux for 3 h. The green solution was then cooled to room tempera-

ture and ﬁltered. Yield: 32 %. m.p: 216.4–220.0 C. IR (ν_max/cm⁻¹):

ʋ(C=N)_imine1486 (s); ʋ(N-C-O)_enolic1184 (m); ʋ(Ru-[PPh₃]₂) 693,

746 (vs). UV-Vis (DCM, ε, M⁻¹cm⁻¹): 359 nm (19480); 418 nm

(sh, 9590); 629 nm (2470). Crystals for elemental analysis were

grown from the slow diffusion of hexane into a chloroform solu-

tion of 2. Anal. Found:₋C, 57.198; H, 3.789; N, 5.186. Anal. Calcd.

(with 1 molecule of PF₆and 2 molecules of chloroform): C, 56.91;

H, 4.41; N, 5.11.

Full optimisations of complexes 1-3 were performed using the

CAM-B3LYP [31] functional in conjunction with the BS1 and BS2

basis sets in dichloromethane (DCM) using the polarisable contin-

uum model (PCM). This was followed by TD-DFT computations to

simulate the electronic spectra and to have a better understand-

ing of the electronic transitions. Transition energies and oscillator

strengths for the electronic excitation of the ﬁrst 70 excited states

of each complex were considered.

2.5. Synthesis of cis-Cl, trans-P-[RuCl₂(PPh₃)₂(cumbh)] (3)

Equimolar amount of cumbh (0.0278 g, 0.104 mmol) and trans-

[RuCl₂(PPh₃)₃] (0.100 g, 0.104 mmol) were heated until reﬂux in

ethanol (20 cm³) for 4 hours. The resulting green solution was

cooled to room temperature and ﬁltered. Green rectangular crys-

tals formed from the slow diffusion methods using a DCM: hexane

All the computations were carried out at 298.15 K and

1 atm. Gaussian 09 [32] was used for the optimisation using the

B3LYP/LANL2DZ method in the gas phase only for which the rel-

atively low Root-Mean-Square-Deviation (RMSD) values between

the optimized conformer and the solid-state structures were gener-

ated, see Fig. S2. All the other computations were performed using

Gaussian16 [33].

solvent system. Yield: 27%. m.p: 259.5–265.0 C. IR (ν_max/cm⁻¹):

ʋ(C=N)_imine1479 (s); ʋ(N-C-O)_enolic1186 (m); ʋ(Ru-[PPh₃]₂) 689,

S. Maikoo, I.N. Booysen, B. Xulu et al.

Journal of Molecular Structure 1244 (2021) 130986

Table 1

Crystal data and structure reﬁnement data for metal compounds 1 - 3.

•

2(PF₆)

Compound

C₇H₈

•

Chemical formula

Formula weight

Temperature (K)

Crystal system

Space group

₂₉H₂₈Cl₃N₃OPRu C₇H₈

C₇₆H₆₈N₆O₂P₂Ru

1550.31

C₅₃H₄₇Cl₂N₂OP₂Ru

765.07

961.83

100(2)

Monoclinic

P2₁/c

Monoclinic

P2₁/n

Monoclinic

P2₁/c

Unit cell dimensions (A,

)

a = 17.1746(14)

b = 10.2679 (9)

c = 19.9184(15)

α = 90

a = 13.8422(13)

b = 14.9830(12)

c = 16.8327(15)

α = 90

a = 22.6852(19)

b = 10.6935(9)

c = 20.1473(15)

α = 90

β = 103.950(3)

γ = 90

β = 104.312(4)

γ = 90

β = 109.933 (3)°

γ = 90

Crystal size (mm)

0.31 × 0.18 × 0.12

3409.0(5)

0.21 × 0.14 × 0.09

3382.7(5)

0.24 × 0.19 × 0.11

4594.6 (6)

V (A )

Density (calc.) (Mg/ m³)

1.491

1.522

1.390

Absorption coeﬃcient (mm⁻¹

)

0.78

0.41

0.57

F (000)

1564

1588

1980

ϴ range for data collection (deg)

Index ranges

2.7°; 26.3°

-21 ≤ h ≤ 20

-8 ≤ k ≤ 12

-23 ≤ l ≤ 24

21296

2.5°; 26.5°

-16 ≤ h ≤ 17

-18 ≤ k ≤ 13

-21 ≤ l ≤ 20

24263

2.7°; 28.3°

-30 ≤ h ≤ 30

-14 ≤ k ≤ 14

-26 ≤ l ≤ 22

44634

Reﬂections measured

Observed reﬂections (I > 2σ (I)

Independent reﬂections

Data/ restraints/ parameters

Goodness of ﬁt on F²

Observed R; wR²

6965

6825

11302

5034

4729

9305

6965/ 2/ 406

1.02

6825/ 0/ 459

1.10

11302 / 0/ 522

1.01

0.043; 0.091

0.053

0.072; 0.150

0.044

0.034; 0.077

0.046

R_int

Table 2

Selected bond lengths [A] and angles [ ] for complex 1.

Table 3

Selected bond lengths [A] and angles [ ] for complex 2.

Experimental

B3LYP/LANL2DZ

B3LYP/BS1

B3LYP/BS2

Experimental

B3LYP/LANL2DZ

B3LYP/BS1

B3LYP/BS2

Ru-Cl1

Ru-Cl2

Ru-Cl3

Ru-P

2.355(1)

2.318(1)

2.342(1)

2.304(1)

2.101(2)

2.201(3)

1.401(4)

1.274(4)

1.438(4)

1.374(4)

1.366(5)

83.3(1)

2.397

2.436

2.432

2.614

2.296

1.910

1.432

1.276

1.371

1.385

1.392

80.3

2.399

2.340

2.393

2.367

2.200

2.243

1.411

1.254

1.428

1.378

1.390

81.4

2.385

2.331

2.380

2.353

2.180

2.223

1.410

1.255

1.429

1.379

1.391

81.8

Ru-O1

2.153(3)

2.112(4)

2.4066(14)

2.4065(14)

1.276(7)

1.390(7)

1.352(7)

1.359(7)

1.352(7)

1.359(7)

1.275(6)

180

2.196

2.137

2.592

1.366

1.439

1.438

1.378

1.438

1.298

180.0

80.6

2.197

2.158

2.505

1.334

1.411

1.405

1.363

1.405

1.475

1.268

180.0

80.2

2.179

2.134

2.484

1.338

1.408

1.405

1.364

1.405

1.474

1.269

180.0

80.6

Ru-O2

Ru-N1

Ru-N2

Ru-O

Ru-P1

Ru-N3

Ru-P2

N2-N3

C1-O

C9-N1

C26-N4

N2-N3

C2-N3

C1-N1

N5-N6

C3-N2

N2-C11

N3-C13

N5-C28

N5-C30

C30-O2

C13-O1

P1-Ru-P2

O1-Ru-O2

N1-Ru-N4

N1-Ru-O1

N4-Ru-O2

N1-Ru-O2

N4-Ru-O1

O-Ru-N3

P-Ru-Cl2

Cl1-Ru-Cl3

P-Ru-N3

O-Ru-Cl2

90.26(4)

169.42(4)

178.61(8)

173.20(7)

88.3

93.2

93.3

161.6

172.1

167.4

165.7

176.9

170.8

166.1

177.3

170.7

180

2.8. UV-Vis spectrophotometric DNA titrations

82.63(14)

97.37(14)

80.6

80.2

80.6

99.4

99.8

99.4

The CT-DNA interactive studies of 1 - 3 were done at a pH

of 7.2 in phosphate-buffered saline (PBS). The CT-DNA solution in

PBS produced a ratio of 1.9:1 at 260 nm and 280 nm, which pro-

poses that the CT-DNA was effectively free of protein. The molar

absorption coeﬃcient (ε₂₆₀= 6600 M⁻¹cm⁻¹) was used in calcu-

lating the CT-DNA concentration per nucleotide [34]. The resulting

99.4

99.8

99.4

CT-DNA stock solution was kept at 4 C and used within a 48 h

window. Solutions of the metal complexes and CT-DNA were incu-

In the overhead equation, [DNA] is equivalent to the concentra-

tion of DNA in base pairs, ε_amatches the extinction coeﬃcient of

the particular absorption band at the speciﬁed DNA concentration

(corresponding to A_obs/[complex]), ε_fcorresponds to the extinction

coeﬃcient of the free compound in solution, and ε_bis equated to

the extinction coeﬃcient of the fully bound compound to DNA. The

plot of [DNA]/[ε_a- ε_f] versus [DNA], produces a slope of 1/[ε_a- ε_f]

and a Y intercept of 1/K_b[ε_b- ε_f]. The ratio of the slope to the

intercept is expected to be the intrinsic binding constant (K_b) [35].

bated at 25 C for 24 h preceding any UV-Vis measurements [35].

Afterwards, UV-Vis spectra of standard solutions for the individ-

ual metal compounds were collected in methanol and observed af-

ter the addition of varying concentrations of CT-DNA in PBS buffer.

The intrinsic binding constant (K_b) was determined by ﬁtting the

titration data into the following equation:

DNA

]

[

]

[

ꢀ

ꢁ

ꢀ

ꢁ

ꢀ

ꢁ

(A)

ε_a− − ε_f

ε_b− − ε_f

K_bε_b− − ε_f

S. Maikoo, I.N. Booysen, B. Xulu et al.

Journal of Molecular Structure 1244 (2021) 130986

Table 4

where A₀and A are equivalent to the absorbances of BSA at

280 nm in the absence and presence of a metal compound, ε_BSA

and ε_Bare the extinction coeﬃcients of BSA and the bound com-

plex (viz. adduct of a metal complex and BSA), K_appis equal to the

apparent association constant and C_compoundis the concentration of

a metal complex. The following double reciprocal plot can be pro-

duced from the equation (C) and the apparent association constant

(K_app) is determined from the ratio of the intercept to the slope

[39].

Selected bond lengths [A] and angles [ ] for complex 3.

Experimental

B3LYP/LANL2DZ

B3LYP/BS1

B3LYP/BS2

Ru-Cl1

2.3449(7)

2.3360(6)

2.029(2)

2.065(2)

2.39365(5)

2.4074(6)

1.289(3)

1.404(2)

1.314(3)

1.281(2)

76.33(6)

99.67(2)

176.51(2)

2.420

2.447

2.054

2.121

2.487

1.305

1.440

1.326

1.321

75.9

2.386

2.383

2.077

2.148

2.503

2.482

1.302

1.376

1.317

1.296

74.5

2.374

2.370

2.058

2.125

2.484

2.463

1.306

1.378

1.319

1.297

75.0

Ru-Cl2

Ru-O

Ru-N1

Ru-P1

Ru-P2

C10-N1

N1-N2

ꢂ

ꢃ

C11-N2

C11-O2

O1-Ru-N1

Cl1-Ru-Cl2

P1-Ru-P2

A − A

C_complex

(

)

99.1

100.4

175.7

99.9

2.10.2. Fluorescence emission spectroscopic titrations

176.0

175.2

The effects on the emission spectrum of BSA upon increasing

the metal compounds’ concentrations were evaluated. Fluorescence

emission spectra were performed at 293 K with the width of ex-

citation and emission slits adjusted to 5 nm. The spectra were

run within a wavelength range of 300–500 nm at an excitation of

280 nm. The data was used to estimate the Stern-Volmer quench-

ing constant (K_SV) by means of the Stern-Volmer relationship [40]:

2.9. Antioxidant studies

The experimental method for the radical scavenging experi-

ments were modiﬁed from literature methods [36,37]. Each experi-

ment was carried out in triplicate to safeguard data reproducibility.

The standard equation (B) was employed to ﬁnd the experimental

percentage radical scavenging activities:

I₀

= 1 + K_SVcomplex

(D)

[

]

ꢂ

ꢃ

A_c− A_f

% Radical scavenging activity =

x 100

(B)

where I₀and I are emission intensities in the absence and presence

of each metal compound. The K_SVvalues were determined from the

slope of:

A_c

where A_cis the control’s absorbance (NO or DPPH radicals) and

A_fis the absorbance following the addition of the different metal

compounds to the control. The IC₅₀values (concentrations which

induce 50% radical scavenging activity) of the metal compounds

were calculated from their distinctive experimental percentage

radical scavenging activities. The experiment for the DPPH radi-

cal assay was performed by running the control’s UV-Vis spectrum

[DPPH (0.2 mM in MeOH)] followed by the addition of 1 cm³of a

solution of metal compound [30 μM in MeOH] and thereafter the

sample solution was shaken to guarantee homogeneity. Then, the

sample solution underwent incubation in the dark for 20 min and

its UV-Vis spectrum was run.

I₀

vs complex

(E)

[

]

The quenching rate constant (K_q) could then be calculated from

equation (F):

K_SV= K_qτ₀

(F)

where τ₀is the lifetime of the protein (10⁻⁸s) without a quencher.

2.10.3. Competitive binding studies

Competitive binding experiments were carried out by employ-

ing two site markers (competitors), viz., warfarin for site I and

ibuprofen for site II. First, an equivalent concentration of the com-

petitor was added to BSA (each at 5 μM) and variations in the ﬂu-

orescence spectra were observed. The ﬂuorescence titration was

then achieved by varying the concentration of each metal com-

pound in the BSA-competitor solution and noting the spectral

changes [41]. The titration data was also ﬁtted to the Stern-Volmer

plot (equation E) and the attained quenching and rate constants

were compared to the values in the absence of the competitors

[42].

The following experimental technique was used for the NO rad-

ical assay: initially, a 10 mM solution of sodium nitroprusside was

made up in PBS buffer and incubated for 3 h at room temperature.

Subsequently, 1 cm³of Griess reagent was added to a 0.5 cm³of

the nitroprusside solution and this resulting solution functioned as

the control. The UV-Vis spectrum of the control was run. The sam-

ple solutions were prepared by the adding each metal compound

(30 μM in MeOH) to a 0.5 cm³aliquot of sodium nitroprusside.

After 3 h of incubation, 1 cm³of Griess reagent was added to each

sample solution and their respective UV-Vis spectra were run.

2.10. BSA binding interaction studies

3. Results and discussion

The BSA stock concentration was determined spectrophotomet-

rically by means of an extinction coeﬃcient of 43824 M⁻¹cm⁻¹

(at 280 nm) and using a solution prepared in PBS buffer at a pH of

7.2 [38]. The sample solutions of 1–5 were prepared in MeOH.

3.1. Synthesis, spectral characterization and redox properties of

complexes 1-3

Equimolar coordination reactions of the respective Schiff

bases: cumap, cinap and cumbh with trans-[RuCl₂(PPh₃)₂] af-

forded the paramagnetic complexes, [RuCl₃(PPh₃)ap (1), trans-P-

[Ru(PPh₃)₂(cinap)₂] (2) and cis-Cl, trans-P-[RuCl₂(PPh₃)₂(cumbh)]

(3) which were isolated in low to moderate yields, see Scheme 2.

It is clearly evident from the spectroscopic data of 1 that the

cumap Schiff base hydrolyzed upon coordination which afforded

the neutral bidentate N_aminoO_ketoniccoordination mode of the ap (4-

aminoantipyrine) moiety whilst the cinap and cumbh Schiff bases

remained intact in their corresponding metal compounds, 2 and 3,

see Figs. 1 - 3. The cinap ligand (for 2) coordinated in a ‘2 + 2’

manner through its monoanionic donor sets (N_imineO_enol) while

2.10.1. Electronic spectrophotometric titrations

The BSA interaction experiments were carried out by keeping

the BSA concentration constant (~20 μM) whilst varying the con-

centrations of the individual metal compounds (0–~320 μM). A

2 min incubation was used for the sample mixtures. Equivalent

volumes of metal compound were added to both the sample and

reference cells. The titration data was ﬁtted to the following equa-

tion:

ꢄ

ꢅ

ꢂ

ꢃ ꢂ

ꢃ

ꢆ

A_o

ε_BSA

ε_B

ε_BSA

ε_B.K_app

(C)

A_o− A

C_compound

S. Maikoo, I.N. Booysen, B. Xulu et al.

Journal of Molecular Structure 1244 (2021) 130986

Fig. 1. ORTEP view of metal complex 1 showing 50 % probability displacement el-

lipsoids and the atom labelling. The solvent molecule of recrystallization has been

omitted for the sake of clarity.

Scheme 2. Synthetic routes for the paramagnetic ruthenium complexes 1 – 3.

In addition, the monomeric metal complexes display also mid

to high solubility in polar aprotic solvents and moderate solubil-

ity in alcoholic media. The experimental and simulated IR spec-

tra of complexes 1-3 are displayed in Figs. S3 – S8. In general,

both the B3LYP/BS1 and B3LYP/BS2 methods are in agreement with

each other. The IR spectrum of 1 show medium-to-strong bands

ascribed to the vibrations of the amino and ketonic bonds while

an intense vibrational band at 692 cm⁻¹is ascribed to (Ru-[PPh₃])

[43]. Computed wavenumbers for the Ru-P stretching are found at

537 cm⁻¹. The most characteristic peak is assigned to stretching

vibrations of C-O and C=C in the region of 1680 cm⁻¹while the

C-N stretching corresponds to the intense peak in the region of

the remaining octahedral coordination sites are occupied by triph-

enylphosphine co-ligands, see Fig. 2. In contrast to 2, only a sin-

gle unit of the cumbh ligand functioned as a monoanionic chelator

(N_imineO_enol) for 3 leading to the stabilization of the paramagnetic

metal centre by the cis-orientated chloride and trans-positioned

triphenylphosphonine co-ligands, see Fig. 3. In our recent study,

we have computationally illustrated that the Schiff base function-

ality of cumap is weaker compared to that of cinap [19]. Conse-

quently, the latter rationalized the formation of 1 and 2. The hy-

drolysis of cumap is caused by water which originates from the

open atmosphere and then diffuses into the reaction mixture.

Fig. 2. ORTEP view of compound 2 showing 50 % probability displacement ellipsoids and the atom labelling. The counterion has been omitted.

S. Maikoo, I.N. Booysen, B. Xulu et al.

Journal of Molecular Structure 1244 (2021) 130986

Electronic spectra of 1 – 3 display several absorption bands

in the UV region below 400 nm, which are readily assigned to

ligand-centred π – π^∗and n – π^∗transitions occurring within

the Schiff base chelates [51, 52], see Figs. S9 – S11. Broad absorp-

tion bands appearing in the visible region in the spectra of 1 and

2, between 418–461 nm, are mainly attributed to Ligand-to-Metal

Charge Transfer (LMCT) bands. Truncated intensity d – d transi-

tions are observed for 2 and 3 at 629 and 624 nm respectively, due

to their low-spin d⁵electronic conﬁgurations. However, no metal-

based electronic transition was observed for 1 which hints at the

ap moiety acting as a strong-ﬁeld ligand that culminates into a

larger crystal-ﬁeld splitting energy [53].

The computed electronic spectra of 1 - 3 are displayed in Figs.

S12 - S14 which allowed for the interpretation of detailed elec-

tronic transitions within the complexes, refer to Tables S2 – S10.

Since the Ru has an unpaired electron in the complexes, the singly

occupied molecular orbitals (SOMOs) result into α and β orbitals.

The simulated UV-Vis spectrum of complex 1 is illustrated in Fig.

S12 indicating two absorption peaks at 234 and 387 nm using the

CAM-B3LYP/BS1 method. The main orbital transitions at 234 nm

are S-2(α)→L+2(α) (21%) and S-3(β)→L+3(β) (29%). The ﬁrst or-

bital transition illustrates a charge transfer from the Ru metal’s d

orbitals and p orbitals of Cl atoms to the cumap chelator; while

the second orbital transition is accounted to electron density re-

distribution of the cumap ligand and p orbitals of Cl atoms to the

d orbitals of the Ru metal. In addition, a secondary orbital elec-

tronic transition of S-2(β)→L+2(β) (19%) mainly concerns the d

orbitals on Ru atom and delocalisation of electron density within

the PPh₃moiety. The peak at 387 nm corresponds to two pri-

mary electronic transitions, S-8(β)→L(β) (44%) and S-3(β)→L(β)

(109%), which have LMCT character, particularly from the cumap

to the d orbitals of the Ru atom. However, an artefact of the CAM-

B3LYP/BS1 method is observed for the orbital contribution of 109%.

Likewise, two absorption peaks at 235 and 348 nm are observed,

with the B3LYP/BS2 method, which are of metal-to-ligand charge

transfer (MLCT) character when considering both α and β orbitals.

The simulated electronic spectrum of complex 2, which is il-

lustrated in Figure S13, has a peak at 293 nm (CAM-B3LYP/BS1)

which mainly arises from the S-7→L in both the α (50%) and β

(37%) orbitals. The transition involving the α orbitals corresponds

to the LMCT while that involving the β orbitals corresponds to

ligand-to-ligand charge transfer. The peak at 430 nm arises due to

the S(β)→L(β) (115%) which leads to MLCT character, originating

Fig. 3. ORTEP view of metal complex 3 showing 50 % probability displacement el-

lipsoids and the atom labelling.

1317 cm⁻¹. The actual trans-[Ru(PPh₃)₂] of 2 and 3 unit appears

dual ﬁnger print bands between 689–746 cm⁻¹

The hydrolysis of the cumap is conﬁrmed by the absence of its

imino signal (at 1595 cm⁻¹) in the IR spectrum of 1 while a shift

of the ketonic vibrational band to a lower frequency, is sugges-

tive of coordinative bonding between the ketonic oxygen and the

metal center. Furthermore, shifts are also observed when compar-

ing the IR spectra of 2 and 3 with their corresponding free-ligands’

IR spectra whereby the v(C=N)_iminosignals occur at lower frequen-

cies in those of the metal complexes than those of their related

free-ligands. In the IR spectrum of 2, the signals associated with

the ketonic C=O bond (originally observed at 1637 cm⁻¹in the IR

spectrum of the free-ligand) disappears which is a distinctive fea-

ture of the enol form of the chelator coordinating [44].

The most intense peak computed in the region of 1500 cm⁻¹

of complex 2 is assigned to the stretching of the C=C bond of the

alkene group while the peaks observed in the region of 1200 cm⁻¹

corresponds to the stretching of the C-N bond. The overlay IR spec-

tra of 3 and its uncoordinated ligand, indicates the loss of the N-

H amide group which is represented by the disappearance of the

infrared stretch at 3236 cm⁻¹and a new infrared stretch (appear-

ing at 1186 cm⁻¹) emanating from the formation of the C-O eno-

lic signal. Hence, the latter IR spectral changes support the con-

version of cumbh to its enol form, and its subsequent deprotona-

tion upon coordination to the metal centre [45, 46]. The experi-

mental C-O stretching frequency is in agreement with the com-

puted wavenumbers at 1183 cm⁻¹. Computationally, the most in-

tense peak in the region of 1550 cm⁻¹in the simulated IR spectra

of complex 3 corresponds to C=N stretching band.

from the d_zorbitals of the Ru metal to the ligand. Once again,

an artefact of the CAM-B3LYP/BS1 method is observed for the or-

bital contribution of 115%. Two peaks are observed using the CAM-

B3LYP/BS2 method at 285 and 374 nm. Both peaks correspond

mainly to LMCT.

The simulated UV-Vis spectrum of complex 3 show two major

transitions with high oscillator strengths as shown in Table S8. The

broad absorption band peak observed with the CAM-B3LYP/BS1

method consists of two peaks at 267 and 317 nm. The peak at 267

nm corresponds to the S-1(β)→L+2(β) (51%) transition consisting

of the electron transfer within the Ru and Cl atoms while the peak

at 317 nm mainly corresponds to S(α)→L(α) (34%) ligand-to-ligand

charge transfer. Two distinct transitions at 257 and 325 nm are ob-

served using the CAM-B3LYP/BS2 method. The peak at 257 nm in-

dicates mainly transition within the Ru and Cl atoms along with

LMCT character, particularly from cumbh to the Ru atom and the

peak at 325 nm consists mainly of the ligand-to-ligand charge

transfer.

The uncorrected computed wavenumbers of the complexes 1 -

3 allowed the assignments of the stretching vibrations related to

the Ru metal centres and these are listed in Table S1. The com-

puted wavenumbers are in agreement with experimental IR ab-

sorptions reported in literature; the Ru-Cl stretching vibrations are

reported within the range of 320–220 cm⁻¹[47,48] and the Ru-N

stretching vibrations are observed in the range of 650–250 cm⁻¹

[49]. The Ru-O and Ru-P stretching vibrations have been experi-

mentally observed within the range of 520–450 cm⁻¹[50,51] and

around 555 cm⁻¹[50], respectively.

The paramagnetic nature of the metal centres for the respec-

tive metal compounds were determined by ambient ESR spec-

troscopy, see Fig. 4. Asymmetry within the octahedrons of 1 and

3 is clearly evident which culminates into poor resolved rhom-

bic patterns (g_x= 2.27 for 1 and 2.26 for 3) [54]. In contrast,

S. Maikoo, I.N. Booysen, B. Xulu et al.

Journal of Molecular Structure 1244 (2021) 130986

fords non-linear bond angles [Cl1-Ru-Cl3

169.42(4)°, P-Ru-

N3 178.61(8)° and 173.20(7)°. Furthermore, the N1-Ru-O1

[82.63(14)°], N4-Ru-O2 [82.63(14)°], N1-Ru-O2 [97.37(14)°] and N4-

Ru-O1 [97.37(14)°] bond angles of 2 that constitute the O2N4O1N1

basal plane differ from the idealized value of 90° but the P1-Ru-

P2, O1-Ru-O2 and N1-Ru-N4 are linear. Contrastingly, the trans-

[Ru(PPh₃)₂]³⁺[176.51(2)°] of 3 is non-linear and it has a wide Cl1-

Ru-Cl2 [99.67(2)°] bond angle.

As expected, the Ru-O_ketonicbond of 1 [2.101(2) A] is shorter

than the Ru-O_enolbond of 2 [Ru-O1/O2 = 2.153(3) A] but the

latter is longer Ru-O_enolbond of 3 [2.029(2) A] which is due

to variable trans-inﬂuence imposed on the by their respective

enolic O-donor atoms. Though, the analogous bonds [1.984(1)

and 2.003(1) A] of the paramagnetic ruthenium compound,

[RuCl(obs)₂(PPh₃)₂] (Hobs = 2-hydroxyphenylbenzothiazole) were

comparable to those of 3 [60]. Indicatively, the hybridization of

the nitrogen donor atoms can be discriminated based on the

respective Ru-N_amino[2.201(3) A for 1] and Ru-N_imino[2.112(4)

Fig. 4. Overlay solution ESR spectra of the respective metal compounds.

A for 2 and 2.201(3) A for 3] bond lengths. As per literature

trend, the higher Lewis acidic character of 2 and 3 affords elon-

gated ruthenium-to-nitrogen bonds than the ‘3+3’ ruthenium(II)

Schiff base complex, [Ru(tpsal)₂] (Htpsal = 3,5-di-tert-butyl-N-(2-

(methylsulfanyl)phenyl)-salicylaldimine) with Ru-N_iminobond dis-

the symmetrical orientation of the co-ligands occupying coordina-

tion sphere of 2 renders three signals, viz. g_x= 2.42, g_y= 2.00

and g_z= 1.78. Overall, the nature of the ESR spectra and g-values

obtained are characteristic of octahedral organoruthenium(III)

complexes. Typical examples in literature includes the param-

agnetic ruthenium compounds trans-[RuCl(AsPh₃)₂(Nap-Nmtsc)]

(Nmtsc = napthaldehyde-N-methylthiosemicarbazide) and trans-

tances of 2.051(5) and 2.053(5) A. However, the Ru^III-N_aminobonds

are especially rare where predominately shorter Ru^II-N_aminobonds

of monomeric diamagnetic ruthenium complexes were found in

the Cambridge Crystallographic Database Centre (CCDC) [61–63].

Trans-axial orientations of the triphenylphosphine co-ligands in 2

[RuCl₂(AsPh₃)₂(L₂)] (HL₂

4-methoxy-2-hydroxybenzophenone)

exhibit respective isotropic and rhombic ESR spectra with g-values

ranging from 1.84 to 2.36 [54, 55].

[Ru-P1 = 2.4066(14) A and Ru-P2 = 2.4065(14) A] and 3 [Ru-

P1 = 2.39365(5) A and Ru-P2 = 2.4074(6) A] affords similar Ru-

Electron-transfer properties of 1–3 were probed in

dichloromethane via cyclic voltammetry (CV) and squarewave

voltammetry (SWV), see Figs. S15 - S17. The CVs of the metal

compounds shows single redox waves which are all considered as

quasi-reversible as their peak-to-peak separations differ from the

standard ferrocene (ꢀE_p= 90 mV vs Ag| AgCl), refer to Table S11.

Notably, sharp peaks are observed within CV of 1 which are typical

of adsorption on working electrode surface [56]. The propensity

of 1 to induce electrode modiﬁcation is tentatively ascribed to

its smaller size which allows its molecules to immobilize as a

P bonds whereas in 1 [Ru-P = 2.304(1) A], the amino nitro-

gen imposes on a stronger trans-effect on the phosphorus donor

atom. These ruthenium-to-phosphorous distances and the chloro-

coordination [Ru-Cl1 = 2.355(1) A, Ru-Cl2 = 2.318(1) A for 1 and

Ru-Cl1 – 2.3449(7) A, Ru-Cl2 = 2.3360(6) A for 2] bonds are com-

parable to other ruthenium(III) complexes found within literature

[64–67].

The comparison between the ketonic bond of

[C1-

O = 1.274(4) A] and the enol bonds of the other metal compounds

[C30-O2 = 1.275(6) A for 2 and C30-O2 = 1.281(2) A for 3]

thin ﬁlm on the GCE. The redox couples of 1 (E = 0 V vs Ag |

unequivocally show that the former exhibit double bond character.

AgCl) and 2 (E = 1.11 V vs Ag | AgCl) can be readily ascribed

Indicatively, the C1-N1 [1.374(4) A], C2-N3 [1.438(4) A], C3-N2

to respective Ru(II)/Ru(III) and Ru(III)/Ru(IV) couples whereas

[1.366(5) A] single bonds of 1 are elongated when compared to

the redox couple of 3 (E = -1.05 V vs Ag | AgCl) is assigned

the imino bonds of 2 [C9-N1/ C26-N4 = 1.276(7) A] and 3 [C10-

to the d⁵/d⁶interconversions [57–59]. These assignments are

based on the similarity of their half-wave potentials with that

of other ruthenium(III) species, e.g. mer-[Ru^III(bpy)(CH₃OH)Cl₃]

(bpy = bipyridine) display redox couples of -0.33 and +1.32 V (vs

Ag| Ag⁺) [57–59].

N1 = 1.374(4) A]. Interestingly, the characteristic N-N intracyclic

antipyrine bonds of 1 [N2-N3 = 1.401(4) A] and 2 [N2-N2/ N5-

N6 = 1.390(7) A] compares well with the corresponding aliphatic

bond of 3 [N1-N2 = 1.404(2) A]. Consequently, the neighbouring

C11-N2 [C11-N2 = 1.314(3) A] bond is a localized pi-bond whereas

those of the C-N antipyrine moieties of 1 and 2 [N2-C11/ N5-

C28 = 1.352(7) A and N3-C13/ N5-C30 = 1.359(7) A] are sigma

bonds. The computed structural parameters of complexes 1–3 are

comparable to those determined by the X-ray diffraction although

they are slightly larger than the experimental values, refer to

Tables 2 - 4 [68]. The root mean square deviations (RMSDs) for

the bond lengths and bond angles between the computed and

experimental structures were also calculated for non-hydrogen

atoms (Table S12) and the RMSDs indicate that the optimised

geometries using both computational methods are in agreement

with the experiment.

3.2. Crystallographic description

The mononuclear compounds 2 and 3 both exhibit a monoclinic

crystal system with 2 co-crystallizing with a PF₆counter-ion in a

P2₁/n space group while 3 crystallizing as a single molecule in a

P2₁/c space group, see Figs. 1 - 3. Hydrogen-bonded dimers of 1

crystallizes along with toluene molecules of recrystallization, see

Fig. S18. Non-classical close contacts between the hexaﬂuorophos-

phate counter-ions and their adjacent molecules of 2 allows these

molecules to pack in columns aligned with the [b]- and [c]-axes.

Similarly, non-polar interactions between neighbouring molecules

of 3 orientate co-planar with respect to the [b] and [c]-axes.

Constrained ﬁve-membered chelate rings [O-Ru-N3 = 83.3(1)°,

O1-Ru-N1/ O₂-Ru-N4 = 82.63(14)° and O1-Ru-N1 = 76.33(6)°]

within the respective ruthenium compounds induce octahedral

3.3. Antioxidant studies

Regulation of reactive oxygen species (ROS) within biological

media is of utmost importance since normal cellular operation is

dependent on the latter [69]. An increase in ROS can cause oxida-

distortion. In particular, the opposing donor atoms of

1 af-

S. Maikoo, I.N. Booysen, B. Xulu et al.

Journal of Molecular Structure 1244 (2021) 130986

Fig. 5. Overlay UV-Vis spectra of compound 1 in the absence and presence of increasing amounts of CT-DNA. A dashed line indicates the initial spectrum. Inset: Plot of

[DNA]/(ε_a- ε_f) x 10⁸vs [DNA] × 10⁵and the linear ﬁt for the titration.

Table 5

binding results in a hyperchromic effect with a signiﬁcant red shift

owing to the robust π – π^∗stacking interaction between the aro-

matic chromophore of a chelator and the base pairs of DNA [12,73].

Antioxidant activities of 1 – 5 and Vitamin C

against the DPPH and NO radicals.

Compound

DPPH Radical

IC₅₀(μM)

NO Radical

IC₅₀(μM)

Absorbance spectra of the metal compounds were distinctively

changed upon the progressive additions of CT-DNA, see Figs. 5, 6,

S19 – S21. Numerous clear isosbestic points are noted in the indi-

vidual electronic spectral proﬁles of 1 - 5 which reveal the pres-

ence of more than one spectroscopically distinct chromophore in

solution (viz. free and bound) and is suggestive of a single bind-

ing mode of the respective metal complexes with DNA [74]. With

the introduction of CT-DNA, hypochromicity along with a slight red

shift of the absorption band at 461 nm is detected. Despite the fact

141

210

Vitamin C

that the intrinsic binding constant (K_b) of 1, 2.67 × 10⁶

⁻¹, is of

tive damage to vital biomolecules or tissue which may contribute

to numerous diseases, such as, cancer, hypertension and Parkin-

son’s disease [70,71]. Antioxidants derivatized from metal coordi-

nation have recently gained attention in safeguarding biological

systems from the negative effects of ROS and the Schiff base com-

plexes of ruthenium are among these [53]. The radical scavenging

capabilities of 1–5 were assessed against the DPPH and NO radicals

and compared to the natural antioxidant, Vitamin C (the standard),

refer to Table 5. The formulated ruthenium compounds are seen to

possess higher DPPH (IC₅₀= 12–61 μM) and NO (IC₅₀= 21–54 μM)

radical scavenging activities than the standard (Vitamin C) and are

within range if the activities seen for other ruthenium compounds

[71,72].

the same magnitude as known traditional intercalators (10⁶M⁻¹

)

[75, 76], it is anticipated that 1 is a DNA groove-binder. This is

largely due to the presence of the bulky triphenylphosphine co-

ligand which impedes the DNA intercalation of 1.

Diminishing metal-based electronic transitions for

(at

620 nm) and 3 (at 625 nm) accompanied with no batchochromic

shifts suggest coordinative bonding between the respective metal

compounds and the phosphate group on the backbone or with the

DNA base pairs within the major or minor grooves of DNA [77–79].

In addition, an appearance of a charge transfer band at 425 nm in

the electronic spectra of 2 is indicative of a new specie forming in

solution due to the binding of the metal complex to DNA. A similar

trend is seen by a disappearance of an absorption band at 317 nm

with a simultaneous appearance of a shoulder at 382 nm in the

overlay electronic spectra of 3. Hypochromism was also observed

in the electronic spectral proﬁle of 5 at 383 nm; whilst that of 4

indicates overall hyperchromism.

3.4. DNA interaction studies

DNA is a critical target for anticancer transition metal com-

plexes and thus their modes of interactions and aﬃnities towards

the CT-DNA is of high importance. Electronic spectroscopy is a

technique that is universally employed to assess the binding in-

teractions between metal compounds and DNA. Generally, this is

achieved by monitoring changes in the UV-Vis spectral proﬁle of

a metal compound when exposed to standardized volumes of Calf

Thymus DNA (CT-DNA). Electrostatic or non-intercalative modes of

binding may result in a hyperchromic- or hypochromic effects with

no signiﬁcant wavelength shifts. However, an intercalative mode of

The intrinsic binding constants (K_b) were found to be be-

10⁵M⁻¹for 2–5, which further cor-

tween 2.75–7.00

roborates the fact that their DNA binding mode is indeed

non-intercalative since the K_bvalues are smaller than for

known intercalators or partial intercalators like ethidium bro-

mide (1.80 × 10⁶

M M

⁻¹) or [Ru(phen)₂(dppz)]²⁺(> 10^{6 −1}),

which is most likely due to the steric hindrance of the bulky

PPh₃co-ligands [74, 80]. Furthermore, groove-binding ruthe-

S. Maikoo, I.N. Booysen, B. Xulu et al.

Journal of Molecular Structure 1244 (2021) 130986

Fig. 6. Overlay UV-Vis spectra of compound 3 in the absence and presence of increasing amounts of CT-DNA. A dashed line indicates the initial spectrum. Inset: Plot of

[DNA]/(ε_a- ε_f) × 10⁷vs [DNA] × 10⁴and the linear ﬁt for the titration.

nium complexes [Ru(dmp)₂PMIP]²⁺(dmp

2,9-dimethyl-1,10-

mote folding of the protein and stabilizing it upon binding by con-

cealing it within the BSA’s hydrophobic pocket [85–88]. Contrar-

ily, a red shift was observed in the UV BSA titration proﬁle of 4

which is suggestive of slight unwinding of the BSA strand by the

metal complex which exposes the tryptophan residue to a more

hydrophilic environment in aqueous media [89, 90].

phenanthroline and PMIP = 2-(4-methylphenyl)imidazo[4,5-f]1,10-

phenanthroline) and [Ru(SPF)(PPh₃)₂Cl₂] (SPF = sparﬂoxacin) have

alike K_bvalues of 2.70 × 10⁵M⁻¹and 8.41 × 10⁶

⁻¹, respec-

tively [81,82]. Furthermore, molecular docking simulations illus-

trated that 2 and 3 dock within the major groove while 1, 4 and 5

docks in close proximity of the B-DNA minor groove, see Figs. S22

– S26.

The apparent association constants (K_app

) were calculated

graphically from the double reciprocal plots of 1/(A₀– A) Ver-

sus 1/C_complexand are shown in Table S14. The metal compounds

are considered as “ideal” binders towards BSA since their associa-

tion constants are within range of 10⁴– 10⁶M⁻¹of other strong

ruthenium-based binders, with 1, 2, and 5 having higher bind-

ing aﬃnities towards BSA [91–93]. The ﬂuorescence quenching of

BSA was investigated in the presence of increasing amount of each

metal compound, see Figs. S32 – S36. A decrease in the ﬂuores-

cence intensities of BSA is noticed upon the addition of the respec-

tive organometal compounds which is indicative of an interaction

between latter and former. Moreover, blue shifts of the emission

maxima are seen for 1, 2, 3 and 5, whilst a red shift is observed

for 4. This validates the UV-Vis study as it suggests that 1, 2, 3

and 5 promotes folding of the BSA strand due to Van der Waals

or hydrogen interactions (i.e. tryptophan residues moved more into

hydrophobic pocket) whilst 4 promotes slight unwinding (i.e. tryp-

tophan group moved towards the hydrophilic surface) [94,95]

The Stern-Volmer quenching constants (K_SV) and quenching rate

constants (K_q) were obtained from the linear Stern-Volmer plots,

refer to Table 6. The K_SVvalues may be used to assess the binding

aﬃnities of the metal compounds towards BSA and the results are

consistent with the ﬁndings in the UV-Vis study, in that 1, 2 and

5 have higher aﬃnities for BSA. A static quenching mechanism for

the ruthenium compounds was conﬁrmed by considerable changes

in spectra of the UV-Vis study as well as the fact that the K_qval-

ues found are greater than the maximum scatter collision constant

3.5. BSA interaction studies

Serum albumins play important roles in metabolism and have

the ability to reversibly bind and hence transport many endo- and

exogenous substances in the mammalian system. Consequently, the

binding properties of serum albumin with organometal species are

often conducted to study the mechanism of interaction between

them, which helps contribute to the development of physiolog-

ically compatible agents. Bovine serum albumin (BSA) is widely

used as the selected protein model due to its structural similarity

with human serum albumin (HSA) [83]. In order to evaluate the

ability of BSA to function as an appropriate transporter of the se-

lected complexes, the extent of folding (or unfolding) of the BSA

strand upon binding with each of the metal compounds was stud-

ied by means of UV-Vis and ﬂuorescence spectroscopy. The distinct

peaks in the spectra of the protein are due to tryptophan residues

within a BSA hydrophobic pocket and therefore, any changes in the

spectra reﬂects conformational changes of the BSA strand. Prefer-

ably, the protein should be able retain its native state (folded) as

much as possible in order to function as an ideal transporter [84].

Analysing the UV-Vis spectra of BSA, in the presence of incre-

menting concentrations of 1 – 5, decreases in the absorbance in-

tensities are observed which indicates that interactions between

BSA and the respective metal compounds occurred, see Figs. S27

- S31 [35,36]. Moreover, slight blue shifts are seen in the absorp-

tion maxima in the electronic spectral proﬁles of 1, 2, 3 and 5

which are characteristic of increased polarity around the trypto-

phan residue in BSA. Consequently, these metal compounds pro-

responsible for dynamic quenching (2 × 10¹⁰M⁻¹

⁻¹) [91, 94].

BSA shows distinct binding sites with various speciﬁcities, the

most signiﬁcant ones being referred to as sites I and II which

are located in hydrophobic subdomains IIA and IIIA, respectively.

S. Maikoo, I.N. Booysen, B. Xulu et al.

Journal of Molecular Structure 1244 (2021) 130986

Table 6

Binding parameters of 1 – 5 from BSA ﬂuorescence experiments.

Compound

No Site marker

Ibuprofen

K_q(M⁻¹s⁻¹

Warfarin

K_SV(M⁻¹

)

K_q(M⁻¹s⁻¹

)

K_SV(M⁻¹

)

K_SV(M⁻¹

)

K_q(M⁻¹s⁻¹

)

1.06 × 10⁵

2.12 × 10⁵

5.59 × 10⁴

3.11 ×10⁴

3.38 × 10⁵

1.06 × 10¹³

2.12 × 10¹³

5.59 × 10¹²

3.11 × 10¹²

3.38 × 10¹³

1.57 × 10⁵

2.11 × 10⁵

3.16 × 10⁴

5.92 × 10⁴

1.26 × 10⁵

1.57 × 10¹³

2.11 × 10¹³

3.16 × 10¹²

5.92 ×ꢀ10¹²

1.26 × 10¹³

4.28 × 10⁴

4.42 × 10⁴

2.79 × 10³

3.21 × 10³

9.38 ×10⁴

4.28 × 10¹²

4.42 × 10¹²

2.79 × 10¹¹

3.21 ×ꢀ 10¹¹

9.38 × 10¹²

Competitive binding experiments were conducted by observing the

ﬂuorescence quenching capabilities of BSA of each metal com-

pound in the presence of a site marker in order to ascertain their

preferred binding site within the BSA structure. Site markers are

small molecules that have precise binding locations within the BSA

structure [96]. In particular, Warfarin was used as a site marker for

site I whilst Ibuprofen was used for site II. As the concentration

of each metal compound was increased, the emission maxima of

BSA were decreased expressively in the presence of both site mark-

ers, see Figs. S37 – S41. The Stern-Volmer plots were then used

to determine the K_SVvalues for the respective BSA-Warfarin and

BSA-Ibuprofen systems. The preferred binding sites of each metal

compound was determined by their ability to displace each site

marker, this was done by comparing the K_SVvalues in the absence

and presence of each site marker. It is seen that the K_SVvalues of

the BSA-Ibuprofen systems are not signiﬁcantly different in magni-

tude relative to the K_SVvalues in the absence of any site markers,

however, a pronounced decrease is seen with the K_SVvalues of the

BSA-Warfarin systems. These experimental trends propose that the

ruthenium compounds are able to displace Warfarin from its bind-

ing site in BSA and therefore assume site I (situated in subdomain

IIA) as the preferred binding site [97].

be largely originating from hydrogen-bonding interactions between

DNA base pairs or the sugar-phosphate backbone within grooves

and the polar functional groups of the respective organic chelators

[106].

Based on the K_appand K_SVvalues, metal compounds 1, 2 and

5 are seen to have higher binding aﬃnities towards BSA, refer to

Table 6. The higher BSA aﬃnities of 2 and 5 are attributed to the

larger sizes of the organic chelators, which encourages increased

interactions at BSA’s hydrophobic surface while the chloride co-

ligands of 1 promote hydrogen-bonding interactions within the hy-

drophilic BSA cavity [107]. On the contrary, metal compound 4 ex-

hibits the weakest binding ability, which are accounted to its fewer

hydrogen bonding sites that culminate into a less stable BSA-metal

complex adduct and hence, it does not encourage folding of the

protein [108, 109].

4. Conclusions

Paramagnetic ruthenium(III) compounds: fac-[RuCl₃(PPh₃)(ap)]

(1) (ap

(2) (cinap

1-ylidene]amino-1,2-dihydro-3H-pyrazol-3-one)

Cl, trans-P-[RuCl₂(PPh₃)₂(cumbh)] (3) (cumbh

4-aminoantipyrine), trans-P-[Ru(PPh₃)₂(cinap)₂](PF₆)

1,5-dimethyl-2-phenyl-4-[3-phenylprop-2-en-

and

cis-

N’-(4-

3.6. Structure-activity relationship

isopropylbenzylidene)benzohydrazide) were synthesized and

characterized by various physicochemical techniques. In addition,

we used DFT method to attain molecular insights of some of the

experimental ﬁndings. Voltammograms of the respective metal

compounds showed quasi-reversible redox processes which were

attributed to metal-based interconversion. Structure-activity corre-

lations were deduced by comparing the stereoelectronic properties

of the metal compounds with their antioxidant properties as

well as DNA and BSA binding aﬃnities. The radical scavenging

experiments revealed that all the metal compounds displayed

better activities than vitamin C where the paramagnetic metal

centres of 1–4 promotes better eﬃcacies to neutralize the DPPH

and NO radicals. The organic chelators of metal compounds 1–5

can serve as proton-donors to render antioxidant responses. The

UV-Vis spectrophotometric titrations of the metal compounds with

CT-DNA show that they are groove-binders and the strength of

the interactions are primarily attributed to the steric factors of

the metal compounds. Stabilization of the BSA-metal compounds

adducts was enforced by different hydrophobic or hydrophilic

functional groups of the metal compounds. Fluorescence and

UV-Vis spectrophotometric titrations of each metal compounds

against BSA revealed that the former as dynamic quenchers with

strong binding aﬃnities towards site I. The larger sizes of 1, 2

and 5 were shown to promote stronger binding aﬃnities towards

the hydrophobic BSA surface, while the chloride co-ligands of 1

induces hydrogen-bonding interactions inside the hydrophilic BSA

cavity.

Correlations between the intrinsic properties of the metal com-

pounds and their activities could be derived. It is apparent that

the unpaired d-electrons of the paramagnetic metal compounds

1 – 4 scavenge the NO and DPPH radicals more eﬃciently than

the diamagnetic metal compound 5, refer to Table 5 [1 7,98]. In

addition, since all the metal compounds contains pharmacophore

derivatives (viz. chromone, antipyrine, cinnamaldehyde and cumi-

naldehyde) that are known radical scavengers via proton donation,

it is evident that these mechanism of antioxidant activity is inher-

ited by the individual organic chelators of the metal compounds

[99–104].

Furthermore, structure-activity relationships could also be de-

duced from the biomolecular interaction activities. CT-DNA titra-

tions monitored by electronic spectrophotometry unequivocally

show that the metal compounds are groove binders which are

attributed to the presence of their triphenylphosphine co-ligands

which impedes the intercalation of these metal compounds be-

tween the DNA base pairs [105]. This is also corroborated by

molecular docking simulations which show that 1 ﬁt well into the

minor groove opposed to the other metal compounds that ﬁts in

the minor groove poorly (for 2 and 5) or occupies the major groove

(for 3 and 4).

An interesting correlation between the steric factors of the

metal compounds and their intrinsic binding constants is observed.

More speciﬁcally, the lower steric demands of 1 affords a higher

K_bvalue (K_bfor 1 = 2.67 × 10⁶

⁻¹) than the other metal com-

pounds. As mentioned before, the other metal compounds have K_b

values in the order of 10⁵M⁻¹which implies that relatively stable

DNA-metal compound adducts. The reinforcing interactions that af-

fords the stabilization of the metal compound-DNA hybrids could

Credit author statement

Dr Sanam Maikoo performed the experimental work under the

supervision of Prof Irvin Noel Booysen. Prof Irvin Noel Booysen and

S. Maikoo, I.N. Booysen, B. Xulu et al.

Journal of Molecular Structure 1244 (2021) 130986

Dr Sanam Maikoo conceptualized the research study and funding

was attained by Prof Irvin Noel Booysen. Dr Lydia Naicker and Prof

Ponnadurai Ramasami conducted the DFT studies and the interpre-

tation thereof.

[17] G. Prakash, R. Manikandan, P . V iswanathamurthi, K. Velmurugan, R. Nand-

hakumar, Ruthenium(III) S-methylisothiosemicarbazone Schiff base com-

plexes bearing PPh3/AsPh3 coligand: synthesis, structure and biological in-

vestigations, including antioxidant, DNA and protein interaction, and in vitro

anticancer activities, J. Photochem. Photobiol. B. 138 (2014) 63–74.

[18] I.N. Booysen, S. Maikoo, M.P. Akerman, B. Xulu, Novel ruthenium(II) and (III)

compounds with multidentate Schiff base chelates bearing biologically signif-

icant moieties, Polyhedron 79 (2014) 250–257.

Declaration of Competing Interest

The authors declare no conﬂict of interests.

Acknowledgements

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