Synthesis, characterization, and in vitro antioxidant and anticancer studies of ruthenium(III) complexes of symmetric and asymmetric tetradentate Schiff bases

Article abstract of DOI:10.1080/00958972.2015.1043127

Ruthenium(III) complexes of three tetradentate Schiff bases with N₂O₂ donors formulated as [RuCl(LL¹)(H₂O)], [RuCl(LL²)(H₂O)] and [RuCl(LL³)(H₂O)] were synthesized and

Full text of DOI:10.1080/00958972.2015.1043127

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Synthesis, characterization, and in
vitro antioxidant and anticancer
studies of ruthenium(III) complexes
of symmetric and asymmetric
tetradentate Schiff bases
Ikechukwu P. Ejidikea & Peter A. Ajibadea
a Faculty of Science and Agriculture, Department of Chemistry,
University of Fort Hare, Alice, South Africa
Accepted author version posted online: 06 May 2015.Published
online: 14 Jun 2015.
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To cite this article: Ikechukwu P. Ejidike & Peter A. Ajibade (2015): Synthesis, characterization,
and in vitro antioxidant and anticancer studies of ruthenium(III) complexes of symmetric
and asymmetric tetradentate Schiff bases, Journal of Coordination Chemistry, DOI:
10.1080/00958972.2015.1043127
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Journal of Coordination Chemistry, 2015
http://dx.doi.org/10.1080/00958972.2015.1043127
Synthesis, characterization, and in vitro antioxidant and
anticancer studies of ruthenium(III) complexes of symmetric
and asymmetric tetradentate Schiff bases
IKECHUKWU P. EJIDIKE and PETER A. AJIBADE*
Faculty of Science and Agriculture, Department of Chemistry, University of Fort Hare, Alice,
South Africa
(Received 12 January 2015; accepted 26 March 2015)
Ruthenium(III) complexes of three tetradentate Schiff bases with N2O2 donors formulated as [RuCl
(LL1)(H2O)], [RuCl(LL2)(H2O)] and [RuCl(LL3)(H2O)] were synthesized and characterized by ele-
mental analyses, molar conductance, FTIR, and electronic spectral measurements. The FTIR data
showed that the tetradentate Schiff base ligands coordinate to Ru ions through the azomethine
nitrogen and enolic oxygen. The antioxidant activities of the complexes were investigated through
scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azinobis-3-ethylbenzothiazo-
line-6-sulfonic acid (ABTS) radicals. The DPPH activity for [RuCl(LL2)(H2O)] with
IC50 = 0.031 mg mL−1 was higher than the values obtained for the other Ru(III) compounds. The
study revealed that the synthesized Ru(III) complexes of the tetradentate Schiff base exhibited strong
scavenging activities against DPPH and moderate against ABTS radicals. In addition, the antipro-
liferative studies of the complexes were also tested against human renal cancer cells (TK10), human
melanoma cancer cells (UACC62), and human breast cancer cells (MCF7) using the SRB assay.
The results indicated that the Ru(III) complexes showed low anticancer activities against the tested
human cancer cell lines.
*Corresponding author. Email: pajibade@ufh.ac.za
© 2015 Taylor & Francis

2
I.P. Ejidike and P.A. Ajibade
Keywords: Ru(III) complexes; Tetradentate Schiff base; Spectral studies; Antioxidant; Anticancer
1. Introduction
Synthesis of biologically active molecules is a vigorous task and the variables affecting bio-
logical activity are diverse [1–4]. Many studies on the molecular structure of metal com-
plexes and their bioactivity have created much awareness in the field of bio-inorganic
chemistry [5, 6]. The interaction of DNA and transition metal complexes containing multi-
dentate aromatic ligands with a prescribed N4 or N2O2 coordination has been studied [7, 8].
Development of new therapeutic agents and DNA probes [9–11] stems from DNA binding
studies, as it has inspired considerable interest in the study of the biochemical behavior of
these metal compounds: interactions with DNA and serum proteins [12–15]. In the quest
for small molecules that can efficiently bind to DNA and cleave it, Schiff bases and their
metal complexes have gained recognition by various researchers and groups [16].
Schiff bases are important class of compounds widely studied for various applications
[17–32]. Ruthenium Schiff base complexes have been widely studied, imperative as bio-
chemical, analytical, and antimicrobial reagents [27, 28]. Metal complexes of Schiff bases
have attracted considerable attention due to their antifungal, antibacterial, and antitumor
activities [33, 34]. Physiological and biochemical processes are a pathway for generation of
reactive oxygen species (ROS) through the living cells in the body [35–37]. Hence, antioxi-
dants become important as they play vital roles toward protecting the human body against
damage by ROS [38–40]. In view of growing interest in oxygenation and azomethination
of Ru(III) complexes for development of new therapeutic agents and DNA probes for dis-
ease defense, we present the synthesis and characterization of some stable Ru(III) Schiff
bases complexes of the type [RuCl(LL′)(H2O)]·2H2O (LL′ = H2LL1, H2LL2, H2LL3) and
their antioxidant and anticancer studies.
2. Experimental
2.1. Materials and methods
All reagents used were analytical grade and used as received, 2,4-pentanedione from Fluka,
ethylenediamine and ascorbic acid from Merck, RuCl2·3H2O, 2′,4′-dihydroxyacetophenone
and 1-phenylbutane-1,3-dione from Aldrich. 1,1-diphenyl-2-picrylhydrazyl (DPPH),
2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), rutin hydrate, and butylated
hydroxytoluene (BHT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Elemental analysis was obtained using a Perkin-Elmer elemental analyzer. Molar conduc-
tance of the Schiff base ligands and their Ru complexes were determined on freshly pre-
pared 10−3 M solutions in CH2Cl2 at room temperature using a Crison EC-Meter Basic 30+
conductivity cell. IR spectra were recorded on an FT-IR spectrometer, Perkin-Elmer System
(Spectrum 2000) from 4000 to 400 cm−1 using the KBr disk method. Electronic spectra
were recorded on a Perkin-Elmer Lambda-25 spectrophotometer from 200 to 900 nm. Melt-
ing points were recorded with a Stuart melting point (SMP 11).

Ruthenium(III) and tetradentate Schiff bases
3
2.2. General procedure for the synthesis of Schiff base ligands (H2LL1–H2LL3)
2.2.1. [OHC6H3OHC(CH3):N(C2H4)N:C(CH3)HOC6H3OH)], H2LL1. The ligand was
prepared by modification of a literature method [41]. The tetradentate Schiff base was syn-
thesized via the method: an ethanolic solution (20 mL) containing ethane-1,2-diamine
(0.01 mol, 0.601 g) was added slowly to a stirring ethanolic solution (50 mL) containing
2′,4′-dihydroxyacetophenone (0.02 mol, 3.043 g). The resulting light brown mixture was
stirred and refluxed for 3 h. The obtained precipitate was filtered and washed with ethanol,
followed by recrystallization in ethanol and air-drying to give a brownish yellow solid
(Yield = 2.51 g, 76.52%).
2.2.2. [OHC6H3OHC(CH3):N(C2H4)N:C(CH3)CH:C(CH3)OH)], H2LL2. The ligand
was prepared by modification of a literature method [42]. A typical procedure for the syn-
thesis was as follows: ethylenediamine (0.015 mol, 0.902 g) in 30 mL ethanol was slowly
added to an ethanolic solution (40 mL) containing 2′,4′-dihydroxyacetophenone (0.015 mol,
2.282 g), followed by slow addition of acetylacetone (0.015 mol, 1.502 g) dissolved in
30 mL ethanol. The resulting colored mixture was refluxed with stirring for 4 h, cooled and
the resultant precipitate was filtered, washed several times with ethanol, followed by recrys-
tallization in ethanol (Yield = 2.53 g, 61.23%).
2.2.3. [OHC6H3OHC(CH3):N(C2H4)N:C(CH3)CH:C(C6H5)OH)], H2LL3. The ligand
was prepared by modification of a literature method [42]. A typical procedure for the syn-
thesis was as follows: ethylenediamine (0.015 mol, 0.902 g) in 30 mL ethanol was slowly
added to an ethanolic solution (40 mL) containing 2′,4′-dihydroxyacetophenone (0.015 mol,
2.282 g), followed by slow addition of 1-phenylbutane-1,3-dione (0.015 mol, 2.4329 g),
(H2LL3) dissolved in 40 mL ethanol. The resulting colored mixture was refluxed with stir-
ring for 4 h, cooled and the resultant precipitate was filtered, washed several times with
ethanol, followed by recrystallization in ethanol (Yield = 3.75 g, 74.17%).
2.3. General procedure for the preparation of the complexes
All operations were carried out under strictly anhydrous conditions. The various complexes
were prepared by addition of RuCl3·3H2O (0.5 mmol) dissolved in 15 mL of absolute etha-
nol, into a hot ethanolic solution (20 mL) of H2LL1–H2LL3 ligands (0.5 mmol) in molar
ratio (1 : 1). The color changed immediately. The resulting mixture was then refluxed for
6 h. The precipitated solids were allowed to cool and filtered off from the reaction mixture,
thoroughly washed with absolute ethanol and then with diethyl ether (3 × 5 mL), and were
dried over anhydrous calcium chloride.
2.4. Antioxidant assay
2.4.1. Scavenging activity of DPPH radical. The antioxidant activities for N2O2 Schiff
base ligands and their synthesized Ru(III) complexes were studied spectrophotometrically
by DPPH method. DPPH is known as a stable commercially available free radical, soluble
in methanol to give a violet solution, which, upon reduction by an antioxidant, changes to a

4
I.P. Ejidike and P.A. Ajibade
corresponding light yellow to yellow. The free radical scavenging effects of the Ru(III)
compounds and Schiff base ligands with the DPPH radical were evaluated as previously
described with slight modification [43, 44]. A solution of 0.4 mM DPPH in methanol was
prepared and 1.0 mL of this solution was mixed with 1.0 mL DMF solutions of Schiff base
ligands and Ru(III) complexes with various concentrations (8, 17, 25, 33, and 42 μg mL−1).
The reaction mixture was stirred thoroughly and left in the dark at room temperature for
30 min. The absorbance of the mixture was measured spectrophotometrically at 517 nm.
Rutin and ascorbic acid (vitamin C) are used as standard drugs. The actual decrease in
absorption was measured against that of the control. All tests and analyses were run in
triplicate and the results obtained were averaged. The ability to scavenge DPPH radical was
calculated by the following equation:
Abscontrol ꢀ Abssample
DPPH radical scavenging activity ð%Þ ¼
ꢁ 100
Abscontrol
where Abscontrol is the absorbance of DPPH radical + DMF, and Abssample is the absorbance
of DPPH radical + sample [test samples/standard].
2.4.2. ABTS radical scavenging assay. ABTS scavenging ability of the Ru(III) com-
pounds and Schiff base ligands was evaluated by the previously described method of Ade-
dapo and co-workers [45]. The working solution was prepared by mixing stock solutions of
7 mM ABTS solution and 2.4 mM potassium persulfate solution in equal amounts (1 : 1)
and allowing the solution to react in the dark for 12 h at room temperature. The resulting
solution was further diluted by mixing 1 mL ABTS+ solution to obtain an absorbance of
0.706 0.001 units at 734 nm using the spectrophotometer. Test samples (1 mL) were
allowed to react with 1 mL of the ABTS+ solution, followed by the absorbance reading at
734 nm after 7 min using the spectrophotometer. The ABTS scavenging capacities of the
Ru(III) compounds and Schiff base ligands were compared with that of rutin and BHT (s-
tandard drugs). All tests were run in triplicate, and the results obtained were averaged. The
percentage inhibition was calculated as ABTS radical scavenging activity using the follow-
ing equation:
Abscontrol ꢀ Abssample
ð%Þ Inhibition ¼
ꢁ 100
Abscontrol
where Abscontrol is the absorbance of ABTS radical + DMF, and Abssample is the absorbance
of ABTS radical + sample [test samples/standard].
2.5. Cell lines and culture conditions
Human renal cancer cell line (TK10), human melanoma cancer cell line (UACC62), and
human breast cancer cell line (MCF7) were obtained from NCI in the framework a collab-
orative research program between CSIR and NCI. Cell lines were routinely maintained as a
monolayer cell culture at 37.0 °C with 5% CO2, 95% air and 100% relative humidity in
RPMI medium which is supplemented with 5% fetal bovine serum, 2 mM L glutamine and
50 μg mL−1 gentamicin.

Ruthenium(III) and tetradentate Schiff bases
5
2.6. Cell viability assay
Cell viability was examined by Sulforhodamine B (SRB) assay as previously described
[46, 47]. The cells (TK10, UACC62, and MCF7) (3–19 passages) were inoculated into
96-well microtiter plates at plating densities of 7–10,000 cells/well and were incubated for
24 h. After 24 h, the cells were treated with the experimental compounds which were previ-
ously dissolved in DMSO and diluted in medium to produce concentrations of 0.01, 0.1, 0,
10, and 100 μM. Cells without drug addition served as control. The blank contains com-
plete medium without cells. Parthenolide was used as a standard. The plates were incubated
for 48 h after addition of the compounds. Viable cells were fixed to the bottom of each well
with cold 50% trichloroacetic acid, washed, dried and dyed by SRB. Thereafter, the
unbound dye was removed, and protein-bound dye was extracted with 10 mM Tris base for
optical density determination at 540 nm using a multiwell spectrophotometer. Data analysis
was performed using GraphPad Prism software; 50% of cell growth inhibition (IC50) was
determined by non-linear regression. The Z′-factor coefficient was adapted to monitor the
quality of immunocytochemical assays such as the SRB.
3. Results and discussion
3.1. Synthesis
Mononuclear ruthenium(III) complexes, [RuCl(LL′)(H2O)] (LL′ = H2LL1, H2LL2, H2LL3)
(LL′ = dibasic tetradentate Schiff base ligand), were synthesized in good yields from reac-
tion of RuCl3·3H2O with Schiff base ligands in 1 : 1 M ratio in absolute EtOH to give six-
coordinate ruthenium(III) Schiff base complexes according to the equation:
RuCl3 ꢂ 3H2O þ H2LL0 ꢀ! ½RuClðLL0ÞðH2OÞꢃ þ 2HCl þ 2H2O
where H2LL = H2LL1 = [OHC6H3OHC(CH3):N(C2H4)N:C(CH3)HOC6H3OH)], H2LL2 =
[OHC6H3OHC(CH3):N(C2H4)N:C(CH3)CH:C(CH3)OH)] and H2LL3 = [OHC6H3OHC
(CH3):N(C2H4)N:C(CH3)CH:C(C6H5)OH)] (scheme 1). The synthesized mononuclear
ruthenium(III) Schiff base complexes (figure 1) are stable in air at room temperature, non-
hygroscopic and insoluble in water, partially soluble in common solvents such as dichloro-
methane, acetonitrile, chloroform, but easily soluble in polar solvents such as DMF and
DMSO producing intense color in their solutions. The solubility of the complexes may be
due to the presence of chlorides [48] and hydroxyl groups on the benzene ring [49]. The
tetradentate N2O2 donor site of LL′ (H2LL1, H2LL2, H2LL3) is capable of forming com-
plexes with ruthenium. The analytical data are listed in table 1 and are in agreement with
the proposed formulations for the complexes.
3.2. Infrared spectra
Important IR absorptions for the complexes are shown in table 2. The observed bands have
been classified into those originating from the ligands and those arising from the bands
formed between ruthenium(III) and the coordinating sites. IR spectra of the free ligands
showed bands at 3475–3479, 2873–3076, 1605–1616, 1470–1588, and 1171–1288 cm−1

6
I.P. Ejidike and P.A. Ajibade
H3C
O
H3C
C
N
NH2
OH
Ethanol
+
H2N
NH2
Stirred, Reflux
OH
OH
HO
H3C
CH3
C
N
N
C
OH
HO
H2LL1
HO
OH
H3C
CH3
C
H3C
C
N
N
C
C
N
NH2
C5H8O2
Ethanol, Reflux
OH
HO
C
OH
CH3
H2LL2
HO
HO
H3C
CH3
C
C
N
N
C
C
OH
HO
HO
H2LL3
Scheme 1. Synthesis of H2LL1–H2LL3.
assignable to ν(OH), ν(CH3/CH2), ν(C=N), ν(C=C), and ν(C–O), respectively [20, 49, 50].
H2LL1, H2LL2 and H2LL3 show a very strong absorption at 1605–1616 cm−1 in their IR
spectra, characteristic of the azomethine ν(C=N) (table 2). In the Schiff base complexes, this
absorption shifted to 1621–1623 cm−1 indicating coordination of the Schiff bases through
nitrogen in accord with coordination of the azomethine function to the metal ion for all the
complexes [28, 50, 51]; this shift of wavenumber is expected due to coordination of nitro-
gen of the azomethine group to ruthenium, thereby reducing electron density in the azome-
thine [50, 52].
A medium band corresponding to phenolic oxygen ν(C–O) is observed at 1171–
1288 cm−1 for the free ligands. Upon chelation, this band shifted to lower frequency
(1470–1543 cm−1) for all the ruthenium(III) Schiff base complexes [28, 53]. This indicates
enolization of >C=O followed by deprotonation and complexation with metal and the
destruction of keto group presumably viz., enolization and ketolization bonding of the ligand
through the resulting enolate and ketolate oxygen. This is further supported by the disap-
pearance of ν(OH) at 3475–3479 cm−1 in the complexes. The presence of coordinated water
at 3419–3435 and 846–861 cm−1, due to ν(O–H) stretching and ν(O–H) rocking vibrations,
respectively, further confirmed the presence of water [49, 54, 55]. In the low frequency

Ruthenium(III) and tetradentate Schiff bases
7
H3C
CH3
Cl
C
N
O
N
O
C
Ru
. 2H2O
H2O
HO
OH
[a]
H3C
CH3
C
Cl
C
N
O
N
O
C
C
Ru
. 2H2O
H2O
CH3
HO
[b]
H3C
CH3
Cl
C
N
O
N
O
C
C
Ru
C
. 2H2O
H2O
HO
[c]
Figure 1. Proposed structure for the Ru(III) complexes (a) [RuCl(LL1)(H2O)]·2H2O, (b) [RuCl(LL2)
(H2O)]·2H2O, and (c) [RuCl(LL3)(H2O)]·2H2O.
region, the observed bands at 519–535 and 415–437 cm−1 are probably due to the
formation of ν(M–N) and ν(M–O) vibrations, respectively [41, 56, 57].
3.3. Molar conductivity measurements
The molar conductivity (Λμ) values of the Ru(III) complexes in 10−3 M DMF
solution (table 1) at room temperature are 23.8–47.4 μS cm−1, indicating the essential
non-electrolytic character of the compounds [1, 58].
3.4. The antioxidant assay
Oxidative reactions of biological molecules induce a variety of pathological events such as
cellular injury and aging process, and these damaging events are caused by free radicals
[59, 60]. Therefore, to prevent free radical damage in the body, it is important to administer
drugs that may be rich in antioxidants. The antioxidant activity of ligands and their metal
complexes have been investigated using the in vitro method [43, 61]. However, the antioxi-

8
I.P. Ejidike and P.A. Ajibade

Ruthenium(III) and tetradentate Schiff bases
9
Table 2. FTIR spectral data of the N2O2 Schiff bases and their Ru complexes (cm−1).
Compound
ν(OH) ν(CH3/CH2) ν(C=N)
ν(C=C)
ν(C–O)
ν(Ru–N) ν(Ru–O)
H2LL1
3475
3435
3475
3419
3479
3430
2929, 2873
2977, 2845
2928, 2911
2959, 2844
3076, 2981
2997, 2901
1616
1622
1612
1621
1605
1623
1588, 1532 1267, 1173
1530, 1479 1244, 1170
1534, 1480 1243, 1171
1543, 1525 1243, 1168
1543, 1470 1288, 1241
1543, 1508 1258, 1138
–
535
–
522
–
–
437
–
428
–
[RuCl(LL1)(H2O)]·2H2O
H2LL2
[RuCl(LL2)(H2O)]·2H2O
H2LL3
[RuCl(LL3)(H2O)]·2H2O
519
415
dant mechanism of the complexes has not been explained so far [62]. The antioxidant assay
study was carried out using different concentrations of the test samples (Schiff base ligands
and the Ru(III) complexes) with DPPH and ABTS radicals, while ascorbic acid, rutin, and
BHT were used as standards in order to establish some structure antioxidant activity rela-
tionship.
3.4.1. DPPH radical scavenging assay. DPPH is a compound widely used to examine the
ability of a given sample to act as free radical scavengers or hydrogen donors and to evalu-
ate antioxidant activity of foods [63]. The results of the DPPH radical scavenging abilities
of H2LL1, H2LL2 and H2LL3 and the Ru complexes were studied and compared with the
standard (ascorbic acid and rutin). The Ru(III) complexes exhibited significant DPPH radi-
cal scavenging ability in all the concentrations used, i.e. chelated Ru(III)-Schiff base com-
plexes were more effective free radical scavengers than the corresponding free H2LL1,
H2LL2, and H2LL3 Schiff bases; this could be attributed to the acquisition of additional
superoxide dismutating centers [64].
However, the Ru(III) complexes showed comparable or higher scavenging activity com-
pared to the standards (ascorbic acid and rutin) with [RuCl(LL2)(H2O)] showing signifi-
cantly higher scavenging ability. The DPPH radical scavenging ability of the Ru(III)
complexes can be ranked, [RuCl(LL2)(H2O)] > [RuCl(LL3)(H2O)] > [RuCl(LL1)(H2O)].
IC50 values of Schiff bases H2LL1, H2LL2, and H2LL3 on DPPH radical are 0.067 0.006,
0.065 0.001, and 0.055 0.002 mg mL−1, respectively, whereas [RuCl(LL1)(H2O)],
[RuCl(LL2)(H2O)], and [RuCl(LL3)(H2O)] showed IC50 values at 0.041 0.003, 0.031
0.006, and 0.036 0.002 mg mL−1, respectively (table 3). Therefore, the scavenging
effect of the free ligand is lower compared to that of their corresponding Ru(III) complexes,
Table 3. DPPH and ABTS radical scavenging capacities
(IC50 SD, mg mL−1) of standard drugs, N2O2 Schiff bases, and
their Ru complexes.
Compound
DPPH
ABTS
H2LL1
0.067 0.006
0.041 0.003
0.065 0.001
0.031 0.006
0.055 0.002
0.036 0.002
0.045 0.005
0.037 0.009
0.008 0.0003
0.011 0.0005
0.009 0.0004
0.036 0.0027
0.009 0.0006
0.025 0.0047
0.023 0.0035
0.004 0.0003
[RuCl(LL1)(H2O)]
H2LL2
[RuCl(LL2)(H2O)]
H2LL3
[RuCl(LL3)(H2O)]
Vitamin C
Rutin
Note: (n = 3, X SEM), IC50 – inhibitory concentration.

10
I.P. Ejidike and P.A. Ajibade
related to chelation of the organic molecules with the metal ions. Also, the violet color of
DPPH radical changed to yellow upon addition of Ru(III) compounds because proton from
the test samples were transferred to DPPH, converting it into the corresponding hydrazine
form [40]. Thus, these compounds could be a promising therapeutic agents to treat
stress-induced pathological conditions such aging, cancer, and cardiovascular and
neurodegenerative diseases.
3.4.2. ABTS radical scavenging activity. ABTS cation radicals (ABTS+) are produced by
oxidation of ABTS with potassium persulfate and, thus, are reduced in the presence of
hydrogen-donating antioxidants [65]. This has been the basis of one of the spectrophoto-
metric methods applied to measurement of the total antioxidant activity of solutions of pure
substances and aqueous extracts [66, 67]. The method described gives a measure of the
antioxidant activity of test samples determined by the decolorization of ABTS+ through
measuring the reduction of the radical cation as the percentage inhibition of absorbance at
734 nm [68]. H2LL1, H2LL2 and H2LL3 and their Ru complexes exhibited low-to-moderate
scavenging ability of the ABTS radical (figure S2) and showed comparable or slightly
lower activity to that of rutin and BHT (standard drugs).
At a concentration of 4.35 μg mL−1, the percentage inhibition was 60.2, 66.4, 59.8, 54.8,
and 50.6% for [RuCl(LL1)(H2O)], [RuCl(LL2)(H2O)], [RuCl(LL3)(H2O)], rutin, and BHT,
respectively. Nevertheless, the ABTS activities of the Ru(III) complexes were significantly
enhanced compared to their corresponding free Schiff base ligands. Lowest concentration of
the H2LL1, H2LL2 and H2LL3 and its Ru complexes were more effective in quenching
ATBS radicals in the system. IC50 value of H2LL1, H2LL2, and H2LL3 on ABTS radical is
0.067 0.006, 0.065 0.001, and 0.055 0.002 mg mL−1, respectively, while the IC50 val-
ues at 0.011 0.0005, 0.036 0.0027, and 0.025 0.0047 mg mL−1 are for [RuCl(LL1)
(H2O)], [RuCl(LL2)(H2O)], and [RuCl(LL3)(H2O)], respectively (table 3). Furthermore, the
synthesized compounds scavenged the ABTS radical in a concentration-dependent pattern.
Generally, the scavenging activities of the DPPH radical by H2LL1, H2LL2, and H2LL3
and the Ru(III) complexes are higher than that of ABTS radical. Wang and co-workers [69]
reported that some compounds which exhibited ABTS scavenging activity did not possess
DPPH scavenging activity. Hence, this study revealed the synthesized tetradentate Schiff
base Ru(III) complexes exhibited strong scavenging activities against DPPH and moderate
activity against ABTS radicals. This result shows that the compounds in this study can
scavenge different free radicals in different systems, indicating that they may be useful as
therapeutic agents for treating pathological damage associated with radical generation.
3.5. Antiproliferative activity evaluation
The in vitro anticancer activities of Ru(III) complexes and parthenolide (at various concen-
trations ranging from 0.01 to 100 μM) were evaluated using three cancer cell lines: human
renal cancer cell (TK10), human melanoma cancer cell (UACC62), and human breast can-
cer cell (MCF7) using the SRB assay and parthenolide was used as standard. Parthenolide
demonstrated high levels of antiproliferative effect against all cell lines, in accord with
previous reports [70]. The values of the concentration of the compounds for 50% inhibition
(IC50) were obtained from non-linear regression analysis of dose response data for the
compounds tested and are presented in table 4. The Ru(III) complexes demonstrate low-
to-moderate in vitro antiproliferative effect compared to parthenolide (standard agent)

Ruthenium(III) and tetradentate Schiff bases
11
Table 4. IC50 values (μM) of Ru(III) complexes and parthenolide against human cell
lines*.
Antiproliferative activity IC50 (μM) 48 h
Compound
TK-10
UACC-62
MCF-7
[RuCl(LL1)(H2O)]·2H2O
[RuCl(LL2)(H2O)]·2H2O
[RuCl(LL3)(H2O)]·2H2O
Parthenolide
>100
>100
>100
>100
>100
>100
>100
90
90
8
5
4.64 1.43
11.37 2.18
3.52 2.02
*Cells were treated with various concentrations of tested compounds for 48 h. IC50 values were calcu-
lated as described in Section 2. Each value represents the mean SD of three independent experiments
(Z′ factor > 0.5).
against selected tumor cell lines. [RuCl(LL1)(H2O)]·2H2O displayed non-selective
antiproliferative activity against all tumor cells tested, while [RuCl(LL2)(H2O)]·2H2O and
[RuCl(LL3)(H2O)]·2H2O had a low antiproliferative effect with IC50 at 90 μM against
MCF-7 (Z′ factor > 0.5). The inhibition effects were enhanced by increasing the concentra-
tion of the Ru(III) complexes. The results showed that [RuCl(LL2)(H2O)]·2H2O was more
active against all the selected tumor cells than [RuCl(LL1)(H2O)]·2H2O and [RuCl(LL3)
(H2O)]·2H2O (Z′ factor > 0.5), which is in agreement to their order of in vitro DPPH
scavenging ability of the Ru(III) complexes. Binding of the three N2O2 Schiff base Ru(III)
complexes to biological targets other than DNA could be responsible for the observed
antiproliferative activity of the complexes.
4. Conclusion
Ru(III) complexes of symmetric and asymmetric Schiff base ligands derived from ethane-
1,2-diamine, 4-acetylresorcinol, acetylacetone, and 1-phenylbutane-1,3-dione were synthe-
sized and characterized. Conductance measurements revealed non-electrolytes. The ligands
are dibasic, ONNO tetradentate, coordinated to the Ru(III) through the phenolic oxygen and
imino nitrogen; octahedral geometry around the Ru(III) ions is completed by H2O and a
Cl−. In vitro anticancer studies of the Ru complexes show that they are inactive against
human cancer cells (TK10) and human melanoma cancer cell (UACC62) but show mild
activity against human breast cancer cell line (MCF7). The synthesized complexes exhibited
low biological activities as potential anticancer agents. Derivatization of the Schiff base by
substituting CH3 on the C2 and C4 position of acetylacetone could increase the activity of
the complexes. Nevertheless, antioxidant activities of the complexes exhibited moderate to
strong free radical inhibitors or scavenger for treating pathological damage associated with
radical generation.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was financially supported by the Govan Mbeki Research and Development (GMRD), University of Fort
Hare.

12
I.P. Ejidike and P.A. Ajibade
Supplemental data
Supplemental data for this article can be accessed here [http://dx.doi.10.1080/00958972.2015.1043127].
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