Aryl variation and anion effect on CT-DNA binding and in vitro biological studies of pyridinyl Ag(I) complexes

Article abstract of DOI:10.1016/j.jinorgbio.2020.111266

Synthesis and spectroscopic characterization of five ligands ((E)-2-((pyridin-2-ylmethylene)amino)phenol L1, 2-(pyridin-2-yl)benzo[d]thiazole L2, (E)-N-(2-fluorophenyl)-1-(pyridin-2-yl)methanimine L3, (E)-1-(pyridin-2-yl)-N-(p-tolyl)methanimine L4 and (E)

Full text of DOI:10.1016/j.jinorgbio.2020.111266

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Aryl variation and anion effect on CT-DNA binding and in vitro
biological studies of pyridinyl Ag(I) complexes
Adesola Abimbola Adeleke, Md. Shahidul Islam, Olakunle Sanni,
Chunderika Mocktar, Sizwe J. Zamisa, Bernard Omondi
PII:
S0162-0134(20)30294-4
DOI:
https://doi.org/10.1016/j.jinorgbio.2020.111266
JIB 111266
Reference:
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
30 April 2020
26 September 2020
27 September 2020
Please cite this article as: A.A. Adeleke, M.S. Islam, O. Sanni, et al., Aryl variation
and anion effect on CT-DNA binding and in vitro biological studies of pyridinyl Ag(I)
complexes, Journal of Inorganic Biochemistry (2018), https://doi.org/10.1016/
j.jinorgbio.2020.111266
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Aryl variation and anion effect on CT-DNA binding and in vitro biological studies of
pyridinyl Ag(I) complexes
Adesola Abimbola Adeleke1, Md. Shahidul Islam2, Olakunle Sanni2, Chunderika Mocktar3
Sizwe J. Zamisa4 and Bernard Omondi*1
1 School of Chemistry and Physics, University of Kwazulu-Natal, Pietermaritzburg Campus, Private Bag X01,
Scottsville 3209, South Africa
2 Discipline of Biochemistry, School of Life Sciences, University of Kwazulu-Natal, Westville Campus, Private
Bag X54001, Durban 4000, South Africa
3 Discipline of Pharmaceutical Sciences, School of Health Sciences, University of Kwazulu-Natal, Westville
Campus, Private Bag X54001, Durban 4000, South Africa
4 School of Chemistry and Physics, University of Kwazulu-Natal, Westville Campus, Private Bag X54001,
Westville 4001, South Africa
*Corresponding author.
E-mail address: owaga@ukzn.ac.za http://orcid.org/0000-0002-3003-6712.
Abstract
Synthesis and spectroscopic characterization of five ligands ((E)-2-((pyridin-2-
ylmethylene)amino)phenol L1, 2-(pyridin-2-yl)benzo[d]thiazole L2, (E)-N-(2-fluorophenyl)-
1-(pyridin-2-yl)methanimine L3, (E)-1-(pyridin-2-yl)-N-(p-tolyl)methanimine L4 and (E)-1-
(pyridin-2-yl)-N-(thiophen-2-ylmethyl)methanimine L5 along with fifteen silver(I)
-
complexes of L1 – L5, with a general formula [AgL2]+X- (L = Schiff base and X = NO3 ,
-
-
ClO4 or CF3SO3 ) is reported. The structures of complexes [Ag(L4)2]NO3, [Ag(L5)2]NO3,
[Ag(L3)2]ClO4, [Ag(L4)2]ClO4 and [Ag(L5)2]CF3SO3 were determined unequivocally by
single crystal X-ray diffraction analysis. calf-thymus deoxyribonucleic acid (CT-DNA),
bovine serum albumin (BSA) binding studies, antioxidant, and antibacterial studies were
performed for all complexes. Complexes [Ag(L2)2]NO3, [Ag(L5)2]NO3, [Ag(L1)2]ClO4 and
[Ag(L3)2]ClO4 whose ligands have an OH- and F- as substituents or with a thiophene or
thiazole moiety showed better antibacterial activities with lower minimum inhibitory
concentration (MIC) values compared to the standard ciprofloxacin, against most of the
bacterial strains tested. Similarly, complexes [Ag(L1)2]NO3, [Ag(L2)2]NO3, [Ag(L3)2]NO3
-
-
and [Ag(L5)2]NO3 with the NO3 anion, [Ag(L1)2]ClO4 and [Ag(L2)2]ClO4 with ClO4
-
anion, and [Ag(L5)2]CF3SO3 with CF3SO3 anion showed higher activities for antioxidant
studies. Complexes [Ag(L4)2]ClO4 and [Ag(L4)2]CF3SO3 with the Methyl substituent and
-
CF3SO3 as the anion, displayed high antioxidant activities in FRAP (ferric reducing
antioxidant power) than the standard ascorbic acid. Spectroscopic studies of all the
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complexes revealed their moderate to high interaction with calf thymus DNA via an
intercalation mode. In addition, the relatively moderate interaction of most of the complexes
with BSA was through a static quenching mechanism.
Keywords: Ag(I) complexes, Pyridinyl Schiff bases, Antimicrobial, Antioxidant, DNA
binding, protein binding.
Introduction
Schiff bases are an important class of ligands in coordination chemistry since they can easily
be synthesised. They can also be electronically and sterically fine-tuned to stabilize various
metal oxidation states. Schiff base ligands can accommodate acyl, aryl, and heteroaryl
moieties making them excellent for transition metal complexation [1]. Heteroaryl with
pharmacophores such as pyridine, [2, 3], and their derivatives can be found in over seven
thousand already existing drugs [4]. In such Schiff bases, the pyridine moiety provides an
extra N atom for coordination to metal ions in addition to the imine N atom and any other
donor atoms such as O, S, and P that might be present on the ligand. This may lead to diverse
modes of coordination with a transition metals, thereby providing an avenue for possible drug
design [5].
Ag(I) complexes of Schiff bases have been widely studied due to their wide therapeutic
applications and broad biological spectrum such as anticancer, antimicrobial, antioxidant,
DNA and protein binding [6]. The choice for silver as antimicrobial agents has been based on
their relatively low toxicity as compared to other transition metals and its ionic or chelated
form exhibits tremendous biological activity [7]. Silver(I) sulfadiazine [8], for example, has
been used in treating severe burns to prevent bacterial infections.
Several Ag(I) complexes [9-13] and their nanoparticles [14-16] have been reported for their
antioxidant activity. Ag(I) complexes have shown to be effective scavengers of free radicals
and reactive oxygen species (ROSs) which damage DNA, RNA, proteins, enzymes, and
lipids, resulting in ailments and disorders such as diabetes mellitus, neurodegenerative
diseases, rheumatoid arthritis, cataracts, cardiovascular diseases, respiratory diseases and
aging to the human body [17]. Thus, the interaction of metal complexes with DNA and
plasma proteins is important in the design of biologically active compounds [18]. The
interaction of a complex with DNA may be via intercalation between base-pairs, electrostatic
between cationic species and negatively charged DNA phosphate residues, or through major
or minor grooves of the DNA via hydrogen bonding or van der Waals interactions [19-21]. It
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can also be by catalytic cleavage of DNA through hydrolytic or oxidative reactions [22].
Through studies involving interactions of protein to metal complexes, important information
like the compound's distribution impact, concentration, metabolism, toxicity, and
pharmaceutical kinetic in the bloodstream are obtained.
In continuation of our work on novel Ag(I) pyridinyl Schiff base complexes, we explored
new ligands derived from thiophene-2-ylmethanamine, 2-fluoroaniline, p-toluidine, 2-
aminophenol, and 2-aminobenzenethiol. We report herein the synthesis, characterization, and
biological studies of N,N-bidentate pyridinyl Schiff base ligands, and their Ag(I) nitrate,
perchlorate, and triflate complexes.
2. Experimental
2.1. Materials and instrumentation
Ethanol 99.5% (Aldrich, USA), diethyl ether 99.8% (Aldrich, USA), DMSO–d6 99.8%
Merck, Germany), 2-pyridinecarboxaldehyde 99% (Aldrich, USA), 2-aminophenol >99%
(Aldrich, USA), 2-aminobenzenethiol 99.5% (Aldrich, USA), 2-thiophenemethylamine
>92% (Merck), p-toluidine 99.5% (Aldrich, USA), 2-fluoroaniline 99.5% (Aldrich, USA),
dichloromethane 99% (Aldrich, USA), calf-thymus DNA (CT-DNA), ethidium bromide 95%
(Aldrich, USA), bovine serum albumin 99% (Aldrich, USA), nitrogen gas, 5.0 technical
grade (Air flex Industrial Gases, South Africa) were purchased from local suppliers. All
chemicals were in analytical grade and used as received, while most of the solvents were
dried using conventional techniques.
13
1H NMR and C NMR spectra were recorded on a BRUKER 400 MHz spectrometer in
DMSO–d6 and acetone-d6. Chemical shift values are reported in parts per million (ppm)
relative to the solvent residual peaks in DMSO-d6 and acetone-d6; 2.5 and 2.05 ppm
1
13
respectively for H NMR and 39.5 and 29.4 ppm respectively for C NMR. The splitting
patterns in 1H NMR spectra are reported as s for singlet, d for doublet, m for multiplet while J
(the coupling constant is given in Hertz). The infrared spectra were recorded using a
PerkinElmer Spectrum 100 FT-IR spectrometer, and the data are reported as percentage
transmittances at the respective wavenumbers (cm-1), between 4000 and 650 cm-1. The mass
spectra were recorded using Shimadzu LCMS-2020 instrument with only molecular ions
(M+) and major fragmentation peaks being reported with intensities quoted as percentages of
the base peak. Elemental analyses were performed on Thermal-Scientific Flash 2000
CHNS/O analyzer. All melting points were determined using the Stuart Scientific melting
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point apparatus. Absorption studies of CT-DNA and protein were recorded using Shimadzu
UV-1800 UV-Vis Spectrophotometer. The photoluminescence studies of CT-DNA and
protein were recorded using PerkinElmer LS 45 Fluorescence spectrometer.
2.2. Synthesis of ligands
L1-L5 have been previously synthesised [23-28] but in this study, a slightly modified
literature procedure [29] was employed in the pyridinyl Schiff base synthesis. To a hot
solution of 2-pyridinecarboxaldehyde (0.091 mL, 1 mmol) in anhydrous ethanol (10 mL) was
added to a hot solution of 2-aminophenol (1 mmol, 0.109 g) , 2-aminothiophenol (0.10 mL, 1
mmol), 2-fluoroaniline (0.097 mL, 1 mmol), p-toluidine (1 mmol, 0.11 g) and 2-
thiophenemethylamine (1 mmol, 0.10 mL) respectively in anhydrous ethanol (10 mL) in the
presence of glacial acetic acid. The resulting reaction was reflux at ca. 80 °C for 4 h. L1, L2,
and L4 solutions were evaporated to one-third of their initial volume under reduced pressure
and ether (10 mL) was added to each solution to afford a precipitate. The precipitates were
isolated via filtration, washed with cold ether (10 mL x 2), recrystallized from ethanol and
dried over anhydrous MgSO4. L3 and L5 solutions, on the other hand, were evaporated
completely under pressure, the oil obtained was recrystallized from ethanol and dried over
anhydrous MgSO4.
2.3. Synthesis of Complexes
Complexes 1‒15 were synthesized by dropwise addition of ethanolic solution (ca. 15 mL) of
each of L1‒L5 (1 mmol) to an ethanolic (ca. 10 mL) solution of silver nitrate (0.5 mmol, ca.
0.085 g), silver perchlorate (0.5 mmol, ca. 0.10 g) and silver trifluoromethanesulfonate (0.5
mmol, ca. 0.13 g) respectively under constant stirring at dark. The reaction was carried out
under nitrogen at ambient temperature for 6 h. The resulting precipitates were isolated using a
vacuum filter. Afterward, the precipitates were washed with cold ethanol (10 mL X2)
followed by cold ether (10 mL X2) and dried in-vacuo. The obtained complexes were
recrystallized by dissolving the complexes in dichloromethane and layering with toluene.
2.3.1 [Ag(L1)2]NO3
1
1
Brown, 0.38 g, 67 %, Melting point: 194–195 °C. H-NMR (400 MHz, DMSO-d6, δ ppm):
10.04 (2H, d, J = 17.28 Hz, Hf-OH), 9.00 (2H, s, He-C=N-), 8.74 (2H, d, J =3.93 Hz, Hd-
Py), 8.17 (4H, m, Ha,b-Py), 7.73 (2H, t, J = 4.89, 4.66 Hz, Hc-Py), 7.43 (2H, d, J = 7.73 Hz,
Hg-C6H4), 7.20 (2H, t, J = 7.72, 6.86 Hz, Hh-C6H4), 6.98 (2H, d, J = 8.22 Hz, Hj-C6H4), 6.89
(2H, t, J = 7.81, 7.40 Hz, Hi- C6H4). 13C-NMR (400 MHz, DMSO-d6, 25 ℃): δ = 159.05 (C6-
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C=N-), 151.43 (C5-Py), 151.14 (C12-C6H4), 150.74 (C4-Py), 139.20 (C7- C6H4), 135.93 (C2-
Py), 129.27 ( C10- C6H4), 127.98 (C3-Py), 127.69 (C1-Py), 121.81 (C8- C6H4), 119.93 (C9-
C6H4), 116.90 (C11- C6H4). FT-IR (cm-1); (OH) 3337, (Ar-CH) 3026, (-C=N-) 1622,
(pyridyl) 1582. UV/Vis (CH3CN): λmax 321 nm. MS: m/z Calcd. for [C24H20AgN4O2]: 504.32;
found [Ag(L1)2 + K + MeOH]+: 575 (12%), [Ag(L1-OH)]+ 288 (100%). Anal. Calcd. (%) for
(C24H20AgN5O5):. C, 50.90; H, 3.56; N, 12.37; found (%): C, 50.99; H, 3.58; N, 12.62.
2.3.2 [Ag(L2)2]NO3
2
1
Yellow solid, 0.40 g, 68 %, Melting point: 94–95 °C. H-NMR (400 MHz, DMSO-d6, δ
ppm): 8.74 (2H, d, J = 4.59 Hz, Hd-Py ), 8.34 (2H, d, J = 7.87 Hz, Hh-C6H4), 8.18 (2H, d, J =
8.09 Hz, He-C6H4), 8.12 (2H, d, J = 8.09 Hz, Ha-Py), 8.05 (2H, m, Hb-Py), 7.59 (4H, m, Hg,
f-C6H4), 7.51 (2H, t, J = 8.09, 7.00 Hz, Hc-Py). 13C-NMR (400 MHz, DMSO-d6, 25 ℃): δ =
168.90 (C5-Py), 153.60 (C7- C6H4), 150.16 (C6- C=N-),150.00 (C4-Py), 137.95 (C2-Py),
135.33 (C8- C6H4), 126.66 (C11- C6H4), 126.19 ( C10- C6H4), 126.01 (C1-Py), 123.28 (C9-
C6H4), 122.57 (C12-C6H4), 120.53 (C3-Py). FT-IR (cm-1): (Ar-CH) 3058, (C-S-C) 754,
(pyridyl) 1584. UV/Vis (CH3CN): λmax 298 nm. MS: m/z Calcd. for [C24H16AgN4S2]: 532,41;
found [Ag(L2)2 + H]+: 533 (100 %), [Ag(L2) + CH3CN + H]+: 359 (61%), Anal. Calcd. (%)
for (C24H16AgN5O3S2): C, 48.50; H, 2.71; N, 11.78; found (%): C, 48.46; H, 2.68; N, 11.73.
2.3.3 [Ag(L3)2]NO3
3
Green solid, 0.52 g, 92 %, Melting point: 84–85 oC, 1H-NMR (400 MHz, DMSO-d6, δ ppm):
8.95 (2H, s, He-C=N), 8.80 (2H, d, J = 4.77 Hz, Hd-Py), 8.20 (2H, m, Hb-Py), 8.14 (2H, d, J
= 7.28 Hz, Ha-Py), 7.77 (2H, m, Hh-C6H4), 7.48 (2H, m, Hc-Py), 7.30 (6H, m, Hi, g, f-C6H4).
13C-NMR (400 MHz, DMSO-d6, 25 ℃): δ = 162.57 (C6-C=N-), 151.20 (C12-C6H4), 150.37
(C5-Py), 149.94 (C4-Py), 139.09 (C2-Py), 137.06 (C7-C6H4), 128.95 ( C10-C6H4), 127.96
(C3-Py), 127.85 (C9-C6H4), 125.14 (C8- C6H4). 122.32 (C1-Py), 116.42 (C11- C6H4). FT-IR
(cm-1): (Ar-CH) 3068, (-C=N-) 1626, (pyridyl) 1586, (C-F) 1490. UV/Vis (CH3CN): λmax 291
nm. MS: m/z Calcd. for [C24H18AgF2N4]: 508.30; found [Ag(L3)2 + H]+: 509 (100%). Anal.
Calcd. (%) for (C24H18AgF2N5O3): C, 50.55; H, 3.18; N, 12.28; found (%): C, 50.21; H, 3.05;
N, 12.15.
2.3.4 [Ag(L4)2]NO3
4
o
1
Yellow solid, 0.51 g, 90 %, Melting point: 182–185 C. H-NMR (400 MHz, DMSO-d6, δ
ppm): 8.79 (2H, s, He-CH=N-), 8.72 (2H, m, Hd-Py ), 8.13 (2H, m, Ha-Py), 8.04 (2H, m, Hb-
Py), 7.60 (2H, m, Hc-Py), 7.30 (4H, m, Hg-C6H4), 7.21 (4H, m, Hf-C6H4), 2.32 (6H, s, Hh-
CH3). 13C-NMR (400 MHz, DMSO-d6, 25 ℃): δ = 159.28 (C6-C=N-), 152.50 (C5-Py),
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150.24 (C4-Py), 146.85 (C7-C6H4), 137.87 (C10-C6H4), 136.97 (C2-Py), 129.83 (C9- C6H4 ,
C12-C6H4), 126.40 (C3-Py), 123.98 (C1-Py), 121.49 (C8-C6H4, C13-C6H4), 20.57 (C11-
CH3), FT-IR (cm-1): (Ar-CH) 3025, (-C=N-) 1623, (pyridyl) 1584, (C-CH3) 2917. UV/Vis
(CH3CN): λmax 292, 321 nm. MS: m/z Calcd. for [C26H24AgN4]: 500,37; found [Ag(L4) +
EtOH + CH3CN]+: 391 (8%), [Ag(L4)2 + EtOH + CH3CN + Na + H]+: 611 (6%). Anal.
Calcd. (%) for (C26H24AgN5O3): C, 55.53; H, 4.30; N, 12.45; found (%): C, 55.26; H, 4.19;
N, 12.37.
2.3.5 [Ag(L5)2]NO3
5
Green solid, 0.60 g, 97 %, Melting point: 81–82 oC. 1H-NMR (400 MHz, DMSO-d6, δ ppm):
8.79 (2H, s, He-CH=N), 8.46 (2H, m, Hd-Py), 8.13 (2H, m, Hb-Py), 7.94 (2H, d, J = 7.68 Hz,
Ha-Py), 7.67 (2H, m, Hc-Py), 7.35 (2H, m, Hi-thio), 6.98 (2H, m, Hg-thio), 6.88 (2H, m, Hh-
thio), 5.04 (4H, s, Hf-CH2). 13C-NMR (400 MHz, DMSO-d6, 25 ℃): δ = 162.10 (C6- C=N-),
150.75 (C5-Py), 149.35 (C4-Py), 140.24 (C8-thio), 139.00 (C2-Py), 127.70 (C10-C4H3S),
127.32 ( C9-C4H3S), 127.04 (C3-Py), 126.54 (C11-C4H3S), 125.96 (C1-Py), 57.31 (C7-CH2).
FT-IR (cm-1): (Ar-CH) 3081, (-C=N-) 1639, (Pyridyl) 1588, (CH2) 1435, (thiophene) 697.
UV/Vis (CH3CN): λmax 289 nm. MS: m/z Calcd. for [C22H20AgN4S2]: 512,42; found [Ag(L5)
+ Lig. Frag. ]+: 400 (100%), 401 (28%), [Ag(L5)2 + Lig. Frag. + EtOH + Na]+: 691 (14%),
692 (7%). Anal. Calcd. (%) for (C22H20AgN5O3S2. 0.25 C3H6O): C, 46.40; H, 3.68; N, 11.89;
found (%): C, 46.22; H, 3.29; N, 11.69.
2.3.6 [Ag(L1)2]ClO4
6
o
1
Brown solid, 0.48 g, 79 %, Melting point: 147–148 C. H-NMR (400 MHz, DMSO-d6): δ
ppm = 9.04 (2H, s, -C=N-), 8.63 (2H, d, J = 4.25 Hz, Hd-Py), 8.11 (2H, t, J = 7.04, 7.66 Hz
Hb-Py), 8.00 (2H, d, J = 7.56 Hz, Ha-Py), 7.63 (2H, t, J = 7.15, 4.88 Hz, Hc-Py), 7.38 (2H, d,
J = 7.73 Hz, Hj-C6H4), 7.14 (2H, t, J = 7.58, 7.44 Hz, Hh-C6H4), 6.84 (4H, t, J = 8.01, 5.00
13
Hz, Hi-C6H4). C-NMR (400 MHz, DMSO-d6, 25 ℃): δ = 158.72 (C6-C=N-), 151.10 (C5-
Py), 150.81 (C12-C6H4), 150.41 (C4-Py), 138.87 (C7- C6H4), 135.60 (C2-Py), 128.94 ( C10-
C6H4), 127.65 (C3-Py), 127.36 (C1-Py), 121.48 (C8- C6H4), 119.60 (C9-C6H4), 116.57 (C11-
C6H4). FT-IR(cm-1); (OH) 3290, (-C=N-) 1624, (pyridyl) 1585. UV/Vis (CH3CN): λmax 291,
347, 357 nm. MS (ESI): m/z Calcd. for [C24H20AgN4O2]: 504.32; found [AgL-OH]+: 288
(100%), [AgL + CH3CN + H]+ 346 (27%), [AgL + Lig. Frag. + Na]+ 346 (47%), [Ag(L1)2]+
503 (29 %). Anal. Calcd. (%) for (C24H20AgClN4O6): C, 47.74; H, 3.34; N, 9.28; found (%):
C, 47.38; H, 3.08; N, 9.03.
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2.3.7 [Ag(L2)2]ClO4
7
1
White solid, 0.57 g, 90 %, Melting point: 195–196 °C. H-NMR (400 MHz, DMSO-d6, δ
ppm): 8.74 (2H, m, Hd-Py ), 8.34 (2H, d, J = 7.87 Hz, Hh-C6H4), 8.17 (2H, d, J = 7.88 Hz,
He-C6H4), 8.11 (2H, m, Ha-Py), 8.05 (2H, m, Hb-Py), 7.59 (4H, m, Hg, f-C6H4), 7.50 (2H,
13
m, Hc-Py). C-NMR (400 MHz, DMSO-d6, 25 ℃): δ = 168.90 (C5-Py), 153.60 (C7-C6H4),
150.15 (C6-C=N-),150.01 (C4-Py), 137.96 (C2-Py), 135.33 (C8-C6H4), 126.67 (C11-C6H4),
126.20 (C10-C6H4), 126.02 (C1-Py), 123.29 (C9-C6H4), 122.57 (C12-C6H4), 120.57 (C3-Py).
FT-IR (cm-1): (Ar-CH) 3067, (thiazole-C=N-) 1490, 1318, (C-S-C) 760, (Pyridyl) 1585.
UV/Vis (CH3CN): λmax 309 nm. MS: m/z Calcd. for [C24H16AgN4S2]: 532,41; found
[Ag(L2)2+H]+: 533 (100%), [Ag(L2) + CH3CN + H]+ 362 (41%). Anal. Calcd. (%) for
(C24H16AgClN4O4S2): C, 45.62; H, 2.55; N, 8.87; found (%): C, 45.51; H, 2.48; N, 8.74.
2.3.8 [Ag(L3)2]ClO4
8
1
Yellow solid, 0.58, 95 %, Melting point: 126–127 °C. H-NMR (400 MHz, DMSO-d6, δ
ppm): 8.94 (2H, s, He-CH=N), 8.80 (2H, m, Hd-Py), 8.17 (4H, m, Hb, a-Py), 7.76 (2H, m,
13
Hh-C6H4), 7.47 (2H, m, Hc-Py), 7.30 (6H, m, Hf, g, i-C6H4). C-NMR (400 MHz, DMSO-
d6, 25 ℃): δ = 162.53 (C6-C=N-), 153.69 (C12-C6H4), 151.22 (C5-Py), 149.94 (C4-Py),
139.10 (C2-Py), 128.96 (C7-C6H4), 127.96 ( C10-C6H4), 125.15 (C3-Py), 125.11 (C9-C6H4),
122.29 (C2-Py), 122.06 (C1-Py), 116.47 (C11-C6H4). FT-IR (cm-1): (Ar-CH) 3073, (-C=N-)
1625, (Pyridyl) 1584, (C-F) 1489. UV/Vis (CH3CN): λmax 292, 321 nm. MS: m/z Calcd. for
[C24H18AgF2N4]: 508,30; found: [L3 + CH3CN + Na]+ 264 (100%), [Ag(L3)2 + H ]+ 509
(98%). Anal. Calcd. (%) for (C24H18AgClF2N4O4): C, 47.43; H, 2.99; N, 9.22; found (%): C,
47.32; H, 2.83; N, 9.02.
2.3.9 [Ag(L4)2]ClO4
9
1
Yellow solid, 0.55 g, 92 %, Melting point: 210–211 °C. H-NMR (400 MHz, DMSO-d6, δ
ppm): 9.09 (2H, s, He-CH=N-), 8.78 (2H, m, Hd-Py ), 8.22 (2H, m, Hb-Py), 8.11 (2H, m, Ha-
Py), 7.76 (2H, m, Hc-Py), 7.41 (4H, m, Hg-C6H4), 7.23 (4H, m, Hf-C6H4), 2.30 (6H, s, Hh-
CH3). 13C-NMR (400 MHz, DMSO-d6, 25 ℃): δ = 158.98 (C6-C=N-), 151.54 (C5-Py),
150.35 (C4-Py), 145.73 (C7-C6H4), 139.59 (C10-C6H4), 138.35 (C2-Py), 130.24 (C9-C6H4 ,
C12-C6H4), 128.70 (C3-Py), 128.18 (C1-Py), 122.45 (C8-C6H4, C13-C6H4), 20.90 (C11-
CH3). FT-IR (cm-1): (Ar-CH) 3028, (-C=N-) 1628, (Pyridyl) 1590. UV/Vis (CH3CN): λmax
290, 324 nm. MS: m/z Calcd. for [C26H24AgN4]: 500,37; found [Ag(L4) + MeOH + Na]+ 359
(100%), [Ag(L4)2 + H]+ 501 (67%). Anal. Calcd. (%) for (C26H24AgClN4O4): C, 52.06; H,
4.03; N, 9.34; found (%): C, 51.86; H, 3.78; N, 9.28.
7

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2.3.10 [Ag(L5)2]ClO4 10
1
Brown, 0.35 g, 57 %, Melting point: 155-156°C. H-NMR (400 MHz, DMSO-d6, δ ppm):
8.74 (2H, s, He-CH=N), 8.56 (2H, d, J = 4.26 Hz Hd-Py), 8.10 (2H, m, Hb-Py), 7.94 (2H, d,
J = 7.77 Hz, Ha-Py), 7.67 (2H, m, Hc-Py), 7.41 (2H, d, J = 5.06 Hz, Hi-thio), 7.04 (2H, m, J
13
= 3.34 Hz, Hg-thio), 6.95 (2H, m, Hh-thio), 5.06 (4H, s, Hf-CH2). C-NMR (400 MHz,
DMSO-d6, 25 ℃): δ = 162.32 (C6-C=N-), 150.60 (C5-Py), 149.83 (C4-Py), 140.41 (C8-
C4H3S), 138.80 (C2-Py), 127.46 (C10-C4H3S), 127.11 (C9-C4H3S), 126.58 (C3-Py), 126.42
(C11-thio), 125.91 (C1-Py), 57.42 (C7- CH2). FT-IR (cm-1): (Ar-CH) 3079, (Pyridyl) 1593,
(thiophene) 705. UV/Vis (CH3CN): λmax 291 nm. MS: m/z Calcd. for [C22H20AgN4S2]:
512.42; found [Ag(L5)2 + Lig. Frag.]+ 613 (100%). Anal. Calcd. (%) for (C22H20AgClN4O4S2
. 0.25 C3H6O): C, 43.62; H, 3.46; N, 8.94; found (%): C, 43.64; H, 3.68; N, 8.68.
2.3.11 [Ag(L1)2]CF3SO3 11
Brown, 0.62 g, 95 %, Melting point: 137-138 °C. 1H-NMR (400 MHz, DMSO-d6): δ ppm =
9.96 (2H, s, Hf-OH), 9.05 (2H, s, He-C=N-), 8.68 (2H, d, J = 4.77 Hz, Hd-Py), 8.16 (4H, m,
Ha,b-Py),7.69 (2H, m, Hc-Py), 7.39 (2H, m, Hg- C6H4), 7.17 (2H, m, Hh- C6H4), 6.94 (2H,
13
m, Hj-C6H4), 6.87 (2H, m, Hi- C6H4). C-NMR (400 MHz, DMSO-d6, 25 ℃): δ = 158.84
(C6-C=N-), 151.26 (C5-Py), 150.68 (C12-C6H4), 150.38 (C4-Py), 138.64 (C7- C6H4), 137.84
(C2-Py), 128.98 ( C10- C6H4), 128.56 (C3-Py), 127.15 (C1-Py), 121.18 (C8- C6H4), 119.73
(C9-C6H4), 116.60 (C11- C6H4). FT-IR (cm-1); (OH) 3251, (Ar-CH) 3060, (-C=N-) 1623,
(pyridyl) 1587. UV/Vis (CH3CN): λmax 292, 347, 358 nm. MS (ESI): m/z Calcd. for
[C24H20AgN4O2]: 503.06; found [AgL1-OH]+: 288 (100%), [AgL1 + CH3CN + H]+ 346
(29%), [AgL1 + Frag. + Na]+ 346 (29%), [Ag(L1)2]+ 503 (29 %). Anal. Calcd. (%) for
(C25H20AgF3N4O5S. 0.25 C3H6O): C, 46.31; H, 3.24; N, 8.39; found (%): C, 46.72; H, 3.18;
N, 8.67.
2.3.12 [Ag(L2)2]CF3SO3 12
1
Orange solid, 0.48 g, 70 %, Decomposes at 183–184 °C, H-NMR (400 MHz, DMSO-d6, δ
ppm): 8.73 (2H, m, Hd-Py ), 8.34 (2H, m, Hh-C6H4), 8.18 (2H, m, He-C6H4), 8.11 (2H, m,
Ha-Py), 8.05 (2H, m, Hb-Py), 7.60 (2H, m, Hg-C6H4), 7.56 (2H, m, Hf-C6H4), 7.50 (2H, m,
13
Hc-Py). C-NMR (400 MHz, DMSO-d6, 25 ℃): δ = 169.92 (C5-Py), 158.86 (C7-C6H4),
152.92 (C6-C=N-),148.49 (C4-Py), 137.86 (C2-Py), 135.99 (C8-C6H4), 135.36 (C11-C6H4),
126.13 ( C10-C6H4), 125.02 (C1-Py), 123.27 (C9-C6H4), 119.61 (C12-C6H4), 119.03 (C3-
Py). FT-IR (cm-1): (Ar-CH) 3065, (thiazole-C=N-) 1461, 1376, (C-S-C) 762, (Pyridyl) 1588.
UV/Vis (CH3CN): λmax 300 nm. MS: m/z Calcd. for [C24H16AgN4S2]: 532,41; found [Ag(L2)2
8

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+ 2H] : 535 (100). Anal. Calcd. (%) for (C25H16AgF3N4O3S3): C, 44.06; H, 2.37; N, 8.22;
found (%): C, 43.73; H, 1.95; N, 8.02
2.3.13 [Ag(L3)2]CF3SO3 13
Green, 0.53, 81 %, Melting point: 106-107 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 8.98
(2H, s, He-CH=N), 8.81 (2H, m, Hd-Py), 8.22 (4H, m, Hb-Py), 8.14 (2H, d, Ha-Py), 7.78
(2H, m, Hh-C6H4), 7.48 (2H, m, Hc-Py), 7.29 (6H, m, Hf, g, i-C6H4). 13C-NMR (400 MHz,
DMSO-d6, 25 ℃): δ = 162.47 (C6-C=N-), 153.71 (C12-C6H4), 151.33 (C5-Py), 149.64 (C4-
Py), 139.26 (C2-Py), 129.03 (C7-C6H4), 128.40 (C10-C6H4), 128.12 (C3-Py), 125.10 (C9-
C6H4), 122.33 (C8-C6H4). 122.21 (C1-Py), 116.46 (C11-C6H4). FT-IR (cm-1): (Ar-CH) 3068,
(-C=N-) 1628, (Pyridyl) 1588, (C-F) 1491. UV/Vis (CH3CN): λmax 290 nm. MS: m/z Calcd.
for [C24H18AgF2N4]: 508,30; found [Ag(L3)2 + H]+: 509 (24%). Anal. Calcd. (%) for
(C25H18AgF5N4O3S): C, 45.68; H, 2.76; N, 8.52; found (%): C, 46.01; H, 2.83; N, 8.72.
2.3.14 [Ag(L4)2]CF3SO3 14
o
1
Yellow solid, 0.46 g, 71 %, Melting point: 99–101 C. H-NMR (400 MHz, DMSO-d6, δ
ppm): 9.05 (2H, s, He-CH=N-), 8.78 (2H, m, Hd-Py ), 8.21 (2H, m, Hb-Py), 8.12 (2H, m, Ha-
Py), 7.75 (2H, m, Hc-Py), 7.39 (4H, m, Hg-C6H4), 7.23 (4H, m, Hf-C6H4), 2.30 (6H, s, Hh-
CH3). 13C-NMR (400 MHz, DMSO-d6, 25 ℃): δ = 159.04 (C6-C=N-), 151.46 (C5-Py),
150.57 (C4-Py), 145.85 (C7-C6H4), 139.46 (C10-C6H4), 138.24 (C2-Py), 130.23 (C9-C6H4
,C12-C6H4), 128.31 (C3-Py), 128.04 (C1-Py), 122.38 (C8-C6H4, C13-C6H4), 20.89 (C11-
CH3), FT-IR (cm-1): (Ar-CH) 3025, (-C=N-) 1626, (Pyridyl) 1587, (C-CH3) 2917. UV/Vis
(CH3CN): λmax 290, 321 nm. MS : m/z Calcd. for [C26H24AgN4]: 500,37; found [Ag(L4)2 + H
]+ 501 (100%). Anal. Calcd. (%) for (C27H24AgF3N4O3S): C, 49.94; H, 3.73; N, 8.63; found
(%): C, 49.67; H, 3.38; N, 8.49.
2.3.15 [Ag(L5)2]CF3SO3 15
Green solid, 0.58 g, 87 %, Melting point: 97–98 oC. 1H-NMR (400 MHz, DMSO-d6, δ ppm):
8.79 (2H, s, He-CH=N), 8.47 (2H, m, Hd-Py), 8.13 (2H, m, Hb-Py), 7.94 (2H, d, J = 7.43 Hz,
Ha-Py), 7.68 (2H, m, Hc-Py), 7.34 (2H, m, Hi-thio), 6.98 (2H, m, Hg-thio), 6.88 (2H, m, Hh-
thio), 5.03 (4H, s, Hf-CH2). 13C-NMR (400 MHz, DMSO-d6, 25 ℃): δ = 162.36 (C6- C=N-),
156.15 (C5-Py), 150.72 (C4-Py), 140.57 (C8-C4H3S), 138.92 (C2-Py), 127.59 (C10-C4H3S),
127.23 ( C9-C4H3S), 126.57 (C3-Py), 126.01 (C11-C4H3S), 125.27 (C1-Py), 57.57 (C7-CH2).
FT-IR (cm-1): (Ar-CH) 3077, (-C=N-) 1622, (Pyridyl) 1591, (CH2) 1474, (thiophene) 703.
UV/Vis (CH3CN): λmax 290 nm. MS : m/z Calcd. for [C22H20AgN4S2]: 512.42; found
9

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[Ag(L5)2] 513 (91%), [Ag(L5)2 + Lig. Frag. + 2EtOH ] 701 (100%). Anal. Calcd. (%) for
(C23H20AgF3N4O3S3): C, 41.76; H, 3.05; N, 8.47; found (%): C, 41.72; H, 2.96; N, 8.27.
2.4 Single-crystal X-ray diffraction
Crystal evaluation and data collection of 4, 5, 8, 9, and 15 were recorded on a Bruker Apex
Duo diffractometer equipped with an Oxford Instruments Cryojet operating at 100(2) K and
an Incoatec microsource operating at 30 W power. The data were collected with Mo Kα (λ =
0.71073 Å) radiation at a crystal-to-detector distance of 50 mm using omega and phi scans.
The data were reduced with the programme SAINT [30] using outlier rejection, scan speed
scaling, as well as standard Lorentz and polarisation correction factors. A SADABS [31]
semi-empirical multi-scan absorption correction was applied to the data.
The structures of 4, 5, 8, 9, and 15 were solved by the direct method using the SHELXS [32]
program and refined. The visual crystal structure information was performed using ORTEP-3
[33], system software. Non-hydrogen atoms were first refined isotropically and then by
anisotropic refinement with a full-matrix least-squares method based on F2 using SHELXL
[34]. All hydrogen atoms were positioned geometrically, allowed to ride on their parent
atoms, and refined isotropically. The crystallographic data and structure refinement
parameters for 4, 5, 8, 9, and 15 are given in Table 1.
Complex 4 is a two component, non-merohedral twin. An HKLF5 data refinement revealled a
mass ratio of 0.741(15):0.259(15) with a twin matrix -1 0 0 0 -1 0 0 0 -1. A disorder was
observed in 5, 9 and 15. The anionic molecular species is severely disordered in 5 because it
resides in voids that form channels between the cationic Ag(I) complexes as depicted in Fig.
S1 (see supplementary info). Attempts to model the disordered ion was unsuccessful and the
contribution of the missing solvent to the calculated structure factors was taken into account
by using a SQUEEZE [35] routine of PLATON [36]. A solvent mask was calculated, and
204 electrons were found in a volume of 540 Å3 in 1 void per unit cell. This is consistent with
the presence of 1.5[NO3] per Asymmetric Unit which accounts for 186 electrons per unit cell.
In 9, one of the perchlorate ions is disordered over a special position. PART -1 and -2
instructions were used to model the disorder with all components having equal site
occupancy factor. The triflate ion is also disordered over a special position in 15 and PART -
1 instruction and 50% site occupancy was used to model the disordered anion.
Table 1: Crystal data and structure refinement for complexes 4, 5, 8, 9 and 15
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4
5
8
9
15
Chemical C52H50Ag2N10O7 C22H20AgN4S2 C24H18AgF2N4·ClO4
formula
C26H24AgClN4O4
C23H22AgF3N4O4S3
Formula
Weight
1142.76
512.41
607.74
599.81
679.49
Crystal
system
Monoclinic
Monoclinic
Monoclinic
Monoclinic
Monoclinic
Space group
a(Å)
Pn
11.6045(7)
17.0924(10)
12.4183(7)
90
I2/a
11.4202(2)
15.9615(3)
13.5887(3)
90
C2/c
18.988(2)
5.9799(7)
13.8799(15)
90
C2/c
25.8553(14)
15.9354(14)
19.6875(12)
90
I2/c
11.5656(8)
15.4463(11)
15.1331(10)
90
b(Å)
c(Å)
α(°)
β(°)
102.270(2)
90
101.925(2)
90
130.737(4)
90
111.464(4)
90
107.439(3)
90
γ(°)
V(Å3)
2406.9(2)
2
2423.54(8)
4
1194.2(2)
2
7549.0(9)
12
2579.2(3)
4
Z
ρcalc(gcm-3)
1.577
1.404
1.690
1.583
1.750
µ (mm−1)
F(000)
0.879
1164
1.02
1.011
608
0.947
3648
1.09
1368
Crystal size
(mm3)
0.40 x 0.18 x 0.40× 0.30 × 0.24 × 0.10 × 0.04
0.37 x 0.28 x 0.22
0.30 × 0.20 × 0.10
0.17
0.20
θ range for
data
2.4 to 56.5
4.5 to 56.8
3.9 to 56.4
3.1 to 55.0
1.9 to 28.2
collection (°)
Reflections
collected
44935
13183
10381
65342
21898
Independent 10584 [R(int) =
2583 [R(int) =
0.018]
8598 [R(int) = 0.027]
3195 [R(int) = 0.024]
2985 [R(int) =
0.016]
reflections
0.029]
Data /
restraints /
parameters
10584 /321 /
792
2985/0/132
2583 / 1 / 164
R1 = 0.018
8598 / 0 / 509
R1 = 0.027
3195 / 164 / 216
R1 = 0.036
R indices
R1 = 0.022
R1 = 0.026
[I>2sigma(I)]
wR2 = 0.051
R1 = 0.024
wR2 = 0.063
R1 = 0.027
wR2 = 0.043
R1 = 0.019
wR2 = 0.065
R1 = 0.033
wR2 = 0.094
R1 = 0.037
R indices (all
data)
wR2 = 0.052
wR2 = 0.064
wR2 = 0.043
wR2 = 0.069
wR2 = 0.095
Largest diff. 0.46 and -0.33 0.39 and -0.56
0.54 and -0.29
1.08 and -0.63
1.80 and -0.72
peak and hole
(e Å-3)
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2.5 In vitro antimicrobial studies
2.5.1 Müeller-Hinton agar test plates preparation
Sterilized Nutrient agar medium was first prepared by dissolving 38 g of Müeller-Hinton agar
(MHA) (Biolab, South Africa) in distilled water (1 L). The resulting Nutrient agar medium
was subjected to sterilization by autoclaving for 15 min at 121 °C and then cooled down to 45
°C in a water bath. The cooled Agar medium was poured in Petri dishes, while ensuring a
uniform 4 mm depth of the medium and cooling further to ambient temperature [29].
2.5.2 Inoculation procedure
Complexes 1‒15 and their ligands were tested against four gram-negative bacteria;
Salmonella typhimurium ATCC 14026, Pseudomonas aeruginosa ATCC 27853, Escherichia
coli ATCC 25922 and Klebsiella pneumoniae ATCC 31488 and two gram-positive bacteria,
viz: Staphylococcus aureus ATCC 700699 (methicillin-resistant) and Staphylococcus aureus
ATCC 25923. These bacteria were inoculated in a sterilized Nutrient Broth (NB) (Biolab,
South Africa) through streak plate technique and incubated at 37 °C for 18 h. The nutrient
broth was sterilized by dissolving 1.3 g in distilled water (100 mL). Ca. 10 mL of the nutrient
broth was transferred into a cotton wool-plugged test tubes and wrapped with an aluminium
foil. The nutrient broth in the test tubes was then autoclaved for 15 min at 121 °C and cooled
to 37 °C. Following this was the isolation of a single colony of the bacteria and inoculated
into a 10 mL sterile NB which was then incubated in a shaking incubator at 37 °C for 18 h.
Each of the bacteria strain concentrations was adjusted with sterile distilled water to get a
final concentration of 1.5 x 108 cfu/mL, i.e., 0.5 Mc Farland’s Standard using a densitometer
(Mc Farland Latvia) [37]. The bacteria were lawn inoculated on the set MHA Petri-dishes
using a sterile cotton swab. Screening of L1‒L5 and 1‒15 for antibacterial activity was first
carried out by spotting 5µL of their solution (prepared from the dissolution of 1000 µg of the
L1‒L5 and 1‒15 in 1 mL dimethyl sulfoxide (DMSO) on an MHA petri dish and incubated
at 37 °C for 18 h. The antibacterial activity was determined by looking out for a clear zone at
the spotting point. The compounds with potential antimicrobial activity were then tested for
their minimum inhibitory concentration (MIC) against six bacterial. 10 serial dilution of L1‒
L5 and 1‒15 was done to obtain 1000 µg/mL to 0.2 µg/mL concentrations, where the
compounds showed lower MICs than 0.2 µg/mL, the solution was further diluted 5 times to
obtain 0.100 µg/mL to 0.00625 µg/mL concentrations. Evaluation of the compounds MIC
was determined by spotting 5 µL of each concentration of L1‒L5 and 1‒15 on the MHA
plates, and the plates were incubated at 37 °C for 18 h. These procedures were done in
triplicate to give the accurate lowest concentration of the compounds where no visible
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bacterial growth was seen after incubation. Ciprofloxacin was used as a standard and DMSO
as a negative control [37-39].
2.6 Antioxidant assay
Antioxidant activity studies of L1‒L5 and 1‒15 was done using two assays; Free radical
scavenging and Ferric reducing antioxidant power (FRAP) assay.
The free radical scavenging ability of L1‒L5 and 1‒15 was determined using the 2,2-
diphenyl-1-picrylhydrazyl (DPPH) method following a procedure by Liyana-Pathirana et al.
[40]. Briefly, a 500 μL of 0.3 mM DPPH solution was mixed with 1 mL of different
concentrations (0.25 mg/mL, 0.5 mg/mL, 1 mg/mL and 2 mg/mL) of the L1‒L5 and 1‒15.
Various concentrations of Ascorbic acid (0.25 mg/mL, 0.5 mg/mL, 1 mg/mL and 2 mg/mL)
were used as reference material. The mixture was vortexed thoroughly and kept in the dark at
room temperature for 30 min. Then the absorbances were measured at 517 nm after 30 min of
incubation at room temperature to evaluate the DPPH reduction. L1‒L5 and 1‒15 ability to
scavenge DPPH radical was calculated by the formula as shown in Equation 1.
(1)
The ferric reducing antioxidant power assay is often used to evaluate the ability of an
antioxidant to donate an electron. The reducing ability of the compounds was measured using
a previously published method [41] after slight modifications where different concentrations
(0.25 mg/mL, 0.5 mg/mL, 1 mg/mL and 2 mg/mL) of L1‒L5 and 1‒15 and standard
(Ascorbic acid) were mixed with 2.5 mL of phosphate buffer (0.2 mol/L, pH 6.6) and 2.5 mL
of 1% w/v potassium ferricyanide respectively. The resulting mixture was incubated at 50 °C
for 20 min, and 2.5 mL of 10% trichloroacetic acid was added to acidify the mixture. After
that, 1 mL of the acidified mixture was added to 1 mL of distilled water and 0.5 mL of 0.1%
FeCl3. The absorbance of the resulting solution was then measured at 700 nm. The
antioxidant power of the compounds was expressed as a percentage of ferric reducing
antioxidant power Ascorbic acid equivalent, as shown in Equation 2.
(2)
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2.7 DNA binding experiments
The interaction of the L1‒L5 and complexes 1‒15 with calf thymus-DNA was carried out in
a Phosphate buffer saline solution pH 7.2 stored at 4 °C. The DNA stock solution was
prepared by dissolving CT-DNA sodium salt in Phosphate buffer saline solution with
continuous stirring overnight. It was filtered and stored at 4 °C and used within four days.
The final concentration of CT-DNA sodium salt was determined by UV-visible absorption
using the absorption coefficient ε260 = 6600 M-1 cm-1 [42]. The ratio of CT-DNA at 260 and
280 nm UV absorbance was 1.93, indicating that it is pure and sufficiently free of protein
contamination. A fixed amount of the compounds (50 µM) were prepared by dissolving an
appropriate amount in DMSO.
2.7.1 DNA Absorption spectral study
The absorption study was done by titrating different concentrations of CT-DNA (0–30 µM)
in phosphate buffer saline solution against constant concentrations (50 µM) of L1–L5 and 1–
15. The compounds-CT-DNA mixture was incubated for 10 mins before measuring the
absorbance using UV-vis absorption spectroscopy [43]. An equal amount of CT-DNA was
also added to the reference solution to get rid of CT-DNA absorbance.
The compounds intrinsic binding constant, Kb were determined using Wolfe-Shimer equation
as follows:
[
]
[
]
(3)
Where [DNA], εa, εb, and εf are DNA concentration, apparent, fully bound complex, and free
complex extinction coefficients, respectively. The ratio of the slope to the intercept from the
plot of [DNA]/(εa - εf) against [DNA] gave the binding constant Kb of the compounds.
2.7.2 Luminescence competitive displacement study
A competitive displacement study was carried out using fluorescence spectroscopy between
CT-DNA pre-treated with ethidium bromide (EB) and complexes 1‒15 to establish the
complexes' actual mode of binding to CT-DNA. Ethidium bromide in buffer solution has low
fluorescence intensity due to fluorescence quenching of the free ethidium bromide by the
solvent molecule, but on binding through intercalation to DNA, its emission intensity is
drastically enhanced [44]. The competitive luminescence displacement assay was done using
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a modified method [45]. Pre-treated 15 µM solution of EB with 15 µM of CT-DNA was
prepared in a phosphate buffer of pH 7.2 and left to equilibrate for 30 mins. Keeping CT-
DNA-EB concentrations constant, different concentrations (0-20 µM) of the complexes were
added at 8 mins interval for equilibration at room temperature. The fluorescence quenching
ability of CT-DNA bound EB by the complexes were recorded in the wavelength range of
530-700 nm with an excitation wavelength at 525 nm at 25 °C.
[ ]
[ ]
(4)
A Stern-Volmer equation 4, was used to evaluate the CT-DNA-EB fluorescence quenching of
the complexes. Where Fₒ and F denote the relative fluorescence intensities of CT-DNA in the
absence and presence of the quencher, Ksv, Stern-Volmer quenching constant, [Q], quencher
concentration, Kq, bimolecular quenching constant and τₒ, average lifetime of the fluorophore
in the absence of the quencher which is typically equal to 10-8 s in biomacromolecules.
2.7.3 Albumin binding assay using absorption spectroscopy titration
Absorbance assay of albumin binding is a very simple method used in identifying the
conformational changes in protein. A stock solution of bovine serum albumin (BSA) was
prepared by dissolving an appropriate amount of BSA in phosphate buffer saline solution (pH
7.2) under constant stirring for 1 h at 25 °C. It was kept at 4 °C and used within four days.
The BSA concentration was determined spectrophotometrically by using ε280 = 44300 M-1
cm-1 absorption coefficient. The stock solution of the complex was prepared by dissolving 1
mmol of the complexes in DMSO. Absorption titration assay was done by adding different
concentrations (0–10 µM) of the complexes to a constant BSA concentration (6 µM). The
samples solution was incubated after each addition of the concentrations for 10 mins at 25 °C
before recording the absorbance at λmax 280 nm. Binding constant Kb was calculated from
the intercept to the slope ratio of 1/[A - A°] vs. 1/[complex] linear curve where A and A°
represent BSA absorbance in the presence and absence of complexes, respectively [46].
2.7.4 Albumin binding studies using fluorescence quenching method
A solution of 1 µM of BSA was titrated against various concentrations of the complexes (0–
35 µM) and incubated for 8 mins at 25 °C. BSA was excited at 280 nm and emitted at 346
nm wavelength [22]. Ksv constant was determined from the Stern-Volmer equation:
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⁄
[ ] [ ]
(5)
Where I0 and I represent the intensities of the fluorescence in the presence and absence of the
quencher respectively while Kq, Ksv, τₒ, [Q] represent the BSA quenching rate constant,
dynamic quenching constant, the average lifetime of BSA without the quencher (τₒ = 10−8 s)
and concentration of quencher respectively.
3.0 Results and Discussion
3.1 Synthesis and Characterization of L1 – L5
L1 - L5 were prepared by the condensation reaction of 2-pyridine carboxaldehyde with five
substituted anilines (2-aminophenol, L1; 2-aminobenzenethiol, L2; 2-fluoroaniline, L3; p-
toluidine, L4; and 2-thiophenemethylamine, L5) in anhydrous ethanol, all in excellent yields.
1
13
The reactions were monitored using IR and were further characterized using H NMR, C
NMR, mass spectrometry, UV-Vis, and melting points. In the IR spectra of ligands L1–L5,
the absence of carbonyl stretching bands and appearance of the imine ѵ(C=N) absorption
band between 1620 and 1625 cm-1, revealed the successful synthesis of the proposed Schiff
base ligands. 1H NMR spectra of L1, L3, L4, and L5 in deuterated acetone exhibited singlets
at 8.81, 8.65, 8.59 and 8.44 ppm respectively attributed to the azomethine (CH=N) proton.
There was no azomethine proton resonance in the spectrum of L2 due to cyclization of the
ligand through S atom to form 2-(pyridin-2-yl)benzo[d]thiazole. All the physical and spectral
data for the known ligands [23-28] were in agreement with the ones previously reported.
3.2 Synthesis and characterization of the complexes
Complexes 1 - 15 were synthesized by reacting two equivalents of the respective ligands with
one equivalent of the silver(I) salts in anhydrous ethanol. The complexes were obtained in
good to excellent yields of between 60 and 97 % as air-stable solids. The formation and
purity of the products were confirmed by various spectroscopic and analytical techniques.
The scheme below represents the synthesis route for complexes 1 to 15.
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Scheme 1: Synthesis of complexes 1 – 15 under constant magnetic stirring in anhydrous ethanol
Coordination of L1‒L5 to the Ag(I) centers were monitored by comparing the FT-IR
spectrum of the free ligands to their respective complexes (see supplementary info Table S1).
The Bands at 1620–1622 cm-1 in the FTIR spectra of L3 and L4 ligands associated with the -
C=N- bond stretching frequencies shifted to higher frequencies between 1625 and 1628 cm-1
band upon coordination to Ag(I), while in the spectra of L1 and L5 bands shifted to lower
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frequencies from between 1625 and 1644 to between 1622 and 1639 cm respectively, on
coordination. In the spectrum of L2 with thiazole moiety, the -C=N- bond shifted from 1457
cm-1 to higher frequency in the spectra of 2, 7 and 12 between ca. 1461–1490 cm-1. The
absorption bands associated with the pyridinyl ring in the range 1579–1585 cm-1 in the
spectra of L1–L5 shifted to 1583–1593 cm-1. Coordination of L1, L3, L4, and L5 to Ag(I)
was via Nim and Npy donor atoms, while for L2 coordination was via Nthiazole and Npy donor
atoms, similar to reports of related structures in literature [47]. Bands attributed to anionic
functional groups were observed in all the complexes with strong sharp bands attributed to
-
-
-
NO3 , ѵ3 asymmetric stretch for ClO4 , and SO3 of the triflate anion, observed between 1273
and 1335 cm-1 in the spectra of 1–5, 1067 and 1096 cm-1 in the spectra of 6–10, and at 1025–
1026 cm-1 in the spectra of 11–15 respectively.
1
The H-NMR spectra of protons close to the donor atoms in L1‒L5 were compared to 1‒15
1H-NMR spectra (see supplementary info Table S1), and a noticeable downfield shift in the
protons around N atoms was observed. In the spectra of complexes with L1, L3, L4, and L5
as the ligands, the azomethine alpha protons and for those with L2 as the ligand, the thiazole
-C=N- alpha protons with respect to their pyridinyl N, shifted downfield relative to those of
their respective free ligand. This is an indication of coordination of the Ag(I) center via the
imine N and pyridinyl N atoms in the complexes of L1, L3, L4, and L5, and to the thiazole N
and pyridinyl N atoms in the complexes of L2.
The mass spectra of 1–15 in acetonitrile or methanol were all obtained in the positive ion
mode and are a confirmation of coordination to Ag(I) centers in all complexes. The spectra
all showed signals, which correspond to species containing the ligands together with the
silver ion and sometimes in the presence of solvent molecules. There were peaks at 503 for
complexes 1, 6 and 11 arising from [Ag(L1)2]+, 533 for 2, 7 and 12 corresponding to
[Ag(L2)2]+ while for 3, 8 and 13 and 4, 9 and 14 at m/z 509 and at m/z 501 corresponding to
[Ag(L3)2]+ and [Ag(L4)2]+ respectively. For complexes 5, 10 and 15, the molecular ion peak
at m/z 513 corresponded to [Ag(L5)2]+.
Complexes 1 ‒ 15 are soluble in most polar solvents, some in water as well. Their molar
conductivities measured in dimethylformamide at 25 °C gave values which range between
1.02 and 3.48 x 104 S m-2 mol-1 (see supplementary info Table S2), indicating that the
complexes act as electrolytes. The electronic behaviours of L1 – L5 and 1 ‒ 15 were studied
using UV-vis spectrophotometry with the spectra recorded in acetonitrile. L1 and L4 show
absorption bands each at 289 and 289 nm, and 362 and 329 nm respectively attributable to π–
π* and n–π* transitions. L2, L3, and L5 each have one absorption band at 309, 284, and 238
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nm, respectively, which can be assigned to π–π* transitions [48-50]. Complex 1 has only one
band, while 6 and 11 have two absorption bands, similar to those of L1. The two bands in the
spectra of 6 and 11 were observed at 323 and 359 nm, 289 and 293 nm, respectively. It is
possible that in the spectrum of 1, the two overlay and are observed in the broad band at 334
nm. These bands attributed to intra-ligand n–π* and π–π*, as well as metal to ligand charge
transfer transitions (MLCT) since no d–d transitions are expected for d10 silver(I) complexes
[51, 52]. There is generally a slight red-shift in the spectra of all complexes relative to those
of ligands associated with a decrease in energy at the excited states upon coordination to
silver(I) [53-55]. The absorption patterns of complexes 4, 6, 8, 9, 11, and 14 are similar
having two absorption bands at 321–359 nm, which may be attributed to the n–π* transition
and 290 – 308 nm allied to π–π* transition in the ligand. Generally, the presence of different
phenyl substituents ranging from electron-donating to electron-withdrawing as well as
different counter ions bring about the different electronics properties of complexes 1-15.
Sterically, the complexes differ slightly due to the great resemblance in the backbone of L1-
L5 and this agrees with their similar Ag(I) complexes spatial geometry structures. Although,
the influence of steric effect of the substituents or the counter ion can be observed from the
discrepancies in the complexes N‒Ag‒N bond lengths and angles. However, the electronic
effect, steric effect and the nature of the counter ion have less influence on the shape of
complexes 1-15.
3.3. Crystal structures of complexes 4, 5, 8, 9 and 15
The ORTEP diagrams, along with the atom numbering schemes for complexes 4, 5, 8, 9, and
15 are shown in Fig. 1. Firstly, all complexes feature two sets of planes. In the first set of
planes, Plane 1 can be described by a five-member metallacycle including the pyridine imine
unit and Plane 2 is described by substituted phenyl ring for 4, 8 and 9. In all complexes, two
ligands coordinate to the metal center thereby resulting in a pseudo-tetrahedral geometry. In
this communication, the dihedral angle between the two planes formed by the metallacycles
is defined as θ. Only 4, 8 and 9 posses plane 2. The dihedral angles between plane 1 and
plane 2 described above are hereby denoted γ. The second set of planes are in the structures
of complexes 5 and 15 and are defined by the thiophene ring along with the methylene linker.
In the asymmetric unit of complex 4, are two molecules of the cation [Ag(L4)2]+ and two
-
molecules of the NO3 anion. The two cationic molecules are not related by symmetry and
display differences in the conformations of the L4 moieties wherein molecule 1, the angle γ
seems to be smaller in molecule 2 (1.41(7) and 9.73(8)°) compared to molecule 1 (24.15(9)
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and 31.60(8)°). Complex 5 has half a molecule of the complex [Ag(L5)] which is neutral
with no dihedral γ angle as Planes 1 and 2 are linked via a CH2 linker. 8 has ½ a molecule of
the cation [Ag(L3)]+ and ½ a molecule of the perchlorate anion with a γ angle of 38.62(7)°. In
the asymmetric unit of 9 are one and a half molecules of the cation [Ag(L4)2]+ and [Ag(L4)]+
and similarly one and a half molecules of the perchlorate anions. In the structure of the full
molecule, the γ angles are different for each set of planes of each ligand coordinated to the
Ag(I) center and whereas one displays near coplanarity (6.77(6)°), the other has a dihedral
angle of 25.23(4)°, and 24.63(4)° in the half molecule. The asymmetric unit of 15 has half a
-
molecule of the cation [Ag(L5)]+, one molecule of the anion CF3SO3 , and a molecule of
water. This fashion of coordination and arrangement in the asymmetric units is similar to
those reported by our group [29].
Secondly, in all complexes, the metal centre coordinates to two ligands via the pyridine N
and the imine N atoms in a bidentate manner with the anion outside the coordination sphere.
Since the geometry around the metal centre is pseudo-tetrahedral in each case, the dihedral
angles between the chelate rings involving the metal center, defined by θ in this
communication, in each complex vary but ranges between 64.16(4) in complex 9 and
88.99(7)° in complex 5 (see supplementary info Table S3). The angles around the Ag(I)
centres are either acute and range between 71.83(10) and 73.14(5)°for the chelate ring
moieties or obtuse and range between 109.53(5) and 153.09(6)°. The obtuse Npy—Ag—Nim
angles of the complexes 4, 5, 8, 9 and 15 range between 113.44(9) and 135.72(10)° (see
supplementary info Table S4) showing their deviation from an ideal tetrahedral geometry.
Geometry index for four coordinates complex τ4 (equation 6) was used to determine the
complexes geometry
Equation (6)
and the τ4 values of the complexes fall within 0.52 – 0.67, which fits in Yang et al. [56]
seesaw geometry description. Complexes 4, 5, 8, 9, and 15 are unsymmetrical structurally
and, therefore, belong to the C1 point group.
The Ag-Nim bond distances in 5 and 15 are generally shorter than the Ag—Npy bond distance
but longer in 4, 8, and 9, but all fall within the expected range for Ag—N bond distances in
literature for related compounds [47, 57].
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(a) 4
(b) 5
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(c) 8
(d) 9
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(e) 15
Figure 1: The ORTEP diagrams showing the atom numbering scheme with the thermal ellipsoids
drawn at the 50% probability level for molecules of (a) complex 4, (b) 5, (c) 8, (d) 9 and (e) 15.
Hydrogen atoms have been omitted for clarity except for the water molecule in 4. Symmetry
operators: Complex 5, (i) = ½ - x, y, -z; Complex 8, (i) = 2 - x, y, 2 – z; Complex 9, (i) = -x, y, 3/2 - z
and Complex 15, (i) = 2 - x, y, ½ - z.
3.4 Biological Application Studies
3.4.1 In-vitro antimicrobial studies
L1 – L5 and 1 – 15, along with ciprofloxacin (used as a standard), were screened for
antimicrobial activity against two Gram-positive bacteria, MRSA and S. aureus, and four
Gram-negative bacteria, E. coli, S. typhi, K. pneumoniae, and P. aeruginosa. L1 – L5 were
screened mainly to establish the contribution of the ligands biologically. The minimum
inhibitory concentrations (MIC) given in Table 2 indicated that complexes 1 – 15 generally
had better antibacterial activity compared to L1 – L5, an indication that complexation to
Ag(I) enhanced the antimicrobial properties of L1 – L5. The complexes probably have a
better ability to penetrate the bacterial cell membranes due to enhanced lipophilicity of the
complexes. In such a process, the lipid layer of the microbe cells gets infiltrated, and the
respiration process gets destroyed, thereby stopping bacterial growth [58]. There is a
reduction in metal ion polarity on chelation to the ligand due to overlapping of the ligand
orbital and partial sharing of the positive charge of the metal ion with donor groups. Also, the
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π⸺electron delocalization over the whole complex improves complex lipophilicity, which
subsequently aids permeation through the lipid layer of the cell membrane [59]. Amongst the
ligands, L1 and L5 were the least active ligands with L1 only active against S. aureus. L5
had MIC values above 1000 µg/mL against all except against E. coli for which it was
inactive. L2 is selectively active against S. aureus and P. aeruginosa, L3 against E. coli, K.
pneumoniae and P. aeruginosa and L4 against the notorious MRSA with activity similar to
that of the standard ciprofloxacin.
The compounds were generally more active against E. coli, S. typhi, K. pneumoniae, and P.
aeruginosa. Gram-negative bacteria often have a thinner cell membrane making accessibility
easier. Complexes 5, 6 and 10 showed the highest antimicrobial activity against all bacteria
tested. The ligands of each of 5, 6 and 10, possess either the thiophene moiety or the hydroxyl
substituent. Significant antimicrobial activity of compounds containing either thiophene
moiety or hydroxyl substituent has been reported [27, 28, 60]. The high lipophilic nature of
the thiophene moiety [61] in 5 and 10 which in turn influence membrane penetration is
assumed to be responsible for their high activities. In 6, the o-position of the hydroxyl
substituent in phenyl ring could have limited its activity [62] as observed in 1 and 11 because
the electron donating group and o-position of a substituent would lead to low lipophilicity but
the presence of perchlorate anion in 6 must have enhanced the activity. Therefore, the counter
anions could possibly also be aiding in lipophilicity [63].
In terms of selectivity, 8 and 10, both with the 2-fluorophenyl and thiophen-2-ylmethyl
moieties were more active against S. aureus each with a value of 0.8 µg/mL MIC. 3, 5 and 7
which have MIC values between 0.025 – 0.4 µg/mL, were active against MRSA.
The metal salts on their own seem to be fairly active. On complexation, only complexes 8 and
10 seemed to show MICs lower (0.8 µg/mL) than those of AgNO3 and AgClO4 (3.125
µg/mL) against S. aureus. L3 is inactive while L5 is least active but when complexed to
AgClO4, the activity increases as observed for complexes 8 and 10 with F and S atoms in the
structures of the ligands. Only 7 had a lower MIC (0.1 µg/mL) than the salts and the standard
-
against MRSA. It is assumed that the ClO4 anion and the presence of S moiety could
probably have a contribution to the lower MIC value. Complexes 7, 9, and 10 are fairly
active, with their MIC values the same as their metal salt against E. coli. The enhanced
activity of 4 with Methyl substituent is almost 4 times and for complex 3, 1000-fold. The
activity of 4 was similar to that of the standard, while that of 3 was much better than that of
the standard, both with the F- substituent. The activities of 6 (with OH-), 13 (with F-), and 14
(with thiophene) against S. typhi are the same as the standard. Complex 2 (with S moiety and
-
NO3 as the anion), the MIC value was half that of the standard but similar to that of AgClO4
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against S. typhi. Complex 6 with the OH group and 9 with the CH3 group had lower MIC
values (0.05 µg/mL) than the standard, while 10 with the thiophene moiety had a MIC value
-
same as that of the standard against P. aeruginosa ( 6, 9, and 10, all have ClO4 as the anion).
-
In general, the substituents OH-, F-, thiazole and thiophene and the anion ClO4 seem to play a
role when incorporated in the complexes leading to enhanced antimicrobial activities when
compared to the ligands and or the salts against the different microbes. A comparison bar
chart representing each salt complexes is shown in Fig. 2a, 2b and 2c. Compounds with 1000
µg/mL MIC value and those without antimicrobial activity are omitted from the bar plot for
clarity.
Table 2: Minimum inhibitory concentration of silver complexes of L1-5 (µg/mL)
Compounds
Gram +ve Bacteria
Gram -ve Bacteria
SA
MRSA
EC
NA
ST
NA
KP
NA
PA
L1
1000
12.50
NA
NA
NA
L2
NA
1000
3.125
1000
NA
NA
NA
6.25
L3
1000
25.00
1000
12.50
12.50
0.40
NA
25.00
1000
1000
6.25
6.25
0.40
1000
0.80
0.20
1000
0.05
1000
12.50
1000
0.40
12.50
3.125
1000
0.80
25.00
50.00
1.60
3.125
1000
1000
12.50
1000
1000
6.25
L4
NA
NA
L5
1000
1000
12.50
25.00
12.50
12.50
12.50
1000
0.80
1000
1000
0.20
0.80
1000
1.60
0.40
NA
1
1000
1000
0.0125
0.20
2
3
4
50.00
0.025
12.50
0.10
5
12.50
6.25
12.50
0.05
6
7
0.80
1000
12.50
0.05
8
12.50
12.50
12.50
12.50
1000
1000
1000
1000
0.20
1000
0.80
12.50
0.80
6.25
1000
50.00
0.40
0.40
1000
1000
0.20
12.50
0.40
9
1000
0.80
10
0.80
0.80
11
25.00
12.50
25.00
1000
1000
3.125
3.125
NA
100
12.50
1000
12.50
1000
1000
1000
1.60
12
13
0.80
25.00
50.00
1000
12.50
0.80
14
15
AgNO3
AgClO4
AgCF3SO3
Ciprofloxacins[37]
6.25
1000
25.00
1000
0.20
1000
0.80
25.00
NA = No activity, s = standard, SA = Salmonella typhimurium, PA = Pseudomonas aeruginosa, EC =
Escherichia coli, KP = Klebsiella pneumoniae, SA = Staphylococcus aureus, MRSA = methicillin-resistant
Staphylococcus aureus
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(a)
(b)
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(c)
Figure 2: (a) Minimum inhibitory concentration (µg/mL) of L1-L5 silver(I) complexes with nitrate
as counter anion, (b) Minimum inhibitory concentration (µg/mL) of L1-L5 silver(I) complexes with
perchlorate as counter anion and (c) Minimum inhibitory concentration (µg/mL) of L1-L5 silver(I)
complexes with triflate as counter anion.
3.4.2 In vitro antioxidant studies
Compounds with antioxidant activity such as dithiocarbamate complexes [37] and
benzimidazole [64] have the abilities to slow down and stop damage to DNA, RNA, protein
substrates and thereby prevent the occurrence of diseases such as cardiovascular disease,
diabetes, rheumatism, atherosclerosis, cancer and aging [65]. Active antioxidant agents act as
single electron transfer agents or as hydrogen atom transfer agents, and can also bind to
transition metals [66]. The free radical scavenging ability of L1 ‒ L5 and 1 ‒ 15 were studied
using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP).
In antioxidant studies, DPPH with absorbance at 517 nm, are frequently used in determining
radical scavenging properties of the compounds. It is known to be a stable free radical with
an odd electron in its structure that acts as an electron acceptor or a radical hydrogen acceptor
to form a stable diamagnetic molecule. DPPH free radical scavenging method relies on its
decolorization in the presence of an antioxidant agent, which leads to a decrease in its
absorbance as the number of electrons accepted increases and can then be quantitatively
evaluated [67] in comparison to a standard. The ferric reducing antioxidant power (FRAP)
assay is often preferred because it is simple, fast, economical, and direct in antioxidant
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measurement. FRAP assay is based on the electron-donating ability of an antioxidant
compound. This involves the reduction of a colourless Fe3+ tripyridyltriazine complex
(Fe3+(TPTZ)) to an intense blue-coloured Fe2+ tripyridyltriazine complex (Fe2+(TPTZ)) by
the electron-donating antioxidants at low pH [68].
Table 3 summarizes the mean values from three independent experiments of in vitro
antioxidant activities of L1 ‒ L5 and Complexes 1 ‒ 15 at different concentrations. The
antioxidant activities of the compounds are expressed as 50 % inhibitory concentration (IC50
in mg/mL). Ascorbic acid with an IC50 value of 1.0 x 10-3 mg/mL and 2.68 mg/mL was used
as a standard for DPPH and FRAP studies, respectively. The standard ascorbic acid value for
DPPH is comparable to those of Borra et al. [69] and Saravanakumar et al. [70].
Table 3: Mean ± standard deviation values of In vitro antioxidant activities of L1 ‒ L5 and
Complexes 1 ‒ 15 from three independent experiments.
Antioxidant(IC50 mg/mL)
COMP.
DPPH
FRAP
8.32 x 10-1 ± 0.002
1.71 x 10-2 ± 0.005
3 x 10-2 ± 0.007
11.732 ± 0.28
9.2 x 10-2 ± 0.014
6.79 x 10-1 ± 0.003
1.33 x 10-1 ± 0.017
2.803 ± 0.029
NA
4.3 x 10-2 ± 0.002
6.1 x 10-2 ± 0.008
7637.256 ± 625.26
306.651 ± 16.59
NA
9.14 x 10-5 ± 0.0005
1.1939 ± 0.0094
1.52 x 10-1 ± 0.002
3.96 ± 0.023
2.64 x 10-2 ± 0.005
7.01x 10-16 ± 0.00
2.086 ± 0.067
8.774 ± 0.78
1,18 ± 0.0088
26.2762 ± 3.34
33.1538 ± 4.48
6.44 ± 0.050
4.14 ± 3.90
1
2
3
4
5
6
7
8
9
10
11
12
13
1.66 ± 0.037
14
0.654 ± 0.003
NA
1 x 10-4 ± 0.0004
15
63.86 ± 9.97
L1
3.81 ± 0.05
18.084 ± 2.07
4.3 x 10-1 ± 0.017
NA
L2
NA
L3
71.607 ± 2.93
82.16 ± 3.23
1 x 10-3 ± 0.0004
L4
NA
L5
As
2.67562 ± 0.11
NA = Not Active, As = Ascorbic Acid
Complexes 1 – 15 were assayed for free radical scavenging activity by DPPH and FRAP
method and are presented graphically as Fig. 3a and 3b, respectively. The IC50 values of L1 –
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L5 and complexes 1 – 15 are shown in Table 3. The complexes displayed higher antioxidant
values compared to their free ligands. Complexes 2, 3, 5, 7, 10 and 11 had good antioxidant
activity between 0.017 – 0.133 mg/mL (Table 3). Compounds with benzo[d]thiazole moiety
were the best scavengers with 2 having the highest amongst them. These are in agreement
with antioxidant activity of structurally related compounds [71, 72]. The S atom acts as a
donor of an electron which stabilizes the free radicals. Similarly, complexes 5 and 10 showed
high activities, probably due to the lone pair of electrons on their thiophene moieties. Similar
activities have been reported with thiophene containing compounds [73]. However,
complexes 12 and 13 had the lowest activities with 306.65 and 7637.26 mg/mL IC50 despite
12 having S on the thiazole moiety. Both complexes have triflate anion, which may be the
reason for the poor effects on the activity of the compounds. Complexes with p-tolyl
substituent generally had low antioxidant activity and even no activity at all, as observed for
4, 9, and 14. Significant antioxidant activities were observed in FRAP for complexes 1, 2, 3,
5, 6, 7, 9, 14 and 15. Most of the complexes showed higher activity than ascorbic acid, whose
IC50 value is 2.68 mg/mL. This implies that the complexes' ability to reduce Fe3+ to Fe2+ was
good. Complex 6, in particular, with an IC50 value of 7.01 x 10-16 mg/mL, had the lowest
ferric reducing power attributable to the hydroxyl group in the compound. This kind of
activity has been observed for compounds of phenolic nature [41].
Figure 3a: DPPH % free radical scavenging vs. concentration (mg/mL) of complexes 1–15 and
Ascorbic Acid. Values represent mean ±standard deviation (n = 3). a-gDifferent alphabets over the bars
29