Investigation of derivatized schiff base ligands of 1,2,4-triazole amine and their oxovanadium(IV) complexes: Synthesis, structure, DNA binding, alkaline phosphatase inhibition, biological screening, and insulin mimetic properties

Article abstract of DOI:10.1134/S1070363215090248

A series of novel azomethine precursors were derivatized by the condensation of 4H-1,2,4-triazole-4-amine with salicylaldehyde (HL ¹) (saltrz), 2,3-dihydroxybenzaldehyde (HL ²) (dhtrz), 2-hydroxy-1-naphthaldehyde (HL ³) (n

Full text of DOI:10.1134/S1070363215090248

ISSN 1070-3632, Russian Journal of General Chemistry, 2015, Vol. 85, No. 9, pp. 2183–2197. © Pleiades Publishing, Ltd., 2015.
Investigation of Derivatized Schiff Base Ligands
of 1,2,4-Triazole Amine
and Their Oxovanadium(IV) Complexes:
Synthesis, Structure, DNA Binding,
Alkaline Phosphatase Inhibition, Biological Screening,
and Insulin Mimetic Properties1
K. S. Munawara, S. Alia, M. N. Tahirb, N. Khalidc,
Q. Abbasd, I. Z. Qureshid, and S. Shahzadia
a Department of Chemistry, Quaid-i-Azam University, Islamabad, Pakistan
e-mail: drsa54@hotmail.com; sairashahzadi@hotmail.com
b Department of Physics, University of Sargodha, Sargodha, Pakistan
c Chemistry Division, Pakistan Institute of Nuclear Science & Technology, P.O. Nilore, Islamabad, Pakistan
d Department of Animal Sciences, Quaid-i-Azam University, Islamabad, Pakistan
Received July 17, 2015
Abstract―A series of novel azomethine precursors were derivatized by the condensation of 4H-1,2,4-triazole-
4-amine with salicylaldehyde (HL1) (saltrz), 2,3-dihydroxybenzaldehyde (HL2) (dhtrz), 2-hydroxy-1-
naphthaldehyde (HL3) (ndtrz) and 5-chloro-2-hydroxybenzaldehyde (HL4) (cltrz). Subsequently, oxovanadium(IV)
complexes of the type [VO(saltrz)2] (1), [VO(dhtrz)2] (2), [VO(ndtrz)2] (3), and [VO(cltrz)2] (4) were
synthesized by the reactions of vanadyl(V) isopropoxide [VO(OCHMe2)3] with the ligands HL1–HL4. The
synthesized compounds were characterized by the melting point method, elemental analysis, FT-IR and 1H and
13C NMR spectroscopies, the molar susceptibility and conductivity methods, and by the thermogravimetry.
Ligands HL3 and HL4 were also characterized by single crystal analysis. The binding modes of SS-DNA with
the compounds were confirmed by the hypochromism and red/blue shift of the UV-Vis spectra. The negative
DG values of the compound-DNA adducts show that the binding is a spontaneous process. The complexes
were found to be persuasive inhibitors of enzyme alkaline phosphatase (ALP). The in vitro screening of the
antimicrobial activities of the synthesized compounds revealed significantly enhanced activities of
oxovanadium(IV) complexes as compared to their respective ligands. The promising results were obtained in
studying the insulin mimetic of the synthesized compounds.
Keywords: 1,2,4-triazole amine derivatives, oxovanadium(IV) complexes, DNA binding, alkaline phosphatase
inhibition, antimicrobial activities, hemolysis, insulin mimetic properties
DOI: 10.1134/S1070363215090248
INTRODUCTION
The vanadium compounds exhibit marked hypo-
glycemic activity and are used as antidiabetic drugs
[5–7]. They are also known as prospective inhibitors of
various enzymes, such as alkaline phosphatase. In-
organic vanadium compounds, like vanadyl sulfate and
vanadate, are poorly diffused through the gastric and
intestinal tracts and are more toxic. Therefore, vana-
dium(IV) compounds are preferred for in vivo
administration [8, 9]. The nature of organic ligands in
vanadyl complexes is related to their pharmaco-
In recent years, coordination chemistry of vana-
dium has aroused interest of researches, since this
compound is involved into several biological pro-
cesses, is present in organisms such as Ascidians and
Amanita mushrooms, and is a constituent of the
cofactors in vanadate-dependent haloperoxidases and
vanadium nitrogenase [1–4].
1 The text was submitted by the authors in English.
2183

2184
MUNAWAR et al.
kinetics. The catalytic, biological, and medicinal pro-
perties of the vanadium complexes have stimulated the
scientists to synthesize new more active model vana-
dium compounds containing O and N donor ligands.
(gallenkamp). Magnetic moment was determined on a
Sherwood magnetic susceptibility balance at ambient
temperature (25±2°C) with Hg[Co(SCN)4] as calibrant.
Thermogravimetric analysis was performed with
Universal V4.3A TA Instruments. The electrical
conductance of 10−3 molar solutions in DMSO : H2O
(7 : 3) was recorded on an Elico CM-180 Conductivity
Bridg. The X-ray diffraction data wasere collected on a
Bruker SMART APEX CCD diffractometer, equipped
with a 4 K CCD detector set 60.0 mm from the crystal.
The crystals were cooled to 296±1 K using the Bruker
KRYOFLEX low temperature device. In measuring
the intensity, a MoKα radiation from a sealed ceramic
diffraction tube (SIEMENS) was used. The structure
was solved by the Patterson methods (DIRDIF or SIR
2004 program). For the refinement of crystal structure
we used a SHELXL-97 program, a modified version of
the PLUTO program (preparation of illustrations) and
PLATON package [12].
Schiff-base ligands prepared by the condensation of
aromatic aldehydes and polyamines are highly promis-
ing compounds, as they exhibit higher efficiency for
the above mentioned processes. The modification of
amino-heterocyclic compounds, such as triazoles, into
Schiff bases might result in the formation of
polydentate ligands with interesting properties. The
derivatives of 1,2,4-triazole are efficient ligands for
coordination with vanadium ions [10, 11].
Based on the properties of existing triazoles, a new
series of triazole derivatives and their oxovanadium
complexes were synthesized and their potency as DNA
binders, alkaline phosphatase inhibitors, antimicrobial
agents, and especially, insulin mimetics was evaluated.
Study of Salmon sperm-DNA binding by the
electronic absorption titration. The interaction of
ligands and their vanadium complexes with SS-DNA
was performed in tris HCl buffer [5 mM of tris
(hydroxylmethyl)aminomethane and 50 mM of NaCl
at pH 7.2]. A solution of SS-DNA in a buffer gave an
absorbance ratio of 1.9 : 1 at 260 and 280 nm,
indicating that the SS-DNA was free from protein [13,
14]. The concentration of SS-DNA was measured by
using an extinction coefficient of 6600 M−1 cm−1 at
260 nm and was found to be 2.0 × 10−4 M. The
compound was dissolved in DMSO : H2O (7 : 3) at a
concentration of 2 mM. In measurements of the UV-
Vis absorption spectra, the SS-DNA concentration was
varied, whereas the concentrations of the compound
and the reference solution were constant. The
compound-SS-DNA solutions were incubated for
30 min at room temperature (25±1°C) before
measuring the absorption.
EXPERIMENTAL
Chemicals and instrumentation. Solvents and
reagents were obtained from commercial sources and
used without further purification unless otherwise
stated. Vanadyl(V) isopropoxide, 4H-1,2,4-triazole-4-
amine, salicylaldehyde, 2,3-dihydroxybenzaldehyde,
2-hydroxy-1-naphthaldehyde,
5-chloro-2-hydroxy-
benzaldehyde, p-nitrophenyl phosphate hexahydrate
(p-NPP), diethanolamine and magnesium chloride
were purchased from Sigma Aldrich. Sodium salt of
Salmon sperm DNA (SS-DNA) was obtained from
Acros Organics and used as received. Human serum
was used as a source of alkaline phosphatase (ALP).
Elemental analysis of the synthesized compounds was
carried out on an Elemental Vario EL elemental
analyzer. FT-IR spectra in the range of 4000–250 cm−1
were obtained on a Thermo Nicolet-6700 FT-IR
spectrophotometer. Multinuclear (1H and 13C) NMR
spectra were recorded on a Bruker-400 MHz FT-NMR
Alkaline phosphatase inhibition .The bioassay of
the substrate was prepared by following the reported
procedure [15], by mixing four parts of reagent A
(diethanolamine pH 9.8, 2 M and magnesium chloride
0.5 mM) and one part of reagent B (p-NPP 50 mM).
The mixture was incubated for 5 min at 25± 1°C. Into
a cuvette, 2 mL of the substrate was poured and 40 µL
of human serum having the activity of 165 IU/L was
added into it. After 1 min of incubation, the
absorbance was measured at 405 nm to confirm
enzyme activity. Different aliquots of a 25 mM stock
solution of oxovanadium complexes were added to the
Spectrometer, using DMSO-d6 as
a
solvent
[δ H (DMSO) = 2.5 ppm and δ 13C (DMSO) =
1
39 ppm]. Chemical shifts are given in ppm and
coupling constants (J), in Hz. The multiplicity of H
1
NMR signals (s = singlet, d = doublet, d.d = doublets
of doublet, t = triplet, d.d.d. = doublet of doublet of
doublets, and m = multiplet) is indicated with chemical
shifts. The absorption spectra were recorded on a
Shimadzu 1800 UV-Vis Spectrophotometer. The
melting points were determined in capillary tubes
using an electrothermal melting point apparatus
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INVESTIGATION OF DERIVATIZED SCHIFF BASE LIGANDS
2185
above substrate after regular intervals, incubated for
3 min, and the absorbance was then recorded from 1–
5 min after each 1 min The average value of these
readings was used to calculate the age inhibition (%).
polystyrene screw-captube, centrifuged for 5 min at
850g, the resulting supernatant was poured off and the
viscous pellet was washed thriply with 5 mL of chilled
(4°C) sterile isotonic phosphate-buffered saline (PBS)
having pH 7.4. The washed cells were suspended in
20 mL of chilled PBS and counted on a hemocyto-
meter. The blood cell suspension (maintained on wet
ice) was diluted with sterile PBS to 7.068 × 108 cells mL−1
for each assay. Aliquots of 20 μL of each sample
solutions were placed aseptically into 2.0 mL micro-
fuge tubes. For each assay, 0.1% Triton X-100 was
used as positive control (100% lysis) and PBS as
negative control (0% lytic). Aliquots of 180 μL diluted
blood cell suspension was aseptically placed into each
2 mL tube and mixed gently three times. The tubes
were incubated for 35 min at 37°C with agitation (at
80 rpm) and then placed on ice for 5 min., followed by
centrifugation for 5 min at 1310 g. Aliquots of 100 μL
of supernatant were collected, placed into a sterile
microfuge tube (volume 1.5 mL) and then diluted with
900 μL of chilled and sterile PBS. All tubes were
maintained on wet ice after dilution. Absorbance at
576 nm was then measured on a Microquant. The
experiment was done in triplicate.
Antimicrobial activities. The fungicidal and
bactericidal activities of the synthesized triazole pre-
cursors and their respective oxovanadium complexes
against various bacteria and fungi were determined by
the following methods:
a. Growth medium, culture, and inoculum prepara-
tion. The bacterial strains (Escherichia coli, Bacillus
subtilis, Staphylococcus aureus and Pasturella multo-
cida) were cultured overnight at 37°C in nutrient agar.
The pure bacterial cultures obtained were stored in the
medium in slants and petri plates. For inoculums
preparation, 13 g of nutrient broth was suspended in
1 L of distilled water, homogenously mixed, and
autoclaved for 15 min at 121°C. Then, 10 µL of pure
culture of a bacterial strain was added to 100 mL of
freshly prepared nutrient broth medium and shacked
(at 140 rpm) for 24 h at 37°C. The prepared inocula
were stored at 4°C. The inocula with 1 × 108 spores/mL
were used for activity measurement [16].
The fungal strains (Alternaria alternata, Ganoderma
lucidum, Aspergillus niger, and Penicillium notatum)
were cultured overnight at 28°C using potato dextrose
agar. The pure cultures were maintained in sabouraud
dextrose agar (SDA) medium in slants and petri plates,
which were presterilized in hot air oven at 180°C for
3 h. These cultured slants were incubated at 28°C for
3–4 days for the multiplication of fungal strains.
Antidiabetic study. a. Animals and maintenance.
Healthy adult male BALB/c mice (n = 50, average
body weight = 35±5 g) were obtained from the
“National Institute of Health,” Islamabad. Five animals
were housed per cage and were given free access to
standard rodent diet and water ad libitum. Photoperiod
was maintained at 12 : 12 hour light/dark cycle.
b. Induction of diabetes. In order to induce diabetes,
a single injection of Alloxan monohydrate (Sigma
Aldrich, USA) was given intraperitoneally (i.p.) at the
dose of 150 mg/kg body weight.The mice with fasting
plasma glucose levels > 200 mg/dL were considered
diabetic and were selected for further studies.
b. Measurement of antimicrobial assay by disc
diffusion method. Antimicrobial activities were
measured by the disc diffusion method [17]. 2.8 g of
nutrient agar (for antibacterial activities) or 3.9 g of
potato dextrose agar (for antifungal activities) was
suspended in 100 mL of distilled water and sterilized
by autoclaving at 121°C for 15 min Then, the
suspension was thoroughly mixed with 100 μL of
inoculum and poured into sterilized petri plates.
Finally, a 9-mm filter paper discs soaked with 100 μL
of a specific solution were laid flat on the growth
medium. The petri plates were then incubated at 37°C
for 24 h or at 28°C for 48 h for the bacteria or fungi
growth, respectively. Clear zones of the inhibition
formed around the discs were measured in millimeters
using a zone reader [18, 19].
c. Experimental design. Animals were divided into
ten groups, five animals each. They were treated with
acute doses (35.9 mg/kg body weight) of compounds
HL1–HL4 and complexes 1–4. Positive control groups
were treated with
a
known antidiabetic drug
Glibenclamide (10 mg/kg body weight in distilled
H2O) [20]. The treatment groups were as follows:
Control groups. Group 1: Negative control
(diabetic, only Alloxan pretreated). Group 2: Positive
control (diabetic, Alloxan pretreated followed by
Glibenclamide treatment).
c. Hemolytic activity. Heparinized fresh human
blood (3 mm) was mixed with 15 mL of sterile in a
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MUNAWAR et al.
Experimental groups. Group 3: Compound HL1
d.d (H8, J[1H–1H] = 8.4, 1.6 Hz); 13C NMR spectrum
(DMSO-d6 101 MHz, δ, ppm): 139.0 (C1, C1'), 156.0
(C2), 118.0 (C3), 158.3 (C4), 116.7 (C5), 134.2 (C6),
119.6 (C7), 127.6 (C8), Λm (DMSO) 2.5 S cm2 mol–1.
treated (diabetic, Alloxan pretreated). Group 4:
Compound 1 treated (diabetic, Alloxan pretreated).
Group 5: Compound HL2 treated (diabetic, Alloxan
pretreated). Group 6: Compound 2 treated (diabetic,
Alloxan pretreated). Group 7: Compound HL3 treated
(diabetic, Alloxan pretreated). Group 8: Compound 3
treated (diabetic, Alloxan pretreated). Group 9: Com-
pound HL4 treated (diabetic, Alloxan pretreated).
Group 10: Compound 4 treated (diabetic, Alloxan
pretreated).
b. Synthesis of (E)-3-[(4H-1,2,4-triazole-4-ylimino)
methyl]benzene-1,2-diol (HL2). HL2 was synthesized
by the procedure used for HL1. However, in this case,
2,3-dihydroxybenzaldehyde was used instead of
salicylaldehyde. White precipitates were immediately
obtained on cooling.Yield 84%, mp 270°C, IR
spectrum, ν, cm–1 3453 (–OH), 1610 (HC=N), 1312
1
d. Glucose determination. Blood was collected
through a caudal venepuncture using a 26 gauge
butterfly cannula. Blood samples were drawn at 0 h
(Pre Alloxan), 1 h (Post Alloxan), and at 1–7 h after
dosing. Plasma glucose was determined using a
dextrostix test strip and an Accu-Check blood glucose
meter (Active, Roche Pharma).
(C–O), HNMR spectrum (DMSO-d6 400 MHz, δ,
ppm): 10.17 s (–OH), 9.61 s (–OH'), 9.12 s (H1, H1'),
9.10 s (HC=N), 6.93 d.d (H6, J[1H–1H] = 8.0, 1.6 Hz),
6.73 t (H7, J[1H–1H] = 8.0 Hz), 7.19 d.d (1H, H8, J[1H–
1H] = 8.0, 1.6 Hz); 13C NMR spectrum (DMSO-d6
101 MHz, δ, ppm): 138.9 (C1, C1'), 155.5 (C2), 117.8
(C3), 147.2 (C4), 146.0 (C5), 118.6 (C6), 118.7 (C7),
119.4 (C8), Λm (DMSO) 3.8 S cm2 mol–1.
e. Determination of total serum cholesterol and
triglycerides. Total Serum cholesterol and triglycerides
were determined through commercially available kits
from Globe Diagnostics (Italy) using Microlab 300
(Germany).
c. Synthesis of (E)-1-[(4H-1,2,4-triazole-4-ylimino)
methyl]naphthalen-2-ol (HL3). The procedure of HL3
preparation was the same as for HL1. The only
difference was that 2-hydroxy-1-naphthaldehyde was
used instead of salicylaldehyde. On cooling and eva-
poration, colorless acicular crystals were collected from
the basic liquor after 2 days. Yield 88%, mp 250°C; IR
spectrum, ν, cm–1 3442 (–OH), 1618 (HC=N), 1299
Statistical analysis. The data were analyzed
through one-way analysis of variance (ANOVA) using
the Statistical Package for Social Sciences (SPSS
version, 16.0 Inc. Chicago, Illinois, USA). Post-hoc
Tukey-Kramer test and normality test failed; ANOVA
on Ranks test was applied. p < 0.05 was considered
statistically significant difference. Data are presented
as line or bar diagrams using the GraphPad Prism 5
(Version 5.01 GraphPad Software Inc., USA).
1
(C–O), H NMR spectrum (DMSO-d6 400 MHz, δ,
ppm): 11.36 s (–OH), 9.30 s (H1, H1'), 9.66 s (HC=N),
8.06 d (H5, J[1H–1H] = 8.8 Hz), 7.29 d (H6, J[1H–1H] =
8.8 Hz), 8.86 d (H8, J[1H–1H] = 8.8 Hz), 7.45 d.d.d
(H9, J[1H–1H] = 8.4, 6.4, 2.4 Hz), 7.63 d.d.d (H10,
J[1H–1H] = 8.4, 6.4, 2.4 Hz), 7.93 d (H11, J[1H–1H] =
8.8 Hz); 13C NMR spectrum (DMSO-d6 101 MHz, δ,
ppm): 138.9 (C1, C1'), 156.5 (C2), 108.4 (C3), 159.4
(C4), 118.4 (C5), 131.6 (C6), 128.9 (C7), 128.3 (C8),
123.4 (C9), 128.0 (C10), 123.9 (C11), 135.4 (C12), Λm
(DMSO) 4.2 S cm2 mol–1.
Syntheses
of
triazole-based
azomethine
precursors. a. Synthesis of (E)-2-[(4H-1,2,4-triazole-
4-ylimino)methyl]phenol (HL1). A dried ethanolic
solution of salicylaldehyde (1.25 mL, 1.18 mmol) was
added to an ethanolic solution of 4H-1,2,4-triazole-4-
amine (1 g, 1.18 mmol). The slightly yellow mixture
obtained was stirred for 15 min and refluxed for 45
min The resulting pale-yellow solution was cooled to
room temperature. The white crystalline solid obtained
was filtered off, washed first with ethanol and then
with diethyl ether, and, finally, was dried under
vacuum. Yield 85%, mp 206°C, IR spectrum, ν, cm–1:
d. Synthesis of (E)-2-[(4H-1,2,4-triazole-4-ylimino)-
methyl]-4-chlorophenol (HL4). The HL4 was prepared
by adopting the same procedure as described for HL1,
by replacing salicylaldehyde with 5-chloro-2-hydroxy-
benzaldehyde. On cooling and subsequent slow
evaporation, colorless acicular crystals were collected
from the mother liquor after 24 h. Yield 88%, mp 230°C,
IR spectrum, ν, cm–1 3455 (–OH), 1614 (HC=N), 1302
1
3435 (–OH), 1602 (HC=N), 1299 (C–O), H NMR
spectrum (DMSO-d6, 400 MHz, δ, ppm): 10.54 s (–
OH), 9.38 s (H1, H1'), 9.19 s (HC=N), 7.02 d.d (H5, J
[1H–1H] = 8.4, 1.6 Hz), 7.44 d.d.d (H6, J[1H–1H] = 8.4,
1.6 Hz), 6.96 d.d.d (H7, J[1H–1H] = 8.4, 1.6 Hz), 7.02
1
(C–O), H NMR spectrum (DMSO-d6 400 MHz, δ,
ppm): 11.43 s (–OH), 8.31 s (H1, H1'), 10.20 s (HC=N),
6.87 d (H5, J[1H–1H] = 8.4 Hz), 7.22 d.d (H6, J[1H–1H] =
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INVESTIGATION OF DERIVATIZED SCHIFF BASE LIGANDS
2187
8.8, 2.8 Hz), 8.10 d (H8, J[1H–1H] = 2.4 Hz); 13C NMR
spectrum (DMSO-d6 101 MHz, δ, ppm): 137.3 (C1,
C1'), 155.1 (C2), 122.3 (C3), 177.8 (C4), 117.6 (C5),
130.3 (C6), 123.4 (C7), 125.4 (C8), Λm (DMSO)
2.3 S cm2 mol–1.
C18H14N8O5V. Found, %: C 45.53; H 3.10; N 23.82; O
16.85. IR spectrum, ν, cm–1 1600 (HC=N), 1313 (C–O),
989 (V=O), 570 (V–O), 461 (V–N). μeff, BM: 1.91,
Λm (DMSO) 12.9 S cm2 mol–1.
Complex 3. Yield 86%, mp > 300°C; MW 541.4,
Calculated, %: C 57.68; H 3.35; N 20.70; O 8.87.
C26H18N8O3V. Found, %: C 57.98; H 3.21; N 20.88; O
8.95. IR spectrum, ν, cm–1 1615 (HC=N), 1319 (C–O),
959 (V=O), 562 (V–O), 458 (V–N). μeff, BM: 1.84,
Λm (DMSO) 3.4 S cm2 mol–1.
e. Synthesis of [VO(saltrz)2] (1), [VO(dhtrz)2] (2),
[VO(ndtrz)2] (3), and [VO(cltrz)2] (4). Oxovanadium
complexes 1–4 were synthesized by the reaction of
vanadyl(V) isopropoxide, [VO(OCHMe2)3], with
HL1–HL4 at the ratio 1 : 2 using acetonitrile (MeCN)
as a solvent. The green colored complexes 1–4 were
precipitated immediately, washed with acetonitrile,
and then dried in air.
Complex 4. Yield 93%, mp > 300°C; MW 510.2.
Calculated, %: C 42.38; H 2.37; N 21.96; O 9.41.
C18H12Cl2N8O3V. Found, %: C 42.45; H 2.35; N 21.85;
O 9.35. IR spectrum, ν, cm–1 1604 (HC=N), 1293 (C–O),
960 (V=O), 553 (V–O), 463 (V–N). μeff, BM: 1.85,
Λm (DMSO) 2.4 S cm2 mol–1.
Complex 1. Yield 85%, mp > 300°C; MW 441.3.
Calculated, %: C 48.99; H 3.20; N 25.39; O 10.88;
C18H14N8O3V. Found, %: C 49.23; H 3.15; N 25.24; O
10.75. IR spectrum, ν, cm–1 1594 (HC=N), 1306 (C–O),
973 (V=O), 554 (V–O), 455 (V–N). μeff, BM: 2.03,
Λm (DMSO) 10.1 S cm2 mol–1.
RESULTS AND DISCUSSION
The general method for the synthesis of triazole
Complex 2. Yield 79%, mp > 300°C; MW 473.3.
Calculated, %: C 45.68; H 2.98; N 23.68; O 16.90.
precursors HL1–HL4 is shown in the scheme (1).
H
N
N
N
EtOH
+
(1)
H2O,
+
N NH2
N N
HC R
O C
N
15 min stirring
45 min reflux
R
Cl
(HL4).
(HL1),
(HL2),
(HL3),
R =
HO
HO
HO
HO
OH'
The oxovanadium complexes 1–4 with the
synthesized triazole ligands HL1–HL4 were prepared
in acetonitrile by the reaction (2).
All synthesized compounds are insoluble in water,
whereas the ligands are soluble in organic solvents,
like ethanol, methanol, acetonitrile, and chloroform,
where DMSO are only soluble among metal com-
plexes. The ligands and their complexes are stable at
room temperature in the solid state. The molar
conductance of the complexes in DMSO : H2O (7 : 3)
lies between 2.4 to 12.9 S cm2mol–1, indicating their
nonelectrolytic nature [21]. The proposed formulae of
the compounds were confirmed by the FT-IR, multi-
nuclear (1H and 13C) NMR, elemental, thermo-
gravimetric, and single crystal analyses.
O
N
O
O
N
MeCN
stirring
1
4
VO(OCHMe ) +
2HL −HL
L
2 3
L
V
(2)
The spectroscopic and analytical data of the
synthesized triazole ligands and their respective oxo-
vanadium complexes are presented in experimental
section. The structural formulae of ligands along with
the labeling scheme of atoms are shown in Fig. 1.
FT-IR spectroscopy. Triazole Schiff bases showed
noticeable peaks in the range from 1602 to 1618 cm–1
corresponding to ν(HC=N), which were shifted to
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MUNAWAR et al.
confirm the formation of HL1–HL4 ligands. In HL2 the
N
N
N
N
N
N
1
non-hydrogen bonded phenolic proton (–OH') gave an
upfield signal at 9.25 ppm. Aromatic protons gave
signals in the range of 6.73–8.86 ppm. The 13C NMR
spectra show characteristic peaks of azomethine
carbon (HC=N) in the range 155.1–156.5 ppm, which
also supports the formation of HL1–HL4 ligands. The
signals of carbons in triazole ring appeared in the
range 137.3–139.0 ppm, whereas those of the aromatic
carbons, in the range 159.4–108.4 ppm. Phenolic
carbon in HL4 showed a downfield signal at 177.8 ppm
(NMR numbering scheme is given in Fig. 1).
1
1'
1'
H
C
2
8
H
C
2
N
8
5
3
N
7
3
7
4
6
4
6
OH'
OH
5
OH
HL1
HL2
N
N
N
N
1
10
1
1'
11
12
N
1'
9
8
N
H
H
C
2
8
5
Cl
C
N
3
N
3
2
7
6
7
Crystal structures of HL3 and HL4. The data
collection and the crystal structure refinement data for
ligands HL3 and HL4 are listed in Table 1, together
with the standard discrepancy indices, R1 and wR2. The
selected interatomic parameters for the ligands HL3
and HL4 are listed in Tables 2 and 3, respectively. The
ORTEP drawing of the ligands HL3 and HL4 is
presented in Figs. 2,3, respectively. The observed bond
lengths and bond angles in the ligand precursors are
similar to those reported in the literature for other
relevant compounds [26]. The XRD data show that
HL3 and HL4 are monomers and azomethine nitrogen
and phenolic oxygen show hydrogen bonding to form a
six-membered ring [27]. For hydrogen bonding, the
unsaturated HC=N bond permits π-electronic coupling
between the acidic hydrogen and basic nitrogen centers
of the molecules. Moreover, this chelate ring is planar
in the both Schiff base ligands, which is in accordance
with the Ito’s et al. data [28]. Ligands HL3 and HL4
showed intramolecular hydrogen bonding as
O__H1···N1 that stabilizes the molecular conformation.
Ligand HL3 is present in the form of a zwitter ion and
exhibits intermolecular hydrogen bonding between
triazole hydrogen and nitrogen as C12__H12···N3 and
4
4
6
OH
OH
5
HL3
HL4
Fig. 1. Scheme of NMR numbering for ligands HL1–HL4.
lower wave numbers, i.e., in the range 1594–1615 cm–1
on complexation. The detected shifts of azomethine
peaks ν(HC=N) on going from the free triazole Schiff
base ligands to the complexes is indicative of bonding
of azomethine nitrogen with vanadium atom. In
addition, the spectra of the HL1–HL4 ligands have
broad weak-intensity bands of the ν–OH) vibrations in
the range 3435–3455 cm–1, which disappear after
chelation, indicating on coordination through the
deprotonated form.
This result was further supported by the
appearance of two new less intense bands in the range
553–570 and 455–463 cm–1, which are due to ν(V–O)
and ν(V–N) vibrations, respectively. Furthermore, the
ν(V=O) bands of the medium intensity were also
appeared in the region 959-989 cm–1. These results are
in agreement with the literature data on these
compounds [22, 23]. Based on the perturbation of the
azomethine and phenolic group peaks in the FT-IR
spectra of the complexes 1–4, we conclude that the
ligands HL1–HL4 behave as bidentate.
C
13__H13···N4. Ligand HL4 shows intermolecular hydro-
gen bonds of the kind C3__H3···O1 and C8__H8···N4.
Thermogravimetric study. Thermogravimetric
analysis of the complexes 1–4 was carried at
temperatures varied from the ambient temperature to
1000°C in the nitrogen atmosphere. The fragments
decomposed in the different temperature ranges are
listed in Table 4. The table data are in agreement with
the results of the elemental analyses and also confirm
the composition of the complexes 1–4. In complexes 1,
3 and 4, the vanadium metal, along with some content
of organic constituents, is present in the form of
residue.
NMR spectroscopy. The H and 13C NMR spectra
1
of the triazole ligands were recorded in deuterated
dimethyl sulphoxide (DMSO). The chemical shifts of
the protons and carbons agree with the reported values
[24, 25] (see experimental section).
1
The H NMR spectra have characteristic peaks of
azomethine protons (HC=N) in the range 9.12–
10.20 ppm and peaks of hydrogen bonded phenolic
protons (–OH) in the range 10.54–11.44 ppm, which
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 9 2015

INVESTIGATION OF DERIVATIZED SCHIFF BASE LIGANDS
Table 1. Crystal and structure refinement data for HL3 and HL4
2189
Ligand
Parameter
Chemical formula
HL3
C13H10N4O
238.25
HL4
C9H7ClN4O
222.64
Molecular weight
T, K
296(2)
296(2)
Wavelength, Å
Crystal system
Space group
0.71073
0.71073
Monoclinic
P21
Monoclinic
PC
a, Å
8.7362(3)
5.8614(2)
10.9722(4)
90
3.8468(4)
14.2942(16)
9.0339(10)
90
b, Å
c, Å
α, deg
β, deg
91.874(2)
90
101.898(4)
90
γ, deg
V, Å3
561.55(3)
2
486.07(9)
2
Z
Absorption coefficient, mm–1
0.095
0.369
F(000)
248
228
Crystal size, mm
θ range for data collection, deg
Reflections collected
Independent reflections
Goodness-of-fit on F2
Final R indices [I > 2σ(I)]
R indices (all data)
Data/restrains/ parameters
Density (calculated), mg/m3
CCDC no.
0.25×0.18×0.16
3.714 to 56.544
5600
0.25×0.14×0.12
2.85 to 54.156
4027
2555 (Rint = 0.0188)
1.000
1841 (Rint = 0.0635)
0.0948
R1 = 0.0330, wR2 = 0.0842
R1 = 0.0400, wR2 = 0.0894
2555/1/170
1.409
R1 = 0.0481, wR2 = 0.0666
R1 = 0.1182, wR2 = 0.0829
1841/2/137
1.521
998780
998781
Magnetic moment study. The effective magnetic
moment per metal atom was calculated by Eq. (3).
The magnetic moments at 298 K for the complexes 1–
4 were found to be 2.03, 1.91, 1.84, and 1.85 BM,
respectively, which confirms their paramagnetic
character and the distorted trigonal bipyramidal geo-
metry around the [VO(IV)] ion [29]. These values are
characteristic of the monomeric 3d1 oxovanadium(IV)
complexes with S = 1/2, where metal ion is not in-
volved in the magnetic exchange with the neighboring
metal ion or atoms [30].
⎯
(3)
μ
eff = 797.5√χmT ,
where χm is the molar magnetic susceptibility of the
complex. Diamagnetic correction was made by the use
of Pascal’s constants for constituent atoms and groups
of the complex and as calibrant was used Hg[Co(SCN)4].
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 9 2015

2190
MUNAWAR et al.
Table 3. Selected bond lengths (Å), bond angles (deg), and
hydrogen bonding in HL4
Table 2. Selected bond lengths (Å), bond angles (deg), and
hydrogen bonding in HL3
Bond length
O1–C1
Bond angle
C1–O1–H1
Bond length
Cl1–C4
Bond angle
N2–N1–C7
1.3533
0.9700
1.3902
1.2744
1.3580
1.3523
1.2959
1.3906
1.2988
0.7000
1.3948
109.00
117.45
132.53
104.68
122.74
107.27
106.56
118.00
122.00
120.95
121.38
1.7475
1.3647
0.8200
1.2888
1.4019
1.3536
1.3623
1.2980
1.3883
1.2923
116.95
132.85
122.45
104.52
107.00
107.10
122.49
117.01
118.92
110.72
O1–H1
N1–N2
N1–C11
N2–C12
N2–C13
N3–C12
N3–N4
N4–C13
N1–H1A
C1–C10
N2–N1–C11
N1–N2–C12
C12–N2–C13
N1–N2–C13
N4–N3–C12
N3–N4–C13
C11–N1–H1A
N2–N1–H1A
C2–C1–C10
N1–C11– C10
O1–C1
O1–H1
N1–C7
N1–N2
N2–C8
N2–C9
N3–C8
N3–N4
N4–C9
N1–N2–C8
N1–N2–C9
C8–N2–C9
N4–N3–C8
N3–N4–C9
O1–C1–C6
O1–C1–C2
Cl1–C4–C5
N2–C8–N3
Hydrogen bonding
d[D–H] d[D···A]
Hydrogen bonding
d[D–H] d[H···A] d[D···A] [DHA] angle
D–H···A
O1–H1···N1
C3–H3···O1
C8–H8···N4
[DHA] angle
D–H···A
O1–H1···N1
0.9700
1.7600 2.6037
143.00
149.00
138.00
0.8200 2.6285
0.9300 3.4162
0.9300 3.2589
145.00
164.00
170.00
C12–H12···N3 0.9300 2.4300 3.2616
C13–H13···N4 0.9300 2.5000 3.2536
Electronic absorption titration. The interaction
of triazole ligands and their oxovanadium complexes
with SS-DNA was studied by the UV absorption
spectroscopy. Generally, DNA binding occurs in three
different modes: (1) Electrostatic interaction with the
negatively charged nucleic sugar-phosphate structure,
which is along the external DNA double helix and do
not possess selectivity (2) binding interaction with
grooves of the DNA double helix, and (3) intercalation
between the stacked base pairs of native DNA [31].
O1
C2
C3
C1
O1
C2
C10
C11
C9
C13
C4
C1
N4
N3
C3
N1
N2
N2
N4
N1
C12
C5
C6
C9
C6
C7
C5
C4
C8
C8
N3
C7
Cl1
Fig. 2. The ORTEP drawing of the ligand HL3 with the
Fig. 3. The ORTEP drawing of the ligand HL4 with the
displacement ellipsoids at the 50% probability level.
displacement ellipsoids at the 50% probability level.
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INVESTIGATION OF DERIVATIZED SCHIFF BASE LIGANDS
Table 4. Thermogravimetric data on the oxovanadium complexes 1–4
2191
Weight loss, %
Complex no. Temperature, °C
Evolved components
experimental
6.17
calculated
6.35
1
53–238
238–362
362–431
431–948
>948
N2
C3H3N2
15.77
22.36
24.72
30.92
7.22
15.20
21.54
26.09
30.82
7.18
C3H3N4
C9H7
Residue (C3HO3V)
2OH
2
3
44–230
230–261
261–951
951
2C2H2N4
C14H8O3V
Residue
N2CH
35.08
56.53
1.01
34.67
58.13
0.02
50–236
236–285
285–458
458–717
717–951
>951
7.44
7.58
C3H3N3
14.76
22.47
11.85
17.75
25.68
10.51
18.13
29.61
19.61
22.01
14.97
22.19
11.84
18.30
25.12
10.59
18.63
29.71
19.31
21.76
C6H6N3
C5H4
C8H3
Residue (C3HO3V)
C2H2N2
4
37–217
217–284
284–502
502–951
>951
C3H3N4
C7H4ClN2
C5H3Cl
Residue (CO3V)
Hyperchromic and hypochromic effects are the spec-
tral features of DNA concerning its double helix struc-
ture. The hypochromism results from the contraction
of DNA in the helix axis, as well as from the change in
conformation on DNA, while the hyperchromism
results from the damage of the DNA double helix
structure [32]. The absorption spectra of the metal
complex bound to DNA through the intercalation
exhibit the hypochromism and show a red/blue shift
due to the strong π–π* stacking interaction between the
aromatic chromophore of the metal complexes and the
base pairs of DNA.
wavelength range 600–250 nm at a constant con-
centration of the complexes. Two representative
absorption spectra of HL3 and complex 3 are shown in
Fig. 4. After 24 h, the spectra were the same, i.e., the
drug-DNA adduct is stable. The data obtained were
substituted into the following Benesi–Hildebrand
relationship [33] to determine the intrinsic binding
constant K from Eq. (4):
εG
A0
εG
1
,
−
+
(4)
εH−G − εG
εH−G − εG
A − A0
K[DNA]
The UV-visible titrations of the ligands HL1–HL4
and oxovanadium complexes 1, 3, and 4, in the
absence (1) and in the presence (2–10) of SS-DNA
have been studied in tris buffer solution in the
where K is the binding constant, A0 and A is the
absorbance of drug and drug-SS-DNA adduct, respec-
tively, and εG, εH–G is the absorption coefficient of drug
and drug-SS-DNA adduct, respectively.
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2192
MUNAWAR et al.
(a)
(b)
1/[DNA], μM–1
0.03 0.05 0.07
λ = 324 nm
1
9
0.5
0.01
1/[DNA], μM–1
0.03 0.05 0.07
λ = 323 nm
1
0.5
–4
–8
K = 1.78 × 103, M–1
ΔG = –18.55 kJ/mol
0.01
–4
10
K = 8.70 × 103, M–1
ΔG = –22.49 kJ/mol
–6
–8
–12
0.0
0.0
300 350 400 450 500 550 600
Wavelength, nm
300 350 400 450 500
Wavelength, nm
Fig. 4. Absorption spectra of the ligand (a) HL3 and (b) its oxovanadium complex 3 in the (a) absence and (b) in the presence of
DNA. DNA concentration (µM): (1) 10, (2) 19, (3) 27, (4) 35, (5) 42, (6) 48, (7) 54, (8) 59, (9) 64, and (10) 68. The arrow
indicates increasing DNA concentrations. The insert is the A0/(A – A0) vs 1/[DNA] plot.
The binding constants were obtained from the
A0/(A – A0) vs. 1/[DNA] plots. The change in the
Gibb’s free energy (ΔG) was determined from Eq. (5):
could be coupled with the π orbital of the base pairs,
thus decreasing the π–π* transition energy. It also
shows a minor interaction with the grooves through
hydrogen bonding with bases, typically to N3 of
adenine and O2 of thymine [34]. These findings are in
agreement with the reported studies on oxovanadium
complexes [35, 36]. On the other hand, the
hypochromism with a red shift exhibited by HL1
shows that the drug-DNA interaction occurs by the
electrostatic and groove binding mechanisms.
ΔG = –RTln K,
(5)
where R is the general gas constant (8.314 J K–1 mol–1),
T, temperature (K), and K, binding constant.
Complex 2 did not interact with SS-DNA and
therefore is not discussed further. The determined
values of the absorption bands’ maxima, λmax, their
shifts, binding constants (K), and the Gibb’s free
energy (ΔG) are listed in Table 5. These spectral
characteristics showed hypochromism and a blue shift
with the increase in the DNA concentration for HL2–
HL4 and for complexes 1, 3, and 4. These compounds
may interact with DNA by the intercalation and groove
binding mechanisms. After intercalating with the base
pairs of DNA, the π* orbital of the intercalated ligand
The determined binding constants of complexes 3
(8.7 × 103) and 4 (6.32 × 103) were higher than those
of their free ligands. This might be due to the presence
of oxovanadium moiety, which assists the stacking
interaction with DNA. However, the binding of HL1
with DNA occurs via the electrostatic interaction, as
mentioned above.
Table 5. Data on the Drug-DNA interaction
Compound λmax, nm Hypochromism, % Bathochromism, nm Hypsochromism, nm K × 103, M−1
ΔG, kJ/mol
–20.66± 0.55
–19.86± 0.36
–22.72± 0.30
–18.55± 0.61
–22.49± 0.29
–17.11± 1.16
–21.60± 0.38
HL1
330
329
292
324
323
342
341
23.18
20.09
27.25
31.15
28.50
29.30
25.71
1
–
–
–
–
–
–
–
4
2
2
1
2
2
4.18± 0.91
3.03± 0.09
9.64± 1.14
1.78± 0.43
8.70± 0.99
1.00± 0.43
6.32± 0.95
1
HL2
HL3
3
HL4
4
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INVESTIGATION OF DERIVATIZED SCHIFF BASE LIGANDS
2193
The Gibb’s free energy of the ligands HL1–HL4
and complexes 1, 3 and 4 lie from –6.9 to –20.6 and
from –15.93 to –22.48 kJ/mol, respectively, which
shows that the drug-DNA interaction is the
spontaneous process.
is isostructural and isoelectronic to phosphate [37, 38].
Like the phosphate ion, it can form stable five-
coordinated species, and therefore, may serve as a
transition-state analog for both the acquisition of
phosphate and its release in the catalytic reaction of
alkaline phosphatase [39]. The ALP hydrolysis of p-
NPP occurs by Eq. (6) to form a yellow-colored p-
nitrophenol that absorbs at 405 nm.
Enzyme inhibition study. The vanadate ion is a
potent competitive inhibitor of phosphatase because it
O−
O
H
N+
O−
O−
−O
N+
O
N+
(6)
O
ALP
−O
O−
+
P
Hydrolysis
O
O−
O
O−
p-Nitrophenol
O
P
O−
Inorganic phosphate
−O
p-NPP
The activity of the enzymes decreases, as the
concentration of the compound increases. The X-ray
crystal structure of Yersinia PTP (protein-tyrosine
phosphatase) complexed with vanadate suggests that
the vanadate occupies the active site of the
phosphatase enzyme to form a thiol-vanadyl ester
linkage with a catalytic cysteine residue [40]. All the
synthesized triazole precursors (HL1–HL4) and the
oxovanadium complexes 1–4 were screened for their
inhibition against ALP. The results are shown in Fig.
5. All the triazole precursors tested are inactive against
ALP inhibition, while their oxovanadium complexes
exhibit activity, depending on concentration. Among
the tested series, the complex 4 is the most potent
inhibitor since it completely inhibits the ALP at the
concentration of 184 mM and above. It was also found
that at a concentration of 0.247 mM the ALP inhibition
is 100% for all complexes. These findings agree with
the fact that the vanadium compounds inhibit over
50% of ALP activity at concentrations above 50 μM
[41]. Probably, the quantitative inhibition of ALP by
the tested compounds could be due to their binding
with the active sites of ALP, which results in the
failure of the enzyme and catalyses the reaction.
Consequently, the absorption decrease is due to the
formation of a lesser amount of the product.
Ganoderma lucidum, Aspergillus niger and
Penicillium notatum) and bacteria (Escherichia coli,
Bacillus subtilis, Staphylococcus aureus and
Pasturella multocida) by disc diffusion method.
Fluconazole and Streptomycin were used as positive
controls for antifungal and antibacterial screening
tests, respectively, whereas DMSO was used as
negative control. Each disc (9 mm) was soaked with a
sample solution (1 mg/mL of drug in DMSO) [42] and
the zones of inhibition formed in the respective media
were measured in millimeters. Percentage of growth
inhibition was calculated with reference to the growth
in a control from Eq. (7).
120
1 2 3
4
100
2
1
80
60
3
1
4
2
3
1
40
20
0
2
4
3
0.0623 0.124
0.184
0.247
Antimicrobial activities. The synthesized free
ligands and their respective oxovanadium complexes
were screened to see their in vitro response against
various strains of fungi (Alternaria alternata,
Concentration of inhibitors, mM
Fig. 5. Inhibition of alkaline phosphatase (ALP) with the
oxovanadium complexes 1–4.
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2194
MUNAWAR et al.
Linear growth in test sample, nm
(7)
.
× 100
Growth inhibition, % = 100 −
Linear growth in control, nm
The data obtained are summarized in Tables 6 and
the antifungal activity of their complexes was also
maximal, as compared to all novel complexes studied,
and even exceeded that of the standard control drug.
7. It was found that, in general, the vanadium
complexes have significantly higher activity towards
all the tested organisms than the respective free ligand
precursors. Among all thetriazol ligands, the activity of
HL3 and HL4 against all fungal species was maximal;
The greater antifungal activity of the tested triazol
ligands and their oxovanadium complexes was accounted
Table 6. Antifungal activity of the ligands HL1–HL4 and their oxovanadium complexes 1–4
Average zone of inhibition, mm
Compound
A. alternata
38.33a,b ± 0.41
20.11c ± 0.10
25.20b,c ± 0.18
29.12b,c ± 0.19
34.04a,b ± 0.07
30.25b,c ± 0.22
39.66a,b ± 0.28
32.24a,b ± 0.16
42.19a ± 0.17
G. lucidum
A. niger
P. notatum
35.44b,c ± 0.27
22.18c ± 0.17
27.21b,c ± 0.14
25.29b,c ± 0.15
37.16b,c ± 0.28
55.37a,b ± 0.33
62.37a ± 0.43
35.29b,c ± 0.19
60.31a,b ± 0.18
Fluconazole
HL1
41.29a,b ± 0.51
18.15c ± 0.12
23.33b,c ± 0.17
23.29b,c ± 0.14
34.26b,c ± 0.18
41.37a,b ± 0.21
56.33a ± 0.14
34.71b,c ± 0.15
52.25a,b ± 0.12
40.33a,b ± 0.36
25.08c ± 0.14
33.25b,c ± 0.20
26.21b,c ± 0.12
31.71b,c ± 0.15
35.39b,c ± 0.27
47.29a,b ± 0.28
32.21b,c ± 0.14
52.36a ± 0.13
1
HL2
2
HL3
3
HL4
4
Concentration is 1 mg/mL in DMSO; 0 = No activity, 5–10 = Activity is present, 11–25 = Moderate activity, 26–40 = Strong activity;
Antibacterial values are the average values obtained in three independent measurements at p < 0.1; Letters in superscripts denote activity
a
b
c
a,b
b,c
level: maximum, intermediate, minimum,
same for Table 7.
between maximum and intermediate, and
between intermediate and minimum; the
Table 7. Antibacterial activity of the ligands HL1–HL4 and their oxovanadium complexes 1–4
Average zone of inhibition (mm)
Compound
E. coli
B. subtilis
S. aureus
P. multocida
30.29a,b ± 0.29
21.33c ± 0.14
23.33b,c ± 0.26
22.13b,c ± 0.20
25.16b,c ± 0.07
26.28a,b ± 0.16
29.87a,b ± 0.21
25.14b,c ± 0.17
30.75a ± 0.13
Streptomycin
30.33a,b ± 0.26
20.37c ± 0.14
26.32b,c ± 0.20
22.17b,c ± 0.13
30.33a,b ± 0.26
28.46a,b ± 0.18
35.31a ± 0.27
28.66a,b ± 0.31
34.27a,b ± 0.18
30.29a ± 0.19
19.29c ± 0.15
25.23a,b ± 0.12
22.09b,c ± 0.18
25.17a,b ± 0.23
27.25a,b ± 0.12
30.42a ± 0.11
24.29b,c ± 0.15
29.21a,b ± 0.14
30.37a ± 0.33
18.21c ± 0.14
23.12b ± 0.19
20.08b,c ± 0.14
27.15a,b ± 0.23
27.37c ± 0.21
28.46b,c ± 0.10
25.23b ± 0.09
29.25a,b ± 0.22
HL1
1
HL2
2
HL3
3
HL4
4
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INVESTIGATION OF DERIVATIZED SCHIFF BASE LIGANDS
2195
for by the fact that their structure is bulky and has
600
400
200
0
electronegative nature, which results in a larger
lipophilicity and assists diffusion through the
microbial cell membrane and, consequently, ensures
larger microbial sensitivity towards these drugs. It was
also found that oxovanadium complexes are more
active inhibitors against fungal culture than bacterial
growth, which is in accordance with the literature data
[42].
2
1
6
7
3
9
8
5
4
10
0
1
2
3
4
5
6
7
8
Time, h
Hemolytic activity. The hemolytic activity was
studied, because the compound having antimicrobial
activity cannot be used in medicine, if its hemolytic
properties are unknown [42].
Fig. 6. Concentration of plasma glucose in mice treated
with the synthesized ligands (HL1–HL4) and their
oxovanadium complexes 1–4. Negative control was treated
with Alloxan, whereas positive control was treated with
Glibenclamide. (*) p < 0.001, q = 4.4. (1) Negative control,
(2) positive control, (3) HL1, (4) complex 1, (5) HL2,
(6) complex 2, (7) HL3, (8) complex 3, (9) HL4,
(10) complex 4.
The hemolytic bioassays of the synthesized
complexes were performed with human red blood cells
in vitro with respect to the Triton X-100 and PBS as
positive control (100% lysis) and negative control (0%
lysis), respectively. The percentage of hemolysis was
calculated by Eq. (8) [43]:
The ligand HL1 showed a significant glucose-
lowering potential, as compared with the negative
control, whereas in comparison with the positive
control, no glucose lowering was observed (p < 0.001;
Fig. 6). This compound led to a significant decrease in
the cholesterol concentration (p < 0.001; Fig. 7a),
whereas caused a significant increase in the total serum
triglycerides’ concentration (p < 0.001; Fig. 7b). The
treatment with the complex 1 led to a significant
lowering of a plasma glucose, as compared to negative
Abssample
(8)
× 100.
Hemolysis, % =
Abscontrol
The values of the hemolytic activity are listed in
Table 8, as the average of three independent measure-
ments. As seen, the activity of all complexes synt-
hesized here is significantly lower than that of
TrilonX-100 and higher than that of PBS. The activity
of complexes 2 and 3 is increased in comparison with
the corresponding ligands HL2 and HL3, whereas the
activity of the vanadium complexes 1 and 4 is de-
creased compared to the non-coordinated free ligands
HL1 and HL4. The compounds showing higher activity
may be used for further study on antitumor activity.
control (p
< 0.001; Fig. 6). The cholesterol
concentration considerably decreased in comparison
with negative control, whereas the concentration of
serum triglyceride increased significantly in
comparison with positive and negative controls (q =
4.450; Fig. 7a) and (p < 0.001; Fig. 7b).
The ligand HL2 caused a significant reduction in
the plasma glucose concentration against negative
control (p < 0.001; Fig. 6), whereas to an increase in
the concentration of serum cholesterol (p < 0.019; Fig.
7a). However, for triglyceride, effect was insignificant
(Fig. 7b). Complex 2 significantly reduced the the
plasma glucose (Fig. 6) and serum cholesterol (q =
4.550; Fig. 7a) concentrations. In contrast, it did not
affect the concentration of serum triglyceride (Fig. 7b).
Insulin mimetic study. Potential antidiabetic
activity of triazole precursors HL1–HL4 and their oxo-
vanadium complexes 1–4 was tested on BALB/c
albino mice. In general, the animals retained health and
activity. Positive control was treated with Gliben-
clamide and negative control, with Alloxan only (see
Figs. 6, 7). The results obtained are in agreement with
the published data on other oxovanadium(IV) com-
plexes [44, 45].
Table 8. Hemolytic activity of the ligands HL1–HL4 and their oxovanadium complexes 1–4
HL1 HL2 HL3
Triton-
X 100
Compound
1
2
3
HL4
4
Hemolysis, % 72.41±0.02 62.24±0.03 41.15±0.05 55.31±0.02 32.79±0.03 42.21±0.04 49.32±0.06 25.12±0.01 99.53±0.72
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 9 2015

2196
MUNAWAR et al.
600
a
(a)
(b)
a
3
600
400
200
0
Control
4
Control
9
400
200
0
a
5
10
9
2
10
6
4
5
2
1
7
8
3
1
6
7
8
Fig. 7. Concentration of serum cholesterol (a) and triglycerides (b) in mice treated with the ligands HL1-HL4 and their
oxovanadium complexes 1-4. Negative control was treated with Alloxan only and positive control, with Glibenclamide. (*) p <
0.001, q = 4.40. (a) p < 0.001. (1) Negative control, (2) positive control, (3) HL1, (4) complex 1, (5) HL2, (6) complex 2, (7) HL3,
(8) complex 3, (9) HL4, (10) complex 4.
In the presence of the ligand HL3, the concentra-
tions of plasma glucose (p < 0.015; Fig. 6) and serum
cholesterol (q = 4.550; Fig. 7a) reduced significantly,
whereas the concentration of serum triglyceride did not
change (Fig. 7b). Administration of the complex 3 led
to a significant lowering of plasma glucose, serum
cholesterol, and triglyceride concentrations (Figs. 6,
7a, and 7b).
configuration of vanadium in the vanadium complexes
was confirmed. The synthesized compounds exhibit
excellent antidiabetic properties.
Crystallographic data for the reported structures are
deposited with the Cambridge Crystallographic Data
Centre, CCDC (998780 and 998781 for HL3 and HL4,
respectively).
The treatment with the ligand HL4 did not decrease
the plasma glucose and cholesterol concentrations,
whereas reduced the triglyceride concentration (q =
4.400; p < 0.05, Figs. 6, 7a, and 7b). The complex 4
led to a significant reduction of the plasma glucose
concentration, but caused the increase in the
concentrations of serum cholesterol and triglyceride
(p < 0.019; p < 0.05, Figs. 6, 7a, and 7b).
ACKNOWLEDGMENTS
K.S. Munawar is thankful to the Higher Education
Commission, Islamabad, Pakistan, for the financial
support of this study.
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