Synthesis and biological activity of the pinostrobin oxime complex compounds with some d-metals

Article abstract of DOI:10.1134/S1070363211010142

The process of the complex formation by the natural flavonoid pinostrobin (5-hydroxy-7-methoxyflavonone) oxime with the Cu(II) and Fe(III) ions in a water-ethanol medium was investigated. The metal: ligand ratio in the complex was determined by the satura

Full text of DOI:10.1134/S1070363211010142

ISSN 1070-3632, Russian Journal of General Chemistry, 2011, Vol. 81, No. 1, pp. 96–101. © Pleiades Publishing, Ltd., 2011.
Original Russian Text © A.A. Mashentseva, T.S. Seytembetov, S.M. Adekenov, B.I. Tuleuov, O.P. Loiko, A.I. Khalitova, 2011, published in Zhurnal Obshchei
Khimii, 2011, Vol. 81, No. 1, pp. 99–105.
Synthesis and Biological Activity of the Pinostrobin Oxime
Complex Compounds with Some d-Metals
A. A. Mashentsevaa, T. S. Seytembetova, S. M. Adekenovb,
B. I. Tuleuovb, O. P. Loikoc, and A. I. Khalitovac
a Gumilev Eurasian National University, Munaitpasova ul. 5, Astana, 010008 Kazakhstan
e-mail: mashentseva.a@gmail.com
b International Holding “Phytochemistry,” Karaganda, Kazakhstan
c
Buketov Karaganda State University, Karaganda, Kazakhstan
Received September 6, 2010
Abstract―The process of the complex formation by the natural flavonoid pinostrobin (5-hydroxy-7-
methoxyflavonone) oxime with the Cu(II) and Fe(III) ions in a water–ethanol medium was investigated. The
metal : ligand ratio in the complex was determined by the saturation method. The results of the analysis of IR
spectra and the data of mass-spectrometric studies of the structure of the obtained complex compounds are
reported. The antiviral activity of the synthesized complexes with respect to the human immunodeficiency virus
HIV-1 IIIB and HIV-2 ROD strains was studied. The antioxidant activity of the complexes, as well as the
parent pinostrobin oxime ligand, was evaluated with respect to the diphenylpicrylhydrazyl (DPPH) radical and
the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical cation.
DOI: 10.1134/S1070363211010142
In recent years the interest of both scientific
community and pharmaceutical companies grew to the
polyphenolic compounds of plant origin. Flavonoids
that belong to the most widespread group of such
compounds show a broad spectrum of biological
activity and are widely used as drugs. The flavonoid
molecule is underlain by the 2-phenylbenzo-γ-pyrone
(I). A great variety of molecular structures among the
natural flavonoids and a wide range of their biological
activity can be attributed to the variety of the types and
position of substituents. More than 4000 different
biologically available flavonoids have been described
[1].
compounds. The transition metal ions are known to
play an important role in the initiation of free radical
processes [5], in particular, the Fenton reaction,
resulting in the formation of hydroxyl radicals. Thus,
the iron(III) ions act as a catalyst in the formation of
lipid peroxides in the process of lipid peroxidation.
Excess of such compounds causes development of
oxidative stress resulting in many serious diseases.
Copper is an essential element, a part of many
vitamins, hormones, enzymes, and respiratory pigments,
and is involved in metabolism, tissue respiration, etc.
Copper-containing compounds (copper sulfate, Bordeaux
mixture) are widely used to control pathogenic fungi
and bacteria in agriculture [6, 7]. However, the high
content of copper compounds in the body is very toxic
to humans. Chelating the copper excess in the body by
natural compounds can significantly reduce its
toxicity, while does not remove its useful properties
[8].
B
O
A
C
OH
O
I
In addition to their direct antiradical effect, the
flavonoids are capable of binding transition metal ions
to form chelates. The formation of such complexes
leads to inhibition of free radical processes. Due to
their chelating properties, the dietary flavonoids can
The intensive study of complexation of natural
flavonoids with the d-metal ions [2–4] indicates the
importance of this area as one of the most promising in
the development of new drugs based on natural
96

SYNTHESIS AND BIOLOGICAL ACTIVITY OF THE PINOSTROBIN OXIME COMPLEX
97
affect ionic balance of metals in the body and oxidative
status of cells and tissues. Therefore, chelating of
metals can be considered as a possible mechanism of
antioxidant protection. A series of studies [9–11]
showed that the formation by flavonoids of complex
compounds significantly extends the range of their
biological activity compared to the original flavonoids.
In addition, the low risk of negative side effects, rapid
absorption, low toxicity, which are an advantage of
flavonoids, suggests the preservation of similar pro-
perties in the chelates.
complex formation by the flavonone derivative
pinostrobin oxime (II) with copper(II) and iron(III)
ions, and carried out a preliminary screening of
biological activity using the PASS program.
As a result of performed reactions we found that
iron(III) and copper(II) ions react with pinostrobin
oxime in a water–ethanol medium to form complex
compounds. The iron complex is purple, and copper
complex is green.
Pinostrobin oxime reacts with the studied metal
salts by the following scheme.
In this study, we investigated a possibility of
H3CO
O
O
NOH
(H2O)n
O
5 H2O
CuSO4
(FeCl3
Me
2
X
6 H2O)
H3CO
O
NOH
O
O
NOH
OCH3
III, IV
III, Me = Cu, X = SO4·(H2O)n; IV, Me = Fe, X = Cl3·(H2O)n.
Electron absorption spectra of the initial pino-
strobin oxime in ethanol and the products of its
reaction with iron(III) and copper(II) ions taken
relative to the solvent in the wavelength range 300–
800 nm differ from each other, which indicates
formation of new compounds in the system.
in 10 min in the case of the iron(III) complex IV, and
then remains almost constant. Taking into account
these findings, we performed further studies after 5
and 10 min delay respectively after preparation of
solutions.
Our experiments showed that pinostrobin oxime is
poorly soluble in water but its solubility increases
greatly in the presence of ethanol. The complex
compounds of the oxime II with the metals also are
limitedly solubile in water, so we used their water–
ethanol solution. We found that both the studied
compounds precipitate at the alcohol content less than
75%. In the case of complex IV with iron(III), at the
increase in the alcohol concentration the absorption of
the solution increases. The complex IV solubility at
the ethanol content about 75% is 1.87×10–3 mol l–1 or
0.30 g l–1.
Complex IV of iron(III) is characterized by two
absorption maxima in the UV and visible ranges of the
spectrum, at 360 (ε 355) and 535 nm (ε 17). The
spectrum of complex compound III with copper(II)
also contains two bands, with the absorption maxima
at 360 (ε 355) and 610 nm (ε 168). In contrast to the
compounds obtained, the organic reagent itself does
not absorb in the visible region.
We found that the absorption of the solutions under
study varies in time. Optical density decreases sharply
in 5 min in the case of the copper(II) complex III and
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MASHENTSEVA et al.
Table 1. Probability of the promising types of biological activity of compounds II–IV according to the PASS programa
Activity type
II
III
IV
Antivirus
0.885 (0.016)
0.885 (0.016)
−
0.908 (0.009)
0.908 (0.009)
0.890
0.908 (0.009)
0.908 (0.009)
−
Antivirus (HIV)
Antivirus (Hepatitis B)
Apoptosis agonist
−
0.873 (0.004)
0.908 (0.009)
0.908 (0.009)
0.908 (0.009)
0.908 (0.009)
−
−
Antiepileptic
0.885 (0.016)
0.885 (0.016)
0.885 (0.016)
0.885 (0.016)
0.885 (0.016)
0.885 (0.016)
0.885 (0.016)
0.783 (0.012)
0.773 (0.008)
−
0.908 (0.009)
0.908 (0.009)
0.908 (0.009)
0.908 (0.009)
−
Antiinflammatory
Agonist of membrane integrity
Chemoprotector
Anesthetic
Radioprotector
0.908 (0.009)
0.908 (0.009)
0.842 (0.005)
0.832 (0.004)
0.782 (0.007)
0.797 (0.026)
0.762 (0.056)
0.754 (0.005)
0.720 (0.004)
0.908 (0.009)
0.908 (0.009)
0.842 (0.005)
0.832 (0.004)
0.782 (0.007)
0.797 (0.026)
0.762 (0.056)
0.754 (0.005)
0.720 (0.004)
Antipruritic
Anti-neurotoxic
NOS2 expression inhibitor
UGT1A9 substrate
Chlordecone reductase inhibitor
Ubiquinol–cytohrom reductase inhibitor
CYP1A inhibitor
0.741 (0.038)
−
−
UGT1A7 substrate
−
a In parentheses the probability is shown of the absence of this type activity.
As to the complex III with copper(II) ions, the
absorption of its solution increases with growing
ethanol content to 85 vol %, and then decreases.
Obviously, at this content of ethanol in the solution the
complex is formed most completely. Further studies
were performed at the ethanol concentration 85 vol %.
Solubility of the complex III in such solution is
1.25×10–3 mol l–1 or 0.17 g l–1.
activity using the online version of the PASS program
(Table 1) [17].
As seen from Table 1, in the case of formation of
complex compounds IV with iron ions and III with
copper ions, the probability to find biological activity
increases. For the iron complex is found nine, and for
the complex with copper six new types of activity of
575 possible, at 70% probability of their occurrence.
The results of this screening suggest the presence of a
wide spectrum of biological activity of the chelates.
Using the method of spectrophotometric saturation
[16], we have determined the metal : ligand ratio in the
complexes, which has been found equal to 1:2 for both
compounds, in consistence with the mass spectrometric
data: molar mass of complex IV is 640.15 g mol–1, and
of complex III, 924.38 g mol–1.
Antiradical activity of these complexes was studied
in respect to the DPPH (diphenylpycrylhydrazyl)
radicals and ABTS•+ [2,2'-azino-bis(3-ethylbenzthia-
zoline-6-sulphonic acid] radical cations.
The stability constants of the iron(III) and copper
(II) complex compounds are (7.11 ± 0.01)×104 and
(8.31 ± 0.03)×106, respectively, indicating a high
stability of the complexes.
Formation of a stable form due to protonation of the
radical forms by active antioxidant leads to a change in
the color of DPPH radical solution in alcohol from a
intense purple to pale yellow. As a reference
compound we used butylated hydroxyanisole (BHA)
widely used in industry as an antioxidant reference. As
shows the graphical dependence in Fig. 1, the anti-
Preliminary to the study of the biological activity of
the pinostrobin oxime complex compounds with metal
ions we carried out a prediction of their biological
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SYNTHESIS AND BIOLOGICAL ACTIVITY OF THE PINOSTROBIN OXIME COMPLEX
99
Table 2. The IC50 coefficients for the pinostrobin oxime
complexes
radical activity is of concentration nature. The pino-
strobin oxime complex with iron ions (Ox–Pb–Fe)
shows a more positive value of antioxidant activity
compared to the initial pinostrobin oxime (Ox–Pb)
ligand. The complex compound with copper (Ox–Pb–
Cu) shows minimal activity toward the studied radical.
Compound
DPPH
2.99
ABTS
4.19
Ox–Pb (II)
Ox–Pb–Fe (IV)
Ox–Pb–Cu (III)
BHA
3.12
1.54
The calculated values of effective thermal
inhibition index IC50 (Table 2) indicates the minimum
amount of respective compound required to inhibit
50% of the radical in the reaction mixture.
26.61
0.315
110.2
0.039
shows lower values of effective thermal inhibition of
free radicals compared with the original ligand and
with the copper complex III, which indicates its higher
antioxidant activity.
We also have studied the effect of the exogenous
antioxidants (concentration of 0.5 mg ml–1) on the
process of inhibition of the radical cation ABTS•+ in
time. The curves in Fig. 2 show that in the case of the
reference substance (BHA) within 2 min occurs a
sharp decrease in optical density of reaction medium
and, consequently, a decrease in the content of the
radical active form, and then it falls lineary.
Several researchers [18, 19] indicate a high
potential of chelate complexes of flavonoids as
antiviral drugs and as the agents possessing anticancer
activity. The obtained complex compounds of
pinostrobin oxime with ions of iron and copper were
also tested for antiviral activity. Table 3 lists the results
of studies of the antiviral activity against strains of the
human immunodeficiency viruses HIV-1 IIIB and
HIV-2 ROD performed in accordance with [20].
Change in optical density of the pinostrobin oxime
iron complex IV is more extreme, but after 5 min of
the reaction, the absorption reaches the same values as
that of the reference. Therewith, the complex IV shows
much better results compared to the initial pinostrobin
oxime ligand II and complex III.
The selectivity index SI (av.IC50/CC50) given in the
Table 3 points to the activity of the compound with
respect to the virus strain. The higher the value of this
index, the more effective is the drug impact on the
cells infected with MT-4. A comparative analysis of
the pinostrobin oxime II and its complexes III and IV
with iron and copper with the reference drugs
To determine the effective coefficient of inhibition
of the radical cation ABTS•+, we investigated the
concentration dependence of inhibition in the
concentration range 0.1–1.0 mg ml–1. The calculated
IC50 values are listed in Table 2. As can be seen from
these data, the pinostrobin oxime iron complex IV
Concentration, mg ml–1
Time, min
Fig. 1. Dynamics of the antiradical activity change of the
Fig. 2. Dynamics of inhibition of the ABTS•+ radical cation
pinostrobin oxime complexes.
in time.
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MASHENTSEVA et al.
Table 3. Biological activity of the complexes against some
HIV strains
density of solutions was measured on a KFK-3 device.
Preparation of pinostrobin oxime II. A mixture
of 200 mg of pinostrobin and 77 mg of hydroxylamine
hydrochloride was dissolved in ethanol and 100 mg of
NaHCO3 was added to the mixture. The reaction was
carried out at 60°C. After 4 h the mixture was treated
with 1% HCl solution and water [2]. Colorless
rhombic crystals were obtained, yield 84%, mp 182–
184°C. IR spectrum (KBr), ν, cm–1: 3431 (OH), 1647
(C=N), 1617, 1578 (C=C). UV spectrum, λmax, nm (ε,
EtOH): 205 (23 442), 251 (4074), 279 (20 417).
a
b
av.IC50
,
av.CC50 ,
Strain
Compound
Ox–Pb (II)
SI
Ug ml–1
Ug ml–1
IIIB
>0.25
0.25
<1
<1
ROD
IIIB
>0.25
0.25
Ox–Pb–Fe (IV)
>0.35
0.35
<1
ROD
IIIB
>0.35
0.35
<1
Ox–Pb–Cu (III)
Nevirapin (NVP)
>51.25
>51.25
0.050
51.25
51.25
>4.00
>4.00
>25.00
>25.00
<1
Iron(III) solution. The solution of the concentra-
tion 5×10–2 mol l–1 was prepared by dissolving
accurately weighed FeCl3·6H2O in water [12]. The
exact concentration of the resulting solution was
determined using permanganatometric method [13].
ROD
IIIB
<1
>80
<1
ROD
IIIB
>4.00
Retrovir (AZT)
0.0022
0.00094
>11587
>26731
Pinostrobin oxime complex with iron(III). Iron(III)
chloride (0.2 ml of 5×10–2 M solution) was mixed
with 2 ml of 1×10–2 M solution of compound I and
1.3 ml of 96% ethanol was added. The resulting
solution is of violet color. After stirring electronic
spectra of the solution were recorded relative to water
in the wavelength range 300–800 nm. To isolate the
substance in solid form, the solution was evaporated at
room temperature. The resulting purple reaction
product had mp 234–236°C. Yield 94%. IR spectrum
(KBr), ν, cm–1: 621.93, 485.15 (C–Fe). UV spectrum,
ROD
a
The average concentration at which the compound protects 50%
of cells from exposure to the virus strain. The average con-
centration at which the compound kills 50% of uninfected cells.
b
neviropin and retrovir showed that the studied pino-
strobin oxime complex compounds with metal ions do
not possess antiviral activity against the studied HIV
infection strains.
Analysis of the computer prediction data of
biological activity of test substances using the PASS
program in comparison with the data obtained from
in vitro and in vivo studies revealed some discrepancy
between theoretical calculations and experimental data,
which may be due to the structural features of the
compounds. It is noteworthy that the dependence
between the increased probability of manifestation of
biological activity going from the structure II to the
structures of complexes III and IV seen in the table is
partially confirmed in the study of the antiradical
activity. Thus, the complex compound with iron ions
shows better results in the inhibition of free radicals
than the original pinostrobin oxime ligand. The very
low activity of the complex with copper ions possibly
relates to both the structural features of the ion chelator
and a synergistic effect of the interaction with the
flavonoid chelate.
λ
max, nm (ε): 360 (355), 535 (17). Mass spectrum:
C 61.89, H 5.04, Fe 8.72, N 4.37, O 19.98.
Pinostrobin oxime complex with copper(II).
Copper(II) sulfate (0.2 ml of 5×10–2 M solution) was
mixed with 2 ml of 1×10–2 M solution of compound I
and 1.3 ml of 96% ethanol was added. The resulting
solution is of green color. After stirring electronic
spectra of the solution were recorded relative to water
in the wavelength range 300–800 nm. To isolate the
substance in solid form, the solution was evaporated at
room temperature. The resulting green reaction
product had mp 240–242°C. Yield 92%. IR spectrum
(KBr), ν, cm–1: 521.93, 485.15 (C–Cu). UV spectrum,
λ
max, nm (ε): 360 (355), 610 (168). Mass spectrum:
C 42.88, H 5.67, Cu 6.87, N 3.03, O 38.08, S 3.47.
Inhibition of 1,1-diphenyl-2-picrylhydrazyl
(DPPH) radical was carried out in accordance with
the procedure [14]. An aliquot of the sample (0.1 ml)
was added to 3 ml of 6×10–5 M solution of the radical
in methanol. After vigorous stirring, the solution was
left in the dark for 30 min. The change in the optical
density was measured at 520 nm.
EXPERIMENTAL
IR spectra were recorded on
a
Vector-22
instrument. Electron absorption spectra were recorded
on a Specord UV-VIZ spectrophotometer. The optical
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SYNTHESIS AND BIOLOGICAL ACTIVITY OF THE PINOSTROBIN OXIME COMPLEX
101
6. Horsfall, J.G., Principles of Fungicidal Action,
Interaction with 2,2'-azinobis-(3-ethyltiazolin-6-
sulfonate cation radical (ABTS•+) was investigated
according to [15]. To generate the radical, 5 ml of
14 mM ABTS solution was mixed with 5 ml of
potassium persulfate (0.0066 g in 5 ml of deionized
water). The mixture was left for 16 h in a dark place.
To determine the antiradical activity, a 10 µl sample
was added to 990 ml of working solution of the radical
previously diluted with ethanol until the optical density
of 0.700 ± 0.020 at 734 nm. The reduction in the
optical density was measured at 734 nm after 1 min.
The antiradical activity value (ARA) was calculated
with the following formula:
Waltham: Chronica Botanica Co., 1956, p. 141.
7. Bukharia, S.B., Shahabuddin, M., Mahroof-Tahirc, M.,
and Bhanger, M.I., Spectrochim. Acta, A, 2009, vol. 71,
p. 1901.
8. Arman, P. and Wain, R.L., Ann. Appl. Biol., 1958,
vol. 46, p. 366.
9. Kostyuk, V.A., Potapovich, A.I., and Strigunova, E.N.,
Arch. Biochem. Biophys., 2004, vol. 428, p. 204.
10. Bukharia, S.B., Shahabuddin, M., Mahroof-Tahirc, M.,
and Bhanger, M.I., J. Mol. Struct., 2008, vol. 892, p. 39.
11. Stavrescu, R.B., Kimura, T., Hayakawa, F., and Ando, T.,
Central Eur. J. Chem., 2003, no. 1, p. 35.
12. Korostelev, P.P., Prigotovlenie rastvorov dlya khimiko-
analiticheskikh rabot (Preparation of Solutions for the
Chemical Analytical Work), Moscow: Akad. Nauk
SSSR, 1962.
13. Osnovy analiticheskoi khimii (Fundamentals of
Analytical Chemistry), Zolotov, Yu.A., Ed., Moscow:
Vysshaya Shkola, 2001.
APA (%) = [(A0 – Aτ)/A0]×100%.
Here A0 is optical density at τ = 0, Aτ is optical density
measured after 1 min keeping.
ACKNOWLEDGMENTS
14. Brand-Williams, W., Cuvelier, M.E., and Berset, C.,
Lebensmittel Wissenschaft und Technologie, 1995,
vol. 28, p. 25.
15. Re, R., Pellegrini, N., Proteggente, A., Pannala, A.,
Yang, M., and Rice-Evans, C., Free Radical Biology
and Medicine, 1999, vol. 26(9), p. 1231.
16. Bulatov, M.I. and Kalinkin, I.P., Prakticheskoe
rukovodstvo po fotometricheskim metodam analiza
(Practical Manual on the Photometric Methods of
Analysis), Leningrad: Nauka, 1976.
17. Lagunin, A., Stepanchikova, A., Filimonov, D., and
Poroikov, V., Bioinformatics, 2000, vol. 16, no. 8,
p. 747.
The authors express great gratitude to Professor
C. Pannecouque and assistant K. Erven (Catholic
University of Leuven, Belgium) for assistance in the
antiviral activity testing.
REFERENCES
1. Kostyuk, V.A. and Potapovich, A.I., Bioradikaly i
bioantioksidanty (Bioradicals and Bioantioxidants),
Moscow: BGU, 2004.
2. Moridani Majid, Y., Pourahmad, J., and Hoang, B.,
Free Radical Biology and Medicine, 2003, vol. 34,
no. 2, p. 243.
18. Kopacz, M., Woznicka, E., and Gruszecka, J., Acta
Polon. Pharm. Drug Research, 2005, vol. 62, no. 1,
p. 65.
3. Perez, C.A., Yong Tong, and Maolin Guo, Curr. Bioact.
Compd., 2008, vol. 4(3), p. 150.
4. Bravo, A. and Anacona, J., Transition Met. Chem.,
19. Cornard, J.P. and Merlin, J.C., J. Inorg. Biochem., 2002,
vol. 92, p. 19.
2001, vol. 26, p. 20
5. Weijun Chen, Shaofang Sun, Wei Cao, Yan Liang, and
20. Pannecouque, C., Daelemans, D., and De Clercq, E.,
Nature Protocols, 2008, no. 3, p. 427.
Jirong Song, J. Mol. Struct., 2009, vol. 918, p. 194.
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