Rev. Med. Chir. Soc. Med. Nat., Iai 2015 vol. 119, no.

PHARMACY ORIGINAL PAPERS

SYNTHESIS AND EVALUATION OF ANTIOXIDANT ACTIVITY OF

SOME HYDRAZONES WITH XANTHINE STRUCTURE

Sandra Constantin 1, Andreea Pnzariu 1, Ioana Vasincu 1, Maria Apotrosoaei 1,

Luminia Confederat 1, F. Buron2, S. Routier2, Lenua Profire 1*
University of Medicine and Pharmacy Grigore. T. Popa - Iai,
Faculty of Pharmacy
1. Departament of Pharmaceutical Chemistry
University of Orlans
2. Institute of Organic and Analytical Chemistry - Orlans, France
*Corresponding author: lenuta.profire@umfiasi.ro

SYNTHESIS AND EVALUATION OF ANTIOXIDANT ACTIVITY OF SOME HYDRA-

ZONES WITH XANTHINE STRUCTURE (Abstract): Aim: To synthesize some hydrazones
with xanthine structure and to evaluate their antioxidant activity. Material and methods: The
synthesis started from theophylline sodium salt, prepared from theophylline and sodium met h-
oxide, which was transformed into its ethyl ester and then in hydrazide. The hydrazo ne deriva-
tives were obtained by the condensation of hydrazide with different aromatic aldehydes (benz -
aldehyde, 4-nitro/3-nitro/3-hydroxy/4-chloro/4-bromo-benzaldehyde). The antioxidant poten-
tial was evaluated in vitro by using the DPPH radical scavenging assay and was compared with
theophylline activity used as parent compound. Results: Six hydrazones with xanthine struc-
ture were obtained. They were characterized in terms of their physical properties and their
structures were confirmed by IR and 1H-NMR spectroscopy. Some of these compounds have an
antioxidant effect more pronounced than theophylline at higher concentrations. The most active
compound was 3-nitro-benzylidene-hydrazino-acetyl-theophylline (compound g), with an
inhibition rate of 65% 0.99 at 30 min and 79% 0.81 at 60 min. Conclusions: The antioxi-
dant activity of synthesized hydrazones depends on the substituents used in structural modul a-
tion. The substitution of aromatic ring with nitro group in meta position had the most favorable
influence. Keywords: THEOPHYLLINE, HYDRAZONE, ANTIOXIDANT ACTIVITY, DPPH.

Xanthine structure is known for its im-
portant biological effects and could be found
in the structure of compounds with vasodila-
tor, bronchodilator, anticancer, anti-
inflammatory and hypoglycemic action (1).
The most important compounds with xan-
thine structure are caffeine, theophylline and
theobromine (2). Theophylline is used for
the treatment of essential asthma, asthmatic
dyspnea and bronchopulmonary disease.
The xanthine moiety is present also in the
structure of Linagliptin (Tradjenta , Trajen-
ta), a DPP-4 inhibitor used for the treat-
ment of diabetes mellitus type 2 (3).
On the other hand, there is increasing
evidence supporting the involvement of
oxidative stress in such diseases as athero-
sclerosis, diabetes, cancer, glaucoma,
rheumatoid arthritis, psoriasis and Alz-
heimer disease (4). Oxidative stress is an
imbalance between production of harmful
reactive oxygen species such as free radi-

910
 Synthesis and evaluation of antioxidant activity of some hydrazones with xanthine structure
cals or peroxides named oxidants and the
ability of the body to fight with their ef-
fects using different endogenous antioxi-
dants (5).
The main objective of this study was the
synthesis of some hydrazones with xan-
thine structure and the evaluation of their
potential antioxidant activity.
MATERIAL AND METHODS
Reagents. Theophylline, sodium, ethyl
chloroacetate, hydrazine hydrate 64%,
aromatic aldehydes (benzaldehyde, 4-
nitro/3-nitro/3-hydroxy/4-chloro/4-bromo-
benzaldehyde), organic solvents (methanol,
ethanol, dimethyl formamide - p.a. quality)
and standard reagents for antioxidant assay
were purchased from Sigma Aldrich Com-
pany. All reagents and solvents were used
without previous purification. For monitor-
ing the reactions Thin Layer Chromatog-
Fig. 1. The synthesis of hydrazone derivatives with xanthine structure.
General procedure for synthesis of hy-
drazones with xanthine structure
Theophylline-ethyl acetate (c). To a so-
lution of sodium methoxide freshly pre-
pared from sodium (0.1 mol) and dry meth-
anol (200 mL), theophylline (0.1 mol) was
added in small parts. The reaction was
raphy (TLC) Silica gel 60 F 254 plates pro-
duced by Merck Company were used. The
Proton Nuclear Magnetic Resonance ( 1H-
NMR) spectra were obtained in dimethyl
sulfoxide deuterated (DMSO-d 6) produced
by Sigma Aldrich Company.
Chemistry. The synthesis of compounds
started from theophylline sodium salt (b),
which was obtained from theophylline (a)
and sodium methoxide, prepared from so-
dium and dry methanol. In the next step,
the sodium salt reacted with ethyl chloro-
acetate when theophylline-ethyl acetate (c)
was formed. Then, the resulted ester was
treated with an excess of hydrazine hydrate
(64%) (6-8) to obtain hydrazide (d), which,
by condensation with different aromatic
aldehydes (benz-aldehyde, 4-nitro/3-
nitro/3-hydroxy/ 4-chloro/ 4-bromo-
benzaldehyde), formed the corresponding
hydrazones (e-j) (fig. 1).
carried out at room temperature with stir-
ring until all the solvent was evaporated.
The resulted theophylline sodium salt (0.1
mol) was added to a mixture of ethanol:
dimethyl formamide (DMFA) (260 mL:
100 mL). The mixture was heated under
reflux and stirred for 1 h and after that
911
 Sandra Constantin et al.
ethyl chloroacetate (0.1 mol, 10.71 mL)
was added dropwise and left refluxing
overnight. The ethyl ester was recrystal-
lized from ethanol. The reaction was moni-
tored by TLC using dichloromethane:
methanol = 9.6: 0.4 as eluent system.
Theophylline-hydrazide (d). The ethyl
ester of theophylline (0.035 mol) in ethanol
(50 mL) was heated under reflux and
stirred until solubilization and then hydra-
zine hydrate 64% (0.11 mol, 5.52 mL) was
added dropwise. The extra amount of etha-
nol (150 mL) was added and the mixture
was heated under reflux for 6 h. The reac-
tion was monitored by TLC using ethyl
acetate: acetone =7.5:3 as eluent system.
The product was separated by filtration and
washed two times with diethyl ether.
Theophylline-hydrazones (e-j). A mix-
ture of acetyl-theophylline-hydrazine (0.006
mol) and ethanol (200 mL) was refluxed for
1 h. Some drops of glacial acetic acid and
the aromatic aldehyde (benzaldehyde, 4-
nitro/ 3-nitro/ 3-hydroxy/ 4-chloro/ 4-
bromo-benzaldehyde) (0.009 mol) were
added and the mixture was refluxed for 4-24
h with continuous stirring. The reaction was
monitored by TLC using ethyl acetate: ace-
tone =7.5:3 as eluent system. The resulted
hydrazones were purified by filtration and
washed two times with diethyl ether and n-
pentane.
Spectral characterization. Fourier trans-
form infrared (FT-IR) spectra were record-
ed on a Nicolet iS50 FT-IR Spectrometer,
at a resolution of 4 cm -1 after 16 scans in
the 4000-600 cm-1 range. The spectra were
interpreted using Omnic Spectra Software.
1
H-NMR spectra were recorded using a
NMR Bruker 400 MHz spectrometer (Ger-
many).
Antioxidant assay
DPPH radical scavenging activity. In
order to evaluate the antiradical capacity
912
against DPPH radical, the hydrazone deriva-
tives (e-j) were dissolved in DMSO to ob-
tain a stock solution with a concentration of
25 mg/mL. From the stock solution different
volumes (50 L, 100 L, 150 L, 200 L,
250 L, 300 L) were taken and diluted
with DMSO in order to obtain 400 L. The
sample solution (400 L) was added to 2.5
mL solution of DPPH in methanol 0.1 mM
and the mixture was left in the dark. After
30 min and 60 min, respectively, the absorb-
ance was measured at 517 nm against a
blank (methanol) using a UV-VIS spectro-
photometer (9, 10). The DPPH radical scav-
enging activity was expressed as percentage
according to the following equation: % In-
hibition = (A0 - A1)/A0 100, where
A0= absorbance of the control (solution
of DPPH in methanol);
A1= absorbance of the sample at 30
min and 60 min respectively.
Statistical analysis. All experiments
were performed in triplicate and the data
were analyzed using the analysis of vari-
ance and were expressed as arithmetic
mean standard deviation (SD).
RESULTS AND DISCUSSION
Chemistry
Theophylline-ethyl acetate (c) is color-
less or white needle crystals, with melting
point (m.p.) = 137-140 C, soluble in etha-
nol on heating.
The hydrazide of theophylline (d) is a
white powder, very electrostatic, with
m.p.=272-275 C, soluble on heating in
DMSO and a mixture of acetone and few
drops of water.
The hydrazones with xanthine structure
(e-j) are fluffy solid compounds, with small
density and cotton structure. They have
electrostatic properties. The compounds are
white or yellow in color. They are soluble
in a mixture of acetone and few drops of
 Synthesis and evaluation of antioxidant activity of some hydrazones with xanthine structure

water, DMSO and DMFA on heating, par- slightly soluble in chloroform, insoluble in
tially soluble in methanol on heating, absolute ethanol (tab. I).

TABLE I
Physical characteristics of hydrazones with xanthine structure (e-j)
Molecular Appearance Yield
Comp. R Formula M.p. (C)
weight (solid) (%)
e H C 16H16N 6O 3 340.34 white 270-272 74 %
f 4-NO 2 C 16H15N 7O 5 385.34 yellow 305-308 87 %
g 3-NO 2 C 16H15N 7O 5 385.34 white 295-296 77 %
h 4-Cl C 16H15ClN 6O 3 374.79 white 294-296 95 %
i 4-Br C 16H15BrN 6O 3 419.24 white 301-303 95 %
j 3-OH C 16H16N 6O 4 356.34 yellowish 295-296 94 %

TABLE II
Spectral characteristics for hydrazones with xanthine structure
FT-IR characteristic 1H-NMR
Comp. R
band (cm-1) (400 MHz, DMSO-d6, ppm)
11.77 (s, 1H, CO-NH-), 8.06 (d, 2H, CH=N), 7.69-7.74 (m, 2H, Ar-H), 7.43-
3078 (-NH-),
7.46 (m, 3H, Ar-H), 5.55 (s, 2H, CH 2-CO), 3.46 (s, 3H, N-CH3), 3.19 (s, 3H,
2966 (CH AR ),
e H N-CH 3 ); tautomer form (20%): 11.77 (s, 1H, CO-NH), 8.22 (s, 1H, CH=N),
1671 (-CO-NH-),
8.07 (s, 1H, CH=N), 7.69-7.74 (m, 2H, Ar-H), 7.43-7.46 (m, 3H, Ar-H), 5.12
1608 (-CH=N)
(s, 2H, CH2-CO), 3.45 (s, 3H, N-CH 3), 3.19 (s, 3H, N-CH3)
3068 (-NH-), 12.07 (s, 1H, CO-NH), 8.29 (d, 2H, Ar-H), 8.16 (s, 1H, CH=N,), 8.06 (s, 1H,
2948 (CH AR ), CH=N), 7.99 (dd, 2H, Ar-H), 5.59 (s, 2H, CH 2-CO), 3.46 (s, 3H, N-CH 3),
f 4-NO 2 1653 (-CO-NH-), 3.19 (s, 3H, N-CH3); tautomer form (23%): 12.07 (s, 1H, CO-NH), 8.33 (s,
1597 (-CH=N), 1H, CH=N), 8.29 (d, 2H, Ar-H), 8.08 (s, 1H, CH=N), 7.99 (dd, 2H, Ar-H),
1551 (-C-N) 5.16 (s, 2H, CH 2-CO), 3.45 (s, 3H, N-CH 3), 3.19 (s, 3H, N-CH 3)
12.01 (s, 1H, CO-NH ), 8.52 (s, 1H, CH=N), 8.25 (d, 1H, Ar-H), 8.18 (d, 2H,
3079 (-NH-),
Ar-H), 8.05 (s, 1H, CH=N), 7.74 (t, 1H, Ar-H), 5.59 (s, 2H, CH 2-CO), 3.45 (s,
2953 (CH AR ),
3H, N-CH 3), 3.18 (s, 3H, N-CH 3); tautomer form (25%): 12.01 (s, 1H, CO-
g 3-NO 2 1653 (-CO-NH-),
NH ), 8.52 (s, 1H, CH=N), 8.34 (s, 1H, Ar-H), 8.25 (d, 1H, Ar-H), 8.14 (d,
1610 (-CH=N),
1H, Ar-H), 8.07 (s, 1H, CH=N), 7.74 (t, 1H, Ar-H), 5.15 (s, 2H, CH 2-CO),
1551 (-C-N)
3.45 (s, 3H, N-CH3), 3.18 (s, 3H, N-CH3)
3109 (-NH-), 11.83 (s, 1H, CO-NH), 8.05 (s, 1H, CH=N), 8.04 (s, 1H, CH=N), 7.76 (d,
2974 (CH AR ), 2H, Ar-H), 7.52 (d, 2H, Ar-H), 5.55 (s, 2H, CH 2-CO ), 3.45 (s, 3H, N-CH 3 ),
h 4-Cl 1635 (-CO-NH-), 3.19 (s, 3H, N-CH3); tautomer form (23%): 11.83 (s, 1H, CO-NH), 8.21 (s,
1594 (-CH=N), 1H, CH=N), 8.07 (s, 1H, CH=N), 7.72 (d, 2H, Ar-H), 7.52 (d, 2H, Ar-H), 5.12
826 (-C-Cl) (s, 2H, CH2-CO), 3.45 (s, 3H, N-CH 3 ), 3.19 (s, 3H, N-CH3)
3109 (-NH-),
11.83 (s, 1H, CO-NH), 8.04 (d, 2H, CH=N), 7.67 (dd, 4H, Ar-H), 5.54 (s, 2H,
2974 (CH AR ),
CH 2-CO), 3.45 (s, 3H, N-CH 3), 3.19 (s, 3H, N-CH 3); tautomer form (22%):
i 4-Br 1635 (-CO-NH-),
11.83 (s, 1H, CO-NH), 8.19 (s, 1H, CH=N), 8.07 (s, 1H, CH=N), 7.67 (dd,
1609 (-CH=N),
4H, Ar-H), 5.12 (s, 1H, CH 2-CO), 3.45 (s, 3H, N-CH 3), 3.19 (s, 3H, N-CH 3)
824 (-C-Br)
11.69 (s, 1H, CO-NH ), 9.62 (s, 1H, C-OH), 8.06 (s, 1H, CH=N), 7.96 (s,
3135 (-NH-),
1H, CH=N), 7.25 (t, 1H, Ar-H), 7.18 7.05 (m, 2H, Ar-H), 6.84 (d, 1H, Ar-
3014 (-OH),
H), 5.53 (s, 2H, CH 2-CO ), 3.46 (s, 3H, N-CH 3), 3.20 (s, 3H, N-CH 3);
2987 (CH AR ),
j 3-OH tautomer form (24%): 11.69 (s, 1H, CO-NH ), 9.62 (s, 1H, C-OH), 8.12 (s,
1652
1H, CH=N ), 8.06 (s, 1H, CH=N ), 7.25 (t, 1H, Ar-H), 7.18 7.05 (m, 2H, Ar-
(-CO-NH-),
H), 6.84 (d, 1H, Ar-H), 5.11 (s, 1H, CH 2-CO), 3.46 (s, 3H, N-CH3), 3.20 (s,
1588 (-CH=N)
3H, N-CH 3)

The structure of the final compounds tives (e-j) were obtained in two tautomer
was confirmed by FT-IR and 1H-NMR forms, the second form being in the ratio of
spectroscopy (tab. II). Hydrazone deriva- 20-25%. The presence of the tautomer

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 Sandra Constantin et al.

forms was demonstrated using 1H-NMR-
temperature at 80 C.
Antioxidant assay
DPPH radical scavenging activity. The
evaluation of antioxidant activity was per-
formed by measurement of radical scaveng-
ing ability of the compounds against free
radicals such as 2,2-diphenyl-1-picryl-
hydrazyl (DPPH), superoxide anion radical,
hydroxyl radical or 2,2-azino-bis(3-ethyl-
benzothiazoline-6-sulphonic acid) cation
radical (ABTS +. ) (11). The DPPH assay is
the most used method to evaluate the anti-
oxidant activity. This method evaluates the
hydrogen atom donating activity using the
spectrophotometric measurement of the
radical concentration. DPPH is a purple-
colored organic nitrogen radical, which
changes its color to pale yellow upon a
reduction reaction with hydrogen donating
compounds (fig.2). The decrease in optical
density expresses a measure of antioxidant
activity (12) (tab. III, IV).
It can be observed that the most active
compound is compound g, which has as
substituent on aromatic ring the nitro group
in meta position. For this compound the
inhibition rate (%) ranged from 31% to
65% (at 30 min) and from 44% to 79% (at
60 min), depending on sample concentra-
tion (0.431 mg/mL, 0.862 mg/mL; 1.293
mg/mL; 1.724 mg/mL; 2.155 mg/mL;
2.586 mg/mL). The antioxidant activity of
compound g was 2-3 times higher than that
of theophylline, used as parent compound
(fig. 3, 4). These results support the favor-
able influence of chemical modulation on
radical scavenging ability. The other com-
pounds have an inhibition percentage al-
most equal to that of theophylline at small
concentrations (0.431 mg/mL, 0.862
mg/mL, 1.293 mg/mL), but at higher con-
centrations the antioxidant effect was more
pronounced.

Fig. 2. The reduction reaction of the DPPH.

TABLE III
DPPH radical scavenging activity (inhibition rate - %) of theophylline and its hydra-
zone derivatives at 30 min
Inhibition rate (%) at different concentrations
Comp. R
C1 C2 C3 C4 C5 C6
a - 19.850.17 22.050.43 23.420.89 21.660.87 22.550.15 23.050.14
e H 18.580.43 22.250.51 25.420.33 26.760.41 29.840.23 33.110.25
f 4-NO 2 19.210.22 21.170.76 23.760.81 25.420.65 26.570.37 27.030.35
g 3-NO 2 31.450.19 41.350.15 47.380.82 58.710.56 62.980.85 65.140.99
h 4-Cl 18.670.18 20.330.52 21.600.06 24.610.84 23.801.89 26.421.44
i 4-Br 18.060.44 19.880.52 21.150.19 22.180.46 23.070.20 24.020.07
j 3-OH 21.611.07 21.360.16 23.410.18 23.800.60 25.270.60 24.470.20
a: theophylline; C 1: 0.431 mg/mL; C 2: 0.862 mg/mL; C 3: 1.293 mg/mL; C 4: 1.724 mg/mL; C 5: 2.155 mg/mL;C 6: 2.586 mg/mL

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 Synthesis and evaluation of antioxidant activity of some hydrazones with xanthine structure

TABLE IV
DPPH radical scavenging activity (inhibition rate - %) of theophylline and its
hydrazone derivatives at 60 min
Inhibition rate (%) at different concentrations
Comp. R
C1 C2 C3 C4 C5 C6
a - 22.880.05 25.890.46 27.770.80 26.030.20 26.280.37 26.550.27
e H 19.490.07 23.690.63 27.411.25 30.230.27 31.860.41 34.270.26
f 4-NO 2 20.230.34 22.870.85 26.520.4 27.020.91 30.760.54 31.320.67
g 3-NO 2 43.940.27 57.200.71 63.930.91 72.420.83 75.830.66 79.260.81
h 4-Cl 20.700.80 23.910.48 25.721.02 27.131.69 29.350.42 31.050.70
i 4-Br 19.960.34 22.921.73 25.010.22 26.271.44 28.440.96 27.660.38
j 3-OH 21.670.46 22.120.39 25.080.24 27.330.79 26.290.49 25.850.22
a: theophylline; C 1: 0.431 mg/mL; C 2: 0.862 mg/mL; C 3: 1.293 mg/mL;C 4: 1.724 mg/mL; C 5: 2.155 mg/mL; C 6: 2.586 mg/mL

Fig. 3. The antiradical effect of theophyl- Fig. 4. The antiradical effect of compound
line at different concentrations g at different concentrations

CONCLUSIONS
In our studies, six hydrazones with xan-
thine structure have been synthesized start-
ing from theophylline, a substance known
for its beneficial effects, especially as
bronchodilator agent. The resulted com-
pounds were characterized in terms of their
physical properties (chemical formula,
molecular weight, yield, melting point,
solubility in different organic solvents,
etc.). Their structure was confirmed by FT-
IR and 1H-NMR spectroscopy. The antioxi-
dant activity of the synthesized compounds
was evaluated in vitro using the DPPH radi-
cal scavenging assay. Compound g (3-nitro-
benzylidene-hydrazino-acetyltheophylline)
was the most active compound, with the
highest inhibition rate compared with
theophylline.
ACKNOWLEDGMENTS
This paper was published under the
frame of European Social Found, Human
Resources Development Operationl Pro-
gramme 2007-2013, project no.
POSDRU/159/1.5/136893 entitled: Stra-
tegic partnership for increasing the quality
of scientific research in medical universi-
ties through doctoral and postdoctoral
scholarships DocMed.Net_2.0.

915
 Sandra Constantin et al.

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