Rev. Med. Chir. Soc. Med. Nat., Iaşi – 2013 – vol. 117, no. 1
PHARMACY
ORIGINAL PAPERS
NEW HYDRAZINES WITH SULPHONAMIDIC STRUCTURE:
SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL ACTIVITY
Oana Maria Parasca (Dragostin) 1, Florentina Lupascu 2,
Cornelia Vasile3, M. Mares 4, V. Nastasa4, Lenuta Profire 2*
University of Medicine and Pharmacy ’’Grigore T. Popa’’- Iasi
Faculty of Pharmacy
1. Ph.D. student
2. Discipline of Pharmaceutical Chemistry
3. Romanian Academy,“P. Poni” Institute of Macromolecular Chemistry
University of Agricultural Sciences and Veterinary Medicine of Iasi
4. Faculty of Veterinary Medicine
NEW HYDRAZINES WITH SULPHONAMIDIC STRUCTURE: SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL ACTIVITY (Abstract): Infections caused by bacterial
species are common in immunocompromised patients and carry significant treatment costs and
mortality. The emerging resistance of microorganisms to some synthetic antimicrobial agents
makes it necessary to continue the research for new antimicrobial drugs. Aim: To design new
sulphonamide compounds with potential antibacterial and antioxidant activity. Material and
methods: New N-hydrazino acetyl-sulphonamides were prepared by condensation of some sulphonamides with chloroacetyl chloride and amination of intermediate compounds with hydrazine hydrate. Results: The synthesized compounds were screened for their antibacterial activity against Gram positive (Klebsiellapneumoniae, Proteus vulgaris, Citrobacter freundii, Enterobacter cloacae, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis)
and Gram negative bacterial strains (Pseudomonas aeruginosa, Escherichia coli). Some of
them were found to have good antibacterial activity. The antioxidant activity of these co mpounds was also tested using different methods: total antioxidant capacity, reducing power, and
DPPH radical scavenging activity. Conclusions: Chemical modulations performed on sulphonamide structure have a good influence on the biological activity of the synthesized compounds,
especially on their antioxidant effects. Keywords: SULPHONAMIDES, HYDRAZINES, ANTIBACTERIAL ACTIVITY, ANTIOXIDANT ACTIVITY.
Sulphonamides and their different derivatives are extensively used in therapy
due to their antibacterial activity. They
interfere with the use of p-aminobenzoic
acid (PABA) in the biosynthesis of tetrahydrofolic acid, which is an essential growth
factor, vital to the bacterial metabolism (1).
The chemistry of sulphonamides has been
recently recognized in the preparation of
238
various valuable biologically active compounds used especially as antibacterial
agents (2). On the other hand the production of free radicals is relatively increased
during infections. Starting from these observations and continuing our research on
bioactive molecules, we synthesized some
novel hydrazine acetyl-sulphonamides in
view of studying their biological activities.
New hydrazines with sulfonamidic structure: synthesis, characterization and biological activity
Given their antibacterial and antioxidant
potential, these compounds could be used
in protecting biological systems from oxidative damage and bacterial infections.
MATERIAL AND METHODS
Sulphonamides, chloroacetyl chloride,
organic solvents (p. aquality), standard
reagents for antioxidant and antimicrobial
tests were purchased from Sigma Aldrich
Company. All solvents and reagents were
used without further purification.
Synthesis of sulphonamide derivatives.
Six sulphonamides (sulfamethoxydiazine,
sulfadiazine,
sulfamerazine,
sulfadimethoxine, sulfisoxazole and sulfamethoxazole) were subjected to reaction with
chloroacetyl chloride in dry acetone in the
presence of anhydrous K 2 CO 3 (Scheme 1).
The resulting N-chloroacetyl-sulphonamides have then reacted with hydrazine
hydrate in anhydrous ethanol. The reactions were carried out by adapting similar
methods used for derivatization of some
compounds with primary aromatic amine
groups (3, 4, 5, 6).
O H2
HN C C Cl
NH2
+
Cl CH2C
O
Cl
H
O2S N R
+
H2N NH2 . H2O
H
O2S N R
H
O2S N R
3a-f
2a-f
1a-f
OCH3
N
R=
N
OCH3
(a)
N
N
O H2 H
HN C C N NH2
(b)
N
N (c)
(e)
N
N
CH3
OCH3
N
N
O
O
H 3C
(d)
(f)
CH3
CH3
Scheme 1. Synthesis of new hydrazine with sulphonamide structure
General procedure for synthesis of Nhydrazinoacetyl-sulphonamides (3a-f):
A mixture of each of the compounds
2a-f (0.04 mole) and hydrazine hydrate
(99%, 0.12 mole) in ethanol (150 ml) was
refluxed for 10 hours. From the resulting
mixture excess ethanol was removed under
reduced pressure. The resulting solid residue was purified by recrystallization using
the proper solvent and separated by filtration.
Characterization by FTIR Spectroscopy.Fourier transform infrared (FTIR) spectrum was recorded on a Bomem MB-104
FT-IR spectrometer (Canada). All spectra
were obtained in the 4000 - 600 cm-1range
at a resolution of 4 cm -1 over 32 scans.
Spectroscopy data were processed with a
Grams/32 software (Galactic Industry Corporation).
Antioxidant assays
Determination of total antioxidant capacity. The antioxidant activity of tested
compounds was evaluated by the phosphomolybdenum method according to the
standard procedure (7) with minor modifications. Briefly 50 µl sample solution (5
mg/ml) in DMSO was mixed with the rea-
239
Oana Maria Parasca (Dragostin) et al.
gent solution (2 ml, 0.6 M sulphuric acid,
28 mM sodium phosphate and 4 mM ammonium molybdate). The samples were
incubated at 95°C for 90 minutes. After the
mixture had cooled to room temperature,
the absorbance of each solution was measured at 695 nm against a blank (DMSO
mixed with reagent solution). Ascorbic acid
(AA) in concentration matching that of the
samples was used as standard antioxidant.
Reducing power.The reducing power of
the compounds was quantified by the
method described in the literature (8) with
minor modifications. Briefly, 1 ml of reaction mixture, containing 5 mg of samples in
DMSO, was mixed with 0.2 M sodium
phosphate buffer (1 ml, pH=6.6). The reaction was initiated by addition of potassium
ferricyanide (1% w/v, 1 ml). After 20 min
of incubation at 50°C, the reaction was
terminated by trichloroacetic acid solution
(10% w/v, 1 ml). The mixture solution was
centrifuged at 4500 rpm for 15 min. 1 ml of
supernatant was diluted with 1 ml of deionised water, and 0.2 ml ferric chloride (0.1%
w/v) solution was added. After 5 min of
incubation, absorbance was measured at
700 nm against a blank (the mixture of
DMSO with the reagents). Higher absorbance indicates higher reducing power. Effective concentration (EC50: the concentration of a sample in which the absorbance is
0.5 and 50% of substrate is reduced) was
calculated.
DPPH radical scavenging activity. The
radical scavenging activity of tested compounds
against
1,1-diphenyl-2-picrylhydrazyl (DPPH) was measured as described by (9) with a few modifications. 50
µl of each sample solution (20 mg/ml in
DMSO) was added to 2950 µl of 0.1 nM
DPPH methanol solutions. The mixtures
were left at room temperature and in the
240
dark for 30 min, and then absorbance was
measured at 517 nm. Ascorbic acid (AA) in
concentration matching that of the samples
was used as standard.
DPPH methanol solution was used as
control sample. The radical scavenging
activity was calculated using the following
formula: % inhibition = [(control absorbance-sample absorbance) /control absorbance] x 100.
Antimicrobial assessment
The determination of minimum inhibitory concentrations (MICs) of samples was
performed by agar dilution method. Nine
reference bacterial strains (K. pneumoniae
CIP 53153, P. vulgaris CIP 104989, C.
freundii CIP 5732, E. cloacae CIP 103475,
S. aureus ATCC 6583, S. epidermidis
ATCC 12228, E. coli ATCC 25922, E.
faecalis ATCC 25912, P. aeruginosa CIP
82118) were included in this study which
was performed according to EUCAST
standard. The stock solutions were prepared by dissolving 200 mg of each compound in 19.5 ml of DMSO. All stock solutions were stored at 4 °C and used within
24 h. Then 20 ml (19 ml of molten agar to
1 ml of sample solution) were used in 9 cm
Petri dishes for agar dilution MICs. The
MIC value was defined as the lowest concentration exhibiting no visible bacterial
growth at 15°C for 44 to 48 hours and/or
68 to 72 hours.
RESULTS AND DISCUSSION
Chemistry
The hydrazines with sulfonamide structure are crystalline light yellow to light
brown powders very soluble in DMF and
DMSO. The characteristic IR spectral
bands of sulphonamid-hydrazines are
shown in table 1. The presence of these
bands confirms the chemical structure of
New hydrazines with sulfonamidic structure: synthesis, characterization and biological activity
the compounds.
Antioxidant asays
Determination of Total Antioxidant Capacity. The total antioxidant capacity assay
is based on the reduction of Mo +6 to Mo +5
by the tested compounds followed by formation of a green phosphate/ Mo +5 complex at acidic pH. The results of the synthesized compounds in comparison with their
parent sulphonamides are presented in fig.
1, 2. It was noticed that the most active
compound as compared to the parent sulphonamide was 3d while as compared to
ascorbic acid 3e.
changing from yellow to green depending
on the reducing power of each sample. The
derivatization of parent sulphonamides
with acetyl-hydrazine chain led to increased antioxidant capacity, all tested
compounds being more active than parent
sulphonamides (fig. 3, 4).
Fig. 3. Reducing power of (1a-f)
Fig. 1. Total antioxidant capacity of (1a-f)
Fig. 4.Reducing power of (3a-f)
Fig. 2. Total antioxidant capacity of (3a-f)
Reducing power. The synthesized compounds (3a-f) caused the reduction of the
Fe3+/ ferricyanide complex to the ferrous
form (Fe 2+), the colour of the test solution
DPPH Radical Scavenging Activity.The
results are presented in figs. 5 and 6. In all
six sulphonamides the percentage of inhibition of free radical DPPH is very low
(I%=0-36.53), while all six hydrazines
have an inhibition percentage (I%=51.25%97.45%) comparable with that of ascorbic
acid (I%=97.59).
Antimicrobial assessment.CMI values of
the tested compounds are presented in fig. 7,
8. According to the obtained results only N 4
hydrazin acetylamino-sulpha dime-thoxine
has a better antimicrobial activity as compared to its parent sulphadimethoxine.
241
Oana Maria Parasca (Dragostin) et al.
Fig. 5. Radical scavenging activity of
(1a-f)
Fig. 6. Radical scavenging activity of
(3a-f)
Fig. 7. MIC values of (1a-f).
Fig. 8.MIC values of (3a-f).
Comp.
no
3a
3b
3c
3d
3e
3f
242
TABLE I
FTIR characterization and physical measurements
of N- hydrazinoacetyl-sulphonamides (3a-f)
Infrared (cm-1)
Physical characterization
HN-NH2
3351
3353
3350
3320
3307
3350
CH2-NH
2853
2850
2850
2886
2843
2875
HN-CO
1686
1674
1684
1703
1670
1688
Molecular formula
C13H16O4N6S
C12H14O3N6S
C14H18O5N6S
C12H15O4N5S
C13H16O3N6S
C13H17O4N5S
m.p (°C)
181
165
102-105
220-222
155-160
135-140
Yield (%)
85,12
65,09
84,75
75,90
75,41
63,10
New hydrazines with sulfonamidic structure: synthesis, characterization and biological activity
CONCLUSIONS
Some new hydrazines with sulphonamide structure have been synthesized and
optimal conditions of reaction were established for obtaining compounds with high
yields and advanced purity. The synthesized compounds have been characterized
by their physical constants (melting point,
yield, molecular formula, molecular
weight, solubility in different organic solvents) and their chemical structure was
confirmed by FT-IR spectroscopy. Their
antibacterial and antioxidant activities have
been evaluated. All compounds have
marked antioxidant activity compared to
the parent sulphonamides and, for some of
them; the antioxidant activity is comparable to that of ascorbic acid at the same
concentration.
ACKNOWLEDGMENTS.
Oana-Maria Dragostin thanks AMPOSDRU for financial support of the research in the project “Doctoral Scholarships for increasing competitiveness in the
medical
and
pharmaceutical
field”
(POSDRU/88/ 1.5/S/58965).
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