Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq.
281
SYNTHESIS AND PHYSIOLOGICAL STUDY OF SCHIFF BASE
DERIVED FROM SULFANILAMIDE AND VANILINE
Hanadi Abadul Gabar Al-Halfi, Wasfi Aboud Al-Masoudi* ,
Ahlam Ali Alrekabi
Department of Physiology, Pharmacology and Chemistry, College of Veterinary
Medicine, University of Basrah,Basrah,Iraq.
Corresponding Author: [email protected]
Keywords: Sulfonalamide, Schiff base,TWBC.
ABSTRACT
The design and production of innovative and safer drugs attracted to organic
chemists is urgently required in order to synthesize new compounds with the potential
of biological and chemotherapeutic activities. We're reporting here, the condensation
of 4-aminobenzene-1-sulfonamide (sulphanilamide drug) with 4-Hydroxy-3-methoxybenzaldehyde
(vanillin), yielded derivative of Schiff base in good yield. Elemental
analysis (CHN), IR, 1H and 13C-NMR spectroscopy were used to characterize the
synthesized compound. Using the Balb/c mouse model, the toxicity of the
synthesized compound was determined. The up and down method of Dixon was
found to have a body weight of 1677.2 mg / kg LD50 and mild toxicity. The results
showed the ability of the prepared compound to improve the TWBC and DWBC
values approach to control, giving HSV a less harmful effect than the sulfanilamide
drug.
INTRODUCTION
As the first drugs used against some bacterial infections, sulfa drugs are know
n as treatments of eye infections, measles, meningitis, and other meningitis such as
actinomycea, infections of the urinary tract as preventive and therapeutic compounds.
(1-3).
Schiff bases are a significant class of organic compounds with biological activ
ities and structural chemical significance, and because of promising antibacterial and
antiviral activities, as well as metal chelating effects and other pharmacological
effects, many Schiff base derivatives have been used in different physiological and
coordination chemistry fields (4-6).
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq.
282
Schiff bases was prepared via indole-3-carbox aldehyde condensation with a
number of sulfa medicinal products, including sulfanilamide, sulfapyridine,
sulfadiazine, sulfamethoxazole, sulfamethoxypyri dazine and sodium sulfacetamide.
(7)The goal of the present study is to synthesize and investigate the 4-aminobenzene-
1-sulfonamide (sulphanilamide drug) and 4-hydroxy-3-methoxy benzaldehyde
(vanillin) of the Schiff base and characterized by using physical and spectral
techniques, including elemental analysis, IR and NMR. Acute toxicity and
hematological Parameters of synthesized compound was performed.
Materials and methods
Instrumentation
The IR spectra were recorded at the Polymer Research Centre, University of Basrah,
Basrah, Iraq, in the range of 4000-200 cm-1 on a Pye-Unicam SP3-300 spectrometer
using KBr disks. The IR, 1H, and 13C-NMR spectra were measured at 600 MHz on
the Bruker, with TMS as internal reference at the University of Konstanz, Germany.
The melting point was determined at the College of Veterinary Medicine, University
of Basrah, Iraq, using a Philip Harris melting point unit.
Acute toxicity (LD50) study
The lethal dose (50%) of the synthesized compound was calculated in Balb/c mice
using the up and down method (8). After a series of studies, male and female mice
aged 4-6 weeks were injected intraperitoneally with different doses of the synthesized
compound. A series of tracks were carried out using this approach with equal spacing
between doses: increased dose following a negative response and decreased dose
following a positive response. Testing continued until the "nominal" sample size
chosen was achieved. After reading the final result (response-dead (X) or nonresponse
alive (O), LD50 was calculated according to the following equation:
LD50 = XF + Kd .
The LD50 approximation is XF + Kd where the final test level is XF and the
distance between the dose levels is K. The tabulated value is d.
Synthesis
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq.
283
Synthesis of 4-{(E)-[(4-hydroxy-3-methoxyphenyl)methylidene]amino}benzene-
1-sulfonamide.
4-aminobenzene-1-sulfonamide (sulphanilamide drug) (5.8 mmol, 1.0 g) in 25
methanol was added to hot ethanolic solution of 4-hydroxy-3-methoxy benzaldehyde
(Vanilline) (5.8 mmol, 0.883 g), glacial acetic acid (3 drops) were added and the
resultng solution was refluxed for 3 h and then lifted over-night. The solid product
obtained was purified by washed with ethanol and the final product was recrystallized
to create yellow crystals by using chloroform: methanol (8:2, v : v).
Yield: 83%.
M.p.: 183-185 °C.
FT-IR (KBr, , cm-1): 3484-3345 (NH), 3254 (OH), 3087 and 3013 (CH-Ar-H); 2965
(CH-Aliph.), 1670-1586 (C=C, C=N).
1H NMR (600 MHz, DMSO-6d, δ, ppm): 3.84 (s, 3H, O-CH3), 5.79 (d, 2H, NH2),
7.84-6.58 (m, 7H, Ar-H), 8.47 (s, 1H, CH=N), 9.77 (s, 1H, OH).
13C NMR (150 MHz, DMSO-6d, δ, ppm), 55.5 (1C, OCH3), 112.4-154.8 (12C, C-Ar),
162.0 (1C, CH=N).
Anal. calcd. for C14H14N2O4S: C, 54.90; H, 4.57; N, 9.15. Found: C, 54.37; H, 4.16;
N, 8.86%.
H2N S
O
NH2
O
+
H O
OH
O
CH3
Glacial HOAc N S
O
NH2
O
HO
O
CH3
Scheme1:Synthesis of 4-{(E)-[(4-hydroxy-3-
methoxyphenyl)methylidene]amino}benzene-1-sulfonamide
RESULTS AND DISCUSSION
Chemistry
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq.
284
Schiff base derived from sulfanilamide drug and vanillin was describes in this
work via reaction of 4-aminobenzene-1-sulfonamide with 4-Hydroxy-3-methoxybenzaldehyde
in 1:1 ratio to yield imine compound, Scheme 1. In certain regions and
characteristic bands in the fingerprint and other regions, IR spectra for the compound
showed typical characteristics. The IR spectrum confirms the existence of the
azomethine group (CH = N) extending about 1586 cm1 with a sharp area .
1H NMR spectrum of synthesized compound show single signal attributed to
azomethine proton (CH=N) at δ 8.47 ppm, 1H NMR spectrum of Schiff base show a
doublet at δ 5.79 ppm due to NH2 protons and singlet at 3.84 ppm due to methoxy
group OCH3. The region at δ 6.58-7.84 ppm due to aromatic protons. 1H NMR
spectra of synthesized compound show singlet at δ 9.77 ppm due to phenolic OH (8).
The 13C NMR spectrum of synthesized compound was measured in
DMSOd6. The spectra revealed the presence of –CH=N group around δ 162.0 ppm.
The signals at the range δ 110.5-154.8 ppm due to C-Ar. These spectra data sports the
structure of synthesized compound (9).
Figure 1: Infra-red of synthesized compound.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq.
285
Figure 2. 1H NMR of synthesized compound.
Figure 3. 13C NMR of synthesized compound.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq.
286
Physiological Study:
Determination of the 50% of lethal dose (LD50) of the synthesized Schiff base invivo
The LD50 of the synthesized compound was detected in mice using the "up-and
down" technique defined by Dixon (10), in the experiment using 10 white mice
animals 7-8 weeks of age. Graded doses of injection were administered to each
animal, a series of concentrations (250,300,350---1600 mg / kg.bw) in 0.1 mL of
dimethyl sulfoxide ( DMSO) were administered and selected with equal spacing
(concentrations).
Mortality was reported after 24 h that each animal received a single dose, and after
24 h that each animal received an O if the animal survived and then increased the dose
treated. While X reported the death of the animal and then reduced the dose according
to the outcome of the animal, the code established as being (OOOX) and Dixon value
was obtained and the LD50 was calculated accordingto the formula used by Dixon:
(LD50 = Xf + Kd; LD50 = 1600 + 1.544 × 50 = 1677.2 mg/kg.bw).
White blood cells study
Animals
30 local male rabbits aged 6 months and weighing 1000-1600 grams were used
and brought to Basra town from the local market. Animals were housed in regular
laboratory-controlled cages at temperatures of 25 ± 2 ° C and 12 hrs. Regular delivery
of light / dark cycle, food and water ad libitum. In the animal house where
experiments were carried out, one week after acclimatization.
Experimental design
Rabbits are divided into 5 groups (6 rabbits in each group) as follows:-
Group I (control): 6 male rabbits were administrated 0.5 ml dimethyl sulphoxide
(DMSO) intrapertoineal for three weeks daily.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq.
287
Group 2 (Treated 1) T1: 1/20 0f LD50 (195 mg / kg) of 0.5 ml dissolved
sulfanilamide drug (DMSO) intrapertoineal was administered to 6 male rabbits for
three weeks daily.
Group 3 (Treated 2) T2: 6 male rabbits got 1/20 0f LD50 (195 mg / kg) of 0.5ml
(DMSO) intrapertoineal dissolved sulfanilamide drug and 0.15 ml / kg of vitamin E
orally administered for three weeks daily.
Group 4 (Treated 3) T3: 1/20 0f LD50 (83.86 mg / kg) of synthesized compound
(HSV) dissolved by 0.5 ml (DMSO) intrapertoineal was given to 6 male rabbits for
three weeks daily.
Group 5 (Treated 4) T4: 1/20 0f LD50 (83.86 mg / kg) of (HSV) dissolved by 0.5 ml
(DMSO) intrapertoineal and 0.15ml / kg vitamin E were administered orally to 6 male
rabbits for three weeks daily.
At the end of study, samples of blood were collected from the heart, 2 ml of blood
was put into a tube containing the EDTA as an anticoagulant for white blood cells
examinations.
White blood cells analysis
The Auto Hematology Analyzer BC5300 was used to obtain total number of
white blood cells (WBC) and the differential count of white blood cells.
Statistical assay
In current experiment ,the data were expressed as mean ± SD and analyzed
by using One -way analysis of Variance (ANOVA), values of p ≤ 0.05 was
considered statistically significant.
RESULTS
The results explained in Table (1) a significant decreased in total, and differential
WBC count of the sulfanilamide group when compared to control group except with
lymphocyte percentage. Whereas, significantly increase of total and differential
WBC count of the sulfanilamide and vitamin E group as comparison to
sulfanilamide except no significant increase in basophile and lymphocyte percentage.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq.
288
The results in the same table showed significant increase of total, and differential
WBC count of (HSV) group when comparison to sulfanilamide and vitamin E group
with except no a significant increase with lymphocyte percentage. Also, there was
significantly increase of WBC count, and neutrophil percentage while no significant
increase in other types of WBC (basophil, eosinophil, monocyte, lymphocyte) with
(HSV) and vitamin E group when comparison to (HSV) group.
Table (1): Total WBC count and differential WBC count in treated and control
Groups.
Data are expressed as mean ± SD (n=6). The different letters refer to significant
difference (p≤ 0.05).
DISCUSSION
parameters
groups
TWBC
X103cell/
mm3
Neutrop
hil %
Basop
hil %
Eosinop
hil %
Monoc
yte %
Lymphoc
yte %
Control group 0.5
ml DMSO
6.49 a
±0.44
42.24 a
±2.24
0.67 a
±0.12
1.78 a
±0.30
2.59 a
±0.25
23.49 a
±2.18
Sulfanilamide
drug 195
mg/kg
4.04 e
±0.18
21.95
d
±1.52
0.08 c
±0.11
0.77 d
±0.31
0.52 d
±0.17
20.55 a
±1.20
Sulfanilamide
195mg/kgand0.15
ml/kg of vitamin
E
4.76 d
±0.46
25.76
c
±1.87
0.22 c
±0.12
1.17 c
±0.14
1.33 c
±0.35
21.06 a
±1.92
HSV
83.86mg/kg
5.49 c
±0.2 0
34.79 b
±1.87
0.42 b
±0.14
1.40 b
±0.07
1.74
b
±0.16
21.65 a
±2.28
HSV 83.86mg/kg
and 0.15ml/kg of
vitamin E
6.01 b
±0.10
40.22 a
±1.45
0.50 b
±0.10
1.45 b
±0.24
1.96 b
±0.22
22.06 a
±1.17
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq.
289
From our results, a significant decrease in total WBC and types of WBC count
with sulfanilamide group only no significant decrease in lymphocyte percentage, this
finding agreed with (11), who showed the drug induced a hypo-cellular bone
marrow like sulfonamide, at the same table observed significantly increased of the
total white blood cell, and some WBC types only no a significant increase of the
basophil and lymphocyte percentages with sulfanilamide group, and vitamin E, this
may be to the antioxidant effect of vitamin E which lead to protect blood cells from
damage.
This results agreed with (12) who found that vitamin E has protective effect
against oxidative damage in the cell membrane from reactive oxygen species attack.
These findings exhibit a significant increase of the total WBC and WBC types count
except in lymphocyte percentage in treated group of (HSV) due to the ameliorative
effect of HSV on WB, this results in agreement with (13) who showed that vanillin
has potential antioxidant activity from ascorbic acid which is interact with
radicals through a self-dimerization mechanism, the dimerization attributed to the
strong reaction stoichiometry of vanillin for ROS, also observed a significant
increase of the neutrophil percentage and total WBC with (HSV) and vitamin E
group from (HSV) treated group caused the Vitamin E has the protective effect on
the blood cells. this results which agreed with (14) demonstrated that the vitamin E
treatment induced decreased the adverse effects of cadmium on the blood indices.
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