SYNTHESIS, CHARACTERIZATION AND PHARMACOLOGICAL STUDY OF NEW SCHIFF BASE DERIVED FROM AMOXICILLIN DRUG

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Wasfi Abood Al-Masoudi et al. Int. Res. J. Pharm. 2015, 6 (6)
Page 386
INTERNATIONAL RESEARCH JOURNAL OF PHARMACY
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ISSN 2230 – 8407
Research Article
SYNTHESIS, CHARACTERIZATION AND PHARMACOLOGICAL STUDY OF
NEW SCHIFF BASE DERIVED FROM AMOXICILLIN DRUG
Wasfi Abood Al-Masoudi*, Hassan Toma Mohammad and Adel Amin Hama
Department of Physiology, Pharmacology and Chemistry, College of Veterinary Medicine, University of Basrah-Iraq
*Corresponding Author Email: almasoudi59@yahoo.com
Article Received on: 21/04/15 Revised on: 29/05/15 Approved for publication: 11/06/15
DOI: 10.7897/2230-8407.06680
ABSTRACT
Condensation of 4-thia-1-azabicyclo[3.2.0] heptane-2-carboxylic acid,6-[[amino- (4-hydroxy phenyl) acetyl]amino]-3,3-dimethyl-7-oxo,t rihydrate with 2-
hydroxy-1- naphthal dehyde yielded a novel Schiff ba se derivative of amoxicilli n in good yield. A new compound was characterized by elemental analysis,
IR, and 1H-NMR spectroscopy. T he synthe sized compound was screened for antibacterial activity against Staphylococcus aureus, Escherichia coli, Bacillus
cereus, Streptococcus, Klebsella, salmonella and Psedumonas spp and fungicidal activity against Aspergillus multi, Aspergillus niger, Candida albicans,
Candida trobicalis and Candida krusi. The toxicity of t he compound was also assayed via the determination of thei r LD50 value by using Dixon,s up and
down method (1980). Studied compound was found to have an LD50 of 492.8 mg / kg of body weight.
Keywords: Amoxicillin, 2-hydroxy-1- naphthaldehyde, Microbial activity, NMR spectroscopy, Acute toxicity.
INTRODUCTION
Antibiotics are chemical substances produced by various species of
microorganisms and other living systems that are capable in small
concentration inhibiting the growth of or killing bacteria and other
microorganisms.
The b-lactams antibiotics are an important type of vital antibiotics
used to treat infectious disease including tetracycle b-lactam atoms1.
NH
O
Figure 1: Structure of bbbb-Lactam
The b-lactam antibiotics inhibit bacteria, exhibiting activities that
differ in pattern and intensity2.
Amoxicillin is a p-hydroxy derivative of ampicillin. The chemical
name of amoxicillin3 is: 4-thia-1-azabicyclo [3.2.0] heptane-2-
carboxylic acid, 6-[[amino (4-hydroxy phenyl) acetyl]amino]-3,3-
dimethyl-7-oxo,trihydrate, (C16H19O5S).3H2O.
.3H2O
Figure 2: Structural formula of amoxicillin trihydrate
Amoxicillin is a bacteriolytic β-lactam antibiotic drug of class
penicillin, active against various gram positive and gram negative
bacteria4,5.
Presence of amide and amino group in structure of amoxicillin
suggest it a better starting compound for the synthesis of new Schiff
base. Compounds containing an azomethine group (imine) are a
class of important compounds in medicinal and pharmaceutical
field.
Some Schiff‐bases were exhibits antibiotic, antiviral and antitumor
agents because of their specific structure. The wide use of
antibiotics resulted in the serious medical problem of drugs
resistance and public health concern. The synthesis of new
derivatives of antibiotics has become an important task to cope with
drug resistance problems6.
Naz and Iqbal7 found that the Schiff base complexes derived from
amoxicillin having good antibacterial activity in good range when
comparison to control (Amoxicillin).
In view of the importance of such imines, we describe here the
synthesis, characterization, invitro anti microbial activity and acute
toxicity of novel Schiff base by reaction of amoxicillin trihydrate
drug with 2-hydroxy-1- naphthaldehyde.
MATERIALS AND METHODS
Physical measurements
Infrared spectra (IR) were recorded as KBr discs in the range of
4000-400 cm-1 using FT-IR spectrophotometer Shimadzu model IR.
Affinity-1 at the department of Chemistry, College of Education for
pure sciences, University of Basrah, Iraq. 1H NMR spectra were
measured on a Brucker at 600 MHz, with TMS as internal reference
at Konstanz University, Germany. Microanalysis for carbon,
hydrogen and nitrogen were carried out by a Perkin-Elmer 240B

Wasfi Abood Al-Masoudi et al. Int. Res. J. Pharm. 2015, 6 (6)
Page 387
Elemental Analyzer. Melting point was measured by a Philip Harris
melting point apparatus and uncorrected.
Antimicrobial activity
The invitro biological screening of the 4-[(E)-(3,4-
dihydroxybenzylidene)amino]-N-(1,3-thiazol-2-yl)
benzenesulfonamide was investigated against various bacterial
species like Staphylococcus aureus, Escherichia coli, Bacillus
cereus, Streptococcu , Klebsella, salmonella and Psedumonas sp
and fungicidal activity against Aspergillus multi, Aspergillus niger,
Candida albicans, Candida trobicalis and Candida krusi using the
disc-agar diffusion technique8. Muller Hinton agar was used as
culture media for antibacterial activity. The antifungal activities
were tested against fungus above by disk diffusion method.
Recommended concentration 50, 100 and 200 μg/ml of the test
samples in DMSO solvent was introduced in the respective method.
Antibiotic drugs Amoxicillin (10 mg) were used as control for
bacteria and fungi. Petri plates containing 20 ml of Mueller Hinton
Agar were used for all the bacteria tested. Aspergillus multi,
Aspergillus niger, Candida albicans, Candida trobicalis and
Candida krusi strains was cultivated in Sabouraud’s dextrose agar.
Sterile Whatman no.1 filter paper disks (6mm in diameter)
impregnated with the solution in DMSO of the test were placed on
the Petri plates. A paper disc impregnated with dimethylsulfoxide
(DMSO) was used as negative control. The plates were incubated
for 24 h in the case of bacteria and 72 h for fungi at 28 °C. The
inhibition zone diameters were measured in millimeters using a
caliper vernia.
Acute toxicity (LD50)
Animals. All experiments were performed on 10-14- weak old male
and female ratus-ratus/rats weighing 200-250 gm at the time of
treatment by using up-and-down method, Dixon9 1980. Male and
female rats were injected intraperitonially with different doses of the
Amoxicillin derivative after conducting series of test levels. With
equal spacing between doses, a series of trails were carried out using
this method: increased dose following a negative response and
decreased dose following a positive response. Testing continued
until chosen "nominal" sample size was reached. LD50 were
determined after reading final result (response-dead (X) or non
response alive (O) , then the following equation was applied LD50
= XF + Kd.
The estimate of LD50 is XF + Kd, where ( XF ) is the final test level
and ( K ) is the interval between dose levels. ( d ) is the tabulated
value (Table 1).
Table 1: Dixon values; Dixon (1980)
K represented serial tests started with :-
OOOO
OOO
OO
O
OXXX
0.154-
0.154-
0.154-
0.157-
XOOO
OXXO
0.860-
0.860-
0.861-
0.878-
XOOX
OXOX
0.741
0.741
0.747
0.701
XOXO
OXOO
0.182
0.181
0.169
0.084
XOXX
OOXX
0.381
0.380
0.372
0.305
XXOO
OOXO
0.142-
0.144-
0.169
0.305-
XXOX
OOOX
1.549-
1.544
1.500
1.288
XXXO
OOOO
1.000
0.985
0.0897
0.555
XXXX
XXXX
XXX
XX
X
K represented serial tests started with :-
LD50 = Xf+Kd
LD50 = Median Lethal Dose
xf = Last dose used in the experiment
k = Factor of change from the table
d = Difference between doses.
Synthesis and Characterization of Novel Schiff Base
A solution of Amoxicilline trihydrate (2.247 gm, 5 mmole) in
methanol (10 ml) was added to a solution of 2-Hydroxy-1-
naphthaledyne (0.870 gm, 5mmole) in methanol (10 ml). The
mixture was refluxed for (5 hours) with stirring. The resulting was
yellow solution allowed to cool and dried at room temperature, then
re-crystallization to the precipitate with ethanol, pale-yellow solid
was obtained by evaporation of ethanol during (24 hours) Scheme
(1).
Yield; 82%, M.P.= 137-1400C. FT-IR(KBr, cm-1), 310-3200(OH,
NH); 3068(C-H, aromatic); 2968,2933(C-
H,aliphatic);1850(C=OCarboxylic);1660(C=O,b-
Lactam);1635(C=N);1616(C=C);
1597, 1369 (COOH ).
1H NMR(DMSO-d6); δ 9.42(s, 1H, COOH ); 8.36(s,1H,CH=N);
8.10(s, 1H,NH amide); 7.10-
6.71(m,10H-Ar-H);5.57,5.68(s,2H-Ar-OH); 4.6(s,1H-CH-
N),4.7(s,1H,CH-COOH) , 3.34(1H,CH-
S), 1.50,1.24(s,6H-2CH3).
Anal. for C27H25N3O6S.3H2O(M.wt 573): Calc. C, 56.54; H, 5.41; N,
7.32; Found: C,56.58; H,
4.78 ; N, 7.31.

Wasfi Abood Al-Masoudi et al. Int. Res. J. Pharm. 2015, 6 (6)
Page 388
OH
NH2
O
NH
O
N
SCH3
CH3
OOH
+
O
H
OH
OH
N
O
NH
O
N
SCH3
CH3
OOH
CH
OH
.3H 2O
.3H 2O
Scheme 1: Synthesis of new Schiff base derived from amoxicillin drug
RESULTS AND DISCUSSION
Chemistry
Isolated yields, melting points, colors and spectral data IR and 1H
NMR of synthesized novel compound was reported. The present
work describes the synthesis of new Schiff base derived from
amoxicillin and aldehyde to produce bioactive Schiff base, thus, the
reaction of 4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylic acid, 6-
[[amino (4-hydroxy phenyl) acetyl]amino]-3,3-dimethyl-7-
oxo,trihydrate with 2-hydroxy naphthaldehyde in 1:1 mole ratio
gave the new organic compound in good yield. IR spectra for
synthesized compound displayed common features in certain
regions and characteristic bands in the fingerprint and other regions.
The IR spectra of new prepared compound show strong and broad
bands in the rang 3310-3200 cm-1 due to ν(O-H) and ν(N-H)
secondary amine stretching vibration and disappeared the band for
the ν (N–H) primary amine stretching vibration. The IR spectra of
synthesized compound displays band at 1635cm-1 is due to
azomethine group ν (-HC=N-) stretching vibrations. The band at
1850 cm-1 is due to ν (C=O) cm-1 stretching vibration for (COOH).
The band at 1660 cm-1 stretching vibration is due to ν (C=O) for β-
Lactam group overlapping with ν (-HC=N-) stretching vibrations.
The bands at 1597 cm-1, and 1369 cm-1 were assigned to stretching
vibration (COOH) asymmetric and symmetric stretching vibration,
respectively. The bands at 1616, 3068, 2968-2933 cm-1 were
assigned to ν (C=C), aromatic, ν (C–H) aromatic and ν (C–H)
aliphatic, respectively10.
The 1H NMR spectra of studied synthesized compound was
recorded in DMSOd6 solution and show all the expected protons
with proper intensity ratio. In 1H NMR spectrum of the synthesized
compound in DMSO-d6, single peaks attributed to methyl groups
appeared at 1.50 and 1.24 ppm (2CH3). The aromatic protons of
compound appeared within the range 7.10-6.71 ppm. It is worthy to
note that the protons of Ar-OH resonate as a singlet at 5.68 and
5.57 ppm. The proton of azomethine (CH=N) resonate as a singlet at
8.36 ppm, single peak attributed to hydroxyl group of carboxylic
appeared at 9.42 ppm. Three groups of double peaks given by (CO–
CH) and (N–CH) on the b-Lactam ring and (NH sec.) amide
appeared at 4.6, 4.7 and 8.10 ppm, respectively11. 1H NMR show
signal at 3.34 ppm attributed to (CH-S) group.
Pharmacological study
Median lethal dose (LD50)
Determination of the 50% of lethal dose (LD50) of the studied
compound in- vivo was detected in the rats by using the "up-and-
down" procedure described by (Dixon, 1980)9. In the experiment
we using 10 animals of white rats 10-14 weeks in age, Graded doses
of injection to each one animal, a series of concentrations (250, 300,
350, 400,450 and 500) mg/k.g b.w) in 0.1 ml (Dimethyl sulphoxide)
DMSO, were administered and chosen with equal spacing
(concentrations) between doses. Mortality was recorded after 24 hrs
that each one animal treated with one dose and after 24 hrs was
recorded as O if the animal lives and then increased the treated dose.
While X recorded for the death of animal and then decreased the
dose according for the result of the animal the code which formed as
being (OOXO) and according for Dixon value was get and the LD50
was determined according to the formula employed by Dixon
(1980).
LD50 =Xf + Kd
LD50 = 500 + (-0.144) x 50
LD50 = 492.8 mg / kg b.w
1/10 LD50= 49.28 mg / kg (1 kg = 6 rats) Depending on the weight
rat about 175 gram).
1/10 LD50= 8.213 mg /rat Depending on the weight rat 175 gram.
Microbial study
The results of the antibacterial activity are shown in Table 2. The
studied compound show no activity against Escherichia coli,
Streptococcus sp and Psedumonas sp but moderate active in
Bacillus cereus and Staphylococcus aureus at all concentrations.
The new compound show high activity against Salmonella sp and
Klebsella sp. The results of antifungal activity of the compound
show not active towards Candida trobicalis, Candida krusi and
Aspergillus niger but moderate active against Candida albicans and
Aspergillus multi at 200 μg/ml compared with amoxicillin, Table 3.

Wasfi Abood Al-Masoudi et al. Int. Res. J. Pharm. 2015, 6 (6)
Page 389
The bacteria and fungi were supplied from department of
Microbiology, College of Veterinary Medicine, University of
Basrah.
Table 2: Antibacterial activity of the Schiff-base derivatives of amoxicillin drug
Comp.
Conc.
µg/ml
E.coli
B.cerus
Strepto.
Staph.
Salmonella
Klebsella
Psedumonas
50 100 200
50 100 200
50 100 200
50 100 200
50 100 200
50 100 200
50 100 200
Amoxicillin
- - -
7 7 7
- - -
7 8 8
10 15 15
15 20 25
- - -
New
compound
- - -
8 8 8
- - -
8 9 9
15 15 15
10 10 12
- - -
Diameter of inhibition zone in mm for different microbial species
Table 3: Antifungal activity of the Schiff-base de rivatives of amoxicillin drug
Comp.
Conc.
µg/ml
C.trobicalis
C.krusii
A.multii
C.albicans
A.niger
50 100 200
50 100 200
50 100 200
50 100 200
50 100 200
Amoxicillin
-
- -
-
- -
- 10 12
10 15 18
-
- -
New compound
- - -
- - -
- - 8
7 8 8
- - -
Diameter of inhibition zone in mm for different microbial species
CONCLUSION
In conclusion the present study was, firstly, to synthesis of novel
derivative of amoxicillin Drug named 4-[(E)-(3,4-
dihydroxybenzylidene)amino]-N-(1,3-thiazol-
2yl)benzenesulfonamide. The molecular structure of new compound
was characterized by spectroscopic methods. The Synthesized
compound was investigated in vivo toxic effects and to find acute
toxic dose (LD50) which have moderate toxicity. And secondly, to
investigate in vitro antimicrobial activity, such as, antibacterial and
anti fungal activity against some bacterial and fungi in hope to
expansion their biological studies in future.
ACKNOWLEDGEMENTS
The authors are grateful to Prof. Dr. Najim Abood Al-Masoudi
(Konstanz University, Germany) for providing some elemental
analysis and NMR spectroscopy. We are also grateful to Department
of Physiology and Chemistry, College of Veterinary Medicine, Al-
Basrah University, Iraq for providing the facilities.
REFERENCES
1. Krik and othmer, "Encyclopedia of chemical technology", 4th
ed., John wiley & Sons Inc., Vol.3, p.1-250, 1992; New York.
2. Florey H.W., Chain E., Heatley N.G., Jennings M.A., Sanders
A.G., Abraham E.P., and Florey M.E., (Antibiotics), Vol. II,
Oxford University Press, 1949; London, New York, Toronto,
3. Newton G.G.F. and Abraham E.P. Isolation of cephalosporin C,
a penicillin-like antibiotic containing d-a-aminoadipic acid,
Biochem. J., 1956; 62, 651.
4. Anacona JR. Synthesis and antibacterial activity of some metal
complexes of Beta-Lactamic antibiotics. Journal of coordination
chemistry, 2001; 54(3-4). http://dx.doi.org/10.1080/0
0958970108022648
5. Sharma VK, Liu F, Tolan S, Sohn M, Kim H, Oturan MA.
Oxidation of β-lactam antibiotics by ferrate(IV). Chemical
Engineering Journal, 2013; 221: 446-451. http://dx.doi.org
/10.1016/j.cej.2013.02.024
6. Kaur S. P., Rao R. and Nanda S. Amoxicillin: A broad Spectrum
Antibiotic, International Journal of Pharmacy and
Pharmaceutical Sciences, 2011; 3 (3),14-19.
7. Naz N. and Iqbal M. Z. Synthesis, Spectroscopic and biological
studies of transition metal complexes of novel Schiff bases
derived from amoxicillin and sugars. J. Chem. Soc. Pak., 2009;
31, 440-446.
8. Wayne, (National Committee for Clinical Laboratory Standards,
NCCLS Approved standard M27- PA), 1997; USA.
9. Dixon, W. J. Efficient analysis of experimental observations,
Ann. Rev. Toxicol.,1980; Vol. 20, P:441 – 462.
http://dx.doi.org/10.1146/annurev.pa.20.040180.002301
10. Al-Noor T. H., Aziz M. R. and AL- Jeboori A.T. Synthesis,
characterization and antimicrobial activities of [Fe(II), Co(II),
Ni(II),Cu(II) and Zn(II)] mixed ligand complexes schiff base
derived from amoxicillin drug and 4-
(dimethylamino)benzaldehyde with nicotinamide J. of Chemical
and Pharmaceutical Research, 2014; 6(4):1225-1231.
11. Lever A.B.P., ( (Inorganic Electronic spectroscopy)), 2rd Ed
Elsevier,1984; New York.
Cite this article as:
Wasfi Abood Al-Masoudi, Hassan Toma Mohammad and Adel
Amin Hama. Synthesis, characterization and pharmacological study
of new Schiff base derived from amoxicillin drug. Int. Res. J.
Pharm. 2015;6(6):386-389 http://dx.doi.org/10.7897/2230-8407.
06680
Source of support: Nil, Conflict of interest: None Declared