Synthesis, characterization and biological activity of Schiff bases based on chitosan and arylpyrazole moiety

2. Materials and methods 
2.1. Materials 
Chitosan from crab shell was purchased from oxford laboratory (India). 
Its
degree of deacetylation 
(DD) is 94%, as determined by titration method [38] and the viscosity-
average molecular weight is 
160,000 Da
. Phenyl hydrazine, acetophenone and its 4-methyl, 4-methoxy, 4-chloro, and 4-nitro 
derivatives all were supplied from Merck. POCl
3
, DMF, Acetic acid, ethanol were obtained from 
Merck and used without purification.
2.2. Synthesis of intermediate 3-(4-substituted-phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde 
3-(4-Substituted-phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde was prepared by the reaction of 
acetophenone and its derivatives with phenyl hydrazine in presence of ethanol as a solvent and 
acetic acid as catalyst, then the resulted hydrazone derivative was treated with the Vilsmeier -Haack 
reagent (DMF–POCl
3
) leading to the corresponding 4-carboxaldehyde functionalized pyrazole ring 
as shown in Scheme 1[39, 40]. The crude products were crystallized twice with anhydrous ethanol. 
The structure of the obtained materials was confirmed by FT-IR and 
1
H NMR analyses
(supplemental data)
. 
Scheme1. Synthesis of 3-(4-substituted-phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde. 
 
 

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2.3. Synthesis of Chitosan Schiff Bases (ChBs) 
Chitosan Schiff bases were synthesized according to Scheme 2. Thus, chitosan (3 g) was dissolved 
in 150 
mL
of a mixed solution of ethanol and 1% acetic acid into three necked flask, and stirred at 
room temperature for 6 h till complete dissolution. The arylpyrazole aldehyde was then dissolved in 
ethanol and added gradually to the mixture. The reaction mixture was reacted at 70°C for 12 h 
under nitrogen with continuous stirring. After cooling, the solution was precipitated and washed 
several times in anhydrous ethanol to remove the unreacted arylpyrazole aldehyde. For further 
purification the traces of unreacted aldehydes were extracted in a soxhlet apparatus with ethanol for 
3 days. The final products were dried at 60
o
C in vacuum for 24 h.
The obtained chitosan Schiff bases are abbreviated here as Ch-H, Ch-CH
3
, Ch-OCH
3
, Ch-
Cl, and Ch-NO
2
as the given order from the following used arylpyrazole aldehydes : [3-phenyl-1-
phenyl-1H-pyrazole-4 carbaldehyde, 3-(4-methyl-phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde, 
3-(4-methoxy-phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde, 3-(4-chloro-phenyl)-1-phenyl-1H-
pyrazole-4-carbaldehyde, 3-(4-nitro-phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde].
The yield % of the prepared chitosan Schiff bases was calculated from the following equation [41]: 
Yield % 
x
x100 (1) 
where m
1
is the quantity of chitosan (g); m
2
is the quantity of Schiff base (g); M
1
is the molecular 
weight of chitosan unit (161 
g mol
-1
); and M
2
is the molecular weight of the corresponding Schiff 
base (
g mol
-1
). The yield % and the elemental analyses of the obtained different chitosan Schiff 
bases are represented in Table 1.
The degree of substitution (DS) of the Schiff bases was estimated from the elemental analyses and 
also reported in (Table 1) [42].
The solubility test of the chitosan Schiff bases was evaluated at a concentration of 5 
mg mL
-1
in 
different solvents including distilled water, DMSO, DMF, THF, acetone and aqueous acetic acid at 
25
o
C (Table 1). Due to low solubility of the products in most organic solvents and water the H- 
NMR data were difficult to evaluate. 

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