Advanced technologies 4(1) (2015) 49-53

THE SYNTHESIS AND ANTIMICROBIAL ACTIVITY

OF ISATIN-3-(4’-HYDROXY)BENZOYLHYDRAZONE

Sandra S. Konstantinović1*, Jelena Tomić1, Jasmina Savić2, Milica Zlatković1,

Jasmina Jovanović Mirković3, Gordana V. Sredojević Dugalić4
(ORIGINAL SCIENTIFIC PAPER)
UDC 547.978
1Faculty of Technology, University of Niš, Leskovac, Serbia
2Vinca Institute of Nuclear Science, University of Belgrade, Belgrade, Serbia
3High Medical School of Professional Studies, Ćuprija, Serbia
4Clinic of Pulmonary Diseases and TBC, Knez Selo, Clinical Center of Niš, Serbia

Isatin is an endogenous compound and acts as a important precursor for the

synthesis of various pharmacologically active heterocyclic compounds. Isatin
and its derivatives possess a different activity such as antimicrobial, antivi-
ral, anti-inflammatory, anticonvulsive, antitumor, anti-HIV, etc. In the present Keywords: Isatin, synthesis, spectroscopic
work, isatin-3-(4`-hydroxy)benzoylhydrazone was synthesized and identified analysis, antimicrobial activity
by elemental microanalysis as well as FTIR, UV/VIS and MS methods. The
compound was tested for antimicrobial activity against Staphylococcus aureus,
Serratia marcescens, Pseudomonas aeruginosa, Klebsiella pneumoniae, En-
terococcus faecalis and Candida albicans.

Introduction

Isatin and itş derivatives as biological and pharma- further purification. They were purchased from Sigma Al-
ceutical active components are very important intermedi- drich (Munich, Germany).
ates in different organic synthesis [1]. Isatin is an endog-
enous compound, which can be found in plants of the Instruments
genus Isatis, in Calanthe discolor LINDL and Couroupita Carbon, hydrogen and nitrogen were determined by
guianensis Aubl [2]. It is a consistent component of the microanalysis using a Carlo Erba 1106 microanalyser.
secretion from the parotid gland of Bufo frogs and a me- The melting point was determined in open capillary tubes
tabolic derivative of adrenaline in humans [2]. in a Thomas-Hoover point apparatus and was uncorrect-
Isatin is a building unit of various numbers of alka- ed. The purity of the compound was obtained by TLC on
loids, drugs, pesticides and dyes [3]. Isatin and its deriv- silica gel using benzene:chloroform = 55:45, the com-
atives possess an antimicrobial, antiviral, anticonvulsive, pound being visualized by iodine vapors. The FTIR spec-
antitumor, anti-HIV, anti-inflammatory and antimycoba- trum was recorded with a MichaelsonBomen MB-series
cterial activity [4]. spectrophotometer, using the KBr pellet (1 mg/100 mg)
Hydrazones also possess antibacterial, anticancer, technique. The UV/VIS spectrum was recorded on a
tuberculostatic and fungicidal properties and have a Perkin-Elmer Lambda 15 UV/Vis spectrophotometer, us-
wide application in medicine and pharmacy [5]. ing 10-5 mol•dm-3 solutions in 95% ethanol. MALDI-TOF
The present paper describes the synthesis and iden- mass spectrum was acquired on a Voyager Biospec-
tification of isatin-3-(4`-hydroxy)benzoylhydrazone. The trometry DE Pro Workstation (Perseptive Biosystems,
synthesized compound was tested for antibacterial activ- Framingham, MA, USA). The instrument is equipped
ity against Staphylococcus aureus ATCC 6538, Entero- with a 20Hz N2 laser emitting at 337 nm. The spectrum
coccus faecalis ATCC 29212, Pseudomonas aeruginosa was acquired without a low mass gate and the spectrum
ATCC 43895, Klebsiella pneumoniae ATTC 13883, Se- represents an average of at least 400 single laser shots.
rratia marcescens ATCC 39006 and Candida albicans The spectrum was recorded from the methanolic solution
ATCC 10231 by the standard minimum inhibitory con- of 1µL, without a matrix.
centration (MIC) of a serial dilution test.
Methods
Experimental Synthesis of isatin-3-(4`-hydroxy)benzoylhydrazone
Isatin-3-(4`-hydroxy)benzoylhydrazone was prepared
Chemicals by the reaction of the equimolar amount of isatin (1.47g)
All chemicals were of reagent grade and used without and 4-hydroxybenzoylhydrazide (1.52g) in 95 % ethanol

*Author address: Sandra Konstantinović, Faculty of Technology, University of Niš,

Bulevar oslobođenja 124, 16000 Leskovac, Serbia
E-mail:[email protected]
The manuscript received: April, 21, 2015.
Paper accepted: May, 15, 2015.

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Advanced technologies 4(1) (2015) 49-53

following the standard procedure [6,7]. The mixture was pound (25 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml and
refluxed at 50 0C for 60 min. The product, precipitated 400 μg/ml) with Mueller Hinton broth for bacteria and
as a yellow solid, was collected by filtration and washed RPMI 1640 medium for the yeast was inoculated and
with using a solvent. incubated at 37 °C for 24 h (bacteria) and 48–72 h for
the yeast [6-9].
Antimicrobial activity
The compound was evaluated for its in vitro antimi- Results and discussion
crobial activity against Gram-positive bacteria Staphylo-
coccus aureus ATCC 25923 and Enterococcus faecalis Isatin-3-(4`-hydroxy)benzoylhydrazone was synthe-
ATCC 29212, Gram-negative bacteria Serratia marc- sized by using the carbonyl-amine condensation rea-
escens ATCC 39006, Pseudomonas aeruginosa ATCC ction of isatin and 4-hydroxybenzoylhydrazide (Figure
43895, Klebsiella pneumoniae ATTC 13883, and the 1). Although the primary amines are suitable nucleo-
yeast Candida albicans ATTC 10231. The minimum in- philic, the reaction required an acid catalyst to activate
hibitory concentration (MIC) was assessed by the agar a carbonyl group, speed reaction and to allow the water
dilution method. The minimum inhibitory concentration elimination. The C-3 position in isatin is very susceptible
(MIC) represents the concentration of antimicrobial at to the nucleophilic attack, while C-2 reacts with nucleo-
which there is a complete inhibition of the growth of or- philes only under specific conditions due to a negative
ganism. A series of different concentrations of the com- inductive effect of the amido group [2].

H O O
H
4 O H2N
2 2`
H 3a 3 C 1 3 4
N C 1`
N H 3a 3 N 3`
5 pH 4.5 5
2 + 4`
H 4 EtOH 2 H
H 6 O 6 6`
7
7a N1 OH -H2O H 6 7a N1 O OH
5 7 5`
H H H H
isatin 4-hydroxybenzoylhydrazide isatin-3-(4`-hydroxy)benzoylhydrazone

Figure 1.Synthesis of isatin-3-(4`-hydroxy)benzoylhydrazone using the carbonyl-amine condensa-

tion reaction

Table 1. Physico-chemical characteristics of isatin-3-(4`-hydroxy)benzoylhydrazone

a – Found (Calculated)

The yield of the synthesized compound is significant tion band for the azomethine (HC=N) group and also the
(94.30%). The experimental and calculated values for confirmation of the isatin derivative formation.
the elemental analysis are in a good accordance, which
confirms the suggested molecular formula C15H11N3O3of
isatin-3-(4`-hydroxy)benzoylhydrazone (Table 1).

UV/VIS spectral analysis of

isatin-3-(4`-hydroxy)benzoylhydrazone
The UV/VIS spectra of isatin, 4-hydroxybenzoyl-
hydrazide and isatin-3-(4`-hydroxy)benzoylhydrazone
were recorded in the solution of 95% ethanol (Figure 2).
The numerical data are given in Table 2. Comparing to
electronic spectra of started components, the spectrum
of isatin-3-(4`-hydroxy)benzoylhydrazone shows a new
band at λmax= 342 nm. This is a characteristic absorp-

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Advanced technologies 4(1) (2015) 49-53

Figure 2. UV/VIS spectra of isatin, 4-hydroxybenzoylhydrazide and isatin-3-(4`-

hydroxy)benzoylhydrazone

Table 2. UV/VIS spectral data of isatin, 4-hydroxybenzoylhydrazide and isatin-

3-(4`-hydroxy)benzoylhydrazone

FTIR spectral analysis of tains a peak near a molecular one which is of low inten-
isatin-3-(4`-hydroxy)benzoylhydrazone sity and belongs to M+1 ion. This is also a characteristic
The main IR spectral bands and their numerical data of the compounds with nitrogen atoms, i.e. isotopes u
are given in Figure 3 and Table 3. Comparing to spec- 14N and 15N. The spectrum contains high-intensity peaks
tra of isatin and 4-hydroxybenzoylhydrazide, the spec- at m/z = 149 [C8H5N3O]+ and m/z = 121 [C7H5NO]+ which
trum of isatin-3-(4`-hydroxy)benzoylhydrazone contains belong to fragmentation ions.
a band at 1610 cm-1, due to a newly formed ν(C=N)
stretching vibration. The spectrum of 4-hydroxybenzoyl-
hydrazide contains symmetrical and asymmetrical NH2 Antimicrobial activity
stretching bands at 3316 cm-1 and 3274 cm-1, which can- Experimental results of the antimicrobial activity are
not be found in the spectrum of isatin-3-(4`-hydroxy)ben- given in Table 4. MIC values show that isatin-3-(4`-hy-
zoylhydrazone. The position of other bands are slightly droxy)benzoylhydrazone possesses a different activity
batochromic shifted due to extended conjugation within against the tested microorganisms.
isatin-3-(4`-hydroxy)benzoylhydrazone The best results are shown against the Gram-positive
bacteria Enterococcus faecalis and yeast Candida al-
MS spectral analysis of bicans in the concentration range of 25-50 μg-cm-3. Isa-
isatin-3-(4`-hydroxy)benzoylhydrazone tin-3-(4`-hydroxy)benzoylhydrazone shows a medium
Mass spectrum of the synthesized compoundis given activity against Gram-negative bacteria Klebsiella pne-
in Figure 4. The spectrum of isatin-3-(4`-hydroxy)ben- umoniae, Serratia marcescens i Pseudomonas aerugi-
zoylhydrazone contains the characteristic molecular ion nosain the concentration range of 50-100 μg-cm-3, while
(M+) at m/z = 281. The position of this peak is in accord- the lowest activity is achieved against the Gram-positive
ance with the molecular mass of the compound with mo- bacteria Staphylococcus aureusin the concentration of
lecular formula C15H11O3N3. It also followed the „nitrogen <100 μg-cm-3.
rule“, by which the odd number of nitrogen means the
odd molecular mass of the molecule. The spectrum con-

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Advanced technologies 4(1) (2015) 49-53

Figure 3. FTIR spectrum of isatin, 4-hydroxybenzoylhydrazide and

isatin-3-(4`-hydroxy)benzoylhydrazone

Figure 4. MS spectrum of isatin-3-(4`-hydroxy)

benzoylhydrazone

Table 3. FTIR spectral data of isatin, 4-hydroxybenzoylhydrazide and

isatin-3-(4`-hydroxy)benzoylhydrazone

Table 4. MIC values ( μg-cm-3)of isatin-3-(4`-hydroxy)benzoylhydrazone

(+) active
(-) neactive

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Advanced technologies 4(1) (2015) 49-53

Conclusion [2] J.F.M. da Silva, S.J. Garden, A. C Pinto, The Chemistry

of Isatins: a Review from 1975 to 1999, Journal of the
The synthesis of isatin-3-(4`-hydroxy)benzoylhydra- Brazilian Chemical Society,12 (2001) 273-324.
zone was done by using the carbonyl-amine condensa- [3] N.Lashgari, G. M. Ziarani, Synthesis of heterocyclic
compounds based on isatin through 1, 3-dipolar
tion reaction of isatin and 4-hydroxybenzoylhydrazide
cycloaddition reactions, ARKIVOC i (2012) 277-320.
in 95% ethanol. The structure and molecular formula [4] G. Mathur, S. Nain, Recent Advancement in Synthesis
C15H11O3N3 of the synthesized compound was esta- of Isatin as Anticonvulsant Agents: A Review, Medicinal
blished by the elemental microanalysis as well as FTIR, Chemistry, 4 (2014) 417-427.
UV/VIS and MS methods. The compound shows a di- [5] S. Konstantinović, B. Radovanović, Ž. Cakić, V. Vasić,
fferent antimicrobial activity against Staphylococcus au- Synthesis and characterization of Co(II), Ni(II), Cu(II)
reus, Serratia marcescens, Pseudomonas aeruginosa, and Zn(II) complexes with 3-salicylidenehydrazono-
Klebsiella pneumoniae, Enterococcus faecalis and Can- 2-indolinone, Journal of Serbian Chemical Society,
dida albicans.The best results were shown against the 68(2003)641–647.
[6] S. Konstantinović, B. Konstantinović, J.
Gram-positive bacteria Enterococcus faecalis and yeast
Jovanović,Synthesis and structure of vanillin azomethines,
Candida albicans in the concentration range of 25-50 μg-cm-3. Chemical Industry & Chemical Engineering Quarterly, 15
(2009) 279−281.
Aknowledgments [7] S. Konstantinović, A. Kapor, B. Radovanović, A. Deak,
Synthesis, X-ray and antimicrobial activity of isatin-
This work has been funded by the Ministry of Edu- 3-phenylhydrazone, Chemical Industry & Chemical
cation, Science and Technological Development of the Engineering Quarterly, 14 (2008) 27−34.
Republic of Serbia (Project III 45001). [8] NCCLS Approval Standard Document M7-A5, National
Committee for Clinical Laboratory Standards, Villanova,
Pa, USA, 2000
References
[9] Reference method for broth dilution antifungal susceptibility
testing for yeasts; approved standard Document M27-A,
[1] P. Ramu, T.A.A.Prasad, S.M., Vithiyac, S. A.Antony, National Committee for Clinical Laboratory Standards,
Synthesis, characterization and biological activity of novel Villanova,PA, USA, 17(1997) 1–29.
spiroheterocycles from isatin derivatives, Der Pharma
Chemica, 6(2014) 30-36.

Izvod

SINTEZA I ANTIMIKROBNA AKTIVNOST

IZATIN-3-(4’-HIDROKSI)BENZOILHIDRAZONA
Sandra S. Konstantinović1*, Jelena Tomić1, Jasmina Savić2, Milica Zlatković1,
Jasmina Jovanović Mirković3, Gordana V. Sredojević Dugalić4
(ORIGINALNI NAUČNI RAD)
UDK 547.978
1Tehnološki fakultet, Univerzitet u Nišu, Leskovac, Srbija
2 INN Vinča, Univerzitet u Beogradu, Beograd, Srbija
3Visoka medicinska škola strukovnih studija, Ćuprija, Srbija
4Klinika za plućne bolesti, Knez Selo, Klinički centar Niš, Srbija

Izatin je endogeno heterociklično jedinjenje koje predstavlja veoma važan prekur-

sor u sintezi farmaceutski aktivnih komponenti. Izatin i njegovi derivati poseduju
različitu biološku aktivnost, kao što je antimikrobna, antiviralna, antiinflamatorna,
antikonvulzivna, antitumorna, anti-HIV, itd. U ovom radu je izvršena sinteza izatin-
3-(4`-hidroksi)benzoilhidrazona i jedinjenje je identifikovano pomoću elementa- Ključne riječi: Izatin, sineza, spektroskopska
analiza, antimikrobna aktivnost
rne mikroanalize, FTIR, UV/VIS i MS metode. Sintetisanom derivatu izatina je
određena antimikrobna aktivnost dilucionom metodom, odnosno određivanjem
MIC vrednosti u odnosu na mikroorganizme Staphylococcus aureus, Serratia
marcescens, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterococcus
faecalis i Candida albicans. Najbolji rezultati su postignuti u odnosu na Gram-
pozitivnu bakteriju Enterococcus faecalis i kvasac Candida albicans u koncen-
traciji 25-50 μg-cm-3.

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