Revue Roumaine de Chimie, 2009 , 54(2), 151156 [601956]

Revue Roumaine de Chimie, 2009 , 54(2), 151–156

SPECTROSCOPIC ANALYSIS AND ANTIMICROBIAL ACTIVITY
OF SOME 4-PHENYLAZO-PHENOXYACETIC ACIDS
Anca MOANȚĂa* and Stelian RADUb
aFaculty of Chemistry, University of Craiova, 107i Calea Bucures ti, 200478 Craiova, Roumania
bFaculty of Farmacy, University of Medicine and Farmacy of Craio va, 66 1 May Avenue, 200638 Craiova, Roumania
Received June 13, 2008
In this paper we present the results obtained in the study of t he structure and the antimicrobial activity of 4-phenylazo-
phenoxyacetic acids synthesised by the condensation of sodium a zobenzene-4-oxydes with monochloroacetic acid. The
structure elucidation of eleven 4-phenylazo-phenoxyacetic acids having general formula Ph=Ph-O-CH 2-COOH is
described. The results of this study have demonstrated the stru cture of all compounds using 1H NMR and mass spectra.
The most intense peaks have been u sed to characterise positive ion mass spectra. The losing of C 2H2O2 from the parent ion
is the dominating reaction. All th ese compounds were evaluated for antibacterial activity against Staphylococcus aureus ,
Streptococcus pyogenes , Escherichia coli , Pseudomonas aeruginosa , Proteus vulgaris by disk diffusion method. The
screening data releaved that th e title compounds carrying methy le group exhibited good antimicrobial activity.
INTRODUCTION
4-Phenylazo-phenoxyacetic acids are azo dyes
of large interest from the point of view of possible
applications in medicine because of their antimicrobial activity and in non-linear optics. We have recently reported
the synthesis and the physical characterization of some 4-phenylazo-
phenoxyacetic acids.1-3 In continuation of our
earlier studies, we report here the 1H NMR spectra,
the mass spectra and the antimicrobial activity of eleven acids (Table 1) having the general formula:

CH 2COOH O NN R5R1R2
R3R4

Table 1
4-Phenylazo-phenoxyacetic acids*
Compound Name R1 R2 R3 R4 R5
I
II
III
IV
V
VI
VII
VIII
IX
X
XI 2-Methyl-4-phenylazo -phenoxyacetic acid
3-Methyl-4-phenylazo -phenoxyacetic acid
4-(2-Methyl-phenylazo)-phenoxyacetic acid
2,3-Dimethyl-4-phenylazo-phenoxyacetic acid
2,6-Dimethyl-4-phenylazo-phenoxyacetic acid
2-Methyl-4-(4-methyl-phenylazo)-phenoxyacetic acid
4-(4-Chloro-phenylazo )-phenoxyacetic acid
2-Methyl-4-(4-chloro-phenylazo)-phenoxyacetic acid
3-Methyl-4-(4-chloro-phenylazo)-phenoxyacetic acid
2-Chloro-4-(4-chloro-pheny lazo)-phenoxyacetic acid
2,5-Dichloro-4-(4-chloro-phenylazo)-phenoxyacetic acid CH 3
H
H
CH 3
CH 3
CH 3
H
CH 3
H
Cl
H H
CH 3
H
CH 3
H
H
H
H
CH 3
H
Cl H
H
H
H
CH 3
H
H
H
H
H
Cl H
H
CH 3
H
H
H
H
H
H
H
H H
H
H
H
H
CH 3
Cl
Cl
Cl
Cl
Cl

* Corresponding author: [anonimizat]

152 Anca Moanță and Stelian Radu
The synthesis of organic compounds having
biological properties using monochloroacetic acid
have received considerable interest in recent years.
Treatment of 6-( p-chlorophenyl)-1,4,5,6-
tetrahydro-s-tetrazine-3(2H)-thione with
chloroacetic acid gives 3-( p-chlorophenyl)-2,3,6,7-
tetrahydro-4H-thiazolo(3,2-b)-s-tetrazin-6-one,
evaluated for their antibacterial and antifungal
activity.4 The condensation of 3-arylrhodanines
with chloroacetic acid and sodium methoxide
affords 3-arylrhodanino(4,5-b)furan-6-(5H)-ones.
All the compounds have been tested for their
fungicidal activity against Aspergillus niger a n d
Aspergillus flavus .5 The reaction of 9-methyl-
1,2,4,5-tetraazaspiro[5.5]undecane-3-thione with
chloroacetic acid results in the facile synhesis of
4-methyl-6'(7'H)-oxospiro(cyclohexane-1,3'(4H)-
(2H)-thiazolo(3,2-b)-s-tetrazine. The antibacterial
and antifungal activies have been determined.6
Several new 5-(4-oxothiazolidin-2-ylidene)-rhodanine were synthesized through the reaction of
5-thiocarbamoylrhodanines with monochloroacetic
acid. Some compounds showed promising
anticancer activity against particular human cell
lines used in the assay.
7 Thiazolo[2,3-
b]quinazolines were obtained in one pot synthesis
by treating octahydroquinazoline with
monochloroacetic acid and aromatic aldehydes.
Antifungal activity was shown for some of the
synthesized compounds.8 Treatment of 1',2',4',5',-
tetrahydrospiro(adamantane-2,3'-tetrazine)-6-
thione with monochloroacetic acid gives
spiro(adamantane-2,3'(4'H)-(2H)-thiazolo(3,2-b)- s-
tetrazin-6'(7')-one. Their antibacterial and
antifungal activities have been evaluated.9
EXPERIMENTAL
Reagents
4-Phenylazo-phenoxyacetic acids were obtained according
to the literature methods by co ndensation of monochloroacetic
acid with different sodium s alts of 4-phenylazo-phenols.1
Apparatus
The 1H NMR spectra were registered on Varian EM-360,
60MHz spectrometer, using CCl 4 as solvent and TMS as
internal standard.
Mass spectra were run with HPGC-MS 5890 Series II and
MSD 5971 Series. We are using an electron ionising energy of
70 eV at 250 șC (the s ource temperature). General procedure
The title compounds were screen ed for their antibacterial
activity using cup pl ate diffusion method.10 The bacterial
organisms used in the present in vestigation were isolated from
human being with characteristic infections and diseases.
All compounds were tested for antimicrobial activity
against 5 microorganisms: Staphylococcus aureus , Streptococcus
pyogenes , Pseudomonas aeruginosa, Proteus vulgaris and
Escherichia coli . Test compounds were dissolved in ethanol.
Concentrations 0.2% of the test compounds were obtained. Each well (diameter 6 mm) was loaded with 0.1 mL of test compound solution.
Tests of different isolates of microorganisms used were
carried out by pouring 15 mL s terile Mueller Hinton agar in
each Petri discs by 9 cm diameter. After solidification, the
plates were placed in an incubator at 37 șC for 30 minutes to remove excessive moisture.
Overnight broth culture was streaked evenly onto medium in
three directions using a wooden stick cotton swab. Excess suspension was removed from the swab by rotating it firmly against the side of the tube before seeding the plate surface u sing
sterile forceps. The plates wer e inoculated aerobically at 37
oC
within 15 minutes. Afte r 24 hours incubation, the diameters of the
inhibition zones were measured (including the 6 mm diameter of the disc) with a rule. The results were compared with [1-(ethoxycarbonyl)-pentadecyl] -trimethylammonium bromide
(Septonex), a comercial antiseptic agent.
RESULTS AND DISCUSSION
1H NMR spectra
Table 2 gives the chemical shifts, δ (ppm) in the
1H NMR spectra of all compounds.
The 4-phenylazo-phenoxyacetic acids exhibited
characteristic 1H NMR spectral data in agreement
with earlier reported data.11-14 The 1H NMR spectra
also showed expected signals.
In the 1H NMR spectra of all compounds the
presence of CH 2 group is confirmed by the
appearance of a signal at δ=4.5-5.5 ppm, an
expected singlet. The presence of a singlet at
δ=10.1-11.1 ppm may be assigned to COOH
protons. The aromatic protons from the two
substituted benzene rings came into resonance as a
multiplet at δ=6.9-8.7 ppm. Also, it is possible to
appear additional signals caused by the substituents. For instance, for 3-methyl-4-
phenylazo-phenoxyacetic acid can be observed on
additional singlet at δ=2.2 ppm, corresponding to
the methyl group protons.

Antimicrobial activity 153
Table 2
Spectral data
Compound Phenyl protons
(δ ppm) CH 2
(δ ppm) COOH
(δ ppm) CH 3
(δ ppm) Base peak
m/z (%)
I
II
III
IV
V
VI
VII
VIII
IX
X
XI 6.9-7.5
7.3-7.9
7.1-7.7
6.9-7.6
7.1-7.6
7.1-7.7
7.6-8.3
7.6-8.2
7.5-8.1
7.9-8.5 8.1-8.7 4.5
4.8
4.6
4.5
4.6
4.5
5.3
5.2
5.2
5.5 5.2 10.4
10.6
10.5
10.1
10.3
10.4
10.7
10.6
10.5
11.0 11.1 2.1
2.2
2.2
2.3
2.6
2.5
–
3.4
3.5
– – 77(100%)
77(100%)
91(100%)
77(100%)
77(100%)
91(100%)
111(100%)
111(100%)
111(100%)
111(100%) 111(100%)

Mass spectra
Further evidence for the 4-phenylazo-
phenoxyacetic acids structure was obtained from mass spectrum. For example, for 2-methyl-4-(4-
methyl-phenylazo)-phenoxyacetic acid, Scheme 1 shows the fragmentation process specific to the
phenoxyacetic acids by the cleavage of the bond between the C of the methylene group and C atoms o f t h e c a r b o x y l g r o u p f o l l o w e d b y C O
2
elimination.

-CO2
m/z284 (I=12.54%)
m/z240 (I=21.73%)NNCH3
OC H2COOH CH3
NNCH3
OC H3 CH3

Scheme 1

The mass spectrum of 2-methyl-4-(4-methyl-
phenylazo)-phenoxyacetic acid, for example,
showed the peak at m/z 226, consistent with molecular formula C 14H14N2O . T h e p e a k a t m / z ,
226 can be assigned due to the loss of C 2H2O2 from
the parent ion (Scheme 2).

-C2H2O2
m/z226 (I=10.03%)NNCH 3
OC H 2COOH H3C
NNCH 3
OH H3Cm/z284 (I=12.54%)

Scheme 2

154 Anca Moanță and Stelian Radu
The cleavage of the C-N bonds in this
fragmentation produced fragments at m/z 135, m/z
91 (base peak), m/z 119 and m/z 107, respectively (Scheme 3). The cleavage at the O-CH
2 bond and the lost N 2 give another fragment at m/z 197
(Scheme 4). In another fragmentation M+ lost N 2
and all radicals to produce diphenyl cation at m/z 151 (Scheme 5).

m/z226(I=10.03%)a b
+
ba
N N + + H3C
m/z107(I=15.13%)m/z91(I=100%)
N NCH 3
OH H3C H3C + N NCH 3
OH
CH 3
OH
m/z119(I=12.03%)b
OHCH 3
H3C N N+ +
a
OHCH 3
+ +N N H3C
m/z135(I=8.47%)

Scheme 3

m/z284 (I=12.54%)
m/z226 (I=10.03%)NNCH 3
OC H 2COOH H3C
NNCH 3
O H3C-CH 2COOH
-N2CH 3
H 3C O
m/z197 (I=4.29%)
Scheme 4

m/z 284 (I=12.54%)
m/z166
I=4.27%m/z209 (I=7.39%)NNCH 3
H3C +
-CH 3
m/z181 (I=5.48%)
-CH 3
-N2
m/z151
I=4.23%NNCH 3
OC H 2COOH H3C
O-CH 2-COOH
–
CH 3
H3C+

Scheme 5

The tropilium ion, m/z 91, obtained by skeletal
transposition, characteristic of the monoalkyl aromatic compounds, could be eliminate acetylene, and formed the fragment m/z 65 (cyclopentadienil-ium), which by itself losing an other acetylene
molecule generate the ion cyclopropenilium (m/z 39) (Scheme 6).

Antimicrobial activity 155
+ +
m/z 91
(I=100%)CH 2+
m/z 65
(I=8.45%)-C2H2
-C2H2
+
m/z 39
(I=7.49%)

Scheme 6

All fragmentation process can support the
structure formula assigned to the compounds and
are in agreement with the literature and with fragmentation of phenoxyacetic acid.
15
The fragmentation described in Schemes 1-6 for
2-methyl-4-(4-methyl-phenylazo)-phenoxyacetic
acid, are characteristic for all compounds
mentioned in Table 1.
Antimicrobial activity
The results of screening antimicrobial activity
are given in Table 3.
The 4-phenylazo-pheno xyacetic acids thus
described were subjected to antibacterial activity screening against two gram-positive bacteria
(Staphylococcus aureus and Streptococcus pyogenes )
and three gram-neg ative bacteria ( Pseudomonas
aeruginosa, Proteus vulgaris and Escherichia coli )
employing the disk diffu sion technique. Test
compounds were dissolved in ethanol.
Concentrations 0.2% of the test compounds were
obtained. Diameter of cup was 6 mm.
The results show that compounds I-VII and XI
present antimicrobial activity against most of the tested species. Compounds VIII-X are inactive
against all six microorganisms tested. The best
efficiency was exhibited by all compounds having methyl substituent.

Table 3
Antimicrobial activ ities of compounds
Diameter of zone of inhibition in mm Compound
Staphylococcus
aureus Streptococcus
pyogenes Escherichia
coli Pseudomonas
aeruginosa Proteus
vulgaris
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
Septonex 15
16 18
8
8
9
18
– – –
22
18 7
18 20 10
7
10
– – – –
20
17 18
7
21 12
9
7 9
– – – –
14 15
13 10 15
20
10 20
– – – –
19 20
15 20
8
20
12
– – – – –
16

CONCLUSIONS
The 1H-NMR and mass spectra confirm the
structure of all 4-phenylazo-phenoxyacetic acids. Six compounds present antimicrobi al activity against all
of the tested species. The investigations on the structure-activity relati onships confirmed the
importance of the nature of substituent in the
antimicrobial activity. The best efficiency was
exhibited by all compounds having methyl as substituent. REFERENCES
1. S. Radu, M. Băniceru, A. Jianu and G. Rău, Rev. Roum.
Chim. , 2001 , 46, 63-68.
2. S. Radu and G. Brătulescu, Rev. Chim. (Bucharest) , 1996 ,
47, 411-415.
3. A. Moanță and S. Radu, Rev. Chim. (Bucharest), 2008, 59,
708-711 .
4. J. Mohan and D. Kiran, Indian J. Chem. Sect. B , 1990 ,
29, 148-151.
5. N. Tiwari and B. Chaturvedi, Indian J. Chem. Sect. B ,
1989 , 28, 796-798.

156 Anca Moanță and Stelian Radu
6. J. Mohan and G. S. R. Anjaneyulu, Indian J. Chem. Sect.
B, 1987 , 26, 473-475.
7. H. T. Y. Fahmy and A. A. Bekhit, Pharmazie , 2002 , 57,
800-803.
8. S. M. Abdel-Gawad, M. S. A. El-Gaby and M. M. Ghorab,
Farmaco , 2000 , 55, 287-292.
9. J. Mohan and S. Kataria, Indian J. Chem. Sect. B , 1994 ,
33, 196-197. 10. F. Kavanagh, “Analytical Microbiology”, Academic
Press, New York, 1963, p.125-126.
11. A. H. Dabbagh and Y. Mausoori, Dyes Pigments , 2002 ,
54, 37-46.
12. E. Yildiz and H. Boztepe, Turk. J. Chem ., 2002 , 26, 897-903.
13. L. A. Fedorov, Russ. Chem. Rev. , 1988 , 57, 941-955.
14. J. H. Liu and H. Y. Wang, J. Appl. Polym. Sci. , 2004 , 91,
789-799.
15. G. Brătulescu, Rev. Roum. Chim. , 2000 , 45, 277-283.