Synthesis and microbiological activity of some N-(o-hydroxyphenyl)benzamides and phenylacetamides as the possible metabolites of antimicrobial active benzoxazoles: Part II

Article abstract of DOI:10.1016/S0014-827X(00)00070-7

The synthesis of some N-(o-hydroxyphenyl)benzamides and benzacetamides (2a-2p) in order to determine their in vitro antimicrobial activity against two Gram-positive bacteria, three Gram-negative bacteria and the fungus Candida albicans is described. The new compounds were compared with several control drugs. The derivative 2g, 4-amino-N-(o-hydroxyphenyl)benzamide, was found active at an MIC value of 25 μg/ml against the Gram-negative microorganism Klebsiella pneumoniae. Most of the compounds exhibited antibacterial activity at an MIC value of 25 μg/ml against Pseudomonas aureginosa. For the antifungal activity against C. albicans, compounds 2e, 2h and 2m were found more active than the other derivatives (MIC 12.5 μg/ml). The antimicrobial activity of some of these benzamide and phenylacetamide derivatives (2a, 2b, 2f, 2g, 2h and 2k), possible metabolites of benzoxazoles, was also compared with that of the cyclic analogues 3-8. Compound 2f possesses two dilutions better antifungal activity than its cyclic analogue the benzoxazole derivative 5 against C. albicans, while having one dilution better antibacterial activity against Streptococcus faecalis and K. pneumoniae. (C) 2000 Elsevier Science S.A.

Full text of DOI:10.1016/S0014-827X(00)00070-7

www.elsevier.nl/locate/farmac
Il Farmaco 55 (2000) 469–476
Synthesis and microbiological activity of some
N-(o-hydroxyphenyl)benzamides and phenylacetamides as the
possible metabolites of antimicrobial active benzoxazoles: part II
a
a
a,
a
:
8
8
Esin Akı S¸ener , Kamuran K. Bingo¨l , Ilkay Oren *, Ozlem Temiz Arpacı ,
a
b
:
Ismaıl Yalc¸ın , Nurten Altanlar
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara Uni6ersity, 06100 Tandog˘an, Ankara, Turkey
b Department of Microbiology, Faculty of Pharmacy, Ankara Uni6ersity, 06100 Tandog˘an, Ankara, Turkey
Received 20 December 1999; accepted 12 June 2000
Abstract
The synthesis of some N-(o-hydroxyphenyl)benzamides and benzacetamides (2a–2p) in order to determine their in vitro
antimicrobial activity against two Gram-positive bacteria, three Gram-negative bacteria and the fungus Candida albicans is
described. The new compounds were compared with several control drugs. The derivative 2g, 4-amino-N-(o-hydroxy-
phenyl)benzamide, was found active at an MIC value of 25 mg/ml against the Gram-negative microorganism Klebsiella
pneumoniae. Most of the compounds exhibited antibacterial activity at an MIC value of 25 mg/ml against Pseudomonas aureginosa.
For the antifungal activity against C. albicans, compounds 2e, 2h and 2m were found more active than the other derivatives (MIC
12.5 mg/ml). The antimicrobial activity of some of these benzamide and phenylacetamide derivatives (2a, 2b, 2f, 2g, 2h and 2k),
possible metabolites of benzoxazoles, was also compared with that of the cyclic analogues 3–8. Compound 2f possesses two
dilutions better antifungal activity than its cyclic analogue the benzoxazole derivative 5 against C. albicans, while having one
dilution better antibacterial activity against Streptococcus faecalis and K. pneumoniae. © 2000 Elsevier Science S.A. All rights
reserved.
Keywords: N-(2,5-Disubstituted)benzamides; Phenylacetamides; Antimicrobial activity; Benzoxazole metabolites
A review of the literature revealed that Phase I
metabolism pathways of benzoxazole and 2-methylben-
zoxazole in the rabbit involved cleavage of the oxazole
ring at the (C–O) linkage on the fused hetero-
cyclic system by mild hydrolysis and produced
o-formamidophenol and o-acetamidophenol respec-
tively [9], as shown in Scheme 1, omitting the intermedi-
ate stages.
1. Introduction
In the last few years we reported the synthesis and
antimicrobial activity of various 2,5-and/or 6-substi-
tuted benzoxazoles of general structure shown below,
possessing significant in vitro antibacterial activity espe-
cially against some enteric Gram-negative rods such as
Klebsiella pneumoniae, Pseudomonas aeruginosa and the
yeast Candida albicans [1–8].
Benzamide derivatives show various types of biologi-
cal properties such as antihelmintic, antihistaminic, an-
tifungal and antibacterial activities [10–13]. Oxyclo-
* Corresponding author. Tel.: +90-312-212 6805; fax: +90-312-
213 1081.
:
8
E-mail address: oren@pharmacy.ankara.edu.tr (I. Oren).
0014-827X/00/$ - see front matter © 2000 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(00)00070-7

470
E.A. S¸ener et al. / Il Farmaco 55 (2000) 469–476
between 25 and 100 mg/ml. In general, antimicrobial
activity of benzoxazole derivatives has been found to be
better than that of their corresponding acetamides, but
some acetamide derivatives possessed either the same or
improved potency with respect to their cyclic analogues.
Scheme 1.
zanide, which has a benzamide structure, was discov-
ered in 1969 as an antihelmintic agent effective against
Fasciola hepatica for the treatment of liver fluke infec-
tion [10]. However, there are few published data on the
antibacterial and antifungal activity of the benzamide
derivatives.
In this study, we report the synthesis and antimicro-
bial activity of several N-(o-hydroxyphenyl)benzamides
and phenylacetamides (2a–2p) and their activity was
compared to that of the cyclic analogues, benzoxazoles
(3–8), assuming that the acetamides would be the pos-
sible metabolites of these heterocyclic compounds.
We recently reported some novel active N-(2-hy-
droxyl-5-substituted phenyl)benzacetamide, phenoxyac-
etamide and thiophenoxyacetamide derivatives with the
general structure shown below as the possible metabo-
lites of antimicrobial active benzoxazoles [14]. Accord-
ing to our previous study, synthesized compounds
showed significant antimicrobial effects at MIC values
2. Chemistry
The synthesis of the compounds 2a–2f, 2h–2k, 2m–
2p was performed by reacting suitable 2-aminophenols
with appropriate carboxylic acid chlorides, obtained in
turn by treating carboxylic acids with thionyl chloride
(Scheme 2). Additionally, the synthesis of the com-
Scheme 2.

E.A. S¸ener et al. / Il Farmaco 55 (2000) 469–476
471
pounds 2g and 2l was accomplished by reduction of
compounds 2d and 2k respectively (Scheme 3).
3.2. Procedure for N-(o-hydroxyphenyl)benzamides and
phenylacetamides (2a–2p)
Compounds 2a–2p are new products except 2d–2f
and 2p which have been already described but were
obtained by a different synthetic route [15–17]. Melting
points of the compounds 2d, 2e and 2p were found to
be 204–206°C (lit. m.p.: 224°C), 152–154°C (lit. m.p.:
163°C), [15] and 224–226°C (lit. m.p.: 232–234°C) [16]
respectively. Elemental analyses and IR spectral data of
2d and 2e were in accordance with the literature [15].
However, the spectral data for the compound 2p and
the physical and spectral data for 2f synthesized as an
intermediate substance were not reported in the litera-
ture [17]. Therefore, the physical and spectral data of
all the synthesized compounds are given in Table 1;
Appropriate carboxylic acid (0.5 mmol) and thionyl
chloride (1.5 ml) were refluxed in benzene (5 ml) at
80°C for 3 h; excess thionyl chloride was then removed
in vacuo. The residue was dissolved in ether (10 ml) and
the solution added during 1 h to a stirred, ice-cooled
mixture of suitable o-aminophenol (0.5 mmol), sodium
bicarbonate (0.5 mmol), ether (10 ml) and water (10
ml). The mixture was stirred overnight at room temper-
ature and filtered. The precipitate was washed with
water, 2 N HCl, again with water and finally with ether
to give 2a–2p except for 2g and 2l. The product was
recrystallized from methanol for 2a–2c, 2e–2f, 2m–2o,
methanol–acetone for 2d, 2i, 2j, 2k, ethanol for 2h, and
methanol–water for 2p and dried in vacuo. Com-
pounds 2g and 2l were synthesized from 2d and 2k
respectively, which (5 mmol) were treated with
NiCl2·6H2O (15 mmol) and Zn (40 mmol) in methanol
(25 ml) refluxing the mixture at 60°C for 4 h. The
precipitate was filtered and the product was recrystal-
lized from methanol.
1
their IR and H NMR spectra are in agreement with
the proposed structures.
3. Experimental
3.1. Chemistry
Kieselgel HF254 chromatoplates (0.3 mm) were used
for TLC and the solvent systems were chloro-
form:methanol (20:1) for 2a, 2b, 2c, 2d, 2e, 2g, 2m, 2n,
chloroform:methanol (20:0.4) for 2f, 2o, chloroform:n-
hexane (20:0.3) for 2h, 2i, 2j, 2l, 2p, and chloro-
form:methanol (20:0.6) for 2k. All the melting points
were taken on a Buchi SMP 20 capillary apparatus and
are uncorrected. IR spectra were recorded by Perkin
Elmer 1330 and Pye Unicam SP-1025 with KBr
3.3. Microbiology
For both antibacterial and antimycotic assays, com-
pounds 2a–2p and 3–8 were dissolved in absolute
ethanol (0.8 mg/ml) [18]. Further dilutions of the com-
pounds and standard drugs in the test medium have
concentrations of 400, 200, 100, 50, 25, 12.5, 6.25, 3.12,
1.56, 0.78 mg/ml. The minimum inhibitory concentra-
tions (MIC) were determined using the method of
two-fold serial dilution [18,19]. In order to ensure that
the solvent ‘per se’ had no effect on bacterial growth, a
control test was also performed containing inoculated
broth supplemented with only ethanol at the same
dilutions used in our experiments and found inactive in
culture medium.
1
discs. H NMR spectra were obtained with a Bruker
NMR type AC-80 MHz spectrometer in d6-dimethylsul-
foxide and TMS was used as an internal standard.
Elemental analyses were carried out with a Hewlett
Packard 185 CHN analyser. The results of the elemen-
tal analyses (C, H, N) were within 90.4% of the
calculated amounts.
Scheme 3.

472
E.A. S¸ener et al. / Il Farmaco 55 (2000) 469–476
Table 1
Physical properties and spectral data of the compounds

E.A. S¸ener et al. / Il Farmaco 55 (2000) 469–476
473
Table 1 (Continued)
All the compounds were tested for their in vitro
growth inhibitory activity against different bacteria and
the fungus Candida albicans RSKK 628. The origins of
bacterial strains were Staphylococcus aureus RSSK 250,
Streptococcus faecalis RSSK 500 as Gram-positive and
Escherichia coli RSSK 313, Klebsiella pneumoniae
RSSK 256, and Pseudomonas aeruginosa RSKK 356 as
Gram-negative bacteria. RSKK strains of the microor-
ganisms used in this study were obtained from the
culture collection of Refik Saydam Health Institution of
Health Ministry, Ankara and maintained at the Micro-
biology Department of the Faculty of Pharmacy of
Ankara University.
Ampicillin, amoxicillin, erythromycine, chloram-
phenicol, streptomycin, tetracycline, oxiconazole, and
haloprogin were used as control drugs. The data on the
antimicrobial activity of the compounds and the con-
trol drugs are given in Table 2.
3.3.1. Antibacterial assay
The cultures were obtained in Mueller–Hinton broth
(Difco) for all the bacteria after 24 h of incubation at
3791°C. Testing was carried out in Mueller–Hinton
broth at pH 7.4 and the two-fold serial dilution tech-
nique was applied. The final inoculum size was 105
CFU/ml. A set of tubes containing only inoculated
Table 2
The in vitro antimicrobial activity of the compounds 2a–2p and standard drugs (MIC in mg/ml)
a
Comp.
Microorganism
Gram-positive
S.a.
Gram-negative
Fungus
S.f.
50
E.c.
K.p.
P.a.
C.a.
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
50
50
50
50
25
25
50
50
25
25
25
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
25
50
50
50
50
50
50
50
50
50
50
50
50
50
25
25
25
50
25
25
25
25
25
25
25
50
25
25
25
25
12.5
25
25
12.5
25
25
25
25
12.5
25
25
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
25
Ampicillin
Amoxicillin
Erythromycine
Chloramphenicol
Streptomycin
Tetracycline
Oxiconazole
Haloprogin
0.39
0.39
25
12.5
3.12
0.78
0.39
1.56
1.56
50
25
1.56
3.12
12.5
12.5
50
12.5
1.56
3.12
\400
\400
25
25
100
50
0.39
1.56
6.25
100
0.78
6.25
6.25
a Abbreviations: S.a., S. aureus; S.f., S. faecalis; E.c., E. coli; K.p., K. pneumoniae; P.a., P. aeruginosa; C.a., C. albicans.

474
E.A. S¸ener et al. / Il Farmaco 55 (2000) 469–476
Table 3
Comparison of the antimicrobial activity of selected amides 2a, 2b, 2f, 2g, 2h, 2k with the cyclic analogues 3-8 (MIC in mg/ml)
Comp. No:
Synthesized amides and their cyclic analogues
Microorganismsa
Gram-positive
Gram-negative
Fungus
S.a.
S.f.
E.c.
K.p.
P.a.
C.a.
2a
50
50
50
50
50
25
3b
2b
50
50
50
50
50
50
25
50
50
50
25
25
4b
2f
50
25
50
50
50
50
25
50
25
25
25
25
5c
12.5
100
50
50
50
100
25
12.5
100
25
2g
50
25
6c
12.5
50
100
50
25
50
12.5
12.5
50
25
2h
50
12.5
7d
2k
8c
50
50
25
50
50
25
50
50
50
25
25
25
50
25
12.5
12.5
12.5
12.5
a Abbreviations: S.a.: Staphylococcus aureus, S.f.: Streptococcus faecalis, E.c. Escherichia coli, K.p.: Klebsiella pneumoniae, P.a.: Pseudomonas
aeruginosa, C.a.: Candida albicans.
b See reference 2.
c See reference 1.
d See reference 7.
broth was kept as controls. After incubation for 24 h at
3791°C, the last tube with no growth of microorgan-
ism was recorded to represent MIC expressed in mg/ml.
3.3.2. Antifungal assay
The yeast Candida albicans was maintained in
Sabouraud dextrose broth (Difco) after incubation for

E.A. S¸ener et al. / Il Farmaco 55 (2000) 469–476
475
24 h at 2591°C. Testing was performed in Sabouraud
dextrose broth at pH 7.4 and the two-fold serial dilution
technique was applied. The final inoculum size was 104
CFU/ml. A set of tubes containing only inoculated
broth was kept as controls. After incubation for 48 h at
2591°C, the last tube with no growth of yeast was
recorded to represent MIC expressed in mg/ml.
2a, 2b, 2f, 2g, 2h and 2k with their heterocyclic ana-
logues 3–8 [1,2,7], assuming that they are the possible
metabolites of benzoxazoles as given in Table 3.
Table 3 reveals that although most of the benzoxazole
derivatives (3–8) show better antimicrobial activity than
the corresponding amides, some amide derivatives pos-
sessed either the same or one-fold improved potency.
This is the case of compound 2f which showed one dilu-
tion better antibacterial activity than the compared ben-
zoxazole derivative 5 against S. faecalis and K.
pneumoniae. Additionally, compounds 2a and 2b
showed the same activity as benzoxazole derivatives 3, 4
against the tested two Gram-positive bacteria and E.
coli.
As far as activity is concerned, most of the synthe-
sized compounds were as potent as the cyclic analogues
3–8, showing a MIC value of 25 mg/ml against C. albi-
cans, except 2f and 2k. While compound 2k showed
one-fold less potency than its cyclic analogue, com-
pounds 2f and 2h showed better antifungal activity than
the compared benzoxazole derivatives.
4. Results and discussion
The antimicrobial activity of these compounds and
the control drugs is shown in Table 2 indicates that the
compounds 2a–2p were able to inhibit in vitro growth
of a number of microorganisms, exhibiting MIC values
between 50 and 12.5 mg/ml.
Table 2 reveals that the synthesized compounds
showed antibacterial activity at an MIC value of 50 mg/
ml against the Gram-positive bacteria S. aureus except
the derivatives 2e, 2f, 2i, 2j, 2k, which were active at 25
mg/ml. All the synthesized compounds possessed the
same potency, 50 mg/ml MIC value, against S. faecalis.
The activity of the compounds 2a–2p was also tested
against E. coli, K. pneumoniae and P. aeruginosa as
Gram-negative bacteria. These compounds, against E.
coli and K. Pneumoniae, exhibited lower potency than
the control drugs tetracycline and streptomycin, but
they showed the same potency as erythromycine. The
In conclusion, antimicrobial activity data reported in
Table 3 suggest that the pharmacophoric groups in
these sets of amides and cyclic analogues could be simi-
lar. If these amides are the possible metabolites of the
corresponding fused heterocyclics, then we can expect
prolonged antimicrobial activity for these derivatives.
compound
4-amino-N-(2%-hydroxyphenyl)benzamide
(2g) was the most active against K. pneumoniae (MIC
value of 25 mg/ml).
Acknowledgements
As regards antibacterial activity against the enter-
obacter P. aeruginosa, the synthesized compounds 2e–
2g, 2i–2o showed significant activity (25 mg/ml MIC
value) and possessed better potency than the control
drugs streptomycin and tetracycline and showed the
same activity as erythromycine and chloramphenicol.
Moreover, the antifungal activity of the synthesized
compounds was tested against C. albicans and MIC val-
ues between 25–12.5 mg/ml were found. Compounds 2e,
2h, 2m were found more active than the other com-
pounds, showing a MIC value of 12.5 mg/ml. However,
the control drugs oxiconazole and haloprogin possessed
one dilution better potency than these compounds.
Benzamides 2d–2p were found more active than
phenylacetamides 2a–2c, in particular against Gram-
positive bacteria S. aureus, Gram-negative bacteria P.
aeruginosa and the fungus C. albicans. When the MIC
values of 2a–2c are compared with the previously syn-
thesized phenylacetamides [14] against C. albicans, it is
observed that substitution with an atom and/or atom
groups at position R3 possessing electronically positive
field effects, such as Cl, Br and NO2, increases their anti-
fungal activity.
We would like to thank the Research Fund of Ankara
University (Grant No. 92-03-00-02) for financial sup-
port of this research.
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