Conjugates with an antimicrobial effect based on new derivatives of mercaptobenzoxazole esterified onto poly(maleic anhydride-alt-vinyl acetate)

Article abstract of DOI:10.1002/jhet.1814

New compounds with biological activity based on hydroxy-amino derivatives of benzoxazolyl-2-mercaptoformic acid, benzoxazolyl-2-mercaptoacetic acid, and chloracetyl-2-mercaptobenzoxazole have been synthesized. The chemical bonding of these compounds to po

Full text of DOI:10.1002/jhet.1814

1
124
Conjugates with an Antimicrobial Effect Based on New Derivatives of
Vol 51
Mercaptobenzoxazole Esterified onto Poly(maleic anhydride-alt-vinyl acetate)
a,b
a
b
c
Radita Aparaschivei, Valeriu Şunel, Marcel Popa, and Jacques Desbrieres *
a
“Al.I.Cuza” University, Department of Organic Chemistry, 11 Carol I Blv., 700506, Iasi Romania
b
“
Gheorghe Asachi” Technical University of Iasi, Department of Natural and Synthetic Polymers, 73, Prof.dr.docent
Dimitrie Mangeron Str, 700050 Iasi Romania
Pau et des Pays de l’Adour University, IPREM, Helioparc Pau Pyrénées, 2 Avenue P. Angot, 64053 Pau cedex 9, France
c
*
E-mail: jacques.desbrieres@univ-pau.fr
Received June 5, 2012
DOI 10.1002/jhet.1814
Published online 30 January 2014 in Wiley Online Library (wileyonlinelibrary.com).
New compounds with biological activity based on hydroxy-amino derivatives of benzoxazolyl-2-mercaptofor-
mic acid, benzoxazolyl-2-mercaptoacetic acid, and chloracetyl-2-mercaptobenzoxazole have been synthesized.
The chemical bonding of these compounds to poly(maleic anhydride-alt-vinyl acetate), through esterification, leads
in obtaining conjugates of polymer biologically active compound type, tests indicating a sustained release of the
active chemical, with time (between 5 and 6 h). Reaction products were characterized through elemental and
1
spectral analysis (FTIR and H NMR). Toxicology and antimicrobial activity tests recommend compounds with
small molecule, as well as their conjugates as therapeutical candidates (antimicrobial inhibitors) for pharma-
cological application.
J. Heterocyclic Chem, 51, 1124 (2014).
INTRODUCTION
concentration levels), the transportation, and protection of
the medicine until it reaches the target organ, increasing the
compliance of the patient to the treatment.
The controlled release of a drug, as a concept, was first
proposed by Dr. Judah Folkman in 1964, when he observed
that a silicone capsule can be implanted in the human
body and that it can release, with a constant speed, the active
matter [1]. In this case, a tank-type system was used. This is
the beginning of the polymer–drug system with sustained/
controlled release research, which has seen explosive growth
over the last five decades.
The benzoxazole and more and more of its derivatives
belong to the heterocyclic compounds category, with a
well-known biological activity, for which scientific
literature mentions antifungal (3-[2-[(2-fluorophenoxy)
methyl]benzoxazol-4-yloxy]-N-(pyridin-3-ylmethyl) propan-
1-amine and other similar structured derivatives [4],
2-tert-butyl-4-[(3S)-3-( dimethylamino) pyrrolidin-1-yl]-
5-phenyl-1,3-benzoxazole-7-carbonitrile [5]), antimicrobial,
tuberculostatic (derivatives of 5,7-di-tert-butylbenzoxazoles
According to the method of obtaining them, systems
with controlled release can be classified as follows:
[
6]), and antitumoral activity (6-[3-benzoxazol-2-ylthio)
a) physical systems, when the active matter is physically
incorporated in a matrix (polymeric or not)
b) chemical systems, when the active matter is tied to the
polymer through chemical hydrolyzable bounds [2].
propoxy)-N-(3-chloro-4-fluoro-phenyl)-7-methoxy-qui-
nazolin-4-amine [7], (6-[3-benzoxazol-2-ylthio)propoxy)-N-
(4-bromo-2-ethylphenyl)-7-methoxy-quinazolin 4- amine
[7], and conjugates of benzoxazole-pyrrolo[2,1-c][1,4] benzo-
By accomplishing these methods, we attempt to increase
the effectiveness [3] (by the sustained and controlled release
of the drug with time, avoiding toxic or under-therapeutical
diazepine [8]).
Also, it is used in treating cognitive dysfunctions, disorders
of the central nervous system [9], Alzheimer’s [9],
©
2014 HeteroCorporation

July 2014
Conjugates with an Antimicrobial Effect Based on New Derivatives
1125
of Mercaptobenzoxazole Esterified onto Poly(maleic anhydride-alt-vinyl acetate)
Scheme 2. Synthesis of 2-mercapto-benzoxazole chloracetyl (IV).
schizophrenia [9] (derivatives of benzoxazolyl-2[benzyl-
-carboxylated piperazine] [9]), HIV [10,11], and inflam-
matory [10] and neurodegenerative [10] diseases (derivatives
of N-aryl and N-alkyl-2-aminobenzoxazole [10], 2-(1,2-
difluoro-2-metoxy-2-phenyl-ethyl) benzoxazole [11], and
1
N
N
Cl CO CH Cl
2
S
CO CH Cl
2
S Na
-
NaCl
O
O
I
IV
2
(
8
-(benzoxazole-2-yl)-2,2 difluoro-1- pyridine-3-ylethyl-3-
trifluoromethyl)benzoate [11] and derivatives of 6,
-dichloro[1,2,4]triazolo[3,4-b][1,3]benzoxazol-3(2H)-thione
that also have antioxidative and anthelmintic activity,
respectively) [12].
A first objective of this study is the synthesis and charac-
terization, in terms of the biological activity, of new deriva-
tives from benzoxazole.
Confirmation of compound structure (II, III, and IV) was
1
carried out by elemental and spectral analysis (FTIR, H
À1
NMR). FTIR spectrum presents at 1728 cm (compound III),
À1
1730 cm (compound II), a characteristic bandwidth for es-
À1
À1
À1
ter C═O group, and at 744 cm (compound III), 745 cm
Its second goal is the synthesis of conjugates through chem-
ical binding of some of the obtained derivatives, with the pur-
pose of obtaining systems capable of sustained/controlled
release of a biologically active matter. To achieve this objec-
tive, we had to select a macromolecular compound, which is
reactive in moderate temperature conditions, without the need
for catalysis. Selection was stopped over vinyl acetate and
maleic anhydride copolymer. A priority is that it is a biocom-
patible [13], nontoxic compound, and it has a high reactivity
imprinted by the anhydrous cycle. It has biological activity
per se namely antitumoral activity, and also, it is involved in
the transformation of the pyruvic acid into lactic acid [14].
The chemical bond of some of the synthesized benzoxa-
zole derivatives on the mentioned copolymer was obtained
by esterification of the macromolecular support to the anhy-
drous cycle by the terminal hydroxyl group of the derived.
(
compound II), and 787cm (compound IV) a specific
bandwidth for C-S group. The absorption band for vibrations
À1
À1
of aromatic ring appears at 3300 cm
(compound II),
À1
3
000 cm (compound III), and 2944 cm (compound IV).
C═N bond vibrations, from the oxazolic heterocycle, were
À1
À1
1
identified at 811 cm (compound II), 3000 cm (com-
À1
pound III), and 2944cm
(compound IV). H NMR
spectra certify the presence of characteristic structural
elements of each compound. At adequate values, methyl
groups are identified in the aliphatic area. Signal of the
CH protons appear at 3.4–3.7 ppm, whereas those of the
2
aromatic protons occur at 7.3–7.7 ppm.
Scientific literature contains data regarding biological
importance of the compounds that include monoethanola-
mine in their molecule. They constitute the objective of
numerous studies on the anti-inflammatory [15] and antican-
cer [15] properties. They are also used in treating cardiovas-
cular disease [16], venous malformations [16], benign
vascular lesions [17], esophageal varicose [17], hemangio-
mas [17], sublingual varicose [17], Shigella flexneri [18]
(Gram negative bacteria that causes diarrhea), pulmonary
cancer [19], and improve metabolism [20].
Considering these facts, we synthesized [(b-hydroxy-
ethyl-amino)-formyl]-2-mercapto-benzoxazole (V) and
[(b-hydroxy-ethyl-amino)-acetyl]-2-mercapto-benzoxazole
(VI) by treating the esters (II) and (III) with monoethanola-
mine in anhydrous dioxane on reflux. Similarly, we obtained
[(b-hydroxy-ethyl-amino)-carboxymethyl]-2-mercapto-
benzoxazole (VII) by treating 2-mercapto-benzoxazole
chloracetyl (IV) with monoethanolamine (Scheme 3).
RESULTS AND DISCUSSION
Chemistry. The esters (II) and (III) are prepared by
condensation of 2-mercapto-benzoxazole and ethyl
chloroformate/chloracetate, in the presence of sodium
ethoxide (Scheme 1).
The advantage of the method is that it eliminates the
separation phase of the sodium salt of solid 2-mercapto-
benzoxazole.
To obtain the compound (IV), we isolate the sodium salt (I)
of 2-mercapto-benzoxazole that is treated with monochloroa-
cetic acid chloride and leads to 2-mercapto-benzoxazole
chloracetyl (IV) (Scheme 2).
Scheme 1. Synthesis of ethyl ester of the 2-mercapto-benzoxazol-yl-formic acid (II) and ethyl ester of the 2-mercapto-benzoxazol-yl-acetic acid (III).
N
Cl COOC2H5
S
COOC H
2 5
-
NaCl
O
II
N
O
N
O
C H O Na
2
5
S
Na
SH
-
C H OH
2 5
N
I
ClCH2 COOC H
2
5
S
CH2 COOC2
-
NaCl
H5
O
III
Journal of Heterocyclic Chemistry
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126
R. Aparaschivei, V. Şunel, M. Popa, and J. Desbrieres
Vol 51
Scheme 3. Synthesis of b-hydroxy-ethyl benzoxazol-yl-2-mercaptoformic
IV), benzoxazolyl-2-mercaptoacetic acid amides (V), and 2-mercapto-
benzoxazole chloracetyl.
the CH group and the NH protons as a singlet form at
2
(
8
4
.36–8.65. The protons from OH group show signals at
.65–4.72 ppm.
N
NH2 CH2 CH2 OH
Starting from the premise that the benzoxazole deriva-
(
II),(III),(IV)
S
R
tives have important biological properties, our research
was directed towards compounds V, VI, and VII immobi-
lization by chemical binding (esterification) on the maleic
anhydride-alt-vinyl acetate copolymer (VIII). Our purpose
was obtaining a number of polymer-active matter systems
with a low level of toxicity, better and longer pharmaco-
logical effects because of the polymeric support, and a
longer release as proven by other studies [21–23].
The nucleophilic character of compounds V, VI, and
VII is quite pronounced, and therefore, they can determine
the decyclization of the anhydride from copolymer (VIII),
leading to [(b-ethyl-amino-formyl)-2-mercapto-benzoxa-
zole-yl]-poly(maleic anhydride-alt-vinyl acetate)-ester
(IX), [(b-ethyl-amino-acetyl)-2-mercapto-benzoxazole-
yl]-poly(maleic anhydride-alt-vinyl acetate)-ester (X), and
[(b-ethyl-amino-carboxymethyl)-2-mercapto-benzoxazole-yl]-
poly(maleic anhydride-alt-vinyl acetate)-ester (XI), respec-
tively (Scheme 4).
-
OH
5
C H
2
O
V,VI,VII
V - R = - CO - NH - CH
VI - R = - CH
VII – R = - CO - CH
2
– CH
2
- OH;
– CH
– CH
2
– CO – NH - CH
–NH - CH
2
2
- OH;
- OH;
2
2
2
The structure of compounds V, VI, and VII was tested
through the results of the elemental and spectral analysis
1
(
FTIR and H NMR).
FTIR spectra of the three synthesized compounds show at
À1
À1
1
069 cm (compound VII), 1079 cm (compound VI),
À1
and 1089 cm (compound V), lines characteristic of the
C-NH group, at 1644 cm
compound VII), and 1616 cm (compound V), lines charac-
teristic of the carbonyl group, and at 3223 cm (compound
VII), 3250 cm (compound V), and 3375 cm (compound
VI) specific absorption bands of CH groups in aromatic ring.
The compounds still show a band at 1459 cm (compound
VII), 1450 cm (compound VI), and 1471 cm (compound
V) because of its C═N connection from the oxazole heterocy-
cle. The OH specific group line appears at 3513 cm
À1
À1
(compound VI), 1649 cm
À1
(
À1
Spectral and elemental analysis (determination of nitro-
gen amount) data confirm the structure of the products
IX, X, and XI.
À1
À1
À1
In the FTIR spectrum, the OH group characteristic absorp-
À1
À1
À1
À1
À1
tion appears at 3114cm
(compound XI), 3318 cm
(compound X), and 3344 cm (compound IX). The amidic
À1
CO-NH group stands out by the absorption band from
À1
À1
À1
À1
(
3
compound VII), 3608 cm
(compound VI), and at
1460 cm
1512 cm
(compound XI), 1485 cm
(compound X),
À1
À1
611 cm (compound V).
(compound IX), and 1726 cm
(compound
1
À1
The H NMR spectra show four signals at 7.28–7.70 ppm
IX); at 1717cm (compound X) and 1703 (compound
assigned to the four hydrogen atoms of the benzene nucleus;
at 3.21–3.75 ppm, we observe signals for the protons from
XI) appear absorption bands characteristic to the C═O
esteric group. At 2922cm (compound X), 2980 cm
À1
À1
Scheme 4. Synthesis of [(b-ethyl-amino-formyl)-2-mercapto-benzoxazole-yl]-PMAVA ester and [(b-ethyl-amino-acetil)-2-mercapto-benzoxazole-yl]-
PMAVA ester.
CH CH CH2 CH
CH CH CH2 CH
(
V)
n
n
C
C
O
HOOC
(IX)
C
O
O
OCOCH
3
O
O
O
CH2 CH2 NH CO
S
N
O
(VIII)
COCH3
(
VI)
CH CH CH2 CH
n
HOOC
X)
C
O
O
OCOCH
N
3
CH2 CH2 NH CO CH2
S
(
O
(VII)
CH CH CH2 CH
n
HOOC
XI)
C
O
O
OCOCH
N
O
3
CH2 CH2 NH CH2 CO S
(
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July 2014
Conjugates with an Antimicrobial Effect Based on New Derivatives
1127
of Mercaptobenzoxazole Esterified onto Poly(maleic anhydride-alt-vinyl acetate)
À1
Table 1
(
compound XI), and 3114 cm (compound IX) absorption
bands similar to the CH groups from the aromatic ring
appeared.
The efficiency of the bonding and the molar ratio of the compounds IX, X,
and XI in the active polymer-principle conjugates obtained.
The C═N group from the oxazole heterocycle causes
Polymer-active matter
conjugates—active
biological compound
Bonding
efficiency
(%)
The active biological
compound/copolymer in b
conjugate ratio (mol/mol)
À1
a
some absorption bands around 1079 cm (compound X),
À1
À1
1
143cm
(compound IX), and 1238cm
(compound
À1
XI), whereas the C-S group is associated with 753 cm
compound XI), 805 cm
compound IX) and the CH -S group with 744 cm (only
IX
X
XI
78.7
86.0
90.0
0.70
0.77
0.80
À1
À1
(
(
(compound X), 865 cm
À1
2
in compound X).
a
Presented as the ratio of the amount of benzoxazolic derivative linked to
the support and the theoretical one (all anhydric rings having reacted).
Calculated from the H NMR.
1
At 7.32–7.63 ppm, the H NMR spectra contain signals
specific to the aromatic ring. The protons from the CH2
aliphatic group slightly stand out at 4.72 ppm. At 1.23 ppm,
b
1
the ester CH group protons appear, whereas the NHCO
3
proton appears at 9.8 ppm. The 12.4–12.53 ppm range is
evaluation of the bonding, 78.66%, 85.98%, and 90.03%,
respectively.
associated only with the COOH protons that show that the
1
decycling took place. An example of H NMR spectrum is
shown in the third image for the X conjugate (Fig. 1).
Among the H NMR spectra of the (IX, X, XI)
On the basis of these results, we also determined the active
molar compound and copolymer; in the resulted conjugate, its
value indicating to which extent the copolymer anhydric rings
participated to the reaction. Both theoretical and experimental
1
conjugates, we have shown the molar ratio between the
active (V, VI, VII) biological compounds coupled with the
maleic anhydride of the copolymer (Table 1), taking into
consideration the integrals for the COOH protons (formed
after the opening of the anhydric cycle of the copolymer)
and the aromatic ring protons.
Comparing the result of the study on the nitrogen atom
from the conjugates (IX 5.21%; X 5.76%; XI 5.78) with
the theoretical one and taking into consideration the fact
that the biological compound opened the anhydric cycles,
these values (IX 6.63%; X 6.42%; XI 6.42%) allow the
1
results using H NMR methods led us to the same results.
The efficiency of the bonding has to be observed. It is
manifested through the high quantity of active product in the
conjugate of the copolymer anhydric cycle on one hand and
on the other of the benzoxazole derivatives (by the -OH group
introduced in the hydroxylamine reaction).
Biological activities
Toxicity. To administer them to a living organism, the
obtained conjugates, with potential biological activity,
1
Figure 1. H NMR spectrum of [(b-ethyl-amino-acetyl)-2-mercapto-benzoxazole-yl]-poly(maleic anhydride-alt-vinyl acetate)-ester (X).
Journal of Heterocyclic Chemistry
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R. Aparaschivei, V. Şunel, M. Popa, and J. Desbrieres
Vol 51
must meet a number of constraints, among which the
biocompatibility and the lack of toxicity.
(b) and 3(a), (b) show the Petri dishes sown with the E. coli
(a) and S. aureus (b) strains.
Toxicity is frequently measured through LD , calculated
by using the Spearman–Karber [24] arithmetic method; the
results are presented in Table 2.
The resulted conjugates (IX, X, XI) show a lower toxicity
level than the synthetic compounds, and they are not hazard-
ous to the organism.
Figure 2(a), (b) demonstrates the inhibition areas for
[(b-hydroxy-ethyl-amino)-acetyl]-2-mercapto-benzoxazole
(VI), and Figure 3(a), (b) shows the conjugate [(b-ethyl-
amino-acetyl)-2-mercapto-benzoxazole-yl]-poly(maleic
anhydride-alt-vinyl acetate)-ester (X).
50
The release of the active principle from the macromolecular
The final results allow us to appreciate that the polymeric
support is characterized by its own biological activity and a
very good biocompatibility that can reduce the toxicity level
of the conjugate, the synthetic products to the lab screening
being recommended.
The antimicrobial activity. The antimicrobial activity tests
carried out on four bacterial cultures show that the germs are
resistant to small concentrations of the conjugates. The
inhibition area diameter is sensitive to the 2 mg/mL
concentration.
The tested products present the ability to inhibit some of our
strains (Staphylococcus aureus and Escherichia coli), making
them “sensitive”. The Enterococcus faecalis and Salmonella
enteritidis strains were not inhibited by these compounds.
The compound activity is similar to the sulphaphenazole
support. In the case of the basic hydrolysis (at pH = 9.64),
we saw evidence of altered pH, and consequently, the release
of active principle took place rapidly, the pH reaching a
stable value in approximately 30–45min because of the
ester break between the polymer and principle and also the
reaction of the side chain of the polymer where there is an
ester linkage. In the case of a side test made only from
copolymer, we saw a relatively rapid pH decrease that gets
to a constant value in approximately 60 min.
Given that our initial objective was not achieved (the
sustained release of the active principle on a larger scale
of time), we abandoned the systematic study of the release
of the active principle in this pH conditions.
We continued by monitoring the kinetics of release for the
IX and XI compounds, within the acid hydrolysis environ-
ment, similar to the stomach and observed that the pH
increased until a constant value that is much slower
(up to 5–6 h).
(5 mg/mL on the culture environment), noting that we used
smaller concentration levels for the V–VII and IX–XI
compounds. The results are shown in Table 3; Figures 2(a),
The variations of the pH of the system with time are due
to the partial consumption of the acid from the reaction
environment through the hydrolysis of the ester groups
and the release of its active compound.
Table 2
LD50 values (mg/kg body) for the V, VI, VII, IX, X, and XI compounds.
Figure 4a shows the variation with time of pH during
the hydrolysis of conjugate IX and Figure 4b for the
compound XI. On the basis of the pH variation curve, we
calculated the number of ester bonds and thus the quantity
Compound
LD50 (mg/kg body)
2
4 h
48 h
8575
8975
4905
9030
9330
4950
7 days
8500
8750
4530
8940
9240
4860
Average
8550
8900
4780
9000
9300
4920
V
8575
8975
4905
9030
9330
4950
VI
VII
IX
X
(mol and mg, compared with the analyzed conjugate
quantity) of released drug.
Figure 5 shows the variation of the quantity of active com-
pound released because of hydrolysis in acid conditions
XI
Table 3
The diameter of the inhibition area for compounds V–VII, IX–XI, and XII–sulphaphenazole.
Diameter of the inhibition area (mm)
Compound
Staphylococcus aureus
Escherichia coli
Enterococcus faecalis
Salmonella enteritidis
V
16
+
+
+
+
+
+
+
17
18
15
18
20
19
19
+
+
+
+
+
+
+
5
4
6
6
4
7
8
À
À
À
À
À
À
À
9
8
À
À
À
À
À
À
+
VI
VII
IX
X
XI
XII
17
16
19
21
17
23
10
11
10
11
18
+
Antimicrobial activity.
Lack of antimicrobial activity.
À
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July 2014
Conjugates with an Antimicrobial Effect Based on New Derivatives
1129
of Mercaptobenzoxazole Esterified onto Poly(maleic anhydride-alt-vinyl acetate)
(
a)
(b)
Figure 2. Photography of a Petri dish sown with the Escherichia coli (a) and Staphylococcus aureus (b) strains probing the antimicrobial activity of compound VI.
(
a)
(b)
Figure 3. Photography of a Petri dish sown with the Escherichia coli (a) and Staphylococcus aureus (b) strains probing the antimicrobial activity of compound X.
3
3
3
3
,8
,6
,4
,2
3
3
3
3
,8
,6
,4
3,2
3
2
2
,8
,6
2,8
2,6
0
100
200
300
400
500
0
100
200
300
400
500
Time (minutes)
Time (minutes)
a
b
Figure 4. The variation as a function of time of pH during the hydrolysis of conjugate IX (a) and XI (b) in an acid hydrolysis environment (initial pH = 2.60,
conjugate subjected to hydrolysis = 0.1211 mg (a) and 0.1213 mg (b)).
(
mg active matter/g conjugate), (a) the efficiency of the release
b-ethyl-amino-acetyl, and b-hydroxy-ethyl-amino-carboxy-
methyl of 2-mercapto-benzoxazole derivatives were obtained.
The new synthesized compounds were immobilized on
maleic anhydride-alt-vinyl acetate copolymer support, result-
ing in new polymer-active principles systems.
(b) for two of the obtained active polymer principle.
The effectiveness of the release after 6 h is 43.7%
compound IX) and 36.0% (compound XI); the peak was
(
reached in approximately 5–6 h, similar to the time food
stays in the body.
The structure of the new compounds synthesized (II–VII,
IX–XI) was confirmed by elemental and spectral analysis
1
(
FTIR and H NMR).
CONCLUSIONS
The toxicity of the products (V–VII, IX–XI) was investi-
Ethyl esters of fatty 2-mercapto-benzoxazol-yl-formic
acid,2-mercapto-benzoxazol-yl-acetic acid, and 2-mercapto-
benzoxazole chloracetyl were synthesized. b-Ethyl-amino-formyl,
gated obtaining LD . It was noted that the restraint on a poly-
meric support determines the loss of the biological active
principles toxicity (V–VII).
50
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R. Aparaschivei, V. Şunel, M. Popa, and J. Desbrieres
Vol 51
1
1
1
4
2
0
8
6
4
2
0
5
0
45
4
0
5
3
30
compound IX
compound XI
compound IX
compound XI
2
5
0
2
15
1
0
5
0
0
100
200
300
400
500
0
100 200 300 400 500
Time (minutes)
Time (minutes)
a
b
Figure 5. The variation of the quantity of active matter released because of hydrolysis in acid conditions (a) and the efficiency of the release of the active
principle from the support (b) for compounds IX and XI.
The antimicrobial activity of synthesized compounds was
researched and determined that some of them present a similar
activity to the one of some reference sulphonamides.
The conjugates (IX–XI) present a more efficient bacterio-
static activity, probably because of the polymeric support
influence.
vacuum to remove the sodium chloride. After cooling, we pour the
resulted solution in thin stream in ice water while stirring.
An abundant precipitate was separated, which we filter under
vacuum and dry. The crude product is purified by recrystallization
from boiling ethyl alcohol.
Product properties: solid creamy (57.41 g, yield %: 87.2), melt-
ꢀ
À1
ing point: 85–87 C. IR; n (cm ): 3300 (CH), 1730 (C═O), 811
1
(
C═N), 745 (-C-S-). H NMR (DMSO-d , 400 MHz), d (ppm):
Because of hydrolysis reaction, the release of the com-
pounds, in an acid medium, is carried out in a 6 to 7-h interval.
6
1
7
5
6
.6 (t, 3H, CH
.70 (d, 2H, Ar CH). Anal. Calcd for C10
3.80; H, 4.06; N, 6.27; S, 14.36; Found: C, 54.11; H, 4.17; N,
.39; S, 14.54.
3
); 4.4–4.5 (d, 2H, CH
2
); 7.40 (d, 2H Ar CH);
NO S (%): C,
H
9
3
EXPERIMENTAL
Ethyl ester of benzoxazol-yl-2-mercaptoacetic acid (III). We
prepared similarly to compound II, starting from 100mL
of anhydrous ethyl alcohol, 0.1 mol metallic sodium, 0.1 mol
Materials.
2-Mercaptobenzoxazole was provided by
2
-mercaptobenzoxazole, and 0.1 mol ethyl chloroacetate.
Merck; monoethanolamine, monochloracetic acid chloride,
metallic sodium, maleic anhydride, and ethyl acetate were
supplied by Aldrich, dioxane by Fulka, ethanol anhydride and
acetone delivered by S.C. Chemical Company S.A. Substances
were used in the form provided by manufacturers, without
additional purifying. Mueller Hinton agar culture medium used
for antimicrobial tests was supplied by OXOID.
Product properties: solid gray (19.1 g, yield %: 80.6), melting point
ꢀ
À1
: 47–48 C. IR; n (cm ): 3000 (CH), 1728 (C═O), 842 (C═N), 744
1
(-CH
CH ), 3.4–3.7 (d, 2H, CH
CH), 7.60 (d, 2H, Ar CH). Anal. Calcd for C11
-S-). H NMR (DMSO-d
, 400 MHz), d (ppm): 1.20 (t, 3H,
S), 7.30 (d, 2H Ar
S (%): C,
2
6
), 4.50 (S, 2H, CH
3
2
2
H
11NO
3
55.68; H, 4.67; N, 5.90; S, 13.51; Found: C, 55.73; H, 4.83; N,
6.17; S, 13.70.
Methods of synthesis
Sodium salt of 2-mercaptobenzoxazole (I). In a volumetric
flask, equipped with an ascendant cooler, 50 mL of anhydrous
ethanol and 0.05 mol of metallic sodium are inserted, little by little,
while stirring continuously. Over the hot sodium ethoxide solution,
2-Mercapto-benzoxazole chloracetyl (IV). We use a flask with
an upper water cooler to introduce 10 mL of acetone ethanol that
contains 0.01 mol of sodium salt from 2-mercaptobenzoxazole. We
add 0.01 mol of chloride of monochloroacetic acid while stirring.
ꢀ
0.05 mol of 2-mercapobenzoxazole is inserted; after which, the
To perfect the reaction, we heat the reaction mixture at 65 C for
mixture is refluxated on the water bath for 90 min. Excess of
ethanol was removed through low pressure distillation, and the
isolated product was vacuum-filtered and dried.
about 90 min. We filter it under vacuum and then let the solution at
room temperature crystallize the final product.
Product properties: solid creamy (1.78 g, yield %: 78.24), melting
ꢀ
À1
Product properties: solid dark gray (16.73 g, yield %: 97.38), melt-
point: 115–118 C. IR; n (cm ): 2944 (CH), 1728 (C═O), 824
ꢀ
À1
1
1
ing point: 299 C. IR; n (cm ): 2950, 3150 (CH), 750 (CH -S-). H
(C═N), 733 (C-Cl), 787 (-C-S-). H NMR (DMSO-d , 400 MHz),
2
6
NMR (DMSO-d , 400 MHz), d (ppm): 7.60 (d, 2H, Ar CH); 7.75
d (ppm): 2.78–4.08 (d, 2H, CH ); 7.20 (d, 2H Ar); 7.30 (d, 2H,
6
2
(
2
d, 2H, Ar CH). Anal. Calcd for C
.56; N, 8.91; S, 20.39; Found: C, 52.23; H, 2.49; N, 8.98; S, 18.37.
Ethylic ester of benzoxazolyl-2-mercaptoformic acid (II). We
7
H
4
NOSNa (%): C, 53.49; H,
Ar); Anal. Calcd for C H NO SCl (%):C, 47.48; H, 2.65; N, 6.15;
S, 14.08; Found: C, 47.29; H, 2.77; N, 6.26; S, 14.25.
UV spectrum in ethanol has two absorption maxima 255 nm
and 298 nm (very intense).
9
6
2
introduce 100 mL of anhydrous ethanol in a flask with an upper
condenser to which we add 0.1 mol of metallic sodium. Stir until all
of the sodium reacts. Thus, we obtain a sodium ethoxide solution in
which we introduce 0.1 mol of 2-mercaptobenzoxazol in small
portions while stirring and slight warming, on water fountain for
homogeneization.
[
(b-Hydroxy-ethyl-amino)-formyl]-2-mercapto-benzoxazole
(
V). In a volumetric flask, equipped with an ascendent
cooler, 0.01 mol of ethyl ester of the 2-mercapto-
benzoxazolyl-formic acid that was previously dissolved in
1
0 mL of anhydrous dioxane is inserted, and then, we added
We add 0.11 mol ethyl chloroformate to the hot alcoholic sodium
derivative of 2-mercaptobenzoxazole (I) and continue heating for
0 min while we separate the sodium chloride. We filter it under
0.06 mol of monoethanolamine. The mixture is refluxed for
8 h on an oil bath. The reaction product is precipitated with
water and purified by recrystallization from alcohol.
6
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

July 2014
Conjugates with an Antimicrobial Effect Based on New Derivatives
1131
of Mercaptobenzoxazole Esterified onto Poly(maleic anhydride-alt-vinyl acetate)
[
(b-Ethyl-amino-acetyl)-2-mercapto-benzoxazole-yl]-poly(maleic
Product properties: white-gray solid (1.08 g, yield %: 45.27),
melting point: 156/158 C. IR; n (cm ): 3250 (CH), 3611
ꢀ
À1
anhydride-alt-vinyl acetate)-ester (X). Compound X is synthesized
using the same procedure that is used for compound IX, starting
from 0.0025 mol of maleic anhydride-alt-vinyl acetate copolymer,
20 mL anhydrous dioxane, and 0.0025 mol compound VI.
(
OH), 1616 (C═O), 811 (-C-S-), 1089 (C-NH), 1471 (C═N).
1
6
H NMR (DMSO-d 400 MHz), d (ppm): 3.21–3.30 (d, 2H,
CH ); 3.62–3.75 (d, 2H, CH ); 4.65 (s,1H, OH); 7.28 (d, 2H
2
2
Ar); 7.70 (d, 2H, Ar); 8.65 (s, 1H, NHCO). Anal. Calcd for
S (%): C, 50.41; H, 4.23; N, 11.75; S, 13.45; Found:
Product properties: solid white (0.70 g, yield %: 64.81), melting
C
10
H
10
N
2
O
3
ꢀ
À1
point: 161/165 C. IR; n ( cm ): 3318, (OH), 1485, (CO-NH),
-S-), 1717 (C═O), 1372 (C-O), 1140 (C-O),
05 (C-S), 2922 (CH). H NMR (DMSO-d , 400 MHz), d (ppm):
C, 50.73; H, 4.81; N, 12.12; S, 13.74.
In an alcoholic solution (ethanol), UV spectrum presents two
maxima of absorption: 255 nm and 298 nm (very intense).
1
8
1
079 (C═N), 744 (-CH1
6
.23 (3H, CH ), 4.04 (S, 2H, CH -S), 5.09 (2H, CH ), 4.16 (2H,
3 2 2
[
(b-Hydroxy-ethyl-amino)-acetyl]-2-mercapto-benzoxazole
CH
COOH) . Anal. Calcd for C19
(b-Ethyl-amino-carboxymethyl)-2-mercapto-benzoxazole-yl]-poly
maleic anhydride-alt-vinyl acetate)-ester (XI). Compound XI is
synthesized using the same method as for the compound IX, using
.0025 mol of maleic anhydride-alt-vinyl acetate copolymer, 20 mL
2
), 7.32 (d, 2H, Ar), 7.63 (d, 2H, Ar), 9.8 (1H, NH), 12.53 (1H,
(
VI). It was prepared similarly to compound V, from 0.01 mol
of ethyl ester of the 2-mercaptobenzoxazolyl-acetic acid, 10 mL
anhydrous dioxane, and 0.02 mol monoethanolamine.
20 2 8
H N O S (%): N, 6.41; Found: N, 5.52.
[
(
Product properties: solid white (1.56 g, yield %: 62.27), melting
ꢀ
À1
point: 127/128 C; IR; n (cm ): 3375 (CH, Ar), 2977 (CH), 3608
0
1
(
OH), 805 (-C-S-), 1644 (C═O), 1079 (C-NH), 1450 (C═N). H
anhydrous dioxane, and 0.0025 mol compound VII.
Product properties: solid cream-colored (0.96 g, yield %: 88.46),
melting point: 167/173 C). IR; n (cm ): 3114 (OH), 2980 (CH),
NMR (DMSO-d , 400 MHz), d (ppm): 3.18–3.35 (d, 2H, CH2);
6
3
(
.42–3.43 (d, 2H, CH2); 4.15 (d, 2H, CH
d, 2H Ar); 7.63 (d, 2H, Ar); 8.36 (s, 1H, NHCO). Anal. Calcd for
S (%): C, 52.36; H, 4.79; N, 11.10; S, 12.70; Found:
2
); 4.72 (s, 1H, OH); 7.33
ꢀ
À1
1
1
(
460 (CO-NH), 1238 (C═N), 753 (C-S-), 1703 (C═O). H NMR
DMSO-d 400 MHz), d (ppm): 1.10 (3H, CH ), 6.35 (S, 2H,
CH ), 4.90 (2H, CH ), 4.20 (2H, CH ), 7.23 (d, 2H, Ar), 7.72
d, 2H, Ar), 4.8 (1H, NH), 12.12 (1H, COOH). Anal. Calcd for
C H N O S (%): N, 6.41; Found: N, 5.78.
11 12 2 3
C H N O
6
3
C, 52.59; H, 4.98; N, 11.41; S, 12.92.
In an alcoholic solution (ethanol), UV spectrum presents two
maxima of absorption: 247 nm (more intense) and 275 nm.
2
2
2
(
1
9 20 2 8
[
(b-Hydroxy-ethyl-amino)-carboxymethyl]-2-mercapto-benzoxazole
VII). In a reaction flask, 0.06 mol monoethanolamine and
.01 mol triethylamine were added on 0.01 mol 2-mercapto-
Methods of characterization
Structural characterization. FTIR spectroscopy was achieved
(
0
using an FTIR (ATR) Brucker Tensor-27 spectrophotometer
benzoxazole chloracetyl (IV) in 10 mL anhydrous dioxane. The
reaction mixture was heated under reflux for 3 h. The final
product was precipitated after cooling. It was purified by
recrystallization from boiling water.
Bruker Optik, Ettlinger, Germany). 1
(
H NMR spectra recordings
were performed using a Brucker Avance DRX 400 spectrometer,
1
13 31 19
equipped with 5 mm QNP H/ C/ P/ F samples, working with
the Silicon Graphics Indigo interface.
Product properties: cream-colored solid (1.70 g, yield %:
Quantitative elemental analysis was assessed with Elemental
Exeter Analytical CE 440 device (Exeter Analytical, Coventry,
UK). The melting points were determined with Mel-Temp machine,
which has a digital thermometer, and the pH determination was
made with a Labcor Consort C831 pH meter (Cole-Parmer/Amex
Export-Import SRL, Bucarest, Romania).
ꢀ
À1
6
3
(
3
7.69), melting point: 153–156 C; IR; n (cm ): 3026 (CH),
223 (CH, Ar), 3513 (OH), 824 (-C-S-), 1069 (C-NH), 1649
1
C═O), 1459 (C═N). H NMR (DMSO-d
6
400 MHz), d (ppm):
.37 (s, H, OH); 3.63 (d, 2H, CH ); 4.44 (s, H, NH); 6.36–6.73
2
(
d, 2H, CH ); 6.9 (d, 2H, Ar); 7.3–7.4 (d, 2H, Ar). Anal. Calcd
2
12 2 3
for C11H N O S (%): C, 52.36; H, 4.79; N, 11.10; S, 12.70;
Found: C, 52.63; H, 5.03; N, 11.40; S, 12.95.
In an alcoholic solution (ethanol), UV spectrum presents two
maxima of absorption: 242 nm (very intense) and 279 nm.
Poly (maleic anhydride-alt-vinyl acetate) copolymer (PMAVA)
Determination of toxicity. White male mice (20 Æ 2 g) were
used to determine the toxicity of the compounds (IV, V, VII, VIII).
The mice were kept under observation for 7 days, at constant
temperature (22 C Æ 1 C), receiving common nutrition. Their
weighing was performed for 2 days at the same time, removing
the ones that have decreased in weight. To determine the acute
toxicity, six lots of mice were used. Tested substances were
administered intraperitoneally, as a stabilized (by Tween 80)
aqueous suspension, and mortality was recorded at 24h, 48 h, and
ꢀ
ꢀ
(
VIII). It was synthesized by maleic anhydride copolymerization
ꢀ
with vinyl acetate at 80 C in the presence of benzoyl peroxide [21].
À1
The copolymer was obtained as a white solid of 10,000 g mol
molecular weight.
[(b-Ethyl-amino-formyl)-2-mercapto-benzoxazole-yl]-poly(maleic
7
days.
anhydride-alt-vinyl acetate)-ester (IX).We use a 50-mL flask, equipped
with an ascendent cooler, to introduce 0.0025 mol maleic anhydride-
alt-vinyl acetate copolymer predissolved in 10 mL of dioxane, and
.0025 mol compound V was also dissolved in 10 mL of dioxane.
The resulted solution is to be refluxed for 90 min. The dioxane excess
will be distilled under low pressure.
The solid glue (IX) is filtered and dried under vacuum, and
then, it is washed with anhydrous ethyl ether.
Determination of antimicrobial activity. Antimicrobial activity
of the synthesized compounds was determined on S. aureus ATCC
5923 (bacil gram “+”) cultures, E. coli ATCC 25922 (coc gram
2
“
0
À”), E. faecalis ATCC 51299 (coc gram “À”), and also
S. enteritidis ATCC 13076 (bacil gram “À”) cultures, in the
serology laboratory of the Public Health Department, Iasi, Romania.
For this, the capacity of inhibition of the compounds, on the
mentioned cultures, was followed measuring the inhibition area
Product properties: solid white (0.76 g, yield %: 72.34), melting
using Kirby–Bauer [25] diffusimetric method.
ꢀ
À1
point: 177/182 C. IR; n (cm ): 3114 (CH), 1726 (C═O), 1512
According to international regulations, the classification in the
1
(
d
CO-NH), 3344 (OH), 1143 (C═N), 865 (C-S). H NMR (DMSO-
400 MHz), d (ppm): 1.20 (3H, CH ), 4.70 (2H, CH ), 4.25 (2H,
CH ), 7.25 (d, 2H, Ar), 7.60 (d, 2H, Ar), 8.8 (1H, NH), 12.13 (1H,
COOH). Anal. Calcd for C18 S (%): N, 6.63; Found: N, 5.21.
category sensitive/resistant of bacterial strains (mentioned previ-
ously) is made depending on the inhibition area diameter as follows:
sensitive ≥17mm, intermediate sensitive = 13–16 mm, and resistant
≤12 mm.
6
3
2
2
18 2 8
H N O
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

1
132
R. Aparaschivei, V. Şunel, M. Popa, and J. Desbrieres
Vol 51
active principle (in this way, we are able to calculate the produced
active biological quantity released).
The samples were dissolved in dimethylformamide (DMF)
while ensuring a complete solubilization.
The germs were grown on Mueller Hinton agar. Their prepara-
tion must be proceeded as follows. Dissolve 38 g of dehydrated
medium in 1 L of distilled water; for complete solubilization,
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you must reach boiling point and then sterilize by autoclaving at
ꢀ
1
21 C for 15 min.
[
[
Approximately 0.5 units McFarland of the strains for testing
are sowed on Petri plates with a diameter of 9 cm in a 4–4.5 mm
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ꢀ
will be heated at 37 C in the thermostat.
3531.
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[
ꢀ
compound (IX, X, XI) suspended in 20 mL NaOH solution, at 37 C.
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intestine, previous studies led to the conclusion that the order of the
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dropped very fast after the suspension in the basic solution of
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[
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were suspended in 25 mL HCl (8.17%) at 37 C. The quantitative
[
evaluation of the bioactive product released by hydrolysis
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analyzed in the hydrolysis environment.
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mer was added has not modified its pH during the experiment.
2
006, 83, S1 499.
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[
[
[
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We conclude that the pH modification is due to the hydrolysis
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet