Polymer conjugates with potential biological activity based on new derivatives of 2-mercaptobenzoxazole-synthesis and characterization

Article abstract of DOI:10.2478/s11532-013-0310-8

New potentially biologically active compounds derived from 2-mercapto-benzoxazole were synthesized and coupled on polymeric support of poly (maleic anhydride-alt-vinyl acetate) for the preparation of polymer-drug conjugates with controlled drug release. All compounds were characterized by elemental and spectroscopy (FT-IR, ¹H-NMR) analysis. The toxicological tests recommend the products for further laboratory screening. [Figure not available: see fulltext.]

Full text of DOI:10.2478/s11532-013-0310-8

Cent. Eur. J. Chem. • 11(11) • 2013 • 1808-1815
DOI: 10.2478/s11532-013-0310-8
Central European Journal of Chemistry
Polymer conjugates with potential
biological activity based on new derivatives
of 2-mercaptobenzoxazole-synthesis
and characterization
Research Article
Rǎdiţa Aparaschivei1,2, Valeriu Şunel1, Mihaela Holban1,
Marcel Popa2*, Jacques Desbrieres3
1Department of Organic Chemistry, Faculty of Chemistry,
”Al.I.Cuza” University, 700506, Iasi Romania
2Department of Natural and Synthetic Polymers,
Faculty of Chemical Engineering and Environmental Protection,
“Gheorghe Asachi” Technical University of Iasi, 700050 Iasi, Romania
3Pau and Adour Countries University (UPPA), IPREM,
Helioparc Pau Pyrenees, 64053 PAU cedex 9, France
Received 20 January 2013; Accepted 2 July 2013
Abstract:
New potentially biologically active compounds derived from 2-mercapto-benzoxazole were synthesized and coupled on polymeric
support of poly (maleic anhydride-alt-vinyl acetate) for the preparation of polymer-drug conjugates with controlled drug release. All
1
compounds were characterized by elemental and spectroscopy (FT-IR, H-NMR) analysis. The toxicological tests recommend the
products for further laboratory screening.
Keywords: 2-mercapto-benzoxazole derivatives • Hydrazides • Drug-polymer conjugates • Poly (maleic anhydride-alt-vinyl acetate)
© Versita Sp. z o.o.
found to be of extreme importance in the chemistry of
vital processes [2].
1.Introduction
The presence of the benzoxazole ring in numerous
naturally occurring organic compounds (i.e., enzymes,
nucleic acids, vitamins, etc.), which has important
therapeutic effects stimulated research in obtaining new
benzoxazole derivatives, more intensely after 1964,
when Harris and Folkers prepared vitamin B6 starting
from a 4, 5-disubstituted oxazole derivative. During
the research evolution of these types of compounds,
some are worth mentioning, such as Flunoxaprofen, a
non-steroidian anti-inflammatory drug with important
applications in modern chemotherapy [1]. Also, the
role of some benzoxazole derivatives in inhibiting
topoisomerase I and reverse-transcriptase HIV-1 was
2-mercaptobenzoxazole and its derivatives are
heterocyclic compounds of strong nucleophilic character
with numerous applications in medicine, such as in the
treatment of schizophrenia (4-7 substituted derivatives
of arylsulfonyl benzoxazole [3]), Alzheimer (5,
6
substituted derivatives of 2-(4-dimethylaminophenyl)-
1,3-benzoxazole [3,4]), diseases of the central nervous
system
[3],
HIV
(3H-pyrido-[2,1-b]-benzoxazole;
2-(4-amino-3-substituted-2-thioxo-2,3-dihydrotiazole-5-
yl) benzoxazole [5]), cancer (isosteric benzoxazole [6];
conjugates based on [2,1-c] pyrrolo-benzoxazole [1,4];
benzodiazine [7,5]), tuberculosis (2-substituted, 5,7-di-
tert-butyl-benzoxazole [4]).
* E-mail: marpopa@yahoo.fr
1808

R. Aparaschivei et al.
support, with biological activity of its own- poly(maleic
anhydride-alt-vinyl acetate) [19]. Besides its biomaterial
characteristics, the copolymer is highly reactive in
normal temperature conditions (due to anhydride ring),
without the need of a catalyst or high temperature
and forms strong hydrophilic groups (-COOH), of the
drug binding to the support. Thus, the present paper
aims to synthesize new 2-mercaptobenzoxazole
derivatives and their corresponding conjugates with
poly(maleic anhydride-alt-vinyl acetate) and evaluate
their physico-chemical characteristics and biological
properties.
Thus, the first aim of this paper is the synthesis
and characterization of new 2-mercaptobenzoxazole
derivatives following both novel and classic methods
and the evaluation of their acute toxicity.
In the last decade, the term of nanomedicine has
been more and more used by physicians, biologists and
scientists activate in human health research. Science
Citation Index (Institute for Scientific Information,
Thompson, Philadelphia, USA) reported the first
publications mentioning this term around the year 2000.
As a unanimously accepted concept, nanomedicine is
considered as the use of nanoscale or nanostructured
materials in medicine that, according to their structure,
have unique medical effects, for example, the ability
to cross biological barriers or the passive targeting of
tissues. This definition does not include traditional small-
2. Experimental procedure
molecule drugs as they are not specifically engineered 2.1. Material
on the nanoscale to achieve therapeutic effects that 2-Mercaptobenzoxazole was provided by Merck,
relate to their nanosize dimension [8-10]. However, ethyl chloroformate, ethyl chloroacetate, ethyl
the term refers to macromolecular drugs, respectively p-aminobenzoate, chloroacetyl chloride, sodium, ethyl
polymers with biological activity per se or polymers acetate from Aldrich, dioxane from Fluka, acetone,
which attain biological effect by chemical binding of anhydrous ethanol from Chemical Company S.A. The
certain low molecular compounds on the main chain, chemical substances were used as provided, without
thus, justifying the already popular term of “polymer any supplementary purification.
therapeutics” (rational design of nanomedicines).
This type of drug-polymer associations, achieved
2.2. Methods
by chemical binding of biologically active sites to
macromolecular support are also called polymer-drug
conjugates, a term first mentioned by Ringsdorf in 1975
[11].According to his concept and the model proposed by
Ringsdorf, a polymer-drug conjugate has triple structure:
a support of macromolecular chain, containing reactive
functional groups (or groups to be further functionalized)
on which the biologically active molecule is bound
through a spacer (generally, biodegradable in order to
determine the release of the drug and having pending
reactive groups-amine, carboxyl, hydroxyl, etc.), a
strong hydrophilic group to ensure water solubility of the
complex macromolecular architecture and a functional
group able to be recognize by the cell receptors specific
for the targeted organs. Of course, the above model
is an ideal one, so for many versions being realized,
sometimes have no functional groups to target to a
certain organ. In the last two decades, the research in
the field of drug-polymer conjugates has shown a real
increasing interest [12], with an impressive number of
papers being published [13-18].
Ethyl ester of benzoxazolyl-2-mercaptoformic acid
(II) was obtained starting from 2-mercaptobenzoxazole
and ethyl chloroformate in anhydrous ethanol, according
to a method described in literature [20].
Cream coloured solid: (57.41 g, 87.2%), m.p. 85-
87oC. IR; ν(cm-1): 2850, 3300 (CH), 1730 (C=O), 745
(-C-S-). 1H-NMR (DMSO-d6, 400 MHz), δ (ppm): 1.6 (t,
3H, CH3); 4.4 – 4.5 (d, 2H, CH2); 7.40 (d, 2H Ar CH);
7.70 (d, 2H, Ar CH). Anal.calcd. for C10H9NO3S (%): C,
53.81; H, 4.03; N, 6.27; S, 14.39; Found: C, 54.20; H,
4.47; N, 6.69; S, 14.76.
Ethyl ester of benzoxazolyl-2-mercaptoacetic
acid (III) was synthesized from 2-mercaptobenzoxazole
and ethyl chloroacetate in anhydrous ethanol [20].
Grey solid: (19.1 g, 80.6%), m.p. 47 – 48 oC. IR; ν (cm-1):
1
2933, 3000 (CH), 1728 (C=O), 744 (-CH2-S-). H-NMR
(DMSO-d6, 400 MHz ), δ (ppm): 1.20 (t, 3H, CH3), 3.4 –
3.7 (d, 2H, CH2), 4.50 (S, 2H, CH2S), 7.30 (d, 2H Ar CH),
7.60 (d, 2H, Ar CH). Anal. Calcd. for C11H11NO3S (%):
C, 55.69; H, 4.64; N, 5.90; S, 13.5; Found: C, 56.10; H,
5.13; N, 6.37; S, 13.90.
Besides the well known advantages of using the
drug-polymer conjugates for the treatment of certain
diseases, the second aim of the paper was the
preparation of such systems by chemical binding of the
synthesized 2-mercaptobenzoxazole derivatives to a
reactive, nontoxic and biocompatible macromolecular
Chloroacetyl-2-mercaptobenzoxazole (IV) was
prepared by the reaction of 2-mercaptobenzoxazole
sodium salt with chloroacetyl chloride in acetone [22].
Cream coloured solid: (1.78g, 78.24%), m.p. : 115-
118oC. IR; ν (cm-1): 2944 (CH), 1728 (C=O), 824 (C=N),
733 (C-Cl), 787 (-C-S-). 1H-NMR (DMSO-d6, 400 MHz),
1809

Polymer conjugates with potential biological activity based on new
derivatives of 2-mercaptobenzoxazole-synthesis and characterization
δ (ppm): 2.78–4.08 (d, 2H, CH2); 7.20 (d, 2H Ar); 7.30 3281 (CH), 1595 (-CO-NH-), 1495 (C=N), 1680 (C=O).
(d, 2H, Ar); Anal.calcd. for C9H6NO2SCl(%): C, 47.48; H, 1H-NMR (DMSO-d6,400 MHz) δ (ppm): 4.14-4.28 (d, 2H,
2.65; N, 6.15; S, 14.08; Found:C, 47.29; H, 2.77; N, NH2), 6.55-6.56 (d, 2H, Ar CH), 7.18-7.26 (d, 4H, Ar CH),
6.26; S, 14.25.
UV spectrum in ethanol presents 2 absorption peaks 10.04 (s, 1H, NHCO). Anal.Calcd. for C15H12N4O3S (%):
at 255 nm and 298 nm (very intense), respectively. C, 54.87; H, 3.65; N, 17.07; S, 9.75; Found: C, 54.30; H,
Hydrazide of benzoxazolyl-2-mercaptoformic 3.23; N, 17.28; S, 9.67.
7.58 – 7.60 (d, 2H, Ar CH), 7.89 – 7.98 (s, 1H, NHCO),
acid (V) was obtained by treating compound (II) with
hydrazine hydrate in absolute ethanol [20].
p-Aminobenzoyl-hydrazide of benzoxazolyl-2-
mercaptoacetic acid (IX) was synthesized in similar
White solid: (1.92 g, 92%), m.p. 168–171oC. FT-IR; manner as compound (VIII), starting from compound
max cm-1: 3289 (NH), 1659 (CO-NH), 614 (-C-S-), 1508 (VI) (0.002 mol ) and ethyl p-aminobenzoate (0.002
ν
(C=N). 1H-NMR (DMSO-d6, 400 MHz ) δ(ppm): 4.50 (s, mol), each dissolved in 10 mL dioxane (10 mL).
2H, NH2), 7.20 – 7.40 (d, 2H, Ar CH), 7.50 – 7.9 (d, 2H,
Light cream solid: (0.34 g, 50.74%), m.p. 172-173oC.
Ar CH), 9.5 (s, 1H, NH). Anal. Calcd. for C8H7N3O2S (%): FT-IR; νmax cm-1: 3304 (NH), 3000 (CH), 1642 (C=O),
1
C, 45.93; H, 3.34; N, 20.09; S, 15.31; Found: C, 46.20; 1454 (C=N), 1536 (-CO-NH-). H-NMR (DMSO-d6, 400
H, 3.65; N, 20.55; S, 15.43.
MHz) δ (ppm): 4.17-4.19 (d, 2H, CH2), 4.20 – 4.22 (d,
Hydrazide of benzoxazolyl-2-mercaptoacetic 2H, NH2), 6.55 – 6.58 (d, 2H, Ar CH), 7.31 – 7.66 (m, 6H,
acid (VI) was synthesized by treating compound (III) Ar CH), 9.31 (s, 1H, NHCO), 9.72 (s, 1H, NHCO). Anal.
with hydrazine hydrate in absolute ethanol [20].
Calcd. for C16H14N4O3S (%): C, 56.14; H, 4.09; N, 16.37;
White solid: (1.62 g, 72.65%), m.p. 173 – 175oC. FT- S, 9.35; Found: C, 56.49; H, 3.93; N, 16.62; S, 9.72.
IR; νmax cm-1: 3202, 3304 (NH), 2994, 3037 (CH), 1645
Benzoxazolyl-2-mercapto-carboxymethyl-
1
(-CO-NH-), 1503 (C=N). H-NMR (DMSO-d6, 400 MHz) hydrazide of p-aminobenzoic acid (X) was prepared
δ (ppm): 4.10 (s, 2H, NH2), 4.37 (s, 2H, CH2-S), 7.32 – following a similar method as compound (VIII). Thus,
7.48 (d, 2H, Ar CH), 7.60 – 8.0 (d, 2H, Ar CH), 9.48 (s, compound (VII) (0.001 mol ) and ethyl p-aminobenzoate
1H, NH). Anal.Calcd. for C9H9N3O2S (%): C, 48.43; H, (0.002 mol) are dissolved in anhydrous dioxane (20 mL)
4.03; N, 18.83; S, 14.34; Found: C, 48.82; H, 4.22; N, and a light grey precipate is obtained and purified by
19.27; S, 14.67.
Benzoxazolyl-2-mercapto-carboxymethyl-
hydrazide (VII)
recrystallization from boiling ethanol.
Light grey solid: (0.29 g, 84.79 %), m.p. 160-162oC.
IR; νmax (cm-1): 3306 NH), 3112 (CH, Ar), 2986 (CH),
Chloroacetyl-2-mercaptobenzoxazole (IV) (0.004 1726 (CO), 1613 (NH), 1571 (C=C), 1494 (C=N), 1336
mol) are suspended in dioxane (10 mL) and hydrazine (C-O), 1212 (C-N), 750 (C-S). 1H-NMR (DMSO-d6,
hydrate (0.012 mol) and triethylamine (0.4 g) are added 400 MHz) δ (ppm): 3.37 (s, 1H, NH), 3.63 (s, 2H, CH2),
under stirring, obtaining a cream coloured precipitate. 4.44 (s, 2H, NH2), 6.36-6.62 (m, 3H, Ar), 7.03 (m, 3H,Ar),
Thestirringiscontinuedfor4hours, atroomtemperature, 7.26 (m, 1H, Ar), 7.52 (m, 1H, Ar), 8.25 (s, 1H, NHCO).
then the product is vacuum filtered and purified by Anal.Calcd. for C16H14N4O3S (%): C. 56,14; H, 4.09; N,
recrystallization from boiling water.
Light cream solid: (0.33 g, 36.26 %), m.p. 190-192oC. 9.60.
FT-IR; νmax cm-1: 2944 (CH), 1728 (C=O), 824 (C=N),
Poly
16.37; S, 9.35. Found: C, 56.21; H, 3.98; N, 16.53; S,
(maleic
anhydride-alt-vinyl
acetate)
733 (C-Cl), 787 (-C-S-). 1H-NMR (DMSO-d6, 400 MHz), poly(MAVA) (XI) with molecular weight of 100,000
δ (ppm): 2.78 – 4.08 (d, 2H, CH2); 7.20 (d, 2H Ar). Anal. g mol-1 (determined by viscosimetric method) was
Calcd. for C9H6NO2SCl (%): C, 42.47; H, 2.63; N, 6.15; obtained by the co-polymerization of maleic anhydride
S, 14.06. Found: C, 43.08; H, 2.56; N, 6.07; S, 14.45.
with vinyl acetate at 80°C, in the presence of benzoyl
p-Aminobenzoyl-hydrazide of benzoxazolyl-2- peroxide [19].
mercaptoformic acid (VIII)
Benzoxazolyl-2-mercaptoformyl-hydrazine-
Compound (V) (0.001 mol) is dissolved in dioxane anylide of poly (maleic acid-alt-vinyl acetate) (XII)
(10 mL) then, separately, in ethyl p-aminobenzoate
Poly
(maleic
anhydride-alt-vinyl
acetate)
(0.002 mol) in dioxane (10 mL). The two solutions are (0.0025 mol) and p-aminobenzoyl-hydrazide of
introduced in a flask provided with ascendant condenser benzoxazolyl-2-mercaptoformic (VIII) (0.0025 mol) are
and kept under refluxion on a water bath for 2 hours. each separately dissolved in dioxane (15 mL). The two
The, dioxane is evaporated under reduced pressure, solutions are mixed together wth stirring in a round
thus obtaining a cream coloured precipitate.
bottom flask. The reaction is carried out at 110-115oC,
Cream coloured solid: (0.214 g, 65.32%), m.p. in an oil bath for 2 hours. A homogenous solution is
127 – 132oC. FT-IR; νmax cm-1: 3329, 3420 (NH), 2983, obtained, and the excess of dioxane is removed by
1810

R. Aparaschivei et al.
distillation under reduced pressure. The final product is
Silicone Graphics Indigoz and elemental analysis
precipitated by repeated washings in anhydrous ether, was made using Exeter Analytical CE 440 elemental
filtered and dried at 50oC for 180 minutes.
analyzer. The melting points of the obtained compounds
Yellow solid: (1.005 g, 78.51%); m.p. 161oC. IR; were determined with a Mel-Temp melting point module,
νmax (cm-1): 3242 (NH), 1703 (CO din COOH), 1684 provided with digital thermometer.
(C=O), 1540 (CO-NH), 1496 (C=N), 743 (C-S). 1H-NMR
(DMSO-d6, 400 MHz) δ (ppm): 1.26 (t, 3H, CH3), 3.20 2.3.1. Determination of toxicity
(d,2H,CH2), 4.19 (s,H, CH), 6.55 (d, 2H, Ar), 7.24 (m, Acute toxicity of the synthesized compounds (V-X and
4H, Ar), 7.46-7.62 (d, 2H, Ar), 7.89 (s, 2H, NHCO), XII-XIV) and copolymer [poly (MAVA)] was evaluated
10.04 (s, H, NHCO), 11.079 (s, H, COOH). Anal.Calcd. usingwhitemalemiceof20±2geach.Themicewerekept
for C23H20N4O8S (%) N, 10.93. Found: N, 5.86.
under observation for 7 days at constant temperature,
Benzoxazolyl-2-mercaptoacetyl-hydrazine- receiving habitual nourishment. Mice weighing was
anylide of poly (maleic acid-alt-vinyl acetate) done every two days, eliminating from the test groups
(XIII) was prepared in a similar manner as compound the animals which presented weight loss. Every group
(XII), from poly(maleic anhydride-alt-vinyl acetate) consisted of 6 animals. The tested compounds were
(0.0025 mol) and p-aminobenzoyl-hydrazide of administered intraperitoneally as aqueous suspensions
benzoxazolyl-2-mercaptoacetic acid (IX) (0.0025 mol) stabilized by Tween 80; mice mortality was registered
in dioxane.
Orange solid: (0.831 g, 63.19%), m.p. 170oC. IR;
after 24 hours, 48 hours and 7 days.
νmax (cm-1): 3307 (NH), 1717 (CO din COOH), 1644 2.3.2. Determination of tuberculostatic activity
(C=O), 1537 (CO-NH), 1454 (C=N), 807 (C-S), 739 Compounds V-X and XII-XIV were tested for their
1
(CH2-S). H-NMR (DMSO-d6, 400 MHz) δ (ppm): 1.25- in vitro biological activity, using the serial dilution
1.28 (t,3H,CH3), 3.57 (d,2H,CH2), 4.18 (s,H, CH), 4.29 technique, and Youmans medium with bovine serum
(d,2H,CH2), 6.55-6.57 (d, 2H, Ar), 7.33-7.73 (d, 6H, Ar), as liquid environment, inoculated with Mycobacterium
7.94-8.35 (s, 2H, NHCO), 12.50 (s, H,COOH). Anal. tuberculosis,var.hominis(strainH37RV),inaconcentration
Calcd. for C24H22N4O8S (%) N, 10.64. Found N, 5.394.
of 10-2 mg mL-1 [20]. The biologically active compounds
Benzoxazolyl-2-mercapto-carboxymethyl were previously dissolved in dimethylsulphoxide, due to
hydrazine-anylide of poly (maleic acid-alt-vinyl their low solubility in saline solutions. Solutions formed
acetate) (XIV) was prepared by a similar method as of 100 μg compound dissolved in 1 mL solvent (DMSO/
compound (XII), starting from poly(maleic anhydride- phosphate buffer solution pH 7 of ¼ (v/v)) were used in
alt-vinyl acetate) (0.0025 mol) and benzoxazolyl-2- concentrations of 5, 10, 20, 30 and 40 μg compound/
mercapto-carboxymethyl-hydrazide of p-aminobenzoic mL culture environment, the results being registered
acid (0.0025 mol) in dioxane.
(Table 3) 6 and 15 days after inoculation.
Cream coloured solid: (0.70 g, 53.23%), m.p. 184oC.
IR; νmax (cm-1): 3123-3313 (NH), 2989 (CH,Ar), 1712 (CO
din COOH), 1613 (CO), 1517 (CO-NH), 1494 (C=N), 3. Results and discussion
786 (C-S). 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 1.23
(D,2H,CH2), 1.91 (s, 1H, NH), 1.93 (t, 3H, CH3), 3.37 (d, Compounds (VIII-X) were synthesized using as
2H, CH2), 3.52 (d, 2H, CH2), 4.203 (s, 1H, CH), 6.55 (d, intermediates ethyl esters of benzoxazolyl-2-
2H, Ar), 6.90 (d, 2H, Ar), 7.10 (d, 2H, Ar), 7.63 (d, 2H, mercaptoformic (II), benzoxazolyl-2-mercaptoacetic (III)
Ar), 8.34 (s, 1H, NHCO), 9.10 (s, 1H, NHCO), 11.4 (s, and chloroacetyl-2-mercaptobenzoxazole (IV), as well
1H, COOH). Anal.Calcd. for C24H22N4O8S (%) N, 10.64. as their hydrazides (V-VII), which were treated with ethyl
Found N, 7.38.
p-aminobenzoate (Scheme 1).
The chemical structure of compounds (VIII-X) was
confirmed by elemental and spectroscopy analysis (FT-
FTIR IR, H-NMR). IR spectra for all evaluated compounds
2.3. Characterizations
1
FT-IR spectra were obtained using
a
spectrophotometer (ATR) Bruker Tensor-27 (KBr disk (VIII - X) present at 1454-1495 cm-1 an absorption
technique);
band specific for C=N and at 1642-1680 cm-1 for C=O.
1H-NMR analysis was performed on a Bruker DRX The aromatic ring is highlighted by the presence of an
400 spectrometer (5mm QNP probe; 1H/13C/31P/19F. absorption band at 3000-3281 cm-1.
The compounds were previously dissolved into DMSO
at a concentration of aprox. 7 mg mL-1. The spectra were structural
run at room temperature.
(VIII-X). Aliphatic and aromatic protons appear at
1H-NMR spectra prove the presence of characteristic
elements specific for compounds
1811

Polymer conjugates with potential biological activity based on new
derivatives of 2-mercaptobenzoxazole-synthesis and characterization
Scheme 1. Synthesis of p-aminobenzoyl-hydrazide of benzoxazolyl-2-mercaptoformic acid (VIII), p-aminobenzoyl-hydrazide of benzoxazolyl-2-
mercaptoacetic acid (IX) and benzoxazolyl-2-mercapto-carboxymethyl-hydrazide of p-aminobenzoic acid (X).
Scheme 2. Synthesis of benzoxazolyl-2-mercaptoformylhydrazine-anilide of poly (maleic anhydride-alt-vinyl acetate) (XII benzoxazolyl-2-
mercaptoacetylhydrazine-anilide of poly (maleic anhydride-alt-vinyl acetate) (XIII) and benzoxazolyl-2-mercapto-carboxymethyl
hydrazine-anilide of poly (maleic anhydride-alt-vinyl acetate) (XIV).
4.17-4.19 ppm and 6.55-7.66 ppm, respectively. NH2 synthesized conjugates are considered as derivative
protons are shown at 4.14-4.28 and NH-CO protons of poly (maleic acid-alt-vinyl acetate) (poly MAVA) and
(compounds VIII and IX) are proved by peaks at 7.89- named as corresponding. The chemical structure
10.04 ppm. In case of compound X, protons can be of conjugates (XII-XIV) is confirmed by elemental
identified at 3.63 ppm-aliphatic, at 3.37 ppm-NH and (nitrogen content) and spectroscopic analysis (FT-IR,
at 4.44 ppm-NH2. The two aromatic protons from the 1H-NMR). FT-IR spectra show absorption peaks and
p-aminobenzoic ring are found at 6.36-6.62 ppm and bands for CO-NH at 1490-1495 cm-1, for C=O (ester) at
the rest of the protons at 7.03-7.55. Amide protons are 1703-1717 cm-1 and for NH at 3242-3307 cm-1,
identified at 8.25 ppm.
respectively. C=N group from oxazole heterocycle is
Due to the fact that benzoxazole derivatives shown by absorption bands at 1454-1496 cm-1, C-S and
generally present important biological properties, CH2-S at 743-807 cm-1 and 739 cm-1.
one of the objectives of the paper was the grafting of
1H-NMR spectra present specific signal for aromatic
the synthesized compounds (VIII-X) onto copolymer rings at 6.55-7.73 ppm. CH3 protons are marked
poly (maleic anhydride-alt-vinyl acetate) (XI) for the by signals at 1.23-1.28 ppm (compounds XII, XIII;
preparation of low toxic water soluble polymer-drug Fig. 1), at 1.93 ppm (compound XIV; Fig. 2), CH2 protons
systems. The alkalinity of compounds (VIII-X) is rather at 3.20 ppm, (compound XII, CH-CH2-CH), at 3.37 ppm
high and can determine the opening of anhydride ring (compound XIV) and at 3.57 ppm (compound XIII).
from copolymer (XI), thus resulting conjugates (XII-XIV) The peak attributed to CH proton appears at 4.03 ppm
(Scheme 2).
(compound XIV), at 4.18 ppm (compound XIII) and at
By opening the anhydride ring, a new amidic 4.19 ppm (compound XII) and CH2 protons from the
derivative of maleic acid is formed; thus, the new low molecular weight compounds appear at 3.53 ppm
1812

R. Aparaschivei et al.
Figure 1. 1H-NMR spectrum of benzoxazolyl-2-mercaptoacetylhydrazine-anilide of poly(maleic acid -alt-vinyl acetate) (XIII).
Figure 2. 1H-NMR spectrum of benzoxazolyl-2-mercapto-carboxymethyl hydrazine-anilide of poly (MAVA) (XIV).
(compound XIV) and at 4.29 ppm (compound XIII), lower yields seem to be obtained. This fact is probably
respectively. Specific peaks for amidic and carboxylic due to the greater chain length of spacers, which
protons are shown at 7.89-10.04 ppm and 11.079-12.50, increases the volume of the substitutes and determines
proving anhydride decyclization.
The yields of immobilizing biologically active solubility of conjugates is different. If, benzoxazolyl-
compounds (XII- XIV) onto copolymer and the molar 2-mercaptocarbonyl-hydrazide
of poly (MAVA),
ratio between active principle and polymer support, benzoxazolyl-2-mercaptoacetyl-hydrazide
of poly
calculated by elemental analysis for determining the (MAVA) and benzoxazolyl-2-mercapto-carboxymethyl-
nitrogen content are shown in Table 1.
hydrazide of poly (MAVA) were found to be practically
important steric hindrance. Moreover, the water
The efficiency of coupling/immobilization of active insoluble in water [20], by increasing the spacer
compound on polymeric support is expressed as a ratio chain length, the compounds (XII-XIV) gain water
between the practical weight of benzoxazole derivative solubility, their behaviour being similar to Ringsdorf’s
immobilized and the theoretical one (considering that model.
all anhydride cycles reacted). Comparing the coupling
efficiency of these compounds with other conjugates 3.1. Biologic activity
reported in previous work [20]: benzoxazolyl-2- 3.1.1. Acute toxicity
mercaptocarbonyl-hydrazide of poly (MAVA) (66.38%), If prepared conjugates are intended to be administered
benzoxazolyl-2-mercaptoacetyl-hydrazide
of
poly into an organism or in contact with it, they are required
(MAVA) (48.59%) and benzoxazolyl-2-mercapto- to present certain characteristics, among which
carboxymethyl-hydrazide of poly (MAVA) (85.16%), biocompatibility and nontoxicity are essential. Lethal
1813

Polymer conjugates with potential biological activity based on new
derivatives of 2-mercaptobenzoxazole-synthesis and characterization
dose 50 (LD50) was determined using Spearman-Karber compounds are almost non-toxic, while the reference
arithmetic method [21] and the results are presented in drug is moderately toxic (LD50 = 176). Therefore, the
Table 2.
The results obtained from the biological tests reveal screening.
that poly(MAVA),the compounds (VIII-X), as well as
new compounds can be used for further laboratory
their corresponding conjugates present lower toxicity, 3.1.2. Tuberculostatic activity
compared to the product previously reported [20], The results obtained following the determination of the
with a slightly higher toxicity shown by the polymeric tuberculostatic activity are presented in Table 3.
support (Table 2). However, compared with isonicotinic
Testingtheinvitrobiologicalactivityoftheinvestigated
hydrazide, the copolymer and the synthesized compounds against the development of Koch bacillus
allowed the establishment of a correlation between their
Table 1. Coupling efficiency and molar ration between compounds
chemical structure and biological potential, as follows:
- Poly (MAVA) has no tuberculostatic potential
(Table 3), but enables the biological activity of the
immobilized compounds, through a new carboxylic
group into the conjugate, appear following the
decyclization of the anhydride ring; therefore, the drug-
polymer conjugates present higher activity than the
small molecules immobilized onto the polymer support
-The enhanced tuberculostatic effect of the
conjugates (XII, XIII, XIV), compared to hydrazides (VIII,
IX, X) is determined by the influence of the carboxy-anilid
support of poly (MAVA) on the rest of benzoxazolyl-2-
mercapto-hydrazide.
(XII- XIV) and poly (MAVA).
Polymer-biologically Coupling
Active compound
active compound
conjugate
efficiency immobilized on poly
(%; wt)
MAVA (mol mol-1)
XII
53.9
50.7
69.4
0.313
0.263
0.436
XIII
XIV
Table 2. Acute toxicity for compounds VIII-X and XII- XIV.
Compound
LD50 (mg kg-1 body)
24 hours 48 hours 7 days Average
-The nature of the side chain from the structure of
2-mercapto-hydrazide derivatives immobilized onto
carboxy anilid support of poly (MAVA) influences their
biologicalactivity.Thus,anincreaseofthetuberculostatic
effect is shown for compound XIV, explained by the
p-π conjugation present at a single CO-NH- group,
in comparison with conjugates XII and XIII. This fact
results in live organisms having an easier cleavage of
benzoxazolyl-2-mercapto-carboxymethyl-hydrazide (X),
responsable for the tuberculostatic activity, of similar
XI
7053
6840
6610
6032
7370
7036
7118
7053
6840
6610
6032
7370
7036
7118
7002
6795
6580
6011
7310
7003
7064
7036
6825
6600
6025
7350
7025
7100
VIII
IX
X
XII
XIII
XIV
Table 3. Tuberculostatic activity of compounds VIII – XIV against M. tuberculosis, vs. isonicotinic hydrazide.
Compound
Active principle concentration
in culture medium (µg mL-1)
MIC*
(µg mL-1)
5
10
20
30
40
6
15
6
15
6
15
6
15
6
15
days
days
days
days
days
days
days
days
days
days
VIII
IX
++
++
++
++
++
++
++
++
++
++
++
++
+
++
++
++
+
++
++
++
+
+
+
+
++
-
-
+
+
++
-
-
-
-
-
-
-
25
30
35
-
-
-
-
X
+
-
-
XI
++
++
++
++
++
XII
XIII
XIV
+
+
-
-
-
-
-
-
-
-
-
-
-
-
-
20
15
10
-
-
-
+
-
-
-
Isonicotinic
hydrazide
-
-
-
-
-
-
-
-
-
-
10
- no microbial growth; + medium microbial growth; ++ high microbial growth* MIC = minimum
inhibitory concentration
1814

R. Aparaschivei et al.
degree as isonicotinic acid, comparing to the hydrazides
The chemical structure of the new compounds
of benzoxazolyl-2-mercaptoformic acid (VIII) and of (VIII-X, XII-XIV) has been confirmed by elemental and
benzoxazolyl-2-mercaptoacetic acid (IX).
spectroscopic analysis (FT-IR, 1H-NMR).
Acute toxicity of VIII-X and XII-XIV has been
evaluated by registering LD50 and revealed that
hydrazides immobilization on copolymer support
determined the decrease of toxicity for compounds
VIII-X. Also, LD50 values classify them as practically
nontoxic, compared to compounds which do not contain
ethyl p-aminobenzoate.
Also, the in vitro biological activity of hydrazides
and conjugates against Mycobacterium tuberculosis,
was evaluated with the conjugates presenting similar
minimum inhibitory concentration as isonicotinic
hydrazide.
4. Conclusions
There have been synthesized three new derivatives from
mercaptobenzoxazole, respectively p-aminobenzoyl-
hydrazide of benzoxazolyl-2-mercaptoformic acid
(VIII), p-aminobenzoyl-hydrazide of benzoxazolyl-2-
mercaptoacetic acid (IX) and benzoxazolyl-2-mercapto-
carboxymethyl-hydrazide of p-aminobenzoic acid (X).
The derivatives have been immobilized by amidic bonds
onto poly(maleic anhydride-alt-vinyl acetate) to obtain
new drug-polymer systems.
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