Immobilization and release studies of triazole derivatives from grafted copolymer based on gellan-carrying betaine units

Article abstract of DOI:10.3390/molecules26113330

New polymer-bioactive compound systems were obtained by immobilization of triazole derivatives onto grafted copolymers and grafted copolymers carrying betaine units based on gellan and N-vinylimidazole. For preparation of bioactive compound, two new types of heterocyclic thio-derivatives with different substituents were combined in a single molecule to increase the selectivity of the biological action. The 5-aryl-amino-1,3,4 thiadiazole and 5-mercapto-1,2,4-triazole derivatives, each containing 2-mercapto-benzoxazole nucleus, were prepared by an intramolecular cyclization of thiosemicarbazides-1,4 disubstituted in acidic and basic medium. The structures of the new bioactive compounds were confirmed by elemental and spectral analysis (FT-IR and¹H-NMR). The antimicrobial activity of 1,3,4 thiadiazoles and 1,2,4 triazoles was tested on gram-positive and gram-negative bacteria. The triazole compound was chosen to be immobilized onto polymeric particles by adsorption. The Langmuir, Freundlich, and Dubinin–Radushkevich adsorption isotherm were used to describe the adsorption equilibrium. Also, the pseudo-first and pseudo-second models were used to elucidate the adsorption mechanism of triazole onto grafted copolymer based on N-vinylimidazole and gellan (PG copolymer) and grafted copolymers carrying betaine units (PGB1 copolymer). In vitro release studies have shown that the release mechanism of triazole from PG and PGB1 copolymers is characteristic of an anomalous transport mechanism.

Full text of DOI:10.3390/molecules26113330

molecules
Article
Immobilization and Release Studies of Triazole Derivatives
from Grafted Copolymer Based on Gellan-Carrying
Betaine Units
Nicolae Baranov 1,2, Stefania Racovita 3, Silvia Vasiliu 3,*, Ana Maria Macsim 3 , Catalina Lionte 4, Valeriu Sunel 2,
Marcel Popa 1,5,
*
, Jacques Desbrieres 6 and Corina Cheptea 7
1
Department of Natural and Synthetic Polymers, Faculty of Chemical Engineering and Environmental
Protection, Gheorghe Asachi Technical University of Iasi, Prof. Dr. Docent Dimitrie Mangeron Street, No. 73,
700050 Iasi, Romania; Baranov_nicolae@yahoo.com
Faculty of Chemistry, Al. I. Cuza University, Carol I Bvd., No. 11, 700506 Iasi, Romania; vsunel@uaic.ro
Department of “Mihai Dima” Functional Polymers, Petru Poni Institute of Macromolecular Chemistry,
Grigore Ghica Voda Alley, No. 41A, 700487 Iasi, Romania; stefania.racovita@icmpp.ro (S.R.);
macsim.ana@icmpp.ro (A.M.M.)
Faculty of Medicine, Gr. T. Popa University of Medicine and Pharmacy, Universitatii Street, No. 16,
700115 Iasi, Romania; clionte@yahoo.com
Academy of Romanian Scientists, Splaiul Independentei Street No. 54, 050085 Bucuresti, Romania
Institut des Sciences Analytiques et de Physico-Chimie pour l’Environnement et les Materiaux (IPREM), Pau
and Pays de l’Adour University (UPPA), UMR CNRS 5254, Helioparc Pau Pyrenees, 2 av. President Angot,
CEDEX 09, 64053 Pau, France; jacques.desbrieres@univ-pau.fr
2
3
4
5
6
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
7
Department of Biomedical Sciences, Faculty of Biomedical Bioengineering, Gr. T. Popa University of Medicine
and Pharmacy, Kogalniceanu Street No. 9–13, 700454 Iasi, Romania; coricheptea@yahoo.com
Correspondence: silvia.vasiliu@icmpp.ro (S.V.); marpopa2001@yahoo.fr (M.P.)
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Baranov, N.; Racovita, S.;
Vasiliu, S.; Macsim, A.M.; Lionte, C.;
Sunel, V.; Popa, M.; Desbrieres, J.;
Cheptea, C. Immobilization and
Release Studies of Triazole
*
Abstract: New polymer-bioactive compound systems were obtained by immobilization of triazole
derivatives onto grafted copolymers and grafted copolymers carrying betaine units based on gellan
and N-vinylimidazole. For preparation of bioactive compound, two new types of heterocyclic thio-
derivatives with different substituents were combined in a single molecule to increase the selectivity
of the biological action. The 5-aryl-amino-1,3,4 thiadiazole and 5-mercapto-1,2,4-triazole derivatives,
each containing 2-mercapto-benzoxazole nucleus, were prepared by an intramolecular cyclization
of thiosemicarbazides-1,4 disubstituted in acidic and basic medium. The structures of the new
bioactive compounds were confirmed by elemental and spectral analysis (FT-IR and 1H-NMR). The
antimicrobial activity of 1,3,4 thiadiazoles and 1,2,4 triazoles was tested on gram-positive and gram-
negative bacteria. The triazole compound was chosen to be immobilized onto polymeric particles by
adsorption. The Langmuir, Freundlich, and Dubinin–Radushkevich adsorption isotherm were used
to describe the adsorption equilibrium. Also, the pseudo-first and pseudo-second models were used
to elucidate the adsorption mechanism of triazole onto grafted copolymer based on N-vinylimidazole
and gellan (PG copolymer) and grafted copolymers carrying betaine units (PGB1 copolymer). In vitro
release studies have shown that the release mechanism of triazole from PG and PGB1 copolymers is
characteristic of an anomalous transport mechanism.
Derivatives from Grafted Copolymer
Based on Gellan-Carrying Betaine
Units. Molecules 2021, 26, 3330.
https://doi.org/10.3390/
molecules26113330
Academic Editor: Roberta Cassano
Received: 29 April 2021
Accepted: 29 May 2021
Published: 1 June 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Keywords: grafted copolymer; triazole derivative; adsorption studies
Copyright:
© 2021 by the authors.
1. Introduction
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
The design of new bioactive compounds that can be immobilized onto a polymeric
support to achieve a controlled/sustained release and to find a way to treat some diseases
represents an important goal in medical and pharmaceutical research [1,2].
Depending on the radicals present on the heterocycle, the organic compounds that
contain in their molecule 1,3,4-thiadiazole and 1,2,4-triazole heterocycles have gained a
remarkable interest due to their different biological properties, such as: antibacterial [3–5]
,
Molecules 2021, 26, 3330. https://doi.org/10.3390/molecules26113330
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3330
2 of 23
antifungal [
6–9
], tuberculostatic [10
,11], analgesic [12
,13], anti-inflammatory [14,15], hyp-
notic and sedative [16], cardioprotective [17], anticancer [18
–22], anti-ulcerative [23], anti-
convulsant [24], antidiabetic agents [25], allosteric modulators [26], and cathepsin B and
tubulin inhibitors [27,28].
At the same time, the addition of a benzoxazole structure can be useful for the im-
provement of the biological activity. An argument in favor of this statement is that the
compounds with benzoxazole heterocycle in their structure have practically the same
biological properties like thiadiazole and triazole derivatives, such as antimicrobial, anti-
fungal, antiviral, anti-inflammatory, antipyretic, analgesic, anticonvulsant, tuberculostatic,
and antitumor agents [29–36]. Benzoxazole and triazole heterocyclic rings played an im-
portant role in the improving of antimicrobial and anticancer activity of the bioactive
compounds [37].
These facts served as an argument in the synthesis of new heterocyclic compounds con-
taining 1,3,4-thiadiazole and 1,2,4-triazole nucleus and the rest of 2-mercaptobenzoxazole,
considering their mutual influence as well as the biological effect that can be generated
throughout the molecule.
Although the researches on compounds with a benzoxazole structure are multiple
and varied [38–41], some aspects of the synthesis and studies of these types of compounds
in which both thiadiazole and triazole structures are grafted on the aromatic heterocycle,
2-mercaptobenzoxazole, have not been studied. Based on this premise, we expanded our
research to the synthesis of new derivatives of 1,3,4-thiadiazole and 1,2,4-triazole classes of
compounds using 1,4-disubstituted thiosemicarbazides as intermediates.
Also, the choice of macromolecular support for immobilization of bioactive com-
pounds is very important. Encouraged by the preliminary results obtained in our previous
study [42], we chose to use for the immobilization of these new bioactive compounds two
types of macromolecular supports: grafted copolymers and grafted copolymers carrying
betaine units based on gellan and N-vinylimidazole (PG and PGB1, respectively). The
structure of PG and PGB1 copolymers are presented in Figure 1.
Figure 1. Chemical structure of PG and PGB1 graft porous copolymers.
In this paper, our investigations were directed to the following directions:
•
Synthesis of new bioactive compounds in which thiadiazole and triazole structures
are grafted onto the aromatic heterocycle, 2-mercaptobenzoxazole, followed by the
choice of the compound with the best biological properties in order to be immobilized
on a polymeric support;
•
•
Synthesis of new polymer-bioactive compound systems; and
Detailed studies on the immobilization of the bioactive compound by adsorption on
grafted copolymers and those containing the betaine structure.

Molecules 2021, 26, 3330
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2. Results and Discussion
2.1. Synthesis of Thiadiazole and Triazole Derivatives
Among the heterocyclic combinations, the 1,3,4-thiadiazole and 1,2,4-triazole deriva-
tives present a remarkable application interest. The synthesis of 1,3,4-thiadiazole and
1,2,4-triazole derivatives were performed in several steps as follows:
Step 1. Synthesis of 1-(benzoxazole-20-yl-mercapto-acetyl)-4-aryl-thiosemicarbazides
(II–VII) was performed in methanol solution by heating a mixture of benzoxazolyl-2-
mercapto-acetic acid hydrazide (I) [43] with various types of isothiocyanate derivatives:
phenyl, p-tolyl, p-methoxyphenyl, p-bromophenyl, p-chlorophenyl, and p-iodofenil, ac-
cording to the procedures described in our previous papers for other types of thiosemicar-
bazides (Figure 2) [4,44,45].
0
Figure 2. Synthesis of 1-(benzoxazole-2 -yl-mercapto-acetyl)-4-aryl-thiosemicarbazides (II-VII).
The structure elucidation of the compounds II–VII was carried out by elemental
analysis as well as by spectroscopic methods (FT-IR and 1H-NMR).
The results obtained from the elemental analysis as well as the chemical formula and
the yield of reaction of all bioactive compounds synthesized in this paper (compounds
II-XIX) are presented in Table 1.
Table 1. Some characteristics of compounds II–XIX.
Elemental Analysis, Calc (Found) (%)
Br
1,4-disubstituted thiosemicarbazides (compounds II–VII)
Chemical
Formula
Yield
(%)
Samples
C
H
N
S
O
Cl
I
53.6
(53.9)
54.8
(55.1)
52.6
(52.9)
43.9
(44.1)
48.9
3.9
(4.2)
4.3
(4.5)
4.1
(4.3)
2.9
(3.2)
3.3
15.6
(16)
15.1
(15.4)
14.4
(14.8)
12.8
(12.9)
14.3
(14.6)
11.6
17.9
(18.2)
17.2
(17.6)
16.4
(16,9)
14.6
(15)
8.9
(7.7)
8.6
(7.4)
12.5
(11.1)
7.4
(6.3)
8.2
(6.8)
6.6
II
III
IV
V
-
-
-
-
-
-
-
-
-
-
-
-
C16H14N4O2S2
C17H16N4O2S2
C17H16N4O3S2
C16H13N4O2S2Br
C16H13N4O2S2Cl
C16H13N4O2S2I
65
75
85
80
78
77
18.3
(18.6)
16.3
(16.6)
13.2
9
(9.5)
VI
VII
-
(49)
39.7
(39.9)
(3.6)
2.7
(2.9)
26.2
(26.6)
-
-
(11.9)
(13.6)
(5.2)
1,3,4-thiadiazoles (compounds VIII–XIII)
56.5
(56.6)
57.6
(58)
52.1
(52.4)
45.8
(46.2)
51.3
(51.4)
41.2
(41.5)
3.5
(3.8)
4
(4.1)
3.8
(4)
2.6
(2.8)
2.9
(3.1)
2.4
16.5
(16.8)
15.8
(16.1)
15.1
(15.4)
13.4
(13.6)
15
18.8
(19.2)
18.1
(18.5)
17.3
(17.6)
15.3
(15.7)
17.1
4.7
(3.6)
4.5
(3.3)
11.7
(10.6)
22.9
(21.7)
13.7
VIII
IX
-
-
-
-
-
-
-
-
-
-
-
-
C16H12N4OS2
C17H14N4OS2
C17H14N4O2S2
C16H11N4OS2Br
C16H11N4OS2Cl
C16H11N4OS2I
89
83
81
79
74
71
X
19
(19.4)
XI
9.5
(9.7)
XII
XIII
-
(15.3)
12
(12.3)
(17.4)
13.7
(14.1)
(12.8)
30.7
(29.2)
27.3
(27.5)
-
-
(2.6)

Molecules 2021, 26, 3330
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Table 1. Cont.
Elemental Analysis, Calc (Found) (%)
Chemical
Formula
Yield
(%)
Samples
C
H
N
S
O
Br
Cl
I
1,2,4-triazoles (compounds XIV–XIX)
56.5
XIV
3.5
(3.9)
4
(4.3)
3.8
(4)
2.6
(3)
2.9
(3.3)
2.4
(2.5)
16.5
(16.9)
15.8
(16.2)
15.1
(15.5)
13.4
(13.7)
15
18.8
(19.2)
18.1
(18.3)
17.3
(17.7)
15.3
(15.7)
17.1
4.7
(3.3)
4.5
(3.4)
8.7
(7.5)
22.9
(21.6)
13.7
(12.7)
30.7
(29.7)
-
-
-
-
-
-
-
-
-
-
-
-
C16H12N4OS2
C17H14N4OS2
C17H14N4O2S2
C16H11N4OS2Br
C16H11N4OS2Cl
C16H11N4OS2I
66
74
88
79
77
74
(56.7)
57.6
(57.8)
XV
55.1
(55.3)
XVI
45.8
(46)
19
(19.3)
XVII
51.3
XVIII
9.5
(9.9)
-
(51.4)
(15.2)
12
(12.4)
(17.4)
13.7
(14)
41.2
XIX
27.3
(27.6)
-
-
(41.4)
In the FT-IR spectra (Figure not shown), the following characteristic absorption bands
can be observed: 700–749 cm−1 assigned to the C-S stretching vibration; 1400–1498 cm−1
attributed to the CH2 bending vibration; 1620–1704 cm−1 assigned to the C=O stretching
vibrations; the C=S stretching vibration was observed at 1120–1150 cm−1; NH stretching
vibration appears at 2880–3150 cm−1; the absorption bands of the mono and disubstituted
aromatic rings were identified in the region of 680–789 cm−1; and the C-Br, C-Cl, and C-I
stretching vibrations appear at 629 cm−1, 725 cm−1, and 630 cm−1, respectively.
In the 1H-NMR spectra, the two methylene protons generate a singlet at δ = 4.25–4.60 ppm
and at δ = 8.67–8.89 ppm, while the protons from the -NH group appear at 10.06–10.85 ppm
.
The aromatic protons in phenyl and benzoxazole appear within the range 6.50–7.92 ppm
.
The protons belonging to the methyl groups are identified at
δ = 2.11 ppm for the compound
III and at δ = 3.38 ppm for the thiosemicarbazide IV (Figure 3).
Step 2. Synthesis of new 1,3,4-thiadiazoles and 1,2,4-triazoles starting from 1,4-
disubstituted-thiosemicarbazides (compounds II–VII). The 5-aryl-amino-2-substituted-
1,3,4-thiadiazoles (compounds VIII–XIII) were obtained by intramolecular cyclization in
concentrated sulfuric acid medium, while the 5-mercapto-3,4-disubstituted -1,2,4-triazoles
(compounds XIV–XIX) were prepared under the catalytic action of hydroxyl ions, according
to our previously work described in literature (Figure 4) [4,44–47].
The idea of synthesis of grafted 1,3,4-thiadiazoles on the 2-mercapto-benzoxazole
molecule is an extremely interesting possibility to find new derivatives, which can be active
against some microbial strains.
The mechanism of the synthesis reaction of these types of heterocyclic compounds is
presented in Figure 5 and contains the following steps:
In case of 1,2,4-triazoles, the mechanism of cyclization in the presence of hydroxyl
ions (Figure 6) begins with the hydrogen extraction from the nitrogen atom situated at
position 4 of 1,4-disubstituted thiosemicarbazide, which has an increased mobility due
to the p-
and the
π
conjugation between the lone pair electrons belonging to the nitrogen atom
π
electrons of thionic bond or of benzene nucleus, respectively. As a result of this
conjugation, a negative charge appears on the nitrogen atom from position 4 that can be
in equilibrium with another anion formed at the sulfur atom. In this mesomer ion, the
negatively charged nitrogen atom (more stable) attacks the carbon belonging to the carbonyl
group simultaneously with
π electron delocalization, leading to the formation of the five-
membered heterocycle, the negative charge being located at the oxygen atom outside
the ring. This structure is stabilized by eliminating the hydroxyl group in the reaction
medium leading to the formation of 5-mercapto-1,2,4-triazole structure. Depending on
the preparation conditions and the reactant nature, as well as due to the presence of
thioamide group in triazole molecule, 5-mercapto-3,4 disubstituted-1,2,4-triazoles show

Molecules 2021, 26, 3330
5 of 23
the phenomenon of double reactivity, reacting as if they had a thionic structure (A) or a
thiol structure (B). This behavior explains the presence of a reaction center at the sulfur
atom from 5-position of five-membered heterocycle [48].
0
Figure 3. 1H-NMR spectra of 1-(benzoxazole-2 -yl-mercapto-acetyl)-4-(p-methoxyphenyl)-thiosemicarbazide (compound IV).
Figure 4. Synthesis of 1,3,4-thiadiazoles (compounds VIII–XIII) and 1,2,4-triazoles (compounds XIV–XIX).
Figure 5. Intramolecular cyclization mechanism of reaction synthesis of 5-aryl-amino-2-substituted-1,3,4-thiadiazoles.

Molecules 2021, 26, 3330
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Figure 6. Cyclization mechanism of reaction synthesis of 5-mercapto-3,4-disubstituted-1,2,4-triazoles.
In order to obtain the information about the structure of 1,3,4-thiadiazoles (compounds
VIII–XIII) and 1,2,4-triazoles (compounds XIV–XIX), the elemental analysis (Table 1) as
well as the FT-IR (Figure 7) and 1H-NMR spectra (Figure 8) were performed.
Generally, in FT-IR spectra of the 1,3,4-thiadiazole (compounds VIII–XIII), t−h1e fol-
lowing absorption bands can be observed: the absorption bands at 3318–2880 cm cor-
responding to the NH stretching vibrations; the absorption band at 1438–1462 cm−1 can
be attributed to the stretching vibration of -N=C-S group; the bending vibration of the
CH2 group has been assigned to the absorption bands situated between 1401–1496 cm−1
;
characteristic CH bands from mono and disubstituted benzene nucleus are assigned to
the absorption band situated in the range of 680–789 cm−1; and the C-Br, C-Cl, and C-I
stretching vibrations appear at 629, 725, and 630 cm−1, respectively.
In the case of 1,2,4-triazole derivatives, the FT-IR spectra of the compounds XIV–X−IX1
present a wide band at 3553 cm−1 due to the NH stretching vibrations; 1613–1628 cm
corresponding to the C=N stretching vibration; the C=S group appears at 1490–1495 cm−1
while the bands at 837–942 cm−1 can be attributed to the para-disubstituted benzene
nucleus; the S-CH2 stretching vibration has been assigned to an adsorption band at
765–785 cm−1; and the C-Br, C-Cl, and C-I stretching vibrations show maximum adsorption
bands at 753, 746, and 748 cm−1, respectively.
For example, in Figure 7, the spectra of compound VIII (a) and compound XVI (b),
respectively, are presented.
1H-NMR spectra bring additional arguments regarding the structure of the obtained
organic compounds. In case of 1,3,4-thiadiazole derivatives, the protons of the CH2 group
bound to the sulfur atom situated at position 20 of the benzoxazole nucleus appear as a
singlet in the region of 4.25–4.53 ppm. The proton bound to the nitrogen could be detected
as a singlet in the region 10.27–10.98 ppm. For all types of 1,3,4-thiadiazoles, aromatic
protons appear at 6.91–7.56 ppm and 8.21–8.57 ppm, respectively. The protons of the
methyl group in 1,3,4-thiadiazoles (compounds IX and X) are found as a triplet at 2.98 and
3.38 ppm, respectively.
The 1H-NMR spectra of 1,3,4-thiadiazoles (compound VIII) and 1,2,4-triazoles (com-
pound XVI) are presented in Figure 8.
The 1H-NMR spectra of the 1,2,4-triazole derivatives (compounds XIV–XIX) present a
singlet due to the protons of the CH2 group at 4.17–4.18 ppm, while at 14.04–14.33 ppm is
observed a singlet specific to the proton belonging to the NH group. For all 1,2,4-triazoles,
the aromatic protons appear at 6.99–7.85 ppm, and for compounds XV and XVI, the protons
of the methyl group appear at 2.43 and 3.43 ppm, respectively.

Molecules 2021, 26, 3330
7 of 23
Figure 7. FT-IR spectrum of (a) 1,3,4-thiadiazoles (compound VIII) and (b) 1,2,4-triazole (compound XVI).
1
Figure 8. H-NMR spectra of (a) 1,3,4-thiadiazoles (compound VIII) and (b) 1,2,4-triazoles (compound XVI).
2.2. Antibacterial Activities of Bioactive Compounds
The antimicrobial activity of 1,3,4 thiadiazoles and 1,2,4 triazoles was tested according
to the National Committee for Clinical Laboratory Standards Institute NCCLS Approval
Standard Document M2-A5, Vilanova, PA, USA (2000), on the following microbial strains:
gram-positive bacteria (Staphylococcus aureus (ATCC-2593), Bacillus subtilis (ATCC-6638),
Bacillus cereus (ATCC-10876)) and gram-negative bacteria (Salmonella enteritidis (P-1131)
and Escherichia coli (ATCC-25922)).

Molecules 2021, 26, 3330
8 of 23
The results regarding the antimicrobial activity of the bioactive compounds synthe-
sized can be seen in Table 2.
Table 2. Antibacterial spectra of 1,3,4-thiadiazoles and 1,2,4- triazoles.
Staphylococcus
aureus
Salmonella
enteritidis
Bacillus subtilis
Bacillus cereus
Escherichia coli
Time for
Thermostating
(h)
Sample Code
mg/mL
mg/mL
mg/mL
mg/mL
mg/mL
1
0.5 0.25
1
0.5 0.25
1
0.5 0.25
1
0.5 0.25
1
0.5 0.25
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
-
-
+
-
•
-
-
-
-
•
•
+
+
-
+
-
+
+
+
+
-
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
-
+
+
+
+
+
+
-
-
-
-
-
•
•
•
-
•
•
•
•
•
•
•
-
•
•
•
•
•
•
•
•
•
•
•
•
-
VIII
IX
-
•
•
•
•
+
+
•
•
•
•
-
•
•
•
•
•
•
•
+
+
+
•
+
-
-
-
•
•
•
•
•
•
•
•
•
•
-
•
•
•
+
•
•
•
•
•
•
-
+
-
-
-
-
•
+
•
+
•
+
-
+
+
+
+
+
+
+
+
+
+
-
+
•
+
•
+
•
+
•
+
•
+
-
•
•
•
•
+
•
•
•
•
•
+
-
-
X
-
+
+
+
+
+
+
+
•
+
•
+
•
+
•
+
•
+
•
+
-
+
+
+
+
+
+
+
•
+
•
+
•
+
•
+
•
+
•
+
-
•
•
•
•
•
•
•
-
-
-
-
-
•
+
+
+
+
+
-
+
-
-
XI
XII
XIII
XIV
•
•
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
XV
-
-
-
•
+
•
+
•
+
-
•
•
-
-
-
•
+
•
+
•
+
-
-
-
-
-
XVI
-
•
•
+
•
+
•
+
-
-
-
-
-
-
-
•
+
•
+
•
+
-
•
+
•
+
•
+
-
•
+
•
+
•
+
-
•
+
•
+
•
+
-
•
+
•
+
•
+
+
+
•
+
•
+
-
+
-
•
+
•
+
•
+
-
•
+
•
+
•
+
-
XVII
XVIII
XIX
Kanamycine
-
-
-
-
-
-
-
-
•
-
-
-
-
+
- no growth; • moderate growth; + normal growth.
Experimental data shows that 1,3,4-thiadiazoles (compounds VIII–XIII) are sensitive
to Staphyloccocus aureus and present a moderate effect on Bacillus subtilis and Escherichia coli,
while the culture of Bacillus cereus and Salmonella enteritidis are resistant to the action of the
1,3,4-thiadiazole test.
The 1,2,4-triazoles (compounds XIV–XVI) have been shown to be strong inhibitors
on the growth of the bacteria Escherichia coli, Salmonella enteritidis, and Bacillus subtilis
in any concentration and regardless of the contact time, while against Staphylococcus
aureus and Bacillus cereus germs, the 1,2,4-triazoles show a moderate action. In contrast,
the 1,2,4-triazoles (compounds XVII–XIX) show a moderate action on all tested germs
only in a short period of contact time and at all concentrations. It can be stated that the
tested 1,3,4-thiadiazoles and 1,2,4-triazoles show appreciable antimicrobial activity against
microbial strains similar to that of the model drug, kanamycin.
2.3. Immobilization Studies of Triazoles
Among the new synthesized compounds, the 1,2,4-triazole derivative obtained with
the highest yield (88%) and the best antimicrobial activity, namely 3-(benzoxazole-20-yl-
mercapto-methyl)-4-(p-methoxyphenyl)-5-mercapto-1,2,4-triazole (compound XVI), which
was selected for immobilization by sorption onto grafted copolymers.
The interaction between PG and PGB1 copolymers and compound XVI was high-
lighted by FT-IR spectroscopy (Figure 9).

Molecules 2021, 26, 3330
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Figure 9. FT-IR spectra for (A) PG-T system; (B) PGB1-T system.
•
For PG—1,2,4-triazole systems (PG-T), it can be observed some new absorption bands
as follows: the absorption band at 3140 cm−1 due to the stretching vibration of -NH
group; 1542 cm−1 assigned to the stretching vibration of C=S grou−p1; 1450 cm−1
corresponding to the stretching vibration of -N=C-S group; and 793 cm assigned to
the stretching vibration of S-CH2 group.
•
For PGB1—1,2,4-triazole systems (PGB1-T), the changes that occur in the FTIR spec-
trum are the following: the band vibration of 3439 cm−1 from PGB1 copolymer is
shifted in PGB1-T spectra to 3445 cm−1 due to the overlap of the absorption band of
-C=N+ group from PGB1 copolymer, with absorption band corresponding to the -NH
group belonging to the 1,2,4-triazole; absorption band at 832 cm−1 is assigned to the
stretching vibration of C-H out of plane bending.
Also, XRD and SEM studies were performed for a better characterization of PG-T
and PGB1-T systems. The XRD patterns of 1,2,4-triazole, PG-T, and PGB1-T are presented
in Figure 10.
The XRD analysis of PG-T and PGB1-T systems showed the combined signals of both
PG/PGB1 copolymers and 1,2,4-triazole, leading to the conclusion that the 1,2,4-triazole
was successfully immobilized onto polymeric supports.
Visualization of surface morphology of PG-T and PGB1-T systems was achieved
through SEM microscopy with a magnification of 1000, and the images are presented
in Figure 11.

Molecules 2021, 26, 3330
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Figure 10. XRD patterns of 1,2,4-triazole, PG-T, and PGB1-T systems (a) and of PG and PGB1 copolymers (b).
Figure 11. SEM images for 1,2,4-triazole, PG-T, and PGB1-T systems.
From SEM images, it can be observed that the PG-T and PGB1-T systems appear to
have porous structures different from those of PG and PGB1 copolymers. The changes
in the surface morphologies of PG-T and PGB1-T systems indicate that the sorption of
1,2,4-triazole onto polymeric support occurred.
Generally, in order to evaluate a sorption process of bioactive compound, it is impor-
tant to consider two physico-chemical aspects, namely the equilibrium and the kinetics
of sorption.
2.3.1. Sorption Isotherms
To describe the interactions between sorbate and sorbent that occurs in sorption
processes, three model isotherms were used, namely: Langmuir, Freundlich, and Dubinin–
Radushkevich.
•
Langmuir isotherm
The Langmuir isotherm describes the sorption in a homogeneous system [49] and is
given by the equation:
qm·KL·Ce
1 + KL·Ce
qe =
(1)

Molecules 2021, 26, 3330
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where:
q
e represents the amount of 1,2,4-triazole sorbed at equilibrium (mg/g);
q
m repre-
sents the maximum amount sorbed (mg/g);
Ce is the concentration of 1,2,4-triazole solution
at equilibrium (mg/L); and
sorbate and sorbent.
KL is the Langmuir constant that reflects the affinity between
To determine whether the sorption system used is favorable or unfavorable to the
sorption, the equilibrium parameter RL, given by the equation [50], was also calculated:
1
RL =
(2)
1 + KL·Ci
where Ci represents the initial concentration. According to the literature [51], if RL < 1,
then the sorption is unfavorable, for RL = 1, the sorption is linear; if 0 < RL < 1, the sorption
is favorable or irreversible if RL = 0.
•
Freundlich isotherm
Another model used in sorption studies is the Freudlich isotherm. This isotherm model
is applied in the case of multilayer sorption of sorbate on a heterogeneous surface [52]. The
Freundlich isotherm is described by the following equation:
q = KF·C1e/n
(3)
f
e
where KF is the Freundlich constant which represents the amount of 1,2,4-triazole sorbed
per gram of sorbent when the equilibrium concentration is equal to unity (L/g); 1/nf
indicates the type of isotherm as follows: favorable 0 < 1/nf < 1 or unfavorable 1/nf >1.
•
Dubinin–Radushkevich isotherm
The Dubinin–Radushkevich isotherm is an empirical model designed to estimate
the apparent free energy of sorption as well as to differentiate between the physical and
chemical sorption process [53]. The Dubinin–Radushkevich model equation is given by the
following mathematical relation:
(
)
ꢀ
ꢁ
ꢂꢃ
2
1
Ce
qe = qDR·exp −β RTln 1 +
(4)
where
q
DR represents the maximum amount sorbed (mg/g);
β
is the Dubinin–Radushkevich
constant (mol2/kJ2);
temperature (K).
R
represents the ideal gas constant (
R
= 8.314 kJ/mol K); and is the
·
T
The Dubinin–Radushkevich isotherm constant, β, is associated with the average free
sorption energy, E (kJ/mol), calculated using the following equation:
E = 1(2β)21
(5)
The value of
The sorption process can be physical when the
exchange for values of between 8 and 16 kJ/mol, and chemical nature for values of
greater that 16 kJ/mol [54].
E
is used to obtain information about the nature of the sorption process.
E
values are between 1 and 8 kJ/mol, ion
E
E
The parameters of the studied isotherms as well as the values of the error functions
R2 and χ2 are presented in Table 3.

Molecules 2021, 26, 3330
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Table 3. Parameter values corresponding to Langmuir, Freundlich, and Dubinin–Radushkevich isotherms calculated in case
of 1,2,4-triazole sorption on PG and PGB1 copolymers at different temperatures.
PG
PGB1
303
298
303
308
298
308
Langmuir model
636
qm (mg/g)
KL (L/mg)
478
0.068
533
0.086
537
0.098
616
0.126
685
0.161
0.109
RL
χ2
R2
0.01–0.29
3.324
0.994
0.01–0.25
2.613
0.993
0.10–0.20
3.429
0.997
Freundlich model
0.617
0.01–0.22
2.700
0.993
0.01–0.18
2.010
0.992
0.004–0.15
2.150
0.996
KF (L/g)
1/nf
0.457
0.939
33.462
0.911
0.512
0.738
23.958
0.920
0.528
0.686
25.213
0.913
0.602
0.540
19.219
0.921
0.673
0.383
18.782
0.914
0.729
38.332
0.915
χ2
R2
Dubinin–Radushkevich model
qDR (mg/g)
463
521
629
532
612
679
E (kJ/mol)
1.099
0.402
0.998
1.561
0.359
0.997
2.171
0.413
0.998
1.357
0.371
0.998
2.608
0.226
0.999
3.714
0.277
0.998
χ2
R2
From data presented in Table 3, the following conclusions can be drawn:
The theoretical values obtained for the maximum sorption capacity ( m) calculated on
the basis of the Langmuir isotherm are close to the experimental values c (457, 512
and 618 mg 1,2,4-triazole /g PG copolymer and 529, 601 and 672 mg of 1,2,4-triazole
/g PGB1 copolymer);
•
•
q
q
The values of the equilibrium parameter,
RL, were in the range between 0 and 1,
thus confirming that the PG and PGB1 copolymers are favorable supports for the
sorption of 1,2,4-triazoleat at the three temperatures studied. It is also observed that
the KL values are higher in the case of the PGB1 copolymer than in the case of the PG
copolymer, which indicates a higher affinity of the PGB1 copolymer for 1,2,4-triazole;
this is in agreement with the highest sorption capacity obtained in the case of the
PGB1 copolymer;
•
•
The values for R2 and χ2 are in the range of 0992–0.997 and 2.010–3.429, respectively,
indicating that the Langmuir isotherm describes well the experimental data;
Although the values of 1/nf are in the range of 0–1, which indicates that the Fre-
undlich isotherm is favorable in the case of 1,2,4-triazolesorption onto PG and PGB1
copolymers, the small values of R2 (0.911 to 0.921) associated with high values of
χ2 (18.782–38.332) shows that the Freundlich isotherm does not describe well the
experimental data;
•
•
The analysis of the parameter values obtained by applying the Dubinin–Radushkevich
isotherm shows that the qDR values are very close to the experimental values, which in-
dicates that this isotherm describes well the experimental data. This is also supported
by the fact that high values for R2 (0.997–0.999) and low values for χ2 (0.226–0.413)
were obtained, confirming that the Dubinin–Radushkevich isotherm describes very
well the sorption of 1,2,4-triazole onto PG and PGB1 copolymers;
The calculated values of the average free energy of 1,2,4-triazole sorption onto PG
and PGB1 copolymers are in the range of 1.099–3.714 kJ/mol, which indicates that the
sorption process studied is physical in nature.
2.3.2. Thermodynamic Parameters
By means of thermodynamic parameters, such as Gibbs free energy change (∆G),
enthalpy change (∆H), and entropy change (∆S), the mechanism and the type of sorption
process can be determined. Thus, according to the literature [55], it is known that in the case

Molecules 2021, 26, 3330
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of physical sorption, the values of ∆H are less than 40 kJ/mol, while in the case of chemical
sorption, the values of ∆H are in the range of 40–120 kJ/mol. The value of ∆H and ∆S can
be determined by means of the Langmuir constant using the Van ’t Hoff equation [56]:
∆S ∆H
lnKL =
−
(6)
R
RT
From the linear representation ln KL versus 1/
T
(Figure 12), the values of ∆S and ∆H
were obtained from the intercept and slope, respectively, and the results are presented in
Table 4. The values of ∆G were obtained using the following equation:
∆G = ∆H − T∆S
(7)
Figure 12. Plots of lnKL versus 1/T for sorption of 1,2,4-triazole derivative onto PG and PGB1 copolymers.
Table 4. Thermodynamic parameters of sorption process.
∆G (kJ/mol)
R2
Sample Code
∆H (kJ/mol)
∆S (J/mol·K)
298 K
303 K
308 K
PG
PGB1
36.49
37.67
100.05
107.14
0.995
0.998
−29.78
−31.89
−30.278
−32.425
−30.78
−32.96
As it can be seen from Table 4, the negative values of ∆G show that the 1,2,4-triazole
sorption onto PG and PGB1 copolymers is spontaneous and favorable. Also, the values
of this thermodynamic parameter decrease with increasing of the temperature, indicating
that there is a higher efficiency of the sorption process at higher temperatures. The values
of ∆H < 40 kJ/mol indicate that the interactions between the PG or PGB1 copolymers
and 1,2,4-triazole are physical in nature. On the other hand, the positive value of the ∆H
shows that the sorption process is endothermic and the increase of the temperature leads
to an increase in the amount of 1,2,4-triazole sorbed. The positive value of ∆S indicates the
affinity of PG and PGB1 copolymers for 1,2,4-triazole as well as the increase randomness at
the solid-solution interface during the sorption process [57]. Also, the positive value of ∆S
can indicate an increase in the degree of freedom of 1,2,4-triazole. Moreover, the positive
values of ∆H and ∆S lead to the conclusion that the sorption process occurs spontaneously
at all temperatures.
2.3.3. Sorption Kinetic Study
The study of sorption kinetic describes the rate of sorption and is very important
because it gives us information about the mechanism of sorption. Two mathematical
models, such as the Lagergren model (pseudo-first order kinetic model) and the Ho model
(pseudo-second order kinetic model), were used to elucidate the mechanism of 1,2,4-triazole

Molecules 2021, 26, 3330
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sorption onto PG and PGB1 copolymers. The two models mentioned above are described
by the following equations:
•
Lagergren model [58]:
ꢄ
ꢅ
qt = qe 1 − e−k t
(8)
1
•
Ho model [59]:
k2·q2t ·t
qt =
(9)
1 + k2·qe·t
where
q
e and
q
t are the amounts of 1,2,4-triazole sorbed at equilibrium and at time
t
(mg/g),
k1 is the rate constant of the pseudo-first order sorption process (min−1), and k2 is the rate
constant of pseudo-second order sorption process (g/mg·min).
Figure 13 presents the plots of the Lagergren and Ho models in the case of 1,2,4-triazole
sorption (C1,2,4-triazole = 15
Table 5 shows the parameter values corresponding to the Lagergren and Ho models.
−3
×
10 g/mL) onto PG and PGB1 copolymers at
T
= 308 K, and
Figure 13. Graphical representation of Lagergren and Ho models in case of 1,2,4-triazole sorption
−3
onto PG and PGB1 copolymers at T = 308 K and C1,2,4-triazole = 15 × 10 g/mL.
Table 5. The parameters corresponding to the Lagergren and Ho models used in the case of 1,2,4-triazole sorption onto PG
and PGB1 copolymers.
PG
303
394
PGB1
303
C1,2,4-triazole
(g/mL)
298
325
308
473
298
412
308
587
qe,exp (mg/g)
qe,calc (mg/g)
496
Lagergren model
415.91
3.01
2.263
0.998
346.29
2.19
1.738
0.998
491.95
3.31
1.119
0.998
Ho model
428.82
3.83
1.119
0.999
511.76
3.92
1.682
0.998
600.94
4.14
1.103
0.999
−1
k1 (×103 min
)
χ2
R2
−4
3.6 × 10
qe,calc (mg/g)
449.72
1.26
4.014
0.997
482.38
1.62
4.686
0.997
564.84
2.43
5.976
0.997
579.40
2.89
2.411
0.998
616.92
3.51
5.499
0.997
696.22
3.92
5.040
0.998
k2
[
×
106 (g/mg
·
min)]
χ2
R2

Molecules 2021, 26, 3330
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Table 5. Cont.
PG
303
394
491
PGB1
303
C1,2,4-triazole
(g/mL)
298
325
432
308
473
593
298
412
507
308
qe,exp (mg/g)
qe,exp (mg/g)
496
587
583
659
Lagergren model
qe,calc (mg/g)
473.63
3.10
1.456
0.998
523.96
622.21
4.01
1.569
0.998
531.87
4.13
1.312
0.997
606.53
4.27
1.852
0.998
679.53
4.87
1.786
0.997
−1
k1 (×103 min
)
3.52
1.446
0.999
χ2
R2
−3
3.6 × 10
Ho model
qe,calc (mg/g)
546.70
2.19
2.071
0.997
621.17
2.46
2.539
0.996
709.71
3.11
2.390
0.997
649.28
3.07
2.772
0.995
694.28
3.72
2.083
0.997
766.03
4.01
2.310
0.996
k2
[
×
106 (g/mg
·
min)]
χ2
R2
qe,exp (mg/g)
449
504
606
519
594
667
Lagergren model
qe,calc (mg/g)
463.71
3.42
1.722
0.997
527.91
622.32
4.43
1.654
0.998
533.86
4.60
1.034
0.998
621.43
4.90
1.326
0.997
681.07
5.10
1.764
0.999
−1
k1 (×103 min
)
3.96
1.949
0.997
χ2
R2
−3
7 × 10
Ho model
qe,calc (mg/g)
567.15
3.09
2.609
0.996
613.04
3.87
2.913
0.997
713.83
4.11
1.895
0.996
621.28
3.35
2.776
0.997
734.63
4.19
2.864
0.997
796.60
5.17
3.775
0.998
k2 [×106 (g/mg min)]
χ2
R2
qe,exp (mg/g)
457
512
618
529
601
672
Lagergren model
qe,calc (mg/g)
483.63
3.67
1.466
0.997
532.96
644.21
4.81
1.691
0.998
544.25
4.92
1.144
0.998
627.05
5.14
1.671
0.999
694.28
5.48
1.024
0.999
−1
k1 (×103 min
)
4.67
1.343
0.999
χ2
R2
−3
15 × 10
Ho model
qe,calc (mg/g)
568.17
3.85
3.050
0.996
624.11
4.38
3.208
0.997
754.83
5.83
3.903
0.996
635.77
4.23
3.887
0.998
729.27
4.97
4.321
0.997
783.34
5.73
3.678
0.997
k2 [×106 (g/mg min)]
χ2
R2
From data presented in Table 5, it is observed that the value of the sorption rate
increases with the increase of the 1,2,4-triazole concentration. The highest amount of
1,2,4-triazole sorbed was obtained in the case of the PGB1 copolymer. The qe values
calculated by applying the Lagergren model are close to the experimental values compared
to the qe values calculated by applying the Ho model. Although the values of the correlation
coefficients R2 are higher when applying the two kinetic models, the χ2 values are higher
when applying the Ho model, which indicates that the Lagergren model describes better
the experimental data. These results indicate that 1,2,4-triazole sorption onto PG and PGB1
copolymer is a physical process.
2.4. In Vitro Release Studies
In vitro release studies were performed on the PG-T and PGB1-T systems with the
highest amount of immobilized 1,2,4-triazole. In vitro 1,2,4-triazole release studies were
realized at pH = 1.2, and the release profiles are shown in Figure 14.

Molecules 2021, 26, 3330
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Figure 14. Release profiles of 1,2,4-triazole from PG-T and PGB1-T systems at pH = 1.2.
The kinetic studies and the release mechanism from PG-T and PGB1-T copolymers
loaded with 1,2,4-triazole were examined using the following mathematical models:
•
Higuchi model [60]:
Qt = kH·t1/2
(10)
where
Q
t is the amount of drug released at time
t
;
kH is the Higuchi dissolution constant;
and t is the time.
•
Korsmeyer–Peppas model [61]:
Mt
M∞
= kr·tn
(11)
where Mt
/
M∞ is the fraction of drug released at time
t
;
kr is the release rate constant
is the diffusion
that is characteristic to bioactive compound-polymer interactions; and
n
coefficient that is characteristic to the release mechanism. The values of the 1,2,4-triazole
release parameters from PG-T and PGB1-T systems are presented in Table 6.
Table 6. Kinetic parameters of 1,2,4-triazole release from PG-T and PGB1-T systems.
Higuchi Model
Korsmeyer–Peppas Model
Sample Code
−1/2
kH (h
0.324
0.294
)
R2
kr (min
n
R2
−n
)
PG-T
PGB1-T
0.993
0.994
0.014
0.017
0.534
0.634
0.995
0.996
The release exponent n from the Korsmeyer–Peppas equation is situated between
0.534 and 0.634. These results suggest that the release mechanism of 1,2,4-triazole from
PG-T and PGB1-T systems was controlled by more than one process, namely diffusion and
swelling.
3. Materials and Methods
3.1. General Information
All reactants and solvents were purchased from Merck Company KGaA (Darmstadt,
Germany) and were used without purification. Elemental analyses were performed by
Exeter Analytical CF-440 Elemental Analyzer (Coventry, United Kingdom). The bioactive
compounds (1,4-disubstituted-thiosemicarbazides, 1,3,4-thiadiazoles, and 1,2,4-triazoles)
were characterized by FT-IR spectroscopy (Bruker Vertex FT-IR spectrometer) as potassium

Molecules 2021, 26, 3330
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−1
bromide pellets in the range of 4000–400 cm and at a resolution of 2 cm−1. The 1H NMR
spectra were recorded in DMSO-d60 on Bruker ARX 400 Spectrometer (400 MHz). X-ray
diffraction analysis was performed in a Rigaku Miniflex 600 diffrac◦tometer using CuK
of 5◦/min. The surface morphologies of PG-T system, PGB1-T system, and 1,2,4-triazole
in powder form were analyzed with an environmental scanning electron microscope type
Quanta 200 at 25kV with secondary electrons in low vacuum.
α-
◦
emission in the angular range 3–90 (2
θ
) with a scanning step of 0.01 and a recording rate
3.2. Synthesis of Benzoxazole-20-yl-mercapto-acetic Acid Hydrazide (Compound I)
0
It was obtained by treating benzoxazol-2 -yl-mercapto-acetic acid ethyl ester with 98%
hydrazine hydrate in anhydrous ethanol at room temperature.
3.3. General Procedure for Synthesis of 1-(Benzoxazole-20-yl-mercapto-acetyl)-4-aryl-
thiosemicarbazide (Compounds II–VII)
A total of 0.005 moles of benzoxazole-20-yl-mercapto-acetic acid hydrazide (I) dis-
solved in 30 mL absolute methyl alcohol was treated with 0.005 moles of aromatic isothio-
cyanate in 5 mL anhydrous methanol. The mixture was refluxed for 2 h. During heating, a
crystalline precipitate appeared and became more and more abundant once the reaction
was complete. After cooling, the precipitate was filtered and washed with anhydrous ethyl
ether. Purification was carried out by recrystallization from anhydrous methanol.
0
1-(Benzoxazole-2 -yl-mercapto-acetyl)-4-phenyl-thiosemicarbazide (Compound II), Physical state:
White, crystalline powder; Mp = 182–183 ◦C. FT-IR (KBr,
ν
, cm−1): 2917, 3150 (NH); 1670
(C=O); 1400 (CH2); 1145 (C=S); 755 (monosubstituted benzene nucleus); 700 (C-S).
1-(Benzoxazole-20-yl-mercapto-acetyl)-4-(p-tolyl)-tiosemicarbazide (Compound III), Physical
state: White-ivory powder, acicular crystals; Mp = 163–164 ◦C. FT-IR (KBr, , cm−1): 2880,
ν
3096 (NH); 1709 (C=O); 1424 (CH2); 720 (C-S); 750 (para-disubstituted benzene nucleus).
1H-NMR (DMSO-d6, 400 MHz,
δ
ppm): 2.11 (t, 3H, CH3); 4.39 (s, 2H, CH2); 6.95–6.98 (d,
2H, CHAr); 7.13–7.18 (d, 2H, CHAr); 7.57–7.63 (d, 2H, CHAr); 7.76–7.79 (d, 2H, CHAr);
9.86 (s, 1H, NHCO); 10.19 (s, 1H, NH); 10.76 (s, 1H, NH).
1-(Benzoxazole-20-yl-mercapto-acetyl)-4-(p-methoxytolyl) thiosemicarbazide (Compound IV),
◦
Physical state: White, crystalline powder; Mp = 186–188 C. FT-IR (KBr,
3096 (NH); 1623 (C=O); 1492 (CH2); 1239 (C=S); 789 (monosubstituted benzene nucleus);
749 (C-S). 1H-NMR (DMSO-d6, 400 MHz,
ppm): 3.38 (t, 3H, OCH3); 4.38 (s, 2H, CH2);
ν
, cm−1): 2918,
δ
6.09–6.11 (d, 2H, CHAr); 7.11–7.13 (d, 2H, CHAr); 7.41–7.43 (d, 2H, CHAr); 7.84–7.92 (d,
2H, CHAr); 8.86 (s, 1H, NHCO); 10.26 (s, 1H, NH); 10.86 (s, 1H, NH).
0
1-(Benzoxazole-2 -yl-mercapto-acetyl)-4-(p-chlorophenyl) thiosemicarbazide (Compound V), Phys-
◦
ical state: White-ivory, crystalline powder; Mp = 147–149 C. FT-IR (KBr,
ν
, cm−1): 3096 (NH)
;
1620 (C=O); 1482 (CH2); 1249 (C=S); 749 (para-disubstituted benzene nucleus); 710 (C-S);
1
629 (C-Br). H-NMR (DMSO-d6, 400 MHz,
δ ppm): 4.27 (d, 2H, CH2); 7.32–7.35 (d, 2H,
CHAr); 7.38–7.40 (d, 2H, CHAr); 7.52–7.56 (d, 2H, CHAr); 7.65–7.67 (d, 2H, CHAr); 9.67 (s,
1H, NHCO); 9.90 (s, 1H, NH); 10.49 (s, 1H, NH).
0
1-(Benzoxazole-2 -yl-mercapto-acetyl)-4-(p-chlorophenyl)-thiosemicarbazide (Compound VI), Phys-
ical state: White-ivory, crystalline powder; Mp = 150–151 ◦C. FT-IR (KBr,
ν
, cm−1): 3080,
3124 (NH); 1620 (C=O); 1498 (CH2); 1250 (C=S); 725 (C-Cl); 708 (C-S); 680 (para-disubstituted
1
benzene nucleus). H-NMR (DMSO-d6, 400 MHz,
δ
ppm): 4.23 (d, 2H, CH2); 6.92–7.04 (d,
2H, CHAr); 7.29–7.32 (d, 2H, CHAr); 7.39–7.41 (d, 2H, CHAr); 8.77 (s, 1H, NHCO); 10.11 (s,
1H, NH); 10.85 (s, 1H, NH).
0
1-(Benzoxazole-2 -yl-mercapto-acetyl)-4-(p-iodophenyl)-thiosemicarbazide (Compound VII). Phys-
ical state: White-yellow, crystalline powder; Mp = 172–179 ◦C. FT-IR (KBr,
ν
, cm−1): 3097,
3140 (NH); 1620 (C=O); 1490 (CH2); 1120 (C=S); 785 (para-disubstituted benzene nucleus);
712 (C-S); 630 (C-I). 1H-NMR (DMSO-d6, 400 MHz,
δ
ppm): 4.25 (d, 2H, CH2); 6.91–7.03 (d,

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2H, CHAr); 7.30–7.31 (d, 2H, CHAr); 7.40–7.42 (d, 2H, CHAr); 7.66–7.69 (d, 2H, CHAr);
8.74 (s, 1H, NHCO); 10.10 (s, 1H, NH); 10.31 (s, 1H, NH).
3.4. General Procedure for Synthesis of 1-(Benzoxazole-20-yl-mercapto-methyl)-5-(aryl-amino)-
1,3,4,thiadiazoles (Compounds VIII–XIII)
A total of 6 mL of concentrated sulfuric acid were added under stirring to 0.005 moles
0
of 1-(benzoxazole-2 -yl-mercapto-acetyl)-4-aryl-thiosemicarbazide (compounds II–VII). The
reaction mixture was stirred at room temperature for 45–50 min to complete the cyclization
and then was poured on crushed ice when an abundant precipitate appeared. The mixture
was neutralized with ammonium hydroxide, and, after 2 h, the microcrystals were filtered
in vacuum and washed with distilled water until the washing water reached pH = 7. Then,
the obtained compounds were dried in vacuum oven at 55–60 ◦C and were purified by
recrystallization from ethyl alcohol.
2-(Benzoxazole-20-yl-mercapto-methyl)-5-(phenyl-amino)-1,3,4-thiadiazole (Compound VIII),
Physical state: White-yellow, crystalline powder; Mp = 194–196 ◦C. FT-IR (KBr, , cm−1):
ν
2945, 3256 (NH); 1496 (CH2); 1454 (N=C-S); 1401, 1535 (thiadiazole nucleus) 1090 (C-S-C);
785, 815 (para-disubstituted benzene nucleus); 750 (C-S). 1H-NMR (DMSO-d6, 400 MHz,
δ
ppm): 4.26 (s, 2H, CH2); 6.91 (t, 1H, CHAr); 6.98–7.00 (d, 2H, CHAr); 7.46–7.49 (d, 2H,
CHAr); 8.35 (d, 2H, CHAr); 8.71–8.72 (d, 2H, CHAr); 10.48 (s, 1H, NH).
2-(Benzoxazole-20-yl-mercapto-methyl)-5-(tolyl-amino)-1,3,4-thiadiazole (Compound IX), Phys-
ical state: Brown-red, amorphous powder; Mp = 154–156 ◦C. FT-IR (KBr, , cm−1): 2945
ν
(NH); 1480 (CH2); 1450 (N=C-S); 1448, 1517 (thiadiazole nucleus); 1070 (C-S-C); 828 (para-
disubstituted benzene nucleus); 730 (C-S). 1H-NMR (DMSO-d6, 400 MHz,
δ
ppm): 2.29 (t,
3H, CH3); 4.49 (s, 2H, CH2); 7.36–7.38 (d, 2H, CHAr); 7.66–7.69 (d, 2H, CHAr); 8.21–8.23 (d,
2H, CHAr); 8.39–8.42 (d, 2H, CHAr); 10.21 (s, 1H, NH).
0
2-(Benzoxazole-2 -yl-mercapto-methyl)-5-(p-methoxyphenyl-amino)-1,3,4-thiadiazole (Compound X), Phys-
ical state: Brown, crystalline powder; Mp = 116–118 ◦C. FT-IR (KBr,
ν
, cm−1): 2500, 3000
(NH); 1475 (CH2); 1438 (N=C-S); 1243, 1615 (thiadiazole nucleus); 1068 (C-S-C); 830 (para-
disubstituted benzene nucleus); 710 (C-S). 1H-NMR (DMSO-d6, 400 MHz),
δ
ppm: 3.38 (t,
3H, OCH3); 4.53 (s, 2H, CH2); 7.31–7.34 (d, 2H, CHAr); 7.53–7.56 (d, 2H, CHAr); 8.50 (d,
2H, CHAr); 8.86 (d, 2H, CHAr); 10.37 (s, 1H, NH).
0
2-(Benzoxazole-2 -yl-mercapto-methyl)-5-(p-bromophenyl-amino)-1,3,4-thiadiazole (Compound XI), Phys-
ical state: Brown, crystalline powder; Mp = 197–197◦C. FT-IR (KBr,
ν
, cm−1): 2880, 2905
(NH); 1480 (CH2); 1449 (N=C-S); 1240, 1518 (thiadiazole nucleus); 1070 (C-S-C); 828 (para-
1
disubstituted benzene nucleus); 750 (C-Br). H-NMR (DMSO-d6, 400 MHz),
δ
ppm: 4.49
(s, 2H, CH2); 7.21-7.23 (d, 2H, CHAr); 7.47-7.51 (d, 2H, CHAr); 8.19-8.21 (d, 2H, CHAr);
8.33-8.35 (d, 2H, CHAr); 10.98 (s, 1H, NH).
0
2-(Benzoxazole-2 -yl-mercapto-methyl)-5-(p-chlorophenyl-amino)-1,3,4-thiadiazole (Compound XII), Phys-
ical state: Brown-white, crystalline powder; Mp = 201–203 ◦C. FT-IR (KBr,
ν
, cm−1): 3318
(NH); 1475 (CH2); 1450 (N=C-S); 1240, 1518 (thiadiazole nucleus); 1071 (C-S-C); 830 (para-
disubstituted benzene nucleus); 755 (C-Cl). 1H-NMR (DMSO-d6, 400 MHz), ppm: 4.32
δ
(s, 2H, CH2); 7.17–7.19 (d, 2H, CHAr); 7.39–7.42 (d, 2H, CHAr); 8.22–8.24 (d, 2H, CHAr);
8.54–8.57 (d, 2H, CHAr); 10.31 (s, 1H, NH).
0
2-(Benzoxazole-2 -yl-mercapto-methyl)-5-(p-iodophenyl-amino)-1,3,4-thiadiazole (Compound XIII),
Physical state: Brown, crystalline powder; Mp = 187 ◦C. FT-IR (KBr,
ν
, cm−1): 2880, 2918
(NH); 1478 (CH2); 1462 (N=C-S); 1438, 1580 (thiadiazole nucleus); 1070 (C-S-C); 825 (para-
disubstituted benzene nucleus); 760 (C-I). 1H-NMR (DMSO-d6, 400 MHz),
δ
ppm: 4.25
(s, 2H, CH2); 7.28–7.32 (d, 2H, CHAr); 7.40–7.42 (d, 2H, CHAr); 8.16–8.18 (d, 2H, CHAr);
8.47–8.50 (d, 2H, CHAr); 10.27 (s, 1H, NH).

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3.5. General Procedure for Synthesis of 3-(Benzoxazole-20-yl-mercapto-methyl)-4-aryl-5mercapto-
1,2,4-triazoles (Compounds XIV–XIX)
0
A total of 0.0027 moles of 1-(Benzoxazole-2 -yl-mercapto-acetyl)-4-aryl-thiosemicarbazide (II-
VII) were treated with 20 mL of 2N sodium hydroxide solution at room temperature. Then,
the reaction mixture was heated under reflux for 60 min. After that, the mixture was cooled,
diluted with distilled water (v/v), and treated with a diluted solution of hydrochloric
acid (1:1) to pH = 4.5. The precipitates [mercapto-1,2,4-triazole (compounds XIV–XIX)
were filtered, washed on filter with 500 mL of distilled water, and finally dried. After
recrystallization from boiling ethyl alcohol, the finished products were crystalline.
3-(Benzoxazole-20-yl-mercapto-methyl)-4-phenyl-5-mercap◦to-1,2,4-triazoles (Compound XIV),
Physical state: White, crystalline powder; Mp = 239–241 C. FT-IR (KBr,
1622 (C=N); 1490 (C=S); 820 (monosubstituted benzene nucleus); 785 (S-CH2). H-NMR
ν
, cm−1): 3095 (NH);
1
(DMSO-d6, 400 MHz),
δ ppm: 4.78 (s, 2H, CH2); 6.99–7.01 (d, 2H, CHAr); 7.13–7.15 (d, 2H,
CHAr); 7.22 (t, 1H, CHAr); 14.06 (s, 1H, NH).
3-(Benzoxazole-20-yl-mercapto-methyl)-4-(p-tolyl)-5-mercapto-1,2,4-triazole (Compound XV),
Physical state: White-yellow, crystalline powder; Mp = 198–200 ◦C. FT-IR (KBr, , cm−1):
ν
3036 (NH); 1623 (C=N); 1492 (C=S); 898 (para-disubstituted benzene nucleus); 782 (S-CH2).
1H-NMR (DMSO-d6, 400 MHz),
δ
ppm: 2.34 (t, 3H, CH3); 4.51 (s, 2H, CH2); 7.07–7.09 (d,
2H, CHAr); 7.28–7.30 (d, 2H, CHAr); 7.51–7.53 (d, 2H, CHAr); 14.04 (s, 1H, NH).
0
3-(Benzoxazole-2 -yl-mercapto-methyl)-4-(p-methoxyphenyl)-5-mercapto-1,2,4-triazole (Compound XVI),
◦
Physical state: White-ivory, crystalline powder; Mp = 213–215 C. FT-IR (KBr,
3100 (NH); 1628 (C=N); 1492 (C=S); 837 (para-disubstituted benzene nucleus); 783 (S-CH2).
1H-NMR (DMSO-d6, 400 MHz),
ppm: 3.46 (t, 3H, OCH3); 4.29 (s, 2H, CH2); 7.09–7.11 (d,
ν
, cm−1):
δ
2H, CHAr); 7.25–7.27 (d, 2H, CHAr); 7.53–7.55 (d, 2H, CHAr); 14.09 (s, 1H, NH).
0
3-(Benzoxazole-2 -yl-mercapto-methyl)-4-(p-bromoyphenyl)-5-mercapto-1,2,4-triazole (Compound XVII),
◦
Physical state: Brown-red, crystalline powder; Mp = 202–204 C. FT-IR (KBr,
(NH); 1617 (C=N); 1494 (C=S); 914 (para-disubstituted benzene nucleus); 780 (S-CH2). H-
ν
, cm−1): 3328
1
NMR (DMSO-d6, 400 MHz), δ ppm: 4.17 (s, 2H, CH2); 7.20–7.23 (d, 2H, CHAr); 7.47–7.50
(d, 2H, CHAr); 7.75–7.77 (d, 2H, CHAr); 14.21 (s, 1H, NH).
0
3-(Benzoxazole-2 -yl-mercapto-methyl)-4-(p-chloroyphenyl)-5-mercapto-1,2,4-triazole (Compound XVIII),
◦
Physical state: White-ivory, crystalline powder; Mp = 189–191 C. FT-IR (KBr,
2894 (NH); 1615 (C=N); 1493 (C=S); 899 (para-disubstituted benzene nucleus); 765 (S-CH2);
746 (c-Cl). 1H-NMR (DMSO-d6, 400 MHz),
ppm: 4.64 (s, 2H, CH2); 7.31–7.33 (d, 2H,
ν
, cm−1):
δ
CHAr); 7.42–7.44 (d, 2H, CHAr); 7.73–7.75 (d, 2H, CHAr); 14.29 (s, 1H, NH).
3-(Benzoxazole-20-yl-mercapto-methyl)-4-(p-iodoyphenyl)-5-mercapto-1,2,4-triazole (Compound XIX),
◦
Physical state: Brown-red, crystalline powder; Mp = 222–224 C. FT-IR (KBr,
(NH); 1614 (C=N); 1495 (C=S); 915 (para-disubstituted benzene nucleus); 770 (S-CH2); 748
(C-I). 1H-NMR (DMSO-d6, 400 MHz),
ppm: 4.23 (s, 2H, CH2); 7.35–7.37 (d, 2H, CHAr);
ν
, cm−1): 3038
δ
7.42–7.45 (d, 2H, CHAr); 7.59–7.61 (d, 2H, CHAr); 14.33 (s, 1H, NH).
3.6. Antibacterial Activity
The microbial strains (Staphylococcus aureus (ATCC-2593), Bacillus subtilis (ATCC-6638),
Bacillus cereus (ATCC-10876), Salmonella entiritidis (P-1131) and Escherichia coli (ATCC-25922))
were grown on Mueller–Hinton agar, with incubation at 37 ◦C for 48 h. Previously, the
tested substances were weighted and dissolved in dimethylsulphoxide (DMSO) to prepare
the solutions with the following concentrations: 1 mg/mL, 0.5 mg/mL, and 0.25 mg/mL
culture medium. The solutions thus prepared were included in 100 mL of culture medium,
previously agarized, melted, and then homogenized. In parallel, the control sample was
prepared consisting of glucose agar. The culture media were then distributed in the test
tubes and sterilized for 20 min at 120 ◦C. The bacterial cultures were prepared according

Molecules 2021, 26, 3330
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to the manufacture recommendations, using suspensions with a concentration of about
5.2 · 107 CFU/mL.
3.7. Immobilization of 1,2,4-Triazole
Immobilization of the 1,2,4-triazole was performed as follows: 0.2 g powder of PG
and PGB1 copolymers were introduced into 50 mL conical flasks over which 20 mL of
1,2,4-triazole solution was added (C1,2,4-triazole = 3.6
×
10−4
—
15
×
10−3 g/mL). Then,
the samples were placed in a thermostated water-bath shaker (Memmert MOO/M01,
Schwabach, Germany) and shaken at 180 strokes/minute until equilibrium was reached.
The 1,2,4-triazole immobilization studies on PG and PGB1 copolymers were performed
at different temperatures: 25, 30, and 35 ◦C and for different contact times. The conical
flasks were removed from the water-bath shaker, and the copolymers were centrifuged
at 78 RCF for 10 min. Then, the samples were separated by filtration, and the amount
of 1,2,4-triazole immobilized on PG and PGB1 copolymers was determined by UV-VIS
spectrophotometry (SPEKOL 1300 spectrophotometer, Analytik Jena, Jena, Germany) at a
wavelength of 226 nm, based on a calibration curve. The retained amount of 1,2,4-triazole
was calculated by the difference between 1,2,4-triazole concentration in the supernatant
before and after sorption process.
The amount of immobilized 1,2,4-triazole was calculated using the following equation:
(C0 − Ce)·V
qe =
(12)
W
where
0 is the initial 1,2,4-triazole concentration (mg/mL),
concentration (mg/mL), is the volume of 1,2,4-triazole solution (mL), and
q
e is the amount of 1,2,4-triazole immobilized onto PG and PGB1 copolymers (mg/g),
e is the equilibrium 1,2,4-triazole
is the
C
C
V
W
amount of copolymers. PG and PGB1 copolymers are in the form of powder consisting of
irregularly shaped particles.
3.8. Release of 1,2,4-triazole
In vitro 1,2,4-triazole release studies were performed by immersing the PG-T and
PGB1-T systems (0.1 g) in 10 mL of simulated gastric fluids of pH = 1.2 for 2 h at 37
◦C. The samples were placed in a thermostated water-bath shaker under gentle stirring
(50 strokes/minute). Samples (1 µL) of supernatant solution were collected at different time
intervals using a microsyringe and then analyzed spectrophotometrically at a wavelength
of 226 nm, using a UV-VIS spectrophotometer (Nanodrop ND 100, Wilmington, DE, USA).
The amount of 1,2,4-triazole released was determined using the calibration curve.
4. Conclusions
The 2-mercapto-benzoxazole molecule was used to achieve a selective support for
some heterocycles with 1,3,4-thiadiazole and 1,2,4-triazole structure, respectively. The
addition reaction of benzoxazolyl-2-mercapto-acetic acid hydrazide to aromatic isoth-
iocyanantes was used to obtain a new series of acyl-thiosemicarbazides (compounds
II–VII). Applying the cyclization reaction, the 4-substituted acyl-thiosemicarbazides were
converted in acid medium into thiadiazole derivatives with benzoxazole residue in the
molecule (compounds VIII–XIII) and in basic medium in mercapto-triazole-3,4-disubstituted
(compounds XIV–XIX). All new compounds were characterized using elemental and spec-
tral analysis (FT-IR and 1H-NMR). Among the active principles synthesized, 3-(benzoxazole-
20-yl-mercapto-methyl)-4-(p-methoxyphenyl)-5-mercapto-1,2,4 triazole was chosen to be
immobilized onto a polymeric support due to its biological properties. The antimicro-
bial studies have shown that 1,2,4 triazole presents very good antimicrobial activities
against several microbial strains, such as Escherichia coli, Salmonella enteritidis, and Bacil-
lus subtilis compared to 1,3,4-thiadiazoles that show good antimicrobial activity against
Staphylococcus aureus.

Molecules 2021, 26, 3330
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Two polymeric supports were used for the immobilization of 1,2,4-triazole derivative
(compound XVI): grafted copolymer and grafted copolymer carrying betaine moieties
based on gellan and N-vinyl imidazole, which were chosen due to the biocompatibility of
the polysaccharide as well as of the polymer/grafts generated by the vinyl monomer and
betaine function.
To describe the interactions that occur in sorption processes between 1,2,4-triazole
and PG and PGB1 copolymers, Langmuir, Freundlich, and Dubinin–Radushkevich model
isotherms were used. Kinetic sorption studies conclude that the Lagergren model best
describes the experimental data and confirm that the sorption of 1,2,4-triazole on the
grafting copolymers is physical in nature.
The thermodynamic study completed by the kinetic one led to the conclusion that the
sorption process of 1,2,4-triazole on the betainized copolymer is more intense than in the
case of the grafted copolymer only. The sorption process of 1,2,4-triazole is spontaneous
and favored by the increase of temperature. The release of 1,2,4-triazole from polymeric
supports proceeds through a complex mechanism controlled by both swelling and diffusion
processes. Similar to the results obtained in case of immobilization of the cefotaxime sodium
salt, these results confirm the potential of the grafted copolymer with betaine structure as a
candidate for developing sustained/controlled drug delivery systems.
Author Contributions: Conceptualization, N.B. and S.R.; methodology, S.R., N.B., and S.V.; valida-
tion, V.S., M.P., and J.D.; formal analysis, N.B., A.M.M., and C.C.; investigation, C.L. and V.S.; data
curation, S.R., N.B., and S.V.; writing—original draft preparation, N.B. and S.V.; writing—review and
editing, M.P. and J.D., visualization and supervision, S.V., M.P., and J.D. All authors have read and
agreed to the published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: The authors thank Marius Zaharia and Ana-Lavinia Vasiliu from Petru Poni
Institute of Macromolecular Chemistry, Iasi, for their great support and help in the XRD studies and
SEM analysis.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.
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