Immobilization and Release Studies of Triazole Derivatives from Grafted Copolymer Based on Gellan-Carrying Betaine Units

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.


Introduction
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].
At the same time, the addition of a benzoxazole structure can be useful for the improvement 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, antifungal, antiviral, anti-inflammatory, antipyretic, analgesic, anticonvulsant, tuberculostatic, and antitumor agents [29][30][31][32][33][34][35][36]. Benzoxazole and triazole heterocyclic rings played an important 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 containing 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][39][40][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 compounds 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. 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 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.
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.  The structure elucidation of the compounds II-VII was carried out by elemental analysis as well as by spectroscopic methods (FT-IR and 1 H-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. 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 CH 2 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 1 H-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).
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-π conjugation between the lone pair electrons belonging to the nitrogen atom and the 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 fivemembered 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 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]. 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][45][46][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-π conjugation between the lone pair electrons belonging to the nitrogen atom and the 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 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 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]. 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 1 H-NMR spectra ( Figure 8) were performed. 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)  Generally, in FT-IR spectra of the 1,3,4-thiadiazole (compounds VIII-XIII), the following absorption bands can be observed: the absorption bands at 3318-2880 cm −1 corresponding 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 CH 2 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-XIX present a wide band at 3553 cm −1 due to the NH stretching vibrations; 1613-1628 cm −1 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-CH 2 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. 1 H-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 CH 2 group bound to the sulfur atom situated at position 2 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 1 H-NMR spectra of the 1,2,4-triazole derivatives (compounds XIV-XIX) present a singlet due to the protons of the CH 2 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. present a wide band at 3553 cm −1 due to the NH stretching vibrations; 1613-1628 cm −1 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.   The 1 H-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.
The results regarding the antimicrobial activity of the bioactive compounds synthe-
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.
The interaction between PG and PGB1 copolymers and compound XVI was highlighted by FT-IR spectroscopy (Figure 9). -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. 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. 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. 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 important to consider two physico-chemical aspects, namely the equilibrium and the kinetics of sorption.  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. 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 important to consider two physico-chemical aspects, namely the equilibrium and the kinetics of sorption. 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 important to consider two physico-chemical aspects, namely the equilibrium and the kinetics of sorption.

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: where: q e represents the amount of 1,2,4-triazole sorbed at equilibrium (mg/g); q m represents the maximum amount sorbed (mg/g); C e is the concentration of 1,2,4-triazole solution at equilibrium (mg/L); and K L is the Langmuir constant that reflects the affinity between sorbate and sorbent.
To determine whether the sorption system used is favorable or unfavorable to the sorption, the equilibrium parameter R L , given by the equation [50], was also calculated: where C i represents the initial concentration. According to the literature [51], if R L < 1, then the sorption is unfavorable, for R L = 1, the sorption is linear; if 0 < R L < 1, the sorption is favorable or irreversible if R L = 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: where K F 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/n f indicates the type of isotherm as follows: favorable 0 < 1/n f < 1 or unfavorable 1/n f >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: where q DR represents the maximum amount sorbed (mg/g); β is the Dubinin-Radushkevich constant (mol 2 /kJ 2 ); R represents the ideal gas constant (R = 8.314 kJ/mol·K); and T is the temperature (K). The Dubinin-Radushkevich isotherm constant, β, is associated with the average free sorption energy, E (kJ/mol), calculated using the following equation: The value of E is used to obtain information about the nature of the sorption process. The sorption process can be physical when the E values are between 1 and 8 kJ/mol, ion exchange for values of E between 8 and 16 kJ/mol, and chemical nature for values of E greater that 16 kJ/mol [54].
The parameters of the studied isotherms as well as the values of the error functions R 2 and χ 2 are presented in Table 3. From data presented in Table 3, the following conclusions can be drawn: The values of the equilibrium parameter, R L , 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 K L 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 R 2 and χ 2  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.

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 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]: From the linear representation ln K L 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:

Thermodynamic Parameters
By means of thermodynamic parameters, such as Gibbs free energy change (ΔG), enthalpy change (ΔH), and entropy change (ΔS), the mechanism and the type of sorption process can be determined. Thus, according to the literature [55], it is known that in the case of physical sorption, the values of ΔH are less than 40 kJ/mol, while in the case of chemical sorption, the values of ΔH are in the range of 40-120 kJ/mol. The value of ΔH and ΔS can be determined by means of the Langmuir constant using the Van 't Hoff equation [56]: From the linear representation ln KL versus 1/T (Figure 12), the values of ΔS and ΔH were obtained from the intercept and slope, respectively, and the results are presented in Table 4. The values of ΔG were obtained using the following equation:  As it can be seen from Table 4, the negative values of ΔG 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 ΔH < 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 ΔH 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 ΔS 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 ΔS can indicate an increase in the degree of freedom of 1,2,4-triazole. Moreover, the positive values of ΔH and ΔS lead to the conclusion that the sorption process occurs spontaneously at all temperatures.  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.

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 sorption onto PG and PGB1 copolymers. The two models mentioned above are described by the following equations:

•
Lagergren model [58]: • Ho model [59]: where q e and q t are the amounts of 1,2,4-triazole sorbed at equilibrium and at time t (mg/g), k 1 is the rate constant of the pseudo-first order sorption process (min −1 ), and k 2 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 (C 1,2,4-triazole = 15 × 10 −3 g/mL) onto PG and PGB1 copolymers at T = 308 K, and Table 5 shows the parameter values corresponding to the Lagergren and Ho models.

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 sorption onto PG and PGB1 copolymers. The two models mentioned above are described by the following equations:

•
Lagergren model [58]: Ho model [59]: where qe and qt 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 × 10 −3 g/mL) onto PG and PGB1 copolymers at T = 308 K, and Table 5 shows the parameter values corresponding to the Lagergren and Ho models. Figure 13. Graphical representation of Lagergren and Ho models in case of 1,2,4-triazole sorption onto PG and PGB1 copolymers at T = 308 K and C1,2,4-triazole = 15 × 10 −3 g/mL.    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 q e values calculated by applying the Lagergren model are close to the experimental values compared to the q e values calculated by applying the Ho model. Although the values of the correlation coefficients R 2 are higher when applying the two kinetic models, the χ 2 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.

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. 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]: where Qt is the amount of drug released at time t; kH is the Higuchi dissolution constant; and t is the time.
• Korsmeyer-Peppas model [61]: where Mt/M∞ is the fraction of drug released at time t; kr is the release rate constant that is characteristic to bioactive compound-polymer interactions; and n is the diffusion 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. 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.

•
Higuchi model [60]: where Q t is the amount of drug released at time t; k H is the Higuchi dissolution constant; and t is the time.
• Korsmeyer-Peppas model [61]: where M t /M ∞ is the fraction of drug released at time t; k r is the release rate constant that is characteristic to bioactive compound-polymer interactions; and n is the diffusion 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. 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.

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 bromide pellets in the range of 4000-400 cm −1 and at a resolution of 2 cm −1 . The 1 H NMR spectra were recorded in DMSO-d 60 on Bruker ARX 400 Spectrometer (400 MHz). X-ray diffraction analysis was performed in a Rigaku Miniflex 600 diffractometer using CuKαemission in the angular range 3-90 • (2θ) with a scanning step of 0.01 • and a recording rate 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.

General Procedure for Synthesis of 1-(Benzoxazole-2 -yl-mercapto-acetyl)-4-arylthiosemicarbazide (Compounds II-VII)
A total of 0.005 moles of benzoxazole-2 -yl-mercapto-acetic acid hydrazide (I) dissolved in 30 mL absolute methyl alcohol was treated with 0.005 moles of aromatic isothiocyanate 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.

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 (C 1,2,4-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: where q e is the amount of 1,2,4-triazole immobilized onto PG and PGB1 copolymers (mg/g), C 0 is the initial 1,2,4-triazole concentration (mg/mL), C e is the equilibrium 1,2,4-triazole concentration (mg/mL), V is the volume of 1,2,4-triazole solution (mL), and W is the amount of copolymers. PG and PGB1 copolymers are in the form of powder consisting of irregularly shaped particles.

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.
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.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.