Synthesis of New Polymeric Composite Materials Derived from 2,2-(1,6-Dioxohexane-1,6-diyl) Dihydrazinecarbothioamide

INTRODUCTION
When metal had been warmed and ceramics were modeled, the industrial production of the first synthetic polymer signaled yet an additional change in the evolution of humanity. As a result, then, the development of polymers has jumped dramatically. It could be proposed that these materials are now used in all aspects of human endeavor. Human curiosity and inventiveness, along with performance and financial factors, led to the development of this new class of materials. Consequently, research on blends of synthetic and natural polymers with possible applications in biological materials and ecology has recently increased [1,2]. In the 1980s, systems consisting solely of a polymer matrix with support from fabric components or inorganic filler materials were called polymer composites [3]. The composite substances are made up of two or more components, one of which may be in the matrix phase and the other may be in the form of a fiber or molecule [4-6]. Construction, mechanical, automotive, marine, biomedical, and aerospace industries have all shown significant benefits from the use of natural or synthetic fibers in composite materials [7,8]. Nanocomposite substances have much better structural, thermal, mechanical, and tribological parameters than many traditional materials, according to studies done over the past 20 years [9,10]. The matrix material, which also includes nanoscale-confirming elements, makes up the structure of nanocomposites. These elements may be fibers, particles, nanotubes, whiskers, or other comparable elements [11]. Polymer nanocomposites are used in many different industrial products, such as those used in the electronics, automotive, and aerospace industries, because of their remarkable thermal properties [12,13]. Additionally, it has been found that triazole heterocyclic structures can form a wide variety of weak nonbond interactions with biological systems’ enzymes and receptors [14]. Scientists from a variety of fields of study, including agricultural, chemical, medicine, supramolecular, polymer, and materials sciences, have gravitated to triazole compounds owing to their fundamental properties, which have made them major chromophores with significant medical potential [15]. Antifungal, antibacterial, anti-inflammatory, antiviral, antitubercular, anticoagulant, antioxidant, antidiabetic, and anticancer drugs based on triazole are among the many therapeutic drugs available [16]. The objective of this study is to first synthesize novel nanocomposites from dimethyl adipate with (ZnNPs, AgNPs, SiNPs, or IONPs), via a green method to increase their potential medical uses and improving their thermal analysis of their nanocomposites and polymers.

MATERIALS AND METHODS
All of the chemicals were supplied by Merck and Aldrich. FTIR spectra have been obtained using the Shimadzu FTIR spectrophotometer (Ir prestige 21) as well as KBr discs on an 8400 s spectrophotometer. The performed proton nuclear magnetic resonance (1H-NMR) spectra were reported in ppm. These spectra were produced by Tehran, Iran’s Bruker Ultra Shield 500MHz Razi Laboratories. The Sharif University of Technology in Tehran, Iran, uses Linseis Origin with platinum assessment V1.0.89 for thermogravimetric analysis (TGA/DSC).

Synthetic Procedures
Figs. 1 and 2 illustrate the procedure to forming the newly nanocomposites.

Synthetic of 2,2’-(1,6-dioxohexane-1,6-diyl)dihydrazinecarbothioamide [C1]
The initial compound was created by combining 0.87g and 0.01 mol of dimethyl adipate and 0.91g and 0.02 mol of thiosemicarbazide.10 ml of ethanol was then added, and the mixture was refluxed for ten hours before the excess solvent was removed [17]. White precipitate with a 93% yield, mp, 180–182°C. 

Synthetic of 5-[4-(5-sulfanyl-1H-1,2,4-triazol-3-yl) butyl]-1H-1,2,4-triazole-3-thiol) [C2]
After dissolving 0.73g,0.01mol of compound [C1] in 10 ml of 10% sodium hydroxide, the mixture was refluxed for a full day. Following the filtering step, the liquid was mixed with diluted hydrochloric acid to create an emulsion [18]. After filtering and washing the emulsion with water, we allowed the white precipitate to dry, yielding 78%, mp, 234–236°C.

Synthetic of 3-hydrazine-5-[4-(5-hydrazine-1H-1,2,4-triazole-3-yl) butyl]-1H-1,2,4-triazole [C3]
5 ml of ethanol was used to dissolve 2.56 g,0.01mol of compound [C2]. Hydrozine hydrate (0.02 mol) was then added dropwise while stirring for 30 minutes [19], and the mixture was refluxed for 24 hours [20]. Following that, allow the sediment to dry to yield 72.5%, mp: 203-205°C.

Synthetic of fourth compound [C4]
Compound [C3] (2.52 g, 0.01 mol) and maleic anhydride (1.96 g, 0.02 mol) were combined in 25 mL of DMF, and the mixture refluxed for approximately 4 hours. After using diethyl ether to clean the result, it was allowed to dry at room temperature [21]. m.p.: 175–177°C, yield 75%.

Synthetic of polymer [C5]
0.42 g of ammonium persulfate and 0.41 g of the compound [C4] were mixed in 15 mL of ethanol as a polymerization initiator. After stirring the mixture for two hours at room temperature, it refluxed for 12 hours [22]. 85% yield, m.p.: 260–262.

Synthetic of Nanocomposites [C6-C9]
50mL of a 250 mg/L solution of the nanoparticles [zinc nanoparticles (ZnNPs), silver nanoparticles (AgNPs), silica nanoparticles (SiNPs), or iron oxide nanoparticles (IONPs)] was combined with 100mg of dried polymer [C5] using a hotplate stirrer for three hours to bond the nano metal in the blend matrix [23].

Molecular Docking Study 
The full Genetic Optimization of the British Cambridge Crystallographic Data Center (CCDC) (GOLD) Hermes 2021.2.0 (Build 327809) made it feasible to conduct molecular docking studies of compounds. This made it possible to show proteins, ligands, short contacts, hydrogen bonding interactions, and bond length computations. The effectiveness of the enzyme (6NE7) was shown using the Protein Data Bank (rcsb.org) [24].

Biological Activity 
The cup-plate agar diffusion method was used to ascertain whether the nanocomposite, a polymer comprising zinc nanoparticles, silver nanoparticles, silica nanoparticles, and iron oxide nanoparticles, possesses antibacterial properties against Escherichia coli and Bacillus cereus. Amoxicillin, which was given at a concentration of 50µg/ml, was a common drug for antibacterial activity. After being sterilized, these agar solutions were placed in petri dishes and left to become solid. A micropipette has been used to sequentially introduce several of the synthesized compounds into the cavities. After that, these substances were given an hour to spread. All of the compounds have been dissolved in DMSO, which has also been used as a management. These plates were heated up for 24 hours at 37°C to test for antibacterial activity. The area of resistance surrounding the cups after the corresponding incubation was measured in millimeters [25-27].

Cytotoxicity Activity 
A human liver cancer cell line (HepG2) was used to test the cytotoxic impact of the polymer [C5] and nanocomposite [C6] (polymer and ZnNPs), and the outcomes were contrasted with those of a normal liver cell line (WRL-68). To investigate the cytotoxic effect, 96-well plates were used for the MTT test. Cells were treated with polymer [C5] and nanocomposite [C6] after 24 hours, or if a nanocomposite was created. Once the cells had been treated for twenty-four hours, the medium was removed, µl/well solutions of MTT were added, and the samples were incubated for four hours at 37°C to assess the viability of the cells. Afterward the removal of the MTT solution, 200µL of DMSO (Dimethyl Sulfoxide) was added to the wells to dissolve the crystals [28-30]. The mixture was then shaken frequently and incubated at 37 degrees Celsius for 15 minutes. The absorbency at a wavelength of 620 nm was measured using a microplate reader. Equations Freshney have been made available to calculate the rate of cell growth inhibition [31-33].

RESULTS AND DISCUSSION 
FTIR and 1HNMR of synthesis of compounds
An overview of the reactions carried out in this investigation is given in Figs. 1 and 2. A new stretching band at 1641 cm-1 that was attributed to the C=O group of the amide moiety appeared in the FT-IR spectrum for compound [C1], replacing the absorption stretching band of the C=O groups ester in the starting materials. Many signals at δ (1.50-2.31) ppm for (m,4H, CH2CH2CH2CH2), multiple signals in the region δ (3.53-3.57) ppm for (m,4H, CH2CH2CH2CH2), at (4.30-4.48) ppm for (b,4H, NH2), and a singlet signal at δ7.18 ppm for (s,2H, NH-C=S) were detected in the 1H-NMR spectrum of compound [C1] (in DMSO-d6 as solvent). Lastly, (s, 2H, NH-C=O) is responsible for a singlet signal at δ 7.54 ppm. When compound [C1] reacted with sodium hydroxide, compound [C2] was created. The FTIR of compound [C2] revealed new absorption stretching bands at 2491 and 1313 cm-1, which are allocated to the υ SH and υ C=S groups, respectively, and the disappearance bands of the NH2 groups. Additionally, a stretching band for the 1,2,4-triazole ring’s υ C=N (endocyclic) was seen at (1602-1636) cm-1. The 1H-NMR spectrum of compound [C2] revealed a singlet signal at 1.43 ppm for (s,2H, SH), numerous signals in the region (δ, ppm): (1.21-1.24) for (m,4H, CH2CH2CH2CH2), and signals (t,4H, CH2CH2CH2CH2) at (0.80-0.85). Finally, for (broad, 2H, NH) at (1.55.157) ppm [34-36].  One mole of compound [C2] was replaced neucleophilically with a sufficient amount of 80% hydrazine hydrate in absolute ethanol as a solvent to create the compound [C3]. The compound [C3]’s FT-IR spectra revealed the emergence of new absorption stretching bands in the region (3300-3353) cm-1 and 3175cm-1, which have been attributed to (NH2, NH), respectively, along with the disappearance of absorption bands due to the (C=S) and (SH) groups.  The compound [C3]’s 1H-NMR spectrum revealed signals (t,4H, CH2CH2CH2CH2) at δ (0.78-0.86) ppm, numerous signals in the δ (1.30-1.33) ppm region for (m,4H, CH2CH2CH2CH2), a singlet signal at (1.19-130) ppm for (s,4 H, NH), and the (broad, 4H, NH2) at (3.70-422) ppm. Compound [C4] was synthesized from compound [C3] and maleic anhydride. Because of the carboxylic acid’s C=O, the compound [C4]’s FTIR shows a new band at 1699 cm-1 and the disappearance of NH2. Compound [C4] reacts with ammonium persulfate as an initiator to produce polymer [C5]. The FTIR of the polymer [C5] shows that the band at 1597 cm-1 is gone because of the υ C=C, Table 1. The 1H-NMR spectrum of compound [C5] shows that there are many signals in the region δ (3.37-3.39) ppm for (m,4H, CH2CH2CH2CH2), a triplet signal at δ (3.7-3.8) ppm is due to (t,8 H. O= C-CH2 –CH2-C=O), a broad signal at δ (4.59-4.70) ppm for (broad,2H, NH of ring), and finally at (6.95-7.29) ppm for (2H, NH-C=O). This polymer was then combined with nano, which showed the bands at (439-450) cm-1 due to NPs bonding with the polymer [C5] [37,38].

Molecular Docking Study
The synthesized polymer [C5] showed notable activity in the examination of the stability of cancer cells in the enzyme (6NE7) due to hydrogen bond interactions with the primary amino acids ARG770, VAL906, and TYR928, [39] (Protein Data Bank, rcsb.org). 
The prepared polymer [C5] Fig. 3 was more efficient and had a higher binding energy of 76.41 with the enzyme than the compound under comparison, Fig. 4, which had a binding energy of 51.58. This was because, as Table 2 shows, the compound was interacting with the primary amino acids ARG770, VAL906, ARG908, and SER909 through hydrogen bonds [40].

Biological activity
With the highest activity comparable to that of amoxicillin, the common antibiotic, the nanocomposites [C6-C9] Fig. 5 showed an extraordinary rate of inhibition against two pathogenic bacterial types (G+) Bacillus cereus and E. coli (G–), Table 3. Because of their high cell affinity and ease of absorption by immune cells, NPs can be precisely delivered to the site of infection, where they can inhibit and harm microbiological pathogens. Since they strongly attach to electron donor groups—such as elements like sulfur, nitrogen, or oxygen—found in microbial cell walls and penetrate the bacterial cell wall, nanoparticles (NPs) have an antibacterial effect. Although they also release free radicals, NPs can harm cells and rupture their membranes [41-43]. Recent research suggests that magnetic nanoparticles could be harmful to microorganisms [44,45].

Cytotoxicity activity
The nanocomposite [C6] and polymer [C5] may be able to enter cancer cells specifically. At concentrations (10, 20, 50, 70, and 100) µg/ml higher than the polymer [C5], the nanocomposite [C6] demonstrated good inhibition, as indicated by Figs. 6- 11 and Tables 4-11. ZnNPs can induce cytotoxicity by increasing ROS levels [40]. One of the recent studies showed that they were effective cytotoxic agents when administered in vivo. It appears that magnetic ZnNPs mediate the DNA lesions in the tumor cells. Accordingly, at concentrations of 100 μg/ml, Nanocomposite [C6]’s anticancer activity showed significant effects against the HepG2 cell line, with an IC50 of 32.06 and an IC50 of 86.91 for (WRL-68) [46-48].

Field Emission Scanning electron microscope studies (FESEM) 
 The dimensions and shape of the polymer [5] and the nanocomposite [C6] have been approved using FESEM. The average size of the polymer [C5] Fig. 12 particles is between 211 and 409 nm. ZnNPs were found to be uniformly distributed across the matrix’s surface; in the nanocomposite [C6] Fig. 13, their nanosize is 17.09 nm. However, when the surface is slightly difficult, some of the formation nanoparticles have also been found. According to several studies [49] the particles in nano-composite films have an almost spherical morphology.

Thermal analysis
For both pure polymers and novel nano-composites, thermo-gravimetric (DSC/TG) curves have been developed at a heating rate of 10 °C min-1 under argon. The temperature ranging from 0°C to 1200°C has been recorded. The thermal breakdown of pure polymer [C5] and nano-composite [C6] is shown in the table. The TGA curves of the polymer [C5] in Fig. 14 demonstrated two primary phases of weight loss. At a temperature of 45–450 °C, the first stage, which involved the evaporation of volatile compounds, primarily water, resulted in weight losses of roughly 73.35 %. At temperatures between 450 and 1020 °C, the polymer main chain disintegrated in the last one, resulting in weight losses of roughly -7.139 %. The polymer film DSC curve displays a glass transition temperature (TG) of (55.99) °C, an exothermic peak (130 °C) that represents the crystalline temperature Tc, and a sizable endothermic peak (Tm) at (377) °C that is associated with the melting of polymers. 
The degradation of polymer (Td) begins at (470) °C. Fig. 15 depicts the nano-composite [C6] TGA thermo-gram, which reveals three stages of decomposition [49,50]. The weight loss from the first occurs between 45 and 200°C and is about 5.166%. The weight loss from the second occurs between 200 and 370°C and is about -45%; and the weight loss from the third occurs between 370 and 1010°C and is about 7.047. The DSC curve shows a degradation point at 550°C, an endothermic melting point (Tm) of 397°C for modified PVA, an exothermic peak at 85.90°C representing the glass transition temperature (Tg), and a crystalline temperature point (Tc) of 170°C [51,52]. 

CONCLUSION 
Molecular docking, antibacterial and anticancer properties, field emission scanning electron microscopy, and thermal analysis of a few novel nanocomposites are all included in this study. The prepared polymer is more effective than the contrasting compound, according to the molecular docking study. Depending on the results, the development inhibition zone was wider for nanocomposites, and nanocomposites [C6] demonstrated outstanding antimicrobial activity against Bacillus cereus and E. coli that was comparable to that of amoxicillin, a common antibiotic. The anticancer activity of polymer [C5] and nanocomposites [C6] against HepG2 (a human liver cancer cell line) and normal cell line WRL68 (a normal liver cell line) was then examined. With IC50 values of 32.06 on HepG2 and 86.91 on WRL-68 cancer cell lines, nanocomposite [C6] demonstrated a high rate of restriction and reduced toxicity. The new bonds between the polymers and nanoparticles caused the surface morphology of the synthesized nanocomposites to change, as demonstrated by FESEM studies. Lastly, the findings showed that the prepared polymer [C5] is less thermally stable than the nanocomposite [C6].

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.