2.1 Materials and strains

Piperazine (S D Fine-Chem Ltd.), 2-aminobenzoic acid, formaldehyde (37% aqueous solution), sodium hydroxide (Merck), and M(CH₃COO)₂·xH₂O, where M = Mn(II), Co(II), Ni(II), Cu(II), and Zn(II) and x = 1,2,4 (Qualigens fine chemicals) were used without further purification. Solvents such as acetone, methanol, diethyl ether, dimethyl formamide (DMF), dimethylsulfoxide (DMSO) (S D Fine-Chem Ltd.), and ethanol (Jiangsu Hauxi International) were purified by standard procedures before use. All the microorganisms were provided by the Culture Collection Center of Microbiology laboratory, Department of Microbiology (AMU, Aligarh).

2.2 Synthesis of AFP ligand

2-aminobenzoic acid (0.01 mol) and formaldehyde (0.01 mol) in 1:1 M ratio were transferred to a 250 ml three-necked round bottom flask equipped with thermometer, condenser, and magnetic stirrer containing DMF (∼50 mL) as a solvent, and the pH was adjusted in alkaline range with 40% aqueous NaOH. The reaction mixture was stirred magnetically at temperature up to 80°C for 3 h. A piperazine solution (0.01 mol) in 25 mL DMF was added to this system and stirred again for about 1 h up to 90–115°C. The progress of reaction was monitored by thin layer chromatography (TLC). The reaction mixture was cooled and precipitated using a 50/50 (v/v) water/acetone mixture. The obtained solid which is light brown in color (AFP–ligand) was filtered, and re-precipitated from DMF and ethanol. It was then filtered and washed repeatedly with distilled water and acetone and dried in a vacuum oven to remove the solvent (DMF and ethanol), yield 82% (2.05 gm).

2.3 Synthesis of polymer–metal complexes of AFP

A series of polymer–metal complexes [Mn(II), Co(II), Ni(II), Cu(II) and Zn(II)] were prepared by using equimolar ratio (1:1) of AFP ligand and metal acetates. A typical synthesis of polymer AFP–Mn(II) is as follows.

The AFP ligand (3.84 gm, 0.01 mol) was dissolved in DMF (20 mL). Mn(II) acetate tetrahydrate (2.45 gm, 0.01 mol) was also dissolved in a minimum quantity of hot DMF (25 mL). Both the solutions were mixed and heated at reflux at 75°C with constant stirring. The resulting viscous dark brown colored product was precipitated by pouring it into excess of distilled water. The AFP–Mn(II) complex gets precipitated, and it was filtered and washed several times with water and acetone and dried at room temperature, yield 76%. Similar procedures were adopted for the synthesis of other polymer–metal complexes.

2.4 Measurements

The elemental analysis of carbon, hydrogen, and nitrogen were carried out on a Perkin–Elmer model-2400 elemental analyzer. The metals were determined by complexometric titration against EDTA after decomposing with concentrated nitric acid. The solubility of ligand and their polymer–metal complexes were investigated in different solvents at room temperature. The IR spectra were recorded on a PerkinElmer infrared spectrometer model 621 using KBr pellets. ¹H NMR spectra were recorded on a JEOL-FX-100 FT-NMR instrument in dimethylsulfoxide (DMSO-d₆) solution with tetramethylsilane as an internal standard. The thermal stability of ligand and their polymer–metal complexes were evaluated with a TA analyzer 2000 at a heating rate of 20°C/min under nitrogen. The electronic spectra of the polymer–metal complexes were recorded on a PerkinElmer Lambda-201, and magnetic susceptibility measurements were carried out with vibrating sample magnetometer. The antimicrobial activity of the ligand and their polymer–metal complexes was tested against different bacterial and fungal strains.

3.1 Composition and chemistry

The AFP ligand is composed of 2-aminobenzoic acid, formaldehyde, and piperazine. The preparation of the AFP ligand was carried out in two steps. In the first step, 2-aminobenzoic acid reacts with formaldehyde in (1:1) molar ratio and forms 2-amino-5-(hydroxymethyl)benzoic acid. In the second step, 2-amino-5-(hydroxymethyl)benzoic acid and piperazine undergo condensation reaction and forms the AFP ligand (Scheme 1). The polymer–metal complexes were obtained by the reaction of the AFP ligand with metal acetate in DMF in (1:1) molar ratio (Scheme 2). The AFP ligand and their polymer–metal complexes were soluble in DMF and DMSO and insoluble in most of the common organic solvents such as benzene, methanol, ethanol, and water.

All the polymer–metal complexes were colored and obtained in good yield. Various observations such as the nature of the AFP ligand, high thermal stability, metal to ligand ratio (1:1), and insolubility of the polymer–metal complexes in common organic solvents suggest their polymeric nature [17]. Elemental analysis, physical properties, and IR data provide good evidence that the compounds are polymeric [18]. The insolubility of the polymer–metal complexes in common organic solvents does not permit the determination of their molecular weight. The elemental analysis [Table 1] showed that AFP ligand to metal ratio in all the polymer–metal complexes was 1:1, in good agreement with the calculated values. The structures of AFP ligand and its polymer–metal complexes were also confirmed by IR, ¹H NMR, ¹³C NMR, UV–visible, and magnetic susceptibility measurements. The magnetic moments were measured by placing the sample inside the uniform magnetic field at 25°C and 1 atm pressure. The vibrating sample magnetometer measures the magnetization of a small sample of magnetic material placed in an external magnetizing field by converting the dipole field of the sample into an AC electrical signal which in turn is equated into magnetic moment values.

3.2 FT-IR spectra

The infrared spectra of the AFP ligand and their polymer–metal complexes are presented in Table 2. They resemble each other in their general shape, but the comparison of the IR spectrum of the ligand and their coordination polymers have revealed certain characteristics differences.

One of the significant differences in the IR spectrum of the AFP ligand and their coordination polymers is the presence of more broadened bands in the range of 3430–3452 cm⁻¹ in the latter due to the formation of coordination bond between the O–H group of the ligand and the metal ions [19–21]. This is explained by the observation that water molecules might have strongly absorbed on to the polymer sample during their formation. In the spectrum of the AFP ligand, an absorption band appearing at 3360 cm⁻¹, which is assigned to the ν(N–H) stretching, [22] is shifted toward higher frequency in the spectra of the coordination polymers, due to the formation of metal–nitrogen bonds. Another noticeable difference is the appearance of band due to the C=O stretching vibration at 1640 cm⁻¹ of the AFP ligand which is shifted to lower frequency. The band at 1280 cm⁻¹ of the AFP ligand assigned to in-plane OH deformation [23] is shifted toward lower frequency in the spectra of the coordination polymers due to the formation of metal–oxygen bond [24,25]. Bands appearing at 526–540 cm⁻¹ and 480–498 cm⁻¹ are attributed to M–O and M–N vibrations and support the involvement of nitrogen and oxygen atoms in complexation with metal ions [26] in polymer–metal complexes. Thus, all these characteristics features of the IR studies suggest the proposed structure of the coordination polymer as shown in Schemes 1 and 2.

3.3 ¹H NMR spectra

The ¹H NMR spectra of AFP ligand and their polymer complex of Zn(II) are shown in Figs. 2 and 3, respectively. The aromatic protons show multiple resonance signals between 6.30–6.65 and 6.40–7.73 ppm for AFP ligand and AFP–Zn(II), respectively [27]. The singlet corresponding to one proton at 11.00 ppm is assigned to the –COOH group, and a resonance signal appeared at 5.5 ppm is due to the proton of the N–H group [28], whereas the resonance signal found at 2.4 ppm indicate the presence of the –CH₂–CH₂– groups of the piperazine moiety, and a resonance signal appearing at 3.35 is due to proton of the benzylic methylene group. The signals for the protons of the –COOH group are absent in the spectrum of AFP–Zn(II), suggesting the participation of the –COOH group to the metal center in the formation of COO–M. Similarly, the protons of the N–H group were shifted toward down field at 5.4 ppm due to the coordination of nitrogen with metal ion. This significant shifting in all the peaks was observed, which confirms the formation of polymer–metal complexes. The peak of aromatic protons became broad due to the intermolecular interaction toward the metal ion and variation in the π-electron density around the protons.

3.4 ¹³C NMR spectra

The ¹³C NMR spectrum of AFP ligand is depicted in Fig. 4. Four prominent peaks are observed in the spectrum. The multiplets observed in the range of 112–140 ppm may be ascribed to the benzylic carbons while the piperazinic carbons were found to resonate in between 44 and 46 ppm. The carboxylic carbons of 2-aminobnzoic acid moiety were observed as a single sharp peak at 160 ppm while the formaldehydic carbons were observed as singlet at 62 ppm corroborating the proposed structure of the AFP ligand.

3.5 Electronic spectra and magnetic moments

Magnetic moments (μ_eff) of the polymer–metal complexes are given in Table 3. The data reveal that all the coordination polymers other than that of Zn(II) are paramagnetic while that of Zn(II) is diamagnetic.

The electronic spectrum of AFP–Mn(II) shows three bands at 14,222 cm⁻¹, 19,431 cm⁻¹, and 25,022 cm⁻¹ which have been assigned to the transitions ⁴T_1g(G)←⁶A_1g, ⁴T_2g(G)←⁶A_1g, and ⁴T_1g(F)←⁶A_1g(F), respectively, suggesting an octahedral structure of AFP–Mn(II) polymer–metal complex. The electronic spectrum of AFP–Co(II) polymer–metal complex shows three bands at 14,095, 18,995, and 24,302 cm⁻¹, which have been assigned to the transitions ⁴T_2g←⁴T_1g(F), ⁴A_2g←⁴T_1g(F), and ⁴T_1g(P)←⁴T_1g(F), respectively, and are consistent with the values observed for an octahedral structure. The Ni(II) polymer–metal complex [29] gave three bands at 11,087, 18,011, and 24,991 cm⁻¹, corresponding to the transitions ³T_2g(F)←³A_2g(F), ³T_1g(F)←³A_2g(F), and ³T_1g(P)←³A_2g(F), respectively, corresponding to octahedral geometry. The electronic spectrum of the AFP–Cu(II) polymer–metal complex [30] exhibits a band at 14,999 cm⁻¹ assigned to the ²T_2g←²E_g transition, implying a distorted octahedral geometry. Besides, a strong charge transfer band is observed at 25,701 cm⁻¹. The value of the ligand field splitting energy (10 Dq), Racah interelectronic repulsion parameter (B), nephelauxetic ratio (β), and β% are presented in Table 3. The Zn(II) coordination polymer is diamagnetic in nature as expected for d¹⁰ system.

3.6 Thermogravimetric analysis

The thermal behavior of the coordination polymers was investigated by thermogravimetric analysis (TGA). The TGA data of all the samples are presented in Table 4, and the thermograms are shown in Fig. 5. The initial degradation temperature of the coordination polymers, as well as the AFP ligand begins from 25 to 50°C. A very slight decrease in weight loss (2–5%) depicted from the thermogram in the temperature range 25–150°C for the AFP ligand and may be attributed to unbound water molecules. However, the gradual weight loss that initially occurred below 150°C in all of the coordination polymers of AFP may be due to removal of hydrate water, whereas the loss obtained in the range of 150–200°C might be due to metal-coordinated water molecules [31]. The rate of decomposition for the entire coordination polymer is initially low up to 150°C and gradually increases to a maximum in the range of 250–600°C at which almost 65–85% loss in weight occurred. A remarkable difference is observed in the thermal degradation of the AFP ligand and its coordination polymer. The AFP ligand follows a two-step [32] mode as compared to a single-step thermal degradation of the coordination polymers. In AFP ligand, the first slow step of thermal degradation in the range of 250–340°C may be due to the decomposition of a more labile aliphatic bridge present between the two 2-aminobenzoic acid units, and the second step of thermal degradation may be in the anthranilic acid, initiated at 500°C, with a rapid weight loss at around 550°C which gets completed at 700°C.

These results revealed that all the polymer–metal complexes show higher thermal stability than the AFP ligand due to the incorporation of metal ions into the polymeric chain as a result of chelation. Another factor that may be responsible for the enrichment of the thermal stability of the polymer–metal complexes is the increase in the molecular weight due to the joining of two different polymer chains. The thermogram confirms that the polymer–metal complexes are thermally more stable than the AFP ligand.

3.7 Antimicrobial studies

The antibacterial and antifungal activities of the AFP ligand and their polymer–metal complexes were identified based on the zone of inhibition for bacterial growth around the wells at one atmospheric pressure and ambient temperature. The results of activity were expressed as inactive, mild, moderate, and high. The results are summarized in Tables 5 and 6. The results reveal that all the compounds exhibited antibacterial and antifungal activities to a certain extent. All the compounds were screened for their antibacterial activity against Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Pseudomonas fluorescens, and Salmonella typhi and for their antifungal activity against Candida albicans, Aspergillus niger, and Microsporum canis. All the compounds generally showed good antibacterial activity, but more significant antifungal activity was observed against most of the strains. A marked enhancement of activity was exhibited in all the polymer–metal complexes against all the bacterial/fungal strains. It was evident from the data that the antimicrobial activity of all the polymeric compounds was increased relative to the AFP ligand. The chelation/coordination reduces the polarity of the metal ion by partial sharing of its positive charge with the donor groups and possibly π-electron delocalization takes place within the whole chelate ring [33]. This process thus increases the lipophilic nature of the central metal atom [34], which, in turn, favors its greater penetration through the bacterial wall of the microorganisms, thus killing them more effectively [35]. It has also been observed that the solubility, conductivity, and dipole moment are also influenced by the presence of metal ions; these could be the significant factors responsible for increasing the hydrophobic character and liposolubility of the molecule, hence enhancing the biological activity.

The results of antimicrobial activity reveal that the AFP–Cu(II) showed the highest antibacterial and antifungal activity than other polymer–metal complexes. This result may have been due to the higher stability constant of Cu(II) than the other polychelates [36,37]. According to the stability constant, the Cu(II) ion has stronger interactions with N and O donor atoms, by which its lipophilic nature was enhanced.