Novel Gallium(III), Germanium(IV), and Hafnium(IV) Folate Complexes and Their Spectroscopic, Thermal Decomposition, Morphological, and Biological Characteristics

In this study, we describe novel gallium(III), germanium(IV), and hafnium(IV) folate complexes, including their synthesis and analyses. The synthesized folate complexes were also subject to thermal analysis (TGA) to better examine their thermal degradation and kinetic properties. The folate complexes had high stability and were nonspontaneous. The Coats–Redfern and Horowitz–Metzger equations were used to determine thermodynamic parameters and describe the kinetic properties. These complexes were synthesized through the chemical interactions in neutralized media between the folic acid drug ligand (FAH2) with GaCl3, GeCl4, and HfCl4 metal salts at 1 : 2 (metal : ligand) molar ratio. The conductance measurements have low values due to their nonelectrolytic behavior. The X-ray powder diffraction solid powder pattern revealed a semicrystalline nature. In vitro, we screened the synthesized folate chelates for antibacterial and antifungal activities. The inhibition of four bacterial and two fungi pathogens (E. coli, B. subtilis, P. aeruginosa, S. aureus, A. flavus, and Candida albicans) was improved using a folic acid drug relative to the control drug.


Introduction
Folic acid (FAH 2 ) is also referred to as pteroylglutamic acid [N-(4{[(2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl] amino}benzoyl)-L-glutamic acid]. Folic acid is a member of the B9 vitamin family [1]. Folic acid consists of three sections of p-aminobenzoic acid (PABA) pteridine ring and glutamic acid moieties (Figure 1). e term folate refers to the deprotonated form of folic acid, which plays an essential role in critical biosynthetic processes in mammalian cells [2]. e folate molecule is made as a coenzyme in the case of DNA, RNA, and protein components synthesized by single carbon transfer reactions [3]. Folate is necessary for the synthesis of methionine, methylation of DNA, histone neurotransmitters, and lipids. e deficiency in folic acid led to DNA strand breaks [4], DNA hypomethylation [5], and abnormal gene expression [6]. Folic acid has a potential anticancer activity in some important cases, such as breast, colorectal, and ovarian carcinomas [7]. e World Health Organization has added folic acid to the list of trusted drugs that are most needed. For humans, this vitamin should be obtained from the diet because the human body cannot produce it. e recommended daily folate intake for adults is 400 μg [8]. Folic acid is one of the major molecules for the synthesis of DNA [9]. Folic acid receptors (FRs) have been considered a target to assimilate the organic molecules associated with folic acid in cells. Application of folic acid receptor-targeted molecules is not new; there have been many articles addressing the conjugation of folates to known organic drug molecules, such as taxol, paclitaxel, and doxorubicin, to improve drug targeting [10][11][12][13].
However, there is a lack of literature concerning the conjugation of folic acid to metal-based molecules and even less regarding their chemotherapeutic potential. Incorporating simple folate ligands into biologically active metal complex systems might offer a simple strategy for improving their selective uptake in FR cells [2]; on the contrary, it was found that the metal complexes of folic acid might provide an advantage by increasing the inhibitory activity of folic acid [7]. Folic acid can form complexes with different metal ions via the pteridine ring or glutamic acid moiety. e pteridine ring binds to metal ions via its nitrogen and oxygen atoms (of a carbonyl or an iminol group) in the coordination process [14]. But this case is not common since the pteridine ring is a highly π-electron-deficient heterocycle, while glutamic acid forms stable complexes with metal ions [15]. ree types of coordination modes have been mentioned in literature on folic acid: bidentate bridging via α and c carboxylate groups [16][17][18][19][20], tridentate (α and c carboxylate groups) and the amide nitrogen of the glutamate moiety [2,21], and chelating via α carboxylate and the amide nitrogen of the glutamate moiety [22]. Many publications focus on the synthesis and characterization of metal-folate complexes. Still, they do not discuss their biological activity although such complexes might have high selectivity towards FR over expressing cells and thus a distinct biological activity and anticancer properties.
Here, we describe the synthesis of new metal-folate complexes as therapeutic agents. New metal-folate complexes were formed from the chemical reactions between FAH 2 and some metal ions like Ga(III), Ge(IV), and Hf(IV). e synthesized folate complexes were discussed by using microanalytical, spectral tools, and thermogravimetric analysis. An antimicrobial assessment for the synthesized folate complexes towards bacteria and fungi species and the anticancer properties have been studied, for instance, on the germanium(IV)-folate complex against the human hepatocellular carcinoma (HepG-2) cell line.

Chemicals and Equipment.
e pure grade chemical materials (GaCl 3 , GeCl 4 , HfCl 4 , and folic acid) were received from Sigma-Aldrich chemical company. e microanalytical, physical, and spectral measurements with corresponding models are listed as follows:  e microanalysis (%) of the elements (C, H, and N) for the three synthesized complexes is summarized as follows:

Antimicrobial Inhibitions.
A modified Kirby-Bauer experiment was used to assess antimicrobial inhibitions [23]. A cytotoxicity assay for the germanium(IV)-folate complex was performed using the human hepatocellular carcinoma  (HepG-2) cell line [24,25]. e HepG-2 cell line was obtained from the VACSERA Tissue Culture Unit.

Infrared Interpretations.
e FTIR spectra for the FAH 2free chelate and the synthesized metal complexes are shown in Figures 2(a) and 2(b). All complexes have similar spectra, which reflect similar structural characteristics for these complexes. e significant infrared spectral bands were assigned and inserted in Table 1. ese assignments can be summarized with the following evidences: (i) e folic acid-free ligand has a stretching vibration band at 1702 cm −1 , which is attributed to ] (C�O) ketonic of the carboxylic group; this band is overlapped with the ] (C�O) amide group [16]. is band was shifted to lower wavenumber in all complexes, with a marked decrease in the spectral intensity. Interestingly, the bands present at 1604 and 1485 cm −1 were assigned to ] as (COO − ) and ] s (COO − ) stretching vibrations in the case of the spectrum of the folic acid ligand. ese bands were shifted to higher and lower frequencies, respectively, under complexation because of the involvement of the oxygen of the carboxylate group in the coordination towards metal ions. e difference between antisymmetric (] as COO) and symmetric (] s COO) stretching vibrations for the COO group gives an impression about the speculated molecular structure of the folate complexes [27]. e coordination mode of the carboxylate group was discussed by Deacon and Phillips [28] according to the e interactions between the metal ions and the carboxylate group were (a) monodentate fashion when Δ] >200 cm −1 , (b) bidentate/chelating fashion when Δ] is smaller than ionic form, and (c) bridging bidentate when Δ] has nearly ionic values. e observed Δ] values for all the complexes exhibited within the 211-217 cm −1 range, as discussed in Table 2. erefore, the carboxylate groups chelated to metal ions as a unidentate mode [28]. (ii) e FAH 2 -free ligand has a distinguished band at 3318 cm −1 that is attributed to the ] (N−H) of the amido group. is band blue-shifted in the case of the spectra of the synthesized complexes, and the characteristic band for δ (NH) amide is downshifted in the complexes relative to the free ligand. is result is in agreement with the HNMR data, which confirmed the participation of the amide nitrogen in the coordination of metal ions. So, this situation confirms the presence of a folate ligand as a tridentate ONO chelate through α-COO, β-COO, and amide groups [2,21]. . ese data assume a tridentate manner of coordination for the folate chelate towards metal ions by deprotonating the two carboxylic groups of glutamic acid and NH of the amide group.

Electronic Spectra.
e UV-visible spectrum of folic acid ( Figure 3) exhibits absorbance bands at 220 and (290 and 380 nm) attributed to π ⟶ π * and n ⟶ π * transitions. e first peak is probably due to the alkyl and aromatic species, while the other bands are assigned to COOH, NH, NH 2 , and C�O groups [32]. ese bands are shifted to longer wavelengths, which support the complexity of metal ions with carboxylic and amide groups (Figure 3). e folate complexes have bands within the 428-454 nm range due to the L ⟶ M CT transitions [33,34].

1 HNMR Study.
e 1 HNMR spectrum of the FAH 2 -free ligand in DMSO-d 6 ( Figure 1S and Table 3) has a quadrature signal obtained at δ 2.317 and 2.293 ppm of the protons H (21) and triplet signal at δ 2.504, 2.501, and 2.495 ppm of the protons H (22) due to the methylene CH 2 group. e signals were shifted downfield in the case of the synthesized folate complexes ( Figures 2S and 3S and Table 3). A signal at δ 8.093 ppm of the proton H (18) is due to the NH amide group. After complexation, this signal was upfield shifted with a chemical shift difference at 0.305-0.338 ppm, thus supporting the amido group's involvement in the chelation process [35]. e triplet signal at δ 4.476 ppm of the proton H (19) is due to the methylene group. is group significantly affected the spectra of the folate complexes, which was shifted to the low field. is was because of the effect of attaching the amide and carboxylic groups. A signal at δ 4.496 ppm of the proton H (9) is due to shifting of the methylene CH 2 group to a low field, a result of the cycling effect from one side and the NH group effect from the other side. A single signal at δ 8.5 ppm of the proton H (7)

ermogravimetric Analysis.
e stabilities of the thermal decompositions of the synthesized metal chelates were discussed based on the TGA analysis ( Figure 4 and Table 4). e thermal decomposition of the NH 4 [Ga(FA) 2 ]·4H 2 O complex (I) takes place through three degradation stages with a mass loss of 8.66% (temperature range between 56 and 181°C), 37.98% (temperature range between 181 and 445°C), and 44.056% (temperature range between 445 and 800°C), respectively. e remaining residual mass is due to the gallium metal contaminated with few unoxidized carbon atoms. e thermal decomposition of the [Ge(FA) 2 ]·3H 2 O complex(II) passes through three steps. e first decomposition step is within the 30-146°C temperature range and has a mass loss of 5.45%. e second step is within the 146-321°C range and has a mass loss of 38.54%. e third step is within the 321-800°C range, with a mass loss of 39.58%. e germanium metal polluted with few unoxidized carbon atoms is a final residue at 800°C. e [Hf(FA) 2 ]·3H 2 O complex(III) was decomposed through three steps within temperature ranges at 34-154°C, 154-380°C, and 358-800°C with mass losses of 4.86%, 33.55%, and 45.60%, respectively. At 800°C, the hafnium metal was mixed with some nonoxidized carbon atoms, which represents the residual material.

Kinetic and ermodynamic Parameters.
Using official integral methods, including Horowitz-Metzger (HM) [36] and Coats-Redfern (CR) methods [37], the parameters of the kinetic thermodynamic process are calculated and listed in Table 5. From the theoretical data, it can be deduced that the activation energies and, by extension, the thermal stability of the folate complexes are ordered as Ga(III) > Ge(IV) > Hf(IV). e results of the two methods used to calculate the thermodynamic parameters are satisfactorily consistent with each other. e pyrolysis steps of the folate complexes are nonspontaneous (∆S * ) with negative data due to the thermal stability of the synthesized complexes.  discussed based on the elemental analysis, conductance, FTIR, UV-Vis, 1 HNMR, and TGA measurements. e various analyses performed in this study deduced that the coordination between the folic acid ligand and central metal ions passes through oxygen atoms of the carboxylate group and nitrogen atoms of amide groups.

XRD, SEM, and TEM Analyses.
In X-ray diffractograms of the folate complexes, major patterns were scanned within the 4°to 80°2θ range. e XRD patterns of the folate complexes ( Figure 6) are completely different from those of folic acid [38] due to the formation of new coordination compounds. e X-ray diffractogram of the three synthesized metal-folate complexes is semicrystalline as well as amorphous in nature. According to the Scherrer equation, the crystallite sizes of the synthesized folate complexes were calculated using full width at half maximum of the diffraction peak. e crystallite sizes of the folate complexes were located at ∼30-100 nm. e SEM images of the synthesized complexes are given in Figure 7. e surface morphology changes with changes in the ionic radius of specific metal ions; both the images have many irregular shapes. TEM micrographs of the Ga 3+ , Ge 4+ , and Hf 4+ complexes (Figure 8) demonstrated particles within a nanosize range. e average particle diameter for folate complexes was in the range of 30-100 nm, in agreement with the XRD data.

Biological Results.
We report the results of in vitro microbial tests of the FAH 2 drug and its gallium(III), germanium(IV), and hafnium(IV) complexes against a panel of bacteria and fungi species (Table 6 and Figure 9):     than free FAH 2 -free drugs. e folic acid ligand and its metal complexes displayed antibacterial and antifungal activities against the tested organisms. According to Tweedy's chelation theory [39], these data support the notion that metal ions enhance the antibacterial and antifungal activities by increasing lipophilicity, thus facilitating the penetration of metal complex across the cell membrane [39,40]. We tested the inhibitory concentration 50% (IC 50 ) of the germanium(IV) complex with human hepatocellular carcinoma (HepG-2) cells (Table 7). e IC 50 was higher than 1000 µg/ mL, indicating that the germanium(IV) complex has a significant efficiency against the HepG-2 cell line.

Conclusions
e metal chelation between Ga(III), Ge(IV), and Hf(IV) metal ions with folic acid with 1 : 2 molar ratio was prepared. e folic acid acts as a tridentate chelate via oxygen and nitrogen atoms of carboxylate and amido groups. e formulas of the synthesized folate complexes were NH 4 1 HNMR, and TGA analyses. e thermal stability behavior of the synthesized folate complexes was confirmed as dependent on the calculation of kinetic thermodynamic parameters. e biological efficiency of the synthesized folate complexes was screened against bacteria and fungi species with significant values. e experimental IC 50 data of the germanium(IV) complex in vitro showed the readiness of the Ge(IV) complex to be used as an antihepatocellular anticancer drug.

Data Availability
e data associated with this study can be accessed from the first author upon a reasonable request.

Disclosure
No funding was obtained for the study.

Conflicts of Interest
e authors declare that they have no conflicts of interest.  : Inhibition zone diameter of DMSO control, "tetracycline" antibacterial agent, "amphotericin B" antifungal agent, and folic acid and its synthesized complexes against some bacteria and fungi species.