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.

1. Introduction

Folic acid (FAH2) 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). The term folate refers to the deprotonated form of folic acid, which plays an essential role in critical biosynthetic processes in mammalian cells [2]. The 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. The 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]. The 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. The 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 [1013].

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. The 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]. Three types of coordination modes have been mentioned in literature on folic acid: bidentate bridging via α and γ carboxylate groups [1620], tridentate (α and γ 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 FAH2 and some metal ions like Ga(III), Ge(IV), and Hf(IV). The 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.

2. Materials and Methods

2.1. Chemicals and Equipment

The pure grade chemical materials (GaCl3, GeCl4, HfCl4, and folic acid) were received from Sigma-Aldrich chemical company. The microanalytical, physical, and spectral measurements with corresponding models are listed as follows:

Analysis techniqueInstruments
Elemental analysisPerkinElmer, CHN 2400
ConductanceJenway 4010 conductivity meter
FTIR spectraBruker FTIR spectrophotometer
Electronic spectraUV2 Unicam UV/Vis spectrophotometer
Magnetic susceptibilityMagnetic susceptibility balance
1HNMR spectraVarian mercury VX-300 NMR spectrometer, 300 MHz
ThermogravimetricTG/DTG–50H, Shimadzu thermogravimetric analyzer
SEMQuanta FEG 250 equipment
XRDX‘Pert PRO PAN analytical, with copper target
TEMJEOL 100 s microscopy
2.2. Preparation of Folate Complexes

The folate complexes with the molecular formulas NH4[Ga(FA)2]·4H2O (1), [Ge(FA)2]·3H2O (2), and [Hf(FA)2]·3H2O (3) were synthesized using the following procedure. Mixtures of 2.0 mmol of GaCl3, GeCl4, or HfCl4 with 4.0 mmol of FAH2 were stirred in 25 mL of CH3OH solvent. The pH of the mixtures was adjusted to about 7.7–8.0 by adding ammonia solution (0.2M) dropwise and refluxed at about ∼60°C for 3 h. The solutions of these mixtures were reduced by leaving them still for 1 week. The solid yields of folate complexes reached 60–66%, with a higher melting point above 300°C. The microanalysis (%) of the elements (C, H, and N) for the three synthesized complexes is summarized as follows:

ComplexesElementsCalc. (%)Found (%)


2.3. 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]. The HepG-2 cell line was obtained from the VACSERA Tissue Culture Unit.

3. Results and Discussion

3.1. Molar Conductance

The molar ratios and conductance behaviors of gallium(III), germanium(IV), and hafnium(IV) folate complexes have been discussed. The elemental analyses are consistent with 1 : 2 (metal : ligand) stoichiometry. The synthesized complexes are insoluble in polar solvents but soluble in common organic solvents like DMF and DMSO. Gallium(III), germanium(IV), and hafnium(IV) folate complex solutions dissolved in DMSO showed slightly low conductance (Λm = 14–30 ohm−1 cm2 mol−1), suggesting a nonelectrolyte behavior [26].

3.2. Infrared Interpretations

The FTIR spectra for the FAH2-free 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. The significant infrared spectral bands were assigned and inserted in Table 1. These assignments can be summarized with the following evidences:(i)The folic acid-free ligand has a stretching vibration band at 1702 cm1, which is attributed to ν (C=O)ketonic of the carboxylic group; this band is overlapped with the ν (C=O) amide group [16]. This 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 νas (COO) and νs (COO) stretching vibrations in the case of the spectrum of the folic acid ligand. These 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. The difference between antisymmetric (νasCOO) and symmetric (νsCOO) stretching vibrations for the COO group gives an impression about the speculated molecular structure of the folate complexes [27]. The coordination mode of the carboxylate group was discussed by Deacon and Phillips [28] according to the Δν = [νas (COO)–νs (COO)] relationship. The interactions between the metal ions and the carboxylate group were (a) monodentate fashion when Δν >200 cm−1, (b) bidentate/chelating fashion when Δν is smaller than ionic form, and (c) bridging bidentate when Δν has nearly ionic values. The observed Δν values for all the complexes exhibited within the 211–217 cm−1 range, as discussed in Table 2. Therefore, the carboxylate groups chelated to metal ions as a unidentate mode [28].(ii)The FAH2-free ligand has a distinguished band at 3318 cm−1 that is attributed to the ν (N−H) of the amido group. This 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. This 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].(iii)There are two vibration bands in the case of the spectrum of FAH2-free ligand located at 3558 and 3411 cm−1, which are assigned to the ν (O–H) vibrations of the carboxylic groups. These bands are present with broadening in the spectra of folate complexes due to the presence of the crystalline water molecules [1, 29, 30].(iv)The weak and very weak intensity bands in the range of 600–400 cm−1 are attributed to stretching vibrations of M–N and M–O [31]. These 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.

3.3. Electronic Spectra

The 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. The first peak is probably due to the alkyl and aromatic species, while the other bands are assigned to COOH, NH, NH2, and C=O groups [32]. These bands are shifted to longer wavelengths, which support the complexity of metal ions with carboxylic and amide groups (Figure 3). The folate complexes have bands within the 428–454 nm range due to the L ⟶ MCT transitions [33, 34].

3.4. 1HNMR Study

The 1HNMR spectrum of the FAH2-free ligand in DMSO-d6 (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 CH2 group. The 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]. The triplet signal at δ 4.476 ppm of the proton H (19) is due to the methylene group. This group significantly affected the spectra of the folate complexes, which was shifted to the low field. This 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 CH2 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) is due to the (CH=N) 2-pyrazine group. These findings strongly support the coordination site through the tridentate fashion of the folate ligand [21, 35].

3.5. Thermogravimetric Analysis

The stabilities of the thermal decompositions of the synthesized metal chelates were discussed based on the TGA analysis (Figure 4 and Table 4). The thermal decomposition of the NH4[Ga(FA)2]·4H2O 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. The remaining residual mass is due to the gallium metal contaminated with few unoxidized carbon atoms. The thermal decomposition of the [Ge(FA)2]·3H2O complex(II) passes through three steps. The first decomposition step is within the 30–146°C temperature range and has a mass loss of 5.45%. The second step is within the 146–321°C range and has a mass loss of 38.54%. The third step is within the 321–800°C range, with a mass loss of 39.58%. The germanium metal polluted with few unoxidized carbon atoms is a final residue at 800°C. The [Hf(FA)2]·3H2O 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.

3.6. Kinetic and Thermodynamic 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). The results of the two methods used to calculate the thermodynamic parameters are satisfactorily consistent with each other. The pyrolysis steps of the folate complexes are nonspontaneous (∆S) with negative data due to the thermal stability of the synthesized complexes.

3.7. Speculated Structure of Folate Complexes

The proposed structures of NH4[Ga(FA)2]·4H2O (1), [Ge(FA)2]·3H2O (2), and [Hf(FA)2]·3H2O (3) folate complexes (Figure 5) were discussed based on the elemental analysis, conductance, FTIR, UV-Vis, 1HNMR, and TGA measurements. The 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.

3.8. XRD, SEM, and TEM Analyses

In X-ray diffractograms of the folate complexes, major patterns were scanned within the 4° to 80° 2θ range. The 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. The 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. The crystallite sizes of the folate complexes were located at ∼30–100 nm. The SEM images of the synthesized complexes are given in Figure 7. The surface morphology changes with changes in the ionic radius of specific metal ions; both the images have many irregular shapes. TEM micrographs of the Ga3+, Ge4+, and Hf4+ complexes (Figure 8) demonstrated particles within a nanosize range. The average particle diameter for folate complexes was in the range of 30–100 nm, in agreement with the XRD data.

3.9. Biological Results

We report the results of in vitro microbial tests of the FAH2 drug and its gallium(III), germanium(IV), and hafnium(IV) complexes against a panel of bacteria and fungi species (Table 6 and Figure 9):

E. coliB. subtilisAspergillus flavus
P. aeruginosaS. aureusCandida albicans

Gallium(III), germanium(IV), and hafnium(IV) folate complexes were more active against B. subtilis and S. aureus bacteria than the free folic acid ligand drug. The hafnium(IV) complex has a bactericidal efficiency against Escherichia coli. All folate metal complexes have antifungal activity against different kinds of fungi understudy rather than free FAH2-free drugs. The 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% (IC50) of the germanium(IV) complex with human hepatocellular carcinoma (HepG-2) cells (Table 7). The IC50 was higher than 1000 µg/mL, indicating that the germanium(IV) complex has a significant efficiency against the HepG-2 cell line.

4. Conclusions

The metal chelation between Ga(III), Ge(IV), and Hf(IV) metal ions with folic acid with 1 : 2 molar ratio was prepared. The folic acid acts as a tridentate chelate via oxygen and nitrogen atoms of carboxylate and amido groups. The formulas of the synthesized folate complexes were NH4[Ga(FA)2]·4H2O, [Ge(FA)2]·3H2O, and [Hf(FA)2]·3H2O. These complexes have been discussed and assigned according to the microanalytical, conductance, FTIR, UV-Vis, 1HNMR, and TGA analyses. The thermal stability behavior of the synthesized folate complexes was confirmed as dependent on the calculation of kinetic thermodynamic parameters. The biological efficiency of the synthesized folate complexes was screened against bacteria and fungi species with significant values. The experimental IC50 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

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


No funding was obtained for the study.

Conflicts of Interest

The authors declare that they have no conflicts of interest.


The author is grateful to “Princess Nourah Bint Abdulrahman University” for supporting this research through the sabbatical leave program.

Supplementary Materials

Supplementary files included in the manuscript are 1HNMR spectra of the FAH2-free ligand (Figure 1S), Ga(III) complex (Figure 2S), and Ge(IV) complex (Figure 3S) in DMSO-d6. (Supplementary Materials)