Synthesis, characterization and metal coordination of a potential β-lactamase inhibitor: 5-Methyl-2-phenoxymethyl-3-H-imidazole-4-carboxylic acid (PIMA)

2.1 Synthesis

PIMA was obtained following Scheme 2.

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Scheme 2

Synthesis of PIMA.

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The imidazole carboxylate (1), precursor of PIMA, can be easily obtained by one-pot reaction of a suitable 1,2-diaza-1,3-diene with 2-phenoxy-acetaldehyde and benzylamine as already described (Preti et al., 2010). The reaction implies the 1,5-electrocyclization of the in situ formed azavinyl azomethine ylide, promoted by microwave irradiation. The described intermediate 1 simply requires two deprotection steps to obtain the final compound: an initial hydrolysis furnishes the carboxy group, while the subsequent debenzylation unmasks the NH of imidazole.

Ethyl 3-benzyl-5-methyl-2-phenoxymethyl-3-H-imidazole-4-carboxylate (1) (840 mg, 2.40 mmol) was treated with a 2 M solution of sodium hydroxide in ethanol (3 mL, 6.00 mmol) and heated at reflux temperature for 2 h. The reaction mixture was then partitioned between water (20 mL) and diethyl ether (20 mL) and the aqueous phase was acidified with HCl 5% w/w. The precipitate formed was filtered and dried in vacuo overnight affording 3-Benzyl-5-methyl-2-phenoxymethyl-3H-imidazole-4-carboxylic acid (2) as a cream-colored solid (635 mg, 82%). M.p. 206–208 °C. ¹H NMR (400 MHz, CD₃OD): δ 2.69 (3H, s, CH₃), 5.42 (2H, s, CH₂O), 5.92 (2H, s, NCH₂), 6.94 (2H, d, J 7.9, H_Arom), 7.05 (1H, t, J 7.6, H_Arom), 7.23 (2H, d, J 6.5, H_Arom), 7.29–7.40 (5H, m, H_Arom). ¹³C NMR (100 MHz, CD₃OD): δ 10.5, 49.9, 59.6, 114.5 (2C), 121.6, 122.3, 126.5 (2C), 128.0, 128.7 (2C), 129.5 (2C), 134.3, 137.8, 144.6, 157.0, 159.5. MS (EI): m/z (%) 278 [M–COOH]⁺ (1), 185 (46), 91 (100), 77 (3), 65 (17).

The previous intermediate 2 (604 mg, 1.87 mmol) was dissolved in methanol (120 mL) and palladium catalyst (Pd(OH)₂, 20 wt.% Pd on carbon, 604 mg) was added. The system was evacuated, purged with hydrogen and vigorously stirred for 16 h. The reaction mixture was then filtered and concentrated under reduced pressure, affording 5-methyl-2-phenoxymethyl-3-H-imidazole-4-carboxylic acid (PIMA) as a cream-colored solid (360 mg, 83%). M.p. 209–210 °C. ¹H NMR (400 MHz, DMSO-d₆): δ 2.42 (3H, s, CH₃), 5.07 (2H, s, CH₂O), 6.98 (1H, t, J 7.5, H_Arom), 7.05 (2H, d, J 7.5, HArom), 7.32 (2H, t, J 7.5, H_Arom). ¹³C NMR (100 MHz, DMSO-d₆): δ 12.3, 62.4, 115.2 (2C), 121.7, 124.2, 130.0 (2C), 138.8, 143.4, 158.3, 163.2. MS (EI): m/z (%) 155 [M–Ph]⁺ (1), 141 (3), 127 (5), 113 (4), 99 (17), 85 (50), 71 (69), 57 (100), 43 (61). Anal. Calculated for C₁₂H₁₂N₂O₃: C 62.00, H 5.21, N 12.12. Found%: C 61.72, H 5.18, N 12.87.

2.2 Potentiometric measurements

The aqueous solution of the PIMA (5.0 × 10⁻³ M) was obtained by water dilution of a methanolic solution (5.0 × 10⁻² M); the concentration was determined potentiometrically. The percentage of methanol in the final solution was 10%. Potentiometric measurements were performed in aqueous solution at 25.0 ± 0.1 °C, using a fully automated ORION 960 Autochemistry System following the general procedures previously reported (Menabue et al., 1998). All experiments were carried out in a nitrogen atmosphere at an ionic strength of 0.1 M (adjusted with solid NaNO₃). Aqueous NaOH (0.01 M) or HNO₃ (0.01 M) was used as titrant. Ten titrations were performed with 40 data points in each titration in the range of pH 1–9. The protonation constants (β_pq) are defined in the following equations:

2.3 Spectrophotometric measurements

UV–vis spectrophotometric measurements were performed using a Jasco V-570 spectrophotometer at 25 ± 0.1 °C in the 200–400 nm spectral range employing 1 cm quartz cells.

Spectrophotometric titrations were performed using 5.0 × 10⁻⁴ M solution of PIMA obtained by water dilution of a freshly prepared methanolic solution (5.0 × 10⁻² M) with addition of an appropriate amount of a aqueous solution of the metal ion (5 × 10⁻² M; 1:1, 1:2 and 1:3 M:L molar ratios). The amount of methanol in the final solution was 1%; hence, it was used as an aqueous solution. The pH-metric titrations of the ligand and metal–ligand systems were obtained varying the pH value by adding small amounts of concentrated NaOH or HCl in the pH range 2–9 (total number of different pH values ranging from 20 to 25 in each titration). A constant ionic strength of 0.1 M (NaNO₃) was maintained in all experiments. Each titration was repeated three times. The overall stability constants (expressed as log β_MLH) were refined from spectrophotometric data by least-squares calculation using the software HypSpec (Gans et al., 1999), and taking into account the presence of the species [Fe(OH)]²⁺; [Fe(OH)₂]⁺; [Fe₂⁻(OH)₂]⁴⁺ (Escandar et al., 1994).

2.4 NMR spectroscopy

NMR spectra were recorded on a Bruker Avance AMX-400 spectrometer with a Broad Band 5 mm probe in inverse detection. Nominal frequencies were 100.13 MHz for ¹³C, and 400.13 MHz for ¹H. For each sample, 0.5 mL of mM solution was prepared in MeOD-d₄. The synthesis of Ga³⁺:PIMA complex in MeOD-d₄ was obtained in situ, by adding to mM ligand solution (0.5 mL) the proper addition of Ga(NO₃)₃ solution to reach the 1:1 metal to ligand molar ratio. Spectra were registered after few minutes from the addition. Typical parameters were used for 2D COSY, HSQC, and HMBC experiments.

2.5 Computational details

All calculations were performed with the Gaussian 03 package of programs (Frisch et al., 2004) and GaussView 03 (Dennington et al., 2003) used as the plotting tool for data visualization. The computations were performed by DFT approaches, and the structures were fully optimized using hybrid-functional B3LYP applied to the 6-311G^∗∗ basis set (B3LYP/6-311G^∗∗).

3.1 Acid–base equilibria

The acid–base equilibria of PIMA are reported in Scheme 3.

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Scheme 3

Neutral and ionic form of PIMA with atom numbering scheme.

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The analysis of potentiometric data yields two pK_a values corresponding to the dissociation of carboxylic and protonated imidazole groups [pK_a1 = 2.78(2), pK_a2 = 5.81(2)] (Fig. 1S).

The spectrophotometric titration of PIMA (Fig. 1) shows a significant increase in absorbance at $\lambda$ 260 nm and $\lambda$ 300 nm with increasing pH; by plotting A vs. pH a titration trend is observed although the unique clearly visible equivalent point is the dissociation of the protonated imidazole group. By processing the data with HySpec program (Gans et al., 1999) two pK_a are obtained [pK_a1 = 2.91(2) pK_a2 = 5.84(2)] whose values are in good agreement with the potentiometrically found data. pK_a1 value is near to that found for IMA (Sanna et al., 1998; Várnagy et al., 1994) while pK_a2 is lower than the value found for IMA (7.34(1), 7.12(4) (Aljahdali et al., 2013) and is lower also with respect to free imidazole (6.95) (Várnagy et al., 1994) or imidazole-4-carboxylic acid (6.28) (Podsiadly, 2008). The greater value of IMA with respect to imidazole was previously attributed to the formation of an intramolecular hydrogen bond involving the protonated imidazole N and the carboxylate group (Aljahdali et al.), even though in the crystal structure of hydrated IMA reported by Okabe and Hayashi (1999) there is no evidence of intramolecular hydrogen bond. Hence, the structure of IMA in solution still remains unclear. In our case, the presence of an electron-withdrawing group such as the phenoxymethyl contributes to increase the acidity of NH⁺ hydrogen.

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Figure 1

Spectrophotometric titration of an aqueous solution of PIMA 5 × 10⁻⁴ M, the inset shows the plot of absorbance (A) vs. pH at $\lambda$  = 260 nm.

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3.2 Metal complexation

The metal complexation was studied by means of ¹H NMR and spectrophotometric titrations while potentiometric analysis was prevented by the poor water solubility of the metal complex species in the investigated pH range.

PIMA shows the typical ¹H NMR spectral pattern of substituted imidazoles (see experimental section). Ga(NO₃)₃ is used as NMR probe for Fe³⁺ (Atkinson et al., 1998; Hara et al., 2000), and its addition to a solution of PIMA at acidic pH, immediately originates a complex species, whose spectral pattern resembles one of the free ligands, but it is downfield shifted (Fig. 2). The greater downfield shift is observed for methylenic (H-6) and methylic (H-14) protons, and this behavior should be principally due to the formation of a metal complex, which is in fast exchange with the free ligand in the NMR timescale.

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Figure 2

¹H NMR titration of PIMA with Ga³⁺ in MeOD-d₄. From bottom (free ligand), spectra obtained by progressive additions of metal ion to the ligand up to metal to ligand 1:1 M ratio (top spectrum).

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By reaching the Ga³⁺:PIMA 1:3 M ratio no further downfield shift is observed but a new set of signals appears, attributed to a complex species at M:L 1:2 M ratio while the M:L 1:3 metal complex, observed at downfield, represents a minor species.

As far as Gallium ion is added a new set of signals which are attributed to the formation of M:L 1:1 species appears at higher field than M:L 1:2 complex species. From this point onward, the metal-complex species present in solution exchange fast between each other with respect to the NMR timescale, then the addition of Ga³⁺ varies speciation distribution as observed by the change in signals’ integrated areas. Upon reaching M:L 1:1 the three complex species at M:L 1:1, 1:2 and 1:3 are simultaneously present with the prevalence of 1:1 and 1:3 M ratio.

The ¹H ¹³C hetero correlated spectra reported in Fig. 3 (Panel A and B) show that the most significant shifts are observed for H-6 protons while the phenyl protons and C8 are only poorly affected. This finding suggests the involvement of both carboxylic group and imidazole nitrogen in metal coordination with the formation of a five-membered metallacycle.

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Figure 3

Panel A: Overlap of ¹H–¹³C HSQC (Hetero-nuclear Single Quantum Coherence) spectra of free PIMA (red) and Ga³⁺:PIMA 1:1 system (black). Panel B: Overlap of ¹H–¹³C HMBC (Hetero-nuclear Multiple Bond Coherence) spectra of free PIMA (red) and Ga³⁺:PIMA 1:1 system (black). All spectra were acquired in MeOD-d₄ at 298 K.

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Spectrophotometric titration was performed by adding Ga³⁺, Fe³⁺ and Zn²⁺ to a methanolic-aqueous solution (1:100 V/V) of the ligand. Fig. 4 reports the spectra of Fe³⁺:PIMA system; at each addition of metal ion there is an increase in absorbance which reaches the maximum value at the M:L 1:1 M ratio (inset Fig. 4).

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Figure 4

Spectrophotometric titration of PIMA 5 × 10⁻⁴ M with Fe³⁺, [Fe³⁺] increasing from bottom to top. Inset shows the plot of absorbance (A) vs. M/L molar ratio at $\lambda$ 300 nm.

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Increasing pH in the M:PIMA 1:2 M ratio system for all metal ions (Fig. 5 reports the titration for Fe³⁺:PIMA system), an increase in absorbance at $\lambda$ 300 nm is observed confirming that the formation of metal complex is pH dependent. At pH 4 the complex species is completely formed as the absorbance reaches its maximum value. This absorption band was attributed to the dissociation of protonated imidazole group; therefore, we can confirm that metal complexation involves imidazole nitrogen and the replacement of hydrogen by the metal ion occurs at a pH value significantly lower compared to the dissociation of the free ligand (pH ∼ 4 vs. pH ∼ 5.8). Over pH 8 the competition with hydroxyl anion becomes significant and, in this working concentration, the formation of metal hydroxide takes place.

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Figure 5

pH-metric spectrophotometric titration of Fe³⁺:PIMA system in 1:2 M ratio, inset shows the plot of absorbance vs. pH at $\lambda$ 300 nm; [Fe³⁺] 2.5 × 10⁻⁴ M.

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From the spectrophotometric titration of Mⁿ⁺:PIMA system in 1:1, 1:2 M ratio for n = 2 and 1:3 M ratio for n = 3 the stability constants of the complex species are evaluated taking into account different types of protonated and neutral species; best convergence of data was reached considering the log β values reported in Table 1.

Fig. 6 reports the species distribution curves for Fe³⁺, Ga³⁺ and Zn²⁺ containing systems. The prevailing species in physiological condition is ML₃ for Fe³⁺ and Ga³⁺ and ZnL_2, in which PIMA coordinates the metal ion through deprotonated carboxylic oxygen and imidazole nitrogen, as it was proposed by NMR data.

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Figure 6

Species distribution curves for M:L systems [M]_tot = 2.5 × 10⁻⁴; [L]_tot = 5 × 10⁻⁴.

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The formation of a chelate complex is consistent with the high $\text{log}{K}_{\text{ZnL}}^{\text{Zn}}$ QUOTE value compared to QUOTE $\text{log}{K}_{\text{HL}}^{\text{H}}$ of PIMA as suggested by Sigel et al. (2000). This coordination behavior was found also for imidazole-4-carboxylic acid toward V³⁺ ion in solution (Podsiadly, 2008). In the solid state IMA forms chelate complexes with Co²⁺ and Mn²⁺ ions (Kurdziel et al., 2003) while with Zn²⁺, in acidic conditions, it was found to give polymeric arrangement in which one anionic ligand molecule bridges two Zn(II) ions and the second neutral ligand acts as monodentate through carboxylic oxygen (Drożdżewski et al., 2002). The stability constants of Zn(II) complexes are comparable to those found for the corresponding species with IMA by Török et al. (1998) (log β₁₁₀ 3.81 and log β₁₂₀ 7.15), in which the suggested N,O coordinating mode was attributed to imidazole nitrogen and carboxylic oxygen. A comparison with similar, but not imidazole-based ligands, such as β-ala, evidences how the imidazole ring greatly increases the stability of metal complexes.

The stability constants of Fe³⁺ PIMA complexes are similar to those observed by Farkas et al. (2007) for the corresponding complexes with imidazole-4-carbohydroxamic acid (log β₁₁₀ 10.46(1) and log log β₁₁₁ 12.1(1)) in which the chelate coordination mode involves only O_hydroxamate atoms. In the investigated system, imidazole-4-carbohydroxamic acid resulted competitive with hydroxide ion in binding Fe(III), and precipitation phenomena were attributed to the formation of low water-soluble polynuclear complexes rather than iron-hydroxo species. The log β₁₁₀ for PIMA is greater than the corresponding one for carboxylate complexes, and this suggests a possible involvement of imidazole nitrogen in stabilizing the metal complex, and particularly a remarkable π-acceptor property of imidazole ring, as suggested by Török et al. (1998). The precipitation of hydroxo species was only observed above pH 9, suggesting the immobilization of iron by PIMA in the experimental conditions applied for spectrophotometric titrations. Differently from the cited system, in our case also a FeL₃ complex is found, which prevails at physiological pH as well as for Ga³⁺, consistently with NMR data. Ga³⁺ ion gives less stable species than Fe³⁺; this trend was previously found for other chelated complexes with O-donor ligands (Ferrari et al., 2013). According to species distribution curves, PIMA seems a good iron chelator at physiological pH, at least in the experimental conditions applied for spectrophotometric measurements. A more proper indicator of the Fe³⁺ sequestering ability in physiological conditions is represented by pFe³⁺ defined as –log[Fe³⁺] (Liu and Hider, 2002).

Table 2 reports the pFe³⁺ values for several hexadentate and bidentate ligands, which are effective iron chelators for therapeutic applications (Liu and Hider, 2002). Hexadentate ligands show greater pFe³⁺ values but they have the disadvantage of a high molecular weight.

Among bidentate ligands hydroxypyridinones present a great effectiveness in iron chelation conjugated with a low molecular mass. The key property for orally active iron chelator is its ability to be efficiently absorbed from the gastrointestinal tract (GIT) and to cross biological membranes. The molecular size is a critical factor, which influences the rate of drug absorption: with respect to the molecular weight ‘‘cut-off’’ (ca. 500 Da) for the paracellular route absorption in the human small intestine (Liu and Hider, 2002) PIMA (232 Da) could reach the target sites at sufficient concentration.

3.3 DFT calculations

Fig. 7 shows the optimized structures for different conformers of PIMA and Table 3 reports the total energy values (E) and relative energy values (ΔE) of the different conformers and relative monoanion. The most stable conformer A is probably stabilized by an attractive interaction involving H(N3) and carboxylic oxygen, in addition to the planarity of the molecule, shown by the dihedral angle N3–C2–C6–O7 (Table 2, atom numbering as in Scheme 2). A similar interaction was found in a theoretical study on different imidazole carboxylic acids (Alkorta and Elguero, 2005).

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Figure 7

Optimized structures at B3LYP/6-31G^∗∗ level of different tautomers of PIMA and its monoanion (Z).

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Previous crystal data of imidazole-4-acetic acid monohydrate showed IMA in the zwitterionic form in the solid state (Okabe and Hayashi, 1999) stabilized by hydrogen bonds involving water molecules. In the anionic form of both A and B conformers the planarity of the molecule is removed being the dihedral angle of 68.80° and 52.72° respectively, even if the hydrogen bond interaction between H(N3) and carboxylic oxygen is retained.

In order to calculate the most plausible structure of PIMA metal complexes, we have taken into account the B(H₋₁) anion, which is the only one able to interact with metal ion leading to the formation of a chelate complex. Actually, the involvement of N3 in metal coordination requires the proton dissociation that cannot be proposed under the pH conditions in which the present study was carried out.

Considering the stoichiometry of the prevailing species at physiological pH and assuming the coordination mode suggested by NMR data we also calculated the structure for Ga(PIMA)₃ shown in Fig. 8, together with the total energy value (E) and the binding energy value (BE) of the complex. Apparently, no examples of DFT calculations on Ga(III) complexes with N,O donor atoms are reported in the literature; however, the BE value of Ga(PIMA)₃ is of the same order of magnitude with respect to that found for Ga(III) complexes with curcumin 1347.73 kcal/mol and curcumin derivatives 1354.22 kcal/mol (Ferrari et al., 2014).

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Figure 8

Optimized structures at B3LYP/6-31G^∗∗ level of Ga(PIMA)₃ complex. TEc – 4320.481825 (a.u.); BE 1452.61 (kcal/mol). Where BE = TE_S – TE_C where TE_S = sum of the total electronic energy of the bare metal ion and the relative isomer of the complexing anion (a.u.); TE_C = total electronic energy of complexes (a.u.).

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