Antibacterial susceptibility of new copper(II) N-pyruvoyl anthranilate complexes against marine bacterial strains – In search of new antibiofouling candidate

2.1 Synthesis of the complexes

Generally, 2.0 mmol of the ligand TPAH₂ or TPXAH₂ (cf. Scheme 1) was dissolved in 15 mL of absolute ethanol with a constant stirring/gentle heating. To this solution, 0.399 g (2.0 mmol) of Cu(OAc)₂·H₂O in 10 mL of water was added dropwise with continuous stirring and the pH of the reaction mixture was adjusted to neutral (pH ≈ 6.8) by use of 0.1 M NaOH solution. The reaction mixture was heated under refluxing for 4 h. After cooling slowly, the green precipitates were separated out. The separated compound was filtered, washed thoroughly with absolute ethanol and water, and then dried in a vacuum desiccator with P₂O₅. Unfortunately, many attempts to grow single crystals of these complexes were unsuccessful.

[Cu(TPA)H₂O](4a): Yield 83%. FT-IR (ATR, cm⁻¹): 3517 (m, br, ν_(O—H), H₂O), 3257 (m, br, ν_(N—H), NHCO), 1675 (s, sh, ν_(C⚌O), amide I), 1576 (w, sh, ν_{(C⚌C—C⚌O)}), 1516 (m, sh, ${\nu }_{\text{as}({\text{COO}}^{-})}$ ), 1496 (s, sh, ν_(CN) + δ_(N—H), amide II), 1460 (m, sh, ${\nu }_{\text{s}({\text{COO}}^{-})}$ ), 1351 (m, sh, ν_(CSC), thiophene), 1062 (m, sh, ν_(C−O), COO⁻), 858, 653, 464; EI-MS m/z calcd for C₁₅H₁₁NO₅S: 396.86, found: 396.78. Anal. Calcd. for C₁₅H₁₁CuNO₆S (M = 396.86 g/mol): C, 45.40; H, 2.79; N, 3.53; S, 8.08, found: C, 45.44; H, 3.00; N, 3.47; S, 8.23%.

[Cu(TPCA)H₂O](4b): Yield 81%. FT-IR (ATR, cm⁻¹): 3444 (m, br, ν_(O—H), H₂O), 3330 (m, br, ν_(O—H), H₂O), 3269 (m, br, ν_(N—H), NHCO), 1592 (s, sh, ν_(C⚌O), amide I), 1565 (w, sh, ν_{(C⚌C—C⚌O)}), 1495 (m, sh, ${\nu }_{\text{as}({\text{COO}}^{-})}$ ), 1445 (m, sh, ${\nu }_{\text{s}({\text{COO}}^{-})}$ ), 1352 (m, sh, ν_(CSC), thiophene), 1083 (m, sh, ν_(C—O), COO⁻), 876, 674, 465; EI-MS m/z calcd for C₁₅H₁₀ClNO₆SCu: 431.30, found: 431.31; Anal. Calcd. for C₁₅H₁₁CuNO₆S (M = 431.30): C, 41.77; H, 2.34; N, 3.25; S, 7.43, found: C, 42.03; H, 2.32; N, 3.23; S, 7.38%.

[Cu(TPBA)H₂O]·H₂O(4c): Yield 77%. FT-IR (ATR, cm⁻¹): 3488 (m, br, ν_(O—H), H₂O), 3330 (m, br, ν_(O—H), H₂O), 3229 (m, br, ν_(N—H), NHCO), 1609 (s, sh, ν_(C⚌O), amide I), 1571 (w, sh, ν_{(C⚌C—C⚌O)}), 1490 (m, sh, ${\nu }_{\text{as}({\text{COO}}^{-})}$ ), 1412 (m, sh, ${\nu }_{\text{s}({\text{COO}}^{-})}$ ), 1350 (m, sh, ν_(CSC), thiophene), 1060 (m, sh, ν_(C—O), COO⁻), 858, 661, 463; EI-MS m/z calcd for C₁₅H₁₂BrNO₇SCu: 431.30, found: 431.31. Anal. Calcd. for C₁₅H₁₂BrCuNO₇S (M = 493.77): C, 36.49; H, 2.45; N, 2.84; S, 6.49, found: C, 36.38; H, 2.43; N, 2.39; S, 6.73%.

[Cu(TPNA)(H₂O)₂](4d): Yield 68%. FT-IR (ATR, cm⁻¹): 3447 (m, br, ν_(O—H), H₂O), 3332 (m, br, ν_(O—H), H₂O), 3176 (m, br, ν_(N—H), NHCO), 1588 (s, sh, ν_(C⚌O), amide I), 1556 (w, sh, ν_{(C⚌C—C⚌O)}), 1489 (m, sh, ${\nu }_{\text{as}({\text{COO}}^{-})}$ ), 1425 (m, sh, ${\nu }_{\text{s}({\text{COO}}^{-})}$ ), 1355 (m, sh, ν_(CSC), thiophene), 1081 (m, sh, ν_(C—O), COO⁻), 846, 665, 459; EI-MS m/z calcd for C₁₅H₁₂CuN₂O₉S: 459.87, found: 459.79. Anal. Calcd. for C₁₅H₁₂CuN₂O₉S (M = 459.87): C, 39.18; H, 2.63; N, 6.09; S, 6.97, found: C, 39.50; H, 2.71; N, 6.10; S, 6.84%.

2.2 X-ray crystallography

A single crystal with the dimensions of 0.06 × 0.21 × 0.17 mm³ of ethyl 2-thienyl-2-(2-carboxyanilino)-4-oxobutenoate (3b) was carefully selected under a polarizing microscope and mounted on a Rigaku R-axis Spider diffractometer equipped with sealed tube graphite-monochromatic Mo Kα radiation sources (λ = 0.71073 Å) and image plate (IP) detector. The structure was solved by direct methods (SHELXS-97) (Sheldrick, 2008) and refined using full-matrix least squares on F² using the SHELXL-97 program (Sheldrick, 2008); empirical (multi-scan) absorption correction with Abscor (Rigaku) (Higashi, 1995). All non-hydrogen atoms were refined with anisotropic temperature factors. At the final stage of the refinement, H-atoms were positioned geometrically and refined using a riding model (AFIX 43 for aromatic CH, AFIX 13 for aliphatic CH and AFIX 23 for CH₂) with U_iso(H) = 1.2U_eq(CH). For CH₃ AFIX 33, 133 or 137 was used with U_iso(H) = 1.5U_eq(CH₃). The carboxylic H atom has been refined with the riding model AFIX 83 and U_iso(H) = 1.5U_eq(O). The imine H atom has been refined with the riding model AFIX 43 while the amino nitrogen atoms of 2-aminobenzoic acid have been refined with the riding model AFIX 93. In both cases the (N—)H atoms have been refined with U_iso(H) = 1.5U_eq(O). The thiophene ring (Th) is disordered about the (O)C—C(Th) bond, depicted in Fig. 1. This thiophene disorder was refined using a PART command.

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Figure 1

(a) Typical sulfur atom or thiophene ring disorder in the planar ligand and (b) resulting superimposed image in an X-ray structure refinement with approximately half-occupation of the given (5-)C- and S-atom positions.

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The asymmetric unit contains half molecule of 2-aminobenzoic acid. The 2-aminobenzoic acid co-crystallite sits on the inversion center as special position. Its unique atoms were refined using PART-n commands because the molecule is disordered on a special position of higher symmetry than the molecule has itself. Thus, special position constraints are suppressed and bonds to symmetry generated atoms with the same or a different non-zero PART number are excluded. The crystallographic data and refinement parameters are listed in Table 1 (Tables S2, S3, supplementary information). Graphics were drawn with DIAMOND (Brandenburg, 2007–2012).

2.3 Microbiological screening

3.1 Synthesis and characterization of ligands

Choosing an appropriate solvent is of crucial importance not only for successful synthesis but also for the effective control of chemoselective reactions. For example, 2-thenoyl-pyruvate 1b reacted with equimolar amounts of anthranilic acid derivatives in HOAc/EtOH (few drops of HOAc) mixed-solvent system to give thenoyl–pyruvoyl-anthranilide products (α-keto anthranilides). However, if HOAc is used alone, the thienyl-carboxyphenylamino-butenoates (enaminones) were obtained as the main products. Under these optimized chemoselective conditions, a series of thenoyl–pyruvoyl-anthranilic acids (2a–d) and enaminones (3a–e) were synthesized via two component reactions of 2-Thenoyl-pyruvate 1b and different anthranilic acid derivatives in a mixed-solvent, HOAc/EtOH and single-solvent, HOAc, respectively (see Scheme 2). These compounds have been characterized based on FT-IR, ¹H and ¹³C NMR, elemental analysis and single crystal XRD.

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Scheme 2

Chemoselective aminolysis of thenoyl-pyruvate with different substituted anthranilic acids.

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In order to extend the present method to synthesize different enaminones, the aminolysis of thenoyl-pyruvate with different substituted anthranilic acids was explored. Interestingly, the presence of an electron-withdrawing group on the phenyl ring requires longer reaction times with slightly decreased yields.

FTIR spectra of thenoylpyruvanthranilic acids (2a–d) revealed the following highlights: (i) Absorptions at the ranges of 3395 ± 25 cm⁻¹ and 1603 ± 27 cm⁻¹ are assignable to the hydroxyl and carbonyl groups stretching vibrations of enolic structural unit (O⚌C—C⚌C—OH), respectively. (ii) A broad peak for the N—H stretch of the oxamido group is observed in the range of 3203–3324 cm⁻¹ due to the involvement of amide proton in hydrogen bonding (Krishnapriya et al., 2008). (iii) The broad band centered in the range of 2517–2645 cm⁻¹ is assigned to H-bonded OH stretching frequency of the carboxyl moiety which exhibits additional band, due to carbonyl moiety, around 1695 cm⁻¹. (iv) Three vibrational bands in the range of 1662 ± 8 cm⁻¹, 1602 ± 28 cm⁻¹ and 1513 ± 5 cm⁻¹ were ascribed to the vibrational stretches of the amide I (ν_C⚌O of oxamide) (Nakamoto, 1997), amide II (δ_N—H + ν_C⚌N) and amide III (ν_C—N + δ_N—H), respectively (Sahni et al., 1997).

The NMR studies of thenoylpyruvanthranilic acids (2a–d) showed that, different substituents on benzoic acid fragment reveal diverse central backbone NMR profiles. For example in TPAH₂ (2a), no substituent, the different central backbone is in the enol-oxo-amide tautomeric form and not in the dioxo-amide (keto-oxo-amide) form (Scheme 3). Furthermore, this tautomer must be considered as (Z)-enol-γ-oxo-amide structure as revealed by ¹H—¹H COSY NMR. The prevalence of the (Z)-enol-γ-oxo-amide resonance structure in TPAH₂ ligand could be attributed to intramolecular hydrogen bonding (C⚌O⋯H—N) of the enol-γ-oxo-amide arms which led to the formation of a stable six-membered ring system. The ¹H NMR spectrum of TPAH₂ (Fig. S2, supplementary information) exhibits resonances at δ 12.07 ppm for enolic C(8)—OH and at 6.70 ppm for C(7)–vinylic proton typical for enol-oxo-amide form (Belyaev et al., 2002). Further evidence for the prevalence of enolic tautomer is provided by ¹³C NMR spectroscopy, where the absence of NMR signal for the methylene carbon in ¹³C NMR spectrum of TPAH₂ and instead a vinylic carbon signal of the enol-oxo-amide form appears at δ ∼ 99 ppm in addition to the downfield signal in the region δ ∼ 188 ppm for the enolic carbon.

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Scheme 3

Possible tautomeric forms and intramolecular hydrogen-bonding in backbone of thenoylpyruvanthranilic acids.

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Contrarily, ¹H NMR spectrum of TPBAH₂ (Fig. S3, supplementary information) reveals two singlets at 12.43 ppm for enolic OH and 7.18 ppm for vinylic proton, typical for oxo-enamide tautomeric form of ligand. The singlet at 4.63 ppm, ascribed to geminal CH₂ protons, is also typical for dioxo-amide form of TPBAH₂. Consequently, this ligand must be considered as a resonance hybrid of the (Z)-enol-γ-oxo-amide resonance structure with some contribution of the dioxo-amide resonance structure (Belyaev et al., 2002) (cf. Scheme 3). ¹³C NMR provides additional evidence supporting the coexistence of the oxoenamide – dioxoamide tautomers. The ¹³C NMR spectrum of TPBAH₂ exhibits a weak resonance at ∼60 ppm for the methylene carbon and strong signal at ∼107 ppm for a vinylic carbon of the enol form, additionally, the downfield signal in the region ∼189 ppm for the enolic carbon and the carbonyl C⚌O signal of the dione form at ∼181 ppm.

The ¹H—¹H COSY NMR (Fig. S4, supplementary information) shows the broadened non-coupled singlet at δ 12.3 ppm, which has been assigned as an enolic OH. This indicates that the enolic proton at a considerable distance from thiophene moiety, which suggests that only one tautomeric enol form of the ligands exists in solution under the experimental conditions (enol-γ-oxo-amide).

3.1.1

3.1.1 Crystallographic structure of (3b)

X-ray crystallographic data indicates the co-crystallization of ethyl 2-thienyl-2-(2-carboxyanilino)-4-oxobutenoate (ETCAOB) with 2-aminobenzoic acid (anthranilic acid, Anth). ETCAOB and Anth form a 2:1 co-crystal of 3b in the triclinic space group P − 1 with Z = 1. The asymmetric unit consists of two ETCAOB and one Anth molecules (Fig. S5, supplementary information). H-bonding is dominant interactions in the single crystal structure of 3b. Major H-bonding interactions (Fig. 2) involved N1—H⋯O1 (distance of 2.14 Å) and N1—H⋯O4 (distance of 2.05 Å) in ETCAOB fragment.

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Figure 2

Ellipsoid plot of the Z-ETCAOB, showing the atom-labeling scheme and H-bonding interactions.

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X-ray analyses of ETCAOB reveal that it exists in the Z form, Noteworthy, the O⚌C—C⚌C—N moiety is planar and the bond lengths indicate electron delocalization. The O⚌C—C⚌C—N plane is twisted with respect to the benzene and thienyl rings by 34.76(48)° and 21.06(87)°, respectively. Furthermore, the O⚌C—C⚌C—N plane and the O⚌C—O plane make a dihedral angle of 34.96(17)°.

3.2 Synthesis and characterization of Cu(II) complexes

Mononuclear Cu(II) complexes of anthranilides (2a–d) have been synthesized from a typical synthetic procedure in which, Cu(OAc)₂·H₂O is reacted with dianionic (Z)-enol-γ-oxo-amide core of anthranilide ligands in aqueous ethanol as shown in Scheme 4. These complexes are soluble in DMSO and DMF, slightly soluble in methanol while fairly insoluble in water. The structural and geometrical features of the new complexes were deduced from the elemental and spectral (FTIR, UV–Vis, EIMS) analysis (see Section 2).

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Scheme 4

Coordination of Cu(II) ion to thenoylpyruvanthranilic acids.

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FTIR spectra of complexes (4a-d) displayed a set of bands at ca. 1588–1560 cm⁻¹ and 1410–1362 cm⁻¹ assignable to the asymmetric ν_as(COO⁻) and symmetric ν_s(COO⁻) vibrations of coordinated carboxylate group, respectively. The frequency gaps (Δν) between these bands have an average of ∼217 cm⁻¹ which is consistent with a monodentate coordination mode (η¹) of carboxylate ligation (Elshaarawy et al., 2014, 2013; Refat et al., 2014; Elshaarawy and Janiak, 2012; Mahmoud and Janiak, 2009). The absence of the characteristic bands at 1730–1690 cm⁻¹ for protonated carboxylate and 1603 ± 27 cm⁻¹ for enolate groups indicates the complete deprotonation of the anthranilides ligands which proof that, all ligands coordinate through carboxylate and enolate oxygen atoms. The absence of any significant change in amide I, νC⚌O, coupled with a negative shift (∼17–52 cm⁻¹) of the amide NH band (relative to the free ligand) proofs the coordination the amide nitrogen. This further confirmed by the movement of amide II stretch upon coordination toward lower frequency by ∼22–28 cm⁻¹ (compared to that of free ligand). Moreover, a wide strong band (3500–3300 cm⁻¹) and new band at 856–866 cm⁻¹ are due to the O—H stretching and ρrOH vibrations, respectively, of the coordinated water molecule. Finally, the ν(CSC) vibration remains almost unperturbed in all complexes indicating the non-participation of the sulfur atom in coordination (Elshaarawy and Janiak, 2012). Consequently, our FTIR data revealed that, the anthranilides (2a–d) ligands act as ONO tridentate dianionic ligands.

In order to explore the ligand to copper stoichiometric ratios, the UV–Vis titration of TPAH₂ ligand with Cu(II) salt was carried out in aqueous DMSO (9:1 v/v) at 25 °C. Here we take TPAH₂ as representative of these tridentate ligands. As shown in Fig. 3, the 1:1 stoichiometry for the Cu(II)-TPAH₂ complexation was elaborated by the profile of the UV titration curve at 406 nm. These results were in good agreement with those deduced from the elemental analysis and EI-MS studies of complexes 4a.

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Figure 3

UV–Vis spectra for titration of TPAH₂ (5 × 10⁻⁵ M) with Cu²⁺ ion (5 × 10⁻³ M) in 10% DMSO aqueous solution at r.t.

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The electronic spectra of mononuclear Cu(II) complexes 4a–c in aqueous DMSO are almost identical. The most characteristic feature of the UV–Vis spectra for these complexes is the broad absorption band in the wavelength range of 369–475 nm which can be assigned to the spin-allowed d-d transition, ²B₁g → ²Eg. This assignment suggests the square planar geometry for Cu(II) complexes under investigation with [CuO₃N] core (Lever, 1984; Khana et al., 2009). On the other hand, the electronic spectrum for a five coordinate Cu(II) compound (4d) exhibits absorption band near 690 nm due to ²B₁g → ²A₁g transition supporting an approximate square pyramidal geometry around Cu(II) ion with [CuO₄N] core (Lever, 1984).

3.3 Antibacterial activity profile

The target anthranilides (2a–d), enaminones (3a–e), complexes (4a–d) and standards drugs were in vitro assessed separately for their capacity to inhibit the growth of two clinically significant pathogenic bacterial strains namely PSSA (S. aureus) and MDREC (E. coli). Examination of the ZOI (mm) (Table S1, supplementary information) (Fig. 4), MIC and MBC (mM) values (Table 2) for the most potent compounds, (4a–d), lead us to highlight the following aspects: (i) All compounds exhibit significant antibacterial efficacy with a higher degree of antistaphylococcal potency than antiescherichia activity and are therefore of interest as potential antistaphylococcal agents. (ii) Noteworthy, the enaminones (3a–e), ester derivatives, are more potent than the anthranilides (2a–d), amide analogues, while less active than the amide complexes (4a–d). (iii) The antibacterial susceptibility was found to depend on the nature of the substituent attached to the anthranilic moiety. For example, the substituent-dependent antibacterial activity of [Cu(TPBA)(H₂O)_n]·mH₂O (4a–d) against S. aureus and E. coli follows the trend below:

• X (MIC/MBC mM)_{S. aureus}: NO₂ (9.25/10.50) > Cl (12.50/14.75) ∼ Br (12.25/15.25) > H (13.75/18.75),

• X (MIC/MBC mM)_{E. coli}: NO₂ (13.25/13.50) > Br (14.75/14.75) ∼ Cl (14.60/15.25) > H (16.55/16.75).

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Figure 4

Graph of zone of inhibition (mm) for target compounds against different bacterial species.

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(iv) Cu(II) complex of 2-thenoyl-pyruv-5-nitroanthranilamide, 4d, exhibits remarkable extra-potent staphylococcalcidal action (MIC/MBC = 9.25/10.50 mM), higher than the standard drug, and thus, can be classified as a new good candidate in the fight against PSSA infections. Further studies are required to explore these complexes as drugs.

In comparison with 2-thenoyl-N-pyruvanthranilic acids (2a–d), the observed enhanced antibacterial impact of enaminone esters (3a–e) could be attributed to faster membrane depolarization by (3a–e) which may be explained by their greater hydrophobicity. This enhanced depolarization facilitates the spontaneous insertion of enaminones into the bacterial membranes, and their conformational flexibility allows better adaptability to the membrane (Bionda et al., 2012).

The enhanced antibacterial activity of the Cu(II) complexes can be explained in terms of Overtone’s concept of cell permeability (Dharamaraj et al., 2001) and Tweedy’s Chelation theory (Malhota et al., 1993). Considering these theories, such antibacterial potency of complexes could be assigned to their higher liposolubility effect and enhanced cell permeability owing to diminishing the polarity of the copper ion upon chelation due to the overlap with the ligand orbitals and partial sharing of the positive charge on the copper ion with donor atoms. Further, the delocalization of charge/electrons over the chelate ring enhances the lipophilicity of the complexes. This increased lipophilicity facilitates penetration of these probes into lipid membranes with blocking metal binding sites in the enzymes of microorganisms. These complexes can also disturb the respiration of bacterial cell and thus inhibit protein synthesis that restricts further bacterial growth.

Although the exact mechanism by which antibacterial Cu(II)-thenoylpyruvanthranilates complexes (4a–d) exert their microbiological toxicity has not fully elucidated, their biocidal mode of action may involve various targets in microorganisms: (i) Inhibition of, or interference with cellular envelope biosynthesis inducing envelope damage that can lead to the alteration of cell permeability or disorganized lipoprotein arrangements which ultimately resulted in cell death (Mott et al., 2008). (ii) Hydrogen-bonding interactions of the complex H-receptor sites (such as anilide, carbonyl and carboxylate fragments) with active binding sites of cell constituents, resulting in interference with the normal cell process (Das, 1990). (iii) The redox cycling between Cu(II) and Cu(I) can catalyze the production of highly reactive oxygen species (ROS) such as hydroxyl radicals, thus targeting numerous biochemical pathways within the cell causing oxidation of thiol proteins, DNA damage, lipid peroxidation and the binding-site displacement of Cu-containing metalloprotein (Harrison et al., 2008; Stohs and Bagchi, 1995). Once the Cu(II) complex penetrates the bacterial cell wall, the cellular oxygen oxidizes the protein thiols (R-SH) followed by a one-electron transfer reduction of copper(II) center to produce Cu(I) complex. Further, oxidation of sulfhydryl group in sulfhydryl-containing proteins (R–S–S–R), such as Cysteine, is another pathway to produce reduced copper complexes. The Cu(II)/Cu(I) complexes system exhibits a relatively high superoxide dismutase (SOD)-like activity, where Cu(I) complex catalyzes the one-electron reduction of normal dioxygen (O₂) to reactive oxygen species (O₂⁻) and peroxide (H₂O₂), while Cu(II) complex may catalyze the dismutation of highly reactive superoxide (O₂⁻) into dioxygen (O₂) (see Scheme 5). The higher reactive oxygen species (ROS) formed during this cycle, superoxide (O₂⁻) and peroxide (H₂O₂), nonspecifically oxidizes lipids, proteins, nucleic acids, and sugars, resulting in staphylococcalcidal effect and E. coli-cidal actions. In addition, the reduced Cu(I) complex may inhibit DNA synthesizer ATP production through inhibition of mitochondrial respiration and destruction of cell viability. In conclusion, the quasi-reversible one-electron Cu^II/Cu^I redox waves are responsible for the mode of antibacterial action of copper complexes.

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Scheme 5

O₂⁻ dismutation mediated by the SOD [Cu^II/Cu^I-complex] redox couple confined in the bacterial species.

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3.4 Biofilm susceptibility

The susceptibility of the bacterial biofilm to serial concentrations of the target complexes was carried out by calculating the percentage reduction of Alamar blue for treated S. aureus and E. coli, spectrophotometrically. Interestingly, from Fig. 5, Cu(II)-TPXA complexes exhibited significant MRSA and MDREC biofilms growth-inhibitory activity at all the examined concentrations and the percentage reduction of Alamar blue varies in a dose-dependent profile.

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Figure 5

Percent reduction of Alamar blue by S. aureus and E. coli biofilms treated with different concentrations of complexes (4a–d).

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Our data demonstrate a remarkable biofilm-producing resistance by the action of complex 2a ((Reduction %)_{S. aureus-biofilm} = 6.43 at 20 mM; (Reduction%)_{E. coli-biofilm} = 10.41 at 20 mM), compared with other complexes. Eventually, refinements of the most active biocidal agent, [Cu(TPNA)(H₂O)₂] (4d) may serve as a platform toward discovery of exceptionally active antibiofouling agent to control the formation of biofilm.