Theoretical Studies of the Tautomerism in 3-( 2R-Phenylhydrazono )-naphthalene-1 , 2 , 4-triones : Synthesis of Copper ( II ) Complexes and Studies of Antibacterial and Antitumor Activities

Instituto de Química, Universidade Federal Fluminense, Campus do Valonguinho, Centro, 24020-150 Niterói-RJ, Brazil Instituto de Química, Universidade Federal do Rio de Janeiro, Ilha do Fundão, 21945-970 Rio de Janeiro-RJ, Brazil Instituto Oswaldo Cruz, FIOCRUZ, CP 926, 21045-900 Rio de Janeiro-RJ, Brazil Universidade Federal do Ceará, Depto de Fisiologia e Farmacologia, Campus do Porangabussu, 60430-270 Fortaleza-CE, Brazil Departamento de Química da Universidade Federal do Paraná, 81531-990 Curitiba-PR, Brazil


Introduction
The 1,2 and 1,4-naphthoquinone nuclei are commonly encountered in natural products 1,2 and their derivatives are found to exhibit an interesting range of pharmacological properties including antibacterial, [3][4][5][6] antiviral, 7,8 trypanocidal, [9][10][11] anticancer, [12][13][14] antimalarial, [15][16][17] antifungal 18,19 and moluscicide 20 activities.Such properties are due to the interference of quinones in the electron transport chain by electron reduction processes, generating semiquinone radical (Q •-) and hydroquinone anion (Q 2-). 21,22zo dyes with ortho and para hydroxy substituents to the azo linker generally exhibit azo-hydrazone tautomerism, which involves transfer of the hydroxyl hydrogen to one of the nitrogens in the azo group. 23Tautomeric forms can be identified from their spectral properties 24 and the stability of such forms in solution may be influenced by the nature of the substituents present in the molecules. 25ncorporation of an azo group into 2-hydroxy-1,4naphthoquinone has led to promising antimalarial and anticancer agents, in which metal complexation with copper(II) resulted in increased cytotoxicity. 26,27The derivatives can be represented by several tautomeric structures illustrated in Figure 1: 3-(2-R-phenylhydrazono)naphthalene-1,2,4-triones, Ia and Ib, 3-R-arylazo-4hydroxy-1,2-naphthoquinones, IIa and IIb, and 3-R-arylazo-2-hydroxy-1,4-naphthoquinone III.A poor X-ray diffraction study of the derivative containing R=3-Me suggested that this compound exists as tautomer IIa in the solid state. 26owledge of the geometry and electronic structures, as well as the relative stabilities of the various tautomeric forms in solution, is of utmost importance as it provides a basis for understanding the pharmacological properties of the molecules and designing new derivatives with improved activity. 26,27In the present work we describe the results of our theoretical calculations using density functional theory (DFT) carried out to investigate the relative stability of the tautomeric forms of 3-(2-R-phenylhydrazono)naphthalene-1,2,4-trione derivatives (Figure 1) as a function of the substituent, both in the gas phase and in solution.Low solubility prevented experimental NMR experiments. 25Some of these compounds have been previously described, 26,27,30,31 but their tautomerism has not yet been investigated.In addition to reporting the synthesis of novel compounds (HL1-HL13), we also present the results of in vitro antitumor screening and antibacterial activity of these compounds and of their copper(II) complexes (Scheme 1) against several cancer cell lines (SF-295, HCT-8, MDAMB-435 and HL-60) and bacteria strains (Bacillus cereus, Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia and Pseudomonas aeruginosa).

Materials and methods
Reagents and solvents were used without further purification, except for triethylamine, which was previously distilled.Microanalyses were performed using a Perkin-Elmer CHN 2400 micro analyser at the Central Analítica -Instituto de Química, USP-São Paulo, Brazil.Melting points were obtained with a Mel-Temp II, Laboratory Devices -USA apparatus and are uncorrected.IR spectra (KBr pellets) were recorded on a FT-IR Spectrum One (Perkin Elmer) spectrophotometer. 1 H and 13 C NMR spectra were recorded with a Varian Unit Plus 300 MHz spectrometer in dmso-d 6 ; coupling constants are reported in Hertz (Hz) and chemical shifts in parts per million (ppm) relative to internal standard Me 4 Si.The hydrogen signals were attributed through coupling constant values and 1 H × 1 H -COSY experiments.Electronic spectra were taken on a Diode Array 8452A (Hewlett Packard -HP) spectrophotometer using spectroscopic grade solvents (Tedia Brazil) in 10 -3 and 10 -4 mol L -1 solutions.Electron paramagnetic resonance (EPR) spectra of the frozen copper(II) samples dissolved in dmso at 77 K were obtained on a Bruker EMX equipment with modulation frequency of 100 kHz operating at about 9.5 GHz (X-band), using quartz tubes accommodated in a quartz Dewar.The EPR parameter values were obtained by treating and simulating the experimental spectra using the Windows software programs WINEPR and SIMFONIA (Bruker), and the WEAK PITCH BRUKER sample pattern.Cyclic voltammograms were obtained on an Epsilon -BAS potentiostat-galvanostat from 1×10 -3 mol L -1 solutions in dmso containing 0.1 mol L -1 of (Bu 4 N)BF 4 as supporting electrolyte, at room temperature and under argon atmosphere.A standard three component system was used: a carbon-glassy working electrode, a platinum wire auxiliary electrode, and an Ag/AgCl reference electrode for organic media.Ferrocene was used as an internal standard (E 1/2 0.40 V vs. NHE, normal hydrogen electrode).Density functional calculations were carried out using the Gaussian03W molecular orbital package. 32Geometries were fully optimized using the B3LYP functional 33 with the standard 6-31G(d) basis set. 34The electronic spectra were calculated using the TD (Time Dependent) methodology available in Gaussian with the PBE1PBE functional and the 6-311+G(2d,p) basis set.For calculation of the electronic spectra, solvent effects (dmso) were included by mean of the continuum solvation model using the conductor-like polarisable continuum model 35 (CPCM).The dielectric constant of dmso, ε, is 46.7.Relative energies are reported at the PBE1PBE/6-311+G(2d,p) level with inclusion of solvent effects.

Synthesis of the hydrazono compounds HL1-HL13
Compounds HL1-HL13 (Figure 2) were synthesised according to the general procedure described in the literature. 26,27,30,31HL1, HL7, HL11, HL12 and HL13 were described previously. 26,30See Supplementary Information for description on the synthesis, analytical and spectroscopic data of the novel products.

Synthesis of complexes [Cu(L1-L13) 2 ] from HL1-HL13
To a suspension of 1 mmol of the hydrazono proligand HL in 30 mL MeOH, was added a solution of CuCl 2 • 2H 2 O (83 mg, 0.5 mmol) in 1 mL MeOH.After addition of Et 3 N (0.14 mL, 1 mmol), the suspension turned into a solution, followed immediately by the formation of a brown solid.The reaction mixture was left under stirring in the dark for 24 h at room temperature.The resulting solids were filtered off, washed with cold methanol and dried under vacuum.Complexes 1-13 (Figure 3) have not been described previously.See Supplementary Information for analytical and spectroscopic data.

Synthesis and characterization of the hydrazono compounds
Compounds HL1-HL13 (Figure 2) were synthesized from the diazonium salts of the respective arylamines, followed by coupling with lawsone C3 in ethanol under stirring at room temperature.After addition of the diazonium salts to the lausonate solution, the orange products precipitated immediately.Following filtration and washing with cold EtOH, the products were washed with hot acetonitrile, dried under vacuum and obtained in pure form, in yields ranging from 71-92%.Compounds HL1-HL13 are stable in the solid state and in solution.Their structures were formulated on the basis of analytical and spectroscopic data (see Supplementary Information).
The 1 H spectra of compounds HL1-HL13 were obtained in hot dmso-d 6 , due to poor solubility in other solvents.The spectra exhibit characteristic signals attributed to the four hydrogens of the naphthoquinone unit (H5-H8), which appear in the d 7.5-8.5 region as dd or bd (H5 and H8) and td (H5 and H7) (see Figure 2 for numbering).The substituted phenylene ring hydrogens appear as broad doublets (substituents in the para position) and multiplets/ triplet (substituents in the meta/ortho positions); in most cases the naphthoquinone hydrogen signals overlap with those of the arylamine ring, resulting in multiplet signals.
Attributions were made on the basis of 1 H × 1 H -COSY experiments, J values and multiplicity.The 13 C NMR spectra of the previously reported 2-, 3-and 4-substituted methyl derivatives 26 have not been described.In the 13 C NMR spectra of compounds HL1-HL13 the number of signals does not correspond to the number of the expected carbons, either because of the high relaxation time of some of the carbon nuclei, or due to the poor solubility of the compounds (see Supplementary Information).For this reason tautomerism studies using this technique could not be carried out as described previously for similar systems; 25 instead, density functional calculations involving relative stability of the possible tautomers and full geometry optimization for the ground state were carried out and are described below.

Theoretical calculations
Calculation of several tautomers of the unsubstituted derivative, e.g., the enol-azo forms II and III and the alternative keto-hydrazone Ia and Ib forms (Figure 1) showed that Ia and Ib are much more stable than the enol-azo forms IIa (presumably the molecular structure determined by an X-ray diffraction study, see below) and IIb, by at least 14 kcal mol -1 (Table 1).Indeed geometry optimization of the enol-azo tautomer IIb directly converges to the keto-hydrazone form Ib. The two rotamers Ia and Ib are almost isoenergetic, with relative energies in the order or below 0.5 kcal mol -1 (see Supplementary Information for detailed theoretical calculations data).This result is in agreement with those reported on a similar system, viz.2-arylazo-1-hydroxycyclohex-1-en-3-one. 39The nature of the substituent in position 4 (electron withdrawing substituents NO 2 and CN versus electron donor OMe) does not affect appreciably this stability order (Ib @ Ia >> II).This agrees with previous calculations on tautomeric equilibrium for 4-anilino-1,2-naphthoquinones 40 and is in contrast with the theoretical and experimental results reported for the analogous tautomeric equilibrium of the azo derivatives of 2-naphthol for which electron withdrawing groups were found to strongly stabilize the keto-hydrazone tautomer, whereas electron donor groups shift the equilibrium toward the enol-azo tautomer. 25cording to the reported X-ray diffraction study of the R = 3-Me (HL-3-Me) derivative, 26 this compound exists in the solid state as the enol-azo tautomer IIa (see Figure 1).The authors reported that the molecule occupies two semipopulated sites related by crystallographic inversion centers, and because many atoms coincided, it was necessary to apply bond length constraints to refine the structure.The cif list does not make clear which atoms presented problems (an analysis of the data would be necessary); however, it shows that C(1) and H( 14) on the one hand, and C(4) and N(2) on the other (see Figure 4 for the numbering) have been refined in the same positions.It is possible, therefore, that the C(1)-O(1), C(4)-O(4) and N(1)-N(2) distances reported were influenced by the applied restrictions.Considering that the structure was proposed to be that of tautomer IIa (1,2-rather than a 1,4-naphthoquinone and enol-azo instead of keto-hydrazone) based on C-O, C-N and N-N bond length analysis, and that our theoretical calculations indicate that this is the highest energy tautomer of all forms investigated, we propose that the structure of this compound is best described as a mixture of the ketohydrazone tautomers (rotamers Ia and Ib).Unfortunately suitable crystals of HL1-HL13 for an X-ray diffraction study could not be obtained in a variety of solvents and conditions to confirm this proposal.

FTIR spectra
The FTIR spectra of compounds HL1-HL13 support the proposed structure in the solid state.The broad band around 3450 cm -1 , previously attributed to the intramolecular hydrogen bond between C(4) hydroxyl group and N(1) of the azo linkage, 26 may be attributed to intramolecular hydrogen bonded N-H stretching present in the two rotamers Ia and Ib, 41 since elemental analyses do not show the presence of water (see Supplementary Information).Calculation of the vibrational spectrum at the B3LYP/6-31G(d) level indicate that OH stretching in the enol-azo tautomers appears at a much lower wavenumber (around 2830 cm -1 for R=H) than the NH stretching in the keto-hydrazone forms which appears at about 3300 cm -1 (R=H). 41The n(C=O) stretches appear as a single band around 1690-1655 cm -1 , 26 as the result of coalescence of the carbonyl bands; in the cases of compounds HL3, HL4 and HL10, in whose spectra this band appears at the lower end of this energy range, an additional n(C=O) band is observed around 1695 cm -1 .The absorption due to n(N=N) vibrations, expected at about 1420-1450 cm -1 according to the literature 40 (calculated 1462 cm -1 ) was not observed, although a very weak band around 970 cm -1 previously attributed to n(C-N=N-C) was observed in the spectra of all compounds and may arise from some tautomerization.

UV-Vis spectra
The UV-Vis spectra of HL1-HL13, obtained in dmso, show two absorption bands: one very intense in the 257-298 nm region and a broad low-energy band in the visible region of the spectrum between 459-411 nm.The high intensity of the band in the 257-298 nm region, attributed to the aromatic and quinone p-p * transitions, is associated to the high conjugation performed by the arylazo group.The low-energy band is influenced by the nature of the substituent on the phenylene ring, electronreleasing groups (-OMe) shifting it to higher wavelengths (bathochromic shift) and electron-withdrawing groups blue shifting it (hypsochromic effect).Furthermore, substituents in ortho and para positions were found to affect λ max more significantly than groups in the meta position.
The calculated electronic spectra of the most stable tautomers (rotamers Ia and Ib) were obtained using the TDDFT methodology together with the PBE1PBE/6-  311+G(2d,p) method.In agreement with the experimental observations, they show two main intense absorption bands due to p → p * transitions (HOMO → LUMO and HOMO → LUMO+1).HOMO is one of the p orbitals of the phenyl ring whereas LUMO and LUMO+1 both are p * orbitals of the quinone ring (see Supplementary Information for details of the calculated electronic spectra and illustrations of these orbitals).

Synthesis and characterization of the copper(II) complexes
The copper(II) complexes of proligands HL1-HL13 were synthesized in order to investigate their antibacterial and antitumor activities, since complexation of the methyl substituted compounds was found to result in increased cytotoxicity. 26,27omplexes 1-13 (Figure 3) were obtained by addition of triethylamine to a methanol suspension of HLn (n = 1-13) and CuCl 2 • 2H 2 O (2:2:1), under stirring at room temperature, for 24 h, in yields varying from 63 to 88%.Elemental analyses confirmed the same formulation, [CuL 2 ], as that of the analogous complexes of the 2-, 3-and 4-substituted methyl derivatives (HL-Me) whose structures were established by an X-ray diffraction study of the 3-Me derivative. 27Due to low solubility in methanol, acetonitrile and water, conductivity measurements could not be carried out.All compounds were characterized by IR, UV-Vis and EPR spectroscopy (see Supplementary Information).
The FTIR spectra of the complexes are in accordance with those described for the complexes of the HL-Me derivatives. 27All spectra exhibit a weak broad absorption band around 3450-3500 cm -1 , assigned to the n(O-H) stretching vibrations of the hydration water molecules in the complexes, whose presence in all samples was confirmed by elemental analyses (see Supplementary Information).As the result of complexation to the oxygen phenolate and the nitrogen bonded to the phenylene ring, two sharp n(C=O) bands (instead of one in the spectra of most proligands) are observed in the spectra of most complexes, except for compounds 3, 11-13 that exhibit a broad absorption band (Table 2).Absorptions due to n(N=N) and n(C-N=N-C) vibrations were not observed in most spectra, due to the apolar nature of these bonds. 40he UV-Vis spectra of complexes 1-13 were recorded in dmso and compared to those of their respective proligands.Upon complexation, the low-energy band in the visible region (416-473 nm) only shifts slightly, except in the cases of compounds 5, 11, 12 and 13, for which high bathochromic shifts are observed.The band around 257-298 nm is not altered by coordination.In general, increase in the intensities of the bands of all complexes is observed.The expected charge transfer L→M band, around 306-370 nm, 42 is not observed and is possibly masked by the broad energy band of the solvent.Only in the spectra of compounds 2, 5, 6, 7 and 8 (10 -3 mol L -1 ) it was possible to observe a very broad and low intensity band, between 500-600 nm, assigned to d-d transitions of the copper(II) ion.As most complexes are poorly soluble, even in dmso, diffuse reflectance spectra of some of the compounds were obtained (complexes 1, 3, 4, 9, 10, 12, 13), which show a band around 586-637 nm.
The EPR spectra of all compounds exhibit g || > g ⊥ > 2 values, in accordance with approximately square-planar geometry, in some cases with some tetrahedral distortion.The EPR parallel parameters ratio (g || /A || ) has been used as a convenient empirical index of tetrahedral distortion in the CuL 4 chromophore units. 43This value ranges from ca. 105 to 135 cm for the square-planar structure, and increases upon increasing tetrahedral distortion.Complexes 3, 4 and 11 have the highest g || /A || ratio values which indicates that these compounds present the weakest ligand field strength in dmso solutions, as expected, due to the presence of electron withdrawing groups in the 4-position (see Supplementary Information for Spin-Hamiltonian parameters).

Cyclic voltammetry studies of HL1-HL13 and complexes 1-13
The redox behavior of compounds HL1-HL13 and their respective complexes 1-13 was evaluated by cyclic voltammetry (CV) at room temperature in dmso/(Bu 4 N)BF 4 (0.1 mol L -1 ).The CVs were obtained in the potential range from +1.5 to -1.8V vs. FcH/FcH + (Table 3).For most cases three quasi-reversible pairs of waves were observed for the hydrazono compounds in the negative region of the CV, which is attributed to the electron transfer of the hydrazono forms Ia/Ib and/or process associated to deprotonation of the hydroxyl group in forms IIa/III 26 (Figure 1).The redox potentials of the naphthoquinone unit are directly influenced by the substituents in the phenylene ring: electron-donor groups present lower E 1/2 when compared to electron-releasing groups.The complexity of the CV observed for HL11 and complex 11 indicated that reduction potentials for the nitro group and the naphthoquinone are similar.Upon complexation, the E 1/2 (1) peak is slightly shifted to more positive potentials.

Antibacterial activity
The antibacterial activity of the hydrazono compounds HL1-HL13 and their copper(II) complexes 1-13 was evaluated against seven strains of bacteria: Bacillus cereus (BC), Bacillus subtilis (BS), Escherichia coli (EC), Enterococcus faecalis (EF), Klebsiella pneumoniae (KP), Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA).The results show that only compound HL5 exhibited significant activity against three strains of bacteria (BC, BS and EC).It inhibited EC growth at 20 mmol L -1 , i.e., at lower concentration than the positive control (chloramphenicol, 40-90 mmol L -1 ) and exhibited similar activity (90 mmol L -1 ) to chloramphenicol against BC and BS strains.Furthermore, this compound was far more active against EC than the analogous 3-hydrazino-naphthoquinones derived from 3-diazo-naphthalene-1,2,4-trione. 6None of the complexes showed significant antibacterial activity (> 200 mmol L -1 ); furthermore, complex 5 was less active than its proligand HL5.Lawsone and CuCl 2 • 2H 2 O, tested for comparison, only inhibited bacterial growth at high concentrations (see Supporting Information for detailed antibacterial assay data).Low activity of both proligands and complexes may be associated to their poor solubility and, therefore, low bioavailability.Same behavior was observed for the copper(II) complexes of aminonaphthoquinone Mannich bases derived from lawsone, 3 and the metal complexes of the anion of 5-amino-8-hydroxy-1,4-naphthoquinone. 44

Antitumor activity
The antitumor screening of proligands HL1-HL13 and complexes 1-6, 9, 10, 12 and 13 was initially carried out against three strains of cancer cell lines: SF-295 (central nervous system), HCT-8 (colon) and MDAMB-435 (breast).Doxorubicin (dox) was used as a positive control.Lawsone was also tested for comparison (see Supplementary Information for antitumor activity data).Only the hydrazono compound HL6 exhibited higher growth inhibition of colon cancer cells HCT-8 (96.03%) than the positive control dox (91.67%); however, its complex, 6, did not show any significant activity.Complex 13 also exhibited higher antitumor activity (96.03 %) than dox.In four cases (HL2, HL4, HL9 and HL13) complexation resulted in increased antitumor activity against all cancer cell lines.The results indicate, in this system, that the presence of NO 2 and I groups in ortho and para positions, respectively, is relevant for the antitumor activity.Due to the high antitumor activity of compounds HL6 and 13, they were selected for IC 50 determination against four cancer cell lines: SF-295 (central nervous system), HCT-8 (colon), MDAMB-435 (breast) and HL-60 (leukemia) and the results are presented in Table 4. Compound 13 (Entry 2) shows moderate cytotoxic activity against leukemia cell line.
Interestingly, the most active compound against HL-60 cell line (human leukemia), complex 13 (R = 2-NO 2 ), also presents the lowest EPR parameter g || /A || ratio, i.e., the weakest field ligand (see Supporting Information).The EPR spectrum of this complex also shows a typical organic free radical line over the perpendicular copper(II) spectrum that is absent in the spectra of complexes 11 (R = 4-NO 2 ) and 12 (R = 3-NO 2 ) and may be responsible for the antitumor activity of complex 13.

Conclusions
The predominance of the keto-hydrazone tautomers Ia/Ib, as opposed to the previously reported enol-azo IIb, was confirmed by theoretical calculations that also established that, differently from the analogous 2-naphthol azo system, the nature of the substituent in the phenylene ring does not affect this stability order (Ib @ Ia >> II).In spite of the higher stability of the keto-hydrazone Ia and Ib forms, deprotonation, followed by reaction with Cu 2+ , results in the formation of enolate complexes of the azo form IIb.
The antibacterial assays revealed that of all compounds only proligand HL5 (R = 3-I) inhibited the growth of EC at lower concentration than the positive control (chloramphenicol), thus structure/reduction potentialactivity correlations could not be attempted.In the antitumor screening, in general, complexes were found to be more active than the respective proligands; however, only complex 13 (R = 2-NO 2 ) showed moderate cytotoxic activity against HL-60 cells.Formation of an organic free radical observed in the EPR spectrum of this complex might be responsible for this cytotoxic behaviour.

Table 4 .
Cytotoxic activity of compounds HL6 and 13, expressed in IC 50 , obtained by MTT assay after incubation of cells for 72 h in the concentrations 0.01-5 mg mL-1