Novel Aminonaphthoquinone Mannich Bases Derived from Lawsone and their Copper ( II ) Complexes : Synthesis , Characterization and Antibacterial Activity

Uma série de novas Bases de Mannich (HL1-HL13) derivadas da 2-hidroxi-1,4-naftoquinona (lausona), benzaldeídos substituídos [C 6 H 2 RRRC(O)H] e várias aminas primárias (NH 2 R, R = n-butil, benzil, alil, 2-furfuril) e seus complexos de Cu, [Cu(L1) 2 ]-[Cu(L13) 2 ], foram sintetizados e caracterizados por métodos analíticos e espectroscópicos. As estruturas dos complexos 1 (R = R = R = H; R = Bu), 2 (R = R = H; R = NO 2 ; R= Bu) e 7 (R = OH; R = R = H; R= Bu) foram determinadas por estudos de difração de raios-X de monocristal. Todos os compostos cristalizam em grupos espaciais centrossimétricos, com um cobre no centro de inversão. Dois L coordenam-se através dos átomos de oxigênio do naftalen-2-olato e do nitrogênio da amina secundária, formando anéis quelatos de seis membros ao redor do átomo de cobre em um ambiente trans-N 2 O 2 . A atividade antimicrobial de todos os compostos foi testada em sete diferentes linhagens de bactérias: Bacillus cereus, Bacillus subtilis, Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa e Staphylococcus aureus. Em geral, as bases de Mannich foram mais ativas que os complexos, sendo HL11 (R = OH; R =H; R = Me; R= Bn) e HL13 (R = OH; R = H; R = Br; R= Bn) os inibidores mais potentes. O MIC para o composto mais ativo HL11 contra S. Coli foi 20 μmol L (8 μg mL), melhor que o cloranfenicol (90 μmol L) e bem abaixo da maioria dos valores descritos para outras naftoquinonas.


Introduction
Natural and synthetic naphthoquinones are known for a wide range of biological activities, 1 amongst which anti-cancer, 2,3 tripanocidal, 4 molluscicidal, 5 antimalarial, 6 leishmaniscide, 7 bacteriostatic and bactericidal. 8,9The most accepted mechanism for the antimicrobial activity of naphthoquinones is based on the generation of reactive oxygen species by two successive reduction processes to form radical anion and dianion species that are toxic to bacteria. 10,119][20][21][22] We therefore evaluated the antimicrobial activity of a novel series of 2-hydroxy-3alkylamine-1,4-naphthoquinones, known as Mannich bases.These compounds were first synthesized over sixty years ago, 23 and their antimalarial 23,24 and molluscicidal 25 activities have been described.In spite of the fact that metal complexation of a number naphthoquinones or naphthoquinone derived compounds has resulted in increased citoxicity, 26 antimalarial 27,28 and anticancer 29 activity, transition metal complexes of Mannich bases derived from 2-hydroxy-1,4-naphthoquinone (lawsone) have not yet been reported.Herein we describe the synthesis of novel Mannich bases HL1-HL13 from lawsone, a number of primary amines and substituted benzaldehydes and of their copper(II) complexes, [Cu(L) 2 ] (1-13) (Scheme 1), their characterization by analytical and spectroscopic techniques, and the X-ray diffraction studies of three complexes.Furthermore, we report the results of antibacterial activity screening of all compounds and discuss growth inhibition as a function of structural changes and metal complexation.

Materials and methods
Reagents and solvents were used without further purification.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 CDCl 3 or d 6 -DMSO; 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 thought 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 solid samples were obtained at liquid nitrogen temperature (77 K), on a Bruker ESP300E equipment with modulation frequency of 100 kHz, operating at 9.5 GHz (X-band).

Synthesis of the Mannich bases HL1-HL13
Compounds HL1-HL13 (Figure 1) were synthesised according to the general procedure described in the literature 24,30 with modifications.They were obtained by reacting a suspension of lawsone (5 mmol, 0.870 g), in 15 mL of ethanol, with the respective amine (5.5 mmol).After formation of the lawsonate solution, the aldehyde (6 mmol) is added and the mixture, left stirring at room Scheme 1. Synthesis of Mannich bases HL1-HL13 and of complexes 1-13.
Novel Aminonaphthoquinone Mannich Bases Derived from Lawsone and their Copper(II) Complexes J. Braz.Chem.Soc.714   temperature for 12 h in the dark.The orange solids were filtered, washed with ethanol, diethyl eter and dried under vacuum.

-[ N -( nb u t y l ) a m i n o b e n z y l ] -2h y d ro x y -1 , 4naphthoquinone (HL1)
From 0.54 mL of butylamine and 0.61 mL of benzaldehyde.Yield:

Synthesis of complexes [Cu(L) 2 ] 1-13 from HL1-HL13, respectively
To a suspension of 1 mmol of the ligand in 10 mL MeOH, was added a solution of CuCl 2 .2H 2 O (83 mg, 0.5 mmol) in 2 mL MeOH.After addition of Et 3 N (0.14 mL, 1 mmol), the suspension was left under stirring in the dark for 12h at room temperature.The resulting solids were filtered off, washed with methanol, diethyl ether and dried under vacuum (Figure 2).

X-ray crystallography
The x-ray diffraction data for compounds were collected using a Bruker KAPPA CCD diffractometer, 31 at 295K and Mo graphite monochromatic radiation.The cell parameters for the molecules were obtained and refined using the PHICHI 32 and DIRAX 33 programs, respectively, catching reflections with random orientation in hkl planes.Intensities were corrected by Lorentz polarization and absorption with the SADABS program. 34The structure was solved by Direct Methods using the SHELXS-97 program. 35The anisotropy parameters of non-H atoms were refined with the SHELXL-97 program. 36In 1, 2 and 7 the aromatic, methyl, methyne and methylene H-atoms were geometrically included in the refinement.Aromatic carbons were refined with U iso (H) = 1.2 Ueq Csp 2 , methylene carbons with U iso (H) = 1.2 Ueq Csp 3 , methine carbons with U iso (H) = 1.2 Ueq Csp 3 and methyl carbons with U iso (H) = 1.5 Ueq Csp 3 .The hydrogen atom of the water molecules, N-H amine in the three compounds and O-H hydroxyl for 2 were localized experimentally in the Fourier map.For 2 the hydrogen atom coordinates corresponding to a water molecule could not be localized experimentally in the Fourier map.In view of this we opted for using the SQUEEZE 37 tool contained in the WinGX 38 package, in order to exclude any electronic density contributions relative to the disordered water molecules.This procedure is in accordance with the elemental analysis of the complex, confirming a species free from any crystallization solvate.Consequently we do not comment in this work on the hydrogen bonds for 2. Vol. 20, No. 4, 2009   The solution and refinement of 1 suggested the presence of disordered C21carbon of the butyl moiety.X-ray data are listed in Table 1 and ORTEP-3 39 for Windows was used to draw the Figures.
Minimum inhibitory concentration (MIC) was determined by the microdilution broth technique according to the M7-A6 document. 40The assays were carried in 96well tissue culture microplates filled with Mueller Hinton broth (100 µL per well). 41The inoculum suspension of each strain was prepared in Mueller Hinton broth (108 bacteria cells per mL, corresponding to O. D. = 0.08-0.1 at 625 nm) and diluted to 1:10.All samples were tested in eighth concentrations from 3 to 0.02 × 10 -3 mol L -1 .The inoculum suspension (5 µL per well) was applied into the microplates which were incubated at 37 °C overnight.An aqueous solution of p-iodonitrotetrazolium violet (p-INT) (Sigma) (20 µL) was added 42 and the microplates were incubated once more for 1-2 hours at 37 °C.The

Syntheses
The Mannich bases HL1-HL13 (Figure 1) were synthesized from the reactions of 2-hydroxy-1,4naphthoquinone (lawsone) with an various primary amines and aldehydes in ethanol under stirring at room temperature.The orange products are stable in the solid state, but undergo decomposition when left in solution for a long period of time.Compounds HL1-HL7 and HL11-HL13 are obtained in a pure state, but HL8-HL10 need to be recrystallised from hot ethanol.They were obtained in yields ranging from 53 (HL4) to 93% and formulated on the basis of analytical and spectroscopic data (see Experimental).
The 1 H spectra of compounds HL1-HL13 exhibit peaks due to the four naphthoquinone aromatic hydrogens H5-H8 that appear in the d 7-8 ppm region as dd or ddd (H5 and H8) and td (H6 and H7) (see Figure 1 for numbering and experimental for data).The other chemical shifts are compatible with the structures proposed for these compounds.In general, the butyl hydrogens appear in the d 0.9 to 3.1 ppm region as multiplets (H19-H21) or triplets (methyl H22); in the case of the benzyl group; the CH 2 hydrogens H19 appear as a doublet around d 4.2 -4.6 and the phenyl H21-H25, as multiplets.The (substituted) phenyl group hydrogens H13-17 appear as expected, depending on the substitution pattern.Attributions were made on the basis of 1 H × 1 H (COSY experiments), J values and multiplicity.All expected resonances were observed in the 13 C NMR spectra of compounds HL1-HL13.The resonances arising from the carbonyl carbons were found around d 185 and 179, and those attributed to C2 bound to the hydroxyl group, at about d 171.
Complexes 1-13 (Figure 2) were obtained by addition of trietylamine to a methanolic suspension of the ligand and CuCl 2 .2H 2 O (2:2:1), under stirring at room temperature for 12 h in yields varying from 60 to 92%, except for complex 12, isolated in 30%.Elemental analysis confirmed the proposed formulation.Due to low solubility in methanol, acetonitrile and water, conductivity measurements could not be carried out.
All compounds were also characterized by EPR and UV-Vis spectroscopy, and the structures of 1, 2 and 7, determined by X-ray diffraction analyses.

Description of the X-ray structures
Good quality crystals suitable for single crystal X-ray diffraction analyses were obtained for compounds 1, 2 and 7.The molecular structures of 1, 2 and 7 are shown in Figures 3, 4 and 5, respectively and selected bond lengths and angles are given in Table 2.
All complexes crystallize in centrosymmetric space groups, with a copper atom in the inversion centre.Two deprotonated ligands (L − ) coordinate through the naphthalen-2-olate oxygen and secondary amine-N atoms, forming two six-membered chelate rings around the copper atom in a trans-N 2 O 2 environment.5][46] In the same molecule, the two ligands have different absolute configurations at the chiral C11carbon.In the structures of compounds 1 and 2 the butyl and phenyl groups block the axial positions preventing further coordination to donor molecules (e.g.coordinating solvent), normally observed in the structures of analogous complexes. 47,48he packing arrangement of 1 exhibits molecules of 1 and water linked by N-H … O and O-H … O classic hydrogen bonds along the [100] crystallographic direction, resulting in a 1D supramolecular arrangement, Figure 6.The water molecules are responsible for the 1D self-assembly formed in the solid state, and the network is cemented by bifurcated and linear H-bonds around these molecules.The O4 # -H4A #… O1 and O4 # -H4A #… O2 bifurcated hydrogen bond forms a five membered ring between complex 1 and water, these interactions providing stabilization of the 1D network [symmetry code for (1): (#) = x, 1+y, z; (##) = 1+x, y, z].In addition the crystalline structure is stabilized by C-H … O and C-H … N intramolecular interactions (Figure 7).Table 3 shows all H-bond parameters.All hydrogen bonds were calculated using PLATON 37 (Table 3) and agree with the literature. 37he crystalline structure of complex 7 shows a 2D selfarrangement governed by O-H … O and C-H … O classical and non classical hydrogen bonds, respectively.The lattice grows in the [100] direction through O6 ii -H6A ii… O3, O6 ii -H6B ii… O2 iii and O4 i -H4 i… O6 ii interactions and in the [001] direction, through C18-H18B … O5 iii interactions, as shown in Figure 8 [symmetry code for 7: 4].The network in the [100] direction is built around the water molecules and in the [001] direction, via the dioxane interactions.In addition, intramolecular interactions via N-H … O and C-H … O, specifically the N1-H1 … O4 hydrogen bond prevents the copper atom from interacting with the hydroxyl O(4), as illustrated in Figure 9. Similar type of interaction has been observed in an analogous copper compound. 45he geometric parameters for these interactions are listed in Table 4.All hydrogen bonds were calculated using PLATON 37 and agree with the literature. 37

FT-IR spectra
The FT-IR spectra of the complexes show a broad band near 3400 cm -1 assigned to O-H stretching of the water molecules present. 499][50] These bands were not observed in the spectra of the free ligands, due to the presence of the broad ν O-H band centered around 3400 cm -1 .This shift in ν N-H frequency confirms complexation to the Cu 2+ center.Several weak bands observed in the 2850-3100 cm -1 range are attributed to aliphatic and aromatic C-H groups. 49The strong carbonyl ν CO band around 1680 cm -1 and aromatic ring ν C=C bands, around 1590 and 1470 cm -1 were not altered by complexation. 48The strong ν C-O band around 1280 cm -1 was attributed to the naphthoquinonato group. 47,51

EPR Spectra
The EPR spectra of complexes 1-13 were measured in the solid state at liquid nitrogen temperature.The Hamiltonian parameters (Table 5) obtained in    Table 3. Geometric parameters for non-classical H bonds for complex 1 (Å, degree) Symmetry code for 1: (a) x, 1+y, z; (b) 1+x, y, z.   that the unpaired electron is predominantly in the dx 2 -y 2 orbital, which gives 2 B 1g as the ground state.The very low parallel and perpendicular components of the hyperfine coupling constant for complexes 2 and 12 (not resolved in the spectra) has been explained by considering a mixture of the Cu 2+ dz 2 and dx 2 -y 2 orbitals as the ground state.It is found that a 10% mixture of dx 2 -y 2 and dz 2 results in a 20% reduction in dipolar anisotropy. 54he relatively low g || values of the complexes are consistent with a N 2 O 2 environment around the Cu 2+ ions. 52s expected, the ligand field strength depends mainly on the nature of the R 4 substituent on the nitrogen, the highest ligand field being observed for complexes of ligands containing R 4 = butyl, independently of the nature of R 2 and R 3 .The presence of the hydroxyl group (R 1 =OH), however, may lead to a decrease in the ligand field, as observed for complex 7, compared with complex 1 (Table 5), due to the presence of a O-H…N1 hydrogen bond that reduces the Lewis basicity of N1.This interaction is evidenced in the supramolecular arrangement of the structure of 7.This interaction may be present in all complexes containing R1 = OH (6-13).

UV-Vis spectra
The electronic spectra of complexes 1-5 and 12, recorded in CHCl 3 solution, are characterized by two   intense absorptions observed in the 425-315 nm range that are presumably due to a charge-transfer band from the naphthquinonato moiety to the metal ion, and to ligand based transitions. 559][50] .The d-d band appears between 530-550 nm and can be attributed to a 2 A 1g ← 2 B 1g transition which supports the square planar geometry for the complexes in solution. 50he spectra of complexes 6-11 and 13 were recorded in DMSO solution due to their low solubility in CHCl 3 .All exhibit two intense absorption bands: the band observed around 450 nm was assigned to charge-transfer from the naphthoquinonate moiety to the metal ion and that at 277 nm, also present in the spectra of the respective free ligands, to π-π* transitions of the naphthoquinone ring.No d-d transition band was observed, which suggests coordination of DMSO molecules and distorted octahedral coordination environment of the Cu 2+ ion.and R 4 = Bu is much less active than HL11 against all strains of bacteria.Solubility differences might be responsible for the changes in activity.
With a few exceptions, the complexes were less active than the respective pro-ligands, which is probably due to their lower solubility.Thus, the activity of HL11 (Entry 11) decreased upon complexation, from 20 to 90 µmol L -1 for 11 (E.coli) and from 20 to 90 µmol L -1 for 11 (S.aureus) (Entry 28), although slight increase in growth inhibition of all the other bacteria strains was observed (from >200 µmol L -1 for HL11 to 180 µmol L -1 for 11).
Improvement of the activity of HL10 and H12 that only inhibited the growth of all strains of bacteria above 180-200 µmol L -1 (Entries 10 and 12) was observed upon complexation: complex 10 (Entry 27) exhibits slight activity against B. cereus, B. subtilis and S. aureus (180 µmol L -1 ) and growth inhibition against E. coli (90 µmol L -1 ), and complex 12, against E. coli, E. faecalis and S. aureus above 90 µmol L -1 (Entry 29).Complexes 10 and 12 were formed from Mannich bases HL10 and HL12 that only differed from the very active ones, HL11 and HL13 with respect to the R 4 group, butyl, instead of benzyl.
The effect of metal complexation on naphthoquinone antimicrobial agents has been discussed in the literature. 56lthough metal chelation of the anion of 5-hydroxy-1,4naphthoquinone (juglone) has resulted in complexes with similar antibacterial effect 57 or higher antibacterial activity, e.g. against Bacillus ssp and S. aureus, than juglone, complexation of the anions of a series of 5-amino-8hydroxy-1,4-naphthoquinones with M 2+ (M = Ni, Co, Fe, Cu and Cr) resulted in reduced bacterial activity or lack of inhibition effect. 55Considering that redox active metals have been shown to be instrumental in naphthoquinone toxicity, 11 the decreased activity observed in our work and by others is probably associated with decreased bioavailability of the aminonaphthoquinones as the result of decreased solubility upon complexation.

Conclusions
In conclusion, of the thirteen novel aminonaphthoquinones HL1-HL13 synthesized from lawsone, via the Mannich reaction, and their respective copper(II) complexes [Cu(L1) 2 ] -[Cu(L13) 2 ], those containing a 2-hydroxyphenyl group (R 1 = OH) which is substituted at the 5-position, either with R 3 = Me or Br, and with R 4 = benzyl or butyl groups have strongly inhibited the growth especially of Escherichia coli and Staphylococcus aureus.In general complexes were found to be less active than the respective pro-ligands, probably due to their lower solubility.Further work is in progress to improve the bioavailability of the complexes of HL10-HL13.

Figure 7 .
Figure 7.View of intramolecular interactions via non classical hydrogen bonds in complex 1.
the simulations of the EPR spectra g || > g ⊥ > 2 and A || = (188 -200) × 10 -4 cm -1 are typical of elongated octahedral or square planar geometry, suggesting copper(II) sites with axial symmetry.52All complexes, except for 2 and 12, show values of g || /A || between 110 and 122 thus confirming the square planar environment around the Cu 2+ centre 53 as established for compounds 1, 2 and 7 by X-ray analysis.The experimental values, g || > g ⊥ , also indicate

Figure 9 .
Figure 9.View of the intramolecular interactions in complex 7.

Table 1 .
Crystallographic data and refinement parameters for 1, 2 and 7 was defined as the lowest concentration of the extracts that inhibited the antibacterial visible growth as indicated by the p-INT colorimetric reagent.For sterility and growth control the Mueller Hinton broth was used without solvent or compounds.All strains were subcultured twice to verify the cell viability.Tests were performed in triplicate.

Table 4 .
Geometric parameters for non-classical H bonds for complex 7 (Å, degree)

Table 5 .
Spin-Hamiltonian parameters used in the simulated spectra of the copper(II) ion in complexes 1-13 in decreasing order of field strength