Novel aminonaphthoquinone mannich bases derived from lawsone and their copper(II) complexes: synthesis, characterization and antibacterial activity

Abstracts

A series of novel Mannich bases (HL1-HL13) derived from 2-hydroxy-1,4-naphthoquinone (lawsone), substituted benzaldehydes [C6H2R¹R²R³C(O)H] and various primary amines (NH2R4, R4 = n-butyl, benzyl, allyl, 2-furfuryl), and their Cu2+ complexes, [Cu(L1)2]-[Cu(L13)2], have been synthesized and fully characterized by analytical and spectroscopic methods. The structures of complexes 1(R¹ = R² = R³ = H; R4 = Bu), 2(R¹ = R³ = H; R² = NO2; R4= Bu) and 7 (R¹ = OH; R² = R³ = H; R4= Bu) were determined by single crystal X-ray diffraction studies. All complexes crystallize in centrosymmetric space groups, with a copper atom in the inversion centre. Two L- coordinate through the naphthalen-2-olate oxygen and secondary amine-N atoms, forming six-membered chelate rings around the copper atom in a trans-N2O2 environment; spectroscopic data confirm that the other complexes exhibit similar molecular arrangement. The antimicrobial activity of all compounds has been tested on seven different strains of bacteria: Bacillus cereus, Bacillus subtilis, Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosaand Staphylococcus aureus. In general, Mannich bases were more active than complexes, HL11(R¹ = OH; R² =H; R³ = Me; R4= Bn) and HL13(R¹ = OH; R² = H; R³ = Br; R4= Bn) being the most potent inhibitors. The MIC for the most active compound HL11against S. Coliwas 20 µmol L-1 (8 µg mL-1), better than Chloramphenicol (90 µmol L-1) and well below most values reported for other naphthoquinones.

aminonaphthoquinones; copper complexes; Mannich bases; crystal structure determination; antibacterial activity

Uma série de novas Bases de Mannich (HL1-HL13) derivadas da 2-hidroxi-1,4-naftoquinona (lausona), benzaldeídos substituídos [C6H2R¹R²R³C(O)H] e várias aminas primárias (NH2R4, R4 = n-butil, benzil, alil, 2-furfuril) e seus complexos de Cu2+, [Cu(L1)2]-[Cu(L13)2], foram sintetizados e caracterizados por métodos analíticos e espectroscópicos. As estruturas dos complexos 1(R¹ = R² = R³ = H; R4 = Bu), 2(R¹ = R³ = H; R² = NO2; R4= Bu) e 7(R¹ = OH; R² = R³ = H; R4= 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-N2O2. 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 aeruginosae Staphylococcus aureus. Em geral, as bases de Mannich foram mais ativas que os complexos, sendo HL11(R¹ = OH; R² =H; R³ = Me; R4= Bn) e HL13(R¹ = OH; R² = H; R³ = Br; R4= Bn) os inibidores mais potentes. O MIC para o composto mais ativo HL11contra S. Colifoi 20 µmol L-1 (8 µg mL-1), melhor que o cloranfenicol (90 µmol L-1) e bem abaixo da maioria dos valores descritos para outras naftoquinonas.

ARTICLE

Novel aminonaphthoquinone mannich bases derived from lawsone and their copper(II) complexes: synthesis, characterization and antibacterial activity

Amanda P. Neves^I; Cláudia C. Barbosa^I; Sandro J. GrecoI, ^#; Maria D. VargasI, ^*; Lorenzo C. Visentin^II; Carlos B. Pinheiro^III; Antônio S. Mangrich^IV; Jussara P. Barbosa^V; Gisela L. da Costa^V

^IInstituto de Química, Universidade Federal Fluminense, Campus do Valonguinho, Centro, 24020-150 Niterói-RJ, Brazil

^IIInstituto de Química, Universidade Federal do Rio de Janeiro, Ilha do Fundão, 21945-970 Rio de Janeiro-RJ, Brazil

IIIDepartamento de Física, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Pampulha, 31270-901 Belo Horizonte-MG, Brazil

^IVDepartamento de Química, Centro Politécnico, Universidade Federal do Paraná, 81531-970 Curitiba-PR, Brazil

^VInstituto Oswaldo Cruz, CP 926, 21045-900 Rio de Janeiro-RJ, Brazil

ABSTRACT

A series of novel Mannich bases (HL1-HL13) derived from 2-hydroxy-1,4-naphthoquinone (lawsone), substituted benzaldehydes [C₆H₂R¹R²R³C(O)H] and various primary amines (NH₂R⁴, R⁴ = n-butyl, benzyl, allyl, 2-furfuryl), and their Cu²⁺ complexes, [Cu(L1)₂]-[Cu(L13)₂], have been synthesized and fully characterized by analytical and spectroscopic methods. The structures of complexes 1(R¹ = R² = R³ = H; R⁴ = Bu), 2(R¹ = R³ = H; R² = NO₂; R⁴= Bu) and 7 (R¹ = OH; R² = R³ = H; R⁴= Bu) were determined by single crystal X-ray diffraction studies. All complexes crystallize in centrosymmetric space groups, with a copper atom in the inversion centre. Two L- coordinate through the naphthalen-2-olate oxygen and secondary amine-N atoms, forming six-membered chelate rings around the copper atom in a trans-N₂O₂ environment; spectroscopic data confirm that the other complexes exhibit similar molecular arrangement. The antimicrobial activity of all compounds has been tested on seven different strains of bacteria: Bacillus cereus, Bacillus subtilis, Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosaand Staphylococcus aureus. In general, Mannich bases were more active than complexes, HL11(R¹ = OH; R² =H; R³ = Me; R⁴= Bn) and HL13(R¹ = OH; R² = H; R³ = Br; R⁴= Bn) being the most potent inhibitors. The MIC for the most active compound HL11against S. Coliwas 20 µmol L^-1 (8 µg mL^-1), better than Chloramphenicol (90 µmol L^-1) and well below most values reported for other naphthoquinones.

Keywords: aminonaphthoquinones, copper complexes, Mannich bases, crystal structure determination, antibacterial activity

RESUMO

Uma série de novas Bases de Mannich (HL1-HL13) derivadas da 2-hidroxi-1,4-naftoquinona (lausona), benzaldeídos substituídos [C₆H₂R¹R²R³C(O)H] e várias aminas primárias (NH₂R⁴, R⁴ = n-butil, benzil, alil, 2-furfuril) e seus complexos de Cu²⁺, [Cu(L1)₂]-[Cu(L13)₂], foram sintetizados e caracterizados por métodos analíticos e espectroscópicos. As estruturas dos complexos 1(R¹ = R² = R³ = H; R⁴ = Bu), 2(R¹ = R³ = H; R² = NO₂; R⁴= Bu) e 7(R¹ = OH; R² = R³ = H; R⁴= 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-N₂O₂. 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 aeruginosae Staphylococcus aureus. Em geral, as bases de Mannich foram mais ativas que os complexos, sendo HL11(R¹ = OH; R² =H; R³ = Me; R⁴= Bn) e HL13(R¹ = OH; R² = H; R³ = Br; R⁴= Bn) os inibidores mais potentes. O MIC para o composto mais ativo HL11contra S. Colifoi 20 µmol L^-1 (8 µg mL^-1), melhor que o cloranfenicol (90 µmol L^-1) 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.^8,9 The 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.^10,11

It has been shown that the incorporation of amino groups or a nitrogen atom into naphthoquinones often results in increased anticancer,^12-14 molluscicidal^15-17 and antibacterial activities.^10,18-22 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 molluscicidal²⁵ 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 anticancer²⁹ 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-HL13from 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.

Experimental

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. ¹H and ¹³C NMR spectra were recorded with a Varian Unit Plus 300 MHz spectrometer in CDCl₃ or d⁶-DMSO; coupling constants are reported in Hertz (Hz) and chemical shifts in parts permillion (ppm) relative to internal standard Me₄Si. The hydrogen signals were attributed thought coupling constant values and ¹H × ¹H-COSY experiments. Electronic spectra were takenonaDiodeArray 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 temperature for 12 h in the dark. The orange solids were filtered, washed with ethanol, diethyl eter and dried under vacuum.

3-[N-(n-butyl)aminobenzyl]-2-hydroxy-1,4 naphthoquinone (HL1)

From 0.54 mL of butylamine and 0.61 mL of benzaldehyde. Yield: 1.258 g, 74%, mp 143-144 ºC. Anal. Calc. for C₂₁H₂₁NO₃: C, 75.20; H, 6.31; N, 4.18%. Found: C, 75.14; H, 6.29; N, 4.23%. IR (KBr) V_max/cm^-1: 3435 (O-H), 3060 (C-H), 2960 (C-H), 2933 (C-H), 1680 (C=O), 1588 (C=C), 1529 (d N-H), 1276 (C-O). ¹H NMR (DMSO-d⁶, 300 MHz): δ(ppm) 8.02 (ddd, J7.60, 1.22, 0.45 Hz, 1H, H5or H8); 7.93 (ddd, J7.57, 1.41, 0.45 Hz, 1H, H8or H5); 7.81 (td, J7.60, 7.60, 1.41 Hz, 1H, H6or H7); 7.73-7.66 (m, 3H, H7or H6and Ph); 7.49-7.38 (m, 3H, Ph); 5.59 (br s, 1H, H11); 2.97 (br t, J7.65, 7.65 Hz, 2H, H19); 1.76-1.64 (m, 2H, H20); 1.48-1.34 (m, 2H, H21); 0.94 (t, J7.36, 7.36 Hz, 3H, H22). ¹³C NMR (DMSO-d⁶, 75 MHz): δ(ppm) 184.6, 178.7, 170.8, 138.9, 134.8, 134.0, 131.7, 131.1, 128.6, 128.1, 127.9, 125.6, 125.3, 111.5, 59.0, 45.6, 27.9, 19.5, 13.7. UV-Vis (CHCl₃) λ/nm, log ε: 267 (4.19), 338 (3.15), 437(3.20).

3-[N-(n-butyl)amino-3-nitrobenzyl]-2-hydroxy-1,4naphthoquinone (HL2)

From 0.54 mL of butylamine and 0.907 g of p-nitrobenzaldehyde.Yield:1.141g,60%,mp 172 ºC.Anal.Calc. for C₂₁H₂₀N₂O₅: C, 66.31; H, 5.30; N, 7.36%. Found: C, 66.20; H, 5.49; N, 7.51%. IR (KBr) V_max/cm^-1: 3437 (O-H), 3086 (C-H), 2962 (C-H), 1683 (C=O), 1588 (C=C), 1520 (δ N-H), 1281 (C-O). ¹H NMR (DMSO-d⁶, 300 MHz): δ(ppm) 8.33 (d, J8.85 Hz, 2H, H14; H16); 8.03 (ddd, J7.62, 1.34, 0.49 Hz, 1H, H5or H8); 7.97 (d, J8.85 Hz, 2H, H13; H17): 7.93 (ddd, J7.62, 1.39, 0.49 Hz, 1H, H8or H5); 7.82 (td, J7.62, 7.62, 1.39 Hz, 1H, H6or H7); 7.70 (td, J7.62, 7.62, 1.34 Hz, 1H, H7or H6); 5.78 (s, 1H, H11); 3.02 (t, J7.67, 7.67 Hz, 2H, H19); 1.73 (t, J7.67, 7.67 Hz, 2H, H20); 1.40 (sx, J7.33 Hz, 2H, H21); 0.95 (t, J 7.33, 7.33 Hz, 3H, H22). ¹³C NMR (DMSO-d⁶, 75 MHz): δ(ppm) 184.2, 178.5, 170.7, 146.8, 146.0, 134.6, 133.9, 131.6, 131.1, 128.6, 125.5, 123.5, 110.3, 57.9, 45.6, 27.8, 19.4, 13.6. UV-Vis (CHCl₃) λ/nm, log ε: 268 (4.36), 336 (3.60), 427(3.26).

3-[N-(benzyl)amino-3-nitrobenzyl]-2-hydroxy-1,4naphthoquinone (HL 3)

From 0.60 mL of benzyllamine and 0.907 g of p-nitrobenzaldehyde. Yield: (1.513 g, 73%); mp 139-140 ºC. Anal. Calc. for C₂₄H₁₈N₂O₅: C, 69.56; H, 4.38; N, 6.76%. Found: C, 68.80; H, 4.39; N, 6.67%. IR (KBr) V_max/cm^-1: 3445 (O-H), 3066 (C-H), 3032 (C-H), 2969 (C-H), 1677 (C=O), 1592 (C=C), 1523 (δ N-H), 1272 (C-O). ¹H NMR (DMSO-d⁶, 300 MHz): δ (ppm) 8.31 (d, J8.86 Hz, 2H, H14; H16); 8.04 (ddd, J7.60, 1.38, 0.40 Hz, 1H, H5or H8); 7.95 (ddd, J7.48, 1.41, 0.40 Hz, 1H, H8or H5); 7.92 (d, J8.86 Hz, 2H, H13; H17); 7.83 (td, J7.60, 7.60, 1.41 Hz, 1H, H6or H7); 7.70 (td, J7.48, 7.48, 1.38 Hz, 1H, H7or H6); 7.52-7.49 (m, 5H, Ph); 5.76 (s, 1H, H11); 4.28 (s, 2H, H19). ¹³C NMR (DMSO-d6, 75 MHz): δ (ppm) 184.1, 178.6, 170.7, 146.9, 145.7, 134.6, 133.8, 132.1, 131.6, 131.1, 130.2, 128.9, 128.8, 128.6, 125.5, 125.2, 123.5, 110.1, 57.8, 49.3. UV-Vis (CH₂Cl₂) λ/nm, log ε: 273 (4.47), 337 (3.74), 389 (3.22).

3-[N-(n-butyl)amino-2,4-diclorobenzyl]-2-hydroxy-1,4naphthoquinone (HL4)

From 0.60 mL of benzylamine and 1.050 g of 2-4-dicloro-benzaldehyde. Yield: 1.071 g, 53%; mp 142143 ºC. Anal. Calc. for C₂₁H₁₉Cl₂NO₃: C, 62.39; H, 4.74; N, 3.46%. Found: C, 62.00; H, 4.64; N, 3.51%. IR (KBr) V_max/cm^-1: 3437 (O-H), 3064 (C-H), 2958 (C-H), 2870 (CH), 1678 (C=O), 1616 (C=C), 1588 (C=C), 1531 (d N-H), 1272 (C-O). ¹H NMR (DMSO-d⁶, 300 MHz): δ (ppm) 8.01 (dd, J7.46, 1.33 Hz, 1H, H5or H8); 7.97 (dd, J7.46, 1.37 Hz, 1H, H8or H5); 7.96 (d, J8.51 Hz, 1H, H17); 7.83 (td, J7.46, 7.46, 1.37 Hz, 1H, H6or H7); 7.76 (d, J2.19, 1H, H14); 7.72 (td, J7.46, 7.46, 1.33 Hz, 1H, H7or H6); 7.55 (dd, J8.51, 2.19, 1H, H16); 5.99 (s, 1H, H11); 3.05 (br t, J7.35, 7.35 Hz, 2H, H19); 1.80-1.65 (m, 2H, H20); 1.38-1.34 (m, 2H, H21); 0.96 (t, J7.28, 7.28 Hz, 3H, H22). ¹³C NMR (DMSO-d⁶, 75 MHz): δ (ppm) 184.1, 178.9, 171.3, 134.8, 134.5, 134.4, 133.9, 133.8, 131.9, 131.6, 131.1, 128.8, 127.7, 125.5, 125.2, 109.6, 55.4, 45.9, 27.7, 19.3, 13.5. UV-Vis (CHCl₃) λ/nm, log ε: 265 (4.29), 337 (3.42), 422 (3.29).

3-[N-(n-benzyl)amino-2,4-diclorobenzyl]-2-hydroxy-1,4naphthoquinone (HL5)

From 0.54 mL of butylamine and 1.050 g of 2-4-diclorobenzaldehyde. Yield: 1.731 g, 79%; mp 145-146 ºC. Anal. Calc. for C₂₄H₁₇Cl₂NO₃: C, 65.77; H, 3.91; N, 3.20%. Found: C, 65.67; H, 3.87; N, 3.24%. IR (KBr) V_max/cm^-1: 3453 (O-H), 3065 (C-H), 3033 (C-H), 2969 (C-H), 1677 (C=O), 1592 (C=C), 1523 (δ N-H), 1271 (C-O). ¹H NMR (DMSO-d⁶, 300 MHz): δ (ppm) 8.02 (ddd, J7.37, 1.50, Hz, 1H, H5or H8); 8.00 (dd, J7.37, 1.50, 0.47 Hz, 1H, H8or H5); 7.86 (d, J 8.63 Hz, 1H, H17); 7.84 (td, J7.46, 7.46, 1.50 Hz, 1H, H6 or H7); 7.74 (td, J7.46, 7.46, Hz, 1H, H7or H6); 7.73 (d, J2.16 Hz, 1H, H14); 7.55-7.49 (m, 6H, H16; Ph); 5.78 (s, 1H, H11); 4.35 (d, J 13.00 Hz, 1H, H19); 4.21 (d, J13.00 Hz, 1H, H19'). ¹³C NMR (DMSO-d⁶, 75 MHz): δ (ppm) 183.9, 179.3, 171.3, 134.6, 134.5, 134.4, 134.0, 133.9, 132.1, 132.0, 131.7, 131.2, 130.3, 128.9, 128.8, 128.6, 127.8, 125.6, 125.2, 109.4, 55.1, 49.7. UV-Vis (CHCl₃) λ/nm, log ε: 275 (4.14), 334 (3.40), 400 (3.10).

3-[N-(alyl)amino-2-hydroxybenzyl]-2-hydroxy-1,4naphthoquinone (HL6)

From 0.41 mL of alylamine and 0.63 mL of 2-hydroxybenzaldehyde. Yield: 1.475 g, 88%; mp 164-165 ºC. Anal. Calc. for C₂₀H₁₇NO₄: C, 71.63; H, 5.11; N, 4.18%. Found: C, 71.50; H, 5.05; N, 4.27%. IR (KBr) V_max/cm^-1: 3255 (O-H), 3072 (C-H), 2980 (C-H), 2950 (C-H), 1678 (C=O), 1593 (C=C), 1561 (δ N-H), 1277 (C-O). ¹H NMR (DMSO-d⁶, 300 MHz): δ(ppm) 8.02 (ddd, J7.61, 1.35, 0.47 Hz, 1H, H5or H8); 7.97 (dd, J7.53, 1.41, 0.47 Hz, 1H, H8or H5); 7.83 (td, J7.47, 7.47, 1.41 Hz, 1H, H6or H7); 7.73 (td, J7.47, 7.47, 1.35 Hz, 1H, H7or H6); 7.42 (dd, J7.69, 1.65 Hz, 1H, H14 or H17); 7.26 (td, J8.06, 8.06, 1.65 Hz, 1H, H16or H15); 6.96 (dd, J8.06, 0.99 Hz, 1H, H17or H14); 6.86 (td, J7.69, 7.69, 0.99 Hz, 1H, H15or H16); 6.10-5.95 (m, 2H, H20); 5.85 (s, H11); 5.50-5.42 (m, 2H, H21); 3.74-3.60 (m, 2H, H19). ¹³C NMR (DMSO-d⁶, 75 MHz): δ(ppm) 184.1, 179.6, 171.5, 155.5, 134.6, 133.9, 131.6, 131.1, 129.8, 129.4, 128.6, 125.5, 125.2, 123.8, 122.0, 119.1, 116.1, 110.1, 53.3, 47.9. UV-Vis (DMSO) λ/nm, log ε: 278 (4.34), 452 (3.33).

3-[N-(n-butyl)amino-2-hydroxybenzyl]-2-hydroxy-1,4naphthoquinone (HL7)

From 0.54 mL of butylamine and 0.63 mL of 2-hydroxybenzaldehyde. Yield: 1.405 g, 80%; mp 137-138 ºC (with dec.). Anal. Calc. for C₂₁H₂₁NO₄: C, 71.78; H, 6.02; N, 3.99%. Found: C, 71.12; H, 6.03; N, 3.92%. IR (KBr) V_max/ cm^-1: 3233 (O-H), 3069 (C-H), 2959 (C-H), 2875 (C-H), 1681 (C=O), 1590 (C=C), 1528 (δ N-H), 1275 (C-O). ¹H NMR (DMSO-d⁶, 300 MHz): δ(ppm) 8.02 (ddd, J7.66, 1.35, 0.50 Hz, 1H, H5or H8); 7.98 (ddd, J7.47; 1.35; 0.50 Hz, 1H, H8or H5); 7.84 (td, J7.47, 7.47, 1.35 Hz, 1H, H6or H7); 7.73 (td, J7.47, 7.47, 1,35 Hz, 1H, H7or H8); 7.44 (dd, J7.74, 1.56 Hz, 1H, H14 or H17); 7.27 (td, J8.03, 8.03, 1.56 Hz, 1H, H16or H15); 6.99 (dd, J8.03, 1.05 Hz, 1H, H17or H14); 6.86 (td, J7.74, 7.74, 1.05 Hz, 1H, H15or H16); 5.86 (s, 1H, H11); 3.03 (t, J7.50, 7.50 Hz, 2H, H19); 1.79-1.66 (m, 2H, H20); 1.50-1.38 (m, 2H, H21); 0.96 (br t, J7.33, 7.33 Hz, 3H, H22). ¹³C NMR (DMSO-d⁶, 75 MHz): δ(ppm) 184.1, 179.6, 171.6, 155.4, 134.5, 133.9, 131.6, 131.2, 129.6, 128.7, 125.5, 125.2, 123.6, 119.1, 115.9, 109.9, 54.1, 45.7, 27.8, 19.4, 13.5. UV-Vis (DMSO) λ/nm, log ε: 277 (4.33), 450 (3.30).

3-[N-(benzyl)amino-2-hydroxybenzyl]-2-hydroxy-1,4naphthoquinone (HL8)

From 0.60 mL of benzylamine and 0.63 mL of 2-hydroxy-benzaldehyde. Yield: 1.792 g, 93%; mp 165166 ºC. Anal. Calc. for C₂₄H₁₉NO₄C, 74.79; H, 4.97; N, 3.63%. Found: C, 74.78; H, 4.90; N, 3.72%. IR (KBr) V_max/cm^-1: 3436 (O-H), 3067 (C-H), 2744 (C-H), 1681 (C=O), 1589 (C=C), 1516 (δ N-H), 1276 (C-O). ¹H NMR (DMSO-d⁶, 300 MHz): δ(ppm) 8.04 (ddd, J7.50, 1.36, 0.48 Hz, 1H, H5or H8); 7.99 (ddd, J7.50, 1.43, 0.48 Hz, 1H, H8or H5); 7.84 (td, J7.50, 7.50, 1.43 Hz, 1H, H6or H7); 7.73 (td, J7.50, 7.50, 1.36 Hz, 1H, H7or H6); 7.51-7.46 (m, 5H, Ph); 7.38 (dd, J 7.71, 1.62 Hz, 1H, H14or H17); 7.24 (td, J8.05; 8.05, 1.62 Hz, 1H, H16or H15); 6.94 (dd, J8.05, 1.00 Hz, 1H, H17or H14); 6.84 (td, J7.71, 7.71, 1.00 Hz, 1H, H15 or H16); 5.82 (s, 1H, H11); 4.26 (d, J13.09, 1H); 4.17 (d, J13.09, 1H). ¹³C NMR (DMSO-d⁶, 75 MHz): δ(ppm) 184.1, 179.7, 171.5, 155.6, 134.6, 133.9, 132.8, 131.7, 131.1, 130.0, 129.4, 128.9, 128.7, 128.5, 125.5, 125.2, 123.6, 119.0, 115.9, 110.0, 53.8, 49.4. UV-Vis (DMSO) λ/nm, log ε: 277 (4.29), 443 (3.25).

3-[N-(furfurylmethyl)amino-2-hydroxybenzyl]-2-hydroxy1,4-naphthoquinone (HL9)

From furfurylamine (0.49 mL) and 2-hydroxybenzaldehyde (0.63 mL). Yield: 1.653 g, 88%; mp 138 ºC. Anal. Calc. for C₂₄H₁₉NO₄.H₂O : C, 67.17; H, 4.87; N, 3.56%. Found: C, 68.94; H, 4.99; N, 3.69%. IR (KBr) V_max/cm^-1: 3234 (O-H), 2943 (C-H), 1683 (C=O), 1591 (C=C), 1551 (δ N-H), 1279 (C-O). ¹H NMR (DMSO-d⁶, 300 MHz): δ(ppm) 8.02 (br d, J7.61 Hz, 1H, H5or H8); 7.97 (br d, J 7.56 Hz, 1H, H8or H5); 7.83 (td, J7.61, 7.61, 1.20 Hz, 1H, H6or H7); 7.72 (td, J7.56, 7.56, 1.20 Hz, 1H, H7or H6); 7.51-7.46 (m, 5H, Ph); 7.32 (br d, J 7.89 Hz, 1H, H14or H17); 7.23 (br td, J7.89; 7.89, 1.62 Hz, 1H, H16or H15); 6.92 (br d, J7.89 Hz, 1H, H17 or H14); 6.82 (br t, J7.48 Hz, 1H, H15or H16); 5.78 (s, 1H, H11); 4.23 (d, J14.51, 1H); 4.17 (d, J14.51, 1H). ¹³C NMR (DMSO-d⁶, 75 MHz): δ(ppm) 184.2, 179.7, 171.4, 155.7, 143.9, 134.6, 133.9, 131.7, 131.2, 129.4, 128.6, 125.5, 125.3, 123.7, 119.2, 119.0, 116.0, 111.6, 111.0, 110.1, 53.9, 41.9. UV-Vis (CHCl₃) λ/nm, log ε: 272 (4.03), 305 (3.93), 366 (3.74).

3-[N-(n-butyl)amino-2-hydroxy-5-methyl-benzyl]-2hydroxy-1,4-naphthoquinone (HL10)

From butylamine (0.54 mL) and 2-hydroxy-5-methylbenzaldehyde (0.817 g). Yield: 1.279 g, 70%; mp 154155 ºC. Anal. Calc. for C₂₂H₂₃NO₄: C, 72.31; H, 6.34; N, 3.83. Found: C, 71.15; H, 6.28; N, 3.90%. IR (KBr) V_max/ cm^-1: 3065, 2959, 2870, 1686, 1591, 1551, 1508, 1477, 1432, 1374, 1278. ¹H NMR (DMSO-d⁶, 300 MHz): δ(ppm) 8.02 (ddd, J7.67; 1.30; 0.40 Hz, 1H, H5or H8); 7.98 (ddd, J7.54; 1.37; 0.40 Hz, 1H, H8or H5): 7.84 (td, J7.54; 7.54; 1.30 Hz, 1H, H6or H7); 7.73 (td, J7.47; 7.47; 1.37 Hz, 1H, H7or H6); 7.25 (d, J1.94 Hz, 1H, H17): 7.07 (dd, J8.22, 1.94 Hz, 1H, H15); 6.87 (d, J8.22 Hz, H14); 5.81 (s, 1H, H11); 3.00 (t, J7.55, 7.55 Hz, 2H, H19); 2.25 (s, 3H, CH₃); 1.77-1.65 (m, 2H, H20); 1.48-1.34 (m, 2H, H21); 0.96 (t, J7.34, 7.34 Hz, 3H, H22). ¹³C NMR (DMSO-d⁶, 75 MHz): δ(ppm) 184.1, 179.5, 171.4, 153.1, 134.5, 133.9, 131.6, 131.1, 129.8, 128.6, 127.5, 125.5, 125.2, 123.5; 115.9; 110.2; 54.1; 45.7; 27.9; 20.3; 19.4; 13.5. UV-Vis (CHCl₃) λ/nm, log ε: 274 (4.10), 325 (3.63), 373 (3.38), 470 (2.93).

3-[N-(benzyl)amino-2-hydroxy-5-methyl-benzyl]-2hydroxy-1,4-naphthoquinone (HL11)

From benzylamine (0.60 mL) and 2-hydroxy-5-methylbenzaldehyde (0.817 g). Yield: 1.338 g, 67%; mp 147148 ºC. Anal. Calc. for C₂₅H₂₁NO₄: C, 75.17; H, 5.30; N, 3.51. Found: C, 74.54; H, 5.36; N, 3.51%. IR (KBr) V_max/ cm^-1:3266; 3065; 2957; 2862; 1686; 1590; 1539; 1274; 1221. ¹H NMR (DMSO-d⁶, 300 MHz): δ(ppm) 8.04 (ddd, J7.61, 1.39, 0.43 Hz, 1H, H5or H8); 7.99 (ddd, J7.54, 1.41, 0.43 Hz, 1H, H8or H5); 7.84 (td, J7.43, 7.43, 1.41 Hz, 1H, H6or H7); 7.73 (td, J7.43, 7.43, 1.39 Hz, 1H, H7or H6); 7.50-7.47 (m, 5H, Ph); 7.20 (d, J2.03 Hz, 1H, H17); 7.04 (dd, J8.16, 2.03 Hz, 1H, H15); 6.83 (d, J8.16 Hz, 1H, H14); 5.77 (s, H11); 4.24 (d, J13.13 Hz, 1H, H19); 4.16 (d, J13.13 Hz, 1H, H19'); 2.23 (s, 3H, CH₃). ¹³C NMR (DMSO-d⁶, 75 MHz): δ(ppm) 184.1, 179.9, 171.5, 153.2, 134.6, 133.9, 132.8, 131.7, 131.2, 130.1, 129.9, 128.9, 128.7, 128.6, 127.6, 125.6, 125.3, 123.5, 116.0, 110.2, 54.0, 49.5, 20.4. UV-Vis (CHCl₃) λ/nm, log ε: 250 (4.46), 310 (4.15), 382 (3.90).

3-[N-(n-butyl)amino-2-hydroxy-5-bromo-benzyl]-2hydroxy-1,4-naphthoquinone (HL12)

From butylamine (0.54 mL) and 2-hydroxy-5-bromobenzaldehyde (1.206 g). Yield: 1.316 g, 61%; mp 166167 ºC. Anal. Calc. for C₂₁H₂₀BrNO₄: C, 58.62; H, 4.68; N, 3.26. Found: C, 58.74; H, 4.54; N, 3.42%. IR (KBr) V_max/cm^-1: 3202; 2961; 2934; 2867; 1679; 1591; 1524; 1273; 1229. ¹H NMR (DMSO-d⁶, 300 MHz: δ(ppm) 8.04 (d, J 7.20 Hz, 1H, H5or H8); 7.98 (d, J7.20 Hz, 1H, H8or H5); 7.84 (t, J7.20, 7.20 Hz, 1H, H6or H7); 7.73 (t, J7.20, 7.20 Hz, 1H, H7or H6); 7.64 (d, J2.15 Hz, 1H, H17); 7.42 (dd, J8.58, 2.15 Hz, 1H, H15); 6.94 (d, J8.58 Hz, 1H, H14); 5.82 (s, 1H, H11); 3.05-2.95 (m, 2H, H19); 1.78-1.65 (m, 2H, H20); 1.49-1.35 (m, 2H, H21); 0.96 (t, J7.04, 7.04 Hz, 3H, H22). ¹³C NMR (DMSO-d⁶, 75 MHz): δ(ppm) 183.9, 179.5, 171.1, 154.8, 134.4, 133.9, 132.0, 131.6, 131.2, 130.9, 126.5, 125.6, 125.2, 118.2, 110.2, 109.8, 53.4, 45.8, 27.8, 19.3, 13.6. UV-Vis (CHCl₃) λ/nm, log ε: 273 (3.12), 354 (2.27), 457 (2.05).

3-[N-(benzyl)amino-2-hydroxy-5-bromo-benzyl]-2hydroxy-1,4-naphthoquinone (HL13)

From benzylamine (0.60 mL) and 2-hydroxy-5-bromobenzaldehyde (1.206 g). Yield: 1.736 g, 75%; mp 160161 ºC. Anal. Calc. for C₂₄H₁₈BrNO₄: C, 62.08; H, 3.91; N, 3.02. Found: C, 62,09; H, 4.01; N, 3.11%. IR (KBr) V_max/cm^-1: 3224 (O-H), 3068 (C-H), 2958 (C-H), 1688 (C=O), 1589 (C=C), 1539 (δ N-H), 1275 (C-O). ¹H NMR (DMSO-d⁶, 300 MHz): δ(ppm) 8.04 (dd, J7.62, 1.36 Hz, 1H, H5or H8); 7.99 (d, J 7.54, 1.41 Hz, 1H, H8or H5); 7.85 (td, J7.44, 7.44, 1.41 Hz, 1H, H6or H7); 7.74 (td, J7.44, 7.44, 1.36 Hz, 1H, H7or H6); 7.54 (d, J2.37 Hz, 1H, H17); 7.50-7.46 (m, 5H, Ph); 7.39 (dd, J8.63, 2.37 Hz, 1H, H15); 6.89 (d, J8.63 Hz, 1H, H14); 5.77 (s, 1H, H11); 4.24 (d, J 13.15 Hz, 1H, H19); 4.15 (d, J13.15 Hz, 1H, H19'). ¹³C NMR (DMSO-d⁶, 75 MHz) δ(ppm): 184.5, 180.3, 172.1, 155.6, 135.1, 134.5, 133.3, 132.5, 132.3, 131.8, 131.5, 130.7, 129.5, 129.3, 129.1, 127.1, 126.2, 125.9, 118.8, 110.7, 110.4, 53.9, 50.1. UV-Vis (CHCl₃) λ/nm, log ε: 271 (3.10), 377 (2.60), 434 (1.94).

Synthesis of complexes [Cu(L)₂]1-13fromHL1-HL13,respectively

To a suspension of 1 mmol of the ligand in 10 mL MeOH, was added a solution of CuCl₂^.2H₂O (83 mg, 0.5 mmol) in 2 mL MeOH. After addition of Et₃N (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).

[Cu( L1 )₂] ( 1 )

From 335 mg of HL1. Yield: 337 mg, 92%; mp 198 ºC. Slow evaporation of a CHCl₃ solution yielded brown crystals suitable for X-ray diffraction analysis. Anal. calc. for C₄₂H₄₀N₂O₆Cu^.2H₂O: C, 65.65; H, 5.77; N, 3.65%. Found: C, 64.69; H, 5.76; N, 3.54%. IR (KBr) V_max/cm^-1: 3468 (O-H), 3281 (N-H), 3064 (C-H), 2958 (C-H), 2928 (C-H), 1674 (C=O), 1621 (C=C), 1591 (C=C), 1273 (CO). UV-Vis (CHCl₃) λ/nm, log ε: 315 (4.13), 425 (3.78), 538 (2.25).

[Cu( L2 )₂] ( 2 )

From 380 mg of HL2. Yield: 280 mg, 68%; mp 208 ºC. Slow evaporation of the complex solution in a methanol/ isopropanol mixture yielded brown crystals suitable for X-ray diffraction analysis. Anal. calc. for C₄₂H₃₈N₄O₁₀Cu^.2H₂O. H₂O: C, 60.03; H, 4.80; N, 6.67%. Found: C, 59.21; H, 4.70; N, 6.82%. IR (KBr) V_max/cm^-1: 3460 (O-H), 3273 (N-H), 3077 (C-H), 2956 (C-H), 2930 (C-H), 1675 (C=O), 1617 (C=C), 1592 (C=C), 1546 (δ N-H), 1272 (C-O). UV-Vis (CHCl₃) λ/nm, log ε: 298 (4.64), 324 (4.05), 415 (3.72), 536 (2.17).

[Cu( L3 )₂] ( 3 )

From 414 mg of HL3. Yield: 280 mg, 63%; mp 176177 ºC. Anal. Calc. for C₄₈H₃₄N₄O₁₀Cu^.2H₂O^.2HO: C, 62.23; H, 4.13; N, 6.05%. Found: C, 61.19; H, 4.12; N, 6.16%. IR (KBr) V_max/cm^-1: 3459 (O-H), 3151 (N-H), 2933 (C-H), 1671 (C=O), 1591 (C=C), 1547 (d N-H), 1274 (C-O). UV-Vis (CHCl₃) λ/nm, log ε: 298 (4.51), 319 (4.16), 413 (3.70), 556 (2.17).

[Cu( L4 )₂] ( 4 )

From 404 mg of HL4. Yield: 322 mg, 74%; mp 171 ºC. Anal. Calc. for C₄₂H₃₆CI₄N₂O₁₀Cu^.0.5H₂O: C, 57.38; H, 4.24; N, 3.19%. Found: C, 56.49; H, 4.28; N, 3.28%. IR (KBr) V_max/cm^-1: 3446 (O-H); 3273 (N-H), 2959 (C-H), 2931 (CH); 2871 (C-H), 1678 (C=O), 1625 (C=C), 1591 (C=C), 1548 (d N-H), 1275 (C-O). UV-Vis (CHCl₃) λ/nm, log ε: 289 (4.40), 318 (3.97), 412 (3.70), 530 (2.22).

[Cu( L5 )₂] ( 5 )

From 438 mg of HL5. Yield: 276 mg, 59%; mp 172173 ºC. Anal. Calc. for C₄₈H₃₂Cl₄N₂O₆Cu.H₂O: C, 60.30; H, 3.58; N, 2.93%. Found: C, 58.87; H, 3.56; N, 3.02%. IR (KBr) V_max/cm^-1: 3428 (O-H), 3266 (N-H), 3066 (C-H), 2926 (C-H), 1676 (C=O), 1625 (C=C), 1591 (C=C), 1549 (δ N-H), 1277 (C-O). UV-Vis (CHCl₃) λ/nm, log ε: 298 (4.43), 320 (4.03), 411 (3.73), 553 (2.16).

[Cu( L6 )₂] ( 6 )

From 335 mg of HL6. Yield: 296 mg, 81%; mp > 310 ºC. Anal. Calc. for C₄₀H₃₂N₂O₈Cu^.0.5H₂O: C, 64.81; H, 4.49; N, 3.78%. Found: C, 63.29; H, 4.60; N, 4.01%. IR (KBr) V_max/cm^-1: 3478 (O-H), 3153 (N-H), 1670 (C=O), 1593 (C=O), 1534 (d N-H), 1279 (C-O). UV-Vis (DMSO) λ/nm, log ε: 276 (4.65), 454 (3.63).

[Cu( L7 )₂] ( 7 )

From 351 mg of HL7. Yield: 306 mg, 80%; mp 201 ºC. Slow evaporation of the complex solution in THF/dioxane yielded brown crystals suitable for X-ray diffraction analysis. Anal. Calc. for C₄₂H₄₀N₂O₈Cu^.2H₂O: C, 63.03; H, 5.54; N, 3.50%. Found: C, 62.40; H, 5.63; N, 3.41%. IR (KBr) V_max/cm^-1: 3260 (N-H), 2954 (C-H), 2866 (C-H), 1683 (C=O), 1593 (C=C), 1530 (δ N-H), 1276 (C-O). UV-Vis (DMSO) λ/nm, log ε: 277 (4.58), 455 (3.58).

[Cu( L8 )₂] ( 8 )

From 385 mg of 8. Yield: 287 mg, 69%; mp 187 ºC. Anal. Calc. for C₄₈H₃₆N₂O₈Cu^.2H₂O: C, 66.39; H, 4.64; N, 3.23%. Found: C, 66.50; H, 4.60; N, 3.31%. IR (KBr) V_max/ cm^-1: 3474 (O-H), 3274 (N-H), 3067 (C-H), 2944 (C-H), 1668 (C=O), 1591 (C=C), 1533 (δ N-H), 1279 (C-O). UV-Vis (DMSO) λ/nm, log ε: 276 (4.63), 449 (3.64).

[Cu( L9 )₂] ( 9 )

From 375 mg of 9. Yield: 260 mg, 64%; mp > 310 ºC. Anal. Calc. for C₄₄H₃₂N₂O₁₀Cu^.1.5H₂O: C, 62.97; H, 4.20; N, 3.34%. Found: C, 61.93; H, 4.01; N, 3.25%. IR (KBr) V_max/cm^-1: 3478 (O-H), 3245 (N-H), 1669 (C=O), 1591 (C=C), 1534 (δ N-H), 1281 (C-O). UV-Vis (DMSO) λ/ nm, log ε: 276 (4.65), 449 (3.61).

[Cu( L10 )₂], ( 10 )

From 365 mg of 10. Yield: 277 mg, 70%; mp 218 ºC. Anal. Calc. for C₄₄H₄₄N₂O₈Cu: C, 66.69; H, 5.60; N, 3.54%. Found: C, 66.27; H, 5.64; N, 3.67%. IR (KBr) V_max/cm^-1: 3254 (N-H), 2954 (C-H), 2868 (C-H), 1693 (C=O), 1590 (C-C), 1500 (δ N-H), 1277 (C-O). UV-Vis (DMSO) λ/nm, log ε: 277 (4.64), 452 (3.69).

[Cu( L11 )₂] ( 11 )

From 399 mg of 11. Yield: 330 mg, 77%; mp 196 ºC. Anal. Calc. for C₅₀H₄₀N₂O₈Cu.H₂O: C, 68.37; H, 4.82; N, 3.19%. Found: C, 67.21; H, 4.68; N, 3.29%. IR (KBr) V_max/ cm^-1: 3400 (O-H); 3251 (N-H); 3028 (C-H), 2918 (C-H), 1674 (C=O), 1590 (C=C), 1535 (δ N-H), 1279 (C-O). UV-Vis (DMSO) λ/nm, log ε: 276 (4.63), 447 (3.63).

[Cu( L12 )₂] ( 12 )

From 430 mg of HL12. Yield: 152 mg, 33%; mp 186187 ºC. Anal. Calc. for C₄₂H₃₈Br₂N₂O₈Cu^.1.5H₂O: C, 53.15; H, 4.35; N, 2.95%. Found: C, 51.75; H, 4.22; N, 3.10%. IR (KBr) V_max/cm^-1: 3422 (O-H), 3253 (N-H), 2959 (C-H), 2933 (C-H), 2869 (C-H), 1675 (C=O), 1589 (C=C), 1532 (δ N-H), 1277 (C-O). UV-Vis (CHCl₃) λ/nm, log ε: 276 (4.59), 347 (3.58), 428 (3.58).

[Cu( L13 )₂] ( 13 )

From 464 mg of HL13. Yield: 294 mg, 60%; mp 202203 ºC. Anal. Calc. for C₄₈H₃₄Br₂N₂O₈Cu^.2H₂O: C, 56.18; H, 3.73; N, 2.73%. Found: C, 55.77; H, 3.67; N, 2.80%. IR (KBr) V_max/cm^-1: 3454 (O-H), 3213 (N-H), 2946 (C-H), 1673 (C=O), 1591 (C=C), 1531 (δ N-H), 1279 (C-O). UV-Vis (DMSO) λ/nm, log ε: 276 (4.65), 450 (3.65).

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 PHICHI³² and DIRAX³³ programs, respectively, catching reflections with random orientation in hkl planes. Intensities were corrected by Lorentz polarization and absorption with the SADABS program.³⁴ The structure was solved by Direct Methods using the SHELXS-97 program.³⁵ The anisotropy parameters of non-H atoms were refined with the SHELXL-97 program.³⁶ In 1, 2and 7the aromatic, methyl, methyne and methylene H-atoms were geometrically included in the refinement. Aromatic carbons were refined with U_iso(H) = 1.2 Ueq Csp², methylene carbons with U_iso(H) = 1.2 Ueq Csp³, methine carbons with U_iso(H) = 1.2 Ueq Csp³ and methyl carbons with U_iso(H) = 1.5 Ueq Csp³. The hydrogen atom of the water molecules, N-H amine in the three compounds and O-H hydroxyl for 2were localized experimentally in the Fourier map. For 2the 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 SQUEEZE³⁷ tool contained in the WinGX³⁸ 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. The solution and refinement of 1suggested the presence of disordered C21carbon of the butyl moiety. X-ray data are listed in Table 1 and ORTEP-3³⁹ for Windows was used to draw the Figures.

Antibacterial assays

The antibacterial evaluation was performed with Gram-positive (Bacillus cereusATCC 33019, Bacillus subtilisATCC 6633, Enterococcus faecalisATCC 29212, Staphylococcus aureusATCC 25923) and Gram-negative (Escherichia coliATCC 25922, Klebsiella pneumoniaeATCC 700603, Pseudomonas aeruginosaATCC 27853) bacteria as test-microorganisms.

Minimum inhibitory concentration (MIC) was determined by the microdilution broth technique according to the M7-A6 document.⁴⁰ The assays were carried in 96well tissue culture microplates filled with Mueller Hinton broth (100 µL perwell).⁴¹ The inoculum suspension of each strain was prepared in Mueller Hinton broth (108 bacteria cells permL, 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 perwell) was applied into the microplates which were incubated at 37 ºC overnight. An aqueous solution of p-iodonitrotetrazolium violet (p-INT) (Sigma) (20 µL) was added⁴² and the microplates were incubated once more for 1-2 hours at 37 ºC. The MIC 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.

Results and Discussion

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-HL7and HL11 HL13are obtained in a pure state, but HL8-HL10need 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 ¹H spectra of compounds HL1-HL13exhibit peaks due to the four naphthoquinone aromatic hydrogens H5H8 that appear in the δ 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 δ 0.9 to 3.1 ppm region as multiplets (H19-H21) or triplets (methyl H22); in the case of the benzyl group; the CH₂ hydrogens H19 appear as a doublet around δ 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 ¹H × ¹H (COSY experiments), Jvalues and multiplicity. All expected resonances were observed in the ¹³C 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 δ 171.

Complexes 1-13(Figure 2) were obtained by addition of trietylamine to a methanolic suspension of the ligand and CuCl₂ ^.2H₂O (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, 2and 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, 2and 7. The molecular structures of 1, 2and 7are 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-N₂O₂ environment. Bond angles O(1)Cu-N(1) (and β parameters:⁴³ 88.43(9), 1, 91.33(10), 2, and 90.67(9)º, 7) and Cu-N_amine and Cu-O_phenolate distances indicate slightly distorted square-planar coordination of the complexes. The Cu-N(2.023(2),1, 2.023(3),2, and 1.996(2) Å,7) and Cu-Odistances (1.945(2),1, 1.928(2),2, and 1.942(2) Å,7) are in the normal range when compared to those observed for other copper(II) complexes containing the same coordination environment.^44-46 In the same molecule, the two ligands have different absolute configurations at the chiral C11carbon. In the structures of compounds 1and 2the 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.^47,48

The packing arrangement of 1exhibits molecules of 1and 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 1and 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 PLATON³⁷ (Table 3) and agree with the literature.³⁷

The crystalline structure of complex 7shows a 2D self-arrangement governed by O-H...O and C-H...O classical and non classical hydrogen bonds, respectively. The lattice grows in the [100] direction through O6ⁱⁱ-H6Aⁱⁱ...O3, O6ⁱⁱH6Bⁱⁱ...O2ⁱⁱⁱand O4ⁱ-H4ⁱ...O6ⁱⁱinteractions and in the [001] direction, through C18-H18B...O5ⁱⁱⁱinteractions, as shown in Figure 8 [symmetry code for 7: (i) = -1-x, -y, 1-z; (ii) = -x, 1-y, 1-z; (iii) = -1+x, y, z, Table 4]. The network in the [100] direction is built around the water molecules and in the [001] direction, viathe dioxane interactions. In addition, intramolecular interactions viaN-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.⁴⁵ The geometric parameters for these interactions are listed in Table 4. All hydrogen bonds were calculated using PLATON³⁷ 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.⁴⁹ The new bands in the 3200-3300 cm^-1 range can be assigned to ν_N-H.^48-50 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 Cu²⁺ center. Several weak bands observed in the 2850-3100 cm^-1 range are attributed to aliphatic and aromatic C-H groups.⁴⁹ The 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.⁴⁸ The strong ν_C-O band around 1280 cm^-1 was attributed to the naphthoquinonato group.^47,51

EPR Spectra

The EPR spectra of complexes 1-13were measured in the solid state at liquid nitrogen temperature. The Hamiltonian parameters (Table 5) obtained in 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.⁵² All complexes, except for 2and 12, show values of g_||/A_|| between 110 and 122 thus confirming the square planar environment around the Cu²⁺ centre⁵³ as established for compounds 1, 2and 7by X-ray analysis. The experimental values, g_||> g⊥, also indicate that the unpaired electron is predominantly in the dx²-y² orbital, which gives ²B_1g as the ground state. The very low parallel and perpendicular components of the hyperfine coupling constant for complexes 2and 12(not resolved in the spectra) has been explained by considering a mixture of the Cu²⁺ dz² and dx²-y² orbitals as the ground state. It is found that a 10% mixture of dx²-y² and dz² results in a 20% reduction in dipolar anisotropy.⁵⁴

The relatively low g_|| values of the complexes are consistent with a N₂O₂ environment around the Cu²⁺ ions.⁵² As expected, the ligand field strength depends mainly on the nature of the R⁴ substituent on the nitrogen, the highest ligand field being observed for complexes of ligands containing R⁴ = butyl, independently of the nature of R² and R³. The presence of the hydroxyl group (R¹=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 1-5and 12, recorded in CHCl₃ 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.⁵⁵ The band around 290 nm was not altered by coordination as it corresponds to π-π* transitions of the naphthoquinone ring.^48-50. The d-d band appears between 530-550 nm and can be attributed to a ²A_1g←²B_1g transition which supports the square planar geometry for the complexes in solution.⁵⁰

The spectra of complexes 6-11and 13were recorded in DMSO solution due to their low solubility in CHCl₃. 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 Cu²⁺ ion.

Antibacterial activity

The antibacterial activity of Mannich bases HL1-HL13and 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 are reported in Table 6 where the MIC values are expressed in µmol L^-1. Cloramphenicol was used as a positive control in all tests. Compounds 2-hydroxy-1,4-naphthoquinone (A), 2-amino-3-hydroxy-1,4-naphthquinone (B) and lapachol (C) were also tested for comparison.

Two Mannich bases and four complexes exhibited similar or higher activity than Chloramphenicol against three strains of bacteria (B. subtilis, E. coli (EC) and S. aureus), whereas the other compounds, including 2-hydroxy-1,4-naphthoquinone (A, Entry 14), 2-amino3-hydroxy-1,4-naphthquinone (B, Entry 15) and lapachol (CEntry 16) only inhibited bacterial growth above 200 µmol L^-1; CuCl₂ .2H₂O only inhibit bacterial growth above 3000 µmol L^-1. Mannich bases HL11(Entry 20) and HL13(Entry 13) strongly inhibited the growth of E. coli(at 20 and 40 µmol L^-1.

Mannich bases HL11 (Entry 20) and HL13 (Entry 13) strongly inhibited the growth of E. coli (at 20 and 40 µmol L-1, i.e. 8 and 22 µg mL^-1, respectively) and S. aureus(at 40 µmol L^-1, i.e.19 and 22 µg mL^-1, respectively). As shown in Figure 1, these compounds contain a 2-hydroxyphenyl group (R¹ = OH) which is substituted at the 5-position with R³ = Me (HL11) or Br (HL13), respectively, and R⁴ = Bn. Thus, the nature of the R³ substituent appears to be of lesser importance than that of the lateral chain R⁴, considering that compound HL10(Entry 10) with R³ = Me (similar to HL11) and R⁴ = Bu is much less active than HL11against 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 HL11to 180 µmol L^-1 for 11).

Improvement of the activity of HL10and H12that 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. faecalisand S. aureusabove 90 µmol L^-1 (Entry 29). Complexes 10and 12were formed from Mannich bases HL10and HL12that only differed from the very active ones, HL11and HL13with respect to the R⁴ group, butyl, instead of benzyl.

The effect of metal complexation on naphthoquinone antimicrobial agents has been discussed in the literature.⁵⁶ Although metal chelation of the anion of 5-hydroxy-1,4naphthoquinone (juglone) has resulted in complexes with similar antibacterial effect⁵⁷ or higher antibacterial activity, e.g.against Bacillus sspand S. aureus, than juglone, complexation of the anions of a series of 5-amino-8hydroxy-1,4-naphthoquinones with M²⁺ (M = Ni, Co, Fe, Cu and Cr) resulted in reduced bacterial activity or lack of inhibition effect.⁵⁵ Considering 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

Inconclusion,ofthethirteennovelaminonaphthoquinones HL1-HL13synthesized from lawsone, viathe Mannich reaction, and their respective copper(II) complexes [Cu(L1)₂] - [Cu(L13)₂], those containing a 2-hydroxyphenyl group (R¹ = OH) which is substituted at the 5-position, either with R³ = Me or Br, and with R⁴ = benzyl or butyl groups have strongly inhibited the growth especially of Escherichia coliand 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.

Supplementary Information

Supplementary data are available free of charge at http:// jbcs.sbq.org.br, as PDF file.

Crystallographic data for the structural analysis of the three complexes have been deposited with the Cambridge Crystallographic Data Center, CCDC No. 703509 (1), 703510 (2) and 703511 (7). Copies of this information may be obtained free of charge from The Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: [phone]; e-mail: [e-mail]).

Acknowledgments

We thank CNPq, CAPES, FINEP, PRONEX-FAPERJ and FAPERJ for financial support, and the X-ray diffraction laboratory (LDRX) of Universidade Federal Fluminense for data collection.

Received: March 3, 2009

Web Release Date: April 24, 2009

Supplementary Information

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Present Address: Universidade Federal do Espírito Santo, Centro Universitário Norte do Espírito Santo, Rua Humberto de Almeida Franklin, 257, Universitário, 29933-480 São Mateus-ES, Brazil

Publication Dates

• Publication in this collection
  10 June 2009
• Date of issue
  2009

History

• Accepted
  24 Apr 2009
• Received
  03 Mar 2009