Experimental
General
Melting points were determined on a Mel-Temp apparatus and are uncorrected. 1H-NMR and 13C-NMR spectra were recorded on a Bruker 300 MHz spectrometer with chemical shift values reported in δ units (ppm) relative to an internal standard (tetramethylsilane). Infrared data were obtained on a Perkin-Elmer 1600 series Fourier transform instrument as KBr pellets. Amino acids are abbreviated and designated following the rules of the IUPAC-IUB Commission of Biochemical Nomenclature (J. Biol. Chem., 1972, 247, 977). The abbreviations: HATU (N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methyl-methanaminium hexafluorophosphate N-oxide); Boc (t-butyloxy-carbonyl); Et3N (triethylamine) and DMF (N,N-dimethylformamide) were used throughout the manuscript. Follow up of the reactions and checking the homogeneity of the compounds were made by TLC on silica gel-protected aluminum sheets (Type 60 F254, Merck) and the spots were detected by exposure to UV-lamp at λ 254 nm for few seconds. Elemental analyses were performed in the Chemistry Department, Faculty of Science, Cairo, University.
General Procedure for the Reaction of N-Boc-L-Amino Acids with Benzoic Acid Hydrazide.
A mixture of N-Boc-L-amino acid (1 mmol), HATU (0.38 g, 1 mmol) and Et3N (0.28 mL, 2 mmol) was stirred at 0°C for 3 minutes in DMF (2 mL). Then benzoic acid hydrazide (3, 0.122 g, 1 mmol) was added. The reaction mixture was stirred at 0°C for 1 hour and left overnight at room temperature. The reaction mixture was diluted with ethyl acetate (80 mL) and the mixture was washed successively with 5% aqueous citric acid solution (2°10 mL), saturated sodium bicarbonate solution (2°10 mL) and saturated sodium chloride solution (2°10 mL). The organic layer was dried over anhydrous sodium sulphate, filtered and the solvent was removed in vacuo. The crude product was crystallized from methylene chloride-hexane. In the case of N-Boc L-(O-benzyl)-tyrosine (5e) and N-Boc-L-phenylalanie (5d) the reaction mixture was poured into ice water, filtered, washed with 5% aqueous citric acid solution, saturated sodium bicarbonate solution and finally with water, dried and recrystallized from methylene chloride-hexane.
Boc-valyl(N`-benzoyl)-hydrazide (5a): White crystals, 0.32 g (95.4 % yield); mp 156-157°C; IR: 3308, 3226 (NH), 1715 (C=O, urethane), 1689 (C=O, amide), 1668 (C=O, amide) cm-1; 1H-NMR (CDCl3): δ 0.87, 0.95 (2d, 6H, 2 CH3), 1.35 (s, 9H, 3 CH3), 1.88 (m, 1H, CH), 4.93 (d, 1H, CH), 7.20-7.77 (m, 6H, 1 NH, aromatic), 9.17, 9.34 (2 br.s, 2H, 2 NH); 13C-NMR (CDCl3): δ 19.61, 19.70, 26.40, 28.62, 50.49, 80.69, 127.40, 129.00, 129.71, 131.79, 132.70, 136.58, 156.00, 164.62, 169.01; Anal. Calcd for C17H25N3O4: C, 60.88; H, 7.51; N, 12.53. Found: C, 60.72; H, 7.68; N, 12.81.
Boc-(o-benzyl)-glutamyl(N`-benzoyl)-hydrazide (5b): White powder, 0.38 g (80 % yield); mp 126°C; IR: 3336, 3253 (NH), 1753 (C=O, ester), 1727 (C=O, urethane), 1686 (C=O, amide), 1663 (C=O, amide) cm-1; 1H-NMR (CDCl3): δ 1.37 (s, 9H, 3 CH3), 1.90, 2.05 (2m, 2H, CH2), 2.60, 2.71 (2m, 2H, CH2-CO), 4.50 (m, 1H, CH), 5.15 m, 2H, CH2-O), 7.20-7.81 (m, 11H, 1 NH, aromatic), 9.17, 9.34 (2 br.s, 2H, 2 NH); 13C-NMR (CDCl3): δ 24.96, 28.16, 28.67, 49.70, 66.94, 80.72, 127.79, 128.65, 128.94, 128.95, 129.02, 131.87, 132.69, 136.10, 136.16, 153.64, 165.20, 169.82, 173.20; Anal. Calcd for C24H29N3O6: C, 63.28; H, 6.42; N, 9.22. Found: C, 63.02; H, 6.21; N, 8.96.
Boc-leucyl(N`-benzoyl)-hydrazide (5c): White crystals, 0.26 g (74 % yield); mp 147°C; IR: 3318, 3234 (NH), 1726 (C=O, urethane), 1689 (C=O, amide), 1672 (C=O, amide) cm-1; 1H-NMR (DMSO-d6): δ 0.91 (d, 6H, 2 CH3), 1.38 (s, 9H, 3 CH3), 1.51 (m, 2H, CH2), 1.72 (m, 1H, CH), 4.12 (m, 1H, CH), 6.85 (d, 1H, 1 NH), 7.2-7.77 (m, 5H, aromatic), 10.10, 10.40 (2 br.s, 2H, 2 NH); 13C-NMR (DMSO-d6): δ 21.88, 23.00, 23.29, 24.47, 28.54, 51.64, 79.20, 126.00, 127.50, 131.65, 153.65, 156.11, 171.15; Anal. Calcd for C18H27N3O4: C, 61.87; H, 7.79; N, 12.03. Found: C, 62.05; H, 7.92; N, 12.26.
Boc-phenylalanyl(N`-benzoyl)-hydrazide (5d): White powder, 0.31 g (81 % yield); mp 168°C; IR: 3314, 3233 (NH), 1725 (C=O, urethane), 1692 (C=O, amide), 1673 (C=O, amide) cm-1; 1H-NMR (DMSO-d6): δ 1.30 (s, 9H, 3 CH3), 2.82, 3.08 (2m, 2H, CH2), 4.30 (m, 1H, CH), 7.04 (d, 1H, 1 NH), 7.18-7.92 (m, 10H, aromatic), 10.18, 10.29 (2 br.s, 2H, 2 NH); 13C-NMR (DMSO-d6): δ 28.50, 37.68, 54.85, 126.58, 127.80, 128.40, 128.81, 129.59, 132.18, 153.62, 164.9, 173.10; Anal. Calcd for C21H25N3O4: C, 65.78; H, 6.57; N, 10.96. Found: C, 65.98; H, 6.78; N, 10.71.
Boc-(o-benzyl)-tyrosyl(N`-benzoyl)-hydrazide (5e): White crystals, 0.47 g (96 % yield); mp 177°C; IR: 3322, 3245 (NH), 1726 (C=O, urethane), 1693 (C=O, amide), 1673 (C=O, amide) cm-1; 1H-NMR (DMSO-d6): δ 1.31 (s, 9H, 3 CH3), 2.85, 3.05 (2m, 2H, CH2), 4.25 (m, 1H, CH), 5.05 (br.s, 2H, CH2), 6.92-7.93 (m, 15H, aromatic, 1NH), 10.15, 10.46 (2s, 2H, 2 NH); 13C-NMR (DMSO-d6): δ 26.52, 32.01, 53.48, 68.50, 77.52, 112.81, 125.90, 126.02, 126.81, 128.69, 130.21, 135.6, 153.62, 155.04, 163.81, 170.00; Anal. Calcd for C28H31N3O5: C, 68.69; H, 6.38; N, 8.58. Found: C, 68.51; H, 6.15; N, 8.73.
General Procedure for the Reaction of N-Boc-L-Amino Acids with Nicotinic Acid Hydrazide.
A mixture of N-Boc-L-amino acid (1 mmol), HATU (0.38 g, 1 mmol) and Et3N (0.15 mL, 1 mmol) was stirred at 0°C for 3 minutes in DMF (2 mL). Then nicotinic acid hydrazide (4, 0.123 g, 1 mmol) was added. The reaction mixture was stirred at 0°C for 1 hour and left overnight at room temperature. The reaction mixture was diluted with methylene chloride (80 mL) and the mixture was washed with 5% aqueous citric acid solution (2°10 mL), saturated sodium bicarbonate solution (2°10 mL) and saturated sodium chloride solution (2°10 mL). The organic layer was dried over anhydrous sodium sulphate, filtered and the solvent was removed in vacuo. The crude product was crystallized from methylene chloride-hexane.
Boc-valyl(N`-nicotinoyl)-hydrazide (6a): White crystals, 0.28 g (83.3 % yield); mp 209°C; IR: 3344, 3242 (NH), 1726 (C=O, urethane), 1708 (C=O, amide), 1661 (C=O, amide) cm-1; 1H-NMR (DMSO-d6): δ 0.86, 0.94 (2d, 6H, 2 CH3), 1.39 (s, 9H, 3 CH3), 1.96 (m, 1H, CH), 3.93 (m, 1H, CH), 6.81 (d, 1H, 1 NH), 7.77 (d, 2H, aromatic), 8.75 (d, 2H, aromatic), 10.12, 10.68 (2 br.s, 2H, 2 NH); 13C-NMR (DMSO-d6): δ 18.7, 19.5, 28.6, 30.7, 58.7, 78.4, 121.7, 139.8, 150.7, 155.7, 164.1, 171.1; Anal. Calcd for C16H24N4O4: C, 57.13; H, 7.19; N, 16.66. Found: C, 57.34; H, 7.35; N, 16.41.
Boc-(o-benzyl)-glutamyl(N`-nicotinoyl)-hydrazide (6b): White powder, 0.42 g (92 % yield); mp 120°C; IR: 3334, 3243 (NH), 1740 (C=O, ester), 1727 (C=O, urethane), 1681 (C=O, amide) cm-1; 1H-NMR (DMSO-d6): δ 1.38 (s, 9H, 3 CH3), 1.84, 2.04 (2m, 2H, CH2), 2.32 (m, 2H, CH2-CO), 4.07 (m, 1H, CH), 5.14 m, 2H, CH2-O), 7.02 (m, 1H, 1 NH), 7.34 (br.s, 5H, aromatic), 7.77 (d, 2H, aromatic), 8.75 (d, 2H, aromatic), 10.13, 10.66 (2 br.s, 2H, 2 NH); 13C-NMR (DMSO-d6): δ 26.6, 28.3, 30.0, 53.6, 66.2, 78.7, 121.6, 128.1, 128.4, 128.5, 128.7, 136.3, 139.7, 150.8, 156.0, 164.3, 171.1, 172.6, 175.8; Anal. Calcd for C23H28N4O6: C, 60.52; H, 6.18; N, 12.27. Found: C, 60.37; H, 6.32; N, 12.45.
Boc-leucyl(N`- nicotinoyl)-hydrazide (6c): White crystals, 0.25 g (71.3 % yield); mp 210°C; IR: 3301, 3222 (NH), 1710 (C=O, urethane), 1662 (C=O, amide) cm-1; 1H-NMR (DMSO-d6): δ 0.91 (d, 6H, 2 CH3), 1.38 (s, 9H, 3 CH3), 1.53 (m, 2H, CH2), 1.72 (m, 1H, CH), 4.13 (m, 1H, CH), 6.85 (d, 1H, 1 NH), 7.77 (d, 2H, aromatic), 8.71 (d, 2H, aromatic), 10.10, 10.70 (2 br.s, 2H, 2 NH); 13C-NMR (DMSO-d6): δ 21.88, 23.00, 23.29, 24.47, 28.54, 51.64, 78.75, 121.64, 139.77, 150.48, 155.55, 163.97, 172.38; Anal. Calcd for C17H26N4O4: C, 58.27; H, 7.48; N, 15.99. Found: C, 58.02; H, 7.21; N, 16.26.
Boc-phenylalanyl(N`-nicotinoyl)-hydrazide (6d): White powder, 0.29 g (75.4 %) yield, mp 145°C; IR: 3309, 3234 (NH), 1726 (C=O, urethane), 1695 (C=O, amide), 1677 (C=O, amide) cm-1; 1H-NMR (DMSO-d6): δ 1.29 (s, 9H, 3 CH3), 2.82, 3.07 (2m, 2H, CH2), 4.31 (m, 1H, CH), 7.05-7.37 (m, 6H, aromatic, 1 NH), 7.81 (d, 2H, aromatic), 8.76 (d, 2H, aromatic), 10.33, 10.82 (2 br.s, 2H, 2 NH)); 13C-NMR (DMSO-d6): δ 28.12, 28.48, 37.82, 54.87, 78.4, 121.68, 126.62, 128.42, 129.57, 138.30, 139.70, 150.77, 155.67, 164.18, 171.70; Anal. Calcd for C20H24N4O4: C, 62.49; H, 6.29; N, 14.57. Found: C, 62.65; H, 6.50; N, 14.83.
Boc-(o-benzyl)-tyrosyl(N`-nicotinoyl)-hydrazide (6e): White crystals, 0.38 g (77.5 %) yield, mp 141°C; IR: 3311, 3237 (NH), 1727 (C=O, urethane), 1695 (C=O, amide), 1677 (C=O, amide) cm-1; 1H-NMR (DMSO-d6): δ 1.33 (s, 9H, 3 CH3), 2.83, 3.05 (2m, 2H, CH2), 4.26 (m, 1H, CH), 5.10 (br.s, 2H, CH2), 6.96-7.47 (m, 10H, aromatic, 1NH), 7.83 (d, 2H, aromatic), 8.78 (d, 2H, aromatic),10.36, 10.63 (2m, 2H, 2 NH); 13C-NMR (DMSO-d6): δ 28.20, 28.54, 37.03, 55.19, 69.50, 78.38, 114.77, 121.70, 127.99, 128.77, 130.63, 137.60, 140.06, 150.72, 155.68, 157.32, 164.08, 171.64; Anal. Calcd for C27H30N4O5: C, 66.11; H, 6.16; N, 11.42. Found: C, 65.82; H, 5.95; N, 11.59.
General Procedure for the Preparation of the Hydrochloride Salts 7 and 8.
5 or 6 (1 mmol) were dissolved in a mixture of methylene chloride (4 mL) and ether (4 mL). HCl gas was passed through the solution for 2 hours. A white precipitate that formed was filtered and washed with anhydrous ether. The crude product was recrystallized from methyl alcohol-anhydrous ether to give white crystals. The amino acid-(N`- nicotinoyl) hydrazide hydrochloride salts 8a-8e were obtained as hygroscopic salts.
General Procedure for the Preparation of the Cu Complexes.
A solution of Cu(NO3)2 (0.48 gm, 2 mmol) in methyl alcohol (2 mL) was added to a solution of 5, 6, 7 or 8 (1 mmol) in methyl alcohol (4 mL). A change was observed in the color of the solution. The Cu complexes were obtained as green crystals. The Cu complexes were later decomposed and their Cu content was analyzed by atomic absorption to determine the ratio of complex formation of Cu to ligand. The atomic absorption analysis showed the formation of complexes Cu:L in the ratio (1:1).
General Procedure for the Preparation of the Cd Complexes
A solution of Cd(CH3COO)2 (0.46 gm, 2 mmol) in methyl alcohol (2 mL) was added to a solution of 5, 6, 7 or 8 (1 mmol) in methyl alcohol (4 mL). The Cd complexes were obtained as white crystals. The Cd complexes were later decomposed and their Cd content was analyzed by atomic absorption to determine the ratio of complex formation of Cd to ligand. The atomic absorption analysis showed the formation of complexes Cd:L in the ratio (1:1).
In vitro antimicrobial activity
The microdilution susceptibility test in Müller-Hinton Broth (Oxoid) and Sabouraud Liquid Medium (Oxoid) were used for the determination of antibacterial and antifungal activity [
17,
18]. The utilized test organisms were:
Escherichia coli (
E. coli) ATCC 25922 as an example of Gram-negative bacteria,
Staphylococcus aureus (
S. aureus) ATCC 19433 as an example of Gram-positive bacteria and
Candida albicans (
C. albicans) as yeast-like fungi. Ampicillin trihydrate and clotrimazole were used as standard antibacterial and antifungal agents, respectively. Solutions of the test compounds, ampicillin trihydrate and clotrimazole were prepared in DMSO to a concentration of 1600 μg/mL. Twofold dilutions of the compounds were prepared (800, 400, … 6.25 μg/mL). Microorganism suspensions at 10
6 CFU/mL (Colony Forming Unit/mL) concentrations were inoculated to the corresponding wells. Plates were incubated at 36°C for 24 h to 48 h. The incubation chamber was kept sufficiently humid. At the end of the incubation period, the minimal inhibitory concentrations (MIC) were determined.