Synthesis and Antimicrobial Evaluation of 3-Hydrazino-Naphthoquinones as Analogs of Lapachol

Vários derivados de 1,4-naftoquinonas contendo um grupo hidrazino como cadeia lateral foram sintetizados a partir do 3-diazo-naftaleno-1,2,4-triona e foram avaliados como potenciais agentes antimicrobianos. Os derivados naftoquinônicos 2-[N’-(1-acetil-2-oxo-propilideno)-hidrazino]-3hidroxi-[1,4]naftoquinona, 2-[(3-hidroxi-1,4-dioxo-1,4-diidro-naftaleno-2-il)-hidrazono]-3-oxobutirato de etila, 2-[(3-hidroxi-1,4-dioxo-1,4-diidro-naftaleno-2-il)-hidrazono]-3-oxo-butirato de t-butila, 3-hidroxi-2-[(di-O-isopropilideno-malonato)-hidrazino]-1,4-naftoquinona e 2-[(3-hidroxi1,4-dioxo-1,4-diidro-naftaleno-2-il)-hidrazono]-malonato de etila mostraram maior atividade antibacteriana, ao nível de teste preliminar em disco, que o lapachol (1), uma 1,4-naftoquinona muito conhecida pelas suas variadas atividades biológicas. Estudo sobre a concentração mínima inibitória (MIC) para o Staphylococcus aureus mostrou que 2-[(3-hidroxi-1,4-dioxo-1,4-diidronaftaleno-2-il)-hidrazono]-malonato de etila tem uma atividade duas vezes maior que 1. Da mesma forma, o estudo da densidade ótica em cultura de S. aureus com esta substância mostrou uma atividade similar à da vancomicina na concentração de 2xMIC.


Introduction
Quinones have been the subject of much interest for a number of years due to their various biological activities.In 1946 Wendel 1 showed that certain 2-hydroxy-3-alkyl-naphthoquinones inhibited the growth of Plasmodium.Further studies proved that the toxicity of naphthoquinones to Plasmodium sp. is due to interaction with the mitochondrial respiratory chain 2 .This observation led Fieser and collaborators to start an extensive search for new quinones 3 aiming to discover new drugs for malaria chemotherapy.These resulted in the discovery of 3-(8-cyclohexyl-octyl-2-hydroxy-1,4-naphthoquinone (menoctone), a potent inhibitor of NADH and succinatecytochrome reductases of Plasmodium lophurae 4 .The antibacterial and antiprotozoan activities of 2-hydroxy-3alkyl-1,4-naphthoquinones have been summarized by several authors 5 ,6 .Besides these biological activities, various other quinones posses activities against several types of cancer cells 7 (i.e.mitomycins, anthracyclines, etc.), virus 8 ,9 and fungi 10 .
Among the naphthoquinones, lapachol (1) and many heterocyclic derivatives have been investigated during the past years, mainly due to their antibacterial 11 , antifungal 12 and anticancer 13 activities.More recently, b-lapachone, an isomer of lapachol, was intensely investigated for clinical use in cancer chemotherapy 14 .
It is well known that there is a relationship between the side chain attached to 2-hydroxy-1,4-naphthoquinone and its toxic effects on several microorganisms 15 ,16 .Fieser and Richardson showed that, as the isoalkyl side chain of hydrolapachol is lengthened by the insertion of more methylene groups the activity against P. lophurae in the duck increased, reached a maximum with a C9-side chain, and then fell off 17 .Stern et al. found that the hydrogen peroxide formation of several 1,4-naphthoquinone derivatives on blood cell metabolism decreased with the increasing alkyl chain length 18 .As the alkyl side chain is an important factor for the activity of 2-hydroxy-1,4naphthoquinone, it seems appropriate to study the antimicrobial activity of naphthoquinone derivatives having new side chains linked to the quinone moiety.
Table 1 reports the inhibition zones (mm) of 2a, 2b, 2e, 1 and DMSO (solvent) determined for three species of Gram-positive and six Gram-negative bacteria, S. aureus, Staphylococcus epidermidis and the nonfermentable Gramnegative bacillus showed inhibition zones ranging from 9 to 24 mm, where significant response requires ³ 12 mm.Other Gram-negatives and Enterococcus faecalis had no significant result (halo < 12 mm).Therefore, our derivatives were active against four Gram-positive strains, but only against one Gram-negative strain.On the other hand, while 1 exhibited no positive response (halo < 12 mm), DMSO was completely inactive (halo = 0).
The determinations of the minimal inhibitory concentrations (MIC) in clinical S. aureus strains are reported in Table 2.These results indicated that 2e has a greater activity than 1, but it is less active than oxacillin and vancomycin, which are currently used as clinical antibiotics.

Effects on the culture's optical density
The effects on the culture's optical density at 560 nm for S aureus strains, were also studied.Cultures in the logarithimic growthphase were exposed to compound 2e and vancomycin, both at 2xMIC, during a 6 h treatment, with measurements being made at each hour.As is shown in Figure 2, 2e and vancomycin exhibited similar responses, despite the lower MIC of vancomycin.

Conclusion
Our study revealed a new class of naphthoquinone derivatives with high antibacterial activity.The antibacterial activity of 2a, 2b and 2e compared with 1 allow the conclusion that, at the level of the preliminary susceptibility testing in disk, they were active mainly against Grampositive bacteria.The minimal inhibitory concentration (MIC) determination on S. aureus for 2e showed an activity twofold greater than 1.However, 2e and 1 had MICs much greater than some clinical antibiotics.The optical density measurement for S. aureus indicated that 2e at 2xMIC had almost the same activity as vancomycin despite their different MICs.

Experimental
General NMR and HRMS confirmed the structures of the substances. 1 H and 13 C NMR spectra were recorded with a Varian Unity Plus VXR spectrometer operating at 300 and 75 MHz respectively, with tetramethylsilane as internal standard.Low resolution electron-impact mass spectra (12 eV) were obtained using a Hewlett Packard 5985 instrument and high resolution fast atom bombardment mass spectra (HRFABMS) were recorded in a 3-NBA matrix in the positive ion mode on a VG ZAB-E mass spectrometer.Infrared spectra were recorded on a Perkin-Elmer 783 spectrophotometer.Melting points were observed on a Reichert micro hotstage and are uncorrected.The solvents used were of analytical grade.Column chromatography was performed with silica gel 60 (Merck 70-230 mesh).Merck silica gel F254 (0.2 mm) was used for TLC plates, detection being carried out by spraying with a 25% aqueous solution of ammonium sulfate, followed by heating.Solvents were dried using the appropriate drying agents and then distilled directly before use 21 .Freshly purified samples were used for measurement of physical constants and spectral data.2-hydroxy-1,4-naphthoquinone was purchased from Aldrich Chemical Co and used without purification.

General procedure for preparing 2a-e
A mixture of the appropriate dicarbonyl compound (0.38 mmol), anhydrous K 2 CO 3 (57 mg, 0.4 mmol) in dry acetone (15 mL) was stirred for 15 min under a nitrogen atmosphere.To this mixture was added slowly through a syringe a solution of 6 (76.7 mg, 0.38 mmol) in dry acetone (5 mL) during 15 min.After stirring for 2 h the reaction was acidified with 5% (V/V) HCl (40 mL), the solid material was collected by filtration and crystallized from ethanol.

Microbial cultures growth conditions
Tested microorganisms included the following Grampositive bacteria: Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis, and for Gramnegative: Enterobacter, Escherichia coli, Klebsiella sp, nonfermentable bacillus, Proteus mirabilis and, Pseudomonas aeruginosa.All bacteria used in this study were isolated from patients at the University Hospital Antônio Pedro/UFF-RJ and grown (at 37 °C) in medium with peptone, yeast extract, sodium chloride and, dibasicsodium phosphate.Lorian disks (7 mm diameter) were soaked in 5 mg mL -1 of substances (2a-e) as solutions in dimethylsulfoxide (DMSO).Disks were put on an exponentially growing plated culture with appropriate dilution to 1.0x10 7 colony forming unit (CFU mL -1 ).The plates were then incubated for 24 h at 37 °C.The results were recorded by measuring the zones surrounding the disk.Control disks containing DMSO, ATCC 29.213 of S. aureus and the antibiotics oxacillin and vancomycin were used as controls in the Significant results: halo ³ 12 mm.

Minimal inhibitory concentration (MIC)
The hydrazino-quinone 2e was tested by using the two fold serial dilution in agar. 1 mL of DMSO/compound solution was added to 19 mL of the medium at 55-60 o C in order to reach a final concentration from 512 to 1 mg mL -1 .These solutions were plated and after medium solidification, a drop of S. aureus in the logarithmic phase (10 5 CFU mL -1 ) was added.The plates were incubated at 37 °C for 24 h, before being read.MIC was defined as the lowest compound concentration preventing visible bacterial growth.All strains were tested at least in duplicate in four separate experiments.MICs of the reference compound used in this study were similar to those found in other reports 23 ,24 .