A New Class of Dihaloquinolones Bearing N ’-Aldehydoglycosylhydrazides , Mercapto-1 , 2 , 4-triazole , Oxadiazoline and α-Amino Ester Precursors : Synthesis and Antimicrobial Activity

A reação das quinolonas 6-8 com hidrazina forneceu as hidrazidas 9-11 em rendimentos moderados. A condensação de 9 e 11 com CS 2 /KOH rendeu os sais de potássio 13 e 14, respectivamente, que espontaneamente forneceram as 3-(1,2,4-triazolil)-quinolonas 15 e 16, respectivamente, quando tratadas com hidrazina. A reação de 9 com CS 2 /KOH sob refluxo resultou na 3-(1,2,4-oxadiazolil)-quinolona 17. Alternativamente, 15 foi preparado a partir de 17. O derivado 12, obtido a partir de 10, forneceu o derivado α-amino ester 18 mediante reação com o éster etílico da glicina. O acoplamento de 10 com vários açúcares forneceu as N’-(aldeidoglicosil-quinolona-3il)carbohidrazidas 19a-e. Os novos compostos sintetizados foram avaliados quanto a sua atividade antibacteriana.


Introduction
The second generation of fluoroquinolones [1][2][3] such as norfloxacin 4 2, the modified analogue of nalidixic acid 5 1, ciprofloxacin 6 3, ofloxacin 7 4, and sparfloxacin 8 5 are known as a major class of antibacterial agents and widely used to treat patient with infections.In recent years, and due to the increasing of resistance of many infections by gram negative and gram positive bacteria to these quinolones, several studies 9 described various modifications in the quinolone ring, for example: substitution with different groups at aromatic ring, [10][11][12][13] replacement of the same ring by thiophene moiety, 13,14 introduction of amido group 14,15 at C-3, as well as substitution at N-1 by sugar [16][17][18][19] or acyclic moieties. 20evertheless, some quinolones cause injury to the chromosome of eukaryotic cells. 21,22These findings prompted us to optimize the substituent at C-3, by introduction of heterocyclic, α-amino acid ester precursors or N'-aldehydoglycosylcarbohydrazide moities to evaluate the effect of these groups on the antibacterial activity.It had been reported 23 that heterocycles such as oxadiazoles, thiadiazoles and mercaptotriazoles are themselves important chemotherapeutic agents and exhibit antitubercular, bacteriostatic, hypoglycemic, antiviral, antifungal, antithyroid, carcinostatic and strong herbicidal activity.Some reported mercaptotriazole derivatives showed potent activity 24 more than streptomycin against Candida albicans, whereas derivatives of 5-substituted 1,2,4-oxadiazole-2-thiones are known to possess interesting pharmacodynamic property and some have exhibited remarkable activity 25 against Mycobacterium tuberculosis.Recently, bactericidal and/or fungicidal or antimicrobial activity was reported for oxadiazolidinethiones. 26Furthermore, a number of 1,2,4-triazole derivatives are angiotensin II receptor antagonists.To the best of our knowledge, only two examples of quinolone 3hydrazides are reported: 1-phenyl-27 and 1-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrazides. 28

General procedures
Melting points are uncorrected.The 1 H-NMR and 13 C-NMR spectra were recorded on DRX 600, VRX 200 and UNITY 300 spectrometers with tetramethylsilane as an internal standard (δ scale in ppm and coupling constants in Hz).The signal assignments in the 1 H-NMR spectra were confirmed by selective proton decoupling or by COSY spectra.Heteronuclear assignments were verified by 1 H-13 C COSY or HMQC experiments.EI and FAB mass were measured on an MAT 312 mass spectrometer using 3nitrophenol (NBOH) or glycerol as matrix.

Results and Discussion
Hydrazides 9-11 were prepared in 67, 53 and 65% yield, respectively, as key intermediates for the synthesis of the target molecules from reaction of the quinolones 6-8 with the hydrated hydrazine at 23 °C for 72 h (Scheme 1).Condensation of the carboxylic acid hydrazides 9 and 11 with CS 2 in ethanolic KOH afforded the unseparable potassium 3-quinolonodithio-carbazates 13 and 14, respectively.These salts were cyclized, at refluxing temperature, with hydrazine followed by acidification with conc.HCl to furnish triazoles 15 and 16 in 60 and 83% yield, respectively.The 1,3,4-oxadiazole-2-thione derivative 17 was prepared from 9, following Young and Wood 29 in 58% yield.Alternatively, 17 could be converted with hydrazine into the triazole 15 in 57% yield.Treatment of 10 with with NaNO 2 in the presence of 2 mol L -1 HCl and AcOH mixture at -10 °C afforded the unstable carboxylic acid azide derivative 12, which was used immediately for the next step.Condensation of the azide derivative 12 with glycine ethyl ester in the presence of base at -10 °C for 1 h afforded the ethyl ester derivative 18 in 50% yield (Scheme 1).The structures of the newly compounds were confirmed by homo-and heteronuclear NMR spectroscopy methods and mass spectrometry.The proton spin systems of compounds 13-16 were elucidated from their DQF-COSY 30  Derivatization of the carboxylic group of the quinolone bases was extended next.The hydrazide key intermediate was used here for the synthesis of the 3-carbohydrazidesugar compounds as promising antibacterial agents.Thus, boiling of 10 with five aldehydosugars (D-arabinose, D-xylose, D-ribose, D-mannose and D-galactose) for 2-4 h afforded, after purification, the yellowish 3-carbohydrazide derivatives 19a-e in 86-98% yield (Scheme 2).The structures of these compounds were confirmed on the basis of their NMR spectra, which were characterized by the presence of a singlet in the region δ H 8.99 -8.78 (H-2) and two doublets at δ H 8.32 -8.24 with large coupling constant (J H8-F ~ 8.5 Hz), and at δ H 8.15 -8.11 with small coupling constant (J H5-F ~ 5.5 Hz), assigned to H-8 and H-5, respectively.The protons of the carbohydrate moiety were resolved, after exchanging the hydroxyl groups with D 2 O, and comparison of their coupling constants with those of the corresponding free sugars.The proton spin system of 19a,c was further confirmed from DFQ-COSY spectrum: H-1' of 19a appeared as doublet at δ H 4.99, while H-2' -H-5' in the region δ H 4.64 -3.46, and these protons correlated to the signals at δ C 151.9, 70.3, 73.3, 70.9 and 63.3 for C-1' -C-5', respectively.Similarly, both protons and carbons of 19b-d were identified, whereas the 13 C-NMR signals of the quinolone residue in 19a-e were analysed by comparison to those of the previously reported quinolone nucleosides. 32

Bioassay
Two factors influence the Minimal Inhibitory Concentration (MIC) of fluoroquinolones: the rate of penetration into bacterial cell and its inhibitory activity of DNA gyrase. 33Although, most of the studies proposed that a substituent at 7-position of the quinoline ring is related to the binding site with enzyme through electrostatic interactions, 34,35 our aim here was to study the influence of the structural change of the carboxylic group of the quinolone ring by N'-aldehydoglycosyl-carbohydrazides as well as a new heterocycle and a-amino acid ester groups on the antibacterial activity.The in vitro antimicrobial activity of compounds 15-18 and 19a-e was tested by using the Escherichia coli K 12 wild-type strain D10, a gram negative bacterium, and wild-type Bacillius subtilius, a gram-positive bacterium, as well as Staphylococcus aureus.10 4 Cells x mL -1 were incubated into LB medium containing the indicated amount of a given compond.After growth overnight at 37 °C, the optical density of the culture was determined.The minimal inhibitory concentrations (MIC) of 13-19 were determined by assaying the effect of each compound at concentrations of 0.1, 0.5, 1, 10, 50, 100, 200 and 500 mg x mL -1 , and none of them showed significant activity.The screening results of compounds 19a-e are summarized in Table 1, using ciprofloxacine (CPR), as standard, for comparison.
The above data showed that the newly prepared compounds have slightly or no activity against the mentioned organisms, except 19a which exhibited slight activity against E. coli.In conclusion, the substitution of the carboxylic group of the quinolones by 1,2,4-triazolyl, Scheme 2. Synthesis of N'-aldehydoglycosyl-dihaloquinolone-carbohydrazides 19a-e from the dihaloquinolone-carbohydrazide 10.

Table 1 .
In vitro antibacterial screening a Numbers indicates the MIC in µg x mL -1 of the cell cultures.1,3,4-oxadiazolyl,α-amino ester or hydrazide derivatives of aldehydosugars does not influence the antibacterial activity, since the new compounds are nearly inactive. a