Synthesis and Antibacterial Properties of New 8-Nitrofluoroquinolone Derivatives

The objective of this research was the preparation of new 8-nitrofluoroquinolone models and investigation of their antibacterial properties. The work initially involved large scale preparation of the synthon 7-chloro-1-cyclopropyl-6-fluoro-8-nitro-4oxo-1,4-dihydroquinoline-3-carboxylic acid (3), followed by introduction of substituted primary amine appendages at the C-7 position to give derivatives 9a-g, in which the amino group is appended to substituted benzenes or aromatic heterocycles, is part of a primary αamino acid or just a simple primary aliphatic amine. This nucleophilic aromatic substitution step was a very simple procedure since the 8-nitro group of the above synthon facilitated the addition of weak nucleophiles at C-7. All compounds prepared were fully identified and characterized using NMR, IR, EA and MS, and were consistent with expected structures. The prepared targets and the intermediates have shown interesting antibacterial activity against gram positive and/or gram negative strains. In particular, the p-toluidine, p-chloroaniline and aniline derivatives showed good activity against S. aureus with MIC range ≈ 2-5 μg/mL. In conclusion, more lipophilic groups seem to enhance activity against gram positive strains. Molecules 2007, 12 1241


Introduction
In recent years, the numbers of life threatening infections caused worldwide by multi-drug resistant Gram-positive and Gram negative pathogenic bacteria have reached an alarming level.The quinolone antibacterials constitute a major addition with a broad spectrum of in-vitro and in-vivo chemotherapeutic efficiency.A number of quinolines like ciprofloxacin (1), norfloxacin (2), levofloxacin and moxifloxacin are being marketed and many are nowadays in clinical trials [1].
In our search for potent fluoroquinolone derivatives, it has been found that although some were based on modifications on other positions, the most successful compounds developed were based on modifications at C-7, and it has been found that the spectrum and level of antibacterial activity is highly affected by the nature of the C-7 substituent group.In addition to the inhibition of DNA gyrase and cell permeability, the C-7 substituents have been proposed as the domain that interacts with the enzyme to further strengthen drug binding [2,3].In general, C-7 substitution should involve an electron donating group in order to increase electron density on the carbonyl oxygen at C-4 position of the standard structure of 4-quinolone-3-carboxylic acid (Figure 1) [2,3].Arylalkyl or haloalkyl substitution on the nitrogen at C-7 gives compounds with acceptable activity.Medium sized N-heterocyclic rings (5-and 6-membered) located at C-7 of the quinolone have contributed most significantly to their antibacterial activity.Linear substituents with one or two heteroatoms (usually N) were investigated and gave compounds that were not very effective.Groups larger than piperazine led to less active compounds [4,5,6].Methyl-, chloro-and acyclic aminogroups at C-7 (e.g.-NHNH 2 , -NHR, -NHCH 2 CH 2 NH 2 ) resulted in moderate to weak biological activity, compared to ciprofloxacin [5,7].
Although substitution on C-7 has been thoroughly investigated, not much information about (substituted aliphatic or aromatic) primary amines has been reported.Most synthesized 7-amino derivatives were appendages consisting of secondary amines, mostly as part of heterocyclic rings.This was thought to be due to the fact that primary amine (aliphatic or aromatic) might not exhibit biological activity and were therefore not worthy of investigation.Moreover, the weak nucleophilcity of the amine, especially in case of aromatic systems, toward C-7 and the harsh reaction conditions needed hindered real attempts to produce large scale active derivatives.Our literature survey revealed that compounds with substituted primary amine derivatives are also potent antimicrobial agents [8].On the basis of above mentioned findings, this work involves the synthesis of novel C-7 substituted derivatives of 1-cyclopropyl-6-fluoro-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3) having an 8-nitro substituent as an electron withdrawing group and the evaluation of the biological activity of the new derivatives (Figure 1).This synthon contains an electron withdrawing group (the 8-nitro group) ortho-to C-7, which is the target position for new substitutions.This nitro group at C-8 allows the increment of the nucleophilicity at position 7 and facilitates amino substitution at C-7 by nucleophilic aromatic substitution (addition-elimination) reactions.
Finally, methanolic HCl hydrolysis gave the target 7-chloro-1-cyclopropyl-6-fluoro-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3) in good yield (Scheme 1).This sequence was amenable to scale-up and fifty grams of synthon 3 were thus prepared and collected.The new modifications involved distillation of the reaction mixture upon preparation of compound 6.This step was carried out to remove excess thionyl chloride at an early stage.Purification of the intermediate 6 also did not require tedious purification of the final product using column chromatography, as carried out in previous reported procedures.The yield figures were significantly improved to about 91-95 %, while reported yields did not exceed 73 %.
This procedure involved dissolving compound 6 in methanol only (to prepare 7) without the need to use a mixture of expensive solvents (ethoxyethane).It was assumed that the previously reported solvent mixtures were used because compound 6 was not pure and a mixture of solvents was needed to dissolve all compounds in the sample.The new procedure developed produced a pure solid 7 which was collected as a white crude solid by filtering off the final precipitate.Washing with small amounts of ethanol was enough to produce the pure product 7 with significantly high yield (93 %).Preparation of ester 8 was reported to be a difficult step that needs column chromatography purification, yielding not more than 65 % in the best experiments.This work requires no chromatographic separation.The cyclization involved heating for a short time (2 h) but at lower temperature (85 °C instead of 160 °C).The reaction was terminated by pouring the reaction mixture onto crushed ice, and precipitate was collected and dried.The purification step in this work was carried out by simple cyclization step to produce the pure ester 8 in more than 89 % yield.The final hydrolysis step was carried out in acidic media to get pure acid 3 in 92 % yield.In conclusion, the proposed procedure gave significantly higher overall yields (above 95 %).It also uses cheaper and safer solvents, involves much simpler techniques and fewer steps, which ultimately save money and efforts.In addition, costly chromatographic techniques are avoided to further reduce the cost.

Introduction of primary amine appendages at C-7
Preparation of the novel quinolone compounds 9a-g was carried out employing a regiospecific nucleophilic aromatic substitution of the 7-chloro-1-cyclopropyl-6-fluoro-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3) with appropriate substituted primary amines (Scheme 2).This was based on introducing different groups regarding the type of substitution on the primary amine group (screening process), utilizing the new procedure which is facilitated by the existence of the C-8 nitro group on synthon 3.
The previous procedures to introduce nucleophiles at C-7 involved using DMF, DMSO, pyridine and heating at very high temperature [10].This involves harsh conditions, long times and produced low yields, due to difficulties in separation and purification.
The aqueous procedure involves adding three molar excess of the substituted primary amine to the synthon 3 in aqueous ethanol and 18 molar excess of the base (NaHCO 3 ).Stirring of the mixture for 2-5 days at moderate temperature (60-80 °C) furnished a yellow to orange crude mixture.After a third extraction process of the aqueous mixture at pH 3-4, the final product precipitated as bright yellow pure compound and in very high yield (greater than 80 %) with most derivatives.Scheme 2. Synthesis of compounds 9a-g from different amines.
The structures of the prepared fluoroquinolone derivatives 9a-g and intermediates were confirmed based on IR, MS, 1 H-and 13 C-NMR spectral data, which were fully consistent with proposed structures.Signal assignments to various proton and carbons were mostly determined following DEPT and 2D COSY, HMQC and HMBC experiments.It was clearly evident that H-5 in all derivatives, which resonate at around 8.0 ppm (d, 3 J H-F ≈ 13 Hz) showed a consistent doubling pattern in all compounds due to coupling with fluorine.This phenomenon was also clear in the neighboring carbons, according to their position.Long range correlations are observed between H-2 and each of C-8a, C-4 and 4a.Corresponding long range correlations are also observed between H-5 and its neighbor carbons.
All skeletal carbons of the fused benzene ring are recognizable by both their signal splitting arising from coupling with fluorine atom (different value of J for each carbon) and from long range coupling with surrounding protons.It was evident from the new broad signal at around 7 to 8 ppm for NH at C-7 that the primary amine constituent was introduced.The cyclopropyl methine proton (H-1´) experienced a downfield shift in all derivatives (3.7 ppm) probably due to de-shielding effect of the adjacent nitrogen at position 1.
Low-resolution mass analysis was carried out for some models, however, the available electron impact (EI) technique failed to detect the exact mass when the compounds analyzed as acids.Only the ester (8) showed the exact mass upon high resolution analysis.Those which showed the molecular ion at low resolution analysis are reported within experimental part.

Antibacterial activity
Evidence in the literature showed that the nature of the functional group at the C-7 position of the quinolone system has a great influence on the spectrum and extent of the antibacterial activity [2,3].It has been demonstrated by other workers that the activity against bacteria was related to the lipophilicity of the side chain at C-7 of the compounds as well as the lipophilicity of the substitution at N-1 [11].It is generally assumed that the more lipophilic quinolones should have best ability to penetrate the lipophilic wall of Gram positive bacteria and thus have better activity against these bacteria.Moreover, it has been reported that reducing the lipophilicity of the fluoroquinolone molecule resulted in urinary recovery of the unchanged compound at a higher ratio [12].
The pure synthesized compounds 9a-g (Scheme 2), side reaction products 10 and 11 (Scheme 3), synthon 3 and ester 8 (Scheme 1) were evaluated for in vitro antibacterial activity against E. coli and S. aureus.These microorganisms were chosen to represent species of Gram negative and Gram positive bacteria, respectively.The initial investigation by agar diffusion method (Table 1) show that all the tested compounds have some antimicrobial activity against either E. coli and/or S. aureus; except for the ester 8 that shows no activity against both types of bacteria.Compound 8 is an ester form of the synthon 3; it is expected for any fluoroquinolone ester not to have any in vitro activity but might have same in vivo activity due to its hydrolysis to the acid form [13].
MIC results have indicated that the full structure of the C-7 appendages played an important role in determination of the activity.It was evident that both lipophilicity and solubility have affected the activity of these compounds and consequently antimicrobial results.Compounds 3 and 9g showed the good antibacterial activity against both, Gram positive and Gram negative bacteria.Both compounds showed higher activity against S. aureus with MIC ≈ 0.97 and 1.2 µg/ml, respectively (Table 2), compared to their activity against E. coli with MIC ≈ 4.7 and 8.8 µg/mL, respectively.Reduction of activity upon introduction of the amino group is possibly due to the electronwithdrawing chloro group on the nucleus at C-7.Such an observation was also reported upon comparing different 6-fluoro-7-substitutions on the 1-ethyl-1,4-dihydro-4-oxo-6-flouroquinoline-3carboxylic acid nucleus [5].
In the same vein, the highly lipophilic groups as in p-toluidine derivative 9d, p-chloro derivative 9e and aniline 9f showed a strong activity against Gram positive bacteria with MIC ≈ 3.5 µg/mL, 4.0 µg/mL and 5 µg/mL, respectively (Table 2).It is evident by those results that addition of more lipophilic groups to the quinolones standard structure has shifted activity against gram positive bacteria since the ability to penetrate the lipophilic wall of Gram positive bacteria has increased.This finding does have precedent in the literature with other fluoroquinolones [12].
Moreover, such results would eliminate the outer layers of Gram negative bacteria as site of action.It is thus concluded that their mechanism of action is similar to other known fluoroquinolones which were reported to have their activity on DNA enzymes.Again, this is supported by previous literature reports of other fluoroquinolones [4,14].

Cytotoxicity on cancerous epithelial cells
Preliminary cytotoxicity studies were carried out for the 7 candidate compounds with MCF-7 cells, a human breast adenocarcinoma cell line, to test whether these compounds are toxic to epithelial cells or they would have a potential as anticancer agents.All compounds did not change the proliferation rate of the cells as compared to controls (cells incubated with media only).This would suggest that these derivatives are not toxic to epithelial cells.Further evaluation should be carried out for exact determination of the IC 50 .

Conclusions
In conclusion, this work has successfully introduced new substituted primary amine appendages at 8-nitro-fluoroquinolone nucleus utilizing new procedure developed within the course of this work.The fluoroquinolone derivatives were fully identified and characterized using NMR, IR, EA and MS.The antimicrobial properties of all pure compounds were evaluated against E. coli and S. aureus bacteria.Although the selected structures did not follow a specific pattern (structure), they were chosen to have different hydrophilic/lipophilic properties as lead models for further antibacterial investigation.
The results indicate clearly that all new compounds, in addition to the side products, have interesting antimicrobial activity ranging from weak to strong against both strains of bacteria.For the targeted compounds, it is concluded that more hydrophilic groups supported antimicrobial activity against Gram negative bacteria; while more lipophilic groups supported antimicrobial activity against Gram positive bacteria as in p-toluidine.This work is consistent with previous findings that an electron-releasing group is still required at C-7, and more lipophilic cyclic structures direct the activity against Gram positive bacteria.Finally, this work has opened the door for new C-7 appendages at 8nitro-fluoroquinolone systems that may produce new clinical antibacterial agents in the future on bulk scale.

Antibacterial screening:
All the chemical compounds were tested for antibacterial activity against human pathogens Gramnegative (E.coli.ATCC 8739) and Gram-positive bacteria (Staphylococcus aureus ATCC 6538).The minimal inhibitory concentration (MICs) of the chemical compounds assays were carried out as described by Foroumadi et al. [2,3], with minor modification.Ciprofloxacin was used as reference antibacterial agent.

a) Determination of inhibition zones (agar diffusion method):
A drop of bacteria was added to sterile nutrient agar (20 mL), poured into a plate (9 cm in diameter) and allowed to solidify to obtain the seeded agar.The final concentration of the microorganisms in the agar plate was 1-4 x 10 5 cfu.mL -1 (checked by viable counting in normal saline).Aliquots of 25 µL of the freshly prepared saturated solutions of the synthesized compounds (Table 1) were poured in wells (7 mm in diameter).Plates were then incubated at 37 °C for 24 h.The zones of inhibition were determined as the diameter of the zone of inhibition around the well solution for each compound at its saturated concentration (Table 1).Solvent (DMSO) was included in every experiment of determining zones of inhibition as a control to ensure that it has no effect on the bacterial growth.Each experiment was done in duplicate.

b) Determination of minimum inhibitory concentration, MIC, (serial dilution method):
Stock solutions were prepared by dissolving each pure compound (5 mg) in 5 mL of DMSO then 1 mL of the compound stock solution was added to nutrient broth (4 mL).Progressive twofold serial dilutions of the stock solutions were made in nutrient broth starting from 100 µg/mL concentrations in the first test tubes and ending with a concentration of 3.05 x 10 -3 µg/mL.
The standardization of bacterial test suspension was carried out according to the McFarland standard method as described by the National Committee for Clinical Laboratories Standard (NCCLS) (1993).One drop of bacterial suspension was added to the test tubes containing graded concentrations of test compounds to yield final consent-ration of 1-4 x 10 6 cfu•mL -1 .Test tubes were incubated at 37 °C for 24 h and were checked for turbidity.Each experiment was done in duplicate.
Control tests for each experiment were performed.Positive growth control was performed by adding one drop of each micro-organism suspensions to a test tube of the culture medium without the test compound.Negative growth control was also performed using un-inoculated tube of medium without the test compound.Both were incubated for 24 h at 37 °C for both types of bacteria following reported procedures.
Positive and negative controls were performed with DMSO at the same dilutions as in the experiment to ensure that it is incapable of inhibiting the growth of bacteria.Test tubes were incubated at 37 °C for 24 h and found to have no effect on microbial growth at tested concentrations.This procedure was modified from reported literature [2,3,16].

Cytotoxicity on cancerous epithelial cells
MCF-7 cells were trypsinizd, seeded in 96 well plates and incubated for 24 hours.The tested derivatives were diluted with RPMI 1640 cell culture media, added to the cells, and incubated at three concentrations of 10, 30, and 100 µg/mL.The cells were incubated with the compounds for 48 hours and sulphrodamine B assay was run afterwards.All tests were done in triplicates and repeated twice using two different passages.

Figure 1 .
Figure 1.General route for introducing substituted primary amine groups.

major 11, minor 10 3 11, major 10, minor + 11, minor 10, major +
which are mentioned in the experimental part and are identified by spectral techniques.Both products were isolated from different experiments and fully identified.Side products obtained with different amines (failed attempts).

Table 1 .
Zones of inhibition of some synthesized compounds (at saturated concentration) against E. coli and S. aureus.

Table 2 .
MIC for the prepared 8-nitrofluoroquinolones against E. coli and S. aureus.
* Note: ND means antibacterial activity was not detected.