Synthesis of Fluoroquinolones Derivatives as Antimicrobial Agents

Introduction

Anti-infective agents played a major role in saving human lives. Among these agents are the fluoroquinolones class, which had risen to be highly appreciated, especially if there was microbial resistance against penicillin and macrolide. Fluoroquinolone pharmacophore (Figure 1) is well known to have antibacterial activity, and since 1980 there were many generation introduced to the market [1]. The fluoroquinolones were found to be effective to combat urinary tract infection [1] ideal in treating Neisseria gonorrhea [1] and highly effective to treat tuberculosis [2]. In addition, fluoroquinolone nucleus  are presented widely in biologically active compounds such as PDE 4 inhibitors [3], PIM kinase inhibitors [4], GSK β inhibitors [5].

Figure 1: Fluoroquinolone general formula. Click here to view figure

 

The encouraging  properties of fluoroquinolones such as; broad spectrum of activity, good oral bioavailability, good tissue penetrability and low incidence of adverse effect [6]  gave them high appreciation and encouraged researcher to investigate their usefulness as a source of potent antibacterial drugs.

In our previous work, we have described various modifications to the main structure of fluoroquinolone, including the introduction of different substituent’s at position 1 and 7 (Figure 1), and  in continuation for obtaining a new fluoroquinolone derivatives with excellent antibacterial activity, our team  reported synthon C (1,2,3and 4) as a potent antibacterial agents.

Materials and Methods

Experimental

Molecular modelling

Computational software 

The following software packages were utilized:

CS ChemDraw Ultra 6.0, Cambridge Soft Corp. (http:// www.cambridgesoft.Com), USA.2D Structure drawing was performed employing.

Discovery Studio 4.5 (DS 4.5) Standalone Applications, including docking Biovia ® (www.3ds.com), USA.

Accelrys Enterprise Platform Server (AEP) (www.accelrys.com), USA.

The crystal structures of gyrase enzyme were obtained from the protein data bank (http://www.rcsb.org/).

Molecular modelling studies

We docked our synthesized molecules using the Dock Ligands (LibDock) docking alogarithim implemented in the DS 4.5 into the binding pocket of the successful DNA gyraseenzyme namely : (PDB code: 5L3J, resolution 2.83 Ǻ).

Chemistry

General

All chemicals, reagents and solvents were of analytical/ synthetic grade that purchased from Sigma-Aldrich and Acros, Belgium, and used directly without further purification.Nuclear magnetic resonance spectra (NMR) were recorded on Bruker, Avance DPX-300 spectrometer.

High-resolution mass spectra (HRMS) were measured in positive or negative ion mode using electrospray ion trap (ESI) technique by collision induced dissociation on a Bruker APEX-4 (7 Tesla) instrument. Melting points (MP) were determined in open capillaries on a Stuart scientific electro-thermal melting point apparatus, and are uncorrected. Infra red (IR) spectra were recorded using Shimadzu 8400F FT-IR spectrophotometer (KBr discs). Microanalyses were performed using EuroVector Elemental Analyser, model (EA3000 A), Jordan University.

Synthesis of synthon (A)

The synthesisof 7-Chloro-1-Alkyl-6-fluoro-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid was previously described by our group [4, 7, 8]. Then adding a substitution at position 7 of (a and b) synthons, was prepared according to reported method [4, 8] provided the nitro derivatives synthon C (Scheme 1). The synthesized compounds gave satisfactory analytical and spectroscopic data in accordance with their depicted structures.

Scheme 1: Synthesis of flurouquinolone derivatives  Click here to view scheme

2-[(3-carboxy-1-(4-fluorophenyl)-6-fluoro-8-nitro-4-oxo-1,4-dihydroquinolin-7-yl)amino]terephthalic acid (C₁)

2-Aminoterphthalic acid (3.2 g, 18mmol) was reacted with synthon a (2.0g, 5.26mmol) and dimethyl sulfoxide (DMSO) 40ml and pyridine 10ml was heated at 70 °C for 10 days under reflux conditions. The mixture was left to cool, then pH was adjusted by 3.5N HCl dried to give the title compound as dark brown solid; Yield ≈ 1.6 g (60%); mp = 264 °C; 1H- NMR (300 MHz,DMSO-d6): δ 7.00 (d, 2H, H-2”,, H-6”), 7.02 (d, 2H, , H-3”,H-5”), 7.77-8.1 (m, 3H, ArH), 8.46 (d, 1H, H-5), 8.90 ( s, 1H, H-2), 9.24 (br s, 1H, NH),13.50 – 15.40 (2 br s, C₍₃₎COOH and C_(2′)COOH);IR (NaCl): ν 3417, 2067, 1701, 1643, 1265 cm1; Anal.Calcd. for C₂₄H₁₃F₂N₃O₉ (525.06), C, 54.87; H, 2.49; F, 7.23; N, 8.0; Found: C, 54.77,H, 2.43; N, 7.03.

2-[(3-carboxy-1-(4-fluorophenyl)-6-fluoro-8-nitro-4-oxo-1,4-dihydroquinolin-7-yl)amino] phthalic acid (C₂)

A stirred mixture of 2-Aminophthalic acid (1.6 g, 9mmol), synthon a (1.0g, 2.63 mmol) and DMSO 20ml and pyridine 5ml was heated at 70 °C for 10 days under reflux conditions. The same procedure carried out as with synthon C1was done and yielded   brownish solid compound ; Yield ≈ 0.8 g (60%); mp=260 °C; 1H- NMR (300MHz, DMSO-d6 ): δ 7.04-7.11(m, 4H, H-2”, H-3”, H-5”, H-6”), 7.94-8.09 (m, 3H, ArH), 8.11 (d, ³J_H-F = 9 Hz, 1H, H-5), 8.90 ( m, 1H, H-2), 9.15 (br s, 1H, NH); IR (NaCl): ν 3417, 2067, 1701, 1643, 1265 cm1; Anal. Calcd. for C₂₄H₁₃F₂N₃O₉ (525.06), C, 54.87; H, 2.49; F, 7.23; N, 8.0; Found: C, 54.77, H, 2.43; N, 7.03.

7-[(2-carboxy-1-phenylethyl)amino]-1-(4-fluorophenyl)-6-fluoro-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (C₃)

A stirred mixture of 3-phenyl β-alanine (4.06 g, 24mmol), synthon a(2.0 g, 5.2mmol) and sodium hydrogen carbonate (3.0 g, 36mmol) in 50 % aqueous ethanol (280mL) was heated at 70-80 °C for 6 days under reflux condition. The mixture was worked up as described for synthon C₁.  Yellow solid was collected; Yield 2.2 g (74.4%); mp: 280°C; 1H- NMR (300MHz, DMSO-d6 ): δ 2.76 (br m, 2H, CH₂-COOH ,4.31 (br s, 1H, CH-NH ), 7.10-7.21 (4H, P-fluorophenyl), 7.46-7.55 ( br m,5H, ArH), 7.86 (d, ³J_H-F = 14.1 Hz, 1H, H-5), 8.36 (s, 1H, H-2), 15.70(br s, 2H, 2 COOH); IR (NaCl): υ 3417, 2098, 1643, 1481, 1411, 1319, 1188, 1010 cm-1; Anal. calcd. for C₂₅H₁₇F₂N₃O₇ (509.1): C, 58.94; H, 3.36; N, 8.25. Found: C, 58.94; H, 3.36; N, 8.25.1.H, 3.36; N, 8.25.

7-(2-Carboxy-ethylamino)-1-(4-chloro-phenyl)-6-fluoro-8-nitro-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid .(C4)

quinoline-3-carboxylic acid .(C4)

A mixture of b-alanine (1.05 g, 11.5 mmol), Synthon b (1.0 g, 2.2mmol) and sodium hydrogen carbonate (3 g, 35.8 mmol) in 50% aqueous ethanol (120 mL) was heated for 6 days under reflux conditions. The product wasworked up as described for synthon C₁ and yielded a yellowish color. 1H NMR (300 MHz, DMSO- d6): δ2.28 (d, J = 8.1, 2H,CH₂-COOH), 3.66 (m, 2H, CH₂-NH), 7.51 (br t, J = 5.7 Hz, 1H, NHCH₂),7.51–7.54 (m, 2H, H-3′, H-5′), 7.61–7.67 (m, 2H, H-2′, H-6′),8.2 (H-5), 8.62 (s, 1H, H-2); HRMS (ESI,_ve): m/z [M_H]_448.04 C₁₉H₁₂ClFN₃O₇ requires 448.0353.

Biological Evaluation

Test microorganisms

Six pathogenic bacterial strains were used in the antimicrobial assays, four Gram-positive (Staphylococcus aureus ATCC29213, Staphylococcus saprophyticus ATCC^RBAA 750 ,Streptococcus agalactiae ATCC13813,Streptococcus pyogenes ATCC19615, and two Gram-negative (Pseudomonas aeruginosa ATCC27853,  Escherichia coli ATCC11775). Those pathogens were chosen based on their clinical and pharmacological importance. Antibacterial activities were evaluated by the agar well diffusion method as recommended by the Clinical and Laboratory Standards Institute (CLSI) [9-11] and the European Committee on Antimicrobial Susceptibility Testing (EUCAST)[12-13].

Measurement of antibacterial activity of the synthetic compounds

Preparation of synthetic compounds for microbiological assay

Stock solutions of 20 and 30 mg of each synthetic compound dissolved in 1mL of dimethyl sulfoxide (DMSO), as solvent. They were sterilized by filtration, and stored at 4^oC. The antimicrobial activity of the synthesized compounds was evaluated by the agar well diffusion method [14].

Determination of antibacterial activity by agar well diffusion method

All the synthetic compounds of different concentrations were screened for their antibacterial activities against the Staphylococcus  aureus, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pyogenes,  Pseudomonas aeruginosa and Escherichia coli by agar well  diffusion  assay.  Isolated pure colonies from fresh grown bacteria were transferred from the plates into sterile normal saline solution and vortexed to form bacterial homogenous suspensions. The turbidity was then adjusted to 0.5 McFarland standard units, and a volume of the inoculum was spread on the entire surface of agar. Then, a hole with a diameter of 6-8 mm was punched aseptically using a sterile cork borer, and a volume (20-100 μl) of the synthetic compound was introduced at the desired concentration into the well. Control experiments were carried out under similar conditions using amoxicillin (20 mg), ciprofloxacin (5 mg) and gentamicin (10 mg), as positive controls, and sterile distilled water as negative control. The zones of growth inhibition were measured in millimeters (mm) after 18-24 hours of incubation at 37^oC. The sensitivities of the microorganisms to the synthetic compounds were determined by measuring the sizes of inhibitory zones, and values <8 mm were considered as being not active against the tested bacterial strains [15].

Results and Discussion

The synthesized derivatives have been obtained in a good yield, and showed good antibacterial activity against both gram positive and gram negative bacteria (Table 1), and it’s well known that position 7 modifications can bring about the major changes in potency. Attachment of aromatic rings having an amino substitution results in improved activity and it also affects the pharmacokinetics of the compound [16].

Table 1: The antibacterial activity of prepared synthetic compounds and standard drugs against bacterial testing strains.

		Zone of inhibition (mm)
Compound	Concentration(mg/ml)	E.coli	P.aeruginosa	Staph.aureus	Staph. saprophyticus	Strep.  agalactiae	Strep. pyogenes
C1	20	17	22	23	12	15	23
	30	20	17	20	17	23	26
C2	20	18	–	23	16	14	25
	30	21	–	20	17	22	21
C3	20	20	20	19	18	20	23
	30	20	20	18	12	17	20
C4	20	22	22	25	–	–	–
	30	–	–	13	10	19	20
Amoxacillin (20 mg)(+ve)		31	20	20	25	19	30
Ciprofloxacin (5 mg)(+ve)		20	16	20	26	25	22
Sterile D.W.(-ve)		–	–	–	–	–	–

 

E.: Escherichia, Staph. Staphylo coccus, P.: Pseudomonas, Strep.: Streptococcus, (-):no inhibition, (+ve): positive control, (-ve): negative control, D.W.: distilled water.

The lipophilic substitution at position 1 led to  synthons with a good  activity against gram positive bacteria (Figure 2), and position 7 substitution with 2-aminophthalic acid and 3-aminoterphthalic acid imparted both gram negative and gram positive activity(Figure 2).

Figure 2: A. Diagram that shows the main interactions of compound C1 inside the bindingpocket of gyrase enzyme  (PDB code: 5L3J),  Click here to view figure

 

B. Diagram that shows the main interactions of compound C₂ inside the binding pocket of gyrase enzyme  (PDB code: 5L3J), C. Diagram that shows the main interactions of compound C₃  inside the binding pocket of gyrase enzyme  (PDB code: 5L3J).

Conclusions

In summary, we have efficiently synthesized a novel series of fluoroquinolone-modified analogues. Biological testing showed that some of the derivatives have good antibacterial activity in a series of primary assays. .

Acknowledgements

This project was funded by the deanship of research in Zarqa University/Jordan, (1/1/307). The authors thank the faculty of pharmacy in Zarqa University for their generous help and support.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper

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