Design, Synthesis, Characterisation and Biological Evaluation of Novel 7- Fluoroquinolone Derivatives

Heterocyclic compounds have different type of pharmacological properties in which fluoroquinolones are most widely useful as antibacterial agents. Several quinolones are related in clinical world, which are Ciprofloxacin, Pefloxacin, Levofloxacin, Norfloxacin, Enoxacin, Sparfloxacin, Tosufloxacin etc., which is widely used in the treatment of Conjunctivitis and other ophthalmological diseases like gonorrhea and syphilis.

Literature survey of quinolone reveals that the several research workers [1, 2, 4, 5] have studied antibacterial activity of quinolones substituted with piperazine at C-7 and carboxylic acid group at C-3 position. Structure activity relationship study of fluoroquinolones and biological importance of phenyl thiourea provided scope to synthesize the thioureido amide of fluoroquinolones and study the effect of different functional groups on antibacterial activity. It was therefore envisaged that a new series of 1-ethyl-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-chloro-3- quinoline carboxylic acid substituted with phenyl thiourea at C-3 and piperazine, piperadine, morpholine, 1H-imidazole and 2-aminoethane thiol at C-7 position were synthesized and screened for antibacterial and antifungal activities.

1. 1. 7- FLUOROQUINOLONE:

"7-Fluoroquinolone" likely refers to a fluoroquinolone antibiotic with a fluorine atom at the 7th position of the quinolone ring system — though this is a bit unusual, since most common fluoroquinolones have a fluorine at the 6-position, not the 7-position. Fluoroquinolones are a class of broad-spectrum synthetic antibiotics that are effective against many Gram-negative and some Gram-positive bacteria. They work by inhibiting bacterial DNA gyrase and topoisomerase IV, enzymes needed for DNA replication.

Fig: 1 Fluoroquinolone

1. 2. DRUG DESIGN:

Drug design is a fundamental aspect of modern medicinal chemistry, aiming to develop new therapeutic agents through a rational and targeted approach. Rather than relying solely on empirical screening, drug design involves understanding the biological targets—typically enzymes, receptors, or nucleic acids at the molecular level, and designing compounds that can interact with these targets to elicit a desired pharmacological response. This process integrates knowledge from disciplines such as chemistry, biology, pharmacology, and computational modeling. Advances in molecular docking, structure-activity relationships (SAR), and high-throughput screening have significantly accelerated the discovery of lead compounds with improved potency, selectivity, and safety profiles. By optimizing interactions between a drug and its target, drug design enhances therapeutic efficacy while minimizing off-target effects, paving the way for the development of more effective and safer medications.

2. SCHEME OF WORK:

Fig: 2 Scheme of Work

2.1 PROCEDURE:

In the present study, 3-chloro-4-fluoroaniline was heated with diethyl ethoxy methylene malonate to form 7-chloro-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylate (1) which was hydrolyzed  to corresponding carboxylic acid (2). Substitution at N-1 position with aliphatic and aromatic moieties in the presence of anhydrous potassium carbonate formed 7-chloro-1-ethyl-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (3) respectively. Further treated with acid chloride to form carbonyl chloride (4). This acid chloride reacted with phenyl thiourea to give 3 [7- chloro - 1 - ethyl - cyclopropy l- 6- fluoro -1,4- dihydro -4- oxoquinolin-3yl) carbony] -1- phenyl thiourea (5). Further substitution at C-7 position with various heterocyclic and aliphatic moieties to enhance the broad-spectrum antimicrobial activity.

2.2 SYNTHESIS SCHEME:

The starting materials (5) for the synthesis of the title compounds have been previously published [1, 2]. A mixture of 3-[7-chloro-1-ethyl/ cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinolin-3yl)carbony]-1-phenyl thiourea (0.01 mol) Pyridine (10 ml), Triethylamine (3 ml) and different heterocyclic compounds like [(Piperazine (0.05mol), Piperadine (0.05 mol), Morpholine (0.05 mol), Imidazole (0.05 mol), 2-aminoethane thiol (0.05 mol)] was taken in a double neck round bottom flask.

The mixture was irradiated under microwave for 6 mins at power (455 watts). After completion of the reaction, the reaction mixture was cooled to room temperature .The mixture was poured into crushed ice and neutralized with dilute hydrochloric acid .The solid product was filtered and dried. The solid recrystallized from dimethyl formamide (scheme-I).

3. MATERIALS AND METHODS:

3.1 CHARACTERIZATION:

The Melting points were taken in open capillary tube and are uncorrected. The IR spectra of the compounds were recorded on JASCO - FTIR spectrometer 460 with KBr pellets. ¹H-NMR spectra were recorded on JEOL GSX 400 spectrometer. The chemical shifts are reported as parts per million downfield from Tetra methyl silane (TMS). Mass spectra were recorded on Shimadzu GC MS QP 5000. Microanalyses for C, H, N were performed in Heraeus CHN Rapid Analyzer. All the compounds gave satisfactory chemical analyses (91.3%). The purity of the compounds was checked by TLC on precoated SiO₂ gel (HF₂₅₄, 200 mesh) aluminum plates (E Merck) using n-hexane : ethyl acetate (8:2) as mobile phase and visualized by iodine vapors.

Table: 1 Characterization

3.2 IN VITRO & IN VIVO ANTIMICROBIAL ACTIVITY:

The antibacterial activity of the synthesized compounds was tested against S. aureus, S. epidermidis, E. coli and K. pneumoniae using nutrient agar medium (Hi-Media Laboratories, India).The antifungal activity of the compounds was tested against A. niger and A. fumigatus using Sabouraud dextrose agar medium (Hi-Media Laboratories, India).

The In-vitro Antibacterial (Staphylococcus aureus ATCC 9144, Staphylococcus epidermidis ATCC 155, Escherichia coli ATCC 25922 and Klebsiella pneumoniae ATCC 11298) and antifungal (Aspergillus niger ATCC9029 and Aspergillus fumigatus ATCC 46645) activities of the compounds were evaluated by paper disc diffusion method [3, 6, 12, 15]. The minimum inhibitory concentrations (MIC) of the compounds were also determined by agar streak dilution method. The In-vivo Antibacterial activity of the compounds against S. aureus and E.coli was also evaluated by mouse protection test. Acute oral toxicity test was performed for all the synthesized compounds as per Organization of Economic Co-operation and Development (OECD) guidelines.

3.2.1 Animals:

Inbred male swiss albino mice (20-25 g) were used for the In-vivo antibacterial activity. They were kept in colony cages at 25 ± 2ºC and relative humidity 45-55% under 12 h light and dark cycle. The animals were fed with standard animal feed and water ad libitum. All the animals were acclimatized for a week before use. The test compounds and the standard drugs were administered orally by gavage in the form of a suspension (1% carboxy methylcellulose as vehicle). Acute oral toxicity test was performed for all the synthesized compounds according to organization of economic co-operation and development (OECD) guidelines. All the animal experimentation was performed as per the recommendations and the protocols of the institutional animals’ ethics committee.

3.2.2 Acute Oral Toxicity:

Acute oral toxicity [17] was performed as per OECD-423 guidelines (acute toxic class method). Swiss albino mice (n = 3) of either sex selected by random sampling technique was used for the study. The animals were kept fasting for 3 - 4 h providing only water ad libitum, after which the test compounds (suspended in olive oil) were administered orally at the dose level of 5 mg kg^-1 by intra gastric tube and observed for 3 days. If mortality was observed in two to three animals, then the dose administered was assigned as toxic dose. If mortality was observed in one animal, then the same dose was repeated again to confirm the toxic dose. In the present study, mortality was not observed and the procedure was repeated for further higher doses such as 50, 300 and 2000 mg kg^-1.

3.2.3 Mouse Protection Test:

The In-vivo antibacterial activity [7] of the compounds against E. coli was determined in male swiss albino mice (n =/6). The mice were infected intraperitoneally with a suspension (10⁵ cfu mL^-1) containing an amount of S. aureus and E. coli greater than its LD₁₀₀. The mice were treated orally with a specified amount of the synthesized compound 1 and 4 h after infection. ED₅₀ values were calculated by extrapolation among survival rate in each group after a week. The ED₅₀ values represent the total dose of the compound (mg kg^-1) required to protect 50% of the mice from an experimentally induced lethal systemic infection of S. aureus and E. coli.

3.2.4 Paper Disc Diffusion Method:

The sterilized [7, 9, 13] (autoclaved at 120 °C for 30 min) medium (40-50°C) was inoculated (1 mL/ 100 mL of medium) with the suspension (10⁵ cfu mL^-1) of the microorganism (matched to McFarland barium sulphate standard) and poured into a petridish to give a depth of 3-4 mm. The paper impregnated with the test compounds (200 µg mL^-1 in dimethyl formamide) was placed on the solidified medium. The plates were pre-incubated for 1 h at room temperature and incubated at 37 °C for 24 and 48 h for antibacterial and antifungal activities respectively. Ciprofloxacin (100 mg/ disc) and ketoconazole (100 mg/ disc) was used as standard for antibacterial and antifungal activities respectively. The observed zone of inhibition is presented in Table 4.

3.2.5 Minimum Inhibitory Concentration (MIC)

MIC [4, 8, 10] of the test compounds were determined by agar streak dilution method. A stock solution of the synthesized compound (100 µg mL^-1) in dimethyl formamide was prepared and graded quantities of the test compounds were incorporated in specified quantity of molten sterile agar (nutrient agar for antibacterial activity and Sabouraud dextrose agar medium for antifungal activity). A specified quantity of the medium (40 - 50 °C) containing the compound was poured into a petridish to give a depth of 3-4 mm and allowed to solidify. Suspension of the microorganism were prepared to contain approximately 10⁵ cfu mL^-1 and applied to plates with serially diluted compounds indimethyl formamide to be tested and incubated at 37 °C for 24 and 48 h for bacteria and fungi respectively. The MIC was considered to be the lowest concentration of the test substance exhibiting no visible growth of bacteria or fungi on the plate. The observed MIC is presented in Table 4.

4. RESULTS AND DISCUSSION:

4.1 PHYSICAL CHARACTERIZATION:

Table: 2 Physical Characterization

4.2 SPECTRUM CHARACTERIZATION:

Table: 3 Spectrum Characterization

Table: 4 Antimicrobial Activity of the Synthesized Compounds