Nalidixic acid

Traditional Synthesis Methods

The classical route involves cyclization of diethyl ethoxymethylenemalonate with 2-amino-6-picoline. This exothermic reaction proceeds at 90°C under mechanical stirring, forming the first intermediate, diethyl{[(6-methyl-2-pyridinyl)amino]methylene}propanedioate (CAS 13250-95-8). Subsequent thermal cyclization in phenyl ether at 200–230°C yields the naphthyridine core. However, traditional methods suffered from prolonged reaction times (12–24 hours) and byproducts like unreacted intermediates and dimeric impurities.

Optimized Industrial Preparation

Microwave-Assisted Synthesis

Recent studies demonstrate microwave irradiation’s efficacy in accelerating this compound derivatization. Hybrid pharmacophores combining nalidixate and chalcone motifs achieve 85–92% yields in 15–30 minutes vs. 6–8 hours conventionally. Microwave parameters (300 W, 100°C) minimize side reactions, enhancing scalability for preclinical batches.

Advanced Modifications and Complexations

Lanthanide complexes of this compound (e.g., La³⁺, Eu³⁺) exhibit enhanced bioactivity. Synthesis involves refluxing nalidixate sodium salts with lanthanide chlorides in 1:2 or 1:3 molar ratios. Europium(III) complexes, for instance, show 3.5-fold greater cytotoxicity against MDA-MB-231 breast cancer cells vs. free this compound.

Comparative Analysis of Methods

Traditional methods prioritize cost-effectiveness for bulk production, whereas microwave and complexation routes cater to niche applications. Table 2 contrasts key metrics:

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Quality Control and Impurity Profiling

High-performance liquid chromatography (HPLC) identifies critical impurities like des-ethyl this compound (0.2–0.5%) and dimeric byproducts (≤0.1%). Fourier-transform infrared spectroscopy (FTIR) confirms carboxylate coordination in metal complexes, with shifts from 1685 cm⁻¹ (free –COOH) to 1590 cm⁻¹ (deprotonated).

Clinical Applications

1.1 Treatment of Urinary Tract Infections

Nalidixic acid is primarily used for treating urinary tract infections (UTIs) caused by susceptible gram-negative microorganisms. It is particularly effective against Escherichia coli, Enterobacter species, and Proteus species. The drug operates effectively across a wide urinary pH range, making it suitable for various patient populations.

1.2 Systemic Infections

In addition to UTIs, this compound has been administered intravenously for systemic infections, especially those involving the urinary tract. Its bactericidal properties are most potent at concentrations between 50 to 200 µg/ml, effectively inhibiting DNA synthesis in bacteria.

Resistance Patterns

3.1 Emergence of Resistance

Resistance to this compound has been documented among various bacterial strains. A study indicated that approximately 38.5% of isolated strains from blood samples showed resistance to this compound. This resistance is concerning as it reflects broader trends in antibiotic resistance among hospital-associated organisms.

3.2 Monitoring Resistance

Monitoring this compound susceptibility is crucial in understanding resistance patterns to other antibiotics like ciprofloxacin. In regions such as the Indian Subcontinent, this compound susceptibility testing has served as an effective method for tracking ciprofloxacin resistance among Salmonella strains.

Case Studies and Research Findings

4.1 Clinical Case Reports

A notable case reported transient hyperglycemia and glycosuria following an overdose of this compound, highlighting potential side effects that clinicians should monitor during treatment.

4.2 Synergistic Effects with Other Antibiotics

Recent research has explored the synergistic effects of this compound when combined with tetracycline against multi-drug resistant strains of Acinetobacter baumannii and E. coli. This combination demonstrated enhanced efficacy in both in vitro and in vivo models, suggesting potential therapeutic strategies to combat resistant infections.

Data Tables

Comparative Analysis with Structurally and Mechanistically Similar Compounds

Synergistic and Antifungal Performance

• Synergy : this compound lacks synergistic effects with benfothiamine, indicating shared targets rather than complementary mechanisms .
• Antifungal Activity : this compound and its derivatives show negligible activity against fungi (e.g., Candida), with clotrimazole being 10–20x more potent .

Pharmacokinetic and Resistance Considerations

• Resistance: Resistance rates vary geographically (10% in Iran vs. 56% in other regions), linked to mutations in gyrA and plasmid-mediated quinolone resistance (PMQR) genes .
• Metabolism : this compound undergoes hepatic hydroxylation and glucuronidation, with renal clearance significantly reduced in patients with renal insufficiency .

Key Findings:

• Bactericidal Concentration : this compound exhibits optimal bactericidal activity against various gram-negative species at concentrations ranging from 50 to 200 µg/ml. At concentrations above this range, it tends to become bacteriostatic rather than bactericidal.
• Inhibition of Synthesis : Studies have shown that this compound inhibits deoxyribonucleic acid (DNA) synthesis without affecting ribonucleic acid (RNA) or protein synthesis at lower concentrations. However, at higher concentrations, it can inhibit both RNA and protein synthesis as well.

In Vitro Studies

Research has demonstrated this compound's effectiveness against several bacterial strains:

These findings highlight this compound's potency against enteric pathogens and its role in treating infections caused by these organisms.

Case Studies

• Urinary Tract Infections : A study involving patients with recurrent urinary tract infections revealed that this compound was effective in reducing bacterial load and improving clinical outcomes. The study reported a significant decrease in Escherichia coli counts post-treatment.
• Resistance Patterns : Recent data indicate a concerning trend in resistance among gram-negative bacteria to this compound. For instance, a retrospective analysis showed that 38.5% of isolated strains from blood cultures demonstrated resistance to this compound, suggesting an increasing prevalence of multi-drug-resistant organisms.

Comparative Studies with New Quinolones

This compound has been compared with newer quinolone derivatives like ciprofloxacin and norfloxacin:

Newer quinolones exhibit superior antibacterial activity due to their improved pharmacokinetic properties and broader spectrum of action against resistant strains.

Basic Research Questions

Q. How should researchers design experiments to assess the bactericidal effects of nalidixic acid on Gram-positive and Gram-negative bacteria?

• Methodological Answer : Use standardized bacterial strains (e.g., Bacillus subtilis for Gram-positive, Escherichia coli for Gram-negative) and measure minimum inhibitory concentrations (MICs) via broth microdilution. Include controls for solvent effects (e.g., DMSO). Monitor DNA synthesis inhibition using radiolabeled thymidine incorporation assays and correlate with bactericidal activity via time-kill curves. Morphological changes (e.g., cell elongation, Gram-negative staining in Gram-positive species) should be documented using microscopy .

Q. What experimental protocols ensure the chemical stability of this compound during long-term storage and in aqueous solutions?

• Methodological Answer : Store this compound in sealed, light-protected containers at 4°C to prevent degradation. For aqueous studies, prepare fresh solutions in pH-buffered solvents (e.g., phosphate buffer, pH 7.4) and avoid exposure to strong oxidizers. Validate stability via UV spectrophotometry (peak absorbance at ~260 nm) or HPLC over 24-hour periods .

Q. How can researchers quantify this compound in biological matrices like plasma while minimizing interference from metabolites?

• Methodological Answer : Employ high-performance liquid chromatography (HPLC) with UV detection (260 nm) or gas chromatography (GC) after derivatization. Validate methods using spiked plasma samples and compare retention times with known standards. For metabolite exclusion (e.g., hydroxymethylthis compound), confirm chromatographic separation via tandem mass spectrometry (MS/MS) .

Advanced Research Questions

Q. What mechanistic approaches elucidate the selective inhibition of bacterial DNA synthesis by this compound?

• Methodological Answer : Use subcellular systems (e.g., toluene-treated E. coli lacking DNA polymerase I) to isolate ATP-dependent DNA synthesis. Apply this compound at concentrations near the MIC (e.g., 25 µg/mL) and quantify DNA degradation via spectrophotometric or fluorometric assays. Compare sensitivity of membrane-bound DNA synthesizing systems (e.g., from B. subtilis) to identify target specificity .

Q. How can researchers resolve contradictions in this compound’s reported effects on eukaryotic systems (e.g., lifespan modulation)?

• Methodological Answer : Conduct microdissection assays in model eukaryotes (e.g., Caenorhabditis elegans) using controlled doses (e.g., 10–100 µM) and standardized viability metrics. Pair with genomic analysis (e.g., RNA sequencing) to distinguish direct DNA-targeting effects from off-pathway interactions. Validate findings against known lifespan-altering compounds (e.g., nicotinamide) .

Q. What strategies optimize the sensitivity and reproducibility of this compound quantification in complex biological environments?

• Methodological Answer : Combine HPLC with fluorescence detection (excitation 325 nm, emission 370 nm) for enhanced sensitivity. For reproducibility, adhere to metrological guidelines (e.g., ISO/IEC 17025) for calibration standards and inter-laboratory validation. Cross-validate results using alternative techniques like capillary electrophoresis or immunoassays .

Q. Experimental Design and Data Analysis

Q. How should researchers address variability in this compound’s antibacterial activity across bacterial strains?

• Methodological Answer : Perform dose-response assays across phylogenetically diverse strains (e.g., Pseudomonas aeruginosa, Staphylococcus aureus). Use statistical models (e.g., ANOVA with post-hoc Tukey tests) to analyze MIC variations. Include genetic profiling (e.g., gyrA mutations) to link resistance mechanisms to activity shifts .

Q. What protocols validate the absence of cytotoxic effects when testing this compound in eukaryotic cell cultures?

• Methodological Answer : Conduct parallel assays with mammalian cell lines (e.g., HEK293) using MTT or resazurin-based viability tests. Compare cytotoxicity thresholds (IC50) with antibacterial MICs to establish selectivity indices. Include positive controls (e.g., doxorubicin) and negative controls (culture medium only) .