Identification of an ethyl 5,6-dihydropyrazolo[1,5-c]quinazoline-1-carboxylate as a catalytic inhibitor of DNA gyrase

Fluoroquinolones are a class of antibacterial agents used clinically to treat a wide array of bacterial infections and target bacterial type-II topoisomerases (DNA gyrase and topoisomerase IV). Fluoroquinolones, however potent, are susceptible to bacterial resistance with prolonged use, which limits their use in the clinic. Quinazoline-2,4-diones also target bacterial type-II topoisomerases and are not susceptible to bacterial resistance similar to fluoroquinolones, however, their potency pales in comparison to fluoroquinolones. To meet the increasing demand for antibacterial development, nine modified quinazoline-2,4-diones were developed to probe quinazoline-2,4-dione structure modification for possible new binding contacts with the bacterial type-II topoisomerase, DNA gyrase. Evaluation of compounds for inhibition of the supercoiling activity of DNA gyrase revealed a novel ethyl 5,6-dihydropyrazolo[1,5-c]quinazoline-1-carboxylate derivative as a modest inhibitor of DNA gyrase, having an IC₅₀ of 3.5 μM. However, this ethyl 5,6-dihydropyrazolo[1,5-c]quinazoline-1-carboxylate does not trap the catalytic intermediate like fluoroquinolones or typical quinazoline-2,4-diones do. Thus, the ethyl 5,6-dihydropyrazolo[1,5-c]quinazoline-1-carboxylate derivative discovered in this work acts as a catalytic inhibitor of DNA gyrase and therefore represents a new structural type of catalytic inhibitor of DNA gyrase.     

临床分离铜绿假单胞菌对氟喹诺酮类抗菌药物的耐药性检测

目的了解临床分离铜绿假单胞菌对喹诺酮类等抗菌药物的耐药性。方法琼脂稀释法测定86株铜绿假单胞菌对5种氟喹诺酮类抗菌药物以及头孢吡肟、美罗培南的耐药性。结果铜绿假单胞菌对诺氟沙星、氧氟沙星、左氧氟沙星、环丙沙星、加替沙星的耐药率分别为50%、61.6%、51.2%、48.8%、51.2%;对头孢吡肟和美罗培南的耐药率分别为30.2%、23.2%。结论铜绿假单胞菌对氟喹诺酮类抗菌药物耐药显著,临床应加强检测和监测。     

Determinations of fluoroquinolones and nonsteroidal anti-inflammatory drugs in urine by extractive spectrophotometry and photoinduced spectrofluorimetry using multivariate calibration

Multivariate calibration methods are chemometric tools that may be applied to the analysis of spectroscopic data with multichannel detection. Two procedures, based on spectrophotometric and fluorimetric signals, are reported for the simultaneous determination of two fluoroquinolones (ciprofloxacin and ofloxacin) and two nonsteroidal anti-inflammatory drugs (diclofenac and mefenamic acid) using first- and second-order multivariate calibration methods. In the spectrophotometric method, an extractive procedure into chloroform using trioctylmethylammonium chloride-adogen as counter ion was optimized, with the object of extracting the analytes from urine samples and eliminating matrix interferences. After separation, the absorption spectrum of the organic phase was used as the analytical signal in a partial least squares method. A photoinduced spectrofluorimetric (PIF) method using excitation-emission fluorescence matrices, is proposed, to apply three-way chemometric calibration, with the aim of analyzing ofloxacin, ciprofloxacin, and diclofenac in urine samples without the previous extractive sample-cleaning step. For both procedures, recoveries around 100% were found for all the analytes. However, the PIF three-way chemometric method provides the most sensitive and selective procedure as the urine interferences are modulated using the three-way chemometric technique.     

氟喹诺酮类药物体外诱导肺炎克雷伯菌耐药后GyrA和ParC的变异

目的了解3种氟喹诺酮类（FQS）体外诱导肺炎克雷伯菌（Klebsiella pneumoniae,Kpn）耐药性的差异,研究肺炎克雷伯菌DNA旋转酶A亚单位（GyrA）和拓扑异构酶ⅣC亚基（ParC）的变异与其耐喹诺酮类药物的关系。方法采用环丙沙星（CW）、左氧氟沙星（LEX）和加替沙星（GAT）对从临床分离8株Kpn进行体外分步诱导,采用琼脂平板二倍稀释法测定CIP、LVF及GAT对Kpn诱导前、后的最低抑菌浓度（MIC）,并对诱导成功的17株Kpn的GyrA的基因（gyrA）和ParC的基因（parC）进行PCR扩增,选取其中8株KpnDNA测序并进行序列分析比较。结果8株耐FQS菌株都存在GyrA变异,同FQS耐药性相关的变异有Ser83（TCC）→Phe（TTC）、Ile（ATC）和Tyr（TAC）,Asp87（GAC）→Ala（GCC）、ma（GCC）和Glu（GAA）,5株Kpn同时存在ParC的变异：丝氨酸Ser80（AGC）→Ile（ATC）。结论本研究体外实验证实了Kpn可在长期低剂量的接触抗菌药物后形成耐药菌株。在高度耐FQS的Kpn中同时存在GyrA和ParC变异。     

肺炎克雷伯杆菌对氟喹诺酮耐药的机制研究

目的研究肺炎克雷伯杆菌对氟喹诺酮类药物（FQNs）的耐药机制。方法筛选临床分离的对环丙沙星耐药的肺炎克雷伯杆菌共10株,采用微量肉汤稀释法检测菌株对5种氟喹诺酮类药物的MIC值;采用PCR方法检测菌株染色体和质粒携带的喹诺酮耐药基因（gyrA基因、parC基因和qnr基因）并测序;质粒接合试验验证qnr基因的转移性。结果 10株肺炎克雷伯杆菌对5种氟喹诺酮类药物均产生耐药性。扩增产物经测序发现10株肺炎克雷伯杆菌染色体的gyrA基因和parC基因均有突变;有2株菌株（K79和K107）携带qnrA基因,这2株菌的接合菌对喹诺酮抗菌药的MIC值上升了5～30倍;未检测到qnrB阳性的菌株。结论 gyrA和parC基因突变是肺炎克雷菌对氟喹诺酮类产生耐药机制的主要原因,质粒上qnrA基因的存在,也是产生喹诺酮耐药的一个重要因素。     

Synthesis and antitubercular evaluation of new fluoroquinolone derivatives coupled with carbohydrates

We describe in this work the synthesis of nine new fluoroquinolone derivatives based on modifications at the C-7 position of the known fluoroquinolones cipro-, gati-, and moxifloxacin, as well as their antitubercular evaluation. The synthesis of these new analogues was improved using microwave irradiation, providing several advantages such as better yields and shorter reaction times, in comparison with classical reaction conditions. Derivatives 4, 5, and 7 exhibited promising antitubercular activities.     

Potentiation of Antibiotic Activity by a Meldrum’s Acid Arylamino Methylene Derivative against Multidrug-Resistant Bacterial Strains

This study aimed to evaluate the intrinsic antibacterial activity and antibiotic-enhancing effect of an arylamino methylene derivative (MAD) in association with fluoroquinolones. The antibacterial activity against multiresistant Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli was analyzed by determining the minimum inhibitory concentration (MIC) using the broth micro dilution method. A reduction in the MIC of the fluoroquinolones against strains treated simultaneously with the MAD was interpreted as an enhanced antibiotic activity. While the MAD exhibited no clinically effective action (MIC ≥ 1.024 µg/mL), it was found to significantly potentiate the activity of norfloxacin, ofloxacin and lomefloxacin against all the strains, which may be related to structural similarities between the MAD and quinolones. Our findings suggest that Meldrum’s acid arylamino derivatives may represent promising molecules in the elaboration of new drugs to reverse resistance to fluoroquinolones.     

Degradation of the antibiotics norfloxacin and ciprofloxacin by a white-rot fungus and identification of degradation products

More than 90% of the antibiotics ciprofloxacin (CIPRO) and norfloxacin (NOR) at 2 mg L⁻¹ were degraded by Trametes versicolor after 7 days of incubation in malt extract liquid medium. In in vitro assays with purified laccase (16.7 nkat mL⁻¹), an extracellular enzyme excreted constitutively by this fungus, 16% of CIPRO was removed after 20 h. The addition of the laccase mediator 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt led to 97.7% and 33.7% degradation of CIPRO and NOR, respectively. Inhibition of CIPRO and NOR degradation by the cytochrome P450 inhibitor 1-aminobenzotriazole suggests that the P450 system also plays a role in the degradation of the two antibiotics. Transformation products of CIPRO and NOR were monitored at different incubation times by triple-quadrupole and quadrupole time-of-flight mass spectrometry, and can be assigned to three different reaction pathways: (i) oxidation of the piperazinyl substituent, (ii) monohydroxylation, and (iii) formation of dimeric products.     

New 1,4-dihydro[1,8]naphthyridine derivatives as DNA gyrase inhibitors

Owing to the growing need for novel antibacterial agents, we synthesized a novel series of fluoroquinolones including 7-substituted-1-(2,4-difluorophenyl)-6-fluoro-4-oxo-1,4-dihydro[1,8]naphthyridine-3-carboxylic acid derivatives, which were tested against clinically relevant Gram positive and Gram negative bacteria. Chemical structures of the synthesized compounds were identified using spectroscopic methods. In vitro antimicrobial effects of the compounds were determined via microdilution assay. Microbiological examination revealed that compounds 13 and 14 possess a good antibacterial profile. Compound 14 was the most active and showed an antibacterial profile comparable to that of the reference drugs trovafloxacin, moxifloxacin, and ciprofloxacin. A significant MIC₉₀ value (1.95 μg/mL) against S. aureus ATCC 25923, E. coli ATCC 35218, and E. coli ATCC 25922 was recorded for compound 14. We observed reduced metabolic activity associated with compounds 13 and 14 in the relevant bacteria via a luminescence ATP assay. Results of this assay supported the antibacterial potency of compounds 13 and 14. An E. coli DNA gyrase inhibitory assay indicated that compound 14 is a potent inhibitor of E. coli DNA gyrase. Docking studies revealed that there is a strong interaction between compound 14 and the E. coli DNA gyrase enzyme. Genotoxicity and cytotoxicity evaluations of compounds 13 and 14 showed that compound 14 is non-genotoxic and less cytotoxic compared to the reference drugs (trovafloxacin, moxifloxacin, and ciprofloxacin), which increases its biological importance.     

氟喹诺酮类药物的光化学性质及其光敏毒性机理

氟喹诺酮类药物(fluoroquinolones,FQs)是目前得到广泛应用的广谱、高效的合成抗菌素。本文通过对FQs光化学研究进展的介绍,详述了具有代表性的FQs的光物理、光化学性质及光化学反应途径,以及其对生物分子的光敏损伤机理等。FQs具有较高的三重激发态产额,其激发态能够发生多种光化学反应,此类反应极易受到溶剂性质和某些盐类的影响,其光敏毒性与其分子结构有着密切的联系,尤其受8位取代基的影响非常大。对该类药物光化学性质和光敏毒性机理的研究,将会对此类药物的合理使用和新一代药物的研发提供很好的理论依据。