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The total synthesis of capuramycin (1), which is a promising anti-tubercular antibiotics, has been accomplished using Ferrier-type I reaction as a key step. This total synthesis is an alternative approach to the synthesis of capuramycin and its analogues. The 3'-O-demethyl analogue (2), which exhibits an equivalent antibacterial activity as capuramycin (1) against Mycobacterium smegmatis and Mycobacterium avium, is suggested to have potential as a lead structure of capuramycin analogues because 2 is more accessible from a synthetic view point., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Coronavirus disease 2019 (COVID-19) is an infectious disease, caused by a newly emerged highly pathogenic virus called novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Targeting the main protease (M pro , 3CL pro ) of SARS-CoV-2 is an appealing approach for drug development because this enzyme plays a significant role in the viral replication and transcription. The available crystal structures of SARS-CoV-2 M pro determined in the presence of different ligands and inhibitor-like compounds provide a platform for the quick development of selective inhibitors of SARS-CoV-2 M pro . In this study, we utilized the structural information of co-crystallized SARS-CoV-2 M pro for the structure-guided drug discovery of high-affinity inhibitors from the PubChem database. The screened compounds were selected on the basis of their physicochemical properties, drug-likeliness, and strength of affinity to the SARS-CoV-2 M pro . Finally, we have identified 6-Deaminosinefungin (PubChem ID: 10428963) and UNII-O9H5KY11SV (PubChem ID: 71481120) as potential inhibitors of SARS-CoV-2 M pro which may be further exploited in drug development to address SARS-CoV-2 pathogenesis. Both compounds are structural analogs of known antivirals, having considerable protease inhibitory potential with improved pharmacological properties. All-atom molecular dynamics simulations suggested SARS-CoV-2 M pro in complex with these compounds is stable during the simulation period with minimal structural changes. This work provides enough evidence for further implementation of the identified compounds in the development of effective therapeutics of COVID-19., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The off-target binding of aminoglycosides (AGs) to the A site of human mitochondrial ribosomes in addition to bacterial ribosomes causes ototoxicity and limits their potential as antibiotics. A fluorescence assay was employed to determine relative binding affinities of classical and improved AG compounds to synthetic RNA constructs representing the bacterial and mitochondrial A sites. Results compared well with previously reported in vitro translation assays with engineered ribosomes. Therefore, the minimal RNA motifs and fluorescence assay are shown here to be useful for assessing the selectivity of new compounds., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Etimicin (ETM), which belongs to the newest generation of aminoglycosides (AGs), has been proven to not only maintain but also strengthen the advantages of former AGs with relatively less toxicity. Now, it is widely applied for the treatment of bacterial infections in the clinic. Nevertheless, nephrotoxicity and ototoxicity are unavoidable issues for AGs, and while ETM is no exception, the seriousness of these issues is different. To explore the reason why ETM exhibits less toxicity and to better direct the optimization and development of new AGs, it is of great necessity and importance to monitor the pharmacokinetic behaviors of ETM in its potential toxicity target organs, the kidney and internal ear, as well as in plasma. Therefore, a novel, sensitive and efficient LC-MS/MS method without derivatization or ion-pairing agents had been developed and validated for quantification of ETM in rat plasma, kidney and internal ear for the first time. This method showed good linearity over the range of 50-2000ng/mL for rat plasma/internal ear and 100-5000ng/mL for rat kidney. The precision was less than 4.4% and the accuracy was below 4.8%. Recovery and matrix effects were 71.3%-82.8% and 97.6%-108.5%, respectively. After intravenous administration of a single dose of ETM, plasma drug concentrations fit well with a two-compartmental model, and the AUC 0-∞ , t 1/2α , t 1/2β , MRT and CL were 127.96±5.52μg*h/mL, 0.53±0.03h, 3.32±1.11h, 1.01±0.03h and 234.80±10.05mL/h/kg, respectively. Particularly, ETM showed a considerably long half-life in kidney and internal ear, up to 155.96±19.95h and 83.11±26.60h, respectively, which might contribute greatly to its toxicity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

There is an increasing demand in developing of multifunctional materials with good antibacterial activity, biocompatibility and drug/gene delivery capability for next-generation biomedical applications. To achieve this purpose, in this work series of hydroxyl-rich hyperbranched polyaminoglycosides of gentamicin, tobramycin, and neomycin (HP and SS-HP with redox-responsive disulfide bonds) were readily synthesized via ring-opening reactions in a one-pot manner. Both HP and SS-HP exhibit high antibacterial activity toward Escherichia coli and Staphylococcus aureus. Meanwhile, the hemolysis assay of the above materials shows good biocompatibility. Moreover, SS-HPs show excellent gene transfection efficiency in vitro due to the breakdown of reduction-responsive disulfide bonds. For an in vivo anti-tumor assay, the SS-HP/p53 complexes exhibit potent inhibition capability to the growth of tumors. This study provides a promising approach for the design of next-generation multifunctional biomedical materials., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This work deals with the preparation of single-labeled bioconjugates of the antibiotic Gentamicin (GT) with the sulforhodamine-derived fluorescence dye Texas Red(®)-X (TR), its purification by high-performance liquid chromatography (HPLC) and its characterization by high-resolution mass spectrometry. Aminoglycosides such as GT are efficient antibiotics, but also problematic due to severe side effects such as nephro- and ototoxicity. Fluorescent labeled GT is used to visualize cellular uptake and distribution of the antibiotic to finally understand the mechanisms of serious adverse drug reactions. Pharmaceutically administered GT is a mixture of mainly four different components, which exhibit three (GT(C1)) or four (GT(C1a), GT(C2), GT(C2a)) primary amino functional groups which can be coupled with the labeling reagent TR. Thus, multiple labeling could be envisaged which was assumed to be detrimental for uptake studies by fluorescence imaging. The proposed synthesis aimed at preparation of single labeled product and together with the employed purification strategy indeed yielded single labeled GT as product. Analytical control of the reaction product was carried out by means of mass spectrometry (UHPLC-ESI-QTOF-MS/MS) to rule out over-labeling of GT, which would alter the physicochemical characteristics of GT and its cellular uptake significantly. Moreover, LC-MS/MS analysis gave valuable insights into structural diversity of single labeled products. Further, high-resolution MS and MS/MS spectra of underivatized GT are provided as well. The analytical information on preparation strategy and structure diversity is valuable for studies with a clinical focus on research of aminoglycoside toxicity. Furthermore, it is deemed to be useful for the development of LC-MS/MS assays for the determination of aminoglycosides or the fast screening of synthetic biology samples from biotechnological drug discovery., (Copyright © 2015 Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression at the post-transcriptional level. It is now well established that the overexpression of some miRNAs (oncogenic miRNAs) is responsible for initiation and progression of human cancers and the discovery of new molecules able to interfere with their production and/or function represents one of the most important challenges of current medicinal chemistry of RNA ligands. In this work, we studied the ability of 18 different antibiotics, known as prokaryotic ribosomal RNA, to bind to oncogenic miRNA precursors (stem-loop structured pre-miRNAs) in order to inhibit miRNAs production. In vitro inhibition, binding constants, thermodynamic parameters and binding sites were investigated and highlighted that aminoglycosides and tetracyclines represent interesting pre-miRNA ligands with the ability to inhibit Dicer processing., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The analysis of highly polar (often charged) compounds which lack a strong UV absorbing chromophore is really challenging. Despite the numerous analytical methods published, the demand for a simple, robust and cheap technique for their analysis still persists. Here, reversed phase (RP) liquid chromatography (LC) with capacitively coupled contactless conductivity detection (C(4)D) was explored for the first time as a possible method for separation and detection of various aminoglycoside (AMG) antibiotics which were taken as typical test compounds: tobramycin (TOB), spectinomycin, streptomycin, amikacin, kanamycin A and kanamycin B. C(4)D was performed using a commercially available as well as a laboratory made cell. As ion-pairing reagents (IPR) four perfluorinated carboxylic acids were used: pentafluoropropionic acid, heptafluorobutyric acid, nonafluoropentanoic acid (NFPA) and pentadecafluorooctanoic acid (PDFOA). 0.125 mM NFPA-acetonitrile (ACN) (90:10) or 0.125 mM PDFOA-ACN (70:30) as mobile phases were suitable to detect TOB with reasonable retention times. However, NFPA was preferred for practical reasons. Its applicable concentration range in the mobile phase was strongly restricted by loss of chromatographic performance at lower levels and excessive background conductivity at higher levels. Overall repeatability and robustness of the method were rather poor which was explained by the relatively low IPR levels. Selectivity between the tested AMGs was mainly influenced by the number of protonated amino groups per molecule making it impossible to separate compounds of equal net charges. Problems encountered with gradient elution, hydrophilic interaction liquid chromatography (HILIC) and separation at high pH without IPRs are also discussed., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Patients infected with pathogenic bacteria have to be treated with antibiotics. When the infection is in the lungs, as for instance in cystic fibrosis, bronchiectasis and tuberculosis, inhaled antibiotics have certain advantages over systemically administered antibiotics. In this study, it is shown that re-designing the Twincer™ high dose disposable inhaler into a device named Cyclops enables effective dispersion of up to 50mg of pure spray dried tobramycin. This proves that spray dried tobramycin powders in the preferred size range for inhalation can be administered without applying complex particle engineering techniques and/or using excipients. Only some coarse sweeper crystals added separately are desired to minimise the inhaler losses to less than 20% at 4 kPa. The fine particle fractions <5 μm of the aerosol obtained from the Cyclops closely resemble the primary particle size distribution of the spray dried tobramycin powder. Moreover, without any further optimisation the Cyclops performs good with other spray dried aminoglycosides such as kanamycin and amikacin too. Therefore, the results of this study show that with an appropriate inhaler design, adapted to the physico-chemical properties of a particular drug or drug class, excellent dispersion can be achieved for high doses of pure (spray dried) drug., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A description of pyrrolo[1,4]benzodiazepine (PBD) biosynthesis is a prerequisite for engineering production of analogs with enhanced antitumor activity. Predicted dioxygenases Orf12 and SibV associated with dihydropyrrole biosynthesis in PBDs anthramycin and sibiromycin, respectively, were expressed and purified for activity studies. UV-visible spectroscopy revealed that these enzymes catalyze the regiospecific 2,3-extradiol dioxygenation of l-3,4-dihydroxyphenylalanine (l-DOPA) to form l-2,3-secodopa (λmax=368 nm). (1)H NMR spectroscopy indicates that l-2,3-secodopa cyclizes into the α-keto acid tautomer of l-4-(2-oxo-3-butenoic-acid)-4,5-dihydropyrrole-2-carboxylic acid (λmax=414 nm). Thus, the dioxygenases are key for establishing the scaffold of the dihydropyrrole moiety. Kinetic studies suggest the dioxygenase product is relatively labile and is likely consumed rapidly by subsequent biosynthetic steps. The enzymatic product and dimeric state of these dioxygenases are conserved in dioxygenases involved in dihydropyrrole and pyrrolidine biosynthesis within both PBD and non-PBD pathways., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Amino carbamate adduct formation from the amino group of an aminoglycoside and carbon dioxide has been postulated as a mechanism for reducing nephrotoxicity in the aminoglycoside class compounds. In this study, sisomicin was used as a model compound for amino carbamate analysis. A high pH based reversed-phase high performance liquid chromatography (RP-HPLC) method is used to separate the amino carbamate from sisomicin. The carbamate is stable as the breakdown is inhibited at high pH and any reactive carbon dioxide is removed as the carbonate. The amino carbamate was quantified and the molar fraction of amine as the carbamate of sisomicin was obtained from the HPLC peak areas. The equilibrium constant of carbamate formation, Kc, was determined to be 3.3 × 10(-6) and it was used to predict the fraction of carbamate over the pH range in a typical biological systems. Based on these results, the fraction of amino carbamate at physiological pH values is less than 13%, and the postulated mechanism for nephrotoxicity protection is not valid. The same methodology is applicable for other aminoglycosides., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The increasing incidence and severity of methicillin- and vancomycin-resistant infections during pregnancy prompted further development of telavancin. The understanding of the pharmacokinetics of telavancin during pregnancy is critical to optimize dosing. Due to ethical and safety concerns the study is conducted on the pregnant baboons. A method using solid-phase extraction coupled with liquid chromatography-single quadrupole mass spectrometry for the quantitative determination of telavancin in baboon plasma samples was developed and validated. Teicoplanin was used as an internal standard. Telavancin was extracted from baboon plasma samples by using Waters Oasis(®) MAX 96-Well SPE plate and achieved extraction recovery was >66% with variation <12%. Telavancin was separated on Waters Symmetry C18 column with gradient elution. Two SIM channels were monitored at m/z 823 and m/z 586 to achieve quantification with simultaneous confirmation of telavancin identification in baboon plasma samples. The linearity was assessed in the range of 0.188μg/mL to75.0μg/mL, with a correlation coefficient of 0.998. The relative standard deviation of this method was <11% for within- and between-run assays, and the accuracy ranged between 96% and 114%., (Published by Elsevier B.V.)

Objective: To determine the effects of pH on the activity of clinically relevant antibiotics against bacterial uropathogens. Numerous factors affect antibiotic efficacy within the urinary tract including pH. Because human urine can substantially vary from acidic (pH 4.5) to alkaline (pH 8) conditions and can be easily clinically manipulated, it would be a great advantage to better understand the role of pH in antibiotic treatment of urinary tract infection., Materials and Methods: This in vitro study investigated the activity of 24 widely used antimicrobial agents against bacterial strains comprising 6 major uropathogenic species (Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis, Staphylococcus saprophyticus, and Staphylococcus epidermidis) over the range of pH 5-8. Standard disk-diffusion and broth-microdilution assays were used. One-way analysis of variance was applied to determine significance (P <.05)., Results: For 18 of the 24 agents, pH was shown to play a significant role in overall inhibitory activity. Although most agents behaved similarly across most or all of the uropathogens tested, several only showed pH-dependent effects against certain organisms. The fluoroquinolones, co-trimoxazole, aminoglycosides, and macrolides all functioned optimally at alkaline pH, whereas the tetracyclines, nitrofurantoin, and many of the β-lactams tested exhibited their highest activity under more acidic conditions. Sulfamethoxazole, oxacillin, amoxicillin and clavulanic acid, vancomycin, imipenem, and clindamycin were largely unaffected by pH., Conclusion: Clinicians should consider the urinary pH of their patients when treating urinary tract infection, especially in complicated scenarios. Future clinical investigations examining urinary pH and antibiotic efficacy may result in the application of decreased antibiotic dosages and regimen durations, potentially reducing antibiotic resistance development., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Etimicin intermediate 3,2″,6″-N,N,N-triacetyl gentamicin C1a (P1), is a key intermediate of etimicin, which is a semi-synthetic aminoglycoside antibiotic effective to both gram-positive and gram-negative bacteria infections. Four major related impurities of P1 were detected by HPLC-ELSD and ESI-MS(n) methods. Weakly acidic cation exchange resin, CM-sephadex and silica gel column chromatography were used for the isolation and purification of four major impurities. By means of ESI-MS(n) and NMR analysis, related impurities were characterized as 3,2″-N,N-diacetyl gentamicin C1a (1), 3,2″,6″-N,N,N-triacetyl gentamicin C2b (2), 2″-N-acetyl gentamicin C1a (3), and 2″,6″-N,N-diacetyl gentamicin C1a (4). Impurities 1, 2, 4 are novel compounds and the NMR data of these isolates were first reported in this paper. The possible mechanism for the formation of these impurities is also discussed., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We describe the combinatorial synthesis and cheminformatics modeling of aminoglycoside antibiotics-derived polymers for transgene delivery and expression. Fifty-six polymers were synthesized by polymerizing aminoglycosides with diglycidyl ether cross-linkers. Parallel screening resulted in identification of several lead polymers that resulted in high transgene expression levels in cells. The role of polymer physicochemical properties in determining efficacy of transgene expression was investigated using Quantitative Structure-Activity Relationship (QSAR) cheminformatics models based on Support Vector Regression (SVR) and 'building block' polymer structures. The QSAR model exhibited high predictive ability, and investigation of descriptors in the model, using molecular visualization and correlation plots, indicated that physicochemical attributes related to both, aminoglycosides and diglycidyl ethers facilitated transgene expression. This work synergistically combines combinatorial synthesis and parallel screening with cheminformatics-based QSAR models for discovery and physicochemical elucidation of effective antibiotics-derived polymers for transgene delivery in medicine and biotechnology., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

RNA is an extremely important target for the development of chemical probes of function or small molecule therapeutics. Aminoglycosides are the most well studied class of small molecules to target RNA. However, the RNA motifs outside of the bacterial rRNA A-site that are likely to be bound by these compounds in biological systems is largely unknown. If such information were known, it could allow for aminoglycosides to be exploited to target other RNAs and, in addition, could provide invaluable insights into potential bystander targets of these clinically used drugs. We utilized two-dimensional combinatorial screening (2DCS), a library-versus-library screening approach, to select the motifs displayed in a 3×3 nucleotide internal loop library and in a 6-nucleotide hairpin library that bind with high affinity and selectivity to six aminoglycoside derivatives. The selected RNA motifs were then analyzed using structure-activity relationships through sequencing (StARTS), a statistical approach that defines the privileged RNA motif space that binds a small molecule. StARTS allowed for the facile annotation of the selected RNA motif-aminoglycoside interactions in terms of affinity and selectivity. The interactions selected by 2DCS generally have nanomolar affinities, which is higher affinity than the binding of aminoglycosides to a mimic of their therapeutic target, the bacterial rRNA A-site., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The characterization of impurities present in vertilmicin by liquid chromatography (LC) coupled with mass spectrometry (MS) is described. A reversed phase (RP)-LC method using a C18 column resistant to basic pH with an alkaline (pH 11) aqueous mobile phase was developed and coupled to MS with an electrospray ionization (ESI) source in the positive ion mode which provides MS(n) capability. In total, 18 impurities were detected in a commercial sample. Eleven impurities described in this work were newly identified., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The characterization of impurities present in micronomicin sulfate injection by liquid chromatography (LC) coupled with mass spectrometry (MS) is described. A reversed phase (RP)-LC method using a C₁₈ column resistant to an alkaline (pH 11) aqueous mobile phase was developed and coupled to MS with an electrospray ionization (ESI) source in the positive ion mode which provides MS(n) capability. A total of thirty six impurities were detected in commercial samples: five impurities were identified by comparison of their fragmentation patterns with those of available related substances, eleven of them were identified in accordance with relevant literature, while the other twenty impurities were newly identified using the MS/MS spectra of the available related reference substances as interpretative templates combined with knowledge of the nature of functional group fragmentation behaviors. This work was applied to evaluate the quality of micronomicin sulfate injection from different manufacturers., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Due to its wide field of application, sensitivity, wide range of linearity and the applicability to gradient elution, the most common detection technique for HPLC nowadays is UV/vis spectrophotometry. However, UV/vis detection comes to its limits when the analytes are lacking suitable chromophors or exhibit very different UV responses. In the past years, different types of evaporation/aerosol based HPLC detectors have been developed to fill this gap in HPLC detection. Amongst those, the corona-charged aerosol detector (CAD) is one of the most powerful and versatile representatives. In the recent past a variety of papers have been published, demonstrating the potential of the CAD in different fields of analytical chemistry. This paper is intended to provide an overview of the key performance characteristics and manifold applications for HPLC-CAD in the field of pharmaceutical analysis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

C1027 is a potent antitumor agent that damages DNA. It has the unusual ability to produce double strand breaks and interstrand cross-links (ICLs) intracellularly, which enable it to initiate concurrent ataxia-telangiestasia mutated (ATM) and Rad-3 related (ATR) independent damage responses. The latter form of damage is not well characterized. We have examined the effect of DNA sequence on C1027 reactivity and found it to be more diverse than previously thought. In addition, analysis of the chemical stability of ICLs suggests that they result from reaction with the deoxyribose ring on one strand but direct addition to a nucleobase on the opposite strand., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Liquid chromatography with tandem mass spectrometry (LC/MS/MS) methods for the determination of gentamicin, lincomycin and spectinomycin in the presence of their impurities were developed and tested. Chromatographic separations were achieved using gradient elution on a C18 column. All components were ionized by positive-ion electrospray and detected by multi reaction monitoring (MRM) with an LC-tandem mass spectrometer. Calibration curves were linear with correlation coefficients better than 0.99. The developed method for the determination of gentamicin provides complete base line separation of components C1, C1a, C2, C2a and C2b mentioned in the European and British Pharmacopoeias. The second developed method makes possible a simultaneous analysis of the active compounds of both lincomycin and spectinomycin. Additionally, all impurities defined in the pharmacopoeias for all three active components were determined and their identities confirmed. The methods were tested in routine quality control analysis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The mitochondrial 12S rRNA is a hot spot for mutations associated with both aminoglycoside-induced and nonsyndromic hearing loss. Of those, the homoplasmic 1555A>G and 1494C>T mutations at the highly conserved decoding region of the 12S rRNA have been associated with hearing loss worldwide. In particular, these two mutations account for a significant number of cases of aminoglycoside ototoxicity. The 1555A>G or 1494C>T mutation is expected to form a novel 1494C-G1555 or 1494U-A1555 base-pair at the highly conserved A-site of 12S rRNA. These transitions make the human mitochondrial ribosomes more bacteria-like and alter binding sites for aminoglycosides. As a result, the exposure to aminoglycosides can induce or worsen hearing loss in individuals carrying one of these mutations. Biochemical characterization demonstrated an impairment of mitochondrial protein synthesis and subsequent defects in respiration in cells carrying the A1555G or 1494C>T mutation. Furthermore, a wide range of severity, age-at-onset and penetrance of hearing loss was observed within and among families carrying these mutations. Nuclear modifier genes, mitochondrial haplotypes and aminoglycosides should modulate the phenotypic manifestation of the 12S rRNA 1555A>G and 1494C>T mutations. Therefore, these data provide valuable information and technology: (1) to predict which individuals are at risk for ototoxicity; (2) to improve the safety of aminoglycoside antibiotic therapy; and (3) eventually to decrease the incidence of hearing loss., (Copyright © 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Based on the structural information of biomacromolecule-aminoglycoside complexes, a series of kanamycin B analogues were rationally designed and synthesized. A convenient approach to the construction of kanamycin derivatives, in which the C4'-position on ring I of neamine moiety was modified, was developed. Most synthetic analogues exhibited good to excellent antibiotic activity against some typical drug-resistant bacteria. The disclosed results suggested that the C4'-position of aminoglycosides such as kanamycin may be an ideal site for modification to gain new modifying enzyme-resistant aminoglycoside antibiotics., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Using allylic azide rearrangement, a convenient method has been developed for the synthesis of 2',3'-dideoxyaminoglycosides that are, otherwise, difficult to be prepared. The antibacterial activity of these novel aminoglycosides also confirms the indispensable role of 2'-NH(2) group for both neomycin and kanamycin classes of aminoglycosides. A novel structural motif containing the hexylaminocarbonyl groups at O-5 and/or O-6 of 2',3'-dideoxyneamine could lead to the production of new aminoglycosides against resistant bacteria., (Copyright 2010 Elsevier Ltd. All rights reserved.)

An HPLC/MS/MS method was developed for identification of impurities in gentamicin. The HPLC was performed on a Synergy Hydro-RP column using 50 mM trifluoroacetic acid (TFA), pH 2 adjusted with ammonium solution and methanol as mobile phase. All impurities in gentamicin were separated from main gentamicin components. Atmospheric pressure chemical ionization (APCI) was used and product mass spectra of protonated molecules were acquired. Seventeen impurities were detected in gentamicin. Reference compounds: gentamicins: C2b, B, B1, G-418, sisomicin, garamine and gentamines: C1, C1a, C2, C2a were used for spectra interpretation and impurities identification. All MS/MS spectra were interpreted and fragmentation transitions for gentamicins and in general for aminoglycoside antibiotics (AG) were proposed. All impurities were identified. More than one isomere were proposed for three impurities.

Continuing from our ongoing effort in modifying aminoglycoside antibiotics with the goal of counteracting drug resistant bacteria, we have further derivatized pyranmycin, a neomycin class aminoglycoside antibiotic, with modifications at O-6 and N-1 positions. The revealed SAR results demonstrated that the antibacterial activity of pyranmycin can be modulated by different acylic substituents at O-6. Among these results, the 6-O-aminoethyl derivative, JT050, showed effective activity against resistant strain Escherichia coli (pTZ19U-3) and E. coli (pSF815), which provides insight into further structural modifications.

Aminoglycoside antibiotic derivatives such as neamine, methyl neobiosaminide B, 2-deoxystreptamine, tetra-azidoneamine, tetra-N-acetylneamine, tetra-N-carboxy-benzylneamine, tetra-N-carboxy-methylneamine and tetra-p-methoxy-benzyliminoneamine were prepared and evaluated as to their cochlear and vestibular toxicity. Methyl neobiosaminide B, the most promising derivative in the series showed selective, cochlea-dissociated vestibulotoxic activity and was considered to be a potential lead compound for the treatment of Ménière's disease. Antimicrobial properties of the compounds, qualitatively evaluated against a group of pathogenic bacteria, indicated that neomycin B sulfate, neamine as a free base and methyl-neobiosaminide B dihydrochloride show a broader range of activity when compared to the other derivatives.

Neamine derivatives bearing a nucleobase, adenine, cytosine, guanine or thymine with a lysine or an arginine as a linker have been synthesized and its potential as the inhibitor for HIV TAR-Tat interaction examined. Among them, modified neamine having an arginine-nucleobase showed a higher inhibition than that of the one having a lysine-nucleobase. The difference of the nucleobase anchor did not characterize inhibition specificity. Also, stereochemistry of the amino acid in the compounds causes no difference in the inhibition for TAR-Tat.

Chemical modification and inactivation of aminoglycosides by many different enzymes expressed in pathogenic bacteria are the main mechanisms of bacterial resistance to these antibiotics. In this work, we designed inhibitors that contain the 1,3-diamine pharmacophore shared by all aminoglycoside antibiotics that contain the 2-deoxystreptamine ring. A discovery library of molecules was prepared by attaching different side chains to both sides of the 1,3-diamine motif. Several of these diamines showed inhibitory activity toward two or three different representative aminoglycoside-modifying enzymes (AGMEs). These studies yielded the first non-carbohydrate inhibitor N-cyclohexyl-N'-(3-dimethylamino-propyl)-propane-1,3-diamine (Compound G,H) that is competitive with respect to the aminoglycoside binding to the enzyme aminoglycoside-2''-nucleotidyltransferase-Ia (ANT2''). Another diamine molecule N-[2-(3,4-dimethoxyphenyl)-ethyl]-N'-(3-dimethylamino-propyl)-propane-1,3-diamine (Compound H,I) was shown to be a competitive inhibitor of two separate enzymes (aminoglycoside-3'-phosphotransferase-IIIa (APH3') and ANT2'') with respect to metal-ATP. Thermodynamic and structural-binding properties of the complexes of APH3' with substrates and inhibitor were shown to be similar to each other, as determined by isothermal titration calorimetry and NMR spectroscopy.

The C5''-OH group in neomycin B was glycosylated with a variety of mono- and di-saccharides to probe the effect of introduction of additional binding elements on antibacterial activity and interaction with the aminoglycosides modifying enzyme APH(3')-IIIa. The designed structures show antibacterial activity superior to that of neomycin B against pathogenic and resistant strains, while in parallel they demonstrate poor substrate activity with APH(3')-IIIa.

C-1027 belongs to the family of chromoprotein antitumor antibiotics, which contain a carrier apoprotein and a highly unstable enediyne chromophore. The enediyne spontaneously aromatizes to generate p-benzyne biradical, and subsequently abstracts hydrogens from the DNA sugar backbone, resulting in cleavage of the double strand. Using spin-trapping methods, we obtained direct proof of radical intermediates during an DNA cleavage, and found intriguing difference in behavior between the trapping agents 2-methyl-2-nitrosopropane (MNP) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO): MNP added to the sugar radicals of the DNA, whereas DMPO directly trapped a phenyl radical or p-benzyne biradical derived from the C-1027 chromophore.

The aglycon, or so-called 'warhead' portion, of several potent 10-membered ring enediyne antitumor antibiotics contain dienonecarbamate and enediyne chromophores in an unusual bicyclic ring structure in which these two subunits are essentially orthogonal to each other. The circular dichroism (CD) spectra of the calicheamicin, esperamicin, and shisijimicin A families, all of which contain this bicyclic ring system, exhibit a characteristic negative exciton coupled CD at about 310 and 270 nm. This signature CD feature suggested the absolute stereochemical relationship between these chromophores as originally assigned and which was later confirmed by stereospecific total synthesis. Because of the unique nature of this chromophoric interaction and the importance of using this CD spectral feature in the assignment of the absolute stereochemistry of other related enediynes, we report here simulations of the CD spectra of the calicheamicin aglycon A, and of two other truncated models, B and C, by using density functional theory (DFT) and the DeVoe coupled oscillator approach. The DFT calculations provide a strong theoretical basis that the planar enediyne chromophore alone gives a negligible CD contribution, while that coming from the twisted dienonecarbamate is much more substantial. However, the shape and the largest part of the intensity of experimental CD spectrum can only be reproduced when the two unsaturated moieties are simultaneously present. Thus, the exciton coupling between the two chromophores provides the most important contribution to the experimental CD spectrum of calicheamicin. This conclusion is in full agreement with the results from the DeVoe calculation.

A simple reversed-phase high performance liquid chromatographic method was developed for the analysis of vertilmicin sulfate, a novel aminoglycoside (AG). UV detection was used to determine vertilmicin sulfate and its related compounds in drug substance and products without sample derivatization. The method was used to determine the content of vertilmicin and its related compounds and test the stability of vertilmicin sulfate as drug substance and in injections, which was required for registration of new drug.

Eleven derivatives (5-13, 15, and 16) of an immunosuppressive and cytotoxic tricyclic terpenoid, brasilicardin A (1), were prepared and assayed for inhibitory effects to the mouse mixed lymphocyte reaction (MLR) and seven human tumor cell lines. The 17N-methyl form (8) of 1 showed the most potent immunosuppressive activity in mouse MLR, while induction of more bulky group for N-17 resulted in significant decrease of the activity. Compound 8 also showed potent cytotoxic activity against DLD-1, Lu-65, A549, K562, and MOLT-4 cells, while the benzyl ester (13) of 1 exhibited potent cytotoxicity against K562, MOLT-4, and jarkat leukemia cell lines. The 17N-acetyl derivative (11) of 1 selectively inhibited the cell growth of DLD-1 cells. The methyl ester (5) of 1 showed potent cytotoxic activity against K562, MOLT-4, and Ball-1 cell lines, the last of which was resistant to 1, 8, and 13.

Three new tricyclic terpenoids, brasilicardins B-D (2-4), were isolated together with brasilicardin A (1), a potent immunosuppressive compound, from the cultured broth of a pathogenic actinomycete Nocardia brasiliensis IFM0406, and the structures and stereochemistry were determined by spectroscopic data and a single crystal X-ray diffraction analysis. The immunosuppressive and cytotoxic activities of 2-4 were examined in the comparison with 1.

Aminoglycoside antibiotics constitute a class of the drugs of high interest, whose therapeutic action is based upon the electrostatic interaction with the variety of RNA molecules. The positive charge of these drugs molecules, located at their amino functions, has a prevailing influence on this process. The potentiometry and (1)H NMR spectroscopy are applied hereby to achieve the characteristics of the acid-base properties of particular protonating groups. We found that the pK values of deprotonation processes cover a wide values range 6-9.8. The correlation spectra of sisomicin, both COSY and TOCSY, allowed attributing unambiguously individual signals to the corresponding protons. These spectra involve a lot of the cross-peaks originating from the B and C rings protons, while the analogous signals originating from A rings protons are less numerous. Molecular modeling provided that the methylated amino group of A ring is located too far from the protonated functions of the remaining rings to affect their pK values. The phenomena observed herein are discussed in line of strength of the analogous processes observed for other aminoglycosides. As the result, four types of amino groups consisted within these antibiotics are distinguished.

A simple generic continuous-flow enzyme immunoassay (CFEIA) for analysis of aminoglycosides in serum has been successfully developed. The developed assay employed a specific monoclonal antibody and beta-galactosidase (beta-GAL) enzyme as label. The assay involves an off-line competitive binding reaction between the analyte and free labelled analyte for the binding sites of the antibody. After equilibrium is reached, the sample was injected into the flow system. The bound antibody complexes with the analyte and the labelled analyte were trapped in a protein G column, while the unbound free labelled analyte was eluted and detected colorimetrically down-stream, after reaction with chlorophenolic red-beta-D-galactopyranoside as a substrate for the beta-GAL enzyme. The concentration of the analyte in a sample was quantified by its ability to inhibit the binding of the analyte-enzyme conjugate to the antibody, and the signal was directly proportional to the concentration of the analyte in the original sample. The optimum conditions for the developed CFEIA were investigated and applied to the analysis of tobramycin, as a representative example of the aminoglycosides, in serum samples. The detection limit of the assay was 0.06 microgml(-1). The assay showed good precision; the coefficients of variation were 2.49-4.33 and 3.30-6.82% for intra- and inter-assay precision, respectively. Serum matrix constituents and the endogenous compounds did not interfere with the assay. Analytical recovery of spiked tobramycin, in the concentration range between 0.5 and 8.0 microgml(-1), was 101.55+/-3.14. The assay results correlated well with those obtained by high-performance liquid chromatography (r=0.991). All the obtained results strongly demonstrate that the developed CFEIA is a suitable method for a rapid and reliable analysis of aminoglycosides in serum.

The 16S bacterial ribosomal A-site decoding rRNA region is thought to be the pharmacological target for the aminoglycoside antibiotics. The clinical utility of aminoglycosides could possibly depend on the preferential binding of these drugs to the prokaryotic A-site versus the corresponding A-site from eukaryotes. However, quantitative aminoglycoside binding experiments reported here on prokaryotic and eukaryotic A-site RNA constructs show that there is little in the way of differential binding affinities of aminoglycosides for the two targets. The largest difference in affinity is 4-fold in the case of neomycin, with the prokaryotic A-site construct exhibiting the higher binding affinity. Mutational studies revealed that decoding region constructs retaining elements of non-Watson-Crick (WC) base pairing, specifically bound aminoglycosides with affinities in the muM range. These studies are consistent with the idea that aminoglycoside antibiotics can specifically bind to RNA molecules as long as the latter have non-A form structural elements allowing access of aminoglycosides to the narrow major groove.

The use of aminoglycoside antibiotics is limited by side effects, the most critical of which are vestibular and cochlear toxicity. Recent evidence indicates that these effects result from an excitotoxic process mediated, at least in part, through a polyamine-like activation of NMDA receptors. This study investigated whether these positive modulatory effects of aminoglycosides at NMDA receptors are dissociable from their antibacterial properties. A group of structurally related apramycin derivatives was evaluated for the ability to enhance [3H]dizocilpine binding to rat brain membranes, and for the ability to augment agonist responses on recombinant (NR1A/2B) NMDA receptors expressed in Xenopus oocytes. Based on the antibacterial potencies of these derivatives against Staphylococcus aureus and Escherichia coli, it is concluded that there is no correlation between the ability of an aminoglycoside to produce a positive modulation of NMDA receptors and minimum inhibitory antibacterial concentrations. These findings indicate that it may be possible to develop an aminoglycoside antibiotic with reduced potential for ototoxicity.

Aminoglycoside antibiotics have recently emerged as an intriguing family of RNA binding molecules and they became leading structures for the design of novel RNA ligands. The demystification of the aminoglycoside-RNA recognition phenomenon is required for the development of superior binders. To explore the existence of multiple binding sites in a large RNA molecule, we have synthesized covalently linked symmetrical and nonsymmetrical dimeric aminoglycosides. These unnatural derivatives were compared to their natural "monomeric" counterparts in their ability to inhibit the Tetrahymena ribozyme. The dimeric aminoglycosides inhibit ribozyme function 20 to 1.2 x 10(3) fold more effectively than their natural parent compounds. The inhibition curves of dimeric aminoglycosides have characteristic shapes suggesting the presence of at least two high affinity-binding sites within the ribozyme's three-dimensional fold. The interaction of a dimeric aminoglycoside with two complementary sites of the RNA molecule is proposed. This binding motif may have implications on the development of new drugs targeting pivotal RNA molecules of bacterial and viral pathogens.