4″-O-(ω-Quinolylamino-alkylamino)propionyl derivatives of selected macrolides with the activity against the key erythromycin resistant respiratory pathogens

Four macrolides—6-O-methyl-8a-aza-8a-homoerythromycin, clarithromycin, azithromycin and azithromycin 11,12-cyclic carbonate, have been selected for the construction of a series of new quinolone derivatives. The quinolone moiety is connected to the macrolide scaffold via a diaminoaklyl 4″-O-propionyl ester chain of varying length. At the terminus the linker is attached via one of the nitrogen atoms in the linker at C(6) or C(7) of the quinolone. Many of compounds described, particularly clarithromycin derivative 37, and azithromycin derivatives 48 and 55, exhibited excellent antibacterial activity against a wide range of clinically relevant macrolide-resistant organisms, with profiles superior to that of telithromycin, an enhanced spectrum ketolide.     

Synthesis and properties of macrolones characterized by two ether bonds in the linker

In this paper synthesis of macrolones 1–18 starting from azithromycin is reported. Two key steps in the construction of the linker between macrolide and quinolone moiety, are formation of central ether bond by alkylation of unactivated OH group, and formation of terminal C–C bond at 6-position of the quinolone unit. Due to the difficulty in formation of these two bonds the study of alternative synthetic methodologies and optimization of the conditions for the selected routes was required. Formation of C-4″-O-ether bond was completed by modified Michael addition, whereas O-alkylation via diazonium cation proved to be the most effective in formation of the central allylic or propargylic ether bond. Comparison of Heck and Sonogashira reaction revealed the former as preferred route to the C–C bond formation at C(6) position of the quinolone unit. Most of the target compounds exhibited highly favorable antibacterial activity against common respiratory pathogens, without significant cytotoxicity profile when tested in vitro on eukaryotic cell lines.     

Synthesis and biological activity of 4″-O-acyl derivatives of 14- and 15-membered macrolides linked to ω-quinolone-carboxylic unit

The synthesis and antimicrobial activity of a new class of macrolide antibiotics which consist of a macrolide scaffold and a quinolone unit covalently connected by an appropriate linker are described. Optimization of several synthetic steps and structural properties of lead compound 26 are discussed. Promising antibacterial properties of this compound and some of its analogues are reported.     

Peptide Derivatives of Antibiotics Tylosin and Desmycosin, Protein Synthesis Inhibitors

Biologically active peptide derivatives of 16-member macrolide antibiotics were synthesized as potential probes for the investigation of nascent peptide chain topography in the ribosomal exit tunnel. The tylosin and desmycosin aldehyde groups at the C6 position of the lactone ring were modified by the aminooxyacetyl-L-alanyl-L-alanine methyl ester.     

Novel nonviral vectors for gene delivery: Synthesis and applications

Summary We have synthesized novel cationic lipids for gene delivery bearing an ester bond between the lipid moiety and the polyamine head. We have found that an intramolecular rearrangement occurs during purification of one of the products. The rearrangement led to a cyclic lipopolyamine which was active for DNA gene transfer. The formation of cyclization products depends on the spacer found between the lipid and the polyamine. The introduction of arginine in the linker position prevents the formation of cyclic products. Linear as well as cyclic analogues showed high-efficiency gene transfer when tested in vitro for luciferase gene expression as compared to dioctadecylamidoglycyl spermine or lipofectamine and also in vivo in the Lewis lung carcinoma model. The introduction of arginine in the linker position promoted increased transfection activity, demonstrating that a diversity of linkers, such as short peptides or glycosides, can be introduced into cationic lipids for targeted gene transfer.     

Photocleavage of DNA by the p-nitrobenzoyl group covalently linked to proflavine.

We have synthesized two novel DNA photocleaving agents,3,6-diamino-10-[6-(4-nitrobenzoyloxy)hexyl]acridinium chloride and 3,6-diamino-10-[6-(4-nitrobenzamido)-hexyl]acridinium chloride, and studied their DNA binding mode and cleavage properties. These compounds contain the photoactive p-nitrobenzoyl group attached to proflavine via an amide or ester linker group and a polymethylene chain. Spectroscopic and viscometric studies have shown that the compounds bind DNA by an intercalative mode. The presence of covalently-bonded intercalator is essential for the UV (310 nm) induced DNA scission. Above a critical ratio, an increase in the relative concentration of compound to DNA did not induce further cleavage. The cleavage efficiency was dependent on the type of linker group. These results are discussed in regard to possible mechanisms for photoinduced DNA breakage.     

Novel nonviral vectors for gene delivery: Synthesis and applications

We have synthesized novel cationiclipids for gene delivery bearing an ester bond betweenthe lipid moiety and the polyamine head. We have foundthat an intramolecular rearrangement occurs duringpurification of one of the products. The rearrangementled to a cyclic lipopolyamine which was active for DNAgene transfer. The formation ofcyclization products depends on the spacer foundbetween the lipid and the polyamine. The introduction ofarginine in the linker position prevents the formation ofcyclic products. Linear as well as cyclicanalogues showed high-efficiency gene transfer whentested in vitro for luciferase gene expressionas compared to dioctadecylamidoglycyl spermineor lipofectamine and also in vivo in the Lewislung carcinoma model. The introduction of arginine in thelinker position promoted increased transfectionactivity, demonstrating that a diversity of linkers,such as short peptides or glycosides, can beintroduced into cationic lipids for targeted gene transfer.     

WQ-3810 exerts high inhibitory effect on quinolone-resistant DNA gyrase of Salmonella Typhimurium

ABSTRACT

The inhibitory effect of WQ-3810 on DNA gyrase was assayed to evaluate the potential of WQ-3810 as a candidate drug for the treatment of quinolone resistant Salmonella Typhymurium infection. The inhibitory effect of WQ-3810, ciprofloxacin and nalidixic acid was compared by accessing the drug concentration that halves the enzyme activity (IC₅₀) of purified S. Typhimurium wildtype and mutant DNA gyrase with amino acid substitution at position 83 or/and 87 in subunit A (GyrA) causing quinolone resistance. As a result, WQ-3810 reduced the enzyme activity of both wildtype and mutant DNA gyrase at a lower concentration than ciprofloxacin and nalidixic acid. Remarkably, WQ-3810 showed a higher inhibitory effect on DNA gyrase with amino acid substitutions at position 87 than with that at position 83 in GyrA. This study revealed that WQ-3810 could be an effective therapeutic agent, especially against quinolone resistant Salmonella enterica having amino acid substitution at position 87.     

Synthesis of 8-hydroxyquinoline glycoconjugates and preliminary assay of their β1,4-GalT inhibitory and anti-cancer properties

8-Hydroxyquinoline scaffold is a privileged structure used in designing a new active agents with therapeutic potential. Its connections with the sugar unit is formed to improve the pharmacokinetic properties. The broad spectrum of activity of quinoline derivatives, especially glycoconjugates, is often associated with the ability to chelate metal ions or with the ability to intercalate into DNA. Simple and effective methods of synthesis glycoconjugates of 8-hydroxyquinoline and 8-hydroxyquinaldine derivatives, containing an O-glycosidic bond or a 1,2,3-triazole linker in their structure, have been developed. The obtained glycoconjugates were tested for their ability to inhibit β-1,4-Galactosyltransferase, as well as inhibit cancer cell proliferation. It was found that used glycoconjugation strategy influenced both improvement of activity and improvement of the bioavailability of 8-HQ derivatives. Their activity depends on type of attached sugar, presence of protecting groups in sugar moiety and presence of a linker between sugar and quinolone aglycone.     

An unexpected interaction between the modular polyketide synthases,erythromycin DEBS1 and pikromycin PikAIV,leads to efficient triketide lactone synthesis

An unusual feature of the 6-module pikromycin polyketide synthase (PikPKS, PikAI-PikAIV) of S. venezuelae is the ability to generate both 12- and 14-membered ring macrolides. The PikAIV component containing the last extension module and a thioesterase domain is responsible for generating both of these products. In the case of the 12-membered ring macrolide, an acyl-enzyme intermediate on PikAIII is able to efficiently "skip" the last extension step and is cyclized by the TE domain of PikAIV, presumably as a result of a PikAIII-PikAIV interaction. Herein we report that plasmid-based expression (pBK3) of DEBS1, which comprises the loading domain and the first two modules of the Saccharopolyspora erythrea 6-deoxyerythronolide B synthase, in S. venezuelae leads to efficient 15 +/- 3 mg/L production of triketide lactone products (TKLs). Comparable levels of TKLs were observed with a plasmid (pBK1) which expressed DEBS1 fused to a TE domain (DEBS1-TE). These results are in stark contrast to previous in vivo and in vitro analyses, where only DEBS1-TE efficiently produces TKLs. Levels of TKLs decreased dramatically with expression of DEBS1 in both pikAIV and pikAIII-pikAIV deletion hosts (0.5 mg/L), but not DEBS1-TE, and could be partially restored by addition of a PikAIV complementation plasmid. These data suggest that PikAIV is able to efficiently catalyze formation of 6-membered lactone ring products from acyl-bound intermediates on DEBS1 in a manner analogous to that observed for 12-membered macrolide products from PikAIII. Significant sequence similarity and length of the C-terminal linker region of PikAIII and DEBS1 suggest that this region may be responsible for the interaction with PikAIV. A replacement of this linker region of DEBS1 with the corresponding region of PikAI led to a 95% decrease in TKL levels in S. venezuelae, consistent with this hypothesis.     

Synthesis of sugar fatty acid esters using a β-xylosidase from <Emphasis Type="Italic">Aspergillus awamori</Emphasis> for transxylosylation

Aspergillus awamori K4 β-xylosidase has broad acceptor specificity. It has been used to synthesize a sugar fatty acid ester via its transxylosylation activity. One xylosyl residue was initially transferred to hexamethylene glycol as a linker with a yield of 0.36 g/g xylobiose. Linoleic acid was subsequently linked to one terminal hydroxyl side of the transfer product hydroxyhexyl xyloside through an esterification reaction catalyzed by a lipase. The synthesis of hexyl linoleoyl xyloside was confirmed by TOF-MS analysis. The binding with a linker improved the esterification reaction because of the hydrophobic hexamethylene chain and also prevented steric hindrance by the xylosyl residue. This sugar fatty acid ester synthesis method using transglycosylation should facilitate the production of emulsifiers or surfactants with various functions.     

Oligonucleotides conjugated to short lysine chains

A new method for synthesizing oligonucleotide peptide conjugates by an in-line approach is presented. A phosphorothioate oligonucleotide with the sequence of bcl-2 targeted Oblimersen by employing a modified 2'-amino-2'-desoxy-uridine nucleotide bearing a succinyl linker at the 2' position was prepared. The carboxyl group was protected for solid-phase synthesis as the benzyl ester. Ester cleavage was afforded by a phase transfer reaction using palladium nanoparticles as catalyst and cyclohexadiene as hydrogen donor. Short tails of up to three lysyl residues were conjugated to the oligonucleotide by an inverse stepwise peptide synthesis. The conjugates were characterized by HPLC, mass spectrometry, and circular dichroism. Influence of lysyl tails on CD spectra were minimal. Melting profiles revealed only minimal destabilizing effects on duplexes by conjugation of peptides.     

Synthesis and biological studies of 5-aminolevulinic acid-containing dendrimers for photodynamic therapy.

Using a convergent growth approach, a series of novel 5-aminolevulinic acid (ALA)-containing dendrimers have been synthesized. In these molecules, ALA residues are attached to the periphery by ester linkages, with amide bonds connecting the dendrons. Three first-generation dendrimers, bearing either 6 or 9 ALA residues, were synthesized by attachment of a tris(Boc-protected ALA)-containing wedge (1) to a di- or tripodent aromatic, or tripodent aliphatic core. Two second generation 18-ALA-containing dendrimers were also synthesized using a 3,3'-iminodipropionic acid spacer unit between wedge 1 and the aromatic core. These compounds differed only in the distance between the core and the linker unit. The Boc-protected dendrimers were deprotected using trifluoroacetic acid and isolated as their TFA salts. The potential of these ALA ester dendrimers as macromolecular prodrugs for photodynamic therapy has been demonstrated in the tumorigenic keratinocyte PAM 212 cell line.     

6-Alkylquinolone-3-carboxylic acid tethered to macrolides synthesis and antimicrobial profile

Two series of clarithromycin and azithromycin derivatives with terminal 6-alkylquinolone-3-carboxylic unit with central ether bond in the linker were prepared and tested for antimicrobial activity. Quinolone-linker intermediates were prepared by Sonogashira-type C(6)-alkynylation of 6-iodo-quinolone precursors. In the last step, 4″ site-selective acylation of 2′-protected macrolides was completed with the EDC reagent, which selectively activated a terminal, aliphatic carboxylic group in dicarboxylic intermediates. Antimicrobial activity of the new series of macrolones is discussed. The most potent compound, 4″-O-{6-[3-(3-carboxy-1-ethyl-4-oxo-1,4-dihydroquinolin-6-yl)-propoxy]-hexanoyl}-azithromycin (10), is highly active against bacterial respiratory pathogens resistant to macrolide antibiotics and represents a promising lead for further investigation.     

Novel 9a-carbamoyl- and 9a-thiocarbamoyl-3-decladinosyl-6-hydroxy and 6-methoxy derivatives of 15-membered macrolides

An efficient method for the synthesis of diverse 9a-carbamoyl- and 9a-thiocarbamoyl-3-decladinosyl-6-hydroxy and 3-decladinosyl-6-methoxy derivatives of 15-membered azalides has been developed. These derivatives bear various alkyl and aryl groups attached to macrolide scaffold through urea or thiourea moieties at 9a position. Chemical transformations of hydroxy group at position C-3 afforded range of ketolides, anhydrolides, hemiketals, cyclic ethers, and acylides. It has been shown that 6-hydroxy and 6-methoxy derivatives undergo different chemical transformations under otherwise identical reaction conditions. Antimicrobial properties of prepared compounds were evaluated.     

Nitroquinolones with broad-spectrum antimycobacterial activity in vitro.

During search on quinolonecarboxylic acids we used a facile, convenient two- or three-step procedure to synthesize new quinolone analogs, bearing at the C-7 position alkylamino substituents, and at the C-6 position a fluorine or alternatively a nitro group. The new derivatives were tested against both Gram-positive and Gram-negative bacteria and against a number of different mycobacteria. In vitro assays showed 1-tert-butyl-7-tert-butylamino-6-nitro-1,4-dihydro-4-quinolone-3-carboxy lic acid to be a potent inhibitor of Streptococcus and Staphylococcus with potencies superior to those of ofloxacin and ciprofloxacin, used as reference drugs. Some 6-nitroquinolones were found to exert good inhibiting activities against Mycobacterium tuberculosis and various atypical mycobacteria, whereas the 6-fluoro counterparts showed poor or no activity against this bacterium.     

Genetic basis of quinolone resistance and epidemiology of resistant and susceptible isolates of porcine Campylobacter coli strains

AIMS: The aims of this study were to investigate the epidemiology of quinolone-resistant and -susceptible porcine isolates of Campylobacter coli and to characterize the genetic basis of quinolone resistance. METHODS AND RESULTS: Penner serotyping and flagellin gene sequence polymorphisms were used to investigate the epidemiology of the C. coli isolates. A total of 55 isolates were included, of which 30 were paired resistant and susceptible isolates from 15 pigs. Amplification of gyrA, gyrB and parC, followed by direct sequencing of amplicons was used to identify mutations in the targets of quinolones. Overall, 31 of the isolates were resistant to ciprofloxacin (minimum inhibitory concentrations (MIC), 2- >or = 32 microg x ml(-1)). Thirteen DdeI-flaA profiles were observed and resistant and susceptible strains were identified for nine profiles. The majority of resistant strains exhibited either profile 1 or 6. While profile 1 comprised susceptible and resistant strains, all of the strains with profile 6 were resistant to ciprofloxacin. The serogroup (O:24) of the profile 6 strains was identical. The only other serogroup to be uniformly associated with quinolone resistance was O:5. Strains with this phenotype comprised a number of genotypes, including profile 1. Only four of the paired isolates from individual pigs had the same profile. The genetic basis of quinolone resistance was investigated in two strains with ciprofloxacin MICs of 2 and > or = 32 miccrog x ml(-1), respectively. The amino acid substitution of isoleucine for threonine at position 86 was identified in the GyrA proteins from both strains. No mutations were identified in the GyrB proteins. CONCLUSIONS: There was an association between two of the genotypes, serotypes 5 and 24, and quinolone resistance. The association between genotype, serotype and resistance in C. coli isolates has not been reported previously. Only the mutation in GyrA associated with quinolone resistance was identified. No mutations in GyrB were identified. Amplification products of parC were not obtained and it may be that this gene is not present in some Campylobacter spp. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides data on the distribution of ciprofloxacin resistance between subtypes of C. coli.     

3-Aminoxypropionate-based linker system for cyclization activation in prodrug design

A novel linker system based on 3-aminoxypropionate was designed and evaluated for drug release using proteolysis as an activation trigger followed by intramolecular cyclization. The hydroxylamine moiety present in the linker system enabled faster release of the parent drug from the linker–drug conjugate at lower pH as compared to an aliphatic amine moiety. Introduction of two methyl groups strategically at the α position to the carboxylate in the linker further improved the rate of cyclization by nearly 2-fold. The 3-aminoxypropionate linker was successfully applied to a model prodrug for protease activation using α-chymotrypsin as the activating enzyme; the activation of the model prodrug bearing the 3-aminoxypropionate linker was 136 times faster than the corresponding model prodrug bearing an amine linker.     

Synthesis of a lipid derivative of the antitumor agent methotrexate

A lipophilic methotrexate prodrug capable of incorporation into membranes of carrier liposomes was synthesized. The conjugate consists of a lipophilic rac-1,2-dioleoylglycerol anchor connected to methotrexate through beta(Ala)-N-carbonylmethylene linker, which should be located in the polar region of the lipid bilayer. The ester bond between the hydrophilic linker and the antitumor agent can be hydrolyzed by intracellular esterases. The liposomal formulation of the prodrug exhibited a cytotoxic activity in vitro. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.     

The juxtamembrane sequence of cytochrome P-450 2C1 contains an endoplasmic reticulum retention signal

The N-terminal signal anchor of cytochrome P-450 2C1 mediates retention in the endoplasmic reticulum (ER) membrane of several reporter proteins. The same sequence fused to the C terminus of the extracellular domain of the epidermal growth factor receptor permits transport of the chimeric protein to the plasma membrane. In the N-terminal position, the ER retention function of this signal depends on the polarity of the hydrophobic domain and the sequence KQS in the short hydrophilic linker immediately following the transmembrane domain. To determine what properties are required for the ER retention function of the signal anchor in a position other than the N terminus, the effect of mutations in the linker and hydrophobic domains on subcellular localization in COS1 cells of chimeric proteins with the P-450 signal anchor in an internal or C-terminal position was analyzed. For the C-terminal position, the signal anchor was fused to the end of the luminal domain of epidermal growth factor receptor, and green fluorescent protein was additionally fused at the C terminus of the signal anchor for the internal position. In these chimeras, the ER retention function of the signal anchor was rescued by deletion of three leucines at the C-terminal side of its hydrophobic domain; however, deletion of three valines from the N-terminal side did not affect transport to the cell surface. ER retention of the C-terminal deletion mutants was eliminated by substitution of alanines for glutamine and serine in the linker sequence. These data are consistent with a model in which the position of the linker sequence at the membrane surface, which is critical for ER retention, is dependent on the transmembrane domain.