New Bicyclic Azalide Macrolides Obtained by Tandem Palladium Catalyzed Allylic Alkylation/Conjugated Addition Reaction.

Unprecedented tandem allylic alkylation/intermolecular Michael addition was used in the preparation of novel bicyclic azalides. NMR spectroscopy was used not only to unambiguously determine and characterize the structures of these unexpected products of chemical reaction but also to investigate the effect the rigid bicyclic modification has on the conformation of the whole molecule. Thus, some of the macrolides prepared showed antibacterial activity in the range of well-known antibiotic drug azithromycin.

A novel class of fast-acting antimalarial agents: Substituted 15-membered azalides.

BACKGROUND AND PURPOSE: Efficacy of current antimalarial treatments is declining as a result of increasing antimalarial drug resistance, so new and potent antimalarial drugs are urgently needed. Azithromycin, an azalide antibiotic, was found useful in malaria therapy, but its efficacy in humans is low. EXPERIMENTAL APPROACH: Four compounds belonging to structurally different azalide classes were tested and their activities compared to azithromycin and chloroquine. in vitro evaluation included testing against sensitive and resistant Plasmodium falciparum, cytotoxicity against HepG2 cells, accumulation and retention in human erythrocytes, antibacterial activity, and mode of action studies (delayed death phenotype and haem polymerization). in vivo assessment enabled determination of pharmacokinetic profiles in mice, rats, dogs, and monkeys and in vivo efficacy in a humanized mouse model. KEY RESULTS: Novel fast-acting azalides were highly active in vitro against P. falciparum strains exhibiting various resistance patterns, including chloroquine-resistant strains. Excellent antimalarial activity was confirmed in a P. falciparum murine model by strong inhibition of haemozoin-containing trophozoites and quick clearance of parasites from the blood. Pharmacokinetic analysis revealed that compounds are metabolically stable and have moderate oral bioavailability, long half-lives, low clearance, and substantial exposures, with blood cells as the preferred compartment, especially infected erythrocytes. Fast anti-plasmodial action is achieved by the high accumulation into infected erythrocytes and interference with parasite haem polymerization, a mode of action different from slow-acting azithromycin. CONCLUSION AND IMPLICATIONS: The hybrid derivatives described here represent excellent antimalarial drug candidates with the potential for clinical use in malaria therapy.

Current Trends in Macrocyclic Drug Discovery and beyond-Ro5.

This chapter will discuss the recent literature of macrocycles and drug-like property space moving beyond the rule of five (bRo5). Trends in chemical classes that fall within this definition are discussed and the impact of the latest technologies in the field assessed. The physicochemical properties, which have provided both successes and challenges, especially in scale-up, are discussed. A recent patent literature is reviewed and the chapter concludes with a perspective on the future of macrocyclic drug discovery.

Around the macrolide - Impact of 3D structure of macrocycles on lipophilicity and cellular accumulation.

The aim of this study was to investigate lipophilicity and cellular accumulation of rationally designed azithromycin and clarithromycin derivatives at the molecular level. The effect of substitution site and substituent properties on a global physico-chemical profile and cellular accumulation of investigated compounds was studied using calculated structural parameters as well as experimentally determined lipophilicity. In silico models based on the 3D structure of molecules were generated to investigate conformational effect on studied properties and to enable prediction of lipophilicity and cellular accumulation for this class of molecules based on non-empirical parameters. The applicability of developed models was explored on a validation and test sets and compared with previously developed empirical models.

Structure and conformational analysis of spiroketals from 6-O-methyl-9(E)-hydroxyiminoerythronolide A.

Three novel spiroketals were prepared by a one-pot transformation of 6-O-methyl-9(E)-hydroxyiminoerythronolide A. We present the formation of a [4.5]spiroketal moiety within the macrolide lactone ring, but also the unexpected formation of a 10-C=11-C double bond and spontaneous change of stereochemistry at position 8-C. As a result, a thermodynamically stable structure was obtained. The structures of two new diastereomeric, unsaturated spiroketals, their configurations and conformations, were determined by means of NMR spectroscopy and molecular modelling. The reaction kinetics and mechanistic aspects of this transformation are discussed. These rearrangements provide a facile synthesis of novel macrolide scaffolds.

Fluorescently labeled macrolides as a tool for monitoring cellular and tissue distribution of azithromycin.

Exceptional therapeutic effects of macrolides in treating various infections and inflammatory conditions can be significantly contributed to their unique pharmacokinetic properties. Macrolides accumulate in cells and tissues, with concentrations usually 10 to more than 100 times higher of those measured in plasma. Intracellular distribution of macrolides has so far been examined using extensive subcellular fractionation techniques, radiolabeled compounds and conventional pharmacokinetic methods. In this study we evaluated four fluorescently labeled macrolides on their applicability to monitor azithromycin distribution in vitro and in vivo. 9-Deoxo-9a-{3-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]propyl}-9a-aza-9a-homoerythromycin A (9a-NBD-azithromycin) was selected as a compound with most similar cellular pharmacokinetics to azithromycin. 9a-NBD-azithromycin demonstrated antimicrobial properties comparable to azithromycin, displayed the same biological activity profile in LPS-stimulated J774A.1 murine macrophage cells and, even though it accumulated in cells almost 50% more than azithromycin, it showed same rate of retention. Identical to azithromycin, 9a-NBD-azithromycin was localized in lysosomes of J774A.1 cells. Two hours after 9a-NBD-azithromycin was administered intraperitonally to mice, a strong fluorescent signal was located in kidneys and liver and slightly weaker in the spleen. In kidneys, the signal was concentrated in tubuli, and glomeruli were negative. Patchy florescence in hepatocytes supports lysosomal cellular localization. Weaker staining of white pulp compared to red pulp of spleen is in agreement with lower accumulation of azithromycin in lymphocytes compared to other cell types present. We conclude that 9a-NBD-azithromycin can be used as a fluorescent analog of azithromycin to visualize its distribution in in vitro systems, and is also suitable for in vivo studies.

N'-substituted-2'-O,3'-N-carbonimidoyl bridged macrolides: novel anti-inflammatory macrolides without antimicrobial activity.

Macrolide antibiotics, like erythromycin, clarithromycin, and azithromycin, possess anti-inflammatory properties. These properties are considered fundamental to the efficacy of these three macrolides in the treatment of chronic inflammatory diseases like diffuse panbronchiolitis and cystic fibrosis. However, long-term treatment with macrolide antibiotics presents a considerable risk for promotion of bacterial resistance. We have examined antibacterial and anti-inflammatory effects of a novel macrolide class: N'-substituted 2'-O,3'-N-carbonimidoyl bridged erythromycin-derived 14- and 15-membered macrolides. A small focused library was prepared, and compounds without antimicrobial activity, which inhibited IL-6 production, were selected. Data analysis led to a statistical model that could be used for the design of novel anti-inflammatory macrolides. The most promising compound from this library retained the anti-inflammatory activity observed with azithromycin in lipopolysaccharide-induced pulmonary neutrophilia in vivo. Importantly, this study strongly suggests that antimicrobial and anti-inflammatory activities of macrolides are independent and can be separated, which raises development plausibility of novel anti-inflammatory therapeutics.

Novel desosamine-modified 14- and 15-membered macrolides without antibacterial activity.

Novel modifications of the desosamine sugar of 14- and 15-membered antibacterial macrolides, in which the desosamine was fused with N-substituted-1,3-oxazolidin-2-ones, were developed in order to completely suppress antibacterial activity and make them promising agents for other biological targets. The synthesis of such bicyclic desosamine derivatives, especially 1,3-oxazolidin-2-one formation, was optimized and conducted under mild conditions without a need for protection/deprotection steps for other functional groups. A focused series of novel desosamine-modified macrolide derivatives was prepared and their antibacterial activities tested. It was shown that these macrolide derivatives do not possess any residual antibacterial activity.

Design, synthesis, and in vitro activity of novel 2'-O-substituted 15-membered azalides.

Malaria remains one of the most widespread human infectious diseases, and its eradication will largely depend on antimalarial drug discovery. Here, we present a novel approach to the development of the azalide class of antimalarials by describing the design, synthesis, and characterization of novel 2'-O-substituted-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A derivatives consisting of different quinoline moieties covalently liked to a 15-membered azalide scaffold at position 2'. By multistep straightforward synthesis, 19 new, stable, and soluble compounds were created and biologically profiled. Most active compounds from the 4-amino-7-chloroquinoline series showed high selectivity for P. falciparum parasites, and in vitro antimalarial activity improved 1000-fold over azithromycin. Antimalarial potency was equivalent to chloroquine against the sensitive strain (3D7A) and up to 48-fold enhanced over chloroquine against the chloroquine-resistant strain (W2). Concurrently, the antibacterial activity of the compounds was eliminated, thus facilitating the development of malaria-specific macrolide agents.

Novel hybrid molecules based on 15-membered azalide as potential antimalarial agents.

Malaria remains the most prevalent tropical disease, and due to the spread of resistant parasites novel therapeutics are urgently needed. Azithromycin has shown potential in malaria treatment so we designed hybrid azalide molecules with the aim to improve activity against and selectivity for the malaria parasite. Novel hybrid molecules comprising 4-aminoquinoline moiety covalently liked to 15-membered azalide scaffold at position C-3' were synthesized and biologically evaluated. Antimalarial testing against Plasmodium falciparum sensitive and resistant strains confirmed the improved in vitro activity over azithromycin and chloroquine. Selectivity of the compounds (HepG2 IC(50)/P. falciparum IC(50) ratio) for the parasite was high (100-2700) and their antibacterial activity diminished. Even though oral bioavailability determined for compound 12 was low, novel quinoline C-3'-substituted 15-membered azalides represent an interesting subclass of antimalarial macrolides that need further research and evaluation.

Valosin containing protein (VCP) interacts with macrolide antibiotics without mediating their anti-inflammatory activities.

In addition to antibacterial activity, some macrolide antibiotics, such as azithromycin and clarithromycin, also exhibit anti-inflammatory properties in vitro and in vivo, although the targets and mechanism(s) of action remain unknown. The aim of the present study was to identify protein targets of azithromycin and clarithromycin which could potentially explain their anti-inflammatory effects. Using chemical proteomics approach, based on compound-immobilized affinity chromatography, valosin containing protein (VCP) was identified as a potential target of the macrolides. Validation studies confirmed the interaction of macrolides and VCP and gave some structural characteristics of this interaction. Cell based assays however, including the use of gene silencing and the study of VCP specific cellular functions in J774.A1 (murine macrophage) and IB3-1 (human cystic fibrotic epithelial) cell lines, failed to confirm an association between the binding of the macrolides to VCP and anti-inflammatory effects. These findings suggest the absence of an abundant high affinity protein target and the potential involvement of other biological molecules in the anti-inflammatory activity of macrolides.

Antimalarial activity of 9a-N substituted 15-membered azalides with improved in vitro and in vivo activity over azithromycin.

Novel classes of antimalarial drugs are needed due to emerging drug resistance. Azithromycin, the first macrolide investigated for malaria treatment and prophylaxis, failed as a single agent and thus novel analogues were envisaged as the next generation with improved activity. We synthesized 42 new 9a-N substituted 15-membered azalides with amide and amine functionalities via simple and inexpensive chemical procedures using easily available building blocks. These compounds exhibited marked advances over azithromycin in vitro in terms of potency against Plasmodium falciparum (over 100-fold) and high selectivity for the parasite and were characterized by moderate oral bioavailability in vivo. Two amines and one amide derivative showed improved in vivo potency in comparison to azithromycin when tested in a mouse efficacy model. Results obtained for compound 6u, including improved in vitro potency, good pharmacokinetic parameters, and in vivo efficacy higher than azithromycin and comparable to chloroquine, warrant its further development for malaria treatment and prophylaxis.

Synthesis of macrolones with central piperazine ring in the linker and its influence on antibacterial activity.

Three macrolides, clarithromycin, azithromycin and 11-O-Me-azithromycin have been selected for the construction of a series of new macrolone derivatives. Quinolone-linker intermediates are prepared by Sonogashira-type C(6)-alkynylation of 6-iodoquinolone precursors. The final macrolones, differing by macrolide moiety and substituents at the position N-1 of the quinolone or by the presence of an ethyl ester or free acid on the quinolone unit attached via a linker. The linker comprises of a central piperazine ring bonded to the 4″-O position of cladinose by 3-carbon ester or ether functionality. Modifications of the linker did not improve antibacterial properties compared to the previously reported macrolone compounds. Linker flexibility seems to play an important role for potency against macrolide resistant respiratory pathogens.

Synthesis and activity of new macrolones: conjugates between 6(7)-(2'-aminoethyl)-amino-1-cyclopropyl-3-carboxylic acid (2'-hydroxyethyl) amides and 4″-propenoyl-azithromycin.

A set of novel macrolones containing the flexible C8 basic linker and quinolone 3-(2'-hydroxyethyl)carboxamido group has been prepared and structurally characterized by NMR and IR spectroscopy, mass spectrometry and molecular modeling. The new compounds were evaluated in vitro against a panel of erythromycin-susceptible and erythromycin-resistant Gram-positive and Gram-negative bacterial strains. Compared to azithromycin, most of the compounds exhibited improved in vitro potency against the key respiratory pathogens.

Synthesis, activity and pharmacokinetics of novel antibacterial 15-membered ring macrolones.

Synthesis, antibacterial activity and pharmacokinetic properties of a novel class of macrolide antibiotics-macrolones-derived from azithromycin, comprising oxygen atom(s) in the linker and either free or esterified quinolone 3-carboxylic group, are reported. Selected compounds showed excellent antibacterial potency towards key erythromycin resistant respiratory pathogens. However, the majority of compounds lacked good bioavailability. The isopropyl ester, compound 35, and a macrolone derivative with an elongated linker 29 showed the best oral bioavailability in rats, both accompanied with an excellent overall microbiology profile addressing inducible and constitutive MLSb as well as efflux mediated macrolide resistance in streptococci, while compound 29 is more potent against staphylococci.

Discovery of novel ureas and thioureas of 3-decladinosyl-3-hydroxy 15-membered azalides active against efflux-mediated resistant Streptococcus pneumoniae.

A series of novel ureas and thioureas of 3-decladinosyl-3-hydroxy 15-membered azalides, were discovered, structurally characterized and biologically evaluated. They have shown good antibacterial activity against selected Gram-positive and Gram-negative bacterial strains. These include N″ substituted 9a-(N'-carbamoyl-γ-aminopropyl)- (6a,c), 9a-(N'-thiocarbamoyl-γ-aminopropyl)- (7a,e), 9a-[N'-(ß-cyanoethyl)-N'-(carbamoyl-γ-aminopropyl)]- (9a-c, 9g) 9a-[N'-(ß-cyanoethyl)-N'-(thiocarbamoyl-γ-aminopropyl)]-derivatives (10d-f) of 5-O-desosaminyl-9-deoxo-9-dihydro-9a-aza-9a-homoerythronolide A (3). Among the synthesized compounds thiourea 7a and urea 9b have shown substantially improved activity comparable to azithromycin (1) and significantly better activity than the 3-decladinosyl-azithromycin (2) and the parent 3-cladinosyl analogues against efflux-mediated resistant S. pneumoniae.

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.

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 omega-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.

Novel 8a-aza-8a-homoerythromycin--4''-(3-substituted-amino)propionates with broad spectrum antibacterial activity.

Fifteen-membered 8a-aza-8a-homoerythromycins derived from either erythromycin or clarithromycin have been acylated to form 4''-O-propenoyl derivative. These functionalized analogues underwent Michael reaction with primary or secondary amines to afford novel 8a-aza-8a-homoerythromycin-4''-(3-substituted-amino)propionates. This preparative sequence was adapted so that analogues could be made by parallel synthesis. Among them, 4-quinolone derivatives show particularly good antibacterial potency against macrolide resistant bacteria, comparable or better than azithromycin and telithromycin.