The Benzothiazine Core as a Novel Motif for DNA-Binding Small Molecules.

A new series of 4H-1,3-benzothiazine dyes were prepared and fully characterized in an aqueous medium. Benzothiazine salts were synthesized either through the classical synthetic pathway using Buchwald-Hartwig amination or through economical and environmentally friendly electrochemical synthesis. The latest synthetic approach employs successful electrochemical intramolecular dehydrogenative cyclization of N-benzylbenzenecarbothioamides to form 4H-1,3-benzothiazines. 4H-1,3-Benzothiazines were evaluated as novel DNA/RNA probes. Through the use of several methods such as UV/vis spectrophotometric titrations, circular dichroism and thermal melting experiments, the binding of four benzothiazine-based molecules to polynucleotides was examined. Compounds 1 and 2 acted as DNA/RNA groove binders, thus suggesting the potential of these compounds as novel DNA/RNA probes. This is a proof-of-concept study and will be expanded to include SAR/QSAR studies.

Unprecedented Epimerization of an Azithromycin Analogue: Synthesis, Structure and Biological Activity of 2'-Dehydroxy-5″-Epi-Azithromycin.

Certain macrolide antibiotics, azithromycin included, possess anti-inflammatory properties that are considered fundamental for their efficacy in the treatment of chronic inflammatory diseases, such as diffuse pan-bronchiolitis and cystic fibrosis. In this study, we disclose a novel azithromycin analog obtained via Barton-McCombie oxidation during which an unprecedented epimerization on the cladinose sugar occurs. Its structure was thoroughly investigated using NMR spectroscopy and compared to the natural epimer, revealing how the change in configuration of one single stereocenter (out of 16) profoundly diminished the antimicrobial activity through spatial manipulation of ribosome binding epitopes. At the same time, the anti-inflammatory properties of parent macrolide were retained, as demonstrated by inhibition of LPS- and cigarette-smoke-induced pulmonary inflammation. Not surprisingly, the compound has promising developable properties including good oral bioavailability and a half-life that supports once-daily dosing. This novel anti-inflammatory candidate has significant potential to fill the gap in existing anti-inflammatory agents and broaden treatment possibilities.

Synthesis of novel adamantyl and homoadamantyl-substituted ß-hydroxybutyric acids.

Several new adamantyl and homoadamantyl-substituted [Formula: see text]-hydroxybutyric acids, 2-[2-(1-adamantyl)ethyl]-3-hydroxybutyric acid (2), 2-(3-homoadamantyl)-3-hydroxybutyric acid (3), and 2-(1-homoadamantyl)-3-hydroxybutyric acid (4), analogues of the 2-(1-adamantyl)-3-hydroxybutyric acid (1), have been prepared as mixtures of diastereoisomers using selective reduction of corresponding [Formula: see text]-keto esters or aldol condensation of the corresponding carboxylic acid and acetaldehyde. The rearrangement of adamantylmethyl and 3-homoadamantyl groups provided entry to both 3-homoadamantyl and 1-homoadamantyl-substituted hydroxy acids 3 and 4, respectively. The relative configurations of diastereoisomers 3 and 4 have been determined by NMR spectroscopy comparing the values of coupling constants. Adamantyl-substituted [Formula: see text]-hydroxybutyric acid 2 has also been prepared in enantiomerically pure form by Evan's asymmetric synthesis and the absolute configuration has been determined by X-ray crystallography. Contrary to the long-chain acid 2, the attempt to prepare short-chain hydroxy acids 1 and 4 by the same method failed indicating pronounced sensitivity of the used method to the vicinity of the bulky cage group.

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.

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.

Impact of stereochemistry on the biological activity of novel oleandomycin derivatives.

A set of 8-methylene-, 8-methyl-, and 8-methyl-9-dihydro-oleandomycin derivatives having different combinations of stereochemistries at positions C-8 and/or C-9 have been prepared in a chemoselective and stereoselective manner and tested in vitro for antibacterial activity and inhibition of IL-6 production. Configurations of the stereocenters at C-8 and C-9 were determined using 2D NMR techniques. We have shown that change of stereochemistry at these positions can exert a major influence on antibacterial activity as well as IL-6 inhibition, providing novel macrolide derivatives with diminished antibacterial and potent anti-inflammatory activity. In addition, the anti-inflammatory activity observed in vitro was confirmed in an in vivo model of lipopolysaccharide-induced inflammation.

Novel 9a,11-bridged azalides: One-pot synthesis of N'-substituted 2-imino-1,3-oxazolidines condensed to an azalide aglycone.

An efficient one-pot method for the synthesis of novel 9a,11-bridged 15-membered 9a-aza-9-deoxo-9a-homoerythromycin A and its 6-O-methyl analogue has been developed. The novel bicyclic azalide scaffold is characterized by an N'-substituted-2-imino-1,3-oxazolidine moiety bound to a macrolactone ring between positions 9a and 11. Removal of the cladinose sugar from the starting compounds allows easy preparation of a small series of such bicyclic 3-keto and 3,6-hemiketal azalide derivatives. A mechanism for the formation of N'-substituted-2-imino-1,3-oxazolidines is discussed. Antibacterial properties of the prepared compounds were evaluated.

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.

Clarithromycin inhibits autophagy in colorectal cancer by regulating the hERG1 potassium channel interaction with PI3K.

We have studied how the macrolide antibiotic Clarithromycin (Cla) regulates autophagy, which sustains cell survival and resistance to chemotherapy in cancer. We found Cla to inhibit the growth of human colorectal cancer (CRC) cells, by modulating the autophagic flux and triggering apoptosis. The accumulation of cytosolic autophagosomes accompanied by the modulation of autophagic markers LC3-II and p62/SQSTM1, points to autophagy exhaustion. Because Cla is known to bind human Ether-à-go-go Related Gene 1 (hERG1) K+ channels, we studied if its effects depended on hERG1 and its conformational states. By availing of hERG1 mutants with different gating properties, we found that fluorescently labelled Cla preferentially bound to the closed channels. Furthermore, by sequestering the channel in the closed conformation, Cla inhibited the formation of a macromolecular complex between hERG1 and the p85 subunit of PI3K. This strongly reduced Akt phosphorylation, and stimulated the p53-dependent cell apoptosis, as witnessed by late caspase activation. Finally, Cla enhanced the cytotoxic effect of 5-fluorouracil (5-FU), the main chemotherapeutic agent in CRC, in vitro and in a xenograft CRC model. We conclude that Cla affects the autophagic flux by impairing the signaling pathway linking hERG1 and PI3K. Combining Cla with 5-FU might be a novel therapeutic option in CRC.

Correction: Clarithromycin inhibits autophagy in colorectal cancer by regulating the hERG1 potassium channel interaction with PI3K.

The financial support for this Article was not fully acknowledged. The acknowledgements should have included the following: We thank M. Lulli (University of Florence, Italy) for acquiring images of immunofluorescence-labeled cells. This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro (#15627, #21510 and #19766 to A.A.); PAR FAS-Linea di Azione 1.1-Azione 1.1.2-Bando FAS Salute. 2014 (DD 4042/ 2014) Project OMITERC to A.A.; FAR 2018 to A.B.

Tumor-cell-targeted methionine-enkephalin analogues containing unnatural amino acids: design, synthesis, and in vitro antitumor activity.

A series of new peptides (8-25) containing different unnatural amino acids of the adamantane type (1-6), was synthesized. Possible cytotoxic activity on human cervical adenocarcinoma (HeLa), larynx carcinoma (HEp-2), colon carcinomas (HT-29, Caco-2), poorly differentiated cells from lymph node metastasis of colon carcinoma (SW-620), mammary gland adenocarcinoma (MCF-7), and melanoma (HBL) cells were tested by the MTT assay. The results were compared with the effect of methionine-enkephalin (Tyr-Gly-Gly-Phe-Met, or opioid growth factor, OGF), and its shorter N-terminal fragments. Peptide analogues containing C(alpha alpha)-dialkylated glycine (Aaa1, 1) or C(alpha)-alkylated glycine (Aaa2, 2) amino acid residues showed antitumor activity against melanoma, larynx carcinoma, colon carcinomas, and colon metastasis cell lines in vitro. The pentapeptide Tyr-(R,S)-Aaa2-Gly-Phe-Met (18) was the most effective analogue especially against the most antitumor drug-resistant cell lines HEp-2 and SW-620. Apoptosis as a mode of cell death was confirmed in these tumor cells after exposure to pentapeptide 18.

The design of novel classes of macrolides for neutrophil-dominated inflammatory diseases.

Neutrophil-dominated inflammatory diseases, like chronic obstructive pulmonary disease, cystic fibrosis, bronchiectasis, bronchiolitis obliteras syndrome and non-eosinophilic asthma, present a significant medical problem lacking adequate therapy. Macrolide antibiotics have been reported to be effective in the treatment of the aforementioned diseases, for reasons unrelated to their antibacterial action. This has resulted in research activities aimed at gaining a better understanding of the immunomodulatory actions of macrolides and the synthesis of various novel anti-inflammatory macrolides without antimicrobial activity. Despite the difficult chemistry and lack of an extensive knowledge for their mechanism of action, several interesting molecules from this class, including potential clinical candidates, are on the horizon.

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.

Could LogP be a principal determinant of biological activity in 18-crown-6 ethers? Synthesis of biologically active adamantane-substituted diaza-crowns.

18-crown-6 ethers are known to exert their biological activity by transporting K(+) ions across cell membranes. Using non-linear Support Vector Machines regression, we searched for structural features that influence antiproliferative activity in a diverse set of 19 known oxa-, monoaza- and diaza-18-crown-6 ethers. Here, we show that the logP of the molecule is the most important molecular descriptor, among ∼1300 tested descriptors, in determining biological potency (R(2)(cv) = 0.704). The optimal logP was at 5.5 (Ghose-Crippen ALOGP estimate) while both higher and lower values were detrimental to biological potency. After controlling for logP, we found that the antiproliferative activity of the molecule was generally not affected by side chain length, molecular symmetry, or presence of side chain amide links. To validate this QSAR model, we synthesized six novel, highly lipophilic diaza-18-crown-6 derivatives with adamantane moieties attached to the side arms. These compounds have near-optimal logP values and consequently exhibit strong growth inhibition in various human cancer cell lines and a bacterial system. The bioactivities of different diaza-18-crown-6 analogs in Bacillus subtilis and cancer cells were correlated, suggesting conserved molecular features may be mediating the cytotoxic response. We conclude that relying primarily on the logP is a sensible strategy in preparing future 18-crown-6 analogs with optimized biological activity.

Modeling cellular pharmacokinetics of 14- and 15-membered macrolides with physicochemical properties.

Macrolides with 14- and 15-membered ring are characterized by high and extensive tissue distribution, as well as good cellular accumulation and retention. Since macrolide structures do not fit the Lipinski rule of five, macrolide pharmacokinetic properties cannot be successfully predicted by common models based on data for small molecules. Here we describe the development of the first models for macrolide cellular pharmacokinetics. By comparison of cellular accumulation and retention in six human primary cell cultures of leukocytic and lung origin, as well as in lung carcinoma cell line NCI-H292, this cell line was found to be an adequate representative cell type for modeling macrolide cellular pharmacokinetics. Accumulation and retention in the NCI-H292 cells, as well as various physicochemical properties, were determined for a set of 48 rationally designed basic macrolide compounds. Classification models for predicting macrolide cellular accumulation and retention were developed using relatively easily determined and conceptually simple descriptors: experimentally determined physicochemical parameters ChromlogD and CHI IAM, as well as a calculated number of positively charged atoms (POS). The models were further tested and improved by addition of 37 structurally diverse macrolide molecules.

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.

Solid-phase synthesis of lipidated peptides.

A new flexible and efficient methodology for the solid-phase synthesis of lipidated peptides has been developed. The approach is based on the use of previously synthesized building blocks and overcomes the limitations of previously reported methods, since long doubly lipidated peptides can be synthesized by using this route. Furthermore, it was thus possible to prepare a large number of N- and H-Ras peptides bearing a wide range of reporter and/or linking groups--efficient tools for the investigation of biological processes. In terms of efficiency and flexibility this solid-phase method is superior to the solution-phase synthesis. It gives pure peptides in multimilligram amounts within a much shorter time and with superior overall yield.

Induction of influenza type A virus-specific resistance by immunization of mice with a synthetic multiple antigenic peptide vaccine that contains ectodomains of matrix protein 2.

Matix protein 2 (M2) is a transmembrane protein of influenza type A virus. It contains a 23 aa long ectodomain (M2e) that is highly conserved amongst human influenza type A viruses. M2e-specific antibodies have been shown to restrict virus growth in vitro and in vivo and thus have the potential of providing cross-reactive resistance to influenza type A virus infection. We attempted to induce M2e-specific protection with synthetic multiple antigen peptide (MAP) constructs that contained covalently linked M2e- and Th-determinant peptides. Mice, vaccinated twice by the intranasal (i.n.) route with adjuvanted M2e-MAPs exhibited significant resistance to virus replication in all sites of the respiratory tract. Compared to mice primed by two consecutive heterosubtypic infections, resistance was of similar strength in nasal and tracheal tissue but lower in pulmonary tissue. Importantly, the protection in M2e-MAP- and infection-immunized mice appeared to be mediated by distinct immune mechanisms. This suggests that stronger protection may be achievable by combining both protective activities.