A few atoms make the difference: synthetic, CD, NMR and computational studies on antiviral and antibacterial activities of glycopeptide antibiotic aglycon derivatives.

Despite the close structural similarity between the heptapeptide cores of the glycopeptide antibiotics teicoplanin and ristocetin, synthetically modified derivatives of their aglycons show significantly different antibacterial and antiviral properties. The teicoplanin aglycon derivatives with one exception proved to be potent antibacterials but they did not exhibit anti-influenza virus activity. In contrast, the aglycoristocetin derivatives generally showed high anti-influenza virus activity and possessed moderate antibacterial activity. A systematic structure-activity relationship study has been carried out on ristocetin and teicoplanin aglycon derivatives, to explore which structural differences are responsible for these markedly different biological activities. According to electronic circular dichroism and in silico conformational studies, it was found that the differences in anti-influenza virus activity are mainly determined by the conformation of the heptapeptide core of the antibiotics controlled by the presence or absence of chloro substituents. Knowledge of the bioactive conformation will help to design new analogs with improved anti-influenza virus activity. For the teicoplanin derivatives, it was shown that derivatization to improve the antiviral efficacy was accompanied by a significant decrease in antibacterial activity.

Intracytoplasmic trapping of influenza virus by a lipophilic derivative of aglycoristocetin.

We report on a new anti-influenza virus agent, SA-19, a lipophilic glycopeptide derivative consisting of aglycoristocetin coupled to a phenylbenzyl-substituted cyclobutenedione. In Madin-Darby canine kidney cells infected with influenza A/H1N1, A/H3N2, or B virus, SA-19 displayed a 50% antivirally effective concentration of 0.60 µM and a selectivity index (ratio of cytotoxic versus antiviral concentration) of 112. SA-19 was 11-fold more potent than unsubstituted aglycoristocetin and was active in human and nonhuman cell lines. Virus yield at 72 h p.i. was reduced by 3.6 logs at 0.8 µM SA-19. In contrast to amantadine and oseltamivir, SA-19 did not select for resistance upon prolonged virus exposure. SA-19 was shown to inhibit an early postbinding step in virus replication. The compound had no effect on hemagglutinin (HA)-mediated membrane fusion in an HA-polykaryon assay and did not inhibit the low-pH-induced refolding of the HA in a tryptic digestion assay. However, a marked inhibitory effect on the transduction exerted by retroviral pseudoparticles carrying an HA or vesicular stomatitis virus glycoprotein (VSV-G) fusion protein was noted, suggesting that SA-19 targets a cellular factor with a role in influenza virus and VSV entry. Using confocal microscopy with antinucleoprotein staining, SA-19 was proven to completely prevent the influenza virus nuclear entry. This virus arrest was characterized by the formation of cytoplasmic aggregates. SA-19 appeared to disturb the endocytic uptake and trap the influenza virus in vesicles distinct from early, late, or recycling endosomes. The aglycoristocetin derivative SA-19 represents a new class of potent and broad-acting influenza virus inhibitors with potential clinical relevance.

A derivate of the antibiotic doxorubicin is a selective inhibitor of dengue and yellow fever virus replication in vitro.

A doxorubicin derivate, SA-17, that carries a squaric acid amide ester moiety at the carbohydrate (α-l-daunosaminyl) group was identified as a selective inhibitor of in vitro dengue virus (DENV) serotype 2 replication (50% effective concentration [EC(50)] = 0.34 ± 0.20 µg/ml [0.52 ± 0.31 µM]). SA-17 is markedly less cytostatic than the parent compound, resulting in a selectivity index value of ∼100. SA-17 also inhibits yellow fever virus 17D (YFV-17D) replication (EC(50) = 3.1 ± 1.0 µg/ml [4.8 ± 1.5 µM]), although less efficiently than DENV replication, but proved inactive against a variety of enveloped and nonenveloped viruses. SA-17 inhibits in vitro flavivirus replication in a dose-dependent manner, as was assessed by virus yield reduction assays and quantification of viral RNA by means of real-time quantitative reverse transcriptase PCR (RT-qPCR) (∼2 to 3 log reduction). The anti-DENV activity was confirmed using a Renilla luciferase-expressing dengue reporter virus. Time-of-drug-addition studies revealed that SA-17 acts at the very early stages of the viral replication cycle (i.e., virus attachment and/or virus entry). This observation was corroborated by the observation that SA-17, unlike the nucleoside analogue ribavirin, does not inhibit the replication of DENV subgenomic replicons. Preincubation of high-titer stocks of DENV or YFV-17D with ≥5 µg/ml SA-17 resulted in 100% inhibition of viral infectivity (≥3 log reduction). SA-17, however, did not prove virucidal.

Click reaction synthesis of carbohydrate derivatives from ristocetin aglycon with antibacterial and antiviral activity.

New sugar derivatives of ristocetin were prepared by copper-catalyzed 1,3-dipolar cycloaddition reaction using azido-ristocetin aglycon and various propargyl glycosides. Some of them were found to be active against gram-positive bacteria and showed favorable antiviral activity against the H1N1 subtype of influenza A virus.

Diazo transfer-click reaction route to new, lipophilic teicoplanin and ristocetin aglycon derivatives with high antibacterial and anti-influenza virus activity: an aggregation and receptor binding study.

Semisynthetic, lipophilic ristocetin and teicoplanin derivatives were prepared starting from ristocetin aglycon and teicoplanin psi-aglycon (N-acetyl-D-glucosaminyl aglycoteicoplanin). The terminal amino functions of the aglycons were converted into azido form by triflic azide. Copper catalyzed 1,3-dipolar cycloaddition reaction with lipophilic alkynes resulted in the title compounds. Two of the teicoplanin derivatives showed very good MIC and MBC values against various Gram-positive bacteria, including vanA enterococci. The aggregation and interaction of a n-decyl derivative with bacterial cell wall components was studied. One of the lipophilic ristocetin derivatives displayed favorable anti-influenza virus activity.

Anti-influenza virus activity and structure-activity relationship of aglycoristocetin derivatives with cyclobutenedione carrying hydrophobic chains.

Previous studies have demonstrated that glycopeptide compounds carrying hydrophobic substituents can have favorable pharmacological (i.e. antibacterial and antiviral) properties. We here report on the in vitro anti-influenza virus activity of aglycoristocetin derivatives containing hydrophobic side chain-substituted cyclobutenedione. The lead compound 8e displayed an antivirally effective concentration of 0.4 microM, which was consistent amongst influenza A/H1N1, A/H3N2 and B viruses, and a selectivity index > or =50. Structural analogues derived from aglycovancomycin were found to be inactive. The hydrophobic side chain was shown to be an important determinant of activity. The narrow structure-activity relationship and broad activity against several human influenza viruses suggest a highly conserved interaction site, which is presumably related to the influenza virus entry process. Compound 8e proved to be inactive against several unrelated RNA and DNA viruses, except for varicella-zoster virus, against which a favorable activity was noted.

N-glycosylthioureido aglyco-ristocetins without platelet aggregation activity.

The water-soluble N-methoxy-PEG-yl-, N-beta-D-glucopyranosyl- and N-beta-D-maltosylthioureido aglyco-ristocetin were prepared which, in contrast to ristocetin A, did not induce thrombocyte aggregation. The antibacterial activity of N-beta-D-maltosylthioureido aglyco-ristocetin A against MRSA was comparable to that of ristocetin A, while its activity against Enterococcus faecalis (VRE, TSE) is somewhat stronger when compared to those of vancomycin and ristocetin A.

A new series of glycopeptide antibiotics incorporating a squaric acid moiety. Synthesis, structural and antibacterial studies.

The aglycones of the antibiotics eremomycin, vancomycin and ristocetin (3, 4 and 6, respectively) were prepared by deglycosidation of the parent antibiotics with hydrogen fluoride, and complete assignation of their 1H, 13C and 15N spectra was performed. The squaric acid amide esters (11-14), were prepared from dimethyl squarate. The corresponding asymmetric diamides (16-19, 22, 23) were also synthesized using 4-phenylbenzylamine and triglycine. The advantage of the method is the high regioselectivity and that no protecting group strategy is required. Electrospray mass spectroscopic method was elaborated for the determination of the site of substitution of the modified antibiotics. The antibacterial activity of the prepared compounds is discussed in detail.

A new class of semisynthetic anthracycline glycoside antibiotics incorporating a squaric acid moiety.

Treatment of the squaric acid amide esters (7, 9) of anthracycline glycoside antibiotics with aliphatic and aromatic primary and secondary amines, amino acids, peptides and aminodeoxy sugars furnished the new asymmetric diamides 16-19, 25-30, 32, 34 and 38-40 in stereoselective reactions which do not require protecting group-manipulations. The IC50 = 0.12 microM value measured for daunorubicin (1) on human leukemia (HL-60) cells is comparable to those obtained for the daunomycin-L-leucyl squaric acid diamide (30, IC50 = 0.18 microM) and the corresponding D-galactosamine derivative (40, IC50=0.22 microM).

Formation of squaric acid amides of anthracycline antibiotics. Synthesis and cytotoxic properties.

The reaction of the anthracycline glycoside antibiotics 1-3 with the squaric acid ester 4 gave the squaric acid amide esters 5-7 under neutral conditions, whereas over pH7 the products are the symmetric diamides (8, 9, 11, and 12). Of the prepared compounds 11 was the most active on MCF-7 human mammary adenocarcinoma cells.

Synthesis of a novel N[bond]O-interglycosidic disaccharide.

The carbohydrate subunits carrying an N-O-interglycosidic bond play a very important role in the biological activity of the enediyne antibiotics. Condensation of O-(alpha- and beta-D-glucopyranosyl)hydroxylamine (5a and 5b) with the hex-3-ulopyranoside (6) furnished methyl 4,6-O-benzylidene-2,3-dideoxy-3-(2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyloxy)imino-alpha- and beta-D-erythro-hexopyranoside (7a and 7b). Stereoselective reduction of the Cz.dbnd6;N bond of 7a and 7b with sodium cyanoborohydride resulted in the formation of the required protected N-O-interglycosidic disaccharides (8a and 8b). Finally, catalytic hydrogenation of 8a afforded methyl 2,3-dideoxy-3-(alpha-D-glucopyranosyloxy)amino-alpha-D-ribo-hexopyranoside (9a). Under similar conditions the beta anomer 8b underwent decomposition.

Osteoadsorptive bisphosphonate derivatives of fluoroquinolone antibacterials.

Bisphosphonates conjugated to fluoroquinolone antibacterials through an intermediate carbon had better activity than conjugates lacking the carbon. Virtually all molar-based activity of these esterified bisphosphonate derivatives was identical to that of its parent. De-esterified free-acid forms retained good activity against most Gram-negative bacteria, but not against Gram-positives. A free-acid derivative remained bound to washed bone and completely inhibited Staphylococcus aureus growth. The more potent parent, ciprofloxacin, failed to bind significantly, and bacterial growth occurred.

Synthesis and mode of action of hydrophobic derivatives of the glycopeptide antibiotic eremomycin and des-(N-methyl-D-leucyl)eremomycin against glycopeptide-sensitive and -resistant bacteria.

Des-(N-methyl-D-leucyl)eremomycin was obtained by Edman degradation of eremomycin. Derivatives with a hydrophobic substituent at the exterior of the molecule were then synthesized, and their antibacterial activities were compared with similar derivatives of eremomycin. Comparison of derivatives of eremomycin containing the n-decyl or p-(p-chlorophenyl)benzyl substituent in the eremosamine moiety (N') and n-decyl or p-(p-chlorophenyl)benzylamides with similar derivatives of eremomycin possessing the damaged peptide core (a defective binding pocket) showed that compounds of both types are almost equally active against glycopeptide-resistant strains of enterococci (GRE), whereas eremomycin derivatives are more active against staphylococci. Hydrophobic 7d-alkylaminomethylated derivatives of eremomycin (9, 10) demonstrated similar antibacterial properties. Since the basic mode of action of glycopeptide antibiotics involves binding to cell wall intermediates terminating in -D-Ala-D-Ala and this interaction is seriously decreased in the hexapeptide derivatives (lacking the critical N-methyl-D-leucine), we suggest that these hydrophobic derivatives may inhibit peptidoglycan synthesis in the absence of dipeptide binding. NMR binding experiments using Ac-D-Ala-D-Ala show that binding constants of these hexapeptide derivativies are decreased in comparison with the corresponding heptapeptides with intact binding pocket. This is in agreement with the decreased biological activity of the hexapeptide derivatives against vancomycin-sensitive strains in comparison with the activity of parent compounds. Binding to the lactate cell wall analogue Ac-D-Ala-D-Lac with decylamide of eremomycin 8 was not observed, demonstrating that the interaction with this target in GRE does not occur. While hydrophobic glycopeptide derivatives retain the ability to inhibit the synthesis of peptidoglycan in manner of natural glycopeptides, biochemical investigation supports the hypothesis that they inhibit the transglycosylase stage of bacterial peptidoglycan biosynthesis even in the absence of dipeptide or depsipeptide binding.