Evaluation of New Antimicrobial Agents Based on tris(1H-Indol-3-yl)methylium Salts: Activity, Toxicity, Suppression of Experimental Sepsis in Mice.

The antimicrobial activity and toxicity of three novel synthetic antibacterial agents containing tris(1H-indol-3-yl)methylium fragment were studied in vitro and in vivo. All compounds in vitro revealed high activity (minimal inhibitory concentration (MIC) 0.13-1.0 µg/mL) against bacteria that were either sensitive or resistant to antibiotics, including multidrug-resistant clinical isolates. The derivatives combining high antimicrobial activity with relatively low cytotoxicity against human donor fibroblasts HPF-hTERT were subjected to further testing on mice. In vivo they revealed fairly good tolerance and relatively low toxicity. Acute toxicity was evaluated, and the main indicators of toxicity, including LD50 and LD10, were determined. A study of compounds in vivo showed their efficiency in the model of staphylococcal sepsis in mice. The efficiency of compounds may be due to the ability of indolylmethylium salts to form pores in the cytoplasmic membrane of microbial cells and thereby facilitate the penetration of molecules into the pathogen.

Gausemycins†A,B: Cyclic Lipoglycopeptides from Streptomyces sp.*.

We report a novel family of natural lipoglycopeptides produced by Streptomyces sp. INA-Ac-5812. Two major components of the mixture, named gausemycins A and B, were isolated, and their structures were elucidated. The compounds are cyclic peptides with a unique peptide core and several remarkable structural features, including unusual positions of d-amino acids, lack of the Ca2+ -binding Asp-X-Asp-Gly (DXDG) motif, tyrosine glycosylation with arabinose, presence of 2-amino-4-hydroxy-4-phenylbutyric acid (Ahpb) and chlorinated kynurenine (ClKyn), and N-acylation of the ornithine side chain. Gausemycins have pronounced activity against Gram-positive bacteria. Mechanistic studies highlight significant differences compared to known glyco- and lipopeptides. Gausemycins exhibit only slight Ca2+ -dependence of activity and induce no pore formation at low concentrations. Moreover, there is no detectable accumulation of cell wall biosynthesis precursors under treatment with gausemycins.

Aminoalkylamides of Eremomycin Exhibit an Improved Antibacterial Activity.

After decades, the glycopeptide vancomycin is still the preferred antibiotic against resistant strains of Gram-positive bacteria. Although its clinical use is strictly regulated, the gradual spread of vancomycin-resistant bacteria, such as glycopeptide-resistant and glycopeptide-intermediate Staphylococcus aureus and vancomycin-resistant Enterococcus spp., is a serious health problem. Based on the literature data and previous studies, our main goal was to assess the antimicrobial potential and to study the structure-activity relationship of new eremomycin aminoalkylamides. We designed and synthesized a series of new eremomycin amides in which eremomycin is conjugated with a hydrophobic arylalkyl group via an alkylenediamine spacer, and tested their antibacterial activities on a panel of Gram-positive strains that were sensitive and resistant to a "gold-standard" vancomycin. Based on the data obtained, the structure-activity relationships were investigated, and a lead compound was selected for in-depth testing. Research carried out using an in vivo model of staphylococcus sepsis, acute toxicity studies, and the estimated therapeutic index also showed the advantage of the selected eremomycin amide derivative in particular, as well as the chosen direction in general.

Synthesis and antibacterial activity of novel arylbis(indol-3-yl)methane derivatives.

A series of new compounds-arylbis(indol-3-yl)methylium derivatives-were synthesized and their antimicrobial activity was evaluated. All the compounds turned out to be highly active, with MIC depending on their structure and the length of N-alkyl residues. The parent triarylmethane compounds possess weaker activity.

Discovery of Amphamide, a Drug Candidate for the Second Generation of Polyene Antibiotics.

Amphotericin B (AmB, 1) is the drug of choice for treating the most serious systemic fungal or protozoan infections. Nevertheless, its application is limited by low solubility in aqueous media and serious side effects such as infusion-related reactions, hemolytic toxicity, and nephrotoxicity. Owing to these limitations, it is essential to search for the polyene derivatives with better chemotherapeutic properties. With the objective of obtaining AmB derivatives with lower self-aggregation and improved solubility, we synthesized a series of amides of AmB bearing an additional basic group in the introduced residue. The screening of antifungal activity in vitro revealed that N-(2-aminoethyl)amide of AmB (amphamide, 6) had superior antifungal activity compared to that of the paternal AmB. Preclinical studies in mice confirmed that compound 6 had a much lower acute toxicity and higher antifungal efficacy in the model of mice candidosis sepsis compared with that of AmB (1). Thus, the discovered amphamide is a promising drug candidate for the second generation of polyene antibiotics and is also prospective for in-depth preclinical and clinical evaluation.

Synthesis and antimicrobial activity of 3,4-bis(arylthio)maleimides.

A series of 3,4-bis(arylthio)maleimides were synthesized and their antimicrobial activity was evaluated against Gram-positive and Gram-negative bacteria, including multidrug resistant (MDR) strains and some fungi. Most compounds turned out to be highly active, activity being dependent on substituents on phenyl rings.

Synthesis and evaluation of biological activity for dual-acting antibiotics on the basis of azithromycin and glycopeptides.

One of the promising directions of the combined approach is the design of dual-acting antibiotics - heterodimeric structures on the basis of antimicrobial agents of different classes. In this study a novel series of azithromycin-glycopeptide conjugates were designed and synthesized. The structures of the obtained compounds were confirmed using NMR spectroscopy and mass spectrometry data including MS/MS analysis. All novel hybrid antibiotics were found to be either as active as azithromycin and vancomycin against Gram-positive bacterial strains or have superior activity in comparison with their parent antibiotics. One compound, eremomycin-azithromycin conjugate 16, demonstrated moderate activity against Enterococcus faecium and Enterococcus faecalis strains resistant to vancomycin, and equal to vancomycin's activity for the treatment of mice with Staphylococcus aureus sepsis.

Synthesis and evaluation of biological activity of benzoxaborole derivatives of azithromycin.

Novel benzoxaborole derivatives of azithromycin in which benzoxaborole residue is attached to the 4″-hydroxy-group of azithromycin have been synthesized. Antibacterial activity of synthesized derivatives in comparison with azithromycin was tested on a panel of Gram-positive and Gram-negative bacterial strains. All the investigated compounds demonstrated broad spectrum of antibacterial activity being in general more active against Gram-positive strains. New benzoxaborole derivatives of azithromycin demonstrated high activity against Streptococcus pyogenes ATCC 19615 and Propionibacterium acnes ATCC 6919 strains. Some of the new compounds were more active than azithromycin against Streptococcus pneumoniae ATCC 49619 strain or Enterococcus faecium strains. Using a reporter construct created on the basis of the transcription attenuator region of the Escherichia coli tryptophan operon pRFPCER-TrpL2A it has been demonstrated that the mechanism of action of azithromycin analogs is blocking nascent peptide in ribosome tunnel.

Eremomycin pyrrolidide: a novel semisynthetic glycopeptide with improved chemotherapeutic properties.

PURPOSE: Development of new semisynthetic glycopeptides with improved antibacterial efficacy and reduced pseudoallergic reactions. METHODS: Semisynthetic glycopeptides 3-6 were synthesized from vancomycin (1) or eremomycin (2) by the condensation with pyrrolidine or piperidine. The minimum inhibitory concentration (MIC) for the new derivatives was measured by the broth micro-dilution method on a panel of clinical isolates of Staphylococcus and Enterococcus. Acute toxicity (50% lethal dose, maximum tolerated doses), antibacterial efficacy on model of systemic bacterial infection with S. aureus and pseudoallergic inflammatory reaction (on concanavalin A) of eremomycin pyrrolidide (5) were evaluated in mice according to standard procedures. RESULTS: The eremomycin pyrrolidide (5) was the most active compound and showed a high activity against Gram-positive bacteria: vancomycin-susceptible staphylococci and enterococci (minimum inhibitory concentrations [MICs] 0.13-0.25 mg/L), as well as vancomycin-intermediate resistant Staphylococcus aureus (MICs 1 mg/L). Antimicrobial susceptibility tested on a panel of 676 isolates showed that 5 had similar activity for the genera Staphylococcus and Enterococcus with MIC90=0.5 mg/L, while vancomycin had MIC90=1-2 mg/L. The number of resistant strains of Enterococcus faecium (vancomycin-resistant enterococci) (MIC =64 mg/L) with this value was 7 (8%) for vancomycin (1) and 0 for the compound 5. In vivo comparative studies in a mouse model of systemic bacterial infection with S. aureus demonstrated that the efficacy of 5 was notably higher than that of the original antibiotics 1 and 2. In contrast to 1, compound 5 did not induce pseudoallergic inflammatory reaction (on concanavalin A). CONCLUSION: The new semisynthetic derivative eremomycin pyrrolidide (5) has high activity against staphylococci and enterococci including vancomycin-resistant strains. Compound 5 has a higher efficacy in a model of staphylococcal sepsis than vancomycin (1) or eremomycin (2). In striking contrast to natural antibiotics, the novel derivative 5 does not induce a pseudoallergic inflammatory reaction to concanavalin A and therefore has no histamine release activity. These results indicate the advantages of a new semisynthetic glycopeptide antibiotic eremomycin pyrrolidide (5) which may be a prospective antimicrobial agent for further pre-clinical and clinical evaluations.

Two approaches to the use of benzo[c][1,2]oxaboroles as active fragments for synthetic transformation of clarithromycin.

Clarithromycin (active against Gram positive infections) and 1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole derivatives (effective for Gram negative microbes) are the ligands of bacterial RNA. The antimicrobial activities of these benzoxaboroles linked with clarithromycin at 9 or 4″ position were compared. Two synthetic pathways for these conjugates were elaborated. First pathway explored the substitution of the C-9 carbonyl group of macrolactone's cycle via oxime linker, the second direction used the modification of the 4″-O-group of cladinose via the formation of carbamates of benzoxaboroles. 4″-O-(3-S-(1-Hydroxy-1,3-dihydro-benzo[c][1,2]oxaborole)-methyl-carbamoyl-clarithromycin showed twofold decrease in MICs for S. epidermidis and S. pneumoniae than clarithromycin. 4″-O-Modified clarithromycin demonstrated an efficacy against Gram positive strains only. Compounds with C-9 substitution were more active than 4″-O-substituted antibiotics for susceptible strains E. coli tolC and did not exceed the activity of initial antibiotics.

Synthesis and Antibacterial Activity of Quaternary Ammonium 4-Deoxypyridoxine Derivatives.

A series of novel quaternary ammonium 4-deoxypyridoxine derivatives was synthesized. Two compounds demonstrated excellent activity against a panel of Gram-positive methicillin-resistant S. aureus strains with MICs in the range of 0.5-2 µg/mL, exceeding the activity of miramistin. At the same time, both compounds were inactive against the Gram-negative E. coli and P. aeruginosa strains. Cytotoxicity studies on human skin fibroblasts and embryonic kidney cells demonstrated that the active compounds possessed similar toxicity with benzalkonium chloride but were slightly more toxic than miramistin. SOS-chromotest in S. typhimurium showed the lack of DNA-damage activity of both compounds; meanwhile, one compound showed some mutagenic potential in the Ames test. The obtained results make the described chemotype a promising starting point for the development of new antibacterial therapies.

Synthesis, anti-MRSA, and anti-VRE activity of hemin conjugates with amino acids and branched peptides.

The increasing prevalence of antibiotic-resistant bacterial strains has necessitated the synthesis of novel antibacterial agents. It was previously shown that naturally occurring metalloporphyrin hemin possesses dark antibacterial activity against Gram-positive bacteria. To improve hemin antibacterial activity, we synthesized a number of hemin conjugates with amino acids and branched peptides. Arginine-containing hemin conjugates demonstrated high antibacterial activity against Gram-positive bacteria including methicillin- and vancomycin-resistant strains in vitro. Most of the synthesized conjugates showed low toxicity against human erythrocytes and leukocytes.

Structure-antifungal activity relationships of polyene antibiotics of the amphotericin B group.

A comprehensive comparative analysis of the structure-antifungal activity relationships for the series of biosynthetically engineered nystatin analogues and their novel semisynthetic derivatives, as well as amphotericin B (AMB) and its semisynthetic derivatives, was performed. The data obtained revealed the significant influence of the structure of the C-7 to C-10 polyol region on the antifungal activity of these polyene antibiotics. Comparison of positions of hydroxyl groups in the antibiotics and in vitro antifungal activity data showed that the most active are the compounds in which hydroxyl groups are in positions C-8 and C-9 or positions C-7 and C-10. Antibiotics with OH groups at both C-7 and C-9 had the lowest activity. The replacement of the C-16 carboxyl with methyl group did not significantly affect the in vitro antifungal activity of antibiotics without modifications at the amino group of mycosamine. In contrast, the activity of the N-modified derivatives was modulated both by the presence of CH3 or COOH group in the position C-16 and by the structure of the modifying substituent. The most active compounds were tested in vivo to determine the maximum tolerated doses and antifungal activity on the model of candidosis sepsis in leukopenic mice (cyclophosphamide-induced). Study of our library of semisynthetic polyene antibiotics led to the discovery of compounds, namely, N-(L-lysyl)-BSG005 (compound 3n) and, especially, L-glutamate of 2-(N,N-dimethylamino)ethyl amide of S44HP (compound 2j), with high antifungal activity that were comparable in in vitro and in vivo tests to AMB and that have better toxicological properties.

Synthesis and study of the antifungal activity of new mono- and disubstituted derivatives of a genetically engineered polyene antibiotic 28,29-didehydronystatin A1 (S44HP).

Mono- and disubstituted novel derivatives of the heptaene nystatin analog 28,29-didehydronystatin A(1) (S44HP, 1) were obtained by chemical modification of the exocyclic C-16 carboxyl and/or an amino group of mycosamine moiety. The strategy of preparation of mono- and double-modified polyene macrolides was based on the use of intermediate hydrophobic N-Fmoc (9-fluorenylmethoxycarbonyl) derivatives that facilitated the procedures of isolation and purification of new compounds. The antifungal activity of the new derivatives was first tested in vitro against yeasts and filamentous fungi, allowing the selection of the most active compounds that were subsequently tested for acute toxicity in mice. 2-(N,N-dimethylamino)ethylamide of 1 (2) and 2-(N,N-dimethylamino)ethylamide of N-fructopyranosyl-28,29-didehydronystatin A(1) (2a) were then selected for further evaluation in a mouse model of disseminated candidosis, and showed high efficacy while being considerably less toxic than amphotericin B (AmB). The compound with improved water solubility (2G, L-glutamic acid salt of 2) showed better chemotherapeutic activity than AmB in the mouse model of candidosis sepsis on a leucopenic background. Very low antifungal effect was seen after treatment with AmB, even if it was used in maximum tolerated dose (2 mg kg(-1)). Unlike AmB, compound 2G exhibited high activity in doses from 0.4 up to 4.0 mg kg(-1), despite leucopenic conditions.

Chemical modification and biological evaluation of new semisynthetic derivatives of 28,29-Didehydronystatin A1 (S44HP), a genetically engineered antifungal polyene macrolide antibiotic.

Twenty-three new derivatives of the heptaene nystatin analogue 28,29-didehydronystatin A(1) (1) (S44HP) were obtained by chemical modification of C16 carboxyl and amino groups of mycosamine. These derivatives comprised 15 carboxamides, 4 N-alkyl derivatives, 3 N-derivatives containing additional N-linked monosaccharide or disaccharide moiety (products of Amadori rearrangement), and 1 N-aminoacyl derivative. The derivatives have been tested in vitro against yeasts Candida albicans, Cryptococcus humicolus, and filamentous fungi (molds) Aspergillus niger and Fusarum oxysporum, as well as for hemolytic activity against human erythrocytes. Structure-activity relationships for the compounds obtained are discussed. The most active and least hemolytic derivative 3-(N,N-dimethylamino)propylamide of S44HP (6) was tested for acute toxicity and antifungal activity in animal model. Whereas amphotericin B and S44HP were active in vivo at doses close to the maximal tolerated dose, 6 was considerably less toxic and more active compared to these two antibiotics.

Improved antifungal polyene macrolides via engineering of the nystatin biosynthetic genes in Streptomyces noursei.

Seven polyene macrolides with alterations in the polyol region and exocyclic carboxy group were obtained via genetic engineering of the nystatin biosynthesis genes in Streptomyces noursei. In vitro analyses of the compounds for antifungal and hemolytic activities indicated that combinations of several mutations caused additive improvements in their activity-toxicity properties. The two best analogs selected on the basis of in vitro data were tested for acute toxicity and antifungal activity in a mouse model. Both analogs were shown to be effective against disseminated candidosis, while being considerably less toxic than amphotericin B. To our knowledge, this is the first report on polyene macrolides with improved in vivo pharmacological properties obtained by genetic engineering. These results indicate that the engineered nystatin analogs can be further developed into antifungal drugs for human use.

N'-(alpha-aminoacyl)- and N'-alpha-(N-alkylamino)acyl derivatives of vancomycin and eremomycin. II. Antibacterial activity of N'-(alpha-aminoacyl)- and N'-alpha-(N-alkylamino)acyl derivatives of vancomycin and eremomycin.

The antibacterial activities of the series of novel N'-(alpha-aminoacyl)- and N'-alpha-(N-akylamino)acyl derivatives of eremomycin and vancomycin containing hydrophobic moieties have been investigated. The N'-(N-alkylglycyl) derivatives of vancomycin are more active against vancomycin-susceptible staphylococci and enterococci and glycopeptide intermediate-resistant Staphylococcus aureus (GISA) than the corresponding eremomycin derivatives, but except for N'-[N-(p-octyloxybenzyl)glycyl-vancomycin] (28) and N'-[N-(p-octyloxybenzyl)-L-alanyl-vancomycin (33)--they are less active against glycopeptide-resistant enterococci (GRE). Derivatives 28 and 33 are the most active compounds (MIC's for glycopeptide-sensitive staphylococci and enterococci are 0.25 approximately 1 microg/ml, for GISA 1 approximately 2 microg/ml, for GRE 2 approximately 6 microg/ml). In in vivo studies, derivative 28 was active against S. aureus infections in mice with ED(50) 1 mg/kg versus 2 mg/kg for vancomycin (iv). In general N'-(N-alkylglycyl)-derivatives of vancomycin and eremomycin were more active than the corresponding N'-aminoacylated derivatives of these antibiotics containing other than glycin amino acids (L-Lys, L-Met, L-Orn, L- and D-Ala) and also L- and D-Phe or benzyl-O-L-Tyr.

Structure-activity relationship studies of a series of antiviral and antibacterial aglycon derivatives of the glycopeptide antibiotics vancomycin, eremomycin, and dechloroeremomycin.

N-(adamantyl-1)methyl, N-(adamantyl-2), and N-(omega-aminodecyl) amides of vancomycin, eremomycin, and dechloroeremomycin aglycons and their des-(N-Me-D-Leu) derivatives were synthesized and their antibacterial and anti-HIV activities were investigated. Carboxamides with an intact peptide core demonstrated activity against glycopeptide-susceptible and -resistant bacteria (1-32 microM). N-(adamantyl-1)methylcarboxamide of eremomycin aglycons had good antiretroviral activity (1.6 microM against HIV-1). Compounds with destroyed peptide core [des-(N-Me-D-Leu)-aglycon amides] were inactive against both glycopeptide-sensitive and -resistant bacteria. (Adamantyl-1)methylamide of des-(N-Me-D-Leu)-eremomycin aglycon had good antiretroviral activity (EC50 of 5.5 microM for HIV-1 and 3.5 microM for HIV-2). (Adamantyl-1)methylamides of eremomycin aglycon and its des-(N-Me-d-Leu)-derivative are the most promising and selective antiretroviral agents. Their ability to induce bacterial resistance to glycopeptide antibiotics during prolonged administration may be expected to be very low or absent. This might make the use of these derivatives feasible in the prolonged therapy or prophylaxis of HIV infections.

Role of the glycopeptide framework in the antibacterial activity of hydrophobic derivatives of glycopeptide antibiotics.

The antibacterial properties of glycopeptide antibiotics are based on their interaction with the d-Ala-d-Ala containing pentapeptide of bacterial peptidoglycan. The hydrophobic amides of vancomycin (1), teicoplanin (2), teicoplanin aglycon (3), and eremomycin (4) were compared with similar amides of minimally or low active des-(N-methyl-d-leucyl)eremomycin (5), eremomycin aglycon (6), des-(N-methyl-d-leucyl)eremomycin aglycon (7), and a teicoplanin degradation product TB-TPA (8). All hydrophobic amides of 1, 3, 4, and 6 were almost equally active against glycopeptide-resistant enterococci (GRE) [minimum inhibitory concentrations (MIC)

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.