Fluorinated captopril analogues inhibit metallo-ß-lactamases and facilitate structure determination of NDM-1 binding pose.

Bacterial resistance to the majority of clinically used ß-lactam antibiotics is a global health threat and, consequently, the driving force for the development of metallo-ß-lactamase (MBL) inhibitors. The rapid evolution of new MBLs calls for new strategies and tools for inhibitor development. In this study, we designed and developed a series of trifluoromethylated captopril analogues as probes for structural studies of enzyme-inhibitor binding. The new compounds showed activity comparable to the non-fluorinated inhibitors against the New Delhi Metallo-ß-lactamase-1 (NDM-1). The most active compound, a derivative of D-captopril, exhibited an IC50 value of 0.3 µM. Several compounds demonstrated synergistic effects, restoring the effect of meropenem and reducing the minimum inhibitory concentration (MIC) values in NDM-1 (up to 64-fold), VIM-2 (up to 8-fold) and IMP-26 (up to 8-fold) harbouring Escherichia coli. NMR spectroscopy and molecular docking of one representative inhibitor determined the binding pose in NDM-1, demonstrating that fluorinated analogues of inhibitors are a valuable tool for structural studies of MBL-inhibitor complexes.

Polymyxin B stabilized DNA micelles for sustained antibacterial and antibiofilm activity against P. aeruginosa.

Nucleic acid-based materials showcase an increasing potential for antimicrobial drug delivery. Although numerous reports on drug-loaded DNA nanoparticles outline their pivotal antibacterial activities, their potential as drug delivery systems against bacterial biofilms awaits further studies. Among different oligonucleotide structures, micellar nanocarriers derived from amphiphilic DNA strands are of particular interest due to their spontaneous self-assembly and high biocompatibility. However, their clinical use is hampered by structural instability upon cation depletion. In this work, we used a cationic amphiphilic antibiotic (polymyxin B) to stabilize DNA micelles destined to penetrate P. aeruginosa biofilms and exhibit antibacterial/antibiofilm properties. Our study highlights how the strong affinity of this antibiotic enhances the stability of the micelles and confirms that antibacterial activity of the novel micelles remains intact. Additionally, we show that PMB micelles can penetrate P. aeruginosa biofilms and impact their metabolic activity. Finally, PMB micelles were highly safe and biocompatible, highlighting their possible application against P. aeruginosa biofilm-colonized skin wounds.

Amphipathic barbiturates as marine product mimics with cytolytic and immunogenic effects on head and neck squamous cell carcinoma cell lines.

The incidence of head and neck squamous cell carcinoma (HNSCC) is increasing and the conventional treatments for this form of cancer can be tough. Despite the success of existing immunotherapies in some HNSCC patients, many do not respond to this type of treatment. Thus, the development of novel anti-cancer therapies should be prioritized. In the current study, the anticancer activity of a panel of novel compounds, herein termed marine product mimics (MPMs), against HNSCC cell lines is explored. The previously reported compound MPM-1, which is structurally related to the novel MPMs, was shown to have promising effects on the HNSCC cell line HSC-3. The results from the current study indicate that the novel MPMs are more potent than MPM-1 but cause a similar type of cell death. The results indicated that the MPMs must cross through the cell membrane to exert their action and that they are lysosomotropic. Further experiments showed that some of the MPMs could induce phosphorylation of eukaryotic initiation factor 2α (eIF2α) in HSC-3 and UT-SCC-24A cells, which indicates that they can activate the integrated stress response that is strongly associated with immunogenic cell death. Cell surface expression of calreticulin and release of HMGB1 and ATP, which are all hallmarks of immunogenic cell death, was also demonstrated in HSC-3 and UT-SCC-24A cells treated with MPMs. This suggests that the MPMs are interesting candidates for future HNSCC cancer therapies.

Investigation of tetrasubstituted heterocycles reveals hydantoins as a promising scaffold for development of novel antimicrobials with membranolytic properties.

Mimics of antimicrobial peptides (AMPs) have been proposed as a promising class of antimicrobial agents. We report the analysis of five tetrasubstituted, cationic, amphipathic heterocycles as potential AMP mimics. The analysis showed that the heterocyclic scaffold had a strong influence on the haemolytic activity of the compounds, and the hydantoin scaffold was identified as a promising template for drug lead development. Subsequently, a total of 20 hydantoin derivatives were studied for their antimicrobial potency and haemolytic activity. We found 19 of these derivatives to have very low haemolytic toxicity and identified three lead structures, 2dA, 6cG, and 6dG with very promising broad-spectrum antimicrobial activity. Lead structure 6dG displayed minimum inhibitory concentration (MIC) values as low as 1 µg/mL against Gram-positive bacteria and 4-16 µg/mL against Gram-negative bacteria. Initial mode of action (MoA) studies performed on the amine derivative 6cG, utilizing a luciferase-based biosensor assay, suggested a strong membrane disrupting effect on the outer and inner membrane of Escherichia coli. Our findings show that the physical properties and structural arrangement induced by the heterocyclic scaffolds are important factors in the design of AMP mimics.

Tailored anti-biofilm activity - Liposomal delivery for mimic of small antimicrobial peptide.

The eradication of bacteria embedded in biofilms is among the most challenging obstacles in the management of chronic wounds. These biofilms are found in most chronic wounds; moreover, the biofilm-embedded bacteria are considerably less susceptible to conventional antimicrobial treatment than the planktonic bacteria. Antimicrobial peptides and their mimics are considered attractive candidates in the pursuit of novel therapeutic options for the treatment of chronic wounds and general bacterial eradication. However, some limitations linked to these membrane-active antimicrobials are making their clinical use challenging. Novel innovative delivery systems addressing these limitations represent a smart solution. We hypothesized that incorporation of a novel synthetic mimic of an antimicrobial peptide in liposomes could improve its anti-biofilm effect as well as the anti-inflammatory activity. The small synthetic mimic of an antimicrobial peptide, 7e-SMAMP, was incorporated into liposomes (~280 nm) tailored for skin wounds and evaluated for its potential activity against both biofilm formation and eradication of pre-formed biofilms. The 7e-SMAMP-liposomes significantly lowered inflammatory response in murine macrophages (~30 % reduction) without affecting the viability of macrophages or keratinocytes. Importantly, the 7e-SMAMP-liposomes completely eradicated biofilms produced by Staphylococcus aureus and Escherichia coli above concentrations of 6.25 µg/mL, whereas in Pseudomonas aeruginosa the eradication reached 75 % at the same concentration. Incorporation of 7e-SMAMP in liposomes improved both the inhibition of biofilm formation as well as biofilm eradication in vitro, as compared to non-formulated antimicrobial, therefore confirming its potential as a novel therapeutic option for bacteria-infected chronic wounds.

The marine natural product mimic MPM-1 is cytolytic and induces DAMP release from human cancer cell lines.

Bioprospecting contributes to the discovery of new molecules with anticancer properties. Compounds with cytolytic activity and the ability to induce immunogenic cell death can be administered as intratumoral injections with the aim to activate anti-tumor immune responses by causing the release of tumor antigens as well as damage-associated molecular patterns (DAMPs) from dying cancer cells. In the present study, we report the cytolytic and DAMP-releasing effects of a new natural product mimic termed MPM-1 that was inspired by the marine Eusynstyelamides. We found that MPM-1 rapidly killed cancer cells in vitro by inducing a necrosis-like death, which was accompanied by lysosomal swelling and perturbation of autophagy in HSC-3 (human oral squamous cell carcinoma) cells. MPM-1 also induced release of the DAMPs adenosine triphosphate (ATP) and high mobility group box 1 (HMGB1) from Ramos (B-cell lymphoma) and HSC-3 cells, as well as cell surface expression of calreticulin in HSC-3 cells. This indicates that MPM-1 has the ability to induce immunogenic cell death, further suggesting that it may have potential as a novel anticancer compound.

A concise SAR-analysis of antimicrobial cationic amphipathic barbiturates for an improved activity-toxicity profile.

An amphipathic barbiturate mimic of the marine eusynstyelamides is reported as a promising class of antimicrobial agents. We hereby report a detailed analysis of the structure-activity relationship for cationic amphipathic N,N'-dialkylated-5,5-disubstituted barbiturates. The influence of various cationic groups, hydrocarbon linkers and lipophilic side chains on the compounds' antimicrobial potency and haemolytic activity was studied. A comprehensive library of 58 compounds was prepared using a concise synthetic strategy. We found cationic amine and guanidyl groups to yield the highest broad-spectrum activity and cationic trimethylated quaternary amine groups to exert narrow-spectrum activity against Gram-positive bacteria. n-Propyl hydrocarbon linkers proved to be the best compromise between potency and haemolytic activity. The combination of two different lipophilic side chains allowed for further fine-tuning of the biological properties. Using these insights, we were able to prepare both, the potent narrow-spectrum barbiturate 8a and the broad-spectrum barbiturates 11lG, 13jA and 13jG, all having low or no haemolytic activity. The guanidine derivative 11lG demonstrated a strong membrane disrupting effect in luciferase-based assays. We believe that these results may be valuable in further development of antimicrobial lead structures.

Amphipathic Barbiturates as Mimics of Antimicrobial Peptides and the Marine Natural Products Eusynstyelamides with Activity against Multi-resistant Clinical Isolates.

We report a series of synthetic cationic amphipathic barbiturates inspired by the pharmacophore model of small antimicrobial peptides (AMPs) and the marine antimicrobials eusynstyelamides. These N,N'-dialkylated-5,5-disubstituted barbiturates consist of an achiral barbiturate scaffold with two cationic groups and two lipophilic side chains. Minimum inhibitory concentrations of 2-8 µg/mL were achieved against 30 multi-resistant clinical isolates of Gram-positive and Gram-negative bacteria, including isolates with extended spectrum ß-lactamase-carbapenemase production. The guanidine barbiturate 7e (3,5-di-Br) demonstrated promising in vivo antibiotic efficacy in mice infected with clinical isolates of Escherichia coli and Klebsiella pneumoniae using a neutropenic peritonitis model. Mode of action studies showed a strong membrane disrupting effect and was supported by nuclear magnetic resonance and molecular dynamics simulations. The results express how the pharmacophore model of small AMPs and the structure of the marine eusynstyelamides can be used to design highly potent lead peptidomimetics against multi-resistant bacteria.

Total Synthesis of Phorbazole B.

Phorbazoles are polychlorinated heterocyclic secondary metabolites isolated from a marine sponge and several of these natural products have shown inhibitory activity against cancer cells. In this work, a synthesis of the trichlorinated phorbazole B using late stage electrophilic chlorination was developed. The synthesis relied on the use of an oxazole precursor, which was protected with an iodine in the reactive 4-position, followed by complete chlorination of all pyrrole positions. Attempts to prepare phorbazole A and C, which contain a 3,4-dichlorinated pyrrole, were unsuccessful as the desired chlorination pattern on the pyrrole could not be obtained. The identities of the dichlorinated intermediates and products were determined using NMR techniques including NOESY/ROESY, 1,1-ADEQUATE and high-resolution CLIP-HSQMBC.

Structural studies of triazole inhibitors with promising inhibitor effects against antibiotic resistance metallo-ß-lactamases.

Metallo-ß-lactamases (MBLs) are an emerging cause of bacterial antibiotic resistance by hydrolysing all classes of ß-lactams except monobactams, and the MBLs are not inhibited by clinically available serine-ß-lactamase inhibitors. Two of the most commonly encountered MBLs in clinical isolates worldwide - the New Delhi metallo-ß-lactamase (NDM-1) and the Verona integron-encoded metallo-ß-lactamase (VIM-2) - are included in this study. A series of several NH-1,2,3-triazoles was prepared by a three-step protocol utilizing Banert cascade reaction as the key step. The inhibitor properties were evaluated in biochemical assays against the MBLs VIM-2, NDM-1 and GIM-1, and VIM-2 showed IC50 values down to nanomolar range. High-resolution crystal structures of four inhibitors in complex with VIM-2 revealed hydrogen bonds from the triazole inhibitors to Arg228 and to the backbone of Ala231 or Asn233, along with hydrophobic interactions to Trp87, Phe61 and Tyr67. The inhibitors show reduced MIC in synergy assays with Pseudomonas aeruginosa and Escherichia coli strains harbouring VIM enzymes. The obtained results will be useful for further structural guided design of MBL inhibitors.

ZN148 Is a Modular Synthetic Metallo-ß-Lactamase Inhibitor That Reverses Carbapenem Resistance in Gram-Negative Pathogens In Vivo.

Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Carbapenemases (ß-lactamases able to inactivate carbapenems) have been identified in both serine ß-lactamase (SBL) and metallo-ß-lactamase (MBL) families. The recent introduction of SBL carbapenemase inhibitors has provided alternative therapeutic options. Unfortunately, there are no approved inhibitors of MBL-mediated carbapenem-resistance and treatment options for infections caused by MBL-producing Gram-negatives are limited. Here, we present ZN148, a zinc-chelating MBL-inhibitor capable of restoring the bactericidal effect of meropenem and in vitro clinical susceptibility to carbapenems in >98% of a large international collection of MBL-producing clinical Enterobacterales strains (n = 234). Moreover, ZN148 was able to potentiate the effect of meropenem against NDM-1-producing Klebsiella pneumoniae in a murine neutropenic peritonitis model. ZN148 showed no inhibition of the human zinc-containing enzyme glyoxylase II at 500 µM, and no acute toxicity was observed in an in vivo mouse model with cumulative dosages up to 128 mg/kg. Biochemical analysis showed a time-dependent inhibition of MBLs by ZN148 and removal of zinc ions from the active site. Addition of exogenous zinc after ZN148 exposure only restored MBL activity by ∼30%, suggesting an irreversible mechanism of inhibition. Mass-spectrometry and molecular modeling indicated potential oxidation of the active site Cys221 residue. Overall, these results demonstrate the therapeutic potential of a ZN148-carbapenem combination against MBL-producing Gram-negative pathogens and that ZN148 is a highly promising MBL inhibitor that is capable of operating in a functional space not presently filled by any clinically approved compound.

Carbonylative Suzuki-Miyaura couplings of sterically hindered aryl halides: synthesis of 2-aroylbenzoate derivatives.

We have developed a carbonylative approach to the synthesis of diversely substituted 2-aroylbenzoate esters featuring a new protocol for the carbonylative coupling of aryl bromides with boronic acids and a new strategy to favour carbonylative over non-carbonylative reactions. Two different synthetic pathways - (i) the alkoxycarbonylation of 2-bromo benzophenones and (ii) the carbonylative Suzuki-Miyaura coupling of 2-bromobenzoate esters - were evaluated. The latter approach provided a broader substrate tolerance, and thus was the preferred pathway. We observed that 2-substituted aryl bromides were challenging substrates for carbonylative chemistry favouring the non-carbonylative pathway. However, we found that carbonylative Suzuki-Miyaura couplings can be improved by slow addition of the boronic acid, suppressing the unwanted direct Suzuki coupling and, thus increasing the yield of the carbonylative reaction.

Formal C-H Carboxylation of Unactivated Arenes.

A formal C-H carboxylation of unactivated arenes using CO2 in green solvents is described. The present strategy combines a sterically controlled Ir-catalyzed C-H borylation followed by a Cu-catalyzed carboxylation of the in situ generated organoboronates. The reaction is highly regioselective for the C-H carboxylation of 1,3-disubstituted and 1,2,3-trisubstituted benzenes, 1,2- or 1,4-symmetrically substituted benzenes, fluorinated benzenes and different heterocycles. The developed methodology was applied to the late-stage C-H carboxylation of commercial drugs and ligands.

Exploration of New Biomass-Derived Solvents: Application to Carboxylation Reactions.

A range of hitherto unexplored biomass-derived chemicals have been evaluated as new sustainable solvents for a large variety of CO2 -based carboxylation reactions. Known biomass-derived solvents (biosolvents) are also included in the study and the results are compared with commonly used solvents for the reactions. Biosolvents can be efficiently applied in a variety of carboxylation reactions, such as Cu-catalyzed carboxylation of organoboranes and organoboronates, metal-catalyzed hydrocarboxylation, borocarboxylation, and other related reactions. For many of these reactions, the use of biosolvents provides comparable or better yields than the commonly used solvents. The best biosolvents identified are the so far unexplored candidates isosorbide dimethyl ether, acetaldehyde diethyl acetal, rose oxide, and eucalyptol, alongside the known biosolvent 2-methyltetrahydrofuran. This strategy was used for the synthesis of the commercial drugs Fenoprofen and Flurbiprofen.

Unequivocal structure confirmation of a breitfussin analog by anisotropic NMR measurements.

Structural features of proton-deficient heteroaromatic natural products, such as the breitfussins, can severely complicate their characterization by NMR spectroscopy. For the breitfussins in particular, the constitution of the five-membered oxazole central ring cannot be unequivocally established via conventional NMR methods when the 4'-position is halogenated. The level of difficulty is exacerbated by 4'-iodination, as the accuracy with which theoretical NMR parameters are determined relies extensively on computational treatment of the relativistic effects of the iodine atom. It is demonstrated in the present study, that the structure of a 4'-iodo breitfussin analog can be unequivocally established by anisotropic NMR methods, by adopting a reduced singular value decomposition (SVD) protocol that leverages the planar structures exhibited by its conformers.

Kinase Chemodiversity from the Arctic: The Breitfussins.

In this work, we demonstrate that the indole-oxazole-pyrrole framework of the breitfussin family of natural products is a promising scaffold for kinase inhibition. Six new halogenated natural products, breitfussin C-H (3 - 8) were isolated and characterized from the Arctic, marine hydrozoan Thuiaria breitfussi. The structures of two of the new natural products were also confirmed by total synthesis. Two of the breitfussins (3 and 4) were found to selectively inhibit the survival of several cancer cell lines, with the lowest IC50 value of 340 nM measured against the drug-resistant triple negative breast cancer cell line MDA-MB-468, while leaving the majority of the tested cell lines not or significantly less affected. When tested against panels of protein kinases, 3 gave IC50 and Kd values as low as 200 and 390 nM against the PIM1 and DRAK1 kinases, respectively. The activity was confirmed to be mediated through ATP competitive binding in the ATP binding pocket of the kinases. Furthermore, evaluation of potential off-target and toxicological effects, as well as relevant in vitro ADME parameters for 3 revealed that the breitfussin scaffold holds promise for the development of selective kinase inhibitors.

Antimicrobial activity of amphipathic α,α-disubstituted ß-amino amide derivatives against ESBL - CARBA producing multi-resistant bacteria; effect of halogenation, lipophilicity and cationic character.

The rapid emergence and spread of multi-resistant bacteria have created an urgent need for new antimicrobial agents. We report here a series of amphipathic α,α-disubstituted ß-amino amide derivatives with activity against 30 multi-resistant clinical isolates of Gram-positive and Gram-negative bacteria, including isolates with extended spectrum ß-lactamase - carbapenemase (ESBL-CARBA) production. A variety of halogenated aromatic side-chains were investigated to improve antimicrobial potency and minimize formation of Phase I metabolites. Net positive charge and cationic character of the derivatives had an important effect on toxicity against human cell lines. The most potent and selective derivative was the diguanidine derivative 4e with 3,5-di-brominated benzylic side-chains. Derivative 4e displayed minimum inhibitory concentrations (MIC) of 0.25-8 µg/mL against Gram-positive and Gram-negative reference strains, and 2-32 µg/mL against multi-resistant clinical isolates. Derivative 4e showed also low toxicity against human red blood cells (EC50 > 200 µg/mL), human hepatocyte carcinoma cells (HepG2: EC50 > 64 µg/mL), and human lung fibroblast cells (MRC-5: EC50 > 64 µg/mL). The broad-spectrum antimicrobial activity and low toxicity of diguanylated derivatives such as 4e make them attractive as lead compounds for development of novel antimicrobial drugs.

Caesium fluoride-mediated hydrocarboxylation of alkenes and allenes: scope and mechanistic insights.

A caesium fluoride-mediated hydrocarboxylation of olefins is disclosed that does not rely on precious transition metal catalysts and ligands. The reaction occurs at atmospheric pressures of CO2 in the presence of 9-BBN as a stoichiometric reductant. Stilbenes, ß-substituted styrenes and allenes could be carboxylated in good yields. The developed methodology can be used for preparation of commercial drugs as well as for gram scale hydrocarboxylation. Computational studies indicate that the reaction occurs via formation of an organocaesium intermediate.

Iron-Catalyzed Carbenoid-Transfer Reactions of Vinyl Sulfoxonium Ylides: An Experimental and Computational Study.

A method for the generation of unprecedented vinyl carbenoids from sulfoxonium ylides has been developed and applied in the synthesis of a diverse array of heterocycles such as indolizines, pyrroles, 3-pyrrolin-2-ones, and furans. The reactions proceed by FeBr2 catalysis under mild reaction conditions with a broad substrate scope. A reaction pathway involving iron carbenoids is proposed based on a series of control experiments and DFT calculations.

An amphipathic cyclic tetrapeptide scaffold containing halogenated ß2,2 -amino acids with activity against multiresistant bacteria.

The present study describes the synthesis and biological studies of a small series of head-to-tail cyclic tetrapeptides of the general structure c(Lys-ß2,2 -Xaa-Lys) containing one lipophilic ß2,2 -amino acid and Lys, Gly, Ala, or Phe as the Xaa residue in the sequence. The peptides were investigated for antimicrobial activity against gram-positive and gram-negative reference strains and 30 multiresistant clinical isolates including strains with extended spectrum ß-lactamase-carbapenemase (ESBL-CARBA) production. Toxicity was determined against human red blood cells. The most potent peptides showed high activity against the gram-positive clinical isolates with minimum inhibitory concentrations of 4-8 µg/mL and low haemolytic activity. The combination of high antimicrobial activity and low toxicity shows that these cyclic tetrapeptides containing lipophilic ß2,2 -amino acids form a valuable scaffold for designing novel antimicrobial agents.