Search

Scalable syntheses of the northern macrobicyclic thiolincosamine fragments of two structurally complex antibiotic candidates, BT-33 and cresomycin, are presented. A key transformation in each route is the highly diastereoselective addition of a putative allenylzinc nucleophile to a common Ellman sulfinimine intermediate using a zinc-promoted Barbier-type propargylation protocol that is detailed herein. These transformations proceed with dynamic kinetic resolution and use just 1.2 equiv of each respective propargyl bromide precursor.

Objective: Antimicrobial resistance is a global pandemic that poses a major threat to vision health as ocular bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA), are becoming increasingly resistant to first-line therapies. Here we evaluated the antimicrobial activity of new synthetic lincosamides in comparison to currently used antibiotics against clinical ocular MRSA isolates., Methods: Antimicrobial susceptibility testing was performed by broth microdilution for two novel synthetic lincosamides (iboxamycin and cresomycin) and eight comparator antibiotics against a collection of 50 genomically characterised ocular MRSA isolates, including isolates harbouring erm genes (n = 25)., Results: Both drugs were active against widespread MRSA clonal complexes CC8 and CC5. The MIC 50 and MIC 90 of iboxamycin were 0.06 and 2 mg/L, respectively. Cresomycin (MIC 50 = 0.06 mg/L) also displayed good activity with an in vitro potency four-fold higher (MIC 90 = 0.5 mg/L) than iboxamycin. In isolates harbouring erm genes, MIC 90 were >16, 2, and 0.5 mg/L for clindamycin, iboxamycin, and cresomycin, respectively. The in vitro potencies of iboxamycin and cresomycin were similar or higher than that of comparator agents and were not impacted by multidrug-resistance phenotypes or by the presence of erm genes when compared with clindamycin., Conclusions: Our results demonstrate that iboxamycin and cresomycin display potent in vitro activity against ocular MRSA isolates, including multidrug-resistant isolates harbouring erm genes., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The bacterial ribosome is an essential drug target as many clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent resistance mechanisms to PTC-acting drugs in Gram-positive bacteria is C8-methylation of the universally conserved A2503 nucleobase by Cfr methylase in 23S ribosomal RNA. Despite its clinical importance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. Here, we report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-transfer RNAs. These structures reveal an allosteric rearrangement of nucleotide A2062 upon Cfr-mediated methylation of A2503 that likely contributes to the reduced potency of some PTC inhibitors. Additionally, we provide the structural bases behind two distinct mechanisms of engaging the Cfr-methylated ribosome by the antibiotics iboxamycin and tylosin., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

An experiment was conducted to evaluate the live performance, processing characteristics, and apparent ileal digestibility (AID) of nutrients and energy (IDE) in broilers under two coccidiosis control programs (CCP) and fed three starter diet energy levels. Treatments were a factorial arrangement of CCP [in-feed diclazuril (ACD) or vaccinated after hatch (VAC)] and three starter diet energy levels [3008 (standard), 3058 (moderate), and 3108 (high) kcal/kg apparent ME n ] achieved with different soybean oil concentrations. Birds were reared in floor pens (12 per pen) and received experimental starter diets from 0 to 18 d and common grower and finisher diets to 43 d. At d 11, VAC birds had higher ( p < 0.05) excreta oocyst counts and lower ( p < 0.05) plasma carotenoids, nutrient AID, and IDE than ACD birds. From 0 to 18 and 0 to 31 d, VAC decreased ( p < 0.05) body weight gain and increased (energy × CCP, p < 0.05) feed conversion ratio of birds fed the moderate and high-energy diets but not for those fed the standard energy diet. From 0 to 43 d, VAC only increased the feed conversion ratio of birds fed the moderate-energy starter diet (energy × CCP, p < 0.05). Carcass yields were lower ( p < 0.05) for VAC birds than for ACD birds, and interactive effects ( p < 0.05) were observed for wing yield. In summary, increasing dietary lipid concentration to account for Eimeria -induced reductions in lipid digestibility during the starter period of coccidiosis-vaccinated broilers may exacerbate, rather than ameliorate, these impacts on bird performance.

We report the design conception, chemical synthesis, and microbiological evaluation of the bridged macrobicyclic antibiotic cresomycin (CRM), which overcomes evolutionarily diverse forms of antimicrobial resistance that render modern antibiotics ineffective. CRM exhibits in vitro and in vivo efficacy against both Gram-positive and Gram-negative bacteria, including multidrug-resistant strains of Staphylococcus aureus , Escherichia coli , and Pseudomonas aeruginosa . We show that CRM is highly preorganized for ribosomal binding by determining its density functional theory-calculated, solution-state, solid-state, and (wild-type) ribosome-bound structures, which all align identically within the macrobicyclic subunits. Lastly, we report two additional x-ray crystal structures of CRM in complex with bacterial ribosomes separately modified by the ribosomal RNA methylases, chloramphenicol-florfenicol resistance (Cfr) and erythromycin-resistance ribosomal RNA methylase (Erm), revealing concessive adjustments by the target and antibiotic that permit CRM to maintain binding where other antibiotics fail.

Hydrogen-tritium exchange is widely employed for radioisotopic labeling of molecules of biological interest but typically involves the metal-promoted exchange of sp 2 -hybridized carbon-hydrogen bonds, a strategy that is not directly applicable to the antibiotic iboxamycin, which possesses no such bonds. We show that ruthenium-induced 2'-epimerization of 2'-epi-iboxamycin in HTO (200 mCi) of low specific activity (10 Ci/g, 180 mCi/mmol) at 80 °C for 18 h affords after purification tritium-labeled iboxamycin (3.55 µCi) with a specific activity of 53 mCi/mmol. Iboxamycin displayed an apparent inhibition constant (K i, app ) of 41 ± 30 nM towards Escherichia coli ribosomes, binding approximately 70-fold more tightly than the antibiotic clindamycin (K i, app  = 2.7 ± 1.1 µM)., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: A.G.M. has filed an international patent application WO/2019/032936 ‘Lincosamide Antibiotics and Uses Thereof’. A.G.M. has filed an international patent application WO/2019/032956 ‘Lincosamide Antibiotics and Uses Thereof’., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The mechanism proposed for the transformation of cyclopentanone to the dienoic acid 1 , as published in this journal, is revealed to be in error. We show that carbon 11 derives not from dimethyl sulfoxide as proposed but from the dichloromethane present in the "quenching" solution. The intermediacy of an α-chloromethyl ketone and its subsequent fragmentation in the presence of a hydroxide ion is supported by experiments described herein and by extensive literature precedent.

Genome-encoded antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F subfamily (ARE-ABCFs) mediate intrinsic resistance in diverse Gram-positive bacteria. The diversity of chromosomally-encoded ARE-ABCFs is far from being fully experimentally explored. Here we characterise phylogenetically diverse genome-encoded ABCFs from Actinomycetia (Ard1 from Streptomyces capreolus, producer of the nucleoside antibiotic A201A), Bacilli (VmlR2 from soil bacterium Neobacillus vireti) and Clostridia (CplR from Clostridium perfringens, Clostridium sporogenes and Clostridioides difficile). We demonstrate that Ard1 is a narrow spectrum ARE-ABCF that specifically mediates self-resistance against nucleoside antibiotics. The single-particle cryo-EM structure of a VmlR2-ribosome complex allows us to rationalise the resistance spectrum of this ARE-ABCF that is equipped with an unusually long antibiotic resistance determinant (ARD) subdomain. We show that CplR contributes to intrinsic pleuromutilin, lincosamide and streptogramin A resistance in Clostridioides, and demonstrate that C. difficile CplR (CDIF630&#95;02847) synergises with the transposon-encoded 23S ribosomal RNA methyltransferase Erm to grant high levels of antibiotic resistance to the C. difficile 630 clinical isolate. Finally, assisted by uORF4u, our novel tool for detection of upstream open reading frames, we dissect the translational attenuation mechanism that controls the induction of cplR expression upon an antibiotic challenge., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The regioselectivity of a [3+2] dipolar cycloaddition reaction of a stabilized azomethine ylide with an electron-deficient dipolarophile was found to be counter to a report published in this journal.

Since antibiotic resistance is often associated with a fitness cost, bacteria employ multi-layered regulatory mechanisms to ensure that expression of resistance factors is restricted to times of antibiotic challenge. In Bacillus subtilis, the chromosomally-encoded ABCF ATPase VmlR confers resistance to pleuromutilin, lincosamide and type A streptogramin translation inhibitors. Here we show that vmlR expression is regulated by translation attenuation and transcription attenuation mechanisms. Antibiotic-induced ribosome stalling during translation of an upstream open reading frame in the vmlR leader region prevents formation of an anti-antiterminator structure, leading to the formation of an antiterminator structure that prevents intrinsic termination. Thus, transcription in the presence of antibiotic induces vmlR expression. We also show that NusG-dependent RNA polymerase pausing in the vmlR leader prevents leaky expression in the absence of antibiotic. Furthermore, we demonstrate that induction of VmlR expression by compromised protein synthesis does not require the ability of VmlR to rescue the translational defect, as exemplified by constitutive induction of VmlR by ribosome assembly defects. Rather, the specificity of induction is determined by the antibiotic's ability to stall the ribosome on the regulatory open reading frame located within the vmlR leader. Finally, we demonstrate the involvement of (p)ppGpp-mediated signalling in antibiotic-induced VmlR expression., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Listeriosis is a food-borne disease caused by the Gram-positive Bacillota (Firmicute) bacterium Listeria monocytogenes . Clinical L. monocytogenes isolates are often resistant to clinically used lincosamide clindamycin, thus excluding clindamycin as a viable treatment option., Objectives: We have established newly developed lincosamide iboxamycin as a potential novel antilisterial agent., Methods: We determined MICs of the lincosamides lincomycin, clindamycin and iboxamycin for L. monocytogenes , Enterococcus faecalis and Bacillus subtilis strains expressing synergetic antibiotic resistance determinants: ABCF ATPases that directly displace antibiotics from the ribosome and Cfr, a 23S rRNA methyltransferase that compromises antibiotic binding. For L. monocytogenes strains, either expressing VgaL/Lmo0919 or lacking the resistance factor, we performed time-kill kinetics and post-antibiotic effect assays., Results: We show that the synthetic lincosamide iboxamycin is highly active against L. monocytogenes and can overcome the intrinsic lincosamide resistance mediated by VgaL/Lmo0919 ABCF ATPase. While iboxamycin is not bactericidal against L. monocytogenes , it displays a pronounced post-antibiotic effect, which is a valuable pharmacokinetic feature. We demonstrate that VmlR ABCF of B. subtilis grants significant (33-fold increase in MIC) protection from iboxamycin, while LsaA ABCF of E. faecalis grants an 8-fold protective effect. Furthermore, the VmlR-mediated iboxamycin resistance is cooperative with that mediated by the Cfr, resulting in up to a 512-fold increase in MIC., Conclusions: While iboxamycin is a promising new antilisterial agent, our findings suggest that emergence and spread of ABCF ARE variants capable of defeating next-generation lincosamides in the clinic is possible and should be closely monitored., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

The dearth of new medicines effective against antibiotic-resistant bacteria presents a growing global public health concern 1 . For more than five decades, the search for new antibiotics has relied heavily on the chemical modification of natural products (semisynthesis), a method ill-equipped to combat rapidly evolving resistance threats. Semisynthetic modifications are typically of limited scope within polyfunctional antibiotics, usually increase molecular weight, and seldom permit modifications of the underlying scaffold. When properly designed, fully synthetic routes can easily address these shortcomings 2 . Here we report the structure-guided design and component-based synthesis of a rigid oxepanoproline scaffold which, when linked to the aminooctose residue of clindamycin, produces an antibiotic of exceptional potency and spectrum of activity, which we name iboxamycin. Iboxamycin is effective against ESKAPE pathogens including strains expressing Erm and Cfr ribosomal RNA methyltransferase enzymes, products of genes that confer resistance to all clinically relevant antibiotics targeting the large ribosomal subunit, namely macrolides, lincosamides, phenicols, oxazolidinones, pleuromutilins and streptogramins. X-ray crystallographic studies of iboxamycin in complex with the native bacterial ribosome, as well as with the Erm-methylated ribosome, uncover the structural basis for this enhanced activity, including a displacement of the [Formula: see text] nucleotide upon antibiotic binding. Iboxamycin is orally bioavailable, safe and effective in treating both Gram-positive and Gram-negative bacterial infections in mice, attesting to the capacity for chemical synthesis to provide new antibiotics in an era of increasing resistance., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

A gram-scale synthesis of iboxamycin, an antibiotic candidate bearing a fused bicyclic amino acid residue, is presented. A pivotal transformation in the route involves an intramolecular hydrosilylation-oxidation sequence to set the ring-fusion stereocenters of the bicyclic scaffold. Other notable features of the synthesis include a high-yielding, highly diastereoselective alkylation of a pseudoephenamine amide, a convergent sp 3 -sp 2 Negishi coupling, and a one-pot transacetalization-reduction reaction to form the target compound's oxepane ring. Implementation of this synthetic strategy has provided ample quantities of iboxamycin to allow for its in vivo profiling in murine models of infection.

The development of a flexible, component-based synthetic route to the amino sugar fragment of the lincosamide antibiotics is described. This route hinges on the application and extension of nitroaldol chemistry to forge strategic bonds within complex amino sugar targets and employs a glycal epoxide as a versatile glycosyl donor for the installation of anomeric groups. Through building-block exchange and late-stage functionalization, this route affords access to a host of rationally designed lincosamides otherwise inaccessible by semisynthesis and underpins a platform for the discovery of new lincosamide antibiotics.

Macrolides are among the most widely prescribed antibiotics, particularly for bacterial lung infections, due to their favorable safety, oral bioavailability, and spectrum of activity against Gram-positive pathogens such as Streptococcus pneumoniae , the most common cause of bacterial pneumonia. Their utility against Gram-negative bacteria is extremely limited and does not include the Enterobacteriaceae or other ESKAPE pathogens. With the increasing development of resistance to current therapies and the lack of safe, oral options to treat Gram-negative infections, extended-spectrum macrolides have the potential to provide valuable treatment options. While the bacterial ribosome, the target of macrolides, is highly conserved across Gram-positive and Gram-negative bacteria, traditional macrolides do not possess the proper physicochemical properties to cross the polar Gram-negative outer membrane and are highly susceptible to efflux. As with most natural product-derived compounds, macrolides are generally prepared through semisynthesis, which is limited in scope and lacks the ability to make the drastic physicochemical property changes necessary to overcome these hurdles.By using a fully synthetic platform technology to greatly expand structural diversity, novel macrolides were prepared with a focus on lowering the MW and increasing the polarity to achieve a physicochemical property profile more similar to that of traditional Gram-negative drug classes. In addition to the removal of lipophilic groups, a critical structural feature for obtaining Gram-negative activity in the macrolide class proved to be the introduction of small secondary or tertiary amines to yield polycationic species potentially capable of self-promoted uptake. Within the azithromycin-like 15-membered azalides, potent activity was seen when small alkyl amines were introduced at the 6'-position of desosamine. The biggest gains, however, were made by replacing the entire C10-C13 fragment of the macrolactone ring with commercially available or readily synthesized 1,2-aminoalcohols, leading to 13-membered azalides. The introduction of a tethered basic amine at the C10-position and systematic optimization of substitution and tether length and flexibility ultimately provided new macrolides that for the first time exhibit clinically relevant antibacterial activity against multi-drug resistant Gram-negative bacteria. A retrospective computational analysis of >1800 fully synthetic macrolides prepared during this effort identified key drivers and optimum ranges for improving permeability and avoiding efflux. In contrast to standard Gram-negative drugs which generally have MWs below 600 and clogD 7.4 values below 0, we found that the ideal ranges for Gram-negative macrolides were MW between 600 and 720 and cLogD 7.4 between -1 and 3. A total charge of between 2.5 and 3 was also required to provide optimal permeability and efflux avoidance. Thus, Gram-negative macrolides occupy a unique physicochemical property space that lies between traditional Gram-negative drug classes and Gram-positive macrolides.

Apart from their antimicrobial properties, tetracyclines demonstrate clinically validated effects in the amelioration of pathological inflammation and human cancer. Delineation of the target(s) and mechanism(s) responsible for these effects, however, has remained elusive. Here, employing quantitative mass spectrometry-based proteomics, we identified human 80S ribosomes as targets of the tetracyclines Col-3 and doxycycline. We then developed in-cell click selective crosslinking with RNA sequence profiling (icCL-seq) to map binding sites for these tetracyclines on key human rRNA substructures at nucleotide resolution. Importantly, we found that structurally and phenotypically variant tetracycline analogs could chemically discriminate these rRNA binding sites. We also found that tetracyclines both subtly modify human ribosomal translation and selectively activate the cellular integrated stress response (ISR). Together, the data reveal that targeting of specific rRNA substructures, activation of the ISR, and inhibition of translation are correlated with the anti-proliferative properties of tetracyclines in human cancer cell lines., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Key building blocks for the production of fully synthetic macrolides have been scaled-up in first time pilot plant and kilo-lab campaigns. These building blocks have supported the discovery of new macrolide antibiotics as well as ongoing preclinical studies.

5-Oxatetracyclines were synthesized from d-arabinose using sequential Michael-Claisen cyclization reactions via a 5-oxa-AB enone substrate. The 5-oxatetracyclines were found to have poor stability in aqueous buffer (pH 7.4, 37 °C) and showed little to no inhibition of bacterial growth (S. aureus, E. coli).

Mutated nucleophosmin 1 (NPM1) acts as a proto-oncogene and is present in ~30% of patients with acute myeloid leukemia (AML). Here we examined the in vitro and in vivo anti-leukemic activity of the NPM1 and chromosome region maintenance 1 homolog (CRM1) interacting natural product avrainvillamide (AVA) and a fully syntetic AVA analog. The NPM1-mutated cell line OCI-AML3 and normal karyotype primary AML cells with NPM1 mutations were significantly more sensitive towards AVA than cells expressing wild-type (wt) NPM1. Furthermore, the presence of wt p53 sensitized cells toward AVA. Cells exhibiting fms-like tyrosine kinase 3 (FLT3) internal tandem duplication mutations also displayed a trend toward increased sensitivity to AVA. AVA treatment induced nuclear retention of the NPM1 mutant protein (NPMc+) in OCI-AML3 cells and primary AML cells, caused proteasomal degradation of NPMc+ and the nuclear export factor CRM1 and downregulated wt FLT3 protein. In addition, both AVA and its analog induced differentiation of OCI-AML3 cells together with an increased phagocytotic activity and oxidative burst potential. Finally, the AVA analog displayed anti-proliferative activity against subcutaneous xenografted HCT-116 and OCI-AML3 cells in mice. Our results demonstrate that AVA displays enhanced potency against defined subsets of AML cells, suggesting that therapeutic intervention employing AVA or related compounds may be feasible., Competing Interests: AGM holds a patent on the synthesis of avrainvillamide and analogs thereof (WO2006102097 A3). BTG and EMC owns shares in KinN Therapeutics AS. The remaining authors declare no conflict of interest.

Tetracyclines have proven to be safe and effective antibiotics over decades but to date all approved members of the class have been discovered and manufactured by chemical modification of fermentation products, which greatly limits the number of new structures that can be explored as future medicines. This review summarizes research leading to the development of a platform synthetic technology that enabled the discovery of the clinical candidate eravacycline, as well as other promising new tetracycline antibiotics, and provides the basis for a practical route for their manufacture. The approach argues for a reassessment of other antibiotic classes based on natural products for which practical, fully synthetic routes have not yet been developed, suggesting that these may represent underdeveloped resources with great potential to offer safer and more effective anti-infective agents., (Published by Elsevier Ltd.)

The chemical modification of structurally complex fermentation products, a process known as semisynthesis, has been an important tool in the discovery and manufacture of antibiotics for the treatment of various infectious diseases. However, many of the therapeutics obtained in this way are no longer effective, because bacterial resistance to these compounds has developed. Here we present a practical, fully synthetic route to macrolide antibiotics by the convergent assembly of simple chemical building blocks, enabling the synthesis of diverse structures not accessible by traditional semisynthetic approaches. More than 300 new macrolide antibiotic candidates, as well as the clinical candidate solithromycin, have been synthesized using our convergent approach. Evaluation of these compounds against a panel of pathogenic bacteria revealed that the majority of these structures had antibiotic activity, some efficacious against strains resistant to macrolides in current use. The chemistry we describe here provides a platform for the discovery of new macrolide antibiotics and may also serve as the basis for their manufacture.

D-Desosamine is synthesized in 4 steps from methyl vinyl ketone and sodium nitrite. The key step in this chromatography-free synthesis is the coupling of (R)-4-nitro-2-butanol and glyoxal (trimeric form) mediated by cesium carbonate, which affords in crystalline form 3-nitro-3,4,6-trideoxy-α-D-glucose, a nitro sugar stereochemically homologous to D-desosamine. This strategy has enabled the syntheses of an array of analogous 3-nitro sugars. In each case the 3-nitro sugars are obtained in pure form by crystallization., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling transcription factors and oncogenes. BRD4 and CDK7 are positive regulators of SE-mediated transcription. By contrast, negative regulators of SE-associated genes have not been well described. Here we show that the Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We report that the natural product cortistatin A (CA) selectively inhibits Mediator kinases, has anti-leukaemic activity in vitro and in vivo, and disproportionately induces upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the transcription factors CEBPA, IRF8, IRF1 and ETV6 (refs 6-8). The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has anti-leukaemic activity. Individually increasing or decreasing the expression of these transcription factors suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to the dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types, and can be pharmacologically targeted as a therapeutic approach to AML.

Nucleophosmin (NPM1) is a multifunctional phosphoprotein localized predominantly within the nucleoli of eukaryotic cells. Mutations within its C-terminal domain are frequently observed in patients with acute myeloid leukemia (AML), are thought to play a key role in the initiation of the disease, and result in aberrant, cytoplasmic localization of the mutant protein. We have previously shown that the electrophilic antiproliferative natural product (+)-avrainvillamide (1) binds to proteins, including nucleophosmin, by S-alkylation of cysteine residues. Here, we report that avrainvillamide restores nucleolar localization of certain AML-associated mutant forms of NPM1 and provide evidence that this relocalization is mediated by interactions of avrainvillamide with mutant NPM1 and exportin-1 (Crm1). Immunofluorescence and mass spectrometric experiments employing a series of different NPM1 constructs suggest that a specific interaction between avrainvillamide and Cys275 of certain NPM1 mutants mediates the relocalization of these proteins to the nucleolus. Avrainvillamide treatment is also shown to inhibit nuclear export of Crm1 cargo proteins, including AML-associated NPM1 mutants. We also observe that avrainvillamide treatment displaces Thr199-phosphorylated NPM1 from duplicated centrosomes, leads to an accumulation of supernumerary centrosomes, and inhibits dephosphorylation of Thr199-phosphorylated NPM1 by protein phosphatase 1. Avrainvillamide is the first small molecule reported to relocalize specific cytoplasmic AML-associated NPM1 mutants to the nucleolus, providing an important demonstration of principle that small molecule induction of a wild-type NPM1 localization phenotype is feasible in certain human cancer cells.

X-ray crystallographic characterization of products derived from natural and fully synthetic trioxacarcins, molecules with potent antiproliferative effects, illuminates aspects of their reactivity and mechanism of action. Incubation of the fully synthetic trioxacarcin analog 3, which lacks one of the carbohydrate residues present in the natural product trioxacarcin A (1) as well as oxygenation at C2 and C4 yet retains potent antiproliferative effects, with the self-complimentary duplex oligonucleotide d(AACCGGTT) led to production of a crystalline covalent guanine adduct (6). Adduct 6 is closely analogous to gutingimycin (2), the previously reported guanine adduct derived from incubation of natural trioxacarcin A (1) with duplex DNA, suggesting that 3 and 1 likely share a common basis of cytotoxicity. In addition, we isolated a novel, dark-red crystalline guanine adduct (7) from incubation of trioxacarcin A itself with the self-complimentary duplex oligonucleotide d(CGTATACG). Crystallographic analysis suggests that 7 is an anthraquinone derivative, which we propose arises by a sequence of guanosine alkylation within duplex DNA, depurination, base-catalyzed elimination of the trioxacarcinose A carbohydrate residue, and oxidative rearrangement to form an anthraquinone. We believe that this heretofore unrecognized chemical instability of natural trioxacarcins may explain why trioxacarcin analogs lacking C4 oxygenation exhibit superior chemical stabilities yet, as evidenced by structure 3, retain a capacity to form lesions with duplex DNA., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The discovery and implementation of antibiotics in the early twentieth century transformed human health and wellbeing. Chemical synthesis enabled the development of the first antibacterial substances, organoarsenicals and sulfa drugs, but these were soon outshone by a host of more powerful and vastly more complex antibiotics from nature: penicillin, streptomycin, tetracycline, and erythromycin, among others. These primary defences are now significantly less effective as an unavoidable consequence of rapid evolution of resistance within pathogenic bacteria, made worse by widespread misuse of antibiotics. For decades medicinal chemists replenished the arsenal of antibiotics by semisynthetic and to a lesser degree fully synthetic routes, but economic factors have led to a subsidence of this effort, which places society on the precipice of a disaster. We believe that the strategic application of modern chemical synthesis to antibacterial drug discovery must play a critical role if a crisis of global proportions is to be averted., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

β-Hydroxy-α-amino acids figure prominently as chiral building blocks in chemical synthesis and serve as precursors to numerous important medicines. Reported herein is a method for the synthesis of β-hydroxy-α-amino acid derivatives by aldolization of pseudoephenamine glycinamide, which can be prepared from pseudoephenamine in a one-flask protocol. Enolization of (R,R)- or (S,S)-pseudoephenamine glycinamide with lithium hexamethyldisilazide in the presence of LiCl followed by addition of an aldehyde or ketone substrate affords aldol addition products that are stereochemically homologous with L- or D-threonine, respectively. These products, which are typically solids, can be obtained in stereoisomerically pure form in yields of 55-98 %, and are readily transformed into β-hydroxy-α-amino acids by mild hydrolysis or into 2-amino-1,3-diols by reduction with sodium borohydride. This new chemistry greatly facilitates the construction of novel antibiotics of several different classes., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Analogs of salinosporamide A with variations of the C2 and C5 substituents are prepared in 8-10 steps using as the first and key transformation a diastereoselective Mukaiyama aldol reaction between the chiral 5-tert-butyldimethylsiloxy-3-methyl-1H-pyrrole-2-carboxylic ester depicted and various aldehyde substrates, promoted by tert-butyldimethylsilyl triflate. In this transformation, the 4-trimethylsilyl-3-butyn-2-ol ester functions to direct the formation of predominantly one of four possible diastereomeric aldol products. Introduction of the C2 appendage by a later-stage, stereocontrolled alkylation reaction permits the construction of analogs variant at this position. Results from in vitro and cell-based assays of proteasomal inhibition are reported. Mass spectrometric studies provide mechanistic details of proteasomal modification by salinosporamide A and analogs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A three-step synthesis of pseudoephenamine suitable for preparing multigram amounts of both enantiomers of the auxiliary from the inexpensive starting material benzil is described. The sequence involves synthesis of the crystalline monomethylimine derivative of benzil, reduction of that substance with lithium aluminum hydride, and resolution of pseudoephenamine with mandelic acid.

The trioxacarcins are polyoxygenated, structurally complex natural products that potently inhibit the growth of cultured human cancer cells. Here we describe syntheses of trioxacarcin A, DC-45-A1 and structural analogues by late-stage stereoselective glycosylation reactions of fully functionalized, differentially protected aglycon substrates. Key issues addressed in this work include the identification of an appropriate means to activate and protect each of the two 2-deoxysugar components, trioxacarcinose A and trioxacarcinose B, as well as a viable sequencing of the glycosidic couplings. The convergent, component-based sequence we present allows for rapid construction of structurally diverse, synthetic analogues that would be inaccessible by any other means, in amounts required to support biological evaluation. Analogues that arise from the modification of four of five modular components are assembled in 11 steps or fewer. The majority of these are found to be active in antiproliferative assays using cultured human cancer cells.

The utility of pseudoephenamine as a chiral auxiliary for the alkylative construction of quaternary α-methyl α-amino acids is demonstrated. The method is notable for the high diastereoselectivities of the alkylation reactions, for its versatility with respect to electrophilic substrate partners, and for its mild hydrolysis conditions, which provide α-amino acids without salt contaminants. Alternatively, α-amino esters can be obtained by direct alcoholysis.

Two routes to the 2,6-dideoxysugar methyl trioxacarcinoside A are described. Each was enabled by an apparent α-chelation-controlled addition of an allylmetal reagent to a ketone substrate containing a free α-hydroxyl group and a β-hydroxyl substituent, either free or protected as the corresponding di-tert-butylmethyl silyl ether. Both routes provide practical access to gram quantities of trioxacarcinose A in a form suitable for glycosidic coupling reactions.

Here we describe chemical innovations that enable the preparation of fully synthetic tetracyclines containing an all-carbon quaternary, stereogenic center at position C5a, a structurally novel class of compounds in this important family of therapeutic agents. In the key transformation and an important extension of the powerful Michael-Claisen cyclization (AB plus D) approach to the construction of fully synthetic tetracyclines, we show that the six-membered C ring comprising a C5a quaternary carbon center can be assembled by highly stereocontrolled coupling reactions of β-substituted AB enones and o-toluate ester anion D-ring precursors. Novel and versatile β-functionalization reaction sequences employing tris(methylthio)methyllithium and 2-lithio-1,3-dithiane have been developed to transform the AB enone 1 (the key precursor to fully synthetic tetracyclines) into a diverse range of β-substituted AB enone products, including a highly efficient, single-operation method for the synthesis of a β-methyl ester-substituted AB enone. A C5a-C11a-bridged cyclopropane tetracycline precursor was found to undergo efficient and regioselective ring-opening reactions with a range of nucleophiles in the presence of magnesium bromide, thus providing another avenue for the preparation of fully synthetic tetracyclines containing an all-carbon quaternary center at position C5a. Two compounds prepared from the bridged cyclopropane intermediate served as (further) diversifiable branch-points, allowing maximally expedient synthesis of C5a-substituted tetracyclines by final-step diversification.

A 9-step synthetic route to the complex carbohydrate methyl α-trioxacarcinoside B from 2-acetylfuran is described. Anomerically activated forms, including 1-phenylthio, 1-O-(4'-pentenyl), 1-fluoro, and 1-O-acetyl derivatives are also prepared., (© 2011 American Chemical Society)

An efficient four-step synthetic route to the useful chiral building block (2R,3S)-dihydroxybutyric acid acetonide in >95% ee is detailed. The sequence is readily scaled, requires no chromatography, and allows for efficient recycling of p-phenylbenzyl alcohol, an expedient for enantio- and diastereoenrichment by recrystallization.

We show that arylpalladium(II) reagents linked to biotin and indocyanine dye residues can be prepared by decarboxylative palladation of appropriately substituted electron-rich benzoic acid derivatives. When prepared under the conditions described, these organometallic intermediates are tolerant of air and water, can be stored for several months in solution in dimethyl sulfoxide, and permit biotin- and indocyanine dye-labeling of functionally complex olefinic substrates in water by Heck-type coupling reactions.

Here we describe a 5-step sequence to prepare the AB enone 1, the key precursor to fully synthetic tetracyclines, that begins with a diastereoselective Michael-Claisen coupling of two simple starting materials, a cyclohexenone (compound 2 or, in a refinement, a substituted variant, vide infra) and the isoxazole ester 3. This advance defines an 8-step linear sequence to 6-deoxytetracycline antibiotics from three components of similar complexity (cyclohexenone 2, isoxazole ester 3, and structurally diverse D-ring precursors) in which sequential diastereoselective Michael-Claisen cyclization reactions form the A- and C-rings, respectively, of the linearly fused ABCD tetracycline skeleton. In addition to providing a readily scalable, practical route to fully synthetic tetracyclines of broad structural diversity, the sequence reported comprises a series of non-obvious stereoselective transformations, including a novel means for C12a hydroxylation.

Many first-line cancer drugs are natural products or are derived from them by chemical modification. The trioxacarcins are an emerging class of molecules of microbial origin with potent antiproliferative effects, which may derive from their ability to covalently modify duplex DNA. All trioxacarcins appear to be derivatives of a nonglycosylated natural product known as DC-45-A2. To explore the potential of the trioxacarcins for the development of small-molecule drugs and probes, we have designed a synthetic strategy toward the trioxacarcin scaffold that enables access to both the natural trioxacarcins and nonnatural structural variants. Here, we report a synthetic route to DC-45-A2 from a differentially protected precursor, which in turn is assembled in just six steps from three components of similar structural complexity. The brevity of the sequence arises from strict adherence to a plan in which strategic bond-pair constructions are staged at or near the end of the synthetic route.

The cortistatins are a recently identified class of marine natural products characterized by an unusual steroidal skeleton, which have been found to inhibit differentially the proliferation of various mammalian cells in culture by an unknown mechanism. We describe a comprehensive route for the synthesis of cortistatins from a common precursor, which in turn is assembled from two fragments of similar structural complexity. Cortistatins A and J, and for the first time K and L, have been synthesized in parallel processes from like intermediates prepared from a single compound. With the identification of facile laboratory transformations linking intermediates in the cortistatin L synthetic series with corresponding intermediates to cortistatins A and J, we have been led to speculate that somewhat related paths might occur in nature, offering potential sequencing and chemical detail for cortistatin biosynthetic pathways.

Methyl alpha-methylene-beta-tert-butyldimethylsilyloxycarboxylate esters are found to undergo diastereoselective epoxidation in the presence of potassium tert-butoxide-tert-butyl hydroperoxide to form anti products. In an effort to better understand mechanistic details of the transformation and the basis of diastereoselectivities observed, we studied the epoxidation of substrates with alpha-methylene groups containing (trans) deuterium labels and discovered that oxygen-atom transfer proceeds with > or = 95% stereospecificity in all cases examined. These and other experiments suggest that the mechanism of epoxidation is not distinguishable from a concerted process.

We describe a synthetic sequence that allows for the preparation of optically active trans-1,2-diol monosilyl ether derivatives from ketones, providing a new means for retrosynthetic simplification of differentiated diol and polyol targets. The sequence involves silyl enol ether formation, Shi asymmetric epoxidation, and then regio- and stereospecific addition of hydride, methide, or higher alkylide. The tactical combination presented has not been integrated in synthetic problem solving, so far as we are aware, but has promise for broad application.

We used multiple imaging assays to test the hypothesis that GPR6, a constitutively active Gs-coupled receptor, is present on the cell surface. A pHluorin tag at the N-terminus of rat GPR6 expressed in human embryonic kidney 293 (HEK293) cells was not accessible to protons, chymotrypsin or anti-green fluorescent protein antibody, demonstrating that GPR6 is primarily located in intracellular compartments. Similar intracellular localization of pHluorin-tagged GPR6 was found in striatal neurons, where endogenous GPR6 is expressed. Confirmation of Gs-mediated constitutive activity in HEK293 cells and striatal neurons led us to conclude that GPR6 can signal from intracellular compartments.

Tetracyclines and tetracycline analogues are prepared by a convergent, single-step Michael-Claisen condensation of AB precursor 1 or 2 with D-ring precursors of wide structural variability, followed by removal of protective groups (typically in two steps). A number of procedural variants of the key C-ring-forming reaction are illustrated in multiple examples. These include stepwise deprotonation of a D-ring precursor followed by addition of 1 or 2, in situ deprotonation of a D-ring precursor in mixture with 1 or 2, and in situ lithium-halogen exchange of a benzylic bromide D-ring precursor in the presence of 1 or 2, followed by warming. The AB plus D strategy for tetracycline synthesis by C-ring construction is shown to be robust across a range of different carbocyclic and heterocyclic D-ring precursors, proceeding reliably and with a high degree of stereochemical control. Evidence suggests that Michael addition of the benzylic anion derived from a given D-ring precursor to enones 1 or 2 is quite rapid at -78 degrees C, while Claisen cyclization of the enolate produced is rate-determining, typically occurring upon warming to 0 degrees C. The AB plus D coupling strategy is also shown to be useful for the construction of tetracycline precursors that are diversifiable by latter-stage transformations, subsequent to cyclization to form the C ring. Results of antibacterial assays and preliminary data obtained from a murine septicemia model show that many of the novel tetracyclines synthesized have potent antibiotic activities, both in bacterial cell culture and in vivo. The platform for tetracycline synthesis described gives access to a broad range of molecules that would be inaccessible by semisynthetic methods (presently the only means of tetracycline production) and provides a powerful engine for the discovery and, perhaps, development of new tetracycline antibiotics.

Protocols for the stereodefined formation of alpha,alpha-disubstituted enolates of pseudoephedrine amides are presented followed by the implementation of these in diastereoselective alkylation reactions. Direct alkylation of alpha,alpha-disubstituted pseudoephedrine amide substrates is demonstrated to be both efficient and diastereoselective across a range of substrates, as exemplified by alkylation of the diastereomeric pseudoephedrine alpha-methylbutyramides, where both substrates are found to undergo stereospecific replacement of the alpha-C-H bond with alpha-C-alkyl, with retention of stereochemistry. This is shown to arise by sequential stereospecific enolization and alkylation reactions, with the alkyl halide attacking a common pi-face of the E- and Z-enolates, proposed to be opposite the pseudoephedrine alkoxide side chain. Pseudoephedrine alpha-phenylbutyramides are found to undergo highly stereoselective but not stereospecific alpha-alkylation reactions, which evidence suggests is due to facile enolate isomerization. Also, we show that alpha,alpha-disubstituted pseudoephedrine amide enolates can be generated in a highly stereocontrolled fashion by conjugate addition of an alkyllithium reagent to the s-cis-conformer of an alpha-alkyl-alpha,beta-unsaturated pseudoephedrine amide, providing alpha,alpha-disubstituted enolate substrates that undergo alkylation in the same sense as those formed by direct deprotonation. Methods are presented to transform the alpha-quaternary pseudoephedrine amide products into optically active carboxylic acids, ketones, primary alcohols, and aldehydes.

Here we present evidence that (+)-avrainvillamide, a naturally occurring alkaloid with antiproliferative effects, binds to the nuclear chaperone nucleophosmin, a proposed oncogenic protein that is overexpressed in many different human tumors. Among other effects, nucleophosmin is known to regulate the tumor suppressor protein p53. A synthetic biotin-avrainvillamide conjugate, nearly equipotent to the natural product in inhibiting the growth of cultured T-47D cells, was used for affinity-isolation of a protein identified as nucleophosmin by MS sequencing and Western-blotting. Affinity-isolation of nucleophosmin was inhibited in the presence of iodoacetamide (10 mM), free (+)-avrainvillamide (100 microM), and a series of closely related structural analogues of (+)-avrainvillamide, the latter with inhibitory effects that appear to correlate with measured growth-inhibitory potencies. Using fluorescence microscopy, a synthetic dansyl-avrainvillamide conjugate was observed to localize within the nucleoli and the cytosol of treated cancer cells. Site-directed mutagenesis of each of the three cysteine residues of a truncated nucleophosmin coexpressed with native nucleophosmin in COS-7 cells revealed that the mutation cys275 --> ala275 effectively and uniquely reduced affinity-isolation of the truncated protein, suggesting that avrainvillamide targets cys275 of nucleophosmin. Finally, we show that treatment of adhered LNCaP or T-47D cells with (+)-avrainvillamide leads to an increase in cellular p53 concentrations, and that siRNA-promoted depletion of nucleophosmin in a population of HeLa S3 cells leads to increased sensitivity of that population toward apoptotic death upon treatment with (+)-avrainvillamide. Although potentially desirable as lead compounds for the development of novel anticancer therapies, nonpeptidic, synthetic small molecules that bind to nucleophosmin have not been described, prior to this report.