Asymmetric Total Synthesis of (+)-Epiibogamine Enabled by Three-Component Domino Michael/Michael/Mannich Annulation of N-Sulfinyl Metallosilylenamines.

The iboga alkaloids are promising antiaddictive and neuroregeneration candidates for medical treatment. There is a lack of studies for C20-epi iboga alkaloids due to the synthetic difficulties. Herein we report the shortest total synthesis of (+)-epiibogamine in seven steps from trimethyl orthobutyrate. The novel N-sulfinyl silylenamine reagent enabled the key step, with three-component domino Michael/Michael/Mannich annulation providing the 1-amino-2,4-diester scaffold with four new chiral centers, and access to the isoquinuclidine in high yield (84%) and diastereoselectivity (>95:5 dr).

Diastereoselective Hydroxylation of N-tert-Butanesulfinyl Imines with 2-(Phenylsulfonyl)-3-phenyloxaziridine (Davis Oxaziridine).

The diastereoselective α-hydroxylation of N-tert-butanesulfinyl metallodienenamine and metalloenamines with Davis oxaziridine affords α-hydroxy N-sulfinyl imines with 50-88% yield and up to 98:2 diastereomeric ratio. Dramatic changes in diastereoselectivity and stereoselectivity were observed by choice of metal bases. The mechanistic understanding for the switch in diastereoselectivity was assisted by DFT computational modeling, which suggests the facial approach is governed by aza-enolate geometry. A one-pot protocol for the asymmetric synthesis of 1,2-amino alcohols is described.

Semisynthesis of Bis-Indole Alkaloid (-)-Melodinine K Enabled by a Combination of Biotransformation and Chemical Synthesis.

Enzymatic biotransformation has become a widely used technique in synthetic chemistry to achieve difficult chemical transformations. Cytochrome P450 monooxygenase enzymes found in nature carry out a wide range of difficult chemical reactions, such as the oxidation of the monoterpene indole alkaloid (-)-tabersonine at the unreactive 16th position on the indoline benzene ring in the biosynthesis of biologically active natural products such as the bis-indole alkaloid (-)-melodinine K. Herein, we describe the first semisynthesis of (-)-melodinine K enabled by a biological gram scale route to the northern fragment, (-)-16-hydroxytabersonine, as well as a chemical route to the southern fragment, (-)-pachysiphine, both derived from (-)-tabersonine and subsequently coupled in only eight linear steps. (-)-16-Hydroxytabersonine is produced through an enzymatic biotransformation with a genetically modified Saccharomyces cerevisiae yeast strain expressing a tabersonine 16-hydroxylase enzyme to enable regioselective oxidation on multigram scale, and (-)-pachysiphine is produced through stereoselective and regioselective epoxidation of the disubstituted alkene.

Semi-syntheses and interrogation of indole-substituted Aspidosperma terpenoid alkaloids.

We demonstrated here a series of Aspidosperma terpenoid alkaloids can be quickly prepared using semisynthesis from naturally sourced tabersonine, featuring multiple oxygen-based substituents on the indole ring such as hydroxy and methoxy groups. This panel of complex compounds enabled the exploration of indole modifications to optimize the indole alkaloids' anticancer activity, generating lead compounds (e.g., with C15-hydroxy, C16-methoxy, and/or C17-methoxy derivatizations) that potently inhibit cancer cell line growth in the single-digit micromolar range. These results can help guide the development of Aspidosperma terpenoid alkaloid therapeutics. Furthermore, this synthetic approach features late-stage facile derivatization on complex natural product molecules, providing a versatile path to indole derivatization of this family of alkaloids with diverse chemical functionalities for future medicinal chemistry and chemical biology discoveries.

Alternative approaches utilizing click chemistry to develop next-generation analogs of solithromycin.

The marked rise in bacterial drug resistance has created an urgent need for novel antibacterials belonging to new drug classes and ideally possessing new mechanisms of action. The superior biological activity of solithromycin against streptococci and other bacteria causative of community-acquired pneumonia pathogens, compared to telithromycin and other macrolides encouraged us to extensively explore this class of antibiotics. We, thus, present the design and synthesis of a novel series of solithromycin analogs. Three main strategies were pursued in structure-activity relationship studies covering the N-11 side chain and the desosamine motif, which are both chief elements for establishing strong interactions with the bacterial ribosome as the molecular target. Minimal inhibitory concentration assays were determined to assess the in vitro potency of the various analogs in relation to solithromycin. Two analogs exhibited improved activity compared to solithromycin against resistant strains, which can be assessed in further pre-clinical studies.

Aspidosperma and Strychnos alkaloids: Chemistry and biology.

Of Nature's nearly 3000 unique monoterpene indole alkaloids derived from tryptophan, those members belonging to the Aspidosperma and Strychnos families continue to impact the fields of natural products (i.e., isolation, structure determination, biosynthesis) and organic chemistry (i.e., chemical synthesis, methodology development) among others. This review covers the biological activity (Section 2), biosynthesis (Section 3), and synthesis of both classical and novel Aspidosperma (Section 4), Strychnos (Section 5), and selected bis-indole (Section 6) alkaloids. Technological advancements in genetic sequencing and bioinformatics have deepened our understanding of how Nature assembles these intriguing molecules. The proliferation of innovative synthetic strategies and tactics for the synthesis of the alkaloids covered in this review, which include contributions from over fifty research groups from around the world, are a testament to the creative power and technical skills of synthetic organic chemists. To be sure, Nature-the Supreme molecular architect and source of a dazzling array of irresistible chemical logic puzzles-continues to inspire scientists across multiple disciplines and will certainly continue to do so for the foreseeable future.

Domino Michael/Mannich Annulation Reaction of N-Sulfinyl Lithiodienamines.

The domino Michael/Mannich (DMM) annulation reaction between an N-sulfinyl lithiodienamine and an electrophilic alkene is developed for the synthesis of chiral 2-amino cyclohexenes, a key building block in asymmetric synthesis. The DMM reaction proceeds at low temperature while maintaining the stereochemical fidelity. The product functionalized amino cyclohexenes, here obtained in 55-82% yield with diastereomeric ratios as high as >19:1.

Synthesis, Biological Evaluation, and Computational Analysis of Biaryl Side-Chain Analogs of Solithromycin.

There is an urgent need for new antibiotics to mitigate the existential threat posed by antibiotic resistance. Within the ketolide class, solithromycin has emerged as one of the most promising candidates for further development. Crystallographic studies of bacterial ribosomes and ribosomal subunits complexed with solithromycin have shed light on the nature of molecular interactions (π-stacking and H-bonding) between from the biaryl side-chain of the drug and key residues in the 50S ribosomal subunit. We have designed and synthesized a library of solithromycin analogs to study their structure-activity relationships (SAR) in tandem with new computational studies. The biological activity of each analog was evaluated in terms of ribosomal affinity (Kd determined by fluorescence polarization), as well as minimum inhibitory concentration assays (MICs). Density functional theory (DFT) studies of a simple binding site model identify key H-bonding interactions that modulate the potency of solithromycin analogs.

Staphylococcus aureus resistance to albocycline can be achieved by mutations that alter cellular NAD/PH pools.

Small molecule target identification is a critical step in modern antibacterial drug discovery, particularly against multi-drug resistant pathogens. Albocycline (ALB) is a macrolactone natural product with potent activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) whose mechanism of action has been elusive to date. Herein, we report biochemical and genomic studies that reveal ALB does not target bacterial peptidoglycan biosynthesis or the ribosome; rather, it appears to modulate NADPH ratios and upregulate redox sensing in the cell consistent with previous studies at Upjohn. Owing to the complexity inherent in biological pathways, further genomic assays are needed to identify the true molecular target(s) of albocycline.

Synthesis and biological evaluation of semi-synthetic albocycline analogs.

Albocycline (ALB) is a unique macrolactone natural product with potent, narrow-spectrum activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate (VISA), and vancomycin-resistant S. aureus (VRSA) strains (MIC = 0.5-1.0 µg/mL). Described herein is the synthesis and evaluation of a novel series analogs derived from albocycline by functionalization at three specific sites: the C2-C3 enone, the tertiary carbinol at C4, and the allylic C16 methyl group. Exploration of the structure-activity relationships (SAR) by means of minimum inhibitory concentration assays (MICs) revealed that C4 ester analog 6 was twice as potent as ALB, which represents a class of lead compound that can be further studied to address multi-drug resistant pathogens.

Synthesis of (-)-Melodinine K: A Case Study of Efficiency in Natural Product Synthesis.

Efficiency is a key organizing principle in modern natural product synthesis. Practical criteria include time, cost, and effort expended to synthesize the target, which tracks with step-count and scale. The execution of a natural product synthesis, that is, the sum and identity of each reaction employed therein, falls along a continuum of chemical (abiotic) synthesis on one extreme, followed by the hybrid chemoenzymatic approach, and ultimately biological (biosynthesis) on the other, acknowledging the first synthesis belongs to Nature. Starting materials also span a continuum of structural complexity approaching the target with constituent elements on one extreme, followed by petroleum-derived and "chiral pool" building blocks, and complex natural products (i.e., semisynthesis) on the other. Herein, we detail our approach toward realizing the first synthesis of (-)-melodinine K, a complex bis-indole alkaloid. The total syntheses of monomers (-)-tabersonine and (-)-16-methoxytabersonine employing our domino Michael/Mannich annulation is described. Isolation of (-)-tabersonine from Voacanga africana and strategic biotransformation with tabersonine 16-hydroxylase for site-specific C-H oxidation enabled a scalable route. The Polonovski-Potier reaction was employed in biomimetic fragment coupling. Subsequent manipulations delivered the target. We conclude with a discussion of efficiency in natural products synthesis and how chemical and biological technologies define the synthetic frontier.

Comparison of micronuclei frequency between smokers and non-smokers: a systematic review / Comparação da frequência de micronúcleos entre indivíduos fumantes e não fumantes: uma revisão sistemática

ABSTRACT Introduction: Cancer results from the accumulation of several modifications in the genetic material and, therefore, it is possible to carry out its cytogenetic detection using biomarkers. Micronuclei (MN) have been addressed in the literature as biomarkers of genotoxic damage. These markers provide information on biological or biochemical changes in a target tissue at early stage, enabling a favorable prognosis. Objective: To compare whether the frequency of MN in active smokers is higher than that in non-smokers. Material and method: The search was performed in the Medical Literature Analysis and Retrieval System (PubMeb), in the Latin American and Caribbean Literature in Health Sciences (LILACS), and in the Scientific Electronic Electronic Library Online (SciELO), for articles published in the last ten years. Randomized clinical trials, with a cross-sectional design, which compared the MN frequency in the oral mucosa of adult smokers and non-smokers, were selected. Results: A total of 52 articles were identified, four of them were removed due to duplicity. From the remaining 48 studies, after reading their titles and abstracts, 14 remained, which had their full texts read. Finally, eight articles remained for the qualitative analysis. Discussion and conclusion: Individuals who use tobacco present genotoxic and cytotoxic damages that interfere in the mitosis process, which leads to MN formation. The hypothesis of smoking as the cause of this genetic alteration is corroborated by the authors when comparing the data obtained in their studies between risk groups and control groups.

RESUMEN Introducción: El cáncer es el resultado de la acumulación de diversas modificaciones en el material genético, por lo tanto, es posible realizar su detección citogenética usando biomarcadores. Micronúcleos (MN) se han abordado por la literatura como biomarcadores de daño genotóxico. Estos marcadores ofrecen información sobre cambios biológicos o bioquímicos en un tejido diana todavía en forma precoz, permitiendo un pronóstico bueno. Objetivo: Determinar si la frecuencia de MN en fumadores activos es superior a la de los no fumadores. Material y método: Hicimos una búsqueda en Medical Literature Analysis and Retrieval System (PubMeb), Literatura Latino-Americana e do Caribe em Ciências da Saúde (LILACS) y Scientific Eletronic Library On-line (SciELO) de artículos publicados en los últimos diez años. Hemos seleccionado ensayos clínicos aleatorizados, de diseño transversal, que compararon la frecuencia de MN en la mucosa bucal de personas adultas fumadoras y no fumadoras. Resultados: Se identificaron 52 artículos; cuatro de ellos fueron eliminados debido a duplicidad. Entre los 48 estudios restantes, tras la lectura de sus títulos y resúmenes, quedaron 14, que tuvieron sus textos completos leídos. Por último, ocho artículos permanecieron para el análisis cualitativo. Discusión y conclusión: Personas que utilizan tabaco presentan daños genotóxicos y citotóxicos que interfieren con el proceso de mitosis, lo que acarrea la formación de MN. La hipótesis del tabaquismo como causa de ese cambio genético es corroborada por los autores al cotejar los datos obtenidos en sus estudios entre grupos de riesgo y grupos de control.

RESUMO Introdução: O câncer é o resultado do acúmulo de diversas modificações no material genético, portanto, é possível realizar sua detecção citogenética utilizando biomarcadores. Micronúcleos (MN) têm sido abordados pela literatura como biomarcadores de danos genotóxicos. Esses marcadores fornecem informações sobre alterações biológicas ou bioquímicas em um tecido-alvo ainda de forma precoce, possibilitando um prognóstico favorável. Objetivo: Comparar se a frequência de MN em indivíduos fumantes ativos é superior à dos não fumantes. Material e método: Realizou-se a busca no Medical Literature Analysis and Retrieval System (PubMeb), no Literatura Latino-Americana e do Caribe em Ciências da Saúde (LILACS) e no Scientific Eletronic Library On-line (SciELO) de artigos publicados nos últimos dez anos. Foram selecionados ensaios clínicos randomizados, com delineamento transversal, que compararam a frequência de MN na mucosa bucal de indivíduos adultos fumantes e não fumantes. Resultados: Cinquenta e dois artigos foram identificados; quatro deles foram removidos devido à duplicidade. Dos 48 estudos restantes, após a leitura dos seus títulos e resumos, restaram 14, os quais tiveram seus textos completos lidos. Por fim, oito artigos permaneceram para a análise qualitativa. Discussão e conclusão: Indivíduos que utilizam tabaco apresentam danos genotóxicos e citotóxicos que interferem no processo de mitose, o que acarreta a formação de MN. A hipótese do tabagismo como causa dessa alteração genética é corroborada pelos autores ao confrontar os dados obtidos em seus estudos entre grupos de risco e grupos-controle.

Total Synthesis of (+)-epi-Condyfoline.

Herein, we report the first asymmetric total synthesis of aspidospermatan indole alkaloid (+)-epi-condyfoline (1) in 15 steps from commercially available 2-methylindole-3-carboxaldehyde. Key steps include (1) our domino Michael/Mannich annulation method of N-sulfinyl metallodienamines to set three contiguous stereocenters, (2) LiHMDS-mediated cyclization of an ω-tosyloxy N-sulfinamide to prepare the signature indole-fused 2-azabicyclo[3.3.1]nonane framework, and (3) DMTSF-promoted spirocyclization of a dithioacetal intermediate to access the final pyrrolidine ring. Functional group manipulations delivered the targeted alkaloid (+)-epi-condyfoline (1) in 13 steps and 1.25% overall yield from N-sulfinylimine (+)-8.

Synthesis and biological evaluation of solithromycin analogs against multidrug resistant pathogens.

Novel antibacterial drugs that treat multidrug resistant pathogens are in high demand. We have synthesized analogs of solithromycin using Cu(I)-mediated click chemistry. Evaluation of the analogs using Minimum Inhibitory Concentration (MIC) assays against resistant Staphylococcus aureus, Escherichia coli, and multidrug resistant pathogens Enterococcus faecium and Acinetobacter baumannii showed they possess potencies similar to those of solithromycin, thus demonstrating their potential as future therapeutics to combat the existential threat of multidrug resistant pathogens.

Ribosome-Templated Azide-Alkyne Cycloadditions Using Resistant Bacteria as Reaction Vessels: in Cellulo Click Chemistry.

In situ click chemistry has been a powerful method for fragment-based drug design since its discovery in 2002. Recently, we demonstrated that the bacterial ribosome can template the azide-alkyne cycloaddition reaction to expedite the discovery of novel antibiotics. We now report this process can be performed in an antibiotic-resistant bacterial cell. The corresponding triazole products formed in cellulo are potent antibiotics that inhibit bacterial growth; moreover, the potency of each cycloadduct can be visualized using the traditional MIC assay in a 96-well plate format. We characterized the in cellulo clicked products by independent chemical synthesis and LC-MS analysis, which showed that mass count percent increase was directly proportional to 1/MIC. In other words, potent compounds detected by MIC were formed in greater amounts. Control experiments unambiguously showed the ribosome was responsible for templating triazole formation. Significantly, our method (1) obviates the need to isolate bacterial ribosomes; (2) could be applied to different bacterial strains, which broadens the scope and facilitates the discovery of narrow-spectrum antibiotics; and (3) does not require the knowledge of mode-of-action and thus could uncover novel antibiotic targets. We believe this method could be expanded and implemented as a novel approach for antibiotic drug discovery.

Two Tabersonine 6,7-Epoxidases Initiate Lochnericine-Derived Alkaloid Biosynthesis in Catharanthus roseus.

Lochnericine is a major monoterpene indole alkaloid (MIA) in the roots of Madagascar periwinkle (Catharanthus roseus). Lochnericine is derived from the stereoselective C6,C7-epoxidation of tabersonine and can be metabolized further to generate other complex MIAs. While the enzymes responsible for its downstream modifications have been characterized, those involved in lochnericine biosynthesis remain unknown. By combining gene correlation studies, functional assays, and transient gene inactivation, we identified two highly conserved P450s that efficiently catalyze the epoxidation of tabersonine: tabersonine 6,7-epoxidase isoforms 1 and 2 (TEX1 and TEX2). Both proteins are quite divergent from the previously characterized tabersonine 2,3-epoxidase and are more closely related to tabersonine 16-hydroxylase, involved in vindoline biosynthesis in leaves. Biochemical characterization of TEX1/2 revealed their strict substrate specificity for tabersonine and their inability to epoxidize 19-hydroxytabersonine, indicating that they catalyze the first step in the pathway leading to hörhammericine production. TEX1 and TEX2 displayed complementary expression profiles, with TEX1 expressed mainly in roots and TEX2 in aerial organs. Our results suggest that TEX1 and TEX2 originated from a gene duplication event and later acquired divergent, organ-specific regulatory elements for lochnericine biosynthesis throughout the plant, as supported by the presence of lochnericine in flowers. Finally, through the sequential expression of TEX1 and up to four other MIA biosynthetic genes in yeast, we reconstituted the 19-acetylhörhammericine biosynthetic pathway and produced tailor-made MIAs by mixing enzymatic modules that are naturally spatially separated in the plant. These results lay the groundwork for the metabolic engineering of tabersonine/lochnericine derivatives of pharmaceutical interest.

In vivo Antimalarial and Antitrypanosomal Activity of Strychnogucine B, a Bisindole Alkaloid from Strychnos icaja.

Strychnogucine B is a bisindole alkaloid previously isolated from Strychnos icaja that possesses promising in vitro antiplasmodial properties. This compound was synthesized in four steps from (-)-strychnine. As no acute toxicity was observed at the highest tested cumulative dose of 60 mg/kg, its in vivo antimalarial activity was determined intraperitoneally at 30 mg/kg/d in a Plasmodium berghei murine model. In the Peters's 4-d suppressive test, this alkaloid suppressed the parasitaemia by almost 36% on day 5 and 60% on day 7 compared to vehicle-treated mice. In addition to this interesting antimalarial activity, it showed moderate in vitro antitrypanosomal activity but no in vivo activity in an acute Trypanosoma brucei model. It was also inactive in vitro on Leishmania mexicana promastigotes. This highlights its selective antimalarial efficacy and leads to further investigation to assess its potential as new antimalarial lead compound.

Elucidating the inhibition of peptidoglycan biosynthesis in Staphylococcus aureus by albocycline, a macrolactone isolated from Streptomyces maizeus.

Antibiotic resistance is a serious threat to global public health, and methicillin-resistant Staphylococcus aureus (MRSA) is a poignant example. The macrolactone natural product albocycline, derived from various Streptomyces strains, was recently identified as a promising antibiotic candidate for the treatment of both MRSA and vancomycin-resistant S. aureus (VRSA), which is another clinically relevant and antibiotic resistant strain. Moreover, it was hypothesized that albocycline's antimicrobial activity was derived from the inhibition of peptidoglycan (i.e., bacterial cell wall) biosynthesis. Herein, preliminary mechanistic studies are performed to test the hypothesis that albocycline inhibits MurA, the enzyme that catalyzes the first step of peptidoglycan biosynthesis, using a combination of biological assays alongside molecular modeling and simulation studies. Computational modeling suggests albocycline exists as two conformations in solution, and computational docking of these conformations to an ensemble of simulated receptor structures correctly predicted preferential binding to S. aureus MurA-the enzyme that catalyzes the first step of peptidoglycan biosynthesis-over Escherichia coli (E. coli) MurA. Albocycline isolated from the producing organism (Streptomyces maizeus) weakly inhibited S. aureus MurA (IC50 of 480 µM) but did not inhibit E. coli MurA. The antimicrobial activity of albocycline against resistant S. aureus strains was superior to that of vancomycin, preferentially inhibiting Gram-positive organisms. Albocycline was not toxic to human HepG2 cells in MTT assays. While these studies demonstrate that albocycline is a promising lead candidate against resistant S. aureus, taken together they suggest that MurA is not the primary target, and further work is necessary to identify the major biological target.

A BAHD acyltransferase catalyzing 19-O-acetylation of tabersonine derivatives in roots of Catharanthus roseus enables combinatorial synthesis of monoterpene indole alkaloids.

While the characterization of the biosynthetic pathway of monoterpene indole alkaloids (MIAs) in leaves of Catharanthus roseus is now reaching completion, only two enzymes from the root counterpart dedicated to tabersonine metabolism have been identified to date, namely tabersonine 19-hydroxylase (T19H) and minovincine 19-O-acetyltransferase (MAT). Albeit the recombinant MAT catalyzes MIA acetylation at low efficiency in vitro, we demonstrated that MAT was inactive when expressed in yeast and in planta, suggesting an alternative function for this enzyme. Therefore, through transcriptomic analysis of periwinkle adventitious roots, several other BAHD acyltransferase candidates were identified based on the correlation of their expression profile with T19H and found to localize in small genomic clusters. Only one, named tabersonine derivative 19-O-acetyltransferase (TAT) was able to acetylate the 19-hydroxytabersonine derivatives from roots, such as minovincinine and hörhammericine, following expression in yeast. Kinetic studies also showed that the recombinant TAT was specific for root MIAs and displayed an up to 200-fold higher catalytic efficiency than MAT. In addition, gene expression analysis, protein subcellular localization and heterologous expression in Nicotiana benthamiana were in agreement with the prominent role of TAT in acetylation of root-specific MIAs, thereby redefining the molecular determinants of the root MIA biosynthetic pathway. Finally, identification of TAT provided a convenient tool for metabolic engineering of MIAs in yeast enabling efficiently mixing different biosynthetic modules spatially separated in the whole plant. This combinatorial synthesis associating several enzymes from Catharanthus roseus resulted in the conversion of tabersonine in tailor-made MIAs bearing both leaf and root-type decorations.

Canvass: A Crowd-Sourced, Natural-Product Screening Library for Exploring Biological Space.

Natural products and their derivatives continue to be wellsprings of nascent therapeutic potential. However, many laboratories have limited resources for biological evaluation, leaving their previously isolated or synthesized compounds largely or completely untested. To address this issue, the Canvass library of natural products was assembled, in collaboration with academic and industry researchers, for quantitative high-throughput screening (qHTS) across a diverse set of cell-based and biochemical assays. Characterization of the library in terms of physicochemical properties, structural diversity, and similarity to compounds in publicly available libraries indicates that the Canvass library contains many structural elements in common with approved drugs. The assay data generated were analyzed using a variety of quality control metrics, and the resultant assay profiles were explored using statistical methods, such as clustering and compound promiscuity analyses. Individual compounds were then sorted by structural class and activity profiles. Differential behavior based on these classifications, as well as noteworthy activities, are outlined herein. One such highlight is the activity of (-)-2(S)-cathafoline, which was found to stabilize calcium levels in the endoplasmic reticulum. The workflow described here illustrates a pilot effort to broadly survey the biological potential of natural products by utilizing the power of automation and high-throughput screening.