Rapid Inhibitor Discovery by Exploiting Synthetic Lethality.

Synthetic lethality occurs when inactivation of two genes is lethal but inactivation of either single gene is not. This phenomenon provides an opportunity for efficient compound discovery. Using differential growth screens, one can identify biologically active compounds that selectively inhibit proteins within the synthetic lethal network of any inactivated gene. Here, based purely on synthetic lethalities, we identified two compounds as the only possible inhibitors of Staphylococcus aureus lipoteichoic acid (LTA) biosynthesis from a screen of ∼230,000 compounds. Both compounds proved to inhibit the glycosyltransferase UgtP, which assembles the LTA glycolipid anchor. UgtP is required for ß-lactam resistance in methicillin-resistant S. aureus (MRSA), and the inhibitors restored sensitivity to oxacillin in a highly resistant S. aureus strain. As no other compounds were pursued as possible LTA glycolipid assembly inhibitors, this work demonstrates the extraordinary efficiency of screens that exploit synthetic lethality to discover compounds that target specified pathways. The general approach should be applicable not only to other bacteria but also to eukaryotic cells.

Saccharomyces cerevisiae Requires CFF1 To Produce 4-Hydroxy-5-Methylfuran-3(2H)-One, a Mimic of the Bacterial Quorum-Sensing Autoinducer AI-2.

Quorum sensing is a process of cell-to-cell communication that bacteria use to orchestrate collective behaviors. Quorum sensing depends on the production, release, and detection of extracellular signal molecules called autoinducers (AIs) that accumulate with increasing cell density. While most AIs are species specific, the AI called AI-2 is produced and detected by diverse bacterial species, and it mediates interspecies communication. We recently reported that mammalian cells produce an AI-2 mimic that can be detected by bacteria through the AI-2 receptor LuxP, potentially expanding the role of the AI-2 system to interdomain communication. Here, we describe a second molecule capable of interdomain signaling through LuxP, 4-hydroxy-5-methylfuran-3(2H)-one (MHF), that is produced by the yeast Saccharomyces cerevisiae Screening the S. cerevisiae deletion collection revealed Cff1p, a protein with no known role, to be required for MHF production. Cff1p is proposed to be an enzyme, with structural similarity to sugar isomerases and epimerases, and substitution at the putative catalytic residue eliminated MHF production in S. cerevisiae Sequence analysis uncovered Cff1p homologs in many species, primarily bacterial and fungal, but also viral, archaeal, and higher eukaryotic. Cff1p homologs from organisms from all domains can complement a cff1ΔS. cerevisiae mutant and restore MHF production. In all cases tested, the identified catalytic residue is conserved and required for MHF to be produced. These findings increase the scope of possibilities for interdomain interactions via AI-2 and AI-2 mimics, highlighting the breadth of molecules and organisms that could participate in quorum sensing.IMPORTANCE Quorum sensing is a cell-to-cell communication process that bacteria use to monitor local population density. Quorum sensing relies on extracellular signal molecules called autoinducers (AIs). One AI called AI-2 is broadly made by bacteria and used for interspecies communication. Here, we describe a eukaryotic AI-2 mimic, 4-hydroxy-5-methylfuran-3(2H)-one, (MHF), that is made by the yeast Saccharomyces cerevisiae, and we identify the Cff1p protein as essential for MHF production. Hundreds of viral, archaeal, bacterial, and eukaryotic organisms possess Cff1p homologs. This finding, combined with our results showing that homologs from all domains can replace S. cerevisiae Cff1p, suggests that like AI-2, MHF is widely produced. Our results expand the breadth of organisms that may participate in quorum-sensing-mediated interactions.

A preparative chiral separation of hydroxychloroquine using supercritical fluid chromatography.

A robust supercritical chromatography (SFC) method using an Enantiocel C2-5 column was developed for the multigram separation of the enantiomers of hydroxychloroquine (HCQ), affirming its use as a scalable technology and ability to provide quantities of each enantiomer for clinical evaluation. The enantiomers of HCQ were collected on a gram scale with greater than 99% enantiomeric excess. The S and R enantiomer elution order was confirmed using optical rotation determinations with comparison to previously determined assignments.

Synthesis and Structures of Polyphenylphenanthrenes.

1,2,3,4,5,6,7,8-Octaphenylphenanthrene (4) and decaphenylphenanthrene (5) were prepared by very short syntheses (two or three steps) from tetraphenylfuran and polybrominated benzene derivatives. The X-ray structures of compounds 4 and 5 show them to be quite crowded, with the phenanthrene cores twisted by about 40° due to the clash of the C4 and C5 phenyl groups. Compound 4 was resolved by chromatography on a chiral support, and its free energy of activation for racemization was determined to be 24.6 kcal mol-1 at 40 °C. Computational studies indicate that compound 5 has a racemization barrier approximately 6 kcal mol-1 lower than 4, and thus 5 would not be configurationally stable at room temperature.

A congested in,in-diphosphine.

The synthesis and characterization of the bis(triarylphosphine) 3 are described. Slow protonation of an inwardly directed phosphine is possible, but the phosphines do not react with larger reagents. X-ray structures of the parent compound, its HCl salt, and the corresponding trisulfone are reported. Compound 3 was resolved by chiral chromatography, but the barrier to racemization is only 20.7 kcal/mol.

Concise, aromatization-based approach to an elaborate C2-symmetric pyrenophane.

A very short synthesis (5 steps), the crystal structure and resolution of an elaborate, inherently chiral [n](1,6)pyrenophane is reported. The synthesis hinges upon two very productive events: a multicomponent reaction and an unprecedented double-McMurry/valence isomerization/dehydrogenation step. Aromatization reactions are involved in the formation of all four of the rings of the pyrene system.

Synthesis, crystal structure, and resolution of [10](1,6)pyrenophane: an inherently chiral [N]cyclophane.

A synthetic approach to a set of three inherently chiral [n]cyclophanes, [n](1,6)pyrenophanes (29a-c, n = 8-10) was investigated. Progress toward 29a was thwarted by the failure of the key dithiacyclophane-forming reaction. For the next higher homologue, the synthesis was completed, but the desired [9](1,6)pyrenophane (29b) could only be partially separated from an isomeric pyrenophane, [9](1,8)pyrenophane (28b), and an unidentified byproduct. Work aimed at the synthesis of the next higher homologue resulted in the isolation of a 7:4 mixture of [10](1,8)pyrenophane (28c) and [10](1,6)pyrenophane (29c), which could not be separated by column chromatography or crystallization. However, single-crystal X-ray structures of 28c and 29c were obtained after manual separation of two crystals with different morphologies from the same batch of crystals obtained from the 7:4 mixture of 28c and 29c. The pyrene system of 29c was found to have a gentle end-to-end bend as well as a significant longitudinal twist. Short intermolecular C(sp(3))-H···π contacts (2.64 to 2.76 Å) between H-atoms on the bridge and the centroids of three of the four six-membered rings of the pyrene system of a neighboring pyrenophane of like chirality give rise to the formation of single enantiomer columns. From a DNMR study of the mixture of 28c and 29c, the bridge in [10](1,8)pyrenophane (28c) was found to undergo a conformational flip from one side of the pyrene system to the other with ΔG(‡) = 14.9 ± 0.2 kcal/mol. A two-stage preparative HPLC protocol was subsequently developed for the separation of 28c and 29c (Chiralpak AD-H column) and then the enantiomers of 29c (Chiralcel OJ-H column). This enabled the measurement of their optical rotations and CD spectra.

Signal production and detection specificity in Vibrio CqsA/CqsS quorum-sensing systems.

Quorum sensing is a process of bacterial cell-cell communication that enables populations of cells to carry out behaviours in unison. Quorum sensing involves detection of the density-dependent accumulation of extracellular signal molecules called autoinducers that elicit population-wide changes in gene expression. In Vibrio species, CqsS is a membrane-bound histidine kinase that acts as the receptor for the CAI-1 autoinducer which is produced by the CqsA synthase. In Vibrio cholerae, CAI-1 is (S)-3-hydroxytridecan-4-one. The C170 residue of V. cholerae CqsS specifies a preference for a ligand with a 10-carbon tail length. However, a phenylalanine is present at this position in Vibrio harveyi CqsS and other homologues, suggesting that a shorter CAI-1-like molecule functions as the signal. To investigate this, we purified the V. harveyi CqsS ligand, and determined that it is (Z)-3-aminoundec-2-en-4-one (Ea-C8-CAI-1) carrying an 8-carbon tail. The V. harveyi CqsA/CqsS system is exquisitely selective for production and detection of this ligand, while the V. cholerae CqsA/CqsS counterparts show relaxed specificity in both production and detection. We isolated CqsS mutants in each species that display reversed specificity for ligands. Our analysis provides insight into how fidelity is maintained in signal transduction systems.

High performance liquid chromatographic evaluation of artemisinin, raw material in the synthesis of artesunate and artemether.

A barrier to the development of artemisinin derivative based combination treatment of malaria is the lack of defined specifications and purity test methods for the raw material artemisinin. An HPLC method previously published in the International Pharmacopoeia to evaluate purity of artemisinin as an active pharmaceutical ingredient is adapted for use. Excellent method precision and linearity are demonstrated along with observations of robustness. In support of the development of specifications major impurities are identified using high resolution HPLC-MS, isolation via preparative HPLC followed by NMR. The identified impurities differ from those previously claimed.

Configurationally stable longitudinally twisted polycyclic aromatic compounds.

Two strategies for the synthesis of configurationally stable twisted polycyclic aromatic compounds (PACs) were pursued. The first approach employed dissymmetrically positioned 1-naphthyl substituents to bias the direction of twist in highly substituted PACs. 2,3-Bis(1-naphthyl)-1,4-diphenyltriphenylene (7) was prepared, and its meso cis-dinaphthyl and enantiomeric trans-dinaphthyl isomers were resolved by preparative supercritical fluid chromatography (SFC) on chiral supports. Similarly, several naphthyl-substituted derivatives of the more highly twisted 9,10,11,12,13,14-hexaphenylbenzo[b]triphenylene (2) were prepared. Of these, 10-(1-naphthyl)-9,11,12,14-tetraphenylbenzo[b]triphenylene (13) was resolved by SFC on a chiral support. The pure enantiomers of trans-7 showed moderately large specific rotations ([alpha]D(25) = -330 and +320 degrees), but the specific rotations for the enantiomers of 13 were unexpectedly small ([alpha]D(25) = -23 and +23 degrees). Computational studies suggest that the latter result is due to presence of a minor conformation of 13 possessing a larger rotation of opposite sign than the major conformation. Both 7 and 13 showed strong circular dichroism and moderately strong circularly polarized luminescence. A byproduct of these syntheses was 9,10,19,21-tetraphenyldiphenanthro[9,10-b:9,10-h]carbazole (15), a very crowded carbazole that exhibits an 81 degree end-to-end twist but is not resolvable. In the second approach, the large, twisted, polycyclic aromatic ligand 9,10,11,12,13,14-hexaphenylbenzo[h]naphtho[2,3-f]quinoline (21, an aza-2) was used to prepare the chiral, cyclometallated iridium(III) complex 4. The ligand 21 was prepared via an unusually stable benzannulated norbornadienone, for which the free energy of activation for decarbonylation was a remarkable 33.5 kcal/mol. The iridium complex 4 proved to be configurationally stable and resolvable by analytical HPLC on chiral supports, but the low solubility of 4 prevented its resolution on a preparative scale. A much more soluble dibutyl analogue of 4 (complex 28) was then prepared, but it was not resolvable on any of the available media.

Thermal C1-C5 diradical cyclization of enediynes.

Computational studies at the BLYP/6-31G(d) level (supplemented by BCCD(T)/cc-pVDZ calculations) suggest that in aryl-substituted 1,2-diethynylbenzenes, steric effects disfavor the thermal C1-C6 diradical cyclization reaction (Bergman) and electronic effects favor the regiovariant C1-C5 cyclization to the extent that the C1-C5 process should become an important reaction pathway in the thermolyses of such compounds. Experimentally, thermolyses of 1,2-bis(2,4,6-trichlorophenylethynyl)benzene, a particularly favorable case, yields only products derived from C1-C5 cyclization [specifically, 1-(2,4,6-trichlorobenzylidene)-2-(2,4,6-trichlorophenyl)-1H-indene and its hydrogenation product 3-(2,4,6-trichlorobenzyl)-2-(2,4,6-trichlorophenyl)-1H-indene], and even for the parent hydrocarbon 1,2-bis(phenylethynyl)benzene, the formation of C1-C5 cyclization products is competitive with the major Bergman reaction. Although some C1-C5 cyclization products are probably formed by transfer hydrogenation from 1,4-cyclohexadiene (commonly included in such reactions), thermolyses in the absence of 1,4-CHD as well as deuterium labeling studies confirm the existence of direct C1-C5 diradical cyclizations for diaryl-substituted enediynes.

Analysis and purification of alcohol-sensitive chiral compounds using 2,2,2-trifluoroethanol as a modifier in supercritical fluid chromatography.

2,2,2-Trifluoroethanol (TFE) is evaluated as an alternative modifier for the analysis and purification of alcohol-sensitive chiral compounds using supercritical fluid chromatography (SFC). Four chiral compounds, selected for their sensitivity to alcohols, in addition to a variety of standard chiral compounds were analyzed by SFC using TFE with polysaccharide and Pirkle-type chiral stationary phases (CSPs) to produce selectivities (alpha) and resolutions (Rs) as high as 1.4 and 7.2. A preparative isolation of 2-phenylglutaric anhydride was achieved using TFE as the mobile phase modifier to produce clean enantiomers.

Synthesis, separation, and circularly polarized luminescence studies of enantiomers of iridium(III) luminophores.

A family of heteroleptic (C;N)2Ir(acac) and homoleptic fac-Ir(C;N)3 complexes have been synthesized and their photophysical properties studied (where C;N = a substituted 2-phenylpyridine and acac = acetylacetonate). The neutral Delta and Lambda complexes were separated with greater than 95% enantiomeric purity by chiral supercritical fluid chromatography, and the solution circular dichroism and circularly polarized luminescence spectra for each of the enantio-enriched iridium complexes were obtained. The experimentally measured emission dissymmetries (gem) for this series compared well with predicted values provided by time-dependent density functional theory calculations. The discovered trend further showed a correlation with the dissymmetries of ionic, enantiopure hemicage compounds of Ru(II) and Zn(II), thus demonstrating the applicability of the model for predicting emission dissymmetry values across a wide range of complexes.

The major Vibrio cholerae autoinducer and its role in virulence factor production.

Vibrio cholerae, the causative agent of the human disease cholera, uses cell-to-cell communication to control pathogenicity and biofilm formation. This process, known as quorum sensing, relies on the secretion and detection of signalling molecules called autoinducers. At low cell density V. cholerae activates the expression of virulence factors and forms biofilms. At high cell density the accumulation of two quorum-sensing autoinducers represses these traits. These two autoinducers, cholerae autoinducer-1 (CAI-1) and autoinducer-2 (AI-2), function synergistically to control gene regulation, although CAI-1 is the stronger of the two signals. V. cholerae AI-2 is the furanosyl borate diester (2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran borate. Here we describe the purification of CAI-1 and identify the molecule as (S)-3-hydroxytridecan-4-one, a new type of bacterial autoinducer. We provide a synthetic route to both the R and S isomers of CAI-1 as well as simple homologues, and we evaluate their relative activities. Synthetic (S)-3-hydroxytridecan-4-one functions as effectively as natural CAI-1 in repressing production of the canonical virulence factor TCP (toxin co-regulated pilus). These findings suggest that CAI-1 could be used as a therapy to prevent cholera infection and, furthermore, that strategies to manipulate bacterial quorum sensing hold promise in the clinical arena.

Estrogen receptor dependent inhibitors of NF-kappaB transcriptional activation-1 synthesis and biological evaluation of substituted 2-cyanopropanoic acid derivatives: pathway selective inhibitors of NF-kappaB, a potential treatment for rheumatoid arthritis.

Pathway selective ligands of the estrogen receptor inhibit transcriptional activation of proinflammatory genes mediated by NF-kappaB. Substituted 2-cyanopropanoic acid derivatives were developed leading to the discovery of WAY-204688, an orally active, pathway selective, estrogen receptor dependent anti-inflammatory agent. This propanamide was shown to be orally active in preclinical models of inflammatory diseases, such as rheumatoid arthritis, without the proliferative effect associated with traditional estrogens.

Enantiomeric separation and determination of absolute stereochemistry of asymmetric molecules in drug discovery: building chiral technology toolboxes.

The application of Chiral Technology, or the (extensive) use of techniques or tools for the determination of absolute stereochemistry and the enantiomeric or chiral separation of racemic small molecule potential lead compounds, has been critical to successfully discovering and developing chiral drugs in the pharmaceutical industry. This has been due to the rapid increase over the past 10-15 years in potential drug candidates containing one or more asymmetric centers. Based on the experiences of one pharmaceutical company, a summary of the establishment of a Chiral Technology toolbox, including the implementation of known tools as well as the design, development, and implementation of new Chiral Technology tools, is provided.

Synthesis, structure, and resolution of exceptionally twisted pentacenes.

9,10,11,20,21,22-hexaphenyltetrabenzo[a,c,l,n]pentacene (2) and a dimethyl derivative (2m) were prepared by the reaction of 1,3-diphenylphenanthro[9,10-c]furan with bisaryne equivalents generated from 1,2,4,5-tetrabromo-3,6-diarylbenzenes in the presence of n-butyllithium, followed by deoxygenation of the double adducts with low-valent titanium. Both are bright red solids with a strong orange fluorescence in solution. The X-ray structures of these compounds show them to be the most highly twisted polycyclic aromatic hydrocarbons known. Compound 2 has an end-to-end twist of 144 degrees , and the two crystallographically independent molecules of 2m have twists of 138 degrees and 143 degrees. Both molecules were resolved by chromatography on chiral supports, and the pure enantiomers have extremely high specific rotations (for 2, [alpha]D = 7400 degrees; for 2m, 5600 degrees), but the molecules racemize slowly at room temperature (DeltaG++rac = 24 kcal/mol). Both the experimental geometry and the observed racemization barrier for 2 are in good agreement with computational studies of the molecule at a variety of levels. Attempts to prepare compound 2 by reaction of tetraphenylbenzyne with 9,10,12,13-tetraphenyl-11-oxacyclopenta[b]triphenylene (3, a twisted isobenzofuran) gave no adducts, and attempts to prepare tetradecaphenylpentacene by reaction of hexaphenylisobenzofuran (11) with bisaryne equivalents gave only monoadducts.

Synthesis of plakortone B and analogs.

[Structure: see text] Use of a palladium-mediated alkoxycarbonylation/lactonization process provides a variable route to analogs of the plakortones. Four different analogs, including natural plakortone B, have been synthesized via this route.

Synthesis and biological evaluation of novel compounds within a class of 3-aminochroman derivatives with dual 5-HT1A receptor and serotonin transporter affinity.

Compounds containing a 5-carbamoyl-8-fluoro-3-amino-3,4-dihydro-2H-1-benzopyran and a 3-alkylindole moiety linked through a common basic nitrogen were prepared and evaluated for 5-HT1A affinity, serotonin rat transporter affinity, and functional antagonist activity in vitro. 26a was found to be the most potent and selective compound in this series and was shown to possess neurochemical activity in vivo by producing acute and rapid increases in 5-HT in the rat frontal cortex.