Anticancer Activities of Novel Nicotinamide Phosphoribosyltransferase Inhibitors in Hematological Malignancies.

Targeting cancer cells that are highly dependent on the nicotinamide adenine dinucleotide (NAD+) metabolite is a promising therapeutic strategy. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme catalyzing NAD+ production. Despite the high efficacy of several developed NAMPT inhibitors (i.e., FK866 (APO866)) in preclinical studies, their clinical activity was proven to be limited. Here, we report the synthesis of new NAMPT Inhibitors, JJ08, FEI191 and FEI199, which exhibit a broad anticancer activity in vitro. Results show that these compounds are potent NAMPT inhibitors that deplete NAD+ and NADP(H) after 24 h of drug treatment, followed by an increase in reactive oxygen species (ROS) accumulation. The latter event leads to ATP loss and mitochondrial depolarization with induction of apoptosis and necrosis. Supplementation with exogenous NAD+ precursors or catalase (ROS scavenger) abrogates the cell death induced by the new compounds. Finally, in vivo administration of the new NAMPT inhibitors in a mouse xenograft model of human Burkitt lymphoma delays tumor growth and significantly prolongs mouse survival. The most promising results are collected with JJ08, which completely eradicates tumor growth. Collectively, our findings demonstrate the efficient anticancer activity of the new NAMPT inhibitor JJ08 and highlight a strong interest for further evaluation of this compound in hematological malignancies.

Structure-based optimization of type III indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors.

The haem enzyme indoleamine 2,3-dioxygenase 1 (IDO1) catalyses the rate-limiting step in the kynurenine pathway of tryptophan metabolism and plays an essential role in immunity, neuronal function, and ageing. Expression of IDO1 in cancer cells results in the suppression of an immune response, and therefore IDO1 inhibitors have been developed for use in anti-cancer immunotherapy. Here, we report an extension of our previously described highly efficient haem-binding 1,2,3-triazole and 1,2,4-triazole inhibitor series, the best compound having both enzymatic and cellular IC50 values of 34 nM. We provide enzymatic inhibition data for almost 100 new compounds and X-ray diffraction data for one compound in complex with IDO1. Structural and computational studies explain the dramatic drop in activity upon extension to pocket B, which has been observed in diverse haem-binding inhibitor scaffolds. Our data provides important insights for future IDO1 inhibitor design.

Synthesis and Applications of Silyl 2-Methylprop-2-ene-1-sulfinates in Preparative Silylation and GC-Derivatization Reactions of Polyols and Carbohydrates.

Trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, and triisopropylsilyl 2-methylprop-2-ene-1-sulfinates were prepared through (CuOTf)2⋅C6H6-catalyzed sila-ene reactions of the corresponding methallylsilanes with SO2 at 50 °C. Sterically hindered, epimerizable, and base-sensitive alcohols gave the corresponding silyl ethers in high yields and purities at room temperature and under neutral conditions. As the byproducts of the silylation reaction (SO2 +isobutylene) are volatile, the workup was simplified to solvent evaporation. The developed method can be employed for the chemo- and regioselective semiprotection of polyols and glycosides and for the silylation of unstable aldols. The high reactivity of the developed reagents is shown by the synthesis of sterically hindered per-O-tert-butyldimethylsilyl-α-D-glucopyranose, the X-ray crystallographic analysis of which is the first for a per-O-silylated hexopyranose. The per-O-silylation of polyols, hydroxy carboxylic acids, and carbohydrates with trimethylsilyl 2-methylprop-2-ene-1-sulfinate was coupled with the GC analysis of nonvolatile polyhydroxy compounds both qualitatively and quantitatively.

1,2,3-Triazoles as inhibitors of indoleamine 2,3-dioxygenase 2 (IDO2).

Indoleamine 2,3-dioxygenase 2 (IDO2) is a potential therapeutic target for the treatment of diseases that involve immune escape such as cancer. In contrast to IDO1, only a very limited number of inhibitors have been described for IDO2 due to inherent difficulties in expressing and purifying a functionally active, soluble form of the enzyme. Starting from our previously discovered highly efficient 4-aryl-1,2,3-triazole IDO1 inhibitor scaffold, we used computational structure-based methods to design inhibitors of IDO2 which we then tested in cellular assays. Our approach yielded low molecular weight inhibitors of IDO2, the most active displaying an IC50 value of 51µM for mIDO2, and twofold selectivity over hIDO1. These compounds could be useful as molecular probes to investigate the biological role of IDO2, and could inspire the design of new IDO2 inhibitors.

Identification of new FK866 analogues with potent anticancer activity against pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal diseases for which chemotherapy has not been very successful yet. FK866 ((E)-N-(4-(1-benzoylpiperidin-4-yl)butyl)-3-(pyridin-3-yl)acrylamide) is a well-known NAMPT (nicotinamide phosphoribosyltransferase) inhibitor with anti-cancer activities, but it failed in phase II clinical trials. We found that FK866 shows anti-proliferative activity in three PDAC cell lines, as well as in Jurkat T-cell leukemia cells. More than 50 FK866 analogues were synthesized that introduce substituents on the phenyl ring of the piperidine benzamide group of FK866 and exchange its buta-1,4-diyl tether for 1-oxyprop-3-yl, (E)-but-2-en-1,4-diyl and 2- and 3-carbon tethers. The pyridin-3-yl moiety of FK866 was exchanged for chlorinated and fluorinated analogues and for pyrazin-2-yl and pyridazin-4-yl groups. Several compounds showed low nanomolar or sub-nanomolar cell growth inhibitory activity. Our best cell anti-proliferative compounds were the 2,4,6-trimethoxybenzamide analogue of FK866 ((E)-N-(4-(1-(2,4,6-trimethoxybenzoyl)piperidin-4-yl)butyl)-3-(pyridin-3-yl)acrylamide) (9), the 2,6-dimethoxybenzamide (8) and 2-methoxybenzamide (4), which exhibited an IC50 of 0.16 nM, 0.004 nM and 0.08 nM toward PDAC cells, respectively.

Structural insights into the specificity of Xyn10B from Paenibacillus barcinonensis and its improved stability by forced protein evolution.

Paenibacillus barcinonensis is a soil bacterium bearing a complex set of enzymes for xylan degradation, including several secreted enzymes and Xyn10B, one of the few intracellular xylanases reported to date. The crystal structure of Xyn10B has been determined by x-ray analysis. The enzyme folds into the typical (beta/alpha)(8) barrel of family 10 glycosyl hydrolases (GH10), with additional secondary structure elements within the beta/alpha motifs. One of these loops -L7- located at the beta7 C terminus, was essential for xylanase activity as its partial deletion yielded an inactive enzyme. The loop contains residues His(249)-Glu(250), which shape a pocket opened to solvent in close proximity to the +2 subsite, which has not been described in other GH10 enzymes. This wide cavity at the +2 subsite, where methyl-2,4-pentanediol from the crystallization medium was found, is a noteworthy feature of Xyn10B, as compared with the narrow crevice described for other GH10 xylanases. Docking analysis showed that this open cavity can accommodate glucuronic acid decorations of xylo-oligosaccharides. Co-crystallization experiments with conduramine derivative inhibitors supported the importance of this open cavity at the +2 subsite for Xyn10B activity. Several mutant derivatives of Xyn10B with improved thermal stability were obtained by forced evolution. Among them, mutant xylanases S15L and M93V showed increased half-life, whereas the double mutant S15L/M93V exhibited a further increase in stability, showing a 20-fold higher heat resistance than the wild type xylanase. All the mutations obtained were located on the surface of Xyn10B. Replacement of a Ser by a Leu residue in mutant xylanase S15L can increase hydrophobic packing efficiency and fill a superficial indentation of the protein, giving rise to a more compact structure of the enzyme.

Concise synthesis of complicated polypropionates through one-pot dissymmetrical two-directional chain elongation.

Herein, a major breakthrough in a sulfur dioxide-mediated oxyallylation cascade reaction is reported that allows the preparation of complex long-chain polyketide fragments with more than ten stereogenic centers through a carefully designed desymmetrization process. An allylbissilane is combined, under the appropriate reaction conditions, with two different 1,3-dioxy-1,3-dienes permitting the construction of a 13-membered polypropionate precursor in one pot. Four stereocenters are selectively created during this process. The so-obtained pseudo-C(2)- or -C(S)-symmetric products are desymmetrized through selective deprotection and can be selectively elongated in both directions using aldol chemistry.

Total asymmetric syntheses of ß-hydroxy-δ-lactones via Umpolung with sulfur dioxide.

Cyclic stereotriads and stereotetrads of the ß-hydroxy-δ-lactone type, e.g. prelactones B and E, common in polyketides and polypropionates, are prepared via SO(2)-induced oxyallylations of enoxysilanes with (1E,3Z)-1-(1-phenylethoxy)penta-1,3-dien-3-yl carboxylates. Using (Z)- or (E)-enoxysilanes both 4,5-cis- or 4,5-trans-δ-lactones are obtained. Depending on the reduction method applied to the obtained aldol intermediates 5,6-trans or 5,6-cis-derivatives are formed. The δ-lactones can be prepared in both their enantiomeric forms depending on the (1R)- or (1S)-configuration of the starting 1-(1-phenylethoxy)penta-1,3-dienes.

Anti-cancer activity of 5-O-alkyl 1,4-imino-1,4-dideoxyribitols.

New derivatives of 1,4-dideoxy-1,4-imino-D-ribitol have been prepared and evaluated for their cytotoxicity on solid and haematological malignancies. 1,4-Dideoxy-5-O-[(9Z)-octadec-9-en-1-yl]-1,4-imino-D-ribitol (13, IC(50) ∼2 µM) and its C(18)-analogues (IC(50) <10 µM) are cytotoxic toward SKBR3 (breast cancer) cells. 13 also inhibits (IC(50) ∼8 µM) growth of JURKAT cells.

Azole-Based Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitors.

The heme enzyme indoleamine 2,3-dioxygenase 1 (IDO1) plays an essential role in immunity, neuronal function, and aging through catalysis of the rate-limiting step in the kynurenine pathway of tryptophan metabolism. Many IDO1 inhibitors with different chemotypes have been developed, mainly targeted for use in anti-cancer immunotherapy. Lead optimization of direct heme iron-binding inhibitors has proven difficult due to the remarkable selectivity and sensitivity of the heme-ligand interactions. Here, we present experimental data for a set of closely related small azole compounds with more than 4 orders of magnitude differences in their inhibitory activities, ranging from millimolar to nanomolar levels. We investigate and rationalize their activities based on structural data, molecular dynamics simulations, and density functional theory calculations. Our results not only expand the presently known four confirmed chemotypes of sub-micromolar heme binding IDO1 inhibitors by two additional scaffolds but also provide a model to predict the activities of novel scaffolds.

Short diastereoselective synthesis of the C1-C13 (AB spiroacetal) and C17-C28 fragments (CD spiroacetal) of spongistatin 1 and 2 through double chain-elongation reactions.

A unique and practical synthetic sequence for rapid access to polyketides and to further the spiroacetals derived from them, which utilizes a bidirectional Hosomi-Sakurai allylation approach around key allylsilanes in the synthesis of the AB and CD ring systems of spongistatin 1 and 2, is reported. The synthesis of the AB spiroacetal 9 requires 13 steps, with a longest linear sequence of seven steps in an overall yield of 27%. The synthesis of the CD spiroacetal 13 requires 15 steps, with a longest linear sequence of 11 steps in an overall yield of 30%. Both syntheses start from but-3-enol.

BCl3-mediated ene reaction of sulfur dioxide and unfunctionalized alkenes.

The first ene reactions of SO(2) and unfunctionalized alkenes are reported. Calculations suggest that the endergonic ene reactions of SO(2) with alkenes can be used to generate beta,gamma-unsaturated sulfinyl and sulfonyl compounds. Indeed, in the presence of one equivalent of BCl(3), the unstable sulfinic acid form stable sulfinic acid.BCl(3) complexes that can be reacted in situ with NCS to generate corresponding sulfonyl chlorides, or with a base to generate corresponding sulfinates. The latter can be reacted with electrophiles to generate sulfones, or with silyl chloride to form beta,gamma-unsaturated silyl sulfinates. The sulfinic acid.BCl(3) complexes can be reacted with ethers that act as oxygen nucleophiles to produce corresponding sulfinic esters. Thus one-pot, three-component synthesis of beta,gamma-unsaturated sulfonamides, sulfinyl esters and sulfones have been developed starting from alkenes and sulfur dioxide (reagent and solvent).

Synthesis of new oxathiazinane dioxides and their in vitro cancer cell growth inhibitory activity.

New oxathiazinane dioxides have been derived from D- and L-serine and tested for their in vitro cell growth inhibitory activity toward SKBR3 breast cancer cells. (5R)-5-(4-(4'-Bromomethyl)phenyl)benzyloxymethyl-[1,3,4]-oxathiazinane-3,3-dioxide showed a cytotoxicity of IC(50) approximately 10 microM.

Novel 2-[(benzylamino)methyl]pyrrolidine-3,4-diol derivatives as alpha-mannosidase inhibitors and with antitumor activities against hematological and solid malignancies.

Novel alpha-mannosidase inhibitors of the type (2R,3R,4S)-2-({[(1R)-2-hydroxy-1-arylethyl]amino}methyl)pyrrolidine-3,4-diol have been prepared and assayed for their anticancer activities. Compound 30 with the aryl group=4-trifluoromethylbiphenyl inhibits the proliferation of primary cells and cell lines of different origins, irrespective of Bcl-2 expression levels, inducing a G2/Mcell cycle arrest and by modification of genes involved in cell cycle progression and survival.

Synthesis and glycosidase inhibitory activity of 1-amino-3,6-anhydro-1-deoxy-D-sorbitol derivatives.

3,6-Anhydro-1-(aryl or alkylamino)-1-deoxy-D-sorbitol derivatives have been prepared in four steps from isosorbide, a by-product from the starch industry. The inhibitory activities of these new compounds have been evaluated towards 13 glycosidases. A first lead-compound was identified, which inhibited beta-N-acetylglucosaminidase from bovine kidney (82% inhibition at 1mM).

Desulfinylation of prop-2-enesulfinic acid: experimental results and mechanistic theoretical analysis.

The potential energy surfaces of the desulfinylation of prop-2-enesulfinic acid (13) in CH(2)Cl(2) solution at -15 degrees C have been explored by quantum calculations and analyzed with kinetic data obtained for the reaction in absence or presence of additives. Monomeric 13 adopts a preferred conformation with gauche S=O/sigma(C(1)-C(2) bond pairs and the O-H bond pointing toward C(3). It equilibrates with the more stable dimer (13)(2) (at -15 degrees C) formed by two O-H...O=S hydrogen bonds and in which the S=O/sigmaC(1)-C(2) are gauche also, but the SOH moieties are antiperiplanar with respect to sigma(C(1)-C(2)). Dimer (13)(2) undergoes desulfinylation into propene + SO(2) + 13 following a one-step, concerted mechanism. The preferred transition state is a six-membered, chairlike transition structure (C...S elongation and S-O...H...C(3) hydrogen transfer occur in concert) in which the S=O/sigma(C(1)-C(2)) bonds are gauche (S=O adopt pseudoaxial positions). There are at least 48 transition states, each one defining a different pathway, all with similar calculated free energies (DeltaG(double dagger) = 25.3-28.6 kcal/mol), which makes the bimolecular (autocatalyzed) retro-ene elimination of SO(2) competing (entropy factor) with a monomolecular process for which the transition state (calculated DeltaG(double dagger) = 24.3 kcal/mol) implies only one molecule of sulfinic acid. This agrees with the experimental rate law of the reaction which is first order in the concentration of dimer (13)(2). SO(2), CF(3)COOH, and BF(3) x Me(2)O do not catalyze the reaction. In the presence of an excess of BF(3) x Me(2)O the desulfinylation is completely inhibited due to the formation of a stable tetramolecular complex of type (CH(2)=CHCH(2)SO(2)H x BF(3))(2) (18), for which quantum calculations show that the S=O/sigma(C(1)-C(2)) bonds are antiperiplanar whereas the S-OH/sigma(C(1)-C(2)) bonds are gauche. Independently of the additive, the retro-ene eliminations of SO(2) are calculated to be concerted and have transition states adopting six-membered cyclic structures in which S=O and sigma(C(1)-C(2)) are gauche, the S=O interacting with the additive. Preliminary experiments suggested that the thermodynamically unfavored ene reaction of SO(2) with propene can occur at low temperature using 1 equiv of BF(3).

Synthesis and conformational analysis of (alpha-D-galactosyl)phenylmethane and alpha-,beta-difluoromethane analogues: interactions with the plant lectin viscumin.

(Alpha-D-galactosyl)phenylmethane (1), (alpha- and beta-D-galactosyl)(difluoro)phenylmethane (2 and 3) have been prepared and their conformations in solution were described by using a combination of force-field calculations and NMR spectroscopic studies. Galactoside 1 adopts a (4)C(1) chair conformation and an exo anomeric orientation, as is the case for natural alpha-galactosides. The X-ray crystal structure of its difluoromethylene derivative 2 similarly shows a (4)C(1) chair conformation. Surprisingly, compound 2 exhibits a different equilibrium between (1)C(4) chair and (1)S(3) skew boat conformations and significant flexibility around the pseudoglycosidic linkage when in solution. The beta-stereoisomer 3 adopts a major (4)C(1) chair conformation. Interestingly, C-galactosides 1, 2, and 3 bind to viscumin (VAA), a galactoside-specific lectin, which is confirmed by NMR experiments and docking calculations.

Synthesis of optically active 5-alkoxy-6-methylcyclohex-2-en-1-ones and 4-alkoxy-5-methylcyclopent-1-enyl benzoate.

The reaction of (-)-(1E,3Z)-2-methyl-1-((1S)-1-phenylethoxy)penta-1,3-dien-3-ol benzoate with allyltrimethylsilane in SO2 and in the presence of a catalytic amount of Tf2NTMS gives a silyl sulfinate intermediate that furnishes (-)-(6Z,1'S,4S,5S)-5-methyl-4-(1'-phenylethoxy)octa-1,6-dien-6-yl benzoate after acidic workup. The latter undergoes ring-closing metathesis producing (-)-(2S,3S)-2-methyl-3-((1S)-1-phenylethoxy)cyclopent-5-en-1-yl benzoate. It has been converted also into the corresponding trimethylsilyl enol ether. After oxidation, an enone is obtained that undergoes ring-closing metathesis giving (-)-(5S,6S)-6-methyl-5-((1S)-1-phenylethoxy)cyclohex-2-en-1-one.

A short and convenient synthesis of 1-deoxymannojirimycin and N-oxy analogues from D-fructose.

Ketonitrone 8 was prepared from D-fructose as an inexpensive starting material and was used in a stereoselective synthesis of 1-deoxymannojirimycin (DMJ, 4), of its previously unknown N-hydroxy analogue 15, and of the polyhydroxylated ketonitrone 14. The latter were assayed as potential glycosidase inhibitors on a panel of 13 selected purified enzymes. Disappointingly, the polyhydroxylated nitrone 14 inhibited none of these enzymes. However, N-hydroxy-DMJ (15) exhibited a moderate and non-selective activity toward the snail beta-mannosidase EC 3.2.1.25.

Synthesis of (E,Z)-1-alkoxy-3-acyloxy-2-methylpenta-1,3-dienes via Danishefsky-type dienes or O-acylation of enones.

1-Alkoxy-2-methyl-3-acyloxy-(E,E)-penta-1,3-dienes have been prepared applying among others a modified Danishefsky's general method, including chiral, racemic, and achiral derivatives.