Synthesis and evaluation of analogs of 5'-(((Z)-4-amino-2-butenyl)methylamino)-5'-deoxyadenosine (MDL 73811, or AbeAdo) - An inhibitor of S-adenosylmethionine decarboxylase with antitrypanosomal activity.

We describe our efforts to improve the pharmacokinetic properties of a mechanism-based suicide inhibitor of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC), essential for the survival of the eukaryotic parasite Trypanosoma brucei responsible for Human African Trypanosomiasis (HAT). The lead compound, 5'-(((Z)-4-amino-2-butenyl)methylamino)-5'-deoxyadenosine (1, also known as MDL 73811, or AbeAdo), has curative efficacy at a low dosage in a hemolymphatic model of HAT but displayed no demonstrable effect in a mouse model of the CNS stage of HAT due to poor blood-brain barrier permeation. Therefore, we prepared and evaluated an extensive set of analogs with modifications in the aminobutenyl side chain, the 5'-amine, the ribose, and the purine fragments. Although we gained valuable structure-activity insights from this comprehensive dataset, we did not gain traction on improving the prospects for CNS penetration while retaining the potent antiparasitic activity and metabolic stability of the lead compound 1.

Identification of Trypanosoma brucei AdoMetDC Inhibitors Using a High-Throughput Mass Spectrometry-Based Assay.

Human African trypanosomiasis (HAT) is a fatal infectious disease caused by the eukaryotic pathogen Trypanosoma brucei (Tb). Available treatments are difficult to administer and have significant safety issues. S-Adenosylmethionine decarboxylase (AdoMetDC) is an essential enzyme in the parasite polyamine biosynthetic pathway. Previous attempts to develop TbAdoMetDC inhibitors into anti-HAT therapies failed due to poor brain exposure. Here, we describe a large screening campaign of two small-molecule libraries (∼400,000 compounds) employing a new high-throughput (∼7 s per sample) mass spectrometry-based assay for AdoMetDC activity. As a result of primary screening, followed by hit confirmation and validation, we identified 13 new classes of reversible TbAdoMetDC inhibitors with low-micromolar potency (IC50) against both TbAdoMetDC and T. brucei parasite cells. The majority of these compounds were >10-fold selective against the human enzyme. Importantly, compounds from four classes demonstrated high propensity to cross the blood-brain barrier in a cell monolayer assay. Biochemical analysis demonstrated that compounds from eight classes inhibited intracellular TbAdoMetDC in the parasite, although evidence for a secondary off-target component was also present. The discovery of several new TbAdoMetDC inhibitor chemotypes provides new hits for lead optimization programs aimed to deliver a novel treatment for HAT.

Improved Synthesis of MDL 73811 - a Potent AdoMetDC Inhibitor and Anti-Trypanosomal Compound.

An improved synthesis of MDL 73811 - a potent AdoMetDC (S-adenosylmethionine decarboxylease) inhibitor and anti-trypanosomal compound with in vivo activity has been completed in four steps from commercially available 2',3'-O-isopropylideneadenosine. Utilization of Mitsunobu chemistry was crucial for the reliable and scalable introduction of the 5'-methylamine moiety, which was problematic using traditional activation/displacement chemistry as previously reported. All reactions in this synthesis were run on gram-scale resulting in a five-fold increase in yield over the original synthesis.

Lysosomal acid lipase and lipophagy are constitutive negative regulators of glucose-stimulated insulin secretion from pancreatic beta cells.

AIMS/HYPOTHESIS: Lipolytic breakdown of endogenous lipid pools in pancreatic beta cells contributes to glucose-stimulated insulin secretion (GSIS) and is thought to be mediated by acute activation of neutral lipases in the amplification pathway. Recently it has been shown in other cell types that endogenous lipid can be metabolised by autophagy, and this lipophagy is catalysed by lysosomal acid lipase (LAL). This study aimed to elucidate a role for LAL and lipophagy in pancreatic beta cells. METHODS: We employed pharmacological and/or genetic inhibition of autophagy and LAL in MIN6 cells and primary islets. Insulin secretion following inhibition was measured using RIA. Lipid accumulation was assessed by MS and confocal microscopy (to visualise lipid droplets) and autophagic flux was analysed by western blot. RESULTS: Insulin secretion was increased following chronic (≥ 8 h) inhibition of LAL. This was more pronounced with glucose than with non-nutrient stimuli and was accompanied by augmentation of neutral lipid species. Similarly, following inhibition of autophagy in MIN6 cells, the number of lipid droplets was increased and GSIS was potentiated. Inhibition of LAL or autophagy in primary islets also increased insulin secretion. This augmentation of GSIS following LAL or autophagy inhibition was dependent on the acute activation of neutral lipases. CONCLUSIONS/INTERPRETATION: Our data suggest that lysosomal lipid degradation, using LAL and potentially lipophagy, contributes to neutral lipid turnover in beta cells. It also serves as a constitutive negative regulator of GSIS by depletion of substrate for the non-lysosomal neutral lipases that are activated acutely by glucose.

Palladium- and nickel-catalyzed alkenylation of enolates.

Transition-metal-catalyzed alkenylation of enolates provides a direct method to synthesize broadly useful ß,γ-unsaturated carbonyl compounds from the corresponding carbonyl compound and alkenyl halides. Despite being reported in the early seventies, this reaction class saw little development for many years. In the past decade, however, efforts to develop this reaction further have increased considerably, and many research groups have reported efficient coupling protocols, including enantioselective versions. These reactions most commonly employ palladium catalysts, but there are also some important reports using nickel. There are many examples of this powerful transformation being used in the synthesis of complex natural products.

Concise, convergent syntheses of (±)-trichostatin A utilizing a Pd-catalyzed ketone enolate α-alkenylation reaction.

Two concise, convergent syntheses of (±)-trichostatin A (1), a potent histone deacetylase inhibitor, have been accomplished. The key step in both is a Pd-catalyzed α-alkenylation reaction between ketone 2 and either dienyl bromide 3 or alkenyl bromide 9 using a modification of cross-coupling conditions described by Negishi and Hartwig. A brief investigation has shown the potential utility of a Ni-catalyzed version of this reaction. The overall synthetic routes are short and amenable to scaleup, providing access to trichostatin A via trichostatic acid as a direct precursor.

Selective oxidation of benzylic and allylic alcohols using Mn(OAc)3/catalytic 2,3-dichloro-5,6-dicyano-1,4-benzoquinone.

A practical, chemoselective oxidation of alcohols employing catalytic quantities of DDQ as the oxidant and Mn(OAc)(3) as the co-oxidant is described. Electron-rich benzylic alcohols are oxidized efficiently to their corresponding carbonyls, but less electron-rich benzylic alcohols remain unchanged. Allylic alcohols are rapidly oxidized to their corresponding aldehyde or ketone counterparts in high yields. This protocol is operationally simple, employs an inexpensive source of Mn(OAc)(3), has short reaction times, and exhibits a significant chemoselectivity favoring allylic alcohols over benzylic alcohols. This procedure also avoids the use of very large excesses of reagents and sometimes poor reproducibility that characterize previously developed reagents such as MnO(2).

Histone deacetylase inhibitor treatment dramatically reduces cholesterol accumulation in Niemann-Pick type C1 mutant human fibroblasts.

Niemann-Pick type C (NPC) disease is predominantly caused by mutations in the NPC1 protein that affect intracellular cholesterol trafficking and cause accumulation of unesterified cholesterol and other lipids in lysosomal storage organelles. We report the use of a series of small molecule histone deacetylase (HDAC) inhibitors in tissue culture models of NPC human fibroblasts. Some HDAC inhibitors lead to a dramatic correction in the NPC phenotype in cells with either one or two copies of the NPC1(I1061T) mutation, and for several of the inhibitors, correction is associated with increased expression of NPC1 protein. Increased NPC1(I1061T) protein levels may partially account for the correction of the phenotype, because this mutant can promote cholesterol efflux if it is delivered to late endosomes and lysosomes. The HDAC inhibitor treatment is ineffective in an NPC2 mutant human fibroblast line. Analysis of the isoform selectivity of the compounds used implicates HDAC1 and/or HDAC2 as likely targets for the observed correction, although other HDACs may also play a role. LBH589 (panobinostat) is an orally available HDAC inhibitor that crosses the blood-brain barrier and is currently in phase III clinical trials for several types of cancer. It restores cholesterol homeostasis in cultured NPC1 mutant fibroblasts to almost normal levels within 72 h when used at 40 nM. The findings that HDAC inhibitors can correct cholesterol storage defects in human NPC1 mutant cells provide the potential basis for treatment options for NPC disease.

Thiadiazole carbamates: potent inhibitors of lysosomal acid lipase and potential Niemann-Pick type C disease therapeutics.

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized at the cellular level by abnormal accumulation of cholesterol and other lipids in lysosomal storage organelles. Lysosomal acid lipase (LAL) has been recently identified as a potential therapeutic target for NPC. LAL can be specifically inhibited by a variety of 3,4-disubstituted thiadiazole carbamates. An efficient synthesis of the C(3) oxygenated/C(4) aminated analogues has been developed that furnishes the products in high yields and high degrees of purity. Common intermediates can also be used for the synthesis of the C(3) carbon substituted derivatives. Herein we tested various thiadiazole carbamates, amides, esters, and ketones for inhibition of LAL. In addition, we tested a diverse selection of commercially available non-thiadiazole carbamates. Our studies show that, among the compounds examined herein, only thiadiazole carbamates are effective inhibitors of LAL. We present a mechanism for LAL inhibition by these compounds whereby LAL transiently carbamoylates the enzyme similarly to previously described inhibition of acetylcholinesterase by rivastigmine and other carbamates as well as acylation of various lipases by orlistat.

Investigation of N-aryl-3-alkylidenepyrrolinones as potential Niemann-Pick type C disease therapeutics.

A five-step synthesis of an array of N-aryl-3-alkylidenepyrrolinones, which are potential Niemann-Pick type C (NPC) disease therapeutics, is described. The synthetic route allows for the production of analogues, including photoaffinity and biotinylated derivatives. Compound 1a increased esterification by acyl-coenzyme A:cholesteryl acyltransferase in NPC1 mutant cells. It also decreased LDL uptake and increased cholesterol efflux in both NPC1-deficient and normal cells.

Substituted hippurates and hippurate analogs as substrates and inhibitors of peptidylglycine alpha-hydroxylating monooxygenase (PHM).

Peptidyl alpha-hydroxylating monooxygenase (PHM) functions in vivo towards the biosynthesis of alpha-amidated peptide hormones in mammals and insects. PHM is a potential target for the development of inhibitors as drugs for the treatment of human disease and as insecticides for the management of insect pests. We show here that relatively simple ground state analogs of the PHM substrate hippuric acid (C(6)H(5)-CO-NH-CH(2)-COOH) inhibit the enzyme with K(i) values as low as 0.5microM. Substitution of sulfur atom(s) into the hippuric acid analog increases the affinity of PHM for the inhibitor. Replacement of the acetylglycine moiety, -CO-NH-CH(2)-COOH with an S-(thioacetyl)thioglycolic acid moiety, -CS-S-CH(2)-COOH, yields compounds with the highest PHM affinity. Both S-(2-phenylthioacetyl)thioglycolate and S-(4-ethylthiobenzoyl)thioglycolic acid inhibit the proliferation of cultured human prostate cancer cells at concentrations >100-fold excess of their respective K(i) values. Comparison of K(i) values between mammalian PHM and insect PHM shows differences in potency suggesting that a PHM-based insecticide with limited human toxicity can be developed.