Guy Fournet

Stereospecific synthesis of arylidene and allylidene cyclopentanes by a palladium-catalyzed cylisation

Abstract Vinyl and aryl halides react with e-acetylenic β-diesters, β-keto esters and βsulfonylesters in the presence of a Pd(o) catalyst leading in good yields to the title compounds. The acetylenic homolog containing an additional carbon leads in the same conditions to a cyclohexane, but this process then competes with the arylation of the terminal acetylenic carbon.

PALLADIUM-MEDIATED CYCLOPENTANATION OF ALKENES BEARING A NUCLEOPHILIC SUBSTITUENT. STEREOCHEMISTRY AND MECHANISM

Abstract The increased facility of the reaction 1 2 simultaneously with the increased nucleophilicity of the malonate, as well as the stereospecificity observed in the case of 3 , led to the conclusion that the cyclisation proceeds by a nucleophilic attack on the double bond activated by a palladium(II) species.

The effect of a novel irreversible inhibitor of aldehyde dehydrogenases 1 and 3 on tumour cell growth and death.

Aldehyde dehydrogenases (ALDHs) are a family of several isoenzymes expressed in various tissues and in all subcellular fractions. In some tumours, there is an increase of ALDH activity, especially that of class 1 and 3. The increase in the activity of these isoenzymes is correlated with cell growth and drug resistance shown by these cells. It has been observed that hepatoma cells expressing low ALDH3 activity are more susceptible to growth inhibition by low concentration of lipid peroxidation products than hepatoma cells expressing high ALDH3 activity. The products of lipid peroxidation are good substrates for ALDH, but when their intracellular levels are increased in hepatoma cells treated repeatedly with prooxidants, they inhibit ALDH3 and bring about growth inhibition or cell death. As a follow up to the work previously reported on S-methyl 4-amino-4-methylpent-2-ynethioate, a synthetic suicide inhibitor of ALDH1, which induced bcl2 overexpressing cells into apoptosis and exhibited an ED50 of 400 microM, a novel broad spectrum inhibitor of ALDH1 and ALDH3 was synthesised. This new compound (ATEM) is a suicide inhibitor of ALDH1, an irreversible inhibitor of ALDH3 and exhibits an ED50 of 10-25 microM on rat cultured hepatoma cells. Four hours after treatment with 25 microM ATEM, ALDH activity using benzaldehyde or propionaldehyde in hepatoma cells was decreased by 40% and cell number by 15% compared with controls. As cell growth did not resume when the inhibitor was removed from the culture medium, it suggested strongly that ALDHs play a pivotal role in mediating cell death.

New palladium mediated cyclopentanation of alkenes bearing a δ nucleophilic substituent

Abstract A δ-alkylpalladium intermediate generated by carbopalladation of a double bond can be intramolecularly trapped by a β-diester or a β-keto ester enolate if a cyclopentane ring is formed. Otherwise, normal β-elimination is observed.

Novel competitive irreversible inhibitors of aldehyde dehydrogenase (ALDH1): restoration of chemosensitivity of L1210 cells overexpressing ALDH1 and induction of apoptosis in BAF3 cells overexpressing bcl2

4-Amino-4-methyl-pent-2-ynthioc acid S-methyl ester (ampal thiolester: ATE) was used as a lead compound to synthesise new amino-substituted derivatives of alpha, beta acetylenic thiolester compounds as inhibitors of aldehyde dehydrogenase 1, (ALDH1). Of these compounds, the dimethyl derivative (DIMATE) was a competitive irreversible inhibitor (K(i) approximately 280 microM) of bakers yeast ALDH1 in vitro showing 80% inhibition at 400 microM when preincubated with the enzyme for 30min, whereas the trimethyl ammonium and the morpholine derivatives showed only 15% inhibition at 600 microM even after 60min preincubation. ATE inhibited ALDH1 activity in ALDH1-transfected L1210 T cells resistant to hydroperoxycyclophosphamide (HCPA) and inhibited growth synergistically in the presence of HCPA. In non-transfected L1210 counterparts ATE did not potentiate growth inhibition by HCPA. DIMATE was a 30-100-fold more effective growth inhibitor than ATE. Endogenous ALDH1 activities of BAF(3) cells over-expressing different levels of bcl(2) (0-100%) were similar (16-20mU/mg protein) and were all inhibited by DIMATE, reaching 20-30% at 4 microM. Up to 4 microM no apoptosis, as measured by DNA-fragmentation was observed, but at 8 and 10 microM DIMATE, DNA-fragmentation increased concomitantly with ALDH1 inhibition. No DNA-fragmentation was observed with ALDH1 irreversible inhibitors devoid of a thiolester group or with thiolesters which were not inhibitors of ALDH1. It was seen only with competitive irreversible inhibitors having the methanethiol and enzyme-inhibitory moieties. The methanethiol putatively released from DIMATE by ALDH1 esterase activity plays a role, albeit undefined, in lowering intramitochondrial glutathione levels which decreased by 47% as DNA-fragmentation increased.

Anaplerotic reactions in tumour proliferation and apoptosis

Our aim in this commentary is to provide evidence that certain oxoacids formed in anaplerotic reactions control cell proliferation/apoptosis. In tumour cells with impaired Krebs cycle enzymes, some anaplerotic reactions do compensate for the deficit in oxoacids. One of these, oxaloacetate, derived from the transamination of asparagine but not of aspartate, is decarboxylated 4-fold more efficiently in polyoma-virus transformed cells than in their non-transformed counterparts. The deamidation of asparagine, in the cell culture medium, to aspartate by asparaginase decreases asparagine transamination and inhibits concomitantly the growth of asparaginase-sensitive lymphoma cells, suggesting a causal relationship between asparagine transamination and growth. Another oxoacid that can provide ATP when metabolised in mitochondria, but by the branched-chain oxoacid dehydrogenase complex (BCOADC), is 2-oxobutanoate. It has two origins: (a) deamination of threonine, and (b) cleavage of cystathionine, a metabolite derived from methionine. 2-Oxobutanoate in the presence of insulin promotes growth in G1/S arrested cells. But methionine also gives rise to another substrate of BCOADC, 4-methylthio-2-oxobutanoate (MTOB), which is synthesised exclusively from methylthioadenosine (MTA) by the action of MTA phosphorylase. In Met-dependent tumour cells with defective MTA phosphorylase, 2-oxobutanoate production would exceed that of MTOB. Further, BCOADC also has 3-fold greater affinity for 2-oxobutanoate than for MTOB; hence, the deficiency in 3-methylthio propionyl CoA, the final product of MTOB decarboxylation, would be exacerbated. Methional, the transient metabolic precursor in 3-methylthio propionyl CoA biosynthesis, is apoptogenic for both normal and bcl(2)-negative transformed cells in culture. Investigations of other causal relationships between the genes/enzymes mediating the homeostasis of anaplerotic oxoacids and cell growth/death may be worthwhile.

A Thioester Analogue of an Amino Acetylenic Aldehyde Is A Suicide Inhibitor of Aldehyde Dehydrogenase and an Inducer of Apoptosis in Mouse Lymphoid Cells Overexpressing the bcl2 Gene

We have previously shown that methional, CH3SCH2CH2CHO, an endogeneous cellular aldehyderived by the oxidative decarboxylation of 4-methylthio-2-oxobutanoic acid (B), an intermediate in the methionine salvage pathway is a potent inducer of apoptosis when added to cultures of mouse lymphoid cells BAF3 bo (Quash et al., 1995). MTOB a00 μM was also capable of alleviating the methionine dependence shown by transfed cells (Ogier et al., 1993) because of its ready transamination to methionine, with gamine as amine donor (Backlund et al., 1982). Further, the inhibition of this MTOB transaminase with novel transition state inhibitors such as methionine-ethyl ester- pyridoxal induced apoptosis in BAF3 bo cells but not in BAF3 bcl2 which had been trans- fected with the human bcl2 gene (Roch et al., 1996). When the reasons for this refractory behaviour of BAF3 bcl2 were investigated using[14C] MTOB it was found that the production of [14methional from [14C] MTOB was reduced by 50% in BAF3 bcl2 and that even the direct addition of 600μM methional could not induce apoptosis in BAF3 bcl2 (Roch and al, 19 Methional is itself further metabolised intracellularly [Figure 1] by 3 pathways: reduction by aldehyde reductase (ALR) to methionol, oxidation by aldehyde dehydro- genaseLDH1) to methyl thiopropionic acid and β-hydroxylation to malondialdehyde. We therefore tried to see whether the inhibition of the reductase or the dehydrogenase would uce apoptosis in methional treated BAF3 bcl2 cells. Using quercitin as an inhibitor of ALR increase in apotosis was observed in BAF3 bcl2, but this result is to be interpreted with caution as quercitin also induces DNA strand breaks directly.With disulfiram as an ibitor of ALDH a small increase was seen but experimentation could be carried out only with low concentrations (< 100μM) due to the intrinsic toxicity of this compound. It was therefore clear that more specific inhibitors of ALDH were required to try to determine if ALDH played a role in the resistance to the apoptosis-inducing activity of methio.

Ring enlargement of 2-oxo-1,3-dithiane : easy access to 4-substituted-7,8-dihydro-2H,6H-1,5-dithiocin-2-one

Abstract the 2-Oxo-1,3-diathiane reacts with various lithium acetylides to furnish a new class of compounds: the 4-substituted-7,8-dihydro-2H,6H-1,5-dithiocin-2-one derivations.

Dose‐dependent activation and block by bisG10, a K+ channel blocker, of mouse and frog skeletal muscle KATP channels

The effects of a K+ channel blocker, bisG10, were examined on ATP‐sensitive K+ (KATP) channels in membrane patches excised from mammalian and amphibian skeletal muscle fibres using the patch‐clamp technique. At micromolar concentrations, bisG10, added on the intracellular side, induced a strong, reversible, flickery block of KATP channels. BisG10, added on the extracellular side, was about 100‐fold less potent at inhibiting channel activity. At 10 nM, intracellular bisG10 increased KATP channel activity. This activation was independent of the presence of internal ATP or Mg2+. The inhibitory effect of bisG10 most likely arose from open‐channel block whereas activation could result from more complex, indirect interactions.

Targeting RNA structure in SMN2 reverses spinal muscular atrophy molecular phenotypes

Modification of SMN2 exon 7 (E7) splicing is a validated therapeutic strategy against spinal muscular atrophy (SMA). However, a target-based approach to identify small-molecule E7 splicing modifiers has not been attempted, which could reveal novel therapies with improved mechanistic insight. Here, we chose as a target the stem-loop RNA structure TSL2, which overlaps with the 5′ splicing site of E7. A small-molecule TSL2-binding compound, homocarbonyltopsentin (PK4C9), was identified that increases E7 splicing to therapeutic levels and rescues downstream molecular alterations in SMA cells. High-resolution NMR combined with molecular modelling revealed that PK4C9 binds to pentaloop conformations of TSL2 and promotes a shift to triloop conformations that display enhanced E7 splicing. Collectively, our study validates TSL2 as a target for small-molecule drug discovery in SMA, identifies a novel mechanism of action for an E7 splicing modifier, and sets a precedent for other splicing-mediated diseases where RNA structure could be similarly targeted.Spinal muscular atrophy (SMA) is an autosomal recessive disorder with no present cure. Here the authors perform an in vitro screening leading to the identification of a small molecule that alters the conformational dynamics of the TSL2 RNA structure and acts as a modulator of SMN exon 7 splicing.