Effect of Fluorination on Skin Sensitization Potential and Fragrant Properties of Cinnamyl Compounds.

A series of three α- and three ß-fluorinated representatives of the family of cinnamate-derived odorants (cinnamaldehyde (1), cinnamyl alcohol (2), and ethyl cinnamate (3)) as used as fragrance ingredients is described. Olfactive evaluation shows that the fluorinated compounds exhibit a similar odor profile to their parent compounds, but the olfactive detection thresholds are clearly higher. In vitro evaluation of the skin sensitizing properties with three different assays indicates that α-fluorination of Michael acceptor systems 1 and 3 slightly improves the skin sensitization profile. α-Fluorocinnamyl alcohol 2b is a weaker skin sensitizer than cinnamyl alcohol 2a by in vitro tests and the fluorinated product drops below the sensitization threshold of the KeratinoSens® assay. On the other hand, ß-fluorination of compounds 1 - 3 results in highly reactive products which display a worsened in vitro skin sensitization profile.

Pharmacophore-based design of novel oxadiazoles as selective sphingosine-1-phosphate (S1P) receptor agonists with in vivo efficacy.

Sphingosine-1-phosphate (S1P) receptor agonists have shown promise as therapeutic agents for multiple sclerosis (MS) due to their regulatory roles within the immune, central nervous system, and cardiovascular system. Here, the design and optimization of novel [1,2,4]oxadiazole derivatives as selective S1P receptor agonists are described. The structure-activity relationship exploration was carried out on the three dominant segments of the series: modification of the polar head group (P), replacement of the oxadiazole linker (L) with different five-membered heterocycles, and the use of diverse 2,2'-disubstituted biphenyl moieties as the hydrophobic tail (H). All three segments have a significant impact on potency, S1P receptor subtype selectivity, physicochemical properties, and in vitro absorption, distribution, metabolism, excretion and toxicity (ADMET) profile of the compounds. From these optimization studies, a selective S1P1 agonist, N-methyl-N-(4-{5-[2-methyl-2'-(trifluoromethyl)biphenyl-4-yl]-1,2,4-oxadiazol-3-yl}benzyl)glycine (45), and a dual S1P1,5 agonist, N-methyl-N-(3-{5-[2'-methyl-2-(trifluoromethyl)biphenyl-4-yl]-1,2,4-oxadiazol-3-yl}benzyl)glycine (49), emerged as frontrunners. These compounds distribute predominantly in lymph nodes and brain over plasma and induce long lasting decreases in lymphocyte count after oral administration. When evaluated head-to-head in an experimental autoimmune encephalomyelitis mouse model, together with the marketed drug fingolimod, a pan-S1P receptor agonist, S1P1,5 agonist 49 demonstrated comparable efficacy while S1P1 -selective agonist 45 was less potent. Compound 49 is not a prodrug, and its improved property profile should translate into a safer treatment of relapsing forms of MS.

BAX channel activity mediates lysosomal disruption linked to Parkinson disease.

Lysosomal disruption is increasingly regarded as a major pathogenic event in Parkinson disease (PD). A reduced number of intraneuronal lysosomes, decreased levels of lysosomal-associated proteins and accumulation of undegraded autophagosomes (AP) are observed in PD-derived samples, including fibroblasts, induced pluripotent stem cell-derived dopaminergic neurons, and post-mortem brain tissue. Mechanistic studies in toxic and genetic rodent PD models attribute PD-related lysosomal breakdown to abnormal lysosomal membrane permeabilization (LMP). However, the molecular mechanisms underlying PD-linked LMP and subsequent lysosomal defects remain virtually unknown, thereby precluding their potential therapeutic targeting. Here we show that the pro-apoptotic protein BAX (BCL2-associated X protein), which permeabilizes mitochondrial membranes in PD models and is activated in PD patients, translocates and internalizes into lysosomal membranes early following treatment with the parkinsonian neurotoxin MPTP, both in vitro and in vivo, within a time-frame correlating with LMP, lysosomal disruption, and autophagosome accumulation and preceding mitochondrial permeabilization and dopaminergic neurodegeneration. Supporting a direct permeabilizing effect of BAX on lysosomal membranes, recombinant BAX is able to induce LMP in purified mouse brain lysosomes and the latter can be prevented by pharmacological blockade of BAX channel activity. Furthermore, pharmacological BAX channel inhibition is able to prevent LMP, restore lysosomal levels, reverse AP accumulation, and attenuate mitochondrial permeabilization and overall nigrostriatal degeneration caused by MPTP, both in vitro and in vivo. Overall, our results reveal that PD-linked lysosomal impairment relies on BAX-induced LMP, and point to small molecules able to block BAX channel activity as potentially beneficial to attenuate both lysosomal defects and neurodegeneration occurring in PD.

Enantioselective synthesis and physicochemical properties of libraries of 3-amino- and 3-amidofluoropiperidines.

The enantioselective syntheses of 3-amino-5-fluoropiperidines and 3-amino-5,5-difluoropiperidines were developed using the ring enlargement of prolinols to access libraries of 3-amino- and 3-amidofluoropiperidines. The study of the physicochemical properties revealed that fluorine atom(s) decrease(s) the pKa and modulate(s) the lipophilicity of 3-aminopiperidines. The relative stereochemistry of the fluorine atoms with the amino groups at C3 on the piperidine core has a small effect on the pKa due to conformationnal modifications induced by fluorine atom(s). In the protonated forms, the C-F bond is in an axial position due to a dipole-dipole interaction between the N-H(+) and C-F bonds. Predictions of the physicochemical properties using common software appeared to be limited to determine correct values of pKa and/or differences of pKa between cis- and trans-3-amino-5-fluoropiperidines.

Titanium mediated olefination of aldehydes with α-haloacetates: an exceptionally stereoselective and general approach to (Z)-α-haloacrylates.

An exceptionally stereoselective and general synthesis of (Z)-α-haloacrylates, ready to undergo various synthetic transformations, has been demonstrated from α-haloacetates and aldehydes in a one-pot manner via the titanium-enolate based asymmetric aldol condensation. Besides being an expedient synthetic procedure, the ready availability of diverse α-haloacetates, exceptional stereoselectivity, and high yields make the process a versatile transformation in organic synthesis. The potential of this method in up-scaling operations has been illustrated.

XtalFluor-E, an efficient coupling reagent for amidation of carboxylic acids.

Amides were produced from carboxylic acids and amines by using XtalFluor-E as an activator. Even poorly reactive carboxylic acids can be transformed to amides. In addition, optically active amines and/or carboxylic acids were not epimerized/racemized during the process.

Identification and optimization of an aminoalcohol-carbazole series with antimalarial properties.

Recent observations on the emergence of artemisinin resistant parasites have highlighted the need for new antimalarial treatments. An HTS campaign led to the identification of the 1-(1-aminopropan-2-ol)carbazole analogues as potent hits against Plasmodium falciparum K1 strain. The SAR study and optimization of early ADME and physicochemical properties direct us to the selection of a late lead compound that shows good efficacy when orally administrated in the in vivo P. berghei mouse model.

Access and regioselective transformations of 6-substituted 4-aryl-2,8-dichloropyrido[3,2-d]pyrimidine compounds.

We report herein an efficient route for the synthesis of 2,4,8-trichloropyrido[3,2-d]pyrimidines 1 with R(1) substituents at C-6. The potential of such scaffolds was demonstrated by the possibility to displace regioselectively each aromatic chloride to introduce diversity. Sequential sulfur nucleophilic addition followed by Liebeskind-Srogl cross-coupling reaction yielded unprecedented aryl introduction at C-4 on a trichloropyrido[3,2-d]pyrimidine derivative. The reactivity difference of the remaining two chlorides toward S(N)Ar reactions was investigated. Amination yielded high C-2 regioselectivity, while thiolation was influenced by C-6 substituents, resulting in medium to high C-2 versus C-8 regioselectivity. The last chloride was efficiently displaced by S(N)Ar, Suzuki-Miyaura cross-coupling reaction, or reduction. C-2 arylation as a final step was also possible by Liebeskind-Srogl cross-coupling reaction on the previously introduced C-2 thioether. A concise and highly divergent synthetic use of 1 was developed, thereby providing an efficient approach to explore the structure-activity relationship of pyrido[3,2-d]pyrimidine derivatives such as 9, 10, 15, and 16.

Regioselective synthesis of 2,8-disubstituted 4-aminopyrido[3,2-d]pyrimidine-6-carboxylic acid methyl ester compounds.

We report herein the synthesis of 4-amino-2,8-dichloropyrido[3,2-d]pyrimidine derivatives 2 and their regioselective diversification through S(N)Ar and metal-catalyzed cross-coupling reactions. While amination of 2 took place selectively at C-2, the regioselectivity of thiol or thiolate addition depended on the reaction conditions. Selective C-8 addition was obtained in DMF with Hünig's base and C-2 addition in (i)PrOH. These C-2 or C-8 regioselective thiolations provided an opportunistic way to selectively activate either of the two positions toward the metal-catalyzed cross-coupling reaction. The chloride could be efficiently substituted by Suzuki-Miyaura reaction and the sulfanyl group by Liebeskind-Srogl cross-coupling reaction, demonstrating the orthogonality of both reactive centers. The development of regioselective conditions for these different transformations yielded the synthesis of 4-amino-2,6,8-trisubstituted pyrido[3,2-d]pyrimidine derivatives, with various substituents.

Discovery of a Novel Series of CRTH2 (DP2) Receptor Antagonists Devoid of Carboxylic Acids.

Antagonism of the CRTH2 receptor represents a very attractive target for a variety of allergic diseases. Most CRTH2 antagonists known to date possess a carboxylic acid moiety, which is essential for binding. However, potential acid metabolites O-acyl glucuronides might be linked to idiosynchratic toxicity in humans. In this communication, we describe a new series of compounds that lack the carboxylic acid moiety. Compounds with high affinity (K i < 10 nM) for the receptor have been identified. Subsequent optimization succeeded in reducing the high metabolic clearance of the first compounds in human and rat liver microsomes. At the same time, inhibition of the CYP isoforms was optimized, giving rise to stable compounds with an acceptable CYP inhibition profile (IC50 CYP2C9 and 2C19 > 1 µM). Taken together, these data show that compounds devoid of carboxylic acid groups could represent an interesting alternative to current CRTH2 antagonists in development.

Discovery of a novel series of CXCR3 antagonists.

The discovery of a novel series of CXCR3 antagonists is described. Starting from an HTS positive, iterative optimization gave potent compounds (IC(50) 15 nM in a chemotaxis assay). The strategy employed to improve the metabolic stability of these derivatives is described.

Discovery of a novel series of potent S1P1 agonists.

The discovery of a novel series of S1P1 agonists is described. Starting from a micromolar HTS positive, iterative optimization gave rise to several single-digit nanomolar S1P1 agonists. The compounds were able to induce internalization of the S1P1 receptor, and a selected compound was shown to be able to induce lymphopenia in mice after oral dosing.

MAC inhibitors suppress mitochondrial apoptosis.

MAC (mitochondrial apoptosis-induced channel) forms in the mitochondrial outer membrane and unleashes cytochrome c to orchestrate the execution of the cell. MAC opening is the commitment step of intrinsic apoptosis. Hence closure of MAC may prevent apoptosis. Compounds that blocked the release of fluorescein from liposomes by recombinant Bax were tested for their ability to directly close MAC and suppress apoptosis in FL5.12 cells. Low doses of these compounds (IC50 values ranged from 19 to 966 nM) irreversibly closed MAC. These compounds also blocked cytochrome c release and halted the onset of apoptotic markers normally induced by IL-3 (interleukin-3) deprivation or staurosporine. Our results reveal the tight link among MAC activity, cytochrome c release and apoptotic death, and indicate this mitochondrial channel is a promising therapeutic target.

GLEPP1/protein-tyrosine phosphatase phi inhibitors block chemotaxis in vitro and in vivo and improve murine ulcerative colitis.

We describe novel, cell-permeable, and bioavailable salicylic acid derivatives that are potent and selective inhibitors of GLEPP1/protein-tyrosine phosphatase . Two previously described GLEPP1 substrates, paxillin and Syk, are both required for cytoskeletal rearrangement and cellular motility of leukocytes in chemotaxis. We show here that GLEPP1 inhibitors prevent dephosphorylation of Syk1 and paxillin in resting cells and block primary human monocyte and mouse bone marrow-derived macrophage chemotaxis in a gradient of monocyte chemotactic protein-1. In mice, the GLEPP1 inhibitors also reduce thioglycolate-induced peritoneal chemotaxis of neutrophils, lymphocytes, and macrophages. In murine disease models, the GLEPP1 inhibitors significantly reduce severity of contact hypersensitivity, a model for allergic dermatitis, and dextran sulfate sodium-induced ulcerative colitis, a model for inflammatory bowel disease. Taken together, our data provide confirmation that GLEPP1 plays an important role in controlling chemotaxis of multiple types of leukocytes and that pharmacological inhibition of this phosphatase may have therapeutic use.

Convergent functional genomics of oligodendrocyte differentiation identifies multiple autoinhibitory signaling circuits.

Inadequate remyelination of brain white matter lesions has been associated with a failure of oligodendrocyte precursors to differentiate into mature, myelin-producing cells. In order to better understand which genes play a critical role in oligodendrocyte differentiation, we performed time-dependent, genome-wide gene expression studies of mouse Oli-neu cells as they differentiate into process-forming and myelin basic protein-producing cells, following treatment with three different agents. Our data indicate that different inducers activate distinct pathways that ultimately converge into the completely differentiated state, where regulated gene sets overlap maximally. In order to also gain insight into the functional role of genes that are regulated in this process, we silenced 88 of these genes using small interfering RNA and identified multiple repressors of spontaneous differentiation of Oli-neu, most of which were confirmed in rat primary oligodendrocyte precursors cells. Among these repressors were CNP, a well-known myelin constituent, and three phosphatases, each known to negatively control mitogen-activated protein kinase cascades. We show that a novel inhibitor for one of the identified genes, dual-specificity phosphatase DUSP10/MKP5, was also capable of inducing oligodendrocyte differentiation in primary oligodendrocyte precursors. Oligodendrocytic differentiation feedback loops may therefore yield pharmacological targets to treat disease related to dysfunctional myelin deposition.

An efficient and expeditious synthesis of di- and trisubstituted amino-phenyl and -benzyl derivatives of tetrazole and [1,3,4]oxadiazol-2-one.

A practical protocol for the parallel synthesis and purification of amino tetrazole and [1,3,4]oxadiazol-2-one derivatives as carboxylic acid bioisosteres is described. Phenyl- and benzyl-amines, substituted with tetrazole or [1,3,4]oxadiazol-2-one, were transformed into functionally diverse and novel compounds, with p K a values ranging from 4.9 to 8.4, by two sequential reductive alkylation reactions. These series of di- and trisubstituted amino-phenyl and -benzyl derivatives were produced in solution using solid-supported reagents and were purified by solid-phase extraction (SPE) techniques.

Improvement of peripheral endothelial dysfunction by protein tyrosine phosphatase inhibitors in heart failure.

BACKGROUND: Chronic heart failure (CHF) induces endothelial dysfunction characterized by a decrease in nitric oxide (NO) production in response to flow (flow-mediated dilatation [FMD]). Because activation of endothelial NO synthase (eNOS) by flow requires tyrosine phosphorylation, we tested whether endothelial dysfunction could be corrected by increasing phosphotyrosine levels using protein tyrosine phosphatase (PTP) inhibitors and especially inhibitors of PTP1B. METHODS AND RESULTS: CHF was induced by coronary ligation in mice, and FMD was assessed in isolated and cannulated mesenteric artery segments (2 mm in length and <300 microm in diameter). CHF almost abolished FMD but only moderately affected the response to acetylcholine. In mice with CHF, the PTP1B inhibitors AS279, AS098, and AS713 restored FMD to levels similar to those of normal mice. This restoration was reduced by inhibitors of eNOS and phosphatidylinositol-3 kinase. Polymerase chain reaction and Western blot showed that arteries express PTP1B, and this expression was not affected by CHF. Immunolocalization revealed the presence of PTP1B in the endothelium and the adventitia. Flow induced a transient eNOS phosphorylation that was absent in CHF. PTP1B inhibition stimulated early eNOS phosphorylation and increased phosphorylation of Akt. CONCLUSIONS: Our results demonstrate for the first time that PTP1B inhibitors may be potent treatments for endothelial dysfunction.

Bax channel inhibitors prevent mitochondrion-mediated apoptosis and protect neurons in a model of global brain ischemia.

Ischemic injuries are associated with several pathological conditions, including stroke and myocardial infarction. Several studies have indicated extensive apoptotic cell death in the infarcted area as well as in the penumbra region of the infarcted tissue. Studies with transgenic animals suggest that the mitochondrion-mediated apoptosis pathway is involved in ischemia-related cell death. This pathway is triggered by activation of pro-apoptotic Bcl-2 family members such as Bax. Here, we have identified and synthesized two low molecular weight compounds that block Bax channel activity. The Bax channel inhibitors prevented cytochrome c release from mitochondria, inhibited the decrease in the mitochondrial membrane potential, and protected cells against apoptosis. The Bax channel inhibitors did not affect the conformational activation of Bax or its translocation and insertion into the mitochondrial membrane in cells undergoing apoptosis. Furthermore, the compounds protected neurons in an animal model of global brain ischemia. The protective effect in the animal model correlated with decreased cytochrome c release in the infarcted area. This is the first demonstration that Bax channel activity is required in apoptosis.

3,6-dibromocarbazole piperazine derivatives of 2-propanol as first inhibitors of cytochrome c release via Bax channel modulation.

There is compelling evidence that Bax channel activity stimulates cytochrome c release leading ultimately to cell death, which is a key event in ischemic injuries and neurodegenerative diseases. Here 3,6-dibromocarbazole piperazine derivatives of 2-propanol are described as the first small and potent modulators of the cytochrome c release triggered by Bid-induced Bax activation in a mitochondrial assay. Furthermore, a mechanism of action is proposed, and fluorescent derivatives allowing the localization of such inhibitors are reported.