Lieven Meerpoel

C-2 arylation of piperidines through directed transition-metal-catalyzed sp3 C-H activation.

The development of transition-metal-catalyzed methods for the direct functionalization of C H bonds has attracted much attention during the past decade. While the direct functionalization of sp C H bonds is an active field of research, the corresponding knowledge on sp C H bonds is still limited and remains one of the current challenges in organic chemistry. Within the area of sp C H activation, the transformation of a C H bond in the a-position to the nitrogen atom of saturated cyclic amines is of particular importance, since such heterocyclic motifs can be found in an impressive number of natural products and marketed drugs. The intermolecular, direct transition-metal-catalyzed functionalization of saturated cyclic amines adjacent to nitrogen offers a simple and efficient synthetic approach towards valuable building blocks. To the best of our knowledge, only six articles are hitherto published on this topic, wherein the main focus is on five-membered cyclic subACHTUNGTRENNUNGstrates.[6,7a] In 2006, Sames and co-workers reported the first direct arylation of saturated cyclic amines through transition-metal-catalyzed sp C H activation, involving arylboronate esters as a coupling partner. Pyrrolidines were successfully arylated adjacent to nitrogen by using a Ru-catalyzed C-H activation process, directed by a pyrroline group and mediated by ketone (used also as a solvent). The authors suggested that the key role of the ketone (pinacolone) is to allow a transmetalation by transforming the initially formed metal hydride species into a metal alkoxide intermediate. The pyrroline directing group could be removed by treatment of the a-arylated pyrrolidines with NH2NH2/ AcOH. One example of a piperidine substrate also appeared in this work, which was C-2 p-methoxyphenylated in moderate yield (38 %). The six-membered piperidine ring is inherently less reactive than its five-membered analogue (pyrrolidine) due to its chair conformation. Consequently, there are very few examples in the literature of direct functionalization of piperidines by means of a transition-metalcatalyzed, sp C H activation process. We report here a novel method for the direct C-2 arylation of piperidines with arylboronate esters, resulting from a mechanistic study of the transmetalation step of the catalytic cycle. As part of our interest in C-2 functionalized 4-substituted piperidines, we first applied the conditions described by Sames and co-workers to 1-(pyrrolin-2-yl)-4-piperidinone ethylene ketal (Scheme 1, 1). Unfortunately, the reaction of 1 with phenylboronic acid pinacol ester resulted in a low conversion to the arylated product, and directing group in[a] Dr. H. Prokopcov , Dr. S. D. Bergman, Dr. K. Aelvoet, V. Smout, Prof. Dr. B. U. W. Maes Organic Synthesis, University of Antwerp Groenenborgerlaan 171, 2020 Antwerp (Belgium) Fax: (+32) 32653233 E-mail : bert.maes@ua.ac.be [b] Prof. Dr. W. Herrebout, Prof. Dr. B. Van der Veken Cryospectroscopy, University of Antwerp Groenenborgerlaan 171, 2020 Antwerp (Belgium) [c] Dr. L. Meerpoel Johnson & Johnson Pharmaceutical Research & Development A Division of Janssen Pharmaceutica N.V. Turnhoutseweg 30, 2340 Beerse (Belgium) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201001887. Scheme 1. 4-Piperidinone ethylene ketal bearing a pyrroline or pyridine directing group: substrates designed to explore the C-2 arylation process.

In Vitro and In Vivo Activities of the Novel Azole Antifungal Agent R126638

ABSTRACT R126638 is a new triazole agent with potent antifungal activity in vitro against various dermatophytes, Candida spp., and Malassezia spp. Its activity against Malassezia spp. in vitro was superior to that of ketoconazole, the agent currently used for the treatment of Malassezia-related infections. R126638 showed activity comparable to or lower than that of itraconazole against dermatophytes in vitro; however, in guinea pig models of dermatophyte infections, R126638 given orally consistently showed antifungal activity superior to that of itraconazole, with 50% effective doses (ED50s) three- to more than eightfold lower than those of itraconazole, depending on the time of initiation and the duration of treatment. The ED50 of R126638 in a mouse dermatophytosis model was more than fivefold lower than that of itraconazole. These data indicate that if the effects of R126638 seen when it is used to treat animals can be extrapolated to humans, the novel compound would be expected to show effects at doses lower than those of existing drugs and, hence, present a lower risk for side effects.

The Novel Azole R126638 Is a Selective Inhibitor of Ergosterol Synthesis in Candida albicans, Trichophyton spp., and Microsporum canis

ABSTRACT R126638 is a novel triazole with in vitro activity similar to that of itraconazole against dermatophytes, Candida spp., and Malassezia spp. In animal models of dermatophyte infections, R126638 showed superior antifungal activity. R126638 inhibits ergosterol synthesis in Candida albicans, Trichophyton mentagrophytes, Trichophyton rubrum, and Microsporum canis at nanomolar concentrations, with 50% inhibitory concentrations (IC50s) similar to those of itraconazole. The decreased synthesis of ergosterol and the concomitant accumulation of 14α-methylsterols provide indirect evidence that R126638 inhibits the activity of CYP51 that catalyzes the oxidative removal of the 14α-methyl group of lanosterol or eburicol. The IC50s for cholesterol synthesis from acetate in human hepatoma cells were 1.4 μM for itraconazole and 3.1 μM for R126638. Compared to itraconazole (IC50 = 3.5 μM), R126638 is a poor inhibitor of the 1α-hydroxylation of 25-hydroxyvitamin D3 (IC50 > 10 μM). Micromolar concentrations of R126638 and itraconazole inhibited the 24-hydroxylation of 25-hydroxyvitamin D3 and the conversion of 1,25-dihydroxyvitamin D3 into polar metabolites. At concentrations up to 10 μM, R126638 had almost no effect on cholesterol side chain cleavage (CYP11A1), 11β-hydroxylase (CYP11B1), 17-hydroxylase and 17,20-lyase (CYP17), aromatase (CYP19), or 4-hydroxylation of all-trans retinoic acid (CYP26). At 10 μM, R126638 did not show clear inhibition of CYP1A2, CYP2A6, CYP2D6, CYP2C8, CYP2C9, CYP2C10, CYP2C19, or CYP2E1. Compared to itraconazole, R126638 had a lower interaction potential with testosterone 6β hydroxylation and cyclosporine hydroxylation, both of which are catalyzed by CYP3A4, whereas both antifungals inhibited the CYP3A4-catalyzed hydroxylation of midazolam similarly. The results suggest that R126638 has promising properties and merits further in vivo investigations for the treatment of dermatophyte and yeast infections.

Liquid chromatography-mass spectrometry with chemiluminescent nitrogen detection for on-line quantitative analysis of compound collections: advantages and limitations

Recently, alternative detection methods such as chemiluminescent nitrogen detection (CLND) have been coupled successfully with HPLC for quantification. This detector produces a signal proportional to the number of moles nitrogen present in the compound. Sample concentration of compounds with a known formula can be determined by use of an external calibration standard such as caffeine. Hence, the CLND can be used without the need for primary standards of the compound with unknown concentration, which enables the use of this detector for high-throughput analysis. In this work, the reliability and pitfalls of this coupled LC-MS/CLND are demonstrated. Nitrogen detection is specific as it only gives a response for nitrogen containing compounds and universal since it only gives a linear response. Nevertheless the lower response for N=N and N-N containing compounds has been evaluated in this study.

A fungicidal piperazine-1-carboxamidine induces mitochondrial fission-dependent apoptosis in yeast

To unravel the working mechanism of the fungicidal piperazine-1-carboxamidine derivative BAR0329, we found that its intracellular accumulation in Saccharomyces cerevisiae is dependent on functional lipid rafts. Moreover, BAR0329 induced caspase-dependent apoptosis in yeast, in which the mitochondrial fission machinery consisting of Fis1 (Whi2), Dnm1 and Mdv1 is involved. Our data are consistent with a prosurvival function of Fis1 (Whi2) and a proapoptotic function of Dnm1 and Mdv1 during BAR0329-induced yeast cell death.

Fragment-Based Discovery of Type I Inhibitors of Maternal Embryonic Leucine Zipper Kinase

Fragment-based drug design was successfully applied to maternal embryonic leucine zipper kinase (MELK). A low affinity (160 μM) fragment hit was identified, which bound to the hinge region with an atypical binding mode, and this was optimized using structure-based design into a low-nanomolar and cell-penetrant inhibitor, with a good selectivity profile, suitable for use as a chemical probe for elucidation of MELK biology.

Structure-Based Design of Type II Inhibitors Applied to Maternal Embryonic Leucine Zipper Kinase

A novel Type II kinase inhibitor chemotype has been identified for maternal embryonic leucine zipper kinase (MELK) using structure-based ligand design. The strategy involved structural characterization of an induced DFG-out pocket by protein-ligand X-ray crystallography and incorporation of a slender linkage capable of bypassing a large gate-keeper residue, thus enabling design of molecules accessing both hinge and induced pocket regions. Optimization of an initial hit led to the identification of a low-nanomolar, cell-penetrant Type II inhibitor suitable for use as a chemical probe for MELK.

Design and Synthesis of a Series of Piperazine‐1‐carboxamidine Derivatives with Antifungal Activity Resulting from Accumulation of Endogenous Reactive Oxygen Species

In this study, we screened a library of 500 compounds for fungicidal activity via induction of endogenous reactive oxygen species (ROS) accumulation. Structure–activity relationship studies showed that piperazine‐1‐carboxamidine analogues with large atoms or large side chains substituted on the phenyl group at the R3 and R5 positions are characterized by a high ROS accumulation capacity in Candida albicans and a high fungicidal activity. Moreover, we could link the fungicidal mode of action of the piperazine‐1‐carboxamidine derivatives to the accumulation of endogenous ROS.

Synthesis and in vitro and in vivo Antifungal Activity of the Hydroxy Metabolites of Saperconazole

Herein we describe the scalable diastereoselective and enantioselective syntheses of eight enantiomers of hydroxy metabolites of saperconazole. The in vitro antifungal activity of the eight stereoisomers (compounds 1–8) was compared against a broad panel of Candida spp. (n=93), Aspergillus spp. (n=10), Cryptococcus spp. (n=19), and dermatophytes (n=27). The four 2S isomers 1–4 of the new agent were generally slightly more active than the four 2R isomers 5–8. All eight isomers were tested in a model of experimental A. fumigatus infection in guinea pigs by intravenous inoculation of the fungal conidia. Treatment doses were 1.25 mg kg−1 and 2.5 mg kg−1 per day. Infection severity was measured in terms of mean survival time (MST) after infection and mean tissue burdens in brain, liver, spleen, and kidney at postmortem examination. Among the eight isomers, the 2S diastereomers 1–4 showed a generally higher level of activity than the 2R diastereomers 5–8, revealing compounds 1 and 4 as the most potent overall in eradicating tissue burden and MST. Compared with reference compounds itraconazole and saperconazole, the hydroxy isomers 1–8 are less potent inhibitors of the growth of A. fumigatus in vitro and of ergosterol biosynthesis in both A. fumigatus and C. albicans.

Abstract DDT02-04: A novel PRMT5 inhibitor with potent in vitro and in vivo activity in preclinical lung cancer models

PRMT5 is a type II methyltransferase that specifically adds methyl groups to arginine as a long-lasting post-translational modification. The PRMT5/MEP50 complex regulates a plethora of cellular processes, such as epigenetics and splicing, which are notable events involved in tumorigenesis. Although not frequently mutated or amplified in tumors, elevated PRMT5 protein levels in lung and hematologic cancers are correlated with poorer survival. The PRMT5 inhibitor JNJ-64619178 has been selected as a clinical candidate based on its high selectivity and potency (subnanomolar range) under different in vitro and cellular conditions, paired with favorable pharmacokinetics and safety properties. JNJ-64619178 binds into the SAM binding pocket and reaches the substrate binding pocket to inhibit PRMT5/MEP50 function in a time-dependent manner. Broad cell line panel profiling of JNJ-64619178 revealed a wide range of sensitivity, which is indicative of a genomic dependency instead of a general cytotoxic on-target consequence of PRMT5 inhibition. Further investigations indicate a synthetic lethal correlation between PRMT5 inhibition and key cancer driver pathways. JNJ-64619178, dosed orally (10 mg/kg, every day), showed selective and efficient blockage of the methylation of SMD1/3 proteins, which are crucial components of the spliceosome and substrates of PRMT5/MEP50. JNJ-64619178 also demonstrated tumor regression in a biomarker-driven human small cell lung cancer xenograft model (NCI-H1048) and prolonged tumor growth inhibition after dosing cessation. In rodent and nonrodent toxicology studies, a tolerated dose of JNJ-64619178 has been identified, with the observed toxicity consistent with on-target activity. In summary, JNJ-64619178 has a favorable preclinical package that supports clinical testing in patients diagnosed with lung cancer and hematologic malignancies. Citation Format: Dirk Brehmer, Tongfei Wu, Geert Mannens, Lijs Beke, Petra Vinken, Dana Gaffney, Weimei Sun, Vineet Pande, Jan-Willem Thuring, Hillary Millar, Italo Poggesi, Ivan Somers, An Boeckx, Marc Parade, Erika van Heerde, Thomas Nys, Carol Yanovich, Barbara Herkert, Tinne Verhulst, Marc Du Jardin, Lieven Meerpoel, Christopher Moy, Gaston Diels, Marcel Viellevoye, Wim Schepens, Alain Poncelet, Joannes T. Linders, Edward C. Lawson, James P. Edwards, Dushen Chetty, Sylvie Laquerre, Matthew V. Lorenzi. A novel PRMT5 inhibitor with potent in vitro and in vivo activity in preclinical lung cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr DDT02-04. doi:10.1158/1538-7445.AM2017-DDT02-04