Search

This study aimed to develop a simple fluorescent probe for detecting glutathione (GSH) over cysteine (Cys) and homocysteine (Hcy). To this end, two fluorescent probes HPY1, 2 that used pyrazoline as the fluorophore and an acrylate group as the recognition group for thiols were designed and synthesized. HPY1, 2 possessed excellent sensitivity, high selectivity and great stability for detecting GSH. The detection limit of HPY1, 2 for GSH was found to be 2.06 × 10-7 M, 1.36 × 10-7 M respectively. The recognition mechanism of HPY1, 2 to GSH were studied by fluorescence change experiments, HRMS and DFT calculation, which elucidated that HPY1, 2 and GSH could undergo Michael addition reaction to generate strongly fluorescent addition products. Additionally, HPY1 was successfully applied to imaging of both endogenous and exogenous GSH in L929 cells based on its low cytotoxicity and good cell permeability. This wok indicated that HPY1, 2 have a great potential for application in detection of GSH.

We describe an efficient synthesis of 2,4-substituted pyrido[4,3-d]pyrimidin-5(6H)-ones, which involves the acid-promoted cyclization of cyano enamine 15 to afford 2,4-bis(methylthio)pyrido[4,3-d]pyrimidin-5(6H)-one 17 as a key intermediate. Selective displacement of the 4-methylthio group by a wide range of anilines followed by oxidation of the 2-methylthio group and subsequent substitution by amines enabled the synthesis of a variety of 2,4-disubstituted pyrido[4,3-d]pyrimidin-5(6H)-ones.

The design, synthesis, and structure-activity relationships (SAR) of a series of novel proline and pyrrolidine based melanocortin receptor (MCR) agonists are described. To validate a conformationally constrained Arg-Nal dipeptide analogue strategy, we first synthesized and evaluated a test set of cis-(2R,4R)-proline analogues (21a-g). All of these compounds showed significant binding and agonist potency at the hMC1R, hMC3R, and hMC4R. Potent cis-(2S,4R)-pyrrolidine based MCR agonists (35a-g) were subsequently developed by means of this design approach. A SAR study directed toward probing the effect of the two chiral centers in the pyrrolidine ring on biological activity revealed the importance of the (S) absolute configuration at the 2-position for binding affinity, agonist potency, and receptor selectivity. Among the four sets of the pyrrolidine diastereomers investigated, analogues with the (2S,4R) configuration were the most potent agonists across the three receptors, followed by those possessing the (2S,4S) configuration.

The first synthesis of Tic-D-Phe Psi[CH(2)-CH(2)] isostere is described, which features diastereoselective alkylation of the tricyclic lactam 14. The use of this novel dipeptide isostere in the development of melanocortin agonists has been demonstrated by the synthesis of peptidomimetic 7 and non-peptidic ligand 27. Both compounds displayed significant binding and agonist potency at the MC4R.

A series of proline based melanocortin ligands has been developed on the basis of initial piperazine leads by using a more conformationally rigid scaffold. A number of these novel ligands showed significant binding affinity for MC3 and MC4 receptors.

The synthesis of the ortho- and para-e isomers in the oxide-bridged 5-phenylmorphan series of rigid tetracyclic compounds was accomplished via rac-5-(2-fluoro-5-nitrophenyl)-2-methyl-2-azabicyclo[3.3.1]nonan-9β-ol ((±)-10), an intermediate containing an aromatic nitro-activated fluorine atom. The fluorine atom was used as the leaving group for the formation of the strained tetracyclic trans-fused 5,6-ring system in rac-(1α,4aα,9aα)-1,3,4,9a-tetrahydro-2-methyl-6-nitro-2H-1,4a-propanobenzofuro[2,3-c]pyridine ((±)-11), although preference for cis ring fusion during the formation of tricyclic tetra- and hexahydrodibenzofurans has been well-documented. Single-crystal X-ray crystallographic study of the desired para-e isomer ((±)-2), as well as of two intermediates in its synthesis, provided assurance of the correct structures. The e-isomers are among the last of the 12 oxide-bridged 5-phenylmorphans to be synthesized. We envisioned the syntheses of these rigid, tetracyclic compounds in order to determine the ...

SummaryEpigenetic dysregulation has emerged as an important mechanism in cancer. Alterations in epigenetic machinery have become a major focus for targeted therapies. The current report describes the discovery and biological activity of a cyclopropylamine containing inhibitor of Lysine Demethylase 1 (LSD1), GSK2879552. This small molecule is a potent, selective, orally bioavailable, mechanism-based irreversible inactivator of LSD1. A proliferation screen of cell lines representing a number of tumor types indicated that small cell lung carcinoma (SCLC) is sensitive to LSD1 inhibition. The subset of SCLC lines and primary samples that undergo growth inhibition in response to GSK2879552 exhibit DNA hypomethylation of a signature set of probes, suggesting this may be used as a predictive biomarker of activity.

Asymmetric Michael reaction of phosphinic or aminophosphinic acids with acrylate derivatives afforded phosphinyl dipeptidomimetics in excellent yields (>90%). Chiral induction of substituents at the α-position of acrylate derivatives of Evans oxazolidinone type auxiliaries was obtained in moderate to excellent diastereomeric and enantiomeric excesses (50–98%). Pure diastereomers and enantiomers of phosphinyl dipeptidomimetics 16 – 19 were also successfully separated by HPLC.

Whole-cell biooxidation of bromobenzene with Pseudomonas putida 39D or the recombinant Escherichia coli JM109 (pDTG601) yields (1S,2S)-3-bromocyclohexa-3,5-diene-1,2-diol (9a), which is protected as the acetonide and converted to vinylaziridines 7, 15a, 63, and 64. Our route to (+)-pancratistatin features the coupling of a higher order cyanocuprate (derived by ortho-metalation from N,N-dimethyl-2-[(tert-butyldimethylsilyl)oxy]-3,4-(methylenedioxy)benzamide) with aziridine 7 to generate 28, which contains the carbon framework of the title alkaloid. Functional group manipulations resulted in the preparation of epoxydiol 50, which was transformed in a unique fashion and under mild conditions (H2O/PhCO2Na) to (+)-pancratistatin, thus completing a concise synthesis of (+)-pancratistatin in 14 steps from bromobenzene (2% overall yield). To improve this first generation attempt, a new route was devised utilizing carbomethoxyaziridine 64 and its coupling to the cuprate of 3,4-(methylenedioxy)bromobenzene. The add...

Asymmetric Michael reaction of phosphinic or aminophosphinic acids with acrylate derivatives afforded phosphinyl dipeptidomimetics in excellent yields (>90%). Chiral induction of substituents at the α-position of acrylate derivatives of Evans oxazolidinone type auxiliaries was obtained in moderate to excellent diastereomeric and enantiomeric excesses (50–98%). Pure diastereomers and enantiomers of phosphinyl dipeptidomimetics 16 – 19 were also successfully separated by HPLC.

A study that was designed to identify plausible replacements for highly basic guanidine moiety contained in potent MC4R agonists, as exemplified by 1, led to the discovery of initial nonguanidine lead 5. Propyl analog 23 was subsequently found to be equipotent to 5, whereas analogs bearing smaller and branched alkyl groups at the 3 position of the oxopiperazine template demonstrated reduced binding affinity and agonist potency for MC4R. Acylation of the NH2 group of the 4F-D-Phe residue of 3-propyl analog 23 significantly increased the binding affinity and the functional activity for MC4R. Analogs with neutral and weakly basic capping groups of the D-Phe residue exhibited excellent MC4R selectivity against MC1R whereas those with an amino acid had moderate MC4R/MC1R selectivity. We have also demonstrated that compound 35 showed promising oral bioavailability and a moderate oral half life and induced significant weight loss in a 28-day rat obesity model.

Lysine specific demethylase 1 (LSD1) is a histone H3K4me1/2 demethylase found in various transcriptional co-repressor complexes. LSD1 mediated H3K4 demethylation can result in a repressive chromatin environment that silences gene expression and has been shown to play a role in hematopoietic differentiation. LSD1 is also overexpressed in multiple tumor types. These studies implicate LSD1 as a key regulator of the epigenome that modulates gene expression through post-translational modification of histones and its presence in transcriptional complexes. The current study describes the anti-tumor effects of a novel, irreversible, GSK LSD1 inhibitor (GSK2879552) in acute myeloid leukemia (AML) and small cell lung cancer (SCLC). GSK2879552 is a potent, selective, mechanism-based inhibitor of LSD1. Screening of over 150 cancer cell lines revealed that AML and SCLC cells have a unique requirement for LSD1. While GSK2879552 treatment did not affect the global levels of H3K4me1 or H3K4me2, local changes in these histone marks were observed near transcriptional start sites of genes whose expression increased with LSD1 inhibition. Treatment of AML cell lines with GSK2879552 increased cell surface expression of CD11b and CD86, markers associated with a differentiated immunophenotype. Six days of GSK2879552 treatment resulted in potent anti-proliferative growth effects in 19 of 25 AML cell lines representing a range of AML subtypes. Treating for longer time periods revealed sensitivity in all AML cell lines. AML blast colony forming ability was also inhibited in 4 of 5 bone marrow samples derived from primary AML patient samples. The effects of LSD1 inhibition were further characterized in vivo using a mouse model of AML induced by transduction of mouse hematopoietic progenitor cells with a retrovirus encoding MLL-AF9 and GFP. Primary AML cells were transplanted into a cohort of secondary recipient mice and were treated upon engraftment. After 17 days of treatment, control mice had 80% GFP+ cells in the bone marrow whereas treated mice had only 2.8% GFP positive cells (p Growth inhibition was also observed in a subset of SCLC cell lines. GSK2879552 treatment of mice engrafted with SCLC lines resulted in greater than 80% tumor growth inhibition. Studies using patient derived primary SCLC showed similar efficacy demonstrating the growth inhibition of SCLC with an LSD1 inhibitor extended beyond cell lines. Together, these data demonstrate that pharmacological inhibition of LSD1 may provide a promising treatment for AML and SCLC. A Phase I clinical trial using GSK2879552 was initiated in March, 2014. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed by the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. Citation Format: Kimberly Smitheman, Monica Cusan, Yan Liu, Michael Butticello, Melissa Pappalardi, James Foley, Kelly Federowicz, Glenn Van Aller, Jiri Kasparec, Xinrong Tian, Dominic Suarez, Jess Schneck, Jeff Carson, Patrick McDevitt, Thau Ho, Charles McHugh, William Miller, Scott Armstrong, Christine Hann, Neil Johnson, Ryan G. Kruger, Helai P. Mohammad, Shekhar Kamat. Inhibition of LSD1 for the treatment of cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3513. doi:10.1158/1538-7445.AM2015-3513

The EZH2 histone methyltransferase is frequently mutated in diffuse large B-cell lymphoma leading to increased trimethylation of histone H3 lysine 27 (H3K27me3). Drug discovery efforts have previously identified a pyridone-based chemical series of EZH2 inhibitors that potently and selectively inhibit EZH2 catalytic activity. These compounds are capable of globally decreasing H3K27me3 levels, de-repressing EZH2 target genes, and inducing growth inhibition of many lymphoma cell lines both in cell culture and in vivo. Through medicinal chemistry optimization, we have developed EZH2 inhibitors with significantly improved potency in both biochemical and cellular assays. These compounds exhibit a prolonged enzyme residence time that can be further extended in vitro through the addition of an H3K27me3 peptide. Herein, we report the biochemical and cellular activity of these new EZH2 inhibitors. Citation Format: Heidi Ott, Glenn van Aller, Jessica Ward, BaoChau Le, Cynthia Rominger, James Foley, Susan Korenchuk, Charles McHugh, Michael Butticello, Charles Blackledge, James Brackley, Joelle Burgess, Celine Duquenne, Neil Johnson, Jiri Kasparec, Louis LaFrance, Mei Li, Kenneth McNulty, Kenneth Newlander, Stuart Romeril, Stanley Schmidt, Mark Schulz, Dai-Shi Su, Dominic Suarez, Xinrong Tian, Christopher Carpenter, Juan Luengo, Ryan Kruger, Steven Knight, Michael T. McCabe. A potent EZH2 inhibitor exhibits long residence time and anti-tumor activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5379. doi:10.1158/1538-7445.AM2015-5379

The design and synthesis of a series of potent 1,3,4-trisubstituted-2-oxopiperazine based MC4 agonists are described. The tripeptidomimetic analogs (12a,b and 23) and the dipeptidomimetic 27 displayed single-nanomolar binding affinity and agonist potency for MC4R and excellent selectivity for MC4R relative to MC1R.

Homochiral vinyloxiranes and vinylaziridines were reacted with a series of organometallic reagents in order to determine the regio- and stereoselectivity of the ring opening with carbon nucleophiles

Lysine specific demethylase 1 (LSD1) is a histone H3K4me1/2 demethylase found in various transcriptional co-repressor complexes. These complexes include Histone Deacetylases (HDAC1/2) and Co-Repressor for Element-1-Silencing Transcription factor (CoREST). LSD1 mediated H3K4 demethylation can result in a repressive chromatin environment that silences gene expression. LSD1 has been shown to play a role in development in various contexts. LSD1 can interact with pluripotency factors in human embryonic stem cells and is important for decommissioning enhancers in stem cell differentiation. Beyond embryonic settings, LSD1 is also critical for hematopoietic differentiation. LSD1 is overexpressed in multiple cancer types and recent studies suggest inhibition of LSD1 reactivates the all-trans retinoic acid receptor pathway in acute myeloid leukemia (AML). These studies implicate LSD1 as a key regulator of the epigenome that modulates gene expression through post-translational modification of histones and through its presence in transcriptional complexes. The current study describes the anti-tumor effects of a novel LSD1 inhibitor (GSK2879552) in AML. GSK2879552 is a potent, selective, mechanism-based, irreversible inhibitor of LSD1. Screening of over 150 cancer cell lines revealed that AML cells have a unique requirement for LSD1. While LSD1 inhibition did not affect the global levels of H3K4me1 or H3K4me2, local changes in these histone marks were observed near transcriptional start sites of putative LSD1 target genes. This increase in the transcriptionally activating histone modification correlated with a dose dependent increase in gene expression. Treatment with GSK2879552 promoted the expression of cell surface markers, including CD11b and CD86, associated with a differentiated immunophenotype in 12 of 13 AML cell lines. For example, in SKM-1 cells, increases in cell surface expression of CD86 and CD11b occurred after as early as one day of treatment with EC50 values of 13 and 7 nM respectively. In a separate study using an MV-4-11 engraftment model, increases in CD86 and CD11b were observed as early as 8 hours post dosing. GSK2879552 treatment resulted in a potent anti-proliferative growth effect in 19 of 25 AML cell lines (average EC50 = 38 nM), representing a range of AML subtypes. Potent growth inhibition was also observed on AML blast colony forming ability in 4 out of 5 bone marrow samples derived from primary AML patient samples (average EC50 = 205 nM). The effects of LSD1 inhibition were further characterized in an in vivo mouse model of AML induced by transduction of mouse hematopoietic progenitor cells with a retrovirus encoding MLL-AF9 and GFP. Primary AML cells were transplanted into a cohort of secondary recipient mice and upon engraftment, the mice were treated for 17 days. After 17 days of treatment, control treated mice had 80% GFP+ cells in the bone marrow whereas treated mice possessed 2.8% GFP positive cells (p Together, these data demonstrate that pharmacological inhibition of LSD1 may provide a promising treatment for AML by promoting differentiation and subsequent growth inhibition of AML blasts. GSK2879552 is currently in late preclinical development and clinical trials are anticipated to start in 2014. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. Disclosures: Kruger: GlaxoSmithKline Pharmaceuticals: Employment. Mohammad:GlaxoSmithKline Pharmaceuticals: Employment. Smitheman:GlaxoSmithKline Pharmaceuticals: Employment. Liu:GlaxoSmithKline Pharmaceuticals: Employment. Pappalardi:GlaxoSmithKline Pharmaceuticals: Employment. Federowicz:GlaxoSmithKline Pharmaceuticals: Employment. Van Aller:GlaxoSmithKline Pharmaceuticals: Employment. Kasparec:GlaxoSmithKline Pharmaceuticals: Employment. Tian:GlaxoSmithKline Pharmaceuticals: Employment. Suarez:GlaxoSmithKline Pharmaceuticals: Employment. Rouse:GlaxoSmithKline Pharmaceuticals: Employment. Schneck:GlaxoSmithKline Pharmaceuticals: Employment. Carson:GlaxoSmithKline Pharmaceuticals: Employment. McDevitt:GlaxoSmithKline Pharmaceuticals: Employment. Ho:GlaxoSmithKline Pharmaceuticals: Employment. McHugh:GlaxoSmithKline Pharmaceuticals: Employment. Miller:GlaxoSmithKline Pharmaceuticals: Employment. Johnson:GlaxoSmithKline Pharmaceuticals: Employment. Armstrong:Epizyme Inc.: Has consulted for Epizyme Inc. Other. Tummino:GlaxoSmithKline Pharmaceuticals: Employment.