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Background and purpose Depression and anxiety are common causes of disability, and innovative tools and potential pharmacological targets are actively sought for prevention and treatment. Therapeutic strategies targeting the relaxin-3 peptide or its primary endogenous receptor, RXFP3, for the treatment of major depression and anxiety disorders have been limited by a lack of compounds with drug-like properties. We proposed that a hydrocarbon-stapled mimetic of relaxin-3, when administered intranasally, might be uniquely applicable to the treatment of these disorders. Experimental approach We designed a series of hydrocarbon-stapled relaxin-3 mimetics and identified the most potent compound using in vitro receptor binding and activation assays. Further, we assessed the effect of intranasal delivery of relaxin-3 and the lead stapled mimetic in rat models of anxiety and depression. Key results We developed an i,i+7 stapled relaxin-3 mimetic that manifested a stabilized α-helical structure, proteolytic resistance, and confirmed agonist activity in receptor binding and activation in vitro assays. The stapled peptide agonist enhanced food intake after intracerebral infusion in rats, confirming in vivo activity. We showed that intranasal delivery of the lead i,i+7 stapled peptide or relaxin-3 had orexigenic effects in rats, indicating a potential clinically translatable route of delivery. Further, intranasal administration of the lead i,i+7 stapled peptide exerted anxiolytic and antidepressant-like activity in anxiety- and depression-related behaviour paradigms. Conclusions and implications Our preclinical findings demonstrate that targeting the relaxin-3/RXFP3 receptor system via intranasal delivery of an i,i+7 stapled relaxin-3 mimetic may represent an effective treatment approach for depression, anxiety, and related neuropsychiatric disorders.

Specifically blocking more than one oncogenic pathway simultaneously in a cancer cell with a combination of different drugs is the mainstay of the majority of cancer treatments. Being able to do this via two targeted pathways without inducing side effects through a general mechanism, such as chemotherapy, could bring benefit to patients. In this work we describe a new dual inhibitor of the JAK-STAT and HDAC pathways through designing and developing two types of molecule based on the JAK2 selective inhibitor XL019 and the pan-HDAC inhibitor, vorinostat. Both series of compounds had examples with low nanomolar JAK2 and HDAC1/6 inhibition. In some cases good HDAC1 selectivity was achieved while retaining HDAC6 activity. The observed potency is explained through molecular docking studies of all three enzymes. One example, 69c had 16–25 fold selectivity against the three other JAK-family proteins JAK1, JAK3 and TYK2. A number of compounds had sub-micromolar potencies against a panel of 4 solid tumor cell lines and 4 hematological cell lines with the most potent compound, 45h, having a cellular IC50 of 70 nM against the multiple myeloma cell line KMS-12-BM. Evidence of both JAK and HDAC pathway inhibition is presented in Hela cells showing that both pathways are modulated. Evidence of apoptosis with two compounds in 4 sold tumor cell lines is also presented.

Inhibition of more than one pathway in a cancer cell with a single molecule could result in better therapies with less complex dosing regimens. In this work multi-component ligands have been prepared by joining together key pharmacophores of two different enzyme inhibitors in a way which increases potency against the individual pathways. Selective JAK1/2 inhibitor, ruxolitinib (3), and pan-HDAC inhibitor vorinostat (4) were linked together by a single nitrogen atom to create a new series of compounds with very potent JAK2 and HDAC6 inhibition with selectivity against HDAC1. A preferred compound, 13b, had unprecedented sub-nanomolar JAK2 potency with an IC50 of 41 pM and a sub-nanomolar IC50 against HDAC6 of 200 pM. Binding models show a good fit into both JAK2 and HDAC6.

Inhibition of multiple signaling pathways in a cancer cell with a single molecule could result in better therapies that are simpler to administer. Efficacy may be achieved with reduced potency against individual targets if there is synergy through multiple pathway inhibition. To achieve this, it is necessary to be able to build multi-component ligands by joining together key pharmacophores in a way which maintains sufficient activity against the individual pathways. In this work, designed triple inhibiting ligands are explored aiming to block three completely different target types: a kinase (JAK2), an epigenetic target (HDAC) and a chaperone (HSP90). Although these enzymes have totally different functions they are related through inter-dependent pathways in the developing cancer cell. Synthesis of several complex multi-inhibiting ligands are presented along with initial enzyme inhibition data against 3 biological target classes of interest. A lead compound, 47, was discovered which had low micromolar activity for all 3 targets. Further development of these complex trispecific designed multiple ligands could result in a 'transient drug', an alternative combination therapy for treating cancer mediated via a single molecule.

Concomitant inhibition of multiple oncogenic pathways is a desirable goal in cancer therapy. To achieve such an outcome with a single molecule would simplify treatment regimes. Herein the core features of ruxolitinib (1), a marketed JAK1/2 inhibitor, have been merged with the HDAC inhibitor vorinostat (2), leading to new molecules that are bispecific targeted JAK/HDAC inhibitors. A preferred pyrazole substituted pyrrolopyrimidine, 24, inhibits JAK1 and HDACs 1, 2, 3, 6, and 10 with IC50 values of less than 20 nM, is

Analysis of interactions between molecules is of fundamental importance in life science research. In this study, we applied weak affinity chromatography, based on high-performance liquid chromatography and mass spectrometry, as a powerful tool for direct analysis of the components of a chemical reaction mixture for their binding to a target protein. As a demonstration of the potential of this method, we analyzed the binding of the compounds of the reaction mixture to the chaperone heat shock protein 90 (Hsp90). It was possible to analyze quantitatively the binding of the components of the mixture to the target independently from each other without any preceding process such as purification. This feature has wide implications in biological sciences as crude mixtures, either natural or synthetic, can be analyzed directly for their possible binding to a target. This method could lead to savings in costs and labor through shortening chemical research project development time.

Herein, we report the discovery of a dual histone deacetylase inhibitor displaying a unique HDAC3/6 selectivity profile. An initial strategy to merge two epigenetic pharmacophores resulted in the discovery of potent HDAC6 inhibitors with selectivity over HDAC1. Screening in an HDAC panel revealed additional low nanomolar inhibition only against HDAC3. Low micromolar antiproliferative activities against two breast cancer and four hematological cancer cell lines was supported by pharmacodynamic studies on a preferred molecule, 24c, substantiating the HDAC inhibitory profile in cells. Apoptosis was identified as one of the main cell death pathways. Modelling studies of 24c against HDAC1,2,3 and 6 further provided insights on the orientation of specific residues relevant to compound potency, explaining the observed HDAC3/6 selectivity. A subset of the compounds also exhibited good antimalarial activities, particularly against the chloroquine-resistant strain K1 of P.falciparum. In vitro studies revealed a favourable DMPK profile warranting further investigation of the therapeutic potential of these compounds.

Methyltransferase enzymes are promising epigenetic oncotargets. Recent efforts toward the development of inhibitors of two methyltransferases, EZH2 and G9a, as potential anticancer therapies are reviewed with a focus on the structure–activity relationships of compounds published from 2012. Benzamide-substituted 2-pyridones are still by far the most popular selective EZH2 inhibitor class but alternative classes are now being reported. There are now three EZH2 inhibitors in clinical development with the first responses in lymphoma patients with tazemetostat. Potent inhibitors of G9a are also published but no examples have yet reached the clinic. Dual blockage of EZH2–G9a is exemplified by one series of compounds. We conclude this review by presenting the three clinical stage compounds with the first clinical response data.

Selective targeting of cancer cells over normal cells is a key objective of targeted therapy. However few approaches achieve true mechanistic selectivity resulting in debilitating side effects and dose limitation. In this work we describe the discovery of A131 (4a), a new agent with an unprecedented dual mechanism of action targeting both mitosis and autophagy. Compound 4a was first identified in a phenotypic screen in which HeLa cells treated with 4a manifested mitotic arrest along with formation of multiple vesicles. Further investigations showed that 4a causes an increase in mitotic marker pH3 and autophagy marker LC3. Importantly 4a induces cell death in cancer cells while sparing normal cells which regrow after 4a is removed. Dual activities against pH3 and LC3 markers are required for cancer cell selectivity. An extensive SAR investigation confirmed 4a as the optimal dual inhibitor with potency against a panel of 30 cancer cell lines (average antiproliferative GI50 1.5 μM). In a mouse model of paclitaxel-resistant colon cancer, 4a showed 74% tumor growth inhibition when administered at a dose of 20 mg/kg IP twice a day.

Chemotherapy remains the most prescribed anti-cancer therapy, despite patients suffering severe side effects and frequently developing chemoresistance. These complications can be partially overcome by combining different chemotherapeutic agents that target multiple biological pathways. However, selecting efficacious drug combinations remains challenging. We previously used fission yeast Schizosaccharomycespombe as a surrogate model to predict drug combinations, and showed that suberoylanilide hydroxamic acid (SAHA) and cisplatin can sensitise gastric adenocarcinoma cells toward the cytotoxic effects of doxorubicin. Yet, how this combination undermines cell viability is unknown. Here, we show that SAHA and doxorubicin markedly enhance the cleavage of two apoptosis markers, caspase 3 and poly-ADP ribose polymerase (PARP-1), and increase the phosphorylation of γH2AX, a marker of DNA damage. Further, we found a prominent reduction in Ser485 phosphorylation of AMP-dependent protein kinase (AMPK), and reductions in its target mTOR and downstream ribosomal protein S6 phosphorylation. We show that SAHA contributes most of the effect, as confirmed using another histone deacetylase inhibitor, trichostatin A. Overall, our results show that the combination of SAHA and doxorubicin can induce apoptosis in gastric adenocarcinoma in a synthetically lethal manner, and that fission yeast offers an efficient tool for identifying potent drug combinations against human cancer cells.

Blockage of more than one oncoprotein or pathway is now a standard approach in modern cancer therapy. Multiple inhibition is typically achieved with two or more drugs. Herein, we describe a pharmacophore merging strategy combining the JAK2/FLT3 inhibitor pacritnib with the pan-HDAC inhibitor, vorinostat, to create bispecific single molecules with both JAK and HDAC targeted inhibition. A preferred ether hydroxamate, 51, inhibits JAK2 and HDAC6 with low nanomolar potency, is100 nM potent against HDACs 2 and 10, submicromolar potent against HDACs 1, 8, and 11, and50-fold selective for JAK2 in a panel of 97 kinases. Broad cellular antiproliferative potency is supported by demonstration of JAK-STAT and HDAC pathway blockade in several hematological cell lines, inhibition of colony formation in HEL cells, and analysis of apoptosis. This study provides new tool compounds for further exploration of dual JAK-HDAC pathway inhibiton achieved with a single molecule.

Hydrogen sulfide (H 2 S) has been investigated for its potential in therapy. Recently, we reported novel H 2 S donor molecules based on a thiophosphorus core, which slowly release H 2 S and have improved anti-proliferative activity in cancer cell lines compared to the most widely studied H 2 S donor GYY4137 ( 1 ). Herein, we have prepared new thiophosphorus organic H 2 S donors with different ring sizes and evaluated them in two solid tumor cell lines and one normal cell line. A seven membered ring compound, 17 , was found to be the most potent with sub-micromolar IC 50s in breast (0.76 μM) and ovarian (0.76 μM) cancer cell lines. No significant H 2 S release was detected in aqueous solution for this compound. However, confocal imaging showed that H 2 S was released from 17 inside cells at a similar level to the widely studied H 2 S donor GYY4137, which was shown to release 10 μM H 2 S after 12 h at a concentration of 400 μM. Comparison of 17 with its non-sulfur oxygen analogue, 26 , provided evidence that the sulfur atom is important for its potency. However, the significant potency observed for 26 (5.94–11.0 μM) indicates that the high potency of 17 is not entirely due to release of H 2 S. Additional mechanism(s) appear to be responsible for the observed activity, hence more detailed studies are required to better understand the role of H 2 S in cancer with potent thiophosphorus agents.

Exogenous hydrogen sulfide (H2S) is known to exert anti-inflammatory effects both in macrophages and in animal models. In this study, we first showed that NaHS caused a concentration dependent reduction in TNFα and IL-6 secretion in LPS-stimulated RAW264.7 macrophages in the absence of cell death. Thereafter, we screened a series of novel slow H2S donors for similar activity. One such compound, FW1256, concentration dependently decreased TNFα, IL-6, PGE2 and NO generation in LPS-stimulated RAW264.7 macrophages and BMDMs. FW1256 also significantly reduced IL-1β, COX-2 and iNOS mRNA and protein in LPS-stimulated RAW264.7 macrophages. Mechanistically, FW1256 decreased NFκB activation as evidenced by reduced cytosolic phospho-IκBα levels and reduced nuclear p65 levels in LPS-stimulated RAW264.7 macrophages treated with FW1256. Using a H2S fluorescent probe in FW1256-treated RAW264.7 macrophages, H2S release from FW1256 was apparent over a period of 24h in these cells. Moreover, the effect of FW1256 on TNFα and IL-6 by FW1256 in LPS-stimulated RAW264.7 macrophages was reversed by treatment with the H2S scavenger, vitamin B12a. FW1256 had no cytotoxic effect on LPS-stimulated RAW264.7 macrophages or BMDMs. In vivo, FW1256 administration also reduced IL-1β, TNFα, nitrate/nitrite and PGE2 levels in LPS-treated mice. We show here a novel slow H2S-releasing compound that exerts anti-inflammatory effects in macrophages and in vivo. FW1256 may be a useful tool to study the biological effects of exogenous H2S and could also have future therapeutic value in inflammatory conditions.

A high-throughput screen against Aurora A kinase revealed several promising submicromolar pyrimidine-aniline leads. The bioactive conformation found by docking these leads into the Aurora A ATP-binding site had a semicircular shape. Macrocycle formation was proposed to achieve novelty and selectivity via ring-closing metathesis of a diene precursor. The nature of the optimal linker and its size was directed by docking. In a kinase panel screen, selected macrocycles were active on other kinase targets, mainly FLT3, JAK2, and CDKs. These compounds then became leads in a CDK/FLT3/JAK2 inhibitor project. Macrocycles with a basic nitrogen in the linker form a salt bridge with Asp86 in CDK2 and Asp698 in FLT3. Interaction with this residue explains the observed selectivity. The Asp86 residue is conserved in most CDKs, resulting in potent pan-CDK inhibition by these compounds. Optimized macrocycles generally have good DMPK properties, and are efficacious in mouse models of cancer. Compound 5 (SB1317/TG02), a pan-CDK/FLT3/JAK2 inhibitor, was selected for preclinical development, and is now in phase 1 clinical trials.

Small molecules offer powerful ways to alter protein function. However, most proteins in the human proteome lack small-molecule probes, including the large class of non-catalytic transmembrane receptors, such as death receptors. We hypothesized that small molecules targeting the interfaces between transmembrane domains (TMDs) in receptor complexes may induce conformational changes that alter receptor function. Applying this concept in a screening assay, we identified a compound targeting the TMD of death receptor p75NTR that induced profound conformational changes and receptor activity. The compound triggered apoptotic cell death dependent on p75NTR and JNK activity in neurons and melanoma cells, and inhibited tumor growth in a melanoma mouse model. Due to their small size and crucial role in receptor activation, TMDs represent attractive targets for small-molecule manipulation of receptor function.

Resistance to antimalarial therapies, including artemisinin, has emerged as a significant challenge. Reversal of acquired resistance can be achieved using agents that resensitize resistant parasites to a previously efficacious therapy. Building on our initial work describing novel chemoreversal agents (CRAs) that resensitize resistant parasites to chloroquine (CQ), we herein report new hybrid single agents as an innovative strategy in the battle against resistant malaria. Synthetically linking a CRA scaffold to chloroquine produces hybrid compounds with restored potency toward a range of resistant malaria parasites. A preferred compound, compound 35, showed broad activity and good potency against seven strains resistant to chloroquine and artemisinin. Assessment of aqueous solubility, membrane permeability, and in vitro toxicity in a hepatocyte line and a cardiomyocyte line indicates that compound 35 has a good therapeutic window and favorable drug-like properties. This study provides initial support for CQ-CRA hybrid compounds as a potential treatment for resistant malaria.

Many diverse, sensitive and structurally novel fluorescent probes have recently been reported for H2S detection. Quantification of H2S requires a selective chemosensor which will react only with H2S against a background of high concentrations of other thiols or reducing agents. Most published probes are able to quantify H2S selectively in a simple in vitro system with the most sensitive probes able to detect H2S at below 100 nM concentrations. A subset of probes also have utility in sensing H2S in living cells, and there are now several with specific sub-cellular localization and a few cases of in vivo applications. Biologists studying H2S now have a wide range of tools to assist them to aid further understanding of the role of H2S in biology.

Hydrogen sulfide (H2S) is now recognized as a physiologically important gasotransmitter. Compounds which release H2S slowly are sought after for their potential in therapy. Herein the synthesis of a series of phosphordithioates based on 1 (GYY4137) are described. Their H2S release profiles are characterized using 2,6-dansyl azide (2), an H2S specific fluorescent probe. Most compounds have anticancer activity in several solid tumor cell lines and are less toxic in a normal human lung fibroblast, WI38. A preferred compound, 14, with 10-fold greater anticancer activity than 1, was shown to release H2S in MCF7 cells using a cell active probe, 21. Both permeability and intracellular pH (pHi) were found to be significantly improved for 14 compared to 1. Furthermore, 14 was also negative in the AMES test for genotoxicity. Cyclization of these initial structures gave a series of 2,3-dihydro-2-phenyl-2-sulfanylenebenzo[d][1,3,2]oxazaphospholes, of which the simplest member, compound 22 (FW1256), was significantly more potent in cells. The improved therapeutic window of 22 in WI38 cells was compared with three other cell types. Potency of 22 was superior to 1 in MCF7 tumor spheroids and the mechanism of cell death was shown to be via apoptosis with an increase in cleaved PARP and activated caspase-7. Evidence of H2S release in cells is also presented. This work provides a "toolbox" of slow-release H2S donors useful for studies of H2S in biology and as potential therapeutics in cancer, inflammation, and cardiovascular disease.

Hydrogen sulfide (H2S) is now recognized as the so called "third gasotransmitter" taking its place alongside nitric oxide and carbon monoxide. In recent years, H2S has been reported to exhibit a diverse range of pharmacological effects in biological systems. Much of this evidence is derived from a combination of conventional pharmacological and genetic approaches coupled with the use of chemical compounds such as sodium hydrosulfide, a rapid H2S releasing donor. Developments in the design of new drug entities which attempt to take into account physicochemical properties, targeting to specific cellular organelles, triggering of H2S release upon specific chemical reactions in the cell, and controlling the release of H2S over extended periods of time have been described. For most of these molecules, little or no work has been conducted to determine their biological activity or possible therapeutic effects. It is therefore not clear whether such molecules have therapeutic potential which highlights the need for further in vivo studies. One exception to the general rule is GYY4137 (morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate), a slow releasing H2S donor, which has been evaluated for activity in a range of pharmacological models both in vitro and in vivo. GYY4137 was first reported to release H2S and exhibit vasodilator activity over 5 years ago and, to date, GYY4137 is becoming increasingly employed as a pharmacological "tool" to explore the biological functions of H2S.

Many diverse, sensitive and structurally novel fluorescent probes have recently been reported for H2S detection. Quantification of H2S requires a selective chemosensor which will react only with H2S against a background of high concentrations of other thiols or reducing agents. Most published probes are able to quantify H2S selectively in a simple in vitro system with the most sensitive probes able to detect H2S at below 100 nM concentrations. A subset of probes also have utility in sensing H2S in living cells, and there are now several with specific sub-cellular localization and a few cases of in vivo applications. Biologists studying H2S now have a wide range of tools to assist them to aid further understanding of the role of H2S in biology.

Consisting of four members, JAK1, JAK2, JAK3 and TYK2, the JAK kinases have emerged as important targets for proliferative and immune-inflammatory disorders. Recent progress in the discovery of selective inhibitors has been significant, with selective compounds now reported for each isoform. This article summarizes the current state-of-the-art with a discussion of the most recently described selective compounds. X-ray co-crystal structures reveal the molecular reasons for the observed biochemical selectivity. A concluding analysis of JAK inhibitors in the clinic highlights increased clinical trial activity and diversity of indications. Selective JAK inhibitors, as single agents or in combination regimens, have a very promising future in the treatment of oncology, immune and inflammatory diseases.

Phosphoinositide 3-kinases (PI3Ks) and the mammalian target of rapamycin (mTOR) act as critical effectors in a commonly deregulated cell signaling pathway in human cancers. The abnormal activation of the PI3K/mTOR pathway has been shown to play a role in initiation, progression, and metastasis of human tumors. Being one of the most frequently activated pathways in cancer, much effort has been directed toward inhibition of the PI3K/mTOR pathway as a novel oncology therapy. Previous work by a number of groups has revealed several selective PI3K and dual mTOR/PI3K inhibitors. However, there are few reports of therapeutic agents with a pan-PI3K/mTOR inhibitory profile within a narrow concentration range. We therefore initiated a drug discovery project with the aim of discovering dual mTOR/PI3K inhibitors which would equipotently inhibit the 4 isoforms of PI3K, α, β, γ, and δ, and mTOR a compelling profile for powerful blockage of the PI3K/mTOR pathway. A pharmacophore model was generated and used for designing a series of novel compounds, based on a purine scaffold, which potently inhibited mTOR and PI3Ks. These compounds contained a phenol headgroup essential for binding to the target proteins. Early efforts concentrated on finding replacements for the phenol as it was rapidly conjugated resulting in a short half-life in vivo. Compounds with a variety of headgroups were docked into the PI3Kα and mTOR ATP-binding sites, and aminopyrimidine and aminopyrazine were found to make excellent phenol replacements. Further structure guided optimization of side chains in the 8- and 9-positions of the purine resulted in potent inhibitors with good PKDM properties. As the PI3 kinases play a role in insulin signaling, it is believed that targeting mTOR selectively may give the benefit of blocking the AKT-pathway while avoiding the potential side effects associated with PI3K inhibition. As a result we designed a further series of selective mTOR kinase inhibitors. The project was successfully concluded by progressing both a dual mTOR/PI3K inhibitor, SB2343, and a selective mTOR inhibitor, SB2602, into preclinical development. SB2343 has since entered phase 1 clinical development as VS-5584.

Janus kinases (JAKs) comprise a family of four enzymes, JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2), centrally implicated in cell signaling processes important in cancer and immune-inflammatory diseases. Progression in the field has taken a recent step forward with the approval of ruxolitinib (Jakafi), a selective inhibitor of JAK1/2 and very recently tofacitinib (Xeljanz), a pan-JAK inhibitor. There are many new JAK family enzyme inhibitors in the clinic now with a range of selectivity profiles. More selective JAK2 or JAK3 compounds are now coming through in considerable numbers and this review attempts to provide an update of the recent patent literature of those new compounds. An overview is given on the diversity of core structures employed for inhibitor design showing that the vast majority of compounds are based on classic ATP-competitive kinase inhibitor heterocycles.This review updates new patents claiming JAK2 and/or JAK3 inhibitors published from 2010 to 2012. Pre-2010 patents have been extensively covered in previous reviews. Comments on the context of each chemical series are given where applicable to orientate the readers on the bewildering array of molecular designs now available. This review does not cover JAK1 or TYK2 inhibitors but mention is made of these where they occur within series of JAK2/3 inhibitors. Given the overlap between many pharmacophores, it was not possible to completely separate inhibitors of JAK2 from JAK3, hence the material is organized by JAK2, JAK3 and JAK2/3 and within each section by alphabetical order of the patent assignee, some companies having published five or more patents, such as Ambit (10), Incyte (9), Galapagos (7), Almirall (6) and Biocryst (5). A total of 98 patents are reviewed herein.JAK inhibitor therapy is entering a significant new era with the advent on the market of the JAK1/2 inhibitor ruxolitinib and the pan-JAK inhibitor tofacitinib, with unprecedented speed of development. Selectivity against the four individual JAK family enzymes, JAK1, 2, 3 and TYK2, is now a key goal since they each play subtly different roles in cytokine-induced cell signaling. The future looks bright for patients as many new drugs are being developed and now combinations of JAK inhibitors with other targeted agents are being studied in the clinic. These advances are expected to lead to further significant progress improving patient outcomes and quality of life.

Herein, we describe the synthesis and SAR of a series of small molecule macrocycles that selectively inhibit JAK2 kinase within the JAK family and FLT3 kinase. Following a multiparameter optimization of a key aryl ring of the previously described SB1518 (pacritinib), the highly soluble 14l was selected as the optimal compound. Oral efficacy in the murine collagen-induced arthritis (CIA) model for rheumatoid arthritis (RA) supported 14l as a potential treatment for autoimmune diseases and inflammatory disorders such as psoriasis and RA. Compound 14l (SB1578) was progressed into development and is currently undergoing phase 1 clinical trials in healthy volunteers.

Macrocycles from our Aurora project were screened in a kinase panel and were found to be active on other kinase targets, mainly JAKs, FLT3 and CDKs. Subsequently these compounds became leads in our JAK2 project. Macrocycles with a basic nitrogen in the linker form a salt bridge with Asp86 in CDK2 and Asp698 in FLT3. This residue is conserved in most CDKs resulting in potent pan CDK inhibition. One of the main project objectives was to achieve JAK2 potency with 100-fold selectivity against CDKs. Macrocycles with an ether linker have potent JAK2 activity with the ether oxygen forming a hydrogen bond to Ser936. A hydrogen bond to the equivalent residues of JAK3 and most CDKs cannot be formed resulting in good selectivity for JAK2 over JAK3 and CDKs. Further optimization of the macrocyclic linker and side chain increased JAK2 and FLT3 activity as well as improving DMPK properties. The selective JAK2/FLT3 inhibitor 11 (Pacritinib, SB1518) has successfully finished phase 2 clinical trials for myelofibrosis and lymphoma. Another selective JAK2/FLT3 inhibitor, 33 (SB1578), has entered phase 1 clinical development for the non-oncology indication rheumatoid arthritis.

A high-throughput screening campaign resulted in the discovery of a highly potent dual cannabinoid receptor 1 (CB1) and 2 (CB2) agonist. Following a thorough SAR exploration, a series of selective CB2 full agonists were identified.

The effect of hydrogen sulfide (H2S) on differentiation of 3T3L1-derived adipocytes was examined. Endogenous H2S was increased after 3T3L1 differentiation. The expression of the H2S-synthesising enzymes, cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), was increased in a time-dependent manner during 3T3L1 differentiation. Expression of genes associated with adipogenesis related genes including fatty acid binding protein 4 (FABP4/aP2), a key regulator of this process, was increased by GYY4137 (a slow-releasing H2S donor compound) and sodium hydrosulfide (NaHS, a classical H2S donor) but not by ZYJ1122 or time-expired NaHS. Furthermore expression of these genes were reduced by aminooxyacetic acid (AOAA, CBS inhibitor), DL-propargylglycine (PAG, CSE inhibitor) as well as by CSE small interference RNA (siCSE) and siCBS. The size and number of lipid droplets in mature adipocytes was significantly increased by both GYY4137 and NaHS, which also impaired the ability of CL316,243 (β3-agonist) to promote lipolysis in these cells. In contrast, AOAA and PAG had the opposite effect. Taken together, we show that the H2S-synthesising enzymes CBS, CSE and 3-MST are endogenously expressed during adipogenesis and that both endogenous and exogenous H2S modulate adipogenesis and adipocyte maturation.

Aims Whether hydrogen sulfide (H2S) exhibits pro- or anti-inflammatory activity is not clear [1] . To date, ‘fast-releasing H2S donors’ like NaHS (release in seconds) and ‘slow-releasing’ H2S donors like GYY4137 (release in hours) have been examined for their effect on inflammation. We have previously shown that GYY4137 exhibited anti-inflammatory properties (c.f. NaHS [2] ). We report here the effect of a range of novel, slow-releasing H2S donors on lipopolysaccharide (LPS) challenged macrophages in vitro. Methods Mouse macrophages (RAW264.7) were pretreated (30 min) with slow-releasing H2S donor compounds prior to 24 h stimulation with Escherichia coli LPS. Supernatant was assayed for inflammatory markers. Drug toxicity was determined by MTT assay. Results 11 novel H2S donor compounds with different H2S release rates were screened for anti-inflammatory activity in LPS-challenged macrophages. Of these, FW1256 reduced TNF α and IL-6 generation. Structural derivatives of the parent compound FW1251 & FW1259 with lower/higher H2S release rates in vitro were synthesized. These compounds not only reduced TNF α and IL-6 generation but also PGE2 and NO and were not toxic. Expression of IL-1 β , COX-2 and iNOS genes were also reduced. Using a H2S specific fluorescent-detection probe, FW1251 released >50% of its H2S payload in 3 days whereas FW1256 released approximately 15% of its payload. As proof of concept that sustained H2S administration elicits an anti-inflammatory effect, repeated dosing (every 2 h for 6 h) of macrophages with NaHS significantly inhibited LPS-evoked TNF α and IL-6 generation. Conclusion These novel H2S donor drugs are non-toxic and show promising anti-inflammatory activity in vitro, warranting further evaluation of their effects in suitable in vivo models.

It has previously been reported that hydrogen sulfide (H2S) exerts both pro- and anti-apoptotic activity in different cultured cells [1] , [2] , [3] . However, the precise effect and mechanism(s) involved remain unclear. We have previously reported that the slow-releasing H2S donor, GYY4137 [4] (morpholin-4-ium 4 methoxyphenyl(morpholino) phosphinodithioate) exerts anti-cancer activity both in vivo and in vitro [5] . In this report, GYY4137 showed a distinct anti-proliferative effect across a broad range of cancer cell lines but did not affect proliferation in normal cell lines. In contrast, NaHS, which releases large amounts of H2S over a short time course, failed to affect proliferation of either cancer or normal cell lines. This work raised the possibility that H2S donors, with differing H2S-releasing kinetics, may be of value to kill cancer (but not normal) cells. To this end, in preliminary compound screening tests, we examined a number of novel compounds all of which are GYY4137 analogues, for the ability to kill both cancer and normal cell lines. We also determined the H2S-releasing profiles of these compounds using the conventional methylene blue spectrophotometric assay to monitor H2S release. In the course of this study, we identified an additional, slow-releasing H2S donor, FW1010. FW1010 can slow release H2S more than GYY4137 over a few days. Incubation of either FW1010 or GYY4137 (400μM) in culture medium led to the generation of low concentrations of H2S over 6 days. Comparing FW1010 to GYY4137, FW1010 exhibited enhanced, concentration-dependent killing of different human cancer cell lines (e.g. MCF7, HepG2, U2OS, HL-60, MDA-MB-231, SKOV3, and OVCAR10) but had little or only minimal killing effect in a normal cell line (WI38).We propose that the ability of GYY4137 and FW1010 to kill cancer cells in vitro is likely due to their ability to release H2S slowly over a period of several days. The precise mechanism of action is not clear but measuring intracellular pH (pHi) by confocal microscopy we noted that FW1010 treatment caused a significant concentration-dependent, decline in intracellular pH in MCF7 cells and that this effect was greater than that of GYY4137 at identical concentrations. In contrast, neither GYY4137 nor FW1010 affected pHi in a normal cell line (i.e. WI38). Moreover, both GYY4137 and FW1010 significantly increased lactate release and glucose uptake in a concentration dependent manner in MCF7 (but not in W138) cells. FW1010 was more potent than GYY4137 in this regard. These experiments provide further evidence of the anti-cancer effect of GYY4137 in vitro and identify a new analogue of this compound with seemingly greater efficacy. Additional studies are ongoing to define further the possible mechanisms of action of both FW1010 and GYY4137.

Introduction: During our quest to find novel kinase inhibitor motifs for our protein kinase research projects we discovered a novel series of macrocyclic structures which possess unique but tunable kinase inhibitory profiles. Here we present the chemical structure of SB1317 and the SAR studies leading to the discovery of this novel CDK/FLT3/JAK2 inhibitor, currently in clinical trials for hematological malignancies. Methods/Results: The unusual structure of SB1317 is characterized by a biaryl connection to the hinge-binding 2-anilinopyrimidine and an allylic tertiary amine – homo allylic ether linker forming an 18-membered macrocyclic ring. Combination of the biaryl motif and unsaturation in the linker confers limited conformational freedom and may explain the specific biological activity observed with this series. Following extensive SAR studies a single preferred compound, SB1317, emerged with potent CDK (IC50 against CDKs 1, 2 and 9 = 9, 5 and 3 nM, respectively), FLT3 (IC50 = 19nM) and JAK2 (IC50 = 19nM) potency. SB1317 has low molecular weight and good physicochemical properties conferring good oral exposure and prompting its further evaluation in vitro and in in vivo cancer models. Following promising efficacy data in these studies, SB1317 was nominated as the preferred candidate for progression to the clinic. SB1317 was synthesized via a ring-closing metathesis (RCM) strategy where the macrocycle was formed from coupling the two halves of the molecule to provide a diene which was then closed by RCM using either Grubbs or Zhan catalysts. Crucially the RCM reaction requires acidic conditions to neutralize the basic centre to allow the metathesis to proceed. Conclusions: SB1317 is a low molecular weight compact macrocyclic structure with a unique spectrum of kinase activity suggesting it may have particular utility in the treatment of hematological malignancies and potential for solid tumor therapy as well. SB1317 is currently being developed in the clinic by Tragara Pharmaceuticals (San Diego, CA) under the name TG02. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3591. doi:10.1158/1538-7445.AM2011-3591

Introduction: Janus kinase 2 (JAK2) and fms-related tyrosine kinase 3 (FLT3) are important tyrosine kinases in hematopoietic progenitor cell differentiation and proliferation. Aberrant signaling of JAK2 or FLT3 is implicated in various myeloid malignancies. SB1518, a new chemical entity, is an inhibitor of both JAK2 (IC50 = 22 nM) and its JAK2V617F mutant (IC50 = 19 nM) as well as FLT3 (IC50 = 22 nM) and its mutant FLT3D835Y (IC50 = 6 nM). It is currently being evaluated in Phase I and II clinical trials for advanced hematologic malignancies and is showing promising therapeutic effects. Here we present a pharmacological evaluation of SB1518 in in vitro and in vivo models of myeloid disease that provide a rationale for its selection for clinical development. Methods: The inhibitory effect of SB1518 on JAK2, FLT3 and STAT signaling was evaluated by Western-blot analysis in cell lines with different JAK2/FLT3 mutation status. Cell proliferation assays using the CellTiterGlo® assay were performed on a panel of different cancer cell lines and primary cells from patients. Selected cells were treated with SB1518 to determine effects on apoptosis (Caspase 3/7 activation) and cell cycle (propidium iodide stain). SET-2 cells (a megakaryoblastic cell line derived from an ET-patient bearing JAK2V617F) and MOLM-13 cells (an AML cell line bearing FLT3-ITD) were used to establish subcutaneous tumors in nude mice to test the anti-tumor efficacy of SB1518 after oral daily dosing. Results: SB1518 inhibits phosphorylation of JAK2 at Y221, FLT3 and of STAT proteins and shows anti-proliferative activity in cell lines driven by mutant or wt JAK2 or FLT3-ITD (IC50 range of 47-348 nM), leading to cell cycle arrest (in G0/1) and induction of apoptosis. Cells bearing JAK2 or FLT-3 mutations or genetic alterations leading to high expression of JAK2, are the most sensitive to the anti-proliferative effects of SB1518, as are primary cells isolated from PV or AML patients. In these cells SB1518 induces cell cycle arrest and apoptosis. SB1518 has favorable physicochemical properties and pharmacokinetics allowing oral dosing. It significantly blocks tumor growth in a subcutaneous model of megakaryoblastic AML (SET-2 cells, TGI = 61% at 150 mg/kg b.i.d.). In a model of late stage, aggressive AML (MOLM-13), SB1518 not only blocked tumor growth (TGI = 81%, at 150 mg/kg b.i.d.), but also significantly reduced lung metastasis. In both models SB1518 was very well tolerated and did not induce cytopenia. Conclusions: SB1518, a novel, orally bio-available small molecule inhibitor of JAK2/FLT3, shows promising activities in various hematological cancer cell lines as well as primary cells from patients. It is efficacious in mouse xenograft models of FLT-3 dependent MOLM-13 cells and JAK2-dependent SET-2 cells. SB1518 is currently undergoing evaluation in Phase 2 clinical trials in myelofibrosis (post-PV, post-ET and primary) and has shown encouraging clinical activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3570. doi:10.1158/1538-7445.AM2011-3570

Introduction: Here we present the unique chemical structure of SB1518 and SAR studies leading to the discovery of this exciting novel JAK2/FLT3 inhibitor, currently in clinical trials for myelofibrosis and lymphoma. Methods/Results: As part of our innovative chemical strategy towards creating novel kinase scaffolds we discovered a series of unusual small molecular weight macrocycles which were optimized for selective JAK2 inhibitory potency culminating in the discovery of SB1518, which is selective for JAK2 (IC50 = 22nM) and its V617F mutant (IC50 = 19nM) over JAK1 (IC50 > 1µM) and JAK3 (IC50 > 0.5µM). FLT3 was identified as an additional kinase target (IC50 = 22nM and 6nM for its D835Y mutant) giving a broader potential in other hematological malignancies. The exciting and novel chemical structures of these compounds led us into a full lead optimization program to take full advantage of the unique nature of these molecules. SAR studies modifying the macrocyclic linker allowed unwanted protein kinase activity to be tuned out, whilst retaining a narrow spectrum of desired JAK2 kinase activity and selectivity over JAK1 and JAK3, a distinguishing feature. SB1518 and analogues were synthesized via a ring-closing metathesis (RCM) strategy: coupling of the two halves of the molecule provided a diene which was closed by RCM using either Grubbs or Zhan catalysts. SAR investigations, led by structure-based design, influenced the choice and position of a critical solubilising side-chain. Preferred compounds had improved aqueous solubility and high permeability. SB1518 is very stable both chemically and metabolically with a long half life in human microsomal preparations. With the desired balance of potency and oral pharmacokinetics, SB1518 was selected for further profiling. SB1518 demonstrated potent activity in cell lines driven by mutant JAK2 or FLT3 and was efficacious at very well tolerated oral doses in tumor models generated with these cell lines. Based on its attractive preclinical profile, SB1518 was selected for clinical development. Conclusions: Discovery of a new series of small molecule macrocycles of a unique and highly novel chemical structure are presented as inhibitors of JAK2 and FLT3 kinases. SAR studies directed us towards an optimal linker and solubilising side chain fine-tuning the molecular structure. SB1518 was selected as a development candidate with an attractive and distinguishing JAK2 selective inhibitory profile and additional activity against FLT3. SB1518 has shown encouraging clinical activity demonstrating that its attractive preclinical profile translates to the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3564. doi:10.1158/1538-7445.AM2011-3564

Kinase inhibitors have found applications in multiple oncology settings due to their ability to target key signaling pathways in many different cancers. In general, the broad-spectrum kinase inhibitors have yielded better clinical outcomes than more selective ones because they block more than one pathway critical for tumor growth. We describe herein the pharmacological profile of TG02, a multi-kinase inhibitor being developed by Tragara Pharmaceuticals, which combines cell cycle regulatory and transcriptional CDK inhibition with activity against kinase targets important in the etiology of various leukemias. In vitro potency against the kinases was evaluated using recombinant enzymes with synthetic substrates. Potency on intracellular signaling pathways was assessed by Western blot analyses of the phosphorylated substrates in cell lysates. Functional potency was assessed using proliferation assays performed on human tumor cell lines as well as hematopoietic progenitors expanded from patients' blood. Evaluation of pharmacokinetics, pharmacodynamic biomarkers and anti-tumor efficacy was performed in nude mouse xenograft models of leukemia. TG02 was shown to be a potent inhibitor of several key CDKs (IC50 = 19, 11, 37 & 10nM vs. CDK1, 2, 7 & 9) as well as two other kinases implicated in hematological malignancies, FLT3 and JAK2, with similar potency. The CDK spectrum includes both cell cycle regulatory and transcriptional CDKs. TG02 potently inhibits proliferation across a broad panel of human liquid tumor cell lines (IC50 from 68 to 230 nM). Comparison with reference inhibitors that block only one of the kinase targets of interest demonstrated the significant added benefit of combined CDK and JAK2 targeting. Inhibition of signaling by the CDK and FLT3-JAK2 pathways in leukemia cell lines was detected at the same range (0.10 to 0.35 μM). TG02 was generally cleared from the blood within 8 hours of oral administration but was retained in tumor masses at supratherapeutic levels for 24-48 hours, depending on dose. Accordingly, pathway-related biomarkers were markedly suppressed for 24-72 hours after dosing. In a murine model of FLT3-mutated leukemia (MV4-11), TG02 induced tumor regression after oral daily dosing (40 mg/kg for 21 days). Furthermore, in an orthotopic model of AML with WT FLT3 and JAK2 (HL60), TG02 significantly prolonged median survival time (59 days vs. 40 days) after treatment at 100 mg/kg p.o. 2d on/5d off. Proliferation assays using hematopoietic progenitors expanded from patients with JAK2 mutations or FLT3 wild-type or mutant acute myeloid leukemia revealed higher efficacy of TG02 compared to reference inhibitors targeting only one of the kinases of interest, again demonstrating the benefits of co-targeting CDKs and additional kinases. TG02 is a multi-kinase inhibitor with a previously unreported spectrum of targets that shows promising preclinical activity for the treatment of hematological malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2542.

Background: Dysregulation of the PI3K signaling pathway, either through activating mutations, amplification of PI3K or deletion of PTEN has been linked to a wide range of hyperproliferative diseases. The development of small molecule modulators of key proteins in this signaling cascade has focused mainly on mTOR or PI3K. Recent investigations have shown a significant overlap in the activity of small molecule inhibitors against these two proteins. Inhibitors that are currently progressing through preclinical and early clinical development have been shown to affect both mTOR and PI3K to some extent. However, most reported inhibitors do not inhibit both of these kinases in a close dose range. Our drug discovery program has focused on equipotent inhibition of both targets. SB2312 is a novel compound with very favorable drug-like properties, giving rise to equivalent targeting of both mTOR and PI3K in cells and tumor tissue and resulting in a more efficient inhibition of this pathway compared to reference compounds currently under evaluation in clinical trials. Methods: In vitro-IC50 for mTOR and PI3K were determined using recombinant enzymes. Inhibitory activity of SB2312 on the mTOR/PI3K signaling pathway was determined by Western-blot analysis, Surefire assays, and proliferation assays in various cancer cell lines. Efficacious doses of SB2312 or reference compounds were selected after pharmacokinetic and biomarker analysis of PC3 tumor-bearing mice, treated with single oral doses. Anti-tumor efficacy of SB2312 and reference compounds was evaluated in mouse xenograft models after continuous daily oral dosing. Results: SB2312 is a potent and selective dual mTOR and PI3K inhibitor (IC50: 55 nM and 18 nM, respectively) and has no significant activity on a large panel of protein kinases or enzymes and receptors. Efficient and equivalent inhibition of mTOR and PI3K in human cell lines was demonstrated by blockade of the phosphorylation of S6KT389 (IC50= 70 nM) and AKTS473 (IC50= 21 nM). SB2312 inhibits the proliferation of a broad range of human tumor cell lines independently of their PI3K pathway mutation status (IC50= 112–882 nM) and is also effective against rapamycin-resistant cell lines. SB2312 has very favorable physicochemical and pharmacokinetic properties, with high oral bioavailability and tumor exposure. Efficient and sustained inhibition of PI3K and mTOR was demonstrated in tumor tissues from a PC3 tumor xenograft model, along with dose-dependent anti-tumor activity at very well tolerated doses. Tumor growth inhibition was 53%, 105%, and 117% at 11, 33, and 80 mg/kg SB2312, respectively, without any body weight loss at any of these doses. Summary: Herein, we report SB2312, a novel, potent and selective dual mTOR/PI3K inhibitor with excellent drug-like properties. Our studies show superior and sustained blockade of the mTOR/PI3K pathway and high antitumor efficacy at well tolerated doses. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B138.

FLT3 is the most common mutated gene in acute myeloid leukemia (AML), and FLT3 mutations are strongly correlated with poor prognosis. Small-molecule inhibitors of FLT3 kinase have been evaluated in clinical trials but with limited success due to the emergence of activating mutants other than FLT3. CDK1, 2, 4 and 6 are well-established anti-cancer targets due to their direct role in cell cycle control. CDK7 and 9 are transcriptional CDKs that are emerging anti-cancer targets as a result of recent advances in the understanding of their effects on apoptotic regulators. Recent evidence suggests that the combined targeting of CDK1, 2 and 9 enhances apoptotic killing of tumor cells. Most CDK active compounds currently in clinical development do not target all of these CDKs and have poor pharmacokinetic/pharmacodynamic properties. Compounds targeting these CDKs, in addition to FLT3, may be more effective in AML and also be effective against other hematological as well as solid tumors. SB1317 is a novel potent inhibitor of FLT3 kinase (IC50 = 45 nM) and CDK2 (IC50 = 11 nM). The CDK spectrum also includes potent inhibition of CDK1 and 9 (IC50 = 19 and 10 nM respectively). SB1317 inhibits proliferation of a broad panel of tumor cell lines, and is particularly potent against the mutant FLT3-dependent AML cell line, MV4-11 (proliferation IC50 = 18 nM). SB1317 reduced phospho-Rb and phospho-FLT3 levels in MV4-11 cells in a dose-dependent manner. It induced apoptosis in MV4-11 cells as well as in primary AML cells from patients. The pharmaceutical, metabolic and pharmacokinetic properties of SB1317 render it amenable to oral dosing. In a nude mouse subcutaneous model of AML (MV4-11), SB1317 induced complete tumor regression after oral daily dosing (40 mg/kg for 21 days). In an orthotopic model of AML (HL60), SB1317 significantly prolonged median survival time (59 days versus 40 days) after treatment at 100 mg/kg p.o. 2d on/5d off. It also showed significant anti-tumor activity in a nude mouse model of B-cell lymphoma (Ramos) with a tumor growth inhibition of 63% (15 mg/kg i.p. 5d on/5d off). These results demonstrate the therapeutic potential of this novel kinase inhibitor for the treatment of hematological malignancies.

Enantioselective [2 + 2] cycloaddition of (trimethylsilyl)ketene to aldehydes catalysed by methylalumino-imidazolines gives 3-(trimethylsilyl)oxetan-2-ones with up to 83% ee.