Henri Mattes

Identification of a potent agonist of the orphan nuclear receptor Nurr1.

The identification of potent small-molecule ligands to receptors and enzymes is one of the major goals of chemical and biological research. Two powerful tools that can be used in these efforts are combinatorial chemistry and structure-based design. Herein we address how to join these methods in a design protocol that produces a lead-finding library of compounds focused on the nuclear receptor target family, as well as a second-generation library directed against a specific member of this family for lead optimization. The nuclear receptor family is a group of structurally related transcription factors that includes receptors for steroid hormones, vitamins, and thyroid hormone, as well as orphan receptors whose cognate ligands, if any, remain to be identified. 2] The ability to respond to such chemicals empowers these receptors with the capacity to coordinate tissue patterning, differentiation, and growth in response to remotely released molecules. This makes them important potential targets for therapeutic intervention. NUR77-related protein 1 (Nurr1) is an orphan member of this superfamily that is expressed predominantly in the central nervous system. The protein exhibits a close structural relationship to the orphan receptors NUR77 and neuron-derived orphan receptor. 5] These three proteins compose the NR4 A subfamily of nuclear receptors that bind to the same cis-acting consensus sequence—the nerve growth factor inducible b-response element (NBRE)—to regulate target gene expression. The essential role of Nurr1 in dopaminergic cell development was dramatically demonstrated in mouse gene knockout experiments in which homozygous mice lacking Nurr1 failed to generate midbrain dopaminergic neurons. Nurr1 was shown to be directly involved in the regulation of genes coding for aromatic amino acid decarboxylase, tyrosine hydroxylase (TH), and the dopamine transporter. These observations triggered the hypothesis that the Nurr1 protein might play a pathophysiological role in distinct conditions ranging from multiple inflammatory responses to dopaminergic nerve function and rescue. The identification of potent and selective agonists of Nurr1 may further clarify the role of Nurr1, and open new therapeutic interventions into central nervous system disorders and inflammatory conditions. Very recently 6-mercaptopurine (6-MP) was reported as a modest nonselective agonist of Nurr1. In addition, isoxazolopyridinone-based agonists (EC50: 40–70 nm) have also been disclosed. To find new ligands for the emerging members of the nuclear receptor family, we undertook the design and synthesis of a library of molecules oriented toward this family. Similar strategies, namely the creation of libraries directed toward families of receptors, have been described recently. 12] Such strategies were used to discover potent kinase inhibitors or G-proteincoupled receptor (GPCR) ligands. However, the vast majority of published examples of the combination of combinatorial chemistry and structure-based drug design are for libraries that were designed for a single target. The choice of the benzimidazole scaffold was based on several criteria, namely: physicochemical properties, modularity, and structural overlap with known nuclear receptor ligands. This scaffold, which represents a common motif found in many compounds of medicinal interest, is present in various GPCR ligands such as angiotensin II antagonists and NK1 antagonists. It is also part of the core structure of retinoic acid receptor antagonists, thrombin inhibitors, and gp IIb/IIIa inhibitors. Because it is composed of a basic functional group, it offers the potential to make salts, which would increase the water solubility of its derivatives. Undesirable physicochemical properties have often hampered the biological testing of firstgeneration combinatorial libraries or have hampered the successful use of screening hits discovered within them. Further, the benzimidazole scaffold can be synthesized from three easily accessible building blocks, o-nitrophenylhalides, primary amines, and carboxylic acids or aldehydes. To focus this library on nuclear receptors, we superposed the benzimidazole scaffold with a set of known nuclear receptor ligands (Figure 1). This experiment defined the substitution pattern and the potential scope of the side chains needed to mimic other known nuclear receptor ligands. A trisubstituted benzimidazole 5-carboxamide structure was finally chosen as scaffold for this library (Figure 2). Manual docking experiments of the designed compounds into 3D (e.g. , estrogen receptors) and homology (e.g. , PPAR) models of nuclear receptors confirmed the choice of the benzimidazole scaffold and further defined the size and shape of the potential substitution patterns. The final building blocks were chosen to allow a systematic assembly of pharmacogenic functions such as hydrophobicity, aromaticity, basicity, acidity, and hydrophilicity. By implementing structural elements that are able to engage in all fundamental nonbonded interactions at each of the variable sites of the core, we expected to partly cover the diversity of functions that are potentially present in nuclear receptor binding sites. This selection led to a virtual library of 4608 compounds. A further selection based on the rule of five was applied to choose compounds with the best drug-like features, and a library of 3840 compounds was finally selected for synthesis. [a] C. Dubois, Dr. H. Mattes Novartis Institutes for Biomedical Research Novartis Pharma AG, WKL-122.243, 4002 Basel (Switzerland) Fax: (+41)616962455 E-mail : henri.mattes@novartis.com [b] Dr. B. Hengerer Boehringer Ingelheim Pharma GmbH&Co. KG 88397 Biberach (Germany) Supporting information for this article is available on the WWW under http://www.chemmedchem.org or from the author: Representative experimental procedures, characterization data for compounds, and a description of the cellular assay used.

Quinazolinedione sulfonamides: A novel class of competitive AMPA receptor antagonists with oral activity

Quinazoline-2,4-diones with a sulfonamide group attached to the N(3) ring atom constitute a novel class of competitive AMPA receptor antagonists. One of the synthesized compounds, 28, shows nanomolar receptor affinity, whereas other examples of the series display oral anticonvulsant activity in animal models.

Design and Synthesis of Selective and Potent Orally Active S1P5 Agonists

The immunomodulatory drug fingolimod (FTY720, 2-amino-2[2-(4-octylphenyl)ethyl]propane-1,3-diol), derived from a fungal metabolite (ISP-1, myriocin), is phosphorylated in vivo by sphingosine kinases to produce (R)-FTY720-phosphate (FTY720-P). 2] FTY720-P activates sphingosine-1-phosphate (S1P) receptors S1P1, S1P3, S1P4, and S1P5 at low nanomolar concentrations and is inactive toward the S1P2 receptor. The FTY720-P-mediated activation of the S1P1 receptor on lymphocytes induces receptor internalization, which attenuates T-cell response to S1P gradients, preventing their egress from secondary lymphoid tissues. In addition to playing a role in the immune system, all S1P receptors except S1P4 are also found differentially expressed in the central nervous system and on various tumor cell types. 6] Although the precise regulation of these receptors by locally released S1P remains unclear, S1P receptors are thought to play a role in such events as astrocyte migration, oligodendrocyte differentiation, and cell survival and neurogenesis. To assess the relevance of individual S1P receptor subtypes for the activity of FTY720-P, selective agonists are required. Because S1P5 receptors are expressed on oligodendrocytes, and S1P5 receptors are thought to play a role in oligodendrocyte differentiation and survival, we focused on the development of S1P5 agonists. By using a highthroughput screening calcium mobilization assay with GPCR priming and FLIPR technology, we discovered benzamide 1, which has good in vitro potency toward the S1P5 receptor (EC50=270 nm), but has modest selectivity against S1P1 (EC50=3140 nm) and S1P4 (EC50=100 nm). Herein we report our studies of various benzamide modifications carried out to improve the selectivity, bioactivity, pharmacokinetic properties, and ancillary profile of 1, ultimately resulting in the discovery of potent and very selective S1P5 agonists. To guide the optimization process, homology models of all S1P receptors were built from a crystal structure of bovine rhodopsin (PDB ID: 1F88). Docking experiments of 1 into these models revealed a possible location of the binding site, some essential features of the interactions, and indicated potential regions for gaining selectivity and improving potency. In these complexes (Figure 1), 1 adopts a twisted conformation with the aniline ring, ~708 out of the benzamide plane and stabilized by a hydrogen bond between the aniline NH group and the amide carbonyl. In the S1P5 receptor complex, the amide group forms a hydrogen bond with OG1-Thr120. The benzamide phenyl ring lies in a large hydrophobic pocket surrounded by Phe196, Phe201, Phe268, Leu119, Trp264, Leu267, and Leu271. The aniline ring undergoes a T-shaped interaction with Phe116 and hydrophobic contacts with Leu271 and Leu292. The ortho-methyl substituents fill a small pocket formed by Tyr89, Val115, and Leu292 on one side, and sit at the face of Phe196 on the other side. Inspection of sequence alignments (Figure 2) revealed two positions, one in transmembrane (TM) helix TM3 (115, S1P5 sequence) and one in TM5 (192), where S1P5 has smaller residues lining the binding site, thus creating putative pockets. We hypothesized that filling these pockets with atoms from our ligands should lead to high selectivity for the S1P5 receptor. Position 2 on the benzamide core, which was closest to the hypothesized pocket around Val115, was therefore extensively modified. Syntheses of derivative 1A–L (Scheme 1) began with 3-fluorobromobenzene 2, which was converted into acid 3 by reaction with lithium diisopropylamide (LDA) and carbon dioxide. Nucleophilic substitution of the fluorine atom with trimethylaniline at 78 8C yielded 4. This intermediate was then used in various ways. Copper-catalyzed nucleophilic substitution of the bromine atom with various alcohols yielded ethers 5D–N, which were amidated with ammonia using chlorodimethoxytriazine for activation to yield 1D–J. Palladium-catalyzed substitution of the bromine atom in acid 4 with various alkylstannanes yielded 6A–C, which were amidated as described above to yield 1A–C. Alternatively, palladium-catalyzed substitution of the bromine atom with tributyl-(1-ethoxyvinyl)stannane yielded 9, which was cyclized to 1L by reaction with hydrazine. Acid 4 was also amidated with allylamine, using chlorodimethoxytriazine for activation, to yield allylamide 10. Palladiumcatalyzed cyclization of this intermediate led to 1K. All compounds were assayed for S1P5 activation in GTPgS assays, which gave more reliable structure–activity results than the FLIPR assays, at concentrations up to 10 mm. EC50 values were determined for all compounds (Table 1). Disrupting the intramolecular hydrogen bond by introducing small alkyl substituents at position 2 (compounds 1A–C), led to a [a] Dr. H. Mattes, Prof. Dr. K. K. Dev, Dr. R. Bouhelal, Dr. C. Barske, Dr. F. Gasparini, Dr. D. Guerini, Dr. A. K. Mir, Dr. D. Orain, M. Osinde, A. Picard, C. Dubois, E. Tasdelen, S. Haessig Novartis Institute for Biomedical Research WKL-122 4002 Basel (Switzerland) Fax: (+41)61 696 2455 E-mail : henri.mattes@novartis.com [b] Prof. Dr. K. K. Dev Molecular Neuropharmacology, Department of Physiology Trinity College Institute of Neuroscience (TCIN) Medical School Trinity College Dublin, Dublin 2 (Ireland) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201000253.

6-Amino quinazolinedione sulfonamides as orally active competitive AMPA receptor antagonists.

A new set of quinazolinedione sulfonamide derivatives as competitive AMPA receptor antagonist with improved properties compared to 1 is disclosed. By modulating physico-chemical properties, compound 29 was identified with a low ED(50) of 5.5mg/kg in an animal model of anticonvulsant activity after oral dosage.

Discovery of novel non-peptidic β-alanine piperazine amide derivatives and their optimization to achiral, easily accessible, potent and selective somatostatin sst1 receptor antagonists

Structural simplification of the core moieties of obeline and ergoline somatostatin sst(1) receptor antagonists, followed by systematic optimization, led to the identification of novel, highly potent and selective sst(1) receptor antagonists. These achiral, non-peptidic compounds are easily prepared and show promising PK properties in rodents.

The serotonin 5-HT4 receptor: Part 3: Design and pharmacological evaluation of a new class of antagonists

Abstract The design, synthesis and pharmacological activity of a new class of potent and selective 5-HT 4 receptor antagonists containing an indole nucleus linked to a carbazimidamide are presented. 4c , a representative member of our new class is a potent competitive antagonist at 5-HT 4 receptors with a pA 2 value of 8.4, displaying selectivity (ranging from 20 to over 1000 fold) versus other serotonin receptor subtypes.

The Crystal Structure of Cancer Osaka Thyroid Kinase Reveals an Unexpected Kinase Domain Fold.

Background: Cancer Osaka thyroid (COT) kinase plays a crucial role in inflammatory diseases and cancer. Results: Production of catalytically competent COT kinase yielded protein suitable for structure guided drug discovery. Conclusion: COT kinase has a unique and structurally versatile active site. Significance: The discovery of a novel variation of the protein kinase fold will impact drug discovery for COT kinase. Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors.

Discovery of Imidazoquinolines as a Novel Class of Potent, Selective, and in Vivo Efficacious Cancer Osaka Thyroid (COT) Kinase Inhibitors.

Cancer Osaka thyroid (COT) kinase is an important regulator of pro-inflammatory cytokines in macrophages. Thus, pharmacologic inhibition of COT should be a valid approach to therapeutically intervene in the pathogenesis of macrophage-driven inflammatory diseases such as rheumatoid arthritis. We report the discovery and chemical optimization of a novel series of COT kinase inhibitors, with unprecedented nanomolar potency for the inhibition of TNFα. Pharmacological profiling in vivo revealed a high metabolism of these compounds in rats which was demonstrated to be predominantly attributed to aldehyde oxidase. Due to the very low activity of hepatic AO in the dog, the selected candidate 32 displayed significant blood exposure in dogs which resulted in a clear prevention of inflammation-driven lameness. Taken together, the described compounds both potently and selectively inhibit COT kinase in primary human cells and ameliorate inflammatory pathologies in vivo, supporting the notion that COT is an appropriate therapeutic target for inflammatory diseases.

Synthesis and Biological Evaluation of New Triazolo- and Imidazolopyridine RORγt Inverse Agonists

Retinoic‐acid‐related orphan receptor γt (RORγt) is a key transcription factor implicated in the production of pro‐inflammatory Th17 cytokines, which drive a number of autoimmune diseases. Despite diverse chemical series having been reported, combining high potency with a good physicochemical profile has been a very challenging task in the RORγt inhibitor field. Based on available chemical structures and incorporating in‐house knowledge, a new series of triazolo‐ and imidazopyridine RORγt inverse agonists was designed. In addition, replacement of the terminal cyclopentylamide metabolic soft spot by five‐membered heterocycles was investigated. From our efforts, we identified an optimal 6,7,8‐substituted imidazo[1,2‐a]pyridine core system and a 5‐tert‐butyl‐1,2,4‐oxadiazole as cyclopentylamide replacement leading to compounds 10 ((S)‐N‐(8‐((4‐(cyclopentanecarbonyl)‐3‐methylpiperazin‐1‐yl)methyl)‐7‐methylimidazo[1,2‐a]pyridin‐6‐yl)‐2‐methylpyrimidine‐5‐carboxamide) and 33 ((S)‐N‐(8‐((4‐(5‐(tert‐butyl)‐1,2,4‐oxadiazol‐3‐yl)‐3‐methylpiperazin‐1‐yl)methyl)‐7‐methylimidazo[1,2‐a]pyridin‐6‐yl)‐2‐methylpyrimidine‐5‐carboxamide). Both derivatives showed good pharmacological potencies in biochemical and cell‐based assays combined with excellent physicochemical properties, including low to medium plasma protein binding across species. Finally, 10 and 33 were shown to be active in a rodent pharmacokinetic/pharmacodynamic (PK/PD) model after oral gavage at 15 mg kg−1, lowering IL‐17 cytokine production in ex vivo antigen recall assays.

Chapter 13 Case History on Tegaserod (Zelnorm®/Zelmac®)

Publisher Summary Tegaserod (HTF919; Zelnorm ® /Zelmac ® ) is an innovative and potent partial agonist at 5-HT 4 receptors, which mediate multiple physiological functions in the gastrointestinal (GI) tract. The compound is a representative of a new class of 5-HT 4 receptor agonists, with regard to both chemistry and pharmacology. As a non-benzamide drug, it lacks 5-HT 3 receptor as well as dopamine D2 receptorblocking properties. Tegaserod has been shown to stimulate the coordinated release of neurotransmitters such as calcitonin gene-related peptide, substance P, vasoactive intestinal peptide (VIP) and acetylcholine from enteric nerves. As a result, tegaserod enhances basal motor activity and normalizes impaired motility throughout the GI tract. Preclinical investigations revealed that tegaserod can stimulate intestinal chloride/water secretion, in line with reports on functions of intestinal 5-HT 4 receptors. Importantly, preclinical and clinical studies demonstrated an attenuation of visceral sensitivity during colorectal distension following treatment with tegaserod. In conclusion, tegaserod restores GI homoeostasis via stimulation of 5-HT 4 receptors. The drug does not cause exaggerated effects associated with full agonism at 5-HT 4 receptors and minimizes receptor desensitization. Tegaserod secures a balanced modulation of 5-HT 4 receptors expressed in the GI tract and, thereby, provides a mechanism to treat functional GI disorders (FGIDs).