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Although multiple approaches have been suggested, treating mild-to-severe fibrosis in the context of metabolic dysfunction associated with liver disease (MASLD) remains a challenging area in drug discovery. Pathogenesis of liver fibrosis is multifactorial, and pathogenic mechanisms are deeply intertwined; thus, it is well accepted that future treatment requires the development of multitarget modulators. Harnessing the 3,4,5-trisubstituted isoxazole scaffold, previously described as a key moiety in Farnesoid X receptor (FXR) agonism, herein we report the discovery of a novel class of hybrid molecules endowed with dual activity toward FXR and the leukemia inhibitory factor receptor (LIFR). Up to 27 new derivatives were designed and synthesized. The pharmacological characterization of this series resulted in the identification of 3a as a potent FXR agonist and LIFR antagonist with excellent ADME properties. In vitro and in vivo characterization identified compound 3a as the first-in-class hybrid LIFR inhibitor and FXR agonist that protects against the development of acute liver fibrosis and inflammation.

The sphingosine-1-phosphate-1 (S1P 1 ) receptor agonists have great potential for the treatment of multiple sclerosis (MS) because they can inhibit lymphocyte egress through receptor internalization. We designed and synthesized triazole and isoxazoline derivatives to discover a novel S1P 1 agonist for MS treatment. Of the two scaffolds, the isoxazoline derivative was determined to have excellent in vitro efficacy and drug-like properties. Among them, compound 21l was found to have superior drug-like properties as well as excellent in vitro efficacies (EC 50 = 7.03 nM in β-arrestin recruitment and EC 50 = 11.8 nM in internalization). We also confirmed that 21l effectively inhibited lymphocyte egress in the peripheral lymphocyte count test and significantly improved the clinical score in the experimental autoimmune encephalitis MS mouse model.

The inhibition of the nuclear receptor retinoic-acid-receptor-related orphan receptor γt (RORγt) is a promising strategy in the treatment of autoimmune diseases. RORγt features an allosteric binding site within its ligand-binding domain that provides an opportunity to overcome drawbacks associated with orthosteric modulators. Recently, trisubstituted isoxazoles were identified as a novel class of allosteric RORγt inverse agonists. This chemotype offers new opportunities for optimization into selective and efficacious allosteric drug-like molecules. Here, we explore the structure-activity relationship profile of the isoxazole series utilizing a combination of structure-based design, X-ray crystallography, and biochemical assays. The initial lead isoxazole ( FM26 ) was optimized, resulting in compounds with a ∼10-fold increase in potency (low nM), significant cellular activity, promising pharmacokinetic properties, and a good selectivity profile over the peroxisome-proliferated-activated receptor γ and the farnesoid X receptor. We envisage that this work will serve as a platform for the accelerated development of isoxazoles and other novel chemotypes for the effective allosteric targeting of RORγt.

Two series of saccharin/isoxazole and saccharin/isoxazoline hybrids were synthesized by 1,3-dipolar cycloaddition. The new compounds showed to be endowed with potent and selective inhibitory activity against the cancer-related human carbonic anhydrase (hCA) IX and XII isoforms in the nanomolar range, while no affinity was encountered for off-targets, such as hCA I and II. Successive enantioseparation on a milligram scale of the most representative compounds led to the discovery that (S)-isomers were more potent than their corresponding (R)-enantiomers. Lastly, molecular modeling studies were conducted to define those structural requirements that were responsible for the discrimination among selected human isoforms of carbonic anhydrases. Two nanomolar hCA IX and XII inhibitors were also screened for their selective toxicity against non tumoral primary cells (fibroblasts) and against a breast adenocarcinoma cell line (MCF7) in hypoxic environment. The efficacious combination of these compounds with doxorubicin on MCF7 cells was demonstrated after 72 h of treatment.

Retinoic acid receptor-related orphan receptor γt (RORγt) is a nuclear receptor associated with the pathogenesis of autoimmune diseases. Allosteric inhibition of RORγt is conceptually new, unique for this specific nuclear receptor, and offers advantages over traditional orthosteric inhibition. Here, we report a highly efficient in silico-guided approach that led to the discovery of novel allosteric RORγt inverse agonists with a distinct isoxazole chemotype. The the most potent compound, 25 ( FM26 ), displayed submicromolar inhibition in a coactivator recruitment assay and effectively reduced IL-17a mRNA production in EL4 cells, a marker of RORγt activity. The projected allosteric mode of action of 25 was confirmed by biochemical experiments and cocrystallization with the RORγt ligand binding domain. The isoxazole compounds have promising pharmacokinetic properties comparable to other allosteric ligands but with a more diverse chemotype. The efficient ligand-based design approach adopted demonstrates its versatility in generating chemical diversity for allosteric targeting of RORγt.

Transcription factors GATA4 and NKX2-5 directly interact and synergistically activate several cardiac genes and stretch-induced cardiomyocyte hypertrophy. Previously, we identified phenylisoxazole carboxamide 1 as a hit compound, which inhibited the GATA4-NKX2-5 transcriptional synergy. Here, the chemical space around the molecular structure of 1 was explored by synthesizing and characterizing 220 derivatives and structurally related compounds. In addition to the synergistic transcriptional activation, selected compounds were evaluated for their effects on transcriptional activities of GATA4 and NKX2-5 individually as well as potential cytotoxicity. The structure-activity relationship (SAR) analysis revealed that the aromatic isoxazole substituent in the southern part regulates the inhibition of GATA4-NKX2-5 transcriptional synergy. Moreover, inhibition of GATA4 transcriptional activity correlated with the reduced cell viability. In summary, comprehensive SAR analysis accompanied by data analysis successfully identified potent and selective inhibitors of GATA4-NKX2-5 transcriptional synergy and revealed structural features important for it.

Tuberculosis remains one of the deadliest infectious diseases in the world, and the increased number of multidrug-resistant and extremely drug-resistant strains is a significant reason for concern. This makes the discovery of novel antitubercular agents a cogent priority. We have previously addressed this need by reporting a series of substituted 2-aminothiazoles capable to inhibit the growth of actively replicating, nonreplicating persistent, and resistant Mycobacterium tuberculosis strains. Clues from the structure-activity relationships lining up the antitubercular activity were exploited for the rational design of improved analogues. Two compounds, namely N-phenyl-5-(2-(p-tolylamino)thiazol-4-yl)isoxazole-3-carboxamide 7a and N-(pyridin-2-yl)-5-(2-(p-tolylamino)thiazol-4-yl)isoxazole-3-carboxamide 8a, were found to show high inhibitory activity toward susceptible M. tuberculosis strains, with an MIC 90 of 0.125-0.25 μg/mL (0.33-0.66 μM) and 0.06-0.125 μg/mL (0.16-0.32 μM), respectively. Moreover, they maintained good activity also toward resistant strains, and they were selective over other bacterial species and eukaryotic cells, metabolically stable, and apparently not susceptible to the action of efflux pumps.

We report here structure-guided optimization of a novel series of NF-κB inducing kinase (NIK) inhibitors. Starting from a modestly potent, low molecular weight lead, activity was improved by designing a type 11/2 binding mode that accessed a back pocket past the methionine-471 gatekeeper. Divergent binding modes in NIK and PI3K were exploited to dampen PI3K inhibition while maintaining NIK inhibition within these series. Potent compounds were discovered that selectively inhibit the nuclear translocation of NF-κB2 (p52/REL-B) but not canonical NF-κB1 (REL-A/p50).

Sphingosine 1-phosphate (S1P) is the endogenous ligand for the sphingosine 1-phosphate receptors (S1P1-5) and evokes a variety of cellular responses through their stimulation. The interaction of S1P with the S1P receptors plays a fundamental physiological role in a number of processes including vascular development and stabilization, lymphocyte migration, and proliferation. Agonism of S1P1, in particular, has been shown to play a significant role in lymphocyte trafficking from the thymus and secondary lymphoid organs, resulting in immunosuppression. This article will detail the discovery and SAR of a potent and selective series of isoxazole based full agonists of S1P1. Isoxazole 6d demonstrated impressive efficacy when administered orally in a rat model of arthritis and in a mouse experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis.

A series of analogues of the (S)-2-Amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propionic acid (AMPA) receptor agonist BnTetAMPA (5b) were synthesized and characterized pharmacologically in radioligand binding assays at native and cloned AMPA receptors and functionally by two-electrode voltage clamp electrophysiology at the four homomeric AMPA receptors expressed in Xenopus laevis oocytes. The analogues 6 and 7 exhibit very different pharmacological profiles with binding affinity preference for the subtypes GluA1 and GluA3, respectively. X-ray crystal structures of three ligands (6, 7, and 8) in complex with the agonist binding domain (ABD) of GluA2 show that they induce full domain closure despite their low agonist efficacies. Trp767 in GluA2 ABD could be an important determinant for partial agonism of this compound series at AMPA receptors, since agonist efficacy also correlated with the location of the Trp767 side chain.

The recent discovery of the role of receptor interacting protein 1 (RIP1) kinase in tumor necrosis factor (TNF)-mediated inflammation has led to its emergence as a highly promising target for the treatment of multiple inflammatory diseases. We screened RIP1 against GSK's DNA-encoded small-molecule libraries and identified a novel highly potent benzoxazepinone inhibitor series. We demonstrate that this template possesses complete monokinase selectivity for RIP1 plus unique species selectivity for primate versus nonprimate RIP1. We elucidate the conformation of RIP1 bound to this benzoxazepinone inhibitor driving its high kinase selectivity and design specific mutations in murine RIP1 to restore potency to levels similar to primate RIP1. This series differentiates itself from known RIP1 inhibitors in combining high potency and kinase selectivity with good pharmacokinetic profiles in rodents. The favorable developability profile of this benzoxazepinone template, as exemplified by compound 14 (GSK'481), makes it an excellent starting point for further optimization into a RIP1 clinical candidate.

Upregulation of pyruvate dehydrogenase kinase (PDHK) has been observed in a variety of cancers. Inhibition of PDHK offers an attractive opportunity for the development of novel cancer therapies. To obtain novel PDHK inhibitors, we took advantage of the homology of the ATP-binding pocket between Heat Shock Protein 90 (HSP90) and PDHK, and utilized 4,5-diarylisoxazole based HSP90 inhibitor for structural design. Our efforts led to the identification of 5k that inhibited PDHK1 with an IC50 value of 17 nM, which, however, showed marginal cellular activity. Further structural optimization resulted in compound 11a with improved cellular activity which could effectively modulate the metabolic profile of cancer cells and lead to the inhibition of cancer cell proliferation, evidenced by the increased oxidative phosphorylation and decreased glycolysis and associated oxidative stress. Our results suggested 11a as an excellent lead compound and a favorable biological tool to further evaluate the therapeutic potential of PDHK and HSP90 dual inhibitors in the treatment of cancer.

Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme that catalyzes the posttranslational modification of glutamine residues on protein or peptide substrates. A growing body of literature has implicated aberrantly regulated activity of TG2 in the pathogenesis of various human inflammatory, fibrotic, and other diseases. Taken together with the fact that TG2 knockout mice are developmentally and reproductively normal, there is growing interest in the potential use of TG2 inhibitors in the treatment of these conditions. Targeted-covalent inhibitors based on the weakly electrophilic 3-bromo-4,5-dihydroisoxazole (DHI) scaffold have been widely used to study TG2 biology and are well tolerated in vivo, but these compounds have only modest potency, and their selectivity toward other transglutaminase homologues is largely unknown. In the present work, we first profiled the selectivity of existing inhibitors against the most pertinent TG isoforms (TG1, TG3, and FXIIIa). Significant cross-reactivity of these small molecules with TG1 was observed. Structure-activity and -selectivity analyses led to the identification of modifications that improved potency and isoform selectivity. Preliminary pharmacokinetic analysis of the most promising analogues was also undertaken. Our new data provides a clear basis for the rational selection of dihydroisoxazole inhibitors as tools for in vivo biological investigation.

The compounds described herein with a spirocyclic architecture fused to a benzisoxazole ring represent a new class of antibacterial agents that operate by inhibition of DNA gyrase as corroborated in an enzyme assay and by the inhibition of precursor thymidine into DNA during cell growth. Activity resided in the configurationally lowest energy (2S,4R,4aR) diastereomer. Highly active compounds against Staphylococcus aureus had sufficiently high solubility, high plasma protein free fraction, and favorable pharmacokinetics to suggest that in vivo efficacy could be demonstrated, which was realized with compound (-)-1 in S. aureus mouse infection models. A high drug exposure NOEL on oral dosing in the rat suggested that a high therapeutic margin could be achieved. Importantly, (-)-1 was not cross-resistant with other DNA gyrase inhibitors such as fluoroquinolone and aminocoumarin antibacterials. Hence, this class shows considerable promise for the treatment of infections caused by multidrug resistant bacteria, including S. aureus.

We developed a new class of covalent inhibitors of Plasmodium falciparum glyceraldehyde-3-phosphate dehydrogenase, a validated target for the treatment of malaria, by screening a small library of 3-bromo-isoxazoline derivatives that inactivate the enzyme through a covalent, selective bond to the catalytic cysteine, as demonstrated by mass spectrometry. Substituents on the isoxazolinic ring modulated the potency up to 20-fold, predominantly due to an electrostatic effect, as assessed by computational analysis.

Sirtuins are a family of NAD(+)-dependent protein deacetylases that play critical roles in epigenetic regulation, stress responses, and cellular aging in eukaryotic cells. In an effort to identify small molecule inhibitors of sirtuins for potential use as chemotherapeutics as well as tools to modulate sirtuin activity, we previously identified a nonselective sirtuin inhibitor called cambinol (IC50 ≈ 50 μM for SIRT1 and SIRT2) with in vitro and in vivo antilymphoma activity. In the current study, we used saturation transfer difference (STD) NMR experiments with recombinant SIRT1 and 20 to map parts of the inhibitor that interacted with the protein. Our ongoing efforts to optimize cambinol analogues for potency and selectivity have resulted in the identification of isoform selective analogues: 17 with >7.8-fold selectivity for SIRT1, 24 with >15.4-fold selectivity for SIRT2, and 8 with 6.8- and 5.3-fold selectivity for SIRT3 versus SIRT1 and SIRT2, respectively. In vitro cytotoxicity studies with these compounds as well as EX527, a potent and selective SIRT1 inhibitor, suggest that antilymphoma activity of this compound class may be predominantly due to SIRT2 inhibition.

Ruthenium catalyzed 1,3-cycloaddition (click chemistry) of an azido moiety installed on dihydroxycumene scaffold with differently substituted aryl propiolates gave a new family of 1,4,5-trisubstituted triazole carboxylic acid derivatives that showed high affinity toward Hsp90 associated with cell proliferation inhibition, both in nanomolar range. The 1,5 arrangement of the resorcinol, the aryl moieties, and the presence of an alkyl (secondary) amide in position 4 of the triazole ring were essential to get high activity. Docking simulations suggested that the triazoles penetrate the Hsp90 ATP binding site. Some 1,4,5-trisubstituted triazole carboxamides induced dramatic depletion of the examined client proteins and a very strong increase in the expression levels of the chaperone Hsp70. In vitro metabolic stability and in vivo preliminary studies on selected compounds have shown promising results comparable to the potent Hsp90 inhibitor NVP-AUY922. One of them, (compound 18, SST0287CL1) was selected for further investigation as the most promising drug candidate.

Today, there are approximately 8 million cases of Chagas disease in the southern cone of South America alone, and about 100 million people are living with the risk of becoming infected. The present pharmacotherapy is sometimes ineffective and has serious side effects. Here, we report a series of 4,5-dihydroisoxazoles incorporating hydroxamate moieties, which act as effective inhibitors of the carbonic anhydrase (CA) from Trypanosoma cruzi (TcCA). One compound (5g) was evaluated in detail and shows promising features as an antitrypanosomal agent. Excellent values for the inhibition of growth for all three developmental forms of the parasite were observed at low concentrations of 5g (IC50 values from 7.0 to <1 μM). The compound has a selectivity index (SI) of 6.7 and no cytotoxicity to macrophage cells. Preliminary in vivo data showed that 5g reduces bloodstream parasites and that all treated mice survived; it was also more effective than the standard drug benznidazole.

3-(5-Chlorofuran-2-yl)-5-methyl-4-phenylisoxazole (P6), a known selective cyclooxygenase-1 (COX-1) inhibitor, was used to design a new series of 3,4-diarylisoxazoles in order to improve its biochemical COX-1 selectivity and antiplatelet efficacy. Structure-activity relationships were studied using human whole blood assays for COX-1 and COX-2 inhibition in vitro, and results showed that the simultaneous presence of 5-methyl (or -CF3), 4-phenyl, and 5-chloro(-bromo or -methyl)furan-2-yl groups on the isoxazole core was essential for their selectivity toward COX-1. 3g, 3s, 3d were potent and selective COX-1 inhibitors that affected platelet aggregation in vitro through the inhibition of COX-1-dependent thromboxane (TX) A2. Moreover, we characterized their kinetics of COX-1 inhibition. 3g, 3s, and 3d were more potent inhibitors of platelet COX-1 and aggregation than P6 (named 6) for their tighter binding to the enzyme. The pharmacological results were supported by docking simulations. The oral administration of 3d to mice translated into preferential inhibition of platelet-derived TXA2 over protective vascular-derived prostacyclin (PGI2).

The discovery and synthesis of potential and novel antipsychotic coumarin derivatives, associated with potent dopamine D2, D3, and serotonin 5-HT1A and 5-HT2A receptor properties, are the focus of the present article. The most-promising derivative was 7-(4-(4-(6-fluorobenzo[d]isoxazol-3-yl)-piperidin-1-yl)butoxy)-4-methyl-8-chloro-2H-chromen-2-one (17m). This derivative possesses unique pharmacological features, including high affinity for dopamine D2 and D3 and serotonin 5-HT1A and 5-HT2A receptors. Moreover, it possesses low affinity for 5-HT2C and H1 receptors (to reduce the risk of obesity associated with chronic treatment) and hERG channels (to reduce the incidence of torsade des pointes). In animal models, compound 17m inhibited apomorphine-induced climbing behavior, MK-801-induced hyperactivity, and the conditioned avoidance response without observable catalepsy at the highest dose tested. Further, fewer preclinical adverse events were noted with 17m compared with risperidone in assays that measured prolactin secretion and weight gain. Acceptable pharmacokinetic properties were also noted with 17m. Taken together, 17m may constitute a novel class of drugs for the treatment of schizophrenia.

The bromodomain protein module, which binds to acetylated lysine, is emerging as an important epigenetic therapeutic target. We report the structure-guided optimization of 3,5-dimethylisoxazole derivatives to develop potent inhibitors of the BET (bromodomain and extra terminal domain) bromodomain family with good ligand efficiency. X-ray crystal structures of the most potent compounds reveal key interactions required for high affinity at BRD4(1). Cellular studies demonstrate that the phenol and acetate derivatives of the lead compounds showed strong antiproliferative effects on MV4;11 acute myeloid leukemia cells, as shown for other BET bromodomain inhibitors and genetic BRD4 knockdown, whereas the reported compounds showed no general cytotoxicity in other cancer cell lines tested.

Virtual screening in a huge collection of virtual combinatorial libraries has led to the identification of two new structural classes of GPR119 agonists with submicromolar in vitro potencies. Herein, we describe the virtual screening process involving feature trees fragment space searches followed by a 3D postprocessing step. The in silico findings were then filtered and prioritized, and finally, combinatorial libraries of target molecules were synthesized. Furthermore the so-called "activity-anchor principle" is introduced as an element to increase the chance to generate true hits. An activity anchor is a structural element expected to provide key contributions to a certain biological activity. Application of this technique has led to the discovery of two new GPR119-agonist hit series, one of which was further optimized to progress as a novel lead class.

This report describes the discovery and optimization of a BACE-1 inhibitor series containing an unusual acyl guanidine chemotype that was originally synthesized as part of a 6041-membered solid-phase library. The synthesis of multiple follow-up solid- and solution-phase libraries facilitated the optimization of the original micromolar hit into a single-digit nanomolar BACE-1 inhibitor in both radioligand binding and cell-based functional assay formats. The X-ray structure of representative inhibitors bound to BACE-1 revealed a number of key ligand:protein interactions, including a hydrogen bond between the side chain amide of flap residue Gln73 and the acyl guanidine carbonyl group, and a cation-π interaction between Arg235 and the isothiazole 4-methoxyphenyl substituent. Following subcutaneous administration in rats, an acyl guanidine inhibitor with single-digit nanomolar activity in cells afforded good plasma exposures and a dose-dependent reduction in plasma Aβ levels, but poor brain exposure was observed (likely due to Pgp-mediated efflux), and significant reductions in brain Aβ levels were not obtained.

D-Alanine:D-alanine ligase (Ddl) is an essential ATP-dependent bacterial enzyme involved in peptidoglycan biosynthesis. Discovery of Ddl inhibitors not competitive with ATP has proven to be difficult because the Ddl bimolecular d-alanine binding pocket is very restricted, as is accessibility to the active site for larger molecules in the catalytically active closed conformation of Ddl. A molecular dynamics study of the opening and closing of the Ddl lid loop informs future structure-based design efforts that allow for the flexibility of Ddl. A virtual screen on generated enzyme conformations yielded some hit inhibitors whose bioactivity was determined.

We report here the design, preparation, and systematic evaluation of a novel cycloalkane[d]isoxazole pharmacophoric fragment-containing androgen receptor (AR) modulators. Cycloalkane[d]isoxazoles form new core structures that interact with the hydrophobic region of the AR ligand-binding domain. To systematize and rationalize the structure-activity relationship of the new fragment, we used molecular modeling to design a molecular library containing over 40 cycloalkane[d]isoxazole derivatives. The most potent compound, 4-(3a,4,5,6,7,7a-hexahydrobenzo[d]isoxazol-3-yl)-2-(trifluoromethyl)benzonitrile (6a), exhibits antiandrogenic activity significantly greater than that of the most widely used antiandrogenic prostate cancer drugs bicalutamide (1) and hydroxyflutamide (2) in reporter gene assays measuring the transcriptional activity of AR (decreasing approximately 90% of the total AR activity) and in competitive AR ligand-binding assays (showing over four times higher potency to inhibit radioligand binding in comparison to bicalutamide). Notably, 6a maintains its antiandrogenic activity with AR mutants W741L and T877A commonly observed and activated by bicalutamide and hydroxyflutamide, respectively, in prostate cancer patients.

The HIV-1 nucleocapsid (NC) is a RNA/DNA binding protein encoded within the Gag polyprotein, which is critical for the selection and chaperoning of viral genomic RNA during virion assembly. RNA/DNA binding occurs through a highly conserved zinc-knuckle motif present in NC. Given the necessity of NC-viral RNA/DNA interaction for viral replication, identification of compounds that disrupt the NC-RNA/DNA interaction may have value as an antiviral strategy. To identify small molecules that disrupt NC-viral RNA/DNA binding, a high-throughput fluorescence polarization assay was developed and a library of 14,400 diverse, druglike compounds was screened. Compounds that disrupted NC binding to a fluorescence-labeled DNA tracer were next evaluated by differential scanning fluorimetry to identify compounds that must bind to NC or Gag to impart their effects. Two compounds were identified that inhibited NC-DNA interaction, specifically bound NC with nanomolar affinity, and showed modest anti-HIV-1 activity in ex vivo cell assays.

A series of betulinic acid (BA) derivatives were designed and synthesized by introducing various fused heterocyclic rings at C-2 and C-3 positions. Their inhibitory effects of RANKL-induced osteoclastogenesis were evaluated by using a cell-based tartrate-resistant acid phosphatase (TRAP) activity assay. To our delight, most of these compounds exhibited a dramatic increase in inhibitory potency, compared with BA. The most potent compound, 20, showed 66.9% inhibition even at the low concentration of 0.1 μM, which was about 200-fold more potent than the lead compound BA. What's more, the cytotoxicity assay on RAW264.7 suggested that the inhibition of 20 on osteoclast differentiation did not result from its cytotoxicity. The primary mechanistic study indicated that 20 could inhibit osteoclastogenesis-related marker gene expression levels of cathepsin K and TRAP. More importantly, 20 could attenuate bone loss of ovariectomy mouse in vivo. Therefore, these BA derivatives could be used as potential leads for the development of a new type of antiosteoporosis agent., (© 2012 American Chemical Society)

The Ras/RAF/MEK/ERK mitogen-activated protein kinase (MAPK) signaling pathway plays a central role in the regulation of cell growth, differentiation, and survival. Expression of mutant BRAF(V600E) results in constitutive activation of the MAPK pathway, which can lead to uncontrolled cellular growth. Herein, we describe an SAR optimization campaign around a series of quinazoline derived BRAF(V600E) inhibitors. In particular, the bioisosteric replacement of a metabolically sensitive tert-butyl group with fluorinated alkyl moieties is described. This effort led directly to the identification of a clinical candidate, compound 40 (CEP-32496). Compound 40 exhibits high potency against several BRAF(V600E)-dependent cell lines and selective cytotoxicity for tumor cell lines expressing mutant BRAF(V600E) versus those containing wild-type BRAF. Compound 40 also exhibits an excellent PK profile across multiple preclinical species. In addition, significant oral efficacy was observed in a 14-day BRAF(V600E)-dependent human Colo-205 tumor xenograft mouse model, upon dosing at 30 and 100 mg/kg BID.

There is considerable evidence to support the hypothesis that the blockade of nAChR is responsible for the antidepressant action of nicotinic ligands. The nicotinic acetylcholine receptor (nAChR) antagonist, mecamylamine, has been shown to be an effective add-on in patients that do not respond to selective serotonin reuptake inhibitors. This suggests that nAChR ligands may address an unmet clinical need by providing relief from depressive symptoms in refractory patients. In this study, a new series of nAChR ligands based on an isoxazole-ether scaffold have been designed and synthesized for binding and functional assays. Preliminary structure-activity relationship (SAR) efforts identified a lead compound 43, which possesses potent antidepressant-like activity (1 mg/kg, IP; 5 mg/kg, PO) in the classical mouse forced swim test. Early stage absorption, distribution, metabolism, excretion, and toxicity (ADME-Tox) studies also suggested favorable drug-like properties, and broad screening toward other common neurotransmitter receptors indicated that compound 43 is highly selective for nAChRs over the other 45 neurotransmitter receptors and transporters tested.

Bromodomains are epigenetic reader modules that regulate gene transcription through their recognition of acetyl-lysine modified histone tails. Inhibitors of this protein-protein interaction have the potential to modulate multiple diseases as demonstrated by the profound anti-inflammatory and antiproliferative effects of a recently disclosed class of BET compounds. While these compounds were discovered using phenotypic assays, here we present a highly efficient alternative approach to find new chemical templates, exploiting the abundant structural knowledge that exists for this target class. A phenyl dimethyl isoxazole chemotype resulting from a focused fragment screen has been rapidly optimized through structure-based design, leading to a sulfonamide series showing anti-inflammatory activity in cellular assays. This proof-of-principle experiment demonstrates the tractability of the BET family and bromodomain target class to fragment-based hit discovery and structure-based lead optimization.

A structural investigation on the isoxazole scaffold led to the discovery of 3,4-isoxazolediamide compounds endowed with potent Hsp90 inhibitory properties. We have found that compounds possessing a nitrogen atom directly attached to the C-4 heterocycle ring possess in vitro Hsp90 inhibitory properties at least comparable to those of the structurally related 4,5-diarylisoxazole derivatives. A group of compounds from this series of diamides combine potent binding affinity and cell growth inhibitory activity in both series of alkyl- and aryl- or heteroarylamides, with IC50 in the low nanomolar range. The 3,4-isoxazolediamides were also very effective in causing dramatic depletion of the examined client proteins and, as expected for the Hsp90 inhibitors, always induced a very strong increase in the expression levels of the chaperone Hsp70. In vivo studies against human epidermoid carcinoma A431 showed an antitumor effect of morpholine derivative 73 comparable to that induced by the reference compound 10.

A series of Δ(2)-isoxazoline constrained analogues of procaine/procainamide (7a-k and 8a-k) were prepared and their inhibitory activity against DNA methyltransferase 1 (DNMT1) was tested. Among them, derivative 7b is far more potent in vitro (IC(50) = 150 μM) than other non-nucleoside inhibitors and also exhibits a strong and dose-dependent antiproliferative effect against HCT116 human colon carcinoma cells. The binding mode of 7b with the enzyme was also investigated by means of a simple competition assay as well as of docking simulations conducted using the recently published crystallographic structure of human DNMT1. On the basis of the findings, we assessed that the mode of inhibition of 7b is consistent with a competition with the cofactor and propose it as a novel lead compound for the development of non-nucleoside DNMT inhibitors.

Depression, a common neurological condition, is one of the leading causes of disability and suicide worldwide. Standard treatment, targeting monoamine transporters selective for the neurotransmitters serotonin and noradrenaline, is not able to help many patients that are poor responders. This study advances the development of sazetidine-A analogues that interact with α4β2 nicotinic acetylcholine receptors (nAChRs) as partial agonists and that possess favorable antidepressant profiles. The resulting compounds that are highly selective for the α4β2 subtype of nAChR over α3β4-nAChRs are partial agonists at the α4β2 subtype and have excellent antidepressant behavioral profiles as measured by the mouse forced swim test. Preliminary absorption, distribution, metabolism, excretion, and toxicity (ADMET) studies for one promising ligand revealed an excellent plasma protein binding (PPB) profile, low CYP450-related metabolism, and low cardiovascular toxicity, suggesting it is a promising lead as well as a drug candidate to be advanced through the drug discovery pipeline.

Histone-lysine acetylation is a vital chromatin post-translational modification involved in the epigenetic regulation of gene transcription. Bromodomains bind acetylated lysines, acting as readers of the histone-acetylation code. Competitive inhibitors of this interaction have antiproliferative and anti-inflammatory properties. With 57 distinct bromodomains known, the discovery of subtype-selective inhibitors of the histone-bromodomain interaction is of great importance. We have identified the 3,5-dimethylisoxazole moiety as a novel acetyl-lysine bioisostere, which displaces acetylated histone-mimicking peptides from bromodomains. Using X-ray crystallographic analysis, we have determined the interactions responsible for the activity and selectivity of 4-substituted 3,5-dimethylisoxazoles against a selection of phylogenetically diverse bromodomains. By exploiting these interactions, we have developed compound 4d, which has IC(50) values of <5 μM for the bromodomain-containing proteins BRD2(1) and BRD4(1). These compounds are promising leads for the further development of selective probes for the bromodomain and extra C-terminal domain (BET) family and CREBBP bromodomains.

The design, synthesis, docking, and biological evaluation of novel potent HDAC3 and HDAC8 isoxazole- and pyrazole-based diazide probes suitable for binding ensemble profiling with photoaffinity labeling (BEProFL) experiments in cells is described. Both the isoxazole- and pyrazole-based probes exhibit low nanomolar inhibitory activity against HDAC3 and HDAC8, respectively. The pyrazole-based probe 3f appears to be one of the most active HDAC8 inhibitors reported in the literature with an IC(50) of 17 nM. Our docking studies suggest that unlike the isoxazole-based ligands the pyrazole-based ligands are flexible enough to occupy the second binding site of HDAC8. Probes/inhibitors 2b, 3a, 3c, and 3f exerted the antiproliferative and neuroprotective activities at micromolar concentrations through inhibition of nuclear HDACs, indicating that they are cell permeable and the presence of an azide or a diazide group does not interfere with the neuroprotection properties, or enhance cellular cytotoxicity, or affect cell permeability.

Herein, we report the development of an antifiloviral screening system, based on a pseudotyping strategy, and its application in the discovery of a novel group of small molecules that selectively inhibit the Ebola and Marburg glycoprotein (GP)-mediated infection of human cells. Using Ebola Zaire GP-pseudotyped HIV particles bearing a luciferase reporter gene and 293T cells, a library of 237 small molecules was screened for inhibition of GP-mediated viral entry. From this assay, lead compound 8a was identified as a selective inhibitor of filoviral entry with an IC(50) of 30 μM. To analyze functional group requirements for efficacy, a structure-activity relationship analysis of this 3,5-disubstituted isoxazole was then conducted with 56 isoxazole and triazole derivatives prepared using "click" chemistry. This study revealed that while the isoxazole ring can be replaced by a triazole system, the 5-(diethylamino)acetamido substituent found in 8a is required for inhibition of viral-cell entry. Variation of the 3-aryl substituent provided a number of more potent antiviral agents with IC(50) values ranging to 2.5 μM. Lead compound 8a and three of its derivatives were also found to block the Marburg glycoprotein (GP)-mediated infection of human cells.

Transient receptor potential melastatin 8 (TRPM8) is a nonselective cation channel that is thermoresponsive to cool to cold temperatures (8-28 °C) and also may be activated by chemical agonists such as menthol and icilin. Antagonism of TRPM8 activation is currently under investigation for the treatment of painful conditions related to cold, such as cold allodynia and cold hyperalgesia. The design, synthesis, and optimization of a class of selective TRPM8 antagonists based on a benzimidazole scaffold is described, leading to the identification of compounds that exhibited potent antagonism of TRPM8 in cell-based functional assays for human, rat, and canine TRPM8 channels. Numerous compounds in the series demonstrated excellent in vivo activity in the TRPM8-selective "wet-dog shakes" (WDS) pharmacodynamic model and in the rat chronic constriction injury (CCI)-induced model of neuropathic pain. Taken together, the present results suggest that the in vivo antagonism of TRPM8 constitutes a viable new strategy for treating a variety of disorders associated with cold hypersensitivity, including certain types of neuropathic pain.

Sphingosine 1-phosphate lyase (S1PL) has been characterized as a novel target for the treatment of autoimmune disorders using genetic and pharmacological methods. Medicinal chemistry efforts targeting S1PL by direct in vivo evaluation of synthetic analogues of 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI, 1) led to the discovery of 2 (LX2931) and 4 (LX2932). The immunological phenotypes observed in S1PL deficient mice were recapitulated by oral administration of 2 or 4. Oral dosing of 2 or 4 yielded a dose-dependent decrease in circulating lymphocyte numbers in multiple species and showed a therapeutic effect in rodent models of rheumatoid arthritis (RA). Phase I clinical trials indicated that 2, the first clinically studied inhibitor of S1PL, produced a dose-dependent and reversible reduction of circulating lymphocytes and was well tolerated at dose levels of up to 180 mg daily. Phase II evaluation of 2 in patients with active rheumatoid arthritis is currently underway.

Human protease activated receptor 2 (PAR2) is a G protein-coupled receptor that is associated with inflammatory diseases and cancers. PAR2 is activated by serine proteases that cleave its N-terminus and by synthetic peptides corresponding to the new N-terminus. Peptide agonists are widely used to characterize physiological roles for PAR2 but typically have low potency (e.g., SLIGKV-NH(2), SLIGRL-NH(2)), uncertain target selectivity, and poor bioavailability, limiting their usefulness for specifically interrogating PAR2 in vivo. Structure-activity relationships were used to derive new PAR2 agonists and antagonists containing nonpeptidic moieties. Agonist GB110 (19, EC(50) 0.28 μM) selectively induced PAR2-, but not PAR1-, mediated intracellular Ca(2+) release in HT29 human colorectal carcinoma cells. Antagonist GB83 (36, IC(50) 2 μM) is the first compound at micromolar concentrations to reversibly inhibit PAR2 activation by both proteases and other PAR2 agonists (e.g., trypsin, 2f-furoyl-LIGRLO-NH(2), 19). The new compounds are selective for PAR2 over PAR1, serum stable, and suitable for modulating PAR2 in disease models.

New antituberculosis (anti-TB) drugs are urgently needed to shorten the 6-12 month treatment regimen and especially to battle drug-resistant Mycobacterium tuberculosis (Mtb) strains. In this study, we have continued our efforts to develop isoxazole-based anti-TB compounds by applying rational drug design approach. The biological activity and the structure-activity relationships (SAR) for a designed series of 5-phenyl-3-isoxazolecarboxylic acid ethyl ester derived anti-TB compounds were investigated. Several compounds were found to exhibit nanomolar activity against the replicating bacteria (R-TB) and low micromolar activity against the nonreplicating bacteria (NRP-TB). The series showed excellent selectivity toward Mtb, and in general, no cytotoxicity was observed in Vero cells (IC(50) > 128 muM). Notably, selected compounds also retained their activity against isoniazid (INH), rifampin (RMP), and streptomycin (SM) resistant Mtb strains. Hence, benzyloxy, benzylamino, and phenoxy derivatives of 5-phenyl-3-isoxazolecarboxylic acid ethyl esters represent a highly potent, selective, and versatile series of anti-TB compounds and as such present attractive lead compounds for further TB drug development.

The actual development and clinical use of new therapeutics for tuberculosis (TB) have remained stagnant for years because of the complexity of the disease process, the treatment of which at present requires the administration of drug combinations over a 6 month period. There is thus an urgent need for the discovery and development of novel, more active, and less toxic anti-TB agents. In this study, we report on the chemistry and biology of a series of potent 5-(2-methylbenzothiazol-5-yloxymethyl)isoxazole-3-carboxamide derivatives, which proved to be active against replicating Mycobacterium tuberculosis (Mtb) H(37)Rv. The most potent compounds 7j and 7s were found to inhibit Mtb growth at micromolar concentrations, with MIC values of 1.4 and 1.9 microM, respectively. Impressively, all active compounds were nontoxic toward Vero cells (IC(50) > 128 microM). Moreover, the best of these compounds were also tested against protozoan parasites, and some of these compounds were found to show activity, especially against Plasmodium falciparum. These studies thus suggest that certain 2-methylbenzothiazole based compounds may serve as promising lead scaffolds for further elaboration as anti-TB drugs and as possible antimalaria drugs.

Tuberculosis (TB) remains as a global pandemic that is aggravated by a lack of health care, the spread of HIV, and the emergence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) strains. New anti-TB drugs are urgently required to shorten the long 6-12 month treatment regimen and to battle drug-resistant Mtb strains. We have identified several potent quinoline-based anti-TB compounds, bearing an isoxazole containing side-chain. The most potent compounds, 7g and 13, exhibited submicromolar activity against the replicating bacteria (R-TB), with minimum inhibitory concentrations (MICs) of 0.77 and 0.95 microM, respectively. In general, these compounds also had micromolar activity against the nonreplicating persistent bacteria (NRP-TB) and did not show toxicity on Vero cells up to 128 microM concentration. Compounds 7g and 13 were shown to retain their anti-TB activity against rifampin, isoniazid, and streptomycin resistant Mtb strains. The results suggest that quinoline-isoxazole-based anti-TB compounds are promising leads for new TB drug development.

In an effort to develop dual PPARalpha/gamma activators with improved therapeutic efficacy, a series of diaryl alpha-ethoxy propanoic acid compounds comprising two aryl groups linked by rigid oxime ether or isoxazoline ring were designed and synthesized and their biological activities were examined. Most of the compounds possessing an oxime ether linker were more potent PPARgamma activators than the lead PPARalpha/gamma dual agonist, tesaglitazar in vitro. Compound 18, one of the derivatives with an oxime ether linker, was found to selectively transactivate PPARgamma (EC 50 = 0.028 microM) over PPARalpha (EC 50 = 7.22 microM) in vitro and lower blood glucose in db/ db mice more than muraglitazar after oral treatment for 11 days.

A new study on terphenyl and diaryl-isoxazole and -isoxazoline derivatives, maintaining a common 3-adamantyl-4-hydroxyphenyl moiety, has been conducted to find compounds with growth supporting and antiapoptotic properties. Unexpectedly, diphenyisoxazole derivatives bearing a nitro group replacing the carboxylic function have been found with the highest cell protective activity within the series, in complete and in serum-free conditions. Inhibition of apoptosis induced by daunorubicin has also been observed for the most active compound.

Drugs used to treat various disorders target GABA A receptors. To develop alpha subunit selective compounds, we synthesized 5-(4-piperidyl)-3-isoxazolol (4-PIOL) derivatives. The 3-isoxazolol moiety was substituted by 1,3,5-oxadiazol-2-one, 1,3,5-oxadiazol-2-thione, and substituted 1,2,4-triazol-3-ol heterocycles with modifications to the basic piperidine substituent as well as substituents without basic nitrogen. Compounds were screened by [(3)H]muscimol binding and in patch-clamp experiments with heterologously expressed GABA A alpha ibeta 3gamma 2 receptors (i = 1-6). The effects of 5-aminomethyl-3 H-[1,3,4]oxadiazol-2-one 5d were comparable to GABA for all alpha subunit isoforms. 5-piperidin-4-yl-3 H-[1,3,4]oxadiazol-2-one 5a and 5-piperidin-4-yl-3 H-[1,3,4]oxadiazol-2-thione 6a were weak agonists at alpha 2-, alpha 3-, and alpha 5-containing receptors. When coapplied with GABA, they were antagonistic in alpha 2-, alpha 4-, and alpha 6-containing receptors and potentiated alpha 3-containing receptors. 6a protected GABA binding site cysteine-substitution mutants alpha 1F64C and alpha 1S68C from reacting with methanethiosulfonate-ethylsulfonate. 6a specifically covalently modified the alpha 1R66C thiol, in the GABA binding site, through its oxadiazolethione sulfur. These results demonstrate the feasibility of synthesizing alpha subtype selective GABA mimetic drugs.

In this paper, the preparation and systematic evaluation of estrogen receptor alpha (ER alpha) and estrogen receptor beta (ER beta) activities of some diaryl-1,3-diones and their synthetic intermediates, diaryl-4,5-dihydroisoxazoles, diaryl-3-hydroxyketones, diaryl-3-methoxyketones, and diaryl-2-(dimethyl-lambda 4-sulfanylidene)-1,3-diones, is described. The set of 72 compounds constitutes a general schematic structure aryl1-linker1-spacer-linker2-aryl2, where the linker1-spacer-linker2 length varies between 4 and 8 carbons. The set of compounds was applied here to map and explore the active sites of subtypes ER alpha and ER beta. The highest activities were obtained with dihydroisoxazole and hydroxyketone spacers, but even the most flexible diones with unsubstituted aryl groups showed some agonism. Most compounds were found to be ER alpha selective or to activate both receptors, but in some cases we saw also clearly stronger ER beta activation.

D-amino acid oxidase (DAAO) catalyzes the oxidation of D-amino acids including d-serine, a full agonist at the glycine site of the NMDA receptor. A series of benzo[ d]isoxazol-3-ol derivatives were synthesized and evaluated as DAAO inhibitors. Among them, 5-chloro-benzo[ d]isoxazol-3-ol (CBIO) potently inhibited DAAO with an IC50 in the submicromolar range. Oral administration of CBIO in conjunction with d-serine enhanced the plasma and brain levels of d-serine in rats compared to the oral administration of d-serine alone.

The two enantiomeric pairs of erythro- and threo-amino-(3'-hydroxy-4',5'-dihydro-isoxazol-5'-yl)-acetic acids were synthesized via the 1,3-dipolar cycloaddition of bromonitrile oxide to ( R)- or ( S)-3-( tert-butoxycarbonyl)-2,2-dimethyl-4-vinyloxazolidine. The pharmacological profiles of the studied amino acids reflect the relationship between the activity/selectivity and the stereochemistry of the two stereogenic centers: while the (2 S,5' S) stereoisomer is an agonist at the AMPA and KA receptors, its (2 R,5' R) enantiomer interacts selectively with the NMDA receptors; the (2 S,5' R) stereoisomer is the only one capable to activate the mGluRs.

Pantothenate synthetase (PS) is one of the potential new antimicrobial targets that may also be useful for the treatment of the nonreplicating persistent forms of Mycobacterium tuberculosis. In this Letter we present a series of 5- tert-butyl- N-pyrazol-4-yl-4,5,6,7-tetrahydrobenzo[ d]isoxazole-3-carboxamide derivatives as novel potent Mycobacterium tuberculosis PS inhibitors, their in silico molecular design, synthesis, and inhibitory activity.