Design and synthesis of functionalized piperazin-1yl-(E)-stilbenes as inhibitors of 17α-hydroxylase-C17,20-lyase (Cyp17).

The synthesis of steroid hormones is critical to human physiology and improper regulation of either the synthesis of these key molecules or activation of the associated receptors can lead to disease states. This has led to intense interest in developing compounds capable of modulating the synthesis of steroid hormones. Compounds capable of inhibiting Cyp19 (Aromatase), a key enzyme in the synthesis of estrogens, have been successfully employed as breast cancer therapies, while inhibitors of Cyp17 (17α-hydroxylase-17,20-lyase), a key enzyme in the synthesis of glucocorticoids, mineralocorticoids and steroidal sex hormones, are a key component of prostate cancer therapy. Inhibition of CYP17 has also been suggested as a possible target for the treatment of Cushing Syndrome and Metabolic Syndrome. We have identified two novel series of stilbene based CYP17 inhibitors and demonstrated that exemplary compounds in these series have pharmacokinetic properties consistent with orally delivered drugs. These findings suggest that compounds in these classes may be useful for the treatment of diseases and conditions associated with improper regulation of glucocorticoids synthesis and glucocorticoids receptor activation.

Design, synthesis, and evaluation of (2S,4R)-Ketoconazole sulfonamide analogs as potential treatments for Metabolic Syndrome.

Metabolic Syndrome, also referred to as 'Syndrome X' or 'Insulin Resistance Syndrome,' remains a major, unmet medical need despite over 30years of intense effort. Recent research suggests that there may be a causal link between this condition and abnormal glucocorticoid processing. Specifically, dysregulation of the hypothalamic-pituitary-adrenocortical (HPA) axis leads to increased systemic cortisol concentrations. Cushing' syndrome, a disorder that is also typified by a marked elevation in levels of cortisol, produces clinical symptomology that is similar to those observed in MetS, and they can be alleviated by decreasing circulating cortisol concentrations. As a result, it has been suggested that decreasing systemic cortisol concentration might have a positive impact on the progression of MetS. This could be accomplished through inhibition of enzymes in the cortisol synthetic pathway, 11ß-hydroxylase (Cyp11B1), 17α-hydroxylase-C17,20-lyase (Cyp17), and 21-hydroxylase (Cyp21). We have identified a series of novel sulfonamide analogs of (2S,4R)-Ketoconazole that are potent inhibitors of these enzymes. In addition, selected members of this class of compounds have pharmacokinetic properties consistent with orally delivered drugs, making them well suited to further investigation as potential therapies for MetS.

Identification and characterization of acidic mammalian chitinase inhibitors.

Acidic mammalian chitinase (AMCase) is a member of the glycosyl hydrolase 18 family (EC 3.2.1.14) that has been implicated in the pathophysiology of allergic airway disease such as asthma. Small molecule inhibitors of AMCase were identified using a combination of high-throughput screening, fragment screening, and virtual screening techniques and characterized by enzyme inhibition and NMR and Biacore binding experiments. X-ray structures of the inhibitors in complex with AMCase revealed that the larger more potent HTS hits, e.g. 5-(4-(2-(4-bromophenoxy)ethyl)piperazine-1-yl)-1H-1,2,4-triazol-3-amine 1, spanned from the active site pocket to a hydrophobic pocket. Smaller fragments identified by FBS occupy both these pockets independently and suggest potential strategies for linking fragments. Compound 1 is a 200 nM AMCase inhibitor which reduced AMCase enzymatic activity in the bronchoalveolar lavage fluid in allergen-challenged mice after oral dosing.

Ion channel screening plates: design, construction, and maintenance.

Ion channels have provided a diverse set of therapeutic targets across all areas of the pharmaceutical industry. Many companies are pursuing this unique class of targets for areas of unmet medical need such as neuropathic and inflammatory pains. In the past, focused library screening sets had been designed for CNS and kinase targets. Our investigations were aimed at creating a similar dynamic screening set enriched for compounds targeting ion channels to aid screening efforts of this important class of targets. The key advantages of this approach for ion channel targets would be: (1) to identify tool compounds for novel targets and assist in assay validation, (2) to serve as a focused screen for non-384-well adaptable targets, and (3) to jump start a particular program, that is, catch-up to competition for validated, well-known targets.

Benzimidazole- and indole-substituted 1,3'-bipyrrolidine benzamides as histamine H3 receptor antagonists.

Using a focused screen of biogenic amine compounds we identified a novel series of H(3)R antagonists. A preliminary SAR study led to reduction of MW while increasing binding affinity and potency. Optimization of the physical properties of the series led to (S)-6n, with improved brain to plasma exposure and efficacy in both water intake and novel object recognition models.

Novel benzofuran derivatives with dual 5-HT1A receptor and serotonin transporter affinity.

Several benzofuran derivatives linked to a 3-indoletetrahydropyridine through an alkyl chain were prepared and evaluated for serotonin transporter and 5-HT(1A) receptor affinities. Their design, synthesis and structure-activity relationships are described.

Novel imidazolopyrimidines as dual PI3-Kinase/mTOR inhibitors.

This article describes the syntheses and SAR of a series of imidazolopyrimidine derivatives, which are evaluated as inhibitors of PI3-Kinase (PI3K) and mTOR. These compounds were found to be ATP competitive with good tumor cell growth inhibition, and suppression of pathway specific biomakers such as phosphorylation of Akt at T308.

Identification of a new class of small molecule C5a receptor antagonists.

C5a is a terminal product of the complement cascade that activates and attracts inflammatory cells including granulocytes, mast cells and macrophages via a specific GPCR, the C5a receptor (C5aR). Inhibition of C5a/C5aR interaction has been shown to be efficacious in several animal models of autoimmune diseases, including RA, SLE and asthma. This account reports the discovery of a new class of C5aR antagonists through high-throughput screening. The lead compounds in this series are selective and block C5a binding, C5a-promoted calcium flux in human neutrophils with nanomolar potency.

Discovery and initial optimization of 5,5'-disubstituted aminohydantoins as potent beta-secretase (BACE1) inhibitors.

8,8-Diphenyl-2,3,4,8-tetrahydroimidazo[1,5-a]pyrimidin-6-amine (1) was identified through HTS, as a weak (micromolar) inhibitor of BACE1. X-Ray crystallographic studies indicate the 2-aminoimidazole ring forms key H-bonding interactions with Asp32 and Asp228 in the catalytic site of BACE1. Lead optimization using structure-based focused libraries led to the identification of low nanomolar BACE1 inhibitors such as 20b with substituents which extend from the S(1) to the S(3) pocket.

Discovery of potent and selective inhibitors of the mammalian target of rapamycin (mTOR) kinase.

The mammalian target of rapamycin (mTOR) is a central regulator of cell growth, metabolism, and angiogenesis and an emerging target in cancer research. High throughput screening (HTS) of our compound collection led to the identification of 3-(4-morpholin-4-yl-1-piperidin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)phenol (5a), a modestly potent and nonselective inhibitor of mTOR and phosphoinositide 3-kinase (PI3K). Optimization of compound 5a, employing an mTOR homology model based on an X-ray crystal structure of closely related PI3Kgamma led to the discovery of 6-(1H-indol-5-yl)-4-morpholin-4-yl-1-[1-(pyridin-3-ylmethyl)piperidin-4-yl]-1H-pyrazolo[3,4-d]pyrimidine (5u), a potent and selective mTOR inhibitor (mTOR IC(50) = 9 nM; PI3Kalpha IC(50) = 1962 nM). Compound 5u selectively inhibited cellular biomarker of mTORC1 (P-S6K, P-4EBP1) and mTORC2 (P-AKT S473) over the biomarker of PI3K/PDK1 (P-AKT T308) and did not inhibit PI3K-related kinases (PIKKs) in cellular assays. These pyrazolopyrimidines represent an exciting new series of mTOR-selective inhibitors with potential for development for cancer therapy.

Biochemical, cellular, and in vivo activity of novel ATP-competitive and selective inhibitors of the mammalian target of rapamycin.

The mammalian target of rapamycin (mTOR) is centrally involved in cell growth, metabolism, and angiogenesis. While showing clinical efficacy in a subset of tumors, rapamycin and rapalogs are specific and allosteric inhibitors of mTOR complex 1 (mTORC1), but they do not directly inhibit mTOR complex 2 (mTORC2), an emerging player in cancer. Here, we report chemical structure and biological characterization of three pyrazolopyrimidine ATP-competitive mTOR inhibitors, WAY-600, WYE-687, and WYE-354 (IC(50), 5-9 nmol/L), with significant selectivity over phosphatidylinositol 3-kinase (PI3K) isofoms (>100-fold). Unlike the rapalogs, these inhibitors acutely blocked substrate phosphorylation by mTORC1 and mTORC2 in vitro and in cells in response to growth factor, amino acids, and hyperactive PI3K/AKT. Unlike the inhibitors of PI3K or dual-pan PI3K/mTOR, cellular inhibition of P-S6K1(T389) and P-AKT(S473) by the pyrazolopyrimidines occurred at significantly lower inhibitor concentrations than those of P-AKT(T308) (PI3K-PDK1 readout), showing mTOR selectivity in cellular setting. mTOR kinase inhibitors reduced AKT downstream function and inhibited proliferation of diverse cancer cell lines. These effects correlated with a strong G(1) cell cycle arrest in both the rapamycin-sensitive and rapamycin-resistant cells, selective induction of apoptosis, repression of global protein synthesis, and down-regulation of angiogenic factors. When injected into tumor-bearing mice, WYE-354 inhibited mTORC1 and mTORC2 and displayed robust antitumor activity in PTEN-null tumors. Together, our results highlight mechanistic differentiation between rapalogs and mTOR kinase inhibitors in targeting cancer cell growth and survival and provide support for clinical development of mTOR kinase inhibitors as new cancer therapy.

Synthesis and activity of tryptophan sulfonamide derivatives as novel non-hydroxamate TNF-alpha converting enzyme (TACE) inhibitors.

A novel series of non-hydroxamate tryptophan sulfonamide derivatives containing a butynyloxy P1' moiety was identified as inhibitors of TNF-alpha converting enzyme (TACE). The structure-activity relationship of the series was examined via substitution on the tryptophan indole ring. Of the compounds investigated, 2-(4-(but-2-ynyloxy)phenylsulfonamido)-3-(1-(4-methoxybenzyl)-1H-indol-3-yl)propanoic acid (12p) has the best in vitro potency against isolated TACE enzyme with an IC(50) of 80 nM. Compound 12p also shows good selectivity over MMP-1, -13, -14.

(S)-N-(5-Chlorothiophene-2-sulfonyl)-beta,beta-diethylalaninol a Notch-1-sparing gamma-secretase inhibitor.

Accumulation of beta-amyloid (Abeta), produced by the proteolytic cleavage of amyloid precursor protein (APP) by beta- and gamma-secretase, is widely believed to be associated with Alzheimer's disease (AD). Research around the high-throughput screening hit (S)-4-chlorophenylsulfonyl isoleucinol led to the identification of the Notch-1-sparing (9.5-fold) gamma-secretase inhibitor (S)-N-(5-chlorothiophene-2-sulfonyl)-beta,beta-diethylalaninol 7.b.2 (Abeta(40/42) EC(50)=28 nM), which is efficacious in reduction of Abeta production in vivo.

Discovery of begacestat, a Notch-1-sparing gamma-secretase inhibitor for the treatment of Alzheimer's disease.

SAR on HTS hits 1 and 2 led to the potent, Notch-1-sparing GSI 9, which lowered brain Abeta in Tg2576 mice at 100 mg/kg po. Converting the metabolically labile methyl groups in 9 to trifluoromethyl groups afforded the more stable analogue 10, which had improved in vivo potency. Further side chain modification afforded the potent Notch-1-sparing GSI begacestat (5), which was selected for development for the treatment of Alzheimer's disease.

Identification, characterization and initial hit-to-lead optimization of a series of 4-arylamino-3-pyridinecarbonitrile as protein kinase C theta (PKCtheta) inhibitors.

The protein kinase C (PKC) family of serine/threonine kinases is implicated in a wide variety of cellular processes. The PKC theta (PKCtheta) isoform is involved in TCR signal transduction and T cell activation and regulates T cell mediated diseases, including lung inflammation and airway hyperresponsiveness. Thus inhibition of PKCtheta enzyme activity by a small molecule represents an attractive strategy for the treatment of asthma. A PKCtheta high-throughput screening (HTS) campaign led to the identification of 4-(3-bromophenylamino)-5-(3,4-dimethoxyphenyl)-3-pyridinecarbonitrile 4a, a low microM ATP competitive PKCtheta inhibitor. Structure based hit-to-lead optimization led to the identification of 5-(3,4-dimethoxyphenyl)-4-(1H-indol-5-ylamino)-3-pyridinecarbonitrile 4p, a 70 nM PKCtheta inhibitor. Compound 4p was selective for inhibition of novel PKC isoforms over a panel of 21 serine/threonine, tyrosine, and phosphoinositol kinases, in addition to the conventional and atypical PKCs, PKCbeta, and PKCzeta, respectively. Compound 4p also inhibited IL-2 production in antiCD3/anti-CD28 activated T cells enriched from splenocytes.

Discovery of a novel series of Notch-sparing gamma-secretase inhibitors.

Using a cell-based assay, we have identified a new series of Notch-sparing gamma-secretase inhibitors from HTS screening leads 2a and 2e. Lead optimization studies led to the discovery of analog 8e with improved gamma-secretase inhibitory potency and Notch-sparing selectivity.

Discovery of benzisoxazoles as potent inhibitors of chaperone heat shock protein 90.

Heat shock protein 90 (Hsp90) is a molecular chaperone that is responsible for activating many signaling proteins and is a promising target in tumor biology. We have identified small-molecule benzisoxazole derivatives as Hsp90 inhibitors. Crystallographic studies show that these compounds bind in the ATP binding pocket interacting with the Asp93. Structure based optimization led to the identification of potent analogues, such as 13, with good biochemical profiles.

Acylguanidine inhibitors of beta-secretase: optimization of the pyrrole ring substituents extending into the S1' substrate binding pocket.

The proteolytic enzyme beta-secretase (BACE-1) produces amyloid beta (Abeta) peptide, the primary constituent of neurofibrillary plaques, implicated in Alzheimer's disease, by cleavage of the amyloid precursor protein. A small molecule inhibitor of BACE-1, (diaminomethylene)-2,5-diphenyl-1H-pyrrole-1-acetamide (1, BACE-1 IC(50)=3.7 microM), was recently described, representing a new small molecule lead. Initial SAR investigation demonstrated the potential of accessing the nearby S(3) and S(1)(') substrate binding pockets of the BACE-1 enzyme by building substituents off one of the phenyl substituents and guanidinyl functional group. We report here the optimization of guanidinyl functional group substituents on 1, leading to potent submicromolar BACE-1 inhibitors.

Acylguanidine inhibitors of beta-secretase: optimization of the pyrrole ring substituents extending into the S1 and S3 substrate binding pockets.

Proteolytic cleavage of amyloid precursor protein by beta-secretase (BACE-1) and gamma-secretase leads to formation of beta-amyloid (A beta) a key component of amyloid plaques, which are considered the hallmark of Alzheimer's disease. Small molecule inhibitors of BACE-1 may reduce levels of A beta and thus have therapeutic potential for treating Alzheimer's disease. We recently reported the identification of a novel small molecule BACE-1 inhibitor N-[2-(2,5-diphenyl-pyrrol-1-yl)-acetyl]guanidine (3.a.1). We report here the initial hit-to-lead optimization of this hit and the SAR around the aryl groups occupying the S(1) and S(2') pockets leading to submicromolar BACE-1 inhibitors.

Discovery of N1-(6-chloroimidazo[2,1-b][1,3]thiazole-5-sulfonyl)tryptamine as a potent, selective, and orally active 5-HT(6) receptor agonist.

N1-Arylsulfonyltryptamines have been identified as 5-HT6 receptor ligands. In particular, N1-(6-chloroimidazo[2,1-b][1,3]thiazole-5-sulfonyl)tryptamine (11q) is a high affinity, potent full agonist (5-HT6 Ki = 2 nM, EC50 = 6.5 nM, Emax = 95.5%). Compound 11q is selective in a panel of over 40 receptors and ion channels, has good pharmacokinetic profile, has been shown to increase GABA levels in the rat frontal cortex, and is active in the schedule-induced polydipsia model for obsessive compulsive disorders.