Discovery of (-)-6-[2-[4-(3-fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl]-3,4-dihydro-2(1H)-quinolinone--a potent NR2B-selective N-methyl D-aspartate (NMDA) antagonist for the treatment of pain

(-)-6-[2-[4-(3-Fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl]-3,4-dihydro-2(1H)-quinolinone was identified as an orally active NR2B-subunit selective N-methyl-d-aspartate (NMDA) receptor antagonist. It has very high selectivity for NR2B subunits containing NMDA receptors versus the HERG-channel inhibition (therapeutic index=4200 vs NR2B binding IC(50)). This compound has improved pharmacokinetic properties compared to the prototype CP-101,606.     

Novel substituted (Z)-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-one and (Z)-(+/-)-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-ol derivatives as potent thermal sensitizing agents

Use of ionizing radiation is essential for the management of many human cancers, and therapeutic hyperthermia has been identified as a potent radiosensitizer. Radiation therapy combined with adjuvant hyperthermia represents a potential tool to provide outstanding local-regional control for refractory disease. (Z)-(±)-2-(N-Benzylindol-3-ylmethylene)quinuclidin-3-ol (2) and (Z)-(±)-2-(N-benzenesulfonylindol-3-ylmethylene)quinuclidin-3-ol (4) were initially identified as potent thermal sensitizers that could lower the threshold needed for thermal sensitivity to radiation treatment. To define the structural requirements of the molecule that are essential for thermal sensitization, we have synthesized and evaluated a series of (Z)-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-one (9), and (Z)-(±)-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-ol (10) analogs that incorporate a variety of substituents in both the indole and N-benzyl moieties. These systematic structure–activity relationship (SAR) studies were designed to further the development and optimization of potential clinically useful thermal sensitizing agents. The most potent analog was compound 10 (R¹ = H, R² = 4-Cl), which potently inhibited (93% inhibition at 50 μM) the growth of HT-29 cells after a 41 °C/2 h exposure.     

Synthesis and sulfatase inhibitory activities of (E)- and (Z)-4-hydroxytamoxifen sulfamates

We report the development of (E)- and (Z)-4-hydroxytamoxifen sulfamates as estrone sulfatase inhibitors, potential therapeutic agents for the treatment of breast cancer. Both compounds competitively inhibit estrone sulfatase isolated from rat liver with apparent Ki of 35.9 microM for (E)-4-hydroxytamoxifen sulfamate and an apparent Ki of > 500 microM for the (Z) isomer.     

Therapeutic efficacy of 4-(adamantyl-1)-1-(1-aminobutyl) benzene in experimental fungal pathology

Antifungal activity of an adamantane derivative, i. e. 4-(adamantyl-1)-1-(1-aminobutyl) benzol was studied in experiments in vivo in a model of generalized infection and local skin injury. In doses of 0.1 LD50, 0.001 LD50, 0.001 LD50 (intraperitoneal administration) it had a therapeutic effect on generalized candidiasis and promoted 60-80-percent survival of the laboratory animals. Its application as 1-percent ointment to Candida albicans infected skin prevented the wound formation in rabbits and showed therapeutic efficacy in the treatment of infected wounds in albino rats.     

Synthesis and in vivo evaluation of (S)-6-(4-fluorophenoxy)-3-((1-[11C]methylpiperidin-3-yl)methyl)-2-o-tolylquinazolin-4(3H)-one, a potential PET tracer for growth hormone secretagogue receptor (GHSR)

The peptide hormone ghrelin mediates through action on its receptor, the growth hormone secretagogue receptor (GHSR), and is known to play an important role in a variety of metabolic functions including appetite stimulation, weight gain, and suppression of insulin secretion. In light of the fact that obesity is one of the major health problems plaguing the modern society, the ghrelin signaling system continues to remain an important and attractive pharmacological target for the treatment of obesity. In vivo imaging of the GHSR could shed light on the mechanism by which ghrelin affects feeding behavior and thus offers a new therapeutic perspective for the development of effective treatments. Recently, a series of piperidine-substituted quinazolinone derivatives was reported to be selective and potent GHSR antagonists with high binding affinities. Described herein is the synthesis, in vitro, and in vivo evaluation of (S)-6-(4-fluorophenoxy)-3-((1-[(11)C]methylpiperidin-3-yl)methyl)-2-o-tolylquinazolin-4(3H)-one ([(11)C]1), a potential PET radioligand for imaging GHSR.     

(R)- and (S)-4-Amino-3-(trimethylsilyl)methylbutanoic acids ameliorate neuropathic pain without central nervous system-related side effects

Neuropathic pain is a chronic pain condition resulting from neuronal damage, and is usually treated with pregabalin or gabapentin, which are structurally related to γ-aminobutyric acid (GABA) and are originally developed as anticonvulsant drugs. Here, we report the synthesis and pharmacology of (R)- and (S)-4-amino-3-(trimethylsilyl)methylbutanoic acids (1a and 1b), which showed analgesic activity as potent as that of pregabalin in the Chung spinal nerve ligation model. However, unlike pregabalin, 1a and 1b do not have antiepileptic effects, and they are therefore promising candidates for selective therapeutic agents to treat neuropathic pain without central nervous system-related side effects.     

Preparation and immunosuppressive activity of 32-(O)-acylated and 32-(O)-thioacylated analogues of ascomycin

A series of 32-(O)-acylated and 32-(O)-thioacylated derivatives of the antibiotic ascomycin (1) have been synthesized. These readily accessible analogues exhibit potent immunosuppressive activity in vitro, as measured by an interleukin-2 reporter gene assay and the mixed lymphocyte reaction. Such molecules are expected to have a therapeutic potential in chronic inflammatory diseases of the airways such as asthma.     

Design and synthesis of novel 3-(benzo[d]oxazol-2-yl)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridin-2-amine derivatives as selective G-protein-coupled receptor kinase-2 and -5 inhibitors

G-protein-coupled receptor kinase (GRK)-2 and -5 are emerging therapeutic targets for the treatment of cardiovascular disease. In our efforts to discover novel small molecules to inhibit GRK-2 and -5, a class of compound based on 3-(benzo[d]oxazol-2-yl)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridin-2-amine was identified as a novel hit by high throughput screening campaign. Structural modification of parent benzoxazole scaffolds by introducing substituents on phenyl displayed potent inhibitory activities toward GRK-2 and -5.     

Therapeutic effect of (Z)-3-(2,5-dimethoxyphenyl)-2-(4-methoxyphenyl) acrylonitrile (DMMA) against Staphylococcus aureus infection in a murine model

Sortase enzymes belong to a family of transpeptidases found in Gram-positive bacteria. Sortase is responsible for the reaction that anchors surface protein virulence factors to the peptidoglycan cell wall of the bacteria. The compound (Z)-3-(2,5-dimethoxyphenyl)-2-(4-methoxyphenyl) acrylonitrile (DMMA) has previously been reported as a novel sortase inhibitor in vitro, but the in vivo effects of DMMA have not been studied. Here, we evaluated the in vivo effects of DMMA against infection by wild-type and sortase A- and/or sortase B-deficient Staphylococcus aureus in Balb/c mice. With DMMA treatment, survival rates increased and kidney and joint infection rates decreased (p < 0.01) in a dose-dependent manner. The rate of kidney infection was significantly reduced in the mice treated with sortase A knock-out S. aureus (p < 0.01). These results indicate that by acting as a potent inhibitor of sortase A and moderate inhibitor of sortase B, DMMA can decrease kidney and joint infection rates and reduce mortality in mice infected with S. aureus. These findings suggest that DMMA is a promising therapeutic compound against Gram-positive bacteria.     

Biological activity and antidiabetic potential of synthetic fragment peptides of glucose-dependent insulinotropic polypeptide, GIP(1-16) and (Pro3)GIP(1-16)

Glucose-dependent insulinotropic polypeptide (GIP) is a key physiological insulin releasing peptide and potential antidiabetic agent. The present study was undertaken in an attempt to develop small molecular weight GIP agonist and antagonist molecules. The bioactivity of two modified C-terminally truncated fragment GIP peptides, GIP(1-16) and (Pro3)GIP(1-16), was examined in terms of insulin secretion and glucose homeostasis using BRIN-BD11 cells and type 2 diabetic mice. In vitro insulin release studies demonstrated that GIP(1-16) and (Pro3)GIP(1-16) possessed weak GIP-receptor agonist and antagonistic properties, respectively. Intraperitoneal administration of GIP(1-16) in combination with glucose to obese diabetic (ob/ob) mice did not effect the glycaemic excursion and had a marginal effect on insulin release. GIP(1-16) was substantially less effective than the native GIP(1-42). (Pro3)GIP(1-16) administration significantly curtailed (P < 0.05) the insulinotropic and glucose lowering effects of native GIP, but was significantly less effective than (Pro3)GIP. Based on the established concept of a therapeutic benefit of GIP receptor antagonism in obesity-diabetes, ob/ob mice received once daily injection of (Pro3)GIP(1-16) for 14 days. No significant effects were observed on food intake, body weight, HbA1c, glucose tolerance, metabolic response to feeding and either insulin secretion or insulin sensitivity following prolonged (Pro3)GIP(1-16) treatment. These data demonstrate that C-terminal truncation of GIP or (Pro3)GIP yields small molecular weight GIP molecules with significantly reduced biological activity that precludes therapeutic utility.     

Synthesis and dopamine transporter affinity of chiral 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(2-hydroxypropyl)piperazines as potential cocaine abuse therapeutic agents

A series of optically pure phenyl-and non-phenyl-substituted 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(2-hydroxypropyl)piperazines was synthesized and their binding affinity for dopamine transporter (DAT) was investigated. The analogues with a hydroxyl group in the S configuration were more selective for the DAT over the serotonin transporter (SERT) than the corresponding R enantiomers. Compound (+)-11 showed high affinity and selectivity for DAT over the SERT and, therefore, is a potential candidate for the development of a long-acting cocaine abuse therapeutic agent.     

Adipocyte enhancer-binding protein 1 (AEBP1) (a novel macrophage proinflammatory mediator) overexpression promotes and ablation attenuates atherosclerosis in ApoE (-/-) and LDLR (-/-) mice

Atherogenesis is a long-term process that involves inflammatory response coupled with metabolic dysfunction. Foam cell formation and macrophage inflammatory response are two key events in atherogenesis. Adipocyte enhancer-binding protein 1 (AEBP1) has been shown to impede macrophage cholesterol efflux, promoting foam cell formation, via peroxisome proliferator-activated receptor (PPAR)-γ1 and liver X receptor α (LXRα) downregulation. Moreover, AEBP1 has been shown to promote macrophage inflammatory responsiveness by inducing nuclear factor (NF)-κB activity via IκBα downregulation. Lipopolysaccharide (LPS)-induced suppression of pivotal macrophage cholesterol efflux mediators, leading to foam cell formation, has been shown to be mediated by AEBP1. Herein, we showed that AEBP1-transgenic mice (AEBP1(TG)) with macrophage-specific AEBP1 overexpression exhibit hyperlipidemia and develop atherosclerotic lesions in their proximal aortas. Consistently, ablation of AEBP1 results in significant attenuation of atherosclerosis (males: 3.2-fold, P = 0.001 [en face]), 2.7-fold, P = 0.0004 [aortic roots]; females: 2.1-fold, P = 0.0026 [en face], 1.7-fold, P = 0.0126 [aortic roots]) in the AEBP1(-/-)/low-density lipoprotein receptor (LDLR )(-/-) double-knockout (KO) mice. Bone marrow (BM) transplantation experiments further revealed that LDLR (-/-) mice reconstituted with AEBP1(-/-)/LDLR (-/-) BM cells (LDLR (-/-)/KO-BM chimera) display significant reduction of atherosclerosis lesions (en face: 2.0-fold, P = 0.0268; aortic roots: 1.7-fold, P = 0.05) compared with control mice reconstituted with AEBP1(+/+)/LDLR (-/-) BM cells (LDLR (-/-)/WT-BM chimera). Furthermore, transplantation of AEBP1(TG) BM cells with the normal apolipoprotein E (ApoE) gene into ApoE (-/-) mice (ApoE (-/-)/TG-BM chimera) leads to significant development of atherosclerosis (males: 2.5-fold, P = 0.0001 [en face], 4.7-fold, P = 0.0001 [aortic roots]; females: 1.8-fold, P = 0.0001 [en face], 3.0-fold, P = 0.0001 [aortic roots]) despite the restoration of ApoE expression. Macrophages from ApoE (-/-)/TG-BM chimeric mice express reduced levels of PPARγ1, LXRα, ATP-binding cassette A1 (ABCA1) and ATP-binding cassette G1 (ABCG1) and increased levels of the inflammatory mediators interleukin (IL)-6 and tumor necrosis factor (TNF)-α compared with macrophages of control chimeric mice (ApoE (-/-)/NT-BM ) that received AEBP1 nontransgenic (AEBP1(NT) ) BM cells. Our in vivo experimental data strongly suggest that macrophage AEBP1 plays critical regulatory roles in atherogenesis, and it may serve as a potential therapeutic target for the prevention or treatment of atherosclerosis.     

Discovery of (S)-2-((S)-2-(3,5-difluorophenyl)-2-hydroxyacetamido)-N-((S,Z)-3-methyl-4-oxo-4,5-dihydro-3H-benzo[d][1,2]diazepin-5-yl)propanamide (BMS-433796): a gamma-secretase inhibitor with Abeta lowering activity in a transgenic mouse model of Alzheimer's disease

We report on the design of benzodiazepinones as peptidomimetics at the carboxy terminus of hydroxyamides. Structure-activity relationships of diazepinones were investigated and orally active gamma-secretase inhibitors were synthesized. Active metabolites contributing to Abeta reduction were identified by analysis of plasma samples from Tg2576 mice. In particular, (S)-2-((S)-2-(3,5-difluorophenyl)-2-hydroxyacetamido)-N-((S,Z)-3-methyl-4-oxo-4,5-dihydro-3H-benzo[d][1,2]diazepin-5-yl)propanamide (BMS-433796) was identified with an acceptable pharmacodynamic and pharmacokinetic profile. Chronic dosing of BMS-433796 in Tg2576 mice suggested a narrow therapeutic window and Notch-mediated toxicity at higher doses.     

Anti-inflammatory and anti-arthritic effects of new synthetic 3-(4-hydroxyphenyl)-4-(4-thiomethoxyphenyl)-1H-pyrrole-2,5-dione

We previously reported that 3-(4-hydroxyphenyl)-4-(4-thiomethoxyphenyl)-1H-pyrrole-2,5-dione (1, HMP) has a strong inhibitory effect on prostaglandin E(2) (PGE(2)) production. In this study, the anti-inflammatory and anti-arthritic effects of HMP were evaluated on LPS-induced RAW 264.7 macrophages and rats with carrageenan-induced paw edema and adjuvant-induced arthritis (AIA). The attenuation of PGE(2) production by HMP was found to be caused by the inhibition of cyclooxygenase-2 (COX-2) activity, but not COX-1 activity. However, HMP did not affect COX-2 at the protein or mRNA levels, whereas it suppressed the releases and expressions of inflammatory cytokines, such as, interleukin-1β (IL-1β) and IL-6 in LPS-induced macrophages. Furthermore, HMP suppressed LPS-induced nitric oxide (NO) production by down regulating the protein and mRNA expressions of inducible nitric oxide synthase (iNOS). In rats with carrageenan-injected acute inflammation, oral administration of HMP (25 or 50mg/kg, po) reduced paw swelling, and PGE(2) release and myeloperoxidase (MPO) activity in tissue. Furthermore, HMP (25 or 50mg/kg, po) significantly reduced paw swelling, arthritic indices and plasma PGE(2) concentrations in rat with AIA. These results show that HMP reduces swelling in a model acute inflammation and inhibits arthritic responses in a model of chronic inflammation via the inhibition of PGE(2) production. These results suggest that HMP is a potential therapeutic agent for the treatment of arthritis and associated disorders.     

1-(sulfonyl)-5-(arylsulfonyl)indoline as activators of the tumor cell specific M2 isoform of pyruvate kinase

Cancer cells preferentially use glycolysis rather than oxidative phosphorylation for their rapid growth. They consume large amount of glucose to produce lactate even when oxygen is abundant, a phenomenon known as the Warburg effect. This metabolic change originates from a shift in the expression of alternative spliced isoforms of the glycolytic enzyme pyruvate kinase (PK), from PKM1 to PKM2. While PKM1 is constitutively active, PKM2 is switched from an inactive dimer form to an active tetramer form by small molecule activators. The prevalence of PKM2 in cancer cells relative to the prevalence of PKM1 in many normal cells, suggests a therapeutic strategy whereby activation of PKM2 may counter the abnormal cellular metabolism in cancer cells, and consequently decreased cellular proliferation. Herein we describe the discovery and optimization of a series of PKM2 activators derived from the 2-((2,3-dihydrobenzo[b][1,4] dioxin-6-yl)thio)-1-(2-methyl-1-(methylsulfonyl)indolin-5-yl) ethanone scaffold. The synthesis, SAR analysis, enzyme active site docking, enzymatic reaction kinetics, selectivity and pharmaceutical properties are discussed.     

Potential Antileukemia Effect and Structural Analyses of SRPK Inhibition by N-(2-(Piperidin-1-yl)-5-(Trifluoromethyl)Phenyl)Isonicotinamide (SRPIN340)

Dysregulation of pre-mRNA splicing machinery activity has been related to the biogenesis of several diseases. The serine/arginine-rich protein kinase family (SRPKs) plays a critical role in regulating pre-mRNA splicing events through the extensive phosphorylation of splicing factors from the family of serine/arginine-rich proteins (SR proteins). Previous investigations have described the overexpression of SRPK1 and SRPK2 in leukemia and other cancer types, suggesting that they would be useful targets for developing novel antitumor strategies. Herein, we evaluated the effect of selective pharmacological SRPK inhibition by N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340) on the viability of lymphoid and myeloid leukemia cell lines. Along with significant cytotoxic activity, the effect of treatments in regulating the phosphorylation of the SR protein family and in altering the expression of MAP2K1, MAP2K2, VEGF and FAS genes were also assessed. Furthermore, we found that pharmacological inhibition of SRPKs can trigger early and late events of apoptosis. Finally, intrinsic tryptophan fluorescence emission, molecular docking and molecular dynamics were analyzed to gain structural information on the SRPK/SRPIN340 complex. These data suggest that SRPK pharmacological inhibition should be considered as an alternative therapeutic strategy for fighting leukemias. Moreover, the obtained SRPK-ligand interaction data provide useful structural information to guide further medicinal chemistry efforts towards the development of novel drug candidates.     

Preparation of alkyl (R)-(-)-3-hydroxybutyrate by acidic alcoholysis of poly-(R)-(-)-3-hydroxybutyrate

An efficient method for the preparation of optically active alkyl (R)-(-)-3-hydroxybutyrates by chemical depolymerization of biopolymer, poly-(R)-(-)-(3-hydroxybutyrate), was established. This method consists of simple recovery of poly-(R)-(-)-(3-hydroxybutyrate) from bacterial cells followed by acidic alcoholysis. When poly-(R)-(-)-(3-hydroxybutyrate) was purified by a simple digestion method that used 0.2 N sodium hydroxide, alkyl (R)-(-)-hydroxybutyrates were most efficiently produced by alcoholysis with anhydrous hydrochloric acid.     

Selective inhibition of heme oxygenase-2 activity by analogs of 1-(4-chlorobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-benzimidazole (clemizole): Exploration of the effects of substituents at the N-1 position

Several analogs based on the lead structure of 1-(4-chlorobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-benzimidazole (clemizole) were synthesized and evaluated as novel inhibitors of heme oxygenase (HO). Many of the compounds were found to be potent and highly selective for the HO-2 isozyme (constitutive), and had substantially less inhibitory activity on the HO-1 isozyme (inducible). The compounds represent the first report of highly potent and selective inhibitors of HO-2 activity, and complement our suite of selective HO-1 inhibitors. The study has revealed many candidates based on the inhibition of heme oxygenases for potentially useful pharmacological and therapeutic applications.     

Design, synthesis, and biological evaluation of 4-(5-dimethylamino-naphthalene-1-sulfon-amido)-3-(4-iodophenyl)butanoic acid as a novel molecular probe for apoptosis imaging

Apoptosis (programmed cell death) plays a crucial role in the pathogenesis of many disorders, thus the detection of apoptotic cells can provide the physician with important information to further therapeutic strategies and would substantially advance patient care. A small molecule, 4-(5-dimethylamino-naphthalene-1-sulfonamido)-3-(4-iodo-phenyl)butanoic acid (DNSBA), was designed as a novel probe for imaging apoptosis and synthesized with good yield. The biological characterization demonstrated that DNSBA can be used to specifically and selectively detect apoptotic cancer cells at all stages. DNSBA is also designed as a potential SPECT and PET probe when labeled with radioiodine (I-123, -124, and -131).     

Triazole analog 1-(1-benzyl-5-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2-(4-bromophenylamino)-1-(4-chlorophenyl)ethanol induces reactive oxygen species and autophagy-dependent apoptosis in both in vitro and in vivo breast cancer models

Autophagy is considered as an important cell death mechanism that closely interacts with other common cell death programs like apoptosis. Critical role of autophagy in cell death makes it a promising, yet challenging therapeutic target for cancer. We identified a series of 1,2,3-triazole analogs having significant breast cancer inhibition property. Therefore, we attempted to study whether autophagy and apoptosis were involved in the process of cancer cell inhibition. The lead molecule, 1-(1-benzyl-5-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2-(4-bromophenylamino)-1-(4-chlorophenyl)ethanol (T-12) induced significant cell cycle arrest, mitochondrial membrane depolarization, apoptosis and autophagy in MCF-7 and MDA-MB-231 cells. T-12 increased reactive oxygen species and its inhibition by N-acetyl-l-cysteine protected breast cancer cells from autophagy and apoptosis. Autophagy inhibitor, 3-methyladenine abolished T-12 induced apoptosis, mitochondrial membrane depolarization and reactive oxygen species generation. This suggested that T-12 induced autophagy facilitated cell death rather than cell survival. Pan-caspase inhibition did not abrogate T-12 induced autophagy, suggesting that autophagy precedes apoptosis. In addition, T-12 inhibited cell survival pathway signaling proteins, Akt, mTOR and Erk1/2. T-12 also induced significant regression of tumor with oral dose of as low as 10 mg/kg bodyweight in rat mammary tumor model without any apparent toxicity. In presence of reactive oxygen species inhibitor (N-acetyl-l-cysteine) and autophagy inhibitor (chloroquine), T-12 induced tumor regression was significantly decreased. In conclusion, T-12 is a potent inducer of autophagy-dependent apoptosis in breast cancer cells both in vitro and in vivo and can serve as an important lead in development of new anti-tumor therapy.