Homology modeling and 3D-QSAR study of benzhydrylpiperazine δ opioid receptor agonists.

The binding affinity of a series of benzhydrylpiperazine δ opioid receptor agonists were pooled and evaluated by using 3D-QSAR and homology modeling/molecular docking methods. Ligand-based CoMFA and CoMSIA 3D-QSAR analyses with 46 compounds were performed on benzhydrylpiperazine analogues by taking the most active compound BW373U86 as the template. The models were generated successfully with q2 value of 0.508 and r2 value of 0.964 for CoMFA, and q2 value of 0.530 and r2 value of 0.927 for CoMSIA. The predictive capabilities of the two models were validated on the test set with R2pred value of 0.720 and 0.814, respectively. The CoMSIA model appeared to work better in this case. A homology model of active form of δ opioid receptor was established by Swiss-Model using a reported crystal structure of active µ opioid receptor as a template, and was further optimized using nanosecond scale molecular dynamics simulation. The most active compound BW373U86 was docked to the active site of δ opioid receptor and the lowest energy binding pose was then used to identify binding residues such as s Gln105, Lys108, Leu125, Asp128, Tyr129, Leu200, Met132, Met199, Lys214, Trp274, Ile277, Ile304 and Tyr308. The docking and 3D-QSAR results showed that hydrogen bond and hydrophobic interactions played major roles in ligand-receptor interactions. Our results highlight that an approach combining structure-based homology modeling/molecular docking and ligand-based 3D-QSAR methods could be useful in designing of new opioid receptor agonists.

A Novel Naphthyridine Derivative, 3u, Induces Necroptosis at Low Concentrations and Apoptosis at High Concentrations in Human Melanoma A375 Cells.

Naphthyridine derivatives are a widely-used class of heterocycles due to their pharmacological activities. A novel compound (10-Methoxy-1,2,3,4-tetrahydrobenzo(g)(1,3) diazepino(1,2-a)-(1,8)naphthyridin-6-yl)(phenyl) methanone (named 3u), showed good anticancer activity in the human malignant melanoma cell line A375 via Thiazolyl Blue Tetrazolium Bromide (MTT) assay. After Western blotting confirmed, we found that 3u induces necroptosis at low concentrations and apoptosis at high concentrations via the upregulation of death receptors and scaffold protein in A375 cells. Furthermore, by combining 3u with the caspase inhibitor zVAD-fmk or Receptor Interacting Serine/Threonine Kinase 1 (RIP1) kinase inhibitor Necrostatin-1 (Nec-1), we found that the activity of caspase-8 was the crucial factor that determined whether either apoptosis or necroptosis occurred. The results indicate that 3u should be considered as a potential chemical substance for melanoma treatment.

The opioid receptor triple agonist DPI-125 produces analgesia with less respiratory depression and reduced abuse liability.

Opioid analgesics remain the first choice for the treatment of moderate to severe pain, but they are also notorious for their respiratory depression and addictive effects. This study focused on the pharmacology of a novel opioid receptor mixed agonist DPI-125 and attempted to elucidate the relationship between the δ-, µ- and κ-receptor potency ratio and respiratory depression and abuse liability. Five diarylmethylpiperazine compounds (DPI-125, DPI-3290, DPI-130, KUST202 and KUST13T02) were selected for this study. PKA fluorescence redistribution assays in CHO cells individually expressing δ-, µ- or κ-receptors were used to measure the agonist potency. The respiratory safety profiles were estimated in rats by the ratio of ED50 (pCO2 increase)/ED50 (antinociception). The abuse liability of DPI-125 was evaluated with a self-administration model in rhesus monkeys. The observed agonist potencies of DPI-125 for δ-, µ- and κ-opioid receptors were 4.29±0.36, 11.10±3.04, and 16.57±4.14 nmol/L, respectively. The other four compounds were also mixed agonists with varying potencies. DPI-125 exhibited a high respiratory safety profile, clearly related to its high δ-receptor potency. The ratio of the EC50 potencies for the µ- and δ-receptors was found to be positively correlated with the respiratory safety ratio. DPI-125 has similar potencies for µ- and κ-receptors, which is likely the reason for its reduced abuse potential. Our results demonstrate that the opioid receptor mixed agonist DPI-125 is safer and less addictive than traditional µ-agonist analgesics. These findings suggest that the development of δ>µâˆ¼κ opioid receptor mixed agonists is feasible, and such compounds could represent a promising class of potent analgesics with wider therapeutic windows.

Overcoming resistance to TRAIL-induced apoptosis in solid tumor cells by simultaneously targeting death receptors, c-FLIP and IAPs.

The discovery of the TRAIL protein and its death receptors DR4/5 changed the horizon of cancer research because TRAIL specifically kills cancer cells. However, the validity of TRAIL-based cancer therapies has yet to be established, as most cancer cells are TRAIL-resistant. In this report, we demonstrate that TRAIL-resistance of many cancer cell lines can be overcome after siRNA- or rocaglamide-mediated downregulation of c-FLIP expression and simultaneous inhibition of IAPs activity using AT406, a pan-antagonist of IAPs. Combined triple actions of the TRAIL, the IAPs inhibitor, AT406, and the c-FLIP expression inhibitor, rocaglamide (ART), markedly improve TRAIL-induced apoptotic effects in most solid cancer cell lines through the activation of an extrinsic apoptosis pathway. Furthermore, this ART combination does not harm normal cells. Among the 18 TRAIL-resistant cancer cell lines used, 15 cell lines become sensitive or highly sensitive to ART, and two out of three glioma cell lines exhibit high resistance to ART treatment due to very low levels of procaspase-8. This study provides a rationale for the development of TRAIL-induced apoptosis-based cancer therapies.

Inhibition of c-FLIP by RNAi enhances sensitivity of the human osteogenic sarcoma cell line U2OS to TRAIL-induced apoptosis.

To study effects of cellular FLICE (FADD-like IL-1ß-converting enzyme)-inhibitory protein (c-FLIP) inhibition by RNA interference (RNAi) on sensitivity of U2OS cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, plasmid pSUPER-c-FLIP-siRNA was constructed and then transfected into U2OS cells. A stable transfection cell clone U2OS/pSUPER-c-FLIP-siRNA was screened from the c-FLIP-siRNA transfected cells. RT-PCR and Western blotting were applied to measure the expression of c-FLIP at the levels of mRNA and protein. The results indicated that the expression of c-FLIP was significantly suppressed by the c-FLIP-siRNA in the cloned U2OS/pSUPER-c-FLIP-siRNA as compared with the control cells of U2OS/pSUPER. The cloned cell line of U2OS/pSUPER-c-FLIP-siRNA was further examined for TRAIL- induced cell death and apoptosis in the presence of a pan-antagonist of inhibitor of apoptosis proteins (IAPs) AT406, with or without 4 hrs pretreatment with rocaglamide, an inhibitor of c-FLIP biosynthesis, for 24 hrs. Cell death effects and apoptosis were measured by the methods of MTT assay with 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide and flow cytometry, respectively. The results indicated that TRAIL-induced cell death in U2OS/pSUPER-c-FLIP-siRNA was increased compared with control cells U2OS/pSUPER in the presence or absence of AT406. Flow cytometry indicated that TRAIL-induced cell death effects proceeded through cell apoptosis pathway. However, in the presence of rocaglamide, cell death or apoptotic effects of TRAIL were similar and profound in both cell lines, suggesting that the mechanism of action for both c-FLIP-siRNA and rocaglamide was identical. We conclude that the inhibition of c-FLIP by either c-FLIP-siRNA or rocaglamide can enhance the sensitivity of U2OS to TRAIL-induced apopotosis, suggesting that inhibition of c-FLIP is a good target for anti-cancer therapy.

Copper-catalyzed cascade addition/cyclization: highly efficient access to phosphonylated quinoline-2,4(1H,3H)-diones.

A novel and facile Cu-catalyzed addition/cyclization cascade of o-cyanoarylacrylamide was developed. The process exhibits significant functional group tolerance, and provides an efficient and straightforward pathway for the synthesis of various phosphonylated quinoline-2,4(1H,3H)-diones.

Association of DR4 (TRAIL-R1) polymorphisms with cancer risk in Caucasians: an updated meta-analysis.

Death receptor 4 (TRAIL-R1 or DR4) polymorphisms have been associated with cancer risk, but findings have been inconsistent. To estimate the relationship in detail, a meta-analysis was here performed. A search of PubMed was conducted to investigate the association between DR4 C626G, A683C and A1322G polymorphisms and cancer risk, using odds ratios (ORs) with 95% confidence intervals. The results suggested that DR4 C626G and A683C polymorphisms were indeed associated with cancer risk (for C626G, dominant model, OR 0.991, 95%CI 0.866-1.133, p=0.015; for A683C, additive model, OR=1.140, 95%CI: 0.948-1.370, p=0.028; dominant model, OR=1.156, 95%CI: 0.950-1.406, p=0.080) in the Caucasian subgroup. However, the association was not significant between DR4 polymorphism A1322G with cancer risk in Caucasians (For A1322G, additive model: OR 1.085, 95%CI 0.931-1.289, p=0.217; dominant model: OR 1.379, 95%CI 0.934-2.035, p=0.311; recessive model: OR 1.026, 95%CI 0.831-1.268 p=0.429.). In summary, our finding suggests that DR4 polymorphism C626G and A683 rather than A1322G are associated with cancer risk in Caucasians.

ARD-353 [4-((2R,5S)-4-(R)-(4-diethylcarbamoylphenyl)(3-hydroxyphenyl)methyl)-2,5-dimethylpiperazin-1-ylmethyl)benzoic acid], a novel nonpeptide delta receptor agonist, reduces myocardial infarct size without central effects.

A novel delta-receptor selective compound, ARD-353 [4-((2R,5S)-4-(R)-(4-diethylcarbamoylphenyl)(3-hydroxyphenyl)methyl)-2, 5-dimethylpiperazin-1-ylmethyl)benzoic acid], was evaluated for activity on infarct size in a rat model of acute myocardial infarction. ARD-353 was characterized as having delta receptor selectivity using radioligand binding and had no apparent selectivity between delta receptor subtypes as determined by [(3)H] cyclic [D-Pen(2),D-Pen(5)]enkephalin (delta(1)) and [(3)H]Deltorphin II (delta(2)) competition binding. ARD-353 also showed selective delta receptor agonist activity in mouse-isolated vas deferens. There was no evidence of any seizure-like convulsions when ARD-353 was administered to mice either i.v. or p.o., implying minimal penetration of the blood-brain barrier. ARD-353 decreased infarct size in a left anterior descending coronary artery (LAD) occlusion model of myocardial infarction. In animals pretreated with ARD-353 (i.v.) and then subjected to 30 min of LAD occlusion followed by 90 min of reperfusion, infarct size was reduced in a dose-dependent manner compared with vehicle-treated controls. The effects of ARD-353 on infarct size were blocked by the delta(1)-opioid selective antagonist 7-benzylidenenaltrexone, indicating a significant role for the delta(1)-opioid receptor in the cardioprotective mechanism of ARD-353. ARD-353 (0.3 mg/kg i.v.) produced significant protection when administered 5 min and 12 and 48 h before ischemic insult or when given immediately after the ischemic insult (at the start of reperfusion). Given the lack of central nervous system effects and beneficial efficacy in the rat model of myocardial ischemia, it is felt that ARD-353 is the first nonpeptide delta-receptor agonist with true potential for clinical use before surgically induced ischemia or in an emergency setting.

DPI-221 [4-((alpha-s)-alpha-((2s,5r)-2,5-dimethyl-4-(3-fluorobenzyl)-1-piperazinyl)benzyl)-N,N-diethylbenzamide]: a novel nonpeptide delta receptor agonist producing increased micturition interval in normal rats.

There is a wealth of information from animal models and clinical opioid-analgesic use that indicates a significant role for opioid receptors in the modulation of bladder activity. The novel benzhydrylpiperazine compound DPI-221 [4-((alpha-S)-alpha-((2S,5R)-2,5-dimethyl-4-(3-fluorobenzyl)-1-piperazinyl)benzyl)-N,N-diethylbenzamide] was characterized as having delta receptor selectivity using radioligand binding (K(i) = 2.0 +/- 0.7 nM, delta receptor; 1800 +/- 360 nM, mu receptor; and 2300 +/- 680 nM, kappa receptor), and agonist activity was demonstrated in the mouse isolated vas deferens where DPI-221 inhibited electrically induced contractions with an IC(50) value of 88 +/- 7.5 nM. In the guinea pig isolated ileum, DPI-221 had no effect on electrically induced contractions at concentrations as high as 1 microM. Sterile saline was infused (7 ml/h) into the bladder of Sprague-Dawley rats, via a transmural catheter; DPI-221 (1.0 to 20 mg/kg p.o.) significantly increased the interval between micturition events, whereas peak void pressure was not significantly decreased by any dose of DPI-221. The micturition effects of 10 mg/kg p.o. DPI-221 were blocked by naltrindole, indicating a delta receptor mechanism of action. In isolated rat bladder strips, DPI-221 was ineffective at relaxing detrusor muscle precontracted with carbachol. The most crucial safety aspect of delta agonist administration is the incidence of seizure-like convulsions in rodents. DPI-221 produced no convulsions at doses up to 100 mg/kg p.o. in mice, although rapid bolus i.v. injection of 5 mg/kg produced convulsions in 3% of mice tested. These findings indicate a good safety profile for DPI-221 administered orally, with potent efficacy in modifying bladder activity.

DPI-3290 [(+)-3-((alpha-R)-alpha-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N-(3-fluorophenyl)-N-methylbenzamide]. II. A mixed opioid agonist with potent antinociceptive activity and limited effects on respiratory function.

Allyl-2,5-dimethyl-1-piperazines have been of interest as analgesic agents for the management of moderate-to-severe pain. In this study, we compared the antinociceptive properties and respiratory depressant activity of one such agent, (+)-3-((alpha-R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N-(3-fluorophenyl)-N-methylbenzamide (DPI-3290), with those of established narcotic analgesics, morphine and fentanyl. Intravenous administration of DPI-3290 in conscious laboratory rats increased antinociception in a dose-dependent manner with a corresponding ED(50) value of 0.05 +/- 0.0072 mg/kg. Simultaneous measurement of arterial blood gas in animals treated with DPI-3290 demonstrated dose-dependent increases in pCO2 with an ED(50) value of 0.91 +/- 0.22 mg/kg. In comparison, morphine and fentanyl increased antinociception in rats with ED(50) values of 2.01 +/- 0.0005 and 0.0034 +/- 0.00024 mg/kg, respectively, and the ED(50) value for morphine-induced changes in pCO2 was 4.23 +/- 0.72 mg/kg, whereas the ED(50) value for fentanyl-induced changes in pCO2 was 0.0127 +/- 0.0035 mg/kg. A separate series of experiments were designed to examine the effects of DPI-3290 on mu-opioid receptor induced antinociception and hypercapnia. Intravenous bolus doses of DPI-3290 that ranged from 0.2 to 1.0 mg/kg had no effect on antinociception mediated by alfentanil (2 microg/kg/min i.v.) but reduced hypercapnia by approximately 50%. Results from these studies demonstrate the equivalent antinociceptive efficacy of DPI-3290, morphine, and fentanyl but dramatic differences in the hypercapnia that antinociceptive doses of these drugs produce. When measured simultaneously, DPI-3290 had an 18.2-fold difference in the ratio comparing the ED(50) value for antinociception with the ED(50) value for changes in pCO2; this ratio was 2.1 for morphine and 3.7 for fentanyl. Furthermore, DPI-3290 reduced the alfentanil-mediated hypercapnia without any effect on antinociception. Together, the balanced opioid agonist activity of DPI-3290 may provide a means of powerful analgesia while mitigating the mu-opioid receptor-mediated hypercapnia.

DPI-3290 [(+)-3-((alpha-R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N-(3-fluorophenyl)-N-methylbenzamide]. I. A mixed opioid agonist with potent antinociceptive activity.

Compound (+)-3-((alpha-R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N-(3-fluorophenyl)-N-methylbenzamide (DPI-3290), is one of a series of novel centrally acting agents with potent antinociceptive activity that binds specifically and with high affinity to opioid receptors. In saturation equilibrium binding studies performed at 25 degrees C using membranes from rat brain or guinea pig cerebellum, the Ki values measured for DPI-3290 at delta-, mu-, and kappa-opioid receptors were 0.18 +/- 0.02, 0.46 +/- 0.05, and 0.62 +/- 0.09 nM, respectively. In vas deferens isolated from laboratory mice, DPI-3290 decreased electrically induced tension development in a concentration-dependent manner with corresponding IC50 values of 1.0 +/- 0.3, 6.2 +/- 2.0, and 25.0 +/- 3.3 nM at delta-, mu-, and kappa-receptors, respectively. The activity of DPI-3290 in isolated vas deferens tissue was approximately 20,000, 175.8, and 1500 times more efficacious than morphine, and 492, 2.5, and 35 times more efficacious than fentanyl at delta-, mu-, and kappa-receptors, respectively. In ileal strips isolated from guinea pigs, DPI-3290 inhibited tension development with a corresponding IC50 value of 3.4 +/- 1.6 nM at mu-opioid receptors and 6.7 +/- 1.6 nM at kappa-opioid receptors. Intravenous administration of 0.05 +/- 0.007 mg/kg DPI-3290 produced a 50% antinociceptive response in rats. The antinociceptive properties of DPI-3290 were blocked by naloxone (0.5 mg/kg s.c.). Compared with morphine, this study demonstrated that DPI-3290 is more potent and elicited a similar magnitude of antinociceptive activity in the rat, actions mediated by its mixed opioid receptor agonist activity. The marked antinociceptive activity of DPI-3290 will likely provide a means for relieving severe pain in patients that require analgesic treatment.

3-(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N-alkyl-N-arylbenzamides: potent, non-peptidic agonists of both the micro and delta opioid receptors.

Opioid analgesics with both micro and delta opioid receptor activation represent a new approach to the treatment of severe pain with an improved safety profile. Compounds with this profile may exhibit strong analgesic properties due to micro agonism, with a reduced side effect profile resulting from delta agonism. Replacing the p-diethylamide of the known potent delta opioid receptor selective agonist BW373U86 with a m-diethylamide resulted in a compound with agonist activity at both the micro and delta opioid receptors. Modifying the amide to an N-methyl-N-phenylamide increased agonist potency at both receptors. A series of 3-(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N-alkyl-N-arylbenzamides have been made to explore the structure-activity relationship (SAR) around the N-methyl-N-phenylamide. Several potent agonists of both the micro and delta opioid receptors have been identified, including (+)-3-((alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N-(4-fluorophenyl)-N-methylbenzamide (23), which has EC50 values of 0.67 and 1.1 nM at the micro (guinea pig ileum assay) and delta (mouse vas deferens assay) opioid receptors, respectively.