The discovery of potent cRaf1 kinase inhibitors.

A series of benzylidene-1H-indol-2-one (oxindole) derivatives was synthesized and evaluated as cRaf-1 kinase inhibitors. The key features of the molecules were the donor/acceptor motif common to kinase inhibitors and a critical acidic phenol flanked by two substitutions. Diverse 5-position substitutions provided compounds with low nanomolar kinase enzyme inhibition and inhibited the intracellular MAPK pathway.

Delta-opioid ligands reverse alfentanil-induced respiratory depression but not antinociception.

Evidence suggests both opioid mu and delta receptors may participate in the regulation of respiration at different central nervous system sites. In the past, the overlapping receptor specificity of various opioid drugs has made it difficult to dissect the receptor subtype-specific activities involved in respiratory regulation. The new family of delta receptor selective agents such as cyclic[D-Pen2, 5]enkephalin, deltorphins, (+)-4-((alpha-R)-alpha-((2S,5R)-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N,N-diethylbenzamide, naltrindole and H-Tyr-Tic(psi)[CH2NH]Phe-Phe-OH have now made it feasible to more clearly define the role of delta receptors in respiratory control. In a series of experiments we observed that systemic infusion of rats with the highly mu receptor-specific opioid alfentanil induced antinociception and hypercapnia, and both of these effects were antagonized by the mu antagonist D-Phe-Cys-Tyr-Orn-Thr-Pen-Thr-NH2. However, peripheral administration of the delta receptor antagonist naltrindole reverses the hypercapnia but not the antinociceptive activity of alfentanil. This differential effect of naltrindole on antinociception and hypercapnia could also be produced with the delta agonist (+)-4-((alpha-R)-alpha-((2S,5R)-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N,N-diethylbenzamide. In addition, intracerebroventricular delivery of a number of peptide delta ligands cyclic[D-Pen2,5]enkephalin, deltorpnin II and H-Tyr-Tic(psi)[CH2NH]Phe-Phe-OH also produced the same differential reversal of hypercapnia without affecting antinociception. Thus, both the traditional delta agonists and antagonists are able to reverse the alfentanil-induced hypercapnia without affecting antinociception. The reversal of alfentanil-induced hypercapnia by these delta ligands was antagonized by a novel synthetic delta antagonist cis-4-(alpha-(4-((Z)-2-butenyl)-3, 5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N,N-diethylbenzamide. We propose that in this experimental respiration model, the delta antagonists naltrindole and H-Tyr-Tic(psi)[CH2NH]Phe-Phe-OH behave like delta agonists with low but sufficient intrinsic activities to reverse alfentanil-induced hypercapnia in rats. The results suggest that a function of the delta receptor is to modulate or counteract the respiratory depression induced by the mu receptor.

Antagonistic modulation between the delta opioid agonist BW373U86 and the mu opioid agonist fentanyl in mice.

This study was performed to assess the interactions that occur between delta- and mu opioid receptors by studying effects of the systemically active nonpeptide delta agonist BW373U86 and the mu agonist fentanyl in mice. Concentrations of the compounds were varied, and analgesic responses were determined by 55 degrees C hot-plate assays. BW373U86 produced hot-plate antinociceptive activity along with convulsive side effects. These effects could be antagonized by the selective delta antagonist naltrindole. Fentanyl produced hot-plate antinociceptive activity with Straub tail and hyperactivity as side effects. When BW373U86 and fentanyl were coadministered, BW373U86 convulsive activity was attenuated by fentanyl in a dose-dependent manner and the fentanyl-induced Straub tail effect was antagonized by BW373U86, also in a dose-dependent manner. Hot-plate analgesic activity was additive between the two compounds. The delta antagonist naltrindole partially antagonized the ability of BW373U86 to block the fentanyl-induced Straub tail effect. The mu antagonist beta-funaltrexamine antagonized the fentanyl-induced blockade of the convulsive effects of BW373U86. These data suggest that complex inhibitory interactions take place between mu and delta receptors in mice. Future studies are clearly needed to study the neuromodulatory effects of mu and delta receptors. The widespread use of mu agonists in the clinic indicates that a large number of patients exist who could greatly benefit from the conjunctive use of delta pharmaceuticals.

Binding of [3H](+)-BW373U86 to delta-opioid receptors in rat brain membranes.

A tritiated form of the non-peptide delta-opioid receptor agonist (+)-BW373U86 ((+)-4-((alpha-R)-alpha-((2S,5R)-4-allyl-2, 5-dimethyl-l-piperazinyl)-3-hydroxybenzyl)-N,N-diethylbenzamide) was synthesized and its binding characteristics studied. [3H](+)-BW373U86 bound with subnanomolar affinity to rat brain membranes and was displaced most effectively by ligands selective for delta-opioid receptors. Naltrindole, naltriben, and 7-benzylidenenaltrexone exhibited apparent inhibition constants of 0.06, 1.54, and 4.49 nM, respectively, while mu- or kappa-selective ligands showed little affinity for this site. [3H](+)-BW373U86 binding was sensitive to the presence of guanine nucleotides; GDP caused a 3-fold decrease and 5'-guanylyl-imidodiphosphate (Gpp[NH]p) caused a 25% increase in binding affinity.

Characterization of delta opioid receptors in lung cancer using a novel nonpeptidic ligand.

Cancer cells are often characterized by the presence of membrane receptors not normally associated with nontransformed cells from the same tissue type. Recent studies have demonstrated increased expression of high-affinity binding sites for opioid receptor-selective ligands in lung cancer cell lines relative to normal lung tissue. We investigated the binding of a nonpeptidic delta opioid receptor ligand in small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells with the aim of developing the ligand as a novel lung cancer imaging agent. The ligand, [3H] (+)-4-[alpha-R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3- hydroxybenzyl)-N,N-diethylbenzamide ([3H](+)BW373U86), bound with high-affinity [Kd (dissociation constant) = 0.066 +/- 0.012 nM] to membranes prepared from six different SCLC cell lines but not to those from seven NSCLC cell lines, including one mesothelioma. The number of biding sites varied from 10 to 300 fmol/mg membrane protein. Competition binding studies demonstrated displacement of [3H](+)BW373U86 binding by the delta-selective antagonists naltriben and 7-benzylidenenaltrexone but not with the mu- and kappa- selective antagonists D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 and trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]ben zeneacetamide methanesulfonate. Mean apparent Kis for naltriben and 7-benzylidenenaltrexone in membranes from two SCLC cell lines were 0.17 and 3.9 nM, respectively, but were >10 microM for the mu and kappa ligands. The nonselective antagonist naloxone displaced [3H](+)BW373U86 binding with an apparent Ki of approximately 29 nM. On the basis of these data, we believe the lung cancer receptor to be similar, if not identical, to the human brain delta opioid receptor. The lack of high-affinity [3H](+)BW373U86 binding in normal mouse lung membranes suggests a potential role for this ligand as a novel therapeutic or imaging agent.

SNC 80, a selective, nonpeptidic and systemically active opioid delta agonist.

The present study has investigated the pharmacology of SNC 80, a nonpeptidic ligand proposed to be a selective delta agonist in vitro and in vivo. SNC 80 was potent in producing inhibition of electrically induced contractions of mouse vas deferens, but not in inhibiting contractions of the guinea pig isolated ileum (IC50 values of 2.73 nM and 5457 nM, respectively). The delta selective antagonist ICI 174,864 (1 microM) and the mu selective antagonist CTAP (1 microM) produced 236- and 1.9-fold increases, respectively, in the SNC 80 IC50 value in the mouse vas deferens. SNC 80 preferentially competed against sites labeled by [3H]naltrindole (delta receptors) rather than against those labeled by [3H]DAMGO (mu receptors) or [3H]U69, 593 kappa receptors) in mouse whole-brain assays. The ratios of the calculated Ki values for SNC 80 at mu/delta and kappa/delta sites were 495- and 248-fold, respectively, which indicates a significant degree of delta selectivity for this compound in radioligand binding assays. SNC 80 produced dose- and time-related antinociception in the mouse warm-water tail-flick test after i.c.v., i.th. and i.p. administration. The calculated A50 values (and 95% C.I.) for SNC 80 administered i.c.v., i.th. and i.p. were 104.9 (63.7-172.7) nmol, 69 (51.8-92.1) nmol and 57 (44.5-73.1) mg/kg, respectively. The i.c.v. administration of SNC 80 also produced dose- and time-related antinociception in the hot-plate test, with a calculated A50 value (and 95% C.I.) of 91.9 (60.3-140.0) nmol.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel, potent and selective nonpeptidic delta opioid receptor agonist BW373U86.

Four different opioid receptor binding assays and three different isolated tissue studies were used to screen for delta receptor-selective nonpeptidic compounds. (+/-)-4-((alpha-R*)-alpha-((2S*,5R*)-4-Allyl-2,5- dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N,N-diethylbenzamide (BW373U86) was a potent delta receptor-selective ligand in receptor binding assays. The Ki values were 1.8 +/- 0.4, 15 +/- 3, 85 +/- 4 and 34 +/- 3 nM for delta, mu, epsilon and kappa receptor binding sites, respectively. BW373U86 inhibited electrically evoked muscle contraction of mouse vas deferens with an ED50 value of 0.2 +/- 0.06 nM. This inhibitory effect of BW373U86 was antagonized by the delta receptor-selective antagonist naltrindole in a competitive manner: the Schild plot indicated a slope of 1 and a pA2 value of 9.43 (Ke = 3.7 x 10(-10) M), which is consistent with the high affinity of naltrindole in delta receptors. BW373U86 did not interact significantly with other receptors. BW373U86 inhibited the acoustic startle reflex after subcutaneous administration from 0.2- to 2-mg/kg doses in rats, and this inhibition was blocked by naltrindole. BW373U86 also induced a dose-dependent increase of locomotor activity in rats at similar doses. This effect was inhibited by naltrindole. These data suggest that BW373U86 is a potent and selective nonpeptidic delta agonist, and it elicits distinct in vivo pharmacological activities.

Antinociceptive actions of BW373U86 in the mouse.

This study explored the antinociceptive properties of (+-)-4-[(alpha- R*)-alpha-[(2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinol]-3-hydroxy benzyl] - N,N-diethyl-benzamide dihydrochloride (BW373U86) a nonpeptidic compound proposed to be a delta opioid agonist, using three models of nociception and four routes of administration in mice. BW373U86 produced dose- and time-dependent, naloxone-sensitive antinociception in both the tail-flick and tail-pinch assays when given intrathecally (i.t.). However, at doses up to 187 nmol/mouse, i.c.v. BW373U86 was inactive in the tail-flick or tail-pinch assays. Additionally, at doses up to 187 mumol/kg, BW373U86 was not active after i.p. or p.o. administration in these endpoints. Further, in the tail-flick test, i.c.v. BW373U86 did not antagonize the antinociceptive effects of i.c.v. [D-Pen2,D-Pen5]enkephalin or [D-Ala2,Glu4]deltorphin, but partially antagonized the effects of i.c.v. morphine. In the acetic-acid abdominal constriction assay, BW373U86 produced dose-dependent antinociceptive effects when given by the i.p., i.c.v. or i.t., but not by the p.o., routes. Intrathecal BW373U86 was 663-fold more potent in the abdominal constriction assay than when given by the same route in the tail-flick test. The effects of an A70 dose of i.p. or i.c.v. BW373U86 in the abdominal constriction assay were partially antagonized by i.c.v. naloxone, but not by i.c.v. N,N-diallyl-Tyr-Aib- Aib-Phe-Leu-OH, where Aib is alpha-aminoisobutyric acid (ICI-174,864) or naltrindole. In contrast, i.t. naloxone, ICI-174,864 or naltrindole antagonized the antinociceptive effect of i.p. or i.t. BW373U86 in the abdominal constriction assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Discriminative stimulus effects of BW373U86: a nonpeptide ligand with selectivity for delta opioid receptors.

Several opioid agonists were evaluated in pigeons trained to discriminate i.m. injections of sterile water from either the mu agonist morphine (5.6 mg/kg), the kappa agonist bremazocine (0.032mg/kg) or (+/-)-4-((alpha-R*)-alpha-((2S*,5R*)-4-allyl-2,5-dimethyl-1- piperazinyl)-3-hydroxybenzyl)-N,N-diethylbenzamide (BW373U86; 0.56 mg/kg). Pigeons were trained to peak one of two keys on a fixed-ratio 20 schedule for food reinforcement. The pattern of substitution of mu, kappa and delta selective agonists in the three groups of birds suggested that the discriminative stimulus effects of morphine, bremazocine and BW373U86 were different; however, a component of the discriminative stimulus effects of BW373U86 appeared to be shared with morphine. Apparent pA2 values for naltrexone with morphine, bremazocine and BW373U86 were 7.6, 6.8 and 6.3, respectively. The apparent pA2 value for naltrindole with BW373U86 was 8.3. Naltrindole (10.0 mg/kg) produced a 3-fold shift to the right in the dose-effect curve for morphine but did not antagonize bremazocine. Although results from the substitutions experiments suggested that a component of the BW373U86 discriminative stimulus was mediated through mu opioid receptors, the fact that naltrindole was 1000-fold more potent and naltrexone was 30-fold less potent in antagonizing BW373U86 than morphine indicated that the discriminative effects of BW373U86 were also mediated through delta opioid receptors.

A nonpeptidic delta opioid receptor agonist, BW373U86, attenuates the development and expression of morphine abstinence precipitated by naloxone in rat.

The effect of (+-)-4-((alpha-R*)-alpha-((2S*,5R*)-4-allyl-2,5-dimethyl-1- piperazinyl)-3-hydroxybenzyl)-N,N-diethylbenzamide (BW373U86), the first potent nonpeptidic, highly selective delta opioid receptor agonist, on morphine dependence was studied in rats. Continuous infusion of BW373U86 by a subcutaneously implanted osmotic minipump did not induce any abnormal behavior. After 6 days of BW373U86 infusion, intraperitoneal injection of a high dose of naloxone or naltrindole did not precipitate morphine-like abstinence syndromes. Furthermore, a single injection of BW373U86 did not induce abstinence syndromes or modulate morphine abstinence precipitated by naloxone in chronic morphine-treated rats. However, naloxone-precipitated abstinence syndromes in morphine-dependent rats were partially suppressed by BW373U86 in a dose-dependent manner when the compound was infused subcutaneously before and throughout morphine treatment. Abstinence signs such as wet-dog shake, forelimb tremor and teeth chattering were either suppressed or the intensity was significantly attenuated in these BW373U86-infused rats. This effect was antagonized by naltrindole. These data show that chronic infusion of BW373U86 does not produce physical dependence and that it attenuates some abstinence behaviors in morphine-dependent rats via delta opioid receptors.

Convulsive effects of systemic administration of the delta opioid agonist BW373U86 in mice.

A systemically active, nonpeptidic delta receptor-selective agonist, (+-)-4-((alpha-R*)-alpha-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl) -3- hydroxybenzyl)-N,N-diethylbenzamide (BW373U86), produced a brief, nonlethal convulsion in mice. The behavioral pattern of convulsion produced by pentylenetetrazol was similar to that produced by systemic administration of BW373U86. Although several episodes of convulsion occurred with pentylenetetrazol, BWB373U86 produced a single, brief episode. Naltrexone (10.0 and 100 mg/kg) and naltrindole (1.0, 3.2 and 10.0 mg/kg), but not midazolam (0.32 mg/kg), produced dose-dependent rightward shifts in the potency of BW373U86 to induce a convulsion. A dose of 3.2 mg/kg of midazolam completely eliminated convulsions induced by BW373U86. Midazolam (0.32 and 3.2 mg/kg), but not naltrindole (3.2 and 32.0 mg/kg), produced parallel rightward shifts in the pentylenetrazol dose-effect curve. Pretreatment with a single injection of BW373U86 (3.2, 10.0, 32.0 or 100 mg/kg) produced a dose-related reduction in the capacity of BW373U86 to induce a second convulsion. Recovery of sensitivity to BW373U86 did not return to control levels for up to 2 weeks after pretreatment with a single injection of 32.0 mg/kg of BW373U86. Naltrindole (3.2 mg/kg) administered within 1 hr, but not at 2 hr, after a pretreatment dose of 10.0 mg/kg of BW373U86 prevented the refractoriness (tolerance) induced by the single dose of BW373U86. These data suggest that the convulsions as well as the tolerance induced by BW373U86 were initiated through delta opioid receptors.

BW373U86: a nonpeptidic delta-opioid agonist with novel receptor-G protein-mediated actions in rat brain membranes and neuroblastoma cells.

BW373U86 is a potent and highly selective nonpeptidic agonist for delta-opioid receptors. To determine its ability to couple with G protein-linked second messenger systems, this study examined the effects of BW373U86 on the inhibition of adenylyl cyclase and the stimulation of low-Km GTPase activity. In rat striatal membranes, BW373U86 inhibited basal adenylyl cyclase activity in a GTP-dependent manner, with maximal inhibition levels similar to those of the prototypic delta agonist [D-Ser2,Thr6]Leu-enkephalin (DSLET). However, BW373U86 was approximately 100 times more potent than DSLET in inhibiting adenylyl cyclase. Analysis of the inhibitory activity across 10 brain regions revealed that both low and high concentrations of BW373U86 inhibited adenylyl cyclase activity in a manner similar to that of DSLET. Inhibition of adenylyl cyclase by BW373U86 was delta receptor selective, because the delta receptor-selective antagonist naltrindole was significantly more potent than naloxone and the mu receptor-selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 was ineffective in blocking BW373U86 inhibition. BW373U86 also inhibited adenylyl cyclase activity in membranes prepared from NG108-15 cells, with an IC50 value 5 times lower than that of DSLET. This increased potency was not observed in concentration-effect curves for agonist-stimulated low-Km GTPase in NG108-15 membranes. BW373U86 is a competitive inhibitor of [3H]diprenorphine at delta receptors of NG108-15 cell membranes. However, unlike DSLET, BW373U86 displacement of [3H]diprenorphine binding to NG108-15 cell membranes was not affected by sodium and guanine nucleotides. This lack of GTP effect on binding apparently produced slow dissociation rates for this agonist, because naltrindole was less potent in blocking BW373U86 inhibition of adenylyl cyclase when membranes were preincubated with this agonist. These results demonstrate the novel finding that the binding of a full agonist to a G protein-coupled receptor is not regulated by GTP, and they also show how the lack of regulation in receptor binding affects agonist potency.

Binding of BW 373U86, a non-peptidic delta opioid receptor agonist, is not regulated by guanine nucleotides and sodium.

BW 373U86 is a novel, non-peptidic delta-opioid receptor ligand with agonist properties in mouse brain and in the mouse isolated vas deferens. The sensitivity of BW 373U86, and of the peptide delta-opioid agonists [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Ala2,Glu4]deltorphin, to regulation by guanine nucleotides and sodium was evaluated in competition studies against the 5 selective radioligand [3H]naltrindole. The IC50 values for DPDPE and [D-Ala2,Glu4]deltorphin were significantly increased in brain and mouse vas deferens in the presence of Gpp(NH)p and NaCl. In contrast, the IC50 values for BW 373U86 were not altered in the presence of Gpp(NH)p and NaCl in either tissue. The data indicate that the agonist properties of BW 373U86 may not be affected by the supposed uncoupling of the alpha-subunit of the G-protein from a receptor thought to be G-protein linked.