Orvinols with mixed kappa/mu opioid receptor agonist activity.

Dual-acting kappa opioid receptor (KOR) agonist and mu opioid receptor (MOR) partial agonist ligands have been put forward as potential treatment agents for cocaine and other psychostimulant abuse. Members of the orvinol series of ligands are known for their high binding affinity to both KOR and MOR, but efficacy at the individual receptors has not been thoroughly evaluated. In this study, it is shown that a predictive model for efficacy at KOR can be derived, with efficacy being controlled by the length of the group attached to C20 and by the introduction of branching into the side chain. In vivo evaluation of two ligands with the desired in vitro profile confirms both display KOR, and to a lesser extent MOR, activity in an analgesic assay suggesting that, in this series, in vitro measures of efficacy using the [(35)S]GTPγS assay are predictive of the in vivo profile.

Fumaroylamino-4,5-epoxymorphinans and related opioids with irreversible µ opioid receptor antagonist effects.

We have previously shown that cinnamoyl derivatives of 14ß-amino-17-cyclopropylmethyl-7,8-dihydronormorphinone and 7α-aminomethyl-6,14-endoethanonororipavine have pronounced pseudoirreversible µ opioid receptor (MOR) antagonism. The present communication describes the synthesis and evaluation of fumaroylamino analogues of these cinnamoylamino derivatives together with some related fumaroyl derivatives. The predominant activity of the new ligands was MOR antagonism. The fumaroylamino analogues (2a, 5a) of the pseudoirreversible antagonist cinnamoylamino morphinones and oripavines (2b, 5b) were themselves irreversible antagonists in vivo. However the fumaroylamino derivatives had significantly higher MOR efficacy than the cinnamoylamino derivatives in mouse antinociceptive tests. Comparison of 2a and 5a with the prototypic fumaroylamino opioid ß-FNA (1a) shows that they have similar MOR irreversible antagonist actions but differ in the nature of their opioid receptor agonist effects; 2a is a predominant MOR agonist and 5a shows no opioid receptor selectivity, whereas the agonist effect of ß-FNA is clearly κ opioid receptor (KOR) mediated.

Reversible, allosteric small-molecule inhibitors of regulator of G protein signaling proteins.

Regulators of G protein signaling (RGS) proteins are potent negative modulators of G protein signaling and have been proposed as potential targets for small-molecule inhibitor development. We report a high-throughput time-resolved fluorescence resonance energy transfer screen to identify inhibitors of RGS4 and describe the first reversible small-molecule inhibitors of an RGS protein. Two closely related compounds, typified by CCG-63802 [((2E)-2-(1,3-benzothiazol-2-yl)-3-[9-methyl-2-(3-methylphenoxy)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]prop-2-enenitrile)], inhibit the interaction between RGS4 and Galpha(o) with an IC(50) value in the low micromolar range. They show selectivity among RGS proteins with a potency order of RGS 4 > 19 = 16 > 8 >> 7. The compounds inhibit the GTPase accelerating protein activity of RGS4, and thermal stability studies demonstrate binding to the RGS but not to Galpha(o). On RGS4, they depend on an interaction with one or more cysteines in a pocket that has previously been identified as an allosteric site for RGS regulation by acidic phospholipids. Unlike previous small-molecule RGS inhibitors identified to date, these compounds retain substantial activity under reducing conditions and are fully reversible on the 10-min time scale. CCG-63802 and related analogs represent a useful step toward the development of chemical tools for the study of RGS physiology.

Proerectile effects of dopamine D2-like agonists are mediated by the D3 receptor in rats and mice.

Dopamine D(2)-like agonists induce penile erection (PE) and yawning in a variety of species, effects that have been suggested recently to be specifically mediated by the D(4) and D(3) receptors, respectively. The current studies were aimed at characterizing a series of D(2), D(3), and D(4) agonists with respect to their capacity to induce PE and yawning in the rat and the proerectile effects of apomorphine [(R)-(-)-5,6,6a,7-tetrahydro-6-methyl-4H-dibenzo-[de,g]quinoline-10,11-diol hydrochloride] in wild-type and D(4) receptor (R) knockout (KO) mice. All D(3) agonists induced dose-dependent increases in PE and yawning over a similar range of doses, whereas significant increases in PE or yawning were not observed with any of the D(4) agonists. Likewise, D(2), D(3), and D(4) antagonists were assessed for their capacity to alter apomorphine- and pramipexole (N'-propyl-4,5,6,7-tetrahydrobenzothiazole-2,6-diamine dihydrochloride)-induced PE and yawning. The D(3) antagonist, PG01037 [N-{4-[4-(2,3-dichlorophenyl)-piperazin-1-yl]-trans-but-2-enyl}-4-pyridine-2-yl-benzamide hydrochloride], inhibited the induction of PE and yawning, whereas the D(2) antagonist, L-741,626 [3-[4-(4-chlorophenyl)-4-hydroxypiperidin-l-yl]methyl-1H-indole], reversed the inhibition of PE and yawning observed at higher doses. The D(4) antagonist, L-745,870 [3-(4-[4-chlorophenyl]piperazin-1-yl)-methyl-1H-pyrrolo[2,3-b]pyridine trihydrochloride], did not alter apomorphine- or pramipexole-induced PE or yawning. A role for the D(3) receptor was further supported because apomorphine was equipotent at inducing PE in wild-type and D(4)RKO mice, effects that were inhibited by the D(3) antagonist, PG01037, in both wild-type and D(4)R KO mice. Together, these studies provide strong support that D(2)-like agonist-induced PE and yawning are differentially mediated by the D(3) (induction) and D(2) (inhibition) receptors. These studies fail to support a role for the D(4) receptor in the regulation of PE or yawning by D(2)-like agonists.

The discriminative stimulus effects of dopamine D2- and D3-preferring agonists in rats.

RATIONALE: Previous research has found the stimulus effects of dopamine D2- and D3-preferring agonists difficult to distinguish in drug discrimination studies. Antagonism studies suggest that the stimulus effects of both types of agonists may be mediated primarily through D2 receptors. OBJECTIVES: The current study was designed to further assess the receptors mediating the stimulus effects of these agonists and to attempt to train rats to discriminate directly between D2- and D3-preferring dopamine agonists. MATERIALS AND METHODS: Four groups of eight rats were trained to discriminate either 0.1 mg/kg of the D3-preferring agonist pramipexole from saline, 1.0 mg/kg of the D2-preferring agonist sumanirole from saline, 0.1 mg/kg pramipexole from either saline or 1.0 mg/kg sumanirole, or 1.0 mg/kg sumanirole from either saline or 0.1 mg/kg pramipexole. RESULTS: Three of eight rats in the 0.1 mg/kg pramipexole vs. 1.0 mg/kg sumanirole or saline failed to meet the training criteria, and the discrimination in this group was tenuous. The D2-preferring antagonist L-741,626 at 1.0 mg/kg was more effective at shifting to the right the pramipexole dose-response curve in pramipexole-trained rats, while 32 mg/kg of the selective D3 antagonist PG01037 had little effect. Quinpirole and 7-OH-DPAT fully or partially substituted for both pramipexole and sumanirole in each group tested, while cocaine did not substitute in any group. CONCLUSIONS: Antagonist data along with the pattern of training and substitution data suggested that D2 receptor activation is primarily responsible for the stimulus effects of both sumanirole and pramipexole with D3 receptor activation playing little or no role.

Selective chemical probe inhibitor of Stat3, identified through structure-based virtual screening, induces antitumor activity.

S3I-201 (NSC 74859) is a chemical probe inhibitor of Stat3 activity, which was identified from the National Cancer Institute chemical libraries by using structure-based virtual screening with a computer model of the Stat3 SH2 domain bound to its Stat3 phosphotyrosine peptide derived from the x-ray crystal structure of the Stat3beta homodimer. S3I-201 inhibits Stat3.Stat3 complex formation and Stat3 DNA-binding and transcriptional activities. Furthermore, S3I-201 inhibits growth and induces apoptosis preferentially in tumor cells that contain persistently activated Stat3. Constitutively dimerized and active Stat3C and Stat3 SH2 domain rescue tumor cells from S3I-201-induced apoptosis. Finally, S3I-201 inhibits the expression of the Stat3-regulated genes encoding cyclin D1, Bcl-xL, and survivin and inhibits the growth of human breast tumors in vivo. These findings strongly suggest that the antitumor activity of S3I-201 is mediated in part through inhibition of aberrant Stat3 activation and provide the proof-of-concept for the potential clinical use of Stat3 inhibitors such as S3I-201 in tumors harboring aberrant Stat3.

Abrogation of heat shock protein 70 induction as a strategy to increase antileukemia activity of heat shock protein 90 inhibitor 17-allylamino-demethoxy geldanamycin.

17-Allylamino-demethoxy geldanamycin (17-AAG) inhibits the chaperone association of heat shock protein 90 (hsp90) with the heat shock factor-1 (HSF-1), which induces the mRNA and protein levels of hsp70. Increased hsp70 levels inhibit death receptor and mitochondria-initiated signaling for apoptosis. Here, we show that ectopic overexpression of hsp70 in human acute myelogenous leukemia HL-60 cells (HL-60/hsp70) and high endogenous hsp70 levels in Bcr-Abl-expressing cultured CML-BC K562 cells confers resistance to 17-AAG-induced apoptosis. In HL-60/hsp70 cells, hsp70 was bound to Bax, inhibited 17-AAG-mediated Bax conformation change and mitochondrial localization, thereby inhibiting the mitochondria-initiated events of apoptosis. Treatment with 17-AAG attenuated the levels of phospho-AKT, AKT, and c-Raf but increased hsp70 levels to a similar extent in the control HL-60/Neo and HL-60/hsp70 cells. Pretreatment with 17-AAG, which induced hsp70, inhibited 1-beta-D-arabinofuranosylcytosine or etoposide-induced apoptosis in HL-60 cells. Stable transfection of a small interfering RNA (siRNA) to hsp70 completely abrogated the endogenous levels of hsp70 and blocked 17-AAG-mediated hsp70 induction, resulting in sensitizing K562/siRNA-hsp70 cells to 17-AAG-induced apoptosis. This was associated with decreased binding of Bax to hsp70 and increased 17-AAG-induced Bax conformation change. 17-AAG-mediated decline in the levels of AKT, c-Raf, and Bcr-Abl was similar in K562 and K562/siRNA-hsp70 cells. Cotreatment with KNK437, a benzylidine lactam inhibitor of hsp70 induction and thermotolerance, attenuated 17-AAG-mediated hsp70 induction and increased 17-AAG-induced apoptosis and loss of clonogenic survival of HL-60 cells. Collectively, these data indicate that induction of hsp70 attenuates the apoptotic effects of 17-AAG, and abrogation of hsp70 induction significantly enhances the antileukemia activity of 17-AAG.

Synthetic applications of electrophilic fluorodesilylation.

Recently, with the appearance of electrophilic sources of fluorine including the commercially available N-F reagents, the concept of electrophilic fluorodesilylation has emerged as a new strategy to prepare a variety of fluorine containing compounds. This paper highlights how this concept has been applied to the preparation of a series of fluorinated molecules including fluoroaromatic compounds, fluoroalkenes, difluoroamides, difluoroalcohols, difluoroethers and alpha-fluorinated carbonyl derivatives.