Discovery, synthesis, and pharmacological evaluation of spiropiperidine hydroxamic acid based derivatives as structurally novel histone deacetylase (HDAC) inhibitors.

New spiro[chromane-2,4'-piperidine] and spiro[benzofuran-2,4'-piperidine] hydroxamic acid derivatives as HDAC inhibitors have been identified by combining privileged structures with a hydroxamic acid moiety as zinc binding group. The compounds were evaluated for their ability to inhibit nuclear extract HDACs and for their in vitro antiproliferative activity on different tumor cell lines. This work resulted in the discovery of spirocycle 30d that shows good oral bioavailability and tumor growth inhibition in an HCT-116 murine xenograft model.

Synthesis and biological evaluation of new N-alkyl 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides as cannabinoid receptor ligands.

A series of N-alkyl 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides were synthesized as new ligands of the human recombinant receptor hCB1. n-Alkyl carboxamides brought out different SARs from the branched subgroup. Unsubstituted pyrrole derivatives bearing a tert-alkyl chain at the 3-carboxamide nitrogen showed greater hCB1 receptor affinity than the corresponding unbranched compounds. In particular, the tert-butyl group as a chain terminal moiety strongly improved hCB1 receptor affinity (compound 24: Ki=45.6 nM; 29: Ki=37.5 nM). Acute administration of either compound 12 or 29 resulted in a specific, dose-dependent reduction in food intake in rats. Such results provide an useful basis for the design of new CB1 ligands.

Synthesis and biological characterization of amidopropenyl hydroxamates as HDAC inhibitors.

A series of amidopropenyl hydroxamic acid derivatives were prepared as novel inhibitors of human histone deacetylases (HDACs). Several compounds showed potency at <100 nM in the HDAC inhibition assays, sub-micromolar IC(50) values in tests against three tumor cell lines, and remarkable stability in human and mouse microsomes was observed. Three representative compounds were selected for further characterization and submitted to a selectivity profile against a series of class I and class II HDACs as well as to preliminary in vivo pharmacokinetic (PK) experiments. Despite their high microsomal stability, the compounds showed medium-to-high clearance rates in in vivo PK studies as well as in rat and human hepatocytes, indicating that a major metabolic pathway is catalyzed by non-microsomal enzymes.

New perspectives in bio-analytical techniques for preclinical characterization of a drug candidate: UPLC-MS/MS in in vitro metabolism and pharmacokinetic studies.

Lead optimization requires rapid bio-analytical turnover for the generation of early absorption, distribution, metabolism, excretion (ADME) and pharmacokinetics (PK) data maintaining a high quality level. Therefore, one of the major challenges in the bio-analytical field is to achieve faster and more sensitive quantification protocols. In the present communication, a comparison between HPLC and ultra performance liquid chromatography (UPLC) performances in terms of sensitivity and resolution is shown using a pharmakokinetic study and a metabolism study as models. The studies highlight the features of the new technology and the resulting impact in the PK throughput and in the characterization of isomeric metabolites using UPLC/MS/MS technique.

Potential antidepressant properties of IDN 5491 (hyperforin-trimethoxybenzoate), a semisynthetic ester of hyperforin.

Hyperforin is one of the possible active principles mediating the antidepressant activity of Hypericum perforatum L. extracts. The ester derivative IDN 5491 (hyperforin-trimethoxybenzoate) showed antidepressant-like properties in the forced swimming test (FST) in rats, with no effect on open-field activity, when given as three intraperitoneal injections in 24 h at 3.125 and 6.25 mg/kg. The plasma concentrations of IDN 5491 were 30-50 microM, and those of hyperforin much lower but still close to those after effective doses of hyperforin-dicyclohexylammonium and Hypericum extract. This suggests that hyperforin plays a role in the antidepressant-like effect of the ester and of Hypericum extract. In vitro binding and uptake data showed that IDN 5491 is inactive on a wide panel of CNS targets at a concentration (14 microM) much higher than that measured in the brain of treated rats (0.3 microM). Like the extract, the antidepressant-like effect of IDN 5491 was blocked by (-)-sulpiride, a selective D2 receptor antagonist and by BD-1047, a selective sigma1 antagonist. Ex-vivo binding studies showed that brain sigma1 receptors are occupied after in vivo treatment with IDN 5491, possibly by an unknown metabolite or by endogenous ligand induced by hyperforin.

Hyperforin contributes to the hepatic CYP3A-inducing effect of Hypericum perforatum extract in the mouse.

This study in mice investigated whether hyperforin accounts for the inductive effects on cytochrome P4503A enzymes of St. John's wort extracts (SJW; Hypericum perforatum), one of the most popular herbal preparations because of its alleged activity in mild to moderate depression. A hydroalcoholic extract containing 4.5% hyperforin was given at a dose of 300 mg/kg, bis in die (b.i.d.), for 4 and 12 days. Hyperforin, its main phloroglucinol component, was given as dicyclohexylammonium (DCHA) salt (18.1 mg/kg, b.i.d.) on the basis of its content in the extract, to ensure comparable exposure to hyperforin. The extract increased hepatic erythromycin-N-demethylase (ERND) activity, which is cytochrome P450 enzyme (CYP) 3A-dependent, about 2.2-fold after 4 days of dosing, with only slightly greater effect after 12 days (2.8 times controls). Hyperforin too increased ERND activity within 4 days, much to the same extent as the extract (1.8 times the activity of controls), suggesting that it behaves qualitatively and quantitatively like the extract as regards induction of CYP3A activity. This effect was confirmed by Western blot analysis of hepatic CYP3A expression. Exposure to hyperforin at the end of the 4-day treatment was still similar to that with SJW extract, although it was variable and lower than after the first dose in both cases, further suggesting that hyperforin plays a key role in CYP3A induction by the SJW extract in the mouse. Standardization of the extracts based on the hyperforin content can be proposed for further evaluation of their potential action on first-pass metabolism and clearance of coadministered CYP3A substrates.

Role of hyperforin in the antidepressant-like activity of Hypericum perforatum extracts.

RATIONALE: Hyperforin has been identified as an active constituent of Hypericum perforatum but its importance in the antidepressant effect of this plant's extracts is not really known. OBJECTIVE: To evaluate the antidepressant-like activity of two extracts in relation to the content of hyperforin and its plasma and whole brain concentrations, compared with a stable salt of hyperforin (dicyclohexylammonium; DCHA). METHODS: The effects of the extracts and hyperforin were evaluated in the rat forced swimming test. The specificity of the effects was demonstrated evaluating the rats' locomotor activity. Plasma and brain concentrations of hyperforin were determined by high performance liquid chromatography. RESULTS: The 4.5% extract (but not the 0.5% extract) given as three IP injections in 24 h (3.12-6.25 mg/kg) reduced the total immobility of rats, yielding dose-related plasma concentrations of hyperforin. These concentrations were of a similar magnitude to those after hyperforin DCHA which also significantly reduced immobility when given on the basis of the hyperforin content of the 4.5% extract (0.14 and 0.28 mg/kg). However, hyperforin was undetectable in rat brain, possibly because of poor passage of the blood-brain barrier. CONCLUSION: These results support the view that hyperforin plays a key role in the antidepressant-like activity of Hypericum p. However, brain concentrations after effective doses are probably far from those active in vitro on the neurotransmitter mechanisms so far investigated.

Stimulation of serotonin1B receptors induces conditioned place aversion and facilitates cocaine place conditioning in rats.

RATIONALE: Changes in serotonin(1B) (5-HT(1B)) receptor function appear to modify the reinforcing properties of cocaine, but the direction of this effect is not completely clear. Pharmacological stimulation of 5-HT(1B) enhanced the rewarding properties of self-administered cocaine while attenuating the threshold-reducing effect of cocaine in the intracerebral brain stimulation procedure. OBJECTIVE: The present study investigates how pharmacological modification of 5-HT(1B) receptor-mediated neurotransmission influence cocaine motivational properties in the conditioned place preference paradigm in rats. METHODS: In separate groups of rats the motivational properties of CP 94,253, a selective 5-HT(1B) agonist, or GR 127935, a 5-HT(1B/D) receptor partial agonist, given alone or in combination, were determined. To evaluate their influence on cocaine-induced place conditioning, CP 94,253, that was found to be aversive, was given every day before each conditioning session, while GR 127935, which given alone had no effect, was administered only before cocaine conditioning sessions. RESULTS: CP 94,253, injected IP at 2.5 and 10 (but not 0.5) mg/kg produced place aversion in the place conditioning paradigm. The aversive effect of 2.5 mg/kg CP 94,253 was completely reversed by 10 mg/kg SC GR 127935. Given before every conditioning session, CP 94,253 did not modify place conditioning by four injections of 10 mg/kg cocaine but at 2.5 mg/kg it potentiated a sub-threshold dose of cocaine. The place preference caused by these two drugs was completely reversed by 10 mg/kg GR 127935. The antagonism by GR 127935 of CP 94,253's effects was shown not to be due to the induction of state-dependent effects. CONCLUSION: The results suggest that stimulation of 5-HT(1B) receptors causes place aversion, and enhances the effect of low doses of cocaine in the conditioned place preference paradigm.