Optimization of the synthesis of a key intermediate for the preparation of glucocorticoids.

A short and efficient synthesis, based on a one-step double elimination, of a key intermediate in the synthesis of various glucocorticosteroids has been developed. This method can be carried out on large scale for further industrial applications. The synthesis allowed us to identify a novel prednisolone derivative 10 and its anti-inflammatory activity was determined in an in vivo model of inflammation. In order to understand the regioselectivity of the double elimination under various conditions, mechanistic studies were undertaken and confirmed the experimental results. We also propose a mechanism for the formation of the new steroid 10 studied by molecular modeling.

[11C]LBT-999: a suitable radioligand for investigation of extra-striatal dopamine transporter with PET.

A new tropane derivative, (E)-N-(4-fluorobut-2-enyl)-2beta-carbomethoxy-3beta-(4'-tolyl)nortropane (LBT-999), was evaluated in baboons as a carbon-11 radioligand for studies of the dopamine transporter (DAT) using positron emission tomography (PET). Brain uptake was high in the striatum (17 and 13% ID/100 mL tissue in the putamen and the caudate, respectively), moderate in the midbrain and thalamus (5 and 3% ID/100 mL tissue, respectively), and low in the cortex and cerebellum (2% ID/100 mL tissue) at 30 min post injection. The striatum-to-cerebellum ratio was high (30 at 110 min post injection). Specific binding was completely blocked following pretreatment with the DAT antagonists GBR12909 (5 mg/kg i.v.) or PE2I (1 mg/kg i.v.). The [(11)C]LBT-999 uptake was decreased by these antagonists in the putamen (-79 and -92%, respectively), caudate (-80 and -91%, respectively), midbrain (-73 and -78%, respectively), and thalamus (-34 and -46%, respectively). The serotonin transporter (SERT) antagonist citalopram (5 mg/kg i.v.) or the norepinephrine transporter antagonist maprotiline (5 mg/kg i.v.) had no effect on LBT specific binding. Pharmacological challenge with PE2I (1 mg/kg i.v.) induced a rapid and almost complete decrease of the specific binding in the putamen (-97%), caudate (-96%), midbrain (-96%), and thalamus (-81%), confirming the reversibility of [(11)C]LBT-999 binding. The high brain uptake of [(11)C]LBT-999 together with its low nonspecific binding (reflected by the very high brain structure-to-cerebellum ratio) indicate that this radiotracer is an excellent candidate for in vivo quantification of the DAT, especially in extrastriatal structures, such as the midbrain.

Synthesis, radiosynthesis and in vivo preliminary evaluation of [11C]LBT-999, a selective radioligand for the visualisation of the dopamine transporter with PET.

LBT-999 (8-((E)-4-fluoro-but-2-enyl)-3beta-p-tolyl-8-aza-bicyclo[3.2.1]octane-2beta-carboxylic acid methyl ester), a cocaine derivative belonging to a new generation of highly selective dopamine transporter (DAT) ligands, and its corresponding carboxylic acid derivative, the latter used as precursor for labelling both with tritium and the positron-emitter carbon-11 (half-life: 20.38 min), were synthesized from (R)-cocaine. [(3)H]LBT-999 (>99% radiochemically pure, specific radioactivity of 3.1 TBq/mmol) was prepared from [(3)H]methyl iodide, allowing its in vitro pharmacological evaluation (K(D): 9 nM for DAT and IC(50) > 1000 nM for SERT and NET). Routine production batches of 4.5-9.0 GBq of iv injectable solutions of [(11)C]LBT-999 (with specific radioactivities ranging from 30 to 45 GBq/mumol) were prepared in 25-30 min (HPLC purification and formulation included) using the efficient methylation reagent [(11)C]methyl triflate. The preliminary in vivo pharmacological evaluation of [(11)C]LBT-999, using both biodistributions in rats and brain imaging in monkeys with positron emission tomography (PET), clearly illustrates that this ligand is an excellent candidate for quantification with PET of DAT in humans.

Pharmacological characterization of (E)-N-(4-fluorobut-2-enyl)-2beta-carbomethoxy-3beta-(4'-tolyl)nortropane (LBT-999) as a highly promising fluorinated ligand for the dopamine transporter.

In the aim to develop an efficient fluorinated probe for positron emission tomography (PET) exploration of the dopamine transporter (DAT), we studied several in vitro and in vivo characteristics of the phenyltropane derivative (E)-N-(4-fluorobut-2-enyl)-2beta-carbomethoxy-3beta-(4'-tolyl)nortropane (LBT-999). In vitro on rat striatal membrane, [(3)H]LBT-999 bound to a single site with a K(d) of 9 nM, B(max) of 17 pmol/mg protein, and a very high selectivity for the DAT [IC(50) for 1-{2-[bis-(4-fluorophenyl)-methoxy]ethyl}-4-(3-phenylpropyl)piperazine (GBR 12909) and (E)-N-(3-iodoprop-2-enyl)-2beta-carbomethoxy-3beta-(4'-methylphenyl)nortropane (PE2I): 2.4 and 18 nM, respectively; IC(50) for paroxetine, citalopram, N,N-dimethyl-2-(2-amino-4-methylphenyl thio)benzylamine, nisoxetine, and desipramine >1 muM]. In vitro on post-mortem human brain sections, LBT-999 bound with high intensity to the caudate-putamen, weakly to the thalamus, and not in the neocortex and cerebellum. This binding was totally abolished in the presence of PE2I. Ex vivo cerebral biodistribution of [(11)C]LBT-999 in rats showed striatum/cerebellum radioactivity ratios of 18 and 25 at 30 and 60 min postinjection, respectively. This accumulation was strongly prevented by preinjection of GBR 12909, whereas paroxetine and nisoxetine had no effect. An in vivo kinetic PET study in three baboons showed a fast and very high uptake in the striatum, with a plateau at 30 min postinjection and a maximal putamen/cerebellum ratio of 30. Taken together, these findings demonstrate that LBT-999 is a highly promising agent for in vivo exploration of the DAT. This probe is currently labeled with (18)F for further characterizations.