Structure optimization of positive allosteric modulators of GABAB receptors led to the unexpected discovery of antagonists/potential negative allosteric modulators.

Positive allosteric modulators (PAMs) of GABAB receptor represent an interesting alternative to receptor agonists such as baclofen, as they act on the receptor in a more physiological way and thus are devoid of the side effects typically exerted by the agonists. Based on our interest in the identification of new GABAB receptor PAMs, we followed a merging approach to design new chemotypes starting from selected active compounds, such as GS39783, rac-BHFF, and BHF177, and we ended up with the synthesis of four different classes of compounds. The new compounds were tested alone or in the presence of 10 µM GABA using [35S]GTPγS binding assay to assess their functionality at the receptor. Unexpectedly, a number of them significantly inhibited GABA-stimulated GTPγS binding thus revealing a functional switch with respect to the prototype molecules. Further studies on selected compounds will clarify if they act as negative modulators of the receptor or, instead, as antagonists at the orthosteric binding site.

Direct Synthesis of 2-Formylpyrrolidines, 2-Pyrrolidinones and 2-Dihydrofuranones via Aerobic Copper-Catalyzed Aminooxygenation and Dioxygenation of 4-Pentenylsulfonamides and 4-Pentenylalcohols.

A new method for the direct conversion of 4-pentenylsulfonamides to 2-formylpyrrolidines and a 2-ketopyrrolidine has been developed. This transformation occurs via aerobic copper-catalyzed alkene aminooxygenation where molecular oxygen serves as both oxidant and oxygen source. The 2-formylpyrrolidines can further undergo oxidative carbon-carbon bond cleavage in situ upon addition of DABCO, providing 2-pyrrolidinones. These transformations have been demonstrated for a range of 4-pentenylsulfonamides. 4-Pentenylalcohols also undergo oxidative cyclization to form γ-lactones predominantly. The reaction is chemoselective, oxidizing one alkene in the presence of others, and is compatible with several functional groups. Application of these reactions to the formal syntheses of baclofen and (+)-monomorine was demonstrated.

Efficient Synthesis of ß-Aryl-γ-lactams and Their Resolution with (S)-Naproxen: Preparation of (R)- and (S)-Baclofen.

An efficient synthesis of enantiomerically-pure ß-aryl-γ-lactams is described. The principal feature of this synthesis is the practical resolution of ß-aryl-γ-lactams with (S)-Naproxen. The procedure is based on the Michael addition of nitromethane to benzylidenemalonates, which was easily obtained, followed by the reduction of the γ-nitroester in the presence of Raney nickel and the subsequent saponification/decarboxylation reaction. The utility of this methodology was highlighted by the preparation of enantiomerically-pure (R)- and (S)-Baclofen hydrochloride.

GABA(B)-agonistic activity of certain baclofen homologues.

Baclofen (1) is a potent and selective agonist for bicuculline-insensitive GABA(B) receptors and is used clinically as an antispastic and muscle relaxant agent. In the search for new bioactive chemical entities that bind specifically to GABA(B) receptors, we report here the synthesis of certain baclofen homologues, namely (R,S)-5-amino-3-arylpentanoic acid hydrochlorides (R,S)-1a-h as well as (R,S)-5-amino-3-methylpentanoic acid [(RS)-1i] to be evaluated as GABA(B)R agonists. Compound 1a is an agonist to GABA(B) receptors with an EC50 value of 46 µM on tsA201 cells transfected with GABA(B1b)/GABA(B2)/Gqz5, being the most active congener among all the synthesized compounds.

Regio- and enantioselective palladium-catalyzed allylic alkylation of nitromethane with monosubstituted allyl substrates: synthesis of (R)-rolipram and (R)-baclofen.

The Pd-catalyzed asymmetric allylic alkylation (AAA) reaction of nitromethane with monosubstituted allyl substrates was realized for the first time to provide corresponding products in high yields with excellent regio- and enantioselectivities. The protocol was applied to the enantioselective synthesis of (R)-baclofen and (R)-rolipram.

Synthesis of (R,S)-[4-11C]baclofen via Michael addition of nitromethane labeled with short-lived 11C.

The synthesis of (R,S)-[4-11C]baclofen, the first 11C-labeled GABAB agonist, was demonstrated via Michael addition of nitro[11C]methane as a key step. A tetrabutylammonium fluoride promoted Michael addition of nitro[11C]methane to methyl p-chlorocinnamate, followed by the nitro-group reduction in the presence of NiCl2 and NaBH4 in aqueous MeOH and alkaline hydrolysis yielded (R,S)-[4-11C]baclofen in 36.4+/-1.8% radiochemical conversion in three steps within 20 min.

Digitization of multistep organic synthesis in reactionware for on-demand pharmaceuticals.

Chemical manufacturing is often done at large facilities that require a sizable capital investment and then produce key compounds for a finite period. We present an approach to the manufacturing of fine chemicals and pharmaceuticals in a self-contained plastic reactionware device. The device was designed and constructed by using a chemical to computer-automated design (ChemCAD) approach that enables the translation of traditional bench-scale synthesis into a platform-independent digital code. This in turn guides production of a three-dimensional printed device that encloses the entire synthetic route internally via simple operations. We demonstrate the approach for the γ-aminobutyric acid receptor agonist, (±)-baclofen, establishing a concept that paves the way for the local manufacture of drugs outside of specialist facilities.

Rh-catalyzed asymmetric hydrogenation of gamma-phthalimido-substituted alpha,beta-unsaturated carboxylic acid esters: an efficient enantioselective synthesis of beta-aryl-gamma-amino acids.

A series of chiral beta-aryl-gamma-amino acid ester derivatives were synthesized in high enantioselectivities (93-97% ee) via the Rh-catalyzed asymmetric hydrogenation of gamma-phthalimido-alpha,beta-unsaturated carboxylic acid esters using highly modular chiral BoPhoz-type phosphine-aminophosphine ligands. The method has been applied successfully to the synthesis of several chiral pharmaceuticals including (R)-baclofen and (R)-rolipram with high enantioselectivities.

Continuous chromatographic separation of a baclofen precursor (N-Boc-4-[p-chloro-phenyl]-2-pyrrolidone) in a simulated moving bed using a polysaccharide carbamate as chiral stationary phase.

Liquid chromatography is known as one of the most flexible, efficient and cost-effective methods to resolve racemic mixture in order to attend the growing demand of the pharmaceutical industry for pure enantiomeric compounds. Cellulose tris(3,5-dimethylphenylcarbamate) is frequently used as a stationary phase for enantiomeric separations because of its attractive properties, including high enantioselectivity, high loading capacity and good mechanical stability. In this study, we investigated the usefulness of cellulose tris(3,5-dimethylphenylcarbamate) as the stationary phase and of ethanol and hexane mixtures as the mobile phases for the chromatographic separation of potential pharmaceutical intermediates. Using adsorption equilibrium data, we determined the optimal operational conditions for the separation of the N-Boc-4-[p-chloro-phenyl]-2-pyrrolidone enantiomers - a baclofen precursor - in a semi-preparative scale simulated moving bed unit. This unit was used to obtain high purity enantiomers on a scale of 1g/day. The outlet streams were analyzed by an on-line system that consisted of a UV-vis spectrophotometric unit, a polarimeter, and HPLC. Enantiomeric purities of up to 97% were obtained for the raffinate stream and up to 90% for the extract stream.

One-Pot Synthesis of (S)-Baclofen via Aldol Condensation of Acetaldehyde with Diphenylprolinol Silyl Ether Mediated Asymmetric Michael Reaction as a Key Step.

An efficient asymmetric total synthesis of (S)-baclofen was accomplished via a one-pot operation from commercially available materials using sequential reactions, such as aldol condensation of acetaldehyde, diphenylprolinol silyl ether mediated asymmetric Michael reaction of nitromethane, Kraus-Pinnick oxidation, and Raney Ni reduction. Highly enantioenriched baclofen was obtained in one pot with a good yield over four reactions.

Baclofen-loaded microspheres: preparation and efficacy testing in a new rabbit model.

Intrathecal baclofen is the reference treatment for severe spasticity. This drug has to be injected chronically in the intrathecal space by implanted pumps which are very expensive, uncomfortable and sometimes lead to side effects. Previous work has been performed by our group to assess the feasibility of encapsulating baclofen into poly(lactide-co-glycolide) (PLGA) microspheres and injecting these preparations in the intrathecal space of rabbits. The aims of the present study were to improve the encapsulation process for industrial application (scale-up), and to set up an animal model to assess the duration of effect of the new formulations. Modifications included the replacement of methylene chloride by a less toxic solvent, ethyl acetate, and the use of high molecular weight polymers to extend the release rate of the drug. The temperature and organic solvent extraction rate were fully controlled during the whole manufacturing process. All these modifications resulted in high quality microsphere batches with a CV inferior to 5% for encapsulation efficiency and drug loading. Encapsulation efficiency and release patterns were dependent on the drug payload and the polymer used. A formulation displaying a sustained release of baclofen over 174 days and a moderate burst effect of 16% in the first day in vitro was evaluated in a new reliable model of baclofen activity based on electrophysiological measurement of H-reflex in the rabbit. The activity of a very low dose of baclofen microspheres in vivo was sustained over 35 days. Furthermore, the preparation was well tolerated. These newly developed preparations are a very promising approach for enhancing the efficacy and comfort of patients undergoing spasticity treatment.

Synthesis and biochemical evaluation of baclofen analogues locked in the baclofen solid-state conformation.

The synthesis of six close analogues of baclofen [3-(4-chlorophenyl)-4-aminobutyric acid] (BAC), a potent GABAB agonist, are reported. The compounds were designed starting from the structural informations contained in the solid state of BAC, regarded as a possible bioactive conformation, in which the p-chlorophenyl ring is perpendicular to the GABA backbone. A similar conformational situation was created by rigidifying the BAC structure by means of methylene (1), ethylene (2 and 6), or propylene (3) units, or by introducing chlorine atoms (4 and 5) into the ortho positions ("ortho effect"). Only compound 5 showed affinity for the GABAB receptor. Compound 6 [1-(aminomethyl)-5-chloro-2,3-dihydro-1H-indene-1-acetic acid], which was initially considered as representing the optimal mimic of the solid-state conformation of BAC, was surprisingly found inactive. An extensive conformational analysis was performed on compounds 1-6 in order to evaluate their flexibility and the overlap of their conformational population with respect to BAC. For this purpose a distance map was generated from three possible pharmacophoric groups: the amino and the carboxylic functions, and the phenyl ring. Finally, several explanations are proposed to account for the poor affinities of the prepared compounds such as steric hindrance or flexibility demand of the receptor.

Synthesis and pharmacological evaluation of gamma-aminobutyric acid analogues. New ligand for GABAB sites.

Baclofen (beta-p-chlorophenyl-GABA) is the only selective agonist for the bicuculline-insensitive GABAB receptor. We report the synthesis of new GABA analogues and baclofen analogues. In vitro, two compounds, 4-amino-3-benzo[b]furan-2-ylbutanoic acid (9g) and 4-amino-3-(5-methoxybenzo[b]furan-2-yl)butanoic acid (9h), showed an affinity for the GABAB receptor. The results obtained with racemic compounds of benzofuran structure, new for this series, and the surprising inactivity of compound 3a (4-amino-3-(4-hydroxyphenyl)butanoic acid) permit the proposal of an hypothesis for the structure-activity relationships with regard to GABAB receptor.

Enantioselective Michael addition of nitromethane to alpha, beta-enones catalyzed by chiral quaternary ammonium salts. A simple synthesis of (R)-baclofen.

[reaction:see text] Enantioselective Michael addition of nitromethane to an alpha, beta-enone is a key step in the synthesis of (R)-baclofen.

Recoverable resin-supported pyridylamide ligand for microwave-accelerated molybdenum-catalyzed asymmetric allylic alkylations: enantioselective synthesis of baclofen.

The syntheses of a series of 4-monosubstituted pyridylamides and a resin-supported pyridylamide are described. The ligands were evaluated in the microwave-accelerated molybdenum-catalyzed asymmetric allylic alkylation. The reaction afforded the product in high yield and with high regio- and enantioselectivity. The heterogeneous ligand could be reused several times with no change in the reaction outcome. The asymmetric allylic alkylation was employed as the key step in the enantioselective synthesis of (R)-baclofen. [reaction: see text]

[Studies of synthesis and structure-activity relationships of analogs of the muscle relaxant--baclofen].

In an effort to study the structure-activity relationship of analogs of baclofen and to search for more potent and less toxic muscle relaxants and analgesics, fifteen 3-substituted pheny-4-aminobutyric acids and their derivatives were synthesized. Preliminary pharmacological tests in albino mice showed that some of the target compounds exhibited muscle relaxant and analgesic effects. Compounds Ib, IIb, IIIa, IIIc and compounds Ib, IIb, IIIa, IIIb were, respectively, more potent in hot plate tests and morphine-induced straub tail reaction tests than other analogs, but they were still less potent than baclofen. The structure-activity relationship showed that the effects of baclofen were strongly structure specific, and the results also suggested that at least three groups in baclofen-phenyl, amino and carboxy--take important parts in the combination of baclofen with GABAB receptor.

[Biocatalytic desymmetric hydrolysis of 3-(4-chlorophenyl)-glutaronitrile to the key precursor of optically pure baclofen].

We produced (S)-4-cyano-3-(4-chlorophenyl)-butyrate by highly stereoselective biocatalyst in this study. A nitrilase-producing strain, named Gibberella intermedia WX12, was isolated by 3-(4-chlorophenyl)-glutaronitrile as substrate in the screening with phenol-sodium hypochlorite method. The fermentation conditions and catalytic properties of this strain were investigated. The preferred carbon and nitrogen sources for nitrilase production were lactose (30 g/L) and peptone (20 g/L). After being cultivated for 96 h, the cells were collected for use in biotransformation. The hydrolysis of 3-(4-chlorophenyl)-glutaronitrile was performed at 30 degrees C in phosphate buffer (pH 8.0, 50 mmol/L) for 24 h to give (S)-4-cyano-3-(4-chlorophenyl)-butyric acid with 90% yield and > 99% of ee, which can be used for the synthesis of (R)- and (S)-baclofen. The configuration of product was determined by chemically converting it to baclofen and comparison with the authentic sample by chiral HPLC analysis.

Asymmetric synthesis of ß-substituted γ-lactams via rhodium/diene-catalyzed 1,4-additions: application to the synthesis of (R)-baclofen and (R)-rolipram.

An efficient rhodium/diene-catalyzed asymmetric addition of arylboronic acids to α,ß-unsaturated γ-lactams has been developed. The power of this methodology is further demonstrated by the concise synthesis of (R)-baclofen and (R)-rolipram.

Synthesis of alpha,beta-unsaturated gamma-lactams via asymmetric iridium-catalysed allylic substitution.

Syntheses of alpha,beta-unsaturated gamma-lactams are described that are based on ring-closing metathesis in combination with enantioselective Ir-catalysed allylic amination using N-Boc-N-(but-2-enoyl)-amine as a pronucleophile. As an example application, the synthesis of a Baclofen analogue is presented.

Enantioselective carbon-hydrogen insertion is an effective and efficient methodology for the synthesis of (R)-(-)-baclofen.

A highly enantioselective methodology for the synthesis of the GABA(B) receptor agonist (R)-(-)-baclofen is described. This synthesis begins with p-chlorophenethyl alcohol and involves a catalytic carbon-hydrogen insertion reaction of a chiral dirhodium(II) carboxamidate with the corresponding diazoacetate (81% yield, 95% ee). Subsequent steps convert the intermediate gamma-lactone to (R)- (-)-baclofen in a 60% overall yield. The amount of catalyst required for the C-H insertion transformation is only 0.5 mol%.