Stuart J. Mickel

GABA and glutamate release affected by GABAB receptor antagonists with similar potency : no evidence for pharmacologically different presynaptic receptors

1 The effects of a series of nine GABAB receptor antagonists of widely varying potencies on electrically stimulated release from cortical slices of [3H]‐GABA in the absence or presence of 10 μm of the GABABagonist, (−)−baclofen and of endogenous glutamate in the presence of (−)−baclofen were compared. 2 The concentrations of the compounds half maximally increasing [3H]‐GABA release (EC50s) at a stimulation frequency of 2 Hz correlated well with the IC50 values obtained from the inhibition of the binding of the agonist, [3H]‐CGP 27492, to GABAB receptors in rat brain membranes (rank order of potency: CGP 56999 A ≥ CGP 55845 A > CGP 52432 ≥ CGP 56433 A > CGP 57034 A > CGP 57070 A ≥ CGP 57976 > CGP 51176 > CGP 35348). 3 Likewise, the concentrations causing half‐maximal increases of [3H]‐GABA in the absence or presence of (−)−baclofen, and of endogenous glutamate in the presence of (−)−baclofen, correlated well with each other. Reports in the literature suggesting the CGP 35348 exhibits a 70 fold preference for inhibition of (−)−baclofens effects on glutamate over [3H]‐GABA release, and that CGP 52432 shows a 100 fold preference in the opposite sense, could not be confirmed in our model. 4 Therefore, our results suggest that, if there are pharmacological differences between GABAB autoreceptors and GABAB heteroreceptors on glutamatergic nerve endings in the rat cortex, they are not revealed by this series of compounds of widely different potencies. 5 In particular, our results with CGP 35348 and CGP 52432 do not support the hypothesis that GABAB autoreceptors and GABAB heteroreceptors on glutamatergic nerve endings represent subtypes with different pharmacology.

The action of new potent GABAB receptor antagonists in the hemisected spinal cord preparation of the rat

CGP 52432 (3-N-(3,4-dichlorobenzyl)aminopropyl-P-diethoxymethylphosphinic acid), CGP 54062 (3-N[1-(R,S)-(3,4-dichlorophenyl)ethyl]amino-2-(S)-hydroxypropyl-P-benzy l- phosphinic acid), CGP 54626 (3-N[[1-(S)-(3,4-dichlorophenyl)ethyl]amino-2-(S)- hydroxypropyl-P-cyclohexylmethylphosphinic acid) and CGP 55845 (3-N[1-(S)-(3,4-dichlorophenyl)ethyl]amino-2-(S)- hydroxypropyl-P-benzyl-phosphinic acid) are novel selective GABAB receptor antagonist. The apparent Kd values for the complex formed between the GABAB receptor and these compounds were determined using the monosynaptic reflex in the hemisected rat spinal cord preparation in vitro. CGP 55845 was found to be the most potent GABAB receptor antagonist tested (apparent Kd = 30 nM). On the same preparation 0.3 microM CGP 55845 was equipotent with 100 microM of CGP 35348 (P-(3-aminopropyl)-P-diethoxymethyl-phosphinic acid) for reversal of the depressant action of (R)-(-)-baclofen.

Chemistry of GABAB Modulators

Nearly 10 yr have passed since the authors review “Chemistry of GABAB Modulators” appeared in the book “The GABA Receptors,” 2nd edition, edited by S. J. Enna and N. G. Bowery, Humana Press, Totowa, 1997 (1). In this update the authors wish to outline only new developments not covered in the 1997 paper. Baclofen, synthesized for the first time in September 1962, is still the only γ-aminobutyric acid (GABAB)-receptor agonist marketed for the treatment of spasticity and trigeminal neuralgia. It is fascinating to learn how many highly competent chemists devised manifold and elegant synthetic procedures for either racemic or (R)-(−)-baclofen and the structurally closely related potent antidepressant (R)-(−)-Rolipram, a selective phosphodiesterase-4 inhibitor. The new syntheses published after 1996 are listed in alphabetical order (2–16). Very recently a prodrug of (R)-(−)-baclofen (Fig. 1) was described which enhanced the oral bioavailability in Cynomolgus monkeys to more than 80% (17). It is planned to test this prodrug in clinical trials for the treatment of spasticity and gastroesophageal reflux disease.

Ligands for expression cloning and isolation of GABAB receptors

Outlined is the rationale behind the syntheses of radioligands [125I]CGP64213 and [125I]CGP71872, which led to the identification of cloned GABA(B) receptors 1a and 1b 17 years after the first pharmacological characterisation of native GABA(B) receptors by Bowery et al. [Nature 283 (1980) 92-94]. More recently it was shown that the N-terminal extracellular domains of GABA(B) receptors 1a and 1b contain the binding sites for agonists and antagonists [B. Malitschek et al., Mol. Pharmacol. 56 (1999) 448-454]. In order to isolate the extracellular domain(s) of GABA(B) receptors 1a (or 1b) and to purify and crystallise these proteins a third ligand [125I]CGP84963 was designed, which combines, in one molecule, a GABA(B) receptor binding part, an azidosalicylic acid as photoaffinity moiety and 2-iminobiotin, which binds to avidin in a reversible, pH-dependent fashion [W. Froestl et al., Neuropharmacology 38 (1999) 1641-1646].

Total synthesis of the marine natural product (+)-discodermolide in multigram quantities

The novel polyketide (+)-discodermolide was isolated in very small quantities from sponge extracts. This compound is one of several microtubule stabilizers showing promise as novel chemotherapeutic agents for the treatment of cancer. The clinical evaluation of this and similar compounds is hampered by lack of material, and at present, the only way to obtain the necessary quantities is total chemical synthesis.

Ligands for the isolation of GABAB receptors : W. Froestl would like to dedicate this work to the first GABAB chemist, Cr Heinrich Keberle, on the occasion of his 77th birthday

Since the discovery that the most abundant inhibitory neurotransmitter in the mammalian brain, GABA (gamma-aminobutyric acid), interacts not only with ionotropic GABA(A) receptors, but also with metabotropic GABA(B) receptors (Bowery et al., 1980) much work has been devoted to the elucidation of the structure of GABA(B) receptors by either affinity chromatography purification or by expression cloning. In 1997 Kaupmann et al. succeeded in cloning two splice variants designated GABA(B) R1a (960 amino acids) and GABA(B) R1b (844 amino acids). Although the amino acid sequences are now known, precise information on the three-dimensional environment of the GABA(B) R1 binding site is still lacking. Recent experiments demonstrated that the amino acids of the seven transmembrane helices are not essential for ligand binding as a soluble GABA(B) receptor fragment is still able to bind antagonists (Malitschek et al., 1999). For the isolation and purification of the soluble N-terminal extracellular domain (NTED) of GABA(B) receptors potent ligands for affinity chromatography were synthesised with the aim of obtaining a crystalline receptor fragment-ligand complex for X-ray structure determination. The most promising ligand [125I]CGP84963 (K(D) = 2 nM) combines, in one molecule, a GABA(B) receptor binding part, an azidosalicylic acid as a photoaffinity moiety separated by a spacer consisting of three GABA molecules from 2-iminobiotin, which binds to avidin in a reversible, pH-dependent fashion.

Determination of rat brain and plasma levels of the orally active GABAB antagonist 3-amino-propyl-n-butyl-phosphinic acid (CGP 36742) by a new GC/MS method

An involvement of GABAergic neurons has been suggested in the process of memory consolidation based on anatomical evidence and increasing physiological and biochemical data. With the advent of orally active GABAB antagonists, such as CGP 36742, the question of their therapeutic value, for example in Alzheimers disease, becomes relevant. Therefore, a new GC/MS method was developed to determine the concentration of CGP 36742 (3-amino-propyl-n-butyl phosphinic acid) in various intra- and extracerebral tissues after different routes of application. The compound was chemically derivatised in a two-step process (acylation of the amino group and esterification of the phosphinic acid). The limit of detection of the method was 0.01 microgram/g tissue and 0.0005 microgram/mL plasma. The time-course after i.p. treatment showed peak levels of CGP 36742 between 30 min and 1 hr after injection. After a dose of 100 mg/kg, the concentration in the brain ranged from 1 to 1.4 microgram/g or 6 to 8 microM, assuming that 1 mg tissue equals 1 microL (i.e., below the IC50 of the interaction with GABAB receptors as measured by [3-3H]-aminopropyl-phosphinic acid binding [35 microM]). These results are discussed in light of the psychopharmacological effects (improvement of cognitive performance of rats) of CGP 36742 observed at very low oral doses.

Broad spectrum chemistry as practised by novartis process research

Three actual examples from the current product palette within Novartis Process Research will demonstrate the some of the variety and challenges encountered in modern chemical development.

GABAB receptor antagonists:New tools and potential new drugs

Publisher Summary This chapter discusses GABA B receptor antagonists. The most abundant inhibitory neurotransmitter in the mammalian central nervous system is γ-aminobutyric acid (GABA), which interacts with two types of receptors designated GABA A and GABA B by Hill and Bowery in 1981. GABA A receptors are linked to chloride channels and transmit fast synaptic inhibition. The actions of GABA B receptors are mediated indirectly through heterotrimeric G-proteins of the subtypes G o and G il . Agonists for GABA B receptors are GABA and the (R)-(-)- enantiomer of the antispastic agent, baclofen, synthesized in 1962. GABA B receptor subtypes are located on postsynaptic as well as on presynaptic sites. Postsynaptic GABA B receptors are coupled via pertussis toxin (PTX) sensitive G-proteins to potassium channels of the GIRK class. One of the physiological roles of the postsynaptic GABA B receptors is to mediate the late inhibitory postsynaptic potential. The chapter discusses GABA B receptor antagonists as tools to elucidate mechanisms of induction of long-term potentiation as well as their therapeutic potential. Extensive in vivo investigations suggest that GABA B receptor antagonists may have therapeutic potential for the treatment of cognition deficits, absence epilepsy, anxiety and depression.

Chapter 9 Adventures in natural product synthesis: From deep sea sponge to pilot plant. The large scale total synthesis of the marine natural product (+)-discodermolide

Publisher Summary This chapter discusses the large scale total synthesis of the marine natural product ( + )-, discodermolide, which is a novel polyketide natural product first isolated in very small quantities from the extracts of the marine sponge, Discodermia dissoluta . The strategy used in the synthesis is to avoid scale-up problems—selecting reagents and reaction conditions to allow safe and efficient manufacture in a pilot plant to ultimately produce material of such a quality that it can be utilized for human clinical trials. Structurally, discodermolide consists of a linear polypropionate chain containing thirteen stereocenters, six of which are hydroxyl bearing, with one of these esterified as a γ-lactone (C 5 ) and another as a carbamate (C 19 ). It also features seven methyl-bearing stereocenters and three Z-configured alkenes, one of these being part of the terminal diene unit and another being a cis -trisubstituted system at C 13 –C 14 . Also present in the structure is a common stereo triad (methyl, hydroxyl, and methyl) that is repeated three times. Attempts to reproducibly isolate a discodermolide-producing microorganism for fermentation have not been successful to date. Therefore, all discodermolide used for late preclinical research and development activities, as well as for the ongoing clinical trial, has to be supplied by total synthesis.