Stephen Robert Fletcher

[3H]L-655,708, a Novel Ligand Selective for the Benzodiazepine Site of GABAA Receptors which Contain the α5 Subunit

A compound (L-655,708) has been identified which has at least 50-fold selectivity for the benzodiazepine site on GABAA receptors containing an alpha 5 subunit over those containing an alpha 1, alpha 2, alpha 3 or alpha 6 subunit in combination with beta 3 and gamma 2. The compound was radiolabelled with tritium and investigated as a novel radioligand which recognizes the benzodiazepine site of GABAA receptors which contain the alpha 5 subunit. [3H]L-655,708 labels one saturable and specific population of binding sites in rat hippocampus with a Kd of 2.4 +/- 0.7 nM and a Bmax of 256 +/- 42 fmol/mg protein. The pharmacology of the binding site labelled was consistent with that of receptors present in cells transfected with alpha 5, beta 2 and gamma 2 and with receptors immunoprecipitated from rat brain with an alpha 5-selective antiserum. It is concluded that [3H]L-655,708 is the first radioligand to date which is selective for any BZ2 subtype of the GABAA receptor and should provide a valuable tool for elucidating the structure and function of the alpha 5-containing GABAA receptor subtype.

In Vitro and In Vivo Inhibition of Inositol Monophosphatase by the Bisphosphonate L‐690,330

Abstract: We have previously described the synthesis of bis‐phosphonate‐containing inhibitors of inositol monophosphatase. In the present study, a more detailed examination of the in vitro and in vivo properties of one of these compounds, L‐690,330, is described. L‐690,330 is a competitive inhibitor of inositol monophosphatase with a K1, depending on the source of IMPase, of between 0.2 and 2 μM. Although ∼1,000‐fold more potent in vitro than lithium, in muscarinic m1 receptor‐transfected Chinese hamster ovary cells prelabelled with [3H]inositol, L‐690,330 only produced 40% of the accumulation of [3H]inositol monophosphates achieved by lithium at the same concentration (10 mM), suggesting that the ability of L‐690,330 to cross the cell membrane is limited. Nevertheless, under conditions of cholinergic stimulation (100 mg/kg of pilocarpine s.c.), high doses of L‐690,330 were able to increase brain inositol(1)phosphate levels in vivo to three‐ to fourfold control levels. This effect was dose dependent (ED50= 0.3 mmol/kg s.c.) and was maximal after 1 h. In peripheral tissues, the effects of L‐690,330 on inositol(1)phosphate levels mimicked those of lithium both qualitatively and quantitatively. However, in the brain, the effects of L‐690,330 were much less than seen with lithium, consistent with the blood‐brain barrier restricting access of the polar L‐690,330 into the CNS, thereby further limiting entry of compound into cells in the brain. In the future, it may be possible to develop prodrugs of this compound, which circumvent many of the cell permeability problems inherent in bisphosphonate compounds.

The synthesis of (+)- and (–)-epibatidine

The synthesis of the alkaloid epibatidine {exo-2-(2-chloro-5-pyridyl)-7-azabicyclo[2.2.1]heptane} in enantiomeric form involving, as the critical step, reaction or 5-lithio-2-chloropyridine with N-tert-butoxycarbonyl-7-azabicyclo[2.2.1]heptan-2-one is described.

Discovery and Optimization of an Azetidine Chemical Series As a Free Fatty Acid Receptor 2 (FFA2) Antagonist: From Hit to Clinic

FFA2, also called GPR43, is a G-protein coupled receptor for short chain fatty acids which is involved in the mediation of inflammatory responses. A class of azetidines was developed as potent FFA2 antagonists. Multiparametric optimization of early hits with moderate potency and suboptimal ADME properties led to the identification of several compounds with nanomolar potency on the receptor combined with excellent pharmacokinetic (PK) parameters. The most advanced compound, 4-[[(R)-1-(benzo[b]thiophene-3-carbonyl)-2-methyl-azetidine-2-carbonyl]-(3-chloro-benzyl)-amino]-butyric acid 99 (GLPG0974), is able to inhibit acetate-induced neutrophil migration strongly in vitro and demonstrated ability to inhibit a neutrophil-based pharmacodynamic (PD) marker, CD11b activation-specific epitope [AE], in a human whole blood assay. All together, these data supported the progression of 99 toward next phases, becoming the first FFA2 antagonist to reach the clinic.

Triazolopyridines as Selective JAK1 Inhibitors: From Hit Identification to GLPG0634

Janus kinases (JAK1, JAK2, JAK3, and TYK2) are involved in the signaling of multiple cytokines important in cellular function. Blockade of the JAK-STAT pathway with a small molecule has been shown to provide therapeutic immunomodulation. Having identified JAK1 as a possible new target for arthritis at Galapagos, the compound library was screened against JAK1, resulting in the identification of a triazolopyridine-based series of inhibitors represented by 3. Optimization within this chemical series led to identification of GLPG0634 (65, filgotinib), a selective JAK1 inhibitor currently in phase 2B development for RA and phase 2A development for Crohns disease (CD).

Identification of a 4-(Hydroxymethyl)diarylhydantoin as a Selective Androgen Receptor Modulator

Structural modification performed on a 4-methyl-4-(4-hydroxyphenyl)hydantoin series is described which resulted in the development of a new series of 4-(hydroxymethyl)diarylhydantoin analogues as potent, partial agonists of the human androgen receptor. This led to the identification of (S)-(-)-4-(4-(hydroxymethyl)-3-methyl-2,5-dioxo-4-phenylimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile ((S)-(-)-18a, GLPG0492) evaluated in vivo in a classical model of orchidectomized rat. In this model, (-)-18a exhibited anabolic activity on muscle, strongly dissociated from the androgenic activity on prostate after oral dosing. (-)-18a has very good pharmacokinetic properties, including bioavailability in rat (F > 50%), and is currently under evaluation in phase I clinical trials.

L-708,474: the C5-cyclohexyl analogue of L-365,260, a selective high affinity ligand for the CCKB/gastrin receptor

Abstract The C5-cyclohexyl analogue of the cholecystokinin type-B (CCKB) receptor antagonist L-365,260 has been prepared. This derivative has significantly higher CCKB affinity and markedly improved CCKB/CCKA receptor selectivity (6,500 v. 87-fold) than the parent compound. It is one of the most potent and selective CCKB ligands reported to date.

POTENT, SELECTIVE, WATER-SOLUBLE BENZODIAZEPINE-BASED CCKB RECEPTOR ANTAGONISTS THAT CONTAIN LIPOPHILIC CARBOXYLATE SURROGATES

Abstract Acylsulphonamide analogues of the meta-tolylurea L-708,474 have been synthesised and evaluated as CCKB receptor antagonists. Such derivatives retain very high affinity and subtype selectivity for the CCKB receptor, and have good aqueous solubility. The ortho-tolyl acylsulphonamide L-736,309 is orally bioavailable and brain penetrant in rat.

Synthesis of α-methylene-β-lactams

The phase-transfer catalysed cyclisation of 3-bromo-2-bromomethylpropionamides provides a simple synthesis of α-methylene-β-lactams.

C5-piperazinyl-1,4-benzodiazepines, water-soluble, orally bioa vailable CCKB/gastrin receptor antagonists

Abstract A novel series of potent, water-soluble benzodiazepine based CCKB/gastrin antagonists has been prepared which incorporate an N-methylpiperazine group at the C5 position of the benzodiazepine ring system. The N1-n-propyl analogue (7b) is a high affinity, selective and potent receptor antagonist in vitro, with good bioavailability and excellent oral absorption providing high plasma levels in vivo.