Synthesis and pharmacological evaluation of Tic-hydantoin derivatives as selective sigma1 ligands. Part 2

Herein is described a new class of selective sigma1 ligands consisting of tetrahydroisoquinoline-hydantoin (Tic-hydantoin) derivatives. Compound 1a has high affinity (IC50 = 16 nM) for sigma1 receptor and is selective in a large panel of therapeutic targets. This study presents structural changes on the side chain of the Tic-hydantoin core. Analogs of higher affinity could be identified (IC50 approximately 2-3 nM).     

NE-100, a novel sigma receptor ligand: In vivo tests

It has been suggested that sigma receptor antagonists may be useful as antipsychotic drugs. N, N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE-100) is a novel compound with high affinity for the sigma receptor (IC50 = 4.16 nM), but low affinity (IC50 > 1000 nM) for D1, D2, 5-HT1A, 5-HT2 and phencyclidine (PCP) receptors. The head-weaving behavior induced by either (+)SKF10047 or PCP was dose-dependently antagonized by NE-100 with oral ED50 at 0.27 and 0.12 mg/kg, respectively. NE-100 did not affect dopamine agonists-induced stereotyped behavior and/or hyperactivity. NE-100 failed to induce catalepsy in rats. These findings indicate that NE-100 may have antipsychotic activity without the liability of motor side effects typical of neuroleptics.     

Purification and properties of Arabidopsis thaliana type 1 protein phosphatase (PP1).

The Arabidopsis thaliana type 1 protein phosphatase (PP1) catalytic subunit was released from its endogenous regulatory subunits by ethanol precipitation and purified by anion exchange and microcystin affinity chromatography. The enzyme was identified by MALDI-TOF mass spectrometry from a tryptic digest of the purified protein as a mixture of PP1 isoforms (TOPP 1-6) indicating that at least 4-6 of the eight known PP1 proteins are expressed in sufficient quantities for purification from A. thaliana suspension cells. The enzyme had a final specific activity of 8950 mU/mg using glycogen phosphorylase a as substrate, had a subunit molecular mass of 35 kDa as determined by SDS-PAGE and behaved as a monomeric protein of approx. 39 kDa on Superose 12 gel filtration chromatography. Similar to the mammalian type 1 protein phosphatases, the A. thaliana enzyme was potently inhibited by Inhibitor-2 (IC(50)=0.65 nM), tautomycin (IC(50)=0.06 nM), microcystin-LR (IC(50)=0.01 nM), nodularin (IC(50)=0.035 nM), calyculin A (IC(50)=0.09 nM), okadaic acid (IC(50)=20 nM) and cantharidin (IC(50)=60 nM). The enzyme was also inhibited by fostriecin (IC(50)=22 microM), NaF (IC(50)=2.1 mM), Pi (IC(50)=9.5 mM), and PPi (IC(50)=0.07 mM). Purification of the free catalytic subunit allowed it to be used to probe protein phosphatase holoenzyme complexes that were enriched on Q-Sepharose and a microcystin-Sepharose affinity matrix and confirmed several proteins to be PP1 targeting subunits.     

Synthesis and pharmacological evaluation of 6-piperidino- and 6-piperazinoalkyl-2(3H)-benzothiazolones as mixed sigma/5-HT(1A) ligands

In an effort to produce new pharmacological probes with mixed sigma/5-HT(1A) affinity, we have synthesized a series of 12 original 6-piperidino- or piperazino-alkyl-2(3H)-benzothiazolones and their receptor binding profile (sigma, 5-HT(1A), 5-HT(2A), 5-HT(3), D(2), H(1), and M(1)) was determined. The best mixed sigma/5-HT(1A) affinity profile was found within the piperidine series with 4-benzyl substitution associated to linker methylene chain n=2 (K(i) 5 and 4nM, respectively). Moreover, a highly selective sigma2 ligand was obtained with a 3,4-dichlorobenzyl substitution associated to n=4 (K(i) 2nM, selectivity ratio sigma1/sigma2=70).     

Discovery of potent and selective succinyl hydroxamate inhibitors of matrix metalloprotease-3 (stromelysin-1)

Structure activity relationships are described for a series of succinyl hydroxamic acids 4a-o as potent and selective inhibitors of matrix metalloprotease-3 (stromelysin-1). Optimisation of P1' and P3' groups gave compound 4j (MMP-3 IC50=5.9nM) which was >140-fold less potent against MMP-1 (IC50=51,000nM), MMP-2 (IC50=1790nM), MMP-9 (IC50=840nM) and MMP-14 (IC50=1900nM).     

Synthesis and benzodiazepine receptor (omega receptor) affinities of 3-substituted derivatives of pyrrolo[2,3-c]pyridine-5-carboxylate, a novel class of omega1 selective ligands.

Based on the structure of ZK91296 (4d), a high affinity partial agonist of the central benzodiazepine (omega) receptor, a series of pyrrolo[2,3-c]pyridine-5-carboxylate derivatives having mainly aralkyl and aralkyloxy substituents at C-3 was synthesized. The in vitro binding affinities of these compounds for three subclasses of the omega receptor (omega1, omega2, omega5) were determined using rat brain tissue. Practically all of these compounds (except the diethyl ester derivative 22c) showed an approximately twofold selectivity for omega1 (IC50's in the 200-500 nM range) compared to omega2 receptors and practically no affinity for omega5 receptors. Compound 22c showed the highest affinity of all the compounds synthesized (IC50 = 70 nM for omega1 receptors) as well as a fivefold selectivity for omega1 versus omega2 receptors but also displayed significant binding to omega5 receptors (IC50 = 250 nM). The absence of appreciable binding of 4-methyl and 4-methoxymethyl derivatives to omega receptors, in contrast to beta-carbolines having these similarly located substituents, suggests that the pyrrolo[2,3-c]pyridine-5-carboxylates may be considered an entirely novel class of selective omega receptor ligands.     

Discovery of 3,5-bis(trifluoromethyl)benzyl L-arylglycinamide based potent CCR2 antagonists

Systematic modification of a screening lead yielded a class of potent glycinamide based CCR2 antagonists. The best compound (55, (2S)-N-[3,5-bis(trifluoromethyl)benzyl]-2-{[2-(1-piperidinyl)ethyl]amino}-2-(3-thienyl)acetamide) displayed good binding affinity (IC50=30 and 39 nM) toward human monocytes and CHO cell expressing human CCR2b, respectively. Functionally, it blocked MCP-1 (CCL2)-induced calcium mobilization (IC50=50 nM) and chemotaxis mediated through the CCR2 receptor (9.6 nM). It is selective against other chemokine receptors tested.     

3H]BHDP as a novel and selective ligand for sigma1 receptors in liver mitochondria and brain synaptosomes of the rat

The binding profile of [(3)H]BHDP ([(3)H]N-benzyl-N'-(2-hydroxy-3,4-dimethoxybenzyl)-piperazine) was evaluated. [(3)H]BHDP labelled a single class of binding sites with high affinity (K(d)=2-3 nM) in rat liver mitochondria and synaptic membranes. The pharmacological characterization of these sites using sigma reference compounds revealed that these sites are sigma receptors and, more particularly, sigma1 receptors. Indeed, BHDP inhibited [(3)H]pentazocine binding, a marker for sigma1 receptors, with high affinity in a competitive manner. BHDP is selective for sigma1 receptors since it did not show any relevant affinity for most of the other receptors, ion channels or transporters tested. Moreover, in an in vitro model of cellular hypoxia, BHDP prevented the fall in adenosine triphosphate (ATP) levels caused by 24 h hypoxia in cultured astrocytes. Taken together, these results demonstrate that [(3)H]BHDP is a potent and selective ligand for sigma1 receptors showing cytoprotective effects in astrocytes.     

Benzofuro[3,2-b]pyridines as mixed ET(A)/ET(B) and selective ET(B) endothelin receptor antagonists

The discovery, synthesis and structure-activity relationships of a series of novel benzofuro[3,2-b]pyridines as non-selective endothelin ET(A)/ET(B) as well as selective ET(B) receptor antagonists are described. The most potent non-selective inhibitor 7s displayed an IC50 of 21 nM and 41 nM for ET(A) and ET(B) receptors, respectively, whereas 7ee merely showed affinity for the ET(B) receptor (IC50 = 3.6 nM).     

Kinetic and pharmacological properties of cloned human equilibrative nucleoside transporters, ENT1 and ENT2, stably expressed in nucleoside transporter-deficient PK15 cells. Ent2 exhibits a low affinity for guanosine and cytidine but a high affinity for inosine

We stably transfected the cloned human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) into nucleoside transporter-deficient PK15NTD cells. Although hENT1 and hENT2 are predicted to be 50-kDa proteins, hENT1 runs as 40 kDa and hENT2 migrates as 50 and 47 kDa on SDS-polyacrylamide gel electrophoresis. Peptide N-glycosidase F and endoglycosidase H deglycosylate hENT1 to 37 kDa and hENT2 to 45 kDa. With hENT1 being more sensitive, there is a 7000-fold and 71-fold difference in sensitivity to nitrobenzylthioinosine (NBMPR) (IC(50), 0.4 +/- 0.1 nM versus 2.8 +/- 0.3 microM) and dipyridamole (IC(50), 5.0 +/- 0.9 nM versus 356 +/- 13 nM), respectively. [(3)H]NBMPR binds to ENT1 cells with a high affinity K(d) of 0.377 +/- 0.098 nM, and each ENT1 cell has 34,000 transporters with a turnover number of 46 molecules/s for uridine. Although both transporters are broadly selective, hENT2 is a generally low affinity nucleoside transporter with 2.6-, 2.8-, 7. 7-, and 19.3-fold lower affinity than hENT1 for thymidine, adenosine, cytidine, and guanosine, respectively. In contrast, the affinity of hENT2 for inosine is 4-fold higher than hENT1. The nucleobase hypoxanthine inhibits [(3)H]uridine uptake by hENT2 but has minimal effect on hENT1. Taken together, these results suggest that hENT2 might be important in transporting adenosine and its metabolites (inosine and hypoxanthine) in tissues such as skeletal muscle where ENT2 is predominantly expressed.     

The mechanism of SR95531 inhibition at GABA receptors examined in human alpha1beta1 and alpha1beta1gamma2S receptors

We examined the interaction of GABA and the competitive inhibitor SR95531 at human alpha1beta1gamma2S and alpha1beta1 GABA(A) receptors expressed in Sf9 cells. The efficacy and potency of inhibition depended on the relative timing of the GABA and SR95531 applications. In saturating (10 mM) GABA, the half-inhibitory concentrations of SR95531 (IC50) when coapplied with GABA to alpha1beta1gamma2S or alpha1beta1 receptors were 49 and 210 microM for the peak and 18 and 130 microM for the plateau current, respectively. Our data are explained by an inhibition mechanism in which SR95531 and GABA bind to two sites on the receptor where the binding of GABA allows channel opening but SR95531 does not. The SR95531 affinity for both receptor types was approximately 200 nM and the binding rate was found to be 10-fold faster than that for GABA. The dual binding-site model gives insights into the differential effects of GABA and SR95531 on the peak and plateau currents. The model predicts the effect of SR95531 on GABA currents in the synapse (GABA concentration approximately mM) and at extrasynaptic (GABA concentration < or = microM) sites. The IC50 (50-100 nM) for the synaptic response to SR95531 was insensitive to the GABA affinity of the receptors whereas the IC50 (50-800 nM) for extrasynaptic inhibition correlated with the GABA affinity.     

Discovery of highly potent and selective benzyloxybenzyl-based peroxisome proliferator-activator receptor (PPAR) delta agonists

A series of 1,4-benzyloxybenzylsulfanylaryl carboxylic acids were prepared and their activities for PPAR receptor subtypes (alpha, delta, and gamma) with potential indications for the treatment of dyslipidemia were investigated. Analog 13a displayed the greatest binding affinity (IC(50)=10nM) and selectivity (120-fold) for PPARdelta over PPARalpha. Many of the analogs investigated were found to be highly selective for PPARdelta and were dependent on the point of attachment of the substituent. In the 1,4-series, analog 28e was found to be the most potent (IC(50)=1.7 nM) and selective (>1000-fold) compound for PPARdelta. None of the compounds tested showed appreciable binding affinity for PPARgamma.     

Structure-Activity relationships of 2-substituted 5,7-Diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acids as a novel class of endothelin receptor antagonists

Synthesis and structure-activity relationships of 2-substituted-5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acids, a novel class of endothelin receptor antagonists, were described. Derivatization of a lead structure 1 (IC(50)=2.4nM, 170-fold selectivity) by incorporating a substituent such as an alkyl, alkoxy, alkylthio, or alkylamino group into the 2-position of the cyclopenteno[1,2-b]pyridine skeleton was achieved via the key intermediate 8. Introduction of an alkyl group led to the identification of potent ET(A)/ET(B) mixed receptor antagonists, a butyl (2d: IC(50)=0.21nM, 52-fold selectivity) and an isobutyl (2f: IC(50)=0.32nM, 26-fold selectivity) analogue. In contrast, installment of a primary amino group resulted in ET(A) selective antagonists, a propylamino 2p (IC(50)=0.12nM, 520-fold selectivity) and an isopropylamino 2q (IC(50)=0.10nM, 420-fold selectivity) analogue. These results suggested that a substituent at the 2-position of the 5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acids played a key role in the binding affinity for both ET(A) and ET(B) receptors.     

1,3-Disubstituted benzazepines as neuropeptide Y Y1 receptor antagonists.

A novel class of potent and selective non-peptide neuropeptide Y (NPY) Y1 receptor antagonists, having benzazepine nuclei, have been designed, synthesized, and evaluated for activity. Through a blind screening we found the compound 1-N-(3-(N'-(tert-butoxycarbonyl)amino)benzyl)-7-methoxy-(3-(3)-methyl ureido)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one (9: IC50 = 1.6 microM). Chemical modifications of 9 gave a potent NPY Y1 antagonist 3-(N-(4-hydroxyphenyl)-N'-methylguanidino)-1-N-(3-(N'-(tert-butoxy carbonyl)amino)benzyl)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one (14c: IC5(0=43 nM), which had no affinity for NPY Y2 and Y5 receptors.     

Conformationally constrained N1-arylsulfonyltryptamine derivatives as 5-HT6 receptor antagonists

Several series of conformationally constrained N1-arylsulfonyltryptamine derivatives were prepared and tested for 5-HT6 receptor binding affinity and ability to modulate cAMP production in a cyclase assay. The 3-piperidin-3-yl-, 3-(1-methylpyrrolidin-2-ylmethyl)-, and 3-pyrrolidin-3-yl-1H-indole arrays (8-13) appear to be able to adopt a conformation that allows high affinity 5-HT6 receptor binding, while the beta-carboline array 14 binds with a significantly weaker (10- to 100-fold) affinity. N1-Benzenesulfonyl-3-piperidin-3-yl-1H-indole 9a is a high affinity full agonist with EC50 = 24 nM. Several of the N1-arylsulfonyl-3-(1-methylpyrrolidin-2-ylmethyl)-1H-indole derivatives behave as very potent antagonists ((S)-11r, (S)-11t; IC50 = 0.8, 1.0 nM).     

The discovery and synthesis of JNJ 31020028, a small molecule antagonist of the Neuropeptide Y Y₂ receptor

A series of small molecules based on a chemotype identified from our compound collection were synthesized and tested for binding affinity (IC(50)) at the human Neuropeptide Y Y(2) receptor (NPY Y(2)). Six of the 23 analogs tested possessed an NPY Y(2) IC(50) ≤ 15 nM. One member of this series, JNJ 31020028, is a selective, high affinity, receptor antagonist existing as a racemic mixture. As such a synthetic route to the desired enantiomer was designed starting from commercially available (S)-(+)-mandelic acid.     

Discovery of novel 3,5-disubstituted indole derivatives as potent inhibitors of Pim-1, Pim-2, and Pim-3 protein kinases

A series of novel 3,5-disubstituted indole derivatives as potent and selective inhibitors of all three members of the Pim kinase family is described. High throughput screen identified a pan-Pim kinase inhibitor with a promiscuous scaffold. Guided by structure-based drug design, SAR of the series afforded a highly selective indole chemotype that was further developed into a potent set of compounds against Pim-1, 2, and 3 (Pim-1 and Pim-3: IC(50)≤2nM and Pim-2: IC(50)≤100nM).     

Synthesis and evaluation of novel 1-(2-acylhydrazinocarbonyl)-cycloalkyl carboxamides as interleukin-1beta converting enzyme (ICE) inhibitors

Novel 1-(2-acylhydrazinocarbonyl)cycloalkyl carboxamides were designed as peptidomimetic inhibitors of interleukin-1beta converting enzyme (ICE). A short synthesis was developed and moderately potent ICE inhibitors were identified (IC(50) values <100 nM). Most of the synthesized examples were selective for ICE versus the related cysteine proteases caspase-3 and caspase-8, although several dual-acting inhibitors of ICE and caspase-8 were identified. Several of the more potent ICE inhibitors were also shown to inhibit IL-1beta production in a whole cell assay (IC(50) < 500 nM).     

Synthesis and structure-activity relationships of N-(3-phenylpropyl)-N'-benzylpiperazines: Potent ligands for sigma1 and sigma2 receptors

Ten N-(3-phenylpropyl)-N'-benzylpiperazines having different substituents on the benzyl moiety were synthesized and evaluated for sigma(1) and sigma(2) receptor binding. The sigma(1) affinities were 0.37-2.80nM, sigma(2) affinities were 1.03-34.3nM, and selectivities, as sigma(2)/sigma(1) affinity ratios, ranged from 1.4 to 52. Three compounds tested in a phenytoin shift binding assay profiled as probable sigma(1) antagonists. Quantitative structure-activity relationships depended on pi(x), MR or E(s) and Hammett sigma values. The hydrophobicity term is negative for sigma(1) binding but positive for sigma(2) binding, indicating a major difference between the pharmacophores.     

The importance of the nine-amino acid C-terminal sequence of exendin-4 for binding to the GLP-1 receptor and for biological activity

Exendin-4, a 39-amino acid (AA) peptide, is a long-acting agonist at the glucagon-like peptide-1 (GLP-1) receptor. Consequently, it may be preferable to GLP-1 as a long-term treatment for type 2 diabetes mellitus. Exendin-4 (Ex-4), unlike GLP-1, is not degraded by dipeptidyl peptidase IV (DPP IV), is less susceptible to degradation by neutral endopeptidase, and possesses a nine-AA C-terminal sequence absent from GLP-1. Here we examine the importance of these nine AAs for biological activity of Ex-4, a sequence of truncated Ex-4 analogs, and native GLP-1 and GLP-1 analogs to which all or parts of the C-terminal sequence have been added. We found that removing these AAs from Ex-4 to produce Ex (1-30) reduced the affinity for the GLP-1 receptor (GLP-1R) relative to Ex-4 (IC50: Ex-4, 3.22+/-0.9 nM; Ex (1-30), 32+/-5.8 nM) but made it comparable to that of GLP-1 (IC50: 44.9+/-3.2 nM). The addition of this nine-AA sequence to GLP-1 improved the affinity of both GLP-1 and the DPP IV resistant analog GLP-1 8-glycine for the GLP-1 receptor (IC50: GLP-1 Gly8 [GG], 220+/-23 nM; GLP-1 Gly8 Ex (31-39), 74+/-11 nM). Observations of the cAMP response in an insulinoma cell line show a similar trend for biological activity.