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CEP-32215 is a new, potent, selective, and orally bioavailable inverse agonist of the histamine H3 receptor (H3R) with drug-like properties. High affinity in human (hH3R Ki = 2.0 ± 0.2 nM) and rat (rH3R Ki = 3.6 ± 0.7 nM) H3R radioligand binding assays was demonstrated. Potent functional antagonism (Kb = 0.3 ± 0.1 nM) and inverse agonism (EC50 = 0.6 ± 0.2 nM) were demonstrated in [(35)S]guanosine 5(')-O-(γ-thio)-triphosphate binding assays. Oral bioavailability and dose-related exposure was consistent among rat, dog, and monkey. After oral dosing, occupancy of H3R by CEP-32215 was estimated by the inhibition of ex vivo binding in rat cortical slices (ED50 = 0.1 mg/kg p.o.). Functional antagonism in brain was demonstrated by the inhibition of R-α-methylhistamine-induced drinking in the rat dipsogenia model (ED50 = 0.92 mg/kg). CEP-32215 significantly increased wake duration in the rat EEG model at 3-30 mg/kg p.o. Increased motor activity, sleep rebound or undesirable events (such as spike wave or seizure activity) was not observed following doses up to 100 mg/kg p.o., indicating an acceptable therapeutic index. CEP-32215 may have potential utility in the treatment of a variety of sleep disorders. This article is part of the Special Issue entitled 'Histamine Receptors'.

A novel series of 3,4-diaza-bicyclo[4.1.0]hept-4-en-2-ones were designed and synthesized as H3R analogs of irdabisant 6. Separation of the isomers, assignment of the stereochemistry by crystallography, and detailed profiling of diastereomers 25 and 26 led to the identification of (1R,6S)-5-{4-[3-((R)-2-methyl-pyrrolidin-1-yl)propoxy]phenyl}-3,4-diaza-bicyclo[4.1.0]hept-4-en-2-one 25 as a potential second generation H3R candidate. Diastereomer 25 had high H3R binding affinity, excellent selectivity, displayed potent H3R functional antagonism and robust wake-promoting activity in vivo, and showed acceptable pharmacokinetic and pharmaceutical profiles for potential further development.

A novel class of benzocinnolinones analogs of irdabisant were designed and synthesized as histamine H3R antagonists/inverse agonists. Modifications to the pyridazinone portion of the core and linker led to the identification of molecules with excellent target potency and selectivity with improved rat pharmacokinetic properties and reduced potential hERG liabilities.

Optimization of a series of aminomethyl ketone diamine H3R antagonists to reduce the brain exposure by lowering the pKa, led to molecules with improved pharmacokinetic properties. Compounds 9, 19, and 25 had high affinity for human H3R and demonstrated in vivo H3R functional activity in the rat dipsogenia model. Compound 9 displayed modest wake-promoting activity in the rat EEG/EMG model.

H3R structure–activity relationships for a new class of 4,5-dihydropyridazin-3-one H3R antagonists/inverse agonists are disclosed. Modification of the 4,5-dihydropyridazinone moiety to block in vivo metabolism identified 4,4-dimethyl-6-{4-[3-((R)-2-methyl-pyrrolidin-1-yl)-propoxy]-phenyl}-4,5-dihydro-2H-pyridazin-3-one 22 as a lead candidate demonstrating potent in vivo functional H3R antagonism in the rat dipsogenia model and robust wake promoting activity in the rat EEG/EMG model.

A novel class of 4-alkoxy-[1′-cyclobutyl-spiro(3,4-dihydrobenzopyran-2,4′-piperidine)] analogues were designed and synthesized as H3R antagonists. Structure–activity relationship identified sulfone 27 with excellent H3R affinities in both humans and rats, and acceptable pharmacokinetic properties. Further, compound 28 achieved single digit nanomolar H3R affinities in both species with minimum hERG activity.

6-{4-[3-(R)-2-Methylpyrrolidin-1-yl)propoxy]-phenyl}-2H-pyridazin-3-one 6 (Irdabisant; CEP-26401) was recently reported as a potent H(3)R antagonist with excellent drug-like properties and in vivo activity that advanced into clinical evaluation. A series of pyridone analogs of 6 was synthesized and evaluated as H(3)R antagonists. Structure-activity relationships revealed that the 5-pyridone regiomer was optimal for H(3)R affinity. N-Methyl 9b showed excellent H(3)R affinity, acceptable pharmacokinetics and pharmaceutical properties. In vivo evaluation of 9b showed potent activity in the rat dipsogenia model and robust wake-promoting activity in the rat EEG model.

A series of pyridazinone-phenethylamine derivatives with moderate to low nanomolar affinity for rat and human H(3)R are described. These analogs exhibited excellent selectivity and metabolic stability, with acceptable rat pharmacokinetic properties. In vivo, 7 and 11 demonstrated potent H(3)R functional antagonism in the rat dipsogenia model and robust wake-promoting activity in the rat electroencephalogram/electromyography (EEG/EMG) model.

Three series of novel 4,5-fused pyridazinones were synthesized as histamine H3 receptor antagonists. The 2,5,6,7-tetrahydrocyclopenta[d]pyridazin-1-one 5q and 5,6,7,8-tetrahydro-2H-phthalazin-1-one 5u displayed high affinity at both rat and human H3 receptors, and showed potent antagonist and full inverse agonist activity in functional assays.

Pyridazinone 1 was recently reported as a potent H(3)R antagonist with good drug-like properties and in vivo activity. A series of constrained amine analogs of 1 was synthesized to identify compounds with improved pharmacokinetic profiles. From these efforts, a new class of (S)-2-pyrrolidin-1-ylmethyl-1-pyrrolidinyl amides was identified.

H3R structure–activity relationships on a novel class of pyridazin-3-one H3R antagonists/inverse agonists are disclosed. Modifications of the pyridazinone core, central phenyl ring and linker led to the identification of molecules with excellent target potency, selectivity and pharmacokinetic properties. Compounds 13 and 21 displayed potent functional H3R antagonism in vivo in the rat dipsogenia model and demonstrated robust wake activity in the rat EEG/EMG model.

Fused dihydroindazolopyrrolocarbazole oximes have been identified as low nanomolar, potent dual TIE-2 and VEGF-R2 receptor tyrosine kinase inhibitors with excellent cellular potency. Development of the structure-activity relationships (SAR) led to identification of compounds 35 and 40 as potent, selective dual TIE-2/VEGF-R2 inhibitors with favorable pharmacokinetic properties. Compound 35 was orally active in tumor models with no observed toxicity.

A novel series of C-3 urea, amide, and carbamate fused dihydroindazolocarbazole (DHI) analogs are reported as highly potent dual inhibitors of TIE-2 and VEGF-R2 receptor tyrosine kinases with excellent cellular potency. Structure-activity relationship (SAR) studies indicate the optimal N-13 alkyl substitutions are n-propyl and i-butyl. The isopropyl carbamate 39 displayed good dual enzyme, cell potency, and rat pharmacokinetic properties for advancement to in vivo evaluation.

A series of novel pyrrolocarbazole lactams was identified as potent PARP-1 inhibitors in vitro and in a PC12 cellular NAD(+) depletion assay. The SAR trends of substituents at the 3-position, as well as the effect of blocking the indole or lactam NH-groups of the template by methylation or formylation, are discussed in relation to molecular modeling studies.

A series of novel pyrrolocarbazoles was synthesized as potential PARP-1 inhibitors. Pyrrolocarbazole 1 was identified as a potent PARP-1 inhibitor (IC50 = 36 nM) from our internal database. Synthesis of analogs around this template with the aid of modeling studies led to the identification of the truncated imide 14. Compound 14 (IC50 = 40 nM), with deleted B-ring, was found to be an equipotent PARP-1 inhibitor.

A novel class of 1'-cyclobutyl-6-(4-piperidyloxy)spiro[benzopyran-2,4'-piperidine] derivatives with low nanomolar affinity for the human and rat histamine-3 receptors (H(3)Rs) are described. The spirobenzopyran piperidine ether analogs demonstrated excellent H(3)R affinity and selectivity against histamine receptor subtypes (H(1)R, H(2)R, and H(4)R), were stable in liver microsomes, and had selectivity against CYP P450 enzymes. Compounds 10, 13, 15, and 16 demonstrated high H(3)R affinity, in vitro liver microsomal stability, selectivity against CYP isoforms, moreover, these ether analogs exhibited acceptable iv pharmacokinetic (PK) properties but had poor oral exposure in rat.

The MLK1-3 activity for a series of analogues of the indolocarbazole K-252a is reported. Addition of 3,9-bis-alkylthiomethyl groups to K-252a results in potent and selective MLK inhibitors. The in vitro and in vivo survival promoting activity of bis-isopropylthiomethyl-K-252a (16, CEP-11004/KT-8138) is reported.

A 96-well microtiter enzyme-linked immunosorbent assay was developed to assay the activity of the cytoplasmic domain of trkA tyrosine kinase. The assay involves immobilization of phospholipase C-γ/glutathione S -transferase fusion protein on a microtiter plate, addition of the kinase reaction mixture, and detection by an antibody to phosphotyrosine followed by an alkaline phosphatase-conjugated second antibody. The substrate used in this system, phospholipase C-γ, is one of several biologically important substrates for the phosphorylation reaction of receptor-linked tyrosine kinases. The assay was then used to characterize kinase inhibitory activities of various small molecules including analogs of K-252a.

Pyrrolo[3,4-c]carbazole derivatives were prepared as potential neurotrophic agents. The compounds were assayed for their ability to stimulate choline acetyltransferase (ChAT) activity in embryonic rat spinal cord cultures. These simplified K252a derivatives, although less potent and efficacious, have led to the identification of minimal structural requirements for K252a neurotrophic activity.

A series of 4-phenylpiperidinyl and 4-phenylpiperazinyl alkyl spaced 5,5-diphenylhydantoins was prepared and evaluated for affinity at σ sites. Increasing the alkyl spacer between the two pharmacophore recognition units resulted in a progressive increase in σ binding affinity. The pentyl 12 and hexyl 13 4-phenylpiperdine derivatives exhibited subnanomolar affinity (0.7 nM and 0.6 nM) for the PENT site.

The M{sub 1}-selective muscarinic antagonists aprophen, caramiphen, carbetapentane, 2-DAEX, dicyclomine, hexahydrosiladifenidol, iodocaramiphen, nitrocaramiphen, oxybutynin and trihexyphenidyl potently inhibited binding to {sigma} sites in brain. Both basic ester and non-ester structural type compounds which exhibit affinity for the muscarinic receptor also demonstrated affinity for the {sigma} site, while the classical antimuscarinic agents atropine and QNB, and the tricyclic pirenzepine, were ineffective in binding to this site. The authors also observed a significant correlation between the K{sub i} values for {sigma}compounds to inhibit ({sup 3}H)pirenzepine binding and their IC{sub 50} values to inhibit carbachol-stimulated phosphoinositide turnover. These observations may aid in elucidating the relationship of {sigma} binding to inhibition of phosphoinositide turnover stimulated by cholinergic agonists.

The potent antimuscarinic benzetimide and its resolved stereoisomers dexetimide and levetimide were tested for their affinities at σ sites labelled by [ 3 H](+)pentazocine or [ 3 H]1,3-di(2-tolyl)guanidine. Levetimide was a potent and stereoselective inhibitor of [ 3 H](+)pentazocine binding, with a K i of 2.2 nM, while dexetimide was nine-fold less potent (K i = 19 nM). Dexetimide and levetimide potently inhibited [ 3 H]DTG binding although without stereoselectivity (K i values of 65 and 103 nM, respectively). Levetimide may be a useful tool with which to investigate σ recognition sites and σ subtypes.

RLH-033 [2-(4-phenylpiperidinyl)ethyl 1-(4-nitrophenyl)cyclopentanecarboxylate HCl] is a rationally designed ligand that was synthesized and evaluated for its binding affinities at sigma 1 and sigma 2 sites in guinea pig brain. RLH-033 has high affinity (Ki = 50 pM) for sigma 1 sites labeled by [3H](+)-pentazocine, while it was over 2000-fold less affinity at sigma 2 sites labeled by [3H]1,3-di(2-tolyl)guanidine (DTG) in the presence of 500 nM (+)-pentazocine (Ki = 105 nM). Unlike its potent sigma activity, the compound has little affinity for dopamine D1 (Ki = 2.9 microM), D2 (Ki = 0.35 microM), muscarinic M1 (Ki = 0.88 microM) or M2 (Ki = 1.7 microM) receptors, and none at all for N-methyl-D-aspartate, phencyclidine and opioid receptors. Thus, RLH-033 is the most potent sigma 1 ligand reported to date, and its very high affinity suggests it may be a useful radioligand to characterize the pharmacology of sigma 1 recognition sites.