Thiazolo[4,5-d]pyrimidine thiones and -ones as corticotropin-releasing hormone (CRH-R1) receptor antagonists.

A series of thiazolo[4,5-d]pyrimidine thiones and -ones was prepared and discovered to have good binding affinity to the CRH-R1 receptor, thus showing promise as a new class of potential anxiolytics and/or antidepressants.

Non-peptide corticotropin-releasing hormone antagonists: syntheses and structure-activity relationships of 2-anilinopyrimidines and -triazines.

Screening of our chemical library using a rat corticotropin-releasing hormone (CRH) receptor assay led to the discovery that 2-anilinopyrimidine 15-1 weakly displaced [125I]-0-Tyr-oCRH from rat frontal cortex homogenates when compared to the known peptide antagonist alpha-helical CRH(9-41) (Ki = 5700 nM vs 1 nM). Furthermore, 15-1 weakly inhibited CRH-stimulated adenylate cyclase activity in the same tissue, but it was less potent than alpha-helical CRH(9-41) (IC50 = 20 000 nM vs 250 nM). Systematic structure-activity relationship studies, using the cloned human CRH1 receptor assay, defined the pharmacophore for optimal binding to hCRH1 receptors. Several high-affinity 2-anilinopyrimidines and -triazines were discovered, some of which had superior pharmacokinetic profiles in the rat. This paper describes the structure-activity studies which improved hCRH1 receptor binding affinity and pharmacokinetic parameters in the rat. Compound 28-17 (mean hCRH1 Ki = 32 nM) had a significantly improved pharmacokinetic profile in the rat (19% oral bioavailability at 30 mg/kg) as well as in the dog (20% oral bioavailability at 5 mg/kg) relative to the early lead structures.

Corticotropin-releasing hormone receptor antagonists: framework design and synthesis guided by ligand conformational studies.

As described in the preceding paper (Arvanitis et al. J. Med. Chem. 1999, 42), anilinopyrimidines I were identified as potent antagonists of corticotropin-releasing hormone-1 receptor (CRH1-R, also referred to as corticotropin-releasing factor, CRF1-R). Our next goal was to understand the receptor-bound conformation of the antagonists and to use this information to help guide preclinical optimization of the series and to develop new leads. Since receptor structural information was not available, we assumed that these small, high-affinity antagonists would tend to bind in conformations at or energetically close to their global minima and that rigid analogues that maintained the important stereoelectronic features of the bound anilinopyrimidine would also bind tightly. Conformational preferences and barriers to rotation of the anilinopyrimidines were determined by semiempirical methods, and X-ray and variable-temperature NMR spectroscopy provided experimental results that correlated well with calculated structures. Using these data, a key dihedral angle was constrained to design fused-ring analogues, substituted N-arylpyrrolopyridines II, synthesis of which provided CRH1 receptor antagonists with potency equal to that of the initial congeneric leads (Ki = 1 nM) and which closely matched the conformation held by the original compound, as determined by crystallography. In addition to providing a useful template for further analogue synthesis, the study unequivocally determined the active conformation of the anilinopyrimidines. Theoretical and spectroscopic studies, synthesis, and receptor binding data are presented.

Synthesis, corticotropin-releasing factor receptor binding affinity, and pharmacokinetic properties of triazolo-, imidazo-, and pyrrolopyrimidines and -pyridines.

The synthesis and CRF receptor binding affinities of several new series of N-aryltriazolo- and -imidazopyrimidines and -pyridines are described. These cyclized systems were prepared from appropriately substituted diaminopyrimidines or -pyridines by nitrous acid, orthoester, or acyl halide treatment. Variations of amino (ether) pendants and aromatic substituents have defined the structure-activity relationships of these series and resulted in the identification of a variety of high-affinity agents (Ki's < 10 nM). On the basis of this property and lipophilicity differences, six of these compounds (4d,i,n,x, 8k, 9a) were initially chosen for rat pharmacokinetic (PK) studies. Good oral bioavailability, high plasma levels, and duration of four of these compounds (4d,i,n,x) prompted further PK studies in the dog following both iv and oral routes of administration. Results from this work indicated 4i,x had properties we believe necessary for a potential therapeutic agent, and 4i1 has been selected for further pharmacological studies that will be reported in due course.

2-Fluoro-4-pyridinylmethyl analogues of linopirdine as orally active acetylcholine release-enhancing agents with good efficacy and duration of action.

In an effort to improve the pharmacokinetic and pharmacodynamic properties of the cognition-enhancer linopirdine (DuP 996), a number of core structure analogues were prepared in which the 4-pyridyl pendant group was systematically replaced with 2-fluoro-4-pyridyl. This strategy resulted in the discovery of several compounds with improved activity in acetylcholine (ACh) release-enhancing assays, in vitro and in vivo. The most effective compound resulting from these studies, 10, 10-bis[(2-fluoro-4-pyridinyl)methyl]-9(10H)-anthracenone (9), is between 10 and 20 times more potent than linopirdine in increasing extracellular hippocampal ACh levels in the rat with a minimum effective dose of 1 mg/kg. In addition to superior potency, 9 possesses an improved pharmacokinetic profile compared to that of linopirdine. The half-life of 9 (2 h) in rats is 4-fold greater than that of linopirdine (0.5 h), and it showed a 6-fold improvement in brain-plasma distribution over linopirdine. On the basis of its pharmacologic, pharmacokinetic, absorption, and distribution properties, 9 (DMP543) has been advanced for clinical evaluation as a potential palliative therapeutic for treatment of Alzheimer's disease.

Two new potent neurotransmitter release enhancers, 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone and 10,10-bis(2-fluoro-4-pyridinylmethyl)-9(10H)-anthracenone: comparison to linopirdine.

Linopirdine (3,3-bis(4-pyridinylmethyl)-1-phenylindolin-2-one, DUP996) is an extensively studied representative of a class of cognition enhancing compounds that increase the evoked release of neurotransmitters. Recent studies suggest that these agents act through the blockade of specific K+ channels. We have recently identified more potent anthracenone analogs of linopirdine: 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE991) and 10,10-bis(2-fluoro-4-pyridinylmethyl)-9(10H)-anthracenone (DMP 543). Although linopirdine possesses an EC50 of 4.2 microM for enhancement of [3H]ACh release from rat brain slices, XE991 and DMP 543 have EC50S of 490 and 700 nM, respectively. In addition to greater in vitro potency relative to linopirdine, both compounds show greater in vivo potency and duration of action. Although 5 mg/kg (p.o.) linopirdine does not lead to statistically significant increases in hippocampal extracellular acetylcholine levels, 5 mg/kg (p.o.) XE991 leads to increases (maximal effect > 90% over baseline) which are sustained for 60 min. Moreover, DMP 543 at 1 mg/kg causes more than a 100% increase in acetylcholine levels with the effect lasting more than 3 hr. At doses relevant to their release-enhancing properties, the only overt symptom consistently observed was tremor, possible via a cholinergic mechanism. These results suggest that XE991 and DMP 543 may prove to be superior to linopirdine as Alzheimer's disease therapeutics. In addition, these agents should be useful pharmacological tools for probing the importance of particular ion channels in the control of neurotransmitter release.

Ion channel modulators that enhance acetylcholine release: potential therapies for Alzheimer's disease.

Enhancing the release of acetylcholine (ACh) in the brain is one approach to increasing neuronal activity, restoring central cholinergic tone and improving attention and cognition. ACh release is modulated by both ligand-gated (gamma-amino butyric acid A receptors/benzodiazepine [GABA(A)/BDZ], nicotinic-acetylcholine and serotonin, 5-HT3) and voltage-gated (calcium and potassium) ion channels. Of the ligand-gated channel modulators, the BDZ receptor (BDZR) inverse agonists (beta-CCM, ZK 93426) enhance activity-dependent release in animals, whereas S-8510, a partial inverse agonist, and the BDZR antagonist, flumazenil, show enhancement regardless of the behavioural state of the animal. Some of these agents have undergone limited clinical evaluation for Alzheimer's disease (AD) (ZK 93426, flumazenil, S-8510), but their potential anxiogenic liability makes their therapeutic use uncertain until more clinical data are available. Within the group of nicotinic agonists, ABT-418, though less potent than nicotine and epibatidine in promoting ACh release in vitro, was clinically evaluated based on its in vivo profile. Its lack of oral bioavailability has limited its acceptability, though transdermal administration has been used to circumvent this deficiency. Serotonin 5-HT3 receptor modulators have not been advanced for clinical evaluation for the treatment of AD. Among the voltage-gated ion channel modulators affecting L- or N-type calcium channels, nefiracetam, a nootropic agent, also increased ACh release in animal studies. It is currently undergoing clinical evaluation for AD, though a need for more potent and brain selective calcium channel blockers exists. Potassium channel modulators have been the most studied ACh release enhancing agents and several of these compounds (4-AP, 3,4-DAP, linopirdine) have been clinically evaluated. In AD patients, 4-AP, an A-type K+ channel blocker, elicited inconsistent and unremarkable effects. Linopirdine, whose enhancement of ACh release correlates with its ability to block M-type K+ channels, also produced disappointing clinical results, which may have been related to its suboptimal pharmacokinetic profile. Further work in this series has provided a compound (DMP 543) that should be a more reliable indicator of whether a blocker of this ion channel can activate the cholinergic system in man. This agent is currently undergoing clinical evaluation for AD.

Mono- and bis(aminomethyl)phenylacetic acid esters as short-acting antiarrhythmic agents. 2.

The synthesis, antiarrhythmic activity, and blood hydrolysis properties of a series of mono- and bis(aminomethyl)phenylacetic acid esters related to a previously reported class Ic antiarrhythmic agent (ACC-9358) are described. Of the various oxa-, aza-, thia-, and carbacyclic esters initially prepared in the bis(pyrrolidinomethyl)-4-hydroxyphenylacetic acid series, the 1,4-benzodioxanyl-2-methyl(3q) and the thienyl-2-methyl(31) esters were evaluated in vivo for antiarrhythmic efficacy. In addition, a number of monoappended phenylacetic esters of 3q with or without the 4-hydroxy group were also prepared for evaluation of antiarrhythmic, lipophilic, and metabolic properties. Of these compounds, 3q possessed the most desirable pharmacological and pharmacokinetic profile.

Synthesis and structure-activity relationships of a new series of antiarrhythmic agents: monobasic derivatives of disobutamide.

Analogues of the dibasic antiarrhythmic agent disobutamide (2) were prepared and evaluated for antiarrhythmic efficacy, myocardial depression, and anticholinergic activity. The replacement of an isopropyl group in disobutamide by an acetyl group led to the monobasic analogue SC-40230, 7a, which demonstrated good antiarrhythmic activity accompanied by less myocardial depressant and anticholinergic activities. In addition, it did not induce clear cytoplasmic vacuoles as did the parent compound. SC-40230 was chosen from among other analogues as a candidate for clinical evaluation. Other compounds prepared and evaluated included indolizidinones and a secondary amine isomer of disobutamide.

Synthesis and structure-activity relationships of a new series of antiarrhythmic agents: 4,4-disubstituted hexahydro-3H-pyrido[1,2-c]pyrimidin-3-ones and related compounds.

A series of 4,4-disubstituted tetrahydro- and 4,4-disubstituted hexahydro-3H-pyrido[1,2-c]pyrimidin-3-ones (4 and 5, respectively) were prepared from 2-aryl-2-(2-piperidinyl)-4-[N,N-bis (1-methylethyl)amino] butanamides (2). Individual racemates of the piperidinyl amides 2 were converted to pure racemic diaza bicyclic compounds that were evaluated for antiarrhythmic activity in the Harris dog model and anticholinergic activity in a muscarinic receptor binding assay. Selected compounds were subsequently evaluated for hemodynamic effects in anesthetized dogs where blood pressure depression and negative inotropic activity were assessed. Of this group, 4a (R = CH3) and 5a (R = CH3) showed the most favorable pharmacological profiles; the former compound was chosen for toxicity testing over the latter due to its lack of noncompetitive inhibition of acetylcholine-induced contractions of guinea pig ileum segments. Clinical evaluation is now under way.

22-Hydroxycholesterol derivatives as HMG CoA reductase suppressors and serum cholesterol lowering agents.

A series of 22-hydroxycholesterol derivatives with a modified side chain terminus was prepared. These agents were evaluated in vitro and in vivo for their ability to suppress HMG CoA reductase, the rate-limiting enzyme of cholesterol biosynthesis. In tissue culture assays, 22-hydroxycholesterol as well as the side chain modified analogues were potent inhibitors of HMG CoA reductase. However, only those sterols with a modified side chain terminus were effective suppressors of liver reductase when administered ig to rats. 22-Hydroxy-25-methylcholesterol (4a) and 25-fluoro-22-hydroxycholesterol (15a) significantly lowered serum cholesterol levels when administered ig to primates; 25-chloro-22-hydroxycholesterol (15b) and the analogue with a cyclopropyl terminus, 20b, were ineffective. The cholesterol-lowering sterols did not significantly alter lipoprotein levels; however, the two compounds have been shown to inhibit acyl-coenzyme A:cholesterol acyl-transferase (ACAT) in tissue culture studies.

Synthesis, benzodiazepine receptor binding, and anticonvulsant activity of 2,3-dihydro-3-oxo-5H-pyrido[3,4-b][1,4]benzothiazine-4-carbonitriles.

A series of oxopyridobenzothiazines (azaphenothiazines) were prepared and evaluated for binding to the benzodiazepine receptor, anticonvulsant activity in the pentylenetetrazole-induced convulsion assay, and, in two cases, ability to increase punished responding in a standard conflict test. While parent compound 1a showed binding affinity comparable to chlorodiazepoxide (CDP), its potency in the anticonvulsant assay and the anticonflict test was considerably weaker than CDP. Of the variety of derivatives synthesized, only the 7-chloro compound 1b showed receptor affinity comparable to 1a with slightly improved in vivo activity. The poor correlation between receptor binding and in vivo activity may be due to variability in absorption or pharmacological responses unrelated to affinity for the benzodiazepine receptor.

Inhibition of cholesteryl ester formation in human fibroblasts by an analogue of 7-ketocholesterol and by progesterone.

The synthesis of cholesteryl esters in cultured human fibroblasts is catalyzed by a microsomal acyl-coenzyme A:cholesterol acyltransferase (EC 2.3.1.26). The acyltransferase activity is enhanced when fibroblasts take up cholesterol contained in plasma low density lipoprotein. In the current studies two steroids, SC-31769 (an analogue of 7-ketocholesterol) and progesterone, were shown to inhibit acyltransferase activity in cell-free extracts of human fibroblasts. When added to intact cells, these steroids inhibited the incorporation of [(14)C]oleate into cellular cholesteryl [(14)C]oleate and reduced the accumulation of cholesteryl esters in fibroblasts exposed to low density lipoprotein. The inhibition of cholesteryl ester formation in intact cells by SC-31769 and progesterone was readily reversible. Neither compound inhibited the incorporation of [(14)C]oleate into [(14)C]triglycerides or [(14)C]phospholipids. When incubated with fibroblast monolayers at a concentration of 1 mug/ml, SC-31769 suppressed the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase [mevalonate:NADP(+) oxidoreductase (CoA-acylating); EC 1.1.1.34], the rate-controlling enzyme in cholesterol synthesis. In contrast, progesterone had no effect on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity at concentrations as high as 25 mug/ml. The availability of two types of steroid compounds that inhibit the acyltransferase activity and cholesteryl ester synthesis in human fibroblasts should prove useful in further studies of the regulatory mechanisms responsible for cholesteryl ester accumulation in human cells under normal and pathologic conditions.