Discovery of a new class of p38 kinase inhibitors.

The MAP kinase p38 has been implicated in cytokine signaling, and its inhibitors are potentially useful for the treatment of arthritis and osteoporosis. Novel small-molecule inhibitors of p38 kinase were derived from a combinatorial chemistry effort and exhibit activity in the nanomolar range. Very steep structure-activity relationships are observed within this class.

1-Phenyl-5-pyrazolyl ureas: potent and selective p38 kinase inhibitors.

Inhibitors of the MAP kinase p38 are potentially useful for the treatment of arthritis and osteoporosis. Several 2,3-dichlorophenyl ureas were identified as small-molecule inhibitors of p38 by a combinatorial chemistry effort. Optimization for cellular potency led to the discovery of a new class of potent and selective p38 kinase inhibitors, exemplified by the 1-phenyl-5-pyrazolyl urea 7 (IC50 = 13 nM).

Novel inhibitors of cytokine-induced IkappaBalpha phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo.

We have identified two compounds that inhibit the expression of endothelial-leukocyte adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. These compounds act by inhibiting tumor necrosis factor-alpha-induced phosphorylation of IkappaB-alpha, resulting in decreased nuclear factor-kappaB and decreased expression of adhesion molecules. The effects on both IkappaB-alpha phosphorylation and surface expression of E-selectin were irreversible and occurred at an IC50 of approximately 10 microM. These agents selectively and irreversibly inhibited the tumor necrosis factor-alpha-inducible phosphorylation of IkappaB-alpha without affecting the constitutive IkappaB-alpha phosphorylation. Although these compounds exhibited other activities, including stimulation of the stress-activated protein kinases, p38 and JNK-1, and activation of tyrosine phosphorylation of a 130-140-kDa protein, these effects are probably distinct from the effects on adhesion molecule expression since they were reversible. One compound was evaluated in vivo and shown to be a potent anti-inflammatory drug in two animal models of inflammation. The compound reduced edema formation in a dose-dependent manner in the rat carrageenan paw edema assay and reduced paw swelling in a rat adjuvant arthritis model. These studies suggest that inhibitors of cytokine-inducible IkappaBalpha phosphorylation exert anti-inflammatory activity in vivo.

A-77636: a potent and selective dopamine D1 receptor agonist with antiparkinsonian activity in marmosets.

A-77636, ((1R,3S) 3-(1'-adamantyl)-1-aminomethyl-3,4-dihydro-5,6-dihydroxy-1H-2-benz opyran hydrochloride), is a selective dopamine D1 receptor agonist. In a battery of receptor binding assays, A-77636 shows the highest affinity (pKi = 7.40 +/- 0.09; Ki = 39.8 nM) for the dopamine D1 receptor. A-77636 is an agonist at the dopamine D1 receptors in the fish retina (pEC50 = 8.13; EC50 = 1.1 nM; intrinsic activity = 102% of dopamine) and the rat caudate-putamen (pEC50 = 8.97; intrinsic activity = 134% of dopamine). The compound is functionally inactive at dopamine D2 receptors (EC50 > 10 microM). In rats with unilateral 6-OHDA (6-hydroxydopamine) lesions of the nigro-striatal dopaminergic pathway, A-77636 elicits prolonged (> 20 h) contralateral turning that is blocked by SCH 23390, a D1 receptor antagonist, but not by haloperidol at doses selective for the dopamine D2 receptor. Higher doses of A-77636 produce forelimb clonus in rats and mice. When tested in marmosets treated with MPTP to induce a parkinsonian-like state, A-77636 increases locomotor activity and decreases the severity of the parkinsonian-like symptoms: the compound is active after either subcutaneous or oral administration. A-77641, the optical antipode of A-77636, has a lower affinity towards the dopamine D1 receptor (pKi = 5.14, Ki = 7200 nM), is less potent as a dopamine D1 receptor agonist (pEC50 = 5.65; EC50 = 2200 nM), fails to elicit turning in the 6-OHDA-lesioned rat, and lacks antiparkinsonian efficacy in the MPTP-treated marmoset.(ABSTRACT TRUNCATED AT 250 WORDS)

A68930: a potent agonist specific for the dopamine D1 receptor.

A68930, [1R, 3S] 1-aminomethyl-5,6-dihydroxy-3-phenylisochroman HCl, is a potent, partial agonist in the dopamine-sensitive adenylate cyclase model of the D1 dopamine receptor in fish retina. In the rat caudate-putamen model of the D1 dopamine receptor, A68930 is a potent (EC50 2.1 nM) full agonist. In contrast, A68930 is a much weaker (EC50 = 3,920 nM) full agonist in a biochemical model of the D2 dopamine receptor. A68930 also displays weak 2 agonist activity but the molecule is virtually inactive at the 1 and beta-adrenoceptors. When tested in rats bearing a unilateral 6-OHDA lesion of the nigro-neostriatal neurons, A68930 elicits prolonged (> 20 hr) contralateral turning.

Synthesis and pharmacological evaluation of 1-(aminomethyl)-3,4-dihydro-5-hydroxy-1H-2-benzopyrans as dopamine D1 selective ligands.

A series of 3-substituted 1-(aminomethyl)-3,4-dihydro-5-hydroxy-1H-2- benzopyrans were prepared as potential D1 selective antagonists. The compounds were evaluated for their affinity and selectivity for the D1 receptor as well as for their functional antagonism of D1-mediated pharmacological events. The compounds show potent D1 antagonist properties in vitro. The optimum nitrogen substitution was found to be the primary amine and the observed order of potency for substitution at the 6-position is OH greater than Br greater than H greater than OMe. Two representative compounds, the 6-methyl and 6-bromo analogues, were also evaluated in vivo for dopaminergic activity. Interestingly, both compounds behave as potent in vivo agonists.

Synthesis and dopaminergic activity of 3-substituted 1-(aminomethyl)-3,4-dihydro-5,6-dihydroxy-1H-2-benzopyrans: characterization of an auxiliary binding region in the D1 receptor.

The synthesis and dopaminergic activity of a series of C3 and nitrogen-substituted 1-(aminomethyl)-3,4-dihydro-5,6-dihydroxy-1H-2-benzopyrans (isochromans) is described. The synthesis of the compounds was stereospecific for the 1,3 cis isomer, and the enantioselective synthesis of both enantiomers of one of the analogues (20) was achieved. It was determined that all of the dopaminergic activity resides in the [1R,3S] isomer. Generally, substitution at the C3 position provided compounds with very high potency (less than 10 nm EC50) and selectivity for the D1 receptor, with a wide range of intrinsic activities (60-160%). Analogues containing C3 substituents including aryl, arylalkyl, and cyclic and acyclic alkyl groups showed a marked enhancement of dopaminergic activity compared to the unsubstituted compound. As a class, the drugs were orally active in the rat rotation model with a very long duration of action.

A68930: a potent agonist selective for the dopamine D1 receptor.

A68930, (1R,3S)-1-aminomethyl-5,6-dihydroxy-3-phenylisochroman HCl, is a potent (EC50 = 2.5 nM), partial (intrinsic activity = 66% of dopamine) agonist in the fish retina dopamine-sensitive adenylate cyclase model of the D1 dopamine receptor. In the rat caudate-putamen model of the D1 dopamine receptor, A68930 is a potent (EC50 = 2.1 nM) full agonist. In contrast, A68930 is a much weaker (EC50 = 3920 nM) full agonist in a biochemical model of the dopamine D2 receptor. The orientation of the 3-phenyl substituent in the molecule is critical for the affinity and selectivity of the molecule towards the dopamine D1 receptor. A68930 also displays weak alpha 2-agonist activity but the molecule is virtually inactive at the alpha 1- and beta-adrenoceptors. When tested in rats bearing a unilateral 6-OHDA lesion of the nigro-neostriatal neurons, A68930 elicits prolonged (greater than 20 h) contralateral turning that is antagonized by dopamine D1 receptor selective doses of SCH 23390 but not by D2 receptor selective doses of haloperidol. In this lesioned rat model, A68930 increases 2-deoxyglucose accumulation in the lesioned substantia nigra, pars reticulata. When tested in normal rats, A68930 elicits hyperactivity and, at higher doses, produces a forelimb clonus.

A68930: a potent and specific agonist for the D-1 dopamine receptor.

A68930 (5,6-dihydroxy-3-phenyl-1-aminomethyl-isochroman) is a potent (EC50 = 2.5 nmol/L) partial agonist at the D-1 dopamine receptor. In contrast, A68930 is a much weaker agonist (EC50 = 3,920 nmol/L) at the D-2 dopamine receptor. The orientation of the 3-phenyl substituent in the molecule is critical for the affinity and selectivity of the molecule towards the D-1 receptor in vitro and in vivo. The role of the D-1 receptor in the functioning of the basal ganglia is discussed.

Preparation of an affinity chromatography matrix for the selective purification of the dopamine D2 receptor from bovine striatal membranes.

A ligand affinity matrix has been developed and utilized to purify the dopamine D2 receptor approx. 2100 fold from bovine striatal membranes. 3-[2-Aminoethyl]-8-[3-(4-fluorobenzoyl)propyl]-4-oxo-1-phenyl-1,3,8- triazaspiro[4.5]decan-4-one (AES) was synthesized and used to prepare the affinity matrix by coupling to epoxy-activated Sepharose 6B (AES-Sepharose). AES (Ki approximately 1.7 nM) is similar in potency to the parent compound, spiperone (Ki approximately 0.8 nM), in competing for [3H]spiperone-binding activity. AES has no significant potency in competing for the dopamine D1 receptor as assessed by competition for [3H]SCH23390 binding (Ki greater than 1 microM). Covalent photoaffinity labeling of the dopamine D2 receptor in bovine striatal membranes with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS) was prevented by AES at nanomolar concentrations. The dopamine D2 receptor was solubilized from bovine striatal membranes using 0.25% cholate in the presence of high ionic strength, followed by precipitation and subsequent treatment with 0.5% digitonin. Nearly 100% of the [3H]spiperone-binding activity in the cholate-digitonin solubilized preparation was absorbed at a receptor-to-resin ratio of 2:1 (v/v). Dopamine D2 receptor was eluted from the affinity resin using a competing dopaminergic antagonist molecule, haloperidol. Recovery of dopamine D2 receptor activity from the affinity matrix was approx. 9% of the activity adsorbed to the resin. The [3H]spiperone-binding activity in AES-Sepharose affinity purified preparations is saturable and of high affinity (0.2 nM). Affinity-purified preparations maintain the ligand-binding characteristics of a dopamine D2 receptor as assessed by agonist and antagonist competition for [3H]spiperone binding.

Phycobiliprotein-bilin linkage diversity. I. Structural studies on A- and D-ring-linked phycocyanobilins.

Phycocyanobilin (PCB) peptides alpha-1 PCB and beta-2T PCB were obtained by proteolytic degradation of Synechococcus 6301 C-phycocyanin. These peptides were found to have the following sequences. alpha-1 PCB Cys(PCB)-Ala-Arg beta-2T PCB Ile-Thr-Gln-Gly-Asp-Cys(PCB)-Ser-Ala. The peptides were examined by 1H NMR, circular dichroism spectroscopy, and secondary ion mass spectrometry. The 1H NMR data confirmed that in each case the bilin was attached through a single linkage, a thioether bond between the cysteinyl residue and the tetrapyrrole moiety. Comparison of the 1H NMR spectra of these peptides with those of appropriate model compounds showed that the thioether linkage in alpha-1 PCB was to the C-3' position and that in beta-2T PCB to the C-18' position on the bilin. Refluxing in neutral methanol under nitrogen led to the release of PCB from alpha-1 PCB but did not release the D-ring-linked tetrapyrrole from beta-2T. The above results together with those of an earlier study (Lagarias, J. C., Glazer, A. N., and Rapoport, H. (1979) J. Am. Chem. Soc. 101, 5030-5037) complete the determination of the mode of linkage of each of the three bilins on C-phycocyanin; two are linked through ring A and one through ring D. This is the first documented report of a singly D-ring-linked bilin.

Bilin attachment sites in the alpha and beta subunits of B-phycoerythrin. Structural studies on a doubly peptide-linked phycoerythrobilin.

A bilin-containing fragment of the beta subunit of Porphyridium cruentum B-phycoerythrin produced by cleavage with thermolysin was shown by sequence analysis (Lundell, D.J., Glazer, A.N., DeLange, R.J., and Brown, D.M. (1984) J. Biol. Chem. 259, 5472-5480) to have the following structure. (Formula: see text) Secondary ion mass spectrometry of this bilin-peptide yielded a protonated molecular ion of 1629 mass units corresponding to that predicted from the composition of the fragment, and indicated that the heptapeptide is linked to ring A and the tripeptide to ring D. NMR spectra provided definitive evidence for a thioether linkage at the C-3' carbon of ring A and a second thioether linkage at teh C-18' carbon of ring D of the bilin. This is the first documented report of a bilin linked through two thioether linkages to a polypeptide.

Bilin attachment sites in the alpha and beta subunits of B-phycoerythrin. Structural studies on the singly linked phycoerythrobilins.

Five unique phycoerythrobilin (PEB) peptides were prepared from Porphyridium cruentum B-phycoerythrin by a combination of tryptic and thermolytic digestion without alteration in the spectroscopic properties of the bilin (Lundell, D.J., Glazer, A.N., DeLange, R.J., and Brown, D.M. (1984) J. Biol. Chem. 259, 5472-5480). alpha-1 Cys(PEB)-Tyr-Arg alpha-2 Leu-Cys(PEB)-Val-Pro-Arg beta-1 Met-Ala-Ala-Cys(PEB)-Leu-Arg beta-2T Phe-Ala-Ala-Gly-Asp-Cys(PEB)-Thr-Ser (Formula: see text) where alpha and beta refer to the subunits from which the peptides were derived High resolution 1H NMR analysis of peptides alpha-2, beta-1, and beta-2T combined with earlier studies of peptide alpha-1 (Schoenleber, R.W., Leung, S.-L., Lundell, D.J., Glazer, A.N., and Rapoport, H. (1983) J. Am. Chem. Soc. 105, 4072-4076) has provided proof that all of the singly linked PEB peptides contain a thioether bond to the 3' position of ring A, and strong evidence in support of a trans-dihydro ring A in each of these chromopeptides. The circular dichroism spectra of the four singly linked PEB peptides show that the configuration at C-16 is R in each instance. The present study coupled with previously reported results on peptide beta-3T (Schoenleber, R.W., Lundell, D.J., Glazer, A.N., Rapoport, H. (1984) J. Biol. Chem. 259, 5481-5484 provides the first comprehensive analysis of the structure of all the polypeptide-linked prosthetic groups on the alpha and beta subunits of B-phycoerythrin.