The solid-phase synthesis of novel 14-membered macrocycles for high throughput screening.

A simple and efficient synthesis of novel 14-membered macrocycles from a resin-bound orthogonally protected lysine residue is described. Reductive alkylation of the lysine alpha-nitrogen introduces the first diversity element. Acylation of the resultant secondary amine with an Fmoc-amino acid introduces the second diversity element providing a resin-bound protected di-peptide precursor. Removal of the Fmoc-group is followed by acylation with a succinic anhydride to introduce the final diversity elements. Removal of the methyltrityl-group from the amino group followed by macrocyclization provides the desired macrocycles, after TFA cleavage, in excellent yield and purity.

In vitro characterization of a novel series of platelet-derived growth factor receptor tyrosine kinase inhibitors.

In this report, we describe the discovery and characterization of a novel biarylhydrazone series of platelet-derived growth factor (PDGF) receptor tyrosine kinase inhibitors typified by the prototype WIN 41662 (3-phenyl-N1-[1-(4-pytidyl)pyrimidine]hydrazone). WIN 41662 inhibited PDGF-stimulated autophosphorylation of PDGF receptors from human vascular smooth muscle cells (hVSMC) with an IC50 value of 60 nM. The inhibitor appeared to be competitive with respect to substrate (Mn(2+)-ATP), having a calculated Ki of 15 +/- 5 nM. WIN 41662 was approximately 500-fold more potent in inhibiting the PDGF receptor tyrosine kinase than the p56lck tyrosine kinase. It was inactive against other serine/threonine and tyrosine kinases tested. WIN 41662 produced concentration-dependent inhibition of PDGF-stimulated receptor autophosphorylation in intact hVSMC with an IC50 < 100 nM. Intracellular Ca2+ mobilization and cell proliferation were events that occurred in hVSMC subsequent to PDGF receptor activation. WIN 41662 inhibited PDGF-stimulated Ca2+ mobilization and cell proliferation ([3H]TdR incorporation) with IC50 values of 430 nM and 2.3 microM, respectively. These effects appeared to be specifically related to PDGF receptor tyrosine kinase inhibition since WIN 41662 was not cytotoxic (in vitro) and since WIN 72039, a close structural analog that does not inhibit PDGF receptor tyrosine kinase, also did not inhibit PDGF-stimulated receptor autophosphorylation, Ca2+ mobilization, or hVSMC proliferation. Thus, WIN 41662 is representative of a novel class of selective PDGF receptor tyrosine kinase inhibitors that inhibit PDGF-regulated secondary events in intact cells.

WIN 17317-3: novel nonpeptide antagonist of voltage-activated K+ channels in human T lymphocytes.

We report the in vitro biological characterization of WIN 17317-3 (1-benzyl-7-chloro-4-n-propylimino-1,4-dihydroquinoline hydrochloride), a novel inhibitor of voltage-activated (n-type) K+ channels in human T lymphocytes. WIN 17317-3 inhibits 125I-charybdotoxin binding to n-type K+ channels with an IC50 value of 83 +/- 4 nM. WIN 17317-3 demonstrates competitive inhibition of 125I-charybdotoxin binding by increasing its dissociation constant without changing the total number of channels bound and by having no effect on its dissociation rate constant. WIN 17317-3 inhibits whole-cell, n-type K+ currents with characteristics indicative of open channel block and has an IC50 value of 335 nM. The compound is 150-fold selective for n-type K+ channels, compared with Ca(2+)-activated, charybdotoxin-sensitive K+ channels in smooth muscle. In purified CD4+ T lymphocytes activated with either anti-CD3 plus phorbol ester or anti-CD3 plus anti-CD28, WIN 17317-3 decreases interleukin-2 production with EC50 values of 0.8 microM and 1 microM, respectively. WIN 17317-3 is a novel, potent, and selective nonpeptide n-type K+ channel antagonist that inhibits interleukin-2 production in human T lymphocytes.

Novel inhibitors of the nuclear factor of activated T cells (NFAT)-mediated transcription of beta-galactosidase: potential immunosuppressive and antiinflammatory agents.

The preparation of a series of quinazoline-2,4-diones, 1-3, and pyrrolo[3,4-d]pyrimidine-2,4-diones, 4-8 is described. A small number of quinazolinedione analogs were identified from random screening to possess low micromolar (1.3-4.4 microM) potency in the nuclear factor of activated T cells-1-regulated beta-galactosidase expression assay. An expanded analog search resulted in identifying pyrrolopyrimidinedione 4b which is 5-10-fold (0.26 microM) more potent than the quinazolinediones. Replacement of the benzyl group with naphthyl led to greater potency and conformationally restricted analogs 4u-w. The naphthyl and acenaphthyl analogs are 10-100 times more potent inhibitors of beta-galactosidase expression than 4b. Binding affinity data for displacement of radiolabeled 4s from Jurkat cell membranes reflected an excellent correlation with the IC50 value for inhibition of beta-galactosidase activity. These products, whose structure-activity relationships are discussed, are of interest as potential agents for preventing interleukin-2 gene transcription.

[(Biaryloxy)alkyl]isoxazoles: picornavirus inhibitors.

A series of biphenyl analogs, 6, of 5-[5-(2,6-dichloro-5-oxazolylphenoxy)pentyl]-3-methylisoxazole (2) have been synthesized and tested in vitro against 10 human rhinovirus serotypes in a TCID50 assay. The most potent compound in the series 6s, 3-[3-[2,6-dimethyl-4-(4-fluorophenyl)-phenoxy]propyl]-3-methylisoxazole , was screened against an additional 84 serotypes. It was found to be active against 64 of the serotypes, while 87 serotypes were sensitive to 2 at < 3 micrograms/mL. On comparison of the active serotypes, 6s exhibited greater potency versus 2. Analogs 6a-c,s were examined for in vitro metabolic stability by monkey liver microsomal assay. These analogs exhibited a greater than 7-fold improvement (t1/2 > 200 min) in metabolic stability compared with 2 (t1/2 > 27 min).

Novel inhibitors of potassium ion channels on human T lymphocytes.

The in vitro biological characterization of a series of 4-(alkylamino)-1,4-dihydroquinolines is reported. These compounds are novel inhibitors of voltage-activated n-type potassium ion (K+) channels in human T lymphocytes. This series, identified from random screening, was found to inhibit [125I]charybdotoxin binding to n-type K+ channels with IC50 values ranging from 10(-6) to 10(-8) M. These analogs also inhibit whole cell n-type K+ currents with IC50 values from 10(-5) to 10(-7) M. The preparation of a series of new 4-(alkylamino)-1,4-dihydroquinolines is described. Structure-activity relationships are discussed. Naphthyl analog 7c, the best compound prepared, exhibited > 100-fold selectivity for inhibition of [125I]charybdotoxin binding to n-type K+ channels compared with inhibition of [3H]dofetilide binding to cardiac K+ channels. These compounds represent a potent and selective series of n-type K+ channel inhibitors that have the potential for further development as anti-inflammatory agents.