Design, synthesis and biological evaluation of a novel series of potent, orally active adenosine A1 receptor antagonists with high blood-brain barrier permeability.

A novel series of 3-(2-substituted-3-oxo-2,3-dihydropyridazin-6-yl)-2-phenylpyrazolo[1,5-a]pyridines (5-38) were synthesized and evaluated for their in vitro adenosine A1 and A(2A) receptor binding activities, and in vitro metabolism by rat liver in order to search for orally active compounds. Most of the test compounds were potent adenosine A1 receptor antagonists with high A1 selectivity and the A1 affinity and A1 selectivity of carbonyl derivatives (5-11) was particularly high. In particular, compound 7 was an extremely potent and selective adenosine A1 antagonist with high A1 selectivity (Ki=0.026 nM, A(2A)/A1=5400). In terms of metabolic stability, 2-oxopropyl (5), 2-hydroxypropyl (12), N-methylacetamide (16), 2-(piperidin-1-yl)ethyl (28) and 1-methylpiperidin-4-yl (32, FR194921) were the most stable compounds in this series of analogues. Further in vivo evaluation indicated that compounds 5, 13, 17, 28 and 32 were detected in both plasma and brain after oral administration in rats. In particular, 32 displayed good plasma and brain concentrations (dose: 32 mg/kg (n=3); after 30 min, plasma conc.=3390+/-651nM, brain conc.=3670+/-496nM; after 60min, plasma conc.=1580+/-348nM, brain conc.=2143+/-434nM), and a good brain/plasma ratio (1.11+/-0.060 (30min), 1.39+/-0.172 (60min)). As a result, we could show that 32 is a good candidate for an orally active adenosine A1 receptor antagonist with high blood-brain barrier permeability and good bioavailability (Ki=6.6nM, A(2A)/A1=820, BA=60.6+/-4.9% (32 mg/kg)).

Novel adenosine A1 receptor antagonists. Synthesis and structure-activity relationships of a novel series of 3-(2-cyclohexenyl-3-oxo-2,3-dihydropyridazin-6-yl)-2-phenylpyrazolo[1,5 -a]pyridines.

A novel series of 3-(2-cyclohexenyl-3-oxo-2,3-dihydropyridazin-6-yl)-2-phenylpyrazol o[1,5-a]pyridines was synthesized and evaluated for in vitro adenosine A1 and A2A receptor binding activities. Most of the cyclohexenyl derivatives (7a-e, 8a-s) were found to be potent adenosine A1 receptor antagonists. In a series of analogues of FR166124 (3a), alcohol 7c, nitrile 7e and amide derivatives (7d, 8c, 8r) were found to be more potent A1 antagonists with higher A2A/A1 selectivity than FR166124. Amongst them, 8r showed considerable water solubility (33.3 mg/mL), but lower than that of the sodium salt of FR166124 (> 200 mg/mL). Additionally, FR166124 had strong diuretic activity by both p.o. and iv administration in rats (minimum effective dose=0.1 and 0.032 mg/kg, respectively).

Discovery of FR166124, a novel water-soluble pyrazolo-[1,5-a]pyridine adenosine A1 receptor antagonist.

Novel 3-(2-cycloalkyl and cycloalkenyl-3-oxo-2,3-dihydropyridazin-6-yl)-2-phenylpyrazo lo [1,5-a]-pyridines were synthesized and evaluated for their adenosine A1 receptor binding activities. In this series, FR166124 (3) was found to be the most potent and selective adenosine A1 receptor antagonist, and the double bond of the cyclohexenyl acetic acid group was essential for selectivity of A1 receptor binding. Furthermore, the solubility in water of the sodium salt of FR 166124 was high.

Restricted transport of cyclosporin A across the blood-brain barrier by a multidrug transporter, P-glycoprotein.

The blood-brain barrier permeability of cyclosporin A (CsA), an immunosuppressive cyclic peptide, is restricted despite its highly lipophilic nature. In this study, the uptake of CsA by primary cultured bovine brain capillary endothelial cells (BCEC) was investigated in order to clarify whether P-glycoprotein (P-gp), an ATP-dependent drug efflux pump expressed in the luminal surface of BCEC, causes the decreased transport of CsA into the brain. P-gp, having a molecular mass of 130-140 kDa, was detected with anti-P-gp monoclonal antibody, C219, using western blot analysis of the membrane fraction isolated from the bovine brain capillary. The uptake of CsA by primary cultured bovine BCEC was time-dependent, and the steady-state uptake of CsA was increased in the presence of several multidrug resistance reversing agents including verapamil and steroid hormones and the substrate of P-gp in BCEC, vincristine. The steady-stage uptake was increased significantly to approximately 4-fold when cellular ATP was depleted by treating with 2,4-dinitrophenol, suggesting that the efflux process is ATP dependent. Furthermore, in the presence of an anti-P-gp monoclonal antibody, MRK16, at a 10 micrograms/mL concentration, the uptake of CsA was increased approximately 3-fold. These results suggest that the low permeability of CsA into the brain is caused by the active efflux from BCEC by P-gp present in the luminal surface of cells.

P-glycoprotein as the drug efflux pump in primary cultured bovine brain capillary endothelial cells.

The expression of a functional P-glycoprotein (P-gp) which pumps drugs out of brain capillary endothelial cells (BCEC) into blood was studied by evaluating the steady-state uptake and efflux of vincristine (VCR) by primary cultured bovine BCEC. The steady-state uptake of VCR was increased in the presence of metabolic inhibitors, and an anti-P-gp monoclonal antibody, MRK16, as well as verapamil and steroid hormones which are known to reverse multidrug resistance in tumor cells. Furthermore, efflux of VCR from BCEC was inhibited by verapamil. By immunohistochemistry, P-gp was localized at the luminal side of the capillary endothelial cells in both gray matter of bovine brain and primary cultured BCEC. These data suggest that P-gp functions as a drug efflux pump at the luminal side of BCEC and regulates the transfer of certain lipophilic drugs from the blood into the brain.