Fluorosulfonyl- and bis-(beta-chloroethyl)amino-phenylamino functionalizedpyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives: Irreversible antagonists at the human A(3) adenosine receptor and molecular modeling studies

A series of pyrazolotriazolopyrimidines was previously reported to be highl y potent and selective human A(3) adenosine receptor antagonists (Baraldi e t al. J. Med. Chem. 2000, 43, 4768-4780). A derivative having a methyl grou p at the N-8 pyrazole combined with a 4-methoxyphenylcarbamoyl moiety at N- 5 position, displayed a K-i value at the hA(3) receptor of 0.2 nM. We now d escribe chemically reactive derivatives which act as irreversible inhibitor s of this receptor. Electrophilic groups, specifically sulfonyl fluoride an d nitrogen mustard (bis-(beta -chloroethyl)amino) moieties, have been incor porated at the 4-position of the aryl urea group. Membranes containing the recombinant hA3 receptor were preincubated with the compounds and washed ex haustively. The loss of ability to bind radioligand following this treatmen t indicated irreversible binding. The most potent compound in irreversibly binding to the receptor was 14, which contained a sulfonyl fluoride moiety and a propyl group at the N-8 pyrazole nitrogen. The bis-(beta -chloroethyl )amino derivatives displayed a much smaller degree of irreversible binding than the sulfonyl fluoride derivatives. A computer-generated model of the h uman A(3) receptor was built and analyzed to help interpret these results. The model of the A(3) transmembrane region was derived using primary sequen ce comparison, secondary structure predictions, and three-dimensional homol ogy building, using the recently published crystal structure of rhodopsin a s a template. According to our model, sulfonyl fluoride derivatives could d ock within the hypothetical TM binding domain, adopting two different energ etically favorable conformations. We have identified two amino acids, Ser24 7 and Cys251, both in TM6, as potential nucleophilic partners of the irreve rsible binding to the receptor.