High throughput screening (HTS) of encoded combinatorial libraries led to the identification of the diaminopyrimidine 13 (PS-020990) (81,82). It is a compound readily dissected into four domains (corresponding to the combinatorial synthetic steps from which it was derived), the central element of which is the heterocyclic pyrimidine core. Compound 13 represents an optimized member of a library and inhibits the binding of [3H]-desArg10-kallidin to IMR-90 cells expressing an endogenous human B1 receptor with a K, value of 6 nM. It also inhibits agonist-induced (desArg10-kallidin) phosphatidyl inositol turnover (KBapp = 0.4 ± 0.2 nM) and calcium mobilization (KBapp = 17 ± 2 nM) in IMR-90 cells. In addition, compound 13 is at least 1000-fold selective for the B1 receptor relative to the B2 receptor.

A patent issued earlier this year claims tetrahydroquinoxalines as B1 receptor antagonists (83). While the number of compounds in this disclosure is large, details relating to their biological activity are sparse. In a functional in vitro assay measuring intracellular calcium mobilization in stabilized CHO cells, compound 14 had an IC5o value of 17 nM. In vivo functional activity is also claimed but not reported. Coincidentally, the discovery of closely related dihydroquinoxalinones was disclosed in a separate report (84). Optimization of a HTS lead employing molecular modeling and following classical SAR trends yielded the potent (human B1, K, = 0.034 nM) B1 receptor antagonist 15. Compound 15 is selective for the B1 versus the B2 receptor (human B2, K, > 10 nM), as well as a number of human opioid receptor subtypes (IC5o: 7.6 nM (n), 3.2 |xM (8), 7.3 nM (*))• In a panel of assays representing 170 enzymes, receptors, and transporters, 15 exhibited over 5000-fold selectivity for the human B1 receptor. Compound 15 also showed potent antinociceptive activity in a rabbit assay of inflammatory hyperalgesia with ID5o values of 3.5 ng/kg and 16.4 ng/kg for low and high intensity stimuli, respectively.

Receptor based screening also identified a 3-ureidobenzodiazepine structure as an antagonist of the human B1 receptor (85). Systematic modifications of the appendages at the 3- and 5-positions of the diazepine ring in the screening lead afforded the optimal compound (16, B1, K, = 0.59 nM (human), 0.92 nM (rat); B2, K, > 10 tiM (rat)). In a rodent hyperalgesia assay, at a dose of 1 and 3 mg/kg (IP), carrageenan-induced hyperalgesia was suppressed 30% and 90%, respectively. By comparison, morphine dosed (IP) at 1 and 3 mg/kg resulted in 75% and 105% inhibition, respectively. That the efficacy of 16 and morphine are comparable in this rodent pain model despite the different target receptors, underscores the potential of selective B1 antagonists as efficacious agents in the amelioration of pain without the deleterious adverse effects associated with the opiates.

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