Synthesis and pharmacology of benzoxazines as highly selective antagonists at M4 muscarinic receptors

Previously, we reported on PD 102807 (41) as being the most selective synthetic M-4 muscarinic antagonist identified to date. Synthesized analogues of 41 showed no improvement in affinity and selectivity at that time. However, several newly synthesized compounds exhibit a 7-fold higher affinity at M-4 receptors and demonstrate a selectivity of at least 100-fold over all other muscarinic receptor subtypes. For example, compound 28 showed an affinity of pK(i) = 9.00 at M-4 receptors and a selectivity of M-1/M-4 = 13 183-fold, M-2,/M-4 = 339-fold, M-3/M-4 = 151-fold, and M-5/M-4 = 11 220-fold. This high selectivity along with high affinity has not been reported for any synthetic muscarinic antagonist, nor for natural occurring M4 antagonists such as the M-4 selective Eastern Green Mamba venom MT3 (M4 pK(b) = 8.7, M-1/M-4 = 40-fold, M-2/M-4 greater than or equal to 500-fold, M-3/M-4 greater than or equal to 500-fold, and M-5/M-4 greater than or equal to 500-fold). Derivative 24, a compound with a high selectivity pattern as well, has been tested for in vivo efficacy. It was able to block the L-3,4-dihydroxyphenylalanine accumulation produced by CI-1017, an M-1/M-4 selective muscarinic agonist, in the mesolimbic region and striatum, which suggests that 24 is capable of crossing the blood-brain barrier and confirms the pharmacokinetic data obtained on this compound. This is evidence that suggests that agonist-induced increase in catecholamine synthesis observed in these regions is mediated by M-4 receptors.