Synthesis of 7-O, 8-aryl- and 8-aryl-substituted 2-morpholino-benzoxazin-4-ones as DNA-PK, PI3K and PDE3A Inhibitors

Compounds comprising a 2-morpholino-1,3-benzoxazine scaffold have been proven to possess important biological activities, especially with substitution at 7 and/or 8 positions and were reported in the literature however, extensive study of this particular substitution has not been done. So, a series of 8-aryl-7-(O-substituted)-2-morpholino-4H-benzo[1,3]oxazin-4-ones was synthesized via a key precursor, 8-bromo-7-hydroxy-2-morpholino-4H-benzo[1,3]oxazin-4- one 107 which was amenable to ether synthesis at the 7-position and Suzuki coupling at the 8- position. Another series of analogues of 8-(dibenzothiophen-4-yl)-2- morpholino‑4H‑benzo[1,3]oxazin-4-ones was also synthesized where various primary or secondary amines were placed at the 1-position of dibenzothiophene through an acetamido/propanamido- linkage. All new compounds were evaluated for their inhibitory activities against DNA-PK, PI3K isoforms and PDE3A enzymes. It was shown that 7, 8- disubstitution has no promising inhibitory effect on DNA-PK and PI3K isoforms except for PI3Kδ which was somewhat more tolerant of this substitution particularly where 8-(4- methoxylphenyl) substituents (114c, 115c, 116c and 118c) were present (IC50 ~ 2-3 μM). Good activities against PDE3A were obtained for this series of compounds, with particular members of the 7-(2-pyridinyl)methoxy series (114a, 114e, 114f and 114g) showing good inhibition (IC50 ~ 2-3 μM). In the case of 8-(dibenzothiophen-1-yl)-substitution series (134a-p), a number of compounds with a significant DNA-PK inhibition at sub-nanomolar range with excellent selectivity over the PI3K isoforms was obtained, such as the compound with 2,6- dimethylmorpholino-acetamido substitution at 1-dibenzothiophene (134d) had IC50 = 0.48 nM (at least 250 times more active against DNA-PK than PI3K isoforms). Several compounds also showed potent inhibition of specific PI3K isoforms. For example, the compound with 2- morpholinoacetamido substitution (134c) showed the most potent PI3Kδ isoform activity of all the compounds tested with IC50 = 50 nM and moderate PI3Kβ isoform inhibition (IC50 =100 nM). Other compounds had a potency in the low micromolar range in certain isoforms, and showed some diverse selectivity profiles. DNA-PK activity of N-(4-(2-morpholino-4-oxo-4Hbenzo[e][1,3]oxazin-8-yl)dibenzo[b,d]thiophen-1-yl)acrylamide 135 and 3-substituted-N-(4- (2-morpholino-4-oxo-4H-benzo[e][1,3]oxazin-8-yl)dibenzo[b,d]thiophen-1-yl)propanamide 136a-d was lower than that for acetamido-analogues 134a-p and the IC50 values were found to be 65.31-221.8 nM. The results indicate that 7, 8-disubstituted compounds have strong activity against PDE3A and PI3Kδ, whereas 8-(dibenzothiophen-1-yl)-substituted products have very high activity against DNA-PK and PI3K (β and δ) with good specificity.

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Pharmacy & Applied Science, College of Science, Health and Engineering, La Trobe University, Bendigo, Victoria.