Discovery and Optimization of Macrocyclic Quinoxaline-pyrrolo-dihydropiperidinones as Potent Pim-1/2 Kinase Inhibitors

Abstract

The identification of Pim-1/2 kinase overexpression in B-cell malignancies suggests that Pim kinase inhibitors will have utility in the treatment of lymphoma, leukemia, and multiple myeloma. Starting from a moderately potent quinoxaline-dihydropyrrolopiperidinone lead, we recognized the potential for macrocyclization and developed a series of 13-membered macrocycles. The structure-activity relationships of the macrocyclic linker were systematically explored, leading to the identification of 9c as a potent, subnanomolar inhibitor of Pim-1 and -2. This molecule also potently inhibited Pim kinase activity in KMS-12-BM, a multiple myeloma cell line with relatively high endogenous levels of Pim-1/2, both in vitro (pBAD IC50 = 25 nM) and in vivo (pBAD EC50 = 30 nM, unbound), and a 100 mg/kg daily dose was found to completely arrest the growth of KMS-12-BM xenografts in mice.

Keywords: KMS-12-BM; Pim kinase inhibitor; macrocycle; multiple myeloma.

Figure 1

Screening hit and structure–activity relationship of 1–3 and potential macrocyclization. Macrocycle minimized by molecular mechanics (MMFF94) implemented in MOE (Chemical Computing Group).

Figure 2

X-ray structure of 9c/Pim-1 (PDB: 5EOL). (a) Compound 9c occupies the ATP-binding site; hydrogen bond contacts are shown with dashed lines; the top of the ATP-binding pocket is omitted for clarity. (b) CPK (Corey–Pauling–Koltun) rendering of 9c illustrating that the macrocyclic linker forms a complementary hydrophobic surface to Leu-44, Gly-45, and Phe-49.

Figure 3

(a) Compound 9c (25, 50, or 100 mg/kg p.o.) is associated with a dose- and concentration-dependent reduction of Ser-112 phosphorylation of BAD in KMS-12-BM xenografts; a single dose of 100 mg/kg inhibits Pim-dependent BAD phosphorylation at Ser-112 by greater than 50% for 16 h. Data represent the mean (n = 3) ± standard deviation, with bars corresponding to %pBAD and the left-hand axis and red circles corresponding to total plasma concentration and the right-hand axis. Statistical significance (* p < 0.05) was evaluated by analysis of variance (ANOVA), followed by Dunnet’s post hoc. (b) Compound 9c (10, 25, 50, or 100 mg/kg p.o.) is associated with a dose-dependent inhibition of KMS-12-BM growth in a xenograft experiment. Data represent the mean (n = 10) ± SEM. Statistical significance (*p < 0.0001) was determined by repeated measures analysis of variance (RMANOVA) followed by Dunnet’s post hoc.