Discovery of 5,7-Dihydro‑6<i>H</i>‑pyrrolo[2,3‑<i>d</i>]pyrimidin-6-ones as Highly Selective CDK2 Inhibitors

A series of exceptionally selective CDK2 inhibitors are described. Starting from an HTS hit, we successfully scaffold hopped to a 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one core structure, which imparted a promising initial selectivity within the CDK family. Extensive further SAR identified additional factors that drove selectivity to above 200× for CDKs 1/4/6/7/9. General kinome selectivity was also greatly improved. Finally, use of in vivo metabolite identification allowed us to pinpoint sulfonamide dealkylation as the primary metabolite, which was ameliorated through the deuterium effect

Preclinical characterization of INCB053914, a novel pan-PIM kinase inhibitor, alone and in combination with anticancer agents, in models of hematologic malignancies

<div><p>The Proviral Integration site of Moloney murine leukemia virus (PIM) serine/threonine protein kinases are overexpressed in many hematologic and solid tumor malignancies and play central roles in intracellular signaling networks important in tumorigenesis, including the Janus kinase–signal transducer and activator of transcription (JAK/STAT) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. The three PIM kinase isozymes (PIM1, PIM2, and PIM3) share similar downstream substrates with other key oncogenic kinases and have differing but mutually compensatory functions across tumors. This supports the therapeutic potential of pan-PIM kinase inhibitors, especially in combination with other anticancer agents chosen based on their role in overlapping signaling networks. Reported here is a preclinical characterization of INCB053914, a novel, potent, and selective adenosine triphosphate-competitive pan-PIM kinase inhibitor. <i>In vitro</i>, INCB053914 inhibited proliferation and the phosphorylation of downstream substrates in cell lines from multiple hematologic malignancies. Effects were confirmed in primary bone marrow blasts from patients with acute myeloid leukemia treated <i>ex vivo</i> and in blood samples from patients receiving INCB053914 in an ongoing phase 1 dose-escalation study. <i>In vivo</i>, single-agent INCB053914 inhibited Bcl-2–associated death promoter protein phosphorylation and dose-dependently inhibited tumor growth in acute myeloid leukemia and multiple myeloma xenografts. Additive or synergistic inhibition of tumor growth was observed when INCB053914 was combined with selective PI3Kδ inhibition, selective JAK1 or JAK1/2 inhibition, or cytarabine. Based on these data, pan-PIM kinase inhibitors, including INCB053914, may have therapeutic utility in hematologic malignancies when combined with other inhibitors of oncogenic kinases or standard chemotherapeutics.</p></div

Structure of INCB053914 and IC₅₀ values for the inhibition of PIM isozymes by INCB053914 in biochemical assays.

<p>Structure of INCB053914 and IC₅₀ values for the inhibition of PIM isozymes by INCB053914 in biochemical assays.</p

Preclinical characterization of INCB053914, a novel pan-PIM kinase inhibitor, alone and in combination with anticancer agents, in models of hematologic malignancies - Fig 6

<p><b>Effects of the selective PI3Kδ inhibitor, INCB050465, on PIM isozyme expression in Pfeiffer DLBCL cells (A). Effects of INCB053914 alone, or in combination with INCB050465, on the <i>in vitro</i> proliferation of Pfeiffer DLBCL cells (B). Effects of INCB053914 alone or in combination with INCB050465 on tumor growth in a DLBCL xenograft model (C); with cytarabine on tumor growth in an AML xenograft model (D); with the JAK1-selective inhibitor, itacitinib, on BAD, STAT3 phosphorylation, and MYC levels (E), and on tumor growth in an INA-6 MM xenograft model (F).</b> Error bars represent standard error of the mean. BW, twice a week; CR, complete regression; IP, intraperitoneally; QD, once a day.</p

Preclinical characterization of INCB053914, a novel pan-PIM kinase inhibitor, alone and in combination with anticancer agents, in models of hematologic malignancies - Fig 2

<p><b>INCB053914 inhibits cellular proliferation in hematologic tumor cell lines (A), inhibits phosphorylation of PIM substrates (B), including pBAD (C), and increases PIM2 expression (D) in hematologic tumor cell lines.</b> For all Western blots, actin controls confirmed equivalent loading. IC₅₀ values for pBAD inhibition were determined by fitting the percent inhibition versus the log [INCB053914] data to sigmoidal dose–response (variable slope) curve. Error bars represent standard deviation. GI₅₀ values >3 μM are not shown. *pP70S6K band intensities in KMS-12-BM (MM) cells were below the limit of detection at all INCB053914 concentrations tested. HL, Hodgkin lymphoma; ND, not determined. Original Western blot images are shown in Supporting Information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199108#pone.0199108.s008" target="_blank">S4 File</a>.</p

Preclinical characterization of INCB053914, a novel pan-PIM kinase inhibitor, alone and in combination with anticancer agents, in models of hematologic malignancies - Fig 5

<p><b>INCB053914 inhibits the phosphorylation of BAD in mice bearing MOLM-16 (AML) (A) or KMS-12 (MM) tumors (B), and inhibits growth of MOLM-16 (AML) (C) and KMS-12 (MM) (D) tumors <i>in vivo</i>. Mean INCB053914 plasma concentrations were determined at 2, 4, 8, and 16 hours post oral administration in mice bearing MOLM-16 tumors (E) or KMS-12-BM tumors (F).</b> EC₅₀ values for pBAD inhibition were determined by fitting data to a sigmoidal dose–response (variable slope) curve. Error bars represent standard error of the mean. BID, twice a day; PO, orally.</p

INCB053914 inhibits erythroid colony formation in patients with JAK2 V617F-positive MPNs.

<p>Error bars represent standard deviation. *p < 0.05; **p < 0.01.</p

Preclinical characterization of INCB053914, a novel pan-PIM kinase inhibitor, alone and in combination with anticancer agents, in models of hematologic malignancies - Fig 3

<p><b>INCB053914 inhibits phosphorylation of PIM substrates and increases PIM2 expression in primary bone marrow (BM) blasts (A), and increases PIM2 expression in PBMCs derived from whole blood samples from patients with AML (B). Pharmacodynamic effects of INCB053914 on PIM2 expression and 4E-BP1 phosphorylation in whole blood samples obtained 0 to 6 hours post-dose from two separate patients with AML enrolled in the ongoing phase 1/2 trial (C).</b> The EC₅₀ for increased PIM2 expression were determined by fitting data to a sigmoidal dose–response (variable slope) curve. Original Western blot images are shown in Supporting Information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199108#pone.0199108.s008" target="_blank">S4 File</a>.</p