Acute myeloid leukemia (AML) is an aggressive malignancy with only a handful of therapeutic options. About 30% of AML patients harbor mutated FLT3 kinase, and thus, this cancer-driver has become a hotly pursued AML target. Herein we report a new class of FLT3 inhibitors, which potently inhibit the proliferation of acute myeloid leukemia (AML) cells at nanomolar concentrations.

The lead aminoisoquinolines potently inhibited FLT3-driven AML cell lines, MV4-11 and MOLM-14. These aminoisoquinoline benzamides represent new kinase scaffolds with high potential to be translated into anticancer agents.

Developing continuous syntheses of lead compounds to support in vivo studies and preclinical evaluation remains an underdeveloped area. We report a telescoped continuous flow synthesis of an alkynylnaphthyridine lead compound for the treatment of FLT3 mutations in acute myeloid leukemia. Different strategies were used to develop the route, including Design of Experiments (DoE), high-throughput experimentation (HTE), and application of desorption electrospray ionization mass spectrometry (DESI-MS) to optimize and telescope the amidation and Sonogashira couplings to prepare the target compound, HSN608, a potent FLT3 inhibitor. Findings from these statistical design and automation studies helped streamline our workflow to achieve 10-fold and 5-fold reductions in the catalyst and cocatalyst loadings, respectively, in the synthesis. The application of high-throughput tools combined with a telescoped continuous synthesis method enabled an efficient and safe synthesis of this lead compound using the hazardous coupling reagent HATU while minimizing byproduct formation.

BackgroundAcute myeloid leukemia (AML) remains one of the most lethal, rarely cured cancers, despite decades of active development of AML therapeutics. Currently, the 5-year survival of AML patients is about 30% and for elderly patients, the rate drops to <10%. About 30% of AML patients harbor an activating mutation in the tyrosine kinase domain (TKD) of Fms-Like Tyrosine kinase 3 (FLT3) or a FLT3 internal tandem duplication (FLT3-ITD). Inhibitors of FLT3, such as Rydapt that was recently approved by the FDA, have shown good initial response but patients often relapse due to secondary mutations in the FLT3 TKD, like D835Y and F691 L mutations.MethodsAlkynyl aminoisoquinoline and naphthyridine compounds were synthesized via Sonogashira coupling. The compounds were evaluated for their in vitro and in vivo effects on leukemia growth.FindingsThe compounds inhibited FLT3 kinase activity at low nanomolar concentrations. The lead compound, HSN431, also inhibited Src kinase activity. The compounds potently inhibited the viability of MV4–11 and MOLM-14 AML cells with IC50 values <1 nM. Furthermore, the viability of drug-resistant AML cells harboring the D835Y and F691 L mutations were potently inhibited. In vivo efficacy studies in mice demonstrated that the compounds could drastically reduce AML proliferation in mice.InterpretationCompounds that inhibit FLT3 and downstream targets like Src (for example HSN431) are good leads for development as anti-AML agents.FundPurdue University, Purdue Institute for Drug Discovery (PIDD), Purdue University Center for Cancer Research, Elks Foundation and NIH P30 CA023168.

The introduction of imatinib into the clinical scene revolutionized the treatment of chronic myelogenous leukemia (CML). The overall eight-year survival rate for CML has increased from about 6 % in the 1970s to over 90 % in the imatinib era. However, about 20 % of CML patients harbor primary or acquired resistance to tyrosine kinase inhibitors. ABL1 point mutations in the BCR-ABL1 fusion protein, such as ABL1(T315I), typically emerge after prolonged kinase inhibitor treatment. Ponatinib (AP24534) is currently the only approved CML drug that is active against the ABL1(T315I) mutation. However, ponatinib has severe cardiovascular toxicities; hence, there have been efforts to find safer CML drugs that work against ABL1 secondary mutations. We reveal that isoquinoline- or naphthyridine-based compounds, such as HSN431, HSN576, HSN459, and HSN608 potently inhibit the enzymatic activities of ABL1, ABL1(T315I), and ABL1(E255K). These compounds inhibit the proliferation of ABL1-driven CML cell lines, K652 and KCL22 as well as the drug-resistant cell line, KCL22-IR, which harbors the secondary mutated ABL1(T315I) kinase.