Discovery of quinolinone derivatives as potent FLT3 inhibitors Hye Jin Chung a , Majid Rasool Kamli a , Hyo Jeong Lee a , Jae Du Ha b , Sung Yun Cho b , Jongkook Lee c , Jae Yang Kong d , Sun-Young Han a,⇑ a College of Pharmacy and Research Institute of Pharmaceutical Sciences, Gyeongsang National University, Jinju, Republic of Korea b Bio-organic Science Division, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea c College of Pharmacy, Kangwon National University, Chuncheon, Republic of Korea d College of Pharmacy, Keimyung University, Daegu, Republic of Korea article info Article history: Received 28 January 2014 Available online 13 February 2014 Keywords: Fms-like tyrosine kinase 3 (FLT3) Acute myeloid leukemia Quinolinone abstract Recently some fms-like tyrosine kinase 3 (FLT3) inhibitors have shown good efficacy in acute myeloid leuke- mia (AML) patients. In an effort to develop anti-leukemic drugs, we investigated quinolinone derivatives as novel FLT3 inhibitors. Two substituted quinolinones, KR65367 and KR65370 were subjected to FLT3 kinase activity assay and showed potent inhibition against FLT3 kinase activity in vitro, with IC 50 of 2.7 and 0.57 nM, respectively. As a measure of selectivity, effects on the activity of other kinases were also tested. Both compounds have negligible activity against Met, Ron, epidermal growth factor receptor, Aurora A, Janus kinase 2, and insulin receptor; with IC 50 greater than 10 lM. KR compounds showed strong growth inhibition in MV4;11 AML cells and increased the apoptotic cell death in flow cytometric analyses. A decrease in STAT5 phosphorylation by KR compounds was observed in MV4;11 cells. Furthermore, in vitro evaluation of compounds structurally related to KR65367 and KR65370 showed a good structure-activity relationship. Ó 2014 Elsevier Inc. All rights reserved. 1. Introduction Fms-like tyrosine kinase 3 (FLT3), also known as fetal liver ki- nase 2, is a receptor tyrosine kinase involved in proliferation and differentiation of hematopoietic stem cells [1]. FLT3 is activated by binding of FLT3 ligand, and results in activation of tyrosine ki- nase activity and downstream signal transduction pathways [2]. FLT3 is overexpressed in most acute myeloid leukemia (AML) cases and activating mutations are found in about one third of AML pa- tients [3]. There are two classes of activating mutations. One is internal tandem duplication (ITD) in the juxtamembrane domain, and the other is point mutation in the tyrosine kinase domain of FLT3. The mutant form of FLT3 aberrantly activates downstream effectors such as STAT5 in a manner different from wild type FLT3 [4]. FLT3 has emerged as an important target for molecular therapy and ITD mutations in FLT3 have been well documented as a drug target in leukemia [5]. AML, most common type of acute leukemia, is characterized by blocking differentiation, and uncontrolled proliferation of cells in- side the bone marrow [6]. There are many reports which show that AML patients with mutated FLT3 genes show poor prognosis [7]. Currently, a combination of cytarabine and anthracycline is mainly used for chemotherapy of AML patients. However, there are high remission rates after the chemotherapy, resulting in a high unmet medical need for a new therapeutic strategy [8,9]. Quite a few small molecules that act as FLT3 inhibitors have been identified for the purpose of developing anti-leukemic agents. Small mole- cules tested in clinical trials include lestaurtinib [10], midostaurin [11], tandutinib [12], sunitinib [13], and quizartinib [14]. In this study we present two quinolinone derivatives as potent FLT3 inhibitors. The compounds KR65367 and KR65370 inhibited FLT3 activity and downstream STAT5 phosphorylation. Both of the compounds exhibited cytotoxicity against AML cells and induced apoptosis in flow cytometric analysis. 2. Materials and methods 2.1. Cell culture and reagents Human MV4;11 and RS4;11 leukemic cells were purchased from the American Type Culture Collection (Rockville, MD). Cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) and 1% http://dx.doi.org/10.1016/j.bbrc.2014.02.029 0006-291X/Ó 2014 Elsevier Inc. All rights reserved. Abbreviations: FLT3, fms-like tyrosine kinase 3; AML, acute myeloid leukemia; ITD, internal tandem duplication; HTRF, homogeneous time-resolved fluorescence; TR-FRET, time resolved-fluorescence resonance energy transfer; VEGFR-2, vascular endothelial cell growth factor receptor 2; STAT5, Signal transducer and activator of transcription 5. ⇑ Corresponding author. Address: College of Pharmacy, Gyeongsang National University, 501 Jinju-daero, Jinju, Gyeongnam 660-751, Republic of Korea. Fax: +82 55 772 2429. E-mail address: syhan@gnu.ac.kr (S.-Y. Han). Biochemical and Biophysical Research Communications 445 (2014) 561–565 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc