Bioorganic & Medicinal Chemistry Letters 23 (2013) 1768–1770

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Flavonoids as receptor tyrosine kinase FLT3 inhibitors

0960-894X/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.bmcl.2013.01.049

⇑ Corresponding author. Tel.: +82 55 772 2423.E-mail address: syhan@gnu.ac.kr (S.-Y. Han).

Young-Won Chin a, Jae Yang Kong b, Sun-Young Han b,⇑a College of Pharmacy, Dongguk University-Seoul, Goyang, Gyeonggi-do 410-820, South Koreab College of Pharmacy and Research Institute of Pharmaceutical Sciences, Gyeongsang National University, Jinju, Gyeongnam 660-701, South Korea

a r t i c l e i n f o

Article history:Received 29 October 2012Revised 31 December 2012Accepted 15 January 2013Available online 26 January 2013

Keywords:Acute myeloid leukemiaFLT3FlavonoidsLuteolin

a b s t r a c t

The Fms-like tyrosine kinase 3 (FLT3), a receptor tyrosine kinase, is involved in the proliferation, differ-entiation and apoptosis of hematopoietic cells. FLT3 is highly overexpressed in acute myeloid leukemia(AML) of the majority of patients. Screening for flavonoids including flavones, flavanones, flavonols,and flavanonols disclosed that luteolin was potent FLT3 enzyme inhibitor. Furthermore, luteolinsuppressed cell proliferation in MV4;11 cells with constitutively activated FLT3.

� 2013 Elsevier Ltd. All rights reserved.

The Fms-like tyrosine kinase 3 (FLT3), expressed at high levelsin acute myeloid leukemia (AML) of the majority of patients, ispresented as a potential therapeutic target.1,2 Clinical trials forsmall-molecule FLT3 inhibitors, such as lestaurtinib (CEP-701),midostaurin (PKC-412), tandutinib (MNL-518), sorafenib, KW-2449, and AC-220, are currently undergoing.3 As part of our ongo-ing investigation for FLT3 inhibitory substances from naturallyoccurring secondary metabolites,4 flavonoids were assessed fortheir FLT3 inhibitory activities using homogenous time-resolvedfluorescence assay.5

Flavonoids are commonly found in plant extracts and consumedas edible vegetables. Flavonoids consist of three ring, C6–C3–C6,called A, C, and B ring, respectively. In the present study, represen-tative skeletons of flavonoids, flavones, flavanone, flavonol, flava-nonols, and flavonol glycosides were tested in in vitro FLT3kinase assay (Fig. 1). Interestingly, only flavones and flavonolswere found active in this assay system while flavanones (naringe-nin 8 and eriodictyol 9) and flavanonols (aromadendrin 10 andtaxifolin 11) deemed inactive. Of flavones, apigenin (2) and luteolin(3) inhibited FLT3 enzyme activities with IC50 1.45 and 0.83 lM,respectively. Kaempferol (5) (IC50 2.32 lM), quercetin (6) (IC50

0.59 lM), and myricetin (7) (IC50 1.68 lM) also demonstrated theirinhibitory activities against FLT3 enzyme. In the aspect of struc-ture–activity relationship, double bond in the C ring is importantsince flavanones and flavanonols are found inactive. Hydroxylgroup at C-3 in active flavones does not make any difference in

their activities as shown in luteolin and quercetin (Fig. 2). It wasobserved that chrysin (1) and F18 (4), compounds without hydro-xyl groups in the B ring, are inactive. Hence, it is suggested that hy-droxyl groups in the B ring may be responsible in part for FLT3enzyme inhibition. Even though compounds with two hydroxylgroups in the B ring are more potent than one hydroxyl group,compound with three hydroxyl groups in the B ring did not givebetter inhibitory activity.

To assess the importance of glycosides, we tested three flavonolglycosides, quercetin-3-O-glucopyranoside (15), quercetin-3-O-galactopyranoside (16), and quercetin-3-O-rhamnopyranoside(17) and found all of them were inactive. Furthermore, baicalein(12) (OH-5,6,7 in the A ring and no hydroxyl group in the B ring),scutellarein (13) (OH-5,6,7 in the A ring and 40-OH in the B ring)and oroxylin (14) (OH-5,7, OCH3-6 in the A ring and no hydroxylgroup in the B ring) were tested to provide more insight for theimportance of free hydroxyls in flavones. When compared toapigenin, scutellarein has one more hydroxyl group at C-6 in theA ring but the presence of hydroxyl group led to the loss of activity.Methylation of OH-6 as shown in oroxylin structure displayedactivity while baicalein was found inactive.

Along with FLT3 kinase activity assay, cytotoxic effects of flavo-noids against AML cell lines were measured using tetrazolium-based cell viability assay (Table 1).6 MV4;11 cells harboring FLT3mutation and RS4;11 cells with wild-type FLT3 were employed.Apigenin (2) and kaempferol (5) showed mild cytotoxic activityagainst MV4;11 cells (GI50 2.81 and 3.34 lM, respectively) reflect-ing their mild FLT3 kinase inhibitory activity. GI50 for myricetinwas more than 50 lM, suggesting weak or no activity in cell-basedassay. Luteolin (3) showed strong activity with GI50 of 1.76 lM in

Figure 1. Structure of flavonoids.

Figure 2. Effects of (A) luteolin (3) and (B) quercetin (6) on FLT3 kinase activity. X-axis shows concentration of compounds in log scale (lM). % inhibition was calculated using1% DMSO treatment as a negative control. Data are mean and SEM of three independent experiments.

Table 1Effects of flavonoids on the kinase activity of FLT3 and growth inhibition on AML cells

Compound FLT3 IC50 (lM) MV4;11 GI50 (lM) RS4;11 GI50 (lM)

1 >102 1.45 ± 0.32 2.81 27.93 0.83 ± 0.12 1.76 7.254 >105 2.32 ± 0.11 3.34 >506 0.59 ± 0.12 13.1 21.67 1.69 ± 0.18 >50 >508 >109 >1010 >1011 >1012 >1013 >1014 1.48 ± 0.12 5.59 40.015 >1016 >1017 >10Lestaurtiniba 0.0082 ± 0.0005

a Positive control.

Y.-W. Chin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1768–1770 1769

MV4;11 cells. When compared with quercetin (6) with similarin vitro kinase activity, luteolin (3) showed better effects in growthinhibition of AML cell lines. As shown in Figure 2, luteolin (3)showed selectivity in growth inhibition between MV4;11 cellsand RS4;11 cells. MV4;11 cells with constitutively activated FLT3are more sensitive to luteolin than RS4;11 cells. Because MV4;11cells are ‘addicted’ to FLT3 signaling, meaning that growth and sur-vival of the cells depend on the FLT3 activity, inhibition of FLT3 byluteolin resulted in growth inhibition of MV4;11 cells.3

In order to evaluate the effects of luteolin on the cell cycle andcell death, MV4;11 cells treated with luteolin (3) were subjectedflow cytometric analyses (Fig. 3).7 Cell population in S phase andG2/M phase decreased with luteolin and cells in sub-G1 populationincreased from 3.86% (control) to 30.76% (luteolin 10 lM), indicat-ing dead cell population.

Based on these data from in vitro kinase assay and cell-basedassay, luteolin (3) seemed to possess selective FLT3 inhibitoryactivity. Therefore, this structure along with active FLT3 inhibitoryflavonoids may provide chemically valuable information in AMLresearch field.

Figure 3. Effect of luteolin on the cell cycle of MV4;11 cells. Cells were treated with luteolin at the indicated concentration for 48 h and stained with propidium iodide forflow cytometric analyses. M1: sub-G1 phase, M2: G1 phase, M3: S phase, M4: G2/M phase.

1770 Y.-W. Chin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1768–1770

Acknowledgments

This work was supported by grants of the NRF (2011-0014225,2011-0010374) funded by the government of Korea (MEST) and agrant (12182KFDA666) from Korea Food & Drug Administrationin 2012.

References and notes

1. Stirewalt, D. L.; Radich, J. P. Nat. Rev. Cancer 2003, 3, 650.2. Smith, C. C.; Wang, Q.; Chin, C.-S.; Salerno, S.; Damon, L. E.; Levis, M. J.; Perl, A. E.;

Travers, K. J.; Wang, S.; Hunt, J. P.; Zarrinkar, P. P.; Schadt, E. E.; Kasarskis, A.;Kuriyan, J.; Shah, N. P. Nature 2012, 485, 260.

3. Pratz, K.; Levis, M. Leuk. Lymphoma 2008, 49, 852.4. Han, S.-Y.; Chin, Y.-W. J. Enzyme Inhib. Med. Chem. 2011, 26, 445.5. In vitro kinase assay. Inhibition of kinase activity against FLT3 kinase was

measured using homogeneous time-resolved fluorescence (HTRF) assays. Inbrief, assays are based on the phosphorylation of peptide substrates in thepresence of ATP. Resulting phosphorylated substrates are detected by a TR-FRET

(time resolved-fluorescence resonance energy transfer) signal. Recombinantproteins containing a kinase domain were purchased from Millipore (Billerica,MA). Optimal enzyme, ATP, and substrate concentrations were established usingHTRF KinEASE kit (Cisbio, France) according to the manufacturer’s instructions.Assays consist of enzymes mixed with serially diluted compounds and peptidesubstrates in a kinase reaction buffer (250 mM HEPES (pH 7.0), 0.5 mMorthovanadate, 0.05% BSA, 0.1% NaN3). Following the addition of reagents fordetection, the TR-FRET signal was measured using an EnVision multi-labelreader (Perkin Elmer, Waltham, MA). IC50 was calculated by a nonlinearregression using Prism version 5.01 (GraphPad, La Jolla, CA). Each partition,chromatographic fractions, and isolated compounds were tested against FLT3kinase.

6. Cytotoxicity assay. MV4;11 and RS4;11 cells were plated in 96-well plates(10,000 cells per well) and serial dilutions of compounds were added. At the endof the incubation period (72 h), cell viability was measured using tetrazolium-based Ez CyTox cell viability assay kit (Daeil, Korea). IC50 was calculated by anonlinear regression using Prism (version 5.01).

7. Cell cycle analysis. MV4;11 cells were treated with luteolin for 48 h. Cells werethan fixed and stained with propidium iodide (Sigma) and subjected to flowcytometry using FACSCalibur (BD Biosciences, Billerica, MA). Data wereanalyzed by CellQuest Pro (BD Biosciences).