Identification of novel FLT3 kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.03.024

FLT3 and PDGFR tyrosine kinases are important targets for therapy of different types of leukemia. Several FLT3/PDGFR inhibitors are currently under clinical investigation for combination with standard therapy for treatment of acute myeloid leukemia (AML), however these agents only induce partial remission and development of resistance has been reported. In this work we describe the identification of potent and novel dual FLT3/PDGFR inhibitors that resulted from our efforts to screen a library of 25,607 small molecules against the FLT3 dependent cell line MOLM-13 and the PDGFR dependent cell line EOL-1. This effort led to the identification of five compounds that were confirmed to be active on additional FLT3 dependent cell lines (cellular EC₅₀ values between 35 and 700 nM), while having no significant effect on 24 other tyrosine kinases.

Full text of DOI:10.1016/j.ejmech.2013.03.024

European Journal of Medicinal Chemistry 63 (2013) 713e721
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Identification of novel FLT3 kinase inhibitors
,
,
,b
Daphnie Pauwels a b, Hugo Klaassenc, Idoya Lahortigaa b, Amuri Kilonda c, Kris Jacobsa
,
Bram Swerona b, Romuald Corbau c, Patrick Chaltinc d, Arnaud Marchand c, Jan Coolsa
,
,
,b,
*
a Center for the Biology of Disease, VIB, Leuven 3000, Belgium
b Center for Human Genetics, KU Leuven, Leuven 3000, Belgium
c Center for Innovation and Stimulation of Drug Discovery (CISTIM), Leuven 3000, Belgium
d Center for Drug Design and Development (CD3), KU Leuven R&D, Leuven 3000, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
FLT3 and PDGFR tyrosine kinases are important targets for therapy of different types of leukemia. Several
FLT3/PDGFR inhibitors are currently under clinical investigation for combination with standard therapy
for treatment of acute myeloid leukemia (AML), however these agents only induce partial remission and
development of resistance has been reported. In this work we describe the identification of potent and
novel dual FLT3/PDGFR inhibitors that resulted from our efforts to screen a library of 25,607 small
molecules against the FLT3 dependent cell line MOLM-13 and the PDGFR dependent cell line EOL-1. This
effort led to the identification of five compounds that were confirmed to be active on additional FLT3
dependent cell lines (cellular EC50 values between 35 and 700 nM), while having no significant effect on
24 other tyrosine kinases.
Received 23 November 2012
Received in revised form
9 March 2013
Accepted 13 March 2013
Available online 21 March 2013
Keywords:
Tyrosine kinase
FLT3
Ó 2013 Elsevier Masson SAS. All rights reserved.
PDGFR
Acute myeloid leukemia
Inhibitors
Cell based drug screening
1. Introduction
when this mutation is found in the presence of internal tandem
duplication (ITD) of FLT3 [9,10].
Acute myeloid leukemia (AML) is a heterogenous disease
resulting from the clonal expansion of immature hematopoietic
cells by the cooperation of various mutations and chromosomal
aberrations. AML represents the most frequent acute leukemia in
adults with a peak of incidence around 65 year [1]. The overall
survival at 4 years is around 37% for pediatric AML and 16% for adult
AML [2]. Cytogenetic analysis of the leukemic cells is used to stratify
the patients in different prognostic subgroups, with CBFB-MYH11
and RUNX-RUNX1T1 showing a good survival, while patients
expressing MLL fusions having a bad prognosis [3e5]. In addition to
the chromosomal aberrations, point mutations in FLT3, ASXL1,
DNMT3A, TET2, IDH1, IDH2 and NPM1 are also recurrently observed
in AML and further influence the prognosis [6]. The presence of
FLT3 mutations is associated with a poor prognosis and an
increased risk of relapse [7,8], while mutations in NPM1 gene
correlate with a high rate of complete remission and a favorable
overall survival [6]. However, this positive prognostic effect is lost
FLT3 is a transmembrane receptor tyrosine kinase, which is
selectively expressed on hematopoietic cells where it mediates
stem cell differentiation and proliferation [11e13]. Activation of the
FLT3 receptor in AML precursor cells is believed to stimulate pro-
liferation and differentiation, and to inhibit apoptosis of leukemia
cells. Internal tandem duplications in the juxtamembrane domain
of FLT3 are the most common mechanism of activation of FLT3 that
is found in up to 30% of all AML cases [14,15]. Additional activating
point mutations in FLT3 occur in the activation loop, most
frequently at position 835 and are identified in 5e10% of AML cases
[16]. The FLT3-ITD and point mutations activate the FLT3 kinase and
its downstream signaling pathways, including STAT5 and PI3K/AKT
pathway, known to give leukemic cells a proliferation and survival
advantage [17,18].
Several kinase inhibitors with activity against FLT3 (PKC412,
Lestaurtinib/CEP701, Sunitinib/SU11248 and Tandutinib/MLN518)
are currently under clinical investigation for combination with
standard therapy of AML. However, the currently available FLT3
inhibitors are not selective, induce only partial and transient re-
sponses. In a phase IIB clinical trial with PKC412, the treatment was
not sufficient to reach complete remission, and even partial
remission was only obtained in 3% of the cases [19]. In a phase II
* Corresponding author. Center for the Biology of Disease, VIB, Herestraat 49, Box
602, Leuven 3000, Belgium. Tel.: þ32 16 3 30082; fax: þ32 16 3 30827.
E-mail address: Jan.cools@cme.vib-kuleuven.be (J. Cools).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.024

714
D. Pauwels et al. / European Journal of Medicinal Chemistry 63 (2013) 713e721
clinical trial with Lestaurtinib a transient reduction in peripheral
blood and bone marrow blasts was observed in 3 out of 5 patients
(60%) with FLT3 mutations and in 5 out of 22 patients (23%) with
wild-type FLT3 [20].
inhibited the growth of EOL-1 and MOLM-13 cells by more than
50% and inhibited the growth of HEL cells by less than 20%. Using
these criteria we selected 56 compounds for further analysis.
Subsequently, the selected compounds were tested against two
additional FLT3 dependent cell lines: a human AML cell line MV4-
11 and the mouse Ba/F3 cells transformed by FLT3-ITD. The FLT3
independent cell lines KG-1 and parental Ba/F3 cells were used as
extra controls. Ba/F3 cells transformed by FLT3-ITD are an inter-
esting cell line for this type of experiments, as they are only
dependent on FLT3-ITD for their proliferation and survival, and can
be compared to Ba/F3 cells grown in the presence of IL-3, which are
independent of FLT3 activity [36]. The EC50 values of the 56 selected
compounds are listed in Supplementary Table 1. By using a cut-off
Quizartinib (AC220), a selective FLT3 inhibitor currently in a
phase II clinical trial, shows activity at low nanomolar concentra-
tions in vitro and in animal models and could represent a more
attractive compound for the treatment of FLT3-ITD positive pa-
tients with relapsed/refractory AML [21]. Indeed, in an initial phase
I study, responses were seen in both FLT3-ITD positive and FLT3-ITD
negative AML patients, with an overall response rate of 56% and
20%, respectively [22]. However, even potent selective FLT3 in-
hibitors are expected to face the development of resistance due to
mutations in the kinase domain [23e25]. Such mutations have
been identified in vitro, and have been observed in AML patients
treated with PKC412 or AC220 [25,26]. Resistance to PKC412 can be
caused by FLT3 point mutations at amino acids N676, F691 and
G697, some of which interfere with drug binding or by modifying
the stability or activity of the kinase [24,27].
value of an EC50 <1
mM on the FLT3 dependent Ba/F3 cells and an
EC50 of >10 M on the wild type Ba/F3 cells, we selected the five
m
most potent compounds for further analysis. Concerning the
chemical structures, there was only a structural relationship be-
tween compounds 1, 2 and 3 (all aminopyrazole structures), while
the chemical structure of all other identified hits were not related.
The cell-based EC50 values of these five identified hits were
determined for all the cell lines used during the screening and are
listed in Table 1. Compounds 1, 2 and 3 were already known as kinase
inhibitors [37], whereas compound 5 belongs to a family of com-
pounds with described effects on stem cell differentiation [38]. The
structures of these compounds are shown in Fig. 1. Compounds 1, 2
and 3 belong to the same family and contain an aminopyrazole
structure. More specifically, compound 1 is a 4-methoxy-N-(3-
phenyl-1H-pyrazol-5-yl)benzamide, compound 2 is a 4-fluoro-N-(3-
(p-tolyl)-1H-pyrazol-5-yl)benzamide and compound 3 is a N-(3-(4-
fluorophenyl)-1H-pyrazol-5-yl)-3-methylbenzamide. Compounds 4
and 5 belong to a different class of molecules with compound
4 being a 4-(3-(3-chloro-4-fluorophenyl)-1H-pyrazol-4-yl)pyridine
and compound 5 a 4-methyl-N-(1-(p-tolyl)-1H-benzo[d]imidazol-5-
yl)phthalazin-1-amine.
PDGFR
a
and PDGFR
b
belong to the same family as the FLT3 ki-
or kinase is observed in
nase, and aberrant activation of PDGFR
a
b
various myeloid malignancies that are now referred to as myeloid
neoplasms with eosinophilia and with chromosomal aberrations
involving PDGFRA, PDGFRB or FGFR1 [28]. The FIP1L1-PDGFRA is the
most common PDGFRA fusion, whereas ETV6-PDGFRB is the most
common PDGFRB fusion, but a large number of other fusion genes
involving PDGFRA or PDGFRB have been described as well [29,30].
Furthermore, it is well established that the inhibitor imatinib tar-
gets PDGFR
PDGFR in several chronic myeloproliferative diseases [29,31].
Similarly as for the FLT3 inhibitors, resistance against imatinib oc-
curs and could be overcome by PKC412 (in the case of PDGFR ) and
sorafenib (in the case of PDGFR ) [27,32].
a in idiopathic hypereosinophilic syndrome and
b
a
b
Here, we have used a cell-based high throughput screen to
identify novel FLT3/PDGFR inhibitors and we have tested their
inhibitory activity on oncogenic FLT3 mutants and on the FIP1L1-
2.2. Chemical synthesis of compounds 1, 2 and 3
PDGFRa fusion.
For the confirmation assays, we reordered compounds 4 and 5
and synthesized compounds 1, 2 and 3, because they were no
longer commercially available. The synthesis was performed as
followed: To a suspension of 5-phenyl-1H-pyrazol-3-amine deriv-
ative 4aec (1.0 mmol) in dichloromethane (5 mL) at 0 ꢀC were
added benzoyl chloride derivative 5aec (1.05 mmol) and triethyl-
amine (1.20 mmol). The reaction mixture was stirred at 0 ꢀC for
5 min and at room temperature for 1 h. It was then diluted with
dichloromethane, washed with water and brine, and concentrated
under reduced pressure. The residue was purified by flash chro-
2. Results
2.1. Compound screening
In order to identify novel dual FLT3/PDGFR kinase inhibitors, we
screened a library of 25,607 compounds for activity against the FLT3
dependent AML cell line MOLM-13 and the PDGFR
AML cell line EOL-1 [33,34]. These cells were treated for 72 h with
an average compound concentration of 10 M (fixed weight). As a
positive control we used 10 M of Ara-C, a known nucleoside
a dependent
m
m
matography on silica gel using a gradient of methanol in
analog marketed for the treatment of various leukemia [35]. At the
end of the 72 h, we calculated the cell number differences between
the drug-treated and DMSO-treated cells. As a negative control we
tested the activity of the compounds against HEL cells, an AML cell
line known to be independent of FLT3/PDGFR signaling for its
proliferation. The compounds were classified as hits when they
dichloromethane to give compounds 1e3 as a white solid (Fig. 2).
1H and 13H spectra are available in Supplementary material.
Confirmatory 1H NMR, TOF-MS and LC-MS analyses were per-
formed for compounds 1 to 3. Analytical data for compounds 4 and
5 were obtained from the commercial suppliers Asinex (Moscow,
Russia) and ChemDiv (San Diego, CA, USA) respectively. Analytical
Table 1
EC50 values (mM) of the top five compounds (Cpd) in the AML and Ba/F3 cell lines used during the screening.
Compound
Cell line
Ba/F3-FLT3-ITD no IL-3
Ba/F3-FLT3-ITD þ IL-3
MOLM-13
MV4-11
EOL-1
HEL
47
1000
62
1000
14
KG-1
1
2
3
4
5
0.036
0.138
0.296
0.405
0.717
18
23
21
16
19
0.292
0.612
0.63
0.258
0.332
0.142
2.821
1.779
2.755
3.414
0.066
0.502
0.202
0.382
0.889
15
28
29
24
20

D. Pauwels et al. / European Journal of Medicinal Chemistry 63 (2013) 713e721
715
Fig. 1. Chemical structures of the identified FLT3 inhibitors. Compound 1: 4-methoxy-N-(3-phenyl-1H-pyrazol-5-yl)benzamide; compound 2: 4-fluoro-N-(3-(p-tolyl)-1H-pyrazol-5-
yl)benzamide; compound 3: N-(3-(4-fluorophenyl)-1H-pyrazol-5-yl)-3-methylbenzamide; compound 4: 4-(3-(3-chloro-4-fluorophenyl)-1H-pyrazol-4-yl)pyridine; compound 5: 4-
methyl-N-(1-(p-tolyl)-1H-benzo[d]imidazol-5-yl)phthalazin-1-amine.
data of these compounds confirmed not only the exact structure of
the compounds used in the biological testing but also their high
level of purity. All the information and NMR spectra as a string for
the different compounds are available in the Supplementary
material.
(CH), 115.68 (CH), 94.76 (CH), 21.24 (CH3); ESI/APCI (þ) m/z 296
(M þ H); ESI/APCI (ꢁ) 294 m/z (M ꢁ H).
2.2.3. N-(3-(4-Fluorophenyl)-1H-pyrazol-5-yl)-3-methylbenzamide
(compound 3)
Prepared by reaction of 5-(4-fluorophenyl)-1H-pyrazol-3-amine
(4c) and 3-methylbenzoyl chloride (5c). Purified using a gradient of
methanol (1e7%) in dichloromethane. Yield 48%; white solid; 1H
2.2.1. 4-Methoxy-N-(3-phenyl-1H-pyrazol-5-yl)benzamide
(compound 1)
Prepared by reaction of 5-phenyl-1H-pyrazol-3-amine (4a) and
4-methoxybenzoyl chloride (5a). Purified using a gradient of
methanol (1e10%) in dichloromethane. Yield 62%; white solid; 1H
NMR (600 MHz, DMSO-d6):
(m, 4H), 7.39 (d, J ¼ 4.5 Hz, 2H), 7.30 (t, J ¼ 8.7 Hz, 2H), 7.02 (s, 1H),
2.39 (s, 3H); 13C NMR (151 MHz, DMSO):
165.03 (HNeC]O),
d 12.91 (s, 1H), 10.80 (s, 1H), 7.88e7.78
d
NMR (600 MHz, DMSO-d6):
d
10.75 (s, 1H), 8.04 (d, J ¼ 8.8 Hz, 2H),
162.20 (]CeF), 148.81 (C), 141.30 (C), 138.04 (C), 134.37 (C), 132.61
(CH) 128.71 (CH), 128.65 (CH), 127.49 (CH), 126.49 (C), 125.28 (CH),
116.32 (CH), 95.21 (CH), 21.36 (CH3); ESI/APCI (þ) m/z 296 (M þ H);
ESI/APCI (ꢁ) m/z 294 (M ꢁ H).
7.77 (dd, J ¼ 8.4, 1.2 Hz, 2H), 7.46 (t, J ¼ 8.4 Hz, 2H), 7.35 (tt, J ¼ 8.4,
1.2 Hz,1H), 7.05 (d, J ¼ 8.9 Hz, 2H), 6.97 (s,1H), 3.84 (s, 3H); 13C NMR
(151 MHz, DMSO):
d 164.36 (HNeC]O), 162.34 (C), 147.55 (C),
143.25 (C), 130.42 (C), 130.09 (CH), 129.37 (CH), 128.42 (CH), 126.45
(C), 125.37 (CH), 113.99 (CH), 94.65 (CH), 55.82 (OCH3); ESI/APCI (þ)
m/z 294 (M þ H); ESI/APCI (ꢁ) m/z 292 (M ꢁ H).
2.3. Confirmation assays
We confirmed the growth inhibitory effects of the five selected
compounds against EOL-1, MOLM-13 and MV4-11 cell lines. Dosee
response curves are depicted in Fig. 3A. From these data it is clear
that compound 1 is the most potent molecule with EC50 of 70e
300 nM. All the compounds showed a similar selectivity toward
2.2.2. 4-Fluoro-N-(3-(p-tolyl)-1H-pyrazol-5-yl)benzamide
(compound 2)
Prepared by reaction of 5-(p-tolyl)-1H-pyrazol-3-amine (4b)
and 4-fluorobenzoyl chloride (5b). Purified using a gradient of
methanol (1e7%) in dichloromethane. Yield 23%; white solid; 1H
FLT3-ITD (MOLM-13 and MV4-11) and PDGFRa (EOL-1).
NMR (600 MHz, DMSO-d6):
d 12.88 (s, 1H), 10.88 (s, 1H), 8.11 (dd,
We also determined the EC50 values in Ba/F3 cells transformed
by FLT3-D835Y, an activated mutant form of FLT3 with a point
mutation in the activation loop of the kinase domain. In addition,
we also tested FLT3-ITD with additional mutations (F691I and
G697R) in its kinase domain. These mutations (D835Y, F691I and
J ¼ 8.7, 5.6 Hz, 2H), 7.65 (d, J ¼ 8.2 Hz, 2H), 7.34 (t, J ¼ 8.7 Hz, 2H),
7.27 (d, J ¼ 7.8 Hz, 2H), 7.00 (s, 1H), 2.34 (s, 3H); 13C NMR (151 MHz,
DMSO):
d 164.50 (CeF), 163.84 (HNeC]O), 148.61 (C), 142.30 (C),
137.96 (C), 130.99 (C), 130.90 (CH), 129.95 (CH), 127.03 (C), 125.30
Fig. 2. Overview of the chemical synthesis of compounds 1, 2 and 3. 5-Phenyl-1H-pyrazol-3-amine derivative 4aec were added to benzoyl chloride derivative 5aec to create
compounds 1, 2 and 3.

716
D. Pauwels et al. / European Journal of Medicinal Chemistry 63 (2013) 713e721
Fig. 3. Doseeresponse curves for the AML cell lines (A) and the Ba/F3 cell lines (B). A) The AML cell lines EOL-1 (PDGFR dependent), MOLM-13 (FLT3 dependent) and MV4-11 (FLT3
dependent) were treated with the five different hit compounds with concentrations ranging between 35 and 700 nM. The AML cell line KG-1 (FGFR1 dependent) served as a control.
B) Same experiments were performed on the Ba/F3 cell lines (FLT3-ITD, FLT3-ITD-F691I, FLT3-ITD-G697R and FLT3-D835Y). Ba/F3 wild type cells (Ba/F3 WT) were used as a control.
Data represent mean þ/ꢁ SEM.

D. Pauwels et al. / European Journal of Medicinal Chemistry 63 (2013) 713e721
717
Fig. 4. Western blot analysis of the effects on phosphorylation. A) Ba/F3-FLT3-ITD cells were treated with different concentrations of the hit compounds. Phosphorylation of FLT3
and STAT5 was measured. FLT3 and STAT5 protein levels were used as loading controls. B) For the EOL-1 cells we determined phosphorylation of PDGFR
STAT5 protein levels were used as loading controls.
a and STAT5. PDGFRa and

718
D. Pauwels et al. / European Journal of Medicinal Chemistry 63 (2013) 713e721
G697R) are known to confer resistance to various FLT3 kinase in-
hibitors [25,27,32]. We tested the activity of the five compounds
against these different FLT3 mutants and the resulting dosee
response curves are shown in Fig. 3B, pointing out mild activities
against some of the mutant FLT3 cell lines.
To confirm that the compounds had a direct effect on FLT3/
PDGFR we analyzed the effects of the compounds on the auto-
phosphorylation of FLT3-ITD and FIP1L1-PDGFRa in the Ba/F3-
FLT3-ITD cells and EOL-1 cells, respectively. Furthermore, we
determined the effect of treatment with the inhibitors on the
phosphorylation of STAT5, a downstream effector of FLT3/PDGFRa.
Western blot analysis confirmed the inhibiting effect of the five
compounds on FLT3 auto-phosphorylation. These data also
confirmed that compound 1 is the most potent inhibitor with an
inhibition of FLT3 auto-phosphorylation starting at a concentration
as low as 50 nM (Fig. 4). The other compounds blocked the auto-
phosphorylation of FLT3 with similar potency at a concentration
of 500 nM. Compound 2 showed a remarkable lower inhibition of
the auto-phosphorylation of PDGFRa, indicating that compound 2
is a more specific for FLT3 than for PDGFR
a
.
2.4. Specificity of the FLT3/PDGFR inhibitors
To determine the specificity of these compounds for FLT3/
PDGFR, we next tested their effect on 24 other tyrosine kinases by
using the Ba/F3 cell system. Each of the 24 respective Ba/F3 cell
lines expresses a specific activated tyrosine kinase and is depen-
dent on this kinase activity for its proliferation and survival.
Interestingly, compounds 1, 2, 3 and 5 did not show any activity
against the 24 other kinases (Fig. 5). For compound 4 we observed
an inhibiting effect on cells dependent on ABL2, EPHA5, EPHA8,
ROS1 and SRC at 5
additional doseeresponse experiment, but no inhibitory effect of
compound 4 below 1 M was seen, indicating the specificity for
mM (Fig. 5). For these kinases we performed an
m
FLT3/PDGFR at lower concentrations (Fig. 6).
3. Discussion
AML is the most frequent acute leukemia in adults and has a low
survival rate of around 37% for pediatric AML and 16% for adult AML
[2]. The chemotherapeutic treatment of AML has been improved
over the last years, but there remains a strong need for the intro-
duction of targeted therapies that are more potent and less toxic
than the current chemotherapy. The high incidence rate of FLT3
mutations in AML patients and its correlation with a poor prognosis
makes FLT3 a very interesting target for therapy [5,10]. FLT3 is
mutated in approximately 30% of AML cases and is known to be
important for the proliferation and survival of the leukemia cells.
In the past ten years, a number of FLT3 kinase inhibitors, such as
PKC412, Lestaurtinib, Semaxanib, Sunitinib and Tandutinib have
been developed, but the clinical results so far have only been
modest, especially in monotherapy setting where these compounds
seem to fail to induce a long lasting remission. In addition, we can
expect that similarly to the development of resistance to BCR-ABL1
inhibitors in blast crisis CML, resistance to FLT3 inhibitors is likely
to develop during therapy with FLT3 inhibitors. Different resistance
mechanisms have been described in tyrosine kinases of which the
most important mechanism is the acquisition of mutations in the
kinase domain [24]. Indeed, in an ongoing clinical trial with the
FLT3 inhibitor AC220 the development of resistance was reported,
due to the acquisition of specific resistance mutations in the FLT3
kinase domain [25].
Fig. 5. Specificity testing on 24 other kinases. Each hit compound (1 or 5 mM) was
tested on Ba/F3 cells transformed by one of the 24 kinases. These Ba/F3 cells are
specifically dependent on the activity of the indicated tyrosine kinase. Percentage of
growth was measured after 24 h. Black bars represent DMSO and gray bars represent
the compound. Data represent mean þ/ꢁ SEM.
very specific, many are targeting multiple kinases (both tyrosine
and serine/threonine kinases), which may be beneficial to target
cancer cells, but also a disadvantage with respect to side effects.
All these current limitations of the existing FLT3 inhibitors
indicate the need to identify novel potent and specific FLT3 in-
hibitors. To this end, we screened a library of 25,607 small mole-
cules on a FLT3 dependent AML cell line (MOLM-13) and a PDGFR
a
dependent AML cell line (EOL-1). Using this screening strategy, we
identified five potentially interesting FLT3/PDFGR inhibitors
(Table 1). The most potent compounds (compounds 1e3) belong to
the same chemical class (3-aminopyrazole) that were previously
described as PDGFR kinase inhibitors in one patent application [37].
Our data now demonstrates that these compounds are selective
inhibitors of the FLT3/PDGFR kinases, since we observed potent
Another potential issue is the poly-pharmacology associated
with the existing FLT3 inhibitors that are responsible for the un-
wanted side effects of the drugs. While some kinase inhibitors are

D. Pauwels et al. / European Journal of Medicinal Chemistry 63 (2013) 713e721
719
fetal bovine serum (Invitrogen, Carlsbad, CA, USA) at 37 ꢀC in a 5%
CO2-incubator. The Ba/F3 cells were cultured in the presence of
mouse IL-3 (1 mg/ml) (Peprotech Inc., Rocky Hill, NJ, USA). Ba/F3
cells transformed by activated kinases do not require IL-3 for their
growth.
5.2. Chemical compounds
The compounds were selected by the Center for Drug Design
and Discovery (CD3). The collection tested consisted of 25,607
small molecules (Mw < 500 g/mol), which were selected based on
(i) chemical diversity, (ii) druglike properties (Lipinski rule of five
compliant) and purchased from multiple commercial suppliers. As
a positive control we used the known FLT3 inhibitor tandutinib
(Selleck Chemicals LLC, Houston, TX, USA) [39].
Fig. 6. Doseeresponse curves of compound 4 for the Ba/F3 cells transformed with
ABL2, EPHA1, EPHA5, EPHA8, ROS1 or SRC. Ba/F3 wild type cells (Ba/F3 WT) were used
as a control. Data represent mean þ/ꢁ SEM.
5.3. Chemical synthesis
inhibition of FLT3 and PDGFR, with no activity against 24 additional
tyrosine kinases (Figs. 5 and 6). Based on both the cellular and the
auto-phosphorylation data, compound 1 was recognized as the
most potent inhibitor, while compound 2 was the most specific
FLT3 inhibitor (Figs. 3 and 4). Compound 4 was found to show some
activity against additional tyrosine kinases, but still the activity
Reagents and anhydrous solvents were purchased from com-
mercial suppliers (Acros, SigmaeAldrich and Alfa Aesar) and used
without further purification. Flash chromatography was performed
with flash chromatography cartridges Biotage SNAP 10 and 25G
either on Biotage Isolera or Biotage SP4 flash chromatography in-
strument. 1H NMR and 13C NMR were recorded at 600 and 151 MHz,
respectively, on a Bruker Avance II-600. Chemical shifts are re-
ported relative to tetramethylsilane at 0.00 ppm. Mass spectral data
were obtained using a Brucker Esquire 6000 (equipped with a
multimode source, ESI/APCI). Mass spectra were acquired in posi-
tive and negative modes. The chemical purity of the target com-
pounds was determined by LC-MS. LC-MS analysis were performed
on a Dionex Ultimate 3000 HPLC system (equipped with a PDA
detector) connected to a mass spectrometer Brucker Esquire 6000
(equipped with a multimode source, ESI/APCI). The purity of each
compound was ꢂ95% in either analysis.
against FLT3 was the only inhibitory activity below 1
mM. It is
interesting to note that this compound also showed good activity
against EPHA5. Compound 5, a benzimidazole-based compound, is
patented for its effect on stem cell differentiation [38], which could
be attributed to its inhibitory effect on FLT3 kinase, a protein
important for hematopoietic stem cells. Furthermore, compounds 1
and 5 retained some inhibitory activity against the D835Y acti-
vating FLT3 mutant and the F691I/G697R resistant mutants, but
none of the other compounds showed an inhibitory effect against
these FLT3 mutants (Fig. 3B).
In conclusion, we identified five FLT3/PDGFR inhibitors from a
library of 25,607 small molecules. All compounds had similar ac-
tivity toward both FLT3 and PDGFRa, although compound 1 was
clearly the most potent one, while compound 2 was the most
specific FLT3 inhibitor. Finally, all five compounds were found to be
selective FLT3/PDGFR inhibitors, as no effect was observed on 24
other tyrosine kinases.
Confirmatory 1H NMR, TOF-MS and LC-MS analyses were per-
formed for compounds 1 to 3. Analytical data for compounds 4 and
5 were obtained from the commercial suppliers Asinex (Moscow,
Russia) and ChemDiv (San Diego, CA, USA) respectively
(Supplementary material).
5.4. Compound treatment
4. Conclusion
Cells (100,000 cells/ml) were seeded in 384-well plates (library
screening) or 96-well plates (doseeresponse experiments) with the
cell dispenser Fluid Xrd-384 (FluidX, Cheshire, UK). Compounds
were added to the wells with the TECAN pipetting robot (TECAN
Schweiz AG, Switzerland). For the library screening we used a final
Even though a large number of FLT3/PDGFR kinase inhibitors
have already been identified, most of these show poor activity
in vivo and the development of resistance has been described.
Therefore, it is important to develop novel FLT3/PDGFR kinase in-
hibitors to overcome these current problems. We performed a cell-
based high throughput screening to identify inhibitors that show
cellular activity against FLT3/PDGFR. The top five compounds were
further characterized and we confirmed their selective activity
against FLT3/PDGFR. These compounds could be considered for
further modification and optimization to obtain more potent and
selective compounds.
concentration of 10
mM compound with dimethyl sulfoxide (DMSO)
as a negative control and 10
m
M 1- -Arabinofuranosylcytosine
b-D
(Ara-C) (Merck KGaA, Darmstadt, Germany) as a positive control.
For doseeresponse experiments we used 8 different concentrations
(50, 100, 200, 400, 800, 1600, 3200 and 6400 nM) each in triplicate
with DMSO as a negative control. The number of viable cells was
counted after a treatment of 72 h (library screening) and 24 h
(doseeresponse experiments) with ATP-lite 1step reagent (Perki-
nElmer, Boston, MA, USA). Luminescence was measured with the
multilabel plate readers Envision and Victor X4 (PerkinElmer,
Boston, MA, USA). EC50 values were calculated with GraphPad
Prism. The specificity of the hit compounds was tested on Ba/F3
cells transformed with ABL2, AXL, EGFR, EPHA1, EPHA2, EPHA5,
EPHA7, EPHA8, ERBB2, FGFR3, FLT4, FYN, HCK, IGFR1, LTK, MATK1,
MERTK, NTRK3, ROS1, SRC, SYK, TNK1, TYRO3 and YES. The cells
5. Experimental
5.1. Cell culture
The human AML cell lines EOL-1, MOLM-13, MV4-11, HEL and
KG-1 (DSMZ, Braunschweig, Germany) and the mouse Ba/F3 wild
type, Ba/F3-FLT3-ITD, Ba/F3-FLT3-D835Y, Ba/F3-FLT3-ITD-F691I
and Ba/F3-FLT3-ITD-G697R (developed in house) were cultured in
RPMI 1640 medium (without phenol red) supplemented with 10%
were incubated for 24 h with 1
M (compounds 4 and 5). Each sample was set up in triplicate.
DMSO was used as a negative control.
mM (compounds 1, 2 and 3) and
5
m

720
D. Pauwels et al. / European Journal of Medicinal Chemistry 63 (2013) 713e721
B. Lowenberg, C.D. Bloomfield, Diagnosis and management of acute myeloid
5.5. Western blotting
leukemia in adults: recommendations from an international expert panel, on
behalf of the European LeukemiaNet, Blood 115 (2010) 453e474.
The cell lines Ba/F3-FLT3-ITD and EOL-1 were treated during
90 min with increasing doses of the drugs. Cells were harvested and
lysed using lysis buffer (Cell signaling, Danvers, MA, USA) supple-
mented with 1 mM NaVO4 and protease inhibitors (Complete,
Roche Applied Science, Penzberg, Germany). Samples were reduced
and gel electrophoresis was performed using NuPage Bis-Tris 4e
12% gels (Invitrogen). Antibodies used were rabbit polyclonal anti-
FLT3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse
monoclonal anti-phosphoFLT3 (Cell Signaling), rabbit polyclonal
[10] R.F. Schlenk, K. Dohner, J. Krauter, S. Frohling, A. Corbacioglu, L. Bullinger,
M. Habdank, D. Spath, M. Morgan, A. Benner, B. Schlegelberger, G. Heil,
A. Ganser, H. Dohner, Mutations and treatment outcome in cytogenetically
normal acute myeloid leukemia, N. Engl. J. Med. 358 (2008) 1909e1918.
[11] O. Rosnet, H.J. Buhring, S. Marchetto, I. Rappold, C. Lavagna, D. Sainty,
C. Arnoulet, C. Chabannon, L. Kanz, C. Hannum, D. Birnbaum, Human FLT3/
FLK2 receptor tyrosine kinase is expressed at the surface of normal and ma-
lignant hematopoietic cells, Leukemia 10 (1996) 238e248.
[12] R.J. Ray, C.J. Paige, C. Furlonger, S.D. Lyman, R. Rottapel, Flt3 ligand supports
the differentiation of early B cell progenitors in the presence of interleukin-11
and interleukin-7, Eur. J. Immunol. 26 (1996) 1504e1510.
[13] L.S. Rusten, S.D. Lyman, O.P. Veiby, S.E. Jacobsen, The FLT3 ligand is a direct
and potent stimulator of the growth of primitive and committed human
CD34þ bone marrow progenitor cells in vitro, Blood 87 (1996) 1317e1325.
[14] M. Nakao, S. Yokota, T. Iwai, H. Kaneko, S. Horiike, K. Kashima, Y. Sonoda,
T. Fujimoto, S. Misawa, Internal tandem duplication of the flt3 gene found in
acute myeloid leukemia, Leukemia 10 (1996) 1911e1918.
anti-PDGFR
phosphoPDGFR
anti-STAT5 (Santa Cruz Biotechnology) and rabbit monoclonal
a
(Santa Cruz Biotechnology), rabbit polyclonal anti-
a
(Santa Cruz Biotechnology), rabbit polyclonal
a
anti-phosphoSTAT5 (Cell Signaling). The LAS-3000 Imaging System
(Fujifilm Global) was used for protein blot analysis.
[15] H. Kiyoi, M. Towatari, S. Yokota, M. Hamaguchi, R. Ohno, H. Saito, T. Naoe,
Internal tandem duplication of the FLT3 gene is a novel modality of elongation
mutation which causes constitutive activation of the product, Leukemia 12
(1998) 1333e1337.
5.6. Statistical analysis
[16] Y. Yamamoto, H. Kiyoi, Y. Nakano, R. Suzuki, Y. Kodera, S. Miyawaki, N. Asou,
K. Kuriyama, F. Yagasaki, C. Shimazaki, H. Akiyama, K. Saito, M. Nishimura,
T. Motoji, K. Shinagawa, A. Takeshita, H. Saito, R. Ueda, R. Ohno, T. Naoe,
Activating mutation of D835 within the activation loop of FLT3 in human
hematologic malignancies, Blood 97 (2001) 2434e2439.
The students t-test was performed to determine significant
differences between two groups. Normality test was applied to test
the normal distribution.
[17] F. Hayakawa, M. Towatari, H. Kiyoi, M. Tanimoto, T. Kitamura, H. Saito,
T. Naoe, Tandem-duplicated Flt3 constitutively activates STAT5 and MAP ki-
nase and introduces autonomous cell growth in IL-3-dependent cell lines,
Oncogene 19 (2000) 624e631.
Acknowledgments
[18] S.M. Kornblau, M. Womble, Y.H. Qiu, C.E. Jackson, W. Chen, M. Konopleva,
E.H. Estey, M. Andreeff, Simultaneous activation of multiple signal trans-
duction pathways confers poor prognosis in acute myelogenous leukemia,
Blood 108 (2006) 2358e2365.
[19] T. Fischer, R.M. Stone, D.J. Deangelo, I. Galinsky, E. Estey, C. Lanza, E. Fox,
G. Ehninger, E.J. Feldman, G.J. Schiller, V.M. Klimek, S.D. Nimer, D.G. Gilliland,
C. Dutreix, A. Huntsman-Labed, J. Virkus, F.J. Giles, Phase IIB trial of oral
Midostaurin (PKC412), the FMS-like tyrosine kinase 3 receptor (FLT3) and
multi-targeted kinase inhibitor, in patients with acute myeloid leukemia and
high-risk myelodysplastic syndrome with either wild-type or mutated FLT3,
J. Clin. Oncol. 28 (2010) 4339e4345.
This work was supported by grants from the KU Leuven
(concerted action grant to J.C.), the FWO-Vlaanderen (G.0287.07,
J.C.) the Foundation against Cancer (SCIE2006-34, J.C.) an ERC-
starting grant (J.C.), the Interuniversity Attraction Poles (IAP)
granted by the Federal Office for Scientific, Technical and Cultural
Affairs, Belgium (J.C.). D.P. is supported by a Ph.D. grant of the
Agency for Innovation by Science and Technology (IWT), Flanders,
Belgium.
[20] S. Knapper, A.K. Burnett, T. Littlewood, W.J. Kell, S. Agrawal, R. Chopra, R. Clark,
M.J. Levis, D. Small, A phase 2 trial of the FLT3 inhibitor lestaurtinib (CEP701) as
first-line treatment for older patients with acute myeloid leukemia not
considered fit for intensive chemotherapy, Blood 108 (2006) 3262e3270.
[21] P.P. Zarrinkar, R.N. Gunawardane, M.D. Cramer, M.F. Gardner, D. Brigham,
B. Belli, M.W. Karaman, K.W. Pratz, G. Pallares, Q. Chao, K.G. Sprankle,
H.K. Patel, M. Levis, R.C. Armstrong, J. James, S.S. Bhagwat, AC220 is a uniquely
potent and selective inhibitor of FLT3 for the treatment of acute myeloid
leukemia (AML), Blood 114 (2009) 2984e2992.
Appendix ASupplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.03.024.
References
[22] J. Cortes, A. Perl, C. Smith, T. Kovacsovics, H. Dombret, H. Döhner, A phase II
open-label, AC220 monotherapy efficacy (ACE) study in patients with Acute
Myeloid Leukemia (AML) with FLT3-ITD activating mutations: interim results,
16th Congress of the European Hematology Association, 2011.
[23] T. Kindler, D.B. Lipka, T. Fischer, FLT3 as a therapeutic target in AML: still
challenging after all these years, Blood 116 (2010) 5089e5102.
[1] B. Deschler, M. Lubbert, Acute myeloid leukemia: epidemiology and etiology,
Cancer 107 (2006) 2099e2107.
[2] T. Buchner, W.E. Berdel, C. Haferlach, T. Haferlach, S. Schnittger, C. Muller-
Tidow, J. Braess, K. Spiekermann, J. Kienast, P. Staib, A. Gruneisen, W. Kern,
A. Reichle, G. Maschmeyer, C. Aul, E. Lengfelder, M.C. Sauerland, A. Heinecke,
B. Wormann, W. Hiddemann, Age-related risk profile and chemotherapy dose
response in acute myeloid leukemia: a study by the German Acute Myeloid
Leukemia Cooperative Group, J. Clin. Oncol. 27 (2009) 61e69.
[3] C.C. Kumar, Genetic abnormalities and challenges in the treatment of acute
myeloid leukemia, Genes Cancer 2 (2011) 95e107.
[4] R.K. Slany, When epigenetics kills: MLL fusion proteins in leukemia, Hematol.
Oncol. 23 (2005) 1e9.
[24] E. Weisberg, M. Sattler, A. Ray, J.D. Griffin, Drug resistance in mutant FLT3-
positive AML, Oncogene 29 (2010) 5120e5134.
[25] C.C. Smith, Q. Wang, C.S. Chin, S. Salerno, L.E. Damon, M.J. Levis, A.E. Perl,
K.J. Travers, S. Wang, J.P. Hunt, P.P. Zarrinkar, E.E. Schadt, A. Kasarskis,
J. Kuriyan, N.P. Shah, Validation of ITD mutations in FLT3 as a therapeutic
target in human acute myeloid leukaemia, Nature 485 (2012) 260e263.
[26] F. Heidel, F.K. Solem, F. Breitenbuecher, D.B. Lipka, S. Kasper, M.H. Thiede,
C. Brandts, H. Serve, J. Roesel, F. Giles, E. Feldman, G. Ehninger, G.J. Schiller,
S. Nimer, R.M. Stone, Y. Wang, T. Kindler, P.S. Cohen, C. Huber, T. Fischer, Clinical
resistance to the kinase inhibitor PKC412 in acute myeloid leukemia by muta-
tion of Asn-676 in the FLT3 tyrosine kinase domain, Blood 107 (2006) 293e300.
[27] J. Cools, E.H. Stover, C.L. Boulton, J. Gotlib, R.D. Legare, S.M. Amaral, D.P. Curley,
N. Duclos, R. Rowan, J.L. Kutok, B.H. Lee, I.R. Williams, S.E. Coutre, R.M. Stone,
D.J. DeAngelo, P. Marynen, P.W. Manley, T. Meyer, D. Fabbro, D. Neuberg,
E. Weisberg, J.D. Griffin, D.G. Gilliland, PKC412 overcomes resistance to ima-
tinib in a murine model of FIP1L1-PDGFRalpha-induced myeloproliferative
disease, Cancer Cell 3 (2003) 459e469.
[5] D.L. Stirewalt, J.P. Radich, The role of FLT3 in haematopoietic malignancies,
Nat. Rev. Cancer 3 (2003) 650e665.
[6] J.P. Patel, M. Gonen, M.E. Figueroa, H. Fernandez, Z. Sun, J. Racevskis, P. Van
Vlierberghe, I. Dolgalev, S. Thomas, O. Aminova, K. Huberman, J. Cheng,
A. Viale, N.D. Socci, A. Heguy, A. Cherry, G. Vance, R.R. Higgins, R.P. Ketterling,
R.E. Gallagher, M. Litzow, M.R. van den Brink, H.M. Lazarus, J.M. Rowe,
S. Luger, A. Ferrando, E. Paietta, M.S. Tallman, A. Melnick, O. Abdel-Wahab,
R.L. Levine, Prognostic relevance of integrated genetic profiling in acute
myeloid leukemia, N. Engl. J. Med. 366 (2012) 1079e1089.
[7] D.L. Stirewalt, K.J. Kopecky, S. Meshinchi, F.R. Appelbaum, M.L. Slovak,
C.L. Willman, J.P. Radich, FLT3, RAS, and TP53 mutations in elderly patients
with acute myeloid leukemia, Blood 97 (2001) 3589e3595.
[28] K. De Keersmaecker, J. Cools, Chronic myeloproliferative disorders: a tyrosine
kinase tale, Leukemia 20 (2006) 200e205.
[8] S. Meshinchi, W.G. Woods, D.L. Stirewalt, D.A. Sweetser, J.D. Buckley, T.K. Tjoa,
I.D. Bernstein, J.P. Radich, Prevalence and prognostic significance of Flt3 in-
ternal tandem duplication in pediatric acute myeloid leukemia, Blood 97
(2001) 89e94.
[9] H. Dohner, E.H. Estey, S. Amadori, F.R. Appelbaum, T. Buchner, A.K. Burnett,
H. Dombret, P. Fenaux, D. Grimwade, R.A. Larson, F. Lo-Coco, T. Naoe,
D. Niederwieser, G.J. Ossenkoppele, M.A. Sanz, J. Sierra, M.S. Tallman,
[29] J. Cools, D.J. DeAngelo, J. Gotlib, E.H. Stover, R.D. Legare, J. Cortes, J. Kutok,
J. Clark, I. Galinsky, J.D. Griffin, N.C. Cross, A. Tefferi, J. Malone, R. Alam,
S.L. Schrier, J. Schmid, M. Rose, P. Vandenberghe, G. Verhoef, M. Boogaerts,
I. Wlodarska, H. Kantarjian, P. Marynen, S.E. Coutre, R. Stone, D.G. Gilliland,
A tyrosine kinase created by fusion of the PDGFRA and FIP1L1 genes as a
therapeutic target of imatinib in idiopathic hypereosinophilic syndrome,
N. Engl. J. Med. 348 (2003) 1201e1214.

D. Pauwels et al. / European Journal of Medicinal Chemistry 63 (2013) 713e721
721
[30] T.R. Golub, G.F. Barker, M. Lovett, D.G. Gilliland, Fusion of PDGF receptor beta
to a novel ets-like gene, tel, in chronic myelomonocytic leukemia with t(5;12)
chromosomal translocation, Cell 77 (1994) 307e316.
[34] J. Cools, H. Quentmeier, B.J. Huntly, P. Marynen, J.D. Griffin, H.G. Drexler,
D.G. Gilliland, The EOL-1 cell line as an in vitro model for the study of FIP1L1-
PDGFRA-positive chronic eosinophilic leukemia, Blood 103 (2004) 2802e2805.
[35] W. Hiddemann, Cytosine arabinoside in the treatment of acute myeloid leu-
kemia: the role and place of high-dose regimens, Ann. Hematol. 62 (1991)
119e128.
[36] M. Warmuth, S. Kim, X.J. Gu, G. Xia, F. Adrian, Ba/F3 cells and their use in
kinase drug discovery, Curr. Opin. Oncol. 19 (2007) 55e60.
[37] N. Giese, N. Lokker, A. Laibelman, R. Scarborough, in: Organization WIP (Ed.),
Pharmaceutical Pyrazole Compositions Useful as Inhibitors of Protein Kinases,
World Intellectual Property Organization, 1996.
[31] J.F. Apperley, M. Gardembas, J.V. Melo, R. Russell-Jones, B.J. Bain, E.J. Baxter,
A. Chase, J.M. Chessells, M. Colombat, C.E. Dearden, S. Dimitrijevic, F.X. Mahon,
D. Marin, Z. Nikolova, E. Olavarria, S. Silberman, B. Schultheis, N.C. Cross,
J.M. Goldman, Response to imatinib mesylate in patients with chronic
myeloproliferative diseases with rearrangements of the platelet-derived
growth factor receptor beta, N. Engl. J. Med. 347 (2002) 481e487.
[32] E. Lierman, I. Lahortiga, H. Van Miegroet, N. Mentens, P. Marynen, J. Cools, The
ability of sorafenib to inhibit oncogenic PDGFRbeta and FLT3 mutants and
overcome resistance to other small molecule inhibitors, Haematologica 92
(2007) 27e34.
[38] M. Mercola, M. Dawson, J. Cashman, in: Organization WIP (Ed.), Small Mole-
cule Compounds for Stem Cell Differentiation, 2010.
[33] S. Yokota, H. Kiyoi, M. Nakao, T. Iwai, S. Misawa, T. Okuda, Y. Sonoda, T. Abe,
K. Kahsima, Y. Matsuo, T. Naoe, Internal tandem duplication of the FLT3 gene
is preferentially seen in acute myeloid leukemia and myelodysplastic syn-
drome among various hematological malignancies. A study on a large series of
patients and cell lines, Leukemia 11 (1997) 1605e1609.
[39] L.M. Kelly, J.C. Yu, C.L. Boulton, M. Apatira, J. Li, C.M. Sullivan, I. Williams,
S.M. Amaral, D.P. Curley, N. Duclos, D. Neuberg, R.M. Scarborough, A. Pandey,
S. Hollenbach, K. Abe, N.A. Lokker, D.G. Gilliland, N.A. Giese, CT53518, a novel
selective FLT3 antagonist for the treatment of acute myelogenous leukemia
(AML), Cancer Cell 1 (2002) 421e432.