Identification of New FLT3 Inhibitors That Potently Inhibit AML Cell Lines via an Azo Click-It/Staple-It Approach

Article abstract of DOI:10.1021/acsmedchemlett.6b00468

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.

Full text of DOI:10.1021/acsmedchemlett.6b00468

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Letter
Identification of new FLT3 inhibitors that potently inhibit
AML cell lines, via an azo click-it/staple-it approach
Jie Zhou, Xiaochu Ma, Changhao Wang, Brandon Carter-Cooper, Elizabeth Larocque, Jonathan
Fine, Genichiro Tsuji, Fan Yang, Gaurav Chopra, Rena G. Lapidus, and Herman O. Sintim
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00468 • Publication Date (Web): 14 Apr 2017
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Identification of new FLT3 inhibitors that potently inhibit AML
cell lines, via an azo click-it/staple-it approach
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Xiaochu Ma†,a,b,d, Jie Zhou†,a,d, Changhao Wang†b, Brandon Carter-Cooperc, Fan Yangb, Elizabeth
Larocquea,d, Jonathan Finea,d, Genichiro Tsuji a,d, Gaurav Chopraa,d, Rena G. Lapidusc and Herman O.
Sintima, b, d, e*
aInstitute for Drug Discovery, dDepartment of Chemistry, ePurdue University Center for Cancer Research, Purdue University,
West Lafayette, IN 47907, USA.
bDepartment of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA.
cTranslational Core Laboratory, University of Maryland Greenebaum Cancer Center, 655 W. Baltimore Street, Baltimore,
MD 21201, USA.
KEYWORDS. FMS-like tyrosine kinase 3 (FLT3), Acute Myeloid Leukemia (AML), inhibitor, azo, click-it/staple-it
ABSTRACT: 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 con-
centrations.
Cancer continues to claim the lives of millions of patients
annually, despite the great advances that have been made in
cancer therapeutics in the last few decades. Whereas the five
year survival rates of cancer patients who are diagnosed with
thyroid, prostate, skin and hormone responsive breast are now
over 90%, those with pancreatic, liver, lung and esophageal
have less than 20% chance of surviving in five years.1 Even
for certain types of cancers such as breast, which generally has
a good prognosis, there are still certain subtypes that record
low five years survival rates. For example the five-year sur-
vival rate for hormone responsive ER+ breast cancer is over
90%1 but for inflammatory breast cancer is at a miserly 44%2
and for metastatic triple negative breast cancer the median
survival is less than two years.3 Another example is leukaemia,
which has a general five-year survival rate of 59%1 but acute
myeloid leukaemia (AML) register a disappointing five-year
survival rate of 26%.4 Clearly there is a need for new chemo-
types that kill cancers that are associated with poor survival
rates.
tients (~30%).6 The FLT3 protein is a member of the class III
receptor-tyrosine kinase (RTK) family.7 Upon binding of
FLT3 ligand, FLT3 receptor dimerizes, resulting in phosphor-
ylation, followed by activation of downstream signaling cas-
cades, including the RAS/MEK, PI3K/AKT/mTOR, and
STAT5 pathways, which in turn regulate cell cycle progres-
sion, apoptosis and other important cellular processes.8
We have focused on finding new chemical entities that in-
hibit the proliferation of AML because there has not been a
breakthrough therapeutic against AML for over two decades
and cytarabine (araC), which is commonly used for treating
AML patients is the most effective when used in combination
with other drugs. Resistance to araC has also been well docu-
mented and 30-50% of patients relapse upon treatment with
araC alone or in combination with other drugs.5 Arguably
there is a need for new chemical entities that are effective
against AML. Recently there has been interest in targeting
FMS-like tyrosine kinase 3 (FLT3), a tyrosine receptor kinase
that is commonly mutated (activating mutations) in AML pa-
Figure 1: “Click it and staple it” strategy for compound library con-
struction and hit optimization. X=CH or N, Y and R1 are substituents. See
Figure S1 for details.
Mutations in FLT3 lead to constitutive autophosphorylation
of FLT3 and thus uncontrolled activation of downstream sig-
naling.9 Examples of FLT3 inhibitors that have been devel-
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oped10-13 and are in clinical trials for treating AML are soraf-
and the compounds were screened against MV4-11 (SI, Table
S1).
To confirm that compounds that were active against MV4-
enib, quizartinib, crenolanib and midostaurin.14, 15, 16 Unfortu-
nately many of the tested FLT3 inhibitors have experienced
limited clinical efficacy, especially on patients who harbor
FLT3 D835Y/V/I/F mutations (these mutations are particular-
ly resistant to type II inhibitors, which target the inactive state
of the active ATP binding site)17. Therefore, despite the avail-
ability of several FLT3 inhibitors, there is still a need for new-
generation kinase inhibitors that could lead to a durable dis-
ease-free survival.
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11 and Molm-14 were also FLT3 inhibitors, we screened (us-
ing Reaction Biology service) the compounds against FLT3 at
concentration of 500 nM, in the presence of 100 µM radio-
labeled ATP (SI, Figure S2). Compounds containing benzami-
dines and isoquinolin-3-amine moieties had superior FLT3
inhibition profile than those containing naphthalen-2-amine or
quinolin-7-amine or quinolin-6-amine or isoquinolin-6-amine
or quinolin-8-amine groups (SI, Figure S2), suggesting that the
position of the ring nitrogen on the isoquinoline or quinoline
and also the substitution pattern of the benzene ring (denoted
as Y in Figure 1) were critical for FLT3 inhibition. The best
azo inhibitor, HSW630-1 (see Figure 2) inhibited FLT3 and
FLT3 ITD (internal tandem duplications mutation) with IC50
values of 18.6 nM and 8.77 nM respectively (SI, Figure S2C).
As stated earlier, several FLT3 point mutations, such as
D835Y, confer resistance to FLT3 inhibitors (especially type
II inhibitors). Interestingly, HSW630-1 could inhibit FLT3
D835Y, 21 with IC50 of 28 nM (SI, Figure S2C). Docking of
HSW630-1 against FLT3, using the CANDOCK program,
suggested that HSW630-1 binds to a site near the ATP binding
site (see SI, Figure S3). Other FLT3 inhibitors that are current-
ly in clinical trials, such as quizartinib and crenalonib also
inhibit c-Kit and TrKC in addition to FLT3 inhibition, see SI,
Table S2. Analogously, HSW630-1 could also inhibit TrKC
(IC50 = 5.9 nM) and c-Kit (IC50 = 6.9 nM). The effect of
HSW630-1 on FLT3-mediated signaling in leukemic cells was
investigated using Western blot analysis (Figure 3). In agree-
ment with the in vitro FLT3 inhibition data, HSW630-1 could
inhibit FLT3 phosphorylation in a dose-dependent manner
(see Figure 3).
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Figure 2. Replacement of azo moiety in hit compound with alkyne and
alkene units.
Figure 3. Western Blot analysis of phospho-FLT3 and total FLT3 pro-
tein expression in MV4-11 after treatment with HSW630-1. MV4-11 cells
were treated with DMSO vehicle (V) and HSW630-1. Scanned images
were analyzed using image J software.
We have developed a simple strategy to identify and devel-
op FLT3 kinase inhibitors by first making compound libraries,
using the facile azo coupling (which is tolerant of many func-
tional groups and operationally simple) and testing the “azo”
library against MV4-11 or Molm-14 cell lines, which are driv-
en by FLT3 ITD mutation (Figure 1). Compounds that inhibit
these FLT3-driven cell lines but not non-FLT3 driven cell
lines, such as THP-1 are likely FLT3 inhibitors (Figure 1). We
were aware that azo moieties are generally not stable in bio-
logical environments18 but the successful development of the
drug eltrombopag19 from an azo hit encouraged us to pursue
the “azo library” strategy, with an eye towards replacing the
azo group with a more stable moiety after a hit molecule had
been found (see Figures 1 and 2). We call this approach “Azo-
Click-it/Staple-it” drug discovery strategy. The “Azo-Click-it”
term denotes the fact that azo coupling is facile and falls with-
in Sharpless’ definition of a Click reaction20 and the “Staple-it”
term reflects the fact that for practical application one has to
change the “labile” azo group with a more stable unit. A first-
generation library of potential kinase inhibitors was therefore
made by conjugating various aromatic diazonium compounds
(generated from the aromatic amines) with quinolines and
isoquinolines (privileged pharmacophores), see SI, Figure SI,
Figure 4. Anti-proliferative effects of HSW630-1 against leukemia and
normal lung (MRC-5A) cell lines.
Having established that HSW630-1 and analogs are FLT3
inhibitors (Figure 3 and SI, Figure S2) and inhibit leukemia
cell line MV4-11 (FLT3-driven), Figure 4 and SI Table S1, we
also tested the HSW630-1 against MOLM-14 (also FLT3-
driven), K-562 (chronic myelogenous leukemia, CML, which
is not driven by FLT3) and THP1 (non-FLT3-driven), using
Alamar Blue assay (see Figure 4). The effect of HSW630-1
was also evaluated against MRC-5A (lung), which is a control
cell line that is typically used to evaluate the effect of a cyto-
toxic agent against normal cells. Excitingly, and in agreement
with the FLT3 inhibition profile, HSW630-1 showed potent
inhibition of FLT3-driven MV4-11 and MOLM-14 cancer
cells (IC50 ~150 nM), but not THP1 (IC50 > 1000 nM) or K-
562 (IC50 = 3000 nM). Compounds, which did not potently
inhibit FLT3 also did not inhibit the proliferation of MV4-11
cells (FLT3-driven), see Figure S2 and Table S1 for details.
Since HSW630-1 (a FLT-3 inhibitor) could slow down the
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proliferation of MV4-11 and MOLM-14 but not K-562 or part, are ineffective anti-proliferative agents in AML cell lines.
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THP1 (both are not FLT3- driven), it is likely that HSW630-1
and analogs thereof that also inhibit FLT3 inhibit MV4-11 and
MOLM-14 proliferation via FLT3 inhibition.
The amidine group also appears to be important for anti-
proliferative activity. The cyano analog HSM1611 is ineffec-
tive against MV4-11 and MOLM-14 whereas the hydroxyam-
idine analog (HSM1819), which is derived from HSM1611,
potently inhibits the proliferation of FLT3-driven AML cell
lines. The hydroxyamidine HSM1819 inhibits FLT3 and FLT3
ITD with IC50 of 217 nM and 240 nM respectively in vitro.
HSM1820, another hydroxyamidine analog inhibits FLT3 and
FLT3 ITD with IC50 of 359 nM and 350 nM respectively in
vitro.
Having achieved our goal of identifying an azo compound
(HSW630-1) that could potently inhibit FLT3 kinase and also
inhibit AML proliferation, we proceeded to identify linkers
that could replace the potentially problematic azo moiety in
HSW630-1.
9
We designed few analogs, which we thought could be readi-
ly synthesized, where the azo moiety was replaced with a
more “stable” group (alkene, alkyne, ether and amines) and
used computational methods (Gausian22 at B3lYP/6-31+G(d)
level of theory) to identify the most stable conformers of these
analogs. From these calculations, it appeared that the alkene
and alkyne moieties were better mimics of the azo unit than
the rest, see SI, Figure S4 for discussion. Alkyne and alkene
analogs of HSW630-1 were readily prepared via Sonogashira
or Suzuki coupling of iodo arenes with alkynes or alkene
boronates, catalysed by Pd(PPh3)2Cl2 (Sonogashira) or
Pd(PPh3)4 (Suzuki), see SI, Scheme 1. In our hands it was
easier to make the Sonogashira products than the Suzuki prod-
ucts therefore the majority of the stable analogs that were syn-
thesized contained the alkyne unit (see Table 1). The non-azo
compounds were also FLT3 inhibitors (see Table 1 for per-
centage enzymatic inhibition at 0.5 M compounds), albeit not
as potent enzyme inhibitors as the azo compounds. Detailed
characterization of one of the non-azo analogs, HSM1651,
revealed that it inhibited FLT3, FLT3 ITD and FLT3 D835Y
with IC50 values of 40 nM, 100 nM and 56 nM respectively
(see Figure 5A).
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Table 1. FLT3 inhibition profile and anti-proliferative activ-
ities of alkyne analogs of HSW630-1 against different leuke-
mia cancer cell lines.
Entry
Structure
% Inhibi-
tion of
kinases
FLT3 at
500 nM
Anti-
proliferative
effects in
leukemia
panel (µM)
MV4-11a
MOLM14b
THP-1c
K-562d
HL60e
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0.15 ± 0.01a
0.15 ± 0.01b
1.20 ± 0.20c
3.00 ± 0.96d
As was in the HSW630-1 case, the “stable” analogs also in-
hibited c-Kit and TrkC enzymes, see SI Table S3. Encourag-
ingly, the “stable” HSW630-1 analogs could also potently
inhibit AML cell lines (see Table 1). Western analysis of
MV4-11, treated with HSM1651, revealed that FLT3 phos-
phorylation as well as the downstream STAT5 phosphoryla-
tion23, 24 were reduced in the presence of HSM1651 (Figure 5
B and C) and so it appears that the stable analogs act analo-
gously to the azo lead compound, HSW630-1. A few of the
alkyne analogs have respectable anti-proliferative properties
(HSM1819 has an IC50 value of 20 nM, which is only four
times less potent than crenolanib (IC50 = 5 nM), which pro-
ceeded to clinical trials). Regarding structure-activity-
relationship studies of the alkyne/alkene analogs, it appears
that modifications to the isoquinoline ring greatly affected
both kinase inhibition and anti-proliferative activity, which is
similar to what was observed in the azo series (see SI, Figures
S1, S2 and Table S1). In the azo series, it was observed that
substitution of the isoquinoline 6-position with halogen or
alkyl groups was beneficial for both kinase inhibition and in-
hibition of MV4-11 proliferation. For example HSW 1107-2
(Cl at position 6 of isoquinoline) inhibited proliferation of
MV4-11 with IC50 of 60 nM whereas HSW308-4 (H at posi-
tion 6 of isoquinoline), inhibited the proliferation of MV4-11
with IC50 of 900 nM (see SI, Figure S1 and Table S1). For the
alkyne series, HSM1813 (H at 3-position and also lacking Cl
at 6-position) was not a potent inhibitor of MV4-11 prolifera-
tion (see Table 1, entry 10). HSM1651 (amino at 3-position
and Cl at 6-position) is a good inhibitor of MV4-11 and
MOLM-14 proliferation whereas compounds HSM1781, 1798
and 1796, which differed from HSM1651 at the isoquinoline
0.05 ± 0.01a
0.11 ± 0.02b
1.85 ± 0.92c
1.39 ± 0.01d
3.05 ± 0.07e
3
4
43
51
0.30 ± 0.20a
0.10 ±
0.02b
0.08 ± 0.02a
0.30 ± 0.08b
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0.005 ± 0.001a
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0.07 ± 0.02a
0.30 ± 0.01b
15
99
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0.004 ±
0.001b
a-e: these correlate the cell lines tested to respective IC50
1
> 5a
> 5b
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Figure 5: A) Dose-response curves of HSW1651 against FLT3 and im-
portant FLT3 mutants. B & C) Western Blot analyses of p-FLT3/total
FLT3 (B) and p-STAT5/STAT5 (C) protein expression in MV4-11 after
treatment with HSM1651 and DMSO vehicle (V) control. Scanned images
were analyzed using image J software.
The azo unit is not strictly forbidden in a drug and an exam-
ple of an azo containing drug is phenazopyridine, which is an
analgesic extensively used to alleviate pain or irritation associ-
ated with the urinary tract.25 Drugs containing azo moiety can
however cause hemolysis in patients with glucose-6-phosphate
dehydrogenase (G-6-PD) deficiency and this patient group is
excluded from taking azo-containing drugs.26 Therefore it is a
good measure to replace the azo unit in drug candidates when
possible. Azo-containing compounds are easy to synthesize so
the “Azo-Click-It/Staple-It” approach is a powerful (yet an
underutilized strategy in drug discovery and optimization)
means to explore chemical space quickly and then convert
“hits” into drug-like molecules. Future work aims to determine
the ADME and in-vivo efficacy properties of our new FLT3
inhibitors. These ongoing studies will be reported in a full
paper in future.
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0.02 ± 0.01a
0.06 ± 0.01b
0.35±1.17 a
14
98
0.001 ± 0.001a
0.001 ±
0.001b
ASSOCIATED CONTENT
Supporting Information
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Detailed synthesis strategies and chemical characterization as well
pteridin-7(8H)-one-based inhibitors targeting FMS-like tyrosine
1
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as biological assays are supplied as Supporting Information
should be included.
kinase 3 (FLT3) and its mutants. J. Med. Chem. 2016, 59, 6187–
6200.
11.
Wander, S. A.; Levis, M. J.; Fathi, A. T. The evolving
The Supporting Information is available free of charge on the
ACS Publications website.
role of FLT3 inhibitors in acute myeloid leukemia: quizartinib and
beyond. Ther. Adv. Hematol. 2014, 5, 65-77.
12.
Warkentin, A. A.; Lopez, M. S.; Lasater, E. A.; Lin, K.;
He, B. L.; Leung, A. Y.; Smith, C. C.; Shah, N. P.; Shokat, K. M.
Overcoming myelosuppression due to synthetic lethal toxicity for
FLT3-targeted acute myeloid leukemia therapy. Elife 2014,
3:e03445.
AUTHOR INFORMATION
Corresponding Author
*Corresponding author email: hsintim@purdue.edu
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13.
Park, S.; Chapuis, N.; Bardet, V.; Tamburini, J.; Gallay,
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Author Contributions
N.; Willems, L.; Knight, Z. A.; Shokat, K. M.; Azar, N.; Viguié,
F.; Ifrah, N.; Dreyfus, F.; Mayeux, P.; Lacombe, C.; Bouscary, D.
PI-103, a dual inhibitor of class IA phosphatidylinositide 3-kinase
and mTOR, has antileukemic activity in AML. Leukemia 2008,
22, 1698-1706.
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manu-
script. †These authors contributed equally.
ACKNOWLEDGMENT
14.
Grunwald, M. R.; Levis, M. J. FLT3 tyrosine kinase
inhibition as a paradigm for targeted drug development in acute
myeloid leukemia. Semin. Hematol. 2015, 52, 193-199.
We thank Purdue University for financial support. NMR and MS
data were acquired by the NMR and MS facilities supported by
NIH P30 CA023168. XCM thanks China Scholarship Council
(No.201406140119) for financial support.
15.
Kadia, T. M.; Ravandi, F.; Cortes, J.; Kantarjian, H.
New drugs in acute myeloid leukemia. Ann. Oncol. 2016, 27, 770-
778.
16.
Levis, M. FLT3 mutations in acute myeloid leukemia:
ABBREVIATIONS
what is the best approach in 2013? Hematology Am. Soc.
Hematol. Educ. Program 2013, 2013, 220-226.
ITD, internal tandem duplication; PI3K, phosphatidylinositide 3-
kinases; mTOR, mechanistic or mammalian target of rapamycin;
STAT5, Signal transducer and activator of transcription 5; DMSO,
dimethyl sulfoxide; AKT, v-akt murine thymoma viral oncogene
homolog 1; TrkC, Tropomyosin receptor kinase C; FMS, feline
McDonough sarcoma viral oncogene homolog; c-Kit, CD117;
RAS, guanine-nucleotide binding protein; MEK, mitogen-
activated protein kinase; ADME, absorption, distribution, metabo-
lism, and excretion.
17.
Smith, C. C.; Lin, K.; Stecula, A.; Sali, A.; Shah, N. P.
FLT3 D835 mutations confer differential resistance to type II
FLT3 inhibitors. Leukemia 2015, 29, 2390-2392.
18.
Ueda, T.; Yamaoka, T.; Miyamoto, M.; Kim, S.-l.
Bacterial reduction of azo compounds as a model reaction for the
degradation of azo-containing polyurethane by the action of
intestinal flora. Bull. Chem. Soc. Jpn. 1996, 69, 1139-1142.
19.
Weisberg, E.; Sattler, M.; Ray, A.; Griffin, J. D. Drug
resistance in mutant FLT3-positive AML. Oncogene 2010, 29,
5120-5134.
REFERENCES
1.
American Cancer Society. Cancer Facts & Figures
20.
Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.;
2014. http://www.cancer.org/acs/groups/content/@research/docu
ments/webcontent/acspc-042151.pdf.
Sharpless, K. B. A stepwise huisgen cycloaddition process:
copper(I)-catalyzed regioselective "ligation" of azides and
terminal alkynes. Angew. Chem. Int. Ed. Engl. 2002, 41, 2596-
2599.
2.
Dawood, S.; Merajver, S. D.; Viens, P.; Vermeulen, P.
B.; Swain, S. M.; Buchholz, T. A.; Dirix, L. Y.; Levine, P. H.;
Lucci, A.; Krishnamurthy, S.; Robertson, F. M.; Woodward, W.
A.; Yang, W. T.; Ueno, N. T.; Cristofanilli, M. International
expert panel on inflammatory breast cancer: consensus statement
for standardized diagnosis and treatment. Ann. Oncol. 2011, 22,
515-523.
21.
Smith, C. C. Disease diversity and FLT3 mutations.
Proc. Natl. Acad. Sci. U S A 2013, 110, 20860-20861.
22. Frisch, M. J. et al. Gaussian 09, Revision D.01; Gaussian,
Inc.: Wallingford, CT, 2013.
23.
Benekli, M.; Baer, M. R.; Baumann, H.; Wetzler, M.
3.
Ovcaricek, T.; Frkovic, S. G.; Matos, E.; Mozina, B.;
Signal transducer and activator of transcription proteins in
leukemias. Blood 2003, 101, 2940-2954.
Borstnar, S. Triple negative breast cancer - prognostic factors and
survival. Radiol. Oncol. 2011, 45, 46-52.
24.
Choudhary, C.; Brandts, C.; Schwable, J.; Tickenbrock,
4.
http://www.cancer.net/cancer-types/leukemia-acute-
L.; Sargin, B.; Ueker, A.; Böhmer, F. D.; Berdel, W. E.; Müller-
Tidow, C.; Serve, H. Activation mechanisms of STAT5 by onco-
genic Flt3-ITD. Blood 2007, 110, 370-374.
25. Deepalatha, C.; Deshpande, N. A comparative study of phena-
zopyridine (pyridium) and cystone as short-term analgesic in un-
complicated urinary tract infection. Int. J. Pharm. Pharm. Sci.
2011, 3, 224-226.
myeloid-aml/statistics,
5.
Cai, J.; Damaraju, V. L.; Groulx, N.; Mowles, D.; Peng,
Y.; Robins, M. J.; Cass, C. E.; Gros, P. Two distinct molecular
mechanisms underlying cytarabine resistance in human leukemic
cells. Cancer Res. 2008, 68, 2349-2357.
6.
Levis, M.; Small, D. FLT3: It does matter in leukemia.
Leukemia 2003, 17, 1738-1752.
26.
Mercieca, J.E.; Clarke, M.F.; Phillips, M.E.; Curtis, J.R.
7.
Matthews, W.; Jordan, C. T.; Wiegand, G. W.; Pardoll,
Acute hemolytic anaemia due to phenazopyridine hydrochloride
in G-6-PD deficient subject. Lancet 1982, 2, 564.
D.; Lemischka, I. R. A receptor tyrosine kinase specific to
hematopoietic stem and progenitor cell-enriched populations. Cell
1991, 65, 1143-1152.
8.
Expert Opin. Ther. Targets 2015, 19, 37-54.
9. Small, D. FLT3 mutations: biology and treatment.
Hematology Am. Soc. Hematol. Educ. Program 2006, 178-184.
10. Sun, D.; Yang, Y.; Lyu, J.; Zhou, W.; Song, W.; Zhao,
Z.; Chen, Z.; Xu, Y.; Li, H. Discovery and rational design of
Konig, H.; Levis, M. Targeting FLT3 to treat leukemia.
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Lay summary: Acute myeloid leukemia is an aggressive cancer with low survival rate. New compounds that inhibit the prolifera-
tion of acute myeloid leukemia cell lines have been developed.
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