Computer-guided design, synthesis, and biological evaluation of quinoxalinebisarylureas as FLT3 inhibitors

Article abstract of DOI:10.1002/cmdc.201402477

Activating mutations of FMS-like tyrosine kinase 3 (FLT3) are present in ～30% of patients with acute myeloid leukemia (AML) and are associated with poor prognosis. Point mutations in the tyrosine kinase domain (TKD) are observed as primary mutations or are acquired as secondary mutations in FLT3 with internal tandem duplications (ITDs) after treatment with tyrosine kinase inhibitors (TKIs). Although dozens of potent inhibitors against FLT3 ITD have been reported, activating TKD point mutations, especially at residues F691 and D835, remain the leading cause for therapy resistance, highlighting the consistent need for new potent inhibitors. Herein we report the identification and characterization of novel quinoxaline-based FLT3 inhibitors. We used the pharmacophore features of diverse known inhibitors as a starting point for a new optimization algorithm for type II TKIs, starting from an in silico library pharmacophore search and induced-fit docking in the known FLT3 structure. This led to the design of a set of diverse quinoxaline-bisarylureas, which were profiled in an FLT3 kinase activity assay. The most promising compounds were further evaluated in a zebrafish embryo phenotype assay.

Full text of DOI:10.1002/cmdc.201402477

DOI: 10.1002/cmdc.201402477
Full Papers
Computer-Guided Design, Synthesis, and Biological
Evaluation of Quinoxalinebisarylureas as FLT3 Inhibitors
Stefan Gçring, Dennis Bensinger, Eva C. Naumann, and Boris Schmidt*[a]
Activating mutations of FMS-like tyrosine kinase 3 (FLT3) are
present in ~30% of patients with acute myeloid leukemia
(AML) and are associated with poor prognosis. Point mutations
in the tyrosine kinase domain (TKD) are observed as primary
mutations or are acquired as secondary mutations in FLT3 with
internal tandem duplications (ITDs) after treatment with tyro-
sine kinase inhibitors (TKIs). Although dozens of potent inhibi-
tors against FLT3 ITD have been reported, activating TKD point
mutations, especially at residues F691 and D835, remain the
leading cause for therapy resistance, highlighting the consis-
tent need for new potent inhibitors. Herein we report the iden-
tification and characterization of novel quinoxaline-based FLT3
inhibitors. We used the pharmacophore features of diverse
known inhibitors as a starting point for a new optimization al-
gorithm for type II TKIs, starting from an in silico library phar-
macophore search and induced-fit docking in the known FLT3
structure. This led to the design of a set of diverse quinoxaline-
bisarylureas, which were profiled in an FLT3 kinase activity
assay. The most promising compounds were further evaluated
in a zebrafish embryo phenotype assay.
Introduction
Acute myeloid leukemia (AML) is a malignant disorder of the
hematopoietic system characterized by uncontrolled prolifera-
tion of cells inside the bone marrow.[1] The five-year survival
rate for patients under 60 years old is only 40%.[2] FMS-like ty-
rosine kinase 3 (FLT3) is a class III receptor tyrosine kinase in-
cluding FMS, c-KIT and two genes encoding platelet-derived
growth factor receptors a and b.[3] It is the most common mu-
tated gene in AML and has been demonstrated to play a crucial
role in the development and growth of hematopoietic and
non-hematopoietic cells.[4] FLT3 is composed of an N-terminal
extracellular ligand binding domain with five immunoglobulin-
like motifs, a transmembrane domain followed by an intracellu-
lar juxtamembrane domain, and a C-terminal tyrosine kinase
domain.[5] The native ligand for FLT3 (FL) was cloned in 1993.[6]
It is a type I transmembrane protein and is expressed in most
tissues including hematopoietic organs (spleen and bone
marrow), prostate, ovary, kidney, lung, heart, and placenta.[7]
After binding of FL, the membrane-bound FLT3 undergoes
a rapid conformational change including homodimerization
and exposure of phosphoryl acceptor sites in the tyrosine
kinase domain. This activates multiple downstream signaling
pathways.[7a,8]
Point mutations at residues D835 or I836 in the activation loop
of FLT3 are less frequent and were found in ~7% of AML
cases.[2,7b,10] It was confirmed that both types of mutations con-
stitutively activate the tyrosine kinase activity.[11]
Smith et al. first described the appearance of drug-resistant
kinase mutations in FLT3 ITD-positive AML patients relapsing
after treatment with quizartinib (AC220).[12] They found muta-
tions at the gatekeeper (F691L) and within the activation loop
(D835Y, D835V, Y842C, Y842H) conferring high degrees of in
vitro quizartinib resistance.
Because of the high frequency of drug-resistant AML, there
is an unmet demand for potent and selective FLT3 inhibi-
tors.[5,7b,8,13] Quinoxalines AG1295 and AG1296 were the first
inhibitors developed for FLT3, exhibiting an I+C50 value of
~1 mm (Figure 1).[14] Consecutively, several small-molecule FLT3
inhibitors, including quizartinib (AC220), sorafenib (BAY-43-
9006), sunitinib (SU-11248), and midostaurin (PKC412), have
been discovered; these were evaluated in vitro and in vivo and
have entered late-stage clinical trials (Figure 1).[8,14b,15] In partic-
ular, the bisarylurea AC220 was identified and evaluated as
a uniquely potent and selective FLT3 inhibitor by Chao et al. in
2009.[16] Despite considerable research efforts in academic and
industrial groups, no FLT3-targeting drug has yet been ap-
proved for the treatment of FLT3-mutant AML.
Two classes of activating FLT3 mutations have been identi-
fied and are present in ~30% of all AML cases. FLT3 internal
tandem duplications (ITDs) occur in the juxtamembrane
domain and were identified in ~23% of all AML patients.[9]
Optimization strategy
[a] S. Gçring,+ D. Bensinger,+ Dr. E. C. Naumann, Prof. Dr. B. Schmidt
Clemens Schçpf—Institute of Organic Chemistry & Biochemistry
Technische Universitꢀt Darmstadt, 64287 Darmstadt (Germany)
E-mail: schmidt_boris@t-online.de
The quinoxaline scaffold can undergo alternating donor–ac-
ceptor interactions with the gatekeeper+1/gatekeeper+3 res-
idues in the hinge region of the kinase domain. This binding
mode has been confirmed by crystallographic observations for
other kinases (Figure 2a). Docking of AG1295 in the FLT3
hinge region explains that the observed high ligand efficiency
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402477.
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the bisarylurea binding motif
(Figure 4). From this database
136 molecules with an IC50 or Ki
value of >1000 nm were select-
ed at random. They were consid-
ered inactive for the preparation
of the pharmacophore model.
A total of 425 pharmacophore
hypotheses were generated and
sorted by ascending p value. The
accuracy of active compounds
(Acc1) or inactive compounds
(Acc0) is the ratio m1/N1 and m0/
N0, respectively, for which N is
the total number of molecules
and m is the number of mole-
cules that fit the model (actives)
or do not fit in the model (inac-
tives). For Acc1 =1, all active
Figure 1. Selected structures of known FLT3 inhibitors.[14–15]
molecules were taken into ac-
count, whereas with Acc0 =1 no
inactive molecule fit the pharmacophore hypothesis.
The statistical significance of these accuracy values
was determined using the c2-derived p value. This is
the base-10 logarithm of the probability of c2 that
the accuracy results Acc1 and Acc0 were achieved by
chance only. Lower p values indicate higher signifi-
cance of the accuracy. The generated pharmaco-
phores A–D contain a donor projection, acceptor
projection, p-ring projections, and aromatic rings
(Table 1). In all structures, the donor groups (NH) of
the urea motif were considered as a projection, but
Figure 2. a) Structure of quinoxaline-based inhibitor in CHK2 (PDB ID: 4BDC) and
b) result of the docking study of AG1295 in the active site of FLT3 reveals donor–accept-
or interactions with the hinge region.
is due to hydrogen bonding to the hinge backbone and p–p
interactions with the gatekeeper F691 or F830 (Figure 2b).
Table 1. Generated pharmacophore hypothesis with lowest p values.[a]
Substitution at the 4’-phenyl position enables interactions with
the DFG motif by a tethered group, which may lead to type II
inhibition. The occupation of the regulatory domain pocket by
aromatic groups is frequently observed for TKIs. Due to the
ubiquitous occurrence of bisarylureas connected to a hinge
binding scaffold, we were interested in a general pharmaco-
phore model for FLT3 type II TKIs binding to a kinase in
a DFGout/JMout conformation. This can be observed by the co-
crystallized sorafenib–VEGFR2 structure (PDB ID: 4ASD), in
which the urea motif binds in the hydrophobic back pocket
followed by a heterocyclic hinge binding motif.[17] This model
was adapted and subsequently used to filter an in silico library
of bisarylureas created by the combination of hypothetic pre-
cursor molecules with amines from our in-house chemical da-
tabase (Figure 3).
Entry Pharmacophore
Acc p value Acc1 Acc0
A
0.9931 ꢀ26.2 0.875 1.0000
B
C
D
0.9861 ꢀ22.6 0.875 0.9926
0.9861 ꢀ22.6 0.875 0.9926
0.9792 ꢀ21.3 1.000 0.9779
A ligand-based pharmacophore represents the essential in-
teractions of a ligand with a receptor. We used a freely accessi-
ble online database of 1675 FLT3 inhibitors with corresponding
in vitro pharmacology listed.[26] For this study, eight highly
potent inhibitors were selected a) because their binding mode
has been solved crystallographically for other kinases or
b) they are likely to exert type II inhibition and also contain
[a] Acc indicates the overall accuracy of active (Acc1) and inactive (Acc0)
compounds covered by the pharmacophore; p value indicates statistical
significance. Generated pharmacophores consist of donor (purple), ac-
ceptor (cyan), and aromatic ring (orange) projections.
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not the carbonyl acceptor. By
using the pharmacophore search
for various scaffolds the generat-
ed hypotheses can be superior
to a single scaffold, as potential
interactions can be covered
more accurately. In pharmaco-
phore hypothesis A each of the
connecting phenyl rings as well
the various head groups are rec-
ognized, which is not the case in
B–D. The terminal phenyl ring of
the bisarylurea motif is covered
by an aromatic ring center (A) or
a projection (B–D) with high su-
perposition of all molecular scaf-
folds. All models show a high ex-
clusion of nearly all inactive mol-
ecules (Acc0 ~1) and include,
except for the quizartinib deriva-
tive 2, the entire set of active
molecules.
The
pharmacophore-based
method offers a rapid assess-
ment of many molecules using
simple parameters. However, in-
formation on potential interac-
tions between ligand and recep-
tor and the assessment of the
size of a binding pocket requires
docking methods. Unfortunately,
just two crystal structures are
available for FLT3. In 2004, Grif-
fith et al. described the structure
of the cytosolic kinase domain,
but neither in complex with an
inhibitor nor ADP.[18] Due to the
position of the juxtamembrane
domain and the activation loop,
these are in the autophosphory-
lated, inactive conformation
(PDB ID: 1RJB). The structure of
the immunoglobulin-like do-
mains in complex with the FLT3
ligand was reported in 2012 by
Verstraete et al. (PDB IDs: 3QS7,
3QS9).[19] In the next step, hits
from the pharmacophore search
were rated by an induced-fit
docking in the kinase structure
of FLT3 to assess binding geom-
etry and potential ligand–recep-
tor interactions.
Figure 3. Flowchart for the optimization of quinoxaline-based TKIs starting from AG1295 using pharmacophore
elucidation, pharmacophore search, and molecular docking.
Subsequently, sterically de-
manding amine fragments (bicy-
clics, bulky side groups, etc.)
were removed, as placement in
Figure 4. Active set of inhibitors in addition to TKIs in clinical trials (sorafenib, quizartinib) to generate pharmaco-
phore hypothesis.
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Figure 5. a),b) Docking study of compound 12b using MOE 2013.08 and the known FLT3 crystal structure (PDB ID: 1RJB). c) Ensemble of compounds that fit
into the hinge region and the DFG pocket (best-scoring pose). d) Compounds that could not be docked properly.[20]
the type II conformation was impossible (Figure 5). Various
functional groups were omitted from the synthesis candidates,
as they represent prodrug concepts and thus do not bind to
the kinase in the present form (carboxylate) or are potentially
toxic (phenols, thiophenol, anilines). Diamine derivatives were
ranked with low priority, as they require regioselective synthe-
sis (formation of regioisomers) and can be oxidized to toxic
para-aminophenols that can modify cellular macromolecules as
benzoquinone imines. Extended Lipinski parameters were used
for the selection; therefore derivatives with logP>6.0 and
a Mr >600 Da were discarded. Finally, 18 derivatives were se-
lected for synthesis (Table 2). As observed for AG1295, the qui-
noxaline motif was oriented toward the protein surface and
the aromatic head groups were located in the DFG pocket for
quinoxalines 12a–m. Thus, similar interactions were identified
within the ATP binding pocket of FLT3. The benzothiazole de-
rivative 12i and the phenoxy derivative 12m are very lipophil-
ic. However, larger hydrophobic and aromatic contact surfaces
may lead to higher activity. The lowest lipophilicities are exhib-
ited by the picolyl derivative 12d and the pyridine derivative
12g. They are likely to display better solvent solubility, as p-
stacking interactions in the crystal are weakened by the ben-
zylic position. Several potential interactions were identified be-
tween the quinoxaline scaffold and the hinge region. Strong
hydrogen bonds were predicted between the nitrogen atom
of the quinoxaline and C694, additionally to the hydrogen
bond with E692. L616 forms a hydrogen–p interaction with
the quinoxaline. Furthermore, the donor–acceptor motif of the
urea group forms multiple interactions with residues D829,
K644, and E661.
Synthesis and in vitro pharmacology
The synthesis of compounds 12a–m started with the prepara-
tion of arylgyloxal 7, which was used for condensation to the
quinoxaline scaffold. Glyoxal 7 was prepared by oxidation of
its corresponding acetophenone 6 by using selenium dioxide
(Scheme 1). The commercial 4,5-dimethylbenzene-1,2-diamine
and glyoxal 7 reacted at 808C in N,N-dimethylformamide
(DMF) within 1 h to form 2-arylquinoxaline derivative 8, which
was obtained in good yield (80%). Reduction of intermediate 8
into its corresponding amine 9 was carried out in the presence
of iron and iron(III) chloride. The final compound 12a was ob-
tained by in situ coupling of amine 9 with isocyanate 10.
Aniline 9 was treated with triphosgene under basic condi-
tions to form isocyanate 13, which was coupled in situ with
the selected amines to obtain the final quinoxaline derivatives
12b–r (Scheme 1). The reference compound AG1295 was syn-
thesized as described by Zall et al. via a-ketoaldehydes starting
from benzaldehyde and 4,5-dimethylbenzene-1,2-diamine.[21]
The obtained quinoxalines 12a–r were tested for their ability
to inhibit FLT3 kinase activity (Table 2). The assay results are in
agreement with our previous docking studies. Thus, seven qui-
noxaline derivatives 12a–g exhibited an FLT3 inhibitory activity
of >50% at 100 nm. Interestingly, five of them, 12c–g, contain
a methylene bridge between the urea motifs and the aromatic
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Table 2. FLT3 in vitro inhibitory activities of quinoxaline derivatives 12a–
r and their calculated chemical properties.
Table 2. (Continued)
Compd
R
logP[a]
tPSA [ꢁ][a]
Inhib. [%][b]
Compd
R
logP[a]
tPSA [ꢁ][a]
24.7
Inhib. [%][b]
37
AG1295
–
4.28
12r
5.67
87.4
0
12a
12b
4.66
4.69
87.4
80
[a] The partition coefficient (logP) and topological polar surface area
(tPSA) were calculated with ChemBioDraw Ultra (version 13.0.2). [b] Re-
sults are expressed as a percentage of the control specific activity (SA)
determined against FLT3 at an inhibitor concentration of 100 nm; percent
inhibition=[(100ꢀSAmeasured)/SAcontrol]ꢂ100.
78.2
76
12c
12d
4.83
3.49
65.8
78.2
76
72
head groups. Comparison of compound 12c and 12l confirms
the importance of the methylene bridge. Compound 12c ex-
hibited an FLT3 inhibition of 76% at 100 nm, whereas com-
pound 12l, which lacks this methylene group, exerts decreased
inhibitory activity of merely 12%. One of the most potent com-
pounds, 12a, fuses structural elements from AG1295 and qui-
zartinib and exhibited an inhibition of 80% at 100 nm. The
high activity of 12b may be attributed to the chlorine substitu-
tion at the para position, which lowers the intramolecular hy-
drogen bonding capacity to the distal urea amine.
12e
12 f
4.70
4.60
75.1
84.3
67
65
12g
4.70
75.1
63
Compounds 12k and 12q, which lack this para substitution,
exert no significant FLT3 inhibitory activity at 100 nm. Notably,
differences in activity regarding closely related compounds
cannot be explained by our modeling, for example, 12e and
12p. Next we investigated the role of the dimethyl quinoxaline
moiety. Based on the most active and promising compound
12a, we replaced the hydrophobic dimethyl groups by solubi-
lizing methoxy or dimethoxy derivatives as present in 18a and
18b (Scheme 2). Thus, both nitro compounds 14a and 14b
were reduced with Pd/C under hydrogen into their corre-
sponding amines 15a and 15b, and were directly coupled
with the previously synthesized glyoxal 7 to obtain the dime-
thoxy (16a) and methoxy (16b) intermediates, respectively.
After reduction with Fe and FeCl3 into their amino counter-
parts 17a and 17b, respectively, a solution of isocyanate 11
was added to give the desired products 18a and 18b.
12h
12i
12j
12k
12l
3.42
5.04
5.71
4.62
4.76
78.2
117.7
78.2
42
41
20
13
12
78.2
65.9
12m
12n
12o
5.73
5.24
4.63
65.9
65.9
75.1
9
9
5
The results of the FLT3 in vitro assay indicated that both
compounds exhibited lower inhibitory activity than that of de-
rivative 12a (Table 3). Nevertheless, the dimethoxy derivative
18a exhibited better FLT3 in vitro inhibitory activity (51% in-
hibition at 100 nm) than compound 18b with an inhibition of
30%.
12p
12q
6.35
4.13
75.1
78.2
3
0
The IC50 values of compounds 12a–c were determined for
better differentiation. IC50 values of 71 nm for 12a, 32 nm for
12b, and 88 nm for compound 12c were observed, confirming
the identification of a series of highly potent FLT3 inhibitors
based on the quinoxaline scaffold (Table 4).
We further evaluated the most potent compound 12b in
a kinase selectivity panel at a concentration of 1 mm, including
the FLT3 mutant D835Y (Figure 6). Remotely related kinases of
other kinome families, such as LRRK2 (TKL family), GSK3-
b (CMGC family), Pim1 kinase (CAMK family), ROCK1 (AGC
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family), and MEK1 (STE family)
are not inhibited, as are single
members of the TK family (e.g.,
IGF1R, VEGFR2, Abl). Closely re-
lated kinases in the type III RTK
family are inhibited, reaching
from relatively weak inhibition of
PDGFRa and PDGFRb kinases to
nearly equipotent inhibition of
c-KIT kinase. PDGFR and espe-
cially KIT activity can be com-
monly observed for FLT3 inhibi-
tors as quizartinib, based on
high sequence identity (62%)
and sequence similarity (77%).
The FLT3 D835Y mutant destabil-
izes the inactive kinase confor-
mation, a prerequisite for type II
inhibitor binding. The mutant
kinase is not inhibited by 12b,
which is indicative of type II ATP-
competitive binding and is in ac-
cordance with in vitro data for
other type II inhibitors.[12] Dual
inhibition of FLT3 and c-KIT may
offer clinical advantages.[22]
Scheme 1. Reactions and conditions: a) SeO2, dioxane/H2O 3:1, 12 h, 1008C; b) diamine, DMF, 1 h, 808C; c) Fe/
FeCl3, AcOH, EtOH, 1 h, reflux; d) triphosgene, NEt3, CH2Cl2, 1 h, ꢀ108C; e) toluene, 2 h, reflux; f) triphosgene, sat.
NaHCO3, CH2Cl2, 20 min, RT; g) amine, THF, 2 h, reflux.
Next, we examined compound
12a and AG1295 for their in
vivo activity on zebrafish
embryo development. In the
past decades the zebrafish
(Danio rerio) has become
a
useful aquatic vertebrate
model to study developmental
mechanisms and to assess toxi-
cological effects of chemicals
and drugs; it is standardized in-
ternationally.[23] Indeed, the ze-
brafish has high fecundity (~60–
200 eggs per spawning), rapid
Scheme 2. Reactions and conditions: a) Pd/C, H2, CH3OH, 4 h, RT; b) glyoxal 7, DMF, 1 h, 808C; c) Fe/FeCl3, AcOH,
CH3OH, 1 h, reflux; d) isocyanate 11, toluene, 2 h, reflux.
embryonic
development
(~3 days) and small size. The
embryos are transparent and develop outside the body.[23c,24]
These features have established the zebrafish as a key model
in toxicological and pharmacological research. Treatment of ze-
brafish with test drugs or chemicals may result in a change in
Table 3. FLT3 in vitro inhibitory activity of compounds 18a and 18b.
Table 4. FLT3 in vitro inhibitory activity of compounds 12a–c.
Compd
R1
logP[a]
Inhibition [%][b]
Compd
IC50 [nm]
Inhibition [%][a]
18a
18b
OMe
H
3.43
3.56
51
30
12a
12b
12c
71
32
88
80
76
76
[a] The partition coefficient (logP) was calculated with ChemBioDraw
Ultra (version 13.0.2). [b] Results are expressed as a percentage of the
control specific activity (SA) determined against FLT3 at an inhibitor con-
centration of 100 nm; percent inhibition=[(100ꢀSAmeasured)/SAcontrol]ꢂ100.
[a] Results are expressed as a percentage of the control specific activity
determined against FLT3 at an inhibitor concentration of 100 nm; percent
inhibition=[(100ꢀSAmeasured)/SAcontrol]ꢂ100.
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Conclusions
We sought an efficient in silico
algorithm to optimize the frag-
ment-sized
FLT3
inhibitor
AG1295. Substitution of the
AG1295 core with bisarylureas
occupying the DFG pocket led
to potent FLT3 inhibitors. Based
on this hypothesis, we created
an optimization algorithm to
refine an in silico library by phar-
macophore filtering using a di-
verse set of known inhibitors.
Subsequent structure-based fil-
tering led to optimized quinoxa-
line-based tyrosine kinase inhibi-
tors. These in silico hits were
synthesized and evaluated in
biological assays resulting in ten-
Figure 6. Kinase selectivity panel of compound 12b at a concentration of 1 mm.
fold increased inhibitory potency
relative to that of AG1295. The
most active compounds exhibit
phenotype. Therefore, conclusions can be drawn concerning
the safety and permeability of the compounds in question.
In our zebrafish assay the embryos were collected and main-
tained in E3 medium at 288C. Compound 12a was added at
24 hours post-fertilization (hpf), and the phenotypes were
compared after 96 hpf (Figure 7). Lower concentrations of
compound 12a from 1 to 20 mm did not reveal any abnormali-
ties relative to control (Figure 7A–C). At concentrations of 30
and 40 mm, the phenotypes changed, and 15% of the zebrafish
displayed stunted and crooked tails (Figure 7D,E). After treat-
ing the zebrafish with compound 12a at higher concentrations
(50 and 100 mm), related but more pronounced phenotypes
were observed. Stunted and crooked tails were observed in ad-
dition to the constrained circular movement. At 100 mm, com-
pound 12a was observed to impart a developmental delay
(50% of the zebrafish did not hatch), and all embryos exhibit-
ed abnormalities relative to the control (Figure 7H). In addition
to phenotypic description, we monitored lethality in the zebra-
fish every 24 h up to 120 hpf by heartbeat observation
(Figure 7). We observed that at lower concentrations (1–40 mm)
the lethality in zebrafish embryos ranged from 0 to 10%. At
100 mm, the survival rate decreased to 20%. Related pheno-
types were observed for AG1295. At concentrations <20 mm
the zebrafish embryos exhibited no abnormalities relative to
control. Upon increasing concentration to 100 mm, both stunt-
ed and crooked tails were more pronounced. AG1295 also dis-
played similar lethality as observed for compound 12a. Never-
theless, the zebrafish assay did not indicate any abnormalities
for compound 12a at concentrations <20 mm, and indicated
no (1 mm) or weak cytotoxicity up to concentrations of 40 mm.
As quizartinib was reported to be nonlethal toward zebrafish
embryos, the observed lethality may be due to off-target ef-
fects.[25]
IC50 values of 71 nm (12a) and 32 nm (12b). Consequently, we
evaluated the toxicity of compound 12a against AG1295 in
our in vivo zebrafish embryo phenotype assay, observing no
toxicity <20 mm for both compounds. Future studies will focus
on the development of quinoxaline-based inhibitors of the
active kinase state to overcome increasing resistance to type II
inhibitors caused by the FLT3 D835Y mutation. Evaluation of
future kinase inhibitors should focus on selectivity over even
near family members and should aim for sustained inhibition
in cellular assays and in vivo models for AML.
Experimental Section
1H NMR spectra were recorded on a Bruker AC 300 spectrometer at
300 MHz and a Bruker AC 500 spectrometer at 500 MHz. 13C NMR
spectra were recorded on a Bruker AC 300 spectrometer at 75 MHz
and a Bruker AC 500 spectrometer at 125 MHz. Chemical shifts are
reported in ppm and are calibrated to the particular solvent. Mass
spectrometry was performed on a Bruker–Franzen Esquire LC mass
spectrometer and a MAT 95 double focusing sector field MS instru-
ment. Microwave experiments were carried out with a Biotage Ini-
tiator microwave apparatus. All microwave experiments were car-
ried out in sealed microwave process vials using the standard ab-
sorbance level (300 W maximum power). Silica gel chromatography
was carried out using Merck silica gel 60 (0.015–0.040 mm). High-
performance liquid chromatography was carried out on an Agilent
1100 (column: reversed-phase, Zorbax Eclipse XDB-C8, 4.6ꢂ
150 mm; 254 nm). The eluent was composed of: H2O (1% TFA) (A)
and CH3CN (1% TFA) (B) with a gradient 30!90% B within 12 min.
Automated flash chromatography was performed on a Teledyne
ISCO Combi Flash RF 4X using H2O and CH3CN solvent mixtures. All
commercially available reagents and solvents were purchased at
ABCR, Acros, Sigma–Aldrich and VWR.
2-(4-Nitrophenyl)-2-oxoacetaldehyde (7): A suspension of SeO2
(671 mg, 6.05 mmol) in dioxane (6 mL) and H2O (2 mL) was stirred
at 558C until the solid dissolved, and then treated with 1-(4-nitro-
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Figure 7. In vivo cytotoxicity toward zebrafish embryos and the survival rate at various time points for compound 12a (top) and AG1295 (bottom). Com-
pound 12a and AG1295 were added 24 hours post-fertilization (hpf) to ten embryos in duplicates, and the phenotypes were compared after 96 hpf. A) Con-
trol embryo in 1% DMSO. B)–H) Zebrafish embryos treated with compound 12a and AG1295 at increasing concentrations of B) 1, C) 10, D) 20, E) 30, F) 40,
G) 50, and H) 100 mm.
phenyl)ethanone 6 (200 mg, 1.21 mmol). The mixture was stirred
overnight at 1008C. The black solid was filtered off and discarded,
and the filtrate was concentrated in vacuo. The residue was diluted
in EtOAc (20 mL), washed with H2O (15 mL) and saturated aq
NaHCO3 (15 mL), dried (MgSO4) and filtered, and the solvent was
removed in vacuo. Purification by silica gel column chromatogra-
phy (cyclohexane/EtOAc, 1:1) gave the title compound as a yellow
solid (148 mg, 68%); HPLC: tR =1.83 min; 1H NMR ([D6]DMSO,
300 MHz): d=8.41–8.21 (m, 4H), 7.05 ppm (d, 2H, J=6.8Hz);
13C NMR ([D6]DMSO, 75 MHz): d=195.3, 149.8, 138.5, 130.8, 123.5,
90.1 ppm.
HPLC: tR =7.83 min; 1H NMR (CDCl3, 500 MHz): d=9.28 (s, 1H),
8.41–8.36 (m, 4H), 7.96 (s, 2H), 2.55 ppm (s, 6H); 13C NMR (CDCl3,
125 MHz): d=148.9, 148.7, 142.8, 142.4, 142.3, 141.7, 141.0, 140.2,
129.0, 128.3, 127.8, 124.4, 20.7, 20.6 ppm.
4-(6,7-Dimethylquinoxalin-2-yl)aniline (9): Acetic acid (1.30 mL),
Fe (190 mg, 3.39 mmol) and FeCl3 (92 mg, 0.57 mmol) were added
to
a solution of 6,7-dimethyl-2-(4-nitrophenyl)quinoxaline 8
(158 mg, 0.57 mmol) in EtOH (7 mL), and the mixture was stirred
for 1 h at 808C. After cooling to RT, the solvent was removed, and
the residue was dissolved in EtOAc (30 mL) and washed with H2O
(20 mL). The organic phase was dried (MgSO4) and filtered, and the
solvent was removed in vacuo. Purification by silica gel column
chromatography (CHCl3/CH3OH, 20:1) gave the title compound as
6,7-Dimethyl-2-(4-nitrophenyl)quinoxaline (8): A solution of 2-(4-
nitrophenyl)-2-oxoacetaldehyde 7 (140 mg, 0.78 mmol) and 4,5-di-
methylbenzene-1,2-diamine (106 mg, 0.78 mmol) in DMF (5 mL)
was stirred for 2 h at 808C. After cooling to RT, H2O (3 mL) was
added, and the obtained solid was isolated by filtration and dried
to give the title compound as a light-yellow solid (174 mg, 80%);
a
yellow solid (70 mg, 50%); HPLC: tR =3.09 min; 1H NMR
([D6]DMSO, 500 MHz): d=9.28 (s, 1H), 8.02 (d, 2H, J=8.6 Hz), 7.77
(s, 2H), 6.71 (d, 2H, J=8.6 Hz), 5.67 (s, 2H), 2.45 ppm (d, 6H, J=
4.2Hz); 13C NMR ([D6]DMSO, 125 MHz): d=151.0, 150.5, 142.0,
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141.9, 140.4, 140.2, 139.1, 139.3, 128.3, 127.7, 123.3, 113.8, 19.8,
19.6 ppm.
0.30 mmol) and using the same procedure as described for 12b,
the title compound 12d was obtained as a colorless solid (84 mg,
78%); HPLC: tR =3.76 min (100%); 1H NMR ([D6]DMSO, 500 MHz):
d=9.39 (s, 1H), 9.00 (s, 1H), 8.58 (d, 1H, J=2.2 Hz), 8.49 (dd, 1H,
J=4.8, 1.6 Hz), 8.26–8.19 (m, 2H), 7.84 (d, 2H, J=8.5 Hz), 7.76 (dt,
1H, J=7.8, 2.0 Hz), 7.68–7.59 (m, 2H), 7.39 (dd, 1H, J=7.8, 4.8 Hz),
6.89 (t, 1H, J=6.0 Hz), 4.39 (d, 2H, J=5.9 Hz), 2.49 ppm (s, 6H);
13C NMR ([D6]DMSO, 125 MHz): d=155.0, 149.8, 148.7, 148.0, 142.4,
142.2, 140.6, 140.4, 139.6, 139.4, 135.7, 134.99, 129.0, 127.9, 127.8,
127.7, 123.4, 117.8, 40.5, 19.8, 19.7 ppm; MS (EI, 70 eV): m/z=383
[M]+.
1-(5-tert-Butylisoxazol-3-yl)-3-(4-(6,7-dimethylquinoxalin-2-yl)-
phenyl)urea (12a): A solution of 5-tert-butylisoxazol-3-amine 10
(17 mg, 0.12 mmol) and triphosgene (10 mg, 0.12 mmol) in CH2Cl2
(2 mL) was treated dropwise at ꢀ108C with a solution of Et3N
(34 mL, 0.22 mmol) in CH2Cl2 (0.5 mL). The mixture was stirred for
1 h at this temperature. Afterward, a solution of 4-(6,7-dimethylqui-
noxalin-2-yl)aniline 9 (30 mg, 0.12 mmol) in toluene (1 mL) was
added, and the mixture was stirred for 2 h at 1108C. After cooling
to RT, H2O (5 mL) was added, and the aqueous mixture was extract-
ed with EtOAc (3ꢂ10 mL). The combined organic layers were dried
(MgSO4) and filtered, and the solvent was removed in vacuo. Purifi-
cation by silica gel column chromatography (CHCl3/CH3OH, 25:1)
gave the title compound as a light-yellow solid (28 mg, 56%);
HPLC: tR =8.00 min (95%); 1H NMR ([D6]DMSO, 500 MHz): d=9.58
(s, 1H), 9.42 (s, 1H), 9.07 (s, 1H), 8.29–8.26 (m, 2H), 7.87 (d, 2H, J=
11.4 Hz), 7.68–7.65 (m, 2H), 6.54 (s, 1H), 2.49 (d, 6H, J=3.0 Hz),
1.31 ppm (s, 9H); 13C NMR ([D6]DMSO, 125 MHz): d=180.3, 158.2,
151.2, 149.6, 142.3, 140.9, 140.7, 140.3, 139.7, 139.7, 130.3, 127.9,
127.7, 118.5, 92.4, 28.3, 19.8, 19.7 ppm; MS (EI, 70 eV): m/z=438
[M+Na]+; HRMS-EI: m/z [M]+ calcd for C24H25N5O2: 415.2009,
found: 415.20082.
1-(4-(6,7-Dimethylquinoxalin-2-yl)phenyl)-3-(4-methoxybenzyl)-
urea (12e): Starting from (4-methoxyphenyl)methanamine (31 mL,
0.24 mmol) and using the same procedure as described for 12b,
the title compound 12e was obtained as a yellow solid (31 mg,
34%); HPLC: tR =7.21 min (97%); 1H NMR ([D6]DMSO, 500 MHz):
d=9.39 (s, 1H), 8.83 (s, 1H), 8.21–8.19 (m, 2H), 7.84 (d, 2H, J=
9.1 Hz), 7.63–7.60 (m, 2H), 7.27–7.24 (m, 2H), 6.92–6.89 (m, 2H),
6.56 (t, 1H, J=5.8 Hz), 4.25 (d, 2H, J=5.8 Hz), 3.74 (s, 3H),
2.48 ppm (d, 6H, J=3.0 Hz); 13C NMR ([D6]DMSO, 125 MHz): d=
158.2, 154.9, 149.9, 142.5, 142.2, 140.6, 140.4, 139.6, 139.4, 132.0,
128.8, 128.5, 127.9, 127.8, 127.7, 117.7, 113.7, 55.1, 42.3, 19.8,
19.7 ppm; MS (EI, 70 eV): m/z=412 [M]+.
1-(4-Chloropyridin-2-yl)-3-(4-(6,7-dimethylquinoxalin-2-yl)-
phenyl)urea (12b): A biphasic system of triphosgene (35 mg,
0.12 mmol) in CH2Cl2 (3 mL) and saturated NaHCO3 solution
(15 mL) was treated dropwise with a solution of 4-(6,7-dimethylqui-
noxalin-2-yl)aniline 9 (84 mg, 0.34 mmol) in CH2Cl2 (10 mL). The or-
ganic phase turned red, and the mixture was stirred until homoge-
nous. The reaction was stopped by separation of the organic
phase, which was dried (MgSO4), filtered and concentrated in
vacuo to give a red solid, which was used directly in the next step.
The red solid was dissolved in THF (4 mL), and the mixture was
added to a solution of 4-chloropyridin-2-amine (48 mg, 0.37 mmol)
in THF (4 mL). The reaction was stirred at reflux for 2 h. After cool-
ing to RT, the solvent was removed, and the residue was dissolved
in a small amount of DMF, and H2O (1 mL) was added. The ob-
tained precipitate was isolated by filtration and dried in vacuo to
give the desired compound as a yellow solid (51 mg, 36%); HPLC:
tR =7.46 min (99%); 1H NMR ([D6]DMSO, 500 MHz): d=10.21 (s,
1H), 9.64 (s, 1H), 9.45 (d, 1H, J=1.2 Hz), 8.35–8.29 (m, 3H), 7.92–
7.86 (m, 2H), 7.80 (d, 1H, J=1.9 Hz), 7.77–7.71 (m, 2H), 7.19 (dd,
1H, J=5.5, 1.9 Hz), 2.51–2.51 (m, 3H), 2.51–2.49 ppm (m, 3H);
13C NMR ([D6]DMSO, 125 MHz): d=179.7, 153.7, 151.7, 149.6, 148.9,
144.1, 142.3, 140.7, 140.4, 139.8, 139.7, 130.4, 128.0, 127.7, 118.8,
118.4, 117.9, 111.3, 19.8, 19.8 ppm; MS (EI, 70 eV): m/z=403 [M]+;
HRMS-EI: m/z [M]+ calcd for C22H18ClN5O: 403.1197, found:
403.12015.
1-(Benzo[d][1,3]dioxol-5-ylmethyl)-3-(4-(6,7-dimethylquinoxalin-
2-yl)phenyl)urea (12 f): The title compound 12 f was obtained as
a yellow solid (35 mg, 37%) using the same procedure as de-
scribed for 12b starting from benzo[d][1,3]dioxol-5-ylmethanamine
1
(36 mg, 0.24 mmol); HPLC: tR =7.21 min 96%); H NMR ([D6]DMSO,
500 MHz): d=9.39 (s, 1H), 8.84 (s, 1H), 8.22–8.19 (m, 2H), 7.84 (d,
2H, J=9.2 Hz), 7.63–7.60 (m, 2H), 6.89 (d, 1H, J=1.6 Hz), 6.87 (d,
1H, J=7.9 Hz), 6.80 (dd, 1H, J=7.9, 1.6 Hz), 6.67 (t, 1H, J=5.9 Hz),
5.98 (s, 2H), 4.23 (d, 2H, J=5.9 Hz), 2.47 ppm (d, 6H, J=3.0 Hz);
13C NMR ([D6]DMSO, 125 MHz): d=154.9, 149.9, 147.2, 146.0, 142.5,
142.2, 140.6, 140.4, 139.6, 139.4, 134.1, 128.8, 127.9, 127.8, 127.7,
120.4, 117.7, 108.0, 107.9, 100.9, 42.6, 19.8, 19.7 ppm; MS (EI,
70 eV): m/z=426 [M]+.
1-(4-(6,7-Dimethylquinoxalin-2-yl)phenyl)-3-(3-methoxybenzyl)-
urea (12g): The title compound 12g was obtained as a yellow
solid (25 mg, 26%) using the same procedure as described for 12b
starting from 3-methoxybenzylamine (32 mg, 0.233 mmol); HPLC:
1
tR =7.27 min (95%); H NMR ([D6]DMSO, 500 MHz): d=9.45 (s, 1H),
8.95 (s, 1H), 8.27 (d, J=8.8 Hz, 2H), 7.92 (s, 1H), 7.90 (s, 1H), 7.68
(d, J=8.8 Hz, 2H), 7.32 (t, J=7.8 Hz, 1H), 7.00–6.92 (m, 1H), 6.91–
6.86 (m, 1H), 6.79 (t, J=6.0 Hz, 1H), 4.37 (d, J=5.9 Hz, 2H), 3.81 (s,
3H), 2.54 (s, 3H), 2.54 ppm (s, 3H); 13C NMR ([D6]DMSO, 125 MHz):
d=159.33, 154.96, 149.87, 142.48, 142.22, 140.37, 139.61, 139.46,
129.40, 128.87, 127.87, 127.80, 127.70, 119.28, 117.74, 116.47,
112.84, 112.09, 54.99, 42.73, 39.82, 19.82, 19.72, 19.57 ppm; MS (EI,
70 eV): m/z=412 [M]+.
1-Benzyl-3-(4-(6,7-dimethylquinoxalin-2-yl)phenyl)urea
(12c):
Starting from phenylmethylamine (26 mL, 0.24 mmol) and using the
same procedure as described for 12b, the title compound was ob-
tained as a yellow solid (35 mg, 42%); HPLC: tR =7.24 min (96%);
1H NMR ([D6]DMSO, 500 MHz): d=9.39 (s, 1H), 8.88 (s, 1H), 8.22–
8.19 (m, 2H), 7.84 (d, 2H, J=9.1 Hz), 7.64–7.61 (m, 2H), 7.37–7.32
(m, 4H), 7.27–7.24 (m, 1H), 6.74 (t, 1H, J=5.9 Hz), 4.33 (d, 2H, J=
5.9 Hz), 2.47 ppm (d, 6H, J=3.0 Hz); 13C NMR ([D6]DMSO, 125 MHz):
d=154.9, 149.8, 142.5, 142.2, 140.6, 140.5, 140.1, 139.6, 139.4,
128.8, 128.3, 127.9, 127.8, 127.7, 127.1, 126.7, 117.7, 42.8, 19.8,
19.7 ppm; MS (EI, 70 eV): m/z=382 [M]+.
1-(4-(6,7-Dimethylquinoxalin-2-yl)phenyl)-3-(pyridin-3-yl)urea
(12h): The title compound 12h was obtained as a yellow solid
(84 mg, 67%) using the same procedure as described for 12b start-
ing from pyridin-3-amine (32 mg, 0.34 mmol); HPLC: tR =4.36 min
1
(94%); H NMR ([D6]DMSO, 500 MHz): d=9.42 (s, 1H), 9.11 (s, 1H),
8.93 (s, 1H), 8.64 (d, 1H, J=2.3 Hz), 8.28–8.25 (m, 2H), 8.21 (dd,
1H, J=4.6, 1.1 Hz), 7.98–7.96 (m, 1H), 7.86 (d, 2H, J=10.6 Hz),
7.70–7.67 (m, 2H), 7.33 (dd, 1H, J=8.3, 4.7 Hz), 2.48 ppm (d, 6H,
J=3.4 Hz); 13C NMR ([D6]DMSO, 125 MHz): d=152.4, 149.7, 143.1,
142.2, 141.4, 140.7, 140.3, 140.2, 139.7, 139.6, 136.2, 129.8, 127.9,
127.7, 125.3, 123.6, 118.4, 19.8, 19.7 ppm; MS (ES, 70 eV): m/z=369
[M]+.
1-(4-(6,7-Dimethylquinoxalin-2-yl)phenyl)-3-(pyridin-3-ylmethyl)-
urea (12d): Starting from pyridin-3-ylmethylamine (33 mg,
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1-(4-(6,7-Dimethylquinoxalin-2-yl)phenyl)-3-(4-methyl-3-nitro-
phenyl)urea (12i): The title compound 12i was obtained as
a light-brown solid (65 mg, 45%) using the same procedure as de-
scribed for 12b starting from 4-methyl-3-nitroaniline (52 mg,
0.34 mmol); HPLC: tR =8.03 min (95%); 1H NMR ([D6]DMSO,
500 MHz): d=9.42 (s, 1H), 9.14 (s, 1H), 9.09 (s, 1H), 8.30 (d, 1H, J=
2.3 Hz), 8.28–8.25 (m, 2H), 7.86 (d, 2H, J=11.9 Hz), 7.70–7.76 (m,
2H), 7.59 (dd, 1H, J=8.3, 2.3 Hz), 7.42 (d, 1H, J=8.5 Hz), 2.48 (d,
6H, J=3.3 Hz), 2.46 ppm (s, 3H); 13C NMR ([D6]DMSO, 125 MHz):
d=152.2, 149.7, 148.8, 142.3, 141.3, 140.7, 140.3, 139.7, 139.6,
138.5, 133.0, 129.9, 127.9, 127.9, 127.7, 125.7, 123.1, 118.5, 113.4,
19.8, 19.7, 18.9 ppm; MS (ES, 70 eV): m/z=469 [M]+.
48%) using the same procedure as described for 12b starting from
o-toluidine (47 mg, 0.44 mmol); HPLC: tR =7.24 min (98%); H NMR
1
([D6]DMSO, 500 MHz): d=9.54 (s, 1H), 9.34 (s, 1H), 8.21–8.17 (m,
2H), 8.14 (s, 1H), 7.78 (m, 2H), 7.65–7.59 (m, 2H), 7.12 (m, 1H),
7.08 (m, 1H), 6.96 (t, J=7.2 Hz, 1H), 6.90 (t, J=7.3 Hz, 1H), 2.42 (s,
3H), 2.41 (s, 3H), 2.22 ppm (s, 3H); 13C NMR ([D6]DMSO, 125 MHz):
d=152.57, 149.80, 142.24, 142.00, 140.68, 140.37, 139.65, 139.53,
137.20, 130.83, 130.19, 129.34, 127.90, 127.70, 126.84, 126.11,
122.87, 121.24, 117.97, 17.98, 17.03, 8.52 ppm; MS (EI, 70 eV): m/z=
382 [M]+.
1-(4-(6,7-Dimethylquinoxalin-2-yl)phenyl)-3-(2-methoxyphenyl)-
urea (12o): The title compound 12o was obtained as a brown
solid (88 mg, 57%) using the same procedure as described for 12b
starting from 2-anisidine (47.9 mg, 0.389 mmol); HPLC: tR =
1-(Benzo[d]thiazol-2-yl)-3-(4-(6,7-dimethylquinoxalin-2-yl)-
phenyl)urea (12j): The title compound 12j was obtained as a red
solid (51 mg, 43%) using the same procedure as described for 12b
starting from benzo[d]thiazol-2-amine (45 mg, 0.30 mmol); HPLC:
1
7.84 min (95%); H NMR ([D6]DMSO, 500 MHz): d=9.66 (s, 1H), 9.46
(s, 1H), 8.38 (s, 1H), 8.31 (d, J=8.8 Hz, 1H), 8.21 (dd, J=7.9, 1.7 Hz,
1H), 7.94–7.89 (m, 1H), 7.73 (d, J=8.7 Hz, 1H), 7.10 (dd, J=8.1,
1.5 Hz, 1H), 7.03 (td, J=7.8, 1.7 Hz, 1H), 6.98 (td, J=7.7, 1.5 Hz,
1H), 3.96 (s, 3H), 2.54 (s, 3H), 2.53 ppm (s, 3H); 13C NMR
([D6]DMSO, 125 MHz): d=152.19, 149.78, 147.75, 142.24, 141.81,
140.70, 140.37, 139.67, 139.57, 129.49, 128.41, 127.94, 127.89,
127.70, 122.07, 120.56, 118.43, 117.96, 110.80, 55.81, 39.99, 19.82,
19.73 ppm; MS (EI, 70 eV): m/z=398 [M]+.
1
tR =7.89 min (96%); H NMR ([D6]DMSO, 500 MHz): d=9.84 (s, 1H),
9.45 (s, 1H), 8.37–8.30 (m, 2H), 8.30 (s, 1H), 7.93 (dd, 1H, J=7.9,
1.1 Hz), 7.89 (d, 1H, J=16.1 Hz), 7.82–7.74 (m, 2H), 7.68 (d, 1H, J=
8.0 Hz), 7.45–7.40 (m, 1H), 7.30–7.25 (m, 1H), 2.50 (s, 3H), 2.49 ppm
(s, 3H); 13C NMR ([D6]DMSO, 125 MHz): d=159.8, 152.5, 149.6,
147.4, 142.3, 140.8, 140.6, 140.35, 139.7, 130.9, 130.6, 128.0, 127.7,
126.0, 122.9, 121.6, 119.1, 118.7, 19.8, 19.8 ppm; MS (EI, 70 eV): m/
z=425 [M]+.
1-(4-(6,7-Dimethylquinoxalin-2-yl)phenyl)-3-(4-(trifluorometh-
oxy)benzyl)urea (12p): The title compound 12p was obtained as
an orange solid (109 mg, 54%) using the same procedure as de-
scribed for 12b starting from 4-trifluromethoxybenzylamine
1-(4-(6,7-Dimethylquinoxalin-2-yl)phenyl)-3-(5-methylpyridin-2-
yl)urea (12k): The title compound 12k was obtained as a yellow
solid (52 mg, 51%) using the same procedure as described for 12b
starting from 5-methylpyridin-2-amine (31 mg, 0.29 mmol); HPLC:
tR =5.44 min (96%); 1H NMR ([D6]DMSO, 500 MHz): d=10.61 (s,
1H), 9.35 (d, 2H, J=5.0 Hz), 8.26–8.17 (m, 2H), 8.10–8.02 (m, 1H),
7.79 (d, 2H, J=13.5 Hz), 7.71–7.62 (m, 2H), 7.54 (dd, 1H, J=8.5,
2.3 Hz), 7.39 (d, 1H, J=8.4 Hz), 2.42 (s, 3H), 2.41 (s, 3H), 2.18 ppm
(s, 3H); 13C NMR ([D6]DMSO, 125 MHz): d=152.1, 150.6, 149.7,
146.5, 142.3, 141.1, 140.7, 140.4, 139.7, 139.6, 139.2, 136.1, 130.1,
127.9, 127.7, 126.5, 118.8, 111.6, 19.8, 19.7, 17.1 ppm; MS (EI, 70 eV):
m/z=384 [M]+.
1
(78 mg, 0.41 mmol); HPLC: tR =7.97 min (95%); H NMR ([D6]DMSO,
500 MHz): d=9.40 (s, 1H), 8.96 (s, 1H), 8.22 (d, J=8.8 Hz, 2H), 7.86
(d, J=9.0 Hz, 2H), 7.64 (d, J=8.8 Hz, 2H), 7.47 (d, J=8.7 Hz, 2H),
7.36 (d, J=8.0 Hz, 2H), 6.84 (t, J=5.9 Hz, 1H), 4.38 (d, J=5.9 Hz,
2H), 2.49 (s, 3H), 2.49 ppm (s, 3H); 13C NMR ([D6]DMSO, 125 MHz):
d=154.99, 149.85, 147.13, 142.41, 142.21, 141.47, 140.65, 140.36,
139.85, 139.61, 139.46, 128.92, 127.87, 127.78, 127.69, 120.92,
117.79, 42.09, 19.81, 19.71 ppm; MS (EI, 70 eV): m/z=466 [M]+.
1-(4-(6,7-Dimethylquinoxalin-2-yl)phenyl)-3-(pyridin-2-yl)urea
(12q): The title compound 12q was obtained as a green solid
(45 mg, 36%) using the same procedure as described for 12b start-
ing from pyridin-2-amine (32 mg, 0.34 mmol); HPLC: tR =5.12 min
1-(4-(6,7-Dimethylquinoxalin-2-yl)phenyl)-3-phenylurea
(12l):
The title compound 12l was obtained as a red solid (35 mg, 32%)
using the same procedure as described for 12b starting from ani-
line (30 mg, 0.32 mmol); HPLC: tR =7.31 min (95%); 1H NMR
([D6]DMSO, 500 MHz): d=9.43 (s, 1H), 9.09 (s, 1H), 8.87 (s, 1H),
8.27 (d, 2H, J=8.8 Hz), 7.88 (dd, 2H, J=11.9, 1.1 Hz), 7.69 (d, 2H,
J=8.8 Hz), 7.54–7.47 (m, 2H), 7.32 (dd, 2H, J=8.5, 7.3 Hz), 7.01 (tt,
1H, J=7.4, 1.2 Hz), 2.50 ppm (s, 6H); 13C NMR ([D6]DMSO,
125 MHz): d=152.4, 149.8, 142.2, 141.8, 140.7, 140.4, 139.7, 139.6,
139.5, 129.5, 128.8, 127.8, 127.7, 122.0, 118.3, 118.2, 19.8, 19.7 ppm;
MS (EI, 70 eV): m/z=386 [M]+.
1
(92%); H NMR ([D6]DMSO, 500 MHz): d=10.73 (s, 1H), 9.52 (s, 1H),
9.43 (s, 1H), 8.33- 8.27 (m, 3H), 7.87 (d, 2H, J=14.6 Hz), 7.79–7.73
(m, 3H), 7.54 (d, 1H, J=8.4 Hz), 7.05–7.03 (m, 1H), 7.49 ppm (d,
6H, J=3.4 Hz); 13C NMR ([D6]DMSO, 125 MHz): d=152.7, 152.0,
149.7, 146.9, 142.3, 141.0, 140.7, 140.3, 139.7, 139.6, 138.6, 130.2,
127.9, 127.7, 118.8, 117.7, 112.0, 19.8, 19.7 ppm; MS (EI, 70 eV): m/
z=369 [M]+.
1-(4-(6,7-Dimethylquinoxalin-2-yl)phenyl)-3-(3-phenoxypyridin-
2-yl)urea (12r): The title compound 12r was obtained as a yellow
solid (63 mg, 42%) using the same procedure as described for 12b
starting from 3-phenoxypyridin-2-amine (64 mg, 0.35 mmol); HPLC:
tR =7.27 min (95%); 1H NMR ([D6]DMSO, 500 MHz): d=11.95 (s,
1H), 9.46 (s, 1H), 8.37–8.25 (m, 3H), 8.00 (dd, 1H, J=5.0, 1.4 Hz),
7.95–7.87 (m, 2H), 7.84 (d, 1H, J=8.8 Hz), 7.59 (d, 1H, J=1.5 Hz),
7.55 (dd, 1H, J=8.1, 1.4 Hz), 7.48–7.41 (m, 2H), 7.40–7.35 (m, 1H),
7.09 (dd, 1H, J=8.0, 5.0 Hz,), 5.31 (s, 2H), 2.52 (s, 3H), 2.51 ppm(s,
3H); 13C NMR ([D6]DMSO, 125 MHz): d=175.1, 171.8, 167.4, 166.1,
161.0, 150.7, 143.1, 142.4, 140.8, 140.0, 137.1, 136.1, 128.5, 128.2,
128.00, 127.8, 123.2, 121.2, 119.9, 119.4, 117.9, 112.9, 109.2, 19.8,
19.8 ppm.
1-(2,6-Dimethylphenyl)-3-(4-(6,7-dimethylquinoxalin-2-yl)pheny-
l)urea (12m): The title compound 12m was obtained as a brown
solid (81 mg, 42%) using the same procedure as described for 12b
starting from 2,6-dimethylaniline (59 mg, 0.49 mmol); HPLC: tR =
1
7.41 min (99%); H NMR ([D6]DMSO, 500 MHz): d=9.42 (s, 1H), 9.24
(s, 1H), 8.25 (dd, J=9.0, 2.8 Hz, 2H), 7.98 (s, 1H), 7.87 (d, J=
10.5 Hz, 2H), 7.72–7.66 (m, 2H), 6.98 (d, J=7.4 Hz, 1H), 6.80 (t, J=
7.5 Hz, 1H), 2.50 (s, 3H), 2.49 (s, 3H), 2.25 ppm (s, 3H); 13C NMR
([D6]DMSO, 125 MHz): d=152.96, 149.85, 142.40, 142.24, 140.67,
140.37, 139.63, 139.48, 135.53, 135.12, 129.05, 128.36, 127.88,
127.81, 127.71, 126.02, 117.88, 19.82, 18.25, 17.69 ppm; MS (EI,
70 eV): m/z=396 [M]+.
1-(4-(6,7-Dimethylquinoxalin-2-yl)phenyl)-3-o-tolylurea
(12n):
6,7-Dimethoxy-2-(4-nitrophenyl)quinoxaline (16a): Pd/C (10%,
The title compound 12n was obtained as a brown solid (81 mg,
30 mg) was added under argon to a stirred solution of 1,2-dime-
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thoxy-4,5-dinitrobenzene 14a (100 mg, 0.44 mmol) in CH3OH
(5 mL). The mixture was flushed with hydrogen and stirred 4 h at
RT. Afterward the suspension was filtered through a pad of Celite
and the solvent was concentrated in vacuo to 1 mL. This solution
was added to a second solution of 2-(4-nitrophenyl)-2-oxoacetal-
dehyde 7 (74 mg, 0.45 mmol) in DMF (1.5 mL) and stirred for 1 h
at 808C. After cooling to RT, H2O (1 mL) was added, and the ob-
tained brown solid was filtered, dried and gave 61 mg (44%);
HPLC: tR =6.87 min; 1H NMR (CDCl3, 500 MHz): d=9.18 (s, 1H),
8.40 (m, 2H), 8.45 (m, 2H), 7.44 (d, 2H, J=3.0 Hz), 4.11 ppm (d,
6H, J=2.5 Hz); 13C NMR (CDCl3, 125 MHz): d=154.0, 154.0, 148.7,
147.2, 143.2, 140.0, 139.8, 139.4, 128.0, 124.4, 107.1, 106.4, 56.7,
56.7 ppm.
added to a solution of 7-methoxy-2-(4-nitrophenyl)quinoxaline
16b (220 mg, 0.78 mmol) in EtOH (10 mL) and stirred for 1 h at
808C. After cooling to RT, the solvent was removed and the residu-
al amount was diluted in CH2Cl2 and was washed with H2O. The or-
ganic layer was dried (MgSO4), filtered and the solvent was re-
moved in vacuo. After purification by silica gel column chromatog-
raphy (cyclohexane/EtOAc 1:1) 153 mg (78%) of yellow solid was
obtained; HPLC: tR =2.43 min; 1H NMR ([D6]DMSO, 500 MHz): d=
9.23 (s, 1H), 8.07–8.04 (m, 2H), 7.89 (d, 1H, J=9.0 Hz), 7.36–7.32
(m, 2H), 6.73–6.70 (m, 2H), 5.72 (s, 2H), 3.95 ppm (s, 3H); 13C NMR
([D6]DMSO, 125 MHz): d=160.4, 151.3, 151.2, 143.3, 140.2, 136.1,
129.7, 128.5, 123.0, 120.7, 113.8, 106.7, 55.7 ppm.
1-(5-tert-Butylisoxazol-3-yl)-3-(4-(7-methoxyquinoxalin-2-yl)phe-
nyl)urea (18b): To solution of 5-tert-butylisoxazol-3-amine 10
(50 mg, 0.36 mmol) in CH2Cl2 (5 mL) and triphosgene (36 mg,
0.12 mmol) was added dropwise at ꢀ108C a solution of Et3N
(100 mL, 0.72 mmol) in CH2Cl2 (1 mL). The mixture was stirred for
1 h at this temperature. Afterward a solution of 4-(7-methoxyqui-
noxalin-2-yl)aniline 17b (90 mg, 0.36 mmol) in toluene (2 mL) was
added and the mixture was stirred for 2 h at 1108C. After cooling
to RT, H2O (10 mL) was added and extracted with CH2Cl2 (3ꢂ
10 mL). The solid was diluted in a small amount of DMF and
heated at 1008C. H2O (1 mL) was added and the obtained light-
yellow solid was filtered to give 66 mg (44%); HPLC: tR =7.65 min
4-(6,7-Dimethoxyquinoxalin-2-yl)aniline (17a): Acetic acid
(0.45 mL), Fe (57 mg, 1.02 mmol) and FeCl3 (57 mg, 0.17 mmol)
were added to a solution of 6,7-dimethoxy-2-(4-nitrophenyl)qui-
noxaline 16a (53 mg, 0.17 mmol) in EtOH (2.5 mL) and stirred for
1 h at 808C. After cooling to RT, the obtained brown solid was fil-
tered and dried. The filtrate was extracted with CH2Cl2 (3ꢂ10 mL).
The combined organic layers were dried (MgSO4), filtered and the
solvent was removed in vacuo to give 46 mg (96%) of brown solid
and was used without further purification; HPLC: tR =1.73 min;
1H NMR ([D6]DMSO, 500 MHz): d=9.17 (s, 1H), 8.00 (d, 2H, J=
8.4 Hz), 7.35 (d, 2H, J=4.8 Hz), 6.70 (d, 2H, J=8.4 Hz), 5.59 (s, 2H),
3,96 ppm (d, 6H, J=8.9 Hz).
1
(99%); H NMR ([D6]DMSO, 500 MHz): d=9.59 (s, 1H), 9.37 (s, 1H),
9.09 (s, 1H), 8.31–8.32 (m, 2H), 7.98 (d, 1H, J=9.0 Hz), 7.69–7.66
(m, 2H), 7.47–7.43 (m, 2H), 6.54 (s, 1H), 3.98 (s, 3H), 1.31 ppm (s,
9H); 13C NMR ([D6]DMSO, 125 MHz): d=180.3, 160.6, 158.3, 151.2,
150.5, 143.2, 141.1, 140.5, 136.8, 130.1, 129.8, 128.1, 122.1, 118.5,
106.9, 92.5, 55.9, 28.3 ppm; MS (EI, 70 eV): m/z=417 [M]+.
1-(5-tert-Butylisoxazol-3-yl)-3-(4-(6,7-dimethoxyquinoxalin-2-yl)-
phenyl)urea (18a): To solution of 5-tert-butylisoxazol-3-amine 10
(30 mg, 0.21 mmol) in CH2Cl2 (2 mL) and triphosgene (16 mg,
0.05 mmol) was added dropwise at ꢀ108C a solution of Et3N
(58 mL, 0.42 mmol) in CH2Cl2 (1 mL). The mixture was stirred for 1 h
at this temperature. Afterward a solution of 4-(6,7-dimethoxyqui-
noxalin-2-yl)aniline 17a (46 mg, 0.16 mmol) in toluene (1.5 mL) was
added and the mixture was stirred for 2 h at 1108C. After cooling
to RT, H2O (10 mL) was added and extracted with EtOAc (3ꢂ
10 mL). The combined organic layers were dried (MgSO4), filtered
and the solvent was removed in vacuo. After purification by flash
chromatography (RediSep Column: 13 g C18 gold, H2O/CH3CN:
0%!95% CH3CN in 12 min) 23 mg (48%) of a yellow solid was ob-
Acknowledgements
The authors thank Christoph Scholz (Technische Universitꢀt
Darmstadt) for help with scripting. D.B. thanks the Stiftung Indus-
trieforschung (Essen, Germany) for support with a personal schol-
arship.
1
tained; HPLC: tR =7.17 min (100%); H NMR ([D6]DMSO, 500 MHz):
d=9.58 (s, 1H), 9.30 (s, 1H), 9.04 (s, 1H), 8.26–8.23 (m, 2H), 7.67–
7.64 (m, 2H), 7.42 (d, 2H, J=7.4 Hz), 6.54 (s, 1H), 3.99 (d, 6H, J=
7.2 Hz), 1.31 ppm (s, 9H); 13C NMR ([D6]DMSO, 125 MHz): d=180.3,
158.3, 152.8, 152.1, 151.2, 148.1, 140.5, 140.1, 138.6, 137.9, 130.5,
127.5, 118.5, 106.7, 106.6, 92.5, 56.0, 28.3 ppm; MS (EI, 70 eV): m/
z=447 [M]+.
Keywords: acute myeloid leukemia · drug discovery · FMS-like
tyrosine kinase 3 · inhibitors · zebrafish
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Received: November 6, 2014
Revised: December 2, 2014
Published online on February 10, 2015
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