Structure-activity relationship studies of pyrazolo[3,4-d]pyrimidine derivatives leading to the discovery of a novel multikinase inhibitor that potently inhibits FLT3 and VEGFR2 and evaluation of its activity against acute myeloid leukemia in vitro and in vivo

Article abstract of DOI:10.1021/jm301537p

We describe the structural optimization of a hit compound, 1-(4-(1H-pyrazolo[3,4-d]pyrimidin-4-ylamino)phenyl)-3-(3-methoxyphenyl)urea (1), which exhibits inhibitory activity but low potency against FLT3 and VEGFR2. A series of pyrazolo[3,4-d]pyrimidine derivatives were synthesized, and structure-activity relationship analysis using cell- and transgenic-zebrafish- based assays led to the discovery of a number of compounds that exhibited both high potency against FLT3-driven human acute myeloid leukemia (AML) MV4-11 cells and a considerable antiangiogenic effect in transgenic-zebrafish-based assays. The compound 1-(4-(1H-pyrazolo[3,4-d]pyrimidin -4-yloxy)phenyl)-3-(4-chloro-3- (trifluoromethyl)phenyl)urea (33), which exhibited the highest activity in preliminary in vivo anti-AML assays, was chosen for further anti-AML studies. The results demonstrated that compound 33 is a multikinase inhibitor that potently inhibits FLT3 and VEGFR2. In an MV4-11 xenograft mouse model, a once-daily dose of compound 33 at 10 mg/kg for 18 days led to complete tumor regression without obvious toxicity. Western blot and immunohistochemical analyses were performed to determine the mechanism of action of compound 33.

Full text of DOI:10.1021/jm301537p

Article
pubs.acs.org/jmc
Structure−Activity Relationship Studies of Pyrazolo[3,4‑d]pyrimidine
Derivatives Leading to the Discovery of a Novel Multikinase Inhibitor
That Potently Inhibits FLT3 and VEGFR2 and Evaluation of Its Activity
against Acute Myeloid Leukemia in Vitro and in Vivo
Ling-Ling Yang,†,‡,§ Guo-Bo Li,†,§ Shuang Ma,† Chan Zou,† Shu Zhou,† Qi-Zheng Sun,† Chuan Cheng,†
Xin Chen,† Li-Jiao Wang,† Shan Feng,† Lin-Li Li,† and Sheng-Yong Yang*,†
‡
†State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, and College of
Chemical Engineering, Sichuan University, Sichuan 610041, China
S
* Supporting Information
ABSTRACT: We describe the structural optimization of a hit
compound, 1-(4-(1H-pyrazolo[3,4-d]pyrimidin-4-ylamino)-
phenyl)-3-(3-methoxyphenyl)urea (1), which exhibits inhibitory
activity but low potency against FLT3 and VEGFR2. A series of
pyrazolo[3,4-d]pyrimidine derivatives were synthesized, and
structure−activity relationship analysis using cell- and trans-
genic-zebrafish-based assays led to the discovery of a number of
compounds that exhibited both high potency against FLT3-
driven human acute myeloid leukemia (AML) MV4-11 cells and
a considerable antiangiogenic effect in transgenic-zebrafish-based
assays. The compound 1-(4-(1H-pyrazolo[3,4-d]pyrimidin −4-
yloxy)phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea (33),
which exhibited the highest activity in preliminary in vivo anti-
AML assays, was chosen for further anti-AML studies. The results demonstrated that compound 33 is a multikinase inhibitor that
potently inhibits FLT3 and VEGFR2. In an MV4-11 xenograft mouse model, a once-daily dose of compound 33 at 10 mg/kg for
18 days led to complete tumor regression without obvious toxicity. Western blot and immunohistochemical analyses were
performed to determine the mechanism of action of compound 33.
A number of small-molecule FLT3 inhibitors have been
reported, and several, such as SU-11248, CHIR-258, PKC412,
CEP-701, MLN518, and AC220, have been investigated in
clinical trials.12−18 However, with few exceptions, such as
AC220, the clinical efficacy of these FLT3 inhibitors in patients
with AML has been unimpressive, with responses characterized
by transient clearance of leukemia blast cells, followed by
progressive disease or resistance to the treatment.9 The causes
of this unimpressive efficacy are complex, but a critical factor
may be aberrant VEGF signaling in the bone marrow of AML
patients, which promotes autocrine AML blast cell prolifer-
ation, survival, and chemotherapy resistance and mediates
paracrine vascular endothelial cell-controlled angiogenesis in
AML.19−24 More recently, researchers focusing on bone
marrow stem cell niches have demonstrated a role for VEGF
signaling in the preservation of several cell types within these
niches.23,25−28 Bone marrow niches have been proposed to be a
protective microenvironment for AML cells that could be
responsible for relapses in AML patients.19 These studies have
indicated that therapeutics targeting VEGF signaling could
1. INTRODUCTION
Acute myeloid leukemia (AML) is a malignant disorder of
hematopoietic cells that is characterized by the rapid growth of
abnormal white blood cells, which accumulate in the bone
marrow and hinder the development of normal blood cells.1
AML has become one of the leading causes of cancer-related
mortality worldwide. Recent studies regarding the pathogenesis
of AML have revealed that mutations and/or aberrant
expression of specific protein tyrosine kinases (PTKs) are
frequently responsible for the development of AML.2−5 Of
special note is the FMS-like tyrosine kinase 3 (FLT3), which is
a class III receptor tyrosine kinase. Activating mutations in
FLT3 kinase are found in up to one-third of AML cases; the
most prevalent activating mutations are “internal tandem
duplications” (ITDs) in the juxtamembrane domain that lead
to constitutive, ligand-independent activation of the kinase.6−8
Numerous studies have demonstrated that FLT3-ITD muta-
tions represent a driving mutation for the development of AML
and are associated with a poor prognosis for overall
survival.9−11 Therefore, FLT3 has been considered a valid
therapeutic target for AML treatment, and many pharmaceut-
ical companies and research institutes have been involved in the
discovery of FLT3 inhibitors.
Received: October 20, 2012
© XXXX American Chemical Society
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potentially prevent relapses of AML. Because VEGFR2 is the
key receptor of VEGF, we hypothesized that a dual FLT3/
VEGFR2 tyrosine kinase inhibitor could function in both
clearing leukemia blast cells and preventing relapses of AML.
To identify potent dual FLT3/VEGFR2 inhibitors, we
recently performed virtual screening against several commercial
chemical databases and an in-house chemical library, followed
by an in vitro kinase inhibition assay. From the hit compounds
obtained, we chose the compound 1-(4-(1H-pyrazolo[3,4-
d]pyrimidin-4-ylamino)phenyl)-3-(3-methoxyphenyl)urea (1;
Figure 1a) for further structural optimization. Compound 1
chloride with commercially available 1H-pyrazolo[3,4-d]-
pyrimidin-4(5H)-one (34) with a yield of 56% (Scheme 1).31
All the target compounds can be classified into two
categories: 4-N-pyrazolopyrimidine derivatives 1−5 and 4-O-
pyrazolopyrimidine derivatives 6−33. Scheme 2 depicts the
general synthetic route for the 4-N-pyrazolopyrimidine
derivatives 1−5. First, intermediates 38−42 were prepared by
a synthetic method similar to that described previously.32
Target compounds 1−5 were then obtained by a classic
nucleophilic substitution reaction between 38−42 and the
previously synthesized intermediate 35 in 1-butanol in the
presence of concentrated HCl in 74−80% yield.
The 4-O-pyrazolopyrimidine derivatives 6−9, 20, and 25−33
were obtained via the two routes outlined in Scheme 3.31,33 In
route 1, condensation of 4-aminophenol with the correspond-
ing isocyanates in THF provided the desired urea derivative
intermediates 62−75 in high yields with short reaction times.
Subsequent nucleophilic substitution of intermediate 35 with
the phenolic hydroxyl group of the urea derivatives 62−75 in
the presence of sodium hydroxide produced the target
compounds 6−9, 20, and 25−33 in low yield (see Scheme
3). In route 2, 4-aminophenol initially forms 4-aminophenolate
in a sodium hydroxide solution. Selective nucleophilic
substitution reaction of phenolate anion (bearing an oxygen
anion) with the chloride of compound 35 resulted in
intermediate 76 in a yield of 32% since the nucleophilicity of
the oxygen anion is relatively stronger than that of nitrogen in
4-aminophenol.33 Then target compounds 6−9, 20, and 25−33
were produced in excellent yield by the reaction of intermediate
76 with the corresponding isocyanates under reflux in
acetonitrile (see Scheme 3). The second route gave high yields
and thus was adopted here. In addition, intermediate 76 was
reacted with commercially available 4-bromobenzoic acid and
2-(4-bromophenyl)acetic acid to give compounds 18 and 19,
respectively, which contain an amide or acetamide moiety
(Scheme 3).
Compounds 10 and 11, which contain a methyl substituent
at the 1-NH of pyrazolopyrimidine, were prepared from the
corresponding analogues 6 and 9 (Scheme 4). In this reaction,
sodium hydroxide was used to deprotonate the NH group in
DMF at ambient temperature. Iodomethane was then added,
and the mixture continued to react overnight.
The 4-O-pyrazolopyrimidine derivatives 12−17 were synthe-
sized as outlined in Scheme 5. Urea compounds 12 and 13
were obtained via the second route (see Scheme 3), as
described above. Compounds 14−17 were obtained following
the condensation of commercially available carboxylic acids and
intermediate 77 in a mixture of 1.0 equiv of HOBT, 1.0 equiv
of EDCI, and 1.5 equiv of DIEA. Compounds 12−17 were all
obtained in high yields.
Figure 1. (a) Structure of compound 1. (b) Schematic showing
subgroups or atoms that were the focus of structural modifications.
was chosen because the 1-(4-(1H-pyrazolo[3,4-d]pyrimidin-4-
ylamino)phenyl)-3-phenylurea scaffold is novel for FLT3 and
VEGFR2 inhibitors (no FLT3 or VEGFR2 inhibitors possess
this scaffold) and has a relatively low molecular weight (375
Da).29,30 Compound 1 inhibits both FLT3 and VEGFR2 with a
half-maximal inhibitory concentration (IC50) of 1.278 μM for
FLT3 and 0.305 μM for VEGFR2. Thus, the potency of
compound 1 against FLT3 and VEGFR2 is poor and must be
optimized further. The purpose of this study was to perform
structural modifications to optimize the potency of compound
1 against both FLT3 and VEGFR2. A series of novel
pyrazolo[3,4-d]pyrimidine derivatives were synthesized and
tested for their in vitro anti-AML activity and antiangiogenic
effect. The most potent derivatives were then subjected to
further in vitro and in vivo assays. In this paper, we report the
chemical synthesis and structure−activity relationship (SAR)
studies of this novel series of compounds, as well as the in vitro
and in vivo anti-AML activities of the most active compounds.
2. CHEMISTRY
To explore the SAR of the pyrazolo[3,4-d]pyrimidine
compound class, a series of derivatives were synthesized
according to the synthetic routes outlined in Schemes 1−6.
All of the target compounds were prepared from the general
intermediate 4-chloro-1H-pyrazolo[3,4-d]pyrimidine (35),
which was obtained through reaction of phosphorus oxy-
Scheme 6 depicts the general reaction route of compounds
21−24. Intermediate 76, which was obtained via route 2 in
Scheme 3, was reacted with various isocyanates to give the final
products with yields ranging from 44% to quantitative.
a
Scheme 1
3. RESULTS AND DISCUSSION
3.1. SAR of Pyrazolo[3,4-d]pyrimidine Derivatives
Using Cell- and Transgenic-Zebrafish-Based Assays.
For SAR analysis, cell- and transgenic-zebrafish-based assays
were used. To screen FLT3 kinase inhibitors, two tumor cell
lines, MV4-11 and HeLa, were chosen. MV4-11 is a human
AML cell line whose viability depends on the activation of
mutated FLT3 kinase (FLT3-ITD).34−37 HeLa is a human
a
Reagents and conditions: (a) phosphorus oxychloride, DIEA,
toluene, reflux, 56%.
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a
Scheme 2
a
Reagents and conditions: (b) triphosgene, Et3N, THF, rt; (c) RNH2, THF, 45 °C, 72−96% for two steps; (d) Fe, NH4Cl, EtOH, H2O, 91−98%;
(e) HCl, 1-BuOH, 100 °C, 74−80%.
a
Scheme 3
a
Reagents and conditions: (f) triphosgene, THF, Et3N, rt; 4-aminophenol, THF, 45 °C, 64−99%; (g) sodium hydroxide, H2O, THF, 60 °C, 21−
34%; (h) 4-aminophenol, sodium hydroxide, H2O, THF, 60 °C, 32%; (i) substituted anilines, triphosgene, THF, Et3N, rt; acetonitrile, reflux, 61−
84%; (j) HOBT, EDCI, DIEA, THF, rt to reflux, 66−73%.
cancerous cervical tumor cell line whose viability is
independent of FLT3; Hela cells were used to rule out activity
due to non-FLT3-mediated effects (“off-target” or cellular toxic
effects). Compounds are likely better FLT3 inhibitors if they
have high potency against MV4-11 cells but low or no activity
against HeLa cells. To screen VEGFR2 kinase inhibitors, an in
vivo live fluorescent transgenic zebrafish (FLK-1:EGFP) assay
was adopted; VEGFR2 is one of the key regulators of
angiogenesis, and the transgenic zebrafish assay has been
utilized as a less costly and more rapid in vivo method for the
screening of agents with antiangiogenic activity.38−41 The
combination of cell- and transgenic-zebrafish-based assays is an
effective method for identifying dual inhibitors of FLT3 and
VEGFR2. There are some advantages of performing SAR
analysis with cell- and transgenic-zebrafish-based assays rather
than enzymatic assays for hit/lead optimization. For example,
SAR analysis based on cellular assays may permit the evaluation
of both the intrinsic activity of the compounds against the
C
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a
a
Scheme 4
Scheme 6
a
Reagents and conditions: (k) iodomethane (MeI), sodium hydroxide,
a
Reagents and conditions: (n) amine, triphosgene, THF, Et3N, rt;
acetonitrile, reflux, 44−77%.
DMF, rt, 65−70%.
protein target and the ability of the compounds to permeate the
cell wall. The transgenic zebrafish assay combines the
physiological complexity of an in vivo vertebrate model with
the speed of high-throughput screening. Thus, SAR studies
based on both cell (in vitro) and zebrafish (in vivo) assays can
potentially accelerate the hit/lead optimization process to
rapidly obtain an agent with bioactivity both in vitro and in
vivo. Certainly, there may exist some possibility of off-target
effects contributing to cell growth inhibitory potency or
antiangiogenic activity, although some strategies, such as the
use of Hela cells, have been adopted to rule out this possibility.
Thus, enzymatic assays were also performed but only on
compounds with higher potency in cell and zebrafish assays.
The most active compounds in the enzyme, cell, and zebrafish
assays were selected for further in vivo experiments to evaluate
their anti-AML activity.
The SAR analysis below focuses on four positions: the 4-
position linker atom (Y), the N-1 position of pyrazolo[3,4-
d]pyrimidine (R1), the bridge group that connects rings A and
B, and the ring B moiety (see Figure 1b).
3.1.1. Impact of the 4-Position Linker Atom (Y) of
Pyrazolo[3,4-d]pyrimidine. Table 1 shows the bioactivities of
two series of 1H-pyrazolo[3,4-d]pyrimidine derivatives, namely,
4-anilino-1H-pyrazolo[3,4-d]pyrimidine and 4-phenoxy-1H-
pyrazolo[3,4-d]pyrimidine derivatives, which contain a nitrogen
(−NH) and oxygen (−O) atom at the 4-position linker site,
respectively. The cell growth inhibitory activities (IC50) against
MV4-11 of the 4-phenoxy-1H-pyrazolo[3,4-d]pyrimidine de-
rivatives 6, 7, 8, 9, and 22 were 0.020, 0.083, 0.037, 0.005, and
1.077 μM, respectively, corresponding to a higher potency than
that of the corresponding 4-anilino-1H-pyrazolo[3,4-d]-
pyrimidine derivatives 1, 2, 3, 4, and 5 (IC50 = 1.65, 1.24,
1.69, 0.26, and 2.05 μM, respectively). Here one may argue that
the lower MV4-11 cell growth inhibitory potency observed with
compounds 1−5 might be due to poorer cell permeability. To
clarify this issue, we tested the enzymatic inhibitory potency of
compounds 1−5 against the FLT3 kinase, and the results
showed that compounds 1−5 had a poor FLT3 kinase
inhibitory potency (1 μM < IC50 < 10 μM). These results
together with the fact that many of their analogues displayed a
higher MV4-11 cell growth inhibitory potency (see below)
demonstrated that the lower MV4-11 cell growth inhibitory
potency observed with compounds 1−5 should not be, at least
not mainly, due to poorer cell permeability. All of the 1H-
pyrazolo[3,4-d]pyrimidine derivatives had IC50 values greater
than 10 μM against HeLa cells (the same phenomenon will be
seen below), indicating that the cell growth inhibitory activities
of these compounds against the MV4-11 cells were not due to
cellular toxicity. By contrast, all of the 4-phenoxy-1H-pyrazolo-
[3,4-d] pyrimidine derivatives also exhibited slightly more
potent antiangiogenic activity in zebrafish assays than did the
corresponding 4-anilino-1H-pyrazolo[3,4-d]pyrimidine deriva-
tives. Thus, we can conclude that the 4-phenoxy-1H-pyrazolo-
[3,4-d]pyrimidine derivatives are more potent than the 4-
anilino-1H-pyrazolo[3,4-d]pyrimidine derivatives, in terms of
their bioactivities in both cell and zebrafish assays.
3.1.2. Substitution Effect of the N-1 Position (R1) of
Pyrazolo[3,4-d]pyrimidine. The possible influence of the
a
Scheme 5
a
Reagents and conditions: (h) 3-aminophenol, sodium hydroxide, THF, H2O, 60 °C, 38%; (i) for 12 and 13, substituted aniline, triphosgene, THF,
Et3N, rt; acetonitrile, reflux, 87−89%; for 14 and 15, HOBT, EDCI, DIEA, THF, rt to reflux, 71−74%; (m) HOBT, EDCI, DIEA, THF, rt to reflux,
79−86%.
D
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Table 1. Cell Growth Inhibitory Activities of Pyrazolo[3,4-d]pyrimidine Derivatives with Different 4-Position Linker Atoms (Y)
and Different Substituents at the N-1 Position (R1) of Pyrazolo[3,4-d]pyrimidine against FLT3-ITD-Dependent MV4-11 Cells
and FLT3-Independent HeLa Cells, as Well as Their Antiangiogenic Activities in Transgenic-Zebrafish-Based Assays
a
Each compound was tested in triplicate; the data are presented as the mean SD.
substitution of the N-1 position (R1) of pyrazolo[3,4-
d]pyrimidine was examined next. The N-1 hydrogen of the
pyrazolo[3,4-d]pyrimidine moiety of the two most potent 4-
phenoxy-1H-pyrazolo[3,4-d]pyrimidine derivatives (6 and 9)
was replaced by a methyl group, yielding compounds 10 and 11
(Table 1), respectively. Compounds 10 and 11 exhibited much
lower antiproliferative and antiangiogenic activities than their
unsubstituted counterparts 6 and 9, implying that substitution
at the N-1 position of pyrazolo[3,4-d]pyrimidine is not
beneficial for activity.
groups connecting ring A and ring B, including p-urea, m-urea,
p-amide, m-amide, p-acetamide, and m-acetamide, are presented
in Table 2.42 Replacement of the urea group by an amide or
acetamide led to a decreased bioactivity (12 vs 14 and 16; 13 vs
15 and 17; 7 vs 18 and 19) in the cellular assay. Furthermore, a
comparison of the bioactivities of the compound pairs [12
(4.53 μM), 9 (0.005 μM)] and [13 (7.82 μM), 7 (0.083 μM)],
in which the urea group is attached to the para- or meta-
position, respectively, indicated that substitution at the para-
position was preferred to substitution at the meta-position to
increase bioactivity in the cellular assay.
3.1.3. Influence of Different Bridge Groups Connecting
Ring A and Ring B. The bioactivities of a series of 4-phenoxy-
1H-pyrazolo[3,4-d]pyrimidine derivatives with different bridge
3.1.4. Influence of Variation of the Ring B Moiety. Table 3
presents the bioactivities of a series of 4-phenoxy-1H-
E
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based assays. The three most potent compounds, 9, 27, and 33,
displayed nanomolar potencies at the cellular level and low
micromolar potencies in the zebrafish assay. The kinase
inhibitory potencies (IC50) against FLT3 and VEGFR2, as
well as the binding affinity (Kd, dissociation constant) for
FLT3-ITD of the three compounds were then measured, and
the results are presented in Table 4. The three compounds are
all potent FLT3 and VEGFR2 inhibitors, and their kinase
inhibitory activities are at least as potent as that of sorafenib,
which is a multikinase inhibitor and can potently inhibit
VEGFR2 and FLT3; sorafenib is currently in clinical studies for
the treatment of AML.43,44 Of note is that compounds 27 and
33 showed lower nanomolar binding affinity (Kd) against the
FLT3-ITD mutant, which is at least 10 times more potent
compared with sorafenib. We subsequently performed a
preliminary in vivo anti-AML study in a xenograft MV4-11
mouse model. Compound 33 was the most active in this study
(see Figure 2). This increased activity could be due to the
potentially increased bioavailability of compound 33 compared
with the other compounds. Further in-depth in vitro (including
enzymatic and cellular assays) and in vivo anti-AML studies
were subsequently performed with compound 33.
Table 2. 4-Phenoxy-1H-pyrazolo[3,4-d]pyrimidine
Derivatives with Different Bridge Groups Connecting Rings
A and B, Together with Their Cell Growth Inhibitory
Activities against FLT3-ITD-Dependent MV4-11 Cells and
FLT3-Independent HeLa Cells and Their Zebrafish-Based
Antiangiogenic Activities
3.2. Enzymatic Activity of Compound 33 against
Various Kinases. The kinase inhibition profile of compound
33 against a panel of selected recombinant human protein
kinases is presented in Table 5. Compound 33 potently
inhibited FLT3 and VEGFR2 kinases (IC50 = 0.039 μM for
FLT3 and 0.012 μM for VEGFR2). Compound 33 also exhibits
considerable potency against several other kinases, including c-
RAF (0.072 μM), PDGFRα (0.223 μM), PDGFRβ (0.408
μM), c-Kit (0.507 μM), and FGFR2 (1.805 μM). Compound
33 displayed almost no inhibitory activity against 23 other
tested protein kinases. These data demonstrate that compound
33 is a multikinase inhibitor that potently inhibits FLT3 and
VEGFR2 kinases.
Furthermore, a primary kinase selectivity assay was
performed through measuring the binding affinities of
compound 33 with a large set of representative kinases. A
total of 131 kinases were selected; these kinases cover all of the
kinase subfamilies, and most of them are common kinases.
Compound 33 was screened against the kinase set at a fixed
concentration of 10 μM. The results, shown in Figure S1 and
Table S1 (see the Supporting Information), indicate that
compound 33 also has a considerable binding affinity for a few
of the kinases in addition to FLT3 and VEGFR2. The
calculated selectivity scores, S(1), S(10), and S(35), whose
definitions are given in the Experimental Section, are 0.076,
0.122, and 0.206, respectively.
3.3. In Vitro Cell Growth Inhibitory Activity of
Compound 33. The cell growth inhibitory potency of
compound 33 against various leukemia and solid tumor cell
lines was examined, and the results are presented in Table 6. As
noted previously, compound 33 potently inhibits the viability of
FLT3-driven AML MV4-11 cells, with an IC50 value of 0.004
μM (also see Figure 3a). It exhibited weak inhibitory activity
against several other cell lines, including Jurkat (leukemia, IC50
= 8.72 μM), MKN45 (gastric cancer, IC50 = 8.23 μM), MDA-
MB-468 (breast cancer, IC50 = 6.68 μM), and TT (thyroid
cancer, IC50 = 3.42 μM). Negligible activity against the
remaining 16 human cancer cell lines was observed. These
results indicate that compound 33 is relatively selective for the
human AML cell line MV4-11.
a
Each compound was tested in triplicate; the data are presented as the
mean SD.
pyrazolo[3,4-d] pyrimidine derivatives containing a variety of
moieties at the ring B position. From Table 3, we can see that,
compared with other moieties at the ring B position, including
a six-membered heteroaromatic ring (21) and other six-
membered cyclic groups (22−24), a phenyl group at the ring
B position yielded a more potent compound (20) in both the
cell and zebrafish assays. We then explored the influence of
different substituents on the phenyl ring (ring B). It is clear
that, compared with compound 20, which has no substituent
on ring B, substitution at the meta- and/or para-position of ring
B considerably increased the potency of cell growth inhibition
against MV4-11. Substitution on ring B also increased the
antiangiogenic potency, with the exception of compound 26,
which contains a morpholine ring. Furthermore, for both the
cell growth inhibitory activity against MV4-11 and antiangio-
genic activity, compounds with substituents at the meta-
position were identically potent or slightly more potent
compared with compounds with substituents at the para-
position (see 27 vs 9, 29 vs 25).
Collectively, the structural optimization and SAR studies led
to the discovery of a number of compounds that exhibited both
high potency against FLT3-driven human AML MV4-11 cells
and a considerable antiangiogenic effect in transgenic-zebrafish-
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Table 3. 4-Phenoxy-1H-pyrazolo[3,4-d]pyrimidine Derivatives Containing Various Moieties at the Ring B Position Together
with Their Cell Growth Inhibitory Activities against FLT3-ITD-Dependent MV4-11 Cells and FLT3-Independent Hela Cells
and Zebrafish-Based Antiangiogenic Activities
a
Each compound was tested in triplicate; the data are presented as the mean SD.
G
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concentration of 0.10 μM, an apoptosis rate of 35.8% was
observed.
Table 4. Kinase Inhibitory Potency against FLT3 and
VEGFR2 for the Most Active Compounds in Cell and
Zebrafish Assays
The ability of compound 33 to inhibit the activation of FLT3
and downstream signaling proteins in intact cells was assessed
by Western blot analysis. After a 5 h treatment with increasing
concentrations of compound 33, MV4-11 cells were harvested
and lysed for an IP/wt assay. As shown in Figure 3c, compound
33 inhibited FLT3 phosphorylation in a dose-dependent
manner. Consistent with the downregulation of the phosphor-
ylation of FLT3, the phosphorylation of the downstream
signaling proteins STAT5 and ERK1/2 was also significantly
inhibited at concentrations of compound 33 >0.003 μM
(Figure 3c). The observed IC50 value was approximately 0.003
μM for phosphorylation of FLT3, 0.003 μM for STAT5, and
0.001 μM for Erk1/2.
compd
FLT3 IC50, μM
VEGFR2 IC50, μM
FLT3-ITD Kd, μM
9
0.032
0.009
0.039
0.033
0.038
0.011
0.012
0.090
0.91
27
0.0066
0.0043
0.079
33
sorafenib
Here we have to point out that inhibition of VEGF signaling
in intact MV4-11 cells was not measured because MV-411 cells
express very low levels of VEGFR2, below that which can be
detected by Western blot. VEGFR2 inhibition should not
significantly contribute to the cell viability of MV4-11 cells. As
mentioned before, aberrant VEGF signaling often exists in the
bone marrow of AML patients, which has been demonstrated
to be associated with drug resistance and relapses of AML after
treatment with FLT3 inhibitors. Blockade of VEGF signaling is
expected to play important roles in preventing drug resistance
and relapses of AML. Thus, we shall in the next section
examine the suppressive ability of compound 33 against the
VEGF signaling using transgenic-zebrafish-based assays.
3.5. Antiangiogenic Activity of Compound 33. The
effect of compound 33 on embryonic angiogenesis in zebrafish
was examined. Figure 4a shows 33hpf zebrafish embryos treated
with 1.25, 2.5, and 5 μM 33, as well as a blank control.
Treatment of live fish embryos with compound 33 completely
blocked the formation of intersegmental vessels (ISVs) at a
concentration of 5 μM while preserving fluorescence in the
doral aorta and major cranial vessels. At 1.25 or 2.5 μM 33, the
formation of intersegmental vessels was considerably inhibited
compared with that of the vehicle control group, indicating a
dose-dependent inhibition pattern. The IC50 value was
approximately 2.5 μM (Figure 4b).
Figure 2. Preliminary in vivo anti-AML assays. Daily oral
administration of compounds 9, 27, and 33 at concentrations of 5,
7.5, and 5 mg/kg/d, respectively, was initiated when the MV4-11
tumors reached approximately 400 mm3 in volume (three mice per
group).
Table 5. Kinase Inhibition Profile for Compound 33 against
Human FLT3/VEGFR2 and a Panel of Other Selected
Protein Kinases
kinase
Flt3
IC50, μM
kinase
IC50, μM
kinase
IC50, μM
0.039
0.012
0.072
0.223
0.408
0.507
1.805
>10
Pim-1
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
DMPK
ERN1
IGF1R
MLK1
MEK1
PAK1
PAK2
PAK4
ERK
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
VEGFR2
c-RAF
Syk
aurora A
aurora B
PLK1
PDGFRα
PDGFRβ
c-Kit
CDK2
CHEK1
CAMK4
CTK
FGFR2
EGFR
ErBB2
>10
ErBB4
>10
DLK
Lck
3.6. In Vivo Effects of 33 against sc MV4-11 Tumor
Xenografts. The in vivo anti-AML activity of compound 33
was evaluated using the FLT3-ITD-positive MV4-11 xenograft
model. When the tumor grew to a volume of 300−500 mm3,
the mice were grouped and treated orally once daily with 1, 3,
or 10 mg/kg/d of compound 33 for 18 days. The tumor
3.4. Apoptosis Assays and Signaling Inhibition in
MV4-11 Cells. A flow cytometry assay was performed to
examine cell apoptosis upon treatment with compound 33.
Compound 33 induced apoptosis in MV4-11 cells in a
concentration-dependent manner (see Figure 3b). At a
Table 6. Cell Growth Inhibitory Potency of Compound 33 against Various Leukemia and Solid Tumor Cell Lines
a
a
tumor type
cell line
MV4-11
IC50, μM
tumor type
liver cancer
cell line
Bel7402
IC50, μM
leukemia, AML
leukemia, ALL
leukemia, APL
leukemia, CML
cervical cancer
colon cancer
0.004 0.0004
8.72 0.440
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
Jurkat
liver cancer
SMMC7721
A549
HL60
lung cancer
K562
>10
lymphoma
Raji
Hela
>10
lymphoma
U937
HCT116
MKN45
MDA-MB-468
MCF-7
TT
>10
lymphoma
Karpass299
LoVo
gastric cancer
breast cancer
breast cancer
thyroid cancer
8.23 0.485
6.68 0.652
>10
colorectal carcinoma
prostate cancer
multiple myeloma
ovarian cancer
ovarian cancer
DU145
U266
3.42
SKOV3
SK
a
Each compound was tested in triplicate; the data are presented as the mean SD.
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Figure 3. (a) Growth inhibitory profile of compound 33 against MV4-11 cells using the MTT assay method. (b) A flow cytometry assay was
performed to examine cell apoptosis. MV4-11 cells (1 × 105) were harvested after treatment with various drug concentrations for 24 h. Cells were
stained with an Annexin V & PI Kit and analyzed by flow cytometry. Percentages in the bottom right quadrants are the ratio of cells in early
apoptosis (annexin V positive and PI negative). Percentages in the upper right quadrants are the ratio of cells in late apoptosis (Annexin V positive
and PI positive). (c) Western blot analysis was used to examine the phosphorylation of FLT3 and downstream signals in MV4-11 cells treated with
different doses of compound 33 for 20 h.
volumes were measured every 3 d. Treatment with compound
33 at 10 mg/kg/d resulted in rapid and complete tumor
regression in all mice in this group. Compound 33 treatment at
3 mg/kg/d stopped tumor growth and resulted in a slight
regression of tumor growth (Figure 5a). Moreover, throughout
the experiment, no significant weight loss or any other obvious
signs of toxicity were observed for any of the compound 33-
treated mice (Figure 5b).
demonstrated an obvious increase in the percentage of
apoptotic cells in a time-dependent manner (Figure 6b). In
addition, the phosphorylation of STAT5, which is an important
downstream signaling protein of FLT3 signaling, was also
significantly reduced in MV4-11 tumor tissues of the
compound 33-treated group compared with the vehicle group
(Figure 6c), indicating that the observed efficacy in vivo was
consistent with FLT3 signaling inhibition.
Compound 33 was also evaluated for its effects on the tumor
mitotic index (Ki67) and apoptosis, as well as the FLT3
signaling, using histological and immunohistochemical techni-
ques. Similar to the tumor xenograft models, a dose of 10 mg/
kg/d of compound 33 was administered by oral gavage for the
MV4-11 model. After treatment for 3 or 4 d, tumors were
collected and analyzed. Tumor tissues from the vehicle group
stained strongly with Ki67, indicating a large number of highly
proliferative cells. Conversely, the tumor tissues from the
compound 33-treated groups exhibited significantly fewer Ki67-
positive cells (Figure 6a). Furthermore, the TUNEL data
It is important to mention again that the MV4-11 cell line
expresses very low levels of VEGFR2. VEGFR2 inhibition
should not have a significant effect on the AML cell viability,
but is expected to contribute to blockade of aberrant VEGF
signaling that often exists in the bone marrow of AML patients,
which may be helpful in preventing relapses of AML. We
acknowledged that the in vivo MV4-11 xenograft model used
here is not suitable for examining the effects of compound 33
on preventing relapses of AML. Other more sophisticated
models such as orthotopic AML models should be adopted for
this purpose; the related studies are still under way.
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Figure 5. (a) Daily oral administration of compound 33 at
concentrations of 1, 3, and 10 mg/kg/d and sorafenib at a dose of 3
mg/kg/d was initiated when the MV4-11 tumors reached approx-
imately 500 mm3 in volume (six mice per group). (b) Average body
weights for 33-treated mouse groups in the MV4-11 xenograft model.
(c) Pharmacokinetics of compound 33 in SD rats (five rats per group).
The rats were treated orally with 10 mg/kg/d of compound 33, and
plasma was collected via jugular vein cannulation. Finally, compound
concentrations in the plasma were detected by liquid−liquid extraction
followed by LC−MS detection.
Figure 4. (a) 33hpf zebrafish embryos treated with blank control or
1.25, 2.5, or 5 μM 33. ISV budding and outgrowth were almost
completely inhibited by treatment with 5 μM 33. (b) Statistics of ISV
length in embryos treated with blank control and compound 33. ISVs
above the yolk extension were counted. Key: columns, mean; bars, SD
(n = 11; ANOVA; **, P < 0.01 vs the control).
once-daily dose of compound 33 at 10 mg/kg for 18 days led to
complete tumor regression in the MV4-11 xenograft model.
Preliminary pharmacokinetic studies indicated that compound
33 possesses good pharmacokinetic properties. By the way,
since the AML MV4-11 cell line used in this study expresses
very low levels of VEGFR2, inhibition of VEGFR2 should not
significantly contribute to the suppression of tumor cell growth,
but might play some important roles in preventing relapses of
AML due to the fact that aberrant VEGF signaling often exists
in the bone marrow of AML patients. Issues related to the
VEGFR2 inhibition and its roles in preventing the relapses of
AML still need further intense studies.
3.7. Pharmacokinetic Characteristics of Compound
33. The preliminary pharmacokinetic characteristics of
compound 33 following po administration to rats were also
analyzed. As shown in Figure 5c, the compound was absorbed
well, achieving a maximum plasma level (Cmax) of 15.12 μg/mL
within 7 h after oral administration at a dose of 10 mg/kg/d.
The measured plasma protein binding value was 87.96%.
Furthermore, the apparent plasma half-life was approximately
12.6 h, the AUC0−24h was approximately 318817.4 μg/(L·h),
and the oral bioavailability was about 66.7%. All of these
indicated that compound 33 has good pharmacokinetic
properties.
5. EXPERIMENTAL SECTION
5.1. Chemistry Methods. All reagents were purchased and used
without further purification unless otherwise indicated. Reactions were
monitored by thin-layer chromatography (TLC) on Merck silica gel 60
F-254 thin-layer plates. All of the final compounds were purified to
>95% purity, as determined by high-performance liquid chromatog-
raphy (HPLC). HPLC analysis was performed on a Waters 2695
HPLC system with the use of a Kromasil C18 column (4.6 mm × 250
4. CONCLUSIONS
In this paper, a series of pyrazolo[3,4-d]pyrimidine derivatives
was synthesized, and an SAR analysis of these compounds
based on cell and zebrafish assays led to the identification of
three pyrazolo[3,4-d]pyrimidine derivatives with considerable
potency. Further in-depth in vitro and in vivo assays were
performed with compound 33, which exhibited the highest in
vivo anti-AML activity in a preliminary in vivo assay. The kinase
inhibition profile revealed that compound 33 was a multikinase
inhibitor that potently inhibits FLT3 and VEGFR2 and showed
good kinase spectrum selectivity. Western blot analysis
demonstrated that compound 33 significantly inhibited FLT3
phosphorylation and the activation of downstream signaling
proteins. In vivo anti-AML activity assays showed that an oral,
1
mm, 5 um). H NMR and 13C NMR spectra were recorded on a
Bruker AV-400 spectrometer at 400 and 100 MHz, respectively.
Chemical shifts (δ) are listed in parts per million (ppm) relative to
tetramethylsilane (TMS) as an internal standard. Multiplicities are
given as s (singlet), d (doublet), dd (double−doublet), t (triplet), q
(quadruplet), m (multiplet), and br s (broad signal). Low-resolution
and high-resolution mass spectral (MS) data were acquired on an
Agilent 1100 series LC−MS instrument with UV detection at 254 nm
in low-resonance electrospray mode (ESI).
5.1.1. 4-Chloro-1H-pyrazolo[3,4-d]pyrimidine (35). To a cooled
solution of 1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (34; 5.0 g, 36.7
mmol) and N,N-diisopropylethylamine (3.0 mL, 18.4 mmol) in
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Figure 6. Ki67, TUNEL, and p-STAT5 detection of MV4-11 tumor tissues after treatment with compound 33 for 72 or 96 h. At 24 h after the final
dose of compound 33, the tumor samples were collected for analysis.
toluene was slowly added phosphorus oxychloride (16.8 mL, 183.5
mmol). The reaction mixture was warmed to room temperature and
refluxed for 5 h. After evaporation of the organic solvent, the residue
was cooled to room temperature and treated with excess ice−water. At
this time, a buff solid was formed and collected by filtration. The crude
product was dried in a vacuum oven to give the title compound 35
(3.2 g, 56%). This product was used in the next step without
purification. 1H NMR (400 MHz, DMSO-d6): δ 14.12 (s, 1H), 9.32 (s,
1H), 7.55 (s, 1H). LC−MS: m/z 155.0 [M + H]+.
5.1.2. 1-(4-(1H-Pyrazolo[3,4-d]pyrimidin-4-ylamino)phenyl)-3-(3-
methoxyphenyl)urea (1). To a mixture of 1-(4-aminophenyl)-3-(3-
methoxyphenyl)urea (180 mg, 0.7 mmol) in 1-butanol (10 mL) was
added 6 N HCl (0.2 mL). The reaction mixture was stirred at room
temperature for 20 min. Then a solution of 35 (108 mg, 0.7 mmol) in
1-butanol (10 mL) was added to the reaction mixture, followed by
heating to 100 °C for 3.5 h. The solvent was removed by rotary
evaporation. The crude mixture was added to 100 mL of water, and
the pH was adjusted to 7−8 with K2CO3. The aqueous layer was
extracted with ethyl acetate (2 × 120 mL). Then the combined organic
layers were dried over MgSO4 and concentrated. The residue was
purified by column chromatography (eluent gradient CH2Cl2:MeOH
= 35:1 to 20:1) and recrystallized from EtOAc and petroleum to afford
the product 1 (205 mg, 78% yield, 98% HPLC purity). 1H NMR (400
MHz, DMSO-d6): δ 13.59 (s, 1H), 9.93 (s, 1H), 8.69 (d, J = 5.6 Hz,
2H), 8.35 (s, 1H), 8.15 (br s, 1H), 7.71 (d, J = 8.0 Hz, 2H), 7.47 (d, J
= 8.8 Hz, 2H), 7.21−7.16 (m, 2H), 6.93 (d, J = 8.0 Hz, 1H), 6.55 (dd,
J = 2.4 Hz, J = 5.6 Hz, 1H), 3.74 (s, 3H). 13C NMR (100 MHz,
DMSO-d6): δ 160.2, 155.8, 155.0, 153.0, 141.5, 133.8, 132.9, 130.0,
122.8, 119.1, 110.9, 107.6, 104.3, 100.8, 55.4. LC−MS: m/z 376.1 [M
+ H]+.
in vacuo. The residue obtained was dissolved in acetone (4 mL), and
upon addition of water, a solid precipitated. This solid was collected,
washed with water and Et2O, and dried under vacuum to give the
products 62−75 (64−99%).
5.1.4. 4-(1H-Pyrazolo[3,4-d]pyrimidin-4-yloxy)aniline (76). To a
solution of 4-aminophenol (3.53 g, 32.4 mmol) in H2O (40 mL) was
added a sodium hydroxide solution (1.30 g, 32.4 mmol in 40 mL of
water). The mixture was stirred at room temperature for 30 min. Then
the intermediate 35 (5.00 g, 32.4 mmol) in THF (40 mL) was slowly
added, and the reaction mixture was heated to 60 °C for 1.5 h. The
solvent was then partially evaporated on a rotary evaporator. The
crude mixture was extracted with ethyl acetate (2 × 120 mL) and
water. The combined organic layers were dried over MgSO4 and
concentrated. The obtained residue was purified by column
chromatography (eluent gradient CH2Cl2:MeOH = 100:1 to 60:1)
without final recrystallization to give the product 76 (2.35 g, 32%) as a
1
brown solid. H NMR (400 MHz, DMSO-d6): δ 14.07 (s, 1H), 8.50
(s, 1H), 7.67 (s, 1H), 6.96 (d, J = 8.8 Hz, 2H), 6.64 (d, J = 8.8 Hz,
2H), 5.20 (s, 2H). LC−MS: m/z 228.1 [M + H]+.
5.1.5. 1-(4-(1H-Pyrazolo[3,4-d]pyrimidin-4-yloxy)phenyl)-3-(3-
methoxyphenyl)urea (6). Two methods were used to obtain the
final product. Method A: A suspension of 1-(4-hydroxyphenyl)-3-(3-
methoxyphenyl)urea (0.35 g, 1.35 mmol) and sodium hydroxide
(0.065 g, 1.62 mmol) in THF (10 mL) and water (2 mL) was stirred
at room temperature for 30 min. Then the intermediate 35 (0.31 g, 2.0
mol) was added, and the resulting mixture was heated to 60 °C for 6 h.
The solvent was concentrated in vacuo, treated with water (100 mL),
and extracted with EtOAc (3 × 60 mL). The combined organic layers
were removed in vacuo. The residue obtained was purified by column
chromatography (eluent gradient CH2Cl2:MeOH = 70:1) and
recrystallized from EtOAc and petroleum ether to provide 6 (0.13 g,
26.3% yield, 98% HPLC purity). Method B: A solution of 3-
methoxyaniline (0.62 g, 5.0 mmol) dissolved in THF (40 mL) was
slowly dripped into a stirred solution of triphosgene (1.49g, 5.0 mmol)
in THF (10 mL) by using a constant-pressure dropping funnel. NEt3
(1.5 mL, 10.5 mmol) was then added slowly to the reaction mixture
after the aniline was added. After evaporation of the solvent, the
residue was taken up in acetonitrile (80 mL), and compound 76 (1.02
g, 4.5 mmol) was added directly to the residue. Next the reaction
mixture was stirred at 90 °C for 6 h, and the solvent was removed in
vacuo. The residue obtained was purified by column chromatography
(eluent gradient CH2Cl2:MeOH = 70:1) and recrystallized from
Compounds 2−5 were synthesized using the same methodology.
Experimental data for compound 2−5 are given in the Supporting
Information.
5.1.3. General Procedure for the Synthesis of Ureas 62−75. A
solution of substituted anilines (10.0 mmol) dissolved in THF (80
mL) was slowly dripped into a stirred solution of triphosgene (2.98 g,
10.0 mmol) in THF (10 mL) by using a constant-pressure dropping
funnel. NEt3 (3 mL, 21.0 mmol) was then added slowly to the reaction
mixture after the anilines were added. After evaporation of the solvent,
the residue was taken up in THF (80 mL), and 4-aminophenol (0.93
g, 8.5 mmol) was added directly to the residue. The reaction mixture
was stirred at 45 °C for 2 h, and the solvent was subsequently removed
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EtOAc and petroleum ether to provide 6 (1.37 g, yield 81%, 98%
HPLC purity). 1H NMR (400 MHz, DMSO-d6): δ 14.14 (s, 1H), 8.79
(s, 1H), 8.73 (s, 1H), 8.51 (s, 1H), 8.03 (s, 1H), 7.60 (d, J = 8.8 Hz
2H), 7.26−7.21 (m, 4H), 6.96 (d, J = 8.0 Hz, 1H), 6.57 (dd, J = 2.0
Hz, J = 6.0 Hz, 1H), 3.74 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ
163.2, 159.6, 156.7, 154.9, 152.5, 146.4, 140.8, 137.5, 131.8, 129.5,
122.2, 119.4, 110.5, 107.2, 104.0, 101.3, 54.9. LC−MS: m/z 377.1 [M
+ H]+.
purity: 98%. 1H NMR (400 MHz, DMSO-d6): δ 14.14 (s, 1H), 9.10 (s,
1H), 8.93 (s, 1H), 8.51 (s, 1H), 8.04 (s, 2H), 7.61−7.51 (m, 4H), 7.32
(d, J = 7.6 Hz, 1H), 7.26 (d, J = 8.8 Hz, 2H). 13C NMR (100 MHz,
DMSO-d6): δ 163.2, 156.7, 154.9, 152.6, 146.6, 140.5, 137.2, 131.8,
129.9, 122.3, 121.8, 119.7, 101.3. HRMS: m/z calcd for C19H13F3N6O2
[M + H]+ 415.1052, found 415.1062.
5.1.11. 1-(4-(1H-Pyrazolo[3,4-d]pyrimidin-4-yloxy)phenyl)-3-(4-
chloro-3-(trifluoromethyl)phenyl)urea (33). The title compound
was prepared from 76 and 4-chloro-3-(trifluoromethyl)aniline using
method B, purified by column chromatography (eluent gradient
CH2Cl2:MeOH = 75:1), and recrystallized from EtOAc and petroleum
5.1.6. 1-(4-(1H-Pyrazolo[3,4-d]pyrimidin-4-yloxy)phenyl)-3-(4-
(trifluoromethyl)phenyl)urea (9). The title compound was prepared
from 76 and 4-(trifluoromethyl)aniline using method B, purified by
column chromatography (eluent gradient CH2Cl2:MeOH = 70:1), and
recrystallized from EtOAc and petroleum ether. Yield: 83%. HPLC
purity: 98%. 1H NMR (400 MHz, DMSO-d6): δ 14.15 (s, 1H), 9.13 (s,
1H), 8.97 (s, 1H), 8.51 (s, 1H), 8.04 (s, 1H), 7.60−7.51 (m, 5H), 7.32
(d, J = 7.2 Hz, 1H), 7.26 (d, J = 8.8 Hz, 2H). 13C NMR (100 MHz,
DMSO-d6): δ 163.2, 155.8, 155.0, 152.6, 146.6, 140.5, 137.3, 131.8,
130.0, 129.6, 122.3, 121.8, 119.7, 118.1, 114.1, 101.3. HRMS: m/z
calcd for C19H13F3N6O2 [M + H]+ 415.1052, found 415.1064.
5.1.7. 1-(3-Methoxyphenyl)-3-(4-((1-methyl-1H-pyrazolo[3,4-d]-
pyrimidin-4-yl)oxy)phenyl)urea (10). A suspension of compound 6
(752 mg, 2.0 mmol) and sodium hydroxide (80 mg, 2.0 mmol) in dry
DMF (10 mL) was stirred at room temperature for 30 min.
Iodomethane (0.15 mL, 2.4 mmol) was added, and the resulting
mixture was stirred overnight. The reaction mixture was treated with
water (100 mL) and extracted with EtOAc (3 × 60 mL). The
combined organic layers were removed in vacuo. The residue obtained
was purified by column chromatography (eluent gradient CH2Cl2:
MeOH = 90:1) and recrystallized from EtOAc and petroleum ether to
1
ether. Yield: 82%. HPLC purity: 99%. H NMR (400 MHz, DMSO-
d6): δ 14.14 (s, 1H), 9.22 (s, 1H), 8.99 (s, 1H), 8.51 (s, 1H), 8.13 (s,
1H), 8.05 (s, 1H), 7.64 (q, J = 6.8 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H),
7.25 (d, J = 8.4 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 163.2,
156.7, 154.9, 152.5, 146.7, 139.3, 137.1, 131.9, 131.8, 123.0, 122.3,
119.9, 101.3. HRMS: m/z calcd for C19H12ClF3N6O2 [M + H]+
449.0662, found 449.0680.
Experimental data for compound 7, 8, 11, 13−17, 19−26, and 28−
32 are given in the Supporting Information.
5.2. Kinase Inhibition, Binding Affinity Assays, and Kinase
Selectivity. The kinase inhibition assays were performed according to
the KinaseProfiler assay protocols of Upstate Biotechnology
(Millipore). The Ambit in vitro KINOMEscan kinase binding assays
(Ambit Biosciences) were employed to determine the binding affinity
of the compounds. The kinase selectivity is described by the selectivity
score, S-score, which is calculated by dividing the number of kinases
that compounds bind to by the total number of distinct kinases tested,
excluding mutant variants. Here S-score is defined as follows: S(X) =
(number of nonmutant kinases with %Ctrl < X)/(number of
nonmutant kinases tested).
5.3. Cell Lines and Cell Culture. All cell lines were obtained from
the American Type Culture Collection (ATCC; Manassas, VA). MV4-
11 was maintained in IMDM culture medium according to ATCC
guidelines. The other cell lines were grown in RPMI 1640 or DMEM
culture medium containing 10% fetal bovine serum (v/v) in 5% CO2
at 37 °C.
5.4. Cell Viability Assays. The MTT assay was used to detect the
viability of cells. Leukemia cells were collected and seeded in a 96-well
plate at (1−4) × 104 cells per well. Different concentrations of
inhibitors were added to the cells and incubated at 37 °C for 72 h.
Then 20 μL of 5 mg/mL MTT reagent was added to each well and
incubated for 2−4 h, and 50 μL of 20% acidified SDS was used to lyse
the oxidative product. The other cell lines were seeded in 96-well
plates at a density of (2−5) × 103 cells per well, and after 24 h, the
medium in the plates was replaced by medium containing serial
dilutions of inhibitors. Following a 72 h incubation, the MTT reagent
was added for a 2−4 h incubation, and 100% DMSO was used to
dissolve the oxidative product. Finally, the light absorption (OD) of
the dissolved cells was measured at 570 nm using a SpectraMAX M5
microplate spectrophotometer (Molecular Devices). All experiments
were performed in triplicate. The IC50 values were calculated using
GraphPad Prism software.
1
provide 10 (546 mg, 70%, 98% HPLC purity). H NMR (400 MHz,
DMSO-d6): δ 8.82 (s, 1H), 8.75 (s, 1H), 8.55 (s, 1H), 8.05 (s, 1H),
7.55 (d, J = 8.8 Hz, 2H), 7.26−7.17 (m, 4H), 6.95 (d, J = 8.4 Hz, 1H),
6.57 (d, J = 8.4 Hz, 1H), 4.04(s, 3H), 3.74(s, 3H). 13C NMR (100
MHz, DMSO-d6): δ 163.3, 159.7, 154.9, 154.8, 152.5, 146.4, 140.8,
137.6, 130.9, 129.5, 122.1, 119.4, 110.5, 107.2, 104.0, 101.7, 54.9, 34.0.
LC−MS: m/z 391.4 [M + H]+.
5.1.8. 1-(3-(1H-Pyrazolo[3,4-d]pyrimidin-4-yloxy)phenyl)-3-(4-
(trifluoromethyl)phenyl)urea (12). The title compound was prepared
from 77 and 4-(trifluoromethyl)aniline using method B, purified by
column chromatography (eluent gradient CH2Cl2:MeOH = 75:1), and
recrystallized from EtOAc and petroleum ether. Yield: 89%. PHLC
purity: 97%. 1H NMR (400 MHz, DMSO-d6): δ 14.16 (s, 1H), 9.17 (s,
1H), 9.03 (s, 1H), 8.53 (s, 1H), 8.10 (s, 1H), 7.66−7.57 (m, 5H), 7.42
(t, J = 8.0 Hz, 1H), 7.33 (d, J = 8.8 Hz, 1H), 6.97 (d, J = 7.2 Hz, 1H).
13C NMR (100 MHz, DMSO-d6): δ 162.8, 156.7, 154.9, 152.3, 152.1,
143.2, 140.8, 131.9, 130.0, 126.1, 123.1, 122.0, 121.7, 117.9, 115.9,
115.5, 111.7, 101.4. LC−MS: m/z 415.1 [M + H]+.
5.1.9. N-(4-(1H-Pyrazolo[3,4-d]pyrimidin-4-yloxy)phenyl)-4-bro-
mobenzamide (18). A mixture of 4-bromobenzoic acid (265 mg,
1.32 mmol), 1-hydroxybenzotriazole (HOBT; 178.8 mg, 1.32 mmol),
1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride
(EDCI; 254.6 mg, 1.32 mmol), and N,N-diisopropylethylamine
(DIEA; 0.33 mL, 2.0 mmol) in THF (25 mL) was stirred at room
temperature for 0.5 h. Next compound 76 (300 mg, 1.32 mmol) was
added to the mixture, which was heated to reflux for 12 h. Then the
mixture was cooled to room temperature, and water (5 mL) was
added. The precipitate was filtered and washed with ice−water and
THF to obtain compound 18 (390 mg, 73%). The white precipitate
was purified by recrystallization from EtOAc and petroleum ether
5.5. Western Blot Analysis. After treatment with a series of
concentrations of compound 33 for 20 h at 37 °C, MV4-11 cells were
harvested, washed with ice-cold physiological saline, and lysed with
RIPA lysis buffer (Beyotime) including 1% cocktail (Sigma-Aldrich).
Cell lysates were separated by SDS−PAGE and electrotransferred onto
PVDF membranes (Millipore). The PVDF membranes were incubated
with each antibody and detected according to the immunoblot analysis
principle. The antibodies were purchased from Cell Signaling
Technology, with the exception of the anti-FLT3 antibody, which
was obtained from Abcam.
1
(97% HPLC purity). H NMR (400 MHz, DMSO-d6): δ 14.18 (s,
1H), 10.49 (s, 1H), 8.53 (s, 1H), 8.11 (br s, 1H), 7.95 (d, J = 8.4 Hz,
2H), 7.88 (d, J = 8.8 Hz, 2H), 7.79 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 9.2
Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ164.7, 162.8, 156.8,
154.9, 152.2, 141.0, 133.7, 131.7, 131.4, 129.8, 125.5, 122.3, 117.7,
117.2, 113.7, 101.4. LC−MS: m/z 411.2 [M + H]+.
5.1.10. 1-(4-(1H-Pyrazolo[3,4-d]pyrimidin-4-yloxy)phenyl)-3-(3-
(trifluoromethyl)phenyl)urea (27). The title compound was prepared
from 76 and 3-(trifluoromethyl)aniline using method B, purified by
column chromatography (eluent gradient CH2Cl2:MeOH = 70:1), and
recrystallized from EtOAc and petroleum ether. Yield: 84%. HPLC
5.6. Apoptosis Analysis in MV4-11 Cells. A total of 4 × 105
MV4-11 cells were plated in a six-well plate and treated with
compound 33 for 24 h at 37 °C. After incubation, the cells were
harvested and washed with PBS. The apoptosis ratio was analyzed
using an Annexin V-FITC Apoptosis Analysis Kit (Tianjin Suangene
Biotech Co.) and BD FACSCalibur.
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5.7. In Vivo Models. For the subcutaneous xenograft model, MV4-
11 cells were harvested and washed three times with serum-free
medium, followed by resuspension at a concentration of 1 × 108 per
mL. A total of 100 μL of cell suspension was injected into NOD-SCID
mice (5−6 weeks) subcutaneously. When the tumors had grown to
300−500 mm3, all the mice were grouped and administered different
doses of compound or vehicle. The compounds were dissolved in
12.5% (v/v) castor oil (Sigma-Aldrich), 12.5% ethanol, and 75%
distilled water. Tumor growth was measured every 3, and the volume
was calculated as follows: tumor size = long diameter × (short
diameter)2/2.
5.8. Histopathology. NOD-SCID mice bearing tumors were
administered compound 33 at a dose of 10 mg/kg/d orally. Two or
three days later, the mice were killed, and the tumors were fixed in
formalin. Proliferation was assessed by immunostaining with Ki67
(Thermo Fisher Scientific, Fremont, CA). Apoptosis was determined
using transferase-mediated dUTP nick-end labeling (TUNEL) staining
(Roche Applied Science). The p-STAT5 staining was performed using
the p-STAT5 antibody from Cell Signaling Technology. Images were
acquired on an Olympus digital camera attached to a light microscope.
5.9. In Vivo Live Fluorescent Zebrafish Assay. Transgenic
zebrafish (FLK-1:EGFP) embryos were grown and maintained
according to the same protocols as given in ref 41. The screen was
carried out in 24-well plates. Each compound was prepared initially as
10 mmol/L stock solution in dimethyl sulfoxide (DMSO). Stock
solution was diluted in the relevant assay concentration with fish water,
and 0.3% DMSO served as a vehicle control. The embryos were
distributed to 24-well plates with 10 embryos placed in each well.
Then a diluted solution of each compound was added. The embryos
were exposed to compound solution and incubated at 28.5 °C from 15
h postfertilization (hpf) until 31 hpf. At 31 hpf, zebrafish were
anesthetized with 0.01% tricaine and imaged under a fluorescence
microscope (Carl Zeiss Microimaging Inc.) equipped with an
AxioCam MRc5 digital CCD camera (Carl Zeiss Microimaging Inc.).
layer chromatography; STAT5, signal transducer and activator
of transcription 5; HOBT, 1-hydroxybenzotriazole; EDCI, 1-(3-
(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride;
DIEA, N,N-diisopropylethylamine
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ASSOCIATED CONTENT
* Supporting Information
■
S
Table S1 listing the binding affinities of compound 33 with
various kinases, Figure S1 showing the TREEspot interaction
maps for compound 33, experimental details of the synthesis
and analytical characterization of compounds 2−5, 7, 8, 11,
13−17, 19−26, and 28−32 described in this paper, and purity
data for the final compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +86-28-85164063. Fax: +86-28-85164060. E-mail:
yangsy@scu.edu.cn.
■
Author Contributions
§These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was supported by the National Natural Science
Foundation of China (Grant 81172987), the 973 Program
(Grant 2013CB967204), and the 863 Hi-Tech Program
(Grants 2012AA020301 and 2012AA0203).
ABBREVIATIONS USED
■
FLT3, FMS-like tyrosine kinase 3; VEGFR2, vascular
endothelial growth factor receptor 2; AML, acute myeloid
leukemia; PTK, protein tyrosine kinase; SAR, structure−activity
relationship; ITDs, internal tandem duplications; TLC, thin-
M
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