Discovery of the novel potent and selective FLT3 inhibitor 1-{5-[7-(3-morpholinopropoxy)quinazolin-4-ylthio]-[1,3,4]thiadiazol-2-yl} -3-p-tolylurea and its anti-acute myeloid leukemia (AML) activities in vitro and in vivo

Article abstract of DOI:10.1021/jm300042x

Structure-activity relationship (SAR) studies of 2-(quinazolin-4-ylthio) thiazole derivatives, which are for optimizing the in vitro and in vivo antiacute myeloid leukemia (AML) activity of a previously identified FLT3 inhibitor 2-(6,7-dimethoxyquinazolin-4-ylthio)thiazole (1), are described. SAR studies centering around the head (thiazole) and tails (6- and 7-positions) of the quinazoline moiety of 1 led to the discovery of a series of compounds that exhibited significantly increased potency against FLT3-driven AML MV4-11 cells. Preliminary in vivo assays were carried out on three highly active compounds, whose results showed that 1-{5-[7-(3-morpholinopropoxy)quinazolin-4-ylthio]-[1, 3,4]thiadiazol-2-yl}-3-p-tolylurea (20c) had the highest in vivo activity. Further in vitro and in vivo anti-AML studies were then performed on 20c; in an MV4-11 xenograft mouse model, a once-daily dose of 20c at 100 mg/kg for 18 days led to complete tumor regression without obvious toxicity. Western blot and immunohistochemical analysis were carried out to illustrate the mechanism of action of 20c.

Full text of DOI:10.1021/jm300042x

Article
pubs.acs.org/jmc
Discovery of the Novel Potent and Selective FLT3 Inhibitor 1-{5-[7-(3-
Morpholinopropoxy)quinazolin-4-ylthio]-[1,3,4]thiadiazol-2-yl}-3-p-
tolylurea and Its Anti-Acute Myeloid Leukemia (AML) Activities in
Vitro and in Vivo
Wei-Wei Li,†,‡,∥ Xiao-Yan Wang,†,§,∥ Ren-Lin Zheng,†,∥ Heng-Xiu Yan,†,∥ Zhi-Xing Cao,†,∥ Lei Zhong,†
Ze-Rong Wang,† Pan Ji,† Ling-Ling Yang,† Li-Jiao Wang,† Yong Xu,† Jing-Jing Liu,† Jiao Yang,†
Chun-Hui Zhang,† Shuang Ma,† Shan Feng,† Qi-Zheng Sun,† Yu-Quan Wei,† and Sheng-Yong Yang*,†
†State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University,
Sichuan 610041, China
‡College of Chemical Engineering, Sichuan University, Sichuan 610041, China
§West China School of Pharmacy, Sichuan University, Sichuan 610041, China
S
* Supporting Information
ABSTRACT: Structure−activity relationship (SAR) studies of
2-(quinazolin-4-ylthio)thiazole derivatives, which are for
optimizing the in vitro and in vivo antiacute myeloid leukemia
(AML) activity of a previously identified FLT3 inhibitor 2-
(6,7-dimethoxyquinazolin-4-ylthio)thiazole (1), are described.
SAR studies centering around the head (thiazole) and tails (6-
and 7-positions) of the quinazoline moiety of 1 led to the
discovery of a series of compounds that exhibited significantly
increased potency against FLT3-driven AML MV4-11 cells. Preliminary in vivo assays were carried out on three highly active
compounds, whose results showed that 1-{5-[7-(3-morpholinopropoxy)quinazolin-4-ylthio]-[1,3,4]thiadiazol-2-yl}-3-p-tolylurea
(20c) had the highest in vivo activity. Further in vitro and in vivo anti-AML studies were then performed on 20c; in an MV4-11
xenograft mouse model, a once-daily dose of 20c at 100 mg/kg for 18 days led to complete tumor regression without obvious
toxicity. Western blot and immunohistochemical analysis were carried out to illustrate the mechanism of action of 20c.
Because of the therapeutic values in AML, the discovery of
FLT3 inhibitors has increasingly attracted much attention in
recent years. To date, several FLT3 inhibitors including SU-
5416,12 SU-11248,13 CHIR-258,14 PKC-412,15 CEP-701,16
MLN-518,17 BAY-43-9006,18 KW-2449,19 and AC22020 have
been advanced to clinical trials. However, except for very few of
them, such as AC220, the clinical efficacy of most of these
FLT3 inhibitors in patients with AML seems unimpressive,
mainly because of their potency and/or adverse events that
stem from their poor target selectivity. A survey analysis of
FLT3 inhibitors that are currently in clinical trials made by us
indicated that most of these inhibitors were not initially
screened for sensitivity and selectivity against the FLT3 kinase
but found an extra inhibitory potency against FLT3 in addition
to the targeting of their designated kinases (see Table S1 in
Supporting Information).21−23 Therefore, the discovery and
development of the next generation of novel FLT3 inhibitors
with higher potency and better selectivity are strongly
demanded at the present time.
INTRODUCTION
■
FMS-like tyrosine kinase 3 (FLT3), a member of the class III
receptor tyrosine kinase family, has been demonstrated to play
an important role in the proliferation, differentiation, and
apoptosis of hematopoietic cells.1−3 Typically, FLT3 is
activated upon the binding of the FLT3 ligand, which
subsequently activates multiple downstream signaling pathways,
including signal transducer and activator of transcription 5
(STAT5), Ras/mitogen-activated protein kinase (MAPK), and
phosphatidylinositol 3-kinase (PI3K)/AKT pathways. Activat-
ing mutations in FLT3 kinase are found in up to 1/3 of acute
myeloid leukemia (AML) cases.4,5 The most prevalent
activating mutation is “internal tandem duplications” (ITD)
in the juxtamembrane domain that leads to constitutive, ligand-
independent activation of the kinase. The prognosis for AML
patients with FLT3-ITD mutations is significantly worse than
that for patients with wt-FLT3 (wild-type FLT3) when treated
with standard therapy.6−8 In addition, activating point
mutations in the kinase activation loop of FLT3 are found in
another subset of AML patients,6,9−11 which are also associated
with a poor prognosis for overall survival. Thus, FLT3 has been
considered as a potential molecular target in the treatment of
AML.
Received: January 11, 2012
Published: March 27, 2012
© 2012 American Chemical Society
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As a part of the efforts in discovering more potent and
selective FLT3 inhibitors for the treatment of AML, we recently
found a new lead compound, namely, 2-(6,7-dimethoxyquina-
zolin-4-ylthio)thiazole (1), with the aid of a restricted de novo
design strategy proposed by us.24 Compound 1 has a
considerable inhibitory potency against FLT3 kinase with a
half maximal inhibitory concentration (IC50) of 0.007 μM. In
addition, compound 1 also displayed a good selectivity for
FLT3 against a panel of selected human kinases (see Table S2
in Supporting Information). Unfortunately, 1 showed poor
antitumor activities in vitro and in vivo in models of FLT3-
driven AML; the IC50 value against the human AML cell line
MV4-11, which is a typical FLT3-ITD-expressing and a FLT3-
driven AML cell line, is 1.14 μM, and the tumor inhibitory rate
is less than 50% in its maximum dose in an MV4-11 xenograft
model. The purpose here is to carry out a structural
modification to optimize the in vitro and in vivo anti-AML
activity of 1. A series of novel 2-(quinazolin-4-ylthio)thiazole
derivatives were synthesized and tested for their in vitro anti-
AML activity. The most potent derivatives were then subjected
to an in vivo assay in a mouse xenograft model of AML. Finally,
a drug candidate, 20c, which shows considerable in vitro and in
vivo efficacy in models of FLT3-driven AML, was identified. In
this account, we report the chemical synthesis, and the
structure−activity relationship (SAR) of these novel series of
compounds as well as in vitro and in vivo anti-AML activities of
20c.
the presence of N,N-diethylaniline, which gave 4-chloro-7-
methoxyquinazolin-6-yl acetate (5). Substitution of 4-chloride
in 5 by thiazole-2-thiol in basic conditions afforded
intermediate 7-methoxy-4-(thiazol-2-ylthio)quinazolin-6-yl ac-
etate (6). Then intermediate 6 was cleanly hydrolyzed by
NH3·H2O in methanol to obtain 7-methoxy-4-(thiazol-2-
ylthio)quinazolin-6-ol (7), which was alkylated with various
chloralkanes in the presence of K2CO3 to provide 6-alkyloxy-7-
methyoxy analogues 8a−e. The synthesis of 6-methoxy-7-
alkyloxy analogues 13a−d (Scheme 3) was commenced with
commercially available 6-methoxy-4-oxo-3,4- dihydroquinazo-
lin-7-yl acetate (9) using conditions similar to those for the
preparation of 8a−e to afford the desired compounds.
The synthesis of analogues 15a−o with substitutive groups in
the 4- and 5-positions of the thiazole ring is accomplished in a
two-step process outlined in Scheme 4. Commercially available
2 and 3 were reacted with 2-(2-mercapto-4-methylthiazol-5-
yl)acetic acid or 2-(2-mercaptothiazol-4-yl)acetic acid at reflux
under basic conditions in butanone overnight to afford
intermediates 4-substitutive-quinazolines 14a, 14b, and 14c.
Then, an amidation reaction of intermediates 14a, 14b, and 14c
with varieties of substitutive anilines was carried out in the
presence of 1-hydroxybenzotriazole, 1-(3-dimethylaminoprop-
yl)-3-ethylcarbodiimide hydrochloride, and N,N-diisopropyle-
thylamine at reflux for 12−24 h in CH2Cl2 to provide the
compounds shown in Table 2.
Finally, we synthesized a series of the 2-(quinazolin-4-
ylthio)-[1,3,4]thiadiazole analogues 17a−h and 20a−e with the
original thiazole ring replaced by the thiadiazole ring (see
Schemes 5 and 6). The intermediate 16a was achieved by
reaction of 5-amino-[1,3,4]thiadiazole-2-thiol with acetic
anhydride under reflux utilizing the synthetic method as
described in ref 25. The intermediate 16b was synthesized by
an amidation reaction of 5-amino-[1,3,4]thiadiazole-2-thiol
with acrylyl chloride in the presence of organic base using
the synthetic procedure as described in ref 26. The
intermediates 16c−i were achieved by the reaction of 5-
amino-[1,3,4]thiadiazole-2-thiol with substituent phenyl iso-
cyanates under reflux in an appropriate solvent overnight using
the methods described in refs 27 and 28. Then, intermediates
16a−h were reacted with commercially available 3 in the
presence of K2CO3 at 100 °C in N,N-dimethylformamide
overnight to provide the 6,7-bis(2-methoxyethoxy) quinazoline
analogues 17a−h (see Scheme 5). The synthesis of 6-H-7-(3-
morpholinopropoxy)quinazoline analogues 20a−e was
achieved via the intermediate 19, which was synthesized
starting from commercially available 7-fluoro-3H-quinazolin-4-
one (18) (see Scheme 6). The fluoro group of 7-fluoro-3H-
quinazolin-4-one was displaced with the anion of 3-
morpholinopropan-1-ol at 100 °C in N,N-dimethylformamide
overnight to give intermediate quinazolin-4-one (19). The
intermediate 19 was then chlorinated by phosphorus oxy-
chloride in the presence of N,N-diethylaniline followed by
substitution with the intermediates 16b, c, g, f, and i using the
same conditions as those for the preparation of 17 described in
Scheme 5 to afford the final products 20a−e.
CHEMISTRY
■
As depicted in Scheme 1, compounds 1, 1a−1d, which have
identical side chains at the C-6 and C-7 positions of the
a
Scheme 1
a
Reagents and conditions: (a) thiazole-2-thiol, [1,3,4]thiadiazole-2-
thiol, or 4-methylthiazole-2-thiol, K2CO3, 2-butanone, 80 °C, 2−4 h.
quinazoline, were prepared by treatment of the commercially
available 4-chloro-6,7-dimethoxyquinazoline (2) or 4-chloro-
6,7- bis(2-methoxyethoxy)quinazoline (3) with thiazole-2-thiol
or its analogues ([1,3,4]thiadiazole-2-thiol and 4-methylthia-
zole-2-thiol).
Synthetic routes of 2-(quinazolin-4-ylthio)thiazole deriva-
tives with nonidentical 6- and 7-position side chains are
illustrated in Schemes 2 and 3. The production of 6-alkyloxy-7-
methyoxyquinazoline analogues 8a−e was started from the
reaction of commercially available 7-methoxy-4-oxo-3,4-dihy-
droquinazolin-6-yl acetate (4) with phosphorus oxychloride in
RESULTS AND DISCUSSION
■
Since our intention here is to optimize the functional activities
of compound 1 both in vitro and in vivo, cell-based assays will
be used to evaluate the bioactivity of synthesized compounds in
the SAR studies.29,30 Two cell lines including FLT3-driven
MV4-11 and FLT3-independent HCT116 were chosen here;
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a
Scheme 2
a
Reagents and conditions: (a) phosphorus oxychloride, toluene, N,N-diethylaniline, reflux, 3 h; (b) thiazole-2-thiol, K2CO3, 2-butanone, 80 °C, 2−4
h; (c) NH3·H2O, MeOH, rt, 5 h; (d) alkyl halides, K2CO3, 2-butanone, 85 °C, 6 h.
a
Scheme 3
a
Reagents and conditions: (a) phosphorus oxychloride, chloroform, Et3N, reflux, overnight; (b) thiazole-2-thiol, K2CO3, 2-butanone, 80 °C, 4 h; (c)
NH3·H2O, MeOH, rt, 5 h; (d) alkyl halides, K2CO3, 2-butanone, 85 °C, 6 h.
the involvement of HCT116 is for ruling 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 HCT116. It should be pointed out from the start that
the SAR obtained from such cell-based assays potentially report
a combination of intrinsic activity of compounds against the
kinase target modulated by the ability of compounds to
permeate the cell wall during the assay. The enzymatic assays
were also performed but just on compounds with higher
potency at the cellular level. The most active compounds in
both enzymatic and cellular levels will be selected for further in
vivo experiments to evaluate their anti-AML activity.
Modifications of 6- and 7-Positions of Quinazoline. As
previously indicated,24 the 6- and 7-substituents of the
quinazoline ring in 1 extend toward the solvent. The
modifications of these positions were thus expected to help
to improve the absorption ability of the cells.29−32 Our attempts
for the modifications of 6- and 7-substituents include increasing
the chain length and varying the polarity of the tail. Various
derivatives of 1 together with their bioactivities are presented in
Table 1. Obviously, the modifications of 6- and 7-substituents
of the quinazoline ring in 1 did not bring an increase in
inhibitory activities against both MV4-11 and HCT116 cells.
We thus started to modify the thiazole head.
Substitutions of 4- and 5-Positions of Thiazole. The
modification toward the thiazole head was initiated to
substitute the 4- and 5-positions of thiazole with different
groups. At first, a single methyl group was introduced to the 4-
position of thiazole. The resulting compound 1a showed a
similar antiproliferative activity against MV4-11 cells and no
activity against HCT116 (unless it is necessary, we shall, later
on, not mention the activity against HCT116 if there is no
activity). We next explored the SAR of 5-position of thiazole;
the methyl group was kept at the 4-position of thiazole due to
the synthetic accessibility. The different substituents on the 5-
position of thiazole (see 15a−15e, Table 2) have considerable
influence on the antiproliferative activity against MV4-11. The
most active compound 15c, in which the 5-position substituent
is N-[3-(trifluoromethyl)phenyl]propionamide, has an IC50
value of 0.02 μM against MV4-11. The cellular activity
increases more than 50 times relative to that of compound 1.
We then replaced the methoxy groups at the 6- and 7-positions
of the quinazoline ring with longer methoxyethoxy groups,
leading to compounds 15f−15k. The bioactivities of the series
of compounds were not improved, which is consistent with the
above result that the modifications of 6- and 7-substituents of
the quinazoline ring did not bring an increase in bioactivity. In
the series of compounds, the most active compounds 15g
(IC50: 0.025 μM) and 15h (IC50: 0.027 μM) again correspond
to the 5-position substitution of N-[3-(trifluoromethyl)phenyl]-
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a
Scheme 4
a
Reagents and conditions: (a) 2-(2-mercapto-4-methylthiazol-5-yl)acetic acid or 2-(2-mercaptothiazol-4-yl)acetic acid, 2-butanone, K2CO3, reflux,
overnight; (b) anilines or amines, HOBT, EDCI, DIEA, CH2Cl2, reflux, 12−24 h.
a
Scheme 5
a
Reagents and conditions: (a) N,N-dimethylformamide, K2CO3, 100 °C, overnight.
propionamide (15g) and its isomer N-[4-(trifluoromethyl)-
phenyl]propionamide (15h). Lastly, in order to explore the
substitution effect of 4-position of thiazole, we synthesized
compounds 15l−15o that contain different substituents at the
4-position of thiazole. The bioactivity of 15l−15o at the cellular
level is clearly decreased relative to those of their counterparts
15f−15i (see Table 2). From here, we can conclude that a
bulkier substituent on the 5-position of thiazole is of benefit to
increasing the bioactivity against MV4-11.
Replacement of Thiazole with Thiadiazole. In order to
further improve the potency, we replaced the thiazole ring with
the thiadizole ring based on the bioisosterism.33 The first
compound (1c) obtained contains a thiadizole head and two
methyoxy groups at the 6- and 7-positions of quinazoline.
Compound 1c has FLT3 kinase inhibitory potency (IC50
=
0.009 μM) very similar to that of 1. The antiproliferative
activity (IC50) of 1c against MV4-11 is 1.20 μM, which is also
similar to that of 1. We then replaced methyoxy groups at the
6- and 7-positions of quinazoline with methoxyethoxy groups.
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a
Scheme 6
a
Reagents and conditions: (a) 3-morpholinopropan-1-ol, NaH (60%), N,N-dimethylformamide, 100 °C, overnight; (b) (i) phosphorus oxychloride,
N,N-diethylaniline, reflux, 3 h; (ii) 16, N,N-dimethylformamide, K2CO3, 100 °C, overnight.
Table 1. 2-(Quinazolin-4-ylthio)thiazole Derivatives with
Different Substituents at the 6- and 7-Positions of
Quinazoline Together with Their Antiviability Activity
against FLT3-ITD-Expressing MV4-11 Cells and FLT3-
Independent HCT116 Cells
(17f, 20c, and 20e), which potently inhibited the proliferation
of AML MV4-11 cells with IC50 values less than 0.01 μM but
displayed very weak or no inhibitory activity against HCT116,
implying that these compounds could possibly have a potential
value in the treatment of AML. Thus, the three compounds
were selected to carry out preliminary in vivo anti-AML
experiments using the MV4-11 tumor xenograft model.
Compound 20c displayed significant anti-AML potency (see
Figure 2a), while 17f and 20e just showed very weak tumor
inhibitory activity (the tumor inhibition rate is less than 50% at
their maximum dose, namely, 50 mg/kg/d, for both of them by
intraperitoneal administration). A plausible explanation for this
is that 20c has better bioavailability relative to 17f and 20e.
Indeed, a comparison of pharmacokinetic profiles of 20c and
20e showed that 20c has much better absorption than 20e
(Cmax values are 5.31 μg/mL for 20c and 2.74 μg/mL for 20e at
a dose of 40 mg/kg by intraperitoneal administration. For
details, see Table S3 and Figure S1 in Supporting Information).
Therefore, further studies on the kinase inhibition profile and
anti-AML activity of 20c both in vitro and in vivo will be carried
out subsequently.
Kinase inhibition profiles of 20c against a panel of
recombinant human protein kinases are shown in Table 4.
Compound 20c potently inhibited FLT3 with an IC50 value of
0.01 μM (see Figure 1a). It just weakly inhibited Aurora A,
FMS, FLT4, and c-Kit (IC50s: 1.5 μM, 2.8 μM, 3.7 μM, and 6.8
μM, respectively). Compound 20c displayed almost no
inhibitory activity against the other 13 selected protein kinases.
All of these demonstrate that 20c is a potent FLT3 inhibitor
with good kinase selectivity.
a
In Vitro Growth Inhibitory Activities of 20c against
Leukemia and Other Cancer Cells. The growth inhibitory
potencies of 20c against various cell lines, including leukemia
and solid tumor cell lines, were examined, and the results are
presented in Table 5. Compound 20c potently inhibited the
growth of MV4-11 cells that express FLT3-ITD, with an IC50
value of 0.006 μM. It just exhibited very weak inhibitory activity
against human T lymphoma Jurkat cells, human Burkitt’s
lymphoma Ramos cells, human lung cancer PC-9 and H292
cells, and human epithelial carcinoma A431 cells (IC50: 3.05
μM, 6.25 μM, 3.72 μM, 6.94 μM, and 8.91 μM, respectively).
For other leukemia and solid tumor cell lines, including K562,
U937, Karpas299, HCC827, A549, H2228, H820, MDA-MB-
231, BT474, MCF-7, HCT116, SW480, LoVo, HeLa, SKOV-3,
SK, DU145, PC-3, A431, and SH-SY5Y, 20c did not exhibit an
obvious growth inhibition effect at the concentration of 10 μM
(Table 5).
Each compound was tested in triplicate; the data are presented as the
mean SD.
The resulting compound 1d has almost the same bioactivity as
1c, which is again consistent with previous observations. We
next optimized the thiadiazole head by replacing the 5-position
with different substituents containing an amide or urea. We
obtained a series of compounds 17a−h, of which, 17f that
contains 3-ethynylphenylurea is the most active compound
(IC50 = 0.005 μM). In order to further optimize the
physicochemical properties, we introduced the polar group 4-
(3-methoxypropyl) morpholine at the 7-position of quinazo-
line, which led to 20a−e (see Table 3). Obviously, the
introduction of a polar group considerably improves the
bioactivity at the cellular level. Of special note are 20c and 20e,
whose IC50 values against MV4-11 are 0.006 μM and 0.0003
μM, respectively (the kinase inhibitory potency against FLT3 is
0.01 μM for 20c and 0.006 μM for 20e, showing the
consistency between cellular and enzymatic activities).
Signaling Inhibition in Intact MV4-11 Cells. The ability
of 20c to inhibit the activation of FLT3 and downstream
signaling proteins in intact cells was assessed using Western
blot analysis. After a 5 h treatment with increasing
Kinase Inhibition Profile of 20c. The chemical
modifications toward compound 1 resulted in three compounds
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Table 2. 2-(Quinazolin-4-ylthio)thiazole Derivatives with Different Substituents at the 4- and 5-Positions of the Thiazole Ring
Together with Their Antiviability Activity against FLT3-ITD-Expressing MV4-11 Cells and FLT3-Independent HCT116 Cells
a
Each compound was tested in triplicate; the data are presented as the mean SD.
concentrations of 20c, MV4-11 cells were harvested and lysed
for an IP/wt assay. As shown in Figure 1b, 20c inhibited FLT3
phosphorylation in a dose-dependent manner. Consistent with
the downregulation of the phosphorylation of FLT3, the
phosphorylation of the downstream signaling proteins STAT5
and ERK1/2 was also significantly inhibited at concentrations
>0.1 μM (Figure 1b). We also observed that 20c did not
modulate the expression of these proteins during the drug
treatment period.
In Vivo Effects of 20c against s.c. MV4-11 Tumor
Xenografts. The in vivo antileukemia activity of 20c was
evaluated using an MV4-11 xenograft model. In the MV4-11
model, when the tumor grew to a volume of 300−500 mm3, the
mice were grouped and treated intraperitoneally once daily with
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Table 3. 2-(Quinazolin-4-ylthio)-1,3,4-thiadiazole Derivatives with Different Substituents at C-6 and C-7 of Quinazoline and the
5-Position of the Thiadiazole Ring Together with Their Anti-vViability Activity against FLT3-ITD-Expressing MV4-11 Cells
and FLT3-Independent HCT116 Cells
a
Each compound was tested in triplicate; the data are presented as the mean SD.
20, 40, or 100 mg/kg/d 20c for 21 days. The tumor volumes
were measured every 3 days. Treatment with 20c at 100 mg/
kg/d resulted in rapid and complete tumor regression in all
mice of this group (see Figure 2a). Compound 20c treatment
at 20 mg/kg/d and 40 mg/kg/d significantly slowed down the
tumor growth; the tumor inhibition rates are 66% and 84%,
respectively. Moreover, during the whole experiment, no
significant weight loss (see Figure 2b) or any other obvious
signs of toxicity were observed for all of the 20c-treated mice.
Compound 20c was also evaluated for its effects on the
tumor mitotic index (Ki67) and apoptosis using histological
and immunohistochemical techniques. Similar to the tumor
xenograft model, a dose of 20 mg/kg/d of 20c was
administered in the MV4-11 model. After treatment for 2 or
3 days, tumors were collected and analyzed. Tumor tissues
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Table 4. Kinase Inhibition Profile for 20c against Human
FLT3 and a Panel of Other Selected Protein Kinases
kinase
FLT3
IC50 (μM)
0.01
6.8
c-Kit
FMS
2.8
PDGFR
PDGFR
FLT1
KDR
>10
>10
>1
>10
3.7
FLT4
Aurora A
IGF-1R
Met
1.5
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
Pim-1
PLK1
Ret
Syk
c-Raf
EGFR
JAK3
Lck
Table 5. Antiproliferative Activities of 20c against Various
Cell Lines
cell lines
MV4−11
tumor type
IC50 (μM)
human acute myeloid leukemia
human chronic myelogenous leukemia
human leukemic monocyte lymphoma
human T lymphoma
0.006
12.65
>20
K562
U937
Figure 1. (a) Dose−response relationship of 20c inhibition against the
human FLT3 kinase (ATP concentration: 200 μM). (b) Western blot
analysis showing that 20c inhibits FLT3 autophosphorylation and the
phosphorylation of downstream signaling proteins STAT5 and ERK1/
2.
Jurkat
3.05
6.25
>20
Ramos
Karpas299
HCC827
A549
human Burkitt’s lymphoma
human anaplastic large cell lymphoma
human lung cancer
12.5
>20
human lung cancer
H2228
H820
human lung cancer
>20
human lung cancer
>20
CONCLUSIONS
■
PC-9
human lung cancer
3.72
6.94
>20
We have described the optimization of the FLT3 inhibitor 2-
(6,7-dimethoxyquinazolin-4-ylthio)thiazole (1) that showed
considerable enzymatic potency but very low anti-AML activity
in vitro and in vivo. SAR studies centering around the head
(thiazole) and tails (6- and 7-positions) of the quinazoline
moiety led to the discovery of compounds displaying
significantly increased potency against FLT3-driven MV4-11
cells but not active against HCT116 that is FLT3-independent.
In-depth in vitro and in vivo assays were then carried out on one
of the most active compounds, 20c, which exhibited a higher
potency against MV4-11 with IC50 = 0.006 μM and the
maximum tumor inhibition rate in an in vivo MV4-11 xenograft
model. Compound 20c displayed very good FLT3 kinase
selectivity against a panel of selected kinases and considerable
sensitivity against FLT3-driven MV4-11 cells. Western blot and
immunohistochemical analysis were carried out to illustrate the
mechanism of action of 20c, which showed that 20c down-
regulated the phosphorylation of FLT3/STAT5/ERK, blocked
cell proliferation, and induced apoptosis in tumor tissue.
H292
human lung cancer
MDA-MB-231
BT474
MCF-7
HCT116
SW480
LoVo
human breast cancer
human breast cancer
>20
human breast cancer
>20
human colorectal carcinoma
human colorectal carcinoma
human colorectal carcinoma
human cervical carcinoma
human ovarian carcinoma
human ovarian carcinoma
human prostate cancer
>20
11.16
>20
HeLa
>20
SKOV-3
SK
>20
>20
DU145
PC-3
18.60
15.72
8.91
>20
human prostate cancer
A431
human epithelial carcinoma
human neurobfastoma
SH-SY5Y
from the vehicle group were stained strongly with Ki67,
indicating a large number of highly proliferative cells (see
Figure 2c). Conversely, the tumor tissues from the 20c-treated
groups showed significantly fewer Ki67-positive cells. Fur-
thermore, the TUNEL data showed an obvious increase in the
percentage of apoptotic cells in a time-dependent manner.
Hematoxylin staining also showed a significant regression of
MV4-11 tumors (Figure 2c).
EXPERIMENTAL SECTION
■
Chemistry Methods. Unless otherwise noted, all materials were
obtained from commercial suppliers and used without further
purification. The synthesis of compounds 1, 8, 13, 15, 17, and 20
was as previously reported. All final compounds were purified to >95%
purity, as determined by high-performance liquid chromatography
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Figure 2. (a) Antitumor efficacy of 20c at different doses in an in vivo MV4-11 xenograft model. (b) Average body weights for 20c-treated mice
groups at different doses (20 mg/kg, 40 mg/kg, and 100 mg/kg) and vehicle group in an in vivo MV4-11 xenograft model. (c) Ki67 and TUNEL
staining of tumor tissues showing the inhibition of cell proliferation and the induction of apoptosis.
(HPLC). HPLC analysis was carried out on a Waters 2695 HPLC
system with the use of a Kromasil C18 column (4.6 mm × 250 mm, 5
um). 1H NMR and 13C NMR spectra were recorded on a Bruker AV-
400 spectrometer at 400 and 100 MHz, respectively. Chemical shifts
are reported in ppm (δ) 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
(HRMS) data were obtained on an Agilent 1100 series LCMS with
UV detection at 254 nm using electrospray ionization. Thin layer
chromatography (TLC) was conducted on aluminum sheet silica gel
Merck 60F254. The spots were visualized using ultraviolet light.
6,7-Dimethoxy-4-(thiazol-2-ylthio)quinazoline (1). A solution of
4-chloro-6,7-dimethoxy-quinazoline (2, 600 mg, 2.67 mmol), thiazole-
2-thiol (330 mg, 2.8 mmol), and K2CO3 (1.1 g, 7.98 mmol) in 2-
butanone was heated under reflux for 3 h. The reaction mixture was
evaporated under reduced pressure. The residue was extracted with
CH2Cl2 and water. The organic layer was collected, washed with brine,
and dried over MgSO4. The solvent was evaporated under reduced
pressure. The residue was decolorized by active carbon and purified by
recrystallization from ethanol yielding 653 mg (80%, 99% HPLC
purity) of 1 as fine white crystals. 1H NMR (400 MHz, DMSO-d6): δ
8.88(s, 1H), 8.06(d, J = 3.2 Hz, 1H), 8.03(d, J = 3.2 Hz, 1H), 7.40(s,
1H), 7.29(s, 1H), 4.00(s, 6H) ppm. 13C NMR (100 MHz, DMSO-d6):
δ 162.4, 156.1, 153.8, 151.6, 150.5, 146.0, 143.0, 125.3, 117.4, 107.1,
100.7, 56.3, 56.1 ppm. MS (ESI, positive ion) m/z: 306.10 [M + H].
6,7-Dimethoxy-4-(4-methylthiazol-2-ylthio)quinazoline (1a). The
title compound was prepared from 2 and 4-methylthiazole-2-thiol
using the procedure previously described for compound 1 and was
purified by column chromatography eluting with 30−50% EtOAc in
petroleum ether to provide 1a (300 mg, 82% yield, 98% HPLC
purity). H NMR (400 MHz, CDCl3): δ 8.88(s, 1H), 7.29(s, 1H),
7.24(s, 1H), 7.15(d, J = 1.2 Hz, 1H), 4.05(s, 6H), 2.53(d, J = 0.8 Hz,
3H) ppm. MS (ESI, negative ion) m/z: 318.30 [M − H].
1
6,7-Bis(2-methoxyethoxy)-4-(thiazol-2-ylthio)quinazoline (1b).
The title compound was prepared from 3 and thiazole-2-thiol using
the procedure previously described for compound 1 and was purified
by column chromatography eluting with 30−50% EtOAc in petroleum
ether to provide 1b (200 mg, 75% yield, 98% HPLC purity). 1H NMR
(400 MHz, CDCl3): δ 8.89(s, 1H), 7.97(d, J = 3.2 Hz, 1H), 7.61(d, J =
3.2 Hz, 1H), 7.33(s, 1H), 4.33(m, 4H), 3.89(m, 4H), 3.50(d, J = 7.6
+
Hz, 6H) ppm. HRMS (m/z): calcd for C17H19N3O4S2 [M + H]
394.0895; found, 393.8950.
4-([1,3,4]Thiadiazol-2-ylthio)-6,7-dimethoxyquinazoline (1c).
The title compound was prepared from 2 and [1,3,4]thiadiazole-2-
thiol using the procedure previously described for compound 1 and
was purified by column chromatography eluting with 30−50% EtOAc
in petroleum ether to provide 1c (350 mg, 86% yield, 98% HPLC
1
purity). H NMR (400 MHz, CDCl3): δ 9.33(s, 1H), 8.97(s, 1H),
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1
7.37(s, 1H), 7.23(s, 1H), 4.09(d, J = 5.6 Hz, 6H) ppm. HRMS (m/z):
calcd for C12H10N4O2S2 [M + H]+ 307.0323; found, 307.2012.
4-([1,3,4]Thiadiazol-2-ylthio)-6,7-bis(2-methoxyethoxy)-
quinazoline (1d). The title compound was prepared from 3 and
[1,3,4]thiadiazole-2-thiol using the procedure previously described for
compound 1 and was purified by column chromatography eluting with
30−50% EtOAc in petroleum ether to provide 1d (300 mg, 79% yield,
ethyl ether. Yield: 100 mg (37%, 96% HPLC purity). H NMR (400
MHz, DMSO-d6): δ 8.88(s, 1H), 8.06(d, J = 3.6 Hz, 1H), 8.03(d, J =
3.2 Hz, 1H), 7.40(s, 1H), 7.36(s, 1H), 4.33(t, J = 5.8 Hz, 2H), 4.00(s,
3H), 3.61(t, J = 4.4 Hz, 4H), 2.80(t, J = 5.8 Hz, 2H), 2.51(t, J = 1.8
Hz, 4H) ppm. HRMS (m/z): calcd for C18H20N4O3S2 [M + H]+
405.1055; found, 405.0109.
7-Methoxy-6-(2-(pyrrolidin-1-yl)ethoxy)-4-(thiazol-2-ylthio)-
quinazoline (8c). The title compound was prepared from 7 and 1-(2-
chloroethyl)pyrrolidine hydrochloride using the procedure previously
described for compound 8c and was purified by recrystallization from
1
98% HPLC purity). H NMR (400 MHz, CDCl3): δ 9.86(s, 1H),
8.94(s, 1H), 7.46(s, 1H), 7.40(s, 1H), 4.38(q, 4H), 3.78(q, 4H),
3.37(d, J = 7.6 Hz, 6H) ppm. HRMS (m/z): calcd for C16H18N4O4S2
[M + Na]+ 417.0667; found, 417.0803.
1
ethyl ether. Yield: 92 mg (42%, 96% HPLC purity). H NMR (400
MHz, DMSO-d6): δ 8.88(s, 1H), 8.06(d, J = 3.2 Hz, 1H), 8.03(d, J =
3.2 Hz, 1H), 7.41(s, 1H), 7.36(s, 1H), 4.31(t, J = 5.6 Hz, 2H), 4.01(s,
3H), 2.91(t, J = 5.6 Hz, 2H), 2.59(s, 4H), 1.71(s, 4H) ppm. MS (ESI,
positive ion) m/z: 388.99 [M + H].
7-Methoxy-6-[2-(piperidin-1-yl)ethoxy]-4-(thiazol-2-ylthio)-
quinazoline (8a). Phosphorus oxychloride (6.0 mL) was added to a
solution of 7-methoxy-4-oxo-3,4-dihydro-quinazolin-6-yl acetate (4,
3.0 g, 12.8 mmol) and N,N-diethylaniline (2.5 mL, 15.4 mmol) in
toluene at room temperature. Then the mixture was stirred in a
preheated oil bath (100 °C) and refluxed for 3 h. The solvent was
removed under reduced pressure. Ice water (50 mL) was added and
the light brown precipitate filtered and washed with ice water (100
mL). The brownish precipitate was dried to give 5 (2.85 g, 88%). MS
(ESI, positive ion) m/z: 253.03 [M + H].
3-[7-Methoxy-4-(thiazol-2-ylthio)quinazolin-6-yloxy]-N,N-dime-
thylpropan-1-amine (8d). The title compound was prepared from 7
and 3-chloro-N,N-dimethylpropan-1-amine hydrochloride using the
procedure previously described for compound 8d and was purified by
recrystallization from ethyl ether. Yield: 90 mg (46%, 96% HPLC
1
purity). H NMR (400 MHz, DMSO-d6): δ 8.87(s, 1H), 8.06(d, J =
3.2 Hz, 1H), 8.03(d, J = 3.2 Hz, 1H), 7.39(s, 1H), 7.28(s, 1H), 4.22(t,
J = 6.6 Hz, 2H), 4.01(s, 3H), 2.41(t, J = 7.0 Hz, 2H), 2.18(s, 6H),
1.96(m, 2H) ppm. MS (ESI, positive ion) m/z: 376.99 [M + H].
7-Methoxy-6-(3-morpholinopropoxy)-4-(thiazol-2-ylthio)-
quinazoline (8e). The title compound was prepared from 7 and 4-(3-
chloropropyl)morpholine using the procedure previously described for
compound 8e and was purified by recrystallization from ethyl ether.
Yield: 150 mg (54%, 96% HPLC purity). 1H NMR (400 MHz,
CDCl3): δ 8.90(s, 1H), 7.97(d, J = 3.2 Hz, 1H), 7.60(d, J = 3.6 Hz,
1H), 7.30(s, 1H), 4.26(t, J = 6.4 Hz, 2H), 4.04(s, 3H), 3.75(s, 4H),
2.60(br s, 2H), 2.51(br s, 4H), 2.15(br s, 2H) ppm. HRMS (m/z):
calcd for C19H22N4O3S2 [M + H]+ 419.1212; found, 419.1217.
6-Methoxy-7-[2-(piperidin-1-yl)ethoxy]-4-(thiazol-2-ylthio)-
quinazoline (13a). The title compound was prepared from 12 and 1-
(2-chloroethyl)piperidine hydrochloride using the procedure pre-
viously described for compound 8a and was purified by recrystalliza-
A solution of 5 (2.0 g, 7.9 mmol), thiazole-2-thiol (927.6 mg, 7.9
mmol), and K2CO3 (3.27 g, 23.7 mmol) in 2-butanone was heated
under reflux for 2 h. The reaction mixture was evaporated under
reduced pressure. The residue was extracted with CH2Cl2 and water.
The organic layer was collected, washed with brine, and dried over
MgSO4. The solvent was evaporated under reduced pressure. The
residue was purified by column chromatography eluting with 30−50%
EtOAc in petroleum ether to provide 6 (2.35 g, 89% yield) as an
1
orange yellow powder. H NMR (400 MHz, CDCl3): δ 8.95(s, 1H),
7.99(d, J = 3.2 Hz, 1H), 7.79(s, 1H), 7.63(d, J = 3.2 Hz, 1H), 7.43(s,
1H), 4.02(s, 3H), 2.41(s, 3H) ppm.
A solution of 7-methoxy-4-(thiazol-2-ylthio)quinazolin-6-yl acetate
(6, 250 mg, 0.75 mmol) in NH3·H2O/MeOH (10 mL, 1.5 N solution)
was stirred at room temperature for 5 h. The reaction mixture was
monitored by TLC. The solvent was evaporated under reduced
pressure to give the crude intermediate 7 (yield by 100%). The residue
was not further purified and directly used for the next step reaction.
A solution of 7-methoxy-4-(thiazol-2-ylthio)quinazolin-6-ol (7,
218.5 mg, 0.75 mmol) and K2CO3 (310.4 mg, 2.25 mmol) in 2-
butanone (10 mL) was stirred at room temperature for 15 min, and
then 2-piperidinoethylchloride hydrochloride (207 mg, 1.13 mmol)
was added. This reaction mixture was heated under reflux for 7 h. The
solvent was evaporated under reduced pressure. The residue was
extracted with CH2Cl2 and water. The organic layer was collected,
washed with brine, and dried over MgSO4. The solvent was evaporated
under reduced pressure. The residue was recrystallized from ethyl
ether yielding 153 mg (50%, 96% HPLC purity) of 8a as a white
1
tion from ethyl ether. Yield: 211 mg (36%, 96% HPLC purity). H
NMR (400 MHz, CDCl3): δ 8.89(s, 1H), 7.97(d, J = 3.6 Hz, 1H),
7.60(d, J = 3.2 Hz, 1H), 7.30(d, J = 6.0 Hz, 2H), 4.34(br s, 2H),
4.04(s, 3H), 2.95(br s, 2H), 2.60(br s, 4H), 1.67(br s, 4H), 1.48(br s,
2H) ppm. HRMS (m/z): calcd for C19H22N4O2S2 [M + H]+ 403.1262;
found, 403.1256.
6-Methoxy-7-(2-morpholinoethoxy)-4-(thiazol-2-ylthio)-
quinazoline (13b). The title compound was prepared from 12 and 4-
(2-chloroethyl)morpholine hydrochloride using the procedure pre-
viously described for compound 8a and was purified by recrystalliza-
1
tion from ethyl ether. Yield: 112 mg (29%, 96% HPLC purity). H
NMR (400 MHz, CDCl3): δ 8.90(s, 1H), 7.97(d, J = 3.6 Hz, 1H),
7.60(d, J = 3.2 Hz, 1H), 7.30(d, J = 2.0 Hz, 2H), 4.35(t, J = 6.4 Hz,
2H), 4.04(s, 3H), 3.78(t, J = 4.4 Hz, 4H), 2.96(t, J = 5.8 Hz, 2H),
2.67(br s, 4H) ppm. HRMS (m/z): calcd for C18H20N4O3S2 [M + H]+
405.1055; found, 405.1059.
1
powder. H NMR (400 MHz, DMSO-d6): δ 8.88(s, 1H), 8.06(d, J =
3.2 Hz, 1H), 8.03(d, J = 3.2 Hz, 1H), 7.40(s, 1H), 7.38(s, 1H), 4.31(t,
J = 6.0 Hz, 2H), 4.00 (s, 3H), 2.75(t, J = 5.8 Hz, 2H), 2.48(br s, 3H),
1.84(s, 1H), 1.50−1.55(m, 4H), 1.39−1.40(m, 2H) ppm. MS (ESI,
positive ion) m/z: 403.03 [M + H].
6-Methoxy-7-[2-(pyrrolidin-1-yl)ethoxy]-4-(thiazol-2-ylthio)-
quinazoline (13c). The title compound was prepared from 12 and 1-
(2-chloroethyl)pyrrolidine hydrochloride using the procedure pre-
viously described for compound 8a and was purified by recrystalliza-
4-Chloro-6-methoxyquinazolin-7-yl acetate (10). Phosphorus
oxychloride (3.0 mL) was added to a solution of 6-methoxy-4-oxo-
3,4-dihydro-quinazolin-7-yl acetate (9, 3.0 g, 12.8 mmol) in chloro-
form at room temperature. Then the mixture was stirred in a
preheated oil bath (70 °C) and refluxed for 1 h, and then triethylamine
(4.8 mL) was added dropwise. The reaction mixture was refluxed for
12 h. The solvent was removed under reduced pressure. Ice water (50
mL) was added, and the aqueous solution was neutralized with
saturated sodium carbonate solution. The aqueous solution was
extracted with CH2Cl2. The organic layer was collected, washed with
brine, and dried over MgSO4. The solvent was evaporated under
reduced pressure to afford 10 (3.18 g, 98%) without further
purification. MS (ESI, positive ion) m/z: 253.03 [M + H].
1
tion from ethyl ether. Yield: 124 mg (38%, 96% HPLC purity). H
NMR (400 MHz, CDCl3): δ 8.89(s, 1H), 7.97(d, J = 3.6 Hz, 1H),
7.60(d, J = 3.2 Hz, 1H), 7.30(d, J = 2.8 Hz, 2H), 4.32(t, J = 6.0 Hz,
2H), 4.05(s, 3H), 3.07(t, J = 6.0 Hz, 2H), 2.70(br s, 4H), 1.85(br s,
4H) ppm. HRMS (m/z): calcd for C18H20N4O2S2 [M + H]+ 389.1106;
found, 389.1101.
6-Methoxy-7-(3-morpholinopropoxy)-4-(thiazol-2-ylthio)-
quinazoline (13d). The title compound was prepared from 12 and 4-
(3-chloropropyl)morpholine using the procedure previously described
for compound 8a and was purified by recrystallization from ethyl
1
ether. Yield: 150 mg (40%, 96% HPLC purity). H NMR (400 MHz,
7-Methoxy-6-(2-morpholinoethoxy)-4-(thiazol-2-ylthio)-
quinazoline (8b). The title compound was prepared from 7 and 4-(2-
chloroethyl)morpholine hydrochloride using the procedure previously
described for compound 8a and was purified by recrystallization from
CDCl3): δ 8.90(s, 1H), 7.97(d, J = 3.2 Hz, 1H), 7.61(d, J = 3.2 Hz,
1H), 7.30(s, 1H), 7.27(s, 1H), 4.27(t, J = 6.6 Hz, 2H), 4.04(s, 3H),
3.76(br s, 4H), 2.62(br s, 2H), 2.53(br s, 4H), 2.16(br s, 2H) ppm.
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HRMS (m/z): calcd for C19H22N4O3S2 [M + H]+ 419.1212; found,
419.1221.
pressure. Ice water (100 mL) was added and the light yellow
precipitate filtered and washed with ice water (100 mL). The light
yellow precipitate was recrystallized from ethanol to give 14b (655 mg,
78%).
N-(3-Chlorophenyl)-2-[2-(6,7-dimethoxyquinazolin-4-ylthio)-4-
methylthiazol-5-yl]acetamide (15a). A solution of 4-chloro-6,7-
dimethoxyquinazoline (2, 500 mg, 2.22 mmol), 2-(2-mercapto-4-
methylthiazol-5-yl)acetic acid (420 mg, 2.22 mmol), and K2CO3 (920
mg, 6.66 mmol) in 2-butanone was refluxed overnight. The reaction
mixture was evaporated under reduced pressure. Ice water (100 mL)
was added and the light yellow precipitate filtered and washed with ice
water (100 mL) and CH2Cl2. The light yellow precipitate was dried to
give 14a (655 mg, 78%). 1H NMR (400 MHz, DMSO-d6): δ 12.77(s,
1H), 8.87(s, 1H), 7.39(s, 1H), 7.28(s, 1H), 4.00(s, 6H), 3.92(s, 2H),
2.34(s, 3H) ppm.
2-[2-(6,7-Dimethoxyquinazolin-4-ylthio)-4-methylthiazol-5-yl]-
acetic acid 14a (250 mg, 0.66 mmol) was reacted with 3-
chlorobenzenamine (0.1 mL, 0.99 mmol) in the presence of 1-
hydroxybenzotriazole (HOBT, 107.4 mg, 0.79 mmol), 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI,
152.4 mg, 0.79 mmol), and N,N-diisopropylethylamine (DIEA, 0.13
mL, 0.79 mmol) in CH2Cl2 (10 mL). The mixture was heated under
reflux for 24 h. Then, the mixture was cooled to room temperature,
water (5 mL) added, and the precipitate filtered and washed with ice
water and CH2Cl2 to obtain 15a (194 mg, 60%). The light white
precipitate was purified by recrystallization from ethanol (>98% HPLC
purity). 1H NMR (400 MHz, DMSO-d6): δ 10.52(s, 1H), 8.86(s, 1H),
7.82(s, 1H), 7.45(d, J = 8.4 Hz, 1H), 7.34−7.39(m, 2H), 7.29(s, 1H),
7.13(d, J = 7.6 Hz, 1H), 4.00(s, 8H), 2.39(s, 3H) ppm. MS (ESI,
negative ion) m/z: 485.00 [M − H].
N-(3,5-Dichlorophenyl)-2-[2-(6,7-dimethoxyquinazolin-4-ylthio)-
4-methylthiazol-5-yl]acetamide (15b). The title compound was
prepared from 14a and 3,5-dichlorobenzenamine using the procedure
previously described for compound 15a and was purified by
recrystallization from ethanol. Yield: 110 mg (33%, > 98% HPLC
purity). 1H NMR (400 MHz, DMSO-d6): δ 10.68(s, 1H), 8.87(s, 1H),
7.67(s, 2H), 7.40(s, 1H), 7.30(d, J = 8.4 Hz, 2H), 4.01(d, J = 7.6 Hz,
6H), 3.96(s, 2H), 2.39(s, 3H) ppm. MS (ESI, positive ion) m/z:
520.91 [M + H].
2-[2-(6,7-Dimethoxyquinazolin-4-ylthio)-4-methylthiazol-5-yl]-N-
[3-(trifluoromethyl)phenyl]acetamide (15c). The title compound was
prepared from 14a and 3-(trifluoromethyl)benzenamine using the
procedure previously described for compound 15a and was purified by
recrystallization from ethanol. Yield: 153 mg (45%, > 98% HPLC
purity). 1H NMR (400 MHz, DMSO-d6): δ 10.67(s, 1H), 8.87(s, 1H),
8.11(s, 1H), 7.78(d, J = 7.6 Hz, 1H), 7.58(t, J = 8.0 Hz, 1H), 7.40−
7.44(m, 2H), 7.29(s, 1H), 4.04(s, 2H), 4.00(s, 6H), 2.40(s, 3H) ppm.
MS (ESI, negative ion) m/z: 519.00 [M − H].
2-{2-[6,7-Bis(2-methoxyethoxy)quinazolin-4-ylthio]-4-methylthia-
zol-5-yl}acetic acid (14b) (250 mg, 0.54 mmol) was reacted with the
3-chlorobenzenamine (0.1 mL, 0.80 mmol) in the presence of 1-
hydroxybenzotriazole (HOBT, 87.0 mg, 0.65 mmol), 1-(3-dimethyla-
minopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 123.4 mg,
0.65 mmol), and N,N-diisopropylethylamine (DIEA, 0.11 mL, 0.65
mmol) in CH2Cl2 (10 mL). The mixture was heated under reflux for
24 h. Then the clear solution was evaporated under reduced pressure,
the residue was chromatographed on a silica gel column (eluting
system: EtOAc) to obtain 15f (200 mg, 62%) as a white powder,
which was purified by recrystallization from ethanol (>98% HPLC
purity). 1H NMR (400 MHz, DMSO-d6): δ 10.52(s, 1H), 8.85(s, 1H),
7.82(t, J = 1.8 Hz, 1H), 7.42−7.46(m, 2H), 7.38(s, 1H), 7.34−7.36(m,
1H), 7.13(dd, J = 0.8 Hz, 0.8 Hz, 1H), 4.35(m, 4H), 4.01(s, 2H),
3.77(m, 4H), 3.36(d, J = 6.0 Hz, 6H), 2.39(s, 3H) ppm. MS (ESI,
negative ion) m/z: 573.02 [M − H].
2-{2-[6,7-Bis(2-methoxyethoxy)quinazolin-4-ylthio]-4-methyl-
thiazol-5-yl}-N-[3-(trifluoromethyl)phenyl]acetamide (15g). The
title compound was prepared from 14b and 3-(trifluoromethyl)-
benzenamine using the procedure previously described for compound
15f and was purified by column chromatography (eluting system:
EtOAc) and recrystallization from ethanol. Yield: 119 mg (37%, > 98%
HPLC purity). 1H NMR (400 MHz, DMSO-d6): δ 10.67(s, 1H),
8.86(s, 1H), 8.11(s, 1H), 7.78(d, J = 8.0 Hz, 1H), 7.58(t, J = 8.0 Hz,
1H), 7.43(d, J = 6.8 Hz, 2H), 7.35(s, 1H), 4.36(t, J = 2.2 Hz, 4H),
4.03(s, 2H), 3.77(t, J = 2.2 Hz, 4H), 3.36(d, J = 5.6 Hz, 6H), 2.40(s,
3H) ppm. MS (ESI, negative ion) m/z: 607.22 [M − H].
2-{2-[6,7-Bis(2-methoxyethoxy)quinazolin-4-ylthio]-4-methyl-
thiazol-5-yl}-N-[4-(trifluoromethyl)phenyl]acetamide (15h). The
title compound was prepared from 14b and 4-(trifluoromethyl)-
benzenamine using the procedure previously described for compound
15f and was purified by column chromatography (eluting system:
EtOAc) and recrystallization from ethanol. Yield: 160 mg (49%, > 98%
HPLC purity). 1H NMR (400 MHz, DMSO-d6): δ 10.69(s, 1H),
8.85(s, 1H), 7.82(d, J = 8.4 Hz, 2H), 7.70(d, J = 8.8 Hz, 2H), 7.42(s,
1H), 7.35(s, 1H), 4.36(m, 4H), 4.05(s, 2H), 3.77(m, 4H), 3.36(d, J =
6.0 Hz, 6H), 2.40(s, 3H) ppm. MS (ESI, negative ion) m/z: 607.22
[M − H].
2-{2-[6,7-Bis(2-methoxyethoxy)quinazolin-4-ylthio]-4-methyl-
thiazol-5-yl}-N-(3,5-dimethylphenyl)acetamide (15i). The title com-
pound was prepared from 14b and 3,5-dimethylbenzenamine using the
procedure previously described for compound 15f and was purified by
column chromatography (eluting system: EtOAc) and recrystallization
N-(4-Acetamidephenyl)-2-[2-(6,7-dimethoxyquinazolin-4-ylthio)-
4-methylthiazol-5-yl]acetamide (15d). The title compound was
prepared from 14a and N-(4-aminophenyl)acetamide using the
procedure previously described for compound 15a and was purified
by recrystallization from ethanol. Yield: 232 mg (57%, > 98% HPLC
purity). 1H NMR (400 MHz, DMSO-d6): δ 10.27(s, 1H), 9.90(s, 1H),
8.86(s, 1H), 7.51(s, 4H), 7.39(s, 1H), 7.28(s, 1H), 4.00(s, 6H), 3.96(s,
2H), 2.40(s, 3H), 2.02(s, 3H) ppm. MS (ESI, negative ion) m/z:
508.00 [M − H].
1
from ethanol. Yield: 210 mg (69%, > 98% HPLC purity). H NMR
(400 MHz, DMSO-d6): δ 10.17(s, 1H), 8.85(s, 1H), 7.42 (s, 2H),
7.35(s, 1H), 7.22(s, 1H), 6.71(s, 1H), 4.36(m, 4H), 3.96(s, 2H),
3.77(m, 4H), 3.36(d, J = 6.0 Hz, 6H), 2.39(s, 3H), 2.23(s, 6H) ppm.
MS (ESI, negative ion) m/z: 567.02 [M − H].
2-{2-[6,7-Bis(2-methoxyethoxy)quinazolin-4-ylthio]-4-methyl-
thiazol-5-yl}-N-cyclopropylacetamide (15j). The title compound was
prepared from 14b and cyclopropanamine using the procedure
previously described for compound 15f and was purified by column
chromatography (eluting system: EtOAc) and recrystallization from
N-(4-Chlorobenzyl)-2-[2-(6,7-dimethoxyquinazolin-4-ylthio)-4-
methylthiazol-5-yl]acetamide (15e). The title compound was
prepared from 14a and (4-chlorophenyl)methanamine using the
procedure previously described for compound 15a and was purified by
recrystallization from ethanol. Yield: 153 mg (48%, > 98% HPLC
1
ethanol. Yield: 90 mg (33%, > 98% HPLC purity). H NMR (400
MHz, CDCl3): δ 8.88(s, 1H), 7.33(s, 1H), 7.30(s, 1H), 5.73(s, 1H),
4.32(t, J = 4.0 Hz, 4H), 3.89(t, J = 4.0 Hz, 4H), 3.71(s, 2H), 3.50(d, J
= 6.4 Hz, 6H), 2.72(m, 1H), 2.42(s, 3H), 0.79(m, 2H), 0.50(m, 2H)
ppm. MS (ESI, positive ion) m/z: 505.00 [M + H].
2-{2-[6,7-Bis(2-methoxyethoxy)quinazolin-4-ylthio]-4-methyl-
thiazol-5-yl}-N-(6-methoxybenzo[d]thiazol-2-yl)acetamide (15k).
The title compound was prepared from 14b and 6-methoxybenzo-
[d]thiazol-2-amine using the procedure previously described for
compound 15f and was purified by column chromatography (eluting
system: EtOAc) and recrystallization from ethanol. Yield: 102 mg
(30%, > 98% HPLC purity). 1H NMR (400 MHz, DMSO-d6): δ
12.60(s, 1H), 8.88(s, 1H), 7.66(d, J = 8.8 Hz, 1H), 7.58(s, 1H), 7.43(s,
1
purity). H NMR (400 MHz, DMSO-d6): δ 8.85(s, 1H), 8.77(s, 1H),
7.39(d, J = 7.2 Hz, 3H), 7.30(d, J = 8.0 Hz, 3H), 4.30(d, J = 4.8 Hz,
2H), 4.00(s, 6H), 3.81(s, 2H), 2.35(s, 3H) ppm. MS (ESI, positive
ion) m/z: 500.96 [M + H].
2-{2-[6,7-Bis(2-methoxyethoxy)quinazolin-4-ylthio]-4-methyl-
thiazol-5-yl}-N-(3-chlorophenyl)acetamide (15f). A solution of 4-
chloro-6,7-bis(2-methoxyethoxy) quinazoline (3, 500 mg, 1.60 mmol),
2-(2-mercapto-4-methylthiazol-5-yl)acetic acid (302.6 mg, 1.60
mmol), and K2CO3 (662 mg, 4.80 mmol) in 2-butanone was refluxed
overnight. The reaction mixture was evaporated under reduced
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1H), 7.35(s, 1H), 7.04(d, J = 7.2 Hz, 1H), 4.36(br s, 4H), 4.17(s, 2H),
3.81(s, 3H), 3.77(br s, 4H), 3.37(br s, 6H), 2.40(s, 3H) ppm. HRMS
(m/z): calcd for C28H29N5O6S3 [M + H]+ 628.1358; found, 628.0308.
2-{2-[6,7-Bis(2-methoxyethoxy)quinazolin-4-ylthio]thiazol-4-yl}-
N-(3-chlorophenyl)acetamide (15l). A solution of 4-chloro-6,7-bis(2-
methoxyethoxy)quinazoline (3, 1.0 g, 3.20 mmol), 2-(2-mercaptothia-
zol-4-yl)acetic acid (561.0 mg, 3.20 mmol), and K2CO3 (1.32 g, 9.60
mmol) in 2-butanone was refluxed overnight. The reaction mixture
was evaporated under reduced pressure. Ice water (100 mL) was
added and the light yellow precipitate filtered and washed with ice
water (100 mL). The light yellow precipitate was recrystallized from
4H), 3.37(d, J = 7.6 Hz, 6H), 2.24(s, 3H) ppm. HRMS (m/z): calcd
for C18H21N5O5S2 [M + H]+ 452.1062; found, 452.1068.
N-{5-[6,7-Bis(2-methoxyethoxy)quinazolin-4-ylthio]-[1,3,4]-
thiadiazol-2-yl}acrylamide (17b). The title compound was prepared
from 3 and N-(5-mercapto-[1,3,4]thiadiazol-2-yl)acrylamide (16b)
using the procedure previously described for compound 17a and was
purified by recrystallization from ethanol. Yield: 144 mg (31%, 96%
HPLC purity). 1H NMR (400 MHz, DMSO-d6): δ 13.07(s, 1H),
8.89(s, 1H), 7.46(s, 1H), 7.40(s, 1H), 6.60(dd, J = 10.0 Hz, 10.0 Hz,
1H), 6.49(d, J = 16.8 Hz, 1H), 6.02(d, J = 10.0 Hz, 1H), 4.38(br s,
4H), 3.78(br s, 4H), 3.36(d, J = 7.6 Hz, 6H) ppm. HRMS (m/z):
calcd for C19H21N5O5S2 [M + H]+ 464.1062; found, 464.1071.
1-{5-[6,7-Bis(2-methoxyethoxy)quinazolin-4-ylthio]-[1,3,4]-
thiadiazol-2-yl}-3-(3-chlorophenyl)urea (17c). The title compound
was prepared from 3 and 1-(3-chlorophenyl)-3-(5-mercapto-[1,3,4]-
thiadiazol-2-yl)urea (16c) using the procedure previously described for
compound 17a and was purified by recrystallization from ethanol.
Yield: 440 mg (60%, 96% HPLC purity). 1H NMR (400 MHz,
DMSO-d6): δ 11.41(s, 1H), 9.34(s, 1H), 8.89 (s, 1H), 7.73(s, 1H),
7.45(s, 1H), 7.38(br s, 3H), 7.14 (s, 1H), 4.38(m, 4H), 3.78(m, 4H),
3.37(d, J = 7.6 Hz, 6H) ppm. MS (ESI, negative ion) m/z: 563.00 [M
− H].
1-{5-[6,7-Bis(2-methoxyethoxy)quinazolin-4-ylthio]-[1,3,4]-
thiadiazol-2-yl}-3-(3-fluorophenyl)urea (17d). The title compound
was prepared from 3 and 1-(3-fluorophenyl)-3-(5-mercapto-[1,3,4]-
thiadiazol-2-yl)urea (16d) using the procedure previously described
for compound 17a and was purified by recrystallization from ethanol.
Yield: 118 mg (46%, 96% HPLC purity). 1H NMR (400 MHz,
DMSO-d6): δ 11.47(s, 1H), 9.66(s, 1H), 8.89(s, 1H), 7.35−7.51(m,
4H), 7.24(d, J = 8.0 Hz, 1H), 6.90(t, J = 7.6 Hz, 1H), 4.37(t, J = 4.2
Hz, 4H), 3.78(t, J = 6.2 Hz, 4H), 3.37(d, J = 7.2 Hz, 6H) ppm. HRMS
(m/z): calcd for C23H23FN6O5S2 [M + H]+ 547.1234; found,
547.1238.
1-{5-[6,7-Bis(2-methoxyethoxy)quinazolin-4-ylthio]-[1,3,4]-
thiadiazol-2-yl}-3-(3-bromophenyl)urea (17e). The title compound
was prepared from 3 and 1-(3-bromophenyl)-3-(5-mercapto-[1,3,4]-
thiadiazol-2-yl)urea (16e) using the procedure previously described
for compound 17a and was purified by recrystallization from ethanol.
Yield: 188 mg (45%, 96% HPLC purity). 1H NMR (400 MHz,
DMSO-d6): δ 11.60(s, 1H), 9.84(s, 1H), 8.90(s, 1H), 7.86(s, 1H),
7.20−7.48(m, 5H), 4.38(br s, 4H), 3.72(br s, 4H), 3.36(d, J = 7.6 Hz,
6H) ppm. HRMS (m/z): calcd for C23H23BrN6O5S2 [M + H]+
607.0433; found, 607.0424.
1-{5-[6,7-Bis(2-methoxyethoxy)quinazolin-4-ylthio]-[1,3,4]-
thiadiazol-2-yl}-3-(3-ethynylphenyl)urea (17f). The title compound
was prepared from 3 and 1-(3-ethynylphenyl)-3-(5-mercapto-[1,3,4]-
thiadiazol-2-yl)urea (16f) using the procedure previously described for
compound 17a and was purified by recrystallization from ethanol.
Yield: 420 mg (67%, 96% HPLC purity). 1H NMR (400 MHz,
DMSO-d6): δ 11.78(s, 1H), 9.47(s, 1H), 8.82(s, 1H), 7.80(s, 1H),
7.58(s, 1H), 7.24−7.43(m, 3H), 7.04(d, J = 5.6 Hz, 1H), 4.41(br s,
4H), 4.24(s, 1H), 3.77(br s, 4H), 3.36(d, J = 6.4 Hz, 6H) ppm. HRMS
(m/z): calcd for C25H24N6O5S2 [M + H]+ 553.1328; found, 553.1024.
1-{5-[6,7-Bis(2-methoxyethoxy)quinazolin-4-ylthio]-[1,3,4]-
thiadiazol-2-yl}-3-p-tolylurea (17g). The title compound was
prepared from 3 and 1-(5-mercapto-[1,3,4]thiadiazol-2-yl)-3-p-tolylur-
ea (16g) using the procedure previously described for compound 17a
and was purified by recrystallization from ethanol. Yield: 90 mg (42%,
96% HPLC purity). 1H NMR (400 MHz, DMSO-d6): δ 11.21(s, 1H),
9.05(s, 1H), 8.88(s, 1H), 7.58(s, 1H), 7.45(s, 1H), 7.40(d, J = 7.6 Hz,
2H), 7.15(d, J = 8.4 Hz, 2H), 4.38(br s, 4H), 3.78(br s, 4H), 3.37(d, J
= 7.6 Hz, 6H), 2.27(s, 3H) ppm. HRMS (m/z): calcd for
C24H26N6O5S2 [M + H]+ 543.1484; found, 543.1220.
1
ethanol to give 14c (1.1 g, 76%). H NMR (400 MHz, DMSO-d6): δ
12.53(s, 1H), 8.89(s, 1H), 7.77(s, 1H), 7.43(s, 1H), 7.34(s, 1H),
4.36(br s, 4H), 3.81(s, 2H), 3.78(br s, 4H), 3.36(br s, 6H) ppm. MS
(ESI, negative ion) m/z: 450.10 [M − H].
2-{2-[6,7-Bis(2-methoxyethoxy)quinazolin-4-ylthio]thiazol-4-yl}-
acetic acid (14c, 450.0 mg, 1.0 mmol) was reacted with the 3-
chlorobenzenamine (0.2 mL, 1.6 mmol) in the presence of 1-
hydroxybenzotriazole (HOBT, 162.2 mg, 1.2 mmol), 1-(3-dimethyla-
minopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 230.0 mg, 1.2
mmol), and N,N-diisopropylethylamine (DIEA, 0.2 mL, 1.2 mmol) in
CH2Cl2 (10 mL). The mixture was heated under reflux for 24 h. Then
the clear solution was evaporated under reduced pressure, and the
residue was chromatographed on a silica gel column (eluting system:
EtOAc) to obtain 15l (360 mg, 64%, > 96% HPLC purity) as a white
1
powder. H NMR (400 MHz, DMSO-d6): δ 10.47(s, 1H), 8.89(s,
1H), 7.85(s, 1H), 7.81(s, 1H), 7.48−7.44(m, 2H), 7.37−7.33(m, 2H),
7.12(d, J = 8.0 Hz, 1H), 4.36(br s, 4H), 3.91(s, 2H), 3.77(br s, 4H),
3.36(br s, 6H) ppm. MS (ESI, negative ion) m/z: 559.16 [M − H].
2-{2-[6,7-Bis(2-methoxyethoxy)quinazolin-4-ylthio]thiazol-4-yl}-
N[3-(trifluoromethyl)phenyl]acetamide (15m). The title compound
was prepared from 14c and 3-(trifluoromethyl)benzenamine using the
procedure previously described for compound 15l and was purified by
column chromatography (eluting system: EtOAc) and recrystallization
1
from ethanol. Yield: 276 mg (47%, > 96% HPLC purity). H NMR
(400 MHz, DMSO-d6): δ 10.62(s, 1H), 8.89(s, 1H), 8.14(s, 1H),
7.83(s, 1H), 7.79(d, J = 8.4 Hz, 1H), 7.57(t, J = 7.8 Hz, 1H), 7.44−
7.41(m, 2H), 7.35(s, 1H), 4.36(br s, 4H), 3.93(s, 2H), 3.76(br s, 4H),
3.36(br s, 6H) ppm. MS (ESI, negative ion) m/z: 593.22 [M − H].
2-{2-[6,7-Bis(2-methoxyethoxy)quinazolin-4-ylthio]thiazol-4-yl}-
N-[4-(trifluoromethyl)phenyl]acetamide (15n). The title compound
was prepared from 14c and 4-(trifluoromethyl)benzenamine using the
procedure previously described for compound 15l and was purified by
column chromatography (eluting system: EtOAc) and recrystallization
1
from ethanol. Yield: 334 mg (56%, > 96% HPLC purity). H NMR
(400 MHz, DMSO-d6): δ 10.64(s, 1H), 8.89(s, 1H), 7.83(d, J = 9.2
Hz, 3H), 7.69(d, J = 8.4 Hz, 2H), 7.44(s, 1H), 7.35(s, 1H), 4.36(br s,
4H), 3.94(s, 2H), 3.76(br s, 4H), 3.36(br s, 6H) ppm. MS (ESI,
negative ion) m/z: 593.22 [M − H].
2-{2-[6,7-Bis(2-methoxyethoxy)quinazolin-4-ylthio]thiazol-4-yl}-
N-(3,5-dimethylphenyl)acetamide (15o). The title compound was
prepared from 14c and 4-(trifluoromethyl)benzenamine using the
procedure previously described for compound 15l and was purified by
column chromatography (eluting system: EtOAc) and recrystallization
1
from ethanol. Yield: 355 mg (64%, > 96% HPLC purity). H NMR
(400 MHz, DMSO-d6): δ 10.10(s, 1H), 8.90(s, 1H), 7.79(s, 1H),
7.44(s, 1H), 7.36(s, 1H), 7.24(s, 2H), 6.70(s, 1H), 4.36(br s, 4H),
3.86(s, 2H), 3.76(br s, 4H), 3.36(br s, 6H), 2.23(s, 6H) ppm. MS
(ESI, negative ion) m/z: 553.24 [M − H].
N-{5-[6,7-Bis(2-methoxyethoxy)quinazolin-4-ylthio]-[1,3,4]-
thiadiazol-2-yl}acetamide (17a). A solution of 4-chloro-6,7-bis(2-
methoxyethoxy)quinazoline (3, 300 mg, 0.96 mmol), N-(5-mercapto-
[1,3,4]thiadiazol-2-yl)acetamide (16a, 185 mg, 1.05 mmol), and
K2CO3 (397 mg, 2.88 mmol) in N,N-dimethylformamide (10 mL)
was heated overnight at 100 °C. The reaction mixture was evaporated
under reduced pressure. Ice water (20 mL) was added and the white
precipitate filtered and washed with ice water (50 mL), which was
recrystallized from ethanol to give 17a (205 mg, 46%, 96% HPLC
purity) as a white powder. 1H NMR (400 MHz, DMSO-d6): δ 12.82(s,
1H), 8.86(s, 1H), 7.44(s, 1H), 7.38(s, 1H), 4.38(br s, 4H), 3.78(br s,
1-{5-[6,7-Bis(2-methoxyethoxy)quinazolin-4-ylthio]-[1,3,4]-
thiadiazol-2-yl}-3-[4-(trifluoromethyl)phenyl]urea (17h). The title
compound was prepared from 3 and 1-(5-mercapto-[1,3,4]thiadiazol-
2-yl)-3-[4-(trifluoromethyl)phenyl]urea (16h) using the procedure
previously described for compound 17a and was purified by
recrystallization from ethanol. Yield: 220 mg (40%, 96% HPLC
1
purity). H NMR (400 MHz, DMSO-d6): δ 11.90(s, 1H), 10.89(s,
1H), 8.89(s, 1H), 7.71−7.74(m, 4H), 7.45(s, 1H), 7.39(s, 1H),
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4.38(br s, 4H), 3.78(br s, 4H), 3.36(d, J = 7.2 Hz, 6H) ppm. HRMS
(m/z): calcd for C24H23F3N6O5S2 [M + Na]+ 619.1021; found,
619.1024.
recrystallization from ethanol. Yield: 355 mg (72%, 95% HPLC
purity). H NMR (400 MHz, DMSO-d6): δ 11.45(s, 1H), 10.19(s,
1H), 8.95(s, 1H), 8.15(d, J = 8.8 Hz, 1H), 7.77(s, 1H), 7.34−7.44(m,
4H), 7.10(d, J = 7.2 Hz, 1H), 4.27(t, J = 5.8 Hz, 2H), 3.61(br s, 4H),
2.55(br s, 2H), 2.48(br s, 4H), 2.00(m, 2H) ppm. HRMS (m/z): calcd
for C24H24ClN7O3S2 [M + H]+ 558.1149; found, 558.1156.
1-(3-Ethynylphenyl)-3-{5-[7-(3-morpholinopropoxy)quinazolin-4-
ylthio]-[1,3,4]thiadiazol-2-yl}urea (20e). The title compound was
prepared from the intermediate yellowish oil and 1-(3-ethynylphenyl)-
3-(5-mercapto-[1,3,4]thiadiazol-2-yl)urea (16f) using the procedure
previously described for compound 20a and was purified by
recrystallization from ethanol. Yield: 368 mg (77%, 95% HPLC
purity). 1H NMR (400 MHz, DMSO-d6): δ 11.85(s, 1H), 9.83(s, 1H),
8.94(s, 1H), 8.14(d, J = 8.8 Hz, 1H), 7.57(d, J = 8.0 Hz, 1H), 7.32−
7.43(m, 3H), 7.15(d, J = 7.6 Hz, 2H), 4.26(t, J = 6.0 Hz, 2H), 4.19(s,
1H), 3.59(br s, 4H), 2.47(br s, 2H), 2.41(br s, 4H), 1.97(m, 2H) ppm.
HRMS (m/z): calcd for C26H25N7O3S2 [M + H]+ 548.1539; found,
548.1553.
1
N-{5-[7-(3-Morpholinopropoxy)quinazolin-4-ylthio]-[1,3,4]-
thiadiazol-2-yl}acrylamide (20a). To a N,N-dimethylformamide
solution (5 mL) of 3-morpholinopropan-1-ol (2.5 mL, 18.3 mmol)
at 0 °C was added 60% NaH (744 mg, 18.6 mmol), and the mixture
was stirred for 30 min. To this cold solution was added a N,N-
dimethylformamide solution (3 mL) of 7-fluoroquinazolin-4(3H)-one
(18, 1.0 g, 6.1 mmol), and the mixture was heated at 100 °C overnight.
The solvent was evaporated, and the residue was purified by ethyl
ether to afford desired product 19 (1.43 g, 81%) as a white powder. 1H
NMR (400 MHz, DMSO-d6): δ 12.11(s, 1H), 8.05(s, 1H), 8.00−
8.02(m, 1H), 7.07−7.10(m, 2H), 4.15(t, J = 6.4 Hz, 2H), 3.58(t, J =
4.4 Hz, 4H), 2.43(t, J = 7.2 Hz, 2H), 2.37(br s, 4H), 1.92(m, 2H)
ppm.
Phosphorus oxychloride (8.0 mL) was added dropwise to N,N-
diethylaniline (1.0 mL) with magnetic stirring followed by the addition
of 7-(3-morpholinopropoxy)quinazolin-4(3H)-one (19, 1.0 g, 3.45
mmol), and the reaction flask was immersed in a preheated oil bath
(70 °C). The temperature was increased to 90 °C and kept at 90 °C
for another 3 h. Most of the excess of phosphorus oxychloride was
then removed under reduced pressure and the resulting dark oil
triturated with toluene (3 × 40 mL). The residue was dissolved in
water (20 mL) and extracted with EtOAc (2 × 15 mL). The pH of the
aqueous phase was adjusted to 9−10 with Na2CO3, and it was
extracted with CH2Cl2 (3 × 150 mL) and dried over Na2SO4, and the
organic solvent was removed to afford the product as yellowish oil
(900 mg, 84%) without further purification to use for the next step.
A solution of the yellowish oil (300 mg, 0.97 mmol), N-(5-
mercapto-[1,3,4]thiadiazol-2-yl)acrylamide (16b, 220 mg, 1.18 mmol),
and K2CO3 (404 mg, 2.93 mmol) in N,N-dimethylformamide (8 mL)
was heated at 100 °C overnight. The reaction mixture was evaporated
under reduced pressure. Ice water (20 mL) was added and the white
precipitate filtered and washed with ice water (50 mL), which was
recrystallized from ethanol to give 20a (225 mg, 50%, 95% HPLC
Kinase Inhibitory Assays. The kinase inhibitory assays were
performed according to the KinaseProfiler assay protocols of Upstate
Biotechnology (Millipore).
Cell Culture. Unless specifically mentioned, the cell lines were
obtained from the American Type Culture Collection (Manassas, VA,
USA). Cells were grown in RPMI 1640 or DMEM culture medium
containing 10% fetal bovine serum (v/v) in 5% CO2 at 37 °C, except
for MV4-11 cells, which were cultured in IMDM culture medium.
Cell Viability Assays. The viability of cells was determined using
the MTT assay method. The leukemia cells were seeded in a 96-well
plate at 1−4 × 104 cells per well, and an equal volume of medium
containing increasing concentrations of inhibitors was added to each
well. At the end of the incubation period (72 h at 37 °C), 20 μL of 5
mg/mL MTT reagent was added per well for 2−4 h of incubation, and
50 μL of 20% acidified SDS per well was used to lyse the cells. The
other cell lines were seeded in 96-well plates at a density of 2−5 × 103
cells/well for 24 h followed by replacement of the medium with serial
dilutions of inhibitors in culture medium. Following a 72-h incubation,
the MTT reagent was added for a 2−4-h incubation, and 100% DMSO
was used to dissolve the cells. 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 percentage of viability was calculated
and compared with that of the control cells treated with DMSO
(0.1%).
Immunoprecipitation and Western Blot Assays. Immunopre-
cipitation and immunoblotting were performed for the analysis of
FLT3 autophosphorylation. After treatment with a series of
concentrations of 20c for 24 h at 37 °C, MV4-11 cells were harvested,
washed in ice-cold PBS, and lysed with RIPA buffer (10 mM Tris-HCl
(pH 7.8), 1% NP40, 0.15 M NaCl, 1 mM EDTA, 10 μM aprotinin, 1
mM NaF, and 1 mM Na3VO4). Samples were incubated overnight at 4
°C with the anti-FLT3 antibody, and immune complexes were
precipitated with protein A agarose (Roche Applied Science) at 4 °C.
The precipitated samples were subjected to immunoblot analysis to
detect FLT3 phosphorylation by probing with the antiphosphotyr-
osine antibody (Upstate Biotechnology, Lake Placid, NY). Total FLT3
was measured with an anti-FLT3 antibody after the PVDF membrane
had been incubated with stripping buffer. STAT5 and Erk1/2
phosphorylation was detected through immunoblot analysis. Briefly,
after 20 h of 20c treatment, MV4-11 cells were harvested and lysed
with RIPA buffer. Samples were analyzed by immunoblotting as
outlined above using antiphospho-STAT5, anti-STAT5, anti-p44/42
MAPK, and antiphospho-p44/42 MAPK antibodies.
1
purity) as a white solid. H NMR (400 MHz, DMSO-d6): δ 13.02(s,
1H), 8.95(s, 1H), 8.15(d, J = 9.2 Hz, 1H), 6.60(dd, J = 10.0 Hz, 10.0
Hz, 1H), 6.49(dd, J = 1.6 Hz, 1.6 Hz, 1H), 6.02(dd, J = 2.0 Hz, 2.0 Hz,
1H), 4.27(t, J = 6.2 Hz, 2H), 3.59(t, J = 4.4 Hz, 4H), 2.48(br s, 2H),
2.40(br s, 4H), 1.97(m, 2H) ppm. HRMS (m/z): calcd for
C20H22N6O3S2 [M + H]+ 459.1273; found, 459.1270.
1-{5-[7-(3-Morpholinopropoxy)quinazolin-4-ylthio]-[1,3,4]-
thiadiazol-2-yl}-3-phenylurea (20b). The title compound was
prepared from the intermediate yellowish oil and 1-(5-mercapto-
[1,3,4]thiadiazol-2-yl)-3-phenylurea (16i) using the procedure pre-
viously described for compound 20a and was purified by
recrystallization from ethanol. Yield: 180 mg (70%, 95% HPLC
purity). 1H NMR (400 MHz, DMSO-d6): δ 11.43(s, 1H), 9.31(s, 1H),
8.94(s, 1H), 8.14(d, J = 9.2 Hz, 1H), 7.53(d, J = 8.0 Hz, 2H), 7.32−
7.44(m, 4H), 7.07(t, J = 7.4 Hz, 1H), 4.27(t, J = 6.4 Hz, 2H), 3.59(t, J
= 4.4 Hz, 4H), 2.48(br s, 2H), 2.42(br s, 4H), 1.98(m, 2H) ppm.
HRMS (m/z): calcd for C24H25N7O3S2 [M + H]+ 524.1539; found,
524.1553.
1-{5-[7-(3-Morpholinopropoxy)quinazolin-4-ylthio]-[1,3,4]-
thiadiazol-2-yl}-3-p-tolylurea (20c). The title compound was
prepared from the intermediate yellowish oil and 1-(5-mercapto-
[1,3,4]thiadiazol-2-yl)-3-p-tolylurea (16g) using the procedure pre-
viously described for compound 20a and was purified by
recrystallization from ethanol. Yield: 210 mg (59%, 95% HPLC
purity). 1H NMR (400 MHz, DMSO-d6): δ 11.40(s, 1H), 9.17(s, 1H),
8.95(s, 1H), 8.15(d, J = 9.2 Hz, 1H), 7.40−7.44(m, 4H), 7.15(d, J =
8.4 Hz, 2H), 4.27(t, J = 6.0 Hz, 2H), 3.60(br s, 4H), 2.48(br s, 2H),
2.42(br s, 4H), 2.27(s, 3H), 1.98(m, 2H) ppm. HRMS (m/z): calcd
for C25H27N7O3S2 [M + H]+ 538.1695; found, 538.1685.
1-(3-Chlorophenyl)-3-{5-[7-(3-morpholinopropoxy)quinazolin-4-
ylthio]-[1,3,4]thiadiazol-2-yl}urea (20d). The title compound was
prepared from the intermediate yellowish oil and 1-(3-chlorophenyl)-
3-(5-mercapto-[1,3,4]thiadiazol-2-yl)urea (16c) using the procedure
previously described for compound 20a and was purified by
In Vivo Models. MV4-11 cells were harvested during the
exponential-growth phase, washed twice with serum-free medium,
and resuspended at the concentration of 1 × 108/mL. One hundred
microliters of cell suspensions was injected subcutaneously into the
hind flank of each female NOD-SCID mouse (6−7 weeks old). The
tumors were allowed to grow to 300−500 mm3 at which point the
mice were randomized into 4 groups (6 mice for each group) and
dosed with 20c (20, 40, or 100 mg/kg/d) or vehicle. The compounds
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Journal of Medicinal Chemistry
Article
were dissolved in 25% (v/v) PEG400 plus 5% DMSO and
administered intraperitoneally at a dose of 5 mL/kg. Tumor growth
was measured every 3 days using Vernier calipers for the duration of
the treatment. The volume was calculated as follows: tumor volume =
a × b2/2 (a, long diameter; b, short diameter).
Histopathology and IHC. NOD-SCID mice bearing tumors or
having undergone bone marrow engraftment were treated with 20c at
a dose of 20 mg/kg/d (ip). At the indicated time after dosing,
individual mice were sacrificed. The tumors or femoral bones were
fixed with formalin and embedded in paraffin (femoral bones were
decalcified). Sections measuring 4−8 μm in thickness were prepared
for histological and immunohistochemical analysis. Proliferation was
detected using immunostaining with the Ki67 antibody (Thermo
Fisher Scientific, Fremont, CA). Apoptosis was determined using
transferase-mediated dUTP nick-end labeling (TUNEL) and staining
(Roche Applied Science). Finally, images were captured with an
Olympus digital camera attached to a light microscope.
Pharmacokinetic Analysis. Male Sprague−Dawley rats (200−
250 g) were used and randomly divided into two groups (n = 3 in each
group). A catheter was surgically placed into the femoral vein for
collection of blood samples. Rats were fasted overnight before dosing.
Compounds 20c and 20e were administered intraperitoneally (ip) at a
dose of 40 mg/kg. Plasma concentrations of each compound were
determined by LC-MS/MS (a 3200 QTRAP system, Applied
Biosystems/MDS Sciex).
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ASSOCIATED CONTENT
* Supporting Information
■
S
IC50 values of 1 against human FLT3 and pharmacokinetic
properties of 20c and 20e. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: +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.
ACKNOWLEDGMENTS
■
This work was supported by the National Natural Science
Foundation of China (81172987), the National S&T Major
Project (2012ZX09102-101-002), and the 863 Hi-Tech
Program (2012AA020301 and 2012AA020308).
(17) DeAngelo, D. J.; Stone, R. M.; Heaney, M. L.; Nimer, S. D.;
Paquette, R. L.; Klisovic, R. B.; Caligiuri, M. A.; Cooper, M. R. J.;
Lecerf, M.; Karol, M. D.; Sheng, S.; Holford, N.; Curtin, P. T.; Druker,
B. J.; Heinrich, M. C. Phase 1 clinical results with tandutinib
(MLN518), a novel FLT3 antagonist, in patients with acute
myelogenous leukemia or high-risk myelodysplastic syndrome: safety,
pharmacokinetics, and pharmacodynamics. Blood 2006, 108, 3674−
3681.
ABBREVIATIONS USED
■
FLT3, FMS-like tyrosine kinase 3; STAT5, signal transducer
and activator of transcription 5; AML, acute myeloid leukemia;
SAR, structure−activity relationship; MAPK, mitogen-activated
protein kinase; PI3K, phosphatidylinositol 3-kinase; ITD,
internal tandem duplications; TLC, thin layer chromatography;
HOBT, 1-hydroxybenzotriazole; EDCI, 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride; DIEA, N,N-diiso-
propylethylamine
(18) Zhang, W.; Konopleva, M.; Shi, Y. X.; McQueen, T.; Harris, D.;
Ling, X.; Estrov, Z.; Quintas-Cardama, A.; Small, D.; Cortes, J.;
́
Andreeff, M. Mutant FLT3: a direct target of sorafenib in acute
myelogenous leukemia. J. Natl. Cancer Inst. 2008, 100, 184−198.
(19) Shiotsu, Y.; Kiyoi, H.; Ishikawa, Y.; Tanizaki, R.; Shimizu, M.;
Umehara, H.; Ishii, K.; Mori, Y.; Ozeki, K.; Minami, Y.; Abe, A.;
Maeda, H.; Akiyama, T.; Kanda, Y.; Sato, Y.; Akinaga, S.; Naoe, T.
KW-2449, a novel multikinase inhibitor, suppresses the growth of
leukemia cells with FLT3 mutations or T315I-mutated BCR/ABL
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