Structural modifications at the 6-position of thieno[2,3- d ]pyrimidines and their effects on potency at FLT3 for treatment of acute myeloid leukemia

Article abstract of DOI:10.1016/j.ejmech.2016.05.022

Fms-like tyrosine kinase 3 (FLT3) is a well-known and important target for the treatment of acute myeloid leukemia (AML). A series of thieno[2,3-d]pyrimidine derivatives from a modification at the 6-position were synthesized to identify effective FLT3 inhibitors. Although compounds 1 and 2 emerged as promising FLT3 inhibitors among the synthesized compounds, both compounds exhibited poor metabolic stability in human and rat liver microsomes. Hence, further optimization was required for the discovery of FLT3 inhibitors, with a focus on improving metabolic stability. Compound 16d, which had structural modifications of the methyl group at the 5-position and the 4-(2-methylaminoethoxy) phenyl group at the 6-position, exhibited good inhibitory activity against FLT3 and showed effective antiproliferative activity against four leukemia cell lines, including MV4-11. Moreover, compound 16d displayed enhanced metabolic stability. The results of this study indicated that 16d could be a promising compound for further optimization and development as a potent FLT3 inhibitor.

Full text of DOI:10.1016/j.ejmech.2016.05.022

European Journal of Medicinal Chemistry 120 (2016) 74e85
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Structural modifications at the 6-position of thieno[2,3-d]pyrimidines
and their effects on potency at FLT3 for treatment of acute myeloid
leukemia
,
,
,
,
Hyuntae Kim a b, Chulho Lee a b, Jee Sun Yang a b, Seonghwi Choi a b, Chun-Ho Park c,
Jong Soon Kang d, Soo Jin Oh d, Jieun Yun d, Myung-Hwa Kim e, Gyoonhee Han a
, b, *
a Department of Integrated OMICS for Biomedical Sciences (WCU Program), Yonsei University, Seoul 120-749, Republic of Korea
b Translational Research Center for Protein Function Control, Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea
c Division of New Drug Development, R&D Center, Jeil Pharmaceutical Co., Ltd., Gyeonggi-Do 449-861, Republic of Korea
d Bioevaluation Center, Korea Research Institute of Bioscience and Biotechnology, Ochang, Cheongwon, Chungbuk 363-883, Republic of Korea
e Korea Drug Development Fund, A502, U-space2, 670 Daewangpangyo-Ro, Bundang-Gu, Seongnam-si, Gyeonggi-Do 463-400, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Fms-like tyrosine kinase 3 (FLT3) is a well-known and important target for the treatment of acute
myeloid leukemia (AML). A series of thieno[2,3-d]pyrimidine derivatives from a modification at the 6-
position were synthesized to identify effective FLT3 inhibitors. Although compounds 1 and 2 emerged
as promising FLT3 inhibitors among the synthesized compounds, both compounds exhibited poor
metabolic stability in human and rat liver microsomes. Hence, further optimization was required for the
discovery of FLT3 inhibitors, with a focus on improving metabolic stability. Compound 16d, which had
structural modifications of the methyl group at the 5-position and the 4-(2-methylaminoethoxy) phenyl
group at the 6-position, exhibited good inhibitory activity against FLT3 and showed effective anti-
proliferative activity against four leukemia cell lines, including MV4-11. Moreover, compound 16d dis-
played enhanced metabolic stability. The results of this study indicated that 16d could be a promising
compound for further optimization and development as a potent FLT3 inhibitor.
Received 2 October 2015
Received in revised form
6 May 2016
Accepted 7 May 2016
Available online 9 May 2016
Keywords:
Fms-like tyrosine kinase 3 (FLT3)
Acute myeloid leukemia (AML)
Thieno[2,3-d]pyrimidine
Metabolic stability
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
predominant FLT3 mutation is an internal tandem duplication
mutation (FLT3-ITD) within the juxtamembrane domain that oc-
Acute myeloid leukemia (AML) is a heterogeneous disorder of
the blood and bone marrow that is characterized by an increased
number of immature myeloid cells in the bone marrow and a loss of
normal hematopoietic function [1,2]. AML develops following the
genetic damage of cell-surface receptors such as Ras, or of receptor
tyrosine kinases such as Fms-like tyrosine kinase 3 (FLT3) and c-KIT
[3]; however, FLT3 damage is a frequent genetic event in AML [4,5].
FLT3 is a member of the class III receptor tyrosine kinase family
that is mainly expressed by immature hematopoietic cells and is
important for hematopoiesis [6]. Further, mutations in the FLT3
gene are present in approximately 30% of AML patients [7,8]. The
curs in approximately 25% of AML patients. The second most
common FLT3 mutation is a point mutation within the tyrosine
kinase domain (FLT3-TKD) most often involving aspartic acid 835
(D835) that occurs in approximately 7% of AML patients. Both
mutations result in constitutive ligand-independent active con-
formations that promote the activation of downstream signaling
pathways [9] and result in a growth advantage for leukemia cells
[10]. In addition, the overexpression of FLT3 is an unfavorable
prognostic factor for overall survival in AML. In particular, FLT3-ITD
causes significantly higher rates of relapse in AML patients after
remission [11,12]. Therefore, the importance of FLT3 in the patho-
genesis and prognosis of AML patients is both evident and
profound.
* Corresponding author. Department of Integrated OMICS for Biomedical Sciences
(WCU Program), Yonsei University, Seoul 120-749, Republic of Korea; Translational
Research Center for Protein Function Control, Department of Biotechnology, Yonsei
University, Seoul 120-749, Republic of Korea.
Due to the importance of FLT3 in the treatment of AML, much
attention has been focused on the development of FLT3 inhibitors
[13]. Currently, several FLT3 inhibitors including Sorafenib [14],
Tandutinib [15], Midostaurin [16], and Quizartinib [17], have
E-mail address: gyoonhee@yonsei.ac.kr (G. Han).
http://dx.doi.org/10.1016/j.ejmech.2016.05.022
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

H. Kim et al. / European Journal of Medicinal Chemistry 120 (2016) 74e85
75
demonstrated effective inhibition of FLT3 and have advanced to
clinical trials. Despite their promising results, these inhibitors also
display unwanted side effects, such as overexpression of the FLT3
receptor, FLT3 ligand stimulation, and secondary mutations [18,19].
As a result, recent studies have centered in the investigation of
highly potent and more selective FLT3 inhibitors with a focus on
overcoming drug resistance.
We recently identified a series of thieno[2,3-d]pyrimidines as a
new class of FLT3 inhibitors that carried a chemical modification of
SPC-839, a well-known IkB kinase b (IKKb) inhibitor, by the intro-
duction of thiophene instead of a phenyl ring on the quinazoline
structure [20]. The structure-based efforts resulted in thieno[2,3-d]
pyrimidine derivatives 1 and 2 (Fig. 1), both of which are potent
inhibitors of FLT3 with significant antiproliferative activity against
MV4-11 cells, which harbor FLT3-ITD [20,21]. Unfortunately, their
poor metabolic stability precluded any further evaluation (1 and 2
showed 17.2% and 6.68% remaining after 30 min incubation with rat
liver microsomes, respectively). However, compound 2 improved
the inhibition of FLT3-D835Y and maintained inhibitory activity
against wild type FLT3 (FLT3-wt) through the introduction of a
methoxy group at the 6-position of the structure of compound 1,
which suggested that thieno[2,3-d] pyrimidine might require
further optimization. Therefore, compound 1 served as the starting
point for optimization to improve a number of properties, including
potency and metabolic stability.
the identification of an optimized substitution pattern of thieno
[2,3-d]pyrimidine for the future discovery of potent FLT3 inhibitors.
2. Chemistry
The synthetic methods for the preparation of the thieno[2,3-d]
pyrimidine derivatives are illustrated below (Schemes 1e5). As
detailed in Scheme 1, 2-aminothiophenes 3ae3i were synthesized
from the corresponding ketone or aldehyde, along with ethyl cya-
noacetate, sulfur, and the appropriate base using the Gewald re-
action [22,23]. The synthesis of the thieno[2,3-d]pyrimidine
derivatives 8ae8l was previously described (Scheme 2) [20,21]. In
addition, 4-chloro-6-(4-hydroxyphenyl)thieno[2,3-d]pyrimidines
6a and 6b were synthesized by demethylation using boron tri-
bromide (Scheme 3).
Compound 11 with an amide group at the 6-position of thieno
[2,3-d]pyrimidine was synthesized according to Scheme 4. Ethyl
thieno[2,3-d]pyrimidine-6-carboxylate 4d was converted to thieno
[2,3-d]pyrimidine-6-carboxylic acid 9 by hydrolysis using sodium
hydroxide. The 4-chlorothieno[2,3-d]pyrimidine compound 10 was
synthesized by heating thieno[2,3-d]pyrimidine-6-carboxylic acid
9 with phosphorous oxychloride. The amide coupling of 4-
chlorothieno[2,3-d]pyrimidine compound 10 with dimethylethy-
lenediamine using 1-ethyl-3-(3-dimethylaminopropyl) carbodii-
mide hydrochloride (EDC) resulted in compound 11. Furthermore,
diverse alkoxy groups at the 4-position of the phenyl group at the
6-position of the thieno[2,3-d]pyrimidine were introduced using
In this study, we describe the synthesis of thieno[2,3-d]pyrim-
idine derivatives and their biological activities. Our results indicate
Fig. 1. Chemical structures of FLT3 inhibitors.

76
H. Kim et al. / European Journal of Medicinal Chemistry 120 (2016) 74e85
Scheme 1. The synthesis of 2-aminothiophene intermediates 3ae3i.
Scheme 2. The synthesis of thieno[2,3-d]pyrimidine derivatives 8ae8l.
Scheme 3. The synthesis of 6-(4-Hydroxyphenyl) thieno[2,3-d]pyrimidine intermediates 6a and 6b.
the Mitsunobu reaction of 4-chlorothieno[2,3-d]pyrimidine 6a with
various alcohols as shown in Scheme 5.
obtained by refluxing 4-chlorothieno[2,3-d]pyrimidines 11 or 14
with hydrazine hydrate. Finally, the treatment of 4-hydrazino
thieno[2,3-d]pyrimidines 12 or 15 with 3-methylfuran-2, 5-dione
Next, the 4-hydrazino thieno[2,3-d]pyrimidines 12 and 15 were

H. Kim et al. / European Journal of Medicinal Chemistry 120 (2016) 74e85
77
Scheme 4. The synthesis of thieno[2,3-d]pyrimidine derivative 13.
Scheme 5. The synthesis of thieno[2,3-d]pyrimidine derivative 16ae16j.
yielded thieno[2,3-d]pyrimidine derivatives 13 or 16ae16j.
contained BCR-ABL expression), FLT3-wt expressing cells (THP-1
and HL-60), and FLT3-ITD harboring cells (MV4-11), was measured
using an XTT assay. The known FLT3 inhibitor AC220 (Quizartinib)
was used as a positive control.
3. Results and discussion
As shown in Table 1, the remaining activity of FLT3-wt at 1 mM of
It has been previously reported that the thiophene at the 2-
position and the 2, 5-dioxo-4-methylpyrroloamino group at the
4-position are important for inhibitory activity against FLT3-wt
[20,21]. Therefore, our initial efforts focused on introducing
diverse substituents at the 6-position of thieno[2,3-d]pyrimidine to
improve inhibitory activity and metabolic stability.
the prepared compound was evaluated for inhibitory activity. As
indicated, most compounds with low remaining activity exhibited
good inhibitory activity against FLT3-wt. The compounds with a
small groups at the 6-position (8f and 8g) exhibited < 20%
remaining activity. The compounds with a relatively large group at
the 6-position (8c and 13) exhibited high remaining activity.
However, the phenyl groups with the substituent in the para po-
sition at the 6-position (8a, 8b, 8h, and 16ae16i) exhibited < 50%
remaining activity. In particular, the compound with the cyclo-
aminoalkoxy or elongated aminoethoxy group in the para position
The percentage of remaining activity of FLT3-wt at 1 mM and the
inhibitory activity (IC50) of the prepared compounds against FLT3-
wt and FLT3-D835Y were evaluated using the KinaseProfiler™
service. Cell proliferation against four representative human leu-
kemia cell lines, including non-FLT3 expressing cells (K562, which

78
H. Kim et al. / European Journal of Medicinal Chemistry 120 (2016) 74e85
Table 1
The remaining activity of FLT3-wt at 1
m
M and inhibitory activities of thieno[2,3-d]pyrimidine derivatives against FLT3-wt and FLT3-D835Y.
Cpd
Structure
R1
Remaining activity (%)a
IC50(nM)b
FLT3-wt
NTc
R2
FLT3-D835Y
NTc
8a
8b
8c
8d
Me
49
41
50
52
Me
Me
Me
NTc
NTc
NTc
NTc
NTc
NTc
8e
Me
20
156.0 1.408
91.23 3.191
8f
8g
Me
Me
Me
7
16
11.38 0.849
118.2 1.337
13.68 0.684
74.97 1.758
8h
8i
Me
H
45
7
NTc
NTc
68.64 1.392
124.0 1.145
47.08 1.393
54.37 1.396
8j
H
13
8k
8l
Me
H
H
4
11
16.78 0.882
75.51 1.510
14.66 1.016
78.71 1.114
13
Me
61
NTc
NTc
16a
16b
16c
16d
16e
16f
Me
Me
Me
Me
Me
Me
Me
Me
5
4
3.769 2.089
31.44 0.664
6.427 0.807
8.026 0.594
5.314 0.643
2.495 3.621
71.87 1.629
191.4 32.82
29.32 1.091
27.20 1.579
16.52 0.825
10.15 0.652
0.334 26.19
35.86 1.116
9.478 2.514
29.41 1.038
9
9
8
7
16g
16h
13
17
16i
16j
Me
Me
32
82
e
NTc
NTc
NTc
NTc
AC220
26.08 1.378
288.7 3.758
a
The percentage of remaining activity was evaluated by the KinaseProfiler™ service at Eurofins Pharma Discovery Services. (average of duplicates).
IC50 values were determined by the remaining activity of nine concentrations (1, 3, 10, 30, 100, 300, 1000, 3000, and 10,000 nM).
NT: not tested.
b
c

H. Kim et al. / European Journal of Medicinal Chemistry 120 (2016) 74e85
79
of the phenyl group on the 6-position (16ae16h) exhibited < 20%
remaining activity. The compounds without a methyl group at the
(MV4-11) than those treated with AC220 (2.125, 1.488, and
1.103 M, respectively), except compounds 8i and 16e. Although
m
5-position (8ie8l) were also evaluated at 1
m
M for their remaining
compound 16e demonstrated significant inhibitory activities
against FLT3-wt and FLT3-D835Y, it did not exhibit remarkable
antiproliferative activity against the leukemia cells. Furthermore,
the compounds without a methyl group at the 5-position (8i, 8k,
and 8l) did not provide significantly improved antiproliferative
activity. Thus, the methyl group at the 5-position could provide
effective antiproliferative activity against the leukemia cells. The
compound with a methyl group at the 6-position (8f) had the
highest antiproliferative activity against most leukemia cells,
including THP-1, HL-60, and MV4-11 cells (0.222, 0.127, and
activity against FLT3-wt. Although no significant effect on FLT3-wt
inhibition was anticipated from these derivatives, the result was
notably different from expected, as these derivatives exhibited
<20% remaining activity. The IC50 against both FLT3-wt and FLT3-
D835Y of the compounds with <30% remaining activity were
evaluated. The data in Table 1 demonstrates that a methyl group (8f
and 8k) and a cycloaminoalkoxy or elongated aminoethoxy group
in the para position of the phenyl group at the 6-position (16ae16f)
provided effective inhibitory activities against FLT3-wt. In addition,
the absence of substituents at the 5-position (8ie8l) displayed
enhanced inhibitory activity against FLT3-wt. As a result, the
compounds with <10% remaining activities of FLT3-wt (8f, 8k, 16a,
and 16ce16f) exhibited higher inhibitory activities against FLT3-wt
than AC220 (26 nM), with the exception of 16b (31 nM).
0.191 mM, respectively). Moreover, the compound with the cyclo-
aminoalkoxy or elongated aminoethoxy group in the para position
of the phenyl group at the 6-position (16ae16d and 16f) demon-
strated effective antiproliferative activity against MV4-11 cells with
GI50 values < 0.600 mM.
The inhibitory activities against FLT3-D835Y of the compounds
with <20% remaining activities of FLT3-wt (8ee8g, 8ie8l, 16ae16h)
were confirmed to be higher than with AC220 (288 nM). Unlike the
inhibitory activities against FLT3-wt, a hydroxymethyl (8e), cyano
(8g), 4-(piperidine-4-yl)phenyl (16b), 4-{2-(N-benzyl)amino-
ethoxy}phenyl (16g), or 4-{2-(N-cyanoethyl)aminoethoxy}phenyl
group (16h) at the 6-position exhibited good inhibitory activity
against FLT3-D835Y with IC50 values < 100 nM. Moreover, when
comparing the IC50 values between FLT3-wt and FLT3-D835Y, these
derivatives were more active against FLT3-D835Y than FLT3-wt.
Aside from this finding, the structure-activity relationships
following the evaluation of the prepared compounds against FLT3-
D835Y were similar to the inhibitory activity against FLT3-wt. As a
result, the compounds with <10% remaining activity of FLT3-wt (8f,
8k, and 16ae16f) showed strong inhibitory activity against FLT3-
wt, as well as its drug-resistant mutant FLT3-D835Y, with IC50 be-
tween 0.3 and 36 nM. Among them, the compound with a methyl
group at the 5-position and a 2-(N-hydroxyethyl)aminoethoxy
group at the 6-position (16e) displayed significant inhibitory ac-
tivities against both FLT3-wt and FLT3-D835Y (5 and 0.3 nM,
respectively).
However, the compounds with small or simple groups at the 6-
positions typically showed low metabolic stability (Table 3). Com-
pound 8f exhibited the highest antiproliferative activity against
FLT3-ITD harboring cells (MV4-11) but showed a low percentage of
remaining human and rat liver microsomes (32.4% and 22.7%
remaining after the 30 min incubation, respectively). Fortunately,
the compounds with a cycloaminoalkoxy or elongated amino-
ethoxy group in the para position of the phenyl group at the 6-
position (16ae16f) exhibited enhanced metabolic stabilities in
human and rat liver microsomes. As shown in Table 3, most of the
compounds with a para-substituted phenyl group at the 6-position
(8b, 8h, 8j, 8l, and 16ae16i) displayed slightly enhanced metabolic
stability with a remaining percentage of >40% after the 30 min
incubation with human liver microsomes. This result suggested
that the presence of a para-substituted phenyl group at the 6-
position partially blocked any metabolic activity. Among them,
the compound with a methyl group on the 5-position and a 4-(2-
methylaminoethoxy) phenyl group at the 6-postion (16d) exhibi-
ted relatively high metabolic stability (79.8% and 67.1% remaining
after the 30 min incubation in the human and rat liver microsomes,
respectively). Despite a methyl group at the 6-position, which
contributed to effective FLT3 inhibition and antiproliferative
The antiproliferative activities (GI50) of the compounds that
displayed the inhibitory activity against FLT3-wt and had IC50
values < 100 nM (8f, 8i, 8k, 8l, and 16ae16f) against the four leu-
kemia cell lines are presented in Table 2. Most of the prepared
derivatives exhibited higher antiproliferative activity against FLT3-
wt expressing cells (THP-1 and HL-60) and FLT3-ITD harboring cells
Table 3
The in vitro metabolic stability of thieno[2,3-d]pyrimidine derivatives.
Cpd
In vitro metabolic stability(%)a
HLMb
RLMc
8b
8e
8f
8g
8h
8j
8k
8l
16a
16b
16c
16d
16e
16f
16g
16h
16i
16j
39.52
24.62
32.37
52.66
71.84
61.04
58.82
74.14
50.79
52.35
72.67
79.79
91.29
66.15
86.23
52.04
41.73
66.86
54.46
20.45
22.69
26.08
28.84
26.91
32.63
28.21
34.21
47.84
41.52
67.09
NDd
Table 2
Cell proliferation assay of thieno[2,3-d]pyrimidine derivatives.
Cpd
GI50(m
M)a
K562b
THP-1c
HL-60d
MV4-11e
8f
8i
8k
8l
16a
16b
16c
16d
16e
16f
AC220
0.105
1.090
1.005
0.506
0.315
0.425
1.045
0.564
1.413
0.489
2.638
0.222
1.271
1.236
0.765
0.227
0.46
0.636
0.559
0.843
0.449
2.125
0.127
0.682
0.422
0.336
0.214
0.377
1.262
0.25
0.191
1.703
1.055
0.927
0.366
0.58
0.585
0.540
1.132
0.278
1.103
39.68
30.41
32.13
41.03
35.65
1.345
0.598
1.488
a
a
Growth inhibition was measured by the XTT assay (average of four replicates).
Non-FLT3 expressing cells.
FLT3-wt expressing cells.
FLT3-wt expressing cells.
FLT3-ITD harboring cells.
Remaining percentage of the compound after the 30 min incubation
(average of triplicate).
b
c
b
Human liver microsomes.
Rat liver microsomes.
ND: Remaining compound was not detected after the 30 min incubation.
d
e
c
d

80
H. Kim et al. / European Journal of Medicinal Chemistry 120 (2016) 74e85
Table 4
activities against the leukemia cells, this compound showed poor
metabolic stability. However, the cycloaminoalkoxy or elongated
aminoethoxy group in the para position of the phenyl group at the
6-position displayed enhanced metabolic stability but no signifi-
cantly improved antiproliferative activities against the leukemia
cells. Compound 16d exhibited higher inhibitory activities against
FLT3-wt and FLT3-D835Y (8 and 10 nM, respectively) and effective
The inhibitory activities and remaining activities against Abl1 and Tie2 kinases of 8f,
16a, and 16d.
Compound
IC50
(
m
M)b
Abl1
Tie2
8f
16a
16d
29% at 10
26% at 10
2.877 0.501
m
m
Ma
Ma
32% at 10
25% at 10
9.750 12.50
m
m
Ma
Ma
antiproliferative activity against MV4-11 cells (0.540 mM).
Interestingly, all of the prepared compounds exhibited higher
antiproliferative activity against non-FLT3 expressing cells (K562)
than against AC220 (2.638 mM). In particular, compounds 8f, 8l, 16a,
16b, 16d, and 16f exhibited good antiproliferative activities against
K562 cells with GI50 values ranging from 105 to 564 nM. The
K562 cells express high BCR-ABL, as well as Abl1 kinase levels.
Accordingly, compound 16d was tested against the remaining ac-
a
The percentage of remaining activity was evaluated by the KinaseProfiler™
service at Eurofins Pharma Discovery Services. (average of duplicates).
b
IC50 values were determined by the remaining activity of nine concentrations (1,
3, 10, 30, 100, 300, 1000, 3000, and 10,000 nM).
Table 5
Rat pharmacokinetic study and solubility of 16d.
tivities of 56 kinases at a 10 mM to determine if 16d inhibited any
IVa (n ¼ 4, 5 mg/kg)
POb (n ¼ 4, 30 mg/kg)
other kinases (Fig. 2). In accordance with the remaining activities of
the 56 kinases, FLT3-wt and FLT3-D835Y were identified as the
primary target of compound 16d. Moreover, as shown in Table 4,
compound 16d had a moderate effect on Abl1 and Tie2 kinases with
c
Tmax (hr)
e
0.25
0.42
2.173
e
d
Cmax
(
m
g/mL)
e
e
AUC0-inf (hr$
CLf (L/hr/kg)
Vssg (L/kg)
m
g/mL)
2.125
2.197
8.217
7.610
e
IC50 values of 2.88
mM and 9.75 mM, respectively. These results
e
h
t1/2 (hr)
15.15
17.04
indicated that thieno[2,3-d]pyrimidine derivatives did not selec-
tively inhibit FLT3 kinases or Abl1 kinase activity, but showed good
antiproliferative activity against K562 cells. Furthermore, AC220
inhibited effectively proliferation of K562 and HL60 cells unlike the
previous results, which showed no antiproliferative activity [24]. It
is thought to be arisen from the differences of the initial density of
cell seeding in the proliferation assays.
The compound with the highest metabolic stability (16d) was
further evaluated in a rat pharmacokinetic study (Table 5). Com-
pound 16d demonstrated acceptable solubility in phosphate buffer
Fi (%)
Solubilityj (
m
M)
2.96
a
Intravenous.
Oral administration.
b
c
d
e
f
Time to maximum plasma concentration.
Maximum plasma concentration after oral administration.
Area under the curve.
Clearance.
g
h
i
Volume of distribution at steady state.
Elimination half life.
Bioavailability.
j
solution (2.96
mM) and relatively favorable oral bioavailability
The solubility was tested in 100 mM pH 7.4 phosphate buffer solution.
(17.04%). Based on these results, 16d appeared to be promising lead
compound for further optimization and potential development as a
Fig. 2. Percentage of 16d kinase activities against 56 kinases at 10 mM.

H. Kim et al. / European Journal of Medicinal Chemistry 120 (2016) 74e85
81
FLT3 inhibitor. Taken together, structural modifications at the 6-
5.2.2. Ethyl 2-amino-5-cyano-4-methylthiophene-3-carboxylate
(3f)
position led to improved potency and metabolic stability, which
provided a good opportunity for further optimization of the PK
profiles.
A mixture of ethyl cyanoacetate (1.25 g, 11.0 mmol), 3-
aminocrotonitrile (0.91 g, 11.0 mmol), sulfur (1.4 g, 11.0 mmol),
and piperidine (0.94 g, 11.0 mmol) in ethanol (15 mL) was refluxed
for 24 h then cooled and concentrated under reduced pressure.
Compound 3d (pale yellow, 0.53 g, 22.9%) was obtained by column
chromatography (EtOAc: hexane ¼ 1: 7), 1H NMR (400 MHz, DMSO-
4. Conclusions
We prepared thieno[2,3-d]pyrimidine derivatives to serve as
potential FLT3 inhibitors for the treatment of AML. The structural
modification of thieno[2,3-d]pyrimidines was determined by the
results of kinase and cell proliferation assays; however, metabolic
stability was also considered. As a result, a methyl group at the 5-
position and a 4-(2-methylaminoethoxy) phenyl group at the 6-
position were associated with effective inhibition of FLT3 kinase
activity, as well as inhibition of the growth of several leukemia cells
with a relatively high metabolic stability. On the basis of the
inhibitory activity, antiproliferative activity and in vitro metabolic
stability, compound 16d we have generated appears to be a well-
balanced potential FLT3 inhibitor. We expect that 16d will pro-
vide the structural basis for further optimization and could prove to
be a promising therapeutic agent for the treatment of AML.
d6):
d
8.10 (s, 2H), 4.18 (q, ¼ 7.2 Hz, 2H), 2.33 (s, 3H), 1.24 (t,
J ¼ 7.0 Hz, 3H).
5.3. General procedure for the synthesis of 2-aminothiophenes
3ge3i
A mixture of ethyl cyanoacetate (13.6 mmol), sulfur (13.6 mmol)
and triethylamine (13.6 mmol) in dimethylformamide (DMF;
15 mL) was stirred at 50 ꢀC. A solution of the corresponding alde-
hyde (13.6 mmol) in DMF (10 mL) was added dropwise over 20 min.
The mixture then was stirred at 50 ꢀC overnight. The reaction
mixture was cooled, poured into water, and the aqueous layer was
extracted with ethyl acetate. The organic layer was separated,
washed with brine, and dried over Na2SO4 then concentrated under
reduced pressure. The title compound was obtained by column
chromatography (EtOAc: hexane ¼ 1: 15).
5. Experimental
5.3.1. Ethyl 2-amino-5-(4-methoxyphenyl)lthiophene-3-
carboxylate(3g)
5.1. Chemistry
The parameters for 3g were: pale yellow solid, 60% yield, 1H
Reagents and solvents were purchased and used without further
purification. The reaction progress was determined by thin-layer
chromatography (TLC) on silica gel 60G F254 plates (Merck, Mum-
bai, India). Flash column chromatography was performed on silica
(Merck EM9385, 230e400 mesh). The spots were visualized using
UV light (254 nm). 1H NMR spectra were recorded at 400 MHz on a
Varian 400 Mercury Plus spectrometer (Varian, Palo Alto, CA, USA).
NMR (400 MHz, CDCl3):
(d, J ¼ 8.8 Hz, 2H), 5.95 (br, 2H), 4.29 (q, J ¼ 6.8 Hz, 2H), 3.81 (s, 3H),
1.37 (t, J ¼ 6.8 Hz, 3H).
d
7.37 (d, J ¼ 8.8 Hz, 2H), 7.01 (s, 1H), 6.87
5.3.2. Ethyl 2-amino-5-(4-fluorophenyl)lthiophene-3-carboxylate
(3h)
The parameters for 3h were: yellow solid, 26% yield, 1H NMR
Chemical shifts are reported in ppm (d) relative to tetramethylsi-
(400 MHz, DMSO-d6):
d 7.45e7.42 (m, 4H), 7.15 (s, 1H), 7.12 (t,
lane (TMS), which was used 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). The synthesis
of 8a, 8e, and 8h were previously reported [20,21]. Low-resolution
mass spectra and high-resolution spectra were obtained on a Shi-
madzu LCMS-2010EV system (Kyoto, Japan) and an Agilent 6530
Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) liquid
chromatography-mass spectrometry (LC/MS) system (Santa Clara,
CA, USA) with electrospray ionization (ESI).
J ¼ 8.8 Hz, 2H), 4.17 (q, J ¼ 6.8 Hz, 2H), 1.24 (t, J ¼ 7.2 Hz, 3H).
5.3.3. Ethyl 2-amino-5-methylthiophene-3-carboxylate (3i)
The parameters for 3i were: yellow solid, 17% yield, 1H NMR
(400 MHz, DMSO-d6):
2H), 1.19 (t, J ¼ 7.0 Hz, 3H).
d
7.06 (s, 2H), 6.45 (s, 1H), 4.10 (q, J ¼ 7.2 Hz,
5.4. General procedure for the synthesis of 4-chlorothieno[2,3-d]
pyrimidine intermediate 5
A mixture of the corresponding ethyl 2-aminothiophene 3
(0.48 mmol) and thiophene-2-carbonitrile (0.62 mmol) in 4M HCl
1,4-dioxane solution (6 ml) was stirred at 60 ꢀC under argon gas
overnight. The reaction mixture was then cooled and concentrated
under reduced pressure and the resulting residue was triturated
with diethyl ether. The resulting thienopyrimidin-4-one 4 was
dried under vacuum and used directly in the next step. A mixture of
the corresponding thienopyrimidin-4-one 4 in phosphoryl chloride
(4 ml) was refluxed for 12 h then the reaction mixture was cooled
and concentrated under reduced pressure. The resulting residue
was diluted with CH2Cl2/saturated aqueous NaHCO3. The organic
layer was separated, washed with brine, dried over Na2SO4 and
concentrated under reduced pressure. The title compound 5 was
then obtained by column chromatography (EtOAc: hexane ¼ 1: 20).
5.2. General procedure for the synthesis of 2-aminothiophene
intermediate 3ae3e
The synthesis of 2-aminothiophenes 3ae3e was previously re-
ported [20,21].
5.2.1. Ethyl 2-amino-5-morpholino-4-methylthiophene-3-
carboxylate (3c)
A mixture of ethyl cyanoacetate (1.54 g, 13.6 mmol), acetone
(0.79 g, 13.6 mmol), and sulfur (3.5 g, 13.6 mmol) in ethanol (15 mL)
was stirred at 60 ꢀC. Morpholine (2.37 g, 27.2 mmol) was added
dropwise over 10 min. The mixture was stirred at 60 ꢀC overnight
and filtered. The filtrate was cooled and evaporated under reduced
pressure. The title compound 3c (yellow solid, 120 mg, 3.3%) was
obtained by column chromatography (EtOAc: hexane ¼ 1: 10), 1H
5.4.1. 4-Chloro-5-methyl-6-morpholino-2-(thiophen-2-yl)thieno
[2,3-d]pyrimidine (5c)
NMR (400 MHz, DMSO-d6):
d
7.23 (s, 2H), 4.11 (q, J ¼ 6.8 Hz, 2H),
3.62 (t, J ¼ 4.4 Hz, 4H), 2.60 (t, J ¼ 4.4 Hz, 4H), 2.08 (s, 3H), 1.20 (t,
The parameters for 5c were: yellow solid, 32% yield, 1H NMR
J ¼ 7.0 Hz, 3H).
(400 MHz, DMSO-d6):
d
7.90 (d, J ¼ 3.6 Hz, 1H), 7.76 (d, J ¼ 4.8 Hz,

82
H. Kim et al. / European Journal of Medicinal Chemistry 120 (2016) 74e85
1H), 7.18 (m, 1H), 3.75 (t, J ¼ 4.4 Hz, 4H), 2.97 (t, J ¼ 4.4 Hz, 4H),
5.6.2. 1-[5-Methyl-6-morpholino-2-(thiophen-2-yl)thieno[2,3-d]
pyrimidin-4-ylamino]-3-methyl-1H-pyrrole-2,5-dione (8c)
The parameters for 8c were: yellow solid, 1H NMR (400 MHz,
2.44(s, 3H).
5.4.2. 4-Chloro-6-cyano-5-methyl-2-(thiophen-2-yl)thieno[2,3-d]
pyrimidine (5f)
DMSO-d6): d 9.43 (s, NH), 7.61e7.60 (m, 2H), 7.08e7.07 (m, 1H), 6.95
(m, 1H), 3.74e3.73 (m, 4H), 2.88 (m, 4H), 2.48 (s, 3H), 2.14 (s, 3H);
ESI (m/z) 442 (MHþ); HRMS (ESI) calculated for C20H19N5O3S2
[MHþ]: 442.1002, detected: 442.0999.
The parameters for 5f were: white solid, 37% yield, 1H NMR
(400 MHz, DMSO-d6):
d
8.05 (d, J ¼ 3.6 Hz, 1H), 7.92 (d, J ¼ 5.2 Hz,
1H), 7.25 (m, 1H), 2.75 (s, 3H).
5.6.3. Ethyl 5-methyl-4-(3-methyl-2,5-dioxo-2,5-dihydro-1H-
pyrrol-1-ylamino)-2-(thiophen-2-yl)thieno[2, 3-d]pyrimidine-6-
carboxylate (8d)
5.4.3. 4-Chloro-6-(4-methoxyphenyl)-2-(thiophen-2-yl)thieno[2,3-
d]pyrimidine (5g)
The parameters for 5g were: yellow solid, 92% yield, 1H NMR
The parameters for 8d were: pale yellow solid, 1H NMR
(400 MHz, CDCl3):
d
8.06 (dd, J ¼ 3.6, 1.2 Hz, 1H), 7.65 (d, J ¼ 8.8 Hz,
(400 MHz, DMSO-d6):
d 9.87 (s, 1H), 7.72e7.71 (m, 2H), 7.14e7.11
2H), 7.50 (dd, J ¼ 5.2, 1.2 Hz, 1H), 7.41 (s, 1H), 7.16 (dd, J ¼ 5.2, 3.6 Hz,
(m, 1H), 6.99e6.98 (m, 1H), 4.31 (q, J ¼ 6.8 Hz, 2H), 2.94 (s, 3H), 2.15
(s, 3H), 1.29 (t, J ¼ 7.2 Hz, 3H); ESI (m/z) 429 (MHþ); HRMS (ESI)
calculated for C19H16N4O4S2 [MHþ]: 429.0686, detected: 442.0682.
1H), 6.98 (d, J ¼ 8.8 Hz, 2H), 3.87 (s, 3H).
5.5. General procedure for the synthesis of 4-Chloro-6-(4-hydroxy)
phenylthieno[2,3-d]pyrimidine intermediate (6a and 6b)
5.6.4. 1-[5,6-Dimethyl-2-(thiophen-2-yl)thieno[2,3-d]pyrimidin-4-
ylamino]-3-methyl-1H-pyrrole-2,5-dione (8f)
The parameters for 8f were: pale yellow solid, 1H NMR
A solution of the corresponding 4-chlorothienopyrimidine 5a
and 5b (1.32 mmol) in CH2Cl2 was stirred at room temp. A 1M boron
tribromide hexane solution (8 ml) was added dropwise to the re-
action mixture followed by stirring at room temperature for 6 h.
The reaction mixture was diluted with ethyl acetate/saturated
aqueous NH4Cl. The organic layer was separated, washed with
brine, dried over Na2SO4 and concentrated under reduced pressure.
The title compound 6 was then obtained by column chromatog-
raphy (EtOAc: hexane ¼ 1: 3).
(400 MHz, DMSO-d6):
d
9.46 (s, 1H), 7.62 (d, J ¼ 4.4 Hz, 2H),
7.10e7.07 (m, 1H), 6.95 (m, 1H), 2.48 (s, 3H), 2.41 (s, 3H), 2.14 (s,
3H); ESI (m/z) 371 (MHþ); HRMS (ESI) calculated for C17H14N4O2S2
[MHþ]: 371.0631, detected: 371.0623.
5.6.5. 1-[6-Cyano-5-methyl-2-(thiophen-2-yl)thieno[2,3-d]
pyrimidin-4-ylamino]-3-methyl-1H-pyrrole-2,5-dione (8g)
The parameters for 8g were: brown solid, 1H NMR (400 MHz,
DMSO-d6): d 10.00 (s,1H), 7.76e7.73 (m, 2H), 7.15e7.13 (m,1H), 6.99
5.5.1. 4-Chloro-6-(4-hydroxyphenyl)-5-methyl-2-(thiophen-2-yl)
thieno[2,3-d]pyrimidine (6a)
(m, 1H), 2.78 (s, 3H), 2.15 (s, 3H); ESI (m/z) 382 (MHþ); HRMS (ESI)
calculated for C17H11N5O2S2 [MHþ]: 382.0427, detected: 371.0418.
The parameters for 6a were: yellow solid, 81% yield, 1H NMR
(400 MHz, DMSO-d6):
d 9.93 (s, 1H), 7.88e7.85 (m, 1H), 7.74e7.73
5.6.6. 1-[6-(4-Methoxyphenyl)-2-(thiophen-2-yl)thieno[2,3-d]
pyrimidin-4-ylamino]-3-methyl-1H-pyrrole-2,5-dione (8i)
The parameters for 8i were: yellow solid, 1H NMR (400 MHz,
(m,1H), 7.29 (d, J ¼ 8.4 Hz, 2H), 7.14e7.12 (m,1H), 6.87 (d, J ¼ 8.4 Hz,
2H), 2.43 (s, 3H).
DMSO-d6):
d 10.6 (s, 1H), 7.82 (s, 1H), 7.66e7.61 (m, 4H), 7.10 (m,
5.5.2. 4-Chloro-6-(4-hydroxyphenyl)-2-(thiophen-2-yl)thieno[2,3-
d]pyrimidine (6b)
1H), 7.06 (d, J ¼ 4.4 Hz, 2H), 6.97 (m, 1H), 3.70 (s, 3H), 2.15 (s, 3H);
ESI (m/z) 449 (MHþ); HRMS (ESI) calculated for C22H16N4O3S2
[MHþ]: 449.0737, detected: 449.0734.
The parameters for 6b were: yellow solid, 92% yield, 1H NMR
(400 MHz, DMSO-d6): d 10.0 (s, 1H), 7.87 (m, 1H), 7.74e7.73 (m, 1H),
7.61 (d, J ¼ 8.0 Hz, 2H), 7.54 (s, 1H), 7.14 (m, 1H), 6.83 (d, J ¼ 8.4 Hz,
5.6.7. 1-[6-(4-Fluorophenyl)-2-(thiophen-2-yl)thieno[2,3-d]
pyrimidin-4-ylamino]-3-methyl-1H-pyrrole-2,5-dione (8j)
The parameters for 8j were: yellow solid, 1H NMR (400 MHz,
2H).
5.6. General procedure for the synthesis of thieno[2,3-d]pyrimidine
derivatives 8ae8k
DMSO-d6):
d
10.7(s, 1H), 7.91(s, 1H), 7.73(dd, J ¼ 5.2, 4.2 Hz, 2H),
7.67e7.65(m, 2H), 7.34 (t, J ¼ 8.6 Hz, 2H), 7.12e7.09 (m, 1H),
6.98e6.97 (m, 1H), 2.15 (s, 3H); ESI (m/z) 437 (MHþ); HRMS (ESI)
calculated for C21H13FN4O2S2 [MHþ]: 437.0537, detected: 437.0534.
A solution of the corresponding 4-chlorothienopyrimidine 5,
6ae6b (0.22 mmol) in tetrahydrofuran (THF) was stirred at room
temperature. Hydrazine hydrate (0.67 mmol) was added dropwise
and the reaction mixture was refluxed overnight. The reaction
mixture was then cooled and concentrated under reduced pressure
and the resulting solid compound 7 was used directly in the next
step. A mixture of the corresponding hydrazino-intermediate 7 and
citraconic anhydride (0.67 mmol) was refluxed overnight. The re-
action mixture was cooled and concentrated under reduced pres-
5.6.8. 1-[6-Methyl-2-(thiophen-2-yl)thieno[2,3-d]pyrimidin-4-
ylamino]-3-methyl-1H-pyrrole-2,5-dione (8k)
The parameters for 8k were: pale yellow solid, 1H NMR
(400 MHz, DMSO-d6): d 10.5 (s, 1H), 7.62e7.61 (m, 2H), 7.28 (s, 1H),
7.09e7.07 (m, 1H), 6.95 (m, 1H), 2.56 (s, 3H), 2.13 (s, 3H); ESI (m/z)
357 (MHþ); HRMS (ESI) calculated for C16H12N4O2S2 [MHþ]:
357.0474, detected: 357.0471.
sure. The title compound
8 was then obtained by column
chromatography (EtOAc: hexane ¼ 1: 2).
5.6.9. 1-[6-(4-Hydroxyphenyl)-2-(thiophen-2-yl)thieno[2,3-d]
pyrimidin-4-ylamino]-3-methyl-1H-pyrrole-2,5-dione (8l)
The parameters for 8l were: yellow solid, 1H NMR (400 MHz,
5.6.1. 1-[6-(4-Fluorophenyl)-5-methyl-2-(thiophen-2-yl)thieno
[2,3-d]pyrimidin-4-ylamino]-3-methyl-1H-pyrrole-2,5-dione (8b)
The parameters for 8b were: yellow solid, 1H NMR (400 MHz,
DMSO-d6): d 10.73 (br, 1H), 7.84 (s, 1H), 7.69e7.67 (m, 2H), 7.56 (dd,
DMSO-d6):
d
9.65 (s, 1H), 7.67e7.66 (m, 2H), 7.61e7.58 (m, 2H), 7.36
J ¼ 8.8 Hz, 2.0 Hz, 2H), 7.16e7.12 (m,1H), 7.01 (d, J ¼ 2.0 Hz,1H), 6.93
(dd, J ¼ 8.8, 2.0 Hz, 2H), 2.18 (d, J ¼ 2.0 Hz, 3H); ESI (m/z) 435 (MHþ);
HRMS (ESI) calculated for C21H14N4O3S2 [MHþ]: 435.0580, detec-
ted: 435.0560.
(t, J ¼ 8.4 Hz, 2H), 7.12 (m,1H), 6.98 (m,1H), 2.59 (s, 3H), 2.16 (s, 3H);
ESI (m/z) 451 (MHþ); HRMS (ESI) calculated for C22H15FN4O2S2
[MHþ]: 451.0693, detected: 451.0685.

H. Kim et al. / European Journal of Medicinal Chemistry 120 (2016) 74e85
83
5.7. 5-Methyl-4-oxo-2-(thiophen-2-yl)-3,4-dihydrothieno[2,3-d]
pyrimidine-6-carboxylic acid (9)
citraconic anhydride was refluxed overnight then the reaction
mixture was cooled and concentrated under reduced pressure. The
resulting residue was purified by column chromatography (EtOAc:
hexane ¼ 1: 1) and dissolved in 4M HCl 1,4-dioxane solution, then
concentrated under reduced pressure. The crude residue was
recrystallized from diethyl ether to obtain the title compound 13 or
16.
A solution of the thieno[2,3-d]pyrimidine-4-one 4d (0.48 g,
1.5 mmol) in 2 N aqueous NaOH (30 ml) was refluxed for 14 h. The
reaction mixture was cooled and washed with ethyl acetate and the
aqueous layer was neutralized with 6 N aqueous HCl. The pre-
cipitates were collected by filtration and washed with water, which
yielded the title compound 9 (white solid, 0.41 g, 94%), 1H NMR
5.9.1. N-[2-(dimethylamino)ethyl]-5-methyl-4-[(3-methyl-2,5-
dioxo-2,5-dihydro-1H-pyrrol-1-yl)amino]-2-thiophen-2-yl)thieno
[2,3-d]pyrimidine-6-carboxamide hydrochloride (13)
(400 MHz, DMSO-d6):
1H), 7.26e7.24 (m, 1H), 2.82 (s, 3H).
d
8.28 (d, J ¼ 4.0 Hz, 1H), 7.94 (d, J ¼ 4.8 Hz,
The parameters for 13 were: yellow solid, 1H NMR (400 MHz,
5.7.1. 4-Chloro-5-Methyl-2-(thiophen-2-yl)-3,4-dihydrothieno[2,3-
d]pyrimidine-6-carboxylic acid-{(2-dimethyl-amino)ethyl}amide
(11)
DMSO-d6):
d
11.34 (br, 1H), 9.55 (br, 1H), 8.78 (t, J ¼ 5.6 Hz, 1H), 7.84
(d, J ¼ 3.6 Hz, 1H), 7.47 (d, J ¼ 4.8 Hz, 1H), 7.10 (t, J ¼ 3.6 Hz, 1H), 6.69
(d, J ¼ 1.2 Hz, 1H), 3.86e3.82 (m, 2H), 3.43e3.39 (m, 2H), 2.97 (s,
3H), 2.94 (d, J ¼ 4.8 Hz, 6H), 2.24 (d, J ¼ 1.2 Hz, 3H); ESI (m/z) 471
(MHþ); HRMS (ESI) calculated for C21H22N6O3S2 [MHþ]: 471.1268,
detected: 471.1254.
A mixture of the 4-oxo-thieno[2,3-d]pyrimidine-6-carboxylic
acid 9 (350 mg, 1.2 mmol) in phosphoryl chloride was refluxed
overnight and the reaction mixture was cooled and concentrated
under reduced pressure. The resulting residue was diluted with
CH2Cl2/saturated aqueous NaHCO3. The organic layer was sepa-
rated, washed with brine, dried over Na2SO4 and concentrated
under reduced pressure. The resulting 4-chloro-thienopyrimidin-
6-carboxylic acid 10 was dried under vacuum and used directly in
the next step. A mixture of 4-chloro-thienopyrimidin-6-carboxylic
acid 10 (60 mg, 0.20 mmol), 1-(3-methylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDC$HCl, 48 mg, 0.26 mmol), 1-
hydroxybenzotriazole hydrate (2 mg, 0.02 mmol) in CH2Cl2 was
5.9.2. 3-Methyl-1-{[5-methyl-6-(4-(pyrrolidin-3-yloxy)phenyl)-2-
(thiophen-2-yl)thieno[2,3-d]pyrimidin-4-yl]amino}-1H-pyrrole-
2,5-dione hydrochloride (16a)
The parameters for 16a were: yellow solid, 1H NMR (400 MHz,
DMSO-d6):
d 9.78 (br, 1H), 9.63 (s, 1H), 9.56 (br, 1H), 7.65 (d,
J ¼ 4.0 Hz, 2H), 7.49 (d, J ¼ 8.4 Hz, 2H), 7.11e7.09 (m, 3H), 6.97 (s,
1H), 3.49e3.45 (m, 1H), 3.34e3.2 (m, 3H), 2.59 (s, 3H), 2.15 (s, 3H);
ESI (m/z) 518 (MHþ); HRMS (ESI) calculated for C26H23N5O3S2
[MNaþ]: 540.1135, detected: 540.1133.
stirred at room temperature. N, N-dimethylethylenediamine (24 mL,
0.22 mmol) was added slowly dropwise and the reaction mixture
was stirred for 2 h then concentrated under reduced pressure. The
title compound 11 (yellow solid, 60 mg, 80%) was obtained by
5.9.3. 3-Methyl-1-{[5-methyl-6-(4-(piperidin-4-yloxy)phenyl)-2-
(thiophen-2-yl)thieno[2,3-d]pyrimidin-4-yl]amino}-1H-pyrrole-
2,5-dione hydrochloride (16b)
column chromatography (CH2Cl2: MeOH
(400 MHz, CDCl3):
7.19e7.17 (m, 1H), 6.84 (br, 1H), 3.54 (q, J ¼ 5.6 Hz, 2H), 2.91 (s, 3H),
2.55 (t, J ¼ 5.6 Hz, 2H), 2.29 (s, 6H).
¼
9: 1), 1H NMR
d
8.10 (d, J ¼ 4.0 Hz, 1H), 7.55 (d, J ¼ 4.8 Hz, 1H),
The parameters for 16b were: yellow solid, 1H NMR (400 MHz,
DMSO-d6): d 9.65 (s, 1H), 8.91 (br, 2H), 7.69 (m, 2H), 7.52 (d,
J ¼ 8.8 Hz, 2H), 7.18e7.02 (m, 3H), 7.01 (s, 1H), 4.77e4.74 (m, 1H),
3.24 (br, 2H), 3.09 (br, 2H), 2.62 (s, 3H), 2.19 (s, 3H), 2.14e2.08 (m,
2H), 1.91e1.87 (m, 2H); ESI (m/z) 532 (MHþ); HRMS (ESI) calculated
for C27H25N5O3S2 [MHþ]: 532.1472, detected: 532.1464.
5.8. General procedure for the synthesis of 4-Chlorothieno[2,3-d]
pyrimidine derivatives 14
A
mixture of the 4-chlorothieno[2,3-d]pyrimidine 6a
5.9.4. 3-Methyl-1-{[5-methyl-6-(4-(2-(pyrrolidin-1-yl)ethoxy)
phenyl)-2-(thiophen-2-yl)thieno[2,3-d]pyrimidin-4-yl]amino}-1H-
pyrrole-2,5-dione hydrochloride (16c)
(0.42 mmol), triphenylphosphine (PPh3; 0.84 mmol) and the cor-
responding alcohol intermediate (0.42 mmol) in THF was stirred at
0
ꢀC. Diisopropyl azodicarboxylate (0.84 mmol) was added drop-
The parameters for 16c were: yellow solid, 1H NMR (400 MHz,
wise over 20 min and the reaction mixture was slowly warmed to
room temperature, stirred overnight and concentrated under
reduced pressure. The title compound 14 was then obtained by
column chromatography (EtOAc: hexane ¼ 1: 7).
DMSO-d6):
d
9.68 (s, 1H), 7.69 (m, 2H), 7.55 (d, J ¼ 8.4 Hz, 2H), 7.15
(m, 3H), 7.02 (d, J ¼ 2.0 Hz, 1H), 4.45 (t, J ¼ 4.8 Hz, 2H), 3.58 (m, 4H),
3.10 (m, 2H), 2.62 (s, 3H), 2.19 (d, J ¼ 2.0 Hz, 3H), 2.00 (m, 2H), 1.89
(m, 2H); ESI(m/z) 546 (MHþ); HRMS (ESI) calculated for
C
28H27N5O3S2 [MHþ]: 546.1628, detected: 546.1622.
5.8.1. The tert-butyl [2-[4-(4-chloro-5-methyl-2-(thiophen-2-yl)
thieno[2,3-d]pyrimidine-6-yl)phenoxy]ethyl]methylcarbamate
(14d)
5.9.5. 3-Methyl-1-{[5-methyl-6-[4-(2-(methylamino)ethoxy)
phenyl]-2-(thiophen-2-yl)thieno[2,3-d]pyrimidin-4-yl]amino}-1H-
pyrrole-2,5-dione hydrochloride (16d)
The parameters for 14d were: yellow solid, 1H NMR (400 MHz,
DMSO-d6):
d
7.88 (d, J ¼ 3.2 Hz, 1H), 7.76 (d, J ¼ 4.8 Hz, 1H), 7.38 (d,
The parameters for 16d were: yellow solid, 1H NMR (400 MHz,
J ¼ 8.4 Hz, 2H), 7.15 (t, J ¼ 4.4 Hz, 1H), 7.03 (d, J ¼ 8.4 Hz, 2H), 4.11
DMSO-d6): d 9.68 (s, 1H), 9.24 (s, 2H), 7.70e7.68 (m, 2H), 7.54 (d,
(m, 2H), 3.52 (m, 2H), 2.83 (m, 3H), 2.45 (s, 3H), 1.30 (s, 9H).
J ¼ 8.8 Hz, 2H), 7.17e7.13 (m, 3H), 7.02 (s,1H), 4.35 (t, J ¼ 4.8 Hz, 2H),
3.36e3.32 (m, 2H), 2.62 (s, 6H), 2.19 (s, 3H); ESI (m/z) 506 (MHþ);
HRMS (ESI) calculated for C25H23N5O3S2 [MHþ]: 506.1315, detected:
506.1311.
5.9. General procedure for the synthesis of thieno[2,3-d]pyrimidine
derivatives 13, 16ae16h
A solution of the corresponding 4-chlorothienopyrimidine 11 or
14 in THF was stirred at room temperature. Hydrazine hydrate was
added dropwise and the reaction mixture was refluxed overnight
then cooled and concentrated under reduced pressure. The
resulting residue 12 or 15 was used directly in the next step. A
mixture of the corresponding hydrazino-intermediate 12 or 15 and
5.9.6. 1-{[6-(4-(2-((2-Hydroxyethyl)amino)ethoxy)phenyl)-5-
methyl-2-(thiophen-2-yl)thieno[2,3-d]pyrimidin-4-yl)]amino}-3-
methyl-1H-pyrrole-2,5-dione hydrochloride (16e)
The parameters for 16e were: yellow solid, 1H NMR (400 MHz,
DMSO-d6):
d 9.67(s, 1H), 8.93(s, 2H), 7.70e7.68 (m, 2H), 7.55 (d,
J ¼ 8.4 Hz, 2H), 7.18e7.14 (m, 3H), 7.02 (s,1H), 4.36 (t, J ¼ 4.8 Hz, 2H),

84
H. Kim et al. / European Journal of Medicinal Chemistry 120 (2016) 74e85
3.72e3.70 (m, 2H), 3.42e3.40 (m, 2H), 3.13e3.10 (m, 2H), 2.62 (s,
3H), 2.19 (s, 3H); ESI (m/z) 536 (MHþ); HRMS (ESI) calculated for
C
HPLC and an Agilent 6490 triple quadrupole LC/MS with an ESI
source. The percentage of the compound remaining was deter-
mined by calculating the ratio between peak areas of the com-
pounds after incubation for 0 min and peak area at 30 min.
26H25N5O4S2 [MHþ]: 536.1421, detected: 536.1407.
5.9.7. 1-{[6-(4-(2-(Isopentylamino)ethoxy)phenyl)-5-methyl-2-
(thiophen-2-yl)thieno[2,3-d]pyrimidin-4-yl]amino}-3-methyl-1H-
pyrrole-2,5-dione hydrochloride (16f)
5.11. The remaining activity and kinase inhibition analysis
The parameters for 16f were: yellow solid, 1H NMR (400 MHz,
The remaining activity and kinase inhibition were determined
using the KinaseProfiler™ assay protocols (Eurofins Pharma Dis-
covery Services, Dundee, UK). The IC50 values of FLT3 and FLT3-
D835Y were determined by evaluating the remaining activity at
10,000, 3000, 1000, 300, 100, 30, 10, 3, and 1 nM with 10 mM ATP
according to the KinaseProfiler™ assay protocol.
DMSO-d6):
d 9.67 (s, 1H), 9.10 (s, 2H), 7.70e7.69 (m, 2H), 7.55 (d,
J ¼ 7.6 Hz, 2H), 7.17e7.14 (m, 3H), 7.02 (s, 1H), 4.37 (t, J ¼ 4.8 Hz, 2H),
3.37 (m, 2H), 3.00 (m, 2H), 2.63 (s, 3H), 2.19 (s, 3H), 1.68e1.63 (m,
1H), 1.62e1.54 (m, 2H), 0.91 (s, 3H), 0.90 (s, 3H); ESI (m/z) 562
(MHþ); HRMS (ESI) calculated for C29H31N5O3S2 [MNaþ]: 584.1761,
detected: 584.1746.
5.12. Cell proliferation assays
5.9.8. 1-{[6-(4-(2-(Benzylamino)ethoxy)phenyl)-5-methyl-2-
(thiophen-2-yl)thieno[2,3-d]pyrimidin-4-yl]amino}-3-methyl-1H-
pyrrole-2,5-dione hydrochloride (16g)
The human leukemia cell lines K562, HL-60, THP-1, and MV4-11
were purchased from American Type Culture Collection (Manassas,
VA, USA) and cultured in RPMI 1640 supplemented with 10% fetal
bovine serum (FBS). Cell cultures were maintained at 37 ꢀC under a
humidified atmosphere of 5% CO2. Cell proliferation assays were
performed using the Cell Proliferation Kit II (XTT) (Roche, Indian-
apolis, IN, USA) according to the manufacturer’s protocol. Briefly,
6000e8000 cells were seeded on 96-well plates and treated the
The parameters for 16g were: yellow solid, 1H NMR (400 MHz,
DMSO-d6):
d 9.66 (s, 1H), 9.50 (br, 2H), 7.69 (m, 2H), 7.59 (m, 2H),
7.54 (m, 2H), 7.48e7.43 (m, 3H), 7.17e7.13 (m, 3H), 7.02 (s, 1H), 4.38
(t, J ¼ 4.7 Hz, 2H), 3.37 (m, 2H), 4.26 (t, J ¼ 5.1 Hz, 2H), 3.36 (br, 2H),
2.62 (s, 3H), 2.19 (s, 3H); ESI (m/z) 582 (MHþ); HRMS (ESI) calcu-
lated for C31H27N5O3S2 [MNaþ]: 604.1448, detected: 604.1439.
folloing day with the test compounds. After 48 h, 50 mL of XTT la-
5.9.9. 3-{[2-(4-(5-Methyl-4-((3-methyl-2,5-dioxo-2,5-dihydro-1H-
pyrrol-1-yl)amino)-2-(thiophen-2-yl)thieno[2,3-d]pyrimidin-6-yl)
phenoxy)ethyl]amino}propanenitrile hydrochloride (16h)
beling mixture, which was prepared by mixing 50 vol of 1 mg/mL
sodium 30-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-
methoxy-6-nitro) benzene sulfonic acid hydrate with 1 volume of
0.383 mg/mL of N-methyldibenzopyrazine methyl sulfate, was
added to each well and incubated for 2 h at 37 ꢀC. The absorbance
was measured at 490 nm with a reference wavelength of 655 nm
using an ELISA plate reader (Molecular Devices, Sunnyvale, CA,
USA).
The parameters for 16h were: yellow solid, 1H NMR (400 MHz,
DMSO-d6):
d 9.67 (s, 1H), 9.58 (br, 2H), 7.69 (m, 2H), 7.56 (d,
J ¼ 8.4 Hz, 2H), 7.18e7.13 (m, 3H), 7.02 (s,1H), 4.36 (t, J ¼ 4.6 Hz, 2H),
3.42e3.36 (m, 4H), 3.06 (t, J ¼ 7.1 Hz, 2H), 2.62 (s, 3H), 2.19 (s, 3H);
ESI (m/z) 545 (MHþ); HRMS (ESI) calculated for C27H24N6O3S2
[MHþ]: 545.1424, detected: 545.1413.
5.13. Solubility test
5.9.10. 3-Methyl-1-[{5-methyl-6-(4-((tetrahydrofuran-2-yl)
methoxy)phenyl)-2-(thiophen-2-yl)thieno[2,3-d]pyrimidin-4-yl}
amino]-1H-pyrrole-2,5-dione (16i)
The hydrochloride salt of the appropriate compound was used
in the study. Compound 16d was suspended in pH 7.4 PBS. The
samples were gently shaken for 1 h at 37 ꢀC, centrifuged for 5 min,
The parameters for 16i were: pale yellow solid, 1H NMR
(400 MHz, DMSO-d6):
d
9.56 (s, 1H), 7.65 (m, 2H), 7.45 (d, J ¼ 8.4 Hz,
and filtered over 0.2
solution was added to 100
Agilent 1260 HPLC and 6490 triple quadruple LC/MS with an ESI
source. The solubility was determined from a standard curve sing
standards that were prepared in acetonitrile/PBS (1:1).
m
m filters. An amount equal to 100
mL of each
2H), 7.11 (t, J ¼ 4.4 Hz, 1H), 7.07 (d, J ¼ 8.8 Hz, 2H), 6.97 (m, 1H),
4.18e4.12 (m, 1H), 4.02e3.93 (m, 2H), 3.80e3.74 (m, 1H), 3.68e3.63
(m, 1H), 2.58 (s, 3H), 2.16 (s, 3H), 2.03e1.95 (m, 1H), 1.92e1.75 (m,
2H), 1.70e1.60 (m, 1H); ESI (m/z) 533 (MHþ); HRMS (ESI) calculated
for C27H24N4O4S2 [MHþ]: 533.1312, detected: 533.1274.
mL of acetonitrile and analyzed by the
5.14. Pharmacokinetics study
5.9.11. 1-[{6-(4-((4-Fluorobenzyl)oxy)phenyl)-5-methyl-2-
(thiophen-2-yl)thieno[2,3-d]pyrimidin-4-yl}amino]-3-methyl-1H-
pyrrole-2,5-dione (16j)
The hydrochloride salt of the appropriate compound was used
in the study. Male Sprague Dawley (SD) rats (8 weeks of age) were
acclimated to the testing facility under controlled temperature and
humidity conditions for approximately 1 week prior to the study. A
jugular vein catheter (Braintree Scientific, Inc., Braintree, MA, USA)
was surgically implanted into the right jugular vein under
ketamine-xylazine anesthesia. Compound 16d was dissolved in
saline and administered orally (30 mg/kg dosing level) and intra-
venously (iv; 5 mg/kg dosing level was) to four male rats per time
point. Blood samples were collected in heparinized micro-tubes at
0.08, 0.25, 0.5, 1, 2, 3, 4, 6, 8, and 24 h after oral administration, and
at 0.05, 0.25, 0.5, 1, 3, 5, 8, and 24 h after iv administration. Blood
samples were centrifuged at 3000 rpm for 10 min to collect plasma
and stored in a freezer until analyzed. Plasma samples were pre-
pared for analysis by the following protein precipitation method.
The parameters for 16j were: pale yellow solid, 1H NMR
(400 MHz, DMSO-d6):
d 9.61 (s, 1H), 7.66e7.65 (m, 2H), 7.52e7.46
(m, 4H), 7.21 (t, J ¼ 8.8 Hz, 2H), 7.15e7.10 (m, 3H), 6.97 (m, 1H), 5.13
(s, 2H), 2.58 (s, 3H), 2.16 (s, 3H); ESI (m/z) 557 (MHþ); HRMS (ESI)
calculated for C29H21FN4O3S2 [MHþ]: 557.1112, detected: 557.1073.
5.10. Liver microsomal stability
The compounds (1
mM) were incubated with human liver
microsomal (1 mg/mL) protein in the presence of nicotinamide
adenine dinucleotide phosphate (NADPH; 1 mM) in phosphate-
buffered saline (PBS; 0.1 mM) at 37 ꢀC for 0 and 30 min. The
microsomal reaction was terminated by adding the same volume of
acetonitrile containing carbamazepine (IS). The resulting mixture
was vortexed for 5 min and centrifuged at 7000 rpm for 15 min at
The plasma samples (100
mL) were transferred to a micro-tube,
three volumes of acetonitrile containing carbamazepine (IS) were
added, and the resulting mixture was vortexed for 5 min. The
4
ꢀC. Supernatants were analyzed by LC/MS using an Agilent 1260

H. Kim et al. / European Journal of Medicinal Chemistry 120 (2016) 74e85
85
properties to multipotent progenitors in a murine model of chronic myelo-
monocytic leukemia, Cancer Cell 12 (2007) 367e380.
[11] S. Scholl, H.J. Fricke, H.G. Sayer, K. Hoffken, Clinical implications of molecular
genetic aberrations in acute myeloid leukemia, J. Cancer Res. Clin. Oncol. 135
(2009) 491e505.
[12] R. Swords, C. Freeman, F. Giles, Targeting the FMS-like tyrosine kinase 3 in
acute myeloid leukemia, Leukemia 26 (2012) 2176e2185.
[13] P.H. Wiernik, FLT3 inhibitors for the treatment of acute myeloid leukemia,
Clin. Adv. Hematol. Oncol. 8 (2010) 429e436, 444.
micro-tube was then centrifuged at 12,000 rpm for 10 min and the
supernatant was analyzed using the API2000 LC/MS/MS system.
Pharmacokinetic
parameters
were
obtained
by
non-
compartmental analysis of the plasma concentration-time pro-
files, using PK Solution 2.0 (Summit Research Services, Montrose,
CO, USA).
[14] D. Auclair, D. Miller, V. Yatsula, W. Pickett, C. Carter, Y. Chang, X. Zhang,
D. Wilkie, A. Burd, H. Shi, S. Rocks, R. Gedrich, L. Abriola, H. Vasavada,
M. Lynch, J. Dumas, P.A. Trail, S.M. Wilhelm, Antitumor activity of sorafenib in
FLT3-driven leukemic cells, Leukemia 21 (2007) 439e445.
[15] I.J. Griswold, L.J. Shen, P. La Rosee, S. Demehri, M.C. Heinrich, R.M. Braziel,
L. McGreevey, A.D. Haley, N. Giese, B.J. Druker, M.W. Deininger, Effects of
MLN518, a dual FLT3 and KIT inhibitor, on normal and malignant hemato-
poiesis, Blood 104 (2004) 2912e2918.
[16] R.M. Stone, T. Fischer, R. Paquette, G. Schiller, C.A. Schiffer, G. Ehninger,
J. Cortes, H.M. Kantarjian, D.J. DeAngelo, A. Huntsman-Labed, C. Dutreix, A. del
Corral, F. Giles, Phase IB study of the FLT3 kinase inhibitor midostaurin with
chemotherapy in younger newly diagnosed adult patients with acute myeloid
leukemia, Leukemia 26 (2012) 2061e2068.
[17] P.P. Zarrinkar, R.N. Gunawardane, M.D. Cramer, M.F. Gardner, D. Brigham,
B. Belli, M.W. Karaman, K.W. Pratz, G. Pallares, Q. Chao, K.G. Sprankle,
H.K. Patel, M. Levis, R.C. Armstrong, J. James, S.S. Bhagwat, AC220 is a uniquely
potent and selective inhibitor of FLT3 for the treatment of acute myeloid
leukemia (AML), Blood 114 (2009) 2984e2992.
Acknowledgments
This research was supported by a grant from the Translational
Research Center for Protein Function Control (Grant NRF-2009-
0083522), the Ministry of Science, ICT and Future Planning (Grant
NRF-2013M3A6A4072536), the Basic Science Research Program
through the National Research Foundation of Korea funded by the
Ministry of Education (Grant NRF-2013R1A1A2008165 and Grant
NRF-2015R1A6A3A01020077), the Korea Health Technology R&D
Project through the Korea Health Industry Development Institute
(KHIDI), funded by the Ministry of Health & Welfare, Republic of
Korea (grant number: HI14C1324), and the KRIBB Research Initia-
tive Program.
[18] M.R. Grunwald, M.J. Levis, FLT3 inhibitors for acute myeloid leukemia: a re-
view of their efficacy and mechanisms of resistance, Int. J. Hematol. 97 (2013)
683e694.
Appendix A. Supplementary data
[19] Y. Alvarado, H.M. Kantarjian, R. Luthra, F. Ravandi, G. Borthakur, G. Garcia-
Manero, M. Konopleva, Z. Estrov, M. Andreeff, J.E. Cortes, Treatment with FLT3
inhibitor in patients with FLT3-mutated acute myeloid leukemia is associated
with development of secondary FLT3-tyrosine kinase domain mutations,
Cancer 120 (2014) 2142e2149.
[20] C.H. Park, C. Lee, J.S. Yang, B.Y. Joe, K. Chun, H. Kim, H.Y. Kim, J.S. Kang, J.I. Lee,
M.H. Kim, G. Han, Discovery of thienopyrimidine-based FLT3 inhibitors from
the structural modification of known IKKbeta inhibitors, Bioorg. Med. Chem.
Lett. 24 (2014) 2655e2660.
[21] J.S. Yang, C.H. Park, C. Lee, H. Kim, C. Oh, Y. Choi, J.S. Kang, J. Yun, J.H. Jeong,
M.H. Kim, G. Han, Synthesis and biological evaluation of novel thieno[2,3-d]
pyrimidine-based FLT3 inhibitors as anti-leukemic agents, Eur. J. Med. Chem.
85 (2014) 399e407.
[22] M.Y. Jang, Y. Lin, S. De Jonghe, L.J. Gao, B. Vanderhoydonck, M. Froeyen,
J. Rozenski, J. Herman, T. Louat, K. Van Belle, M. Waer, P. Herdewijn, Discovery
of 7-N-piperazinylthiazolo[5,4-d]pyrimidine analogues as a novel class of
immunosuppressive agents with in vivo biological activity, J. Med. Chem. 54
(2011) 655e668.
[23] A.I. Sanchez, R. Meneses, J.M. Minguez, A. Nunez, R.R. Castillo, F. Filace,
C. Burgos, J.J. Vaquero, J. Alvarez-Builla, A. Cortes-Cabrera, F. Gago,
E. Terricabras, V. Segarra, Microwave-assisted synthesis of potent PDE7 in-
hibitors containing a thienopyrimidin-4-amine scaffold, Org. Biomol. Chem.
12 (2014) 4233e4242.
[24] K.M. Kampa-Schittenhelm, M.C. Heinrich, F. Akmut, H. Dohner, K. Dohner,
M.M. Schittenhelm, Quizartinib (AC220) is a potent second generation class III
tyrosine kinase inhibitor that displays a distinct inhibition profile against
mutant-FLT3, -PDGFRA and -KIT isoforms, Mol. Cancer 12 (2013) 19.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.05.022.
References
[1] E. Estey, H. Dohner, Acute myeloid leukaemia, Lancet 368 (2006) 1894e1907.
[2] J.L. Shipley, J.N. Butera, Acute myelogenous leukemia, Exp. Hematol. 37 (2009)
649e658.
[3] C.C. Kumar, Genetic abnormalities and challenges in the treatment of acute
myeloid leukemia, Genes Cancer 2 (2011) 95e107.
[4] D. Small, Targeting FLT3 for the treatment of leukemia, Semin. Hematol. 45
(2008) S17eS21.
[5] D.G. Gilliland, J.D. Griffin, The roles of FLT3 in hematopoiesis and leukemia,
Blood 100 (2002) 1532e1542.
[6] D.L. Stirewalt, J.P. Radich, The role of FLT3 in haematopoietic malignancies,
Nat. Rev. Cancer 3 (2003) 650e665.
[7] H. Quentmeier, J. Reinhardt, M. Zaborski, H.G. Drexler, FLT3 mutations in acute
myeloid leukemia cell lines, Leukemia 17 (2003) 120e124.
[8] M. Levis, D. Small, FLT3: ITDoes matter in leukemia, Leukemia 17 (2003)
1738e1752.
[9] M. Levis, FLT3 mutations in acute myeloid leukemia: what is the best
approach in 2013? Hematol. Am. Soc. Hematol. Educ. Program 2013 (2013)
220e226.
[10] B.H. Lee, Z. Tothova, R.L. Levine, K. Anderson, N. Buza-Vidas, D.E. Cullen,
E.P. McDowell, J. Adelsperger, S. Frohling, B.J. Huntly, M. Beran, S.E. Jacobsen,
D.G. Gilliland, FLT3 mutations confer enhanced proliferation and survival