Synthesis and biological evaluation of novel thieno[2,3-d]pyrimidine-based FLT3 inhibitors as anti-leukemic agents

Article abstract of DOI:10.1016/j.ejmech.2014.08.001

The most common mutations in acute myeloid leukemia (AML) are those that cause the activation of FMS-like tyrosine kinase 3 (FLT3). Therefore, FLT3 is regarded as a potential target for the treatment of AML. A novel series of thieno[2,3-d]pyrimidine-based analogs was designed and synthesized as FLT3 inhibitors. All synthesized compounds were assayed for the tyrosine kinase activity of FLT3 and growth inhibitory activity in four human leukemia cell lines (THP1, MV4-11, K562, and HL-60). Among these compounds, compound 17a, which possesses relatively short and simple substituents at the C₆ position of thieno[2,3-d]pyrimidine, emerged as the most promising anti-leukemic agent. Compound 17a exhibited potent inhibition of FLT3-positive leukemic cell growth and of the FLT3 D835Y kinase; such inhibition is required for the successful treatment of AML. The data supports the further investigation of this class of compounds as potential anti-leukemic agents.

Full text of DOI:10.1016/j.ejmech.2014.08.001

European Journal of Medicinal Chemistry 85 (2014) 399e407
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of novel thieno[2,3-d]pyrimidine-
based FLT3 inhibitors as anti-leukemic agents
,
,
Jee Sun Yang a 1, Chun-Ho Park b 1, Chulho Lee a, Hwan Kim a, Changmok Oh a,
Yejoo Choi a, Jong Soon Kang c, Jieun Yun c, Jin-Hyun Jeong d, Myung-Hwa Kim e,
Gyoonhee Han a
, f, *
a Translational Research Center for Protein Function Control, Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea
b Graduate Program in Biomaterials Science & Engineering, Yonsei University, Seoul 120-749, Republic of Korea
c Bioevaluation Center, Korea Research Institute of Bioscience and Biotechnology, Ochang, Cheongwon, Chungbuk 363-883, Republic of Korea
d College of Pharmacy and Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Yeonsu-gu, Incheon 406-840, Republic of Korea
e Korea Drug Development Fund, 14th Fl, KT&G SeodaemunTower, 21-1, Migeun-dong, Seodaemun-gu, Seoul, Republic of Korea
f Department of Integrated OMICS for Biomedical Sciences (WCU Program), Yonsei University, Seoul 120-749, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
The most common mutations in acute myeloid leukemia (AML) are those that cause the activation of
FMS-like tyrosine kinase 3 (FLT3). Therefore, FLT3 is regarded as a potential target for the treatment of
AML. A novel series of thieno[2,3-d]pyrimidine-based analogs was designed and synthesized as FLT3
inhibitors. All synthesized compounds were assayed for the tyrosine kinase activity of FLT3 and growth
inhibitory activity in four human leukemia cell lines (THP1, MV4-11, K562, and HL-60). Among these
compounds, compound 17a, which possesses relatively short and simple substituents at the C6 position
of thieno[2,3-d]pyrimidine, emerged as the most promising anti-leukemic agent. Compound 17a
exhibited potent inhibition of FLT3-positive leukemic cell growth and of the FLT3 D835Y kinase; such
inhibition is required for the successful treatment of AML. The data supports the further investigation of
this class of compounds as potential anti-leukemic agents.
Received 16 April 2014
Received in revised form
29 July 2014
Accepted 1 August 2014
Available online 1 August 2014
Keywords:
FMS-like tyrosine kinase 3 (FLT3)
Acute myeloid leukemia (AML)
Thieno[2,3-d]pyrimidine
Internal tandem duplications (ITD)
D835Y
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
(ITDs) within the juxtamembrane (JM) domain and point mutations
within the tyrosine kinase domain (TKD). Approximately 23% of
Acute myeloid leukemia (AML) is a clonal hematopoietic stem
cell disorder characterized by the abnormal proliferation and dif-
ferentiation of immature blast cells in the bone marrow and pe-
ripheral blood [1]. Several studies have explored abnormalities in
the FMS-like tyrosine kinase 3 (FLT3), RAS, and p53 genes in search
of clues relating to the pathogenesis of AML [2e7]. The FLT3 gene is
found to have mutations in 30% of adult AML cases, whereas
approximately 20% and 5% of adult AML cases reported mutations
in RAS and p53, respectively. The most common molecular abnor-
malities in AML are activating mutations of FLT3, which are linked
with poor patient prognosis [8]. FLT3-activating mutations are
divided into two broad categories: internal tandem duplications
patients with de novo AML harbor FLT3/ITD mutations, which
represent the most frequent activating mutation. This portends a
poor prognosis and these patients experience a high relapse rate
[8e13]. The other major FLT3-activating mutations are FLT3 TKD
mutations comprising about 7% of de novo AML cases. Point mu-
tations in which the aspartate residue at position 835 (D835) is
replaced by various amino acids occur most commonly, but with
lower frequency than the ITD mutations [14,15]. Another method
by which FLT3 is constitutively activated in AML is the over-
expression of wild-type FLT3 proteins. Although overexpression of
wt-FLT3 is relatively uncommon in AML cases, it is reported that it
could function as an unfavorable prognostic factor in cases that do
not exhibit the FLT3/ITD mutation [7]. Since FLT3 activating muta-
tions are the most common mutations in AML, inhibition of FLT3
has proved to be a therapeutic target in AML. In particular, because
FLT3/ITD mutations have emerged as a negative prognostic factor in
AML, FLT3/ITD has become an attractive target for the treatment of
AML.
* Corresponding author. Translational Research Center for Protein Function
Control, Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of
Korea.
E-mail addresses: gyoonhee@yonsei.ac.kr, gyoonhee@gmail.com (G. Han).
These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2014.08.001
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

400
J.S. Yang et al. / European Journal of Medicinal Chemistry 85 (2014) 399e407
Several small-molecule inhibitors of FLT3 tyrosine kinase are
prepared according to literature precedent [22]. As illustrated in
Schemes 1e3, the final compounds were synthesized from various
2-aminothiophenes by three or four-step procedures. Thienopyr-
imidine derivatives (2, 9, and 14) were conveniently synthesized by
heating various 2-aminothiophenes (1, 8, and 13) with the appro-
priate carbonitrile under acidic conditions. Compound 2 was con-
verted to chloride (3) by a Sandmeyer reaction, while compounds 9
and 14 were chlorinated using POCl3 at 100 ꢀC to generate 4-chloro-
thieno[2,3-d]pyrimidine 10 and 15. Thieno[2,3-d]pyrimidin-4-
amine derivatives 5, 12, and 17 were then prepared in a stepwise
fashion by first treating 10 or 15 with hydrazine hydrate in tetra-
hydrofuran, followed by treatment with 3-methylfuran-2,5-dione.
The structures of the newly synthesized compounds were charac-
terized by 1H and 13C nuclear magnetic resonance (NMR), low-
resolution mass spectroscopy (LRMS), and high-resolution mass
spectroscopy (HRMS). The compounds were screened for their
in vitro biological activities.
under development, and are being evaluated in early-phase clinical
trials (Fig. 1). The first-generation of FLT3 inhibitors, such as sor-
afenib (BAY-43-9006) [16], lestaurtinib (CEP-701) [17], sunitinib
(SU11248) [18], and tandutinib (MLN518) [19], were discovered
during studies of other receptor tyrosine kinase inhibitors. Initially,
these compounds were not developed or optimized for FLT3 inhi-
bition; thus, they inhibited not only FLT3 but also other multiple
kinases, resulting in off-target effects. To evaluate more focused and
potent FLT3 inhibitors for AML therapy, a second-generation in-
hibitor, quizartinib (AC220), was specifically designed [20]. All of
these reported drugs efficaciously inhibited FLT3 in cellular assays
and in vivo models of FLT3-ITD AML. Clinical activity was also seen
in all of them, but only a few patients had complete remission in the
trials due to insufficient efficacy of FLT3 inhibition and/or several
mutations on FLT3 kinase that impart resistance. Therefore, resis-
tance to therapy has become a significant barrier to the develop-
ment of successful FLT3 inhibitors [21].
Thieno[2,3-d]pyrimidine derivatives are a novel class of FLT3 in-
hibitors for the treatment of AML. A series of compounds were inci-
dentally found to have selective activity against FLT3 while
3. Results and discussion
3.1. Biological evaluation of final compounds
developing IKKb inhibitors from the quinazoline analog SPC-839, as
reported in our previous study [22]. Herein, we report the synthesis
and biological activities of a novel series of thieno[2,3-d]pyrimidine
derivatives based on our previous work. We demonstrate that novel
thieno[2,3-d]pyrimidine-based compounds inhibit FLT3 with great
potency, and also have potent inhibitory activities against AML cell
lines with FLT3/ITD mutations compared with AC220 and MLN518.
We furtherdemonstrate that the high potencyexhibited by this series
of compoundsonFLT3/ITDand FLT3D835Y(the mostcommonkinase
domain mutant in AML [23]) indicates the feasibility of developing
successful FLT3 inhibitors by reducing resistance. Our results suggest
that novel thieno[2,3-d]pyrimidine derivatives represent promising
chemotherapeutic agents for the treatment of AML.
All synthesized compounds were assayed for tyrosine kinase
activity of FLT3 and growth inhibitory activity in four human leu-
kemia cell lines that harbored either wt-FLT3 (THP1), a mutated
FLT3 kinase (MV4-11), or were FLT3-null (K562 and HL-60). The
results are tabulated as IC50 and GI50 values in the micromolar
range (Table 1). AC220 and MLN518 were used as positive refer-
ences for the comparison of in vitro activities.
As shown in Table 1, the primary in vitro kinase assay results
revealed that five compounds exhibited moderate tyrosine kinase
inhibitory activities against human FLT3. In particular, compounds
5a and 17a showed superior inhibitory activities (0.069 and
0.055
mM, respectively) compared with the positive controls, AC220
and MLN518 (0.12 and 0.102
m
M, respectively). Most of the evalu-
2. Chemistry
ated compounds also showed favorable growth inhibitory activities
against the MV4-11 and THP1 cell lines, which represent FLT3/ITD
mutations and wt-FLT3 in AML, respectively. Notably, compounds
17b, 17c, and 17d exhibited excellent inhibitory activities in the
MV4-11 cell line, with GI50 values of 86, 91, and 92 nM, respectively.
As indicated in Table 1, most of the synthesized compounds
Based on the thieno[2,3-d]pyrimidine structures of the com-
pounds described in our previous work, we designed and synthe-
sized 22 analogs with diverse substitutions on the thieno[2,3-d]
pyrimidine moiety at the C2, C4, and C6 positions. 5a and 12b were
Fig. 1. Chemical structures of FLT3 inhibitors.

J.S. Yang et al. / European Journal of Medicinal Chemistry 85 (2014) 399e407
401
Scheme 1. 1) Schematic for the synthesis of final compounds 5ae5d and 2) Schematic for the synthesis of final compound 6.
Scheme 2. Schematic for the synthesis of final compounds 12ae12e.
inhibited FLT3/ITD and wt-FLT3 more effectively than the known
selective inhibitor, AC220.
synthesized compounds inhibited the tyrosine kinase activity of
FLT3 with IC50 values of 0.47, 0.069, and 0.521 M, respectively.
m
Among these three compounds, only 5a inhibited FLT3 kinase ac-
tivity and the growth of MV4-11 cells significantly better than did
AC220.
3.2. SAR study
Initially, in order to assess the effects of different substituents at
the C4 position on thieno[2,3-d]pyrimidine, we synthesized three
compounds, 4a, 5a, and 6. The 2-thienyl group at the C2 and C5
position of the compounds was fixed and various substituents, such
as 3-methyl-1H-pyrrole-2,5-dione, hydrazine, or piperazine, were
introduced at the C4 position of thienopyrimidine. These newly
To evaluate the effect of different substituents at the C2 position
of the compounds, nine compounds were prepared containing 3-
methyl-1H-pyrrole-2,5-dione at the C4 position of the thienopyr-
imidine core, since this moiety was the only active compound in the
MV4-11 cell line. The nine compounds were divided into two
groups, one containing a phenyl group (12be12e) and the other

402
J.S. Yang et al. / European Journal of Medicinal Chemistry 85 (2014) 399e407
Scheme 3. Schematic for the synthesis of final compounds 17ae17k.
containing a 2-thienyl group (5ae5d and 12a) at the C5 position of
thieno[2,3-d]pyrimidine with diverse C2-substituents. All of these
compounds, except 12a, showed good growth inhibitory activity
superior to the positive control AC220 (3.310 mM) on the MV4-11
Table 1
Biological activities of thieno[2,3-d]pyrimidine derivatives.
cell line, while they did not exhibit favorable FLT3 kinase activity
as much as AC220. However, these series of compounds, except 5a
and 12b, showed good growth inhibition not only in FLT3-positive
cell lines but also in FLT3-negative cell lines. Additionally, they
displayed almost no activity on FLT3 kinase. These results might
imply that these compounds inhibit the growth of AML cells by
their effects on kinases other than FLT3. Therefore, compounds
containing a 2-(thiophen-2-yl)thieno[2,3-d]pyrimidine moiety (5a
and 12b) emerged as potent compounds in this series that exhibi-
ted good activity for both wt-FLT3 and FLT3/ITD.
Based on compounds 5a and 12b, which showed potent anti-
proliferative activity in FLT3-positive AML cell lines and inhibitory
activity on FLT3 kinase, eleven compounds with an array of sub-
stituents at the C6 position of thieno[2,3-d]pyrimidine were also
synthesized. Based on results from our previous study, we fixed a
methyl group at the C5 position, as this had exhibited excellent
inhibitory activities. To assess the effects of different substituents at
the C6 position of the compounds, small substituents (17ae17f),
such as methoxy, carboxylate, methanol, or carboxamide, and
substituted phenyl or benzyl (17ge17k) moieties were introduced.
These groups of compounds displayed outstanding GI50 values of
86e193 nM which are 17- to 39-fold better than AC220 for the
FLT3/ITD mutated cell line. Interestingly, the potency of the com-
pounds with small substituents on FLT3 kinase and the MV4-11 cell
line surpassed that of the phenyl- or benzyl-substituted com-
pounds. Moreover, 17a exhibited the most potent kinase activity
Cpd
FLT3 kinase
SD
GI50
(
m
M)b
MV4-11
assay IC50 (m
M)a
K562
HL60
THP1
AC220
MLN518
4a
5a
5b
5c
5d
6
12a
12b
12c
12d
12e
17a
17b
17c
17d
17e
17f
17g
17h
17i
0.12
0.102
0.47
0.069
1.603
0.611
NAc
0.521
NA
0.030
0.303
0.111
0.208
0.124
0.085
e
>10
>10
>10
5.551
6.302
1.916
>10
3.310
8.756
>10
0.320
0.836
0.775
2.009
>10
3.829
0.233
0.407
0.755
2.100
0.100
0.086
0.091
0.092
0.115
0.193
0.135
0.131
0.123
0.112
0.103
8.014
>10
>10
0.746
0.862
0.403
0.725
>10
1.517
0.758
1.313
2.188
3.714
0.240
0.136
0.656
0.712
0.266
7.802
0.624
0.221
0.383
0.418
0.213
5.574
>10
>10
2.673
3.728
2.805
6.261
>10
6.658
0.155
2.205
1.628
4.112
0.906
0.172
2.902
2.595
1.781
2.686
0.902
0.600
0.107
0.846
0.948
0.367
e
>10
3.517
0.074
0.703
1.742
8.159
1.457
0.030
0.483
3.843
1.256
2.777
0.154
0.145
0.003
1.882
0.880
0.208
NA
NA
0.161
e
e
0.546
0.055
0.787
0.866
0.175
0.282
0.131
0.846
0.721
0.633
0.301
3.286
0.201
0.073
0.079
0.063
0.167
0.031
0.064
0.040
0.061
0.040
0.145
0.416
17j
17k
a
IC50 was determined by using the KinaseProfiler service at Millipore (average of
duplicates).
b
c
Growth inhibition was measured by XTT assay (average of four replicates).
NA) not active.
(IC50 ¼ 0.055
mM) and the kinase activity of 17d and 17f were also

J.S. Yang et al. / European Journal of Medicinal Chemistry 85 (2014) 399e407
403
comparable to the positive controls. The relatively simple and short
groups at the C6 position of thieno[2,3-d]pyrimidine transpired to
be the most favorable substituent for FLT3 inhibitory activity.
conducted using Agilent analytic column eclipse-XDB-C18
(150*4.6 mm, 5 m) on Shimazu HPLC 2010 instruments. Condi-
tions and retention times are described in the Supporting
Information.
m
3.3. D835Y
4.1. General procedure for the preparation of title compound 4a
Many researchers have reported that intrinsic and acquired
resistance to therapy might be surmounted by targeting not only
the FLT3/ITD mutations, but also the acquired TKD point mutations.
Therefore, the IC50 values of some of the synthesized compounds
on the most common kinase domain mutant, FLT3 D835Y, were also
determined. Based on the results of FLT3 kinase activity and anti-
proliferative activity, compounds 17a, 17c, 17d, 17f, and 17j were
selected for evaluation. Results are tabulated as IC50 values in the
micromolar range in Table 2. All of these selected compounds
showed excellent IC50 values of 46 nMe587 nM. Among these
compounds, three compounds 17a, 17d, and 17f exhibited superior
activities (46, 112, and 119 nM, respectively) compared with the
positive control AC220 (137 nM). Remarkably, compound 17a
showed high potency against wt-FLT3, FLT3-ITD, and FLT3 D835Y.
We have designed and synthesized 22 thieno[2,3-d]pyrimidine-
based analogs for the treatment of AML, based on our previous
study. We then evaluated the effects of diversity at the C2, C4, and C6
positions of the thieno[2,3-d]pyrimidine moiety on FLT3 kinase and
four human leukemia cell lines. Most of the synthesized thieno[2,3-
d]pyrimidine analogs inhibited FLT3/ITD more effectively than did
AC220. As weak and transient inhibitions were considered major
obstacles to the development of FLT3 inhibitors, this series of
compounds showed potential to overcome barriers to optimal FLT3
inhibition. Moreover, three compounds (17a, 17d, and 17f) potently
inhibited the TKD point mutant (D835Y) together with FLT3/ITD. In
conclusion, compound 17a emerged as the most promising anti-
leukemic agent, exhibiting potent inhibition of FLT3-positive
leukemic cell growth and the FLT3 D835Y kinase; such inhibition
is required for the successful treatment of AML. Our results show
that synthetic thieno[2,3-d]pyrimidine derivatives represent
promising chemotherapeutic agents for the treatment of AML.
According to the reported procedures [22], the key intermediate
and title compound 4a was synthesized by a three-step process, as
shown in Scheme 1.
4.1.1. 2,5-Di(thiophen-2-yl)thieno[2,3-d]pyrimidin-4-amine (2a)
50-Amino-2,30-bithiophene-40-carbonitrile
1.45 mmol) and 2-thiophene carbonitrile (162
1
(300
mg,
m
l, 1.75 mmol) were
dissolved in 4 M HCl 1,4-dioxane solution (6 ml), and the resulting
mixture was stirred at 110 ꢀC for 24 h. After cooling, the reaction
mixture was poured into ice-cold water and extracted with EtOAc.
The combined organic layer was washed with saturated NaHCO3
aqueous solution, dried over Na2SO4, and concentrated under
reduced pressure to afford the title compound as a brown solid
(260 mg, 56%). 1H NMR (400 MHz, CDCl3):
d 7.95 (s, 1H), 7.41 (t,
J ¼ 3.6 Hz, 1H), 7.13 (m, 4H), 5.49 (br, 2H).
4.1.2. 4-Chloro-2,5-di(thiophen-2-yl)thieno[2,3-d]pyrimidine (3a)
CuCl2 (174 mg, 1.29 mmol) and t-BuONO (192 l, 1.62 mmol)
m
were dissolved in MeCN (5 ml), and the resulting mixture was
stirred at 70 ꢀC for 30 min. Into this mixture, 2,5-di(thiophen-2-yl)
thieno[2,3-d]pyrimidin-4-amine 2a (340 mg, 1.08 mmol) in tetra-
hydrofuran (THF; 2 ml) was added dropwise and the reaction
mixture was stirred at the same temperature for 3 h. The cooled
mixture was poured into ice-cold water and extracted with EtOAc.
The combined organic layer was washed with brine, dried over
Na2SO4, and concentrated to dryness. The residue was then purified
by flash column chromatography (n-Hx:EtOAc ¼ 3:1) to afford the
title compound 3a as a yellow oil (75 mg, 21%). 1H NMR (400 MHz,
CDCl3):
d
8.06 (d, J ¼ 4.0 Hz, 1H), 7.50 (d, J ¼ 5.6 Hz, 1H), 7.46 (s, 1H),
7.40 (d, J ¼ 5.6 Hz, 1H), 7.16 (m, 2H), 7.12 (dd, 1H, J ¼ 3.2 Hz,
J ¼ 5.2 Hz).
4. Experimental section
4.1.3. 4-Hydrazinyl-2,5-di(thiophen-2-yl)thieno[2,3-d]pyrimidine
hydrochloride (4a)
All chemicals were obtained from commercial suppliers and
used without further purification. All reactions were monitored by
thin-layer chromatography (TLC) on pre-coated silica gel 60 F254
(mesh) (Merck, Mumbai, India), and spots were visualized under
UV light (254 nm). Flash column chromatography was performed
with silica (Merck EM9385, 230e400 mesh). 1H and 13C NMR
spectra were recorded at 400 MHz and 100 MHz on a Varian 400
To a stirred solution of 3 (0.22 mmol) in THF (3 ml), hydrazine
monohydrate (0.67 mmol) was added dropwise, and the mixture
was stirred at 80 ꢀC for 4 h. After cooling, the title compound 4a was
obtained by removing solvent under reduced pressure and
concentrating in vacuo. Yellow solid (76%); 1H NMR (400 MHz,
DMSO-d6):
d
9.04 (s, 1H), 8.25 (d, J ¼ 3.6 Hz, 1H), 7.81 (d, J ¼ 5.2 Hz,
1H), 7.77 (s, 1H), 7.69 (d, J ¼ 5.2 Hz, 1H), 7.29e7.18 (m, 3H); 13C NMR
Mercury plus spectrometer; chemical shifts are reported in d (ppm)
(100 MHz, DMSO-d6):
d 155.68, 155.05, 152.17, 141.80, 135.32,
units relative to the internal reference tetramethylsilane (TMS).
Mass spectra were obtained on an Applied Biosystems API 2000
mass spectrometer and an Agilent 1200 series LC system. HPLC
(high performance liquid chromatography) experiments were
131.26, 130.79, 128.52, 128.29, 128.14, 127.24, 126.92, 124.54,
111.73 ppm; ESI (m/z) 331 (MHþ); HRMS (ESI) calculated for
C
14H10N4S3 [MHþ]: 331.0146, found: 331.0138.
4.2. General procedure for the preparation of title compounds
5ae5d
Table 2
FLT3 D835Y kinase assay of thieno[2,3-d]pyrimidine derivatives.
Cpd
FLT3 (D835Y) activity (
m
M)a
SD
To a stirred solution of 3 (0.22 mmol) in THF (3 ml) hydrazine
monohydrate (0.67 mmol) was added dropwise, and the mixture
was stirred at 80 ꢀC for 4 h. After cooling, the solvent was removed
under reduced pressure and concentrated in vacuo. The residue 4
was then dissolved in CHCl3 (3 ml), and citraconic anhydride
(0.67 mmol) was slowly added. The resulting mixture was heated to
80 ꢀC and stirred for 18 h. The reaction mixture was cooled to room
temperature and washed with water. The organic layer was dried
over Na2SO4 and concentrated to dryness. The residue was then
AC220
MLN518
17a
17c
17d
0.137
2.444
0.046
0.587
0.112
0.119
0.505
0.060
0.099
0.028
0.049
0.159
0.042
0.321
17f
17j
a
IC50 was determined by using the KinaseProfiler service at Millipore (average of
duplicates).

404
J.S. Yang et al. / European Journal of Medicinal Chemistry 85 (2014) 399e407
purified by flash column chromatography (n-Hx:EtOAc ¼ 2:1) to
afford the title compound 5.
DMSO-d6):
d
10.66 (br s, 1H), 8.01 (d, J ¼ 3.6 Hz, 1H), 7.81 (s, 1H),
7.78 (d, J ¼ 5.2 Hz, 1H), 7.68 (d, J ¼ 4.8 Hz, 1H), 7.28e7.27 (m, 1H),
7.24e7.21 (m, 2H), 3.97e3.93 (m, 2H), 3.28e3.15 (m, 4H), 2.86e2.78
(m, 2H), 2.69e2.68 (m, 2H); ESI (m/z) 385 (MHþ); HRMS (ESI)
calculated for C18H16N4S3 [MHþ]: 385.0615, found: 385.0612; HPLC
(tR : purity ¼ 4.39 min, 99.82%).
4.2.1. 1-[2,5-Di(thiophen-2-yl)thieno[2,3-d]pyrimidin-4-ylamino]-
3-methyl-1H-pyrrole-2,5-dione (5a)
Pale yellow solid (48%); 1H NMR (400 MHz, DMSO-d6): 8.68 (s,
1H), 7.75 (s, 1H), 7.69 (m, 3H), 7.30 (d, J ¼ 3.6 Hz, 1H), 7.20 (t,
J ¼ 4.0 Hz, 1H), 7.14 (t, J ¼ 4.8 Hz, 1H), 6.93 (s, 1H), 2.12 (s, 3H); 13
C
4.4. Procedure for the preparation of intermediate compound 10
NMR (100 MHz, DMSO-d6): d 169.92, 169.10, 168.84, 155.91, 155.53,
145.40, 142.68, 135.57, 131.28, 129.03, 128.97, 128.76, 128.48, 127.67,
127.28, 127.05, 124.73, 111.30, 11.64 ppm; ESI (m/z) 425 (MHþ);
HRMS (ESI) calculated for C19H12N4O2S3 [MHþ]: 425.0201, found:
425.0189; HPLC (tR : purity ¼ 8.11 min, 93.26%).
According to the following procedures, the key intermediate
compound 10 was synthesized by a four-step process, as shown in
Scheme 2.
4.4.1. Methyl 2-cyano-3-(thiophen-2-yl)but-2-enoate (7a)
4.2.2. 3-Methyl-1-[2-phenyl-5-(thiophen-2-yl)thieno[2,3-d]
To a stirred solution of 2-acetylthiophene (1.0 g, 7.92 mmol) in
toluene (20 ml), methyl cyanoacetate (0.84 ml, 9.51 mmol),
ammonium acetate (1.80 g, 23.77 mmol), and acetic acid (1.36 ml,
23.77 mmol) were added. After refluxing for 16 h, the reaction
mixture was poured into ice-cold water and extracted with EtOAc.
The combined organic layer was washed with 1 N HCl aqueous
solution, dried over Na2SO4, and concentrated to dryness. The
residue was then purified by flash column chromatography (n-
Hx:EtOAc ¼ 5:1) to afford the title compound 7a as a yellow oil
pyrimidin-4-ylamino]-1H-pyrrole-2,5-dione (5b)
White solid (96%); 1H NMR (400 MHz, CDCl3):
d 8.23 (d,
J ¼ 7.2 Hz, 2H), 7.45 (d, J ¼ 4.4 Hz, 1H), 7.41e7.38 (m, 4H), 7.34 (s,
1H), 7.20e7.17 (m, 2H), 6.59 (s, 1H), 2.22 (s, 3H); 13C NMR (100 MHz,
DMSO-d6):
d 169.50, 169.01, 168.40, 158.21, 155.80, 145.03, 136.49,
135.23, 130.81, 128.66 (2), 128.36, 128.09, 127.60 (2), 127.27, 126.91,
126.49, 124.80, 111.23, 11.18 ppm; ESI (m/z) 419 (MHþ); HRMS (ESI)
calculated for C21H14N4O2S2 [MHþ]: 419.0636, found: 419.0627;
HPLC (tR : purity ¼ 6.15 min, 94.48%).
(810 mg, 53%). 1H NMR (400 MHz, CDCl3):
d
8.03 (d, J ¼ 4.0 Hz, 1H),
7.80 (d, J ¼ 4.8 Hz, 1H), 7.78 (t, J ¼ 4.4 Hz, 1H), 3.88 (s, 3H) 2.71 (s,
4.2.3. 3-Methyl-1-[2-(5-methylthiophen-2-yl)-5-(thiophen-2-yl)
3H).
thieno[2,3-d]pyrimidin-4-ylamino]-1H-pyrrole-2,5-dione (5c)
Pale yellow solid (73%); 1H NMR (400 MHz, DMSO-d6):
d
8.63 (s,
4.4.2. Methyl 50-amino-2,30-bithiophene-40-carboxylate (8a)
To a stirred solution of methyl 2-cyano-3-(thiophen-2-yl)but-2-
enoate 7a (250 mg, 1.21 mmol) and sulfur (46 mg, 1.45 mmol) in
1H), 7.74 (s, 1H), 7.68 (d, J ¼ 4.8 Hz, 1H), 7.55 (d, J ¼ 3.2 Hz, 1H), 7.30
(d, J ¼ 3.2 Hz, 1H), 7.21 (m, 1H), 6.94 (s, 1H), 6.86 (d, J ¼ 3.6 Hz, 1H),
2.47 (s, 3H), 2.14 (s, 3H); 13C NMR (100 MHz, DMSO-d6):
d
169.52,
EtOH (10 ml) was added piperidine (143 ml, 1.45 mmol), and the
168.66,168.43,155.50,155.14,144.98,144.82,139.85,135.24,128.94,
128.36, 128.06, 127.26, 127.14, 126.90, 126.64, 124.01, 110.66, 15.45,
11.23 ppm; ESI (m/z) 439 (MHþ); HRMS (ESI) calculated for
resulting mixture was stirred at 80 ꢀC for 18 h. After cooling to room
temperature, the reaction mixture was poured into ice-water and
extracted with EtOAc. The combined organic layer was washed
with saturated NH4Cl aqueous solution, dried over Na2SO4, and
concentrated to dryness. The residue was then purified by flash
column chromatography (n-Hx:EtOAc ¼ 5:1) to afford the title
compound 8a as a yellow solid (88 mg, 31%). 1H NMR (400 MHz,
C
20H14N4O2S3 [MHþ]: 439.0357, found: 439.0351; HPLC (tR
:
purity ¼ 6.17 min, 95.66%).
4.2.4. 3-Methyl-1-[5-(thiophen-2-yl)-2-(thiophen-2-ylmethyl)
thieno[2,3-d]pyrimidin-4-ylamino]-1H-pyrrole-2,5-dione (5d)
CDCl3):
2H), 3.67 (s, 3H).
d
7.25 (d, J ¼ 3.6 Hz, 1H), 7.01 (m, 2H), 6.22 (s, 1H), 6.11 (s,
Brown solid (71%); 1H NMR (400 MHz, DMSO-d6):
d
8.87 (s, 1H),
8.17 (s,1H), 8.09 (d, J ¼ 4.8 Hz,1H), 7.75 (d, J ¼ 4.8 Hz,1H), 7.69e7.68
(m, 1H), 7.61 (t, J ¼ 4.2 Hz, 1H), 7.33e7.31 (m, 1H), 7.28 (br s, 1H),
7.22 (s, 1H), 4.62 (s, 2H), 2.49 (s, 3H); ESI (m/z) 433 (MHþ); HRMS
4.4.3. 2-Benzyl-5-(thiophen-2-yl)thieno[2,3-d]pyrimidin-4-ol (9a)
Methyl 50-amino-2,30-bithiophene-40-carboxylate 8a (100 mg,
0.41 mmol) and phenylacetonitrile (140 ml, 1.25 mmol) were dis-
solved in 4 M HCl 1,4-dioxane solution (3 ml), and the resulting
mixture was stirred at 100 ꢀC for 1 day. After cooling, the reaction
mixture was poured into ice-cold water and extracted with EtOAc.
The combined organic layer was washed with saturated NaHCO3
aqueous solution, dried over Na2SO4, and concentrated under
reduced pressure to afford the title compound 9a as a yellow oil
(110 mg, 83%).
(ESI) calculated for
433.0788.
C
22H16N4O2S2 [MHþ]: 433.0793, found:
4.3. Procedure for the preparation of the title compound 4-
(piperazin-1-yl)-2,5-di(thiophen-2-yl)thieno[2,3-d]pyrimidine
hydrochloride (6)
To a stirred solution of 4-chloro-2,5-di(thiophen-2-yl)thieno
[2,3-d]pyrimidine 3a (50 mg, 0.15 mmol) in EtOH (5 ml), TEA (31 ml,
0.22 mmol) and 1-Boc-piperazine (56 mg, 0.30 mmol) were
sequentially added at 0 ꢀC. The resulting mixture was refluxed for 1
day. After cooling, the solvent was removed under reduced pres-
sure and the residue was extracted with CH2Cl2. The combined
organic layer was dried over Na2SO4 and concentrated to dryness.
The residue was then purified by flash column chromatography (n-
Hx:EtOAc ¼ 2:3) to afford t-butyl 4-[2,5-di(thiophen-2-yl)thieno
[2,3-d]pyrimidin-4-yl]piperazine-1-carboxylate (40 mg, 55%).
4 M HCl 1,4-dioxane solution (0.5 ml) was added to a stirred
solution of 4-(piperazin-1-yl)-2,5-di(thiophen-2-yl)thieno[2,3-d]
pyrimidine in 1,4-dioxane (3 ml) at 0 ꢀC. After stirring for 1 h, the
reaction mixture was concentrated in vacuo to afford the title
compound 6 as a white solid (40 mg, 87%). 1H NMR (400 MHz,
4.4.4. 2-Benzyl-4-chloro-5-(thiophen-2-yl)thieno[2,3-d]pyrimidine
(10a)
2-Benzyl-5-(thiophen-2-yl)thieno[2,3-d]pyrimidin-4-ol
9a
(110 mg, 0.33 mmol) was dissolved in POCl3 (1 ml) and the resulting
mixture was heated for 3 h at 110 ꢀC. After cooling to room tem-
perature, the reaction mixture was poured into ice-cold water and
extracted with CHCl3. The combined organic layer was washed with
saturated NaHCO3 aqueous solution, dried over Na2SO4 and
concentrated to dryness. The residue was then purified by flash
column chromatography (n-Hx:EtOAc ¼ 4:1) to afford the title
compound 10a as a white solid (26 mg, 22%). 1H NMR (400 MHz,
CDCl3):
d 7.50 (s, 1H), 7.44e7.40 (m, 3H), 7.33e7.21 (m, 3H),
7.13e7.08 (m, 2H), 4.36 (s, 2H).

J.S. Yang et al. / European Journal of Medicinal Chemistry 85 (2014) 399e407
405
4.5. General procedure for the preparation of title compounds
12ae12e
4.6. Procedure for the preparation of intermediate compounds 15
According to the following procedures, the key intermediate
compound 10 was synthesized by a three-step process, as shown in
Scheme 3.
To a stirred solution of 10 (0.06 mmol) in THF (3 ml), hydrazine
monohydrate (0.19 mmol) was added dropwise, and the mixture
was stirred at 80 ꢀC for 7 h. After cooling, the solvent was removed
under reduced pressure, and the residue 11 was then dissolved in
CHCl3 (5 ml). Citraconic anhydride (0.19 mmol) was then slowly
added and the resulting mixture was heated to 80 ꢀC for 2 h. The
reaction mixture was cooled to room temperature and washed with
water. The organic layer was then dried over Na2SO4, concentrated
to dryness, and purified by flash column chromatography (n-
Hx:EtOAc ¼ 2:1) to afford the title compound 12.
4.6.1. Ethyl 2-amino-5-(4-methoxyphenyl)-4-methylthiophene-3-
carboxylate (13g)
To a stirred solution of 4-methoxyphenylacetone (3.08 ml,
20 mmol), ethyl cyanoacetate (2.13 ml, 22 mmol) and sulfur
(704 mg, 20 mmol) in EtOH (15 ml), diethylamine (2.07 ml,
20 mmol) was added dropwise, and the resulting was stirred at
room temperature for 36 h. The reaction mixture was poured into
ice-cold water and extracted with EtOAc. The combined organic
layer was washed with brine, dried over Na2SO4, and concentrated
to dryness. The residue was then purified by flash column chro-
matography (n-Hx:EtOAc ¼ 10:1) to afford the title compound 13g
as a pale yellow solid (3.79 g, 65%). 1H NMR (400 MHz, CDCl3):
4.5.1. 1-[2-Benzyl-5-(thiophen-2-yl)thieno[2,3-d]pyrimidin-4-
ylamino]-3-methyl-1H-pyrrole-2,5-dione (12a)
Yellow solid (79%); 1H NMR (400 MHz, DMSO-d6):
d 8.42 (s, 1H),
7.73 (s, 1H), 7.67e7.65 (m, 1H), 7.27e7.16 (m, 8H), 6.78 (s, 1H), 3.98
(s, 2H), 2.05 (s, 3H); ESI (m/z) 433 (MHþ); HRMS (ESI) calculated for
d
7.39 (br, 2H), 7.23 (d, J ¼ 8.8 Hz, 2H), 6.96 (d, J ¼ 8.8 Hz, 2H), 4.19
C
22H16N4O2S2 [MHþ]: 433.0793, found: 433.0788; HPLC (tR
:
(qt, J ¼ 6.8 Hz, 2H), 3.76 (s, 3H), 2.19 (s, 3H), 1.26 (t, J ¼ 6.8 Hz, 3H).
purity ¼ 7.09 min, 94.62%).
4.5.2. 3-Methyl-1-[5-phenyl-2-(thiophen-2-yl)thieno[2,3-d]
pyrimidin-4-ylamino]-1H-pyrrole-2,5-dione (12b)
4.6.2. 6-(4-Methoxyphenyl)-5-methyl-2-(thiophen-2-yl)thieno
[2,3-d]pyrimidin-4-ol (14g)
Pale yellow solid (64%); 1H NMR (400 MHz, DMSO-d6):
d
8.41 (s,
Ethyl
carboxylate 13g (874 mg, 3.0 mmol) and 2-thiophene carbonitrile
(420 l, 4.50 mmol) were dissolved in 4 M HCl 1,4-dioxane solution
2-amino-5-(4-methoxyphenyl)-4-methylthiophene-3-
1H), 7.72 (br, 3H), 7.51 (m, 5H), 7.16 (s, 1H), 6.93 (s, 1H), 2.12 (s, 3H);
13C NMR (100 MHz, DMSO-d6):
d
169.48, 168.98, 168.39, 155.40,
m
(10 ml), and the resulting mixture was stirred at 110 ꢀC for 36 h.
After cooling, the reaction mixture was poured into saturated
NaHCO3 aqueous solution and the resulting solid was filtered. The
filter cake was then washed with H2O and EtOAc to afford the title
compound 14g as a yellow solid (950 mg, 93%). 1H NMR (400 MHz,
154.89, 146.28, 144.90, 142.36, 134.67, 134.49, 130.69, 128.92,
128.63, 128.49, 128.41, 128.30, 127.39, 126.81, 123.17, 110.90,
11.18 ppm; ESI (m/z) 419 (MHþ); HRMS (ESI) calculated for
C
21H14N4O2S2 [MHþ]: 419.0636, found: 419.0615; HPLC (tR
:
purity ¼ 8.39 min, 93.71%).
DMSO-d6):
d
8.25 (d, J ¼ 3.6 Hz, 1H), 7.89 (d, J ¼ 4.8 Hz, 1H), 7.46 (d,
J ¼ 8.8 Hz, 2H), 7.25 (t, J ¼ 4.8 Hz,1H), 7.07 (d, J ¼ 8.8 Hz, 2H), 3.81 (s,
4.5.3. 1-[2-(4-Methoxyphenyl)-5-phenylthieno[2,3-d]pyrimidin-4-
3H), 2.54 (s, 3H).
ylamino]-3-methyl-1H-pyrrole-2,5-dione (12c)
Pale yellow solid (61%); 1H NMR (400 MHz, DMSO-d6):
d 8.28
(br, 1H), 8.10 (d, J ¼ 8.8 Hz, 2H), 7.66 (s, 1H), 7.58 (d, J ¼ 7.7 Hz, 2H),
7.52 (t, J ¼ 7.5 Hz, 2H), 7.47e7.44 (m, 1H), 7.02 (d, J ¼ 8.4 Hz, 2H),
6.94 (s, 1H), 3.81 (s, 3H), 2.13 (s, 3H); 13C NMR (100 MHz, DMSO-
4.6.3. 4-Chloro-6-(4-methoxyphenyl)-5-methyl-2-(thiophen-2-yl)
thieno[2,3-d]pyrimidine (15g)
6-(4-Methoxyphenyl)-5-methyl-2-(thiophen-2-yl)thieno[2,3-
d]pyrimidin-4-ol 14g (950 mg, 2.80 mmol) was dissolved in POCl3
(5 ml) and the resulting mixture was heated for 3 h at 100 ꢀC. After
cooling to room temperature, the reaction mixture was poured into
saturated NaHCO3 aqueous solution and the resulting solid was
filtered. The filter cake was then washed with H2O and n-hexane to
afford the title compound 15g as a yellow solid (950 mg, 91%).
d6):
d 169.93, 169.66, 168.83, 161.89, 158.33, 156.06, 145.39, 135.26,
134.79, 129.70 (2), 129.37 (2), 129.33, 129.07 (2), 128.71, 127.27,
123.36, 114.45 (2), 111.22, 55.77, 11.59 ppm; ESI (m/z) 443 (MHþ);
HRMS (ESI) calculated for C24H18N4O3S [MHþ]: 443.1178, found:
443.1168; HPLC (tR : purity ¼ 9.76 min, 99.09%).
4.5.4. 1-{2-[4-(Dimethylamino)phenyl]-5-phenylthieno[2,3-d]
pyrimidin-4-ylamino}-3-methyl-1H-pyrrole-2,5-dione (12d)
Yellow solid (94%); 1H NMR (400 MHz, DMSO-d6):
d 8.21 (s, 1H),
4.7. General procedure for the preparation of title compounds
17ae17k
8.06 (d, J ¼ 8.8 Hz, 2H), 7.60e7.58 (m, 3H), 7.53e7.45 (m, 3H), 6.93
(s, 3H), 3.01 (s, 6H), 2.14 (s, 3H); ESI (m/z) 456 (MHþ); HRMS (ESI)
calculated for C25H21N5O2S [MHþ]: 456.1494, found: 456.1474;
HPLC (tR : purity ¼ 10.86 min, 94.24%).
To a stirred solution of 15 (0.15 mmol) in THF (5 ml) hydrazine
monohydrate (0.44 mmol) was added dropwise, and the resulting
mixture was stirred at 80 ꢀC for 10 h. After cooling to room tem-
perature, the solvent was removed under reduced pressure and
concentrated in vacuo. The residue 16 was then dissolved in CHCl3
(5 ml), and citraconic anhydride (0.44 mmol) was slowly added. The
mixture was heated to 80 ꢀC and stirred for 20 h. After cooling to
room temperature, the reaction mixture was extracted with CHCl3
and the combined organic layer was washed with brine. After
drying over Na2SO4, the mixture was filtered and the filtrate was
concentrated under reduced pressure. The residue was then puri-
fied by flash column chromatography (n-Hx:EtOAc ¼ 1:2) to afford
the title compound 17.
4.5.5. 1-[2-(4-Aminophenyl)-5-phenylthieno[2,3-d]pyrimidin-4-
ylamino]-3-methyl-1H-pyrrole-2,5-dione (12e)
Yellow solid (79%); 1H NMR (400 MHz, DMSO-d6):
d 8.35 (s, 1H),
8.16 (d, J ¼ 8.4 Hz, 2H), 7.71 (s, 1H), 7.59 (d, J ¼ 7.2 Hz, 2H), 7.52 (t,
J ¼ 7.2 Hz, 2H), 7.47 (d, J ¼ 6.4 Hz,1H), 7.26 (d, J ¼ 8.0 Hz, 2H), 6.94 (s,
1H), 2.13 (s, 3H); 13C NMR (100 MHz, DMSO-d6):
d 169.52, 169.15,
168.38, 157.30, 155.78, 145.01, 134.73, 134.42, 128.98 (4), 128.92 (2),
128.70 (2), 128.36, 126.92, 123.66, 121.79, 111.22, 11.21 ppm; ESI (m/
z) 428 (MHþ); HRMS (ESI) calculated for C23H17N5O2S [MHþ]:
428.1181, found: 428.1163; HPLC (tR : purity ¼ 5.16 min, 96.53%).

406
J.S. Yang et al. / European Journal of Medicinal Chemistry 85 (2014) 399e407
4.7.1. 1-[6-Methoxy-5-methyl-2-(thiophen-2-yl)thieno[2,3-d]
4.7.7. 1-[6-(4-Methoxyphenyl)-5-methyl-2-(thiophen-2-yl)thieno
pyrimidin-4-ylamino]-3-methyl-1H-pyrrole-2,5-dione (17a)
[2,3-d]pyrimidin-4-ylamino]-3-methyl-1H-pyrrole-2,5-dione (17g)
Yellow solid (73%); 1H NMR (400 MHz, DMSO-d6):
d
9.43 (s, 1H),
Yellow solid (70%); 1H NMR (400 MHz, DMSO-d6):
d 9.66 (s, 1H),
7.65e7.61 (m, 2H), 7.12 (t, J ¼ 6.0 Hz, 1H), 6.98 (d, J ¼ 1.7 Hz, 1H),
7.70e7.68 (m, 2H), 7.49 (d, J ¼ 8.8 Hz, 2H), 7.16e7.09 (m, 3H), 7.01 (d,
4.00 (s, 3H), 2.42 (s, 3H), 2.17 (s, 3H); 13C NMR (100 MHz, DMSO-
J ¼ 2.0 Hz, 1H), 3.82 (s, 3H), 2.62 (s, 3H), 2.18 (d, J ¼ 2.0 Hz, 3H); 13
C
d6):
d
170.51, 169.46, 160.29, 155.80, 154.77, 153.78, 145.45, 143.14,
NMR (100 MHz, DMSO-d6): d 170.42, 169.36, 166.90, 159.89, 156.07,
130.22, 128.78, 127.86, 127.38, 113.97, 108.12, 62.93, 11.72,
154.83, 145.52, 142.94, 134.65, 131.40 (2), 130.90, 128.89, 128.60,
127.44, 125.22, 124.45, 115.67, 114.89 (2), 55.74, 15.73, 11.68 ppm;
ESI (m/z) 463 (MHþ); HRMS (ESI) calculated for C23H18N4O3S2
[MHþ]: 463.0899, found: 463.0883; HPLC (tR : purity ¼ 10.06 min,
100%).
11.66 ppm; ESI (m/z) 387 (MHþ); HRMS (ESI) calculated for
C
17H14N4O3S2 [MHþ]: 387.0586, found: 387.0565; HPLC (tR
:
purity ¼ 5.61 min, 94.37%).
4.7.2. Methyl 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 (17b)
4.7.8. 1-(6-(4-Hydroxyphenyl)-5-methyl-2-(thiophen-2-yl)thieno
[2,3-d]pyrimidin-4-ylamino)-3-methyl-1H-pyrrole-2,5-dione (17h)
Yellow solid (72%); 1H NMR (400 MHz, DMSO-d6):
d 9.64 (s, 1H),
White solid (58%); 1H NMR (400 MHz, DMSO-d6):
d
9.96 (s, 1H),
7.75 (m, 2H), 7.16 (t, J ¼ 4.8 Hz, 1H), 7.02 (s, 1H), 3.87 (s, 3H), 2.98 (s,
3H), 2.18 (s, 3H); 13C NMR (100 MHz, DMSO-d6):
170.13, 169.06,
7.69e7.67 (m, 2H), 7.38 (d, J ¼ 8.4 Hz, 2H), 7.14 (t, J ¼ 4.4 Hz, 1H),
7.01 (d, J ¼ 2.0 Hz, 1H), 6.92 (d, J ¼ 8.4 Hz, 2H), 2.61 (s, 3H), 2.18 (s,
3H); ESI (m/z) 449 (MHþ); HRMS (ESI) calculated for C22H16N4O3S2
[MHþ]: 449.0742, found: 447.0722; HPLC (tR : purity ¼ 4.93 min,
99.37%).
d
168.46, 162.78, 157.57, 157.30, 145.64, 142.32, 139.64, 132.14, 129.79,
129.12, 127.51, 122.20, 115.19, 53.00, 16.10, 11.70 ppm; ESI (m/z) 415
(MHþ); HRMS (ESI) calculated for C18H14N4O4S2 [MHþ]: 415.0535,
found: 415.0513; HPLC (tR : purity ¼ 6.04 min, 99.43%).
4.7.9. 1-[6-(3-Hydroxyphenyl)-5-methyl-2-(thiophen-2-yl)thieno
[2,3-d]pyrimidin-4-ylamino]-3-methyl-1H-pyrrole-2,5-dione (17i)
Pale yellow solid; 1H NMR (400 MHz, DMSO-d6):
d 9.79 (s, 1H),
4.7.3. Methyl 2-[5-methyl-4-(3-methyl-2,5-dioxo-2,5-dihydro-1H-
pyrrol-1-ylamino)-2-(thiophen-2-yl)thieno[2,3-d]pyrimidin-6-yl]
acetate (17c)
9.66 (s, 1H), 7.70 (d, J ¼ 4.2 Hz, 2H), 7.34 (t, J ¼ 7.9 Hz, 1H), 7.17e7.13
(m, 1H), 7.03e6.97 (m, 2H), 6.95 (s, 1H), 6.87 (d, J ¼ 8.1 Hz, 1H), 2.65
(s, 3H), 2.19 (s, 3H); 13C NMR (100 MHz, DMSO-d6):
d 170.00,
Yellow solid (82%); 1H NMR (400 MHz, DMSO-d6):
d 9.58 (s, 1H),
168.95, 166.73, 157.70, 155.80, 154.62, 145.14, 142.50, 134.31, 133.80,
130.58, 130.16, 128.51, 128.27, 127.04, 124.64, 120.35, 116.27, 115.69,
115.22, 15.42, 11.28 ppm; ESI (m/z) 449 (MHþ).
7.67 (d, J ¼ 5.0 Hz, 2H), 7.13 (t, J ¼ 4.0 Hz, 1H), 7.00 (s, 1H), 4.06 (s,
2H), 3.68 (s, 3H), 2.52 (s, 3H), 2.18 (s, 3H); 13C NMR (100 MHz,
DMSO-d6):
d 170.11, 170.02, 168.97, 166.95, 155.32, 154.43, 145.11,
142.52, 130.41, 128.45, 128.13, 127.33, 127.01, 126.87, 114.27, 52.18,
32.71,14.29,11.26 ppm; ESI (m/z) 429 (MHþ); HRMS (ESI) calculated
for C19H16N4O4S2 [MHþ]: 429.0691, found: 429.0667.
4.7.10. 3-Methyl-1-[5-methyl-6-(4-nitrophenyl)-2-(thiophen-2-yl)
thieno[2,3-d]pyrimidin-4-ylamino]-1H-pyrrole-2,5-dione (17j)
Yellow solid; 1H NMR (400 MHz, DMSO-d6):
d 9.81 (s, 1H), 8.36
(d, J ¼ 7.2 Hz, 2H), 7.86 (d, J ¼ 7.6 Hz, 2H), 7.72 (d, J ¼ 3.6 Hz, 2H), 7.16
(m, 1H), 7.03 (m, 1H), 2.69 (s, 3H), 2.20 (s, 3H); 13C NMR (100 MHz,
4.7.4. 1-[6-(Hydroxymethyl)-5-methyl-2-(thiophen-2-yl)thieno
[2,3-d]pyrimidin-4-ylamino]-3-methyl-1H-pyrrole-2,5-dione (17d)
DMSO-d6):
d 170.34, 169.27, 167.79, 156.54, 155.57, 147.35, 145.60,
Orange solid (100%); 1H NMR (400 MHz, DMSO-d6):
d
9.54 (s,
1H), 7.66 (m, 2H), 7.13 (m, 1H), 7.00 (s, 1H), 4.73 (s, 2H), 2.45 (s, 3H),
2.17 (s, 3H); 13C NMR (100 MHz, DMSO-d6):
170.11, 169.05, 166.50,
142.67, 139.89, 131.88, 131.38, 131.27 (2), 129.06, 128.97, 127.89,
127.49, 124.53 (2), 115.55, 16.05, 11.70 ppm; ESI (m/z) 478 (MHþ);
HPLC (tR : purity ¼ 9.44 min, 99.70%).
d
155.54, 153.96, 145.18, 142.49, 138.88, 130.54, 128.57, 128.24, 127.11,
123.21, 114.96, 56.79, 14.33, 11.36 ppm; ESI (m/z) 387 (MHþ); HRMS
4.7.11. 1-[6-(4-Hydroxybenzyl)-5-methyl-2-(thiophen-2-yl)thieno
(ESI) calculated for
C
17H14N4O3S2 [MHþ]: 387.0586, found:
[2,3-d]pyrimidin-4-ylamino]-3-methyl-1H-pyrrole-2,5-dione (17k)
387.0563; HPLC (tR : purity ¼ 10.19 min, 99.05%).
Brown solid; 1H NMR (400 MHz, DMSO-d6):
d 9.42 (s, 1H),
7.66e7.63 (m, 2H), 7.12 (t, J ¼ 4.0 Hz, 1H), 7.08 (d, J ¼ 8.0 Hz, 2H),
7.00 (s, 1H), 6.72 (d, J ¼ 8.0 Hz, 2H), 4.10 (s, 2H), 2.59 (s, 3H), 2.18 (s,
4.7.5. 1-[6-(Methoxymethyl)-5-methyl-2-(thiophen-2-yl)thieno
3H); 13C NMR (100 MHz, DMSO-d6):
d 170.07, 169.02, 166.59,
[2,3-d]pyrimidin-4-ylamino]-3-methyl-1H-pyrrole-2,5-dione (17e)
156.05, 155.16, 154.06, 145.08, 142.62, 136.39, 130.23 (2), 129.44,
128.41, 127.93, 127.00, 124.25, 115.41 (2), 114.73, 32.31, 30.72, 14.25,
11.27 ppm; ESI (m/z) 463 (MHþ).
Orange solid; 1H NMR (400 MHz, DMSO-d6):
d 9.68 (br, 1H), 7.45
(t, J ¼ 8.0 Hz, 1H), 7.16 (m, 1H), 7.06 (d, J ¼ 8.0 Hz, 1H), 7.01 (s, 1H),
3.69 (s, 2H), 3.31 (s, 3H), 2.65 (s, 3H), 2.19 (s, 3H); ESI (m/z) 401
(MHþ); HRMS (ESI) calculated for C18H16N4O3S2 [MHþ]: 401.0742,
found: 401.0741; HPLC (tR : purity ¼ 10.19 min, 100%).
4.8. Kinase inhibition analysis
Kinase assays were conducted using Millipore's KinaseProfiler
according to the protocols detailed at http://www.millipore.com/
drugdiscovery/dd3/KinaseProfiler. The title compounds were
tested against human FLT3 and FLT3 (D835Y) at five decreasing
4.7.6. 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-carboxamide
(17f)
Pale yellow solid (49%); 1H NMR (400 MHz, DMSO-d6):
d
10.09
concentrations (e.g., 10, 1, 0.1, 0.01, and 0.01 mM) with 10 mM ATP
(s, 1H), 7.76 (m, 2H), 7.50 (br, 2H), 7.17 (d, J ¼ 4.0 Hz, 1H), 7.03 (s,
according to the Millipore protocol, and their corresponding IC50
values were obtained.
1H), 2.81 (s, 3H), 2.18 (s, 3H); 13C NMR (100 MHz, DMSO-d6):
d
169.98, 169.78, 168.90, 157.98, 157.17, 145.70, 144.16, 141.91,
132.63, 130.22, 129.21, 127.53, 114.34, 112.74, 101.26, 17.57,
4.9. Proliferation assays
11.72 ppm; ESI (m/z) 400 (MHþ); HRMS (ESI) calculated for
C
17H13N5O3S2 [MHþ]: 400.0538, found: 400.0515; HPLC (tR
:
Human leukemia cell lines MV4-11, THP-1, K562 and HL-60 were
purchased from American Type Culture Collection (Manassas, VA).
purity ¼ 5.72 min, 97.66%).

J.S. Yang et al. / European Journal of Medicinal Chemistry 85 (2014) 399e407
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Acknowledgments
This research was supported by a grant from the Translational
Research Center for Protein Function Control (NRF-2009-0083522),
the Basic Science Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education
(NRF-2013R1A1A2008165), Ministry of Health
(A120478), and the Ministry of Science, ICT & Future Planning
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&
Welfare
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Appendix A. Supplementary data
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