Design, synthesis and docking studies of novel thienopyrimidine derivatives bearing chromone moiety as mTOR/PI3Kα inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.01.061

Two series of thienopyrimidine derivatives (10a-k, 16a-j) bearing chromone moiety were designed and synthesized. All the compounds were evaluated for inhibitory activity against mTOR kinase at a concentration of 10uM. Four selected compounds were further evaluated for the IC₅₀ values against mTOR kinase, PI3Kα kinase and two cancer cell lines. Some of the target compounds exhibited moderate to excellent mTOR/PI3Kα kinase inhibitory activity and cytotoxicity. The most promising compound 16i showed good inhibitory activity against mTOR/PI3Kα kinase and good antitumor potency for H460 and PC-3 cell lines with IC₅₀ values of 0.16 ± 0.03 μM, 2.35 ± 0.19 μM, 1.20 ± 0.23 μM and 0.85 ± 0.04 μM, which were 8.6, >5, 7.9 and 19.1 times more active than compound I (1.37 ± 0.07 μM, >10 μM, 9.52 ± 0.29 μM, 16.27 ± 0.54 μM), respectively. Structure-activity relationships (SARs) and docking studies indicated that the chromone moiety is necessary for the potent antitumor activity and cytotoxicity of these compounds. Substitution of the chromone moiety at the 6-position has a significant impact to the inhibitory activity, in particular a carboxylic acid group, produced the best potency.

Full text of DOI:10.1016/j.ejmech.2015.01.061

European Journal of Medicinal Chemistry 93 (2015) 64e73
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and docking studies of novel thienopyrimidine
derivatives bearing chromone moiety as mTOR/PI3K inhibitors
a
,
b
,
*
,
,
,
Wufu Zhu a
1, Chen Chen a 1, Chengyu Sun a 1, Shan Xu a, Chunjiang Wu a, Fei Lei a,
Hui Xia a, Qidong Tu a, Pengwu Zheng a
,
**
a School of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang, 330013, PR China
b Key Laboratory of Original New Drugs Design and Discovery of Ministry of Education, Shenyang Pharmaceutical University, 103 Wenhua Road,
Shenhe District, 110016, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of thienopyrimidine derivatives (10aek, 16aej) bearing chromone moiety were designed and
synthesized. All the compounds were evaluated for inhibitory activity against mTOR kinase at a con-
centration of 10uM. Four selected compounds were further evaluated for the IC50 values against mTOR
Received 1 August 2014
Received in revised form
25 January 2015
Accepted 31 January 2015
Available online 31 January 2015
kinase, PI3K
excellent mTOR/PI3K
showed good inhibitory activity against mTOR/PI3K
PC-3 cell lines with IC50 values of 0.16 0.03 M, 2.35 0.19
which were 8.6, >5, 7.9 and 19.1 times more active than compound I (1.37
9.52 0.29 M, 16.27 0.54
a
kinase and two cancer cell lines. Some of the target compounds exhibited moderate to
kinase inhibitory activity and cytotoxicity. The most promising compound 16i
kinase and good antitumor potency for H460 and
a
a
m
m
M, 1.20 0.23
m
M and 0.85 0.04
0.07 M, >10
m
m
M,
M,
Keywords:
Thienopyrimidine
Chromone
Synthesis
Docking
m
m
m
M), respectively. Structureeactivity relationships (SARs) and docking
studies indicated that the chromone moiety is necessary for the potent antitumor activity and cyto-
toxicity of these compounds. Substitution of the chromone moiety at the 6-position has a significant
impact to the inhibitory activity, in particular a carboxylic acid group, produced the best potency.
© 2015 Elsevier Masson SAS. All rights reserved.
mTOR
PI3Ka
Cytotoxicity
1. Introduction
(Fig. 1) is a triazine-hydrazone derivative which exhibited potent
anti-tumor activity with an mTOR IC50 value of 0.27 mM [6]. To our
The PI3K-Akt-mTOR signaling pathway plays a key role in cell
proliferation, migration, survival, and angiogenesis, and developing
mTOR/PI3Ka inhibitors is one of the research hotspots in molecular
knowledge, few studies based on pyrimidine- or triazine-
hydrazones as mTOR inhibitors have been reported to date.
Importantly, the hydrazinyl group or its mimics are key pharma-
cophores in the design of different potential anticancer agents [7].
Therefore, the research on this aspect seemed to be an attractive
task.
targeted therapy for the treatment of human cancer [1,2].
In recent years, a growing number of studies based on the
(fused-)pyrimidine/triazine scaffolds towards antitumor activity
have been carried out [3e7]. Among them, GDC-0941 (Fig.1), which
is a potent, orally bioavailable inhibitor of PI3K, exerted antitumor
activity against an array of human tumor cell lines and is currently
undergoing Phase II clinical trials [5]. Further optimization of GDC-
0941 led to several backup clinical candidates with similar struc-
tures, such as GNE-490 and GNE-493 [4] (Fig. 1). Compound I
In our previous research [8e10], several series of 2-hydrazinyl-
4-morpholinothieno[3,2-d]pyrimidines
which
demonstrated
potent antitumor activity were designed and synthesized, and SARs
were discussed and summarized. The results showed that most of
them exhibited excellent cytotoxicity but possessed disappointing
in vivo antitumor activity, such as the representative compounds II
[9] and III [10] (Fig. 1). This work also showed that introduction of
indole-hydrazone to the target compound benefitted the antitumor
activity.
In continuation of our previous research towards compounds
which possessed excellent antitumor activity, further indole-
hydrazone studies were carried out and are reported herein.
Through analysis of the SARs and the results of docking with mTOR/
* Corresponding author. School of Pharmacy, Jiangxi Science
Normal University, Nanchang, 330013, PR China.
** Corresponding author.
& Technology
E-mail addresses: zhuwufu-1122@163.com (W. Zhu), zhengpw@126.com
(P. Zheng).
1
These authors contribute equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2015.01.061
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

W. Zhu et al. / European Journal of Medicinal Chemistry 93 (2015) 64e73
65
Fig. 1. Structures of some reported (fused-)pyrimidine/triazines and compounds reported in previous research.
PI3K
a
kinase, a new compound library was built with structural
cytotoxicity against H460 (lung) and PC-3 (prostate) cancer cell
lines. Further evaluation was carried out against PI3K kinase to
confirm the types of target compounds, selective or mixed type
inhibitors. Moreover, docking studies are presented providing a
rationale for the observed activity.
modifications at thienopyrimidine moiety and indole-hydrazone
groups (Fig. 1). Firstly, a series of thieno[3, 2-d]pyrimidines
10aek (Fig. 2) were designed by replacing the N-benzyl indole or N-
benzyl benzimidazole moieties of compounds II and III with active
pharmacophore chromone moiety which was proved in our pre-
viously research [11]. However, this series of compounds showed
poor cytotoxicity [12] and moderate mTOR kinase inhibitory ac-
tivity. Further investigations were carried out in detail to study the
effects of the thienopyrimidine scaffold on the antitumor activity.
Therefore, the fusion between the thienyl and pyrimidine in the
scaffold was changed according to the theory of isostere principle,
resulting in the series of thieno[2, 3-d]pyrimidines 16aej (Fig. 2).
The inhibitory activity against mTOR kinase and cytotoxicity of
compounds 16aej were better than that of compounds 10aek.
Considering the high potency of GDC-0941 and its analogous on
lung and prostate cancer cells, herein we disclose the design, syn-
thesis and inhibitory activity of novel thieno[2,3-d]pyrimidine
bearing a chromone moiety against mTOR kinase, as well as the
a
2. Chemistry
The preparation of target compounds 10aek and 16aej is
described in Schemes 1 and 2. The structures and the predicted
cLogp values of target compounds are shown in Table 1.
The substituted 4-oxo-4H-chromene-3-carbaldehydes 4aek
were synthesized through the procedures reported previously by
our group (Scheme 1) [11,13].
The synthesis of compounds 10aek was similar to the pro-
cedures previously reported [8e10,12]. The key intermediate 9
condensed with substituted 4-oxo-4H-chromene-3-carbaldehydes
4aek to afford target compounds 10aek, respectively.
Compounds 16aej were synthesized from methyl 2-
Fig. 2. Structures and design strategy for target compounds 10aek and 16aej.

66
W. Zhu et al. / European Journal of Medicinal Chemistry 93 (2015) 64e73
Scheme 1. Synthetic route of substituted 4-oxo-4H-chromene-3-carbaldehydes (4aek) Reagents and conditions: (a) Ac2O, conc. H2SO4, r.t.; (b) anhydrous AlCl3, 120 ꢀC; (c) DMF/
POCl3, r.t.
aminothiophene-3-carboxylate 11 through five steps. The
commercially available methyl 2-aminothiophene-3-carboxylate
11 was condensed with urea at 180 ꢀC for 2 h to give thienopyr-
imidinedione 12. Chlorination of 12 with POCl3 and DMF (cat.) for
8 h at 115 ꢀC afforded 13 as yellow solid. Substitution reaction of 13
with morpholine at room temperature to yielded 14 which was
then condensed with 4aej to furnish compounds 16aej,
respectively.
Scheme 2. Synthetic routes of target compounds 10aek and 16aej. Reagents and conditions: (a) 5 eq urea, 180 ꢀC, 2 h; (b) POCl3, DMF (cat.), reflux, 8 h; (c) 2.1 eq morpholine,
MeOH, 0 ꢀC, 30 min, r.t., 1e2 h; (d) 80% NH2NH2$H2O,reflux, 8e10 h; (e) EtOH, reflux, 2e4 h.

W. Zhu et al. / European Journal of Medicinal Chemistry 93 (2015) 64e73
67
Table 1
Structures and inhibition rates of target compounds 10aek, 16aej against mTOR/PI3K
a
kinase at 10 mM
Compd.
R
cLogP (predicted)d
(10
m
M inhibitory %)a
Compd.
R
cLogP (predicted)d
(10 m
M inhibitory %)a
mTOR
PI3K
a
mTOR
PI3Ka
10a
10b
10c
10d
10e
10f
10g
10h
10i
Fluoro
Chloro
Bromo
Methyl
Ethyl
Tert-butyl
Hydroxy
Nitro
Carboxyl
H
Isopropyl
e
4.2
4.8
4.9
4.6
5.1
5.9
4.0
3.8
4.0
4.1
5.5
<10%
e
e
e
e
e
e
e
16a
16b
16c
16d
16e
16f
16g
16h
16i
fluoro
chloro
bromo
methyl
ethyl
tert-butyl
hydroxy
nitro
4.0
4.6
4.7
4.3
5.1
5.7
3.8
3.6
3.8
3.8
29.4 5.5
30.8 3.5
31.0 0.3
41.5 0.8
26.7 1.4
34.9 0.8
No active
81.0 0.7
97.2 0.4
42.9 2.0
e
e
e
e
e
e
e
28.8 6.6
41.5 9.4
49.5 0.5
32.2 3.4
25.4 3.1
44.9 2.8
54.3 0.6
61.4 0.5
36.2 3.3
21.7 2.7
36.9 2.8
33.6 3.3
58.8 3.2
95.0 7.9
e
e
e
e
91.2 11.7
90.6 9.6
e
carboxyl
H
10j
16j
10k
IIb,c
IIIb,c
Ib
e
e
76.1 0.3
98.6 0.5
e
e
PI-103b
98.5 3.8
a
The values are an average of two separate determinations.
Used as a positive controls.
Compounds reported in our previous research.
cLogP values are predicted in sybyl 6.9.1.
b
c
d
The relative stereochemistry of the target compounds was easily
confirmed as E isomeric form according to our previously published
method [8e10,12].
In the first series, thieno[3, 2-d]pyrimidines 10aek were
designed by replacing the N-benzyl indoles or N-benzyl benz-
imidazole moieties of compounds II and III with substituted
chromone moieties. The inhibitory activity on mTOR kinase of
compounds 10aek were increased in varying degrees. The results
showed that variations in substitution at 6-position of the chro-
mone moiety had a marked impact on the activity. For compounds
10aek, substitution of the chromone moiety at the 6-position was
well tolerated and 6-NO2 (10h) and 6-COOH (10i) substitution were
more preferred.
Further investigations were carried out in detail to study the
effects of the thienopyrimidine scaffold on the activity. Therefore,
the fusion isomer of thienopyrimidine scaffold was changed,
resulting in the other series thieno[2, 3-d]pyrimidines 16aej. The
mTOR inhibition and cytotoxicity of this series of compounds were
3. Results and discussion
3.1. Biological evaluation
The target compounds were evaluated for their inhibitory ac-
tivity against mTOR kinase at a concentration of 10uM in vitro
together with reference compounds I, II, III and PI103 by LANCE®
Ultra time-resolved fluorescence resonance energy transfer (TR-
FRET) assay. Four selected compounds also measured the PI3K
a
kinase inhibition rate at a concentration of 10 mM with reference
PI103 by Kinase-Glo® Luminescent Kinase Assay. In addition, the
higher than that of compounds 10aek, but most of them the PI3K
a
four selected compounds 10hei and 16hei were further evaluated
inhibition has little difference. It was noticeable that compound 16i
for their IC50 values against mTOR kinase, PI3Ka kinase and two
cancer cell lines H460 (human lung cancer) and PC-3 (human
prostate cancer). The results were expressed as percent inhibition
at single concentration or IC50 values are summarized in Tables 1
and 2 and the values are the average of at least two independent
experiments.
As illustrated in Tables 1 and 2, most of the synthesized com-
pounds showed moderate inhibitory activity against mTOR kinase
and were more potent than the compounds (II and III) reported
previously, suggesting that the replacement of N-benzyl indole or
N-benzyl benzimidazole moiety with chromone moiety enhanced
the anti-tumor activity. Four of them (compounds 10hei and
16hei) showed excellent inhibitory activity with IC50 values from
Table 2
mTOR/PI3K
controls.
a kinase activity and cytotoxicity of selected compounds and positive
Compounds no. IC50
mTOR
(m
M)a
PI3Ka
H460
PC-3
10h
10i
4.62 0.54
3.31 0.12
0.92 0.14
0.16 0.03
1.37 0.07
16.62 0.39
>30
>10
1.81 0.35
1.53 0.21
2.35 0.19
>10
e
ND
ND
6.90 1.22
6.26 0.61
16h
16i
3.24 1.12 1.42 0.19
1.20 0.23 0.85 0.04
9.52 0.29 16.27 0.54
ND
ND
Ib
IIb,c
IIIb,c
PI-103b
ND
ND
ND
e
4.62 0.54
1.80 M to 2.35
inhibition activity were little better than PI3K
m
M to 0.16 0.03
m
M against mTOR kinase and from
kinase. And overall their mTOR
inhibition activity.
0.019 0.004 0.0075 0.0018 ND
m
mM against PI3K
a
Bold values show IC50 of <1
mM.
a
a
The values are an average of two separate determinations.
Used as a positive controls.
Compounds reported in our previous research.
b
c
The inhibitory activity against mTOR kinase of compounds 16h and
16i were 1.49 and 8.56 times more active than lead compound I.

68
W. Zhu et al. / European Journal of Medicinal Chemistry 93 (2015) 64e73
(with negative charge group eCOOH at C-6 position) showed
excellent antitumor activity with the inhibitory activity equal to the
positive control PI-103 (97.2 0.4% vs 98.6 0.5%), which suggested
that the negative charge character of the most active groups has a
more predominant role. This significant increase in the mTOR po-
tency could be ascribed to the increased hydrogen bonds between
the 6-COOH and receptors necessary for activity or the increased
polar interactions with the mTOR.
3.2. Molecular docking study
To explore the binding modes of target compounds with the
active site of mTOR and PI3Ka, molecular docking simulation
studies were carried out by using SURFLEX-DOCK module of SYBYL
package version. Based on the in vitro inhibition results, we selected
compound 16i, our best mTOR/PI3K
ligand example, and the structures of mTOR (PDB ID code:4JT6 [15])
a inhibitor in this study, as
The mTOR/PI3K
a
kinase activity and cytotoxicity of the four
and PI3Ka (PDB ID code:4L23 [16]) were selected as the docking
selected compounds 10hei and 16hei as well as the four lead
models.
compounds are shown in Table 2. Obviously, these four compounds
exhibited good activity towards mTOR/PI3Ka kinase and cytotox-
icity. All of them display an IC50 for mTOR inhibition superior to that
of lead compounds II and III, two of them (16hei) were superior to
compound I. Three compounds (10i,16hei) are more active than
The binding modes of compound 16i and lead compounds were
shown in Fig. 3aed. As depicted in Fig. 3a, b, compound 16i and
PI103 can overlap in the position of morpholine group in the
binding model and the eCOOH group of chromone moiety formed
two hydrogen bonds with residues TRP2239 and ARG2348 in the
docking model of mTOR, respectively. The morpholine group
formed one hydrogen bond with residues VAL2240. Similarly, the
compound I against PI3K
16i showed good inhibitory activity against mTOR, PI3K
and good cytotoxicity for H460 and PC-3 cell lines with IC50 values
a
kinase. The most promising compound
a
kinase
hydrogen bonds were found in the docking model of PI3K
a in
of 0.16
0.85 0.04
0.03
m
M, 2.35
0.19
m
M, 1.20
0.23
m
M
and
Fig. 3c, d, such as the hydrogen bonds between morpholine group
and residue VAL851, the chromone moiety and the residues
mM, which were 8.6, 7.9 and 19.1 times more active than
compound I (1.37 0.07
mM, 9.52 0.29
mM, 16.27 0.54
mM),
SER773, SER919. The activity against mTOR/PI3Ka kinase of com-
respectively.
pound 16i was difference, which was attributed to the difference of
the hydrogen bonds between the chromone moiety and amino acid
residues. According to the docking model of mTOR (Fig. 3b), eCOOH
group of chromone moiety formed two hydrogen bonds with res-
idues TRP2239 and ARG2348, respectively. However, only one
hydrogen bond was formed between eCOOH group of chromone
moiety and amino acid residues in the docking model of
As described above, chromone moieties were necessary for
these thienopyrimidine derivatives. Variations in substitutions of
the chromone moieties had a significant impact on the activity.
Substitution of the chromone moieties at the 6-position with
negatively charged groups was preferred and 6-COOH substitution
produced the best potency.
Fig. 3. aed. Binding models of compounds 16i (shown in Capped Sticks) and native ligand PI 103 (shown in yellow lines) target into active site of mTOR （Fig. 3a, b） and PI3K
a
（Fig. 3c, d）, respectively. Hydrogen bonds were showed in dashed lines (yellow). (For interpretation of the references to color in this figure legend, the reader is referred to the
web version of this article.)

W. Zhu et al. / European Journal of Medicinal Chemistry 93 (2015) 64e73
69
PI3Ka
(Fig. 3c). Analysis of compound 16i's binding mode in the
5.4. General procedure for the preparation of compounds 10aek
active binding site and the results of activity suggested that the
several hydrogen bonds, formed in morpholine group and the
chromone moiety, really plays an important role in increasing the
A mixture of 4-(2-hydrazinylthieno[3,2-d]pyrimidin-4-yl)mor-
pholine 9 (0.12 g, 0.48 mmol), substitued 4-oxo-4H-chromene-3-
carbaldehyde 4aek and a drop of glacial acetic acid in absolute
ethanol (8 mL) was refluxed for 4e8 h. The mixture was cooled,
separated by filtration and washed with EtOH to give off a white to
light yellow solid. The crude product was recrystallized from
ethanol or purified by chromatography on silica gel using MeOH/
CH2Cl2 to afford the solids 10aek.
inhibitory potency of chromone derivatives against mTOR/PI3K
a
kinase. The docking results indicated that these compounds have
plausible binding modes to both enzymes.
Furthermore, the docking results also give us a new direction to
design new mTOR/PI3Ka inhibitors that can interact with VAL2240,
TRP2239, ARG2348, VAL851 and SER773. The above-mentioned
results of SAR analysis and molecular docking study may allow
the rational design of more potent mTOR/PI3K
a
inhibitors.
5.4.1. (E)-6-Fluoro-3-((2-(4-morpholinothieno[3,2-d]pyrimidin-2-
yl)hydrazono)methyl)-4H-chromen-4-one (10a)
4. Conclusions
This compound was obtained as yellow solid in 85% yield. m.p.
228e231 ꢀC. ESI-MS m/z:426.1 IR (KBr) cmꢁ1: 3432, 3086, 2992, 2958,
1629, 1428, 1382, 1255, 1196, 1174, 1115, 933, 881, 788. 1H NMR
In summary, two series of thieno-pyrimidine derivatives bearing
chromone moieties were designed, synthesized and evaluated for
(400 MHz, DMSO)
d 11.07 (s, 1H, NH), 8.73 (s, 1H, NCH), 8.24 (s, 1H,
inhibitory activity against mTOR kinase, PI3K
a
kinase and cytotox-
AreH), 8.05 (d, J ¼ 8.6 Hz,1H, AreH), 7.62(d, J ¼ 7.9 Hz, 2H, AreH), 7.48
(d, J ¼ 6.2 Hz, 1H, AreH), 7.23 (d, J ¼ 6.1 Hz, 1H, AreH), 3.85 (s, 4H,
OCH2), 3.75 (d, J ¼ 4.3 Hz, 4H, NCH2). 13C NMR (101 MHz, DMSO)
icity of two cancer cell lines in vitro. The pharmacological results
indicated that most of the synthesized compounds displayed more
mTOR inhibition than compounds reported in previous articles and
two compounds (16hei) inhibited mTOR at lower doses and were
more cytotoxic than lead compound I. And three compounds also
d
163.1(C), 158.5(C), 157.6(C), 156.8(C), 154.5(CH), 154.1(CH), 151.9(C),
138.2(C),135.3(CH),133.8(CH),124.9(C),124.0(CH),120.2(CH),117.5(C),
111.6(CH), 106.7(C), 66.4(CH2), 66.3(CH2), 46.3(CH2), 45.8(CH2).
Analytical HPLC on an Agilent (1100) using a 250 mm ꢂ 4.6 mm SD-
C18 analytical column, H2O/MeOH (15%H2O) eluent at 1 mL/min flow,
monitored by UV absorption at 325 nm, showed 96.5% purity.
displayed moderate to excellent PI3K
with IC50 values from 1.805 M to 2.352
compound 16i showed good inhibitory activity against mTOR/PI3K
kinase and good antitumor potencyfor H460 and PC-3 cell lines with
IC50 values of 0.16 0.03 M, 2.35 0.19 M, 1.20 0.23 M and
0.85 0.04 M, which were 8.6, >5, 7.9 and 19.1 times more active
than compound I (1.37 0.07 M, >10 M, 9.52 0.29 M,
16.27 0.54 M), respectively. The initial SARs and docking studies
a
kinase inhibition activity
m
mM. The most promising
a
m
m
m
5.4.2. (E)-6-Chloro-3-((2-(4-morpholinothieno[3,2-d]pyrimidin-2-
yl)hydrazono)methyl)-4H-chromen-4-one (10b)
m
m
m
m
This compound was obtained as yellow solid in 88% yield. m.p.
236e238 ꢀC ESI-MS m/z:442.0 IR (KBr) cmꢁ1: 3434, 3092, 2998,
2986, 1651, 1632, 1561, 1545, 1467, 1429, 1379, 1116, 858, 824, 790.
m
showed that the chromone moieties were necessary for the activity
of these compounds. Variations in substitutions of the chromone
moieties had a significant impact on the activity. Substitutions of
chromone moiety at the 6-position have a significant impact to the
inhibitory activity, in particular a carboxylic acid group, produced
the best potency.
1H NMR (400 MHz, DMSO)
d 11.11 (s,1H, NH), 8.77 (s,1H, NCH), 8.24
(d, J ¼ 18.4 Hz, 1H, AreH), 8.05 (d, J ¼ 2.4 Hz, 1H, AreH), 7.99e7.85
(m, 1H, AreH), 7.79 (d, J ¼ 8.9 Hz, 1H, AreH), 7.48 (d, J ¼ 6.2 Hz, 1H,
AreH), 7.23 (d, J ¼ 6.1 Hz, 1H, AreH), 3.86 (s, 4H, OCH2), 3.75 (d,
J ¼ 4.1 Hz, 4H, NCH2). 13C NMR (101 MHz, DMSO)
d 188.2(C),
163.1(C), 162.6(C), 158.6(CH), 158.4(C), 157.6(CH), 155.4(C),
154.5(CH), 154.0(CH), 152.5(CH), 143.2(C), 135.9(CH), 135.4(C),
124.9(CH), 124.8(C), 106.7(C), 66.3(2CH2), 46.2(2CH2). Analytical
HPLC on an Agilent (1100) using a 250 mm ꢂ 4.6 mm SD-C18
analytical column, H2O/MeOH (15%H2O) eluent at 1 mL/min flow,
monitored by UV absorption at 325 nm, showed 95.2% purity.
5. Experimental
5.1. Chemistry
All melting points were obtained on a Büchi Melting Point B-540
apparatus (Büchi Labortechnik, Flawil, Switzerland) and were un-
corrected. NMR spectra were performed using Bruker 400 MHz
spectrometers (Bruker Bioscience, Billerica, MA, USA) with TMS as
an internal standard. Mass spectra (MS) were taken in ESI mode on
Agilent 1100 LCeMS (Agilent, Palo Alto, CA, USA). All the materials
were obtained from commercial suppliers and used without puri-
fication, unless otherwise specified. Yields were not optimized. TLC
analysis was carried out on silica gel plates GF254 (Qindao Haiyang
Chemical, China). All the materials were obtained from commercial
suppliers and used without purification, unless otherwise specified.
Yields were not optimized.
5.4.3. (E)-6-Bromo-3-((2-(4-morpholinothieno[3,2-d]pyrimidin-2-
yl)hydrazono)methyl)-4H-chromen-4-one (10c)
This compound was obtained as yellow solid in 84% yield. m.p.
267e269 ꢀC. ESI-MS m/z:448.0 IR (KBr) cmꢁ1: 3430, 3112, 2962,
1644, 1464, 1377, 1305, 1161, 1116, 858, 822, 789. 1H NMR (400 MHz,
DMSO)
d
10.54 (s, 1H, NH), 8.68 (s, 1H, NCH), 8.21 (d, J ¼ 5.5 Hz, 1H,
AreH), 8.04 (s, 1H, AreH), 7.76 (s, 1H, AreH), 7.62 (d, J ¼ 9.0 Hz, 1H,
AreH), 7.37 (d, J ¼ 2.6 Hz, 1H, AreH), 7.16 (d, J ¼ 5.5 Hz, 1H, AreH),
3.82e3.76 (m, 4H, OCH2), 3.67 (d, J ¼ 3.9 Hz, 4H, NCH2). 13C NMR
(101 MHz, DMSO) d 163.2(C), 158.4(C), 157.7(C), 155.0(CH), 154.0(C),
140.3(CH), 138.2(C), 138.0(CH), 135.8(CH), 135.3(CH), 133.2(C),
132.8(CH), 124.9(C), 117.9(CH), 111.6(C), 110.2(C), 66.4(2CH2),
5.2. General procedure for the preparation of compounds 4aek
46.3(2CH2). Analytical HPLC on an Agilent (1100) using
a
Compounds 4aek was synthesized from compounds 1aek ac-
cording to the procedures in our previous research [11,13].
250 mm ꢂ 4.6 mm SD-C18 analytical column, H2O/MeOH (15%
H2O) eluent at 1 mL/min flow, monitored by UV absorption at
325 nm, showed 91.9% purity.
5.3. Preparation of 4-(2-hydrazinylthieno[3,2-d]pyrimidin-4-yl)
morpholine (9)
5.4.4. (E)-6-Methyl-3-((2-(4-morpholinothieno[3,2-d]pyrimidin-2-
yl)hydrazono)methyl)-4H-chromen-4-one (10d)
Compound 9 was synthesized from compound 5 according to
the procedures in our previous research [8e10,12].
This compound was obtained as light yellow solid in 80% yield.
m.p. 223e225 ꢀC. ESI-MS m/z:422.3 IR (KBr) cmꢁ1: 3412, 3100,

70
W. Zhu et al. / European Journal of Medicinal Chemistry 93 (2015) 64e73
2956, 2920, 1620, 1563, 1497, 1428, 1377, 1170, 1113, 825, 789. 1H
NMR (400 MHz, DMSO) 10.96 (s,1H, NH), 8.74 (s,1H, NCH), 8.22 (s,
5.4.9. (E)-3-((2-(4-Morpholinothieno[3,2-d]pyrimidin-2-yl)
hydrazono)methyl)-4-oxo-4H-chromene-6-carboxylic acid (10i)
This compound was obtained as yellow solid in 87% yield. m.p.
280e282 ꢀC. ESI-MS m/z:452.1 IR (KBr) cmꢁ1: 3424, 3106, 2962,
2926, 1626, 1545, 1428, 1377, 1161, 1113, 828, 789. 1H NMR
d
1H, AreH), 8.10 (d, J ¼ 6.1 Hz, 1H, AreH), 7.92 (s, 1H, AreH), 7.66 (s,
1H, AreH), 7.24 (d, J ¼ 5.3 Hz, 1H, AreH), 7.06 (d, J ¼ 15.2 Hz, 1H,
AreH), 3.89 (d, J ¼ 14.2 Hz, 4H, OCH2), 3.77 (s, 4H, NCH2), 2.45 (s,
3H, CH3). Analytical HPLC on an Agilent (1100) using
a
(400 MHz, DMSO) d 10.59 (s, 1H, COOH), 10.46 (s, 1H, NH), 8.27 (d,
250 mm ꢂ 4.6 mm SD-C18 analytical column, H2O/MeOH (15%
H2O) eluent at 1 mL/min flow, monitored by UV absorption at
325 nm, showed 96.2% purity.
J ¼ 5.5 Hz, 1H, NCH), 8.11 (s, 1H, AreH), 8.06 (d, J ¼ 5.1 Hz, 1H,
AreH), 7.86 (d, J ¼ 8.3 Hz, 1H, AreH), 7.79 (d, J ¼ 6.2 Hz, 1H, AreH),
7.44 (d, J ¼ 5.6 Hz, 1H, AreH), 7.23 (d, J ¼ 5.3 Hz, 1H, AreH), 3.85 (s,
4H, OCH2), 3.72 (s, 4H, NCH2). 13C NMR (101 MHz, DMSO)
d 167.3(C),
163.1(C), 159.6(C), 158.4(C), 157.6(C), 154.0(CH), 138.2(CH), 135.2(C),
133.6(CH), 132.8(CH), 132.0(CH), 124.9(CH), 124.0(C), 122.0(C),
120.2(CH), 119.1(C), 115.8(C), 66.4(CH2), 66.2(CH2), 46.2(CH2),
5.4.5. (E)-6-Ethyl-3-((2-(4-morpholinothieno[3,2-d]pyrimidin-2-
yl)hydrazono)methyl)-4H-chromen-4-one (10e)
This compound was obtained as yellow solid in 82% yield. m.p.
45.7(CH2).
ESI-HRMS
m/z:
calcd
for
C21H17N5O5S
202e205 ꢀC. ESI-MS m/z:436.1 1H NMR (400 MHz, DMSO)
d 10.62
[MþH]þ:452.0950; found 452.0567. Analytical HPLC on an Agilent
(1100) using a 250 mm ꢂ 4.6 mm SD-C18 analytical column, H2O/
MeOH (39%H2O) eluent at 1 mL/min flow, monitored by UV ab-
sorption at 325 nm, showed 97.4% purity.
(s, 1H, NH), 9.30 (s, 1H, NCH), 8.73 (s, 1H, AreH), 8.23 (s, 1H, AreH),
7.93 (s, 1H, AreH), 7.64 (s, 1H, AreH), 7.48 (d, J ¼ 5.3 Hz, 1H, AreH),
7.09 (s, 1H, AreH), 3.88 (s, 4H, OCH2), 3.77 (s, 4H, NCH2), 2.76 (d,
J ¼ 6.7 Hz, 2H, CH2), 1.23 (dd, J ¼ 14.9, 7.4 Hz, 3H, CH3). Analytical
HPLC on an Agilent (1100) using a 250 mm ꢂ 4.6 mm SD-C18
analytical column, H2O/MeOH (15%H2O) eluent at 1 mL/min flow,
monitored by UV absorption at 325 nm, showed 97.4% purity.
5.4.10. (E)-3-((2-(4-Morpholinothieno[3,2-d]pyrimidin-2-yl)
hydrazono)methyl)-4H-chromen-4-one (10j)
This compound was obtained as light yellow solid in 79% yield.
m.p. 280e282 ꢀC. ESI-MS m/z:430.2 IR (KBr) cmꢁ1: 3424, 3112,
2962, 2914, 1647, 1563, 1545, 1464, 1158, 852, 789. 1H NMR
5.4.6. (E)-6-(Tert-butyl)-3-((2-(4-morpholinothieno[3,2-d]
pyrimidin-2-yl)hydrazono)methyl)-4H-chromen-4-one (10f)
This compound was obtained as light yellow solid in 79% yield.
m.p. 225e227 ꢀC. ESI-MS m/z:464.1 1H NMR (400 MHz, DMSO)
(400 MHz, DMSO)
d 11.12 (s, 1H, NH), 8.79 (s, 1H, NCH), 8.27 (s, 1H,
AreH), 8.16 (d, J ¼ 7.9 Hz, 1H, AreH), 7.89 (d, J ¼ 7.8 Hz, 1H, AreH),
7.76 (d, J ¼ 8.4 Hz, 1H, AreH), 7.58 (t, J ¼ 7.4 Hz, 1H, AreH), 7.51 (d,
J ¼ 6.1 Hz, 1H, AreH), 7.26 (d, J ¼ 5.9 Hz, 1H, AreH), 3.89 (s, 4H,
OCH2), 3.78 (s, 4H, NCH2). Analytical HPLC on an Agilent (1100)
using a 250 mm ꢂ 4.6 mm SD-C18 analytical column, H2O/MeOH
(15%H2O) eluent at 1 mL/min flow, monitored by UV absorption at
325 nm, showed 98.4% purity.
d
10.96 (s, 1H, NH), 8.74 (s, 1H, NCH), 8.22 (s, 1H, AreH), 8.08 (d,
J ¼ 10.3 Hz, 2H, AreH), 7.63 (s, 2H, AreH), 7.26 (s, 1H, AreH), 3.91 (s,
4H, OCH2), 3.77 (s, 4H, NCH2), 1.36 (s, 9H, CH3). Analytical HPLC on
an Agilent (1100) using a 250 mm ꢂ 4.6 mm SD-C18 analytical
column, H2O/MeOH (15%H2O) eluent at 1 mL/min flow, monitored
by UV absorption at 325 nm, showed 94.5% purity.
5.4.11. (E)-6-Isopropyl-3-((2-(4-morpholinothieno[3,2-d]
5.4.7. (E)-6-Hydroxy-3-((2-(4-morpholinothieno[3,2-d]pyrimidin-
2-yl)hydrazono)methyl)-4H-chromen-4-one (10g)
This compound was obtained as yellow solid in 85% yield. m.p.
250e251 ꢀC. ESI-MS m/z:424.1 IR (KBr) cmꢁ1: 3432, 3102, 2976,
1628, 1444, 1370, 1322, 1249, 1160, 1114, 879, 825, 788. 1H NMR
pyrimidin-2-yl)hydrazono)methyl)-4H-chromen-4-one (10k)
This compound was obtained as light yellow solid in 87% yield.
m.p. 163e165 ꢀC. ESI-MS m/z:450.1 IR (KBr) cmꢁ1: 3442, 3076,
2962, 1638, 1542, 1482, 1110, 825, 786, 702. 1H NMR (400 MHz,
DMSO)
d 10.92 (s, 1H, NH), 8.69 (s, 1H, NCH), 8.20 (s, 1H, AreH),
(400 MHz, DMSO)
d 10.89 (s, 1H, NH), 10.09 (s, 1H, OH), 8.66 (s, 1H,
8.07 (d, J ¼ 5.6 Hz, 1H, AreH), 7.93 (s, 1H, AreH), 7.79e7.69 (m, 1H,
AreH), 7.64 (s, 1H, AreH), 7.21 (d, J ¼ 5.4 Hz, 1H, AreH), 3.89 (s,
4H, OCH2), 3.74 (s, 4H, NCH2), 3.07e2.99 (m, 1H, CH), 1.24 (d,
NCH), 8.21 (s, 1H, AreH), 8.08 (d, J ¼ 5.5 Hz, 1H, AreH), 7.58 (d,
J ¼ 9.0 Hz, 1H, AreH), 7.38 (s, 1H, AreH), 7.24 (dd, J ¼ 13.4, 7.3 Hz,
2H, AreH), 3.90 (s, 4H, OCH2), 3.76 (s, 4H, NCH2). Analytical HPLC on
an Agilent (1100) using a 250 mm ꢂ 4.6 mm SD-C18 analytical
column, H2O/MeOH (15%H2O) eluent at 1 mL/min flow, monitored
by UV absorption at 325 nm, showed 100% purity.
J ¼ 7.0 Hz, 6H, CH3). 13C NMR (101 MHz, DMSO)
d 175.1(C),
162.8(C), 158.1(C), 157.2(CH), 154.2(C), 152.7(CH), 146.1(C),
133.4(CH), 133.1(CH), 132.1(CH), 123.6(C), 123.0(CH), 121.6(C),
119.2(CH), 118.6(C), 118.5(C),65.8(2CH2), 45.8(2CH2), 32.9(C),
23.7(2CH3). Analytical HPLC on an Agilent (1100) using
a
5.4.8. (E)-3-((2-(4-Morpholinothieno[3,2-d]pyrimidin-2-yl)
hydrazono)methyl)-6-nitro-4H-chromen-4-one (10h)
This compound was obtained as yellow solid in 83% yield. m.p.
286e289 ꢀC. ESI-MS m/z:451.2 IR (KBr) cmꢁ1: 3418, 3100, 2956,
2920,1626,1545, 1431, 1380, 1116, 828, 789. 1H NMR (400 MHz,
250 mm ꢂ 4.6 mm SD-C18 analytical column, H2O/MeOH (15%
H2O) eluent at 1 mL/min flow, monitored by UV absorption at
325 nm, showed 90.1% purity.
DMSO)
d
10.66 (s, 1H, NH), 8.30 (d, J ¼ 5.4 Hz, 1H, NCH), 8.20e8.14
5.5. Thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (12)
(m, 1H, AreH), 8.10 (d, J ¼ 5.5 Hz, 1H, AreH), 7.88 (s, 1H, AreH), 7.44
(d, J ¼ 5.5 Hz, 1H, AreH), 7.25 (d, J ¼ 5.4 Hz, 1H, AreH), 6.98 (d,
J ¼ 9.0 Hz, 1H, AreH), 3.87 (s, 4H, OCH2), 3.76 (d, J ¼ 4.3 Hz, 4H,
Urea (30 g, 0.5 mol) was added to methyl 2-aminothiophene-3-
carboxylate (11, 10 g, 0.06 mol). Heated to 180 ꢀC and stirred for 2 h.
After cooling to 140 ꢀC, reactant was dissolved with 1 N sodium
hydroxide solution. After filtration, 2 N hydrochloric acid was
added to the filtrate until pH 7. After filtration, the filtrate was
refrigerated for 12 h and white solid appeared in the filtrate. The
product was filtered off, washed with water and dry to afford 6.0 g
thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (12). Yield:56.1%,1H
NCH2). 13C NMR (101 MHz, DMSO)
d 185.5(C), 163.0(C), 162.2(C),
157.7(C), 149.1(CH), 135.8(CH), 135.4(C), 133.4(C), 128.5(CH),
127.1(CH), 126.3(CH), 124.8(C), 124.7(CH), 118.2(CH), 116.2(C),
106.5(C), 66.3(CH2), 66.1(CH2), 46.3(CH2), 45.8(CH2). ESI-HRMS m/
z: calcd for C20H16N6O5S [MþH]þ:453.0903; found 453.0975.
Analytical HPLC on an Agilent (1100) using a 250 mm ꢂ 4.6 mm
SD-C18 analytical column, H2O/MeOH (15%H2O) eluent at 1 mL/min
flow, monitored by UV absorption at 325 nm, showed 91.6% purity.
NMR (400 MHz, DMSO)
d 11.94 (s, 1H,CONH), 11.18 (s, 1H, CONH),
7.11 (d, J ¼ 16.0, 5.6 Hz, 2H,2 AreH) ESI-MS [MꢁH] m/z:167.2.

W. Zhu et al. / European Journal of Medicinal Chemistry 93 (2015) 64e73
71
5.6. 2,4-Dichlorothieno[2,3-d]pyrimidine (13)
Thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione
0.012 mol) and a drop of DMF were added to 20 mL POCl3. Heated to
115 ꢀC for 8 h. After cooling to room temperature, the reactant was
added to 500 g ice-water mixture slowly. The product was filtered
off and dissolved in CH2Cl2. The insoluble was filtered off and the
filtrate was distilled under reduced pressure to afford 1.7 g 2,4-
dichlorothieno[2,3-d]pyrimidine (13). Yield: 56.0%, 1H NMR
134.6(C), 132.5(CH), 130.7(CH), 124.8(CH), 124.6(C), 121.8(CH),
121.6(C), 119.8(CH), 117.8(C), 111.0(C), 66.5(2CH2), 47.0(2CH2).
Analytical HPLC on an Agilent (1100) using a 250 mm ꢂ 4.6 mm
SD-C18 analytical column, H2O/MeOH (15%H2O) eluent at 1 mL/min
flow, monitored by UV absorption at 335 nm, showed 92.4% purity.
(12)
(2.5
g,
5.9.3. (E)-6-Bromo-3-((2-(4-morpholinothieno[2,3-d]pyrimidin-2-
yl)hydrazono)methyl)-4H-chromen-4-one (16c)
This compound was obtained as yellow solid in 72% yield. m.p.
222.8e223.9 ꢀC. ESI-MS m/z:488.0 IR (KBr) cmꢁ1: 3426, 3086, 2960,
1650, 1571, 1536, 1462, 1364, 1195, 1016, 866, 776. 1H NMR
(400 MHz, DMSO)
1H, AreH). ESI-MS m/z:205.1.
d
8.16 (d, J ¼ 3.4 Hz,1H, AreH), 7.62 (d, J ¼ 3.4 Hz,
(400 MHz, DMSO)
d 11.12 (s, 1H, NH), 8.78 (s, 1H, NCH), 8.20 (d,
5.7. 4-(2-Chlorothieno[2,3-d]pyrimidin-4-yl)morpholine (14)
J ¼ 9.4 Hz, 2H, AreH), 8.00 (d, J ¼ 9.9 Hz, 1H, AreH), 7.73 (d,
J ¼ 9.0 Hz, 1H, AreH), 7.48 (s, 1H, AreH), 7.22 (s, 1H, AreH), 3.85 (s,
4H, OCH2), 3.74 (s, 4H, NCH2). Analytical HPLC on an Agilent (1100)
using a 250 mm ꢂ 4.6 mm SD-C18 analytical column, H2O/MeOH
(10% H2O) eluent at 1 mL/min flow, monitored by UV absorption at
335 nm, showed 89.5% purity.
2,4-Dichlorothieno[2,3-d]pyrimidine (13, 2.8 g, 0.014 mol) was
dissolved in 20 mL CH3OH. 2.8 mL morpholine was added slowly at
room temperature. Stirred for 1.5 h. The product was filtered off and
dry to afford 3.1 g 4-(2-chlorothieno[2,3-d]pyrimidin-4-yl)mor-
pholine (14). Yield: 86.1%. 1H NMR (400 MHz, DMSO)
d 7.70 (s, 1H,
AreH), 7.69e7.65 (m, 1H, AreH), 3.96e3.86 (m, 4H, OCH2), 3.75 (d,
J ¼ 4.4 Hz, 4H, NCH2). ESI-MS [MþH] m/z:256.1.
5.9.4. (E)-6-Methyl-3-((2-(4-morpholinothieno[2,3-d]pyrimidin-2-
yl)hydrazono)methyl)-4H-chromen-4-one (16d)
This compound was obtained as light yellow solid in 88% yield.
m.p.231.7~233.2 ꢀC. ESI-MS m/z:422.1 IR (KBr) cmꢁ1: 3424, 3104,
2964,1638,1545,1482,1444,1367,1318,1179,1109, 861, 810, 719. 1H
5.8. 4-(2-Hydrazinylthieno[2,3-d]pyrimidin-4-yl)morpholine (15)
Compound 15 was synthesized from compound 14 according to
NMR (400 MHz, DMSO) d 11.07 (s,1H, NH), 8.73 (s,1H, NCH), 8.23 (s,
the procedures in our previous research [6,7]. 1H NMR (400 MHz,
1H, AreH), 7.92 (s, 1H, AreH), 7.65 (dd, J ¼ 18.4, 8.7 Hz, 2H, AreH),
7.48 (d, J ¼ 6.1 Hz, 1H, AreH), 7.22 (d, J ¼ 6.1 Hz, 1H, AreH), 3.85 (s,
4H, OCH2), 3.74 (s, 4H, NCH2), 2.45 (s, 3H, CH3). 13C NMR (101 MHz,
DMSO)
d
7.73 (s, 1H,NH), 7.38 (d, J ¼ 6.2 Hz, 1H, AreH), 7.07 (d,
J ¼ 6.1 Hz, 1H, AreH), 4.11 (s, 2H,NH2), 3.76 (d, J ¼ 4.9 Hz, 4H, OCH2),
3.71 (d, J ¼ 4.8 Hz, 4H, NCH2).ESI-MS [MþH] (m/z):252.1.
DMSO) d 175.5(C), 171.7(C), 159.1(C), 156.3(CH), 154.5(C), 153.3 (2C),
135.9(CH), 133.1(C), 124.8(CH), 123.5(C), 121.9(CH), 119.6(C),
118.9(CH), 117.7(CH), 111.0(C), 66.5(2CH2), 47.0(2CH2), 20.9(CH3).
Analytical HPLC on an Agilent (1100) using a 250 mm ꢂ 4.6 mm
SD-C18 analytical column, H2O/MeOH (15%H2O) eluent at 1 mL/min
flow, monitored by UV absorption at 335 nm, showed 96.8% purity.
5.9. General procedure for the preparation of compounds 16aej
A mixture of 4-(2-hydrazinylthieno[2,3-d]pyrimidin-4-yl)mor-
pholine 15 (50 mg, 0.20 mmol), substitued 4-oxo-4H-chromene-3-
carbaldehyde 4aej (0.20 mmol) and a drop of glacial acetic acid in
absolute ethanol (5 mL) was refluxed for 4e8 h. The mixture was
cooled, separated by filtration and washed with EtOH to afford the
solids 16aej.
5.9.5. (E)-6-Ethyl-3-((2-(4-morpholinothieno[2,3-d]pyrimidin-2-
yl)hydrazono)methyl)-4H-chromen-4-one (16e)
This compound was obtained as yellow solid in 77% yield. m.p.
215.8e216.8 ꢀC. ESI-MS m/z:435.5 IR (KBr) cmꢁ1: 3428, 3258, 2958,
2854, 1621, 1527, 1480, 1308, 1159, 1114, 862, 822, 782, 720. 1H NMR
5.9.1. (E)-6-Fluoro-3-((2-(4-morpholinothieno[2,3-d]pyrimidin-2-
yl)hydrazono)methyl)-4H-chromen-4-one (16a)
(400 MHz, DMSO)
d 11.08 (s, 1H, NH), 8.74 (s, 1H, NCH), 8.24 (s, 1H,
This compound was obtained as light yellow solid in 83% yield.
m.p. 228.3e229.1 ꢀC. ESI-MS m/z:426.1. IR (KBr) cmꢁ1: 3380,
3288,3018, 2964,1640,1530,1480,1318,1188, 887, 783, 725.1H NMR
AreH), 7.93 (s, 1H, AreH), 7.72 (d, J ¼ 8.6 Hz, 1H, AreH), 7.66 (s, 1H,
AreH), 7.47 (s, 1H, AreH), 7.23 (d, J ¼ 6.3 Hz, 1H, AreH), 3.86 (s, 4H,
OCH2), 3.74 (s, 4H, NCH2), 2.09 (s, 2H, CH2), 1.24 (t, J ¼ 7.5 Hz, 3H,
(400 MHz, DMSO)
d
11.13 (s, 1H, NH), 8.80 (s, 1H, NCH), 8.23 (s, 1H,
CH3). 13C NMR (101 MHz, DMSO)
d 175.0(C), 171.2(C), 158.6(C),
AreH), 7.96e7.66 (m, 3H, AreH), 7.49 (d, J ¼ 6.2 Hz, 1H, AreH), 7.24
157.2(CH), 155.9(CH), 154.2(C), 152.8(C), 141.6(C), 134.4(CH),
132.7(CH), 123.1(CH), 121.4(C), 119.1(C), 118.5(CH), 117.2(CH),
110.5(C), 66.0(2CH2), 46.5(2CH2), 27.5(CH2), 15.3(CH3). Analytical
HPLC on an Agilent (1100) using a 250 mm ꢂ 4.6 mm SD-C18
analytical column, H2O/MeOH (15%H2O) eluent at 1 mL/min flow,
monitored by UV absorption at 335 nm, showed 90.6% purity.
(d, J ¼ 6.2 Hz, 1H, AreH), 3.86 (d, J ¼ 4.4 Hz, 4H, OCH2), 3.76 (d,
J ¼ 4.5 Hz, 4H, NCH2). 13C NMR (101 MHz, DMSO)
d 174.9(C),
171.7(C), 160.7(C), 159.1(C), 156.3(CH), 153.7(CH), 152.7(C), 132.6(C),
123.1(CH), 122.9(CH), 121.9(C), 119.2(CH), 117.7(CH), 111.0(C),
110.3(CH), 110.1(C), 66.5(2CH2), 47.0(2CH2). Analytical HPLC on an
Agilent (1100) using a 250 mm ꢂ 4.6 mm SD-C18 analytical col-
umn, H2O/MeOH(15%H2O) eluent at 1 mL/min flow, monitored by
UV absorption at 335 nm, showed 93.9% purity.
5.9.6. (E)-6-(Tert-butyl)-3-((2-(4-morpholinothieno[2,3-d]
pyrimidin-2-yl)hydrazono)methyl)-4H-chromen-4-one (16f)
This compound was obtained as yellow solid in 79% yield. m.p.
131.2e132.5 ꢀC. ESI-MS m/z:464.2 IR (KBr) cmꢁ1: 3440, 3032, 2960,
1642, 1532, 1484, 1366, 1116, 866, 706. 1H NMR (400 MHz, DMSO)
5.9.2. (E)-6-Chloro-3-((2-(4-morpholinothieno[2,3-d]pyrimidin-2-
yl)hydrazono)methyl)-4H-chromen-4-one (16b)
This compound was obtained as yellow solid in 80% yield.
m.p.207.4~208.2 ꢀC. ESI-MS m/z:442.0 IR (KBr) cmꢁ1: 3416, 3054,
2958, 1628, 1537, 1481, 1391, 1336, 1199, 1020, 865, 783, 720. 1H
d 11.10 (s, 1H, NH), 8.74 (s, 1H, NCH), 8.24 (s, 1H, AreH), 8.04 (s, 1H,
AreH), 7.91 (s, 1H, AreH), 7.65 (d, J ¼ 8.7 Hz1, H, AreH), 7.47 (d,
J ¼ 5.6 Hz, 1H, AreH), 7.22 (d, J ¼ 5.6 Hz, 1H, AreH), 3.84 (s, 4H,
OCH2), 3.73 (s, 4H, NCH2), 1.34 (s, 9H, CH3). 13C NMR (101 MHz,
NMR (400 MHz, DMSO) d 11.09 (s,1H, NH), 8.77 (s,1H, NCH), 8.22 (s,
1H, AreH), 8.05 (s, 1H, AreH), 7.87 (t, J ¼ 10.1 Hz,1H, AreH), 7.80 (d,
J ¼ 9.1 Hz, 1H, AreH), 7.47 (s, 1H, AreH), 7.22 (d, J ¼ 6.2 Hz, 1H,
AreH), 3.85 (s, 4H, OCH2), 3.74 (s, 4H, NCH2). 13C NMR (101 MHz,
DMSO) d 175.6(C), 171.1(C), 159.0(C), 156.1(CH), 154.4(CH), 153.4(C),
148.9(C), 133.4(C), 132.7(CH), 123.1(CH), 121.9(CH), 120.8(C),
119.5(C), 118.8(CH), 117.8(CH), 111.0(C), 66.5(2CH2), 47.0(2CH2),
35.0(C), 31.4(3CH3). Analytical HPLC on an Agilent (1100) using a
DMSO) d 174.5(C), 171.6(C), 159.0(C), 156.2(CH), 154.8(C), 153.7(CH),

72
W. Zhu et al. / European Journal of Medicinal Chemistry 93 (2015) 64e73
250 mm ꢂ 4.6 mm SD-C18 analytical column, H2O/MeOH (15%
H2O) eluent at 1 mL/min flow, monitored by UV absorption at
335 nm, showed 99.3% purity.
(15%H2O) eluent at 1 mL/min flow, monitored by UV absorption at
335 nm, showed 97.6% purity.
5.10. mTOR Kinase Assay [10]
5.9.7. (E)-6-Hydroxy-3-((2-(4-morpholinothieno[2,3-d]pyrimidin-
2-yl)hydrazono)methyl)-4H-chromen-4-one (16g)
The mTOR kinase activity of all the compounds were deter-
mined using LANCE® Ultra time-resolved fluorescence resonance
energy transfer (TR-FRET) assay following the manufacturer's in-
structions, with compounds I, II, III and PI-103 as positive controls.
Briefly, mTOR enzyme (10 nM), ATP (21.6 uM), ULight-4E-BP1
Peptide (100 nM) and test compounds were diluted in kinase buffer
(50 mM HEPES pH 7.5.1 mM EGTA, 3 mM MnCl2, 10 mM MgCl2,
2 mM DTT and 0.01%Tween-20). The reactions were performed in
white 384-well Optiplates (PerkinElmer, MA, USA) at room tem-
perature for 1 h and stopped by adding EDTA to 16 mM. Eu-anti-
phospho-4E-BP1 (Thr37/46) Antibody (PerkinElmer, MA, USA)
was then added to each well to a final concentration of 2 nM. The
intensity of the light emission was measured with an EnVision®
Multilabel Reader (PerkinElmer, MA, USA) in TR-FRET mode (exci-
tation at 320 nm and emission at 665 nm). All of the compounds
were tested two times. The results expressed as IC50 (inhibitory
concentration 50%) were the averages of two determinations.
This compound was obtained as light yellow solid in 86% yield.
m.p. 265.3e266.3 ꢀC. ESI-MS m/z:446.1 IR (KBr) cmꢁ1: 3428, 3096,
2960, 2896, 1628, 1546, 1476, 1366, 1320, 1188, 1114, 1022, 870, 820,
784, 730. 1H NMR (400 MHz, DMSO)
d 11.03 (s, 1H, NH), 10.11 (s, 1H,
OH), 8.68 (s, 1H,NCH), 8.21 (s, 1H, AreH), 7.57 (d, J ¼ 9.5 Hz, 1H,
AreH), 7.46 (d, J ¼ 4.0 Hz, 1H, AreH), 7.36 (s, 1H, AreH), 7.22 (dd,
J ¼ 16.6, 7.7 Hz, 2H, AreH), 3.83 (s, 4H, OCH2), 3.73 (s, 4H, NCH2).
Analytical HPLC on an Agilent (1100) using a 250 mm ꢂ 4.6 mm
SD-C18 analytical column, H2O/MeOH (15% H2O) eluent at 1 mL/
min flow, monitored by UV absorption at 335 nm, showed 100%
purity.
5.9.8. (E)-3-((2-(4-Morpholinothieno[2,3-d]pyrimidin-2-yl)
hydrazono)methyl)-6-nitro-4H-chromen-4-one (16h)
This compound was obtained as yellow solid in 79% yield. m.p.
254.7e255.4 ꢀC. ESI-MS m/z:251.1 IR (KBr) cmꢁ1: 3424, 3104, 2952,
2852, 1628, 1546, 1422, 1336, 1116, 878, 716. 1H NMR (400 MHz,
DMSO)
d
10.68 (s, 1H, NH), 8.20 (d, J ¼ 2.3 Hz, 1H, NCH), 7.88 (s, 1H,
5.11. PI3Ka Kinase Assay [14]
AreH), 7.63 (d, J ¼ 3.4 Hz, 2H, AreH), 7.45 (d, J ¼ 6.1 Hz, 1H, AreH),
7.24e7.15 (m, 1H, AreH), 7.01 (d, J ¼ 9.0 Hz, 1H, AreH), 3.81 (d,
J ¼ 4.2 Hz, 4H, OCH2), 3.73 (d, J ¼ 4.3 Hz, 4H, NCH2). 13C NMR
The selected compounds (10h, 10i, 16h, 16i) are tested for their
activity against PI3K
using a Kinase-Glo® Luminescent Kinase
a
(101 MHz, DMSO)
d
168.1(C), 162.1(C), 161.5(C), 159.0(C), 156.7(CH),
Assay, with compounds I and PI103 as positive controls. The kinase
139.4(CH), 137.9(C), 127.1(C), 126.4(CH), 121.9(CH), 121.5(C),
120.7(C), 120.5(CH), 117.6(CH), 115.7(CH), 66.3(2CH2), 47.0(2CH2).
ESI-HRMS m/z: calcd for C20H16N6O5S [MþH]þ:453.0903; found
reaction is done in 384-well black plate. Each well is loaded with
50
m
L of test items (in 90% DMSO) and 5
g/mL PI substrate ( -phosphatidylinositol; Avanti Polar Lipids;
protein 10 nM is then
added to it. The reaction is started by the addition of 5 L of 1 M ATP
prepared in the reaction buffer and is incubated for 60 min for p110
It is terminated by the addition of 10 L Kinase-Glo buffer. The plates
mL reaction buffer containing
10
m
L-a
453.3333. Analytical HPLC on an Agilent (1100) using
a
prepared in 3% octyl-glucoside) and the PI3Ka
250 mm ꢂ 4.6 mm SD-C18 analytical column, H2O/MeOH (25%
H2O) eluent at 1 mL/min flow, monitored by UV absorption at
335 nm, showed 92.4% purity.
m
m
a
.
m
are then read in a Synergy 2 reader for luminescence detection. All of
the compounds were tested two times.
5.9.9. (E)-3-((2-(4-Morpholinothieno[2,3-d]pyrimidin-2-yl)
hydrazono)methyl)-4-oxo-4H-chromene-6-carboxylic acid (16i)
This compound was obtained as light yellow solid in 85% yield.
m.p. 237.7e238.8 ꢀC. ESI-MS m/z:452.1 IR (KBr) cmꢁ1: 3420, 3100,
2960, 2924, 1698, 1620, 1544, 1422, 1198, 1116, 866, 772. 1H NMR
5.12. Cytotoxicity assay in vitro
The cytotoxic activity of four selected compounds (10hei,16hei)
were evaluated with H460, PC-3 cell lines by the standard MTTassay
in vitro, with compound I as positive control. The cancer cell lines
were cultured in minimum essential medium (MEM) supplement
with 10% fetal bovine serum (FBS). Approximately 4 ꢂ 103 cells,
suspended in MEM medium, were plated onto each well of a 96-well
plate and incubated in 5% CO2 at 37 ꢀC for 24 h. The test compounds
at indicated final concentrations were added to the culture medium
and the cell cultures were continued for 72 h. Fresh MTT was added
(400 MHz, DMSO)
d 12.88 (s, 1H, COOH), 10.70 (s, 1H, NH), 8.78 (s,
1H, NCH), 8.67 (d, J ¼ 11.0 Hz, 1H, AreH), 8.17 (s, 1H, AreH), 7.89 (d,
J ¼ 8.5 Hz, 1H, AreH), 7.50e7.42 (m, 2H, AreH), 7.19 (d, J ¼ 5.6 Hz,
1H, AreH), 3.82 (s, 4H, OCH2), 3.74 (s, 4H, NCH2). 13C NMR
(101 MHz, DMSO)
d 175.3(C), 171.6(C), 166.6(C), 159.0(C), 158.2(C),
155.7(CH), 153.7(CH), 151.9(C), 134.8(CH), 132.5(CH), 132.2(CH),
131.4(C), 127.5(C), 121.9(CH), 120.2(C), 119.8(CH), 117.8(C),
66.51(2CH2), 46.99(2CH2).ESI-HRMS m/z: calcd for C21H17N5O5S
[MþH]þ:452.0950; found 452.1018. Analytical HPLC on an Agilent
(1100) using a 250 mm ꢂ 4.6 mm SD-C18 analytical column, H2O/
MeOH (50%H2O) eluent at 1 mL/min flow, monitored by UV ab-
sorption at 335 nm, showed 95.9% purity.
to each well at a terminal concentration of 5
with cells at 37 ꢀC for 4 h. The formazan crystals were dissolved in
100 L DMSO each well, and the absorbency at 492 nm (for absor-
mg/mL and incubated
m
bance of MTT formazan) and 630 nm (for the reference wavelength)
was measured with the ELISA reader. All of the compounds were
tested three times in each of the cell lines. The results expressed as
inhibition rates or IC50 (half-maximal inhibitory concentration)
were the averages of two determinations and calculated by using
the Bacus Laboratories Incorporated Slide Scanner (Bliss) software.
5.9.10. (E)-3-((2-(4-Morpholinothieno[2,3-d]pyrimidin-2-yl)
hydrazono)methyl)-4H-chromen-4-one (16j)
This compound was obtained as light yellow solid in 78% yield.
m.p.221.2~222.0 ꢀC. ESI-MS m/z:430.1 IR (KBr) cmꢁ1: 3444, 3072,
1718, 1640, 1546, 1474, 1186, 1114, 1024, 864, 774, 726. 1H NMR
(400 MHz, DMSO)
d
11.08 (s, 1H, NH), 8.75 (s, 1H, NCH), 8.23 (s, 1H,
5.13. Docking studies
AreH), 8.12 (d, J ¼ 7.9 Hz, 1H, AreH), 7.84 (t, J ¼ 7.9 Hz, 1H, AreH),
7.71 (d, J ¼ 8.4 Hz, 1H, AreH), 7.53 (t, J ¼ 7.4 Hz, 1H, AreH), 7.47 (d,
J ¼ 6.1 Hz, 1H, AreH), 7.21 (d, J ¼ 5.9 Hz, 1H, AreH), 3.84 (s, 4H,
OCH2), 3.73 (s, 4H, NCH2). Analytical HPLC on an Agilent (1100)
using a 250 mm ꢂ 4.6 mm SD-C18 analytical column, H2O/MeOH
For docking purposes, the three-dimensional structures of the
mTOR (PDB code: 4JT6) and PI3Ka (PDB code: 4L23) were obtained
from RCSB Protein Data Bank [15,16]. Hydrogen atoms were added
to the structure allowing for appropriate ionization at physiological

W. Zhu et al. / European Journal of Medicinal Chemistry 93 (2015) 64e73
143e153.
73
pH and waters were removed. The protonated state of several
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important residues, such as GLY2238, ASP2357, VAL2240 and
TYR2225, were adjusted by using SYBYL6.9.1 (Tripos, St. Louis, USA)
in favor of forming reasonable hydrogen bond with the ligand.
Molecular docking analysis was carried out by the SURFLEX-DOCK
module of SYBYL 6.9.1 package to explore the binding model for the
active site of mTOR with its ligand. All atoms located within the
range of 5.0 Å from any atom of the cofactor were selected into the
active site, and the corresponding amino acid residue was, there-
fore, involved into the active site if only one of its atoms was
selected. Other default parameters were adopted in the SURFLEX-
DOCK calculations. All calculations were performed on Silicon
Graphics workstation.
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Acknowledgments
[8] W. Zhu, X. Zhai, Q. Fu, F. Guo, M. Bai, J. Wang, H. Wang, P. Gong, Chem. Pharm.
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We gratefully acknowledge the generous support provided by
The National Natural Science Funds of China (No. 80140357),
Project supported by the Natural Science Foundation of Jiangxi
Province of China (No. 20142BAB215020), Doctoral Scientific
Research Foundation of Jiangxi Science & Technology Normal
University and Program of Key Laboratory of Drug Design and
Optimization,Jiangxi Science & Technology Normal University
(300098010306).
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Appendix A. Supplementary data
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Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.01.061.
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