Design, synthesis, anticancer activity and docking studies of novel 4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine derivatives as mTOR inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.11.003

A series of 7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine derivatives (7a-q, 10a-q) were designed, synthesized and their chemical structures were confirmed by ¹H NMR, ¹³C NMR, MS and HRMS spectrum. All the compounds were evaluated for t

Full text of DOI:10.1016/j.bmc.2014.11.003

Bioorganic & Medicinal Chemistry 22 (2014) 6746–6754
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, anticancer activity and docking studies of
novel 4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine
derivatives as mTOR inhibitors
Wufu Zhu a,b, , , Chengyu Sun a, , Shan Xu a, Chunjiang Wu a, Jielian Wu a, Mengze Xu a, He Zhao a,
⇑
Le Chen a, Weipeng Zeng a, Pengwu Zheng a,
⇑
a School of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang 330013, China
b Key Laboratory of Original New Drugs Design and Discovery of Ministry of Education, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, 110016, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine derivatives (7a–q, 10a–q) were designed, syn-
thesized and their chemical structures were confirmed by 1H NMR, 13C NMR, MS and HRMS spectrum.
Received 15 August 2014
Revised 2 November 2014
Accepted 3 November 2014
Available online 13 November 2014
All the compounds were evaluated for the inhibitory activity against mTOR kinase at 10
selected compounds (7b, 7e, 7h, 10b and 10e) were further evaluated for the inhibitory activity against
PI3K at 10 M level, and the IC50 values against mTOR kinase and two cancer cell lines. Twelve of the
lM level. Five
a
l
target compounds exhibited moderate antitumor activities. The most promising compound 7e showed
strong antitumor activities against mTOR kinase, H460 and PC-3 cell lines with IC50 values of
Keywords:
7,8-Dihydro-5H-thiopyrano
[4,3-d]pyrimidine
Synthesis
0.80 0.15
l
M, 7.43 1.45
lM and 11.90 0.94
l
M, which were 1.28 to 1.71-fold more active than
BMCL-200908069-1 (1.37 0.07
l
M, 9.52 0.29
l
M, 16.27 0.54 M), respectively. Structure–activity
l
relationships (SARs) and docking studies indicated that the thiopyrano[4,3-d]pyrimidine scaffolds
exerted little effect on antitumor activities of target compounds. Substitutions of aryl group at C-4 posi-
tion had a significant impact on the antitumor activities, and 4-OH substitution produced the best
potency.
Docking
mTOR
Antitumor activity
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
properties, further studies on analogous of BMCL-200908069-1
were carried out in this research. Prompted by potent mTOR inhib-
The PI3K-Akt-mTOR signal pathway plays a key role in cell
proliferation, migration, survival, and angiogenesis. Therefore,
developing mTOR inhibitors is one of the research hotspots in
molecular targeted therapy for the treatment of human cancer.1,2
In recent years, many (fused-)pyrimidine/triazine derivatives
were reported as PI3K-Akt-mTOR signal pathway inhibitors
(Fig. 1).3–6 Compound BMCL-200908069-1 (Fig. 1) is a triazine-
hydrazone derivative which exhibits potent anti-tumor activity
with an mTOR IC50 value (half-maximal inhibitory concentration)
itors reported recent years (Fig. 1),3–6 we speculated that only one
morpholine moiety was necessary, and the other one was fused to
the triazine nucleus according to the theory of scaffold hopping.
Meanwhile, we replaced the oxygen atom with sulfur atom and
introduced different substituents to the aryl moiety, resulting in
compounds 7a–q. Further investigations were carried out in details
to study the effect of oxidation of sulfur atom on the antitumor
activity. Therefore, sulfur was oxidized to sulfone to afford com-
pounds 10a–q. The design strategy for all target compounds is
shown in Figure 2.
of 0.27 l
M.6 Structure–activity relationships (SARs) showed that
aryl hydrazones and morpholine-triazine moieties were essential
for the mTOR activity. However, whether both of the two morpho-
line moieties were necessary remains indeterminate.
Herein we disclosed the design, synthesis and antitumor activ-
ity against H460, PC-3 cancer cell lines, PI3K
a kinase and mTOR
kinase of novel thiopyrano[4,3-d]pyrimidine derivatives. More-
In order to screen compounds which possess excellent in vitro/
in vivo anti-tumor activity as well as improved pharmacokinetic
over, docking studies were presented in this paper as well.
2. Chemistry
⇑
Corresponding authors. Tel./fax: +86 791 83802393.
E-mail addresses: zhuwufu-1122@163.com (W. Zhu), zhengpw@126.com
(P. Zheng).
The preparation of target compounds 7a–q and 10a–q was
described in Scheme 1.
These authors contribute equally to this work.
http://dx.doi.org/10.1016/j.bmc.2014.11.003
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

W. Zhu et al. / Bioorg. Med. Chem. 22 (2014) 6746–6754
6747
Figure 1. Structures of some reported (fused-)pyrimidine/triazines.
Figure 2. Structures and design strategy for target compounds 7a–q and 10a–q.
Compounds 7a–q were synthesized from commercially avail-
able methyl dimethyl 3,30-thiodipropionate through six steps.
Intramolecular cyclization reaction of dimethyl 3,30-thiodipropio-
nate in the present of NaH (60%) afforded compound 1 as a yellow
viscous oil. Subsequently, compound 1 was condensed with urea at
reflux for 24 h to give thiopyrano[4,3-d]pyrimidine-2,4-dione
(compound 2). Chlorination of 2 with POCl3 and DMF (cat.) for
3 h at 115 °C achieved 3 as yellow solid. Regioselective nucleo-
philic displacement of the 4-chloride with morpholine, followed
by hydrazinolysis gave access to the key intermediate 5. Finally,
5 condensed with substituted aromatic aldehydes 6a–q to furnish
target compounds 7a–q, respectively.
Luminescent Kinase assay, and the IC50 values against mTOR kinase
and two cancer cell lines H460 (human lung cancer) and PC-3
(human prostate cancer). The results expressed as inhibition rates
or IC50 values were 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, twelve of the synthesized com-
pounds showed moderate to excellent inhibitory activity on mTOR
kinase. Five of them (7b, 7e, 7h, 10b, 10e) showed excellent inhib-
itory activity on mTOR kinase with the inhibition rates from
64.1 2.8% to 89.5 1.8% (IC50 values from 6.93 0.54
0.80 0.03 M), especially compound 7e was 1.71-fold more
active than lead compound BMCL-200908069-1.
lM to
l
On the other hand, compound 4 treated with sodium tungstate
dihydrate and 30% hydrogen peroxide to furnish oxidate 8 which
was then used in a substitution reaction with NH2NH2ꢀH2O (80%)
to generate another key intermediate 9. Similarly, intermediate 9
condensed with the corresponding aromatic aldehydes 6a–q to
afford target compounds 10a–q, respectively.
The relative stereochemistry of the target compounds (7a–q,
10a–q) was easily confirmed as E isomeric form according to the
method in our previous researches.7–9
The results suggested that replacement of the triazine scaffold
with thiopyrano[4,3-d]pyrimidine scaffold enhanced the anti-
tumor activity. It also told us that not both the two morpholine
moieties were necessary and the application of scaffold hopping
was feasible. The oxidation of sulfur atom had little effect on the
antitumor activity, as we could not find significant improved in
antitumor activity of compounds 10a–q.
The inhibitory activities on mTOR kinase of compounds 7a–q
were increased in varying degrees because of the different substit-
uents of aryl moiety. It was noticeable that hydroxyl group at C-4
position significantly increased the activity, such as compounds 7e
and 10e with the antitumor activity equal to the positive control
BMCL-200908069-1. This dramatic boost in the mTOR potency by
the hydroxyl group could be ascribed to the increased hydrogen
bonds between the 4-OH and receptors necessary for activity.
The results showed that variations in substitution at 4-position
of the aryl moiety had a marked impact on the activity. For com-
pounds 7a–q and 10a–q, 4-OH substitution of the aryl moiety
was more preferred.
3. Results and discussion
3.1. Biological evaluation
The target compounds (7a–q and 10a–q) were evaluated for
their inhibitory activity against mTOR kinase at 10 lM level
in vitro together with reference compounds BMCL200908069-1
and PI103 by LANCEÒ Ultra time-resolved fluorescence resonance
energy transfer (TR-FRET) assay. In addition, the five selected com-
pounds (7b, 7e, 7h, 10b and 10e) were further evaluated for the
The antitumor activities of the five selected compounds (7b, 7e,
7h, 10b and 10e) as well as the two lead compounds against PI3Ka,
inhibitory activity against PI3Ka at 10 l
M level using Kinase-GloÒ

6748
W. Zhu et al. / Bioorg. Med. Chem. 22 (2014) 6746–6754
O
N
O
O
O
Cl
COOMe
COOMe
c
b
a
d
S
S
O
S
NH
S
N
S
N
N
H
O
N
3
Cl
N
4
Cl
2
1
O
N
O
N
CHO
R
e
6a-6q
S
N
R
S
N
N
f
N
N
H
N
NHNH2
7a-7q
5
O
N
O
O
N
N
N
O
S
6a-6q
h
O
O
g
O
O
N
S
S
N
R
f
O
N
N
N
H
N
NHNH2
N
Cl
8
9
10a-10q
6a,7a,10a: R=3,4-Methylenedioxy
6j,7j,10j:
6k,7k,10k: R=3,5-dimethoxy
R=2,5-dimethoxy
6b,7b,10b: R=3,5-dibromo-4-hydroxy
6c,7c,10c: R=4-chloro-2-fluoro
6d,7d,10d: R=2,3-dichloro
6l,7l,10l:
R=3,4,5-trimethoxy
6m,7m,10m: R=2,3,4-trimethoxy
6n,7n,10n: R=4-methoxy
6o,7o,10o: R=2,4-dimethoxy
6p,7p,10p: R=2-nitro
6e,7e,10e: R=4-hydroxy-3,5-dimethoxy
6f,7f,10f: R=4-cyano
6g,7g,10g:
6h,7h,10h:
6i,7i,10i:
R=3,5-di-tert-butyl-2-hydroxy
R=3-bromo-4-hydroxy
R=2,4-dihydroxy
6q,7q,10q: R=4-nitro
Scheme 1. Synthetic routes of target compounds 7a–q and 10a–q. Reagents and conditions: (a) NaH, THF, rt, 3 h; (b) 3 equiv urea, C2H5ONa, EtOH, reflux, 24 h; (c) POCl3, DMF
(cat.), reflux, 3 h; (d) 2.4 equiv morpholine, MeOH, rt, 1.5 h; (e) 80% NH2NH2ꢀH2O, 85 °C, 1 h; (f) EtOH, reflux, 1–3 h; (g) Na2WO4ꢀ2H2O, 30% H2O2, 20 °C, 3 h; (h) 80%
NH2NH2ꢀH2O, EtOH, 75 °C, 1 h.
Table 1
Structures and of compounds 7a–q, 10a–q
O
O
N
N
N
O
S
O
N
R
S
N
R
N
N
N
N
H
N
H
10a-10q
7a-7q
Compounds No.
R
mTORa (10
l
M inhibitory %)
Compounds No.
R
mTORa (10
lM inhibitory %)
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
3,4-Methylenedioxy
NAc
78.2 0.6
NA
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
PI-103b
3,4-Methylenedioxy
3,5-Dibromo-4-hydroxy
4-Chloro-2-fluoro
2,3-Dichloro
4-Hydroxy-3,5-dimethoxy
4-Cyano
3,5-Di-tert-butyl-2-hydroxy
3-Bromo-4-hydroxy
2,4-Dihydroxy
2,5-Dimethoxy
3,5-Dimethoxy
3,4,5-Trimethoxy
2,3,4-Trimethoxy
4-Methoxy
44.8 0.3
64.1 4.4
NA
3,5-Dibromo-4-hydroxy
4-Chloro-2-fluoro
2,3-dichloro
4-Hydroxy-3,5-dimethoxy
4-Cyano
3,5-Di-tert-butyl-2-hydroxy
3-Bromo-4-hydroxy
2,4-Dihydroxy
2,5-Dimethoxy
3,5-Dimethoxy
3,4,5-Trimethoxy
2,3,4-Trimethoxy
4-Methoxy
NA
NA
89.5 1.8
61.0 4.5
NA
76.6 2.2
44.2 6.9
NA
56.9 5.8
44.6 7.2
10.9 4.3
33.6 4.7
13.2 4.9
NA
20.3 7.1
44.6 3.2
NA
13.6 6.4
98.6 0.5
70.4 2.8
54.0 4.0
29.3 3.2
39.7 3.3
11.8 15.3
10.3 1.8
12.5 2.4
43.7 2.9
NA
2,4-Dimethoxy
2-Nitro
4-Nitro
2,4-Dimethoxy
2-Nitro
4-Nitro
7q
NA
76.1 0.3
BMCL-200908069-1b
—
—
a
b
c
The values are an average of two separate determinations.
Used as a positive control.
NA: not active (inhibitory rate <10%).
mTOR kinase and two cancer cell lines were shown in Table 2.
Obviously, the activities of these five compounds were equal to
BMCL-200908069-1, or even better, which further confirmed their
strong antitumor activities. However, all these compounds showed
bad inhibitory activity against PI3K . The most promising com-
pound 7e showed strong antitumor activities against mTOR kinase,
a

W. Zhu et al. / Bioorg. Med. Chem. 22 (2014) 6746–6754
6749
Table 2
PI3K /mTOR kinase activity and cytotoxicity of selected compounds and positive controls
a
a
Compounds No.
IC50 (lM)
mTOR
PI3K
a
(10
l
M inhibitory %)
H460
PC-3
>20
11.90 0.94
14.4 1.69
>20
7b
7e
7h
10b
2.67 1.32
0.80 0.15
1.94 1.15
6.93 0.63
2.20 0.77
1.37 0.07
0.019 0.004
21.6 7.0
12.0 5.5
18.8 1.9
24.9 7.6
6.2 3.5
—
NDc
7.43 1.45
10.75 3.19
NDc
10e
NDc
>20
BMCL-200908069-1b
PI-103b
9.52 0.29
16.27 0.54
0.011 0.002 (IC50
)
NDc
NDc
a
b
c
The values are an average of two separate determinations.
Used as a positive control.
Not determinated.
Figure 3. Binding model of compound 7e target into active site of mTOR and hydrogen bonds were showed in dashed lines (yellow). Compounds (3a–3d): binding model of
compound 7e (shown in Capped Sticks), BMCL-200908069-1 (shown in Ball and Stick) and native ligand PI103 (shown in yellow) with mTOR, repectively.
H460 and PC-3 cell lines with IC50 values of 0.80 0.15
l
M,
moiety had a significant impact on the activity, and 4-OH substitu-
tion was the most preferred.
7.43 1.45
more active
9.52 0.29
l
M and 11.90 0.94
l
M, which were 1.28 to 1.71-fold
than
BMCL-200908069-1
(1.37 0.07 l
M,
lM, 16.27 0.54
l
M), respectively.
3.2. Molecular docking study
In view of above, thiopyrano[4,3-d]pyrimidine scaffolds exerted
little effect on the antitumor activities of these thiopyrano[4,3-
d]pyrimidine derivatives. Variations in substitutions of the aryl
To explore the binding modes of target compounds with the
active site of mTOR, molecular docking simulation studies were

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W. Zhu et al. / Bioorg. Med. Chem. 22 (2014) 6746–6754
carried out by using SURFLEX-DOCK module of SYBYL package ver-
sion. Based on the in vitro inhibition results, we selected com-
pound 7e, our best mTOR inhibitor in this study, as ligand
example, and the structure of mTOR was selected as the docking
model (PDB ID code: 4JT610).
(31.8 g, 0.154 mol) in anhydrous THF (40 mL) was added via a
dropping funnel over 1 h (the dropping funnel was rinsed with
15 mL of THF) at room temperature under N2. After stirring for
2 h at rt, the reaction was completed by TLC analysis (20% EtOAc
in light petroleum (60–90 °C). The mixture was transferred to a
beaker, dilute hydrochloric acid was added slowly with stirring
maintaining the temperature below 20 °C; the final pH was 6–7.
The organic layer was separated, and the aqueous layer was
extracted with CH2Cl2 (3 ꢁ 50 mL). The combined organic layers
were washed with brine (3 ꢁ 50 mL), then dried over Na2SO4, the
filtrate was concentrated under reduced pressure to afford 1 as a
yellow viscous oil (26.8 g, 99.8%) and was used for next step with-
out further purification. ESI-MS m/z: [M+H]+ 175.2.
The binding modes of compounds 7e and lead compounds were
shown in Figure 3a–d. As depicted in Figure 3a–d, compound 7e
and BMCL-200908069-1 can nearly overlap in the binding model
and morpholine group, methoxy group, hydroxyl group formed
three hydrogen bonds with residues VAL2240, TYR2225,
ASP2195, respectively. The H-bond distances and donor-H-accep-
tor angles are 2.107 Å, 144.60° and 2.146 Å, 173.61° and 2.362 Å,
82.82°, respectively. The results showed that the quality of these
three H-bonds is well. Analysis of compound 7e’s binding mode
in the active binding site demonstrated that the docking mode of
the 7e is similar to the lead compound BMCL-200908069-1 and
native ligand PI-103 with the same H-bond between morpholine
group and residues VAL2240. The three hydrogen bonds really play
an important role in increasing the inhibitory potency of thiopyr-
ano[4,3-d]pyrimidine derivatives against mTOR kinase according
to the docking results and the activity. Furthermore, the docking
results also give us a new direction to design new mTOR inhibitors
that can interact with VAL2240, TYR2225 and ASP2195. The above-
mentioned results of SAR analysis and molecular docking study
may allow the rational design of more potent mTOR inhibitors.
5.3. Preparation of 7,8-dihydro-1H-thiopyrano[4,3-d]pyrimidine-
2,4(3H,5H)-dione (2)
Finely cut sodium metal (16.2 g, 0.704 mol) was added into
300 mL anhydrous ethanol at ice bath with stirring. After the
sodium metal was completely consumed, urea (32.7 g, 0.545 mol)
and methyl 4-oxotetrahydro-2H-thiopyran-3-carboxylate
1
(31.3 g, 0.180 mol) was added to the solution. The mixture was
heated and refluxed for 24 h, and the reaction was monitored by
TLC. The reaction mixture was concentrated under reduced pres-
sure and the residue was poured into ice water, then adjusted
the pH to 6–7 with acetic acid under ice bath, filtration, the filter
cake was washed with ice-water, dried to obtain 2 as white pow-
dery solid (13.6 g, 41.2%). Mp >300 °C. ESI-MS m/z: [MꢂH]ꢂ
183.2, 1H NMR (400 MHz, DMSO) d 11.10 (s, 1H), 10.75 (s, 1H),
3.39 (s, 2H), 2.80 (t, J = 5.8 Hz, 2H), 2.57 (t, J = 5.6 Hz, 2H).
4. Conclusions
In summary, a series of thiopyrano[4,3-d]pyrimidine derivatives
were designed, synthesized and evaluated for antitumor activity
against mTOR kinase and two cancer cell lines in vitro. The phar-
macological results indicated that twelve of the synthesized com-
pounds displayed moderate antitumor activity and five selected
compounds showed equal to more potency than lead compound
BMCL-200908069-1. The most promising compound 7e exhibited
strong antitumor activities against mTOR kinase, H460 and PC-3
cell lines, which were 1.28 to 1.71-fold more active than BMCL-
200908069-1. The initial SARs and docking studies showed that
the thiopyrano[4,3-d]pyrimidine scaffold had little effect on the
antitumor activities, while variations in substitutions of the aryl
moieties had a significant impact on the activities and 4-OH substi-
tution produced the best potency. Further studies will be carried
out in near future.
5.4. Preparation of 2,4-dichloro-7,8-dihydro-5H-thiopyrano-
[4,3-d]pyrimidine (3)
A
mixture of 7,8-dihydro-1H-thiopyrano[4,3-d]pyrimidine-
2,4(3H,5H)-dione 2 (3.03 g, 0.016 mol), POCl3 (20 mL) was heated
at reflux for 3 h and the reaction was monitored by TLC. The reac-
tion mixture was slowly added to ice/water with vigorous stirring
yielding a precipitate. The mixture was then filtered to yield 2,4-
dichloro-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine 3 as yellow
solid (3.12 g, 85.7%). Mp 87.1–87.9 °C. 1H NMR (400 MHz, CDCl3)
d 3.81 (s, 2H), 3.23 (t, J = 5.9 Hz, 2H), 2.96 (t, J = 6.0 Hz, 2H).
5.5. Preparation of 4-(2-chloro-7,8-dihydro-5H-thiopyrano-
[4,3-d]pyrimidin-4-yl)morpholine (4)
5. Experimental
5.1. Chemistry
To the mixture of 2,4-dichloro-7,8-dihydro-5H-thiopyrano[4,3-
d]pyrimidine 3 (7.40 g, 0.033 mol) and MeOH (150 mL), morpho-
line (7 mL, 0.08 mol) was added drop-wise at room temperature.
The reaction mixture then was stirred at room temperature for
1.5 h. After completion of reaction as indicated by TLC, the mixture
was then filtered, washed with water and MeOH, to yield the title
compound as white solid (6.84 g, 75.3%). Mp 161.1–162.4 °C. ESI-
MS m/z: [M+H]+ 272.1. 1H NMR (400 MHz, CDCl3) d 3.81 (s, 2H),
3.23 (t, J = 5.9 Hz, 2H), 2.96 (t, J = 6.0 Hz, 2H).
All melting points were obtained on a Büchi Melting Point B-
540 apparatus (Büchi Labortechnik, Flawil, Switzerland) and were
uncorrected. 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 LC–MS (Agilent, Palo Alto, CA, USA). All the mate-
rials were obtained from commercial suppliers and used without
purification, 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 other-
wise specified. Yields were not optimized.
5.6. Preparation of 4-(2-hydrazinyl-7,8-dihydro-5H-thiopyrano-
[4,3-d]pyrimidin-4-yl)morpholine (5)
A solution of 80% hydrazine hydrate (NH2NH2ꢀH2O, 110 mL) and
compound 4 (3.47 g, 0.013 mol) was stirred at 85 °C for 1 h. The
precipitate was collected by filtration and washed with water,
and dried to give 5 as white solid (1.73 g, 50.7%). Mp 172.1–
173.2 °C. ESI-MS m/z: [M+H]+ 268.2. 1H NMR (400 MHz, DMSO) d
7.66 (s, 1H), 4.07 (s, 2H), 3.73–3.66 (m, 4H), 3.54 (s, 2H), 3.18 (s,
4H), 2.89 (t, J = 6.1 Hz, 2H), 2.82 (t, J = 6.0 Hz, 2H).
5.2. Preparation of methyl 4-oxotetrahydro-2H-thiopyran-3-
carboxylate (1)
To the mixture of NaH (60%, 8.19 g, 0.205 mol) and anhydrous
THF (100 mL),
a
solution of dimethyl 3,30-thiodipropionate

W. Zhu et al. / Bioorg. Med. Chem. 22 (2014) 6746–6754
6751
5.7. Preparation of 2-chloro-4-morpholino-7,8-dihydro-5H-
5.9.4. (E)-4-(2-(2-(2,3-Dichlorobenzylidene)hydrazinyl)-7,8-
thiopyrano[4,3-d]pyrimidine 6,6-dioxide (8)
dihydro-5H-thiopyrano[4,3-d]pyrimidin-4-yl)morpholine (7d)
This compound was obtained as yellow solid in 82% yield. Mp
239.3–241.3 °C. ESI-MS m/z: [M+H]+ 424.1. 1H NMR (400 MHz,
DMSO) d 11.28 (s, 1H), 8.51 (s, 1H), 7.94 (d, J = 7.7 Hz, 1H), 7.61
(d, J = 7.6 Hz, 1H), 7.41 (t, J = 7.8 Hz, 1H), 3.73 (s, 4H), 3.61 (s,
2H), 3.28 (s, 4H), 2.93 (s, 4H). 13C NMR (126 MHz, DMSO) d
165.85, 165.55, 157.26, 136.15, 135.69, 132.67, 130.44, 130.12,
128.71, 125.25, 110.35, 66.47(2C), 49.37(2C), 33.87, 26.23(2C).
Compound 4 (4.03 g, 0.015 mol) was first dissolved in metha-
nol (60 mL), sodium tungstate dihydrate (0.447 g, 1.36 mmol)
and 30% hydrogen peroxide (5.0 mL, 0.048 mol) were succes-
sively dropped into the solution and stirred at 20 °C for 3 h.
Then, the reaction mixture was filtered, washed with acetone,
dried and gave compound
8 as pale yellow solid (3.92 g,
87.0%). Mp 145.7–146.8 °C. ESI-MS m/z: [M+Na]+ 326.1, [MꢂH]ꢂ
302.1. 1H NMR (400 MHz, DMSO) d 4.38 (s, 2H), 3.77–3.69 (m,
4H), 3.56 (t, J = 6.6 Hz, 2H), 3.45–3.39 (m, 4H), 3.31 (t,
J = 6.7 Hz, 2H).
5.9.5. (E)-2,6-Dimethoxy-4-((2-(4-morpholino-7,8-dihydro-5H-
thiopyrano[4,3-d]pyrimidin-2-yl)hydrazono)methyl)phenol (7e)
This compound was obtained as yellow solid in 75% yield. Mp
141.6–146.2 °C. ESI-MS m/z: [M+H]+ 432.2. 1H NMR (400 MHz,
DMSO) d 10.74 (s, 1H), 8.67 (s, 1H), 7.95 (s, 1H), 6.89 (s, 2H),
3.80 (s, 6H), 3.71 (s, 4H), 3.59 (s, 2H), 3.44–3.30 (m, 4H), 2.91 (s,
4H). 13C NMR (126 MHz, DMSO) d 166.83, 166.83, 162.40, 160.03,
159.62, 157.87, 144.03, 131.41, 111.58, 107.70, 103.28, 66.35(2C),
49.34, 49.15(2C), 46.95, 32.34. ESI-HRMS m/z: calcd for C20H25N5-
O4S [M+H]+: 432.1706; found 432.1686.
5.8. Preparation of 2-hydrazinyl-4-morpholino-7,8-dihydro-5H-
thiopyrano[4,3-d]pyrimidine 6,6-dioxide (9)
The synthesis of compound 9 was similar to the compound 5. A
mixture of 80% hydrazine hydrate (NH2NH2ꢀH2O, 70 mL), C2H5OH
(70 mL) and compound 8 (3.40 g, 0.011 mol) was stirred at 75 °C
for 1 h. The precipitate was collected by filtration and washed with
water, and dried to give 9 as white solid (2.54 g, 75.7%). Mp 230.2–
231.9 °C. ESI-MS m/z: [M+H]+ 300.1. 1H NMR (400 MHz, DMSO) d
7.95 (s, 1H), 4.13 (s, 2H), 4.10 (s, 2H), 3.70 (s, 4H), 3.43 (t,
J = 6.4 Hz, 2H), 3.17 (s, 4H), 3.11 (t, J = 6.7 Hz, 2H).
5.9.6. (E)-4-((2-(4-Morpholino-7,8-dihydro-5H-thiopyrano[4,3-
d]pyrimidin-2-yl)hydrazono)methyl)benzonitrile (7f)
This compound was obtained as yellow solid in 84% yield. Mp
281.6–283.2 °C. ESI-MS m/z: [M+H]+ 381.1. 1H NMR (400 MHz,
DMSO) d 11.23 (s, 1H), 8.10 (s, 1H), 7.82 (dd, J = 24.4, 8.1 Hz, 4H),
3.77 (d, J = 32.9 Hz, 4H), 3.61 (s, 2H), 3.28 (s, 4H), 2.93 (s, 4H).
13C NMR (126 MHz, DMSO) d 165.84, 165.57, 157.26, 140.56,
138.44(2C), 133.10(2C), 127.03, 119.45, 110.68, 110.35,
66.48(2C), 49.37(2C), 33.86, 26.23(2C).
5.9. General procedure for the preparation of compounds 7a–7q
and 10a–10q
A mixture of 4-(2-hydrazinyl-7,8-dihydro-5H-thiopyrano[4,3-
d]pyrimidin-4-yl)morpholine 5 (53 mg, 0.20 mmol) or 2-hydrazi-
nyl-4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine
6,6-dioxide 9 (60 mg, 0.20 mmol), substituted aryl aldehydes 6a–q
(0.20 mmol) and one drop of glacial acetic acid in absolute ethanol
(10 mL) was refluxed for 1–3 h. The mixture was cooled, separated
by filtration and washed with EtOH to afford the solids 7a–q and
10a–q.
5.9.7. (E)-2,6-Di-tert-butyl-4-((2-(4-morpholino-7,8-dihydro-5H-
thiopyrano[4,3-d]pyrimidin-2-yl)hydrazono)methyl)phenol (7g)
This compound was obtained as pale yellow solid in 83% yield.
Mp 147.5–150.3 °C. ESI-MS m/z: [M+H]+ 484.2. 1H NMR (400 MHz,
DMSO) d 10.68 (s, 1H), 7.96 (s, 1H), 7.41 (s, 2H), 7.20 (s, 1H), 3.71 (s,
4H), 3.57 (d, J = 13.3 Hz, 2H), 3.29–3.22 (m, 4H), 2.90 (d, J = 6.2 Hz,
4H), 1.39 (d, J = 10.7 Hz, 18H).
5.9.1. (E)-4-(2-(2-(Benzo[d][1,3]dioxol-5-ylmethylene)hydrazi-
nyl)-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4-yl)morpholine
(7a)
This compound was obtained as white solid in 82% yield. Mp
243.6–244.8 °C. ESI-MS m/z: [M+H]+ 400.2. 1H NMR (400 MHz,
DMSO) d 10.67 (s, 1H), 8.00 (s, 1H), 7.21 (s, 1H), 7.03 (d,
J = 7.7 Hz, 1H), 6.93 (d, J = 8.1 Hz, 1H), 6.05 (s, 2H), 3.72 (s, 4H),
3.59 (s, 2H), 3.27–3.22 (m, 4H), 2.91 (s, 4H).
5.9.8. (E)-2-Bromo-4-((2-(4-morpholino-7,8-dihydro-5H-thio-
pyrano[4,3-d]pyrimidin-2-yl)hydrazono)methyl)phenol (7h)
This compound was obtained as pale yellow solid in 88% yield.
Mp 292.6–294.6 °C. ESI-MS m/z: [M+H]+ 452.1. 1H NMR (400 MHz,
DMSO) d 10.76 (s, 1H), 10.58 (s, 1H), 7.94 (d, J = 10.9 Hz, 1H), 7.74
(s, 1H), 7.44 (d, J = 8.4 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 3.72 (s, 4H),
3.59 (s, 2H), 3.25 (s, 4H), 2.92 (s, 4H). 13C NMR (126 MHz, DMSO) d
165.69, 165.56, 157.55, 155.07, 139.57, 130.74, 128.76, 127.37,
116.98, 110.23, 109.53, 66.48(2C), 49.41(2C), 33.85, 26.27, 26.14.
ESI-HRMS m/z: calcd for C18H20BrN5O2S [M+H]+: 450.0599; found
450.0580,452.0588.
5.9.2. (E)-2,6-Dibromo-4-((2-(4-morpholino-7,8-dihydro-5H-
thiopyrano[4,3-d]pyrimidin-2-yl)hydrazono)methyl)phenol
(7b)
This compound was obtained as yellow solid in 85% yield. Mp
166.8–171.5 °C. ESI-MS m/z: [M+H]+ 529.9. 1H NMR (400 MHz,
DMSO) d 10.91 (s, 1H), 7.97 (s, 1H), 7.80 (s, 2H), 4.35 (s, 1H),
3.74 (s, 4H), 3.62 (s, 2H), 3.29 (s, 4H), 2.95 (s, 4H). 13C NMR
5.9.9. (E)-4-((2-(4-Morpholino-7,8-dihydro-5H-thiopyrano[4,3-
d]pyrimidin-2-yl)hydrazono)methyl)benzene-1,3-diol (7i)
This compound was obtained as yellow solid in 78% yield. Mp
252.5–253.7 °C. ESI-MS m/z: [M+H]+ 388.2. 1H NMR (400 MHz,
DMSO) d 12.14 (s, 1H), 11.04 (s, 1H), 9.76 (s, 1H), 8.08 (s, 1H),
7.10 (d, J = 8.3 Hz, 1H), 6.30 (d, J = 8.4 Hz, 1H), 6.28 (s, 1H), 3.72
(s, 4H), 3.59 (s, 2H), 3.28 (s, 4H), 2.90 (dd, J = 14.9, 5.1 Hz, 4H).
13C NMR (126 MHz, DMSO) d 165.78, 165.54, 159.81, 159.55,
157.07, 143.16, 131.21, 111.72, 109.40, 107.59, 103.28, 66.48(2C),
49.30(2C), 33.85, 26.21, 26.17.
(126 MHz, DMSO)
d
165.51, 138.00, 137.86, 130.10(2C),
112.81(2C), 109.84, 66.46(2C), 49.38(2C), 33.83, 26.23, 26.14. ESI-
HRMS m/z: calcd for C18H19Br2N5O2S [M+H]+: 527.9704; found
527.9689, 529.9667, 531.9645.
5.9.3. (E)-4-(2-(2-(2-Chloro-4-fluorobenzylidene)hydrazinyl)-7,
8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4-yl)morpholine (7c)
This compound was obtained as yellow solid in 83% yield. Mp
234.1–236.9 °C. ESI-MS m/z: [M+H]+ 408.1. 1H NMR (400 MHz,
DMSO) d 11.13 (s, 1H), 8.44 (s, 1H), 7.98 (d, J = 7.5 Hz, 1H), 7.47
(d, J = 9.3 Hz, 1H), 7.30 (s, 1H), 3.72 (s, 4H), 3.60 (s, 2H), 3.27 (s,
4H), 2.92 (s, 4H).
5.9.10. (E)-4-(2-(2-(2,5-Dimethoxybenzylidene)hydrazinyl)-7,8-
dihydro-5H-thiopyrano[4,3-d]pyrimidin-4-yl)morpholine (7j)
This compound was obtained as yellow solid in 86% yield. Mp
133.5–135.9 °C. ESI-MS m/z: [M+H]+ 416.2. 1H NMR (400 MHz,

6752
W. Zhu et al. / Bioorg. Med. Chem. 22 (2014) 6746–6754
DMSO) d 10.93 (s, 1H), 8.37 (s, 1H), 7.37 (s, 1H), 6.99 (d, J = 8.9 Hz,
1H), 6.90 (d, J = 8.6 Hz, 1H), 3.81 (d, J = 24.8 Hz, 4H), 3.73 (d,
J = 6.6 Hz, 6H), 3.59 (s, 2H), 3.27 (s, 4H), 2.92 (s, 4H).
1H), 7.87 (d, J = 8.8 Hz, 2H), 3.74 (d, J = 3.7 Hz, 4H), 3.62 (s, 2H),
3.30 (d, J = 4.0 Hz, 4H), 2.94 (s, 4H).
5.9.18. (E)-2-(2-(Benzo[d][1,3]dioxol-5-ylmethylene)hydrazinyl)-
4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine
6,6-dioxide (10a)
This compound was obtained as pale yellow solid in 79% yield.
Mp 285.3–286.0 °C. ESI-MS m/z: [M+H]+ 432.1. 1H NMR (400 MHz,
DMSO) d 10.96 (s, 1H), 8.02 (s, 1H), 7.23 (s, 1H), 7.06 (d, J = 8.1 Hz,
1H), 6.95 (d, J = 8.0 Hz, 1H), 6.07 (s, 2H), 4.20 (s, 2H), 3.73 (d,
J = 4.4 Hz, 4H), 3.48 (s, 2H), 3.24 (s, 4H), 3.20 (s, 2H). 13C NMR
5.9.11. (E)-4-(2-(2-(3,5-Dimethoxybenzylidene)hydrazinyl)-7,8-
dihydro-5H-thiopyrano[4,3-d]pyrimidin-4-yl)morpholine (7k)
This compound was obtained as pale yellow solid in 79% yield.
Mp 252.2–254.2 °C. ESI-MS m/z: [M+H]+ 416.2. 1H NMR (400 MHz,
DMSO) d 10.71 (s, 1H), 8.32 (s, 1H), 7.74 (d, J = 9.0 Hz, 1H), 6.59 (s,
2H), 3.85 (dd, J = 32.9, 12.8 Hz, 6H), 3.72 (s, 4H), 3.59 (s, 2H), 3.24
(s, 4H), 2.90 (d, J = 5.2 Hz, 4H). 13C NMR (126 MHz, DMSO) d
165.65, 165.55, 161.71, 158.66, 157.73, 136.60, 126.58, 116.94,
109.19, 106.64, 98.66, 66.49(2C), 56.15, 55.81, 49.42(2C), 33.84,
26.29, 26.12.
(126 MHz, DMSO)
d 166.88, 162.33, 158.42, 148.57, 148.33,
141.52, 130.24, 122.38, 108.89, 105.19, 102.86, 101.80, 66.34(2C),
49.35, 49.25(2C), 47.02, 32.35.
5.9.12. (E)-4-(2-(2-(3,4,5-Trimethoxybenzylidene)hydrazinyl)-
7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4-yl)morpholine
(7l)
5.9.19. (E)-2-(2-(3,5-Dibromo-4-hydroxybenzylidene)hydrazinyl)-
4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine
6,6-dioxide (10b)
This compound was obtained as pale yellow solid in 74% yield.
Mp 111.6–113.8 °C. ESI-MS m/z: [M+H]+ 446.2. 1H NMR (400 MHz,
DMSO) d 10.91 (s, 1H), 7.98 (s, 1H), 6.93 (s, 2H), 3.81 (s, 6H), 3.71 (s,
4H), 3.68 (s, 3H), 3.59 (s, 2H), 3.29 (s, 4H), 2.92 (s, 4H).
This compound was obtained as white solid in 68% yield. Mp
276.4–277.1 °C. ESI-MS m/z: [MꢂH]ꢂ 559.9. 1H NMR (400 MHz,
DMSO) d 11.14 (s, 1H), 10.27 (s, 1H), 7.96 (s, 1H), 7.80 (s, 2H),
4.21 (s, 2H), 3.73 (s, 4H), 3.46 (d, J = 6.9 Hz, 2H), 3.24 (s, 6H). 13C
NMR (126 MHz, DMSO) d 166.82, 162.38, 158.23, 151.59, 138.78,
130.44, 130.25(2C), 112.76(2C), 103.30, 66.32(2C), 49.31,
49.23(2C), 46.99, 32.38. ESI-HRMS m/z: calcd for C18H19Br2N5O4S
[M+H]+: 559.9603; found 561.9567, 562.0833.
5.9.13. (E)-4-(2-(2-(2,3,4-Trimethoxybenzylidene)hydrazinyl)-
7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4-yl)morpholine
(7m)
This compound was obtained as pale yellow solid in 73% yield.
Mp 120.5–124.6 °C. ESI-MS m/z: [M+H]+ 446.2. 1H NMR (400 MHz,
DMSO) d 10.77 (s, 1H), 8.27 (s, 1H), 7.54 (d, J = 8.9 Hz, 1H), 6.90 (d,
J = 8.7 Hz, 1H), 3.85–3.75 (m, 9H), 3.72 (s, 4H), 3.59 (s, 2H), 3.25 (s,
4H), 2.91 (s, 4H).
5.9.20. (E)-2-(2-(2-Chloro-4-fluorobenzylidene)hydrazinyl)-4-
morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine 6,6-
dioxide (10c)
This compound was obtained as white solid in 90% yield. Mp
262.3–262.9 °C. ESI-MS m/z: [MꢂH]ꢂ 438.1. 1H NMR (400 MHz,
DMSO) d 11.35 (s, 1H), 8.46 (s, 1H), 8.07–7.95 (m, 1H), 7.51 (d,
J = 8.8 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 4.22 (s, 2H), 3.73 (s, 4H),
3.48 (t, J = 6.4 Hz, 2H), 3.31–3.15 (m, 6H).
5.9.14. (E)-4-(2-(2-(4-Methoxybenzylidene)hydrazinyl)-7,8-
dihydro-5H-thiopyrano[4,3-d]pyrimidin-4-yl)morpholine (7n)
This compound was obtained as yellow solid in 77% yield. Mp
120.5–124.6 °C. ESI-MS m/z: [M+H]+ 386.2. 1H NMR (400 MHz,
DMSO) d 10.72 (s, 1H), 8.03 (s, 1H), 7.56 (d, J = 8.3 Hz, 2H), 6.98
(d, J = 8.3 Hz, 2H), 3.79 (s, 3H), 3.73 (s, 4H), 3.60 (s, 2H), 3.25 (s,
4H), 2.92 (dd, J = 7.4, 4.3 Hz, 4H).
5.9.21. (E)-2-(2-(2,3-Dichlorobenzylidene)hydrazinyl)-4-morph-
olino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine 6,6-dioxide
(10d)
This compound was obtained as yellow solid in 87% yield. Mp
275.8–276.9 °C. ESI-MS m/z: [MꢂH]ꢂ 454.1. 1H NMR (400 MHz,
DMSO) d 11.50 (s, 1H), 8.56 (s, 1H), 7.97 (d, J = 7.9 Hz, 1H), 7.66
(d, J = 7.9 Hz, 1H), 7.45 (t, J = 8.3 Hz, 1H), 4.26 (s, 2H), 3.76 (s,
4H), 3.52 (s, 2H), 3.27 (d, J = 14.9 Hz, 6H).
5.9.15. (E)-4-(2-(2-(2,4-Dimethoxybenzylidene)hydrazinyl)-7,8-
dihydro-5H-thiopyrano[4,3-d]pyrimidin-4-yl)morpholine (7o)
This compound was obtained as white solid in 87% yield. Mp
248.6–249.2 °C. ESI-MS m/z: [M+H]+ 416.2. 1H NMR (400 MHz,
DMSO) d 10.71 (s, 1H), 8.32 (s, 1H), 7.75 (d, J = 5.8 Hz, 1H), 6.60
(s, 2H), 3.81 (d, J = 11.0 Hz, 6H), 3.72 (s, 4H), 3.59 (s, 2H), 3.25 (s,
4H), 3.00–2.84 (m, 4H).
5.9.22. (E)-2-(2-(4-Hydroxy-3,5-dimethoxybenzylidene)hydraz-
inyl)-4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidi-
ne 6,6-dioxide (10e)
5.9.16. (E)-4-(2-(2-(2-Nitrobenzylidene)hydrazinyl)-7,8-dihydro-
5H-thiopyrano[4,3-d]pyrimidin-4-yl)morpholine (7p)
This compound was obtained as orange-yellow solid in 89%
yield. Mp 259.3–260.8 °C. ESI-MS m/z: [M+H]+ 401.2. 1H NMR
(400 MHz, DMSO) d 11.30 (s, 1H), 8.48 (s, 1H), 8.10 (d, J = 7.9 Hz,
1H), 7.99 (d, J = 8.2 Hz, 1H), 7.76 (t, J = 7.6 Hz, 1H), 7.56 (t,
J = 7.5 Hz, 1H), 3.73 (s, 4H), 3.61 (s, 2H), 3.29 (s, 4H), 2.93 (s, 4H).
13C NMR (126 MHz, DMSO) d 165.84, 165.49, 157.25, 147.98,
135.18, 133.75, 130.08, 129.55, 127.84, 124.97, 110.35, 66.48(2C),
49.34(2C), 33.87, 26.23(2C).
This compound was obtained as white solid in 88% yield. Mp
223.5–224.6 °C. ESI-MS m/z: [M+Na]+ 486.1, [MꢂH]ꢂ 462.2. 1H
NMR (400 MHz, DMSO) d 10.96 (s, 1H), 8.74 (s, 1H), 7.98 (s, 1H),
6.92 (s, 2H), 4.19 (s, 2H), 3.80 (s, 6H), 3.72 (s, 4H), 3.46 (s, 2H),
3.23 (d, J = 20.2 Hz, 6H). 13C NMR (126 MHz, DMSO) d 166.81,
162.30, 158.45, 148.59(2C), 142.11, 137.34, 126.15, 104.36(2C),
102.62, 66.33(2C), 56.40(2C), 49.36, 49.22(2C), 47.04, 32.31. ESI-
HRMS m/z: calcd for
464.1595.
C
20H25N5O6S [M+H]+: 464.1640; found
5.9.23. (E)-4-((2-(4-Morpholino-6,6-dioxido-7,8-dihydro-5H-
thiopyrano[4,3-d]pyrimidin-2-yl)hydrazono)methyl)benzonitrile
(10f)
5.9.17. (E)-4-(2-(2-(4-Nitrobenzylidene)hydrazinyl)-7,8-dihydro-
5H-thiopyrano[4,3-d]pyrimidin-4-yl)morpholine (7q)
This compound was obtained as orange-yellow solid in 86%
yield. Mp 268.6–269.8 °C. ESI-MS m/z: [M+H]+ 401.2. 1H NMR
(400 MHz, DMSO) d 11.30 (s, 1H), 8.26 (d, J = 8.8 Hz, 2H), 8.17 (s,
This compound was obtained as yellow solid in 85% yield. Mp
276.6–281.3 °C. ESI-MS m/z: [M+H]+ 413.2. 1H NMR (400 MHz,
DMSO) d 11.38 (s, 1H), 8.13 (s, 1H), 7.83 (dd, J = 21.0, 8.4 Hz, 4H),

W. Zhu et al. / Bioorg. Med. Chem. 22 (2014) 6746–6754
6753
4.22 (s, 2H), 3.77–3.70 (m, 4H), 3.52–3.46 (m, 2H), 3.26 (s, 4H), 3.22
(t, J = 6.8 Hz, 2H).
6H), 3.72 (s, 4H), 3.68 (s, 3H), 3.48 (t, J = 6.3 Hz, 2H), 3.27 (s, 4H),
3.20 (t, J = 6.5 Hz, 2H).
5.9.24. (E)-2-(2-(3,5-Di-tert-butyl-4-hydroxybenzylidene)hydra-
zinyl)-4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimi-
dine 6,6-dioxide (10g)
5.9.30. (E)-4-Morpholino-2-(2-(2,3,4-trimethoxybenzylidene)-
hydrazinyl)-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine 6,6-
dioxide (10m)
This compound was obtained as white solid in 87% yield. Mp
277.6–278.0 °C. ESI-MS m/z: [M+H]+ 516.2, [MꢂH]ꢂ 514.3. 1H
NMR (400 MHz, DMSO) d 10.88 (s, 1H), 7.95 (s, 1H), 7.39 (s, 2H),
7.23 (s, 1H), 4.17 (s, 2H), 3.69 (s, 4H), 3.44 (s, 2H), 3.24 (s, 4H),
3.15 (s, 2H), 1.37 (s, 18H). 13C NMR (126 MHz, DMSO) d 166.74,
162.29, 158.53, 155.49, 139.63(2C), 127.26, 126.68, 123.43(2C),
102.43, 66.33(2C), 49.38, 49.16(2C), 47.04, 35.01(2C), 32.18,
30.67(6C).
This compound was obtained as pale yellow solid in 69% yield.
Mp 243.4–244.0 °C. ESI-MS m/z: [M+Na]+ 500.1, [MꢂH]ꢂ 476.2. 1H
NMR (400 MHz, DMSO) d 10.99 (s, 1H), 8.31 (s, 1H), 7.56 (d,
J = 8.9 Hz, 1H), 6.92 (d, J = 8.8 Hz, 1H), 4.21 (s, 2H), 3.91–3.76 (m,
9H), 3.72 (s, 4H), 3.50–3.45 (m, 2H), 3.22 (s, 6H).
5.9.31. (E)-2-(2-(4-Methoxybenzylidene)hydrazinyl)-4-
morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine 6,6-
dioxide (10n)
5.9.25. (E)-2-(2-(3-Bromo-4-hydroxybenzylidene)hydrazinyl)-4-
morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine 6,6-
dioxide (10h)
This compound was obtained as white solid in 70% yield. Mp
279.1–280.2 °C. ESI-MS m/z: [M+H]+ 484.0, [MꢂH]ꢂ 482.1. 1H
NMR (400 MHz, DMSO) d 10.98 (s, 1H), 10.65 (s, 1H), 7.98 (s,
1H), 7.76 (s, 1H), 7.45 (d, J = 8.0 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H),
4.20 (s, 2H), 3.73 (s, 4H), 3.47 (s, 2H), 3.22 (s, 6H). 13C NMR
This compound was obtained as white solid in 81% yield. Mp
282.9–283.1 °C. ESI-MS m/z: [M+H]+ 418.8. 1H NMR (400 MHz,
DMSO) d 10.94 (s, 1H), 8.05 (s, 1H), 7.58 (d, J = 8.4 Hz, 2H), 6.98
(d, J = 8.4 Hz, 2H), 4.20 (s, 2H), 3.79 (s, 3H), 3.73 (s, 4H), 3.47 (s,
2H), 3.23 (s, 4H), 3.20 (s, 2H).
5.9.32. (E)-2-(2-(2,4-Dimethoxybenzylidene)hydrazinyl)-4-
morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine 6,6-
dioxide (10o)
(126 MHz, DMSO)
d 166.87, 162.36, 158.38, 155.27, 140.55,
130.93, 128.53, 127.52, 117.00, 110.24, 102.96, 66.33(2C), 49.32,
49.26(2C), 47.02, 32.37.
This compound was obtained as pale yellow solid in 77% yield.
Mp 256.3–257.1 °C. ESI-MS m/z: [M+H]+ 448.2. 1H NMR (400 MHz,
DMSO) d 10.92 (s, 1H), 8.34 (s, 1H), 7.74 (d, J = 9.0 Hz, 1H), 6.60 (s,
2H), 4.19 (s, 2H), 3.83 (s, 3H), 3.80 (s, 3H), 3.73 (s, 4H), 3.46 (d,
J = 6.4 Hz, 2H), 3.22 (s, 4H), 3.19 (s, 2H).
5.9.26. (E)-2-(2-(2,4-Dihydroxybenzylidene)hydrazinyl)-4-
morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine 6,6-
dioxide (10i)
This compound was obtained as pale yellow solid in 78% yield.
Mp >300 °C. ESI-MS m/z: [MꢂH]ꢂ 418.1. 1H NMR (400 MHz, DMSO)
d 12.03 (s, 1H), 11.22 (s, 1H), 9.79 (s, 1H), 8.11 (s, 1H), 7.12 (d,
J = 8.3 Hz, 1H), 6.33–6.29 (m, 1H), 6.28 (s, 1H), 4.20 (s, 2H), 3.72
(s, 6H), 3.26 (s, 4H), 3.18 (d, J = 6.7 Hz, 2H).
5.9.33. (E)-4-Morpholino-2-(2-(2-nitrobenzylidene)hydrazinyl)-
7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine 6,6-dioxide (10p)
This compound was obtained as pale yellow solid in 84% yield.
Mp 276.3–276.9 °C. ESI-MS m/z: [M+H]+ 433.1. 1H NMR (400 MHz,
DMSO) d 11.46 (s, 1H), 8.49 (s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 8.00 (d,
J = 8.1 Hz, 1H), 7.76 (s, 1H), 7.58 (s, 1H), 4.22 (s, 2H), 3.73 (s, 4H),
3.48 (s, 2H), 3.27 (s, 4H), 3.22 (t, J = 6.4 Hz, 2H).
5.9.27. (E)-2-(2-(2,5-Dimethoxybenzylidene)hydrazinyl)-4-
morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine 6,6-
dioxide (10j)
5.9.34. (E)-4-Morpholino-2-(2-(4-nitrobenzylidene)hydrazinyl)-
7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine 6,6-dioxide (10q)
This compound was obtained as orange-yellow solid in 77%
yield. Mp 281.1–282.2 °C. ESI-MS m/z: [MꢂH]ꢂ 431.1. 1H NMR
(400 MHz, DMSO) d 11.46 (s, 1H), 8.26 (d, J = 8.7 Hz, 2H), 8.19 (s,
1H), 7.88 (d, J = 8.6 Hz, 2H), 4.23 (s, 2H), 3.74 (s, 4H), 3.49 (s, 2H),
3.30–3.21 (m, 6H).
This compound was obtained as pale yellow solid in 82% yield.
Mp 239.1–240.0 °C. ESI-MS m/z: [M+Na]+ 470.1, [MꢂH]ꢂ 446.2. 1H
NMR (400 MHz, DMSO) d 11.13 (s, 1H), 8.39 (s, 1H), 7.38 (s, 1H),
7.00 (d, J = 8.8 Hz, 1H), 6.92 (d, J = 9.5 Hz, 1H), 4.20 (s, 2H), 3.78
(s, 3H), 3.74 (s, 4H), 3.73–3.70 (m, 3H), 3.47 (d, J = 6.5 Hz, 2H),
3.26 (s, 4H), 3.21 (d, J = 6.2 Hz, 2H). 13C NMR (126 MHz, DMSO) d
166.80, 162.33, 158.41, 153.70, 152.00, 136.87, 124.61, 116.23,
113.64, 109.82, 102.94, 66.32(2C), 56.67, 55.78, 49.36, 49.20(2C),
47.01, 32.34.
5.10. mTOR kinase assay9
The mTOR kinase activity of all the compounds (7a–q, 10a–q)
was determined using LANCEÒ Ultra time-resolved fluorescence
resonance energy transfer (TR-FRET) assay following the manufac-
turer’s instructions, with compounds mTOR inhibitors BMCL-
200908069-1 and PI103 as positive controls. Briefly, mTOR enzyme
5.9.28. (E)-2-(2-(3,5-Dimethoxybenzylidene)hydrazinyl)-4-
morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine 6,6-
dioxide (10k)
This compound was obtained as white solid in 80% yield. Mp
260.1–261.5 °C. ESI-MS m/z: [M+H]+ 448.2, [MꢂH]ꢂ 446.2. 1H
NMR (400 MHz, DMSO) d 10.95 (s, 1H), 8.38 (s, 1H), 7.78 (d,
J = 9.2 Hz, 1H), 6.63 (s, 2H), 4.22 (s, 2H), 3.86 (s, 3H), 3.83 (s, 3H),
3.76 (s, 4H), 3.52–3.47 (m, 2H), 3.28–3.18 (m, 6H).
(10 nM), ATP (21.6 lM), 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 Opti-
plates (PerkinElmer, MA, USA) at room temperature 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 EnVisionÒ Multilabel Reader
(PerkinElmer, MA, USA) in TR-FRET mode (excitation at 320 nm
and emission at 665 nm). All of the compounds were tested two
5.9.29. (E)-4-Morpholino-2-(2-(3,4,5-trimethoxybenzylidene)-
hydrazinyl)-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine 6,6-
dioxide (10l)
This compound was obtained as pale yellow solid in 75% yield.
Mp 260.6–261.2 °C. ESI-MS m/z: [M+H]+ 478.2. 1H NMR (400 MHz,
DMSO) d 11.13 (s, 1H), 8.01 (s, 1H), 6.95 (s, 2H), 4.21 (s, 2H), 3.82 (s,

6754
W. Zhu et al. / Bioorg. Med. Chem. 22 (2014) 6746–6754
times. The results expressed as IC50 (inhibitory concentration 50%)
were the averages of two determinations.
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 SUR-
FLEX-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 res-
idue was, therefore, 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.
5.11. PI3Ka
kinase assay10
The selected compounds (7b, 7e, 7h, 10b and 10e) are tested for
their activities against PI3K
using a Kinase-GloÒ Luminescent
Kinase Assay, with PI103 as positive control. The kinase reaction
is done in 384-well black plate. Each well is loaded with 50 L of
test items (in 90% DMSO) and 5 L reaction buffer containing
10 g/mL PI substrate (l- -phosphatidylinositol; Avanti Polar Lip-
ids; prepared in 3% octyl-glucoside) and the PI3K protein 10 nM
is then added to it. The reaction is started by the addition of 5
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
a
l
l
l
a
a
lL
Acknowledgments
l
a
lL
We gratefully acknowledge the generous support provided by
The National Natural Science Funds of China (No. 80140357), Sci-
ence and Technology Project Founded by the Education Depart-
ment of Jiangxi Province of China (No. GJJ13578), 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).
Kinase-Glo buffer. The plates are then read in a Synergy 2 reader
for luminescence detection. All of the compounds were tested
two times.
5.12. Cytotoxicity assay in vitro
The cytotoxic activities of five selected compounds (7b, 7e, 7h,
10b and 10e) were evaluated with H460, PC-3 cell lines by the
standard MTT assay in vitro, with compounds mTOR inhibitors
BMCL-200908069-1 and PI103 as positive controls. The cancer cell
lines were cultured in minimum essential medium (MEM) supple-
ment with 10% fetal bovine serum (FBS). Approximately 4 ꢁ 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 to each well at a terminal concentration of
References and notes
1. Rodon, J.; Dienstmann, R.; Serra, V.; Tabernero, J. Nat. Rev. Clin. Oncol. 2013, 10,
143.
2. Markman, B.; Tao, J. J.; Scaltriti, M. Curr. Pharm. Des. 2013, 19, 895.
3. Pei, Z.; Blackwood, E.; Liu, L.; Malek, S.; Belvin, M.; Koehler, M. F.; Ortwine, D.
F.; Chen, H.; Cohen, F.; Kenny, J. R.; Bergeron, P.; Lau, K.; Ly, C.; Zhao, X.;
Estrada, A. A.; Truong, T.; Epler, J. A.; Nonomiya, J.; Trinh, L.; Sideris, S.; Lesnick,
J.; Bao, L.; Vijapurkar, U.; Mukadam, S.; Tay, S.; Deshmukh, G.; Chen, Y. H.; Ding,
X.; Friedman, L. S.; Lyssikatos, J. P. Med. Chem. Lett. 2013, 4, 103.
4. Liu, K. K.; Bailey, S.; Dinh, D. M.; Lam, H.; Li, C.; Wells, P. A.; Yin, M. J.; Zou, A.
Bioorg. Med. Chem. Lett. 2012, 22, 5114.
5. Koehler, M. F.; Bergeron, P.; Blackwood, E.; Bowman, K. K.; Chen, Y. H.;
Deshmukh, G.; Ding, X.; Epler, J.; Lau, K.; Lee, L.; Liu, L.; Ly, C.; Malek, S.;
Nonomiya, J.; Oeh, J.; Ortwine, D. F.; Sampath, D.; Sideris, S.; Trinh, L.; Truong,
T.; Wu, J.; Pei, Z.; Lyssikatos, J. P. J. Med. Chem. 2012, 55, 10958.
6. Menear, K. A.; Gomez, S.; Malagu, K.; Bailey, C.; Blackburn, K.; Cockcroft, X. L.;
Ewen, S.; Fundo, A.; Le Gall, A.; Hermann, G.; Sebastian, L.; Sunose, M.; Presnot,
T.; Torode, E.; Hickson, I.; Martin, N. M.; Smith, G. C.; Pike, K. G. Bioorg. Med.
Chem. Lett. 2009, 19, 5898.
5
lg/mL and incubated with cells at 37 °C for 4 h. The formazan
crystals were dissolved in 100 L DMSO each well, and the absor-
l
bency at 492 nm (for absorbance 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.
7. Zhu, W.; Zhai, X.; Fu, Q.; Guo, F.; Bai, M.; Wang, J.; Wang, H.; Gong, P. Chem.
Pharm. Bull. 2012, 60, 1037.
8. Zhu, W.; Liu, Y.; Zhao, Y.; Wang, H.; Tan, L.; Fan, W.; Gong, P. Arch. Pharm.
(Weinheim) 2012, 345, 812.
9. Zhu, W.; Liu, Y.; Zhai, X.; Wang, X.; Zhu, Y.; Wu, D.; Zhou, H.; Gong, P.; Zhao, Y.
Eur. J. Med. Chem. 2012, 57, 162.
5.13. Docking studies
For docking purposes, the three-dimensional structure of the
mTOR (PDB code: 4JT6) were obtained from RCSB Protein Data
Bank.11 Hydrogen atoms were added to the structure allowing for
appropriate ionization at physiological pH. The protonated state
of several important residues, such as GLY2238, VAL2240 and
10. Roper, J.; Richardson, MP; Wang, W. V.; Richard, L. G.; Chen, W.; Coffee, E. M.;
Sinnamon, M. J.; Lee, L.; Chen, P. C.; Bronson, R. T.; Martin, E. S.; Hung, K. E. PloS
One 2011, 6, e25132.
11. Yang, H.; Rudge, D. G.; Koos, J. D.; Vaidialingam, B.; Yang, H. J.; Pavletich, N. P.
Nature 2013, 497, 217.