Design, synthesis, biological evaluation and docking studies of novel 2-substituted-4-morpholino-7,8-dihydro-5 H -thiopyrano[4,3- d ]pyrimidine derivatives as dual PI3Kα/mTOR inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.03.033

Four series of 2-substituted-4-morpholino- 7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine derivatives (9-28) were designed, synthesized and their structures were confirmed by 1H NMR, 13C NMR and MS spectrum. All compounds were evaluated for the IC₅₀ values against three cancer cell lines (A549, PC-3 and MCF-7). And four selected compounds (10, 11, 24, 27) were further evaluated for the IC₅₀ values against PI3Kα and mTOR kinases. Seven of the target compounds exhibited moderate to excellent antitumor activities against these three cancer cell lines. The most promising compound 11 showed good antitumor potency for A549, PC-3 and MCF-7 cell lines with IC₅₀ values of 0.52 ± 0.10 1/4M, 1.41 ± 0.10 1/4M, 4.82 ± 0.24 1/4M and moderate antitumor activities against PI3Kα/mTOR with IC₅₀ values of 6.72 ± 0.30 1/4M and 0.94 ± 0.10 1/4M. Structure-activity relationships (SARs) and docking studies indicated that aryl urea scaffolds had a significant impact on the antitumor activities, and aryl pyridine urea scaffolds produced the best potency. Variations in substitutions of the aryl group had a significant impact on the activity and 3-Cl-4-F or 3-CF₃-4-Cl substitution was more preferred.

Full text of DOI:10.1016/j.ejmech.2016.03.033

Accepted Manuscript
Design, synthesis, biological evaluation and docking studies of novel 2-substituted-4-
morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine derivatives as dual PI3Kα/
mTOR inhibitors
Fei Lei, Chengyu Sun, Shan Xu, Qinqin Wang, Yiqiang OuYang, Chen Chen, Hui Xia,
Linxiao Wang, Pengwu Zheng, Wufu Zhu
PII:
S0223-5234(16)30207-0
10.1016/j.ejmech.2016.03.033
EJMECH 8458
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 November 2015
Revised Date: 11 March 2016
Accepted Date: 13 March 2016
Please cite this article as: F. Lei, C. Sun, S. Xu, Q. Wang, Y. OuYang, C. Chen, H. Xia, L. Wang, P.
Zheng, W. Zhu, Design, synthesis, biological evaluation and docking studies of novel 2-substituted-4-
morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine derivatives as dual PI3Kα/mTOR inhibitors,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.03.033.
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ACCEPTED MANUSCRIPT
Design, synthesis，biological evaluation and docking studies of novel
2-substituted-4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine derivatives as
dual PI3Kα/mTOR inhibitors
Fei Lei1,†, Chengyu Sun1,†, Shan Xu1, Qinqin Wang1, Yiqiang OuYang1, Chen Chen1, Hui Xia1,
Linxiao Wang1, Pengwu Zheng1 , Wufu Zhu1,2,
1 School of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang 330013, P.
R. China.
2
Key Laboratory of Original New Drugs Design and Discovery of Ministry of Education,
Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016,
P. R. China.
Graphical Abstract
Four series of 2-substituted-4-morpholino- 7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine
derivatives were synthesized and evaluated for their activity against PI3Kα and mTOR kinase
† These authors contribute equally to this work
Corresponding author. Tel./fax: +86 791 83802393.
E-mail address: zhuwufu-1122@163.com (W. Zhu), zhengpw@126.com (P. Zheng).

ACCEPTED MANUSCRIPT
and cancer cell lines. The most promising compound 11 showed good antitumor potency for
A549, PC-3 and MCF-7 cell lines with IC50 values of 0.52 ± 0.10ꢀM, 1.41 ± 0.10ꢀM, and
4.82±0.24ꢀM, and strong antitumor activities against PI3Kα/mTOR with IC50 values of 6.72 ±
0.30 ꢀM and 0.94 ± 0.10 ꢀM.

ACCEPTED MANUSCRIPT
Design, synthesis，biological evaluation and docking studies of novel 2-substituted-4-morpholino-
7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine derivatives as dual PI3Kα/mTOR inhibitors
Fei Lei1,†, Chengyu Sun1,†, Shan Xu1, Qinqin Wang1, Yiqiang Ouyang1, Chen Chen1, Hui Xia1, Linxiao Wang1,
Pengwu Zheng1 , Wufu Zhu1,2,
1 School of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang 330013, P. R. China.
2
Key Laboratory of Original New Drugs Design and Discovery of Ministry of Education, Shenyang
Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016, P. R. China.
*Correspondence author.
Tel/Fax: +86 791 8380-2393;
E-mail address: zhuwufu-1122@163.com (W. Zhu), zhengpw@126.com (P. Zheng).
Four series of 2-substituted-4-morpholino- 7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine derivatives (9–28)
were designed, synthesized and their structures were confirmed by 1H NMR, 13C NMR and MS spectrum . All
compounds were evaluated for the IC50 values against three cancer cell lines (A549, PC-3 and MCF-7). And
four selected compounds (10, 11, 24, 27) were further evaluated for the IC50 values against PI3Kα and mTOR
kinases. Seven of the target compounds exhibited moderate to excellent antitumor activities against these
three cancer cell lines. The most promising compound 11 showed good antitumor potency for A549, PC-3 and
MCF-7 cell lines with IC50 values of 0.52 ± 0.10 ꢀM, 1.41 ± 0.10 ꢀM, 4.82 ± 0.24ꢀM and moderate antitumor
activities against PI3Kα/mTOR with IC50 values of 6.72 ± 0.30 ꢀM and 0.94 ± 0.10 ꢀM. Structure–activity
relationships (SARs) and docking studies indicated that aryl urea scaffolds had a significant impact on the
antitumor activities, and aryl pyridine urea scaffolds produced the best potency. Variations in substitutions of
the aryl group had a significant impact on the activity and 3-Cl-4-F or 3-CF3-4-Cl substitution was more
preferred.
Key words: Thiopyrano[4,3-d]pyrimidine; Synthesis; Docking; PI3Kα/mTOR inhibitors
1. Introduction
† These authors contribute equally to this work
Corresponding author. Tel./fax: +86 791 83802393.
E-mail address: zhuwufu-1122@163.com (W. Zhu), zhengpw@126.com (P. Zheng).
1

ACCEPTED MANUSCRIPT
The phosphatidylinositol 3-kinase (PI3K) / protein kinase B (Akt) / the mammalian target of rapamycin
(mTOR) pathway is an intracellular signaling pathway important in regulating various cellular processes,
including growth regulation, apoptosis, and survival in many cancers [1-3]. In recent years, many
(fused-)pyrimidine/triazine derivatives were reported as PI3K-Akt-mTOR signal pathway inhibitors (Figure 1)
[4-9]. Among them, GDC-0941, 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 [10]. PF-05139962
[6], compound [7], PKI-402[11] and PKI-587[12] (Figure 1) are several aryl urea derivatives which exhibit
potent anti-tumor activity. Structure-activity relationships (SARs) showed that aryl urea moieties were essential
for the anti-tumor activity.
In our previous research, we reported several series of analogous of PF-05139962,
7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine hydrazone (Figure 1) which demonstrated excellent antitumor
activity. Biological evaluation results showed that thiopyrano[4,3-d]pyrimidines were important for the activity of
the target compounds.
In continuation of our previous research, and in order to screen compounds which possess excellent in
vitro / in vivo anti-tumor activity as well as improved pharmacokinetic properties, further studies on analogous
of PF-05139962 were carried out in this research. The design strategy for all target compounds is shown in
Figure 2 and is described as follow. Firstly, the urea group was kept unchanged, and a series of phenyl pyridine
urea compounds 9–12 was designed by replacing the ethyl group of PF-05139962 with aryl group and
replacing the phenyl group with pyridine group. What’s more, sulfur atom was oxidized to study the influence to
the target compounds. This series of compounds showed excellent cytotoxicity and moderate PI3Kα/mTOR
kinases inhibitory activity. Furthermore, investigations were carried out in details to study the effect of urea to
the activity and therefore urea was replaced by sulfonylurea group to yield compounds 13–18 and 19–22.
However, this two series of compounds showed poor cytotoxicity and moderate mTOR kinase inhibitory activity.
Thus, we shifted our focus back to the phenyl pyridine urea series. To investigate the influences of pyridine
urea to the activity and inspired by previous compounds 7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine hydrazone
(Compound Ⅱ in Figure 2), phenyl pyridine urea was replaced by piperazine phenylurea to afford compounds
23–28. The activity of 23–28 was similar to that of 9–12 and was much better than that of compounds 13–18
and 19–22.
The studies of Wentao Yue showed that PI3K, Akt and mTOR gene of the PI3K-Akt-mTOR signaling
pathway expression in the non-small cell lung cancer were (61±23)%, (77±32)%, (43±21)%
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respectively[13].And the level of this pathway in breast cancer also up to 70%[14]. In the prostate cancer cells,
the missing of tumor suppressor gene (PTEN) resulted in the high expression of PI3K-Akt-mTOR signaling
pathway. In addition, the studies of GDC-0941 showed it has strong inhibitory activities against A549(lung
cancer cell lines), PC-3 (prostate cancer cell lines)and MCF-7(breast cancer cell lines) , suggesting that these
three cells have a high sensitivity for PI3K-Akt-mTOR signaling pathway inhibitors[15-17].Therefore,
A549,PC-3 and MCF-7 cell lines were chose as the test cell line for in vitro antitumor experiments in this study.
Herein we disclosed the design, synthesis and antitumor activity of four series of compounds against A549,
PC-3, MCF-7 and PI3Kα, mTOR and c-Met kinases. Moreover, docking studies were presented in this paper
as well.
(Fig. 1. should be listed here.)
(Fig. 2. should be listed here.)
2. Chemistry
The preparation of target compounds 9–28 was described in Schemes 1 and 2.
Compounds 7a–d and 8a–e were synthesized according to the procedures reported previously by our group
[18-19]. The different substituent of phenylamine reacted with triphosgene to get compound compounds 7a–d.
8a–e was synthesized from the different substituent of chlorobenzene through three steps.
The key intermediates 4-(2-chloro-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4-yl)morpholine (4a) and
2-chloro-4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine 6,6-dioxide(4b) were synthesized according
to the procedures reported previously by our group [5].
Compounds 5a and 5b were synthesized according to the following procedure. 4a was reacted with
5-bromopyridin-2-amine through Suzuki-coupling reaction using bis(triphenylphosphine)palladium(II) dichloride
as catalyst to generate compound 5a. Similarly, Compound 5b was synthesized from 4b through
Suzuki-coupling reaction. And compound 4a was reacted with 1-bromo-4-nitrobenzene through
Suzuki-coupling reaction and then reduction by NH2NH2▪H2O (80%) yielding compound
4-(4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-2-yl)aniline
5c.
At
the
same
way,
2-(4-aminophenyl)-4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine 6,6-dioxide 5d could be
synthesized from compound 4b through the same methods.
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Substitution of 4a–b with piperazine achieved 6a–b.
Finally, key intermediates 5a–d and 6a–b condensed with 7a–d or 8a–e to afford target compounds 9–28,
respectively.
(Scheme 1. should be listed here.)
(Scheme 2. should be listed here.)
3. Results and discussion
3.1 Biological evaluation
The target compounds (9–28) were evaluated for the cytotoxicity against three cancer cell lines A549, PC-3
and MCF-7 by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) cell proliferation assay,
together with reference compounds sorafenib and GDC-0941. In addition, four selected compounds 10, 11, 24
and 27 were further evaluated for their IC50 values against mTOR, PI3Kα, c-Met kinases by LANCE® Ultra
time-resolved fluorescence resonance energy transfer (TR-FRET) assay, Kinase-Glo® Luminescent Kinase
Assay or by Mobility shift assay against c-Met with ATP concentration at Km. The results expressed as IC50
values are summarized in Tables 1 and 2 and the values are the average of at least two independent
experiments.
(Table 1. should be listed here.)
As illustrated in Table 1, seven of the synthesized compounds showed moderate to significant cytotoxic
activities with IC50 valuables in single-digit ꢀM to nanomole range and four of them ( 10, 11, 24, 27 ) showed
excellent cytotoxic activity with IC50 values from 6.13 ± 0.62 µM to 0.52 ± 0.10 µM which were equal to more
potent than sorafenib and GDC-0941.
Furthermore, we can easily find that the phenyl pyridine urea compounds 9–12 showed excellent cytotoxic
activity and but sulfonylurea derivatives 13–22 showed no activity. It promoted us that replacing the ethyl group
of PF-05139962 with aryl groups and replacing the phenyl urea scaffold with pyridine urea scaffold didn’t
decrease the activity. However, when the urea group was replaced by sulfonylurea, the target compounds lost
biological activity. When the phenyl pyridine urea was replaced by piperazine phenyl urea, the activity of 23–28
were similar to that of 9–12 and were much better than that of compounds 13–22.The results suggested that
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aryl urea scaffold played an important role in the antitumor activity of target compounds and the pyridine group
was not essential for the target compounds. No matter the sulfur atom was oxidized or not, the activity wasn’t
changed remarkablely. What’s more, variations in substitutions of the aryl group had a significant impact on the
activity, and for the piperazine phenyl urea and phenyl pyridine urea series, 3-Cl-4-F (10–11) or 3-CF3-4-Cl (12,
24, 27) substitution were more preferred.
The antitumor activities of the four selected compounds (10, 11, 24, 27) as well as the lead compound
GDC-0941 against PI3Kα, mTOR and c-Met kinases were shown in Table 2. All the four selected compounds
showed well antitumor activity toward PI3Kα, mTOR kinases, especially compounds 10 and 11 with IC50 values
in nanomole range. However, the activity against c-Met kinase is bad. This result told us that these compounds
showed high selectivity toward PI3Kα/mTOR to c-Met kinases. What’s more, the excellent kinases activity of
10, 11, 24, 27 prompt us that linker urea group is essential for the target compounds and piperazine urea or
pyridylurea group were more preferred.
(Table 2. should be listed here.)
3.2 Molecular docking study
To explore the binding modes of target compounds with the active site of mTOR and PI3Kα, 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 four representative compounds 10, 11, 24 and 27, our best
mTOR/PI3Kα inhibitor in this study, as ligand examples, and the structures of mTOR (PDB ID code:4JT6 [20])
and PI3Kα (PDB ID code:4L23 [21]) were selected as the docking models. In this study, we chose compound
11 to explaining the docking model.
(Fig. 3. should be listed here.)
The binding modes of compound 11 and lead compound were shown in Fig. 3a-b. The docking scores were
shown in Table 2. As depicted in Fig. 3a, compound 11 and PI103 can overlap in the position of morpholine
group in the binding model and the pyridine and urea group of bis-aryl urea moiety formed four hydrogen bonds
with residues ASP2195 and ASP2357 in the docking model of mTOR, respectively.The morpholine group
formed one hydrogen bond with residues VAL2240.Similarly, the hydrogen bonds were found in the docking
model of PI3Kα in Fig. 3b, such as the hydrogen bonds between morpholine group and residue VAL851, the
bis-aryl urea moiety and the residues LYS802 and ASP810 and the double oxide thiopyran moiety can also
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formed one hydrogen bonds with residue GLN859. The activity against mTOR/PI3Kα kinases of compound 11
was different, which was attributed to the difference of the hydrogen bonds between the bis-aryl urea moiety
and amino acid residues. Analysis of compound 11’s binding mode in the active binding site and the results of
activity suggested that the several hydrogen bonds, formed in morpholine group and the bis-aryl urea moiety,
really
plays
an
important
role
in
increasing
the
inhibitory
potency
of
2-substituted-4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine derivatives against mTOR/PI3Kα
kinases. The docking results indicated that these compounds have plausible binding modes to both enzymes.
The molecular docking scores in Table 2 show that docking score of pyridine aryl urea compounds is better
than that of pyridine sulfonyl urea compounds and benzene sulfonyl urea compounds. The docking score of
piperizine aryl urea compounds is the lowest. Generally, the docking results were agreed with the biological
evaluation results of target compounds.
Furthermore, the docking results also give us a new direction to design new mTOR/PI3Kα inhibitors that can
interact with ASP2195, ASP2357, LYS802, ASP810 and GLN859. The above-mentioned results of SAR
analysis and molecular docking study may allow the rational design of more potent mTOR/Pi3kα inhibitors.
Conclusions
In summary, four series of 4-(2-chloro-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4-yl)morpholine
derivatives were designed, synthesized and evaluated for antitumor activity against three cancer cell lines and
mTOR, PI3Kα and c-Met kinases in vitro. The pharmacological results indicated that seven of the synthesized
compounds displayed moderate antitumor activity and four selected compounds showed equal to more
potency than lead compound sorafenib and GDC-0941. The most promising compound 11 exhibited strong
antitumor activities against A549, PC-3 and MCF-7 cell lines, with IC50 values of 0.52 ± 0.10 ꢀM, 1.41 ± 0.10
ꢀM,4.82±0.24ꢀM and moderate antitumor activities against PI3Kα/mTOR with IC50 values of 6.72 ± 0.30 ꢀM
and 0.94 ± 0.10 ꢀM. The initial SARs and docking studies showed that aryl urea moieties were necessary for
the activity of these compounds and aryl pyridine urea scaffolds produced the best potency. Variations in
substitutions of the aryl urea moieties had a significant impact on the activity and 3-Cl-4-F or 3-CF3-4-Cl
substitution was more preferred. Further study will be carried out in near future.
5. Experimental
5.1. Chemistry
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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 LCMS (Agilent, Palo Alto, CA, USA). All the materials 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 otherwise specied. Yields were not
optimized.
5.2. Preparation of compounds 5a–d and 6a–b
Compounds 5a–d and 6a–b were synthesized from compound 1 according to the procedures in our previous
research [5].
5.3. General procedure for Compounds 7a–d and 8a–e
Compounds 7a–d and 8a–e were synthesized according to the procedures in our previous research
[18-19] .
5.3 General procedure for the preparation of compounds 9–12
A mixture of 5-(4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-2-yl)pyridin-2-amine 5a (0.10g,
0.30mmol) or 2-(6-aminopyridin-3-yl)-4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine 6,6-dioxide 5b
(0.10g, 0.28mmol), substitued isocyanatobenzene 7a–d in 1,4-dioxane(15mL) was refluxed for 10-30 mins.
The mixture was washed with H2O and separated by filtration to afford white solids 9–12.
5.3.1 1-(5-(4-Morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-2-yl)pyridin-2-yl)-3-phenylurea (9)
This compound was obtained as white solid in 85.2% yield. m.p.: 261.2–262.3 ; ESI-MS m/z: 449.2[M+H]+;
1H NMR (400 MHz, CDCl3) δ 10.46 (s, 1H, NH), 9.68 (s, 1H, NH), 9.19 (s, 1H, NCH), 8.55 (d, J = 8.5 Hz, 1H,
Ar-H), 7.62 (d, J = 8.7 Hz, 1H, Ar-H), 7.56 (d, J = 7.8 Hz, 2H, Ar-H), 7.32 (t, J = 7.5 Hz, 2H, Ar-H), 7.32-7.02 (m,
1H, Ar-H), 3.82–3.76 (m, 6H, OCH2/SCH2), 3.40 (s, 4H, NCH2), 3.07 (d, J = 5.2 Hz, 2H, SCH2), 3.01 (d, J = 5.5
Hz, 2H, CH2).
5.3.2
1-(3-Chloro-4-fluorophenyl)-3-(5-(4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-2-yl)pyridin-2-yl)ure
a (10)
This compound was obtained as white solid in 87.2% yield. m.p.: 245.2–247.1 ; ESI-MS m/z: 535.1[M+Cl] -;
1H NMR (400 MHz, DMSO) δ 10.88 (s, 1H, NH), 9.82 (s, 1H, NH), 9.20 (s, 1H, NCH), 8.55 (d, J = 8.6 Hz, 1H,
7

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Ar-H), 7.93 (d, J = 6.4 Hz, 1H, Ar-H), 7.56 (d, J = 8.6 Hz, 1H, Ar-H), 7.51 – 7.46 (m, 1H, Ar-H), 7.36 (t, J = 9.0
Hz, 1H, Ar-H), 3.80–3.74 (m, 6H, OCH2/SCH2), 3.39 (s, 4H, NCH2), 3.06 (d, J = 5.6 Hz, 2H, CH2), 3.01 (d, J =
5.3 Hz, 2H, SCH2).
5.3.3
1-(3-Chloro-4-fluorophenyl)-3-(5-(4-morpholino-6,6-dioxido-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-2-yl)pyri
din-2-yl)urea (11)
This compound was obtained as white solid in 86.1% yield. m.p.: 285.2–286.1 ; ESI-MS m/z: 533.2[M+H]+;
1H NMR (400 MHz, DMSO) δ 10.82 (s, 1H), 9.85 (s, 1H, NH), 9.22 (s, 1H, NH), 8.57 (d, J = 8.6 Hz, 1H, NCH),
7.93 (d, J = 4.5 Hz, 1H, Ar-H), 7.58 (d, J = 8.7 Hz, 1H, Ar-H), 7.48 (s, 1H, Ar-H), 7.37 (t, J = 9.0 Hz, 1H, Ar-H),
4.37 (s, 2H, SCH2), 3.77 (s, 4H, OCH2), 3.57 (s, 2H, SCH2), 3.39 (s, 6H, NCH2/CH2).
5.3.4
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(5-(4-morpholino-6,6-dioxido-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidi
n-2-yl)pyridin-2-yl)urea (12)
This compound was obtained as white solid in 89.1% yield. m.p.: 223.1–224.2 ; ESI-MS m/z:
583.0[M+H]+; 1H NMR (400 MHz, DMSO) δ 11.01 (s, 1H, NH), 10.01 (s, 1H, NH), 9.22 (s, 1H, NCH), 8.60 (d,
J = 8.7 Hz, 1H, Ar-H), 8.21 (s, 1H, Ar-H), 7.78 (d, J = 8.4 Hz, 1H, Ar-H), 7.78 –7.68 (m, 2H, Ar-H), 4.39 (s, 2H,
SCH2), 3.77 (s, 4H, OCH2), 3.57 (s, 2H, SCH2), 3.47-3.43 (m, 6H, NCH2/CH2).
5.4. General procedure for the preparation of compounds 13–22
A
mixture
of
5a
(0.10g,
0.30mmol),
or
5b
(0.10g,
0.27
mmol),
or
4-(4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-2-yl)aniline 5a (0.10g, 0.30mmol), or 5b(0.10g,
0.28mmol), substitued sulfonylurea 8a–e in methylbenzene(10 mL) was refluxed for 4-8h. The mixture was
separated by filtration to afford the solids 13–22
5.4.1
N-((5-(4-Morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-2-yl)pyridin-2-yl)carbamoyl)thiophene-2-sulfon
amide (13)
This compound was obtained as white solid in 66.2% yield. m.p.: 226.4–227.5
;
ESI-MS m/z: 519.1[M+H]+;
1H NMR (400 MHz, DMSO) δ 9.66 (s, 1H, NH), 8.83 (s, 1H, Ar-H), 7.83 (d, J = 4.0 Hz, 1H, Ar-H), 7.55 (s, 3H,
Ar-H), 7.14 (d, J = 2.7 Hz, 1H, Ar-H), 6.72 (s, 1H, NH), 3.83 – 3.63 (m, 8H, NCH2/OCH2), 3.10 – 2.93 (m, 6H,
SCH2/CH2).
5.4.2
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4-Chloro-N-((5-(4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-2-yl)pyridin-2-yl)carbamoyl)benzenes
ulfonamide (14)
This compound was obtained as white solid in 75.3% yield. m.p.: 216.1–217.5 ; ESI-MS m/z: 548.1[M+H]+; 1H
NMR (400 MHz, DMSO) δ 9.70 (s, 1H, NH), 8.83 (s, 1H, Ar-H), 7.82 (d, J = 8.2 Hz, 2H, Ar-H), 7.65 (d, J = 8.3
Hz, 2H, Ar-H), 7.60 (d, J = 8.2 Hz, 1H, Ar-H), 7.37 (d, J = 8.2 Hz, 1H, Ar-H), 6.75 (s, 1H, NH), 3.80 – 3.64 (m,
8H, NCH2/OCH2), 3.11 – 2.95 (m, 6H, SCH2/CH2).
5.4.3
4-Fluoro-N-((5-(4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-2-yl)pyridin-2-yl)carbamoyl)benzenes
ulfonamide (15)
This compound was obtained as white solid in 80.1% yield. m.p.: 216.8–218.2 ; ESI-MS m/z: 531.2[M+H]+;
1H NMR (400 MHz, DMSO) δ 9.69 (s, 1H, NH), 8.85 (s, 1H, Ar-H), 7.88 (dd, J = 7.8, 5.8 Hz, 2H, Ar-H), 7.52
–7.45 (m, 1H, Ar-H), 7.43 –7.35 (m, 2H, Ar-H), 7.15 (dd, J = 15.6, 7.1 Hz, 1H, Ar-H), 6.74 – 6.44 (m, 1H, NH),
3.84 – 3.63 (m, 8H, NCH2/OCH2 ), 3.10 – 2.89 (m, 6H, SCH2/CH2).
5.4.4
2,4-Difluoro-N-((5-(4-morpholino-6,6-dioxido-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-2-yl)pyridin-2-yl)carba
moyl)benzenesulfonamide (16)
This compound was obtained as white solid in 60.1% yield. m.p.: 190.4–191.2 ; ESI-MS m/z: 581.2[M+H]+;
1H NMR (400 MHz, DMSO) δ 9.66 (s, 1H, NH), 8.86 (s, 1H, Ar-H), 7.86 (dd, J = 15.2, 8.1 Hz, 1H, Ar-H), 7.71 (s,
1H, Ar-H), 7.41 (s, 1H, Ar-H), 7.10 (s, 2H, Ar-H), 6.80 (s, 1H, NH), 4.40–4.34 (m, 2H, SCH2), 3.75 (s, 4H, OCH2),
3.54 (d, J = 5.7 Hz, 2H, SCH2), 3.38 (d, J = 5.0 Hz, 6H, NCH2/CH2).
5.4.5
4-Methyl-N-((5-(4-morpholino-6,6-dioxido-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-2-yl)pyridin-2-yl)carbamo
yl)benzenesulfonamide (17)
This compound was obtained as white solid in 65.3% yield. m.p.: 202.8–203.2 ; ESI-MS m/z: 559.1[M+H]+;
1H NMR (400 MHz, DMSO) δ 9.69 (s, 1H, NH), 8.88 (s, 1H, Ar-H), 7.87 (d, J = 7.9 Hz, 1H, Ar-H), 7.71 (d, J =
7.9 Hz, 2H, Ar-H), 7.36 (d, J = 7.7 Hz, 2H, Ar-H), 7.17 (d, J = 7.3 Hz, 1H, Ar-H), 6.56 (s, 1H, NH), 4.34 (d, J =
17.8 Hz, 2H, SCH2), 3.75 (s, 4H, OCH2), 3.54 (d, J = 6.4 Hz, 2H, SCH2),3.45–3.39 (m, 6H, NCH2/CH2), 2.38 (d,
J = 9.2 Hz, 3H, CH3).
9

ACCEPTED MANUSCRIPT
5.4.6
4-Chloro-N-((5-(4-morpholino-6,6-dioxido-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-2-yl)pyridin-2-yl)carbamo
yl)benzenesulfonamide (18)
This compound was obtained as white solid in 75.2% yield. m.p.: 203.8–204.4 ; ESI-MS m/z: 579.2[M+H]+;
1H NMR (400 MHz, DMSO) δ 9.76 (s, 1H, NH), 8.86 (s, 1H, Ar-H), 7.96 (d, J = 8.3 Hz, 1H, Ar-H), 7.83 (d, J =
8.3 Hz, 2H, Ar-H), 7.65 (d, J = 8.1 Hz, 2H, Ar-H), 7.37 (d, J = 8.2 Hz, 1H, Ar-H), 6.78 (s, 1H, NH), 4.40–4.34 (m,
2H, SCH2), 3.75 (s, 4H, OCH2), 3.55 (d, J = 6.1 Hz, 2H, SCH2), 3.46–3.38 (m, 6H, NCH2/CH2).
5.4.7
4-Chloro-N-((4-(4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-2-yl)phenyl)carbamoyl)Benzenesulfo
namide (19)
This compound was obtained as white solid in 79.2% yield. m.p.: 214.5–216.2 ; ESI-MS m/z: 547.1[M+H]+;
1H NMR (400 MHz, DMSO) δ 9.24 (s, 1H, NH), 8.31 (d, J = 7.5 Hz, 1H, Ar-H), 8.12 (d, J = 7.7 Hz, 1H, Ar-H),
7.92 (d, J = 8.0 Hz, 1H, Ar-H), 7.79 (d, J = 8.3 Hz, 1H, Ar-H), 7.69 (d, J = 6.8 Hz, 1H, Ar-H), 7.54 (s, 2H, Ar-H),
7.46 (d, J = 7.9 Hz, 1H, Ar-H), 6.70 (d, J = 8.0 Hz, 1H, NH), 3.84 (s, 4H, OCH2), 3.78 (d, J = 6.1 Hz, 2H, SCH2),
3.45 (s, 4H, NCH2), 3.20 – 3.05 (m, 4H, SCH2/CH2).
5.4.8
4-Fluoro-N-((4-(4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-2-yl)phenyl)carbamoyl)
benzenesulfonamide (20)
This compound was obtained as white solid in 70.2% yield. m.p.: 220.1–222.3 ; ESI-MS m/z: 530.1[M+H]+;
1H NMR (400 MHz, DMSO) δ 9.12 (s, 1H, NH), 8.31 (d, J = 8.5 Hz, 1H, Ar-H), 8.12 (d, J = 8.4 Hz, 1H, Ar-H),
7.95 (d, J = 8.0 Hz, 1H, Ar-H), 7.80 (d, J = 7.9 Hz, 1H, Ar-H), 7.53 (d, J = 4.8 Hz, 2H, Ar-H), 7.45 (d, J = 7.8 Hz,
1H, Ar-H), 7.34 (s, 1H, Ar-H), 6.69 (d, J = 8.4 Hz, 1H, NH), 3.84 (s, 4H, OCH2), 3.78 (d, J = 8.3 Hz, 2H, SCH2),
3.43 (s, 4H, NCH2), 3.08 (d, J = 5.3 Hz, 4H, SCH2/CH2).
5.4.9
4-Chloro-N-((4-(4-morpholino-6,6-dioxido-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-2-yl)phenyl)carbamoyl)be
nzenesulfonamide (21)
This compound was obtained as white solid in 65.1% yield. m.p.: 232.4–233.1 ; ESI-MS m/z: 578.2[M+H]+;
1H NMR (400 MHz, DMSO) δ 9.33 (s, 1H, NH), 8.33 (d, J = 8.1 Hz, 1H, Ar-H), 8.14 (d, J = 8.1 Hz, 1H, Ar-H),
7.92 (d, J = 8.2 Hz, 1H, Ar-H), 7.81 (d, J = 8.3 Hz, 1H, Ar-H), 7.69 (d, J = 8.2 Hz, 1H, Ar-H), 7.62–7.56 (m,
10

ACCEPTED MANUSCRIPT
2H,Ar-H), 7.46 (d, J = 8.2 Hz, 1H, Ar-H), 6.71 (d, J = 7.9 Hz, 1H, NH), 4.41–4.34 (m, 2H, SCH2), 3.85 (s, 4H,
OCH2), 3.63 (d, J = 6.0 Hz, 2H, SCH2), 3.44 (s, 6H, NCH2/CH2).
5.4.10
4-Chloro-N-((4-(4-morpholino-6,6-dioxido-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-2-yl)phenyl)carbamoyl)be
nzenesulfonamide (22)
This compound was obtained as white solid in 76.1% yield. m.p.: 254.1–255.2 ; ESI-MS m/z: 562.1[M+H]+;
1H NMR (400 MHz, DMSO) δ 9.23 (s, 1H, NH), 8.31 (d, J = 16.0 Hz, 2H, Ar-H), 8.14 (d, J = 8.2 Hz, 2H),
7.60–7.54 (m, 4H, Ar-H), 6.70 (d, J = 8.2 Hz, 1H, NH),4.50–4.41 (m, 2H, SCH2), 3.84 (s, 4H, OCH2), 3.63 (d,
J = 6.4 Hz, 2H, SCH2), 3.43 (s, 6H, NCH2/CH2)
5.5 General procedure for the preparation of compounds 23–28
A
mixture of 4-(2-(piperazin-1-yl)-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4-yl)morpholine 6a (0.10g,
0.31mmol) or 4-morpholino-2-(piperazin-1-yl)-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine 6,6-dioxide 6b
(0.10g, 0.28mmol), substituted isocyanatobenzene 7a–c in 1,4-dioxane(10mL) was refluxed for 10-30 min.
The mixture was washed with 10mL H2O and separated by filtration to afford the solids 23–28
5.5.1
4-(4-Morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-2-yl)-N-(p-tolyl)piperazine-1-Carboxamide
(23)
This compound was obtained as white solid in 85.2% yield. m.p.: 233.1–234.4 ; ESI-MS m/z: 441.2 [M+H]+;
1H NMR (400 MHz, DMSO) δ 8.57 (s, 1H,NH), 7.47 (d, J = 8.4 Hz, 2H, Ar-H), 7.24 (t, J = 7.8 Hz, 2H, Ar-H),
6.94 (t, J = 7.3 Hz, 1H,Ar-H), 3.71 (d, J = 3.9 Hz, 8H, NCH2), 3.57 (d, J = 5.4 Hz, 2H,CH2), 3.51 (d, J = 5.0 Hz,
4H, CH2), 3.22 (s, 4H,OCH2), 2.88 (dd, J = 16.6, 5.4 Hz, 4H, NCH2); 13C NMR (101 MHz, DMSO) δ 165.47(C),
165.18(C), 159.17(C), 155.58(C), 140.96(C), 128.77(2CH), 122.24(C), 120.15(2CH), 107.34(CH), 66.43(2CH2),
49.36(2CH2), 44.02(2CH2), 43.83(2CH2), 34.01(CH2), 26.19(CH2), 25.93(CH2).
5.5.2
N-(4-Chloro-3-(trifluoromethyl)phenyl)-4-(4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-2-yl)piperazi
ne-1-carboxamide (24)
This compound was obtained as white solid in 88.1% yield. m.p.: 218.2–220.4 ; ESI-MS m/z: 543.2[M+H]+;
1H NMR (400 MHz, DMSO) δ 9.47 (s, 1H,NH), 8.17 (s, 1H,Ar-H), 7.90 –7.82 (m, 1H, Ar-H), 7.56 (d, J = 8.8 Hz,
1H, Ar-H), 3.87 (s, 4H,OCH2), 3.69 (s, 8H, NCH2), 3.67 (s, 2H, SCH2), 3.64 (s, 4H,NCH2), 3.12 (t, J = 5.2 Hz,
11

ACCEPTED MANUSCRIPT
2H, CH2), 2.93 (t, J = 5.9 Hz, 2H, SCH2); 13C NMR (101 MHz, DMSO) δ 163.26(C), 154.95(C), 140.78(C),
131.97(C), 126.90(C), 126.60(C), 124.76(C), 124.42(C), 122.59(CH), 122.05(C), 118.44(CH), 106.81(CH),
66.41(2CH2),48.80(2CH2), 45.23(2CH2), 43.59(2CH2), 29.96(CH2), 26.30(CH2), 24.74(CH2).
5.5.3
N-(4-Bromophenyl)-4-(4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-2-yl)piperazine-1-
Carboxamide (25)
This compound was obtained as white solid in 88.9% yield. m.p.: 209.5–212.2 ; ESI-MS m/z: 521.1[M+H]+;
1H NMR (400 MHz, DMSO) δ 8.84 (s, 1H, NH), 7.49 (d, J = 8.3 Hz, 2H, Ar-H), 7.40 (d, J = 8.2 Hz, 2H, Ar-H),
3.74 (s, 6H, 2NCH2/SCH2), 3.69 (s, 4H, OCH2), 3.64 – 3.47 (m, 8H, NCH2), 2.91 (s, 4H,SCH2/ CH2); 13C NMR
(101 MHz, CDCl3) δ 164.44(C), 155.25(C), 140.48(C), 140.48(C), 136.97(C), 131.52(2CH), 121.92(2CH),
113.73(C), 107.16(C), 66.43(2CH2), 49.16(2CH2), 44.26(2CH2), 43.84(2CH2), 26.09(2CH2), 25.76(CH2).
5.5.4
4-(4-Morpholino-6,6-dioxido-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-2-yl)-N-(p-tolyl)piperazine-1-carboxami
de (26)
This compound was obtained as white solid in 83.1% yield. m.p.: 248.2–249.9 ; ESI-MS m/z: 495.2[M+Na]+;
1H NMR (400 MHz, DMSO) δ 8.59 (s, 1H, NH), 7.47 (d, J = 8.5 Hz, 2H, Ar-H), 7.24 (t, J = 7.8 Hz, 2H, Ar-H),
6.94 (t, J = 7.3 Hz, 1H, Ar-H), 4.16 (s, 2H, SCH2), 3.72 (d, J = 3.7 Hz, 8H, NCH2), 3.51 (s, 4H, OCH2), 3.44 (t, J
= 6.5 Hz, 2H, SCH2), 3.20 (s, 4H, NCH2), 3.15 (t, J = 6.7 Hz, 2H, CH2)
5.5.5
N-(4-Chloro-3-(trifluoromethyl)phenyl)-4-(4-morpholino-6,6-dioxido-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-
2-yl)piperazine-1-carboxamide (27)
This compound was obtained as white solid in 89.1% yield. m.p.: 254.1–256.3 ; ESI-MS m/z: 597.1[M+Na]+;
1H NMR (400 MHz, DMSO) δ 9.19 (s, 1H, NH), 8.10 (s, 1H, Ar-H), 7.83 (d, J = 8.8 Hz, 1H, Ar-H), 7.54 (d, J =
8.8 Hz, 1H, Ar-H), 4.14 (s, 2H, SCH2), 3.80–3.71 (m, 8H, NCH2), 3.55 – 3.50 (m, 4H, OCH2), 3.42 (d, J = 6.3 Hz,
2H,SCH2), 3.18 (s, 4H, NCH2), 3.13 (t, J = 6.6 Hz, 2H, CH2).
5.5.6
N-(4-Bromophenyl)-4-(4-morpholino-6,6-dioxido-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-2-yl)piperazine-1-c
arboxamide (28)
This compound was obtained as white solid in 90.3% yield. m.p.: 264.1–265.5 ; ESI-MS m/z: 573.1[M+Na]+;
1H NMR (400 MHz, DMSO) δ 8.73 (s, 1H, NH), 7.45 (d, J = 7.9 Hz, 2H, Ar-H), 7.39 (d, J = 7.9 Hz, 2H,Ar-H),
12

ACCEPTED MANUSCRIPT
4.14 (s, 2H, SCH2), 3.70 (s, 8H, NCH2), 3.50 (s, 4H, OCH2), 3.42 (s, 2H, SCH2), 3.18 (s, 4H, NCH2), 3.14 (d, J =
6.1 Hz, 2H, CH2).
5.6 Cytotoxicity assay in vitro
The cytotoxicity of target compounds (9–28) were evaluated with A549, PC-3, MCF-7 cell lines by the
standard MTT assay in vitro, with Sorafenib and GDC-0941 as positive controls. The cancer cell lines were
cultured in minimum essential medium (MEM) supplement 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
5 mg/mL and incubated with cells at 37°C for 4 h. T he formazan crystals were dissolved in 100 mL DMSO each
well, and the absorbency at 492 nm (for absorbance of MTT formazan) and 630 nm (for the
referencewavelength) 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.
5.7 mTOR Kinase Assay
The mTOR kinase activity of four selected compounds (10, 11, 24, 27) were determined using LANCE®
Ultra time-resolved fluorescence resonance energy transfer (TR-FRET) assay following the manufacturer's
instructions, with GDC-0941 and PI103 as positive controls [22]. Briefly, mTOR enzyme (10 nM), ATP (21.6
µM), 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 temperature for 1 h and stopped by adding EDTA
to 16 mM. Eu-antiphospho-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 (excitation 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.
13

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5.8 PI3Kα kinase assay
Four selected compounds (10, 11, 24, 27) are tested for their activity against PI3Kα using a Kinase-Glo®
Luminescent Kinase Assay, with GDC-0941 and PI103 as positive controls[23]. The kinase reaction is done in
384-well black plate. Each well is loaded with 50 mL of test items (in 90% DMSO) and 5 mL reaction buffer
containing 10 mg/mL PI substrate (L-a-phosphatidylinositol; Avanti Polar Lipids; prepared in 3% octyl-glucoside)
and the PI3kα protein 10 nM is then added to it. The reaction is started by the addition of 5 mL of 1 mMATP
prepared in the reaction buffer and is incubated for 60 min for p110α.It is terminated by the addition of 10 mL
Kinase-Glo buffer. The plates are then read in a Synergy 2 reader for luminescence detection. All of the
compounds were tested two times. The results expressed as IC50 (inhibitory concentration 50%) were the
averages of two determinations.
5.9 c-Met kinase assay
Four selected compounds (10, 11, 24, 27) are tested for their activity against c-Met kinase through the mobility
shift assay [24]. All kinase assays were performed in 96-well plates in a 50 ꢀL reaction volume. The kinase
buffer contains 50 mM HEPES, pH 7.5, 10 mM MgCl2, 0.0015% Brij-35 and 2 mM DTT. The stop buffer
contains 100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3 and 50 mM EDTA. Dilute the
compounds to 500 ꢀM by 100% DMSO, then transfer 10 ꢀL of compound to a new 96-well plate as the
intermediate plate, add 90 ꢀL kinase buffer to each well. Transfer 5 ꢀL of each well of the intermediate plate to
384-well plates. The following amounts of enzyme and substrate were used per well: kinase base buffer,
FAM-labeled peptide, ATP and enzyme solution. Wells containing the substrate, enzyme, DMSO without
compound were used as DMSO control. Wells containing just the substrate without enzyme were used as low
control. Incubate at room temperature for 10 min. Add 10 ꢀL peptide solution to each well. Incubate at 28 °C for
specified period of time and stop reaction by 25 ꢀL stop buffer. At last collect data on Caliper program and
convert conversion values to inhibition values. Percent inhibition = (max − conversion)/(max − min) × 100.
“max” stands for DMSO control; “min” stands for low control.
5.10 Docking studies
For docking purposes, we prepared the receptor proteins PDB ID code:4JT6(mTOR) and and PDB ID
code:4L23(PI3Kα). The three-dimensional structures of the mTOR (PDB code: 4JT6) and PI3Kα (PDB ID
code:4L23) [20-21] were obtained from RCSB Protein Data Bank. We built a small organic molecules set
14

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(compound 10, 11, 24, 27) and used Gasteiger-Huckel to optimized the molecular force field and structure.
Hydrogen atoms were added to the structure allowing for appropriate ionization at physiological pH. First of all,
extract ligand substructure, then remove water and excess structure, finally, add hydrogens and fix sidechain
amides. The protonated state of several important residues 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 or PI3Kα 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, 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. Lastly,
docking resultsand the optimized molecular docking model with the receptor proteins was obtained.
Acknowledgments
We gratefully acknowledge the generous support provided by The National Natural Science Funds (No.
81460527), Project supported by the Natural Science Foundation of Jiangxi Province (No. 20142BAB215020) ,
Program of Key Laboratory of Drug Design and Optimization, Jiangxi Science & Technology Normal University
(300098010306) , College Students' Science and Technology Innovation Project (20140802034) and Graduate
Students' Science and Technology Innovation Project of Jiangxi Province (YC2015-X25).
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Legends
Fig. 1 Structures of some reported (fused-)pyrimidine/triazines
Fig. 2 Structures and design strategy for target compounds 9–28
Fig. 3a-b. Binding models of compound 11 and native ligand PI 103 target into active site of mTOR(Fig.3a) and
PI3Kα(Fig.3b). The proteins were displayed by cyan and white ribbon. Compound 11 and lead compound were
displayed by orange and green sticks, respectively. H-bonding interactions between the 11, lead compound
and mTOR/PI3Kα were indicated with dashed lines in yellow.
Scheme 1. Synthetic routes of key intermediates 5a–d and 6a–b
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) Na2WO4·2H2O, 30% H2O2, 20℃, 3 h; (f)
1)bis(pinacolato)diboron, KAc, Pd(PPh3)2Cl2, 1,2-dimethoxyethane, reflux, 2 h; 2) H2O, Na2CO3, Pd(PPh3)2Cl2,
reflux, 6 h; (g) NH2NH2·H2O, FeCl3·6H2O, actived C, EtOH, 1 h; (h) EtOH, K2CO3, reflux, 2–3 h;
Scheme 2. Synthetic routes of target compounds 9–12, 13–18, 19–22 and 23–28
Reagents and conditions: (a) 1,4-dioxane, H2O, rt, 10–30mins; (b) methylbenzene, rt, 4–8h.
Table 1 Structures and cytotoxicity of target compounds 9–28
17

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a The values are an average of two separate determinations.
b Used as a positive control
c NA: Not active (IC50 > 50 µM)
dND: Not determined
Table 2 Melocular docking scores and mTOR/PI3Kα kinases activity of selected compounds and positive
controls.
a The values are an average of two separate determinations.
b Used as a positive control
cND: Not determined
18

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Table 1 Structures and cytotoxicity of target compounds 9–28
IC50a（µM）
Comp.
No.
R(9–12,23–28)
/Ar(13–22)
A549
PC-3
NA c
MCF-7
46.62±1.12
1.27±0.32
H
9
20.52±0.92
11.47±0.76
4.82±0.24
4.67±0.42
NA
6.07±0.61
0.52±0.10
NA
3-Cl-4-F
3-Cl-4-F
10
11
1.41±0.10
3-CF3-4-Cl
thiophene
4-Cl-phenyl
32.24±0.96
12
13
14
NA
NA
46.88±1.05
NA
NA
NA
NA
NA
4-F-phenyl
36.17±0.98
22.67±0.89
NA
15
16
2,4-diF-phenyl
19.65±0.88
4-CH3- phenyl
4-Cl-phenyl
42.46±1.02
NA
NA
NA
NA
NA
17
18
19
20
21
NA
NA
NA
NA
NA
NA
NA
NA
4-Cl-phenyl
4-F-phenyl
4-Cl-phenyl
4-F-phenyl
H
NA
NA
NA
NA
NA
NA
22
23
3-CF3-4-Cl
26.73±0.85
24
3.83±0.32
5.80±0.52
4-Br
H
39.90±0.89
NA
NA
NA
25
26
27
4.68±0.41
NA
3-CF3-4-Cl
28.73±0.80
6.13±0.62
5.94±0.50
4-Br
19.01±0.87
NA
45.57±0.95
28

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Sorafenibb
GDC-0941 b
-
-
6.53±0.82
1.2 ± 0.11
8.08±0.91
0.8 ± 0.44
4.21±0.62
NDd
a The values are an average of two separate determinations.
b Used as a positive control
c NA: Not active (IC50 > 50 µM)
dND: Not determined
Table 2 Melocular docking scores and mTOR/PI3Kα kinases activity of selected compounds and positive
controls.
Melocular
Compound
NO.
IC50 a (µM)
PI3Kα
Docking Scores
mTOR PI3Kα
mTOR
0.86±0.10
0.94±0.10
6.26±0.52
9.43±0.80
0.58
c-Met
>10
>10
>10
>10
NDc
9.58
9.40
6.27
7.92
-
9.05
9.37
6.96
8.57
-
0.69±0.10
6.72±0.30
>10
10
11
24
27
4.23±0.20
0.003
GDC-0941b
0.019 ±
0.004
PI103b
-
-
0.011 ± 0.002
ND
a The values are an average of two separate determinations.
b Used as a positive control
cND: Not determined

ACCEPTED MANUSCRIPT
(Fig. 1)
Fig.1. Structures of some reported (fused-)pyrimidine/triazines
(Fig. 2)
Fig.2. Structures and design strategy for target compounds 9–28

ACCEPTED MANUSCRIPT
(Fig. 3.)
Fig.3a-b
Fig. 3a-b. Binding models of compound 11 and native ligand PI 103 target into active site of mTOR(Fig.3a)
and PI3Kα(Fig.3b). The proteins were displayed by cyan and white ribbon. Compound 11 and lead
compound were displayed by orange and green sticks, respectively. H-bonding interactions between the 11,
lead compound and mTOR/PI3Kα were indicated with dashed lines in yellow.

ACCEPTED MANUSCRIPT
(Scheme 1.)
Scheme 1. Synthetic routes of key intermediates 5a–5d and 6a–6b
Cl
O
O
COOMe
S
N
S
O
S
NH
S
b
c
a
COOMe
N
3
Cl
O
N
H
O
1
2
O
N
Br
n(O)S
n(O)S
n(O)S
N
N
f
NH2
N
5a n=0
5b n=2
N
NH2
O
O
Br
N
N
NO2
n(O)S
e
N
N
d
N
Cl
N
f,g
5c n=0
5d n=2
4a n=0
4b n=2
NH2
O
N
HN
NH
N
h
N
N
NH
6a n=0
6b n=2
8a:Ar=thiophene
NCO
R
7a:R=H
7b:R=Br
7c:R=3-CF3-4-Cl
7d:R=3-Cl-4-F
O
O
8b:Ar=4-Cl-phenyl
8c:Ar=4-F-phenyl
8d:Ar=4-CH3-phenyl
O
Et
S
O
N
Ar
H
8e:Ar=2,4-diF-phenyl
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) Na2WO4·2H2O, 30% H2O2, 20℃, 3 h; (f)
1)bis(pinacolato)diboron, KAc, Pd(PPh3)Cl2, 1,2-dimethoxyethane, reflux,
2 h; 2) H2O, Na2CO3,
Pd(PPh3)2Cl2, reflux, 6 h; (g) 80% NH2NH2·H2O, FeCl3.6H2O, actived C, EtOH, 78 ℃, 1 h; (h) EtOH, K2CO3,
reflux, 2–3 h;

ACCEPTED MANUSCRIPT
(Scheme 2.)
Scheme 2. Synthetic routes of target compounds 9–28
O
N
9:R=H
7a,7c,7d
10:R=3-Cl-4-F
11:R=3-Cl-4-F
12:R=3-CF3-4-Cl
n(O)S
N
9,10,n=0
11,12,n=2
a
N
O
R
N
N
N
H
H
5a,5b
O
N
13:Ar=thiophene
14:Ar=4-Cl-phenyl
15:Ar=-4-F-phenyl
16:Ar=2,4-diF-phenyl
17:Ar=4-CH3-phenyl
18:Ar=4-Cl-phenyl
8a-8e
13-15:n=0
16-18:n=2
n(O)S
N
b
N
O
O
S
O
N
N
N
Ar
H
H
O
N
19:Ar=4-Cl-phenyl
n(O)S
N
8b,8c
19,20:n=0 20:Ar=4-F-phenyl
5c,5d
6a,6b
21,22:n=2
21:Ar=4-Cl-phenyl
22:Ar=4-F-phenyl
b
N
O
O
S
O
N
N
H
Ar
H
O
N
N
23,26:R=H
24,27:R=3-CF3-4-Cl
25.28:R=4-Br
7a-7c
n(O)S
N
23-25:n=0
26-28:n=2
a
N
H
N
N
R
O
Reagents and conditions: (a) 1,4-dioxane, rt, 10–30mins; (b) methylbenzene, rt, 4–8h.

ACCEPTED MANUSCRIPT
Research Highlights
►
Four series of 2-substituted-4-morpholino- 7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine derivatives were
designed and synthesized
►
►
►
►
Most of the synthesized compounds showed moderate to significant antitumor activity.
3-Cl-4-F (10–11) or 3-CF3-4-Cl (12, 24, 27)substitution were more preferred.
Compound 11 was a similar antitumor activities level of GDC-0941.
Docking study was investigated to explore the binding modes of compounds with mTOR/PI3Ka