Synthesis and structure-activity relationships of 4-morpholino-7,8-dihydro-5h-thiopyrano[4,3-d] pyrimidine derivatives bearing pyrazoline scaffold

Article abstract of DOI:10.3390/molecules22111870

Phosphatidylinositol 3-kinase/Protein kinase B/Mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway is abnormally active in the growth and proliferation of cancer cells. The inhibition of PI3K kinase can effectively block the conduction of sign

Full text of DOI:10.3390/molecules22111870

molecules
Article
Synthesis and Structure–Activity Relationships of
4-Morpholino-7,8-Dihydro-5H-Thiopyrano[4,3-d]
pyrimidine Derivatives Bearing Pyrazoline Scaffold
Qinqin Wang 1, Xiaojing Li 2, Chengyu Sun 3, Binliang Zhang 1, Pengwu Zheng 1, Wufu Zhu 1,
*
and Shan Xu 1,
*
1
Jiangxi Provincial Key Laboratory of Drug Design and Evaluation, School of Pharmacy,
Jiangxi Science & Technology Normal University, Nanchang 330013, China;
m15180134379_2@163.com (Q.W.); zbl1045762244@163.com (B.Z.); zhengpw@jxstnu.edu.cn (P.Z.)
College of Service, Naval Logistics Academy of PLA, Tianjin 300450, China; 13970892404@163.com
Department of Pharmacy, The Affiliated Hospital of Chongqing Three Gorges Medical College, Wanzhou,
Chongqing 404000, China; sunchengyu0902@163.com
2
3
*
Correspondence: zhuwf@jxstnu.edu.cn (W.Z.); xush@jxstnu.edu.cn (S.X.); Tel./Fax: +86-791-8380-2393 (W.Z.)
Received: 6 September 2017; Accepted: 25 October 2017; Published: 31 October 2017
Abstract: Phosphatidylinositol 3-kinase/Protein kinase B/Mammalian target of rapamycin
(PI3K/Akt/mTOR) signaling pathway is abnormally active in the growth and proliferation
of cancer cells.
signaling pathways and is an ideal target for drug design.
of 4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine derivatives bearing pyrazoline
moiety (7a 8a ) were synthesized; and their cytotoxicity in vitro were evaluated by
The inhibition of PI3K kinase can effectively block the conduction of
In this paper; two series
–
l;
–l
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method against four human
cancer cell lines including A549; PC-3; MCF-7; and HepG2 cell lines. The activity of the most
promising compound 8d against PI3Kα kinase was further evaluated. The results indicated that
most of the target compounds showed moderate to excellent cytotoxicity and the most promising
compound 8d showed excellent cytotoxicity against four cancer cell lines with half maximal inhibitory
concentration (IC50) values of 6.02–10.27
a moderate inhibitory activity in the PI3K
µ
M. In addition; the compound 8d was found to have
α
enzyme assay. What’s more; the compounds of which the
substituents of benzene ring at the C-4 position are electron-withdrawing groups such as substituents
(Cl; F; Br) have better activity than the compounds containing the electron donating groups (OCH3; H).
However; the exact action mechanism is not quite clear right now. Further study will be carried out
to identify the exact target in near future.
Keywords: thiopyrano[4,3-d]pyrimidine; pyrazoline; synthesis; cytotoxicity activity; PI3Kα kinase
1. Introduction
Cancer is the main killer of human health, causing high mortality, posing a serious threat to
human health. Chemotherapy has been widely applied in various cancer treatments. The toxicity
and resistance of traditional chemotherapeutic drugs make it urgent to find new targets and novel
drugs for the cancer therapy. The family of lipid kinases termed phosphatidylinositol 3-kinases (PI3Ks)
catalyze the phosphorylation of the 3-hydroxyl position of phosphatidylinositides and has been found
to play a regulatory role in many cellular processes, including cell growth, proliferation, differentiation,
motility, and survival [1]. The PI3K family consists of at least eight proteins that share sequence
homology within their kinase domains and yet have distinct substrate specificities and modes of
regulation. In addition, PI3K signaling pathway is negatively regulated by phosphatase and tensin
Molecules 2017, 22, 1870; doi:10.3390/molecules22111870
www.mdpi.com/journal/molecules

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homolog deleted on chromosome ten (PTEN), which is one of the most commonly mutated proteins
in human malignancy, providing further evidence for the role of the PI3K pathway in cancer [ ].
Many industrial and academic groups paid attention to selective PI3K inhibitors [ ] and several of
these inhibitors have entered to Phase II clinical trials, such as Pictilisib dimethanesulfonate (GDC-0941,
Figure 1). GDC-0941 is the first selective PI3K inhibitor developed by Genentech with a half maximal
inhibitory concentration (IC50) value of 3 nM [4].
2
3
α
Figure 1.
Structures of representative compounds of two kinds of inhibitors and target
compounds. Pictilisib dimethanesulfonate (GDC-0941), 4-((2-(4,6-dimorpholino-1,3,5-triazin-2-yl)
hydrazono)methyl)-2,6-dimethoxyphenol (BMCL-200908069-1).
In our previous study, a series of thiopyrano-pyrimidine compounds containing aromatic
hydrazone structures were designed and synthesized, and Structure-Activity Relationships
(SARs) were discussed and summarized. The results showed that these compounds had strong
cytotoxicity activity. The activity of the most active compound I (Figure 1) is superior to the
lead compound 4-((2-(4,6-dimorpholino-1,3,5-triazin-2-yl)hydrazono)methyl)-2,6-dimethoxyphenol
(BMCL-200908069-1, Figure 1). The SARs results of target compounds demonstrated that the
construction of thiopyrano-pyrimidine nucleus and the introduction of hydrazine group are beneficial
to the cytotoxicity activity of target compounds. However, the activity of these compounds is not as
good as GDC-0941, and the inhibitory activity against PI3K kinase is not ideal. This is inconsistent with
the expected goal of obtaining good PI3Kα kinase inhibitors [5]. It is necessary to further optimize the
previous synthesized compounds. In order to study the effect of changing the structure of the aromatic
hydrazone on antitumor activity, we plan to optimize the structure of compound I. As we know,
many pyrazoline derivatives have been reported to exhibit various pharmacological activities, such as
compounds II and III [6] (Figure 1), and pyrazoline unit was proved to be a good pharmacophore.
Therefore, inspired by pyrazoline derivatives, we decided to retain the thiopyrano-pyrimidine nucleus
structure, the hydrazine group is incorporated into the ring to construct pyrazoline unit. Finally,
we designed and synthesized a series of pyrazole-containing thiopyrano pyrimidine compounds 7a–l
.
The different substituents were introduced into the C-4 position of the benzene ring to investigate
their effect on the activity. In order to further investigate the effect of oxidation of the sulfur atom
on cytotoxicity activity, compounds 8a–l were prepared via oxidize reaction. As a result, two series
of 4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine derivatives (7a–l and 8a–l) bearing
pyrazoline moiety were designed and synthesized (Figures 1 and 2).

Molecules 2017, 22, 1870
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Figure 2. Structures and design strategy for the target compounds (7a–l and 8a–l).
Herein, we disclosed the synthesis and antitumor activity of all the target compounds against four
cancer cell lines human lung cancer cell lines A549, human prostate cancer cell lines PC-3, human breast
cancer cell lines MCF-7, and human hepatoma cell lines HepG2 of thiopyrano[4,3-d]pyrimidine bearing
pyrazoline moiety, as well as the activity against PI3Kα kinase. In addition, we also introduce the
docking studies reulsts.
2. Results and Discussion
The synthetic routes to obtain the compounds 7a–l and 8a–l are outlined in Scheme 1.
Scheme 1. Synthetic route of target compounds. Reagents and conditions: (
a
) 80% NH2NH2
·
H2O, EtOH,
◦
◦
78 C 1 h; (b) 10% NaOH, EtOH, r.t., 24 h; (c) Na2WO4·2H2O, 30% H2O2, 20 C, 3 h; (d) Glacial acetic
◦
acid, H2SO4; 100 C.
The key intermediates 4a and 4b were synthesized from commercially available
0
3,3 -thiomalo-dimethyl malonate through five steps, which were reported in our previously research [
Compounds 4a and 4b were each treated with 80% hydrazine monohydrate in refluxing ethanol,
generating 5a and 5b ]. Then, 5a and 5b were each reacted with chalcone 6a , all of which
were synthesized according to reported procedures [ ] to yield the target compounds (7a and
8a ). The relative stereochemistry of the target compounds (7a 8a ) was confirmed as R isomeric
according to the method in our previous research [ ]. The target compounds 7a and 8a were
established on the basis of 1H-NMR. In a typical example, in the 1H-NMR spectrum of compound 8d
5].
[
7
–l
6
–l
–
l
–l
,
–l
6
–
l
–l
,
the chiral proton of the pyrazoline ring appeared as a doublet of a doublet centered at d 5.63 with
a coupling constant J = 11.9 Hz and J = 5.7 Hz integrating for one proton. The pro-chiral CH2 protons
of pyrazoline ring appeared as one distinct doublet of a doublet centered at d 3.87 (due to geminal and

Molecules 2017, 22, 1870
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vicinal coupling and having a coupling constant of 17.7 Hz and 12.4 Hz), and one of the hydrogen of
methylene groups is mixed in morpholine.
2.1. Biological Evaluation
Taking GDC-0941 as the reference compound, the target compounds (7a–l and 8a–l) were
evaluated for the cytotoxicity against four cancer cell lines A549, PC-3, MCF-7, and HepG2 by
3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay. In order to
confirm the types of target compound, selective or mixed-type inhibitors, the selected compound, 8d
was measured for its PI3K kinase inhibitory activity together with reference compounds GDC-0941
and PI103 via the Kinase-Glo® Luminescent time-resolved fluorescence resonance energy transfer
(TR-FRET) assay [ ]. The results expressed as inhibition rates or IC50 values are summarized in Tables 1
,
α
7
and 2, where the values are the average of at least two independent experiments.
Table 1. Structures and cytotoxicity of Compounds 7a–l and 8a–l.
IC50 (µM) a
Compd.
n
R1
R2
A549
PC-3
HepG2
MCF-7
7a
7b
7c
7d
7e
7f
7g
7h
7i
0
0
0
0
0
0
0
0
0
0
0
0
2
2
2
2
2
2
2
2
2
2
2
2
H
H
CH3
Br
F
H
OCH3
F
OCH3
CH3
F
H
OCH3
H
CH3
Br
F
H
OCH3
F
OCH3
CH3
F
H
OCH3
21.68 ± 0.76
24.17 ± 0.74
13.02 ± 0.42
6.02 ± 1.22
NA
28.8 ± 2.40
14.09 ± 3.45
12.879 ± 1.56
8.91 ± 0.72
37.76 ± 3.64
NA
15.15 ± 1.54
29.82 ± 0.89
25.43 ± 0.74
14.75 ± 1.02
17.83 ± 0.55
28.25 ± 0.14
NA
17.80 ± 1.18
37.58 ± 0.22
19.06 ± 0.87
NA
38.65 ± 0.18
21.84 ± 0.67
26.55 ± 0.33
31.89 ± 0.25
31.86 ± 0.92
33.52 ± 1.67
37.38 ± 0.23
0.20 ± 0.08
20.34 ± 1.15
27.81 ± 0.99
15.36 ± 0.03
8.39 ± 1.91
NA
21.74 ± 0.78
42.65 ± 1.75
22.98 ± 0.19
10.27 ± 0.94
35.89 ± 0.71
NA
26.35 ± 0.37
30.26 ± 0.88
27 .29 ± 1.85
28.25 ± 0.14
35.68 ± 0.74
NA
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
F
F
Br
Br
Br
H
H
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
F
NA
NA
16.92 ± 0.66
26.72 ± 0.88
16.41 ± 0.43
19.25 ± 1.25
31.19 ± 0.59
36.38 ± 0.62
27.58 ± 0.85
35.65 ± 1.47
27.80 ± 1.18
23.84 ± 0.67
NA
20.27 ± 0.37
20.47 ± 0.67
NA
23.83 ± 0.55
32.73 ± 0.36
NA
7j
7k
7l
8a
8b
8c
8d
8e
8f
8g
8h
8i
32.73 ± 0.39
41.25 ± 1.25
42.46 ± 0.03
43.86 ± 0.15
NA
NA
0.06 ± 0.01
30.67 ± 0.09
NA
35.01 ± 0.18
36.01 ± 0.18
32.91 ± 2.82
24.68 ± 0.74
27.76 ± 0.64
35.49 ± 0.33
NA
35.31 ± 0.49
NA
NA
29.08 ± 0.49
34.97 ± 0.74
28.08 ± 0.89
29.66 ± 0.32
34.99 ± 0.47
NA
35.32 ± 0.39
33.87 ± 0.45
NA
46.63 ± 0.89
32.61 ± 0.49
NA
F
Br
Br
Br
8j
8k
8l
H
GDC-0941 b
6.99 ± 0.21
0.07 ± 0.03
4.30 ± 0.84
a
The half maximal inhibitory concentration (IC50) values are an average of two separate determinations; b Used as
a positive control; NA: No Activity.
Table 2. Structures and cytotoxicity of Compounds 7a–l and 8a–l.
a
IC50 (µM)
Compound
PI3Kαc
8d
8.38 ± 0.21
GDC-0941 b
PI-103 b
0.003
0.075 ± 0.018
a
b
c
The IC50 values are an average of two separate determinations;
Phosphatidylinositol 3-kinase α.
Used as a positive control; PI3Kα,

Molecules 2017, 22, 1870
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The results of the cytotoxicity assay of the compounds are summarized in Table 1. As expected,
we can see that most of the target compounds showed moderate cytotoxicity against the four tested
cancer cell lines and most of them showed better activities against PC-3 and HepG2 cancer cell lines
than the other two cancer cell lines (A549, MCF-7). Compared with compounds 7a
activities of compounds 8a were decreased or lost. This result shows that the oxidation of sulfur
atom was not benefical to the cytotoxicity. However, the cytotoxicity against the four cancer cell lines
of compounds 7a and 8a varied in degree because of the different substituents of the benzene
ring at the C-4 position. As seen in Table 1, the electron-withdrawing group-containing compounds
7c 7d 7g, and 7j) have better antitumor activity than those compounds containing electron-donating
groups (7h 7l). This result indicates that compounds of which the substituents of benzene ring at
–l, the cytotoxicity
–l
–l
–l
(
,
,
,
the C-4 position are electron-withdrawing groups such as substituents (Cl, F, Br) have better activity
than that of the compounds containing the electron donating groups (OCH3, H). The most promising
compound, 8d, showed moderate cytotoxicity activity to GDC-0941 with the IC50 values of 6.02 ± 1.22 µM
,
8.91 0.72 M, 8.39 1.91 M, and 10.27 0.94 M. The results show that halogen atom is
±
µ
±
µ
±
µ
a strong electron-withdrawing group, and the introduction of halogen atom can affect the drug
charge distribution, thereby enhancing the electrical interaction with the receptor.
The activities of the selected compound 8d as well as the lead compounds against PI3K
are shown in Table 2. The results showed that compound 8d exhibited moderate inhibitory rate against
PI3K kinase, and it indicated that the replacement of aromatic hydrazone with pyrazoline moiety
α kinase
α
increased the activity against PI3Kα kinase.
2.2. Molecular Docking Study
To explore the binding modes of target compounds with the active site of PI3K
α
, molecular
] of the SYBYL
package. Based on the in vitro inhibition results, we selected compound 8d as the ligand example,
docking simulation studies were carried out using a SURFLEX-DOCK module [
8
and the structures of PI3Kα (Protein Data Bank (PDB) ID code: 4L23 [7]) were selected as the
docking models.
The binding mode of compound 8d with the active site of PI3K
As depicted in Figure 3, the morpholine groups of compound 8d and PI103 were almost completely
overlapped. In the docking model of compound 8d with PI3K , we can see the pyrimidine on
α molecular is shown in Figure 3.
α
the 2-position nitrogen formed one hydrogen bond with residue Asp2357. In addition, a nitrogen
atom on a pyrazole structure formed one hydrogen bond with residue Asn2343 and the fluorine
atom of the benzene group also formed one hydrogen bond with residue His2340, respectively.
The above-mentioned results of SARs analysis and molecular docking study may allow for the rational
design of PI3Kα inhibitors.
Figure 3. Binding models of Compound 8d (shown in orange sticks) and native ligand PI103 (shown
in blue sticks) with PI3K
α. The proteins are represented by green ribbon. Hydrogen bonds are showed
in dashed lines (yellow).

Molecules 2017, 22, 1870
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3. Experimental Section
3.1. General Information
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 electrospray ionization (ESI) mode on Agilent 1100 Liquid chromatography–mass
spectrometry (LC-MS) (Agilent, Palo Alto, CA, USA). Thin layer chromatography (TLC) analysis was
carried out on silica gel plates GF254 (Qindao Haiyang Chemical, Qingdao, China). All materials were
obtained from commercial suppliers and used without purification, unless otherwise specified. Yields
were optimized.
3.2. Chemistry
3.2.1. General Procedure for Preparation of Compounds 4a–b and 5a–b
Compounds 4a–b and 5a–b were synthesized according to the reported procedures by our
research group [5,7].
3.2.2. General Procedure for the Preparation of Compounds 6a–l
Suitably substituted acetophenone (0.1 mol) and substituted aromatic aldehyde (0.1 mol) were
dissolved in ethanol (30 mL). To the clear reaction mixture, 10% NaOH was added dropwise.
The reaction mixture was agitated and allowed to stand at room temperature for 24 h. The precipitated
crystals of 6a–l were collected by filtration, washed, and recrystallized from EtOH.
3.2.3. General Procedure for the Preparation of Target Compounds 7a–l and 8a–l
Substituted chalcone (0.001 mol) and substituted 4-(2-hydrazinyl-7,8-dihydro-5H-thiopyrano
[4,3-d]pyrimidin-4-yl)morpholine (0.001 mol) or 2-hydrazinyl-4-7,8-dihydro-5H-thiopyrano[4,3-d]
pyrimidine 6,6-dioxide (0.001 mol) were dissolved in 20 mL of glacial acetic acid. Catalytic amount
of conc. H2SO4 was added. We then obtained the target compounds, 7a–l and 8a–l, according to the
method reported in the literature [6].
(R)-4-(2-(3,5-Diphenyl-4,5-dihydro-1H-pyrazol-1-yl)-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4-yl)
morpholine (7a). A brown yellow solid. Yield: 70%. m.p. 139–142 ◦C. ESI-MS m/z: [M + H]+ 457.6;
1H-NMR (400 MHz, DMSO)
δ 7.76 (d, J = 7.9 Hz, 2H, Ar-H), 7.43 (d, J = 7.8 Hz, 3H, Ar-H), 7.27
(d, J = 7.7 Hz, 2H, Ar-H), 7.20 (d, J = 7.6 Hz, 3H, Ar-H), 5.63 (dd, J = 11.9, 5.5 Hz, 1H, pyrazoline),
3.87 (dd, J = 17.7, 12.3 Hz, 1H, pyrazoline), 3.55 (d, J = 14.9 Hz, 3H, thiopyrano and morpholine
hydrogen), 3.52–3.43 (m, 3H, thiopyrano and morpholine hydrogen), 3.40 (s, 1H, pyrazoline), 3.10
(dd, J = 17.3, 5.4 Hz, 3H, thiopyrano and morpholine hydrogen), 2.99–2.88 (m, 5H, thiopyrano and
morpholine hydrogen).13C-NMR (100 MHz, DMSO)
δ 165.44, 164.41, 155.51, 151.31, 144.43, 132.53,
129.78 129.13(2C), 128.97(2C), 127.34, 126.69(2C), 126.02(2C), 108.86, 66.28(2C), 62.72, 49.14(2C), 42.36,
33.81, 26.19, 26.06. Purity: 98.27% by high performance liquid chromatography (HPLC) (80:20
MeOH/H2O).
(R)-4-(2-(5-(3,4-Dichlorophenyl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-7,8-dihydro-5H◦-thiopyrano[4,3-d]
pyrimidin-4-yl)morpholine (7b). A pale yellow solid. Yield: 72%. m.p. 194–196 C. ESI-MS m/z:
[M + H]+ 540.5; 1H-NMR (400 MHz, DMSO)
δ 7.64 (d, J = 7.9 Hz, 2H, Ar-H), 7.56–7.49 (m, 2H, Ar-H ),
7.24 (d, J = 7.9 Hz, 2H, Ar-H), 7.17 (d, J = 8.3 Hz, 1H, Ar-H), 5.60 (dd, J = 12.1, 6.1 Hz, 1H, pyrazoline),
3.82 (dd, J = 17.8, 12.2 Hz, 1H, pyrazoline ), 3.51 (dt, J = 23.0, 13.2 Hz, 7H, thiopyrano, morpholine
and pyrazoline hydrogen), 3.17–3.07 (m, 3H, thiopyrano and morpholine hydrogen), 2.95–2.86
(m, 5H, thiopyrano and morpholine hydrogen), 2.33 (s, 3H, –CH3). Purity: 97.50% by HPLC (80:20
MeOH/H2O).

Molecules 2017, 22, 1870
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(R)-4-(2-(3-(4-Bromophenyl)-5-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-7,8-dihydro-5H-thiopyrano
[4,3-d]pyrimidin-4-yl)morpholine (7c). A pale yellow solid. Yield: 78%. m.p. 185–186 ◦C. ESI-MS m/z:
[M + H]+ 605.38;1H-NMR (400 MHz, DMSO)
δ 7.68 (d, J = 8.5 Hz, 2H, Ar-H), 7.63 (d, J = 8.6 Hz, 2H,
Ar-H), 7.54 (d, J = 8.3 Hz, 2H, Ar-H), 7.18 (d, J = 8.1 Hz, 1H, Ar-H), 5.63 (dd, J = 12.2, 6.2 Hz, 1H,
pyrazoline ), 3.84 (dd, J = 17.8, 12.4 Hz, 1H, pyrazoline), 3.67–3.32 (m, 7H ,thiopyrano, morpholine and
pyrazoline hydrogen), 3.19–3.05 (m, 3H, thiopyrano and morpholine hydrogen), 2.98–2.87 (m, 5H,
thiopyrano and morpholine hydrogen ).13C-NMR (100 MHz, DMSO)
δ 166.02, 164.52, 162.57, 155.66,
150.31, 145.42, 132.08, 131.63, 131.34, 129.84(2C), 128.829(2C), 126.46, 123.04, 109.43, 66.30(2C), 62.02,
49.22(2C), 41.78, 33.97, 26.22, 26.09. Purity: 98.60% by HPLC (80:20 MeOH/H2O).
(R)-4-(2-(5-(3,4-Dichlorophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-7,8-dihydro-5H-thiopyrano
[4,3-d]pyrimidin-4-yl)morpholine (7d). A pale yellow solid. Yield: 71%. m.p. 207–211 ◦C. ESI-MS m/z:
1
[M + H]+ 544.47; H-NMR (400 MHz, DMSO)
δ 7.68 (d, J = 8.5 Hz, 2H, Ar-H), 7.63 (d, J = 8.6 Hz, 2H,
Ar-H), 7.54 (d, J = 8.3 Hz, 2H, Ar-H), 7.18 (d, J = 8.1 Hz, 1H, Ar-H), 5.63 (dd, J = 12.2, 6.2 Hz, 1H,
pyrazoline), 3.84 (dd, J = 17.8, 12.4 Hz, 1H, pyrazoline), 3.67–3.32 (m, 7H, thiopyrano, morpholine
and pyrazoline hydrogen), 3.19–3.05 (m, 3H, thiopyrano and morpholine hydrogen), 2.98–2.87 (m, 5H,
thiopyrano and morpholine hydrogen). Purity: 95.31% by HPLC (80:20 MeOH/H 2O).
(R)-4-(2-(5-(3,4-Dichlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-7,8-dihydro-5H-thiopyrano[4,3-d]
pyrimidin-4-yl)morpholine (7e). A yellow solid. Yield: 73%. m.p. 288–289 ◦C. ESI-MS m/z: [M + H]+
556.5; 1H-NMR (400 MHz, DMSO)
δ 7.75 (d, J = 6.7 Hz, 2H, Ar-H), 7.55 (d, J = 8.2 Hz, 2H, Ar-H), 7.44 (d,
J = 7.7 Hz, 3H, Ar-H), 7.18 (d, J = 8.3 Hz, 1H, Ar-H ), 5.63 (dd, J = 12.1, 6.1 Hz, 1H, pyrazoline), 3.86 (dd,
J = 17.7, 12.0 Hz, 1H, pyrazoline), 3.63–3.42 (m, 7H, thiopyrano, morpholine and pyrazoline hydrogen),
3.13 (d, J = 14.0 Hz, 3H, thiopyrano and morpholine hydrogen), 3.00–2.88 (m, 5H, thiopyrano and
morpholine hydrogen). Purity: 97.40% by HPLC (80:20 MeOH/H2O).
(R)-4-(2-(5-(3,4-Dichlorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-7,8-dihydro-5H
-thiopyrano[4,3-d]pyrimidin-4-yl)morpholine (7f). A yellow solid. Yield: 75%. m.p. 197–209 ◦C. ESI-MS
m/z: [M + H]+ 464.1; 1H-NMR (400 MHz, DMSO)
δ 7.81 (d, J = 8.7 Hz, 2H, Ar-H), 7.60–7.55 (m, 2H,
Ar-H), 7.22 (dd, J = 8.4, 1.8 Hz, 1H, Ar-H), 7.04 (d, J = 8.8 Hz, 2H, Ar-H), 5.65 (dd, J = 11.8, 6.1 Hz, 1H,
pyrazoline), 3.93 (dd, J = 18.1, 11.9 Hz, 1H, pyrazoline), 3.81 (s, 3H,–OCH3), 3.56 (dd, J = 3.6 Hz, 4H,
morpholine), 3.42 (d, J = 14.1, 6.9 Hz, 3H, thiopyrano and pyrazoline hydrogen), 3.26–2.83 (m, 8H,
thiopyrano and morpholine hydrogen). Purity: 97.04% by HPLC (80:20 MeOH/H2O).
(R)-4-(2-(3,5-Bis(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4
-yl)morpholine (7g). A pale yellow solid. Yield: 70%. m.p. 144–146◦C.. ESI-MS m/z: [M + H]+ 493.58;
1H-NMR (400 MHz, DMSO)
δ 8.08 (d, J = 2.8 Hz, 2H, Ar-H), 7.60–7.50 (m, 4H, Ar-H), 7.39 (s, 2H,
Ar-H), 5.63 (dd, J = 12.1, 6.1 Hz, 1H, pyrazoline), 3.86 (dd, J = 17.7, 12.0 Hz, 1H, pyrazoline), 3.63–3.42
(m, 7H, thiopyrano, morpholine and pyrazoline hydrogen), 3.13 (d, J = 14.0 Hz, 3H, thiopyrano and
morpholine hydrogen), 3.00–2.88 (m, 5H, thiopyrano and morpholine hydrogen). 13C-NMR (100 MHz,
DMSO)
δ 165.87, 164.55, 162.75, 161.92, 160.33, 155.77, 150.21, 140.53, 129.16, 128.90, 128.83, 128.11,
128.04, 116.25, 116.03, 115.78, 115.57, 109.03, 66.28(2C), 62.10(2C), 49.17, 42.32, 26.25, 26.07. Purity:
95.20% by HPLC (80:20 MeOH/H2O).
(R)-4-(2-(5-(4-Fluorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-7,8-dihydro-5H-thiopyrano
◦
[4,3-d]pyrimidin-4-yl)morpholine (7h). A yellow solid. Yield: 68%. m.p. 116–118 C. ESI-MS m/z:
[M + H]+ 505.6; 1H-NMR (400 MHz, DMSO) δ 8.06 (d, J = 8.3 Hz, 2H, Ar-H), 7.58–7.52 (m, 2H, Ar-H),
7.41 (t, J = 8.6 Hz, 2H,Ar-H), 7.30 (d, J = 8.4 Hz, 2H, Ar-H), 5.92 (dd, J = 11.7, 5.6 Hz, 1H, pyrazoline),
4.18 (dd, J = 17.9, 12.1 Hz, 1H, pyrazoline), 4.08 (s, 3H, –OCH3), 3.84–3.72 (m, 7H, thiopyrano,
morpholine and pyrazoline hydrogen), 3.50 (dd, J = 26.7, 8.6 Hz, 4H, thiopyrano and morpholine
hydrogen), 3.22 (dd, J = 29.1, 5.3 Hz, 4H, thiopyrano and morpholine hydrogen). Purity: 95.80% by
HPLC (80:20 MeOH/H2O).

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(R)-4-(2-(5-(4-Bromophenyl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-7,8-dihydro-5H-thiopyrano[4,3-d]
pyrimidin-4-yl)morpholine (7i). A yellow solid. Yield: 77%. m.p. 142–145 ◦C. ESI-MS m/z: [M + H]+
475.59; 1H-NMR (400 MHz, DMSO)
δ 7.64 (d, J = 7.2 Hz, 2H, Ar-H), 7.47 (d, J = 7.2 Hz, 2H, Ar-H), 7.24
(d, J = 7.4 Hz, 2H, Ar-H), 7.15 (d, J = 7.2 Hz, 2H, Ar-H), 5.63 (dd, J = 12.1, 6.1 Hz, 1H, pyrazoline),
3.86 (dd, J = 17.7, 12.0 Hz, 1H, pyrazoline), 3.63–3.42 (m, 7H, thiopyrano, morpholine and pyrazoline
hydrogen), 3.13 (d, J = 14.0 Hz, 3H, thiopyrano and morpholine hydrogen), 3.00–2.88 (m, 5H,
thiopyrano and morpholine hydrogen), 2.32 (s, 3H, CH3).13C-NMR (100 MHz, DMSO)
δ 165.91, 164.53,
155.78, 151.17, 144.00, 139.50, 131.84(2C), 129.72(2C), 128.41(2C), 126.67(2C), 120.18, 108.94, 66.29(2C),
62.24, 116.03, 49.19(2C),42.16, 33.97, 26.25, 26.05, 21.45. Purity: 95.54% by HPLC (80:20 MeOH/H2O).
(R)-4-(2-(5-(4-Bromophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-7,8-dihydro-5H-thiopyrano
[4,3-d]pyrimidin-4-yl)morpholine (7j). A pale yellow solid. Yield: 71%. m.p. 220–221 ◦C. ESI-MS m/z:
1
[M + H]+ 550.5; H-NMR (400 MHz, DMSO)
δ 7.79 (s, 2H, Ar-H), 7.30–7.20 (m, 4H, Ar-H), 7.11 (d,
J = 7.9 Hz, 2H, Ar-H), 5.68–5.57 (m, 1H, pyrazoline), 3.83 (dd, J = 16.9, 12.6 Hz, 1H, pyrazoline),
3.64–3.41 (m, 7H, thiopyrano, morpholine and pyrazoline hydrogen), 3.08 (d, J = 12.2 Hz, 3H,
thiopyrano and morpholine hydrogen), 2.95–2.85 (m, 5H, thiopyrano and morpholine hydrogen).
Purity: 97.34% by HPLC (80:20 MeOH/H2O).
(R)-4-(2-(5-(4-Bromophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-7,8-dihydro-5H-thiopyrano[4,3-d]
pyrimidin-4-yl)morpholine (7k). A yellow solid. Yield: 69%. m.p. 167–168 ◦C. ESI-MS m/z: [M + H]+
1
536.49; H-NMR (400 MHz, DMSO)
δ 7.79–7.71 (m, 2H, Ar-H), 7.48–7.37 (m, 3H, Ar-H), 7.24 (dd,
J = 8.5, 5.6 Hz, 2H, Ar-H), 7.10 (t, J = 8.8 Hz, 2H, Ar-H), 5.65 (dd, J = 12.1, 5.6 Hz, 1H, pyrazoline),
3.85 (dd, J = 17.7, 12.2 Hz, 1H, pyrazoline), 3.67–3.37 (m, 7H, thiopyrano, morpholine and pyrazoline
hydrogen), 3.16–3.05 (m, 3H, thiopyrano and morpholine hydrogen), 2.97–2.87 (m, 5H, thiopyrano and
morpholine hydrogen). Purity: 96.03% by HPLC (80:20 MeOH/H2O).
(R)-4-(2-(3-(4-Methoxyphenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-7,8-dihydro-5H-thiopyrano[4,3-d]
pyrimidin-4-yl)morpholine (7l). A pale yellow solid. Yield: 67%. m.p. >300 ◦C. ESI-MS m/z: [M + H]+
487.6; 1H -NMR (400 MHz, DMSO)
δ 7.72 (d, J = 8.4 Hz, 2H, Ar-H), 7.28 (d, J = 7.0 Hz, 2H, Ar-H), 7.21
(d, J = 7.6 Hz, 3H, Ar-H), 7.01 (d, J = 8.5 Hz, 2H, Ar-H), 5.63 (s, 1H, pyrazoline), 3.86 (d, J = 17.3 Hz,
1H, pyrazoline), 3.81 (s, 3H, –OCH3), 3.56 (dd, J = 3.6 Hz, 4H, morpholine), 3.42 (d, J = 14.1, 6.9 Hz,
3H, thiopyrano and pyrazoline hydrogen), 3.26–2.83 (m, 8H, thiopyrano and morpholine hydrogen).
Purity: 98.86% by HPLC (80:20 MeOH/H2O).
(R)-2-(3,5-Diphenyl-4,5-dihydro-1H-pyrazol-1-yl)-4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine
◦
6,6-dioxide (8a). A pale yellow solid. Yield: 70%. m.p. 219–220 C. ESI-MS m/z: [M + H]+ 489.5;
1H-NMR (400 MHz, DMSO)
δ 7.90 (d, J = 6.3 Hz, 2H, Ar-H), 7.40 (d, J = 7.3 Hz, 4H, Ar-H), 7.28 (d,
J = 3.2 Hz, 2H, Ar-H), 7.23 (d, J = 7.6 Hz, 2H, Ar-H ), 5.60 (dd, J = 11.9, 6.2 Hz, 1H, pyrazoline),4.12
(dd, J = 39.3, 15.5 Hz, 2H, thiopyrano), 3.87 (dd, J = 17.7, 12.4 Hz, 1H, pyrazoline), 3.56 (s, 2H,
morpholine), 3.51 (s, 2H, morpholine), 3.43 (s, 2H, thiopyrano), 3.25–3.15 (m, 3H, morpholine and
pyrazoline hydrogen), 3.08 (s, 2H, morpholine), 2.97 (s, 2H, thiopyrano). Purity: 97.60% by HPLC
(80:20 MeOH/H2O).
(R)-2-(5-(3,4-Dichlorophenyl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-morpholino-7,8-dihydro-5H
◦
-thiopyrano[4,3-d]pyrimidine 6,6-dioxide (8b). A yellow solid. Yield: 63%. m.p. 225–226 C. ESI-MS
m/z: [M + H]+ 572.50; 1H-NMR (400 MHz, DMSO)
δ 7.65 (d, J = 8.0 Hz, 2H,Ar-H), 7.54 (d, J = 8.8 Hz,
2H, Ar-H), 7.25 (d, J = 8.0 Hz, 2H, Ar-H), 7.16 (d, J = 8.2 Hz, 1H, Ar-H), 5.62 (dd, J = 12.1, 5.9 Hz,
1H, pyrazoline), 4.16 (d, J = 15.6 Hz, 1H, thiopyrano), 4.06 (d, J = 15.5 Hz, 1H, thiopyrano), 3.85 (dd,
J = 17.9, 12.3 Hz, 1H, pyrazoline), 3.56 (s, 2H, morpholine), 3.49 (d, J = 9.0 Hz, 2H, morpholine), 3.43
(s, 2H, thiopyrano), 3.16 (dd, J = 17.9, 5.9 Hz, 3H, morpholine and pyrazoline hydrogen), 3.12–3.05
(m, 2H, morpholine), 2.97 (s, 2H, thiopyrano), 2.33 (s, 3H, –CH3). Purity: 99.45% by HPLC (80:20
MeOH/H2O).

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(R)-2-(3-(4-Bromophenyl)-5-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-morpholino-7,8◦-dihydro-5H
-thiopyrano[4,3-d]pyrimidine 6,6-dioxide (8c). A yellow solid. Yield: 59%. m.p. 162–163 C. ESI-MS
m/z: [M + H]+ 637.37; 1H-NMR (400 MHz, DMSO)
δ 7.68 (d, J = 8.4 Hz, 2H, Ar-H), 7.62 (d, J = 8.3
Hz, 2H, Ar-H), 7.54 (d, J = 6.9 Hz, 2H,Ar-H), 7.17 (d, J = 8.4 Hz, 1H, Ar-H), 5.62 (dd, J = 12.1, 6.0 Hz,
1H, pyrazoline), 4.16 (d, J = 15.6 Hz, 1H, thiopyrano), 4.08 (d, J = 15.6 Hz, 1H,thiopyrano), 3.86 (dd,
J = 17.8, 12.3 Hz, 1H, pyrazoline), 3.56 (d, J = 5.3 Hz, 2H,morpholine), 3.53–3.46 (m, 2H, morpholine),
3.43 (s, 2H, thiopyrano), 3.24–3.14 (m, 3H, morpholine and pyrazoline hydrogen), 3.09 (d, J = 11.8 Hz,
2H,morpholine), 2.97 (s, 2H, thiopyrano). Purity: 96.89% by HPLC (80:20 MeOH/H2O).
(R)-2-(5-(3,4-Dichlorophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-morpholino-7,8-dihydro-5H
-thiopyrano[4,3-d]pyrimidine 6,6-dioxide (8d). A yellow solid. Yield: 61%. m.p. 150–153 ◦C. ESI-MS m/z:
[M + H]+ 576.47; 1H-NMR (400 MHz, DMSO)
δ 7.85–7.77 (m, 2H, Ar-H), 7.55 (d, J = 6.6 Hz, 2H, Ar-H),
7.28 (t, J = 8.4 Hz, 2H, Ar-H), 7.18 (d, J = 8.1 Hz, 1H, Ar-H), 5.63 (dd, J = 11.9, 5.7 Hz, 1H, pyrazoline),
4.12 (dd, J = 39.3, 15.5 Hz, 2H, thiopyrano), 3.87 (dd, J = 17.7, 12.4 Hz, 1H, pyrazoline), 3.56 (s, 2H,
morpholine), 3.51 (s, 2H, morpholine), 3.43 (s, 2H, thiopyrano), 3.25–3.15 (m, 3H, morpholine and
pyrazoline hydrogen), 3.08 (s, 2H, morpholine), 2.97 (s, 2H, thiopyrano). 13C-NMR (100 MHz, DMSO)
δ
165.77, 164.58, 162.55, 162.12, 156.38, 151.39, 145.22, 131.42, 129.91, 129.16, 129.16(2C), 129.08, 128.80,
126.38(2C), 116.31, 116.10, 102.61, 66.17(2C), 61.83, 49.04(2C), 47.03, 32.48. Purity: 95.14% by HPLC
(80:20 MeOH/H2O).
(R)-2-(5-(3,4-Dichlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-4-morpholino-7,8-dihydro-5H
-thiopyrano[4,3-d]pyrimidine 6,6-dioxide (8e). A yellow solid. Yield: 57%. m.p. 245–249 ◦C. ESI-MS m/z:
[M + H]+ 588.5; 1H-NMR(400 MHz, DMSO)
δ 7.85–7.77 (m, 2H, Ar-H), 7.55 (d, J = 6.6 Hz, 2H, Ar-H),
7.28 (t, J = 8.4 Hz, 2H,Ar-H), 7.18 (d, J = 8.1 Hz, 2H, Ar-H), 5.63 (dd, J = 11.9, 5.7 Hz, 1H, pyrazoline ),
4.12 (dd, J = 39.3, 15.5 Hz, 2H, thiopyrano), 3.87 (dd, J = 17.7,12.4 Hz, 1H, pyrazoline), 3.56 (s, 2H,
morpholine), 3.51 (s, 2H, morpholine), 3.43 (s, 2H, thiopyrano), 3.25–3.15 (m, 3H, morpholine and
pyrazoline hydrogen), 3.08 (s, 2H, morpholine), 2.97 (s, 2H, thiopyrano). Purity: 96.53% by HPLC
(80:20 MeOH/H2O).
(R)-2-(5-(3,4-Dichlorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-morpholino-7,8-dihydro
◦
-5H-thiopyrano[4,3-d]pyrimidine 6,6-dioxide (8f). A pale yellow solid. Yield: 55%. m.p. 203–205 C.
1
ESI-MS m/z: [M + H]+ 558.4; H-NMR (400 MHz, DMSO)
δ 7.70 (d, J = 8.0 Hz, 2H, Ar-H), 7.53 (d,
J = 10.9 Hz, 2H, Ar-H), 7.17 (d, J = 8.0 Hz, 1H, Ar-H), 6.99 (d, J = 8.0 Hz, 2H, Ar-H), 5.59 (dd, J = 11.4,
5.3 Hz, 1H, pyrazoline), 4.11 (dd, J = 35.3, 15.4 Hz, 2H, thiopyrano), 3.89–3.82 (m, 1H, pyrazoline), 3.79
(s, 3H, –OCH3), 3.54 (d, J = 17.8 Hz, 4H, morpholine), 3.42 (s, 2H, thiopyrano), 3.19 (d, J = 5.6 Hz, 2H,
morpholine), 3.15–3.05 (m, 3H, morpholine and pyrazoline hydrogen), 2.97 (s, 2H, thiopyrano). Purity:
98.66% by HPLC (80:20 MeOH/H2O).
(R)-2-(3,5-bis(4-Fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]
pyrimidine 6,6-dioxide (8g). A yellow solid. Yield: 53%. m.p. 246–249 ◦C. ESI-MS m/z: [M + H]+ 525.5;
1H-NMR (400 MHz, DMSO)
δ 7.71 (d, J = 8.0 Hz, 2H, Ar-H), 7.23 (s, 2H, Ar-H), 7.11 (t, J = 8.1 Hz, 2H,
Ar-H), 7.00 (d, J = 8.0 Hz, 2H, Ar-H), 5.62 (d, J = 7.0 Hz, 1H, pyrazoline), 4.12 (dd, J = 39.3, 15.5 Hz,
2H, thiopyrano), 3.87 (dd, J = 17.7, 12.4 Hz, 1H, pyrazoline), 3.56 (s, 2H, morpholine), 3.51 (s, 2H,
morpholine), 3.43 (s, 2H, thiopyrano), 3.25–3.15 (m, 3H, morpholine and pyrazoline hydrogen), 3.08 (s,
2H, morpholine), 2.97 (s, 2H, thiopyrano). Purity: 98.38% by HPLC (80:20 MeOH/H2O).
(R)-2-(5-(4-Fluorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-morpholino-7,8-dihydro-5H
-thiopyrano[4,3-d]pyrimidine 6,6-dioxide (8h). A yellow solid. Yield: 50%. m.p. 140–142 ◦C. ESI-MS m/z:
[M + H]+ 537.6; 1H-NMR (400 MHz, DMSO)
δ 7.71 (d, J = 8.0 Hz, 2H,Ar-H), 7.23 (s, 2H, Ar-H), 7.11 (t,
J = 8.1 Hz, 2H,Ar-H), 7.00 (d, J = 8.0 Hz, 2H, Ar-H), 5.62 (d, J = 7.0 Hz, 1H, pyrazolin), 4.16 (d, J = 15.4
Hz, 1H, thiopyrano), 4.04 (d, J = 15.5 Hz, 1H, thiopyrano), 3.85 (d, J = 17.6 Hz, 1H, pyrazolin), 3.79 (s,
3H, –OCH3), 3.57 (s, 2H, morpholine), 3.51 (s, 2H, morpholine), 3.43 (s, 2H, thiopyrano), 3.18 (s, 2H,
morpholine), 3.08 (d, J = 17.9 Hz, 3H, morpholine and pyrazoline hydrogen), 2.95 (s, 2H, thiopyrano).

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13C-NMR (100 MHz, DMSO)
δ
165.82, 162.76, 162.43, 160.91, 160.35, 156.45, 152.00, 140.51, 128.43,
128.06, 127.99, 124.91, 115.84, 115.63(2C), 114.65(2C), 102.07, 66.16(2C), 61.81, 55.79, 49.29, 49.05, 47.11,
42.47, 32.46. Purity: 99.04% by HPLC (80:20 MeOH/H2O).
(R)-2-(5-(4-Bromophenyl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-morpholino-7,8-dihydro-5H-thiopyrano
[4,3-d]pyrimidine 6,6-dioxide (8i). A yellow solid. Yield: 48%. m.p. 282–283 ◦C. ESI-MS m/z: [M + H]+
507.5; 1H-NMR (400 MHz, DMSO)
δ 7.65 (d, J = 7.9 Hz, 2H, Ar-H), 7.47 (d, J = 8.2 Hz, 2H, Ar-H), 7.24
(d, J = 7.9 Hz, 2H, Ar-H), 7.16 (d, J = 8.3 Hz, 2H, Ar-H), 5.60 (dd, J = 11.9, 5.4 Hz, 1H, pyrazolin),
4.17 (d, J = 15.5 Hz, 1H, thiopyrano), 4.05 (d, J = 15.5 Hz, 1H, thiopyrano), 3.84 (dd, J = 17.7, 12.2
Hz, 1H, pyrazolin), 3.56 (s, 2H, morpholine), 3.51 (d, J = 3.4 Hz, 2H, morpholine), 3.42 (d, J = 5.2 Hz,
2H, thiopyrano), 3.19 (d, J = 5.1 Hz, 2H, morpholine), 3.11–3.03 (m, 3H, morpholine and pyrazoline
hydrogen), 2.98 (s, 2H, thiopyrano), 2.32 (s, 3H, –CH3). Purity: 98.63% by HPLC (80:20 MeOH/H2O).
(R)-2-(5-(4-Bromophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-morpholino-7,8-dihydro-5H
◦
-thiopyrano[4,3-d]pyrimidine 6,6-dioxide (8j). A yellow solid. Yield: 49%. m.p. 158–163 C. ESI-MS
m/z: [M + H]+ 582.5; 1H-NMR (400 MHz, DMSO)
δ 7.71 (d, J = 8.0 Hz, 2H, Ar-H), 7.23 (s, 2H, Ar-H),
7.11 (t, J = 8.1 Hz, 2H, Ar-H), 7.00 (d, J = 8.0 Hz, 2H, Ar-H), 5.62 (d, J = 7.0 Hz, 1H, pyrazoline),4.12
(dd, J = 39.3, 15.5 Hz, 2H, thiopyrano), 3.87 (dd, J = 17.7, 12.4 Hz, 1H, pyrazoline), 3.56 (s, 2H,
morpholine), 3.51 (s, 2H, morpholine), 3.43 (s, 2H, thiopyrano), 3.25–3.15 (m, 3H, morpholine and
pyrazoline hydrogen), 3.08 (s, 2H, morpholine), 2.97 (s, 2H, thiopyrano). Purity: 98.37% by HPLC
(80:20 MeOH/H2O).
(R)-2-(5-(4-Bromophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-4-morpholino-7,8-dihydro-5H-thiopyrano
◦
[4,3-d]pyrimidine 6,6-dioxide (8k). A pale yellow solid. Yield: 52%. m.p. 163–167 C. ESI-MS m/z:
[M + H]+ 568.4; 1H-NMR (400 MHz, DMSO)
δ 7.76 (d, J = 6.8 Hz, 2H, Ar-H ), 7.45 (dd, J = 21.0, 6.9
Hz, 5H, Ar-H), 7.17 (d, J = 7.4 Hz, 2H, Ar-H), 5.62 (dd, J = 11.7, 5.1 Hz, 1H, pyrazoline), 4.17 (d,
J = 15.4 Hz, 1H,thiopyrano), 4.05 (d, J = 15.5 Hz, 1H, thiopyrano), 3.87 (dd, J = 17.4, 12.4 Hz, 1H,
pyrazoline), 3.57 (s, 2H, morpholine), 3.49 (d, J = 11.4 Hz, 4H, morpholine and thiopyrano hydrogen),
3.22–3.12 (m, 3H, morpholine and pyrazoline hydrogen), 3.11–3.04 (m, 2H, morpholine), 2.97 (s, 2H,
thiopyrano).13C-NMR (100 MHz, DMSO)
δ 165.75, 162.41, 156.34, 152.17, 143.60, 132.24, 131.93(2C),
130.04, 129.17(2C), 128.39(2C), 126.82(2C), 120.35, 102.38, 66.16(2C), 62.09, 49.29(2C), 49.01, 47.03, 42.17,
32.44. Purity: 96.29% by HPLC (80:20 MeOH/H2O).
(R)-2-(3-(4-Methoxyphenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-4-morpholino-7,8-dihydro-5H-thiopyrano
◦
[4,3-d]pyrimidine 6,6-dioxide (8l). A pale yellow solid. Yield: 50%. m.p. 109–114 C. ESI-MS m/z:
[M + H]+ 519.6; 1H-NMR (400 MHz, DMSO)
δ 7.71 (d, J = 8.7 Hz, 2H, Ar-H), 7.31–7.24 (m, 2H, Ar-H ),
7.19 (d, J = 6.9 Hz, 3H, Ar-H), 6.99 (d, J = 8.8 Hz, 2H, Ar-H), 5.59 (dd, J = 11.9, 5.4 Hz, 1H, pyrazoline),
4.15 (d, J = 15.5 Hz, 1H, thiopyrano), 4.02 (d, J = 15.4 Hz, 1H, thiopyrano), 3.89–3.82 (m, 1H, pyrazoline),
3.78 (s, 3H, –OCH3), 3.53 (d, J = 5.8 Hz, 2H, morpholine), 3.49–3.38 (m, 4H, morpholine and thiopyrano
hydrogen), 3.22–3.15 (m, 2H, morpholine), 3.12–3.00 (m, 3H, morpholine and pyrazoline hydrogen),
2.93 (s, 2H, thiopyrano). Purity: 98.22% by HPLC (80:20 MeOH/H2O).
3.3. Cytotoxicity Assay In Vitro
The in vitro cytotoxic activities of Compounds 7a–l and 8a–l were evaluated with A549, PC-3,
HepG2, and MCF-7 cell lines by the standard MTT assay, with GDC-0941 as a positive control.
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 the
indicated final concentrations were added to the culture medium, and cell cultures continued for
72 h. Fresh MTT was added to each well at a terminal concentration of 5
cells at 37 ◦C for 4 h. The formazan crystals were dissolved in 100
L of DMSO in each well, and the
absorbency at 492 nm (for absorbance of MTT formazan) and 630 nm (for the reference wavelength)
µg/mL and incubated with
µ

Molecules 2017, 22, 1870
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was measured with an enzyme linked immunosorbent assay (ELISA) reader (MR-96A Mindray Elisa
Microplate Reader, Guangzhou, China). All compounds were tested three times in each of the cell
lines. The results expressed as inhibition rates or IC50 were the averages of two determinations and
calculated using the Bacus Laboratories Inc. Slide Scanner (Bliss) software (the Bacus Laboratories Inc.
Slide Scanner (BLISS) system, Lombard, IL, USA) [7].
3.4. PI3Kα Kinase Assay
The selected compound 8d was tested for its activity against PI3K
α
using
a Kinase-Glo®Luminescent Kinase Assay (Promega, Madison, WI, USA), with GDC-0941 and
PI103 as positive controls. The kinase reaction occurred in a 384-well black plate. Each well was
loaded with 50
PI substrate (L-
AL, USA); prepared in 3% octyl-glucoside), and the PI3K
The reaction was started by the addition of 5 L of 1
the reaction buffer and incubated for 60 min for p110
µ
L of test items (in 90% DMSO) and 5
-phosphatidylinositol; Avanti Polar Lipids (Avanti Polar Lipids, Inc., Alabaster,
protein (10 nM) was then added to it.
M Adenosine triphosphate (ATP) prepared in
. It was terminated by the addition of 10 L of
µL of reaction buffer containing 10 µg/mL
α
α
µ
µ
α
µ
Kinase-Glo buffer. The plates were then read in a Synergy 2 reader (BioTek, Winooski, VT, USA) for
luminescence detection. This assay was repeated twice [7].
3.5. Docking Studies
For docking purposes, we prepared the receptor proteins PDB ID code: 4L23 (PI3K
α).
The three-dimensional structure of the PI3K was obtained from the Research Collaboratory for
α
Structural Bioinformatics (RCSB) Protein Data Bank. We built a small organic molecule set (compound
8d) and used the Gasteiger–Huckel method to optimize the molecular force field and structure.
Hydrogen atoms were added to the structure allowing for appropriate ionization at physiological pH.
First, ligand substructure was extracted, water and excess structure were then removed, and hydrogens
and fix sidechain amides were finally added. The protonated state of several important residues, such
as Asp2357, Asn2343, and His2340 were adjusted using SYBYL6.9.1 (Tripos, St. Louis, MO, USA) in
favor of forming a reasonable hydrogen bond with the ligand. Molecular docking analysis was carried
out by the SURFLEX-DOCK module of the SYBYL 6.9.1 package to explore the binding model for the
active site of 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 a Silicon Graphics
workstation (package version 6.9.1 on silicon graphics origin300 workstation with IRIX 6.5 operating
system, San Francisco, CA, USA). Lastly, docking results and the optimized molecular docking model
with the receptor proteins were obtained [7].
4. Conclusions
In summary, two series of 4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine derivatives
bearing pyrazoline scaffolds were designed, synthesized, and evaluated for cytotoxicity activity against
four cancer cell lines and the PI3Kα kinase in vitro. The pharmacological results indicated that most of
the compounds showed moderate cytotoxicity activity against the four cancer cell lines. In particular,
the results indicated that the most promising compound 8d showed excellent cytotoxicity against
four cancer cell lines with IC50 values of 6.02–10.27
a moderate inhibitory activity toward PI3K enzyme. The initial SARs and docking studies with
PI3K molecular showed that the morpholine group and the pyrazoline scaffold were necessary for
µM. In addition, compound 8d was found to have
α
α
these compounds to possess potent cytotoxicity activities. What’s more, various types of substituents
impacted differently on the activity of the two series of compounds. The compounds in which the
substituents of benzene ring at the C-4 position are electron-withdrawing groups such as substituents

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(Cl, F, Br) have better activity than the compounds containing the electron donating groups (OCH3, H).
Further studies will be carried out in the near future.
Acknowledgments: We gratefully acknowledge the generous support provided by the National Natural Science
Funds (No. 81460527), Natural Science Funds for Distinguished Young Scholar of Jiangxi Province, China
(20171BCB23078), the Project Supported by Natural Science Foundation of Jiangxi, China (20161BAB215216),
Key Projects of the Youth Fund, Natural Science Funds of Jiangxi Province (20171ACB21052), the Department
Education, Science and Technology Research Project of Jiangxi, China (GJJ160787), and the Jiangxi Provincial Key
Laboratory of Drug Design and Evaluation (20171BCD40015).
Author Contributions: Q.W. and C.S. synthesized all novel compounds. B.Z., C.W., S.X., and P.Z. ran the bioassay
evaluation and statistics analysis. W.Z. started the project and designed the molecules, and S.X. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 7a–l and 8a–l are available from the authors.
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