Design, synthesis, and biological evaluation of novel thienopyrimidine derivatives as PI3Kα inhibitors

Article abstract of DOI:10.3390/molecules24193422

Three series of novel thienopyrimidine derivatives 9a-l, 15a-l, and 18a-h were designed and synthesized, and their IC₅₀ values against four cancer cell lines HepG-2, A549, PC-3, and MCF-7 were evaluated. Most compounds show moderate cytotoxicity against the tested cancer cell lines. The most promising compound 9a showed moderate activity with IC₅₀ values of 12.32 ± 0.96, 11.30 ± 1.19, 14.69 ± 1.32, and 9.80 ± 0.93 μM, respectively. The inhibitory activities of compounds 9a and 15a against PI3Kα and mTOR kinase were further evaluated. Compound 9a exhibited PI3Kα kinase inhibitory activity with IC₅₀ of 9.47 ± 0.63 μM. In addition, docking studies of compounds 9a and 15a were also investigated.

Full text of DOI:10.3390/molecules24193422

molecules
Article
Design, Synthesis, and Biological Evaluation of Novel
Thienopyrimidine Derivatives as PI3Kα Inhibitors
Lide Yu 1,2,†, Qinqin Wang 1,†, Caolin Wang 1, Binliang Zhang 1, Zunhua Yang 2, Yuanying Fang 2,
Wufu Zhu 1,
*
and Pengwu Zheng 1,
*
1
Jiangxi Provincial Key Laboratory of Drug Design and Evaluation, School of Pharmacy, Jiangxi Science &
Technology Normal University, 605 Fenglin Road, Nanchang 330013, China; 20020680@jxutcm.edu.cn (L.Y.);
m15180134379_2@163.com (Q.W.); wangcllw@163.com (C.W.); zbl1045762244@163.com (B.Z.)
College of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China;
mtdzcool@163.com (Z.Y.); fangyuanying@163.com (Y.F.)
2
*
†
Correspondence: zhuwf@jxstnu.edu.cn (W.Z.); zhengpw@126.com (P.Z.); Tel.: +86-791-8380-2393(W.Z.)
These authors contributed equally to the paper.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 23 August 2019; Accepted: 19 September 2019; Published: 20 September 2019
Abstract: Three series of novel thienopyrimidine derivatives 9a
–l
,
15a–l, and 18a–h were designed
and synthesized, and their IC50 values against four cancer cell lines HepG-2, A549, PC-3, and MCF-7
were evaluated. Most compounds show moderate cytotoxicity against the tested cancer cell lines.
The most promising compound 9a showed moderate activity with IC50 values of 12.32
1.19, 14.69 1.32, and 9.80 0.93
15a against PI3K and mTOR kinase were further evaluated. Compound 9a exhibited PI3K
±
0.96, 11.30
±
±
±
µ
M, respectively. The inhibitory activities of compounds 9a and
α
α
kinase
inhibitory activity with IC50 of 9.47
were also investigated.
±
0.63 µM. In addition, docking studies of compounds 9a and 15a
Keywords: Thienopyrimidine; Pyrazole; PI3Kα inhibitor
1. Introduction
The PI3K-Akt-mTOR signaling pathway plays an important role in tumorigenesis and
development [1,2]. The activation of related proteins in this pathway is closely related to the
occurrence and development of tumors. In recent years, the development of small molecule drugs
to effectively inhibit the overexpression of this pathway has become a research hotspot in cancer
therapy [
Ligand also promoted the development of new drugs [
PI3K-Akt–mTOR signaling pathway have entered clinical studies, and some of them were even
approved by FDA, such as PI3K inhibitor GDC-0941 [ ]; AKT inhibitor GSK2110183 [ ]; mTOR
inhibitor AZD2014 (Figure 1) [ ], Rapamycin, Deforolimus. PI3K is the most important isoform in
cell proliferation in response to growth factor-tyrosine kinase pathway activation. There are currently
more than ten PI3K inhibitors undergoing clinical trials. Many research groups are attempting to
3]. Moreover, the study of the complex co-crystals structure of protein and small molecular
4–6]. Many small molecule inhibitors targeting
7
8
9
α
α
develop some more PI3Kα inhibitors.
Molecules 2019, 24, 3422; doi:10.3390/molecules24193422
www.mdpi.com/journal/molecules

Molecules 2019, 24, 3422
2 of 14
Figure 1. Structures of representative clinical PI3K/mTOR dual inhibitors and target compounds.
The thienopyrimidine core is widely used in small molecule inhibitors of the PI3K-Akt-mTOR
signaling pathway. Among them, thienopyrimidine derivative GDC-0941 developed by Genentech, is
the first PI3K inhibitor entering the clinical stage. Right after, several analogues of GDC-0941 were
obtained. Two of them, GNE-477 and GDC-0980 (Figure 1), exhibited excellent activities and were
regarded as potent PI3K/mTOR inhibitors [10,11]. Research showed that thienopyrimidines core were
very important to the activity of these compounds, and it was considered to be an active pharmacophore.
In our previous research, several series of thienopyrimidine-containing compounds were designed
and synthesized [12–14], one representative compound EJMC20159364-16i (Figure 1) exhibited the
best in vitro cytotoxic activity and kinase inhibitory activity (PI3K, mTOR). SARs of this series of
compounds were summarized. The results showed that the introduction of the flexible hydrazinyl
linker at the pyrimidine 2 position helps to increase the cytotoxic activity of the target compounds.
Continuous to this work, the hydrazinyl linker was kept unchanged in the new designed compounds,
and we constructed a pyrazole ring on the hydrazinyl as a new flexible linker to connect with the
aryl group, hoping to increase the compounds’ inhibitory activity by improving the interaction of
the compounds with the receptor (Figure 2). As a result, two series of thieno[2,3-d]pyrimidines
and thieno[3,2-d]pyrimidines containing pyrazole unit (9a–l, 15a–l) were designed and synthesized.
Inspired by GDC-0084, in order to investigate the influence of the thienopyrimidine core to the
activity, a flexible cyclohexane was introduced to the target compounds, resulting in a series of tricyclic
thienopyrimidine compounds 21a
–h. It is expected to increase the cytotoxic activity and increase
the interaction of the compounds with the enzyme to enhance the activity and selectivity. Finally,
three series of thienopyrimidine derivatives containing pyrazole structure (9a–l, 15a–l, 21a–h) were
designed, synthesized and evaluated for their cytotoxic activity.

Molecules 2019, 24, 3422
3 of 14
Figure 2. The design concept based on the co-crystal structure of GDC-0941 with protein.
2. Results and Discussion
2.1. Chemistry
The synthetic routes for thienopyrimidine derivatives 9a
–
l,
15a
–
l, and 21a
–
h
are outlined in
Schemes 1 and 2. Condensation of the 4-substituted benzaldehydes (1a
acetophenones (2a ) produced the corresponding chalcones 3a (Scheme 1). Treatment of
POCl 3 afforded 2,4-dichlorothieno[2,3-d]pyrimidine , which was then treated with morpholine to
give 4-(2-chlorothieno[2,3-d]pyrimidin-4-yl)morpholine . Treatment of with hydrazine gave the key
intermediate . Intermediate condensed with the corresponding chalcones 3a to afford the target
compounds 9a . The synthesis of compounds 15a was similar to that of compounds
9a (Scheme 2). Generated by condensation reaction between chalcone 3a l, there appears a chiral
–
d) with the 4-substituted
–
e
–
l
5
with
6
7
7
8
8
–l
–
l
–
l
and 21a
–
h
–l
8
–
carbon atom at C-5 position of pyrazole of all of the compounds. The similar reaction was reported
in previous research and the structures of target compounds can be easily identified. The structures
1
of target compounds were confirmed by H-NMR, 13C-NMR, and TOF MS (ESI+), which were in
agreement with the structures depicted.

Molecules 2019, 24, 3422
4 of 14
Scheme 1. Synthetic routes of chalcones 3a–l. Reagents and conditions: a 10% NaOH, EtOH, r.t., 24 h.
Scheme 2. Synthetic routes of target compounds 9a
–
l
,
15a
–l, and 21a–h. Reagents and conditions: a 5
eq urea, 180 ◦C, 2h;
b
POCl3, DMF (cat.), reflux, 8 h;
c
2.1 eq morpholine, MeOH, 0 ◦C, 30 min, r.t., 2h;
d
80% hydrazine monohydrate, reflux, 8h; e Glacial acetic acid, H2SO4 (78%), 100 ◦C.
2.2. Biological Discussion
All target compounds were evaluated for their cytotoxic activities against A549 (human lung
cancer), PC-3 (human prostate cancer), MCF-7 (human breast cancer), HepG2 (human hepatoma) cell
lines. In addition, the activity against PI3Kα/mTOR kinase of the most promising compounds 9a and
15a was further evaluated. The results expressed as IC50 were summarized in Tables 1–3.

Molecules 2019, 24, 3422
5 of 14
Table 1. In vitro cytotoxic activities against different cancer cell lines of 9a–l and 15a–l.
IC50 (µM) a
ClogP b
tPSA b
Compd.
R1
R2
PC-3
A549
MCF-7
HepG2
9a
9b
9c
9d
9e
9f
9g
9h
9i
4-H
4-H
4-F
4-F
4-Br
4-H
4-OCH3
4-F
4-OCH3
4-CH3
4-F
4-H
4-H
4-F
4-Br
4-OCH3
4-CH3
4-H
4-OCH3
4-F
4-OCH3
4-CH3
4-F
4-H
4-H
4-F
4-Br
12.32 ± 0.96
15.36 ± 1.26
18.21 ± 1.55
22.15 ± 1.67
23.16 ± 1.96
>50
11.30 ± 1.19
21.22 ± 2.75
24.23 ± 0.15
>50
14.69 ± 1.32
14.12 ± 2.1
22.37 ± 0.17
20.62 ± 1.90
30.06 ± 2.86
27.72 ± 2.71
28.46 ± 3.9
26.81 ± 2.13
29.53 ± 1.87
39.34 ± 2.51
25.30 ± 2.20
>50
9.80 ± 0.93
13.08 ± 1.20
17.36 ± 0.98
17.98 ± 2.12
21.63 ± 2.35
13.49 ± 1.69
28.85 ± 2.34
23.70 ±2.08
23.70 ± 2.11
>50
20.88 ± 1.98
30.70 ± 0.15
13.14 ± 1.48
34.62 ± 2.82
18.90 ± 1.86
18.02 ± 1.30
24.41 ± 1.14
19.90 ± 1.10
21.36 ± 1.86
21.15 ± 1.97
15.56 ± 1.30
28.97 ± 2.49
19.86 ± 1.88
21.59 ± 1.86
0.07 ± 0.03
6.67
6.59
6.95
6.73
8.03
7.67
7.53
7.97
8.12
8.83
7.89
8.47
6.67
6.58
6.95
6.73
8.03
7.67
7.53
7.97
8.12
8.83
7.89
8.47
3.20
28.07
37.3
28.07
37.3
>50
28.07
28.07
28.07
28.07
28.07
28.07
37.3
28.07
28.07
37.3
4-Br
4-Br
15.37 ± 0.85
22.78 ± 2.15
38.53 ± 3.14
17.75 ± 1.64
>50
30.12 ± 3.01
32.13 ± 2.96
27.65± 2.34
28.32 ± 2.16
33.54 ± 3.26
28.36 ± 2.58
15.53 ± 1.21
25.45 ± 2.32
25.31 ± 2.39
22.13 ± 2.13
37.12 ± 3.16
26.45 ± 2.57
24.22 ± 2.26
27.15 ± 2.53
18.48 ± 1.73
32.97 ±3.22
26.57 ± 2.38
23.68 ±1.94
4.35 ± 0.33
3,4-di Cl
3,4-di Cl
3,4-di Cl
3,4-di Cl
3,4-di Cl
4-H
4-H
4-F
4-F
4-Br
9j
9k
9l
40.65 ± 3.85
>50
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
15l
GDC-0941c
16.90 ± 1.61
>50
17.03 ± 1.68
>50
39.03 ± 3.32
25.83 ± 1.99
45.78 ± 0.81
42.09 ± 0.08
21.73 ± 1.47
33.23 ± 2.14
>50
41.78 ± 0.81
31.05 ± 0.39
31.79 ± 0.52
6.99 ± 0.21
37.12 ± 2.99
35.15 ± 2.04
>50
36.17 ± 1.13
32.14 ± 0.89
>50
28.07
37.3
28.07
28.07
28.07
28.07
28.07
28.07
37.3
4-Br
4-Br
3,4-di Cl
3,4-di Cl
3,4-di Cl
3,4-di Cl
3,4-di Cl
-
>50
>50
>50
>50
4-OCH3
4-CH3
-
28.07
76.85
0.20 ± 0.08
a
The value “>50” indicates that no inhibitory effect at 50
µ
M compound concentration. The values are an average
of two separate determinations; b Calculated by ChemBioDraw Ultra 15.0; c used as a positive control.
Table 2. In vitro cytotoxic activities against different cancer cell lines of 21a–h.
IC50 (µM) a
ClogP b
tPSA b
Compd.
R1
R2
PC-3
A549
MCF-7
HepG2
21a
21b
21c
21d
21e
21f
3,4-di Cl
4-Br
4-H
4-F
4-Br
4-Br
3,4-diCl
3,4-diCl
4-OCH3
4-CH3
4-H
4-OCH3
4-F
4-H
4-Br
4-H
31.75 ± 0.95
20.64 ± 0.63
>50
27.68 ± 0.10
11.59 ± 0.11
41.99 ± 1.49
17.75± 1.1
>50
>50
>50
23.53 ± 0.82
6.99 ± 0.21
8.23
7.44
8.56
8.79
7.86
8.84
8.63
7.95
28.07
28.07
28.07
28.07
28.07
28.07
28.07
28.07
>501
15.29 ± 0.83
>50
32.37 ± 1.84
12.43 ± 0.96
6>50
23.70 ± 0.11
38.71 ± 1.72
>50
25.28 ± 0.75
29.53 ± 1.87
>50
>50
>50
>50
>50
>50
>50
>50
21g
21h
>50
>50
GDC-0941
-
-
4.35 ± 0.33
0.20 ± 0.08
0.07 ± 0.03
3.20
76.85
c
a
The value “>50” indicates that no inhibitory effect at 50
µ
M compound concentration. The values are an average
of two separate determinations; b Calculated by ChemBioDraw Ultra 15.0; c used as a positive control.

Molecules 2019, 24, 3422
6 of 14
Table 3. Enzymatic activities of compounds 9a and 15a against PI3Kα and mTOR (IC50, µM).
IC50 (µM) a
Compd.
R1
R2
PI3Kα
mTOR
9a
15a
4-H
4-H
-
4-H
4-H
-
9.47 ± 0.63
39.9 ± 7.6
25.68 ± 2.33
>50
PI-103 b
a
0.019 ± 0.004
0.011 ± 0.002
b
The values are an average of two separate determinations. used as a positive control.
As illustrated in Tables 1 and 2, most of the target compounds exhibited moderate cytotoxic
activities. The compounds whose skeleton are thieno[2,3-d]pyrimidine (9a ) has slightly stronger
inhibitory activity against the tested cancer cell lines than the compounds whose skeleton are
thieno[3,2-d]pyrimidine (15a and 21a ). The most promising compound 9a showed moderate to
–
1
–
l
–h
well inhibitory activity with IC50 values of 12.32
µM, respectively.
±
0.96, 11.30
±
1.19, 14.69
±
1.32, and 9.80
±
0.93
It is worth noting that when R1 of the aryl group is an electron-withdrawing group (EWG) and R2
is a H atom, the inhibitory activity of the compounds (9g–h, 15g–h, and 21a–h) against cancer cells is
decreased, especially for MCF-7 and A549 cells. The effect is greater than that of HepG2 and PC-3
cancer cell lines. Among them, the inhibitory activity of the compound 15h against MCF-7 cells was
IC50 > 50 µM, which means that the inhibitory activity was almost lost. When the C-3 and C-4 positions
of the aryl group were both replaced by chlorine, the activity of the compounds (9h
lower than that of the compounds (9b–g, 15b–g) substituted by F or Br atoms.
–l, 15h–l) is slightly
When R1 of the aryl group was hydrogen atom and R2 was an electron-donating group, the
inhibitory activities of the compounds 9b and 15b against the tested cancer cells decreased. The
compound 15b which contained the core skeleton thieno[3,2-d]pyrimidine was more obvious in this
respect, and its IC values for A549 and MCF-7 were both higher than 50 µM. When R of the
1
50
aryl group was an electron withdrawing group, the inhibitory activity against cancer cells of the
compounds 9c and 15c tended to decrease irrespective whether the substituent of R2 was an electron
withdrawing group or an electron donating group. Comparing the difference in activity between 9e
and 15e , it is easily to find that the effect of the electron withdrawing group on the activity is slightly
less than that of the electron-donating group. As shown in Table 3, compounds 21a exhibited poor
–l
–l
–
f
–f
–h
cytotoxic activity against the tested cell lines. This may attributed to the introduction of the tricyclic
thieno-pyrimidine structure, resulting in an increase in steric hindrance which reduces the solubility of
the compound at physiological pH and the relative molecular mass of the compound exceeds 500.
Finally, the inhibitory activity against PI3K
and 15a were further examined. As shown in Table 3, enzymatic activity results of compounds 9a and
15a exhibited a moderate to excellent inhibitory activity against PI3K kinase. The inhibitory activity
α kinase and mTOR kinase of selected compounds 9a
α
of the compounds against PI3Kα kinase is better than that of mTOR kinase.
2.3. Molecular Docking Study of Compounds 9a and 15a.
To further explore the binding modes of target compounds (9a and 15a) with the active site of
PI3K
in vitro inhibition results, we selected compounds 9a and 15a as ligand examples, and the structures
of PI3K (PDB code: 3TL5) were selected as the docking models. The best-scoring ligand–protein
complex (Figure 3) was used for binding site analysis.
α, molecular docking simulation studies were carried out by the AutoDock 4.2 software. Based on
α

Molecules 2019, 24, 3422
7 of 14
Figure 3. Docking modes of 9a and 15a. (
A
) Overview of the binding site of compound 9a with PI3K
(3TL5) kinase. ( ) Overview of the
α (3TL5) kinase. (D) Binding model of compound 15a with
α
(3TL5) kinase. ( ) Binding model of compound 9a with PI3K
B
α
C
binding site of compound 15a with PI3K
PI3Kα (3TL5) kinase.
The binding models of compounds 9a and 15a to the 3TL5 protein are approximately the same as
our previously assumed binding model (Figure 3A,C). The detailed binding models of compounds 9a
and 15a with the active site of PI3K
α kinase are shown in Figure 3. In the docking model of compound
9a with PI3K (Figure 3B), we can easily see that the oxygen atom on the morphine ring formed a
α
hydrogen bond with the hinge region residue VAL882. After careful observation of the binding model
of compound 15a with 3TL5 protein kinase (Figure 3D), it is not difficult to find that the N atom on the
pyrimidine ring of thieno[3,2-d]pyrimidine formed a hydrogen bond interaction with residue SER806.
Compared with the original ligand GDC-0980, the hydrogen bonding interaction between compounds
9a and 15a with protein is reduced, which may be one of the reasons why this series of compounds
failed to achieve excellent cytotoxic activity.
3. Experimental Section
3.1. General Information
Unless otherwise required, all reagents used in the experiment were purchased as commercial
analytical grade and used without further purification. Frequently used solvents (Ethanol, petroleum
ether, ethyl acetate, dichloromethane, etc.) were absolutely anhydrous. All actions were monitored
through GF254 thin-layer chromatography plate (Qingdao Haiyang Chemical Co., Ltd., Qingdao,
China) and spots were visualized with iodine or light (in 254 nm or 365 nm). The structure of the target
compound was confirmed by 1H-NMR and 13C-NMR spectra at room temperature on Bruker 400 MHz
spectrometer (Bruker Bioscience, Billerica, MA, USA) with tetramethylsilane (TMS) as an internal
standard. Mass spectrometry (MS) was performed on Waters High Resolution Quadrupole Time of
Flight Tandem Mass Spectrometry (QTOF) (Waters Corporation, Milford, MA, USA). The purity of the

Molecules 2019, 24, 3422
8 of 14
compound was determined by Agilent 1260 liquid chromatograph (Agilent Technologies Inc., Palo
Alto, CA, USA) fitted with an Inertex-C18 column. All target compounds had the purity of ≥95%.
3.2. Chemistry
3.2.1. General Procedure for the Preparation of Compounds 3a–l
Compounds 3a–l were synthesized according to the reported procedures by our research group [15].
3.2.2. General Procedure for the Preparation of Compounds 8, 14 and 20
Compound was obtained by a cyclization reaction starting from commercially available
methyl 2-aminothiophene-3-carboxylate ( ) and urea. Subsequently, compound is subjected
to a chlorination reaction with POCl3 to give compound Compound was substituted by
morphine and hydrazine hydrate respectively to obtain key intermediate . Similarly, we used
5
4
5
6.
6
8
commercially available methyl 2-aminothiophene-3-carboxylate (10) or commercial available methyl
2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (16) as the starting material to obtain
intermediates 14 or 20 through a similar reaction condition. Detailed synthesis of key intermediates 8,
14, and 20, and their precursors 7, 11, 12, and 13. can be found in the article reported by our research
group [13].
3.2.3. General Procedure for the Preparation of Target Compounds 9a–l, 15a–l, and 21a–h.
A mixture of different substituted chalcone 3a (1.5 mmol) and 4-(2-mercaptothieno[2,3-d]
pyrimidin-4-yl) morpholine ( ) or 4-(2-mercaptothieno[3,2-d]pyrimidin -4-yl)morpholine (14
or 4-(2-hydrazinyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)morpholine (20) were
dissolved (1.5 mmol, 14 20) in 25 mL of glacial acetic acid. Subsequently, the reaction solution was
–
l
8
)
8
/
/
stirred at 100–110 ◦C for 1.5 h under the action of 2–3 drops concentrated H2SO4 (78%) as a catalyst
and monitored by thin-layer chromatography (TLC). The reaction mixture was concentrated under
reduced pressure. After the completion of concentrated, the mixture was filtered and the precipitate
was washed with ethanol. If the purify of the compound is not high enough, recrystallization by 95%
EtOH or column chromatography (EtOAc:PE = 1:3) are needed. The obtained solids were then dried
to give the target compounds 9a–l, 15a–l, and 18a–h with the yield ranging from 30%–60%.
4-(2-(3-(3,4-dichlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]pyrimidin-4-yl)
morpholine (9a). Light yellow solid, mp 202–205 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 8.42 (d,
J = 5.3 Hz, 1H), 7.95 (d, J = 8.7 Hz, 2H), 7.73 (s, 1H), 7.62 (m, J = 16.2, 7.0 Hz, 2H), 7.33 (d, J = 8.1 Hz,
1H), 7.12 (d, J = 8.4 Hz, 2H), 5.85–5.75 (m, 1H), 4.08 (m, J = 17.7, 11.6 Hz, 1H), 3.85 (s, 3H), 3.84–3.75 (m,
3H), 3.67 (s, 3H), 3.60–3.54 (m, 1H), 3.50–3.46 (m, 2H). HRMS (ESI): m/z calcd for (C27H25N3OS + H)+:
440.1797; found: 440.1834.
4-(2-(5-(4-bromophenyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]pyrimidin-4-yl)
morpholine (9b). Light yellow solid, m.p. 280–282 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 8.18 (d,
J = 5.5 Hz, 1H), 7.90 (dd, J = 8.4, 5.5 Hz, 2H), 7.63 (dd, J = 8.4, 5.0 Hz, 2H), 7.37 (dd, J = 13.9, 7.0 Hz,
3H), 7.30–7.26 (m, 1H), 5.79 (dd, J = 12.1, 6.1 Hz, 1H), 3.95 (dd, J = 17.8, 12.2 Hz, 1H), 3.82–3.65 (m, 6H),
3.59 (d, J = 7.7 Hz, 3H). HRMS (ESI): m/z calcd for (C28H27N3O2S + H)+: 470.1902; found: 470.1946.
4-(2-(3,5-bis(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]pyrimidin-4-yl)Morpholine (9c).Yellow
solid, m.p. 225–227 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 8.47 (d, J = 5.6 Hz, 1H), 8.13 (s, 2H),
7.63 (s, 1H), 7.50–7.42 (m, 4H), 7.25 (t, J = 8.5 Hz, 2H), 5.88 (s, 1H), 4.13 (d, J = 12.1 Hz, 1H), 3.87 (s,
4H), 3.70 (s, 3H), 3.49 (s, 1H), 3.22 (s, 1H). HRMS (ESI): m/z calcd for (C27H23F2N3OS + H)+: 476.1608;
found: 476.1598.
4-(2-(3-(3,4-dichlorophenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]pyrimidin-4-yl)
◦
1
morpholine (9d).Yellow solid, m.p. 286–289 C; H NMR (400 MHz, DMSO-d6)
δ (ppm): 7.73 (d,

Molecules 2019, 24, 3422
9 of 14
J = 7.5 Hz, 2H), 7.54 (d, J = 7.9 Hz, 2H), 7.31 (d, J = 6.6 Hz, 3H), 7.25 (d, J = 7.8 Hz, 2H), 5.74 (d,
J = 6.6 Hz, 1H), 3.95–3.84 (m, 1H), 3.76–3.63 (m, 6H), 3.59 (s, 2H), 3.49 (d, J = 5.9 Hz, 1H). HRMS (ESI):
m/z calcd for (C28H26FN3O2S + H)+: 488.1808; found: 488.1853.
4-(2-(3-(3,4-Dichlorophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]pyrimidine-4-yl)morpholine
(
9e). Yellow solid, m.p. 228.3~229.1 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 8.07 (d, J = 5.4 Hz, 1H),
7.77 (d, J = 7.1 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 7.6 Hz, 3H), 7.25 (d, J = 5.5 Hz, 2H), 5.72 (dd,
J = 12.1, 5.7 Hz, 1H), 3.87 (dd, J = 18.0, 12.3 Hz, 1H), 3.71 (s, 2H), 3.66–3.58 (m, 4H), 3.54 (s, 2H), 3.18
(dd, J = 18.1, 5.8 Hz, 1H). HRMS (ESI): m/z calcd for (C28H26BrN3OS + H)+: 534.1041; found: 534.0991.
4-(2-(5-(4-methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]pyrimidine-4-yl)morpholine (9f).
Orange solid, m.p. 237.6~239.1 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 8.17 (d, J = 4.9 Hz, 1H),
7.79 (d, J = 8.1 Hz, 2H), 7.35 (d, J = 4.7 Hz, 1H), 7.29 (d, J = 7.3 Hz, 2H), 7.24 (d, J = 7.7 Hz, 3H), 7.03 (d,
J = 8.3 Hz, 2H), 5.69 (d, J = 6.4 Hz, 1H), 3.98–3.87 (m, 2H), 3.80 (s, 3H), 3.68 (s, 4H), 3.57 (s, 4H). HRMS
(ESI): m/z calcd for (C27H23BrFN3OS + H)+: 538.0790; found: 538.0882.
4-(2-(3-(3,4-dichlorophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]pyrimidin-4-yl)morpholine
(
9g). Yellow solid, m.p. 207.4~208.2 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 8.43 (d, J = 5.4 Hz,
1H), 7.90 (d, J = 7.6 Hz, 2H), 7.74 (s, 1H), 7.62 (dd, J = 15.4, 6.7 Hz, 2H), 7.41–7.29 (m, 3H), 5.80 (dd,
J = 11.6, 6.2 Hz, 1H), 4.09 (dd, J = 18.2, 11.5 Hz, 1H), 3.82 (s, 4H), 3.67 (s, 3H), 3.48 (s, 2H), 2.40 (s, 3H).
HRMS (ESI): m/z calcd for (C27H24BrN3OS + H)+: 520.0884; found: 520.0865.
4-(2-(3-(4-bromophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]pyrimidine-4-yl)morpholine (9h).
Light yellow solid, m.p. 222.8~223.9 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 8.11 (d, J = 5.0 Hz,
1H), 7.73 (d, J = 7.5 Hz, 2H), 7.54 (d, J = 7.9 Hz, 2H), 7.31 (d, J = 6.6 Hz, 3H), 7.25 (d, J = 7.8 Hz, 2H),
5.74 (d, J = 6.6 Hz, 1H), 3.95–3.84 (m, 1H), 3.76–3.63 (m, 6H), 3.59 (s, 2H), 3.13 (d, J = 13.7 Hz, 1H),
2.40 (s, 3H). 13C NMR (101 MHz, DMSO-d6)
δ 163.89, 157.95, 156.29, 150.82, 145.81, 134.03, 132.46,
131.45, 131.41, 129.81, 129.71(2,C), 129.17, 128.43, 126.68(2,C), 126.29, 124.43, 106.60, 66.29(2,C), 61.90,
46.26(2,C).HRMS (ESI): m/z calcd for (C27H23Cl2N3OS + H)+: 508.1017; found: 508.1059.
4-(2-(3,5-diphenyl-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]pyrimidin-4-yl)morpholine (9i). Light
yellow solid, m.p. 231.7~233.2 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 8.04 (d, J = 5.5 Hz, 1H),
7.77 (d, J = 7.6 Hz, 2H), 7.48–7.37 (m, 3H), 7.30–7.17 (m, 6H), 5.71 (dd, J = 12.0, 5.3 Hz, 1H), 3.87 (dd,
J = 17.4, 12.2 Hz, 1H), 3.63 (dt, J = 19.1, 10.7 Hz, 6H), 3.49 (d, J = 13.7 Hz, 2H), 3.45–3.40 (m, 1H). HRMS
(ESI): m/z calcd for (C27H22Cl2FN3OS + H)+: 528.0898; found: 528.0891.
4-(2-(3-(4-fluorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-◦1H-pyrazol-1-yl)thieno[2,3-d]pyrimidin-4-yl)
1
morpholine (9j). Light yellow solid, m.p. 215.8~216.8 C; H NMR (400 MHz, DMSO-d6)
δ
(ppm):
7.91–7.86 (m, 2H), 7.50 (t, J = 5.0 Hz, 1H), 7.39–7.24 (m, 4H), 7.16 (t, J = 8.8 Hz, 1H), 7.06 (d, J = 8.6 Hz,
2H), 5.72 (dd, J = 11.0, 5.3 Hz, 1H), 4.07–3.96 (m, 1H), 3.82 (s, 3H), 3.74 (s, 4H), 3.65–3.56 (m, 3H), 3.48 (s,
2H). HRMS (ESI): m/z calcd for (C27H22BrCl2N3OS + H)+: 588.0100; found: 588.0110.
4-(2-(3-(4-bromophenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]Pyrimidin-4-yl)
morpholine (9k). Yellow solid, m.p. 221.2~222.0 ◦C; 1H -NMR (400 MHz, DMSO-d6)
δ (ppm): 8.14 (d,
J = 5.6 Hz, 1H), 7.93–7.85 (m, 2H), 7.55 (d, J = 8.2 Hz, 2H), 7.39–7.30 (m, 3H), 7.27 (d, J = 8.3 Hz, 2H),
5.77 (dd, J = 11.9, 5.4 Hz, 1H), 3.93 (dd, J = 17.7, 12.3 Hz, 1H), 3.80–3.65 (m, 6H), 3.59 (d, J = 10.2 Hz,
2H), 3.19 (dd, J = 17.9, 5.8 Hz, 1H). HRMS (ESI): m/z calcd for (C28H25Cl2N3O2S + H)+: 538.1123;
found: 538.1199.
4-(2-(3-(4-bromophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]pyrimidine-4-Morpholine (9l).
Light yellow solid, m.p. 221.2~222.0 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 8.05 (d, J = 5.5 Hz,
1H), 7.76 (d, J = 7.3 Hz, 2H), 7.50 7.39 (m, 5H), 7.21 (dd, J = 17.0, 6.8 Hz, 3H), 5.70 (d, J = 6.7 Hz, 1H),
3.86 (d, J = 5.2 Hz, 1H), 3.69 (t, J = 10.6 Hz, 2H), 3.64–3.57 (m, 4H), 3.53 (t, J = 9.8 Hz, 2H), 3.12 (d,
J = 5.5 Hz, 1H). HRMS (ESI): m/z calcd for (C28H25Cl2N3OS + H)+: 522.1174; found: 522.1203.

Molecules 2019, 24, 3422
10 of 14
4-(2-(3-(3,4-dichlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]pyrimidin-4-yl)
morpholine (15a). Light yellow solid, m.p. 131.2~132.5 ◦C; 1H NMR (400 MHz, DMSO-d6)
(ppm): 7.73
δ
(d, J = 8.4 Hz, 2H), 7.57 (d, J = 8.1 Hz, 2H), 7.40 (d, J = 6.2 Hz, 1H), 7.19 (d, J = 6.0 Hz, 2H), 7.02 (d,
J = 8.5 Hz, 2H), 5.69 (dd, J = 12.0, 5.5 Hz, 1H), 3.92–3.84 (m, 1H), 3.81 (s, 3H), 3.62 (dd, J = 22.9, 8.6 Hz,
6H), 3.51 (d, J = 8.6 Hz, 2H), 3.17 (dd, J = 17.7, 5.6 Hz, 1H). HRMS (ESI): m/z calcd for(C27H25N3OS +
H)+: 440.1797; found: 440.1808.
4-(2-(5-(4-bromophenyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]pyrimidin-4-yl)
morpholine (15b). Light yellow solid, m.p. 265.3~266.3 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 7.69
(d, J = 7.8 Hz, 2H), 7.50 (d, J = 8.1 Hz, 2H), 7.40 (d, J = 6.2 Hz, 1H), 7.27 (d, J = 7.8 Hz, 2H), 7.20 (d,
J = 7.8 Hz, 2H), 5.69 (dd, J = 12.2, 5.1 Hz, 1H), 3.93–3.82 (m, 1H), 3.62 (dd, J = 23.2, 8.0 Hz, 6H), 3.51 (d,
J = 7.4 Hz, 2H), 3.12 (d, J = 18.1 Hz, 1H). HRMS (ESI): m/z calcd for (C28H27N3O2S + H)+: 470.1902;
found: 470.1939.
4-(2-(3,5-bis(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]pyrimidin-4-yl)Morpholine
(15c).
Yellow solid, m.p. 254.7~255.4 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ
(ppm): 7.88–7.82 (m, 2H), 7.39 (d,
J = 6.1 Hz, 1H), 7.29 (dd, J = 16.3, 8.0 Hz, 4H), 7.19 (d, J = 6.1 Hz, 1H), 7.14 (d, J = 8.5 Hz, 2H), 5.73 (dd,
J = 12.2, 5.2 Hz, 1H), 3.92–3.83 (m, 1H), 3.62 (dd, J = 20.8, 8.0 Hz, 6H), 3.51 (d, J = 8.0 Hz, 2H), 3.13 (dd,
J = 18.0, 5.0 Hz, 1H). HRMS (ESI): m/z calcd for (C27H23F2N3OS + H)+: 476.1608; found: 476.1640.
4-(2-(3-(3,4-dichlorophenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]pyrmidin-4-yl)
morpholine (15d). Yellow solid, m.p. 237.7~238.8 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 7.60
(d, J = 7.9 Hz, 2H), 7.48 (d, J = 6.8 Hz, 2H), 7.31 (d, J = 5.8 Hz, 1H), 7.19 (d, J = 7.8 Hz, 2H), 7.11 (d,
J = 6.4 Hz, 2H), 5.62 (d, J = 6.1 Hz, 1H), 3.82–3.73 (m, 1H), 3.55 (s, 2H), 3.51 (d, J = 7.4 Hz, 4H), 3.43
(d, J = 8.8 Hz, 2H), 3.08 (d, J = 12.8 Hz, 1H). 13C NMR (101 MHz, DMSO-d6)
δ 162.70, 162.39, 161.13,
160.78, 157.90, 156.38, 140.04, 128.77, 128.03, 121.75, 115.91(2,C), 115.77(2,C), 114.63(3,C), 110.46, 66.28,
62.03, 55.83, 47.11, 42.77. HRMS (ESI): m/z calcd for (C28H26FN3O2S + H)+: 488.1808; found: 488.1842.
4-(2-(3-(3,4-Dichlorophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]pyrimidine-4-yl)morpholine
(
15e). Yellow solid, m.p. 263–265 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 7.80 (d, J = 6.9 Hz,
2H), 7.61–7.55 (m, 2H), 7.49–7.44 (m, 3H), 7.43–7.39 (m, 1H), 7.21 (d, J = 6.2 Hz, 2H), 5.73 (dd, J = 12.2,
5.8 Hz, 1H), 3.89 (dd, J = 17.7, 12.2 Hz, 1H), 3.69–3.56 (m, 6H), 3.53–3.48 (m, 2H), 3.20 (dd, J = 17.8,
5.8 Hz, 1H). HRMS (ESI): m/z calcd for (C28H26BrN3OS + H)+: 534.1041; found: 534.1008.
4-(2-(5-(4-methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]pyrimidine-4-y)morpholine
◦
1
(
15f). Yellow solid, m.p. 235–237 C; H NMR (400 MHz, DMSO-d6) δ (ppm): 7.78 (d, J = 8.5 Hz,
2H), 7.42 (d, J = 6.1 Hz, 1H), 7.30 (d, J = 7.2 Hz, 2H), 7.25 (d, J = 6.2 Hz, 4H), 7.04 (d, J = 8.6 Hz, 2H),
5.73–5.65 (m, 1H), 3.91 (dd, J = 18.0, 12.1 Hz, 1H), 3.82 (s, 3H), 3.66–3.53 (m, 6H), 3.46 (s, 2H), 3.16 (d,
J = 17.8 Hz, 1H). HRMS (ESI): m/z calcd for (C27H23BrFN3OS + H)+: 538.0790; found: 538.0832.
4-(2-(3-(3,4-dichlorophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]pyrimidin-4-yl)morpholine
(
15g). Yellow solid, m.p. 267–268 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 7.69 (d, J = 7.7 Hz, 2H),
7.57 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 6.2 Hz, 1H), 7.28 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 6.3 Hz, 2H), 5.70 (dd,
J = 11.9, 5.6 Hz, 1H), 3.91–3.82 (m, 1H), 3.65–3.56 (m, 6H), 3.51 (d, J = 8.4 Hz, 2H), 3.21–3.13 (m, 1H),
2.35 (s, 3H). HRMS (ESI): m/z calcd for (C27H24BrN3OS + H)+: 517.0823; found: 517.0798.
4-(2-(3-(4-bromophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]pyrimidine-4-yl)morpholine
(
15h). Yellow solid, m.p. 255–257 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 7.68 (d, J = 8.1 Hz, 2H),
7.50 (d, J = 8.3 Hz, 2H), 7.40 (t, J = 6.6 Hz, 1H), 7.29 (d, J = 10.6 Hz, 2H), 7.22 – 7.17 (m, 3H), 5.69 (dd,
J = 11.7, 4.7 Hz, 1H), 3.92–3.83 (m, 1H), 3.63–3.55 (m, 6H), 3.50 (d, J = 11.1 Hz, 2H), 3.09 (dd, J = 17.6,
5.7 Hz, 1H), 2.35 (s, 3H). 13C NMR (101 MHz, DMSO-d6)
δ 171.77, 158.31, 154.66, 151.51, 145.64, 132.28,
131.46, 131.41, 129.95, 129.76, 129.17(2,C), 128.46, 126.76(2,C), 126.26, 121.64, 117.97, 110.41, 66.32(2,C),
61.88, 46.96(2,C), 42.01.HRMS (ESI): m/z calcd for (C27H23Cl2N3OS + H)+: 508.1017; found: 508.1044.

Molecules 2019, 24, 3422
11 of 14
4-(2-(3,5-diphenyl-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]pyrimidin-4-yl)morpholine (15i). Yellow solid,
m.p. 266–267 ◦C; 1H NMR (400 MHz, DMSO-d6)
(ppm): 8.04 (d, J = 5.5 Hz, 1H), 7.77 (d, J = 7.6 Hz,
δ
2H), 7.56–7.35 (m, 3H), 7.32–7.15 (m, 6H), 5.71 (dd, J = 12.0, 5.3 Hz, 1H), 3.87 (dd, J = 17.4, 12.2 Hz, 1H),
3.70–3.54 (m, 6H), 3.50 (s, 2H), 3.09 (dd, J = 17.6, 5.3 Hz, 1H). HRMS (ESI): m/z calcd for (C27H22Cl2FN3OS
+ H)+: 526.0923; found: 526.0889.
4-(2-(3-(4-fluorophenyl)-5-(4-methoxyphenyl)-4,5◦-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]pyrimidin-4-yl)
1
morpholine (15j). Yellow solid, m.p. 271–272 C; H NMR (400 MHz, DMSO-d6)
δ
(ppm): 7.74 (d,
J = 7.9 Hz, 2H), 7.35 (d, J = 6.0 Hz, 1H), 7.26–7.17 (m, 3H), 7.07 (d, J = 8.8 Hz, 2H), 6.95 (d, J = 8.6 Hz,
2H), 5.63 (dd, J = 11.8, 5.6 Hz, 1H), 3.85 (d, J = 17.8 Hz, 1H), 3.73 (s, 3H), 3.55 (dd, J = 25.4, 8.6 Hz,
7H), 3.09 (d, J = 16.8 Hz, 2H). HRMS (ESI): m/z calcd for (C27H22BrCl2N3OS + H)+: 588.0100; found:
588.0132.
4-(2-(3-(4-bromophenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]Pyrimidin-4-yl)morpholine
(
15k). Yellow solid, m.p. 251–252 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 7.75 (s, 2H), 7.41 (d,
J = 7.6 Hz, 2H), 7.31 (d, J = 5.5 Hz, 1H), 7.21 (t, J = 8.0 Hz, 2H), 7.12 (d, J = 7.9 Hz, 3H), 5.62 (d,
J = 6.8 Hz, 1H), 3.85–3.74 (m, 1H), 3.53 (d, J = 13.2 Hz, 6H), 3.44 (d, J = 8.9 Hz, 2H), 3.05 (d, J = 17.7 Hz,
1H). HRMS (ESI): m/z calcd for (C28H25Cl2N3O2S + H)+: 538.1123; found: 538.1161.
4-(2-(3-(4-bromophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]pyrimidine-4-Morpholine (15l).
Yellow solid, m.p. 244–245 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 7.79 (d, J = 7.3 Hz, 2H), 7.50 (d,
J = 8.3 Hz, 2H), 7.44 (t, J = 8.0 Hz, 3H), 7.40 (d, J = 6.2 Hz, 1H), 7.24–7.18 (m, 3H), 5.71 (dd, J = 12.1,
5.7 Hz, 1H), 3.89 (dd, J = 17.5, 12.6 Hz, 1H), 3.62 (dd, J = 23.5, 8.5 Hz, 6H), 3.51 (d, J = 8.0 Hz, 2H), 3.13
(dd, J = 17.7, 5.6 Hz, 1H). HRMS (ESI): m/z calcd for (C28H25Cl2N3OS + H)+: 522.1174; found: 522.1201.
4-(2-(3-(3,4-dichlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-5,6,7,8-tetrahydrobenzo
[4,5]thieno[2,3-d]pyrimidin-4-yl)morpholine (21a). Dark blue solid, m.p. 252–253 ◦C; ESI-MS [M + H]+
m/z: 594.5; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 7.73 (d, J = 8.7 Hz, 2H), 7.56 (d, J = 7.0 Hz, 2H),
7.20 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 7.0 Hz, 2H), 5.67 (d, J = 5.7 Hz, 1H), 3.88 (d, J = 17.1 Hz, 1H), 3.81 (s,
3H), 3.62 (d, J = 5.4 Hz, 2H), 3.58 (s, 2H), 3.18 (d, J = 12.1 Hz, 3H), 2.99 (s, 2H), 2.73 (s, 4H), 1.81 (s, 4H).
4-(2-(3-(4-bromophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-5,6,7,8-tetrahdrobenzo[4,5]thieno
◦
[2,3-d]pyrimidin-4-yl)morpholine (21b). Dark blue solid, m.p. 243–244 C; ESI-MS [M + H]+ m/z:
588.5; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 7.69 (d, J = 7.9 Hz, 2H), 7.50 (d, J = 8.3 Hz, 2H), 7.27
(d, J = 7.7 Hz, 2H), 7.19 (d, J = 8.3 Hz, 2H), 5.70 (dd, J = 12.3, 5.4 Hz, 1H), 3.88 (dd, J = 17.7, 12.3 Hz,
1H), 3.64 (s, 2H), 3.57 (s, 2H), 3.15 (d, J = 16.2 Hz, 3H), 2.99 (s, 2H), 2.74 (s, 4H), 2.35 (s, 3H), 1.78 (d,
J = 13.4 Hz, 4H).
4-(2-(3,5-diphenyl-4,5-dihydro-1H-pyrazol-1-yl)-5,6,7,8-tetrahydrobenzo[4,5]thiophene[2,3-d]pyramidin
◦
1
-4-yl)morpholine (21c). Dark blue solid, m.p. 212–213 C; ESI-MS [M + H]+ m/z: 495.6; H NMR
(400 MHz, DMSO-d6)
δ (ppm): 7.82–7.79 (m, 2H), 7.46 (d, J = 7.6 Hz, 3H), 7.29 (d, J = 7.3 Hz, 2H), 7.23
(dd, J = 9.2, 1.9 Hz, 3H), 5.73 (dd, J = 12.1, 5.5 Hz, 1H), 3.92 (dd, J = 17.7, 12.1 Hz, 1H), 3.65–3.60 (m,
2H), 3.55 (d, J = 6.5 Hz, 2H), 3.14 (dd, J = 17.7, 5.7 Hz, 3H), 3.02–2.94 (m, 2H), 2.74 (s, 4H), 1.80 (d,
J = 28.8 Hz, 4H).
4-(2-(3-(4-fluorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-5,6,7,8-tetrahydrobenzo
[4,5]thieno[2,3-d]pyrimidin-4-yl)morpholine (21d). Dark blue solid, m.p. 189-193 ◦C; ESI-MS [M + H]+
m/z: 543.6; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 7.74 (d, J = 8.7 Hz, 2H), 7.28–7.23 (m, 2H), 7.12 (t,
J = 8.8 Hz, 2H), 7.02 (d, J = 8.8 Hz, 2H), 5.71 (dd, J = 12.0, 5.4 Hz, 1H), 3.87 (d, J = 5.0 Hz, 1H), 3.81 (s,
3H), 3.68–3.61 (m, 2H), 3.57 (d, J = 6.4 Hz, 2H), 3.20–3.12 (m, 3H), 3.00 (s, 2H), 2.73 (s, 4H), 1.79 (d,
J = 29.8 Hz, 4H).
4-(2-(3-(4-bromophenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-5,6,7,8-tetrahydrobenzo[4,5]
thieno[2,3-d]pyrimidin-4-yl)morpholine (21e). Dark blue solid, m.p. 272–273 ◦C; ESI-MS [M + H]+ m/z:
592.5; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 7.85 (d, J = 6.4 Hz, 2H), 7.50 (d, J = 7.3 Hz, 2H), 7.30 (s,

Molecules 2019, 24, 3422
12 of 14
2H), 7.20 (d, J = 8.0 Hz, 2H), 5.77–5.66 (m, 1H), 3.91 (t, J = 16.3 Hz, 1H), 3.63 (s, 2H), 3.58 (s, 2H), 3.18 (s,
3H), 3.01 (s, 2H), 2.74 (s, 4H), 1.79 (d, J = 18.0 Hz, 4H).
4-(2-(3-(4-bromophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-yl)morpholine (21f). Dark blue solid, m.p. 257–259 ◦C; ESI-MS [M + H]+ m/z: 574.5;
1H NMR (400 MHz, DMSO-d6)
δ (ppm): 7.80 (d, J = 7.8 Hz, 2H), 7.48 (dd, J = 17.6, 7.9 Hz, 5H), 7.20 (d,
J = 8.4 Hz, 2H), 5.73 (dd, J = 11.9, 5.5 Hz, 1H), 3.91 (dd, J = 17.9, 12.1 Hz, 1H), 3.64 (d, J = 6.0 Hz, 2H),
3.57 (s, 2H), 3.15 (dd, J = 17.8, 5.5 Hz, 3H), 3.02 (s, 2H), 2.74 (s, 4H), 1.78 (d, J = 18.3 Hz, 4H).
4-(2-(5-(4-bromophenyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-5,6,7,8-tetrahydrobenzo
[4,5]thieno[2,3-d]pyrimidin-4-yl)morpholine (21g). Dark blue solid, m.p. 231–234 ◦C; ESI-MS [M + H]+
m/z: 643.4; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 7.71 (d, J = 8.3 Hz, 2H), 7.63 (d, J = 8.3 Hz, 2H),
7.57 (d, J = 11.3 Hz, 2H), 7.22 (d, J = 8.8 Hz, 1H), 5.71 (dd, J = 11.9, 5.9 Hz, 1H), 3.89 (dd, J = 18.0,
12.5 Hz, 1H), 3.62 (s, 2H), 3.57 (s, 2H), 3.21 (dd, J = 17.9, 6.2 Hz, 3H), 2.99 (s, 2H), 2.73 (s, 4H), 1.78 (d,
J = 37.6 Hz, 4H).
4-(2-(3-(3,4-dichlorophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
◦
[2,3-d]pyrimidin-4-yl)morpholine (21h). Dark blue solid, m.p. 293–294 C; ESI-MS [M + H]+ m/z:
563.1; H NMR (400 MHz, DMSO-d6)
1
δ (ppm): 7.80 (d, J = 6.5 Hz, 2H), 7.58 –7.55 (m, 2H), 7.46 (d,
J = 7.6 Hz, 3H), 7.23–7.19(m, 1H), 5.73 (dd, J = 12.1, 5.9 Hz, 1H), 3.92 (dd, J = 17.8, 12.2 Hz, 1H), 3.63 (d,
J = 6.2Hz, 2H), 3.58 (d, J = 6.4Hz, 2H), 3.21 (dd, J = 18.1, 6.3 Hz, 3H), 3.01 (s, 2H), 2.74 (s, 4H), 1.79 (d,
J = 23.9 Hz, 4H).
3.3. Cytotoxicity Assay In Vitro
The in vitro cytotoxic activities of Compounds 9a–l, 15a–l, and 21a–h 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
×
10 3 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
µg/mL and incubated with
cells at 37 ◦C for 4 h. The formazan crystals were dissolved in 100
µL of DMSO in each well, and the
absorbency at 492 nm (for absorbance of MTT formazan) and 630 nm (for the reference wavelength)
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 was 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).
3.4. mTOR Kinase Assay
The potent compounds 9a and 15a were tested for their activities against mTOR enzyme using
Kinase-Glo® Luminescent Kinase Assay (Promega, Madison, WI, USA), with NVPBEZ-235 and
PI103 as positive controls. The kinase reaction was done in a 384-well black plate. Each well was
loaded with 50
substrate (l- -phosphatidylinositol); Avanti Polar Lipids (Avanti Polar Lipids, Inc., Alabaster, AL,
USA); prepared in 3% octyl-glucoside) and the mTOR protein 2.5 nM was then added to it. The
reaction was started by the addition of 5 L of 10 M ATP prepared in the reaction buffer (50 mM
HEPES pH 7.5, 1 mM EGTA, 3 mM MnCl2, 10 mM MgCl2, 2 mM DTT and 0.01% Tween-20) and was
incubated for 60 min. It was terminated by the addition of 10 L Kinase-Glo buffer. The plates were
µL of test items (in 90% DMSO) and 5 µL reaction buffer containing 10 µg/mL PI
α
µ
µ
µ
then read in Synergy 2 readers (BioTek, Winooski, VT, USA) for luminescence detection. The assay was
repeated two times and the results expressed as IC50 (inhibitory concentration 50%) were the averages
of two determinations.

Molecules 2019, 24, 3422
13 of 14
3.5. PI3Kα Kinase Assay
The potent compounds 9a and 15a were tested for their 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 L of test items
(in 90% DMSO) and 5 L of reaction buffer containing 10 g/mL PI substrate (l- -phosphatidylinositol;
Avanti Polar Lipids (Avanti Polar Lipids, Inc.); prepared in 3% octyl-glucoside), and the PI3K protein
(10 nM) was then added to it. The reaction was started by the addition of 5 L of 1 M Adenosine
triphosphate (ATP) prepared in the reaction buffer and incubated for 60 min for p110 . It was
terminated by the addition of 10 L of Kinase-Glo buffer. The plates were then read in a Synergy 2
µ
µ
µ
α
α
µ
µ
α
µ
reader (BioTek, Winooski, VT, USA) for luminescence detection. The assay was repeated two times and
the results expressed as IC50 (inhibitory concentration 50%) were the averages of two determinations.
3.6. Docking Studies
Molecular docking simulation studies were carried out by the AutoDock 4.2 software (The Scripps
Research Institute, San Diego, CA, USA). The docking tutorial we used and the detailed AutoDock
basic operational methods can be found at: http://autodock.scripps.edu/faqs-help/tutorial. The protein
preparation process of flexible docking mainly includes fixing the exact residues, adding hydrogen
atoms, removing irrelevant water molecules, adding charges, etc. The potent compounds were selected
as ligand examples, and the structures of PI3Kα (PDB code: 3TL5, http://www.pdb.org/) were selected
as the docking models. Only the best-scoring ligand–protein complexes were used for the binding site
analyses. All the docking results were processed and modified in Open-Source PyMOL 1.8.x software
(https://pymol.org).
4. Conclusions
Three series of thienopyrimidine derivatives containing pyrazole structure were designed and
synthesized. In addition, we evaluated their cytotoxic activities against four cancer cell lines in vitro
The pharmacological indicated that most of the compounds showed moderate cytotoxic activity against
the tested cancer cell lines. What is more, the inhibitory activities of the compounds 9a and 15a
.
against PI3K
α
and mTOR kinase were further investigated. Compound 9a exhibited moderate levels
kinase. Although compound 9a did
of inhibition activity against four cancer cell lines and PI3K
α
not reach the same level of inhibition as the positive control drug, it also gave us new direction for
developing novel thienopyrimidines containing the pyrazole linker group as PI3Kα inhibitor.
Author Contributions: W.Z., Z.Y., and P.Z. provided and managed the project and supervise the progress of the
project; L.Y., Q.W., B.Z., and Y.F. designed the chemical synthesis route and performed the experiments; L.Y., Q.W.,
and C.W. conducted experimental data analysis and software processing; L.Y., W.Z., and P.Z. started the project,
and wrote and revised the paper.
Funding: We gratefully acknowledge the generous support provided by The National Natural Science Funds
(No. 21967009); The Project Supported by Natural Science Foundation of Jiangxi, China (20171ACB21052
and 2018BBG70003); Natural Science Funds for Distinguished Young Scholar of Jiangxi Province, China
(20171BCB23078); Key projects of the youth fund, Natural Science Funds of Jiangxi Province (20171ACB21052).
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Dehnhardt, C.M.; Venkatesan, A.M.; Delos Santos, E.; Chen, Z.; Santos, O.; Ayral-Kaloustian, S.;
Brooijmans, N.; Mallon, R.; Hollander, I.; Feldberg, L.; et al. Lead optimization of N-3-substituted
7-morpholinotriazolopyrimidines as dual phosphoinositide 3-kinase/mammalian target of rapamycin
inhibitors: Discovery of PKI-402. J. Med. Chem. 2009, 53, 798–810. [CrossRef] [PubMed]
2.
Guba, M.; von Breitenbuch, P.; Steinbauer, M.; Koehl, G.; Flegel, S.; Hornung, M.; Bruns, C.J.; Zuelke, C.;
Farkas, S.; Anthuber, M.; et al. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis:
Involvement of vascular endothelial growth factor. Nat. Med. 2002, 8, 128. [CrossRef] [PubMed]

Molecules 2019, 24, 3422
14 of 14
3.
4.
5.
Marone, R.; Cmiljanovic, V.; Giese, B.; Wymann, M.P. Targeting phosphoinositide 3-kinase—Moving towards
therapy. Biochim. Biophys. Acta (BBA) Proteins Proteom. 2008, 1784, 159–185. [CrossRef] [PubMed]
Lv, X.; Ma, X.; Hu, Y. Furthering the design and the discovery of small molecule ATP-competitive mTOR
inhibitors as an effective cancer treatment. Expert Opin. Drug Dis. 2013, 8, 991–1012. [CrossRef] [PubMed]
Peng, W.; Tu, Z.C.; Long, Z.J.; Liu, Q.; Lu, G. Discovery of 2-(2-aminopyrimidin-5-yl)-4-morpholino
-N-(pyridin-3-yl) quinazolin-7-amines as novel PI3K/mTOR inhibitors and anticancer agents. Eur. J. Med.
Chem. 2016, 108, 644–654. [CrossRef] [PubMed]
6.
7.
Smith, M.C.; Mader, M.M.; Cook, J.A.; Iversen, P.; Ajamie, R.; Perkins, E.; Bloem, L.; Yip, Y.Y.; Barda, D.A.;
Waid, P.P.; et al. Characterization of LY3023414, a novel PI3K/mTOR dual inhibitor eliciting transient target
modulation to impede tumor growth. Mol. Cancer ther. 2016, 15, 2344–2356. [CrossRef] [PubMed]
Sarker, D.; Ang, J.E.; Baird, R.; Kristeleit, R.; Shah, K.; Moreno, V.; Clarke, P.A.; Raynaud, F.I.;
Levy, G.; Ware, J.A.; et al. First-in-human phase I study of pictilisib (GDC-0941), a potent pan–class
I phosphatidylinositol-3-kinase (PI3K) inhibitor, in patients with advanced solid tumors. Clin. Cancer Res.
2015, 21, 77–86. [CrossRef] [PubMed]
8.
9.
Spencer, A.; Yoon, S.S.; Harrison, S.J.; Morris, S.; Smith, D.; Freedman, S.J.; Brigandi, R.; Oliff, A.; Opalinska, J.B.;
Chen, C. Novel AKT inhibitor GSK2110183 shows favorable safety, pharmacokinetics, and clinical activity in
multiple myeloma. Preliminary results from a phase I first-time-in-human study. Blood. 2011, 118, 1856.
Huo, H.; Zhou, Z.; Wang, B.; Qin, J.; Liu, W.Y.; Gu, Y. Dramatic suppression of colorectal cancer cell growth
by the dual mTORC1 and mTORC2 inhibitor AZD-2014. Biochem. Bioph. Res. Commun. 2014, 443, 406–412.
[CrossRef] [PubMed]
10. Sutherlin, D.P.; Bao, L.; Berry, M.; Castanedo, G.; Chuckowree, I.; Dotson, J.; Folks, A.; Friedman, L.;
Goldsmith, R.; Gunzner, J.; et al. Discovery of a potent, selective, and orally available class I
phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) kinase inhibitor (GDC-0980)
for the treatment of cancer. J. Med. Chem. 2011, 54, 7579–7587. [CrossRef] [PubMed]
11. Heffron, T.P.; Berry, M.; Castanedo, G.; Chang, C.; Chuckowree, I.; Dotson, J.; Folkes, A.; Gunzner, J.;
Lesnick, J.D.; Lewis, C.; et al. Identification of GNE-477, a potent and efficacious dual PI3K/mTOR inhibitor.
Bioorg. Med. Chem. Lett. 2010, 20, 2408–2411. [CrossRef] [PubMed]
12. Zhu, W.; Zhai, X.; Fu, Q.; Guo, F.; Bai, M.; Wang, J.; Wang, H.; Gong, P. Design, synthesis and anticancer
activity of 4-morpholinothieno [3, 2-d] pyrimidine derivatives bearing arylmethylene hydrazine moiety.
Chem. Pharm. Bull. 2012, 60, 1037–1045. [CrossRef] [PubMed]
13. Zhu, W.; Chen, C.; Sun, C.; Xu, S.; Wu, C.; Lei, F.; Xia, H.; Tu, Q.; Zheng, P. Design, synthesis and docking
studies of novel thienopyrimidine derivatives bearing chromone moiety as mTOR/PI3K
α inhibitors. Eur. J.
Med. Chem. 2015, 93, 64–73. [CrossRef] [PubMed]
14. Zhu, W.; Liu, Y.; Zhai, X.; Wang, X.; Zhu, Y.; Wu, D.; Zhou, H.; Gong, P.; Zhao, Y. Design, synthesis and
3D-QSAR analysis of novel 2-hydrazinyl-4-morpholinothieno [3, 2-d] pyrimidine derivatives as potential
antitumor agents. Eur. J. Med. Chem. 2012, 57, 162–175. [CrossRef] [PubMed]
15. Wang, Q.; Li, X.; Sun, C.; Zhang, B.; Zheng, P.; Zhu, W.; Xu, S. Synthesis and Structure–Activity Relationships
of 4-Morpholino-7, 8-Dihydro-5H-Thiopyrano [4, 3-d] pyrimidine Derivatives Bearing Pyrazoline Scaffold.
Molecules 2017, 22, 1870. [CrossRef]
Sample Availability: Samples of the compounds 9a–l, 15a–l, and 21a–h are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).