Design, synthesis and biological evaluation of thieno[3,2-d]pyrimidine derivatives containing aroyl hydrazone or aryl hydrazide moieties for PI3K and mTOR dual inhibition

Article abstract of DOI:10.1016/j.bioorg.2020.104197

Recently, PI3K and mTOR have been regarded as promising targets for cancer treatment. Herein, we designed and synthesized four series of novel thieno[3,2-d]pyrimidine derivatives that containing aroyl hydrazone or aryl hydrazide moieties. These derivatives act as PI3K/mTOR dual inhibitors, suggesting that they can be used as cancer therapeutic agents. All compounds were tested for anti-proliferative activity against four cancer cell lines. The structure-activity relationship (SAR) studies were conducted by varying the moieties at the C-6 and C-2 positions of the thieno[3,2-d]pyrimidine core. It indicated that aryl hydrazide at C-6 position and 2-aminopyrimidine at C-2 position are optimal fragments. Compound 18b showed the most potent in vitro activity (PI3Kα IC₅₀ = 0.46 nM, mTOR IC₅₀ = 12 nM), as well as good inhibition against PC-3 (human prostate cancer), HCT-116 (human colorectal cancer), A549 (human lung adenocarcinoma) and MDA-MB-231 (human breast cancer) cell lines. Furthermore, Annexin-V and propidium iodide (PI) double staining confirmed that 18b induces apoptosis in cytotoxic HCT-116 cells. Moreover, the influence of 18b on cell cycle distribution was assessed on the HCT-116 cell line, and a cell cycle arrest was observed at the G₁/S phases.

Full text of DOI:10.1016/j.bioorg.2020.104197

BioorganicChemistry104(2020)104197
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and biological evaluation of thieno[3,2-d]pyrimidine
derivatives containing aroyl hydrazone or aryl hydrazide moieties for PI3K
and mTOR dual inhibition
Yufei Hana, Ye Tiana, Ruxin Wanga, Siyu Fua, Jia Jianga, Jiawen Donga, Mingze Qina,c,
⁎
⁎
Yunlei Houa,b, , Yanfang Zhaoa,
a Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang
110016, China
b Yangtze River Pharmaceutical Group Pharmaceutical Co., Ltd, 1 South Yangtze River Road Taizhou, Jiangsu 225321, China
c Chinese People’s Liberation Army Logistics Support Force No. 967 Hospital, Dalian 116021, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Recently, PI3K and mTOR have been regarded as promising targets for cancer treatment. Herein, we designed
and synthesized four series of novel thieno[3,2-d]pyrimidine derivatives that containing aroyl hydrazone or aryl
hydrazide moieties. These derivatives act as PI3K/mTOR dual inhibitors, suggesting that they can be used as
cancer therapeutic agents. All compounds were tested for anti-proliferative activity against four cancer cell lines.
The structure-activity relationship (SAR) studies were conducted by varying the moieties at the C-6 and C-2
positions of the thieno[3,2-d]pyrimidine core. It indicated that aryl hydrazide at C-6 position and 2-amino-
pyrimidine at C-2 position are optimal fragments. Compound 18b showed the most potent in vitro activity
(PI3Kα IC50 = 0.46 nM, mTOR IC50 = 12 nM), as well as good inhibition against PC-3 (human prostate cancer),
HCT-116 (human colorectal cancer), A549 (human lung adenocarcinoma) and MDA-MB-231 (human breast
cancer) cell lines. Furthermore, Annexin-V and propidium iodide (PI) double staining confirmed that 18b in-
duces apoptosis in cytotoxic HCT-116 cells. Moreover, the influence of 18b on cell cycle distribution was as-
sessed on the HCT-116 cell line, and a cell cycle arrest was observed at the G1/S phases.
PI3K/mTOR
Dual inhibitors
Anti-proliferative activities
Thieno[3,2-d]-pyrimidine derivatives
1. Introduction
tumour development, many inhibitors have been developed. Among
them, the PI3K inhibitors are generally classified into three categories:
Mutations within the phosphoinositide 3-kinase (PI3K)/protein ki-
nase B (Akt)/ mammalian target of rapamycin (mTOR) signalling
pathway are one of the most common causes of many human cancers
[1–4]. When cells are stimulated by stimulators such as growth factors,
they activate receptor tyrosine kinases (RTKs), G-protein-coupled re-
ceptors, Ras proteins, and PI3K kinase. PI3K kinase then generates
phosphorylated phosphatidylinositol from phosphatidylinositol; with
the participation of phosphatidylinositol-dependent protein kinase
(PKD), the PI3K kinase activates Akt by binding to its pH domain. After
activation, Akt can directly or indirectly activate mTOR kinase by
phosphorylating the mTOR molecule or the TSC1/2-complex, respec-
tively. Subsequently, the mTOR kinase then activates downstream
molecules, promoting cell proliferation, differentiation, protein synth-
esis, and metastasis [5–9].
pan-PI3K inhibitors, subtype-specific PI3K inhibitors, and dual pan-
PI3K/mTOR inhibitors [10–19]. Compared to single-target small-mo-
lecule inhibitors, dual PI3K/mTOR inhibitors not only inhibit cell
proliferation, but also promote cell apoptosis. These dual pan-PI3K/
mTOR inhibitors show high potency and little drug-resistance even at
low doses [20]. Dual PI3K/mTOR inhibitors are considered to be of
great value in cancer therapy, and are pursued in clinical trials [21].
The structures of some dual PI3K/mTOR inhibitors with different
scaffolds are shown in Fig. 1 [22–26].
In our previous works, a series of 2,4-dimorpholinylthieno[3,2-d]
pyrimidine derivatives were identified to be PI3K inhibitors. Among
them, compound A12 (Fig. 2) showed an IC50 value of 200 nM against
PI3Kα, and 10% inhibition against mTOR at 100 nM. The docking mode
of compound A12 with PI3Kγ (PDB:3DBS) indicated that only one hy-
drogen bond was formed between A12 and PI3Kγ. However, according
Given the important role played by the PI3K/Akt/mTOR pathway in
⁎ Corresponding authors.
E-mail addresses: houyunlei901202@163.com (Y. Hou), yanfangzhao@126.com (Y. Zhao).
https://doi.org/10.1016/j.bioorg.2020.104197
Received 12 March 2020; Received in revised form 3 July 2020; Accepted 17 August 2020
Availableonline28August2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

Y. Han, et al.
BioorganicChemistry104(2020)104197
Fig. 1. Structures of dual pan-PI3K/mTOR inhibitors.
Fig. 2. The design strategy for the target compounds.
to the docking result, C-6 position of the thieno[3,2-d]pyrimidine
moiety did not generate any hydrogen-bonding interactions with the
residues and the active protein cavity was not entirely occupied by the
moiety residing in the solvent exposure region. In this study, we re-
tained the 2,4-dimorpholinothieno[3,2-d]pyrimidine scaffold and in-
troduced some linkers to the C-6 position of thieno[3,2-d]pyrimidine
moiety, which was expected to occupy the solvent exposure region with
additional interactions.
novel 2,4-dimorpholinothieno[3,2-d]pyrimidine derivatives with aroyl
hydrazone and aryl hydrazide moieties (series I and II, respectively)
were designed and synthesized. The in vitro results of these compounds
showed that N'-benzoyl-2,4-dimorpholinothieno[3,2-d]pyrimidine-6-
carbohydrazide derivatives were beneficial to inhibitory activities.
We continued to analyze the binding mode of A12 with PI3Kγ, and
found that the morpholino group at the C-2 position of pyrimidine lo-
cated in a hydrophobic cavity which is surrounded by some acidic
amino acids (such as Asp 841 and Asp 964). Therefore, we plan to re-
place morpholino with some aromatic and basic fragments. By referring
to the structures of GDC0941 [32] and Apitolisib [33], 2-(1H-indazol-5-
yl)-4-morpholinothieno[3,2-d]pyrimidine (series III) and 2-(2-amino-
In our previous works, small molecules bearing aroyl hydrazone
moiety showed superior anticancer activities due to the presence of
hydrogen bond donors and acceptors as well as its flexible skeleton
[27–29]. Accumulating evidence have illustrated that hydrazone and
hydrazide fragments are important pharmacophores in the area of drug
design, as these groups readily bind with various enzymes and receptors
through hydrogen bond interactions. For example, PAC-1 [30] (Fig. 2),
bearing a hydrazone moiety, was found to be a highly active anticancer
drug. Compound 55 [31] (Fig. 2), containing a hydrazide group, dis-
played insecticidal and bactericidal activity. Therefore, we utilized the
bioisosterism to introduce aroyl hydrazone or aryl hydrazide fragments
and obtained compounds 8a and 10a. By comparing their docking
modes with lead compound A12 in PI3Kγ (Fig. 3), we found that com-
pounds 8a and 10a could form additional hydrogen bonds with Lys 802
and Ala 885 in solvent exposure region, respectively. Thus, two series of
pyrimidine-5-yl)-4-morpholinothieno[3,2-d]pyrimidine
(series IV) were designed and synthesized, respectively.
derivatives
2. Results and discussion
2.1. Chemistry
The overall synthetic routes for generating the target compounds
are illustrated in Schemes 1–5. The key intermediates 2-chloro-4-mor-
pholinothieno[3,2-d]pyrimidine-6-carbaldehyde (5) and 2-chloro-4-
morpholinothieno[3,2-d]pyrimidine-6-carboxylic acid (6) were
2

Y. Han, et al.
BioorganicChemistry104(2020)104197
Fig. 3. Docking modes of two compounds with PI3Kγ. (A) compound 8a with aroyl hydrazone in the binding site of PI3Kγ. (B) compound 10a with aryl hydrazide in
the binding site of PI3Kγ.
2.2. Biological evaluation
2.2.1. Enzymatic and anti-proliferative assays in vitro
All the target compounds were evaluated for their in vitro inhibitory
activities against PI3Kα kinase by a mobility shift assay, with the ATP
concentration at 1.0 μM. The PI3K inhibitor GDC0941 was used as
positive control. The cytotoxicity of the prepared compounds were
evaluated in vitro against four cancer cell lines: PC-3 (human prostate
cancer), HCT-116 (human colorectal cancer), A549 (human lung ade-
nocarcinoma) and MDA-MB-231 (human breast cancer). The results of
the mean values of experiments performed in triplicate, expressed as
half maximal inhibitory concentration (IC50) values, were summarized
Scheme 1. Synthesis of important intermediates 5–6. (a) urea, 190 °C; (b)
in Table 1.
Most of the compounds showed more potent activities than that of
°
POCl3, MeCN, 90 C; (c) morpholine, MeOH, r.t; (d) i-PrMgCl2, n-BuLi, DMF,
THF, −10 °C; (e) AgNO3, NaOH, THF, r.t.
GDC0941 against PI3Kα; however, some derivatives displayed superior
performance over others. Firstly, compounds bearing the hydrazone
moiety (8a-8b) exhibited lower inhibitory activity on PI3Kα at 1 μM
than the hydrazide derivatives (10a-e), which showed more than a 10-
fold improvement in the enzymatic activity compared to the hydrazone
(8a) derivatives, indicating that the hydrazide moiety was preferred by
the solvent exposure region of PI3Kα. In the following modifications,
we paid attention to the 2-position of the pyrimidine unit. Firstly, the
morpholino group was replaced by an indazole in compounds 16a–16k,
and it was found that these compounds increased the activity against
PI3Kα. Further, it was suggested that the indazole moiety has a sig-
nificant effect on inhibitory activity. Secondly, compounds with elec-
tron-donating groups (EDGs), such as 10b and 16b, showed more po-
tent inhibitory activities against PI3Kα than those with electron-
withdrawing groups (EWGs), such as 10c, 16c, and 16g.
achieved as shown in Scheme 1. Commercially available methyl 3-
aminothiophene-2-carboxylate (1) reacted with urea at 190 °C to afford
intermediate 2, which was then chlorinated with phosphorus oxy-
chloride in acetonitrile to provide intermediate 3 [29]. Further, inter-
mediate 3 was treated with morpholine in methanol, which resulted in
the formation of 4-(2-chlorothieno[3,2-d]pyrimidin-4-yl)morpholine
(4); this was then formylated to obtain 2-chloro-4-morpholinothieno
[3,2-d]pyrimidine-6-carbaldehyde (5) [34]. Oxidation of 5 with a so-
lution of sodium hydroxide and silver nitrate in THF gave 2-chloro-4-
morpholinothieno[3,2-d]pyrimidine-6-carboxylic acid (6) [35].
The commercially available substituted aryl hydrazides were
treated separately with intermediates 5 and 6 to afford 7a, 7b and 9a-
9e, respectively; these were then reacted with morpholine in n-BuOH to
generate the target compounds 8a, 8b and 10a-10e, respectively
(Schemes 2 and 3).
Interestingly, the trend seen for the enzymatic activities of these
compounds was also observed for their anti-proliferative activities.
Compounds with strong PI3Kα inhibitory activity (16a, 16f, and 16j)
also showed more potent anti-proliferative activities compared to de-
rivatives with relatively weak PI3Kα inhibitory activities (such as 10a-e
and 16g). Besides, by comparing the anti-proliferative activities of
series I and II, it can be found that compounds bearing aryl hydrazone
had lower cytotoxicity than aryl hydrazide (8a vs 10a). However, the
activities of both two series compounds were not satisfactory enough.
By comparing series II with III, it was observed that compounds
with indazole on C-2 position led to obvious improvement in anti-tumor
activities against PC-3, HCT-116 and A549, such as 16a, 16f, 16i and
16j. Among them, compound 16f with a methoxy group on the 2-
As described in Scheme 4, intermediate 12 was obtained via a
protection reaction of 11 with THP, followed by two Suzuki coupling
reactions using bis(pinacolato)diboron and Pd(PPh3)2Cl2 to yield in-
termediates 13 and 14, respectively. Then, 14 was subjected to acyla-
tion with HATU in DMF, followed by deprotection with methane-
sulfonic acid, resulting in target compounds 16a–16k.
Compounds 18a and 18b were synthesized from intermediate 6
through Suzuki coupling with PdCl2(dppf)2 with subsequent acylation
using substituted aryl hydrazides (Scheme 5).
Scheme 2. Synthesis of target compounds 8a,
8b. (f) AcOH, EtOH, 80 °C; (g) morpholine,
CF3COOH, n-BuOH, 115 °C.
3

Y. Han, et al.
BioorganicChemistry104(2020)104197
Scheme 3. Synthesis of target compounds
10a-10e. (h) EDCI, DMF, r.t; (i) morpholine,
CF3COOH, n-BuOH, 115 °C.
position of terminal phenyl ring showed the most potent anti-pro-
liferative activity. Though compound 16b showed poor antitumor ac-
tivity, it exhibited the most potent inhibitory activity against PI3Kα.
Compounds 18a-18b were obtained by replacing indazole with 2-
aminopyrimidine. We were pleased to find out that compound 18b
showed significant improvement in anti-proliferative activities
(IC50 = 1.95 μM, 2.02 μM, 1.62 μM and 1.55 μM against PC-3, HCT-
116, A549 and MDA-MB-231 cell lines, respectively) and maintained
the PI3Kα inhibitory activity. Then we conducted a statistical analysis
of compound 18b and confirmed that it can exert effective anti-pro-
liferative activities against tumor cell lines (Fig. 4). Therefore, we
thought that 16b and 18b can be used as promising compounds for
further biological evaluations.
mTOR). Thus, 18b was selected as a candidate for further studies.
2.2.3. Effect of compound 18b on cell cycle phases
The effect of compound 18b on various phases of cell cycle pro-
gression was tested on HCT-116 cell lines for 24 h at varied con-
centrations. The cells were fixed and stained with propidium iodide
(PI), and the DNA content was analyzed by flow cytometry. The ob-
tained results were compared with those of non-treated HCT-116 cells
as a control. As shown in Fig. 5, treatment of HCT-116 cells with 18b at
0.333 μM, 1.0 μM, 3.0 μM, and 9.0 μM concentrations increased the
percentage of G1-phase cells from 63.42% (control) to 65.36%, 69.46%,
71.19%, and 74.24%, respectively. These results confirm that com-
pound 18b arrested the cell cycle progression of the cell line in the G1/S
phases in a dose-dependent manner.
In conclusion, four series of compounds were designed and syn-
thesized, of which the most potent compound 18b showed promising
cytotoxicity against PC-3, HCT-116, A549 and MDA-MB-231 cell lines
with IC50 values of 1.95 μM, 2.02 μM, 1.62 μM and 1.55 μM, respec-
tively. The SAR of these compounds indicated that (i) aryl hydrazide on
C-6 position was optimal fragments; (ii) indazole or 2-aminopyrimidine
group on C-2 position exhibited more potent inhibitory activity than
morpholino; compounds with 2-aminopyrimidine on C-2 position
showed more anti-proliferation activities than indazole; (iii) introdu-
cing a methoxy group on the terminal phenyl ring could increase the
inhibitory activity. These encouraging results highlighted the potential
for further development of 2-(1H-indazol-5-yl)-4-morpholinothieno
[3,2-d]pyrimidine (series III) and 2-(2-aminopyrimidine-5-yl)-4-mor-
pholinothieno[3,2-d]pyrimidine derivatives (series IV) derivatives
containing aryl hyrazide moiety on C-6 position and indazole or 2-
aminopyrimidine on C-2 position as potent antitumor agents.
2.2.4. Cell apoptosis study of compound 18b
To characterize the mode of cell death induced by compound 18b,
biparametric-flow-cytometric analysis with Annexin-V and propidium
iodide (PI) was performed. Flow cytometry was used to investigate
whether the anti-proliferative effect of 18b was caused by the activa-
tion of cellular apoptosis in HCT-116 cells. Quantitative analysis of
early-apoptotic, advanced-apoptotic, and necrotic cells was performed
via an Annexin V-FITC and PI assay. HCT-116 cells were stimulated
with 0.333–9 μM concentrations of compound 18b for 24 h. DMSO was
used as negative control. As shown in Fig. 6, the apoptosis induced by
compound 18b was greater than that of the control. Furthermore, the
HCT-116 cells underwent both early apoptosis (Annexin V+ and PI−,
lower right), as well as advanced apoptosis and necrosis (Annexin V+
and PI+, upper right). Compound 18b proved to induce apoptosis by
22.4% at 9 μM compared to 13.7% at 0 μM of apoptotic cells in the
untreated control.
2.2.2. PI3K and mTOR enzymatic assays in vitro
The results of PI3K and mTOR inhibitory activity are summarized in
Table 2. In parallel with cellular results, 18b, bearing a 2-aminopyr-
imidine group at the C-2 position of the thieno[3,2-d]pyrimidine core,
was found to be more favourable than the related 16b derivative,
bearing an indazole group in the 2-position. In summary, compound
18b exhibited potent inhibitory activities against PI3K isoforms and
mTOR (IC50 = 0.46 nM for PI3Kα, IC50 = 55 nM for PI3Kβ,
IC50 = 13 nM for PI3Kγ, IC50 = 32 nM for PI3Kδ, and IC50 = 12 nM for
2.2.5. Wound-healing assay
As migration was an important characteristic for metastatic cancers,
the effect of compound 18b on the migration of cancer cells was in-
vestigated using a wound-healing assay. As shown in Fig. 7, treatment
of HCT-116 cells with compound 18b at indicated concentrations
markedly suppressed the wound healing in a concentration-dependent
manner.
Scheme 4. Synthesis of target compounds 16a-
16k. (j) 3,4-2H-dihydropyran (THP), PTSA,
ethyl acetate, r.t; (k) bis(pinacolato)diboron,
KOAc, Pd(PPh3)2Cl2, dioxane, 80 °C; (l) 2-
chloro-4-morpholinothieno[3,2-d]pyrimidine-6-
carboxylic acid (6), Pd(PPh3)2Cl2, Na2CO3, di-
oxane, 100 °C; (m) HATU, DMF, r.t; (n) metha-
nesulfonic acid, MeOH, r.t.
4

Y. Han, et al.
BioorganicChemistry104(2020)104197
Scheme 5. Synthesis of target compounds 18a
and 18b. (o) 5-(4,4,5,5-Tetramethyl-1,3,2-
dioxaborolan-2-yl)pyrimidin-2-amine, Cs2CO3,
PdCl2(dppf)2, dioxane, 90 °C; (p) EDCI, DMF, r.t.
2.2.6. Binding mode analysis
position of 18b and the Asp 964 and Asp 841 residues in the solvent-
exposed region. Docking structure of compound 18b overlapped with
GDC0941 in mTOR showed that the conformation and the binding
mode of compound 18b were well consistent with GDC0941. These
docking results further confirm the rationality of our design strategy.
It was also observed that both in PI3K and mTOR, the indazole
group of GDC0941 and the 2-aminopyrimidine group of compound 18b
could form hydrogen bonds and potential salt-bridges with Asp 841 or
Asp 964, proving the importance of basic group at C-2 position.
Compound 18b showed optimal PI3K and mTOR inhibitory activ-
ities, hence, a modelling study of 18b docked in PI3Kγ and mTOR was
performed. As shown in Fig. 8B, compound 18b occupied the PI3Kγ
kinase domain in a similar manner to GDC0941. Compound 18b
(Fig. 8A) was found to form one H-bond between the oxygen atom of
the morpholino group with the Val 882 residue. In addition, the aryl
hydrazide moiety was exposed in the solvent area, and the NH of aryl
hydrazide formed two H-bonds with residue Asp 950, while another
hydrogen bond was formed between the carbonyl of the aryl hydrazide
and the Thr 887 residue. Furthermore, the methoxy at the 4-position of
the terminal phenyl ring formed one H-bond with the Lys 890 residue,
and the 2-aminopyrimidine fragment at the C-2 position formed two
hydrogen bonds with the Asp 841 and Asp 964 residues. These hy-
drogen bonding interactions provided a significant contribution to PI3K
binding and inhibition. Compared to GDC0941, it was found to form
four hydrogen bonds with Val 882, Ala 805, Asp 841 and Tyr
867(Fig. 8C)
2.3. Physicochemical and ADME parameters
Compound 18b and GDC0941 were evaluated for their adaptability
with Lipinski's rule of five in free online website (http://www.
swissadme.ch/index.php).
As shown in Table 3, compound 18b conformed not well to the
Lipinski's rule of five. Concerning oral bioavailability, GDC0941 ex-
pected 0.55 of the probability of oral bioavailability score > 10% in
rats for all compounds, > 18b (0.17). Based on the potent antitumor
activity of compound 18b, further studies on optimization of drug-
likeness properties of this series derivatives are currently underway in
our laboratory.
Similar hydrogen bond interactions were observed when 18b
docked in mTOR (Fig. 8D). In contrast to three hydrogen bonds found
in the structure of GDC0941 with mTOR (Fig. 8F), hydrogen bonds
were observed between the morpholino group of 18b and the Val 882
residue, as well as between the 2-aminopyrimidine group at the C-2
Table 1
PI3Kα inhibitory activities and anti-proliferation activities of target compounds.
O
O
N
O
N
O
N
N
O
HN NH
O
S
O
HN NH
O
O
HN NH
O
S
S
S
N
NH
N
N
N
N
N
HN
O
N
N
N
N
N
N
N
Ar
Ar
Ar
Ar
O
O
N
NH2
Series I: 8a,8b
Series II:10a-e
Series III:16a-16k
Series IV:18a,18b
Compd.
Ar
IC50 a (μmol/L)
PC-3
SD
PI3Kα% inhibition at 1 μMd
HCT-116
A549
MDA-MB-231
8a
Ph
33.51
> 50
34.22
21.32
32.74
> 50
25.21
5.20
0.14
> 50
> 50
43.01
32.73
29.15
> 50
32.72
8.31
> 50
> 50
> 50
15.12
20.40
> 50
17.02
8.11
N.D. c
N.D. c
N.D. c
N.D. c
N.D. c
N.D. c
N.D. c
< 0
< 0
8b
2-Py
Ph
10a
10b
10c
10d
10e
16a
16b
16c
16d
16e
16f
0.24
0.53
0.18
0.13
0.66
0.21
50.5
86.4
45.8
85.2
56.6
94.3
99.7
95.5
92.6
99.6
93.7
85.9
97.9
97.9
97.5
96.7
95.0
99.7
83.5
2.0
2.0
1.1
0.2
1.1
0.5
0.0
0.3
0.8
0.1
1.2
0.7
0.3
0.4
0.2
0.2
3.2
0.3
0.4
4-OCH3-Ph
4-F-Ph
0.12
0.10
3-Py
4-CH3-Ph
Ph
0.23
0.12
0.14
0.01
0.03
0.01
5.71
0.03
0.14
4-OCH3-Ph
4-COCH3-Ph
3-F-Ph
14.64
12.91
10.80
> 50
3.21
0.09
0.13
0.10
7.70
0.01
0.24
0.02
0.04
0.03
0.01
> 50
28.34
31.62
24.94
9.92
N.D. c
N.D. c
N.D. c
N.D. c
9.72
30.22
6.91
0.22
0.13
0.02
4-CH3-Ph
2-OCH3-Ph
4-CN-Ph
3-Py
23.21
4.63
0.01
0.01
0.02
16g
16h
16i
> 50
3.90
27.21
> 50
2.05
> 50
> 50
8.02
N.D. c
N.D. c
16.30
6.45
2-F-Ph
10.71
2.61
0.03
0.03
0.01
0.02
0.54
0.13
16j
4-F-Ph
0.02
0.05
0.03
0.02
0.01
3.52
18.70
14.91
10.02
1.62
0.14
16k
18a
18b
GDC0941b
3-OCH3-Ph
Ph
6.21
20.62
2.70
0.10
0.15
0.03
16.01
5.26
0.25
6.12
0.01
0.01
0.04
0.05
0.01
0.03
4-OCH3-Ph
–
1.95
2.02
0.01
1.55
0.72
0.91
2.20
0.06
5.01
a
b
c
Data presented is the mean
Used as positive control.
Not determined.
SD value of three independent determinations.
d
All compounds were assayed at least twice, and the values were averaged.
5

Y. Han, et al.
BioorganicChemistry104(2020)104197
Fig. 4. Compound 18b showed potent anti-proliferative effects against four tumor cell lines. (A) Chemical structure of compound 18b. (B, C, D, E, F) A549, HCT-116,
PC-3 and MDA-MB-231 cell lines were treated compound 18b at the indicated concentrations for 96 h. The results were shown as mean
independent experiments. ***p < 0.001, compared with non-treated group.
SD from three
from Qingdao Ocean Chemicals (Qingdao, Shandong, China). All
melting points were obtained on a Büchi Melting Point B-540 apparatus
(Büchi Labortechnik, Flawil, Switzerland) and were uncorrected. Mass
spectra (MS) were taken in ESI mode on Agilent 1100 LC–MS (Agilent,
Table 2
Enzymatic activities of compound 16b and 18b against PI3Ks and mTOR (IC50
,
nM).
Compd.
PI3Kαa
PI3Kβa
PI3Kγa
PI3Kδa
mTOR a
1
Palo Alto, CA, USA). H NMR and 13C NMR spectra were recorded on
16b
18b
10
576
55
206
13
85
32
30
12
Bruker ARX-400, 400 MHz spectrometers (Bruker Bioscience, Billerica,
MA, USA) with TMS as an internal standard.
0.46
a
All compounds were assayed at least twice, and the values were averaged.
4.1.1. General procedure for preparation of compounds (2–6)
4.1.1.1. thieno[3,2-d]pyrimidine-2,4-diol (2). 46.0 g (0.78 mol) of urea
and 20.0 g (0.13 mol) of 3-aminothiophene-2-methyl formate (1) was
admixed with each other, and the resulted mixture was heated to
190 °C. After completion of the reaction as indicated by TLC, the
mixture was added to 200 mL of 20% sodium hydroxide aqueous
solution, and the resulting solution was decolorized with charcoal and
then acidified to pH = 3 with 2 M hydrochloric acid. The crystalline
precipitate formed thereby was collected by vacuum filtration, dried
under reduced pressure to obtain 18.5 g of 2 as a light yellow solid with
the yield of 86.4%. MS (ESI) m/z (%): 167.1 [M−H]-.
3. Conclusion
In the present study, four series of novel thieno[3,2-d]pyrimidine
derivatives, containing aroyl hydrazone or aryl hydrazide moieties
were designed and synthesized. Exploration of SARs culminated in 18b,
which contained a 4-methoxybenzohydrazide group at the C-6 position
and 2-aminopyrimidine group at C-2 position of the thieno[3,2-d]pyr-
imidine core. 18b showed the most potent anti-proliferative activity
against PI3K and mTOR. It also showed good inhibition against HCT-
116, PC-3, A549 and MDA-MB-231 cell lines. (IC50 = 2.02 μM,
1.95 μM, 1.62 μM and 1.55 μM, respectively). Furthermore, cell cycle
analysis of 18b by flow cytometry showed cell cycle arrest in the G1/S
phases, which induced apoptosis of the HCT-116 cells in a dose-de-
pendent manner. In summary, all these reported results suggest that
compound 18b could be served as a “hit” compound for PI3K/mTOR
dual inhibitors. Further studies on the structural optimization of 18b
are currently underway in our laboratory.
4.1.1.2. 2, 4-dichlorothieno[3,2-d]pyrimidine (3). 16.0 g (0.095 mol) of
intermediate 2 was slowly added to 44 mL (0.048 mol) of phosphorus
oxychloride, and the reaction was heated to reflux for 8 h. The solvent
was concentrated in vacuum and the residue was poured into ice water,
the resulting precipitate was filtered and dried to obtain 15.8 g of 3 as a
yellow solid. Yield: 81.4%. MS (ESI) m/z (%): 205.1 [M+H]+.
4.1.1.3. 4-(2-chlorothieno[3,2-d]pyrimidin-4-yl)morpholine (4). 15.0 g
(0.074 mol) of intermediate 3 was dissolved in 150 mL of methanol,
and 16.8 mL (0.17 mol) of morpholine was added slowly under stirring
at room temperature. After the completion of the dropwise addition, the
mixture was filtered and the filter cake was washed with ethanol
(100 mL) and water (20 mL) in turn. The crude product was
recrystallized with 1,4-dioxane (100 mL) to provide 15.6 g of 4 as a
light yellow solid. Yield: 83.0%. MS (ESI) m/z (%): 256.0 [M+H]+.
4. Experimental
4.1. Chemistry
Unless otherwise specified, all materials were obtained from com-
mercial suppliers and were used without further purification. Reactions’
time and purity of the products were monitored by TLC on FLUKA silica
gel aluminum cards (0.2 mm thickness) with fluorescent indicator
254 nm. Column chromatography was run on silica gel (200–300 mesh)
4.1.1.4. 2-chloro-4-morpholinothieno[3,2-d]pyrimidine-6-carbaldehyde
6

Y. Han, et al.
BioorganicChemistry104(2020)104197
Fig. 5. Effect of compound 18b on the cell cycle distribution of HCT-116 cells.
(5). Under the protection of nitrogen, a solution of 15.0 g (0.059 mol)
4-(2-chlorothieno[3,2-d]pyrimidin-4-yl)morpholine (4) in 150 mL of
tetrahydrofuran was cooled to below −10 °C, 14.8 mL (2 M, 0.03 mol)
of isopropylmagnesium chloride tetrahydrofuran solution was slowly
added to the mixture. After stirred for 1.5 h, 44.3 mL (1.6 M, 0.071 mol)
of n-butyl lithium hexane solution was slowly added to the mixture and
stirred for 2 h. 11.4 mL (0.148 mol) N, N-dimethylformamide was
added to the reaction solution slowly for 1 h at −10 °C, and the
reaction mixture was stirred for 4 h at −10 °C. The mixture was slowly
poured into 750 mL (0.25 M) hydrochloric acid and stirred at room
temperature for 1 h, then heated to 50 °C for 4 h, the resulting
precipitate was filtered and dried to obtain 11.4 g yellow solid with
yield of 68.7%. MS (ESI) m/z (%): 282.4 [M + H]-.
4.1.1.5. 2-chloro-4-morpholinothieno[3,2-d]pyrimidine-6-carboxylic acid
(6). 2 M NaOH aqueous solution was slowly added to the aqueous
solution of silver nitrate in ice-bath. After stirring for 10 min, a solution
of 2.0 g (0.007 mol) 2-chloro-4-morpholinothieno[3,2-d]pyrimidine-6-
Fig. 6. Cell apoptosis induced by compound 18b. HCT-116 cells were treated with 18b at 0.333 μM – 9 μM for 24 h. Apoptosis was examined by flow cytometer.
7

Y. Han, et al.
BioorganicChemistry104(2020)104197
trifluoroacetic acid (0.0014 mol) and morpholine (0.0009 mol) were
added. The resulted mixture was heated to 125 °C and stirred for 6 h.
When the mixture was cooled to room temperature, brown precipitate
was filtered off and washed with water, the crude products were
purified by column chromatography to afford 8a-b as yellow solid.
4.1.2.2.1. N'-((2,4-dimorpholinothieno[3,2-d]pyrimidin-6-yl)
methylene)benzo-hydrazide (8a). Yellow solid; Yield: 30.2%; MS (ESI)
m/z (%): 451.0 [M−H]-; 1H NMR (600 MHz, DMSO‑d6) δ 12.09 (s, 1H),
8.74 (s, 1H), 7.92 (d, J = 7.0 Hz, 2H), 7.62 (t, J = 7.0 Hz, 1H), 7.56 –
7.52 (m, 3H), 3.84 (m, 4H), 3.75 (m, 4H), 3.66 (s, 8H). 13C NMR
(151 MHz, DMSO‑d6) δ 163.55, 163.31, 160.17, 158.31, 144.45,
142.46, 133.42, 132.40, 128.93, 128.08, 127.07, 105.52, 66.45,
46.28, 44.81.
4.1.2.2.2. N'-((2,4-dimorpholinothieno[3,2-d]pyrimidin-6-yl)
methylene)picolino-hydrazide (8b). Yellow solid; Yield: 42.5%; MS (ESI)
m/z (%): 452.1 [M−H]-. 1H NMR (400 MHz, DMSO‑d6) δ 12.49 (s, 1H),
8.93 (s, 1H), 8.73 (d, J = 4.6 Hz, 1H), 8.13 (d, J = 7.7 Hz, 1H), 8.07
(dt, J = 7.7, 1.7 Hz, 1H), 7.69 (dd, J = 7.5, 4.6, 1.7 Hz, 1H), 7.46 (s,
1H), 3.89 – 3.83 (m, 4H), 3.78 – 3.74 (m, 4H), 3.66 (s, 8H). 13C NMR
(151 MHz, DMSO‑d6) δ 165.93, 160.99, 158.36, 142.15, 131.87,
133.90, 130.19, 120.07, 118.33, 112.91, 107.67, 66.28, 55.71, 46.26,
44.76.
Fig. 7. In vitro wound-healing assay on HCT-116 cells. Phase-contrast images
were obtained by the treatment of compounds 18b at indicated concentrations
for 0, 24 and 48 h.
carbaldehyde (5) was dropped into the above mixed solution. After the
completion of the dropwise addition, the reaction mixture was warmed
to room temperature for 5 h. The mixture was filtered through a celite
bed and concentrated in vacuum, the filtrate was acidified to pH = 1
and filtered to provide 1.5 g yellow solid with yield of 71.4%. MS (ESI)
m/z (%): 298.1 [M−H]-.
4.1.3. General procedure for preparation of compounds (10a-e)
4.1.3.1. General procedure for preparation of the intermediate 9a-e. To a
stirred solution of 2-chloro-4-morpholinothieno[3,2-d]pyrimidine-6-
carboxylic acid (6) (2.0 g, 0.007 mol) in 20 mL DMF was added
substituted benzoyl hydrazine (1.0 g, 0.007 mol) and EDCI (3.0 g,
0.014 mol). The mixture was stirred at room temperature for 12 h and
then poured into ice-water. The yellow solid was filtered and dried
under reduced pressure to obtain 9a-e, which were used without
further purification.
4.1.2. General procedure for preparation of compounds (8a-b)
4.1.2.1. General procedure for preparation of the intermediate 7a-b. A
solution of 2.0 g (0.007 mol) of 2-chloro-4-morpholinothieno[3,2-d]
pyrimidine-6-carbaldehyde (5) in 10 mL ethanol was warmed to 80 °C,
then added 1 drop of acetic acid. After stirred for 30 min, substituted
benzoyl hydrazine (0.0073 mmol) was added and stirred for 4 h at
80 °C. The resulting precipitate was filtered and dried to obtain 7a-b.
4.1.2.1.1. N'-((2-chloro-4-morpholinothieno[3,2-d]pyrimidin-6-yl)
methylene)benzo-hydrazide (7a). Yellow solid; Yield: 80.8%.
4.1.3.1.1. N'-benzoyl-2-chloro-4-morpholinothieno[3,2-d]pyrimidine-
6-carbo-hydrazide (9a). Yellow solid; Yield: 60.5%.
4.1.3.1.2. 2-chloro-N'-(4-methoxybenzoyl)-4-morpholinothieno[3,2-d]
pyrimidine-6-carbohydrazide (9b). Yellow solid; Yield: 72.8%.
4.1.3.1.3. 2-chloro-N'-(4-fluorobenzoyl)-4-morpholinothieno[3,2-d]
pyrimidine-6-carbohydrazide (9c). Yellow solid; Yield: 65.6%.
4.1.3.1.4. 2-chloro-4-morpholino-N'-nicotinoylthieno[3,2-d]
4.1.2.1.2. N'-((2-chloro-4-morpholinothieno[3,2-d]pyrimidin-6-yl)
methylene)picolino-hydrazide (7b). Yellow solid; Yield: 90.2%.
pyrimidine-6-carbo-hydrazide (9d). Yellow solid; Yield: 55.4%.
4.1.3.1.5. 2-chloro-N'-(4-methylbenzoyl)-4-morpholinothieno[3,2-d]
pyrimidine-6-carbohydrazide (9e). Yellow solid; Yield: 59.9%.
4.1.2.2. General procedure for preparation of target compounds 8a-8b. To
a mixture of 0.2 g (0.0004 mol) of 7a-b in 2 mL n-butanol,
Fig. 8. The proposed binding mode of 18b with the PI3Kγ kinase domain (PDB code:3DBS) and mTOR kinase domain (PDB code:3L16). H-Bonding interactions were
presented with green lines. (A) Compound 18b docked into PI3Kγ kinase (PDB code:3DBS). (B) Overlapping images of compound 18b (pink) and GDC0941 (white)
complexed with PI3Kγ. (C) GDC0941 docked into PI3Kγ kinase. (D) Compound 18b docked into mTOR (PDB code:3L16). (E) Overlapping images of compound 18b
(pink) and GDC0941 (white) complexed with mTOR. (F) GDC0941 docked into mTOR. Images were generated using PyMol. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
8

Y. Han, et al.
BioorganicChemistry104(2020)104197
Table 3
Physicochemical properties and ADME properties of most active compounds.
Compd
MW (g/mol)
Rotatable bonds < 10 H-bond acceptors < 10 H-bond donors
aTPSA (Å2)
< 140
Log P o/w
< 5
Violation Lipinski Rule of bBS
5
< 500
< 5
18b
506.54
8
5
8
8
3
1
185.72
144.17
3.35
3.01
2
1
0.17
0.55
GDC-0941 513.64
a
b
TPSA: topological polar surface area.
BS: Bioavailability Score.
4.1.3.2. General procedure for preparation of the target compounds 10a-
e. A mixture of 0.2 g (0.0004 mol) 9a-e in 2 mL n-butanol, and
trifluoroacetic acid (0.0014 mol) and morpholine (0.0009 mol) was
heated to 125 °C and stirred for 6 h. The mixture was cooled to room
temperature and then the brown precipitate was filtered off and washed
with water. The crude products were purified by column
chromatography to afford 10a-e as a yellow solid.
flash column chromatography using dichloromethane/methanol as
eluent to afford 12 as a white solid. Yield: 70.3%.
4.1.4.2. 1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-indazole (13). 14.0
g
(0.050 mol) of
intermediate 12, 19.1 g (0.075 mol) of bis(pinacolato) diboron,
14.7 g (0.15 mol) of potassium acetate were dissolved in 140 mL 1,
4-dioxane, 1.4 g (0.002 mol) of Pd (PPh3)2Cl2 was added under the
protection of nitrogen to the mixture and heated to 80 °C for 12 h. The
reaction solution was filtered through a celite bed and the filtrate was
concentrated and purified by flash column chromatography to provide
13 as a brown solid. Yield: 60.5%.
4.1.3.2.1. N'-benzoyl-2,4-dimorpholinothieno[3,2-d]pyrimidine-6-
carbohydrazide (10a). Yield: 30.2%; MS (ESI) m/z (%): 467.0 [M−H]-.
1H NMR (600 MHz, DMSO‑d6) δ 10.89 (s, 1H), 10.66 (s, 1H), 7.93 (d,
J = 1.2 Hz, 1H), 7.92 (d, J = 1.2 Hz, 1H), 7.88 (s, 1H), 7.64 – 7.60 (m,
1H), 7.56 – 7.52 (m, 2H), 3.87 – 3.83 (m, 4H), 3.76 – 3.73 (m, 4H), 3.67
(s, 8H). 13C NMR (151 MHz, DMSO‑d6) δ 166.20, 163.14, 161.02,
160.22, 158.41, 142.13, 132.52, 128.99, 127.85, 124.98, 107.64, 66.45
, 46.25, 44.75.
4.1.4.3. 4-morpholino-2-(1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)
thieno[3,2-d]pyrimidine-6-carboxylic acid (14). Pd (PPh3)2Cl2 (0.5 g,
0.0008 mol) was added to a solution of intermediate 13 (5.0 g,
4.1.3.2.2. N'-(4-methoxybenzoyl)-2,4-dimorpholinothieno[3,2-d]
pyrimidine-6-carbohydrazide (10b). Yield: 50.2%; MS (ESI) m/z (%):
497.0 [M−H]-. 1H NMR (400 MHz, DMSO‑d6) 10.88 (s, 1H), 10.63 (s,
1H), 7.87 (s, 1H), 7.51 (d, J = 7.8 Hz, 2H), 7.44 (d, J = 7.8 Hz, 2H),
3.85 (m, 4H), 3.83 (s, 3H), 3.75 (m, 4H), 3.67 (s, 8H). 13C NMR
(151 MHz, DMSO‑d6) δ 165.93, 160.99, 159.63, 158.36, 142.15,
133.90, 130.19, 120.07, 118.33, 112.91, 107.67, 66.28, 55.71, 46.26,
44.76.
0.015
mol),
2-chloro-4-morpholinothieno[3,2-d]pyrimidine-6-
carboxylic acid (6) (9.12 g, 0.03 mol) and sodium carbonate (3.3 g,
0.03 mol) in 60 mL of mixed solution (5:1 = 1, 4-dioxane:H2O) at room
temperature. The resulted mixture was refluxed under nitrogen until
completion determined by TLC. The reaction solution was cooled,
filtered through a celite bed and the filtrate was concentrated in
vacuum. The residue was extracted with DCM and the water phase
was retained. and acidified with 2 M hydrochloric acid solution to
pH = 3 and filtered to afford the key intermediate 14 as a white solid.
Yield: 75.5%.
4.1.3.2.3. N'-(4-fluorobenzoyl)-2,4-dimorpholinothieno[3,2-d]
pyrimidine-6-carbohydrazide (10c). Yield: 42.5%; MS (ESI) m/z (%):
485.0 [M−H]-. 1H NMR (400 MHz, DMSO‑d6) δ 10.89 (s, 1H), 10.69 (s,
1H), 7.99 (d, J = 8.8 Hz, 2H), 7.87 (s, 1H), 7.39 (d, J = 8.8 Hz, 2H),
3.85 (m, 4H), 3.75 (m, 4H), 3.67 (s, 7H). 13C NMR (151 MHz,
DMSO‑d6) δ 165.18, 163.88, 161.02, 158.39, 142.05, 130.61, 130.03
(s, 1H), 129.03, 124.98, 116.11, 115.96, 107.65, 66.28, 46.25, 44.75.
4.1.3.2.4. 2,4-dimorpholino-N'-nicotinoylthieno[3,2-d]pyrimidine-6-
carbohydrazide (10d). Yield: 42.5%; MS (ESI) m/z (%): 468.1 [M−H]-.
1H NMR (600 MHz, DMSO‑d6) δ 10.95 (s, 1H), 10.90 (s, 1H), 9.08 (s,
1H), 8.80 (s, 1H), 8.27 (d, J = 7.0 Hz, 1H), 7.88 (s, 1H), 7.59 (dd,
J = 7.0, 5.1 Hz, 1H), 3.85 (s, 1H), 3.75 (s, 1H), 3.67 (s, 8H). 13C NMR
(151 MHz, DMSO‑d6) δ 164.85, 163.11, 160.99, 160.22, 158.41,
153.10, 148.80, 141.86, 135.67, 125.13, 124.19, 107.68, 66.29,
46.24, 44.74.
4.1.4.4. General procedure for preparation of the intermediates 15a-k. To
a stirred mixture of intermediate 14 (0.2 g, 0.0004 mol) and substituted
benzoyl hydrazine (0.0004 mol) in 3 mL dichloromethane was added
HATU (0.0003 mol) followed by triethylamine (0.0005 mol). The
mixture was stirred at room temperature for 15 h. The resulting
precipitate was filtered and dried to obtain intermediates 15a-k.
4.1.4.4.1. N'-benzoyl-4-morpholino-2-(1-(tetrahydro-2H-pyran-2-yl)-
1H-indazol-4-yl)thieno[3,2-d]pyrimidine-6-carbohydrazide (15a). Yellow
solid; Yield: 73.2%.
4.1.4.4.2. N'-(4-methoxybenzoyl)-4-morpholino-2-(1-(tetrahydro-2H-
pyran-2-yl)-1H-indazol-4-yl)thieno[3,2-d]pyrimidine-6-carbohydrazide
(15b). Yellow solid; Yield: 55.3%.
4.1.3.2.5. N'-(4-methylbenzoyl)-2,4-dimorpholinothieno[3,2-d]
pyrimidine-6-carbohydrazide (10e). Yield: 45.6%; MS (ESI) m/z (%):
481.1 [M−H]-. 1H NMR (600 MHz, DMSO‑d6) δ 10.84 (s, 1H), 10.57 (s,
1H), 7.87 (s, 1H), 7.83 (d, J = 8.1 Hz, 2H), 7.34 (d, J = 8.1 Hz, 2H),
3.85 (m, 4H), 3.75 (m, 4H), 3.67 (s, 8H), 2.39 (s, 3H). 13C NMR
(151 MHz, DMSO‑d6) δ 166.04, 161.01, 158.42, 142.48, 129.48,
127.88, 124.93, 107.63, 66.46, 66.29, 46.25, 44.75, 21.42.
4.1.4.4.3. N'-(4-acetylbenzoyl)-4-morpholino-2-(1-(tetrahydro-2H-
pyran-2-yl)-1H-indazol-4-yl)thieno[3,2-d]pyrimidine-6-carbohydrazide
(15c). Yellow solid; Yield: 57.8%.
4.1.4.4.4. N'-(3-fluorobenzoyl)-4-morpholino-2-(1-(tetrahydro-2H-
pyran-2-yl)-1H-indazol-4-yl)thieno[3,2-d]pyrimidine-6-carbohydrazide
(15d). Yellow solid; Yield: 70.3%.
4.1.4.4.5. N'-(4-methylbenzoyl)-4-morpholino-2-(1-(tetrahydro-2H-
pyran-2-yl)-1H-indazol-4-yl)thieno[3,2-d]pyrimidine-6-carbohydrazide
(15e). Yellow solid; Yield: 66.9%.
4.1.4. General procedure for preparation of compounds 16a-k
4.1.4.1. 4-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (12). 10 g
(0.051 mol) of 4-bromo-1H-indazole (11) and 8.57 g (0.10 mol) of
3,4-dihydropyran were dissolved in 100 mL ethyl acetate, and then
0.88 g (0.005 mol) of p-toluenesulfonic acid was added to the mixture.
The resulted mixture was stirred at room temperature for 10 h, and then
was extracted with ethyl acetate and the organic phase was washed
with water, dried over anhydrous Na2SO4. The combined organic layer
was concentrated under reduced pressure and was further purified by
4.1.4.4.6. N'-(2-methoxybenzoyl)-4-morpholino-2-(1-(tetrahydro-2H-
pyran-2-yl)-1H-indazol-4-yl)thieno[3,2-d]pyrimidine-6-carbohydrazide
(15f). Yellow solid; Yield: 63.5%.
4.1.4.4.7. N'-(4-cyanobenzoyl)-4-morpholino-2-(1-(tetrahydro-2H-
pyran-2-yl)-1H-indazol-4-yl)thieno[3,2-d]pyrimidine-6-carbohydrazide
(15 g). Yellow solid; Yield: 43.2%.
4.1.4.4.8. 4-morpholino-N'-nicotinoyl-2-(1-(tetrahydro-2H-pyran-2-
9

Y. Han, et al.
BioorganicChemistry104(2020)104197
yl)-1H-indazol-4-yl)thieno[3,2-d]pyrimidine-6-carbohydrazide
(15 h). Yellow solid; Yield: 75.3%.
1H), 7.71 (d, J = 8.1 Hz, 1H), 7.50 (m, 1H), 7.36 (d, J = 8.0 Hz, 2H),
4.08 (m, 4H), 3.86 (m, 4H), 2.40 (s, 3H). 13C NMR (151 MHz,
DMSO‑d6) δ 166.05, 162.07, 160.85, 158.34, 142.92, 142.53, 131.08,
129.96, 129.75, 129.50, 127.92, 126.24, 126.06, 121.84, 121.32,
115.59, 114.96, 66.34, 46.52, 21.43.
4.1.4.4.9. N'-(2-fluorobenzoyl)-4-morpholino-2-(1-(tetrahydro-2H-
pyran-2-yl)-1H-indazol-4-yl)thieno[3,2-d]pyrimidine-6-carbohydrazide
(15i). Yellow solid; Yield: 63.8%.
4.1.4.4.10. N'-(4-fluorobenzoyl)-4-morpholino-2-(1-(tetrahydro-2H-
pyran-2-yl)-1H-indazol-4-yl)thieno[3,2-d]pyrimidine-6-carbohydrazide
(15j). Yellow solid; Yield: 80.6%.
4.1.4.5.6. 2-(1H-indazol-4-yl)-N'-(2-methoxybenzoyl)-4-
morpholinothieno[3,2-d]-pyrimidine-6-carbohydrazide (16f). White solid;
1
Yield: 36.7%; MS (ESI) m/z (%): 528.2 [M−H]-; H NMR (600 MHz,
4.1.4.4.11. N'-(3-methoxybenzoyl)-4-morpholino-2-(1-(tetrahydro-
2H-pyran-2-yl)-1H-indazol-4-yl)thieno[3,2-d]pyrimidine-6-carbohydrazide
(15 k). Yellow solid; Yield: 77.5%.
DMSO‑d6) δ 13.25 (s, 1H), 11.16 (s, 1H), 10.26 (s, 1H), 8.86 (s, 1H),
8.32 (s, 1H), 8.25 (d, J = 7.4 Hz, 1H), 7.78 (d, J = 7.2 Hz, 1H), 7.70 (d,
J = 8.2 Hz, 0H), 7.55 (d, J = 7.2 Hz, 1H), 7.50 (m, 0H), 7.21 (d,
J = 8.2 Hz, 1H), 7.10 (d, J = 7.4 Hz, 1H), 4.08 (m, 4H), 3.93 (s, 3H),
3.86 (m, 4H). 13C NMR (151 MHz, DMSO‑d6) δ 162.12, 160.80, 160.30,
158.35, 157.52, 141.16, 135.28, 133.32, 131.11, 130.89, 129.97,
126.07, 121.81, 121.33, 120.94, 114.92, 112.78, 112.52, 66.35,
56.25, 46.53.
4.1.4.5. General procedure for preparation of the target compounds 16a-
16 k. To a solution of 0.15 g (0.0004 mol) of intermediates 15a-15 k in
10 mL methanol was dropped methanesulfonic acid (0.0024 mol), and
the resulting mixture was kept at room temperature for 24 h. The
solvent was concentrated in vacuum, then water was added and
alkalified with ammonia water to pH = 8. The resulting precipitate
was filtered off and further purified by flash column chromatography to
afford the target compounds 16a-16 k.
4.1.4.5.7. N'-(4-cyanobenzoyl)-2-(1H-indazol-4-yl)-4-
morpholinothieno[3,2-d]-pyrimidine-6-carbohydrazide (16 g). White
solid; Yield: 17.4%; MS (ESI) m/z (%): 523.2 [M−H]-; 1H NMR
(400 MHz, DMSO‑d6) δ 13.26 (s, 1H), 11.10 (s, 2H), 8.88 (s, 1H),
8.34 (s, 1H), 8.26 (d, J = 7.2 Hz, 1H), 8.10 (d, J = 8.7 Hz, 2H), 8.06 (d,
J = 8.7 Hz, 2H), 7.71 (d, J = 8.3 Hz, 1H), 7.51 (t, J = 7.8 Hz, 1H),
4.09 (m, 4H), 3.87 (m, 4H). 13C NMR (151 MHz, DMSO‑d6) δ 162.06,
160.79, 158.34, 141.16, 139.64, 136.38, 135.30, 133.89, 131.07,
129.97, 128.80, 128.24, 126.29, 126.07, 121.84, 121.33, 119.33,
114.96, 112.81, 66.35, 46.53.
4.1.4.5.1. N'-benzoyl-2-(1H-indazol-4-yl)-4-morpholinothieno[3,2-d]
pyrimidine-6-carbohydrazide (16a). White solid; Yield: 30.3%; MS (ESI)
m/z (%): 498.1 [M−H]-; 1H NMR (400 MHz, DMSO‑d6) δ 13.25 (s, 1H),
11.08 (s, 1H), 10.73 (s, 1H), 8.87 (s, 1H), 8.35 (s, 1H), 8.25 (d,
J = 7.2 Hz, 1H), 7.95 (d, J = 7.2 Hz, 1H), 7.70 (d, J = 8.3 Hz, 1H),
7.62 (d, J = 8.4 Hz, 1H), 7.57 (s, 1H), 7.55 (d, J = 7.2 Hz, 1H), 7.51 (d,
J = 7.2 Hz, 1H), 4.08 (m, 1H), 3.86 (m, 3H). 13C NMR (151 MHz,
DMSO‑d6) δ 162.21, 160.84, 160.63, 158.31, 144.19, 142.77, 141.16,
135.29, 133.89, 132.13, 131.15, 130.69, 129.96, 128.85, 126.04,
121.76, 121.29, 115.98, 114.42, 66.35, 46.51.
4.1.4.5.8. 2-(1H-indazol-4-yl)-4-morpholino-N'-nicotinoylthieno[3,2-
d]pyrimidine-6-carbohydrazide (16 h). White solid; Yield: 25.9%; MS
1
(ESI) m/z (%): 499.1 [M−H]-; H NMR (600 MHz, DMSO‑d6) δ 13.26
(s, 1H), 11.18 (s, 1H), 10.98 (s, 1H), 9.11 (s, 1H), 8.87 (s, 1H), 8.81 (d,
J = 3.7 Hz, 1H), 8.36 (s, 1H), 8.30 (d, J = 7.2 Hz, 1H), 8.25 (d,
J = 7.2 Hz, 1H), 7.71 (d, J = 7.4 Hz, 1H), 7.61 (dd, J = 7.4, 3.7 Hz,
1H), 7.50 (t, J = 7.7 Hz, 1H), 4.08 (s, 4H), 3.86 (s, 4H). 13C NMR
(151 MHz, DMSO) δ 164.82, 162.03, 160.83, 158.34, 153.14, 148.86,
142.60, 141.16, 135.71, 135.29, 131.06, 129.96, 128.28, 126.44,
126.07, 124.19, 121.85, 121.32, 115.01, 112.82, 66.34, 46.52.
4.1.4.5.9. N'-(2-fluorobenzoyl)-2-(1H-indazol-4-yl)-4-
4.1.4.5.2. 2-(1H-indazol-4-yl)-N'-(4-methoxybenzoyl)-4-
morpholinothieno[3,2-d]-pyrimidine-6-carbohydrazide
(16b). White
solid; Yield: 40.6%; MS (ESI) m/z (%): 528.0 [M−H]-; 1H NMR
(600 MHz, DMSO‑d6) δ 13.26 (s, 1H), 11.01 (s, 1H), 10.58 (s, 1H),
8.34 (s, 1H), 8.25 (d, J = 7.2 Hz, 1H), 8.07 (s, 1H), 7.94 (d, J = 8.7 Hz,
2H), 7.70 (d, J = 7.2 Hz, 1H), 7.50 (m, 1H), 7.08 (d, J = 8.7 Hz, 2H),
4.66 (s, 2H), 4.08 (m, 4H), 3.86 (d, J = 4.8 Hz, 2H), 3.85 (s, 0H). 13
C
NMR (151 MHz, DMSO‑d6) δ 165.64, 162.59, 162.08, 160.86, 158.35,
142.98, 141.17, 133.90, 131.08, 129.97, 129.83, 126.21, 126.07,
124.69, 121.85, 121.32, 114.95, 114.22, 66.35, 55.83, 46.53.
4.1.4.5.3. N'-(4-acetylbenzoyl)-2-(1H-indazol-4-yl)-4-
morpholinothieno[3,2-d]-pyrimidine-6-carbohydrazide (16i). White solid;
Yield: 32.4%; MS (ESI) m/z (%): 518.0 [M−H]+; 1H NMR (600 MHz,
DMSO‑d6) δ 13.25 (s, 1H), 11.16 (s, 1H), 10.59 (s, 1H), 8.87 (s, 1H),
8.31 (s, 1H), 8.25 (d, J = 7.2 Hz, 1H), 7.73 (d, J = 7.2 Hz, 1H), 7.70 (d,
J = 8.1 Hz, 1H), 7.63 (d, J = 7.7 Hz, 1H), 7.50 (t, J = 7.7 Hz, 1H),
7.39 (d, J = 8.5 Hz, 1H), 7.36 (d, J = 8.5 Hz, 1H), 4.08 (m, 4H), 3.86
(m, 4H). 13C NMR (151 MHz, DMSO‑d6) δ 162.08, 160.80, 160.51,
158.85, 158.34, 141.16, 135.29, 133.67, 131.10, 130.60, 126.07,
125.10, 122.30, 121.83, 121.33, 116.83, 116.69, 114.94, 112.79,
66.35, 46.53.
morpholinothieno[3,2-d]-pyrimidine-6-carbohydrazide
(16c). White
solid; Yield: 23.6%; MS (ESI) m/z (%): 540.1 [M−H]-; 1H NMR
(600 MHz, DMSO‑d6) δ 13.26 (s, 1H), 11.13 (s, 1H), 10.97 (s, 1H),
8.87 (s, 1H), 8.34 (s, 1H), 8.25 (d, J = 7.2 Hz, 1H), 8.11 (d, J = 8.4 Hz,
2H), 8.07 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 8.3 Hz, 1H), 7.53 – 7.47 (m,
1H), 4.13 – 4.04 (m, 4H), 3.91 – 3.81 (m, 4H), 2.66 (s, 3H). 13C NMR
(151 MHz, DMSO‑d6) δ 198.09, 162.06, 160.79, 158.34, 141.16,
139.64, 136.38, 135.30, 133.89, 131.07, 129.97, 128.80, 128.24,
126.29, 126.07, 121.84, 121.33, 114.96, 112.81, 66.35, 46.53, 27.40.
4.1.4.5.4. N'-(3-fluorobenzoyl)-2-(1H-indazol-4-yl)-4-
4.1.4.5.10. N'-(4-fluorobenzoyl)-2-(1H-indazol-4-yl)-4-
morpholinothieno[3,2-d]-pyrimidine-6-carbohydrazide (16j). White solid;
1
Yield: 50.6%; MS (ESI) m/z (%): 516.1 [M−H]-; H NMR (400 MHz,
DMSO‑d6) δ 13.25 (s, 1H), 10.32 (s, 1H), 8.85 (s, 1H), 8.24 (d,
J = 8.2 Hz, 2H), 8.15 (s, 1H), 8.07 (s, 1H), 8.03 (m, 1H), 7.69 (d,
J = 8.2 Hz, 1H), 7.49 (d, J = 8.2 Hz, 2H), 4.06 (m, 4H), 3.86 (m, 4H).
13C NMR (151 MHz, DMSO‑d6) δ 165.14, 162.21, 160.84, 160.63,
158.31, 144.19, 142.77, 141.16, 135.29, 133.89, 131.15, 130.69,
129.96, 126.04, 124.60, 121.76, 121.29, 115.98, 114.42, 66.35, 46.51.
4.1.4.5.11. 2-(1H-indazol-4-yl)-N'-(3-methoxybenzoyl)-4-
morpholinothieno[3,2-d]-pyrimidine-6-carbohydrazide
(16d). White
solid; Yield: 30.8%; MS (ESI) m/z (%): 516.1 [M−H]-; 1H NMR
(400 MHz, DMSO‑d6) δ 13.26 (s, 1H), 11.15 (s, 1H), 10.87 (s, 1H),
8.88 (s, 1H), 8.35 (s, 1H), 8.26 (d, J = 7.4 Hz, 1H), 7.82 (d, J = 7.9 Hz,
1H), 7.74 (d, J = 9.9 Hz, 1H), 7.71 (d, J = 7.4 Hz, 1H), 7.63 (dd,
J = 14.0, 7.9 Hz, 1H), 7.50 (t, J = 7.8 Hz, 2H), 4.08 (s, 4H), 3.86 (s,
4H). 13C NMR (151 MHz, DMSO‑d6) δ 164.85, 163.19, 162.04, 161.57,
160.85, 158.33, 142.66, 141.16, 135.29, 134.79, 131.36, 131.06,
126.39, 126.06, 124.11, 121.85, 115.00, 114.78, 114.62, 112.81,
66.34, 46.52.
morpholinothieno[3,2-d]-pyrimidine-6-carbohydrazide (16k). White solid;
Yield: 53.5%; MS (ESI) m/z (%): 528.2 [M−H]-; 1H NMR (600 MHz,
DMSO‑d6) δ 13.26 (s, 1H), 11.08 (s, 1H), 10.74 (s, 1H), 8.88 (s, 1H), 8.35
(s, 1H), 8.25 (d, J = 7.2 Hz, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.54 (d,
J = 7.8 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.49 (d, J = 7.2 Hz, 1H), 7.46
(d, J = 7.8 Hz, 1H), 4.08 (m, 4H), 3.86 (m, 4H), 3.85 (s, 1H). 13C NMR
(151 MHz, DMSO‑d6) δ 165.85, 162.06, 160.82, 159.66, 158.33, 142.88,
141.16, 135.30, 133.93, 131.08, 130.19, 126.27, 126.06, 121.84, 121.32,
120.12, 118.37, 114.96, 112.94, 112.80, 66.34, 55.73, 46.52.
4.1.4.5.5. 2-(1H-indazol-4-yl)-N'-(4-methylbenzoyl)-4-
morpholinothieno[3,2-d]-pyrimidine-6-carbohydrazide
(16e). White
solid; Yield: 45.6%; MS (ESI) m/z (%): 512.2 [M−H]-; 1H NMR
(600 MHz, DMSO‑d6) δ 13.26 (s, 1H), 11.05 (s, 1H), 10.66 (s, 1H),
8.87 (s, 1H), 8.35 (s, 1H), 8.25 (d, J = 8.0 Hz, 2H), 7.86 (d, J = 8.1 Hz,
10

Y. Han, et al.
BioorganicChemistry104(2020)104197
4.1.5. General procedure for preparation of target compounds 18a-b
4.1.5.1. 2-(2-aminopyrimidin-5-yl)-4-morpholinothieno[3,2-d]pyrimidine-
6-carboxylic acid (17). 0.6 g (0.0008 mol) PdCl2 (dppf)2 was added to a
solution of 1.0 g (0.003 mol) 2-chloro-4-morpholinothieno[3,2-d]
Briefly, 20 μg/mL poly (Glu, Tyr) 4:1 (Sigma) was preloaded as a
substrate in 384-well plates. Then 50 μL of 10 mM ATP (Invitrogen)
solution diluted in kinase reaction buffer (50 mM HEPES, pH 7.0, 1 mM
DTT,1 mM MgCl2, 1 mM MnCl2, 0.1% NaN3) was added to each well.
Various concentrations of compounds were diluted in 10 μL of 1%
DMSO (v/v), with blank DMSO solution as the negative control. The
kinase reaction was initiated by the addition of purified tyrosine kinase
proteins diluted in 39 μL of kinase reaction buffer solution. Reactions
were incubated for 30 min at 25 °C and stopped by the addition of 5 μL
Streptavidin-XL665 and 5 μL Tk Antibody Cryptate working solution to
all of wells. The plate was read by Envision (PerkinElmer) at 320 nm
and 615 nm. IC50 values were calculated from the inhibition curves.
pyrimidine-6-carboxylic acid (6), 2.0
g (0.008 mol) 5-(4,4,5,5-
tetramethyl-1,3,2-dioxoborane-2-yl)pyrimidine-2-amine and cesium
carbonate (2.5 g, 0.007 mol) in 12 mL of mixed solution (5:1 = 1, 4-
dioxane:H2O) at room temperature. The resulted mixture was refluxed
under nitrogen until completion determined by TLC. The reaction
solution was cooled, filtered through a celite bed and the filtrate was
concentrated in vacuum. The residue was extracted with DCM and the
water phase was retained. and acidified with 2 M hydrochloric acid
solution to pH = 3 and filtered to provide 2-(2-aminopyrimidin-5-yl)-4-
morpholinothieno[3,2-d]-pyrimidine-6-carboxylic acid (17) as a yellow
solid. Yield: 60.5%.
4.2.3. Flow cytometry
The HCT-116 cells were seeded in 6-well plates at a seeding density
of 105 cells per mL. Twelve hours later, various concentrations of
compound 18b were added. Cells were treated with compound 18b for
24 h. Then cells were transferred to EP tubes and washed three times
with PBS buffer. Then the procedures according to the operating in-
structions of the kit were followed. Ultimately, cell apoptosis was
analyzed using Annexin-V and propidium iodide (PI) double staining by
flow cytometry. Early apoptotic cells were defined as Annexin-V posi-
tive/PI negative, late apoptotic cells as Annexin-V/PI-double positive
and necrotic cells as Annexin-V positive/PI positive.
4.1.5.2. General procedure for preparation of target compounds 18a-
b. To a stirred solution of 0.2 g (0.0004 mol) intermediate 17 in 2 mL
DMF was added substituted benzoyl hydrazine (0.0005 mol)
followed by EDCI (0.0008 mol). The mixture was stirred at room
temperature for 12 h. The resulting mixture was poured into ice-
water, then the precipitate was filtered and washed with water. The
crude product was dried in vacuum desiccator and further purified
by column chromatography on silica using mobile phase
dichloromethane
−
methanol (30:1, v/v) to obtain target
compounds 18a-b.
4.2.4. Cell cycle distribution analysis
4.1.5.2.1. 2-(2-aminopyrimidin-5-yl)-N'-benzoyl-4-morpholinothieno
[3,2-d] pyrimidine-6-carbohydrazide (18a). White solid; Yield: 77.5%;
MS (ESI) m/z (%): 475.4 [M−H]-; 1H NMR (400 MHz, DMSO‑d6) δ
11.05 (s, 1H), 10.71 (s, 1H), 9.14 (s, 2H), 8.18 (s, 1H), 7.94 (m, 2H),
7.63 (t, J = 7.2 Hz, 1H), 7.57 (s, 1H), 7.54 (d, J = 7.2 Hz, 1H), 7.15 (s,
2H), 4.02 (m, 4H), 3.81 (m, 4H). 13C NMR (151 MHz, DMSO‑d6) δ
166.18, 164.54, 161.86, 161.77, 160.77, 158.39, 158.11, 158.01,
132.56, 132.50, 129.01, 127.86, 125.71, 120.14, 114.32, 66.34, 46.32.
4.1.5.2.2. 2-(2-aminopyrimidin-5-yl)-N'-(4-methoxybenzoyl)-4-
The effects of compounds on cell cycle progression were determined
using a standard propidium iodide (PI) staining procedure followed by
flow cytometry analysis. Briefly, HCT-116 cells were seeded in six-well
plates (5 × 104/well) and then treated with different concentrations of
18b for 24 h. The cells were collected and washed twice with ice cold
PBS, then fixed in ice-cold 70% (v/v) ethanol overnight at 4 °C. The
cells were washed again by PBS, and then the cell DNA was stained with
400 μL PI (Beyotime) for 10 min. Data acquisition and analysis were
performed using a flow cytometer.
morpholinothieno[3,
2-d]pyrimidine-6-carbohydrazide
(18b). White
solid; Yield: 67.9%; MS (ESI) m/z (%): 505.1 [M−H]-; 1H NMR
(600 MHz, DMSO‑d6) δ 10.99 (s, 1H), 10.57 (s, 1H), 9.15 (s, 2H),
8.19 (s, 1H), 7.94 (d, J = 7.4 Hz, 2H), 7.15 (s, 2H), 7.08 (d, J = 7.4 Hz,
2H), 4.02 (s, 4H), 3.85 (s, 3H), 3.81 (s, 4H). 13C NMR (151 MHz,
DMSO‑d6) δ 165.64, 164.59, 162.58, 161.90, 160.82, 158.39, 158.12,
158.03, 142.91, 130.03, 129.81, 125.68, 124.68, 120.12, 114.21,
66.36, 55.83, 46.33.
4.2.5. Cell migration assay
Cell migration assay were carried out using wound-healing assay.
HCT-116 cells were seeded in six-well plates (5 × 104/well) and were
allowed to grow to 100% confluence. A linear wound was made by a
pipet tips across the confluent cell layer. HCT-116 cells were washed
twice with PBS, and then incubated with culture medium containing
DMSO or different concentrations of compound 18b. After treatment
for 0, 24 and 48 h, the images were taken by fluorescence microscope
(Olympus, Tokyo, Japan).
4.2. Biological section
4.2.1. In vitro antiproliferative assays
4.2.6. Molecular docking
MTT assay was carried out on A549, PC-3, HCT116 cells to evaluate
antiproliferative activity of the target compounds. The cancer cell lines
were cultured in minimum essential medium (MEM) supplement with
10% fetal bovine serum (FBS).
The molecules were built using Maestro, version 8.0.308, or con-
verted to 3D structures from the 2D structure using LigPrep, version
2.1.207. The PI3Kγ (PDB entry 3DBS), and mTOR (PDB entry 3L16) X-
ray structures were downloaded from the Protein Data Bank (PDB,
http://www.rcsb.org/). The protein structures were prepared using the
protein preparation wizard in Maestro with standard settings. Grids
were generated using Glide, version 4.5.208, following the standard
procedure recommended by Schrödinger. The conformational en-
sembles were docked flexibly using Glide with standard settings in both
standard and extra precision mode. Only poses with low energy con-
formations and good hydrogen bond geometries were considered.
Figures were drawn using PyMOL (version 1.7).
Approximate 5 × 103 cells, suspended in MEM medium, were
plated in a 96-well plate and incubated in 5% CO2 at 37 °C for 24 h. The
tested compounds were added to the culture medium and the cell cul-
tures were incubated for another 72 h. The cells were incubated at 37 °C
for 4 h with 10 μL of 5 mg/mL MTT solution. The formazan crystals in
each well were dissolved in 100 μL dimethyl sulfoxide, and their ab-
sorbance were measured at 492 nm (absorbance of MTT formazan) and
630 nm (reference wave length) with an ELISA reader. The results,
expressed as IC50, are averages of at least three determinations and
calculated using the Bacus Laboratories Incorporated Slide Scanner
(Bliss) software.
Declaration of Competing Interest
4.2.2. In vitro enzymatic assays
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
The in vitro enzymatic assays of the tested compounds were eval-
uated by homogeneous time-resolved fluorescence (HTRF) assay.
11

Y. Han, et al.
BioorganicChemistry104(2020)104197
Acknowledgements
methylmorpholino)-6-morpholino-1,3,5-triazin-2-yl) pyridin-2-amine (PQR530), a
Potent, Orally Bioavailable, and Brain-Penetrable Dual Inhibitor of Class I PI3K and
mTOR Kinase, J. Med. Chem. 62 (2019) 6241–6261.
This work was supported by National Natural Science Foundation of
China (No. 81573296) and LiaoNing Revitalization Talents Program
(XLYC1808037).
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Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104197.
[23] A.M. Venkatesan, C.M. Dehnhardt, E. Delos Santos, Z. Chen, O. Dos Santos, S. Ayral-
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