Discovery of novel akt1 inhibitor induces autophagy associated death in hepatocellular carcinoma cells

Article abstract of DOI:10.1016/j.ejmech.2020.112076

In this study, a series of thieno [2,3-d]pyrimidine derivatives were designed, synthesized and evaluated as novel AKT1 inhibitors. In vitro antitumor assay results showed that compounds 9d-g and 9i potently suppressed the enzymatic activities of AKT1 and

Full text of DOI:10.1016/j.ejmech.2020.112076

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Discovery of novel akt1 inhibitor induces autophagy associated death in
hepatocellular carcinoma cells
Meng Yu, Minghui Zeng, Zhaoping Pan, Fengbo Wu, Li Guo, Gu He
PII:
S0223-5234(20)30043-X
DOI:
https://doi.org/10.1016/j.ejmech.2020.112076
EJMECH 112076
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 November 2019
Revised Date: 8 January 2020
Accepted Date: 15 January 2020
Please cite this article as: M. Yu, M. Zeng, Z. Pan, F. Wu, L. Guo, G. He, Discovery of novel akt1
inhibitor induces autophagy associated death in hepatocellular carcinoma cells, European Journal of
Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112076.
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Discovery of novel akt1 inhibitor induces autophagy associated death
in hepatocellular carcinoma cells
Meng Yu,1,2,† Minghui Zeng,3,† Zhaoping Pan,2 Fengbo Wu,2 Li Guo1,* and Gu He2,*
1 Department of Medicinal Chemistry, West China School of Pharmacy, Sichuan
University, Chengdu, Sichuan 610041, PR China
2 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital,
Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu,
Sichuan 610041, PR China
3 Department of Pharmacy, Qionglai Medical Center Hospital of Sichuan Province,
Chengdu, Sichuan 611500, PR China
† These authors contributed equally to this work.

Abstract
In this study, a series of thieno[2,3-d]pyrimidine derivatives were designed,
synthesized and evaluated as novel AKT1 inhibitors. In vitro antitumor assay results
showed that compounds 9d-g and 9i potently suppressed the enzymatic activities of
AKT1 and potently inhibited the proliferation of HepG2, Hep3B, Huh-7 and
SMMC-7721 cancer cell lines. Among these derivatives, the compound 9f
demonstrated the best inhibitory activities on AKT1 (IC50 = 0.034µM) and Huh-7
cell (IC50 = 0.076µM). A panel of biological assays showed that compound 9f
suppressed the cellular proliferation of Huh-7 through Akt/mTOR signaling pathway
mediated autophagy mechanism. Furthermore, the antitumor capacity of 9f was
validated in the subcutaneous Huh-7 xenograft models. Together, our results
demonstrate that a novel small-molecule Akt1 inhibitor induces autophagy associated
death in hepatocellular carcinoma, which may afford a potential drug candidate for
targeted cancer therapy.
Keywords: hepatocellular carcinoma, Akt1, inhibitor, autophagy

1. Introduction
Hepatocellular carcinoma (HCC) is a malignant tumor with a high incidence
worldwide.[1-3] It is closely related to liver diseases arising from viral infection, such
as hepatitis B virus (HBV) and hepatitis C virus (HCV), alcoholic liver disease, toxic
substances, parasites, heredity and aflatoxin, etc.[4] Currently, the main clinical care
include surgery, intervention, ablation, targeted drugs, radiotherapy and other
methods according to the stage of the tumor; however, they all have limited
efficacy.[5-7]
AKT, also known as protein kinase B (PKB), is a Ser/Thr kinase with high
homology to PKA and PKC, belongs to AGC kinases family. Akt includes three
closely
related
isoforms, comprising Akt1,
Akt2,
and
Akt3,
and
these three isoforms share more than 80% sequence identity and similar downstream
targets.[8-10] It plays a key role in many cell growth processes, such as glucose
metabolism, apoptosis, cell proliferation, transcription and cell migration.[11, 12] It is
over expressed and / or over activated in many tumor cells.[13] This abnormally high
expression of Akt in cancer,[14] can be used to design therapeutics against it for
restraining the growth and progression of the tumor. It can be used alone as a
therapeutic or in combination with other chemotherapeutics.[15-17] Nowadays, there
are several Akt inhibitors, which are undergoing clinical trials for efficacy (Figure 1
and Table S1). Many immunotherapies have been in development for different types
of cancers. Particularly, Ipatasertib (GDC-0068) is currently in a phase-II study to
check its efficacy as a monotherapy for the treatment of triple-negative breast cancer
(TNBC).[18] Further, the use of AZD5363 in combination with paclitaxel to treat
triple-negative breast cancer has also been reported, which demonstrates that this

treatment can prolong the overall survival of patients significantly and improve their
quality of life.[19] Another drug, GSK690693 which has been used to treat acute
lymphoblastic leukemia clinically, has been withdrawn due to some adverse reactions,
like hyperglycemia.[20]
Figure 1. Chemical structures of representative Akt1 inhibitors.
GSK2141795 (Uprosertib), a ATP competitive Akt inhibitor, which is used as a
therapeutic in different types of cancer (such as melanoma, breast cancer, etc).[21]
Another monotherapy, MK-2206 used against for the clinical management of
myelogenous leukemia, has only limited topical use as the patients experienced
hemolytic toxicity after intravenous injection.[22] Therefore, this indicates that Akt
inhibitors might have broad application prospects as anticancer drugs.
In the current study, we have investigated the structured-based design, synthesis
and in vitro mechanistic studies of thieno[2,3-d]pyrimidine based Akt1 inhibitors. The
substituted phenylalanine amino acids was linked to the thieno[2,3-d]pyrimidine
scaffold resulting in better kinase inhibition. The in vitro kinase inhibition and
selectivity assays, cell proliferation, apoptosis and autophagy assays were measured to
understand the molecular mechanisms of these novel Akt1 inhibitors. The results
suggests that the novel Akt1 inhibitor 9f suppress the activation of the
PI3K/Akt/mTOR signaling pathway and cellular proliferation in human HCC cells.

Furthermore, 9f induces autophagy and apoptotosis of the HCC cells. This suggests
novel molecular mechanism of these Akt inhibitors which needs to be studied further
for the development of more efficient therapeutics.
2. Results and discussion
Akt1 is hyperactivated in hepatocellular carcinoma
The mRNA-sequencing data of the hepatocellular carcinoma cohort in TCGA
(The Cancer Genomics Atlas) database showed that there was no significant
difference on the Akt1 mRNA levels between the cancer and the normal tissues
(Figure 2A).[23-25] However, the expression level of Akt1 was a significant
prognostic factor for both, overall survival (p = 0.013, HR = 1.6) and disease-free
survival (p < 0.01, HR = 1.6) of the HCC patients (Figure 2B and 2C). Moreover, the
IHC images in ProteinAtlas database suggests that the Akt1 protein levels were not
changed in both the HCC tissues and the adjacent normal tissues (ANT).[26] As there
was no significant difference in the mRNA and protein levels of Akt1 in the HCC and
the ANT tissues, we speculated that it was probably hyperactivated in the HCC tissues.
Therefore, we performed IHC analysis of phosphorylated antibody of Akt1 (Ser473)
on a HCC tissue microarray. As shown in Figure 2D and Table S2 the pAkt1 (Ser473)
levels in HCC tissues were remarkably higher than that of adjacent normal tissues,
and the expression profiles of pAkt1 (Ser473) were significantly related to the
primary tumor stage, regional lymph nodes and distant metastasis.[27-29] Thus, from
this results, we hypothesize that the hyperactivated Akt1 might be a potential
therapeutic target in HCC.

Figure 2. (A) The Akt1 mRNA expression levels of HCC and normal liver tissues in
TCGA database; The Kaplan-Meier plots of overall survival (B) and disease-free
survival (C) ratio in HCC cohort of TCGA database; (D) The protein expression levels
of total Akt1 and p-Akt1(S473) in HCC and adjacent normal tissues by IHC; (E) The
3D contour of the binding mode of GDC-0068 in ATP site of Akt1; (F) The scaffold
hopping strategy of novel Akt1 inhibitors.

Design and Synthesis of novel Akt1 Inhibitors.
There were many Akt1 inhibitors reported with diverse structural scaffold,
however, the kinase selectivity of some Akt1 inhibitors remained limited. We utilized
protein kinase A (PKA), a highly homology proteins of Akt1, to analyze the specific
binding mechanisms of Akt1.[30, 31] After aligned and superposed the crystallized
structures of PKA and Akt1 together (Figure S1A), we could find that they were very
similar to each other. The only difference between the ATP sites of PKA and Akt1 was
depicted in Figure 2E and Figure S1B and S1C. The steric tolerance in the ATP site
of Akt1 was higher than that of PKA, which was also validated by the previous
reported SAR analysis of GDC-0068 analogues. Therefore, at the hand of rational
drug design strategy, we proposed to change we a thieno[2,3-d]pyrimidine based
tricycle scaffold as the core structure to replace the cyclopenta[d]pyrimidine scaffold
of GDC-0068 or pyrrolo[2,3-d]pyrimidine scaffold of AZD5363.[32-35] The
α-substituted phenylacetyl piperidine group, serving as key pharmacophore, were
retained and variants at 4-position of pyrimidine ring could enrich the diversity in the
drug-like skeleton. The resulting compounds may serve as the basis for exploring
novel and efficient Akt1 inhibitors (Figure 2F).
The synthesis routes of the compounds 5a-o, 9a-k, and 13 are shown in Scheme
1-3. The synthesis of thieno[2,3-d]pyrimidine derivatives 2a-f followed our
established methods.[36, 37] Briefly, we used N-substituted piperidinones and an
α-cyanoester as raw materials to synthesize 2-aminothiophenes by the Gewald
synthesis in the presence of rhombic sulfur and a base.[38, 39] Next, these
intermediates were incubated in formamidine acetate in DMF for 12 hours at 100°C to
obtain a crystal (2a-f) requiring no further purification, and then the compounds 2a-f

were converted to an intermediate form, 4-chloro-pyrimidines by treatment with
POCl3. The piperazine analogue 3a-f was synthesized through the SNAr reaction using
the commercially available N-Boc piperazine and 4-chloro-pyrimidines under alkaline
condition in NMP as outlined (Scheme 1) to afford the N-Boc piperazine intermediate
crystals requiring no further purification, after that the standard trifluoroacetic
acid-based N-Boc de-protection reaction was followed to obtain 3a-f.[40] The
substituted phenylalanine amino acids derivatives 4a-j was synthesized through four
steps, as shown in Scheme S1. In the first stage, phenyl acetate esters were converted
to the acrylates as previously reported.[41] Then through Michael addition of amines
following by N-Boc protection and hydrolysis of the methyl ester, the compound 4a-j
was obtained for overall yield of 13-17%. With the essential thieno[2,3-d]pyrimidine
scaffolds and phenylalanine acids derivatives 4a-j in hand, the synthesis of the target
compounds 5a-o was completed by amide coupling under the presence of the peptide
coupling agent HATU, followed by the N-Boc de-protection under acid conditions, as
shown in Scheme 1. Similarly, we used cyclohexanone, cyclopentanone and
tetrahydropyranone (8a-c) as raw materials to synthesize the compounds 9a-k through
the same steps as above (Scheme 2).
The preparation of the compound 13 is shown in Scheme 3. We choose
N-Boc-4-piperidone 10 as the starting materials with similar reaction conditions to
synthesize the compound 11, followed by the N-Boc de-protection of 11 in the
presence of trifluoroacetic acid and the obtained compound was treated with
dimethylcarbamoyl chloride under the presence of DMAP as a catalyst.[42] Then
through the same SNAr reaction with N-Boc piperazine, N-Boc de-protection and
amide coupling with compound 4j, as above in Scheme 1, we finally synthesized the
target compound 13. The synthesized target compounds were characterized by 1H

NMR, 13C NMR and HR-MS (high-resolution mass spectrometry) and the
detailed experimental operation, process and characterization are shown in the
experimental sections.
Table 1. The remained kinase activities (%) after 1µM compounds 5a-f, 9a-c and 13
incubation on Akt1-3 and PKA.
% controla @ 1µM
X
n
R1
R2
R3
No.
PKA(h) Akt1(h) Akt2(h)
Akt3(h)
69
-
-
-
CH3
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
-
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
-
72
87
88
80
92
84
78
45
78
80
50
73
75
71
77
76
55
11
78
86
91
82
89
77
71
81
72
57
81
95
5a
5b
5c
5d
5e
5f
-
Et
71
-
-
n-Pr
79
-
-
Bn
71
-
-
-
cyclopropyl
70
-
isopropyl
78
CH2
CH2
O
1
0
1
-
-
-
-
-
63
9a
9b
9c
13
19
77
-
87
a % control = kinase activity remained.
Scheme 1. Synthesis of compound 5a-o.

Reagents and conditions: (a) NCCH2CO2Et, S8, EtOH, Et3N, reflux, 12h; (b) formamidine acetate,
DMF 100 °C, 1h; (c) POCl3, DIPEA, toluene, 80 °C, overnight; (d) 1-Boc-piperazine, DIPEA, NMP; (e)
TFA/DCM, room temperature, 30mins; (f) HATU, ET3N, DCM.
Scheme 2. Synthesis of compound 9a-k.
Reagents and conditions: (a) NCCH2CO2Et, S8, EtOH, Et3N, reflux, 12h; (b) formamidine acetate,
DMF 100 °C, 1h; (c) POCl3, DIPEA, toluene, 80 °C, overnight; (d) 1-Boc-piperazine, DIPEA, NMP; (e)
TFA/DCM, room temperature, 30mins; (f) HATU, ET3N, DCM.
Reagents and conditions: (a) NCCH2CO2Et, S8, EtOH, Et3N, reflux, 12h; (b) formamidine acetate,
DMF 100 °C, 1h; (c) POCl3, DIPEA, toluene, 80 °C, overnight; (d) TFA/DCM, room temperature,
30mins; (e) dimethylcarbamoyl chloride, pyridine, DMAP, DMSO, 23 °C, 16h; (f) 1-Boc-piperazine,
DIPEA, NMP; (g) HATU, ET3N, DCM.

2.4 The SAR (structure-activity relationship) analysis and binding modes of
novel Akt1 inhibitors
The kinase inhibitory abilities of the compounds 5a-o, 9a-k, and 13 are shown in
Table 1 and 2, including the percentage of the 1.0 µM inhibitor incubated Akt1, Akt2,
Akt3 and PKA kinase activity. Table 1 shows the comparison between the
bioactivities of the different ring types that fuse to the thieno[2,3-d]pyrimidine
scaffold. The results of the kinase inhibitory assays suggest that a bigger steric
volume is an unfavorable to all kinase inhibitory assays, and Akt1 and Akt3 have
relatively lower sensitivity than that of PKA. Only two compounds, 5a and 9b could
suppress half or more of the Akt1 kinase activities at 1.0 µM. The other compounds in
Table 1 with larger substitution to pocket 1, resulted in only marginal inhibition of
PKA and Akts, depicting low kinase selectivity. We further optimized the substituted
groups on the phenylacetyl piperidine fragment based on the cyclopenta[4,5]thieno
[2,3-d]pyrimidine scaffold of 9b and N-methyl-pyridothieno[2,3-d]pyrimidine
scaffold of 5a (Table 2) and observed that any changes to the R2 or R3 groups on the
N-methyl-pyridothieno[2,3-d]pyrimidine scaffold resulted in a loss of the kinase
inhibition activities. For the cyclopenta[4,5]thieno[2,3-d]pyrimidine scaffold, the
larger substituted groups on R3 group is favorable, 4-Cl and 4-Br substitution on R2
group is favorable, while the ortho-substitution on the R2 group could diminish the
kinase inhibitory activities. Compounds 9d-g and 9i almost totally suppressed the
kinase activity of Akt1 at 1.0µM concentration. The IC50 values of these compounds
on Akt1, Akt2 and Akt3, and the IC50 of cellular proliferation on HCC cell lines
HepG2, Hep3B, Huh-7 and SMMC-7721 are listed in Table 3. Compound 9f
demonstrated the best inhibitory activities on Akt1, Akt2 and Akt3 with IC50 value of

0.034, 1.709 and 0.129µM, respectively. And all the five compounds exhibit potent
cellular proliferation inhibitory capacities on the HCC cell line. The Huh-7 cell is
relatively more sensitive than the other cell lines, with an IC50 value of 0.076, 0.159
and 0.112 µM to 9f, 9g and 9i, respectively. Moreover, the pan-cancer kinase
profiling of compound 9f was performed to identify its selectivity to a panel of 104
kinases by the KINOMEscan® method. As listed in Table S3, with 1.0 µM
concentration of the inhibitor, only Akt1 was totally suppressed while the other
kinases showed over 40% activity. Thus, compound 9f was selected for further studies
to understand its role in the involved molecular mechanism of the novel AKT1
induced autophagy in the hepatocellular carcinoma with in vivo xenograft
evaluations.
Table 2. The remained kinase activities (%) after 1µM compounds 5g-o and 9d-k incubation
on Akt1-3 and PKA.
% controla @ 1µM
X
n
R1
R2
R3
No.
PKA(h) Akt1(h) Akt2(h)
Akt3(h)
74
-
-
-
CH3
CH3
CH3
CH3
CH3
CH3
CH3
4-Cl
4-Cl
4-Cl
4-Cl
4-F
Et
n-Bu
91
81
82
95
97
88
90
95
86
31
37
95
81
82
84
88
69
78
81
96
1
107
93
81
88
111
86
73
78
83
58
56
5g
5h
5i
-
84
-
-
Cyclopentyl
Cyclohexyl
i-Pr
76
-
-
69
5j
-
-
87
5k
5l
-
-
4-Br
4-Me
i-Pr
72
-
-
-
i-Pr
73
5m
5n
5o
9d
9e
-
CH3 4-OMe
i-Pr
76
-
-
CH3
2-Cl
4-Cl
4-Cl
i-Pr
76
CH2
CH2
0
0
-
-
Et
21
n-Bu
1
15

CH2
CH2
CH2
CH2
CH2
CH2
0
0
0
0
0
0
-
-
-
-
-
-
4-Cl
4-Cl
Cyclopentyl
Cyclohexyl
i-Pr
35
29
77
23
66
95
1
1
63
51
22
11
45
20
42
92
9f
9g
9h
9i
4-F
26
1
104
67
4-Br
4-OMe
2-Cl
i-Pr
i-Pr
18
88
86
9j
i-Pr
101
9k
a % control = kinase activity remained.
Table 3. The IC50 values (µM) of compounds 9d-g and 9i on Akt1-3 and HCC cell lines.
Kinase activities (IC50, µM)a
Akt1 Akt2 Akt3
Anti-proliferative activities(IC50, µM)b
No.
9d
HepG2
0.580±0. 0.541±0. 0.283±0.
068 084 029
0.914±0. 0.833±0. 0.367±0.
077 120 057
0.183±0. 0.131±0. 0.076±0.
019 014 010
0.386±0. 0.294±0. 0.159±0.
040 025 026
0.202±0. 0.228±0. 0.112±0.
018 026 009
0.268±0. 0.389±0. 0.167±0.
037 041 018
Hep3B
Huh-7
SMMC-7721
1.249±0.12
0.125± 3.05±0. 0.101±0.008
0.019 31
0.166± 3.78±0. 0.137±0.011
0.022 54
0.034± 1.71±0. 0.129±0.010
0.003 18
0.051± 1.30±0. 0.045±0.007
0.006 21
0.039± 1.36±0. 0.095±0.011
0.005 16
0.019± 0.032±0 0.026±0.007
0.006 .04
1.354±0.19
0.386±0.05
0.978±0.08
0.521±0.05
0.661±0.14
9e
9f
9g
9i
GDC-
0068
a
IC50 values for enzymatic inhibition of Akt1-3; Data displayed is the average of at least three
independent replicates ± standard deviation; IC50 values were determined from KinaseProfiler of
Eurofins.
b IC50 = compound concentration required to inhibit tumor cell proliferation by 50%; Data displayed is
the average of at least three independent replicates ± standard deviation.
The 3D and 2D contours of compound 9f bound to the ATP sites of Akt1 are
shown in Figure 3A and 3B, respectively. It can be observed that the N1 and S atoms
of the thieno[2,3-d]pyrimidine ring form stable hydrogen bonds with the Ala230
residue, and the nitrogen atom of the cyclopentamine group forms a hydrogen bond

with Lys158 and electrostatic interaction to Glu278 of Akt1. Moreover, the
cyclopenta[d]thieno[2,3-d]pyrimidine fragment could form hydrophobic interactions
with the inner pocket of Akt1, including Met281 and Lys159, which might be a
potential reason of kinase selectivity of the compound 9f to Akt1 to the other kinases.
Figure 3. The 3D contour (A) and 2D contour (B) of the binding mode of compound
9f in ATP site of Akt1.
2.5 Novel Akt1 inhibitor induces lethal autophagy in HCC cells
The cytotoxicity assay showed that the compound 9f suppress the cellular
proliferation of a panel of HCC cell lines at submicromolar levels. The potential cell
death subroutines of the Huh-7 cells under 9f incubation were determined by
apoptosis and autophagy assays. These include the Annexin-V/PI dual-staining
apoptosis assay, GFP-LC3 transfected autophagic puncta assay, as well as the western
blotting of the apoptosis and the autophagy related proteins (Figure 4). It was
observed that 9f remarkably induced apoptosis of the Huh-7 cells in a
dosage-dependent manner, and the number of the autophagosome puncta also
increased with 9f incubation in both Huh-7 and Hep3B cells (Figure 4B and
S2).[43-46] WB analysis demonstrates that 9f treatment declined the phosphorylation
levels of Akt1 and GSK3β without interfering with the protein expression of total
Akt1 and GSK3β; however, the expression of Bcl-2, an apoptosis inhibitor, was

suppressed after 9f incubation. To further detect the influence of autophagy flux of 9f
incubation, the mCherry-GFP-LC3 plasmid was transfected into Huh-7 cells, and
there were massive autophagy puncta formed after 0.2μM 9f incubation (Figure 4E).

Figure 4. (A) The apoptosis in Huh-7 cells induced by compound 9f detected by
Annexin V/PI dual staining; (B) The formation of autophagosome in GFP-LC3
transfected Huh-7 cells induced by 0.2μM 9f, Scale bar: 6 µm; (C) Western blotting
of proteins associated in Akt/mTOR signaling pathway, apoptosis and autophagy with
or without compound 9f incubation; (D) The quantitative analysis of differential
expression proteins in western blotting; (E) The TEM images of Huh-7 cells with or
without compound 9f incubation,Scale bar: 5.0 µm; (F) The fluorescent images of
mCherry-GFP-LC3 plasmid transfected Huh-7 cells induced by 0.2μM compound 9f,
Scale bar: 6 µm; (G) The co-localization analysis of LC3 and LAMP1 in Huh-7 cells
with or without 0.2μM 9f incubation, Scale bar: 6 µm; *, P < 0.05; **, P < 0.01.

Figure 5. (A) The cell viabilities of Huh-7 cell after incubation with compound 9f
and/or autophagy inhibitors; (B) The intracellular Cathepsin B/D activities after
incubation with 0.2μM 9f and/or autophagy inhibitors; (C) The accumulation of
autophagosomes in GFP-LC3 transfected Huh-7 cells induced by 0.2μM 9f and
autophagy inhibitors. Scale bar: 6 µm; **, P < 0.01.
Upregulation in the expression of cytochrome C and cleaved caspase-3 were
observed after 9f treatment, which suggests that the compound 9f triggers the
mitochondrial apoptosis pathway, as shown by the increased levels of LC3-II and
declined levels of p-mTOR indicating the initiation of the autophagic flux after the 9f
incubation. It is surprising that the 9f induced accumulation of p62/SQSTM1, a
marker of protein digestion by autopahgolysosome, is observed. The autophagy flux
arrest by 9f incubation was also confirmed by the immunofluorescent staining of LC3
and LAMP1, a marker of lysosome (Figure 4F). The fusion of autophagosome
(stained by LC3) and lysosome (stained by LAMP1) was suppressed by 9f treatment.
In addition, there was slightly autophagy flux arrest observed after GDC-0068
incubation (Figure S3). In support of our observation, there are several other reports
about the Akt1 allosteric inhibitor MK2206 triggered cytoprotective autophagy in the
melanoma and the HCC cells.[47-49] We further determined the potential biological
functions of the 9f-induced autophagy by addition of the autophagy inhibitor 3-MA
and bafilomycin-A1 (Baf-A1), which 3-MA decreased the cytotoxicity of 9f; Baf-A1
displayed a synergistic effect to the compound 9f in Huh-7 cells (Figure 5A). The
activity assays of cathepsin B and cathepsin D suggests that Baf-A1 suppressed the
activities of cathepsins rather than 3-MA (Figure 5B). These results suggest that 9f
induces the initiation of autophagosome and disrupts the autophagic flux. Moreover,
the fluorescent microscopy images of the GFP-LC3 transfected Huh-7 cells indicate

that the addition of 3-MA inhibited the formation of the autophagosome induced by 9f,
and there was accumulation of autophagosomes after Baf-A1 incubation (Figure 5C).
The potential signaling pathway involved in the crosstalk between Akt1 inhibition by
9f and autophagic flux regulation were studied further.
2.6 In vivo xenograft models
A subcutaneous xenograft model of the Huh-7 cells in nude mice was established
to investigate the antitumor effect of 9f in vivo. As shown in Figure 6A,
intraperitoneal administration of 25 or 50mg/Kg 9f significantly suppressed the
growth of the Huh-7 xenografts (p < 0.01). The average weight of the tumor tissues of
the treatment group was significantly lower than that of the control group (Figure 6B).
The inhibition potentials of the xenograft tumor volume and weight in the 25mg/Kg
9f-treated group are slightly lower than those of the 50mg/Kg treated group. Moreover,
there were no significant changes of the body weight of mice in both the 9f-treated
and control groups. As shown in Figure 6C, we used immunofluorescence and IHC to
analyze the tumor tissues further to verify whether the tumor growth suppression
regulated by 9f was related to the inhibition of Akt1 and the apoptosis/autophagy
related signaling pathways. The number of the apoptotic positive cells determined by
TUNEL staining of the 9f-treated tumors was significant. In addition, the IHC
analysis showed that the phosphorylated levels of Akt decreased, and there was no
significant difference between the total Akt levels. The expression levels of the
proliferative marker Ki-67 and the autophagy marker p62/SQSTM1 declined,
however, the expression of LC3-II elevated. The results of these in vivo experiments
demonstrate that 9f exhibits inhibitory effects on Akt1 to trigger lethal autophagy in
vivo. Thus, the results of the in vivo experiments are in accordance with those of in
vitro experiments.

Figure 6. Compound 9f suppressed the growth of Huh-7 xenograft models in vivo. (A)
Changes in tumor volume in each group were determined from 1 to 15 days; (B)
Analysis of the xenograft tumor tissue weight in each mouse in (A); (D) Protein
expression levels of Ki67, TUNEL, total AKt, p-Akt (S473), LC3-II, and
p62/SQSTM1 in each group were determined by IHC or IF. Scale bar: 50 µm.
In conclusion, we reported the design, synthesis and biological evaluation of the
novel Akt1 inhibitors with the tricycle thieno[2,3-d]pyrimidine scaffold as a novel
Akt1 inhibitor in the Huh-7 cells. The most potent compound 9f remarkably
suppressed the Huh-7 cell proliferation and induced lethal autophagy and apoptotic
cell death. The detailed binding modes of 9f to Akt1 were probed to elucidate its
kinase selectivity. Furthermore, the antitumor capacity of 9f was validated in the
subcutaneous Huh-7 xenograft models. Collectively, these results indicate that 9f
suppressed the Huh-7 cell proliferation by inducing lethal autophagy mediated by the
Akt/mTOR signaling pathway, suggesting novel molecular mechanism of these novel
Akt inhibitors worthy for further study.
3. Experimental section
Reagents, Antibodies and Cell Cultures
GDC-0068, 3-MA and Bafilomycin-A1 were obtained from Selleckchem Co. Ltd. (Shanghai,
China). The antibodies recognizing Bcl-2, Caspase-3, Caspase-9, Cytochrome C, LC-3, p62/SQSTM1
and GAPDH were purchased from Proteintech (Wuhan, China). The antibody recognizing Akt, p-Akt,
mTOR and p-mTOR were purchased from Abcam (Cambridge, MA, USA). The malignant melanoma
cell lines, including HepG2, Hep3B, Huh-7 and SMMC-7721 were obtained from the Chinese Center for
Type Culture Collection (Wuhan, China) and cultured in DMEM (Dulbecco’s modified Eagle’s

medium) with 10% of FBS (fetal bovine serum) and streptomycin. The cytotoxicity assay was performed
with MTT method as previously described.
Kinase inhibition assay
AKT1, AKT2, AKT3 and PKA kinase assay used the Kinase Profiler services supplied by
Eurofins in the light of our previous report.[50-52] The KINOMEScan® selectivity profiling was
implemented by DiscoveRx Co. Ltd. and on the baisis of the company's protocol.
Apoptosis and autophagy assay
The apoptosis of cells caused by the compound 9f is tested by Annexin V/PI dual-staining flow
cytometry method on SMMC-7721 cells. The experimental method and procedure have been described
detailedly in our previous reports. And the autophagy assay is relied on SMMC-7721 cells transfected
by GFP-LC3. Briefly speaking, the SMMC-7721 cells after transfecting are processed by saline or
compound 9f for 6 h, after that the cells are going to fix by paraformaldehyde and the phenomenon of
autophagosome punctures can be observed through the accumulated of GFP-LC3.
Western Blotting analysis
For western blotting, the extracts of total protein from each sample were treated in accordance with
the method we described previously. In short, the total proteins were loaded into the SDS-PAGE
(sodium dodecyl sulfate-polyacrylamide) gel and separated through the different of their molecular
weight in electrophoresis (PAGE), after that the separated proteins were shifted into a PVDF membrane
and incubated through corresponding membrane react pathways. (Millipore, Burlington, MA, USA).
The resulted membranes reacted with major antibodies and HRP (horseradish peroxidase)-conjugated
secondary antibodies (1:10,000). The expression profiles of target proteins can be identified through an
enhanced chemiluminescence (ECL) WB substrate (Millipore, MA. USA).
Animal models
The research on anti-tumor activity of 9f in vivo was implemented based on the Guidelines for the
Care. All animal experimental studies were authorized by the Animal Ethics Committee of Animal
Experimentation of West China Hospital, Sichuan University. The 6-8 weeks old SPF (specific
pathogen-free) nude mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. The
preliminary safety evaluation of the anti-tumor activity of 9f in vivo was performed using the models of
SMMC-7721 subcutaneous xenograft. SMMC-7721 cells, a single cell suspension (5 × 105/100 µL) in

PBS, were injected into the dorsal subcutaneous of mice. The size of the tumor was measured every two
days from the time of injection. The volume of tumor is calculated in the light of Vol = a×b2×0.5 (a, b
represent the large and the small diameter tumor tissue, respectively). The tumor tissues were stripped,
fixed with formalin, embedded in paraffin and sliced, after that the sections were stained by TUNEL,
LC3-II, Akt, pAkt473 and Ki67 for further histological research.
Tissue microarray (TMA), Immunohistochemistry and Immunofluorescent assays
The histological sections of tumor tissue were immersed into EDTA antigen recovery buffer (pH
8.0) or citrate buffer (pH 6.0), and then microwave was used to recover antigen. Then the slide was
incubated with the corresponding primary antibody at 37 °C for 30-40 minutes. Normal anti-rabbit or
anti-mouse IgG was used as a negative control group. Immunohistochemical analysis was performed
with diaminobenzidine solution after the sections was treated with HRP polymer as well as the second
antibody for 30 minutes. Immunofluorescence (IF) test was carried out by treating slides with
fluorescein combined with secondary antibody and detecting under fluorescence microscope. A human
liver cancer TMA was purchased from Shanghai Outdo Biotech. The IHC staining method is to use the
primary antibody of rabbit against p-akt1 s473, as we mentioned before, unless otherwise stated.
Chemical synthesis section.
The whole chemical reagents including anhydrous reagents were commercially available and used
without further purification. The reactions were monitored using TLC purchasing from commercial
company. The silica gel for column chromatography was buying from Qingdao Haiyang Chemical Co.,
1
Ltd (Pingdu, Qingdao, Shandong, China). H NMR spectra were collected on a Bruker Avance III
400MHz apparatus and 13C NMR data were measured at 100MHz in the same instrument. TMS
(tetramethylsilane) was used as the internal standard and CDCl3 was used to dissolve samples. The
chemical shifts (δ) was marked in ppm. ESI-HRMS spectrum was collected using Waters TOF-MS
instrument (Waters, Milford, MA, USA).
2-(4-chlorophenyl)-3-(isopropylamino)-1-(4-(7-methyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2
,3-d]pyrimidin-4-yl)piperazin-1-yl)propan-1-one (5a) as a white solid, m.p. 89.6-91.6 °C, yield 77%.

1H NMR (400 MHz, CDCl3) δ 8.49 (s, 1H), 7.32 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 8.4 Hz, 2H), 4.06 (dd,
J = 9.2, 5.2 Hz, 1H), 3.96 - 3.86 (m, 1H), 3.71 (s, 2H), 3.69 - 3.60 (m, 1H), 3.59 - 3.50 (m, 1H), 3.49 -
3.40 (m, 2H), 3.36 - 3.31 (m, 1H), 3.30 - 3.25 (m, 1H), 3.24 - 3.18 (m, 1H), 3.01 - 2.93 (m, 2H), 2.89 -
2.78 (m, 2H), 2.76 - 2.71 (m, 1H), 2.71 - 2.66 (m, 2H), 2.52 (s, 3H), 2.24 - 2.17 (m, 1H), 1.09 (d, J =
6.4 Hz, 3H), 1.05 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 170.36, 168.78, 161.69, 151.75,
136.57, 133.46, 133.28, 129.41, 129.08, 124.78, 120.68, 52.26, 51.56, 50.52, 49.79, 49.29, 48.97, 45.59,
45.01, 41.53, 29.69, 27.34, 22.64, 22.58. HRMS: m/z calculated for C26H33ClN6OS [M + Na]+:
535.2023, found: 535.2019.
2-(4-chlorophenyl)-1-(4-(7-ethyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidin-4-yl)pi
1
perazin-1-yl)-3-(isopropylamino)propan-1-one (5b) as a white solid, m.p. 84.8-85.9 °C, yield 65%. H
NMR (400 MHz, CDCl3) δ 8.47 (s, 1H), 7.30 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 8.4 Hz, 2H), 4.09 (dd, J
= 9.2, 5.2 Hz, 1H), 3.95 - 3.85 (m, 1H), 3.75 (s, 2H), 3.67 - 3.58 (m, 1H), 3.58 - 3.49 (m, 1H), 3.48 -
3.38 (m, 2H), 3.36 - 3.31 (m, 1H), 3.30 - 3.24 (m, 1H), 3.24 - 3.17 (m, 1H), 2.95 (s, 2H), 2.90 - 2.78 (m,
2H), 2.82 - 2.77 (m, 1H), 2.76 (d, J = 4.8 Hz, 1H), 2.72 (t, J = 5.6 Hz, 2H), 2.65 (q, J = 7.2 Hz, 2H),
1.19 (t, J = 7.2 Hz, 3H), 1.10 (d, J = 6.4 Hz, 3H), 1.06 (d, J = 6.4Hz, 3H). 13C NMR (100 MHz, CDCl3)
δ 170.30, 168.79, 161.62, 151.70, 136.43, 133.51, 133.49, 129.43, 129.08, 125.14, 120.65, 52.38, 51.96,
51.42, 50.45, 50.23, 49.75, 49.10, 45.02, 41.55, 27.43, 22.48, 22.41, 12.43. HRMS: m/z calculated for
C27H35ClN6OS [M + Na]+: 549.2179, found: 549.2172.
2-(4-chlorophenyl)-3-(isopropylamino)-1-(4-(7-propyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2
,3-d]pyrimidin-4-yl)piperazin-1-yl)propan-1-one (5c) as a white solid, m.p. 79.5-81.6 °C, yield 70%.
1H NMR (400 MHz,CDCl3) δ 8.46 (s, 1H), 7.33 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 8.4 Hz, 2H), 4.32 (br s,
1H), 4.27 (dd, J = 10.0, 4.4 Hz, 1H), 3.93 - 3.85 (m, 1H), 3.74 (s, 2H), 3.71 - 3.63 (m, 1H), 3.54 - 3.45

(m, 2H), 3.41 - 3.31 (m, 3H), 3.28 - 3.20 (m, 1H), 3.16 - 3.05 (m, 1H), 2.99 - 2.91 (m, 3H), 2.89 - 2.80
(m, 1H), 2.76 - 2.67 (m, 2H), 2.56 - 2.49 (m, 2H), 1.67 - 1.56 (m, 2H), 1.24 (d, J = 6.4 Hz, 3H), 1.22 (d,
J = 6.4 Hz, 3H), 0.96 (t, J = 7.6 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 169.93, 168.77, 161.52,
151.64, 135.21, 134.05, 133.61, 129.71, 129.08, 125.23, 120.68, 60.09, 52.81, 50.59, 50.49, 50.33,
49.42, 47.64, 44.99, 41.66, 27.37, 21.23, 21.20, 20.46, 11.90. HRMS: m/z calculated for C28H37ClN6OS
[M + Na]+: 563.2336, found: 563.2327.
1-(4-(7-benzyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidin-4-yl)piperazin-1-yl)-2-(4
-chlorophenyl)-3-(isopropylamino)propan-1-one (5d) as a white solid, m.p. 77.7-87.2 °C, yield 66%.
1H NMR (400 MHz, CDCl3) δ 8.49 (s, 1H), 7.41 - 7.32 (m, 5H), 7.31 (d, J = 8.4 Hz, 2H), 7.23 (d, J =
8.4 Hz, 2H), 4.10 (dd, J = 9.2, 5.2 Hz, 1H), 3.96 - 3.86 (m, 1H), 3.74 (d, J = 3.2 Hz, 4H), 3.68 - 3.60
(m, 1H), 3.59 - 3.51 (m, 1H), 3.50 - 3.40 (m, 2H), 3.38 - 3.32 (m, 1H), 3.31 - 3.26 (m, 1H), 3.26 - 3.20
(m, 1H), 2.96 (t, J = 5.6 Hz, 2H), 2.88 - 2.80 (m, 2H), 2.80 - 2.76 (m, 2H), 2.73 (d, J = 5.2 Hz, 1H),
2.63 (br s, 1H), 1.11 (d, J = 6.3 Hz, 3H), 1.07 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
170.34, 168.81, 161.65, 151.68, 137.57, 136.50, 133.47, 133.45, 129.42, 129.11, 129.09, 128.49,
127.50, 125.17, 120.72, 62.44, 52.70, 51.48, 50.58, 50.18, 49.69, 49.13, 49.01, 45.01, 41.55, 27.36,
22.55, 22.48. HRMS: m/z calculated for C32H37ClN6OS [M + H]+: 589.2516, found: 589.2512.
2-(4-chlorophenyl)-1-(4-(7-cyclopropyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidin
-4-yl)piperazin-1-yl)-3-(isopropylamino)propan-1-one (5e) as a white solid, m.p. 69.3-70.6 °C, yield
1
64%. H NMR (400 MHz, CDCl3) δ 8.41 (s, 1H), 7.24 (d, J = 8.4 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H),
3.94 (dd, J = 8.8, 4.8 Hz, 1H), 3.90 - 3.86 (m, 1H), 3.84 (s, 2H), 3.62 - 3.44 (m, 2H), 3.44 - 3.32 (m,
2H), 3.31 - 3.24 (m, 1H), 3.21 (m, 1H), 3.24 - 3.17 (m, 1H), 2.86 (t, J = 3.6 Hz, 4H), 2.80 - 2.69 (m,
2H), 2.65 (dd, J = 11.6, 5.2 Hz, 1H), 1.95 (br s, 1H), 1.85 - 1.78 (m, 1H), 1.00 (d, J = 6.4 Hz, 3H), 0.96

(d, J = 6.4 Hz, 3H), 0.54 - 0.45 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 170.31, 168.71, 161.59, 151.58,
136.70, 133.33, 133.25, 129.27, 129.04, 125.01, 120.66, 52.68, 51.68, 50.54, 50.48, 49.69, 49.42, 48.73,
44.93, 41.45, 37.80, 27.16, 22.81, 22.75, 6.28. HRMS: m/z calculated for C28H35ClN6OS [M + H]+:
539.2360, found: 539.2355.
2-(4-chlorophenyl)-1-(4-(7-isopropyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidin-4-
yl)piperazin-1-yl)-3-(isopropylamino)propan-1-one (5f) as a white solid, m.p. 65.2 - 67.0 °C, yield
1
55%. H NMR (400 MHz, CDCl3) δ 8.48 (s, 1H), 7.32 (d, J = 8.4 Hz, 2H), 7.23 (d, J = 8.4 Hz, 2H),
4.05 (dd, J = 8.8, 4.8 Hz, 1H), 3.96 - 3.88 (m, 1H), 3.85 (s, 2H), 3.70 - 3.60 (m, 1H), 3.59 - 3.51 (m,
1H), 3.51 - 3.40 (m, 2H), 3.40 - 3.33 (m, 1H), 3.32 - 3.26 (m, 1H), 3.26 - 3.20 (m, 1H), 3.00 - 2.92 (m,
3H), 2.90 - 2.80 (m, 2H), 2.80 - 2.72 (m, 3H), 2.58 (br s, 1H), 1.17 (s, 3H), 1.15 (s, 3H), 1.11 (d, J = 6.4
Hz, 3H), 1.07 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 170.28, 168.74, 161.50, 151.59,
136.45, 134.09, 133.44, 129.38, 129.07, 125.32, 120.64, 54.17, 51.50, 50.41, 49.64, 49.14, 49.08, 48.31,
46.20, 44.99, 41.54, 28.09, 22.53, 22.48, 18.54, 18.49. HRMS: m/z calculated for C28H37ClN6OS [M +
H]+: 541.2516, found: 541.2514.
2-(4-chlorophenyl)-3-(ethylamino)-1-(4-(7-methyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d
1
]pyrimidin-4-yl)piperazin-1-yl)propan-1-one (5g) as a white solid, m.p. 95.4-96.2 °C, yield 65%. H
NMR (400 MHz, CDCl3) δ 8.48 (s, 1H), 7.30 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 8.4 Hz, 2H), 4.05 (dd, J
= 8.8, 5.2 Hz, 1H), 3.95 - 3.86 (m, 1H), 3.70 (s, 2H), 3.68 - 3.60 (m, 1H), 3.60 - 3.50 (m, 1H), 3.48 -
3.38 (m, 2H), 3.35 - 3.26 (m, 2H), 3.26 - 3.18 (m, 1H), 3.03 - 2.92 (m, 2H), 2.88 - 2.79 (m, 1H), 2.75 -
2.70 (m, 1H), 2.70 - 2.66 (m, 3H), 2.66 - 2.59 (m, 1H), 2.51 (s, 3H), 2.29 (s, 1H), 1.09 (t, J = 7.2 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ 170.34, 168.77, 161.69, 151.74, 136.54, 133.41, 133.28, 129.38,

129.07, 124.77, 120.68, 54.63, 53.90, 52.25, 50.56, 49.80, 49.22, 45.59, 45.01, 44.19, 41.52, 27.34,
15.00. HRMS: m/z calculated for C25H31ClN6OS [M + Na]+: 521.1866, found: 521.1859.
3-(butylamino)-2-(4-chlorophenyl)-1-(4-(7-methyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d
1
]pyrimidin-4-yl)piperazin-1-yl)propan-1-one (5h) as a white solid, m.p. 84.8-86.5 °C, yield 74%. H
NMR (400 MHz, CDCl3) δ 8.47 (s, 1H), 7.29 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 8.4 Hz, 2H), 4.04 (dd, J
= 8.8, 5.2 Hz, 1H), 4.08 - 3.99 (m, 1H), 3.69 (s, 2H), 3.66 - 3.59 (m, 1H), 3.57 - 3.49 (m, 1H), 3.47 -
3.37 (m, 2H), 3.35 - 3.16 (m, 3H), 2.95 (t, J = 5.4 Hz, 2H), 2.87 - 2.77 (m, 1H), 2.71 (d, J = 5.2 Hz,
1H), 2.69 - 2.65 (m, 2H), 2.64 - 2.54 (m, 2H), 2.49 (s, 3H), 2.22 (s, 1H), 1.49 - 1.39 (m, 2H), 1.34 -
1.25 (m, 3H), 0.87 (t, J = 7.6 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 170.35, 168.75, 161.67, 151.72,
136.57, 133.37, 133.26, 129.35, 129.06, 124.77, 120.66, 54.61, 54.12, 52.24, 50.54, 49.80, 49.74, 49.21,
45.58, 45.00, 41.50, 31.94, 27.33, 20.39, 13.95. HRMS: m/z calculated for C27H35ClN6OS [M + Na]+:
549.2179, found: 549.2170.
N-(2-(4-chlorophenyl)-3-(4-(7-methyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidin-4
-yl)piperazin-1-yl)but-3-en-1-yl)cyclopentanamine (5i) as a white solid, m.p. 92.2-94.3 °C, yield 79%.
1H NMR (400 MHz, CDCl3) δ 8.47 (s, 1H), 7.29 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 8.4 Hz, 2H), 4.04 (dd,
J = 8.8, 5.2 Hz, 1H), 3.93 - 3.85 (m, 1H), 3.69 (d, J = 2.2 Hz, 2H), 3.67 - 3.57 (m, 1H), 3.57 - 3.48 (m,
1H), 3.47 - 3.38 (m, 2H), 3.34 - 3.17 (m, 3H), 3.09 - 3.01 (m, 1H), 2.98 - 2.92 (m, 2H), 2.86 - 2.76 (m,
1H), 2.71 - 2.64 (m, 3H), 2.50 (s, 3H), 2.33 (br s, 1H), 1.87 - 1.77 (m, 2H), 1.71 - 1.57 (m, 2H), 1.55 -
1.44 (m, 2H), 1.35 - 1.26 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 170.37, 168.75, 161.67, 151.72,
136.67, 133.36, 133.25, 129.36, 129.07, 124.77, 120.65, 60.02, 54.62, 52.95, 52.24, 50.52, 49.79, 49.39,
45.58, 45.00, 41.50, 32.99, 32.79, 27.33, 24.05, 24.01. HRMS: m/z calculated for C28H35ClN6OS [M +
Na]+ 561.2179, found: 561.2177.

2-(4-chlorophenyl)-3-(cyclohexylamino)-1-(4-(7-methyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[
2,3-d]pyrimidin-4-yl)piperazin-1-yl)propan-1-one (5j) as a white solid, m.p. 84.9-86.2 °C, yield 81%.
1H NMR (400 MHz, CDCl3) δ 8.50 (s, 1H), 7.31 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 8.4 Hz, 2H), 4.06 (dd,
J = 8.8, 5.2 Hz, 1H), 3.97 - 3.87 (m, 1H), 3.72 (s, 2H), 3.69 - 3.61 (m, 1H), 3.60 - 3.51 (m, 1H), 3.50 -
3.40 (m, 2H), 3.38 - 3.28 (m, 2H), 3.27 - 3.18 (m, 1H), 2.98 (s, 2H), 2.88 - 2.74 (m, 3H), 2.69 (t, J =
5.2 Hz, 2H), 2.52 (s, 3H), 2.49 - 2.42 (m, 1H), 1.94 - 1.82 (m, 2H), 1.76 - 1.66 (m, 2H), 1.64 - 1.56 (m,
1H), 1.24 - 1.21 (m, 1H), 1.20 - 1.12 (m, 2H), 1.12 - 1.01 (m, 2H). 13C NMR (100 MHz, CDCl3) δ
170.35, 168.74, 161.66, 151.71, 136.60, 133.38, 133.24, 129.34, 129.06, 124.77, 120.64, 56.97, 54.61,
52.24, 51.13, 50.50, 49.78, 49.35, 45.57, 44.99, 41.50, 33.21, 33.13, 27.33, 26.00, 24.95, 24.93. HRMS:
m/z calculated for C29H37ClN6OS [M + Na]+: 575.2336, found: 575.2328.
2-(4-fluorophenyl)-3-(isopropylamino)-1-(4-(7-methyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2
,3-d]pyrimidin-4-yl)piperazin-1-yl)propan-1-one (5k) as a white solid, m.p. 108.8-110.0 °C, yield 65%.
1H NMR (400 MHz, CDCl3) δ 8.48 (s, 1H), 7.28 (dd, J = 8.8, 5.4 Hz, 2H), 7.05 (t, J = 8.6 Hz, 2H),
4.42 (br s, 1H), 4.34 (dd, J = 9.2, 4.4 Hz, 1H), 3.99 - 3.89 (m, 1H), 3.72 (s, 2H), 3.69 - 3.61 (m, 1H),
3.60 - 3.52 (m, 1H), 3.52 - 3.41 (m, 2H), 3.36 (dd, J = 11.2, 9.2 Hz, 2H), 3.27 - 3.18 (m, 1H), 3.07 -
3.00 (m, 1H), 2.99 (t, J = 5.6 Hz, 2H), 2.88 (dd, J = 11.6, 4.8 Hz, 1H), 2.84 - 2.87 (m, 1H), 2.69 (t, J =
5.6 Hz, 2H), 2.52 (s, 3H), 1.21 (d, J = 6.4 Hz, 3H), 1.20 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 170.26, 168.70, 162.15(d, JCF = 245.7 Hz), 161.63, 151.68, 133.24, 133.07(d, JCF = 3.2 Hz),
129.36(d, JCF = 7.9 Hz), 124.78, 120.64, 116.33(d, JCF = 21.4 Hz), 54.59, 52.21, 50.73, 50.37, 49.71,
49.67, 47.95, 45.55, 45.02, 41.58, 27.29, 21.61, 21.55. HRMS: m/z calculated for C26H33FN6OS [M +
Na]+: 519.2318, found: 519.2311.