Discovery of pyrazole-thiophene derivatives as highly Potent, orally active Akt inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.07.017

A series of pyrazole-thiophene derivatives exhibiting good Akt inhibitory activities were obtained on the basis of conformational restriction strategy, leading to the discovery of compound 1d and 1o which showed excellent in vitro antitumor effect against a variety of hematologic cancer cells and their potential of inducing apoptosis, blocking the cell cycles at S phase and significantly inhibiting the phosphorylation of downstream biomarkers of Akt kinase of cancer cells. Amongst, compound 1o also exhibited good PK profiles and inhibited about 40% tumor growth in MM1S xenograft model. Compound 1o might be a potential candidate for further development.

Full text of DOI:10.1016/j.ejmech.2019.07.017

Accepted Manuscript
Discovery of pyrazole-thiophene derivatives as highly Potent, orally active Akt
inhibitors
Wenhu Zhan, Jinxin Che, Lei Xu, Yizhe Wu, Xiaobei Hu, Yubo Zhou, Gang Cheng,
Yongzhou Hu, Xiaowu Dong, Jia Li
PII:
S0223-5234(19)30638-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.07.017
EJMECH 11514
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 May 2019
Revised Date: 5 July 2019
Accepted Date: 5 July 2019
Please cite this article as: W. Zhan, J. Che, L. Xu, Y. Wu, X. Hu, Y. Zhou, G. Cheng, Y. Hu, X. Dong, J.
Li, Discovery of pyrazole-thiophene derivatives as highly Potent, orally active Akt inhibitors, European
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.07.017.
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ACCEPTED MANUSCRIPT

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Discovery of Pyrazole-thiophene Derivatives as Highly Potent, Orally
Active Akt Inhibitors
Wenhu Zhan,a, ‡, # Jinxin Che,a, ‡ Lei Xu,b Yizhe Wu,a Xiaobei Hu,b Yubo Zhou,b Gang Cheng,c
Yongzhou Hu,a Xiaowu Dong,a,* Jia Lib,*
a
ZJU-ENS Joint Laboratory of Medicinal Chemistry, Zhejiang Province Key Laboratory of
Anti-Cancer Drug Research, Hangzhou Institute of Innovative Medicine, College of Pharmaceutical
Sciences, Zhejiang University, Hangzhou, 310058, P.R. China
b
National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, P.R. China
c College of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou, 311402, P.R.
China
‡ These authors contributed equally to this work.
# Present address: Department of Microbiology & Immunology, Weill Cornell Medicine, 1300 York
Avenue, New York, NY 10065, United States.
* Corresponding authors: X. Dong: dongxw@zju.edu.cn; J. Li: jli@simm.ac.cn
Abstract:
A series of pyrazole-thiophene derivatives exhibiting good Akt inhibitory activities were obtained
on the basis of conformational restriction strategy, leading to the discovery of compound 1d and 1o
which showed excellent in vitro antitumor effect against a variety of hematologic cancer cells and
their potential of inducing apoptosis, blocking the cell cycles at S phase and significantly inhibiting
the phosphorylation of downstream biomarkers of Akt kinase of cancer cells. Amongst, compound
1o also exhibited good PK profiles and inhibited about 40% tumor growth in MM1S xenograft
model. Compound 1o might be a potential candidate for further development.
Keywords: pyrazole-thiophene derivatives; Akt inhibitor; antitumor
1. Introduction

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Serine/threonine kinase Akt (also known as protein kinase B or PKB) has become a major focus
of attention due to its essential role in diverse cellular processes.[1] As we known, PI3K/Akt
signaling pathway is abnormal in cancer cells and plays a pivotal role in neoplastic
transformation.[2, 3] Akt is considered to be a key mediator of the PI3K pathway, and the inhibition
of Akt can decrease the tumor growth effectively.[4-9] So far, a number of Akt inhibitors have been
under clinical evaluation (Figure 1), demonstrating its potential value for cancer therapy.
In particular, a phase III clinical trial is ongoing to investigate the effectiveness of GDC0068 in
the treatment of triple-negative breast cancer (TNBC) (NCT03337724). Besides, AZD5363 in
combination with paclitaxel for the treatment of TNBC also provide a positive outcome in a phase
II clinical trial.[10] GSK2141795 (GSK-795 or Uprosertib) and GSK2110183 (GSK-183 or
Afuresertib) are ATP-competitive Akt inhibitors developed by the GSK company, which are
currently in clinical trials for the evaluation of its therapeutic effect against different types of cancer,
such as melanoma, breast cancer, etc. Thus, it is fully demonstrated that the promising prospect of
Akt inhibitors as anti-cancer agents.
Figure 1. Chemical structures of representative Akt inhibitors.
Conformational restriction is a common methodology for drug design, which has been widely
applied in drug discovery in a variety of diseases,[11, 12] including cancer and Alzheimer’s disease,
as well as in the development of antibacterial or antifungal agents. In our previous studies,[13-17] a
series of 4,4-disubstituted or 3,4-disubstituted/3,4,6-trisubstituted (unpublished data) piperidine
derivatives featuring more restricted conformation in contrast to GSK-795 were rationally designed
and synthesized, exhibiting excellent Akt inhibitory activities and good in vitro or in vivo properties.
Thus, we thought that conformational restriction strategy may also be suitable to be applied in
GSK-183. Herein, as a continuing effort, compound A bearing (3S,4S) piperidine skeleton was

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synthesized as shown in Figure 2. Since the synthetic method we used [18] gave trace (3S,4R)- or
(3R,4S)-product, and we have demonstrated that (3S, 4S)-product was more favorable for binding
with Akt1 than that of (3R,4R)-product (unpublished data), the (3S, 4S)-configuration was chosen
for further study. Whereas, the Akt inhibitory activity of A decreased dramatically, exhibiting an
IC50 value of 102 nM. Following research indicated that different link mode of thiophen ring can
upregulate the Akt inhibitory potential of this series compound. Furthermore, the antitumor
mechanism, PK profiles, in vitro or in vivo antitumor activities of the most promising compound 1d
and 1o were studied.
Figure 2. Design rationale based on the structure of GSK-183.
2. Results and discussion
2.1 Chemistry
Synthesis of biaryl carboxylic acids and 3,4-disubstituted piperidines. The synthetic route for
the preparation of the acid fragments was similar to the literature conditions (patent:
WO2008098104, Figure S1). The synthesis of 3,4-disubstituted piperidine derivatives was shown
in Scheme 1, compounds 11a-c underwent S Jorgensen-Hayashi reagent catalyzed cyclization [18]
with compound 12 to provide the (3S,4S) compound 13a-c. Treatment of the compounds with
Et3SiH, and following in the presence of Fe/NH4Cl provided 3-amino-4-aryl piperidine (15a-c).

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Scheme 1. (a) (S)-2-(diphenyl((trimethylsilyl)oxy)methyl)pyrrolidine, dichloromethane; ii. TFA,
dichloromethane; (b) i. EtSiH, TFA; ii. Boc2O, TEA, dichloromethane; (g) Fe, NH4Cl, EtOH/H2O.
Synthesis of target compounds. The synthetic route for the preparation of the target compounds
was shown in Scheme 2. Treatment of the acids with the amines in the presence of EDCI and HOBt
provided the condensed intermediates. Then, deprotection with HCl in ethyl acetate yielded the
target compounds 1a-f and 1n-p.
Scheme 2. (a) EDCI, HOBt, DIPEA; (b) EA, HCl.
Condensation
of
pyrazol-thiophene
carboxylic
acid
10c
and
(3S,
4S)-N-Boc-3-amino-4-arylpiperidine 15a afforded intermediate 16 which subsequently coupled
with different boronic acids and de-Boc protected to give the title compound 1g, 1l and 1m. In
addition, the intermediate 16 can be coupled with an ethylene boronic anhydride pyridine complex
to obtain an ethylene intermediate 17 which then deprotected to remove Boc to obtain compound 1h,
or reduced with H2 to obtain compound 1i. Compound 18 was obtained by oxidizing compound 17
with OsO4/NaIO4/NMO system. Then further oxidized and deprotected to get compound 1j, or
reduced and deprotected to get compound 1k.

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Scheme 3. (a) EDCI, HOBt, DIPEA; (b) substituted boronic acids or vinylboronic anhydride
pyridine complex, K2CO3, Pd2(dba)3, dioxane; (c) EA, HCl; (d) H2, Pd/C, MeOH; (e) OsO4/NaIO4,
NMO, THF/H2O; (f) RuCl3, NaIO4, CH3CN/H2O; (g) NaBH4, THF/MeOH.
2.2 In vitro activity evaluation
We investigated the 2,5-substituted thiophene linkage at first. As shown in Table 1, replacement
of the hydrogen of R1 group with chlorin resulted in a 10-folds increasement of potency (1a vs 1b,
1c vs 1d). Whereas, after introducing bromine to R1 (compound 1e), a loss of inhibitory activity
was observed. The Akt inhibitory activity of compounds with 4-Cl or 3-F substituent on the phenyl
of 4-piperidine performed better than that of compounds with 4-Cl-3-CF3 substituent (1b or 1d vs
1f). Hence, 3-F substituent was chosen as the privileged group for further exploration. Other groups
on 4-pyrazole (R1) were also investigated, the IC50 value could still remain below 10 nM unless the
size of the substituent was small such as methyl group (1g). Introducing other large groups such as
vinyl (1h), ethyl (1i), carboxyl (1j), hydroxymethyl (1k), phenyl (1l) or 4-pyridyl (1m) would lead

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to a dramatically loss of Akt1 inhibitory potency of compounds, indicating that the tolerance of this
position was limited. Besides, proliferation inhibitory activities of compounds against OVCAR-8
and HCT116 cells were also evaluated. Most of the compounds exhibiting better Akt1 inhibitory
potency performed better anti-proliferation activity against OVCAR-8 and HCT116 cells.
Compounds with R1 of acid or basic group, the anti-proliferation activity decreased dramatically
such as compounds 1j and 1m, which IC50 values were > 20 µM.
Table 1. Akt1 enzymatic and cancer cell line activities for compounds 1a-1m.
IC50 (µM)a
Akt1
Compd.
R1
R2
IC50 (nM)
10.2
1.8
OVCAR-8
1.39
HCT116
8.87
3.02
1.37
0.44
0.89
1.41
1.78
6.85
2.83
>50
H
Cl
4-Cl
4-Cl
3-F
1a
1.47
1b
H
10.6
1.0
1.59
1c
Cl
3-F
0.23
1d
Br
3-F
5.46
65.2
9.0
NTb
1e
Cl
4-Cl-3-CF3
3-F
0.64
1f
Me
vinyl
Et
1.74
1g
3-F
46.9
48.9
101.0
62.2
70.4
99.9
9.6
3.19
1h
3-F
2.70
1i
carboxyl
CH2OH
Ph
3-F
>50
1j
3-F
7.17
7.42
4.42
24.01
1.75
NTb
1k
1l
3-F
2.51
4-Py
-
3-F
27.99
0.54
1m
-
GSK2141795
GSK2110183
-
-
16.9
NTb
a. The IC50 values are an average of three independent determinations.
b. NT =Not Tested.

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Furthermore, the compounds with 2,4-substituted thiophene linkage were also tested. As shown
in Table 2, similar to the SAR of compounds with 2,5-substituted thiophene linkage, 3-F
substituents (1o) showed better activity than those of 4-Cl or 4-Cl-3-CF3 substituents (1n or 1p),
respectively. The anti-proliferation activity of compound 1o was better than those of 1n and 1p,
respectively. Amongst, compound 1d and 1o exhibited better Akt inhibitory or anti-proliferation
activity than that of the positive GSK2141795 and GSK2110183.
Table 2. Akt1 enzymatic and cancer cell line activities for compounds 1n-1p.
IC50 (µM)a
Akt1
Compd.
R1
IC50 (nM)
13.7
OVCAR-8
5.69
HCT116
1.21
4-Cl
1n
1o
3-F
1.26
NTb
0.95
4-Cl-3-CF3
35.3
0.58
1.85
1p
-
-
9.6
0.54
1.75
GSK2141795
GSK2110183
16.9
NTb
NTb
a. The IC50 values are an average of three independent determinations.
b. NT =Not Tested.
2.3 In vitro anti-proliferation activity
The activity of compound 1d or 1o was measured by its ability to inhibit growth of a panel of 11
cell lines derived from hematologic tumors, by a standard proliferation assay. As shown in Table 3,
compound 1o showed better inhibitory potency against MM1S, RMPI8226, CEM-C1and MOLT-4
cell line than compound 1d. Among the cell lines, MM1S, CEM-C1 and CCRF-CEM exhibited
highly sensitive to compound 1o, which IC50 values were < 10 nM. Whereas, both of the
compounds performed the lowest sensitivity against K562 cell line. Moreover, compound 1d and 1o
showed better anti-proliferation activity than that of GSK2141795 against some cell lines such as

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MM1S, RMPI8226, MV4-11 et al.
Table 3. hematologic cancer cell line activities for compounds 1d and 1o.
IC50 (nM)a
Cell lines
1d
21
1o
2
GSK2141795
32
538
30
MM1S
RMPI8226
CEM-C1
MOLT-4
MV4-11
HUT78
JVM-2
263
28
49
7
50
14
66
135
298
208
78
441
NTb
NTb
188
8
635
378
293
101
NTb
NTb
>3000
U937
NTb
NTb
> 3000
CCRF-CEM
Mino
531
> 3000
K562
a. The IC50 values are an average of three independent determinations.
b. NT =Not Tested.
2.4 Compound 1d and 1o induce apoptosis and cell cycles blockage of cancer cells
To determine whether the antiproliferative activity of 1d and 1o on colon cancer cell (HCT116)
proliferation was accompanied by enhanced cancer cell apoptosis, the percentages of apoptotic cells
were tested by conducting a flow cytometry assay. The results indicated that compound 1d and 1o
both had an apoptosis-inducing effect in a dose-dependent manner, with 89.62 % and 24.58 % of
apoptotic cells were detected at the concentration of 10 µM respectively (Figure 3A and B).
Compound 1o showed weaker apoptosis-inducing effect than 1d. Besides, western blot analysis was
further performed to test the Akt inhibitory ability of compound 1d and 1o in tumor cells. The
results demonstrated that, upon treatment with the inhibitor E22 in vitro, a dose-dependent
significant reduction in the phosphorylation of GSK3β and PRAS40 was observed in HCT116 cells.
Consistent with other ATP-competitive Akt kinase depressors,[19-21] both of 1d and 1o showed a
concentration-dependent feedback increase in Akt phosphorylation (Figure 3C). In addition, it was

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further demonstrated that compound 1d and 1o can also arrest cell cycle progression of HCT116
cells into the S phase staring from a concentration of 0.4 µM (Figure 4A and B).
Figure 3. Compound 1d and 1o induce apoptosis in vitro. (A) Apoptosis was assessed upon
treatment with the indicated concentrations (0.4-10 µM) by flow cytometry using the FL1-H
channel (Annexin-V) and FL2-H channel (PI) of a Becton Dickinson FACS Calibur. Representative
dot plots are shown, and the results are summarized in (B). (C) Western blot analysis of total AKT,
p-AKT, PRAS40, p-PRAS40, GSK3β and p- GSK3β in the colon cancer cell line HCT116. Results
are presented as the mean ± SD from three independent experiments.

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Figure 4. Compound 1d (A) and 1o (B) arrested cell cycle progression in HCT116 cells. Results are
presented as the mean ± SD from three independent experiments.
2.5 Pharmacokinetic study
On the basis of Akt inhibitory and anti-cancer cell proliferation potency, compound 1o was
chosen to further test its pharmacokinetic properties. As shown in Table 4, after 10 mg/kg oral
dosing (compound was dissolved in saline) in rats, a good absorption and exposure were observed,
which t1/2, Cmax and AUC0-t values were 2.75 h, 423 µg/L and 3886 µg /L·h respectively. The
bioavailability of 1o was 52.5 %, which was favorable for in vivo efficacy study. Furthermore,
compound 1o also exhibited moderate hERG inhibition (40 % @ 3 µM). The in vitro stability of 1o
was also evaluated in liver microsomes and plasma. The stability of compound 1o was good in the
plasma, but showed less stable in the liver microsomes of rat.
Table 4. Pharmacokinetic profile of compound 1o
1o
Parameters
p.o.
i.v.
(10mg/kg)
(2mg/kg)

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2.75
4.74
0.083
3.52
Tmax (h)
T1/2 (h)
423.0
3886.0
3940.5
1439.0
1481.1
1507.7
Cmax (µg/L)
AUC0-t (µg /L·h)
AUC0-∞(µg/L·h)
52.5
40.0
F(%)
hERG inhibition rate (%, @ 3 µM)
Liver microsome stability (%, remaining percent @1.5 h)
[mouse, rat, dog, human]
82.7, 63.7, 81.3, 88.3
> 99, > 99
Plasma stability (%, remaining percent @1.5 h) [mouse, human]
2.6 In vivo xenograft tumor inhibition
The in vivo anti-tumor activity of compound 1o was determined in the MM1S xenografts model
in nude mice. After 14 days treatment with compound 1o (15 or 30 mg/kg, p.o., q.d.), body weight
loss of mice was observed (Figure 5A). In addition, both dosages showed remarkable tumor growth
inhibition and tumor weight control (Figure 5B, C and D). The tumor growth inhibition rate of
compound 1o at the dose of 15 mg/kg was comparable to that of 30 mg/kg (40.70% vs. 42.07%
inhibition respectively), and slightly better than that of GSK2141795 (36.77% inhibition),
demonstrating a suitable point for further development.

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Figure 5. Compound 1o develops an anti-tumor effect through inhibiting the kinase activity of Akt.
(A, B, C and D) The mice transplanted with MM1S xenografts were randomly divided into four
groups (N = 5 mice/group) and gave an injection of 1o at 15 or 30 mg/kg and GSK2141795 at 30
mg/kg or vehicle daily for a period of 14 days. The mice body weight (A), tumor volume (B), tumor
weight (C) and tumor image (D) were recorded. Data represent the mean ± SD.
2.7 Molecular docking
In order to explore the possible binding mode of compound 1o with Akt protein, computational
docking and molecular dynamics were performed. First, the complex of 4GV1 (PDB code) [22]
with 1o was simulated for 20 ns. The RMSD value of the protein backbone was calculated in a
trajectory from 0 to 20 ns. As shown in Figure 6A, after about 500 ps of simulation, the RMSD
became stable, indicating that dynamic equilibrium was attained. The amino acid residues such as
Val164, Met227, Ala230 and Thr291 contributed most for the binding between Akt protein and 1o
(Figure 6B). The hydrogen bond distance between 1o and Asp292 indicated that the system reached
equilibrium conditions (Figure 6C). Based on the relative stable conformation, 1o was hydrogen
bonded with the Akt residue Ala230 and Asp292. Besides, the 4-phenyl group inserted into the

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hydrophobic pocket formed by the residues of Gly162, Val164, Lys179 and Leu181 (Figure 6D).
Figure 6. MD simulations and analysis of the interaction of compound E22 with 4GV1. (A) RMSD
of Akt backbone during the 20 ns simulation time; (B) Plot of the inhibitor-residue interaction
spectrum of the two hydrogen bonds (purple and red) formed between the amide-NH of 1o and the
Asp-292 amino acid residue; (C) Distance plot of Asp292 with 1o; (D) 3D plot of the binding
pattern.
3. Conclusion
Based on the conformational restriction strategy, the activities of compounds with thiophene
linkage were explored, leading to the discovery of compound 1d and 1o. Both of the compounds
inhibited a variety of hematologic cancer cells and showed their potential of inducing apoptosis and
blocking the cell cycles at S phase of cancer cells. Compound 1o exhibited excellent PK profiles
and in vivo tumor inhibition activity, demonstrating 1o would be a promising candidate for further
development.
4. Materials and Methods

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4.1 Chemistry
1H NMR and 13C NMR spectra were recorded at 500 MHz using a Bruker AVANCE III
spectrometer in CDCl3, or DMSO-d6 solution, with tetramethylsilane (TMS) serving as internal
standard. Chemical shift values (δ) were reported in ppm. Multiplicities are recorded by the
following abbreviations: s, singlet; d, double; t, triplet; q, quartet; m, multiplet; J, coupling constant
(Hz). High resolution mass spectrum (HRMS) was obtained from Agilent Technologies 6224 TOF
LC/MS. The purities of compounds for biological testing were assessed by NMR and HPLC, and
the purities were ≥95 %. The analytical HPLC was performed on a Agilent 1260 Infinity II (LC03)
machine and a C18 reversed-phase column (Agilent Eclipse XDB-C18, 4.6*250 mm, 5 µm), with a
flow rate of 1.0 mL/min, the detection by UV absorbance at a wavelength of 254 nm, the column
temperature was 25 , eluting with water (0.1% trifluoroacetic acid) as A phase and methanol as B
phase (0 min, A phase: 90%, B phase: 10%; 8 min, A phase: 10%, B phase: 90%; 13 min, A phase:
10%, B phase: 90%; 15 min, A phase: 90%, B phase: 10%; 20 min, A phase: 90%, B phase: 10%).
The melting point was recorded from Büchi M-560. The specific optical rotation was obtained from
o
an Insmark IP-cligi 300 machine, with a temperature of 20 C, the detection wavelength was 589
nm, the compound concentration was 0.1 g/100 mL and the length of the polarization tube was 100
mm. The optical data was an average of three independent determinations. Unless otherwise noted,
reagents and solvents were obtained from commercial suppliers and without further purification.
General procedure A: (for the synthesis of compounds 3 and 9) Tetrakistriphenylphosphane Pd (0)
(3.45 g, 3 mmol) was added to a stirred suspension of 4-halogen substituted aromatic ester or
aldehyde (30 mmol), 1-methyl-5-(4,4,5,5-tetramethyl-[1,3,2] dioxaborolan-2-yl)-1H-pyrazole (7.5 g,
36 mmol) and potassium phosphate (12g, 45 mmol) in dimethylformamide (100 mL) at 0 °C under
nitrogen protection. The reaction mixture was heated at 100 °C for 10 h, then poured into H2O (300
mL) and extracted with ethyl acetate (100 mL × 3). The combined organic layers were washed with
saturated brine (200 mL × 3), dried over Na2SO4, and concentrated under vacuum to afford the
crude product, then purified by column chromatography.
General procedure B: (for the synthesis of compounds 10a-c) To a solution of biaryl ester (10
mmol) in dry THF/DMF (v/v, 10: 1) at 0 °C was added NCS or NBS (11 mmol) in one portion. The
reaction mixture was stirred at r.t. for 5 h. After it was fully reacted, the mixture was poured into
H2O (50 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were
washed with saturated brine (50 mL × 2), dried over Na2SO4 and concentrated under vacuum. The

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residue was purified by column chromatography.
To a solution of the above intermediate products (8.8 mmol) in dry THF/EtOH (v/v, 4: 1) was
slowly added aqueous NaOH (3 N, 10 mL, 30 mmol) and the mixture was stirred at r.t. for 5 h.
After it was fully reacted, the solvent was removed under vacuum and the resulting crude mixture
was dissolved in H2O (30 mL) and acidified with 1 N hydrochloric acid until pH 2~3, and extracted
with ethyl acetate (30 mL × 3). The combined organic layers were washed with saturated brine,
dried over anhydrous Na2SO4, and then concentrated to dryness under vacuum to provide the white
solid.
General procedure C: (for the synthesis of compounds 13a-c) To a solution of compound 12 (1.5
mmol), (S)-2-(diphenyl((trimethylsilyl)oxy)methyl)pyrrolidine (33 mg, 0.1 mmol) and benzoicacid
(25 mg, 0.2 mmol) in dry dichloromethane (2 mL) was slowly added substituted cinnamyl aldehyde
11a-c (1 mmol) at 0 °C. After the addition was complete, the reaction was warmed up to room
temperature and stirred overnight. The stirring was continued until aldehyde was consumed
(monitored by TLC). The solution was diluted to 10 mL with dichloromethane and trifluoroacetic
acid (148 µL, 2 mmol) was added dropwise, then the reaction mixture was stirred for another 5 h.
The reaction was quenched by the dropping of 1N aqueous sodium bicarbonate solution (10 mL)
and stirred for another 10 min. Water phase was extracted with ethyl acetate (10 mL × 3), and the
combined organic layers were washed with saturated brine (20 mL × 2) dried over anhydrous
sodium sulfate and concentrated. Purification by flash column chromatography afforded yellow
solid.
General procedure D: (for the synthesis of compounds 14a-c) To a solution of 3,4-disubstituted
piperidine compound (5.4 mmol), triethylsilane (1.8 mL, 11 mmol) in dichloromethane (25 mL)
was added dropwise trifluoroacetic acid (3.7 mL, 50 mmol) at 0 °C. After the addition was complete,
the reaction was warmed up to room temperature and stirred overnight. Then the reaction was
quenched by the dropping of saturated aqueous sodium bicarbonate (50 mL) and stirred for another
10 min. Water phase was extracted with dichloromethane (20 mL × 3), and the combined organic
layers were washed with saturated brine (20 mL × 2) dried over anhydrous sodium sulfate and
concentrated in vacuo to give an oil. To a suspension of the residue and triethylamine (1.4 mL, 10
mmol) in tetrahydrofuran (45 mL), di-tert-butyl dicarbonate (3.7 mL, 50 mmol) was added in
batches at 0 °C. After the addition was complete, the reaction was warmed up to room temperature
and stirred for 5 h. After solvent removal, the residue was dissolved in ethyl acetate (50 mL),

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washed by 0.5N hydrochloric acid (20 mL × 2) and saturated brine (20 mL × 2) and dried over
anhydrous sodium sulphate. After solvent removal, the residue was purified by column
chromatography to afford product as a white solid.
General procedure E: (for the synthesis of compounds 15a-c) To a suspension of 3,4-disubstituted
piperidine compound (4.2 mmol) in ethyl alcohol - water (v/v, 3: 1, 40mL) was added iron powder
(2.82 g, 50.4 mmol) and ammonium chloride (1.0 g, 16.8 mmol), then the mixture was refluxed for
5 h with mechanical stirring. After it is fully reacted, the mixture was filtered and filtrate was
concentrated under vacuum. The residue was dissolved in ethyl acetate (50 mL), washed by
saturated aqueous sodium bicarbonate (20 mL × 2) and saturated brine (20 mL × 2) and dried over
anhydrous sodium sulphate. After solvent removal, the residue was dried in vacuum to give gray
solid.
General procedure F: (for the synthesis of compounds 1a-f, 1n-p and 16) To a suspension of the
acid (0.2 mmol), EDCI (69 mg, 0.36 mmol), HOBT (49 mg, 0.36 mmol) and DIPEA (87 µL, 0.5
mmol) in dichloromethane was added the amine (58.8 mg, 0.2 mmol), then the mixture was reacted
for 5 h at room temperature with mechanical stirring. After it was fully reacted, the mixture was
concentrated under vacuum. The residue was dissolved in ethyl acetate (50 mL), washed by 1N HCl
(20 mL × 2) and saturated brine (20 mL × 2) and dried over anhydrous sodium sulphate. After
solvent removal, the residue was purified by column chromatography to afford the product.
To a solution of the above product in ethyl acetate was added HCl saturated ethyl acetate, stirred
at room temperature for 6 h. After it was fully reacted, the mixture was filtered, the resulted solid
was washed with diethyl ether. The solid was dried in vacuum to afford target product as white
solid.
General procedure G: (for the synthesis of compounds 1g, 1l, 1m) To a suspension of substituted
boric acid (0.5 mmol) and K2CO3 (41 mg, 0.3 mmol) was added Pd2(dba)3 (10 mg, 0.01 mmol) and
10% tri-tert-butylphosphine pentane solution (60 µL, 0.03 mmol) in dioxane and H2O (v/v, 5:1).
o
The mixture was heated to 70 C and stirred overnight. After the reaction was completed, the
reaction solution was poured into hydrogen peroxide solution (10 mL), extracted with ethyl acetate
(10 mL × 3), and the combined organic layers were washed with saturated brine (20 mL × 2) dried
over anhydrous sodium sulfate and concentrated. Purification by flash column chromatography
afforded the product.
To a solution of the above product in ethyl acetate was added HCl saturated ethyl acetate, stirred

ACCEPTED MANUSCRIPT
at room temperature for 6 h. After it was fully reacted, the mixture was filtered, the resulted solid
was washed with diethyl ether. The solid was dried in vacuum to afford target product as white
solid.
After the SAR study, 16 compounds were obtained in total, the compound information of the
intermediates can be found in the Supporting Information.
4-(1-Methyl-1H-pyrazol-5-yl)thiophene-2-carbaldehyde (3)
General procedure A, Yield: 81%; 1H NMR (500 MHz, CDCl3) δ 9.98 (d, J = 1.1 Hz, 1H), 7.85 (d, J
= 1.1 Hz, 1H), 7.77 (s, 1H), 7.53 (d, J = 1.9 Hz, 1H), 6.39 (d, J = 1.9 Hz, 1H), 3.97 (s, 3H). ESI-MS:
m/z = 193 [M + H]+.
4-(4-Chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-carbaldehyde (4)
General procedure B, Yield: 90%; 1H NMR (500 MHz, CDCl3) δ 10.01 (d, J = 1.1 Hz, 1H), 7.92 (d,
J = 1.1 Hz, 1H), 7.90 (s, 1H), 7.52 (s, 1H), 3.91 (s, 3H). ESI-MS: m/z =227 [M + H]+.
4-(4-Chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxylic acid (5)
To a solution of compound 4 (2 mmol) in acetone(15 mL) was added KMnO4 (0.38 g, 2.4 mmol) in
o
batches at 0 C. The mixture was stirred at r.t. for 3 h. After it was fully reacted, the mixture was
filtered and the filtrate was concentrated under vacuum. The residue was purified by column
chromatography. Yield: 85%; 1H NMR (500 MHz, DMSO-d6) δ 13.35 (s, 1H), 8.20 (d, J = 1.5 Hz,
1H), 7.93 (d, J = 1.5 Hz, 1H), 7.64 (s, 1H), 3.84 (s, 3H). ESI-MS: m/z = 241 [M − H]−.
5-Bromothiophene-2-carboxylic acid (7)
To a solution of compound 6 (2 mmol) in acetone (15 mL) was added KMnO4 (0.38 g, 2.4 mmol) in
o
batches at 0 C. The mixture was stirred at r.t. for 3h. After it was fully reacted, the mixture was
filtrated and filtrate was concentrated under vacuum. The residue was purified by column
chromatography. Yield: 85%; 1H NMR (500 MHz, CDCl3) δ 7.64 (d, J = 4.5 Hz, 1H), 7.11 (d, J =
4.5 Hz, 1H). ESI-MS: m/z = 205 [M − H]−.
Methyl 5-bromothiophene-2-carboxylate (8)
To a solution of compound 7 (5.2 g, 25 mmol) in 50 mL anhydrous MeOH was slowly added SOCl2
(2.6 mL, 37.5 mmol) at 0 , then 1 mL anhydrous DMF was added and refluxed for 3 hours. After
the reaction was completed, the mixture was concentrated under vacuum. The residue was dissolved
in EtOAc and washed with saturated NaHCO3 solution (20 mL × 3), brine (20 mL × 3) and dried
over Na2SO4 and concentrated. Purification by flash column chromatography afforded the white
solid (5.0 g, 22.7 mmol). Yield: 91%; 1H NMR (500 MHz, CDCl3) δ 7.54 (d, J = 4.0 Hz, 1H), 7.07

ACCEPTED MANUSCRIPT
(d, J = 4.0 Hz, 1H), 3.87 (s, 3H). ESI-MS: m/z = 221 [M + H]+.
Methyl 5-(1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxylate (9)
General procedure A, Yield: 73%; 1H NMR (500 MHz, CDCl3) δ 7.78 (d, J = 3.9 Hz, 1H), 7.49 (d, J
= 1.8 Hz, 1H), 7.16 (d, J = 3.9 Hz, 1H), 6.47 (d, J = 1.8 Hz, 1H), 4.02 (s, 3H), 3.91 (s, 3H). ESI-MS:
m/z = 223 [M + H]+.
5-(1-Methyl-1H-pyrazol-5-yl)thiophene-2-carboxylic acid (10a)
General procedure B, Yield: 88%; 1H NMR (500 MHz, DMSO-d6) δ 13.28 (s, 1H), 7.74 (d, J = 3.9
Hz, 1H), 7.47 (d, J = 2.0 Hz, 1H), 7.44 (d, J = 3.9 Hz, 1H), 6.61 (d, J = 2.0 Hz, 1H), 3.96 (s, 3H).
ESI-MS: m/z = 207 [M − H]−.
5-(4-Chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxylic acid (10b)
General procedure B, Yield: 91%; 1H NMR (500 MHz, DMSO-d6) δ 13.44 (s, 1H), 7.83 (d, J = 3.9
Hz, 1H), 7.72 (s, 1H), 7.52 (d, J = 3.9 Hz, 1H), 3.92 (s, 3H). ESI-MS: m/z = 241 [M − H]−.
5-(4-Bromo-1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxylic acid (10c)
General procedure B, Yield: 87%; 1H NMR (500 MHz, DMSO-d6) δ 13.41 (s, 1H), 7.83 (d, J = 3.9
Hz, 1H), 7.71 (s, 1H), 7.50 (d, J = 3.9 Hz, 1H), 3.91 (s, 3H). ESI-MS: m/z = 285 [M − H]−.
Tert-butyl (3S,4S)-4-(3-fluorophenyl)-3-nitro-3,4-dihydropyridine-1(2H)-carboxylate (13a)
General procedure C, yield: 47%; Specific optical rotation: -181.015o; 1H NMR (500 MHz, CDCl3)
δ 7.35 – 7.28 (m, 1H), 7.19 – 6.93 (m, 4H), 4.93 and 4.82 (d, J = 4.3 Hz, and d, J = 5.3 Hz, 1H,
1:1.3 ratio due to amide rotamers), 4.69 – 4.59 (m, 1H), 4.26 – 4.22 (m, 1H), 4.17 – 4.06 (m, 1H),
3.92 and 3.99 (d, J = 12.5 Hz, and d, J = 12.2 Hz, 1H, 1:1.3 ratio due to amide rotamers), 1.52 (s,
13
9H). C NMR (125 MHz, CDCl3, 1:1.3 ratio due to amide rotamers) δ 163.20 (d, J = 246.3 Hz),
151.71 and 151.57, 142.42 (d, J = 6.6 Hz), 130.73 (d, J = 8.7 Hz), 126.67 and 126.98, 123.85 (d, J =
2.5 Hz), 115.23, 115.07, 103.48 and 103.07, 84.73, 82.54, 42.62 and 42.56, 42.32 and 41.56, 28.31.
ESI-MS: m/z = 345 [M + Na]+.
Tert-butyl (3S,4S)-4-(4-chlorophenyl)-3-nitro-3,4-dihydropyridine-1(2H)-carboxylate (13b)
General procedure C, yield: 55%; 1H NMR (500 MHz, CDCl3) δ 7.32 (d, J = 8.4 Hz, 2H), 7.18 (d, J
= 8.4 Hz, 2H), 7.14 and 6.99 (d, J = 7.5 Hz, and d, J = 7.6 Hz, 1H, 1:1.2 ratio due to amide
rotamers), 4.91 and 4.81 (d, J = 6.5 Hz, and d, J = 6.0 Hz, 1H, 1:1.2 ratio due to amide rotamers),
4.64 – 4.56 (m, 1H), 4.23 – 4.19 (m, 1H), 4.09 – 3.91 (m, 2H), 1.52 (s, 9H). ESI-MS: m/z = 361 [M
+ Na]+.
Tert-butyl

ACCEPTED MANUSCRIPT
(3S,4S)-4-(4-chloro-3-(trifluoromethyl)phenyl)-3-nitro-3,4-dihydropyridine-1(2H)-carboxylate
(13c)
General procedure C, yield: 72%; 1H NMR (500 MHz, CDCl3) δ 7.56 (d, J = 1.9 Hz, 1H), 7.49 (d, J
= 8.3 Hz, 1H), 7.36 (dd, J = 8.3, 1.9 Hz, 1H), 7.18 and 7.03 (d, J = 7.9 Hz, and d, J = 7.8 Hz, 1H,
1:1.2 ratio due to amide rotamers), 4.89 and 4.78 (d, J = 6.0 Hz, and d, J = 6.0 Hz, 1H, 1:1.2 ratio
due to amide rotamers), 4.66 – 4.58 (m, 1H), 4.29 – 4.26 (m, 1H), 4.11 – 3.97 (m, 2H), 1.53 (s, 9H).
ESI-MS: m/z = 429 [M + Na]+.
Tert-butyl (3S,4S)-4-(3-fluorophenyl)-3-nitropiperidine-1-carboxylate (14a)
1
General procedure D, yield: 85%; H NMR (500 MHz, CDCl3) δ 7.32 – 7.26 (m, 1H), 7.00 – 6.95
(m, 2H), 6.93 – 6.89 (m, 1H), 4.84 – 4.55 (m, 2H), 4.27 (m, 1H), 3.30 (td, J = 12.2, 4.1 Hz, 1H),
3.19 (m, 1H), 2.88 (m, 1H), 1.96 (d, J = 13.7 Hz, 1H), 1.79 – 1.73 (m, 1H), 1.50 (s, 9H). 13C NMR
(125 MHz, CDCl3) δ 163.13 (d, J = 246.3 Hz), 154.09, 141.62 (d, J = 7.5 Hz), 130.75 (d, J = 7.5
Hz), 123.03 (d, J = 2.5 Hz), 115.12 (d, J = 20.0 Hz), 114.40 (d, J = 21.3 Hz), 86.78, 81.29, 46.83,
44.35, 43.81, 31.80, 28.47.
Tert-butyl (3S,4S)-4-(4-chlorophenyl)-3-nitropiperidine-1-carboxylate(14b)
General procedure D, yield: 91%; 1H NMR (500 MHz, CDCl3) δ 7.29 (d, J = 8.4 Hz, 2H), 7.13 (d, J
= 8.4 Hz, 2H), 4.84 – 4.57 (m, 2H), 4.28 – 4.25 (m, 1H), 3.34 – 3.08 (m, 2H), 2.89 – 2.87 (m, 1H),
1.93 (d, J = 13.8 Hz, 1H), 1.81 – 1.70 (m, 1H), 1.49 (s, 9H). ESI-MS: m/z = 325 [M − CH3]+.
Tert-butyl
(3S,4S)-4-(4-chloro-3-(trifluoromethyl)phenyl)-3-nitropiperidine-1-carboxylate(14c)
General procedure D, yield: 92%; 1H NMR (500 MHz, CDCl3) δ 7.51 (d, J = 2.1 Hz, 1H), 7.46 (d, J
= 8.3 Hz, 1H), 7.32 (dd, J = 8.3, 2.1 Hz, 1H), 4.67 (dd, J = 10.6, 7.3 Hz, 1H), 4.31 – 4.28 (m, 1H),
3.78 – 3.71 (m, 1H), 3.38 – 3.31 (m, 1H), 3.29 – 3.07 (m, 1H), 2.90 – 2.87 (m, 1H), 1.95 (d, J =
13.5 Hz, 1H), 1.81 – 1.70 (m, 1H), 1.49 (s, 9H).
Tert-butyl (3S,4S)-3-amino-4-(3-fluorophenyl)piperidine-1-carboxylate(15a)
General procedure E, yield: 81%; 1H NMR (500 MHz, DMSO-d6) δ 7.38 (td, J = 7.9, 6.3 Hz, 1H),
7.23 (dd, J = 10.3, 2.0 Hz, 1H), 7.17 (d, J = 7.7 Hz, 1H), 7.10 – 7.06 (m, 1H), 4.37 (d, J = 10.6 Hz,
1H), 4.04 – 3.94 (m, 1H), 3.18 (dd, J = 10.4, 6.5 Hz, 1H), 2.90 – 2.61 (m, 3H), 1.79 – 1.70 (m, 1H),
1.67 – 1.57 (m, 1H), 1.43 (s, 9H). ESI-MS: m/z = 295 [M + H]+.
Tert-butyl (3S,4S)-3-amino-4-(4-chlorophenyl)piperidine-1-carboxylate(15b)
General procedure E, yield: 85%; ESI-MS: m/z = 311 [M + H]+.

ACCEPTED MANUSCRIPT
Tert-butyl
(3S,4S)-3-amino-4-(4-chloro-3-(trifluoromethyl)phenyl)piperidine-1-carboxylate(15c)
General procedure E, yield: 96%; ESI-MS: m/z = 379 [M + H]+.
Tert-butyl
(3S,4S)-3-(5-(4-bromo-1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxamido)-4-(3-fluorophenyl
)piperidine-1-carboxylate (16)
General procedure F, Yield: 82%; 1H NMR (500 MHz, CDCl3) δ 7.49 (s, 1H), 7.30 – 7.23 (m, 2H),
7.10 (d, J = 3.9 Hz, 1H), 7.04 (d, J = 7.8 Hz, 1H), 6.97 – 6.94 (m, 1H), 6.92 – 6.88 (m, 1H), 6.00 (br
s, 1H), 4.55 – 4.48 (m, 1H), 4.31 – 4.11 (m, 2H), 3.86 (s, 3H), 2.94 – 2.69 (m, 3H), 1.99 – 1.90 (m,
1H), 1.80 – 1.71 (m, 1H), 1.49 (s, 9H). ESI-MS: m/z = 585 [M + Na]+.
Tert-butyl
(3S,4S)-4-(3-fluorophenyl)-3-(5-(1-methyl-4-vinyl-1H-pyrazol-5-yl)thiophene-2-carboxamido)
piperidine-1-carboxylate (17)
Tris(dibenzylideneacetone)dipalladium
(Pd2(dba)3,10
mg,
0.01
mmol)
and
10%
tri-tert-butylphosphine pentane solution (60 µL, 0.03 mmol) were dissolved in 1 mL anhydrous
DMF. After stirring at room temperature for 3 h, a mixed solution (5 mL) of dioxane and H2O (v/v,
5:1) was added slowly under N2 protection. To the above solution was added compound 16 (56.3
mg, 0.1 mmol), vinylboronic anhydride pyridine complex (120 mg, 0.5 mmol) and K2CO3 (41 mg,
0.3 mmol) at 0 , and then heated to 70 for 12 h. After the reaction was completed, the mixture
was poured into hydrogen peroxide (10 mL), and then extracted with ethyl acetate (10 mL × 3), the
combined organic layers were washed with saturated brine (20 mL × 2), dried over anhydrous
sodium sulfate and concentrated. Purification by flash column chromatography (petroleum ether:
1
EtOAc = 3:1-1:1) afforded the product. Yield: 83%; H NMR (500 MHz, CDCl3) δ 7.69 (s, 1H),
7.33 – 7.27 (m, 2H), 7.06 (d, J = 8.0 Hz, 1H), 6.99 – 6.90 (m, 3H), 6.42 (dd, J = 17.6, 11.1 Hz, 1H),
5.68 (s, 1H), 5.51 (dd, J = 17.6, 1.2 Hz, 1H), 5.09 (dd, J = 11.1, 1.2 Hz, 1H), 4.53 (d, J = 9.7 Hz,
1H), 4.37 – 4.10 (m, 2H), 3.82 (s, 3H), 2.87 – 2.71 (m, 3H), 2.01 – 1.90 (m, 1H), 1.82 – 1.74 (m,
1H), 1.51 (s, 9H). ESI-MS: m/z = 533 [M + Na]+.
Tert-butyl
(3S,4S)-4-(3-fluorophenyl)-3-(5-(4-formyl-1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxamido
)piperidine-1-carboxylate(18)
Compound 17 (102 mg, 0.2 mmol) was dissolved in a mixture of 10 mL THF and H2O (v/v, 3:1) on

ACCEPTED MANUSCRIPT
the ice bath. Osmium tetroxide (5 mg) and N-methylmorpholine oxide (47 mg, 0.4 mmol) were
added, and then stirred at room temperature overnight. NaIO4 (107 mg, 0.5 mmol) was added to the
reaction and stirred for 3 hours at room temperature. After the reaction was completed, the reaction
liquid was poured into the saturated Na2S2O3 solution (20 mL). Extracted with ethyl acetate (10 mL
× 3), the combined organic layers were washed with saturated brine (20 mL × 2), dried over
anhydrous sodium sulfate and concentrated. Purification by flash column chromatography afforded
the white solids (94 mg, 0.184 mmol). Yield: 92%; 1H NMR (500 MHz, CDCl3) δ 9.72 (s, 1H), 8.02
(s, 1H), 7.32 – 7.27 (m, 2H), 7.18 (d, J = 3.8 Hz, 1H), 7.05 (d, J = 7.2 Hz, 1H), 7.00 – 6.91 (m, 2H),
5.76 (br s, 1H), 4.57 – 4.50 (m, 1H), 4.34 – 4.14 (m, 2H), 3.89 (s, 3H), 2.94 – 2.71 (m, 3H), 1.98 –
1.91 (m, 1H), 1.85 – 1.75 (m, 1H), 1.51 (s, 9H). ESI-MS: m/z = 535 [M + Na]+.
N-((3S,4S)-4-(4-chlorophenyl)piperidin-3-yl)-5-(1-methyl-1H-pyrazol-5-yl)thiophene-2-carbox
amide hydrochloride (1a)
1
General procedure F, yield: 72%; Retention time: 9.373 min, purity: 96.62 %; H NMR (500 MHz,
DMSO-d6) δ 9.55 (d, J = 9.3 Hz, 1H), 9.46 (d, J = 10.5 Hz, 1H), 9.04 (d, J = 8.9 Hz, 1H), 7.89 (d, J
= 3.9 Hz, 1H), 7.45 (d, J = 1.9 Hz, 1H), 7.37 – 7.29 (m, 5H), 6.53 (d, J = 1.9 Hz, 1H), 4.54 – 4.45
(m, 1H), 3.92 (s, 3H), 3.36 (dd, J = 9.2, 4.7 Hz, 2H), 3.23 – 3.18 (m, 1H), 2.99 – 2.92 (m, 2H), 2.11
13
– 1.99 (m, 1H), 1.97 – 1.92 (m, 1H). C NMR (125 MHz, DMSO-d6) δ 160.14, 140.86, 139.43,
138.13, 135.36, 134.94, 131.27, 129.43, 129.27, 128.33, 127.46, 106.85, 47.39, 46.41, 43.93, 43.25,
38.08, 29.31. ESI-MS: m/z = 401 [M + H]+. HRMS (ESI) (m/z): calcd for C20H22ClN4OS [M + H]+
401.1197, found 401.1212.
5-(4-Chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(4-chlorophenyl)piperidin-3-yl)thiophene
-2-carboxamide hydrochloride (1b)
General procedure F, yield: 80%; 1H NMR (500 MHz, DMSO-d6) δ 9.45 (d, J = 9.5 Hz, 1H), 9.36
(d, J = 10.3 Hz, 1H), 9.04 (d, J = 8.9 Hz, 1H), 7.92 (d, J = 3.9 Hz, 1H), 7.66 (s, 1H), 7.40 (d, J = 3.9
Hz, 1H), 7.36 – 7.28 (m, 5H), 4.57 – 4.49 (m, 1H), 3.86 (s, 3H), 3.36 (dd, J = 14.0, 7.0 Hz, 2H),
3.21 (td, J = 11.7, 4.0 Hz, 1H), 2.97 (q, J = 10.9 Hz, 2H), 2.09 – 1.94 (m, 2H). 13C NMR (125 MHz,
DMSO-d6) δ 160.03, 141.13, 140.76, 136.67, 132.09, 131.27, 130.97, 129.97, 129.35, 128.75,
128.31, 108.49, 47.37, 46.35, 43.85, 43.24, 38.96, 29.37. ESI-MS: m/z = 435 [M + H]+. HRMS (ESI)
(m/z): calcd for C20H21Cl2N4OS [M + H]+ 435.0808, found 435.0828.
N-((3S,4S)-4-(3-fluorophenyl)piperidin-3-yl)-5-(1-methyl-1H-pyrazol-5-yl)thiophene-2-carbox
amide hydrochlorid (1c)

ACCEPTED MANUSCRIPT
General procedure F, yield: 54%; Retention time: 9.191 min, purity: 96.23 %; Melting point:
206~208 oC; 1H NMR (500 MHz, MeOD) δ 8.02 (d, J = 2.6 Hz, 1H), 7.74 (d, J = 4.0 Hz, 1H), 7.46
(d, J = 3.9 Hz, 1H), 7.29 (dd, J = 14.0, 7.9 Hz, 1H), 7.19 (d, J = 7.8 Hz, 1H), 7.14 (dd, J = 10.0, 1.9
Hz, 1H), 6.92 (td, J = 8.5, 2.3 Hz, 1H), 6.82 (d, J = 2.6 Hz, 1H), 4.65 (td, J = 11.6, 4.3 Hz, 1H),
4.10 (s, 3H), 3.64 – 3.61 (m, 1H), 3.56 (d, J = 12.7 Hz, 1H), 3.30 – 3.18 (m, 3H), 2.22 – 2.08 (m,
2H). 13C NMR (125 MHz, MeOD) δ 164.22 (d, J = 243.8 Hz), 162.60, 144.90 (d, J = 6.3 Hz),
142.59, 140.68, 136.93, 133.53, 131.40 (d, J = 7.5 Hz), 131.19, 130.46, 124.71 (d, J = 2.5 Hz),
115.47 (d, J = 21.3 Hz), 115.03 (d, J = 21.3 Hz), 109.01, 49.38, 47.92, 46.29, 45.07, 38.06, 31.12.
ESI-MS: m/z = 385 [M + H]+.
5-(4-Chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(3-fluorophenyl)piperidin-3-yl)thiophene
-2-carboxamide hydrochloride (1d)
o
1
General procedure F, yield: 78%; Melting point: 200~202 C; H NMR (500 MHz, MeOD) δ 7.62
(d, J = 3.8 Hz, 1H), 7.52 (s, 1H), 7.35 – 7.28 (m, 2H), 7.16 (d, J = 7.8 Hz, 1H), 7.10 (d, J = 10.1 Hz,
1H), 6.95 (td, J = 8.5, 2.2 Hz, 1H), 4.58 (td, J = 11.6, 4.2 Hz, 1H), 3.87 (s, 3H), 3.65 – 3.59 (m, 1H),
3.54 (d, J = 12.7 Hz, 1H), 3.20 (t, J = 13.0 Hz, 2H), 3.13 (t, J = 12.0 Hz, 1H), 2.20 (d, J = 12.4 Hz,
1H), 2.12 – 2.02 (m, 1H). 13C NMR (125 MHz, MeOD) δ 164.35 (d, J = 247.5 Hz), 163.15, 144.73
(d, J = 6.3 Hz), 141.50, 138.11, 133.94, 133.57, 131.51 (d, J = 7.5 Hz), 131.00, 129.97, 124.56,
115.41 (d, J = 22.5 Hz), 115.19 (d, J = 21.3 Hz), 111.29, 49.41, 48.06, 46.42, 45.17, 39.19, 31.33.
ESI-MS: m/z = 419 [M + H]+. HRMS (ESI) (m/z): calcd for C20H21ClFN4OS [M + H]+ 419.1109,
found 419.1113.
5-(4-Bromo-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(3-fluorophenyl)piperidin-3-yl)thiophene-
2-carboxamide hydrochlorid (1e)
General procedure F, yield: 89%; Retention time: 9.860 min, purity: 95.58 %; Melting point:
189~192 oC; 1H NMR (500 MHz, MeOD) δ 7.66 (d, J = 3.9 Hz, 1H), 7.54 (s, 1H), 7.33 – 7.28 (m,
1H), 7.26 (d, J = 3.9 Hz, 1H), 7.18 (d, J = 7.7 Hz, 1H), 7.12 (d, J = 9.9 Hz, 1H), 6.94 (td, J = 8.5,
2.2 Hz, 1H), 4.61 (td, J = 11.5, 4.1 Hz, 1H), 3.85 (s, 3H), 3.65 – 3.59 (m, 1H), 3.55 (d, J = 12.3 Hz,
13
1H), 3.28 – 3.12 (m, 3H), 2.20 (d, J = 13.7 Hz, 1H), 2.15 – 2.03 (m, 1H). C NMR (125 MHz,
CDCl3) δ 164.30 (d, J = 243.8 Hz), 163.06, 144.80 (d, J = 6.3 Hz), 141.70, 140.36, 135.61, 133.95,
131.47 (d, J = 7.5 Hz), 131.39, 129.95, 124.61, 115.43 (d, J = 21.3 Hz), 115.13 (d, J = 21.3 Hz),
96.03, 49.34, 48.01, 46.33, 45.14, 39.12, 31.27. ESI-MS: m/z = 463 [M + H]+. HRMS (ESI) (m/z):
calcd for C20H21BrFN4OS [M + H]+ 463.0603, found 463.0649.

ACCEPTED MANUSCRIPT
5-(4-Chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(4-chloro-3-(trifluoromethyl)phenyl)pipe
ridin-3-yl)thiophene-2-carboxamide hydrochlorid (1f)
General procedure F, yield: 77%; Retention time: 10.630 min, purity: 95.89 %; 1H NMR (500 MHz,
DMSO-d6) δ 9.45 (d, J = 9.0 Hz, 1H), 9.26 (d, J = 9.8 Hz, 1H), 9.12 (d, J = 8.9 Hz, 1H), 7.92 (d, J
= 3.6 Hz, 1H), 7.79 (s, 1H), 7.70 – 7.63 (m, 2H), 7.59 (d, J = 8.1 Hz, 1H), 7.42 (d, J = 3.7 Hz, 1H),
4.58 – 4.48 (m, 1H), 3.86 (s, 3H), 3.42 – 3.27 (m, 3H), 3.02 – 2.91 (m, 2H), 2.15 – 1.97 (m, 2H).
13C NMR (125 MHz, DMSO-d6) δ 160.09, 141.63, 140.83, 136.60, 132.76, 132.00, 131.61, 130.98,
129.86, 128.86, 128.70, 127.31 (q, J = 5.1 Hz), 126.24 (q, J = 30.1 Hz), 122.74 (q, J = 271.4 Hz),
108.44, 47.28, 46.25, 43.72, 43.10, 38.83, 28.68. ESI-MS: m/z = 503 [M + H]+. HRMS (ESI) (m/z):
calcd for C21H20Cl2F3N4OS [M + H]+ 503.0687, found 503.0711.
5-(1,4-Dimethyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(3-fluorophenyl)piperidin-3-yl)thiophene-2-car
boxamide hydrochloride (1g)
1
General procedure G yield: 51%; H NMR (500 MHz, CDCl3) δ 7.73 (s, 1H), 7.68 (d, J = 3.8 Hz,
1H), 7.38 – 7.25 (m, 2H), 7.17 (d, J = 7.4 Hz, 1H), 7.11 (d, J = 8.8 Hz, 1H), 6.96 (dd, J = 11.5, 5.3
Hz, 1H), 4.63 – 4.55 (m, 1H), 3.90 (s, 3H), 3.68 – 3.50 (m, 3H), 3.25 – 3.10 (m, 2H), 2.20 (d, J =
14.0 Hz, 1H), 2.11 (s, 3H), 2.09 – 2.02 (m, 1H). 13C NMR (125 MHz, CDCl3) δ 164.33 (d, J =
242.5 Hz), 162.92, 144.79 (d, J = 7.5 Hz), 142.37, 137.55, 137.39, 133.64, 131.84, 131.52 (d, J =
8.8 Hz), 130.20, 124.61, 118.99, 115.44 (d, J = 21.3 Hz), 115.16 (d, J = 21.3 Hz), 49.28, 48.02,
46.40, 45.13, 37.71, 31.33, 9.38. ESI-MS: m/z = 399 [M + H]+.
N-((3S,4S)-4-(3-fluorophenyl)piperidin-3-yl)-5-(1-methyl-4-vinyl-1H-pyrazol-5-yl)thiophene-2
-carboxamide hydrochlorid (1h)
To a solution of compound 17 in ethyl acetate was added HCl saturated ethyl acetate, stirred at
room temperature for 6 h. After it was fully reacted, the mixture was filtered, the resulted solid was
washed with diethyl ether. The solid was dried in vacuum to afford target product as white solid.
Yield: 89%; Melting point: 238~240 oC; 1H NMR (500 MHz, CDCl3, 3:2 ratio due to atropisomers)
δ 7.93 and 7.85 (s, 1H), 7.70 (s, 1H), 7.35 – 7.06 (m, 4H), 6.94 (t, J = 8.0 Hz, 1H), 6.43 (dd, J =
17.5, 11.1 Hz, 1H), 5.59 (d, J = 17.5 Hz, 1H), 5.16 (d, J = 11.1 Hz, 1H), 4.67 – 4.55 (m, 1H), 3.86
and 3.83 (s, 3H), 3.67 – 3.51 (m, 2H), 3.29 – 3.15 (m, 3H), 2.23 – 2.04 (m, 2H). ESI-MS: m/z = 411
[M + H]+. HRMS (ESI) (m/z): calcd for C22H24FN4OS [M + H]+ 411.1655, found 411.1654.
5-(4-Ethyl-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(3-fluorophenyl)piperidin-3-yl)thiophene-2
-carboxamide hydrochlorid (1i)

ACCEPTED MANUSCRIPT
To a solution of compound 17 (51 mg, 0.1 mmol) in 3 mL MeOH was added 10% Pd/C (5 mg) at
0
. The atmosphere of the reaction system was replaced by hydrogen three times and reacted
overnight. After the reaction was completed, Pd/C was removed by filtrated, and the filtrate was
concentrated to afford gray solid. To a solution of the above solid in ethyl acetate was added HCl
saturated ethyl acetate, stirred at room temperature for 6 h. After it was fully reacted, the mixture
was filtered, the resulted solid was washed with diethyl ether. The solid was dried in vacuum to
afford target product as white solid. Yield: 72%; Retention time: 9.876 min, purity: 95.64 %;
o
1
Melting point: 226~228 C; H NMR (500 MHz, MeOD) δ 7.90 (s, 1H), 7.74 (d, J = 3.8 Hz, 1H),
7.34 – 7.27 (m, 2H), 7.19 (d, J = 7.7 Hz, 1H), 7.13 (d, J = 10.0 Hz, 1H), 6.94 (td, J = 8.5, 2.2 Hz,
1H), 4.62 (td, J = 11.6, 4.3 Hz, 1H), 3.91 (s, 3H), 3.64 – 3.60 (m, 1H), 3.55 (d, J = 12.6 Hz, 1H),
13
3.30 – 3.15 (m, 3H), 2.52 (q, J = 7.5 Hz, 2H), 2.23 – 2.05 (m, 2H), 1.15 (t, J = 7.5 Hz, 3H). C
NMR (125 MHz, MeOD) δ 164.28 (d, J = 242.5 Hz), 162.78, 144.86 (d, J = 6.3 Hz), 142.94,
137.35, 135.84, 132.75, 132.37, 131.46 (d, J = 7.5 Hz), 130.26, 126.08, 124.65 (d, J = 2.5 Hz),
115.48 (d, J = 21.3 Hz), 115.10 (d, J = 21.3 Hz), 49.38, 47.96, 46.32, 45.11, 37.47, 31.24, 18.27,
15.07. ESI-MS: m/z = 413 [M + H]+. HRMS (ESI) (m/z): calcd for C22H26FN4OS [M + H]+
413.1811, found 413.1838.
5-(5-(((3S,4S)-4-(3-fluorophenyl)piperidin-3-yl)carbamoyl)thiophen-2-yl)-1-methyl-1H-pyrazo
le-4-carboxylic acid hydrochloride (1j)
To a 10 mL mixture of CH3CN and hydrogen peroxide (v/v, 1:1) was added compound 18 (102 mg,
0.2 mmol), ruthenium trichloride (4 mg, catalytic capacity) and NaIO4 (214 mg, 1 mmol) at 0
,
and then reacted overnight at room temperature. After the reaction was completed, the black
insoluble substances were removed by filtration, and the filtrate was extracted with ethyl acetate (10
mL × 3), the combined organic layers were washed with saturated brine (20 mL × 2), dried over
anhydrous sodium sulfate and concentrated. Purification by flash column chromatography (DCM:
1
MeOH = 30:1-10:1) afforded the N-Boc protective products as white solid; H NMR (500 MHz,
CDCl3) δ 8.01 (s, 1H), 7.31 – 7.23 (m, 2H), 7.09 – 7.01 (m, 2H), 6.98 (d, J = 9.8 Hz, 1H), 6.93 –
6.88 (m, 1H), 6.13 (brs, 1H), 4.52 – 4.35 (m, 1H), 4.26 – 4.13 (m, 2H), 3.79 (s, 3H), 2.99 – 2.75 (m,
3H), 2.02 – 1.91 (m, 1H), 1.86 – 1.74 (m, 1H), 1.49 (s, 9H). ESI-MS: m/z = 567 [M + K]+.
To a solution of the above solid in ethyl acetate was added HCl saturated ethyl acetate, stirred at
room temperature for 6 h. After it was fully reacted, the mixture was filtered, the resulted solid was
washed with diethyl ether. The solid was dried in vacuum to afford target product as white solid.

ACCEPTED MANUSCRIPT
o
1
Yield: 55%; Melting point: 181~183 C; Specific optical rotation: -40.687o; H NMR (500 MHz,
MeOD) δ 7.94 (s, 1H), 7.61 (d, J = 3.1 Hz, 1H), 7.37 – 7.28 (m, 1H), 7.22 (d, J = 3.2 Hz, 1H), 7.19
– 7.07 (m, 2H), 6.96 (t, J = 7.6 Hz, 1H), 4.65 – 4.55 (m, 1H), 3.75 (s, 3H), 3.61 (d, J = 7.0 Hz, 1H),
3.53 (d, J = 11.0 Hz, 1H), 3.26 – 3.07 (m, 3H), 2.20 – 2.02 (m, 2H). 13C NMR (125 MHz, MeOD) δ
165.45, 163.32 (d, J = 242.5 Hz), 163.19, 144.80 (d, J = 7.5 Hz), 142.35, 142.23, 139.29, 134.30,
132.43, 131.53 (d, J = 7.5 Hz), 129.56, 124.63, 115.59, 115.40 (d, J = 21.3 Hz), 115.16 (d, J = 21.3
Hz), 49.27, 48.07, 46.35, 45.22, 37.99, 31.41. ESI-MS: m/z = 429 [M + H]+. HRMS (ESI) (m/z):
calcd for C21H22FN4O3S [M + H]+ 429.1397, found 429.1380.
N-((3S,4S)-4-(3-fluorophenyl)piperidin-3-yl)-5-(4-(hydroxymethyl)-1-methyl-1H-pyrazol-5-yl)t
hiophene-2-carboxamide hydrochloride (1k)
Compound 18 (94 mg, 0.184 mmol) was dissolved in a 5 mL mixture of THF and MeOH (v/v, 10:1).
Then NaBH4 (11 mg, 0.3 mmol) was added slowly to the mixture at 0 , and reacted at room
temperature for 2 h. After the reaction was completed, the mixture was cooled to 0 and quenched
with 10% NH4Cl solution (5 mL). The mixture was extracted with ethyl acetate (10 mL × 3), the
combined organic layers were washed with saturated brine (20 mL × 2), dried over anhydrous
sodium sulfate and concentrated. Purification by flash column chromatography afforded the foam
solid. Yield: 88%;1H NMR (500 MHz, MeOD, 1:9 ratio due to atropisomers) δ 7.84 and 7.78 (s,
1H), 7.67 (d, J = 3.9 Hz, 1H), 7.35 – 7.27 (m, 2H), 7.17 (d, J = 7.7 Hz, 1H), 7.11 (d, J = 10.0 Hz,
1H), 6.96 (dd, J = 11.8, 5.1 Hz, 1H), 4.59 (td, J = 11.6, 4.3 Hz, 1H), 4.45 and 4.27 (s, 2H), 3.92 and
3.92 (s, 3H), 3.64 – 3.60 (m, 1H), 3.54 (d, J = 12.6 Hz, 1H), 3.26 – 3.10 (m, 3H), 2.20 (d, J = 12.9
13
Hz, 1H), 2.11 – 2.04 (m, 1H). C NMR (125 MHz, MeOD) δ 164.33 (d, J = 242.5 Hz), 163.02,
144.80 (d, J = 7.5 Hz), 142.39, 139.18, 137.67, 133.74, 131.89, 131.51 (d, J = 8.8 Hz), 130.12,
124.60 (d, J = 2.5 Hz), 120.09, 115.44 (d, J = 21.3 Hz), 115.16 (d, J = 21.3 Hz), 65.19, 49.28, 48.01,
46.39, 45.13, 37.92, 31.33. ESI-MS: m/z = 415 [M + H]+.
N-((3S,4S)-4-(3-fluorophenyl)piperidin-3-yl)-5-(1-methyl-4-phenyl-1H-pyrazol-5-yl)thiophene-
2-carboxamide hydrochloride (1l)
General procedure G yield: 76%; Melting point: 190~193 oC; 1H NMR (500 MHz, MeOD) δ 7.90 (s,
1H), 7.67 (d, J = 3.2 Hz, 1H), 7.31 – 7.06 (m, 9H), 6.94 (t, J = 7.5 Hz, 1H), 4.63 – 4.53 (m, 1H),
13
3.83 (s, 3H), 3.58 – 3.44 (m, 2H), 3.25 – 3.07 (m, 3H), 2.22 – 2.03 (m, 2H). C NMR (125 MHz,
MeOD) δ 164.27 (d, J = 243.8 Hz), 162.91, 144.81 (d, J = 7.5 Hz), 142.51, 137.36, 134.75, 134.66,
132.48, 132.41, 131.46 (d, J = 7.5 Hz), 130.31, 129.68, 128.89, 128.36, 124.89, 124.59, 115.46 (d, J

ACCEPTED MANUSCRIPT
= 22.5 Hz), 115.09 (d, J = 21.3 Hz), 49.34, 47.96, 46.27, 45.11, 37.59, 31.20. ESI-MS: m/z = 461
[M + H]+. HRMS (ESI) (m/z): calcd for C26H26FN4OS [M + H]+ 461.1811, found 461.1846.
N-((3S,4S)-4-(3-fluorophenyl)piperidin-3-yl)-5-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-5-yl)thio
phene-2-carboxamide hydrochloride (1m)
General procedure G yield: 46%;1H NMR (500 MHz, MeOD) δ 8.59 (d, J = 6.7 Hz, 2H), 8.27 (s,
1H), 7.83 (d, J = 6.7 Hz, 2H), 7.78 (d, J = 3.8 Hz, 1H), 7.36 – 7.31 (m, 2H), 7.19 (d, J = 7.7 Hz, 1H),
7.12 (d, J = 9.9 Hz, 1H), 6.98 (td, J = 8.4, 2.1 Hz, 1H), 4.59 (dt, J = 11.6, 5.8 Hz, 1H), 3.81 (s, 3H),
3.61 – 3.57 (m, 1H), 3.56 – 3.51 (m, 1H), 3.29 – 3.10 (m, 3H), 2.21 – 2.16 (m, 1H), 2.11 – 2.04 (m,
1H). 13C NMR (125 MHz, MeOD) δ 164.33 (d, J = 243.8 Hz), 162.69, 152.06, 145.04, 144.89 (d, J
= 7.5 Hz), 144.08, 142.37, 140.41, 136.89, 133.48, 133.24, 131.57 (d, J = 8.8 Hz), 130.84, 127.15,
124.62, 124.28, 118.99, 115.50 (d, J = 21.3 Hz), 115.18 (d, J = 21.3 Hz), 49.40, 47.95, 46.26, 45.10,
37.94, 31.37. ESI-MS: m/z = 462 [M + H]+. HRMS (ESI) (m/z): calcd for C25H25FN5OS [M + H]+
462.1764, found 462.1765.
4-(4-Chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(4-chlorophenyl)piperidin-3-yl)thiophene
-2-carboxamide (2R,3R)-2,3-dihydroxysuccinate (1n)
General procedure F, yield: 47%; 1H NMR (400 MHz, DMSO-d6) δ 8.76 (d, J = 7.8 Hz, 1H), 8.03
(s, 1H), 7.88 (s, 1H), 7.62 (s, 1H), 7.39 – 7.25 (m, 4H), 4.55 – 4.39 (m, 1H), 4.17 (s, 2H), 3.81 (s,
3H), 3.46 – 3.31 (m, 2H), 3.08 – 2.87 (m, 3H), 2.06 – 1.91 (m, 2H). 13C NMR (100 MHz,
DMSO-d6) δ 174.03, 160.10, 140.80, 139.95, 136.39, 133.96, 131.55, 131.28, 129.35, 128.45,
128.33, 127.33, 107.34, 72.11, 47.66, 46.55, 44.20, 43.21, 38.60, 29.72. HRMS (ESI) (m/z): calcd
for C20H21Cl2N4OS [M + H]+ 435.0813, found 435.0737.
4-(4-Chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(3-fluorophenyl)piperidin-3-yl)thiophene
-2-carboxamide hydrochloride (1o)
General procedure F, yield: 69%; Retention time: 9.735 min, purity: 96.10 %; Melting point:
189~192 oC; Specific optical rotation: -88.742o; 1H NMR (500 MHz, MeOD) δ 7.89 (d, J = 1.0 Hz,
1H), 7.79 (s, 1H), 7.52 (s, 1H), 7.32 – 7.27 (m, 1H), 7.17 (d, J = 7.7 Hz, 1H), 7.11 (d, J = 10.0 Hz,
1H), 6.97 – 6.92 (m, 1H), 4.59 (td, J = 11.5, 4.1 Hz, 1H), 3.83 (s, 3H), 3.64 – 3.59 (m, 1H), 3.57 –
3.50 (m, 1H), 3.26 – 3.13 (m, 3H), 2.22 – 2.03 (m, 2H). 13C NMR (125 MHz, MeOD) δ 164.31 (d, J
= 243.8 Hz), 163.06, 144.76 (d, J = 7.5 Hz), 140.44, 137.78, 136.00, 133.02, 131.48 (d, J = 7.5 Hz),
130.08, 129.10, 124.62 (d, J = 2.5 Hz), 115.45 (d, J = 21.3 Hz), 115.15 (d, J = 21.3 Hz), 110.12,
49.46, 48.05, 46.39, 45.14, 38.86, 31.18. ESI-MS: m/z = 419 [M + H]+. HRMS (ESI) (m/z): calcd

ACCEPTED MANUSCRIPT
for C20H21ClFN4OS [M + H]+ 419.1109, found 419.1115.
4-(4-Chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(4-chloro-3-(trifluoromethyl)phenyl)pipe
ridin-3-yl)thiophene-2-carboxamide hydrochloride (1p)
General procedure F, yield: 49%; Retention time: 10.568 min, purity: 95.69 %; 1H NMR (500 MHz,
DMSO-d6) δ 9.34 (s, 1H), 9.20 (s, 1H), 9.09 (d, J = 8.9 Hz, 1H), 8.04 (d, J = 1.2 Hz, 1H), 7.79 (d, J
= 1.5 Hz, 1H), 7.70 – 7.63 (m, 3H), 7.50 (d, J = 8.3 Hz, 1H), 4.54 – 4.46 (m, 1H), 3.82 (s, 3H), 3.31
– 3.27 (m, 2H), 3.02 – 2.93 (m, 2H), 2.72 (q, J = 11.1 Hz, 1H), 2.14 – 1.94 (m, 2H). 13C NMR (125
MHz, DMSO-d6) δ 168.81, 160.10, 141.74, 139.71, 136.36, 133.95, 132.86, 131.64, 128.92, 128.67,
127.51 (q, J = 4.6 Hz), 127.28, 126.24 (q, J = 32.5 Hz), 122.88 (q, J = 271.2 Hz), 107.36, 47.43,
46.38, 44.05, 42.95, 38.59, 29.06. ESI-MS: m/z = 503 [M + H]+. HRMS (ESI) (m/z): calcd for
C21H20Cl2F3N4OS [M + H]+ 503.0687, found 503.0680.
4.2 Akt1 inhibitory activity assay
The Akt1 inhibitory activities were determined using the homogeneous time-resolved fluorescence
(HTRF) KinEASE-STK S1 kit (Cat.#62ST2PEC, Cisbio) according to the manufacturer’s
instructions. Akt1 kinase was expressed and purified from Escherichia coli system in-house. In
short, the kinase reactions were carried out in a 384 ProxiPlate with 10 µL reaction volume per well
containing 0.25 µg/mL Akt1, 1 µM peptide substrate, test compound and 20 µM ATP in assay
buffer (50 mM HEPES pH 7.5, 0.01% BRIJ-35, 5 mM MgCl2, 1 mM EGTA). After incubation for 1
h at r.t., the reaction was stopped by the addition of 5µL Sa-XL665 and 5 µL STK Antibody-Eu(K)
in EDTA. The plate was sealed and incubated for 1 h at r.t., and the resulting TR-FRET signal was
measured on Envision-PerkinElmer. The fluorescence emission was measured at 615 nm (cryptate)
and 665 nm (XL665). An Emission Ratio was calculated (665/615) for each well and the Percent
inhibitions were expressed as follows: Percent inhibition = (max-sample Ratio)/ (max-min)*100
(“Min” means the Ratio of no enzyme control and “max” means the Ratio of DMSO control).
Compounds were initially tested at a fixed concentration (1 µg/mL), and those displaying more than
50% inhibition were further tested for dose–response IC50 values.
4.3 Cell culture
Human ovarian carcinoma cells (OVCAR8) and human colon cancer cells (HCT116) were obtained
from the Cell Bank of the China Science Academy, Shanghai and were cultured in DMEM, F-12K
or RPMI-1640 medium (Gibco Laboratories), supplemented with 10% fetal bovine serum (Gibco
Laboratories), 105 U/L penicillin, and 100 mg/L streptomycin at 37 °C in an atmosphere containing

ACCEPTED MANUSCRIPT
5% CO2. Cells were grown to 70% to 80% confluency in dishes or cell culture plates and treated
under various conditions as indicated.
4.4 Anti-proliferative assay
OVCAR8 and HCT116 cells were treated with the compounds at various doses for 72h, and cell
proliferation was tested by a sulforhodamine B (SRB) protein assay (Sigma, S1402). Cells
(5000/well) were incubated with 10% trichloroacetic acid (TCA) for 1 h (4 °C) and then stained
with SRB for 20 min. The SRB was washed away with 1% glacial acetic acid, and 100 µ l of 1%
Tris-base was added to each well. The optical density (OD) was determined at 515 nm by a
Multiskan Spectrum plate reader (Thermo Electron Corporation, Marietta, OH, USA).
4.5 Western blot assay
Whole protein extracts from cultured cells or tissues were prepared and subjected to western
blotting. Protein samples were size-fractionated by 10% SDS-PAGE, analysed by immunoblotting
and visualized by enhanced chemiluminescence (ECL, Amersham Biosciences, Castle Hill,
Australia) and then exposed on X-ray film. Proteins in the lysates were equalized and then analyzed
by specific antibodies. The primary antibodies were listed as follows: AKT (CST, 9272S),
pAKT-Thr308 (Santa, sc-16646), pAKT-Ser473 (Santa, sc-7985), PRAS40 (CST, 2691P),
p-PRAS40 (CST, 13175P), GSK-3β (Santa, sc-7291), p-GSK-3β (CST, 8566S), β-Actin (Santa,
SC-1615). Appropriate secondary antibodies and ECL were used to visualize the protein signaling.
4.6 Flow cytometry
HCT116 cells were seeded in 6-well dish plates at 1×105 cells/well and exposed to different
concentrations of compounds. After 72 h, the cells were harvested and stained with Annexin-V/PI
Solution using the FITC Annexin V Apoptosis Detection Kit I (BD Bioscience, 556547) for 10–15
min and resuspended in binding buffer. Then the fluorescence emission at 530 nm and 585 nm using
488 nm excitation were measured by flow cytometry (BD FACSCalibur).
4.7 hERG inhibition assay
Currents were recorded from HEK-293 cells, using the whole-cell patch-clamp technique. The cells
were transferred to a perfusion chamber and the perfusion was performed with extracellular fluid.
The extracellular fluid (mM): K Aspartate, 130; MgCl2, 5; EGTA, 5; HEPES, 10; Tris-ATP, 4; pH
7.2. Electrodes were pulled using a dual-stage glass micropipette puller (Narishige PC-10, Japan).
Current traces of hERG channels were elicited by applying a pulse from -80 mV to +40 mV for 4 s
followed by a step to -40 mV for 2 s. The procedure was repeated every 20 seconds. After the