Design, synthesis and biological evaluation of AKT inhibitors bearing a piperidin-4-yl appendant

Article abstract of DOI:10.1039/c8md00197a

A series of AKT inhibitors possessing a piperidin-4-yl side chain was designed and synthesized. Some of them showed high AKT1 inhibitory activity and potent anti-proliferative effect on PC-3 prostate cancer cells in the preliminary screening. Further studies revealed the most potent compound, 10h, as a pan-AKT inhibitor. Compound 10h was able to inhibit the cellular phosphorylation of AKT effectively and induce apoptosis of PC-3 cells. It also showed high metabolic stability in human liver microsomes. Preclinical characterization of 10h, a promising lead AKT inhibitor, as a potential anti-prostate cancer therapeutic needs to be further investigated.

Full text of DOI:10.1039/c8md00197a

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Page 1 of 26
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DOI: 10.1039/C8MD00197A
Design, synthesis and biological evaluation of AKT inhibitors bearing
a piperidinꢀ4ꢀyl appendant
Daoguang Zhang1,†, Dongdong Tong2,†, Dezhi Yang3, Jing Sun4, Fenghe Zhang2,*,
Guisen Zhao1,*
1Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry
of Education), School of Pharmaceutical Sciences, Shandong University, Jinan,
Shandong 250012, PR China
2Department of Oral and Maxillofacial Surgery, School of Stomatology, Shandong
University, Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Jinan,
Shandong 250012, PR China
3National Pharmaceutical Experimental Teaching Demonstration Center, School of Ph
armacy, Zunyi Medical University, Zunyi, Guizhou 563003, PR China
4Department of Bone Metabolism, School of Stomatology, Shandong University,
Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Jinan, Shandong
250012, PR China
†These authors contributed equally to this work.
*Corresponding author:
Fenghe Zhang: Department of Oral and Maxillofacial Surgery, Shandong Provincial
Key Laboratory of Oral Tissue Regeneration, School of Stomatology Shandong
University, Jinan, Shandong 250012, PR China. Tel & Fax: +86ꢀ531ꢀ88382961.
Eꢀmail: zfengh@sdu.edu.cn
Guisen Zhao: Department of Medicinal Chemistry, Key Laboratory of Chemical
Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong
University, Jinan, Shandong 250012, PR China. Tel & Fax: +86ꢀ531ꢀ88382009.
Eꢀmail: guisenzhao@sdu.edu.cn
Abstract
A series of AKT inhibitors possessing a piperidinꢀ4ꢀyl side chain was designed
and synthesized. Some of them showed high AKT1 inhibitory activity and potent

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antiꢀproliferative effect on PCꢀ3 prostate cancer cells in the preliminary screening.
Further studies revealed the most potent compound, 10h, as a panꢀAKT inhibitor.
Compound 10h was able to inhibit the cellular phosphorylation of AKT effectively
and induce apoptosis of PCꢀ3 cells. It also showed high metabolic stability in human
liver microsomes. As a promising lead AKT inhibitor, preclinical characterization of
10h as a potential antiꢀprostate cancer therapeutic needs to be further investigated.
1. Introduction
Hyperactive aberration of PI3KꢀAKT signaling is observed in a wide variety of
cancer types, which makes components of this signaling possible targets for cancer
therapy1. The serine/threonine kinase AKT, also known as protein kinase B (PKB),
plays a critical role in this signaling and is hyperꢀactivated in over 50% of human
tumors2. It regulates by phosphorylation over one hundred downstream substrates that
are involved in the regulation of various cellular events, including cell survival,
proliferation and metabolism. Glycogen synthase kinase 3 (GSK3) is the first reported
substrate of AKT3, and evaluation of the phosphorylation level of its β isoform is
typically used as a tool assessing the intracellular inhibition level of AKT by small
molecule inhibitors.
Given that AKT is tightly linked to cell physiology and pathology, it has been
catching attention of the pharmaceutical world in the past decades. A number of
AKTꢀtargeted agents have entered clinical trials for an exploration of their therapeutic
potential against various tumor types. For instance, AZD5363 (Figure 1) was
investigated for its safety and efficacy in prostate cancer in a phase 1 clinical trial
(NCT01692262). Particularly, since activation of the PI3KꢀAKT signaling through
loss of the tumor suppressor PTEN was proven as a potential mechanism for the
pathology of castrationꢀresistant prostate cancer (CRPC)4, AKT inhibitors have been
utilized in combination therapy for the treatment of CRPC, such as a phase 2 trial of
AZD5363 in combination with enzalutamide in metastatic CRPC (NCT02525068).

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Figure 1. ATPꢀcompetitive inhibitors of AKT in clinical trials
Among all the AKT inhibitors reported, a specific type of ATPꢀcompetitive
inhibitors, including GSK690693, AZD5363 and GDC0068, was well developed
(Figure 1)5. Analysis of the coꢀcrystals of AZD53636 or GDC00687 in complex with
AKT revealed that these agents bond to the hinge region of AKT. The modes of this
ligandꢀprotein interaction can be capitalized in the discovery of novel AKT inhibitors.
Our laboratory previously reported a series of AKT inhibitors with general
structure shown in figure 28. Represent compounds 5q and 5t in this series showed
high AKT1 inhibition at the concentration of 50 nM with inhibitory rates of 69.7%
and 65.5%, respectively. Both compounds also exhibited moderate antiꢀproliferative
effect in a prostate cancer cell line of PCꢀ3 with IC50 values of 21.1 ꢁM and 31.5 ꢁM,
respectively. In this work, structural modification was further performed. First, since
the hydrogen bond interactions, formed between the hydroxyethyl group of AZD5363
or isopropylaminomethyl group of GDC0068 and the carboxyl residues of two
glutamic acids (Glu 234 and Glu 278) near the hinge region, were critical for their
ligandꢀprotein binding affinity6, 7. So, one modification to our lead structure was to
introduce an appendant to the bridging methylene between the carbonyl and phenyl
groups (Figure 2). The criteria for this appendant was that it should provide a
hydrogen bond donor to generate similar hydrogen bond interactions without
introduction of chirality. After a literature survey, a piperidinꢀ4ꢀyl group which had
been adopted by previously reported AKT inhibitors AIꢀ1 (AKT IC50 = 18 nM) and
AIꢀ2 (AKT IC50 = 20 nM) was adopted (Figure 2)9, 10. This was a symmetric cyclic

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appendant with a terminal imine group as the hydrogen bond donor. The other
modification focused on the hingeꢀbinding pyrrolopyrimidine group, three
nitrogenꢀcontaining bicyclic systems were attempted in this work. These
modifications led to a series of Nꢀheterocyclic based AKT inhibitors.
Figure 2. Design of novel ATK inhibitors based on our and others’ previous studies
2. Results and discussion
2.1. Chemistry
Compounds 10aꢀ10l were prepared following the route as shown in scheme 1.
Halogen groups like chloride or bromide were first introduced at Cꢀ5 position of a
commercially available 4ꢀchloropyrazolopyrimidine (1) via
a
free radical
halogenation. Aromatic nucleophilic substitution of the chloride from Cꢀ4 position of
intermediate 2 by tertꢀbutyl piperazineꢀ1ꢀcarboxylate under microwave irradiation
afforded intermediate 3, which was deꢀprotected with trifluoroacetic acid (TFA) to
give intermediate 4. Finally, condensation with a proper carboxylic acid (5) followed
by deꢀprotection of the Boc protective group afforded compounds 10aꢀ10l.
This synthetic route was successfully applied to the preparation of compounds
14aꢀ14d and 18aꢀ18d using 6ꢀchloroꢀ9Hꢀpurine (11) and 4ꢀchloroquinazoline (15) as
starting materials, respectively (Scheme 1).

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Scheme 1. Reagents and conditions: (a) NCS or NBS, DMF, r.t., 12ꢀ18 h; (b)
tertꢀbutyl piperazineꢀ1ꢀcarboxylate, DMF, microwave, 150 °C, 20 min; (c) TFA,
DCM, r.t., 4 h; (d) HBTU, DIPEA, DMF, r.t., 6 h; (e) TFA, DCM, r.t., 4 h.
4ꢀchlorobenzylcyanide (6) was used as a starting material for the preparation of
building block 5 (Scheme 2). The two hydrogen atoms of the methylene group of 6
were first abstracted by sodium hydride, followed by replacing of the two chlorine
atoms from Bocꢀprotected bis(2ꢀchloroethyl)amine (8) to afford the substituted
4ꢀphenylpiperidineꢀ4ꢀcarbonitrile as intermediates 9. A subsequent hydrolysis
generated the carboxylic acid as intermediate 5.

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Scheme 2. Reagents and conditions: (a) aq. NaOH, (Boc)2O, DCM, r.t., 12 h; (b)
NaH, DMF, 0ꢀ5 °C; 80 °C, 12ꢀ18 h; (c) NaOH, ethanol, 60 °C, 48 h.
2.2. AKT inhibitory activity and antiꢀproliferative effect on PCꢀ3 cells
Preliminary screenings were carried out for the newly synthesized compounds,
including the evaluation of AKT inhibitory activity at a compound concentration of 1
ꢁM and of antiꢀproliferative effect on a Prostate cancer cell line of PCꢀ3 (Table 1).
Compounds (14aꢀ14d) containing a purine ring exhibited higher AKT1
inhibitory activity than their pyrazolopyrimidine analogues (10aꢀ10d) and quinazoline
analogues (18aꢀ18d), but lacked antiꢀproliferative activity. Compounds (18aꢀ18d)
bearing
a
quinazoline structure showed low AKT1 inhibition but high
antiꢀproliferation, while pyrazolopyrimidineꢀderived compounds (10aꢀ10d) lacked
both AKT1 inhibitory activity and cytotoxicity. However, introduction of a halogen
substituent to the Cꢀ3 position of the pyrazolopyrimidine ring significantly elevated
inhibitory activity against both AKT1 and PCꢀ3 cells. Compounds (10eꢀ10l) with a
halogen substitution (Cl or Br) at the Cꢀ3 position of the pyrazolopyrimidine ring
exhibited both high AKT inhibition and high growth inhibition in PCꢀ3 cells than did
their hydrogen substituted counterparts (10aꢀ10d).
For compounds (10aꢀ10l) based on a pyrazolopyrimidine ring with or without
halogen substituent on Cꢀ3 position, substitutions on the terminal phenyl ring had
different influences. All of the 4ꢀF substituted compounds (10a, 10e and 10i) showed
weaker AKT1 inhibition at 1 ꢁM than their chlorine or bromine substituted analogues.
This tendency was confirmed at a diluted concentration of 0.1 ꢁM as compounds (10e
and 10l) showed inferior AKT1 inhibition than their chlorine or bromine substituted
analogues 10fꢀ10h and 10jꢀ10l. Similar trend was also observed on the potency

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against PCꢀ3 cells, as compounds (10fꢀ10h and 10jꢀ10l) with chlorine or bromine
substitution all showed higher antiꢀproliferative activity than their 4ꢀF substituted
analogues (10e and 10i).
Based on the preliminary data on both AKT1 inhibition and PCꢀ3 cell growth
inhibition, compounds (10fꢀ10h and 10jꢀ10l) were selected for the determination of
AKT1 inhibitory IC50 values (Table 1). In comparison with the positive drug
GSK690693, these compounds all showed slightly lower AKT1 inhibitory activity.
Among them, compound 10h showed the highest AKT1 inhibitory activity with an
IC50 value of 24.3 nM, and was most potent against PCꢀ3 cells with an IC50 value of
3.7 ꢁM. This cellular potency was nearly 4ꢀfold more effective than GSK690693
(IC50 = 14.1 ꢁM).
Table 1. Preliminary screenings for the new compounds: AKT1 inhibition and
antiꢀproliferation in PCꢀ3 cells.
Structure
AKT1 inhibition
Antiꢀproliferative
effect on PCꢀ3
cells (IC50/
> 40
Code
R1
R2
%, @1 ꢁMa %, @0.1 ꢁMa
IC50/nMb
µ
M)b
H
H
H
4ꢀF
4ꢀCl
4ꢀBr
7.4
ꢀ
ꢀ
ꢀ
10a
10b
10c
10d
38.7
11.6
52.1
86.7
91.9
95.4
94.3
ꢀ
> 40
ꢀ
ꢀ
> 40
H 3,4ꢀdiCl
4ꢀF
ꢀ
ꢀ
> 40
10e Cl
60.9
83.4
84.5
83.7
ꢀ
37.4 ± 2.2
19.4 ± 1.6
32.4 ± 2.1
3.7 ± 0.9
Cl 4ꢀCl
Cl 4ꢀBr
Cl 3,4ꢀdiCl
66.6 ± 2.1
32.9 ± 1.9
24.3 ± 1.6
10f
10g
10h

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Br
4ꢀF
93.2
96.7
96.5
93.8
94.2
45.6
68.0
51.4
23.0
34.2
6.1
53.3
ꢀ
36.6 ± 2.4
10i
10j
Br 4ꢀCl
Br 4ꢀBr
Br 3,4ꢀdiCl
86.7
26.9 ± 1.7
9.8 ± 1.2
15.0 ± 1.3
10.3 ± 1.1
> 40
90.6
51.9 ± 2.1
10k
10l
90.9
30.5 ± 1.8
14a
14b
14c
14d
18a
18b
18c
18d
ꢀ
ꢀ
ꢀ
4ꢀF
4ꢀCl
4ꢀBr
ꢀ
ꢀ
ꢀ
ꢀ
> 40
ꢀ
ꢀ
> 40
ꢀ 3,4ꢀdiCl
ꢀ
ꢀ
33.9 ± 2.2
13.7 ± 1.3
> 40
ꢀ
ꢀ
ꢀ
4ꢀF
4ꢀCl
4ꢀBr
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
22.8 ± 1.6
14.0 ± 1.8
14.1 ± 1.6
ꢀ 3,4ꢀdiCl
21.9
99.5
ꢀ
98.5
21.3 ± 2.1
GSK690693
an = 3
bIC50 data are mean of three independent experiments
2.3. Kinase selectivity study of selected compound 10h
Compound 10h was further evaluated for its selectivity against a panel of 16
kinases (Figure 3) at the concentration of 1 µM. This compound showed high AKT1
selectivity over 11 kinases (Abl, BꢀRaf, CDK1, CHK1, GSK3β, JAK1, PDK1, PKCα,
RAFꢀ1 and ROCK1). However, compound 10h showed moderate inhibitory activity
against MNK1 (47%) and Aurora A (64%), and high inhibitory activity against RSK1
(83%), PKA (99%) and p70S6K (99%). The lack of selectivity against the latter three
kinases was mainly due to high overall ATP binding site homology (>70%) of these
kinases with AKT111.
At the concentration of 10 nM, compound 10h showed panꢀinhibitory activity to
all three AKT isoforms with moderate to high inhibition of AKT2, AKT1 and AKT3
by 38%, 83% and 93%, respectively (Figure 3). A more than 2ꢀfold selectivity of
AKT1 and 3 against AKT2 was also observed.

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Figure 3. Selectivity assay for 10h in 16 human kinases and three AKT kinase
isoforms (n = 3).
2.4. Influence of 10h on cellular AKT and GSK3β phosphorylation
Compound 10h was further evaluated for its impact on cellular AKT signaling in
PCꢀ3 cells via an immunoblotting assay (Figure 4A). Contrary to GSK690693 which
enhanced AKT activation in this assay, a high inhibition of AKT phosphorylation by
10h was observed after 24 hours of treatment at all three concentrations. Similar
phenomenon also occurred on reported AKT inhibitors based on
a
diphenylmethylamine scaffold12, and indicated an involvement of additional
functional mechanism for compound 10h to overcome the positive feedback loop
which usually was evoked to fight back the AKT inhibition within the cells. In the
meantime, 10h showed weaker inhibition on the phosphorylation of GSK3β, a
downstream effector of AKT, compared with efficient inhibition of GSK3β
phosphorylation by GSK690693. This was consistent with the distinct function of the
two compounds on AKT phosphorylation as GSK3β was directly downstream and
suppressed by activated AKT within cells.

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Figure 4. (A) Effects of 10h on AKT signaling within PCꢀ3 cells; (BꢀC) Apoptosis
induction by 10h in PCꢀ3 cells.
2.5. Apoptotic induction of 10h in PCꢀ3 cells
Compound 10h was further evaluated for its apoptosis inductive effect in PCꢀ3
cells via an annexin VꢀFITC/propidium iodide (PI)ꢀbinding assay (Figure 4BꢀC). After
48 hours of the treatment, a mild degree of apoptosis was observed in a
doseꢀdependent manner. The percentage of apoptotic cells increased by 1.32%ꢀ27.83%
when compared to the DMSO control.
2.6. Stability in human liver microsomes
Liver microsomes are subcellular fractions derived from the endoplasmic
reticulum of liver, they contain a variety of metabolic enzymes (cytochrome P450)
and are used as an in vitro model for the prediction of in vivo metabolic stability in
liver. Compound 10h showed high metabolic stability with a more than 80% total
remaining after an oneꢀhour treatment in human liver microsomes. The calculated
halfꢀlife (T1/2) and clearance rate (CLint(mic)) were more than 145 min and less than 9.6
ꢁL/min/mg, respectively (Table 2).
Table 2. Metabolic stability of 10h in human liver microsomes.
CLint(mic)
CLint(liver)
Remaining
Code
T1/2 (min)
(ꢁL/min/mg) (mL/min/kg) (T = 60min)

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> 145
16.9
10.2
7.7
< 9.6
82.2
< 8.6
74.0
81.8%
8.1%
1.5%
0.5%
10h
Testosterone
Diclofenac
Propafenone
136.2
180.9
122.6
162.8
3. Conclusion
In this work, the discovery of a potent AKT targeted inhibitor 10h showing
efficient antiꢀprostate cancer potency in PCꢀ3 cells was described. This compound
belonged to a series of
Nꢀheterocyclic based AKT1 inhibitors with the structural
feature of a piperidinꢀ4ꢀyl side chain. Compounds based on a 3ꢀhalogenic
pyrazolopyrimidine ring in this series showed high AKT1 inhibition and potent
antiꢀproliferative effect in PCꢀ3 cells, of which 10h showed the highest potency on
both AKT1 inhibition (IC50 = 24.3 nM) and PCꢀ3 cell proliferation (IC50 = 3.7 ꢁM). In
a further characterization of kinase selectivity profile, compound 10h showed potent
panꢀAKT inhibitory activity, as well as inhibitory activity against kinases of AGC
family. 10h could moderately inhibit cellular AKT phosphorylation and induce
apoptosis of PCꢀ3 cells. It also showed high metabolic stability in human liver
microsomes. Therefore, further evaluation of 10h was warranted for its potential
application as an antiꢀprostate cancer therapeutic.
4. Materials and methods
4.1. Chemistry
All reagents and solvents were purchased from commercial suppliers and used
without further purification. All reactions were monitored by thin layer
chromatography (TLC), silica gel GF254 plates were used and visualized with UV
light. Column chromatography was performed with silica gel using the solvent
systems as indicated in the synthetic procedures. NMR spectra were recorded on a
Bruker AVANCE DRX400 or DRX600 spectrometer using tetramethylsilane (TMS)
as an internal standard in DMSOꢀ
(ppm). Coupling constants ( ) are given in Hz. The mass spectra (MS) were measured
with an API 4000. All of the melting points were determined using a Büchi capillary
d6. Chemical shifts are reported in parts per million
J

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melting point apparatus and were uncorrected.
4.1.1. General procedure for the synthesis of intermediate 2
To a solution of 4ꢀchloroꢀ1 ꢀpyrazolo[3,4ꢀ ]pyrimidine (1, 3.1 g, 20.0 mmol) in
N,Nꢀdimethylformamide (DMF, 15 ml), ꢀchlorosuccinimide (NCS, 2.8 g, 21.0 mmol)
or ꢀbromosuccinimide (NBS, 3.7 g, 21.0 mmol) was added. This mixture was stirred
H
d
N
N
at room temperature for 12ꢀ18 hours. Icy water (150 ml) was poured into the reaction
mixture, and the precipitate was filtered, washed with water (2 × 20 ml), and dried to
give intermediate 2.
3,4ꢀDichloroꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidine (2a)
1
Light yellow solid, yield 74%. H NMR (400 MHz, DMSOꢀd6) δ 14.77 (s, 1H), 8.88
(s, 1H).
3ꢀBromoꢀ4ꢀchloroꢀ1
H
ꢀpyrazolo[3,4ꢀd]pyrimidine (2b)
1
Offꢀwhite solid, yield 94%. H NMR (400 MHz, DMSOꢀ
1H).
d6) δ 14.13 (s, 1H), 8.06 (s,
4.1.2. General procedure for the synthesis of intermediates 3, 12 and 16
A solution of intermediate 2 (15.2 mmol), ꢀdiisopropylethylamine (DIPEA,
N,N
4.0 g, 30.4 mmol) and tertꢀbutyl piperazineꢀ1ꢀcarboxylate (3.1 g, 16.7 mmol) in DMF
(15 ml) was heated at 150 °C under microwave irradiation for 20 min. This reaction
mixture was cooled down and poured into iceꢀcold water (150 ml), followed by
extraction of ethyl acetate (EA, 3 × 30 ml). The organic extracts were combined,
washed by saturated aqueous NH4Cl (2 × 30 ml) and brine (2 × 30 ml), dried over
anhydrous Na2SO4, filtered, condensed under reduced pressure, and purified by
column chromatography (petroleum ether (PE)/EA = 9/1) to get intermediate 3.
Intermediates 12 and 16 were prepared in a similar procedure as described above
using the commercially available 6ꢀchloroꢀ9Hꢀpurine (11) and 4ꢀchloroquinazoline
(15) as starting materials, respectively.
Tertꢀbutyl 4ꢀ(9
H
ꢀpurinꢀ6ꢀyl)piperazineꢀ1ꢀcarboxylate (12)
1
Offꢀwhite solid, yield 91%. H NMR (400 MHz, DMSOꢀ
d
6) δ 13.09 (s, 1H), 8.23 (s,
1H), 8.15 (s, 1H), 4.21 (br s, 4H), 3.44 (br s, 4H), 1.43 (s, 9H).
Tertꢀbutyl 4ꢀ(quinazolinꢀ4ꢀyl)piperazineꢀ1ꢀcarboxylate (16)

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Offꢀwhite solid, yield 88%. 1H NMR (400 MHz, DMSOꢀ
d6) δ 8.88 (s, 1H), 8.24 (d, J
= 8.4 Hz, 1H), 8.09–7.93 (m, 2H), 7.72 (t,
4.14 (m, 4H), 3.62 (br s, 4H), 1.44 (s, 9H).
J = 7.7 Hz, 1H), 7.49–7.15 (m, 1H), 4.26–
4.1.3. General procedure for the synthesis of intermediates 4, 13 and 17
To a solution of intermediate 3 (1.0 mmol) in dichloromethane (DCM, 6 ml),
trifluoroacetic acid (TFA, 2 ml) was added. This mixture was stirred at room
temperature for 4 hours, and the precipitate formed was collected by filtration and
dried to give intermediate 4. Intermediates 13 and 17 were prepared in a similar
procedure as described above.
3ꢀChloroꢀ4ꢀ(piperazinꢀ1ꢀyl)ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidine (4a)
Offꢀwhite solid, yield 68%. 1H NMR (400 MHz, DMSOꢀ
d6) δ 8.36 (d, J = 5.7 Hz, 1H),
3.93–3.79 (m, 4H), 3.12–3.00 (m, 4H).
3ꢀBromoꢀ4ꢀ(piperazinꢀ1ꢀyl)ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidine (4b)
Offꢀwhite solid, yield 88%. 1H NMR (400 MHz, DMSOꢀ
d6) δ 8.38 (s, 1H), 3.96–3.74
(m, 4H), 3.20–3.07 (m, 4H).
4ꢀ(Piperazinꢀ1ꢀyl)ꢀ1
H
ꢀpyrazolo[3,4ꢀd]pyrimidine (4c)
1
Offꢀwhite solid, yield 84%. H NMR (400 MHz, DMSOꢀ
1H), 8.63 (s, 1H), 4.32 (s, 4H), 3.35 (s, 4H).
d6) δ 9.99 (s, 2H), 8.97 (s,
6ꢀ(Piperazinꢀ1ꢀyl)ꢀ9Hꢀpurine dihydrochloride (13)
Offꢀwhite solid, yield 88%. 1H NMR (400 MHz, DMSOꢀ
d6) δ 9.71 (br s, 2H), 8.45 (s,
1H), 8.39 (s, 1H), 4.52 (s, 4H), 3.28 (s, 4H).
4ꢀ(Piperazinꢀ1ꢀyl)quinazoline dihydrochloride (17)
1
Offꢀwhite solid, yield 84%. H NMR (400 MHz, DMSOꢀ
d
6) δ 10.02 (s, 2H), 8.95 (s,
= 8.4, 5.6, 2.7 Hz, 1H),
1H), 8.25 (d,
J
= 8.4 Hz, 1H), 8.10–8.02 (m, 2H), 7.74 (ddd,
J
4.37 (s, 4H), 3.34 (s, 4H).
4.1.4. General procedure for the synthesis of intermediate 8
To a solution of bis(2ꢀchloroethyl)amine hydrochloride (7, 8.0 g, 44.8 mmol) in
DCM (20 ml) at room temperature, aqueous NaOH (6 M, 20 ml) was added, followed
by addition of Boc2O (9.8 g, 44.8 mmol). After addition, the reaction mixture was
stirred for 12 hours, and extracted with DCM (2 × 30 ml). The organic extracts were

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DOI: 10.1039/C8MD00197A
combined, washed by brine (30 ml), dried over Na2SO4, filtered and concentrated
under reduced pressure to give a colorless oil as intermediate 8 which was used
directly for next step without further purification.
4.1.5. General procedure for the synthesis of intermediate 9
To a solution of substituted phenylacetonitrile 6 (14.8 mmol) and intermediate 8
(16.3 mmol) in DMF (25 mL) on an ice bath, sodium hydride (1.1 g, 44.4 mmol) was
added in portion. After addition, the reaction mixture was heated at 80 °C for 12ꢀ18
hours. This mixture was then cooled down and quenched by dropwise addition of H2O
(20 ml). After extraction with EA (3 × 30 ml), the organic extracts were combined,
washed with brine (2 × 30 ml), dried over Na2SO4, filtered, concentrated under
reduced pressure, and purified using column chromatography (PE/EA = 20/1) to
afford intermediate 9.
Tertꢀbutyl 4ꢀcyanoꢀ4ꢀ(4ꢀfluorophenyl)piperidineꢀ1ꢀcarboxylate (9a)
1
White solid, yield 43%. H NMR (400 MHz, DMSOꢀ
d
6) δ 7.43 (s, 4H), 3.76 (d,
J =
13.6 Hz, 2H), 2.98 (br s, 2H), 2.33 (d,
9H).
J = 13.6 Hz, 2H), 1.76–1.68 (m, 2H), 1.39 (s,
Tertꢀbutyl 4ꢀ(4ꢀchlorophenyl)ꢀ4ꢀcyanopiperidineꢀ1ꢀcarboxylate (9b)
1
White solid, yield 52%. H NMR (600 MHz, DMSOꢀ
d
6) δ 7.57 (d,
J
= 8.4 Hz, 2H),
7.52 (d,
1.90 (td,
J
= 8.4 Hz, 2H), 4.13 (br s, 2H), 3.01 (br s, 2H), 2.11 (d,
= 12.9, 4.2 Hz, 2H), 1.41(s, 9H).
J = 13.2 Hz, 2H),
J
Tertꢀbutyl 4ꢀ(4ꢀbromophenyl)ꢀ4ꢀcyanopiperidineꢀ1ꢀcarboxylate (9c)
1
White solid, yield 40%. H NMR (400 MHz, DMSOꢀ
d
6) δ 7.64 (d,
= 12.8 Hz, 2H), 3.01 (br s, 2H), 2.12 (d,
= 13.1, 4.2 Hz, 2H), 1.41 (s, 9H).
4.1.6. General procedure for the synthesis of intermediate 5
J
= 8.6 Hz, 2H),
7.53 (d,
J
= 8.6 Hz, 2H), 4.12 (d,
J
J
= 12.8
Hz, 2H), 1.90 (td,
J
To a solution of intermediate 9 (6.6 mmol) in ethanol (10 ml), aqueous NaOH
(10 M, 10 ml) was added. The reaction mixture was stirred at 60 °C for 48 hours,
cooled down and concentrated under reduced pressure. The residue was adjusted by
aqueous HCl (1 M) to pH 5ꢀ6, and white solid precipitate was collected by filtration,
washed with H2O (2 × 30 ml), and dried to afford the intermediate 5.

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1ꢀ(Tertꢀbutoxycarbonyl)ꢀ4ꢀ(4ꢀfluorophenyl)piperidineꢀ4ꢀcarboxylic acid (5a)
White solid, yield 86%. 1H NMR (400 MHz, DMSOꢀ
3.77 (d, = 13.6 Hz, 2H), 2.97 (br s, 2H), 2.34 (d,
2H), 1.39 (s, 9H).
1ꢀ(Tertꢀbutoxycarbonyl)ꢀ4ꢀ(4ꢀchlorophenyl)piperidineꢀ4ꢀcarboxylic acid (5b)
d
6) δ 12.82 (s, 1H), 7.43 (s, 4H),
J
J = 13.6 Hz, 2H), 1.77–1.69 (m,
White solid, yield 75%. 1H NMR (400 MHz, DMSOꢀ
3.76 (d, = 13.6 Hz, 2H), 2.98 (br s, 2H), 2.33 (d,
2H), 1.40 (s, 9H).
d
6) δ 12.81 (s, 1H), 7.42 (s, 4H),
J
J = 13.6 Hz, 2H), 1.74–1.68 (m,
4ꢀ(4ꢀBromophenyl)ꢀ1ꢀ(tertꢀbutoxycarbonyl)piperidineꢀ4ꢀcarboxylic acid (5c)
1
White solid, yield 87%. H NMR (600 MHz, DMSOꢀ
d
6) δ 12.80 (s, 1H), 7.54 (d,
= 13.8 Hz, 2H), 2.97 (br s, 2H), 2.34
= 13.8 Hz, 2H), 1.72–1.66 (m, 2H), 1.41 (s, 9H).
J =
8.4 Hz, 2H), 7.36 (d,
(d,
J = 8.4 Hz, 2H), 3.76 (d, J
J
4.1.7. General procedure for the synthesis of compounds 10, 14 and 18
To a solution of intermediate 5 (1.0 mmol) in DCM (6 ml), HBTU (417 mg, 1.1
mmol) and DIPEA (388 mg, 3.0 mmol) were added. After stirring at room
temperature for 15 min, intermediate 4 (1.0 mmol) was added. This reaction mixture
was stirred for 6 hours and washed with saturated NH4Cl (3 × 20 ml) and brine (30
ml). The organic layer was concentrated under reduced pressure to give an oily
residue. This residue was dissolved in DCM (4 ml), and TFA (2 ml) was added. After
stirring for 4 hours at room temperature, the mixture was concentrated under reduced
pressure, adjusted with saturated Na2CO3 to pH = 9, and extracted with DCM (3 × 20
ml). The organic extracts were combined, washed with brine (2 × 20 ml), and
concentrated under reduced pressure to give a solid residue which was purified by
column chromatography (DCM/MeOH = 50/1 ramping to 20/1) to give compounds
10aꢀ10l. Compounds 14aꢀ14d and 18aꢀ18d were prepared in a similar procedure as
mentioned above.
(4ꢀ(1
ꢀ4ꢀyl)methanone (10a)
White solid, yield 64%. Mp: 112–115 °C. 1H NMR (400 MHz, CD3OD) δ 8.20 (s, 1H),
8.11 (s, 1H), 7.36 (dd, = 8.5, 5.2 Hz, 2H), 7.14 (t, = 8.6 Hz, 2H), 3.69 (br s, 8H),
Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀyl)piperazinꢀ1ꢀyl)(4ꢀ(4ꢀfluorophenyl)piperidin
J
J

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2.38 (d, J
= 13.3 Hz, 2H), 2.05–1.84 (m, 2H). 13C NMR (100 MHz, CD3OD) δ 173.46,
157.08, 155.04, 154.69, 140.40, 133.41, 126.93, 115.80, 115.58, 99.78, 49.11, 43.92,
42.77, 35.57. HRMS (ESI) m/z calculated for C21H25FN7O [M+H]+: 410.2099, found
410.2098.
(4ꢀ(1
nꢀ4ꢀyl)methanone (10b)
White solid, yield 67%. Mp: 120–123 °C. H NMR (400 MHz, CD3OD) δ 8.21 (s,
1H), 8.12 (s, 1H), 7.42 (d, = 8.3 Hz, 2H), 7.34 (d, = 8.3 Hz, 2H), 4.05–3.45 (m,
8H), 3.20–3.04 (m, 4H), 2.42 (d,
= 13.5 Hz, 2H), 2.01 (br s, 2H). 13C NMR (100
Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀyl)piperazinꢀ1ꢀyl)(4ꢀ(4ꢀchlorophenyl)piperidi
1
J
J
J
MHz, MeOD) δ 172.82, 157.09, 155.04, 154.70, 142.69, 133.40, 132.97, 129.19,
126.69, 99.79, 48.88, 43.84, 42.43, 34.52. HRMS (ESI) m/z calculated for
C21H25ClN7O [M+H]+: 426.1804, found 426.1799.
(4ꢀ(1
nꢀ4ꢀyl)methanone (10c)
White solid, yield 62%. Mp: 149–152 °C. H NMR (400 MHz, CD3OD) δ 8.21 (s,
1H), 8.12 (s, 1H), 7.55 (d, = 8.6 Hz, 2H), 7.27 (d, = 8.6 Hz, 2H), 3.67 (br s, 8H),
3.04 (br s, 4H), 2.37 (d, = 12.9 Hz, 2H), 1.95 (t,
= 14.7 Hz, 2H). 13C NMR (100
Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀyl)piperazinꢀ1ꢀyl)(4ꢀ(4ꢀbromophenyl)piperidi
1
J
J
J
J
MHz, MeOD) δ 173.17, 157.09, 155.04, 154.69, 143.66, 133.42, 132.12, 127.06,
120.63, 99.79, 49.31, 43.33, 41.81, 35.35. HRMS (ESI) m/z calculated for
C21H25BrN7O [M+H]+: 472.1278, found 472.1282.
(4ꢀ(1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀyl)piperazinꢀ1ꢀyl)(4ꢀ(3,4ꢀdichlorophenyl)piper
idinꢀ4ꢀyl)methanone (10d)
White solid, yield 52%. Mp: 174–177 °C. 1H NMR (400 MHz, CD3OD) δ 8.20 (d,
J
=
10.6 Hz, 1H), 8.13 (s, 1H), 7.55 (d,
J
= 8.5 Hz, 1H), 7.51 (d,
J
= 1.9 Hz, 1H), 7.26 (dd,
J
= 8.4, 1.9 Hz, 1H), 3.80 (br s, 4H), 3.55 (br s, 4H), 3.14–3.01 (m, 4H), 2.39 (d,
J
=
13.3 Hz, 2H), 2.02–1.92 (m, 2H). 13C NMR (100 MHz, MeOD) δ 172.43, 157.08,
155.04, 154.70, 144.94, 133.47, 132.98, 131.14, 130.92, 127.07, 125.18, 99.80, 49.12,
45.46, 42.53, 34.91. HRMS (ESI) m/z calculated for C21H24Cl2N7O [M+H]+:
460.1414, found 460.1409.
(4ꢀ(3ꢀChloroꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀyl)piperazinꢀ1ꢀyl)(4ꢀ(4ꢀfluorophenyl

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)piperidinꢀ4ꢀyl)methanone (10e)
1
Offꢀwhite solid, yield 53%. Mp: 135–138 °C. H NMR (400 MHz, DMSOꢀ
d
6) δ 8.21
(s, 1H), 7.35–7.27 (m, 2H), 7.21 (t,
Hz, 4H), 2.18 (d,
= 12.8 Hz, 2H), 1.84–1.67 (m, 2H). 13C NMR (100 MHz,
DMSOꢀ 6) δ 172.62, 162.37, 159.95, 158.41, 154.14, 141.96, 129.69, 127.67, 116.29,
J = 8.7 Hz, 2H), 3.42 (br s, 8H), 2.82 (d, J = 13.2
J
d
116.08, 98.88, 49.51, 48.06, 43.76, 41.09, 37.08. HRMS (ESI) m/z calculated for
C21H24ClFN7O [M+H]+: 444.1709, found 444.1710.
(4ꢀ(3ꢀChloroꢀ1
)piperidinꢀ4ꢀyl)methanone (10f)
White solid, yield 51%. Mp: 184–188 °C. 1H NMR (400 MHz, DMSOꢀ
Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀyl)piperazinꢀ1ꢀyl)(4ꢀ(4ꢀchlorophenyl
d
6) δ 8.23 (s,
1H), 7.44 (d,
15.6 Hz, 4H), 2.17 (d,
DMSOꢀ
J
= 8.5 Hz, 2H), 7.30 (d,
J
= 8.5 Hz, 2H), 3.44 (br s, 8H), 2.82 (d,
J =
J
= 12.9 Hz, 2H), 1.76 (d, J
= 7.5 Hz, 2H). 13C NMR (100 MHz,
d
6) δ 172.52, 158.34, 158.10, 154.50, 144.71, 131.60, 130.10, 129.43, 127.68,
98.94, 49.65, 47.96, 43.70, 41.08, 36.84. HRMS (ESI) m/z calculated for
C21H24Cl2N7O [M+H]+: 460.1414, found 460.1416.
(4ꢀ(4ꢀBromophenyl)piperidinꢀ4ꢀyl)(4ꢀ(3ꢀchloroꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀyl
)piperazinꢀ1ꢀyl)methanone (10g)
1
White solid, yield 56%. Mp: 181–185 °C. H NMR (400 MHz, DMSOꢀ
d
6) δ 8.10 (s,
1H), 7.57 (d,
J
= 8.1 Hz, 2H), 7.24 (d,
J
= 8.2 Hz, 2H), 3.41 (br s, 8H), 2.82 (br s, 4H),
6) δ
2.15 (d, = 12.7 Hz, 2H), 1.73 (d,
J
J
= 9.7 Hz, 2H). 13C NMR (100 MHz, DMSOꢀ
d
172.45, 160.45, 158.41, 152.79, 144.79, 135.71, 132.41, 128.05, 127.84, 98.77, 49.70,
48.26, 43.84, 40.83, 36.99. HRMS (ESI) m/z calculated for C21H24ClBrN7O [M+H]+:
504.0909, found 504.0904.
(4ꢀ(3ꢀChloroꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀyl)piperazinꢀ1ꢀyl)(4ꢀ(3,4ꢀdichloroph
enyl)piperidinꢀ4ꢀyl)methanone (10h)
Offꢀwhite solid, yield 49%. Mp: 195–199 °C. 1H NMR (400 MHz, CD3OD) δ 8.22 (d,
J
= 15.1 Hz, 1H), 7.56 (d,
J
= 8.4 Hz, 1H), 7.51 (s, 1H), 7.27 (d,
J = 6.8 Hz, 1H), 3.61
(br s, 8H), 3.08 (br s, 4H), 2.39 (d,
J
= 13.4 Hz, 2H), 1.96 (br s, 2H). 13C NMR (100
MHz, MeOD) δ 172.31, 158.46, 154.59, 144.99, 132.97, 131.17, 131.14, 130.94,
127.14, 125.19, 122.38, 98.78, 49.19, 49.05, 42.55, 42.47, 34.84. HRMS (ESI) m/z

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calculated for C21H23Cl3N7O [M+H]+: 494.1024, found 494.1024.
(4ꢀ(3ꢀBromoꢀ1
)piperidinꢀ4ꢀyl)methanone (10i)
Offꢀwhite solid, yield 32%. Mp: 194–196 °C. 1H NMR (400 MHz, DMSOꢀ
(s, 1H), 7.31ꢀ7.21 (m, 4H), 3.42 (br s, 8H), 2.87 (br s, 4H), 2.17 (s, 2H), 1.78 (s, 2H).
13C NMR (100 MHz, DMSOꢀ
6) δ 172.63, 158.88, 157.61, 154.74, 141.82, 127.65,
Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀyl)piperazinꢀ1ꢀyl)(4ꢀ(4ꢀfluorophenyl
d
6) δ 8.27
d
118.64, 117.03, 116.23, 101.59, 49.40, 48.75, 43.63, 36.77. HRMS (ESI) m/z
calculated for C21H24BrFN7O [M+H]+: 490.1184, found 490.1186.
(4ꢀ(3ꢀBromoꢀ1
)piperidinꢀ4ꢀyl)methanone (10j)
Offꢀwhite solid, yield 43%. Mp: 180–184 °C. 1H NMR (400 MHz, CD3OD) δ 8.27 (s,
1H), 7.48 (d, = 8.4 Hz, 2H), 7.37 (d, = 8.5 Hz, 2H), 3.96–3.59 (m, 4H), 3.43 (d,
= 12.9 Hz, 2H), 3.36 (d, = 12.0 Hz, 2H), 3.21 (q, = 7.3 Hz, 4H), 2.57 (d, = 13.9
Hz, 2H), 2.17 (t, 6) δ 171.16, 158.80,
= 11.6 Hz, 2H). 13C NMR (100 MHz, DMSOꢀ
Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀyl)piperazinꢀ1ꢀyl)(4ꢀ(4ꢀchlorophenyl
J
J
J
J
J
J
J
d
157.01, 155.13, 142.52, 132.36, 129.77, 127.52, 119.08, 101.59, 48.02, 45.91, 41.54,
32.21. HRMS (ESI) m/z calculated for C21H24BrClN7O [M+H]+: 506.0888, found
506.0898.
(4ꢀ(3ꢀBromoꢀ1
H
ꢀpyrazolo[3,4ꢀ
d
]pyrimidinꢀ4ꢀyl)piperazinꢀ1ꢀyl)(4ꢀ(4ꢀbromophenyl
)piperidinꢀ4ꢀyl)methanone (10k)
1
White solid, yield 52%. Mp: 175–178 °C. H NMR (400 MHz, CD3OD) δ 8.26 (s,
1H), 7.62 (d,
J
= 8.5 Hz, 2H), 7.32 (d,
J
= 8.5 Hz, 2H), 3.96 – 3.59 (m, 4H), 3.46 –
3.35 (m, 4H), 3.21 (q,
J
= 7.3 Hz, 4H), 2.56 (d,
J
= 13.8 Hz, 2H), 2.19 (t, = 11.5 Hz,
J
2H). 13C NMR (100 MHz, MeOD) δ 171.48, 158.91, 156.41, 154.62, 141.99, 132.44,
126.96, 121.34, 119.03, 101.53, 47.93, 46.51, 41.70, 38.90, 32.07. HRMS (ESI) m/z
calculated for C21H24Br2N7O [M+H]+: 550.0383, found 550.0393.
(4ꢀ(3ꢀBromoꢀ1
enyl)piperidinꢀ4ꢀyl)methanone (10l)
Hoary solid, yield 34%. Mp: 195–198 °C. H NMR (400 MHz, CD3OD) δ 8.27 (s,
1H), 7.60 (d, = 8.5 Hz, 1H), 7.53 (d, = 2.2 Hz, 1H), 7.29 (dd, = 8.5, 2.2 Hz, 1H),
3.66 (br s, 8H), 3.28 (br s, 2H), 3.21 (t, = 11.8 Hz, 2H), 2.49 (d, = 13.4 Hz, 2H),
Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀyl)piperazinꢀ1ꢀyl)(4ꢀ(3,4ꢀdichloroph
1
J
J
J
J
J

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2.08 (t,
J
= 15.2 Hz, 2H). 13C NMR (100 MHz, MeOD) δ 171.49, 158.97, 156.50,
154.56, 144.10, 133.13, 131.31, 127.08, 125.16, 118.95, 101.57, 48.36, 47.05, 45.26,
41.94, 33.24. HRMS (ESI) m/z calculated for C21H23BrCl2N7O [M+H]+: 540.0499,
found 540.0510.
(4ꢀ(9
H
ꢀpurinꢀ6ꢀyl)piperazinꢀ1ꢀyl)(4ꢀ(4ꢀfluorophenyl)piperidinꢀ4ꢀyl)methanone
(14a)
1
White solid, yield 59%. Mp: 173–175 °C. H NMR (400 MHz, DMSOꢀ
d
6) δ 8.19 (s,
= 8.4 Hz, 2H), 3.90 (br s, 4H),
= 11.7 Hz, 2H), 2.29 (d, = 13.3
= 11.0 Hz, 2H). 13C NMR (100 MHz, DMSOꢀ
6) δ 171.63, 153.55,
1H), 8.11 (s, 1H), 7.61 (d,
3.39 (br s, 4H), 3.10 (d,
Hz, 2H), 1.99 (t,
J
= 8.4 Hz, 2H), 7.25 (d,
J
J
= 12.5 Hz, 2H), 2.96 (t,
J
J
J
d
152.07, 143.98, 138.97, 132.51, 127.97, 120.46, 119.26, 48.77, 44.51, 44.42, 44.18,
42.47, 34.17. HRMS (ESI) m/z calculated for C21H25FN7O [M+H]+: 410.2099, found
410.2097.
(4ꢀ(9
H
ꢀpurinꢀ6ꢀyl)piperazinꢀ1ꢀyl)(4ꢀ(4ꢀchlorophenyl)piperidinꢀ4ꢀyl)methanone
(14b)
1
White solid, yield 63%. Mp: 186–188 °C. H NMR (400 MHz, DMSOꢀ
d
6) δ 8.18 (s,
= 8.8 Hz, 2H), 4.28–3.69
= 12.8 Hz, 2H), 3.03 (t, = 11.7 Hz, 2H), 2.36
= 12.3 Hz, 2H). 13C NMR (100 MHz, DMSO) δ 171.52,
1H), 8.10 (s, 1H), 7.34 (dd,
(m, 6H), 3.58–3.41 (m, 2H), 3.21 (d,
(d, = 13.7 Hz, 2H), 2.09 (t,
J = 8.8, 5.3 Hz, 2H), 7.26 (t, J
J
J
J
J
162.65, 160.22, 153.48, 152.14, 140.24, 138.96, 127.66, 119.26, 116.59, 116.38,
48.15, 46.08, 44.59, 44.40, 43.12, 41.96, 33.25. HRMS (ESI) m/z calculated for
C21H25ClN7O [M+H]+: 426.1804, found 426.1806.
(4ꢀ(9
H
ꢀpurinꢀ6ꢀyl)piperazinꢀ1ꢀyl)(4ꢀ(4ꢀbromophenyl)piperidinꢀ4ꢀyl)methanone
(14c)
1
White solid, yield 67%. Mp: 234–236 °C. H NMR (400 MHz, DMSOꢀ
d
6) δ 8.18 (s,
= 8.4 Hz, 2H), 3.88 (br s, 4H),
= 13.1 Hz, 2H), 1.86 (t, = 10.0 Hz,
6) δ 172.08, 153.48, 152.07, 144.18, 138.97,
1H), 8.10 (s, 1H), 7.46 (d,
3.39 (br s, 4H), 3.01–2.83 (m, 4H), 2.23 (d,
2H). 13C NMR (100 MHz, DMSOꢀ
J = 8.4 Hz, 2H), 7.31 (d, J
J
J
d
131.75, 129.50, 127.67, 119.25, 49.18, 44.82, 44.49, 44.28, 43.12, 35.66. HRMS (ESI)
m/z calculated for C21H25BrN7O [M+H]+: 472.1278, found 472.1279.

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(4ꢀ(9
e (14d)
White solid, yield 69%. Mp: 203–205 °C. H NMR (400 MHz, DMSOꢀ
1H), 8.11 (s, 1H), 7.64 (d, = 8.4 Hz, 1H), 7.50 (s, 1H), 7.24 (d, = 9.9 Hz, 1H), 3.93
(br s, 8H), 2.95–2.78 (m, 4H), 2.20 (d, = 13.0 Hz, 2H), 1.79 (d, = 13.4 Hz, 2H).
13C NMR (100 MHz, DMSOꢀ
6) δ 171.84, 153.46, 152.08, 146.64, 139.03, 132.14,
Hꢀpurinꢀ6ꢀyl)piperazinꢀ1ꢀyl)(4ꢀ(3,4ꢀdichlorophenyl)piperidinꢀ4ꢀyl)methanon
1
d
6) δ 8.19 (s,
J
J
J
J
d
131.63, 129.72, 127.66, 126.52, 119.27, 49.58, 46.15, 44.61, 43.44, 36.39. HRMS
(ESI) m/z calculated for C21H24Cl2N7O [M+H]+: 460.1414, found 460.1411.
(4ꢀ(4ꢀFluorophenyl)piperidinꢀ4ꢀyl)(4ꢀ(quinazolinꢀ4ꢀyl)piperazinꢀ1ꢀyl)methanone
(18a)
1
White solid, yield 67%. Mp: 145–147 °C. H NMR (400 MHz, DMSOꢀ
d
6) δ 8.61 (s,
= 8.4 Hz, 2H), 7.53 (s, 1H), 7.24
= 8.4 Hz, 2H), 3.91–3.45 (m, 6H), 3.41–3.35 (m, 2H), 3.25 (d, = 12.6 Hz, 2H),
3.05 (t, = 12.1 Hz, 2H), 2.34 (d, = 13.6 Hz, 2H), 2.14 (t,
= 11.7 Hz, 2H). 13C
NMR (100 MHz, DMSOꢀ 6) δ 171.08, 164.10, 153.95, 151.69, 143.15, 133.31,
1H), 7.94 (d,
(d,
J = 8.3 Hz, 1H), 7.81 (s, 2H), 7.63 (d, J
J
J
J
J
J
d
132.74, 128.50, 127.71, 126.28, 125.54, 120.72, 116.06, 49.09, 48.06, 41.66, 32.56.
HRMS (ESI) m/z calculated for C24H27FN5O [M+H]+: 420.2194, found 420.2189.
(4ꢀ(4ꢀChlorophenyl)piperidinꢀ4ꢀyl)(4ꢀ(quinazolinꢀ4ꢀyl)piperazinꢀ1ꢀyl)methanone
(18b)
Offꢀwhite solid, yield 64%. Mp: 106–108 °C. 1H NMR (400 MHz, DMSOꢀ
d6) δ 8.60
(s, 1H), 7.93 (d, = 8.2 Hz, 1H), 7.80 (s, 2H), 7.52 (s, 1H), 7.38–7.16 (m, 4H), 3.80–
J
3.41 (m, 6H), 3.26–3.17 (m, 4H), 3.03 (t,
J
= 11.9 Hz, 2H), 2.34 (d,
J = 13.3 Hz, 2H),
13
2.08 (t,
J
= 11.8 Hz, 2H). C NMR (100 MHz, DMSOꢀ
d
6) δ 171.53, 164.11, 153.95,
151.65, 140.21, 133.30, 128.50, 127.54, 126.27, 125.69, 116.65, 116.44, 116.29,
48.99, 48.83, 48.20, 41.97, 33.27. HRMS (ESI) m/z calculated for C24H27ClN5O
[M+H]+: 436.1899, found 436.1902.
(4ꢀ(4ꢀBromophenyl)piperidinꢀ4ꢀyl)(4ꢀ(quinazolinꢀ4ꢀyl)piperazinꢀ1ꢀyl)methanone
(18c)
1
White solid, yield 72%. Mp: 189–191 °C. H NMR (400 MHz, DMSOꢀ
d
6) δ 8.61 (s,
= 8.6
1H), 7.94 (d,
J
= 8.3 Hz, 1H), 7.84–7.78 (m, 2H), 7.57–7.45 (m, 3H), 7.31 (d,
J

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Hz, 2H), 3.92–3.38 (m, 6H), 3.26 (br s, 4H), 3.07 (t,
J = 12.0 Hz, 2H), 2.36 (d, J =
13.8 Hz, 2H), 2.17 (t,
J
= 11.8 Hz, 2H). 13C NMR (100 MHz, DMSO) δ 171.15,
164.09, 153.94, 151.66, 142.67, 133.29, 132.29, 129.77, 128.50, 127.44, 126.27,
125.69, 116.29, 48.92, 48.11, 41.58, 32.38. HRMS (ESI) m/z calculated for
C24H27BrN5O [M+H]+: 482.1373, found 482.1374.
(4ꢀ(3,4ꢀDichlorophenyl)piperidinꢀ4ꢀyl)(4ꢀ(quinazolinꢀ4ꢀyl)piperazinꢀ1ꢀyl)methano
ne (18d)
1
White solid, yield 76%. Mp: 254–256 °C. H NMR (400 MHz, DMSOꢀ
d
6) δ 8.61 (s,
= 8.5 Hz, 1H), 7.56–7.48
= 8.5, 2.2 Hz, 1H), 3.94–3.31 (m, 7H), 3.27 (br s, 3H), 3.06 (t,
= 12.0 Hz, 2H), 2.37 (d, = 13.8 Hz, 2H), 2.18 (t,
= 11.6 Hz, 2H). 13C NMR (100
1H), 7.95 (d,
J = 8.3 Hz, 1H), 7.83–7.79 (m, 2H), 7.69 (d, J
(m, 2H), 7.25 (dd,
J
J
J
J
MHz, DMSO) δ 170.67, 164.09, 153.94, 151.68, 144.67, 133.29, 132.44, 131.96,
130.47, 128.51, 127.43, 126.32, 126.29, 125.69, 116.31, 49.00, 48.98, 48.17, 41.56,
32.31. HRMS (ESI) m/z calculated for C24H26Cl2N5O [M+H]+: 470.1509, found
470.1506.
4.2. In vitro kinase assay
In vitro AKT1 kinase inhibitory activity was evaluated via a Homogeneous
TimeꢀResolved Fluorescence (HTRF) assay (LANCE) using an AKT kinase kit
(Cisbio) in a 384ꢀwell plate. Each well was added subsequently with AKT1, STK
substrateꢀbiotin, tested compounds and ATP in kinase buffer (50 mM HEPES, 5
mM MgCl2, 1 mM DTT, 0.02% NaN3 and 0.01% BSA, pH 7.0), and incubated for
45 minutes at 25 °C. Finally, SaꢀXL665 and STK AbꢀCryptate were added to stop
the enzymatic step and further incubated for 2 hours to finish the detection process.
The ratio (665 nm/620 nm) was obtained using a microplate reader (Perkin Elmer,
USA).
4.3. Cell proliferation assays (MTT)
Human prostate cancer cell line PCꢀ3 was purchased from the American type
culture collection (ATCC), and cultured in RPMI 1640 medium containing 10% fetal
bovine serum (Gibco, USA) and 100 IU/mL penicillin and 100 ꢁg/mL streptomycin in
a humidified atmosphere with 5% CO2 at 37°C. All the compounds were evaluated for

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their antiꢀproliferative activity against PCꢀ3 cells using an MTT assay. PCꢀ3 cells
were seeded in a 96ꢀwell plate and cultured for 10 hours at 37 °C. Thereafter, cells
were treated with tested compounds at serial concentrations and DMSO (control), and
incubate for 72 hours. MTT (5 mg/mL, 10 ꢁL) was added to the culture and incubated
for another 4 hours. Subsequently, the formazan crystal was extracted with DMSO
and the absorbance (OD570) was detected with a microplate reader (BioꢀRad 680,
USA). The IC50 values was calculated with GraphPad Prism (version 5.0).
4.4. Kinase selectivity assay
Compounds are prepared to 50× final assay concentration in 100% DMSO, and
the working stock of compound is added to the assay well as the first component in
the reaction, followed by the remaining components. Kinases are diluted in specific
buffer prior to addition to the reaction mix. There is no preꢀincubation step between
the compound and the kinase prior to initiation of the reaction. The reaction is
initiated by the addition of the Mg/ATP mix. After incubation for 40 minutes at room
temperature, the reaction is stopped by the addition of phosphoric acid to a
concentration of 0.5%. 10 µL of the reaction is then spotted onto a P30 filtermat and
washed four times for 4 minutes in 0.425% phosphoric acid and once in methanol
prior to drying and scintillation counting.
The positive control wells contain all components of the reaction, except the
compound of interest, and DMSO (at a final concentration of 2%) is included in these
wells to control for solvent effects. The blank wells contain all components of the
reaction, with a reference inhibitor replacing the compound of interest. This abolishes
kinase activity and establishes the baseꢀline (0% kinase activity remaining).
4.5. Western blotting
Western blot analysis was carried out using a wholeꢀcell lysate. PCꢀ3 cells were
treated with DMSO, 10h (1, 5 and 10 ꢁM) or GSK690693 (1, 5 and 10 ꢁM) for 4
hours, respectively. Afterward, the cells were rinsed twice with iceꢀcold PBS and
lysed in a cell lysis buffer (50 mM Tris–HCl, pH 8.0; 1% Nonidet Pꢀ40; 0.5% sodium
deoxycholate; 0.1% SDS; 150 mM NaCl; 1 mM PMSF; and a protease inhibitor
cocktail). Protein concentration of each sample was measured by using bicinchoninic

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acid (BCA) protein assay reagent (Thermo Pierce, IL). 20 ug of lysate was loaded into
each lane. Antibodies for GSK3β and AKT (1:1000) were purchased from Santa
(Santa Cruz, USA). The secondary antibodies were horseradish peroxidase
(HRP)ꢀlinked goatꢀanti rabbit IgG (Santa Cruz, USA). After SDS–PAGE, proteins
were electroblotted onto a PVDF membrane. The blots were blocked with 5%
BSA/TBST for 1 hour at room temperature, probed with specific ABS overnight at
4°C, washed, incubated with linked goatꢀanti rabbit IgG (Santa Cruz, USA), and
probed with ECLꢀplus (GE Healthcare, Sweden). The immunoreactive bands were
detected using an image analyzer (LAS1000; Fuji Photo Film, Japan).
4.6. Cell apoptosis assay
Apoptosis was quantified by annexin VꢀFITC/propidium iodide (PI)ꢀbinding
assay. Cells were seeded in a 6ꢀwell plate with 1 ꢁM, 5 ꢁM or 10 ꢁM of 10h for 48
hours. Treated cells were washed twice with cold phosphateꢀbuffered saline (PBS)
and reꢀsuspended in binding buffer (100 µL), to which 2 µL of annexin VꢀFITC and 5
µL of PI were added. The samples were gently vortexed and incubated for 15 minutes
at room temperature in the dark. After addition of 200 ꢁL of binding buffer, samples
were immediately analyzed by flow cytometry using a FACScan flow cytometer
(Becton Dickinson, CA). The percentage of apoptotic cells was analysed using a
Flowjo software.
4.7. Human liver microsomes metabolic stability assay
Testosterone, diclofenac and propafenone were tested as control drugs in this
assay as all of them were substrates of cytochrome P450 enzymes. A DMSO solution
of the tested compound (10 mM, 10 ꢁl/well) and solution of microsome (80 ꢁl/well)
were added to a 96 well plate, this mixture was first incubated at 37
℃ for 10 min.
Then potassium phosphate buffer (100 mM, 10 ꢁl/well) was added and No CoꢀFactor
(NCF) remaining was evaluated after a further incubation of 60 min. After this
preꢀwarming process, NADPH regenerating system (10 ꢁl/well) was added and
remaining of each compound was tested at 5 time points (5 min, 10 min, 20 min, 30
min and 60 min). At each time point, stop solution (Including 100 ng/ml Tolbutamide

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and 100 ng/ml Labetalol, cold in 4
reaction. The sampling plates were shaked for approximate 10 min, and then samples
were centrifuged at 4000 rpm for 20 min under 4 to afford the supernatant (100 ꢁl)
for LC/MS test. Intrinsic clearance (CLint) and halfꢀlife (T1/2) values were then
calculated. CLint(mic) = 0.693/halfꢀlife/mg microsome protein per ml. CLint(liver)
℃, 300 ꢁl/well) was added to terminate the
℃
=
CLint(mic) × (45 mg microsomal protein/g liver weight) × (20 g liver weight/kg body
weight).
Conflict of interest
The authors declare no conflict of interest.
Acknowledgement
This research is funded by Key research and development project of Shandong
Province (No. 2017CXGC1401).
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Compound 10h is a potent pan-AKT inhibitor showing cellular AKT inhibition and
apoptosis induction in PC-3 prostate cancer cells.