Structure-based design, synthesis and biological evaluation of diphenylmethylamine derivatives as novel Akt1 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.11.036

A series of diphenylmethylamine derivatives were rationally designed, synthesized and biologically evaluated. Most of them exhibited moderate to good Akt1 inhibitory activities, as well as promising anti-proliferative efficacy against cancer cell lines. Besides, molecular docking studies were carried out to probe their binding modes with Akt1. Further kinase selectivity studies of compound 22c were performed, indicating its excellent selectivity against Aurora A, Drak, IKKβ, GSK3β, SYK and JAK2, and moderate selectivity against PKC and BRAF. Finally, a refined pharmacophore model was generated using the most active compounds 2, 12c and 22c via application of HipHop program.

Full text of DOI:10.1016/j.ejmech.2013.11.036

European Journal of Medicinal Chemistry 73 (2014) 167e176
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structure-based design, synthesis and biological evaluation of
diphenylmethylamine derivatives as novel Akt1 inhibitors
,
Tao Liu a, Wenhu Zhan a, Yanming Wang a, Liangren Zhang b, Bo Yang c, Xiaowu Dong a
**
*
,
,
Yongzhou Hu a
a ZJU-ENS Joint Laboratory of Medicinal Chemistry, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences,
Zhejiang University, Hangzhou 310058, China
b State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100083, China
c Department of Pharmacology, Zhejiang University, Hangzhou 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of diphenylmethylamine derivatives were rationally designed, synthesized and biologically
evaluated. Most of them exhibited moderate to good Akt1 inhibitory activities, as well as promising anti-
proliferative efficacy against cancer cell lines. Besides, molecular docking studies were carried out to
probe their binding modes with Akt1. Further kinase selectivity studies of compound 22c were per-
Received 29 August 2013
Received in revised form
15 November 2013
Accepted 24 November 2013
Available online 19 December 2013
formed, indicating its excellent selectivity against Aurora A, Drak, IKKb, GSK3b, SYK and JAK2, and
moderate selectivity against PKC and BRAF. Finally, a refined pharmacophore model was generated using
the most active compounds 2, 12c and 22c via application of HipHop program.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Akt1 inhibitor
Diphenylmethylamine
Pharmacophore
Molecular docking
Anti-proliferative activity
1. Introduction
AZD5363 [19] and AT13148 [20]). However, there are still some
issues that need to be resolved, such as selectivity and safety.
PKB/Akt, a serine/threonine kinase, functions as a key signaling
node in the PI3K/Akt/mTOR pathway that plays a critical role in cell
growth, proliferation, motility and survival [1e6]. Frequent acti-
vation of Akt kinases was observed in a wide assortment of human
solid tumors and hematological malignancies [7,8]. Akt consists of
three isoforms, known as Akt1, Akt2 and Akt3. Although these three
isoforms are encoded by separate genes, the identity of their overall
sequence is very high (w80%) [9,10]. Up to now, accumulating re-
searches have validated that the inhibition of Akt1, Akt2 and Akt3
activities is closely linked to the decrease of tumor growth, the
apoptosis of cancer cells, as well as the reverse of tumor resistance
[11,12]. Neverthlessly, a strong interest has been stimulated in tar-
geting Akt for cancer therapy, leading to the discovery of a number
of Akt inhibitors [13e15]. Currently, several Akt inhibitors are being
evaluated in clinical trials (Fig. 1), including phosphatidylinositol
(3,4,5)-trisphosphate mimics (e.g. Perifosine) [16,17], allosteric in-
hibitors (e.g. MK-2206) [18] and ATP-competitive inhibitors (e.g.
Despite these, there continues to be a great interest in the devel-
opment of novel Akt inhibitors. Owing to the availability of high
resolution structures of Akt [16,19e21], structure-based drug
design is a promising method to accelerate the discovery process of
drug candidates.
In our previous studies, a virtual evaluation system, which
consists of pharmacophore and molecular docking models, has
been developed and demonstrated as a good tool in identification
of Akt1 inhibitors [21]. As a continuous study (Fig. 2), the analogs of
1 [22] were generated “in silico” and submitted to virtual evalua-
tion. The preliminary results revealed that both of 2 and 3 still
showed good scores in pharmacophore and molecular docking
models, when bulky alkyl group was introduce onto primary amine
of 1 (e.g. isopropyl for 2 and cyclopropyl for 3). Thus, there would
be a novel optimization site for compound 1, which is much
different from that of others’ work [22e24]. Moreover, the hy-
pothesis was validated by the good Akt1 inhibitory activities of
compounds 2 and 3 (IC50, 2: 0.16 mM; 3: 0.47 mM), after these two
compounds were synthesized and biologically evaluated. Accord-
ingly, more extensively modification of diphenylmethylamine de-
rivatives was performed, in attempt to improve their potency and/
* Corresponding author. Tel./fax: þ86 571 88981051.
** Corresponding author. Tel./fax: þ86 571 88208460.
E-mail addresses: dongxw@zju.edu.cn (X. Dong), huyz@zju.edu.cn (Y. Hu).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.11.036

168
T. Liu et al. / European Journal of Medicinal Chemistry 73 (2014) 167e176
Afterward, all of the designed compounds were synthesized and
biologically evaluated. Insight into the structureeactivities rela-
tionship (SAR), molecular docking studies were further performed
and discussed. Moreover, a refined pharmacophore model was
obtained by using newly synthesized compounds.
2. Computational methods
2.1. General methodology
Docking studies were achieved using Discovery Studio 2.5/
Ligandfit module (Accelrys Inc., San Diego, CA). Pharmacophore
mapping was performed using Discovery Studio 2.5/Pharmaco-
phore module (Accelrys Inc., San Diego, CA). Diverse conforma-
tional models for each compound were generated by Discovery
Studio 2.5/Pharmacophore module (Accelrys Inc., San Diego, CA)
using an energy range of 20 kcal/mol of the calculated potential
energy minimum. Specify 250 as the maximum number of con-
formers for each molecule to ensure maximum coverage of the
conformational space. The refined pharmacophore model (HipHop)
was also generated using the Discovery Studio 2.5/Pharmacophore
module (Accelrys Inc., San Diego, CA).
2.2. Molecular docking with LigandFit
The docking site of Akt1 enzyme (PDB entry code: 3OCB) was
derived using the LigandFit site search utility in Discovery studio
2.5 [25]. For the generation of the ligand’s conformations, we used
variable numbers of Monte Carlo simulations. All the calculations
during the docking steps were performed under the PLP Force Field
formalism. A short rigid body minimization was then performed
and 50 poses for each ligand were saved. Scoring was performed
with a set of scoring functions (including Dock_score, LigScore2,
PLP1, Jain and PMF) implemented in LigandFit module. The
Fig. 1. The structure of representative Akt inhibitors under clinical trials.
or selectivity. Different substituents, including hydrophobic, hy-
drophilic, aliphatic and aromatic groups were introduced to pri-
mary amine of diphenylmethylamine skeleton, while replacing the
purine moiety of the parent compound with pyridine and pyrazole,
which are privileged fragments of numerous kinase inhibitors.
Fig. 2. The evolution of novel diphenylmethylamine derivatives as Akt inhibitors.

T. Liu et al. / European Journal of Medicinal Chemistry 73 (2014) 167e176
169
combination of consensus scoring method and the interaction
mode was applied to select the preferable output conformation.
water under microwave irradiation [28]. Afterward the 6-
chloropurine 13 and 4-iodopyrazole 18 were protected by 3,4-
dihydro-2H-pyran in presence of p-toluenesulfonic acid in CH2Cl2
to give 14 and 19, respectively. Compounds 15 and 20 were ob-
tained by Suzuki coupling of 14 and 19 with 6 and 5, respectively.
Then, chlorination of 9, 15 and 20 with thionyl chloride in CH2Cl2
gave compounds 10, 16 and 21, respectively. Alkalization of 10, 16
and 21 with appropriate amine under microwave irradiation yiel-
ded target compounds 2, 3, 11, 12, 17 and 22.
2.3. Generation of pharmacophore hypotheses with HipHop
Discovery Studio 2.5/HipHop module [26] was employed to
evaluate the common features required for highly potency of Akt1
inhibitors. A training set consisting of compounds 2, 12c and 22c
was submitted for pharmacophore model generation based on
common chemical features. All the parameters were kept at the
default setting except the “best method” for the conformation
generation and fitting.
3.2. Pharmacological activities
The Akt1 inhibitory activities of compounds 11aec, 12aeg, 17ae
c and 22aej were determined via a competitive fluorescence po-
larization kinase activity assay. H-89 [29] and staurosporine [30]
were employed as positive control. Table 1 presents the biological
results of tested compounds. With an insight into the modification
of purine moiety of compound 1, except for 12e and 22f, com-
pounds 12, 17, and 22 bearing 4-pyridine, purine and pyrazole
moieties showed much more potent Akt1 inhibitory activities than
that of compound 11 bearing 3-pyridine moieties. It is suggested
that the hydrogen bond interactions of heterocyclic moiety with
Akt1 are essential. Comparing with the substituent on amino group
of 2 and 22c, the more bulky substituent (e.g. N-cyclohexyl for 17b
and N-benzyl for 17c) and the longer substituent (e.g. N-isobutyl for
17a, N-(2-hydroxy)ethyl for 22h and N-(2-amino)ethyl for 22i)
3. Results and discussion
3.1. Chemistry
The synthetic route for compounds 2, 3, 11aec, 12aeg, 17aec
and 22aej is outlined in Scheme 1. Treatment of 4 with 4-
bromophenyl magnesium bromide in Et2O yielded diphenylme-
thanol 5, which was successively transformed to the corresponding
borate 6 by reaction with bis(pinacolato)diboron in presence of
Pd(PPh3)2Cl2 and KOAc in dioxane. Reaction of pyridine 7 with n-
BuLi and triisopropyl borate in THF, followed by hydrolysis, yielded
boronic acid 8 [27]. Compound 9 was obtained by Suzuki coupling
of 8 with 5 in the presence of Pd(PPh3)4 and NaHCO3 in DMF and
Scheme 1. The synthetic route for target compounds 2, 3, 11aec, 12aeg, 17aec and 22aej. Reagents and conditions: (a) 4-bromophenyl magnesium bromide, Et2O; (b) Pd(PPh3)2Cl2,
bis(pinacolato)diboron, KOAc, dioxane; (c) n-BuLi, triisopropyl borate, THF; (d) Pd(PPh3)4, NaHCO3, DMF/H2O, microwave; (e) SOCl2, CH2Cl2; (f) appropriate amine, K2CO3, KI, CH3CN,
microwave; (g) 3,4-dihydro-2H-pyran, TsOH$H2O, CH2Cl2; (h) Pd(PPh3)4, Na2CO3, dioxane/H2O, microwave.

170
T. Liu et al. / European Journal of Medicinal Chemistry 73 (2014) 167e176
Table 1
significant changes in overall total GSK3b and GAPDH protein levels
were observed (Fig. 3B).
The Akt1 inhibitory and in vitro anti-proliferative activities of compounds 2, 3, 11ae
c, 12aeg, 17aec and 22aej, against OVCAR-8, HL60 and HCT-116 cells.
Compd.
Akt1 inhibitory
activity
Anti-proliferative activity (IC50, mM)
3.3. Kinase selectivity assay
OVCAR-8
HL60
HCT-116
IC50, mM
The selectivity profile of 22c is summarized in Table 2. The non-
selective kinase inhibitor staurosporine was used as a control.
Overall, compound 22c demonstrates excellent selectivity against
distinct families of kinases including Aurora A, Drak, IKK
SYK and JAK2, for which the IC50 values were greater than 2.5 m
However, selectivity versus other closely related kinases such as
BRAF1 (22-fold) and PKC (85-fold) are moderate, which may be
attributed to the high degree of homology in the ATP-binding
pocket.
(inhibition, %)a
Staurosporine
H-89
2
0.04
1.72
0.16
0.47
(10%)a
(19%)a
(20%)a
0.22
1.43
0.17
1.26
(26%)a
1.25
0.74
0.64
0.74
0.37
0.90
0.038
0.48
1.1
/
/
/
10.5
17.0
30.6
N.T.b
N.T.b
N.T.b
22.8
16.3
16.1
15.6
N.T.b
18.5
9.9
11.4
13.2
11.9
N.T.b
N.T.b
N.T.b
13.9
3.3
15.7
8.5
b
, GSK3
b,
3
13.4
N.T.b
N.T.b
N.T.b
17.3
15.5
14.4
12.4
N.T.b
16.3
7.7
M.
11a
11b
11c
12a
12b
12c
12d
12e
12f
17a
17b
17c
22a
22b
22c
22d
22e
22f
22g
22h
22i
3.9
3.2
3.4. Molecular docking studies using LigandFit
N.T.b
12.5
5.3
With the aim to further probe the SAR of tested compounds, the
interaction mode between Akt1 and the good Akt1 inhibitors 12a,
12c, 17aec, 22aed and 22geh, the IC50 value of which were lower
8.6
11
14.4
16.2
11.6
14.1
8.9
12.5
12.2
8.1
12.1
10.7
5.4
than 1 mM, were proposed and simulated by Discovery studio 2.5/
LigandFit module. All of the scoring functions (DOCK_Score [25],
LigScore2 [31], ePLP1 [32] and ePMF [33]) available in LigandFit
program were applied for the evaluation of accuracy and reliability
of molecular docking. Besides, the key interactions formed between
Akt1 and the docked ligands were also emphasized as essential
scoring parameters. Table 3 presents the molecular docking results
of synthesized compounds. Most of the simulated compounds
showed good consensus results, with good performance in more
than three scoring functions. As exemplified by 22c, the scores from
DOCK_Score, LigScore2, PLP1 and PMF of 22c (DOCK_Score: 83.6;
LigScore2: 5.0; ePLP1: 88.0; ePMF: 70.7) are preferable. Further,
the interactions with the key amino acid residues of Akt1 were
highlighted and the distances were recorded. The interaction mode
between Akt1 and 2, 12c and 22c which exhibited the most potent
inhibitory activities are presented in Fig. 4AeC. Two hydrogen
bonds are formed between 22c (or 2) and Glu228 and Ala 230 of
Akt1, respectively, while one hydrogen bond is formed between 12c
and Ala 230 of Akt1. All of the secondary amine of 2,12c and 22c can
form ionic interaction with Glu234 of Akt1. In addition, the hy-
drophobic force between 4-chlorophenyl group of 2, 12c and 22c
and Phe161 of Akt1 serves to increase the thermal stability of the
complex. Overall, the conformation and orientation of 2, 12c and
22c are similar when their structure were mapping together
(Fig. 4D).
8.1
5.3
7.9
5.5
7.8
15.6
26.8
N.T.b
8.3
27.59
10.73
2.1
(29%)a
1.32
0.65
0.82
N. T.b
11.9
34.68
11.05
N.T.b
5.3
N.T.b
N.T.b
a
The inhibition rate of Akt1 activities was determined at a concentration of
10
m
M.
b
N.T. means no test.
decreased the Akt1 inhibitory activities. In addition, compounds
12e and 22f bearing N-phenyl group even showed no Akt1 inhibi-
tory activity (inhibition rate of 12e and 22f were 26% and 29%,
respectively, at a concentration of 10
mM), suggesting that aryl
substituent on amino group of diphenylmethylamine skeleton is
unfavorable. Overall, the N-tert-butyl group on diphenylmethyl-
amine is the best substituent in both of pyridine and pyrazole series
for Akt1 inhibitory activity (IC50, 12c IC50; 0.17
0.038 M).
mM, 22c IC50:
m
Furthermore, compounds 12aed, 12f, 17aec, 22aee and 22gei
with good Akt1 inhibitory activities were tested for their in vitro
anti-proliferative activities against three human cancer cell lines
including OVCAR-8 (Ovarian Adenocarcinoma), HL-60 (Promyelo-
cytic Leukemia) and HCT116 (Human Colon Cancer). The results of
them together with compounds 2 and 3 are summarized in Table 2.
As expected, all of them exhibited moderate to potent anti-
proliferative activities, especially for HL60 cell lines. Compound
22c, the most potent one, also showed promising anti-proliferative
activities against OVCAR-8 (IC50 ¼ 8.1
HCT-116 (IC50 ¼ 8.9 M) cells, which are better than that of com-
pound 2 against OVCAR-8 (IC50 ¼ 17.0 M), HL60 (IC50 ¼ 13.2 M),
HCT-116 (IC50 ¼ 8.5 M) cells. The preliminary mechanisms on the
3.5. Refined pharmacophore model established by HipHop program
With aim to improve the prediction ability of the pharmaco-
phore model for diphenylmethylamine derivatives, three highly
active compounds 2, 12c and 22c with different heterocyclic
mM), HL60 (IC50 ¼ 5.3
mM),
m
Table 2
m
m
Selectivity profile of 22c in comparison with that of staurosporine.
m
Test kinases
Compd. 22c IC50
,
m
M (inhibition, %)a
Staurosporine IC50, mM
anti-proliferative activities against Ovcar-8 cells of 22c were stud-
ied. At first, the effect of 22c on the apoptosis of OVCAR-8 cells
assessed using propidium iodide (PI) staining of the sub-G1 cell
population. As shown in Fig. 3A, exposure of OVCAR-8 cells to 22c
for 48 h led to the apoptosis of 66.8% cells, while the negative
control only induced the apoptosis of 1.2% cells. Then, the effect of
22c on Akt phosphorylation was tested on OVCAR-8 cells. Treat-
ment of OVCAR-8 cells with 22c resulted in down-regulation of
Akt1
0.038
0.040
0.015
0.015
0.001
0.026
0.001
0.072
0.060
0.002
Aurora A
BRAF1
PKC
(28.3%)a
0.88
3.4
Drak
(12.0%)a
(1.8%)a
(7.2%)a
(0.98%)a
(19.5%)a
IKKb
GSK3
SYK
JAK2
b
phosphorylated Akt (Ser-473), as well as phosphorylated GSK3
which is one of the major downstream targets of Akt, while no
b
a
The inhibition rate was determined at a concentration of 0.8 mg/mL.

T. Liu et al. / European Journal of Medicinal Chemistry 73 (2014) 167e176
171
Fig. 3. (A) cell apoptotic index analyses of OVCAR-8 cells exposed to 22c; (B) treatment with 22c-caused decrease of Akt and GSK3b phosphorylation in OVCAR-8 cells.
moieties attached to diphenylmethylamine skeleton were chosen
as a training set to generate the refined pharmacophore model.
Ten pharmacophore models were obtained and the best phar-
macophore model (Hypo1) with the highest score 32.76 con-
tained four chemical features: one pos-ionizable (PI), two
hydrophobic (HP), and one hydrogen bond acceptor (HBA)
(Fig. 5A). Compound 22c, the most potent one, mapped well with
all of the four features of Hypo1 (Fig. 5B). It is suggested that the
refined pharmacophore would be good tool not only in further
structural optimization of diphenylmethylamine derivatives as
potent and selective Akt1 inhibitors, but also in the identification
of novel Akt1 inhibitors.
5. Experimental
5.1. Chemistry
Melting points were obtained on a B-540 Büchi melting-point
apparatus and are uncorrected. 1H NMR spectra were recorded on
a 500 MHz 1H and 400 MHz. 13C NMR were recorded on a 125 MHz
and 100 MHz spectrometer, respectively (chemical shifts are given
in ppm (d) relative to TMS as internal standard, coupling constants
(J) are in hertz (Hz), and signals are designated as follows: s, singlet;
d, doublet; t, triplet; m, multiplet; br s, broad singlet, etc.). Mass
spectral data were obtained on an Esquire-LC-00075 spectrometer.
High resolution mass spectra were measured on an Agilent 1290
HPLC-6224 Time of Fight Mass Spectrometer.
4. Conclusion
5.1.1. (4-Bromophenyl)(4-chlorophenyl)methanol 5
The rational strategy combined with virtual evaluation and
experimental feedback were performed for the structural optimi-
zation of diphenylmethylamine derivative as potent Akt1 in-
hibitors. Particularly, the modification on the novel site of
diphenylmethylamines was extensively carried out. The biological
assay reveals that most of the synthesized compounds possessed
both of good Akt1 inhibitory activities and anti-proliferative ac-
tivities against cancer cells. Favorable selectivity profile of repre-
sentative compound 22c was also observed. Therefore, the most
promising compounds 2, 12c and 22c would be interesting leads for
further structureeactivity relationship exploration and structurally
optimization. Furthermore, the HipHop pharmacophore model was
constructed based on the common features of highly active com-
pounds 2, 12c and 22c. The application of this refined pharmaco-
phore model is undergoing for the development of Akt1 inhibitors
with novel skeleton.
To
a cooled solution of 4-bromobenzaldehyde (10.4 g,
56 mmol) in dry THF (30 ml) was added dropwise 4-
chlorophenylmagnesium bromide (60 ml, 1 N solution in THF,
60 mmol). The solution was stirred for 2 h, and then saturated
ammonium chloride (300 ml) was added, followed by ethyl ac-
etate (250 ml). The layers were separated, and the organic frac-
tion was washed with water (100 ml), then dried, concentrated
and purified by flash column chromatography to afford the
desired product (yield, 62%) as a white solid, mp 100e102 ꢀC; 1H
NMR (500 MHz, CDCl3)
d
7.47 (d, J ¼ 8.3 Hz, 2H), 7.31 (q,
J ¼ 8.4 Hz, 4H), 7.24 (d, J ¼ 8.2 Hz, 2H), 5.78 (s, 1H). ESI-MS (m/z):
297 [M þ 1]þ.
5.1.2. (4-Chlorophenyl)(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)phenyl)methanol 6
To a solution of compound 5 (297 mg, 1 mmol) in dioxane (5 ml)
was added bis(pinacolato)diboron (280 mg, 1.1 mmol),
Pd(PPh3)2Cl2(23.65 mg, 0.0337 mmol), and KOAc (252 mg, 3 mmol).
The reaction mixture was heated to 85 ꢀC in a sealed tube for 12 h.
The reaction solution was poured into H2O (20 ml) and extracted
with ethyl acetate (15 ml ꢁ 3). The combined organic layers were
dried over anhydrous Na2SO4, concentrated under vacuum and
purified on silica gel to afford the title compound as a white solid
Table 3
The molecular docking results of 2, 3, 12a, 12c, 17aec, 22aed and 22geh.
ꢀ
Compd. DOCK_ LigScore2 ePLP1 ePMF Key interaction (distance, A)
Score
Ala232
Glu230
Glu234
2
3
96.3
96.8
92.5
89.0
97.6
99.1
104.8
84.3
83.4
83.6
86.0
87.7
89.7
2.9
4.0
3.5
4.4
3.3
4.28
3.1
4.7
4.91
5.0
4.6
5.1
5.0
103.8 100.0 2.08
2.08
2.06
/
4.42
4.44
4.03
3.45
4.14
4.40
4.19
3.02
3.26
2.81
2.64
3.03
4.32
103.7
97.5
93.3
104.1
100.9
108.8
87.7
91.2
87.1
90.8
92.6
92.3
78.0 2.10
(yield, 61%), mp 116e118 ꢀC. 1H NMR (500 MHz, DMSO)
d 7.64e7.59
12a
12c
17a
17b
17c
22a
22b
22c
22d
22h
22i
77.8 2.08
72.3 1.97
83.0 1.92
78.9 2.07
70.1 2.59
63.6 2.00
61.3 2.21
66.1 1.91
74.5 1.88
69.5 2.09
66.7 2.05
(m, 2H), 7.41e7.33 (m, 6H), 5.72 (d, J ¼ 3.8 Hz, 1H), 1.27 (s, 12H). ESI-
/
MS (m/z): 345 [M þ 1]þ.
1.82
2.03
2.73
2.23
2.59
2.25
2.25
2.48
2.37
5.1.3. General procedure for the synthesis of compound 8
To a solution of compound 5 (100 mg, 0.33 mmol) in 4 ml
dioxane/H2O (3:2, v/v) was added compound 7 (62 mg, 0.5 mmol),
tetrakistriphenylphosphane Pd (0) (5 mol%), and NaHCO3 (126 mg,
1.5 mmol). The reaction mixture was heated to 100 ꢀC in a sealed
tube for 6 h. The reaction solution was poured into H2O (25 ml) and

172
T. Liu et al. / European Journal of Medicinal Chemistry 73 (2014) 167e176
Fig. 4. Interaction mode between Akt1 and 22c (A), 2 (B), 12c (C) with Akt1, hydrogen bond interaction was highlighted using yellow dash line, hydrophobic force was highlighted
using yellow dash line; (D) the aliment of 2, 12c and 22c. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
extracted with ethyl acetate (15 ml ꢁ 3). The combined organic
5.1.3.2. Pyridin-4-ylboronic acid 8b. Yellow solid (yield, 78%), mp:
layers were dried over Na2SO4, concentrated under vacuum and
153e155 ꢀC. 1H NMR (500 MHz, CDCl3)
d 8.62 (s, 2H), 7.55 (dd,
purified on silica gel to afford title compounds.
J ¼ 6.5, 1.7 Hz, 2H), 7.51e7.44 (m, 4H), 7.41e7.34 (m, 2H), 7.24 (d,
J ¼ 8.4 Hz, 2H), 5.03 (s, 1H). ESI-MS (m/z): 296 [M þ 1]þ.
5.1.3.1. Pyridin-3-ylboronic acid 8a. Pale yellow solid (yield, 91%),
mp: 152e155 ꢀC. 1H NMR (500 MHz, DMSO)
d
8.89 (d, J ¼ 1.9 Hz,
5.1.4. General procedure for the synthesis of compounds 14 and 19
A suspension of 6-chloro-9H-purine (or 4-iodo-1H-pyrazole)
(13 mmol) and 4-methylbenzenesulfonic acid (0.12 g, 0.65 mmol) in
EtOAc (25 ml) was treated with 3,4-dihydro-2H-pyran (3.54 ml,
1H), 8.58 (dd, J ¼ 4.8, 1.3 Hz, 1H), 8.16e8.05 (m, 1H), 7.69 (d,
J ¼ 8.3 Hz, 2H), 7.54e7.48 (m, 3H), 7.44 (d, J ¼ 8.5 Hz, 2H), 7.41e7.36
(m, 2H), 5.76 (s, 1H). ESI-MS (m/z): 296 [M þ 1]þ.
Fig. 5. Hypo1 and its mapping to representative compounds. (A) The topological features of Hypo1: the cyan contour represents hydrophobic (HY), green contour represents
hydrogen bond acceptor (HBA), and red contour represents pos-ionizable (PI) features; (B) compound 22c mapped with Hypo1. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)

T. Liu et al. / European Journal of Medicinal Chemistry 73 (2014) 167e176
173
39 mmol). The mixture was heated at 90 ꢀC and the solid slowly
dissolved over 1 h. The flask was removed from the oil bath and the
cloudy yellow solution was filtered and concentrated under vac-
uum. The pale yellow residue was purified by flash chromatography
to give title compound.
J ¼ 4.8 Hz, 1H), 7.86e7.80 (m, 1H), 7.55e7.48 (m, 4H), 7.41 (d,
J ¼ 8.0 Hz, 2H), 7.35 (dd, J ¼ 8.0, 4.8 Hz, 1H), 7.29 (d, J ¼ 8.0 Hz, 2H),
4.85 (s, 1H), 2.48e2.38 (m, 2H), 1.81 (dq, J ¼ 13.3, 6.6 Hz, 1H), 0.94
(d, J ¼ 6.6 Hz, 6H). ESI-MS (m/z): 351 [M þ 1]þ, HRMS (ESI): m/z
calcd for (C22H23ClN2 þ H)þ: 351.1628; found: 351.1630.
5.1.4.1. 6-Chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine
14.
5.1.6.2. N-((4-Chlorophenyl)(4-(pyridin-3-yl)phenyl)methyl)-2-
White solid (yield, 89%), mp 70e71 ꢀC. 1H NMR (500 MHz, CDCl3)
methylpropan-2-amine 11b. Yellow solid (yield, 29%), mp: 100e
102 ꢀC. 1H NMR (500 MHz, CDCl3)
d 8.80 (s, 1H), 8.57 (s, 1H), 7.86e
d
8.76 (s, 1H), 8.35 (s, 1H), 5.80 (dd, J ¼ 10.5, 2.4 Hz, 1H), 4.24e4.15
(m, 1H), 3.80 (td, J ¼ 11.7, 2.7 Hz, 1H), 2.22e2.15 (m, 1H), 2.14e2.01
(m, 2H), 1.86e1.73 (m, 2H), 1.71e1.64 (m, 1H). ESI-MS (m/z): 239
[M þ 1]þ.
7.80 (m, 1H), 7.55e7.46 (m, 4H), 7.43 (d, J ¼ 8.0 Hz, 2H), 7.34 (dd,
J ¼ 8.0, 4.8 Hz, 1H), 7.27 (d, J ¼ 8.0 Hz, 2H), 5.08 (s, 1H), 1.11 (s, 9H).
ESI-MS (m/z): 351 [M
þ
1]þ. HRMS (ESI): m/z calcd for
(C22H23ClN2 þ H)þ: 351.1628; found: 351.1635.
5.1.4.2. 4-Iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole
19.
Yellow oil (yield, 80%). 1H NMR (500 MHz, CDCl3)
d
7.67 (s, 1H), 7.55
5.1.6.3. N-((4-Chlorophenyl)(4-(pyridin-3-yl)phenyl)methyl)cyclo-
hexanamine 11c. Yellow solid (yield, 39%), mp: 104e105 ꢀC. 1H
(s, 1H), 5.38 (dd, J ¼ 8.2, 3.7 Hz, 1H), 4.08e3.99 (m, 1H), 3.73e3.63
(m, 1H), 2.14e1.96 (m, 3H), 1.75e1.58 (m, 3H). ESI-MS (m/z): 279
[M þ 1]þ.
NMR (500 MHz, CDCl3)
d
8.81 (d, J ¼ 1.9 Hz, 1H), 8.57 (d, J ¼ 3.6 Hz,
1H), 7.88e7.80 (m, 1H), 7.52 (d, J ¼ 8.0 Hz, 2H), 7.47 (d, J ¼ 8.0 Hz,
2H), 7.37 (d, J ¼ 8.0 Hz, 2H), 7.35e7.32 (m, 1H), 7.30e7.27 (m, 2H),
5.07 (s, 1H), 2.41 (dd, J ¼ 11.6, 8.1 Hz, 1H), 1.97 (d, J ¼ 10.1 Hz, 2H),
1.70 (d, J ¼ 6.0 Hz, 2H), 1.22e1.03 (m, 6H). ESI-MS (m/z): 377
[M þ 1]þ. HRMS (ESI): m/z calcd for (C24H25ClN2 þ H)þ: 377.1785;
found: 377.1795.
5.1.5. General procedure for the synthesis of compounds 15 and 20
To a solution of compound 14 (or 19) (0.44 mmol) in 15 ml DMF/
H2O (3:2, v/v) was added compound 11 (207 mg, 0.44 mmol), tet-
rakistriphenylphosphane palladium (5 mol %), and Na2CO3 (138 mg,
1.3 mmol). The reaction mixture was heated to 110 ꢀC in a sealed
tube for 6 h. The reaction solution was poured into H2O (50 ml) and
extracted with ethyl acetate (25 ml ꢁ 3). The combined organic
layers were dried over Na2SO4, concentrated under vacuum and
purified on silica gel to afford the title compound.
5.1.6.4. N-((4-Chlorophenyl)(4-(pyridin-4-yl)phenyl)methyl)propan-
2-amine 12a. Yellow solid (yield, 21%), mp: 75e77 ꢀC. 1H NMR
(500 MHz, CDCl3)
d
8.64 (d, J ¼ 5.7 Hz, 2H), 7.58 (d, J ¼ 8.0 Hz, 2H),
7.49 (d, J ¼ 8.0 Hz, 2H), 7.47 (d, J ¼ 5.8 Hz, 2H), 7.37 (d, J ¼ 7.8 Hz,
2H), 7.28 (d, J ¼ 8.0 Hz, 2H), 5.01 (s, 1H), 2.76 (dt, J ¼ 12.6, 6.2 Hz,
1H), 1.10 (dd, J ¼ 6.1, 1.0 Hz, 6H). ESI-MS (m/z): 337 [M þ 1]þ. HRMS
(ESI): m/z calcd for (C21H21ClN2 þ H)þ: 337.1472; found: 337.1467.
5.1.5.1. (4-Chlorophenyl)(4-(9-(tetrahydro-2H-pyran-2-yl)-9H-
purin-6-yl)phenyl)methanol 15. White solid (yield, 67%), mp: 151e
153 ꢀC. 1H NMR (500 MHz, CDCl3)
d 7.84 (s, 1H), 7.80 (s, 1H), 7.48 (d,
J ¼ 8.0 Hz, 2H), 7.38 (d, J ¼ 8.0 Hz, 2H), 7.32 (d, J ¼ 8.0 Hz, 2H), 7.25
(d, J ¼ 8.0 Hz, 2H), 5.82 (dd, J ¼ 9.2, 3.0 Hz, 1H), 4.25e4.12 (m, 1H),
5.77 (s, 1H), 3.83e3.72 (m, 1H), 2.20e2.00 (m, 3H), 1.82e1.58 (m,
3H). ESI-MS (m/z): 421 [M þ 1]þ.
5.1.6.5. N-((4-Chlorophenyl)(4-(pyridin-4-yl)phenyl)methyl)-2-
methylpropan-1-amine 12b. Yellow solid (yield, 39%), mp: 91e
93 ꢀC. 1H NMR (500 MHz, CDCl3)
d 8.65 (s, 2H), 7.60 (s, 4H), 7.47 (s,
4H), 7.31 (d, J ¼ 7.7 Hz, 2H), 4.93 (s, 1H), 2.47 (s, 1H), 1.91e1.82 (m,
1H), 1.26 (s, 1H), 0.94 (d, J ¼ 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3)
5.1.5.2. (4-Chlorophenyl)(4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyr-
d
148.6, 148.4, 136.3, 134.4, 130.9, 130.7, 129.0, 128.9, 128.2, 127.6,
azol-4-yl)phenyl)methanol 20. Yellowish solid (yield, 76%), mp:
123.7, 66.6, 55.9, 27.8, 20.9. ESI-MS (m/z): 351 [M þ 1]þ. HRMS (ESI):
125e127 ꢀC. 1H NMR (500 MHz, CDCl3)
d 7.84 (s, 1H), 7.80 (s, 1H),
m/z calcd for (C22H23ClN2 þ H)þ: 351.1628; found: 351.1635.
7.46 (d, J ¼ 8.0 Hz, 2H), 7.38 (d, J ¼ 8.0 Hz, 2H), 7.32 (d, J ¼ 8.0 Hz,
2H), 7.25 (d, J ¼ 8.0 Hz, 2H), 5.82 (s,1H), 5.41 (dd, J ¼ 9.2, 3.0 Hz,1H),
4.11e4.05 (m, 1H), 3.73e3.63 (m, 1H), 2.22e2.01 (m, 3H), 1.80e1.57
(m, 3H). ESI-MS (m/z): 369 [M þ 1]þ.
5.1.6.6. N-((4-Chlorophenyl)(4-(pyridin-4-yl)phenyl)methyl)-2-
methylpropan-2-amine 12c. Yellow solid (yield, 46%), mp: 85e
87 ꢀC. 1H NMR (500 MHz, CDCl3)
d
8.62 (s, 2H), 7.56 (dd, J ¼ 6.5,
1.7 Hz, 2H), 7.51e7.44 (m, 4H), 7.41e7.34 (m, 2H), 7.26 (d, J ¼ 8.4 Hz,
2H), 5.05 (s, 1H), 1.09 (s, 9H). 13C NMR (100 MHz, CDCl3)
150.4,
5.1.6. General procedure for the synthesis of compounds 10, 16 and
21
d
148.1, 136.7, 132.6, 131.6, 130.8, 128.9, 128.7, 128.2, 127.3, 121.6, 60.8,
52.2, 30.2. ESI-MS (m/z): 351 [M þ 1]þ. HRMS (ESI): m/z calcd for
(C22H23ClN2 þ H)þ: 351.1628; found: 351.1618.
To a solution of 9 (15, or 20) (0.2 mmol) in anhydrous CH2Cl2
(4 ml) at 0 ꢀC was added SOCl2 (1.2 mmol) in one portion. The so-
lution stirred at 25 ꢀC for 6 h and was then poured into ice-water
and extracted with ethyl acetate (20 ml ꢁ 3). The organic phase
was washed with brine, dried over anhydrous Na2SO4, and then
concentrated under vacuum to title compound without further
purification. Then, it was immediately dissolved in CH3CN (2 ml)
was added the appropriate amine (2 mmol), K2CO3 (55 mg,
0.4 mmol) and KI (1.66 mg, 0.01 mmol). The reaction mixture was
irradiated with microwaves for 20 min at 140 ꢀC and was then
poured into water (20 ml). The mixture was extracted with ethyl
acetate (10 ml ꢁ 3). The combined organic layer was dried over
anhydrous NaSO4, concentrated and purified on silica gel to afford
the title compound.
5.1.6.7. N-((4-Chlorophenyl)(4-(pyridin-4-yl)phenyl)methyl)cyclo-
hexanamine 12d. Orange solid (yield 36%), mp: 73e75 ꢀC. 1H NMR
(500 MHz, CDCl3)
d
8.64 (dd, J ¼ 4.7, 1.4 Hz, 2H), 7.58 (d, J ¼ 8.1 Hz,
2H), 7.48 (t, J ¼ 7.8 Hz, 4H), 7.36 (d, J ¼ 8.3 Hz, 2H), 7.28 (d, J ¼ 8.4 Hz,
2H), 5.07 (s, 1H), 2.40 (t, J ¼ 9.7 Hz, 1H), 1.96 (s, 2H), 1.70 (d,
J ¼ 5.6 Hz, 2H), 1.57 (s, 2H), 1.20e1.09 (m, 4H). ESI-MS (m/z): 377
[M þ 1]þ. HRMS (ESI): m/z calcd for (C24H25ClN2 þ H)þ: 376.1706;
found: 376.1710.
5.1.6.8. N-((4-Chlorophenyl)(4-(pyridin-4-yl)phenyl)methyl)aniline
12e. Orange solid (yield, 42%), mp: 114e116 ꢀC. 1H NMR (500 MHz,
5.1.6.1. N-((4-Chlorophenyl) (4-(pyridin-3-yl)phenyl)methyl)-2-
CDCl3)
d
8.66 (s, 2H), 7.62 (d, J ¼ 7.4 Hz, 2H), 7.53 (d, J ¼ 4.0 Hz, 2H),
methylpropan-1-amine 11a. White solid (yield, 38%), mp: 86e
7.47 (d, J ¼ 7.2 Hz, 2H), 7.33 (s, 4H), 7.15 (t, J ¼ 7.2 Hz, 2H), 6.74 (t,
88 ꢀC. 1H NMR (500 MHz, CDCl3)
d
8.81 (d, J ¼ 1.7 Hz, 1H), 8.57 (d,
J ¼ 7.3 Hz, 1H), 6.56 (d, J ¼ 7.7 Hz, 2H), 5.53 (s, 1H). ESI-MS (m/z):

174
T. Liu et al. / European Journal of Medicinal Chemistry 73 (2014) 167e176
371 [M þ 1]þ. HRMS (ESI): m/z calcd for (C24H19ClN2 þ H)þ:
73 ꢀC. 1H NMR (500 MHz, CDCl3)
d
7.82 (s, 2H), 7.44 (d, J ¼ 8.2 Hz,
371.1315; found: 371.1318.
2H), 7.37 (dd, J ¼ 8.2, 5.7 Hz, 4H), 7.24 (d, J ¼ 8.4 Hz, 2H), 4.77 (s, 1H),
2.44e2.32 (m, 2H), 1.76 (dt, J ¼ 13.3, 6.7 Hz, 1H), 0.92 (dd, J ¼ 6.6,
5.1.6.9. N-Benzyl-1-(4-chlorophenyl)-1-(4-(pyridin-4-yl)phenyl)
1.3 Hz, 6H). 13C NMR (100 MHz, CDCl3)
d 142.1, 141.6, 132.80, 131.5,
methanamine 12f. Yellow solid (yield, 46%), mp: 77e79 ꢀC. 1H NMR
130.9, 129.5, 128.7, 128.7, 127.8, 126.0, 122.4, 66.6, 54.7, 28.3, 20.7.
(500 MHz, CDCl3)
d
8.63 (dd, J ¼ 4.7, 1.4 Hz, 2H), 7.59 (d, J ¼ 8.2 Hz,
ESI-MS (m/z): 340 [M
þ
1]þ. HRMS (ESI): m/z calcd for
2H), 7.53 (d, J ¼ 8.3 Hz, 2H), 7.47 (dd, J ¼ 4.6, 1.6 Hz, 2H), 7.42e7.39
(C20H22ClN3 þ H)þ: 340.1581; found: 340.1580.
(m, 2H), 7.37e7.27 (m, 7H), 4.90 (s, 1H), 3.76 (s, 2H). 13C NMR
(100 MHz, CDCl3)
d 150.2, 147.9, 147.2, 137.2, 133.2, 130.8, 129.5,
5.1.6.17. N-((4-(1H-Pyrazol-4-yl)phenyl)(4-chlorophenyl)methyl)-2-
128.9, 128.6, 128.4, 128.1, 127.5, 127.4, 127.3, 121.5, 65.2, 51.5. ESI-MS
(m/z): 385 [M þ 1]þ. HRMS (ESI): m/z calcd for (C25H21ClN2 þ H)þ:
385.1472; found: 385.1486.
methylpropan-2-amine 22c. White solid (yield, 33%), mp: 85e87 ꢀC.
1H NMR (500 MHz, CDCl3)
d
7.82 (s, 2H), 7.42 (d, J ¼ 8.2 Hz, 2H), 7.39
(d, J ¼ 8.4 Hz, 2H), 7.35 (d, J ¼ 8.2 Hz, 2H), 7.24 (d, J ¼ 8.4 Hz, 2H),
5.00 (s,1H),1.08 (s, 9H). 13C NMR (100 MHz, DMSO-d6)
138.7,137.1,
133.3, 132.6, 131.3, 130.2, 129.3, 128.8, 126.1, 121.3, 60.9, 59.9, 27.0.
d
5.1.6.10. N-((4-(9H-Purin-6-yl)phenyl)(4-chlorophenyl)methyl)
propan-2-amine 2. Yellow solid (yield, 20%), mp: 109e111 ꢀC. 1H
ESI-MS (m/z): 340 [M
þ
1]þ. HRMS (ESI): m/z calcd for
NMR (500 MHz, CDCl3)
d
8.93 (s, 1H), 8.64 (d, J ¼ 7.9 Hz, 2H), 8.26 (s,
(C20H22ClN3 þ H)þ: 340.1581; found: 340.1570.
1H), 7.62 (d, J ¼ 8.2 Hz, 2H), 7.44 (d, J ¼ 8.3 Hz, 2H), 7.29 (d,
J ¼ 8.3 Hz, 2H), 5.16 (s, 1H), 3.02e2.89 (m, 1H), 1.21 (t, J ¼ 6.8 Hz,
6H). ESI-MS (m/z): 378 [M þ 1]þ. HRMS (ESI): m/z calcd for
(C21H20ClN5 þ H)þ: 378.1485; found: 378.1480.
5.1.6.18. N-((4-(1H-Pyrazol-4-yl)phenyl)(4-chlorophenyl)methyl)
cyclopropanamine 22d. White solid (yield, 21%), mp: 62e64 ꢀC. 1H
NMR (500 MHz, CDCl3)
7.25 (m, 6H), 4.90 (s,1H), 2.12e2.05 (m, 1H), 0.45e0.35 (m, 4H). ESI-
d
7.83 (s, 2H), 7.45 (d, J ¼ 8.0 Hz, 2H), 7.35e
5.1.6.11. N-((4-(9H-Purin-6-yl)phenyl)(4-chlorophenyl)methyl)cyclo-
MS (m/z): 324 [M
þ
1]þ. HRMS (ESI): m/z calcd for
propanamine 3. White solid (yield, 47%), mp: 79e81 ꢀC. 1H NMR
(C19H18ClN3 þ H)þ: 324.1268; found: 324.1266.
(500 MHz, CDCl3)
d
8.98 (s, 1H), 8.74 (d, J ¼ 8.4 Hz, 2H), 8.28 (s, 1H),
7.55 (d, J ¼ 8.3 Hz, 2H), 7.32 (d, J ¼ 8.5 Hz, 2H), 7.27 (d, J ¼ 8.5 Hz,
2H), 4.99 (s, 1H), 2.11e2.08 (m, 1H), 0.48e0.36 (m, 4H). ESI-MS (m/
z): 376 [M þ 1]þ. HRMS (ESI): m/z calcd for (C21H18ClN5 þ H)þ:
376.1329; found: 376.1331.
5.1.6.19. N-((4-(1H-Pyrazol-4-yl)phenyl)(4-chlorophenyl)methyl)
cyclohexanamine 22e. White solid (yield, 54%), mp: 69e71 ꢀC. 1H
NMR (500 MHz, CDCl3)
d
7.81 (s, 2H), 7.43 (d, J ¼ 8.2 Hz, 2H), 7.35
(dd, J ¼ 8.5, 2.5 Hz, 4H), 7.29e7.24 (m, 2H), 5.01 (s, 1H), 2.44e2.32
(m, 1H), 1.95 (d, J ¼ 6.6 Hz, 2H), 1.69 (dd, J ¼ 9.2, 3.4 Hz, 2H), 1.21e
1.03 (m, 6H). ESI-MS (m/z): 366 [M þ 1]þ. HRMS (ESI): m/z calcd for
(C22H24ClN3 þ H)þ: 366.1737; found: 366.1696.
5.1.6.12. N-((4-(9H-Purin-6-yl)phenyl)(4-chlorophenyl)methyl)-2-
methylpropan-1-amine 17a. White solid (yield, 39%), mp: 87e
90 ꢀC. 1H NMR (500 MHz, CDCl3)
d
8.96 (s, 1H), 8.72 (d, J ¼ 8.1 Hz,
2H), 8.27 (s, 1H), 7.60 (d, J ¼ 8.3 Hz, 2H), 7.38 (d, J ¼ 8.5 Hz, 2H), 7.27
(d, J ¼ 8.3 Hz, 2H), 4.87 (s, 1H), 2.42 (d, J ¼ 6.6 Hz, 2H), 1.86e1.74 (m,
1H), 0.93 (dt, J ¼ 9.8, 4.9 Hz, 6H). ESI-MS (m/z): 392 [M þ 1]þ. HRMS
(ESI): m/z calcd for (C22H22ClN5 þ H)þ: 392.1642; found: 392.1652.
5.1.6.20. N-((4-(1H-Pyrazol-4-yl)phenyl)(4-chlorophenyl)methyl)an-
iline 22f. Yellow solid (yield, 25%), mp: 69e71 ꢀC. 1H NMR
(500 MHz, CDCl3)
d
7.85 (s, 2H), 7.47 (d, J ¼ 8.2 Hz, 2H), 7.35e7.29
(m, 6H), 7.13 (t, J ¼ 7.9 Hz, 2H), 6.72 (t, J ¼ 7.3 Hz, 1H), 6.55 (d,
J ¼ 8.2 Hz, 2H), 5.47 (s,1H). ESI-MS (m/z): 360 [M þ 1]þ. HRMS (ESI):
m/z calcd for (C22H18ClN3 þ H)þ: 359.1189; found: 359.1200.
5.1.6.13. N-((4-(9H-Purin-6-yl)phenyl)(4-chlorophenyl)methyl)cyclo-
hexanamine 17b. Yellow solid (yield, 32%), mp: 72e74 ꢀC. 1H NMR
(500 MHz, CDCl3)
d
9.02 (s, 1H), 8.71 (d, J ¼ 8.1 Hz, 2H), 8.30 (s, 1H),
7.58 (d, J ¼ 8.3 Hz, 2H), 7.37 (d, J ¼ 8.4 Hz, 2H), 7.26 (d, J ¼ 8.5 Hz,
2H), 5.12 (s, 1H), 2.44 (s, 1H), 2.03e1.91 (m, 2H), 1.69 (s, 2H), 1.20e
1.07 (m, 6H). ESI-MS (m/z): 418 [M þ 1]þ. HRMS (ESI): m/z calcd for
(C24H24ClN5 þ H)þ: 418.1798; found: 418.1801.
5.1.6.21. 1-(4-(1H-Pyrazol-4-yl)phenyl)-N-benzyl-1-(4-chlorophenyl)
methanamine 22g. Yellow solid, yield 39%, mp: 63e69 ꢀC. 1H NMR
(500 MHz, CDCl3)
(m, 4H), 7.38e7.26 (m, 7H), 4.85 (s, 1H), 3.76 (s, 2H). 13C NMR
(100 MHz, CDCl3) 142.3, 141.8, 140.0, 132.8, 131.4, 128.9, 128.7,
d
7.83 (s, 2H), 7.46 (d, J ¼ 7.6 Hz, 2H), 7.44e7.38
5.1.6.14. 1-(4-(9H-Purin-6-yl)phenyl)-N-benzyl-1-(4-chlorophenyl)
d
methanamine 17c. White solid (yield, 33%), mp: 118e120 ꢀC. 1H
128.6, 128.5, 128.2, 127.8, 127.1, 126.1, 122.5, 65.4, 51.1. ESI-MS (m/z):
374 [M þ 1]þ. HRMS (ESI): m/z calcd for (C23H20ClN3 þ H)þ:
374.1424; found: 374.1454.
NMR (500 MHz, CDCl3)
d
8.97 (s, 1H), 8.74 (d, J ¼ 8.3 Hz, 2H), 8.28 (s,
1H), 7.62 (d, J ¼ 8.3 Hz, 2H), 7.39 (t, J ¼ 6.7 Hz, 2H), 7.36e7.32 (m,
4H), 7.28 (d, J ¼ 8.4 Hz, 3H), 4.93 (s, 1H), 3.78 (d, J ¼ 3.1 Hz, 2H). ESI-
MS (m/z): 426 [M
þ
1]þ. HRMS (ESI): m/z calcd for
5.1.6.22. 2-(((4-(1H-Pyrazol-4-yl)phenyl)(4-chlorophenyl)methyl)
(C25H20ClN5 þ H)þ: 426.1485; found: 426.1478.
amino)ethanol 22h. Yellow syrup (yield, 35%), 1H NMR (500 MHz,
CDCl3)
d
7.78 (s, 2H), 7.43 (d, J ¼ 7.6 Hz, 2H), 7.37e7.30 (s, 4H), 7.27
5.1.6.15. N-((4-(1H-Pyrazol-4-yl)phenyl)(4-chlorophenyl)methyl)
(s, 1H), 4.85 (s, 1H), 3.73 (s, 2H), 2.79 (s, 2H). ESI-MS (m/z): 328
[M þ 1]þ. HRMS (ESI): m/z calcd for (C18H18ClN3O þ H)þ: 328.1217;
found: 328.1212.
propan-2-amine 22a. Yellow solid (yield, 27%), mp: 78e80 ꢀC. 1H
NMR (500 MHz, CDCl3)
d
7.84e7.79 (m, 2H), 7.44 (d, J ¼ 8.1 Hz, 2H),
7.35 (dd, J ¼ 8.4, 3.1 Hz, 4H), 7.24 (d, J ¼ 8.4 Hz, 2H), 4.95 (s,1H), 2.75
(dt, J ¼ 12.7, 6.3 Hz, 1H), 1.09 (dd, J ¼ 6.2, 3.6 Hz, 6H). 13C NMR
(100 MHz, CDCl3)
d
141.5, 140.9, 132.9, 131.5, 130.9, 128.8, 128.7,
5.1.6.23. N1-((4-(1H-Pyrazol-4-yl)phenyl)(4-chlorophenyl)methyl)
128.0, 126.0, 122.3, 63.4, 46.7, 22.6. ESI-MS (m/z): 326 [M þ 1]þ.
HRMS (ESI): m/z calcd for (C19H20ClN3 þ H)þ: 326.1424; found:
326.1456.
ethane-1,2-diamine 22i. Yellow syrup (yield, 42%), 1H NMR
(500 MHz, CDCl3)
d
7.78 (s, 2H), 7.43 (d, J ¼ 8.0 Hz, 2H), 7.35 (t,
J ¼ 8.0 Hz, 4H), 7.28 (d, J ¼ 8.0 Hz, 2H), 4.81 (s, 1H), 3.72 (q,
J ¼ 7.0 Hz, 1H), 2.86 (t, J ¼ 5.6 Hz, 2H), 2.67 (t, J ¼ 5.6 Hz, 2H), 1.26 (t,
J ¼ 7.0 Hz, 2H). ESI-MS (m/z): 327 [M þ 1]þ. HRMS (ESI): m/z calcd
for (C18H19ClN4 þ H)þ: 327.1376; found: 327.1380.
5.1.6.16. N-((4-(1H-Pyrazol-4-yl)phenyl)(4-chlorophenyl)methyl)-2-
methylpropan-1-amine 22b. Yellow solid (yield, 40%), mp: 71e

T. Liu et al. / European Journal of Medicinal Chemistry 73 (2014) 167e176
175
5.2. Pharmacological assay
5.2.1. Akt1 assay
This study was financially supported by National Natural Science
Foundation of China (No. 81001359), China Postdoctoral Science
Foundation (No. 201003734), Zhejiang province Provincial Natural
Science Foundation of China (LY13H300002), and Program for
Zhejiang Leading Team of S&T Innovation.
AKT1/PKBa Kinase Assay Kit (catalog No. 32-021) and active
recombinant AKT1 kinase were commercially available from UP-
STATE (acquired by Merck Millipore, Billerica, MA). 4 ng of Akt1 in
8
m
L of 2.5ꢁ kinase buffer [62.5 mM TriseHCl (pH 7.5), 25 mM
MgCl2, 12.5 mM -glycerophosphate, 0.25 mM Na3VO4, 5 mM
dithiothreitol (DTT)], was mixed with 2 L of dimethyl sulfoxide
(DMSO) vehicle or each of the compound (indicated concentra-
tions), incubated at room temperature for 5 min and 10 L of ATP/
substrate cocktail (20 mM ATP, 3 mM eNOS served as substrate) was
added. After incubation at room temperature for 30 min, add 20
Appendix A. Supplementary data
b
m
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.11.036.
m
References
mL
[1] S.P. Staal, Molecular cloning of the Akt oncogene and its human homologues
AKT1 and AKT2: amplification of AKT1 in a primary human gastric adeno-
carcinoma, Proc. Natl. Acad. Sci. U. S. A. 84 (1987) 5034e5037.
[2] J.Q. Cheng, B. Ruggeri, W.M. Klein, G. Sonoda, D.A. Altomare, D.K. Watson,
J.R. Testa, Amplification of AKT2 in human pancreatic cancer cells and inhi-
bition of AKT2 expression and tumorigenicity by antisense RNA, Proc. Natl.
Acad. Sci. U.S.A. 93 (1996) 3636e3641.
[3] R.T. Joseph, B. Alfonso, AKT plays a central role in tumorigenesis, Proc. Natl.
Acad. Sci. U.S.A. 98 (2001) 10983e10985.
[4] L.C. Cantley, The phosphoinositide 3-kinase pathway, Science 296 (2002)
1655e1657.
[5] K.M. Nicholson, N.G. Anderson, The protein kinase B/Akt signalling pathway in
human malignancy, Cell. Signal. 14 (2002) 381e395.
[6] J.Q. Cheng, C.W. Lindsley, G.Z. Cheng, H. Yang, S.V. Nicosia, The Akt/PKB
pathway: molecular target for cancer drug discovery, Oncogene 24 (2005)
7482e7492.
[7] T.F. Franke, PI3K/Akt: getting it right matters, Oncogene 27 (2008) 6473e
6488.
[8] L. Salmena, A. Carracedo, P.P. Pandolfi, Tenets of PTEN tumor suppression, Cell
133 (2008) 403e414.
of 50 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0) and
terminate the reaction. Then, PKB/Akt1 kinase activity was
analyzed according to the manufacturer’s instructions.
5.2.2. Anti-proliferative activity assay
The cytotoxic activity of the tested compounds in OVCAR-8
(Ovarian Adenocarcinoma), HL-60 (Promyelocytic Leukemia) and
HCT116 (Human Colon Cancer) cells was measured using the MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a
tetrazole) method. Cells were seeded in 96-well microtiter plates
(at a density of 4000 cells per well) for overnight attachment and
exposed to each of the compound (1.0e100.0
solution (5.0 mg/mL in RPIM 1640 medium; SigmaeAldrich) was
added (20.0 l/well), and plates were incubated for a further 4 h at
37 ꢀC. The purple formazan crystals were dissolved in 100.0
L of
mM) for 72 h. The MTT
m
m
DMSO. After 5 min, the plates were read on an automated micro-
plate spectrophotometer (Bio-Tek Instruments, Winooski, VT) at
570 nm. Assays were performed in triplicate on three independent
experiments. The concentration of drug inhibiting 50% of cells
(IC50) was calculated using the software of dose-effect analysis with
microcomputers.
[9] S.R. Datta, A. Brunet, M.E. Greenberg, Cellular survival: a play in three Akts,
Genes Dev. 13 (1999) 2905e2927.
[10] M.P. Scheid, J.R. Woodgett, PKB/AKT: functional insights from genetic models,
Nat. Rev. Mol. Cell Biol. 2 (2001) 760e768.
[11] E. Tokunaga, A. Kataoka, Y. Kimura, E. Oki, K. Mashino, K. Nishida, T. Koya,
M. Morita, Y. Kakeji, H. Baba, S. Ohno, Y. Maehara, The association between
Akt activation and resistance to hormone therapy in metastatic breast cancer,
Eur. J. Cancer 42 (2006) 629e635.
[12] E. Oki, H. Baba, E. Tokunaga, T. Nakamura, N. Ueda, M. Futatsugi, K. Mashino,
M. Yamamoto, M. Ikebe, Y. Kakeji, Y. Maehara, AKT phosphorylation associates
with LOH of PTEN and leads to chemoresistance for gastric cancer, Int. J.
Cancer 117 (2005) 376e380.
[13] M.E. Mattmann, S.L. Stoops, C.W. Lindsley, Inhibition of Akt with small mol-
ecules and biologics: historical perspective and current status of the patent
landscape, Expert. Opin. Ther. Pat. 21 (2011) 1309e1338.
[14] C.W. Lindsley, S.F. Barnett, M.E. Layton, M.T. Bilodeau, The PI3K/Akt pathway:
recent progress in the development of ATP-competitive and allosteric Akt
kinase inhibitors, Curr. Cancer Drug Targets 8 (2008) 7e18.
5.2.3. Apoptosis assay
To analysis apoptosis, OVCAR-8 cells (5 ꢁ 104 cells/ml, 5 ml)
were cultured in 25 cm2 flasks and treated with vehicle to test
compounds (20 mM). After 48 h of treatments, cells were harvested
and quickly washed twice with ice-cold PBS. Detection of apoptosis
by FACSCalibur flow cytometer (Becton Dickinson, Lincoln Park, NJ)
was performed using the Annexin V-FITC/Propidium iodide (PI)
apoptosis detection kit (BioVision, Mountain View, CA).
[15] E.J. Meuillet, Novel inhibitors of AKT: assessment of a different approach
targeting the Pleckstrin homology domain, Curr. Med. Chem. (2011).
[16] W. Marsh Rde, C.M. Rocha Lima, D.E. Levy, E.P. Mitchell, K.M. Rowland Jr.,
A.B. Benson 3rd, A phase II trial of perifosine in locally advanced, unresectable,
or metastatic pancreatic adenocarcinoma, Am. J. Clin. Oncol. 30 (2007) 26e31.
[17] N.B. Leighl, S. Dent, M. Clemons, T.A. Vandenberg, R. Tozer, D.G. Warr,
R.M. Crump, D. Hedley, G.R. Pond, J.E. Dancey, M.J. Moore, A phase 2 study of
perifosine in advanced or metastatic breast cancer, Breast Cancer Res. Treat.
108 (2008) 87e92.
[18] T.A. Yap, L. Yan, A. Patnaik, I. Fearen, D. Olmos, K. Papadopoulos, R.D. Baird,
L. Delgado, A. Taylor, L. Lupinacci, R. Riisnaes, L.L. Pope, S.P. Heaton,
G. Thomas, M.D. Garrett, D.M. Sullivan, J.S. de Bono, A.W. Tolcher, First-in-man
clinical trial of the oral pan-AKT inhibitor MK-2206 in patients with advanced
solid tumors, J. Clin. Oncol. 29 (2011) 4688e4695.
[19] B.R. Davies, H. Greenwood, P. Dudley, C. Crafter, D.H. Yu, J. Zhang, J. Li, B. Gao,
Q. Ji, J. Maynard, S.A. Ricketts, D. Cross, S. Cosulich, C.C. Chresta, K. Page,
J. Yates, C. Lane, R. Watson, R. Luke, D. Ogilvie, M. Pass, Preclinical pharma-
cology of AZD5363, an inhibitor of AKT: pharmacodynamics, antitumor ac-
tivity, and correlation of monotherapy activity with genetic background, Mol.
Cancer Ther. 11 (2012) 873e887.
[20] T.A. Yap, M.I. Walton, K.M. Grimshaw, R.H. Te Poele, P.D. Eve, M.R. Valenti,
A.K. de Haven Brandon, V. Martins, A. Zetterlund, S.P. Heaton, K. Heinzmann,
P.S. Jones, R.E. Feltell, M. Reule, S.J. Woodhead, T.G. Davies, J.F. Lyons,
F.I. Raynaud, S.A. Eccles, P. Workman, N.T. Thompson, M.D. Garrett, AT13148
is a novel, oral multi-AGC kinase inhibitor with potent pharmacodynamic and
antitumor activity, Clin. Cancer Res. 18 (2012) 3912e3923.
5.2.4. Western blotting assay
OVCAR-8 cells were harvested, washed with ice-cold 1ꢁ
phosphate-buffered saline (PBS), and lysed in immunoprecipitation
assay buffer [150 mM NaCl, 50 mM Tris, 2 mM ethyleneglycol-bis(b-
aminoethylether), 2 mM EDTA, 25 mM NaF, 25 mM b-glycer-
ophosphate, 0.2% TritonX-100, 0.3% Nonidet P-40, and 0.1 mM
phenylmethylsulfonyl fluoride]. Cellular debris was pelleted by
centrifugation at 13,000 rpm for 30 min at 4 ꢀC. The concentrations
of the total lysate protein were measured using a standard Bradford
assay (Bio-Rad, San Diego, CA). For Western blot analysis, 40e80 mg
of protein from the total cell lysate was electrophoresed by SDS-
PAGE. The proteins were then transferred to nitrocellulose mem-
brane (Pierce Chemical) and probed with primary antibodies fol-
lowed by horseradish peroxidase-labeled secondary antibodies.
Proteins were visualized using enhanced chemiluminescence
(Pierce Chemical).
Acknowledgment
[21] X. Dong, X. Zhou, H. Jing, J. Chen, T. Liu, B. Yang, Q. He, Y. Hu, Pharmacophore
identification, virtual screening and biological evaluation of prenylated fla-
vonoids derivatives as PKB/Akt1 inhibitors, Eur. J. Med. Chem. 46 (2011)
5949e5958.
We thank Mrs. Jianyang Pan (Pharmaceutical Informatics Insti-
tute, Zhejiang University) for performing the NMR spectrometry.

176
T. Liu et al. / European Journal of Medicinal Chemistry 73 (2014) 167e176
[22] A. Donald, T. McHardy, M.G. Rowlands, L.J. Hunter, T.G. Davies, V. Berdini,
R.G. Boyle, G.W. Aherne, M.D. Garrett, I. Collins, Rapid evolution of 6-
phenylpurine inhibitors of protein kinase B through structure-based design,
J. Med. Chem. 50 (2007) 2289e2292.
[23] K.D. Freeman-Cook, C. Autry, G. Borzillo, D. Gordon, E. Barbacci-Tobin,
V. Bernardo, D. Briere, T. Clark, M. Corbett, J. Jakubczak, S. Kakar, E. Knauth,
B. Lippa, M.J. Luzzio, M. Mansour, G. Martinelli, M. Marx, K. Nelson, J. Pandit,
F. Rajamohan, S. Robinson, C. Subramanyam, L. Wei, M. Wythes, J. Morris,
Design of selective, ATP-competitive inhibitors of Akt, J. Med. Chem. 53 (2010)
4615e4622.
[24] M. Addie, P. Ballard, D. Buttar, C. Crafter, G. Currie, B.R. Davies, J. Debreczeni,
H. Dry, P. Dudley, R. Greenwood, P.D. Johnson, J.G. Kettle, C. Lane, G. Lamont,
A. Leach, R.W. Luke, J. Morris, D. Ogilvie, K. Page, M. Pass, S. Pearson, L. Ruston,
Discovery of 4-amino-N-[(1S)-1-(4-chlorophenyl)-3-hydroxypropyl]-1-(7H-
pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide (AZD5363), an orally
bioavailable, potent inhibitor of Akt kinases, J. Med. Chem. 56 (2013) 2059e
2073.
[27] W.J. Li, D.P. Nelson, M.S. Jensen, R.S. Hoerrner, D.W. Cai, R.D. Larsen, P.J. Reider,
An improved protocol for the preparation of 3-pyridyl- and some arylboronic
acids, J. Org. Chem. 67 (2002) 5394e5397.
[28] S. Lucas, R. Heim, C. Ries, K.E. Schewe, B. Birk, R.W. Hartmann, In vivo active
aldosterone synthase inhibitors with improved selectivity: lead optimization
providing
a series of pyridine substituted 3,4-dihydro-1H-quinolin-2-one
derivatives, J. Med. Chem. 51 (2008) 8077e8087.
[29] C.C. Kumar, V. Madison, Akt crystal structure and Akt-specific inhibitors,
Oncogene 24 (2005) 7493e7501.
[30] M.W. Karaman, S. Herrgard, D.K. Treiber, P. Gallant, C.E. Atteridge,
B.T. Campbell, K.W. Chan, P. Ciceri, M.I. Davis, P.T. Edeen, R. Faraoni, M. Floyd,
J.P. Hunt, D.J. Lockhart, Z.V. Milanov, M.J. Morrison, G. Pallares, H.K. Patel,
S. Pritchard, L.M. Wodicka, P.P. Zarrinkar, A quantitative analysis of kinase
inhibitor selectivity, Nat. Biotechnol. 26 (2008) 127e132.
[31] A. Krammer, P.D. Kirchhoff, X. Jiang, C.M. Venkatachalam, M. Waldman, Lig-
Score: a novel scoring function for predicting binding affinities, J. Mol. Graph.
Model. 23 (2005) 395e407.
[25] C.M. Venkatachalam, X. Jiang, T. Oldfield, M. Waldman, LigandFit: a novel
method for the shape-directed rapid docking of ligands to protein active sites,
J. Mol. Graph. Model. 21 (2003) 289e307.
[26] R. Dayam, T. Sanchez, O. Clement, R. Shoemaker, S. Sei, N. Neamati, Beta-
diketo acid pharmacophore hypothesis. 1. Discovery of a novel class of HIV-1
integrase inhibitors, J. Med. Chem. 48 (2005) 111e120.
[32] D.K. Gehlhaar, G.M. Verkhivker, P.A. Rejto, C.J. Sherman, D.B. Fogel, L.J. Fogel,
S.T. Freer, Molecular recognition of the inhibitor AG-1343 by HIV-1 protease:
conformationally flexible docking by evolutionary programming, Chem. Biol.
2 (1995) 317e324.
[33] I. Muegge, Y.C. Martin, A general and fast scoring function for protein-ligand
interactions: a simplified potential approach, J. Med. Chem. 42 (1999) 791e804.