Discovery of 4-(Piperazin-1-yl)-7H-pyrrolo[2,3-d]pyrimidine Derivatives as Akt Inhibitors

Article abstract of DOI:10.1002/ardp.201500427

A series of 4-(piperazin-1-yl)-7H-pyrrolo[2,3-d]pyrimidine derivatives was synthesized and evaluated as Akt inhibitors by optimization of a weak screening lead (1). Typically, compounds 5q and 5t significantly improved the Akt1 inhibitory potency with IC<

Full text of DOI:10.1002/ardp.201500427

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Arch. Pharm. Chem. Life Sci. 2016, 349, 356–362
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Full Paper
Discovery of 4-(Piperazin-1-yl)-7H-pyrrolo[2,3-d]pyrimidine
Derivatives as Akt Inhibitors
1
1
1
2
2
1
2
ꢀ
Yang Liu , Yanzhen Yin , Jingya Zhang , Krystle Nomie , Liang Zhang , Dezhi Yang , Michael L. Wang ,
1
and Guisen Zhao
1
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School
of Pharmaceutical Sciences, Shandong University, Jinan, Shandong, P. R. China
The University of Texas MD Anderson Cancer Center, Houston, TX, USA
2
A series of 4-(piperazin-1-yl)-7H-pyrrolo[2,3-d]pyrimidine derivatives was synthesized and evaluated as
Akt inhibitors by optimization of a weak screening lead (1). Typically, compounds 5q and 5t
significantly improved the Akt1 inhibitory potency with IC50 values of 18.0 and 21.3 nM, respectively,
with desirable antiproliferative effect against the cell lines LNCaP and PC-3. The inhibitors 5q and 5t
might serve as lead compounds for further exploration of Akt inhibitors as anticancer agents.
Keywords: Akt / Anticancer / Docking / Pyrrolopyrimidines
Received: November 27, 2015; Revised: February 23, 2016; Accepted: February 25, 2016
DOI 10.1002/ardp.201500427
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
3
08
Introduction
kinase 1 (PDK1) at Thr
and by mammalian target of
4
73
rapamycin complex 2 (mTORC2) at Ser , Akt phosphorylates
a wide range of effectors that regulate tumor-associated cell
survival and proliferation [4]. These effectors, including
glycogen synthase kinase 3 (GSK-3), proapoptotic proteins
Bad and BAX, tuberous sclerosis protein complex 1/2 (TSC1/2)
and others, block apoptosis and maintain the proliferation of
tumor cells when phosphorylated [5]. Ectopic expression of
constitutively active Akt has been observed in many cancers
such as breast cancer [6], ovarian carcinoma [7], and prostate
cancer [8]. Furthermore, aberrant activation of Akt renders
tumor cells resistant to chemotherapeutic drugs such as
monoclonal antibodies [9]. Overall, these findings suggest
that Akt is a well-validated target for anticancer drug
discovery.
Significant efforts have been made to explore Akt
inhibitors [10]. A self-established compounds library compris-
ing different structural scaffolds has been established for
random screening of anticancer candidates, in which the
novel pyrrolopyrimidine-based lead 1 was discovered to
exhibit inhibitory activity against Akt1 with an inhibition
The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is
an important signal transduction pathway that regulates
multiple cellular processes, including cell proliferation,
transformation, and differentiation [1]. Akt, also known as
protein kinase B (PKB), is a serine/threonine kinase that
functions as a critical junction in the PI3K-Akt signaling
cascade. To date, three closely related Akt isoforms have been
identified: Akt1 (PKBa), Akt2 (PKBb), and Akt3 (PKBg) [2]. All
of these homologous isoforms have 97–100% sequence
identity in the ATP-binding site and share a common
structural organization comprising three functional domains:
the N-terminal pleckstrin homology (PH) domain, the
central catalytic domain, and the C-terminal regulatory
domain [3].
As a pivotal node in the PI3K/Akt pathway, when dually
phosphorylated by 3-phosphoinositide-dependent protein
Correspondence: Prof. Guisen Zhao, Department of Medicinal
Chemistry, School of Pharmaceutical Sciences, Shandong
University, Jinan, Shandong 250012, P. R. China.
E-mail: guisenzhao@sdu.edu.cn
ꢀ
Additional correspondence: Prof. Michael L. Wang,
Fax: þ86-531-88382009
E-mail: wang@mdanderson.org
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Arch. Pharm. Chem. Life Sci. 2016, 349, 356–362
Pyrrolopyrimidine Derivatives as Akt Inhibitors
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rate of 70.1% at 5 mM, making compound 1 the potential lead
compound for further exploration of Akt inhibitors. Herein
we describe the structural modifications of compound 1, with
halogen atoms at the 5-position on the pyrrolopyrimidine
core and substituted phenyls or naphthyls linked to the
C5 on the pyrrolopyrimidine core produced potent enzyme
inhibition (5l, 5p vs. 5e). Insight into the R
unsubstituted phenyl compound 5n or replacement of the
phenyl ring with naphthyl moiety (5u) resulted in
2
substitution,
a
significantly decreased potency versus Akt1. Furthermore,
the addition of para-substituents of the phenyl group
yielded an improved potency in Akt1 inhibition than
meta-substituents (5k vs. 5l, 5o vs. 5p). The para-chloro/
bromo-phenyl (5l, 5p, and 5q) and the 3,4-dichloro substitu-
tion (5t) is essential for Akt1 inhibitory potency, methoxyl
substitution (5r) led to moderately decreased Akt1 inhibitory
activity, while the tert-butyl derivative (5s) led to a substantial
reduction in Akt1 potency by more than 10-fold.
4
-piperazine moiety through an acetamide bond, which led
to a series of 4-(piperazin-1-yl)-7H-pyrrolo[2,3-d]pyrimidine
derivatives evaluated as Akt inhibitors (Fig. 1).
Results and discussion
Chemistry
The synthesis of the pyrrolopyrimidine derivatives is
depicted in Scheme 1. Briefly, treatment of the readily
available 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (2a) with
N-chlorosuccinimide (NCS) or N-bromosuccinimide (NBS)
caused clean chlorination or bromination at C5 to
give 4,5-dichloro-7H-pyrrolo[2,3-d]pyrimidine (2b) or
Compounds with over-half inhibition at 50 nM were further
selectively investigated for their IC50 values against Akt1 and
cellular potency against the LNCaP and PC-3 cell lines. As
shown in Table 2, in comparison with compound 1,
compounds 5l, 5p, 5q, and 5t significantly improved the
enzyme inhibitory potency and antiproliferative activity
against LNCaP and PC-3 cell lines, indicating that these
compounds blocked proliferation in prostate cancer cells
partly through inhibiting Akt activity. Notably, the most
potent compound 5q inhibited Akt1 with an IC50 value of
18.0 nM, and the 3,4-dichloro substitution (5t) exhibited
comparable potency (IC50 ¼ 21.3 nM).
5
-bromo-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (2c), respec-
tively. Subsequent incorporation of the Boc-protected piper-
azine via aromatic nucleophilic substitution delivered 3a–c.
Acid-promoted Boc deprotection provided key intermediates
4
4
a–c as dihydrochloride salts. Finally, the designed
-(piperazin-1-yl)-7H-pyrrolo[2,3-d]pyrimidine derivatives
(
5a–u) were achieved by amide coupling with the requisite
cores 4 and commercially available substituted acetic acids.
Molecular modeling
Compound 5q was chosen for molecular modeling to
rationalize the observed activity against Akt1 (PDB: 4EKL)
using Sybyl 1.1 software package. As depicted in Fig. 2,
compound 5q nicely occupied the ATP cleft, the para-
chlorophenyl group entered the hydrophobic pocket B
formed by Gly159, Phe161, Gly162, and Leu181. The
pyrrolopyrimidine core formed a pair of key hydrogen bonds
to residues Ala230 and Glu228 in pocket A. Though no
obvious interaction between the bromine atom and the
protein was observed, the C-5 bromine substitution, with
improved size and hydrophobicity, was in proximity to the
lipophilic side chains of Ala177, Val164, and Met227, which
might be responsible for the improved potency.
Biology
Twenty-one
4-(piperazin-1-yl)-7H-pyrrolo[2,3-d]pyrimidine
derivatives were prepared. Their Akt1 inhibitory activities
were detected using a homogeneous time-resolved fluores-
cence (HTRF) kinase activity assay, the inhibition ratios were
determined at dual concentrations of 50 nM and 5 mM,
respectively (Table 1).
As depicted in Table 1, most of the synthesized compounds
showed over-half inhibition potency at 5 mM against Akt1.
Considering the enzyme inhibitory activity at 50 nM, almost
no Akt1 inhibitory activity was observed for C5 hydrogen
analogs (5a–j). Introduction of a chlorine or bromine atom at
Conclusion
In summary, optimization of the pyrrolopyrimidine lead
compound 1 as novel Akt1 inhibitors was performed. Among
the synthesized compounds, compounds 5q and 5t inhibited
Akt1 kinase activity with IC50 values of 18.0 and 21.3 nM,
respectively, and displayed desirable antiproliferative effect
against LNCaP and PC-3 cell lines with IC50 values at low micro-
molar level. Structure–activity relationship analysis revealed
that the chloro- or bromo-substitution at the 5-position of
the pyrrolopyrimidine core and the 4-Cl/3,4-diCl-substituted
phenyl moiety linked to piperazine ring is critical to the
enzyme inhibitory activity and cell potency. The compounds
5
q and 5t might serve as lead compounds for further
Figure 1. Structures of the lead 1 and designed compounds.
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exploration of Akt inhibitors as anticancer agents.
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Arch. Pharm. Chem. Life Sci. 2016, 349, 356–362
Y. Liu et al.
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Scheme 1. Synthesis of compounds 5a–u. Reagents and conditions: (a) NCS, DMF, rt, 72 h; (b) NBS, DMF, rt, 72 h; (c) N-Boc-piperazine,
DMF, 110°C, 16 h; (d) 4 M HCl in dioxane, CH
3
OH, rt, 24 h; (e) substituted acetic acid, EDCI, HOBT, DIEA, THF, rt, 24 h.
Experimental
Chemistry
monitored by thin layer chromatography (TLC), and silica gel
GF254 plates were used and visualized with UV light. Column
chromatography was performed with silica gel using the
1
13
All of the materials were obtained from commercial suppliers
indicated solvents. H NMR and
C NMR spectra were
and used without further purification. All reactions were
recorded on a Varian Inova 600 (Switzerland) or Bruker
Table 1. The inhibitory effects of pyrrolopyrimidine derivatives on Akt1 activity.
Inhibition of Akt1
activity (%)
Inhibition of Akt1
activity (%)
Code
R
1
R
2
50 nM
5 mM
Code
R
1
R
2
50 nM
5 mM
5
5
5
5
5
5
5
5
5
5
5
a
b
c
H
H
H
H
H
H
H
H
H
H
Cl
Ph
9.2
5.0
34.3
80.5
88.9
99.4
98.5
39.3
76.1
66.2
29.4
92.4
98.1
5l
5m
5n
5o
5p
5q
5r
Cl
Cl
Br
Br
Br
Br
Br
Br
Br
Br
Cl
4-Br-Ph
naphth-2-yl
Ph
71.5
14.3
NA
99.8
95.0
88.3
98.5
100.0
96.6
99.2
82.4
100.0
97.0
70.1
3-Br-Ph
4-F-Ph
NA
d
e
f
4-Cl-Ph
4-Br-Ph
4-tBu-Ph
12.4
16.0
NA
3-Br-Ph
4-Br-Ph
4-Cl-Ph
18.7
69.5
69.7
36.6
5.3
g
h
i
3
4-CH O-Ph
NA
3
4-CH O-Ph
2,4-diCl-Ph
naphth-1-yl
naphth-2-yl
3-Br-Ph
NA
5s
5t
4-tBu-Ph
3,4-diCl-Ph
naphth-2-yl
Ph
5.2
65.5
NA
j
8.3
5u
1
k
15.0
NA
NA, not active.
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Arch. Pharm. Chem. Life Sci. 2016, 349, 356–362
Pyrrolopyrimidine Derivatives as Akt Inhibitors
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Table 2. The inhibitory effects of pyrrolopyrimidine derivatives on Akt1 activity and prostate cancer cells.
a)
a)
a)
Code
Akt1, IC50 (nM)
LNCaP, IC50 (mM)
PC-3, IC50 (mM)
5
5
5
5
1
l
45.3 ꢂ 0.6
31.6 ꢂ 2.2
18.0 ꢂ 0.7
21.3 ꢂ 1.4
>1000
6.6 ꢂ 2.0
9.8 ꢂ 1.1
9.3 ꢂ 1.9
7.0 ꢂ 0.5
45.0 ꢂ 0.9
25.3 ꢂ 1.3
38.7 ꢂ 0.6
21.1 ꢂ 1.6
31.5 ꢂ 0.2
70.4 ꢂ 1.5
p
q
t
a)
IC50 data are the mean of three independent measurements.
Avance III 400 (Germany) spectrometer using TMS as an
internal standard in DMSO-d . Chemical shifts are reported in
4,5-Dichloro-7H-pyrrolo[2,3-d]pyrimidine (2b)
Gray powder, yield 95%, mp: 287–290°C, H NMR (400 MHz,
1
6
parts per million (ppm). Coupling constants (J) are given in
Hertz. The mass spectra (MS) were measured with an API 4000.
All of the melting points were determined on a capillary
melting point apparatus RY-1G (TianGuang Optical Instru-
ments, Inc., China) and are uncorrected. Human Akt1 was
obtained from Carna Biosciences, Inc. (Canada) and the HTRF
assay kit was purchased from Cisbio Bioassays, Inc. (France).
Please see the Supporting Information for the InChI codes
of the new compounds.
DMSO) d (ppm): 12.87 (s, 1H), 8.63 (s, 1H), 7.91 (s, 1H). MS (ESI)
m/z: 188 [MþH] .
þ
5-Bromo-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (2c)
1
Pale powder, yield 95%, mp: 290–292°C, H NMR (400 MHz,
DMSO) d (ppm): 13.00 (s, 1H), 8.64 (s, 1H), 7.97 (s, 1H). MS (ESI)
þ
m/z: 232 [MþH] .
Preparation of 3a–c
To a solution of 2a–c (3.2 mmol) in DMF (3 mL) were added
General methods for preparation of the compounds
N-Boc-piperazine (3.6 mmol, 750 mg) and triethylamine
(
3
Et N) (4.9 mmol, 0.65 mL). The reaction mixture was heated
Preparation of 2b,c
for 16 h at 110°C, then treated with water (300 mL) and
extracted with ethyl acetate (EtOAc) (4 ꢁ 60 mL). The
combined organic phases were washed with brine
To a solution of 2a (3.0 g, 20 mmol) in N,N-dimethylforma-
mide (DMF) (30 mL) was added NCS/NBS (21 mmol). The
mixture was stirred at room temperature for 72 h. The mixture
was poured into ice water (300 mL) and the precipitate was
filtered, washed with water (3 ꢁ 10 mL), and dried to obtain
(2 ꢁ 50 mL), dried over sodium sulfate (Na
2 4
SO ), filtered
and concentrated in vacuo. The crude residue was purified by
column chromatography with petroleum ether/ethyl acetate
(1:3) to obtain 3a–c.
2
b,c.
Tert-butyl-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazine-
1
-carboxylate (3a)
1
Offwhite solid, yield 59%, H NMR (600 MHz, DMSO) d (ppm):
1
1.73 (s, 1H), 8.15 (s, 1H), 7.20 (d, J ¼ 0.6 Hz, 1H), 6.64 (d,
J ¼ 1.8 Hz, 1H), 3.86 (s, 4H), 3.48 (s, 4H), 1.43 (s, 9H). MS (ESI)
þ
m/z: 304 [MþH] .
Tert-butyl 4-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-
piperazine-1-carboxylate (3b)
1
Offwhite solid, yield: 51%. H NMR (400 MHz, DMSO) d (ppm):
1
1
2.23 (s, 1H), 8.29 (s, 1H), 7.52 (d, J ¼ 2.8 Hz, 1H), 3.52 (s, 8H),
þ
.42 (s, 9H). MS (ESI) m/z: 338 [MþH] .
Tert-butyl 4-(5-bromo-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-
piperazine-1-carboxylate (3c)
Pale yellow solid, yield 48%, H NMR (400 MHz, DMSO) d
1
(
(
ppm): 12.21 (s, 1H), 8.37 (s, 1H), 7.52 (d, J ¼ 2.6 Hz, 1H), 3.52
þ
s, 8H), 1.42 (s, 9H). MS (ESI) m/z: 382 [MþH] .
Preparation of 4a–c
To 4 M HCl in dioxane (5 mL) was added 3a–c (1 mmol). The
reaction mixture was stirred for 24 h at room temperature.
The precipitate was isolated by filtration, washed with diethyl
Figure 2. The predicted binding mode of 5q at the ATP-binding
site of Akt1.
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Arch. Pharm. Chem. Life Sci. 2016, 349, 356–362
Y. Liu et al.
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ether (2 ꢁ 5 mL), and dried to obtain 4a–c, which was used for
the next step without further purification.
J
2
¼ 1.68 Hz, 1H), 3.87 (t, J ¼ 4.64 Hz, 4H), 3.78 (s, 2H), 3.69–3.64
1
3
(m, 4H). C NMR (100 MHz, DMSO) d (ppm): 169.3, 156.8,
1
52.4, 150.9, 135.3, 131.6 (2C), 131.5, 128.6 (2C), 122.0, 102.8,
þ
Preparation of 5a–u
To a solution of substituted acetic acids (0.365 mmol) in
101.2, 45.3 (4C), 41.5. MS (ESI) m/z: 356 [MþH] .
dry tetrahydrofuran (THF) (6 mL) at 0°C were added
1-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)-2-(4-
bromophenyl)ethanone (5e)
1
-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochlo-
ride (EDCI) (77 mg, 0.4 mmol), 1-hydroxybenzotriazole
Pale yellow solid, yield 75%, purity 98.5%, mp: 175–177°C,
H NMR (400 MHz, DMSO) d (ppm): 11.73 (s, 1H), 8.16 (s, 1H),
1
(
(
HOBt) (61 mg, 0.4 mmol) and N,N-diisopropylethylamine
DIEA) (141 mg, 1.1 mmol). The reaction mixture was stirred
7.50 (d, J ¼ 8.4 Hz, 2H), 7.21 (d, J ¼ 8.4 Hz, 3H), 6.62 (s, 1H),
13
for 30 min before 4a–c (0.365 mmol) was added portionwise.
Then the reaction was warmed to room temperature and
stirred for another 24 h. The mixture was diluted with ethyl
acetate (20 mL) and washed with sodium bicarbonate
solution (10%, 20 mL), water (20 mL), and brine (20 mL).
3.92–3.82 (m, 4H), 3.77 (s, 2H), 3.72–3.59 (m, 4H). C NMR
(100 MHz, DMSO) d (ppm): 169.5, 156.8, 152.4, 150.9, 131.5,
131.4, 122.0, 115.5 (2C), 115.3 (2C), 102.8, 101.2, 45.3 (4C),
þ
41.5. MS (ESI) m/z: 400 [MþH] .
The organic layer was dried over Na
2
SO
4
, filtered, and
1-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)-2-(4-
concentrated in vacuo. The crude residue was purified by
recrystallization using petroleum ether/ethyl acetate to
obtain 5a–u.
(tert-butyl)phenyl)ethanone (5f)
White solid, yield 82%, purity 96.2%, mp: 222–225°C, H NMR
1
(400 MHz, DMSO) d (ppm): 11.74 (s, 1H), 8.15 (s, 1H), 7.33 (d,
J ¼ 12 Hz, 2H), 7.20–7.16 (m, 3H), 6.61 (s, 1H), 3.85 (s, 4H), 3.72
1
3
1
-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)-2-
phenylethanone (5a)
White solid, yield 80%, purity 98.2%, mp: 169–172°C, H NMR
(s, 2H), 3.65 (d, J ¼ 12 Hz, 4H), 1.26 (s, 9H). C NMR (100 MHz,
DMSO) d (ppm): 169.8, 156.8, 152.4, 150.9, 149.1, 133.1, 129.1
(2C), 125.5 (2C), 122.0, 102.8, 101.2, 46.1 (4C), 41.5, 34.5, 31.6
1
þ
(
(
3
1
1
400 MHz, DMSO) d (ppm): 11.71 (s, 1H), 8.15 (s, 1H), 7.33–7.19
m, 6H), 6.61 (t, J ¼ 1.6 Hz, 1H), 3.87–3.83 (m, 4H), 3.77 (s, 2H),
(3C). MS (ESI) m/z: 378 [MþH] .
13
.68–3.64 (m, 4H). C NMR (100 MHz, DMSO) d (ppm): 169.6,
1-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)-2-(4-
methoxyphenyl)ethanone (5g)
Yellow solid, yield 87%, purity 97.3%, mp: 170–174°C, H NMR
(400 MHz, DMSO) d (ppm): 11.74 (s, 1H), 8.15 (s, 1H), 7.20–7.16
56.8, 152.4, 150.9, 136.2, 129.5 (2C), 128.7 (2C), 126.8, 122.0,
1
02.8, 101.2, 45.3 (2C), 45.3 (2C), 41.5. MS (ESI) m/z: 322
þ
[
MþH] .
(
m, 3H), 6.87 (d, J ¼ 8.4 Hz, 2H), 6.61 (s, 1H), 3.85–3.82 (m, 4H),
13
1
-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)-2-(3-
3.72 (s, 3H), 3.69 (s, 2H), 3.64 (s, 4H). C NMR (100 MHz, DMSO)
d (ppm): 169.9, 158.3, 156.8, 152.4, 150.9, 130.4 (2C), 128.0,
122.0, 114.2 (2C), 102.8, 101.2, 55.4, 45.4 (2C), 45.3 (2C), 41.5.
MS (ESI) m/z: 352 [MþH] .
bromophenyl)ethanone (5b)
White solid, yield 89%, purity 97.1%, mp: 150–152°C,
1
þ
H NMR (400 MHz, DMSO) d (ppm): 8.16 (s, 1H), 7.47–7.43
(
(
m, 2H), 7.30–7.26 (m, 2H), 7.21 (s, 1H), 6.63 (s, 1H), 3.88
s, 4H), 3.80 (s, 2H), 3.69–3.60 (m, 4H). C NMR (100 MHz,
13
1-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)-2-
DMSO) d (ppm): 169.2, 156.8, 152.4, 151.0, 139.1, 132.6,
(2,4-dichlorophenyl)ethanone (5h)
White solid, yield 84%, purity 97.0%, mp: 162–165°C, H NMR
(400 MHz, DMSO) d (ppm): 11.73 (s, 1H), 8.17 (s, 1H), 7.59 (s,
1
1
30.7, 129.7, 128.9, 122.0, 121.8, 102.8, 101.2, 45.3 (4C), 41.5.
þ
MS (ESI) m/z: 400 [MþH] .
1
H), 7.37 (s, 2H), 7.21 (s, 1H), 6.64 (s, 1H), 3.97 (s, 2H), 3.88
1
3
1
-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)-2-(4-
(s, 4H), 3.74 (s, 2H), 3.65 (s, 2H). C NMR (100 MHz, DMSO) d
(ppm): 168.0, 156.8, 152.4, 151.0, 135.2, 134.1, 133.7, 132.4,
128.8, 127.5, 122.0, 102.8, 101.3, 45.3 (2C), 45.2 (2C), 41.6. MS
(ESI) m/z: 390 [MþH] .
fluorophenyl)ethanone (5c)
Pale yellow solid, yield 81%, purity 97.8%, mp: 194–197°C,
1
þ
H NMR (400 MHz, DMSO) d (ppm): 11.67 (s, 1H), 8.16 (s, 1H),
7
(
(
.28 (m, 2H), 7.21–7.17 (m, 1H), 7.14–7.10 (m, 2H), 6.61
dd, J ¼ 1.7 Hz, 1H), 3.86 (d, J ¼ 5.2 Hz, 4H), 3.76
¼ 3.4 Hz, J
s, 2H), 3.69–3.62 (m, 4H). C NMR (100 MHz, DMSO) d (ppm):
69.3, 159.6, 152.1, 151.1, 135.4, 131.9, 131.5 (2C), 129.1, 128.6
1
2
1-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)-2-
(naphthalen-2-yl)ethanone (5i)
13
1
White solid, yield 83%, purity 98.4%, mp: 219–221°C,
1
(
[
2C), 124.8, 104.9, 50.4 (2C), 50.2 (2C), 41.6. MS (ESI) m/z: 340
MþH] .
H NMR (400 MHz, DMSO) d (ppm): 11.66 (s, 1H), 8.15
þ
(s, 1H), 7.87–7.85 (m, 3H), 7.76 (s, 1H), 7.48 (d, J ¼ 3.5 Hz, 2H),
7
.42 (d, J ¼ 8.4 Hz, 1H), 7.18 (s, 1H), 6.60 (s, 1H), 3.95 (s, 2H),
13
1
-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)-2-(4-
chlorophenyl)ethanone (5d)
White solid, yield 87%, purity 98.2%, mp: 175–178°C, H NMR
400 MHz, DMSO) d (ppm): 11.72 (s, 1H), 8.15 (s, 1H), 7.36 (d,
3.87 (s, 4H), 3.70 (d, J ¼ 18.4 Hz, 4H). C NMR (100 MHz,
DMSO) d (ppm): 169.6, 156.8, 152.4, 150.9, 134.0, 133.5,
132.2, 128.2, 128.1, 127.9, 127.8, 127.7, 126.5, 126.0, 122.0,
102.8, 101.2, 45.5 (2C), 45.3 (2C), 41.5. MS (ESI) m/z: 372
1
(
þ
J ¼ 8.36 Hz, 2H), 7.27 (d, J ¼ 8.36 Hz, 2H), 6.61 (dd, J
1
¼ 3.32 Hz,
[MþH] .
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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360

Arch. Pharm. Chem. Life Sci. 2016, 349, 356–362
Pyrrolopyrimidine Derivatives as Akt Inhibitors
ARC HH PHARM
Archiv der Pharmazie
1
(
-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)-2-
naphthalen-1-yl)ethanone (5j)
White solid, yield 88%, purity 97.7%, mp: 205–208°C, H NMR
7.59 (s, 1H), 7.47–7.43 (m, 2H), 7.30–7.26 (m, 2H), 3.81 (s, 2H),
1
3
3.71 (d, J ¼ 16 Hz, 4H), 3.50 (s, 4H). C NMR (100 MHz, DMSO) d
(ppm): 169.1, 159.6, 152.1, 151.1, 139.2, 132.5, 130.7, 129.7,
128.8, 124.8, 121.8, 104.9, 86.3, 50.4 (2C), 50.2 (2C), 41.6. MS
1
(
(
1
400 MHz, DMSO) d (ppm): 11.67 (s, 1H), 8.17 (s, 1H), 7.99
d, J ¼ 6.8 Hz, 1H), 7.92 (d, J ¼ 8.8 Hz, 1H), 7.82 (d, J ¼ 8.1 Hz,
þ
(ESI) m/z: 480 [MþH] .
H), 7.57–7.42 (m, 3H), 7.37 (d, J ¼ 6.7 Hz, 1H), 7.19 (s, 1H), 6.62
(
s, 1H), 4.22 (s, 2H), 3.93 (s, 4H), 3.73 (d, J ¼ 36.1 Hz, 4H).
1-(4-(5-Bromo-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-
1-yl)-2-(4-bromophenyl)ethanone (5p)
1
3
C NMR (100 MHz, DMSO) d (ppm): 169.6, 156.8, 152.4, 151.0,
1
1
33.8, 133.0, 132.6, 128.8, 127.6, 127.5, 126.4, 126.1, 125.9,
Pale yellow solid, yield 89%, purity 98.1%, mp: 176–178°C,
H NMR (400 MHz, DMSO) d (ppm): 12.31 (s, 1H), 8.31 (s, 1H),
1
24.8, 122.0, 102.8, 101.3, 45.4 (2C), 45.3 (2C), 41.5. MS (ESI)
þ
m/z: 372 [MþH] .
7.58 (s, 1H), 7.50 (d, J ¼ 8.4 Hz, 2H), 7.21 (d, J ¼ 8.4 Hz, 2H), 3.77
(
s, 2H), 3.70 (t, J ¼ 6.28 Hz, 4H), 3.48 (d, J ¼ 3.36 Hz, 4H).
1
3
2
-(3-Bromophenyl)-1-(4-(5-chloro-7H-pyrrolo[2,3-d]-
pyrimidin-4-yl)piperazin-1-yl)ethanone (5k)
White solid, yield 86%, purity 97.5%, mp: 194–196°C, H NMR
C NMR (100 MHz, DMSO) d (ppm): 169.2, 159.6, 152.1, 151.1,
135.8, 131.9 (2C), 131.5 (2C), 124.8, 119.9, 104.9, 86.2, 50.4
1
þ
(4C), 50.2 (4C), 41.6. MS (ESI) m/z: 480 [MþH] .
(
(
(
(
400 MHz, DMSO) d (ppm): 12.24 (s, 1H), 8.30 (s, 1H), 7.54
s, 1H), 7.47–7.43 (m, 2H), 7.28–7.26 (m, 2H), 3.81 (s, 2H), 3.69
1-(4-(5-Bromo-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-
1-yl)-2-(4-chlorophenyl)ethanone (5q)
13
d, J ¼ 24 Hz, 4H), 3.55 (s, 4H). C NMR (100 MHz, DMSO) d
ppm): 169.1, 159.1, 151.5, 151.2, 139.2, 132.5, 130.7, 129.6,
Pale yellow solid, yield 70%, purity 97.9%, mp: 193–195°C,
1
1
28.8, 121.9, 121.8, 103.3, 101.6, 49.8 (2C), 49.5 (2C), 41.6. MS
H NMR (400 MHz, DMSO) d (ppm): 12.32 (s, 1H), 8.31 (s, 1H),
þ
(
ESI) m/z: 434 [MþH] .
7.59 (s, 1H), 7.37 (d, J ¼ 8.4 Hz, 2H), 7.27 (d, J ¼ 8.4 Hz, 2H), 3.79
1
3
(
s, 2H), 3.76–3.65 (m, 4H), 3.49 (d, J ¼ 3.5 Hz, 4H). C NMR
2
-(4-Bromophenyl)-1-(4-(5-chloro-7H-pyrrolo[2,3-d]-
pyrimidin-4-yl)piperazin-1-yl)ethanone (5l)
White solid, yield 82%, purity 98.1%, mp: 189–191°C, H NMR
(100 MHz, DMSO) d (ppm): 169.2, 156.8, 152.4, 150.9, 135.8,
132.0 (2C), 131.5 (2C), 129.1, 122.0, 119.9, 102.8, 101.2, 45.3
1
þ
(4C), 41.5. MS (ESI) m/z: 434 [MþH] .
(
(
(
400 MHz, DMSO) d (ppm): 12.24 (s, 1H), 8.30 (s, 1H), 7.53
s, 1H), 7.50 (d, J ¼ 8.4 Hz, 2H), 7.21 (d, J ¼ 8.4 Hz, 2H), 3.77
1-(4-(5-Bromo-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-
1-yl)-2-(4-methoxyphenyl)ethanone (5r)
13
s, 2H), 3.68 (d, J ¼ 16.0 Hz, 4H), 3.53 (s, 4H). C NMR (100 MHz,
DMSO) d (ppm): 169.2, 159.1, 151.5, 151.2, 135.8, 131.9 (2C),
Pale yellow solid, yield 88%, purity 98.5%, mp: 170–173°C,
1
1
31.5 (2C), 121.9, 119.9, 103.3, 101.6, 49.8 (2C), 49.5 (2C), 41.6.
H NMR (400 MHz, DMSO) d (ppm): 12.33 (s, 1H), 8.30 (s, 1H),
þ
MS (ESI) m/z: 434 [MþH] .
7.58 (s, 1H), 7.17 (d, J ¼ 8.0 Hz, 2H), 6.88 (d, J ¼ 8.0 Hz, 2H),
1
3
3
.73–3.70 (m, 9H), 3.45 (d, J ¼ 20 Hz, 4H). C NMR (100 MHz,
1
1
-(4-(5-Chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-
-yl)-2-(naphthalen-2-yl)ethanone (5m)
DMSO) d (ppm): 169.9, 159.6, 158.3, 152.1, 151.1, 130.4
(2C), 128.0, 124.8, 114.2 (2C), 104.9, 86.3, 55.4, 50.3 (2C), 50.3
þ
Pale yellow solid, yield 79%, purity 97.0%, mp: 150–153°C,
(2C), 41.5. MS (ESI) m/z: 430 [MþH] .
1
H NMR (400 MHz, DMSO) d (ppm): 12.15 (s, 1H), 8.27 (s, 1H),
7
.87 (t, J ¼ 7.9 Hz, 3H), 7.76 (s, 1H), 7.50–7.46 (m, 3H), 7.42 (dd,
¼ 8.5 Hz, J ¼ 1.5 Hz, 1H), 3.96 (s, 2H), 3.73 (d, J ¼ 18.6 Hz,
H), 3.54 (d, J ¼ 17.8 Hz, 4H). C NMR (100 MHz, DMSO) d
1-(4-(5-Bromo-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-
1-yl)-2-(4-(tert-butyl)phenyl)ethanone (5s)
J
1
2
13
4
Pale yellow solid, yield 85%, purity 99.2%, mp: 113–116°C,
1
(
ppm): 169.6, 159.1, 151.5, 151.2, 134.0, 133.5, 132.2, 128.2,
H NMR (400 MHz, DMSO) d (ppm): 12.24 (s, 1H), 8.29 (s, 1H),
1
4
28.1, 127.9, 127.9, 127.7, 126.5, 126.0, 121.9, 103.3, 101.6,
9.8 (2C), 49.6 (2C), 41.6. MS (ESI) m/z: 406 [MþH] .
7.53 (s, 1H), 7.33 (d, J ¼ 8.0 Hz, 2H), 7.17 (d, J ¼ 8.0 Hz, 2H),
þ
3.73 (s, 2H), 3.68 (s, 4H), 3.52 (d, J ¼ 12 Hz, 4H), 1.27 (s, 9H).
1
3
C NMR (100 MHz, DMSO) d (ppm): 169.7, 159.1, 151.2,
1
1
-(4-(5-Bromo-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-
-yl)-2-phenylethanone (5n)
149.0, 133.2, 129.4, 129.1, 125.5, 121.9, 103.2, 101.6, 49.8
(2C), 49.5 (2C), 41.5, 34.5, 31.6 (3C). MS (ESI) m/z: 456
þ
Pale yellow solid, yield 81%, purity 98.4%, mp: 120–124°C,
[MþH] .
1
HNMR(400MHz,DMSO)d(ppm):12.33(s,1H),8.31(s,1H),7.58
(
s, 1H), 7.34–7.23 (m, 5H), 3.78 (s, 2H), 3.70 (s, 4H), 3.46 (d,
1-(4-(5-Bromo-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-
1-yl)-2-(3,4-dichlorophenyl)ethanone (5t)
13
J ¼ 20Hz, 4H). C NMR (100 MHz, DMSO) d (ppm): 169.6, 159.6,
1
8
52.1, 151.1, 136.3, 129.4 (2C), 128.8 (2C), 126.8, 124.8, 104.9,
Yellow solid, yield 78%, purity 97.7%, mp: 149–152°C,
þ
1
6.3, 50.3 (2C), 50.2 (2C), 41.5. MS (ESI) m/z: 400 [MþH] .
H NMR (400 MHz, DMSO) d (ppm): 12.34 (s, 1H), 8.32
(
s, 1H), 7.59–7.53 (m, 3H), 7.24 (d, J ¼ 8.4 Hz, 1H), 3.83
13
1
1
-(4-(5-Bromo-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-
-yl)-2-(3-bromophenyl)ethanone (5o)
(s, 2H), 3.71 (d, J ¼ 20 Hz, 4H), 3.52 (s, 4H).
C NMR
(100 MHz, DMSO) d (ppm): 169.9, 158.3, 156.8, 152.4, 150.9,
Pale white solid, yield 83%, purity 97.8%, mp: 184–186°C,
H NMR (400 MHz, DMSO) d (ppm): 12.33 (s, 1H), 8.32 (s, 1H),
130.4 (2C), 128.0, 122.0, 114.2 (2C), 102.8, 101.2, 55.4, 45.4
(2C), 45.3 (2C), 41.5. MS (ESI) m/z: 468 [MþH] .
1
þ
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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361

Arch. Pharm. Chem. Life Sci. 2016, 349, 356–362
Y. Liu et al.
ARC HH PHARM
Archiv der Pharmazie
1
1
-(4-(5-Bromo-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-
-yl)-2-(naphthalen-2-yl)ethanone (5u)
preparation was performed by extracting the ligand,
removing water molecules, adding hydrogen atoms, and
assigning AMBER7 FF99 charges to the protein. All of the
target compounds were docked into Akt1 and formed
hydrogen bonds and hydrophobic interactions in the
model. After docking calculations, 20 binding poses per
ligand were generated, and the best conformation with the
highest CScore was selected for subsequent interaction
analysis.
Pale yellow solid, yield 80%, purity 98.3%, mp: 144–146°C,
1
HNMR(400MHz,DMSO)d(ppm):12.25(s,1H),8.29(s,1H),7.87
(
(
(
t, J ¼ 7.8 Hz, 3H), 7.77 (s, 1H), 7.55 (s, 1H), 7.52–7.44
m, 2H), 7.42 (dd, J ¼ 1.6 Hz, 1H), 3.96 (s, 2H), 3.75
¼ 8.4 Hz, J
d, J ¼ 16.0 Hz, 4H), 3.49 (d, J ¼ 19.7 Hz, 4H). C NMR (100 MHz,
1
2
13
DMSO) d (ppm): 169.5, 159.6, 152.1, 151.1, 134.0, 133.5, 132.2,
1
5
28.2, 128.1, 128.0, 127.9, 127.7, 126.5, 126.0, 124.8, 104.9, 86.3,
0.4 (2C), 50.3 (2C), 41.6. MS (ESI) m/z: 450 [MþH] .
þ
Biology
This work was supported by the National Natural Science
Foundation of China (no. 21272140) and Projects of Shandong
Science and Technology (no. 2014GSF119003).
In vitro Akt1 kinase activity assay
In vitro Akt1 kinase activity was evaluated in 384-well plates
1
using the HTRF assay (LANCE ), which has been well validated
and applied to a number of enzymatic activity assays [11]. The
assay format involves two steps. Kinase reaction step: each well
was added Akt1 (0.075ng/mL), STK Substrate-biotin (0.25 mM),
gradient-diluted compound (0.064–5000 nM) in kinase buffer
The authors have declared no conflicts of interest.
(
2 3
50 mMHEPES, 5 mMMgCl , 1 mMDTT,0.02%NaN ,and0.01%
References
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During this step, only the active kinase phosphorylated the
substrate. Detection step: Sa-XL665 conjugate (0.0625mM) and
STK Ab-Cryptate (5 mL) were added to the wells and incubated
for 2 h. During this step, EDTA in the detection buffer stopped
the enzymatic reaction, the detection reagents caught the
phosphorylated substrate. Then the fluorescence was
measured at 620 and 665nm using a microplate reader
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The antiproliferative effects of the target compounds on the
LNCaP and PC-3 cell lines were tested using the MTT assay. The
3
cells were seeded in 96-well microplates at a density of 5 ꢁ 10
cells per well and cultured in a humidified atmosphere with
5
% CO
treated with compounds at final concentrations ranging from
to 80 mM for PC-3 cells (2.5–40 mM LNCaP cells). The final
concentration of DMSO in medium was less than 0.5%. After
2 h of incubation, 5 mg/mL MTT (10 mL) was added followed
2
at 37°C. When attached to the bottom, the cells were
5
7
by another 4 h incubation, then the supernatant was removed
and the formazan crystal was extracted by DMSO. The optical
density (OD570 nm) value was detected using a microplate
reader (Bio-Rad 680, USA). The IC50 was calculated using
PRISM version 5.0 software from the non-linear curve.
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Molecular docking was performed using the Sybyl 1.1
program and the Akt1 crystal structure (PDB: 4EKL). Protein
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ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
362