Structural optimization elaborates novel potent Akt inhibitors with promising anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2017.06.067

Targeting of Akt has been validated as a well rationalized approach to cancer treatment, and represents a promising therapeutic strategy for aggressive hematologic malignancies. We describe herein an exploration of novel Akt inhibitors for cancer therapy through structural optimization of previously described 4-(piperazin-1-yl)-7H-pyrrolo[2,3-d]pyrimidine derivatives. Our studies yielded a novel series of pyrrolopyrimidine based phenylpiperidine carboxamides capable of potent inhibition of Akt1. Notably, 10h exhibited robust antiproliferative effects in both mantle cell lymphoma cell lines and primary patient tumor cells. Low micromolar doses of 10h induced cell apoptosis and cell cycle arrest in G₂/M phase, and significantly downregulated the phosphorylation of Akt downstream effectors GSK3β and S6 in Jeko-1 cells.

Full text of DOI:10.1016/j.ejmech.2017.06.067

Accepted Manuscript
Structural optimization elaborates novel potent Akt inhibitors with promising
anticancer activity
Yang Liu, Yanzhen Yin, Zhen Zhang, Carrie J. Li, Hui Zhang, Daoguang Zhang,
Changying Jiang, Krystle Nomie, Liang Zhang, Michael L. Wang, Guisen Zhao
PII:
S0223-5234(17)30521-4
10.1016/j.ejmech.2017.06.067
EJMECH 9562
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 February 2017
Revised Date: 18 April 2017
Accepted Date: 29 June 2017
Please cite this article as: Y. Liu, Y. Yin, Z. Zhang, C.J. Li, H. Zhang, D. Zhang, C. Jiang, K. Nomie, L.
Zhang, M.L. Wang, G. Zhao, Structural optimization elaborates novel potent Akt inhibitors with promising
anticancer activity, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.06.067.
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Graphic Abstract
Structural optimization produced a novel series of pyrrolopyrimidine based phenylpiperidine carboxamides as potent
Akt inhibitors. Notably, 10h exhibited robust antiproliferative effects in both mantle cell lymphoma cell lines and
primary patient tumor cells.

ACCEPTED MANUSCRIPT
Structural optimization elaborates novel potent Akt inhibitors with promising
anticancer activity
Yang Liu1, Yanzhen Yin1, Zhen Zhang1, Carrie J Li2, Hui Zhang2, Daoguang Zhang1, Changying Jiang2,
Krystle Nomie2, Liang Zhang2, Michael L. Wang2,* and 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;
2The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
Corresponding authors
Guisen Zhao*
Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, Jinan,
Shandong 250012, PR China;
Tel/ Fax: +86 531 88382009;
E-mail: guisenzhao@sdu.edu.cn.
Michael L. Wang*
The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
Tel: +1 7137922860
Fax: +1 7137945656
E-mail: miwang@mdanderson.org

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Abstract
Targeting of Akt has been validated as a well rationalized approach to cancer treatment, and
represents a promising therapeutic strategy for aggressive hematologic malignancies. We describe
herein an exploration of novel Akt inhibitors for cancer therapy through structural optimization of
previously described 4-(piperazin-1-yl)-7H-pyrrolo[2,3-d]pyrimidine derivatives. Our studies yielded a
novel series of pyrrolopyrimidine based phenylpiperidine carboxamides capable of potent inhibition of
Akt1. Notably, 10h exhibited robust antiproliferative effects in both mantle cell lymphoma cell lines
and primary patient tumor cells. Low micromolar doses of 10h induced cell apoptosis and cell cycle
arrest in G2/M phase, and significantly downregulated the phosphorylation of Akt downstream effectors
GSK3β and S6 in Jeko-1 cells.
Keywords: Akt, Anticancer, Docking, Mantle cell lymphoma, Piperidyl, Pyrrolopyrimidines
1. Introduction
Akt, also called protein kinase B or PKB, is a serine/threonine kinase that acts as a central junction
in the phosphoinositide 3-kinase (PI3K) − Akt signaling pathway [1]. This cascade plays a critical role
in regulating cell survival and proliferation, oncogenesis and mechanisms of drug resistance [2, 3].
There are three closely related Akt isoforms, Akt1 (PKBα), Akt2 (PKBβ) and Akt3 (PKBγ), all of
which share significant sequence identity in the ATP-binding site [4]. Akt promotes cell proliferation
through the phosphorylation of various substrates such as glycogen synthase kinase 3 (GSK-3), TSC1/2,
NF-κB, Bcl-2 family proteins and mammalian target of rapamycin complex 1 (mTORC1) [5, 6].
Aberrant Akt activation has been confirmed in various cancers, including both hematological
neoplasms and solid tumors, often in correlation with resistance to chemotherapy [7, 8]. Therefore,
targeting Akt is a well rationalized approach to cancer treatment [9].
Mantle cell lymphoma (MCL) is a subtype of Non-Hodgkin’s Lymphoma (NHL) with poor
prognosis [10]. Although MCL typically responds to conventional chemotherapy, most patients
eventually experience disease progression [11]. Aberrant Akt activation has been implicated in MCL
pathogenesis and survival [12, 13]. While no PI3KCA mutations have been reported as contributors to
constitutive Akt activation, loss of tumor suppressor PTEN may be an underlying drive of constitutive
Akt signaling in MCL [14]. Such findings support the possibility of Akt inhibition as a novel strategy
for the treatment of MCL.
Multiple strategies have been adopted for the discovery of small molecules that target Akt,

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including ATP- or substrate-competitive inhibitors and allosteric inhibitors [15]. Of these,
ATP-competitive inhibitors have yielded the most promising results. Common interactions between the
ATP-competitive inhibitors and Akt include a heteroaromatic ring which forms bidentate hydrogen
bonds to residues Glu228 and Ala230 in the hinge region, a basic amino group that can form hydrogen
binding interactions with the acid hole formed by Glu234 and Glu278, and an aromatic group
positioned in a hydrophobic pocket under the P-loop [16, 17].
Our laboratory has reported the development of a weak hit 1 from self-library screening into lead
2, an ATP-competitive inhibitor with desirable antiproliferative effects in prostate cancer cell lines [18].
For further modification of 2, we decided to introduce an amino functionality at the benzylic position,
which was inspired by the common structural platform of ATP-competitive inhibitors. In this regard,
using a well-established conformational restriction drug design strategy, a piperidyl moiety with more
conformational rigidity was deliberately chosen and incorporated, elaborating a novel series of
(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)(4-phenylpiperidin-4–yl)methanones
enhanced inhibitory potency in both enzyme activity and cancer cell growth (Fig. 1).
with

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Fig. 1. Design of the targeted compounds. A) Binding site of the ATP-competitive inhibitors; B) common features of
ATP-competitive inhibitors; C) Design of the targeted compounds from the initial hit 1
2. Results and discussion
2.1. Chemistry
The general synthetic route for the pyrrolopyrimidine derivatives is outlined in Scheme 1.
Chlorination or bromination at the 5-position of the readily available 3a afforded compound 3b or 3c,
subsequent introduction of the Boc-protected piperazine linker via SNAr delivered 4a-4c, followed by
removal of the Boc group affording 5a-5c as dihydrochloride salts. Commercially available 6 reacted
with di-tert-butyl dicarbonate to produce the Boc-protected 7, which was further used in the
dialkylation of the substituted benzeneacetonitrile to afford intermediate 8a-8g, basic hydrolysis of the
nitrile 8a-8g gave the carboxylic acid 9a-9g. Amide coupling between 5a-5c and 9a-9g and
deprotection of the Boc group achieved the target compounds 10a-10n.
Scheme 1. Synthetic route for the designed derivatives. Reagents and conditions: (a) NCS, DMF, r.t.; (b) NBS, DMF, r.t.; (c)
1-Boc-piperazine, DIEA, DMF, 110℃; (d) HCl-dioxane, CH3OH, r.t.; (e) (Boc)2O, CH2Cl2, 10% NaOH (aq), r.t. (f) substituted
benzeneacetonitrile, NaH, DMF, 60℃; (g) CH3CH2OH, NaOH(aq, 10M), 78℃; (h) (i) EDCI, HOBT, DIEA, DMF, r.t.; (ii)
HCl-dioxane, CH3OH, r.t..
2.2. Akt inhibition and cell antiproliferative activity
The fourteen newly synthesized pyrrolopyrimidine analogs were evaluated for their activity
against Akt1 via a Homogeneous Time-Resolved Fluorescence (HTRF) kinase activity assay. Their
effects on proliferation were further assessed in MCL cell lines. The first Akt inhibitor to enter clinical
trials, GSK690693, served as a positive control. GSK690693 was previously shown to induce growth
inhibition and apoptosis in acute lymphoblastic leukemia [19].

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Table 1. The inhibitory effects of pyrrolopyrimidine derivatives on Akt1 activity
Akt1,
IC50(nM) a
Akt1,
Code
R1
R2
Code
R1
R2
IC50 (nM) a
2.9 ± 0.9
20.3 ± 1.6
7.6 ± 2.0
14.1 ± 3.3
1.5 ± 1.3
4.3 ± 0.4
1.7± 1.1
18.0b
H
H
4-tBu
4-OCH3
4-Cl
> 40
Cl
3,4-di-Cl
H
10a
10b
10c
10d
10e
10f
10i
10j
35.6 ± 3.2
26.5 ± 0.7
27.1 ± 1.6
> 40
Br
H
Br
3-Br
10k
10l
H
4-Br
Br
4-OCH3
4-Cl
H
3,4-di-Cl
3-Br
Br
10m
10n
Cl
Cl
Cl
5.6 ± 2.5
1.9 ± 1.6
1.8 ± 1.2
Br
3,4-di-Cl
4-Cl
10g
GSK690693
2
4-Br
10h
aValues are means of three independent experiments;
bValue for lead 2 was previously reported [18].
As Table 1 shows, introduction of the piperidyl moiety at the benzylic position represented a
substantial improvement in Akt1 inhibition over the initial lead (2 vs 10m). C5-substitution for chloro
and bromo substituents produced an equivalent profile, with more than a 10-fold increase in enzyme
inhibitory activity (10c vs 10g and 10m). Examination of substituents on the aromatic ring indicated
that the chlorine or bromine substituent (10k, 10m and 10n) increased potency relative to the
unsubstituted phenyl group of 10j. The small decrease in enzyme potency for 10f vs 10h indicated that
the meta-position was potentially less promising for manipulation; however, in the para-position,
chloro and bromo analogues (10g, 10h and 10m) displayed similar levels of efficacy against Akt1 and
showed the greatest enzyme potency comparable to that of GSK690693. A reduction in Akt1 potency
was observed in the case of methoxy substitution (10l), whereas 3, 4-di-chloro substitution (10i and
10n) was somewhat less active than the p-substitution (10g and 10m).
The promising candidates 10h and 10m were further explored in a panel of MCL cell lines. As
shown in Table 2, in comparison with the previous lead 2, both compounds 10h and 10m demonstrated
improved antiproliferative activities in MCL cell lines compared with GSK690693. 10h was the most
potent inhibitor in MCL cells with an IC50 value lower than 1 µM.

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Table 2. Cell proliferation assay assessing the effects of 10h and 10m on MCL cell lines
Cell proliferation assay, IC50 ± SD/µMa
Code
JVM-2
9.9±0.7
0.7±0.4
2.3±1.3
1.6±0.7
Maver-1
18.5±2.2
0.3±0.1
1.8±0.4
5.1±0.5
SP-49
Z-138
Mino
Jeko-1
8.7±1.3
0.3±0.1
1.2±0.1
3.0±1.5
2
10h
7.8±1.0
1.5±0.3
4.7±0.1
4.8±0.1
6.8±0.5
0.2±0.1
1.7±0.3
3.1±0.2
18.1±2.6
0.7±0.1
3.5±0.3
20.8±3.5
10m
GSK690693
aValues are means of three independent experiments
2.3. Cell apoptosis and cell cycle assay
Activated Akt influences many factors that mediate apoptosis and the cell cycle [20]. Accordingly,
apoptosis and cell cycle assays were performed after 10h treatment in MCL Jeko-1 cells, which are
characterized by constitutive phosphorylation of Akt and PTEN loss [21]. Annexin V/PI staining
showed that 10h induced cell apoptosis in Jeko-1 cells in a dose- and time-dependent manner (Fig. 2).
Caspase-3 and PARP cleavage were measured to further confirm the initiation of apoptotsis, and the
results revealed dose-dependent activation of cleaved PARP and caspase-3 in Jeko-1 cells, indicating
that 10h induced cell apoptosis through the caspase 3-mediated PARP apoptotic pathway. The cell
cycle assay demonstrated that treatment with 10h dose dependently induced the cell cycle arrest in the
G2/M phase after 24 hours of treatment, effectively preventing cell cycle progression and promoting
cell death.

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Fig. 2. Cell apoptosis and cell cycle assay for 10h in Jeko-1 cells. A/C) Apoptosis assay of 10h at indicated concentrations in
Jeko-1 cells for 48 hours; B/D) Apoptosis assay of 10h at 1 µM in Jeko-1 cells for the indicated treatment periods; E)
Dose-dependent activation of cleaved caspase-3 and PARP after 24 hours treatment with 10h; F) Cell cycle analysis of 10h at
indicated concentrations in Jeko-1 cells for 24 hours.
2.4. Inhibitory effects on Akt signaling
To further examine the inhibitory effects of 10h on Akt signaling, immunoblotting was performed
to detect the phosphorylation of Akt and its downstream effectors GSK3β and S6 in Jeko-1 cells (Fig.
3A&B). After 24 hours of 10h treatment, phosphorylated GSK3β and S6 levels were downregulated in
a dose-dependent manner, while Ser473 and Thr308 phosphorylation of Akt was observed. These
results are consistent with reported mechanisms of other Akt inhibitors, which point to a positive
feedback mechanism as a mechanism for increased Akt phosphorylation [22-24].

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2.5. Cell viability assay in primary MCL cells
The antiproliferative efficacy of the lead 2, 10h, 10m and GSK690693 were investigated in
primary MCL patient cells. As shown in Fig. 3C, consistent with MCL cell lines, 10h and 10m
dramatically decreased the cell viability of these cells in comparison to GSK690693.
Fig. 3. Western blot analysis, cell viability assay in patient cells and molecular modeling. A) Effects of 10h on Akt signaling in
Jeko-1 cells after 24 hours treatment; B) Relative protein expression was determined using Image J. Values were normalized to
loading control; C) Cell viability assay of 10h in primary MCL cells. Values were normalized to control; Unpaired student’s t-test
between 10h and GSK690693-treated cells is statistically significant (* P < 0.01); D) Proposed binding mode of 10h at the ATP
binding site of Akt1 (PDB: 4EKL).
2.6. Molecular modeling
The binding mode of 10h was explored by molecular modeling using Sybyl 2.0 (Fig. 3D). As
expected, the pyrrolopyrimidine core formed a pair of hydrogen bonds with the hinge region through
Ala230 and Glu228, the 4-chlorophenyl group entered into a hydrophobic pocket under the P-loop.
Instead of forming hydrogen bonds between Glu234 and Glu278 respectively within the acid hole, a
different hydrogen bond was formed between the introduced piperidyl moiety and Lys158 to contribute
to the binding potency to Akt enzyme.
3. Conclusion
In summary, we have described the optimization of previously reported pyrrolopyrimidine Akt

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inhibitors to a series of advanced pyrrolopyrimidine based phenylpiperidine carboxamides.
Introduction of piperidyl moiety at the benzylic position resulted in a substantial boost in Akt1
inhibitory potency and antiproliferative effects in cancer cell growth, supporting further evaluation and
development of the promising derivative 10h in cancer therapy.
4. Experimental section
4.1. Chemistry
All solvents and reagents were purchased from commercial suppliers and used without further
purification. All reactions were 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 solvents indicated. NMR spectra were recorded on a Bruker avance DRX600 or 400
Spectrometer using TMS as an internal standard in DMSO-d6. Chemical shifts are reported in parts per
million (ppm). Coupling constants (J) are given in Hz. The mass spectra (MS) were measured with an
API 4000. All of the melting points were determined in a Büchi capillary melting point apparatus and
are uncorrected. Human Akt1 was obtained from Carna Biosciences, Inc. (Canada). The HTRF assay
kit was purchased from Cisbio assays, Inc. (France).
4.2. General synthesis of compounds
4.2.1. General synthesis of compounds 3b and 3c
To a solution of readily available 3a (20 mmol) in N, N-dimethylformamide (DMF) (15 mL) was
added NCS/NBS (21 mmol) and the reaction mixture was stirred at room temperature for 72 h. Then
ice water (150 mL) was poured into the mixture, the precipitate was filtered, washed with water (3×100
mL), and dried to give 3b and 3c.
4.2.1.1. 4, 5-Dichloro-7H-pyrrolo[2,3-d]pyrimidine (3b)
Grey powder, yield 95%, mp: 287～290℃, 1H NMR (400 MHz, DMSO) δ (ppm): 12.87 (s, 1H), 8.63
(s, 1H), 7.91 (s, 1H). MS (ESI) m/z: 188 [M+H]+.
4.2.1.2. 5-Bromo-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (3c)
Pale powder, yield 95%, mp: 290～292℃, 1H NMR (400 MHz, DMSO) δ (ppm): 13.00 (s, 1H), 8.64 (s,
1H), 7.97 (s, 1H). MS (ESI) m/z: 232 [M+H]+.
4.2.2. General synthesis of compounds 4a-4c
To a solution of 3a-3c (3.2 mmol) and N, N-diisopropylethylamine (DIEA) (4.8 mmol) in DMF (3
mL) was added N-Boc-piperazine (3.6 mmol), the resulting mixture was heated at 110℃ for 16 h. The

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reaction mixture was poured into ice water (30 mL) and extracted with ethyl acetate (3×30 mL), the
combined organic layers were washed with brine (2×30 mL), dried over sodium sulfate, filtered and
concentrated under reduced pressure. The crude residue was purified by silica gel chromatography with
petroleum ether/ethylacetate (1 : 3) to obtain 4a-4c.
4.2.2.1. Tert-butyl 4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazine-1-carboxylate (4a)
Off-white solid, yield 59%, 1H NMR (600 MHz, DMSO) δ (ppm): 11.73 (s, 1H), 8.15 (s, 1H), 7.20 (d,
J = 0.6 Hz, 1H), 6.64 (d, J = 1.8 Hz, 1H), 3.86 (s, 4H), 3.48 (s, 4H), 1.43 (s, 9H). MS (ESI) m/z: 304
[M+H]+.
4.2.2.2. Tert-butyl 4-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazine-1-carboxylate (4b)
Off-white solid, yield: 51%, 1H NMR (400 MHz, DMSO) δ (ppm): 12.23 (s, 1H), 8.29 (s, 1H), 7.52 (d,
J = 2.8 Hz, 1H), 3.52 (s, 8H), 1.42 (s, 9H). MS (ESI) m/z: 338 [M+H]+.
4.2.2.3. Tert-butyl 4-(5-bromo-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazine-1-carboxylate (4c)
Paleyellow solid, yield 48%, 1H NMR (400 MHz, DMSO) δ (ppm): 12.21 (s, 1H), 8.37 (s, 1H), 7.52 (d,
J = 2.6 Hz, 1H), 3.52 (s, 8H), 1.42 (s, 9H). MS (ESI) m/z: 383 [M+H]+.
4.2.3. General synthesis of compounds 5a-5c
To a solution of 4a-4c (1 mmol) suspended in methanol (1 ml) was added 4M HCl in dioxane (5
ml) and the reaction mixture was stirred for 24 h at room temperature. The precipitate was filtrated,
washed with diethyl ether (2×5 ml), and dried to obtain 5a-5c, which were used for the next step
without further purification.
4.2.4. Tert-butyl bis(2-chloroethyl)carbamate (7)
To a solution of 6 (28.0 mmol) in dichloromethane (150 mL), 10% sodium hydroxide solution
(33.6 mmol) was added. This was followed by addition of di-tert-butyl dicarbonate (33.6 mmol) over a
period of 10 min at 0℃. After completion of the reaction, the organic layer was separated, washed with
brine, dried over sodium sulfate and evaporated under reduced pressure. The crude product was
purified by silica gel chromatography with petroleum ether/ethylacetate (8 : 1) to yield 7 as a pale
1
yellow oil. Yield: 95%, H NMR (600 MHz, DMSO) δ (ppm): 3.71-3.67 (m, 4H), 3.52 (t, J = 1.8 Hz,
4H), 1.40 (s, 9H). MS (ESI) m/z: 242 [M+H]+.
4.2.5. General synthesis of compounds 8a-8g
To a solution of 7 (1.66 mmol) and substituted benzeneacetonitrile (1.66 mmol) in anhydrous
DMF (5 mL) was added NaH (60% dispersion in mineral oil, 8.4 mmol) portion wise at 0℃ over 1 h.

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The reaction mixture was heated at 60℃ for 16 h. The reaction mixture was poured into ice-water (50
mL) and extracted with ethyl acetate (3×50 mL). The combined organic layers were washed with brine
(2×50 mL), dried over sodium sulfate, filtered, and concentrated. The crude product was purified by
silica gel chromatography eluting with ether/ethyl ether (15:1) to give 8a-8g.
4.2.5.1. Tert-butyl 4-cyano-4-phenylpiperidine-1-carboxylate (8a)
1
White solid, yield 30%, H NMR (400 MHz, DMSO) δ (ppm): 7.55 (m, 2H), 7.44 (t, J = 7.8 Hz, 2H),
7.38 (d, J = 7.2 Hz, 1H), 4.13 (d, J = 13.2 Hz, 2H), 3.02 (br, 2H), 2.12 (d, J = 11.8 Hz, 2H), 1.92 (td, J1
= 12.9 Hz, J2 = 4.3 Hz, 2H), 1.42 (s, 9H). MS (ESI) m/z: 287 [M+H]+.
4.2.5.2. 1Tert-butyl 4-(3-bromophenyl)-4-cyanopiperidine-1-carboxylate (8b)
White solid, yield 63%, 1H NMR (400 MHz, DMSO) δ (ppm): 7.74 (t, J = 1.76 Hz, 1H), 7.58 (td, J1 =
7.76 Hz, J2 = 1.5 Hz, 2H), 7.41 (t, J = 7.9 Hz, 1H), 4.13 (d, J = 13.0 Hz, 2H), 3.00 (br, 2H), 2.14 (d, J =
12.1 Hz, 2H), 1.90 (td, J1 = 13.0 Hz, J2 = 4.4 Hz, 2H), 1.42 (s, 9H). MS (ESI) m/z: 365 [M+H]+ .
4.2.5.3. Tert-butyl 4-(4-(tert-butyl)phenyl)-4-cyanopiperidine-1-carboxylate (8c)
Pale yellow solid, yield 59%, 1H NMR (400 MHz, DMSO) δ (ppm): 7.45 (s, 4H), 4.12 (d, J = 6.5 Hz,
2H), 3.01 (br, 2H), 2.10 (d, J = 6.2 Hz, 2H), 1.89 (td, J1 = 13.1 Hz, J2 = 4.2 Hz, 2H), 1.42 (s, 9H), 1.28
(s, 9H). MS (ESI) m/z: 343 [M+H]+.
4.2.5.4. Tert-butyl 4-cyano-4-(4-methoxyphenyl)piperidine-1-carboxylate (8d)
Pale yellow solid, yield 61%, 1H NMR (400 MHz, DMSO) δ (ppm): 7.44 (d, J = 8.8 Hz, 2H), 6.98 (d, J
= 8.76 Hz, 2H), 4.11 (d, J = 12.9 Hz, 2H), 3.76 (s, 3H), 3.01 (br, 2H), 2.09 (d, J = 12.8 Hz, 2H), 1.86
(td, J1 = 13.0 Hz, J2 = 4.1 Hz, 2H), 1.42 (s, 9H). MS (ESI) m/z: 317 [M+H]+.
4.2.5.5. Tert-butyl 4-(4-chlorophenyl)-4-cyanopiperidine-1-carboxylate (8e)
White solid, yield 51%, 1H NMR (600 MHz, DMSO) δ (ppm): 7.58 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4
Hz, 2H), 4.12 (br, 2H), 3.00 (br, 2H), 2.12 (d, J = 13.2 Hz, 2H), 1.91 (td, J1 = 12.9 Hz, J2 = 4.2 Hz, 2H),
1.42 (s, 9H). MS (ESI) m/z: 321 [M+H]+.
4.2.5.6. Tert-butyl 4-(4-bromophenyl)-4-cyanopiperidine-1-carboxylate (8f)
White solid, yield 50%, 1H NMR (400 MHz, DMSO) δ (ppm): 7.65 (d, J = 8.6 Hz, 2H), 7.52 (d, J = 8.6
Hz, 2H), 4.13 (d, J = 12.8 Hz, 2H), 3.01 (br, 2H), 2.12 (d, J = 12.8 Hz, 2H), 1.91 (td, J1 = 13.1 Hz, J2 =
4.2 Hz, 2H), 1.42 (s, 9H). MS (ESI) m/z: 365 [M+H]+.
4.2.5.7. Tert-butyl 4-cyano-4-(3,4-dichlorophenyl)piperidine-1-carboxylate (8g)
Pale yellow solid, yield 58%, 1H NMR (400 MHz, DMSO) δ (ppm): 7.82 (d, J = 2.3 Hz, 1H), 7.72 (d, J

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= 8.5 Hz, 1H), 7.57 (dd, J1 = 6.2 Hz, J2 = 2.3 Hz, 1H), 4.13 (d, J = 13.1 Hz, 2H), 3.02 (br, 2H), 2.15 (d,
J = 12.2 Hz, 2H), 1.96 (m, 2H), 1.42 (s, 9H). MS (ESI) m/z: 355 [M+H]+.
4.2.6. General synthesis of compounds 9a-9g
A solution of 8a-8g (1.15 mmol) in ethanol (3 mL) and 3 mL 10 N aqueous sodium hydroxide was
refluxed for 48 hours, cooled to room temperature, then poured into 1 N aqueous hydrochloric acid,
and extracted by ethyl acetate. The organic extracts were washed with brine, dried over sodium sulfate,
and concentrated under reduced pressure. The crude product was recrystallized using ethyl acetate to
obtain 9a-9g.
4.2.6.1. 1-(Tert-butoxycarbonyl)-4-phenylpiperidine-4-carboxylic acid (9a)
1
White solid, yield 73%, H NMR (400 MHz, DMSO) δ (ppm): 12.70 (s, 1H), 7.40-7.33 (m, 4H),
7.28-7.24 (m, 1H), 3.80 (d, J = 13.6 Hz, 2H), 2.96 (br, 2H), 2.36 (d, J = 13.2 Hz, 2H), 1.73-1.67 (m,
2H), 1.39 (s, 9H). MS (ESI) m/z: 304 [M+H]+.
4.2.6.2. 4-(3-Bromophenyl)-1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid (9b)
White solid, yield 78%, 1H NMR (400 MHz, DMSO) δ (ppm): 12.87 (s, 1H), 7.52 (t, J = 18.0 Hz, 1H),
7.50-7.47 (m, 1H), 7.42-7.40 (m, 1H), 7.33 (t, J = 7.8 Hz, 1H), 3.79 (d, J = 5.6 Hz, 2H), 2.99 (br, 2H),
2.34 (d, J = 13.6 Hz, 2H), 1.75-1.67 (m, 2H), 1.39 (s, 9H). MS (ESI) m/z: 382 [M+H]+.
4.2.6.3. 1-(Tert-butoxycarbonyl)-4-(4-(tert-butyl)phenyl)piperidine-4-carboxylic acid (9c)
White solid, yield 67%, 1H NMR (400 MHz, DMSO) δ (ppm): 12.62 (br, 1H), 7.36 (d, J = 8.4 Hz, 2H),
7.30 (d, J = 8.4 Hz, 2H), 3.80 (d, J = 13.6 Hz, 2H), 2.93 (br, 2H), 2.35 (d, J = 13.6 Hz, 2H), 1.71-1.64
(m, 2H), 1.38 (s, 9H), 1.25 (s, 9H). MS (ESI) m/z: 360 [M+H]+.
4.2.6.4. 1-(Tert-butoxycarbonyl)-4-(4-methoxyphenyl)piperidine-4-carboxylic acid (9d)
1
White solid, yield 57%, H NMR (400 MHz, DMSO) δ (ppm): 12.58 (s, 1H), 7.30 (m, 2H), 6.91 (m,
2H), 3.78-3.72 (m, 5H), 2.95 (br, 2H), 2.33 (d, J = 13.6 Hz, 2H), 1.63-1.70 (m, 2H), 1.38 (s, 9H). MS
(ESI) m/z: 334 [M+H]+.
4.2.6.5. 1-(Tert-butoxycarbonyl)-4-(4-chlorophenyl)piperidine-4-carboxylic acid (9e)
White solid, yield 80%, 1H NMR (400 MHz, DMSO) δ (ppm): 12.80 (s, 1H), 7.41 (s, 4H), 3.77 (d, J =
13.6 Hz, 2H), 2.97 (br, 2H), 2.34 (d, J = 13.6 Hz, 2H), 1.74-1.67 (m, 2H), 1.39 (s, 9H). MS (ESI) m/z:
338 [M+H]+.
4.2.6.6. 4-(4-Bromophenyl)-1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid (9f)
White solid, yield 68%, 1H NMR (600 MHz, DMSO) δ (ppm): 12.81 (s, 1H), 7.55 (d, J = 8.4 Hz, 2H),

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7.35 (d, J = 8.4 Hz, 2H), 3.77 (d, J = 13.8 Hz, 2H), 2.96 (br, 2H), 2.33 (d, J = 13.8 Hz, 2H), 1.72-1.67
(m, 2H), 1.39 (s, 9H). MS (ESI) m/z: 382 [M+H]+.
4.2.6.7. 1-(Tert-butoxycarbonyl)-4-(3,4-dichlorophenyl)piperidine-4-carboxylic acid (9g)
White solid, yield 81%, 1H NMR (400 MHz, DMSO) δ (ppm): 12.97 (s, 1H), 7.63 (d, J = 8.2 Hz, 1H),
7.59 (d, J = 3.6 Hz, 1H), 7.40 (dd, J1 = 8.2 Hz, J2 = 2.0 Hz, 1H), 3.76 (d, J = 21.0 Hz, 2H), 2.97 (br, 2H),
2.33 (d, J = 20.4 Hz, 2H), 1.77-1.70 (m, 2H), 1.39 (s, 9H). MS (ESI) m/z: 372 [M+H]+.
4.2.7. General synthesis of compounds 10a-10n
To a solution of 9a-9g (0.30 mmol) and DIEA (3.0 mmol) in DMF (2 mL) was added
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (0.33 mmol) and
1-hydroxybenzotriazole (HOBt) (0.33 mmoL). The reaction mixture was stirred at 0℃ for 1 hour,
followed by addition of 5a-5c (0.30 mmol). After completion of addition, the final mixture was stirred
at room temperature for another 12 hours, then poured into ice water (20 mL), washed with saturated
aqueous sodium bicarbonate (20×3 mL) and brine (20×2 mL), dried over sodium sulfate and solvent
was evaporated under reduced pressure. The residue was suspended in methanol (0.5 mL), after
addition of the 4M HCl in dioxane (3 ml), the reaction mixture was stirred for 24 hours at room
temperature. The precipitate was filtrated, the obtained solid was recrystallized from methanol to give
the desired 10a-10n.
4.2.7.1. (4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)(4-(4-(tert-butyl)phenyl)piperidin-4
yl)met- hanone (10a)
Palewhite solid, yield 69%, mp: 223～228℃, 1H NMR (400 MHz, D2O) δ (ppm): 8.14 (s, 1H), 7.47 (d,
J = 8.8 Hz, 1H), 7.26-7.24 (m, 3H), 6.57 (d, J = 3.6 Hz, 1H), 3.98-3.81 (m, 4H), 3.41-3.23 (m, 8H),
2.52 (d, J = 14.4 Hz, 2H), 2.20 (t, J = 11.6 Hz, 2H), 1.19 (s, 9H). 13C NMR (100 MHz, D2O) δ (ppm):
174.07, 152.03, 151.74, 144.73, 142.35, 138.57, 126.59 (2C), 125.07 (2C), 124.11, 103.93, 102.07,
66.54 (2C), 62.49 (2C), 47.73 (2C), 41.54, 33.89, 31.52 (2C), 30.40 (3C). MS (ESI) m/z: 447 [M+H]+.
4.2.7.2.
(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)(4-(4-methoxyphenyl)piperidin-4-yl)metha- none
(10b)
Palewhite solid, yield 69%, mp: 215～220℃, 1H NMR (400 MHz, D2O) δ (ppm): 8.11 (s, 1H), 7.24 (d,
J = 3.6 Hz, 1H), 7.19 (d, J= 8.8Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H), 6.55 (d, J = 3.6 Hz, 1H), 3.94-3.81
(m, 4H), 3.69 (s, 3H), 3.51-3.19 (m, 10H), 2.51 (d, J = 10.8 Hz, 2H), 2.16 (t, J = 15.6 Hz, 2H). 13C

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NMR (100 MHz, D2O) δ (ppm): 174.24, 158.29, 151.91, 144.81, 142.37, 133.89, 126.63(2C), 124.18,
114.86 (2C), 103.89, 101.95, 66.61 (2C), 55.43 (3C), 47.54 (2C), 44.86 (2C), 41.63, 31.64 (2C). MS
(ESI) m/z: 421 [M+H]+.
4.2.7.3. (4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)(4-(4-chlorophenyl)piperidin-4-yl)metha-
none (10c)
1
Palewhite solid, yield 67%, mp: 189～194℃, H NMR (400 MHz, D2O) δ (ppm): 8.12 (s, 1H),
7.52-7.22 (m, 5H), 6.55 (s, 1H), 3.95-3.83 (m, 4H), 3.45-3.24 (m, 8H), 2.50 (d, J = 10.8 Hz, 2H), 2.13
(s, 2H). 13C NMR (100 MHz, D2O) δ (ppm): 173.55, 152.03, 144.51, 142.38, 140.19, 133.22, 129.56
(2C), 126.81 (2C), 124.30, 103.86, 101.92, 66.62 (2C), 62.60 (2C), 47.81 (2C), 41.57, 31.52 (2C). MS
(ESI) m/z: 425 [M+H]+.
4.2.7.4. (4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)(4-(4-bromophenyl)piperidin-4-yl)methan-
one (10d)
White solid, yield 65%, mp: 253～257℃, 1H NMR (400 MHz, D2O) δ (ppm): 8.13 (s, 1H), 7.43 (d, J =
8.4 Hz, 2H), 7.27 (d, J= 4.0 Hz, 1H), 7.17 (d, J = 8.4 Hz, 2H), 6.56 (dd, J= 3.6Hz, 1H), 3.96-3.83 (m,
4H), 3.47-3.38 (m, 6H), 3.27 (t, J = 11.6 Hz, 2H), 2.51 (d, J = 14.0 Hz, 2H), 2.16 (t, J = 12.0 Hz, 2H).
13C NMR (100 MHz, D2O) δ (ppm): 173.51, 152.13, 144.60, 142.52, 140.70, 132.54 (2C), 127.14 (2C),
124.27, 121.37, 103.81, 101.95, 66.61 (2C), 62.54 (2C), 47.93 (2C), 41.53, 31.45 (2C). MS (ESI) m/z:
471 [M+H]+.
4.2.7.5. (4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)(4-(3,4-dichlorophenyl)piperidin-4-yl)met-
hanone (10e)
Palewhite solid, yield 68%, mp: 195～199℃, 1H NMR (400 MHz, D2O) δ (ppm): 8.12 (s, 1H), 7.35 (d,
J = 8.4 Hz, 1H), 7.32 (d, J= 2.4 Hz, 1H), 7.24 (d, J = 3.6 Hz, 1H), 7.16 (d, J1 = 8.4 Hz, J2 = 2.4 Hz, 1H),
6.49 (d, J = 3.6 Hz, 1H), 3.96-3.86 (m, 4H), 3.52-3.38 (m, 6H), 3.26 (t, J = 12.4 Hz, 2H), 2.51 (d, J =
14.4 Hz, 2H), 2.12 (t, J = 12.0 Hz, 2H). 13C NMR (100 MHz, D2O) δ (ppm): 172.96, 152.07, 144.48,
142.47, 141.94, 132.95, 131.40 (2C), 127.27 (2C), 124.99, 124.39, 103.60, 101.76, 66.62 (2C), 47.98
(2C), 41.49 (2C), 34.60, 31.39 (2C). MS (ESI) m/z: 459 [M+H]+.
4.2.7.6.
(4-(3-bromophenyl)piperidin-4-yl)(4-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)methan
one (10f)
1
Paleyellow solid, yield 80%, mp: 257～261℃, H NMR (400 MHz, D2O) δ (ppm): 8.12 (s, 1H),

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7.45-7.41 (m, 2H), 7.31 (s, 1H), 7.28-7.26 (m, 2H), 3.79 (br, 4H), 3.42-3.22 (m, 8H), 2.50 (t, J = 14.0
Hz, 2H), 2.18 (t, J = 12.4 Hz, 2H). 13C NMR (100 MHz, D2O) δ (ppm): 172.85, 153.46, 145.54, 144.12,
143.20, 139.15, 131.44, 130.96, 128.24, 123.51, 123.03, 104.89, 101.03, 57.43 (2C), 48.47 (2C), 48.02
(2C), 41.46, 31.56 (2C). MS (ESI) m/z: 505 [M+H]+.
4.2.7.7. (4-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)(4-(4-chlorophenyl)piperidin-4-yl)
methanone (10g)
Palewhite solid, yield 66%, mp: 200～205℃, 1H NMR (400 MHz, D2O) δ (ppm): 8.11 (s, 1H), 7.33 (d,
J = 8.8 Hz, 2H), 7.29 (s, 1H), 7.25 (d, J = 8.4 Hz, 2H), 3.85-3.69 (m, 4H), 3.41-3.22 (m, 8H), 2.50 (t, J
= 14.4 Hz, 2H), 2.18 (t, J = 12.0 Hz, 2H). 13C NMR (100 MHz, D2O) δ (ppm): 173.26, 153.65, 145.83,
143.34, 140.48, 133.25, 129.63 (2C), 126.99 (2C), 123.65, 104.99, 101.26, 66.62 (2C), 62.61 (2C),
47.82 (2C), 41.55, 31.64 (2C). MS (ESI) m/z: 459 [M+H]+.
4.2.7.8. (4-(4-bromophenyl)piperidin-4-yl)(4-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)
methanone (10h)
1
Paleyellow solid, yield 61%, mp: 203～208℃, H NMR (400 MHz, D2O) δ (ppm): 8.10 (s, 1H), 7.47
(d, J = 8.6 Hz, 2H), 7.28 (s, 1H), 7.19 (d, J = 8.6 Hz, 2H), 3.76 (s, 3H), 3.67 (s, 2H), 3.46–3.38 (m, 3H),
3.26 (t, J = 11.9 Hz, 3H), 3.11 (s, 2H), 2.49 (d, J = 14.4 Hz, 2H), 2.21–2.10 (m, 2H). 13C NMR (100
MHz, D2O) δ (ppm): 173.10, 153.92, 146.02, 143.77, 141.00, 132.57 (2C), 127.24 (2C), 123.45, 121.37,
104.74, 101.30, 66.58 (2C), 62.55, 47.84 (2C), 41.48 (2C), 31.55 (2C). MS (ESI) m/z: 505 [M+H]+.
4.2.7.9.
(4-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)(4-(3,4-dichlorophenyl)piperidin-4-yl)met
hanone (10i)
1
Palewhite solid, yield 65%, mp: 260～263℃, H NMR (400 MHz, D2O) δ (ppm): 8.11 (s, 1H),
7.44-7.40 (m, 2H), 7.28 (s, 1H), 7.20 (d, J = 7.2 Hz, 1H), 3.76 (br, 4H), 3.48-3.24 (m, 8H), 2.49 (d, J =
10.8 Hz, 2H), 2.13 (s, 2H). 13C NMR (100 MHz, D2O) δ (ppm): 172.67, 153.92, 145.98, 143.79, 142.28,
133.02, 131.55, 131.47, 127.41, 125.20, 123.56, 104.71, 101.19, 66.62 (2C), 62.06 (2C), 47.84 (2C),
41.47, 31.54 (2C). MS (ESI) m/z: 493 [M+H]+.
4.2.7.10. (4-(5-bromo-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)(4-phenylpiperidin-4-yl)methan-
one (10j)
Palewhite solid, yield 63%, mp: 258～261℃, 1H NMR (400 MHz, D2O) δ (ppm): 8.10 (s, 1H), 7.36 (s,
1H), 7.23 (d, J = 8.8 Hz, 2H), 6.92-6.90 (m, 3H), 3.74-3.08 (m, 12H), 2.50 (d, J = 14.8 Hz, 2H), 2.16 (t,

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J = 11.6 Hz, 2H). 13C NMR (100 MHz, D2O) δ (ppm): 173.88, 158.35, 154.08, 146.98, 143.36, 134.19,
126.76 (2C), 126.56, 114.98 (2C), 103.02, 88.94, 66.61 (2C), 55.50 (2C), 47.47 (2C), 41.60, 31.78 (2C).
MS (ESI) m/z: 471 [M+H]+.
4.2.7.11.
(4-(5-bromo-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)(4-(3-bromophenyl)piperidin-4-yl)methan
one (10k)
Paleyellow solid, yield 63%, mp: 240～245℃, 1H NMR (400 MHz, D2O) δ (ppm): 8.11 (s, 1H), 7.45 (s,
1H), 7.42 (dd, J1 = 6.0Hz, J2 = 2.4 Hz, 1H), 7.35 (s, 1H), 7.28-7.25 (m, 2H), 3.78-3.16 (m, 12H), 2.49
(d, J = 14.4 Hz, 2H), 2.19 (t, J = 12.0 Hz, 2H). 13C NMR (100 MHz, D2O) δ (ppm): 172.94, 154.32,
146.98, 144.17, 143.65, 131.50, 131.10, 128.34 (2C), 126.49 (2C), 124.21, 123.12, 103.05, 88.90,
66.62 (2C), 62.60 (2C), 48.06 (2C), 41.51, 31.62 (2C). MS (ESI) m/z: 549 [M+H]+.
4.2.7.12.
(4-(5-bromo-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)(4-(4-methoxyphenyl)piperidin-4-yl)meth
anone (10l)
Palewhite solid, yield 62%, mp: 218～222℃, 1H NMR (400 MHz, D2O) δ (ppm): 8.06 (s, 1H), 7.29 (s,
1H), 7.20 (d, J = 8.8 Hz, 2H), 6.88 (d, J = 8.8 Hz, 2H), 3.72-3.01 (m, 15H), 2.48 (d, J = 14.0 Hz, 2H),
2.16 (t, J = 12.0 Hz, 2H). 13C NMR (100 MHz, D2O) δ (ppm): 173.79, 158.33, 154.29, 146.98, 143.76,
134.21, 126.73 (2C), 126.37, 114.95 (2C), 102.95, 88.74, 66.61 (2C), 62.60 (2C), 55.50 (3C), 47.46
(2C), 41.62, 31.80 (2C). MS (ESI) m/z: 501 [M+H]+ .
4.2.7.13.
(4-(5-bromo-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)(4-(4-chlorophenyl)piperidin-4-yl)methan
one (10m)
Palewhite solid, yield 67%, mp: 252～257℃, 1H NMR (400 MHz, D2O) δ (ppm): 8.11 (s, 1H), 7.33 (s,
1H), 7.31 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.4Hz, 2H), 3.76-3.07 (m, 12H), 2.49 (d, J = 13.6 Hz, 2H),
2.17 (t, J = 11.6 Hz, 2H). 13C NMR (100 MHz, D2O) δ (ppm): 173.11, 154.25, 146.92, 143.64, 140.61,
133.19, 129.63 (2C), 127.03 (2C), 126.51, 102.99, 88.87, 66.62 (2C), 62.60 (2C), 47.80 (2C), 41.55,
31.66 (2C). MS (ESI) m/z: 505 [M+H]+.
4.2.7.14.
(4-(5-bromo-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)(4-(3,4-dichlorophenyl)piperidin
-4-yl)methanone (10n)

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1
Paleyellow solid, yield 68%, mp: 248～251℃, H NMR (400 MHz, D2O) δ (ppm): 8.10 (s, 1H),
7.44-7.42 (m, 2H), 7.31 (s, 1H), 7.20 (d, J = 7.2 Hz, 1H), 3.78-3.19 (m, 12H), 2.48 (d, J = 13.2 Hz, 2H),
2.16 (t, J = 11.6 Hz, 2H). 13C NMR (100 MHz, D2O) δ (ppm): 172.47, 154.65, 147.01, 144.04, 142.38,
133.03, 131.54, 131.46, 127.42, 126.36, 125.22, 102.99, 88.60, 66.60 (2C), 62.58 (2C), 47.80 (2C),
41.44, 31.56 (2C). MS (ESI) m/z: 539 [M+H]+.
4.3. In vitro Akt1 kinase activity assay
The in vitro Akt1 kinase activity was evaluated via an HTRF assay (LANCER○) using the Akt
kinase kit (Cisbio Bioassays, No.62ST3PEB) in 384-well plates. Each well was added Akt1, STK
Substrate-biotin, 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) in sequence, then incubated for 45 min at 25°C.
Finally, Sa-XL665 and STK Ab-Cryptate were added to stop the enzymatic step and incubated for 2
hours to finish the detection process. The ratio (665 nm/620 nm) was obtained using a microplate
reader (Perkin Elmer, USA).
4.4. Cell lines and primary MCL cells
MCL cell lines JVM-2, Maver-1, SP-49, Z-138, Mino and Jeko-1 were purchased from the
American Type Culture Collection (ATCC). Human MCL cells were purified from the apheresis of
MCL patients after obtaining informed consent and approval by the Institutional Review Board at The
University of Texas MD Anderson Cancer Center. Cells were cultured in RPMI-1640, supplemented
with 10% heat-inactivated fetal bovine serum, 2% HEPES buffer and penicillin (10,000 units/mL;
Sigma), streptomycin (10 mg/mL; Sigma).
4.5. Cell proliferation assay
Cell proliferation assay was performed on a panel of MCL cell lines and primary MCL cells using
the CellTiter-Glo Luminescent cell viability assay kit (Promega) following the manufacturer’s protocol.
In short, cells were plated in 96-well plates at a density of 1×104 cells/well for cell lines and 12.5×104
cells/well for primary MCL cells, then treated with DMSO and different concentrations of the
synthesized compounds and incubated in a humidified atmosphere with 5% CO2 at 37°C for 72 hours
on MCL cell lines and 24 hours on primary MCL cells. IC50 values were calculated using the Graphpad
prism 6 software.
4.6. Cell apoptosis assay
Apoptosis was quantified by Annexin V/Propidium Iodide (PI)-binding assay. Cells were seeded

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in 6-well plates with 0.5 µM, 1 µM and 5 µM of 10h for 48 hours, or 1 µM of 10h for 24, 48 and 72
hours. Treated cells were washed twice with cold phosphate-buffered saline (PBS) and then
resuspended in100 µL binding buffer, to which 2 µL of Annexin V-FITC and 5 µL of PI were added.
The samples was gently vortexed and incubated for 15 minutes at room temperature in the dark. After
addition of 200 µL binding buffer, samples were immediately analyzed by flow cytometry using a
FACScan flow cytometer (Becton Dickinson, San Jose, CA). The number of apoptotic cells was
determined using the Flowjo software.
4.7. Cell cycle assay
Cell cycle arrest was measured using PI staining by flow cytometry. Cells was seeded in 6-well
plates with 0.5 µM, 1 µM and 5 µM of 10h for 24 hours, then cells were harvested, washed twice with
cold PBS, and subsequent fixing in cold 70% ethanol overnight at 4 ℃. Then samples were washed
twice with PBS, followed by further treatment with 50 uL of 100 ug/mL ribonuclease and 200 uL 50
ug/mL PI, and finally analyzed by flow cytometry.
4.8. Western blotting
Jeko-1 cells were cultured with 0.5 µM, 1 µM and 5 µM of 10h for 24 hours. Then cells were
harvested and lysed in a lysis buffer (Cell Signaling, Danvers, MA). The cell lysates were kept on ice
for 30 minutes and centrifuged at 12,000 × rpm for 20 minutes at 4oC. The protein concentration was
determined by the Bradford assay (Bio-Rad, Hercules, CA). Twenty micrograms of sample proteins
were mixed with the loading buffer and separated by 10% SDS-PAGE. The proteins were then
transferred onto methanol equilibrated PVDF membrane (BIO-RAD Laboratories, 162-0177), which
was blocked for 1 hour in 5% nonfat dry milk in TBST (BD Bioscience, San Jose, CA). The
membranes were incubated with a primary antibody overnight at 4 ℃. Secondary antibodies were
added for 1 hour at room temperature. Finally, the membrane was visualized by ECL (Perkin Elmer
Life Sciences, NE104001EA). Antibodies against PARP, cleaved caspase 3, p-AKT-308, p-AKT-473,
pan-AKT, p-GSK3β, total- GSK3β and GAPDH were obtained from Cell Signaling.
4.9. Molecular docking
Molecular docking was performed using the Sybyl 2.0 software package and the Akt1 crystal
structure (PDB: 4EKL) was retrieved from the Protein Data Bank. Protein preparation was performed
by extracting the ligand, removing water molecules, adding hydrogen atoms and assigning AMBER7
FF99 charges to the protein. Compound 10h was docked into Akt1 and the hydrogen bonds and
hydrophobic interactions were observed in the model, the best conformation with the highest CScore

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was selected for interaction analysis.
Acknowledgement
This work was supported by the National Natural Science Foundation of China (No. 21272140)
and Projects of Shandong Science and Technology (No. 2014GSF119003).
Conflict of interest
The authors declare no conflict of interest.
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Figure captions
Fig. 1. Design of the targeted compounds. A) Binding site of the ATP-competitive inhibitors; B) common features of
ATP-competitive inhibitors; C) Design of the targeted compounds from the initial hit 1
Fig. 2. Cell apoptosis and cell cycle assay for 10h in Jeko-1 cells. A/C) Apoptosis assay of 10h at indicated concentrations in
Jeko-1 cells for 48 hours; B/D) Apoptosis assay of 10h at 1 µM in Jeko-1 cells for the indicated treatment periods; E)
Dose-dependent activation of cleaved caspase-3 and PARP after 24 hours treatment with 10h; F) Cell cycle analysis of 10h at
indicated concentrations in Jeko-1 cells for 24 hours.
Fig. 3. Western blot analysis, cell viability assay in patient cells and molecular modeling. A) Effects of 10h on Akt signaling in
Jeko-1 cells after 24 hours treatment; B) Relative protein expression was determined using Image J. Values were normalized to
loading control; C) Cell viability assay of 10h in primary MCL cells. Values were normalized to control; Unpaired student’s t-test
between 10h and GSK690693-treated cells is statistically significant (* P < 0.01); D) Proposed binding mode of 10h at the ATP
binding site of Akt1 (PDB: 4EKL).

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Highlights
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Pyrrolopyrimidine based phenylpiperidine carboxamides as potent Akt inhibitors
Antiproliferative effects in mantle cell lymphoma cell lines and patient cells
Induced cell apoptosis and cell cycle arrest in G2/M phase
Downregulated the phosphorylation of Akt downstream effector GSK3β and S6