Discovery of 3,4,6-Trisubstituted Piperidine Derivatives as Orally Active, Low hERG Blocking Akt Inhibitors via Conformational Restriction and Structure-Based Design

Article abstract of DOI:10.1021/acs.jmedchem.9b00891

A series of 3,4-disubstituted piperidine derivatives were obtained based on a conformational restriction strategy and a lead compound, A12, that exhibited potent in vitro and in vivo antitumor efficacies; however, obvious safety issues limited its further development. Thus, systematic exploration of the structure-activity relationship of compound A12, involving the phenyl group, hinge-linkage, and piperidine moiety, led to the discovery of the superior 3,4,6-trisubstituted piperidine derivative E22. E22 showed increased potency in Akt1 and cancer cell inhibition, remarkably reduced human ether-a-go-go-related gene blockage, and significantly improved safety profiles. Compound E22 also exhibited good kinase selectivity, had a good pharmacokinetic profile, and displayed very potent in vivo antitumor efficacy, with over 90% tumor growth inhibition in the SKOV3 xenograft model. Further mechanistic studies were conducted to demonstrate that compound E22 could significantly inhibit the phosphorylation of proteins downstream of Akt kinase in cells and tumor tissue from the xenograft model.

Full text of DOI:10.1021/acs.jmedchem.9b00891

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Article
Discovery of 3,4,6-Trisubstituted Piperidine Derivatives
as Orally Active, Low hERG Blocking Akt Inhibitors via
Conformational Restriction and Structure-based Design
Xiaowu Dong, Wenhu Zhan, Mengting Zhao, Jinxin Che, Xiaoyang Dai, Yizhe
Wu, Lei Xu, Yu-Bo Zhou, Yanmei Zhao, Tian Tian, Gang Cheng, Zegao Jin,
Jia Li, Yanfei Shao, Qiaojun He, Bo Yang, Qinjie Weng, and Yongzhou Hu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00891 • Publication Date (Web): 12 Jul 2019
Downloaded from pubs.acs.org on July 13, 2019
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Discovery of 3,4,6-Trisubstituted Piperidine
Derivatives as Orally Active, Low hERG Blocking
Akt Inhibitors via Conformational Restriction and
Structure-based Design
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Xiaowu Dong,a, ‡ Wenhu Zhan,a,#, ‡ Mengting Zhao,b, ‡ Jinxin Che, Xiaoyang Dai, Yizhe
a
b
a
c
c
a
a
d
a
c
Wu, Lei Xu, Yubo Zhou, Yanmei Zhao, Tian Tian, Gang Cheng, Zegao Jin, Jia Li,
e
b
b
b,*
a,*
Yanfei Shao, Qiaojun He, Bo Yang, Qinjie Weng, Yongzhou Hu
a
ZJU-ENS Joint Laboratory of Medicinal Chemistry, Hangzhou Institute of Innovative
Medicine, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058,
China
b
Institute of Pharmacology & Toxicology, Zhejiang Province Key Laboratory of Anti-
Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou, 310058, China
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c
National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
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College of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou,
3
11402, China
e
Department of Pharmacy, Zhejiang Provincial People's Hospital, Hangzhou, 310014,
China
#
Present Address: Department of Microbiology & Immunology, Weill Cornell Medicine,
1300 York Avenue, New York, NY 10065, United States.
‡
These authors contributed equally to this work.
*
Corresponding authors: Q. Weng: wengqinjie@zju.edu.cn; Y. Hu: huyz@zju.edu.cn
ABSTRACT: A series of 3,4-disubstituted piperidine derivatives were obtained based on
a conformational restriction strategy and a lead compound, A12, that exhibited potent in
vitro and in vivo anti-tumor efficacy; however, obvious safety issues limited its further
development. Thus, systematic exploration of the structure-activity relationship of
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compound A12, involving the phenyl group, hinge-linkage and piperidine moiety, led to
the discovery of the superior 3,4,6-trisubstituted piperidine derivative E22. E22 showed
increased potency in Akt1 and cancer cell inhibition, remarkably reduced hERG
blockage, and significantly improved safety profiles. Compound E22 also exhibited good
kinase selectivity, had a good pharmacokinetic profile, and displayed very potent in vivo
anti-tumor efficacy, with over 90% tumor growth inhibition in the SKOV3 xenograft
model. Further mechanistic studies were conducted to demonstrate that compound E22
could significantly inhibit the phosphorylation of proteins downstream of Akt kinase in
cells and tumor tissue from the xenograft model.
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KEYWORDS: Akt inhibitors; Conformational restriction; Structure-based design; hERG
blockage; Maximum tolerated dose.
1. Introduction
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Akt, also known as protein kinase B or PKB, is a serine/threonine kinase that belongs to
the AGC family of kinases, sharing high homology with PKA and PKC. Akt can be
classified into three different isozymes, including Akt1, Akt2 and Akt3; all three share a
high degree of overall homology and similar downstream targets.1, 2 It is very common
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that the PI3K/Akt signaling pathway is overactivated in various cancer cells, and tumor
growth can be effectively decreased via the inhibition of Akt alone or in combination with
other chemotherapeutics.4-9 Currently, the efficacy of a number of Akt inhibitors is
evaluated in clinical trials (Figure 1).
10
In particular, a phase II clinical trial of GDC0068 was completed , and a phase III
clinical trial is currently investigating the effectiveness of GDC0068 in the treatment of
triple-negative breast cancer (TNBC) (NCT03337724) and metastatic prostate cancer
(NCT03072238). In addition, the results of clinical studies on the use of AZD5363 in
combination with paclitaxel for the treatment of TNBC have also been disclosed,
indicating that this therapy resulted in a positive outcome of significantly longer
progression-free survival and overall survival,11 thus fully demonstrating the promising
prospect of Akt as an anti-cancer target.12-18 Additionally, GSK2141795 (also known as
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GSK-795 or Uprosertib) is another ATP-competitive Akt inhibitor developed by
GlaxoSmithKline, which is currently in early clinical trials for evaluation of its therapeutic
effect against different types of cancer, such as multiple myeloma (NCT01989598),
breast cancer (NCT01964924), and others. In the discovery stage of AZD5363 and
GDC0068,18,19 the druggability involving in vitro and in vivo anti-cancer efficacy, safety
issues (hERG), PK profiles and kinase selectivity were primarily explored and
optimized. In comparison with the preclinical data, some of their advantages are clear,
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such as low hERG blockage of AZD5363, high selectivity profiles of GDC0068, low
20
effective dose and excellent PK profiles of GSK-795, the data pertaining to which have
also provided a criterion for searching for novel Akt inhibitors.
OH
NH
F
O
O
F
Cl
N
NH2
N
N
O
Cl
H
NH2
O
Cl
NH2
N
N
H
N
N
N
N
N
O
N
Cl
HN
N
N
N
N
HO
H
GDC-0068
AZD5363
GSK2141795
MK-2206
Figure 1. Chemical structures of representative Akt inhibitors.
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Conformational restriction is one of the most effective methodologies for drug design,
since it can help improve the PK profiles of compounds and stabilize a favorable binding
pattern in order to improve the binding affinities or kinase isoform selectivity.21,22 This
strategy has been widely applied in drug discovery.22,23 In fact, some FDA approved
drugs were discovered and developed by using conformational restriction, such as
Dolutegravir.24 In our previous study, the discovery of a series of 4,4-disubstituted
piperidine derivatives featuring more restricted conformations in contrast to GSK-795
were successfully achieved,25-29 which proved that conformational restriction strategy
could be used for discovery of new Akt inhibitors with novel skeletons. As a continuation
of this earlier work, herein, the structural modifications of 3,4-disubstituted piperidine
derivatives were reported, which exhibit excellent potency and good PK profiles. Further
structure-based drug design was used to introduce a side chain, leading to the
discovery of a series of novel and highly potent 3,4,6-trisubstituted derivatives of E22
with good PK profiles, lower hERG blockage and improved safety profiles. Furthermore,
the molecular mechanisms of E22 were studied, confirming the blockage of the PI3K-
Akt pathway in cancer cells and tumor tissue.
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. Results and Discussion
2.1 Discovery of 3,4-disubstituted piperidine derivatives as Akt inhibitors
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On the basis of multiple previous research projects,27-32 most of the ATP-competitive
Akt inhibitors were found to share common pharmacophore characteristics, including a
hinge-binding group, a basic center and a hydrophobic group. These three
pharmacophores generate a “Y” shape through a linkage. Although the cocrystal
structure of GSK-795 with Akt1 has not been reported, the docking results (Figure S1)
disclosed that the amino and pyrazole groups were hydrogen-bonded to the amino acid
residues of Glu278 and Ala230. The 3,4-difluorophenyl group inserted into the
hydrophobic pocket and π-π interacted with Phe161. Therefore, we hypothesized that
cyclization of the amino group with the methylene moiety of the benzyl group based on
the conformational restriction strategy may facilitate conformational stability of the
phenyl group and basic center, and therefore would improve the Akt inhibitory potency
(Figure 2).
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Figure 2. Design rationale based on the structures of GSK-795.
In the present study, the racemic 3,4-disubstituted piperidine or pyrrolidine derivatives
were synthesized for further exploration. The linkage and hinge groups selected were
the privileged furan moiety and N-methyl pyrazole, respectively, according to the
structure of GSK-795. As shown in Table 1, when R , R and R substituents were all
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hydrogens, compound A1 resulted in an IC50 value of 709.6 nM. After introducing a
halogen to A1, the Akt1 inhibitory potency was reinforced, as shown in compounds A2,
A3 and A4, demonstrating the importance of halogens in maintaining the compound
activity. Notably, when R was 4-CF or 3,4-difluoro, compounds A5 and A6 both
3
3
exhibited comparable Akt1 inhibitory potency to that of GSK-795. However, after the
replacement of piperidine with pyrrolidine, the activities of compounds A7-A9 were
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dramatically reduced. Therefore, highly potent 3,4-disubstituted compounds A5 and A6
were selected for further chiral resolution to determine the optimal configuration.
Table 1. Akt1 inhibitory activities of compounds A1-A9
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R1
R3
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O
NH
H
A1~A6
A7~A9
Compd.
A1
R1
H
R2
R3
H
Akt1 IC (nM)
50
H
H
709.6
191.0
17.6
17.3
7.2
A2
H
3,4-difluoro
H
A3
Cl
Cl
Cl
Cl
H
Cl
Cl
Cl
Cl
Cl
Br
Cl
-
A4
4-Cl
A5
4-CF3
A6
3,4-difluoro
3,4-difluoro
3,4-difluoro
4-CF3
6.4
A7
1566
3646
538
A8
H
A9
Cl
-
GSK-795
-
4.7
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According to the asymmetric synthetic method for constructing 3,4-disubstituted
piperidine derivatives,33 the trans-conformations (3S,4S or 3R,4R) of 3,4-disubstituted
piperidine were the main products resulting from using (S)- or (R)-Jorgensen-Hayashi
reagent catalysts, respectively. As shown in Table 2, the activity of the (3S,4S)-
enantiomer (compounds A10 and A12) was significantly better than that of the (3R,4R)-
enantiomer (compounds A11 and A13), including their Akt1 inhibitory activities and anti-
proliferative activities against different cancer cell lines, such as OVCAR-8 and
HCT116. In addition, one of the cis-conformation products, the (3R,4S)-enantiomer, was
further separated during the asymmetric synthesis and afforded the compound (3R,4S)-
A6, exhibiting a good IC50 value of 2.5 nM (Figure S2). Considering that the product
yield of the cis-conformation of 3,4-disubstituted piperidine was minimal and difficult to
obtain, we only focused on the (3S,4S)-enantiomer of 3,4-disubstituted piperidine
derivatives in the following studies.
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Table 2. Akt1 enzymatic and cancer cell line activities for compounds A10-A13
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Cl
R
N
O
N
H
(3S, 4S)-A5 R=4-CF3
3S, 4S)-A6 R=3,4-diF
N
(
Cl
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cl
O
Cl
R
N
H
N
O
N
H
A5 R=4-CF3
(3R, 4R)-A5 R=4-CF3
3R, 4R)-A6 R=3,4-diF
N
A6 R=3,4-diF
(
Cl
O
N
H
(3R, 4R)
Compd.
Akt1 IC (nM)
OVCAR-8 (μM)
HCT116 (μM)
0.28
5
0
(3S, 4S)-A5 (A10)
(3R, 4R)-A5 (A11)
(3S, 4S)-A6 (A12)
(3R, 4R)-A6 (A13)
GSK-795
9.3
127.4
3.0
0.12
0.90
0.04
0.49
0.24
1.05
0.21
51.2
8.3
1.09
0.72
As shown in Figure 3, compound A12 exhibited good pharmacokinetics and long t1/2
(
10.7 hours) and excellent bioavailability (F = 97.9%) in mice. This compound was
slightly unstable in the liver microsomes of monkeys (39.9% remaining after incubating
for 1.5 hours) and had a high hERG inhibition rate (62.1% inhibition at 3 μM). Next, the
safety profile of A12 in mice was assessed. After 7 days of intragastric administration of
A12 (10 mg/kg, 20 mg/kg and 40 mg/kg), toxic effects can be obviously observed in
moderate and high dose groups (Figure S3 and Table S1): three of five and two of five
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mice died at the dosages of 40 mg/kg and 20 mg/kg, respectively. Some of the serum
hematological and biochemical parameters such as RBC, HGB, HCT and CREA were
obviously downregulated at the high dose (Figure S3 B and C). Thus, we reasoned that
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
10 mg/kg may be a tolerated dose for an in vivo study. The effect of A12 in vivo was first
characterized by measuring its anti-tumor activity in an MM1S multiple myeloma
xenograft model. Following single oral doses of 10 mg/kg, the impact on tumor growth
was assessed over 22 days. In total, 87.5% inhibition of tumor growth was observed,
indicating that the in vivo efficacy of A12 was strong (Figure S4 A and B). However,
significant body weight loss (up to 25%) was observed in the mice (Figure S4 C), and
one of the four mice died during the experiment (Figure S4 D); the death may be
attributed to lower tolerance to the dose in nude mouse. It is demonstrated that the MTD
of A12 was lower than 10 mg/kg, and dose escalation of A12 was highly limited,
suggesting that potential safety issues must be further resolved.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. The profiles of in vitro and in vivo biological activities and safety issues of
compound A12.
2.2 Discovery of Akt inhibitors with improved safety profiles
After careful evaluation of the safety profile, we found that obvious deficiencies of
compound A12 existed: 1) the MTD dose of A12 was quite low; 2) some toxicity was
indicated by serum hematological and biochemical parameters; 3) heavy
gastrointestinal hemorrhage caused the death of mice in autopsy analysis; 4) high
hERG inhibition of A12 was observed (62.1% inhibition at 3 μM). To address these
issues, subsequent investigation primarily focused on structure optimization, including
the hinge-linkage moiety and phenyl substituents. In particular, on the basis of the
docking results of compound A12 to the ATP bound pocket of Akt1 kinase, it was found
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that the 5- or 6-position of the piperidine ring may be oriented to the solvent area,
providing us with a suitable point for exploration (Figure 4).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Strategies for further optimization of compound A12.
Modification of the hinge-linkage moiety and phenyl substituents. As shown in Table
3, compounds B1 and B2, but not compound B3, showed IC50 values below 10 nM
when the phenyl group was substituted with different halogens. Furthermore, different
hinge binders and linkages were evaluated. The 4-position of the N-methyl pyrazole
hinge binder linked to the 2- or 3-position of the furan linkage (compounds C1 and C3)
lead to a dramatic loss of enzyme activity. Removal of the methyl group of C1 to yield
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2
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compound C2 resulted in an increase in potency. Other hinge binders and linkages
were also tested, such as 3-amino-pyrimidine (compound C4) and cyclopentane-
pyrimidine (compound C5), and the activities of both of these compounds were
dramatically reduced. Furthermore, the anti-proliferation potencies of compounds
against OVCAR-8 and HCT116 cells were determined in accordance with the Akt IC50
values. Compounds A12, B1 and B2 exhibited good Akt IC50 values of <10 nM and
good anti-proliferation activities superior to that of GSK-795. Although compounds A12
and B1 showed good Akt1 activity, they also exhibited disappointingly high hERG
inhibitory potencies at the concentration of 3 μM (62% and 87%, respectively), even
higher than that of GSK-795.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Akt1 enzymatic and cancer cell line activities for compounds B1-B3 and C1-C5.
Cl
Cl
R1
N
O
N
H
H
N
R1
N
Cl
O
O
N
N
H
H
A12, B1~B3
C1~C5
a
a
Compd.
R1
Akt1 IC (nM)
IC (µM)
50
50
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OVCAR-8
0.04
HCT116
0.21
A12
B1
3,4-diF
3,4-diCl
3-CF3
4-Cl
3.0
2.9
0.11
0.28
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
B2
8.0
0.12
0.39
B3
17.3
1.32
1.87
N
N
C1
C2
C3
>1000
50.3
4.41
5.00
15.07
21.78
23.65
O
O
N
HN
O
N
N
>1000
10.01
HN
C4
529.5
12.27
37.11
N
O
N
HO
C5
>1000
8.3
24.05
1.32
22.96
1.27
N
S
N
GSK-795
-
a
The Inhibition% or IC values are an average of three independent determinations.
50
Since we cannot identify better hinge-linkage fragments, N-methyl pyrazole was
chosen as hinge binder and the linkage was further investigated. Different five-
membered ring linkages were first evaluated, including furan, thiophene and N-methyl
pyrrole (Table 4). In the case of the furan linkage, removal of the chloro group on N-
methyl pyrazole yielded the unsubstituted compound D1 or D2, which resulted in a
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
slight reduction in potency (D1 vs. B3; D2 vs. A12). In addition, introducing a bromo
group to the pyrazole did not favor activity improvement (D3 vs. D1). After changing
from the 2,4-disubstituted to 2,5-substituted furan linkage, the activities of the obtained
compounds were reduced (compounds D4-D9).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
When the linkage was between thiophene and N-methyl pyrrole, the IC values of the
50
resulting compounds (D10-D18) were over 100 nM. In contrast to the structure-activity
relationship (SAR) when the linkage included furan, introduction of a chloro group to the
5-position of the thiophene linkage was unfavorable to the compound activity, which
even suffered a significant loss of potency (D10 vs. D11). After introducing a chloro or
bromo group to the N-methyl pyrrole, the inhibitory potency of compounds improved
(D15 vs. D17; D16 vs. D18).
The six-membered ring linkage was also evaluated, including substituted phenyl and
pyridyl. Although the activities of compounds with six-membered ring linkages were not
better than that of compounds with furan linkages, some of the IC50 values were still
lower than 100 nM. Compound D19 showed the most potent IC value of 21.6 nM. The
50
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activities of compounds with pyridine linkages were inferior to those of compounds with
phenyl linkages (D19 vs. D23; D20 vs. D24; D21 vs. D25; and D22 vs. D27).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The cellular potencies of the compounds against OVCAR-8 and HCT-116 cells were
relatively insensitive, with most of the compounds exhibiting IC values of greater than
50
1
μM. Compound D2, exhibiting an Akt1 IC50 value of 5.1 nM, only showed
approximately 1 μM anti-proliferative activity, which is comparable to that of GSK-795.
Compound D16, with lower Akt1 inhibitory activity, also exhibited high hERG inhibition,
demonstrating no relationship between Akt1 and hERG inhibition. Despite the low
hERG inhibition observed for compounds D19 and D22, in which each linkage consisted
of phenyl groups, the Akt1 inhibitory potency was unsatisfactory.
Table 4. Akt1 enzymatic and cancer cell line activities for compounds D1-D28
R2
N
N
H
Linkage
N
R1
O
N
H
D1~D28
a
IC (µM)
5
0
hERG
Akt1 IC
5
0
Compd. Linkage R1
R2
inhibition at
a
(nM)
OVCAR-
8
a
3
μM
HCT116
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
D1
D2
D3
D4
D5
D6
D7
D8
D9
H
Cl
Br
H
4-Cl
N.T. b
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
29.4
5.1
3.50
1.40
9.01
6.09
4.28
3.68
15.00
6.05
4.54
1.56
0.99
O
3,4-diF
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
35.6
230.2
13.7
31.2
162.5
40.0
29.8
2.31
9.56
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cl
Br
H
2.56
O
3.76
13.66
11.68
10.80
Br
Cl
Br
O
S
D10
H
4-Cl
N.T.
187.9
12.62
8.51
D11
D12
D13
D14
D15
Cl
Cl
Cl
4-Cl
3-F
N.T.
N.T.
N.T.
N.T.
N.T.
448.9
101.6
222.2
321.6
211.9
2.51
8.99
3.51
1.15
6.20
2.44
9.43
2.81
1.75
3.04
Cl
S
3,4-diF
Cl 4-Cl-3-CF3
N
Cl
Br
4-Cl
4-Cl
D16
63.8%
255.3
3.03
8.70
Cl
Br
N
N
D17
Cl
Br
4-Cl
4-Cl
N.T.
149.3
12.36
7.33
D18
N.T.
199.1
4.92
7.42
D19
D20
D21
D22
D23
H
Cl
Br
Cl
H
3,4-diF
3,4-diF
3,4-diF
3-F
25.0%
N.T.
21.6
27.3
73.5
72.3
57.7
5.33
2.18
2.90
5.82
2.11
3.72
9.88
22.17
-
N.T.
10.7%
N.T.
3,4-diF
24.16
N
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7
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D24
D25
Cl
Br
H
3,4-diF
N.T.
N.T.
N.T.
N.T.
N.T.
215.0
269.8
76.6
6.12
6.15
7.40
3.46
1.92
34.66
26.93
5.63
3,4-diF
3-F
D26
D27
Cl
Br
3-F
104.1
181.8
4.72
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
D28
3-F
6.25
GSK-
-
-
-
50.2%
8.3
1.32
1.27
7
95
a
The Inhibition% or IC values are an average of three independent determinations.
50
b
N.T. = Not Tested.
,4,6-Trisubstituted piperidine derivatives. Following the previous SAR studies of 3,4-
3
disubstituted piperidine derivatives, we found that the Akt and hERG inhibitory activities
cannot be well balanced. Since the 5- or 6-position of the piperidine may be oriented to
the solvent area, this may offer an overall balance, particularly with respect to Akt
inhibitory potency. As shown in Table 5, a range of introduced side chains could be well
tolerated in both synthesized (3S,4S,6S)-trisubstituted piperidine derivatives.
Additionally, the length of the side chains showed minor influence on the Akt1 inhibitory
activity of compounds, and the IC50 values of most compounds were below 10 nM.
Compounds with hydrophilic side chains performed better in terms of Akt1 inhibitory
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
potency compared with those with hydrophobic side chains (compounds E1 and E2)
and the positive control compounds GSK-795 and AZD5363.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
As for cellular potency, most of the compounds showed good anti-proliferation activity
against LNCaP, OVCAR-8 and HCT116 cells. Comparing these cell lines, LNCaP cells
were more sensitive to the inhibitors than the OVCAR-8 or HCT116 cells. The IC50
values of several compounds against LNCaP cells were below 0.1 μM (compounds
A12, B1, E5, E12, E15, E17, E18, E20, E22 and E23).
The hERG inhibition of compounds with lipophilic side chains still exhibited high
potency: for example, compound E1. Introduction of a hydroxyl group to increase the
hydrophilicity of the side chain (compound E3) led to a decrease in hERG inhibition.
Furthermore, the removal of one carbon to obtain a shortened side chain (compound
E4) resulted in lower hERG activity. Introduction of two hydroxyls to continue increasing
the hydrophilicity of the side chain (compound E5) resulted in significantly reduced
hERG activity.
Further exploration of the SAR around the basic group analogues, including N,N-
dimethyl (E7), piperidine (E9) and morpholine (E10), led to compounds with increased
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hERG affinity. By contrast, introducing a hydroxyl group to the piperidine analogue side
chain (compound E11) led to an obvious reduction in hERG inhibition. When the side
chains were analogues with carboxyl (compound E13) and carbamoyl (compound E14)
substitution, hERG inhibition was significantly lowered, exhibiting only 9.0% and 15.7%
of the activity, respectively.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
When the phenyl group on piperidine was 3,4-difluoro substituted, the hERG affinity
values of compounds were lower than those of 3,4-dichloro substituted compounds
(E20 vs. E4; E21 vs. E5; and E22 vs. E15). The hERG inhibition rates of compounds
E20-E24 were all below 10%, which was comparable to the value for AZD5363 but
much lower than the values of GSK-795 and A12.
Following the exploration of the compounds’ reduced hERG inhibition, other ion
channels were also evaluated to exclude potential off-target effects. The inhibition rates
of compound E22 against 16 different ion channels, including chloride (CFTR), sodium
(Nav1.7) and potassium ion channels (Kir1.1, 2.1; Kv1.2~1.8; HCN 1~4), were quite low
at a concentration of 3 μM (Table S2); these results suggest a safer ion channel profile
and significant advancement over the disubstituted piperidine lead compound, A12.
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1
1
1
1
1
1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 5. Akt1 enzymatic and cancer cell line activities for compounds E1-E25
Cl
F
Cl
F
Cl
Cl
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
O
N
O
N
H
N
H
N
N
Cl
O
Cl
O
N
H
R1
N
H
R1
B1, E1~E19
A12, E20~E25
a
IC (μM)
5
0
Akt1 IC
hERG inhibition
5
0
Compd.
R1
a
a
(nM)
at 3 μM
OVCAR-
8
LNCaP
HCT116
A12
B1
E1
E2
E3
E4
H
H
3.0
2.9
62%
87%
0.06
0.10
N.T. b
0.16
0.21
0.16
0.04
0.11
1.62
0.99
0.60
0.61
0.21
0.28
1.02
0.78
0.51
0.34
10.44
12.41
2.56
85.3%
N.T.
OH
72.0%
51.8%
OH
2.74
OH
E5
E6
E7
OH
3.79
7.01
3.40
17.0%
N.T.
0.07
0.25
1.93
1.61
1.06
1.51
1.11
0.47
1.71
O
N
73.4%
N
N
E8
E9
6.41
5.65
8.78
N.T.
1.92
1.78
0.41
1.84
1.66
1.76
1.94
1.02
1.56
93.1%
82.3%
N
E10
O
N
E11
E12
5.99
4.47
12.0%
50.1%
1.14
0.06
15.86
1.56
13.68
0.56
OH
N
N
N
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9
O
O
E13
E14
1.94
9.1%
0.64
0.16
5.12
1.00
4.02
0.65
OH
3.24
2.64
3.95
7.98
1.41
15.7%
NH2
O
E15
E16
E17
E18
45.9%
70.4%
N.T.
0.04
0.19
0.08
0.04
1.13
0.58
0.61
1.45
0.45
0.74
0.43
0.67
N
H
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
N
H
N
H
O
OH
6.7%
N
H
O
E19
N
OH
3.13
N.T.
0.66
5.90
7.09
H
OH
OH
E20
E21
0.98
3.31
9.0%
4.7%
0.06
0.10
0.46
0.69
0.36
0.80
OH
O
OH
E22
E23
1.37
3.29
4.0%
8.0%
0.05
0.05
0.80
1.53
1.10
0.92
N
H
O
S
N
H
O
O
O
E24
E25
2.49
3.40
7.4%
N.T.
0.13
N.T.
0.88
4.96
0.92
0.69
N
H
O
GSK-795
AZD5363
a
/
/
7.64
50.0%
7.1
0.07
1.32
7.27
1.27
5.20
25.43
0.178
The IC values or Inhibition% are an average of three independent determinations.
50
b
N.T. = Not Tested.
For preliminary assessment of the selectivity profiles of compounds, the IC values or
50
inhibition rates of compounds A12, E20 and E22 were assayed against a panel of
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1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
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5
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6
representative kinases (20), including AGC kinase family and tyrosine kinases (TK),
among others (Table 6). Both compounds exhibited IC50 values of < 10 nM against
Akt1-3. In addition, the tested compounds also exhibited good inhibitory activity against
other AGC family kinases, such as PKA, PKC, ROCK1, RSK1, P70S6K and SGK, while
no significant inhibition was observed against other non-AGC family kinases.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 6. Results of preliminary assessment of compounds’ kinase selectivity profiles
a
IC (nM) / Inhibition (%, 0.5 μM)
5
0
Kinases
A12
3.0
E20
0.98
0.54
0.12
0.2
E22
1.4
GSK-795
9.6
AKT1
AKT2
AKT3
PKA
< 0.64
0.1
1.2
1.0
1.7
1.3
7
0.3
0.6
PKC
156
20
16
94
753
ROCK1
RSK1
38
64
125
AGC kinase
family
48
32
131
8.9
339
P70S6K
SGK
7.0
7.1
60
59
102
4.2%
4.0%
145
-1.0%
12.9%
211
MSK1
PDK1
88.8%
3.7%
87.4%
-6.1%
TSSK1
ABL
-3.9%
-3.0%
-1.8%
6.1%
-10.4%
-0.8%
6.2%
2.4%
TK
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N.I. b
N.I.b
N.I.b
N.I.b
JAK-2
ALK
TKL
20.8%
12.7%
N.I.b
13.8%
6.3%
N.I.b
8.5%
4.3%
N.I.b
5.8%
8.4%
N.I.b
JNK2
CDK2
CMGC
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CAMK
CHK1
PI3Ka
mTOR
1.3%
2.2%
-9.6%
-2.0%
6.3%
-26.8%
5.9%
-7.9%
0.8%
NT b
Atypical
-5.7%
-15.7%
a
b
The IC values or Inhibition% are an average of three independent determinations.
5
0
N.I. = No Inhibition.
To fully understand the selectivity profile of compound E22, inhibition was further
assessed using the KINOMEscan methodology against a full panel of kinases
(
nonmutant, mutant, lipid, atypical and pathogen kinases; total number: 468) at a single
dose concentration of 1 μM. The KINOMEscan selectivity score of S was 0.032
1
(
13/468; S = (number of nonmutant kinases with %Ctrl <1)/(number of nonmutant
1
kinases tested), %Ctrl = (test compound signal – positive control signal)/(negative
control signal – positive control signal)×100); for full screening results, refer to Table
S3). Additionally, the interaction of compound E22 with PRKD, PRKG, HASPIN,
CSNK1E, etc., was detected, showing a comparable selectivity profile to GSK-795
(Figure S5).
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2
.3 Compound E22 exhibited a good PK profile
Based on the results of SAR studies, compounds E20 and E22 were chosen for
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
further testing of their PK profiles. As shown in Table 7, after 40 mg/kg oral dosing of
E22 in mice and rats (compounds were dissolved in saline), we found that the
absorption of E22 in mice was better than in rats, but the compound was more easily
cleared in mice. Furthermore, E22 showed a good bioavailability value of 48.4% in
mice. The high PPB rate (93%) may indicate a long residence time for E22. The dosage
of compound E20 was 25 mg/kg, which indicated good absorption in rats, and a good
clearance rate was observed. In addition, the in vitro stability of E20 and E22 was also
evaluated in liver microsomes and plasma. The stability of these two compounds was
good in the plasma, whereas compound E20 was less stable than E22 in rat and
monkey microsomes.
Table 7. Pharmacokinetic profiles of compounds E20 and E22
E22
E20
Parameters
Mouse (40
Rat (40
mg/kg, i.g.)
Rat (25
mg/kg, i.g.)
mg/kg, i.g.)
Tmax (h)
1.0
1.8
1.1
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T (h)
3.3
3620.0
1.9
6.4
407.4
14.2
2442.2
3092.2
5.8
12.7
145.2
26.0
1
/2
Cmax (μg/L)
CL (L/h/kg)
AUC (μg /L·h)
21091.9
21221.5
5.2
1014.0
1657.0
5.1
0
-t
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
AUC0-∞ (μg /L·h)
MRT (h)
0
-t
MRT0-∞ (h)
Vd (L/kg)
F (%)
5.3
11.4
147.2
-
12.3
9.0
300.9
-
48.4
Plasma Protein Binding rate (%, 300 ng/mL)
mouse]
93
-
[
Liver microsome stability (%, remaining
percent @ 1.5 h) [mouse, rat, dog, monkey]
>99, 69, 96,
18
> 99, 89, 96, 84
> 99, > 99
Plasma stability (%, remaining percent @ 1 h)
>99, >99
[
mouse, human]
2.4 Compound E22 induces apoptosis and inhibits metastasis of cancer cells
Compound E22 was very effective at inhibiting the phosphorylation of downstream Akt
substrates in the LNCaP cell line, and it exhibited potent inhibition against
phosphorylated PRAS40, which is a direct marker of Akt cell activity, with an observed
IC value of 44 nM. To examine the influence of compound E22 on ovarian cancer cell
50
survival prior to conducting an in vivo study, SKOV3, A2780 and OVCAR8 cells were
incubated with different concentrations of E22 for 72 h. A concentration-dependent
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