Discovery of novel ceramide analogs with favorable pharmacokinetic properties and combination with AKT inhibitor against colon cancer

Article abstract of DOI:10.1016/j.ejmech.2021.113274

Ceramides have emerged as potential therapeutic option with novel mechanism to affect the proliferation, differentiation, senescence, and apoptosis of cancer cells through regulation of multiple signal transduction. Aiming at the improvement of the apopto

Full text of DOI:10.1016/j.ejmech.2021.113274

European Journal of Medicinal Chemistry 215 (2021) 113274
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of novel ceramide analogs with favorable pharmacokinetic
properties and combination with AKT inhibitor against colon cancer
,
b
,
,
b
,
c
,
,
,
,
, b
Feng Gao a 1, Xiaoxu Chen a
1, Junyan Lu a b, Shulei Hu a b, Hui Xu a b, Yuqiang Shi a
,
Mingshun Feng a c, Jian Ding a c, Hong Liu a c, Cheng Luo a ***, Zuoquan Xie a
,
,
,
b,
,
b,
,
b,
, b, **
Jiang Wang a
, b, *
a State Key Laboratory of Drug Research and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, China
b University of Chinese Academy of Sciences, Beijing, 100049, China
c School of Life Science and Technology, ShanghaiTech University, Shanghai, 200031, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Ceramides have emerged as potential therapeutic option with novel mechanism to affect the prolifer-
ation, differentiation, senescence, and apoptosis of cancer cells through regulation of multiple signal
transduction. Aiming at the improvement of the apoptosis activity and pharmacokinetic profiles of
ceramides, a novel series of ceramide analogs were developed through structure simplification and
conformation restriction. Among them, compound 12 bearing an alkoxyl naphthyl motif, with favorable
rat pharmacokinetic properties, showed better anti-proliferative activity against various colon cancer
Received 17 August 2020
Received in revised form
1 February 2021
Accepted 1 February 2021
Available online 7 February 2021
cells (IC50 ~20 mM) than other ceramide analogues, as well as the synergistic effect combined with AKT
Keywords:
inhibitor MK2206. Additionally, we demonstrated that this combination therapy promoted caspase 3-
dependent apoptotic pathway and intensified cell cycle arrest in the G0/G1 phase. Furthermore, the
combination of compound 12 and MK2206 displayed synergistic anti-tumor effect in vivo.
© 2021 Published by Elsevier Masson SAS.
Ceramide analogs
AKT inhibitor
MK2206
Colon cancer
Combination therapy
1. Introduction
such as BH3 interacting-domain death agonist (Bid), B-cell lym-
phoma 2 (Bcl-2), Protein kinase B (AKT), or c-Myc [6e8], as well as
Ceramides are composed of a sphingosine backbone and a fatty
acyl chain that varying in length from C14 to C26 (Fig. 1a) [1].
Ceramides have been demonstrated as powerful tumor suppressors
in signal transduction, affecting the proliferation, differentiation,
senescence, and apoptosis [2,3]. Increased ceramides subsequently
activate cascade of caspases and dysfunction of multiple organelles,
which lead to endoplasmic reticulum stress and apoptosis [4].
Specifically, ceramides induce cell death by activating protein
phosphatase-1 (PP1) and protein phosphatase-2A (PP2A) [5],
which result in the inactivation of several pro-apoptotic proteins,
dephosphorylation of retinoblastoma protein (RB), giving rise to
cell cycle arrest [9]. Furthermore, up-regulation of cellular cer-
amide levels during apoptosis has been shown to occur prior to
mitochondrial outer membrane (MOM) permeabilization [10].
The balance of ceramide and sphingosine-1-phosphate (S1P)
levels determines cell fate, in which ceramide acts as a tumor-
suppressor lipid, whereas S1P acts as a tumor-promoting lipid
(Fig. 1a). Defects in ceramide biosynthesis can facilitate cancer cell
survival and drug resistance, thus, targeting ceramide metabolic
pathway is a promising strategy for anti-tumor therapy [11]. The
* Corresponding author. State Key Laboratory of Drug Research and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai, 201203, China.
** Corresponding author. State Key Laboratory of Drug Research and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai, 201203, China.
*** Corresponding author. State Key Laboratory of Drug Research and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai, 201203, China.
E-mail addresses: cluo@simm.ac.cn (C. Luo), zqxie@simm.ac.cn (Z. Xie), jwang@simm.ac.cn (J. Wang).
These authors made equal contributions to this work.
1
https://doi.org/10.1016/j.ejmech.2021.113274
0223-5234/© 2021 Published by Elsevier Masson SAS.

F. Gao, X. Chen, J. Lu et al.
European Journal of Medicinal Chemistry 215 (2021) 113274
Fig. 1. Chemical structure of ceramides, S1P and MK2206.
ceramide-mediated anticancer effects have been demonstrated in
various cancers such as breast [12e14], gastric [15], colon [16e18],
pancreatic [19e22] and leukemia [23]. Besides, ceramides can also
sensitize cancer cell lines to chemotherapy [24e26] or radiotherapy
[27]. Indeed, ceramide-generating agents, modulators of ceramide
metabolism or exogenous ceramides, have been chosen for the
combination therapy with chemotherapeutic drugs [28,29].
AKT, acting as an oncogenic protein to regulate cell survival,
proliferation, and apoptosis, is recognized as a promising thera-
peutic target for cancer therapy [30,31]. However, the efficacy of
AKT inhibitors in clinical trial is limited due to drug resistance, such
as MK2206, the first AKT inhibitor entering clinical trial, which has
been shown to enhance antitumor efficacy of chemotherapeutic
agents or molecular targeted drugs in vitro and in vivo models[32],
but failed in most clinical trials (Fig. 1b). Thus, it is critical to find a
rational combination strategy for its clinical application [33e37].
Previously, we established a knowledge-based network for the
prediction of the development of colitis-associated colon cancer by
integrating the extracellular microenvironment and intracellular
signaling pathways [38]. In silico mutation studies and experi-
mental validations revealed that concurrently targeting ceramide
and PI3K/AKT pathway achieved synergistic anti-cancer effects[38].
As an endogenous molecule, ceramide also has some obvious
drawbacks, such as poor pharmacokinetic properties, e.g. very low
solubility in an aqueous environment, potentially metabolized via
ceramide-sphigosine-S1P (proliferation-promoting lipid) and
finally degraded by S1P-lyase [39], and preferential accumulation
by enhanced permeability and retention (EPR) effect [11], which
drove us to optimize the structure of ceramide to obtain a novel
ceramide analog with good drug-like properties for in vivo appli-
cation. Herein, we reported a series of novel ceramide derivatives
with favorable pharmacokinetic properties for the treatment of
colon cancer with the combination of MK2206 in vitro and in vivo,
which provided a novel strategy of combination therapy for colon
cancer.
amine group with the aid of S1P-lyase, generating hexadecenal and
phosphoethanolamine [39]. Therefore, inspired by the develop-
ment of FTY720 (fingolimod) [40], we simplified the polar head of
ceramide by removing the chiral center and the double bond. We
conjectured that such structure simplification could improve the
metabolic stability in the precondition of activity retention. On the
other hand, introduction of rigid structure to restrict the active
conformation, increasing the aromatic properties, might be bene-
ficial for the improvement of apoptosis activity as well as phar-
macokinetic properties. Variations in the ceramide acyl chains
potentially alter membrane bilayer dynamics or differential
signaling properties by recruitment of different binding partners
[41], driving us to amply explore the acyl chain (R1). Furthermore,
introducing different length of alkyl or alkoxyl chain on aromatic
ring may potentially improve water solubility. Therefore, a series of
novel compounds (1e20) bearing naphthyl-substituted amino diol
as novel ceramide analogs were designed (Fig. 2).
2.2. Chemistry
The general synthetic routes of title compounds are illustrated
in Scheme 1 and 2. Alkyl or alkoxyl substituted naphthalene (21a-e)
underwent Friedel-Crafts reaction with bromoacetyl bromide in
the presence of AlCl3 at ꢀ20 ꢁC to produce satisfactory yields of
22a-e. Subsequent nucleophilic substitution with diethyl acet-
amidomalonate in a Cs2CO3/acetonitrile system produced 23a-e.
After treating intermediates 23a-e with triethylsilane in trifluoro-
acetic acid, compound 24a-e were obtained in high yields. The
further reduction reaction with LiAlH4 in THF at ꢀ20 ꢁC generated
corresponding compounds 1 and 11e14 with moderate yields. Then
hydrolysis of compound 1 and 12 by LiOH in MeOH/THF/H2O as co-
solvent at 60 ꢁC yielded key intermediates 25a and 25e, which were
treated with different acyl chlorides or anhydrides to afford the
target compounds 2e8 and 15e20. The synthetic route of com-
pounds 9 and 10 is depicted in Scheme 2. Intermediate 23e was
reduced by LiAlH4 in THF at ꢀ20 ꢁC to produce target compound 9
with moderate yield. Compound 10 was prepared via demethyla-
tion of compound 11 in the presence of boron tribromide at ꢀ20 ꢁC.
2. Results and discussion
2.1. Design
2.3. In vitro anti-proliferation against colon cancer cell line
Considering the poor pharmacokinetic properties of ceramides,
we aimed to design a novel series of ceramide derivatives with less-
toxicity and improved pharmacokinetic properties. It has been well
demonstrated that degradation of sphingolipids occurs at diol
Ceramides can target a series of proteins, such as Cathepsin D,
Protein kinase C (PKC), and Caspase, showing pro-apoptotic activity
against tumor cells. Thus, we examined the anti-proliferative
2

F. Gao, X. Chen, J. Lu et al.
European Journal of Medicinal Chemistry 215 (2021) 113274
Fig. 2. Design of novel ceramide analogs.
Scheme 1. Reagents and conditions: (a) bromoacetyl bromide, AlCl3, DCM, ꢀ20 ꢁC, 4 h; (b) diethyl acetamidomalonate, Cs2CO3, MeCN, reflux, overnight; (c) Et3SiH, TFA, reflux, 6 h;
(d) LiAlH4, THF, ꢀ20 ꢁC, 4 h; (e) LiOH, MeOH/THF/H2O, 60 ꢁC, overnight; (f) acyl chloride or anhydride, Et3N, DCM, rt, 4 h.
analogs bearing different acyl chains, which could also be critical
for the physical and chemical properties of these compounds as
well. Acetyl-bearing compound 1 showed moderate inhibitory ac-
tivity (IC50 ¼ 36.50
m
M), which was slightly weaker than the
reference compound C2-ceramide (IC50 ¼ 25.90
mM). Extending the
length of acyl chain to 4e8 carbons could improve the anti-
proliferative activities significantly (2e4), while further extension
of acyl chain resulted in the loss of activities (5 and 6). Introduction
of branch or aromatic substitution on acyl chain could maintain the
inhibitory activities (7 and 8). Alternatively, introduction of the
hydroxyl group on the linker also showed moderate inhibitory
activity (9). On the other hand, we tested a series of compounds
bearing alkoxyl chain on naphthalene ring, which could probably
improve the solubility in the precondition of activity retention.
Compounds bearing hydroxyl or methoxy group completely lost
the inhibitory activities against HT-29 cell line (10 and 11), while
heptyloxy-substituted compound 12 exhibited superior activity
Scheme 2. Reagents and conditions: (a) LiAlH4, THF, ꢀ20 ꢁC,
DCM, ꢀ20 ꢁC, 4 h.
4 h; (b) BBr3,
(IC50 ¼ 17.59
mM), which was better than the reference compound.
However, further introduction of rigid phenyl ring led to the
decrease of inhibitory activity (13 and 14). Thus, heptyloxy group
was selected as dominant fragment and the influence of different
acyl chain was tested again, including aliphatic chain, alkoxyl chain,
and alkenyl chain. However, all these compounds 15e20 showed
IC50 values equal to or less than that of compound 12 (Table 1). The
potency of the newly synthesized compounds on colon cancer cell
line HT-29. HT-29 cells were treated with ceramide analogs at serial
concentrations for 72 h, and the IC50 was calculated by the four-
parameter curve for each compound (Table 1). Considering the
variations in the ceramide acyl chains potentially altering signaling
properties, we tested the anti-proliferative activities of ceramide
3

F. Gao, X. Chen, J. Lu et al.
European Journal of Medicinal Chemistry 215 (2021) 113274
Table 1
may be other more sensitive cancer cells in response to compound
12. For this, we expanded the evaluation of anti-proliferative ac-
tivity of compound 12 on several other cancer cell lines, including
liver, stomach, breast, lung and lymphoma cell lines (Table S2).
Interestingly, compound 12 showed a higher potency on liver
Cell cytotoxicity (IC50) of ceramides derivatives in HT-29 cancer cells.
cancer cells SNU878 (IC50 ¼ 6.68
m
M) and B-cell non-Hodgkin’s
lymphoma Mino cells (IC50 ¼ 13.33
mM), demonstrating a wider
therapeutic window in these cancer cells, implicating the potential
for the treatment of liver cancer or B-cell non-Hodgkin’s lym-
phoma. It is likely that much more sensitive cancer cells would be
found in the future.
Compd.
R1
R2
X
R3
IC50 (m
M)a
1
2
3
4
5
6
7
CH3
H
H
H
H
H
H
H
CH2
CH2
CH2
CH2
CH2
CH2
CH2
C7H15
C7H15
C7H15
C7H15
C7H15
C7H15
C7H15
36.50 1.14
26.43 1.05
20.27 0.17
22.01 0.95
>80
C3H7
C5H11
C7H15
C9H19
2.4. In vitro combination effects on cell proliferation
C13H27
>80
25.57 0.22
Given ceramides are bioactive pro-apoptotic lipids, and AKT
inhibitor also could promote cell apoptosis, we thus proposed if
there were combinatory effect on cell apoptosis. We tested the
combination effect of ceramide analog 12 with AKT inhibitor
MK2206. To determine if these two compounds have synergistic
effect, we treated colon cancer cells with these two compounds
alone or in combination at constant ratio in serial concentrations
for 72 h, and the combination index (CI) was calculated according to
the Chou-Talalay method [42], which denotes nearly additive (CI:
0.90e1.10), synergism (CI: < 0.90), and antagonism (CI: > 1.10). The
Fa-CI plot were shown in Fig. 3, and the results showed that the
combination of compound 12 and MK2206 has either additive or
synergistic inhibitory effect on cell proliferation at different dose
levels. For HT-29 cells, low dose combination of compound 12
8
9
10
11
12
13
Ph
H
OH
H
H
H
CH2
CH2
O
O
O
C7H15
C7H15
H
CH3
C7H15
22.96 1.93
77.92 6.66
>80
>80
17.59 1.37
>80
CH3
CH3
CH3
CH3
CH3
H
O
14
CH3
H
O
27.08 1.02
15
16
17
C5H11
C7H15
H
H
H
O
O
O
C7H15
C7H15
C7H15
21.89 0.79
18.82 1.16
22.40 2.71
(10 mM) and MK2206 (2.5 mM) have additive effect, while medium
18
H
O
C7H15
21.40 2.97
and high dose combination of compound 12 and MK2206 have
synergistic effect on cell proliferation (Fig. 3A). For COLO-678 and
T84 cells, low and medium dose combination of compound 12 and
MK2206 has additive effect, while high dose combination of com-
pound 12 and MK2206 has synergistic effect on cell proliferation
(Fig. 3B and C). For SW-480 cells, low and high dose combination of
compound 12 and MK2206 have additive effects, while medium
dose combination of compound 12 and MK2206 has synergistic
effect on cell proliferation (Fig. 3D). These results demonstrated
that compound 12 and MK2206 have either additive or synergistic
effect on colon cancer cell proliferation.
19
20
H
H
/
O
O
/
C7H15
C7H15
/
60.50 5.59
36.42 0.59
25.90 0.70
C2-ceramide
a
IC50 (mM) was shown as the mean value and standard error of the mean (SEM)
which derived from three independent experiments, n ¼ 3, 72 h treatment.
structure-activity relationship study revealed that short acyl chain
(2e8 carbons) led to the most potent anti-proliferative activities
while introducing a hydroxyl group in the linker would result in the
decreasing activity. On the other hand, replacement of alkyl chain
with alkoxyl chain on the aromatic ring could further improve the
anti-proliferative activities.
2.5. Mechanism studies of combination therapy
To explore the underlying mechanism of the combination of
compound 12 and MK2206, the cell apoptosis was determined. HT-
To further assess the anti-tumor activity of ceramides de-
rivatives, we selected representative potent compounds (2, 3, 4, 8,
12, 17 and 18) and compared their anti-proliferative activity in
different colon cancer cell lines, including T84, HCT-8, SW480, etc.
(Table S1). As shown, consistent to the anti-tumor activity of
compounds in HT-29 cells (Table 1), compound 12 showed a litter
better anti-proliferative activity than the other compounds in T84,
HCT-8 and SW480 cancer cells, though it was a little lower activity
than compound 3 and 4 in Caco2, COLO-205 and COLO-678. In
consideration of anti-proliferation activity and solubility, com-
pound 12 bearing short acyl chain (R1) and alkoxyl chain (R3),
which potentially improve the aqueous solubility, was selected for
further test towards a variety of colon cancer cells and non-
cancerous cell lines (Table 2). Anti-proliferative experiment
revealed that compound 12 showed potent inhibitory to all these
colon cancer cells with some selectivity, which displayed more
sensitive on T84 and COLO-205 cells. For the non-cancerous cell
lines (GES-1, GM00637, WI-38), compound 12 exhibited lower
29 cells were treated with low dose of compound 12 (15 mM) and
Table 2
The anti-proliferative activity of compound 12 against various colon cancer and non-
cancerous cell lines.
Cell lines
IC50 (m
M)a
Colon cancer cells
T84
14.94 2.20
18.29 2.68
20.73 0.60
21.76 0.39
22.66 2.49
25.76 7.40
26.35 1.74
28.02 3.77
29.56 1.26
COLO-205
COLO-678
HCT-8
SW-480
HCT-116
Lovo
Caco2
SW620
Non-cancerous cells
GES-1
GM00637
WI-38
41.81 8.42
47.89 20.67
50.94 6.99
a
inhibitory activity with IC50 value more than 40
mM, though the
IC50 (mM) was shown as the mean value and standard error of the mean (SEM)
selectivity index is less than 3 in colon cancer cells, yet that there
which derived from three independent experiments, n ¼ 3, 72 h treatment.
4

F. Gao, X. Chen, J. Lu et al.
European Journal of Medicinal Chemistry 215 (2021) 113274
Fig. 3. Combination effects of 12 and MK2206 on cell proliferation. HT-29 (A), COLO-678 (B), T84 (C) and SW-480 (D) cells were treated with various concentrations of
compound 12 and MK2206 at constant ratio for 72 h, and the combination effect of compound 12 and MK2206 on the proliferation of colon cancer cells were determined by
calculating the CI values (Chou-Talalay method) for each data point.
MK2206 (7.5
m
M) alone or in combination for 48 h, the cell
MK2206 (Fig. 4E). These results indicated that the combination of
compound 12 and MK2206 promoted ROS production, caspase-3
dependent apoptotic pathway.
apoptosis was measured by Annexin V and PI dual staining (Fig. 4).
The results showed that low dose of compound 12 and MK2206
alone did not induce significant cell apoptosis as compared to
vehicle control, however, their combination had dramatic effect in
inducing HT-29 cell apoptosis (~40%) (Fig. 4A and B). We also
examined the production of intracellular reactive oxygen species
Ceramide regulates multiple pathways to induce G0/G1 cell
cycle arrest. The AKT signaling pathway also involved in the regu-
lation of the cell cycle, thus we determine if cell-cycle arrest con-
tributes to the anti-proliferative effect of their combination. We
used PI staining to detect the cell cycle of HT-29 cells under the
treatment of compound 12 or MK2206 or their combination for
24 h (Fig. 5A). The results showed that the G0/G1 phase arrest was
induced by MK2206 but slightly by compound 12, and their com-
bination induced a stronger G0/G1 cell cycle arrest (Fig. 5A and B).
Next, the expression of G1 cell cycle regulators was detected by
western blot, including c-Myc, P21, p-Rb/Rb, Cyclin-dependent ki-
nases (CDKs) and Cyclins. c-Myc and the CDK inhibitor P21 are
upstream positive and negative regulators of the cell cycle, and we
found that the expression of c-Myc was down-regulated and P21
was up-regulated in the combination group, but not dramatic
changed in either compound group, suggesting a combinatory ef-
fect on cell cycle arrest. It has been demonstrated that ceramide
induced the dephosphorylation of the retinoblastoma gene product
(RB) through the activation of PP1 [43], and we found that the
expression of p-RB was down-regulated slightly in compound 12 or
MK2206 group but with a dramatic reduction in the combination
group. Cyclin A and cyclin E1 which in association with CDK2 are
(ROS). HT-29 cells were treated with compound 12 (20
mM) and
MK2206 (10 M) alone or in combination for 16 h, then the ROS was
m
determined for each group. The results showed that HT-29 cells
treatment with MK2206 alone but not compound 12 induced the
generation of ROS, while the combination of compound 12 and
MK2206 induced a much higher ROS production (Fig. 4C). To
further determine the signaling pathway, which may be involved in
inducing cell apoptosis, we tested the AKT phosphorylation, poly
(ADP-ribose) polymerase (PARP) and caspase-3 cleavage and acti-
vation. Indeed, treatment with MK2206 alone and in combination
with compound 12 blocked the AKT phosphorylation, while treat-
ment with compound 12 alone only led to a slight decrease of p-
AKT (Fig. 4D). Interestingly, the combination of compound 12 and
MK2206 induces a much higher cleavage of PARP and caspase-3
than their alone, suggesting they have synergistic effect on cell
apoptosis. Moreover, we found combined treatment of MK2206
and compound 12 significantly increased the activity of caspase-3
compared to the individual treatment of compound 12 or
5

F. Gao, X. Chen, J. Lu et al.
European Journal of Medicinal Chemistry 215 (2021) 113274
Fig. 4. Combination of compound 12 and MK2206 induces increased cell apoptosis. A. HT-29 cells under the treatment with vehicle (0.1% DMSO), MK2206 (7.5
or MK2206 (7.5 M) plus 12 (15 M) for 48 h, and then the cells were analyzed by Annexin V and PI dual staining. B. Apoptotic ratio of HT-29 cells of A (upper right and lower right),
and the statistical analysis was performed between groups. C. HT-29 cells under the treatment with vehicle (0.1% DMSO), MK2206 (10 M), 12 (20 M), or MK2206 (10 M) plus 12
(20 M) for 16 h, H2O2 (10 M) for 1 h (positive control) and then the cells were analyzed using Reactive Oxygen Species Assay Kit. D. Western blotting analysis of expression of
mM), 12 (15 mM),
m
m
m
m
m
m
m
apoptotic proteins induced by MK2206, 12 or their combination in HT-29 cells after 48 h treatment. E. The activity of caspase-3 was determined for HT-29 cells under the treatment
of 12 and MK2206 alone or in combination for 24 h. Error bars represent means SD from triplicates or three independent experiments, *p < 0.01, **p < 0.001, as compared to
vehicle group.
required for the G1/S transition, and Cyclin Dl which in association
with CDK4 and CDK6 are considered to be key regulators of G1
progression [44]. We found that the expression of cyclin A and
CDK2, 4, 6 showed a much more reduction in the combination
group compared to each compound alone, suggesting that the
combination of compound 12 and MK2206 intensified cell cycle
arrest, which may be one of the mechanisms that contribute to the
inhibition of the cell proliferation. It is worth mentioning that some
regulators of the cell cycle including c-Myc, P21, p-Rb/Rb and CDKs
are also involved in the apoptotic pathway. For example, P21 and p-
Rb are targets for cleavage by caspases, which causes cell cycle
progression to stop during apoptosis [45]. The important decisions
of cell death or cell proliferation are likely to be controlled by more
than just one signal, and the combination of compound 12 and
MK2206 may lead the cell to death though multiple pathways
mentioned above.
compound for the in vivo anti-tumor study. The mice were injected
with compound at single dose of 10 mg/kg (IV) and 50 mg/kg (IP),
and the pharmacokinetic parameters were determined, such as
T
max, Cmax, AUC, t1/2, and CLz (Table 3). Compound 12 showed a
much higher AUC0-t (3e4 folds) and Cmax (2e3 folds) than C2-
ceramide, indicating a more exposure in vivo. These pharmacoki-
netic parameters indicated that compound 12 has superior profiles
for in vivo study as compared to C2-ceramide, suggesting it could be
a valuable chemical probe of ceramides.
2.7. In vivo study
We further explore whether the synergistic effect of compound
12 and MK2206 could be achieved in vivo. Anti-tumor effect was
evaluated in subcutaneous tumors generated by injection of HT-
29 cells into the flanks of BALB/c nude mice. To observe the syn-
ergistic effect, mice with tumors of the equal volumes were treated
with low dose of compound 12, MK2206, or in combination. The
result showed that either MK2206 or compound 12 exerted sig-
nificant inhibitory effect on tumor growth, while their combination
exerted much more dramatic inhibition on tumor growth as
compared to single agent (Fig. 6A), indicating they have synergistic
inhibitory effect on tumor growth. The tumor growth inhibition
(TGI) of the combination group was 47.5%, while the TGI of the
2.6. Pharmacokinetics study
C2-ceramide is a good chemical probe of ceramide analogs for
the study of anti-tumor activity and mechanisms at cellular level,
but not appropriate for the in vivo anti-tumor study due to the poor
pharmacokinetics. Therefore, we characterized the pharmacoki-
netics of compound 12 in BALB/c mice, to evaluate if it is a good
6

F. Gao, X. Chen, J. Lu et al.
European Journal of Medicinal Chemistry 215 (2021) 113274
Fig. 5. Combination of compound 12 and MK2206 exhibits enhanced cell cycle arrest. A. HT-29 cells under the treatment with vehicle (0.1% DMSO), MK2206 (10
mM), 12
(20 M), or MK2206 (10 M) plus 12 (20 M) for 24 h, and the cell cycle was analyzed by PI staining. B. Cell cycle distribution of HT-29 cells under the treatment A. C. Expression of
m
m
m
cell cycle associated proteins in HT-29 cells after the treatment for 24 h.
Table 3
Pharmacokinetics of compound 12 and C2-ceramide in mice.
Compd.
Route
Dose (mg/kg)
T
max h
Cmax ng/mL
AUC0-t ng/mL*h
AUC0-∞ ng/mL*h
t1/2
h
CLz mL/min/kg
12
IP
IV
IP
IV
50
10
50
10
0.75
/
0.417
/
6026
/
2744
/
15578
3061
4064
1620
15678
3063
4094
1632
3.30
3.12
3.99
5.08
/
55.2
/
113
C2-ceramide
compound 12 and MK2206 group alone was 25.4% and 30.9%
respectively (Fig. 6A). Consistently, the tumor weight was signifi-
cantly decreased in the combination group as compared to vehicle
group (Fig. 6B). The body weight of mice did not have significant
difference among vehicle, single agent and combination groups
(Fig. 6C), indicating there is no safety issue under the tested dosage.
These results indicated that the combination of compound 12 and
MK2206 has a synergistic anti-tumor effect in vivo.
3. Conclusion
In summary, a novel series of ceramide analogs via structure
simplification and configuration restriction were developed. Com-
pounds were optimized for improved potency of anti-proliferative
activity and better pharmacokinetics. This led to the discovery of
representative compound 12, which showed potent anti-
proliferative activity against various colon cancer cells
7

F. Gao, X. Chen, J. Lu et al.
European Journal of Medicinal Chemistry 215 (2021) 113274
Fig. 6. Effect of combination of 12 and MK2206 on tumor growth in vivo. HT-29 xenograft model was treated with 12 (100 mg/kg/d) and MK2206 (50 mg/kg/Q2D) alone or in
combination. Tumor volume was measured every 3 days, and the statistical difference at each time point between each group was analyzed by Two-Way ANOVA. Tumor weight was
compared between each group by One-Way ANOVA. *p < 0.001, **p < 0.0001, as compared to vehicle group; #p < 0.001, ##p < 0.0001, as compared to single agent group.
(IC50 ¼ 15.90e29.56
m
M). Additionally, the synergistic effect of
slowly at room temperature. The reaction was cooled to ꢀ20 ꢁC in
refrigerated/heated bath circulator and AlCl3 (5.34 g, 40 mmol) was
added portion wise and the resulting mixture was stirred at the
same temperature for 4 h. Then the reaction was poured into
200 mL ice-water and extracted with DCM twice. The combined
organic layers were washed with saturated brine, dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The
residue was purified by flash column chromatography with hex-
anes/ethyl acetate (20/1) as the eluent to afford the title compound
compound 12 and MK2206 have been demonstrated on colon
cancer cell proliferation. Cell apoptosis studies revealed that com-
bination therapy promoted ROS production and caspase-3 depen-
dent apoptotic pathway. Further cell cycle studies demonstrated
that combination therapy intensified cell cycle arrest in the G0/G1
phase. Besides, the pharmacokinetic parameters indicated that
compound 12 had superior characteristics (higher Cmax and AUC)
for in vivo study than the reference compound C2-ceramide.
Collectively, our study provided a preliminary structure activity
relation for ceramide analogs and developed potent ceramide an-
alogs such as compound 12, which demonstrated the synergistic
anti-tumor effect with the AKT inhibitor MK2206 both in vitro and
in vivo study, and it would be an important reference for the
translation of this class of compounds in the future.
(6.15 g, 85%) as a white solid. 1H NMR (600 MHz, CDCl3)
d 8.96 (dt,
J ¼ 8.7, 0.9 Hz, 1H), 8.35 (ddd, J ¼ 8.5, 1.5, 0.7 Hz, 1H), 8.03 (d,
J ¼ 8.2 Hz, 1H), 7.65 (ddd, J ¼ 8.6, 6.8, 1.4 Hz, 1H), 7.54 (ddd, J ¼ 8.2,
6.8, 1.2 Hz, 1H), 6.79 (d, J ¼ 8.3 Hz, 1H), 4.54 (s, 2H), 4.22 (t,
J ¼ 6.4 Hz, 2H), 2.01e1.93 (m, 2H), 1.62e1.52 (m, 3H), 1.45e1.38 (m,
2H), 1.37e1.31 (m, 4H), 0.95e0.88 (m, 3H). 13C NMR (125 MHz,
CDCl3)
d 192.0, 159.3, 132.2, 132.1, 128.7, 125.6, 125.6, 125.6, 123.0,
121.9, 102.2, 68.3, 32.8, 31.3, 28.6, 28.6, 25.7, 22.2, 13.6. HRMS (EI)
4. Experimental section
[M]þ calcd for C19H23O2Br: 362.0876; found: 362.0873.
4.1. Chemistry
4.1.2. Diethyl 2-acetamido-2-(2-(4-(heptyloxy)naphthalen-1-yl)-2-
General procedure. Unless otherwise noted, the reagents
(chemicals) were purchased from commercial sources, and used
without further purification. Water was deionized before used.
Analytical thin layer chromatography (TLC) was HSGF 254
(0.15e0.2 mm thickness). Compound spots were visualized by UV
light (254 nm). Column chromatography was performed on silica
gel FCP 200e300. NMR spectra was run on 400, 500 or 600 Hz
instrument. Chemical shifts were reported in parts per million
oxoethyl)malonate (23a)
To
a solution of 2-bromo-1-(4-(heptyloxy)naphthalen-1-yl)
ethan-1-one 22a (5.00 g, 13.76 mmol) in dry acetonitrile (80 mL)
were added cesium carbonate (9.87 g, 30.28 mmol) and diethyl 2-
acetamidomalonate (4.48 g, 20.64 mmol). The resulting mixture
was stirred under reflux overnight. After the consumption of
starting material, the reaction was cooled to room temperature and
filtered. The filtrate was concentrated under vacuum and the res-
idue was purified by flash column chromatography with hexanes/
ethyl acetate (4/1) as the eluent to afford the title compound
(ppm, d) downfield from tetramethylsilane. Proton coupling pat-
terns are described as singlet (s), doublet (d), triplet (t), quartet (q),
multiplet (m), and broad (br). Low- and high-resolution mass
spectra (LRMS and HRMS) were measured on spectrometer.
(4.90 g, 71%) as a white solid. 1H NMR (400 MHz, CDCl3)
d 9.01 (d,
J ¼ 8.6 Hz, 1H), 8.36 (d, J ¼ 8.3 Hz, 1H), 8.14 (d, J ¼ 8.3 Hz, 1H), 7.62
(ddd, J ¼ 8.6, 6.9, 1.4 Hz, 1H), 7.53 (ddd, J ¼ 8.4, 6.9, 1.4 Hz, 1H), 7.22
(s, 1H), 6.80 (d, J ¼ 8.2 Hz, 1H), 4.37 (s, 2H), 4.32 (q, J ¼ 7.1, 4H), 4.23
(t, J ¼ 6.4 Hz, 2H), 2.00 (s, 3H), 1.99e1.92 (m, 2H), 1.63e1.53 (m, 2H),
1.48e1.39 (m, 2H), 1.39e1.32 (m, 4H), 1.29 (t, J ¼ 7.1, 6H), 0.96e0.89
4.1.1. 2-Bromo-1-(4-(heptyloxy)naphthalen-1-yl)ethan-1-one
(22a)
A solution of
a-substituted naphthalene (4.85 g, 20 mmol) in dry
DCM (100 mL) was added bromoacetyl bromide (1.74 mL, 20 mmol)
(m, 3H). 13C NMR (125 MHz, CDCl3)
d 198.3, 169.5, 167.6, 159.4,
8

F. Gao, X. Chen, J. Lu et al.
European Journal of Medicinal Chemistry 215 (2021) 113274
132.8, 132.1, 128.9, 125.9, 125.9, 125.8, 125.5, 122.3, 102.8, 68.7, 64.4,
62.8, 44.1, 31.8, 29.1, 26.2, 23.0, 22.6, 14.1, 14.0. HRMS (ESI) [M þ H]þ
calcd for C28H38NO7: 500.2643; found: 500.2655.
butyryl chloride (0.18 g, 1.68 mmol) at 0 ꢁC. The resulting mixture
was stirred at room temperature for 4 h. The reaction was quenched
by water and extracted with DCM. The organic phase was washed
with brine, dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The residue was purified by flash column chro-
matography with DCM/MeOH (20/1) as the eluent to afford the title
compound (1.92 g, 89%) as a white solid. 1H NMR (400 MHz, MeOD)
4.1.3. Diethyl 2-acetamido-2-(2-(4-(heptyloxy)naphthalen-1-yl)
ethyl)malonate (24a)
Diethyl 2-acetamido-2-(2-(4-(heptyloxy)naphthalen-1-yl)-2-
oxoethyl)malonate 23a (3.00 g, 6.00 mmol) was dissolved in tri-
fluoroacetic acid (40 mL), followed by the triethylsilane (4.78 mL,
30.00 mmol). The resulting mixture was stirred under reflux for 6 h.
After cooling to room temperature, the solvent was removed under
reduced pressure and the residue was purified by flash column
chromatography with hexanes/ethyl acetate (4/1) as the eluent to
afford the title compound (2.75 g, 94%) as a white solid. 1H NMR
d
8.18 (dd, J ¼ 7.4, 2.2 Hz, 1H), 8.01 (dd, J ¼ 7.4, 2.3 Hz, 1H), 7.51e7.42
(m, 2H), 7.25 (d, J ¼ 7.2 Hz, 1H), 7.17 (d, J ¼ 7.2 Hz, 1H), 3.88 (d,
J ¼ 11.2 Hz, 2H), 3.77 (d, J ¼ 11.2 Hz, 2H), 3.09e3.02 (m, 2H),
3.01e2.93 (m, 2H), 2.32e2.24 (m, 2H), 2.19e2.10 (m, 2H), 1.75e1.64
(m, 4H), 1.41e1.34 (m, 2H), 1.31e1.24 (m, 8H), 1.01 (t, J ¼ 7.4 Hz, 3H),
0.89 (t, J ¼ 6.9 Hz, 3H). 13C NMR (125 MHz, MeOD)
d 175.0, 136.5,
136.1, 131.8, 131.7, 124.9, 124.9, 124.4, 124.3, 123.9, 123.6, 62.4, 60.7,
37.8, 32.2, 31.8, 31.2, 30.3, 28.9, 28.8, 28.6, 26.2, 21.9, 18.7, 12.6, 12.3.
HRMS (ESI) [M þ H]þ calcd for C27H42NO3: 428.3159; found:
428.3154.
(400 MHz, CDCl3)
d
8.34 (d, J ¼ 8.2 Hz, 1H), 7.87 (d, J ¼ 8.3 Hz, 1H),
7.57e7.44 (m, 2H), 7.18 (d, J ¼ 7.8 Hz, 1H), 6.92 (s, 1H), 6.72 (d,
J ¼ 7.8 Hz, 1H), 4.30e4.16 (m, 4H), 4.12 (t, J ¼ 6.4 Hz, 2H), 2.93e2.84
(m, 2H), 2.84e2.76 (m, 2H), 2.10 (s, 3H), 1.99e1.89 (m, 2H),
1.61e1.52 (m, 2H), 1.47e1.39 (m, 2H), 1.39e1.32 (m, 4H), 1.27e1.22
4.1.7. N-(1-hydroxy-2-(hydroxymethyl)-4-(4-octylnaphthalen-1-
yl)butan-2-yl)hexanamide (3)
(m, 6H), 0.93 (t, J ¼ 6.5 Hz, 3H). 13C NMR (125 MHz, CDCl3)
d 169.1,
168.2, 153.9, 132.5, 128.0, 126.4, 126.1, 126.1, 124.7, 123.2, 122.8,
104.1, 68.2, 66.5, 62.6, 33.0, 31.8, 29.3, 29.1, 26.9, 26.3, 23.1, 22.6,
14.1, 14.0. HRMS (ESI) [M þ H]þ calcd for C28H40NO6: 486.2850;
found: 486.2863.
The title compound was prepared using general method B.
Waxy solid. 1H NMR (400 MHz, MeOD)
d 8.21e8.15 (m, 1H),
8.08e8.00 (m, 1H), 7.53e7.43 (m, 2H), 7.30e7.23 (m, 1H), 7.23e7.16
(m, 1H), 3.88 (d, J ¼ 11.2 Hz, 2H), 3.77 (d, J ¼ 11.2 Hz, 2H), 3.14e2.93
(m, 4H), 2.34e2.26 (m, 2H), 2.19e2.10 (m, 2H), 1.76e1.61 (m, 4H),
1.39e1.27 (m, 14H), 0.94e0.88 (m, 6H). 13C NMR (125 MHz, MeOD)
4.1.4. 2-Amino-2-(2-(4-(heptyloxy)naphthalen-1-yl)ethyl)propane-
1,3-diol (25a)
d 175.2, 136.5, 136.1, 131.8, 131.7, 124.9, 124.8, 124.4, 124.3, 123.9,
To
a
solution of N-(4-(4-(heptyloxy)naphthalen-1-yl)-1-
123.6, 62.4, 60.6, 35.8, 32.2, 31.7, 31.2, 30.8, 30.3, 29.0, 28.8, 28.6,
hydroxy-2-(hydroxymethyl)butan-2-yl)acetamide 12 (2.40 g,
6.01 mmol) in 50 mL MeOH/THF/H2O (1:1:1, v/v/v) was added LiOH
(0.57 g, 20.04 mmol). The resulting mixture was stirred at 50 ꢁC
overnight. After cooling to room temperature, the solvent was
removed under vacuum. The residue was dissolved in DCM (20 mL)
and filtered. The filtrate was concentrated under vacuum and pu-
rified by flash column chromatography with DCM/MeOH (20/1) as
the eluent to afford the title compound (1.92 g, 89%) as a white
26.1, 25.1, 21.9, 21.7, 12.6, 12.5. HRMS (ESI) [M þ H]þ calcd for
C
29H46NO3: 456.3472; found: 456.3477.
4.1.8. N-(1-hydroxy-2-(hydroxymethyl)-4-(4-octylnaphthalen-1-
yl)butan-2-yl)octanamide (4)
The title compound was prepared using general method B.
Waxy solid. 1H NMR (400 MHz, MeOD)
d 8.21e8.14 (m, 1H),
8.08e8.03 (m, 1H), 7.52e7.47 (m, 2H), 7.29e7.19 (m, 2H), 3.87 (d,
J ¼ 11.1 Hz, 2H), 3.76 (d, J ¼ 11.1 Hz, 2H), 3.24e3.19 (m, 2H),
3.07e3.03 (m, 2H), 2.30 (t, J ¼ 7.5 Hz, 2H), 2.17e2.09 (m, 2H),
1.76e1.66 (m, 4H), 1.34e1.27 (m, 18H), 0.92e0.87 (m, 6H). 13C NMR
solid. 1H NMR (400 MHz, MeOD)
d
8.26 (d, J ¼ 8.4 Hz, 1H), 8.10 (d,
J ¼ 8.4 Hz, 1H), 7.55e7.48 (m, 1H), 7.48e7.41 (m, 1H), 7.26 (d,
J ¼ 7.7 Hz,1H), 6.79 (d, J ¼ 7.8 Hz,1H), 4.11 (t, J ¼ 6.3 Hz, 2H), 3.61 (d,
J ¼ 10.8 Hz, 2H), 3.55 (d, J ¼ 10.8 Hz, 2H), 3.11e3.01 (m, 2H),
1.95e1.86 (m, 2H), 1.82e1.74 (m, 2H), 1.64e1.52 (m, 2H), 1.49e1.41
(m, 2H), 1.40e1.32 (m, 4H), 0.98e0.87 (m, 3H). 13C NMR (125 MHz,
(125 MHz, MeOD)
d 174.6, 135.9, 135.5, 131.2, 131.1, 124.3, 124.3,
123.8, 123.8, 123.4, 123.1, 61.9, 60.1, 35.3, 31.6, 31.1, 30.6, 30.5, 29.7,
28.4, 28.2, 28.0, 27.8, 25.6, 24.8, 21.3, 21.2, 12.0, 12.0, 6.8. HRMS (ESI)
[M þ H]þ calcd for C31H50NO3: 484.3785; found: 484.3788.
MeOD)
d 153.3, 132.6, 130.3, 126.1, 125.7, 125.3, 124.1, 123.5, 122.1,
104.1, 67.8, 65.3, 55.6, 35.6, 31.6, 29.1, 28.8, 26.0, 25.8, 22.3, 13.0.
HRMS (ESI) [M þ H]þ calcd for C22H34NO3: 360.2533; found:
360.2527.
4.1.9. N-(1-hydroxy-2-(hydroxymethyl)-4-(4-octylnaphthalen-1-
yl)butan-2-yl)decanamide (5)
The title compound was prepared using general method B.
4.1.5. N-(1-hydroxy-2-(hydroxymethyl)-4-(4-octylnaphthalen-1-
yl)butan-2-yl)acetamide (1)
Waxy solid. 1H NMR (400 MHz, MeOD)
d 8.21e8.15 (m, 1H),
8.09e8.03 (m,1H), 7.51e7.47 (m, 2H), 7.27 (d, J ¼ 7.3 Hz,1H), 7.22 (d,
J ¼ 7.3 Hz, 1H), 3.88 (d, J ¼ 11.2 Hz, 2H), 3.76 (d, J ¼ 11.2 Hz, 2H),
3.09e3.01 (m, 4H), 2.30 (t, J ¼ 7.4 Hz, 2H), 2.17e2.10 (m, 2H),
1.75e1.66 (m, 4H), 1.33e1.23 (m, 22H), 0.93e0.88 (m, 6H). 13C NMR
The title compound was prepared using general method A.
White solid, Mp: 113e115 ꢁC. 1H NMR (400 MHz, MeOD)
d
8.22e8.14 (m, 1H), 8.09e8.02 (m, 1H), 7.53e7.46 (m, 2H),
7.29e7.26 (m, 1H), 7.24e7.19 (m, 1H), 3.88 (d, J ¼ 11.2 Hz, 2H), 3.79
(125 MHz, MeOD) d 174.6, 135.9, 135.5, 131.2, 131.1, 124.3, 124.3,
(d, J ¼ 11.2 Hz, 2H), 3.13e2.96 (m, 4H), 2.17e2.08 (m, 2H), 2.06 (s,
123.8, 123.7, 123.4, 123.1, 61.9, 60.1, 35.3, 31.6, 31.1, 30.6, 30.6, 29.7,
28.4, 28.2, 28.2, 28.1, 28.0, 28.0, 27.9, 25.6, 24.8, 21.3, 21.2, 12.0, 12.0.
HRMS (ESI) [M þ H]þ calcd for C33H54NO3: 512.4098; found:
512.4099.
3H), 1.77e1.64 (m, 2H), 1.48e1.21 (m, 10H), 0.96e0.84 (m, 3H). 13
C
NMR (150 MHz, MeOD)
d 172.7, 136.9, 136.6, 132.2, 132.2, 125.4,
125.3, 124.9, 124.8, 124.4, 124.1, 62.5, 61.3, 32.7, 32.1, 31.6, 30.8, 29.4,
29.2, 29.1, 26.6, 22.3, 22.1, 13.0. HRMS (ESI) [M þ H]þ calcd for
C
25H38NO3: 400.2846; found: 400.2851.
4.1.10. N-(1-hydroxy-2-(hydroxymethyl)-4-(4-octylnaphthalen-1-
yl)butan-2-yl)tetradecanamide (6)
4.1.6. N-(1-hydroxy-2-(hydroxymethyl)-4-(4-octylnaphthalen-1-
yl)butan-2-yl)butyramide (2)
The title compound was prepared using general method B.
Waxy solid. 1H NMR (400 MHz, MeOD)
d 8.20e8.16 (m, 1H),
General Method B. Waxy solid. To a solution of 2-Amino-2-(2-(4-
octylnaphthalen-1-yl)ethyl)propane-1,3-diol (0.50 g, 1.40 mmol) in
dry DCM was added triethylamine (0.58 mL, 4.20 mmol) and
8.09e8.04 (m, 1H), 7.52e7.47 (m, 2H), 7.27 (d, J ¼ 7.3 Hz, 1H), 7.23
(d, J ¼ 7.3 Hz, 1H), 3.88 (d, J ¼ 11.2 Hz, 2H), 3.76 (d, J ¼ 11.2 Hz, 2H),
3.09e3.03 (m, 4H), 2.30 (t, J ¼ 7.4 Hz, 2H), 2.17e2.10 (m, 2H),
9

F. Gao, X. Chen, J. Lu et al.
European Journal of Medicinal Chemistry 215 (2021) 113274
1.76e1.67 (m, 4H), 1.33e1.21 (m, 30H), 0.91e0.88 (m, 6H). 13C NMR
(20/1) as the eluent to afford the title compound (0.16 g, 43%) as a
(125 MHz, MeOD)
d
174.6, 135.9, 135.5, 131.2, 124.3, 124.3, 123.8,
white solid. Mp: 117e118 ꢁC. 1H NMR (400 MHz, MeOD)
d 8.26 (d,
123.7, 123.4, 123.0, 61.9, 60.1, 35.3, 31.6, 31.1, 30.6, 30.6, 29.7, 28.4,
28.4, 28.3, 28.3, 28.2, 28.2, 28.2, 28.1, 28.0, 28.0, 27.9, 25.6, 24.8, 21.3,
21.3, 12.0, 12.0. HRMS (ESI) [M þ H]þ calcd for C37H62NO3:
568.4724; found: 568.4737.
J ¼ 8.3 Hz, 1H), 8.01 (d, J ¼ 8.4 Hz, 1H), 7.57e7.50 (m, 1H), 7.48e7.42
(m, 1H), 7.25 (d, J ¼ 7.8 Hz, 1H), 6.80 (d, J ¼ 7.9 Hz, 1H), 4.03e3.96
(m, 4H), 3.16e3.06 (m, 1H), 3.03e2.93 (m, 1H), 2.04 (s, 3H),
2.02e1.92 (m, 1H), 1.89e1.76 (m, 1H). 13C NMR (125 MHz, MeOD)
d
171.9, 154.1, 132.4, 129.4, 125.8, 125.8, 125.5, 124.2, 123.2, 122.0,
4.1.11. N-(1-hydroxy-2-(hydroxymethyl)-4-(4-octylnaphthalen-1-
yl)butan-2-yl)-2-methylbutanamide (7)
103.0, 63.6, 54.4, 51.3, 31.9, 28.6, 21.3. HRMS (ESI) [M þ H]þ calcd for
C
20H18NO2: 304.1332; found: 304.1295.
The title compound was prepared using general method B.
Waxy solid. 1H NMR (400 MHz, MeOD)
d
8.21e8.15 (m, 1H), 8.04 (d,
4.1.15. N-(1-hydroxy-2-(hydroxymethyl)-4-(4-
J ¼ 7.3 Hz, 1H), 7.52e7.43 (m, 2H), 7.26 (d, J ¼ 7.2 Hz, 1H), 7.19 (d,
J ¼ 7.2 Hz, 1H), 3.91e3.84 (m, 2H), 3.82e3.75 (m, 2H), 3.11e3.03 (m,
2H), 3.02e2.97 (m, 2H), 2.46e2.31 (m, 1H), 2.25e2.09 (m, 2H),
1.76e1.63 (m, 4H), 1.45e1.38 (m, 2H), 1.32e1.25 (m, 8H), 1.18 (d,
methoxynaphthalen-1-yl)butan-2-yl)acetamide (11)
The title compound was prepared using general method A.
While solid. Mp: 111e113 ꢁC. 1H NMR (400 MHz, MeOD)
d 8.30 (d,
J ¼ 7.9 Hz, 1H), 8.15 (d, J ¼ 8.4 Hz, 1H), 7.58e7.54 (m, 1H), 7.52e7.46
(m, 1H), 7.31 (d, J ¼ 7.9 Hz, 1H), 6.82 (d, J ¼ 7.8 Hz, 1H), 4.00 (s, 3H),
3.97e3.91 (m, 2H), 3.87e3.81 (m, 2H), 3.10e3.01 (m, 2H), 2.19e2.13
J ¼ 6.8 Hz, 3H), 0.98 (t, J ¼ 7.4 Hz, 3H), 0.90 (t, J ¼ 6.8 Hz, 3H). 13
C
NMR (125 MHz, MeOD)
d 178.4, 136.5, 136.1, 131.8, 131.7, 124.9,
124.8,124.4,124.3,123.9,123.6, 62.7, 62.6, 60.5, 42.3, 32.2, 31.9, 31.2,
30.3, 28.9, 28.8, 28.6, 26.6, 26.2, 21.9, 16.4, 12.6, 10.5. HRMS (ESI)
[M þ H]þ calcd for C28H44NO3: 442.3316; found: 442.3333.
(m, 2H), 2.12 (s, 3H). 13C NMR (150 MHz, MeOD)
d 172.7,154.0,132.6,
130.2, 125.8, 125.8, 125.5, 124.2, 123.5, 122.0, 103.1, 62.6, 61.3, 54.5,
32.1, 26.1, 22.1. HRMS (ESI) [M þ H]þ calcd for C18H24NO4: 318.1700;
found: 318.1704.
4.1.12. N-(1-hydroxy-2-(hydroxymethyl)-4-(4-octylnaphthalen-1-
yl)butan-2-yl)benzamide (8)
4.1.16. N-(4-(4-(heptyloxy)naphthalen-1-yl)-1-hydroxy-2-
The title compound was prepared using general method B.
(hydroxymethyl)butan-2-yl)acetamide (12)
Waxy solid. 1H NMR (400 MHz, MeOD)
d
8.22e8.15 (m, 1H),
General Method A. To a solution of diethyl 2-acetamido-2-(2-(4-
(heptyloxy)naphthalen-1-yl)ethyl)malonate 24 (2.00 g, 4.12 mmol)
in THF (50 mL) was added LiAlH4 (0.31 g, 8.24 mmol) portion wise
at e 20 ꢁC. The mixture was stirred for 4 h. The reaction was
quenched by water and the solvent was removed under vacuum.
The residue was purified by flash column chromatography with
DCM/MeOH (50/1) as the eluent to afford the title compound
(1.06 g, 64%) as a white solid. Mp: 84e86 ꢁC. 1H NMR (400 MHz,
8.03e7.97 (m, 1H), 7.87e7.82 (m, 2H), 7.53e7.40 (m, 5H), 7.24 (d,
J ¼ 7.2 Hz, 1H), 7.14 (d, J ¼ 7.2 Hz, 1H), 4.01 (d, J ¼ 11.2 Hz, 2H), 3.89
(d, J ¼ 11.2 Hz, 2H), 3.16e3.07 (m, 2H), 2.98e2.91 (m, 2H),
2.33e2.24 (m, 2H), 1.68e1.60 (m, 2H), 1.40e1.32 (m, 2H), 1.28e1.22
(m, 8H), 0.88 (t, J ¼ 6.9 Hz, 3H). 13C NMR (125 MHz, MeOD)
d 169.2,
136.5, 136.1, 134.8, 131.8, 131.7, 130.7, 127.7, 126.5, 125.0, 124.9, 124.4,
124.3, 123.9, 123.6, 62.4, 61.1, 32.2, 31.9, 31.2, 30.3, 29.0, 28.8, 28.6,
26.4, 21.9, 12.6. HRMS (ESI) [M þ H]þ calcd for C30H40NO3:
462.3003; found: 462.3014.
MeOD)
d
8.30e8.22 (m, 1H), 8.09 (d, J ¼ 8.4 Hz, 1H), 7.55e7.48 (m,
1H), 7.47e7.41 (m, 1H), 7.25 (d, J ¼ 7.8 Hz, 1H), 6.79 (d, J ¼ 7.9 Hz,
1H), 4.13 (t, J ¼ 6.3 Hz, 2H), 3.87 (d, J ¼ 11.2 Hz, 2H), 3.78 (d,
J ¼ 11.2 Hz, 2H), 3.06e2.95 (m, 2H), 2.15e2.07 (m, 2H), 2.06 (s, 3H),
1.97e1.87 (m, 2H), 1.66e1.53 (m, 2H), 1.50e1.41 (m, 2H), 1.41e1.34
4.1.13. N-(1,4-dihydroxy-2-(hydroxymethyl)-4-(4-octylnaphthalen-
1-yl)butan-2-yl)acetamide (9)
Diethyl
2-acetamido-2-(2-(4-octylnaphthalen-1-yl)-2-
(m, 4H), 0.96e0.92 (m, 3H). 13C NMR (125 MHz, MeOD)
d 171.6,
oxoethyl)malonate (0.60 g, 1.21 mmol) was dissolved in dry THF
(20 mL) and the reaction was cooled to ꢀ20 ꢁC. Then LiAlH4 (0.18 g,
4.82 mmol) was added slowly and the resulting mixture was stirred
for 4 h. The reaction was quenched by water and filtered through a
pad of Celite. The filtrate was concentrated under reduced pressure
and purified by flash column chromatography with DCM/MeOH
(20/1) as the eluent to afford the title compound (0.28 g, 56%) as a
152.4, 131.6, 129.0, 125.0, 124.7, 124.5, 123.1, 122.5, 121.0, 103.1, 66.7,
61.7, 60.2, 31.2, 30.6, 28.0, 27.8, 25.0, 25.0, 21.3, 21.1,12.0. HRMS (ESI)
[M þ H]þ calcd for C24H36NO4: 402.2639; found: 402.2645.
4.1.17. N-(4-(4-(4-butylphenoxy)naphthalen-1-yl)-1-hydroxy-2-
(hydroxymethyl)butan-2-yl)acetamide (13)
The title compound was prepared using general method A.
white solid. Mp: 66e68 ꢁC. 1H NMR (400 MHz, MeOD)
d
8.20e8.16
Waxy solid. 1H NMR (400 MHz, MeOD)
d
8.17 (dd, J ¼ 12.1, 8.4 Hz,
(m, 1H), 8.09e8.04 (m, 1H), 7.52e7.47 (m, 2H), 7.27 (d, J ¼ 7.3 Hz,
1H), 7.23 (d, J ¼ 7.3 Hz, 1H), 3.88 (d, J ¼ 11.2 Hz, 2H), 3.76 (d,
J ¼ 11.2 Hz, 2H), 3.09e3.03 (m, 4H), 2.30 (t, J ¼ 7.4 Hz, 2H), 2.17e2.10
(m, 2H), 1.76e1.67 (m, 4H), 1.33e1.21 (m, 31H), 0.91e0.88 (m, 6H).
2H), 7.55 (dd, J ¼ 8.5, 6.8 Hz, 1H), 7.50e7.41 (m, 1H), 7.25 (d,
J ¼ 7.9 Hz, 1H), 7.12 (d, J ¼ 8.0 Hz, 2H), 6.87 (d, J ¼ 8.0 Hz, 2H), 6.78
(d, J ¼ 7.8 Hz, 1H), 3.88 (d, J ¼ 11.2 Hz, 2H), 3.78 (d, J ¼ 11.1 Hz, 2H),
3.10e2.96 (m, 2H), 2.57 (t, J ¼ 7.8 Hz, 2H), 2.17e2.07 (m, 2H), 2.06 (s,
13C NMR (125 MHz, MeOD)
d
174.6, 135.9, 135.5, 131.2, 124.3, 124.3,
3H), 1.63e1.52 (m, 2H), 1.41e1.28 (m, 2H), 0.94 (t, J ¼ 7.3 Hz, 3H). 13
C
123.8, 123.7, 123.4, 123.0, 61.9, 60.1, 35.3, 31.6, 31.1, 30.6, 30.6, 29.7,
28.4, 28.3, 28.2, 28.1, 28.0, 27.9, 25.6, 24.8, 21.3, 12.0. HRMS (ESI)
[M þ H]þ calcd for C25H36NO4: 414.2650; found: 414.2643.
NMR (125 MHz, MeOD) d 171.6, 154.9, 150.8, 136.4, 132.8, 132.0,
128.2, 126.1, 125.1, 124.4, 124.0, 123.0, 121.1, 116.8, 111.6, 61.7, 60.2,
33.4, 32.7, 31.2, 25.3, 21.1, 20.9, 11.9. HRMS (ESI) [M þ H]þ calcd for
C
27H34NO4: 436.2482; found: 436.2489.
4.1.14. N-(1-hydroxy-2-(hydroxymethyl)-4-(4-hydroxynaphthalen-
1-yl)butan-2-yl)acetamide (10)
4.1.18. N-(4-(4-((4-butylbenzyl)oxy)naphthalen-1-yl)-1-hydroxy-
2-(hydroxymethyl)butan-2-yl)acetamide (14)
To
a
solution of N-(1-hydroxy-2-(hydroxymethyl)-4-(4-
methoxynaphthalen-1-yl)butan-2-yl)acetamide 11 (0.40 g,
1.26 mmol) in dry DCM (10 mL) was added dropwise BBr3 (0.14 mL,
1.51 mmol) at ꢀ20 ꢁC. The resulting mixture was stirred at ꢀ20 ꢁC
for 4 h. The reaction was quenched by water and extracted with
DCM three times. The combined organic layers were dried over
anhydrous Na2SO4 and concentrated under vacuum. The residue
was purified by flash column chromatography with DCM/MeOH
The title compound was prepared using general method A.
While solid. Mp: 129e131 ꢁC. 1H NMR (600 MHz, MeOD)
d 8.29 (dd,
J ¼ 8.7, 1.2 Hz, 1H), 8.11e8.07 (m, 1H), 7.54e7.49 (m, 1H), 7.45e7.41
(m, 1H), 7.40 (d, J ¼ 8.0 Hz, 2H), 7.23 (d, J ¼ 7.9 Hz, 1H), 7.20 (d,
J ¼ 8.0 Hz, 2H), 6.85 (d, J ¼ 7.9 Hz, 1H), 5.16 (s, 2H), 3.86 (d,
J ¼ 11.2 Hz, 2H), 3.77 (d, J ¼ 11.2 Hz, 2H), 3.03e2.95 (m, 2H), 2.62 (t,
J ¼ 7.7 Hz, 2H), 2.11e2.06 (m, 2H), 2.04 (s, 3H), 1.64e1.57 (m, 2H),
10

F. Gao, X. Chen, J. Lu et al.
European Journal of Medicinal Chemistry 215 (2021) 113274
1.39e1.34 (m, 2H), 0.95 (t, J ¼ 7.4 Hz, 3H). 13C NMR (150 MHz,
4.1.23. Benzyl (4-(4-(heptyloxy)naphthalen-1-yl)-1-hydroxy-2-
(hydroxymethyl)butan-2-yl)carbamate (19)
MeOD)
d 172.7, 153.1, 142.3, 134.7, 132.7, 130.4, 128.2, 127.2, 126.1,
125.9, 125.5, 124.3, 123.6, 122.2, 104.8, 69.7, 62.6, 61.3, 35.0, 33.6,
The title compound was prepared using general method B.
32.2, 26.1, 22.1, 22.0, 12.9. HRMS (ESI) [M þ H]þ calcd for
Waxy solid. 1H NMR (600 MHz, MeOD)
d 8.27e8.21 (m, 1H), 8.02 (d,
C
28H36NO4: 450.2639; found: 450.2650.
J ¼ 8.1 Hz, 1H), 7.46e7.38 (m, 4H), 7.34 (t, J ¼ 7.6 Hz, 2H), 7.31e7.26
(m, 1H), 7.17 (d, J ¼ 7.7 Hz, 1H), 6.78e6.73 (m, 1H), 5.11 (s, 2H), 4.10
(t, J ¼ 5.4 Hz, 2H), 3.85 (d, J ¼ 11.1 Hz, 2H), 3.73 (d, J ¼ 11.1 Hz, 2H),
3.04e2.93 (m, 2H), 2.07e2.01 (m, 2H), 1.94e1.84 (m, 2H), 1.62e1.54
(m, 2H), 1.47e1.40 (m, 2H), 1.39e1.32 (m, 5H), 0.96e0.89 (m, 3H).
4.1.19. N-(4-(4-(heptyloxy)naphthalen-1-yl)-1-hydroxy-2-
(hydroxymethyl)butan-2-yl)hexanamide (15)
13C NMR (150 MHz, MeOD)
d 156.3, 153.3, 137.1, 132.6, 130.1, 128.1,
The title compound was prepared using general method B.
Waxy solid. 1H NMR (600 MHz, MeOD)
d
8.25 (dd, J ¼ 8.4, 1.4 Hz,
127.6, 127.5, 126.0, 125.7, 125.5, 124.1, 123.6, 122.0, 104.0, 67.7, 65.8,
62.5, 59.6, 53.4, 32.1, 31.6, 29.1, 28.9, 26.1, 25.9, 22.3, 13.1. HRMS
(ESI) [M þ H]þ calcd for C30H40NO5: 494.2901; found: 494.2903.
1H), 8.07 (dd, J ¼ 8.3, 1.0 Hz, 1H), 7.53e7.47 (m, 1H), 7.46e7.41 (m,
1H), 7.24 (d, J ¼ 7.8 Hz,1H), 6.79 (d, J ¼ 7.8 Hz,1H), 4.12 (t, J ¼ 6.3 Hz,
2H), 3.86 (d, J ¼ 11.1 Hz, 2H), 3.76 (d, J ¼ 11.1 Hz, 2H), 3.03e2.96 (m,
2H), 2.32e2.27 (m, 2H), 2.14e2.08 (m, 2H), 1.96e1.88 (m, 2H),
1.72e1.65 (m, 2H), 1.63e1.57 (m, 2H), 1.48e1.42 (m, 2H), 1.40e1.33
4.1.24. (E)-N-(4-(4-(heptyloxy)naphthalen-1-yl)-1-hydroxy-2-
(hydroxymethyl)butan-2-yl)but-2-enamide (20)
(m, 8H), 0.95e0.91 (m, 6H). 13C NMR (150 MHz, MeOD)
d 175.6,
153.4,132.6, 130.0, 126.0, 125.8, 125.5, 124.1, 123.5, 122.0, 104.0, 67.7,
62.8, 61.1, 36.3, 32.1, 31.6, 31.3, 29.1, 28.9, 26.1, 26.1, 25.6, 22.3, 22.1,
13.0, 12.9. HRMS (ESI) [M þ H]þ calcd for C28H44NO4: 458.3265;
found: 458.3270.
The title compound was prepared using general method B.
White solid. 94e96 ꢁC. 1H NMR (600 MHz, MeOD)
d
8.24 (dd, J ¼ 8.4,
1.3 Hz, 1H), 8.10e8.04 (m, 1H), 7.51e7.46 (m, 1H), 7.46e7.41 (m, 1H),
7.23 (d, J ¼ 7.8 Hz, 1H), 6.86e6.79 (m, 1H), 6.77 (d, J ¼ 7.8 Hz, 1H),
6.14e6.09 (m, 1H), 4.11 (t, J ¼ 6.3 Hz, 2H), 3.88 (d, J ¼ 11.2 Hz, 2H),
3.79 (d, J ¼ 11.2 Hz, 2H), 3.01e2.95 (m, 2H), 2.16e2.09 (m, 2H),
1.95e1.87 (m, 5H), 1.62e1.55 (m, 2H), 1.47e1.41 (m, 2H), 1.39e1.34
4.1.20. N-(4-(4-(heptyloxy)naphthalen-1-yl)-1-hydroxy-2-
(hydroxymethyl)butan-2-yl)octanamide (16)
(m, 4H), 0.97e0.88 (m, 3H). 13C NMR (150 MHz, MeOD)
d 167.7,
The title compound was prepared using general method B.
153.4, 139.4, 132.6, 130.0, 126.0, 125.8, 125.5, 125.2, 124.1, 123.5,
122.0, 104.0, 67.7, 62.9, 61.3, 32.3, 31.6, 29.1, 28.9, 26.1, 26.1, 22.3,
16.4, 13.1. HRMS (ESI) [M þ H]þ calcd for C26H38NO4: 428.2801;
found: 428.2742.
Waxy solid. 1H NMR (400 MHz, MeOD)
d
8.26 (d, J ¼ 8.4 Hz, 1H),
8.08 (d, J ¼ 8.4 Hz,1H), 7.54e7.48 (m,1H), 7.46e7.42 (m,1H), 7.25 (d,
J ¼ 7.8 Hz,1H), 6.80 (d, J ¼ 7.9 Hz,1H), 4.14 (t, J ¼ 6.2 Hz, 2H), 3.87 (d,
J ¼ 11.1 Hz, 2H), 3.76 (d, J ¼ 11.1 Hz, 2H), 3.04e2.96 (m, 2H), 2.30 (t,
J ¼ 7.5 Hz, 2H), 2.16e2.08 (m, 2H), 1.97e1.88 (m, 2H), 1.69e1.58 (m,
4H), 1.50e1.43 (m, 2H), 1.38e1.29 (m, 12H), 0.95e0.86 (m, 6H). 13
C
4.2. Biology
NMR (125 MHz, MeOD)
d 174.6, 152.4, 131.6, 129.0, 125.0, 124.7,
124.4, 123.1, 122.5, 121.0, 103.0, 66.7, 61.9, 60.1, 35.3, 31.1, 30.6, 30.5,
28.0, 28.0, 27.8, 27.8, 25.1, 25.0, 24.8, 21.3, 21.2, 12.0, 12.0. HRMS
(ESI) [M þ H]þ calcd for C30H48NO4: 486.3578; found: 486.3582.
4.2.1. Cell culture
HT-29, LOVO, SW620, HCT-116, COLO-678, T47D, NCIeH441 and
T-84 cells were purchased from ATCC. HCT-8, COLO-205, SW480,
SNU-878 and Mino cells were obtained from the cell bank of the
Chinese Academy of Sciences. Caco2 cells were obtained from
Ocean University of China. PLC/PRF-5 cells were purchased from
CrownBio. NUGC-3 cells were purchased from Japanese Collection
of Research Bioresources Cell Bank (JCRB). JIMT-1 and L-428 cells
were purchased from German Collection of Microorganisms and
Cell Cultures (DSMZ). HCC827 were kindly gifted by Dr. Pasi A.
4.1.21. Butyl (4-(4-(heptyloxy)naphthalen-1-yl)-1-hydroxy-2-
(hydroxymethyl)butan-2-yl)carbamate (17)
The title compound was prepared using general method B.
Waxy solid. 1H NMR (400 MHz, MeOD)
d
8.26 (d, J ¼ 8.6 Hz, 1H),
8.07 (d, J ¼ 8.5 Hz, 1H), 7.50 (t, J ¼ 7.6 Hz, 1H), 7.44 (t, J ¼ 7.5 Hz, 1H),
7.24 (d, J ¼ 7.8 Hz,1H), 6.79 (d, J ¼ 7.8 Hz, 1H), 4.13 (t, J ¼ 6.3 Hz, 2H),
4.08 (t, J ¼ 6.6 Hz, 2H), 3.84 (d, J ¼ 11.1 Hz, 2H), 3.73 (d, J ¼ 11.1 Hz,
2H), 3.06e2.94 (m, 2H), 2.11e2.00 (m, 2H), 1.97e1.87 (m, 2H),
1.68e1.55 (m, 4H), 1.51e1.41 (m, 4H), 1.41e1.29 (m, 6H), 0.95e0.86
€
Janne (Dana-Farber Cancer Institute, Boston, MA). HGC-27 cells
were purchased from Riken Bioresource Center (RBRC). HCT-8,
COLO-678, COLO-205, T47D, NUGC-3 and SNU878 cells were
cultured in RPMI-1640 Medium supplemented with 10% fetal
bovine serum (FBS) and 1% penicillin/streptomycin. HT-29 and
HCT-116 cells were cultured in McCoy’s 5A Medium supplemented
with 10% FBS and 1% penicillin/streptomycin. Lovo cells were
cultured in Ham’s Fe12K (Kaighn’s) Medium supplemented with
10% FBS and 1% penicillin/streptomycin. T-84 cells were cultured in
DMEM/F-12 Medium with 5% FBS and 1% penicillin/streptomycin.
SW620 and SW480 cells were cultured in Leibovitz’s L-15 Medium
supplemented with 10% FBS and 1% penicillin/streptomycin. Caco2,
PLC/PRF-5 and JIMT-1 cells were cultured in DMEM Medium with
10% FBS and 1% penicillin/streptomycin. L-428 and Mino cells were
cultured in RPMI-1640 Medium supplemented with 15% inacti-
vated FBS and 1% penicillin/streptomycin. HCC827 and
NCIeH441 cells were cultured in RPMI-1640 Medium supple-
mented with 2.5 g/L glucose, 10% FBS and 1% penicillin/strepto-
mycin. HGC-27 cells were cultured in MEM Medium supplemented
with 10% FBS and 1% penicillin/streptomycin. Cells were cultured at
37 ꢁC in a humidified atmosphere containing 5% CO2 and 95% air,
except SW620 and SW480 cultured in 37 ꢁC air incubators.
(m, 6H). 13C NMR (125 MHz, MeOD)
d 152.3, 131.6, 129.1, 125.0,
124.7, 124.5, 123.1, 122.5,121.0, 103.1, 66.7, 61.7, 58.4, 31.3, 30.6, 29.9,
28.0, 27.8, 25.0, 25.0, 21.3, 17.8, 12.0, 11.7. HRMS (ESI) [M þ H]þ calcd
for C27H42NO5: 460.3057; found: 460.3061.
4.1.22. Tert-butyl (4-(4-(heptyloxy)naphthalen-1-yl)-1-hydroxy-2-
(hydroxymethyl)butan-2-yl)carbamate (18)
The title compound was prepared using general method B.
White solid. Mp: 92e94 ꢁC. 1H NMR (400 MHz, MeOD)
d 8.26 (d,
J ¼ 8.2 Hz, 1H), 8.10 (d, J ¼ 8.3 Hz, 1H), 7.53e7.40 (m, 2H), 7.24 (d,
J ¼ 7.8 Hz,1H), 6.77 (d, J ¼ 7.8 Hz,1H), 4.10 (t, J ¼ 6.4 Hz, 2H), 3.81 (d,
J ¼ 11.0 Hz, 2H), 3.70 (d, J ¼ 11.0 Hz, 2H), 3.06e2.96 (m, 2H),
2.08e2.00 (m, 2H), 1.96e1.84 (m, 2H), 1.63e1.55 (m, 2H), 1.52 (s,
9H), 1.47e1.33 (m, 6H), 0.97e0.89 (m, 3H). 13C NMR (125 MHz,
MeOD)
d 155.3, 152.3, 131.6, 129.2, 125.0, 124.7, 124.6, 123.1, 122.5,
121.0, 103.1, 66.7, 62.1, 58.2, 31.4, 30.6, 28.0, 27.8, 26.4, 25.1, 25.0,
21.3, 12.0. HRMS (ESI) [M þ H]þ calcd for C27H42NO5: 460.3057;
found: 460.3060.
11

F. Gao, X. Chen, J. Lu et al.
European Journal of Medicinal Chemistry 215 (2021) 113274
4.2.2. Cell proliferation assay
4.2.8. Animal studies
The inhibitory effects of 12, MK2206, and the combination of 12
and MK2206 on cell growth were determined by using the sulfo-
rhodamine B assay (SRB), and the inhibitory effects of compound 12
on lymphoma cell growth were determined by Cell Counting Kit-8
(CCK8). Each experiment was performed in quadruplicate and
repeated at least three times. Synergy between 12 and MK2206 was
determined by the combination index using CompuSyn (Chou-
Talalay method).
Animal experiments were performed according to the institu-
tional ethical guidelines on animal care and approved by the
Institute Animal Care and Use Committee at Shanghai Institute of
Materia Medica.
Female BALB/c nude mice were used when they were 4- to 6-
weeks old. HT-29 cells (4 ꢂ 106) were injected subcutaneous into
the armpit of each nude mice. Once the tumors grew to the volume
of about 100 mm3, the mice were randomly divided into 4 groups
(n ¼ 10): Vehicle group (30% PEG300), 12 (100 mg/kg/d, IP),
MK2206 (50 mg/kg/Q2D, PO), and 12 plus MK2206. Tumor di-
ameters and body weight were recorded with an electronic caliper
every 3 days and tumor volume was calculated with the following
formula: tumor volume (mm3) ¼ 0.5 ꢂ length (mm) ꢂ width (mm)
[2].
4.2.3. Apoptosis assay
HT-29 cells (2 ꢂ 105) were seeded in a 12-well plate and then
treated with vehicle (0.1% DMSO), 12 (15 mM), MK2206 (7.5 mM), 12
(15 mM) plus MK2206 (7.5 mM). After treatment, cells were digested
by trypsin and washed with cold PBS for twice. Cells were resus-
pended in binding buffer and incubated with propidium iodide and
Annexin V-FITC for 10 min at room temperature. The proportion of
apoptosis were analyzed on FACScan flow cytometer (Becton
Dickinson).
4.2.9. Statistical analysis
Comparisons among three or more groups were carried out by
One-Way ANOVA using SPSS 19.0 software. A p value of <0.01 was
considered statistically significant.
4.2.4. ROS measurement
HT-29 cells (4 ꢂ 105) were seeded in a 6-well plate and then
Declaration of competing interest
treated with vehicle (0.1% DMSO), 12 (20 mM), MK2206 (10 mM), 12
(20 mM) plus MK2206 (10 mM), H2O2 (10 mM). In addition to the
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
treatment of H2O2 for 1h, the other groups were treated for 16 h.
ROS was determined by Reactive Oxygen Species Assay Kit (Beyo-
time) according to manufacturer’s instructions, and measured by a
FACScan flow cytometry (Becton Dickinson).
Acknowledgements
4.2.5. Cell cycle assay
We gratefully acknowledge the National Natural Science Foun-
dation of China (91953108, 81620108027, 21632008, and
21877118), and the “Personalized MedicinesddMolecular
Signature-based Drug Discovery and Development”, Strategic Pri-
ority Research Program of the Chinese Academy of Sciences, Grant
No. XDA12020341, and sponsored by Shanghai Pujiang Program
(18PJD052).
HT-29 cells (4 ꢂ 105) were seeded in a 6-well plate and then
treated with vehicle (0.1% DMSO), 12 (20 mM), MK2206 (10 mM), 12
(20 mM) plus MK2206 (10 mM). After treatment, cells were digested
by trypsinization, washed twice in cold PBS, fixed with ice cold 70%
methanol, after that cells were incubated at ꢀ20 ꢁC overnight. Cells
were then washed with PBS and incubated with 25 mg/mL propi-
dium iodide containing 30 mg/mL ribonuclease for 30 min at room
temperature. Cells were analyzed on FACScan flow cytometer
(Becton Dickinson).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113274.
4.2.6. Western blot analysis
Antibodies against AKT (#4691), p-AKT (Ser473, #4060), PARP
(#9542), cleaved-caspase 3 (#9661), P21 (#2947), c-Myc (#5605),
CDK2 (#2546), CDK4 (#12790), CDK6 (#3136), Cyclin A (#4656),
Cyclin D1 (#2922), and Cyclin E1 (#20808) were obtained from Cell
Signalling Technology. The secondary antibodies were obtained
from Jackson ImmunoResearch (#111-035-003). HT-29 cells were
washed with PBS and total protein was extracted with lysis buffer
(1 ꢂ loading buffer containing 2%SDS, 0.01% bromophenol blue,
50 mM Tris-HCL, 10% glycerol and 100 mM DTT). Equal amount of
total protein was submitted to SDS-PAGE on 10% or 12.5% poly-
acrylamide gel. After transfer onto nitrocellulose membranes,
samples were blocked for 1 h with 3% BSA and incubated with the
primary antibodies overnight at 4 ꢁC. After incubated with sec-
ondary antibodies for 1 h, bands were developed with enhanced
chemiluminescence (ECL Plus; Pierce) using GE LMAGE QUANT
LAS4.
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