Design, synthesis and biological evaluation of new pyrrolidine carboxamide analogues as potential chemotherapeutic agents for hepatocellular carcinoma

Article abstract of DOI:10.1016/j.ejmech.2017.08.054

The successful targeting of different malignancies by OSU-2S, encouraged us to design and synthesize a novel series of pyrrolidine aryl carboxamide derivatives. In this context, we found that, the amide nature and tether length were found to be key determinant elements for the anticancer activity of these new and rigid analogues of OSU-2S. The most effective analogues induced apoptosis in cancer cells by a similar mechanism to that of OSU-2S, possibly via the activation of PKCδ in addition to their ability to induce cell cycle arrest and inhibition of cancer cell migration. Compound 10m, possesses anticancer potency comparable to that of OSU-2S when tested against cancer cell lines under study, and was found to be safer on normal cells. Furthermore, compound 10m, was found to be about 2-folds more potent than the anticancer drug Sorafenib in hepatocellular carcinoma (HCC). The newly developed compounds represent a therapeutically promising approach for the treatment of HCC.

Full text of DOI:10.1016/j.ejmech.2017.08.054

Accepted Manuscript
Design, synthesis and biological evaluation of new pyrrolidine carboxamide
analogues as potential chemotherapeutic agents for hepatocellular carcinoma
Hany A. Omar, Dana M. Zaher, Vunnam Srinivasulu, Fatema Hersi, Hamadeh Tarazi,
Taleb H. Al-Tel
PII:
S0223-5234(17)30665-7
10.1016/j.ejmech.2017.08.054
EJMECH 9698
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 July 2017
Revised Date: 22 August 2017
Accepted Date: 23 August 2017
Please cite this article as: H.A. Omar, D.M. Zaher, V. Srinivasulu, F. Hersi, H. Tarazi, T.H. Al-Tel,
Design, synthesis and biological evaluation of new pyrrolidine carboxamide analogues as potential
chemotherapeutic agents for hepatocellular carcinoma, European Journal of Medicinal Chemistry
(2017), doi: 10.1016/j.ejmech.2017.08.054.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Design, Synthesis and Biological Evaluation of New Pyrrolidine Carboxamide Analogues as
Potential Chemotherapeutic Agents for Hepatocellular Carcinoma
a,b,c
a
a
a
a, b
Hany A. Omar, Dana M. Zaher, Vunnam Srinivasulu, Fatema Hersi, Hamadeh Tarazi,
a,b,*
and Taleb H. Al-Tel
a
Sharjah Institute for Medical Research, University of Sharjah, Sharjah 27272, United Arab Emirates
b
College of Pharmacy, University of Sharjah, Sharjah 27272, United Arab Emirates
c
Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of new pyrrolidine carboxamide analogues as
potential chemotherapeutic agents for hepatocellular carcinoma
a, b, c
a
a
a
a, b
Hany A. Omar,
Dana M. Zaher, Vunnam Srinivasulu, Fatema Hersi, Hamadeh Tarazi,
a, b, *
Taleb H. Al-Tel
a
Sharjah Institute for Medical Research, University of Sharjah, Sharjah 27272, United Arab
Emirates
b
College of Pharmacy, University of Sharjah, Sharjah 27272, United Arab Emirates
c
Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt
*
To whom correspondence should be addressed: Tel.: +97165057417, Fax: +97165585812,
Email: taltal@sharjah.ac.ae (T.H.A.)
1

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ABSTRACT
The successful targeting of different malignancies by OSU-2S, encouraged us to design and
synthesize a novel series of pyrrolidine aryl carboxamide derivatives. In this context, we found
that, the amide nature and tether length were found to be key determinant elements for the
anticancer activity of these new and rigid analogues of OSU-2S. The most effective analogues
induced apoptosis in cancer cells by a similar mechanism to that of OSU-2S, possibly via the
activation of PKCδ in addition to their ability to induce cell cycle arrest and inhibition of cancer
cell migration. Compound 10m, possesses anticancer potency comparable to that of OSU-2S
when tested against cancer cell lines under study, and was found to be safer on normal cells.
Furthermore, compound 10m, was found to be about 2-folds more potent than the anticancer
drug Sorafenib in hepatocellular carcinoma (HCC). The newly developed compounds represent
a therapeutically promising approach for the treatment of HCC.
Keywords: Anticancer, OSU-2S
,
Pyrrolidine carboxamide, Hepatocellular carcinoma, PKCδ
2

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1
.
Introduction
Hepatocellular carcinoma (HCC) accounts for most liver cancer and is a leading cause of cancer
related death worldwide [1, 2]. Furthermore, HCC is associated with a wide range of risk factors
that increase its incidence as cirrhosis, which is the major risk factor caused by chronic hepatitis
B, hepatitis C, alcohol abuse or nonalcoholic fatty liver diseases [3]. Treatment of HCC is quite
challenging due to its complexity, heterogeneity and the several complications associated with it
as neutropenia, thrombocytopenia and ascites with intravascular hypovolemia.
Sorafenib is an anti-angiogenic and anti-proliferative drug emerged to provide a
treatment option for patients with advanced unresectable HCC [4]. Sorafenib is a tyrosine kinase
inhibitor, that targets tyrosine kinases of pathways involved in the molecular pathogenesis of
HCC as RAF kinases, vascular endothelial growth factor receptor (VEGFR-1, VEGFR-3) and
platelet-derived growth factor receptor-β (PDGFR-β), thus, it is recommended as the first-line
therapy for advanced HCC patients [5]. However, sorafenib promotes overall survival by only
about 3 months and it may lead to resistance in many patients through increased expression of
ATP binding cassette proteins [6], epidermal growth factor receptor and HER-3 [1]. Therefore,
the low efficacy of sorafenib raised the need for new agents targeting HCC.
On the other side, FTY720 which is a synthetic sphingosine immunosuppressant derived from a
fungal metabolite was FDA-approved for treating relapsing multiple sclerosis through targeting
sphingosine-1-phosphate (S1P) receptors [7, 8]. Furthermore, FTY720 was reported to have
antitumor effects against multiple types of cancers such as those of bladder, breast, ovarian,
colorectal, blood and liver cancers [9, 10]. Different studies showed that FTY720 mediates
caspase-3 apoptotic death in hepatocellular carcinoma cells through the activation of the reactive
oxygen species (ROS)/protein kinase C delta (PKCδ) signaling without any effects on S1P
receptors [11, 12]. To overcome the immunosuppressive effect of FTY720 associated with its
antitumor activity, OSU-2S, a non-immunosuppressive FTY720 analogue was developed [13-
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1
6]. OSU-2S demonstrated high in vitro antiproliferative efficacy compared to FTY720 against
HCC cells [12]. In addition, OSU-2S maintained the ability of FTY720 to induce PKCδ
cleavage and caspase dependent apoptosis [13, 14]. PKCδ is a phospholipid- and diacylglycerol
(DAG)-dependent serine/threonine-protein kinase that belongs to the PKC subfamily [17]. The
anti-tumor activity of OSU-2S through PKCδ cleavage represents a potential strategy for
targeting several cancer types possessing high expression levels of PKCδ, such as colorectal and
lung cancers [18]. Many analogues of FTY720 as potential new anticancer agents were reported
in the literature. Elegant contributions from Hanessian group, described the synthesis and
biological evaluations of constrained pyrrolidine analogs that were found to be potent against
leukemia cell lines [19, 20].
In continuation of our efforts directed toward the design and synthesis of new lead drug
candidates [21-25], we report herein the synthesis and biological evaluations of novel and
enantiomerically pure pyrrolidine-2-carboxamide derivatives. The ultimate goal of our
initiatives is to investigate the anticancer activity and mechanism of action of the newly
developed motifs as a potential therapeutic option for the treatment of HCC.
2
. RESULTS AND DISCUSSION
2
.1. Chemistry
Our synthetic plan was commenced as contemplated in Scheme 1. We first assembled the (4-
oct-1-yn-1-yl)phenyl)methanol (3) scaffold using Sonogashira coupling between (4-
iodophenyl)methanol (1) and oct-1-yne (2) in the presence of Pd(PPh ) Cl and CuI in
(
3
2
2
triethylamine at room temperature to produce the desired compound in 73% yield after column
chromatography. Next, bromination of 3 was achieved using PBr in DCM to deliver 1-
3
(bromomethyl)-4-(oct-1-yn-1-yl) benzene (4). Due to the chemical instability of the latter
compound, it was used in the next step without further purification. Thus, the crude product 4
4

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was reacted with 1-(tert-butyl) 2-methyl (2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate (5)
using NaH in DMF to produce 1-(tert-butyl) 2-methyl (2S,4R)-4-((4-(oct-1-yn-1-
yl)benzyl)oxy)pyrrolidine-1,2-dicarboxylate (6) in 85% yield. Hydrolysis of compound 6 with
2
.5 equivalents of LiOH yielded quantitatively and high purity, the key intermediate (2S,4R)-1-
(tert-butoxycarbonyl)-4-((4-(oct-1-yn-1-yl)benzyl)oxy)pyrrolidine-2-carboxylic acid (7). With
the latter key building block in hand, it allows for diverse structure-activity relationship (SAR)
explorations through coupling with different aryl amines. Thus, the preparation of compounds
needed to delineate the structure activity relationship (SAR) for this study was proceeded as
described in Scheme 1 (vide infra). Thus, N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-
yl)uronium hexafluorophosphate
(HBTU) mediated coupling between acid 7 and various amines
8
a-s using diisopropylamine (DIPEA) in DMF was found to be the most efficient procedure to
produce the corresponding N-boc-prolinamide compounds 9a-s. As an example, the
1
characteristic peaks resonated at 4.61–4.03 ppm in the H NMR spectrum of compound 9a,
unambiguously confirm its structure. For the deprotection of the BOC group, compounds 9a-s
were subjected to excess amount of trifluroacetic acid (TFA) in DCM to produce the desired
target compounds 10a-s in good overall yields (32-81%). The structural elucidation of
compounds 10a-s was unambiguously confirmed from their NMR and Q-TOF high-resolution
mass spectrometer LC-MS spectra.
5

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Br
OH
OH
PBr , DCM
Pd(PPh ) Cl , CuI
TEA, rt, 12 h, 76%
3
3
2
2
0 oC to rt, 1 h
I
1
2
3
4
HO
O
O
o
NaH, DMF, 0 C
1
1
1
1
1
1
0a: R = H, n = 1
10g: R1 = 3-OMe, n = 1
10h: R1 = 3-Cl, n = 1
0c: R = 4-OMe, n = 1 10i: R1 = H, n = 2
1
N
to rt, 12 h, 85%
0b: R = 4-F, n = 1
1
Boc
1
1
0j: R = 4-OMe, n = 2
5
0d: R = 4-Cl, n = 1
1
n
1
R1
O
1
0k: R = H, n = 3
O
0e: R = 4-CF , n = 1
1
1
3
0f: R = 3-F, n = 1
1
O
N
Boc
N
R=
N
N
H
N
N
H
1
0n
10o
1
0l
10m
H
N
O
N
6
HN
O
N
N
H
1
0p
10q
H
10r
LiOH, THF:water:
1
0s
o
MeOH (3:1:1), 0 C
to rt, 8 h, 91%
R
R
NH
NH
O
O
OH
O
NH2
O
NH
TFA, DCM
R
O
N
Boc
0 oC to rt, 3 h
(8a-s)
O
N
Boc
3
2 to 81%
HBTU, DIPEA,
o
DMF, 0 C to rt, 12 h
1
0a-s
9a-s
7
Scheme 1. Synthesis of OSU-2S based pyrrolidine-2-carboxamides derivatives 10a-s.
.2.Biological evaluation
.2.1. In Vitro biological evaluations and SAR Analysis
2
2
Preliminary 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell-based
screening assay of the synthesized compounds 10a-s was performed to evaluate their anticancer
activities against a panel of human cancer cell lines, which included HepG2 (liver), Hep3B
(liver), A-549 (lung) and HT-29 (colon) cell lines. The results, expressed as IC50 values, are
6

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presented in Table 1 using doxorubicin, OSU-2S and sorafenib as positive controls.
Accordingly, most of the synthesized compounds 10a-s, exhibited appreciable cytotoxicity
against cancer cells, with IC values ranging from 2.0 µM to 20 µM. The SAR analysis was
5
0
started by first synthesizing a series of substituted benzamide derivatives, 10a-h. Unfortunately,
none of these analogues exhibited the desired anticancer activities with IC50 values of
approximately 7 µM. However, the 4-methoxy benzyl amide derivative 10c showed promising
activity against four cancer cell lines compared to derivatives that possesses 4-fluoro, 4-chloro,
4
-trifluromethyl, 3-methoxy, 3-fluoro and 3-chloro substituents (compounds 10a-h). To test the
effect of the amide tether length on the activity profile, we prepared phenethyl and phenyl
propyl amide congeners 10i, 10j and 10k. These derivatives indicated no pronounced
improvement of the anticancer activity compared to 10c. To further improve the anticancer
activity of this series, we next turned our attention to replace the aromatic ring of the amide
appendage with heteroaryl moieties. Therefore, compounds 10l-s were prepared and found to
have considerable improvement of the anticancer activities. The first task in gaining access to
these scaffolds was started by the synthesis of the pyridyl derivative 10l, however, no
appreciable enhancement of the anticancer activity was noticed compared to compound 10c.
Therefore, we then synthesized the indolyl bearing analogue 10m. Interestingly, compound 10m
with 2-indolyl methyl amid appendage not only showed potent anticancer activities against the
four cancer cell lines under study (Fig. 1A), but also was two-folds safer than OSU-2S when
tested against F180 normal cell line (Table 1). Noteworthy, compound 10m was found to be
two-folds more potent compared to the anticancer drug Sorafenib. Based on the improved
potency of compound 10m, we envisaged to study the effect of the heteroaryl type as well as the
tether length on the anticancer activity. Therefore, derivatives 10n-s with variable heterocyclic
systems and tether lengths were synthesized. Thus, substituting the indolyl nitrogen with a
methyl group, compound 10n, led to a slight decrease in the anticancer activity against HepG2,
He3B and HT29, while the activity against A549 was lost. Furthermore, including extra nitrogen
7

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in the heteroaryl system, exemplified in the benzimidazole derivative 10o, indicated no
improvement of the anticancer activity compared to 10m. On the other hand, amide linkers
bearing monocyclic systems, compounds 10p and 10q, indicated no enhancement of the
anticancer activities. Moreover, increasing the amide tether length to two carbons, e.g. 10r and
1
0s, led to loss of the anticancer activity compared to 10m. Based on the above results,
compounds 10c, 10l, 10m, 10n, 10o, 10p and 10q were selected for further validation of the
anticancer activities of these constrained analogues.
The first step of these initiatives was to test the safety profile of these derivatives on
normal cells. In this context, normal fibroblast cells (F180) were found to be more viable when
treated with different concentrations of compounds 10c, 10m and 10n compared to OSU-2S.
The appreciable safety profile of these compounds toward normal fibroblasts might be attributed
to their specific effect on PKCδ protein. To further test the validity of these findings, Western
blotting and molecular modeling studies of the new compounds on PKCδ, Akt and ERK were
carried out as described below.
Table 1. Cytotoxicity of compounds 10a-s against human cancer and normal cell lines.
a
IC (µM)
5
0
Compound
HepG2
Hep3B
A549
HT29
F180
NT
1
0a
0b
0c
0d
0e
0f
0g
7.9±0.9 9.7±2.9
8.7±2.3 7.9±1.1
9.3±2.5 7.8±2.1
19.3±2.5 18.6±2.5
1
13.9±2.7
8.6±1.4
9.4±1.7
8.2±1.6
>20
NT
1
>40
NT
1
>20
9.5±1.9
17.9±2.6
1
7.9±1.1 8.5±2.3
8.3±1.2 9.3±1.3
8.9±1.9 8.5±2.7
7.3±1.4 8.3±2.4
7.4±0.7 8.1±1.6
12.2±2.1 19.5±2.9
15.4±2.4 >20
13.4±1.6 19.3±3.1
NT
1
NT
1
NT
1
0h
15.3±2.3
19.6±3.4
15.1±3.1
18.1±3.7
10.3±1.7
4.9±0.9
>20
>20
>20
NT
1
0i
NT
1
0j
>20
>20
9.1±1.6
9.7±1.7
>20
NT
1
0k
0l
0m
0n
>20
NT
1
7.2±2.1 9.7±1.4
4.1±0.3 2.1±0.4
6.2±0.9 4.8±0.7
9.6±2.4
3.7±0.9
5.8±1.1
26.7± 1.9
>40
>40
1
1
8

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1
0o
13.9±1.4 9.9±1.2
5.8±1.1 5.9±0.7
>20
>20
NT
1
1
0p
0q
11.6±1.0 10.1±0.8 37.2± 3.2
11.8±2.3
6.1±0.91
6.4±0.74
7.2
.8±1.0
2
>20
9.9
NT
NT
7
1
0r
0s
11.6±0.97
NT
1
>20
>20
NT
NT
NT
NT
Dox
4.4±1.3 4.5±0.5
2.3±0.5 2.8±0.8
6.2±1.7 9.4±1.2
0.7±0.1
4.2±0.9
6.7±0.8
OSU-2S
Sorafenib
2.7±0.7
5.9±1.2
18.6± 2.1
>40
a
Half-maximal inhibitory concentration (IC ) values are the mean ± SD of
5
0
three individual experiments performed in 6 replicates. Dox = Doxorubicin,
OSU-2S and sorafenib were used as positive controls. NT = Not tested.
2
.2.2. In vitro validation of the anticancer effects of the new compounds
To explore whether the synthesized compounds maintain the ability to activate caspases 3 and 7
13], the effect of the most potent compounds 10c, 10l, 10m, 10n, 10o, 10p and 10q on these
[
executioner caspases in Hep3B cells was tested. OSU-2S and sorafenib were used as positive
controls. Accordingly, the tested compounds significantly induced the activity of caspases 3 and
7
, which was observed after 24 and 48 h of treatment (Fig. 1B). The activation of caspases by
the new compounds highlighted the involvement of apoptosis as a possible mechanism of action
for cancer cell death.
2
.2.3. Studies of the best hits on the kinases PCKδ, Akt and ERK
According to many reports, OSU-2S induces cancer cell apoptosis via the activation of the
proteolytic cleavage of PKCδ in addition to its ability to alter the expression and functional
status of major cell signaling proteins such as Akt and ERK1/2 [13]. Therefore, to validate the
anticancer activity of our new motifs, we examined the expression of PKCδ, Akt, phospho-Akt
(Ser473), phospho-Akt (Thr 308), p44/42 MAPK (ERK1/2) and phospho-p44/42 MAPK
(ERK1/2) proteins by Western blot analysis after treatment with compounds 10c, 10l, 10m, 10n,
1
0o, 10p or 10q. Interestingly, compound 10m showed the same activity profile comparable to
9

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that of OSU-2S in HepG2 (Fig. 1C) and Hep3B (Fig. 1D) cells including PKCδ cleavage in
addition to partial inhibition of Akt and ERK 1/2 phosphorylation.
Fig. 1. Evidence of the anticancer mechanism of action of the most potent compounds. (A) Dose
response curve showing the effect of 10m on cancer cell viability in HepG2, Hep3B, A549 and
HT-29 cells after 48 hours of treatment. (B) The activity of caspase 3 and caspase 7 after 24 and
4
8 h of treatment with 10c, 10l, 10m, 10n, 10o, 10p, or 10q. OSU-2S and Sorafenib were used
as the positive controls. The data represent mean ± SD (n=3). *P<0.05, **P<0.01, ***P<0.001,
one-way ANOVA. (C) and (D) Western blots of the cell lysates of Hep3B and HepG2 cells
respectively, treated with OSU-2S, 10c, 10l, 10m, 10n, 10o, 10p, or 10q. The cells were
immunoblotted for PKCδ, Akt, phospho-Akt (Ser473), phospho-Akt (Thr 308), p44/42 MAPK
(ERK1/2) and phospho- p44/42 MAPK (ERK1/2).
As reported, PKCδ has a transcriptional control on cyclin D1, which may interfere with
it’s expression and promotes G1/S cell cycle arrest [17, 26]. In addition, several findings have
validated the role of p53 tumor suppressor gene in G1 checkpoint [27, 28]. Based on these
findings, the effect of the new compounds on cell cycle checkpoints was explored. Propiduim
iodide staining of HCC cells treated with OSU-2S, 10c, 10l, 10m, 10n, 10o, 10p or 10q showed
cell cycle arrest at G1 phase (Fig. 2A and 2B). The highest effect on cell cycle distribution was
1
0

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observed after treatment with 10l compound. The activation of PKCδ by these new compounds
caused a significant cell cycle arrest only in HepG2 cells, which have a wild type p53 [29, 30].
The cell cycle arrest was not observed in Hep3B cells which are p53-deficient.[29] These results
suggested that the cell cycle arrest induced by PKCδ activation is dependent on the p53 status of
the cancer cells. Furthermore, the role of cyclin D1 in the induced cell cycle arrest at G1 phase
was confirmed by immunoblotting of cyclin D1 in HepG2 cells after treatment with compounds
1
0c, 10l, 10m, 10n, 10o, 10p and 10q. A decrease in the cyclin D1 expression was observed
after the treatment with the most potent compounds. However, no significant change was
observed in the expression of CDK6, cyclin D3 and p21 Waf1/Cip1 proteins (Fig. 2C). These
results might indicate the involvement of cyclin D1 downregulation by the new compounds-
induced cell cycle modulation.
2
.3.4. Wound healing assay
Liver cancer cells exhibit high migration rates, which increases the severity and aggressiveness
of cancer [31]. In this study, the effect of the new compounds on the migration ability of HepG2
was examined using the wound healing migration assay. The treatment of HCC cells with 10c,
1
0n, 10m, and 10p for 24h reduced cancer cell migration compared to control cells (Fig. 2D and
E). Compound 10c was found to possess the highest anti-migratory activity among the tested
2
compounds. Such activities highlight the clinical promise of our new compounds in targeting
cancer cells metastasis and thereby decreasing the severity and aggressiveness of HCC.
1
1

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Fig. 2. Effect of the new compounds on cell cycle distribution and migration of HepG2 cells.
(A) Representative histograms of the cell cycle distributions of HepG2 cells after 24h of the
indicated treatments. The cells were stained with PI and analyzed by flow cytometry. (B) The
percentages of cells in sub-G1, G0/G1, S and G2/M phases analyzed using FlowJo V.10
software. (C) Western blot analysis showing the effect of the tested compounds on the
expression of the indicated cell cycle regulating proteins (cyclin D1, CDK6, cyclin D3, p21
Waf1/Cip1) in HepG2 cells after treatment with test compounds. (D) Wound healing migration
assay of HepG2 cells after 24 h of treatment with the indicated compounds. (E) Bar chart
presenting the relative wound recovery of HepG2 cells after 24h treatment compared to the
control.
1
2

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2
.3.
Molecular Modeling
Molecular modeling was used to investigate the possible binding modes of the active
compounds in PKCδ, ERK and Akt sites. The models were derived by energy minimization of
reported PDB structures. OSU-2S and its analogues are known to target PKCδ, which in turn
affects the extracellular signal-regulated kinases ERK and Akt [13]. Therefore, the synthesized
derivatives 10a-s showed in vitro cytotoxicity against various cancer cell lines and appreciable
cleavage of PKCδ through inhibition of ERK/AKT. In this context, we were interested in
forecasting the probable binding pattern of the most potent compounds 10m, 10n and 10p
against the protein targets involved. To achieve this goal, a homology model for PKCδ kinase
was developed (Figure 3). Accordingly, we carried out in silico screening of the active
compounds for their best docking poses within the binding sites of PKCδ kinase homology
model (blue), Akt-1 catalytic and allosteric sites (purple), Akt-2 catalytic site (magenta), ERK-1
catalytic site (brown) and ERK-2 catalytic site (yellow) (Fig. 3). The obtained docking scores
were expressed as binding free energies (in kcal/mol) against the different macromolecular
targets under study (Table 2).
Fig. 3. Sequence alignment for the PKCδ kinase domain over the template PDB (1XJD).
Identical residues highlighted in cyan, similar residues highlighted in yellow.
1
3

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Table 2. Docking scores for OSU-2S, 10m, 10n and 10p expressed as binding free energy
against the different macromolecular targets.
Compound
E (Kcal/mol)
PKCδ
Kinase-
Domain
-6.7
-8.7
-8.6
Akt-1
Catalytic
Akt-1
Allosteric
Akt-2
Catalytic
ERK-1
Catalytic
ERK-2
Catalytic
OSU-2S
-6.1
-8.9
-7.4
-8.4
-7.5
-10.3
-10.0
-9.4
-6.0
-9.5
-9.5
-8.4
-8.1
-10.7
-10.7
-9.2
-5.9
-7.8
-8.0
-6.9
10m
10n
10p
-8.0
An overview of the docking results suggested that compounds 10m and 10n adopted binding
modes within the PKCδ kinase domain that are consistent with the reference molecule OSU-2S
and were able to interact with the residue Asp-477 (Please see SI). On the other hand, compound
1
0p appeared to have a different binding mode by interacting with the residues Asp-434 and
Asn-478. Furthermore, these compounds showed preferential binding pattern towards the
serine/threonine-protein kinase Akt-1 over Akt-2, which is evidenced by their superior binding
energy scores (Table 2). Moreover, analysis of the binding energies of the best-docked poses
suggested that the allosteric pattern of binding within the Akt-1 enzyme is supported by the fact
that these compounds showed no apparent binding interactions within the Akt-1 catalytic site.
Additionally, these compounds appeared to be engaged in a number of interactions within the
Akt-1 allosteric site, especially with the critical polar residue Thr-82 [32]. Similarly, these
compounds also showed preferential conformations and binding profile towards the extracellular
signal-regulated kinases ERK-1 over ERK-2, which is evident from their superior binding
energy scores (Table 2) and the binding modes adapted (Please see SI). Indeed, these
compounds also showed interaction with the critical DFG-motif residue Asp-184 of the
activation segment. These important in silico findings support the data obtained from the
biological studies.
1
4

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3
. CONCLUSION
In summary, we have synthesized a new series of rigid and enantiomerically pure pyrrolidine
carboxamide analogues based on the structure of the anticancer drug OSU-2S. The designed
motifs showed enhanced anticancer activity and were found to be safer than OSU-2S when
tested against normal cells. The anticancer activity of the new motifs was further validated
through their effects on the kinases, PKCδ, Akt and ERK. Furthermore, the effect of the new
compounds on cell cycle was also studied which confirmed that these derivatives induce cell
cycle arrest mainly at G1 phase. Moreover, the synthesized pyrrolidine derivatives were found to
induce apoptosis through the activation of caspases 3 and 7. Interestingly, compound 10c was
found to possess a potent anti-migratory activity that might have the potential of decreasing the
severity and aggressiveness of HCC. Additionally, a homology model of PKCδ was developed
and the obtained biological results were further supported by in silico studies.
4. Experimental Section
4.1. Chemistry
4.1.1. General methods
Purchased chemical reagents and anhydrous solvents were used without further purification.
Solvents for extraction and column chromatography were distilled prior to use. TLC analysis
was performed with silica gel plates (0.25 mm, 60 F254) using iodine and a UV lamp for
1
13
visualization. H and C NMR experiments were performed on a 400 or 500 MHz instrument,
respectively. Chemical shifts are reported in parts per million (ppm) downstream from the
internal tetramethylsilane standard. Spin multiplicities are described as s (singlet), bs (broad
singlet), d (doublet), dd (doubledoublet), t (triplet), q (quartet), or m (multiplet). Coupling
constants are reported in Hertz (Hz). ESI mass spectrometry was performed on a Q-TOF high
1
5

ACCEPTED MANUSCRIPT
resolution mass spectrometer or Q-TOF Ultim LC-MS. MW irradiation was performed on CEM
synergy microwave synthesizer (version; DSCA02.22). Optical rotations were measured with a
digital polarimeter using a 100 mm cell of 10 ml capacity. All final compounds were determined
to be >95% pure by high-performance liquid chromatography (HPLC). The purity of the
compounds was determined on a Phenomenex Luna C18- (2), 3 mm column, 4.6 mm i.d.× 30
mm length, with 30-75% acetonitrile/water/0.1% trifluoroacetic acid, 1.0 mL/min elution at rt
using 210, 254, or 280 nm wavelength.
4
.1.2. (4-(Oct-1-yn-1-yl)phenyl)methanol (3)
Pd(PPh ) Cl (5 mol%), CuI (5 mol%) and compound 1(10 mmol) were mixed in triethylamine (120 mL)
3
2
2
at room temperature under nitrogen atmosphere. After 10 min, compound 2 was added dropwise and
stirring continued for 12 h. After completion, the reaction mixture was diluted with ethylacetate (500
mL) and washed with saturated NH Cl solution (2 x 150 mL). The organic layer was separated, dried
4
over Na SO and concentrated under vacuum. The crude product was purified on flash chromatography
2
4
1
using 100 % DCM to obtain the compound (3) as colorless liquid: 1.62g (76% yield); H NMR (400
MHz, CDCl ) δ 7.41 (d, J = 8.2 Hz, 2H), 7.30 (d, J = 8.2 Hz, 2H), 4.70 (d, J = 4.1 Hz, 2H), 2.42 (t, J= 7.1
3
Hz, 2H), 1.71 (t, 4.1 Hz, 1H), 1.75 – 1.57 (m, 2H), 1.52 – 1.42 (m, 2H), 1.40 – 1.28 (m, 4H), 0.93 (t, J =
1
3
7
.0 Hz, 3H); C NMR (100 MHz, CDCl ) δ 140.6, 132.2, 127.2, 123.9, 91.1, 80.8, 65.5, 31.8, 29.2, 29.1,
3
+
2
3.0, 19.9, 14.5; LCMS (ESI): m/z 216 [M+H] .
4
.1.3. 1-(Bromomethyl)-4-(oct-1-yn-1-yl)benzene (4)
Compound 3 (7.5 mmol) was dissolved in DCM (30 mL) and a solution of PBr (9 mmol) in DCM (10
3
ml) was added dropwise at 0 °C. The reaction mixture was slowly warmed to room temperature and
stirring continued for 1 h. After completion, the reaction mixture was quenched with cold water (10 mL)
and then diluted with DCM (500 mL). The organic layer was separated, dried over Na SO and
2
4
concentrated to obtain crude product. The latter was used in the next step without further purification.
.1.4. 1-(Tert-Butyl) 2-methyl (2S,4R)-4-((4-(oct-1-yn-1-yl)benzyl)oxy)pyrrolidine-1,2-dicarboxylate
6)
4
(
1
6

ACCEPTED MANUSCRIPT
Compound 4 (6 mmol)and 1-(tert-butyl) 2-methyl (2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate (6,
5
.5 mmol) were mixed in anhydrous DMF (20 mL) at 0 °C. Then, NaH (6.05 mmol) was added portion
wise at 0 °C and slowly warmed to room temperature and stirring was continued for 12 h. After
completion, the reaction mixture was quenched with crushed ice and then diluted with diethylether (400
mL). The organic layer was separated, dried over Na SO and concentrated under vacuum. The crude
2
4
was purified on flash chromatography using 40% EtOAc/hexane as eluent to produce compound 6 as
1
colorless viscus liquid: 2.07g (85% yield); H NMR (400 MHz, CDCl ) mixture of rotamers δ 7.36 (d, J
3
=
8.2 Hz, 2H), 7.22 (d, J = 8.2 Hz, 2H), 4.54 – 4.30 (m, 3H), 4.21 – 4.05 (m, 1H), 3.75 – 3.48 (m, 5H),
2
0
8
.43 – 2.28 (m, 3H), 2.10 – 2.01 (m, 1H), 1.64 – 1.55 (m, 2H), 1.50 – 1.38 (m, 11H), 1.37 – 1.28 (m, 4H),
1
3
.90 (t, J = 6.9 Hz, 3H); C NMR (100 MHz, CDCl ) δ 173.7, 153.9, 137.1, 131.6, 127.3, 123.6, 90.7,
3
0.3, 76.1, 70.7, 58.0, 52.1, 51.3, 36.7, 31.4, 28.7, 28.6, 28.4, 28.3, 22.6, 19.4, 14.1; HRMS (ESI-TOF):
+
m/z calcd for C H NO Na, 466.2569, found 466.2560[M+Na] .
2
6
37
5
4
.1.5. (2S,4R)-1-(Tert-butoxycarbonyl)-4-((4-(oct-1-yn-1-yl)benzyl)oxy)pyrrolidine-2-carboxylic acid
7)
(
Compound 6 (3 mmol) was dissolved in THF:MeOH (3:1 mL) and a solution of LiOH (7.5 mmol) in
water (1.5 mL) was added dropwise at 0 °C and stirring was continued at rt for 8 h. After completion,
most of the MeOH and THF were removed; the aqueous phase was acidified with NH Cl (12 mmol)
4
o
solution at 0 C and extracted into EtOAc (3 x 50 mL). Then, the organic layer was dried over Na SO
2
4
and concentrated under vacuum to obtain the compound 7 as colorless viscus liquid:1.16g (91% yield);
1
H NMR (500 MHz, CDCl ) δ mixture of rotamers 7.36 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.0 Hz, 2H),
3
4
1
.54 – 4.33 (m, 3H), 4.18 – 4.11 (m, 1H), 3.60 – 3.45 (m, 2H), 2.45 – 2.35 (m, 3H), 2.28 – 2.19 (m, 1H),
1
3
.64 – 1.55 (m, 2H), 1.49 – 1.39 (m, 11H), 1.37 – 1.28 (m, 4H), 0.90 (t, J = 7.0 Hz, 3H); C NMR (125
MHz, CDCl ) δ 176.7 154.2, 137.3, 131.8, 127.5, 123.7, 90.8, 81.3, 80.4, 71.0, 52.2, 51.4, 35.0, 31.5,
3
+
2
8.9, 28.7, 28.5, 28.4, 22.7, 19.6, 14.2; LCMS (ESI): m/z 430 [M+H] .
4
.1.6. General reaction procedure for the preparation of compounds 9a-s
Compound 7 (0.5 mmol) and DIPEA (0.7 mmol) were mixed in anhydrous DMF (4 mL) at 0 °C. After
5 minutes, HBTU (0.6 mmol) followed by the amine partner (8a-s; 0.55 mmol) were added and stirring
4
1
7

ACCEPTED MANUSCRIPT
was continued at room temperature for 10-13 h. After completion, the reaction mixture was diluted with
EtOAc and washed with cold water and followed by brain solution. Then, the organic layer was dried
over Na SO and concentrated under the vacuum to obtain the crude product. Then, the residue was
2
4
purified on flash chromatography using EtOAc/hexane or MeOH /DCM as eluent to obtain the title
compounds 9a-s.
4
.1.6.1. Tert-butyl
(2S,4R)-2-(benzylcarbamoyl)-4-((4-(oct-1-yn-1-yl)benzyl)oxy)pyrrolidine-1-
carboxylate (9a)
1
Colorless viscus liquid (194 mg, yield 75%); H NMR (500 MHz, CDCl ) δ mixture of rotamers 7.45 –
3
7
.17 (m, 9H), 4.61 – 4.03 (m, 6H), 3.94 – 3.77 (m, 0.4H), 3.65 – 3.40 (m, , 1.6H), 2.66 – 2.54 (m, 0.6 H),
1
3
2
.52 – 2.36 (m, 2.3H), 2.25 – 1.99 (m, 1.1H), 1.69 – 1.21 (m, 17H), 0.93 (t, J = 6.9 Hz, 3H); C NMR
(
125 MHz, CDCl ) δ 171.4, 156.0, 138.2, 137.2, 131.6, 128.6, 128.0, 127.7, 127.4, 123.5, 90.7, 80.8,
3
8
0.3, 71.0, 58.8, 51.8, 43.4, 33.7, 31.4, 29.7, 28.7, 28.6, 28.3, 22.6, 19.4, 14.1; HRMS (ESI-TOF): m/z
+
calcd for C H N O , 519.3222, found 519.3173 [M+H] .
32
43
2
4
4
.1.7. General reaction procedure for the preparation of compounds 10a-s
Compounds 9a-s (0.25 mmol) was dissolved in DCM (2.0 mL) and excess amount of TFA (0.1 mL) was
added portion wise at 0 °C. The reaction mixture was slowly warmed to room temperature and stirring
continued for 3-5 h. After completion, DCM and excess TFA were removed under vacuum. Then, the
crude was diluted with DCM (20 mL) and washed with NaHCO solution (1N, 5 mL). The organic layer
3
was separated, dried over Na SO and concentrated under vacuum. The residue was purified on flash
2
4
chromatography using MeOH in DCM as eluent to obtain the title compounds 10a-s.
.1.7.1. (2S,4R)-N-Benzyl-4-((4-(oct-1-yn-1-yl)benzyl)oxy)pyrrolidine-2-carboxamide (10a)
4
2
0
1
Colorless viscous oil (84 mg, yield 81%); [α]D +2.7 (c 0.0018, acetone); H NMR (500 MHz, CDCl ) δ
3
8
.01 (s, 1H), 7.52 – 7.13 (m, 9H), 4.55 – 4.40 (m, 4H), 4.14 – 4.06 (m, 1H), 4.03 (t, J = 8.2 Hz, 1H), 3.16
(
d, J = 12.4 Hz, 1H), 2.75 (dd, J = 12.4, 3.5 Hz, 1H), 2.54 – 2.47 (m, 1H), 2.42 (t, J = 7.1 Hz, 2H), 2.07 –
1
3
1
.95 (m, 1H), 1.70 – 1.56 (m, 2H), 1.54 – 1.43 (m, 2H), 1.40 – 1.30 (m, 4H), 0.93 (t, J = 6.8 Hz, 3H); C
NMR (125 MHz, CDCl ) δ 174.4, 138.5, 137.4, 131.6, 128.7, 127.6, 127.4 (2C), 123.5, 90.7, 80.4, 80.3,
3
1
8

ACCEPTED MANUSCRIPT
0.3, 59.9, 52.7, 42.9, 36.3, 31.4, 28.7, 28.6, 22.6, 19.4, 14.1; HRMS (ESI-TOF): m/z calcd for
7
+
C H N O , 419.2698, found 419.2709 [M+H] .
27
35
2
2
4
.1.7.2. (2S,4R)-N-(4-Fluorobenzyl)-4-((4-(oct-1-yn-1-yl)benzyl)oxy)pyrrolidine-2-carboxamide (10b)
2
0
1
Colorless viscous oil (78 mg, yield 72%); [α]D +5.3 (c 0.0009, acetone); H NMR (500 MHz, CDCl ) δ
3
8
4
2
1
1
3
4
.00 (s, 1H), 7.36 (d, J = 8.0 Hz, 2H), 7.27-7.20 (m, 4H), 7.04-6.96 (m, 2H), 4.48 (d, J = 12.0 Hz, 1H),
.44 – 4.33 (m, 3H), 4.10-4.05 (m, 1H), 4.04-3.97 (m, 1H), 3.14 (d, J = 12.5 Hz, 1H), 2.72 (dd, J = 12.5,
.7 Hz, 1H), 2.53-2.44 (m, 1H), 2.39 (t, J = 7.1 Hz, 2H), 2.00 – 1.91 (m, 1H), 1.63 – 1.56 (m, 2H), 1.50-
1
3
.40 (m, 2H), 1.37-1.27 (m, 4H), 0.90 (t, J = 6.8 Hz, 3H); C NMR (100 MHz, CDCl ) δ 174.5, 162.1(d,
3
JC-F = 244 Hz), 134.4, 131.6, 129.3, 127.4, 123.6, 115.6, 115.4, 90.7, 80.4, 80.3, 70.3, 59.9, 52.7, 42.2,
6.3, 31.4, 28.7, 28.6, 22.6, 19.4, 14.1; HRMS (ESI-TOF): m/z calcd for C H FN O , 437.2604, found
27
34
2
2
+
37.2606 [M+H] .
4
.1.7.3. (2S,4R)-N-(4-Methoxybenzyl)-4-((4-(oct-1-yn-1-yl)benzyl)oxy)pyrrolidine-2-carboxamide (10c)
2
0
1
Colorless viscous oil (84 mg, yield 75%); [α]D +8.3 (c 0.0006, acetone); H NMR (400 MHz, CDCl ) δ
3
7
4
1
0
1
.88 (s, 1H), 7.36 (d, J = 8.1 Hz, 2H), 7.24–7.16 (m, 4H), 6.89 – 6.83 (m, 2H), 4.52 – 4.32 (m, 4H),
.09–4.04 (m, 1H), 4.02-3.95 (m, 1H), 3.79 (s, 3H), 3.12 (d,J = 12.5 Hz, 1H), 2.70 (dd, J = 12.5, 3.5 Hz,
H), 2.53–2.35 (m, 3H), 2.01-1.92 (m, 1H), 1.64 – 1.55 (m, 2H), 1.50–1.40 (m, 2H), 1.37 – 1.27 (m, 4H),
1
3
.90 (t, J = 6.9 Hz, 3H); C NMR (100 MHz, CDCl ) δ 174.3, 158.9, 137.4, 131.6, 130.7, 129.0, 127.4,
3
23.5, 114.0, 90.6, 80.4, 80.3, 70.3, 59.9, 55.3, 52.7, 42.4, 36.3, 31.4, 28.7, 28.6, 22.6, 19.4, 14.1; HRMS
+
(
ESI-TOF): m/z calcd for C H N O , 449.2804, found 449.2814 [M+H] .
2
8
37
2
3
4
.1.7.4. (2S,4R)-N-(4-Chlorobenzyl)-4-((4-(oct-1-yn-1-yl)benzyl)oxy)pyrrolidine-2-carboxamide (10d)
2
0
1
Colorless viscous oil (88 mg, yield 78%); [α]D +3.1 (c 0.0016, acetone); H NMR (500 MHz, CDCl ) δ
3
8
.03 (s, 1H), 7.43 – 7.15 (m, 8H), 4.54 – 4.35 (m, 4H), 4.13 – 4.06 (m, 1H), 4.02 (t, J = 8.2 Hz, 1H), 3.16
(
d, J = 12.5 Hz, 1H), 2.72 (dd, J = 12.5, 3.4 Hz, 1H), 2.55 – 2.45 (m, 1H), 2.42 (t, J = 7.1 Hz, 2H), 2.01 –
1
3
1
.92 (m, 2H), 1.67 – 1.57 (m, 2H), 1.52 – 1.41 (m, 2H), 1.40 – 1.29 (m, 4H), 0.93 (t, J = 6.8 Hz, 3H); C
NMR (125 MHz, CDCl ) δ 174.6, 137.4, 137.1, 133.2, 131.6, 129.0, 128.8, 127.4, 123.6, 90.7, 80.35,
3
8
0.31, 70.3, 59.9, 52.7, 42.2, 36.3, 31.4, 28.7, 28.6, 22.6, 19.4, 14.1; HRMS (ESI-TOF): m/z calcd for
+
C H ClN O , 453.2308, found 453.2316 [M+H] .
27
34
2
2
1
9

ACCEPTED MANUSCRIPT
.1.7.5. (2S,4R)-4-((4-(Oct-1-yn-1-yl)benzyl)oxy)-N-(4-(trifluoromethyl)benzyl)pyrrolidine-2-
4
carboxamide (10e).
2
0
1
Colorless viscous oil (82 mg, yield 68%); [α]D +10 (c 0.0005, acetone); H NMR (500 MHz, CDCl ) δ
3
8
4
2
2
1
4
.19 (s, 1H), 7.60 (d, J = 8.0 Hz, 2H), 7.41 – 7.36 (m, 4H), 7.24 (d, J = 8.0 Hz, 2H), 4.54 – 4.47 (m, 3H),
.44 (d, J = 12.0 Hz, 1H), 4.15 – 4.04 (m, 2H), 3.20 (d, J = 12.6 Hz, 1H), 2.77 (dd, J = 12.6, 3.5 Hz, 1H),
.57 – 2.49 (m, 1H), 2.42 (t, J = 7.1 Hz, 2H), 2.02 – 1.94 (m, 1H), 1.66 – 1.58 (m, 2H), 1.51 – 1.44 (m,
1
3
H), 1.38 – 1.31 (m, 4H), 0.93 (t, J = 7.0 Hz, 3H); C NMR (125 MHz, CDCl ) δ 174.3, 142.6, 137.2,
3
31.7, 129.8, 127.8, 127.4, 126.4 (q, 1JC-F = 270 Hz),125.6, 123.6, 90.7, 80.3, 80.1, 70.4, 59.8, 52.6,
2.5, 36.2, 31.4, 28.7, 28.6, 22.6, 19.4, 14.1; HRMS (ESI-TOF): m/z calcd for C H F N O , 487.2572,
2
8
34
3
2
2
+
found 487.2582 [M+H] .
4
.1.7.6. (2S,4R)-N-(3-Fluorobenzyl)-4-((4-(oct-1-yn-1-yl)benzyl)oxy)pyrrolidine-2-carboxamide (10f).
2
0
1
Colorless viscous oil (73 mg, yield 67%); [α]D +9.0 (c 0.0011, acetone); H NMR (500 MHz, CDCl ) δ
3
8
.10 (s, 1H), 7.39 (d, J = 8.2 Hz, 2H), 7.34 – 7.28 (m, 1H), 7.25 (d, J = 8.2 Hz, 2H), 7.06 (d, J = 7.9 Hz,
H), 6.98 (d, J = 8.8 Hz, 2H), 4.55 – 4.41 (m, 4H), 4.14 – 4.03 (m, 2H), 3.19 (d, J = 12.5 Hz, 1H), 2.77
1
(
(
dd, J = 12.5, 3.5 Hz, 1H), 2.58 – 2.48 (m, 1H), 2.42 (t, J = 7.1 Hz, 2H), 2.04 – 1.95 (m, 1H), 1.67 – 1.58
13
m, 2H), 1.50 – 1.43 (m, 2H), 1.39– 1.31 (m, 4H), 0.93 (t, J = 7.0 Hz, 3H); C NMR (100 MHz, CDCl )
3
δ 174.6, 163.1 (d, 1JC-F = 245 Hz), 141.2, 137.4, 131.6, 130.1, 128.3, 127.4, 123.6, 123.1, 114.4, 90.7,
8
0.4, 80.3, 70.3, 59.9, 52.7, 42.4, 36.3, 31.4, 28.7, 28.6, 22.6, 19.4, 14.0; HRMS (ESI-TOF): m/z calcd
+
for C H FN O , 437.2604, found 437.2622 [M+H] .
2
7
34
2
2
4
.1.7.7. (2S,4R)-N-(3-Methoxybenzyl)-4-((4-(oct-1-yn-1-yl)benzyl)oxy)pyrrolidine-2-carboxamide (10g).
2
0
1
Colorless viscous oil (72 mg, yield 65%); [α]D +5.0 (c 0.001, acetone); H NMR (400 MHz, CDCl ) δ
3
7
4
.98 (s, 1H), 7.36 (d, J = 8.1 Hz, 2H), 7.27 – 7.19 (m, 3H), 6.87 – 6.77 (m, 3H), 4.53 – 4.45 (m, 1H),
.44 – 4.37 (m, 3H), 4.11 – 4.05 (m, 1H), 4.04 – 3.98 (m, 1H), 3.79 (s, 3H), 3.18 – 3.11 (m, 1H), 2.73
(
(
dd, J = 12.5, 3.5 Hz, 1H), 2.53 – 2.43 (m, 1H), 2.39 (t, J = 7.1 Hz, 2H), 2.02 – 1.92 (m, 1H), 1.65 – 1.54
13
m, 2H), 1.50 – 1.40 (m, 2H), 1.38 – 1.27 (m, 4H), 0.90 (t, J = 6.9 Hz, 3H); C NMR (100 MHz, CDCl )
3
δ 174.3, 159.9, 140.1, 137.4, 131.6, 129.7, 127.4, 127.3, 123.5, 119.9, 113.3, 112.8, 90.7, 80.3, 70.3,
2
0

ACCEPTED MANUSCRIPT
9.9, 55.2, 52.7, 42.9, 36.3, 31.4, 28.7, 28.6, 22.6, 19.4, 14.1; HRMS (ESI-TOF): m/z calcd for
5
+
C H N O , 449.2804, found 449.2814 [M+H] .
28
37
2
3
4
.1.7.8. (2S,4R)-N-(3-Chlorobenzyl)-4-((4-(oct-1-yn-1-yl)benzyl)oxy)pyrrolidine-2-carboxamide (10h)
2
0
1
Colorless viscous oil (74 mg, yield 66%); [α]D +5.5 (c 0.0009, acetone); H NMR (500 MHz, CDCl ) δ
3
8
4
2
2
1
3
4
.09 (s, 1H), 7.41 (d, J = 8.0 Hz, 2H), 7.33 – 7.24 (m, 5H), 7.21 – 7.16 (m, 1H), 4.56 – 4.41 (m, 4H),
.15 – 4.11 (s, 1H), 4.07 (t, J = 8.2 Hz, 1H), 3.20 (d, J = 12.5 Hz, 1H), 2.77 (dd, J = 12.5, 3.4 Hz, 1H),
.57 – 2.50 (m, 1H), 2.44 (t, J = 7.1 Hz, 2H), 2.04 – 1.96 (m, 1H), 1.68 – 1.60 (m, 2H), 1.53 – 1.44 (m,
1
3
H), 1.41 – 1.31 (m, 4H), 0.94 (t, J = 6.8 Hz, 3H); C NMR (125 MHz, CDCl ) δ 174.6, 140.8, 137.5,
3
34.6, 131.8, 130.1, 127.8, 127.7, 127.5, 125.9, 123.7, 90.8, 80.44, 80.42, 70.5, 60.0, 52.8, 42.5, 36.4,
3
5
1.5, 28.8, 28.7, 22.7, 19.6, 14.2; HRMS (ESI-TOF): m/z calcd for C H ClN O , 453.2308, found
2
7
34
2
2
+
53.2307 [M+H] .
4
.1.7.9. (2S,4R)-4-((4-(Oct-1-yn-1-yl)benzyl)oxy)-N-phenethylpyrrolidine-2-carboxamide (10i)
2
0
1
Colorless viscous oil (76 mg, yield 71%); [α]D +5.0 (c 0.001, acetone); H NMR (400 MHz, MeOD) δ
.41 – 7.16 (m, 9H), 4.48 (s, 2H), 4.17 – 4.05 (s, 1H), 3.80 (t, J = 8.0 Hz, 1H), 3.53 – 3.38 (m, 2H), 3.06
d, J = 12.2 Hz, 1H), 2.95 – 2.86 (m, 1H), 2.82 (t, J = 7.0 Hz, 2H), 2.41 (t, J = 6.8 Hz, 2H), 2.29 (dd, J =
7
(
1
0
1
2.9, 8.4 Hz, 1H), 1.77 – 1.67 (m, 1H), 1.66 – 1.54 (m, 2H), 1.55 – 1.45 (m, 2H), 1.43 – 1.31 (m, 4H),
1
3
.94 (t, J = 6.4 Hz, 3H); C NMR (100 MHz, MeOD) δ 175.4, 138.9, 137.9, 131.0, 128.5, 128.1, 127.3,
26.0, 123.4, 89.6, 80.1, 80.0, 70.0, 59.4, 51.8, 40.0, 36.7, 35.1, 31.1, 28.5, 28.3, 22.2, 18.6, 13.0; HRMS
+
(
ESI-TOF): m/z calcd for C H N O , 433.2855, found 433.2869 [M+H] .
2
8
37
2
2
4
.1.7.10. (2S,4R)-N-(4-Methoxyphenethyl)-4-((4-(oct-1-yn-1-yl)benzyl)oxy)pyrrolidine-2-carboxamide
10j)
(
2
0
1
Colorless viscous oil (69 mg, yield 60%); [α]D +9.1 (c 0.0011, acetone); H NMR (400 MHz, CDCl ) δ
3
7
.68 (s, 1H), 7.38 (d, J = 8.0 Hz, 2H), 7.23 (d, J = 8.0 Hz, 2H), 7.11 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 8.5
Hz, 2H), 4.53 – 4.37 (m, 2H), 4.09 – 4.01 (s, 1H), 3.93 (t, J = 8.2 Hz, 1H), 3.80 (s, 3H), 3.57 – 3.38 (m,
2
H), 3.11 (d, J = 12.4 Hz, 1H), 2.77 (t, J = 6.9 Hz, 2H), 2.65 (dd, J = 12.4, 3.4 Hz, 1H), 2.50 – 2.37 (m,
H), 1.95 – 1.83 (m, 1H), 1.68 – 1.56 (m, 2H), 1.53 – 1.42 (m, 2H), 1.40 – 1.28 (m, 4H), 0.92 (t, J = 6.7
3
2
1

ACCEPTED MANUSCRIPT
1
3
Hz, 3H); C NMR (100 MHz, CDCl ) δ 174.3, 158.2, 137.4, 131.6, 131.0, 129.7, 127.4, 123.5, 113.9,
3
9
0.6, 80.3 (2C), 70.3, 59.8, 55.3, 52.5, 40.2, 36.2, 34.9, 31.4, 28.7, 28.6, 22.6, 19.4, 14.1; HRMS (ESI-
+
TOF): m/z calcd for C H N O , 463.2960, found 463.2948 [M+H] .
29
39
2
3
4
.1.7.11.
10k)
(2S,4R)-4-((4-(Oct-1-yn-1-yl)benzyl)oxy)-N-(3-phenylpropyl)pyrrolidine-2-carboxamide
(
2
0
1
Colorless viscous oil ( 83 mg, yield 75%); [α]D +2.9 (c 0.0017, acetone); H NMR (400 MHz, MeOD) δ
7
.36 – 7.24 (m, 6H), 7.23 – 7.14 (m, 3H), 4.50 (s, 2H), 4.19 – 4.13 (m, 1H), 3.84 (t, J = 8.3 Hz, 1H), 3.23
(
(
(
t, J = 7.0 Hz, 2H), 3.13 – 2.98 (m, 2H), 2.65 (t, J = 8.0 Hz, 2H), 2.41 (t, J = 7.0 Hz, 2H), 2.37 – 2.30
m, 1H), 1.92 – 1.74 (m, 3H), 1.65 – 1.56 (m, 2H), 1.54 – 1.45 (m, 2H), 1.41 – 1.33 (m, 4H), 1.33 – 1.28
1
3
m, 1H), 0.94 (t, J = 7.0 Hz, 3H). C NMR (100 MHz, MeOD) δ 175.2, 141.5, 137.9, 131.0, 128.0, 128.0,
1
1
27.3, 125.5, 123.4, 89.6, 80.1, 80.0, 70.0, 59.5, 51.9, 38.4, 36.8, 32.8, 31.1, 30.8, 28.5, 28.3, 22.2, 18.6,
+
3.0.HRMS (ESI-TOF): m/z calcd for C H N O , 447.3011, found 447.3022 [M+H] .
2
9
39
2
2
4
.1.7.12. (2S,4R)-4-((4-(Oct-1-yn-1-yl)benzyl)oxy)-N-(2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide
10l)
(
2
0
1
Colorless viscous oil (66 mg, yield 61%);[α]D +9.1 (c 0.0011, acetone); H NMR (400 MHz, CDCl ) δ
3
8
.52 (d, J = 4.5 Hz, 1H), 7.99 – 7.90 (m, 1H), 7.59 (td, J = 7.7, 1.8 Hz, 1H), 7.35 (d, J = 8.1 Hz, 2H),
.20 (d, J = 8.1 Hz, 2H), 7.17 – 7.09 (m, 2H), 4.49 – 4.34 (m, 2H), 4.03 – 3.97 (m, 1H), 3.88 (t, J = 8.1
7
Hz, 1H), 3.68 – 3.59 (m, 2H), 3.12 – 3.04 (m, 1H), 2.99 (t, J = 6.7 Hz, 2H), 2.63 (dd, J = 12.5, 3.6 Hz,
1
2
1
2
H), 2.43 – 2.34 (m, 3H), 2.16 – 2.04 (m, 1H), 1.92 – 1.80 (m, 1H), 1.64 – 1.54 (m, 2H), 1.50 – 1.39 (m,
1
3
H), 1.37 – 1.26 (m, 4H), 0.89 (t, J = 6.9 Hz, 3H). C NMR (100 MHz, CDCl ) δ 174.5, 159.4, 149.3,
3
37.5, 136.4, 131.6, 127.4, 123.5, 123.3, 121.5, 90.6, 80.4, 80.3, 70.3, 59.9, 52.6, 38.3, 37.6, 36.3, 31.4,
8.7, 28.6, 22.6, 19.4, 14.0.HRMS (ESI-TOF): m/z calcd for C H N O , 434.2807, found 434.2821
2
7
36
3
2
+
[
M+H] .
4.1.7.13. (2S,4R)-N-((1H-Indol-2-yl)methyl)-4-((4-(oct-1-yn-1-yl)benzyl)oxy)pyrrolidine-2-carboxamide
(10m)
2
2

ACCEPTED MANUSCRIPT
Colorless viscous oil (62 mg, yield 55%); [α]D +8.3 (c 0.0006, acetone); H NMR (400 MHz, CDCl ) δ
2
0
1
3
9
.13 (s, 1H), 8.26 (s, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.43 – 7.33 (m, 3H), 7.24 (d, J = 8.0 Hz, 2H), 7.18 (t,
J = 7.3 Hz, 1H), 7.09 (t, J = 7.4 Hz, 1H), 6.34 (s, 1H), 4.57 – 4.39 (m, 4H), 4.11 – 4.05 (m, 1H), 4.01 (t, J
8.3 Hz, 1H), 3.15 (d, J = 12.5 Hz, 1H), 2.71 (dd, J = 12.5, 3.3 Hz, 1H), 2.55 – 2.46 (m, 1H), 2.42 (t, J =
.1 Hz, 2H), 1.97 – 1.89 (m, 1H), 1.67 – 1.58 (m, 2H), 1.54 – 1.42 (m, 2H), 1.40 – 1.29 (m, 4H), 0.93 (t,
=
7
1
3
J = 6.8 Hz, 3H). C NMR (100 MHz, CDCl ) δ 176.5, 137.3, 136.7, 136.4, 131.6, 127.7, 127.4, 123.6,
3
1
1
21.9, 120.3, 119.6, 111.1, 100.4, 90.7, 80.4, 80.3, 70.3, 59.8, 52.6, 36.9, 36.2, 31.4, 28.7, 28.6, 22.6,
+
9.4, 14.1.HRMS (ESI-TOF): m/z calcd for C H N O , 458.2807, found 458.2813 [M+H] .
2
9
36
3
2
4
.1.7.14. (2S,4R)-N-((1-Methyl-1H-indol-3-yl)methyl)-4-((4-(oct-1-yn-1-yl)benzyl)oxy)pyrrolidine-2-
carboxamide (10n)
2
0
1
Colorless viscous oil (38 mg, yield 32%); [α]D +10 (c 0.0005, acetone); H NMR (400 MHz, CDCl ) δ
3
7.81 (s, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.38 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 8.2 Hz, 1H), 7.29 – 7.26 (m,
1H), 7.26 – 7.21 (m, 2H), 7.17 – 7.12 (m, 1H), 7.04 (s, 1H), 4.60 (dd, J = 5.4, 3.4 Hz, 2H), 4.49 (d, J =
12.0 Hz, 1H), 4.41 (d, J = 12.0 Hz, 1H), 4.08 – 4.04 (m, 1H), 3.99 (t, J = 8.2 Hz, 1H), 3.78 (s, 3H), 3.09
(
d, J = 12.5 Hz, 1H), 2.68 (dd, J = 12.4, 3.6 Hz, 1H), 2.53 – 2.45 (m, 1H), 2.42 (t, J = 7.1 Hz, 2H), 2.03 –
1
3
1
.95 (m, 1H), 1.66 – 1.58 (m, 2H), 1.51 – 1.42 (m, 2H), 1.40 – 1.31 (m, 4H), 0.93 (t, J = 6.9 Hz, 3H); C
NMR (100 MHz, CDCl ) δ 174.2, 137.5, 137.1, 131.6, 127.7, 127.4, 127.1, 123.5, 121.9, 119.3, 119.0,
3
1
11.5, 109.3, 90.6, 80.4, 76.7, 70.3, 59.9, 52.6, 36.3, 34.4, 32.7, 31.4, 28.7, 28.6, 22.6, 19.4, 14.1; HRMS
+
(
ESI-TOF): m/z calcd for C H N O , 472.2964, found 472.2975 [M+H] .
3
0
38
3
2
4
.1.7.15. (2S,4R)-N-((1H-Benzo[d]imidazol-2-yl)methyl)-4-((4-(oct-1-yn-1-yl)benzyl)oxy)pyrrolidine-2-
carboxamide (10o)
2
0
1
Colorless viscous oil (51 mg, yield 45%); [α]D +4.1 (c 0.0012, acetone); H NMR (400 MHz, CDCl ) δ
3
7
.80 – 7.42 (m, 2H), 7.39 – 7.33 (m, 2H), 7.29 – 7.21 (m, 4H), 4.67 – 4.43 (m, 4H), 4.10 (dd, J = 9.2, 5.0
Hz, 1H), 3.97 (t, J = 4.4 Hz, 1H), 3.09 (dd, J = 10.3, 1.3 Hz, 1H), 2.51 – 2.38 (m, 3H), 2.38 – 2.23 (m,
1
3
2
H), 1.67 – 1.56 (m, 2H), 1.50 – 1.41 (m, 2H), 1.35 – 1.28 (m, 4H), 0.92 (t, J = 7.0 Hz, 3H); C NMR
(
100 MHz, CDCl ) δ 177.2, 151.8, 150.7, 137.2, 131.66, 131.58, 127.6, 127.4, 123.6, 123.5, 122.7,
3
2
3

ACCEPTED MANUSCRIPT
11.1, 90.7, 80.1, 70.5, 62.4, 53.5, 38.9, 32.7, 31.4, 28.7, 27.5, 22.6, 22.2, 19.4, 14.1; HRMS (ESI-TOF):
1
+
m/z calcd for C H N O , 459.2760, found 459.2772 [M+H] .
2
8
35
4
2
4
.1.7.16.
(2S,4R)-4-((4-(Oct-1-yn-1-yl)benzyl)oxy)-2-((((S)-pyrrolidin-1-ium-2-
yl)methyl)carbamoyl)pyrrolidin-1-ium 2,2,2-trifluoroacetate (10p)
2
0
1
White solid (99 mg, yield 62%); [α]D +25 (c 0.0008, acetone); H NMR (500 MHz, CDCl ) δ 9.49 (bs,
3
3
H), 7.37 (d, J = 8.1 Hz, 1H), 7.22 (d, J= 8.1 Hz, 1H), 4.58 – 4.42 (m, 3H), 4.30 (s, 1H), 3.92 – 3.75 (m,
H), 3.59 – 3.43 (m, 2H), 3.34 – 3.16 (m, 3H), 2.58 – 2.47 (m, 1H), 2.40 (t, J= 7.1 Hz, 1H), 2.21 – 1.93
2
1
3
(
m, 4H), 1.68 – 1.57 (m, 3H), 1.49 – 1.42 (m, 2H), 1.39 – 1.28 (m, 4H), 0.92 (t, J = 6.9 Hz, 3H); C
NMR (125 MHz, CDCl ) δ 168.6, 162.8, 136.2, 131.8, 127.8, 124.1, 91.2, 80.2, 70.9, 59.2, 58.8, 51.1,
3
4
5.1, 41.2, 34.6, 31.5, 28.7, 27.9, 24.0, 22.7, 19.5, 14.2; HRMS (ESI-TOF): m/z calcd for C H N O ,
25 38 3 2
+
4
12.2964, found 412.2968 [M] .
4
.1.7.17.
(2S,4R)-N-((3-(tert-Butyl)-1H-pyrazol-5-yl)methyl)-4-((4-(oct-1-yn-1-
yl)benzyl)oxy)pyrrolidine-2-carboxamide (10q)
2
0
1
Colorless viscous oil (71 mg, yield 62%); [α]D +2.5 (c 0.002, acetone); H NMR (400 MHz, CDCl ) δ
3
8
4
2
–
1
1
.14 (s, 1H), 7.38 (d, J = 8.2 Hz, 2H), 7.23 (d, J = 8.2 Hz, 2H), 5.99 (s, 1H), 4.53 – 4.32 (m, 4H), 4.11 –
.06 (m, 1H), 4.00 (t, J = 8.2 Hz, 1H), 3.15 (d, J = 12.5 Hz, 1H), 2.77 (dd, J = 12.5, 3.6 Hz, 1H), 2.51 –
.45 (m, 1H), 2.41 (t, J = 7.1 Hz, 2H), 2.0 – 1.92(m, 1H), 1.67 – 1.56 (m, 2H), 1.51 – 1.42 (m, 2H), 1.39
1
3
1.29 (m, 13H), 0.92 (t, J = 7.0 Hz, 3H); C NMR (100 MHz, CDCl ) δ 175.0, 156.6, 147.1, 137.4,
3
31.6, 127.4, 123.5, 100.1, 90.6, 80.4, 80.3, 70.3, 59.9, 52.6, 36.2, 31.4, 31.2, 30.3, 28.7, 28.6, 22.6, 19.4,
+
4.1; HRMS (ESI-TOF): m/z calcd for C H N O , 465.3229, found 465.3244 [M+H] .
2
8
41
4
2
4
.1.7.18.
(2S,4R)-N-(2-(1H-Indol-3-yl)
ethyl)-4-((4-(oct-1-yn-1-yl)benzyl)oxy)pyrrolidine-2-
carboxamide (10r)
2
0
1
Colorless viscous oil (76 mg, yield 65%); [α]D +3.1 (c 0.0016, acetone); H NMR (500 MHz, CDCl ) δ
3
8
.11 (s, 1H), 7.73 (s, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.42 – 7.36 (m, 3H), 7.26 – 7.19 (m, 3H), 7.14 (t, J =
.3 Hz, 1H), 7.04 (s, 1H), 4.48 (d, J = 12.0 Hz, 1H), 4.40 (d, J = 12.0 Hz, 1H), 4.04 – 3.99 (m, 1H), 3.92
7
(
t, J = 8.2 Hz, 1H), 3.64 – 3.56 (m, 2H), 3.06 (d, J = 12.4 Hz, 1H), 3.00 (t, J = 6.9 Hz, 2H), 2.59 (dd, J =
2
4

ACCEPTED MANUSCRIPT
2.4, 3.6 Hz, 1H), 2.47 – 2.39 (m, 3H), 1.96 – 1.85 (m, 1H), 1.68 – 1.58 (m, 2H), 1.51 – 1.43 (m, 2H),
1
1
1
3
4
1
3
.40 – 1.31 (m, 4H), 0.98 – 0.89 (m, 3H); C NMR (125 MHz, CDCl ) δ 174.5, 137.5, 136.3, 131.6,
3
27.5, 127.4, 123.5, 122.1, 121.8, 119.4, 118.8, 113.3, 111.2, 90.7, 80.3, 70.3, 59.9, 52.5, 39.2, 36.2,
1.4, 28.7, 28.6, 25.4, 22.6, 19.4, 14.1; HRMS (ESI-TOF): m/z calcd for C H N O , 472.2964, found
3
0
38
3
2
+
72.2964 [M+H] .
4
.1.7.19. Methyl ((2S,4R)-4-((4-(oct-1-yn-1-yl)benzyl)oxy)pyrrolidine-2-carbonyl)-L-tryptophanate (10s)
2
0
1
Colorless viscous oil (76 mg, yield 58%); [α]D +9.3 (c 0.0018, acetone); H NMR (500 MHz, CDCl ) δ
3
8
.10 (two bs, 2H), 7.55 (d, J = 7.9 Hz, 1H), 7.41 – 7.34 (m, 3H), 7.24 – 7.16 (m, 3H), 7.13 – 7.07 (m,
H), 6.99 (s, 1H), 4.96 – 4.89 (m, 1H), 4.43 (d, J = 12.0 Hz, 1H), 4.36 (d, J = 12.0 Hz, 1H), 3.93 – 3.84
1
(
m, 2H), 3.73 (s, 3H), 3.39 – 3.26 (m, 2H), 2.95 (d, J = 12.4 Hz, 1H), 2.42 (t, J = 7.1 Hz, 2H), 2.39 – 2.31
m, 2H), 1.74 – 1.67 (m, 1H), 1.66 – 1.58 (m, 2H), 1.52 – 1.43 (m, 2H), 1.38 – 1.31 (m, 4H), 0.93 (t, J =
(
1
3
6
1
2
.8 Hz, 3H); C NMR (125 MHz, CDCl ) δ 174.6, 172.6, 137.6, 136.1, 131.7, 127.9, 127.5, 123.6,
3
22.6, 122.3, 119.6, 118.8, 111.3, 110.6, 90.8, 80.4, 80.3, 70.4, 59.8, 52.6, 52.5, 52.4, 36.2, 31.5, 28.8,
8.7, 27.8, 22.7, 19.6, 14.2; HRMS (ESI-TOF): m/z calcd for C H N O , 530.3019, found 530.3020
3
2
40
3
4
+
[
M+H] .
4
.2.
Development of a homology model
The homology model for the human PKCδ kinase domain was developed employing the
following procedure: The domain of the 326 kinase amino acid sequence was obtained from the
Uniprot server (http://www.uniprot.org/) under entry number Q05655. The sequence was then
subjected to blast analysis [33] to search for homologous protein using Blosum62 scoring metric
and Protein Data Bank (http://www.rcsb.org/pdb/home/home.do) as database. The best template
was selected based on its corresponding E-value, sequence identity and coverage. Protein kinase
C theta (PKC-δ, PDB-ID: 1XJD) was found to be the most appropriate template, which
exhibited 73.6% sequence identity and 86.2% sequence similarity. The target/template
sequences were aligned (Figure 3) using ClustalW [34] setting up the gap opening and extension
penalties to 10 and 0.02, respectively. Generation of the human PKCδ kinase domain homology
2
5

ACCEPTED MANUSCRIPT
model was carried out employing the version 9.15 of Modeller program [35, 36]. The refinement
of the developed model was accomplished by conjugate gradient optimization and molecular
dynamic simulations. The model was evaluated by means of WHAT-CHECK subset within the
WHATIF [37, 38] as well as the ProSA web servers [39, 40].
4
.3.
Molecular modeling
The program Autodock Vina [41] was employed during docking experiments. X-ray crystal
structures of the target macromolecules namely; Akt-1, Akt-2, ERK-1 and ERK-2 were obtained
from the RSCB protein data bank (http://www.rcsb.org/pdb/home/home.do). Subsequently, the
complexed inhibitors and water molecules were extracted from the initial X-ray structures.
Then, the polar hydrogens and Gastieger charges were generated using the MGL Tools. The
compounds under study were drawn using the software ChemDraw Ultra 11.0 (Cambridge Soft
Corporation, USA) and were optimized for energy and geometry using MMFF94 force field. A
grid boxes were established to cover the desired site of each target with a spacing of 1.0 Å
between the grid points. The exhaustiveness and the number of poses were set to 12 and 10
respectively.
All compounds were treated employing the preparation procedure mentioned earlier and
were docked into the target macromolecules as well as the developed PKCδ kinase homology
model. Finally, results visualization and the 3D-best docked poses were achieved using the
PyMOL molecular viewer (Schrödinger Inc., New York, NY, USA).
4
4
.4.
Biological evaluation
.4.1. Cell culture
The used cancer cell lines in this study were human hepatoma (Hep3B and HepG2), human
colorectal adenocarcinoma (HT-29) and human lung carcinoma (A549) which were obtained
from European Collection of Cell Cultures (ECACC, UK). The normal fibroblasts (F180) were
2
6

ACCEPTED MANUSCRIPT
kindly provided by professor Ekkehard Dikomey (University Cancer Center, Hamburg
University, Hamburg, Germany). HepG2 and Hep3B cells were maintained in Dulbecco’s
Modified Eagle Medium (DMEM: Sigma-Aldrich) while HT-29 and A549 cells were miniated
in Roswell Park Memorial Institute medium (RPMI, Sigma-Aldrich, St. Louis, MO, USA). The
fibroblast cells were maintained in Minimum Essential Media (MEM). All media were
supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich) and 1%
o
penicillin/streptomycin (Sigma-Aldrich). All incubations were done at 37 C in a humidified
atmosphere of 5 % CO2.
4
.4.2. Cell viability studies
Cell viability was assessed using the MTT assay as described before [42]. In brief, cancer cell
4
lines or normal fibroblasts were seeded at the density of 4x10 /well in 96 well flat-bottom plates,
and incubated in 10% FBS-supplemented media for 24h. Cells were treated with the new
compounds, doxorubicin or sorafenib for 48h. The vehicle (DMSO) was used as a negative
control. Following the treatment, the media were aspirated from each well and replaced by 200
µL fresh media containing 0.5 mg/mL of MTT tetrazolium dye (Sigma-Aldrich). Cells were
o
then incubated for 2h at 37 C. The formed formazan crystals were solubilized in 200 µl/well of
DMSO and absorbance was measured at 570 nm using a microplate reader (Thermo Scientific).
4
.4.3. Caspase 3/7 activity studies
Caspase-3/7 activities in HCC cells treated with 5 µM of OSU-2S, 10c, 10l, 10m, 10o, 10p, 10q
and 10n were measured using Caspase-Glo® 3/7 assay kit (Promega, Madison, WI, USA)
4
following the manufacturer’s instructions. In summary, 1×10 Cells were seeded into 6-well
plates in triplicates. Following treatment with 5 µM of the test compounds, the media were
removed and the cells were washed twice with PBS. RIPA lysis buffer as then used for cell lysis
and 50 µL of caspase substrate was added to each well. Aliquots from each treatment were
transferred to white opaque 96-well plate and the luminescence was measured by GloMax®-96
2
7

ACCEPTED MANUSCRIPT
Microplate Luminometer (Promega, Madison, WI) and the data were expressed as relative light
unit (RLU). The vehicle of the compounds (DMSO) was used as a negative control.
4.4.4. Western blotting
Cells were treated with OSU-2S, 10c, 10l, 10m, 10o, 10p, 10q and 10n at 10 µM. After 48 h of
treatment, cells were lysed and proteins were quantified using DC protein assay kit (Biorad)
according to the manufacturer’s instructions. Proteins were resolved in 12% SDS-poly-
acrylamide gels, then transferred onto nitrocellulose membrane using semi-dry transfer cell.
o
Membranes were incubated with primary antibodies overnight at 4 C. The following antibodies
were used: anti-β actin (Cell Signaling #4970), anti-PKC-δ (Cell Signaling #2058), anti-Akt
3
08
(
Cell Signaling #9272), anti-phospho-Akt (Thr , Cell Signaling #9275), anti-phospho-Akt (Ser
4
73
,
Cell Signaling #9271), anti-p44/42 MAPK (ERK 1/2, Cell Signaling #9102), anti-phospho-
p44/42 MAPK (ERK½, Cell Signaling #4370), cyclin D1 (Cell Signaling #2978), CDK6 (Cell
Signaling#3136), cycling D3 (Cell Signaling #2936) and p21 Waf1/Cip1 (Cell Signaling #2947).
Then membranes were incubated with horseradish peroxidase-conjugated secondary antibodies
and proteins were detected via chemiluminescence.
4
.4.5. Cell cycle analysis
Cell cycle was analyzed by flow cytometry through staining of cellular DNA content of HepG2
cells with propidium iodide (Sigma-Aldrich). After treatment with 5µM of OSU-2S or the most
potent compounds (10c, 10l, 10m, 10o, 10p, 10q and 10n) for 24 h, cells were harvested and
o
fixed with 70% ethanol for 24h at 4 C. The fixed cells were then stained with staining solution
containing 5µg/ml propidium iodide and 50 µg/ml RNase. Stained cells were then analyzed
using BD FACSAria Flow cytometer (San Jose, CA, USA). DNA histograms were obtained
using FlowJo V.10 software (Ashland, OR, USA). DMSO was used as a negative control.
4
.4.6. Wound healing migration assay
2
8