Design and synthesis of pregnenolone/2-cyanoacryloyl conjugates with dual NF-κB inhibitory and anti-proliferative activities

Article abstract of DOI:10.1016/j.bmcl.2017.09.013

Twenty-five novel pregnenolone/2-cyanoacryloyl conjugates (6–30) were designed and prepared, with the aim of developing novel anticancer drugs with dual NF-κB inhibitory and anti-proliferative activities. Compounds 22 and 27–30 showed inhibition against T

Full text of DOI:10.1016/j.bmcl.2017.09.013

Accepted Manuscript
Design and synthesis of pregnenolone/2-cyanoacryloyl conjugates with dual
NF-κB inhibitory and anti-proliferative activities
Jia-Li Song, Juan Zhang, Chang-Liang Liu, Chao Liu, Kong-Kai Zhu, Fei-Fei
Yang, Xi-Gong Liu, João Paulo Figueiró Longo, Luis Alexandre Muehlmann,
Ricardo Bentes Azevedo, Yu-Ying Zhang, Yue-Wei Guo, Cheng-Shi Jiang, Hua
Zhang
PII:
S0960-894X(17)30895-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.09.013
BMCL 25277
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
28 July 2017
2 September 2017
5 September 2017
Please cite this article as: Song, J-L., Zhang, J., Liu, C-L., Liu, C., Zhu, K-K., Yang, F-F., Liu, X-G., Longo, J.P.F.,
Alexandre Muehlmann, L., Azevedo, R.B., Zhang, Y-Y., Guo, Y-W., Jiang, C-S., Zhang, H., Design and synthesis
of pregnenolone/2-cyanoacryloyl conjugates with dual NF-κB inhibitory and anti-proliferative activities,
Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.09.013
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Graphical Abstract
Design and synthesis of pregnenolone/2-cyanoacryloyl conjugates with dual NF-κB
inhibitory and anti-proliferative activities
a,†
Jia-Li Song , Juan Zhang , Chang-Liang Liu , Chao Liu , Kong-Kai Zhu , Fei-Fei Yang ,
b,c†
d
e
a
a
a
b,c
b,c
Xi-Gong Liu , João Paulo Figueiró Longo , Luis Alexandre Muehlmann , Ricardo
b
Bentes Azevedo , Yu-Ying Zhang *, Yue-Wei Guo , Cheng-Shi Jiang , Hua Zhang *
a,
f
a,*
a,
Privileged
Fragment
Combination
Linkage
O
Michael acceptor
H
CN
H
O
H
H
N
O N
2
N
H
O
O
electron-withdrawing
at meta-substitution
Lead compound 30
1
2
3
) NF-κB inhibitory activity with IC50 = 2.5 µM
) Cytotoxicity against MCF-7, A549, H157, and HL-60 cell lines with IC50 = 6.5~36.2 µM
) Effect of electron-withdrawing group at meta-substitution increased their activity
1

Design and synthesis of pregnenolone/2-cyanoacryloyl conjugates with dual
NF-κB inhibitory and anti-proliferative activities
a,†
Jia-Li Song , Juan Zhang , Chang-Liang Liu , Chao Liu , Kong-Kai Zhu , Fei-Fei
b,c†
d
e
a
a
a
b,c
b,c
Yang , Xi-Gong Liu , João Paulo Figueiró Longo , Luis Alexandre Muehlmann ,
c
Ricardo Bentes Azevedo , Yu-Ying Zhang *, Yue-Wei Guo , Cheng-Shi Jiang *, Hua
a,
f
a,
a,
Zhang *
a
School of Biological Science and Technology, University of Jinan, Jinan 250022, China
b
Faculty of Ceilandia, University of Brasilia, Brasilia 72220275, Brazil
c
Institute of Biological Sciences, University of Brasília, Brasília 70910900, Brazil
d
Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA
e
Institute of Agro-Food Science and Technology, Shandong Academy of Agricultural Sciences,
Jinan 250100, China
f
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
Abstract: Twenty-five novel pregnenolone/2-cyanoacryloyl conjugates (6-30) were
designed and prepared, with the aim of developing novel anticancer drugs with dual
NF-κB inhibitory and anti-proliferative activities. Compounds 22 and 27-30 showed
inhibition against TNF-α-induced NF-κB activation in luciferase assay, which was
confirmed by Western blotting. Among them, compound 30 showed potent NF-κB
inhibitory activity (IC50 = 2.5 µM) and anti-proliferative against MCF-7, A549, H157,
and HL-60 cell lines (IC50 = 6.5~36.2 µM). The present study indicated that
pregnenolone/2-cyanoacryloyl conjugate I can server as a novel scaffold for
developing NF-κB inhibitors and anti-proliferative agents in cancer chemotherapy.
Keywords: Pregnenolone, NF-κB, 2-Cyanoacryloyl, Michael acceptor,
Anti-proliferative activity.
*
Corresponding authors. E-mail addresses: yuyingzhang2008@163.com (Y.-Y. Zhang),
jiangchengshi-20@163.com (C.-S. Jiang), bio_zhangh@ujn.edu.cn (H. Zhang).
†
These authors contributed equally to this work.
2

Cancer is a group of diseases characterized by uncontrolled cell growth, which
1
has became a major public health concern over the last several decades. The unmber
of new diagnosed cancer cases reached nearly 14.5 million in 2014 and is expected to
2
rise by about 30% in the next decade. The abnormal cell growth can be a result of
gene mutations induced by DNA damage or aberrant activation of cell signaling
3
-5
pathways, such as hormones cytokines and chemokines.
Among the factors
involved in cancer, the nuclear factor-κB (NF-κB) as a ubiquitous eukaryotic
transcription factor plays an important role in regulating the expression of more than
6
50 genes associated with inflammation, immunity, and cell growth. In particular,
1
the aberrant activation of NF-κB has been frequently observed in various types of
human cancers, and suppression of NF-κB can limit the proliferation of cancer
7
cells. Therefore, NF-κB has been pointed as a therapeutical target in cancer, and
,8
inhibitors of NF-κB function could be developed into new anticancer drugs or
9
-11
leads.
It is generally believed that the Michael acceptors in bioactive compounds can
form adducts with reactive thiol groups of proteins to induce protein modification and
misfolding, which might be responsible for their various biological effects, such as
1
2-14
anti-proliferative activity.
As a Michael acceptor, 2-cyanoacryloyl moiety has
been extensively applied in the design of anticancer drugs. For example, tyrphostin
AG490 (1a, Fig. 1) is the first small molecular Jak2 inhibitor that is clinically used as
an anticancer agent and is also effective in various models of inflammatory and
1
autoimmune diseases; indole/2-cyanoacryloyl hybrid 1b was reported to show
5
1
6
anti-proliferative activity against a range of cancer cells; CDDO-Me (1c) is a
semi-synthetic triterpenoid acting as an inhibitor of NF-κB pathway, which was
shown to be a drug candidate for treating cancer, chronic kidney disease, and other
1
7
diseases.
Pregnenolone (2a, Fig. 1) is an important naturally occurring endogenous steroid
and known as a precursor to most of steroid hormones like estrogen, progesterone,
1
8
testosterone, and glucocorticoids.
The unique structural features and the
broad-spectrum bioactivites of 2a make it to be a promising drug leads, attracting
3

1
9-21
numerous interest in structural modification from the fielf of medicinal chemistry.
2
2,23
As part of our project to develop new drugs derived from pregnenolone,
a new
NF-κB inhibitor, derivative 2b (Fig. 1) with an IC50 value of 12.2 µM, was recently
screened out from our in-house compound library using NF-κB pathway luciferase
assay. In anti-proliferation assay, 2b did not show significant activity against A549
and MCF7 cancer cell lines.
Figure 1. 2-cyanoacryloyl derivatives 1a-1c and steroids 2a and 2b
In our continuous effort to discover anticancer drugs with dual NF-κB inhibitory
and anti-proliferative activities, a privileged fragment combination (PFC) strategy was
recently employed to further modify the structure of 2b. Since pregnenolone is a
privileged molecular skeletion and 2-cyanoacryloyl moiety as Micheal acceptor is a
very useful moiety in the design of anticancer drugs, both pharmacophores was
combined to generate conjugates I, as shown in Fig. 2. In addition, the ethanediamine
linker was preserved since numerous bioactive compounds or drugs contain the
2
4
N-CH
2
-CH
2
-N
moiety.
Herein,
we
reported
the
synthesis
of
pregnenolone/2-cyanoacryloyl conjugate I, and the biological evaluation for their dual
NF-κB inhibitory and anti-proliferative activities.
N-CH -CH -N linkage
2
2
O
Michael acceptor
H
R1
R2
CN
O
H
N
H
H
N
H
O
O
I
Privileged Fragment Combination
4

Figure 2. Proposed novel pregnenolone/2-cyanoacryloyl conjugate I
The synthesis for target compounds 6-30 is depicted in Scheme 1. Briefly,
pregnenolone (1a) was reacted with p-nitrophenyl chloroformatein dichloromethane
using pyridine as the base to obtain the intermediate 3, which was then reacted with
2
5
N-Boc-ethylenediamine in the presence of Et
3
N to yield compound 4. N-Boc
protection in 4 was removed by treating with TFA followed by coupling with
2
6 , 27
cyanoacetic acid in the presence of EDCI and HOBT to give amide 5.
Knoevenagel condensation reaction of 5 with various substituted benzaldehydes
2
catalyzed by piperidine finally afforded pregnenolone/2-cyanoacryloyl conjugates
8
6
-30. The general procedures and spectrascopic data of all synthetic compounds were
described in Supplementary data.
Scheme 1. Synthesis of 6-30. Reagents and conditions: (a) p-nitrophenyl
chloroformate, pyridine, CH
Et N, CH Cl , rt, overnight, 84%; (c) i. trifluoroacetic acid, CH
cyanoacetic acid, EDCI, HOBT, DIEPA, CH Cl , 36 h, 84%; (d) substituted
2
Cl
2
, rt, overnight, 79%; (b) N-Boc-ethylenediamine,
3
2
2
2
Cl , rt, overnight; ii.
2
2
2
benzaldehyde, piperidine, EtOH, 19%-76%.
Table 1
NF-κB inhibitory activity and cytoxicity of analogs 4-30 against HEK293/NF-κB cells.
%
Inhibition on NF-κB
at 20 µg/mL
76.6
b
Viability at 20 µg/mL (%) IC50 (NF-κB, µM)
No.
R
4
5
85.5
87.9
12.2±0.2
12.4±0.1
74.4
6
7
46.7
44.9
5

8
9
22.3
22.2
17.8
59.3
34.6
14.5
5.8
1
0
1
2
3
4
5
6
1
1
1
1
1
1
4.2
22.6
17
5.0
1
8
9
2.1
1.2
1
20
0.5
21
22
23
24
25
26
27
28
43.1
64.2
18.8
46.4
15.9
30.9
64.4
81.4
60.8
7.1±0.5
64.4
59.6
7.5±1.2
3.3±0.1
6

2
9
0
78.3
83.9
74.5
54.7
10.3±0.2
3
2.5±0.1
PS-341
-
0.030±0.007
a
IC50 values are taken as a mean from three independent experiments.
The NF-κB inhibitory activity of target compounds 6-30 and their synthetic
intermediates 4 and 5 was evaluated in TNF-α-stimulated HEK293/NF-κB cells,
2
9
according to a previously reported luciferase assay. TNF-α (tumor necrosis factor
3
alpha) is a known inducer of NF-κB activity, and PS-341 (bortezomib) as known
0
3
1
NF-κB inhibitor was used as reference compound. In brief, The HEK293/NF-κB
cells were inoculated into 96-well plates. After 24 h, the cells were treated with
TNF-α and then incubated with tested compounds. Signal strength of luciferase was
detected by EnVision. The results for these tests were summarized as percentage of
inhibition or IC50 values in Table 1.
At first, the inhibitory activity of compounds against NF-κB was tested at the
concentration of 20 µg/mL (equal to 42 ~ 31 µM for each different compound). The
percentage inhibition of synthetic intermediates 4 and 5 was 76.6% and 74.4%. For
most target compounds, especially those (6-20) with electron-donating substituents in
benzene moiety, showed very weak activity (<50%) against NF-κB at the
concentration of 20 µg/mL, while 22 and 27-30 possessing electron-withdrawing
2
groups (such as F, CN, and NO ) displayed significant inhibitory effect on NF-κB at
the same tested concentration. These results indicated that electron-withdrawing effect
of substituent in benzene ring plays an important role in their bioactivity. The
cytoxicity of selected compounds including 4, 5, 22 and 27-30 was evaluated at 20
µg/mL. From the results, these compounds did not show significant cytotoxicity
against HEK293/NF-κB cells at the tested concentration.
Then, the IC50 values of 4, 5, 22 and 27-30 for NF-κB inhibition were further
tested. The synthetic intermediates 4 and 5 showed NF-κB inhibitory activity with
IC50 values ~12 µM. Among these compounds, 28 and 30 showed the strongest
inhibition against NF-κB (IC50 ~ 3 µM) with around 2- and 4-fold increased activity
7

compared with 27 and 29, respectively, indicating that meta-substitution of
electron-withdrawing group was superior to its para-substitution. A similar result was
also observed by comparing the activity of compounds 21-26. In addition, it was
obvious that the inhibitory activity increased as the effect of electron-withdrawing
2
substituents increased (30, NO > 28, CN> 22, F > 24, Cl >26, Br).
To further confirm these compounds do inhibit NF-kB in cell, the inhibitory
effects of compound 29 on NF-κB pathway activation was examined by Western
3
2
blotting. This compound was selected to be tested due to it showing the lowest
cytotoxicity. The result from Fig. 3 indicates the inhibitory effect of 10 µM 29 was
obvious.
Figure 3. Western blotting analysis of the key proteins in the NF-κB pathway after
compound 29 treatment for 4 h.
The introduction of 2-cyanoacryloyl moiety was expected to increase
anti-proliferative activity, thus these above bioactive compounds were furthermore
selected to be tested for anti-proliferative activity against a panel of cancer cell lines,
including MCF-7 (human breast adenocarcinoma), A549 (human lung carcinoma),
H157 (human oral squamous cell carcinoma), HepG2 (human hepatocellular
carcinoma), and HL-60 (human acute promyelocytic leukemia) using the MTT
3
method with doxorubicin as positive control. The results are shown in Table 2.
3
Table 2
a
In vitro anti-proliferative activity of 4, 5, 22, and 27-30 presented as IC50 (µM)
No.
4
R
MCF-7
b
>50
A549
>50
H157
>50
HepG2
>50
HL-60
>50
5
>50
>50
>50
>50
>50
2
2
6.1±0.2
>50
17.9±0.8
>50
45.6±2.2
8

2
2
2
3
7
8
9
0
>50
8.8±0.8
>50
>50
30.9±0.5
>50
24.5±0.6
21.3±1.4
>50
>50
>50
>50
>100
15.6±1.7
10.1±0.1
6.5±0.1
4.4±0.5
36.2±0.5
11.0±0.2
30.6±3.2
>50
11.2±1.3
Doxorubicin
0.61±0.02
1.0±0.1
0.91±0.14
a
IC50 value was taken as a mean from three independent experiments.
b
IC50 value higher than 50 µM was considered inactive.
From the results, both intermediates 4 and 5 lacking the 2-cyanoacryloyl moiety
were inactive against all the tested cancer cell lines. Compared with 27 and 29 with
weak anti-proliferative activity, compounds 28 and 30 showed potent activity against
most tested cancer cell lines, including MCF-7, A549, H157, and HL-60 with IC50
values ranging from 6.5 to 36.2 µM. These results confirmed the vital importance of
the meta-substitution of electron-withdrawing group in benzene ring for their activity.
However, their anti-proliferative activity seems not directly rely on NF-κB inhibitory
activities.
In conclusion, a series of pregnenolone/2-cyanoacryloyl conjugates were
synthesized and evaluated for their NF-κB inhibitory activity and in vitro
anti-proliferative against different cancer cell lines. Several compounds showed
significant inhibitory activity on TNF-α-induced NF-κB activation and exhibited
potent anti-proliferative against MCF-7, A549, H157, and HL-60 cell lines. The
preliminary SAR studies suggested that the electron-withdrawing effect of substituent
in this series of conjuates plays an important role in their bioactivity. More studied
was needed to clearify their anticancer mechanism, and further inversitgation on the
effect of diamine linkage and heterocycles, instead of benzene, on their bioactivity are
in progress and will be reported in due course.
Acknowledgements
This research work was financially supported by the Natural Science Foundation
of China (No. 21672082), Shandong Key Development Project (No.
9

2016GSF201209), Young Taishan Scholars Program (No. tsqn20161037), Shandong
Talents Team Cultivation Plan of University Preponderant Discipline (No. 10027),
Natural Science Foundation of Shandong Province (Nos. BS2015YY021,
ZR2016HB43), and by the Brazilian Government Agencies FAP/DF and CNPq.
Supplementary data
Supplementary data related to this article can be found, in the online version, at
doi:#.
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Sin. 2013, 34, 939-950.). In detail, HEK293/NF-κB cells, which are HEK293 cells
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and pcDNA3.1 were co-transfected into HEK293 cells using Lipofectamine 2000 at a
concentration of 10:1 (Invitrogen, Carlsbad, CA, USA). Stable recombinant cells were
11

selected for resistance to 1 mg/mL G418 and for a strong luciferase signal. The cells
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nine different concentrations (0.78-20 µM) for 6 h. A 25 µL amount of complexed
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5 µL of zymolyte for 30 min. Signal strength of luciferase was detected by EnVision
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2 Western blotting analysis: anti-p65, anti-phospho-p65 (Ser536), anti-IκBα(L35A5)
were purchased from Cell Signaling Technology (Boston, Massachusetts, USA). All
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subjected to electrophoresis on 10% SDS-PAGE gels and blotted with the indicated
antibodies. Relative expression was determined by densitometric comparison with an
internal control such as β-actin, and the treatment with compounds 29 or PS-341 at
varying concentrations was estimated from IC50 value in luciferase assay.
3
3 Mosmann, T. J. Immunol. Methods 1983, 65, 55.
12

Legends
Fig. 1. 2-cyanoacryloyl derivatives 1a-1c and steroids 2a and 2b
Fig. 2. Proposed novel pregnenolone/2-cyanoacryloyl conjugate I.
Fig. 3. Western blotting analysis
Scheme 1. Synthesis of 6-30.
Supporting Information.
13