Novel 4,5-dihydrospiro[benzo[c]azepine-1,1′-cyclohexan]-3(2H)-one derivatives as PARP-1 inhibitors: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bioorg.2021.104840

To further explore the research of novel PARP-1 inhibitors, we designed and synthesized a series of novel amide PARP-1 inhibitors based on our previous research. Most compounds displayed certain antitumor activities against four tumor cell lines (A549, HepG2, HCT-116, and MCF-7). Specifically, the candidate compound R8e possessed strong anti-proliferative potency toward A549 cells with the IC₅₀ value of 2.01 μM. Compound R8e had low toxicity to lung cancer cell line. And the in vitro enzyme inhibitory activity of compound R8e was better than rucaparib. Molecular docking studies provided a rational binding model of compound R8e in complex with rucaparib. The following cell cycle and apoptosis assays revealed that compound R8e could arrest cell cycle in the S phase and induce cell apoptosis. Western blot analysis further showed that compound R8e could effectively inhibit the PAR's biosynthesis and was more effective than rucaparib. Overall, based on the biological activity evaluation, compound R8e could be a potential lead compound for further developing novel amide PARP-1 inhibitors.

Full text of DOI:10.1016/j.bioorg.2021.104840

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Bioorganic Chemistry 111 (2021) 104840
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Novel 4,5-dihydrospiro[benzo[c]azepine-1,1′-cyclohexan]-3(2H)-one
derivatives as PARP-1 inhibitors: Design, synthesis and
biological evaluation
Shuai Li, Xin-yang Li, Ting-jian Zhang, Ju Zhu, Kai-li Liu, De-pu Wang, Fan-hao Meng*
School of Pharmacy, China Medical University, 77 Puhe Road, North New Area, Shenyang 110122, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
To further explore the research of novel PARP-1 inhibitors, we designed and synthesized a series of novel amide
PARP-1 inhibitors based on our previous research. Most compounds displayed certain antitumor activities
against four tumor cell lines (A549, HepG2, HCT-116, and MCF-7). Specifically, the candidate compound R8e
PARP-1 inhibitor
Antitumor
Apoptosis
possessed strong anti-proliferative potency toward A549 cells with the IC50 value of 2.01 μM. Compound R8e
Drug discovery
had low toxicity to lung cancer cell line. And the in vitro enzyme inhibitory activity of compound R8e was better
than rucaparib. Molecular docking studies provided a rational binding model of compound R8e in complex with
rucaparib. The following cell cycle and apoptosis assays revealed that compound R8e could arrest cell cycle in
the S phase and induce cell apoptosis. Western blot analysis further showed that compound R8e could effectively
inhibit the PAR’s biosynthesis and was more effective than rucaparib. Overall, based on the biological activity
evaluation, compound R8e could be a potential lead compound for further developing novel amide PARP-1
inhibitors.
1. Introduction
cancer treatment. Many compounds from medicinal plants with poten-
tial antitumor activities have been reported [17]. Homoerythrina, as a
In recent years, due to the high morbidity and mortality of malignant
tumors, researchers have been committed to the research and develop-
ment of novel antitumor drugs. At present, targeted DNA damage repair
pathways have become a very effective treatment method in cancer
treatment [1]. Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear
protein related to various cellular functions. It plays a vital role in
repairing DNA breaks and participates in the regulation of DNA repli-
cation, transcription, and cell death [2–5]. PARP-1 inhibitors have
become current effective drugs for treating malignant tumors and crit-
ical targets for the design of novel antitumor drugs [6–9].
plant of the genus Cephalotaxus, had been reported to have antitumor
activity [18,19]. Also, its unique seven-membered spiro ring skeleton
structure has aroused our great interest. 5H-dibenzo[b,e]azepine-6,11-
dione derivatives are a new type of PARP-1 inhibitor previously reported
by our team [20]. Through structural analysis, we found that these
compounds all have similar structures (Color parts in Fig. 1). Therefore,
we tried to use homoerythrina as a lead compound to explore novel
amide PARP-1 inhibitors (Fig. 2).
In short, hybridization of two or more scaffolds to create a new
hybrid molecule is a useful approach for developing new therapeutic
agents [21]. Herein, we report the design and synthesis of a novel series
of 4,5-dihydrospiro[benzo[c]azepine-1,1′-cyclohexan]-3(2H)-one de-
rivatives. And we further explored their anti-proliferative activity and
the mechanism of action of the representative compound R8e. We hope
to discover a new type of amide PARP-1 inhibitor to further enrich the
structural types of PARP-1 inhibitors and provide a new possibility for
future drug development.
As monotherapy or combination therapy drugs, PARP-1 inhibitors
have broad-spectrum therapeutic significance for human malignant tu-
mors [10,11]. At present, most of the PARP-1 inhibitors have an amide
structure, such as rucaparib, olaparib and pamiparib, etc. [12–16]
(Fig. 1). The amide structure could produce hydrogen bond interactions
with the key amino acid residues Ser904 and Gly863 of PARP-1.
Therefore, we tried to discover a novel type of amide PARP-1 inhibitor
and further enrich the structural types of PARP-1 inhibitors.
Plant-derived products have always been an essential source of
* Corresponding author.
E-mail address: fhmeng@cmu.edu.cn (F.-h. Meng).
https://doi.org/10.1016/j.bioorg.2021.104840
Received 4 December 2020; Received in revised form 21 February 2021; Accepted 17 March 2021
Available online 22 March 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

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Bioorganic Chemistry 111 (2021) 104840
2. Results and discussion
compounds with electron-donating groups, when R was substituted by
–
OH (R8q-R8s), the anti-proliferative activity of the compounds was
–
–
2.1. Chemistry
weaker than that of compounds substituted by CH3 and OCH3 (R8h-
R8m). When R was substituted by H (R8a), its anti-proliferative activity
was better than that of meta-substituted compounds in the electron-
donating groups (R8i, R8l, R8r).
The synthetic methods for the preparation of target compounds 4,5-
dihydrospiro[benzo[c]azepine-1,1′-cyclohexan]-3(2H)-one derivatives
were summarized as follows (Scheme 1).
Next, to further verify the toxicity of the candidate compound R8e,
we evaluated it by the selectivity index (SI). As shown in Table 2, the
compound R8e had a higher SI than the other three cells for HPAEpiC,
and the values of compound R8e were significantly higher than that of
rucaparib. This indicated that compound R8e had low toxicity on lung
cancer cells, so we mainly studied the effect of compound R8e in A549
cells.
The preparation of 1-phenylcyclohexan-1-amine (compound 4) was
prepared according to the method reported by Theodoros S [22,23] and
our previous studies [24,25]. Compound 4 underwent alkylation reac-
tion and Friedel-Crafts acylation reaction to produce compound 6. Then
compound 6 reacted with bromine to produce compound 7. Finally,
compound 7 and phenylboronic acid with different substituents under-
went the Suzuki reaction to form target compounds. The Suzuki reaction
has a wide range of applications in organic synthesis due to its strong
substrate adaptability and functional group tolerance [26]. And the
Suzuki reaction is a special place because of its versatility, compatibility
and critical contributions to drug synthesis [27].
2.2.2. Investigation of PARP-1 enzyme inhibition by compound R8e
The above results indicated that compound R8e had the best anti-
proliferative activity and low toxicity against lung cancer cell line.
Therefore, we further explored the PARP-1 enzyme inhibitory activity
and the enzyme selectivity between PARP-1 and PARP-2 of the candi-
date compound R8e. As shown in Table 3, the results showed that
compound R8e had PARP-1 enzyme inhibitory effect in vitro, and the
selective inhibitory activity of compound R8e on PARP-1 was better
than PARP-2.
2.2. Biological evaluation
2.2.1. MTT assay and SAR analysis
All the synthesized 4,5-dihydrospiro[benzo[c]azepine-1,1′-cyclo-
hexan]-3(2H)-one derivatives were screened against A549 (human lung
carcinoma cell line), HepG2 (human liver cancer cell line), HCT-116
(human colon cancer cell line), and MCF-7 (human breast cancer cell
line) by standard MTT assay to evaluate their anti-proliferative activities
in terms of IC50 values (Table 1). By comparison, rucaparib was selected
as the positive reference.
2.2.3. Molecular docking study of compound R8e
To further explored the possible interaction of compound R8e with
PARP-1, molecular simulation of compound R8e in the PARP-1 binding
pocket was performed using MOE (PDB code: 4BJC). Simultaneously,
rucaparib was used as control.
It is reported that there are three crucial hydrogen bond interactions
between the carboxamide moiety of PARP-1 inhibitors and two critical
amino acid residues in the PARP-1 catalytic site, Ser1068 and Gly1032.
According to the docking results (Fig. 3), rucaparib showed better in-
teractions and higher score value than compound R8e (rucaparib:
ꢀ 7.0924; compound R8e: ꢀ 5.9961). Simultaneously, the docking result
of compound R8e showed that the amide structure provided the
According to the MTT analysis results, we found that compound R8e
had the best anti-proliferative activity on A549 cells (IC50 = 2.01 ± 0.98
μ
M). At the same time, compound R8e also had certain anti-proliferative
activity against other cancer cell lines. Among the compounds with
electron-withdrawing groups, when R was substituted by F (R8e-R8g),
its anti-proliferative activity on A549 cells line was better than that of
compounds substituted by Cl (R8b-R8d) and Br (R8n-R8p). In
Fig. 1. The structures of PARP-1 inhibitors, Homoerythrina and 5H-dibenzo[b,e]azepine-6,11-dione derivatives.
2

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S. Li et al.
Bioorganic Chemistry 111 (2021) 104840
hydrogen-bonding interactions with the key residues Ser1068 and
Gly1032, and the benzospiro ring structure provided arene-arene in-
teractions with residue Tyr1071. Though the docking diagram of com-
pound R8e did not show more intermolecular interactions distinctly
than rucaparib, the interactions shown are consistent with the reported
essential effects required for PARP-1 inhibitors. The preliminary dock-
ing results showed that compound R8e conformed to the structural
characteristics of PARP-1 inhibitor.
further inducing apoptosis [25]. The results of western blotting showed
that as the concentration of compound R8e increased, the ratio of PAR/
PARP-1 decreased in a concentration-dependent manner (Fig. 5B).
Simultaneously, at the same 4.0 μM concentration, compound R8e was
more potent than rucaparib (Fig. 5A). The result is significantly different
(P < 0.001).
2.2.7. Effect of compound R8e on the expression of cyclin A protein and
apoptosis-related protein in A549
2.2.4. Compound R8e could arrest cell cycle in the S phase
In this study, to elucidate the underlying mechanism of cell cycle and
apoptosis induced by compound R8e, western blot analysis was per-
formed to study the related protein expression levels. Cyclin A is a
critical protein in the S phase checkpoint. And western blot analysis
showed that when the concentration of compound R8e increases, its
expression decreases in a concentration-dependent manner (Fig. 5C). At
the same time, we further verified the expression of related proteins in
the apoptosis signaling pathway. The results showed that the levels of
bax were increased in the presence of compound R8e, whereas levels of
bcl-2 were decreased. Simultaneously, the activation of caspase 3 caused
an increase in cleaved-caspase 3 protein levels, and ultimately induced
apoptosis. The result is significantly different (P < 0.001).
This is a known fact that PARP-1 inhibitors could affect the cell cycle
by preventing the DNA damage and repair of cancer cells, leading to
tumor cell apoptosis and achieving antitumor purpose [24,28]. To
explore whether compound R8e had a similar pharmacology function,
the effects of compound R8e on cell cycle distribution were determined
by flow cytometry analysis (Fig. 4A). The results of flow cytometry
analysis showed that with an increase in the concentration of compound
R8e present in the cells for 24 h, the percentage of the S phase cells
increased (Fig. 4B). A large number of cells could not enter the G2/M
phase through the S phase, causing cell cycle arrest in the S phase. The
result is significantly different (P < 0.001).
2.2.5. Compound R8e inhibited the proliferation of A549 cells by inducing
apoptosis
3. Conclusions
Apoptosis, a programmed cell death, is a systematic process [29,30].
Our previous studies had reported that PARP-1 inhibitors could further
induce apoptosis [24,25]. To verify whether compound R8e could
induce apoptosis, A549 cells were treated with different concentrations
of compound R8e for 24 h, and flow cytometry analysis was performed.
Flow cytometry analysis results showed that the apoptotic rate of A549
cells was 4.5%, 6.7%, 7.4%, and 24.1% for each concentration,
respectively (Fig. 4A). And, with the increase in concentration, the late
apoptotic rate increased in a concentration-dependent manner (Fig. 4B)
The results indicated that compound R8e could inhibit cell prolif-
eration by inducing apoptosis. The result is significantly different (P <
0.001).
In conclusion, a series of 4,5-dihydrospiro[benzo[c]azepine-1,1′-
cyclohexan]-3(2H)-one derivatives were designed and synthesized in
this research. All compounds studied on the anti-proliferative activity of
cancer cell lines, and compound R8e showed significant anti-
proliferative activity. The SI of compound R8e was higher than ruca-
parib. Simultaneously, the results of in vitro enzyme activity test and
western blot analysis both showed that compound R8e had more po-
tential than rucaparib. Molecular docking studies were employed to
support the experimental results, and the fundamental interactions were
identified. The possible antitumor mechanism of compound R8e was to
arrest the S phase of A549 cells by down-regulating the expression of
cyclin A, and induce apoptosis by down-regulating the ratio of bcl-2/bax
and up-regulating the ratio of cleaved-caspase 3/caspase 3.
2.2.6. Verification of the target of compound R8e at the protein level
PAR (Poly (ADP-ribose)) is the active product of PARP-1. When
PARP-1 is inhibited, PAR’s amount of biosynthesis will decrease, and
In this study, we further enriched the structure types of PARP-1 in-
hibitors by modifying natural products’ structure and obtained a
promising amide PARP-1 inhibitor. The candidate compound R8e
Fig. 2. Design strategy of the target compounds.
3

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Bioorganic Chemistry 111 (2021) 104840
Scheme 1. Reagents and conditions: (i) Mg, cyclohexanone, ethoxyethane, 35 ◦C, 3 h, 95.5%; (ii) NaN3, 20 ◦C, 12 h; (iii) LiAlH4, 40 ◦C, 12 h, 89.43%; (iv) 3-chlor-
opropionyl chloride, K2CO3, 90 ◦C, 12 h, 93.7%; (v) AlCl3, 0 ◦C, 5 h, 50.2%; (vi) Br2, 6 h, 70.9%; (vii) Corresponding phenylboronic acid, Pd, K2CO3, 90 ◦C, 12 h,
75.4%-86.3%.
Table 1
Table 2
The IC50 values of target compounds and rucaparib against A549, HepG2, HCT-
116, and MCF-7 cell lines.
The SI of compound R8e and rucaparib against HPAEpiC, LO2, HIEC and HTB-
125 cell lines.
Compounds
Compounds
IC50 [a](µM)
SI [b]
A549
HepG2
HCT-116
MCF-7
HPAEpiC [c]
LO2 [d]
HIEC [e]
HTB-125 [f]
R8a
R8b
R8c
R8d
R8e
R8f
3.30 ± 0.29
6.23 ± 1.51
17.00 ± 3.31
15.78 ± 8.85
2.01 0.98
4.84 ± 1.17
5.23 ± 2.03
13.56 ± 1.98
16.55 ± 0.45
15.54 ± 3.05
19.48 ± 0.23
14.59 ± 2.63
16.29 ± 1.91
8.99 ± 1.03
10.00 ± 1.82
7.20 ± 0.79
17.85 ± 2.89
14.83 ± 3.01
18.09 ± 4.65
17.63 ± 3.19
>50
5.36 ± 2.14
3.48 ± 0.20
17.64 ± 5.44
3.35 ± 0.28
3.67 0.19
5.47 ± 0.05
4.87 ± 0.90
8.20 ± 0.24
8.99 ± 1.21
4.42 ± 1.20
3.12 ± 0.31
3.45 ± 0.05
4.64 ± 0.32
2.94 ± 0.76
3.37 ± 0.46
4.29 ± 0.06
13.88 ± 3.99
11.90 ± 2.10
16.36 ± 1.89
19.91 ± 3.92
>50
3.43 ± 1.57
9.83 ± 1.23
10.54 ± 3.21
8.35 ± 0.87
4.25 1.65
7.84 ± 2.98
6.98 ± 1.92
9.99 ± 4.13
17.90 ± 2.67
10.88 ± 3.06
13.71 ± 2.10
14.34 ± 5.51
16.70 ± 6.70
10.51 ± 4.01
18.83 ± 3.61
4.64 ± 2.15
19.27 ± 3.23
18.54 ± 0.98
19.75 ± 2.07
18.73 ± 4.01
>50
7.43 ± 0.32
5.40 ± 0.78
15.32 ± 2.07
7.68 ± 0.78
3.94 1.25
7.90 ± 0.72
5.65 ± 0.02
5.68 ± 0.07
4.88 ± 0.10
5.53 ± 0.90
9.29 ± 2.43
8.01 ± 1.77
10.53 ± 3.20
14.62 ± 4.16
12.33 ± 3.09
7.97 ± 1.09
18.01 ± 4.37
16.23 ± 3.57
15.98 ± 4.18
19.95 ± 2.78
>50
R8e
14.87
8.32
7.18
5.98
5.74
3.98
6.68
4.32
Rucaparib
b
SI = CC50/IC50
.
c
Human normal alveolar epithelial cells.
Human normal liver cells.
d
e
f
R8g
R8h
R8i
Human normal intestinal epithelial cells.
Human normal breast cell. From MTT assay after 24 h of treatment; the
values were average from at least 3 independent experiments.
R8j
R8k
R8l
Table 3
R8m
R8n
R8o
R8p
R8q
R8r
The IC50 values of compound R8e against PARP-1/2 enzyme.
Compounds
IC50 [g] (nM)
PARP-1
PARP-2
R8e
19.90 ± 1.48
23.88 ± 2.90
30.45 ± 2.56
25.79 ± 3.17
R8s
Rucaparib
R8t
g
R8u
R8v
Rucaparib
IC50 values are the mean ± S.D. of three separate experiments.
>50
>50
>50
>50
4.91 ± 0.11
8.01 ± 1.02
13.51 ± 0.17
12.98 ± 0.28
aluminum cards (0.2 mm thickness) with fluorescent indicator 254 nm.
All target compounds were purified via silica gel column chromatog-
raphy. All melting points were obtained on the RD-1 melting apparatus
and were uncorrected. HR-MS spectral data were obtained on Agilent
Accurate-Mass Q-TOF 6540 (Agilent, Santa Clara, CA, USA).
a
IC50 values were used to express inhibition effect (IC50 = Mean ± SD). From
MTT assay after 24 h of treatment; the values were average from at least 3 in-
dependent experiments.
provided a new perspective to promote the discovery of PARP-1
inhibitors.
4.1.1. The synthesis of 1-phenylcyclohexan-1-amine
The synthesis methods of the compound 4 mainly refer to our pre-
vious researches [24,25].
4. Experimental procedures
4.1. General methods
4.1.2. The synthesis of 3-chloro-N-(1-phenylcyclohexyl) propionamide
The compound 4 (10 g, 0.057 mol), K2CO3 (15.77 g, 0.114 mol), 3-
chloropropionyl chloride (7.24 g, 0.057 mol) and acetonitrile (500 mL)
were added to a 1000 mL reaction flask at 90 ◦C for 12 h. After the re-
action was completed, 300 mL of water was added to the reaction sys-
tem. Next, the mixture was extracted with EA (3 × 100 mL). After phase
separation, the organic layer was washed with brine, dried over Na2SO4,
Unless otherwise indicated, all reagents and solvents were obtained
from commercial sources and used as received. 1H and 13C NMRs were
obtained on a Bruker Ascend at 600 and 150 MHz, respectively, in the
solvent indicated with tetramethyl silane as an internal standard. TLC
monitored reactions’ time and purity of the products on FLUKA silica gel
4