Design, synthesis and biological evaluation of erythrina derivatives bearing a 1,2,3-triazole moiety as PARP-1 inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.103575

Inhibitors of poly (ADP-ribose) polymerase-1 (PARP-1) have shown to be promising in clinical trials against cancer, and many researchers are interested in the development of new PARP-1 inhibitors. Herein, we designed and synthesized 44 novel erythrina derivatives bearing a 1,2,3-triazole moiety as PARP-1 inhibitors. MTT assay results indicated that compound 10b had the most potent anti-proliferative activity against A549 cells among five cancer cells. The enzyme inhibitory activity in vitro of compound 10b was also significantly better than rucaparib. Furthermore, the selectivity index of compound 10b was higher than rucaparib for lung cancer cells. Flow cytometry analysis showed that compound 10b induced apoptosis of A549 cells by the mitochondrial pathway. Western blot analysis indicated that compound 10b was able to inhibit the biosynthesis of PAR effectively, and it was more potent than rucaparib. Also, compound 10b was able to up-regulate the ratio of bax/bcl-2, activate caspase-3, and ultimately induced apoptosis of A549 cells. The combined results revealed that the discovery of novel non-amide based PARP-1 inhibitors have great research significance and provide a better choice for the future development of drugs.

Full text of DOI:10.1016/j.bioorg.2020.103575

Journal Pre-proofs
Design, synthesis and biological evaluation of erythrina derivatives bearing a
1
,2,3-triazole moiety as PARP-1 inhibitors
Shuai Li, Xin-yang Li, Ting-jian Zhang, Ju Zhu, Wen-han Xue, Xin-hua Qian,
Fan-hao Meng
PII:
S0045-2068(19)32091-7
DOI:
https://doi.org/10.1016/j.bioorg.2020.103575
YBIOO 103575
Reference:
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Bioorganic Chemistry
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6 December 2019
7 January 2020
8 January 2020
Please cite this article as: S. Li, X-y. Li, T-j. Zhang, J. Zhu, W-h. Xue, X-h. Qian, F-h. Meng, Design, synthesis
and biological evaluation of erythrina derivatives bearing a 1,2,3-triazole moiety as PARP-1 inhibitors,
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Design, synthesis and biological evaluation of erythrina derivatives
bearing a 1,2,3-triazole moiety as PARP-1 inhibitors
Shuai Li, Xin-yang Li, Ting-jian Zhang, Ju Zhu, Wen-han Xue, Xin-hua
Qian, Fan-hao Meng*
School of Pharmacy, China Medical University, 77 Puhe Road, North New
Area, Shenyang 110122, China
Correspondence: Professor Fan-hao Meng, School of Pharmacy, China
Medical University, Shenyang 110122, P. R. China.
E-mail: fhmeng@cmu.edu.cn
Fax: +86-24-31939448
Abstract
Inhibitors of poly (ADP-ribose) polymerase-1 (PARP-1) have shown to
be promising in clinical trials against cancer, and many researchers are
interested in the development of new PARP-1 inhibitors. Herein, we
designed and synthesized 44 novel erythrina derivatives bearing a 1,2,3-
triazole moiety as PARP-1 inhibitors. MTT assay results indicated that
compound 10b had the most potent anti-proliferative activity against A549
cells among five cancer cells. The enzyme inhibitory activity in vitro of
compound 10b was also significantly better than rucaparib. Furthermore,
the selectivity index of compound 10b was higher than rucaparib for lung
cancer cells. Flow cytometry analysis showed that compound 10b induced
apoptosis of A549 cells by the mitochondrial pathway. Western blot
1

analysis indicated that compound 10b was able to inhibit the biosynthesis
of PAR effectively, and it was more potent than rucaparib. Also, compound
1
0b was able to up-regulate the ratio of bax/bcl-2, activate caspase-3, and
ultimately induced apoptosis of A549 cells. The combined results revealed
that the discovery of novel non-amide based PARP-1 inhibitors have great
research significance and provide a better choice for the future
development of drugs.
Keywords: Erythrina; 1,2,3-Triazole; PARP-1 inhibitor; Apoptosis
1
. Introduction
Poly (ADP-ribose) polymerases (PARPs) are a family of enzymes that
catalyze the transfer of ADP-ribose from nicotinamide adenine
+
dinucleotide (NAD ) onto acceptor proteins. Among this family, PARP-1
is the most abundant and well-characterized member, and it is a promising
antitumor drug target [1-5]. The development of PARP-1 inhibitors has
been a research focus of antitumor drugs in recent years. PARP-1 plays a
significant role in the DNA repair process and regulating cell survival and
death [6-10]. Several PARP-1 inhibitors have been developed and used in
clinical treatment, such as rucaparib (Fig. 1). These inhibitors usually
possess an amide fragment that could interact with Ser904 and Gly863
residues and considered as the key pharmacophore for the PARP-1
inhibition.
2

Fig.1. The structures of rucaparib, B-10n, erythrina and spiro
[quinazoline-2, 1'-cyclohexane] derivatives.
Nature is an attractive source of new therapeutic candidate compounds
as a tremendous chemical diversity, and natural products have proven the
utility as sources of novel structures, particularly in the area of cancer
[11,12]. The genus erythrina belongs to the Fabaceae family (subfamily
Faboideae and tribe Phaseoleae), with more than 100 species distributed
throughout tropical and warm regions of the world (Fig. 1). The isolated
extracts of this genus had been studied by many investigators and were
well documented as a good source of biologically active compounds
[13,14]. At present, a large number of studies had reported that erythrina
derivatives had antitumor activity [15-19]. Besides, it was reported that
some erythrina analogs characterized by a spiro ring fragment are potential
PARP-1 inhibitors [20], such as spiro [quinazoline-2, 1'-cyclohexane]
3

derivatives (Fig. 1). Therefore, searching for novel PARP-1 inhibitors with
erythrina as a lead compound is an attracting work.
Our team is committed to the discovery of novel PARP-1 inhibitors and
had reported a series of homoerythrina alkaloid derivatives bearing a 1,2,3-
triazole moiety with potent PARP-1 inhibition [21]. Among them,
compound B-10n was considered to be the most promising one (Fig. 1)
[21]. Compared with classic PARP-1 inhibitors, although these compounds
do not contain classic amide fragments, they are a new class of promising
PARP-1 inhibitors. Here, we got inspiration from previous research,
hoping to use erythrina as a lead compound to discover novel non-amide
based PARP-1 inhibitors, and enriched the structural types of PARP-1
inhibitors. As an essential nitrogen heterocyclic compound, 1,2,3-triazole
was widely used in the construction of drug molecules, especially in the
field of anticancer [22,23]. Because of the chemical features, 1,2,3-triazole
was proposed to be aggressive pharmacophores that participate in drug–
receptor interactions while maintaining an excellent chemical and
metabolic profile [23]. So, we had retained 1,2,3-triazole moiety in order
to improve the activity of the compound.
In this work, we had synthesized two series of erythrina derivatives
bearing a 1,2,3-triazole moiety by the molecular assembly principle (Fig.
2
). By studying their biological activities and action mechanisms, a novel
PARP-1 inhibitor, compound 10b, was identified to be a promising
4

compound for research, and our work further enriched the structure types
of PARP-1 inhibitors.
Fig. 2. Design strategy of the target compounds.
2
. Results and discussion
2
.1 Chemistry
The routes to synthesize the target compounds 10a-10v and 12a-12v
were outlined in Scheme 1.
The preparation of 1-phenylcyclohexan-1-amine (compound 4) was
prepared according to the method reported by Theodoros S [24,25] and our
previous study [21]. Based on the literature, compound 4 underwent the
Alkylation reaction, the Hydrolysis reaction, the Friedel-Crafts reaction,
and the Alkylation reaction to form compound 9 [26-29], and compound 9
was subjected to the Reduction reaction to obtain compound 11. Finally,
they were reacted with the corresponding azidebenzene by the click
chemistry to give the target compounds.
5

The click chemistry is a practical approach for the generation of 1,2,3-
triazole. The copper(I)-catalyzed 1,2,3-triazole-forming reaction between
azides and terminal alkynes has become the gold standard of click
chemistry due to its reliability, specificity, and biocompatibility [30-32].
The yields and operational simplicity of this method made it a particularly
attractive protocol for laboratory-scale synthesis [21]. In order to speed up
the reaction rate, we used a microwave reaction generator, which is a
simple, efficient and pollution-free instrument, in the process of
synthesizing the target compounds (10a-10v, 12a-12v), and obtained
excellent results. The synthesis and post-treatment methods of the target
compounds had explicitly been described in the general methods.
6

Br
ⅰ
ⅱ
ⅲ
ⅳ
ⅶ
OH
NH2
N3
2
3
4
1
ⅴ
ⅵ
HN
HN
HN
7 O
O
OH
Cl
O
6
O
5
N
ⅷ
N
ⅸ
N
NH
N
N
click chemistry
O
O
O
9
8
1
0a-10v
R
x
N
N
N
ⅸ
N
N
click chemistry
OH
12a-12v
OH
R
11
b, R=2-F
e, R=2-CH3 h, R=2-Cl k, R=2-OCH3
n, R=2-Br q, R=2-OH t, R=2-CN
f, R=3-CH3 i, R=3-Cl
g, R=4-CH3 j, R=4-Cl
l, R=3-OCH3
o, R=3-Br
r, R=3-OH
u, R=3-CN
v, R=4-CN
a, R=H c, R=3-F
d, R=4-F
m, R=4-OCH3 p, R=4-Br s, R=4-OH
Scheme 1. Reagents and conditions: (ⅰ) Mg, cyclohexanone,
ethoxyethane, 35 ℃, 3 h, 95.75%; (ⅱ) NaN , 20 ℃, 12 h; (ⅲ) LiAlH , 40
3
4
℃
, 12 h, 89.6%; (ⅳ) Ethyl 2-bromoacetate, K CO , 80 ℃, 15 h, 86.86%;
2
3
(
ⅴ) NaOH, water, 50 ℃, 2 h, 96.89%; (ⅵ) SOCl , 35 ℃, 2 h; (ⅶ) AlCl ,
2
3
3
0 ℃, 24 h, 47.2%; (ⅷ) 3-bromoprop-1-yne, 70 ℃, 12 h, 87.78%; (ⅸ)
Corresponding azidebenzene, 35 ℃, 9 minutes, 75%-88%; (x) LiAlH4,
6
0 ℃, 12 h.
2
.2 Biological evaluation
7

2
.2.1 MTT assay and SAR analysis.
The synthesized target compounds were evaluated for anti-proliferative
activities against five cancer cell lines A549, OVCAR-3, HepG2, A375,
and SW-620 by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium
bromide (MTT) method (Table 1). For comparison, one FDA-approved
drug rucaparib was used as the positive control.
Table 1
The IC values of compounds and rucaparib against A549, OVCAR-3,
5
0
HepG2, A375, and SW-620 cell lines.
Compounds
IC50 [a](µM)
A549
OVCAR-3
>50
HepG2
A375
SW-620
Compound 8
29.69 ± 0.48
1.26 ± 0.11
0.23 ± 0.16
0.94 ± 0.08
1.11 ± 0.16
1.86 ± 0.66
1.21 ± 0.02
2.64 ± 0.41
1.71 ± 0.33
2.13 ± 0.25
4.29 ± 0.22
0.98 ± 0.46
1.53 ± 0.31
1.92 ± 0.19
1.69 ± 0.30
2.29 ± 0.02
1.51 ± 0.30
2.09 ± 1.31
3.98 ± 0.72
1.56 ± 0.23
5.88 ± 2.27
6.10 ± 0.12
6.05 ± 1.90
1.29 ± 0.21
>50
>50
>50
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0a
0b
0c
0d
0e
0f
1.89 ± 0.19
1.13 ± 0.06
2.89 ± 0.47
3.02 ± 0.54
4.69 ± 0.12
12.55 ± 4.67
4.68 ± 1.30
4.79 ± 3.63
5.91 ± 0.60
9.23 ± 0.51
2.54 ± 0.97
3.86 ± 0.44
1.77 ± 0.37
1.90 ± 0.20
5.45 ± 1.49
3.07 ± 0.31
2.17 ± 0.35
6.28 ± 0.89
1.98 ± 0.34
8.00 ± 1.32
9.70 ± 1.04
2.37 ± 0.88
12.74 ± 1.56
0.94 ± 0.17
0.79 ± 0.17
0.84 ± 0.09
1.13 ± 0.15
0.99 ± 0.05
2.94 ± 0.52
3.39 ± 1.59
2.09 ± 0.10
6.54 ± 2.30
1.15 ± 0.12
0.88 ± 0.04
>50
2.97 ± 0.75
0.89 ± 0.07
2.56 ± 0.60
1.05 ± 0.09
1.72 ± 0.13
1.21 ± 0.09
1.59 ± 0.25
5.39 ± 0.89
14.65 ± 0.39
1.30 ± 0.17
3.79 ± 0.32
1.31 ± 0.09
1.95 ± 0.56
1.46 ± 0.20
1.02 ± 0.12
1.05 ± 0.12
2.96 ± 0.10
2.82 ± 0.06
1.02 ± 0.03
3.57 ± 0.61
1.13 ± 0.10
1.86 ± 0.18
5.24 ± 0.77
1.57 ± 0.19
0.77 ± 0.54
1.49 ± 0.14
1.57 ± 0.54
3.16 ± 0.68
2.08 ± 0.19
2.57 ± 0.60
3.29 ± 0.41
3.09 ± 0.53
9.49 ± 2.81
3.44 ± 1.32
5.50 ± 3.32
1.17 ± 0.63
3.18 ± 0.68
4.64 ± 0.54
14.25 ± 10.12
3.08 ± 0.96
1.38 ± 0.51
>50
0g
0h
0i
0j
0k
0l
0m
0n
0o
0p
0q
0r
0s
2.22 ± 0.15
4.10 ± 0.16
>50
1.67 ± 0.24
4.05 ± 1.01
12.69 ± 2.03
15.51 ± 1.42
1.87 ± 0.17
1.09 ± 0.08
2.95 ± 0.24
0.38 ± 0.05
0t
>50
0u
0v
2a
>50
3.07 ± 0.52
2.11 ± 0.25
8

1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2b
2c
2d
2e
2f
1.61 ± 0.08
1.50 ± 0.06
2.00 ± 0.45
3.03 ± 0.24
3.30 ± 0.63
5.33 ± 0.24
1.67 ± 0.57
1.65 ± 0.41
1.44 ± 0.27
2.58 ± 0.71
5.99 ± 1.37
1.42 ± 0.59
3.87 ± 1.03
2.43 ± 0.36
2.28 ± 0.08
6.86 ± 0.23
1.18 ± 0.78
9.94 ± 11.14
2.34 ± 0.14
3.79 ± 1.57
1.26 ± 0.21
4.69 ± 0.26
3.75 ± 0.29
3.96 ± 1.00
5.09 ± 2.03
3.80 ± 1.01
>50
3.13 ± 0.18
2.58 ± 0.83
2.03 ± 0.16
4.81 ± 2.34
1.94 ± 0.08
2.65 ± 0.06
4.43 ± 0.23
1.66 ± 0.07
13.67 ± 7.00
14.59 ± 6.96
41.30 ± 25.05
2.20 ± 0.45
1.31 ± 0.04
8.33 ± 0.75
1.92 ± 0.22
2.51 ± 0.07
5.51 ± 1.20
4.98 ± 0.36
7.12 ± 1.86
5.32 ± 0.62
1.20 ± 0.39
8.01 ± 1.02
3.25 ± 0.48
3.86 ± 0.78
15.98 ± 1.72
18.00 ± 1.57
11.75 ± 2.95
2.37 ± 0.50
4.43 ± 0.30
4.55 ± 0.95
5.80 ± 1.10
6.59 ± 0.31
6.69 ± 0.13
4.12 ± 0.18
5.02 ± 0.67
4.93 ± 1.02
2.80 ± 0.25
2.73 ± 0.17
3.20 ± 0.36
2.83 ± 0.29
8.50 ± 1.41
3.81 ± 1.16
1.60 ± 0.08
12.59 ± 0.64
3.32 ± 0.88
2.13 ± 1.01
2.39 ± 0.11
2.04 ± 0.20
1.56 ± 0.09
2.70 ± 0.04
1.97 ± 0.57
3.48 ± 0.34
1.88 ± 0.24
3.77 ± 0.14
1.30 ± 0.22
4.84 ± 0.11
1.54 ± 0.07
1.36 ± 0.04
2.77 ± 0.33
5.98 ± 0.03
1.99 ± 0.18
>50
2g
2h
2i
>50
2.83 ± 1.88
3.40 ± 1.22
5.98 ± 0.31
7.64 ± 1.43
11.15 ± 6.17
4.27 ± 1.64
10.97 ± 0.06
9.60 ± 1.78
23.28 ± 3.66
2.45 ± 0.70
3.53 ± 0.67
3.61 ± 0.23
3.74 ± 0.09
4.51 ± 0.51
2.92 ± 0.44
2.58 ± 0.09
2j
2k
2l
2m
2n
2o
2p
2q
2r
2s
2t
>50
2u
2v
>50
1.72 ± 0.02
13.82 ± 1.21
Rucaparib
[
a] IC values were used to express inhibition effect (IC = Mean ± SD).
5
0
50
From MTT assay after 24 h of treatment; the values were average from at
least 3 independent experiments.
The results in Table 1 indicated that some of the target compounds
displayed moderate to potent inhibitory activities against malignant tumor
cell growth. According to these data, the preliminary SARs of novel
erythrina derivatives be summarized.
We found that in the target compounds 10a-10v, when R was replaced
by 2-F, the compound 10b had the best anti-proliferative activity (IC =
5
0
0
.23 μM) against A549 cells. Simultaneously, for the electron-withdrawing
group, the F group (10b-10d) was superior to the Cl group (10h-10j) and
9

the Br group (10n-10p). In the electron- withdrawing group, when R was
replaced by -CN (10t-10v), the anti-proliferative activity in A549 cells was
significantly weaker than other substituents. When R was replaced by 2-
OCH , the compound 10k had better anti-proliferative activity than other
3
ortho substituent compounds in the electron-donating group.
In the target compounds 12a-12v, for the electron-withdrawing group,
when R was replaced by -Br (12n-12p), the anti-proliferative activity in
A549 cells was significantly weaker than the F group (12b-12d) and the Cl
group (12h-12j). When R was replaced by -CN (12t-12v), the anti-
proliferative activity of compound 12v was significantly better than
compound 12t and compound 12u. When R was replaced by 2-F,
compound 10b had better anti-proliferative activity than compound 12b.
In summary, we found that the compound 10b had the most potent anti-
proliferative activity in A549 cells (IC = 0.23 μM). And the anti-
5
0
proliferative activity of compound 10b was better than rucaparib.
Compound 10b was expected to be a new potential anticancer drug.
2
.2.2 Investigation of PARP-1 enzyme inhibition by candidate compounds.
In order to further verify whether the target compounds had enzyme
inhibitory activity in vitro, we selected some compounds with better anti-
proliferative activity and different substituents from all the target
compounds as representatives and performed in vitro enzyme experiments.
As shown in Table 2, we found that the candidate compounds had certain
1
0

enzyme inhibitory activities. Moreover, the activity of most candidate
compounds was better than rucaparib. The compound 10b was
significantly better than the positive drug. When R was substituted with
2
-F, the enzyme inhibitory activity of compound 10b was markedly better
than that of compound 12b, and this corresponded to the MTT results.
The results showed that more than half of the candidate compounds had
better enzyme inhibitory activity than the positive drug, and compound 10b
was the most prominent. Therefore, we used the compound 10b as a hit
compound and conducted a more in-depth study.
Table 2
PARP-1 enzyme inhibitory activity of candidate compounds.
Compounds
0a
0b
0f
IC50 [b] (nM)
18.70 ± 2.43
14.42 ± 1.23
18.19 ± 2.05
15.47 ± 1.79
24.72 ± 3.57
21.52 ± 2.17
Compounds
12j
IC50 [b] (nM)
39.10 ± 5.06
25.85 ± 3.81
28.53 ± 4.02
20.45 ± 2.69
23.88 ± 2.90
1
1
1
12m
12r
12v
1
1
1
0k
0p
2b
Rucaparib
[
b] IC values are the mean ± S.D. of three separate experiments.
5
0
Besides, the representative compound 10b was further selected for
exploring the enzyme selectivity between PARP-1 and PARP-2. As shown
in Table 3, the results showed that the selective inhibitory activity of
compound 10b on PARP-1 was better than PARP-2.
Table 3
The IC values of compound 10b against PARP-1/2 enzyme.
5
0
Compounds
IC50 [c] (nM)
1
1

PARP-1
PARP-2
1
0b
14.42 ± 1.23
23.88 ± 2.90
38.96 ± 3.49
25.79 ± 3.17
Rucaparib
[
c] IC values are the mean ± S.D. of three separate experiments.
5
0
2
.2.3 Molecular docking study of compound 10b
To further predicted the possible interaction of compound 10b with
PARP-1, molecular simulation of compound 10b in the PARP-1 binding
pocket was performed using MOE (Molecular Operating Environment,
version 2016.08, Canadian Chemical Computing Group Corporation). In
the docking calculation, the crystal structure of PARP-1 with rucaparib
(PDB code: 4BJC) was used. According to the docking results (Fig. 3), the
fragments with benzospirocyclic features provided the hydrogen-bonding
interactions with residues Ser1068 and Gly1032, and arene-arene
interactions with residue Tyr1071. These interactions are consistent with
the effects shown by rucaparib. And, the phenyl-triazole side chain of
compound 10b provided the arene-H interaction with H O. Also, the
2
phenyl-triazole side chain provided the arene-cation interaction with
residue Lys1067.
From the molecular docking results, we found that although compound
1
0b lacked a carboxamide pharmacophore compared to clinically validated
PARP-1 inhibitors, there are essential binding interactions between
compound 10b and PARP-1. And, the results were consistent with the
effects shown by rucaparib. The results showed that the benzospiro ring
fragment of compound 10b had the same interaction mode with the
1
2

carboxamide structure during the process of molecular docking. The
preliminary docking results could support the fact that the compound 10b
was a PARP-1inhibitor.
Fig. 3. The 2D ligand interactions of compound 10b with key residues of
PARP-1. The interaction mode was obtained by molecular docking (PDB
code: 4BJC) and depicted using MOE 2016.08.
2
.2.4 The selectivity index of compound 10b.
To further verify the safety of compound 10b, MTT assay was performed
on healthy cells corresponding to these five cancer cells and used the
selectivity index (SI) to represent. As shown in Table 4, the compound 10b
had a higher SI for HPAEpiC (SI = 19.35), and its SI even higher than the
positive drug.
The above results indicated that compound 10b had high safety on lung
1
3

cancer cells, so we mainly studied the effect of compound 10b in A549
cells.
Table 4
Selectivity index of compound 10b and rucaparib against HPAEpiC,
IOSE80, LO2, HaCat and NCM460 cell lines.
Compound
SI (Selectivity index) [d]
HPAEpiC [e] IOSE80 [f] LO2 [g]
HaCat [h] NCM460 [i]
1
0b
19.35
8.97
7.22
8.15
5.98
8.04
6.01
6.87
9.57
Rucaparib
11.57
[
d] SI = CC / IC ; [e] Human normal alveolar epithelial cells; [f] Human
5
0
50
normal ovarian epithelial cells; [g] Human normal liver cells; [h] Human
normal skin cells; [i] Human normal colonic epithelial cells.
2
.2.5 Compound 10b inhibited the proliferation of A549 cells by inducing
apoptosis.
As a process of programmed cell death, apoptosis is triggered when
healthy cells exceeded life limits or suffered irreversible damage. But
tumor cells were able to suppress apoptosis considerably to gain constant
growth. PARP-1 is a well-known enzyme that participates in DNA repair
and cell death. According to previous research, when PARP-1 was
inhibited, it caused apoptosis, thereby achieving the purpose of inhibiting
cell proliferation [21]. To investigate whether compound 10b was capable
of inducing apoptosis, flow cytometry was performed on A549 cells with
Annexin V/PI staining, which treated with different concentrations of the
compound 10b.
1
4

Flow cytometry showed that the apoptotic rate increased as the
concentration of the compound 10b increased (Fig. 4 (A)). And, the early
apoptotic rate and the late apoptotic rate up-regulated significantly. The
trend of early apoptotic rate was more significant than the late apoptotic
rate.
The results indicated that the compound 10b was able to inhibit cell
proliferation by inducing apoptosis in A549 cells. The result is
significantly different (P < 0.001).
1
5

Fig. 4. Compound 10b induced apoptosis in A549 cells. The effect of
compound 10b on A549 cells death was evidenced by Annexin V-FITC/PI
double staining and FACS analysis. Three independent experiments were
performed, each column represented the mean ± SD of triplicate
1
6

determinations (*P < 0.05; **P < 0.01 and ***P < 0.001. Compound 10b-
treated group VS control).
2
.2.6 Compound 10b caused changes in mitochondrial membrane potential,
thereby inducing apoptosis in A549 cells.
Mitochondria, as the energy center of the cell, are closely connected with
cell apoptotic events. According to related literature reports, when PARP-
1
was inhibited, mitochondrial membrane potential changes, leading to
apoptosis [33-36]. Mitochondria play an essential role in the process of
apoptosis. Mitochondrial function is a critical indicator of cell survival, and
most of the antitumor drugs induce cell apoptosis by the mitochondria-
mediated intrinsic pathway [37]. To further verify whether compound 10b
induced apoptosis via the mitochondrial pathway, we tested mitochondrial
membrane potential changes by flow cytometry.
As shown in Fig. 4 (B), we found that the compound 10b was able to
significantly cause a change in mitochondrial membrane potential,
resulting in a marked increase in apoptosis rate, and the difference was
concentration-dependent.
The results showed that compound 10b effectively induced changes in
mitochondrial membrane potential, thereby inducing apoptosis and
ultimately inhibiting cell proliferation. The result is significantly different
(P < 0.001).
2
.2.7 The effect of candidate compounds on PARP-1 enzyme activity in
1
7

A549 cells.
PARP-1 is termed a ‘guardian of the genome’, because it senses DNA
damage and helps its repair. When PARP-1 is inhibited, DNA function is
impaired, thereby achieving the purpose of treating tumors. PAR (Poly
(ADP-ribose)) is the active product of PARP-1. When PARP-1 is inhibited,
the amount of biosynthesis of PAR will decrease, thereby inducing
apoptosis and achieving the purpose of inhibiting cell proliferation [38-42].
We directly tested the expression of PAR and PARP-1 by western blot.
From the results, we found that these compounds effectively inhibited the
biosynthesis of PAR. At the same 1.0 μM concentration, compound 10b
was considered to be the most effective inhibitor, and it was more potent
than rucaparib in inhibiting PARP-1 enzyme activity (Fig. 5 (A)). Also,
with the increase of the concentration of compound 10b, the biosynthesis
of PAR decreased in a concentration-dependent manner (Fig. 5 (B)).
Combined with the above results, the compound 10b was able to inhibit
the enzymatic activity of PARP-1 more effectively, thereby reducing the
biosynthesis of PAR, and achieving the purpose of inhibiting the
proliferation of A549 cells. This further demonstrated that compound 10b
had more potential research value as a novel PARP-1 inhibitor in anti-
tumor. The result is significantly different (P < 0.001).
1
8

Fig. 5. Western blot analysis: (A): The effect of rucaparib and candidate
compounds on the expression of PAR and PARP-1 in A549 at a
concentration of 1.0 μM. (B): Expression of PAR and PARP-1 at different
1
9

concentrations of compound 10b in A549; Effect of compound 10b for the
apoptotic protein expression of caspase-3, bax/bcl-2 and cleaved caspase-
3
in A549. Each column represented the mean ± SD of triplicate
determinations (*P < 0.05; **P < 0.01 and ***P < 0.001. Compound 10b
treated group VS control).
.2.8 Effect of compound 10b for the apoptotic protein expression of
-
2
caspase-3, bax/bcl-2, and cleaved caspase-3 in A549.
Studies had shown that when PARP-1 is inhibited, DNA function is
impaired, leading to activation of the apoptotic signaling pathway and
ultimately inhibition of cancer cell proliferation [21,43]. Therefore, in
order to further verify the specific mechanism by which compound 10b
inhibits cancer cell proliferation, we explored the expression of apoptosis-
related proteins.
Western blot results showed that in Fig. 5 (B), the protein expression of
bax was significantly increased in a concentration-dependent manner,
while the protein expression of bcl-2 was significantly decreased in a
concentration-dependent manner. Moreover, the ratio of cleaved caspase-
3
/caspase-3 increased as the concentration of compound 10b increased.
The results showed that compound 10b was able to up-regulate the ratio
of bax/bcl-2, activated caspase-3, and ultimately induced to apoptosis. The
result is significantly different (P < 0.001).
3
. Conclusion
2
0

In summary, based on the combination principle of drug structure, we
designed and synthesized two series, a total of 44 novel PARP-1 inhibitors.
Further biological analysis showed that compound 10b had vigorous
inhibitory activity against A549 cells (IC = 0.23 μM). Compound 10b
5
0
also had better enzyme inhibitory activity in vitro than rucaparib. And its
selectivity index was also significantly better than the positive drug. Also,
compound 10b induced apoptosis by the mitochondrial pathway to inhibit
the proliferation of A549 cells. Western blot analysis showed that
compound 10b effectively inhibited the enzymatic activity of PARP-1 in
A549 cells, thereby preventing the biosynthesis of PAR. And, we found
that compound 10b was able to up-regulate the ratio of bax/bcl-2, activate
caspase-3, and ultimately achieved the purpose of inducing apoptosis.
In conclusion, we enriched the structure types of PARP-1 inhibitors and
obtained a more promising non-amide based PARP-1 inhibitor. Compound
1
0b provided a better source for the future development of new PARP-1
inhibitors.
4
. Experimental procedures
4
.1 General methods
Unless otherwise indicated, reagents and solvents were purchased from
commercial sources and applied directly in the experiments without further
purification. Reactions were monitored by TLC, performed on silica gel
glass plates containing 60 GF-254, and visualization on TLC was achieved
2
1

by UV light. All target compounds were purified via silica gel column
chromatography. All melting points were obtained on the RD-1 melting
1
apparatus and were uncorrected. H-NMR spectra were recorded on a
1
3
Bruker Ascend 600 MHz spectrometer, and C-NMR spectra were run at
1
50 MHz. Chemical shifts (δ) were reported in ppm downfield from
internal TMS standard and used DMSO-d as solvents. HR-MS spectral
6
data were obtained on Agilent Accurate-Mass Q-TOF 6540 (Agilent, Santa
Clara, CA, USA). All target compounds were found to have > 95% purity.
4
.1.1 The synthesis of 1-phenylcyclohexan-1-amine
The synthesis methods of the compound 4 mainly refer to our previous
research [21]. And we also detailed the process in the supplementary data.
4
.1.2 The synthesis of ethyl (1-phenylcyclohexyl)glycinate
K CO (31.54 g, 0.228 mol), ethyl 2-bromoacetate (19.06 g, 0.114 mol),
2
3
and compound 4 (20.00 g, 0.114 mol) were simultaneously added to
acetonitrile, and the temperature was raised to 80 °C and reacted for 15 h.
After the reaction was completed, 500 ml of water was added to the
reaction system. Next, the mixture was extracted with EA (3 × 100 mL).
The combined organic layers were dried over anhydrous Na SO4,
2
evaporated in vacuo, and purified by column chromatography to give
compound 5 as white oil. Yield: 86.86%.
4
.1.3 The synthesis of target compound 10a-10v
The synthesis methods of the target compound 10a-10v mainly refer to
2
2

our previous research [21]. And we also detailed the process in the
supplementary data.
4
.1.4
The
synthesis
of
2'-(prop-2-yn-1-yl)-3',4'-dihydro-2'H-
spiro[cyclohexane-1,1'-isoquinolin]-4'-ol
Lithium aluminum hydride (0.45 g, 0.0112mol) and compound 9 (1.00
g, 0.004 mol) were simultaneously added to tetrahydrofuran at low
temperature, and the temperature was raised to 60 °C and reacted for 12 h
to give crude compound 11 as brown powder, which was used directly in
the next step. The post-treatment process was consistent with the method
of compound 4.
4
.1.5 The synthesis of target compound 12a-12v
The experimental and post-treatment procedures in this section were
consistent with the methods of target compounds 10a-10v.
.1.5.1 2'-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-2',3'-dihydro-4'H-
spiro[cyclohexane-1,1'-isoquinolin]-4'-one (10a)
4
1
A white powder, yield: 85.2%. Mp: 154.6-157.2 ℃. H NMR (600 MHz,
DMSO-d ) δ 8.58 (s, 1H), 7.85–7.82 (m, 2H), 7.77 (dd, J = 7.7, 1.3 Hz,
6
1
H), 7.67–7.56 (m, 4H), 7.47 (dd, J = 7.4, 5.3 Hz, 2H), 3.66 (s, 2H), 2.23
(s, 2H), 1.99 (d, J = 5.0 Hz, 2H), 1.91 (s, 2H), 1.78 (d, J = 12.2 Hz, 1H),
1
1
1
.57 (s, 2H), 1.36 (d, J = 12.8 Hz, 1H), 1.24 (s, 1H), 1.17 (d, J = 7.1 Hz,
1
3
H); C NMR (150 MHz, DMSO-d ) δ 197.81, 148.60, 146.30, 137.12,
6
35.17, 130.39, 130.27, 128.96, 127.22, 125.78, 125.44, 122.40, 120.49,
2
3

5
4.35, 43.66, 25.90, 21.25, 14.56. ESI-HRMS calcd. for C H N O [M +
2
3
25
4
+
H] 373.2023, found: 373.2033.
4
.1.5.2 2'-((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2',3'-
dihydro-4'H-spiro[cyclohexane-1,1'-isoquinolin]-4'-one (10b)
1
A brown powder, yield: 83.4%. Mp: 156.2-155.8 ℃. H NMR (600 MHz,
DMSO-d ) δ 8.35 (d, J = 2.0 Hz, 1H), 7.85 (td, J = 7.8, 1.6 Hz, 2H), 7.79–
6
7
1
1
1
1
.72 (m, 2H), 7.47–7.41 (m, 4H), 3.67 (s, 2H), 2.23 (s, 2H), 1.88 (s, 2H),
.76 (d, J = 12.4 Hz, 2H), 1.56 (s, 2H), 1.36 (m, 2H), 1.23 (s, 1H), 1.17 (s,
1
3
H); C NMR (150 MHz, DMSO-d ) 197.79, 170.82, 162.78, 154.18,
6
48.64, 145.85, 142.24, 135.16, 132.00, 131.94, 130.41, 126.54, 126.38,
26.07 (d, J = 3.7 Hz), 125.81, 125.38, 55.91, 43.60, 25.87, 21.24, 14.56.
+
ESI-HRMS calcd. for C H FN O [M + H] 391.1929, found: 391.1920.
2
3
24
4
4
.1.5.3
2'-((1-(3-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2',3'-
dihydro-4'H-spiro[cyclohexane-1,1'-isoquinolin]-4'-one (10c)
1
A brown powder, yield: 84.1%. Mp: 155.5-156.7 ℃. H NMR (600 MHz,
DMSO-d ) δ 8.67 (s, 1H), 7.82–7.74 (m, 3H), 7.66–7.61 (m, 2H), 7.46 (d,
6
J = 7.7 Hz, 1H), 7.38–7.29 (m, 2H), 3.65 (s, 2H), 2.21 (s, 2H), 1.98 (d, J =
4
1
1
1
.8 Hz, 2H), 1.95–1.86 (m, 2H), 1.77 (d, J = 12.6 Hz, 1H), 1.56 (s, 2H),
1
3
.36 (m, 1H), 1.17 (t, J = 7.1 Hz, 2H); C NMR (150 MHz, DMSO-d ) δ
6
97.75, 170.80, 163.68, 162.06, 148.51, 146.55, 142.91, 135.17, 130.35,
27.24, 125.81, 125.45, 124.03, 122.55, 116.30 (d, J = 2.9 Hz), 115.69,
2
4

5
4.30, 43.62, 25.90, 21.22, 14.54. ESI-HRMS calcd. for C H FN O [M
2
3
24
4
+
+
H] 391.1929, found: 391.1939.
4
.1.5.4
2'-((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2',3'-
dihydro-4'H-spiro[cyclohexane-1,1'-isoquinolin]-4'-one (10d)
1
A brown powder, yield: 84.7%. Mp: 153.1-154.2 ℃. H NMR (600 MHz,
DMSO-d ) δ 8.57 (s, 1H), 7.91–7.87 (m, 2H), 7.77 (dd, J = 7.7, 1.3 Hz,
6
1
H), 7.65 (td, J = 7.7, 1.5 Hz, 1H), 7.47–7.43 (m, 3H), 7.36 (td, J = 7.6, 1.0
Hz, 1H), 3.67 (s, 2H), 2.22 (s, 2H), 1.99–1.98 (m, 1H), 1.90 (d, J = 12.9
Hz, 2H), 1.77 (d, J = 12.5 Hz, 1H), 1.57 (s, 2H), 1.41–1.32 (m, 2H), 1.23
1
3
(
s, 1H), 1.18 (t, J = 7.1 Hz, 1H); C NMR (150 MHz, DMSO-d ) δ 197.79,
6
1
1
1
3
4
70.81, 162.80, 161.17, 148.57, 146.36, 135.16, 130.37, 127.22, 125.78,
25.43, 122.79 (d, J = 8.7 Hz), 122.63, 117.01, 54.34, 43.64, 25.90, 21.24,
+
4.56. ESI-HRMS calcd. for C H FN O [M + H] 391.1929, found:
2
3
24
4
91.1930.
.1.5.5 2'-((1-(o-tolyl)-1H-1,2,3-triazol-4-yl)methyl)-2',3'-dihydro-4'H-
spiro[cyclohexane-1,1'-isoquinolin]-4'-one (10e)
1
An off-white solid, yield: 77.2%. Mp: 151.3-152.5 ℃. H NMR (600
MHz, DMSO-d ) δ 8.22 (s, 1H), 7.77 (dd, J = 7.7, 1.3 Hz, 1H), 7.64 (td, J
6
=
7.7, 1.4 Hz, 1H), 7.46 (m, 3H), 7.41–7.36 (m, 2H), 7.32 (d, J = 7.6 Hz,
1
H), 3.70 (s, 2H), 2.23 (s, 2H), 2.10 (s, 3H), 1.89 (d, J = 8.2 Hz, 3H), 1.76
1
3
(
m, 2H), 1.56 (s, 2H), 1.40–1.31 (m, 2H), 1.18 (t, J = 7.1 Hz, 1H); C
NMR (150 MHz, DMSO-d ) δ 197.74, 148.84, 145.25, 136.78, 135.12,
6
2
5

1
1
33.39, 131.75, 130.44, 130.07, 127.35, 127.17, 126.36, 125.78, 125.66,
25.37, 58.16, 54.60, 25.88, 21.24, 17.89, 14.56. ESI-HRMS calcd. for
+
C H N O [M + H] 387.2179, found: 387.2169.
2
4
27
4
4
.1.5.6 2'-((1-(m-tolyl)-1H-1,2,3-triazol-4-yl)methyl)-2',3'-dihydro-4'H-
spiro[cyclohexane-1,1'-isoquinolin]-4'-one (10f)
1
An off-white solid, yield: 76.4%. Mp: 153.5-154.2 ℃. H NMR (600
MHz, DMSO-d ) δ 8.54 (s, 1H), 7.77 (dt, J = 8.5, 4.2 Hz, 1H), 7.70–7.61
6
(m, 3H), 7.47–7.43 (m, 2H), 7.38–7.35 (m, 1H), 7.29 (m, 1H), 3.65 (s, 2H),
2
2
.41 (s, 3H), 2.21 (s, 2H), 1.98 (d, J = 5.3 Hz, 2H), 1.90 (m, 2H), 1.77 (m,
1
3
H), 1.56 (s, 2H), 1.35 (m, 1H), 1.17 (t, J = 7.1 Hz, 1H); C NMR (150
MHz, DMSO-d ) δ 197.78, 148.57, 146.23, 139.99, 137.09, 135.16, 130.38,
6
1
4
30.04, 129.52, 127.23, 125.79, 125.44, 122.30, 120.87, 117.56, 54.30,
3.64, 25.90, 21.38, 14.55, 4.18. ESI-HRMS calcd. for C H N O [M +
2
4
27
4
+
H] 387.2179, found: 387.2189
.1.5.7 2'-((1-(p-tolyl)-1H-1,2,3-triazol-4-yl)methyl)-2',3'-dihydro-4'H-
spiro[cyclohexane-1,1'-isoquinolin]-4'-one (10g)
4
1
An off-white solid, yield: 79.1%. Mp: 151.3-152.4 ℃. H NMR (600
MHz, DMSO-d ) δ 8.51 (s, 1H), 7.79–7.76 (m, 1H), 7.72–7.70 (m, 2H),
6
7
3
2
.63 (td, J = 7.6, 1.4 Hz, 1H), 7.43 (d, J = 7.7 Hz, 1H), 7.38–7.34 (m, 3H),
.66 (s, 2H), 2.36 (s, 3H), 2.20 (s, 2H), 1.98 (d, J = 5.2 Hz, 2H), 1.89 (m,
1
3
H), 1.76 (m, 2H), 1.55 (s, 2H), 1.34 (m, 1H), 1.16 (s, 1H); C NMR (150
MHz, DMSO-d ) δ 197.78, 170.79, 148.57, 146.16, 138.48, 135.12, 134.90,
6
2
6

1
2
3
4
30.57, 127.19, 125.78, 125.39, 122.20, 120.32, 54.32, 43.64, 25.90, 21.21,
+
1.02, 14.53. ESI-HRMS calcd. for C H N O [M + H] 387.2179, found:
2
4
27
4
87.2185.
.1.5.8
2'-((1-(2-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2',3'-
dihydro-4'H-spiro[cyclohexane-1,1'-isoquinolin]-4'-one (10h)
1
A yellow powder, yield: 80.2%. Mp: 148.6-149.2 ℃. H NMR (600
MHz, DMSO-d ) δ 8.32 (s, 1H), 7.79 (dd, J = 7.7, 1.2 Hz, 1H), 7.74 (d, J
6
=
7.7 Hz, 1H), 7.64–7.56 (m, 4H), 7.45 (d, J = 7.7 Hz, 1H), 7.39 (t, J = 7.2
Hz, 1H), 3.66 (s, 2H), 2.22 (s, 2H), 1.89 (m, 3H), 1.75 (d, J = 12.5 Hz, 2H),
1
3
1
1
1
.55 (s, 3H), 1.39–1.30 (m, 2H); C NMR (150 MHz, DMSO-d ) δ 197.74,
6
48.70, 145.40, 135.18, 135.06, 131.96, 130.96, 130.40, 128.92, 128.85,
28.78, 127.23, 126.26, 125.86, 125.43, 58.24, 54.46, 43.67, 25.89, 21.53.
+
ESI-HRMS calcd. for C H ClN O [M + H] 407.1633, found: 407.1646.
2
3
24
4
4
.1.5.9
2'-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2',3'-
dihydro-4'H-spiro[cyclohexane-1,1'-isoquinolin]-4'-one (10i)
1
A yellow powder, yield: 79.8%. Mp: 149.1-149.6 ℃. H NMR (600
MHz, DMSO-d ) δ 8.68 (s, 1H), 7.99 (t, J = 2.0 Hz, 1H), 7.87 (m, 1H),
6
7
.76 (dd, J = 7.7, 1.3 Hz, 1H), 7.64–7.58 (m, 2H), 7.53 (m, 1H), 7.44 (d, J
7.7 Hz, 1H), 7.35 (td, J = 7.6, 0.9 Hz, 1H), 3.64 (s, 2H), 2.20 (s, 2H),
.97 (d, J = 4.8 Hz, 1H), 1.93–1.85 (m, 2H), 1.76 (d, J = 12.4 Hz, 2H), 1.55
=
1
1
3
(
s, 2H), 1.35 (m, 2H), 1.16 (t, J = 7.1 Hz, 1H); C NMR (150 MHz,
DMSO-d ) δ 197.75, 170.81, 154.11, 148.52, 146.55, 142.91, 135.17,
6
2
7

1
4
4
4
34.59, 132.00, 130.34, 128.73, 127.24, 125.80, 125.46, 122.57, 54.30,
+
3.63, 25.91, 14.55, 4.19. ESI-HRMS calcd. for C H ClN O [M + H]
2
3
24
4
07.1633, found: 407.1621.
.1.5.10 2'-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2',3'-
dihydro-4'H-spiro[cyclohexane-1,1'-isoquinolin]-4'-one (10j)
1
A yellow powder, yield: 80.6%. Mp: 146.5-147.2 ℃. H NMR (600
MHz, DMSO-d ) δ 8.61 (s, 1H), 7.89 (d, J = 8.9 Hz, 2H), 7.76 (dd, J = 7.7,
6
1
1
1
.2 Hz, 1H), 7.68–7.61 (m, 3H), 7.43 (d, J = 7.8 Hz, 1H), 7.37–7.32 (m,
H), 3.65 (s, 2H), 2.20 (s, 2H), 1.98–1.83 (m, 4H), 1.76 (d, J = 12.4 Hz,
1
3
H), 1.55 (s, 3H), 1.38–1.28 (m, 1H), 1.18 (dt, J = 14.2, 4.7 Hz, 1H); C
NMR (150 MHz, DMSO-d ) δ 197.74, 148.53, 146.50, 135.91, 135.13,
6
1
5
4
4
33.17, 130.36, 130.20, 127.20, 125.79, 125.40, 122.44, 122.08, 58.26,
+
4.33, 43.63, 25.90, 14.54. ESI-HRMS calcd. for C H ClN O [M + H]
2
3
24
4
07.1633, found: 407.1617.
.1.5.11 2'-((1-(2-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-2',3'-
dihydro-4'H-spiro[cyclohexane-1,1'-isoquinolin]-4'-one (10k)
1
An off-white solid, yield: 77.9%. Mp: 148.6-149.3 ℃. H NMR (600
MHz, DMSO-d ) δ 8.17 (s, 1H), 7.78 (dd, J = 7.7, 1.3 Hz, 1H), 7.63 (m,
6
1
H), 7.54–7.49 (m, 2H), 7.44 (m, 1H), 7.38 (td, J = 7.6, 0.9 Hz, 1H), 7.31
(m, 1H), 7.13 (m, 1H), 3.73 (m, 2H), 2.23 (s, 2H), 1.99 (s, 3H), 1.88 (m,
2
3
H), 1.76 (d, J = 12.5 Hz, 2H), 1.56 (s, 2H), 1.38–1.32 (m, 1H), 1.17 (s,
1
3
H); C NMR (150 MHz, DMSO-d ) δ 197.82, 170.79, 151.99, 148.69,
6
2
8

1
1
44.95, 135.09, 130.97, 130.44, 126.24, 126.11, 125.93, 125.80, 125.35,
21.26, 113.39, 60.22, 54.45, 43.65, 25.87, 21.20, 14.53. ESI-HRMS calcd.
+
for C H N O [M + H] 403.2129, found: 403.2142.
2
4
27
4
2
4
.1.5.12
2'-((1-(3-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-2',3'-
dihydro-4'H-spiro[cyclohexane-1,1'-isoquinolin]-4'-one (10l)
1
An off-white solid, yield: 78.9%. Mp: 149.1-149.4 ℃. H NMR (600
MHz, DMSO-d ) δ 8.59 (s, 1H), 7.79–7.75 (m, 1H), 7.67–7.62 (m, 1H),
6
7
.44 (m, 5H), 7.04 (dd, J = 8.2, 1.8 Hz, 1H), 3.65 (s, 2H), 2.22 (s, 2H), 1.99
(s, 3H), 1.90 (d, J = 9.3 Hz, 3H), 1.77 (m, 2H), 1.56 (s, 3H), 1.39–1.33 (m,
1
3
1
1
1
1
4
4
H), 1.17 (t, J = 7.1 Hz, 1H); C NMR (150 MHz, DMSO-d ) δ 197.79,
6
70.82, 160.59, 148.58, 146.19, 138.17, 135.16, 131.20, 130.38, 127.23,
25.78, 125.45, 122.51, 114.62, 112.49, 56.07, 54.31, 43.62, 25.89, 21.24,
+
4.56. ESI-HRMS calcd. for C H N O [M + H] 403.2129, found:
2
4
27
4
2
03.2137.
.1.5.13
2'-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-2',3'-
dihydro-4'H-spiro[cyclohexane-1,1'-isoquinolin]-4'-one (10m)
1
An off-white solid, yield: 77.6%. Mp: 147.1-148.3 ℃. H NMR (600
MHz, DMSO-d ) δ 8.45 (s, 1H), 7.75 (m, 3H), 7.63 (m, 1H), 7.42 (d, J =
6
7
2
.8 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 7.11 (m, 2H), 3.81 (s, 3H), 3.67 (s,
H), 2.20 (s, 2H), 1.97 (d, J = 4.7 Hz, 1H), 1.88 (s, 3H), 1.75 (d, J = 12.3
1
3
Hz, 2H), 1.54 (s, 2H), 1.34 (m, 1H), 1.17 (m, 1H); C NMR (150 MHz,
DMSO-d ) δ 197.79, 159.57, 148.57, 146.04, 135.11, 130.59, 130.39,
6
2
9