Synthesis and in vitro evaluation of novel spiroketopyrazoles as acetyl-CoA carboxylase inhibitors and potential antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2020.113036

Acetyl-CoA carboxylase (ACC) is a rate-limiting enzyme in de novo fatty acid synthesis, which plays a critical role in the growth and survival of cancer cells. In this study, a series of spiroketopyrazole derivatives bearing quinoline moieties were synthesized, and in vitro anticancer activities of these compounds as ACC inhibitors were evaluated. The biological evaluation showed that compound 7j exhibited the strongest enzyme inhibitory activity (IC₅₀ = 1.29 nM), while compound 7m displayed the most potent anti-proliferative activity against A549, HepG2, and MDA-MB-231 cells with corresponding IC₅₀ values of 0.55, 0.38, and 1.65 μM, respectively. The preliminary pharmacological studies confirmed that compound 7m reduced the intracellular malonyl-CoA and TG levels in a dose-dependent manner. Moreover, it could down-regulate cyclin D1 and CDK4 to disturb the cell cycle and up-regulate Bax, caspase-3, and PARP along with the suppression of Bcl-2 to induce apoptosis. Notably, the combination of 7m with doxorubicin synergistically decreased the HepG2 cell viability. These results indicated that compound 7m as a single agent, or in combination with other antitumor drugs, might be a promising therapeutic agent for the treatment of hepatocellular carcinoma.

Full text of DOI:10.1016/j.ejmech.2020.113036

Journal Pre-proof
Synthesis and in vitro evaluation of novel spiroketopyrazoles as acetyl-CoA
carboxylase inhibitors and potential antitumor agents
Tonghui Huang, Xin Wu, Shirong Yan, Tianya Liu, Xiaoxing Yin
PII:
S0223-5234(20)31008-4
DOI:
https://doi.org/10.1016/j.ejmech.2020.113036
EJMECH 113036
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 September 2020
Revised Date: 6 November 2020
Accepted Date: 14 November 2020
Please cite this article as: T. Huang, X. Wu, S. Yan, T. Liu, X. Yin, Synthesis and in vitro evaluation of
novel spiroketopyrazoles as acetyl-CoA carboxylase inhibitors and potential antitumor agents, European
Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.113036.
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Graphical Abstract
Compound 7m exhibited significant anti-proliferative activity in HepG2 cells.

Synthesis and in vitro evaluation of novel spiroketopyrazoles
as acetyl-CoA carboxylase inhibitors and potential
antitumor agents
Tonghui Huanga, b, 1, *, Xin Wua, 1, Shirong Yana, Tianya Liuc, Xiaoxing Yina, *
a
Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou
Medical University, 221004 Xuzhou, Jiangsu, China.
b
Xuzhou Medical University Science Park, 221000, Xuzhou, Jiangsu, People’s
Republic of China.
c
Department of Pharmacy, The Affiliated Hospital of Xuzhou Medical University,
221002 Xuzhou, Jiangsu, China.
Graphical Abstract
Compound 7m exhibited significant anti-proliferative activity in HepG2 cells.
1 These two authors contributed equally to this work.
∗
Corresponding authors: Xiaoxing Yin, yinxx@xzhmu.edu.cn; Tonghui Huang,
tonghhuang@xzhmu.edu.cn.
1

Abstract
Acetyl-CoA carboxylase (ACC) is a rate-limiting enzyme in de novo fatty acid
synthesis, which plays a critical role in the growth and survival of cancer cells. In this
study, a series of spiroketopyrazole derivatives bearing quinoline moieties were
synthesized, and in vitro anticancer activities of these compounds as ACC inhibitors
were evaluated. The biological evaluation showed that compound 7j exhibited the
strongest enzyme inhibitory activity (IC50 = 1.29 nM), while compound 7m displayed
the most potent anti-proliferative activity against A549, HepG2, and MDA-MB-231
cells with corresponding IC50 values of 0.55, 0.38, and 1.65 µM, respectively. The
preliminary pharmacological studies confirmed that compound 7m reduced the
intracellular malonyl-CoA and TG levels in a dose-dependent manner. Moreover, it
could down-regulate cyclin D1 and CDK4 to disturb the cell cycle and up-regulate
Bax, caspase-3, and PARP along with the suppression of Bcl-2 to induce apoptosis.
Notably, the combination of 7m with doxorubicin synergistically decreased the
HepG2 cell viability. These results indicated that compound 7m as a single agent, or
in combination with other antitumor drugs, might be a promising therapeutic agent for
the treatment of hepatocellular carcinoma.
Keywords: acetyl-CoA carboxylase; anticancer; apoptosis; drug combination.
2

1. Introduction
Malignant cells usually have an extraordinary demand for cellular building
blocks, such as proteins, nucleotides, and lipids, to support continuous proliferation [1,
2]. Therefore, they tend to reprogram their metabolic pathways, which have been
denoted to be the emerging hallmark of cancer [3-5]. One of the most prominent
features of metabolic reprogramming is the elevated rate of de novo fatty acid
synthesis (FASyn) [6]. Unlike normal cells that primarily uptake energy-providing
lipids from the diet, malignant cells principally use lipids coming from FASyn [7, 8].
Moreover, numerous cancers, including those of the lung, liver, breast, prostate, and
colon, have demonstrated an enhancement in the rate of FASyn, and the modulation
of the lipogenic enzymes involved in the FASyn process has shown significant
inhibition in cell division and proliferation [9-11].
Acetyl-CoA carboxylase (ACC) is a crucial enzyme in fatty acid metabolism,
which catalyzes the ATP-dependent carboxylation of acetyl-CoA to produce
malonyl-CoA [12]. There are two well-characterized isoforms of mammalian ACC,
namely ACC1 and ACC2, with disparate subcellular distributions and physiological
significance. ACC1 is a cytosolic isoform that primarily responsible for FASyn,
whereas ACC2, an isoform embedded in the outer membrane of mitochondria, is
mainly in charge of the fatty acid oxidation (FAOxn) [13]. Due to the importance of
ACC in fatty acid metabolism, it has emerged as an attractive target for the treatment
of various diseases, including type 2 diabetes mellitus (T2DM), bacterial infections,
and most recently, cancer [14-16]. The research on cancer initiation and progression
mainly focuses on the ACC1 isoform due to the up-regulation of ACC1 mRNA
involved in most of the cancers [17-21]. In addition, the ACC1 silencing or deletion
was documented to significantly decrease FASyn, induce cell cycle arrest and
apoptosis, and ultimately lead to tumor cell death, while supplementation with
3

exogenous palmitate could completely rescue this effect [22, 23].
Although a variety of ACC inhibitors have been identified, only a few were
found to exhibit antitumor activities (Fig. 1) [24-30]. Compound PF-1 (ACC1 IC50 =
17 nM, Fig. 2) is a potent and orally bioavailable ACC inhibitor that was originally
designed by Pfizer for the treatment of T2DM [31], and in 2018, its derivative PF-2
was found to demonstrate significant cytotoxicity toward A549 and H1975 cells [29].
Based on this research, the anti-proliferative activity of PF-1 was assessed (Table 2).
However, the inhibitory effect of PF-1 on cancer cells was not in par with its strong
enzyme activity, which was speculated to be attributed to the low lipophilicity and
passive permeability of this compound. Furthermore, the co-crystal structure of PF-1
bound to the carboxyl transferase (CT) domain of humanized yeast ACC revealed that
the pyrazolopyranone moiety was located in a hydrophobic groove and the structural
rigidity of the spirocycle was essential for the binding [32]. Therefore, this study
focused on seeking a scaffold that would provide an optimal fit in the binding pocket,
while improving the overall lipophilicity.
Compound AZ-1 is a moderate ACC inhibitor, as identified from the high
throughput screening (IC50 = 3.6 µM, Fig. 2) [33]. In contrast to the
5-carboxyindazole group of PF-1, the quinoline carboxylic acid group of AZ-1 is
more lipophilic and can be conveniently optimized through the established synthetic
pathways [34]. Taking these advantages into account, a spiroketopyrazole scaffold
possessing quinoline moieties was designed, and various aryl or alkoxy substituents
were introduced to occupy the hydrophobic pocket (Fig. 2). It was proposed that the
hybridization could enhance the ligand affinity and passive permeability, ultimately
leading to an increase in ACC inhibitory as well as antitumor activities.
Herein, a series of spiroketopyrazole derivatives bearing quinoline moieties were
designed and synthesized to act as novel ACC inhibitors. The synthesized compounds
4

were evaluated for their biological activity toward the ACC1 enzyme and three
different cancer cell lines. Moreover, the underlying receptor-ligand interactions and
preliminary pharmacological studies were performed on representative compounds to
elucidate the mechanistic action of spiroketopyrazoles.
Fig. 1 Representative of ACC inhibitors with antitumor activity.
Fig. 2 The design strategy of spiroketopyrazoles bearing quinoline moieties as ACC inhibitors.
5

2. Results and discussion
2.1 Chemisry
Scheme 1. Synthesis of target compounds 7a~7u. Reagents and conditions: (a) H2O, r.t., 2 h, 85%;
(b) glyoxal (40 wt.% solution in H2O), H2O, reflux, 5 h, 80%; (c) THF, MeOH,
N-(tert-Butoxycarbonyl)-4-piperidone, r.t. to reflux, 24 h, 60%; (d) TFA, CH2Cl2, r.t., 2 h, 95%; (e)
HATU, triethylamine, DMF, r.t., 12 h, 45-75%; (f) amines, reflux, 2 h, 30-60%.
The synthetic route of the spiroketopyrazole derivatives 7a~7u is shown in
Scheme 1. To brief, the intermediate 3 was readily generated by the nucleophilic
addition of the commercially available compounds 1 and 2. Later, the hydrazone 3
was reacted with glyoxal in an aqueous solution to provide the intermediate 4, which
was finally converted into the key intermediate 5 under a base-catalyzed
spiro-cyclization reaction in the presence of N-(tert-Butoxycarbonyl)-4-piperidone
[31]. The Boc-protecting group was removed using trifluoroacetic acid. The final
compounds 7a~7m and 7q~7u were conveniently prepared by the condensation of
intermediate 6 with different quinoline-4-carboxylic acid derivatives. Furthermore,
6

the nucleophilic substitution reaction of compound 7r with nitrogen heterocycles
offered the desired compounds 7n~7p in a straightforward manner.
2.2 In vitro ACC inhibitory activity
To evaluate the enzymatic inhibitory activity of the target compounds, a
well-established luminescent ADP detection assay against ACC1 enzyme was
performed. Firsocostat (formerly ND-630), an ACC inhibitor under Phase II trial, was
selected as the reference compound along with PF-1 [35, 36]. As shown in Table 1,
the initial screening results of the spiroketopyrazole derivatives were generally
gratifying. All the compounds showed inhibition rates (IR) of greater than 90% under
a concentration of 5 µM, and when the concentration was reduced to 0.1 µM, most
aryl or pyridine derivatives also displayed significant inhibition toward ACC1 (IR >
80%). However, the inhibitory effect decreased remarkably for the compounds with
non-aromatic substituents (7n~7u, IR: 36.66% ~ 67.97%), which highlighted the
importance of aromaticity in ACC inhibition. Besides, the potency of compounds 7c
and 7e were weaker than the other substituted-phenyl derivatives, indicating that the
o-substitution on the phenyl was not conducive to the protein-small molecule
interactions.
Encouraged by the above results, the IC50 values of the synthesized compounds
were subsequently calculated to further explore the structure-activity relationship
(SAR). A total of eleven compounds with stronger potencies in comparison with PF-1
were identified, which were unsurprisingly substituted by aromatic groups. Among
them, the pyridine substituted compounds 7l and 7m were found to exhibit similar
inhibitory activities as that of PF-1, with the IC50 values of 13.84 nM and 11.31 nM,
respectively. In contrast, the IC50 value of the phenyl-substituted compound 7a (IC50 =
6.00 nM) was twice lower than PF-1, indicating the unique advantage of phenyl in
inhibiting the enzymatic activity. Moreover, on substituting the phenyl with
7

electron-neutral (4-CH3) or electron-rich (4-OCH3, 3,4-OCH3, 4-NH2, 4-N(CH3)2)
groups, the IC50 values of the corresponding compounds (7b, 7h~7k) were further
decreased, with the p-aminophenyl substituted compound 7j exhibiting the best ACC
inhibitory activity (IC50 = 1.29 nM).
Table 1. The ACC1 inhibitory activity and calculated physicochemical properties of 7a~7u.
IR (%) a
Compounds
IC50 (nM) a
cLogP b
LLE c
5 μM
100.69
101.35
95.53
103.89
94.39
100.46
102.23
100.55
101.95
101.46
99.63
103.07
100.31
97.56
97.43
97.67
93.60
96.74
93.76
93.88
99.37
101.35
---
0.1 μM
90.54
93.76
41.84
93.22
43.61
87.26
93.25
94.76
95.58
94.14
96.39
84.04
86.25
59.62
52.77
56.89
55.03
59.08
36.66
38.40
67.97
82.46
---
7a
7b
6.00
2.22
3.99
4.49
4.19
4.71
4.46
4.14
4.86
4.01
3.72
2.95
4.27
2.79
2.58
1.90
2.46
1.88
2.39
2.64
2.66
3.19
3.50
1.57
---
4.23
4.16
ND
3.51
ND
3.89
3.39
4.49
4.73
5.94
4.29
5.07
5.37
ND
ND
ND
ND
ND
ND
ND
ND
6.17
---
7c
> 100
6.09
7d
7e
> 100
9.40
7f
7g
5.67
7h
3.18
7i
3.57
7j
1.29
7k
2.72
7l
13.84
11.31
> 20
> 20
> 20
> 20
> 20
> 100
> 100
> 20
17.28
4.34
7m
7n
7o
7p
7q
7r
7s
7t
7u
PF-1
ND-630
a The data represent the mean values of two independent experiments.
b cLogP values were estimated by Molsoft
.
8

c LLE values were calculated according to the formula LLE = pIC50 – cLogP; ND: not determined.
2.3 Molecular docking
To gain insight into the binding modes of the synthesized compounds with ACC
enzyme, the molecular docking studies of representative compounds (7a, 7b, 7j, and
7l) were performed using the software Sybyl-X 2.1, with the results visualized by
PyMOL [37]. The initial configuration of the binding pocket was determined based on
the co-crystal structure of the ACC CT domain with PF-1 (PDB ID: 4WYO) [32], and
the docking protocol was validated by re-docking the energy-minimized PF-1 into the
theoretical binding pocket. As illustrated in Fig. 3A, the post-optimized ligand was
effectively overlapped with the original ligand, and a reasonable root-mean-square
deviation (RMSD) value was obtained (1.493 Å), which implied the reliability of the
docking settings used in this study.
Based on the above information, the selected compounds were energy-minimized
as described in the experimental section, and docked into the hydrophobic binding
pocket. As expected, compounds 7a, 7b, and 7l exhibited similar binding modes as
that of PF-1. These compounds were oriented at the active site of the CT dimer
interface with constant hydrogen-bonded interactions (Fig. 3B). However, the binding
mode of the most potent derivative 7j was obviously distinct from the rest. The ketone
carbonyl of 7j was closer to Arg-1954' rather than Glu-2026’, and two new hydrogen
bonds were observed between the ketone and Arg-1954', which partly explained its
excellent ACC inhibitory activity (Fig. 3C). Notably, it is presumed from this finding
that the shift in the ketone position may provide a promising option for the design of
ACC inhibitors in the future.
9

Fig. 3 Molecular docking studies for representative compounds (PDB ID: 4WYO). (A) Overlay of
the original PF-1 (green) with optimized PF-1 (magenta). (B) Superimposition docking
conformation of compounds PF-1 (green), 7a (yellow), 7b (magenta), and 7l (grayish) with ACC
CT domain. Surface of the binding site is colored in cyan for the N domain and white for the C
domain. (C) Docking model of compound 7j (green) with the active site of the CT domain. All
hydrogen bonds are highlighted in red dashed lines.
2.4 In vitro anticancer evaluation
To identify the potential ACC inhibitors for cancer treatment, the cytotoxicity of
the synthesized compounds against A549, HepG2, and MDA-MB-231 cells was
evaluated using the MTT assay. ACC1 is highly expressed in these cells, which
facilitates a proper assessment of the bioactivity of ACC inhibitors [22, 27, 28]. The
preliminary screening assay was performed at a fixed concentration of 10 µM, with
doxorubicin (DOX) serving as the positive control (Fig. S1). Overall, the
spiroketopyrazole derivatives exhibited varying degrees of cytotoxicity against the
10

three cancer cells. Most of them showed high activity against HepG2 cells, moderate
activity against A549 cells, and low activity against MDA-MB-231 cells. Moreover,
certain compounds were shown to be effective against the three cancer cell lines, in
which 7m exhibited the best inhibition on A549, HepG2, and MDA-MB-231 cells,
with IR values of 79.29%, 80.13%, and 72.20%, respectively.
During the initial screening process, it was observed that the antitumor effects of
the spiroketopyrazoles were not completely consistent with their ACC inhibitory
activities. It was supposed to be attributed to the difference in the lipophilicity of
target compounds. Therefore, the logarithm of the octanol-water partition coefficient
(LogP) was predicted using the Molsoft online software [38]. Moreover, the lipophilic
ligand efficiency (LLE), a metric used to evaluate the affinity of a ligand relative to its
lipophilicity, was calculated by subtracting the cLogP from the negative log10 of the
potency against ACC [39]. The IC50 values of spiroketopyrazoles against A549,
HepG2, and MDA-MB-231 cancer cells, as well as their cLogP and LLE values, are
summarized in Tables 1 and 2.
Similar to the initial screening results, the spiroketopyrazoles containing
different substituent groups exhibited significantly different inhibitory effects on the
cancer cells. Most of the aromatic substituted compounds (7a~7k) were found to
display reasonable anti-proliferative activity toward A549 and HepG2 cell lines.
However, only two compounds (7i and 7j) with relatively low cLogP values exhibited
marginally satisfactory performance toward the inhibition of MDA-MB-231 cells,
indicating the crucial role of lipophilicity in the growth inhibition of these cells.
Besides, compound 7j was found to comply with the physicochemical profiles of oral
drugs (cLogP = 2.95, LLE = 5.94) and exhibit considerable antitumor activity against
the three cancer cell lines [40], with IC50 values of 0.97, 0.63, and 4.96 µM,
respectively.
11

The pyridine-substituted derivatives 7l and 7m were potent antitumor agents for
the three cancer cell lines, with 7m exhibiting the strongest anti-proliferative effect
among the synthesized compounds. The IC50 values of 7m toward A549, HepG2, and
MDA-MB-231 cells were 0.55, 0.38, and 1.65 µM, respectively, which were
apparently superior to those of PF-1. Moreover, the cytotoxicity of 7m against A549
and HepG2 cells was better than DOX, suggesting the ability of the compound to
serve as a promising candidate for cancer treatment. The enhanced bioactivity for 7m
may be attributed to the reasonable balance between the enzyme activity, lipophilicity,
and LLE value of the compound. On the other hand, the substitution of the
compounds with non-aromatic groups (7n~7u) caused a significant decrease in the
antitumor activity against A549 and HepG2 cells, which was consistent with their
moderate ACC1 inhibitory effects. However, the anti-proliferative activity of these
compounds toward MDA-MD-231 cells was satisfactory. This could be the result of
the low lipophilicity of compounds 7n~7u.
To further investigate the selectivity of the target compounds toward cancer cells,
the human umbilical vein endothelial cells (HUVEC) were chosen for further testing
[41]. As shown in Table 2, most of the compounds were essentially non-toxic to
HUVEC, and those with strong antitumor activities (IC50 < 1 µM), namely 7d, 7 g, 7j,
and 7m, exhibited less than 20% IR toward HUVEC, which demonstrated the good
cancer cell selectivity of spiroketopyrazoles. The abnormal toxicity of 7b, 7c, and 7p
could be attributed to the off-target effects of these compounds.
Table 2. Anti-proliferative activity of 7a~7u against three cancer cell lines and HUVEC a.
IC50 (μM)
Cell viability (%)
Compounds
of HUVEC b
A549
HepG2
2.11±0.49
1.44±0.34
>10
MDA-MB-231
7a
7b
7c
1.94±0.49
5.30±1.16
1.88±0.12
>10
>10
>10
97.34±3.42
49.56±5.00
38.62±4.31
12

7d
7e
2.33±0.48
2.18±0.15
5.77±0.29
9.30±1.31
3.74±0.89
2.03±0.47
0.97±0.18
1.15±0.10
7.72±0.93
0.55±0.08
>10
0.63±0.15
>10
>10
>10
87.33±3.85
102.7±5.17
98.69±3.33
84.14±11.08
102.3±3.14
93.67±1.37
84.85±7.73
91.32±4.63
103.7±7.46
95.23±4.59
102.2±3.71
93.45±4.82
34.43±5.29
101.0±5.43
99.27±6.25
98.58±3.09
96.06±3.90
101.8±2.72
101.2±6.86
14.42±4.44
7f
2.93±0.35
0.82±0.12
2.78±0.36
1.56±0.32
0.63±0.09
1.73±0.22
1.50±0.09
0.38±0.05
8.57±1.04
9.55±0.51
4.57±0.46
9.48±3.33
4.72±0.16
>10
>10
7g
>10
7h
7i
>10
7.13±1.00
4.96±0.32
>10
7j
7k
7l
8.10±1.23
1.65±0.32
4.20±0.76
8.13±0.28
3.18±0.19
4.86±0.19
9.22±1.12
>10
7m
7n
7o
>10
7p
7q
7r
>10
>10
7.17±0.30
>10
7s
7t
>10
>10
>10
7u
PF-1
DOX
>10
3.15±0.23
1.88±0.21
0.69±0.10
>10
3.52±0.49
0.59±0.09
5.93±0.82
0.44±0.03
a Data were expressed as mean ± SD (n= 3).
b Cell viability was assessed after incubation of various compounds at a concentration of 100 μM.
2.5 Compound 7m reduces intracellular malonyl-CoA and triglyceride (TG) levels
Given the best antitumor activity of spiroketopyrazoles toward HepG2 cells, this
cell line was selected as a model for further biological research. Initially, the
antiproliferative activities of the representative compounds 7d, 7g, 7j, and 7m were
evaluated at different concentrations and time points. All the compounds were found
to inhibit HepG2 cell proliferation in a concentration- and time-dependent manner, as
illustrated in Fig. S2. Later, the effect of 7m on the malonyl-CoA and TG levels in
HepG2 cells was investigated because the inhibition of ACC leads to a decrease in the
FASyn process. The result showed that the treatment of the cells with different
13

concentrations of 7m decreased the intracellular malonyl-CoA and TG levels
dose-dependently, with an approximate reduction of 31.38% and 36.71% compared to
the control group at a concentration of 10 µM, respectively (Fig. 4).
Fig. 4 Intracellular malonyl-CoA (A) and TG (B) levels after 7m treatment. HepG2 cells were
incubated with indicated concentrations of 7m for 48 h, with the intracellular malonyl-CoA and
TG extracted and quantified. Data were expressed as mean ± SD (n= 3). **P < 0.01 and ***P <
0.001 vs. control group.
2.6 Palmitate rescues the cell death induced by 7m
To demonstrate that the cell proliferation inhibition was indeed caused by the
lack of fatty acids, a palmitate rescue assay was performed. Palmitate is one of the
most abundant dietary saturated fatty acids. Theoretically, the supplementation of the
culture medium with exogenous palmitate could improve the viability of the cancer
cells [25, 26]. Herein, the concentrations of palmitate were determined to be 10 µM
and 25 µM due to the inhibition of cell growth at higher concentrations (Fig. 5A). On
incubating the HepG2 cells with compound 7m in a medium supplemented with
palmitate for five days, the viability was significantly increased from 33.08% to 68.92%
(10 µM palmitate) and 87.12% (25 µM palmitate), respectively. These data indicated
that the palmitate completely eradicated the cell death induced by 7m, strongly
supporting the relationship between the antitumor activity of 7m and its inhibitory
effect on FASyn (Fig. 5B and 5C).
14

Fig. 5 Palmitate rescued the cell death induced by 7m. (A) Cytotoxicity of palmitate on HepG2
cells. HepG2 cells were incubated with palmitate at indicated concentrations for 1, 3 and 5 days,
respectively. (B) Palmitate rescued the cell death induced by 7m. HepG2 cells were incubated
with 7m (0.5 µM) and various concentration of palmitate (0, 10, and 25 µM) for 1, 3 and 5 days,
respectively. (C) Representative microphotographs of HepG2 cells after indicated treatments for 5
days (Scale bars = 100 μm). In A and B, the cell viability (% control) was determined by MTT
assay. Data were expressed as mean ± SD (n= 3). *P < 0.05, **P < 0.01, and ***P < 0.001 vs.
control group.
2.7 Compound 7m induces G0/G1 arrest in HepG2 cells
To further investigate the underlying mechanism of cytotoxicity, a DNA-based
cell cycle analysis was performed using the flow cytometry technique. On treating the
HepG2 cells with diverse concentrations of 7m (1, 5, 10 µM) for 48 h, representative
changes were detected in the cell cycle distribution. As shown in Fig. 6, compound
7m resulted in a significant increase in the accumulation of cells at the G0/G1 phase
from 56.24% to 88.61%, accompanied by a decrease in the cells at the S and G2/M
phases from 30.18% and 13.58% to 6.48% and 4.90%, respectively. These data
implied that 7m arrested the HepG2 cells at the G0/G1 phase in a dose-dependent
manner, which partly accounted for its potent anticancer activity.
15

Fig. 6 Compound 7m induced cell cycle arrest in HepG2 cells. (A) 7m increased the distribution
of cells in G0/G1 phase. After treatment with specified concentrations of 7m for 48 h, HepG2 cells
were harvested and stained with propidium iodide for flow cytometric analysis. (B) Histograms
displayed the percentage of cell cycle distribution. Data were expressed as mean ± SD (n= 3). *P
< 0.05, **P < 0.01, and ***P < 0.001 vs. control group.
2.8 Effect of compound 7m on cell cycle regulatory proteins
Based on the results of cell cycle distribution, the expressions of proteins, such
as Cyclin D1 and its associated protein CDK4, that were significant for the transition
from the G1 phase to the S phase were investigated using Western blot [42]. As can be
seen in Fig.7, the expressions of Cyclin D1 and its associated CDK4 were decreased
in a concentration-dependent manner, with approximately 43.92% and 60.76%
16

suppression at a concentration of 10 µM, respectively. These results suggested that the
7m-induced G0/G1 arrest might be attributed to the down-regulation of Cyclin D1
and CDK4 proteins.
Fig. 7 Effect of compound 7m on cell cycle regulatory proteins. (A) Western blotting analysis on
the effect of 7m on Cyclin D1 and CDK4. After treatment with indicated concentrations of 7m for
48 h, HepG2 cells were collected and lysed to detect Cyclin D1 and CDK4, with β-actin as an
internal control. (B, C) Histograms displayed the relative expression of Cyclin D1 and CDK4.
Data were expressed as mean ± SD (n= 3). **P < 0.01 and ***P < 0.001 vs. control group.
2.9 Compound 7m induces apoptosis in HepG2 cells
Based on the correlation between the antitumor activities of ACC inhibitors and
apoptosis [25, 27], the Hoechst 33258 staining was performed to primarily investigate
the nuclear morphological changes. As illustrated in Fig. 8A, the HepG2 cells treated
with 7m displayed characteristic apoptosis features, including chromatin shrinkage,
nuclear fragmentation, and an increase in the brightness. Furthermore, the vehicle- or
7m-treated HepG2 cells were further stained with Annexin V and PI to further
quantitatively measure the effect of 7m on cell apoptosis (Fig. 8B). It was found that
the incubation of the HepG2 cells with 7m induced a concentration-dependent
increase in both early (from 0.19% to 41.6%) and late stages of apoptosis (from 4.03%
to 45.5%) of these cells. An increase in the percentage of apoptotic cells revealed the
17

ability of compound 7m in the induction of apoptosis.
Fig. 8 Compound 7m induced cell apoptosis. (A) Apoptotic assay by Hoechst 33258. After
treatment with varying concentrations of 7m for 48 h, HepG2 cells were stained with Hoechst
33258 and then visualized (Scale bars = 100 μM). Cells with obvious apoptosis features were
marked with red arrows. (B) Apoptosis analysis using flow cytometry. HepG2 cells were exposed
to 7m for 48 h prior to staining with Annexin V-FITC/PI for flow cytometric analysis. (C)
Histograms displayed the percentage of apoptotic cells. Data were expressed as mean ± SD (n= 3).
**P < 0.01 and ***P < 0.001 vs. control group.
2.10 Effect of compound 7m on apoptosis related proteins
To elucidate the mechanism of apoptosis induced by compound 7m, the
expressions of several apoptosis-related proteins were investigated. It has been
documented that PARP, an enzyme involved in DNA repair and gene integrity
monitoring, was involved in the apoptosis process induced by the inhibition of ACC
18

[27]. Therefore, the HepG2 cells were treated with different concentrations of 7m (1,
5, 10 µM) to determine the protein expression of PARP in these cells. As a result,
there was a remarkable increase in PARP cleavage on treatment with compound 7m
(Fig. 9E).
Since PARP is an important substrate of caspase-3, the expression of caspase-3
and Bcl-2 family proteins (Bcl-2 and Bax) were further determined [43, 44]. It was
observed that the incubation of the cells with 7m decreased the expression of
anti-apoptotic Bcl-2 protein in a dose-dependent manner (Fig. 9B), while the
pro-apoptotic Bax protein expression increased accordingly (Fig. 9C). Besides, the
expression levels of the two cleaved forms of caspase-3 were elevated, indicating the
involvement of caspase-3 activation in cell apoptosis (Fig. 9D). Therefore,
compound 7m may induce apoptosis in HepG2 cells via the mitochondrial apoptotic
pathway.
Fig. 9 Effect of compound 7m on apoptosis related proteins. (A) Western blotting analysis on the
effect of 7m on Bcl-2, Bax, caspase-3, and PARP proteins. After treatment with 7m for 48 h,
HepG2 cells were collected and lysed to detect above-mentioned proteins, with β-actin as an
internal control. (B-E) Histograms displayed the relative protein expression of Bcl-2, Bax, cleaved
caspase-3, and cleaved PARP. Data were expressed as mean ± SD (n= 3). *P < 0.05, **P < 0.01,
and ***P < 0.001 vs. control group.
19

2.11 Combinations of 7m and DOX lead to synergistic inhibition effects
In the course of cancer treatment, a combination of drugs is widely used to
achieve synergistic therapeutic effect, dose and toxicity reduction, or drug resistance
alleviation [45]. Therefore, the potential of 7m in the combination therapy was
determined by analyzing the effects of the combination of 7m and DOX on HepG2
cell viability using the CompuSyn software[46]. As illustrated in Fig. 10, the
combination of 7m with DOX inhibited the viability of HepG2 cells in a
dose-dependent manner and exhibited synergistic effects (CI < 1) at different
concentrations, which highlighted the potential of compound 7m in combination or
adjuvant therapies.
Fig. 10 Effect of 7m and DOX combinations on HepG2 cells. Cell viability was examined after 72
h incubation of HepG2 cells with 7m and DOX, as single agents or in combination, at the
specified concentrations. CI values were calculated by CompuSyn software.
20

3. Conclusion
In this work, a series of new spiroketopyrazoles containing quinoline moieties
were synthesized and evaluated for their anticancer activities as ACC inhibitors. At
the enzyme level, compound 7j exhibited the strongest ACC1 inhibitory activity,
which is attributed to the two hydrogen bonds present between its carbonyl oxygen
atom and Arg-1954’. The cellular assays showed that most of the target compounds
were cytotoxic to the three cancer cells while they were essentially non-toxic to
HUVEC. Among them, compound 7m showed the highest anti-proliferative activity.
The preliminary pharmacological investigation revealed that the presence of 7m
significantly reduced the levels of malonyl-CoA and TG in the HepG2 cells, and
supplementation with exogenous palmitate could completely rescue the cytotoxicity,
which highlighted the causal relationship between FASyn and tumor cell proliferation.
Moreover, compound 7m induced cell cycle arrest and apoptosis by regulating the
cycle checkpoint proteins and mitochondrial apoptosis-related proteins, respectively.
Interestingly, the combination of 7m and DOX led to synergistic effects on the
inhibition of HepG2 cell growth, suggesting its potential in combination or adjuvant
therapies. Overall, these results demonstrated that the compound 7m, acting as an
ACC inhibitor, is a promising candidate for the treatment of hepatocellular carcinoma.
21

4. Experimental section
4.1 Chemistry
All reagents and solvents were purchased from commercial sources. The target
compounds were purified by column chromatography separations on silica gel (200–
300 mesh size). The melting points were measured on YRT-3 melting point apparatus
1
and uncorrected. H NMR and 13C NMR spectra were obtained with a JNM-ECZR
400 MHz spectrometer. High-resolution mass spectra (HRMS) were recorded on a
(UHR-TOF) maXis 4G instrument.
4.1.1 General procedure for the preparation of 7a~7u
(A) Pyruvic aldehyde (2, 25 mmol) was added dropwise to a solution of 1 (30
mmol) in H2O (100 mL), and after stirring at room temperature for 2 h, the mixture
was extracted with CH2Cl2, dried with anhydrous Na2SO4, and concentrated in vacuo
to afford hydrazine 3. Then, a 40% aqueous solution of glyoxal (25 mL) was added to
a solution of 3 (62 mmol) in H2O (100 mL). The mixture was heated at reflux for 5 h
and extracted with ethyl acetate. The organic phase was dried, filtered, and
concentrated to give 4 as yellow solid. Tetrahydropyrrole (8 mmol) was added to a
solution of 4 (24 mmol) in MeOH (45 mL), and after stirring at room temperature for
2 h, 1-(N-Boc)-4-piperidone (28 mmol) was subsequently added, with the mixture
heated at reflux for 24 h. The mixture was concentrated in vacuo and purified via
column chromatography to give 5. The Boc-protecting group was removed using
trifluoroacetic acid in CH2Cl2 to provide 6. Finally, appropriate
quinoline-4-carboxylic acid derivatives (1 mmol) was added to the mixture of 6 (1.1
mmol), HATU (1.2 mmol), and triethylamine (0.2 mL) in DMF (10 mL). The mixture
was stirred at room temperature overnight, and then added to ice water. The resulting
precipitate was filtered, dried, and purified by column chromatography to give target
compounds 7a~7m and 7q~7u.
22

(B) A mixture of 7r (1mmol) and amine solution (2 mL) was stirred at reflux for
2 h. After cooling to room temperature, the mixture was added to water and extracted
with CH2Cl2. The organic layers were dried, filtered, and concentrated in vacuo. The
crude product was purified by column chromatography to yield target compounds
7n~7p.
2'-(tert-butyl)-1-(2-phenylquinoline-4-carbonyl)-2'H-spiro[piperidine-4,5'-pyrano[3,2
-c]pyrazol]-7'(6'H)-one (7a)
The product was obtained as white solid, yield 85%, m.p. 161.5 – 162.5 °C. 1H
NMR (400 MHz, CDCl3): δ 8.24 (d, J = 8.5 Hz, 1H), 8.16 (dd, J = 6.6, 4.6 Hz, 2H),
7.84 (dd, J = 8.2, 3.3 Hz, 1H), 7.77 (dd, J = 9.5, 4.7 Hz, 2H), 7.64 – 7.45 (m, 4H),
7.23 (d, J = 3.0 Hz, 1H), 4.74 (t, J = 8.0 Hz, 1H), 3.44 – 3.35 (m, 2H), 3.25 (t, J =
11.3 Hz, 1H), 2.76 – 2.64 (m, 2H), 2.38 – 2.26 (m, 1H), 1.98 (t, J = 15.2 Hz, 1H),
1.89 – 1.79 (m, 1H), 1.64 (s, 1H), 1.59 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 185.5,
167.1, 157.1, 156.9, 148.3, 147.0, 143.2, 138.8, 138.8, 134.2, 130.5, 130.4, 130.3,
129.9, 129.8, 129.0, 127.5, 127.4, 124.3, 124.0, 123.2, 123.0, 115.7, 112.3, 80.6, 80.5,
60.9, 49.2, 42.8, 42.7, 37.1, 34.8, 34.5, 34.0, 33.8, 29.6. HRMS m/z calcd for
C30H30N4O3 [M + H]+ 495.2391, found 495.2388.
2'-(tert-butyl)-1-(2-(p-tolyl)quinoline-4-carbonyl)-2'H-spiro[piperidine-4,5'-pyrano[3
,2-c]pyrazol]-7'(6'H)-one (7b)
The product was obtained as yellow solid, yield 80%, m.p. 164.5 – 165.5 °C. 1H
NMR (400 MHz, CDCl3): δ 8.22 (d, J = 8.3 Hz, 1H), 8.06 (dd, J = 8.1, 4.4 Hz, 2H),
7.82 – 7.74 (m, 3H), 7.56 (dd, J = 16.4, 8.0 Hz, 1H), 7.34 (dd, J = 7.9, 4.0 Hz, 2H),
7.23 (d, J = 2.8 Hz, 1H), 4.75 (t, J = 8.0 Hz, 1H), 3.47 – 3.31 (m, 2H), 3.24 (t, J =
12.2 Hz, 1H), 2.76 – 2.64 (m, 2H), 2.44 (s, 3H), 2.38 – 2.27 (m, 1H), 1.97 (t, J = 14.7
Hz, 1H), 1.88 – 1.80 (m, 1H), 1.64 (s, 1H), 1.59 (s, 9H). 13C NMR (100 MHz, CDCl3):
δ 185.6, 167.2, 157.1, 156.9, 148.4, 147.0, 142.9, 140.0, 135.9, 134.1, 130.4, 130.2,
23

129.7, 127.3, 127.2, 127.1, 124.2, 123.9, 123.0, 122.8, 115.5, 112.3, 80.6, 80.5, 60.9,
49.1, 42.8, 42.6, 37.1, 34.7, 34.5, 33.9, 33.8, 29.5, 21.3. HRMS m/z calcd for
C31H32N4O3 [M + H]+ 509.2547, found 509.2557.
2'-(tert-butyl)-1-(2-(o-tolyl)quinoline-4-carbonyl)-2'H-spiro[piperidine-4,5'-pyrano[3
,2-c]pyrazol]-7'(6'H)-one (7c)
The product was obtained as yellow solid, yield 75%, m.p. 159.5 – 160.5 °C. 1H
NMR (400 MHz, CDCl3): δ 8.24 (d, J = 8.2 Hz, 1H), 7.91 – 7.74 (m, 2H), 7.63 (dd, J
= 16.0, 8.6 Hz, 1H), 7.48 (t, J = 12.5 Hz, 2H), 7.40 – 7.30 (m, 3H), 7.24 (d, J = 2.8 Hz,
1H), 4.74 (t, J = 13.5 Hz, 1H), 3.54 – 3.22 (m, 3H), 2.79 – 2.61 (m, 2H), 2.43 (s, 3H),
2.32 (t, J = 12.2 Hz, 1H), 2.01 (t, J = 16.0 Hz, 1H), 1.89 – 1.74 (m, 1H), 1.64 (s, 1H),
1.59 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 185.5, 167.0, 159.9, 148.0, 147.0, 142.3,
139.7, 135.9, 134.1, 131.0, 130.4, 130.3, 130.2, 129.7, 128.9, 127.6, 127.4, 126.1,
124.4, 124.1, 122.8, 122.6, 118.8, 112.3, 80.6, 80.5, 60.9, 49.1, 42.8, 42.7, 37.1, 34.8,
34.4, 33.9, 29.5, 20.4. HRMS m/z calcd for C31H32N4O3 [M + H]+ 509.2547, found
509.2561.
2'-(tert-butyl)-1-(2-(4-chlorophenyl)quinoline-4-carbonyl)-2'H-spiro[piperidine-4,5'-
pyrano[3,2-c]pyrazol]-7'(6'H)-one (7d)
1
The product was obtained as white solid, yield 78%, m.p. 222.5 – 223.5 °C. H
NMR (400 MHz, CDCl3): δ 8.22 (d, J = 8.3 Hz, 1H), 8.12 (dd, J = 8.5, 5.7 Hz, 2H),
7.86 – 7.71 (m, 3H), 7.60 (dd, J = 16.8, 7.7 Hz, 1H), 7.51 (dd, J = 8.6, 4.1 Hz, 2H),
7.23 (d, J = 4.8 Hz, 1H), 4.75 (t, J = 11.3 Hz, 1H), 3.49 – 3.31 (m, 2H), 3.23 (t, J =
12.2 Hz, 1H), 2.77 – 2.65 (m, 2H), 2.34 (t, J = 11.4 Hz, 1H), 1.99 (t, J = 14.5 Hz, 1H),
1.88 – 1.74 (m, 1H), 1.64 (s, 1H), 1.59 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 185.5,
166.9, 162.1, 155.8, 155.6, 148.1, 147.0, 143.4, 136.1, 134.1, 130.7, 130.5, 130.3,
129.1, 128.7, 127.7, 127.5, 124.3, 124.0, 123.2, 123.0, 115.3, 115.2, 112.3, 80.6, 80.4,
24

60.9, 49.1, 42.8, 42.7, 37.1, 34.7, 34.5, 34.0, 33.8, 29.5. HRMS m/z calcd for
C30H29ClN4O3 [M + H]+ 529.2001, found 529.1991.
2'-(tert-butyl)-1-(2-(2-chlorophenyl)quinoline-4-carbonyl)-2'H-spiro[piperidine-4,5'-
pyrano[3,2-c]pyrazol]-7'(6'H)-one (7e)
The product was obtained as yellow solid, yield 68%, m.p. 216.5 – 217.5 °C. 1H
NMR (400 MHz, CDCl3): δ 8.27 (d, J = 8.1 Hz, 1H), 7.94 – 7.77 (m, 2H), 7.76 – 7.60
(m, 3H), 7.52 (d, J = 7.1 Hz, 1H), 7.48 – 7.38 (m, 2H), 7.24 (d, J = 3.5 Hz, 1H), 4.74
(t, J = 5.6 Hz, 1H), 3.49 – 3.34 (m, 3H), 2.80 – 2.61 (m, 2H), 2.33 (t, J = 12.1 Hz, 1H),
13
2.04 (t, J = 16.0 Hz, 1H), 1.92 – 1.77 (m, 1H), 1.65 (s, 1H), 1.59 (s, 9H). C NMR
(100 MHz, CDCl3): δ 185.5, 166.8, 156.9, 148.2, 147.0, 141.7, 138.8, 134.1, 132.1,
131.7, 131.6, 130.3, 130.1, 128.0, 127.8, 127.4, 124.6, 124.2, 123.2, 123.1, 119.4,
112.3, 80.7, 80.5, 60.9, 49.2, 42.7, 37.2, 35.0, 34.3, 33.9, 29.5. HRMS m/z calcd for
C30H29ClN4O3 [M + H]+ 529.2001, found 529.1996.
2'-(tert-butyl)-1-(2-(4-fluorophenyl)quinoline-4-carbonyl)-2'H-spiro[piperidine-4,5'-p
yrano[3,2-c]pyrazol]-7'(6'H)-one (7f)
The product was obtained as white solid, yield 75%, m.p. > 240 °C. 1H NMR
(400 MHz, CDCl3): δ 8.22 (d, J = 8.0 Hz, 1H), 8.20 – 8.13 (m, 2H), 7.86 – 7.70 (m,
3H), 7.59 (dd, J = 16.3, 9.2 Hz, 1H), 7.24 (d, J = 5.0 Hz, 3H), 4.75 (t, J = 12.1 Hz,
1H), 3.49 – 3.31 (m, 2H), 3.23 (t, J = 12.1 Hz, 1H), 2.77 – 2.65 (m, 2H), 2.34 (t, J =
11.9 Hz, 1H), 1.99 (t, J = 14.5 Hz, 1H), 1.87 – 1.80 (m, 1H), 1.64 (s, 1H), 1.59 (s, 9H).
13C NMR (100 MHz, CDCl3): δ 185.6, 167.0, 162.8, 155.8, 148.2, 147.0, 143.3, 134.8,
134.1, 130.7, 130.5, 130.2, 129.4, 127.6, 127.4, 124.3, 124.0, 123.0, 116.1, 115.8,
115.4, 112.4, 80.6, 80.5, 60.9, 49.1, 42.9, 42.7, 37.1, 34.7, 34.5, 34.0, 33.8, 29.5.
HRMS m/z calcd for C30H29FN4O3 [M + H]+ 513.2296, found 513.2307.
25

1-(2-(4-bromophenyl)quinoline-4-carbonyl)-2'-(tert-butyl)-2'H-spiro[piperidine-4,5'-
pyrano[3,2-c]pyrazol]-7'(6'H)-one (7g)
The product was obtained as yellow solid, yield 70%, m.p. 171.5 – 172.5 °C. 1H
NMR (400 MHz, CDCl3): δ 8.22 (d, J = 8.3 Hz, 1H), 8.10 – 8.00 (m, 2H), 7.85 – 7.70
(m, 3H), 7.67 (dd, J = 7.8, 2.7 Hz, 2H), 7.60 (dd, J = 15.9, 8.2 Hz, 1H), 7.23 (d, J =
4.0 Hz, 1H), 4.74 (t, J = 11.0 Hz, 1H), 3.50 – 3.32 (m, 2H), 3.22 (t, J = 11.2 Hz, 1H),
2.82 – 2.61 (m, 2H), 2.34 (t, J = 8.0 Hz, 1H), 1.98 (t, J = 13.6 Hz, 1H), 1.91 – 1.79 (m,
13
1H), 1.74 (s, 1H), 1.59 (s, 9H). C NMR (100 MHz, CDCl3): δ 185.5, 167.0, 155.9,
155.7, 148.2, 147.0, 143.3, 137.6, 134.1, 132.1, 130.7, 130.5, 130.3, 129.0, 127.7,
127.6, 124.3, 124.0, 123.2, 123.0, 115.3, 112.3, 80.6, 80.5, 60.9, 49.1, 42.8, 42.7, 37.2,
34.7, 34.5, 34.0, 33.8, 29.5. HRMS m/z calcd for C30H29BrN4O3 [M + H]+ 573.1496,
found 573.1493.
2'-(tert-butyl)-1-(2-(4-methoxyphenyl)quinoline-4-carbonyl)-2'H-spiro[piperidine-4,5'
-pyrano[3,2-c]pyrazol]-7'(6'H)-one (7h)
1
The product was obtained as white solid, yield 73%, m.p. 218.5 – 219.5 °C. H
NMR (400 MHz, CDCl3): δ 8.19 (d, J = 8.3 Hz, 1H), 8.13 (dd, J = 8.6, 5.1 Hz, 2H),
7.85 – 7.69 (m, 3H), 7.55 (dd, J = 15.4, 8.3 Hz, 1H), 7.23 (d, J = 3.3 Hz, 1H), 7.05
(dd, J = 8.7, 3.8 Hz, 2H), 4.74 (t, J = 8.6 Hz, 1H), 3.89 (s, 3H), 3.50 – 3.31 (m, 2H),
3.25 (t, J = 11.4 Hz, 1H), 2.76 – 2.64 (m, 2H), 2.33 (t, J = 9.6 Hz, 1H), 1.97 (t, J =
13
15.1 Hz, 1H), 1.89 – 1.76 (m, 1H), 1.64 (s, 1H), 1.58 (s, 9H). C NMR (100 MHz,
CDCl3): δ 185.6, 167.2, 161.2, 161.1, 156.6, 156.5, 148.3, 147.0, 142.9, 134.1, 131.3,
131.2, 130.4, 130.3, 130.1, 128.9, 127.1, 126.9, 124.2, 123.9, 122.8, 122.6, 115.2,
114.3, 114.3, 112.3, 80.6, 80.5, 60.9, 55.4, 49.2, 42.8, 42.6, 37.1, 34.7, 34.5, 34.0,
33.8, 29.5. HRMS m/z calcd for C31H32N4O4 [M + H]+ 525.2496, found 525.2489.
2'-(tert-butyl)-1-(2-(3,4-dimethoxyphenyl)quinoline-4-carbonyl)-2'H-spiro[piperidine
-4,5'-pyrano[3,2-c]pyrazol]-7'(6'H)-one (7i)
26

The product was obtained as yellow solid, yield 85%, m.p. 169.5 – 170.5 °C. 1H
NMR (400 MHz, CDCl3): δ 8.23 (s, 1H), 7.87 (d, J = 9.7 Hz, 1H), 7.82 – 7.71 (m,
3H), 7.66 (d, J = 8.4 Hz, 1H), 7.59 – 7.52 (m, 1H), 7.23 (d, J = 6.3 Hz, 1H), 7.01 (dd,
J = 8.4, 5.5 Hz, 1H), 4.75 (t, J = 12.2 Hz, 1H), 4.06 (s, 3H), 3.97 (s, 3H), 3.48 – 3.32
(m, 2H), 3.24 (t, J = 12.1 Hz, 1H), 2.79 – 2.62 (m, 2H), 2.34 (t, J = 12.0 Hz, 1H), 1.98
13
(t, J = 14.3 Hz, 1H), 1.88 – 1.79 (m, 1H), 1.64 (s, 1H), 1.59 (s, 9H). C NMR (100
MHz, CDCl3): δ 185.6, 167.2, 156.6, 156.4, 150.7, 150.7, 149.4, 149.4, 148.2, 147.0,
142.9, 134.1, 131.6, 131.5, 130.4, 130.3, 130.1, 127.2, 127.0, 124.2, 123.9, 122.9,
122.7, 120.3, 120.3, 115.4, 115.3, 112.4, 111.0, 110.2, 80.6, 80.5, 60.9, 56.0, 56.0,
49.1, 42.8, 42.7, 37.1, 34.7, 34.4, 34.0, 33.8, 29.5. HRMS m/z calcd for C32H34N4O5
[M + H]+ 555.2602, found 555.2586.
1-(2-(4-aminophenyl)quinoline-4-carbonyl)-2'-(tert-butyl)-2'H-spiro[piperidine-4,5'-p
yrano[3,2-c]pyrazol]-7'(6'H)-one (7j)
1
The product was obtained as yellow solid, yield 55%, m.p. > 240 °C. H NMR
(400 MHz, CDCl3): δ 8.23 (d, J = 10.0 Hz, 1H), 8.03 (dd, J = 8.4, 6.9 Hz, 2H), 7.78 –
7.69 (m, 3H), 7.53 (dd, J = 15.9, 7.7 Hz, 1H), 7.24 (d, J = 2.7 Hz, 1H), 6.81 (dd, J =
8.6, 3.4 Hz, 2H), 4.74 (t, J = 12.0 Hz, 1H), 3.48 – 3.31 (m, 2H), 3.25 (t, J = 11.7 Hz,
1H), 2.76 – 2.63 (m, 2H), 2.33 (t, J = 16.0 Hz, 1H), 1.97 (t, J = 15.9 Hz, 1H), 1.88 –
1.77 (m, 1H), 1.64 (s, 1H), 1.59 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 185.8, 167.5,
157.1, 156.9, 148.5, 148.4, 147.1, 142.8, 142.7, 134.2, 130.4, 130.3, 130.0, 128.9,
126.8, 126.7, 124.3, 124.0, 122.8, 122.6, 115.2, 112.5, 80.8, 80.6, 61.0, 49.3, 42.9,
42.8, 37.2, 34.9, 34.6, 34.1, 33.9, 29.7. HRMS m/z calcd for C30H31N5O3 [M + H]+
510.2500, found 510.2504.
2'-(tert-butyl)-1-(2-(4-(dimethylamino)phenyl)quinoline-4-carbonyl)-2'H-spiro[piperi
dine-4,5'-pyrano[3,2-c]pyrazol]-7'(6'H)-one (7k)
27

1
The product was obtained as red solid, yield 55%, m.p. 199.5 – 200.5 °C. H
NMR (400 MHz, CDCl3): δ 8.19 (d, J = 9.1 Hz, 1H), 8.10 (dd, J = 8.6, 4.8 Hz, 2H),
7.78 – 7.66 (m, 3H), 7.49 (dd, J = 15.6, 8.1 Hz, 1H), 7.23 (d, J = 1.9 Hz, 1H), 6.83
(dd, J = 9.0, 3.5 Hz, 2H), 4.73 (t, J = 7.6 Hz, 1H), 3.44 – 3.35 (m, 2H), 3.26 (t, J =
11.9 Hz, 1H), 3.06 (s, 6H), 2.79 – 2.61 (m, 2H), 2.32 (t, J = 9.6 Hz, 1H), 1.96 (t, J =
13
15.9 Hz, 1H), 1.88 – 1.75 (m, 1H), 1.64 (s, 1H), 1.59 (s, 9H). C NMR (100 MHz,
CDCl3): δ 185.6, 167.5, 157.0, 156.9, 151.6, 148.4, 147.0, 142.5, 134.2, 130.1, 129.8,
128.5, 126.5, 126.3, 124.2, 123.9, 122.6, 115.0, 112.3, 112.1, 80.7, 80.6, 60.9, 49.2,
42.8, 42.6, 40.3, 37.1, 34.8, 34.5, 34.0, 33.9, 29.6. HRMS m/z calcd for C32H35N5O3
[M + H]+ 538.2813, found 538.2811.
2'-(tert-butyl)-1-(2-(pyridin-2-yl)quinoline-4-carbonyl)-2'H-spiro[piperidine-4,5'-pyr
ano[3,2-c]pyrazol]-7'(6'H)-one (7l)
1
The product was obtained as white solid, yield 62%, m.p. 229.5 – 230.5 °C. H
NMR (400 MHz, CDCl3): δ 8.81 – 8.65 (m, 2H), 8.55 (d, J = 23.0 Hz, 1H), 8.23 (d, J
= 8.4 Hz, 1H), 7.97 – 7.73 (m, 3H), 7.61 (dd, J = 16.3, 8.4 Hz, 1H), 7.46 – 7.37 (m,
1H), 7.25 (d, J = 8.3 Hz, 1H), 4.76 (t, J = 8.0 Hz, 1H), 3.58 – 3.21 (m, 3H), 2.82 –
2.58 (m, 2H), 2.32 (t, J = 8.7 Hz, 1H), 1.99 (t, J = 9.6 Hz, 1H), 1.90 – 1.76 (m, 1H),
1.64 (s, 1H), 1.59 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 185.7, 167.1, 155.6, 155.4,
149.2, 149.1, 148.1, 148.0, 147.0, 142.9, 137.1, 134.1, 130.4, 130.3, 130.2, 127.9,
127.7, 124.5, 124.4, 124.3, 124.1, 121.8, 115.7, 112.4, 112.3, 80.8, 80.5, 60.9, 49.2,
42.9, 42.6, 37.1, 34.7, 34.3, 34.0, 33.8, 29.5. HRMS m/z calcd for C29H29N5O3 [M +
H]+ 496.2343, found 496.2362.
2'-(tert-butyl)-1-(2-(pyridin-4-yl)quinoline-4-carbonyl)-2'H-spiro[piperidine-4,5'-pyr
ano[3,2-c]pyrazol]-7'(6'H)-one (7m)
The product was obtained as yellow solid, yield 65%, m.p. 154.5 – 155.5 °C. 1H
NMR (400 MHz, CDCl3): δ 8.81 (s, 2H), 8.25 (d, J = 8.8 Hz, 1H), 8.07 (dd, J = 11.5,
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