Design, synthesis, and biological evaluation of novel tetrahydroprotoberberine derivatives to reduce SREBPs expression for the treatment of hyperlipidemia

Article abstract of DOI:10.1016/j.ejmech.2021.113522

Statins play an important role in the treatment of hyperlipidemia, but drug resistance and adverse effects greatly limits their application. To discover new lipid-lowering drugs, three different series of tetrahydroprotoberberine derivatives (THPBs) were designed and synthesized. These compounds were first tested for their effects on viability of HepG2 cells and 21 compounds with the percent of cell viability over 90% were further screened to evaluate their ability to reduce total cholesterol (TC) and triglyceride (TG) levels. Among these derivatives, two compounds displayed significant down-regulation both intracellular of TC and TG content, especially compound 49 exhibited the greatest efficacy. Mechanistically, compound 49 promoted proteasomal degradation of SREBPs. Importantly, compound 49 displayed superior bioavailability (F = 65.1%) and obvious efficacy in the treatment of high fat diet induced obesity in vivo. Therefore, compound 49 is a promising candidate to develop new treatment of hyperlipidemia.

Full text of DOI:10.1016/j.ejmech.2021.113522

European Journal of Medicinal Chemistry 221 (2021) 113522
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis, and biological evaluation of novel
tetrahydroprotoberberine derivatives to reduce SREBPs expression for
the treatment of hyperlipidemia
,
,
c
,
,
c
,
,
, c
Haixia Ge a 1, Weitao Zhang b 1, Kai Yuan b 1, Hanyue Xue b c, Hao Cheng b
,
Weijiao Chen b c, Yishi Xie b c, Jian Zhang d, Xiaojun Xu b **, Peng Yang b
,
,
,
, c, *
a School of Life Sciences, Huzhou University, Huzhou, 313000, China
b State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, 210009, China
c Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 210009, China
d State Key Laboratory of Natural Medicines, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Statins play an important role in the treatment of hyperlipidemia, but drug resistance and adverse effects
greatly limits their application. To discover new lipid-lowering drugs, three different series of tetrahy-
droprotoberberine derivatives (THPBs) were designed and synthesized. These compounds were first
tested for their effects on viability of HepG2 cells and 21 compounds with the percent of cell viability
over 90% were further screened to evaluate their ability to reduce total cholesterol (TC) and triglyceride
(TG) levels. Among these derivatives, two compounds displayed significant down-regulation both
intracellular of TC and TG content, especially compound 49 exhibited the greatest efficacy. Mechanisti-
cally, compound 49 promoted proteasomal degradation of SREBPs. Importantly, compound 49 displayed
superior bioavailability (F ¼ 65.1%) and obvious efficacy in the treatment of high fat diet induced obesity
in vivo. Therefore, compound 49 is a promising candidate to develop new treatment of hyperlipidemia.
© 2021 Elsevier Masson SAS. All rights reserved.
Received 26 February 2021
Received in revised form
21 April 2021
Accepted 28 April 2021
Available online 5 May 2021
Keywords:
Tetrahydroprotoberberine derivatives
SREBPs
Hyperlipidemia
1. Introduction
proliferator-activated receptor a) [6], statins (3-hydroxy-3-
methylglutaryl-coenzyme A reductase enzyme inhibitors) [7], and
proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors
[8]. But even statins, the mainstay treatment for hyperlipidemia,
still have many limitations including drug resistance and intoler-
ance due to adverse effects. Therefore, it is an urgent need to
develop new drugs with unique mechanism of action.
Sterol regulatory element-binding proteins (SREBPs), including
SREBP-1a, SREBP-1c and SREBP-2, play important roles in the up-
take and synthesis of cholesterol and fatty acids [9]. SREBP-1a and
-1c are processed in Golgi to generate the mature protein, which
mainly regulate the expression of genes required for fatty acid
synthesis. SREBP-2 is responsible for the synthesis of cholesterol
[10]. Accordingly, regulation of SREBPs is a potential strategy to
develop novel lipid-lowing drugs [11].
With the development of the society and improvement of
people's living standards, the diet structure has been remarkably
changed, resulting in significantly increased prevalence of hyper-
lipidemia. Hyperlipidemia is one of the most important risk factors
responsible for many cardiovascular and cerebrovascular diseases
[1,2], such as atherosclerosis [3], hypertension, stroke, coronary
heart disease and so on [4,5]. Hyperlipidemia is characterized by
high levels of cholesterol and increased levels of serum triglyceride.
Currently, drugs against hyperlipidemia mainly consist of fibrates
that promote fatty acid oxidation by PPARa (Peroxisome
* Corresponding author. State Key Laboratory of Natural Medicines, China Phar-
maceutical University, 210009, China.
** Corresponding author. State Key Laboratory of Natural Medicines, Jiangsu Key
Laboratory of Drug Discovery for Metabolic Diseases, China Pharmaceutical Uni-
versity, 210009, Nanjing, Jiangsu, China.
Tetrahydroprotoberberine derivatives (THPBs) are the primary
active components originally isolated from the Chinese herb
Corydalis yanhusuo and various species of Stephania [12]. The pre-
vious study about THPBs mainly focused on central nervous system
diseases [13], while lipid-lowering effects were rarely reported [14].
E-mail addresses: xiaojunxu2000@163.com (X. Xu), pengyang@cpu.edu.cn
(P. Yang).
1
These authors made equal contributions to this work.
https://doi.org/10.1016/j.ejmech.2021.113522
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

H. Ge, W. Zhang, K. Yuan et al.
European Journal of Medicinal Chemistry 221 (2021) 113522
But within a high-throughput screening to discover novel lipid-
lowering ligands [15], compound GZ-1 (Fig. 1A) bearing a THPB
scaffold was identified as a top hit, which could reduce total
cholesterol (TC) and triglyceride (TG) levels in HepG2 cells (Fig. 1B
and C). This result provided us a good lead compound to develop
new lipid-lowering ligands. In this work, a series of THPBs with
various substituents were synthesized and screened based on their
ability to reduce TC and TG levels in HepG2 cells. Compound 49 was
discovered as the top candidate which exhibited the most signifi-
cant effects of reducing TC and TG levels in HepG2 cells. Further
mechanistic investigation demonstrated that compound 49
reduced lipid contents through degrading SREBPs. Meanwhile, 49
displayed excellent pharmacokinetic profiles including superior
bioavailability and high stability in vivo.
explore their structureeactivity relationships. The replacement of
oxacyclopentene with two methoxy groups provided compounds
16e23, 26, and 28 (Table 1), which displayed lower inhibition on
cell viability than compounds 3e10, 13, and 15, respectively.
Especially, cell viability reminded 99.3% at a concentration of 10 mM
of compound 26, which indicated its extremely low toxicity.
After these carboxylate derivatives 3e28 were synthesized, a
series of sulfonate substituent groups were introduced on the ar-
omatic ring D. Overall, most of THPBs sulfonate derivatives (Table 2)
showed stronger inhibition on cell viability when compared to the
THPBs carboxylate derivatives, and only compounds 31, 33, 42, 43,
and 45 displayed less than 10% inhibition on the cell viability, which
were selected for the further studies. Besides the introduction of a
carboxylic ester group and a sulfonate group on the aromatic ring D,
a new series of ether derivatives 46e50 (Table 3) were designed
and synthesized. Generally, these THPB ether derivatives showed
little inhibition on cell viability and compounds 46, 48, and 49
retained cell viability more than 90%. Such as compound 46,
bearing a propyl ether, displayed almost no toxicity on HepG2 cells
2. Results and discussion
2.1. Chemistry
at a concentration of 10 mM.
The synthetic routes for target compounds are illustrated in
Scheme 1. The key intermediates 1 and 2 were synthesized through
pyrolysis monodemethylation and reduction reactions of palmatine
hydrochloride and berberine hydrochloride, respectively, and both
has a rotation value of 0, as the previous report [16]. Compounds
3e28 and 29e45 were synthesized according to the general acet-
ylation reaction procedure by intermediates 1 or 2 and selected acyl
chlorides or sulfonyl chlorides. Compounds 46e50 were obtained
by alkylation reaction of compound 1 and corresponding bromides.
2.2.2. Cellular lipid levels and expression of SREBPs
According to the cell viability assay, 20 compounds with the
percent of cell viability over 90% were further assessed for their
regulatory efficacy on cellular TC and TG levels (Tables 1e3). HepG2
cells were first treated with these 20 compounds at the concen-
tration of 10 mM, and their TC and TG content were subsequently
measured and shown in Fig. 2A and B. Among these 20 compounds,
there are 12 THPB carboxylate derivatives. Compared with the
dioxymethylene substitution on the ring A, THPB carboxylate de-
rivatives with two methoxy groups are more effective in reducing
TC levels. But the overall effect of THPB carboxylate derivatives in
reducing TC levels was minimal. Only compound 22 displayed
similar effects in reducing TC levels with lead compound GZ-1.
For THPB sulfonate derivatives, most of them did not exhibit
obvious activity on decreasing TC levels, but compound 43 with
two methoxy groups on the ring A displayed potent activity and
reduced the TC level by 15%. Compared with compound 33 bearing
a dioxymethylene substitution on the ring A, compound 43 with
two methoxy groups showed much better activity. This provides us
a good strategy for further chemical modification.
2.2. Biological evaluation
2.2.1. Evaluation of all compounds on cell viability
As obese patients tend to take medicine for a long time, the
compound toxicity is critical. Firstly, all synthesized THPBs de-
rivatives were screened at a concentration of 10 mM to test their
cytotoxicity in HepG2 cells. The structures of THPB derivatives and
their corresponding effects on cell viability are summarized in
Tables 1e3. The study of structureeactivity relationships (SARs)
was first focused on the substituent groups of the aromatic ring D.
Compounds 3e15 (Table 1) bearing different carboxyl substituent
groups were designed and synthesized, which displayed little in-
fluence on the cell viability in the range of 80.9%e99.6%. Especially,
compound 14 bearing a chloro-substituted tert-butyl group has
almost no toxicity on HepG2 cells. Therefore, compounds 3, 9, 11,
and 14 with more 90% of cell viability were chosen for further
evaluation on cellular lipid levels and expression of SREBPs.
Besides the modification of ring D, oxacyclopentene of ring A
was also opened, and two methoxy groups were introduced to
Interestingly, THPB ether derivatives showed much better TC-
lowering activity than THPB carboxylate and sulfonate de-
rivatives. Especially, compounds 46 and 49 were more effective
than lead compound GZ-1, which reduced the TC level by 18% and
17%, respectively.
Similar as the regulation of TC levels, THPB carboxylate de-
rivatives with two methoxy groups on the ring A showed more
Fig. 1. (A) The structure of compound GZ-1. (B) Intracellular TC levels assayed in HepG2 cells administered with DMSO or compound GZ-1. (C) Intracellular TG levels assayed in
HepG2 cells administered with DMSO or compound GZ-1.
2

H. Ge, W. Zhang, K. Yuan et al.
European Journal of Medicinal Chemistry 221 (2021) 113522
Scheme 1. Synthesis of compounds 3e50. Reagents and conditions: (a) (1) 195e205 ꢀC, 30 min; (2) EtOH-HCl (95:5), rt, 1 h; (b) NaBH4, MeOH, rt, 3 h; (c) Et3N, CH2Cl2, rt, 4 h; (d)
K2CO3, DMF, 80 ꢀC, 4 h.
remarkable activity on the down-regulation of TG levels than
dioxymethylene substitution on the ring A. Among these THPB
carboxylate derivatives, only compound 20 displayed better effects
in reducing TG levels compared with lead compound GZ-1. Com-
pound 43, which displayed potent activity in reducing TC levels,
also demonstrated better TG-lowering effects than lead compound
GZ-1. All THPB ether derivatives 46, 48 and 49 had great effects in
the down-regulation of TG levels and compound 49 showed the
best effect.
SREBPs compared to cells treated with cycloheximide and vehicle,
which meant compound 49 increased SREBPs degradation (Fig. 2F).
MG-132, a proteasome inhibitor, reversed the reduction of SREBPs
in HepG2 cells treated with compound 49 (Fig. 2G). These results
indicated that compound 49 promoted SREBPs proteasomal
degradation. Moreover, the immunoprecipitation results demon-
strated that compound 49 increased the ubiquitination of SREBPs,
leading to accelerated degradation of SREBPs though the ubiquitin
proteasome system (Fig. 2H).
In conclusion, compounds 43 and 49 reduced both intracellular
TC and TG content, especially compound 49 exhibited the greatest
effects. Therefore, compound 49 was chosen for further in vitro and
in vivo biological evaluation and pharmacokinetic studies.
2.3. Pharmacokinetic properties of Compound 49
The pharmacokinetic evaluation of compound 49 was con-
ducted to further examine its drugability. SD rats were orally
administrated (10 mg/kg) or intravenous injected (1 mg/kg) 49, and
the pharmacokinetic results are shown in Table 4. When compound
49 was administered by intravenous injection, its maximum
plasma concentration (Cmax) was 900.7 ng/mL and AUC0-∞ value
was 658.9 h*ng/mL. The oral administration of compound 49
demonstrated Cmax was 428.3 ng/mL and AUC0-∞ value was
4290 h*ng/mL. Meanwhile, compound 49 displayed great half-life
(t1/2) both through intravenous and oral administration, with the
values of 4.95 and 5.36 h, respectively. Moreover, compound 49
exhibited a good oral bioavailability of 65.1%. All these pharmaco-
kinetic parameters indicated the good drugability of compound 49,
which deserved further research.
After confirming the favorable effects on cell viability and lipid
regulation of compound 49, its underlying mechanism of down-
regulation in intracellular cholesterol and triglyceride levels were
next investigated. The Western blot analysis manifested that
compound 49 downregulated the precursor and mature forms of
SREBP-1 and SREBP-2, in a concentration dependent manner at
protein levels in HepG2 cells (Fig. 2C). In addition, compound 49
showed no effects on the expression of PCSK9 (Fig. 2D). As com-
pound 49 reduced the SREBPs in the nucleus, the expression of
their target genes, 1-aminocyclopropane-1-carboxylic acid (ACC),
fatty acid synthase (FASN), low-density lipoprotein receptor (LDLR)
and stearoyl-CoA desaturase (SCD) were also significantly reduced
(Fig. 2E), which means compound 49 can decrease lipid synthesis.
To further elucidate the mechanism of compound 49 in reducing
SREBPs expression, HepG2 cells were treated with cycloheximide,
an inhibitor of synthesis on both DNA and protein, then SREBPs
amount was measured in different time point to calculate SREBPs
degradation rate. Compound 49 displayed notable reduction of
2.4. Effect of compound 49 on high fat diet induced obesity
Due to the good in vitro activity and favorable drugability, we
further investigated the lipid-lowing effects of compound 49
3

H. Ge, W. Zhang, K. Yuan et al.
European Journal of Medicinal Chemistry 221 (2021) 113522
Table 1
Table 1 (continued )
Effects of THPB carboxylate derivatives on HepG2 cell viability.
ID
R1
R2
R3
Cell Viability
95.1%
20
-CH3
-CH3
21
22
-CH3
-CH3
-CH3
-CH3
95.4%
92.1%
ID
R1
R2
R3
Cell Viability
90.6%
3
-CH2-
23
24
-CH3
-CH3
-CH3
-CH3
84.6%
88.3%
4
5
-CH2-
-CH2-
88.2%
85.5%
25
26
-CH3
-CH3
-CH3
-CH3
81.7%
99.3%
6
7
-CH2-
-CH2-
80.9%
87.7%
27
28
-CH3
-CH3
-CH3
-CH3
91.2%
97.9%
8
9
-CH2-
-CH2-
85.0%
90.7%
in vivo. C57BL/6J mice were fed with chow or high fat diet (HFD) for
15 weeks. After that, HFD-fed mice were treated with vehicle (1%
CMC-Na solution) or compound 49 (low-dose 170 mg/kg/day and
high-dose 340 mg/kg/day) for additional 6 weeks (Fig. 3A). During
the treatment, no obvious toxicity was observed and mice fed with
HFD had similar food intake (Fig. 3B and D). Compound 49
dramatically reduced bodyweight of HFD-fed mice, and high
dosage of compound 49 treated mice were even lighter than the
chow diet-fed mice (Fig. 3B and C). Notably, liver weight in com-
pound 49 treated mice decreased (Fig. 2B and E). Consistently,
epididymal fat/body weight ratio also dropped in compound 49
treated mice, while brown fat/body weight ratio slightly raised only
in high dosage of compound 49 treated mice (Fig. 3B, F and 3G).
H&E and Oil red O section staining revealed that the livers of
compound 49 treated mice contained lower lipid accumulation
compared with the livers of vehicle treated mice (Fig. 3H). Histo-
logical analysis showed that compound 49 reduced the cell size of
both white adipocyte tissue (WAT) and brown adipocyte tissue
(BAT) (Fig. 3H). The H&E staining of heart, spleen, lung, and kidney
showed none obvious side effects on compound 49 treated mice
(Fig. 3I).
Glucose tolerance and insulin resistance were markedly
ameliorated in compound 49 treated HFD-fed mice (Fig. 4A and B).
The serum TC and TG levels in compound 49 treated mice were
significantly lower than those of vehicle treated mice (Fig. 4C and
D). Consistent with the results in HepG2 cells above, SREBPs were
significantly decreased in livers of compound 49 treated mice,
compared to vehicle treated mice (Fig. 4E). The expression of their
target genes, Acc, Fasn, Ldlr and Scd were also reduced (Fig. 4F).
Collectively, these data demonstrated that compound 49 improves
high fat diet induced hyperlipidemia in C57BL/6J mice through
reducing SREBPs.
10
11
-CH2-
-CH2-
83.9%
90.1%
12
13
-CH2-
-CH2-
85.0%
85.6%
14
15
-CH2-
-CH2-
99.6%
86.3%
16
-CH3
-CH3
96.0%
17
18
-CH3
-CH3
-CH3
-CH3
97.8%
84.3%
19
-CH3
-CH3
80.2%
4

H. Ge, W. Zhang, K. Yuan et al.
European Journal of Medicinal Chemistry 221 (2021) 113522
Table 2
Table 2 (continued )
Effects of THPB sulfonate derivatives on HepG2 cell viability.
ID
R1
R2
R4
Cell Viability
81.4%
44
-CH3
-CH3
45
-CH3
-CH3
94.7%
ID
R1
R2
R4
Cell Viability
88.8%
29
-CH2-
Table 3
30
-CH2-
80.7%
Effects of THPB ether derivatives on HepG2 cell viability.
31
32
33
-CH2-
-CH2-
-CH2-
93.4%
86.8%
92.8%
ID
46
47
R1
R2
R5
Cell Viability
101.5%
-CH2-
-CH2-
87.7%
48
49
-CH2-
-CH2-
98.1%
93.0%
34
-CH2-
89.5%
50
-CH2-
87.7%
35
36
-CH2-
-CH3
84.5%
84.8%
-CH3
3. Conclusions
In this study, three different series of THPBs were designed,
synthesized and preliminary screened based on effects on viability
of HepG2 cells. The further screen by examining ability of
decreasing TC and TG in HepG2 cells led to the identification of
compound 49, which exhibited greatest downregulating effects on
TC and TG and reduced SREBPs expression through promoting
proteasomal degradation of SREBPs. In particularly, compound 49
displayed an excellent pharmacokinetic profile and great effects in
improving high fat diet induced obesity in vivo. In conclusion,
compound 49 deserves further research in battle with
hyperlipidemia.
37
38
-CH3
-CH3
-CH3
-CH3
73.3%
86.5%
39
40
41
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
80.2%
87.7%
89.2%
42
43
-CH3
-CH3
-CH3
-CH3
93.4%
91.1%
4. Experimental section
4.1. 4.1 Chemistry
Thin-layer chromatography (TLC) was performed on silica gel
GF254 plates of 0.5 mm of thickness for analysis. Layers were air
dried and activated at 110 ꢀC for 0.5 h before use. NMR spectra (1H
NMR, 13C NMR) were recorded on a Bruker Avance 500 or 400 or
300 MHz spectrometer with tetramethylsilane as the internal
(continued on next page)
standard, and chemical shifts were expressed in
d (parts per
million). HR-ESI-MS experiments were performed on Waters Xevo
G2-XS QTOF mass spectrometer.
5

H. Ge, W. Zhang, K. Yuan et al.
European Journal of Medicinal Chemistry 221 (2021) 113522
Fig. 2. (A) Intracellular TC levels assayed in HepG2 cells. (B) Intracellular TG levels assayed in HepG2 cells. (C) The expression of SREBP-1 and SREBP-2 in HepG2 cells with
compound 49 at the indicated doses for 24 h was detected by Western blot. -actin was used as an endogenous reference. (D) The expression of PCSK9 in HepG2 cells with
compound 49 at the indicated doses for 24 h was detected by Western blot. -actin was used as an endogenous reference. (E) The expression of ACC, FASN, LDLR and SCD was
assessed by qRT-PCR in HepG2 cells. Expression was normalized to GAPDH. Error bars are represented as mean SEM. Statistical analyses were done with one-way ANOVA.
*p < 0.05, **p < 0.01, ***p < 0.001 vs control. (F) After incubation with 50 M cycloheximide for 1 h, cells were switched to medium supplemented with 50 M cycloheximide plus
vehicle, or 10 M compound 49 for indicated period of time. Cells were lysed and SREBPs degrading rate were measured by Western blot. (G) After HepG2 cells were treated with
10 M MG-132 for 1 h, cells were switched to medium supplemented with 10 M MG-132 plus vehicle, or 10 M compound 49 for 4 h. Cells were lysed and SREBPs were detected by
b
b
m
m
m
m
m
m
Western blot. (H) 293T cells were transfected with flag-SREBP-1 or flag-SREBP-2 and HA-ubiquitin for 24 h, after the treatment, the cells were incubated with medium containing
compound 49 for another 24 h. Cells were lysed and pulled down by flag antibody. Ubiquitylated SREBPs were detected by Western blot.
Table 4
Pharmacokinetic parameters of compound 49 in SD rats.
Compd.
49
Admin.
C
max (ng/mL)
AUC0-∞ (h*ng/mL)
MRT0-∞ (h)
Tmax (h)
t1/2 (h)
CL (mL/h/kg)
F (%)
IV
PO
900.7
428.3
658.9
4290
3.18
8.03
0.033
6.67
4.95
5.36
1545
511
e
65.1
aValues are the average of three runs. Cmax, maximum concentration; AUC, area under the plasma concentrationtime curve; MRT, mean residence time; t1/2, half-life; CL,
clearance; F, oral bioavailability. Dose: p.o. at 10 mg/kg. Dose: i.v. at 1 mg/kg.
4.1.1. General synthetic procedures for the target compounds 3e28
(compound 3 as the example)
vacuum. The residues were purified via silica gel chromatography
(CH2Cl2/MeOH) to give the product 3 (0.46 g, 71%).
1H NMR (500 MHz, DMSO‑d6)
d 1.41e1.25 (m, 3H), 1.63e1.49 (m,
4.1.1.1. 10-Methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]iso-
3H), 1.76e1.72 (m, 2H), 2.00e1.97 (m, 2H), 2.49e2.43 (m, 1H),
2.66e2.53 (m, 3H), 2.92e2.86 (m, 1H), 3.06e3.03 (m, 1H),
3.25e3.28 (m, 1H), 3.37e3.33 (m, 1H), 3.42e3.40 (m, 1H), 3.72 (s,
3H), 3.82 (d, J ¼ 16.0 Hz, 1H), 5.94 (d, J ¼ 2.0 Hz, 2H), 6.66 (s,
quinolino[3,2-a]isoquinolin-9-yl
cyclohexanecarboxylate
(3).
The intermediate 1 (0.5 g, 1.5 mmol) was added to dichloromethane
(DCM, 30 mL) and stirred for dissolving at room temperature,
Cyclohexanecarboxylic acid chloride (0.2 mL, 1.5 mmol), 2 mL
triethylamine and slight 4-dimethylamiopryidine were added to
the solution. The mixture was stirred for 4 h at room temperature,
then washed three times by water, saturated NaHCO3 solution and
saturated NaCl solution, respectively. The organic layer was dried
over sodium sulfate, filtered, the solvent was evaporated in
1H),6.92 (s, 1H), 6.95 (d, J ¼ 8.5 Hz, 1H), 7.01 (d, J ¼ 8.5 Hz, 1H). 13
C
NMR (75 MHz, DMSO)
d 172.66, 148.60, 145.67, 145.41, 135.48,
130.64, 127.88, 127.43, 127.39, 126.02, 110.71, 107.99, 105.65, 100.47,
58.82, 55.86, 52.76, 50.47, 41.83, 40.33, 35.42, 28.90, 28.56, 28.56,
25.22, 24.59, 24.59. HR-ESI-MS: (m/z) 436.2138 [MþH] þ, (Calcd:
6

H. Ge, W. Zhang, K. Yuan et al.
European Journal of Medicinal Chemistry 221 (2021) 113522
Fig. 3. (A) Male C57BL/6J mice at 6 weeks of age were fed with chow or HFD for 15 weeks. Then vehicle, 49(low-dose) and 49(high-doge) were administrated to mice by oral gavage
for another 6 weeks. (B) Macroscopic images of mice, their respective livers, WAT and BAT. (C) Body weight per mouse, (D) Food intake per mouse, (E) Liver weight, (F) epididymal
fat/body weight ratio and (G) brown fat/body weight ratio during the experiments. (H) Histologic analysis of liver, white adipose tissue (WAT), and white adipose tissue (BAT). Liver
tissues sections were stained with oil red O or H&E to visualize lipid contents. WAT and BAT were stained with H&E. Bars ¼ 50
mm. (I) Histologic analysis of heart, spleen, lung, and
kidney. The tissues sections were stained with H&E. Bars ¼ 50
mm.
436.2118);
yellow solid, Yield: 24%. 1H NMR (500 MHz, DMSO‑d6)
d 2.45e2.40
(m, 1H), 2.62e2.56 (m, 2H), 2.91e2.85 (m, 1H), 3.04e3.02 (m, 1H),
3.35e3.32 (m, 1H), 3.36e3.40 (m, 1H), 3.43e3.45 (m, 1H), 3.74 (s,
3H), 3.90 (d, J ¼ 16.0 Hz, 1H), 5.95 (d, J ¼ 2.0 Hz, 2H), 6.65 (s, 1H),
4.1.1.2. 10-Methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]iso-
quinolino[3,2-a]isoquinolin-9-yl furan-2-carboxylate (4). Light
7

H. Ge, W. Zhang, K. Yuan et al.
European Journal of Medicinal Chemistry 221 (2021) 113522
Fig. 4. (A) Effect of compound 49 on oral glucose tolerance in HFD-fed mice as determined by oral glucose tolerance test (OGTT). Quantification of the area under the curve (AUC)
from the OGTT. (B) Effect of compound 49 on insulin resistance as determined by insulin tolerance test (ITT). Quantification of the AUC of the ITT. (C) TC levels in serum. (E) TG levels
in serum. (G) The Western blot analysis of SREBPs in mouse liver tissues. (H) The relative levels of SREBPs target genes in mouse liver tissues. Error bars are represented as
mean SEM. Statistical analyses were done with one-way ANOVA (Dunnett's post test). *p < 0.05, **p < 0.01, ***p < 0.001 vs HFD.
6.81e6.80 (m, 1H), 6.93 (s, 1H), 7.02 (d, J ¼ 8.0 Hz, 1H), 7.09 (d,
J ¼ 8.0 Hz, 1H), 7.58 (d, J ¼ 3.0 Hz, 1H), 8.10 (s, 1H). 13C NMR
[MþH] þ, (Calcd: 444.1805).
(125 MHz, DMSO‑d6)
d: 155.29, 148.68, 145.69, 145.41, 142.56,
4.1.1.4. 10-Methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]iso-
quinolino[3,2-a]isoquinolin-9-yl 3-bromobenzoate (6). White solid,
134.61, 130.58, 128.89, 128.15, 127.39, 127.62, 107.98, 126.60, 120.21,
112.67, 110.80, 105.65, 100.47, 58.83, 55.85, 52.69, 50.35, 48.59,
35.42. HR-ESI-MS: (m/z) 420.1437 [MþH] þ, (Calcd: 420.1442).
Yield: 68%. 1H NMR (500 MHz, DMSO‑d6)
d 2.42e2.39 (m, 1H),
2.63e2.55 (m, 2H), 2.91e2.85 (m, 1H), 3.04e3.02 (m, 1H),
3.46e3.37 (m, 3H), 3.73 (s, 3H), 3.96 (br, 1H), 5.95 (d, J ¼ 1.0 Hz, 2H),
6.65 (s, 1H), 6.93 (s, 1H), 7.03 (d, J ¼ 8.5 Hz, 1H), 7.10 (d, J ¼ 8.5 Hz,
1H), 7.6 (t, J ¼ 8.0 Hz, 1H), 7.97 (d, J ¼ 8.0 Hz, 1H), 8.15 (d, J ¼ 8.0 Hz,
4.1.1.3. 10-Methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]iso-
quinolino[3,2-a]isoquinolin-9-yl 3-methylbenzoate (5). White solid,
1H), 8.25 (s, 1H). 13C NMR (125 MHz, DMSO‑d6)
d: 162.27, 148.52,
Yield: 56%. 1H NMR (500 MHz, DMSO‑d6)
d 2.43e2.39 (m, 1H),
145.69, 145.42, 136.79, 135.22, 132.08, 131.28, 130.61, 130.60, 128.83,
128.06, 127.63, 127.41, 126.61, 122.05, 110.78, 107.99, 105.66, 100.48,
58.84, 55.89, 52.59, 50.33, 35.44, 28.89. HR-ESI-MS: (m/z) 508.0746
[MþH] þ, (Calcd: 508.0754).
2.63e2.55 (m, 2H), 2.91e2.85 (m, 1H), 3.04e3.02 (m, 1H),
3.46e3.34 (m, 3H), 3.72 (s, 3H), 3.92 (br, 1H), 5.94 (d, J ¼ 2.0 Hz, 2H),
6.65 (s, 1H), 6.93 (s, 1H), 7.02 (d, J ¼ 8.5 Hz, 1H), 7.09 (d, J ¼ 8.5 Hz,
1H), 7.50 (t, J ¼ 8.0 Hz, 1H), 7.57 (t, J ¼ 8.0 Hz, 1H), 7.94 (d, J ¼ 8.0 Hz,
1H), 7.96 (s, 1H). 13CNMR (75 MHz, DMSO)
d 163.63, 148.69, 145.70,
145.43, 138.52, 135.45, 134.62, 130.62, 130.12, 128.88, 128.41, 128.09,
127.57, 127.42, 126.98, 126.41, 110.76, 108.01, 105.68, 100.50, 58.88,
55.84, 52.76, 50.41 35.49, 28.91, 20.72. HR-ESI-MS: (m/z)444.1808
4.1.1.5. 10-Methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]iso-
quinolino[3,2-a]isoquinolin-9-yl 4-fluorobenzoate (7). White solid,
Yield: 88%. 1H NMR (500 MHz, DMSO‑d6)
d 2.43e2.39 (m, 1H),
8

H. Ge, W. Zhang, K. Yuan et al.
European Journal of Medicinal Chemistry 221 (2021) 113522
2.63e2.55 (m, 2H), 2.91e2.84 (m, 1H), 3.02e3.00 (m, 1H),
3.46e3.37 (m, 3H), 3.73 (s, 3H), 3.92 (br, 1H), 5.95 (s, 2H), 6.65 (s,
1H), 6.93 (s, 1H), 7.03 (d, J ¼ 8.5 Hz, 1H), 7.09 (d, J ¼ 8.5 Hz, 1H), 7.45
(d, J ¼ 8.5 Hz, 2H), 8.23 (d, J ¼ 8.0 Hz, 2H). 13CNMR (125 MHz,
3.42e3.40 (m, 1H), 3.72 (s, 3H), 3.82 (m, 1H), 5.94 (d, J ¼ 2.0 Hz, 2H),
6.15 (dd, J ¼ 1.2 Hz, 12.5 Hz, 1H), 6.65 (s, 1H), 6.92 (s, 1H), 6.96 (d,
J ¼ 8.5 Hz, 1H), 7.03 (d, J ¼ 8.5 Hz, 1H), 7.12 (m, 1H). 13C NMR
(126 MHz, DMSO)
d 163.66, 149.25, 148.39, 146.24, 145.97, 135.83,
DMSO‑d6)
d
: 166.5, 162.6, 148.6, 145.7, 145.4, 135.3, 132.8, 132.7,
131.17, 128.56, 127.99, 127.95, 126.70, 121.55, 111.18, 108.54, 106.21,
101.05, 59.42, 56.30, 53.39, 51.01, 36.02, 29.47, 18.43. HR-ESI-MS:
(m/z) 394.1631 [MþH] þ, (Calcd: 394.1649).
130.6, 128.1, 127.6, 127.4, 126.5, 125.0, 116.2, 116.1, 110.8, 107.9, 105.6,
100.5, 58.8, 55.9, 52.7, 50.4, 35.4, 28.9. HR- ESI-MS: (m/z) 448.1567
[MþH] þ, (Calcd: 448.1555);
4.1.1.11. 10-Methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]
isoquinolino[3,2-a]isoquinolin-9-yl (3r,5r,7r)-adamantane-1-
carboxylate (13). White solid, Yield: 66%. 1H NMR (500 MHz,
4.1.1.6. 10-Methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]iso-
quinolino[3,2-a]isoquinolin-9-yl methacrylate (8). Light yellow
solid, Yield: 56%. 1H NMR (500 MHz, DMSO‑d6)
d
2.01 (s, 3H),
DMSO‑d6) d1.74 (s, 6H), 2.01 (s, 6H), 2.04 (s, 3H), 2.46e2.42 (m, 1H),
2.44e2.41 (m, 1H), 2.60e2.55 (m, 2H), 2.92e2.86 (m, 1H),
3.06e3.03 (m, 1H), 3.43e3.31 (m, 3H), 3.73 (s, 3H), 3.84 (br, 1H),
5.89 (s, 1H), 5.94 (d, J ¼ 2.0 Hz, 2H), 6.30 (s, 1H), 6.66 (s, 1H), 6.92 (s,
1H), 6.98 (d, J ¼ 8.5 Hz, 1H), 7.05 (d, J ¼ 8.5 Hz, 1H). 13C NMR
2.60e2.55 (m, 2H), 2.92e2.87 (m, 1H), 3.05e3.03 (m, 1H),
3.27e3.26 (m, 1H), 3.36e3.33 (m, 1H), 3.42e3.40 (m, 1H), 3.71 (s,
3H), 3.78 (br, 1H), 5.94 (d, J ¼ 2.0 Hz, 2H), 6.66 (s, 1H), 6.92 (s, 1H),
6.94 (d, J ¼ 8.5 Hz, 1H), 7.01 (d, J ¼ 8.5 Hz, 1H). 13C NMR (126 MHz,
(75 MHz, DMSO)
d
164.21, 148.65, 145.69, 145.42, 135.41, 134.76,
DMSO) d 174.65,149.16,146.24,145.97,136.08,131.21,128.41,127.99,
130.62, 127.96, 127.72, 127.50, 127.39, 126.24, 110.71, 107.99, 105.65,
100.49, 58.85, 55.83, 52.70, 50.43, 35.45, 28.92, 18.03. HR-ESI-MS:
(m/z) 394.1631 [MþH] þ, (Calcd: 394.1649).
127.96, 126.52, 111.29, 108.55, 106.21, 101.04, 59.38, 56.51, 53.25,
51.09, 49.08, 41.02, 38.85, 38.85, 38.85, 36.38, 36.38, 35.97, 29.46,
27.81, 27.81, 27.81. HR-ESI-MS: (m/z) 488.2433 [MþH] þ, (Calcd:
488.2431).
4.1.1.7. 10-Methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]iso-
quinolino[3,2-a]isoquinolin-9-yl
cyclopropanecarboxylate
(9).
4.1.1.12. 10-Methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]
i s o q u i n o l i n o [ 3 , 2 - a ] i s o q u i n o l i n - 9 - y l 3 - c h l o r o - 2 , 2 -
dimethylpropanoate(14). White solid, Yield: 62%. 1H NMR
White solid, Yield: 46%. 1H NMR (500 MHz, DMSO‑d6)
d
1.07e1.01
(m, 4H), 1.88e1.93 (m, 1H), 2.47e2.43 (m, 1H), 2.61e2.53 (m, 2H),
2.93e2.87 (m, 1H), 3.07e3.05 (m, 1H), 3.28e3.30 (m, 1H),
3.36e3.33 (m, 1H), 3.42e3.40 (m, 1H), 3.73 (s, 3H), 3.82 (d,
J ¼ 16.0 Hz, 1H), 5.94 (d, J ¼ 2.0 Hz, 2H), 6.66 (s, 1H), 6.91 (s, 1H),
6.95 (d, J ¼ 8.5 Hz, 1H), 7.01 (d, J ¼ 8.5 Hz, 1H). 13C NMR (75 MHz,
(500 MHz, DMSO‑d6)
d 1.39 (s, 6H), 2.47e2.42 (m, 1H), 2.61e2.54
(m, 2H), 2.89e2.86 (m, 1H), 3.01e2.98 (m, 1H), 3.42e3.32 (m, 3H),
3.72 (s, 3H), 3.93e3.89 (m, 3H), 5.94 (s, 2H), 6.66 (s,1H), 6.92 (s,1H),
6.96 (d, J ¼ 8.5 Hz, 1H), 7.04 (d, J ¼ 8.5 Hz, 1H). 13C NMR (126 MHz,
DMSO)
d
171.73, 148.73, 145.69, 145.42, 135.37, 130.62, 127.99,
DMSO) d 172.68, 149.05, 146.24, 145.98, 135.88, 131.17, 128.50,
127.43, 127.39, 126.12, 110.67, 108.00, 105.65, 100.49, 58.81, 55.81,
52.75, 50.45, 35.44, 28.92, 12.25, 8.71, 8.71. HR-ESI-MS: (m/z)
394.1631 [MþH] þ, (Calcd: 394.1649).
128.01, 127.95, 126.86, 111.31, 108.56, 106.21, 101.05, 59.38, 56.49,
53.48, 52.23, 51.14, 45.16, 35.94, 29.44, 23.37, 23.37. HR-ESI-MS: (m/
z) 444.1592 [MþH] þ, (Calcd: 444.1572).
4.1.1.8. 10-Methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]iso-
quinolino[3,2-a]isoquinolin-9-yl 2-ethoxybenzoate (10). Light yel-
4.1.1.13. 10-Methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]
isoquinolino[3,2-a]isoquinolin-9-yl
Light yellow solid, Yield: 51%. 1H NMR (300 MHz, DMSO)
0.97e0.92 (m, 6H), 1.56e1.38 (m, 6H), 1.70e1.64 (m, 2H),
2-propylpentanoate
(15).
low solid, Yield: 72%. 1H NMR (500 MHz, DMSO‑d6)
d 1.37 (t,
J ¼ 7 Hz, 3H), 2.48e2.43 (m, 1H), 2.63e2.57 (m, 2H), 2.94e2.87 (m,
1H), 3.04e3.02 (m, 1H), 3.47e3.37 (m, 3H), 3.75 (s, 3H), 3.96 (br,
1H), 4.18 (q, 2H, J ¼ 7.0 Hz), 5.95 (d, J ¼ 2.0 Hz, 2H), 6.66 (s, 1H), 6.93
(s, 1H), 7.0 (d, J ¼ 8.5 Hz, 1H), 7.08 (m, 2H), 7.21 (d, J ¼ 8.5 Hz, 1H),
7.61 (m, 1H), 7.85 (d, J ¼ 8.0 Hz, 1H).13C NMR (75 MHz, DMSO)
d
2.49e2.40 (m, 1H), 2.65e2.55 (m, 3H), 2.99e2.92 (m, 2H),
3.41e3.24 (m, 3H), 3.70 (s, 3H), 3.84e3.79 (m, 1H), 5.94 (s, 2H), 6.65
(s, 1H), 6.91 (s, 1H), 6.94 (d, J ¼ 8.5 Hz, 1H), 7.02 (d, J ¼ 8.5 Hz, 1H).
13C NMR (75 MHz, DMSO)
d 172.92, 148.55, 145.68, 145.42, 135.43,
d
163.28, 157.92,148.74,145.67,145.39,135.56,134.10,131.11,130.60,
130.55, 127.73, 127.41, 127.32, 126.12, 110.68, 107.97, 105.62, 100.48,
58.76, 55.67, 53.06, 50.56, 44.43, 35.41, 34.15, 34.15, 28.86, 19.90,
19.84, 13.76, 13.76. HR-ESI-MS: (m/z) 452.2417 [MþH]þ, (Calcd:
452.2431).
128.05, 127.50, 127.34, 126.18, 120.12, 119.12, 113.66, 110.74, 107.96,
105.64, 100.46, 64.08, 58.86, 55.84, 53.06, 50.50, 35.47, 28.89, 14.59.
HR-ESI-MS: (m/z) 474.1912 [MþH] þ, (Calcd: 474.1911).
4.1.1.9. 10-Methoxy-5,8,13,13a-tetrahydro-6H-
isoquinolino[3,2-a]isoquinolin-9-yl 4-(chloromethyl)benzoate (11).
White solid, Yield: 26%. 1H NMR (400 MHz, DMSO‑d6)
2.44e2.39
[1,3]dioxolo[4,5-g]
4.1.1.14. 2,3,10-Trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
[3,2-a]isoquinolin-9-ylcycloh exanecarboxylate (16). White solid,
d
Yield: 42%. 1H NMR (500 MHz, DMSO‑d6)
d 1.41e1.23 (m, 3H),
(m, 1H), 2.63e2.51 (m, 2H), 2.92e2.85 (m, 1H), 3.04e3.01 (m, 1H),
3.46e3.40 (m, 3H), 3.73 (s, 3H), 3.95 (br, 1H), 4.91 (s, 2H), 5.96 (d,
J ¼ 2.0 Hz, 2H), 6.67 (s, 1H), 6.96 (s, 1H), 7.04 (d, J ¼ 8.5 Hz, 1H), 7.11
1.64e1.51 (m, 3H), 1.76e1.73 (m, 2H), 2.00e1.98 (m, 2H), 2.48e2.43
(m, 1H), 2.66e2.58 (m, 3H), 2.95e2.89 (m, 1H), 3.06e3.04 (m, 1H),
3.28e3.26 (m, 1H), 3.43e3.40 (m, 2H), 3.72 (s, 6H), 3.75 (s, 3H), 3.83
(d, J ¼ 16.0 Hz, 1H), 6.68 (s, 1H), 6.88 (s, 1H), 6.95 (d, J ¼ 8.5 Hz, 1H),
(d, J ¼ 8.5 Hz, 1H), 7.68 (d, J ¼ 8.2 Hz, 2H), 8.17 (d, J ¼ 8.2 Hz, 2H). 13
C
NMR (126 MHz, DMSO)
d
163.68, 149.19, 146.26, 145.99, 144.22,
7.04 (d, J ¼ 8.5 Hz, 1H). 13CNMR (125 MHz, DMSO‑d6)
d: 172.57,
135.89, 131.15, 130.76, 130.76, 130.76, 129.85, 129.85, 129.85, 128.72,
127.95, 127.04, 111.32, 108.55, 106.23, 101.06, 59.43, 56.41, 53.3þ0,
148.57, 147.18, 135.48, 129.46, 127.94, 127.54, 126.27, 125.95, 111.72,
110.70, 109.45, 58.54, 55.84, 55.69, 55.36, 52.83, 50.64, 41.80, 35.30,
28.53, 28.49, 28.43, 25.18, 24.55, 24.54. HR-ESI-MS: (m/z) 452.2417
[MþH] þ,(Calcd: 452.2431).
50.93, 45.65, 36.01, 29.44. HR-ESI-MS: (m/z) 478.1423 [MþH]
,
(Calcd: 478.1416).
4.1.1.10. 10-Methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]
isoquinolino[3,2-a]isoquinolin-9-yl (E)-but-2-enoate(12). White
solid, Yield: 59%. 1H NMR (500 MHz, DMSO‑d6)
1.96e1.95 (m, 3H),
4.1.1.15. 2,3,10-Trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
[3,2-a]isoquinolin-9-yl furan-2-carboxylate(17). White solid, Yield:
d
22%. 1H NMR (500 MHz, DMSO‑d6)
d 2.45e2.41 (m, 1H), 2.63e2.57
2.45e2.40 (m, 1H), 2.59e2.54 (m, 2H), 2.88e2.86 (m, 1H),
3.04e3.02 (m, 1H), 3.27e3.25 (m, 1H), 3.37e3.34 (m, 1H),
(m, 2H), 2.93e2.88 (m, 1H), 3.05e3.03 (m, 1H), 3.36e3.33 (m, 1H),
3.46e3.44 (m, 2H), 3.72 (s, 3H), 3.74 (s, 3H), 3.76 (s, 3H), 3.91 (d,
9

H. Ge, W. Zhang, K. Yuan et al.
European Journal of Medicinal Chemistry 221 (2021) 113522
J ¼ 15.0 Hz, 1H), 6.67 (s, 1H), 6.81e6.80 (m, 1H), 6.89 (s, 1H), 7.02 (d,
J ¼ 8.5 Hz, 1H), 7.11 (d, J ¼ 8.5 Hz, 1H), 7.58 (d, J ¼ 3.5 Hz, 1H), 8.10 (s,
13C NMR (101 MHz, DMSO)
d 172.30, 149.25, 147.73, 135.91, 129.99,
128.61, 128.10, 126.82, 126.63, 112.24, 111.22, 109.97, 59.12, 56.35,
56.25, 55.92, 53.39, 51.20, 35.91, 29.03,12.79, 9.29, 9.24. HR-ESI-MS:
(m/z) 410.1971 [MþH] þ, (Calcd: 410.1962).
1H). 13C NMR (101 MHz, DMSO)
d 155.87, 149.19, 149.16, 147.73,
147.73, 143.10, 135.15, 129.95, 128.78, 128.30, 127.12, 126.83, 120.80,
113.24, 112.23, 111.31, 109.97, 59.14, 56.37, 56.25, 55.92, 5þ3.36, 51.11,
35.91, 29.01. HR-ESI-MS: (m/z) 436.1753 [MþH]
,
(Calcd:
4.1.1.21. 2,3,10-Trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
[3,2-a]isoquinolin-9-yl 2-ethoxy benzoate (23). White solid, Yield:
436.1755).
81%. 1H NMR (500 MHz, DMSO‑d6)
d
1.38 (t, J ¼ 7 Hz, 3H), 2.48e2.45
4.1.1.16. 2,3,10-Trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
[3,2-a]isoquinolin-9-yl 3-methylbenzoate (18). White solid, Yield:
(m, 1H), 2.64e2.59 (m, 2H), 2.96e2.91 (m, 1H), 3.06e3.04 (m, 1H),
3.40e3.37 (m, 1H), 3.48e3.444 (m, 2H), 3.73 (s, 3H), 3.75 (s, 3H),
3.76 (s, 3H), 3.97 (br, 1H), 4.20e4.16 (m, 2H), 6.68 (s, 1H), 6.90 (s,
1H), 7.01 (d, J ¼ 8.5 Hz, 1H), 7.11e7.07 (m, 2H), 7.22 (d, J ¼ 8.5 Hz,
1H), 7.62e7.59 (m, 1H), 7.84 (d, J ¼ 8.0 Hz, 1H). 13C NMR (101 MHz,
78%. 1H NMR (500 MHz, DMSO‑d6)
d 2.42e2.40 (m, 1H), 2.43 (s, 1H),
2.64e2.56 (m, 2H), 2.92e2.88 (m, 1H), 3.03e3.01 (m, 1H),
3.47e3.35 (m, 3H), 3.72 (s, 3H), 3.73 (s, 3H), 3.76 (s, 3H), 3.91 (br,
1H), 6.67 (s, 1H), 6.90 (s, 1H), 7.03 (d, J ¼ 8.5 Hz, 1H), 7.12 (d,
J ¼ 8.5 Hz, 1H),7.50 (t, J ¼ 7.5 Hz, 1H), 7.57 (d, J ¼ 7.5 Hz, 1H), 7.95 (d,
DMSO)
d 163.88, 158.50, 149.28, 147.71, 147.71, 136.12, 134.72,
131.72, 129.98, 128.70, 128.19, 126.78, 126.73, 120.70, 119.63, 114.22,
112.19, 111.28, 109.94, 64.63, 59.20, 56.40, 56.24, 55.90, 53.72, 51.28,
35.97, 29.03, 15.19. HR-ESI-MS: (m/z) 490.2209 [MþH] þ, (Calcd:
490.2224).
J ¼ 7.5 Hz, 1H), 7.97 (s, 1H) .13C NMR (101 MHz, DMSO)
d 164.16,
149.20, 147.74, 147.74, 139.06, 136.00, 135.17, 130.66, 129.99, 129.42,
128.95, 128.71, 128.24, 127.52, 126.90, 126.83, 112.23, 111.27, 109.97,
59.17, 56.36, 56.25, 55.92, 53.40, 51.14, 35.95, 29.02, 21.25. HR-ESI-
MS: (m/z) 460.2126 [MþH] þ, (Calcd: 460.2118).
4.1.1.22. 2,3,10-trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
[3,2-a]isoquinolin-9-yl 4-(chloro methyl)benzoate (24). White solid,
4.1.1.17. 2,3,10-trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
[3,2-a]isoquinolin-9-yl 3-bromo benzoate (19). White solid, Yield:
Yield: 74%. 1H NMR (500 MHz, DMSO‑d6)
d 2.44e2.41 (m, 1H),
2.64e2.59 (m, 2H), 2.90e2.88 (m, 1H), 3.03e3.02 (m, 1H),
3.44e3.32 (m, 1H), 3.48e3.45 (m, 2H), 3.72 (s, 3H), 3.73 (s, 3H), 3.76
(s, 3H), 4.02e3.83 (m, 1H), 4.89 (s, 2H), 6.67 (s, 1H), 6.90 (s, 1H), 7.03
(d, J ¼ 8.0 Hz,1H), 7.12 (d, J ¼ 8.0 Hz,1H), 7.68 (d, J ¼ 8.0 Hz, 2H), 8.16
63%. 1H NMR (500 MHz, DMSO‑d6)
d: 2.48e2.44 (m, 1H), 2.67e2.59
(m, 2H), 2.93e2.91 (m, 1H), 3.08e3.07 (m, 1H), 3.51e3.48 (m, 3H),
3.76 (s, 3H), 3.77 (s, 3H), 3.79 (s, 3H), 3.99 (br, 1H), 6.70 (s, 1H), 6.93
(s, 1H), 7.07 (d, J ¼ 8.5 Hz, 1H), 7.16 (d, J ¼ 8.5 Hz, 1H), 7.63 (t,
J ¼ 8.0 Hz,1H), 8.01 (d, J ¼ 8.0 Hz,1H), 8.18 (d, J ¼ 8.0 Hz,1H), 8.28 (s,
(d, J ¼ 8.0 Hz, 2H). 13C NMR (126 MHz, DMSO)
d 163.69, 149.18,
147.75, 144.24, 135.92, 130.77, 130.77, 130.77, 129.86, 129.86, 129.86,
129.86,128.73, 128.27, 127.01, 126.88, 112.23, 111.32, 109.95, 59.20,
56.41, 56.26, 55.94, 53.37, 51.12, 45.65, 35.95, 29.00. HR-ESI-MS: (m/
z) 494.1744 [MþH] þ, (Calcd: 494.1729).
1H). 13C NMR (101 MHz, DMSO)
d 162.86, 148.89, 147.68, 137.28,
135.80, 133.70, 132.54, 131.86, 131.10, 129.92, 129.39, 128.64, 128.30,
127.13, 126.83, 122.64, 112.25, 111.21, 109.98, 66.65, 59.16, 56.42,
56.26, 55.90, 53.31, 36.00, 29.01. HR-ESI-MS: (m/z) 524.1049 [MþH]
þ, (Calcd: 524.1067).
4.1.1.23. 2,3,10-Trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
[3,2-a]isoquinolin-9-yl (E)-but-2-enoate (25). Light yellow solid,
4.1.1.18. 2,3,10-trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
[3,2-a]isoquinolin-9-yl 4-fluorobenzoate (20). White solid, Yield:
Yield: 66%. 1H NMR (300 MHz, DMSO‑d6)
d
1.99 (d, J ¼ 6.9 Hz, 3H),
2.52e2.47 (m, 1H), 2.65e2.60 (m, 2H), 2.98e2.89 (m, 1H),
3.09e3.06 (m, 1H), 3.34e3.28 (m, 1H), 3.54e3.45 (m, 2H), 3.75 (s,
6H), 3.78 (s, 3H), 3.75 (s, 3H), 3.85 (d, J ¼ 15.0 Hz, 1H), 6.19 (dd,
J ¼ 16.0 Hz, 1.6 Hz, 1H), 6.71 (s, 1H), 6.92 (s, 1H), 7.01 (d, J ¼ 8.5 Hz,
83%. 1H NMR (500 MHz, DMSO‑d6)
d 2.44e2.40 (m, 1H), 2.64e2.56
(m, 2H), 2.92e2.88 (m, 1H), 3.04e3.01 (m, 1H), 3.47e3.34 (m, 3H),
3.72 (s, 3H), 3.73 (s, 3H), 3.76 (s, 3H), 3.93 (br, 1H), 6.67 (s, 1H), 6.90
(s, 1H), 7.03 (d, J ¼ 8.5 Hz, 1H), 7.12 (d, J ¼ 8.5 Hz, 1H), 7.45 (t,
J ¼ 8.5 Hz, 2H), 8.24e8.21 (m, 2H). 13C NMR (101 MHz, DMSO)
1H), 7.22e7.09 (m, 2H). 13C NMR (126 MHz, DMSO)
d 163.68, 149.24,
148.41, 147.74, 147.74, 135.84, 129.97, 128.63, 128.10, 126.81, 126.67,
d
167.34, 164.83, 163.14, 149.16, 147.74, 135.88, 133.38, 133.28,
121.55, 112.22, 111.20, 109.95, 59.18, 56.29, 56.25, 55.93, 53.48,
þ
129.97, 128.70, 128.27, 127.00, 126.83, 125.57, 116.83, 116.61, 112.23,
111.28, 109.96, 59.15, 56.37, 56.24, 55.91, 53.35, 51.12, 35.94, 29.01.
HR-ESI-MS: (m/z) 464.1889 [MþH] þ, (Calcd: 464.1868).
51.20, 35.93, 29.02, 18.44. HR-ESI-MS: (m/z) 410.1971 [MþH]
,
(Calcd: 410.1962).
4.1.1.24. 2,3,10-Trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
[3,2-a]isoquinolin-9-yl (3r,5r,7r)-adamantane-1-carboxylate (26).
4.1.1.19. 2,3,10-Trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
[3,2-a]isoquinolin-9-ylmethacry late (21). Light yellow solid, Yield:
White solid, Yield: 61%. 1H NMR (500 MHz, DMSO‑d6)
d 1.74 (s, 6H),
21%. 1H NMR (500 MHz, DMSO‑d6)
d
2.01 (s, 3H), 2.45e2.42 (m, 1H),
1.99 (s, 6H), 2.05 (s, 3H), 2.48e2.43 (m, 1H), 2.62e2.56 (m, 2H),
2.91e2.88 (m, 1H), 3.06e3.04 (m, 1H), 3.27e3.26 (m, 1H),
3.43e3.3.40 (m, 2H), 3.71 (s, 3H), 3.72 (s, 3H), 3.75 (s, 3H), 3.78 (br,
1H), 6.68 (s, 1H), 6.88 (s, 1H), 6.95 (d, J ¼ 8.5 Hz, 1H), 7.04 (d,
2.61e2.58 (m, 2H), 2.93e2.88 (m, 1H), 3.07e3.05 (m, 1H),
3.45e3.42 (m, 3H), 3.72 (s, 3H), 3.73 (s, 3H), 3.75 (s, 3H), 3.86 (br,
1H), 5.90 (s, 1H), 6.31 (s, 1H), 6.68 (s, 1H), 6.89 (s, 1H), 6.98 (d,
J ¼ 8.0 Hz, 1H), 7.07 (d, J ¼ 8.0 Hz, 1H). 13C NMR (101 MHz, DMSO)
J ¼ 8.5 Hz, 1H). 13C NMR (126 MHz, DMSO)
d 174.65, 149.14, 147.72,
d
164.76, 149.17, 147.74, 135.96, 135.30, 129.98, 128.57, 128.31, 128.17,
147.72, 136.07, 130.02, 128.49, 128.12, 126.83, 126.48, 112.24, 111.30,
109.96, 59.14, 56.52, 56.24, 55.93, 53.38, 51.29, 41.03, 38.85, 38.85,
38.85, 36.38, 36.38, 35.89, 29.03, 27.80, 27.80, 27.80. HR-ESI-MS: (m/
z) 504.2724 [MþH] þ, (Calcd: 504.2744).
126.82, 126.76, 112.24, 111.26, 109.97, 59.15, 56.37, 56.25, 55.93,
53.34, 51.17, 35.90, 29.02, 18.58. HR-ESI-MS: (m/z) 410.1971 [MþH]
þ, (Calcd: 410.1962).
4.1.1.20. 2,3,10-Trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
[3,2-a]isoquinolin-9-yl cyclopro panecarboxylate (22). Light yellow
4.1.1.25. 2,3,10-Trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
[3,2-a]isoquinolin-9-yl
3-chloro-2,2-dimethylpropanoate
(27).
solid, Yield: 46%. 1H NMR (300 MHz, DMSO‑d6)
d
1.10e1.05 (m, 4H),
White solid, Yield: 69%. 1H NMR (500 MHz, DMSO‑d6)
d
1.42 (s, 6H),
1.96e1.89 (m,1H), 2.52e2.49 (m,1H), 2.60e2.53 (m, 2H), 3.00e2.91
(m, 1H), 3.10e3.09 (m, 1H), 3.34e3.29 (m, 1H), 3.48e3.44 (m, 2H),
3.75 (s, 3H), 3.76 (s, 3H), 3.77 (s, 3H), 3.85 (d, J ¼ 16.0 Hz, 1H), 6.71
(s, 1H), 6.91 (s, 1H), 6.98 (d, J ¼ 8.5 Hz, 1H), 7.08 (d, J ¼ 8.5 Hz, 1H).
2.2.53e2.45 (m, 1H), 2.66e2.61 (m, 2H), 3.07e2.92 (m, 2H),
3.34e3.26 (m, 1H), 3.51e3.46 (m, 2H), 3.75 (s, 6H), 3.79 (s, 3H),
4.02e3.91 (m, 3H), 6.71 (s, 1H), 6.93 (s, 1H), 7.01 (d, J ¼ 8.0 Hz, 1H),
7.11 (d, J ¼ 8.0 Hz, 1H). 13C NMR (126 MHz, DMSO)
d 172.67, 149.03,
10

H. Ge, W. Zhang, K. Yuan et al.
European Journal of Medicinal Chemistry 221 (2021) 113522
147.72, 147.72, 135.88, 129.98, 128.57, 128.13, 126.82, 126.82, 112.23,
111.31, 109.94, 59.14, 56.49, 56.24, 55.92, 53.58, 52.24, 51.34, 45.17,
35.86, 29.00, 23.39. HR-ESI-MS: (m/z) 460.1907 [MþH] þ, (Calcd:
460.1885).
134.49, 130.49, 129.66, 127.91, 127.91, 127.27, 111.16, 111.16, 108.00,
105.60, 100.49, 58.76, 55.57, 53.29, 50.39, 35.21, 28.83. HR-ESI-MS:
(m/z) 472.0862 [MþH] þ, (Calcd: 472.0883).
4.1.2.4. 10-Methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]iso-
4.1.1.26. 2,3,10-Trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
[3,2-a]isoquinolin-9-yl 2-propyl pentanoate (28). Light yellow solid,
quinolino[3,2-a]isoquinolin-9-yl
4-fluorobenzenesulfonate
(32).
White solid, Yield: 67%. 1H NMR (500 MHz, DMSO‑d6)
d
2.46e2.41
Yield: 46%. 1H NMR (300 MHz, DMSO)
d
0.97e0.92 (m, 6H),
(m, 1H), 2.63e2.53 (m, 2H), 2.90e2.86 (m, 1H), 3.01e2.99 (m, 1H),
3.42e3.35 (m, 3H), 3.44 (s, 3H), 4.01 (d, J ¼ 16.0 Hz, 1H), 5.95 (d,
J ¼ 2.5 Hz, 2H), 6.67 (s, 1H), 6.91 (s, 1H), 6.93 (d, J ¼ 8.5 Hz, 1H), 7.08
(d, J ¼ 8.5 Hz, 1H), 7.56e7.53 (m, 2H), 8.03e8.01 (m, 2H). 13C NMR
1.59e1.36 (m, 6H), 1.73e1.65 (m, 2H), 2.49e2.43 (m, 1H), 2.66e2.58
(m, 3H), 3.02e2.88 (m, 2H), 3.31e3.26 (m, 1H), 3.45e3.41 (m, 2H),
3.71 (s, 3H), 3.72 (s, 3H), 3.75 (s, 3H), 3.85e3.79 (m,1H), 6.68 (s, 1H),
6.88 (s, 1H), 6.96 (d, J ¼ 8.5 Hz, 1H),7.05 (d, J ¼ 8.5 Hz, 1H). 13C NMR
(75 MHz, DMSO)
d 167.00, 163.64, 149.17, 145.70, 145.46, 134.51,
(75 MHz, DMSO)
d
172.97, 148.55, 147.21,147.21, 135.45, 129.40,
132.84, 131.26, 131.13, 130.49, 129.73, 127.95, 127.87, 127.29, 116.68,
127.83, 127.56, 126.24, 126.11, 111.71, 110.72, 109.43, 58.54, 55.70,
55.7, 55.37, 53.18, 50.79, 44.44, 35.34, 34.17, 34.17, 28.44, 19.92,
19.85, 13.79, 13.79. HR-ESI-MS: (m/z) 468.2753 [MþH] þ, (Calcd:
468.2744).
116.38, 111.07, 108.01, 105.59, 100.51, 58.76, 55.30, 53.46, 50.42,
þ
35.23, 28.86. HR-ESI-MS: (m/z) 484.1245 [MþH]
, (Calcd:
484.1225).
4.1.2.5. 10-Methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]iso-
4.1.2. General synthetic procedures for the target compounds
29e45 (compound 29 as example)
quinolino[3,2-a]isoquinolin-9-yl 2,4,6-triisopropylbenzenesulfonate
(33). White solid, Yield: 27%. 1H NMR (500 MHz, DMSO‑d6)
d 1.20
4.1.2.1. 10-Methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]iso-
(d, J ¼ 6.5 Hz, 6H), 1.25 (d, J ¼ 6.5 Hz, 6H), 1.90 (d, J ¼ 6.5 Hz, 6H),
2.37e2.32 (m, 1H), 2.59e2.53 (m, 2H), 2.86e2.82 (m, 2H),
3.01e2.98 (m, 1H), 3.27e3.26 (m, 1H), 3.33 (s, 3H), 3.38e3.35 (m,
2H), 3.85 (d, J ¼ 16.0 Hz, 1H), 4.03e3.97 (m, 2H), 5.94 (d, J ¼ 2.0 Hz,
2H), 6.65 (s, 1H), 6.90 (s, 1H), 6.98 (d, J ¼ 8.0 Hz, 1H), 7.05 (d,
quinolino[3,2-a]isoquinolin-9-yl
2-nitrobenzenesulfonate
(29).
The starting material 1 (0.4501 g, 1.4 mmol) was added to DCM
(20 mL) and stirred for dissolving at room temperature, 2-
nitrobenzenesulfonyl chloride (0.3270 g, 1.6 mmol), 2 mL triethyl-
amine and slight 4-dimethylamiopryidine were added to the so-
lution. The mixture was stirred for 4 h at room temperature, then
washed three times by water, saturated NaHCO3 solution and
saturated NaCl solution, respectively. The organic layer was dried
over sodium sulfate, filtered, the solvent was evaporated in vac-
uum. The residue was purified via silica gel chromatography
(CH2Cl2/MeOH) to give the product 29 (0.4003 g, 56%).
J ¼ 8.5 Hz, 1H), 7.35 (s, 2H). 13C NMR (75 MHz, DMSO)
d 153.54,
149.54, 149.16, 149.16, 145.68, 145.42, 134.96, 132.36, 130.45, 129.62,
127.72, 127.50, 127.23, 123.50, 123.50, 110.83, 107.98, 105.61, 100.47,
58.80, 55.20, 53.52, 50.48, 35.23, 33.46, 29.36, 29.36, 28.79, 24.14,
24.14, 24.14, 24.14, 23.33, 23.33. HR-ESI-MS: (m/z) 592.2731 [MþH]
þ, (Calcd: 592.2727).
White solid. 1H NMR (500 MHz, DMSO‑d6)
d
2.45e2.41 (m, 1H),
4.1.2.6. 10-Methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]iso-
quinolino[3,2-a]isoquinolin-9-yl 2,5-dimethylbenzenesulfonate (34).
2.62e2.54 (m, 2H), 2.92e2.87 (m, 1H), 2.99e2.97 (m, 1H), 3.38 (s,
3H), 3.45e3.38 (m, 3H), 3.99 (d, J ¼ 16.0 Hz, 1H), 5.95 (d, J ¼ 3.0 Hz,
2H), 6.67 (s, 1H), 6.91 (s, 1H), 6.96 (d, J ¼ 8.5 Hz, 1H), 7.12 (d,
J ¼ 8.5 Hz, 1H), 7.96 (t, J ¼ 8.0 Hz, 1H), 8.07 (t, J ¼ 8.0 Hz, 1H), 8.11 (d,
J ¼ 8.0 Hz, 1H), 8.19 (d, J ¼ 8.0 Hz, 1H). 13CNMR (125 MHz, DMSO‑d6)
White solid, Yield: 54%. 1H NMR (500 MHz, DMSO‑d6)
d 2.34 (s, 3H),
2.40e2.37 (m, 1H), 2.61e2.54 (m, 2H), 2.65 (s, 3H), 2.91e2.85 (m,
1H), 2.94e2.93 (m, 1H), 3.31 (m, 1H), 3.33 (s, 3H), 3.40e3.37 (m,
2H), 3.91 (d, J ¼ 16.0 Hz, 1H), 5.95e5.94 (m, 2H), 6.66 (s, 1H), 6.89 (s,
1H), 6.9 (d, J ¼ 8.5 Hz, 1H), 7.05 (d, J ¼ 8.5 Hz, 1H), 7.44 (d, J ¼ 8.0 Hz,
1H), 7.49 (d, J ¼ 8.0 Hz, 1H), 7.61 (s, 1H). 13C NMR (75 MHz, DMSO)
d
: 148.90, 147.44, 145.69, 145.47, 136.03, 134.78, 132.81, 130.73,
130.40, 129.36, 129.23, 128.20, 128.08, 127.30, 124.95, 111.24, 100.49,
108.00, 105.60, 58.68, 55.41, 53.19, 50.35, 35.15, 28.80. HR-ESI-MS:
(m/z) 511.1182 [MþH] þ, (Calcd: 511.1170).
d
149.40, 145.67, 145.43, 135.85, 135.83, 134.79, 134.79, 134.51,
132.32, 130.48, 129.68, 128.78, 127.85, 127.65, 127.26, 111.10, 107.98,
105.59, 100.48, 58.76, 55.27, 53.33, 50.39, 35.19, 28.82, 20.09, 19.54.
HR-ESI-MS: (m/z) 494.1653 [MþH] þ, (Calcd: 494.1632).
4.1.2.2. 10-Methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]iso-
quinolino[3,2-a]isoquinolin-9-yl 2,4,6-trimethylbenzenesulfonate
(30). White solid, Yield: 39%. 1H NMR (500 MHz, DMSO‑d6)
d
2.32
4.1.2.7. 10-Methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]iso-
(s, 3H), 2.42e2.38 (m, 1H), 2.53 (s, 6H), 2.61e2.58 (m, 2H),
2.92e2.86 (m,1H), 2.97e2.94 (m,1H), 3.35e3.33 (m, 4H), 3.41e3.37
(m, 2H), 3.98 (d, J ¼ 16.0 Hz,1H), 5.94 (d, J ¼ 3.0 Hz, 2H), 6.66 (s, 1H),
6.89 (d, J ¼ 8.5 Hz, 1H), 6.90 (s, 1H), 7.05 (d, J ¼ 8.5 Hz, 1H), 7.15 (s,
quinolino[3,2-a]isoquinolin-9-yl 4-chloro-3-nitrobenzenesulfonate
(35). White solid, Yield: 61%. 1H NMR (500 MHz, DMSO‑d6)
d:
2.50e2.48 (m, 1H), 2.64e2.55 (m, 2H), 2.91e2.88 (m, 1H),
3.06e3.04 (m, 1H), 3.41e3.37 (m, 1H), 3.45 (s, 3H), 3.48e3.46 (m,
2H), 4.05 (d, J ¼ 16.0 Hz, 1H), 5.95 (d, J ¼ 3.0 Hz, 2H), 6.98 (s, 1H),
6.91 (s, 1H), 6.97 (d, J ¼ 8.5 Hz, 1H), 7.12 (d, J ¼ 8.5 Hz, 1H), 8.12 (d,
J ¼ 8.5 Hz, 1H), 8.23 (dd, J ¼ 2.0 Hz, 8.5 Hz, 1H), 8.66 (d, J ¼ 2.0 Hz,
2H).13C NMR (125 MHz, DMSO‑d6)
d: 149.45, 145.68, 145.44, 143.16,
138.82, 134.75, 132.99, 131.25, 130.53, 129.81, 127.82, 127.55, 127.30,
110.94, 107.99, 105.62, 100.48, 58.82, 55.30, 53.4, 50.42, 35.22,
28.85, 22.08, 20.44. HR-ESI-MS: (m/z) 508.1806 [MþH] þ, (Calcd:
508.1788).
1H). 13C NMR (75 MHz, DMSO)
d 148.76, 147.65, 145.69, 145.47,
136.36, 134.33, 132.94, 132.37, 131.18, 130.35, 129.57, 128.23, 128.13,
127.25, 125.04, 111.16, 108.00, 105.55, 100.50, 58.69, 55.31, 53.32,
50.36, 35.15, 28.80. HR-ESI-MS: (m/z) 545.0790 [MþH]þ, (Calcd:
545.0780).
4.1.2.3. 10-Methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]iso-
quinolino[3,2-a]isoquinolin-9-yl
thiophene-2-sulfonate
(31).
White solid, Yield: 57%. 1H NMR (500 MHz, DMSO‑d6)
d
2.44e2.39
(m, 1H), 2.55e2.53 (m, 1H), 2.62e2.56 (m, 1H), 2.92e2.85 (m, 1H),
3.00e2.97 (m, 1H), 3.36e3.33 (m, 2H), 3.42e3.90 (m, 1H), 3.55 (s,
3H), 3.96 (d, J ¼ 16.0 Hz,1H), 5.95e5.94 (m, 2H), 6.66 (s, 1H), 6.91 (s,
1H), 6.97 (d, J ¼ 8.0 Hz, 1H), 7.10 (d, J ¼ 8.0 Hz, 1H), 7.32e7.30 (m,
1H), 7.86 (dd, J ¼ 15.0 Hz, 4 Hz, 1H), 8.21 (d, J ¼ 5.0 Hz, 1H). 13C NMR
4.1.2.8. 2,3,10-Trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
[3,2-a]isoquinolin-9-yl 2-nitrobenzenesulfonate (36). White solid,
Yield: 62%. 1H NMR (500 MHz, DMSO‑d6)
d 2.46e2.42 (m, 1H),
2.63e2.58 (m, 2H), 2.95e2.89 (m, 1H), 3.00e2.98 (m, 1H), 3.38 (s,
3H), 3.45e3.40 (m, 3H), 3.73 (s, 3H), 3.75 (s, 3H), 3.98 (d, J ¼ 16 Hz,
1H), 6.68 (s, 1H), 6.88 (s, 1H), 6.96 (d, J ¼ 8.5 Hz, 1H), 7.15 (d,
(75 MHz, DMSO)
d 149.47, 145.69, 145.45, 136.34, 135.67, 135.48,
11

H. Ge, W. Zhang, K. Yuan et al.
European Journal of Medicinal Chemistry 221 (2021) 113522
J ¼ 8.5 Hz, 1H), 7.97 (t, J ¼ 8.0 Hz, 1H), 8.07 (t, J ¼ 8.0 Hz, 1H), 8.11 (d,
J ¼ 8.0 Hz, 1H), 8.19 (d, J ¼ 8.0 Hz, 1H). 13C NMR (101 MHz, DMSO)
136.24,136.04,135.06,130.29,129.87,128.59, 128.49,126.73,112.23,
111.73, 109.93, 59.08, 56.25, 56.12, 55.92, 53.94, 51.16, 35.70, 28.96.
HR-ESI-MS: (m/z) 488.1212 [MþH] þ, (Calcd: 488.1196).
d
149.43, 147.99, 147.78, 147.74, 136.60, 135.35, 133.38, 131.28,
129.98, 129.80, 129.75, 128.76, 128.73, 126.74, 125.51, 112.23, 111.77,
109.93, 59.01, 56.25, 55.95, 55.92, 53.86, 51.13, 35.65, 28.94. HR-ESI-
MS: (m/z) 527.1491 [MþH]þ, (Calcd: 527.1483).
4.1.2.14. 2,3,10-Trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
[3,2-a]isoquinolin-9-yl 4-fluoro benzenesulfonate (42). Light yellow
solid, Yield: 38%. 1H NMR (500 MHz, DMSO‑d6)
d 2.45e2.43 (m, 1H),
4.1.2.9. 2,3,10-Trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
2.64e2.55 (m, 2H), 2.95e2.90 (m, 1H), 3.02e2.99 (m, 1H),
3.45e3.39 (m, 3H), 3.45 (s, 3H), 3.73 (s, 3H), 3.75 (s, 3H), 4.01 (d,
J ¼ 16.0 Hz, 1H), 6.68 (s, 1H), 6.87 (s, 1H), 6.94 (d, J ¼ 8.5 Hz, 1H), 7.11
[3,2-a]isoquinolin-9-yl
2,4,6-trimethylbenzenesulfonate
(37).
2.33
Light yellow solid, Yield: 67%. 1H NMR (500 MHz, DMSO‑d6)
d
(s, 3H), 2.43e2.39 (m, 1H), 2.53 (s, 6H), 2.63e2.56 (m, 2H),
2.98e2.91 (m, 2H), 3.34 (s, 3H), 3.44e3.38 (m, 3H), 3.73 (s, 3H), 3.75
(s, 3H), 3.98 (d, J ¼ 16.0 Hz, 1H), 6.68 (s, 1H), 6.88 (s, 1H), 6.90 (d,
J ¼ 8.0 Hz, 1H), 7.09 (d, J ¼ 8.0 Hz, 1H), 7.51 (s, 2H). 13C NMR
(d, J ¼ 8.5 Hz, 1H), 7.55 (t, J ¼ 9.0 Hz, 1H), 8.04e8.01 (m, 2H). 13
C
NMR (101 MHz, DMSO)
d 167.13, 164.60, 149.70, 147.77, 147.74,
135.07, 133.38, 131.81, 131.71, 130.34, 129.86, 128.63, 128.41, 126.75,
117.24, 117.01, 112.24, 111.66, 109.94, 59.08, 56.25, 55.92, 55.8þ8,
(101 MHz, DMSO)
d
149.98, 147.74, 143.73, 139.38, 139.38, 135.29,
54.10, 51.18, 35.71, 28.97. HR-ESI-MS: (m/z) 500.1551 [MþH]
,
133.52, 131.82, 131.82, 131.82, 130.44, 129.88, 128.49, 128.08, 126.73,
112.22, 111.46, 109.94, 59.16, 56.23, 55.92, 55.83, 54.10, 51.19, 35.7þ4,
(Calcd: 500.1538).
28.98, 22.65, 22.65, 21.00. HR-ESI-MS: (m/z) 524.2126 [MþH]
,
4.1.2.15. 2,3,10-Trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
(Calcd: 524.2101).
[3,2-a]isoquinolin-9-yl
2,4,6-triisopropylbenzenesulfonate
(43).
1.20 (d,
White solid, Yield: 37%. 1H NMR (500 MHz, DMSO‑d6)
d
4.1.2.10. 2,3,10-Trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
[3,2-a]isoquinolin-9-yl 4-bromo benzenesulfonate (38). White solid,
J ¼ 6.5 Hz, 12H), 1.25 (d, J ¼ 6.5 Hz, 6H), 2.37e2.32 (m, 1H),
2.59e2.52 (m, 2H), 2.89e2.83 (m, 2H), 3.01e2.98 (m, 1H),
3.27e3.24 (m, 1H), 3.34 (s, 3H), 3.44e3.41 (m, 2H), 3.72 (s, 3H), 3.74
(s, 3H), 3.85 (d, J ¼ 16.0 Hz, 1H), 4.03e3.98 (m, 2H), 6.67 (s, 1H), 6.87
(s, 1H), 6.91 (d, J ¼ 8.0 Hz, 1H), 7.08 (d, J ¼ 8.0 Hz, 1H), 7.35 (s, 2H).
Yield: 66%. 1H NMR (500 MHz, DMSO‑d6)
d 2.46e2.43 (m, 1H),
2.64e2.54 (m, 2H), 2.95e2.90 (m, 1H), 3.01e2.99 (m, 1H),
3.42e3.37 (m, 3H), 3.44 (s, 3H), 3.73 (s, 3H), 3.75 (s, 3H), 3.99 (d,
J ¼ 16.0 Hz,1H), 6.69 (s, 1H), 6.87 (s, 1H), 6.94 (d, J ¼ 8.5 Hz, 1H), 7.12
13C NMR (101 MHz, DMSO)
d 154.14, 150.12, 149.74, 149.74, 147.75,
(d, J ¼ 8.5 Hz, 1H), 7.87 (d, J ¼ 8.5 Hz, 2H), 7.93 (d, J ¼ 8.5 Hz, 2H). 13
C
147.75, 135.53, 132.92, 130.24,129.82, 128.42, 128.03, 126.67, 124.09,
124.09, 112.22, 111.41, 109.92, 59.14, 56.23, 55.92, 55.77, 54.18, 51.27,
35.74, 34.03, 29.94, 28.92, 24.75, 24.75, 24.68,24.68, 23.90, 23.86.
HR-ESI-MS: (m/z) 608.3036 [MþH] þ, (Calcd: 608.3040).
NMR (101 MHz, DMSO)
d 149.65, 147.77, 147.74, 136.25, 135.02,
132.97, 132.97, 130.37, 130.37, 130.28, 129.84, 129.14, 128.66, 128.47,
126.74,112.23,111.68,109.93, 59.07, 56.25, 55.92, 55.85, 5þ4.05, 51.17,
35.69, 28.98. HR-ESI-MS: (m/z) 560.0745 [MþH]
, (Calcd:
560.0737).
4.1.2.16. 2,3,10-Trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
[3,2-a]isoquinolin-9-yl
2,5-dimethylbenzenesulfonate
(44).
2.37
4.1.2.11. 2,3,10-Trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
[3,2-a]isoquinolin-9-yl propane-1-sulfonate (39). Light yellow solid,
Light yellow solid, Yield: 46%. 1H NMR (300 MHz, DMSO‑d6)
d
(s, 3H), 2.45e2.39 (m, 1H), 2.59e2.54 (m, 2H), 2.60 (s, 3H),
2.97e2.89 (m, 2H), 3.34e3.30 (m, 1H), 3.36 (s, 3H), 3.48e3.36 (m,
2H), 3.75 (s, 3H), 3.77 (s, 3H), 3.95 (d, J ¼ 16.0 Hz, 1H), 6.71 (s, 1H),
6.90 (s, 1H), 6.94 (d, J ¼ 8.5 Hz, 1H), 7.12 (d, J ¼ 8.5 Hz, 1H), 7.48 (d,
J ¼ 8.0 Hz, 1H), 7.54 (d, J ¼ 8.0 Hz, 1H), 7.64 (s, 1H). 13C NMR
Yield: 45%. 1H NMR (500 MHz, DMSO‑d6)
d
1.08 (t, J ¼ 7.5 Hz, 3H),
1.95e1.90 (m, 2H), 2.49e2.47 (m, 1H), 2.64e2.51 (m, 2H), 2.93e2.90
(m, 1H), 3.08e3.05 (m, 1H), 3.45e3.41 (m, 2H), 3.50 (d, J ¼ 16.0 Hz,
1H), 3.58 (t, J ¼ 7.5 Hz, 2H), 3.73 (s, 3H), 3.75 (s, 3H), 3.83 (s, 3H),
4.08 (d, J ¼ 16.0 Hz, 1H), 6.68 (s, 1H), 6.88 (s, 1H), 7.03 (d, J ¼ 8.5 Hz,
(101 MHz, DMSO)
d 149.94, 147.71, 136.42, 136.42, 135.34, 135.34,
1H), 7.12 (d, J ¼ 8.5 Hz, 1H). 13C NMR (101 MHz, DMSO)
d
149.72,
135.11, 132.91, 130.30, 129.84, 129.33, 128.54, 128.20, 126.70, 112.18,
111.67, 109.89, 59.10, 56.23, 55.91, 55.83, 53.98, 51.17, 35.68, 28.95,
20.65, 20.11. HR-ESI-MS: (m/z) 510.1935 [MþH]þ, (Calcd: 510.1945).
147.76, 147.74, 135.29, 130.58, 129.90, 128.67, 127.97, 126.77, 112.24,
111.75, 109.95, 59.06, 56.48, 56.24, 55.92, 54.16, 51.22, 35.76, 29.00,
17.67,13.03. HR-ESI-MS: (m/z) 448.1783 [MþH] þ, (Calcd: 448.1788).
4.1.2.17. 2,3,10-Trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
4.1.2.12. 2,3,10-Trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
[3,2-a]isoquinolin-9-yl ethane sulfonate (40). White solid, Yield:
[3,2-a]isoquinolin-9-yl
4-chloro-3-nitrobenzenesulfonate
(45).
White solid, Yield: 23%. 1H NMR (500 MHz, DMSO‑d6)
d
2.50e2.46
47%. 1H NMR (500 MHz, DMSO‑d6)
d
1.45 (t, J ¼ 7.5 Hz, 3H),
(m, 1H), 2.65e2.58 (m, 2H), 2.93e2.90 (m, 1H), 3.06e3.04 (m, 1H),
3.48e3.45 (m, 6H) 3.73 (s, 3H), 3.75 (s, 3H), 4.04 (d, J ¼ 16.0 Hz, 1H),
6.69 (s, 1H), 6.88 (s, 1H), 6.97 (d, J ¼ 8.5 Hz, 1H), 7.15 (d, J ¼ 8.5 Hz,
1H), 8.12 (d, J ¼ 8.5 Hz, 1H), 8.25 (dd, J ¼ 2.0 Hz, 8.5 Hz, 1H), 8.67 (d,
J ¼ 2.0 Hz, 1H). HR-MS: (m/z) 561.1111 [MþH] þ, (Calcd: 561.1093).
2.50e2.48 (m,1H), 2.65e2.54 (m, 2H), 2.97e2.91 (m,1H), 3.10e3.06
(m, 1H), 3.53e3.44 (m, 3H), 3.62 (q, J ¼ 7.5 Hz, 2H), 3.73 (s, 3H), 3.76
(s, 3H), 3.83 (s, 3H), 4.10 (d, J ¼ 16.0 Hz, 1H), 6.70 (s, 1H), 6.95 (s, 1H),
7.06 (d, J ¼ 8.5 Hz, 1H), 7.13 (d, J ¼ 8.5 Hz, 1H). 13C NMR (101 MHz,
DMSO)
d 149.72, 147.76, 147.74, 135.27, 130.58, 129.91, 128.66,
127.98, 126.78, 112.24, 111.74, 109.96, 59.06, 56.45, 56.25, 55.92,
54.17, 51.22, 47.44, 35.77, 29.01, 8.70. HR-ESI-MS: (m/z) 434.1651
[MþH] þ, (Calcd: 434.1632).
4.1.3. General synthetic procedures for the target compounds
46e50 and GZ-1 (compound 46 as the example)
4.1.3.1. 10-Methoxy-9-propoxy-5,8,13,13a-tetrahydro-6H-[1,3]diox-
olo[4,5-g]isoquinolino[3,2-a] isoquinoline (46). The starting material
1 (0.5004 g, 1.5 mmol) was added to dimethylformamide (DMF,
20 mL) and stirred for dissolving at room temperature, 1-
bromopropane (0.14 mL, 1.5 mmol) was added slowly, followed
by anhydrous K2CO3 (0.4228 g, 3 mmol). The mixture was heated at
80 ꢀC for 4 h, then cooled. The mixture was partitioned between
water and DCM. The organic layers were washed three times by
water and saturated NaCl solution, respectively. The extracts were
dried over sodium sulfate, filtered, the solvent was evaporated in
4.1.2.13. 2,3,10-Trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino
[3,2-a]isoquinolin-9-yl thiophene -2-sulfonate (41). Light yellow
solid, Yield: 58%. 1H NMR (500 MHz, DMSO‑d6)
d 2.47e2.44 (m, 1H),
2.66e2.54 (m, 2H), 2.98e2.91 (m, 1H), 3.37 (d, J ¼ 16.0 Hz, 1H),
3.49e3.45 (m, 3H), 3.59 (s, 3H), 3.76 (s, 3H), 3.78 (s, 3H), 4.01 (d,
J ¼ 16.0 Hz, 1H), 6.72 (s, 1H), 6.91 (s, 1H), 7.01 (d, J ¼ 8.5 Hz, 1H), 7.16
(d, J ¼ 8.5 Hz, 1H), 7.35e7.33 (m, 1H), 7.90e7.89 (m, 1H), 8.26e8.24
(m, 1H). 13C NMR (101 MHz, DMSO)
d 150.01, 147.76, 147.74, 136.93,
12

H. Ge, W. Zhang, K. Yuan et al.
European Journal of Medicinal Chemistry 221 (2021) 113522
vacuum. The residue was purified via silica gel chromatography
(CH2Cl2/MeOH) to give the product 46 (0.1984 g, 36%).
4.1.3.6. 10-Methoxy-9-((4-nitrobenzyl)oxy)-5,8,13,13a-tetrahydro-
6H-[1,3]dioxolo[4,5-g]isoquino
Light yellow solid, Yield: 38%. 1H NMR (400 MHz, DMSO‑d6)
d 2.44e2.41 (m, 1H), 2.60e2.52 (m, 2H), 2.91e2.86 (m, 1H),
lino[3,2-a]isoquinoline
(GZ-1).
Light yellow solid. 1H NMR (500 MHz, DMSO‑d6)
d 0.99 (t,
J ¼ 7.5 Hz, 3H), 1.71e1.65 (m, 2H), 2.46e2.44 (m, 1H), 2.61e2.54 (m,
2H), 2.94e2.88 (m, 1H), 3.08e3.05 (m, 1H), 3.31e3.29 (m, 1H),
3.39e3.36 (m, 2H), 3.76 (s, 3H), 3.90e3.82 (m, 2H), 4.06 (d,
J ¼ 16.0 Hz, 1H), 5.94 (d, J ¼ 2.0 Hz, 2H), 6.66 (s, 1H), 6.82 (d,
J ¼ 8.5 Hz, 1H), 6.86 (d, J ¼ 8.5 Hz, 1H), 6.89 (s, 1H). 13C NMR
3.05e3.03 (m, 1H), 3.39e3.3.33 (m, 3H), 3.80 (s, 3H), 4.08 (d,
J ¼ 16.0 Hz, 1H), 5.11 (s 2H), 5.94 (d, J ¼ 2.0 Hz, 2H), 6.65 (s, 1H),
6.95e6.90 (m, 3H), 7.75 (d, J ¼ 8.5 Hz, 2H), 8.26 (d, J ¼ 8.5 Hz, 2H).
13C NMR (126 MHz, DMSO)
d 150.25, 147.48, 146.23, 145.94, 143.43,
(126 MHz, DMSO)
d
150.34, 146.20, 145.91, 144.12, 131.39, 128.82,
131.33, 128.94, 128.94, 128.94, 128.18, 127.97, 124.69, 123.96, 123.96,
123.96, 111.67, 108.55, 106.21, 101.03, 72.65, 59.46, 56.31, 53.89,
51.14, 36.18, 29.47. HR-ESI-MS: (m/z) 461.1700 [MþH] þ, (Calcd:
461.1707).
128.09, 127.97, 123.93, 111.72, 108.53, 106.19, 101.01, 73.95, 59.56,
56.25, 54.07, 51.27, 36.26, 29.52, 23.70, 10.97. HR-ESI-MS: (m/z)
368.1868 [MþH]þ, (Calcd: 368.1856).
4.1.3.2. 9-Ethoxy-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo
[4,5-g]isoquino lino[3,2-a]isoquinoline (47). Light yellow solid,
4.2. Biological assays
Yield: 35%. 1H NMR (500 MHz, DMSO‑d6)
d
1.27 (t, J ¼ 7.0 Hz, 3H),
4.2.1. Cell culture
2.46e2.44 (m,1H), 2.61e2.54 (m, 2H), 2.93e2.87 (m,1H), 3.10e3.07
(m, 1H), 3.31e3.29 (m, 1H), 3.38e3.35 (m, 2H), 3.76 (s, 3H),
4.00e3.94 (m, 2H), 4.06 (d, J ¼ 16.0 Hz, 1H), 5.94 (d, J ¼ 2.0 Hz, 2H),
6.66 (s, 1H), 6.83 (d, J ¼ 8.5 Hz, 1H), 6.87 (d, J ¼ 8.5 Hz, 1H), 6.90 (s,
The cell line HepG2 was maintained in eagle's medium (DMEM,
Gibco, UK), supplemented with 10% fetal bovine serum, 100 units/
ml penicillin, and 100 mg/mL streptomycin and cultured in a 37 ꢀC
incubator with 5% CO2 in the air.
1H). 13C NMR (126 MHz, DMSO)
d 150.36, 146.20, 145.91, 143.94,
131.40, 128.94, 128.06, 127.97, 123.97, 111.64, 108.53, 106.19, 101.01,
67.95, 59.58, 56.22, 54.09, 51.23, 36.25, 29.52,16.20. HR-ESI-MS: (m/
z) 354.1701 [MþH]þ, (Calcd: 354.1700).
4.2.2. Animals and treatment
Animal experiments were conducted in accordance with the
Guidelines for Animal Experimentation of China Pharmaceutical
University, and the protocols were approved by the Animal Ethics
Committee of this institution. Six-week-old male C57BL/6J mice
were fed with high fat diet (HFD, 45% calories from fat, Medicience,
China) for 15 weeks to induce obesity. The obese animals were then
randomly separated into obesity model, low-dose 49 treatment
(170 mg/kg, dissolved in 0.1% (w/v) carboxymethyl cellulose-Na
(CMC-Na); purity > 98%, HPLC) and high-dose 49 treatment
(340 mg/kg) group. The mice in both normal control and obesity
model groups were given the same volume of 0.1% (w/v) CMC-Na.
Mice were administered by gavage at the same time in each day
for 6 weeks. During the gavage period, mice were continually given
free access to water and normal diet (13.5% of energy from fat,
normal diet, Qinglong Mountain, China) for normal control mice or
high fat diet for the mice in model and drug treated groups. Body
weight and food intake were recorded every week.
4.1.3.3. 9-Butoxy-10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo
[4,5-g]isoquinolino[3,2-a]isoquinoline (48). Light yellow solid, Yield:
32%. 1H NMR (500 MHz, DMSO‑d6)
d
0.94 (t, J ¼ 7.0 Hz, 3H),
1.49e1.41 (m, 2H), 1.68e1.63 (m, 2H), 2.47e2.43 (m, 1H), 2.61e2.53
(m, 2H), 2.93e2.87 (m, 1H), 3.08e3.05 (m, 1H), 3.27e3.26 (m, 1H),
3.38e3.35 (m, 2H), 3.75 (s, 3H), 3.95e3.86 (m, 2H), 4.06 (d,
J ¼ 16.0 Hz, 1H), 5.94 (d, J ¼ 2.0 Hz, 2H), 6.65 (s, 1H), 6.82 (d,
J ¼ 8.5 Hz, 1H), 6.86 (d, J ¼ 8.5 Hz, 1H), 6.89 (s, 1H). 13C NMR
(126 MHz, DMSO)
d 150.36, 146.25, 145.91, 144.13, 131.41, 128.84,
128.10, 127.98, 123.96, 111.74, 108.54, 106.20, 101.02, 71.99, 59.57,
56.26, 54.05, 51.27, 36.25, 32.48, 29.51, 19.21, 14.24. HR-ESI-MS: (m/
z) 382.1994 [MþH]þ, (Calcd: 382.2013).
4.1.3.4. 9-(Cyclohexylmethoxy)-10-methoxy-5,8,13,13a-tetrahydro-
6H-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinoline
(49).
1.10e1.03
White solid, Yield: 45%. 1H NMR (500 MHz, DMSO‑d6)
d
4.2.3. Cell viability assay
(m, 2H), 1.31e1.17 (m, 3H), 1.73e1.64 (m, 4H), 1.84e1.82 (m, 2H),
2.46e2.44 (m, 1H), 2.62e2.53 (m, 2H), 2.93e2.88 (m, 1H),
3.06e3.04 (m, 1H), 3.30e3.29 (m, 1H), 3.39e3.36 (m, 2H),
3.71e3.67 (m, 2H), 3.75 (s, 3H), 4.05 (d, J ¼ 16.0 Hz, 1H), 5.94 (d,
J ¼ 2.0 Hz, 2H), 6.66 (s, 1H), 6.82 (d, J ¼ 8.5 Hz, 1H), 6.86 (d,
HepG2 cells were plated in 96-well plates at the density of
2 ꢁ 104 cells/well (n ¼ 6) containing 100
L of DMEM þ10% FBS
m
treated with 10 mM compounds and cultured for 24 h, then incu-
bated with CCK8 solution (10 mL/well) for 2 h. The absorbance was
detected with a microplate reader at a test wavelength of 450 nm.
J ¼ 8.5 Hz, 1H), 6.89 (s, 1H). 13C NMR (126 MHz, DMSO)
d 150.38,
146.21, 145.92, 144.24, 131.41, 128.78, 128.13, 127.97, 123.96, 111.82,
108.54, 106.20, 101.02, 77.61, 59.52, 56.31, 54.04, 51.30, 38.71, 36.24,
29.83, 29.83, 29.51, 26.61, 25.83, 25.83. HR-ESI-MS: (m/z) 422.2306
[MþH]þ, (Calcd: 422.2326).
4.2.4. TC and TG measurement
For the experimental high-throughput screening of intracellular
TC and TG measurement, 480 in-house compounds were screened
with PerkinElmer explorer G3 workstation iX20 incl. combined
with full-automatic hander Flex 750 and JANUS G3 automatic liquid
processing platform. The HepG2 cells were cultured in 96-well
plates for 24 h, and then compounds at the concentration of
4.1.3.5. 9-((2,5-Difluorobenzyl)oxy)-10-methoxy-5,8,13,13a-tetrahy-
dro-6H-[1,3]dioxolo[4,5-g]isoqu
inolino[3,2-a]isoquinoline
(50).
White solid, Yield: 42%. 1H NMR (400 MHz, DMSO‑d6)
d
2.43e2.38
10 mM were incubated with cells for another 24 h. Before mea-
(m, 1H), 2.60e2.54 (m, 2H), 2.91e2.85 (m, 1H), 3.02e2.99 (m, 1H),
3.29e3.26 (m, 1H), 3.35e3.30 (m, 2H), 3.80 (s, 3H), 4.02 (d,
J ¼ 16.0 Hz, 1H), 5.02 (q, 2H), 5.94 (d, J ¼ 2.0 Hz, 2H), 6.65 (s,
1H),6.93e6.87 (m, 3H), 7.32e7.23 (m, 2H), 7.39e7.37 (m, 1H). 13C
surement, cell culture medium was removed and washed by PBS.
Cells were lysed for 1 h with 2% Triton X-100 solution. The TC or TG
measurement solution (Jiancheng, China) was added in cell lysis,
cultured in 37 ꢀC for 10 min, and measured at 510 nm wavelength.
For TC and TG measurement of newly synthesized compounds,
NMR (126 MHz, DMSO)
d 159.47, 157.80, 157.56, 155.87, 150.28,
146.21, 145.93, 143.21, 131.33, 128.99, 128.11, 127.93, 127.27, 127.21,
127.13, 127.07, 124.66, 117.47, 117.43, 117.35, 117.27, 117.23, 117.16,
117.06, 116.99, 116.87, 116.80, 111.66, 108.53, 106.20, 101.02, 67.08,
59.46, 56.29, 53.82, 51.13, 36.14, 29.46. HR-ESI-MS: (m/z) 452.1677
[MþH] þ, (Calcd: 452.1668).
the cells were cultured in six-well plates and collected in 200 mL
PBS. After ultrasonic disruption of cells (energy 30%, on 5 s, off 10s,
total 60s), the concentrations of TC or TG were measured using the
TC or TG determination kit according to the manufacturer's in-
structions, respectively (SKHB, China). And the protein
13

H. Ge, W. Zhang, K. Yuan et al.
European Journal of Medicinal Chemistry 221 (2021) 113522
quantification was operated after the manufacturer's instructions
of BCA kit (Beyotime, China). TC and TG levels in blood were
measured according to the manufacturer's instructions (SKHB,
China).
compound 49 in intravenous and oral administration groups,
respectively. Blood samples of intravenous administration groups
were collected at 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h,
and 12 h after intravenous administration. Blood samples of oral
administration groups were collected at 5 min, 15 min, 30 min, 1 h,
2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after oral administration. The
concentrations of compound 49 in serum were measured by LC/
MS/MS [17e19].
4.2.5. Quantitative PCR
Total RNA was extracted from cells and tissue samples using
TRIzol reagent according to the manufacturer's instructions (Invi-
trogen, USA). Equal amounts of RNA samples were used for cDNA
synthesis with a kit (HiScript Q RT SuperMix for qPCR, Vazyme,
China). Quantitative PCR analysis was carried out using qRT-PCR
(Roche, Basel, Switzerland) with SYBR-green (AceQ qPCR SYBR
Green Master Mix, Vazyme, China).
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
4.2.6. Western blot analysis
Protein from cells or mice liver tissues was extracted with RIPA
lysis buffer (65 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1%
NP-40, 0.5% sodium deoxycholate and 0.1% SDS) together with
protease inhibitor cocktail tablets (#04693132001, Roche) and
quantified with a BCA Protein Assay Kit (Beyotime, China). The
protein samples were separated using 10% SDS-PAGE gels and then
transferred to nitrocellulose filter (NC) membrane. After blocking
with 5% skim milk, the membranes were incubated with the indi-
cated primary antibodies overnight at 4 ꢀC and then with secondary
antibodies for 1 h at room temperature. The protein expression
signals were detected on an Imaging System (Tanon 5200, China).
Acknowledgement
This study was supported by National Natural Science Founda-
tion (21302052, 81773957, 82073701), National Science & Tech-
nology Major Project Key New Drug Creation and Manufacturing
Program (2019ZX09201001-001-001), the Project Program of State
Key Laboratory of Natural Medicines, China Pharmaceutical Uni-
versity (SKLNMZZRC07, SKLNMZZ202013 and SKLNMZZCX201820),
and “Double First-Class” University Project (CPU2018GF04).
References
4.2.7. Immunoprecipitation
[1] J.I. Mann, B. Lewis, J. Shepherd, A.F. Winder, B. Morgan, Blood lipid concen-
trations and other cardiovascular risk factors: distribution, prevalence, and
detection in Britain, Br. Med. J. 296 (1988) 1702e1706.
[2] M. Malaguarnera, M. Vacante, C. Russo, G. Malaguarnera, A. Frigiola, Lip-
oprotein(a) in cardiovascular diseases, BioMed Res. Int. 2013 (2013), 650989.
[3] M. Drechsler, R.T.A. Megens, M. Van Zandvoort, C. Weber, O. Soehnlein,
Hyperlipidemia-triggered neutrophilia promotes early Atherosclerosis Clinical
perspective, Circulation 122 (2010) 1837.
[4] A. Steven Grover, The benefits of treating hyperlipidemia to prevent coronary
heart disease, J. Am. Med. Assoc. 267 (1992) 816e822.
[5] G. Assmann, H. Schulte, The Prospective Cardiovascular Münster (PROCAM)
study: prevalence of hyperlipidemia in persons with hypertension and/or
diabetes mellitus and the relationship to coronary heart disease, Am. Heart J.
116 (1988) 1713e1724.
[6] J.C. Fruchart, P. Duriez, Mode of action of fibrates in the regulation of tri-
glyceride and HDL-cholesterol metabolism, Drugs Today 42 (2006) 39e64.
[7] E. Istvan, Statin inhibition of HMG-CoA reductase: a 3-dimensional view,
Atherosclerosis Suppl. 4 (2003) 3e8.
[8] M. McDonagh, K. Peterson, B. Holzhammer, S. Fazio, A systematic review of
PCSK9 inhibitors alirocumab and evolocumab, J. Manag. Care Spec. Pharm. 22
(2016) 641e653.
293T cells were seeded in three 100-mm dishes (3 ꢁ 106 cells/
dish) on day 0. The cells were transfected with indicated plasmids
(flag-SREBP1, flag-SREBP2 and HA-ubiquitin) on day 1. DMSO or 49
was mixed with fresh medium to replace former culture medium
on day 2. The cells were harvested on day 3 in PBS and lysed by IP
lysis buffer (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 5 mM EDTA, 1%
NP-40, 10% glycerol, 50 mM NaF, 1 mM Na3VO4 and protease in-
hibitor PMSF). The cell lysate was centrifuged for 10 min at
12,000 rpm at 4 ꢀC. About 10% of the supernatant was used for
Western blot as inputs, while the rest of homogenates were incu-
bated with indicated antibodies (flag and IgG) overnight at 4 ꢀC.
Protein A/G plus agarose beads (Santa Cruz) were added at 4 ꢀC for
another 2 h. The beads were washed with cold PBS for five times,
followed by western blotting analysis.
4.2.8. Oral glucose tolerance and insulin tolerance tests
[9] X.D. Wang, R. Sato, M.S. Brown, X.X. Hua, J.L. Goldstein, Srebp-1, a membrane-
bound transcription factor released by sterol-regulated proteolysis, Cell 77
(1994) 53e62.
[10] J.D. Horton, J.L. Goldstein, M.S. Brown, SREBPs: activators of the complete
program of cholesterol and fatty acid synthesis in the liver, J. Clin. Invest. 109
(2002) 1125e1131.
[11] S.H. Lee, J.H. Lee, S.S. Im, The cellular function of SCAP in metabolic signaling,
Exp. Mol. Med. 52 (2020) 724e729.
[12] H.Y. Chu, G.Z. Jin, E. Friedman, X.C. Zhen, Recent development in studies of
tetrahydroprotoberberines: mechanism in antinociception and drug addic-
tion, Cell. Mol. Neurobiol. 28 (2008) 491e499.
Oral glucose tolerance and insulin tolerance tests were per-
formed on mice fasted overnight, and insulin tolerance tests were
performed on mice fasted for 4 h. Mice were injected with either
2 g/kg glucose by i.g. or 0.75 U/kg insulin (Sigma) by i.p.. Glucose
levels were measured from tail blood before and 15, 30, 60, or
120 min after the injection. Insulin tolerance and oral glucose
tolerance tests were performed 1 and 2 weeks before their sacrifice.
[13] J. Mo, Y. Guo, Y.S. Yang, J.S. Shen, G.Z. Jin, X.C. Zhen, Recent developments in
studies of l-stepholidine and its analogs: chemistry, pharmacology and clinical
implications, Curr. Med. Chem. 14 (2007) 2996e3002.
[14] C.L. Wu, C. Xi, J.H. Tong, J. Zhao, H.L. Jiang, J. Wang, Y.P. Wang, H. Liu, Design,
synthesis, and biological evaluation of novel tetrahydroprotoberberine de-
rivatives (THPBs) as proprotein convertase subtilisin/kexin type 9 (PCSK9)
modulators for the treatment of hyperlipidemia, Acta Pharmacol. Sin. B 9
(2019) 1216e1230.
[15] K. Yuan, W. Min, X. Wang, J. Li, W. Kuang, F. Zhang, S. Xie, P. Yang, Discovery of
novel and selective CDK4/6 inhibitors by pharmacophore and structure-based
virtual screening, Future Med. Chem. 12 (2020) 1121e1136.
[16] H.X. Ge, J. Zhang, L. Chen, J.P. Kou, B.Y. Yu, Chemical and microbial semi-
synthesis of tetrahydroprotoberberines as inhibitors on tissue factor procoa-
gulant activity, Bioorg. Med. Chem. 21 (2013) 62e69.
4.2.9. Histopathologic analysis
WAT, BAT, heart, liver, spleen, lung and kidney were fixed in 4%
paraformaldehyde at 4 ꢀC overnight and embedded in paraffin wax.
Paraffin sections were mounted on glass slides for H&E staining.
Cryosections of livers were stained by oil red O and counterstained
with hematoxylin to visualize the lipid droplets.
4.2.10. Pharmacokinetic profiles in SD rats
The PK study of compound 49 was conducted on SD rats. Six SD
rats were divided into two groups, intravenous and oral adminis-
tration group. SD rats were administrated 1 and 10 mg/kg of
[17] Y. Ma, P. Li, D. Chen, T. Fang, H. Li, W. Su, LC/MS/MS quantitation assay for
14

H. Ge, W. Zhang, K. Yuan et al.
European Journal of Medicinal Chemistry 221 (2021) 113522
pharmacokinetics of naringenin and double peaks phenomenon in rats
plasma, Int. J. Pharm. 307 (2006) 292e299.
[18] X.L. Zhang, H.R. Xu, W.L. Chen, N.N. Chu, X.N. Li, G.Y. Liu, C. Yu, Matrine
determination and pharmacokinetics in human plasma using LC/MS/MS,
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 877 (2009) 3253e3256.
[19] A. Hillisch, K.M. Gericke, S. Allerheiligen, S. Roehrig, M. Schaefer,
A. Tersteegen, S. Schulz, P. Lienau, M. Gnoth, V. Puetter, R.C. Hillig,
S. Heitmeier, Design, synthesis, and pharmacological characterization of a
neutral, non-nrodrug thrombin inhibitor with good oral pharmacokinetics,
J. Med. Chem. 63 (2020) 12574e12594.
15