Design, synthesis, and anticancer properties of isocorydine derivatives

Article abstract of DOI:10.1016/j.bmc.2017.10.027

Isocorydine (ICD), an aporphine alkaloid, is widely distributed in nature. Its ability to target side population (SP) cells found in human hepatocellular carcinoma (HCC) makes it and its derivative 8-amino-isocorydine (NICD) promising chemotherapeutic agents for the treatment of HCC. To improve the anticancer activity of isocorydine derivatives, twenty derivatives of NICD were designed and synthesized through chemical structure modifications of the aromatic amino group at C-8. The anti-proliferative activities of all synthesized compounds against human hepatocellular (HepG2), cervical (HeLa), and gastric (MGC-803) cancer cell lines were evaluated using an MTT assay. The results showed that all the synthetic compounds had some tumor cell growth inhibitory activity. The compound COM33 (24) was the most active with IC₅₀ values under 10 μM (IC₅₀ for HepG2 = 7.51 μM; IC₅₀ for HeLa = 6.32 μM). FICD (12) and COM33 (24) were selected for further investigation of their in vitro and in vivo activities due to their relatively good antiproliferative properties. These two compounds significantly downregulated the expression of four key proteins (C-Myc, β-Catenin, CylinD1, and Ki67) in HepG2 cells. The tumor inhibition rate of COM33 (24) in vivo was 73.8% after a dose 100 mg/kg via intraperitoneal injection and the combined inhibition rate of COM33 (24) (50 mg/kg) with sorafenib (50 mg/kg) was 66.5%. The results indicated that these isocorydine derivatives could potentially be used as targeted chemotherapy agents or could be further developed in combination with conventional chemotherapy drugs to target cancer stem cells (CSCs) and epithelial-to-mesenchymal transition (EMT), the main therapeutic targets in HCC.

Full text of DOI:10.1016/j.bmc.2017.10.027

Accepted Manuscript
Design, synthesis, and anticancer properties of isocorydine derivatives
Qian Yan, Ruxia Li, Aiyi Xin, Yin Han, Yanxia Zhang, Junxi Liu, Wenguang
Li, Duolong Di
PII:
S0968-0896(17)31822-9
https://doi.org/10.1016/j.bmc.2017.10.027
BMC 14032
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Accepted Date:
14 September 2017
13 October 2017
20 October 2017
Please cite this article as: Yan, Q., Li, R., Xin, A., Han, Y., Zhang, Y., Liu, J., Li, W., Di, D., Design, synthesis, and
anticancer properties of isocorydine derivatives, Bioorganic & Medicinal Chemistry (2017), doi: https://doi.org/
10.1016/j.bmc.2017.10.027
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Design, synthesis, and anticancer properties of isocorydine
derivatives
Qian Yana, c, 1, Ruxia Lia, b, 1, Aiyi Xina, c, Yin Hana, Yanxia Zhanga, Junxi Liua, *
Wenguang Lib, Duolong Di a
,
a
CAS Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for
Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of
Sciences (CAS), Lanzhou 730000, P. R. China
b Gansu Key Laboratory of Preclinical Studies for New Drugs, Institute of Pharmacology, School
of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, P. R. China
c University of Chinese Academy of Sciences, Beijing 10049, P. R. China
ABSTRACT: Isocorydine (ICD), an aporphine alkaloid, is widely distributed in
nature. Its ability to target side population (SP) cells found in human hepatocellular
carcinoma (HCC) makes it and its derivative 8-amino-isocorydine (NICD) promising
chemotherapeutic agents for the treatment of HCC. To improve the anticancer activity
of isocorydine derivatives, twenty derivatives of NICD were designed and synthesized
through chemical structure modifications of the aromatic amino group at C-8. The
anti-proliferative activities of all synthesized compounds against human
hepatocellular (HepG2), cervical (HeLa), and gastric (MGC-803) cancer cell lines
were evaluated using an MTT assay. The results showed that all the synthetic
compounds had some tumor cell growth inhibitory activity. The compound COM33
(24) was the most active with IC50 values under 10 µM (IC50 for HepG2 = 7.51 µM;
IC50 for HeLa = 6.32 µM). FICD (12) and COM33 (24) were selected for further
investigation of their in vitro and in vivo activities due to their relatively good
*
Corresponding author. E-mail: liujx@licp.cas.cn (Junxi Liu). Tel.: +86 931 4968 212,
Fax: +86 931 8277 088. CAS Key Laboratory of Chemistry of Northwestern Plant
Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou
Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Lanzhou 730000,
P. R. China.
1 Co-first author: These authors contributed equally to this work.
1

antiproliferative properties. These two compounds significantly downregulated the
expression of four key proteins (C-Myc, β-Catenin, CylinD1, and Ki67) in HepG2
cells. The tumor inhibition rate of COM33 (24) in vivo was 73.8% after a dose 100
mg/kg via intraperitoneal injection and the combined inhibition rate of COM33 (24)
(50 mg/kg) with sorafenib (50 mg/kg) was 66.5%. The results indicated that these
isocorydine derivatives could potentially be used as targeted chemotherapy agents or
could be further developed in combination with conventional chemotherapy drugs to
target cancer stem cells (CSCs) and epithelial-to-mesenchymal transition (EMT), the
main therapeutic targets in HCC.
Keywords: Isocorydine, Aporphine alkaloid, Structure modification, Anticancer
activity, Hepatocellular carcinoma
1. Introduction
Aporphines are a group of tetrahydroisoquinoline alkaloids which possessed a
lots of markedly biological activities, such as central nervous system activity,
antitumor activity, et al [1,2]. Isocorydine (1), approved as an antispasmodic analgesic
by the China Food and Drug Administration (CFDA), is a typical natural aporphine
alkaloid and is widely distributed in many plants, including plants of the papaveraceae,
menispermanceae, and annonaceae families. It has antispasmodic, analgesia,
antimalarial, antiarrhythmic, and antitumor properties along with other
pharmacological effects [3-6]. Recent studies have shown that isocorydine not only
inhibits cell proliferation in hepatocellular carcinoma cell (HCC) lines by inducing
G2/M cell cycle arrest and apoptosis, but also targets drug-resistant cellular side
populations (or cancer stem cells, CSCs) by inducing PDCD4-related apoptosis.
Furthermore, isocorydine could selectively reduce the size and weight of side
population cell-induced tumor masses in nude mice, which suggested that isocorydine
is a potential therapeutic drug for targeting side population cancer cells in
hepatocellular carcinoma [7,8]. CSCs possess self-renewal properties and are
chemoresistant to the majority of anticancer agents, which is a major challenge to
clinical chemotherapy. Isocorydine could not only significantly reduce the percentage
of CD133+- and EpCAM-expressing cells, but also suppress the ability of primary
liver carcinoma PLC/PRF/5 CD133+ cells to form hepatospheres and tumor-like
spheres in vitro. To improve the anticancer activity of ICD, 8-amino-isocorydine
(NICD, 6) was prepared through chemical structure modifications [9]. A study on its
2

antitumor activity and the mechanism of action has shown that NICD could inhibit
HCC growth, particularly of the CD133+ subpopulation, and render HCC more
sensitive to sorafenib treatment [10]. In particular, NICD inhibited the expression of
IGF2BP3 in HCCs, which could enrich the CD133+ CSC subpopulation in HCCs.
CD133+ overexpression could then induce the overexpression of drug
resistance-related genes such as ABCB1 and ABCG2. NICD could also induce G2/M
cycle arrest in HCCs via DNA damage 45 alpha (GADD45A) and the p21 pathway
[11]. NICD also could inhibit HCC cell migration and invasion partly by
downregulating E2F1/ITGA1 expression [12].
NICD oxidizes easily in water due to its p-aminophenol sub-structure, which
limits it as an effective antitumor agent [9]. In addition, more effective and selective
antitumor agents are urgently required, especially for the treatment of HCC. Sorafenib
(Fig. 1) is the only successful multi-target tyrosine kinase inhibitor currently available
for the treatment of advanced liver cancer that is ineffective or intolerant to standard
therapy [13].
Completed research in our group supported that the S absolute configuration of
the hydrogen atom at C-6a is a key factor in the anticancer activity of aporphine
alkaloids and the different substituent at C-8 position can significantly affect the
anticancer activities of isocorydine derivatives [9-12]. So amide- and urea-type
derivatives of NICD were designed and synthesized. Following the chemical structure
design philosophy of sorafenib and CHMFL-KIT-8140 [13-15], NICD was considered
as the lead compound, and the C-8 amino group and the C-11 phenolic hydroxyl
group were chemically combined with the pharmacophore of above mentioned
anticancer agents. The anti-proliferative activities of all synthesized compounds
against human hepatocellular (HepG2), cervical (HeLa) and gastric (MGC-803)
cancer cell lines were evaluated via MTT assays. FICD (12) and COM33 (24) were
selected from the obtained compounds because of their better anti-proliferative
activity, especially COM33, and their in vitro and in vivo activities were investigated.
3

Fig. 1. Structures of sorafenib and CHMFL-KIT-8140.
Emerging research data have indicated that epithelial-to-mesenchymal transition
(EMT) and CSCs may be related to a high risk for recurrence and poor prognosis [16].
Recent studies in HCCs have shown that the EMT phenotype and CSC biology are
intricately linked and patients manifesting both CSC and EMT phenotypes are
unresponsive to standard chemotherapies and had low progression-free survival rates.
[16-19]. Therefore, the oncogene proteins C-myc, cell cycle-related protein Cyclin D1,
cell proliferation related protein Ki67, the main EMT marker vementin, and the
cellular differentiation-related signaling pathway Wnt/β-catenin were selected to
investigate the anticancer mechanism via western blot analysis. The results indicated
that NICD derivatives could target CSCs and EMT, which are the main therapeutic
targets in HCC. COM33 showed potential as a target chemotherapy agent to be used
in combination with conventional chemotherapy to target CSCs and EMT.
2. Results and discussion
2.1. Chemistry
To obtain enough aporphine alkaloids, isocorydine (1), corydine (2),
corytuberine (3), and isocorytuberine (4) were selectively isolated from
Dicranostigma leptopodum (Maxim.) Fedde and Stephania yunnanensis Lo via
column chromatography with silica gel in our laboratory (Fig 2) [20,21]. Isocorydione
and NICD were prepared from isocorydine according to the method reported in the
literature [9]. Nuclear magnetic resonance (NMR), High-resolution electrospray
ionization mass spectrometry (HR-ESI-MS) and X-ray crystallography (Fig. 3) were
performed to confirm four chemical structures [22-24].
Fig. 2. Structures of compounds 1-6.
4

Fig. 3. X-ray crystal structures of compounds 1-4.
To synthesize enough NICD derivatives with an aromatic amino functional group
at C-8, three strategies were applied. A nucleophilic substitution reaction between the
aromatic amino group of NICD and an aromatic carboxylic acid/aroyl chloride was
the first applied method, which produced compounds 7-11 with synthetic yields of
53%–81% (Scheme 1). The second strategy was a nucleophilic addition reaction of
aromatic isocyanate with the aromatic amino group of NICD, which formed the key
urea functional group of the HCC target drugs, such as sorafenib and regorafenib
(Scheme 2). Compounds 12-21 were prepared with synthetic yields of 3%–72%. The
aromatic isocyanates have different electrophilic reactivities for the different
substituents at -R1, -R2 and -R3. Consequently, the nucleophilic addition reaction could
occur at the C-11 hydroxy group or at the C-8 amino group in NICD. The key urea
functional group could also be synthesized through a symmetrical nucleophilic
additional-condensational reaction between triphosgene and two different kinds of
aromatic amino groups (Scheme 3), which was performed to obtain compounds 23
and 27. The key intermediates 22 and 26 were prepared via a nucleophilic substitution
reaction
between
Boc-protected-4-aminopiperidine
and
2-chloro-5-
nitrobenzotrifluoride, which was achieved in two steps with 66% yield. Next, under
acidic conditions, the protectional Boc group was removed to afford compounds 24
and 28. Subsequently, amide bond formation with acyl chloride led to the formation of
compounds 25 and 29. The structures of these target compounds were characterized
by 1H NMR, 13C NMR and high-resolution mass spectra (HR-ESI-MS).
5

Scheme 1. Synthesis of compounds 7-11. Reagents and reaction conditions: (a) carboxylic acid, HBTU, DIPEA,
DMF, rt, 8 h.
Scheme 2. Synthesis of compounds 12-21. Reagents and reaction conditions: (a) Phenyl isocyanate, CH2Cl2,
0 °C, 1 h.
Scheme 3. Synthesis of compounds 22-29. Reagents and reaction conditions: (a) K2CO3, DMF, 100 °C, 8 h; (b)
H2, 10% Pd/C, MeOH, rt, 3 h; (c) triphosgene, Et3N, DMAP, DCM, 0 °C to rt, 4 h; (d) 4 M HCl in ethyl acetate, rt,
2 h; (e) propionyl chloride, Et3N, DMF, 0 °C, 0.5 h.
2.2. Biological activity
2.2.1. In vitro anticancer activity
6

The anti-proliferative activities of all obtained compounds (1-29) were evaluated
against human hepatocellular (HepG2), cervical (HeLa), and gastric (MGC-803)
cancer cell lines via MTT assays with sorafenib as a positive control. The IC50 values
obtained are summarized in Table 1. Natural aporphine alkaloids (such as compounds
1-4) had poor inhibitory activities with IC50 values above 100 µM, consistent with the
literature [25]. In the chemical structure of compounds 1-4, the methoxyl and
hydroxyl groups although have different substituted positions, such as C-1, C-2, C-10,
or C-11, their antitumor activity was limited which means the natural aporphine
alkaloids have weakly antitumor activity. So it was necessary to chemical
modifications about natural aporphine alkaloids in order to improve their activities
and effective useage. All synthesized compounds (5-29) showed significant
improvements in anti-proliferative activity with IC50 values in micromolar levels
(9.12–75.37 µM) over all cell lines. FICD (12) and COM33 (24) showed better
anti-proliferative activity against three cancer cell lines, especially COM33. Their
IC50 values less than 10 µM was superior to those of sorafenib, which indicated that
the structure modification of NICD was effective. Pharmacophores of sorafenib and
CHMFL-KIT-8140, such as the urea, CF3, and 4-aminomenthyl-piperdine groups,
were introduced into these test compounds to increase their biological activities. The
results showed that a urea group at C-8 was superior to an amino group, such as in
compounds 7-11 where the antitumor activity was less than that of compound 12. An
aromatic amine substitution of the hydroxyl group at C-11 also increased the cytotoxic
activity, such as in compound 14 (IC50: HepG2, 16.15µM) and compound 17 (IC50:
HepG2, 17.25 µM). The activity of the 4-aminomenthyl-substituted compound (24,
IC50: HepG2, 7.51µM) was superior to that of the propionyl-protected compound (25,
IC50: HepG2, 20.69µM), although the amino group needs to be protected to improve
the pharmacokinetics in compound CHMFL-KIT-8140 [14]. Thus, five nitrogen atoms
introduced into the chemical structure of compound 24 (COM33) increased the
hydrogen bond, hydrophilic, and Van der Walls interactions between the compound
and target proteins or drug metabolic enzymes in the cancer cell, which was useful to
increase the antitumor activity of NICD derivatives.
Table 1
The antiproliferative activities (IC50: µM) of compound 1-29 and sorafenib against human cancer cell lines in vitro.
IC50 (µM)
Compounds
HepG2
>200
Hela
>200
MGC-803
>200
1
7

2
>200
>200
153.9
40.02
51.12
21.53
63.37
30.19
69.75
75.37
17.16
28.36
16.15
53.17
36.74
17.25
31.35
45.86
44.11
26.67
18.94
7.51
>200
>200
109.1
31.34
44.10
16.55
74.49
29.56
49.58
62.11
26.52
21.73
19.10
46.64
36.94
27.05
43.89
48.99
47.67
26.83
22.12
6.32
>200
>200
124.1
49.57
62.08
21.89
37.86
27.20
34.66
56.63
16.64
30.02
25.62
47.79
35.17
26.90
22.83
57.27
39.32
25.08
23.18
11.14
20.41
28.74
32.30
32.48
19.92
3
4
5
6 (NICD)
7
8
9
10
11
12 (FICD)
13
14
15
16
17
18
19
20
21
23
24 (COM33)
25
27
20.69
25.91
28.61
28.52
15.00
32.52
26.72
33.74
33.37
12.01
28
29
Sorafenib
2.2.2. The synergistic effect of FICD (12) and sorafenib for HepG2 growth inhibition
The anti-proliferative activity of FICD (12) against U251, HeLa, MGC-803,
SMMC-7721, HepG2, and MAD-MB-231 cells lines was evaluated (Fig. 4A). During
the concentration ranged from 5 µg/mL to 40 µg/mL, the growth inhibitory activities
of FICD (12) against six human cancer cells were all dose and time dependent. Of the
six cancer cell lines, FICD exhibited the best anticancer activity in HepG2. Thus,
HepG2 was selected to evaluate the detailed anti-cancer mechanism exhibited by
FICD.
8

Fig. 4. Antiproliferative activities of FICD (12) and FICD/sorafenib combination against human cancer cells in
vitro. A: The growth inhibitory activity of FICD against six human cancer cells is dose dependent with
concentrations ranging from 5.0 µg/mL to 40.0 µg/mL. B: FICD has a dose- and time-dependent inhibitory effect
on the proliferation of HepG2. C (FICD: 2.5 µg/mL + sorafenib), D (FICD: 5.0 µg/mL + sorafenib), and E (FICD:
10.0 µg/mL + sorafenib) support the hypothesis that FICD has a synergistic effect with sorafenib. (*P < 0.01, ** P
< 0.01).
As shown in Fig. 4B, FICD had a dose- and time-dependent inhibitory effect on
the proliferation of HepG2. The inhibition rate increased when the incubation time
was prolonged, whereas the inhibition rate gradually increased with the concentration
of FICD.
As sorafenib is a common, clinically used anti-HCC agent, we analyzed the
effect of low-dose FICD (2.5 µg/mL, 5.0 µg/mL, and 10.0 µg/mL) combined with
different doses of sorafenib in HepG2 cells to determine whether these compounds act
synergistically. MTT assays performed 24 h after treatment showed that the HepG2
cells were more sensitive to this combination than to sorafenib treatment alone, which
indicated that a low dose of FICD enhances the cytotoxic effect of sorafenib on HCC
cells in vitro (Table 2, Figs 4C–E). As shown in Fig 4E, at a FICD concentration of
10.0 µg/mL, the growth inhibitory activity of the combination against HepG2
significantly increased (P < 0.01) compared to that of sorafenib at 2.0 µg/mL, 4.0
µg/mL, and 6.0 µg/mL. The values of the combination index (CI) were all less than
0.9, and the synergistic multiple achieved was 2.04.
9

Table 2
The synergistic effect (IC50：µg/mL) and synergistic multiple of FICD (12) combined with sorafenib against
HepG2 growth inhibition.
Treatment group
Sorafenib (µg/mL)
IC50 (Synergistic multiple)
12.40 0.044
Treatment group
S + 5.00 µg/mL FICD
S + 2.50 µg/mL FICD
IC50 (Synergistic multiple)
8.97 (1.25)
5.44 (2.04)
11.20 (0.91)
S + 10.00 µg/mL FICD
Notes: S means sorafenib
2.2.3. Cell cycle analysis of HepG2 with FICD treatment FICD (12)
The cell cycle distribution of HepG2 under treatment with different
concentrations of FICD for 48 h was also evaluated. Untreated cells were used as a
negative control and HepG2 cells were treated with FICD (10.0 µg/mL), sorafenib
(4.0 µg/mL), and the combination (sorafenib 4.0 µg/mL + FICD 10.0 µg/mL) for 48 h.
After the cells were harvested, the cell cycle phases were examined via flow
cytometry. As shown in Fig. 5A, the sorafenib group showed a significant increase in
the number of cells in the G0/G1 phase, whereas the number of cells in the S phase
decreased significantly. In the FICD group, the number of cells in the G2/M phase
increased significantly, and the number of cells in the G0/G1 phase decreased. In the
combination group, the number of cells in the G0/G1 phase decreased compared to
that of the sorafenib group which means that FICD and sorafenib have a completely
different mechanism of pharmacological action.
Fig. 5. The effect of FICD (12) and FICD/sorafenib on cell cycle distribution and apoptosis in HepG2 cells. A:
The cell cycle distribution of HepG2 cells treated with FICD (12) for 48 h via flow cytometric analysis. Control
group, FICD (10.0 µg/mL), sorafenib (4.0 µg/mL), and FICD (10.0 µg/mL) and sorafenib (4.0 µg/mL). B:
10

Apoptosis in HepG2 cells incubated with FICD (12) or sorafenib for 24 h via flow cytometric analysis. Control
group (PBS), FICD (5.0 µg/mL), FICD (10.0 µg/mL), sorafenib (4.0 µg/mL), and FICD (10.0 µg/mL) combined
with sorafenib (4.0 µg/mL).
2.2.4. FICD (12) induces apoptosis in HepG2 cells
As shown in Fig. 5B, FICD induced apoptosis (both early and late) in HepG2
cells after treatment with FICD (5.0 µg/mL or 10.0 µg/mL), sorafenib (4.0 µg/mL), or
the combination (FICD: 10.0 µg/mL + sorafenib: 4.0 µg/mL) for 24 h. Apoptosis in
HepG2 cells increased gradually in a dose-dependent manner. The apoptosis ratios of
FICD (low and high doses), sorafenib，and the combination were found to be 8.94%,
11.89%, 28.86%, and 63.67%, respectively. These results showed that FICD,
especially the combination of FICD with sorafenib, effectively induced apoptosis in
HepG2 cells.
2.2.5. FICD (12) and COM33 (24) prevent HCC proliferation through induced
differentiation and reverse the epithelial-to-mesenchymal transition by regulating
related protein expression
The onco-inducing protein C-myc, cell cycle-related protein Cyclin D1, cell
proliferation-related protein Ki67, EMT marker vementin, and cell
differentiation-related signaling proteins Wnt/β-catenin were selected to investigate
the underlying anticancer mechanism of FICD and COM33 via western blot analysis.
The results indicated that the proteins levels of C-myc, β-catenin, Cyclin D1, and
Ki67 decreased in a dose-dependent manner after 48 h of treatment with FICD,
COM33, or sorafenib. The results were significantly different between the treatment
groups and the control group, as shown in Figs. 6A–B. Compared with other groups,
the combination group (FICD or COM33 with sorafenib) showed clear a decrease in
the levels of four typical proteins (C-myc, β-catenin, Cyclin D1 and Ki67) which are
closely related to highly aggressive tumor invasiveness, intrahepatic spread, and
extrahepatic metastasis, which occurs in HCC. This supports the hypothesis that
isocorydine derivatives have a synergistic effect with sorafenib, a multikinase
inhibitor that has shown efficacy against a wide variety of HCC subtypes.
11

Fig. 6. Western blot analysis of β-Catenin, C-Myc, vimentin, Ki-67, and Cyclin D1 after treatment of HepG2 cells
with FICD (12) (Fig. 6A), COM33 (24) (Fig. 6B), and their combination with sorafenib. Notes: β-actin antibody
was used as the reference control. A: Sorafenib (4.0 µg/mL), FICD (5.0 µg/mL), FICD (10.0 µg/mL), FICD (20.0
µg/mL), and the combination (FICD: 10.0 µg/mL + Sorafenib: 4.0 µg/mL). B: Sorafenib (4.0 µg/mL), COM33 (2.5
µg/mL), COM33 (5.0 µg/mL), COM33 (10.0 µg/mL), and the combination (COM33: 5.0 µg/mL + Sorafenib: 4.0
µg/mL).
In contrast, the expression of the mesenchymal marker vementin increased after
treatment with either sorafenib or NICD derivatives. This was a disappointing result
with respect to the classic targeted therapy, as it meant that the induction of EMT in
HCC resulted in the expression of CSC markers or the creation of CSCs, which leads
to self-renewal, drug resistance, enhanced invasiveness, and increased migratory
potential. Fortunately, as shown in Fig. 6A, the combination of sorafenib with FICD
markedly suppressed the increased expression of vementin. The combination of
sorafenib with COM33, however, had limited effects.
Overall, COM33 showed stronger antitumor activity than FICD. NICD
derivatives could suppress the expression of the oncogene protein C-myc, induce
apoptosis and cell cycle arrest, restrain HCC proliferation, and reverse EMT. The
anticancer mechanism is closely related to the Wnt/β-Catenin signaling pathway. The
detailed mechanism should be explored in the future studies.
2.2.6. In vivo anticancer activity
Tumor growth inhibition was evaluated in tumor-bearing mice after treatment
with various dosages of FICD and COM33. The results are detailed in Tables 3 and 4,
Fig.7, S Table 1, and S Fig 1. The inhibitory rate and tumor weight of the treatment
group were significantly different from those of the blank control group
(physiological saline). When the dosage increased, the FICD inhibitory rates
increased significantly. The tumor inhibition rates exceeded 40%, and the results were
12

statistically significant (Fig. 7A/a–b). Combination with sorafenib significantly
increased the inhibitory effect of FICD, indicating that FICD and sorafenib are
synergistic, such that the inhibitory rate of the high combination dosage group (50
mg/kg sorafenib + 150 mg/kg FICD) exceeded 70.6%. The synergistic effect of FICD
with sorafenib was supported by the results of in vitro experiments. To investigate the
oral absorption and bioavailability of FICD, the pharmacological properties, and the
choice of administrational method, a tumor growth inhibition experiment with
Kunming mice was performed. The results are showed in S Table 1 and S Fig. 1.
There was no statistically significant difference between the different modes of
administration (ip vs ig), indicating that FICD could be absorpted under ig
administration of FICD.
Fig. 7. Inhibitory effect of FICD (12) (Fig. 7A), COM33 (24) (Fig. 7B), and their combinations with sorafenib on
murine hepatoma H22-bearing mice. (*P < 0.01, ** P < 0.01).
Table 3
Inhibitory effect of FICD (12) and the combination with Sorafenib on H22-bearing mice.
Tumor weight
Treatment group
Control
Mice number
10
Inhibition ratio (%)
-
Dose（mg/kg/d)
(g, Mean SD)
2.62 0.92
-
13

FICD
FICD
10
10
10
10
10
10
10
150
100
1.29 0.74**
1.43 0.61**
1.60 0.65**
0.77 0.36**
0.92 0.36**
0.97 0.47**
1.16 0.54**
50.8
45.6
39.1
70.6
65.1
63.1
55.8
FICD
50
FICD+Sorafenib
FICD+Sorafenib
FICD+Sorafenib
Sorafeinb
50 + 150
50+ 100
50 + 50
50
VS Control group, ** P < 0.01.
The tumor inhibition rate of sorafenib was approximately 56.5% according to
three batches of different experiments, which indicated that the murine H22-bearing
mice could be used to evaluate the targeted therapeutic effect of NICD derivatives and
sorafenib.
The inhibitory effect of COM33 is detailed in Table 4 and Fig. 7B. The inhibitory
rate of high-dose COM33 (100 mg/kg/d) on tumor growth was 73.8%, and its tumor
inhibition rate was significantly higher than that of sorafenib (56.5%), (Fig. 7B/b).
The effect of this compound on the body weight of mice was small, which indicated
that COM33 had better tumor inhibition properties in vivo. The combination also
increased the inhibitory effect of COM33, which indicated that COM33 and sorafenib
act synergistically. The inhibitory rate of the combination dosage group (50 mg/kg
sorafenib + 50 mg/kg COM33) exceeded 66.5%.
Table 4
Inhibitory effect of COM33 (24) on H22-bearing mice.
Mice
Tumor weight
Treatment group
Inhibition ratio (%)
Dose（mg/kg/d)
number
(g, Mean SD)
Control
COM33
10
10
10
10
10
-
100
1.89 0.49
0.49 0.35**
1.37 0.61*
0.63 0.49**
0.82 0.66**
-
73.8
27.2
66.5
56.5
COM33
50
Combination
Sorafeinb
50 + 50
50
VS Control group *p < 0.05, ** P < 0.01.
3. Conclusion
In summary, four structurally similar natural isocorydine analogs were extracted
from plants, and more than twenty isocorydine derivatives were prepared through
chemical structure modification. Through the preliminary antitumor screening of the
obtained compounds, FICD (12) and COM33 (24) were selected for further anticancer
14

research using in vitro and in vivo experiments. The results suggested that structural
modifications could significantly improve the anticancer activity of isocorydine.
Identifying the mechanisms by which EMT-transformed CSCs initiate relapse
could facilitate the development of new or enhanced personalized therapeutic
regimens. The association of EMT and CSC may also form the basis for identifying
novel targeting agents to improve clinical outcomes in HCC patients. The results of
this study and previous investigations about isocorydine and NICD indicated that
NICD derivatives could target CSCs and EMT, which are the main therapeutic targets
in HCC. COM33 could be a potential targeted chemotherapy drug or be further
developed into a combination with conventional chemotherapy drugs to target CSCs
and EMT.
4. Experimental
1H NMR and 13C NMR spectral data were recorded in CDCl3, acetone-d6,
CD3OD, or DMSO-d6 using Bruker AVANCE HD 400 NMR spectrometers.
High-resolution accurate mass spectra (HR-ESI-MS) of all final target compounds
were obtained on a Bruker Micromass time flight mass spectrometer equipped with an
electrospray ionization (ESI) detector. X-ray crystallographic data were collected on a
Bruker D8 SMART APEX II. Column chromatography was performed on silica gel
(200-300 mesh, Qingdao Puke Parting Materials Co., Ltd., Qingdao, China). TLC was
also carried out on silica gel GF254 (Qingdao Marine Chemical Ltd., Qingdao, China).
Other chemical solvents and reagents used in synthesis were of analytical grade and
provided by Aladdin Industrial Corporation and used without further purification,
unless noted specifically.
4.1. General procedure for the preparation of compounds 1-6, 7-11, 12-21, and 22-29.
Isocorydine (1), corydine (2), corytuberine (3), and isocorytuberine (4) were
extracted from the plants and identified as D. leptopodum (Maxim) Fedde. S.
yunnanensis Lo was identified by Professor Zhigang Ma (Lanzhou University), and
voucher specimens of both plants were deposited at Key Laboratory of Chemistry of
Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu
Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences,
Lanzhou, China. The synthesis of compounds 5 and 6 from isocorydine and the
identification of compound 1, 5 and 6 have been reported previously [9].
1
Compound 2: H NMR (400 MHz, CDCl3) δ 6.95 (1H, s, H-3), 3.00 (2H, m,
15

H-4), 2.62 (2H, m, H-5), 3.69 (1H, dd, J = 16. 4, 3. 6 Hz, H-6a), 3.16 (1H, m, H-7α),
3.11 (1H, m, H-7β), 6.88 (1H, d, J = 8.0 Hz, H-8), 6.98 (1H, d, J = 8.0 Hz, H-9), 3.15
(3H, s, N-CH3), 3.64 (3H, s, 2-OCH3), 3.79 (3H, s, 10-OCH3), 3.85 (3H, s, 11-OCH3).
13C NMR (101 MHz, CDCl3) δ 143.7 (C-1), 122.9 (C-1a), 124.7 (C-1b), 152.4 (C-2),
111.6 (C-3), 126.2 (C-3a), 25.5 (C-4), 51.2 (C-5), 62.0 (C-6a), 31.7 (C-7), 126.9
(C-7a), 118.8 (C-8), 111.4 (C-9), 144.0 (C-10), 148.7 (C-11), 118.9 (C-11a), 61.6
(2-OCH3), 55.9 (10-OCH3), 61.2 (11-OCH3), 43.4 (N-CH3).
Compound 3: 1H NMR (400 MHz, DMSO-d6) δ 6.92 (1H, s, H-3), 3.40 (2H, m,
H-4), 3.08 (2H, m, H-5), 4.08 (1H, m, H-6a), 3.71 (1H, m, H-7a), 2.97 (1H, m, H-7b),
6.94 (1H, d, J = 8.2 Hz, H-8), 7.00 (1H, d, J = 8.2 Hz, H-9), 3.17 (3H, s, N-CH3), 3.83
(3H, s, 2-OCH3), 3.84 (3H, s, 10-OCH3). 13C NMR (101 MHz, DMSO-d6) δ 141.8
(C-1), 120.5 (C-1a), 127.0 (C-1b), 149.7 (C-2), 111.1 (C-3), 122.0 (C-3a), 25.9 (C-4),
51.8 (C-5), 62.2 (C-6a), 32.2 (C-7), 122.8 (C-7a), 120.1 (C-8), 111.8 (C-9), 149.1
(C-10), 142.7 (C-11), 120.2 (C-11a), 56.5 (2-OCH3), 56.3 (10-OCH3), 49.1 (N-CH3).
Compound 4:1H NMR (400 MHz, DMSO-d6) δ 6.68 (1H, s, H-3), 3.14 (2H, m,
H-4), 3.05 (2H, m, H-5), 2.67 (1H, m, H-6a), 2.50 (1H, m, H-7a), 2.30 (1H, m, H-7b),
6.68 (1H, d, J = 8.0 Hz, H-8), 6.80 (1H, d, J = 8.0 Hz, H-9), 2.60 (3H, s, N-CH3), 3.74
(3H, s, 2-OCH3), 3.74 (3H, s, 11-OCH3). 13C NMR (101 MHz, DMSO-d6) δ 142.9
(C-1), 120.9 (C-1a), 128.5 (C-1b), 148.5 (C-2), 109.7 (C-3), 126.0 (C-3a), 27.2 (C-4),
51.9 (C-5), 62.3 (C-6a), 34.1 (C-7), 120.5 (C-7a), 121.4 (C-8), 110.4 (C-9), 144.2
(C-10), 148.6 (C-11), 121.3 (C-11a), 55.4 (2-OCH3), 55.6 (11-OCH3), 42.3 (N-CH3).
Synthesis of compounds 7-11. To a solution of carboxylic acid (1.17 mmol),
HBTU (0.4495 g, 1.18 mmol), and DIPEA (0.5 mL, 2.21 mmol) in anhydrous DMF
(20 mL), a solution of NICD (0.4186 g 1.17 mmol) in anhydrous DMF (10 mL) was
added at 44 °C. After the mixture was stirred for 6 h, it was diluted with CH2Cl2 (100
mL) and extracted with water (250 mL×3). The organic phase was dried with Na2SO4
and concentrated under reduced pressure. The residue was purified via column
chromatography on silica gel with ethyl acetate/ methanol (V: V: = 8:1) to afford the
compound as dark green solids.
Compound 7: yield 53.2%, 1H NMR (400 MHz, CDCl3) δ 7.71(dd, J = 8.0, 2.0,
1H), 7.58 (dd, J = 8.0 2.0Hz, 1H), 7.52 (s, 1H), 7.34 (dd, J = 2.0, 2.0, Hz, 1H), 6.68 (s,
1H), 3.88 (s, 3H), 3.87 (s, 3H), 3.82 (s, 1H), 3.70 (s, 3H), 3.48 (s, 1H), 3.42 (d, J =
12.0 Hz 1H), 3.20-2.88 (m, 2H), 2.68 (d, J = 16.0 Hz, 1H), 2.50 (s, 3H), 2.15 (t, J =
12.0 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 164.1, 152.7, 151.6, 149.6, 145.0, 142.4,
138.0, 137.6, 132.2, 131.8, 131.8, 126.4, 125.2, 124.8, 123.2, 122.9, 122.3, 117.1,
16

112.5, 111.6, 62.2, 61.4, 56.2, 55.9, 52.5, 43.9, 29.7, 28.6. HRMS (ESI, m/z):
calculated for C28H27ClF3N2O5 [M + H] + 563.1555, found 563.1541.
1
Compound 8: yield, 81.5%, H NMR (400 MHz, acetone-d6) δ 7.75 (d, J = 8.0
Hz, 1H), 7.68 (d, J = 16.0 Hz, 1H), 7.61 (dd, J = 8.0, 2.0 Hz, 1H), 7.52 (dd, J = 8.0,
2.0 Hz, 1H), 7.38 (m, 1H), 7.29 (s, 1H), 7.22(m, 1H), 7.04 (d, J = 16.0 Hz, 1H), 6.79
(s, 1H), 4.06 (dd, J = 16.0, 8.0 Hz, 1H), 3.86 (s, 3H), 3.84 (s, 3H), 3.66 (s, 3H), 3.42
(dd, J = 16.0, 2.0 Hz, 1H), 3.36 - 3.24 (m, 2H), 2.82 (m, 2H), 2.71 (s, 3H), 2.42 (t, J =
12.0 Hz, 1H). 13C NMR (101 MHz, acetone-d6) δ 164.4, 152.0, 148.9, 143.3, 142.9,
142.3, 140.6, 135.2, 129.6, 128.9, 128.6, 127.9, 127.8, 127.0, 125.1, 123.6, 121.9,
118.5, 111.4, 110.4, 110.1, 62.1, 61.3, 59.7, 55.7, 55.4, 52.1, 41.8, 27.2. HRMS (ESI,
m/z): calculated for C29H31N2O5 [M + H] + 487.2227, found 487.2219.
1
Compound 9: yield, 60.1%, H NMR (400 MHz, CDCl3) δ 8.46 (d, J = 8.0 Hz,
1H), 8.36 (d, J = 7.8.0 Hz, 1H), 8.16 (dd, J = 7.8, 0.8 Hz, 1H), 7.77 (s, 1H), 7.44 –
7.39 (d, J = 8.0 Hz, 1H), 7.36 (s, 1H), 7.28 (s, 1H), 7.12 (dd, J = 8.0, 4.0 Hz, 1H),
6.72 (s, 1H), 4.34 (s, H-N), 3.94 (s, 3H), 3.91 (s, 3H), 3.71 (s, 3H), 3.27 (dd, J = 12.0,
4.0 Hz, 1H), 3.23 – 3.14 (m, 1H), 3.05 (m, 1H), 2.92 (m, 1H), 2.71 (m, 2H), 2.50 (s,
3H), 2.23 (t, J = 12.0 Hz, 1H), 2.05 (s, 1H), 1.26 (t, J = 8.0 Hz, 1H). 13C NMR (101
MHz, CDCl3) δ 161.8, 160.7, 160.6, 158.3, 151.4, 149.1, 145.6, 142.6, 142.4, 134.6,
133.7, 132.2, 131.5, 129.7, 129.5, 128.7, 128.3, 127.2, 125.7, 125.0, 123.7, 121.5,
120.6, 116.8, 116.5, 111.4, 109.0, 62.2, 60.4, 56.2, 55.9, 52.5, 43.6, 37.8, 29.7, 29.0.
HRMS (ESI, m/z): calculated for C36H34FN4O6 [M + H] + 637.2457, found 637.2445.
1
Compound 10: yield, 69.1%, H NMR (400 MHz, CDCl3) δ 7.48 (m, 1H), 7.40
(d, J = 8.0 Hz, 1H), 7.31 (s, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.96 (s, 1H), 6.64 (s, 1H),
3.88 (s, 3H), 3.83 (s, 3H), 3.65 (s, 3H), 3.33 (d, J = 12.0 Hz, 1H), 3.18 - 3.04 (m, 1H),
13
2.70 (m, 1H), 2.37 (s, 3H), 2.36 (m, 2H), 2.07 (m, 2H). C NMR (101 MHz, CDCl3)
δ 164.4, 152.0, 148.9, 143.3, 142.9, 142.3, 140.6, 135.2, 129.6, 128.9, 128.6, 128.0,
127.8, 127.01, 125.13, 123.6, 121.9, 118.5, 62.1, 61.3, 55.7, 55.4, 52.1, 41.8, 29.0,
+
27.3. HRMS (ESI, m/z): calculated for C26H28N3O5 [M + H] 462.2023, found
462.2016.
Compound 11: yield, 56.1%, 1H NMR (400 MHz, CDCl3) δ 7.75 (dd, J = 8.0, 2.0
Hz, 1H), 7.75 (7.8, 8.0 1H), 7.68 (s, 1H), 7.52 (d, J = 7.8, 8.0 Hz, 1H), 7.04 (d, J = 8.0
Hz, 1H), 6.79 (s, 1H), 4.14 (dd, J = 14.0, 7.2 Hz, 1H), 3.95 (s, 3H), 3.93(s, 3H), 3.72
(s, 3H), 3.54 (d, J = 13.8 Hz, 1H), 3.33 - 3.21 (m, 1H), 3.12 (m, 1H), 3.00 (m, 1H),
2.75 (m, 1H), 2.58 (s, 3H), 2.06 (m, 1H). 13C NMR (101 MHz, CDCl3) δ 154.1,
151.6, 149.9, 142.5, 135.3, 129.5, 128.8, 127.7, 125.4, 125.1, 125.1, 123.4, 122.3,
17

121.1, 120.6, 120.5, 111.6, 111.1, 110.9, 62.5, 62.2, 56.3, 55.9, 52.6, 43.5, 29.7, 29.6.
HRMS (ESI, m/z): calcd for C27H30N3O5 [M + H] + 476.1947, found 476.2186.
Synthesis of compounds 12-21. To a solution of NICD (0.7915 g, 2.23 mmol) in
anhydrous CH2Cl2 (50 mL), a solution of phenyl isocyanate (2.33 mmol) in anhydrous
CH2Cl2 (50 mL) was added at 0 °C under Nitrogen. After the mixture was stirred for 1
h, it was extracted with 250 mL of water three times. The organic phase was dried
with Na2SO4 and concentrated under reduced pressure. The residue was purified
through column chromatography on silica gel with petroleum ether/ethyl acetate/
methanol (V: V: V = 6:2:1) to afford the compound as a brown solid.
1
Compound 12 (FICD): yield, 72.3%, H NMR (400 MHz, CDCl3) δ 7.71 (s, 1H),
7.60 (d, J = 8.0 Hz,, 1H), 7.37 (d, J = 8.0 Hz, 1H), 6.99 (s, 1H), 6.70 (s, 1H), 3.89 (s,
6H), 3.71 (s, 3H), 3.41 (dd, J = 13.2, 1.2 Hz, 1H), 3.19 (t, J = 14.0 Hz, 1H), 3.04 -
2.98 (m, 1H), 2.83 (m, 1H), 2.70 (m, 1H), 2.47 (s, 3H), 2.15 (t, J = 14.0 Hz, 1H), 1.91
(s, 1H). 13C NMR (101 MHz, CDCl3) δ 154.1, 152.7, 151.5, 149.6, 144.9, 142.4,
138.0, 137.6, 132.2, 131.8, 131.8, 126.4, 125.2, 124.8, 123.2, 122.8, 122.3, 117.1,
112.5, 111.6, 62.2, 61.4, 56.2, 55.9, 52.5, 43.9, 29.7, 28.6. HRMS (ESI, m/z): calcd for
C28H28ClF3N3O5 [M + H] + 578.1651, found 578.1670.
Compound 13: yield, 46.9%, 1H NMR (400 MHz, CDCl3) δ 7.60 (s, 1H), 7.27 (d,
J = 8.0 Hz, 1H), 7.24 (s, 1H), 7.19 (d, J = 8.0 Hz, 1H), 6.75 (s, 1H), 3.89 (s, 6H), 3.70
(s, 3H), 3.43 (d, J = 14.0 Hz, 1H), 3.22 (m, 1H), 3.08 (m, 1H), 2.83 (m, 1H), 2.74 (m,
1H), 2.52 (s, 3H), 2.18 (t, J = 13.6 Hz, 1H), 2.06 (d, J = 3.8 Hz, 1H). 13C NMR (101
MHz, CDCl3) δ 154.0, 151.6, 149.7, 142.5, 135.3, 129.5, 128.8, 127.7, 125.4, 125.1,
125.0, 123.4, 122.3, 121.1, 120.6, 120.5, 111.6, 111.0, 110.9, 62.5, 62.2, 56.2, 55.9,
52.6, 43.5, 29.7, 26.9. HRMS (ESI, m/z): calculated for C27H28Cl2N3O5 [M + H] +
544.1401, found 544.1386.
1
Compound 14: yield, 30.7%, H NMR (400 MHz, CDCl3) δ 7.44 (d, J = 8.0 Hz,
2H), 7.33 (d, J = 8.0 Hz, 2H), 7.28 (s, 1H), 7.12 (d, J = 8.0 Hz, 2H), 6.93 (d, J = 8.0
Hz, 2H), 6.61 (s, 1H), 3.87 - 3.80 (m, 3H), 3.68 (brs, 6H), 3.08 (s, 2H), 3.05 (d, J =
2.4 Hz, 1H), 2.99 (s, 1H), 2.79 (d, J = 14.0 Hz, 2H), 2.47 (s, 3H), 2.35 (s, 3H), 2.29 (s,
13
3H), 1.99 (s, 1H). C NMR (101 MHz, CDCl3) δ 153.4 (2 × C), 151.0, 150.1, 145.1,
136.8, 134.8, 133.9, 133.6, 132.8, 131.3, 129.6 (2 × C), 129.0 (2 × C), 127.8, 126.2,
126.2, 123.3, 120.2, 118.7 (2 × C), 112.4, 107.7, 62.2, 61.2, 56.0, 55.5, 52.6, 43.9,
28.7, 26.9, 20.8, 20.7. HRMS (ESI, m/z): calculated for C36H38N4NaO6 [M + Na] +
645.2684, found 645.2645.
Compound 15: yield, 43.9%; 1H NMR (400 MHz, CDCl3) δ 7.16 (d, J = 8.0 Hz,
18

2H), 7.08 (d, J = 8.0 Hz, 2H), 6.96 (s, 1H), 6.69 (s, 1H), 3.88 (s, 3H), 3.83 (s, 3H),
3.65 (s, 3H), 3.33 (d, J = 16.0 Hz, 1H), 3.18 - 3.04 (m, 1H), 2.95 (dd, J = 11.2, 5.6 Hz,
1H), 2.72 (d, J = 8.0, 1H), 2.63 (d, J = 8.0, 1H), 2.37 (s, 3H), 2.36 (s, 3H), 2.07 (d, J =
13
8.0 Hz, 2H). C NMR (101 MHz, CDCl3) δ 154.7, 151.4, 149.4, 142.9, 142.3, 136.1,
132.5, 129.8, 128.7, 128.3 (2 × C), 125.7, 125.4, 121.0, 120.6 (2 × C), 111.5, 110.5,
62.3, 62.2, 60.4, 56.2, 55.9, 52.5, 43.5, 29.7, 28.9, 16.8. HRMS (ESI, m/z): calculated
for C28H32N3O5S [M + H] + 522.2057, found 522.2066.
1
Compound 16: yield, 3.9%, H NMR (400 MHz, CDCl3) δ 8.32 (d, J = 8.0 Hz,
1H), 7.28 (s, 1H), 7.25 (dd, J = 8.0, 2.0 Hz, 1H), 7.01(d, J = 8.0 Hz, 1H), 6.95 (dd, J =
8.0, 2.0 Hz, 1H), 6.75 (s, 1H), 4.14 (dd, J = 16.0, 8.0 Hz, 1H), 3.95 (s, 3H), 3.93(s,
3H), 3.72 (s, 3H), 3.54 (d, J = 12.0 Hz, 1H), 3.33 - 3.21 (m, 1H), 3.12 (d, J = 12.0 Hz,
1H), 3.00 (brs, 1H), 2.75 (dd, J = 16.0, 2.0 Hz, 1H), 2.58 (s, 3H), 2.30 (t, J = 16.0 Hz,
1H), 2.06 (d, J = 3.8 Hz, 1H), 1.35 - 1.21 (m, 2H). 13C NMR (101 MHz, CDCl3) δ
154.1, 151.6, 149.9, 142.5, 135.3, 129.5, 128.8, 127.7, 125.4, 125.1, 125.0, 123.4,
122.3, 121.1, 120.6, 120.5, 111.6, 111.0, 110.9, 62.5, 62.2, 56.2, 55.9, 52.6, 43.5, 29.7,
+
29.6. HRMS (ESI, m/z): calculated for C27H29ClN3O5 [M + H] 510.1790, found
510.1790.
Compound 17: yield, 43.0%, 1H NMR (400 MHz, CDCl3) δ 7.65 (s, 1H), 7.40 (d,
J = 8.0 Hz, 1H), 7.28 (s, 1H), 7.25 - 7.10 (m, 4H), 7.01 (t, J = 8.0 Hz, 1H), 6.92 (d, J
= 8.0 Hz, 1H), 6.68 (s, 1H) , 3.93 (s, 1H), 3.85 (s, 3H), 3.67 (brs, 6H), 3.19 (d, J =
12.0 Hz, 1H), 3.03 (m, 1H), 2.86 (d, J = 12.0 Hz, 2H), 2.52 (s, 3H), 2.27 – 2.09 (m,
2H), 2.07 (s, 1H). 13C NMR (101 MHz, CDCl3) δ 152.9, 152.4, 151.4, 150.5, 145.8,
135.9, 134.4, 132.6, 129.3, 129.1, 128.8, 127.8, 127.6, 127.3, 126.9, 124.2, 123.4,
123.2, 122.9, 122.8, 122.5, 120.9, 120.7, 113.4, 107.7, 62.3, 61.2, 60.4, 56.3, 55.9,
52.7, 43.8, 28.6, 28.5. HRMS (ESI, m/z): calculated for C34H32Cl2N4NaO6 [M + Na]+
685.1591, found 685.1588.
1
Compound 18: yield, 61.6%, H NMR (400 MHz, CDCl3) δ 7.28 (s, 1H), 7.21
(m, 5H), 7.05 (m, 2H), 6.94 (m, 1H), 6.77 (d, J = 8.0 Hz, 1H), 6.58 (s, 1H), 3.85 (s,
3H), 3.61 (m, 2H), 3.31 - 2.82 (m, 2H), 2.28 (s, 6H), 2.26 (m, 2H), 2.18 (s, 3H), 1.28
(s, 1H). 13C NMR (101 MHz, CDCl3) δ 153.2, 152.4, 151.4, 150.5, 145.4, 139.1,
138.8, 138.3, 129.0, 128.8, 128.5, 127.8, 127.6, 126.1, 125.0, 124.1, 123.3, 123.0,
122.9, 122.8, 122.5, 120.5, 120.3, 119.2, 117.0, 116.7, 115.8, 107.7, 62.2, 61.3, 55.8,
55.6, 52.6, 43.8, 29.7, 28.6. HRMS (ESI, m/z): calculated for C36H39N4O6 [M + H] +
623.2864, found 623.2848.
1
Compound 19: yield, 9.1%, H NMR (400 MHz, CDCl3) δ 7.51 (s, 3H), 7.28 (s,
19

2H), 6.73 (s, 1H), 3.92 (s, 6H), 3.73 (s, 3H), 3.47 (d, J = 16.0 Hz, 1H), 3.24 (m, 1H),
3.09 (m, 1H), 2.93 (m, 1H), 2.75 (d, J = 16.0Hz, 1H), 2.53 (s, 3H), 2.29 - 2.15 (m,
1H), 1.27 (s, 1H). 13C NMR (101 MHz, CDCl3) δ 154.0, 151.8, 151.7, 149.8, 142.6,
141.8, 136.8, 136.6, 129.5, 126.2, 126.2, 125.3, 124.8, 122.8, 121.0, 120.5, 120.4,
118.6 (2 × C), 111.6, 110.5, 62.6, 62.2, 56.3, 55.9, 52.5, 43.2, 29.7, 29.6. HRMS (ESI,
m/z): calculated for C28H29F3N3O5 [M + H] + 544.2054, found 544.2050.
1
Compound 20: yield, 44.5%, H NMR (400 MHz, CDCl3) δ 7.65-7.56 (m, 5H),
7.37 (m, 3H), 7.28 (s, 1H), 6.62 (s, 1H), 3.90 - 3.78 (m, 1H), 3.64 - 3.49 (s, 6H), 3.04
- 2.96 (s, 3H), 2.82 - 2.72 (m, 2H), 2.51 (s, 2H), 2.28 - 2.19 (m, 1H), 2.13 - 2.08 (m,
1H). 13C NMR (101 MHz, CDCl3) δ 152.7 (2 × C), 151.2, 150.2, 145.0, 144.2, 142.3,
140.3, 133.5, 132.7, 126.5 (2 × C), 125.9 (2 × C), 125.6 (2 × C), 125.3, 124.4, 124.1,
123.0, 122.9, 122.7, 119.5 (2 × C), 117.9 (2 × C), 112.4, 107.5, 62.1, 61.3, 55.8, 55.7,
52.5, 43.8, 29.7, 28.5. HRMS (ESI, m/z): calculated for C36H33F6N4O6 [M + H] +
731.2299, found 731.2286.
1
Compound 21: yield, 64.5%, H NMR (400 MHz, acetone-d6) δ 7.63-7.50 (m,
5H), 7.08-7.03 (m, 4H), 6.80 (s, 1H), 3.91 (s, 1H), 3.85 (s, 3H), 3.80 (s, 3H), 3.68 (d,
J = 4.0 Hz, 1H), 3.45 (s, 3H), 3.36 (dd, J = 14.0, 2.0 Hz, 1H), 3.05 - 2.98 (m, 1H),
13
2.79 (m, 1H), 2.68 (m, 1H), 2.50 (s, 3H), 1.29 (s, 1H). C NMR (101 MHz, acetone
d6) δ 151.8, 151.3, 150.1, 148.0, 145.1, 140.3, 135.8, 134.3, 133.2, 132.5, 129.2,
127.9, 125.8, 124.4, 121.9, 121.3, 120.5, 124.3, 120.1, 118.9, 115.2, 115.1, 115.0,
114.9, 112.6, 107.5, 62.9, 60.5, 55.6, 55.6, 52.7, 43.3, 30.1, 29.8. HRMS (ESI, m/z):
calculated for C34H33F2N4O6 [M + H] + 631.2363, found 631.2354.
Synthesis of compound 22. To a solution of 2-chloro-5-nitrobenzotrifluoride
(2.5363 g, 11.27 mmol) and K2CO3 (4.2203 g, 30.58 mmol) in DMF (60 mL),
tert-butyl (piperidin-4-ylmethyl) carbamate (2.4127 g, 11.27 mmol) was added. The
mixture was stirred at 100 °C for 8 h. The solvent was removed under vacuum after it
was allowed to cool down to 25 °C. The residue was dissolved in ethyl acetate (100
mL), washed with water (250 mL×3) and brine (250 mL×3), dried with Na2SO4, and
then concentrated to afford the crude nitrobenzene product as a light yellow solid.
This was used in the following steps without further purification. To a solution of the
crude nitrobenzene in methanol (50 mL), 10% Pd/C (2.0 g, 20% w/w) was added at
25°C. The reaction mixture was stirred under hydrogen in a high-pressure reactor (0.3
MPa) for 3 h and then filtered through busher funnel. The filtrate was concentrated to
obtain the residue, which was purified through column chromatography on silica gel
with ethyl acetate/methanol (V: V = 8:1) to afford compound 22 as a light brown solid.
20

1
Yield, 65.4% in two steps, H NMR (400 MHz, CDCl3) δ 7.17 (d, J = 8.0 Hz, 1H),
6.92 (d, J = 2.0 Hz, 1H), 6.81 (dd, J = 8.0, 2.0 Hz, 1H), 4.63 (s, H-N, 1H), 3.94 (brs,
H-N, 1H), 3.06 (t, J = 12.0 Hz, 2H), 2.95 (dd, J = 12.0, 2.0 Hz, 2H), 2.63 (t, J = 12.0
Hz, 2H), 1.70 (dd, J = 12.0, 2.0 Hz, 2H), 1.45 (s, 9H), 1.38 (dd, J = 12.0, 4.0 Hz, 2H).
13
1.23 (m, 1H) C NMR (101 MHz, CDCl3) δ 156.1, 144.5, 142.9, 125.6, 125.3, 120.8
118.8, 113.3, 79.1, 54.1 (2 × C), 46.4, 36.2, 30.5 (2 × C), 29.8 (3 × C). HRMS (ESI,
m/z): calculated for C18H27F3N3O2 [M + H] + 374.2050, found 374.2038.
Synthesis of compound 23. To a solution of triphosgene (0.7537 g, 2.54 mmol) in
anhydrous CH2Cl2 (30 mL), a solution of NICD (2.0870 g, 5.86 mmol), Et3N (1 mL,
7.30 mmol), and DMAP (0.0753 g, 0.62 mmol) in anhydrous CH2Cl2 (30 mL) was
added at 0 °C under Nitrogen. After stirring for 2 h, a solution of 22 (2.1862 g, 5.86
mmol), Et3N (1 mL, 7.30 mmol), and DMAP (0.0757 g, 0.62 mmol) in anhydrous
CH2Cl2 (30 mL) was added to the above-mentioned reaction mixture at 0°C under
nitrogen. The mixture was warmed to room temperature and stirred for 2 h. The
reaction mixture was washed with water (250 mL×3) and brine (250 mL×3), dried
with Na2SO4, filtered, and concentrated under reduced pressure. The residue was
purified using column chromatography on silica gel with dichloromethane/methanol
1
(V: V = 20:1) to afford compound 23 as a brown solid. Yield, 58.7%, H NMR (400
MHz, CDCl3) δ 7.50 (d, J = 8.0 Hz, 1H), 7.31 (brs, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.96
(s, 1H), 6.64 (s, 1H), 4.83 (s, 1H), 3.83 (s, 3H), 3.76 (s, 3H), 3.58 (s, 3H), 3.26 (d, J =
8.0 Hz, 1H), 2.99 (m, 3H), 2.91 - 2.74 (m, 4H), 2.62 (m, 3H), 2.48 (m, 2H), 2.33 (s,
3H), 2.07 - 1.95 (m, 1H), 1.62 (m, 2H), 1.42 (s, 9H), 1.36 - 1.21 (m, 2H). 13C NMR
(101 MHz, CDCl3) δ 156.4, 154.7, 151.2, 149.1, 148.1, 142.2, 135.5, 129.9, 128.9,
127.6, 127.3, 125.9, 125.4, 125.0, 124.6, 123.6, 122.3, 120.8, 118.1, 111.4, 110.2, 79.3,
63.2, 62.2, 62.0, 56.0, 55.8, 53.8, 52.5, 46.3, 43.5, 37.3, 36.2, 30.4, 29.7, 28.9, 28.4
(3×C). HRMS (ESI, m/z): calculated for C39H49F3N5O7 [M + H] + 756.3579, found
756.3571.
Synthesis of compound 24. Compound 23 (1.4590 g, 1.93 mmol) was dissolved
in 4 N HCl in ethyl acetate (20 mL) at 25°C. The resulting mixture was stirred for 2 h
and then filtered to obtain compound 24 as an off-white solid. Yield, 91.6%, 1H NMR
(400 MHz, CD3OD) δ 8.03 (d, J = 2.0 Hz, 1H), 7.75 (dd, J = 8.0, 2.0 Hz, 1H), 7.67 (d,
J = 8.0 Hz, 1H), 7.16 (s, 1H), 6.98 (s, 1H), 4.02 (dd, J = 12.0, 2.0 Hz, 2H), 3.91 (s,
3H), 3.90 (s, 3H), 3.81 - 3.75 (m, 2H), 3.71 (s, 3H), 3.58 (dd, J = 14.0, 2.0 Hz, 2H),
3.47 (m, 1H), 3.45 (s, 1H), 3.41 (d, J = 4.0 Hz, 1H), 3.18 (s, 4H), 3.08 (m, 2H), 2.95
(d, J = 8.0 Hz, 3H), 2.52 (t, J = 12.0 Hz, 1H), 2.01 (d, J = 12.0 Hz, 4H), 1.67 (d, J =
21

10.7 Hz, 2H). 13C NMR (101 MHz, CD3OD) δ 156.2, 154.5, 150.3, 145.4, 143.3,
140.4, 138.2, 128.1, 127.6, 126.9, 126.6, 126.4, 125.7, 124.3, 123.7, 122.7, 120.7,
118.4, 112.6, 111.4, 63.8, 62.3, 56.8, 56.6, 56.2, 53.5, 47.0, 45.4, 42.4, 34.2, 30.1, 29.0,
28.9, 26.9. HRMS (ESI, m/z): calculated for C34H41F3N5O5 [M + H] + 656.3054, found
656.3039.
Synthesis of compound 25. To a solution of compound 24 (0.1903 g, 0.29 mmol)
and Et3N (0.13 mL, 0.95 mmol) in anhydrous DMF (3 mL) at 0 °C under nitrogen,
propionyl chloride (0.03 mL, 0.31 mmol) was added. After being stirred for 0.5 h, the
reaction mixture was quenched with water, diluted with CH2Cl2 (10 mL), and washed
with water (20 mL×3) and brine (20 mL×3). The organic phase was dried with
Na2SO4, filtered, and concentrated under vacuum to obtain the residue, which was
purified through column chromatography on silica gel with petroleum
dichloromethane/methanol (V: V = 20:1) to afford the compound as a white solid.
1
Yield, 48.6%. H NMR (400 MHz, DMSO-d6) δ 7.81 (dd, J = 8.0, 2.0 Hz, 1H), 7.59
(d, J = 2.0 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.07 (s, 1H), 6.93 (s, 1H), , 3.89 (s, 3H),
3.80 (s, 3H), 3.69 (s, 3H), 3.55 (m, 2H), 3.44 (dd, J = 14.0, 2.0 Hz, 2H), 2.65 (d, J =
3.8 Hz, 1H), 2.41 (s, 3H), 2.35 (s, 1H), 2.24 (d, J = 4.0 Hz, 1H), 2.12 (m, 4H), 2.08
(dd, J = 14.0, 2.0 Hz, 2H), 1.86 (t, J = 6.0 Hz, 3H), 1.67 (t, J = 14.0 Hz, 1H), 1.20 (m,
5H), 1.01 (t, J = 8.0, 2.0 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 173.3, 153.9,
151.5, 148.1, 147.1, 143.1, 141.4, 137.7, 129.4, 127.3, 126.6, 126.3, 125.8, 125.6,
125.3, 123.8, 123.1, 123.0, 120.4, 116.4, 111.9, 109.5, 62.9, 61.7, 56.26, 56.2, 54.1,
52.7, 44.5, 44.1, 40.6, 40.4, 40.2, 40.0, 39.8, 39.6, 39.4, 35.9, 30.8, 30.2, 29.0, 10.6.
HRMS (ESI, m/z): calculated for C37H45F3N5O6 [M + H] + 712.3322, found 712.3308.
Compound 26 was prepared following the synthetic procedure for compound 22.
1
Yield, 66.7% in two steps; H NMR (400 MHz, CDCl3) δ 7.15 (d, J = 8.0 Hz, 1H),
6.89 (d, J = 2.0 Hz, 1H), 6.78 (dd, J = 8.0, 2.0 Hz, 1H), 4.54 (s, 1H, H-N), 2.90 (d, J =
12.0 Hz, 2H), 2.72 (t, J = 8.0 Hz, 2H), 1.95 (d, J = 12.0 Hz, 2H), 1.54 (ddd, J = 14.0,
12.0, 4.0 Hz, 3H), 1.46 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 155.3, 143.7, 143.5,
125.2, 122.5, 118.6, 113.0, 100.1, 79.2, 53.1, 47.7, 33.2, 28.4 (3×C). HRMS (ESI,
m/z): calculated for C17H24F3N3NaO2 [M + H] + 382.1713, found 382.1718.
Compound 27 was prepared following the synthetic procedure for compound 23.
1
Yield, 43.2%, H NMR (400 MHz, CDCl3) δ 7.51 (s, 1H), 7.40 (dd, J = 8.0, 2.0, Hz,
1H), 7.04 (d, J = 8.0 Hz, 1H), 6.95 (s, 1H), 6.62 (s, 1H), 4.64 (s, 1H), 3.81 (s, 3H),
3.72 (s, 3H), 3.55 (s, 3H), 3.33 (s, 1H), 3.23 (d, J = 12.0 Hz, 2H), 3.04 (s, 2H), 2.81
(m, 4H), 2.60 (m, 4H), 2.31 (s, 3H), 1.99 (t, J = 12.0 Hz, 1H), 1.86 (s, 2H), 1.41 (s,
22

13
9H). C NMR (101 MHz, CDCl3) δ 155.5, 154.7, 151.2, 148.9, 147.4, 142.2, 135.9,
129.8, 128.8, 127.6, 127.3, 126.1, 125.4, 125.0, 124.5, 123.4, 122.3, 120.7, 117.9,
111.3, 110.1, 79.3, 62.1, 62.0, 55.9, 55.8, 52.7, 52.4, 50.2, 47.6, 43.4, 33.0, 29.6, 28.8,
28.4 (3×C). HRMS (ESI, m/z): calculated for C38H47F3N5O7 [M + H] + 742.3422,
found 742.3430.
Compound 28 was prepared following the synthetic procedure for compound 24.
Yield, 92.8%; 1H NMR (400 MHz, CD3OD) δ 7.91 (d, J = 2.0 Hz, 1H), 7.63 (dd, J =
8.0, 2.0 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.14 (s, 1H), 6.97 (s, 1H), 4.00 (d, J = 12.0
Hz, 2H), 3.90 (s, 3H), 3.89 (s, 3H), 3.80 - 3.72 (m, 2H), 3.70 (s, 3H), 3.61 - 3.53 (m,
2H), 3.50 - 3.37 (m, 3H), 3.28 - 3.21 (m, 2H), 3.17 (s, 3H), 3.11 - 2.97 (m, 4H), 2.87
(m, 2H), 2.51 (t, J = 12.0 Hz, 1H), 2.07 (m, 2H), 1.81 (m, 2H). 13C NMR (101 MHz,
CD3OD) δ 155.0, 153.2, 149.0, 146.0, 144.0, 141.8, 137.3, 126.9, 126.2, 125.2, 124.9,
122.8, 122.6, 122.3, 121.4, 119.3, 116.8, 111.2, 110.1, 68.1, 62.5, 60.9, 55.4, 55.2,
52.2, 51.8, 41.0, 30.5, 29.7, 27.6, 27.4, 25.6. HRMS (ESI, m/z): calculated for
C33H39F3N5O5 [M + H] + 642.2898, found 642.2891.
Compound 29 was prepared following the synthetic procedure for compound 25.
Yield, 48.3%;1H NMR (400 MHz, CD3OD) δ 7.78 (d, J = 2.0 Hz, 1H), 7.59 (d, J = 8.0
Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.18 (s, 3H), 6.86 (s, 3H), 3.84 (s, 3H), 3.80 (s, 3H),
3.62 (s, 3H), 3.19 (d, J = 2.0 Hz, 1H), 3.01 (m, 4H), 2.86 (m, 2H), 2.63 (m, 3H), 2.41
(m, 2H), 2.35(s, 3H), 2.10 (m, 2H), 1.85 (m, 1H), 1.67 (m, 1H), 1.24 (m, 1H), 1.01 (t,
J = 8.0 Hz, 3H). 13C NMR (101 MHz, CD3OD) δ 173.3, 153.9, 151.4, 147.1, 141.4,
137.7, 127.3, 127.0, 126.9, 126.3, 126.2, 126.3, 125.6, 123.8, 123.0, 120.4, 116.4,
111.9, 109.5, 79.6, 62.9, 61.7, 56.3, 56.2, 54.1, 52.7, 44.5, 44.1, 39.0, 35.9, 30.8, 30.2,
29.0, 10.6. HRMS (ESI, m/z): calculated for C36H43F3N5O6 [M + H] + 698.3160, found
698.3158.
4.2. Biological evaluation
4.2.1. Materials
All human cancer cell lines used in this study were provided by the Cell Bank of
the Biochemistry and Cell Biology at the China Academy of Sciences (Shanghai,
China). Murine hepatoma cell line H22 was obtained from the cell library of the
Institute of Cancer Biology and Drug Discovery, Lanzhou University. All cell lines
were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal
bovine serum (FBS), 100 units/mL of penicillin, and 100 g/mL of streptomycin in a
humidified atmosphere of 5% CO2 at 37 °C. Kunming male mice weighing 18.0–22.0
23

g were purchased from the Experimental Animal Center at Lanzhou University. The
use and treatment of mice were in accordance with institutional guidelines for
LaboratoryAnimal Care.
4.2.2. Cell growth inhibition assay
The anticancer activities of all compounds were investigated in three human
cancer cell lines (HepG2, HeLa, and MGC-803). The in vitro proliferative response of
the cancer cells was estimated using an MTT dye assay. Cancer cells were maintained
in RPMI 1640 and in DMEM medium, respectively. After counting the number of
cells with a hemocytometer by using the 0.04% Trypan Blue dye exclusion technique,
the cells were cultured in 96-well microplates (5×103 cells/100 µL in each well) with
RPMI 1640 medium and incubated for 24 h at 37 °C in 5% CO2. Inhibitor solutions
were prepared by serial dilution of 5 mM DMSO stock solution with the culture
medium. The final amount of DMSO per well was maintained below 0.1% V/V. The
cells were then treated with different concentrations of the test compounds, positive
control sorafenib, and medium alone with or without 0.1% DMSO as negative
controls for 48 h at 37 °C with 5% CO2. Next, 10 µL MTT solution diluted in culture
medium (5 mg/mL) was added to each well and incubated for 4 h. The supernatants
were carefully extracted, and formazan precipitates were dissolved in 100 µL of
DMSO. Plates were then shaken vigorously (300 rpm) for 5 min. Finally, the
difference in absorbance at 570 nm was measured using a Microplate
Spectrophotometer (DNM-9602). Each assay was performed in triplicate. The
inhibition percentage of test compounds with concentrations of 10–100 µg/mL was
calculated as follows: (1- ∆OD wells treated with compounds/∆OD wells treated with medium alone) ×100.
Next, IC50 values were calculated by nonlinear regression analysis using SPSS 19
(Version 6.0) software and expressed as mean values. The CI data were calculated by
using the computer software from CompuSyn for drug combination (ComboSyn, Inc).
4.2.3. Cell cycle distribution detected via flow cytometry assay
For cell cycle analysis, 2×105 cells were plated in a 6-well culture plates and
allowed to grow for 24 h. The HepG2 cells were then incubated with 1 mM thymidine
(Sigma-Aldrich) for 24 h to synchronize cells at the G1/S boundary. The HepG2 cells
were treated with fresh media containing different concentrations of FICD, positive
control sorafenib, and the combination of sorafenib and FICD for 24 h. After
incubation, cells were harvested and washed twice with cold PBS, and fixed with cold
70% ethanol at -20 °C overnight. The cells were then treated with 100 mg/mL RNase
24

A for 30 min at 37 °C after being washed twice with cold PBS, and finally stained
with 1 mg/mL propidium iodide (PI) in the dark at 4 °C for 1 h. Cells were
subsequently analyzed by flow cytometric analysis. Analyses were performed with the
system software (Cell Quest; BD Biosciences).
4.2.4. Apoptosis analysis
Apoptosis was examined via flow cytometry analysis of Annexin V/PI staining.
HepG2 cells were seeded into each well of 96-well plates at a density of 1×105
cells/mL, and DMEM with 10% FBS was added to obtain a final volume of 2 mL.
The plates were then incubated overnight and treated with different concentrations of
FICD, positive control sorafenib, and the combination of sorafenib and FICD for 24 h.
Briefly, cells were harvested and washed three times with ice-cold PBS, and then
suspended in Annexin-binding buffer at a concentration of 5×105 cells/mL. Cells were
incubated with 5 µL annexin V-FITC and 5 µL PI for 30 min at 25 °C in the dark. The
cells were analyzed by system software (Cell Quest; BD Biosciences).
4.2.5. Western blot analysis
Western blot analyses were performed as described previously [23]. HepG2 cells
were treated with different concentrations of FICD (12), COM33 (24), and the
combination for 48 h. Following this, cells were harvested, centrifuged, and washed
twice with ice-cold PBS. The pellet was then resuspended in lysis buffer. After the
cells were lysed on ice for 20 min, the lysates were centrifuged at 12000 rpm at 4 °C
for 5 min. The protein concentration in the supernatant was analyzed using BCA
protein assay reagents (Solarbio, China). Equal amounts of protein per line were
separated by 12% SDS polyacrylamide gel electrophoresis and transferred to PVDF
Hybond-P membranes (GE Healthcare). The membranes were incubated with 5%
skim milk in Tris-buffered saline with Tween 20 (TBST) buffer for 1 h. Next, the
membranes were gently rotated overnight at 4 °C. The membranes were then
incubated with primary antibodies against β-Catenin, C-myc, CyclinD1, Ki-67, and
Vimentin (Abcam, UK) overnight at 4 °C, followed by incubation with
peroxidase-labeled secondary antibodies for 2 h. All membranes were washed with
TBST three times for 15 min each, and the protein blots were analyzed with
chemiluminescence reagent (Thermo Fischer Scientifics Ltd.). The X-ray films were
developed with a developer and fixed by using a fixer solution.
25

4.2.6. Anticancer Activity in Vivo
Model mice ascites (0.2 mL), including 1 × 107 H22 cells/mL, were injected into
the right axilla of mice. From the second day after the implantation of cancer cells,
tumor-bearing mice were grouped randomly as follows: the blank control group was
treated with physiological saline (0.9%), and the positive control group was treated
with sorafenib by intraperitoneal injection (ip) at a dose of 50 mg/Kg. Medicated
groups were treated by ip or intragastric administration (ig) with targeted compounds
at 50 mg/kg, 100 mg/kg, and 150 mg/kg (except for COM33). Subsequently, mice
were treated daily for the following 10 days. Twenty-four hours after the last
administration, mice were killed, and their tumors were totally excised and accurately
weighed. The anticancer activity of isocorydine derivatives in vivo is expressed as
inhibitory rate (IR), which was calculated using the following formula:
IR (%) = (1 - mean tumor weight of dose group/mean tumor weight of control
group) × 100%.
4.2.7. Statistical analysis
All statistical analyses were performed using SPSS Version 9.0. Data were
analyzed using one-way ANOVA. Student's t-tests were used to analyze the
significance of the differences between treated and control groups. All data are
presented as mean values with their standard deviation (S.D), where p ≤ 0.01
indicated statistical significance.
Acknowledgments
This study was supported by the National Natural Science Foundation of China
(NSFC No. 21672225).
Appendix A. Supplementary data
Supplementary data related to this article can be found XXXXX.
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29