Synthesis of novel SN38-aspirin prodrugs for the treatment of hepatocellular carcinoma

Article abstract of DOI:10.3906/kim-1801-21

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and is a leading cause of cancer-related death worldwide. However, there is no effective chemotherapeutic treatment for HCC and its prognosis remains poor. Consequently, it is urgent to find an efficient antitumor agent to treat HCC. In this study, 7-ethyl-10-hydroxycamptothecin (SN38), the bioactive metabolite of the anticancer drug irinotecan (CPT-11), which is 100–1000 times more potent than CPT-11, was coupled with aspirin to give 4 prodrugs. Their structures were characterized by ¹ H NMR and elemental analysis. The in vitro anticancer activities of these compounds on two human hepatocellular carcinoma cell lines (BEL-7404 and HepG2) and preliminary mechanisms of action were explored. Our data indicated that these compounds decreased the viability of cancer cell lines in a concentration- and time-dependent manner. Among them, compound 4b significantly inhibited cell viability of HepG2 cells (IC50 = 0.1208 μM) when compared with CPT-11 (IC50 = 18.4267 μM). Furthermore, compound 4b blocked HepG2 cell migration and invasion in vitro. These findings suggest that compound 4b may be used as a promising anticancer agent against HCC.

Full text of DOI:10.3906/kim-1801-21

Turk J Chem
Turkish Journal of Chemistry
(2018) 42: 929 – 939
¨
http://journals.tubitak.gov.tr/chem/
˙
c
⃝ TUBITAK
doi:10.3906/kim-1801-21
Research Article
Synthesis of novel SN38-aspirin prodrugs for the treatment of hepatocellular
carcinoma
Zhimin CHEN1, Yi LUO2,∗, Aiping FANG2, Chen FAN2, Chenjuan ZENG2
1Department of Hepatobiliary & Gastrointestinal Surgery, Chongzhou People’s Hospital, Chongzhou,
Sichuan, P.R. China
2Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy,
Chengdu, Sichuan, P.R. China
Received: 21.01.2018
•
Accepted/Published Online: 24.04.2018
•
Final Version: 01.06.2018
Abstract: Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and is a leading cause of
cancer-related death worldwide. However, there is no effective chemotherapeutic treatment for HCC and its prognosis
remains poor. Consequently, it is urgent to find an efficient antitumor agent to treat HCC. In this study, 7-ethyl-10-
hydroxycamptothecin (SN38), the bioactive metabolite of the anticancer drug irinotecan (CPT-11), which is 100–1000
times more potent than CPT-11, was coupled with aspirin to give 4 prodrugs. Their structures were characterized by
1 H NMR and elemental analysis. The in vitro anticancer activities of these compounds on two human hepatocellular
carcinoma cell lines (BEL-7404 and HepG2) and preliminary mechanisms of action were explored. Our data indicated
that these compounds decreased the viability of cancer cell lines in a concentration- and time-dependent manner. Among
them, compound 4b significantly inhibited cell viability of HepG2 cells (IC50 = 0.1208 µM) when compared with CPT-
11 (IC50 = 18.4267 µM). Furthermore, compound 4b blocked HepG2 cell migration and invasion in vitro. These findings
suggest that compound 4b may be used as a promising anticancer agent against HCC.
Key words: 7-Ethyl-10-hydroxycamptothecin, aspirin, prodrug, hepatocellular carcinoma
1. Introduction
Hepatocellular carcinoma (HCC) is the sixth most common cancer and the second leading cause of cancer-
related deaths worldwide.1 In contrast to most solid cancers, the incidence of HCC and HCC-related deaths
have increased over the last several decades in many parts of the world, including the United States. In the
United States, more than 39,230 new cases of HCC and more than 27,170 HCC-related deaths were predicted
in 2016.2 The prognosis of HCC is extremely poor, with a median survival of 9 months and a 5-year survival
rate of 9.1%.3 Therefore, it is urgent to discover an efficient drug to overcome HCC.
CPT-11, inhibiting topoisomerase I, is one of the key anticancer drugs in chemotherapy for several cancers
such as colorectal cancer, lung cancer, gastric cancer, and gynecologic cancers.4−7 CPT-11 was also an attractive
drug to evaluate in hepatomas and biliary tract malignancies for several reasons. First, there are in vitro data
indicating the utility of irinotecan in a variety of gastrointestinal cell lines, including human HCC.8−10 Second,
CPT-11 undergoes activation in the liver to its active minor metabolite, 7-ethyl-10-hydroxycamptothecin (SN38),
which leads to high local concentrations of SN38 in the biliary system.11 SN38 also undergoes enterohepatic
∗Correspondence: orthop luoyi@163.com
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recycling, which may further contribute to sustained local SN38 levels in the hepatobiliary tree. Therefore, a
phase II clinical trial was conducted to determine the response rate, toxicity, and overall patient survival for
irinotecan in advanced hepatocellular carcinoma.12 Based on these characteristics of irinotecan, it was evaluated
in phase II studies in patients with advanced HCC. Unfortunately, the antitumor activity of single-agent CPT-11
was not significant in advanced HCC.13
SN38 has high antiproliferation potency for many kinds of tumor cells such as ovarian cancer, mammary
cancer, colon cancer, and lung cancer. According to in vitro results, SN38 is 100–1000 times as potent as CPT-
11.14−17 However, because of the very narrow therapeutic window of SN38, which results in clinical side effects,
and its poor water solubility in any physiologically compatible and pharmaceutically acceptable solvents, SN38
could not be directly applied to clinical therapy of cancer.18 Thus, discovery of novel derivatives based on SN38
for HCC treatment has significant clinical application value.
Aspirin (acetylsalicylic acid) is the best-known salicylate and belongs to the pharmacologic category of
nonsteroidal antiin?ammatory drugs (NSAIDs). It has been widely used for the treatment of inflammation,
fever, and pain for over a century.19 Recently, aspirin has received another boost due to its potential benefits
in the prevention/treatment of cancers and cancer prevention.20,21 Furthermore, aspirin may reduce the risk
of HCC,22 inhibit HepG2 cell growth and induce HepG2 apoptosis,23−25 increase the sensitivity of anticancer
drugs, and overcome anticancer drug resistance in hepatocellular carcinoma cells.26,27
Prompted by the above-mentioned encouraging results, we designed and synthesized novel SN38-aspirin
prodrugs to simultaneously release SN38 and aspirin. The structures of all compounds were confirmed by 1 H
NMR and elemental analysis. Then the in vitro anticancer activity against HCC cancer cell lines and preliminary
mechanisms of action were explored.
2. Results and discussion
2.1. Chemistry
Commercially available acetylsalicylic acid was first reacted with oxalyl chloride in anhydrous dichloromethane
at room temperature to give compound 1. Then compound 1 was reacted with ethylene glycol, diethylene glycol,
triethylene glycol, and tetraethylene glycol, respectively, in the presence of triethylamine to produce compounds
2a–2d. Compounds 3a–3d were prepared by reacting compounds 2a–2d with 4-nitrophenyl chloroformate
using triethylamine as a base. We tried to purify compounds 3a–3d with column chromatography, but no pure
products were obtained. Therefore, crude compounds 3a–3d were used directly for the next preparations, which
were carried out in the same flask. SN38 and N, N -diisopropylethylamine were added to the above-mentioned
flask and the reactions were stirred at room temperature overnight. Compounds 4a–4d were obtained by column
chromatography (dichloromethane : methanol = 100:1 to 50:1). The HPLC purities of compounds 4a–4d are
greater than 97%.
2.2. In vitro cytotoxicity screening and antiproliferative properties
The anticancer activities of compounds 4a–4d were evaluated in vitro on two human hepatocellular carcinoma
cell lines (BEL-7404 and HepG2) by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay according to previous studies,28,29 with SN38, CPT-11, and aspirin as positive controls. Data are expressed
as IC50 (50% inhibitory concentration) and are shown in the Table. As can be seen in the Table, all synthesized
compounds exhibited marked anticancer activity against BEL-7404 and HepG2 cell lines, and especially HepG2
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cells. Among these compounds, 4b has an IC50 of 0.1208 µM, while SN38 and CPT-11 have IC50 values of
0.1450 µM and 18.4267 µM, respectively. Compound 4 showed 152-fold increase in activity when compared
with CPT-11. However, aspirin indicates weak anticancer activity at the experimental concentration. The
above-mentioned results suggest that compound 4b has potent antitumor activity.
Table. Anticancer activities of compounds 4a–4d.a
IC50 (µM)b
HepG2
Compounds
BEL-7404
4a (n = 1)
4b (n = 2)
4c (n = 3)
4d (n = 4)
SN38
0.1676 ꢀ 0.0257
0.1208 ꢀ 0.0081
0.1707 ꢀ 0.0245
0.2028 ꢀ 0.0021
0.1450 ꢀ 0.0468
4.0600 ꢀ 0.2701
2.5295 ꢀ 0.7884
5.0250 ꢀ 1.0757
3.0255 ꢀ 0.8549
0.2553 ꢀ 0.0458
CPT-11
Aspirin
18.4267 ꢀ 1.6279 > 40
> 40
> 40
a Cell lines were treated with gradient concentrations of compounds for 72 h. All assays were carried out in triplicate,
and the results were expressed as mean ꢀ SD.
b 50% growth inhibition as determined by MTT assay.
To further determine whether compound 4b has direct effects on cancer cells’ viability, gradient con-
centrations (0, 0.0391, 0.0781, 0.1563, 0.3125, 0.6250, 1.2500, 2.5000 µM) of compound 4b were used to treat
HepG2 cells for 24, 48, and 72 h, respectively. As shown in Figure 1, significant decreases in cell viability were
observed as the concentration of compound 4b increased from 0.0391 to 2.5000 µM. Meanwhile, treatment with
compound 4b for 24, 48, and 72 h decreased the HepG2 cells’ viability in a time-dependent manner. These data
suggested that compound 4b inhibited HepG2 cells viability in a concentration- and time-dependent manner.
120
24 h 48 h 72 h
100
80
60
40
20
0
Concentrations of compound 4b (µM)
Figure 1. Proliferation of HepG2 cells treated with gradient concentrations (0–2.5000 µM) of compound 4b for 24, 48,
and 72 h, respectively. Cell viability was evaluated by MTT assay. Values represent mean ꢀ SD (n = 3).
2.3. Compound 4b inhibits HepG2 migration and invasion
HCC is a highly aggressive form of carcinoma with poor prognosis, and HCC-associated mortality primarily
occurs due to migration and invasion of HCC cells.30 Tumor metastasis begins with the breakdown of epithelial
integrity, followed by malignant cells invading into the surrounding stroma and lymphovascular space, by which
cancer cells travel to distant target organs.31 Hence, inhibition of tumor cell migration and invasion is a critical
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approach in anticancer treatment.32 Therefore, we performed assays to detect the antimetastatic ability of
compound 4b in vitro. As shown in Figure 2, the number of migrated HepG2 cells decreased with increasing
concentration of the drug in a dose-dependent manner. At a dose of 0.125 µM, only about 33% of cells
passed through the 8-µm bottom membrane of the chamber compared with the control group. The Matrigel
matrix mimics the environment of the basilar membrane in vivo and it is a structure that must pass through
during distant metastasis of cancer cells. Only about 45% cells were inhibited from being invaded when the
concentration was 0.5 µM. The results showed that compound 4b had the ability to inhibit metastasis of HepG2
cells, but the ability of inhibiting invasion of HepG2 cells was not significant.
Figure 2. Compound 4b inhibits HepG2 cells migration (A and C) and invasion (B and D). Migrated and invaded cells
were fixed, stained, photographed, and quantified (20×). *P < 0.05, ***P < 0.001 compared with control.
2.4. Conclusions
In this work, four novel SN38-aspirin prodrugs were synthesized and the in vitro antitumor activities against
BEL-7404 and HepG2 cancer cell lines were assessed by MTT assay. Among them, compound 4b exhibited
the most potent activity in a concentration- and time-dependent manner with an IC50 value of 0.1208 µM
against HepG2 cells, which displayed a 152-fold increase in activity when compared with CPT-11. The results
of preliminary mechanisms of action showed that compound 4b can block HepG2 cells’ migration and invasion.
These results imply that compound 4b might be a potential therapeutic agent for blocking HCC growth and
metastasis.
3. Experimental
3.1. Materials and methods
All chemicals were commercially available and used without further purification unless otherwise stated. All
solvents were dried according to the standard methods prior to use. Column chromatography was carried out on
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silica gel (200–300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China). Thin-layer chromatography (TLC)
was performed on TLC silica gel 60 F254 plates. The purity of the compound screened in biological assays was
determined to be ≥97% by HPLC analysis with a photodiode array detector (Waters, Milford, MA, USA). An
Atlantis C18 (150 mm × 4.6 mm, i.d. 5 µm) (Waters, Milford, MA, USA) was used with a gradient elution
of methanol (MeOH) and HPLC-grade water as mobile phase at a flow rate of 1 mL/min. 1 H NMR spectra
were measured on a Bruker AV-400 (400 MHz) NMR spectrometer and chemical shifts (δ) are reported in parts
per million relative to tetramethylsilane (TMS) used as an internal standard, where (δ) TMS = 0.00 ppm.
Elemental analyses were carried out on a Carlo Erba-1106 analyzer.
3.2. Chemistry
The synthetic route for SN38-aspirin prodrugs is shown in the Scheme.
Scheme. Synthesis route for SN38-aspirin prodrugs. a: n = 1; b: n = 2; c: n = 3; d: n = 4.
3.2.1. Synthesis of compound 1
To a solution of acetylsalicylic acid (5 g, 27.8 mmol) in 50 mL of anhydrous dichloromethane (DCM), 3.3 mL
of oxalyl chloride and a drop of dimethylformamide were added slowly and the mixture was stirred for 3 h at
room temperature. Then the mixture was concentrated and 5.4 g of compound 1 was obtained (yield: 98.5%).
3.2.2. General procedure for the synthesis of compounds 2a–2d
Ethylene glycol (5.39 g, 86.9 mmol) and 10 mL of triethylamine were dissolved in 100 mL of DCM, and newly
prepared compound 1 (5.4 g) in the first step was added slowly. The mixture was stirred overnight at room
temperature and washed with water (2 × 50 mL), and the organic phase was dried over anhydrous sodium
sulfate and concentrated. The residue was purified by column chromatography (DCM : MeOH = 100:1 to 70:1)
to give 1.7 g of compound 2a with a yield of 27.9%. Compounds 2b–2d were prepared using the same method
described above. Diethylene glycol, diethylene glycol, and tetraethylene glycol were used instead of ethylene
glycol.
3.2.3. General procedure for the synthesis of compounds 4a–4d
Compound 2a (224 mg, 1 mmol) and 202 mg of triethylamine (2 mmol) were dissolved in 5 mL of DCM.
After the mixture was cooled to 0 ◦ C, 400 mg of 4-nitrophenyl chloroformate (2 mmol) was added slowly to the
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mixture. The reaction was complete after 24 h as monitored by TLC (petroleum ether : ethyl acetate = 1:1).
SN38 (392 mg, 1 mmol) and 258 mg of N, N -diisopropylethylamine (2 mmol) were added to the above reaction.
The mixture was stirred overnight at room temperature. Water (10 mL) was added to the reaction, and then
the solution was partitioned by DCM and water. The inorganic layer was then extracted by DCM twice (2 ×
25 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was purified
by column chromatography (DCM : MeOH = 100:1 to 70:1) to give 129.7 mg of compound 4a with a yield of
20.2%. Compounds 4b–4d were synthesized according to the method described above. The 1 H NMR spectra
of compounds 4b–4d are shown in Figures 3–6.
Figure 3. 1 H NMR spectrum of compound 4a.
Compound 2a: n = 1, light yellow liquid, yield: 27.9%; 1 H NMR (400 Hz, DMSO-d6) δ 7.99 (dd,
J = 8.0 Hz, 1.6 Hz, 1H), 7.66–7.70 (m, 1H), 7.40–7.44 (m, 1H), 7.24 (dd, J = 8.2 Hz, 1.0 Hz, 1H), 4.92 (t,
J = 5.6 Hz, 1H), 4.22–4.24 (m, 2H), 3.65–3.69 (m, 2H), 2.29 (s, 3H).
Compound 2b: n = 2, light yellow liquid, yield: 27.4%; 1 H NMR (400 Hz, DMSO-d6) δ 7.95 (dd,
J = 8.0 Hz, 1.6 Hz, 1H), 7.67–7.71 (m, 1H), 7.40–7.44 (m, 1H), 7.24 (dd, J = 8.0 Hz, 0.8 Hz, 1H), 4.61 (t,
J = 5.4 Hz, 1H), 4.33–4.35 (m, 2H), 3.69–3.72 (m, 2H), 3.46–3.52 (m, 4H), 2.29 (s, 3H).
Compound 2c: n = 3, light yellow liquid, yield: 29.4%; 1 H NMR (400 Hz, DMSO-d6) δ 7.95 (dd,
J = 7.8 Hz, 1.8 Hz, 1H), 7.67–7.71 (m, 1H), 7.40–7.44 (m, 1H), 7.24 (dd, J = 8.0 Hz, 0.8 Hz, 1H), 4.58 (t,
J = 5.4 Hz, 1H), 4.33–4.35 (m, 2H), 3.69–3.72 (m, 2H), 3.52–3.59 (m, 4H), 3.46–3.50 (m, 2H), 3.40–3.43 (m,
2H), 2.29 (s, 3H).
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Figure 4. 1 H NMR spectrum of compound 4b.
Compound 2d: n = 4, light yellow liquid, yield: 18.5%; 1 H NMR (400 Hz, DMSO-d6) δ 7.95 (dd,
J = 8.0 Hz, 1.2 Hz, 1H), 7.67–7.71 (m, 1H), 7.43 (t, J = 7.6 Hz, 1H), 7.24 (d,J = 8.0 Hz, 1H), 4.52–4.65 (m,
1H), 4.35 (t, J = 4.6 Hz, 2H), 3.71 (t, J = 4.4 Hz, 2H), 3.47–3.58 (m, 10H), 3.39–3.42 (m, 2H), 2.30 (s, 3H).
Compound 4a: n = 1, light yellow solid, yield: 20.2%; HPLC purity: 98.2%. 1 H NMR (400 Hz,
DMSO-d6) δ 8.22 (d, J = 9.2 Hz, 1H), 8.15 (d, J = 2.5 Hz, 1H), 7.99 (dd, J = 7.8, 1.6 Hz, 1H), 7.77 (dd,
J = 9.2, 2.5 Hz, 1H), 7.69–7.74 (m, 1H), 7.42–7.46 (m, 1H), 7.33 (s, 1H), 7.28 (dd, J = 8.0, 0.8 Hz, 1H), 6.54
(s, 1H), 5.44 (s, 2H), 5.33 (s, 2H), 4.59 (s, 4H), 3.14–3.18 (m, 4H), 2.33 (s, 3H), 1.84-1.91 (m, 2H), 1.27 (t,
J = 7.6 Hz, 3H), 0.88 (t, J = 7.3 Hz, 3H). Anal. Calcd. for C34 H30 N2 O11 : C, 63.55; H, 4.71; N, 4.36. Found:
C, 63.58; H, 4.67; N, 4.29.
Compound 4b: n = 2, light yellow solid, yield 20.5%; HPLC purity: 98.7%. 1 H NMR (400 Hz, DMSO-
d6) δ 8.22 (d, J = 9.2 Hz, 1H), 8.15 (d, J = 2.3 Hz, 1H), 7.94 (dd, J = 7.8, 1.4 Hz, 1H), 7.77 (dd, J = 9.2,
2.4 Hz, 1H), 7.65–7.69 (m, 1H), 7.40 (t, J = 7.4 Hz, 1H), 7.34 (s, 1H), 7.22 (d, J = 8.1 Hz, 1H), 6.54 (s, 1H),
5.45 (s, 2H), 5.34 (s, 2H), 4.35–4.39 (m, 4H), 3.72–3.75 (m, 4H), 3.63 (s, 4H), 3.18–3.19 (m, 2H), 2.30 (s, 3H),
1.84–1.91 (m, 2H), 1.29 (t, J = 7.5 Hz, 3H), 0.88 (t, J = 7.3 Hz, 3H). Anal. Calcd. for C36 H34 N2 O12 : C,
62.97; H, 4.99; N, 4.08. Found: C, 62.85; H, 4.91; N, 4.17.
Compound 4c: n = 3, light yellow solid, yield: 20.5%; HPLC purity: 97.9%. 1 H NMR (400 Hz,
DMSO-d6) δ 8.22 (d, J = 9.2 Hz, 1H), 8.15 (d, J = 2.3 Hz, 1H), 7.94 (dd, J = 7.8, 1.4 Hz, 1H), 7.77 (dd,
J = 9.2, 2.4 Hz, 1H), 7.65–7.69 (m, 1H), 7.40 (t, J = 7.4 Hz, 1H), 7.34 (s, 1H), 7.22 (d, J = 8.1 Hz, 1H), 6.54
(s, 1H), 5.45 (s, 2H), 5.34 (s, 2H), 4.35–4.39 (m, 4H), 3.72–3.75 (m, 4H), 3.63 (s, 4H), 3.18–3.19 (m, 2H), 2.30 (s,
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Figure 5. 1 H NMR spectrum of compound 4c.
3H), 1.84–1.91 (m, 2H), 1.29 (t, J = 7.5 Hz, 3H), 0.88 (t, J = 7.3 Hz, 3H). Anal. Calcd. for C38 H38 N2 O13
:
C, 62.46; H, 5.24; N, 3.83. Found: C, 62.41; H, 5.29; N, 3.75.
Compound 4d: n = 4, light yellow solid, yield: 15.5%; HPLC purity: 99.1%. 1 H NMR (400 Hz,
DMSO-d6) δ 8.22 (d, J = 9.2 Hz, 1H), 8.15 (d, J = 2.6 Hz, 1H), 7.93 (dd, J = 7.8, 1.6 Hz, 1H), 7.77 (dd,
J = 9.1, 2.5 Hz, 1H), 7.64–7.68 (m, 1H), 7.38–7.42 (m, 1H), 7.33 (s, 1H), 7.21 (dd, J = 8.1, 1.0 Hz, 1H),
6.53 (s, 1H), 5.44 (s, 2H), 5.34 (s, 2H), 4.32–4.39 (m, 4H), 3.70–3.73 (m, 4H), 3.55–3.58 (m, 8H), 3.16–3.20 (m,
2H), 2.28 (s, 3H), 1.84–1.89 (m, 2H), 1.28 (t, J = 7.6 Hz, 3H), 0.88 (t, J = 7.3 Hz, 3H). Anal. Calcd. for
C40 H42 N2 O14 : C, 62.01; H, 5.46; N, 3.83. Found: C, 62.08; H, 5.37; N, 3.80.
3.3. Cell lines and culture
The HepG2 cell line was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA).
The BEL-7404 human hepatoma cell line was obtained from the Cell Bank of the Chinese Academy of Sciences
(Shanghai, China). Cells were propagated in DMEM medium containing 10% heat-inactivated fetal bovine
serum (FBS; HyClone, Logan, UT, USA) and 1% antibiotics (penicillin and streptomycin) in 5% CO2 at 37
◦ C.
3.4. Cell viability assay
The cell viability of compound-treated cells was assessed by MTT assay. Briefly, exponentially growing cells
(2 × 10 3 cells/well) were plated in 96-well plates (100 µL/well). After 24 h of incubation, the cells were
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Figure 6. 1 H NMR spectrum of compound 4d.
treated with different concentrations of compounds. After treatment for 24, 48, and 72 h, respectively, 20 µL
of 5 mg/mL MTT was added to each well and the plates were incubated at 37 ◦ C for an additional 4 h. The
medium was subsequently removed and the purple-colored precipitates of formazan were dissolved in 150 µL of
DMSO. The color absorbance was recorded at 570 nm using a Spectra MAX M5 microplate spectrophotometer
(Molecular Devices, San Jose, CA, USA). All experiments were performed in triplicate.
3.5. Boyden chamber migration and invasion assay
A modified Boyden chamber (8-µm pore size) migration assay was performed according to previous reports33
with some modification. Briefly, a total of 1 × 105 HepG2 cells in 100 µL of serum-free medium were
added in the upper chamber and 600 µL of medium containing 10% FBS was added at the bottom. Different
concentrations of compound 4b were added in both chambers. Cells were allowed to migrate for approximately
24 h. Nonmigrated cells in the upper chamber were discarded using a cotton swab. The migrated cells were
fixed in methanol and stained with 0.5% crystal violet. Migrated cells in 6 randomly selected fields were counted
and photographed under a light microscope. Invasion assay was conducted as previously described.34 Briefly,
the upper surface of the transwell membrane was coated with serum-free diluted Matrigel (1:1, 60 µL/well,
BD Biosciences). After Matrigel polymerization, the bottom chambers were filled with 600 µL of medium
containing 10% FBS. HepG2 cells (1 × 105) in 100 µL of serum-free medium were added in the upper part
of each transwell and treated with 0.1% DMSO or different concentrations of compound 4b. After incubation
for 24 h, nonmigrated cells on the upper side of the filter were removed and migrated cells were fixed with
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4% paraformaldehyde and stained with 0.5% crystal violet; then migrated cells were counted under a light
microscope. The percentage inhibition of migrated cells by compound 4b was expressed using 100% as the
value assigned for the control group.
3.6. Statistical analysis
Results were represented as mean ꢀ SD of three independent experiments. Statistical comparisons were made
by 2-tailed Student t-tests. Statistically significant P-values were labeled as follows: *P < 0.05, **P < 0.01,
***P < 0.001.
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