Design, synthesis and anticancer activity of naphthoquinone derivatives

Article abstract of DOI:10.1080/14756366.2020.1740693

Basis on molecular docking and pharmacophore analysis of naphthoquinone moiety, a total of 23 compounds were designed and synthesised. With the help of reverse targets searching, anti-cancer activity was preliminarily evaluated, most of them are effective against some tumour cells, especially compound 12: 1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl-4-oxo-4-((4-phenoxyphenyl)amino) butanoate whose IC₅₀ against SGC-7901 was 4.1 ± 2.6 μM. Meanwhile the anticancer mechanism of compound 12 had been investigated by AnnexinV/PI staining, immunofluorescence, Western blot assay and molecular docking. The results indicated that this compound might induce cell apoptosis and cell autophagy through regulating the PI3K signal pathway.

Full text of DOI:10.1080/14756366.2020.1740693

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Design, synthesis and anticancer activity of
naphthoquinone derivatives
Xiao-bao Shen, Yang Wang, Xuan-zhen Han, Liang-quan Sheng, Fu-fang Wu &
Xinhua Liu
To cite this article: Xiao-bao Shen, Yang Wang, Xuan-zhen Han, Liang-quan Sheng, Fu-
fang Wu & Xinhua Liu (2020) Design, synthesis and anticancer activity of naphthoquinone
derivatives, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1, 773-785, DOI:
10.1080/14756366.2020.1740693
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 773–785
https://doi.org/10.1080/14756366.2020.1740693
SHORT COMMUNICATION
Design, synthesis and anticancer activity of naphthoquinone derivatives
Xiao-bao Shena,b, Yang Wanga, Xuan-zhen Hanb, Liang-quan Shengb, Fu-fang Wua,b and Xinhua Liua
b
aSchool of Pharmacy, Anhui Medical University, Hefei, PR China; Engineering Research Center of Biomass Conversion and Pollution Prevention
of Anhui Educational Institutions, Fuyang Normal University, Fuyang, PR China
ABSTRACT
ARTICLE HISTORY
Received 5 December 2019
Revised 9 February 2020
Accepted 2 March 2020
Basis on molecular docking and pharmacophore analysis of naphthoquinone moiety, a total of 23 com-
pounds were designed and synthesised. With the help of reverse targets searching, anti-cancer activity
was preliminarily evaluated, most of them are effective against some tumour cells, especially compound
12: 1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl-4-oxo-4-((4-phenoxyphe-
nyl)amino) butanoate whose IC50 against SGC-7901 was 4.1 2.6 lM. Meanwhile the anticancer mechanism
of compound 12 had been investigated by AnnexinV/PI staining, immunofluorescence, Western blot assay
and molecular docking. The results indicated that this compound might induce cell apoptosis and cell
autophagy through regulating the PI3K signal pathway.
KEYWORDS
Naphthoquinone moiety;
anticancer activ-
ity; autophagy
1. Introduction
study structure–activity relationship. Many reports showed that
the introduction of suitable substituents on the hydroxyl group
could improve activity28–30 and reduce toxicity31. Furthermore,
Ahn32 and Sankawa33 showed that the side chain hydroxyl was
not essential for activity. Both acylation and etherification could
maintain antitumor activity at low concentration and the introduc-
tion of double bond should increase the activity obviously.
So, based on the above, combined with the results of reverse
target finding, a series of new naphthoquinone-esterification/
etherification derivatives targeting PI3K were designed and syn-
thesised (Figure 1). Their inhibitory activities against SGC-7901,
MGC-803, SMMC-7721, and U-87 cells were determined by MTT
assay. The molecular mechanism of title compound was studied
by influencing the signal pathway of PI3K/AKT/mTOR. Meanwhile
molecular docking was used to determine the interaction between
the compound and PI3K protein.
Gastric cancer (GC) is a severe malignant tumour associated with
high mortality, especially in Asia1,2. Due to the non-specific symp-
toms in early appearance and the poor prognosis, the 5-year rela-
tive survival rate for GC was at most 20%3. Because of GC difficult
diagnosis and chemotherapy resistance, it is necessary to investi-
gate the new therapeutic drugs and deeply explore its anti-
tumour mechanism4.
Autophagy is a pathway participated in the degradation of
lysosomal, which contributed to renew the needed energy of cell
survival during starvation5,6 and plays an important role in GC.
Autophagy is expected to be a new target for molecular therapy7
which can inhibit the generation of tumour, on the other hand,
can promote the survival and transfer of tumour cells8. A large
number of studies have shown that autophagy is closely related
to DC. For example, the gene polymorphism of autophagyis
related to the susceptibility of GC9,10. Since lots of compounds
regulating autophagy are discovered, but, the tumour microenvir-
onments are complexity, the role of it in the tumour cells is not
very clear. Therefore, it is necessary to reveal the title compounds’
anticancer mechanism through rational regulating of autophagy.
Compounds with naphthoquinone moiety show good activity
against breast cancer, liver cancer, human cervical carcinoma and
GC11–14 through inducing cell apoptosis15–21. Inducing autophagy
2. Experimental section
2.1. Chemistry
Adriamycin was purchased from Aladdin Chemical Reagent Co.,
Ltd (Shanghai, China) and other reagents were purchased from
Abcam, Cell Signalling Technology (Boston, MA) and Beyotime
(Shanghai, China). 1H NMR spectra were recorded on 400 MHz or
600 MHz and 13 C NMR spectra were obtained at 100 MHz or
150 MHz (Supplementary material).
is also one of the main mechanisms for anti-cancer activity13,22,23
Among them, PI3K and mTOR signalling pathways have been
proved to be the main signal pathways to regulate autoph-
agy24–27. However, for these kinds of derivatives, the poor selectiv-
.
ity and high cytotoxicity limit their clinical application. Therefore, 2.1.1. General procedure for preparation of compounds 1 and 2
how to reduce the toxicity and increase the selectivity is the main To a solution of shikonin in DMF was added iodomethane (CH3I,
trend. Because the hydroxyl group of naphthoquinone is easily 2.0 equiv) orbenzyl bromide and K2CO3 (2.0 equiv) and the mix-
oxidised, and the polyhydroxy is not conducive to selectivity, so ture was stirred at 60 ꢀC for 6 h. The mixture was filtered and the
modification of the hydroxyl group is one of the main methods to residue was washed with EA, the filtrate was extracted by EA and
CONTACT Wu Fufang
Supplemental data for this article can be accessed here.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
wff05001@fync.edu.cn; Liu Xinhua
xhliuhx@163.com
School of Pharmacy, Anhui Medical University, Hefei, 230032, PR China
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

774
X.-B. SHEN ET AL.
Figure 1. The general design strategy in this study.
combined organic layers were washed with water and brine. 4.94–4.91 (m, 1H), 2.68–2.62 (m, 1H), 2.39–2.33 (m, 1H), 1.76 (s,
3H), 1.66 (s, 3H). 13 C NMR (150 MHz, CDCl3) d 180.74, 179.95,
Evaporate the solvent and the products were purified by column
chromatography to give product as a solid.
1: 2-(1-hydroxy-4-methylpent-3-en-1-yl)-5,8-dimethoxynaphtha-
167.72, 165.38, 164.77, 153.99, 151.40, 137.52, 132.42, 132.27,
131.83, 130.91, 128.81, 128.26, 118.39, 112.02, 111.54, 103.67,
65.57, 35.67, 25.97, 18.09. HR-ESI-MS: m/z [M þ H]þ calcd
for 419.1482.
lene-1,4-dione, Brown solid. Yield: 92.3%. m.p. 94.3 ꢀC. 1H NMR
(600 MHz, CDCl3) d ¼ 7.30 (s, 2H), 6.79 (s, 1H), 5.18–5.13 (m, 1H),
4.77–4.72 (m, 1H), 3.95 (s, 6H), 2.62–2.54 (m, 1H), 2.50 (s, 1H),
2.39–2.33 (m, J ¼ 14.7, 7.5, 1H), 1.71 (s, 3H), 1.61 (s, 32H).
5: 1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-meth-
ylpent-3-en-1-yl (E)-4-(3,4,5-trimethoxyphenyl)but-3-enoate, red solid.
Yield: 42.5%. m.p. 54.0 ꢀC. 1H NMR (600 MHz, CDCl3) d ¼ 12.60 (s,
1H), 12.41 (s, 1H), 7.62 (d, J ¼ 15.9, 1H), 7.17 (s, 2H), 7.04 (s, 1H),
6.76 (s, 2H), 6.39 (d, J ¼ 15.9, 1H), 6.14–6.11 (m, 1H), 5.16 (t, J ¼ 6.8,
1H), 3.89 (s, 6H), 3.87 (s, 3H), 1.67 (s, 3H), 1.57 (s, 3H). 13 C NMR
(150 MHz, CDCl3) d 178.16, 176.67, 167.51, 166.98, 165.60, 153.42,
148.24, 145.89, 140.33, 136.08, 132.88, 132.74, 131.50, 129.56,
117.69, 116.45, 111.82, 111.56, 105.33, 69.60, 60.96, 56.16, 32.90,
25.77, 18.00, 11.20. HR-ESI-MS: m/z [M þ H]þ calcd for 523.1964.
6: 1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-meth-
ylpent-3-en-1-yl-2-(4-chlorophenoxy)acetate, deep red solid. Yield:
85.2%. m.p. 102.0 ꢀC. 1H NMR (600 MHz, CDCl3) d ¼ 12.57 (s, 1H),
12.39 (s, 1H), 7.26–7.22 (m, J ¼ 10.1, 2H), 7.19–7.16 (m, 2H), 6.93 (s,
1H), 6.84 (d, J ¼ 8.9, 2H), 6.15 (dd, J ¼ 7.2, 4.7, 1H), 5.03 (t, J ¼ 7.2,
1H), 4.68 (s, 2H), 2.66–2.60 (m, 1H), 2.51–2.47 (m, 1H), 1.67 (s, 3H),
1.56 (s, 3H). 13 C NMR (150 MHz, CDCl3) d 176.20, 174.62, 169.24,
168.73, 167.64, 156.20, 146.63, 136.59, 133.56, 133.35, 131.17,
129.54, 126.93, 117.14, 115.86, 111.75, 111.53, 70.53, 65.40, 32.76,
25.76, 17.95. HR-ESI-MS: m/z [M þ H]þ calcd for 457.1032.
2:
5,8-bis(benzyloxy)-2-(1-hydroxy-4-methylpent-3-en-1-yl)naph-
thalene-1,4-dione, Deep red solid. Yield: 37.6%. m.p. 60.0 ꢀC.1H
NMR (600 MHz, CDCl3) d ¼ 12.57 (s, 1H), 12.42 (s, 1H), 7.31–7.17
(m, 7H), 6.86 (s, 1H), 6.02 (dd, J ¼ 6.7, 4.9, 1H), 5.06 (t, J ¼ 7.2, 1H),
2.97 (t, J ¼ 7.7, 2H), 2.79–2.67 (m, 2H), 2.62–2.54 (m, 1H), 2.48–2.42
(m, 1H), 1.67 (s, 3H), 1.56 (s, 3H). 13 C NMR (150 MHz, CDCl3) d
178.18, 176.68, 171.69, 167.39, 166.87, 148.11, 140.04, 136.08,
132.85, 132.68, 131.46, 128.56, 128.21, 126.43, 117.62, 111.78,
111.53, 69.48, 35.75, 32.83, 30.80, 25.76, 17.95. HR-ESI-MS: m/z
[M þ H]þ calcd for 469.2064
2.1.2. General procedure for preparation of compounds 3–24
To a solution of amine (1 equiv) in toluene (dichloromethane or
furanidine according the amine’s solubility) was added succinic
anhydride (1.1 equiv) or maleic anhydride (1.1 equiv). Then the
solution was refluxed for 0.5 h. After TLC shows the reaction was
completed, the solvent was cooled to room temperature. After fil-
tration, cooled toluene was used washing the precipitate to give
the crude product, if the product was dissolved in the solvent, the
solvent was then removed under reduced pressure to crude prod-
uct for next step directly.
7: 1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-meth-
ylpent-3-en-1-yl-4-oxo-4-(piperidin-1-yl)butanoate, red solid. Yield:
65.2%. m.p. 120.0 ꢀC.1H NMR (600 MHz, CDCl3) d ¼ 12.56 (s, 1H),
12.43 (s, 1H), 7.19–7.14 (m, 2H), 7.06 (s, 1H), 6.03–5.98 (m, 1H),
5.16-5.08 (m, 1H), 3.57–3.47 (m, 2H), 3.42–3.33 (m, 2H), 2.73 (t,
J ¼ 6.4, 2H), 2.66–2.57 (m, J ¼ 18.5, 11.9, 3H), 2.52–2.44 (m, 1H),
1.67 (s, 3H), 1.64–1.60 (m, J ¼ 4.3, 2H), 1.57–1.48 (m, 7H). 13 C NMR
(150 MHz, CDCl3) d 194.47, 178.95, 177.49, 172.20, 168.93, 166.70,
166.18, 148.19, 135.96, 132.58, 132.35, 131.97, 117.71, 111.57,
69.62, 65.57, 46.29, 42.87, 32.77, 29.41, 27.92, 26.32, 25.77, 24.48,
17.96. HR-ESI-MS: m/z [M þ H]þ calcd for 456.2042.
At 0 ꢀC, dicyclohexyl carbodiimide and 4-dimethylaminopyri-
dine (DMAP) were added and stirred in the solution of above-
mentioned carboxylic acid and dichloromethane (DCM) for about
15 min. Then solution of shikonin in DCM was dropped, the com-
bined solution was stirred in ice bath for 6 h, slowly to room tem-
perature. After TLC showed the reaction was completed, the
solution was concentrated and cooled to ꢁ10 ꢀC and filtered to
removal most of the dicyclohexylurea (DCU), the filtrate was
evaporated, the product was purified via prepared TLC to give
deep red solid.
8: 1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-meth-
ylpent-3-en-1-yl 4-morpholino-4-oxobutanoate, red solid. Yield:
26.4%. m.p. 105.0 ꢀC.1H NMR (600 MHz, CDCl3) d ¼ 12.56 (s, 1H),
12.43 (s, 1H), 7.19–7.14 (m, 2H), 7.05 (s, 1H), 6.01 (dd, J ¼ 6.3, 5.3,
1H), 5.12 (t, J ¼ 7.2, 1H), 3.68–3.63 (m, 4H), 3.61–3.58 (m, 2H),
3.48–3.45 (m, 2H), 2.75 (t, J ¼ 6.6, 2H), 2.67–2.58 (m, 3H), 2.53–2.43
(m, 1H), 1.68 (s, 3H), 1.56 (s, 3H). 13 C NMR (150 MHz, CDCl3) d
178.62, 177.13, 172.00, 169.50, 167.02, 166.50, 148.01, 136.03,
132.71, 132.49, 131.85, 117.65, 111.80, 111.56, 69.77, 66.83, 66.46,
45.61, 42.06, 32.76, 29.17, 27.71, 25.78, 17.96. HR-ESI-MS: m/z
[M þ H]þ calcd for 458.1831.
3: 1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-meth-
ylpent-3-en-1-yl-3-phenylpropanoate, deep red solid. Yield: 37.6%.
m.p. 60.0 ꢀC. 1H NMR (600 MHz, CDCl3) d ¼ 12.57 (s, 1H), 12.42 (s,
1H), 7.31–7.17 (m, 7H), 6.86 (s, 1H), 6.02 (dd, J ¼ 6.7, 4.9, 1H), 5.06
(t, J ¼ 7.2, 1H), 2.97 (t, J ¼ 7.7, 2H), 2.79–2.67 (m, 2H), 2.62–2.54 (m,
1H), 2.48–2.42 (m, 1H), 1.67 (s, 3H), 1.56 (s, 3H). 13 C NMR
(150 MHz, CDCl3) d 178.18, 176.68, 171.69, 167.39, 166.87, 148.11,
140.04, 136.08, 132.85, 132.68, 131.46, 128.56, 128.21, 126.43,
117.62, 111.78, 111.53, 69.48, 35.75, 32.83, 30.80, 25.76, 17.95. HR-
ESI-MS: m/z [M þ H]þ calcd for 421.1632.
9: 1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-meth-
ylpent-3-en-1-yl-4-(3,4-dihydroquinolin-1(2H)-yl)-4-oxobutanoate, red
solid. Yield: 44.1%. m.p. 92.0 ꢀC. 1H NMR (600 MHz, CDCl3)
d ¼ 12.57 (s, 1H), 12.44 (s, 1H), 7.19–7.07 (m, 6H), 7.05 (s, 1H),
6.02–5.98 (m, 1H), 5.11 (t, J ¼ 7.0, 1H), 3.83–3.73 (m, 2H), 2.82–2.78
4: 1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-meth-
ylpent-3-en-1-yl cinnamate, red solid. Yield: 64.6%. m.p. 85.0 ꢀC. 1H
NMR (600 MHz, CDCl3) d ¼ 12.61 (s, 1H), 12.50 (s, 1H), 7.75 (d,
J ¼ 16.3, 1H), 7.73–7.70 (m, 1H), 7.54–7.50 (m, 2H), 7.44–7.39 (m,
2H), 7.21–7.16 (m, 3H), 6.45 (d, J ¼ 16.2, 1H), 5.23–5.19 (m, 1H), (m, 2H), 2.77–2.75 (m, 2H), 2.72–2.69 (m, 2H), 2.64–2.58 (m, 1H),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
775
2.52–2.45 (m, 1H), 1.98–1.88 (m, 2H), 1.67 (s, 3H), 1.55 (s, 3H). 13 C 70.12, 32.78, 32.00, 30.92, 29.41, 25.73, 17.94. HR-ESI-MS: m/z
[M þ H]þ calcd for 542.0732.
NMR (150 MHz, CDCl3) d 178.72, 177.25, 171.93, 166.89, 166.38,
156.74, 148.11, 136.00, 132.65, 132.43, 131.88, 128.54, 126.10,
124.68, 117.66, 111.80, 111.56, 69.69, 49.12, 33.92, 32.77, 29.62,
26.77, 25.76, 25.59, 24.93, 23.96, 17.95. HR-ESI-MS: m/z [M þ H]þ
calcd for 504.2024.
15:
1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl-4-((2-methoxyphenyl)amino)-4-oxobutanoate,
red solid. Yield: 87.3%. m.p. 113.0 ꢀC. 1H NMR (600 MHz, CDCl3)
d ¼ 12.57 (s, 1H), 12.42 (s, 1H), 8.32 (d, J ¼ 7.8, 1H), 7.85 (br, 1H),
7.18–7.14 (m, 2H), 7.05–7.04 (m, 1H), 7.02–6.98 (m, 1H), 6.95–6.91
(m, 1H), 6.83 (d, J ¼ 8.2, 1H), 6.05–6.02 (m, 1H), 5.13–5.08 (m, 1H),
3.85 (s, 3H), 2.83 (t, J ¼ 6.6, 2H), 2.72 (t, J ¼ 6.5, 2H), 2.64–2.57 (m,
1H), 2.51–2.45 (m, 1H), 1.66 (s, 3H), 1.55 (s, 3H). 13 C NMR
(150 MHz, CDCl3) d 207.02, 177.72, 176.24, 172.01, 169.05, 167.84,
167.31, 156.36, 147.66, 136.25, 133.00, 132.77, 131.63, 130.80,
121.54, 117.49, 114.07, 111.74, 111.56, 69.99, 55.44, 32.80, 31.82,
30.93, 29.57, 25.74, 17.95. HR-ESI-MS: m/z [M þ H]þ calcd
for 494.1853.
10:
1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl-4-((4-methoxyphenyl)amino)-4-oxobutanoate,
red solid. Yield: 72.9%. m.p. 115.0 ꢀC. 1H NMR (600 MHz, CDCl3)
d ¼ 12.57 (s, 1H), 12.41 (s, 1H), 7.36 (d, J ¼ 8.8, 2H), 7.34 (s, 1H),
7.19–7.15 (m, 2H), 7.04 (s, 1H), 6.82 (d, J ¼ 8.8, 2H), 6.04 (dd,
J ¼ 6.4, 5.3, 1H), 5.11 (t, J ¼ 7.0, 1H), 3.77 (s, 3H), 2.83 (t, J ¼ 6.6,
2H), 2.65 (t, J ¼ 6.5, 2H), 2.62–2.58 (m, 1H), 2.52–2.46 (m, 1H), 1.66
(s, 3H), 1.56 (s, 3H). 13 C NMR (150 MHz, CDCl3) d 207.18, 177.88,
176.40, 172.17, 169.21, 168.00, 167.47, 156.52, 147.82, 136.41,
133.16, 132.93, 131.79, 130.96, 121.70, 117.65, 114.23, 111.96,
111.72, 70.15, 55.60, 32.96, 31.98, 31.09, 29.73, 25.90, 18.11. HR-
ESI-MS: m/z [M þ H]þ calcd for 494.1839.
16:
1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl-4-(naphthalen-1-ylamino)-4-oxobutanoate, red
solid. Yield: 74.8%. m.p. 127.0 ꢀC.1H NMR (600 MHz, CDCl3)
d ¼ 12.54 (s, 1H), 12.26 (s, 1H), 7.94 (d, J ¼ 7.1, 1H), 7.89 (br, 1H),
7.82–7.76 (m, 2H), 7.64 (d, J ¼ 8.2, 1H), 7.47–7.37 (m, 3H), 7.17–7.10
(m, 2H), 6.97 (s, 1H), 6.11–6.04 (m, 1H), 5.14–5.05 (m, 1H), 2.93 (t,
J ¼ 5.5, 2H), 2.87–2.79 (m, 1H), 2.63–2.57 (m, 2H), 2.51–2.43 (m,
1H), 1.63 (s, 3H), 1.54 (s, 3H). 13 C NMR (150 MHz, CDCl3) d 177.20,
172.38, 169.95, 167.43, 156.69, 147.43, 136.30, 133.90, 133.00,
132.72, 132.06, 131.41, 130.89, 128.81, 128.65, 126.66, 126.15,
125.85, 125.70, 125.63, 120.45, 117.43, 111.73, 70.18, 33.92, 32.77,
25.58, 24.91, 17.93. HR-ESI-MS: m/z [M þ H]þ calcd for 514.1846.
11:
1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl-4-((4-fluoro-3-(trifluoromethyl)phenyl)amino)-
4-oxobutanoate, red solid. Yield: 63.2%. m.p. 137.0 ꢀC. 1H NMR
(600 MHz, CDCl3) d ¼ 12.56 (s, 1H), 12.39 (s, 1H), 7.76–7.72 (m, 1H),
7.70–7.63 (m, 2H), 7.19–7.13 (m, 2H), 7.11 (t, J ¼ 9.3, 1H), 7.03 (s,
1H), 6.07–6.02 (m, 1H), 5.10 (t, J ¼ 6.9, 1H), 2.85–2.81 (m, 2H),
2.69–2.58 (m, 3H), 2.52–2.45 (m, 1H), 1.65 (s, 3H), 1.56 (s, 3H). 13 C
NMR (150 MHz, CDCl3) d 207.09, 177.19, 175.67, 172.03, 169.54,
168.32, 167.80, 147.36, 136.32, 136.29, 133.89, 133.20, 132.96,
131.44, 124.85, 118.30, 117.39, 117.22, 111.76, 111.51, 70.21, 32.78,
31.78, 30.92, 29.30, 25.71, 17.93. HR-ESI-MS: m/z [M þ H]þ calcd
for 550.1437.
17:
1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl-4-((3-chlorophenethyl)amino)-4-oxobutanoate,
red solid. Yield: 46.3%. m.p. 91.8 ꢀC. 1H NMR (600 MHz, CDCl3)
d ¼ 12.57 (s, 1H), 12.41 (s, 1H), 7.23–7.14 (m, 5H), 7.05 (d, J ¼ 7.2,
1H), 7.00 (s, 1H), 6.01 (dd, J ¼ 6.3, 5.1, 1H), 5.61 (s, 1H), 5.10 (t,
J ¼ 7.3, 1H), 3.49–3.42 (m, 2H), 2.77–2.72 (m, 4H), 2.62–2.54 (m,
1H), 2.48–2.38 (m, 3H), 1.67 (s, 3H), 1.56 (s, 3H). 13 C NMR
(150 MHz, CDCl3) d 177.84, 176.33, 171.85, 171.02, 167.74, 167.20,
147.82, 140.82, 136.17, 134.34, 132.97, 132.74, 131.54, 129.84,
128.85, 126.91, 126.71, 117.53, 111.78, 111.54, 69.86, 40.53, 35.29,
32.81, 30.81, 29.44, 25.76, 17.95. HR-ESI-MS: m/z [M þ H]þ calcd
for 526.1632.
12:
1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl-4-oxo-4-((4-phenoxyphenyl)amino)butanoate,
red solid. Yield: 82.1%. m.p. 133.0 ꢀC. 1H NMR (600 MHz, CDCl3)
d ¼ 12.59–12.55 (m, 1H), 12.40 (s, 1H), 7.47–7.38 (m, 3H), 7.30 (t,
J ¼ 7.8, 2H), 7.19–7.13 (m, 2H), 7.07 (t, J ¼ 7.4, 1H), 7.04 (s, 1H),
6.95 (t, J ¼ 9.5, 4H), 6.06–6.02 (m, 1H), 5.10 (t, J ¼ 7.2, 1H), 2.84 (t,
J ¼ 6.5, 2H), 2.66 (t, J ¼ 6.5, 2H), 2.64–2.57 (m, 1H), 2.49–2.42 (m,
1H), 1.66 (s, 3H), 1.56 (s, 3H). 13 C NMR (150 MHz, CDCl3) d 177.60,
176.09, 172.01, 169.20, 167.94, 167.42, 153.39, 147.59, 136.26,
133.21, 133.05, 132.82, 131.57, 129.68, 123.02, 121.42, 119.57,
118.36, 117.46, 111.78, 111.55, 70.04, 32.80, 31.83, 29.48, 25.73,
17.95. HR-ESI-MS: m/z [M þ H]þ calcd for 556.1958.
18:
1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl-4-((2-chlorophenethyl)amino)-4-oxobutanoate,
red solid. Yield: 76.6%. m.p. 97.8 ꢀC. 1H NMR (600 MHz, CDCl3)
d ¼ 12.57 (s, 1H), 12.41 (s, 1H), 7.34–7.32 (m, 1H), 7.20–7.14 (m,
5H), 7.01 (s, 1H), 6.01 (dd, J ¼ 6.5, 5.2, 1H), 5.62 (s, 1H), 5.10 (t,
J ¼ 7.3, 1H), 3.51 (dd, J ¼ 13.2, 6.8, 2H), 2.92 (t, J ¼ 7.0, 2H),
2.79–2.70 (m, 2H), 2.64–2.58 (m, 1H), 2.50–2.41 (m, 3H), 1.68 (s,
3H), 1.56 (s, 3H). 13 C NMR (150 MHz, cdcl3) d 177.85, 176.34,
171.84, 170.98, 167.73, 167.20, 147.84, 136.45, 136.17, 134.07,
132.96, 132.73, 131.55, 131.00, 129.59, 128.04, 126.97, 117.54,
111.79, 111.55, 69.81, 39.28, 33.29, 32.82, 30.81, 29.44, 25.76,
17.95. HR-ESI-MS: m/z [M þ H]þ calcd for 526.1662.
13:
1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl-4-oxo-4-((4-(trifluoromethoxy)phenyl)amino)bu-
tanoate, red solid. Yield: 58.6%. m.p. 134.0 ꢀC. 1H NMR (600 MHz,
CDCl3) d ¼ 12.56 (s, 1H), 12.40 (s, 1H), 7.56 (br, 1H), 7.50 (d, J ¼ 8.7,
2H), 7.18–7.11 (m, 4H), 7.04 (s, 1H), 6.05–6.02 (m, 1H), 5.10 (t,
J ¼ 7.0, 1H), 2.83 (t, J ¼ 6.4, 2H), 2.67 (t, J ¼ 6.3, 2H), 2.64–2.58 (m,
1H), 2.52–2.45 (m, 1H), 1.65 (s, 3H), 1.55 (s, 3H). 13 C NMR
(150 MHz, CDCl3) d 177.38, 175.87, 171.99, 169.27, 168.15, 167.62,
147.45, 136.75, 136.29, 133.14, 132.89, 131.88, 131.53, 121.13,
117.42, 116.79, 111.76, 111.54, 70.12, 32.78, 32.00, 30.92, 29.41,
25.73, 17.94. HR-ESI-MS: m/z [M þ H]þ calcd for 548.1546.
19:
1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl-4-((4-bromophenethyl)amino)-4-oxobutanoate,
red solid. Yield: 69.4%. m.p. 134.0 ꢀC. 1H NMR (600 MHz, CDCl3)
d ¼ 12.58 (s, 1H), 12.41 (s, 1H), 7.40 (d, J ¼ 8.2, 2H), 7.16 (s, 2H),
7.04 (d, J ¼ 8.2, 2H), 7.00 (s, 1H), 6.06–5.95 (m, 1H), 5.58 (s, 1H),
5.10 (t, J ¼ 7.1, 1H), 3.49–3.43 (m, 2H), 2.77–2.70 (m, 4H), 2.61–2.56
(m, 1H), 2.52–2.47 (m, 1H), 2.42 (t, J ¼ 6.7, 2H), 1.67 (s, 3H), 1.56 (s,
3H). 13 C NMR (150 MHz, CDCl3) d 177.77, 176.25, 171.85, 170.99,
167.81, 167.28, 147.80, 137.72, 136.18, 132.99, 132.78, 131.65,
14:
1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl-4-((4-bromophenyl)amino)-4-oxobutanoate, red
solid. Yield: 77.6%. m.p. 105.0 ꢀC. 1H NMR (600 MHz, CDCl3)
d ¼ 12.56 (s, 1H), 12.40 (s, 1H), 7.47 (br, 1H), 7.40–7.33 (m, 4H),
7.19–7.14 (m, 2H), 7.03 (s, 1H), 6.06–6.01 (m, 1H), 5.09 (t, J ¼ 7.2,
1H), 2.82 (t, J ¼ 6.4, 2H), 2.68–2.57 (m, 3H), 2.53–2.44 (m, 1H), 1.65
(s, 3H), 1.55 (s, 3H). 13 C NMR (150 MHz, CDCl3) d 177.38, 175.87,
171.99, 169.27, 168.15, 167.62, 147.45, 136.75, 136.29, 133.14,
132.89, 131.88, 131.53, 121.13, 117.42, 116.79, 111.76, 111.54, 131.51, 130.46, 120.35, 117.52, 111.78, 111.54, 69.85, 40.55, 35.05,

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X.-B. SHEN ET AL.
33.92, 32.81, 30.79, 29.42, 25.76, 25.58, 17.95. HR-ESI-MS: m/z 2.2. Cell culture
[M þ H]þ calcd for 570.1137.
MGC-803, SGC-7901, U87 and SMMC-7721 cell lines were cultured
in DMEM supplemented with 10% foetal bovine serum, 100 IU/mL
penicillin/streptomycin. The cells were incubated in an atmos-
phere containing 5% CO2 at 37 ꢀC.
20:
1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl 4-((4-fluorophenethyl)amino)-4-oxobutanoate,
red solid. Yield: 54.2%. m.p. 116.0 ꢀC. 1H NMR (600 MHz, CDCl3)
d ¼ 12.57 (s, 1H), 12.41 (s, 1H), 7.16 (s, 2H), 7.14–7.10 (m, 2H), 7.00
(s, 1H), 6.97 (t, J ¼ 8.6, 2H), 6.05–6.00 (m, 1H), 5.59 (br, 1H), 5.10 (t,
J ¼ 7.3, 1H), 3.51–3.44 (m, 2H), 2.77–2.70 (m, J ¼ 6.8, 4H), 2.66–2.55
(m, 1H), 2.52–2.45 (m, 1H), 2.42 (t, J ¼ 6.8, 2H), 1.67 (s, 3H), 1.56 (s,
3H). 13 C NMR (150 MHz, CDCl3) d 177.80, 176.28, 171.86, 170.96,
167.78, 167.25, 162.42, 160.79, 147.81, 136.18, 132.98, 132.76,
131.52, 130.13, 130.08, 117.52, 115.45, 115.31, 111.78, 111.54,
69.84, 40.81, 34.82, 32.81, 30.80, 29.44, 25.75, 17.94. HR-ESI-MS: m/
z [M þ H]þ calcd for 510.1936.
2.3. Cell viability assay
MGC-803, SGC-7901, U87 and SMMC-7721 cells were plated in 96-
well plate at 7000 per well. Cells were incubated with the different
concentrations of the compounds for 48 h. Subsequently, MTT
(0.5 mg/mL) were used to test the cell viability, then 150 lL DMSO
was added to each well after cells were incubated for 4 h and the
absorbance was measured at 492 nm.
21:
1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl 4-((2-fluorophenethyl)amino)-4-oxobutanoate,
red solid. Yield: 75.5%. m.p. 91.0 ꢀC. 1H NMR (600 MHz, CDCl3)
d ¼ 12.57 (s, 1H), 12.41 (s, 1H), 7.21–7.15 (m, 4H), 7.06 (t, J ¼ 7.4,
1H), 7.03–6.98 (m, 2H), 6.04–5.98 (m, 1H), 5.64 (br, 1H), 5.10 (t,
J ¼ 7.2, 1H), 3.49 (q, J ¼ 6.7, 2H), 2.83 (t, J ¼ 6.9, 2H), 2.75–2.69 (m,
2.4. Apoptosis assay
After the indicated treatments for 48 h, cells were collected,
washed three times with cold PBS, centrifuged and resuspended
2H), 2.64–2.58 (m, 1H), 2.50–2.46 (m, 1H), 2.43 (t, J ¼ 6.9, 2H), 1.67 with 400 lL AnnexinV binding buffer per tube. Then the cells
(s, 3H), 1.56 (s, 3H). 13 C NMR (150 MHz, CDCl3) d 177.92, 176.41, were stained with 5lLAnnexinV-FITC and incubated in the dark
171.83, 170.98, 167.67, 167.14, 162.03, 160.41, 147.86, 136.16, for 10 min on ice. Finally, 10 lL PI staining solution was added,
132.94, 132.71, 131.56, 131.14, 131.11, 128.35, 128.30, 125.74, and cells were detected by the flow cytometry after incubated in
125.63, 124.21, 124.19, 117.55, 115.40, 115.26, 111.79, 111.54, the dark for 5 min. Flowjo 7.6.1 software was used to analyse the
69.80, 39.67, 32.81, 30.79, 29.45, 29.09, 25.75, 17.94. HR-ESI-MS: m/
cell apoptosis.
z [M þ H]þ calcd for 510.1966.
22:
1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
2.5. Colony formation assay
methylpent-3-en-1-yl 4-(diethylamino)-4-oxobutanoate, red solid.
Yield: 78.5%. m.p. 77.0 ꢀC.1H NMR (600 MHz, CDCl3) d ¼ 12.57 (s,
1H), 12.43 (s, 1H), 7.19–7.14 (m, 2H), 7.05 (s, 1H), 6.02–5.99 (m,
1H), 5.12 (t, J ¼ 7.2, 1H), 3.36 (q, J ¼ 7.1, 1H), 3.31 (q, J ¼ 7.1, 2H),
2.75 (t, J ¼ 6.7, 2H), 2.65–2.58 (m, 3H), 2.52–2.47 (m, 1H), 1.67 (s,
3H), 1.56 (s, 3H), 1.18 (t, J ¼ 7.1, 3H), 1.08 (t, J ¼ 7.1, 3H). 13 C NMR
(150 MHz, CDCl3) d 178.85, 177.39, 172.17, 169.81, 166.79, 166.27,
148.18, 135.95, 132.60, 132.37, 131.92, 117.70, 111.82, 111.57,
69.65, 41.73, 40.28, 32.78, 29.43, 27.85, 25.75, 17.94, 14.12, 13.04.
HR-ESI-MS: m/z [M þ H]þ calcd for 444.2024.
SGC-7901 cells (8000/well) were seeded in 6-well plate, and
treated with indicated concentrations of compound 12 (2 lM,
4 lM, 8 lM) for 12 d. The medium was removed and methanol
(500 lL/well) was added to fix at the cells for 3 min. Then cells
were stained with giemsa working fluid for 15 min and washed
with PBS. The number of colonies was counted.
2.6. Acridineorange (AO) staining
23:
1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
4-(dihexylamino)-4-oxobutanoate1-(5,8-dihy-
PH-sensitive Acridine orange (AO) was used to label acidic
vesicles. Human gastric cancer cells were seeded in laser confocal
dishes at 10,000/ml. After indicated treatment of compound 12
(1 lM, 2 lM, 4 lM) for 48 h, cells were stained with AO (1 lg/ml)
for 15 min at the cell culture incubator. After the cells were
washed by PBS, pictures were acquired under laser con-
focal microscope.
methylpent-3-en-1-yl
droxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl
4-(dihexylamino)-4-oxobutanoate, red solid. Yield: 53.8%. m.p.
78.3 ꢀC. 1H NMR (600 MHz, CDCl3) d ¼ 12.56 (s, 1H), 12.44 (s, 1H),
7.19–7.14 (m, 2H), 7.05 (s, 1H), 6.00 (dd, J ¼ 6.3, 5.0, 1H), 5.12 (t,
J ¼ 7.2, 1H), 3.29–3.25 (m, 2H), 3.22–3.18 (m, 2H), 2.74 (t, J ¼ 6.7,
2H), 2.65–2.58 (m, 3H), 2.51–2.45 (m, 1H), 1.67 (s, 3H), 1.55 (s, 3H),
1.32–1.21 (m, 16H), 0.90–0.83 (m, 6H). 13 C NMR (150 MHz, CDCl3) d
178.92, 177.47, 172.18, 170.11, 166.71, 166.19, 148.19, 135.94,
132.58, 132.34, 131.96, 117.71, 111.81, 111.56, 69.62, 47.82, 46.17,
32.78, 31.60, 31.50, 29.69, 29.48, 28.87, 27.90, 27.71, 26.70, 26.60,
25.77, 22.59, 17.95, 14.04, 13.99. HR-ESI-MS: m/z [M þ H]þ calcd
for 556.3284.
2.7. GFP-LC3 transfection
A plasmid GFP-tagged LC3 reporter gene (0.8 lg/500 lL) was
transfected into SGC-7901 cells by Lipofectamine 2000, then cells
were incubated for 24 h. After different treatments for 24 h, cells
were fixed with 4% paraformaldehyde for 20 min, and washed
three times with PBS. Finally, Laser confocal dishes were analysed
using confocal microscope.
24:
1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-
methylpent-3-en-1-yl (Z)-4-morpholino-4-oxobut-2-enoate, red solid.
Yield: 49.6%. m.p. 104.0 ꢀC. 1H NMR (600 MHz, CDCl3) d ¼ 12.57 (s,
1H), 12.40 (s, 1H), 7.17 (s, 2H), 7.02 (s, 1H), 6.61 (d, J ¼ 11.9, 1H),
6.12 (d, J ¼ 11.9, 1H), 6.09 (dd, J ¼ 6.3, 4.9, 1H), 5.10 (t, J ¼ 7.1, 1H),
3.74–3.58 (m, 4H), 2.91 (dd, J ¼ 16.5, 4.5, 2H), 2.76 (dd, J ¼ 16.6,
4.6, 2H), 2.67–2.61 (m, 1H), 2.53–2.45 (m, 1H), 1.67 (s, 3H), 1.55 (s,
2.8. Western blot analysis
RIPA lysis buffer was used to dissociate the treated cells. Cells
3H). 13 C NMR (150 MHz, CDCl3) d ¼ 180.62, 179.13, 174.00, 171.50, were lysed and the supernatant was harvested after centrifuged
169.02, 168.50, 150.01, 138.03, 137.29, 134.71, 134.49, 133.85, for 30 min. The cell proteins were separated using Sodium dodecyl
124.11, 119.65, 113.80, 113.56, 71.77, 68.83, 68.46, 47.61, 44.06, sulphate-polyacrylamide gel electrophoresis, and then they were
29.71, 27.78, 19.96. HR-ESI-MS: m/z [M þ H]þ calcd for 456.1628.
transferred to the PVDF membranes. The membranes were

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
777
Figure 2. Ligand profiler and docking result of naphthoquinone moiety.
Figure 3. Docking result of compound 12 with Akt.

778
X.-B. SHEN ET AL.
Scheme 1. Synthesis of title compounds 1–24.
blocked with skimmed milk solution (5%) for 2 h, then incubated
with primary antibodies (1:1000, cst) for 20 h at 4 ꢀC. The mem-
branes were detected by chemiluminescence reagent after probed
with the secondary antibody for 1 h.
3. Results
3.1. Scaffold design
Discovery Studio 2018 was used as computation programme and
naphthoquinone moiety as ligand to execute reverse targets
searching programme, targets were searched to suit the pharma-
cophores of protein active sites in the database. Comparison with
protein data bank, the results indicated the PI3K/Akt signal path-
way was the target of naphthoquinone moiety. The lower most
2.9. Statistical analysis
Data were represented as the mean SD (standard deviation).
GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA) CDDOCKER energy of all conformations is 6.13917 kcal/m. The
were used to analyse significance by one-way ANOVA. p < 0.05 moiety interacted with protein active site through those forces,
was expressed a significant difference.
containing van der Waals force, conventional hydrogen bond,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
779
carbon hydrogen bond, alkyl and Pi-alkyl interactions. Van der it. The differences of CDDOCKER energy also proved this. (The
Waals force formed between naphthoquinone and the receptors
lower most CDDOCKER Energy of compound 12 is ꢁ15.7597 kcal/
(Thr312, Tyr315, Leu347, Gln352). There was a hydrogen bond
interaction between Asn351 and compound. Alkyl and Pi-Alkyl
interacts with the aryl ring and the alkyl group via Leu357 and
Pro313 (Figure 2).
m and naphthoquinone is 6.13917 kcal/m). These data indicated
that the combination of title compound 12 with the cavity site of
Akt (PDB ID:3CQW) is spontaneous, which was more easier than
that of naphthoquinone moiety (Figure 3).
Title compound 12 has five kinds of interactions, such as
hydrogen bonds, Pi-Anion force, Pi-alkyl interactions, and conven-
tional van der Waals force which were formed with Phe309,
Phe349, Phe358, Asp353, Asn351, Thr312, Lys276, Leu277, Glu341
and Phe236. Between the nitrogen of the compound and Gln352
formed the hydrogen bond. There also existed a Pi-anion inter-
action between aryl ring and the Glu355. Alky and Pi-Alkyl interac-
tions were formed between aryl rings and Pro313, Tyr315, Leu357,
Tyr350 and Leu347. Compared with naphthoquinone moiety,
there was more stronger interaction of compound 12 than that of
3.2. Chemistry
Compounds 1 and 2 can be obtained by substitution of halides
with phenol hydroxyls of naphthoquinone. The condensation reac-
tion between hydroxyl group on side-chain of naphthoquinone
and the related acids can get compounds 3–24. Compounds
25e–u were obtained from succinic anhydride or maleic anhydride
after ring-opening reaction (Scheme 1).
Table 1. In vitro anticancer activities of compounds 1–24 against U 87, SMMC-
7721, SGC-7901, MGC-803 cell lines.
3.3. Evaluation of anti-proliferation activity
Compounds 1–24 were detected for anticancer activities against
SGC-7901, MGC-803, SMMC-7721, U-87 cell lines in vitro by the
MTT assay, and ADM was used as the positive control34. From
Table 1, when the phenolic hydroxyls of naphthoquinone moiety
were converted to ether, the activity was missing. Compounds 9,
11, 12, 13, 17 and 20 have good activity which showed that
hydroxyl group on side-chain of naphthoquinone moiety was
modifiable and the introducing of hydrogen-bond donor, acceptor
and hydrophobic group could benefit the activity. Compound 12
displayed high activity against the MGC-803, SGC-7901 and U87
cells with the IC50s of 4.07, 4.09 and 3.85 lM, respectively. So col-
ony formation assay of this compound (Figure 4) was used to
investigate the effect of it on cell proliferation in SGC-7901 cells.
The experiment examined the ability for producing colonies after
the cells were treated with the cell death agents35,36. The results
exhibited that the colonies were decreased with the increased
concentration of the title compound compared with the control
group, which revealed this compound could inhibit the cell
proliferation.
IC50 lM (n ¼ 3)
Compounds
U87
SMMC-7721
SGC-7901
MGC-803
L02
a
1
2
3
4
5
6
7
8
–
–
–
–
–
–
–
–
–
–
8.0 0.85
65.7 2.7
–
15.6 3.3
10.6 4.3
75.1 0.4
–
4.5 1.1
–
13.6 1.5
–
–
–
–
–
–
–
–
–
9.68 2.3
10.0 1.6
4.45 0.66
12.3 2.8
3.75 0.77
3.8 0.2
3.46 0.31
–
14.4 1.5
16.7 1.8
12.3 0.1
12.4 2.6
9.5 0.8
20.6 0.4
16.9 2.2
14.8 0.1
–
24.3 1.7
15.9 0.2
17.9 1.6
17.9 1.0
18.6 3.6
15.4 0.4
46.5 1.7
–
33.6 2.1
20 3.9
8.6 2.5
11.1 1.2
2.25 1.5
4.1 2.6
7.8 3.7
10.7 1.8
–
5.6 2.6
20.8 0.5
23.7 0.5
7.6 2.3
13.3 0.6
14.8 1.6
19.8 1.8
–
9.7 2.3
10.4 0.7
3.1 0.1
7.9 1.8
8.4 3.1
4.1 0.6
10.2 2.9
10.2 1.3
–
5.9 2.6
6.7 2.4
6.6 4.7
5.7 2.4
3.5 1.4
6.9 3.9
9.4 0.4
–
36.8 4.1
>50
12.1 0.8
11.9 0.6
4.0 0.1
>50
15.8 1.1
16.2 1.7
–
13.0 2.4
11.4 1.8
20.2 2.0
8.5 0.4
12.3 0.5
16.0 2.2
>50
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
ADM
–
5.41 0.67
3.8 1.7
7.7 4.4
9.1 2.5
5.2 1.4
6.1 0.6
40.7 4.7
–
–
–
–
3.4. Metabolic stability assay in human liver microsomes
20.6 0.6
0.57 0.32
50.1 0.8
0.46 0.63
38.5 0.7
0.72 0.12 0.48 0.06
40.8 1.9
To initially evaluate the stability of compound 12, we then tested
the liver microsome stability of this compound. The Mean %
aInactive at 100 mM (highest concentration tested).
ꢂꢂ
Figure 4. The SGC-7901 cells formed colonies (counted with Image-ProPlus) after treated with various concentrations of the compound 12 for 12 d. p < 0.01,
ꢂꢂꢂ
p < 0.001 compared with the control group.

780
X.-B. SHEN ET AL.
Figure 5. (A) Apoptosis ratio was detected by Annexin V/PI staining on the SGC-7901 cells treated with various concentration of title compound 12 (1, 2, 4 lM). (B)
ꢂꢂꢂ ꢂꢂ
Western blots were used to detect the Bax, Bcl-2, cleaved PARP and full length PARP protein expression.
p < 0.001, p < 0.01 compared with the control group.
Parent Remaining of compound 12 in metabolic stability test 3.6. Induces autophagy
were 100, 95, 85, 80, and 70 at 0, 15, 30, 45, and 60 min, respect-
Autophagy is a process of lysosomal degradation that can deliver
ively. The calculated clearance rate was >110 lL/min/mg and the
half-life (t1/2) was more than 60 min which indicated the accept-
able stability.
the cytoplasmic cargo to the lysosome42. To assess whether com-
pound 12 could promote autophagy on SGC-7901 cells, the
expressions of autophagy-related proteins were examined.
Autophagy will be switched on when LC3-I was converted into
LC3-II, and the level of LC3-II involves in the formation of auto-
phagosome. Meanwhile p62 is a marker of the degradative lyso-
some43,44. As shown in Figure 6(A), compound can decrease the
level of P62 and increase the expressions of LC3II and Beclin1. The
preliminary results suggest that title compound may pro-
mote autophagy.
3.5. Induces apoptosis
Apoptosis is one of the main methods that result in cell
death37–39. In this study, Annexin V-FITC/PI kit was used to evalu-
ate the cell apoptosis. From Figure 5(A), it can be see that after
the cells were treated with the compound 12 (1, 2, and 4 lM) for
48 h, the total apoptosis was increased to 6.45%, 9.72%, and
19.9%, respectively, compared to the control group (4.34%). These
results indicated that compound 12 induced apoptosis was associ-
ated with a dose dependent. To further, investigate whether the
cell apoptosis induced by title compound, Western blot analysis
was used to measure the effect of the compound on apoptosis of
SGC-7901 cell. As reported that apoptosis pathway could be regu-
lated by the activation of PARP and Bcl2 family proteins40,41. It
can be found that the levels of Bax and cleaved PARP were up-
regulated and the level of Bcl2 was down-regulated by the
increased concentration of compound 12, which suggested that
compound could promote apoptosis in SGC-7901l cells
(Figure 5(B)).
Another typical feature of autophagy is the accumulation of
the acidic autophagic vacuoles (AVOs)45,46. To verify the develop-
ment of AVOs, Acridine Orange (AO) staining was used to examine
whether the acidic vesicular organelles in SGC-7901 cells were
increased. AO produces red fluorescence when it accumulates in
acidic regions such as autophagy lysosomes and lysosomes and
produces bright green fluorescence in the cytoplasm and the
nucleus47. As it can be seen in Figure 6(B), red fluorescently
labelled vesicle acidic accumulated obviously as the increased
concentrations of title compound (1, 2, and 4 lM). GFP-LC3 indi-
cating technique was also used to detect the autophagy at the
same time. When autophagy is formed, multiple bright green
fluorescent spots will form as the GFP-LC3 fusion protein can
translate to autophagosome membrane48. From Figure 6(C),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
781
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ꢂꢂ
ꢂꢂꢂ
Figure 6. (A) Western blots assay examined the expressions of LC3-II/LC3-, P62, Beclin1. p < 0.05, p < 0.01, p < 0.001 compared with the control group. (B)
SGC-7901 cells were stained with AO after exposed to compound 12 for 48h, then detected by the confocal microscopy at 200ꢃ. (C) SGC-7901 cells were transfected
with GFP-LC3 plasmid, and treated with compound 12 alone (4 lM), 3MA (500 lM, pro-incubated for 1 h), compound 12 and 3MA, then observed under a confocal
microscopy at 200ꢃ.
compared to the blank control group, GFP-LC3 puncta were and the down-regulation of Bcl-2 was inhibited in the group with
increased after cells treated with compound 12 (4 lM) meanwhile 3-MA. These results indicated that cell autophagy induced by
decreased after treated with compound 12 (4 lM) and 3-MA compound 12 might promote the apoptosis of SGC-7901 cells.
(500 lM). This further proved that title compound can pro-
mote autophagy.
3.8. Suppression of activation of the PI3K/AKT/MTOR pathway
As a therapeutic target for cancer, the PI3K/AKT/MTOR signalling
3.7. Role of autophagy regulation
pathway participates in regulating autophagy53–56. Consequently,
The connection between apoptosis and autophagyis complicated Western blot experiment was used to explore the impact of com-
because autophagy can promote apoptosis49,50, and can also sup- pound 12 on the level of phospho-PI3K, phospho-AKT and phos-
press apoptosis51,52. To investigate whether autophagy has an pho-mTOR. After cells were treated with title compound for 48h,
impact on cell apoptosis induced by title compound, the cells the expression of p-PI3K, p-AKT and p-MTOR were decreased in a
were treated with compound 12 (4 lM) for 48 h co-treatment with dose-dependent manner (Figure 8(A)). 3-MA (3-methyladenine) is
or absence of the 3-MA (500 lM) in the apoptosis assays (Figure also a PI3K inhibitor57, so compound 12 (4 lM) with 3-MA (500 lM)
7(A)) detected by the flow cytometry. Compared with compound were used to treated the cells. The experimental data showed that
12, the apoptosis rate of treatment group cells in the presence of the ratio of p-PI3K/PI3K, p-AKT/AKT and p-MTOR/MTOR increased in
3-MA decreased. At the same time, the levels of apoptosis-related the experiment of compound 12 with 3-MA compared with com-
proteins were examined in Western blot assay (Figure 7(B)), it was pound 12 alone (Figure 8(B)). These results revealed title compound
found that the up-regulation of apoptosis protein Bax and cl-PARP could induce cell autophagy via inhibiting PI3K pathway.

782
X.-B. SHEN ET AL.
Figure 7. (A) Cells were treated with compound 12 alone (4 lM), 3MA (500lM, pro-incubated for 1 h), compound 12 combined with 3MA and the Annexinv-FITC
staining was used to evaluate the apoptosis ratio. (B) The activation of Bax, Bcl2, cl-PARP were determined by the western blot after SGC-7901 cells were exposed to
ꢂ
ꢂꢂꢂ
p < 0.001 compared with the control group.
compound 12 for 48 h with or without of 3MA (500 lM). p < 0.05,
in Western blot and GFP-LC3 studies, the level of LC3-II/LC3-I
4. Conclusion
decreased and autophagosome puncta was reduced after pre-
treatment with 3-MA, which further verify our view. In addition,
the cell apoptosis induced by this compound was inhibited and
the cell viability was increased when the cell autophagy was
blocked by 3-MA. Sequentially, the levels of p-MTOR, p-AKT, and
p-PI3K were suppressed after incubated with compound, and
these indicated that title compound could negatively regulate the
According to result of ligand profiler and docking, a series of
naphthoquinone derivatives were designed and synthesised. The
preliminary activity results showed that several compounds had
good anticancer activity. The anticancer mechanism of one com-
pound against SGC-7901 cells was investigated further. The
expression of LC3-II and Beclin1increased and the expression of
P62 decreased after treated with this compound, which means PI3K pathways. In conclusion, title compound could inhibit cancer
that this compound helps promote the cell autophagy. Moreover, cell growth by promoting autophagy.

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Figure 8. (A) Western blots were performed to observe the PI3K, p-PI3K, AKT, p-AKT, mTOR, p-MTOR protein expression incubated with compound 12 (1 lM, 2 lM,
ꢂꢂ ꢂꢂꢂ
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4 lM) for 48h. p < 0.05, p < 0.01,
lysed by western blotting assay with or without pre-treatment of 3MA (500 lM). p < 0.05, p < 0.001 compared with the control group.
p < 0.001 compared with the control group. (B) The expression levels of LC3-II, Beclin-1, P62 and PI3K signalling were ana-
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ꢂꢂꢂ
[HX2019033], and Technological Fund of Fuyang normal univer-
sity [2017FSKJ16].
Disclosure statement
No potential conflict of interest was reported by the author(s).
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Funding
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XDHX2016026], Anhui Province Foundation [No. 18030701213],
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