Synthesis and biological evaluation of lipophilic 1,4-naphthoquinone derivatives against human cancer cell lines

Article abstract of DOI:10.3390/molecules200711994

To examine the effect of hydrophobicity on the anticancer activity of 1,4-naphthoquinone derivatives, a series of compounds bearing a 2-O-alkyl-, 3-C-alkyl- or 2/3-N-morpholinoalkyl group were synthesized and evaluated for their anticancer activity against five human cancer cell lines in vitro. The cytotoxicity of these derivatives was assayed against HT-29, SW480, HepG2, MCF-7 and HL-60 cells by the MTT assay. Among them, 2-hydroxy-3-farnesyl-1,4-naphthoquinone (11a) was found to be the most cytotoxic against these cell lines. Our results showed that the effectiveness of compound 11a may be attributed to its suppression of the survival of HT-29. Secondly, in the Hoechst 33258 staining test, compound 11a-treated cells exhibited nuclear condensation typical of apoptosis. Additionally, cell cycle analysis by flow cytometry indicated that compound 11a arrested HT-29 cells in the S phase. Furthermore, cell death detected by Annexin V-FITC/propidium iodide staining showed that compound 11a efficiently induced apoptosis of HT-29 in a concentration-dependent manner. Taken together, compound 11a effectively inhibits colon cancer cell proliferation and may be a potent anticancer agent.

Full text of DOI:10.3390/molecules200711994

Molecules 2015, 20, 11994-12015; doi:10.3390/molecules200711994
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Biological Evaluation of Lipophilic
1,4-Naphthoquinone Derivatives against Human
Cancer Cell Lines
Shao-Hung Wang 1, Chih-Yu Lo 2, Zhong-Heng Gwo 1, Hong-Jhih Lin 1, Lih-Geeng Chen 1,
Cheng-Deng Kuo 3 and Jin-Yi Wu 1,*
1
Department of Microbiology, Immunology and Biopharmaceuticals, College of Life Sciences,
National Chiayi University, Chiayi 60004, Taiwan; E-Mails: shwang@mail.ncyu.edu.tw (S.-H.W.);
s0990613@mail.ncyu.edu.tw (Z.-H.G.); s1030730@mail.ncyu.edu.tw (H.-J.L.);
lgchen@mail.ncyu.edu.tw (L.-G.C.)
2
Department of Food Science, College of Life Sciences, National Chiayi University, Chiayi 60004,
Taiwan; E-Mail: chihyulo@mail.ncyu.edu.tw
3
Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei 11217,
Taiwan; E-Mail: cdkuo23@gmail.com
* Author to whom correspondence should be addressed; E-Mail: jywu@mail.ncyu.edu.tw;
Tel: +886-5-271-7925; Fax: +886-5-271-7778.
Academic Editors: Jean Jacques Vanden Eynde, Annie Mayence and Tien L. Huang
Received: 29 May 2015 / Accepted: 25 June 2015 / Published: 30 June 2015
Abstract: To examine the effect of hydrophobicity on the anticancer activity of
1,4-naphthoquinone derivatives, a series of compounds bearing a 2-O-alkyl-, 3-C-alkyl- or
2/3-N-morpholinoalkyl group were synthesized and evaluated for their anticancer activity
against five human cancer cell lines in vitro. The cytotoxicity of these derivatives was
assayed against HT-29, SW480, HepG2, MCF-7 and HL-60 cells by the MTT assay. Among
them, 2-hydroxy-3-farnesyl-1,4-naphthoquinone (11a) was found to be the most cytotoxic
against these cell lines. Our results showed that the effectiveness of compound 11a may be
attributed to its suppression of the survival of HT-29. Secondly, in the Hoechst 33258
staining test, compound 11a-treated cells exhibited nuclear condensation typical of apoptosis.
Additionally, cell cycle analysis by flow cytometry indicated that compound 11a arrested
HT-29 cells in the S phase. Furthermore, cell death detected by Annexin V-FITC/propidium
iodide staining showed that compound 11a efficiently induced apoptosis of HT-29 in a

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concentration-dependent manner. Taken together, compound 11a effectively inhibits colon
cancer cell proliferation and may be a potent anticancer agent.
Keywords: 1,4-naphthoquinone; terpenoids; anticancer activity; human colon cancer cells
HT-29; cell cycle distribution; apoptosis
1. Introduction
Millions of lives are lost because of cancer in the world each year and the number is increasing.
However, greater than 30% of cancers are preventable [1] and some forms of cancer are curable
regardless of when they are diagnosed, while many others are only curable if discovered at an early
stage. Thus cancer remains a life-threatening disease and represents a serious peril to human health [2].
Chemotherapy is vital to cancer treatment and the majority of clinical antitumor agents have originated
from natural products. Plants produce a wide range of secondary metabolites such as flavonoids,
terpenoids, alkaloids, quinones, polyacetylene, and sugar as part of their defense mechanism against
insects [3] and microbials [4]. Among these, 1,4-naphthoquinones, in particular, plumbagin (1), lawsone
(2), juglone (3), and shikonin (4) [5] (Figure 1), are an important group of natural products, which have
been employed in the development of potent phytochemicals because of their high abundance and
relatively non-toxic nature. The 1,4-naphthoquinone pharmacophore is known to impart anticancer
activity in a number of drugs, such as streptonigrin [6], mitomycins [7], and doxorubicin [8] among
others. The anticancer activity of the 1,4-naphthoquinone skeleton has been the focus of recent research in
the field [9,10].
Figure 1. Chemical structures of plumbagin (1); lawsone (2); juglone (3) and shikonin (4).
Among 1,4-naphthoquinones, there are numerous interesting biologically active compounds such as
atovaquone, plumbagin, lapachol, and menadione [11]. Nevertheless, a literature precedence revealed
that plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a wide type quinonoid, found in the plants
of the Plumbaginaceae, Droseraceae, Ancestrocladceae, and Dioncophyllaceae families, possesses
significant antimicrobial [12], antimalarial [13], antifilarial [14], antiprotozoal [15], anti-inflammatory [16],
and anticancer properties [17–19].
Further, it was reported that monoterpenyl- and diterpenylnaphthoquinones derivatives had IC50
values in the micromolar range against several tumor cell lines [20]. These derivatives were synthesized
through Diels-Alder addition of natural terpenoids and p-benzoquinones, followed by side chain
transformation [21]. Additionally, several 1,4-naphthoquinone derivatives incorporating with hydroxyl
groups at C-5 can provide enhanced anti-proliferative activity in in vivo and in vitro [22].

Molecules 2015, 20
11996
It was also reported that 1,4-naphthoquinones interfere with the electron transport and oxidative
phosphorylation processes and play roles in enzyme inhibition, and DNA cross linking as well as
have antifungal, antibacterial, and anticancer activities [7]. Nonetheless, many reported amino-1,4-
naphthoquinones did not contain substituents on the benzene ring, although some studies [21,22] have
shown that derivatives with side chains (alkyl or alkenyl) on the benzene ring considerably improved
the cytotoxicity of the 1,4-naphthoquinone moiety.
In this study, we synthesized and evaluated the effect of incorporation of new alkyl or terpenyl
moieties in 1,4-naphthoquinones, including lawsone substituted at the 2-C/3-O-position with alkyl or
terpenyl groups and juglone substituted at position 2/3-N-morpholinoalkyl groups, with the aim to
analyze the influence of these substituents on the cytotoxicity of these derivatives against human cancer
cell lines. We prepared and evaluated C-2 amino substituted 1,4-naphthoquinones, a core structure present
in numerous naturally occurring bioactive quinones, such as mitomycin C, as well as some synthetic
compounds [23].
2. Results and Discussion
2.1. Chemistry
In general, 1,4-naphthoquionone derivatives were synthesized from lawsone (2) with alkyl, terpenyl,
alkylamino, or morpholinealkylamino moieties through 2-O- or 3-C-alkylation. As shown in Scheme 1,
synthesis of 5a,b–11a,b were carried out by reacting lawsone with selected alkyl or terpenyl bromide
in dimethylformamide (DMF) using triethylamine as the base and sodium iodide (NaI) as the catalyst
under N2 for 1 h according to the reported procedure [24]. The addition of an alkyl or terpenyl group
to lawsone (2) gave two products at the 2-O-postion and 3-C-position in 40.0%–72.3% yield. The
3-C-substituted compounds were the major products while the 2-O-substituted compounds were
obtained with a yield of less than 10%.
Reagents and conditions: (a) NaI, triethylamine, DMF, N2, 1 h; (b) alkyl or terpenyl bromide, 40 °C, 3–7 days.
Scheme 1. Synthesis of compounds 5a,b‒11a,b.
The synthesis of juglone (3) was via oxidation of commercially available 1,5-dihydroxy-naphthalene
(12) in air, and dark, in the presence of periodic acid with a yield of 67.6%. Chemical transformation
of juglone (3) to compounds 13a,b‒19a,b was then carried out according to the reported methods with
some modifications [25]. Aminated juglone (3) was prepared by stirring it with a long chain amine in
ethanol for 1 h under reflux to give 2-alkylaminojuglone and 3-alkylaminojuglone as major and minor

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products, respectively. Lastly, once the alkylamino substituted 1,4-naphthoquinone derivatives were
obtained, other alkyl- or morpholinealkylamino-juglones were prepared by adding primary alkyl amines
and morpholinealkyl amines to juglone (3) in ethanolic solution at room temperature. The two-step
reaction and end products are illustrated in Scheme 2.
Reagents and conditions: (a) HIO4, THF/H2O, rt, 30 min; (b) alkyl amine, EtOH, reflux, 1–5 h.
Scheme 2. Synthesis of compounds 13a,b‒19a,b.
2.2. In Vitro Anti-Proliferative Activity by MTT Assay
The cytotoxic effects of the 31 1,4-naphthoquinone derivatives were tested in vitro against five human
cancer cell lines: HT29 (colorectal adenocarcinoma), SW480 (colorectal adenocarcinoma), HepG2
(hepatocellular carcinoma), HL60 (leukemia), MCF-7 (breast adenocarcinoma) and normal murine
embryonic liver BNL CL.2 cells, using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) assay [26]. 5-Fluorouracil (5-Fu), cisplatin and doxorubicin were used as positive controls. The
concentration (in μM) of the test compounds which induced 50% inhibition of cell growth (IC50) is
shown in Table 1.
Of the 31 compounds tested, eleven compounds were bioactive and nine (5b, 6b, 7b, 10a, 11a, 18a,
18b, 19a and 19b) showed significant cell growth inhibition in four human cancer cells except MCF-7
cells. In addition, the anticancer activity of these compounds increased with the higher chain length of
the terpenyl substituents, but not in long chain alkyl groups. This result suggests that the lipophilicity
of terpenyl moiety of the 1,4-naphthoquinone derivatives at the 3-C-substituent affected the bioactivity
of the compound (11a). In particular, among the bioactive ones, compound 11a exhibited the highest
activity against the human colorectal HT-29 cell lines with an IC50 value of 1.99 ± 0.04 μM for 48 h.
Compared to plumbagin (1)’s IC50 value of 3.67 ± 0.46 μM, derivative 11a was slightly more cytotoxic
against human colorectal carcinoma HT-29 cells. No significant cell death was detected in normal
murine embryonic liver BNL CL.2 cells treated with plumbagin (1) and 11a. Furthermore, in BNL CL.2
cells, only a marked effect on cell death (under 20%) was observed at the maximum concentration
(20 μM) of these two compounds after 48 h treatment (see also Figure S1). It thus appeared that both
plumbagin (1) and compound 11a are cytotoxic to human colorectal cancer cells with no significant
adverse side effects toward normal cells. Namely, the lipophilic characteristics of the substituent at the
3-C-position of the farnesyl moieties of 1,4-naphthoquinone derivative 11a can remarkably enhance
their anticancer activity against human cancer HT-29 cell lines. In other words, a higher anticancer

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activity because the longer terpenyl moiety of the 1,4-naphthoquinone derivative 11a enhanced their
cell membrane permeability and bioavailability, thus increasing their cytotoxic activities.
Table 1. IC50 values of plumbagin (1), lawsone (2) and its derivatives 5a,b–11a,b,
13a,b‒19a,b on the growth of human cancer cell lines for 48 h.
IC50 (μM) b (Mean ± SD)
Compound
cLogP a
HT-29 Colon SW480 Colon HepG2 Liver HL60 Leukemia MCF-7 Breast
Plumbagin (1)
Lawsone (2)
Juglone (3)
5a
2.78
1.68
2.26
2.19
1.78
3.79
3.22
5.90
5.34
7.46
8.02
3.70
3.34
5.73
5.37
6.82
7.40
3.65
3.65
4.99
4.99
6.05
6.05
7.11
7.11
8.17
8.17
2.51
2.51
2.80
2.80
-
3.67 ± 0.46
>100
2.19 ± 0.65
>100
2.32 ± 0.18
>100
5.90 ± 1.21
45.12 ± 7.90
46.95 ± 0.58
>100
20.89 ± 0.70
>100
>50
>50
>50
>50
>100
97.09 ± 2.36
4.23 ± 0.50
>100
94.08 ± 2.63
3.32 ± 0.66
>100
>100
5b
8.16 ± 1.16
>100
16.40 ± 2.84
>100
>25
6a
>100
6b
14.81 ± 0.65
33.44 ± 1.48
28.01 ± 3.52
>25
2.02 ± 0.20
9.73 ± 0.04
8.51 ± 0.53
>25
3.67 ± 0.84
20.20 ± 3.52
9.66 ± 0.50
>25
20.04 ± 0.79
>100
31.03 ± 0.32
>50
7a
7b
23.42 ± 1.24
>25
20.81 ± 3.67
>25
8a
8b
>25
>25
>25
>25
>25
9a
>100
>100
74.18 ± 2.61
35.56 ± 1.13
19.10 ± 0.26
29.10 ± 1.21
14.01 ± 2.70
32.33 ± 1.46
9.39 ± 0.12
17.89 ± 0.14
>50
>100
>100
9b
>50
20.72 ± 1.19
14.14 ± 0.96
44.23 ± 1.06
2.02 ± 0.31
37.16 ± 2.21
16.17 ± 0.72
8.28 ± 0.12
>50
>50
>50
10a
12.90 ± 2.13
42.90 ± 1.13
1.99 ± 0.04
40.37 ± 0.75
9.82 ± 1.51
18.11 ± 1.68
>50
9.12 ± 0.69
29.56 ± 0.88
5.41 ± 0.88
35.10 ± 1.52
45.69 ± 2.48
6.50 ± 0.32
>50
>100
10b
>100
11a
47.01 ± 0.47
>100
11b
13a
>100
13b
23.71 ± 0.82
>50
14a
14b
34.53 ± 1.20
>50
15.13 ± 0.49
>50
17.82 ± 2.29
>50
>50
>50
>50
15a
>50
15b
>50
20.23 ± 2.18
>50
47.06 ± 0.63
>50
>50
>50
>50
16a
>50
>50
16b
>50
>50
>50
>50
>50
17a
>20
>20
>20
>20
>20
17b
>20
>20
>20
>20
>20
18a
9.42 ± 0.45
8.59 ± 0.67
9.44 ± 0.32
11.38 ± 1.51
24.07 ± 0.03
>100
8.86 ± 0.50
5.19 ± 0.44
4.66 ± 0.25
3.86 ± 0.61
40.72 ± 1.18
32.72 ± 8.32
0.53 ± 0.07
11.72 ± 0.42
8.84 ± 0.07
10.08 ± 0.57
7.90 ± 0.16
36.07 ± 3.11
40.18 ± 7.63
0.30 ± 0.02
8.42 ± 0.86
6.49 ± 0.84
6.35 ± 0.24
7.76 ± 0.54
>100
>50
18b
>50
19a
>50
19b
>50
Cisplatin
5-Fu
>100
-
>100
>100
Doxorubicin
-
1.70 ± 0.20
14.26 ± 0.89
0.39 ± 0.06
a
b
Calculated log value of partition coefficient by ChemDraw Ultra 8.0; IC50 = compound concentration
required to inhibit tumor cell proliferation by 50%. Data are expressed as the mean ± SD from the dose response
curves of at least three independent experiments.

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2.3. Cytotoxic Activity of Plumbagin (1) and Compound 11a was Exhibited in a Time-Dependent Manner
In light of the cytotoxicity findings described above, we extended our study on the anti-proliferation
efficacy of plumbagin (1) and lawsone derivative 11a against HT-29 cells to 24 h, 48 h and 72 h
periods. The data on plumbagin (1) was used here as base case. As shown in Table 2, first of all, the
cytotoxicity of compound 11a demonstrated, once again stronger bioactivity than that of plumbagin (1)
in vitro. In addition, the growth inhibition of HT-29 cells induced by these two samples was in a
time-dependent manner. The IC50 values of plumbagin (1) and 11a in HT-29 cells for 24 h, 48 h and
72 h incubation times were 4.72, 3.67, 2.10 μM and >20, 1.99, 0.84 μM, respectively. The results revealed
that plumbagin (1) and 11a inhibited the cell viability of HT-29 cells in a time-dependent manner at
longer time.
Table 2. Cytotoxicity of plumbagin (1) and 11a on HT-29 for 24 h, 48 h, and 72 h.
IC50 (μM) a (Mean ± SD)
Compound
24 h
4.72 ± 0.07 3.67 ± 0.46 2.10 ± 0.23
>20 1.99 ± 0.04 0.84 ± 0.12
48 h
72 h
Plumbagin (1)
11a
a
IC50 = compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as
the mean ± SD from the dose response curves of at least three independent experiments.
2.4. Nuclear Morphological Changes of HT-29 Cells Treated with Plumbagin (1) and Compound 11a
Apoptosis, shown by morphological changes, were detected through Hoechst 33258 staining in
the HT-29 cells. The classical characteristics of apoptotic cells are chromatin condensation and nuclear
fragmentation [27]. To further investigate the role apoptosis plays in the superior cytotoxicity of
plumbagin (1) and the potent compound 11a, we incubated HT-29 cells, with plumbagin (1) and 11a,
separately, for 48 h. The cells were then stained with Hoechst 33258, and examined by fluorescence
microscopy for topical morphological changes. As shown in Figure 2, while the nuclei of the cells
were round in shape and stained homogenously in the control without testing compound, those treated
with plumbagin (1) and 11a showed typical morphological features of apoptotic cells based on cell
shrinkage, chromatin condensation and nuclear fragmentation [28]. Evidently, the proliferation of the
cancer cell is inhibited by the testing compounds in vitro.
Figure 2. Cont.

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Figure 2. The morphological changes of HT-29 cells treated with 0–2.5 μM plumbagin (1)
(A) and 11a (B) for 48 h. (magnification, 200). (A,B) The upper panels showed the cell
morphology under phase-contrast microscopy, and the lower panels display the Hoechst
33258-stained nuclear patterns take by fluorescence microscopy (magnification, 200).
2.5. Cell Cycle Distribution Analysis Using Flow Cytometry
To probe the apoptotic effects of plumbagin (1) and 11a on cell cycle progression, HT-29 cells were
treated once again with these compounds, separately, at different concentrations for 48 h. The cell cycle
distribution of the cancer cells was analyzed by flow cytometry, and the sub-G1 phase analyzed by flow
cytometry with propidium iodide (PI) staining [28]. The control of the experiment was the untreated
cells. As illustrated in Figure 3, only a small fraction of apoptotic cells (0.8%) was detected in the control
as well as in plumbagin (1) at a low concentration of 2.5 μM. However, as plumbagin (1) dosage
increased from 0.5 to 2.5 μM, the fraction of apoptotic cell went up substantially from 4.5% to 7.6% in
a concentration-dependent manner. As for compound 11a, the apoptotic effect meagerly laid between
10.7% to 27.8% across the treatment range of 0.5–2.5 μM and induced cell accumulation in the
S phase.
Figure 3. Cont.

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Figure 3. (A) The effects of the 48 h treatments with 0–2.5 μM plumbagin (1) and 11a on
cell cycle distribution of HT-29. After treatment, cells were fixed and stained with PI, and the
cell cycle distribution was examined by flow cytometry; (B) Quantitative data of cell cycle
analysis of HT-29 treated with 0‒2.5 μM plumbagin (1) and 11a for 48 h; (C) Compared
with control, the treated with plumbagin (1) and 11a shows the obvious increase in
sub-G1 fraction. Each value represents the mean ± SD of three independent experiments.
* p < 0.05, ** p < 0.01 vs. control.
2.6. Apoptotic Analyses-Annexin V-FITC/PI Double Staining and Flow Cytometry Analyses
Quantitative analysis of apoptotic effects of plumbagin (1) and 11a on HT-29 cells was conducted
by flow cytometry using Annexin V-FITC and PI double staining. This was to study in depth the
bioactivities of plumbagin (1) and 11a against HT-29 cells. Thus, the cancer cells were treated with
vehicle alone as control or with one of the two testing compounds at different concentrations (0.5–2.5 μM).
After 48 h, the samples were double-stained with Annexin V-FITC and PI [29]. The percentages of cell
populations at various stages of apoptosis were exhibited in Figure 4. The total apoptosis rates were
1.08%, 8.65%, 13.21%, and 21.02% at concentrations of 0, 0.5, 1.0, and 2.5 μM of compound 11a,
respectively. Though the data pointed out that the distributions of apoptotic cell death resulting from
the treatment of lawsone derivative 11a were concentration-dependent, this was not the case for plumbagin
(1). Starting from a dosage of 0.5 μM, compound 11a induced higher frequency of HT-29 cells
apoptosis, as well as cytotoxic effects at both early and late stages. For plumbagin (1) though, the only
discernible effect was seen at a higher threshold (2.5 μM). We attribute this finding to and confirmed
that the superior efficiency of lawsone derivative 11a in its cytotoxicity and inhibitive function on human
colorectal cell proliferation.

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Figure 4. (A) The effects of the 48 h treatments with 0–2.5 μM plumbagin (1) and 11a on
apoptotic percentage distribution of HT-29 cells by Annexin V-FITC/PI staining. (B) The
apoptosis rate was calculated by flow cytometry and cell apoptosis was defined in early
and late apoptosis treatment with 0–2.5 μM plumbagin (1) and 11a for 48 h. Each value
represents the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 and
*** p < 0.001 vs. control.
3. Experimental Section
3.1. General
All chemical reagents of commercial quality were used as received (Sigma-Aldrich, St. Louis, MO,
USA) and were used without further purification. Solvents were dried and the synthesized compounds
were purified using standard techniques. The progression of reactions was monitored by TLC on
aluminum plates coated with silica gel with a fluorescent indicator (Merck, Darmstadt, Germany)
unless otherwise stated. Melting points were determined using open capillaries using the Fargo MP-2D

Molecules 2015, 20
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apparatus (Prosperous instrument, Chaiyi, Taiwan, ROC) and are reported uncorrected. NMR spectra
were recorded using TMS as an internal standard in CDCl3 at 500 MHz for 1H and at 125 MHz for 13C
(Bruker Biospin GmbH AVANCE III 500 MHz, Rheinstetten, Germany). The mass spectra were
acquired using a Thermo Finnigan model LXQ (Thermo Electron Co., Waltham, MA, USA) ion trap
mass spectrometer equipped with ESI source interference and controlled by Xcalibur 2.06 (Thermo
Electron Co., Waltham, MA, USA). The mass spectra were acquired in a positive ion mode or a negative
ion mode. ESI high-resolution mass spectra (HRMS) were recorded on a Finnigan MAT 95S mass
spectrometry (Thermo Fisher Scientific Co. Ltd., Waltham, MA, USA). Column chromatography was
performed with silica gel SiliaFlash G60 (60–200 μm, Quebec City, QC, Canada) purchased from
SiliCycle Inc. (Quebec City, QC, Canada). In general, the reactions were carried out under anhydrous
conditions in dry solvent and nitrogen atmosphere. The purity of the compounds was more than 98%
based on the analysis of HPLC (Hitachi High-Technologies, Tokyo, Japan) using a RP-C18 column
(4.6 mm  250 mm, 5 μm, Merck, Darmstadt, Germany).
3.2. General Synthetic Procedure for 5a,b‒11a,b
A mixture of lawsone (2.87 mmol), NaI (3.16 mmol), triethylamine (3.16 mmol) and alkyl or
terpenyl bromide (2.87 mmol) were stirred in DMF (5 mL) at 40 °C for three days. After the reaction
was cooled to room temperature, 1 N HCl was added and the solution was poured into a separatory
funnel with water, dichloromethane was added and the organic layer was separated from the aqueous layer.
The organic layer was collected, dried over anhydrous magnesium sulfate (MgSO4) and concentrated
under reduced pressure to remove the solvent, and then was subjected to column chromatography
(ethyl acetate/n-hexane = 1/7) to produce yellow solid compounds, which were purified by recrystallization
from n-hexane.
2-Hydroxy-3-methyl-1,4-naphthoquinone (5a). The reaction produced 5a in 45.3% as a yellow solid;
mp 173.4–174.1 °C (lit. [30] 173–174 °C). 1H-NMR (500 MHz, CDCl3) δH 2.09 (s, 3H, CH3), 7.29 (bs,
1H, 2-OH), 7.66 (dt, J = 1.2, 7.5 Hz, 1H, H-7), 7.73 (dt, J = 1.4, 7.6 Hz, 1H, H-6), 8.06 (dd, J = 1.2,
13
7.7 Hz, 1H, H-8), 8.10 (dd, J = 0.7, 7.7 Hz, 1H, H-5); C-NMR (125 MHz, CDCl3) δC 8.91 (CH3),
120.75 (C-3), 126.36 (C-8), 126.97 (C-5), 129.64 (C-9), 133.13 (C-6), 132.94 (C-10), 134.83 (C-7),
153.13 (C-2) 181.19 (C=O), 185.02 (C=O); LC-MS (ESI−, m/z) calculated for C11H8O3: 188.05, found for
186.98 [M − H]−; HRMS-ESI: m/z calculated for C11H8O3: 188.0473 [M]+, found for 188.0471.
2-Methoxy-1,4-naphthoquinone (5b). The reaction produced 5b in 27.0% as a yellow solid; mp
187.0–187.8 °C (lit. [31] 186–189 °C). 1H-NMR (500 MHz, CDCl3) δH 3.89 (s, 3H, CH3), 6.16 (s, 1H,
H-3), 7.70 (dt, J = 1.3, 7.5 Hz, 1H, H-7), 7.74 (dt, J = 1.3, 7.5 Hz, 1H, H-6), 8.07 (dd, J = 1.2, 7.5 Hz,
1H, H-8), 8.11 (dd, J = 1.2, 7.5 Hz, 1H, H-5); 13C-NMR (125 MHz, CDCl3) δC 56.65 (CH3), 110.14 (C-3),
126.43 (C-8), 126.96 (C-5), 131.29 (C-9), 132.28 (C-10), 133.58 (C-6), 134.57 (C-7), 160.67 (C-2),
180.36 (C=O), 185.08 (C=O); LC-MS (ESI+, m/z) calculated for C11H8O3: 188.05, found for 189.09
[M + H]+; HRMS-ESI: m/z calculated for C11H8O3: 188.0473 [M]+, found for 188.0476.
2-Hydroxy-3-butyl-1,4-naphthoquinone (6a). The reaction produced 6a in 36.6% as a yellow solid; mp
97.1–98.0 °C (lit. [32] 93–94 °C). 1H-NMR (500 MHz, CDCl3) δH 0.92 (t, J = 7.5 Hz, 3H, CH3), 1.39

Molecules 2015, 20
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(p, J = 7.5 Hz 2H, H-3′), 1.50 (p, J = 7.5 Hz, 2H, H-2′), 2.58 (t, J = 7.5 Hz, 2H, H-1′), 7.30 (s, 1H,
2-OH), 7.65 (t, J = 7.5 Hz, 1H, H-7), 7.73 (t, J = 7.5 Hz, 1H, H-6), 8.05 (d, J = 7.5 Hz, 1H, H-8), 8.10
13
(d, J = 7.5 Hz, 1H, H-5); C-NMR (125 MHz, CDCl3) δC 14.12 (CH3), 23.09 (C-1′), 29.91 (C-3′),
30.64 (C-2′), 125.04 (C-3), 126.26 (C-8), 126.99 (C-5), 129.67 (C-9), 133.06 (C-6), 133.18 (C-10), 135.04
(C-7), 153.23 (C-2), 181.70 (C=O), 184.96 (C=O); LC-MS (ESI−, m/z) calculated for C14H14O3:
230.09, found for 229.05 [M − H]−; HRMS-ESI: m/z calculated for C14H14O3: 230.0943 [M]+, found
for 230.0948.
2-Butyloxy-1,4-naphthoquinone (6b). The reaction produced 6b in 5.5% as a yellow solid; mp
1
111.5–112.1 °C (lit. [33] 97–98 °C). H-NMR (500 MHz, CDCl3) δH 0.99 (t, J = 7.4 Hz, 3H, CH3),
1.53 (p, J = 7.5 Hz 2H, H-3′), 1.88 (p, J = 7.1 Hz, 2H, H-2′), 4.02 (t, J = 6.6 Hz, 2H, H-1′), 6.16 (s, 1H,
H-3), 7.70 (dt, J = 1.0, 7.5 Hz, 1H, H-7), 7.74 (dt, J = 1.0, 7.5 Hz, 1H, H-6), 8.07 (d, J = 7.3 Hz, 1H,
H-8), 8.11 (d, J = 7.3 Hz, 1H, H-5); 13C-NMR (125 MHz, CDCl3) δC 13.70 (CH3), 19.13 (C-3′), 30.25
(C-2′), 69.39 (C-1′), 110.19 (C-3), 126.11 (C-8), 126.68 (C-5), 131.20 (C-9), 132.04 (C-10), 133.26 (C-6),
134.21 (C-7), 159.93 (C-2), 180.18 (C=O), 185.10 (C=O); LC-MS (ESI−, m/z) calculated for C14H14O3:
230.09, found for 229.52 [M − H]−; HRMS-ESI: m/z calculated for C14H14O3: 230.0943 [M]+, found
for 230.0941.
2-Hydroxy-3-octyl-1,4-naphthoquinone (7a). The reaction produced 7a in 36.2% as a yellow solid; mp
1
91.1–91.6 °C (lit. [34] 88–89 °C). H-NMR (500 MHz, CDCl3) δH 0.86 (t, J = 7.5 Hz, 3H, CH3),
1.23–1.36 (m, 8H, H-4′, H-5′, H-6′, H-7′), 1.39 (p, J = 8.0 Hz, 2H, H-3′), 1.51 (p, J = 7.6 Hz, 2H, H-2′),
2.58 (t, J = 7.7 Hz, 2H, H-1′), 7.26 (s, 1H, 2-OH), 7.66 (dt, J = 1.0, 7.6 Hz, 1H, H-7), 7.73 (dt, J = 1.0,
13
7.6 Hz, 1H, H-6), 8.05 (d, J = 7.5 Hz, 1H, H-8), 8.10 (d, J = 7.6 Hz, 1H, H-5); C-NMR (125 MHz,
CDCl3) δC 14.32 (CH3), 22.88 (C-1′), 28.53 (C-7′), 29.46 (C-2′), 29.64 (C-3′), 29.92 (C-4′), 30.41 (C-5′),
32.10 (C-6′), 125.09 (C-3), 126.27 (C-8), 127.01 (C-5), 129.69 (C-9), 133.06 (C-6), 133.21 (C-10),
135.04 (C-7), 153.20 (C-2), 181.72 (C=O), 184.94 (C=O); LC-MS (ESI+, m/z) calculated for C18H22O3:
286.16, found for 309.35 [M + Na]+; HRMS-ESI: m/z calculated for C18H22O3: 286.1569 [M]+, found
for 286.1568.
2-Octyloxy-1,4-naphthoquinone (7b). The reaction produced 7b in 3.8% as a yellow solid; mp
94.1–95.8 °C. 1H-NMR (500 MHz, CDCl3) δH 0.86 (t, J = 6.9, 3H, CH3), 1.23–1.35 (m, 8H, H-4′, H-5′,
H-6′, H-7′), 1.44 (p, J = 7.6 Hz, 2H, H-3′), 1.87 (p, J = 7.2 Hz, 2H, H-2′), 3.98 (t, J = 6.7 Hz, 2H,
H-1′), 6.12 (s, 1H, H-3), 7.68 (dt, J = 1.5, 7.5 Hz, 1H, H-7), 7.71 (dt, J = 1.0, 7.5 Hz, 1H, H-6), 8.05
(dd, J = 1.0, 7.0 Hz, 1H, H-8), 8.10 (dd, J = 1.5, 7.5 Hz, 1H, H-5); 13C-NMR (125 MHz, CDCl3) δC 14.08
(CH3), 22.63 (C-7′), 25.85 (C-2′), 28.26 (C-3′), 29.14 (C-4′), 29.20 (C-5′), 31.75 (C-6′), 69.69 (C-1′),
110.19 (C-3), 126.11 (C-8), 126.67 (C-5), 131.20 (C-9), 132.05 (C-10), 133.24 (C-6), 134.20 (C-7),
159.92 (C-2), 180.18 (C=O), 185.09 (C=O); LC-MS (ESI+, m/z) calculated for C18H22O3: 286.16,
found for 309.25 [M + Na]+; HRMS-ESI: m/z calculated for C18H22O3: 286.1569 [M]+, found
for 286.1564.
2-Hydroxy-3-dodectyl-1,4-naphthoquinone (8a). The reaction produced 8a in 56.3% as a yellow solid;
1
mp 88.4–88.9 °C (lit. [35] 85–87 °C). H-NMR (500 MHz, CDCl3) δH 0.86 (t, J = 7.0 Hz, 3H, CH3),
1.23–1.35 (m, 18H, H-3′, H-4′, H-5′, H-6′, H-7′, H-8′, H-9′, H-10′, H-11′), 1.51 (p, J = 7.5 Hz, 2H, H-2′),

Molecules 2015, 20
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2.58 (t, J = 7.7 Hz, 2H, H-1′), 7.26 (s, 1H, 2-OH), 7.61 (dt, J = 0.8, 7.5 Hz, 1H, H-7), 7.73 (dt, J = 0.8,
13
7.5 Hz, 1H, H-6), 8.00 (d, J = 7.5 Hz, 1H, H-8), 8.05 (d, J = 7.5 Hz, 1H, H-5); C-NMR (125 MHz,
CDCl3) δC 14.33 (CH3), 22.91 (C-1′), 23.62 (C-11′), 28.53 (C-2′), 29.57 (C-3′), 29.68 (C-4′), 29.80 (C-5′),
29.86 (C-6′), 29.88 (C-7′), 29.92 (C-8′), 30.02 (C-9′), 32.14 (C-10′), 125.09 (C-3), 126.26 (C-8),
127.00 (C-5), 129.69 (C-9), 133.05 (C-6), 133.21 (C-10), 135.04 (C-7), 153.20 (C-2), 181.71 (C=O),
184.93 (C=O); LC-MS (ESI−, m/z) calculated for C22H30O3: 342.22, found for 341.38 [M − H]−;
HRMS-ESI: m/z calculated for C22H30O3: 342.2195 [M]+, found for 342.2189.
2-Dodectyloxy-1,4-naphthoquinone (8b). The reaction produced 8b in 6.9% as a yellow solid; mp
96.1–96.7 °C. 1H-NMR (500 MHz, CDCl3) δH 0.86 (t, J = 7.0 Hz, 3H, CH3), 1.24–1.33 (m, 16H, H-4′,
H-5′, H-6′, H-7′, H-8′, H-9′, H-10′, H-11′), 1.44 (p, J = 7.5 Hz, 2H, H-3′), 1.87 (p, J = 7.1 Hz, 2H, H-2′),
3.98 (t, J = 6.7 Hz, 2H, H-1′), 6.13 (s, 1H, H-3), 7.68 (t, J = 7.3 Hz, 1H, H-7), 7.72 (t, J = 7.3 Hz, 1H,
13
H-6), 8.05 (d, J = 7.4 Hz, 1H, H-8), 8.10 (d, J = 7.5 Hz, 1H, H-5); C-NMR (125 MHz, CDCl3) δC
14.33 (CH3), 22.90 (C-7′), 26.06 (C-2′), 28.48 (C-3′), 29.46 (C-4′), 29.56 (C-5′), 29.69 (C-6′), 29.77
(C-7′), 29.83 (C-8′), 29.84 (C-9′), 32.13 (C-10′), 69.90 (C-1′), 110.40 (C-3), 126.32 (C-8), 126.89
(C-5), 131.42 (C-9), 132.26 (C-10), 133.46 (C-6), 134.42 (C-7), 160.13 (C-2), 180.40 (C=O), 185.31
(C=O); LC-MS (ESI−, m/z) calculated for C22H30O3: 342.22, found for 341.30 [M − H]−; HRMS-ESI:
m/z calculated for C22H30O3: 342.2195 [M]+, found for 342.2197.
2-Hydroxy-3-isoprenyl-1,4-naphthoquinone (9a). The reaction produced 9a in 58.2% as a yellow solid;
mp 142.5–144.0 °C (lit. [36] 141–143 °C). 1H-NMR (500 MHz, CDCl3) δH 1.66 (s, 3H, CH3), 1.77 (s,
3H, CH3), 3.29 (d, J = 7.4 Hz, 2H, H-1′), 5.17–5.21 (m, 2H, H-2′), 7.27 (s, 1H, 2-OH), 7.66 (dt,
J = 1.1, 7.4 Hz, 1H, H-7), 7.73 (dt, J = 1.4, 7.6 Hz, 1H, H-6), 8.05 (dd, J = 1.0, 7.4 Hz, 1H, H-8), 8.10
(dd, J = 1.0, 7.7 Hz, 1H, H-5); 13C-NMR (125 MHz, CDCl3) δC 18.12 (CH3), 22.84 (CH3), 25.98
(C-1′), 119.85 (C-3), 123.68 (C-2′), 126.28 (C-8), 127.01 (C-5), 129.65 (C-9), 133.09 (C-6), 133.14
(C-10), 134.09 (C-3ʹ), 135.08 (C-7), 152.87 (C-2), 181.93 (C=O), 184.79 (C=O); LC-MS (ESI−, m/z)
calculated for C15H14O3: 242.09, found for 241.16 [M − H]–; HRMS-ESI: m/z calculated for C15H14O3:
242.0943 [M]+, found for 242.0948.
2-Isoprenyloxy-1,4-naphthoquinone (9b). The reaction produced 9b in 4.8% as a yellow solid; mp
158.0–159.6 °C (lit. [37] 150–151 °C). 1H-NMR (500 MHz, CDCl3) δH 1.74 (s, 3H, CH3), 1.79 (s, 3H,
CH3), 4.57 (d, J = 7.8 Hz, 2H, H-1′), 5.46–5.49 (m, 2H, H-2′), 6.14 (s, 1H, H-3), 7.68 (dt, J = 1.5, 7.5 Hz,
1H, H-7), 7.72 (dt, J = 1.5, 7.5 Hz, 1H, H-6), 8.06 (dd, J = 1.5, 7.5 Hz, 1H, H-8), 8.10 (dd, J = 1.5,
7.5 Hz, 1H, H-5); 13C-NMR (125 MHz, CDCl3) δC 18.58 (CH3), 26.05 (CH3), 66.63 (C-1′), 110.73 (C-3),
117.41 (C-2′), 126.33 (C-8), 126.92 (C-5), 131.41 (C-9), 132.26 (C-10), 133.46 (C-6), 134.43 (C-7),
140.73 (C-3′), 159.80 (C-2), 180.51 (C=O), 185.28 (C=O); LC-MS (ESI−, m/z) calculated for C15H14O3:
242.09, found for 241.23 [M − H]−; HRMS-ESI: m/z calculated for C15H14O3: 242.0943 [M]+, found
for 242.0941.
2-Hydroxy-3-geranyl-1,4-naphthoquinone (10a). The reaction produced 10a in 50.8% as a yellow
1
solid; mp 112.0–112.5 °C [38]. H-NMR (500 MHz, CDCl3) δH 1.59 (s, 3H, CH3), 1.65 (s, 3H, CH3),
1.81 (s, 3H, CH3), 1.98–2.01 (m, 2H, H-5′), 2.06–2.09 (m, 2H, H-6′), 3.34 (d, J = 7.5 Hz, 2H, H-1′),
5.06–5.09 (m, 1H, H-7′), 5.23 (dt, J = 1.0, 7.5 Hz, 1H, H-2′), 7.31 (s, 1H, 2-OH), 7.70 (dt, J = 1.0, 7.5 Hz,

Molecules 2015, 20
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1H, H-7), 7.78 (dt, J = 1.0, 7.5 Hz, 1H, H-6), 8.10 (dd, J = 1.5, 7.5 Hz, 1H, H-8), 8.15 (dd, J = 1.0, 7.5 Hz,
1H, H-5); 13C-NMR (125 MHz, CDCl3) δC 16.47 (CH3), 17.88 (CH3), 22.77 (CH3), 25.87 (C-1′), 26.82
(C-6′), 39.96 (C-5′), 119.67 (C-3), 123.76 (C-2′), 124.41 (C-7′), 126.28 (C-8), 127.00 (C-5), 129.65
(C-8′), 131.61 (C-9), 133.08 (C-6), 133.15 (C-10), 135.08 (C-7), 137.61 (C-3′), 152.88 (C-2), 181.94
(C=O), 184.74 (C=O); LC-MS (ESI−, m/z) calculated for C20H22O3: 310.16, found for 309.12 [M − H]−;
HRMS-ESI: m/z calculated for C20H22O3: 310.1569 [M]+, found for 310.1572.
2-Geranyloxy-1,4-naphthoquinone (10b). The reaction produced 10b in 5.2% as a pale yellow liquid.
1H-NMR (500 MHz, CDCl3) δH 1.59 (s, 3H, CH3), 1.65 (s, 3H, CH3), 1.81 (s, 3H, CH3), 1.99–2.02 (m,
2H, H-5′), 2.07–2.10 (m, 2H, H-6′), 4.58 (d, J = 6.6 Hz, 2H, H-1´), 5.06–5.10 (m, 1H, H-7´), 5.46 (dt,
J = 0.8, 6.6 Hz, 1H, H-2′), 6.14 (s, 1H, H-3), 7.68 (dt, J = 1.0, 7.5 Hz, 1H, H-7), 7.74 (dt, J = 1.0,
13
7.5 Hz, 1H, H-6), 8.06 (dd, J = 1.5, 7.5 Hz, 1H, H-8), 8.10 (dt, J = 1.0, 7.5 Hz, 1H, H-5); C-NMR
(125 MHz, CDCl3) δC 16.47 (CH3), 17.68 (CH3), 25.70 (CH3), 26.71 (C-6′), 39.65 (C-5′), 66.51 (C-1′),
110.56 (C-3), 117.22 (C-2′), 124.37 (C-7′), 126.23 (C-8), 126.88 (C-5), 131.18 (C-8′), 132.06 (C-9), 133.18
(C-6), 134.26 (C-10), 135.73 (C-7), 141.56 (C-3′), 159.56 (C-2), 180.29 (C=O), 185.06 (C=O); LC-MS
(ESI−, m/z) calculated for C20H22O3: 310.16, found for 309.06 [M − H]−; HRMS-ESI: m/z calculated
for C20H22O3: 310.1569 [M]+, found for 310.1574.
2-Hydroxy-3-farnesyl-1,4-naphthoquinone (11a). The reaction produced 11a in 35.2% as a yellow
solid; mp 84.4–84.9 °C (lit. [39] 68–69 °C). 1H-NMR (500 MHz, CDCl3) δH 1.54 (s, 3H, CH3), 1.54 (s,
6H, CH3), 1.77 (s, 3H, CH3), 1.92–1.95 (m, 2H, H-5′), 1.97–2.00 (m, 4H, H-9′, H-10′) 2.02–2.07 (m, 2H,
H-6′), 3.30 (d, J = 7.5 Hz, 2H, H-1′), 5.00–5.05 (m, 2H, H-7′, H-12′) 5.20 (t, 1H, J = 7.3 Hz, 1H, H-2′),
7.28 (s, 1H, 2-OH), 7.65 (t, J = 7.5 Hz, 1H, H-7), 7.73 (t, J = 7.5 Hz, 1H, H-6), 8.05 (d, J = 7.5 Hz, 1H,
H-8), 8.11 (d, J = 7.5 Hz, 1H, H-5); 13C-NMR (125 MHz, CDCl3) δC 16.22 (CH3), 16.48 (CH3), 17.86
(CH3), 22.77 (C-1′), 25.90 (CH3), 26.69 (C-6′), 26.94 (C-11′), 39.89 (C-5′), 39.95 (C-9′), 119.69 (C-3),
123.75 (C-2′), 124.24 (C-7′), 124.57 (C-11′), 126.27 (C-8), 127.00 (C-5), 129.66 (C-9), 131.43 (C-10),
133.06 (C-6), 133.14 (C-12′), 135.06 (C-7), 135.24 (C-8′), 137.60 (C-3′), 152.89 (C-2), 181.92 (C=O),
184.74 (C=O); LC-MS (ESI−, m/z) calculated for C25H30O3: 378.22, found for 377.23 [M − H]−;
HRMS-ESI: m/z calculated for C25H30O3: 378.2195 [M]+, found for 378.2193.
2-Farnesyloxy-1,4-naphthoquinone (11b). The reaction produced 11b in 7.4% as a yellow liquid.
1H-NMR (500 MHz, CDCl3) δH 1.56 (s, 3H, CH3), 1.58 (s, 6H, CH3), 1.74 (s, 3H, CH3), 1.92–1.95 (m,
2H, H-5′), 2.00–2.03 (m, 2H, H-6′) 2.08–2.12 (m, 4H, H-9′, H-10′), 4.60 (d, J = 6.6 Hz, 2H, H-1′),
5.04–5.08 (m, 2H, H-7′, H-12′) 5.47 (dt, 1H, J = 0.8, 6.6 Hz, 1H, H-2′), 6.14 (s, 1H, H-3), 7.69 (dt,
J = 1.5, 7.5 Hz, 1H, H-7), 7.72 (dt, J = 1.5, 7.5 Hz, 1H, H-6), 8.06 (dd, J = 1.5, 7.5 Hz, 1H, H-8), 8.10 (dd,
13
J = 1.5, 7.5 Hz, 1H, H-5); C-NMR (125 MHz, CDCl3) δC 16.05 (CH3), 16.88 (CH3), 17.68 (CH3),
25.70 (CH3), 26.09 (C-6′), 26.71 (C-11′), 39.53 (C-5′), 39.67 (C-9′), 66.50 (C-1′), 110.55 (C-3), 116.91
(C-2′), 123.39 (C-7′), 124.28 (C-11′), 126.12 (C-8), 126.70 (C-5), 131.20 (C-9), 131.33 (C-10), 132.04
(C-12′), 133.24 (C-6), 134.21 (C-7), 135.72 (C-8′), 143.63 (C-3′), 159.58 (C-2), 180.31 (C=O), 185.04
(C=O); LC-MS (ESI−, m/z) calculated for C25H30O3: 378.22, found for 377.26 [M − H]−; HRMS-ESI:
m/z calculated for C25H30O3: 378.2195 [M]+, found for 378.2190.

Molecules 2015, 20
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3.3. 5-Hydroxy-1,4-Naphthoquinone (Juglone, 3)
A suspension of 1,5-dihydroxynaphthene (1 g, 6.25 mmol) in THF/H2O (50 mL, 1/1, v/v) was
placed in a 100 mL, three-necked flask fitted with a mechanical stirrer and a gas inlet tube and a strong
current of air was bubbled through it. Then, periodic acid (3.12 g, 13.7 mmol) was added with vigorous
stirring at room temperature in the dark over 1 h, and the resultant mixture was stirred for 4 h. The
mixture was filtered, washed with acetonitrile, and the solvent removed under reduced pressure. The
crude product was purified using silica gel with ethyl acetate/n-hexane (1:5) as the solvent to afford
5-hydroxy-1,4-naphthoquinone (juglone, 3) (735 mg, 4.22 mmol, 67.6%) as a brown-red solid; mp
1
146.0–148.2 °C (lit. [40] 152 °C). H-NMR (500 MHz, CDCl3) δH 6.93 (s, 2H, H-2, H-3), 7.26 (dd,
13
J = 1.5, 7.5 Hz, 1H, H-6), 7.59–7.63 (m, 2H, H-7, H-8), 11.88 (s, 1H, 5-OH); C-NMR (125 MHz,
CDCl3) δC 114.97 (C-10), 119.15 (C-6), 124.49 (C-8), 131.76 (C-9), 136.56 (C-7), 138.65 (C-3),
139.59 (C-2), 161.45 (C-5), 184.25 (C=O), 190.28 (C=O); LC-MS (ESI−, m/z) calculated for C10H6O3:
174.03, found for 173.10 [M − H]−; HRMS-ESI: m/z calculated for C10H6O3: 174.0317 [M]+, found
for 174.0313.
3.4. General Proceed Amination of Juglone with Alkyl Amine
A suspension of juglone (200 mg, 1.15 mmol) and alkyl amine (1.15 mmole) in 50 mL of ethanol
was stirred for 1 h under reflux. The mixture was poured into water, 2 mL of 1 N hydrochloric acid
was added, the precipitate was filtered off, washed with water, dried, and dissolved in chloroform, and
the solution was applied to a column charged with silica gel. The column was eluted with ethyl
acetate/n-hexane (1/5) to give the 2-amination and 3-amination products, respectively.
2-Butylamino-5-hydroxy-1,4-naphthoquinone (13a). The reaction produced 13a in 13.9% as an orange
red solid; mp 154.6–155.4 °C (lit. [41] 156–157 °C). 1H-NMR (500 MHz, CDCl3) δH 0.96 (t, J = 7.4 Hz,
3H, CH3), 1.43 (p, J = 7.5 Hz, 2H, H-2′), 1.67 (p, J = 7.5 Hz, 2H, H-3′), 3.17 (q, J = 6.9 Hz, 2H, H-1′),
5.61 (s, 1H, H-3), 6.04 (s, 1H, -NH), 7.22 (dd, J = 0.8, 8.3 Hz, 1H, H-8), 7.44 (t, J = 8.0 Hz, 1H, H-7),
7.56 (dd, 1H, J = 0.8, 7.6 Hz, H-6), 13.08 (s, 1H, 5-OH); 13C-NMR (125 MHz, CDCl3) δC 13.90 (CH3),
20.40 (C-2′), 30.43 (C-3′), 42.63 (C-1′), 99.81 (C-3), 115.19 (C-9), 119.25 (C-6), 126.18 (C-8), 130.70
(C-10), 134.05 (C-7), 148.90 (C-2), 161.33 (C-5), 181.41 (C=O), 189.14 (C=O); LC-MS (ESI+, m/z)
calculated for C14H15NO3: 245.11, found for 268.24 [M + Na]+; HRMS-ESI: m/z calculated for
C14H16O3N: 246.1125 [M + H]+, found for 246.1120.
3-Butylamino-5-hydroxy-1,4-naphthoquinone (13b). The reaction produced 13b in 6.9% as an orange
red solid; mp 125.3–126.5 °C (lit. [41] 111–112 °C). 1H-NMR (500 MHz, CDCl3) δH 0.96 (t, J = 7.5 Hz,
3H, CH3), 1.42 (h, J = 7.5 Hz, 2H, H-3′), 1.67 (p, J = 7.5 Hz, 2H, H-2′), 3.17 (q, J = 6.9 Hz, 2H, H-1′),
5.70 (s, 1H, H-3), 5.85 (bs, 1H, -NH), 7.12 (dd, J = 2.3, 7.0 Hz, 1H, H-8), 7.57–7.62 (m, 2H, H-6, H-7),
13
11.52 (s, 1H, 5-OH); C-NMR (125 MHz, CDCl3) δC 13.91 (CH3), 20.42 (C-2′), 30.45 (C-3′), 42.56
(C-1′), 101.50 (C-3), 114.31 (C-9), 118.95 (C-6), 122.37 (C-8), 133.82 (C-10), 137.91 (C-7), 147.97
(C-2), 161.89 (C-5), 182.42 (C=O), 186.36 (C=O); LC-MS (ESI+, m/z) calculated for C14H15NO3:
245.11, found for 268.29 [M + Na]+; HRMS-ESI: m/z calculated for C14H16O3N: 246.1125 [M + H]+,
found for 246.1121.

Molecules 2015, 20
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2-Hexylamino-5-hydroxy-1,4-naphthoquinone (14a). The reaction produced 14a in 15.1% as an orange
red solid; mp 126.0–127.5 °C. 1H-NMR (500 MHz, CDCl3) δH 0.88 (t, J = 7.5 Hz, 3H, CH3), 1.30–1.32 (m,
4H, H-4′, H-5′), 1.36–1.40 (m, 2H, H-3′), 1.67 (p, J = 7.3 Hz, 2H, H-2′), 3.16 (q, J = 6.8 Hz, 2H, H-1′),
5.60 (s, 1H, H-3), 6.05 (bs, 1H, -NH), 7.22 (d, J = 8.4 Hz, 1H, H-8), 7.44 (t, J = 7.9 Hz, 1H, H-7), 7.56
(d, J = 7.2 Hz, 1H, H-6), 13.08 (s, 1H, 5-OH); 13C-NMR (125 MHz, CDCl3) δC 14.19 (CH3), 22.72 (C-5′),
26.88 (C-2′), 28.38 (C-3′), 31.59 (C-4′), 42.93 (C-1′), 99.78 (C-3), 115.18 (C-9), 119.24 (C-6), 126.16
(C-8), 130.68 (C-10), 134.03 (C-7), 148.87 (C-2), 161.31 (C-5), 181.40 (C=O), 189.12 (C=O); LC-MS
(ESI+, m/z) calculated for C16H19NO3: 273.14, found for 296.29 [M + Na]+; HRMS-ESI: m/z calculated
for C16H20O3N: 274.1438 [M+H]+, found for 274.1435
3-Hexylaminp-5-hydroxy-1,4-naphthoquinone (14b). The reaction produced 14b in 24.1% as an orange
red solid; mp 110.2–111.1 °C. 1H-NMR (500 MHz, CDCl3) δH 0.88 (t, J = 6.4 Hz, 3H, CH3), 1.31–1.32
(m, 4H, H-4′, H-5′), 1.39 (p, J = 6.9 Hz, 2H, H-3′), 1.69 (p, J = 7.3 Hz, 2H, H-2′), 3.16 (q, J = 6.6 Hz,
2H, H-1′), 5.69 (s, 1H, H-2), 5.87 (bs, 1H, -NH), 7.11 (dd, J = 2.0, 7.2 Hz, 1H, H-6), 7.57–7.61 (m, 2H,
H-7, H-8), 11.52 (s, 1H, 5-OH); 13C-NMR (125 MHz, CDCl3) δC 13.97 (CH3), 22.50 (C-5′), 26.68 (C-2′),
28.17 (C-3′), 31.38 (C-4′), 42.63 (C-1′), 101.24 (C-2), 114.07 (C-9), 118.71 (C-6), 122.14 (C-8),
133.58 (C-10), 137.67 (C-7), 147.73 (C-3), 161.66 (C-5), 182.18 (C=O), 186.11 (C=O); LC-MS (ESI+,
m/z) calculated for C16H19NO3: 273.14, found for 296.26 [M + Na]+; HRMS-ESI: m/z calculated for
C16H20O3N: 274.1438 [M + H]+, found for 274.1433.
2-Octylamino-5-hydroxy-1,4-naphthoquinone (15a). The reaction produced 15a in 14.1% as an orange
red solid; mp 128.2–129.5 °C. 1H-NMR (500 MHz, CDCl3) δH 0.87 (t, J = 6.6 Hz, 3H, CH3), 1.26–1.29 (m,
6H, H-5′, H-6′, H-7′), 1.36–1.39 (m, 4H, H-3′, H-4′), 1.67 (p, J = 7.3 Hz, 2H, H-2′), 3.16 (q, J = 6.6 Hz,
2H, H-1′), 5.60 (s, 1H, H-3), 6.04 (bs, 1H, -NH), 7.22 (d, J = 8.0 Hz, 1H, H-8), 7.44 (t, J = 7.9 Hz, 1H,
H-7), 7.57 (d, J = 7.5 Hz, 1H, H-6), 13.08 (s, 1H, 5-OH); 13C-NMR (125 MHz, CDCl3) δC 14.29
(CH3), 22.83 (C-7′), 27.22 (C-2′), 28.43 (C-3′), 29.35 (C-4′), 29.92 (C-5′), 31.95 (C-6′), 42.94 (C-1′),
99.80 (C-3), 115.20 (C-9), 119.25 (C-6), 126.18 (C-8), 130.70 (C-10), 134.04 (C-7), 148.88 (C-2),
161.33 (C-5), 181.42 (C=O), 189.14 (C=O); LC-MS (ESI+, m/z) calculated for C18H23NO3: 301.17,
found for 324.34 [M + Na]+; HRMS-ESI: m/z calculated for C18H24O3N: 302.1751 [M + H]+, found
for 302.1746.
3-Octylamino-5-hydroxy-1,4-naphthoquinone (15b). The reaction produced 15b in 31.4% as an orange
1
red solid; mp 119.5–120.3 °C. H-NMR (500 MHz, CDCl3) δH 0.87 (t, J = 6.3 Hz, 3H, CH3), 1.23–1.29
(m, 6H, H-5′, H-6′, H-7′), 1.37–1.39 (m, 2H, H-4′), 1.65–1.70 (m, 4H, H-2′, H-3′), 3.16 (q, J = 6.4 Hz,
2H, H-1′), 5.70 (s, 1H, H-2), 5.87 (bs, 1H, -NH), 7.11 (d, J = 7.3, 1H, H-8), 7.57–7.61 (m, 2H, H-6, H-7),
13
11.52 (s, 1H, 5-OH); C-NMR (125 MHz, CDCl3) δC 14.29 (CH3), 22.84 (C-7′), 27.24 (C-2′), 28.45
(C-3′), 29.36 (C-4′), 29.42 (C-5′), 31.96 (C-6′), 42.90 (C-1′), 101.46 (C-2), 114.31 (C-9), 118.98 (C-6),
122.40 (C-8), 133.81 (C-10), 137.93 (C-7), 148.02 (C-3), 161.93 (C-5), 182.40 (C=O), 186.33 (C=O);
LC-MS (ESI+, m/z) calculated for C18H23NO3: 301.17, found for 324.36 [M + Na]+; HRMS-ESI: m/z
calculated for C18H24O3N: 302.1751 [M + H]+, found for 302.1749.
2-Decylamino-5-hydroxy-1,4-naphthoquinone (16a). The reaction produced 16a in 19.2% as an orange
red solid; mp 124.5–125.7 °C. 1H-NMR (500 MHz, CDCl3) δH 0.86 (t, J = 6.8 Hz, 3H, CH3), 1.23–1.30

Molecules 2015, 20
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(m, 8H, H-6′, H-7′, H-8′, H-9′), 1.35–1.40 (m, 6H, H-3′, H-4′, H-5′), 1.67 (p, J = 7.3 Hz, 2H, H-2′),
3.17 (q, J = 6.7 Hz, 2H, H-1′), 5.61 (s, 1H, H-3), 6.05 (bs, 1H, -NH), 7.22 (dd, J = 2.2, 7.3 Hz, 1H, H-8),
13
7.44 (dt, 1H, J = 1.0, 7.5 Hz, H-7), 7.56 (dd, 1H, J = 1.0, 7.5 Hz, H-6), 13.08 (s, 1H, 5-OH); C-NMR
(125 MHz, CDCl3) δC 14.09 (CH3), 22.65 (C-9′), 26.98 (C-2′), 28.19 (C-3′), 29.20 (C-4′), 29.25 (C-5′),
29.46 (C-6′), 29.47 (C-7′), 31.85 (C-8′), 42.71 (C-1′), 99.57 (C-3), 114.97 (C-9), 119.02 (C-6), 125.95
(C-8), 130.47 (C-10), 133.82 (C-7), 148.66 (C-2), 161.10 (C-5), 181.20 (C=O), 188.91 (C=O); LC-MS
(ESI+, m/z) calculated for C20H29NO3: 329.20, found for 352.47 [M + Na]+; HRMS-ESI: m/z calculated
for C20H28O3N: 330.2064 [M + H]+, found for 330.2058.
3-Decylamino-5-hydroxy-1,4-naphthoquinone (16b). The reaction produced 16b in 39.5% as an orange
1
red solid; mp 95.8–96.9 °C. H-NMR (500 MHz, CDCl3) δH 0.86 (t, J = 6.8 Hz, 3H, CH3), 1.23–1.31
(m, 10H, H-5′, H-6′, H-7′, H-8′, H-9′), 1.35–1.40 (m, 4H, H-3′, H-4′,), 1.67 (p, J = 7.3 Hz, 2H, H-2′′),
3.16 (q, J = 6.7 Hz, 2H, H-1′), 5.68 (s, 1H, H-2), 5.86 (bs, 1H, -NH), 7.11 (dd, J = 2.2, 7.3 Hz, 1H, H-8),
7.57–7.61 (m, 2H, H-6, H-7), 11.51 (s, 1H, 5-OH); 13C-NMR (125 MHz, CDCl3) δC 14.32 (CH3),
22.88 (C-9′), 27.24 (C-2′), 28.44 (C-3′), 29.45 (C-4′), 29.48 (C-5′), 29.70 (C-6′), 29.92 (C-7′), 32.08
(C-8′), 42.89 (C-1′), 101.45 (C-2), 114.30 (C-9), 118.97 (C-6), 122.39 (C-8), 133.80 (C-10), 137.91 (C-7),
148.02 (C-3), 161.92 (C-5), 182.38 (C=O), 186.31 (C=O); LC-MS (ESI+, m/z) calculated for
C20H29NO3: 329.20, found for 330.26 [M + H]+; HRMS-ESI: m/z calculated for C20H28O3N: 330.2064
[M + H]+, found for 330.2059.
2-Dodecylamino-5-hydroxy-1,4-naphthoquinone (17a). The reaction produced 17a in 15.5% as an
1
orange red solid; mp 121.8–122.2 °C. H-NMR (500 MHz, CDCl3) δH 0.86 (t, J = 6.8 Hz, 3H, CH3),
1.24–1.31 (m, 14H, H-5′, H-6′, H-7′, H-8′, H-9′, H-10′, H-11′), 1.34–1.40 (m, 4H, H-3′, H-4′), 1.67
(p, J = 7.3 Hz, 2H, H-2′), 3.16 (q, J = 6.6 Hz, 2H, H-1′), 5.60 (s, 1H, H-3), 6.05 (bs, 1H, -NH), 7.22 (d,
J = 8.4 Hz, 1H, H-8), 7.44 (t, 1H, J = 7.9 Hz, H-7), 7.56 (d, 1H, J = 7.5 Hz, H-6), 13.08 (s, 1H, 5-OH);
13C-NMR (125 MHz, CDCl3) δC 14.34 (CH3), 22.90 (C-11′), 27.22 (C-2′), 28.42 (C-3′), 29.44 (C-4′),
29.55 (C-5′), 29.69 (C-6′), 29.75 (C-7′), 29.83 (C-8′), 29.92 (C-9′), 32.12 (C-10′), 42.94 (C-1′), 99.80
(C-3), 115.20 (C-9), 119.25 (C-6), 126.18 (C-8), 130.70 (C-10), 134.04 (C-7), 148.88 (C-2), 161.33 (C-5),
181.42 (C=O), 189.13 (C=O); LC-MS (ESI+, m/z) calculated for C22H31NO3: 357.23, found for 380.44
[M + Na]+; HRMS-ESI: m/z calculated for C22H32O3N: 358.2377 [M + H]+, found for 358.2371.
3-Dodecylamino-5-hydroxy-1,4-naphthoquinone (17b). The reaction produced 17b in 24.6% as an
1
orange red solid; mp 98.0–98.5 °C. H-NMR (500 MHz, CDCl3) δH 0.86 (t, J = 6.7 Hz, 3H, CH3),
1.24–1.31 (m, 12H, H-6′, H-7′, H-8′, H-9′, H-10′, H-11′), 1.35–1.39 (m, 4H, H-4′, H-5′), 1.65–1.70 (m,
4H, H-2′, H-3′), 3.15 (t, J = 6.1 Hz, 2H, H-1′), 5.70 (s, 1H, H-2), 5.87 (bs, 1H, -NH), 7.11 (d, J = 7.4 Hz,
13
1H, H-8), 7.57–7.61 (m, 2H, H-6, H-7), 11.52 (s, 1H, 5-OH); C-NMR (125 MHz, CDCl3) δC 14.34
(CH3), 22.90 (C-11′), 27.24 (C-2′), 28.44 (C-3′), 29.45 (C-4′), 29.55 (C-5), 29.70 (C-6′), 29.76 (C-7′), 29.83
(C-8′), 29.92 (C-9′), 32.13 (C-10′), 42.87 (C-1′), 101.49 (C-2), 114.32 (C-9), 118.95 (C-6), 122.37 (C-8),
133.83 (C-10), 137.91 (C-7), 147.97 (C-3), 161.90 (C-5), 182.42 (C=O), 186.34 (C=O); LC-MS (ESI+,
m/z) calculated for C22H31NO3: 357.23, found for 380.46 [M + Na]+; HRMS-ESI: m/z calculated for
C22H32O3N: 358.2377 [M + H]+, found for 358.2374.

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2-[(2-Aminoethyl)-morpholine]-5-hydroxy-1,4-naphthoquinone (18a). The reaction produced 18a in
38.9% as a red solid; mp 122.0–122.5 °C. 1H-NMR (500 MHz, CDCl3) δH 2.48 (t, J = 4.2 Hz, 4H, H-2′′,
H-6′′), 2.67 (t, J = 6.0 Hz, 2H, H-2′), 3.22 (q, J = 5.7 Hz, 2H, H-1′), 3.72 (t, J = 4.6 Hz, 4H, H-3′′, H-5′′),
5.59 (s, 1H, H-3), 6.66 (bs, 1H, -NH), 7.22 (dd, J = 0.9, 8.4 Hz, 1H, H-8), 7.45 (t, J = 7.9 Hz, 1H,
H-7), 7.57 (dd, J = 0.9, 7.2 Hz, 2H, H-6), 13.05 (s, 1H, 5-OH); 13C-NMR (125 MHz, CDCl3) δC 38.68
(C-1′), 53.39 (C-2′′, C-6′′), 55.68 (C-2′), 67.10 (C-3′′, C-5′′), 100.12 (C-2), 115.18 (C-9), 119.22 (C-6),
126.13 (C-8), 130.76 (C-10), 134.13 (C-7), 148.89 (C-3), 161.32 (C-5), 181.28 (C=O), 189.17 (C=O);
LC-MS (ESI−, m/z) calculated for C16H18N2O4: 302.13, found for 301.08 [M − H]−; HRMS-ESI: m/z
calculated for C16H18N2O4: 302.1267 [M]+, found for 302.1264.
3-[(2-Aminoethyl)-morpholine]-5-hydroxy-1,4-naphthoquinone (18b). The reaction produced 18b in
1
16.8% as a red solid; mp 150.2–151.4 °C. H-NMR (500 MHz, CDCl3) δH 2.49 (t, J = 8.5 Hz, 4H,
H-2′′, H-6′′), 2.69 (t, J = 6.0 Hz, 2H, H-2′), 3.20 (q, J = 5.5 Hz, 2H, H-1′), 3.74 (t, J = 5.0 Hz, 4H, H-3′′,
H-5′′), 5.66 (s, 1H, H-3), 6.49 (bs, 1H, -NH), 7.12 (dd, J = 2.7, 6.8 Hz, 1H, H-8), 7.57–7.61 (m, 2H,
H-6, H-7), 11.56 (s, 1H, 5-OH); 13C-NMR (125 MHz, CDCl3) δC 38.36 (C-1′), 53.16 (C-2′′, C-6′′),
55.49 (C-2′), 66.65 (C-3′′, C-5′′), 99.97 (C-2), 114.94 (C-9), 119.05 (C-6), 125.92 (C-8), 130.55
(C-10), 133.96 (C-7), 148.65 (C-2), 161.11 (C-5), 181.01 (C=O), 188.97 (C=O); LC-MS (ESI−, m/z)
calculated for C16H18N2O4: 302.13, found for 301.16 [M − H]−; HRMS-ESI: m/z calculated for
C16H18N2O4: 302.1267 [M]+, found for 302.1261.
2-[(3-Aminopropyl)-morpholine]-5-hydroxy-1,4-naphthoquinone (19a). The reaction produced 19a in
29.2% as a red solid; mp 84.5–85.6 °C. 1H-NMR (500 MHz, CDCl3) δH 1.84 (p, J = 5.9 Hz, 2H, H-2′),
2.49 (bs, 4H, H-2′′, H-6′′), 2.54 (t, J = 5.8 Hz, 2H, H-3′), 3.27 (q, J = 5.7 Hz, 2H, H-1′), 3.84 (t, J = 4.6 Hz,
4H, H-3′′, H-5′′), 5.55 (s, 1H, H-3), 7.20 (dd, J = 0.8, 8.0 Hz, 1H, H-8), 7.43 (t, J = 7.9 Hz, 1H, H-7),
13
7.56 (dd, J = 0.9, 7.6 Hz, 1H, H-6), 8.08 (bs, 1H, -NH), 13.17 (s, 1H, 5-OH); C-NMR (125 MHz,
CDCl3) δC 23.21 (C-2′), 43.15 (C-1′), 53.83 (C-2′′, C-6′′), 57.91 (C-3′), 66.76 (C-3′′, C-5′′), 99.04 (C-2),
115.12 (C-9), 118.89 (C-6), 125.70 (C-8), 130.69 (C-10), 133.70 (C-7), 149.46 (C-3), 161.06 (C-5),
181.21 (C=O), 188.84 (C=O); LC-MS (ESI−, m/z) calculated for C17H20N2O4: 316.14, found for 315.10
[M − H]−; HRMS-ESI: m/z calculated for C17H20O4N2: 316.1423 [M]+, found for 316.1428.
2-[(3-Aminopropyl)-morpholine]-5-hydroxy-1,4-naphthoquinone (19b). The reaction produced 19b in
30.2% as a red solid; mp 101.6–102.7 °C. 1H-NMR (500 MHz, CDCl3) δH 1.84 (p, J = 6.0 Hz, 2H, H-2′),
2.50 (bs, 4H, H-2′′, H-6′′), 2.55 (t, J = 5.8 Hz, 2H, H-3′), 3.25 (q, J = 5.5 Hz, 2H, H-1′), 3.84 (t, J = 4.6 Hz,
4H, H-3′′, H-5′′), 5.63 (s, 1H, H-3), 7.10 (dd, J = 2.2, 7.3 Hz, 1H, H-8), 7.56–7.60 (m, 2H, H-6, H-7),
7.99 (bs, 1H, -NH), 11.63 (s, 1H, 5-OH); 13C-NMR (125 MHz, CDCl3) δC 23.15 (C-2′), 43.24 (C-1′),
53.83 (C-2′′, C-6′′), 58.05 (C-3′), 66.78 (C-3′′, C-5′′), 100.77 (C-3), 114.22 (C-9), 118.60 (C-6), 122.05
(C-8), 133.76 (C-10), 137.55 (C-7), 148.55 (C-2), 161.69 (C-5), 182.17 (C=O), 186.51 (C=O); LC-MS
(ESI−, m/z) calculated for C17H20N2O4: 316.14, found for 315.11 [M − H]−; HRMS-ESI: m/z calculated
for C17H20O4N2: 316.1423 [M]+, found for 316.1428.

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3.5. Biological Activity
3.5.1. Cell Lines and Cell Culture Conditions
All cells were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan).
Human colorectal adenocarcinoma cells (HT-29) and human promyelocytic leukemia cells (HL-60)
cells were cultured in RPMI 1640 (HyClone, Logan, USA) medium, supplemented with 10% fetal bovine
serum (FBS, Gibco, Life Technologies, New York, NY, USA) and antibiotics (100 μg/mL streptomycin and
100 units/mL penicillin, Gibco, Life Technologies). Human colorectal adenocarcinoma cells (SW480),
human breast adenocarcinoma cells (MCF-7), human hepatocellular carcinoma cells (HepG2), and
normal murine embryonic liver cells (BNL CL.2) were cultured in Dulbecco’s modified Eagle’s medium
(DMEM, HyClone, Logan, USA), supplemented with 10% fetal bovine serum (FBS) and antibiotics.
Human cancer cells were maintained in humidified atmosphere with 5% CO2 and 95% air at 37 °C in a
carbon dioxide incubator (SANYO, CO2 incubator, Osaka, Japan).
3.5.2. Cell Viability Assay
The cytotoxic effect of 34 1,4-naphthoquinone derivatives on five human cancer cell lines was measured
by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Bionovas Biotechnology
Co., Ltd., Toronto, ON, Canada) as described by Mosmann [26]. The MTT assay depends on the
mitochondrial enzyme reduction of tetrazolium dye to detect and determine cell viability. Briefly, the
cancer cells or normal cells were plated at 5 × 103–1 × 104 cells into 96-well plates and HL-60 cells were
plated at 1 × 105 cells/mL into 12-well plates. After overnight growth, cells were treated with various
concentrations of 1,4-naphthoquinone derivatives for 24 h, 48 h and 72 h. At the end of treatment, final
concentration of 0.5 mg/mL MTT was added, and the cells were incubated for a further 1.5 h. The
absorbance was recorded on an ELISA plate reader (SpectraMax 340PC384, Molecular Devices, Sunnyvale,
CA, USA) at 540 nm. Cell viability was determined as the test compound concentration required to
inhibit tumor cell proliferation by 50% (IC50) from the dose-response curves. All data are reported as
average values from triplicate samples and the experiments were repeated at least three times.
3.5.3. Nuclear Staining with Hoechst 33258
HT-29 cells were plate at 7 × 105 cells per 6-cm dish. After overnight, cells were treated with
0.5–2.5 μM plumbagin (1) and 11a for 48 h. The cells of each dish were stained with 5 mM Hoechst 33258
(Sigma-Aldrich, St. Louis, MO, USA). After the cells were incubated in a dark room for 5 min, the cells
were washed with PBS and then were observed using a fluorescent microscope (Zeiss, Axio Observer A1,
Göttingen, Germany).
3.4.4. Cell Cycle Distribution Analysis
HT-29 cells were plated at a 1 × 106 cells per 6-cm dish and cultured overnight. Then various
concentration of 0.5–2.5 μM plumbagin (1) and 11a were added for 48 h. The cells of each dish were
harvested, then washed once with PBS and fixed with methanol at 4 °C. After 18 h, the cells of each
dish were harvested, washed once with PBS. The cells were resuspended in 473 μL of PBS containing

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40 μg/mL propidium iodide (PI) and 40 μg/mL RNase. Cells were incubated in a dark room for 30 min
at room temperature then subjected to cell cycle analysis using a FACScan flow cytometer (Becton
Dickinson, San Jose, CA, USA) and analyzed using the ModFit 3.0 software (Verity Software House,
Topsham, ME, USA, 2008).
3.5.5. Assessment of Apoptotic Analysis
HT-29 cells were plated at a concentration of 1 × 106 cells per 6-cm dish. After overnight growth, cells
were treated with 0.5–2.5 μM plumbagin (1) and 11a for 48 h. The cells of each dish were harvested,
then washed once with PBS. The cells were resuspended in 500 μL of PBS containing 4 μg/mL PI and
4 μg/mL Annexin-V-FITC (apoptosis detection kit; R & D Systems, Taipei, Taiwan). Cells were incubated
in a dark room for 30 min at room temperature then subjected to cell cycle analysis using a FACScan
flow cytometer (Becton Dickinson, San Jose, CA, USA). Annexin V-FITC and PI emission were detected
in the FL1 and FL2 channels of the flow cytometer, respectively. Flow cytometer data showed three distinct
populations of HT-29 cells. The normal healthy cells, early apoptotic, late apoptotic, and necrosis
cells were represented by the Annexin V-negative/PI-negative population, Annexin V-positive/PI-negative,
Annexin V-positive/PI-positive, and Annexin V-negative/PI-positive cells, respectively. The data were
analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA) requipped with
ModFit 3.0 software (Verity Software House, Topsham, ME, USA).
3.5.6. Statistical Analysis
The values shown are the mean ± SD of three independent experiments. Data are statistically
evaluated by the Student’s test from SigmaPlot 11.0 (Systat Software Inc., San Jose, CA, USA) and
shown as significantly different when * p < 0.05, ** p < 0.01 and *** p < 0.001.
4. Conclusions
In summary, we have synthesized a series of 2-position and 3-position lipophilic substituted
lawsone and juglone derivatives and shown their enhanced their anti-cancer activity. Among these
compounds, a lawsone derivative 11a showed a superior cytotoxicity to all others. Furthermore, 11a
has better cytotoxicity against human colorectal adenocarcinoma HT-29 cells, over other cell lines,
with an IC50 value of 1.99  0.04 μM for 48 h. Cell cycle distribution and apoptotic analysis by flow
cytometry indicated that 11a was arrested cell cycle in S phase and induced apoptosis in HT-29. The
Hoechst 33258 staining study showed the nuclear condensation of apoptotic with 11a treatment.
Furthermore, compound 11a has markedly induced apoptotic morphological changes and nuclear
DNA fragmentation. The mechanism of cell growth inhibition of the compound remains the target of
future investigations.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/07/11994/s1.

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Acknowledgments
Gratitude is extended to Christopher Liu for his valuable editorial revision. This study is supported by
grants V101E2-001 and V103E2-002 from the Taipei Veterans General Hospital, Taiwan.
Author Contributions
Shao-Hung Wang suggested the research work and discussed the experimental data; Chih-Yu Lo
determined and analyzed the LC-MS data of all compounds; Zhong-Heng Gwo and Hong-Jhih Lin
finalized the experimental work, interpreted the results, and prepared tables and figures; Lih-Geeng Chen
designed the research and discussed the results; Jin-Yi Wu and Cheng-Deng Kuo wrote the paper,
edited and revised manuscript. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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