Synthesis of ergosterol peroxide conjugates as mitochondria targeting probes for enhanced anticancer activity

Article abstract of DOI:10.3390/molecules24183307

Inspired by the significant bioactivity of ergosterol peroxide, we designed and synthesized four fluorescent coumarin and ergosterol peroxide conjugates 8a-d through the combination of ergosterol peroxide with 7-N,N-diethylamino coumarins fluorophore. The cytotoxicity of synthesized conjugates against three human cancer cells (HepG2, SK-Hep1, and MCF-7) was evaluated. The results of fluorescent imaging showed that the synthesized conjugates 8a-d localized and enriched mainly in mitochondria, leading to significantly enhanced cytotoxicity over ergosterol peroxide. Furthermore, the results of biological functions of 8d showed that it could suppress cell colony formation, invasion, and migration; induce G2/M phase arrest of HepG2 cells, and increase the intracellular ROS level.

Full text of DOI:10.3390/molecules24183307

molecules
Article
Synthesis of Ergosterol Peroxide Conjugates as
Mitochondria Targeting Probes for Enhanced
Anticancer Activity
Ming Bu *, Hongling Li , Haijun Wang , Jing Wang , Yu Lin and Yukun Ma 2
1
,
1
1
1
1
1
College of Pharmacy, Qiqihar Medical University, Qiqihar 161006, China; LHL28@qmu.edu.cn (H.L.);
wanghaijun@qmu.edu.cn (H.W.); qyyx305@qmu.edu.cn (J.W.); linyu7373@163.com (Y.L.)
Research Institute of Medicine & Pharmacy, Qiqihar Medical University, Qiqihar 161006, China;
2
mayukun@qmu.edu.cn
*
Correspondence: buming@qmu.edu.cn; Tel.: +86-0452-2663-881
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 12 August 2019; Accepted: 10 September 2019; Published: 11 September 2019
Abstract: Inspired by the significant bioactivity of ergosterol peroxide, we designed and synthesized
four fluorescent coumarin and ergosterol peroxide conjugates 8a through the combination of
–
d
ergosterol peroxide with 7-N,N-diethylamino coumarins fluorophore. The cytotoxicity of synthesized
conjugates against three human cancer cells (HepG2, SK-Hep1, and MCF-7) was evaluated. The
results of fluorescent imaging showed that the synthesized conjugates 8a–d localized and enriched
mainly in mitochondria, leading to significantly enhanced cytotoxicity over ergosterol peroxide.
Furthermore, the results of biological functions of 8d showed that it could suppress cell colony
formation, invasion, and migration; induce G2/M phase arrest of HepG2 cells, and increase the
intracellular ROS level.
Keywords: ergosterol peroxide; target probe; mitochondria; fluorescence imaging; antitumor activity
1
. Introduction
Sterols are the important active ingredients of plants or fungal secondary metabolites. Steroidal
compounds have drawn attention not only due to unusual and interesting chemical structures but also
due to their widespread application as anti-inflammatory, diuretic, anabolic, and anticancer agents [ ].
Sterol 5 ,8 -endoperoxides are a kind of important active substances in drug discovery, which all
have a unique 5 ,8 -peroxy moiety (peroxidic bond or peroxide bridge) in the parent structure [ 10].
Ergosterol peroxide (5 ,8 -epidioxiergosta-6,22-dien-3 -ol, EP, ) is a member of a class of fungal
secondary metabolites of sterol endoperoxide derivatives and has been constantly discovered and
extracted from different sources of medicinal fungi (Figure 1A) [11 14]. It has been reported that
1–4
α
α
α
α
5–
α
α
β
1
–
ergosterol peroxide can inhibit tumor cells growth by anti-angiogenesis or cytotoxicity [15–19].
In our previous study, we obtained pure ergosterol peroxide by chemical extraction and separation
from Ganoderma lucidum. As the isolated amount of ergosterol peroxide from natural sources is not
sufficient for an in-depth study on it, we got an effective chemical method to synthesize ergosterol
peroxide (Figure 1). The synthesis of ergosterol peroxide was achieved by the treatment of natural
ergosterol with oxygen in the presence of visible light and photosensitive catalysis [20–22]. Also, we
have proved that ergosterol peroxide could inhibit forkhead-box O3 transcription factor (Foxo3a)
functions by the inhibition of phosphorylate protein kinase (pAKT) to induce tumor cell death [23].
Molecules 2019, 24, 3307; doi:10.3390/molecules24183307
www.mdpi.com/journal/molecules

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A
O
O
HO
Ergosterol peroxide (EP, 1)
Ganoderma lucidum
HO
Ergosterol
B
Reactive
functional group
Targeting mitochondria
O
O
Homolytic
O-O
cleavage
N
O
O
Enhance
cytotoxicity
ROS
8
Localize at mitochondria
Coumarin-1 Conjugates
Figure 1. The chemical structures of compounds and design strategy. (
source of ergosterol peroxide ( ). ( ) The chemical structure of designed coumarin-
the proposed mechanism of 8 for intracellular tracking and cancer-cell therapy.
A
) The chemical structure and
1
B
1
conjugates , and
8
Natural ergosterol, which is lacking a peroxidic bond (O-O), has been proved with no significant
activity against most of the cancer cells [24]. Hence, it is generally accepted that the peroxide bridge is
a crucial functional group to the biological activities [25,26]. The hemolytic cleavage of the peroxide
bridge in a reducing environment induces reactive oxygen species (ROS), which could be cytotoxic to
cancer cells [27–30].
Selective delivery of drug probes to subcellular organelles in tumor cells has emerged as an effective
and attractive strategy for cancer therapy. It exerts synchronous tumor targeting and molecular imaging
of drug delivery. Therefore, various theranostic probes that target different subcellular organelles
in cancer cells, including the endoplasmic reticulum, lysosomes, and mitochondria, have been
continuously reported in recent years [31–33]. Among them, mitochondria represent the most attractive
target for effective theranostic probe development due to their key role in controlling many essential
functions in cancer cells. The specific motivation of inducing mitochondrial dysfunctions has been
acknowledged as an effective method in cancer therapy [34–37].
Herein, we designed four fluorescent coumarin and ergosterol peroxide conjugates 8a–d through
the combination of ergosterol peroxide with different targeting coumarin-3-carboxamide analogs. We
postulated that the mitochondria-targeting coumarin analogs could efficiently take ergosterol peroxide
selective accumulated in mitochondria, in which subsequent production of ROS from the hemolytic
cleavage of the endoperoxide bond in ergosterol peroxide would induce mitochondrial dysfunction
and kill tumor cells (Figure 1B).
2
. Results and Discussion
2
.1. Chemistry
First, using ergosterol (EG) as material, we achieved a chemical synthesis of ergosterol peroxide
by the treatment of ergosterol in the presence of eosin Y, oxygen, and visible light (Scheme 1) [22].

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Scheme 1. Synthesis of coumarin analogs 4a–c and 6.
There is a hydroxyl group at the C-3 position of ergosterol peroxide that is also a feasible functional
site. We designed and synthesized four ergosterol peroxide-coumarin conjugates 8a to evaluate the
–d
biological activities of various groups of linker at the C-3 position. The reason for the use of amino
acids as spacers is that the amide bond is fairly stable, and the synthesis of compounds in which the
amide bond is formed by coupling reagents is simple. Thus, three different amino acids (glycine,
β-alanine, or γ-aminobutyric acid) were selected as the linker, to increase the space distance between
ergosterol peroxide and coumarin fluorophore.
The synthetic routes of different coumarin fluorophore analogs are presented in Scheme 2.
2
.7-(N,N-Diethylamino)coumarin-3-carboxylic acid (
procedures [38]. Three different amino acid esters were introduced to the carboxyl group of compound
for amide bond formation by the same acylation reaction to give compounds 3a . Then, the ester
protecting a group of compounds 3a could be hydrolyzed with an aqueous solution of hydrochloric
acid to give three coumarin fluorophore carboxylic acid analogs 4a . Besides, as a comparison, we
also synthesized an ergosterol peroxide and fluorophore conjugate 6 with piperazine as the linker.
2) was generated according to known
2
–c
–c
–
c
Scheme 2. Synthesis of coumarin and ergosterol peroxide conjugates 8a–d.

Molecules 2019, 24, 3307
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Finally, conjugates 8a
group of 4a
ergosterol peroxide was reacted with 4-nitrophenyl chloroformate using pyridine as the base in
–
c
were obtained by introducing ergosterol peroxide directly to the carboxylic
–c, using dicyclohexylcarbodi imide (DCC) as the coupling reagent (Scheme 2). Also,
dichloromethane to get intermediate 7. Then, 7 reacted with intermediate 6 to obtain the desired
conjugate 8d.
2
.2. Optical Properties and Subcellular Localization
First, the optical properties of four ergosterol peroxide conjugates 8a
–d were investigated (Table 1).
In general, all four conjugates possess typical optical properties [39]. Especially, probe 8d with a
maximum excitation wavelength (λex) of about 469.5 nm and an emission wavelength (λem) of about
4
04 nm. The large Stokes shift (65.5 nm) of probe 8d ensured ideal photophysical properties for living
cells fluorescence imaging studies. Thus, probe 8d was chosen for further subcellular localization study.
Table 1. Photophysical data for probes 8a–d and 2 [a]
.
Entry
Probe
λex
λem
[c]
Stokes Shift
(nm)
nm) [
b]
(nm)
422.5
(
1
2
3
4
5
8a
8b
8c
8d
2
479
475
476
469.5
470.5
56.5
53
54.5
65.5
64.5
422
421.5
404
406
[a]
Determined in DMSO (1 µM in DMSO). [b] Excitation maxima. [c] Emission maxima.
The subcellular localization of probe 8d in living HepG2 cells was carried with the
green mitochondria-specific dye Rhodamine 123 (Rh123) as a comparison (λex = 488 nm,
λem = 515–530 nm) [40]. The intracellular microscopic fluoroscope imaging was captured by Carl Zeiss
710M confocal laser scanning microscopy. As shown in Figure 2A,B, HepG2 cells were universality
stained by 8d (5 M and 10 M) after 2 h of incubation. Bright blue fluorescence was captured
µ
µ
successfully in HepG2 cells (λex = 430–500 nm), which demonstrated both satisfactory permeability
into the cell membrane and high cellular uptake of 8d. What’s more, the extensive merged blue-green
color fluorescence images illustrated that probe 8d could strongly co-localize with specific dye Rh123
in mitochondria. Also, as shown in Figure 2C,D, MCF-7 cells were successfully stained by 8c or 8d
(
10
µM). The results suggested that the fluorescent coumarin-3-carboxamide could successfully deliver
ergosterol peroxide to mitochondria.
Figure 2. Cont.

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Figure 2. Confocal laser scanning microscopic images of cancer cells treated with 8c or 8d in the
presence of Rh123 (0.5 M, 20 min) in PBS buffer. ( ) Fluorescence images of HepG2 cells stained with
(5 M). ( ) Fluorescence images of HepG2 cells stained with 8d (10 M). ( ) Fluorescence images of
MCF-7 cells stained with 8d (10 M). ( ) Fluorescence images of MCF-7 cells stained with 8c (10 M).
) Cell images in bright-field. ( ) Fluorescence images of cells stained with 8c or 8d. ( ) Fluorescence
). All images were acquired under
µ
A
8
d
µ
B
µ
C
µ
D
µ
(
a
b
c
images of cells stained with Rh123. (d) Merged images of (b) and (c
a 20× objective lens.
2
.3. Biological Evaluation
2
.3.1. Cytotoxic Activity
The newly synthesized conjugates 8a
cytotoxicities against human hepatic carcinoma cells (HepG2, Sk-Hep2) and human breast cancer cells
MCF-7) by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cisplatin
was chosen as a positive control drug. The results in Table 2 show that all conjugates have ideal
cytotoxicity to these three tumor cell lines, with half maximal inhibitory concentration (IC ) values at
–d and ergosterol peroxide were evaluated for their
(
50
the sub-micromolar levels. Among them, probes 8c and 8d were the most potent to the tested cancer
cells. Probe 8c with a longer linker ( -aminobutyric acid) had around twofold improved potency
γ
against three tested cancer cells relative to that of 8a. Besides, probe 8d with a special pyridine moiety
as linker exhibited significant cytotoxicity against human liver cancer cells (HepG2, SK-Hep1), with
IC50 values of 6.60
weaker cytotoxicities were measured for either
were associated with the synergistic effect of these two components. Overall, three probes 8b
and 8d exhibited significant cytotoxicities against HepG2 cell lines, with IC50 values lower than 10
µ
M for HepG2, which is nearly equivalent to 8c for Sk-Hep1. Moreover, much
or , which suggested that the cytotoxicities of probes
8c,
M.
1
2
8
a
–
d
,
µ
To understand the mechanism of cancer cells death caused by treatment with coumarin-1 conjugates,
we chose probe 8d for the series of biological functions experiments.
Table 2. Cytotoxicities of compounds against three cancer cell lines.
IC50 (µM) [a]
Comp.
HepG2
SK-Hep1
MCF-7
8
a
12.34 ± 0.49
9.46 ± 0.35
6.74 ± 0.32
6.60 ± 0.16
23.15 ± 0.18
>100
10.43 ± 0.88
11.70 ± 0.28
5.92 ± 0.53
8.10 ± 0.28
19.25 ± 0.60
>100
14.80 ± 0.28
13.70 ± 0.28
7.45 ± 0.50
10.62 ± 0.10
26.06 ± 0.46
>100
8
b
8
c
8
d
EP(1)
2
Cisplatin
0.65 ± 0.05
2.42 ± 0.06
6.64 ± 0.16
[a]
Data represent the mean ± SD values of three independent determinations.

Molecules 2019, 24, 3307
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2
.3.2. Effect of Probe 8d on the Cell Cycle Distribution
The effect of probe 8d on tumor cell cycle distribution was carried by flow cytometry. We treated
HepG2 cells with probe 8d (3 M) for 24 h. The low concentration of 8d was used to avoid induction of
HepG2 cell death. As shown in Figure 3A, in HepG2 cells, the cell phase distribution was 49.30 1.03%
vs. 53.12 1.12% in G0/G1 phase, 25.09 1.18% vs. 33.49 1.05% in S phase, and 22.89 1.17% vs.
2.31 2.05% in G2/M phase before and after the effect of 8d. The G0/G1 and S phases were increased
µ
±
±
±
±
±
1
±
significantly before and after the effect of 8d, suggesting a chemical effect of 8d on cell cycle progression.
Also, the number of cells decreased in G2/M phases (Figure 3).
Figure 3. The effect of probe 8d on the HepG2 cell cycle. (
with or without 8d (3 M). ( ) Statistical analysis of cells distributed in each phase of cell cycles.
p < 0.05, ** p < 0.01.
A) Typical distribution of cell cycles treated
µ
B
*
2
.3.3. Effect of Probe 8d on the Cell Colony, Migration, and Invasion
The clonogenic assay is an effective method to evaluate the neoplastic transformation indirectly.
Hence, we tested the effect of probe 8d on HepG2 cells colony formation. The cells were treated with
M probe 8d for 20 days. The colony formation was observed under light microscopy. The colony
numbers were 61.3 3.1 vs. 9.1 1.4 in HepG2 cells before and after the effect of 8d (Figure 4). The
results suggested that probe 8d had a significant inhibitory activity to HepG2 cells growth.
7
µ
±
±
Figure 4. Compound 8d inhibited cell colony formation. (
colonies after treatment with 8d (7 M). The plates were stained with Coomassie Blue. (
presented as the mean ± SD of three experiments. ** p < 0.01.
A
) Representative images of HepG2 cells
µ
B
) Data are
To determine if 8d can prevent cancer cell invasion and migration, transwell assays were performed.
Under the transwell assay, the number of migratory cells was 120.4 4.2 vs. 63.2 3.5 for HepG2 cells
before and after the effect of 8d (6 M). The HepG2 cell number was reduced significantly after the
effect of 8d. The results suggested that 8d suppressed HepG2 cancer cells migration (Figure 5A,B). The
number of invasive cells was 107.1 5.2 vs. 52.7 4.6 for HepG2 cells before and after the effect of 8d
M). The HepG2 cell number was reduced significantly after the effect of 8 . The results suggested
that 8d suppressed HepG2 cancer cell migration and invasion (Figure 5C,D).
±
±
µ
±
±
(
6
µ
d

Molecules 2019, 24, 3307
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Figure 5. Compound 8d suppressed HepG2 cell invasion and migration (Transwell). (
in HepG2 cells before and after the effect of 8d (6 M). ( ) The migratory cell number is presented
by bar diagram. ( ) Cell invasion in HepG2 cells before and after the effect of 8d (6 M). ( ) The
A) Cell migration
µ
B
C
µ
D
number of cells invading to the lower chamber is presented by bar diagram. Data are presented as the
mean ± SD of three experiments ** p < 0.01. All images were acquired under a 10× objective lens.
2
.3.4. Effect of 8d on the Level of ROS
To comprehend the mechanism of tumor cells death caused by treatment with probe 8d
,
the level of intracellular ROS in HepG2 cells was monitored by using 5-(and-6)-chloromethyl-
’,7’-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) as an indicator and detected
2
by flow cytometry [41]. Figure 6 shows the effect of probe 8d on ROS generation in HepG2 cells. A
concentration-dependent increase in the ROS level was observed for HepG2 cell lines after incubation
with probe 8d (5 and 20
µM) for 24 h. The relative ROS level in HepG2 cancer cells, resulting from
incubation with 20 M probe 8d, was about twofold higher than that of control cells without 8d. This
µ
suggested that the intracellularly generated ROS was responsible for cell death.
Figure 6. The intracellular ROS (reactive oxygen species) level of HepG2 cells was increased by treating
with different concentrations of probe 8d. Data are presented as the mean
p < 0.05.
±
SD of three experiments.
*
3
. Materials and Methods
3
.1. Chemistry
Reagents were commercially available and used without purification. The H- and 13C-NMR
1
spectra were recorded using the Bruker Avance DRX400 spectrometer (400 MHz and 100 MHz) in
CDCl or DMSO-d . Tetramethylsilane (TMS) was used as internal standards. Melting points were
3
6

Molecules 2019, 24, 3307
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measured by an MP120 point apparatus. Mass spectra were measured in electrospray (ESI) mode on a
mass spectrometer (Esquire 6000). Flash chromatography was performed using 400 mesh silica gel.
1
13
H- and C-NMR spectra of new compounds are available online (See Supplementary Materials).
3
.1.1. Synthesis of Ergosterol Peroxide (1)
Ergosterol (150 mg), eosin (1 mg), and pyridine (20 mL) were added into a quartz tube. The
mixture was kept in a water-cooled bath and vigorously stirred by the gas bubbling (O ), radiating
2
with an iodine tungsten lamp (500 W, 220 V) for 0.5 h. After the reaction, the mixture was poured into
ice-water (20 mL) and then extracted with 50 mL ethyl acetate twice. The ethyl acetate phase was
washed with 50 mL saturated brine twice and then dried with anhydrous Na SO . The crude product
2
4
was purified by chromatographic column (ethyl acetate/petroleumether = 1/5) to get pure ergosterol
peroxide as white solid [22].
3
.1.2. Synthesis of 7-(Diethylamino)-2-Oxo-2H-Chromene-3-Carboxylic Acid (2)
,2-Dimethyl-1,3-dioxane-4,6-dione (1.5 g, 10.36 mmol), 4-(diethylamino)-2-hydroxy-
2
benzaldehyde (2 g, 10.36 mmol), piperidine (0.08 g, 1.04 mmol), and acetic acid (two drops) in
ethanol (20 mL) was stirred for 0.5 h at room temperature, and then the mixture was heated to reflux
for 4 h. After the reaction, the mixture was poured into 50 mL of ice water. The orange precipitate was
collected and washed with ethanol to give 2 the crystalline solid form (63%) [38].
3
.1.3. Synthesis of Amino-Acid Ester Derivatives
◦
Under N , dry methanol or ethanol (100 mL) was cooled down to
−
5 C, and SOCl (21 mL,
2
2
0
.3 mmol) was added dropwise. Glycine, β-alanine, or γ-aminobutyric acid (0.1 mmol) was added to
this solution, and stirring was continued at room temperature for 3 h. The solvent was removed under
reduced pressure to obtain the correspondent pure product as colorless crystals.
3
.1.4. Synthesis of 2-(7-(Diethylamino)-2-Oxo-2H-Chromene-3-Carboxamido Ester Derivatives 3a–c
To a stirred solution of (2.42 g, 9.27 mmol) in dry dichloromethane (DCM) (160 mL),
2
N-methylmorpholine (4.08 mL, 37.1 mmol) and 2-(1H-Benzotriazole-1-yl)-1,1,3,3- tetramethyluronium
tetrafluoroborate (TBTU) (4.46 g, 13.9 mmol) were added. After stirring for 20 min, methyl
2
-aminoacetate chloride salt (ethyl 3-aminopropanoate or methyl 4-aminobutanoate) (13.9 mmol)
was added. The reaction was continued at room temperature for approximately 1.5 h under argon
atmosphere. The reaction was followed on TLC chromatography plates using CH Cl /CH OH (5/1) as
2
2
3
the eluent. The reaction mixture was washed with a saturated water solution of NaHSO (8
×
40 mL)
4
and then dried with anhydrous Na SO . The crude product was purified by chromatographic column
2
4
(10% MeOH in DCM) to give the desired products.
Methyl 2-(7-(Diethylamino)-2-Oxo-2H-Chromene-3-Carboxamido)Acetate Ester (3a): Yellow crystals (62%):
◦
1
mp 176–178 C. H-NMR (CDCl , 400 MHz)
δ
(ppm): 9.20 (1H, t, J = 5.60 Hz, -CON
H
-CH -), 8.70
3
2
4
5
6
(
1H, s, H -Ar), 7.40 (1H, d, J = 8.40 Hz, H -Ar), 6.65 (1H, dd, J = 2.60, 8.20 Hz, H -Ar), 6.50 (1H, d,
J = 3.60 Hz, H -Ar), 4.24 (2H, d, J = 6.20 Hz, -CONH-CH2-), 3.78 (3H, s, -COO-CH3), 3.44 (4H, q,
J = 7.10 Hz, 2
ppm): 170.54, 163.91, 162.93, 158.14, 153.09, 148.70, 131.59, 110.40, 110.04, 108.67, 96.97, 52.621, 45.48,
1.93, 12.80. MS (ESI) m/z: [M + H] 333.1.
8
13
×
-CH2CH ), 1.25 (6H, t, J = 7.10 Hz, 2
×
-CH CH3). C-NMR (CDCl , 100 MHz) δ
2 3
3
(
+
4
Ethyl 3-(7-(Diethylamino)-2-Oxo-2H-Chromene-3-Carboxamido)Propanoate (3b): Yellow crystals (65%): mp
◦
1
1
8
(
29–133 C. H-NMR (CDCl , 400 MHz)
δ
(ppm): 9.09 (1H, t, J = 7.20 Hz, -CON
H
-CH CH -COO-),
3
2
2
4
5
6
.71 (1H, s, H -Ar), 7.44 (1H, d, J = 8.90 Hz, H -Ar), 6.64 (1H, dd, J = 4.20, 7.70 Hz, H -Ar), 6.51
1H, d, J = 3.60 Hz, H -Ar), 4.20 (2H, m, -COO-CH2CH ), 3.72–3.77 (2H, m, -CONH-CH2CH -COO-),
3 2
8
3
.48 (4H, q, J = 7.20 Hz, 2
×
-CH2CH ), 2.67 (2H, m, -CH CH2-COO-), 1.24–1.33 (9H, m, 2
×
3
2
13
-
CH CH3, -COO-CH CH3). C-NMR (CDCl , 100 MHz)
δ
(ppm): 172.16, 163.48, 162.83, 157.95, 152.88,
2
2
3

Molecules 2019, 24, 3307
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1
48.28, 131.41, 110.46, 110.27, 108.60, 96.83, 60.97, 45.40, 35.59, 34.82, 30, 14.54, 12.78. MS (ESI) m/z:
+
[M + H] 361.2.
Methyl 4-(7-(Diethylamino)-2-Oxo-2H-Chromene-3-Carboxamido)Butanoate (3c): Yellow crystals (70%): mp
◦
1
1
50–152 C. H-NMR (CDCl , 400 MHz)
δ
(ppm): 8.82 (1H, t, J = 7.70 Hz, -CON
H
-CH -), 8.70 (1H, s,
3
2
4
5
6
H -Ar), 7.43 (1H, d, J = 8.60 Hz, H -Ar), 6.65 (1H, dd, J = 4.20, 7.60 Hz, H -Ar), 6.49 (1H, d, J = 4.60 Hz,
H -Ar), 3.67 (3H, s, -COO-CH3), 3.42–3.52 (6H, m, 2
2H, t, J = 8.20 Hz, -CONH-CH CH CH2-COO-), 1.91–2.00 (2H, m, -CONH-CH CH2CH -COO-), 1.24
6H, t, J = 7.40 Hz, 2
8
×
-CH2CH , -CONH-CH2CH CH -COO-), 2.42
3
2
2
(
(
1
[
2
2
3
2
2
1
×
-CH CH3). C-NMR (CDCl , 100 MHz)
δ
(ppm): 173.87, 163.61, 163.09, 157.99,
2
3
52.92, 148.41, 131.50, 110.61, 110.34, 108.73, 96.94, 51.97, 45.44, 39.22, 31.91, 25.38, 12.80. MS (ESI) m/z:
+
M + H] 361.2.
3
.1.5. Synthesis of 2-(7-(Diethylamino)-2-Oxo-2H-Chromene-3-Carboxamido)Carboxylic Acids 4a–c
Four molar HCl (60 mL) was added dropwise into a stirred solution of 3a (6 mmol) in 1,4-dioxane
120 mL). Stirring was continued at room temperature for 2 h. The reaction course was followed on
TLC. The pH was adjusted to 3 with NaOH. The mixture was extracted with DCM (3 50 mL) and
then dried with anhydrous Na SO . The crude product was purified by chromatographic column
–c
(
×
2
4
(DCM to 10% MeOH in DCM) to give the desired products 4a–c.
2
2
-(7-(Diethylamino)-2-Oxo-2H-Chromene-3-Carboxamido)Acetic Acid (4a): Yellow crystals (69%): mp
◦
1
83–287 C. H-NMR (CDCl , 400 MHz)
δ
(ppm): 12.74 (1H, s, -COO
H
), 8.98 (1H, t, J = 5.80 Hz,
3
4
5
6
-
CON
H
-CH -), 8.71 (1H, s, H -Ar), 7.73 (1H, d, J = 8.00 Hz, H -Ar), 6.84 (1H, m, H -Ar), 6.67 (1H, d,
2
8
J = 2.30 Hz, H -Ar), 4.07 (2H, d, J = 8.20 Hz, -CONH-CH2-), 3.51 (4H, m, 2
J = 7.20 Hz, 2
×
-CH2CH ), 1.18 (6H, t,
3
13
×
-CH CH3). C-NMR (CDCl , 100 MHz)
δ
(ppm): 172.95, 161.53, 161.41, 157.20, 152.33,
2
3
+
1
47.53, 131.48, 109.99, 109.45, 107.61, 95.84, 44.31, 43.53, 12.29. MS (ESI) m/z: [M + H] 317.1.
3
2
-(7-(Diethylamino)-2-Oxo-2H-Chromene-3-Carboxamido)Propanoic Acid (4b): Yellow crystals (71%): mp
◦
1
02–205 C. H-NMR (DMSO-d , 400 MHz)
δ
(ppm): 12.30 (1H, s, -COO
H
), 8.82 (1H, d, J = 8.20 Hz,
6
4
5
6
-CON
H
-CH -), 8.65 (1H, s, H -Ar), 7.65–7.71 (1H, m, H -Ar), 6.65-6.83 (1H, m, H -Ar), 6.60 (1H, s,
2
8
H -Ar), 3.41–3.61 (6H, m, 2
×
-CH2CH , -CONH-CH2-), 2.45 (2H, m, -CONH-CH CH2-COOH), 1.14
3
2
(6H, t, J = 7.80 Hz, 2
×
-CH CH3). 13C-NMR (DMSO-d , 100 MHz)
δ
(ppm): 174.02, 163.02, 162.51,
2
6
158.06, 153.26, 148.58, 132.40, 110.92, 110.08, 108.52, 96.69, 45.21, 35.76, 34.80, 13.16. MS (ESI) m/z:
+
[M + H] 333.1.
4
-(7-(Diethylamino)-2-Oxo-2H-Chromene-3-Carboxamido)Butanoic Acid (4c): Yellow crystals (70%): mp
◦
1
198–200 C. H-NMR (DMSO-d , 400 MHz)
δ
(ppm): 12.06 (1H, s, -COO
H
), 8.62–8.66 (2H, m,
6
4
5
6
-
CON
H
-CH -, H -Ar), 7.67 (1H, d, J = 8.00 Hz, H -Ar), 6.80 (1H, dd, J = 3.40, 7.20 Hz, H -Ar), 6.61 (1H,
2
8
d, J = 4.60 Hz, H -Ar), 3.48 (4H, m, 2
d, J = 7.80 Hz, -CONH-CH CH CH2-COOH), 1.69–1.79 (2H, m, -CONH-CH CH2CH -COOH), 1.14
×
-CH2CH ), 3.27–3.33 (2H, m, -CONH-CH2CH CH -), 2.26 (2H,
3 2 2
2
2
2
2
13
(6H, d, J = 7.40 Hz, 2
×
-CH CH3). C-NMR (DMSO-d , 100 MHz) δ (ppm): 175.02, 163.11, 162.59,
2 6
158.04, 153.24, 148.45, 132.36, 110.93, 110.40, 108.53, 96.72, 45.20, 32.11, 25.65, 13.17. MS (ESI) m/z:
+
[M − H] 345.1.
3
.1.6. Synthesis of Intermediate 6
To a DCM solution (20 mL) of coumarin acid 2 (992.8 mg, 3.8 mmol) and mono-t-Boc-piperazine
(1061.3 mg, 5.7 mmol), DCC (1030.0 mg, 5.0 mmol) and DMAP (6.1 mg, 0.05 mmol) were added at
room temperature [41]. The solution was stirred at room temperature overnight, and then the solvent
was removed under reduced pressure. The crude product was purified by chromatographic column
(
DCM) to give
5
as yellow solid powder (87%). Compound
6
was obtained by treatment of
5
with
1
trifluoroacetic acid: DCM (1:1) solution. H-NMR (400 MHz, DMSO-d )
8
δ
(ppm): 8.89 (1H, s, NH),
6
4
5
6
.03 (1H, s, H -Ar), 7.51 (1H, d, J = 8.80 Hz, H -Ar), 6.77 (1H, dd, J = 8.00, 3.2 Hz, H -Ar), 6.55 (1H,

Molecules 2019, 24, 3307
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8
d, J = 2.2 Hz, H -Ar), 3.67 (4H, m, 2
×
-CH2CH ), 3.52 (4H, m, 2
×
-CH2CH NH-), 3.20 (4H, m, 2
×
3
2
+
-
CH CH NH-), 1.13 (6H, t, J = 7.2 Hz, 2 × -CH CH ,). MS (ESI) m/z: [M + H] 320.2.
2
2
2
3
3
.1.7. Synthesis of Intermediate 7
To a stirred solution of ergosterol peroxide (200 mg, 0.56 mmol) and 4-nitrophenyl chloroformate
258 mg, 1.28 mmol) in DCM (5 mL) under N gas, pyridine (100 mg, 1.28 mmol) was added, and
(
2
stirring continued at room temperature for 3 h. The reaction course was followed on TLC. Then, DCM
20 mL) and H O (20 mL) were added into the mixture. The DCM phase was combined, washed with
(
2
brine, and then dried with anhydrous Na SO . The solution was concentrated to give yellow residue
7
2
4
for the next step.
3
.1.8. Synthesis of Novel Conjugates 8a–d
General synthesis procedure of conjugates 8a
, or ) (0.20 mmol), DCC (40 mg, 0.20 mmol), and DMAP (10%) were added into the solution of
–d: Coumarin-3-carboxylic acid derivatives 4a (4b,
4
c
7
ergosterol peroxide (68 mg, 0.13 mmol) in DCM (20 mL). The mixture was stirred continuously at
room temperature for 24~36 h. The filtrate was collected and concentrated, and the crude product was
purified by chromatographic column to give target probes 8a–d.
2
-[7-(Diethylamino)-2-Oxo-2H-Chromene-3-Carboxamido]-Glycine-3-(Ergosterol Peroxide) Probe 8a: Yellow
1
powder (52%). H-NMR (400 MHz, CDCl )
δ (ppm): 9.20 (1H, t, J = 5.4 Hz, -CONH-CH -), 8.71 (1H, s,
2
3
4
5
6
8
H -Ar), 7.42 (1H, dd, J = 8.8, 4.2 Hz, H -Ar), 6.71–6.56 (1H, m, H-6), 6.50 (2H, m, H -Ar, H -Ar), 6.25
1H, d, J = 8.7 Hz, H-7), 5.25–5.14 (2H, m, H-22, H-23), 5.11 (1H, m, -O ), 4.20 (2H, m, NHCH2CO),
-CH2CH ), 2.22–2.16 (1H, m), 2.11–2.06 (1H, m), 2.02 (2H, dd, J = 9.8,
(
H
3
3
1
.46 (4H, dd, J = 8.2, 3.6 Hz, 2
.3 Hz), 1.98–1.90 (2H, m), 1.85 (1H, dd, J = 12.9, 6.8 Hz), 1.72 (1H, m), 1.65 (4H, m), 1.58 (1H, m),
.50 (2H, dd, J = 10.7, 7.2 Hz), 1.43 (1H, m), 1.34 (2H, m), 1.28-1.24 (9H, m), 1.11 (1H, m), 1.01 (3H,
×
3
1
3
d, J = 6.6 Hz, -CH3), 0.90 (6H, m, 2
×
-CH CH3), 0.82 (9H, dd, J = 8.3, 4.5 Hz, 3
×
-CH3). C-NMR
2
(
100 MHz, CDCl3)
δ
(ppm): 168.6, 163.4, 162.6, 157.8, 152.7, 148.3, 136.2, 135.1, 132.3, 131.2, 130.8, 109.9,
1
2
09.8, 108.3, 96.7, 81.8, 79.4, 70.75, 56.2, 51.6, 51.0, 45.1, 44.6, 45.1, 44.5, 42.8, 39.7, 36.9, 34.2, 33.1, 29.7,
8.6, 26.2, 23.4, 20.9, 20.6, 19.9, 19.6, 18.1, 17.6, 12.9,12.4. MS (ESI) m/z: 727.4 [M + H] .
+
2
-[7-(Diethylamino)-2-Oxo-2H-Chromene-3-Carboxamido]-Alanine-3-(Ergosterol Peroxide) Probe 8b: Yellow
1
powder (45%). H-NMR (400 MHz, CDCl )
H -Ar), 7.42 (1H, d, J = 8.2 Hz, H -Ar), 6.65 (1H, m, H-6), 6.50 (2H, t, J = 3.6 Hz, H -Ar, H -Ar), 6.25 (1H,
δ
(ppm): 9.06 (1H, t, J = 5.0 Hz, -CON
H
-CH -), 8.68 (1H, s,
3
2
4
5
6
8
d, J = 8.4 Hz, H-7), 5.21–5.15 (2H, m, H-22, H-23), 5.05 (1H, m, -OH), 3.72 (2H, m, -NHCH2CH CO-),
2
3
2
.46 (4H, dd, J = 8.0, 3.6 Hz, 2
.01 (1H, m), 1.99–1.94 (4H, m), 1.71–1.60 (4H, m), 1.50 (2H, m), 1.43 (2H, m), 1.34 (2H, m), 1.28–1.24 (9H,
-CH CH3), 0.81 (9H, dd, J = 8.2, 4.4 Hz,
×
-CH2CH ), 2.62 (2H, t, J = 8.0, 3.8 Hz, -NHCH CH2CO-), 2.11 (1H, m),
3
2
m), 1.11 (1H, m), 1.01 (3H, d, J = 6.6 Hz, -CH3), 0.91 (6H, m, 2
3
×
2
13
×
-CH3). C-NMR (100 MHz, CDCl3) δ (ppm): 170.8, 163.2, 162.5, 157.7, 152.5, 148.1, 135.2, 134.1,
1
32.3, 131.1, 130.8, 110.2, 109.9, 108.3, 96.6, 81.8, 79.3, 69.9, 56.2, 51.6, 51.0, 45.1, 44.6, 42.7, 39.7, 39.3, 36.9,
+
3
4.7, 33.1, 33.0, 28.6, 25.6, 25.0, 20.9, 19.9, 19.6, 18.1, 17.6, 12.9, 12.4. MS (ESI) m/z: 741.4 [M + H] .
2
-[7-(Diethylamino)-2-Oxo-2H-Chromene-3-Carboxamido]-Threonine-3-(Ergosterol Peroxide) Probe 8c: Yellow
1
4
powder (52%). H-NMR (400 MHz, CDCl )
7
H -Ar), 6.23 (1H, d, J = 8.5 Hz, H-7), 5.18 (2H, qd, J = 15.3, 7.7 Hz, H-22, H-23), 5.05–4.94 (1H, m, -OH),
δ
(ppm): 8.84 (1H, s, -CON
H
-CH -), 8.69 (1H, s, H -Ar),
3
2
5
6
.42 (1H, d, J = 9.0 Hz, H -Ar), 6.65 (1H, dd, J = 9.0, 2.5 Hz, H-6), 6.50 (2H, dd, J = 5.4, 3.0 Hz, H -Ar,
8
4
2
0
.12 (6H, q, J = 7.1 Hz, -NHCH2
.05 (3H, m), 1.95–1.92 (4H, m), 1.72–1.32 (16H, m), 1.30-1.22 (6H, m, 2
C
H2
C
H2CO-), 3.54–3.40 (4H, m, 2
×
-CH2CH ), 2.36 (2H, d, J = 7.7 Hz),
3
×
-CH3), 1.14–1.08 (4H, m),
13
.92–0.88 (6H, m, 2
×
-CH CH3), 0.86–0.81 (6H, m, 2
×
-CH3). C-NMR (100 MHz, CDCl ) δ (ppm):
3
2
173.6, 164.7, 163.8, 157.6, 157.0, 153.0, 148.1, 135.2, 135.1, 132.3, 131.1, 130.8, 109.9 108.5, 96.6, 81.7, 79.4,
6
2
9.5, 56.2, 51.6, 51.0, 49.1, 45.1, 44.5, 42.8, 39.7, 39.3, 36.7, 33.9, 33.1, 32.1, 29.7, 26.4, 25.6, 24.9, 23.4, 22.8,
0.9, 19.9, 19.6, 18.1, 17.6, 12.9, 12.4. MS (ESI) m/z: 755.4 [M + H] .
+

Molecules 2019, 24, 3307
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2
-[7-(Diethylamino)-2-Oxo-2H-Chromene-3-Carboxamido]-Piperazine-3-(Ergosterol Peroxide) Probe 8d:
1
4
Yellow powder (49%). H-NMR (400 MHz, CDCl3)
J = 8.80 Hz, H -Ar), 6.60 (1H, dd, J = 9.00, 2.60 Hz, H-6), 6.48 (2H, dd, J = 12.0, 5.20 Hz, H -Ar, H -Ar),
δ (ppm): 7.88 (1H, s, H -Ar), 7.31 (1H, d,
5
6
8
6.23 (1H, d, J = 8.6 Hz, H-7), 5.19 (2H, m, H-22, H-23), 4.98–4.86 (1H, m, -OH), 3.71 (2H, s, -CH2CH3),
3
.54 (4H, s, -NCH2CH N-), 3.45 (4H, q, J = 7.20 Hz, -NCH CH2N-), 3.37 (2H, s, -CH2CH ), 2.18 (1H,
2
2
3
d, J = 4.60 Hz), 2.02 (4H, m), 1.85 (1H, d, J = 7.00 Hz), 1.76-1.69 (1H, m), 1.63 (4H, m), 1.59 (2H, dd,
J = 13.0, 5.8 Hz), 1.52 (2H, t, J = 6.2 Hz), 1.39–1.35 (1H, m), 1.25 (4H, d, J = 5.20 Hz), 1.23 (6H, m, 2
×
13
-
C
H3), 1.01 (3H, m, -CH3), 0.90 (6H, m, 2
CDCl3) (ppm): 165.2, 159.2, 157.3, 154.6, 151.8, 135.1, 135.1, 132.4, 130.9, 130.0, 115.7, 109.4, 107.8, 97.0,
1.9, 79.4, 70.9, 56.3, 51.7, 51.1, 50.0, 44.6, 42.8, 39.7, 39.4, 37.0, 34.4, 33.5, 33.1, 28.7, 23.3, 21.0, 19.8, 19.6,
8.0, 17.6, 12.9, 12.4. MS (ESI) m/z: 784.5 [M + H]+
×
-CH CH3), 0.81 (9H, m, 3
×
-CH3). C-NMR (100 MHz,
2
δ
8
1
3
.2. Biological Evaluation
3
.2.1. Cell Culture
Cancer cell lines (HepG2, SK-Hep1, and MCF-7) were obtained from the College of Pharmacy,
Qiqihar Medical University. Cells were cultured in DMEM medium (HepG2 and SK-Hep1) or RPMI
1
640 medium (MCF-7) supplemented with 10% FBS and 1% penicillin/streptomycin and maintained in
◦
an incubator with a humidified atmosphere of 5% CO at 37 C [23].
2
3
.2.2. MTT Assay
The effects of compounds on cell viabilities of HepG2, SK-Hep1, and MCF-7 cells were evaluated
4
by an MTT assay. Exponentially growing cells were seeded at a density of 2
×
10 cells/mL into 96-well
plates and cultured for 24 h. The tested samples at gradient concentrations (0.5, 1, 10, 20, 50, and
00 M) were solubilized in DMSO. Then, the cells were treated with samples for 48 h and 10 L of
1
µ
µ
MTT (KeyGEN Biotech, Nanjing, China) was added for an extra 2 h. Control cells were treated with
vehicle alone. The formazan dye product was measured on a Spectra Max 340 microplate reader at
490 nm (Thermo Multiskan MK3, Thermo Electron Corporation, Waltham, MA, USA). The IC50 values
of all compounds were calculated using SPSS 17.0 software (SPSS, Chicago, IL, USA).
3
.2.3. Living Cell Staining for Subcellular Localization
5
HepG2 cells were incubated on glass cover-slips in 6-well plate (4
×
10 cells/2 mL of medium
M and 10 M)
in 2 mL of HBSS buffer per well) for 2 h, and then Rh123 (100 nM) was added in the last 30 min.
for per well). After removing the medium, the cells were incubated with probe 8d (5
µ
µ
(
After removing of HBSS buffer and three times of wash with PBS, the cells were fixed with 4%
paraformaldehyde for 10 minutes and washed with PBS for three times. At last, the cover-slips were
sealed strictly with nail polish for imaging.
3
.2.4. Confocal Microscopic Imaging
Confocal images were obtained with confocal microscopy (LSM 510, Carl Zeiss, Oberkochen,
Germany), original magnification 100
×
. The excitation at 579 nm was used for Rh123 dye. For probe
8
d
, the excitation laser was 404 nm, and the images were collected at the emission of 470 nm. The
colocalization analysis of probe 8d and Rh123 on images were processed by LSM 510 software (Carl
Zeiss, Oberkochen, Germany).
3
.2.5. Cell Cycle Analysis
5
The HepG2 cells were cultivated at a concentration of 5
probe 8d (3
×
10 cells/mL into 6-well plates with
µM) for 24 h. The cells were washed in PBS and centrifuged for 5 min at 350 g (Eppendorf
804R, Hamburg, Germany) The collected pellets were incubated at a concentration of 10 cells/mL
6
5

Molecules 2019, 24, 3307
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with propidium iodide (PI, BD Pharmingen, BD Biosciences, San Jose, CA, USA) staining solution
50 µg/mL in PBS) for 30 min. The cells cycle was analyzed with flow cytometry (BD Biosciences).
(
3
.2.6. Colony, Invasion, And Migration Analysis
Colony formation: HepG2 cells (500 cells/well) were mixed with low melting agarose gel (0.25%).
Then, probe 8d (7 µM) was added into 6-well plates and maintained in 5% CO incubator for two
2
weeks. The colonies of cells were counted and stained, and then the photograph was analyzed under
4
an optics microscope. Migration: HepG2 cells (3
×
10 ) were mixed in RPMI 1640 media (200
µ
L) and
m pore size, COSTAR transwell, Corning, NY,
M) for 20 h. Cells that migrated to the lower chamber were
stained using crystal violet, and then the number of cells in three different high-power fields ( 100) was
added to the upper chamber of transwell apparatus (8
USA). The cells were treated with 8d (7
µ
µ
×
counted. Invasion: the upper chamber of transwell was coated using a layer of the extracellular matrix.
10 ) were added to the upper chamber and treated with 8d (7
cells were stained and counted that invaded to the lower chamber in the same way as migration.
5
HepG2 cells (1
×
µM) for 24 h. Then, the
3
.2.7. Measurement of Intracellular ROS in HepG2 Cells
The production of ROS was measured using CM-H DCFDA (Sigma-Aldrich, Milan, Italy). Briefly,
2
5
HepG2 cells (5
concentrations (0, 5, and 20
×
10 ) were seeded in 6 cm dishes and exposed to compound 8d for 24 h at serial
µ
M), after which the medium was removed, and washed with PBS,
◦
followed by incubation with 10
µ
M CM-H DCFDA for 30 min at 37 C in serum-free medium. Then,
2
the incubation medium containing CM-H DCFDA was removed, washed with PBS, and the cells were
2
trypsinized and resuspended in PBS buffer. The quantity of ROS was monitored by flow cytometer
(BD Biosciences), with dual channes fluorescence (DCF) luorescence detection channel.
4
. Conclusions
In summary, we have successfully designed and prepared four mitochondria-targeting theranostic
probes 8a by direct conjugation of a coumarin-3-carboxamide fluorophore with ergosterol peroxide
. The cytotoxicities of synthesized conjugates against three human tumor cell lines (HepG2, SK-Hep1,
and MCF-7) were investigated. The results of fluorescent imaging showed that the synthesized
conjugates 8a localized and enriched mainly in mitochondria, leading to significantly enhanced
–
d
1
–
d
cytotoxicity over ergosterol peroxide. The biological functions of probe 8d showed that it inhibited cell
colony formation, invasion, and migration; induced G2/M phase arrest of HepG2 cells, and increased
the intracellular ROS level. The present work provides new information to the potential of ergosterol
peroxide as a drug lead compound for the versatile interest of research on steroidal anticancer agents.
Besides, the strategy of natural products to mitochondria with enhanced cytotoxicity is in contrast
to the well-established methods of using drug-mediated fluorophore delivery and may represent a
feasible method for the design of theranostic probes.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/18/3307/s1,
1
13
Supplementary materials for H- and C-NMR spectra of new compounds are available online.
Author Contributions: M.B. and H.L. conceived and designed the experiments; H.W., H.L., Y.L., and J.W.
performed the chemistry experiments; Y.M. performed the biology experiments; H.W. and M.B. wrote the paper;
all authors analyzed the data and revised the manuscript.
Funding: This research was funded by the Administration of Traditional Chinese Medicine of Heilongjiang
Province Project (ZHY18-164) and Qiqihar Academy of Medical Sciences Project (QMSI2017B-09).
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2019, 24, 3307
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Abbreviations
HepG2, SK-Hep1 Human liver carcinoma cell line
MCF-7
DCC
human breast adenocarcinoma cell line
dicyclohexylcarbodi imide
HOBT
1-hydroxylbenzotriazole
DMAP
EDCI
PNPFC
CM-H2DCFDA
TBTU
4-dimethylaminopyridine
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
4-nitrophenyl chloroformate
5-(and-6)-chlorometry-2’,7’-dichlorodihydro-fluorescein diacetate acetyl ester
2-(1H-Benzotriazole-1- yl)-1,1,3,3-tetramethyluronium tetrafluoroborate
N,N-diisopropylethylamine
DIPEA
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Sample Availability: Samples of the compounds 8a–d are available from the authors.
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