Synthesis of 5α,8α-Ergosterol Peroxide 3-Carbamate Derivatives and a Fluorescent Mitochondria-Targeting Conjugate for Enhanced Anticancer Activities

Article abstract of DOI:10.1002/cmdc.201700021

Inspired by the significant anticancer activity of our previously screened natural ergosterol peroxide (1), we synthesized and characterized a series of novel ergosterol peroxide 3-carbamate derivatives. The antiproliferative activities of the synthesized

Full text of DOI:10.1002/cmdc.201700021

Accepted Article
Title: Synthesis of 5α,8α-ergosterol peroxide 3-carbamate derivatives
and fluorescent mitochondria-targeting conjugate for enhanced
anticancer activities
Authors: Ming Bu, Tingting Cao, Hongxia Li, Mingzhou Guo, Burton B.
Yang, Chengchu Zeng, and Liming Hu
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ChemMedChem
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Synthesis of 5α,8α-ergosterol peroxide 3-carbamate derivatives
and fluorescent mitochondria-targeting conjugate for enhanced
anticancer activities
[
a]
[a]
[a]
[c]
[d]
[a]
Ming Bu, Tingting Cao, Hongxia Li, Mingzhou Guo, Burton B. Yang, Chengchu Zeng and
[a,b]
Liming Hu*
Abstract: By inspiration of significant anticancer activity of our
previously screened natural ergosterol peroxide (1), a series of novel
ergosterol peroxide 3-carbamate derivatives were synthesized and
characterized. The anti-proliferative activity of synthesized
compounds against human hepatocellular carcinoma cells (HepG2,
SK-Hep1) and human breast cancer cells (MCF-7, MDA-MB231)
were investigated. Compound 3d, 3f and their hydrochloride
exhibited significant in vitro anti-proliferative activity against the
use today are semi-synthetic compounds and widely used in
traditional medicines by the modification of the steroid ring
[8]
system and side chains.
The interesting structural and
stereochemical features of the steroid nucleus provide additional
fascination to the researchers, and thereby the introduction of
heteroatom, heterocycle, amides or replacement of one or more
carbon atoms in the steroidal skeleton has been envisaged to
discover new chemical entities with a potential to afford some
tested tumor cell lines, with IC50 values ranging from 0.85 to 4.62 μM. promising drugs of the future and brings notable modifications of
[
9]
Furthermore, fluorescence mitochondria-targeting images showed
that the designed coumarin-3d conjugate (5) localized mainly in
mitochondria, leading to enhanced anticancer activities over the
parent structure (1). As a whole, it appeared that substituent
changes to the C-3 position could serve as a promising launch point
for further design of this type of steroidal anticancer agents.
its biological activity.
Introduction
Endoperoxides (EPOs) are cyclic organic compounds, with an
[
1]
O-O single bond as a peroxidic bridge. EPOs include 1,2-
dioxanes, 1,2,4-trioxanes and 1,2,4,5-tetraoxanes, all of which
[
2]
possess the critical peroxide linkage. They play an important
[3]
role in drug synthesis as well as in medicine, and represent the
central part of artemisinin, outstanding antimalarial drugs,
Figure 1. Structures of natural endoperoxides with bioactivities
[
4]
honored with the Noble Price in Medicine 2015. Although best
[
5]
known as potent antimalarials, cyclic peroxides also exhibit a
range of activities which encompasses antifungal, antiviral and
Among natural EPOs, 1,2-dioxanes are the most frequent
constituents of naturally occuring endoperoxides, such as sterol
[
6]
anticancer activity (Figure 1).
5
α,8α-endoperoxides are the important active lead compounds
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,
[
10]
in drug discovery. Ergosterol peroxide (5α,8α-epidioxiergosta-
,22-dien-3β-ol, EP, 1) (Figure 2), is a member of a class of
6
[
7]
fungal secondary metabolites of sterol 5α,8α-endoperoxide
derivatives. It can be isolated from many medicinal fungi, such
as Sarcodon aspratus, Hericium erinaceum, Armillariella mellea,
contraceptive, and anticancer agents. Most steroidal drugs in
[
11]
lactarius hatsudake, hypsizigus marmoreus, etc.
Ubiquitous
[
a]
b]
M. Bu, T. T. Cao, H.X. Li, Prof. Dr. C. C. Zeng, Prof. Dr. L. M. Hu
College of Life Science and Bioengineering
Beijing University of Technology
Beijing, 100124, China
E-mail: huliming@bjut.edu.cn
Prof. Dr. L. M. Hu
EP (1) continues to be isolated from a number of natural
sources. A number of biological activities have been attributed
to ergosterol peroxide, such as antitumor activity,
immunomodulatory activity, inhibitory hemolytic activity, anti-
[
[12]
Beijing Key Laboratory of Environmental and Viral Oncology
Beijing University of Technology
Beijing, 100124, China
inflammatory activity, and antiviral activity.
In our previous study, we found that
1
purified from
Ganoderma lucidum, induced cell death and inhibited cell
[
c]
d]
Prof. Dr. M. Z. Guo
Chinese PLA General Hospital,
Beijing, 100853, China
migration, cell cycle progression, and colony growth of human
[
13]
hepatocellular carcinoma cells.
We further examined the
[
Prof. Dr. B. B. Yang
mechanism associated with this effect and found that treatment
with 1 increased expression of Foxo3a mRNA and protein in
HepG2 cells. The levels of Puma and Bax, pro-apoptotic
proteins, were effectively enhanced. Our results suggest that 1
stimulated Foxo3 activity by inhibiting pAKT and c-Myc and
Institute of Medical Science
University of Toronto
Toronto, M4N3M5, Canada
Supporting information for this article is given via a link at the end of
the document.
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activating pro-apoptotic protein Puma and Bax to induce cancer
cell death. With further clinical development, 1 represents a
promising new reagent that can overcome the drug-resistance of
tumor cells. Since the amount of purified 1 is not sufficiently to
perform in vivo experiments and clinical application, we
developed approach to synthesize 1 chemically. The synthesis
crystal structure of 1 was obtained the first time and presented in
Figure 3. The derivatives 3a-3j were synthesized from 1 by the
method as shown in Scheme 1. In the first step, 1 was reacted
with 4-nitrophenyl chloroformate in DCM using pyridine as the
base to obtain intermediate 2. Then, 2 reacted with different
[
14]
amines in the presence of Et
3a-3j. The courses of reactions were monitored by thin layer
chromatography (TLC) plates and/or by staining with H SO
ethanol solution (5% H SO in ethanol). The chemical structures
of the compounds synthesized were elucidated on the basis of
3
N to obtain the desired products
of 1 achieved by the reaction of ergosterol with singlet oxygen
1
(
O
2
), which can be conveniently generated photochemically
2
4
-
[
15]
from molecular oxygen (photooxygenation).
2
4
1
13
H NMR, C NMR, MS and X-ray. The detailed physical and
analytical data are given in experimental part. As shown in Table
, although asymmetric diamine was used as substrate (Table 1,
1
entry 6), the compound mentioned in Table 1 was the main
product.
Figure 2. The structure of ergosterol peroxide (EP, 1).
Acknowledging the limited structure-activity relationship
studies for 1, it is obvious that structurally simpler polar
substituent could be advantageously grafted on C-3 position of 1
to increase anticancer activity while simultaneously increasing
solubility. N-Heterocycles are found in a variety of biologically
active compounds and are easily to form water-soluble salts with
[
16]
organic or inorganic acids.
It is also noticeable that most of
the drugs used in medicine behave as weak acid or base in
solution. N-Heterocycles drugs with a lone pair of electrons on
nitrogen available for protonation are recognized as basic
[
17]
drugs.
On the basis of the above details, we designed and
series of ergosterol peroxide 3-carbamate
synthesized
a
Scheme 1. Synthesis of EP 3-carbamate analogues 3a-3j. Reagents and
derivatives. Meanwhile, their biological activities (IC50 values)
were compared using a MTT assay with four human cancer cell
lines. We hope to get valuable information for further design of
novel steroidal anticancer agents.
conditions: (a) O , eosine, pyridine, hv,
chloroformate, DCM, pyridine, RT; (c) different amines, Et N, DCM, RT.
0
°C, 0.5 h; (b) 4-nitrophenyl
2
3
Mitochondria-targeting theranostic probes that enable the
simultaneously reporting of and triggering of mitochondrial
dysfunctions in cancer cells are highly attractive for cancer
X-ray crystal structure of 1 was obtained and is presented in
Figure 3. The structure confirms the α-stereochemistry of the
peroxy bond at C-5 and C-8 positions. Crystallographic data for
1 has been deposited at the Cambridge Crystallographic Data
Center with CCDC numbers 1442028.
[
18]
diagnosis and therapy.
fluorescent mitochondria-targeting theranostic probe through the
conjugation of synthesized derivatives with coumarin-3-
We postulated that the
Herein, we also designed the
a
[
19]
carboxylic acid fluorophore.
fluorescent conjugates as mitochondria targeting theranostic
probes could efficiently leading to enhance anticancer
[
20]
activities.
Results and Discussion
Figure 3. X-ray crystal structure of 1.
Chemistry
Here a plausible reaction mechanism for the formation of
α,8α-peroxy moiety from ergosterol is depicted. First, singlet
oxygen is generated from sensitization by eosine. Then, the
Using natural and commercially available ergosterol as the
starting material, we performed chemical synthesis and
purification of 1 as described in our previous work. The X-ray
5
[14]
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clear regionselectivity of the singlet oxygen attacks to the C-5
and C-8 positions of the conjugated double bond system in the
Biological evaluation
[
4+2] cycloaddition manner. What’s more, the α-π-facial stereo-
The newly synthesized compounds 3a-3j, 3d·HCl, 3f·HCl and
1 were evaluated for their anti-proliferative activities against
human cancer cell lines derived from various human cancer
types, including human hepatocellular carcinoma cell lines
HepG2, SK-Hep1 and human breast cancer cell lines MDA-
MB231, MCF-7. In vitro cell-based evaluation of the anti-
proliferative activities of the synthesized compounds was carried
out using MTT assay. Cisplatin was employed as the positive
control. The anticancer potency of the compounds was indicated
by IC50 values. The results were summarized in Table 2.
5
,7
selectivity of the reaction of singlet oxygen with △ -diene is
well accepted owing to the fact that the methyl group at C-10 is
β (axial).
[
21]
Table 1. Ergosterol peroxide 3-carbamate derivatives 3a-3j.
[
a]
Entry
1
Compound
R
Yield (%)
85
3a
Table 2. The anti-proliferative activities of compounds.
2
3
3b
3c
56
62
[
a]
Compound
IC50 (μM)
SK-Hep1
HepG2
>40
MCF-7
>40
MDA-MB231
>40
3a
3b
3c
3d
>40
9.76±0.37
13.60±0.88
0.85±0.06
0.79±0.12
19.52±0.47
2.83±0.11
1.96±0.17
29.41±0.61
>40
8.21±0.04
10.23±0.32
1.75±0.29
1.78±0.08
15.23±0.16
0.92±0.04
0.87±0.07
20.66±0.24
>40
11.27±0.06
17.70±0.67
3.26±0.11
4.00±0.21
12.24±0.35
4.62±0.28
3.78±0.20
>40
8.79±0.40
14.55±0.36
4.12±0.38
3.24±0.14
16.08±0.53
3.99±0.20
3.27±0.09
33.05±0.49
>40
4
5
6
3d
3e
3f
60
69
66
3d·HCl
3e
3f
7
8
3g
3h
82
85
3f·HCl
3
g
h
3
>40
3
i
24.25±0.23
22.62±0.18
23.50±0.43
<0.50
20.34±0.33
21.45±0.17
19.71±0.49
2.39±0.14
>40
29.87±0.35
>40
9
3i
3j
80
77
3j
30.80±0.41
26.06±0.18
6.63±0.27
1
24.75±0.26
5.50±0.45
10
Cisplatin
[
a] Each data point and error represents the mean
±
SD of three
[
a] Isolated yields.
experiment results.
X-ray crystal structure of 3c was obtained and is presented in
The IC50 values presented in Table 2 indicate that as
compared to the parent 1, most of the carbamate derivatives
(3a-3j) possess strong growth inhibition activity against all the
four tested adherent cancer cell lines. The screening results
Figure 4. The structure confirms the carbamate structure at C-3
position. Crystallographic data for 3c has been deposited with
CCDC numbers 1514530.
suggested
a rough SAR: among the nitrogen-contained
carbocycle carbamates, the piperazinyl, and piperidinyl moieties
were effective in increasing cytotoxicity. Compounds 3d and 3f
contained piperazinyl and piperidinyl moieties displayed the
most significant anti-proliferative activity with the IC50 values of
0.85-4.62 μM, which were between 6- and 28-fold more potent
than 1. The compounds with N-methyl, N-ethyl and hydroxyl
substituents (3b, 3c, 3e, and 3g) at the nitrogen-contained
carbocycle demonstrated lower activities. The results also
showed that both 3d·HCl and 3f·HCl displayed strong anti-
Figure 4. X-ray crystal structure of 3c.
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proliferative activity, therefor, 3d, 3d·HCl, 3f and 3f·HCl were
chosen for further solubility studies. We further analyzed the
impact of substituents at 4’-nitrogen atom in piperidine and
piperazine ring on cytotoxicities. Compared with 3d and 3f, we
observed that incorporation of Boc-group on 3i and 3j resulted in
Among derivatives 3a-3j, piperazinyl (3d) and piperidinyl (3f)
are two of the distinctive N-heterocycles those are readily
available for protonation in solution and provide opportunity to
design new drug structures. The water solubility of 1, 3d, 3d·HCl,
3f and 3f·HCl were tested, and the solubilities were 0.05, 0.12,
a significant loss of potency against four cell lines (IC50 > 20 μM). 2.00, 0.15 and 2.20 mg/mL, respectively. The results indicated
The results clearly indicated that 4’-NH was favorable to their
cytotoxic activity.
that the solubility of 3d·HCl and 3f·HCl were about 40 times
greater than that of 1 (Figure 5).
Compounds 3b, 3d, 3d·HCl, 3f, 3f·HCl and 1 with good anti-
proliferative activities against cancer cells were selected for
further in vitro cytotoxic evaluation using a non-cancer cell line
HepG2 cells were treated with different concentrations of 3f as
indicated for 48 h. Cells were fixed with cold methanol for 15 min
and stained with the Diff-Quik-Stain kit, followed by microscopic
examination. Typical photos of cell death are shown in Figure 6.
HepG2 cells were also treated with 3f at the concentration of 1
μM for 24 h. Cells were fixed with cold methanol for 15 min and
stained with the Diff-Quik-Stain kit, followed by microscopic
examination. Careful examination of the cells treated with 3f
revealed the typical vacuoles (Figure 6B). The phenomenon
shown that cellular structure integrity has been destroyed by 3f,
such accelerated the cancer cell apoptosis.
293T (human kidney epithelial cell line) as a control.
Exponentially growing 293T cells were treated with the
compounds at different concentrations for 48 h. Cell-growth
inhibition was analyzed by the MTT assay. The results are
summarized in Table 3. As shown in Table 3, the median
cytotoxic concentration (CC50) showed that most of the tested
compounds displayed relative lower cytotoxicity in vitro against
293T cells. They exhibited appropriate selectivity between
cancer cell line and non-cancer cell line.
Table 3. Cytotoxic activity data against 293T cell lines.
[
a]
Compound
CC50 (μM)
93T
2
1
56.82±0.48
61.62±0.79
36.49±0.40
32.50±0.33
23.48±0.65
25.86±0.42
24.22±0.16
3
b
d
3
3
d·HCl
3f
3
f·HCl
Cisplatin
[
a] Each data point and error represents the mean ± standard deviation of
three experiment results.
Figure 6. 3f induced cell death of HepG2. (A)Typical photos of cell death were
shown. (B) The cells treated with 3f revealed the typical vacuoles
Epithelial cell migration is
a regulated and harmonized
mechanism in the cancer cells. It requires their chemotactic
migration which is further guided by the protrusive activity of the
cellular membrane. Conventional scratch wound motility assay
exhibited the ability of 3f to modulate the migration of HepG2
cells in a denuded area. Cell migration was monitored using
scratch assay. Typical photos of cell motility are shown in Figure
7A. It was found that 3f-treated HepG2 cells displayed lower
speeds of motility than the control cells, thereby indicating the
inhibition of proliferation of HepG2 cells by 3f (Figure 7B).
Figure 5. The water solubility of 1, 3d, 3d•HCl, 3f and 3f•HCl. Error bars, SD
(n = 3).
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Scheme 2. Synthetic of the probe coumarin-3d (5). Reagents and conditions:
DCC, DMAP, DCM, RT, 36 h.
Figure 8. Fluorescence excitation spectra and emission spectra for probes 4
and 5 (1.0 μM in DMSO).
We next examined the subcellular localization of the designed
conjugates 5 in living human liver cancer cells (HepG2 and
MCF-7) with the commercially available mitochondria-specific
Figure 7. Cell motility studies with compound 3f. (A) Typical photos of cell
motility were shown. (B) Migration rates were measured and calculated. Error
bars, SD (n = 3).
green dye Rhodamine 123 (Rh123) (λEx = 488 nm and λEm
=
515-530 nm). The intracellular fluoroscope imaging was
explored on a 710M confocal laser scanning microscopy (Carl
Zeiss, Germany). As shown in Figure 9b, cells were extensively
stained by 5 (5 μM) after 2 h of incubation, and strong blue
fluorescence was detected inside the cells with excitation at λ =
Fluorescent mitochondria-targeting probe
4
30-500 nm, which indicated good cell membrane permeability
Based on significant anticancer activity of 3d, we designed a
fluorescent mitochondria-targeting probe coumarin-3d (5)
through the conjugation of 3d with a coumarin-3-carboxamide
fluorophore. Designed conjugate probe 5 was synthesized via
the routes outlined in Scheme 2.
With the probe 5 in hand, we first investigated its optical
properties (Figure 8). In general, it possess typical optical
properties of the courmarin-3-carboxamide fluorophore, with
maximum excitation wavelength (λEx) of about 469.5 nm and
emission wavelength (λEm) of about 404 nm. The large Stokes
shift of probe 65.5 nm ensured good photophysical properties for
fluorescence imaging studies in living cells.
and high cellular uptake of 5. The subcellular localization study
showed that compound 5 could colocalize strongly with Rh123 in
mitochondria, with an extensive blue-green colour in the merged
fluorescence images of 5 and Rh123 (Figure 9d).
Having proved mitochondrial localization of our designed
conjugate 5, we then measured the anti-proliferative activities of
5
against four selected human cancer cell lines (HepG2, Sk-
Hep1 MCF-7 and MDA-MB231). The results in Table 4 show
that designed coumarin-3d (5) conjugate localized mainly in
mitochondria, leading to enhanced anticancer activities over
ergosterol peroxide (1), but lower than 3d. We therefore
postulated that, upon cell uptake, the mitochondria-targeting 5
could efficiently trigger the compound localized in mitochondria,
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in which subsequent triggering of mitochondrial dysfunctions in
cancer cells and induce cell apoptosis
3f, 3d·HCl and 3f·HCl were the most promising derivatives, with
IC50 values ranging from 0.85-4.62 µM against all the four
cancer cell lines. Among them, the solubility of 3d·HCl and
3f·HCl was about 40 times greater than that of 1. In an overall
view, the 5α,8α-ergosterol peroxide 3-carbamate derivatives will
provide better insight into the effect of the C-3 site on anticancer
activity for designing potential steroidal endoperoxide anticancer
agents. What’s more, fluorescence images showed that the
designed coumarin-3d (5) conjugate localized mainly in
mitochondria, leading to enhanced anticancer activities over the
parent structure 1. This study highlights the potential of using
mitochondria-targeting fluorophore to directly visualize subcellular
drug delivery in living cells. Future work will also focus on the
synthesis of additional candidate structures with different
substituents to address specific cancer cell lines. It appeared
that substituent changes to the steroidal C-3 position could
serve as a promising launch point for further design of this type
of steroidal anticancer agents.
Figure 9. Confocal laser scanning microscopic images of HepG2 cells treated
with 5 (5 μM, 2 h) in the presence of Rh123 (0.5 μM, 20 min) in PBS buffer. a)
Cell images in bright field. b) Fluorescence images of HepG2 cells stained with
5. c) Fluorescence images of cells stained with Rh123. d) Merged images of b)
and c).
Experimental Section
Table 4. The anti-proliferative activities of compounds.
Chemistry
[
a]
Compound
IC50 (μM)
SK-Hep1
All commercially available reagents were used without further purification.
Melting points (uncorrected) were determined on a MP120 auto point
HepG2
>100
MCF-7
>100
MDA-MB231
>100
1
apparatus (Hanon instruments Corp., Jinan, China). The H NMR and
13
4
5
>100
C
NMR spectra were measured on
a Bruker Avance DRX400
spectrometer with TMS and solvent signals allotted as internal standards.
7.86±0.49
9.54±0.62
10.46±0.36
8.28±0.52
1
13
The chemical shifts of the H NMR and C NMR were expressed in ppm
δ). ESI mass spectra were obtained on an Esquire 6000 Mass
(
[
a] Each data point and error represents the mean ± standard deviation of
Spectrometer. X-ray diffraction data were collected on CrysAlis PRO,
Agilent Technologies. Silica gel (300-400 mesh) was used for analytical
three experiment results.
and flash chromatography. DCM refers to dichloromethane, Et
to triethylamine, and RT refers to room temperature.
3
N refers
We further analyzed the impact of substituents at 4’-nitrogen
atom in piperazine ring on anti-proliferative activities. Compared
with 3d and probe 5, we observed that incorporation of coumarin-
[14]
1
Ergosterol peroxide (1). White needles; m.p. 181.5-183 °C. H NMR
3-carboxylic acid on 3d resulted in a loss of potency against four
3
(400 MHz, CDCl ) δ (ppm) 0.82 (d, J = 6.8 Hz, 3H), 0.83 (s, 3H), 0.84 (d,
cell lines. The results further indicated that 4’-NH of 3d was the
essential to the cytotoxic activity. In contrast, much weaker anti-
proliferative activities were measured for 4 and 1, which
suggested that the anti-proliferative activities of compound 5 was
associated with the synergistic effect of these two components.
Even so, this study highlights the potential of using mitochondria-
targeting fluorophore to directly visualize subcellular drug delivery
in living cells.
J = 6.8 Hz, 3H), 0.89(s, 3H), 0.91 (d, J = 6.9 Hz, 3H), 1.00 (d, J = 6.4 Hz,
3H), 3.97 (m, 1H), 5.12 (dd, J = 8.0, 15.2 Hz, 1H), 5.23 (dd, J = 7.6, 15.2
13
Hz, 1H), 6.24 (d, J = 8.4 Hz, 1H), 6.51 (d, J = 8.4 Hz, 1H). C NMR (100
MHz, CDCl ) δ (ppm) 12.9, 17.6, 18.2, 19.6, 19.9, 20.6, 20.9, 23.4, 28.6,
0.1, 33.1, 34.7, 37.0, 37.0, 39.3, 39.7, 42.8, 44.6, 51.1, 51.7, 56.2, 66.4,
3
3
7
4
+
9.4, 82.2, 130.7, 132.3, 135.2, 135.4. ESI-MS m/z: 451.4 [M+Na] ,
67.3 [M+K] .
+
General procedure for the synthesis of compounds 3a-3j. To a
stirred solution of 1 (100 mg, 0.28 mmol) and 4-nitrophenyl chloroformate
(
129 mg, 0.64 mmol, 2.3 eq) in anhydrous DCM (2 mL) at room
temperature under nitrogen gas was added pyridine (50 mg, 0.64 mmol,
.3 eq), and stirring continued for 2 h. After completion (by TLC), DCM
10 mL), and H O (10 mL) were added. The organic phase was
separated, washed with aq. Na CO , and brine, dried over Na SO , and
concentrated in vacuo to afford yellow residue 2. To a solution of the
residue in DCM (2.5 mL) was added amine (3 eq) and Et N (3 eq), and
Conclusions
2
(
2
In summary, a series of novel ergosterol peroxide 3-carbamate
derivatives from ergosterol by a three-step reaction route were
synthesized and evaluated for their anticancer activities.
Preliminary structure-activity relationships were put forward
based on the biological results. Some of the synthesized
compounds exhibited potent anticancer activities against the
four tested cancer cell lines in vitro. In particular, compound 3d,
2
3
2
4
3
stirring continued at room temperature for 2 h. After completion (by TLC),
DCM (15 mL) and H O (15 mL) were added. The organic phase was
2
separated, washed with aq. Na
2 3 2 4
CO and brine, dried over Na SO , and
concentrated in vacuo to give yellow residue, and the residue was
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subjected to chromatographic separation on silica gel with petroleum
ether/acetone/triethylamine.
5α,8α-epidioxyergosta-3-yl-(piperidin-4-methylamine)carbamate (3f).
1
Yellow solid, m.p. 204.4-206.3 °C; H NMR (400 MHz, CDCl
3
) δ 6.50 (d,
J = 8.5 Hz, 1H), 6.23 (d, J = 8.5 Hz, 1H), 5.18 (qd, J = 15.3, 7.7 Hz, 2H),
.93-4.81 (m, 1H), 4.20 (t, J = 15.8 Hz, 1H), 3.03-2.91 (m, 1H), 2.88-2.74
(m, 3H), 2.16 (dd, J = 10.9, 3.3 Hz, 1H), 2.05-1.97 (m, 4H), 1.92 (m, 1H),
.81 (m, 3H), 1.69 (dd, J = 10.5, 3.5 Hz, 2H), 1.59-1.49 (m, 6H), 1.38 (dd,
J = 9.4, 4.9 Hz, 3H), 1.26-1.18 (m, 5H), 1.00 (d, J = 6.6 Hz, 3H), 0.91 (d,
4
5
1
=
1
1
2
1
α,8α-epidioxyergosta-3-yl-butylcarbamate(3a). White solid, m.p. 165.9-
67.4°C; H NMR (400 MHz, CDCl ) δ 6.50 (d, J = 8.5 Hz, 1H), 6.22 (d, J
3
1
1
8.5 Hz, 1H), 5.18 (d, J = 15.3, 7.7 Hz, 2H), 4.94-4.81 (m, 1H), 4.54 (s,
H), 3.22-3.07 (m, 2H), 2.15 (dd, J = 13.1, 4.0 Hz, 1H), 1.99 (m, 5H),
.85 (dd, J = 13.0, 6.8 Hz, 1H), 1.75 (d, J = 9.2 Hz, 1H), 1.71-1.64 (m,
H), 1.63-1.53 (m, 3H), 1.56-1.41 (m, 6H), 1.34 (qd, J = 14.1, 6.9 Hz, 4H),
13
J = 6.7 Hz, 6H), 0.82 (m, 9H); C NMR (100 MHz, CDCl
32.3, 130.8, 125.1, 122.3, 81.8, 79.3, 70.3, 56.2, 51.6, 51.0, 48.7, 48.2,
44.5, 42.8, 42.7, 39.7, 39.3, 36.9, 35.3, 33.1, 28.6, 26.8, 23.3, 20.8, 20.6,
3
) δ 154.7, 135.2,
1
13
.28-1.16 (m, 3H), 0.99 (s, 3H), 0.91 (m, 9H), 0.82 (m, 9H); C NMR
) δ 155.7, 135.2, 132.3, 130.8, 81.8, 79.3, 69.6, 56.2,
+
1
9.9, 19.6, 18.1, 17.5, 12.9; MS (ESI) m/z: 555.6 [M+H] .
(100 MHz, CDCl
3
5
2
1.6, 51.0, 44.5, 42.8, 39.7, 39.3, 36.9, 34.3, 33.5, 33.1, 32.1, 28.6, 26.7,
3.4, 20.9, 20.6, 19.9, 19.6, 18.1, 17.5, 13.7, 12.9; MS (ESI) m/z: 528.8
5α,8α-epidioxyergosta-3-yl-(4-(diethylamino)piperidine-1)carbamate
+
1
[M+H] .
(3g). White solid, m.p. 158.9-161.8 °C; H NMR (400 MHz, CDCl
3
) δ 6.50
(d, J = 8.5 Hz, 1H), 6.23 (d, J = 8.5 Hz, 1H), 5.18 (qd, J = 15.3, 7.7 Hz,
2
H), 4.92-4.82 (m, 1H), 4.14 (d, J = 31.6 Hz, 2H), 2.76-2.62 (m, 3H), 2.55
5
α,8α-epidioxyergosta-3-yl-(2-(diethylamino)ethyl)carbamate(3b).
1
(q, J = 7.1 Hz, 4H), 2.20-2.11 (m, 1H), 1.99 (td, J = 10.2, 9.8, 5.7 Hz, 4H),
White solid, m.p. 154.8-156.4 °C; H NMR (400 MHz, CDCl
8.5 Hz, 1H), 6.24 (d, J = 8.5 Hz, 1H), 5.26-5.17 (m, 2H), 5.14 (d, J = 8.0
Hz, 1H), 4.96-4.82 (m, 1H), 3.25 (d, J = 5.3 Hz, 2H), 2.40 (m, 2H), 2.23
m, 6H), 2.17 (dd, J = 10.3, 4.1 Hz, 1H), 2.07-1.94 (m, 6H), 1.87 (dd, J =
3
) δ 6.51 (d, J
1.85 (m, 1H), 1.78-1.67 (m, 5H), 1.60-1.36 (m, 10H), 1.26-1.20 (m, 3H),
=
13
1
.06-0.98 (m, 9H), 0.89 (m, 6H), 0.82 (m, 9H); C NMR (100 MHz,
) δ 154.6, 135.3, 135.2, 132.3, 130.8, 81.8, 79.3, 70.2, 58.1, 56.2,
1.6, 51.0, 44.5, 43.6, 43.5, 42.8, 39.7, 39.3, 36.9, 34.3, 33.6, 33.1, 28.6,
6.8, 20.9, 20.6, 19.9, 19.6, 18.1, 17.5, 13.6, 12.9; MS (ESI) m/z: 611.6
CDCl
5
2
3
(
13.0, 6.8 Hz, 1H), 1.79-1.66 (m, 2H), 1.63-1.46 (m, 6H), 1.44-1.34 (m,
2H), 1.29-1.21 (m, 3H), 1.01 (d, J = 6.6 Hz, 3H), 0.95-0.88 (m, 6H), 0.84
+
13
[M+H] .
(
m, 9H). C NMR (100 MHz, CDCl
3
) δ 155.8, 135.3, 135.2 132.3, 130.8,
8
3
1
1.8, 79.3, 69.6, 58.2, 56.2, 51.6, 51.0, 45.1, 44.5, 42.8, 39.7, 39.3, 38.2,
6.9, 34.3, 33.5, 33.1, 28.6, 26.7, 23.3, 20.9, 20.6, 19.9, 19.6, 18.1, 17.5,
5α,8α-epidioxyergosta-3-yl-((4-methyl)imidazole-1)carbamate(3h).
+
1
2.9; MS (ESI) m/z: 571.5 [M+H] .
White solid, m.p. 166.4-167.6 °C; H NMR (400 MHz, CDCl
3
) δ 8.01 (s,
1
5
2
3
0
1
5
2
H), 7.10 (s, 1H), 6.55 (d, J = 8.5 Hz, 1H), 6.26 (d, J = 8.5 Hz, 1H), 5.26-
.13 (m, 3H), 2.30 (m, 1H), 2.21 (t, J = 3.1 Hz, 3H), 2.13-2.07 (m, 2H),
.00 (m, 3H), 1.86 (m, 1H), 1.80-1.74 (m, 2H), 1.65 (m, 2H), 1.59-1.51 (m,
H), 1.49-1.43 (m, 1H), 1.39 (m, 1H), 1.25 (m, 4H), 1.02-0.99 (m, 3H),
5
α,8α-epidioxyergosta-3-yl-(2-(dimethylamino)ethyl)carbamate (3c).
1
White solid, m.p. 153.9-155.2 °C; H NMR (400 MHz, CDCl
3
) δ 6.50 (d, J
=
1
8.5 Hz, 1H), 6.22 (d, J = 8.5 Hz, 1H), 5.24-5.12 (m, 2H), 4.90-4.82 (m,
H), 3.21 (d, J = 5.1 Hz, 2H), 2.59-2.48 (m, 6H), 2.19-2.10 (m, 1H), 1.98
13
3
.91 (m, 6H), 0.85-0.81 (m, 9H); C NMR (100 MHz, CDCl ) δ 147.7,
36.5, 135.1, 134.6, 132.4, 131.2, 113.1, 81.7, 79.5, 74.1, 56.2, 51.8,
6.2, 51.6, 51.0, 44.6, 42.8, 39.7, 39.2, 36.9, 34.2, 33.1, 33.0, 28.6, 26.2,
(dd, J = 15.8, 7.5 Hz, 4H), 1.85 (dd, J = 13.0, 6.8 Hz, 1H), 1.77-1.64 (m,
4H), 1.57 (dd, J = 15.2, 5.4 Hz, 2H), 1.52-1.47 (m, 2H), 1.39-1.31 (m, 2H),
3.4, 20.9, 20.6, 19.9, 19.6, 18.1, 17.5, 13.5, 12.9; MS (ESI) m/z: 537.7
1
.27-1.19 (m, 5H), 1.01 (m, 9H), 0.93-0.88 (m, 6H), 0.84-0.79 (m, 9H);
+
1
3
[M+H] .
3
C NMR (100 MHz, CDCl ) δ 155.9, 135.3, 135.2, 132.3, 130.8, 81.8,
79.3, 69.6, 56.2, 51.8, 51.6, 51.0, 46.8, 44.6, 42.8, 39.7, 39.3, 36.9, 34.3,
3
3.5, 33.1, 28.6, 26.8, 23.4, 20.9, 20.6, 19.9, 19.6, 18.1, 17.6, 12.9, 11.6;
5α,8α-epidioxyergosta-3-yl-((4-tert-butyl)piperazine-1)carbamate (3i).
+
1
MS (ESI) m/z: 543.7 [M+H] .
White solid, m.p. 208.9-210.4 °C; H NMR (400 MHz, CDCl
=
3
3
) δ 6.51 (d, J
8.5 Hz, 1H), 6.23 (d, J = 8.5 Hz, 1H), 5.18 (m, 2H), 4.95-4.84 (m, 1H),
.40 (s, 8H), 2.17 (dd, J = 13.7, 4.1 Hz, 1H), 1.99 (m, 5H), 1.85 (m, 1H),
5α,8α-epidioxyergosta-3-yl-(piperazine-1)carbamate (3d). Yellow solid,
1
1.73-1.68 (m, 2H), 1.58 (m, 2H), 1.50 (m, 5H), 1.46 (s, 9H), 1.37 (m, 1H),
m.p. 200.9-202.1 °C; H NMR (400 MHz, CDCl
H), 6.23 (d, J = 8.5 Hz, 1H), 5.18 (qd, J = 15.3, 7.7 Hz, 2H), 4.96-4.83
m, 1H), 3.47-3.39 (m, 4H), 2.83 (s, 4H), 2.20-2.14 (m, 2H), 2.05-1.98 (m,
3
) δ 6.50 (d, J = 8.5 Hz,
1
9
1
3
.26-1.19 (m, 3H), 1.00 (d, J = 6.6 Hz, 3H), 0.93-0.90 (m, 6H), 0.82 (m,
H); C NMR (100 MHz, CDCl ) δ 154.6, 135.2, 135.1, 132.3, 130.8,
3
25.1, 122.7, 81.8, 80.5, 80.1, 79.3, 70.7, 56.2, 51.6, 51.0, 44.5, 42.8,
1
(
13
4
4
6
1
5
2
H), 1.86-1.81 (m, 1H), 1.78-1.68 (m, 2H), 1.59 (m, 2H), 1.54-1.44 (m,
H), 1.38 (m, 1H), 1.28-1.19 (m, 4H), 1.00 (d, J = 6.6 Hz, 3H), 0.90 (m,
H), 0.82 (m, 9H); C NMR (100 MHz, CDCl ) δ 154.7, 135.2, 135.1,
3
32.2, 130.8, 126.2, 125.1, 122.2, 115.7, 81.8, 79.4, 70.5, 56.2, 51.6,
1.0, 45.6, 44.5, 42.8, 39.7, 39.3, 36.9, 34.3, 33.6, 33.1, 28.6, 26.8, 23.4,
9.7, 39.3, 36.9, 34.3, 33.5, 33.1, 28.6, 28.4, 26.8, 20.9, 20.6, 19.9, 19.6,
+
13
18.1, 17.5, 12.9; MS (ESI) m/z: 641.8 [M+H] .
5α,8α-epidioxyergosta-3-yl-((4-tert-butyl)piperidine-1)carbamate(3j).
+
1
0.9, 20.6, 19.9, 19.6, 18.1, 17.5, 12.9; MS (ESI) m/z: 541.6[M+H] .
White solid, m.p. 209.3-210.6 °C; H NMR (400 MHz, CDCl
3
) δ 6.51 (d, J
=
4
2
1
8.5 Hz, 1H), 6.23 (d, J = 8.5 Hz, 1H), 5.19 (qd, J = 15.3, 7.7 Hz, 2H),
.88 (s, 1H), 4.49 (s, 1H), 4.00 (s, 2H), 3.63 (s, 1H), 2.85 (t, J = 11.8 Hz,
H), 2.16 (dd, J = 13.9, 4.4 Hz, 1H), 2.04-1.96 (m, 4H), 1.95-1.87 (m, 3H),
.85 (m, 1H), 1.69 (m, 3H), 1.55 (m, 6H), 1.46 (s, 9H), 1.38 (m, 1H), 1.26
5
α,8α-epidioxyergosta-3-yl-((4-hydroxyethyl)piperazine-1)carbamate
1
(
3e). White solid, m.p. 201.5-203.2 °C; H NMR (400 MHz, CDCl
3
) δ 6.51
(d, J = 8.5 Hz, 1H), 6.23 (d, J = 8.5 Hz, 1H), 5.18 (qd, J = 15.3, 7.7 Hz,
(
m, 6H), 1.01 (d, J = 6.6 Hz, 3H), 0.93-0.88 (m, 6H), 0.84-0.81 (m, 9H);
2
2
1
1
0
1
4
1
H), 4.95-4.84 (m, 1H), 3.68-3.58 (m, 2H), 3.47 (d, J = 4.4 Hz, 4H), 2.59-
.53 (m, 2H), 2.52-2.41 (m, 4H), 2.22-2.13 (m, 1H), 2.10-1.92 (m, 5H),
.85 (dd, J = 13.0, 6.8 Hz, 1H), 1.80-1.66 (m, 3H), 1.62-1.43 (m, 6H),
.41-1.32 (m, 2H), 1.23 (dd, J = 9.7, 5.7 Hz, 3H), 1.00 (d, J = 6.6 Hz, 3H),
.91 (m, 6H), 0.82 (m, 9H); C NMR (100 MHz, CDCl ) δ 154.6, 135.2,
3
35.1, 132.3, 130.8, 81.8, 79.3, 70.5, 59.4, 57.7, 56.2, 52.6, 51.6, 51.0,
4.5, 42.7, 39.7, 39.3, 36.9, 34.3, 33.6, 33.1, 28.6, 26.8, 23.4, 20.9, 20.6,
1
3
3
C NMR (100 MHz, CDCl ) δ 154.6, 154.7, 135.2, 135.1, 132.3, 130.8,
8
3
1
1.8, 79.6, 79.3, 69.9, 56.2, 51.6, 51.0, 48.2, 44.5, 42.8, 39.7, 39.3, 36.9,
4.3, 33.5, 33.1, 32.4, 28.6, 28.4, 23.4, 20.9, 20.6, 19.9, 19.6, 18.1, 17.6,
2.9; MS (ESI) m/z: 555.7 [M+H] .
+
13
Compounds 3d·HCl & 3f·HCl. To a stirred solution of 1% HCl (5 mL)
was added compound 3d or 3f (1 mmol). The mixture was stirred at RT
for 15 min. Then, the precipitate was collected by filtration and washed
by cold 50% aq EtOH (5 mL) to afford 3d·HCl or 3f·HCl.
+
9.9, 19.6, 18.1, 17.5, 12.9; MS (ESI) m/z: 585.7 [M+H] .
For internal use, please do not delete. Submitted_Manuscript
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ChemMedChem
10.1002/cmdc.201700021
FULL PAPER
[
22]
Coumarin-3-carboxylic acid (4). A suspension of 4-(diethylamino)-2-
Distance travelled from the initial scratch site was measured and the
migration distance was quantified.
hydroxybenzaldehyde (1.00 g, 5.18 mmol, 1.00 equiv), meldrum’s acid
(
0.75 g, 5.18 mmol, 1.00 equiv), piperidine (0.04 g, 0.52 mmol, 0.10
equiv) and two drops of acetic acid in ethanol (abs, 8 mL) was stirred for
0 min at room temperature and heated to reflux for 3 h. The reaction
Living cell staining for subcellular localization. HepG2 cell lines were
grown on glass cover-slips in 6-well plate (0.4 million cells in 2 mL of
medium per well). For colocalization in mitochondria, the cells were
incubated with 5 (5 µM) for 30 min at 37 °C and mitochondria-specific
green dye Rh123 (100 nM) was added in the last 10 min of incubation.
After removal of HBSS buffer and three times of wash with PBS, the cells
were fixed with 4% para-formaldehyde for 15 min, and then washed with
PBS for three times. The cover-slips were sealed with paraffin for image.
3
mixture was concentrated in vacuum and poured on 15 mL of ice water.
The resulting precipitate was collected and washed with ethanol to give
1
8
54 mg (63%) of an orange crystalline solid. H NMR (400 MHz, CDCl
3
)
δ 12.50 (br s, 1H, –COOH), 8.58 (s, 1H, CH=C), 7.63 (d, 1H, J= 9.0 Hz,
5
H-ph), 6.79 (d, 1H, J= 9.0 Hz, 6H-ph), 6.57 (s, 1H, 8H-ph), 3.48 (q, 4H,
13
J = 7.0 Hz, N(CH
2 3
–CH )
2
), 1.14 (t, 6H, J = 6.9 Hz, N(CH
) δ 165.5, 164.5, 158.1, 153.8, 150.2, 132.0,
10.0, 108.5, 105.5, 96.8, 77.4, 77.1, 76.7, 45.3, 12.4. MS (ESI) m/z:
2 3
–CH )
2
).
C
NMR (100 MHz, CDCl
1
2
3
Confocal microscopic imaging. Confocal images were captured with a
-
60.0 [M-H] .
710M confocal microscope (Carl Zeiss, Germany), original magnification
1
00×. 520 nm was used for excitation of Rh123. For compound 5, the
Synthesis of compound 5. Coumarin-3-carboxylic acid 4 (44 mg, 0.15
mmol, 13 equiv), DCC (31 mg, 0.15 mmol, 13 equiv), and DMAP (2.0 g,
excitation laser was 405-410 nm and images were collected at emission
of 465-470 nm. After image acquisition, colocalization analysis of
compound 5 and the Mito specific dyes on images were processed by
using LSM 510 software.
10%) were added to a solution of 3d (54 mg, 0.1 mmol, 1.0 equiv) in
anhydrous DCM (15 mL). The reaction mixture was stirred at RT for 36 h
and filtrated. The filtrate was removed under vacuum, and was purified
1
by flash column chromatography to provide 5 (32 mg). Yellow crystals. H
NMR (400 MHz, CDCl
J = 8.9, 2.3 Hz, 1H), 6.49 (dd, J = 11.9, 5.3 Hz, 2H), 6.23 (d, J = 8.5 Hz,
H), 5.18 (qd, J = 15.3, 7.7 Hz, 2H), 4.97-4.85 (m, 1H), 3.72 (s, 2H), 3.54
s, 4H), 3.44 (q, J = 7.1 Hz, 4H), 3.38 (s, 2H), 2.17 (d, J = 4.8 Hz, 1H),
3
) δ 7.87 (s, 1H), 7.30 (d, J = 8.9 Hz, 1H), 6.60 (dd,
Acknowledgements
1
(
The authors would like to acknowledge financial support from
the Chinese Natural Science Foundation Project (21272020),
Beijing Key Laboratory of Environmental and Viral Oncology and
Beijing Key Laboratory for Green Catalysis and Separation.
2
1
1
0
1
1
3
1
.03 (dd, J = 16.1, 8.3 Hz, 4H), 1.85 (d, J = 6.5 Hz, 1H), 1.75-1.70 (m,
H), 1.63 (m, 4H), 1.58 (dd, J = 14.9, 5.6 Hz, 2H), 1.51 (t, J = 5.8 Hz, 2H),
.40-1.36 (m, 1H), 1.25 (d, J = 4.8 Hz, 4H), 1.23 (m, 6H), 1.01 (m, 3H),
13
3
.90 (m, 6H), 0.82 (m, 9H). C NMR (100 MHz, CDCl ) δ 165.2, 159.1,
57.3, 154.5, 151.8, 135.2, 135.1, 132.3, 130.8, 129.9, 115.8, 109.4,
07.7, 96.9, 81.8, 79.3, 70.8, 56.2, 51.6, 51.0, 44.9, 44.5, 42.7, 39.7,
9.3, 36.9, 34.3, 33.5, 33.0, 28.6, 23.3, 20.9, 19.9, 19.6, 18.1, 17.5, 12.8,
2.4.
Keywords: ergosterol peroxide • carbamate • fluorescence
image• coumarin • mitochondria-targeting
[
1]
M. Bauch, M. Klaper, T. Linker, J. Phys. Org. Chem. 2016. DOI:
0.1002/poc.3607.
1
Biology
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[
3]
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77-383; b) M. Axelrod, V. L. Gordon, M. Conaway, A. Tarcsafalvi, D. J.
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bromide assay. Human hepatic carcinoma cells (HepG2, SK-Hep1) and
human breast cancer cells (MCF-7, MDA-MB231) were used in the study.
The cells were cultured in DMEM/RPMI1640 supplemented with 10%
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159-165; b) M. Jung, H. Kim, K. Lee, M. Park, Mini-Rev. Med. Chem.
5% CO
2
. Cells (1 × 10 cells/mL) were seeded into 96-well plates and
2
003, 3, 159-165; c) Y. Imamura, M. Yukawa, M. Ueno, K. i. Kimura, E.
were incubated at 37 °C overnight in a humidified incubator containing
% CO . Cells were dosed with compounds at final concentrations
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H. Huang, J. Jiang, X. Zhu, Y. Liu, L. Liu, Y. Lu, M. Li, Y. Lin, Z. She, J.
Nat. Prod. 2011, 74, 1230-1235; e) S. Noori, Z. M. Hassan, Tumor Biol.
5
2
ranging from 0.2 μM to 40 μM in each well of the plates. The cells were
incubated for various periods and analyzed by MTT assay to analyze
rates of cell proliferation as described. Cell survival was determined by
measuring the absorbance at 490 nm using a microplate reader. A
calibration curve was prepared using the SPSS to determine the IC50 of
the target compounds. Cytotoxicity as IC50 for each cell line is the
concentration of compound which reduced by 50% the optical density of
treated cells (48 h) with respect to untreated cells using the MTT assay.
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[
7]
Cell migration assay. Migration assay was performed by wound scratch
5
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Huang, Bioresour. Technol. 2012, 123, 86-91; b) B. Yu, E. Zhang, X. N.
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was monitored and photographed. Microscope images of the scratched
cultures were captured at the beginning and at different intervals later.
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FULL PAPER
[
a]
[a]
[a]
Ming Bu, Tingting Cao, Hongxia Li,
[
c]
[d]
Mingzhou Guo, Burton B. Yang,
Chengchu Zeng and Liming Hu*
[
a]
[a,b]
Page No. – Page No.
Synthesis of 5α,8α-ergosterol
peroxide 3-carbamate derivatives and
fluorescent mitochondria-targeting
conjugate for enhanced anticancer
activities
Small changes, big difference! Through structural changes to synthesized a
series of 5α,8α-ergosterol peroxide 3-carbamate derivatives (3a-3j). Compound 3d,
3f and their hydrochloride exhibited significant in vitro anti-proliferative activity
against the tested tumor cell lines. Fluorescence mitochondria-targeting conjugate 5
was obtained by linking coumarin-3-carboximide with 3d. The probe localized in
mitochondria, leading to enhanced anticancer activity over the parent structure.
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