Syntheses of 7-dehydrocholesterol peroxides and their improved anticancer activity and selectivity over ergosterol peroxide

Article abstract of DOI:10.1039/c7nj04100d

7-Dehydrocholesterol peroxide (5α,8α-epidioxycholest-6-ene-3β-ol, CEP) and its acetate and hemisuccinate derivatives were synthesized and isolated for the first time, which exhibit improved anticancer activity and selectivity over ergosterol peroxide (5α,8α-epidioxy-22E-ergosta-6,22-dien-3β-ol, EEP), showing potential as new chemotherapeutic agents.

Full text of DOI:10.1039/c7nj04100d

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Syntheses of 7-dehydrocholesterol peroxides and
their improved anticancer activity and selectivity
over ergosterol peroxide†
Cite this:DOI: 10.1039/c7nj04100d
Received 23rd October 2017,
Accepted 11th November 2017
ab
a
ab
a
a
Na-na Tian, Chao Li,* Na Tian, Qian-xiong Zhou, Yuan-jun Hou,
a
ab
Bao-wen Zhang and Xue-song Wang
*
DOI: 10.1039/c7nj04100d
rsc.li/njc
7
-Dehydrocholesterol peroxide (5a,8a-epidioxycholest-6-ene-3b-ol,
CEP) and its acetate and hemisuccinate derivatives were synthesized
and isolated for the first time, which exhibit improved anticancer
activity and selectivity over ergosterol peroxide (5a,8a-epidioxy-22E-
ergosta-6,22-dien-3b-ol, EEP), showing potential as new chemo-
therapeutic agents.
Endoperoxides are promising pharmacophores for antimalarial,
1
anticancer, and antiviral agents. One of the most prominent
examples is artemisinin, which is a sesquiterpene lactone endo-
peroxide and exhibits excellent antimalarial activity through
homolytic cleavage of the O–O bond induced by endogenous
2
reductants. Similar to artemisinin, ergosterol peroxide (5a,8a-
epidioxy-22E-ergosta-6,22-dien-3b-ol, EEP) has also shown a variety
3
4
of biological effects, such as anti-inflammatory, antibacterial,
5
and antitumor activities, and as a result has received much
Scheme 1 Photochemical conversions of ergosterol and 7-DHC and
biological effects of the products.
attention as a new drug candidate. Although EEP is widely found
6
7
8
in mushrooms, plants, and lichens, the most facile and
economic way to obtain it in large quantity is photosensitized
5
c,9
oxidation of ergosterol (Scheme 1).
one may expect that 7-dehydrocholesterol peroxide (5a,8a-
As an analogue of ergosterol, 7-dehydrocholesterol (7-DHC) epidioxycholest-6-ene-3b-ol, CEP) may present different bio-
plays important roles in animals. It is the bio-synthetic pre- logical effects with respect to its analogue of EEP. It is really a
cursor of vitamin D
trations in skin, in which it is converted to vitamin D
irradiation (Scheme 1). Ergosterol may also undergo similar of research on the synthesis and isolation of EEP and its bio-
3
and is present in relatively high concen- surprise that there are very few studies on the photosensitized
upon UV oxidation of 7-dehydrocholesterol compared to a large quantity
3
1
0
photochemical conversion to vitamin D upon exposure to UV logical effects. P. W. Albro and L.-Z. Wu’s groups reported the
2
light. Interestingly, though vitamin D and vitamin D are very synthesis of CEP from 7-DHC, however, a 3 : 1 mixture of CEP
3
2
similar in the treatment of rickets for people and mammalian and CHP (5a-7-hydroperoxy-5,8-diene-3b-ol) was obtained and
1
1
3
animals, the therapeutic effect of vitamin D is much higher no further isolation of CEP was performed. Up to now, there
than that of vitamin D
2
for poultry and birds. As a result, is no report associated with the biological effects of CEP. Such a
gap inspired us to explore the photochemical synthesis and
a
biological effect of CEP. Moreover, the 3b-hydroxyl group of
Key Laboratory of Photochemical Conversion and Optoelectronic Materials,
Technical Institute of Physics and Chemistry Chinese Academy of Sciences,
Beijing 100190, P. R. China. E-mail: xswang@mail.ipc.ac.cn,
lychaof@mail.ipc.ac.cn
sterols provides a versatile handle for chemical modification to
finely tune their biological properties, for example, glucose
conjugation of EEP led to an improved proliferation inhibition
b
University of Chinese Academy of Sciences, Beijing 100049, P. R. China
1
2
1
over many tumor cell lines, and cholesterol hemisuccinate was
found to alter the function of P-glycoprotein (Pgp), a protein
that has been linked to the emergence of multidrug resistant
†
Electronic supplementary information (ESI) available: Synthesis, H NMR,
13
C NMR, COSY, HMBC, ESI-MS spectra, reaction optimization for synthesis of
0
0
EEP, 2 and 3 . See DOI: 10.1039/c7nj04100d
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performance may mainly due to their bleaching during the
photoreaction. In sharp contrast, the recycled TPP from the
entry 6 can be reused to give a yield of 73% (entry 13). As
expected, elevated reaction temperature is unfavourable for the
production of CEP (entry 14).
A similar trend was also found in the preparation of EEP
from ergosterol (Table S1, ESI†). The use of n-hexane/methanol
(
3 : 1) as solvent led to an enhanced yield of EEP with respect to
5c,11a
0
0
the literature results.
Besides a higher isolated yield of CEP
Scheme 2 Structures of 1–3, 1 –3 , and EEP.
or EEP, the mixed solvent of n-hexane/methanol we used is more
economic and environmentally friendly than the solvents, such
1
3
(
(
MDR) cancer. Thus, CEP acetate and CEP hemisuccinate as pyridine, benzene, and CH Cl , that have been widely used in
2 2
0
0
5c,11a
2 and 3 in Scheme 2) were also photochemically synthesized the photochemical syntheses of EEP.
7-Dehydrocholesterol
and their anticancer activity was compared with that of CEP acetate and 7-dehydrocholesterol hemisuccinate (2 and 3 in
and EEP. Encouragingly, CEP and its hemisuccinate derivative Scheme 2) were also synthesized and their corresponding
0
0
display much improved in vitro anticancer activity against human peroxides 2 and 3 were prepared photochemically in the yields
breast cancer cells SKOV-3, cervical carcinoma cells HeLa, lung of 78% and 81%, respectively (Table S1, ESI†).
0
cancer cells A549 and prostatic carcinoma cells DU145 and
The chemical structure of CEP (1 ) and its acetate and
0
0
diminished cytotoxicity toward human normal liver cells L-02 hemisuccinate derivatives (2 and 3 ) were fully characterized
1
13
with respect to EEP.
by high-resolution ESI-MS, H NMR, C NMR, HMBC, and
The photooxidation reaction of 7-DHC was carried out at COSY through comparison with EEP and other structurally
1
4
0
2
1C in different O -saturated solvents using meso-tetraphenyl- related sterol endoperoxides (Fig. S1–S30, ESI†). Taking CEP
porphyrin (TPP) as the photosensitizer and a high-pressure Hg as an example, the ESI-MS ion peak of CEP at m/z 439.3161 is
+
light equipped with a 400 nm cut-off filter as the light source. in good agreement with [M + Na] . The assignments of all
1
3
1
As shown in Table 1, after full consumption of 7-DHC, the
isolated yields of CEP followed an order of pyridine 4 n-hexane 4 Tables S2 and S3 (ESI†), and those of EEP are in good agreement
C signals and partial H signals of CEP and EEP are compiled in
14c
2 2
benzene 4 CH Cl (entries 1–4). Notably, when a mixed solvent of with the reported values. These chemical shift assignments get
n-hexane and methanol was used as the medium, improved yields full support from the HBMC spectra. For example, one olefin
were obtained. At the volume ratio of 3 :1 for n-hexane/methanol, proton at 6.24 ppm (H7, see atom numbering in Fig. 1) of CEP
the isolated yield of CEP was as high as 81% (entries 5–8). While showed correlations with C5, C6, C8, C9 and C14 in its HMBC
increasing TPP did not give rise to further improvement for the spectrum, while the other olefin proton at 6.51 ppm (H6) corre-
photoreaction, the reduced TPP from 0.1 to 0.05 equiv. led to a lated with C4, C5, C7, and C8. The chemical shifts of both olefin
lower yield (entry 9). Other single oxygen photosensitizers, such protons and their coupling constant of 8.4 Hz, in combination
as hematoporphyrin, eosin Y, and methylene blue, can also with two oxygenated quaternary carbons at C-5 (82.3 ppm) and C-8
realize the transformation in n-hexane/methanol (3: 1), but with (79.5 ppm), support the 5a,8a-epidioxy structure in the B ring.1
lower yields than the case of TPP (entries 10–12). Their poor Furthermore, the HMBC spectrum may aid in the differentiation
of H-18 (singlet) from H-19 (singlet) by its coupling to C17, an
4a
atom that is also coupled to H-21 (doublet).
MTT assay was used to evaluate the cytotoxicity of EEP, CEP
0 0 0
T (1C) t (h) Yield (%) (1 ) and its two derivatives (2 and 3 ) toward human lung cancer
a
Table 1 Reaction optimization for the synthesis of CEP
b
c
Entry Photosensitizer (mol%) Solvent
cells A549, breast cancer cells SKOV-3, cervical carcinoma
cells HeLa and prostatic carcinoma cells DU145, as well as
human normal liver cells L-02. 7-DHC (1) and its two derivatives
1
2
3
4
5
6
7
8
9
1
1
1
1
1
TPP (0.1)
TPP (0.1)
TPP (0.1)
TPP (0.1)
TPP (0.1)
TPP (0.1)
TPP (0.1)
TPP (0.1)
Pyridine
Benzene
CH Cl
2 2
0
0
0
0
0
0
0
0
0
0
0
0
0
2
2
1
3
3
3
3
3
3
3
3
3
3
3
63
58
43
62
69
81
74
70
64
56
40
62
73
51
n-Hexane
Mix (4 : 1)
Mix (3 : 1)
Mix (2 : 1)
Mix (1 : 1)
Mix (3 : 1)
Mix (3 : 1)
Mix (3 : 1)
Mix (3 : 1)
Mix (3 : 1)
d
d
d
d
d
d
d
d
d
d
(2 and 3) were also tested as negative controls. Fig. 2 shows the
TPP (0.05)
0
1
2
3
4
Hematoporhyrin (0.1)
Eosin Y (0.1)
Methylene blue (0.1)
e
TPP (0.1)
TPP (0.1)
Mix (3 : 1) 30
a
Reaction conditions: 1.56 mmol 7-DHC and 1.9 mM photocatalyst in
2
0 mL of solvent was subjected to visible light irradiation (Z400 nm)
b
under magnetic stirring and bubbling with oxygen. By which all
substrate was consumed. Isolated yield. Mixed solvent of n-hexane/
methanol with varied volume ratio. Recycled TPP from entry 6 was
c
d
e
reused.
Fig. 1 Atom numbering and key COSY and HMBC correlations of CEP.
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0
0
0
0
0
Fig. 2 Cytotoxicity of 1–3, 1 –3 and EEP against A549 (a), SKOV-3 (b),
HeLa (c), and DU145 (d) cells. Data are presented as means ꢀ S.D. of
triplicate samples.
Fig. 3 Cytotoxicity selectivity of 1 (a), 2 (b), 3 (c) and EEP (d) against
various tumor cells over normal L-02 cells. Data are presented as means ꢀ
S.D. of triplicate samples.
cell viability of the examined tumor cells at 37 1C after 48 h of
In summary, CEP and its two derivatives were synthesized and
incubation with varied concentrations of the examined com- isolated in high yields for the first time by the photosensitized
pounds, and the IC50 values are collected in Table 2. While both oxidation approach in a mixed solvent of n-hexane/methanol
CEP and EEP showed cytotoxicity against the examined tumor (3 : 1, v/v). Compared to EEP, CEP and its hemisuccinate derivative
cells, the in vitro anti-cancer activity of CEP is significantly larger exhibit much improved anticancer activity and selectivity, showing
than that of EEP. Taking A549 cells as an example, they may be promising application potential in cancer chemotherapy.
totally inactivated at around 40 mM of CEP, however, the cell
viability was still as high as 80% when incubated with EEP at the
same concentration. Such results reveal the important role of the
Experimental
side chain of the sterol endoperoxides on their biological effects.
Additionally, the lack of cytotoxicity of 7-DHC implies that the
Syntheses of CEP and its derivatives. A solution of 1, 2, or 3
(
(
1.56 mmol) and TPP (1.9 mmol) in 20 mL hexane/methanol
3 : 1) was irradiated for 3 h at 0 1C under magnetic stirring and
anticancer activity of both CEP and EEP may result from their
endoperoxide moieties, which was further confirmed by compar-
continuous bubbling with oxygen. A 500 W high-pressure Hg lamp
in combination with a 400 nm cut-off glass filter was employed as
the light source. The crude product was purified on silica gel using
n-hexane/ethyl acetate (3 : 1 in volume ratio) as eluent.
0
0
ing the cytotoxicity of 2 and 3 with respect to that of 2 and 3.
0
Interestingly, though the acetate derivative of CEP (2 ) generally
showed lower anticancer activity than CEP, its hemisuccinate
derivative indeed exhibited an improved inactivation ability over
the all examined tumor cell lines, demonstrating the importance
of the chemical modification by way of the 3b-OH group. Notably,
both CEP and its hemisuccinate derivative display improved
cancer selectivity than EEP, as evidenced by much diminished
cytotoxicity toward L-02 cells as shown in Fig. 3, suggesting their
promising potential as new chemotherapeutic agents.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was financially supported by Ministry of Science and
Technology (2013CB933801), NSFC (21390400, 21571181 and
0
0
Table 2 IC50 values of 1–3, 1 –3 and EEP
2
1773277), and the Strategic Priority Research Program of the
IC50 (mM)
Chinese Academy of Sciences (XDB17000000).
Compound
A549
SKOV-3
HeLa
DU145
1
2
3
1
2
3
EEP
4100
4100
4100
25.6 ꢀ 0.1
34.4 ꢀ 0.1
16.1 ꢀ 0.02
4100
4100
4100
4100
28.5 ꢀ 0.1
26.6 ꢀ 0.1
9.9 ꢀ 0.02
92.3 ꢀ 0.8
4100
4100
4100
41.1 ꢀ 0.2
83.0 ꢀ 0.2
16.5 ꢀ 0.02
51.9 ꢀ 0.2
4100
4100
4100
20.4 ꢀ 0.2
33.2 ꢀ 0.2
14.4 ꢀ 0.02
50.0 ꢀ 0.5
Notes and references
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