Highly efficient green synthesis and photodynamic therapeutic study of hypericin and its derivatives

Article abstract of DOI:10.1039/c8ra03732a

A highly efficient synthetic pathway for hypericin (7a) was achieved under mild conditions with an overall yield over two steps of 92% using emodinanthrone as a starting material, where protohypericin, a key precursor of hypericin, was synthesized in water with microwave assistance, which was then photocyclized to hypericin with a high yield via 1 h irradiation in a visible light reactor equipped with 575 nm monochromatic lamps. In addition, the method could be used to synthesize hypericin derivatives (7b-d) with similar overall yields. Furthermore, their effects of photodynamic therapy (PDT) were evaluated on A431, HepG-2, and MCF-7 cell lines. The PDT of 7b was better than that of 7a, whereas 7c and 7d were worse. Unlike other cell lines, MCF-7 was not sensitive to any of 7a-d at the same concentrations.

Full text of DOI:10.1039/c8ra03732a

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Highly efficient green synthesis and photodynamic
therapeutic study of hypericin and its derivatives†
Cite this: RSC Adv., 2018, 8, 21786
Ying Zhang, Kun Shang, Xiaowen Wu, Siyu Song, Zebo Li, Zhichao Pei
and Yuxin Pei
*
A highly efficient synthetic pathway for hypericin (7a) was achieved under mild conditions with an overall
yield over two steps of 92% using emodinanthrone as a starting material, where protohypericin, a key
precursor of hypericin, was synthesized in water with microwave assistance, which was then
photocyclized to hypericin with a high yield via 1 h irradiation in a visible light reactor equipped with
5
75 nm monochromatic lamps. In addition, the method could be used to synthesize hypericin derivatives
Received 1st May 2018
Accepted 2nd June 2018
(7b–d) with similar overall yields. Furthermore, their effects of photodynamic therapy (PDT) were
evaluated on A431, HepG-2, and MCF-7 cell lines. The PDT of 7b was better than that of 7a, whereas 7c
and 7d were worse. Unlike other cell lines, MCF-7 was not sensitive to any of 7a–d at the same
concentrations.
DOI: 10.1039/c8ra03732a
rsc.li/rsc-advances
temperature, pressure and as having a very long reaction time
Introduction
(up to 10 days). Besides the total synthesis, semi-synthesis of
Hypericin is a natural polycyclic aromatic dianthraquinone hypericin was proposed by Falk and coworkers using emodin
1
1
present in the St. John's Wort plant (Hypericum perforatum). It extracted from Cortex frangulae as the starting material. Falk's
has been used as a traditional herbal medicine since ancient method only required 3 steps, where emodin was rst reduced
2
times for its anti-viral, anti-bacterial, and anti-inammatory to emodinanthrone by SnCl in concentrated HCl with a high
2–7
properties as well as to treat depression. In recent years, it yield of 90%, and then emodinanthrone took place oxidative
has attracted increasing interest due to its potential as an dimerization in pyridine to form protohypericin, which was
effective photosensitizer used in photodynamic therapy (PDT) nally photocyclized to hypericin upon irradiation of visible
8–10
to ght various cancers.
light overnight in a moderate yield of 56% (overall yield was
The isolation of pure hypericin from Hypericum perforatum is 51.6%). Although the overall yield was later increased to 74% by
a tedious procedure due to its very low content in the plant (0.03 the same group by introducing microwave assistance in the
11
1
to 0.09%) and poor solubility in solvents. To meet the needs synthesis of protohypericin, the reaction was carried out in
of hypericin demand both from academic and industrial areas, a toxic organic solvent DMF and in the presence of a corrosively
16
great efforts have been made in the past to develop a way for it to organic base potassium t-butoxide. Additionally, the photo-
be chemically synthesized. The rst step towards the successful cyclization step of protohypericin to hypericin took place over-
17
chemical synthesis of hypericin dates back to 1957, when night with a 500 W halogen lamp. Therefore, it is necessary to
Brockmann and his co-workers for the rst time reported a total nd an efficient pathway for the synthesis of hypericin, where
synthetic pathway of hypericin. Their pathway started from 3,5- the required reactions can be performed under mild conditions,
dimethoxybenzoic acid methyl ester and involved 12 steps in the solvents involved in each step are less or non-toxic, easy to
total that resulted in a low overall yield of hypericin of 6% to handle aer the reactions, and would preferably be water based
12
9
%. Other total synthetic methods that were later developed to comply with the standpoints of green chemistry.
started with ethyl formate, chlorinated ketene or 2-methyl
In this paper, as shown in Scheme 1, a greatly more efficient,
anthraquinone, and shortened the synthetic pathway to 6 to 8 facile and environmentally friendly method for the synthesis of
steps, although none of these methods reached an overall yield hypericin (7a) was developed. This method started with the
6,13–15
that was higher than 10%.
In addition, these methods were easily available emodinanthraquinone, from where its
generally characterized by the usage of extremely high precursor protohypericin was obtained in water by using
microwaves and the irradiation of protohypericin in acetone
with 575 nm visible light. This method resulted in a high
Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry
&
Pharmacy, Northwest A&F University, Yangling, Shaanxi 712100, PR China. E-mail: hypericin overall yield. Furthermore, we delightedly found that
peiyx@nwafu.edu.cn
the strategy could be successfully adapted for the synthesis of
the derivatives of hypericin (7b–d), which are especially
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c8ra03732a
1
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1
,3,8-Trihydroxy-6-methylanthracen-9(10H)-one (5a). Compound
22
5
a was synthesized by following a published procedure. To
a 250 mL ask, emodin (1.36 g, 5.0 mmol) was stirred in 100 mL
ꢀ
glacial acetic acid (98%) at 60 C. Then SnCl
2
$2H
2
O (7.9 g, 35
mmol) dissolved in 50 mL concentrated hydrochloric acid was
added dropwise into the ask. Then this resulting solution was
reuxed for 2 h, which was then poured into ice water. The
precipitate formed was ltered, washed with deionized water,
and then vacuum dried. The crude was puried by ash column
Scheme 1 General synthetic pathway of hypericin (7a) and its deriv- chromatography (eluent: petroleum ether/ethyl acetate ¼ 4 : 1,
1
atives (7b–d). Reagents and conditions: (1) 1.5% NaOH/H
2
O, pyridine-
v/v) to give 5a as a pale yellow solid (1.22 g, 96%). H NMR (500
ꢀ
N-oxide, FeSO
4
$7H
2
O, 105 C, 10 W, 70 min, N
2
by microwave reactor;
6
MHz, DMSO-d ) d 12.37 (s, 1H), 12.21 (s, 1H), 10.82 (s, 1H), 6.77
(
2) 575 nm monochromatic light, acetone, N
2
, 60 min.
(
2
s, 1H), 6.67 (s, 1H), 6.42 (s, 1H), 6.22 (d, J ¼ 4.3 Hz, 1H), 4.29 (s,
23
H), 2.32 (s, 3H) ppm.
,5,7-Trihydroxy-10-oxo-9,10-dihydroanthracene-2-carboxylic acid
5b). Compound 5b was synthesized by following a published
4
18,19
signicant in new photosensitizer discovery
used in cancer
(
photodynamic therapy.
22
procedure. To a 50 mL ask, compound 4 (300 mg, 1.0 mmol) was
dissolved in 20 mL glacial acetic acid at 60 C. To this solution,
ꢀ
2 2
SnCl $2H O (1.59 g, 7.0 mmol) dissolved in 11 mL concentrated
hydrochloric acid was added drop wise. Then this resulting solution
was reuxed for 2 h, which was then poured into ice water. The
precipitate formed was ltered, washed with deionized water, and
Experimental
General remarks
All reagents and solvents were purchased from commercial vacuum dried. The crude was puried by ash column chromatog-
suppliers and used without further purication unless specied. raphy (eluent: petroleum ether/ethyl acetate ¼ 4 : 1, v/v) to give 5b as
1
Dimethyl sulfate, N-bromosuccinimide (NBS), 18-crown-6 and a pale yellow solid (0.3 g, 92%). H NMR (500 MHz, DMSO-d ) d 13.24
6
benzoyl peroxide (BPO) were purchased from Energy Chemical (br, 1H), 12.29 (s, 1H), 12.22 (s, 1H), 10.99 (s, 1H), 7.46 (s, 1H), 7.27 (s,
24
Reagent Co. Triphenyl phosphine and tin(II) chloride dehydrate 1H), 6.445–6.439 (m, 1H), 6.25 (d, J ¼ 2.7 Hz, 1H), 4.42 (s, 2H) ppm.
SnCl $2H O) were purchased from Sinopharm Chemical Reagent
1,3,8-Trihydroxy-6-pentylanthracen-9(10H)-one (5c). Compound
Co. Butyraldehyde was purchased from TCI. Benzaldehyde was 5c was synthesized by following a published procedure with modi-
(
2
2
22
purchased from Aladdin. Ferrous sulfate heptahydrate (FeSO4- cation. To a 25 mL ask, 12 (55.6 mg, 0.15 mmol) was dissolved
7H O) was purchased from Tianjin Bodi Chemical Co., Ltd. 3-(4,5- in 7 mL glacial acetic acid. To this solution, SnCl $2H O (270 mg, 1.2
$
2
2
2
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was mmol) dissolved in 4 mL 33% HBr was added drop wise. Aer being
purchased from Kehao Biotech Co., Ltd. Flash chromatography reuxed for 1.5 h, the reaction mixture was then poured into ice
was performed using silica gel with a grain size of 40–63 mm water. The precipitate formed was ltered, washed with deionized
(Qingdao Haiyang Co., Ltd). RPMI 1640 medium, Dulbecco's water, and vacuum dried. The crude product was puried by ash
Modied Essential Medium (DMEM) and trypsin–EDTA solution column chromatography (eluent: petroleum ether/ethyl acetate ¼
1
were obtained from Gibco BRL Co., Ltd. Penicillin/streptomycin 6 : 1, v/v) to give 5c as a pale yellow solid (33.6 mg, 71%). H NMR
was purchased from Nanjing KeyGen Biotech, fetal bovine serum (500 MHz, DMSO-d ) d 12.37 (s, 1H), 12.20 (s, 1H), 10.81 (s, 1H), 6.77
6
was purchased from YuanhengJinma Co., Ltd.
(s, 1H), 6.66 (s, 1H), 6.41 (d, J ¼ 1.3 Hz, 1H), 6.23 (d, J ¼ 2.2 Hz, 1H),
1
13
H NMR and C NMR spectra were recorded on a Bruker 500 4.29 (s, 2H), 2.55 (t, J ¼ 7.45 Hz, 2H), 1.60–1.54 (m, 2H), 1.34–1.23
1
13
MHz Spectrometer with working frequencies of 500 MHz for H (m, 4H), 0.86 (t, J ¼ 7.0 Hz, 3H) ppm; C NMR (125 MHz, DMSO-d )
6
1
3
6 4
and 125 MHz for C, respectively, in DMSO-d , MeOH-d or d 191.0, 165.0, 164.5, 161.7, 151.7, 145.0, 141.9, 119.1, 114.4, 113.0,
1
13
CDCl
3 6
. The residual signals from DMSO-d ( H: d 2.50 ppm; C: 108.4, 107.3, 100.9, 35.4, 32.3, 30.9, 29.8, 21.9, 13.9 ppm. HRMS: m/z
1
13
+
d 39.52 ppm), or MeOH-d
4
( H: d 3.31 ppm; C: d 49.00 ppm), calcd for C H O [M + H] 313.1440, found 313.1439. M.p.: 161.4–
19 20 4
1
13
ꢀ
CDCl
standards. HRMS (High Resolution Mass Spectrometer) anal-
ysis was performed on Agilent 1290-6540 UHPLC Q-TOF-HRMS. Compound 5d was synthesized by following a published
3
( H: d 7.26 ppm; C: d 77.00 ppm) were used as internal 162.0 C.
1,3,8-Trihydroxy-6-phenethylanthracen-9(10H)-one
(5d).
22
Microwave assisted syntheses were performed with Discover SP procedure with modication. To a 100 mL ask, 14 (40 mg,
USA). Photochemical reaction was performed with a Rayonet 0.85 mmol) was dissolved in 30 mL acetic acid. To this resulting
Chamber Reactor (Model RPR-100) equipped with 16 of 575 nm solution, SnCl $2H O (1.53 g, 6.8 mmol) dissolved in 20 mL
(
2
2
monochromatic lamps (25 W per lamp, 400 W in total, USA). 33% HBr was added drop wise. Aer being reuxed for 1.5 h, the
The UV-Vis spectra were recorded with a Shimadzu 1750 UV- reaction mixture was then poured into 50 mL ice water. The
Visible spectrophotometer (Japan) at 298 K. The melting precipitate formed was ltered, washed with deionized water,
points were measured by a WRS-2 melting point apparatus and vacuum dried. The crude was puried by ash column
ꢀ
(
Shanghai, China) with a range of room temperature to 300 C. chromatography (eluent: petroleum ether/ethyl acetate ¼ 6 : 1,
1
The compounds 4, 12, 14 were prepared according to the pub- v/v) to give 5d as a pale yellow solid (265 mg, 90%). H NMR (500
20,21
lished procedures
(details can be found in ESI†).
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MHz, DMSO-d
6
) d 12.37 (s, 1H), 12.21 (s, 1H), 10.85 (s, 1H), 7.30– chromatography on silica gel by using petroleum ether/ethyl
7.23 (m, 4H), 7.20–7.15 (m, 1H), 6.85 (s, 1H), 6.74 (s, 1H), 6.43 (d, acetate/methanol (2 : 8 : 0.5, v/v/v) as eluent to give respective
J ¼ 1.2 Hz, 1H), 6.22 (d, J ¼ 2.1 Hz, 1H), 4.30 (s, 2H), 2.93–2.85 7a–d.
1
3
1
(
m, 4H) ppm. C NMR (125 MHz, DMSO-d ) d 191.1, 165.0,
Hypericin (7a). Purple solid (122 mg, 96%). H NMR
64.6, 161.7, 150.7, 145.0, 142.0, 141.2, 128.4, 128.3, 126.0, (500MHz, DMSO-d ) d 14.66 (s, 2H), 14.03 (s, 2H), 7.31 (s, 2H),
19.3, 114.6, 113.2, 108.4, 107.4, 101.0, 37.2, 36.0, 32.4 ppm. 6.44 (s, 2H), 2.65 (s, 6H) ppm.
6
1
1
6
25
+
HRMS: m/z calcd for C22
3
H
18
ꢀ
O
4
[M + H] 347.1283, found
1,6,8,10,11,13-Hexahydroxy-7,14-dioxo-7,14-dihydrophenanthro
[1,10,9,8-opqra]perylene-3,4-dicarboxylic acid (7b). Purple solid
47.1281. M.p.: 196.7–197.1 C.
1
(135 mg, 96%). H NMR (500 MHz, DMSO-d
6
) d 14.73 (s, 2H),
24
1
4.11 (s, 2H), 7.51 (s, 2H), 6.61 (s, 2H) ppm.
General procedure for the synthesis of protohypericin and its
derivatives
1,3,4,6,8,13-Hexahydroxy-10,11-dipentylphenanthro[1,10,9,8-
1
opqra]perylene-7,14-dione (7c). Purple solid (144 mg, 94%). H
To a microwave 10 mL tube, 5a, 5b, 5c, or 5d (0.5 mmol, 1 NMR (500 MHz, MeOD) d 7.24 (s, 2H), 6.69 (s, 2H), 1.40–1.12 (m,
equiv.), pyridine-N-oxide (2.5 mmol, 5 equiv.), FeSO $7H 6H), 1.08–0.94 (m, 2H), 0.93–0.80 (m, 4H), 0.74–0.61 (m, 6H),
4
2
O
1
3
(
10 mg, 36 mmol), and NaOH (40 mg, 1.0 mmol) were dissolved 0.47 (t, J ¼ 7.2 Hz, 6H) ppm. C NMR (125 MHz, DMSO-d
6
)
in 2 mL ultrapure water. The mixture was placed in a microwave d 191.5, 165.4, 165.0, 162.2, 152.1, 145.4, 142.4, 119.6, 114.9,
ꢀ
reactor at 10 W, 105 C under argon atmosphere for 70 min. The 113.5, 108.9, 107.8, 101.4, 40.5, 40.3, 40.2, 40.0, 39.8, 39.7, 39.5,
reaction mixture was then cooled to room temperature, and was 35.8, 32.8, 31.4, 30.3, 22.4, 14.4 ppm. HRMS: m/z calcd for
+
ꢀ
acidied with 3% hydrochloric acid. The precipitate was
ltered, washed with deionized water, and vacuum dried. The
C
38
H
32
O
8
[M + H] , 617.2175, found: 617.2176. M.p. > 300 C.

3,4-Dibenzyl-1,6,8,10,11,13-hexahydroxyphenanthro[1,10,9,8-
purication with column chromatography on silica gel by using opqra]perylene-7,14-dione (7d). Purple solid (160.6 mg, 94%). H
petroleum ether/ethyl acetate/methanol (4 : 8 : 1, v/v/v) as NMR (500 MHz, MeOD) d 7.34 (s, 2H), 6.75 (s, 2H), 6.35–6.21 (m,
1
eluent gave 6a–d, respectively.
Protohypericin (6a). Purple solid (122 mg, 96%). H NMR 2.76–2.67 (m, 2H), 2.13–2.03 (m, 2H) ppm. C NMR (125 MHz,
500 MHz, DMSO-d ) d: 14.36 (br, 2H), 12.86 (br, 2H), 7.20 (s, MeOD) d 185.89, 172.4, 167.9, 162.6, 147.1, 139.1, 127.6, 127.0,
H), 6.74 (s, 2H), 6.33 (s, 2H), 2.05 (s, 6H) ppm.
6H), 6.12–6.00 (m, 4H), 3.80–3.69 (m, 2H), 3.54–3.45 (m, 2H),
1
13
(
2
6
25
126.7, 125.7, 124.4, 121.9, 120.9, 118.1, 117.2, 108.9, 105.6,
+
1
,3,4,6,8,15-Hexahydroxy-7,16-dioxo-7,16-dihydrodibenzo 103.1, 39.0, 38.9 ppm. HRMS: m/z calcd for C44
H
28
O
8
[M + H] ,
ꢀ
[
(
a,o]perylene-10,13-dicarboxylic acid (6b). Purple solid 685.1857, found: 685.1858. M.p. > 300 C.
1
270 mg, 96%). H NMR (500 MHz, DMSO-d ) d 14.69 (s, 2H),
6
1
6
4.47 (s, 2H), 7.77 (s, 2H), 7.73–7.69 (m, 1H), 7.68–7.63 (m, 1H),
Cell culture
The breast cancer cell line (MCF-7), human hepatoma cell line
HepG-2) and the human skin basal cell carcinoma (A431) were
25
.62 (s, 2H) ppm.
,3,4,6,8,15-Hexahydroxy-10,13-dipentyldibenzo[a,o]perylene-
1
(
1
7
,16-dione (6c). Purple solid (140 mg, 90%). H NMR (500 MHz,
obtained from American Type Culture Collection. MCF-7 and
DMSO-d
6
) d 14.37 (s, 2H), 12.84 (s, 2H), 7.19 (s, 2H), 6.73 (s, 2H),
ꢀ
A431 cells were cultured at 37 C under a humidied 5% CO
2
in
6
6
1
1
.33 (s, 2H), 2.39–2.21 (m, 4H), 1.38–1.07 (m, 12H), 0.80 (t, J ¼
DMEM (Dulbecco's Modied Eagle Medium) medium supple-
mented with 10% fetal bovine serum, 1% penicillin/
streptomycin; and HepG-2 cells were cultured in the same way
except the culture medium used was RPMI 1640.
1
3
.9 Hz, 6H) ppm. C NMR (125 MHz, DMSO-d ), d 184.4, 174.0,
6
68.3, 160.0, 147.5, 136.4, 129.8, 127.9, 125.3, 119.6, 115.4,
13.5, 104.2, 99.7, 35.3, 30.8, 29.6, 21.8, 13.6 ppm. HRMS: m/z
ꢁ
calcd for C38
3
H
34
O
8
[M ꢁ H] , 617.2176, found: 617.2171. M.p. >
ꢀ
24
00 C.
Cell viability assay
1
0,13-Dibenzyl-1,3,4,6,8,15-hexahydroxydibenzo[a,o]perylene-
1
7
,16-dione (6d). Purple solid (97 mg, 94%). H NMR (500 MHz, The relative cell viability of compound 7a (7b, 7c, or 7d) was
DMSO-d
6
) d 14.40 (s, 2H), 12.89 (s, 2H), 7.33 (s, 2H), 7.24–7.18 evaluated in vitro by MTT assay. HepG-2, MCF-7 or A431 cells
3
(
(
m, 4H), 7.16–7.04 (m, 6H), 6.82 (s, 2H), 6.36 (s, 2H), 2.70–2.54 were seeded respectively in 96-well plates at a density of 5 ꢂ 10
1
3
m, 8H) ppm. C NMR (125 MHz, DMSO-d ) d 184.1, 174.3, cells per well in 100 mL RPMI 1640 or DMEM medium, and grew
6
ꢀ
1
1
68.0, 162.1, 147.5, 139.9, 127.6, 127.5, 127.2, 125.5, 125.1, for 24 h at 37 C. Then the medium was replaced by 90 mL fresh
21.6, 120.4, 119.0, 117.9, 108.6, 105.7, 102.3, 21.2, 18.7 ppm. medium and 10 mL 7a (7b, 7c, or 7d) in PBS (containing 0.2%
ꢁ
HRMS: m/z calcd for C44
H
30
O
8
[M ꢁ H] , 685.1863, found: DMSO-d
6
) at different concentrations (0.2, 0.4, 0.6, 0.8, 1.0 mM).
Aer 4 h incubation, the cells were washed with PBS for 3 times,
ꢀ
685.1871. M.p. > 300 C.
and then fresh medium was added. The cells were exposed to
ꢁ
2
a LED array photosource (l ¼ 595–600 nm, 8.6 mW cm ) for
General procedure for the synthesis of hypericin and its derivatives
ꢀ
3
0 min, and further cultured for 24 h at 37 C. Then 10 mL fresh
ꢁ
1
To a 50 mL ask, 6a, 6b, 6c, or 6d (0.25 mmol, 1.0 equiv.) dis- medium containing MTT (5 mg mL ) was added to each well.
solved in 20 mL acetone was irradiated for 60 min by means of Aer 4 h incubation, 100 mL DMSO was added to each well to
575 nm monochromatic lamps. The color of the mixture dissolve formazam crystals. Finally, the plate was gently shaken
changed from hyacinthine to prunosus. The solvent was for 10 min and the absorbance at 490 nm was recorded with
removed under vacuum. The crude was puried by ash a microplate reader.
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emodinanthrone was performed in a microwave reactor by
using cheap and easy to handle inorganic bases (such as NaOH,
KOH, and LiOH) as a replacement for the organic bases, and
Results and discussion
Reaction optimization for synthesis of protohypericin
Protohypericin was the key precursor to the synthesis of water as solvent instead of the organic solvents. The reaction
hypericin, therefore the dimerization of emodinanthrone conditions were varied to study their effect on the yield, and the
leading to protohypericin was recognized as the key step in this results were summarized in Table 1.
1
6
method. The synthesis of protohypericin has been extensively
studied, which can be summarized in two methods based on N-oxide (PNO), catalyst, FeSO
The various parameters, including the amount of pyridine
$7H O, base, reaction tempera-
by using potassium ture and time, were thoroughly investigated. As shown in
4
2
20,26
starting materials, (1) emodin-type:
hydroxide as a base, protohypericin can be synthesized in the Table 1, the presence of pyridine N-oxide (PNO) and a suitable
presence of hydroquinone in water. The drawbacks are redox catalyst in the reaction were crucial for the reaction, with
extremely long reaction time (7–20 days) and low yield (25– either absence leading to failure. For example, in the absence
1
6,17
7
2%); (2) emodinanthrone-type:
oxide or piperidine as a base, protohypericin can be obtained in strated that PNO was an indispensible oxygen transfer reagent
a moderate yield (70–78%) in a short time. While the drawbacks in this reaction system. While the absence of FeSO $7H O led
here are the requirement of notorious organic solvents (DMF or to no reaction at all. In addition, ferric chloride could be used
by using potassium t-but- of PNO, 6a was obtained with only 6% yield, which demon-
4
2
3
4
4 2
pyridine) and a still unsatisfying yield. Based on the discoveries to substitute FeSO $7H O without compromise of the yield
previously reported in the literature, we chose emodinanthrone but copper chloride could not. The variation of the amount of
5
as a starting material for our method in order to pursue the PNO did not inuence the yield much provided that the other
1
,7,8
development of an economic, green, and efficient synthetic reaction conditions were kept unchanged,
where the best
1
,11
method of protohypericin. In our method, the dimerization of yield of 96% was obtained when PNO was 5 equivalents; The
Table 1 Reaction optimization for synthesis of 6a.
Reaction conditions
b
ꢀ
c
Entry
PNO (equiv.)
Catalyst (equiv.)
Base
T ( C)
t (min)
Yield (%)
a
1
5.0
—
5.0
3.2
5.2
—
0.08
0.08
—
1.5% NaOH
1.5% NaOH
1.5% NaOH
6.0% NaOH
6.0% NaOH
6.0% NaOH
1.5% NaOH
1.5% NaOH
0.5% NaOH
1.0% NaOH
2.0% NaOH
5.0% NaOH
1.5% LiOH
3.6% LiOH
1.5% KOH
105
105
105
105
105
105
105
105
105
105
105
105
105
105
105
105
90
70
70
70
70
70
70
70
70
70
70
70
70
70
70
70
70
70
70
70
50
60
80
90
96
6
2
3
4
5
6
7
8
9
N.D
94
Very low
N.D
93
91
77
94
96
76
71
95
77
93
72
90
88
d
0.08e
0.08
d,f
3.0
3.5
4.5
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
0.08
0.08
0.08
0.08
0.08
0.08
0.08
0.08
0.08
0.08
0.08
0.08
0.08
0.08
0.08
0.08
0.08
1
1
1
1
1
1
1
1
1
1
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
2.1% KOH
1.5% NaOH
1.5% NaOH
1.5% NaOH
1.5% NaOH
1.5% NaOH
1.5% NaOH
1.5% NaOH
100
120
105
105
105
105
86
90
89
90
a
b
The optimized conditions: 5a, 120 mg, 1 equivalent; microwave, 10 W; water (2 mL); N
2
atmosphere. The catalyst was FeSO
4
$7H
2
O unless
c
d
e
f
specied. Isolated yield. N.D: no detection. The catalyst was FeCl
3
.
2 2
The catalyst was CuCl . O atmosphere.
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1
reaction time varied from 10 min claimed by Falk et al. to 24 h
20
by Kim et al. as well as the yield from 31% to 92%. We spec-
ulated the enormous variation of the reaction time and yield
might result from the emission difference of the light sources.
The UV-Vis spectra of 6a and 7a were shown in Fig. 1a.
20,27,28
Considering
protohypericin has a strong absorption band
at 525–590 nm while no obvious absorption for 7a at 575 nm, we
chose monochromatic lamps with a maximum emission peak at
Fig. 1 (a) UV-Vis absorption spectra of 6a and 7a (0.04 M in acetone); 575 nm as visible light source (Fig. 1b) to perform the photo-
(
b) emission spectrum of the monochromatic lamp with a maximum
cyclization of 6a. The monitoring of the reaction by TLC dis-
played that the reaction was completed at 1 h, where an isolated
yield of 96% was obtained by ash chromatography. The reac-
tion has very good reproducibility and can be performed on
large scale with little compromise to the yield (89.8%, see ESI†).
To study the applicability of the synthetic pathway developed
above, the derivatives of hypericin with different substituents
were synthesized accordingly, where methyl groups on 10- and
peak at 575 nm.
base, such as NaOH, helps to dissolve emodinanthrone in the
reaction mixture and has obvious inuence on the yield. A
concentration of NaOH lower than 0.5% or higher than 5%
decreased the yield from over 90% to about 76%. It's worth to
mention that LiOH and KOH, which have similar chemo-
physical properties as NaOH, gave more or less equally good
yields (95% for LiOH and 93% for KOH ), although the
concentration of the two hydroxides used was slightly higher
than that of NaOH. The study on the effect of reaction
temperature disclosed that the yield increased with the
temperature and reached to a peak value (96% at 105 C, see
Falk et al. ); further increase of the temperature did not
beneted a higher yield, for instance, 88% was obtained at
1
temperature led to side reactions, such as the oxidation of
hydroxyl groups in emodinanthrone. The screening on reac-
tion time proved that the yield increased with the reaction
1
1- positions in hypericin (7a) were replaced with carboxyl
groups for 7b, pentyl groups for 7c, and phenethyl groups for
d, respectively. As descripted in Scheme 1, starting with 5b–c,
1
3
15
7
the analogues of emodinanthrone 5a, respective 6b–d were
successfully synthesized with satisfying to excellent isolated
yields (90–96%) under the optimized conditions screened for
ꢀ
5a, which were subjected to irradiation of visible light to give
1
corresponding 7b–d with excellent yields (94–96%). This indi-
cates that the synthetic pathway developed in the present work
has good applicability for the derivatives of hypericin, which is
signicant in photosensitizer discovery based on natural
bioactive molecules.
ꢀ
17
20 C. This might be ascribed to the possibility that high
1
8,19
1
time
and reached the best yield of 96% at 70 min. Aer
Photodynamic therapy study
this point, the yield decreased slightly with the reaction time
with about 6%,
protohypericin in the hot basic solution.
Notably, the reaction can be carried out in gram-scale with Hypericin has been regarded as an effective natural photosen-
9
2
0,26
which may due to the decomposition of Photodynamic therapy (PDT) is a noninvasive technique that is
29–34
used to treat and detect small and supercial tumors.
35–37
1.6% yield under the optimized conditions (see ESI†).
sitizer,
and has gained increasing attention as a potential
alternative treatment for various cancers due to its unique
9,38–41
photochemical properties and photobiological activities.
To explore the potential of the three derivatives 7b–d as
Synthesis of hypericin and its derivatives
It was known that a major method to obtain hypericin was the photosensitizer for PDT, A431, HepG-2, and MCF-7 cell lines
photocyclization of protohypericin irradiated with visible light. were used for cell cytotoxicity evaluation at a concentration
2
7
The light sources reported previously include sunlight,
range of 0.2–1.0 mM with MTT assay. For comparison, hypericin
17
20
1
halogen, glow, and high-/low-pressure mercury lamps. The was used as a control. As displayed in Fig. 2, 7b showed better
Fig. 2 Cell viability of A431 cells (a); HepG2 cells (b); and MCF-7 cells (c) determined by MTT cell viability assay. The cells were incubated with
compound 7a–d, respectively, and irradiated with 595-600 nm light for 30 min.
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cytotoxicity to the test cell lines than 7a, while the cytotoxicity of
6 N. Adolf and B. Veronika, Lessons learned from herbal
medicinal products: the example of St. John's Wort, J. Nat.
Prod., 2010, 73, 1015–1021.
7 K. Michalska, H. Fernandes, M. Sikorski and M. Jaskolski,
Crystal structure of Hyp-1, a St. John's Wort protein
implicated in the biosynthesis of hypericin, J. Struct. Biol.,
2010, 169, 161–171.
8 A. Kamuhabwa, P. Agostinis, B. Ahmed, W. Landuyt, B. Van
Cleynenbreugel, H. Van Poppel and P. de Witte, Hypericin as
a potential phototherapeutic agent in supercial transitional
cell carcinoma of the bladder, Photochem. Photobiol. Sci.,
2004, 3, 772–780.
9 K. Gyuraszova, J. Mikes, A. Halaburkova, R. Jendzelovsky and
P. Fedorocko, YM155, a small molecule inhibitor of survivin
expression, sensitizes cancer cells to hypericin-mediated
photodynamic therapy, Photochem. Photobiol. Sci., 2016, 15,
812–821.
7
c and 7d were much less than that of 7a at the same concen-
trations. This may be attributed to the better water solubility of
b. The results imply that the properties of the substituents on
7
position of 10- and 11- in hypericin molecules are strongly
related to its photocytotoxicity: changing methyl groups to
hydrophilic ones (such as –COOH for 7b) can enhance the
hydrophilicity of a resulting compound and hence create
a better photocytotoxicity; on contrary, the more hydrophobic
and steric the substituents, the worse the photocytotoxicity (for
instance, comparing methyl groups in 7a with pentyl in 7c or
1
0,42–46
phenethyl in 7d).
These founding are in line with those
21,40,47
reported in the literature.
Conclusions
In summary, we developed an economic, green and highly
efficient synthetic pathway for preparing hypericin and its 10 J. T. Wessels, A.-C. Busse, M. Rave-Fraenk, S. Zaenker,
derivatives under mild reaction conditions. Water was used as
the solvent in the key step of emodinanthrone dimerization.
The reaction time of the photocyclization of protohypericin was
greatly shortened to 1 h. Furthermore, the PDT effect of 7b is
R. Hermann, E. Grabbe and G.-A. Mueller, Photosensitizing
and radiosensitizing effects of hypericin on human renal
carcinoma cells in vitro, Photochem. Photobiol., 2007, 84, 228–
235.
better than those of 7a, 7c and 7d. These results indicate the 11 P. Mauri and P. Pietta, High-performance liquid
derivatives of hypericin synthesized in the present work have
chromatography/electrospray
mass
spectrometry
of
the potential as photosensitizers for ghting cancers.
Hypericum perforatum extracts, Rapid Commun. Mass
Spectrom., 2000, 14, 95–99.
1
2 H. Brockmann, F. Kluge and H. Muxfeldt, Total synthesis of
hypericin, Chem. Ber., 1957, 90, 2302–2318.
Conflicts of interest
The authors declare no competing nancial interest.
13 J. Banville and P. Brassard, Reactions of ketene acetals. 8.
Simple syntheses of the methyl ester-ethers of the
anthraquinones endocrocin, ptilometric acid, and
clavorubin, J. Org. Chem., 1976, 41, 3018–3020.
Acknowledgements
This research work was supported by the National Natural Science 14 Y. Hirose, M. Kuroiwa, H. Yamashita, T. Tanaka and
Foundation of China (21772157, 21572181 and 21174113) to the
Project of Science and Technology of Social Development in
Shaanxi Province (2016SF-029) and Yangling Demonstration Zone
T. Megumi, Chemical studies on the natural anthraquinones.
I. Syntheses of munjistin, emodin, and 3-hydroxy-2-
methylanthraquinone, Chem. Pharm. Bull., 1973, 21, 2790–
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(2016SF-029).
1
5 J. Zhao, Z. Zhang, H. Chen and X. Chen, Studies on synthesis
and anti-HIV RT activity of hypericin and ethylhypericin,
Yaoxue Xuebao, 1998, 33, 67–71.
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