The design, synthesis and biological evaluation of pro-EGCG derivatives as novel anti-vitiligo agents

Article abstract of DOI:10.1039/c6ra23172a

With the aim of overcoming the instability and poor membrane permeability of epigallocatechin-3-gallate (EGCG), a series of prodrugs of EGCG and its derivatives (pro-EGCGs) were designed and synthesized, and their protective effect on melanocytes against

Full text of DOI:10.1039/c6ra23172a

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The design, synthesis and biological evaluation of
pro-EGCG derivatives as novel anti-vitiligo agents†
Cite this: RSC Adv., 2016, 6, 106308
a
b
b
a
a
*
Siyu Wang,‡ Rong Jin,‡ Ruiquan Wang, Yongzhou Hu, Xiaowu Dong
and Ai e Xu
b
*
With the aim of overcoming the instability and poor membrane permeability of epigallocatechin-3-gallate
(EGCG), a series of prodrugs of EGCG and its derivatives (pro-EGCGs) were designed and synthesized, and
their protective effect on melanocytes against H2O2-induced cell damage was biologically evaluated. The
enhanced potency of pro-EGCGs could be clearly observed, and the most potent compound, 3c, showed
excellent protective effect of melanocytes against H2O2-induced cell death, and good effect on lactate
dehydrogenase (LDH) release from melanocytes under oxidant stress. In addition, compounds 14b and
14c, which are the parent compounds of 3b and 3c, also showed good inhibitory activities against JAK1,
JAK2 and JKA3, which are potential therapeutic targets for anti-vitiligo agents, demonstrating the
promising perspective of 3b and 3c as both anti-oxidant and immuno-related agents for anti-vitiligo
treatment.
Received 17th September 2016
Accepted 21st October 2016
DOI: 10.1039/c6ra23172a
www.rsc.org/advances
assessed the role of EGCG in treatment of vitiligo induced by
monobenzone in mice, demonstrating that EGCG can signi-
cantly delay and reduce the prevalence of depigmentation.16 In
1 Introduction
Vitiligo is a depigmenting skin disease caused by a dermato-
logical disorder of the epidermis and hair follicles, manifesting
clinically as expanding hypopigmented lesions of the skin.1 The
prevalence of vitiligo is about 1% around the world.2 So far, the
pathogenesis of vitiligo is not very clear, but it appears to involve
immunologic factors, oxidative stress, sympathetic neurogenic
disturbance and other factors.3,4 In particular, the deleterious
effects of epidermal free radical (e.g. H2O2) overproduction and
total antioxidant status have been extensively demonstrated in
lesional and non lesional epidermis, as well as in melanocytes
of vitiligo patients,5–10 raising the possibility that antioxidants
could be useful to reduce the oxidative stress and the conse-
quent damage to melanocytes.11,12 Moreover, clinical and labo-
ratory ndings have also demonstrated the major role of
autoimmune factors in the progress of vitiligo.13,14
addition, we also proved that EGCG directly inhibited the
kinase activity of Janus kinase 2 (JAK2) to affect the inamma-
tory signaling pathways. In primary cultured human melano-
cytes, EGCG pre-treatment attenuated interferon (IFN)-g-
induced phosphorylation of JAK2 and its downstream signal
transducer and activator of transcription STAT1 and STAT3.17
Therefore, EGCG is a promising anti-vitiligo agent showing both
anti-oxidation activities and immuno-related anti-inammatory
effect.
However, EGCG is notably unstable and is known to have
poor bioavailability, owing to the presence of eight unpro-
tected phenolic hydroxyl groups and a metabolically labile
ester.18 A synthetic derivative of EGCG, obtained by acylation
of EGCG, can act as a prodrug of EGCG (pro-EGCG) with
enhanced stability, improved bioavailability, and increased
cell membrane penetration.19 As part of our continued interest
in the area of vitiligo treatment, herein we investigated the
potential of pro-EGCG derivatives, i.e. prodrugs of EGCG and
its derivatives (Fig. 1), as novel anti-vitiligo agents. These pro-
EGCG derivatives can remarkably protect the melanocytes
from H2O2-induced apoptosis via reducing the intercellular
reactive oxygen species (ROS) level. Although the anti-oxidant
biological activities of EGCG derivatives have been widely
studied, to the best of our knowledge this is the rst example
of a relatively systematic structure–activity relationship (SAR)
study for anti-vitiligo activities of a series of EGCG derivatives
Epigallocatechin-3-gallate (EGCG) is one of the main chem-
ical constituents of green tea, which has been used as an
important traditional Chinese medicine for its biological
activities including anti-oxidation, anti-inammatory, anti-
cancer, and neuroprotection.15 In our previous study, we
aZJU-ENS Joint Laboratory of Medicinal Chemistry, Zhejiang Province Key Laboratory
of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou, 310058, People's Republic of China. E-mail: dongxw@zju.edu.cn; Tel:
+86-57188981051
bDepartment of Dermatology, The Third People's Hospital of Hangzhou, Hangzhou
310009, P. R. China. E-mail: xuaiehz@msn.com; Tel: +86-57187827535
† Electronic supplementary information (ESI) available: Table S1. See DOI:
10.1039/c6ra23172a
via
a combination of prodrug strategy and structural
optimization.
‡ These authors contributed equally to these work.
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HF–pyridine (HF–Py) provided 14. Finally, acyl EGCG deriva-
tives 3a–c were obtained by direct acylation reaction of 14
(Scheme 1).
2.3 Pro-EGCG derivatives protected melanocytes from
H2O2-induced cell damage
It was investigated whether these synthesized pro-EGCG deriv-
atives protect melanocytes from H2O2-induced cell death, which
can mimic the oxidative damage of the physiological vitiligo
state. An in vitro screening model was used for evaluating the
active anti-vitiligo agents.20 In this model, prior to treatment
with 1 mM H2O2, the melanocytes were treated with EGCG or
pro-EGCG derivatives for 1 hour. Subsequently, the culture
solution was removed to eliminate the effect of residual
compounds in the culture. As shown in Fig. 2, the survival of
melanocytes in the negative control group (0.1% DMSO) was
remarkably decreased, while the survival of melanocytes in the
EGCG group was moderate in a dose-dependent manner,
ranging from 36.2% to 67.6%, which would be caused by the low
membrane penetration ability of EGCG. To our delight, the
protective effect of pro-EGCG with full acyl protection was
signicantly improved compared with that of EGCG, maybe due
to the increased cellular drug concentration in the prodrug
strategy. In particular, peracetylated EGCG derivative 1a
exhibited the most potent protecting capacity for melanocytes
against H2O2-induced oxidative stress, and the survival of
melanocytes in the presence of 1a (40 mM) reached up to 91%. In
addition, as the length of acyl group attached to EGCG was
further increased, as exemplied in compounds 1b and 1c, the
Fig. 1 The structures of designed pro-EGCG derivatives.
2 Results and discussion
2.1 The design of pro-EGCG derivatives
Initially, acyl groups with different length were introduced onto
the skeleton of EGCG, affording compounds 1a–c (Fig. 1). It was
demonstrated that the cellular membrane penetration of 1a–c
was increased compared with that of EGCG owing to their
remarkably enhanced lipophilicities. Then, adjustment of the
number of hydroxyl groups on the galloyl moiety of EGCG was
considered as well as the introduction of the hydroxy-cinnamoyl
moiety into EGCG, affording six pro-EGCG derivatives 2a–c and
3a–c (Fig. 1).
2.2 The synthesis of pro-EGCG derivatives
EGCG was chosen as the starting material since it is the most potency of the protecting capacity decreased. Next, the number
abundant catechin from green tea and commercially available. of hydroxy groups on the gallic acid moiety of EGCG derivatives
First, direct acylation of EGCG by use of appropriate acid was reduced to investigate the effect on their capacity to protect
anhydrides afforded acylated EGCG derivatives 1a–c. Then, melanocytes from H2O2-induced oxidative stress. Obviously, the
benzylation of all of the hydroxyl groups of EGCG in the potency of their protecting capacity was signicantly related
presence of benzyl bromide (BnBr) and potassium carbonate with the number of gallic acid moieties; indeed, compounds 2a–
in anhydrous dimethylformamide (DMF) gave perbenzylated c only showed moderate activities at a concentration of 10 mM
EGCG, 4, which was further alkaline hydrolyzed to afford and 20 mM, while the high dose (40 mM) group of 2a and 2c
pentabenzylated epigallocatechin, 5. Subsequently, esterica- showed promising activities, which were better than that of
tion of 5 using various hydroxyl-substituted benzoyl chlorides EGCG.
afforded compounds 6a–c. Further hydrogenolysis of 6a–c
Furthermore, we replaced gallic acid with caffeic acid
using Pd/C at room temperature provided hydroxyl- derivatives. The resulting peracetylated compounds, 3a–c,
substituted benzoates of EGC, 7a–c respectively. Then, acyla- exhibited promising activities in a dose-dependent manner. Of
tion of 7a–c with acetic anhydride afforded 2a–c. In addition, interest, all of these three compounds at a concentration of 25
the preparation of the hydroxyl-substituted caffeate of the mM showed excellent protective effect of melanocytes from
3-OH of EGC started with the protection of the hydroxyl groups H2O2-induced cell death with almost 100% cell viabilities,
of EGCG by tert-butyldimethylsilyl chloride (TBDMSCl) in signicantly more potent than that of their parent compounds
anhydrous dimethylformamide (DMF) in the presence of 14a–c and peracetylated EGCG derivative 1a. It is indicated that
imidazole at room temperature, affording key intermediate 8. the promising activities of 3a–c would also be caused by the
Then reduction of 8 using LiAlH4 in tetrahydrofuran (THF) diverse and potent biological properties of caffeic acid,
gave 5TBS-epigallocatechin, 9. In addition, TBS-protected including anti-inammatory activity, anti-oxidative activity, and
hydroxyl-substituted caffeic acids 12 were obtained by TBS so on.21
protection and subsequent Knoevenagel condensation from
hydroxyl-substituted benzaldehydes 10. Furthermore, also evaluated to ensure their non-toxicity at the biological assay
condensation reaction of with various TBS-protected concentration. Obviously, all of the compounds exhibited
The cytotoxic activities of these pro-EGCG derivatives were
9
hydroxyl-substituted caffeic acids afforded compound 13. a non-cytotoxic effect against melanocytes, demonstrating their
Next, deprotection of the TBS protecting group of 13 by use of excellent safe proles (Table S1†).
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Scheme 1 Synthesis of hydroxyl-substituted benzoates of EGCG. Reagents and conditions: (a) appropriate acid anhydride, EtOAc, TEA; (b) BnBr,
KOH, EtOH/H2O; (c) K2CO3, MeOH; (d) appropriate benzoyl chloride, DMAP; (e) Pd/C, H2; (f) Ac2O, DMAP; (g) TBDMSCl, imidazole, DMF; (h)
LiAlH4, THF; (i) DCC, DMAP, CH2Cl2; (j) pyridine, piperidine, malonic acid; (k) HF–Py complex, THF.
increased to 47.19% on exposure to 1 mM H2O2. As expected,
H2O2-induced leakage of LDH can be remarkably diminished in
a dose-dependent manner by the pretreatment of melanocytes
with EGCG or 3c; in fact, the potency of 3c is much higher than
that of EGCG, which is consistent with their protective effect
against H2O2-induced cell damage of melanocytes.
2.5 Inhibition of JAK kinase assays
In addition to the anti-oxidant properties of pro-EGCG deriva-
tives, we also paid close attention to another potential
Table 1 The effect of pro-EGCG 3c on lactate dehydrogenase (LDH)
Fig. 2 The protective effect of EGCG and pro-EGCG derivatives by
release from melanocytes under oxidant stress
measurement of melanocyte viability under H2O2-induced cell
apoptosis. Compounds 1a–c protect cells from H2O2-induced cell
death.
Concentration
(mM)
LDH release
(% of total LDH)
Compd
Blank
H2O2
—
—
7.58
47.19
45.27
39.30
34.33
21.99
15.37
13.17
2.4 Lactate dehydrogenase (LDH) release assay
EGCGa
6.25
12.5
25
6.25
12.5
25
With the aim of explaining the mechanism of the protection
action of 3c, the most potent compound, the amount of LDH
release was analysed, because of the important role of LDH in
cell destruction under oxidant stress.22 As shown in Table 1, the
percentage release of total LDH in the blank control group was
7.58%, which is very low under normal physiological condi-
tions, while the amount of LDH released was signicantly
3ca
a
Prior to exposure to 1 mM H2O2, melanocytes were pre-treated with the
indicated compound for 1 hour.
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Table 2 The inhibitory activities of compounds 14b and 14c against
JAKs
abbreviations: s, singlet; d, double; t, triplet; q, quartet; m,
multiplet; J, coupling constant (Hz). Electrospray ionization
mass spectroscopy (ESI-MS) spectra were obtained with a Shi-
madzu LCMS-2020 mass spectrometer. (ꢀ)-Epigallocatechin-3-
gallate (EGCG) was purchased from Nanjing Guangrun
Biotechnology Co., Ltd. All the ne chemicals were obtained
from commercial suppliers and used without purication.
IC50 (mM)
Compd
JAK1
JAK2
JAK3
14b
14c
30.2
53.5
15.4
22.6
1.7
0.5
4.2 The synthesis of (2R,3R)-5,7-diacetoxy-2-(3,4,5-
triacetoxyphenyl)chroman-3-yl-3,4,5-triacetoxybenzoate (1a)
therapeutic target (Janus kinases, JAKs) for anti-vitiligo.23 We
anticipated that the excellent protective effect on melanocytes
was also related to the inhibition of Janus kinases (JAKs). To our
delight, compounds 14b and 14c, which are the parent drugs of
compounds 3b and 3c, respectively, showed good inhibitory
activities against all JAK subtypes, JAK1, JAK2, and JAK3, with
IC50 at micromolar concentrations (Table 2). Of interest, both
compounds 14b and 14c showed promising selective proles
against different JAKs, for example, the selectivity of 14c for
JAK3 as against JAK1 and JAK2 was 45.2- and 107-fold, respec-
tively. Compounds 3b and 3c would be hydrolyzed in the
endochylema to release 14b and 14c, resulting in the blockade
of the JAKs pathway which is an important immune-related
signal pathway for the development of vitiligo.
Triethylamine (TEA, 2.2 g, 21.8 mmol) and acetic anhydride
(2.2 g, 21.8 mmol) were added to a solution of EGCG (1 g, 2.18
mmol) in EtOAc in an ice bath, and the mixture was stirred for 4
hours. The reaction was quenched with water, extracted with
EtOAc and then washed with Na2CO3(aq). The organic layer was
concentrated under reduced pressure and the residue was
puried by column chromatography (ethyl acetate : petroleum
ether (EA : PE) ¼ 1 : 1) to provide compound 1a as a white solid.
Analytical data: yield 90%; 1H NMR (500 MHz, CDCl3) d 7.62 (s,
2H), 7.23 (s, 2H), 6.73 (d, J ¼ 2.0 Hz, 1H), 6.60 (d, J ¼ 2.0 Hz, 1H),
5.63 (d, J ¼ 1.5 Hz, 1H), 5.17 (s, 1H), 3.02 (m, 2H), 2.31–2.22 (m,
24H). The data are consistent with those in the literature.24
4.3 The identical method to that for 1a was used to
synthesize and purify (2R,3R)-5,7-dipropionyloxy-2-(3,4,5-
tripropionyloxyphenyl)chroman-3-yl-3,4,5-
3 Conclusion
tripropionyloxybenzoate (1b) as a white solid
In summary, prodrug strategy was investigated to overcome the
instability and poor membrane permeability of EGCG. Several
key structural factors in EGCG were considered in our study and
their relative structure–activity relationships (SAR) were also
explored systematically. Nine pro-EGCGs were synthesized and
biologically evaluated; the potency of these compounds in
protecting melanocytes from H2O2-induced cell death can be
clearly observed, and some of them showed signicantly
improved activities compared with that of EGCG. A preliminary
study of the protection mechanism of compound 3c against
oxidant-stress-caused cell damage to melanocytes was carried
out, which suggested that compound 3c can remarkably
diminish H2O2-induced leakage of LDH. In addition to the anti-
oxidant properties of pro-EGCG derivatives, compounds 14b
and 14c, the parent compounds of 3b and 3c, showed good
inhibitory activities against JAKs which are another potential
immuno-related therapeutic target for anti-vitiligo, demon-
strating the promising perspective of 3b and 3c as both anti-
oxidant and immuno-related agents for anti-vitiligo treatment.
Analytical data: yield 75%; 1H NMR (500 MHz, CDCl3) d 7.63 (s,
2H), 7.25 (s, 2H), 6.76 (d, J ¼ 2.0 Hz, 1H), 6.64 (d, J ¼ 2.0 Hz, 1H),
5.71–5.66 (m, 1H), 5.20 (s, 1H), 3.04 (m, 2H), 2.61–2.50 (m, 16H),
1.29–1.21 (m, 24H). The data are consistent with those in the
literature.24
4.4 The identical method to that for 1a was used to
synthesize and purify (2R,3R)-5,7-dibutyryloxy-2-(3,4,5-
tributyryloxyphenyl)chroman-3-yl-3,4,5-tributyryloxybenzoate
(1c) as a white solid
Analytical data: yield 80%; 1H NMR (400 MHz, CDCl3) d 7.59 (s,
2H), 7.21 (s, 2H), 6.73 (d, J ¼ 2.2 Hz, 1H), 6.60 (d, J ¼ 2.2 Hz, 1H),
5.67 (s, 1H), 5.18 (s, 1H), 3.02 (m, 2H), 2.70–2.28 (m, 16H), 1.96–
1.64 (m, 16H), 1.13–0.81 (m, 24H). The data are consistent with
those in the literature.24
4.5 The synthesis of (2R,3R)-5,7-bis(benzyloxy)-2-(3,4,5-
tris(benzyloxy)phenyl)chroman-3-yl-3,4,5-tris(benzyloxy)
benzoate (4)
4 Experimental section
4.1 Chemistry
EGCG (2 g, 4.3 mmol) was dissolved in anhydrous dimethyl
formamide (DMF) (30 mL). Potassium carbonate (12 g, 87
Proton NMR spectra were obtained on a Bruker AVII 500 or 400 mmol) and benzyl bromide (6.2 mL, 52.4 mmol) were added
NMR spectrometer (500 or 400 MHz) with use of CDCl3, CD3OD, successively. The mixture was stirred at room temperature (rt)
or DMSO-d6 as solvent. Carbon-13 NMR spectra were obtained for 48 h. The reaction mixture was added to water and extracted
on a Bruker spectrometer (125 MHz) by use of CDCl3, CD3OD or with EtOAc. The organic layer was further washed with water
DMSO-d6 as solvent. Carbon-13 chemical shis were referenced and brine, then dried with anhydrous Na2SO4, and evaporated.
to the center peak of CDCl3 (d 77.0 ppm) or DMSO-d6 (d 39.5 Finally, the residue was puried by ash column chromatog-
ppm). Multiplicities are recorded by the following raphy, and recrystallized in petroleum ether–EtOAc (10 : 1) to
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afford 4 as a white solid. Analytical data: yield 45%; H NMR 156.5, 156.4, 155.8, 145.3, 132.3, 131.5, 129.4, 120.9, 114.7,
(500 MHz, CDCl3) d 7.48–7.18 (m, 42H), 6.77 (d, J ¼ 9.5 Hz, 2H), 105.3, 97.9, 95.1, 94.4, 77.1, 68.8, 48.5, 25.3; ESI-MS m/z 427
6.43 (d, J ¼ 2.0 Hz, 1H), 6.38 (d, J ¼ 2.0 Hz, 1H), 5.70 (s, 1H), (M + H)+.
5.12–4.66 (m, 17H), 3.16 (dd, J ¼ 17.5, 4.5 Hz, 1H), 3.10 (dd, J ¼
17.5, 2.0 Hz, 1H). The data are consistent with those in the
literature.25
4.9 The identical method to that for 7a was used to
synthesize and purify (2R,3R)-5,7-dihydroxy-2-(3,4,5-
trihydroxyphenyl)chroman-3-yl 3,4-dihydroxybenzoate (7c)
4.6 The synthesis of (2R,3R)-5,7-bis(benzyloxy)-2-(3,4,5-
tris(benzyloxy)phenyl)chroman-3-ol (5)
Analytical data: yield 69%; 1H NMR (500 MHz, CD3OD) d 7.34 (d,
J ¼ 1.5 Hz, 2H), 6.80–6.71 (m, 1H), 6.53 (d, J ¼ 4.5 Hz, 2H), 5.98
(d, J ¼ 3.0 Hz, 2H), 5.53 (dd, J ¼ 9.0, 7.5 Hz, 1H), 5.00 (s, 1H),
Compound 4 (500 mg, 0.4 mmol) was dissolved in a solution of
methanol (5 mL) and ethylene glycol dimethyl ether (5 mL), and
then potassium carbonate (193 mg, 1.4 mmol) was added. The
resulting mixture was stirred at rt for 5 h until thin layer chro-
matography (TLC) showed the reaction had been completed.
Then the solvent was evaporated, and the residue was puried
by ash chromatography on silica gel to afford compound 5 as
a viscous liquid. Analytical data: yield 85%; 1H NMR (400 MHz,
CDCl3) d 7.58–7.17 (m, 25H), 6.81 (s, 2H), 6.29 (s, 2H), 5.22–4.86
(m, 10H), 4.25 (d, J ¼ 29.8 Hz, 1H), 3.02 (d, J ¼ 17.2 Hz, 1H), 2.93
(dd, J ¼ 17.2, 3.8 Hz, 1H), 1.68 (d, J ¼ 5.2 Hz, 1H). The data are
consistent with those in the literature.25
3.01 (dd, J ¼ 17.0, 4.5 Hz, 1H), 2.88 (dd, J ¼ 17.5, 2.5 Hz, 1H); 13
C
NMR (125 MHz, CD3OD) d 166.2, 156.5, 155.8, 150.3, 145.3,
144.5, 132.4, 129.4, 122.6, 121.3, 116.1, 114.5, 105.4, 98.0, 95.1,
94.4, 77.2, 68.7, 60.2, 25.4, 19.5, 13.1; ESI-MS m/z 443 (M + H)+.
4.10 The identical method to that for 1a was used to
synthesize and purify 5-((2R,3R)-5,7-diacetoxy-3-(benzoyloxy)
chroman-2-yl)benzene-1,2,3-triyl triacetate (2a)
Analytical data: yield 76%;1 H NMR (400 MHz, DMSO-d6) d 7.71
(t, J ¼ 11.4 Hz, 2H), 7.56 (m, 1H), 7.56–7.34 (m, 3H), 7.37–7.07
(m, 1H), 6.91–6.59 (m, 2H), 5.76–5.38 (m, 2H), 3.15 (dd, J ¼ 17.6,
4.0 Hz, 1H), 2.94 (d, J ¼ 17.6 Hz, 1H), 2.65–2.06 (m, 15H).
4.7 The synthesis of (2R,3R)-5,7-dihydroxy-2-(3,4,5-
trihydroxyphenyl)chroman-3-yl benzoate (7a)
4.11 The identical method to that for 1a was used to
synthesize and purify 5-((2R,3R)-5,7-diacetoxy-3-((4-
acetoxybenzoyl)oxy)chroman-2-yl)benzene-1,2,3-triyl
triacetate (2b)
Analytical data: yield 83%; 1H NMR (500 MHz, DMSO-d6) d 7.86–
7.73 (m, 2H), 7.41 (s, 2H), 7.28–7.16 (m, 2H), 6.73 (dd, J ¼ 74.0,
2.0 Hz, 2H), 5.61 (d, J ¼ 40.5 Hz, 2H), 3.16 (dd, J ¼ 17.5, 4.0 Hz,
1H), 2.95 (d, J ¼ 16.5 Hz, 1H), 2.39–2.15 (m, 18H). The data are
consistent with those in the literature.26
Benzoylchloride (240 mg, 0.174 mmol) was dissolved in dry
CH2Cl2 (10 mL) and added dropwise into a solution of
compound 5 (100 mg, 0.116 mmol) and dimethylaminopyridine
ꢁ
(DMAP) (35.36 mg, 0.28 mmol) in CH2Cl2 (10 mL) at 0 C. The
mixture was stirred at rt overnight, the solvent was then evap-
orated, and the resulting mixture was extracted with EtOAc, and
washed with water. The organic layer was dried using anhy-
drous Na2SO4, and evaporated. The crude product 6a was used
directly in the next step without further purication. Then,
under a hydrogen atmosphere, 10% Pd/C (150 mg) was added to
a solution of the above-obtained compound 6a in ethyl acetate
(10 mL). The resulting mixture was stirred at rt overnight. Then
the mixture was evaporated to remove the solvent in vacuum.
The residue was puried by ash column chromatograph to
4.12 The identical method to that for 1a was used to
synthesize and purify 5-((2R,3R)-5,7-diacetoxy-3-((3,4-
diacetoxybenzoyl)oxy)chroman-2-yl)benzene-1,2,3-triyl
triacetate (2c)
Analytical data: yield 90%; 1H NMR (500 MHz, DMSO-d6) d 7.67
(dd, J ¼ 8.5, 1.5 Hz, 1H), 7.61 (d, J ¼ 1.5 Hz, 1H), 7.41 (s, 2H),
7.36 (d, J ¼ 8.5 Hz, 1H), 6.80 (d, J ¼ 2.0 Hz, 1H), 6.68 (t, J ¼
13.5 Hz, 1H), 5.64 (s, 1H), 5.56 (s, 1H), 3.16 (dd, J ¼ 17.5, 4.0 Hz,
1H), 2.96 (d, J ¼ 17.5 Hz, 1H), 2.47–2.03 (m, 21H).
1
afford 7a as a yellow solid. Analytical data: yield 82%; H NMR
(400 MHz, DMSO-d6) d 9.29 (s, 1H), 9.05 (s, 1H), 8.77 (s, 2H), 7.99
(s, 1H), 7.80 (d, J ¼ 8.0 Hz, 2H), 7.61 (t, J ¼ 7.6 Hz, 1H), 7.47 (t, J
¼ 7.6 Hz, 2H), 6.44 (s, 2H), 5.92 (s, 1H), 5.84 (s, 1H), 5.41 (s, 1H),
5.02 (s, 1H), 2.99 (dd, J ¼ 17.4, 4.2 Hz, 1H), 2.75 (d, J ¼ 16.8 Hz,
1H); 13C NMR (125 MHz, CD3OD) d 166.0, 156.6, 156.5, 155.8,
145.4, 132.7, 132.3, 130.0, 129.4, 129.2, 128.1, 105.3, 97.8, 95.1, 4.13 The synthesis of (ꢀ)-epigallocatechin-3-gallate
94.4, 77.1, 69.4, 60.2, 25.3, 19.5, 13.1; electrospray ionization octa(tert-butyldimethylsilyl) ether (8)
mass spectrometry (ESI-MS) m/z 411 (M + H)+.
To a solution of EGCG (20.0 g, 43.6 mmol, 1.0 equiv.) in dry DMF
(100 mL) was added imidazole (29.8 g, 438 mmol) followed by
4.8 The identical method to that for 7a was used to
synthesize and purify (2R,3R)-5,7-dihydroxy-2-(3,4,5-
trihydroxyphenyl)chroman-3-yl 4-hydroxybenzoate (7b)
TBDMSCl (65.8 g, 436 mmol, 10 equiv.) at 0 ꢁC. The solution was
stirred overnight at room temperature. The reaction mixture
was quenched with water (100 mL) and extracted with hexane
Analytical data: yield 76%; 1H NMR (500 MHz, DMSO-d6) d 10.30 (100 mL ꢂ 3). The combined organic layers were washed with
(s, 1H), 9.28 (s, 1H), 9.05 (s, 1H), 8.77 (s, 2H), 8.00 (s, 1H), 7.67 water (50 mL ꢂ 3) and saturated aqueous NaCl (50 mL ꢂ 3),
(d, J ¼ 8.5 Hz, 2H), 6.80 (d, J ¼ 8.5 Hz, 2H), 6.44 (s, 2H), 5.88 (m, dried over NaSO4(s), ltered, and concentrated under reduced
2H), 5.32 (s, 1H), 5.00 (s, 1H), 2.95 (dd, J ¼ 17.0, 4.5 Hz, 1H), 2.72 pressure to provide crude white compound 8, which was used
(d, J ¼ 15.5 Hz, 1H); 13C NMR (125 MHz, CD3OD) d 166.1, 162.0, directly for the next step.
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d 7.74 (t, J ¼ 17.0 Hz, 1H), 7.52–7.41 (m, 2H), 6.92–6.84 (m, 2H),
6.32 (t, J ¼ 16.0 Hz, 1H), 1.01 (d, J ¼ 2.0 Hz, 9H), 0.27–0.23 (m,
6H); ESI-MS m/z 279 (M ꢀ H)ꢀ
4.14 The synthesis of (ꢀ)-epigallocatechin penta(tert-
butyldimethylsilyl) ether (9)
To (ꢀ)-epigallocatechin-3-gallate octa(tert-butyldimethylsilyl)
ether (8, 13.0 g, 9.4 mmol) in THF (100 mL) was slowly added
LiAlH4 (0.72 g, 18.9 mmol) at 0 ꢁC. The solution was warmed to
room temperature and stirred for 3 hours. The reaction was
quenched with water in an ice bath and ltered. The cake was
washed with EtOAc (50 mL) and the combined ltrates were
condensed under reduced pressure. The residue was puried by
column chromatography (1.0% EtOAc in petroleum ether) to
provide (ꢀ)-epigallocatechin penta(tert-butyldimethylsilyl)
4.19 The identical method to that for 12a was used to
synthesize and purify (E)-3-(3,4-bis((tert-butyldimethysilyl)
oxy)phenyl)acrylic acid (12b)
Analytical data: yield of 51%. 1H NMR (500 MHz, CDCl3) d 7.69
(d, J ¼ 15.5 Hz, 1H), 7.07 (dd, J ¼ 7.0, 1.5 Hz, 2H), 6.92–6.81 (m,
1H), 6.26 (t, J ¼ 11.5 Hz, 1H), 1.03–1.01 (m, 18H), 0.25 (d, J ¼
4.5 Hz, 12H). ESI-MS m/z 409 (M ꢀ H)ꢀ.
1
ether, 9, as a white solid. Analytical data: yield 80%. H NMR
(500 MHz, CDCl3) d 6.65 (s, 2H), 6.15 (d, J ¼ 2.0 Hz, 1H), 6.01 (d, J
¼ 2.0 Hz, 1H), 4.88 (s, 1H), 4.19 (d, J ¼ 29.5 Hz, 1H), 2.88 (qd, J ¼
16.5, 4.0 Hz, 2H), 1.04 (d, J ¼ 1.5 Hz, 18H), 1.02–1.00 (m, 9H),
0.97 (s, 18H), 0.29–0.26 (m, 6H), 0.24 (s, 12H), 0.23 (s, 6H), 0.16
(t, J ¼ 3.5 Hz, 6H). The data are consistent with those in the
literature.15
4.20 The identical method to that for 12a was used to
synthesize and purify (E)-3-(3,4,5-tris((tert-butyl dimethylsilyl)
oxy)phenyl)acrylic acid (12c)
Analytical data: yield 43%; 1H NMR (500 MHz, CDCl3) d 7.61 (d, J
¼ 15.5 Hz, 1H), 6.74 (s, 2H), 6.21 (d, J ¼ 15.5 Hz, 1H), 1.07–0.91
(m, 27H), 0.27–0.14 (m, 18H); ESI-MS m/z 539 (M ꢀ H)ꢀ.
4.15 The identical method to that for 8 was used to
synthesize and purify 4-((tert-butyldimethylsilyl)oxy)
benzaldehyde (11a)
Analytical data: yield 75%; 1H NMR (500 MHz, CDCl3) d 9.91
(s, 1H), 7.98–7.65 (m, 2H), 7.12–6.79 (m, 2H), 1.02–1.01 (m, 9H),
0.28–0.26 (m, 6H). The data are consistent with those in the
literature.27
4.21 The synthesis of (2R,3R)-5,7-di((tert-butyldimethysilyl)
oxy)-2-(3,4,5-tri((tert-butyldimethylsilyl)oxy)phenyl)chroman-
3-yl (E)-3-(4-((tert-butyldimethylsilyl)oxy)phenyl)acrylate (13a)
To a solution of compound 12a (1.0 equiv.), N,N0-dicyclohex-
ylcarbodiimide (DCC, 2.2 equiv.), and 4-dimethylaminopyridine
(DMAP, 2.2 equiv.) in dry CH2Cl2 was added with hydroxyl-
ꢁ
substituted caffeic acid (2.0 equiv.) at 0 C. Then the solution
was stirred at room temperature for 3 h, the insoluble urea was
ltered, and the ltrate was concentrated under reduced pres-
sure. The residue was puried by column chromatography
4.16 The identical method to that for 8 was used to
synthesize and purify 3,4-bis((tert-butyldimethylsilyl)oxy)
benzaldehyde (11b)
(1.0% EtOAc in petroleum ether) to provide 13a as a white solid.
Analytical data: yield 82%; 1H NMR (500 MHz, CDCl3) d 9.83 (s, Analytical data for 13a: yield 40%; H NMR (500 MHz, CDCl3)
1H), 7.39 (q, J ¼ 1.5 Hz, 2H), 6.97 (d, J ¼ 7.5 Hz, 1H), 1.02 (d, J ¼ d 7.53–7.47 (m, 1H), 7.35 (t, J ¼ 5.5 Hz, 2H), 6.84–6.80 (m, 2H),
1.5 Hz, 18H), 0.31–0.19 (m, 12H). The data are consistent with 6.62 (s, 2H), 6.23–6.17 (m, 2H), 6.00 (t, J ¼ 2.5 Hz, 1H), 5.57–5.51
1
those in the literature.27
(m, 1H), 5.02 (s, 1H), 1.02–0.91 (m, 54H), 0.25–0.09 (m, 36H).
4.17 The identical method to that for 8 was used to
synthesize and purify 3,4,5-tris((tert-butyldimethylsilyl)oxy)
benzaldehyde (11c)
Analytical data: yield 87%; 1H NMR (500 MHz, CDCl3) d 9.75 (s,
1H), 7.06 (d, J ¼ 11.5 Hz, 2H), 1.02–0.96 (m, 27H), 0.15–0.07 (m,
18H). The data are consistent with those in the literature.28
4.22 The identical method to that for 13a was used to
synthesize and purify (2R,3R)-5,7-di((tert-butyldimethylsilyl)
oxy)-2-(3,4,5-tri((tert-butyldimethylsilyl)oxy)phenyl)chroman-
3-yl (E)-3-(3,4-di((tert-butyldimethylsilyl)oxy)phenyl)acrylate
(13b)
1
Analytical data for 13b: yield 53%; H NMR (500 MHz, CDCl3)
d 7.43 (d, J ¼ 15.5 Hz, 1H), 7.01–6.88 (m, 2H), 6.83–6.75 (m, 1H),
6.66–6.57 (m, 2H), 6.18 (t, J ¼ 7.5 Hz, 1H), 6.12 (d, J ¼ 15.5 Hz,
1H), 5.99 (dd, J ¼ 7.5, 2.0 Hz, 1H), 5.55 (s, 1H), 5.01 (s, 1H), 2.95
(ddd, J ¼ 20.5, 17.5, 4.0 Hz, 2H), 1.02–0.88 (m, 63H), 0.30–0.06
(m, 42H).
4.18 The synthesis of (E)-3-(4-((tert-butyldimethylsilyl)oxy)
phenyl)acrylic acid (12a)
To 20 mL of pyridine containing 2 drops of piperidine was
added 4-((tert-butyldimethylsilyl)oxy)benzaldehyde (11a, 3 g,
12.7 mmol) and malonic acid (2.6 g, 25.4 mmol). The mixture
4.23 The identical method to that for 13a was used to
synthesize and purify (2R,3R)-5,7-di((tert-butyldimethylsilyl)
oxy)-2-(3,4,5-tri((tert-butyldimethylsilyl)oxy)phenyl)chroman-
3-yl (E)-3-(3,4,5-tri((tert-butyldimethylsilyl)oxy)phenyl)acrylate
ꢁ
was heated at 85 C for 5 hours, cooled to room temperature,
and stirred overnight. The reaction mixture was quenched with
2 N HCl (100 mL) and extracted with EtOAc (100 mL ꢂ 3). The
combined the residue was puried by column chromatography
(dichloromethane : MeOH organic layers were washed with
(13c)
1
water and brine, dried over NaSO4(s), ltered, and concentrated Analytical data for 13c: yield 65%; H NMR (500 MHz, CDCl3)
under reduced pressure. ¼ 10 : 1) to provide 12a as a white d 7.36 (d, J ¼ 15.5 Hz, 1H), 7.17 (s, 1H), 6.62 (s, 1H), 6.61 (s, 1H),
solid. Analytical data: yield 37%; 1H NMR (500 MHz, CDCl3) 6.20 (d, J ¼ 2.0 Hz, 1H), 6.06 (d, J ¼ 15.5 Hz, 1H), 6.00 (d, J ¼
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2.0 Hz, 1H), 5.66 (d, J ¼ 13.0 Hz, 1H), 5.58 (s, 1H), 5.43 (s, 1H), quenched with water and Na2CO3(aq) in an ice bath and
2.99–2.90 (m, 2H), 1.04–0.86 (m, 72H), 0.26–0.08 (m, 48H).
extracted with EtOAc. The combined organic layer was
concentrated under reduced pressure and the residue was
puried by column chromatography (EA : PE ¼ 1 : 1) to provide
4.24 The synthesis of (2R,3R)-5,7-dihydroxy-2-(3,4,5-
trihydroxyphenyl)chroman-3-yl (E)-3-(4-hydroxyphenyl)
acrylate (14a)
1
compound 3a as a white solid. Analytical data: yield 69%; H
NMR (500 MHz, CDCl3) d 7.55 (dd, J ¼ 12.5, 6.5 Hz, 3H), 7.29–
7.28 (m, 2H), 7.11 (d, J ¼ 8.5 Hz, 2H), 6.73 (d, J ¼ 2.0 Hz, 1H),
6.61 (d, J ¼ 2.0 Hz, 1H), 6.32 (d, J ¼ 16.0 Hz, 1H), 5.55 (s, 1H),
5.19 (s, 1H), 3.01 (d, J ¼ 20.0 Hz, 2H), 2.41–2.15 (m, 18H).
To a solution of compound 13a (100 mg, 0.088 mmol) in
a plastic beaker was added HF–Py complex (60 equiv.) and Py (60
equiv.). The reaction was stirred at room temperature. Aer the
reaction was completed, the solution was quenched with 1 N
HCl and extracted with EtOAc. The combined organic layer was
concentrated under reduced pressure and the residue was
puried by column chromatography (DCM : MeOH ¼ 10 : 1) to
4.28 The identical method to that for 14a was used to
synthesize and purify 5-((2R,3R)-5,7-diacetoxy-3-(((E)-3-(3,4-
diacetoxyphenyl) acryloyl)oxy)chroman-2-yl)benzene-1,2,3-
triyl triacetate (3b)
provide compound 14a as a yellow solid. Analytical data: yield
1
1
56%; H NMR (500 MHz, CD3OD) d 7.51 (d, J ¼ 16.0 Hz, 1H), Analytical data: yield 78%; H NMR (500 MHz, CDCl3) d 7.53–
7.46–7.38 (m, 2H), 6.82–6.71 (m, 2H), 6.56–6.48 (m, 2H), 6.27 (d, 7.48 (m, 1H), 7.41 (dd, J ¼ 8.5, 2.0 Hz, 1H), 7.37 (d, J ¼ 2.0 Hz,
J ¼ 16.0 Hz, 1H), 5.97 (dd, J ¼ 8.5, 2.5 Hz, 2H), 5.52–5.43 (m, 1H), 1H), 7.28 (s, 2H), 7.21 (t, J ¼ 5.5 Hz, 1H), 6.73 (d, J ¼ 2.0 Hz, 1H),
4.96 (s, 1H), 2.98 (dd, J ¼ 17.0, 4.5 Hz, 1H), 2.85 (dd, J ¼ 17.0, 6.61 (dd, J ¼ 4.5, 2.0 Hz, 1H), 6.32 (d, J ¼ 16.0 Hz, 1H), 5.55 (s,
2.5 Hz, 1H); 13C NMR (125 MHz, CD3OD) d 171.6, 167.2, 159.7, 1H), 5.18 (s, 1H), 3.02 (m, 2H), 2.36–2.20 (m, 21H).
156.5, 156.4, 155.7, 145.4, 145.3, 129.8, 129.4, 125.9, 115.3,
113.9, 105.5, 98.0, 95.2, 94.4, 77.1, 68.4, 60.1, 25.3, 19.4, 13.1;
4.29 The identical method to that for 14a was used to
synthesize and purify 5-((E)-3-(((2R,3R)-5,7-diacetoxy-2-(3,4,5-
triacetoxyphenyl)chroman-3-yl)oxy)-3-oxoprop-1-en-1-yl)
benzene-1,2,3-triyl triacetate (3c)
Analytical data: yield 81%; 1H NMR (500 MHz, CDCl3) d 7.47 (t, J
¼ 13.5 Hz, 1H), 7.28 (s, 2H), 7.27 (s, 2H), 6.67 (dd, J ¼ 54.0,
2.0 Hz, 2H), 6.31 (d, J ¼ 16.0 Hz, 1H), 5.55 (s, 1H), 5.18 (s, 1H),
ESI-MS m/z 453 (M + H)+.
4.25 The identical method to that for 14a was used to
synthesize and purify (2R,3R)-5,7-dihydroxy-2-(3,4,5-
trihydroxyphenyl)chroman-3-yl (E)-3-(3,4-dihydroxyphenyl)
acrylate (14b)
Analytical data: yield 68%; 1H NMR (500 MHz, CD3OD) 1H NMR 3.09–2.92 (m, 2H), 2.38–2.20 (m, 24H).
(500 MHz, CD3OD) d 7.44 (d, J ¼ 16.0 Hz, 1H), 7.01 (d, J ¼ 2.0 Hz,
1H), 6.92 (dd, J ¼ 8.0, 2.0 Hz, 1H), 6.76 (d, J ¼ 8.0 Hz, 1H), 6.52
4.30 Materials and cell culture
(s, 2H), 6.20 (d, J ¼ 16.0 Hz, 1H), 5.98 (dd, J ¼ 8.0, 2.0 Hz, 2H),
Melanocytes were purchased from Cellbio Biological Engi-
5.52–5.40 (m, 1H), 4.96 (s, 1H), 2.98 (dd, J ¼ 17.0, 4.5 Hz, 1H),
2.84 (dd, J ¼ 17.0, 2.5 Hz, 1H); 13C NMR (125 MHz, CD3OD)
d 167.2, 156.5, 156.4, 155.7, 148.1, 145.8, 145.3, 132.4, 129.3,
126.4, 121.6, 115.0, 113.8, 113.7, 105.4, 97.9, 95.1, 94.4, 77.1,
68.4, 60.1, 25.3, 19.4, 13.1; ESI-MS m/z 469 (M + H)+.
neering Limited Company (Shanghai, China) and cultured at
37 ꢁC in an incubator with 5% CO2 and 95% humidity. All
culture media and supplements used for the isolation and
cultivation of melanocytes were obtained from Gibco (Carlsbad,
CA, USA) with the exception of recombinant human basic
broblast growth factor (bFGF; Pepro-Tech, Rocky Hill, NJ, USA)
and isobutylmethylxanthine (IBMX; Sigma-Aldrich, St Louis,
MO, USA). The CytoTox 96 Non-Radioactive Cytotoxicity Assay
kit and Lactate Dehydrogenase Assay kit were purchased from
Promega (Madison, WI, USA). Cells were cultured in Hu16
4.26 The identical method to that for 14a was used to
synthesize and purify (2R,3R)-5,7-dihydroxy-2-(3,4,5-
trihydroxyphenyl)chroman-3-yl (E)-3-(3,4,5-trihydroxyphenyl)
acrylate (14c)
Analytical data: yield 67%; 1H NMR (500 MHz, CD3OD) d 7.36 (d, medium (F12 supplemented with 10% fetal bovine serum (FBS),
J ¼ 15.5 Hz, 1H), 6.55 (m, 4H), 6.16 (d, J ¼ 15.5 Hz, 1H), 5.52– 20 ng mLꢀ1 bFGF and 20 mg mLꢀ1 IBMX) and cultured at 37 ꢁC
5.43 (m, 1H), 4.96 (s, 1H), 2.98 (dd, J ¼ 17.0, 4.5 Hz, 1H), 2.84 with 5% CO2 and 95% humidity. The cells were used between
(dd, J ¼ 17.0, 2.5 Hz, 1H); 13C NMR (125 MHz, CD3OD) d 171.7, passages 2 and 5. Melanocytes were initially plated at a density
167.3, 156.5, 156.4, 155.7, 146.1, 145.6, 145.4, 136.1, 132.4, of 1 ꢂ 104 cells per well in 96-well plates or at a density of 3 ꢂ
129.3, 125.4, 113.9, 107.3, 105.5, 98.0, 77.0, 68.4, 60.1, 25.1, 19.4, 105 cells per well in 6-well plates and incubated overnight before
12.9; ESI-MS m/z 485 (M + H)+.
each experiment.
4.27 The synthesis of 5-((2R,3R)-5,7-diacetoxy-3-(((E)-3-(4-
acetoxyphenyl)acryloyl)oxy)chroman-2-yl)benzene-1,2,3-triyl
triacetate (3a)
4.31 Cell viability assay
Cell viability was measured using the Non-Radioactive Cell
Proliferation Assay (MTS) kit in accordance with the manufac-
To a solution of 14a (20 mg, 0.044 mmol) in EtOAc were added turer's protocol. To examine the cell viability, 10 mL of MTS
DMAP (27 mg, 0.22 mmol) and acetic anhydride (36 mg, 0.35 solution was added to each well of 96-well plates and incubated
ꢁ
mmol) in an ice bath and stirred for 4.0 h. The reaction was at 37 C for 1 h. The absorbance at 490 nm was measured by
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a microplate spectrophotometer (SpectraMax190; MDC, Sun-
nyvale, CA USA). All experiments were performed in triplicate in
three independent experiments.
5 K. U. Schallreuter, J. M. Wood and J. Berger, J. Invest.
Dermatol., 1991, 97, 1081–1085.
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2226.
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2007, 360, 70–75.
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4.32 Lactate dehydrogenase (LDH) release assay
Melanocytes were initially plated into a 96-well plate. Aer
pretreatment, supernatant from each well of the plate was
transferred to the corresponding well of a 96-well enzymatic
assay plate and incubated with reconstituted substrate mix for
30 min. The reaction was stopped by the addition of stop
solution, and the absorbance was measured with a microplate
spectrophotometer at 490 nm. The LDH leakage was then
calculated as percentage (%) of the total LDH activity (LDH in
the total cell lysate) according to the equation: % LDH released
¼ (LDH activity in the supernatant/total LDH activity) ꢂ 100%.
4.33 In vitro JAK inhibition assays
14 Y. Lili, W. Yi, Y. Ji, S. Yue, S. Weimin and L. Ming, PLoS One,
2012, 7, e37513.
The inhibitory activity of compounds against JAK1/2/3 enzyme
was evaluated according to a published procedure.29 The incu-
bation mixtures contained the following components: 1.1 nM
JAK1/2/3, 1.5 mM peptide substrate (5-FAM-KKKKEEIYFFFG-OH
for JAK2) and 30 mM ATP. Aer incubation for 180 min, the
reaction mixture was analyzed on a Caliper LabChip 3000
(Caliper LifeSciences, Hopinkton, MA, USA) by electrophoretic
separation of the uorescent substrate and phosphorylated
product. The virtual screening hits were rstly evaluated at the
concentration of 20 mg mLꢀ1, and further tested for seven
concentrations prepared by 3- or 4-fold serial dilution if the
preliminary activity was more than 60%. The IC50 values were
generated by the analytical soware Prism 5.0 (GraphPad So-
ware Pte Ltd).
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Acknowledgements
This work was supported by the Key Scientic Innovation
Program of Hangzhou (Grant No: 20122513A02), the Science
Foundation of the Ministry of Health (Grant No: WKJ2012-2-
036), the National Natural Science Foundation of China
(Grant No: 81271758 and 81472887), and the National Key
Clinical Specialty Construction Project of China.
´
27 C. A. Faler and M. M. Joullie, Org. Lett., 2007, 9, 1987–1990.
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29 H. H. Wan, G. M. Schroeder, A. C. Hart, J. Inghrim,
J. Grebinski, J. S. Tokarski, M. V. Lorenz, D. You,
T. McDevie, B. Penhallow, R. Vuppugalla, Y. P. Zhang,
X. M. Gu, R. Iyer, L. J. Lombardo, G. L. Trainor, S. Ruepp,
J. Lippy, Y. Blat, J. S. Sack, J. A. Khan, K. Stefanski,
B. Sleczka, A. Mathur, J. H. Sun, M. K. Wong, D. R. Wu,
P. Li, A. Gupta, P. N. Arunachalam, B. Pragalathan,
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