Design and synthesis of aloe-emodin derivatives as potent anti-tyrosinase, antibacterial and anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmcl.2015.10.004

Twenty aloe-emodin derivatives were designed, synthesized, and their biological activities were evaluated. Some compounds displayed potent tyrosinase inhibitory activities, especially, compounds with thiosemicarbazide moiety showed more potent inhibitory

Full text of DOI:10.1016/j.bmcl.2015.10.004

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of aloe-emodin derivatives as potent
anti-tyrosinase, antibacterial and anti-inflammatory agents
⇑
Jinbing Liu , Fengyan Wu, Changhong Chen
Department of Biology and Chemical Engineering, Shaoyang University, Shao Shui Xi Road, Shaoyang 422100, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty aloe-emodin derivatives were designed, synthesized, and their biological activities were evalu-
ated. Some compounds displayed potent tyrosinase inhibitory activities, especially, compounds with
thiosemicarbazide moiety showed more potent inhibitory effects than the other compounds. The struc-
ture–activity relationships (SARs) were preliminarily discussed. The inhibition mechanism of selected
compounds 1 and 13 were investigated. The results showed compound 1 was reversible inhibitor, how-
ever, compound 13 was irreversible. Kinetic analysis indicated that compound 1 was competitive tyrosi-
nase inhibitor. Furthermore, the antibacterial activities and anti-inflammatory activities of some selected
compounds were also screened. The results showed that compound 3 exhibited more potent antibacterial
activity than the aloe-emodin, compounds 5 and 6 possessed more potent anti-inflammatory activities
than the diacerein.
Received 7 August 2015
Revised 16 September 2015
Accepted 3 October 2015
Available online xxxx
Keywords:
Aloe-emodin derivatives
Tyrosinase inhibitory activities
Antibacterial activity
Anti-inflammatory activity
Ó 2015 Elsevier Ltd. All rights reserved.
Tyrosinase (EC 1.14.18.1) is a kind of oxidoreductase with din-
uclear copper ions, belong to the type-3 copper protein family,
namely the two copper ions of tyrosinase active center linked
together with three specific histidine residues form a tetrahedron
structure.1,2 Tyrosinase is widely distributed in microorganisms,
animals, and plants.3 Tyrosinase is a key enzyme in the biosynthe-
sis of melanin pigments, it could catalyze two distinct reactions
involving molecular oxygen in the hydroxylation of monophenols
to o-diphenols (monophenolase) and in the oxidation of o-diphe-
nols to o-quinones (diphenolase).4 Due to the high reactivity,
quinines could polymerize spontaneously to form high molecular
weight brown-pigments (melanins), which widely spreads in skin,
hair and eyes of mammals, or react with amino acids and proteins
to enhance brown color of the pigment produced.5 Melanin plays
an essential role in protection against UV injury under normal
physiological conditions.6,7 Previous reports confirmed that tyrosi-
nase not only was involved in melanizing in animals, but also was
one of the main causes of most fruits and vegetables quality loss
during post-harvest handling and processing, leading to faster
degradation and shorter shelf life.8,9 This can cause a decrease in
nutritional values and economic loss in food industry. Thus, pre-
venting this unfavorable browning reaction has always been a
challenge in food science.10 Recently, investigation also demon-
strated that various dermatological disorders, such as age spots
and freckle, were caused by the accumulation of an excessive level
of epidermal pigmentation.11,12 Skin health and appearance is a
major concerned issue for people globally. As early as last century,
pharmaceuticals and traditional remedies have become incremen-
tally popular for this purpose.13 Tyrosinase has also been linked to
Parkinson’s and other neurodegenerative diseases.14,15 In insects,
tyrosinase is uniquely associated with three different biochemical
processes, including sclerotization of cuticle, defensive encapsula-
tion and melanization of foreign organism, and wound healing.16,17
These processes provide potential targets for developing safer and
effective tyrosinase inhibitors as insecticides and ultimately for
insect control. Thus, the development of safe and effective tyrosi-
nase inhibitors is of great concern in the medical, agricultural, food
and cosmetic industries in relation to hyperpigmentation.18 So far,
some natural and synthetic compounds as tyrosinase inhibitors
were reported, such as hydroquinone, ascorbic acid, arbutin, kojic
acid, aromatic aldehydes, aromatic acids, aromatic alcohol, tropo-
lone, and polyphenols.19 However, only a few such as kojic acid,
arbutin, tropolone, and 1-phenyl-2-thiourea (PTU) are used as cos-
metic products.20 Furthermore, it has been reported that arbutin is
decomposed by temperature (10% decomposition at 20 °C for
15 days) and it has shown very low activity. Arbutin and tropolone
have not been demonstrated to be clinically efficacious when crit-
ically analyzed in carefully controlled studies.21 The use of kojic
acid is controversial due to its toxicity. The use of the most popular
drug hydroquinone in cosmetic products was banned in the
European Union and is under scrutiny by the United States Food
⇑
Corresponding author. Tel./fax: +86 739 5431768.
E-mail address: syuliujb@163.com (J. Liu).
http://dx.doi.org/10.1016/j.bmcl.2015.10.004
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
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2
J. Liu et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
and Drug Administration (FDA) because it can cause DNA damage
and carcinogenic effect.13 Therefore, it is still necessary to search
and discover novel tyrosinase inhibitors with higher activities
and lower side effects.
On the other hand, it was reported that phenyl thioureas and
alkyl thioureas could exhibit weak to moderate depigmenting
activities.22 Because of tyrosinase belongs to catechol oxidase with
the type-3 copper protein group, the sulfur atom of phenyl thiour-
eas or alkyl thioureas can bind to both copper ions in the active site
of the enzyme.5 Ley and Bertram also reported that benzaldoximes
and benzaldehyde-o-alkyloximes possessed higher tyrosinase inhi-
bitory ability.23 The above information was beneficial to design
new tyrosinase inhibitor.
Natural products (NPs), including semi-synthetic NPs and NP
derived compounds, with great characteristics of high chemical
diversity and biochemical specificity, are all through major entities
among the FDA-approved drugs as anticancer, anti-inflammation,
anti-infective agents and so on.24 Rheum officinale Baill, is one of
the most popular traditional medicinal herbs that is officially listed
in the Chinese Pharmacopoeia.25 There has been great clinical
interest in using Rheum officinale Baill in treatment of many kinds
of diseases. Aloe-emodin (AE, 1,8-dihydroxy-3-(hydroxymethyl)
anthracene-9,10-dione), is one of major bioactive anthraquinone
derivatives from the roots of Rheum officinale Baill, which has
been reported to possess antibacterial, antiviral, anticancer, hep-
atoprotective, antiproliferative activity, anti-angiogenic effects,
laxative and anti-inflammation effects.26–28 In addition, aloe-
emodin, as an antioxidant, can reduce oxidative damage caused
by free radicals.
Taking advantage of above information, in order to find some
new bioactivities of aloe-emodin derivatives, such as inhibitory
activities of tyrosinase, antibacterial activities and anti-inflamma-
tory activities. Based on the structural feature of aloe-emodin, a
series of aloe-emodin derivatives were designed and synthesized.
Evaluations of inhibitory activities of tyrosinase, antibacterial
activities and anti-inflammatory activities of these compounds
were carried out. Meanwhile, the structure–activity relationships
between these compounds and inhibitory activities of tyrosinase
were also primarily discussed.
Our results showed that some compounds exhibited certain
inhibition activities on mushroom tyrosinase with IC50 values ran-
ged from 24.52 to 108.62 lM. Especially, compounds 13, 16, 20
bearing thiosemicarbazide showed more potent inhibitory activi-
ties than most of the other compounds. In addition, compounds
13, 16 demonstrated similar inhibitory activities to the reference
standard inhibitor kojic acid, 2-((4,5-dihydroxy-9,10-dioxo-9,10-
dihydroanthracen-2-yl)methylene)hydrazine-1-carbothioamide
(13) exhibited the most potent tyrosinase inhibitory activity with
IC50 value of 24.52 lM. The results showed that the group of
thiosemicarbazide played important role in determining activities
against tyrosinase. These may be related to the structure of tyrosi-
nase contained a type-3 copper center with a coupled dinuclear
copper active site in the catalytic core. Tyrosinase inhibition ability
of compounds 13, 16, 20 depended on the competency of the sulfur
atom to chelate with the dicopper nucleus in the active site, and
tyrosinase would lose its catalyzing ability after forming complex.
These results are consistent with our previous research.29 From the
result of Table 1, the other Schiff bases showed weak tyrosinase
inhibitory activities except for the compound 17 (4,5-bis(ally-
loxy)-9,10-dioxo-9,10-dihydroanthracene-2-carbal-dehyde oxime)
with IC50 value of 83.17 lM. However, it still showed lower inhibi-
tory activity than the Schiff bases with thiosemicarbazide moiety.
Comparing to the tyrosinase inhibitory activities of compounds
1–4, with the increase of lipophilicity of compound, the inhibitory
activities increased gradually, 1,8-dibutoxy-3-(hydroxymethyl)
anthracene-9,10-dione (1) exhibited the most potent tyrosinase
inhibitory activity with IC50 value of 32.81 lM. These results
showed that the inhibitory activities of this kind of compounds
with alkoxyl group at the 1-position and 8-position of anthracene
ring might be relate to the lipophilicity. Because lipophilic groups
can affect both the inhibition potency, as well as the ability of
the tyrosine to compete with the inhibitor. Lipophilic group may
interact with the enzyme hydrophobic pocket and augment bind-
ing affinity. According to the inhibitory activities of compounds
5–8, with the increase of lipophilicity of compounds, the inhibitory
activities also increased gradually.
The inhibition mechanism of compounds 1 and 13 on mush-
room tyrosinase for the oxidation of L-DOPA was determined.
We hope that these findings can lead to the discovery of poten-
tial pharmacological agents for treating the tyrosinase-related dis-
orders and also offer key and useful information for future design
of highly potent tyrosinase inhibitors, antibacterial and anti-
inflammatory reagents.
The general procedure for the synthesis of aloe-emodin deriva-
tives are described in Scheme 1. Aloe-emodin could be alkylated
smoothly by using a simple procedure to give 1,8-alkoxy substi-
tuted aloe-emodin (1–4), but the yields were low to moderate.
Aloe-emodin and the 1,8-alkoxy substituted aloe-emodin (1–4)
were oxidized with silica supported PCC to provide the corre-
sponding 4,5-disubstituted-9,10-dioxo-9,10-dihydroanthracene-
2-carbaldehyde (5–8). These aldehyde compounds reacted with
thiosemicarbazide or the other substituted amines in absolute
ethanol to provide the corresponding Schiff base compounds in
poor to moderate yields. All the target compounds were character-
ized by chemical and spectral methods.
Figure 1 showed the relationship between enzyme activity and
concentration in the presence of different concentrations of com-
pound 1, respectively. The results demonstrated that the plots gave
a family of straight lines, which all passed through the origin.
Increasing the inhibitor concentration resulted in a decrease in
the slope of the line. This result indicated that the inhibition of
compound 1 on mushroom tyrosinase was reversible. Figure 2
showed the relationship between enzyme activity and concentra-
tion in the presence of different concentration of compound 13.
The results displayed that the plots of V versus [E] gave a family
of parallel straight lines with the same slopes. It demonstrated that
the inhibitory effect of compound 13 on the tyrosinase was
irreversible.
To further insight into the inhibition mechanism, finally the
inhibitory type of the selected compound 1 on mushroom tyrosi-
nase for the oxidation of L-DOPA was determined by the Line
Weaver–Burk double reciprocal plots. Figure 3 showed the dou-
ble-reciprocal plots of the enzyme inhibited by compound 1. The
result displayed that the plots of 1/V versus 1/[S] gave three
straight lines with different slopes, but they intersected at the
same point on the ordinate. The values of Vmax remained the
same and the values of Km increased with increasing concentra-
tions of the inhibitor, which indicated that compound 1 was the
competitive inhibitor of tyrosinase. The result showed that com-
pound 1 could only bind with the free enzyme.
For evaluating the tyrosinase inhibitory activity, all the synthe-
sized compounds were subjected to tyrosinase inhibition assay
with L-DOPA as substrate, according to the method reported by
our groups with some slight modifications.29 The tyrosinase inhibi-
tory activity of kojic acid was ever reported, therefore, it was
selected as comparing substance. The IC50 values of aloe-emodin
derivatives against tyrosinase were summarized in Table 1, and
IC50 values of all these compounds were determined from logarith-
mic concentration–inhibition curves and given as means of three
experiments.
Three compounds were selected to test the antibacterial
activities,30 and the results were summarized in Table 2. The MIC
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J. Liu et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
OH
O
O
OH
OR1
O
OR2
OR1
O
OR2
ii
i
OH
OH
O
O
O
6-8
1-4
O
ii
iii
OR2
OR1
O
O
OH
OH
OH
O
OH
iii
R3
R3
O
O
O
5
14-20
9-13
1. R1 = R2 = n-Bu
2. R1 = R2 = CH3
6. R1 = R2 = CH3
3. R1 = R2
7. R1 = R2
=
=
O
4. R1 = R2 = Allyl
8. R1 = R2 = Allyl
O
9. R3
=
10. R3 = NOH
N
O
S
11. R3 = NOCH3
12. R3 = NOCH2CH3
13. R3 = NNHCNH2
14. R1 = R2 = CH3, R3 = NOCH3
15. R1 = R2 = CH3, R3 = NOCH2CH3
S
17. R1 = R2 = Allyl, R3 = NOH
16. R1 = R2 = CH3, R3 = NNHCNH2
18. R1 = R2 = Allyl, R3 = NOCH3
19. R1 = R2 = Allyl, R3 = NOCH2CH3
S
20. R1 = R2
=
R
3 = NNHCNH2
,
O
Scheme 1. The synthesis of aloe-emodin derivatives. Reagents and conditions: (i) RX/K2CO3/DMF/55 °C, or 2-methoxyethanol/tosyl chloride/THF/0 °C/K2CO3/80 °C; (ii) silica
supported PCC/acetone/reflux; (iii) thiosemicarbazide/ethanol/reflux, or substituted amine/ethanol/room temperature.
Table 1
Tyrosinase inhibitory activities of the synthesized compounds
Inhibition ratioa (%)
IC50
(lM)
b
Compounds
C log P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
5.63
2.46
2.24
4.01
3.30
2.98
2.76
4.53
4.84
3.39
3.84
4.37
3.50
3.60
4.13
3.45
4.83
5.15
5.67
3.23
73.57 1.48
9.86 0.37
7.45 0.43
58.63 2.12
49.37 1.38
NAc
32.81 1.06
>200
>200
90.99 4.58
108.62 4.26
NA
55.39 2.85
NA
NA
12.89 1.05
18.33 0.87
78.59 3.67
NA
92.37 3.91
>200
>200
24.52 1.24
NA
76.38 3.35
56.97 2.55
NA
28.05 1.73
83.17 2.61
17
18
19
20
NA
Figure 1. Determination of the inhibitory effect of compound 1 on mushroom
tyrosinase for the oxidation of -DOPA. The concentrations of compound 1 for
curves 1–4 were 0, 10, 30 and 50 M, respectively.
65.27 2.83
58.06 1.85
23.6 0.52
L
Kojic acid
l
a
Percent inhibition at a concentration of 100
Inhibitor concentration (mean SD of three independent experiments) required
l
M. Mean values SD.
b
Due to rhein and diacetylrhein, low molecular weight anthra-
quinone derivatives, have been shown to be useful anti-inflamma-
tory agents in the treatment of rheumatoid arthritis, compounds 2,
5 and 6 were selected for further investigation in vivo in the croton
oil-induced mouse ear swelling test.31 The results of the inhibition
caused by intragastric administration of the selected compounds at
a dose of 0.105 mmol/kg on croton oil-induced mouse ear edema
are shown in Table 3. All the selected compounds showed potent
activity, especially, compound 5 exhibited the highest activity with
the inhibition percentage of 50.6%. Compounds 5 and 6 possessed
more potent activity than the diacerein. The results also
for 50% inactivation of tyrosinase.
c
NA = not active.
data in Table 2 indicated that the selected compounds possessed
moderate antibacterial activities, especially compound 3 exhibited
more potent antibacterial activity than the aloe-emodin using as
positive control drug at the same conditions, MIC value of the com-
pound was 1
lg/mL. This result showed the antibacterial activities
of aloe-emodin derivatives were worth further studying.
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J. Liu et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 3
The effect of the selected synthesized compounds on croton oil-induced ear swelling
in mice (mean SD, n = 8)
Compounds
Doses (mmol/kg)
Swelling degree (mg)
Inhibition (%)
2
5
6
0.105
0.105
0.105
0.105
2.75 1.20**
2.10 1.12**
2.44 0.91**
2.58 0.84**
4.25 0.91
35.2
50.6
42.6
39.1
Diacerein
Control
**
p < 0.01.
1 exhibited inhibitory effects on tyrosinase by acting as the
reversible and competitive inhibitor, and the compound 13 was
irreversible inhibitor. These results suggested that tyrosinase inhi-
bitory activities of aloe-emodin derivatives were worth further
studying. Compound 3 exhibited more potent antibacterial activity
than the aloe-emodin, the MIC value of the compound was
1 lg/mL. This compound might be used as one lead compound
Figure 2. Determination of the inhibitory effect of compound 13 on mushroom
with antibacterial activity. Compounds 5 and 6 possessed more
potent anti-inflammatory activities than the diacerein. Therefore,
these compounds may be considered as the agents with potential
anti-inflammatory activities and good candidates for more
advanced screening.
tyrosinase for the oxidation of
L
-DOPA. The concentrations of compound 13 for
M, respectively.
curves 1–4 were 0, 10, 30 and 50
l
Acknowledgments
We thank the innovation team of Shaoyang University, China
(2012) for funding this project. This work was also financially
supported by the Foundation of Education Department of Hunan
Province, China (15A172).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.10.
004.
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