Regulatory effect of hydroquinone-tetraethylene glycol conjugates on zebrafish pigmentation

Article abstract of DOI:10.1016/j.bmcl.2015.09.059

We synthesized two hydroquinone-tetraethylene glycol conjugates (HQ-TGs) and investigated their log P, photophysical stability, and redox chemical stability. HQ-TGs are a little more hydrophilic than hydroquinone (HQ) and show an enhanced photophysical an

Full text of DOI:10.1016/j.bmcl.2015.09.059

Accepted Manuscript
Regulatory effect of hydroquinone-tetraethylene glycol conjugates on zebrafish
pigmentation
Hoa Thi Le, Bin Na Hong, Young Ro Lee, Ji Hyun Cheon, Tong Ho Kang, Tae
Woo Kim
PII:
S0960-894X(15)30093-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.09.059
BMCL 23140
Reference:
Bioorganic & Medicinal Chemistry Letters
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18 September 2015
24 September 2015
Please cite this article as: Le, H.T., Hong, B.N., Lee, Y.R., Cheon, J.H., Kang, T.H., Kim, T.W., Regulatory effect
of hydroquinone-tetraethylene glycol conjugates on zebrafish pigmentation, Bioorganic & Medicinal Chemistry
Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.09.059
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Graphical Abstract
Regulatory effect of hydroquinone-
tetraethylene glycol conjugates on zebrafish
pigmentation
Leave this area blank for abstract info.
Hoa Thi Lea,†, Bin Na Hongb,†, Young Ro Leeb,c, Ji Hyun Cheona, Tong Ho Kangb,c,*, Tae Woo Kima,*

Bioorganic & Medicinal Chemistry Letters
Regulatory effect of hydroquinone-tetraethylene glycol conjugates on zebrafish
pigmentation
Hoa Thi Lea,†, Bin Na Hongb,†, Young Ro Leeb,c, Ji Hyun Cheona, Tong Ho Kangb,c,*, Tae Woo Kima,*
a Graduate School of East-West Medical Science, Kyung Hee University, Gyeonggi-do 449-701, Republic of Korea
b Department of Oriental Medicinal Materials & Processing, College of Life Sciences, Kyung Hee University, Gyeonggi-do 449-701, Republic of Korea
c Graduate School of Biotechnology, Kyung Hee University, Gyeonggi, Gyeonggi-do 449-701, Republic of Korea
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
We synthesized two hydroquinone-tetraethylene glycol conjugates (HQ-TGs) and investigated
their logP, photophysical stability, and redox chemical stability. HQ-TGs are a little more
hydrophilic than hydroquinone (HQ) and show an enhanced photophysical and redox chemical
stability compared with HQ. In addition we studied the effect of HQ-TGs on cell viability and
on zebrafish pigmentation. MTT assay in HF-16 cells showed HQ-TGs are less cytotoxic than
HQ. The phenotype-based image analysis of zebrafish larvae suggests that HQ-TGs suppress the
pigmentation of zebrafish in a dose-dependent manner. The comparative experiments on
stability, cytotoxicity, and zebrafish pigmentation between HQ and HQ-TGs suggest that mono
tetraethylene glycol-functionalization of HQ is an alternative solution to overcome the adverse
effect of HQ.
Keywords:
hydroquinone
hydroquinone-tetraethylene glycol conjugate
zebrafish
pigmentation
whitening effect
2009 Elsevier Ltd. All rights reserved.
Hyperpigmentation is the darkening of an area of skin caused
by inflammatory skin disorders, allergic/irritant contact
dermatitis, sun damage, or other skin injuries.1 Several
compounds with bleaching properties have been used for the
treatment of pigmentary disorders of the skin. The bleaching or
depigmentation agents are divided into two groups, phenolic and
nonphenolic compounds.2 The phenolic group includes
hydroquinone (HQ), monobenzyl ether of hydroquinone
(MBEH), 4-methoxyphenol (mequinol), 4-isopropylcatechol, N-
acetyl-4-S-cystaminylphenol (NCAP), arbutin, etc. Meanwhile,
azelaic acid, tretinoin, L-ascorbic acid, kojic acid, and N-
acetylcysteine, etc. are belong to the nonphenolic group.
Zebrafish (Danio rerio) has emerged as an important
vertebrate model organism in neurobiological and biomedical
research during the last decades.5 Its value for studying vertebrate
development and human diseases has been recognized because of
its ex utero, optically transparent embryogenesis and amenability
to in vivo manipulation.6 The embryos rapidly absorb low
molecular weight compounds, diluted in the surrounding media,
through skin and gills. In contrast, late stage zebrafish, from 7
days post fertilization (dpf) to the adult stage, absorb compounds
orally.7 Therefore, early stage zebrafish provide another
advantage of testing percutaneous effects of medicinal and/or
cosmetic compounds. In addition, melanin pigments of zebrafish
are easy to observe without complicated experimental
procedures. T. J. Yoon et al. validated zebrafish as a model for
phenotype-based screening of melanogenic regulatory
compounds.8
In the above bleaching or depigmenting agents, HQ has been
the gold standard for treatment of hyperpigmentation for over 50
years.3 2% HQ preparations are the only agents considered safe
and effective for over-the-counter treatment of cutaneous
hyperpigmentation.4 However, it also has some adverse reactions.
It may be irritating and unstable at high concentrations (5~10%).
Even though not satisfying, other phenolic agents have been
developed to overcome the conventional disadvantages of HQ.
Chemically modified HQs are still attractive approach to develop
a price-competitive skin lighter.
Scheme 1. Molecular structures of phenolic depigmenting agents. HQ =
hydroquinone, MBEH = monobenzyl ether of hydroquinone, mequinol = 4-
hydroxyanisole, NCAP = N-acetyl-4-S-cysteaminylphenol, arbutin = 4-
hydroxyphenyl-β-glucopyranoside.
———
*Corresponding authors. Tel.: +82-31-201-3704; fax: +82-31-204-
8119; e-mail: tw1275@khu.ac.kr (T. W. Kim), panjae@khu.ac.kr (T.
H. Kang). †First two authors contributed equally to this work.

Our study commenced with the synthesis of HQ derivatives,
which can suppress pigmentation but simultaneously overcome
the disadvantages of HQ. Compared with HQ, the hydroquinone
derivatives should reduce cytotoxicity and improve chemical
stability against oxidation by light, air, or oxidant. In addition the
compounds should be easy to synthesize and to be scalable.
Scheme 1 represents the molecular structures of depigmenting
agents on market. The depigmentation agents, except HQ itself,
share a common structural motif, mono ether-functionalized HQ.
PEGylation (the covalent attachment of polyethylene glycol
(PEG) to another molecule) of biologics improves their
pharmacokinetic, pharmacodynamic, and immunological
profiles.9 In addition, the recent clinical development of PEG-
naloxol (NKTR-118 from Nektar Therapeutics, San Carlos, CA)
spotlighted the benefits and challenges of small molecular weight
PEGylation of small molecules.10,11 Small molecular weight
PEGylation of HQ was studied in viewpoint of hydrolyzable
prodrug concept,12 but the depigmentation effect of HQ
derivatives attaching small molecular weight PEG was not fully
addressed.
In order to apply HQ-small molecular weight PEG conjugates
as a depigmentation agent, should be taken into account the next
issues; synthesis, stability, safety, and melanogenesis inhibitory
effect. As a proof of concept, we synthesized two HQ-
tetraethylene glycol conjugates (HQ-TGs) and investigated the
logP, photophysical stability, redox chemical stability, and
cytotoxicity of HQ-TGs. We also confirmed the depigmentation
of HQ-TGs using zebrafish phenotype-based evaluation.
Scheme 2. Reagents and conditions. a) p-Toluenesulfonyl chloride, TEA,
CH2Cl2; b) ethyl bromoacetate, NaH, THF; c) NaOH, CH3CN, then HCl; d)
1b, NaH, THF; e) 2c, EDCHCl, DMAP, CH2Cl2; f) H2, Pd/C, THF. Bn =
benzyl, EDCHCl
=
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride, DMAP = 4-(dimethylamino)pyridine.
1 and 2 were prepared by following Scheme 2. The
tetraethylene glycol precursor for ether bond formation (1b) was
prepared by tosylation of tetraethyleneglycol monomethyl ether.
The other tetraethylene glycol precursor for ester bond formation
(2c) was prepared by two step reactions: 1) Williamson ether
synthesis of triethyleneglycol monomethyl ether and ethyl
bromoacetate and 2) basic hydrolysis of ester and acidification.
The tetraethylene glycol-hydroquinone conjugates having ether
linkage (1c, 1) were reported by N. N. Ekwuribe et al. but were
not characterized.12 The tetraethylene glycol-hydroquinone
conjugates having ester linkage (2d) was prepared by
esterification using EDC as a coupling reagent. 1 and 2 were
prepared from debenzylation of 1c and 2d (H2 balloon, 10 wt.%
palladium on carbon). The synthesis and characterization of 1
and 2 was reported in Supplementary Data S1. The synthesis and
NMR, MS characterization of 1c, 1, 2d, and 2 were reported in
SD S1.
Figure 1. Surface area vs. logP correlation of ethylene glycol and
hydrocarbon alkyl chain. The surface area (Å2) was calculated using Spartan
’08 (B3LYP calculation with 6-31G (d) basis set in vacuum, Wavefunction,
Inc.). The logP values were calculated using Advanced Chemistry
Development (ACD/Labs) Software V12.01.
Ethylene glycol chain (-(CH2CH2O)n-) has unique physical
properties compared with hydrocarbon alkyl chain (-(CH2)n-).
The oxygen in ethylene glycol unit makes the chain more
hydrophilic and the ethylene moiety provides hydrophobicity to
the chain. The balance between hydrophilicity and
hydrophobicity of ethylene glycol unit makes polyethylene
glycols (PEGs) miscible in broad range of solvents; from water to
many organic solvents. The surface area vs. logP correlation of
ethylene glycol and hydrocarbon alkyl chains clearly shows the
characteristic hydrophilicity of ethylene glycol chain compared
with hydrocarbon alkyl chain. The longer hydrocarbon alkyl
chains are the more hydrophobic, but the longer ethylene glycol
chains are the more hydrophilic (Fig. 1). In general, the ethylene
glycol modification of a molecule enhances both aqueous and
organic solubility of the molecule at the same time. Usually
ethylene glycol modification has been used for resolving the poor
water-solubility problem of hydrophobic mother molecules.13
It will be worth to mention the melting points of HQ and its
derivatives in Scheme 1; HQ = 172.3, MBEH = 122, mequinol =
57, arbutin = 199.5 C.14 We failed to find out the literature
melting point of NCAP. If considered structural similarity and
molecular weight, it would be reasonable to suggest that NCAP
has higher melting point than mequinol. The physical appearance
of them is solid at ambient temperature. However, we observed
that 1 and 2 are oily at ambient temperature. The liquid-like
property of 1 and 2 would provide better opportunity for
pharmaceutical and/or cosmetic formulation.
To test the effect of tetraethylene glycol moiety on HQ’s
hydrophobicity, logP values were measured by
a UV
spectrometer (Shake-flask method) (Supplementary Data S2).
The tetraethylene glycol moiety made HQ more hydrophilic
(logP (HQ) = 0.25  logP (1) = 0.026, logP (2) = 0.036), but
logPs are within 0.3 (Supplementary Data Table S2). The
linkage type of 1 and 2 (ether or ester) did not effect on the logP

difference of them (logP (1 and 2)  0.01). Though the
measured and the calculated logPs are not coincident, the logP
values show a same trend; HQ > 1  2.
pH is an important factor to determine the chemical
transformation of a compound when it was treated in a living
organism. We designed two HQ-TGs with different bonds to
inquire into the relation between the pH stability and the
melanogenic efficacy of HQ-TGs. Ether bonds are more robust
than ester bonds in acidic or basic conditions. The HPLC
monitoring of hydrolysis reaction at different pH condition shows
that 1 (ether linkage) was not hydrolyzed at the pH 3 ~ 9, but 2
(ester linkage) was hydrolyzed at basic condition (Supplementary
Data Fig. S3).
Figure 3. Cell viability of HQ, 1, and 2. Picture: 0.1% of DMSO (()
control), 200 M of HQ, 1 and 2 were treated in HF-16 cells and the pictures
were taken after 24 hr. The MTT data are expressed as means ± SEM. ***
p< 0.001 indicates a significant difference from () control group.
We investigated the cytotoxicity effect of 1 and 2 on the
viability of HF (human fetal foreskin fibroblast)-16 cells. The
viability of cultured HF-16 cells was evaluated using the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. The cell viability of HF-16 cells was significantly
decreased in HQ and 2 treatment (***, p < 0.001), but 1 did not
show any cytotoxicity in the cells at 200 M (Fig. 3). These data
indicate that 1 is less cytotoxic than HQ or 2.
Figure 2. A, B, C) UV spectrum change before (red circle) and after (black
square) UV exposure at ambient environment for 18 h. D, E, F) Time course
UV measurement after treating Cu2+ (20 M). [sample] = 0.1 mM in 1X PBS
(pH 7.4).
The facile oxidation of HQ to benzoquinone (BQ) under
ambient factors (light, oxygen, etc.) has been well-known.15 HQ
has UV absorption band at 289 nm (Fig. 2A red arrow). After UV
exposure (maximum irradiation at 365 nm) at ambient
environment, the characteristic UV absorption of HQ at 289 nm
decreased and a new UV absorption at 244 nm increased (Fig. 2A
black arrow). The new UV absorption after UV exposure agrees
with the known UV absorption band (244 nm) of BQ.16
Compared with HQ and 2, 1 is more stable under UV exposure at
ambient environment (Fig. 2B). We could not observe detectable
UV changes of 1 after long UV duration (18 h). 2 was more
labile than 1, but still more stable than HQ (Fig. 2C). The results
suggest that the HQ-TGs have better photophysical stability than
HQ under UV exposure.
HQ itself is a reductant and can be easily oxidized by
oxidizing chemicals.
For example, the oxidation of
hydroquinone was strongly accelerated by copper ions and
afforded BQ, which is considered to be more toxic than HQ.17
The Cu2+-mediated oxidation of HQ also generates hydrogen
peroxide (H2O2)18 and the H2O2 may interact with copper and
generate other reactive oxygen species (ROS) such as hydroxyl
radical (OH), single oxygen (1O2). The generation of ROS
results in HQ-induced DNA damage.19 HQ in the presence of
Cu2+ ions was oxidized to BQ in a time-dependent manner (Fig.
2D). However, 1 did not show any detectable changes (Fig. 2E).
2 showed just small UV change, but the change will be ignorable
when it is compared with HQ (Fig. 2F). The redox chemistry of
HQ is a complicate chemical process. Thus we cannot generalize
the effect of tetraethylene glycol conjugation on HQ redox
chemical stability from the above experiment. However, it has to
be considered that the tetraethylene glycol modification of HQ
would be a way to enhance redox chemical stability of HQ.
Figure 4. Phenotype evaluation of zebrafish laevae pigmentation. A) A
schematic representation for the schedule of pigmentation study.
Synchronized embryos were treated with HQ, 1, and 2 at the indicated
concentrations. Test compounds were dissolved in 0.1 % DMSO, and then
added to the embryo medium, B) 0.1% of DMSO, C) 100 M of HQ, D), E),
F) treatment of 1 at 50, 100, and 200 M, G), H), I) treatment of 2 at 50,
100, and 200 M.

We evaluated the depigmentation effect of HQ-TGs in
zebrafish larvae (Fig. 4). When HQ was treated as a positive
control, the black spots of zebrafish larvae faded away and the
eye colors of zebrafish larvae changed from black to gray (Fig.
4C). When 1 and 2 were treated, the black spots of zebrafish
larvae also faded away and the eye colors of zebrafish larvae
changed from black to gray (Fig. 4D-4F for 1 and 4G-4I for 2).
The degree of depigmentation of HQ at 100 M was similar to
that of 1 and 2 at 200 M (Fig. 4C, 4F, 4I). The inhibitory effects
of 1 and 2 on zebrafish pigmentation were dose-dependent. These
phenotype-based observations suggest that 1 and 2 have the
inhibitory effect on zebrafish pigmentation.
whiter image and the more efficient inhibition of pigmentation.
The BT analysis showed that 1 and 2 inhibited the pigmentation
of zebrafish in a dose-dependent manner (*** p < 0.001) (Fig.
5A). Histogram converts each RGB pixel in the image to gray
scale and gives color tone information. As the color tone
gradients move dark to light, the histogram of black spot (HB)
value increases. The histogram analysis also showed that 1 and 2
inhibited the pigmentation of zebrafish (** p < 0.01, *** p <
0.001) (Fig. 5B). The area summation of the black spots of
zebrafish larvae showed that the treatments of 1 and 2 downsized
the size of black spot (SB) in a dose-dependent manner (* p <
0.05, ** p < 0.01, *** p < 0.001) (Fig. 5C). These results suggest
that 1 or 2 suppress the pigmentation of zebrafish depending on
the concentration.
To quantitative comparison, the microscope images of
zebrafish larvae were evaluated by image analysis software. The
mock treatment (0.1% DMSO) was used as a negative control
and the HQ treatment (100 M) was used as a positive control. In
dorsal view pictures the zebrafish pigment spots, except eyes and
yolk, were manually selected and the binary threshold, the
histogram, and the size of the black spots were analyzed.
The bond type (ether or ester) between HQ and tetraethylene
glycol did not have no detectable influence on the binary
threshold, the black spot histogram, and the black spot size in
zebrafish pigmentation. This observation may be related with the
zebrafish early embryo’s characteristics: the embryos directly
absorb chemicals through skin and gills at early development
stage instead of oral absorption. Skin and gills do not form a
dynamic pH barrier compared with digestive organs. Meanwhile,
2 showed higher cytotoxicity than 1 in MTT assay (Fig. 3). If
considered both depigmentation effect and cytotoxicity, 1 (ether
linkage) would be a better option than 2 (ester linkage) as a
potent depigmentation agent.
Figure 6. Inhibitory effect of 1 on -MSH induced pigmentation. A) A
schematic representation for the schedule of -MSH pigmentation study.
Synchronized embryos were pretreated with 0.2 mM 1 at 9 hpf, then washed
intensively and bathed immediately in the fresh medium at 35 hpf.
Chemicals were added and incubated for a further 37 h. B) Representative
morphology of 72 hpf zebrafish larvae. C, D, E) Pigmentation image
analysis of 72 hpf zebrafish larvae by binary threshold, black spot histogram,
and black spot size. Data are expressed as means ± SEM. * p < 0.05, ** p <
0.01 and *** p < 0.001 indicate a significant difference between groups.
Figure 5. Pigmentation inhibitory effect on zebrafish. A negative control
group was exposed to 0.1% DMSO. 100 M of HQ used a positive control
of whitening. 1 or 2 at 50, 100, and 200 M were treated respectively. Data
are expressed as means ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001
indicate a significant difference from (-) control group.
Binary threshold (BT) means a black and white threshold digit
where the black spots disappear. The BT value is inversely
proportional to black color intensity. The higher BT indicates the

16. Wilke, T.; Schneider, M.; Kleinermanns, K. Open J. Phys. Chem.
2013, 3, 97.
17. Thomas, D. J.; Sadler, A.; Subrahmanyam, V.V.; Siegel, D.;
Reasor, M.J.; Wierda, D.; Ross, D. Mol. Pharmacol. 1990, 37,
255.
18. Li, Y.; Trush, M.A. Arch Biochem. Biophys. 1993, 300, 346.
19. Luo, L.; Jiang, L.; Geng, C.; Cao, J.; Zhong, L. Chem. Biol.
Interact. 2008, 173, 1.
Melanocyte stimulating hormones (MSHs) play a key role in
producing coloured pigmentation found in the skin, hair and eyes.
-Melanocyte stimulating hormone (α-MSH), an endogenous
tridecapeptide hormone produced by melanocortin is the most
important one of the MSHs in stimulating melanogenesis. All
groups were exposed with 1 during 9 ~ 35 hpf and then treated
with mock (group I), 1 (group II), α-MSH (group III), and 1 + α-
MSH co-treatment (group IV) (Fig. 6). The removal of 1 (group
I) recovered the pigmentation (low BT, HB & moderate SB). As
expected, the treatment of 1 (group II) suppressed the
pigmentation (high BT, HB & low SB) and the treatment of α-
MSH (group III) stimulated the pigmentation (low BT, HB &
high SB). Interestingly, the co-treatment of 1 and α-MSH (group
IV) showed the similar pattern with group II; a suppressed
pigmentation (high BT, HB & low SB). These data indicate that
Supplementary Material
Supplementary data associated with this article (synthesis and
characterization of 1 and 2, experiment details of stability test,
logP measurement, MTT assay, phenotype-based evaluation of
zebrafish melanogenesis) can be found, in the online version, at
http://dx.doi.org/xxxx/yyyyy.
1
acts as
a competitive inhibitor of -MSH induced
melanogenesis.
In summary, we synthesized two hydroquinone-tetraethylene
glycol conjugates, 1 and 2. The logP, photophysical stability, and
redox chemical stability of them were compared with HQ. The
mono tetraethylene glycol-functionalization of HQ improved the
photophysical and Cu2+ redox chemical stability. MTT assay in
HF-16 cells showed 1 is less cytotoxic than HQ or 2. We
observed the regulatory effect of 1 and 2 on zebrafish
pigmentation. The quantitative image analysis of zebrafish larvae
showed that 1 and 2 suppressed the pigmentation of zebrafish in
a dose-dependent manner. The comparative experiments on
stability, cytotoxicity, and zebrafish pigmentation between HQ,
1, and 2 suggest that mono tetraethylene glycol-functionalization
of HQ is an alternative solution to overcome the conventional
disadvantages of HQ.
Acknowledgments
This work was supported by a grant from the Kyung Hee
University in 2011 (KHU-20110478).
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