Identification of new arylsulfide derivatives as anti-melanogenic agents in a zebrafish model

Article abstract of DOI:10.1016/j.bmcl.2020.127201

A series of aryl sulfide derivatives was synthesized and evaluated for their anti-melanogenic activities. Several compounds, including 3e, 3i and 3q exhibited good anti-melanogenic activities. Among the derivatives, compound 3i showed good inhibitory effe

Full text of DOI:10.1016/j.bmcl.2020.127201

Bioorganic&MedicinalChemistryLetters30(2020)127201
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of new arylsulfide derivatives as anti-melanogenic agents in a
zebrafish model
Se Hwan Ahna,1, Kyu-Seok Hwangb,1, Dae-Seop Shinb, Seong Soon Kimb, Jung Yoon Yangb,
⁎
⁎
Byung Hoi Leeb, Eun Jung Baea, Byeong Wook Choia, Myung Ae Baeb, , Jin Hee Ahna,
a Department of Chemistry, Gwangju Institute of Science and Technology, Gwangju 61005, Republic of Korea
b Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of aryl sulfide derivatives was synthesized and evaluated for their anti-melanogenic activities. Several
compounds, including 3e, 3i and 3q exhibited good anti-melanogenic activities. Among the derivatives, com-
pound 3i showed good inhibitory effects against melanin synthesis and showed no toxicity in reconstituted
human eye and skin tissues.
Zebrafish
Phenotype-based screening
Reconstituted human skin
Skin-whitening agent
Cosmetic
Melanin is a cluster of dark polymer pigments responsible for eye,
hair, and skin color and these pigments are commonly present in almost
all animals.1 Melanin is synthesized by melanocytes in the basal layer of
the epidermis via a process called melanogenesis.2,3 Complex enzymatic
and signaling pathways are involved in melanin synthesis. Tyrosinase
(TYR), tyrosinase-related protein-1 (TRP-1), and tyrosinase-related
protein-2 (TRP-2) are three key regulators of melanin synthesis. Mela-
nogenesis is mediated by a series of complex signaling pathways in-
itiated by internal and external factors, including ultraviolet radiation
(UVR), nitric oxide (NO), and stress.1 Each of the signaling pathways is
associated with a master regulator called MITF, which controls the
expression of genes such as TYR, TRP-1, and TRP-2.
carcinogenicity and instability, and arbutin shows reduced efficacy in
vivo.16,17 Thus, there is a keen interest in the identification of new anti-
melanogenic compounds.
In this study, to identify anti-melanogenic agents, we screened
~1000 compounds in the Korea Chemical Bank library by phenotype
screening using live zebrafish. We positively identified compound KDZ-
003 as a hit. It exhibited moderate anti-melanogenic activity in the
zebrafish model. The initial outcome encouraged us to perform scaffold
modification. We wish to report the synthesis and biological evaluation
of arylsulfide derivatives as anti-melanogenic agents
A series of sulfide derivatives were synthesized according to Scheme
1. Commercially available 1-chloroisoquionoline (1a) reacted with di-
verse thiols (2) such as benzothiazole, imidazole, pyridine, pyrimidine,
and phenyl in ethanol to provide 1-thio-isoquinoline derivatives
(3a~q). Subsequently, not only 1-chloroisoquinoline, but also purine,
pyrrolo[2.3-d]pyrimidine, pyrrolo[3.2-d]pyrimidine, and thieno[3,2-d]
pyrimidine (1b-e) were coupled with 4-fluorophenyl thiols (2a) to
obtain sulfide compounds (3n~q). Compounds 4a and 4b were syn-
thesized by the treatment of 1-chloroisoquinoline (1a) with 4-fluor-
ophenol (2b) and 4-fluoroaniline (2c) as depicted in Scheme 2
In our phenotypic screening assay, we screened ~ 1000 compounds
to identify anti-melanogenic agents. After treatment for 24 h (between
10 and 34 hpf), KDZ-003 affected melanin synthesis with no develop-
mental defects. In an effort to increase its anti-melanogenic activity,
KDZ-003 was optimized, and the results were summarized in Tables
1–4. Compound 3a showed better anti-melanogenic efficacy than KDZ-
Normal melanin pigmentation has several beneficial effects, in-
cluding protection of skin cells from damage from UV light, toxic drugs,
and other chemicals.4,5 In contrast, abnormal production of melanin
causes acute dermatological problems, including senile lentigines,
freckles, melasma, post-inflammatory hyperpigmentation, and age
spots.6–8 These dermatological problems can lead to severe emotional
issues; therefore, the development of anti-melanogenic agents is ne-
cessary.
The most common commercially available cosmetics or skin
whitening agents such as hydroquinone9,10, kojic acid11, and arbutin
are tyrosinase inhibitors.12 Although they are recommended world-
wide, they are associated with certain drawbacks and side effects. Hy-
droquinone is toxic to mammalian cells13,14 and associated with a series
of side effects.15 The usage of kojic acid has been restricted due to its
⁎ Corresponding authors.
E-mail addresses: mbae@krict.re.kr (M.A. Bae), jhahn@gist.ac.kr (J.H. Ahn).
1 These authors equally contributed to this work.
https://doi.org/10.1016/j.bmcl.2020.127201
Received 6 February 2020; Received in revised form 15 April 2020; Accepted 16 April 2020
Availableonline18April2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

S.H. Ahn, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127201
Table 1
Anti-melanogenic efficacy of compounds 3a-e against zebrafish embryos.
a
No
Compound
Structure
Whitening efficacy test based on
zebrafish embryos (10 μM)
1
KDZ-003
+
2
3
3a
3b
++
–
Scheme 1. Reagents and condition: a) R1SH, EtOH, room temperature, 16 h; b)
trimethylamine, 1-butanol, reflux, 16 h; Note: Substituents (R1, X and Y) are
indicated in Tables 1-4.
4
5
6
3c
3d
3e
+
++
++
a
Excellent (+++), Good (++), Moderate (+), No (-).
Scheme 2. Reagents and condition: a) CuI, K2CO3, DMF, 150 °C, 6 h; b) HCl,
EtOH, 90 °C, 8 h.
whitening efficacy. We synthesized more fluorine-containing com-
pounds, via addition (3j) or repositioning (3k), and found that the
mono 4-fluoro derivative is more favorable for in vitro activity.
In addition, we tried to expand the sulfide into ether and amine
derivatives. Interestingly, when the thioether group is changed into the
ether (4a) and amine (4b), the anti-melanogenic effect disappeared;
indicating that the sulfide group is essential for the anti-melanogenic
efficacy.
003, however, compound 3b, which has benzothiazole, was not active.
Therefore, we sought monocyclic aromatic moiety. Pyrimidine (3c),
pyridine (3d), phenyl derivatives (3e) were synthesized and evaluated.
Compound 3c showed lesser anti-melanogenic effect than 3a. Com-
pound 3d and 3e exhibited good anti-melanogenic effect. However,
compound 3d caused cell death. Therefore, we further modified phenyl
derivative with diverse substituents.
Based on these results, we fixed the 4-fluorophenyl sulfide group
and changed the 1-isoquinoline group into different kinds of fused
heterocycles. As shown in Table 4, modification to purine (3n), pyrrolo
[2.3-d]pyrimidine (3o) and, pyrrolo[3.2-d]pyrimidine (3p) decreased
the anti-melanogenic effect compared to that of 3i. Whereas, thieno
[3,2-d]pyrimidine (3q) showed potent anti-melanogenic effect.
From the zebrafish screening results, we chose active compounds
Diverse phenyl derivatives were synthesized and the results are
summarized in Table 2. The substituents showed increased in vitro
activity, especially those with halide groups (3h, 3i) unlike compounds
with ethyl ester (3l) and trifluoromethyl (3m) groups. Among the ha-
lide derivatives, the 4-fluoro compound (3i) showed the most potent
2

S.H. Ahn, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127201
Table 2
Table 3
Anti-melanogenic efficacy of compounds 3f-m against zebrafish embryos.
Anti-melanogenic efficacy of compounds 4a-b against zebrafish embryos.
a
a
No
Compound
Structure
Whitening efficacy test
based on zebrafish embryos
(10 μM)
No
Compound
Structure
Whitening efficacy test based on
zebrafish embryos (10 μM)
1
4a
–
–
1
3f
++
++
++
+++
++
++
+
2
a
4b
2
3
4
5
6
7
3g
3h
3i
Excellent (+++), Good (++), Moderate (+), No (−).
Table 4
Anti-melanogenic efficacy of compounds 3n-q against zebrafish embryos.
a
No
Compound
Structure
Whitening efficacy test based on
zebrafish embryos (10 μM)
1
3n
–
3j
2
3o
+
3k
3l
3
4
3p
3q
++
+++
8
a
3m
–
a
Excellent (+++), Good (++), Moderate (+), No (−).
Based on the anti-melanogenic effects (Fig 1) as well as the side
effects including edema (data not shown), we chose compound 3i as a
prototype. The effect of 3i on melanogenesis-related proteins and genes
level in the HMV-Ⅱ cell lines was investigated. We examined the color
in the pellets after 3i treatment, in which the melanin changed from
black to brown (Fig 2A). Treatment of 3i reduced melanin contents in a
dose-dependent manner. HMV-Ⅱ cell lysates showed 67.6%, 31.3%, and
14.6% of the melanin contents of the DMSO control after treatment
Excellent (+++), Good (++), Moderate (+), No (−).
(3e, 3i and 3q) for a dose-dependency study using the zebrafish model.
We confirmed that 3i, 3q, and 3e treatment in zebrafish embryos re-
sulted in anti-melanogenic effects in a dose-dependent manner (Fig. 1C-
K) compared to DMSO, and PTU-treated control embryos (Fig. 1A, B).
3

S.H. Ahn, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127201
with 1, 10, and 20 µM 3i, respectively (Fig. 2B). Compound 3i more
potently decreased the melanin content than that of arbutin and kojic
acid, which were used as a positive control (Fig. 3).
The effect of 3i on the expression of melanogenesis-related genes
was assessed by real-time RT-PCR. As shown in Fig 2C, 3i treatment
markedly inhibited mRNA transcriptional levels of TRP-1, TRP-2, and
MITF in a dose-dependent manner in HMV-Ⅱ cells. Although the level of
tyrosinase mRNA was also reduced by treatment of 10 or 20 μM 3i,
howerver was not as clear as other genes and there was no concentra-
tion-dependent decrease. These findings demonstrate that the in-
hibitory effects of 3i on melanogenesis in HMV-Ⅱ cells might be
mediated through the downregulation of melanogenic genes TRP-1,
TRP-2, and MITF.
Cytotoxic effects of 3i on HMV-II cells were measured with CytoXTM
solution. When the HMV-II cells were treated with 20 μM 3i, the de-
crease in cell survival rates was less than 10%. This result suggests that
3i is less cytotoxic and safer in the range of the whitening effect. (Fig
3G)
The safety of 3i was tested in artificial ocular and skin tissue,
Neoderm-CD and Neoderm-ED, respectively. We determined the via-
bility of artificial tissues in the presence of 3i using an MTT assay. As a
result, 3i showed no toxic effects up to 50 μM in both Neoderm-CD
(Fig. 4A, B) and Neoderm-ED (Fig. 4C, D), which would indicate that 3i
is safe for general cytotoxicity.
In summary, a new series of aryl sulfide derivatives was synthesized
and evaluated for their anti-melanogenic effect using zebrafish and
mammalian cells. Here, compounds 3e, 3i and 3q showed significant
anti-melanogenic effect in the zebrafish model. Among the derivatives,
compound 3i showed good inhibition of melanin contents and no
toxicity with reconstituted human eye and skin tissue.
Fig. 1. Assessment of anti-melanogenic effect in zebrafish embryos.
Synchronized embryos were treated with (A) 0.1% DMSO and (B) 0.2 mM PTU.
(C-E) 3i, (F-H) 3q, and (I-K) 3e treatments exhibited anti-melanogenic effects in
a dose-dependent manner.
Fig. 2. 3i treatment blocks melanin synthesis in
human melanoma cell line HMV-Ⅱ (A). Melanin
pellets from cell lysates. (B) Total melanin
content compared with the DMSO control. (C)
Effect of 3i melanin production-related genes in
melanoma cells. The expression of genes after
treatment with 3i for 24 h was assessed by real-
time RT-PCR.
4

S.H. Ahn, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127201
Fig. 3. Cytotoxic effect by 3i was minimal in HMV-Ⅱ cells up to 10 μM treatment. Cells were cultured 5 days with (A) DMSO, (B) Arbutin, (C) Kojic acid and (D-F) 3i.
HMV-II cells were seeded in 96-well plates at a density of 7,500 cells/well and cultured for 24 h. The cells were treated with each sample for (G) 5 days. CytoX
reagent was used to measure cell vability.
Fig. 4. Effects of 3i on the viability of artificial ocular and skin tissue. (A) The artificial ocular tissues were stained with MTT solution (1 mg/mL) after each treatment
for 30 min. (B) Formazan was extracted with isopropanol from the tissues and absorbance was measured at 570 nm using a microplate reader (M1000pro; Tecan). (C)
The artificial skin tissues were stained with MTT solution (0.3 mg/mL) after each treatment for 3 h. (D) Formazan was extracted with 0.04 N isopropanol from the
tissues and quantified with a microplate reader.
5

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Bioorganic&MedicinalChemistryLetters30(2020)127201
Acknowledgements
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