1,2,3-Dithiazoles-new reversible melanin synthesis inhibitors: A chemical genomics study

Article abstract of DOI:10.1039/c5md00052a

A chemical genomic screen of an in-house library of small molecule heterocycles was carried out using Xenopus laevis embryos. This led to the identification of N-(4-chloro-5H-1,2,3-dithiazol-5-ylidene)-4-methoxyaniline (1c), which elicits loss of pigmenta

Full text of DOI:10.1039/c5md00052a

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1,2,3-Dithiazoles – new reversible melanin
synthesis inhibitors: a chemical genomics study
Cite this: DOI: 10.1039/c5md00052a
a
b
a
a
Anna Charalambous, Maria Koyioni, Ioanna Antoniades, Despoina Pegeioti,
a
b
c
Iro Eleftheriou, Sophia S. Michaelidou, Stanislav A. Amelichev,
Lidia S. Konstantinova, Oleg A. Rakitin, Panayiotis A. Koutentis*
and Paris A. Skourides*
c
c
b
a
A chemical genomic screen of an in-house library of small molecule heterocycles was carried out using
Xenopus laevis embryos. This led to the identification of N-IJ4-chloro-5H-1,2,3-dithiazol-5-ylidene)-4-
methoxyaniline (1c), which elicits loss of pigmentation in melanophores and the retinal pigment epithelium
(RPE) of developing embryos, independent of the developmental stage of initial exposure. The phenotype
was reversible, since pigmentation returned upon compound removal while analysis of neural crest cell
markers (Pax7) and melanophore markers (Dct/Xtrp2) revealed that both neural crest precursors and fully
differentiated melanophores were present in the dithiazole 1c treated embryos. A subsequent focused
structure–activity relationship (SAR) study identified the more active dithiazole 4-benzyloxy-N-IJ4-chloro-
5H-1,2,3-dithiazol-5-ylidene)-aniline (1l) and the need for a chlorine substituent at the dithiazole C-4 posi-
tion. Both the initial chemical genomic screen and the focused SAR study highlighted the toxicity of
(dithiazolylidene)aminoazines, and also of methoxyaniline (anisidine) analogues that hosted strong
electron-withdrawing or electronegative substituents or acidic hydroxyl groups on the anisidine moiety.
This study suggests that 1,2,3-dithiazoles can act as reversible melanin synthesis inhibitors, revealing a new
biological activity for this class of compounds. The inhibition of melanin synthesis is medically relevant as a
potential treatment for pigmentation disorders such as melasma.
Received 6th February 2015,
Accepted 26th March 2015
DOI: 10.1039/c5md00052a
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d) unprecedented spatial and temporal control over molecu-
lar targets. Small molecules can be administered at any time
1
Introduction
7
Chemical genomics is a rapidly evolving discipline that
depends on both chemical synthesis and molecular biology.
during the cell cycle or at any developmental stage and at any
desired location of the cell or organism, making this
approach pivotal for studying biological mechanisms and
1
,2
It entails high-throughput organic synthesis of small, non-
peptide based molecules, which are phenotypically screened
in model organisms to dissect complex biological pathways
7
drug discovery.
Various model organisms have been used for phenotypic-
3–5
and processes.
based screening of compound libraries, including Drosophila,
8
–11
This discipline is complementary to classical and condi-
tional genetic approaches, generating loss- or gain-of-
function mutations in specific molecular targets, as a means
of delineating the sequence of events in complex, multi-
component biological processes, such as embryonic develop-
C. elegans, zebrafish and more recently Xenopus.
The latter
display advantages since they represent the highest order
vertebrate model used to date and they are also easy to image
and dissect, allowing reproducible phenotypic and bio-
chemical readouts. Whole animal genomic screens are funda-
mental in identifying compounds that affect survival (toxico-
logical studies), perturb the biological process of interest (i.e.
pigment cell development and function) and aid with drug
6
ment and cell signaling. The former approach is gaining
ground over the latter, as it offers advantages: a) technical
simplicity, b) inexpensive reagents, c) low start-up costs, and
4
,9,10,12–22
target discovery and validation.
Compounds that modulate pigment cell development and
function include: a) 4-ij(4-morpholinobutyl)thio]phenol [also
known as IJ2-morpholinobutyl)-4-thiophenol, MoTP or 33N14]
which specifically ablates embryonic melanophores (melanin
producing cells) and also affects melanogenesis by blocking
neural crest cell (NCC) differentiation and melanophore
a
Department of Biology, University of Cyprus, P.O. Box 20537, 1678 Nicosia,
Cyprus
b
Department of Chemistry, University of Cyprus, P.O. Box 20537, 1678 Nicosia,
Cyprus. E-mail: koutenti@ucy.ac.cy
c
2
3
N.D. Zelinsky Institute of Organic Chemistry, RAS, Leninsky Prospekt 47, Moscow
1
19991, Russia
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1
2
multiplication, b) phenylthiourea (PTU) which acts as a
non-toxic melanin synthesis inhibitor by disrupting tyrosi-
and inactivation of the glutamine/amino acid transporter
ASCT2 (ref. 56) have been reported.
24
nase activity but not Dct and Tyrp1 function (cf. hydroquinone,
kojic acid and triazines),
aurovertin which promote melanin synthesis,
β-aminoethyl disulfides, such as cystamine, which affect pig-
mentation of human melanocytes and melanoma cells by
reducing tyrosinase activity in a reversible manner.
In the developing Xenopus embryo there are two main
types of melanin producing pigment cells: a) neural crest cell
The 4-methoxyanilino dithiazole 1c elicited loss of pig-
mentation in both melanophores and the retinal pigment
epithelium (RPE). The pigment loss was elicited irrespective
of what developmental stage the compound was added, and
was reversible since pigmentation returned upon compound
removal. Analysis of neural crest and melanophore markers
revealed that both neural crest precursors and fully differenti-
ated melanophores were present in dithiazole 1c treated
embryos. The data together with the developmental stage
independence of the phenotype and its reversibility suggested
that compound 1c inhibited melanin synthesis. We further
performed a small structure–activity study (SAR) that identi-
fied the more active dithiazole 4-benzyloxy-N-IJ4-chloro-5H-
1,2,3-dithiazol-5-ylidene)aniline (1l).
2
5
c) melanogenin, oligomycin,
2
6,27
and d)
2
8
2
9
derived melanophores and b) diencephalon derived retinal
3
0
pigment epithelium (RPE) cells. The primary melanophore
function is melanin synthesis, which protects the skin from
free radicals and UV radiation and also offers a survival
3
1,32
advantage as it facilitates camouflage.
The melanophores
are committed to the melanogenic lineage before migration
and this specification process is governed primarily by the
transcription factors Mitf, Wnt and BMP, although other sig-
naling pathways involving Kit, Endothelin 3 and the repres-
sor FoxD3 also play a role.
4–30, melanoblasts (melanophore precursors) initiate active
migration in a progressive anterior to posterior wave under
the guidance of various matrix metalloproteinases (MMPs)
and P-rex1, a Rac-specific Rho GTPase, to form the lateral
and dorsal pigment stripes in the embryo.
which functions as a signal centre for histogenesis, layer for-
3
3,34
At developmental stages
2
3
5
2
Results and discussion
3
6
3
7,38
2.1 4-Methoxyanilino dithiazole 1c induces severe pigment
loss in melanophores of the dorsal pigment stripe and
partial pigment loss in the RPE
The RPE,
3
9
mation and maintenance of the overlying photoreceptors, is
also under the direct control of Mitf.
4
0,41
A small library of ij(4-chloro-5H-1,2,3-dithiazol-5-ylidene)amino]arenes
was evaluated in a chemical genomics screen using Xenopus
laevis as an in vivo model system (see Fig. 1), due to their
impact on survival and embryonic development, so as to obtain
information regarding the potential toxicity and biological
activities of these compounds.
Stage 10 Xenopus laevis embryos were cultured with
increasing concentrations (5 and 10 μM) of the tested com-
pound and assessed for survival and elicitation of a distinc-
tive phenotype. From the compounds tested, the 4-methoxy-
anilino dithiazole 1c induced a dramatic phenotype of
The delineation of the developmental pathway undertaken
by pigment cells and the capability for intervention are
medically relevant as numerous diseases can arise in the case
of a defect, including albinism (a congenital disorder attrib-
uted to the absence or defect of tyrosinase, the enzyme
responsible for melanin synthesis), piebaldism (an autoso-
mal disorder leading to localized hypopigmentation), viti-
ligo (autoimmune disease leading to destruction of melano-
4
2
4
3
4
4
cytes),
and melanoma (due to uncontrolled growth of
4
4–46
melanocytes).
Recently, during a toxicological study of an in-house library
of small molecule heterocycles {150 compds: descriptors
[min–max (ave)]: molinspiration predicted log P (milog P)
[0.415–6.668 (3.68)]; topological polar surface area (TPSA)
[12.89–134.72 (55.53); number of atoms (natoms) [9–35
(18.84)]; molecular weight (mw) [157.18–459.55 (297.24)];
number of hydrogen bond acceptors (nON) [1–8 (4.03)]; num-
ber of hydrogen bond donors (nOHNH) [0–5 (0.44)]; number
of violations (nViol) [0–1 (0.19)]; number of rotatable bonds
(nrotb) [0–7 (2.21)]} by a chemical genomic screen of
Xenopus, we serendipitously discovered that the presence of
N-IJ4-chloro-5H-1,2,3-dithiazol-5-ylidene)-4-methoxyaniline (1c)
led to severe pigment loss on the developing embryo, which
is a new biological activity for 1,2,3-dithiazoles. Early studies
on the biological activity of ij(4-chloro-5H-1,2,3-dithiazol-5-
ylidene)amino]arenes focused on agrochemical properties
4
7–49
50–53
such as antibacterial,
antifungal,
and herbicidal
Fig. 1 Schematic representation of the chemical genomic screening
strategy using Xenopus laevis embryos as a model organism.
5
4
55
activities. More recently, interesting antitumor activity
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severe pigment loss in the melanophores of the dorsal pig-
ment stripe and the retinal pigment epithelium (RPE), when
applied to embryos at concentrations as low as 10 μM (Fig. 2).
The severity of the phenotype was dose dependent; doses
pigment loss in the melanophores of the dorsal pigment
stripe and in the RPE, at all developmental stages examined,
albeit less efficiently when the embryos were first exposed at
stage 31 (Fig. 3). This suggested that the dithiazole acts
directly on melanin synthesis rather than on melanophore
differentiation.
>10 μM of 1c led to increased toxicity (data not shown).
Furthermore, the effects were reversible, and pigmentation
returned in both neural crest derived melanophores and the
RPE, 14–16 h after the removal of compound 1c, irrespective
of the stage the embryos were originally exposed to the
dithiazole 1c. These results further support the fact that
compound 1c acts as a reversible enzyme inhibitor of the
melanin synthesis pathway rather than interfering with mela-
nophore survival and/or differentiation.
2
.2 4-Methoxyanilino dithiazole 1c elicited pigment loss is
reversible and can be elicited after melanophore differentiation
Pigment loss can be attributed to several factors including
interference with melanophore differentiation, survival and
inhibition of melanin synthesis. A small molecule that affects
cell fate can lose its activity after melanophore differentiation
and/or potentially lead to an irreversible defect, if for exam-
ple the melanoblasts are pushed towards another fate. There-
fore, to identify the underlying cause of the observed pigment
loss, the dithiazole 1c (10 μM) was applied at three different
developmental stages: 10 (gastrula), 21 (neurula), and 31 (tad-
pole). It should be noted that by stage 31 melanoblasts differ-
entiate into melanophores, migrate to the dorsal pigment
2.3 4-Methoxyanilino dithiazole 1c does not affect the
expression of the neural crest marker Pax7 or the
melanophore marker Dct (Xtrp2)
Pax3 and Pax7 paralogs are evolutionarily conserved transcrip-
tion factors that have overlapping yet different expressions
and roles in amphibian embryos. Both are widely used as neu-
3
7
stripe and begin melanin synthesis. Embryos were allowed
to develop to stage 40 and were visually scored for pigmenta-
tion. The 4-methoxyanilino dithiazole 1c (10 μM) induced
5
7
ral crest markers and it has been shown using whole mount
Fig. 2 Pigment loss attributed to the addition of the 4-methoxyanilino dithiazole 1c (5 and 10 μM) to stage 10 Xenopus embryos that were
allowed to develop to stage 40.
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Fig. 3 The effect of 4-methoxyanilino dithiazole 1c (10 μM) when applied to Xenopus embryos at developmental stages 10, 21 and 31 and allowed
to develop to stage 40. The last panel shows an image of rescued stage 21 exposed embryos (14–16 h after removal of compound 1c).
in situ hybridization and immunostaining that both Pax3 and
Pax7 are expressed in the melanophores of stage 45 embryos.
Further analysis indicated a striking colocalization of Pax7
was unaffected and developed, differentiated and migrated
normally, further supporting the fact that the dithiazole 1c
acts as a reversible melanin synthesis inhibitor.
5
8
mRNA with eumelanin in melanosomes. Therefore, to fur-
ther characterize the biological activity of the dithiazole 1c
we examined the expression pattern and levels of Pax7 using
immunofluorescence staining. Embryos were cultured in com-
pound 1c (10 μM) from stage 10 and allowed to develop to
stage 41, by which point they had already presented the pig-
ment loss phenotype. As shown in Fig. 4 (top panels), both
control embryos and embryos cultured in compound 1c
displayed normal expression levels of Pax7 protein and the
expression patterns in treated and control tadpoles were indis-
tinguishable. This showed that the pluripotent neural crest
cell population, from which the melanophores are derived,
Since the above data suggested that the neural crest was
not affected by 1c, we then examined the expression of a spe-
cific molecular marker of melanoblasts and differentiated
melanophores Xtrp2 (Dct). Dct is a melanogenic enzyme
which plays a central role in the melanin synthesis pathway.
Dct exhibits DOPAchrome tautomerase activity and isomer-
izes the pigmented intermediate DOPAchrome to 5,6-
dihydroxyindole-2-carboxylic acid (DHICA) rather than to 5,6-
dihydroxyindole (DHI). This enzyme thus regulates the
switch that controls the proportion of carboxylated subunits
5
9
in the melanin biopolymer. Melanophore specific expres-
sion of Dct is under the direct synergistic control of the tran-
6
0
scription factors Sox10 and Mitf and its expression can be
detected by stage 25 in melanoblasts and the RPE. To exam-
ine if the dithiazole 1c affects the RPE and melanophore
lineages, embryos were cultured in 1c (10 μM) at stage 10
and allowed to develop to stage 32, by which point they
clearly displayed loss of pigmentation in both the melano-
phores of the dorsal pigment stripe and the RPE compared to
controls. Both control and treated embryos were then
processed for whole mount in situ hybridization using the
previously characterized Dct probe. As shown in Fig. 4 (bot-
tom panels), Dct expression was unaffected in embryos cul-
tured in compound 1c suggesting that melanophore and RPE
differentiation, proliferation and survival were unaffected by
1c, providing further evidence that the 4-methoxyanilino
dithiazole 1c is a melanin synthesis inhibitor.
2.4 Structure–activity relationship (SAR) study of 1,2,3-dithiazoles
In an effort to identify more active analogues, a small struc-
ture–activity relationship (SAR) study was performed on a
series of 1,2,3-dithiazoles of general structure 1. For the prep-
aration of the 1,2,3-dithiazoles 1, primary aromatic or
heteroaromatic amines were reacted with 4,5-dichloro-1,2,3-
dithiazolium chloride (Appel salt), followed by treatment with
2,6-lutidine (2 equiv.) to give the corresponding ij(4-chloro-
Fig. 4 Whole mount immunofluorescence and in situ hybridization
(
WISH) show similar expression of the neural crest cell marker Pax7
and the melanocyte marker Xtrp2 (Dct), respectively, between control
embryos and 4-methoxyanilino dithiazole (1c) treated embryos. (Top
panels) Both control embryos and embryos cultured in compound 1c
display normal expression levels of Pax7 protein and the expression
patterns in the treated and control tadpoles at stage 40 are
indistinguishable. (Bottom panels) Dct expression levels and pattern
are unaffected in embryos cultured in dithiazole 1c to stage 32,
compared to control embryos. This experiment was also performed
using stage 40 embryos and the results were identical (data not
shown). Experiments were repeated three times, with at least 10
embryos per condition.
5
H-1,2,3-dithiazol-5-ylidene)amino]arenes 1 in moderate to
61–63
excellent yields (Scheme 1).
In the initial chemical genomic screen the parent com-
pound (Ar = Ph) and a series of 4′ (para) substituted N-IJ4-
chloro-5H-1,2,3-dithiazol-5-ylidene)anilines were studied and
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the methoxyphenyl group to give the 2-methoxypyrid-3-yl
analogue 1d led to toxicity; it was worth noting that in the
initial chemical genomic screen the substitution of the
aniline moiety by aminoazines (pyridines, pyrimidines and
pyrazines) led to the formation of toxic compounds (data not
shown). Next, the nature of the heteroatom linking the aryl
and the alkyl group was investigated. Replacement of the
Scheme
1 Synthesis and general structure of 1,2,3-dithiazoles 1
studied in the SAR with dithiazole (blue) and arene (red) atom
numbering.
4-methoxy (MeO) group with the more lipophilic 4-methylthio
(
MeS) group gave the analogue 1e that caused developmental
those with either inductively (e.g., halogens, Ar = 4-HalC H ;
delay and developmental defects (kinked tail phenotype). The
role of the alkyl substituent on the ether oxygen was probed.
When the methyl group attached to the oxygen atom was
replaced with a hydrogen atom (H) to give the phenol ana-
6
4
Hal
=
F, Cl, Br or I) and/or mesomerically electron-
, Ar = 4-O NC ) did not
withdrawing groups (e.g., NO
2
2
6 4
H
induce pigment loss. The p-tolyl analogue that hosts an
inductively electron-releasing methyl group (Ar = 4-MeC H )
logue 1f, or a trifluoromethyl group (F C) to give the benzo-
6
4
3
also failed (data not shown). This suggested that the presence
of the mesomerically electron-releasing methoxy group was
important. As such, in the focused SAR screen (see Table 1)
we first investigated the position of the methoxy group. Ana-
logues having the MeO group on the arene ring at position 2′
trifluoride analogue 1g, pronounced toxicity was observed;
6
4
phenols are acidic (pK 9.99), while F C groups are strongly
a
3
inductively electron-withdrawing and lipophilic. Nevertheless,
the analogues of 1c where the methyl group was replaced
with larger alkyl chains (e.g., ethyl, n-propyl, n-butyl) re-
mained active (Table 1, compounds 1h, 1i and 1j, respec-
tively). Interestingly, displacement of the alkyl chain with a
phenyl group (compound 1k) led to high toxicity, however,
(ortho) or 3′ (meta) (Table 1, compounds 1a and 1b, respec-
tively) showed no pigment loss and were therefore inactive.
2
The introduction of an electronegative sp ring nitrogen in
Table 1 Focused SAR study of dithiazoles 1a–t at 5 and 10 μM concentrations (− = no pigment loss, + = minor pigment loss, ++ = moderate pigment
loss, +++ = severe pigment loss, ++++ = complete pigment loss)
Pigment loss
Pigment loss
No.
Ar
Toxicity
5 μM
10 μM
No.
Ar
Toxicity
5 μM
10 μM
1
1
1
1
1
1
1
a
b
c
d
e
f
Non toxic
−
−
1k
Very toxic
−
−
Non toxic
Non toxic
Toxic
−
−
1l
Non toxic
++++
++++
+
++
−
++++
1m
1n
1o
1p
1q
Non toxic
−
−
−
−
−
Non toxic
++
−
+++
−
Toxic
−
Very toxic
Toxic
−
Mildly toxic at 10 μM
Mildly toxic at 10 μM
−
−
g
Very toxic
−
+
++
1
1
h
i
Non toxic
Non toxic
+
++
1r
1s
Mildly toxic at 10 μM
+++
+++
++
+++
Very toxic
−
−
1j
Non toxic
++
+++
1t
Very toxic
−
−
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Fig. 5 Direct comparison of the effect of the methoxyanilino and 4-benzyloxyanilino dithiazoles (1c and 1l (5 μM), respectively) on Xenopus
embryos at developmental stages 21, 25 and 31. The last panels on top and bottom rows are representative images of embryos rescued (14–16 h
after removal of dithiazoles 1c and 1l) after initial exposure to dithiazoles 1c and 1l at stage 21, showing the reversibility of the effect.
when the phenyl group was separated from the oxygen atom
by a methylene spacer, as in the benzyloxy (BnO) analogue
1
l, this led to enhanced pigment loss in comparison with 1c
(Fig. 5). As shown in the last panel of Fig. 5, the effects of the
benzyloxy analogue 1l were also reversible, and pigmentation
returned in both neural crest derived melanophores and
the RPE, irrespective of the stage of initial exposure of the
embryos (representative image shown from embryos initially
exposed to compound 1l at stage 21). Lastly, introduction of
additional electron-donating substituents at the 2′ or 3′ posi-
tion of the arene ring of the dithiazole 1c (compounds 1m,
1
n and 1p–r) was, in some cases, detrimental to the activity
Scheme 2 Synthesis of N-IJ4-phenyl-5H-1,2,3-dithiazol-5-ylidene)aniline
observed. The data tentatively showed that the activity
decreased with increasing bulk at these positions. Introduc-
tion, however, of an acidic hydroxy group (3′-OH) or of an
electron-withdrawing and lipophilic fluorine atom (3′-F), ana-
logues (1s and 1t, respectively) led to severe toxicity effects in
accordance with the mono-substituted analogues (1f and 1g),
further supporting the fact that groups with acidic protons or
strong electronegative groups were associated with toxicity.
From this screen the most active compound was the
(
3) and N-IJ4,5-dichloro-3H-1,2-dithiol-3-ylidene)aniline (4).
Interestingly, replacing the dithiazole 4-chlorine substitu-
ent with a phenyl group gave the 4-phenyl-1,2,3-dithiazole 3
which displayed no phenotypic activity. It was worth noting
that the chlorine, a good nucleofuge, at the dithiazole C-4
position facilitates the ring opening of the 1,2,3-dithiazole
system.
nucleofuge hampers this facile cleavage of the dithiazole
5
6,67
Replacing the chlorine with a group that is not a
4
-benzyloxyanilino dithiazole 1l. A direct comparison of
dithiazole 1l with the 4-methoxyanilino dithiazole 1c (active
compound from initial genomic screen) supported the fact
that the dithiazole 1l treated embryos displayed a more
severe pigment loss phenotype at lower doses and at all
developmental stages examined compared to the 4-methoxy-
anilino dithiazole 1c treated embryos (Fig. 5).
6
8–70
ring.
On the other hand, the 1,2-dithiole 4, which main-
tains the nucleofuge chlorine at the C-4 position, did induce
mild activity at 5 μM, while at 10 μM mild toxicity and some
developmental defects were observed. These results tenta-
tively suggest that a nucleofuge is needed at the dithiazole/
dithiole C-4 position which implies that the mechanism of
With the more potent structural motif for the arene part
elucidated, structural modifications were introduced to the
dithiazole part of the 4-benzyloxy analogue 1l. These included
replacing the dithiazole 4-chlorine substituent with a phenyl
group to give compound 3 and also replacing the dithiazole
ring nitrogen with a chlorocarbon to give compound 4. Both
5
6
action may involve dithiazole or dithiole ring opening.
3
Conclusions
4-Methoxyanilino and 4-benzyloxyanilino dithiazoles (1c and
1l, respectively) were identified in a chemical genomic screen
as compounds affecting the pigmentation of melanophores
5
5,65
compounds were synthesized starting from the oxime 2
and Boberg salt, respectively (Scheme 2).
6
6
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of the dorsal pigment stripe and the retinal pigment epithe-
lium (RPE) in developing Xenopus embryos. This biological
activity of 1,2,3-dithiazoles has not been reported previously.
Unlike other compounds shown to affect levels of melano-
(TOF) mass spectrometer. 4,5-Dichloro-1,2,3-dithiazolium
6
3
chloride (Appel salt), 3,4,5-trichloro-1,2-dithiazolium chlo-
7
3
ride (Boberg salt), N-IJ4-chloro-5H-1,2,3-dithiazol-5-ylidene)-2-
4
8
methoxyaniline (1a), N-IJ4-chloro-5H-1,2,3-dithiazol-5-ylidene)-
2
8,71
61
phore pigmentation in vivo, at doses >40 μM,
com-
3-methoxyaniline (1b),
N-IJ4-chloro-5H-1,2,3-dithiazol-5-ylid-
6
1
pounds 1c and 1l elicit a phenotype of severe pigment loss at
extremely low doses (<10 μM), with higher doses being
increasingly toxic. In addition to potency, these compounds
can have an effect even at later developmental stages (up to
stage 31) and do so reversibly. Reversibility is an extremely
important feature of melanin synthesis inhibitors which
ensures that their molecular targets are not permanently
inactivated and thus avoids cytotoxicity and disruption of
other pathways in which they might be involved. Finally,
these compounds inhibit melanin synthesis without affecting
the survival, differentiation and migration of neural crest cells,
melanophores or RPE, as indicated by unaltered expression
levels of Pax7 and Dct following exposure to the 4-methoxy-
anilino dithiazole 1c. In conclusion, 1,2,3-dithiazoles com-
prise a new class of potent and reversible melanin synthesis
inhibitors. Further work is needed to examine cross-species
activity, to determine the mechanism of action and to identify
the molecular target of these compounds.
ene)-4-methoxyaniline (1c),
ylidene)-4-IJtrifluoromethoxy)aniline (1g), N-IJ4-chloro-5H-1,2,3-
dithiazol-5-ylidene)-4-phenoxyaniline (1k),
1,2,3-dithiazol-5-ylidene)-3,4-dimethoxyaniline (1p), N-(4-chloro-
5H-1,2,3-dithiazol-5-ylidene)-2,3-dihydrobenzo[b][1,4]dioxine-
N-IJ4-chloro-5H-1,2,3-dithiazol-5-
56
56
N-IJ4-chloro-5H-
74
7
5
6-amine(1q),
N-(4-chloro-5H-1,2,3-dithiazol-5-
5
6
ylidene)benzo[d][1,3]dioxol-5-amine (1r), and N-(4-chloro-
56
5H-1,2,3-dithiazol-5-ylidene)-3-hydroxy-4-methoxyaniline (1s) were
prepared according to literature procedures. For the newly
synthesized compounds 1d–f, 1h–j, 1l–o and 1t, analytical,
physicochemical and spectroscopic data are reported below.
4.2 Synthesis of 4-chloro-1,2,3-dithiazoles (general procedure)
To a stirred suspension of 4,5-dichloro-1,2,3-dithiazolium chlo-
ride (Appel salt) (100 mg, 0.48 mmol) in dichloromethane
(4 mL) at ca. 20 °C, the corresponding aminoarene (0.48 mmol)
was added. After 1 h, 2,6-lutidine (111.2 μL, 0.96 mmol) was
added to the reaction mixture dropwise. After a further 2 h
the reaction mixture was adsorbed onto silica and purified
by dry flash chromatography to afford the corresponding
ij(4-chloro-5H-1,2,3-dithiazol-5-ylidene)amino]arene 1.
4
Experimental procedures
4
.1 General synthetic methods and materials
Reactions were protected from atmospheric moisture by
CaCl2 drying tubes. Anhydrous Na SO was used for drying
N-IJ4-Chloro-5H-1,2,3-dithiazol-5-ylidene)-6-methoxypyridin-
3-amine (1d). Obtained as yellow needles (113 mg, 91%),
2
4
organic extracts, and all volatiles were removed under
reduced pressure. All reaction mixtures and column eluents
were monitored by TLC using commercial glass backed thin
layer chromatography (TLC) plates (Merck Kieselgel 60 F254).
The plates were observed under UV light at 254 and 365 nm.
Dry flash chromatography was used throughout for all
non-TLC scale chromatographic separations using Merck
mp 117–119 °C (from c-hexane); R
70 : 30); (found: C, 36.76; H, 2.56; N, 16.01; C H ClN OS
2
f
0.48 IJn-hexane/t-BuOMe,
8
6
3
requires C, 37.00; H, 2.33; N, 16.18%); λmaxIJDCM) 244
(log ε 3.07), 300 (2.74), 378 inf (2.92), 394 (2.93), 411 inf
−
1
(2.84); νmax/cm
1599m, 1562m, 1553m, 1537w, 1520w,
1506w, 1483s, 1470m, 1441w, 1429m, 1375s, 1296s, 1281m,
1254m, 1231m, 1182m, 1153m, 1128s, 1028s, 1009m, 945w,
916m, 862s, 816s, 804s, 770s, 748m; δ (500 MHz; CDCl ) 8.23
7
2
Silica Gel 60 (less than 0.063 mm). Melting points were
determined using a PolyTherm-A, Wagner & Munz, Koefler-
Hotstage Microscope apparatus or a TA Instruments DSC
Q1000 with samples hermetically sealed in aluminum pans
under an argon atmosphere, using heating rates of 5 °C/min.
Solvents used for recrystallization are indicated after the
melting point. UV spectra were obtained using a Perkin-
Elmer Lambda-25 UV/vis spectrophotometer and inflections
are identified by the abbreviation “inf”. IR spectra were
recorded on a Shimadzu FTIR-NIR Prestige-21 spectrometer
with a Pike Miracle Ge ATR accessory and strong, medium
and weak peaks are represented by s, m and w, respectively.
H
3
(1H, d, J 3.0, ArH), 7.58 (1H, dd, J 9.0, 3.0, ArH), 6.85 (1H, d,
J 8.5, ArH), 3.98 (3H, s, CH O); δ (125 MHz; CDCl ) 162.5 (s),
3
C
3
157.3 (s), 148.3 (s), 140.2 (s), 138.6 (d), 131.8 (d), 111.6 (d),
+
53.9 (q); MALDI-TOF MS IJm/z) 262 (MH + 1, 45%), 260
+
(MH , 100).
N-IJ4-Chloro-5H-1,2,3-dithiazol-5-ylidene)-4-methylthioaniline
(1e). Obtained as yellow needles (122 mg, 98%), mp 127–128 °C
f
(from c-hexane); R 0.55 IJn-hexane/DCM, 40: 60); (found: C,
39.41; H, 2.46; N, 10.28; C H ClN S requires C, 39.34; H,
9
7
2 3
2.57; N, 10.19%); λmaxIJDCM) 262 (log ε 3.09), 354 inf (2.76),
−1
398 inf (2.92), 411 (2.94), 431 inf (2.83); νmax/cm 3003w,
2920w, 1589m, 1568m, 1545m, 1504m, 1489m, 1479m,
1429m, 1400m, 1321w, 1298w, 1265w, 1221m, 1179m,
1142m, 1111m, 1092m, 1005m, 968m, 857m, 945m, 860s,
833m, 824m, 775m; δ (500 MHz; CDCl ) 7.33 (2H, d, J 8.5,
1
13
H and C NMR spectra were recorded on a Bruker Avance
00 machine (at 300 and 75 MHz, respectively) or on a Bruker
3
Avance 500 machine (at 500 and 125 MHz, respectively).
Deuterated solvents were used for homonuclear lock and the
signals are referenced to the deuterated solvent peaks. Low
resolution (EI) mass spectra were recorded on a Shimadzu
Q2010 GC-MS with a direct inlet probe. MALDI-TOF MS
were conducted on a Bruker AUTOFLEX III time-of-flight
H
3
ArH), 7.21 (2H, d, J 8.5, ArH), 2.52 (3H, s, CH S); δ (125 MHz;
3
C
3
CDCl ) 157.3 (s), 148.2 (s), 147.5 (s), 137.1 (s), 127.5 (d), 120.5
+
+
(d), 15.9 (q); m/z (EI) 276 (M + 2, 39), 274 (M , 79), 259 (4),
229 (10), 227 (21), 210 (6), 181 (27), 175 (17), 166 (26), 149
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(100), 134 (15), 123 (11), 116 (33), 108 (31), 104 (9), 90 (11),
930m, 903s, 856m, 829m, 800w, 762m; δ (500 MHz; CDCl )
H
3
8
2 (8), 77 (11), 64 (31).
N-IJ4-Chloro-5H-1,2,3-dithiazol-5-ylidene)-4-hydroxyaniline
7.27 (2H, d, J 9.0, ArH), 6.98 (2H, d, J 9.0, ArH), 4.00 (2H, t, J
7.0, CH O), 1.79 (2H, tt, J 7.0, 7.0, CH ), 1.51 (2H, qt, J 7.8,
7.8, CH ), 0.99 (3H, t, J 7.5, CH ); δ (125 MHz; CDCl ) 157.9
2
2
(
1f). Obtained as orange plates (63 mg, 54%), mp (DSC)
onset: 130.0 °C, peak max: 136.9 °C (from c-hexane/1,2-DCE);
0.56 (DCM/t-BuOMe, 90 : 10); (found: C, 39.38; H, 1.97; N,
1.33; C H ClN OS requires C, 39.27; H, 2.06; N, 11.45%);
2
3
C
3
(s), 155.3 (s), 148.5 (s), 142.7 (s), 121.8 (d), 115.2 (d), 68.0 (t),
+
+
R
1
f
31.3 (t), 19.2 (t), 13.8 (q); m/z (EI) 302 (M + 2, 26%), 300 (M ,
51), 244 (59), 227 (11), 183 (18), 151 (28), 145 (23), 119 (100),
93 (16), 64 (35), 57 (20).
8
5
2
2
λmaxIJDCM) 251 (log ε 3.10), 280 inf (2.70), 398 (2.87), 414 inf
−
1
(2.80);
νmax/cm
3121m (OH), 1605m, 1585m, 1547m,
4-Benzyloxy-N-IJ4-chloro-5H-1,2,3-dithiazol-5-ylidene)aniline
(1l). Obtained as yellow plates (158 mg, 98%), mp 109–111 °C
(from c-hexane/1,2-DCE); Rf 0.48 IJn-hexane/DCM, 40 : 60);
1
1
8
504m, 1495m, 1441m, 1364m, 1364m, 1346w, 1269m,
231m, 1215m, 1153m, 1107m, 1011w, 935w, 887s, 858m,
29s, 812m, 768m; δ
H
(500 MHz; CDCl
3
) 7.23 (2H, d, J 8.5,
(found: C, 53.71; H, 3.25; N, 8.25; C15
2 2
H11ClN OS requires C,
ArH), 6.92 (2H, d, J 9.0, ArH), 4.97 (1H, s, OH); δ (125 MHz;
53.81; H, 3.31; N, 8.37%); λmaxIJDCM) 251 (log ε 3.20), 256 inf
C
−
1
CDCl ) 156.0 (s), 154.2 (s), 148.4 (s), 143.4 (s), 121.9 (d), 116.3
(3.18), 307 (2.67), 401 (2.98), 418 inf (2.90); νmax/cm 3038w,
2866w, 1603m, 1576m, 1562m, 1535w, 1495m, 1466m,
1454m, 1416w, 1381m, 1325w, 1304m, 1294m, 1275w, 1242s,
1227m, 1177m, 1157w, 1130m, 1111m, 1082w, 1030w,
3
+
+
(
d); m/z (EI) 246 (M + 2, 21%), 244 (M , 54), 227 (4), 209 (2),
1
9
83 (9), 180 (3), 151 (16), 145 (21), 125 (4), 122 (3), 119 (100),
6 (3), 15 (93), 91 (11), 70 (4), 64 (26).
N-IJ4-Chloro-5H-1,2,3-dithiazol-5-ylidene)-4-ethoxyaniline (1h).
H
1007m, 993m, 959w, 922m, 856s, 835s, 799m, 766s; δ (500
Obtained as yellow needles (119 mg, 91%), mp 106–107 °C
from c-hexane); R 0.48 IJn-hexane/DCM, 40 : 60); (found: C,
3.98; H, 3.26; N, 10.39; C10 ClN OS requires C, 44.03; H,
.33; N, 10.27%); λmaxIJDCM) 251 (log ε 3.05), 309 (2.57), 402
MHz; CDCl ) 7.45 (2H, d, J 7.5, ArH), 7.41 (2H, dd, J 7.5, 7.5,
ArH), 7.35 (1H, dd, J 7.3, 7.3, ArH), 7.28 (2H, d, J 9.0, ArH),
7.07 (2H, d, J 9.0, ArH), 5.10 (2H, s, CH O); δ (125 MHz;
2 C
3
(
4
3
f
H
9
2
2
CDCl ) 157.4 (s), 155.7 (s), 148.4 (s), 143.3 (s), 136.6 (s), 128.6
3
−
1
(2.88), 419 inf (2.80); νmax/cm 3032w, 2978w, 2928w, 1605m,
(d), 128.1 (d), 127.5 (d), 121.7 (d), 115.7 (d), 70.3 (t); m/z (EI)
+
+
1
1
9
576m, 1551m, 1526w, 1501m, 1481m, 1454w, 1439w, 1393m,
296m, 1244s, 1225m, 1171m, 1134m, 1115m, 1043m, 1005w,
336 (M + 2, 4%), 334 (M , 10), 245 (13), 243 (32), 154 (4), 150
(11), 122 (5), 91 (100), 65 (18).
57w, 918m, 858s, 833m, 822m, 791m, 760m; δ
) 7.27 (2H, d, J 8.5, ArH), 6.98 (2H, d, J 8.5, ArH), 4.07
2H, q, J 7.0, CH O), 1.44 (3H, t, J 7.0, CH ); δ (125 MHz; CDCl )
H
(500 MHz;
N-IJ4-Chloro-5H-1,2,3-dithiazol-5-ylidene)-2,4-dimethoxy-
aniline (1m). Obtained as orange cotton fibres (119 mg,
86%), mp 70–71 °C (from n-hexane at ca. −20 °C);
CDCl
(
3
2
3
C
3
1
57.7 (s), 155.4 (s), 148.5 (s), 142.8 (s), 121.8 (d), 115.2 (d), 63.7
λ
maxIJDCM)/nm 258 inf (2.91), 346 inf (2.60), 381 inf (2.67),
+
+
−1
(t), 14.8 (q); m/z (EI) 274 (M + 2, 25%), 272 (M , 60), 245 (12),
412 (2.72), 442 inf (2.59); νmax/cm 1599s, 1580s, 1558w,
1499s, 1468w, 1452m, 1431w, 1414w, 1306s, 1263s, 1211s,
1155s, 1117s, 1038s, 1026s, 984w, 959w, 924m, 856s, 829s,
2
1
43 (27), 227 (6), 209 (3), 183 (3), 179 (10), 173 (14), 151 (26),
47 (50), 122 (14), 119 (100), 102 (4), 91 (18), 70 (6), 64 (34).
N-IJ4-Chloro-5H-1,2,3-dithiazol-5-ylidene)-4-n-propoxyaniline
800w, 789w, 770s, 752m, 733m; δ
(1H, d, J 8.5, ArH), 6.57 (1H, d, J 2.5, ArH), 6.54 (1H, dd,
J 8.5, 2.5, ArH), 3.87 (3H, s, CH O), 3.84 (3H, s, CH O);
(125 MHz; CDCl ) 159.5 (s), 157.9 (s), 151.9 (s), 147.8
(s), 132.7 (s), 119.5 (d), 104.3 (d), 99.9 (d), 55.8 (q), 55.6
H 3
(500 MHz; CDCl ) 7.16
(
(
1i). Obtained as yellow needles (125 mg, 91%), mp 71–72 °C
from c-hexane); R 0.67 IJn-hexane/t-BuOMe, 70 : 30); (found:
11ClN OS requires C, 46.07; H,
.87; N, 9.77%); λmaxIJDCM)/nm 251 (3.14), 309 (2.66), 343 inf
f
3
3
C, 46.14; H, 3.96; N, 9.77; C11
3
(
H
2
2
δ
C
3
−
1
+
+
2.71), 382 inf (2.96), 402 (2.99). 424 inf (2.89); νmax/cm
(q); m/z (EI) 290 (M + 2, 39%), 288 (M , 100), 195 (33),
188 (87), 180 (14), 174 (14), 163 (67), 152 (24), 137 (11),
134 (38), 120 (19), 107 (14), 93 (11), 79 (20), 64 (30),
51 (17).
2
1
1
8
972w, 2943w, 2913w, 2878w, 1605m, 1574m, 1562m, 1531w,
499m, 1472m, 1391w, 1288w, 1250s, 1219m, 1171w, 1136s,
115m, 1047m, 1013s, 970w, 953w, 941w, 912w, 899w, 860s,
31s, 802m, 766s; δ
H
(500 MHz; CDCl
3
) 7.27 (2H, d, J 9.0,
N-IJ4-Chloro-5H-1,2,3-dithiazol-5-ylidene)-4-methoxy-2-
methylaniline (1n). Obtained as orange needles (93 mg, 71%),
ArH), 6.98 (2H, d, J 9.0, ArH), 3.96 (2H, t, J 6.8, CH O), 1.83
2
(
2H, qt, J 7.0, 7.0, CH
2
), 1.05 (3H, t, J 7.3, CH
3
); δ
C
(125 MHz;
mp 86–87 °C (from c-hexane); R
30); (found: C, 43.93; H, 3.39; N, 10.21; C10
C, 44.03; H, 3.33; N, 10.27%); λmaxIJDCM)/nm 254 (3.03), 309
f
0.62 IJn-hexane/t-BuOMe, 70:
ClN OS requires
CDCl ) 157.9 (s), 155.4 (s), 148.5 (s), 142.8 (s), 121.8 (d), 115.2
3
H
9
2
2
+
(
(
(
d), 69.8 (t), 22.6 (t), 10.5 (q); m/z (EI) 288 (M + 2, 23%), 286
M , 56), 244 (37), 183 (12), 151 (27), 145 (19), 119 (100), 93
13), 64 (30).
+
inf (2.54), 349 inf (2.67), 382 inf (2.81), 405 (2.84), 431 inf
−
1
(2.73);
ν
max/cm
3013w, 2965w, 2941w, 2924w, 2839w,
4
-n-Butoxy-N-IJ4-chloro-5H-1,2,3-dithiazol-5-ylidene)aniline
1j). Obtained as yellow cotton fibres (124 mg, 86%), mp 61–62 °C
from c-hexane); R 0.71 IJn-hexane/t-BuOMe, 70 : 30); (found:
1607m, 1591w, 1578w, 1489m, 1450w, 1431w, 1373w, 1306s,
1281m, 1250s, 1221s, 1165m, 1146m, 1109s, 1049m, 991w,
(
(
f
928w, 922w, 854s, 820m, 802m, 775m, 762s, 723m; δ
H
(300
C, 48.03; H, 4.30; N, 9.28; C H ClN OS requires C, 47.91;
MHz; CDCl ) 7.15 (1H, d, J 8.7, ArH), 6.86 (1H, d, J 2.7, ArH),
1
2
13
2
2
3
H, 4.36; N, 9.31%); λmaxIJDCM)/nm 252 (3.16), 309 (2.68), 343
inf (2.72), 382 inf (2.98), 402 (3.01), 425 inf (2.91); νmax/cm
6.80 (1H, dd, J 8.6, 2.9, ArH), 3.82 (3H, s, CH
CH ); δ (75 MHz; CDCl ) 158.1 (s), 155.4 (s), 148.4 (s), 142.3
(s), 133.9 (s), 116.8 (d), 116.6 (d), 111.5 (d), 55.4 (q), 18.1 (q);
3
O), 2.31 (3H, s,
−
1
3
C
3
2
1
963w, 2940w, 2872w, 1607m, 1570w, 1562w, 1501m, 1470w,
395w, 1296w, 1252s, 1175w, 1121s, 1096s, 1036m, 1007m,
+
+
m/z (EI) 274 (M + 2, 30%), 272 (M , 64), 257 (10), 237 (9),
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1
81 (10), 179 (20), 173 (36), 147 (100), 136 (13), 132 (17), 117
868w, 843m, 327m, 810m, 781m, 770m; δ (500 MHz; ace-
H
(18), 104 (19), 91 (21), 77 (63), 64 (46), 51 (32).
tone-d ) 8.20 (2H, dd, J 8.0, 1.5, ArH), 7.54–7.48 (5H, m, ArH),
6
N-IJ4-Chloro-5H-1,2,3-dithiazol-5-ylidene)-2-methyl-4-
7.41 (2H, dd, J 7.5, 7.5, ArH), 7.34 (1H, dd, J 7.3, 7.3, ArH),
7.25 (2H, d, J 9.0, ArH), 7.16 (2H, d, J 9.0, ArH), 5.17 (2H, s,
CH O); δ (125 MHz; acetone-d ) 164.2 (s), 160.7 (s), 157.9 (s),
trifluoromethoxyaniline (1o). Obtained as yellow prisms
41 mg, 26%), mp 49–50 °C (from c-hexane); (found: C, 36.62;
H, 1.94; N, 8.65; C10 ClF OS requires C, 36.76; H, 1.85;
N, 8.57%); λmaxIJDCM)/nm 274 (log ε 3.54), 372 (2.93);
(
2
C
6
H
6
3
N
2
2
146.8 (s), 138.3 (s), 134.0 (s), 131.1 (d), 130.0 (d), 129.3 (d),
129.0 (d), 128.7 (d), 128.5 (d), 121.8 (d), 116.7 (d), 70.8 (t);
-1
+
νmax/cm 2955w, 2924w, 2853w, 1607w, 1591w, 1570w,
MALDI-TOF MS IJm/z) 377 (M + 1, 100%), 345 (18), 285 (24),
1
1
485m, 1381w, 1250s, 1200s, 1157s, 1138w, 1107m, 1040w,
009w, 976w, 891w, 878m, 847m, 822m, 793m, 773s, 723m;
91 (4).
4-Benzyloxy-N-IJ4,5-dichloro-3H-1,2-dithiol-3-ylidene)aniline
(4). A solution of pyridine (151 mg, 1.92 mmol) in DCM
(10 mL) was added dropwise at ca. 20 °C to a stirred mixture
of Boberg salt (154 mg, 0.64 mmol) and 4-benzyloxyaniline
hydrochloride (172 mg, 0.73 mmol) in DCM (10 mL). The
reaction mixture was stirred at ca. 20 °C for 2 h, filtered,
washed with 10% HCl (2 × 10 mL) and water (2 × 10 mL),
dried IJNa SO ), and evaporated. The residue was adsorbed
δ (500 MHz; CDCl ) 7.16 (1H, s, ArH), 7.13–7.10 (2H, m,
H
3
ArH), 2.28 (3H, s, CH
3
); δ
C
(125 MHz; CDCl
3
) 159.0 (s), 148.6
3
(
(
s), 147.8 (s), 146.9 (q, JCF 1.3), 132.5 (s), 123.7 (d), 120.5
q, J 255.0), 119.6 (d), 117.2 (d), 17.7 (q); m/z (EI) 328 (M +
1
+
CF
+
2
2
, 25%), 326 (M , 50), 291 (5), 265 (6), 241 (10), 227 (37),
01 (100), 169 (13), 147 (12), 136 (10), 132 (15), 125 (17), 116
(12), 109 (22), 104 (12), 83 (19), 77 (33), 69 (64), 64 (62), 51 (21).
2
4
N-IJ4-Chloro-5H-1,2,3-dithiazol-5-ylidene)-4-fluoro-3-
onto silica and chromatographed IJn-hexane/DCM) to give the
title compound 4 as orange needles (70 mg, 26%), mp (DSC)
onset: 104.1 °C, peak max: 105.0 °C, decomp. onset: 136.1 °C,
methoxyaniline (1t). Obtained as yellow prisms (86 mg, 65%),
mp 87–88 °C (from c-hexane); R 0.43 IJn-hexane/t-BuOMe,
0 : 30); (found: C, 38.95; H, 2.12; N, 10.17; C ClFN OS
requires C, 39.06; H, 2.19; N, 10.12%); λmaxIJDCM)/nm 248
f
7
9
H
6
2
2
peak max: 147.3 °C (from c-hexane); R
40 : 60); (found: C, 52.22; H, 2.95; N, 3.86; C H Cl NOS
2
f
0.38 IJn-hexane/DCM,
16
11
2
(
(
log ε 3.17), 306 inf (2.63), 340 inf (2.73), 382 (2.97), 399
2.98), 424 inf (2.84); νmax/cm 2970w, 2938w, 2907w, 2843w,
requires C, 52.18; H, 3.01; N, 3.80%); λmaxIJDCM) 312 (log ε
2.70), 365 (2.76); νmax/cm 3034w, 2928w, 2914w, 2866w,
−
1
−1
1
1
9
616m, 1572m, 1564m, 1541w, 1514s, 1468w, 1454m, 1447m,
429w, 1315s, 1279s, 1227s, 1188m, 1161s, 1109s, 1022s,
64m, 905w, 866s, 856s, 799s, 781m, 766w, 748s; δ (500 MHz;
H
1601m, 1593m, 1576m, 1501S, 1496m, 1454m, 1379m,
1287m, 1242m, 1231S, 1184m, 1168w, 1159w, 1113m, 1080w,
1009w, 997m, 978m, 962w, 943w, 922w, 872m, 858s, 835m,
820w, 808w, 799m, 758s; δ (500 MHz; CDCl ) 7.45 (2H, d,
CDCl ) 7.11–7.02 (3H, m, ArH), 3.92 (3H, s, CH O); δ (125
3
3
C
H
3
1
MHz; CDCl
3
) 157.1 (s), 152.5 (d, JCF 247.0), 148.3 (s), 146.3 (d,
J 7.0, ArH), 7.40 (2H, dd, J 7.5, 7.5, ArH), 7.34 (1H, dd, J 7.3,
7.3, ArH), 7.06 (2H, d, J 9.0, ArH), 7.02 (2H, d, J 9.0, ArH),
5.07 (2H, s, CH O); δ (125 MHz; CDCl ) 158.2 (s), 156.8 (s),
2
3
3
4
J
CF, 10.9), 146.4 (d, JCF 8.0), 115.9 (d, JCF 3.5), 113.8 (d, JCF
2
+
2
2
1
6
.6), 109.1 (d, J 19.6), 56.4 (q); m/z (EI) 278 (M + 2, 43%),
CF
2
C
3
+
76 (M , 45), 263 (12), 245 (12), 183 (14), 168 (23), 151 (100),
142.99 (s), 142.95 (s), 136.8 (s), 128.6 (d), 128.1 (d), 127.5 (d),
40 (13), 136 (23), 125 (19), 108 (22), 96 (11), 85 (29), 71 (42),
4 (21), 57 (56).
122.7 (s), 121.3 (d), 115.9 (d), 70.3 (t); MALDI-TOF MS IJm/z)
+
+
+
372 (M + 5, 17%), 370 (M + 3, 78), 368 (M + 1, 100), 340
(15), 301 (33), 277 (51), 90 (41).
4
-Benzyloxy-N-IJ4-phenyl-5H-1,2,3-dithiazol-5-ylidene)aniline
3). Pyridine (237 mg, 0.24 mL, 3 mmol) was added dropwise
at −5 to 0 °C to a stirred solution of acetophenone oxime (2)
135 mg, 1 mmol) and disulfur dichloride (0.16 mL, 2 mmol)
(
4.3 General biological methods and materials
(
in acetonitrile (10 mL) under argon. The mixture was stirred
at 0 °C for 15 min, and a mixture of 4-benzyloxyaniline hydro-
chloride (235 mg, 1 mmol) and triethylamine (101 mg,
University of Cyprus animal ethics guidelines. Research
using animals is rigorously defined and controlled by the
Animal Protection and Welfare Act that was enacted in 1994,
and is carried out under licenses granted by the Animal
Health and Welfare Division of the Veterinary Services
Department of the Ministry of Agriculture, Natural Resources
and Environment of the Republic of Cyprus.
Stock solutions. Compounds were dissolved in DMSO to
make a stock solution of 1 mM concentration and then
diluted in 0.1× Marc's Modified Ringer's (MMR) for the indi-
cated working concentrations (usually 5 or 10 μM).
1
mmol) in acetonitrile (2 mL) was added. The mixture was
stirred at this temperature for another 0.5 h. Pyridine
158 mg, 0.16 ml, 2 mmol) was added and the reaction mix-
(
ture was stirred at ca. 20 °C for 1 h. Then the mixture was
heated at reflux for 5 h and filtered, and the solvent was
evaporated under vacuum. The residue obtained was
adsorbed onto silica and chromatographed IJn-hexane/DCM)
to give the title compound 3 as yellow needles (55 mg, 15%),
mp (DSC) onset: 65.8 °C, peak max: 68.2 °C, decomp. onset:
Embryos. Xenopus laevis embryos from induced
7
6
77
6
9.3 °C, peak max: 69.7 °C (from c-hexane); R
hexane/DCM, 40 : 60); (found: C, 66.93; H, 4.36; N, 7.51;
OS requires C, 67.00; H, 4.28; N, 7.44%); λmaxIJDCM)
76 (log ε 3.58), 231 (3.66); νmax/cm 3068w, 3034w, 2907w,
864w, 1903m, 1486m, 1454m, 1439m, 1331m, 1288m, 1244s,
207m, 1173m, 1165m, 1111m, 1065m, 1020m, 828w, 912w,
f
0.63 IJn-
spawning were staged according to Nieuwkoop and Faber.
Xenopus oocytes were fertilized in vitro and dejellied using
1.8% cysteine–HCl, pH 7.8, then maintained in 0.1× MMR
until they reach the required stage for use in the experi-
ments. Five to ten Xenopus laevis embryos of the appropriate
stage were arrayed in individual wells of a 6-well plate in
C
21
H
16
N
2
2
−
1
2
2
1
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either 0.1× MMR alone (control embryos) or supplemented
with the reported concentrations of the tested compound.
The embryos were allowed to develop and assessed for sur-
vival and elicitation of a distinctive phenotype. The embryos
were rinsed multiple times in fresh media and a freshly solu-
bilized compound was added every day, since the compound
stability, i.e. rates of compound uptake, distribution and
metabolism within embryos, was not known, so as to exclude
the possibility of compound degradation and ensure activity.
All experiments were repeated three times with at least 10
embryos for each condition.
Quantification of pigmentation loss phenotype. Embryos
were visually scored for loss of pigmentation. Embryos were
all initially photographed using a stereomicroscope under
the same conditions and then visually scored by two indi-
viduals one at a time after setting criteria for each category:
methanol dehydration (Murray's Clearing Medium, BB : BA).
The refractive index of BB : BA closely matches the refractive
index of yolk thereby rendering Xenopus embryos nearly
transparent.
In vitro transcription. An anti-sense Digoxigenin (DIG)
labelled Xtrp2/Dct (in pGEMT) probe was synthesized by
in vitro transcription from NcoI linearized plasmid using
RNA polymerase SP6 or T3 and a ribonucleotide mixture
which results in RNA transcripts containing Dig-UTP (Roche).
The manufacturer's protocol was followed with a modifica-
tion in the total reaction volume which was scaled down to
20 μL. RNA probes from these reactions were purified using
isopropanol/LiCl precipitation.
Whole-mount in situ hybridization (WISH). The DIG
labeled Xtrp2/Dct RNA probe (transcribed as described above)
was used to perform in situ hybridization using the protocol
7
8
−
= no pigment loss (no pigment change compared to con-
reported by Harland, R. M. with some modifications. Meth-
anol was substituted with ethanol and 4% paraformaldehyde
in PBS was used to fix the embryos instead of formaldehyde.
After proteinase K treatment (5 min) embryos were blocked
with 0.1% BSA, 10% sheep serum in 0.1% Tween in 1× PBS
(PBT) solution and then washes were performed in PBS. After
blocking, embryos were re-fixed for 1 h in 4% paraformalde-
hyde followed by pre-hybridization at 65 °C (6 h). Subse-
quently the RNA probe was added to the fixed embryos and
incubated at 65 °C (12 h). Several washes using 0.2× SSC at
65 °C followed. After the last wash the embryos were incu-
bated in Rnase and then blocked with 2% BMB blocking
reagent + MAB for 1 h and then transferred to a new vial
which contained 1 mL of 1 : 2000 dilution anti-DIG AP conju-
gates in blocking solution for 4 h at room temperature. After
incubation the embryos are washed extensively with MAB
(12 h) and AP is added, followed by the substrate BM purple.
The reaction stops with addition of MAB. Finally embryos
were washed in PBT (4 × 30 min) at room temperature.
Embryos were then cleared in BB : BA (as described above).
The chromogenic signal remains localized after clearing and
this allows data acquisition from different planes within the
embryo without the need for sectioning.
trols in either the melanophores or the RPE), + = minor pig-
ment loss (minor pigment reduction in the melanophores,
no visible change in the RPE), ++ = moderate pigment loss
(moderate pigment reduction in the melanophores and the
RPE), +++ = severe pigment loss (severe pigment reduction in
the melanophores and the RPE), ++++ = complete pig-
ment loss (no visible pigment in either the melanophores
or the RPE)
Rescue experiments. To test if pigmentation in the devel-
oping embryos would return after compounds were removed
the following experiment was performed. Briefly, 5–10
embryos (developmental stages 10, 21 and 31) were cultured
in 0.1× MMR (3 mL), supplemented with compound 1c or 1l
(5 or 10 μM) in a 6 well plate. The embryos were allowed to
present the pigmentation loss phenotype and develop to
stage 40. At this point, the media was removed; the embryos
were washed extensively and then incubated with 0.1× MMR.
Pigment levels were visually assayed after 14 h.
Whole mount immunofluorescence. Xenopus laevis
embryos were fixed in 3.7% formaldehyde in MEMFA (2 h at
room temperature) and the vitelline envelope was manually
removed with forceps. Permeabilization of embryos was car-
ried out in several ways: 1) overnight in 1× PBS supplemented
with 0.5% Triton and 1% DMSO (PBDT), 2) 2 h in 1× PBS
supplemented with 0.2% SDS, 3) for 4 h in 1× PBS
Imaging. The embryos were imaged with a Zeiss Axio
Imager Z1 using a Zeiss Axiocam MR3 and Axiovision soft-
ware 4.7. Optical sectioning was achieved using a Zeiss
Apotome structured illumination system.
−
1
supplemented with 0.2% SDS or 4) for 25 min in 10 μg mL
proteinase K. Embryos were then blocked for 2 h in 1× PBS
with 0.5% Triton, 5% BSA and 1% normal goat serum. Pri-
mary antibody staining followed. Embryos were incubated
with mouse anti-Xenopus Pax7 antibody overnight at 4 °C at a
dilution of 1 : 100 (in block solution). Embryos were then
washed (3 × 10 min) in PBDT and incubated for 2 h at room
temperature with Cy3 conjugated anti-mouse IgG antibody at
Acknowledgements
The authors thank Dr A. Afantitis (Novamechanics Ltd)
for helpful discussions, the Cyprus Research Promotion
Foundation [grants no. NEAYΠOΔOMH/NEKYΠ/0308/02,
TEXNOΛOΓIA/ΘEΠIΣ/0308IJBE)/08 and ΠENEK KINHT/0308/
06] and the following organizations in Cyprus for their gener-
ous donations of chemicals and glassware: the State General
Laboratory, the Agricultural Research Institute, the Ministry
of Agriculture, Medochemie Ltd. and Biotronics Ltd. Further-
more, we thank the A.G. Leventis Foundation for helping to
establish the NMR facility in the University of Cyprus.
1
: 500 dilution in fresh block solution. After incubation,
embryos were washed in PBDT (3 × 10min). For negative con-
trol experiments, embryos were blocked and then incubated
with secondary conjugates only. Clearing of embryos was
performed by immersion of the embryos in two parts benzyl
benzoate (BB) and one part benzyl alcohol (BA) after
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