Synthesis of Acetoxy Stabilised Indolequinones as Precursors to Model Eumelanin Polymers

Article abstract of DOI:10.1055/s-2003-41409

We report an efficient synthetic route to obtaining a stable analogue of 5,6-dihydroxyindole. These analogues can be used to build controlled composition model melanin biopolymers for solid state and spectroscopic studies of this important biomolecule.

Full text of DOI:10.1055/s-2003-41409

LETTER
1853
Synthesis of Acetoxy Stabilised Indolequinones as Precursors to Model
Eumelanin Polymers
Synthesis of
A
o
cetoxy Stabi
v
lised
I
ndolequinon
Aes tkinson, Paul Meredith*
Department of Physics, Centre for Biophotonics and Laser Science, University of Queensland, 4072, Brisbane, Queensland, Australia
Fax +61(7)33651242; E-mail: meredith@physics.uq.edu.au
Received 2 May 2003
the need to handle the unstable DHI. DAI monomer and
Abstract: We report an efficient synthetic route to obtaining a sta-
the carboxylated form of DHI, can be used to prepare
model synthetic eumelanin analogues with controlled ra-
ble analogue of 5,6-dihydroxyindole. These analogues can be used
to build controlled composition model melanin biopolymers for sol-
tios of DHI and DHICA, and controlled molecular
weights. Synthesis of the acetoxy protected indole is the
subject of this letter. Preparation and characterisation of
model biopolymers through controlled oxidation or
electropolymerisation of DAI and DHICA will be the sub-
jects of future publications.
id state and spectroscopic studies of this important biomolecule.
Key words: 5,6-dihydroxyindole, 5,6-diacetoxyindole, melanin
biopolymers, melanin precursors, nitration of activated aromatic
compound
Various routes to DAI can be found in the literature.5,6
However, these routes involve at least 5 steps prior to the
reductive cyclisation of (E)-4,5-diacetoxy-2,b-dinitr-
ostyrene (4) to form DAI. Here we report a four-step syn-
thetic route to DAI, which significantly simplifies the
process. Our new pathway combines several useful as-
pects of Prota et al’s DHI five step synthesis6, and Mur-
phy’s seven step DAI synthesis.5 The route is summarised
in Scheme 1.
The melanins are a class of biopolymer found throughout
nature.1 It is well known that their main functions in hu-
mans are as pigments and photoprotectants. They are
broadband UV and visible light absorbers, and potent free
radical scavengers and antioxidants. Despite significant
scientific attention over the past fifty years, the structure-
property-function relationships that dictate the behaviour
of these important biological macromolecules are still
poorly understood.
Natural melanins are hetero-biopolymers with non-
specific molecular weights. They have proven somewhat
intractable as far as compositional characterisation is con-
cerned due to their inherent stability and insolubility. A
common strategy for studying melanins is to create suit-
able synthetic analogues. These synthetic forms have been
show to behave in a similar manner to material extracted
from native pigment containing tissues.2
Eumelanin is one of the most common forms of the
biopolymer. It is the material that colours the skin, hair
and eyes of humans. It is also the material implicated in
melanoma skin cancers. It is usually accepted that eumel-
anin is a random hetero-polymer of 5,6-dihydroxindole
(DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHI-
CA).3 In nature, eumelanin is formed enzymatically via
the action of tyrosinase upon dihydroxyphenylalanine
(DOPA). For this reason, eumelanins are often referred to
as dopamelanins. Eumelanins can also be formed synthet-
ically via the auto-oxidation of DOPA or DHI and DHI-
CA. These processes are simple, but produce polymers of
uncontrolled composition and molecular weight. DHI is
inherently unstable and very oxygen sensitive.
Scheme 1 Efficient synthetic route to DAI
Of several strategies that have been cited in literature for
the preparation of DHI or DAI, the most convenient ones
centre on the classical 2, b-dinitrostyrene approach illus-
trated by Benigni and Minnis.7 Improvements to this
popular approach have been substantial.5,6 Our strategy
is based upon one of the refinements published by Prota
et al.6 Prota’s methodology capitalises on the Zn (II)-
5,6-Diacetoxyindole (DAI) is a well known stable and
protected analogue of DHI.4 The diacetate form is of in-
terest because it can be hydrolysed in situ and eliminates
SYNLETT 2003, No. 12, pp 1853–1855
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Advanced online publication: 28.08.2003
DOI: 10.1055/s-2003-41409; Art ID: D10003st.pdf
© Georg Thieme Verlag Stuttgart · New York

1854
S. Atkinson, P. Meredith
LETTER
and bright orange crystals were precipitated by the addition of hex-
ane (17.4350 g, 95%), mp 129–131 °C (lit6. 131–132.2 °C).
ene. The first intermediate in this strategy is nitrostyrene 1H NMR (acetone-d6) d = 6.96 (d, 1 H, J = 8.4 Hz), 7.25 (dd, 1 H,
assisted nitration of 3,4-dihydroxy-b-nitrostyrene (2) and
features an improved reductive cyclisation of dinitrosyr-
J = 8.4 Hz), 7.31 (d, 1 H, J = 2 Hz), 7.77 (d, 1 H, J = 13.6 Hz), 7.98
(d, 1 H, J = 13.6 Hz).
13C NMR (acetone-d6) d = 116.4, 116.7, 123.3, 124.7, 135.8, 140.4,
146.7, 150.6.
2, which we prepared using Henry condensation of 3,4-di-
hydroxybenzaldehyde (1) with nitromethane and with ex-
cellent yield of 95%.
Since our strategy was focused on creating stable acetoxy
precursors to melanin, the next (second) step was
acetylation of the hydroxyl groups. In our improved meth-
od, the acetylation was carried out using acetic anhydride
with a catalytic amount of sulfuric acid. Full acetylation
was achieved with a yield of 99% in less than 15 min at
r.t. compared to the 45 min at a higher temperature report-
ed by Murphy.5
EI-MS: m/z calcd for C12H11NO2 (M+): 181.15, found: 181.0.
(E)-3,4-Diacetoxy-b-nitrostyrene (3)
To a suspension of 2 (5 g, 0.027 mol) and acetic anhydride (5.09 g,
0.054 mol), a catalytic amount of concd sulfuric acid was added.
The mixture was stirred gently for 5–10 min while heat was devel-
oped. The solution was then poured onto crushed ice (200 mL) to
yield a pale yellow powder. The product was filtered and washed re-
peatedly with ice water before being dried. 3,4-Diacetoxy-b-ni-
trostyrene (3) was obtained in 99% yield (6.803 g); mp 110–112 °C
(lit8. 113.5–115 °C).
The third step involved the nitration of 3 at the sixth posi-
tion. Prota et al. used tetranitromethane as the regioselec-
tive nitrating agent in the presence of Zn(II) ions to nitrate
an activated phenolic. However, this particular nitrating
agent is not readily available, so an alternative nitrating
agent was selected. Nitric acid, in the presence of sulfuric
acid is a common nitrating reagent. However, since aceta-
1H NMR (acetone-d6): d = 2.32 (s, 3 H), 2.32 (s, 3 H), 7.28 (d, 1 H,
J = 8.3 Hz), 7.39 (br s, 1 H), 7.42 (d, 1 H, J = 8.3 Hz), 7.51 (d, 1 H,
J = 13.7 Hz), 7.93 (d, 1 H, J = 13.7 Hz).
13C NMR (acetone-d6): d = 16.91, 16.93, 119.5, 121.7, 123.4, 131.6,
132.1, 134.9, 145.2, 145.9, 168.0, 168.12.
–
tes are susceptible to nucleophilic attack by HSO4 ions,
EI-MS: m/z cald for C12H11NO2 (M+): 265.22, found: 265.33.
(arising from the sulfuric acid during the generation of
nitronium ions NO2+ from nitric acid), fuming nitric acid
was used instead. Murphy has also demonstrated this ni-
trating method.5 In our synthesis, this step was used to
prepare 4 in 85% yield.
(E)-4,5-Diacetoxy-2-b-dinitrostyrene (4)
Fuming nitric acid was added to a –30 °C solution of 3 in 100 mL
of 1,2-dichloroethane. The reaction was stirred at ca. –15 °C or be-
low for around 6 h. The mixture was poured onto 500 mL of ice
slurry, extracted with EtOAc and dried over MgSO4. The solvent
was evaporated to yield yellow crystals (4.651 g, 85%); mp 127–
129 °C (lit5. 129–132 °C).
The final step of our synthesis involved the reductive
cyclisation of 4. This was achieved by using Prota’s meth-
od,6 where biphasic conditions [created using acetate
buffer (pH 4) instead of phosphate buffer and CH2Cl2]
were essential to prevent deacetylation by the nucleo-
philic S2O42– ions. The final product (DAI) was produced
with a 60% yield.
1H NMR (DMSO-d6): d = 2.33 (s, 3 H), 2.35 (s, 3 H), 7.97 (s, 1 H),
8.04 (d, 1 H, J = 13.45Hz), 8.26 (s, 1 H), 8.41 (d, 1 H, J = 13.47 Hz).
13C NMR (DMSO-d6): d = 20.78, 122.15, 125,27, 125.71, 134.61,
141.48, 141.38, 146.22, 146.55, 168.17.
EI-MS: m/z calcd for C12H10N2O8 (M+): 310.22, found: 310.2233.
In summary, we have synthesised DAI, a stable precursor
of eumelanin using only four steps. This methodology is
more efficient than any previously reported. Our four step
synthesis had a 48% overall yield compared to Prota et al.
44% yield (six steps) and Murphy’s 66% yield (seven
steps).
5,6-Diacetoxyindole (DAI)
To a solution of 4 (4.00 g, 0.013 mol) in CH2Cl2 (250 mL) was add-
ed a solution of sodium dithionite (33.66 g, 0.194 mol) and ZnSO4
(67.94 g, 0.240 mol) in 0.1 M acetate buffer (pH 4, 250 ml). The
mixture was stirred under N2 atmosphere for ca 4 h. It was left to
cool to r.t. and the organic phase was separated and retained. The
aqueous phase was then extracted with EtOAc (3 × 100 mL). Both
organic phases were combined and dried over sodium dithionite and
concentrated under vacuum. Hexane was added to precipitate out
the greyish solid (1.81 g, 60%); mp 131–133 °C (lit6. mp 132 °C).
1H NMR (DMSO-d6): d = 2.27 (s, 6 H), 6.45 (d, 1 H, J = 2.6 Hz),
7.27 (s, 1 H), 7.36 (s, 1 H), 7.41 (d, 1 H, J = 2.6 Hz), 11.24 (br s, 1
H).
13C NMR (DMSO-d6): d = 21.00, 101.56, 106.12, 113.75, 125.59,
127.55, 133.52, 136.28, 137.84, 169.47.
EI-MS: m/z calcd for C12H11NO4 (M+): 233.22, found: 233.23.
All commercial reagents and solvents were used without further pu-
rification. NMR spectra were recorded in acetone-d6, DMSO or
CDCl3 using a BRUKER AC 200 MHz spectrometer, and chemical
shifts are reported in ppm. All melting points were determined on a
hot stage apparatus without corrections. Mass spectra were recorded
on a Kratos MS25 RFA using the Electron Impact method, and a Di-
rect Insertion Probe ionizing at 70 eV.
(E)-3,4-Dihydroxy-b-nitrostyrene (2)
3,4-Dihydroxybenzaldehyde, (1) (10.00 g, 0.072 mol) was added to
a solution of ammonium acetate (22.34 g, 0.029 mol) and ni-
tromethane (26.53 g, 0.435 mol) in HOAc (100 mL) and refluxed
for 25 min. The solvent was evaporated under vacuum, resulting in
a dark red oily residue. This was taken up in water (50 mL) and ex-
tracted with Et2O (3 × 100 mL). The organic layer was concentrated
Acknowledgment
This work was funded by the Australian Research Council (Disco-
very Grant #DP0210458), and through the University of Queens-
land Research Infrastructure Fund.
Synlett 2003, No. 12, 1853–1855 © Thieme Stuttgart · New York

LETTER
Synthesis of Acetoxy Stabilised Indolequinones
1855
(5) Murphy, B. P. J. Org. Chem. 1985, 50, 5873.
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Synlett 2003, No. 12, 1853–1855 © Thieme Stuttgart · New York