Reduction of the nitro group to amine by hydroiodic acid to synthesize o-aminophenol derivatives as putative degradative markers of neuromelanin

Article abstract of DOI:10.3390/molecules19068039

Neuromelanin (NM) is produced in dopaminergic neurons of the substantia nigra (SN) and in noradrenergic neurons of the locus coeruleus (LC). The synthesis of NM in those neurons is a component of brain aging and there is the evidence that this pigment can be involved in the pathogenesis of neurodegenerative diseases such as Parkinson's disease. NM is believed to derive from the oxidative polymerization of dopamine (DA) or norepinephrine (NE) with the participation of cysteine, dolichols and proteins. However, there are still unknown aspects in the chemical structure of NM from SN (SN-NM) and LC (LC-NM). In this study, we designed a new method to synthesize o-aminophenol compounds as putative degradation products of catecholamines and their metabolites which may be incorporated into NM. Those compounds are aminohydroxyphenylethylamine (AHPEA) isomers, aminohydroxyphenylacetic acid (AHPAA) isomers and aminohydroxyethylbenzene (AHEB) isomers, which are expected to arise from DA or NE, 3,4-dihydroxyphenylacetic acid (DOPAC) or 3,4-dihydroxyphenylmandelic acid (DOMA) and 3,4-dihydroxyphenylethanol (DOPE) or 3,4- dihydroxyphenylethyleneglycol (DOPEG), respectively. These o-aminophenol compounds were synthesized by the nitration of phenol derivatives followed by reduction with hydroiodic acid (HI), and they could be identified by HPLC in HI hydrolysates of SN-NM and LC-NM. This degradative approach by HI hydrolysis allows the identification of catecholic precursors unique to SN-NM and LC-NM, which are present in catecholaminergic neurons.

Full text of DOI:10.3390/molecules19068039

Molecules 2014, 19, 8039-8050; doi:10.3390/molecules19068039
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Reduction of the Nitro Group to Amine by Hydroiodic Acid to
Synthesize o-Aminophenol Derivatives as Putative Degradative
Markers of Neuromelanin
1
,
1
1
2
3
Kazumasa Wakamatsu *, Hitomi Tanaka , Keisuke Tabuchi , Makoto Ojika , Fabio A. Zucca ,
3
1
Luigi Zecca and Shosuke Ito
1
Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake,
Aichi 470-1192, Japan; E-Mails: hmatsuoka@u-gifu-ms.ac.jp (H.T.);
8
2013103@fujita-hu.ac.jp (K.T.); sito@fujita-hu.ac.jp (S.I.)
2
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences,
Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan;
E-Mail: ojika@agr.nagoya-u.ac.jp
3
Institute of Biomedical Technologies, National Research Council of Italy, Via Cervi, 93, Segrate,
Milano 20090, Italy; E-Mails: fabio.zucca@itb.cnr.it (F.A.Z.); luigi.zecca@itb.cnr.it (L.Z.)
*
Author to whom correspondence should be addressed; E-Mail: kwaka@fujita-hu.ac.jp;
Tel.: +81-562-93-2518; Fax: +81-562-93-4595.
Received: 4 May 2014; in revised form: 5 June 2014 / Accepted: 6 June 2014 /
Published: 16 June 2014
Abstract: Neuromelanin (NM) is produced in dopaminergic neurons of the substantia
nigra (SN) and in noradrenergic neurons of the locus coeruleus (LC). The synthesis of
NM in those neurons is a component of brain aging and there is the evidence that this pigment
can be involved in the pathogenesis of neurodegenerative diseases such as Parkinson’s disease.
NM is believed to derive from the oxidative polymerization of dopamine (DA) or
norepinephrine (NE) with the participation of cysteine, dolichols and proteins. However, there
are still unknown aspects in the chemical structure of NM from SN (SN-NM) and LC
(LC-NM). In this study, we designed a new method to synthesize o-aminophenol compounds
as putative degradation products of catecholamines and their metabolites which may be
incorporated into NM. Those compounds are aminohydroxyphenylethylamine (AHPEA)
isomers, aminohydroxyphenylacetic acid (AHPAA) isomers and aminohydroxyethylbenzene
(
AHEB) isomers, which are expected to arise from DA or NE, 3,4-dihydroxyphenylacetic
acid (DOPAC) or 3,4-dihydroxyphenylmandelic acid (DOMA) and 3,4-dihydroxyphenylethanol
DOPE) or 3,4-dihydroxyphenylethyleneglycol (DOPEG), respectively. These o-aminophenol
(

Molecules 2014, 19
compounds were synthesized by the nitration of phenol derivatives followed by
8040
reduction with hydroiodic acid (HI), and they could be identified by HPLC in HI
hydrolysates of SN-NM and LC-NM. This degradative approach by HI hydrolysis allows
the identification of catecholic precursors unique to SN-NM and LC-NM, which are
present in catecholaminergic neurons.
Keywords: neuromelanin; aminohydroxyphenylethylamine; aminohydroxyphenylacetic acid;
aminohydroxyethylbenzene; Parkinson’s disease; HI reduction
1
. Introduction
There are two chemically distinct types of peripheral melanins, black to brown pigments called
eumelanin, and yellow to reddish-brown pigments called pheomelanin [1–3]. Eumelanin is derived
from the oxidative polymerization of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic
acid (DHICA), while pheomelanin consists of benzothiazine and benzothiazole units arising from
cysteinyl-3,4-dihydroxyphenylalanine (Cys-DOPA) isomers. On the other hand, neuromelanin (NM)
consists of black to brown pigments mainly found in neurons of the substantia nigra (SN) and locus
coeruleus (LC) in the central nervous system of human and other mammalian species [4]. Previous
studies from our group and other groups have shown that NM consists of complex polymers derived
from eumelanic DHI and pheomelanic benzothiazine units along with additional aliphatic and proteic
components [5–8]. NM accumulates during normal aging in neurons of different brain areas but,
interestingly, SN and LC that are the main target regions of Parkinson’s disease (PD) are those with
the highest pigment concentrations [9,10]. NM is formed by the oxidation of catecholamines,
dopamine (DA) and norepinephrine (NE), in the presence of cysteine (Cys). The synthesis of NMs in
the various regions of the human brain is an important protective process because the melanic
components are generated through the removal of reactive or toxic o-quinones that would otherwise
cause neurotoxicity. Another important aspect of the protective role of NM is the ability to bind toxic
metals forming stable complexes thereby blocking their toxicity [10]. On the other hand, due to its
biochemical properties, NM has also been discussed as a critical factor underlying neuronal
vulnerability in PD [5,9–11]. In PD the NM released by degenerating neurons can cause microglia
activation and neurodegeneration. In cell culture and in vivo studies it was shown that human NM
induces microglia activation and neuronal death [11–14]. In SN and LC, catecholamine neurons
containing NM highly express major histocompatibility complex-I which triggers neuronal death
mediated by cytotoxic T cells [15].
To study the structure and function of peripheral (cutaneous) melanin, we have developed several
chemical methods to quantify eumelanin and pheomelanin [16–19]. Alkaline H
2 2
O oxidation affords
pyrrole-2,3,5-tricarboxylic acid (PTCA) as a eumelanin marker and thiazole-2,4,5-tricarboxylic acid
(
TTCA) as a pheomelanin marker. Pheomelanin can also be analyzed as 4-amino-3-hydroxyphenylalanine
4-AHP) after hydroiodic acid (HI) hydrolysis. To study the structure and function of NM, we used HPLC
(
to analyze 4-amino-3-hydroxyphenylethylamine (4-AHPEA) and 3-amino-4-hydroxyphenylethylamine
3-AHPEA) after HI hydrolysis, in addition to pyrrole-2,3-dicarboxylic acid (PDCA), TTCA and
(

Molecules 2014, 19
8041
thiazole-4,5-dicarboxylic acid (TDCA) after H O oxidation. Several markers analyzed by HPLC
2
2
are currently used for melanin analysis, but only two, PTCA and 3-amino-4-hydroxyphenylalanine
3-AHP), are commercially available. Thus, most of those HPLC markers must be prepared by simple
(
chemical processes [20]. 4-AHP, a major degradation product of pheomelanin upon HI hydrolysis, can
be prepared by HI hydrolysis of 5-S-cysteinyldopa-melanin [20,21]. However, we found that this
amino acid can be more readily obtained in a 100 mg scale by the nitration of commercially available
m-tyrosine followed by HI reduction of the resulting 3-hydroxy-4-nitrophenylalanine along with other
possible isomers [20]. Authentic samples of 4-AHPEA and 3-AHPEA used in previous studies were
prepared by reductive HI hydrolysis of 5-S-cysteinyldopamine-melanin (5-S-CysDA-melanin) and
2
-S-cysteinyldopamine-melanin (2-S-CysDA-melanin) according to established methods [6,7,22,23].
However, this method requires the preparation of 5-S-CysDA and 2-S-CysDA in advance followed by
their oxidation to melanin, which is time-consuming and requires the skillful separation of 5-S-CysDA
and 2-S-CysDA. Thus, an alternative chemical method was needed to prepare these o-aminophenol
derivatives in more convenient and generally applicable manner. In this study, we developed a
convenient reduction of the nitro group to amine with HI to synthesize o-aminophenol derivatives as
putative degradative markers of human NM.
2
. Results and Discussion
2
.1. Chemistry
The biosynthesis of NM, oxidative polymerization of catecholamines, suggests the possibility
that various catecholic metabolites would also participate in NM genesis (Scheme 1). In addition to
1
and 2, 3,4-dihydroxyphenylalanine (DOPA, 3), 3,4-dihydroxyphenylacetic acid (DOPAC, 4),
,4-dihydroxymandelic acid (DOMA, 5), 3,4-dihydroxyphenylethanol (DOPE, 6) and 3,4-dihydroxy-
3
phenylethyleneglycol (DOPEG, 7) can be incorporated into NM. These compounds are metabolites of
and 2 formed by oxidative deamination by monoamine oxidase followed by oxidation/reduction. We
1
also reported that melanin pigments derived from Cys-DOPA can be identified in NMs isolated from
other important regions of human brain, such as putamen, premotor cortex, and cerebellum [10]. We
have already established methods to determine eumelanin and pheomelanin in peripheral (cutaneous)
melanin that can be used for the characterization of NM [7,8,16–20]. These methods are based on
melanin degradation by alkaline H O oxidation and reductive HI hydrolysis. Upon HI hydrolysis,
2
2
NM afforded 4-AHPEA 8c and its isomer 3-AHPEA 9c along with benzothiazole derivatives. These
o-aminophenol compounds are derived from Cys-DA and Cys-NE melanins. HI hydrolysis of NMs,
derived from the above catechol derivatives, 1–7, is expected to afford the corresponding
o-aminophenol derivatives, AHPEAs (8c and 9c), aminohydroxyphenylacetic acid (AHPAAs, 10c and
1
1c), and aminohydroxyethylbenzene (AHEBs, 12c and 13c) (Scheme 2).

Molecules 2014, 19
Scheme 1. Possible participation in NM biosynthesis of various catecholic metabolites
8042
known to be present in various brain regions of the human brain. Purple circles depict NM
accumulation in neurons of the SN. (O) represents oxidants.
Scheme 2. Possible degradation products afforded by the reductive HI hydrolysis of human NM.
Thus, we planned a synthetic scheme involving the o-nitration of the corresponding phenols with
6
0% HNO followed by reduction with 57% HI. The desired o-aminophenols c were separated on a
3
cation exchange resin and purified by recrystallization of the HCl salts (Scheme 3).
The synthesis of the o-aminophenol derivatives c comprises a series of two known reactions
(
Scheme 3). The first step (aromatic nitration) and the second step (reduction of the nitro group to
amine) are common to all products c. In the first step, the nitration of phenols a was carried out
by 60% HNO at room temperature for 10 min—overnight followed by separation of the nitrated
3
phenols with a cation exchange resin or extraction with ethyl acetate. o- and p-Oriented nitrophenol
isomers were subjected to reduction of the nitro group without further purification. The nitration of
p-substituted phenols afforded only o-nitrophenol derivatives b, so the yields were nearly quantitative.
On the other hand, the nitration of m-substituted phenols afforded isomeric o- and p-oriented

Molecules 2014, 19
8043
nitrophenols and dinitrophenol derivatives. Thus, the yields of the target compounds were much lower
than those from p-substituted phenol derivatives.
Scheme 3. Synthetic scheme of the nitration of m- and p-substituted phenol derivatives
followed by reduction of the nitro group to amine with reductive HI hydrolysis. Parenthesis
is a yield.
In the literature, several methods for the reduction of nitro groups to amines have been reported.
Aromatic amines are known to be produced by catalytic hydrogenation under continuous high
pressure [24–27]. Nitro compounds can also be reduced to amines in good yields with iron and
hydrochloric acid in the Béchamp reaction [25], which is the oldest commercial process for preparing
amines, and is still used in the dyestuff industry. The reduction of o-nitrophenol b to o-aminophenol c
must be done under a reductive environment because aminophenol is susceptible to oxidation. Thus,
3 2
we have developed the HI/H PO method for reduction of the nitro group. HI has a high acidity and
potential reducing ability. Compared with other reduction methods in the literature, this method is easy
because the work-up after the reaction requires only the removal of HI under reduced pressure without
further filtration of the catalyst or extraction of the solution. After the evaporation of HI, the reaction
mixture was chromatographed on an ion-exchange resin. The target compound as the HCl salt was
further recrystallized from ethanol plus ether. 3-Amino-4-hydroxyphenyl derivatives 9c, 11c and 13c
were obtained in moderate to high overall yields (84%, 52% and 42%, respectively). On the other hand,
4
-amino-3-hydroxyphenyl derivatives 8c, 10c and 12c were obtained in relatively low overall yields
11%, 12% and 4.7%, respectively), because of the production of other possible isomers and diamino
derivatives. The poor yield of 12c may be due to the low purity (80%) of the starting material,
-ethylphenol (12a) which contains the isomers 4-ethylphenol (13a) and 2-ethylphenol making the
(
3
separation of the desired product difficult. As the isolation of 10b, 12b, and 13b were difficult, the
reaction products were subjected to the following reaction without the further purification.

Molecules 2014, 19
.2. Chemical Analyses of NM Isolated from the SN and LC of Human Brain
The reductive HI hydrolysis of NM isolated from the SN and LC of human midbrains was
8044
2
performed, and the degradation products were identified by HPLC using newly synthesized
o-aminophenol compounds c as standards (Table 1). It should be emphasized that LC-NM has never
been studied before by chemical degradative methods, which have been mainly employed on NM
isolated from other areas of human brain [6,7,10]. We have previously reported that the chemical
degradation of SN with HI affords 8c and 9c in high yields, and 14c and 15c in low yield [6,7,10].
AHP isomers 14c and 15c are derived from Cys-DOPA melanin [18]. In this study, we could detect
AHEBs for the first time as a mixture of 12c and 13c, and AHPAA isomers 10c and 11c in SN-NM. In
LC-NM, we could also now detect 8c, 9c, 10c and 11c together with AHEBs 12c and 13c. These
results suggest that SN-NM and LC-NM are derived not only from DA 1 and NE 2, but also from
DOPA 3, DOPAC 4, DOMA 5, DOPE 6 and DOPEG 7, because AHPEA isomers 8c, 9c, AHPAA
isomers 10c, 11c, AHEB isomers 12c, 13c and AHP isomers 14c, 15c are expected to arise from DA 1
or NE 2, DOPAC 4 or DOMA 5, DOPE 6 or DOPEG 7 and DOPA 3, respectively. We could also
detect DA 1, DOPA 3, and DOPAC 4 after HI hydrolysis. This result indicated that the protein-bound
derivatives of DA 1, DOPA 3, and DOPAC 4 were incorporated into SN-NM and LC-NM [10].
Table 1. Chemical characterization with reductive HI hydrolysis of human NM isolated
from SN and LC tissues (values in ng/mg).
DA
1)
DOPA
(3)
DOPAC
(4)
4-AHPEA
(8c)
3-AHPEA
(9c)
4-AHPAA
(10c)
3-AHPAA
(11c)
AHEBs
(12c, 13c)
32
4-AHP
(14c)
62
3-AHP
(15c)
66
Sample
(
SN-NM
LC-NM
1,224
955
722
161
428
588
735
571
624
368
806
695
596
310
75
52
114
Values were obtained in a single determination.
3. Experimental Section
3.1. General Information
1
1
13
H-NMR spectra (400 MHz for H-NMR and 100 MHz for C-NMR) were recorded on a Bruker
AVANCE 400 spectrometer and are reported as the chemical shifts δ (ppm) downfield from sodium
,2-dimethyl-2-silapentane-5-sulfonate (DSS) or t-butyl alcohol used as an internal reference.
Coupling constants (J) are expressed in Hz and signals are expressed as s (singlet), d (doublet) or t
2
(
triplet). Elemental analyses were carried out by the Organic Elemental Analysis Research Center
Kyoto University, Japan). For ion-exchange column chromatography, a Dowex 50W-X2 (200–400 mesh)
(
column was used. UV spectrometry was monitored using a JASCO V-630 UV-VIS spectrometer
JASCO Co., Tokyo, Japan). HPLC systems consisted of a JASCO 880-PU pump, a JASCO C18
(
column (JASCO Catecholpak; 4.6 mm × 150 mm; 7 µm particle size) and an EICOM ECD-300
electrochemical detector (EICOM, Kyoto, Japan), and a Shiseido C18 column (Shiseido Capcell Pak
MG; 4.6 mm × 250 mm; 5 µm particle size) and a JASCO UV detector (JASCO Co.). DA 1, NE 2,
DOMA 5, DOPEG 7, 3-amino-4-hydroxyphenylalanine (3-aminotyrosine, 15c), 57% HI and
mushroom tyrosinase (4,276 U/mg) were purchased from Sigma Aldrich Japan (Tokyo, Japan),

Molecules 2014, 19
8045
3
-ethylphenol 12a (80% purity), 4-ethylphenol 13a and DOPE 6 were purchased from Tokyo Chemical
Industry Co. (Tokyo, Japan), and 3-hydroxyphenylethylamine 8a, 4-hydroxyphenylethylamine 9a,
-hydroxyphenylacetic acid 10a, 4-hydroxyphenylacetic acid 11a and Dowex 50W-X2 were purchased
3
from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals were of the highest
purity available.
Reductive HI hydrolysis was performed as described previously [6,7,18,20]. Briefly, a mixture of
1
00 μL suspension of a sample (containing 0.1 mg melanin), 30 μL 30% H PO and 500 μL 57% HI
3 2
was heated in a screw-capped tube at 130 °C for 20 h, after which the mixture was cooled. An aliquot
100 μL) of each hydrolysate was transferred to a test tube and evaporated to dryness using a vacuum
(
pump connected to a dry ice-cooled vacuum trap and two filter flasks containing NaOH pellets. The
residue was dissolved in 200 μL 0.1 M HCl. An aliquot (10 μL) of each solution was analyzed by
HPLC as described in Section 3.2.
Human NMs were isolated from SN and LC tissues and used in the experiments as described
previously [6,7,10,28]. This study was approved by the Ethical Committee of the National Research
Council of Italy-Institute of Biomedical Technologies (Segrate, Milan, Italy) and was carried out in
agreement with the Policy of the National Research Council of Italy. SN-NM was isolated from a pool
of five subjects with age range 73–85 years old. LC-NM was isolated from a pool of 35 subjects with
age range 62–94 years old.
3.2. General Procedure for the Synthesis of 4-Amino-3-hydroxy and 3-Amino-4-hydroxyphenyl
Derivatives 8c–13c
An appropriate phenol compound 8a–13a (1 mmol) was mixed with 60% HNO (4.8 mmol). The
3
resulting suspension was stirred at room temperature for 10 min for 8a and 9a, for 30 min for 10a, 12a
and 13a, or overnight for 11a, leading eventually to an intensively dark red solution. HPLC analysis
confirmed the absence of starting material and the presence of 3 major products corresponding most
likely to nitro derivatives resulting from nitration at the o- and p-positions of the hydroxy group from
the m-substituted phenols 8a, 10a and 12a, but only one product from the p-substituted phenols 9a,
1
1a and 13a. The crude nitrophenol 8b or 9b was diluted with water (20 mL) and applied onto a
+
Dowex 50W-X2 (200–400 mesh) column (1.5 cm × 6 cm, H form, equilibrated with water).
The column was washed with water (100 mL) and then the product was eluted with 2 M HCl
(
20 mL/fraction). Fractions containing the target compound were combined and evaporated to dryness
to give a yellow powder. After the nitration of phenols 10a, 12a and 13a, the reaction mixture (10b,
2b and 13b) was diluted with H O (20 mL) and extracted with ethyl acetate (2 × 100 mL). The
1
2
2 4
combined organic extract was washed with water (2 × 50 mL), dried with Na SO and evaporated
under reduced pressure. The nitration of 11a afforded 11b as an orange powder in the reaction mixture.
After the filtration, the powder was washed with water and dried under reduced pressure to give a dark
red powder 11b.
The nitrophenol compounds 8b–13b were then heated under reflux with 57% HI (10 mL) in the
presence of 30% H PO (1.0 mL). After 60 min, HPLC analysis confirmed the absence of the nitrated
3
2
compounds and the appearance of products corresponding most likely to amino derivatives resulting
from reduction to amine at the o- (and p-) positions of the hydroxy group. The mixture was evaporated

Molecules 2014, 19
8046
to dryness, taken up in 0.1 M HCl (3 mL) and chromatographed on a Dowex 50W-X2 (200–400 mesh)
column (1.8 cm × 31 cm, equilibrated and eluted with 2 M HCl). Fractions of 20 mL were collected
and analyzed by UV spectrophotometry (200–400 nm) and by HPLC. The analytical conditions of
HPLC are described in Section 3.2. After fractions of the target compound were collected,
recrystallization of compounds 8c–13c from ethanol (5 mL) plus ether (25 mL) and compound 14c
from 6 M HCl (3 mL) plus acetone (60 mL) gave colorless crystals as HCl salts.
3
.3. HPLC Analyses of Aminophenols 8c–13c
The HPLC system consisted of a JASCO C column (JASCO Catecholpak; 4.6 mm × 150 mm;
1
8
7
µm particle size) and an EICOM ECD-300 electrochemical detector. The mobile phase used
was 0.1 M sodium citrate buffer, pH 3.0, containing 1 mM sodium octanesulfonate and 0.1 mM
Na EDTA: methanol, 92: 8 (v/v) at 40 °C at a flow rate of 0.7 mL/min. The electrochemical detector
was set at +500 mV versus the Ag/AgCl reference electrode. The retention times were 9.2, 9.7, 15.2,
6.2, 93.2 and 93.5 min for 10c, 11c, 8c, 9c, 12c and 13c, respectively. The retention times for 12c
2
1
and 13c were shortened to 43.6 min using the above buffer: methanol, 80:20. It was impossible to
separate 12c and 13c under the above HPLC conditions.
4
-Amino-3-hydroxyphenylethylamine (4-AHPEA) (8c). Colorless crystals. Yield: 12%. Purity 99% by
1
HPLC. H-NMR (DCl): δ = 2.85 (t, 2H, J = 8.0 Hz, CH ), 3.15 (t, 2H, J = 8.0 Hz, CH ), 6.80 (d, 1H,
2
2
1
3
J = 4.0 Hz, CH), 6.86 (s, 1H, CH), 7.19 (d, 1H, J = 4.0 Hz, CH). C-NMR (D
2
O, t-butyl alcohol was
added as an internal reference): δ = 28.34, 38.96, 115.45, 115.98, 119.60, 122.90, 137.74, 148.68.
Elemental analysis: calculated for C .2HCl. C, 42.68%; H, 6.27%; N, 12.44%; Cl, 31.50%;
found: C, 43.25%; H, 6.07%; N, 12.43%; Cl, 32.76%. UV (0.1 M HCl) λmax 274 nm (ε 2240) and 221 nm
ε 6,700).
8
12 1 2
H O N
(
3
-Amino-4-hydroxyphenylethylamine (3-AHPEA) (9c). Colorless crystals. Yield: 84%. Purity 99% by
1
HPLC. H-NMR (DCl): δ = 2.83 (t, 2H, J = 8.0 Hz, CH
2
), 3.13 (t, 2H, J = 8.0 Hz, CH
2
), 6.93 (d, 1H,
O, t-butyl alcohol
was added as an internal reference): δ = 28.99, 39.87, 116.24, 117.60, 123.55, 128.25, 129.95, 148.29.
Elemental analysis: calculated for C .2HCl. C, 42.68%; H, 6.27%; N, 12.44%; Cl, 31.50%;
found: C, 42.29%; H, 6.17%; N, 12.30%; Cl, 32.08%. UV (0.1 M HCl) λmax 276 nm (ε 1,970) and
1
3
J = 12.0 Hz, CH), 7.13 (s, 1H, CH), 7.15 (d, 1H, J = 12.0 Hz, CH). C-NMR (D
2
8
12 1 2
H O N
2
21 nm (ε 7,720).
4
-Amino-3-hydroxyphenylacetic Acid (4-AHPAA) (10c). Colorless crystals. Yield: 12%. Purity 99% by
1
HPLC. H-NMR (DCl): δ = 3.71 (s, 2H, CH
2
), 6.92 (d, 1H, J = 8.0 Hz, CH), 7.00 (s, 1H, CH), 7.33 (d,
.HCl. C, 47.19%; H, 4.95%;
N, 6.88%; Cl, 17.41%; found: C, 46.95%; H, 5.07%; N, 6.60%; Cl, 17.52%. UV (0.1 M HCl) λmax
7 9 3 1
1H, J = 8.0 Hz, CH). Elemental analysis: calculated for C H O N
2
74 nm (ε 2,110) and 212 nm (ε 6,350).
3
-Amino-4-hydroxyphenylacetic Acid (3-AHPAA) (11c). Colorless crystals. Yield: 52%. Purity 99% by
1
HPLC. H-NMR (DCl): δ = 3.71 (s, 2H, CH
2
), 7.05 (d, 1H, J = 8.4 Hz, CH), 7.27 (d, 1H, J = 8.4 Hz),
7
.28 (s, 1H). Elemental analysis: calculated for C H O N .HCl. C, 47.19%; H, 4.95%; N, 6.88%;
7 9 3 1

Molecules 2014, 19
8047
Cl, 17.41%; found: C, 46.09%; H, 4.89%; N, 6.64%; Cl, 18.05%. UV (0.1 M HCl) λmax 276 nm
ε 1,880) and 212 nm (ε 7,060).
(
4
-Amino-3-hydroxyethylbenzene (4-AHEB) (12c). Colorless crystals. Yield: 4.7%. Purity 95% by
1
1
H-NMR. H-NMR (DCl): δ = 1.18 (t, 3H, J = 7.6 Hz, CH
3
), 2.61 (dd, 2H, J = 7.6, 7.6 Hz, CH
2
), 6.91
1
3
(
dd, 1H, J = 8.0, 1.6 Hz, CH), 6.95 (d, 1H, J = 1.6 Hz, CH), 7.27 (d, 1H, J = 8.0 Hz, CH). C-NMR
DCl): δ = 16.57, 29.78, 116.83, 117.56, 121.79, 125.61, 149.62, 151.51. Elemental analysis: calculated
(
for C H O N .HCl.1/4H O. C, 53.94%; H, 7.07%; N, 7.86%; Cl, 19.90%; found: C, 54.07%;
8
11
1
1
2
H, 6.79%; N, 7.88%; Cl, 19.33%. UV (0.1 M HCl) λmax 272 nm (ε 2,250) and 212 nm (ε 6,750).
3
-Amino-4-hydroxyethylbenzene (3-AHEB) (13c). Colorless crystals. Yield: 42%. Purity 99% by
1
HPLC. H-NMR (DCl): δ = 1.18 (t, 3H, J = 7.6 Hz, CH ), 2.60 (dd, 2H, J = 7.6, 7.6 Hz, CH ), 7.00 (d,
3
2
1H, J = 8.0 Hz, CH), 7.22 (s, 1H, CH), 7.24 (d, 1H, J = 8.0 Hz, CH). Elemental analysis: calculated for
8 11 1 1
C H O N
.HCl. C, 55.33%; H, 6.98%; N, 8.07%; Cl, 20.41%; found: C, 55.16%; H, 6.91%; N,
8
.07%; Cl, 20.64%. UV (0.1 M HCl) λmax 277 nm (ε 2,270) and 220 nm (ε 7,610).
4
-Amino-3-hydroxyphenylalanine (4-AHP) (14c). Colorless crystals. Yield: 23%. Purity 99% by
1
HPLC. H-NMR (DCl): δ = 3.11 (m, 2H, CH
2
), 4.07 (m, 1H, CH), 7.00 (d, 1H, J = 8.0 Hz, CH), 6.79
1
3
(d, 1H, J = 4.0 Hz, CH), 6.85 (s, 1H, CH), 7.22 (d, 1H, J = 4.0 Hz, CH). C-NMR (D
2
O, t-butyl
alcohol was added as an internal reference): δ = 36.46, 55.33, 118.35, 118.60, 122.52, 125.70, 138.08,
1
6
λ
9 12 3 2 3 3
51.37, 172.74. Elemental analysis: calculated for C H O N .2HCl.CH COCH . C, 44.05%; H,
.16%; N, 8.56%; Cl, 21.67%; found: C, 44.77%; H, 5.98%; N, 8.49%; Cl, 20.47%. UV (0.1 M HCl)
max 215 nm (ε 6,840) and 275 nm (ε 2,370).
4
. Conclusions
A general procedure for the synthesis of 4-amino-3-hydroxy- and 3-amino-4-hydroxyphenyl
derivatives 8c–13c was established based on the reduction of the nitro group to amine on the aromatic
ring using 57% HI/H PO . This method is more convenient and generally applicable compared with
3
2
other methods reported in the literature. Some degradation products of human NMs, SN-NM and
LC-NM, were identified with these newly synthesized o-aminophenol compounds in HPLC
determinations: the reductive HI hydrolysis of SN-NM and LC-NM afforded AHPEAs 8c, 9c,
AHPAAs 10c, 11c, AHEBs 12c, 13c and AHPs 14c, 15c as degradation products. Then, by
using this new degradative approach, we have found that SN-NM and LC-NM are derived not
only from DA and NE, but also from several other catecholic precursors which are present in
catecholaminergic neurons.
Acknowledgments
This work was supported in part by a Japan Society for the Promotion of Science (JSPS)
KAKENHI Grant number 24500450 and in part by the Italian Ministry of Education, University, and
Research (MIUR)—Medical Research in Italy (MERIT) Project RBNE08ZZN7. Authors also thank
the Section of Legal Medicine and Insurances, Department of Human Morphology and Biomedical
Sciences, University of Milano, for providing brain tissues.

Molecules 2014, 19
8048
Author Contributions
Conceived and designed the experiments: KW, SI. Performed the experiments: HT, KT, SI.
Analyzed the data: HT, KT, MO, SI. Prepared neuromelanin samples: FZ, LZ. Wrote the paper: KW, SI.
Conflicts of Interest
The authors declare no conflict of interest.
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distributed under the terms and conditions of the Creative Commons Attribution license
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(