Oxidation chemistry of (-)-norepinephrine in the presence of L-cysteine

Article abstract of DOI:10.1021/jm960016t

The noradrenergic neurotransmitter (-)-norepinephrine (1) is very easily oxidized at physiological pH to an o-quinone (2) that normally cyclizes and subsequently oxidatively polymerizes to black melanin. In this investigation it is demonstrated that L-cysteine (CySH) can divert the melanin pathway by efficiently scavenging o-quinone 2 to give, initially, 5-S- cysteinylnorepinephrine (6) and 2-S-cysteinylnorepinephrine (7). These cysteinyl conjugates are appreciably more easily oxidized than 1 to o- quinones that, in part, are further attacked by CySH to give 2,5-bi-S- cysteinylnorepinephrine (8), an even more easily oxidized compound. The o- quinone intermediates formed upon oxidation of 6-8 can also undergo facile intramolecular cyclizations to bicyclic o-quinone imines that oxidize the cysteinyl conjugates from which they are derived in a reaction sequence that leads initially to a number of dihydrobenzothiazines. At least two of these compounds, 7-(1-hydroxy-2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4- benzothiazine-3-carboxylic acid (9) and 8-(1-hydroxy-2-aminoethy])-3,4- dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (10) are lethal when administered into the brains of mice. The in vitro chemical pathways elucidated in this investigation might be of relevance to the depigmentation and degeneration of neuromelanin-pigmented noradrenergic cell bodies in the locus ceruleus in Parkinson's Disease and to the degeneration of noradrenergic nerve terminals in Alzheimer's Disease and following transient cerebral ischemia (stroke).

Full text of DOI:10.1021/jm960016t

2018
J . Med. Chem. 1996, 39, 2018-2029
Oxid a tion Ch em istr y of (-)-Nor ep in ep h r in e in th e P r esen ce of L-Cystein e
Xue-Ming Shen and Glenn Dryhurst*
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019
Received J anuary 5, 1996X
The noradrenergic neurotransmitter (-)-norepinephrine (1) is very easily oxidized at physi-
ological pH to an o-quinone (2) that normally cyclizes and subsequently oxidatively polymerizes
to black melanin. In this investigation it is demonstrated that L-cysteine (CySH) can divert
the melanin pathway by efficiently scavenging o-quinone 2 to give, initially, 5-S-cysteinyl-
norepinephrine (6) and 2-S-cysteinylnorepinephrine (7). These cysteinyl conjugates are
appreciably more easily oxidized than 1 to o-quinones that, in part, are further attacked by
CySH to give 2,5-bi-S-cysteinylnorepinephrine (8), an even more easily oxidized compound.
The o-quinone intermediates formed upon oxidation of 6-8 can also undergo facile intramo-
lecular cyclizations to bicyclic o-quinone imines that oxidize the cysteinyl conjugates from which
they are derived in a reaction sequence that leads initially to a number of dihydrobenzothiazines.
At least two of these compounds, 7-(1-hydroxy-2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-
benzothiazine-3-carboxylic acid (9) and 8-(1-hydroxy-2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-
1,4-benzothiazine-3-carboxylic acid (10) are lethal when administered into the brains of mice.
The in vitro chemical pathways elucidated in this investigation might be of relevance to the
depigmentation and degeneration of neuromelanin-pigmented noradrenergic cell bodies in the
locus ceruleus in Parkinson’s Disease and to the degeneration of noradrenergic nerve terminals
in Alzheimer’s Disease and following transient cerebral ischemia (stroke).
The primary neuropathological feature of Parkinson’s
Disease (PD) is the profound degeneration of nigrostri-
atal dopaminergic neurons caused by toxic processes
that occur in their pigmented cell bodies in the sub-
stantia nigra (SN).1,2 Dopaminergic SN cells are pig-
mented as a result of the autoxidation of cytoplasmic
dopamine (DA) to DA-o-quinone that polymerizes to
black neuromelanin.3,4 The pathological processes that
occurs in these neurons include oxidative stress5 and a
defect in mitochondrial respiration6 although the un-
derlying mechanisms are unknown. However, the Par-
kinsonian SN is characterized by a massive loss of
glutathione (GSH) without a corresponding increase in
GSSG,5,7,8 an increased 5-S-cysteinyldopamine (5-S-
CyS-DA)/DA concentration ratio,9 increased γ-glutamyl
transpeptidase (γ-GT) activity,10 and depigmentation of
these cells.11,12 Such observations have led to the
hypothesis13 that an early step in the pathogenesis of
PD is an elevated γ-GT-mediated translocation of cys-
teine (CySH) or GSH14,15 into pigmented SN cell bodies
that normally contain little or none of these sulfhydryl
compounds.16,17 In vitro studies13,17 suggest that in-
creased cytoplasmic CySH levels would divert the
neuromelanin pathway by scavenging DA-o-quinone to
give soluble cysteinyl conjugates of DA with concomitant
progressive depigmentation of SN cells and an increased
5-S-CyS-DA/DA ratio. Subsequent autoxidation of cys-
teinyldopamines has further been proposed to lead to
products that might include endotoxins that contribute
to nigral cell death and PD.13
that occur in the LC in PD. However, LC and SN
neurons are similar in that they both contain relatively
high cytoplasmic levels of the catecholamine neuro-
transmitters 1 and DA, respectively, that are easily
oxidized to o-quinone intermediates and thence to
neuromelanin, and both depigment and degenerate in
PD. Taken together, such observations might indicate
that similar mechanisms underlie the pathological
processes that occur in both SN and LC cell bodies in
PD. Interestingly, noradrenergic neurons that project
from the LC to the cortex and hippocampus also
degenerate in Alzheimer’s Disease (AD)21 and following
a transient ischemic insult.22 However, unlike PD, in
which the neuropathological processes occur in SN and
LC cell bodies,2,23 the neurodegenerative processes that
occur in AD and following transient cerebral ischemia
are believed to occur in terminal regions of noradren-
ergic and other neurons.24-26 Furthermore, nerve ter-
minals differ from cell bodies in that they contain
significant concentrations of GSH and, presumably to
a lesser extent, its biosynthetic precursor CySH.16
Many lines of evidence indicate that terminal regions
of the brain vulnerable to degeneration in AD and
transient cerebral ischemia, such as the cortex, are
subject to oxidative stress, i.e., damage deriving from
abnormally high levels of oxygen radical species.27-33
Ischemia/reperfusion also results in significant depletion
of GSH in the cortex without a corresponding increase
in GSSG.34,35
The neurodegeneration that occurs in PD also affects
noradrenergic neurons that project from their pig-
mented cell bodies in the locus ceruleus (LC).18,19 These
cell bodies are pigmented because of autoxidation of
norepinephrine (1) to an o-quinone that polymerizes to
neuromelanin.3,20 Little is known about the changes
Oxygen radical-mediated and other oxidative damage
in brain tissue is widely believed to primarily affect
membrane lipids, proteins, and nucleic acids.36 How-
ever, noradrenergic terminals that degenerate in AD
and following an ischemic insult employ a neurotrans-
mitter, 1, that is a very easily oxidized compound. It is
not inconceivable, therefore, that in these brain disor-
ders 1 is oxidized within noradrenergic terminals to an
* Corresponding author.
X Abstract published in Advance ACS Abstracts, April 15, 1996.
S0022-2623(96)00016-7 CCC: $12.00 © 1996 American Chemical Society

Oxidation Chemistry of (-)-Norepinephrine
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10 2019
F igu r e 1. Cyclic voltammograms at the PGE of 0.5 mM norepinephrine (1) in pH 7.4 phosphate buffer (µ ) 1.0) in the presence
of (A) 0 mM CySH, (B) 0.25 mM CySH, (C) 0.5 mM CySH, and (D) 1.0 mM CySH. Sweep rate: 50 mV s-1. All voltammograms
were initiated at -400 mV.
Sch em e 1
o-quinone that is scavenged by CySH and that the
resulting cysteinyl conjugates of the neurotransmitter
are further oxidized to endotoxins that might contribute
to the degeneration of these neurons. In essence, it is
hypothesized that the vulnerability of pigmented nor-
adrenergic LC cell bodies, which normally contain little
or no GSH or CySH, to degeneration in PD is linked to
the fact that 1 is autoxidized in these neurons. The
pathological processes in PD are hypothesized to be
triggered by an aberrant increase in cytoplasmic levels
of CySH or GSH that divert the neuromelanin pathway
to give endotoxic metabolites. By contrast, the vulner-
ability of certain noradrenergic nerve terminals to
degeneration in AD or following an ischemic insult
might be related to aberrant oxidation of 1 to an
o-quinone that reacts with endogenous CySH or GSH
to give the same or similar endotoxins. It was clearly
of interest, therefore, to explore the influence of CySH
on the oxidation chemistry of 1 at physiological pH. A
number of reasons led us to employ electrochemical
techniques in this initial study. Thus, controlled po-
tential electrolysis provides a way of maintaining very
precise control of oxidizing conditions. Furthermore,
cyclic voltammetry often permits considerable insights
into reaction intermediates and mechanisms and pro-
vides an extremely convenient method to measure redox
potentials of both intermediates and products. Because
of the fact that the chemical3 (e.g., autoxidation) and
electrochemical20 oxidations of 1 (and related catechol-
amines) both generate an o-quinone intermediate as the
proximate reaction product, it was anticipated that the
results of this investigation might ultimately provide
valuable insights into more biologically relevant oxida-
tions of this neurotransmitter in the presence of CySH
mediated by molecular oxygen or oxygen radical species.
The principal goals of this investigation were to eluci-
date the influence of CySH on the oxidation chemistry
of 1 with particular emphasis on mechanistic pathways
and characterizations of major reaction intermediates
and products. Furthermore, reaction products were
screened for neurobiological activity following admin-
istration into the brains of mice.
Resu lts
Cyclic Volta m m etr y. A cyclic voltammogram of 1
(0.5 mM) at a pyrolytic graphite electrode (PGE) in pH
7.4 phosphate buffer at a sweep rate (ν) of 50 mV s-1 is
presented in Figure 1A. On the initial anodic sweep,
peak Ia appears at a peak potential (Ep) of 140 mV and
corresponds to the oxidation (2e, 2H+) of 1 to o-quinone
2 (Scheme 1).20,37 An indistinct second peak also ap-
pears at ca. 700 mV (Figure 1A) that might correspond
to oxidation of the side chain residue of o-quinone 2.38
Following scan reversal, peak Ic (Ep ) 111 mV) corre-
sponds to the reversible reduction of o-quinone 2 to 1.
Previous investigators20,37 have established that at pH
7.4 deprotonation of 2 yields 3 which cyclizes to 3,5,6-
trihydroxyindoline (4) that is further oxidized (2e, 2H+)
by 2 at the electrode surface to give the aminochrome 5
and 1. Aminochrome 5 is the species responsible for

2020 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10
Shen and Dryhurst
peak IIc (Ep ) -235 mV) observed in cyclic voltammo-
grams of 1 and corresponds to its reduction (2e, 2H+) to
4. Peak IIa (Ep ) -214 mV), observed on the second
anodic sweep, corresponds to the reversible oxidation
of 4, formed in the peak IIc reduction, to 5. At slow
sweep rates (v e 50 mV s-1) the peak current (ip) for
reduction peak Ic is small compared to that for oxidation
peak Ia owing to the relatively rapid reaction between
2 and 4 (Scheme 1). However, with increasing sweep
rates ip for peak Ic grows relative to that for peak Ia until
the two peaks become of equal size (ν > 10 V s-1).
Correspondingly, the peak IIc/peak IIa couple decreases
and at ν > 10 V s-1 disappears.
Cyclic voltammograms (ν ) 50 mV s-1) of 1 (0.5 mM)
in the presence of CySH (0.25-1.0 mM) at pH 7.4 are
presented in Figure 1B-D. The presence of CySH
clearly results in some significant alterations in the
cyclic voltammetric behaviors of 1. Thus, with increas-
ing concentrations of CySH, reduction peak Ic and the
peak IIc/peak IIa couple decrease and at a concentration
of g0.5 mM disappear (Figure 1C,D). Furthermore, at
the lowest CySH concentration studied (0.25 mM;
CySH/1 mole ratio ) 0.5) two new major oxidation peaks
appear. Peak IIIa (Ep ) 101 mV; ν ) 50 mV s-1) appears
at more negative potentials than peak Ia of 1 (Ep ) 138
mV) and peak IVa (Ep ) 578 mV) at more positive
potentials (Figure 1B). With increasing CySH concen-
trations, peak IIIa becomes the predominant peak
(Figure 1C,D) and shifts toward slightly more negative
potentials. Thus, Ep values (ν ) 50 mV s-1) for peak
IIIa measured with 0.5, 1.0, and 2.5 mM CySH were 96,
90, and 86 mV, respectively. Optimal resolution be-
tween peaks IIIa and Ia was obtained with 0.5 mM 1
and 0.25 mM CySH using a slow sweep rate (10 mV
s-1). Under these conditions, Ep for peak IIIa was 76
mV and Ep for peak Ia was 136 mV (i.e., ∆Ep ) 60 mV).
With increasing sweep rate, Ep for peak IIIa shifts
toward more positive potentials and at ν g 500 mV s-1
peaks IIIa and Ia merge to give a single peak. Taken
together, the above results suggest that there is a rapid
reaction between the proximate oxidation product of 1,
o-quinone 2, and CySH. This reaction not only scav-
enges 2, as demonstrated by the decrease and disap-
pearance of reduction peak Ic with increasing CySH
concentrations (Figure 1B-D) but also blocks formation
of 4 and 5 as shown by the elimination of the peak IIc/
peak IIa couple. Over the potential ranges employed
in cyclic voltammetric experiments, CySH exhibits no
oxidation or reduction peaks at the PGE. Accordingly,
the reaction between 2 and CySH must also yield the
product(s) responsible for oxidation peak IIIa and sub-
sequent oxidation of this (these) compound(s) must yield
the species responsible for oxidation peak IVa.
F igu r e 2. HPLC chromatogram (method I) of the product
solution formed following controlled potential electro-oxidation
of 0.5 mM norepinephrine (1) in the presence of 1.0 mM CySH
in pH 7.4 phosphate buffer for 30 min at 70 mV.
In the absence of free CySH, controlled potential
electrolyses of 1 (0.5 mM) at 70 mV and pH 7.4 were
very slow. After 30 min the solution had a pale orange
color. HPLC analysis (method I) of this product solution
revealed that less than 10% of 1 had been oxidized, and
a single significant product was formed having a reten-
tion time (tR) of 28.5 min. In the HPLC mobile phase
(pH 2.15) this orange product exhibited a UV-visible
spectrum with λmax ) 488, 290, and 216 nm and a
thermospray mass spectrum with an intense pseudo-
molecular ion (MH+) at m/ e ) 166 (40%) expected for
aminochrome 5. Controlled potential electro-oxidation
of 1 (0.5 mM) in the presence of CySH (0.5-1.0 mM) at
pH 7.4 and 70 mV caused the initially colorless solution
to become a very pale yellow color. HPLC analysis
(method I) of the solution formed after 30 min revealed
that a complex mixture of products was formed (Figure
2). Using preparative HPLC (method II) to isolate
individual compounds, and an independent electro-
chemical synthesis, eight of the major products were
isolated and spectroscopically identified (see the Ex-
perimental Section). The structures of these products
are presented in Figure 2 and include 5-S-cysteinyl-
norepinephrine (6), 2-S-cysteinylnorepinephrine (7), and
2,5-bi-S-cysteinylnorepinephrine (8) in addition to di-
hydrobenzothiazines 9-13. When electrolyses were
permitted to proceed for periods greater than 30 min,
the chromatographic peaks for 6-13 systematically
Con tr olled P oten tia l Electr o-Oxid a tion Stu d ies.
At ν ) 10 mV s-1 the Ep values for oxidation peak IIIa
observed in voltammograms of 0.5 mM 1 in the presence
of 0.25, 0.5, 1.0, and 2.5 mM CySH at pH 7.4 were 76,
74, 72, and 70 mV, respectively. Accordingly, controlled
potential electro-oxidations of 1 in the presence of free
CySH at pH 7.4 were carried out at 70 mV. Because
the major goal of this investigation was to identify the
initial steps in the oxidation chemistry of the 1/CySH
system, all controlled potential electrolyses were ter-
minated after e30 min in order to minimize secondary
oxidation reactions.
decreased, and correspondingly, several peaks at tR
>
65 min increased in height. These observations indicate
that the compounds responsible for the latter chromato-
graphic peaks are secondary products and, hence, will
not be discussed further.
Cyclic Volta m m etr y of Cystein yl Con ju ga tes
6-8 a n d Dih yd r oben zoth ia zin es 9-13. Cyclic vol-
tammograms (ν ) 50 mV s-1) of 6-13 in pH 7.4

Oxidation Chemistry of (-)-Norepinephrine
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10 2021
F igu r e 3. Cyclic voltammograms at the PGE of 0.5 mM solutions of 6-13, in pH 7.4 phosphate buffer. Sweep rate: 50 mV s-1
.
phosphate buffer (Figure 3) reveal that each of these
compounds exhibit three major oxidation peaks (Ia, IIa,
and IIIa) on the initial anodic sweep. After scan
reversal, compounds 6, 7, 9, and 10 exhibited reduction
peaks that were reversibly coupled to oxidation peak
IIa. A summary of Ep values for peaks Ia, IIa, IIc, and
IIIa for compounds 6-13 at pH 7.4 at sweep rates of 10
and 50 mV s-1 is presented in Table 1. These results
reveal that the Ep values for the primary oxidation peak
Ia of cysteinyl conjugates 6-8 and dihydrobenzothi-
azines 9-13 all occur at more negative potentials than
that for peak Ia of 1, indicating that these products are
appreciably more easily oxidized than the latter neu-
rotransmitter. With increasing sweep rate, peak IIIa
observed in cyclic voltammograms of 1 (0.5 mM) in the
presence of free CySH (0.25-2.5 mM) shifts toward
more positive potentials whereas peak Ia (corresponding
to oxidation of 1) remains virtually constant (Table 1).
Similarly, peaks 6Ia-13Ia also shift to more positive
potentials with increasing ν.

2022 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10
Shen and Dryhurst
Ta ble 1. Peak Potentials (Ep) for Oxidation and Reduction
Peaks Observed in Cyclic Voltammograms of 1, 1 + CySH, and
Compounds 6-13 at pH 7.4a
F igu r e 4. HPLC chromatograms (method I) of the product
solutions formed following controlled potential electro-oxida-
tion of 0.5 mM solutions of (A) 6, (B) 7, and (C) 8 in pH 7.4
phosphate buffer at 70 mV for 25 min.
larly, 12. When the oxidation reactions of 6-8 were
continued for longer periods of time, the chromato-
graphic peaks corresponding to their various dihy-
drobenzothiazine products systematically decreased and
disappeared and the relatively minor peaks having tR
values g 60 min in Figure 4A-C correspondingly grew
larger and the solution developed a bright yellow color.
These yellow secondary oxidation products, however,
remain to be identified.
Oxid a tion s of Cystein yl Con ju ga tes 6-8. Figure
4 A presents a chromatogram (HPLC method I) of the
product solution formed following controlled potential
electro-oxidation of 6 (0.5 mM) at 70 mV for 25 min in
pH 7.4 buffer and clearly indicates that the major initial
product is dihydrobenzothiazine 9. Similarly, controlled
potential electro-oxidation of 7 (0.5 mM) at 70 mV for
25 min yields dihydrobenzothiazine 10 as the major
initial product (Figure 4B). Oxidation of the 2,5-bi-S-
cysteinyl conjugate 8 under similar conditions yields
dihydrobenzothiazine 12 as the major product and
dihydrobenzothiazine 11 as a minor product (Figure 4C).
These results indicate that cysteinyl conjugates 6 and
7 are precursors of dihydrobenzothiazines 9 and 10,
respectively, and that the bi-S-cysteinyl conjugate 8 is
the precursor of dihydrobenzothiazines 11 and, particu-
Controlled potential electro-oxidation of dihydroben-
zothiazine 9 in the presence of excess free CySH at 70
mV and pH 7.4 initially gave its 6-S-cysteinyl conjugate
11 as the major product (Figure 5A). Oxidation of
dihydrobenzothiazine 10 in the presence of free CySH
under identical conditions gave its 6-S-cysteinyl conju-
gate 12 as the major initial product along with smaller
yields of its 6,7-bi-S-cysteinyl conjugate 13 (Figure 5B).
Oxidation of 12 in the presence of free CySH under the
same conditions gave 13 as the major initial product
(data not shown). More prolonged electro-oxidations of
9, 10, and 12 in the presence of CySH resulted in the
formation of a bright yellow solution containing many
secondary products (which appear as minor products in
Figure 5A,B) that remain to be identified.

Oxidation Chemistry of (-)-Norepinephrine
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10 2023
Rea ction P a th w a ys
The results of cyclic voltammetry experiments indi-
cate that CySH scavenges o-quinone 2, the proximate
oxidation product of 1 (Figure 1A-C). A key observa-
tion in cyclic voltammograms of 1 in the presence of free
CySH is the appearance of oxidation peak IIIa at less
positive potentials than peak Ia of 1. The ip for peak
IIIa compared to that for peak Ia in cyclic voltammo-
grams of 1 even in the presence of relatively small
amounts of CySH (Figure 1B) reveals that the reaction
between 2 and CySH is extensive at potentials corre-
sponding to the foot of peak Ia to form a product(s) that
is more easily oxidized than 1. Ep for peak IIIa (70-76
mV, ν ) 10 mV s-1, pH 7.4) observed in cyclic voltam-
mograms of 1 (0.5 mM) in the presence of free CySH
(0.25-2.5 mM) is very close to the primary oxidation
peaks of cysteinyl conjugates 6 (Ep for peak 6Ia ) 66
mV) and 7 (Ep for peak 7Ia ) 72 mV). Since 6 and 7
must logically be the initial products formed between 2
and CySH it is reasonable to conclude that peak IIIa
corresponds in part to oxidation of 6 and 7. At poten-
tials corresponding to the foot of peak Ia of 1, CySH must
rapidly scavenge 2 to give cysteinyl conjugates 6 and 7,
thus momentarily depleting the surface concentration
of this o-quinone. Nernstian considerations, therefore,
demand that additional 1 is oxidized to 2 at these low
potentials with the result that CySH facilitates oxida-
tion of the neurotransmitter. The fact that 6 and 7 are
also more easily oxidized than 1 accounts for further
enhancement of the current observed at peak IIIa.
F igu r e 5. HPLC chromatograms (method I) of the product
solutions obtained following controlled potential electro-oxida-
tion of 0.1 mM solutions of dihydrobenzothiazines (A) 9 and
(B) 10 in pH 7.4 phosphate buffer in the presence of 0.5 mM
CySH for 25 min at 70 mV.
Controlled potential electrolysis of 1 in the presence
of free CySH at potentials corresponding to peak IIIa
results in the rapid oxidation of the neurotransmitter
compared to its rate of oxidation in the absence of CySH.
However, only rather low yields of 6, 7, and 8 are formed
(Figure 2) because of the ease of further oxidation of
these cysteinyl conjugates of 1 (Table 1). The major
initial products of this electrochemically driven oxida-
tion reaction include dihydrobenzothiazines 9 and 10
and their cysteinyl conjugates 11 and 12 + 13, respec-
tively (Figure 2). This result was somewhat surprising
because (1) intramolecular cyclization of o-quinones
formed upon oxidation of 6 and 7 would be expected to
yield o-quinone imines not their corresponding reduced
forms, i.e., dihydrobenzothiazines 9 and 10, respectively;
(2) dihydrobenzothiazines 9 and 10 (and 11-13) are also
very easily oxidized compounds (Table 1) and would
therefore be expected to be further oxidized at controlled
potentials corresponding to peak IIIa (70 mV). In an
earlier investigation13 it was proposed that structurally
related o-quinone imines were maintained in their
reduced (dihydrobenzothiazine) state by CySH. How-
ever, in this investigation it has been demonstrated that
even in the absence of free CySH cysteinyl conjugates
6 and 7 are initially oxidized to dihydrobenzothiazines
9 and 10, respectively (Figure 4) at peak IIIa potentials
(along with several minor unidentified products). These
observations imply that upon cyclization of the o-
quinones formed by oxidation of 6 and 7 a chemical
pathway must exist whereby the resulting bicyclic
o-quinone imines are reduced to the corresponding
dihydrobenzothiazines. Insights into such a pathway
can be derived from the cyclic voltammetric behaviors
of cysteinyl conjugates such as 6 and 7 and dihydroben-
zothiazines 9 and 10 at pH 7.4. To illustrate, Figure
6A presents a slow sweep rate (ν ) 10 mV s-1) cyclic
voltammogram of dihydrobenzothiazine 10. On the
initial anodic sweep two well-defined oxidation peaks
10Ia (Ep ) 84 mV) and 10IIa (Ep ) 162 mV) appear.
Following scan reversal reduction peak 10IIc (Ep ) 130
mV) forms a reversible couple with oxidation peak 10IIa.
With increasing sweep rates, however, the ip values for
the peak 10IIa/peak 10IIc couple decrease (Figure 6B),
and at ν g 5 V s-1 this couple disappears (Figure 6C).
Under the latter conditions a new reduction peak 10Ic
(Ep ) 102 mV; ν ) 5 V s-1) appears that forms a
reversible couple with peak 10Ia (Figure 6C). Such
observations suggest that at slow sweep rates the
proximate product of the peak 10Ia reaction undergoes
a chemical reaction to give the species responsible for
the peak 10IIa/peak 10IIc couple. However, at fast
sweep rates there is insufficient time for this chemical
reaction to occur with the result that the peak 10IIa/
peak 10IIc couple does not appear and peak 10Ic
corresponds to the reduction of the proximate product
formed at peak 10Ia. These observations are rational-
ized by proposing that dihydrobenzothiazines 10 un-
dergoes a one-electron oxidation in the peak 10Ia
reaction to give radical cation 14 (Scheme 2). At fast
sweep rates, therefore, peak 10Ic corresponds to the
reversible one-electron reduction of 14 to 10. However,
at slower sweep rates deprotonation of 14 yields the
neutral radical 15 that is further oxidized (1e) to
o-quinone imine 16 in the peak 10IIa reaction. After
scan reversal, peak 10IIc therefore corresponds to the
reversible reduction of 16 to 15.

2024 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10
Shen and Dryhurst
F igu r e 6. Cyclic voltammograms at the PGE of a 0.5 mM solution of dihydrobenzothiazine 10 in pH 7.4 phosphate buffer at a
sweep rate of (A) 10 mV s-1, (B) 50 mV s-1, and (C) 5.0 V s-1 and of 0.5 mM 2-S-cysteinylnorepinephrine (7) at a sweep rate of
(D) 10 mV s-1, (E) 100 mV s-1, (F) 200 mV s-1, and (G) 10 V s-1
.
Sch em e 2
Sch em e 3
A slow sweep rate (ν ) 10 mV s-1) cyclic voltammo-
gram of cysteinyl conjugate 7 at pH 7.4 is presented in
Figure 6D. On the initial anodic sweep oxidation peak
7Ia (Ep ) 72 mV) and peak 7IIa (Ep ) 159 mV) appear.
After scan reversal, reduction peak 7IIc (Ep ) 132 mV)
forms a reversible couple with oxidation peak 7IIa. The
Ep values for oxidation peaks 7IIa and 10IIa and for
peaks 7IIc and 10IIc are virtually identical. With
increasing sweep rate, ip for oxidation peak 7IIa de-
creases relative to that for oxidation peak 7Ia (Figure
6E) and ultimately disappears (Figure 6F). A parallel
decrease in ip for reduction peak 7IIc does not occur.
However, at ν g 5 V s-1 peak 7IIc disappears and a new
reduction peak 7Ic appears and forms a reversible couple
with oxidation peak 7Ia (Figure 6G). Under the latter
conditions, peak 7Ia corresponds to the oxidation (2e,
2H+) of cysteinyl conjugate 7 to o-quinone 17 and peak
7Ic to the reverse reaction (Scheme 3). At slower sweep
rates, however, there is sufficient time for intramolecu-
lar cyclization of 17 to o-quinone imine 16. Thus,
oxidation of 7 leads directly to o-quinone imine 16, the
species responsible for reduction peak 10IIc in cyclic
voltammograms of cysteinyl conjugate 7 (Figure 6D-
F). The Ep for peak 7IIc is clearly at more positive
values than that for oxidation peak 7Ia. Thus, o-quinone
imine 16 must be capable of chemically oxidizing
cysteinyl conjugate 7 in a reaction that generates
o-quinone 17 and radical 15 as conceptualized in
Scheme 3. This conclusion is supported by the observa-
tion that at slow sweep rates cyclic voltammograms of
7 exhibit peak 7IIa (equivalent to peak 10IIa in cyclic
voltammograms of 10) corresponding to oxidation of
radical 15, formed in this chemical reaction, to o-
quinone imine 16 (Figure 6D). At faster sweep rates,
however, reaction between 16 and 7 to generate radical
15 is less extensive, and hence ip for oxidation peak 7IIa
decreases whereas peak 7IIc (corresponding to reduction
of 16) remains a significant feature of cyclic voltammo-
grams of 7 (Figure 6E,F). The major initial product of
controlled potential electro-oxidation of 7 at peak IIIa
potentials is dihydrobenzothiazine 10 (Figure 4B). In

Oxidation Chemistry of (-)-Norepinephrine
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10 2025
Sch em e 4
view of this observation, radical 15 must disproportion-
ate to give 10 and 16. The pathway conceptualized in
Scheme 3 predicts that once oxidation of cysteinyl
conjugate 7 is initiated the reaction should become self-
sustaining (autocatalytic). Experimentally, however,
this is not the case indicating that o-quinone imine 16
probably undergoes additional reactions that lead to
several other as yet unidentified products (Figure 4B).
The cyclic voltammetric behaviors of cysteinyl conjugate
6 and dihydrobenzothiazine 9 are very similar to those
described for 7 and 10, respectively. Furthermore,
controlled potential electro-oxidation of 6 at peak IIIa
potentials gives 9 as the major initial product (Figure
4A). Accordingly, it was concluded that the oxidation
chemistry of 6 and 9 is similar to that proposed for 7
(Scheme 3) and 10 (Scheme 2), respectively.
On the basis of the foregoing discussion, the reaction
sequences presented in Scheme 4 are proposed to
account for the major initial products of the controlled
potential electro-oxidation of 1 at pH 7.4 in the presence
of CySH at peak IIIa potentials. Thus, as noted previ-
ously, oxidation of 1 (2e, 2H+) leads to o-quinone 2.
Nucleophilic addition of CySH to 2 then forms cysteinyl
conjugates 6 and 7. Addition of CySH (2.0-4.0 mM) to
o-quinone 2 (ca. 1.0 mM), synthesized by controlled
potential electro-oxidation of 1 at 1.0 V in 0.1M HCl,
results in formation of conjugates 6 and 7 in relative
yields of approximately 3:1 (see the Experimental Sec-
tion). Similar studies could not be carried out at pH
7.4 because of the instability of 2. Nevertheless, chro-
matograms of product solutions formed following oxida-
tion of 1 in the presence of CySH always indicated that
yields of 6 were significantly greater than 7 (Figure 2).
Such observations suggest that at pH 7.4 nucleophilic
addition of CySH to 2 occurs predominantly, but not
exclusively, at the C(5)-position. Further oxidation (2e,
2H+) of 6 and 7 leads to o-quinones 18 and 17,
respectively, that can each react by at least three
pathways. The first of these pathways results from
intramolecular cyclizations of 17 and 18 to 16 and 19,
respectively (Scheme 4). The latter quinone imines then
diffuse away from the electrode and chemically oxidize
cysteinyl conjugates 6 and 7 and hence provide routes
to dihydrobenzothiazines 9 and 10 as conceptualized in
Scheme 4 and discussed in detail in connection with
Schemes 2 and 3.
A second pathway involves nucleophilic addition of
CySH to o-quinone imines 19 and 16 to give the
cysteinyl dihydrobenzothiazine conjugates 11 and 12,
respectively (Scheme 4). These pathways are experi-
mentally supported by the observation that controlled
potential electro-oxidation of dihydrobenzothiazines 9
and 10 in the presence of free CySH at peak IIIa
potentials yield cysteinyl conjugates 11 (Figure 5A) and
12 (Figure 5B), respectively, as the major initial prod-
ucts. However, in these reactions formation of o-
quinone imines 16 and 19 results from a multistep
pathway. To illustrate, at peak IIIa potentials, dihy-
drobenzothiazine 10, for example, is initially oxidized
to radical cation 14 (Scheme 2). Following deprotona-
tion, radical 15 then disproportionates to o-quinone
imine 16 and 10 (Scheme 3).
The third reaction pathway deriving from o-quinones
17 and 18 involves nucleophilic addition of CySH to give
the 2,5-bi-S-cysteinyl conjugates of 1, i.e., 8 (Scheme 4;
Figure 2). Controlled potential electro-oxidation of 8 at
peak IIIa potentials even in the absence of free CySH
yields the cysteinyl dihydrobenzothiazines 11 (minor
product) and 12 (major product) (Figure 4C). Accord-
ingly, by analogy with reaction pathways discussed
previously, it can be concluded that 8, a very easily
oxidized compound (Table 1), is chemically oxidized by
16/19 to o-quinone 21 that undergoes intramolecular
cyclization to o-quinone imines 22 (major route) and 24
(minor route). Subsequent oxidation of 8 by 22 and 24
then leads to radicals 23 and 25, respectively. Dispro-

2026 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10
Shen and Dryhurst
portionations of 23 and 25 then yield dihydrobenzothi-
azines 12 (and 22) and 11 (and 24), respectively.
In view of the fact that dihydrobenzothiazine 12 is
the major initial product of oxidation of 1 in the presence
of CySH (Figure 2) under the conditions employed in
this investigation, o-quinone imine 22 must represent
a major intermediate species. This no doubt accounts
for the appearance of 13, formed by nucleophilic addition
of CySH to 22 (Scheme 4). This conclusion is further
confirmed by the fact that oxidation of 12 in the
presence of free CySH yields 13.
as major initial products. This unusual observation can
be traced to the fact that o-quinone imines 19, 16, and
22/24 are capable of oxidizing cysteinyl conjugates 6,
7, and 8, respectively, and are therefore reduced to the
corresponding dihydrobenzothiazines. Indeed, the for-
mation of such dihydrobenzothiazines provides evidence
to support the conclusion that o-quinone imines such
as 19, 16, and 22/24 catalyze the oxidation of the
cysteinyl conjugates 6, 7, and 8, respectively, from which
they are derived.
The reaction pathways and products determined in
this investigation relate specifically to the electrochemi-
cal oxidation of 1 in the presence of CySH at pH 7.4. It
remains to be experimentally determined whether this
oxidation chemistry mimics more biologically relevant
reactions mediated by molecular oxygen or oxygen
radicals. However, recent reports have demonstrated
that the Fe2+-promoted autoxidation, hydroxyl-radical-
and peroxidase/H2O2-mediated40 and electrochemical13,41
oxidations of the related catecholamine DA are all
potentiated by CySH and 5-S-CyS-DA and 2-S-CyS-DA
are formed as initial products. Moreover, on the basis
of the yields of these cysteinyl conjugates, compared to
the DA consumed in the former chemical and enzymatic
oxidation reactions and HPLC analysis of products40 it
might reasonably be concluded that 5-S-CyS-DA and
2-S-CyS-DA undergo further oxidations perhaps to
dihydrobenzothiazines as predicted from more detailed
electrochemical studies.13,41 These apparent similarities
between the electrochemical, chemical, and enzymatic
oxidations of DA in the presence of CySH suggest that
the present investigation might ultimately provide
insights into the influence of CySH on the autoxidation
and oxygen-radical-mediated oxidations of 1. On the
basis of this assumption it is possible to speculate about
the potential neurobiological relevance of the results of
this investigation. Thus, intraneuronal oxidation of 1
in the presence of CySH, both of which are cytoplasmic
constituents, would be expected to lead to metabolites
that are both lethal (9 and 10) and evoke a profound
neurobehavioral response (7, 9-11). Dihydrobenzothi-
azines 9 and 10 are relatively potent toxins in the brains
of mice having LD50 values of 17.8 µg (66 nmol) and 23.4
µg (87 nmol), respectively. These LD50 values can be
contrasted with that of the catecholaminergic neuro-
toxin 6-hydroxydopamine (6-OHDA), 134 µg (794 nmol),42
a compound that has been considered as a possible
endotoxin in PD.43 Dihydrobenzothiazines 9-11 and
cysteinyl conjugate 7 also evoke a profound neuro-
behavioral response in mice whereas 6-OHDA does
not.42 The mechanisms that underlie the in vivo neu-
robehavioral and/or lethal effects evoked by 7 and 9-11
remain to be elucidated. Furthermore, it must be
stressed that at the current stage of this investigation
there is no evidence that these compounds, other
cysteinyl conjugates of 1, or other dihydrobenzothiazines
identified are either toxic toward noradrenergic or any
other neurons or occur in the brain in neurodegenerative
disorders. Nevertheless, it is known that 1 undergoes
oxidation in the cytoplasm of noradrenergic LC cell
bodies as evidenced by their pigmentation with neu-
romelanin polymer.3,20 Futhermore, the results of this
investigation tend to provide confirmation that LC cell
bodies probably do not normally contain significant
concentrations of CySH or GSH because these sulfhy-
Biologica l Stu d ies
Preliminary in vivo experiments were carried out to
assess the toxicity and behavioral responses evoked
when 6-13 were administered into the brains of labora-
tory mice. In these experiments, each compound was
dissolved in 5 µL of isotonic saline and injected into the
vicinity of the left lateral ventricle while animals were
under a light ether anesthesia. The LD50 value, used
as a measure of toxicity and defined as the dose of
injected compound (expressed as free base) at which
50% of the treated animals died within 1 h, was
determined using the statistical method of Dixon.39
Dihydrobenzothiazines 9 and 10 were lethal, having
experimental LD50 values of 17.8 ( 1.3 µg (mean (
standard deviation) and 23.4 ( 1.3 µg, respectively.
Doses of 9 ranging from 5 to 100 µg and of 10 ranging
from 10 to 100 µg were employed to determine these
LD50 values. The behavioral responses evoked by 9 and
10 were very similar. The responses described below
refer to those evoked at the LD50 dose. Following
recovery from the ether anesthetic, animals initially
either circled contralateral to the site of injection or
moved slowly in a backward direction for ca. 2-10 min.
Subsequently, experimental animals exhibited episodes
of rapid running, jumping, rolling repetitively along the
head-tail axis, and squeaking. Episodes of severe
shivering were also observed. Animals treated with 11
(60-100 µg) and 7 (100 µg) initially circled contralateral
to the site of injection followed by episodes of rapid
running. At the 100 µg dose level 6, 8, 12, and 13
evoked no obvious behavioral response and all animals
survived. Control animals treated with 5 µL of isotonic
saline exhibited none of the above behavioral effects
noted with 7, 9, 10, and 11 and all survived.
Discu ssion
The results of this investigation reveal that CySH can
divert the oxidation pathway of 1 to black melanin
polymer (Scheme 1) by scavenging o-quinone 2 to form
6 and 7 (Scheme 4). However, these cysteinyl conju-
gates of 1 are appreciably more easily oxidized than the
neurotransmitter (Table 1, Figure 3A,B) to give o-
quinones 17 and 18 that are the precursors of dihy-
drobenzothiazines 9-13 (Scheme 4). The key step in
the pathways leading to these dihydrobenzothiazines is
the intramolecular cyclization of o-quinones 17, 18, and
21 to give bicyclic o-quinone imines 16, 19, and 22/24,
respectively. It is conceivable that these o-quinone
imines might be reduced to the corresponding dihy-
drobenzothiazines by CySH.13 However, the present
results demonstrate that, even in the absence of free
CySH, oxidation of cysteinyl conjugates 6, 7, and 8 leads
to dihydrobenzothiazines 9, 10, and 11/12, respectively,

Oxidation Chemistry of (-)-Norepinephrine
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10 2027
NJ ). Voltammograms were obtained at a pyrolytic graphite
electrode (PGE; Pfizer Minerals, Pigments and Metals Divi-
sion, Easton, PA) having an approximate surface area of 6
mm2. A conventional three-electrode voltammetric cell was
used with a platinum wire counter electrode and a saturated
calomel reference electrode (SCE). Cyclic voltammograms
were obtained using a BAS-100A (Bioanalytical Systems, West
Lafeyette, IN) electrochemical analyzer. All voltammograms
were corrected for iR drop. Controlled potential electrolyses
employed a Princeton Applied Research Corporation (Prince-
ton, NJ ) model 173 potentiostat. A three-compartment cell
was used in which the working, counter, and reference
electrode compartments were separated with Nafion mem-
branes (type 117, DuPont Co., Wilmington, DE). The working
electrode compartment had a capacity of 30 mL. The working
electrode consisted of several plates of pyrolytic graphite
having a total surface area of approximately 180 cm2. The
counter electrode was platinum gauze and the reference
electrode a SCE. The solution in the working electrode
dryl compounds would block the formation of neuro-
melanin pigment by scavenging o-quinone 2.
The vulnerability of noradrenergic LC neurons to
degeneration in PD might, similar to pigmented dopa-
minergic SN cells,11,44 be linked to the fact that they
sustain high basal levels of autoxidation of 1 and DA,
respectively. The fact that both of these cell bodies
degenerate in the Parkinsonian brain11,12 and, at least
in the SN, this is accompanied by a massive irreversible
loss of GSH,5,7,8 increased γ-GT activity,10 and an
increased 5-S-CyS-DA/DA ratio9 all point to the idea
that elevated cytoplasmic levels of CySH might be
responsible for diverting the neuromelanin pathways
forming, initially, cysteinyl conjugates of 1 (in the LC)
and of DA (in the SN). Over the course of many years
such a CySH-mediated diversion of these neuromelanin
pathways would be expected to significantly reduce the
amount of pigment deposited in these cell bodies and
account for their apparent depigmentation in the Par-
kinsonian brain.9 From a chemical perspective oxida-
tion of cysteinyl conjugates of 1 should be inevitable
under conditions where 1 is oxidized, i.e., in the cyto-
plasm of pigmented LC neurons, to yield lethal dihy-
drobenzothiazines such as 9 and 10. Work is currently
in progress to determine whether cysteinyl conjugates
of 1 or dihydrobenzothiazines that result from their
facile oxidation (Table 1) are toxic toward noradrenergic
or other LC neurons in order to provide support for the
hypothesis that these compounds might include endo-
toxins that contribute to the neurodegeneration that
occurs in PD.
Noradrenergic terminals also undergo profound de-
generation in certain regions of the brain in AD21 and
as a consequence of transient cerebral ischemia.22
Unlike noradrenergic cell bodies in the LC these ter-
minals are rich in GSH and, presumably, its biosyn-
thetic precursor CySH.16 Furthermore, many lines of
evidence indicate that in both AD27-31 and transient
cerebral ischemia32,33 highly oxidizing conditions, per-
haps mediated by oxygen radical species, develop in
areas of the brain that include terminal regions of
noradrenergic neurons, for example in the cortex.24-26
Furthermore, following ischemia/reperfusion a signifi-
cant loss of GSH occurs without a corresponding in-
crease in GSSG levels.34,35 This suggests that GSH is
not lost as a result of its function as an oxygen radical
scavenger or, in conjunction with glutathione peroxi-
dase, detoxification of H2O2 because both of these
processes should result in elevated levels of GSSG.
Perhaps, therefore, the irreversible loss of GSH results
in part from aberrant oxidation of 1 in noradrenergic
terminals with resultant formation of glutathionyl and
cysteinyl conjugates of the neurotransmitter followed
by further oxidation to dihydrobenzothiazines by path-
ways similar to those conceptualized in Scheme 4. Such
cysteinyl conjugates of 1 and/or resultant DHBTs might,
therefore, represent endotoxins that contribute to the
degeneration of noradrenergic terminals in selected
regions of the AD brain and following an ischemic insult.
compartment was continuously bubbled with
a vigorous
stream of N2 and stirred with a Teflon-coated magnetic stirring
bar. All potentials are referenced to the SCE at ambient
temperature (22 ( 2 °C).
1H NMR spectra were recorded on a Varian (Palo Alto, CA)
XL-300 spectrometer. Low- and high-resolution fast atom
bombardment mass spectrometry (FAB-MS) employed a VG
Instruments (Manchester, UK) model ZAB-E spectrometer.
Thermospray mass spectra were obtained with a Kratos
(Manchester, UK) model 25/RFA instrument equipped with a
thermospray source. Samples collected from preparative scale
HPLC were injected into the thermospray source via a Rheo-
dyne model 7125 loop injector equipped with a 2.0 mL loop.
UV-visible spectra were recorded on a Hewlett-Packard (Palo
Alto, CA) model 8452A diode array spectrophotometer.
High-performance liquid chromatography (HPLC) employed
a Gilson (Middleton, WI) gradient system equipped with dual
model 302 pumps (10 mL pump heads), a Rheodyne (Cotati,
CA) model 7125 loop injector, and a Waters (Milford, MA)
model 440 UV detector set at 254 nm. Two mobile phase
solvents were employed. Solvent A was prepared by adding
concentrated TFA to deionized water until the pH was 2.15.
Solvent B was prepared by adding TFA to a mixture of 2 L of
deionized water and 2 L of HPLC grade acetonitrile until the
pH was 2.15. HPLC method I was employed to monitor the
course of the electrochemical oxidation of NE in the presence
of CySH and to purify reaction products and used a reversed
phase column (Bakerbond C18, 10 µm, 250 × 21.2 mm, P. J .
Cobert Associates, St. Louis, MO) and the following gradient
profile: 0-5 min, 100% solvent A; 5-40 min, linear gradient
to 12% solvent B; 40-80 min, linear gradient to 50% solvent
B; 80-85 min, linear gradient to 100% solvent B; 85-97 min,
100% solvent B. The flow rate was 7.0 mL min-1
. HPLC
method II was used to isolate reaction products and employed
a Bakerbond (J . T. Baker, Phillipsburg, NJ ) reversed phase
column (C18, 10 µm particle size, 250 × 21.2 mm) and the
following gradient profile: 0-60 min, linear gradient from
100% solvent A to 40% solvent B; 60-70 min, linear gradient
to 100% solvent B; 70-82 min, 100% solvent B. The flow rate
was 7.0 mL min-1
.
An im a ls. Outbred adult male mice of the HSD:ICR albino
strain (Harlan Sprague-Dawley, Madison, WI) weighing 28-
32 g were employed. Experimental animals were treated with
test drugs dissolved in 5 µL of isotonic saline vehicle (0.9%
NaCl in deionized water). Control animals were treated with
5 µL of vehicle alone. Animals were anesthetized with ether,
and then drugs or vehicle were injected by means of a 10 µL
microsyringe. Injections were performed freehand with the
point of puncture being 3 mm anterior to the interaural line
and 1 mm left lateral of the midline to a depth of 3 mm
perpendicular to the scalp as described previously.45 Animal
procedures employed were approved by the Institutional
Animal Care and Use Committee of the University of Okla-
homa.
Exp er im en ta l Section
(-)-Norepinephrine hydrochloride (NE‚HCl) and L-cysteine
(CySH) were obtained from Sigma (St. Louis, MO) and were
used without additional purification. Trifluoroacetic acid
(TFA) was obtained from Aldrich (Milwaukee, WI). HPLC
grade acetonitrile was obtained from EM Science (Gibbstown,
Oxid a tion Rea ction P r oced u r es. In a typical controlled
potential electro-oxidation reaction 1‚HCl (3.1 mg; 0.5 mM)
and CySH (3.65 mg; 1.0 mM) were dissolved in 30 mL of pH

2028 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10
Shen and Dryhurst
7.4 phosphate buffer (µ ) 0.2). The resulting solution was
electrolyzed at 70 mV for 30 min. Upon termination of the
reaction the entire pale yellow solution was introduced onto
the prepared reversed phase column via one of the HPLC
pumps, and reactants and products were separated using
method II. The solutions eluted under each of the major
chromatographic peaks were collected individually and im-
mediately frozen and stored at -80 °C. Following several
repetitive experiments, the combined solutions containing each
product were purified using HPLC method I. The solution of
each product so obtained was then freeze-dried.
Electr och em ica l Syn th esis of Cystein yl Con ju ga tes 6
a n d 7. 1‚HCl (6.2 mg; 1.0 mM) was dissolved in 30 mL of 0.1
M HCl and electrolyzed at 1.0 V for 30 min. The reaction
solution changed from initially colorless to a bright yellow color
characteristic of 2. HPLC analysis (method I) revealed that
g90% of 1 was converted to o-quinone 2. Addition of CySH
(7.3-14.6 mg; 2.0-4.0 mM) to the solution of 2 caused the
bright yellow solution to rapidly become a very pale yellow
color. The pH of the solution was adjusted to 8.0 with an
aqueous solution of KOH and then to pH 2.15 with concen-
trated TFA. The total volume of solution was then directly
pumped onto the preparative reversed phase column, and
components were separated using an isochratic method that
employed solvent A (flow rate: 7.0 mL min-1). Cysteinyl
conjugates 7 and 6 eluted at tR values of 15 and 18 min,
respectively. This procedure was repeated several times, the
eluents containing 6 and 7 being collected individually. The
combined solutions containing 6 and 7 were then freeze-dried.
The resulting solid residues were dissolved in 2-10 mL of
deionized water and adjusted to pH 2.15 with TFA and purified
using the same isochratic HPLC method. The solutions
containing 6 and 7 were then freeze-dried.
C(b′)-H), 3.51 (dd, J ) 15.0, 5.7 Hz, 1H, C(a)-H), 3.50 (dd, J )
15.0, 5.7 Hz, 1H, C(a)-H), 3.34 (dd, J ) 15.0, 4.5 Hz, 1H, C(a′)-
H), 3.23 (dd, J ) 13.2, 3.6 Hz, 1H, C(â)-H), 3.20 (dd, J ) 15.0,
4.5 Hz, 1H, C(a′)-H), 3.12 (dd, J ) 13.2, 8.4 Hz, 1H, C(â)-H).
7-(1-H yd r oxy-2-a m in oet h yl)-3,4-d ih yd r o-5-h yd r oxy-
2H-1,4-ben zoth ia zin e-3-ca r boxylic Acid (9). Compound 9
was a very light yellow hygroscopic solid. Anal. (C11H14
-
N2O4S‚CF3COOH). Calcd: C, 40.63; H, 3.91; N, 7.29; S, 8.33;
F, 14.84. Found: C, 40.35; H, 3.84; N, 6.78; S, 8.14; F, 15.49.
At pH 7.4, λmax, nm (log ꢀmax, M-1 cm-1), 304 (3.40), 238 (4.32).
FAB-MS (3-nitrobenzyl alcohol matrix) gave m/ e ) 271.0753
(MH+, 22, C11H15N2O4S; calcd m/ e ) 271.0753). 1H NMR
(Me2SO-d6) gave δ 9.70 (bs, 1H, C(5)-OH), 7.86 (bs, 3H, NH3+),
6.52 (d, J ) 2.1 Hz, 1H, C(6)-H), 6.41 (d, J ) 2.1 Hz, 1H, C(8)-
H), 5.82 (bs, 1H, C(R)-OH), 5.31 (bs, 1H, N(4)-H), 4.52 (dd, J
) 9.9, 3.0 Hz, 1H, C(R)-H), 4.35 (dd, J ) 5.7, 3.3 Hz, 1H, C(3)-
H), 3.15 (dd, J ) 12.6, 3.3 Hz, 1H, C(2)-H), 3.07 (dd, J ) 12.6,
5.7 Hz, 1H, C(2)-H), 2.90 (dd, J ) 12.3, 3.0 Hz, 1H, C(â)-H),
2.74 (dd, J ) 12.3, 9.9 Hz, 1H, C(â)-H). 1H NMR (D2O) gave
δ 6.66 (s, 2H, C(6)-H and C(8)-H), 4.73 (dd, J ) 8.7, 3.9 Hz,
1H C(R)-H), 4.66 (dd, J ) 5.7, 3.6 Hz, 1H, C(3)-H), 3.37 (dd, J
) 14.1, 3.6 Hz, 1H, C(2)-H), 3.31 (dd, J ) 14.1, 5.7 Hz, 1H,
C(2)-H), 3.11 (dd, J ) 13.2, 3.9 Hz, 1H, C(â)-H), 2.98 (dd, J )
13.2, 8.7 Hz, 1H, C(â)-H).
8-(1-H yd r oxy-2-a m in oet h yl)-3,4-d ih yd r o-5-h yd r oxy-
2H-1,4-ben zoth ia zin e-3-ca r boxylic Acid (10). Compound
10 was a very pale pink hygroscopic solid. At pH 7.4, λmax
,
nm (log ꢀmax, M-1 cm-1), 306 (3.39), 232 (4.33), calculated for
the TFA salt. FAB-MS (3-nitrobenzyl alcohol matrix) gave
m/ e 271.0741 (MH+, 30, C11H15N2O4S; calcd m/ e ) 271.0753).
1H NMR (D2O) gave δ 6.79 (d, J ) 8.4 Hz, 1H, C(7)-H), 6.70
(d, J ) 8.4 Hz, 1H, C(6)-H), 5.11 (dd, J ) 8.1, 3.9 Hz, 1H, C(R)-
H), 4.56 (dd, J ) 4.2, 3.6 Hz, 1H, C(3)-H), 3.31 (dd, J ) 13.2,
4.2 Hz, 1H, C(2)-H), 3.16 (dd, J ) 13.2, 3.9 Hz, 1H, C(â)-H),
3.12 (dd, J ) 13.2, 3.6 Hz, 1H, C(2)-H), 3.08 (dd, J ) 13.2, 8.1
Hz, 1H, C(â)-H).
Spectroscopic evidence in support of the proposed structures
of major products formed following oxidation of 1 in the
presence of CySH are presented below. The assignments of
various proton resonances observed in 1H NMR spectra of
products were based on comparisons with the spectra of 1 and
CySH and were confirmed in all cases by two-dimensional
correlated spectroscopy (COSY) experiments.
6-S-Cystein yl-7-(1-h yd r oxy-2-a m in oeth yl)-3,4-d ih yd r o-
5-h yd r oxy-2H-1,4-ben zoth ia zin e-3-ca r boxylic Acid (11).
Compound 11 was a very pale yellow solid. At pH 7.4, λmax
,
nm (log ꢀmax, M-1 cm-1), 324 (3.48), 282 sh (3.76), 252 (4.26),
calculated for 2TFA salt. FAB-MS (thioglycerol/glycerol ma-
trix) gave m/ e ) 390.0775 (MH+, 26, C14H20N3O6S2; calcd m/ e
) 390.0794). 1H NMR (D2O) gave δ 6.86 (s, 1H, C(8)-H), 5.41
(dd, J ) 8.7, 3.6 Hz, 1H, C(R)-H), 4.61 (dd, J ) 4.5, 3.6 Hz,
1H, C(3)-H), 3.94 (t, J ) 5.7 Hz, 1H, C(b)-H), 3.32 (dd, J )
13.2, 4.5 Hz, 1H, C(2)-H), 3.26-3.19 (m, 3H, C(2)-H, C(a)-H2),
3.16 (dd, J ) 12.9, 3.6 Hz, 1H, C(â)-H), 3.13 (dd, J ) 12.9, 8.7
Hz, 1H, C(â)-H).
5-S-Cyst ein yln or ep in ep h r in e (6). Compound
6 was
isolated as a white solid. Anal. (C11H16N2O5S‚2CF3COOH)
Calcd: C, 34.88; H, 3.49; N, 5.43; S, 6.20; F, 22.09. Found:
C, 34.71; H, 3.50; N, 5.25; S, 6.45; F, 20.66. The UV spectrum
of 6 at pH 7.4 exhibited bands, λmax, nm (log ꢀmax, M-1 cm-1),
at 314 sh (3.13), 294 (3.46) and 254 (3.66). FAB-MS (glycerol/
TFA matrix) gave m/ e ) 289.0875 (MH+, 100, C11H17N2O5 S;
calcd m/ e ) 289.0858). 1H NMR (D2O) gave δ 7.07 (d, J 2,6
)
2.1 Hz, 1H, C(2)-H), 6.93 (d, J 2,6 ) 2.1 Hz, 1H, C(6)-H), 4.84
(dd, J ) 8.4, 4.2 Hz, 1H, C(R)-H), 4.08 (dd, J ) 6.3, 4.8 Hz,
1H, C(b)-H), 3.46 (dd, J ) 15.0, 6.3 Hz, 1H, C(a)-H), 3.38 (dd,
J ) 15.0, 4.8 Hz, 1H, C(a)-H), 3.24 (dd, J ) 13.2, 4.2 Hz, 1H,
C(â)-H), 3.15 (dd, J ) 13.2, 8.4 Hz, 1H, C(â)-H).
6-S-Cystein yl-8-(1-h yd r oxy-2-a m in oeth yl)-3,4-d ih yd r o-
5-h yd r oxy-2H-1,4-ben zoth ia zin e-3-ca r boxylic Acid (12).
Compound 12 was a very pale yellow solid. Anal. (C14H19
-
N3O6S2‚2CF3COOH). Calcd: C, 35.01; H, 3.40; N, 6.81; S,
10.37; F, 18.48. Found: C, 35.13; H, 3.81; N, 6.75; S, 9.90; F,
17.61. At pH 7.4, λmax, nm (log ꢀmax, M-1 cm-1), 322 (3.44),
280 sh (3.74), 250 (4.30). FAB-MS (thioglycerol/glycerol
matrix) gave m/ e ) 390.0792 (MH+, 77, C14H20N3O6S2; calcd
m/ e ) 390.0794). 1H NMR (D2O) gave δ 7.02 (s, 1H, C(7)-H),
5.14 (dd, J ) 7.8, 3.6 Hz, 1H, C(R)-H), 4.64 (t, J ) 3.9 Hz, 1H,
C(3)-H), 3.94 (t, J ) 5.7 Hz, 1H, C(b)-H), 3.36 (dd, J ) 13.2,
3.9 Hz, 1H, C(2)-H), 3.30 (d, J ) 5.7 Hz, 2H, C(a)-H2), 3.21
(dd, J ) 13.2, 3.9 Hz, 1H, C(2)-H), 3.12 (dd, J )13.2, 7.8 Hz,
1H, C(â)-H), 3.11 (dd, J ) 13.2, 3.6 Hz, 1H, C(â)-H).
2-S-Cyst ein yln or ep in ep h r in e (7). Compound
7 was
isolated as a white solid. At pH 7.4 the UV spectrum exhibited
bands at λmax, nm (log ꢀmax, M-1 cm-1), 318 sh (3.22), 298 (3.49),
256 (3.42) calculated as the 2TFA salt. FAB-MS (glycerol
matrix) gave m/ e ) 289.0837 (MH+, 30, C11H17N2O5S; calcd
m/ e ) 289.0858). 1H NMR (D2O) gave δ 6.96 (s, 2H, C(5)-H
and C(6)-H), 5.48 (dd, J ) 8.7, 3.3 Hz, 1H, C(R)-H), 4.05 (dd,
J ) 6.9, 4.8 Hz, 1H, C(b)-H), 3.37 (dd, J ) 14.7, 6.9 Hz, 1H,
C(a)-H), 3.25 (dd, J ) 14.7, 4.8 Hz, 1H, C(a)-H), 3.20 (dd, J )
13.2, 3.3 Hz, 1H, C(â)-H), 3.12 (dd, J ) 13.2, 8.7 Hz, 1H, C(â)-
H). 1H NMR (CD3OD) gave δ 7.07 (d, J ) 8.4 Hz, 1H, C(5)-
H), 6.97 (d, J ) 8.4 Hz, 1H, C(6)-H), 5.48 (dd, J ) 9.9, 3.3 Hz,
1H, C(R)-H), 3.94 (dd, J ) 9.0, 4.2 Hz, 1H, C(b)-H), 3.41 (dd,
J ) 14.7, 4.2 Hz, 1H, C(a)-H), 3.25 (dd, J ) 14.7, 9.0 Hz, 1H,
C(a)-H), 3.19 (dd, J ) 12.9, 3.3 Hz, 1H, C(â)-H), 3.03 (dd, J )
12.9, 9.9 Hz, 1H, C(â)-H).
2,5-Bi-S-cystein yln or ep in ep h r in e (8). This compound
was a white solid. At pH 7.4 λmax, nm (log ꢀmax, M-1 cm-1),
320 (3.56), 270 (3.80), 246 (4.10), calculated for the 2TFA salt.
FAB-MS (glycerol/TFA matrix) gave m/ e ) 408.0895 (MH+,
100, C14H22N3O7S2; calcd m/ e ) 408.0899). 1H NMR (D2O)
gave δ 7.25 (s, 1H, C(6)-H), 5.52 (dd, J ) 8.4, 3.6 Hz, 1H, C(R)-
H), 3.96 (t, J ) 5.7 Hz, 1H, C(b)-H), 3.96 (t, J ) 4.5 Hz, 1H,
6,7-Bi-S-cyst ein yl-8-(1-h yd r oxy-2-a m in oet h yl)-3,4-d i-
h ydr o-5-h ydr oxy-2H-1,4-ben zoth ia zin e-3-ca r boxylic Acid
(13). Compound 13 was a very pale pink solid. At pH 7.4,
λ
max, nm (log ꢀmax, M-1 cm-1), 332 sh (3.58), 304 sh (3.64), 264
(4.29), calculated for the 2TFA salt. FAB-MS (thioglycerol/
glycerol matrix) gave m/ e ) 509.0841 (MH+, 100, C17H25N408S3;
calcd m/ e ) 509.0835). 1H NMR (D2O, 50 °C) gave δ 6.28 (dd,
J ) 10.2, 4.2 Hz, 1H, C(R)-H), 5.03 (t, J ) 3.6 Hz, 1H, C(3)-
H), 4.27 (t, J ) 5.7 Hz, 1H, C(b)-H), 4.22 (t, J ) 5.7 Hz, 1H,
C(b′)-H), 4.17 (dd, J ) 13.2, 10.2 Hz, 1H, C(â)-H), 3.70 (dd, J
) 14.4, 5.7 Hz, 1H, C(a′)-H), 3.60 (dd, J ) 13.5, 3.6 Hz, 1H,
C(2)-H), 3.59 (dd, J ) 14.4, 5.7 Hz, 1H, C(a′)-H), 3.59 (d, J )
5.7 Hz, 2H, C(a)-H2), 3.51 (dd, J ) 13.2, 4.2 Hz, 1H, C(â)-H),
3.17 (dd, J ) 13.5, 3.6 Hz, 1H, C(2)-H).

Oxidation Chemistry of (-)-Norepinephrine
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Additional support was provided by the Vice President
for Research and the Research Council at the University
of Oklahoma.
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