Synthesis and neurotoxic potential of racemic and chiral dihydroxytetrahydroquinoline derivatives

Article abstract of DOI:10.1016/S0040-4020(00)00470-1

The synthesis and preliminary neurotoxic investigation of (±), (+) and (-)-3-amino-6,7-dihydroxy-1,2,3,4-tetrahydroquinoline, (±)-3-amino-6,8-dihydroxy-1,2,3,4-tetrahydroquinoline and (±)-3-aminomethyl-6,7-dihydroxy-1,2,3,4-tetrahydroquinoline are described. While exhibiting relatively no dopamine and only moderate norepinephrine depletions, these compounds elicit serotonin depletions equal to those provided by the well-known serotonergic neurotoxin 5,7-dihydroxytryptamine in whole mouse brain. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00470-1

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 5345±5352
Synthesis and Neurotoxic Potential of Racemic and Chiral
Dihydroxytetrahydroquinoline Derivatives
Russell J. Lewis,a Charles A. Francis,b Roland E. Lehrb and C. LeRoy Blankb,
*
aCivil Aeromedical Institute, Federal Aviation Administration, Toxicology and Accident Research Laboratory, 6500 South MacArthur Blvd,
Oklahoma City, OK 73125, USA
bDepartment of Chemistry & Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, OK 73019, USA
Received 2 February 2000; accepted 30 May 2000
AbstractÐThe synthesis and preliminary neurotoxic investigation of (^), (1) and (2)-3-amino-6,7-dihydroxy-1,2,3,4-tetrahydroquino-
line, (^)-3-amino-6,8-dihydroxy-1,2,3,4-tetrahydroquinoline and (^)-3-aminomethyl-6,7-dihydroxy-1,2,3,4-tetrahydroquinoline are
described. While exhibiting relatively no dopamine and only moderate norepinephrine depletions, these compounds elicit serotonin
depletions equal to those provided by the well-known serotonergic neurotoxin 5,7-dihydroxytryptamine in whole mouse brain. q 2000
Elsevier Science Ltd. All rights reserved.
Introduction
oxidase blocking agents to delay onset of Idiopathic
Parkinson's Disease following investigation of the mode
of action of MPTP.4,5
Neurotoxins, such as 6-hydroxydopamine (6-HDA) and 5,7-
dihydroxytryptamine (5,7-DHT), have been employed for
more than 25 years to study behavioral, physiological and
pharmacological phenomena associated with catechol-
aminergic and serotonergic neuronal pathways. However,
while the chemical lesioning afforded by such agents is
highly selective compared with surgical or electrolytic
methods, currently employed toxins still exhibit a high
degree of non-target tissue destruction. Considering this, it
is not surprising that there is an ongoing search for agents
which exhibit a higher degree of toxicity toward targeted
neuronal populations while eliciting little or no non-target
tissue damage. Additionally, the mode(s) of action of
6-HDA,1 5,7-DHT2 and related neurotoxins3 remains rela-
tively unknown; thus, examination of novel, but similar,
neurotoxins will hopefully shed light on this important
area as well.
This paper describes the synthesis and a preliminary exami-
nation of central nervous system (CNS) toxicity for three
putative neurotoxic agents: 3-amino-6,7-dihydroxy-1,2,3,4-
tetrahydroquinoline (9 and its enantiomers), 3-amino-6,8-
dihydroxy-1,2,3,4-tetrahydroquinoline (18), and 3-amino-
methyl-6,7-dihydroxy-1,2,3,4-tetrahydroquinoline
(26).
Synthetic schemes starting from easily available dimethoxy-
benzaldahydes are presented for each of these agents. Pre-
liminary investigations have shown that these compounds,
while exhibiting relatively no dopamine and only moderate
norepinephrine depletions, elicit serotonin depletions
approximately equivalent to those achieved by the estab-
lished 5,7-dihydroxytryptamine in mouse whole brain.
Additionally, these preliminary experimental results, par-
ticularly for neurotoxin 9, suggest that there is no apparent
stereochemical selectivity associated with such agents.
We have accordingly designed ®xed side chain analogs of
6-HDA with the intent to (1) provide neurotoxins which are
superior to those in use, and (2) provide further information
concerning the destructive mode(s) of action of such
species. A more complete understanding of how these and
other toxins elicit neurodestruction might also provide
insight into the underlying mechanisms of neurodegenera-
tive disorders and, ultimately, aid in the postponement of
onset and/or alleviation of these disorders. Such an
approach has already led to the employment of monoamine
Results and Discussion
Syntheses of 3-amino-6,7-dihydroxy-1,2,3,4-tetrahydro-
quinoline hydrobromide and 3-amino-6,8-dihydroxy-
1,2,3,4-tetrahydroquinoline hydrobromide
The syntheses of 3-amino-6,7-dihydroxy-1,2,3,4-tetrahydro-
quinoline hydrobromide (9) and 3-amino-6,8-dihydroxy-
1,2,3,4-tetrahydroquinoline hydrobromide (18) followed
similar pathways, as depicted in Scheme 1. The ®rst step
in the synthesis was the condensation of dimethoxybenz-
aldehyde 1 or 10 with hippuric acid in the presence of
Keywords: asymmetry; cyclization; catecholamines; indoleamines.
* Corresponding author. Tel.: 11-405-325-2967; fax: 11-405-325-6111;
e-mail: lblank@ou.edu
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00470-1

5346
R. J. Lewis et al. / Tetrahedron 56 (2000) 5345±5352
Scheme 1. For compounds 1±8, R1ˆOCH3, R2ˆH. For compounds 10±17, R1ˆH, R2ˆOCH3.
powdered sodium acetate and acetic anhydride. This reac-
tion afforded the azlactones 2 in a 62% yield and 11 in an
86% yield. Basic methanolysis provided opening of the
azlactone ring and addition of the desired methoxy group.
This reaction provided 3 in a 70% yield and 12 in an 88%
yield. The product analyses for both 2 and 3 agreed well
with the literature.6
referee has proposed a modi®ed approach to compounds 9
and 26 to avoid some of these nitration problems using
commercially available 6-nitropiperonal or 6-nitro-
veratraldehyde. We believe this approach would be success-
ful, but was not attempted.)
The quinoline derivatives were formed through catalytic
hydrogenation, using palladium on carbon. The reduction
of the nitro functionality on 5 and 14 resulted in the forma-
tion of their corresponding amines, which then underwent
intramolecular cyclization to afford the lactam derivatives 6
and 15 in a 100 and 90% yield, respectively. This ring-
formation reaction proved to be a very useful and ef®cient
reaction for the production of the tetrahydroquinoline ring
structures. A discussion of the use of this general approach
to the synthesis of quinonlines can be found in the report of
G. Jones;7 a closely related example is given in Narang et
al.8 Utilizing BH3/THF, the amide functionalities were
converted to the corresponding amine derivatives 7 (89%)
and 16 (90%). The benzoyl amide functionality notably
experienced reduction at a much slower rate than did the
aliphatic amide due to its characteristically low electro-
philicity. Catalytic hydrogenation effected N-debenzylation
providing 8 and 17 in 94 and 83% yields, respectively. To
avoid poisoning of the palladium catalyst, these reactions
were conducted in the presence of acetic acid. The ®nal step
in the synthetic scheme was deprotection of the methyl-
ethers. An excess of 48% hydrobromic acid under re¯ux
conditions yielded the desired products 9 (95%) and 18
(86%). The 8-methoxy cleavage required 26 h, which was
approximately ®ve times longer than that needed to cleave
Reduction of the vinyl moiety provided the key step by
which chirality was able to be conveniently introduced in
this synthetic pathway. Conversion of propene derivatives 3
and 12 to their corresponding propane derivatives 4 and 13
was accomplished through catalytic hydrogenation (10%
Pd/C) to afford clean products in quantitative yields. Nitra-
tion of 4, employing fuming nitric acid, resulted in the
corresponding 6-nitro analog 5 in a 99% yield. Nitration
of 13, however, was more dif®cult. Using fuming nitric
acid (2±6 fold excess), various solvents (chloroform, acetic
acid, and a mixture of these), and a wide range of reaction
temperatures (278 to 258C), the yields of nitrated product
14 varied from 4±16%. These reactions resulted in products
which were frequently not readily characterized. Thus,
milder nitration reactions were subsequently attempted.
These incorporated the use of reagent combinations like
ammonium nitrate and tri¯uoroacetic anhydride, leading
to multiple side products but no discernible desired product
and, ®nally, tetranitromethane with pyridine, which resulted
in the desired product 14, but only in an 8% yield. Ulti-
mately, by applying a simplex optimization procedure
using 70.4% nitric acid in an acetic acid solvent, a maximal
yield of 19% was achieved for the formation of 14. (A

R. J. Lewis et al. / Tetrahedron 56 (2000) 5345±5352
Table 1. Optical rotation values for (R) and (S) 4±9
5347
The stereochemistry of the (R)-4 and (S)-4 enantiomers
were initially assessed by determining the optical rotation
values, as shown in Table 1, and comparing to the reported
literature value of [a]2D3ˆ183 (c 2.4, CHCl3) for (S)-4.11 As
can be seen, the results compare reasonably well to the
previously reported value for the (S)-isomer. The optical
rotation values for the enantiomeric forms of 5±9, also
shown in Table 1, could not be employed to assess enantio-
meric purity, since optical rotation values have not been
reported for these compounds. However, enantiomeric
purity, or enantiomeric excess (ee), was determined for 8
using diastereomeric derivatives. Using (R)-(2)-1-(1-
naphthyl)ethyl isocyanate as a chiral derivatizing agent,
(R)-8 and (S)-8 were converted to their diurea derivatives.
These derivatives were then independently separated by
HPLC, and the diastereomeric ratios were determined
from peak areas. The average of the ratios was found to
be 96:4 in both cases, which corresponds to a 92% enantio-
meric excess. The considerable variability in the optical
rotation values for the (R)-9 and (S)-9 enantiomers
listed in Table 1 is assumed to be due to the susceptibility
of these species to rapid autoxidation in aqueous solution;
thus, these are not recommended for use in assessment of
optical purity.
Compound
R
S
[a]2D5
c (g/ml)a
[a]2D5
c (g/ml)a
4
5
6
7
8
9
279
169
238
230
226
116
1.35
1
1.5
1.3
0.5
0.9
181
278
141
136
130
26
2.5
1.6
1.3
1.7
0.8
0.9
a Compounds 4±8 were in CH2Cl2, 9 in H2O.
the 6-methoxy and/or 7-methoxy functionalities. This
excessive reaction time for 18 did not, however, pose a
problem since the resultant dihydroxy compound was rela-
tively stable under these reaction conditions. Caution was
taken in the ®nal work-up of the hydrobromide salts, 9, 18
and 26, to minimize exposure to moisture and oxygen due to
their ease of autoxidation.
Enantiomeric syntheses
The inclusion of chirality into the 3-amino-6,8-dihydroxy-
1,2,3,4-tetrahydroquinoline product was achieved through
introduction of asymmetrical hydrogenation of the a-acyl-
amino methylacrylate 3. This was accomplished utilizing
two enantiomeric forms of a rhodium±DUPHOS complex,
which were previously used by Burk9 in the reduction of
substituted acetamidoacrylates at advantageously low
hydrogen pressures and ambient temperatures. The active
catalysts were prepared as reported10 (Caution: handling
and storage of the active catalysts needs to be performed
in a dry-box). Successful asymmetrical hydrogenation of
prochiral ole®n 3 yielded (R)-4 and (S)-4 in quantitative
amounts. The subsequent synthetic pathways for these
enantiomers leading to the desired dihydroxyquinolines
followed those described for the racemic compound.
Synthesis of 3-aminomethyl-6,7-dihydroxy-1,2,3,4-tetra-
hydroquinoline dihydrobromide
The synthesis of 3-amino-6,7-dihydroxy-1,2,3,4-tetrahydro-
quinoline dihydrobromide (26) is shown in Scheme 2. The
®rst step in this synthesis was the condensation of 3,4-
dimethoxybenzaldehyde (19) with dimethylmalonate in
the presence of piperidine resulting in product 20 with an
88% yield. Catalytic hydrogenation provided reduction of
the benzylidene derivative leading to product 21 in an 86%
yield. Spectral analyses showed products 20 and 21 to be in
agreement with the literature.12,13 Nitration of 21 using
fuming nitric acid provided product 22 in a quantitative
yield. Reduction of the nitro group to the corresponding
Scheme 2.

5348
R. J. Lewis et al. / Tetrahedron 56 (2000) 5345±5352
Table 2. Depletion of whole mouse brain neurotransmitters seven days
following toxin administration (signi®cant differences compared to control
values (t test): *P,0.05, **P,0.01, ***P,0.001. Amounts used: (^)- and
R-(1)-9, 83 nmol; S-(2)-9 and 26, 52 nmol; 18, 333 nmol; 5,7-DHT,
52 nmol; 6-HDA, 300 nmol)
Long-term depletion of endogenous neurotransmitters
Table 2 shows the depletion of norepinephrine (NE), dopa-
mine (DA) and serotonin (5-HT) in whole mouse brains at
seven days following intracerebroventricular administration
of these agents. The NE depletions produced by 9 and its
enantiomers were expected due to their structural similari-
ties to 6-hydroxydopamine (6-HDA) and a-methyl-6-
aminodopamine (a-6-ADA).14 It was surprising, however,
that 18 and 26 elicited only moderate NE depletions (30%).
Additionally, while 6-HDA provides DA depletions greater
than 50%, the racemic and enantiomeric forms of 9
produced only minimal (12±17%) DA alterations.
Compound 18 affected DA levels even less, and 26 did
not alter DA levels at all, reminiscent of the a-6-ADA
results. These modest to minimal NE and minimal to
nonexistent DA depletions may be due to hindered neuronal
uptake at endogenous neurotransmitter uptake sites.
Completely unexpected, however, were the 5-HT depletions
elicited by these quinoline species. The racemic and
enantiomeric forms of 9 and, separately, compound 18
produced approx. 30% depletions in whole brain 5-HT
levels. While these appear on the surface to be relatively
modest, they are, in fact, relatively substantial when
compared to results obtained from the best known alterna-
tive serotonergic agents when employed in mouse brain.
These 5-HT depletions are virtually identical to those
reported15,16for the well known and widely employed 5,6-
DHT and 5,7-DHT neurotoxins.
Compound
n
Levels, % controls^SEM
NE
DA
5-HT
9
11
14
16
16
17
13
17
54^2***
52^2***
55^2***
68^1***
68^2***
84^2***
35^6***
83^3***
86^3***
88^3***
91^3*
74^3***
72^2***
71^3***
73^2***
81^2***
70^2***
88^3*
R-(1)-9
S-(2)-9
18
26
5,7-DHTa
6-HDAb
100^1
71^6**
a Data reported by Herman and Bonczek16 for mouse brain levels seven
days after i.c.v. injection of 5,7-DHT. Percent controls reported above
are the maximum depletions obtained among the four examined
regions, which included cerebral hemispheres, pons/medulla, dience-
phalon, and striatum.
b Data from Ma et al.14 for whole mouse brain seven days after i.c.v.
injection of 6-HDA.
amine, as above, led to intramolecular cyclization of the
side chain via nucleophilic substitution. This reaction
proceeded rapidly and resulted in product 23 with a yield
of 72%.
While appearing straightforward, the production of
carboxamide 24 from the methylester 23 proved to be
challenging. This conversion was attempted using many
different approaches, most of which were not successful.
The unsuccessful reagents and conditions attempted
included ammonium hydroxide and ammonium chloride,
ammonia and ammonium chloride in a pressurized vessel
with heat, and a saturated ammonia solution in a pressurized
vessel with heat. All these attempts yielded the amide from
the methylester, but the major product was the oxidized
species, 3-carboxamide-6,7-dimethoxy-2-hydroxyquinoline,
and small amounts, if any, of desired 24. We found that
exposure of relatively pure 24, isolated from these mixtures,
to similar reaction conditions exhibited a time dependent
conversion to the undesirable oxidized product. Thus, the
time for the reaction was optimized to provide maximal
formation of 24 with minimal formation of the oxidation
product. The optimal approach determined employed an
elevated temperature ammonolysis of the methylester with
ammonia and ammonium hydroxide in a pressurized vessel.
Under these conditions, the major product remained the
oxidized form of 24; however, due to solubility differences
in methanol, 24 was isolable in an acceptable yield of 24%.
(A referee has suggested the possibility of avoiding this
problem through the use of methylmalonate monoamide
as a condensing reagent. Although this pathway was not
tried, we believe it a worthy approach for future work in
this synthesis.) Simultaneous conversion of the two amide
groups to the corresponding amines was achieved via
diborane reduction, resulting in diamine 25 in an 88%
yield. The ®nal step in the synthesis involved cleavage of
the methyl ethers to their corresponding hydroxy function-
alities using 48% hydrobromic acid under re¯ux conditions.
Compound 26 was obtained in high purity with a yield of
74%.
The enantiomers of 9 were investigated to evaluate the
effects of chirality in the catecholaminergic side chain on
the observed neurotoxicity. Based on the whole mouse brain
depletion values, there is no signi®cant difference observed
between the individual enantiomers and/or the racemic
form. Thus, it would appear that there is no advantage to
be gained from using the enantiomers of this compound for
neurodegeneration studies.
However, from the preliminary biological investigations, it
appears that these dihydroxy-tetrahydroquinoline deriva-
tives, and possibly other such analogs, may prove useful
in various neurological and biochemical investigations.
Studies are currently being carried out to determine the
mode of action(s) of these toxic agents.
Experimental
General methods
Proton and carbon NMR spectra were acquired using a
Varian XL-300 MHz spectrometer. Mass spectra were
recorded using a VG-ZAB-E instrument. Optical rotation
was determined using a Autopol III automatic polarimeter.
Melting points were determined using a Mel-Temp II
apparatus and were uncorrected. Tetrahydrofuran (THF)
was re¯uxed over calcium hydride and freshly distilled
prior to use. Acetic anhydride was distilled from phosphorus
pentoxide. Methanol was re¯uxed over Mg turnings and
iodine, distilled, and stored over molecular sieves. Ethyl
acetate was re¯uxed over sodium and benzophenone and

R. J. Lewis et al. / Tetrahedron 56 (2000) 5345±5352
5349
freshly distilled prior to use. All other reagents were
employed without further puri®cation.
3-Benzylamino-6,7-dimethoxy-1,2,3,4-tetrahydroquino-
line (7). To 6 (2.17 g, 7 mmol) in THF (10 mL) was added
BH3/THF (1 M, 120 mL, 120 mmol) at room temperature
under a N2 atmosphere. This solution was heated at re¯ux
for 8 h, allowed to cool, and slowly hydrolyzed with 6 M
HCl until no more hydrogen evolved. This mixture was
stirred for an additional 30 min, cooled, basi®ed with a
saturated sodium hydroxide solution, and extracted with
chloroform (4£50 mL). The combined organic extracts
were dried (sodium sulfate), ®ltered, and concentrated to a
residue which was puri®ed by column chromatography
(SiO2, 99:1 EtOAc/Et3N). The yield of puri®ed N-benzyl-
4-(3,4-Dimethoxybenzylidene)-2-phenyl-5-oxazolone (2).
Prepared as previously described.6
Methyl-2-benzoylamino-3-(3,4-dimethoxyphenyl)-2-pro-
penoate (3). Prepared as previously described.6
(^) Methyl 2-benzoylamino-3-(3,4-dimethoxyphenyl)-
propanoate (4). Compound 3 (18.07 g, 53 mmol) was
dissolved in hot methanol (100 mL) and to this was added
10% palladium on carbon (1.81 g) and glacial acetic acid
(1.81 mL). Hydrogenation was carried out at 45 psi for 12 h
in a Parr shaker apparatus. The solution was bubbled with
N2, ®ltered through a bed of Celite, and the Celite was
washed with 10 mL of hot methanol. The solvent was
stripped to yield 18 g (99%) of hydrogenated product 4;
mp 105±1078C (Lit.11 105±1068C). 1H NMR (CDCl3):
dˆ7.75 (d, Jˆ7.2 Hz, 2H), 7.53±7.40 (m, 3H), 6.80 (d,
Jˆ8.1 Hz, 1H), 6.69 (d, Jˆ1.8 Hz, 1H), 6.65 (dd,
Jˆ4.2 Hz, 1H), 6.7 (d, Jˆ7.2 Hz, 1H, exch. CD3OD), 5.08
(m, 1H), 3.87 (s, 3H), 3.79 (s, 3H), 3.77 (s, 3H), 3.26±3.21
(m, 2H). MS (70 eV-DIP) m/z (relative intensity) 343 (M1,
14), 222 (100), 151 (48), 77 (25).
1
amine derivative 8 was 1.76 g (89%). H NMR (CDCl3):
dˆ7.39±7.26 (m, 5H), 6.54 (s, 1H), 6.14 (s, 1H), 3.93 (s,
2H), 3.80 (s, 6H), 3.37±3.32 (m, 1H), 3.16±3.12 (m, 2H),
2.94 (dd, Jˆ15.9 and 4.4 Hz, 1H), 2.69 (dd, Jˆ15.8 and
7.7 Hz, 1H). MS (70 eV-DIP) m/z (relative intensity) 298
(M1, 100), 192 (43), 91 (44).
3-Amino-6,7-dimethoxy-1,2,3,4-tetrahydroquinoline (8).
Compound 7 (1.16 g, 4 mmol) was suspended in glacial
acetic acid (34 mL), and 10% palladium on carbon
(239 mg) was added. Using a Brown hydrogenation appa-
ratus, the reaction chamber was evacuated, pressurized to
,1 atm H2, and stirred at room temperature for 42 h. The
solution was ®ltered through a bed of Celite and the solvent
evaporated to afford an oil. The oil was dissolved in chloro-
form (100 mL) and washed with a 10% sodium hydroxide
solution followed with water, separated, dried (Na2SO4),
and concentrated. The crude compound was puri®ed using
a column of silica gel (100 g) and 5% MeOH/CHCl3. The
Methyl-2-benzoylamino-3-(3,4-dimethoxy-6-nitrophenyl)-
propanoate (5). Benzylamidomethyl cinnamate (4) (2.69 g,
8 mmol) was dissolved in chloroform (26 mL), and the
temperature was lowered to 08C. Fuming nitric acid
(1.59 mL) was added dropwise. The reaction was stirred
at 08C for 6 h. The reaction mixture was diluted with chloro-
form (20 mL) and neutralized with a sodium hydroxide
solution to a neutral pH. The layers were separated, and
the organic layer was dried (Na2SO4) and evaporated to
1
puri®ed amine was obtained in 94% yield (755 mg). H
NMR (DMSO-d6): dˆ8.25 (bs, 2H, exch. CD3OD), 6.56
(s, 1H), 6.22 (s, 1H), 3.65 (s, 3H), 3.63 (s, 3H), 3.50±3.48
(m, 1H), 3.29 (dd, Jˆ11.6 and 1.7 Hz, 1H), 3.11 (dd,
Jˆ11.7 and 5.4 Hz, 1H), 2.96 (dd, Jˆ16.4 and 5.3 Hz,
1H), 2.69 (dd, Jˆ16.4 and 6.0 Hz, 1H). MS (70 eV-DIP)
m/z (relative intensity) 208 (M1, 100), 193 (51), 150 (12).
1
give a yellow solid in a 99% yield. H NMR (CDCl3):
dˆ7.74 (d, Jˆ6.9 Hz, 2H), 7.57 (s, 1H), 7.53±7.41 (m,
3H), 7.07 (d, Jˆ7.5 Hz, 1H, exch. CD3OD), 6.83 (s, 1H),
5.09 (q, Jˆ7.2 Hz, 1H), 3.94 (s, 3H), 3.88 (s, 3H), 3.80 (s,
3H), 3.59 (d, Jˆ6.9 Hz, 2H). MS (70 eV-DIP) m/z (relative
intensity) 388 (M1, 6), 250 (28), 105 (100).
3-Amino-6,7-dihydroxy-1,2,3,4-tetrahydroquinoline di-
hydrobromide salt (9). To the dimethoxy derivative 8
(0.35 g, 2 mmol) in a pressure bottle was added 48% hydro-
bromic acid (1.85 mL), the solution was ¯ushed with argon
and heated for 6.5 h at re¯ux. The solution was lyophilized,
and the crude solid obtained was washed with ether
(6£2 mL) and acetonitrile (6£2 mL) to obtain 0.55 g
3-Benzoylamino-6,7-dimethoxy-1,2,3,4-tetrahydro-2-oxo-
quinoline (6). Compound 5 (403 mg, 1.04 mmol) was
dissolved in dry methanol (40 mL) by heating. The solution
was cooled to room temperature, and 10% palladium on
carbon (49 mg) and glacial acetic acid (1 mL) were added.
The reaction mixture was pressurized to 50 psi H2 in a Parr
shaker apparatus. The reaction was complete at 18 h. The
solution was bubbled with N2 and ®ltered through Celite.
The Celite was washed with hot chloroform (10 mL), and
the solvent was evaporated from the ®ltrate to yield a
gummy solid. This solid was dissolved in 50 mL CHCl3,
washed with saturated sodium bicarbonate solution and
water. The organic layer was separated, dried over sodium
sulfate, and concentrated to give an off-white solid in quan-
titative yield. 1H NMR (CDCl3): dˆ7.90 (d, Jˆ6.6 Hz, 2H),
7.8 (br, 1H, exch. CD3OD), 7.56±7.46 (m, 3H), 7.34 (d,
Jˆ4.5 Hz, 1H, exch. CD3OD), 6.78 (s, 1H), 6.40 (s, 1H),
4.76±4.68 (m, 1H), 3.88 (s, 3H), 3.66 (dd, Jˆ15.2 and
6.5 Hz, 1H), 2.85 (d, Jˆ14.6 Hz, 1H). MS (70 eV-DIP) m/
z (relative intensity) 326 (M1, 1), 205 (100).
1
(95%) of a tan solid. H NMR (D2O); dˆ6.76 (s, 1H),
6.68 (s, 0.5H, part. exch.), 3.93±3.90 (m, 1H), 3.76 (d,
Jˆ12.3 Hz, 1H), 3.40 (t, Jappˆ11.0 Hz, 1H), 3.18 (dd,
Jˆ16.4 and 5.1 Hz, 1H), 2.86 (dd, Jˆ16.4 and 9.0 Hz,
1H). MS (70 eV-DIP) m/z (relative intensity) 180 (M1,
16), 82 (83), 80 (100).
Rhodium complex [(COD)Rh(1,2-Bis((2S, 5S)-dimethyl-
phospholano)-benzene)]1 OTf2.10 To bis-(cycloocta-
diene)-rhodium tri¯ate (153 mg, 326 mmol) in dry THF
(10 mL) was added 1,2-bis-((2S, 5S)-2,5-dimethylphospha-
lano)-benzene (100 mg, 326 mmol) in THF (3 mL). The
initial yellow solution turned wine-red upon phosphine
addition. The solution was stirred for 15 min followed by
the slow addition of dry ether (30 mL). A bright orange
crystalline compound precipitated; this was ®ltered, dried,
1
and weighed (130 mg, 60%). H NMR (CD2Cl2): dˆ7.75

5350
R. J. Lewis et al. / Tetrahedron 56 (2000) 5345±5352
(m, 4H), 5.62 (br, 2H), 5.05 (br, 2H), 2.75 (m, 2H), 2.65 (m,
2H), 2.20±2.60 (m, 12H), 1.95 (m, 2H), 1.55 (m, 2H), 1.45
(dd, Jˆ18.2 and 7.1 Hz, 6H), 1.01 (dd, Jˆ15.0 and 6.8 Hz,
6H).
was [(COD)Rh-(1,2-bis-((2S, 5S)-dimethylphospholano)-
benzene)]1OTf2. The optical rotation was measured to be
[a]2D5ˆ181 (c 2.5, CH2Cl2).
(S)-(2)-Methyl-2-benzoylamino-3-(3,4-dimethoxy-6-
nitrophenyl)-propanoate [(S)-(2)-5]. The experimental
procedure followed was the same as that described for 5;
[a]2D5ˆ278 (c 1.6, CH2Cl2).
The second rhodium complex, [(COD)Rh-(1,2-bis-
((2R,5R)-dimethylphospholano)-benzene)]1 OTf2, was pre-
pared in the same manner as that stated above with the
exception that the phosphine ligand employed was 1,2-
bis-((2R,5R)-2,5-dimethylphospholano)-benzene.
(S)-(1)-3-Benzoylamino-6,7-dimethoxy-1,2,3,4-tetra-
hydro-2-oxoquinoline [(S)-(1)-6]. The experimental
procedure followed was the same as that described for 6;
[a]2D5ˆ141 (c 1.3, CH2Cl2).
General procedure for enantioselective hydrogenations
To the alkene 3 (0.68 g, 2 mmol) dissolved in dry methanol
(8 mL) was added the rhodium catalyst (2 mg). All the
reagents were handled in a dry box. The reaction was pres-
surized to 35 psi H2 and stirred at room temperature for 6 h.
The solvent was stripped to afford a crude hydrogenated
product which was puri®ed by a short silica gel column
(2% MeOH/CHCl3) to yield 0.67 g of pure hydrogenated
product. The proton NMR and mass spectral data obtained
for the product matched the structural assignments. The
proton NMR and mass spectral data obtained for all (R)
and (S) compounds were in agreement with that of their
racemic congeners.
(S)-(1)-3-Benzoylamino-6,7-dimethoxy-1,2,3,4-tetra-
hydroquinoline [(S)-(1)-7]. The experimental procedure
followed was the same as that described for 7; [a]2D5ˆ136
(c 1.7, CH2Cl2).
(S)-(1)-3-Amino-6,7-dimethoxy-1,2,3,4-tetrahydroquino-
line [(S)-(1)-8]. The experimental procedure followed the
same was the same as that described for 8; [a]2D5ˆ130 (c
0.8, CH2Cl2).
(S)-(2)-3-Amino-6,7-dihydroxy-1,2,3,4-tetrahydroquino-
line [(S)-(2)-9]. The experimental procedure followed was
the same as that described for 9; [a]2D5ˆ26 (c 0.9, H2O).
(R)-(2)-Methyl-2-benzoylamino-3-(3,4-dimethoxyphenyl)-
propanoate [(R)-(2)-4]. Compound (R)-(2)-4 was
prepared following the procedure described above for
enantioselective hydrogenations. The rhodium catalyst
used was [(COD)Rh-(1,2-bis-((2R, 5R)-dimethylphospho-
lano)-benezene)]1 OTf2. The measured optical rotation
was [a]2D5ˆ279(c 1.35, CH2Cl2).
4-(3,5-Dimethoxybenzylidene)-2-phenyl-5-oxazolone (11).
3,5-dimethoxybenzaldehyde (17 g, 102 mmol), hippuric
acid (19.25 g, 107 mmol), anhydrous sodium acetate
(8.39 g, 102 mmol) and acetic anhydride (50 mL,
530 mmol) were combined and heated at 858C with con-
tinuous stirring for 2.5 h. During the reaction, the color of
the mixture turned from white to a bright yellow. The reac-
tion mixture was cooled to room temperature, cold ethanol
(65 mL) was added, and the resultant yellow crystalline
product was further cooled, then ®ltered with suction. The
product was washed with cold ethanol (2£25 mL), boiling
water (3£20 mL) and ice-cold ether (5 mL). The yield was
(R)-(1)-Methyl-2-benzoylamino-3-(3,4-dimethoxy-6-
nitrophenyl)-propanoate [(R)-(1)-5]. The experimental
procedure followed was the same as that described for 5;
[a]2D5ˆ169 (c 1.0, CH2Cl2).
(R)-(2)-3-Benzoylamino-6,7-dimethoxy-1,2,3,4-tetra-
hydro-2-oxoquinoline [(R)-(2)-6]. The experimental
procedure followed was the same as that described for 6;
[a]2D5ˆ238 (c 1.5, CH2Cl2).
1
86%. H NMR (CDCl3): dˆ8.14 (bd, Jappˆ6.9 Hz, 2H),
7.63±7.49 (m, 3H), 7.40 (d, Jˆ2.4 Hz, 2H), 7.15 (s, 1H),
6.65 (t, Jˆ2.4 Hz, 1H), 3.87 (s, 6H).
Methyl-2-benzoylamino-3-(3,5-dimethoxyphenyl)-2-pro-
penoate (12). Compound 11 (26.3 g, 85 mmol), anhydrous
sodium carbonate (26.82 g, 255 mmol), and dry methanol
(263 mL) were re¯uxed for 45 min, during which time the
yellow color of the reaction mixture disappeared. The reac-
tion mixture was ®ltered hot, and the solid was washed with
hot methanol (2£15 mL). The collected ®ltrate was cooled
in the refrigerator for at least 3 h. The white crystalline
product which formed was collected by ®ltration and
washed with 1:1 methanol/water (2£10 mL). The yield
(R)-(2)-3-Benzoylamino-6,7-dimethoxy-1,2,3,4-tetra-
hydroquinoline [(R)-(2)-7]. The experimental procedure
followed was the same as that described for 7; [a]2D5ˆ230
(c 1.3, CH2Cl2).
(R)-(2)-3-Amino-6,7-dimethoxy-1,2,3,4-tetrahydro-
quinoline [(R)-(2)-8]. The experimental procedure
followed was the same as that described for 8; [a]2D5ˆ226
(c 0.5, CH2Cl2).
1
was 88%. H NMR (CDCl3): dˆ7.85 (bd, Jappˆ7.2 Hz,
(R)-(1)-3-Amino-6,7-dihydroxy-1,2,3,4-tetrahydro-
quinoline [(R)-(1)-9]. The experimental procedure
followed was the same as that described for 9; [a]2D5ˆ116
(c 0.9, H2O).
2H), 7.7 (bs, 1H), 7.56±7.43 (m,3H), 7.36 (s, 1H), 6.64
(d, Jˆ2.1 Hz, 2H), 6.40 (t, Jˆ2.1 Hz, 1H), 3.85 (s,3H),
3.65 (s, 6H).
Methyl-2-benzoylamino-3-(3,5-dimethoxyphenyl)pro-
panoate (13). Compound 12 (9.89 g, 29 mmol) was
dissolved in hot THF (55 mL, dry). To this solution was
added dry methanol (55 mL), 10% palladium on carbon
(S)-(1)-Methyl-2-benzoylamino-3-(3,4-dimethoxyphenyl)-
propanoate [(S)-(1)-4]. (S)-(1)-4 was prepared following
the procedure described above for enantioselective hydro-
genations. The rhodium catalyst used for this conversion

R. J. Lewis et al. / Tetrahedron 56 (2000) 5345±5352
5351
(1.50 g) and glacial acetic acid (0.75 mL). The hydrogena-
tion was carried out at 60 psi H2 for 10 h in a Parr shaker
apparatus. The solution was ®ltered through Celite. The
Celite was washed with hot methanol/chloroform (1:3,
3£10 mL), and the solvent was evaporated, providing a
white solid in quantitative yield. 1H NMR (CDCl3):
dˆ7.73 (d, Jˆ7.8 Hz, 2H), 7.52±7.38 (m, 3H), 6.59 (d,
Jˆ7.2 Hz, 1H), 6.33 (d, Jˆ2.4 Hz, 1H), 6.26 (d,
Jˆ2.4 Hz, 2H), 5.06 (dd, Jˆ12.9 and 5.4 Hz, 1H), 3.77 (s,
3H), 3.69 (s, 6H), 3.25±3.12 (m, 2H).
3-Amino-6,8-dihydroxy-1,2,3,4-tetrahydroquinoline
hydrobromide (18). The experimental procedure followed
was the same as described for 9. Compound 17 was re¯uxed
for 26 h, providing a tan solid with a yield of 86%. 1H NMR
(D2O): dˆ6.38 (d, Jˆ2.7 Hz, 0.5H, part, exch.), 6.3 (d,
Jˆ2.4 Hz, 1H), 3.98±3.88 (m, 1H) [alternatively, this multi-
plet could be reported as 3.93 (dddd, Jˆ9.9, 9.6, 5.3 and
3.5 Hz, 1H)], 3.83 (ddd, Jˆ12.3, 3.5 and 1.7 Hz, 1H) 3.42
(dd, Jˆ12.3 and 10.2 Hz, 1H), 3.23(bdd, Jappˆ16.8 and
5.4 Hz, 1H), 2.94 (dd, Jˆ16.8 and 9.3 Hz, 1H). MS
(70 eV-DIP) m/z (relative intensity) 180 (M1, 100), 164
(22), 82 (53), 80 (55).
Methyl-2-benzoylamino-3-(3,5-dimethoxy-6-nitrophenyl)-
propanoate (14). Compound 13 (3.41 g, 9.9 mmol) was
dissolved in glacial acetic acid (136 mL), and purged with
N2. To this solution was added, dropwise, 70.4% nitric acid
(2.25 mL, 39.6 mmol) mixed with glacial acetic acid
(4 mL). The reaction was stirred at 158C for 24 h. The
reaction mixture was neutralized with a sodium hydroxide
solution to a neutral pH. Chloroform (50 mL) was added,
the layers separated, the chloroform layer dried (Na2SO4),
and the solvent evaporated to give a blackish product which
was puri®ed by column chromatography (SiO2, 75:25
CHCl3/EtOAc, 40 g SiO2/g crude). Yield of the pure
Dimethyl-3,4-dimethoxybenzylidenemalonate (20). Pre-
pared as previously described.12,13
Dimethyl-3,4-dimethoxybenzylmalonate (21). Prepared as
previously described.12,13
Dimethyl-3,4-dimethoxy-6-nitrobenzylmalonate
(22).
Experimental procedures employed to synthesize 5 were
followed. Fuming nitric acid (16.3 mL, 354 mmol) was
added dropwise to a solution of dimethyl-3,4-dimethoxy-
benzylmalonate 21 (16.6 g, 58.9 mmol) dissolved in chloro-
form (160 mL) at 08C with a nitrogen atmosphere. The
reaction was stirred at 08C for 4 h. The reaction mixture
was neutralized and washed with a saturated sodium
hydroxide solution followed by water. The organic layer
was dried (Na2SO4), and solvent stripped to give a yellow
1
nitrated product, which was off-white, was 19%. H NMR
(CDCl3): dˆ7.78 (bd, Jappˆ6.9 Hz, 2H), 7.51±7.38 (m, 3H),
7.1 (d, Jˆ7.0 Hz, 1H), 6.43 (d, Jˆ2.4 Hz, 1H), 6.39 (d,
Jˆ2.4 Hz, 1H), 4.98 (ddd, Jˆ9.3 and 7.1 and 5.1 Hz, 1H),
3.83 (s, 3H), 3.77 (s, 3H), 3.76 (s, 3H), 3.23 (dd, Jˆ14.4 and
5.1 Hz, 1H), 3.03 (dd, Jˆ14.4 and 9.3 Hz, 1H).
1
solid in quantitative yield. H NMR (CDCl3): dˆ7.63 (d,
3-Benzoylamino-6,8-dimethoxy-1,2,3,4-tetrahydro-2-oxo-
quinoline (15). The experimental procedure for 6 was
followed with the exception of the solvent and the reaction
time. The nitro compound 14 (4.25 g, 10.9 mmol) was
dissolved in a hot mixture of dry methanol (140 mL), dry
THF (6 mL), 1,4-dioxane (120 mL) and reacted for 48 h.
This resulted in a yield of 90%. 1H NMR (CDCl3):
dˆ7.86 (bd, Jappˆ6.9 Hz, 2H), 7.71 (bs,1H), 7.54±7.41
(m, 3H), 7.33 (d, Jˆ4.3 Hz, 1H), 6.39 (d, Jˆ2.7 Hz, 1H),
6.37 (d, Jˆ2.1 Hz, 1H), 4.66 (ddd, Jˆ14.1 and 6.3 and
4.4 Hz, 1H), 3.84 (s, 3H), 3.78 (s, 3H), 3.69 (dd, Jˆ15.3
and 6.3 Hz, 1H), 2.83 (dd, Jˆ15.3 and 14.1 Hz, 1H).
Jˆ2.1 Hz, 1H), 6.78 (d, Jˆ2.1 Hz, 1H), 3.96±3.93 (m, 1H),
3.92 (s, 6H), 3.69 (s, 6H), 3.49 (dd, Jˆ7.5 and 2.1 Hz, 2H).
3-Methylester-6,7-dimethoxy-1,2,3,4-tetrahydro-2-oxo-
quinoline (23). The experimental procedure followed was
the same as described for 6 with slight modi®cation. The
nitrophenyl derivative 22 (1.2 g, 3.67 mmol) was dissolved
in a mixture of warm THF (15 mL, dry) and methanol
(60 mL, dry), then 10% Pd/C and glacial acetic acid
(0.6 mL) were added. The white precipitate, collected by
suction ®ltration, was pure product and was obtained in a
72% yield. 1H NMR (CDCl3): dˆ7.69 (bs, 1H), 6.68 (s, 1H),
6.31 (s, 1H), 3.83 (s, 6H), 3.74 (s, 3H), 3.60 (dd, Jˆ8.4 and
6.3 Hz, 1H), 3.30 (dd, Jˆ15.9 and 8.4 Hz, 1H), 3.05 (dd,
Jˆ15.9 and 6.3 Hz, 1H). MS (70 eV-DIP) m/z (relative
intensity) 265 (M1, 21), 206 (100). MS (FAB) m/z (relative
intensity) 266 (M11, 100), 265 (98), 206 (54).
3-Benzylamino-6,8-dimethoxy-1,2,3,4-tetrahydroquino-
line (16). The experimental procedure followed was the
same as that described for 7. Reaction time was 3 h. The
yield of 16 obtained was 90%. 1H NMR (CDCl3): dˆ7.38±
7.22 (m, 5H), 6.28 (d, Jˆ2.4 Hz, 1H), 6.17 (d, Jˆ2.4 Hz,
1H), 3.89 (s, 2H), 3.78 (s, 3H), 3.72 (s, 3H), 3.36 (bt,
Jappˆ7.2 Hz, 1H), 3.15±3.08 (m, 2H), 2.95 (dd, Jˆ16.5
and 3.9 Hz, 1H), 2.69 (dd, Jˆ16.5 and 7.5 Hz, 1H). MS
(70 eV-DIP) m/z (relative intensity) 298 (M1, 100), 192 (88).
3-Carboxamide-6,7-dimethoxy-1,2,3,4-tetrahydro-2-oxo-
quinoline (24). To a pressure bottle was added the methyl-
ester derivative 23 (200 mg, 0.755 mmol), methanol (8 mL)
and THF (8 mL). This mixture was heated to dissolve the
ester, then cooled to 08C and degassed with argon. To this
08C solution was added ammonium hydroxide (4 mL), then
saturated with ammonia gas, and the pressure bottle sealed
and heated to 658C for 22 h. A white precipitate began to
form as the reaction proceeded. The solution was cooled to
room temperature and the precipitate was collected by
suction ®ltration. Any further puri®cation is not recom-
mended due to the instability of the product. A yield of
3-Amino-6,8-dimethoxy-1,2,3,4-tetrahydroquinoline (17).
The experimental procedure followed was the same as
described for 6 with slight modi®cation. A reaction time
of 98 h in a Parr shaker apparatus (65 psi H2) was employed.
The product was obtained in 83% yield. 1H NMR (CDCl3):
dˆ6.28 (d, Jˆ2.4 Hz, 1H), 6.16 (d, Jˆ2.7 Hz, 1H), 3.78 (s,
3H), 3.71 (s, 3H), 3.39±3.32 (m, 1H), 3.30 (bd,
Jappˆ11.7 Hz, 1H), 3.06±2.95 (m, 2H), 2.46 (dd, Jˆ16.2
and 6.3 Hz, 1H). MS (70 eV-DIP) m/z (relative intensity)
208 (M1, 100), 193 (34), 192 (23).
1
24% was obtained. H NMR (DMSO-d6): dˆ7.43 (s, 1H),
7.07 (s, 1H), 6.81 (s, 1H), 6.49 (s, 1H), 3.69 (s, 3H), 3.67 (s,
3H), 3.28 (dd, Jˆ9.9 and 6.6 Hz, 1H), 3.08 (dd, Jˆ15.9 and

5352
R. J. Lewis et al. / Tetrahedron 56 (2000) 5345±5352
Related Catecholaminergic Neurotoxins: Molecular Mechanisms.
In Highly Selective Neurotoxins: Basic and Clinical Applications;
Kostrzewa, R. M., Ed.; Humana Press: Totowa, NJ, 1998, pp 1±8.
2. Lew, R.; Malberg, J. E.; Ricuarte, G. A.; Seiden, L. S. Evidence
For and Mechanism of Action of Neurotoxicity of Amphetamine
Related Compounds. Highly Selective Neurotoxins: Basic and
Clinical Applications; Kostrzewa, R. M., Ed.; Humana Press:
Totowa, NJ, 1998, pp 235±268.
9.9 Hz, 1H), 2.88 (dd, Jˆ15.9 and 6.6 Hz, 1H). MS (70 eV-
DIP) m/z (relative intensity) 250 (M1, 32), 206 (100). MS
(12 eV-DIP) m/z (relative intensity) 250 (M1, 30), 206
(100). MS (FAB) m/z (relative intensity) 251 (M11, 100),
250 (63), 206 (81).
3-Aminomethyl-6,7-dimethoxy-1,2,3,4-tetrahydroquino-
line (25). The experimental procedure followed was the
same as described for 7 with slight modi®cation. The
carboxamide compound 24 (100 mg, 0.4 mmol) and BH3/
THF (1 M, 9.6 mL, 9.6 mmol) was heated at re¯ux for 3.5 h
with continuous stirring. This reaction gave a pure product
with a yield of 88%. 1H NMR (CDCl3): dˆ6.50 (s, 1H), 6.09
(s, 1H), 3.76 (s, 6H), 3.33 (dd, Jˆ10.8 and 3.3 Hz, 1H), 2.94
(dd, Jˆ10.8 and 8.7 Hz, 1H), 2.76 (dd, Jˆ15.9 and 5.1 Hz,
1H), 2.69 (dd, Jˆ7.2 and 2.4 Hz, 2H), 2.39 (dd, Jˆ15.9 and
8.7 Hz, 1H), 2.00±1.86 (m, 1H). MS (70 eV-DIP) m/z (rela-
tive intensity) 222 (M1, 100), 192 (12), 190 (70).
3. Royland, J. E.; Langston, J. W. MPTP: a Dopaminergic Neuro-
toxin. In Highly Selective Neurotoxins: Basic and Clinical Appli-
cations; Kostrzewa, R. M., Ed.; Humana Press: Totowa, NJ, 1998,
pp 141±194.
4. Bloem, B. R.; Irwin, I.; Buruma, O. J. S.; Haan, J.; Roos, R. A.
C.; Tetrud, J. W.; Langston, J. W. J. Neurol. Sci. 1990, 97, 273±
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5. Youdim, M. B. H.; Lavie, L. Life Sci. 1994, 55, 2077±2082.
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230±237.
7. Jones, G. Quinolines. Part I. In The Chemistry of Heterocyclic
Compounds, Jones, G., Ed.; Wiley: New York, 1997; Vol. 32,
pp 207±220.
3-Aminomethyl-6,7-dihydroxy-1,2,3,4-tetrahydroquino-
line hydrobromide (26). The experimental procedure
followed was the same as that described for 9. A light tan
solid was isolated for this reaction in a 74% yield. 1H NMR
(D2O); dˆ6.79 (s, 1H), 6.75 (s, 0.5H, part. exch.), 3.67 (bd,
Jappˆ11.1, 1H), 3.19±3.03 (m, 3H), 2.94 (dd, Jˆ15.6 and
3.9 Hz, 1H), 2.59 (dd, Jˆ15.6 and 10.8 Hz, 1H), 2.54±2.44
(m, 1H). MS (12 eV-DIP) m/z (relative intensity) 194 (M1,
76), 176 (26).
8. Narang, K. S.; Ray, J. N.; Sachdeva, T. D. J. Indian Chem. Soc.
1936, 13, 260±266.
9. Burk, M. J. J. Am. Chem. Soc. 1991, 113, 8518±8519.
10. Burk, M. J. Chem. Abstr. 1993, 118, 25118s.
11. O'Reilly, N. J.; Derwin, W. S.; Lin, H. C. Synthesis 1990, 7,
550±556.
12. Buckley III, T. F.; Rapoport, H. J. Am. Chem. Soc. 1980, 102,
3056±3062.
Mouse brain neurotransmiter levels
13. Irie, H.; Shiina, A.; Fushimi, T.; Katakawa, J.; Fujii, N.;
Yajima, H. Chem. Lett. 1980, 1980, 875±878.
ICR:Hsd male mice, weighing approximately 30 g at the
time of experiment, from Harlan Sprague-Dawley
(Madison, WI) were employed. Animals had access to
food and water ad libitum, and a minimum of one week
was allowed between receipt of animals and commence-
ment of the depletion studies. To observe the maximal
depletion capabilities for each tested toxin, we employed
dosages which provided approximately 50% survival rates
for the tested animals. Bilateral intracerebroventricular
injections (3.5 mL total volume, isotonic saline containing
the test compound) were performed under a light ether
anesthesia.17 Control animals were injected with the same
volume of isotonic saline. Animals were sacri®ced seven
days following injection by microwave irradiation (7 kW,
150 ms) using a NJE model 2603±10 kW (New Japan
Radio, Tokyo, Japan).18,19 Brains were removed and
prepared for analysis as previously described.20 Brains
weights ranged from 450±525 mg. Transmitter levels
were determined utilizing liquid chromatography with
electrochemical detection.20,21 Typical control values for
the neurotransmitters determined were (nmol/g, mean^
SEM): NE, 2.98^0.08; DA, 8.19^0.2; and 5-HT,
4.5^0.06.
14. Ma, S.; Lin, L.; Rhagavan, R.; Cohenour, P.; Lin, P. Y. T.;
Bennet, J.; Lewis, R. J.; Kostrzewa, R.; Lehr, R. E.; Blank, C. L.
J. Med. Chem. 1995, 38, 4087±4097.
15. Baumgarten, H. G.; Bjorklund, A.; Lachenmayer, L.; Nobin,
A.; Stenevi, U. Acta Physiol. Scand. Suppl. 1971, 373, 1±15.
16. Herman, Z. S.; Bonczek, A. Pharmacology 1978, 17, 8±14.
17. Blank, C. L.; Murrill, E.; Adams, R. N. Brain Res. 1972, 45,
635±637.
18. Blank, C. L.; Sasa, S.; Isernhagen, R.; Meyerson, L. R.;
Wassil, D.; Wong, P.; Modak, A. T.; Stavinoha, W. B. J. Neuro-
chem. 1979, 33, 213±219.
19. Blank, C. L.; Abdullah, H.; Modak, A. T.; Stavinoha, W. B.
Microwave Heating and Serotonin Determinations. In Microwave
Irradiation as a Tool to Study Labile Metabolites in Tissue;
Blank, C. L., Stavinoha, W. B., Maruyama, Y., Eds.; Pergamon:
London, 1983, pp 63±73.
20. Freeman, K.; Lin, P. Y. T.; Lin, L.; Blank, C. L. Monoamines
and Metabolites in the Brain. In High Performance Liquid
Chromatography in the Neurosciences, Monograph in the Inter-
national Brain Research Organisation (IBRO) Handbook Series,
Holman, R. B., Joseph, M. H., Cross, A. J., Eds.; Wiley: London,
1993, pp 27±55.
21. Lin, P. Y. T.; Bulawa, M. C.; Wong, P.; Lin, L.; Scott, J.;
Blank, C. L. J. Liq. Chromatogr. 1984, 7, 509±538.
References
1. Blank, C. L.; Lewis, R. J.; Lehr, R. E. 6-Hydroxydopamine and