Bromal-derived tetrahydro-β-carbolines as neurotoxic agents: Chemistry, impairment of the dopamine metabolism, and inhibitory effects on mitochondrial respiration

Article abstract of DOI:10.1016/S0968-0896(00)00073-0

The mammalian alkaloids tryptoline (1) and eleagnine (2) as well as the highly halogenated (X=F, Cl, Br) tetrahydro-β-carbolines (THβCs) 3-5, structurally similar to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 6), were found to have a common feature of inducing a severe impairment of the nigrostriatal dopamine metabolism and inhibiting complex I of the mitochondrial respiratory chain highly selectively. Within the series of compounds tested, 1-tribromomethyl-1,2,3,4-tetrahydro-β-carboline ('TaBro', 5), which was prepared in high yields from the biogenic amine tryptamine ('Ta', 7) and the unnatural aldehyde bromal ('Bro', 8) by a Pictet-Spengler cyclization reaction, turned out to be the most potent toxin in vitro and in vivo. As demonstrated by voltammetric measurements on rats, for all the THβCs 1-5 investigated, intranigral application of a single dose of 10μg resulted in a significant reduction of the dopaminergic activity in the striatum, with the strongest effect being observed for TaBro (5). Using rat brain homogenates, again 5 (IC₅₀=200μM) as well as its dehydrohalogenation product 11 (IC₅₀=150μM) exhibited the most pronounced inhibitory potential on mitochondrial respiration. The halogen-free THβCs 1 and 2 as well as the MPTP metabolite 1-methyl-4-phenylpyridinium ion (MPP⁺), by contrast, showed only a moderate inhibition at concentrations in the millimolar range (e.g. for MPP⁺: IC₅₀=3.5mM). For an elucidation of the role of hydrophobic portion in the inhibitory action against complex I activity, several N-acyl derivatives (15-21) of 5 were synthesized and tested. An X-ray diffraction study on the 3-dimensional structure of trifluoroacetylated highly halogenated THβCs (12-14) revealed the tetrahydropyrido part to adopt a nearly planarized half-chair conformation. Because of the steric demand of the trihalogenmethyl moiety (CF₃33), the N-substituent is dramatically pushed out of that ring 'plane'. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00073-0

Bioorganic & Medicinal Chemistry 8 (2000) 1467±1478
Bromal-derived Tetrahydro-ꢀ-carbolines as Neurotoxic Agents:
Chemistry, Impairment of the Dopamine Metabolism, and
y
Inhibitory E€ects on Mitochondrial Respiration
a,
a
a
a
Gerhard Bringmann, * Doris Feineis, Ralph Bru
È
ckner, Michael Blank,
c
b
b
c
d
Karl Peters, Eva-Maria Peters, Heinz Reichmann, Bernd Janetzky, Christoph Grote,
d
d
Hans-Willi Clement and Wolfgang Wesemann
a
Institut fuÈr Organische Chemie, UniversitaÈt WuÈrzburg, Am Hubland, D-97074 WuÈrzburg, Germany
Max-Planck-Institut fuÈr FestkoÈrperforschung, Heisenbergstraûe 1, D-70506 Stuttgart, Germany
b
c
UniversitaÈts-Klinikum Carl Gustav Carus, Klinik und Poliklinik fuÈr Neurologie, Technische UniversitaÈt Dresden,
Fetscherstraûe 74, D-01307 Dresden, Germany
d
Institut fuÈr Physiologische Chemie, Abteilung Neurochemie, Philipps-UniversitaÈt Marburg, Hans-Meerwein-Straûe,
D-35033 Marburg, Germany
Received 14 September 1999; accepted 25 February 2000
AbstractÐThe mammalian alkaloids tryptoline (1) and eleagnine (2) as well as the highly halogenated (X=F, Cl, Br) tetrahydro-
b-carbolines (THbCs) 3±5, structurally similar to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP, 6), were found to have a common feature of inducing a severe impairment of the nigrostriatal dopamine metabolism and
inhibiting complex I of the mitochondrial respiratory chain highly selectively. Within the series of compounds tested, 1-tri-
bromomethyl-1,2,3,4-tetrahydro-b-carboline (`TaBro', 5), which was prepared in high yields from the biogenic amine tryptamine
(
`Ta', 7) and the unnatural aldehyde bromal (`Bro', 8) by a Pictet±Spengler cyclization reaction, turned out to be the most potent
toxin in vitro and in vivo. As demonstrated by voltammetric measurements on rats, for all the THbCs 1±5 investigated, intranigral
application of a single dose of 10 mg resulted in a signi®cant reduction of the dopaminergic activity in the striatum, with the stron-
gest e€ect being observed for TaBro (5). Using rat brain homogenates, again 5 (IC50=200 mM) as well as its dehydrohalogenation
product 11 (IC50=150 mM) exhibited the most pronounced inhibitory potential on mitochondrial respiration. The halogen-free
+
THbCs 1 and 2 as well as the MPTP metabolite 1-methyl-4-phenylpyridinium ion (MPP ), by contrast, showed only a moderate
+
inhibition at concentrations in the millimolar range (e.g. for MPP : IC =3.5 mM). For an elucidation of the role of hydrophobic
50
portion in the inhibitory action against complex I activity, several N-acyl derivatives (15±21) of 5 were synthesized and tested. An
X-ray di€raction study on the 3-dimensional structure of tri¯uoroacetylated highly halogenated THbCs (12±14) revealed the tet-
rahydropyrido part to adopt a nearly planarized half-chair conformation. Because of the steric demand of the trihalogenmethyl
3 3 3
moiety (CF <CCl <CBr ), the N-substituent is dramatically pushed out of that ring `plane'. # 2000 Elsevier Science Ltd. All
rights reserved.
Introduction
The b-carbolines are an interesting class of psychoactive
induction of Parkinsonian-type symptoms in rodents
and primates. The identi®cation of some tetrahydro-
3,6,7
b-carbolines (THbCs) in the human body,
among
1�3
compounds,
with a structural resemblance to the
them the simple formaldehyde- and acetaldehyde-derived
compounds tryptoline (1) and eleagnine (2), respec-
tively, has stimulated increased attention on the
putative role of these alkaloid-type heterocycles as caus-
ative factors in the pathogenesis of neurodegenerative
disorders.8
molecular framework of the well-known dopaminergic
neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
4
,5
(MPTP, 6) (Fig. 1), which has become an ecient and
widely used experimental tool in neurosciences for the
,9
*
4
Corresponding author. Tel.: +49-931-888-5323; fax: +49-931-888-
755; e-mail: bringman@chemie.uni-wuerzburg.de
We have recently reported the highly lipophilic 1-tri-
chloromethyl-1,2,3,4-tetrahydro-b-carboline (`TaClo',
y
Part 32 in the series, Endogenous Alkaloids in Man; for part 31, see
ref 13.
10�12
4)
(Fig. 1) to be formed in small amounts in elderly
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00073-0

1
468
G. Bringmann et al. / Bioorg. Med. Chem. 8 (2000) 1467±1478
Figure 1. The well-established mammalian alkaloids 1 and 2 as well as the highly halogenated tetrahydro-b-carbolines 3±5 in comparison to the
structurally closely related dopaminergic neurotoxin MPTP (6). We have coined the acronyms `TaFlu' (3), `TaClo' (4) and `TaBro' (5) for this new
THbCs because they chemically originate from tryptamine (`Ta') and the aldehydes ¯uoral (`Flu'), chloral (`Clo'), and bromal (`Bro'), respectively.
patients therapeutically treated with the hypnotic agent,
have started to determine the in¯uence of ¯uorine versus
chlorine versus bromine on the neuropharmacological
properties of 1-trihalogenmethyl-THbCs in comparison
to the halogen-free mammalian alkaloids tryptoline (1)
and eleagnine (2) (Fig. 1). In the present study, the
tryptamine-derived heterocycles `TaFlu' (3), `TaClo' (4),
and `TaBro' (5) have been evaluated for dopaminergic
neurotoxicity in vivo, and for respiratory inhibition
in vitro. Furthermore, we report on the synthesis and
X-ray di€raction analysis of various highly halogenated
THbCs, among them 5 and related N-acylated com-
pounds (15±21), which were tested for their inhibitory
e€ects on complex I activity.
1
3
chloral hydrate. This ®nding is of special importance
because 4 was shown to exhibit a pronounced neuro-
toxicity towards dopaminergic neurons.11,12,14 The toxic
potency of TaClo (4), however, seems to be di€erent
from that of MPTP (6) or its active metabolite
+
1-methyl-4-phenylpyridinium ion (MPP ): While
MPTP acutely produces a severe and irreversible syn-
drome similar to Parkinsonism,15 TaClo is capable of
inducing a slowly developing dopaminergic denervation
on rats resulting in a Parkinsonian-type diminished
locomotion several months after the end of a 7 week
1
6,17
treatment.
This chloral-derived THbC thus appears
to be a promising model toxin to study the various
stages of a slowly progressing neurodegeneration as
typical for idiopathic Parkinson's disease. The neuro-
toxic potential of 4 was con®rmed by in vivo pulse vol-
tammetric analysis of the nigrostriatal dopamine
Results and Discussion
Synthesis
1
8
metabolism, and by histological as well as biochemical
1
9
studies on primary mesencephalic cell cultures: these
investigations clearly revealed 4 to cause marked dis-
turbances on dopaminergic activity, and to be respon-
sible for morphological changes in neurons such as
swelling of axons or loss of dendrites.20 Furthermore, 4
and related highly chlorinated analogues were found to
be potent selective inhibitors of complex I (NADH-ubi-
quinone reductase) of the mitochondrial respiratory
chain, thus giving rise to a signi®cant decrease of neu-
ronal energy production.1
The Pictet±Spengler cyclization of tryptamines and
reactive aldehydes or a-keto acids is an ecient syn-
3
,23
thetic pathway for the preparation of THbCs,
among
them the alkaloids tryptoline (1)24 and eleagnine (2)
as well as related heterocycles. Within the series of
highly halogenated THbCs, on which we mainly focus
in this paper, the ¯uoral-derived compound TaFlu (3)
25,26
2
7
has already been synthesized by Maki et al., while
the 1-trichloromethyl substituted THbC TaClo (4) has
been introduced by our group. We have established a
9,21,22
10
rational two-step route for the preparation of 4 via its
formamide, giving rise to analytically completely pure,
In a previous paper,18 we have also described the merely
synthetic TaClo analogue, 1-tribromomethyl-1,2,3,4-
tetrahydro-b-carboline (`TaBro', 5) to severely a€ect the
dopamine metabolism of the rat after its unilateral
injection into the substantia nigra pars compacta (SNpc).
Interestingly, the neurotoxic e€ect was observed to be
even more pronounced for this certainly unnatural bro-
mal-derived heterocycle 5 compared with the endoge-
nously occurring agent 4.
crystalline material of 4, even on a 100 g scale.1
1,22
The
novel highly brominated TaClo analogue TaBro (5) has
now been successfully synthesized (see Scheme 1) by an
adaption of this procedure: At room temperature, tryp-
tamine (7) was condensed with tribromoacetaldehyde
(bromal, 8) in formic acid as the solvent, in the presence
of the formylating reagent trimethylacetic formic anhy-
dride,28 leading to N-formyl-TaBro (9). A smooth N-
deformylation of 9 in methanolic hydrochloric acid
®nally gave the desired target molecule TaBro (5) as its
colorless hydrochloride salt in an overall yield of 46%,
without formation of any by-products. Di€erent from
the corresponding chloral-derived THbC 4, which was
shown to be formed rapidly (e.g. in re¯uxing toluene)
within 2 h from both of its precursors tryptamine and
chloral,10 all attempts to obtain 5 directly by carrying
out the cyclization reaction of 7 and 8 in aprotic solvents
Since a comprehensive structure-neurotoxicity study on
highly halogenated THbCs has not been performed so
far, we ®rst aimed at a more detailed investigation
whether the highly lipophilic (and possibly radical-
inducing) trichloromethyl and tribromomethyl sub-
stitutents at C1 might be associated with the toxic
potential of TaClo (4) and TaBro (5), respectively,
towards the dopaminergic system. For this purpose, we

G. Bringmann et al. / Bioorg. Med. Chem. 8 (2000) 1467±1478
1469
ring plane, occupying a pseudo-axial position. The three
halogen atoms at C14 of all of these THbCs show
a perfectly staggered orientation with respect to the C1±
C14 bond, thus minimizing their steric interactions with
C13 and N2.
The in¯uence of the large van der Waals radius of bro-
Ê
mine (2.0 A) or chlorine (1.8 A) in comparison to the
Ê
Ê
Ê
small radius of ¯uorine (1.4 A) or hydrogen (1.0 A) on
the conformation of the tetrahydropyrido moiety is best
visualized by a matched plot (Fig. 3) of the crystal
structures of 12±14 (and of the eleagnine-derived tri-
¯
uoroacetamide, on which we have reported pre-
29
viously ). Depending on the degree of steric hindrance
due to the steric demand of the respective trihalogen-
methyl group (CF <CCl <CBr ) at C1, the substituent
Scheme 1. Bromal-derival tetrahydro-b-carbolines.
3
3
3
at N2 is increasingly pushed upwards: slightly for the
halogen-free 1-CH analogue, and ®nally dramatically
(
e.g. in acetone, tetrahydrofurane, dichloromethane,
3
toluene or formic acid without addition of trimethyla-
cetic formic anhydride) resulted in less favorable yields
or failed completely.
for the N-tri¯uoroacetyl moiety of the TaClo and
TaBro derivatives 13 and 14.
Striatal dopamine metabolism in the rat after intranigral
application of the THꢀCs 1±5
Following a similar reaction sequence (see Scheme 2),
the 1-dibromomethylene-substituted THbC 11 was also
synthesized via its formamide: Thus, the intermediate 10
was obtained from 9 in methanol at re¯ux by elim-
ination of hydrobromic acid under mild basic condi-
tions. Here as well, similar to the preparation of 5, the
attempt to synthesize 11 directly, by performing the
dehydrohalogenation reaction with 5 as the starting
material, failed completely, resulting in a multi-compo-
nent mixture.
With the THbCs 1±5 in hand, we could now start
investigating their neurotoxic potential. Di€erential
pulse voltammetry using carbon ®bre electrodes repre-
sents a very sensitive tool for the in vivo investigation of
a neurotoxin-induced impairment of the dopaminergic
neurotransmission in rat striatum, as previously demon-
strated for iron,30 MPTP, and its metabolite MPP .
The ability of the THbCs 1±5 to disturb neuronal
activity in the dopaminergic system (Fig. 4) was deter-
mined by assaying in both striata for the extracellular
levels of the dopamine metabolite 3,4-dihydroxy-phe-
nylacetic acid (DOPAC), a reliable indicator of dopa-
mine metabolism.
+ 31
X-Ray investigations
In addition to the constitutions of these novel agents, as
anticipated from syntheses and spectroscopic data, the
knowledge of steric e€ects exhibited by a trihalogen-
methyl group at C1 on the conformation of a tetra-
hydro-b-carboline ring system was highly desirable.
Acetylation of 3±5 with tri¯uoroacetic anhydride pro-
vided crystalline material of the respective derivatives
As reported previously,18 4 and 5 are able to severely
a€ect the striatal dopaminergic functions. TaClo (4) was
measured to signi®cantly reduce the DOPAC signal at
the lesioned side to about 43% (SEMÆ10%) of the
intact side (P<0.05) 1 week after a single unilateral
toxin injection into the right SNpc. The biochemical
lesions of the nigrostriatal system found for TaBro (5)
were distinctly stronger: the DOPAC content in the
ipsilateral striatum decreased to ca. 17% (SEMÆ5.6%)
of the contralateral, i.e. intact side (P<0.01). A systemic
administration of the dopamine precursor l-3,4-di-
hydroxyphenylalanine (l-DOPA), which is used for the
therapy of Parkinsonism, led to a fully restoration of
the DOPAC signal in TaClo-lesioned rats with no
di€erences observable between the striata of both
hemispheres (data not shown). By contrast, the TaBro-
induced impairment of the dopamine metabolism could
not completely be compensated by l-DOPA. The
DOPAC content remains signi®cantly reduced to about
12±14, which proved to be well-suited for single-crystal
X-ray di€raction analyses (Fig. 2a±c). These investiga-
tions clearly revealed the tetrahydropyrido part to
adopt a largely planarized half chair conformation, with
only C3 and N2 being located distinctly out of the ring
plane (best illustrated in Fig. 2c showing a horizontal
view of the molecular structure of 14). The trihalogen-
methyl substituents are twisted out of the b-carboline
4
9% (SEMÆ10%).
In our rat intranigral application protocol, the acetal-
dehyde-derived alkaloid eleagnine (2) and the 1-tri-
¯uoromethyl substituted THbC TaFlu (3) appeared to
Scheme 2. Bromal-derival tetrahydro-b-carbolines.
be about as neurotoxic as TaClo (4). Similar to 4, a

1
470
G. Bringmann et al. / Bioorg. Med. Chem. 8 (2000) 1467±1478
Figure 3. Joint plot of the structures of the eleagnine-derived N-tri-
uoroacetyl derivative (yellow) (cf. lit.29), 12 (red-brown), 13 (green),
¯
and 14 (blue) in the crystal, matched with respect to the indole part of
the molecules (hydrogen atoms omitted for reasons of clarity). The
molecules are viewed from two directions: peripendicular (top) and
horizontal (bottom) to the tetrahydropyrido ring planes.
THbC 1 exhibited an even more pronounced dopami-
nergic toxicity than 2, 3 and 4. Tryptoline (1) reduced
the dopamine metabolism to about 24% (SEMÆ8.5%)
of that of the untreated side (P<0.01). As already
observed for 4, intraperitoneal administration of
l-DOPA fully restored the DOPAC signal in tryptoline-,
eleagnine-and TaFlu-treated rats, thus showing the res-
titution capacity of the nigrostriatal system after toxin
treatment.
Summarizing, severe disturbances of the striatal dopa-
mine metabolism were shown in the rat one week after a
single unilateral injection of the THbCs 1±5 into the
SNpc, with the strongest e€ect being measured for
TaBro (5). In a quantitative sense, this highly bromi-
nated compound 5 caused nigral lesions that nearly
approached the size of the strong impairment of the
dopaminergic system produced by the Parkinsonism-
inducing model toxin MPTP and its metabolite,
Figure 2. Schakal plots of the crystal structures of the N-tri-
uoroacetyl derivatives 12 (a), 13 (b), and 14 (c) with a guide of the
atomic numbering system adopted in the X-ray investigations. The
molecule of the bromal-derived THbC 14 is viewed from two direc-
tions: peripendicular (top) and horizontal (bottom) to the tetra-
hydropyrido ring plane. All these compounds are racemic in the
¯
crystal; for presentation, only those enantiomers with the CX
X=F, Cl, Br) below the graphical plane, have been chosen, arbi-
trarily (hydrogens atoms omitted for reasons of clarity).
3
group
(
+ 31
MPP .
Inhibition of complex I of the mitochondrial respiration
unilateral injection of 2 and 3 signi®cantly (P<0.05)
decreased the voltammetric DOPAC signal in the ipsi-
lateral striatum to about 38% (SEMÆ12.0%) for 2, and to
Since the mechanism of nigrostriatal degeneration in
idiopathic Parkinson's disease as well as in MPTP-
induced Parkinsonism33 was found to be associated with
32
4
2% (SEMÆ12%) for 3. Interestingly, the 1-unsubstituted

G. Bringmann et al. / Bioorg. Med. Chem. 8 (2000) 1467±1478
1471
The concentrations of 5 and its derivatives required for
a complete inhibition of complex I (IC100) in rat brain
homogenates or in SMP were determined to be 10-fold
(
for 5) up to nearly 40-fold (e.g. for 17±19) lower than
+
that of MPP (cf. Tables 1 and 2). Nonetheless, it
should be mentioned that the high neurotoxic potential
of MPP for dopaminergic neurons resides in at least
+
two e€ects: the induction of a selective inhibition of the
mitochondrial respiration, and the energy-driven uptake
of MPP+ into intact mitochondria resulting in an
accumulation of this bioactive MPTP metabolite to high
concentrations.36 Numerous b-carboline cations related
to 1 or 2, by contrast, were all shown to have con-
siderably lower anities for this active transport system
Figure 4. THbC-induced impairment of the striatal dopamine meta-
bolism in the rat demonstrated by the reduction of the DOPAC signal
after an unilateral injection of the halogen-free compounds 1 and 2 (a),
and the 1-trihalogenmethyl THbCs 3±5 (b) into the right SNpc (dose
applied: 10 mg THbC in 2 mL of 10% ethanol). After 1 week, extra-
neuronal DOPAC concentrations (shown as percentage of intact side)
in rat striatum were assayed by in vivo pulse voltammetry (n=5±6,
mean ÆSEM; *P<0.05, **P<0.01, paired Student's t-test, lesioned
side versus intact side). As clearly shown by control experiments,
intranigral application of 2 mL of the solvent ethanol itself did not
result in an impairment of the striatal dopamine metabolism of the rat.
For further details, see Experimental.
+
37
than MPP . It can be assumed that due to the lacking
permanent positive charge (unless by reversible proto-
nation), TaClo (4) or TaBro (5) will not be actively
taken up into mitochondria. Indeed, uptake studies with
radiolabeled 4 into human neuroblastoma cells expres-
sing the dopamine transporter, revealed 4 to be trans-
38
ported through the cell membranes mainly passively.
Since the extent of lipophilicity of the THbCs presented
in this paper seems to be strongly correlated with their
inhibitory capacities against complex I activity, parti-
tion coecients P (given as log P) were determined to
assess the ability of these compounds to enter cells. As
expected, partitioning of 1-substituted THbCs into the
organic layer markedly increased starting from eleag-
nine (2) and TaFlu (3), followed by TaClo (4) and
TaBro (5) (cp. Table 1). An approximate 600- and 300-
fold increase in partitioning was observed between the
halogen-free compounds 1 and 2, respectively, and the
most lipophilic THbC 5. In the series of halogenated
THbCs, a nearly 25-and 65-fold di€erence was deter-
mined between the partition coecients of 3 to 4 and 3
to 5, respectively. Between 2 and 4 there was a two
order of magnitude increase (factor: ca. 100) of parti-
tioning into the organic layer, while only a 2.5-fold dif-
ference was found between the P values of 4 and 5.
Regarding the IC50 values of the THbCs 2±5 against
mitochondrial complex I activity in relation to their
1-octanol/water partition coecients P, it became
indeed evident that the extent of the increasing inhibi-
tory potency of 2 to 5 correlates very well with the
enhanced lipophilicity of these compounds. Thus, in
comparison to the likewise polar and only moderately
active eleagnine (2), the highly lipophilic TaClo (4) and
TaBro (5) molecules were found to be ca. 5- to 6-times
more potent inhibitors.
mitochondrial complex I de®ciency in dopaminergic
neurons, the toxic e€ects of MPTP-like substances such
as b-carboline alkaloids and their analogues have been
studied more closely with respect to their inhibitory
potential against mitochondrial respiration. Among
these compounds, only some representatives of
N-methylated fully aromatic b-carbolines derived from
acetaldehyde (e.g. N-methylharmine, N-methylharmol)
were shown to cause a selective and highly ecient
reduction of complex I activity with a magnitude of
+
inhibition quite similar to that of MPP , the oxidation
3
4,35
product of MPTP (6). The N-methyl derivatives of
norharman and harman, however, proved to be nearly
3
4
ine€ective as inhibitors. Interestingly, in a previous
2
2
study, we obtained similar results for the THbCs
tryptoline (1) and eleagnine (2), which were both
demonstrated to only moderately a€ect complex I
activity (cf. Table 1). By contrast, the chloral-derived
THbC TaClo (4) signi®cantly inhibited complex I of the
mitochondrial respiratory chain (cf. Table 1) in rat
brain homogenates and submitochondrial particles
+
(
e€ectively.
SMP), similar to MPP , but about 10 times more
2
1
As obvious from Tables 1 and 2, the by far most active
compounds within the series of THbCs presented in this
paper are the bromal-derived heterocycles. Comparable
to TaBro (5) itself, which totally inhibited complex I at
a concentration similar to that of TaClo (4), all of the
TaBro derivatives tested, among them 11 (see Scheme 2)
and the N-acyl derivatives 14±21, were shown to exhibit
pronounced inhibitory capacities towards complex I
mediated oxidative phosphorylation. The IC50 and
IC100 values of these compounds vary between 650 mM
As mentioned above, a whole series of N-acyl deriva-
tives of TaBro (5) with increasing alkyl chain length
(C ±C ) have been synthesized (cp. Table 2) in order to
1
6
evaluate the in¯uence of an additional hydrophobic
portion on the inhibitory potential of highly brominated
THbCs towards complex I. Expectedly, maximum
partitioning into cyclohexane was achieved with the
N-hexanoyl compound 21, but in comparison to the
parent compound 5, only a ca. 3-fold increase of lipo-
philicity was attained. Interestingly, with alkyl chain
lengths smaller than three carbon atoms (compounds 9,
15±17), no enhancement of lipophilicity was observed
(
IC100 of 5) and 150 mM (IC50 for 11). By contrast, like
the halogen-free heterocycles 1 and 2, the ¯uoral-
derived THbC TaFlu (3) turned out to display only a
weak inhibitory e€ect on the mitochondrial respiratory
chain.

1
472
G. Bringmann et al. / Bioorg. Med. Chem. 8 (2000) 1467±1478
Table 1. Inhibitory potential of halogen-free and highly halogenated THbCs on mitochondrial NADH-ubiquinone reductase (complex I) activity
in rat brain homogenates and SMPs, and determination of the lipophilicity of these compounds
Compound
R
log P
log P
log P
Inhibition of complex I
IC100 (mM) IC50 (mM)
cyclohexane/H
2
Oa
1-octanol/H
2
Oa
1-octanol/H
2
Ob
c
1
2
3
4
5
H
CH
Ð
Ð
0.631Æ0.039
0.913Æ0.026
Ð
Ð
IC25=1000 mM
IC25=800 mM
c
Ðc
3
1200
c
CF
CCl
CBr
3
0.985Æ0.0007
2.371Æ0.122
2.806Æ0.071
Ð
Ð
1.595Æ0.007
2.981Æ0.122
3.416Æ0.071
Ð
c
3
700
650
350
7500
250
200
150
3500
c
3
Ð
Ð
c
c
11
MPP
=CBr
Ð
2
Ð
+
c
c
Ð
Ð
Ð
aLog partition coecients experimentally determined by the HSCCC technique.
48�50
Data represents the average value ÆSEM of four separate
experiments. Using an alkane/water system instead of the 1-octanol/water mixture to measure the partititon coecients P for the halogenated THbC
representatives (see also Table 2) was necessary in order to attain reasonable retention times in relation to the standard anthracene. For further
details, see Experimental.
b
=0.61,51
For a systematic comparison of the lipophilicity of the halogen-free compounds 1 and 2 with the data obtained for 3±5, the parameter I
H
conceived as a measure of the hydrogen-bonding ability of the given solute, was used allowing the interconversion of the P values from the cyclo-
hexane/water to the 1-octanol/water system. Log 1-octanol/water partition coecients were calculated according to the equation: log Poctanol=log
P
H
cyclohexane+I .
c
Not determined.
Table 2. Inhibitory e€ects of N-acyl derivatives of the bromal-
derived THbC 5 on complex I of the mitochondrial respiration, and
partition coecients P (given as log P values) for these compounds in
a cyclohexane/water mixture
the mitochondrial respiratory chain (see section above),
or to the generation of reactive oxygen species, such as
hydroxyl radicals. Indeed, a progressive increase in
hydroxyl radical formation in the striatum was recently
3
9
shown after a systemic adminstration of TaClo to rats.
The increase of lipophilicity andÐsimultaneouslyÐ
neurotoxicity of a THbC molecule by introducing a
large trichloro- or tribromomethyl group at C1, may
explain the ability of 4 or 5 to stimulate damaging pro-
cesses on a more long-term basis presumably triggered
Compound
R
log P
Inhibition of complex I
Oa IC100 (mM) IC50 (mM)
16�18
cyclohexane/H
2
by a slow and continuous uptake of these agents.
Current research is now focussing on a more detailed
investigation concerning the role of highly halogenated
THbCs in the manifestation of a slowly developing
Parkinsonian-type neurodegeneration.
9
H
CF
CH
2.137Æ0.039
350±400
150
Ð
350
200
200
200
400
200
200
b
b
14
15
16
17
18
19
20
21
3
Ð
Ð
Ð
c
c
3
2.205Æ0.015
2.596Æ0.073
2.724Æ0.010
C
C
2
3
H
H
5
>200
>150
>150
>150
250
7
b
CH(CH
3
)
2
Ð
b
C(CH
C
3
)
3
Ð
3.112Æ0.053
4
H
9
Experimental
C
5
H
11
3.252Æ0.028
150
General methods
aFor the HSCCC method used to determine partition coecients P
refer to Experimental. Data represents the average value ÆSEM of
three separate experiments.
Melting points (uncorrected) were determined on a
Reichert-Jung Thermovar hot-stage apparatus. Infrared
spectra (IR) were obtained on a Perkin±Elmer Model
b
Not determined.
The compound was not suciently soluble in the test system.
c
1
420 spectrophotometer. KBr refers to a potassium
relative to 5. Obviously, due to this e€ect, the IC100 and
IC50 values of the N-acyl compounds 9 and 12±21 are
nearly identical. In general, an increase of inhibitory
activity (cf. IC100 values of 9 and 12±21) by a factor of
up to 3 as compared to 5 was observed for all of the
TaBro derivatives.
bromide disk for infrared spectra. Proton and Carbon-
13 spectra were recorded on a Bruker AC 250 spectro-
1
meter at 250 MHz (for H NMR), and at 63 MHz (for
C NMR). Chemical shifts (d) are reported in parts per
million (ppm) with the proton or carbon-13 signals of
13
1
1
chloroform ( H, d=7.26 ppm), methanol ( H, d=
13
1
3
2
.33 ppm;
C, d=49.02 ppm), acetone ( H, d=
3
1
.01 ppm; C, d=29.85 and 205.9 ppm), or dimethyl-
Outlook
13
sulfoxide ( C, d=39.43 ppm) in the deuterated solvents
as internal reference. Coupling constants (J) are given
in Hertz (Hz). The following abbreviations are used: s
(singlet), d (doublet), dd (double doublet), t (triplet), mc
The mode of action of TaClo (4) and TaBro (5) on the
dopaminergic system is as yet unknown. It might be
ascribed to either the strong inhibition of complex I of

G. Bringmann et al. / Bioorg. Med. Chem. 8 (2000) 1467±1478
1473
(
central multiplet). Electron impact mass spectral data
temperature a€ording a colorless crude solid. Crystal-
.
lization from methanol gave 3 HCl (2.72 g, 9.83 mmol,
were obtained on either a Finnigan MAT 8200 or a
Finnigan MAT 90 mass spectrometer. The peaks listed
ꢁ
28
147±149 C, free base). IR (KBr, cm ) 3380 (indole
96% yield) as white crystals: mp 205±207 C (dec) (lit.
+
ꢀ
ꢁ
NH), 2880, 2580, 2560 (CH), 1560 (NH), 1190 (CF); H
� 1
are those arising from the molecular ion [M ] , those
+
1
attributable to loss of certain fragments (M minus a
fragment), and some other prominent peaks. Elemental
analyses were conducted by the Microanalysis Labora-
3
NMR (CD OD): d 3.17 (t, J =6.11 Hz, 2H, 4-H),
3
3,4
3
3.58±3.82 (m, 2H, 3-H), 5.78 (q, J1,F=6.72 Hz, 1H, 1-
H), 7.08±7.14 (m, 1H, 6- or 7-H), 7.20±7.27 (m, 1H, 7-
or 6-H), 7.41±7.45 (m, 1H, 5- or 8-H), 7.54±7.58 (m, 1H,
È
tory of the University of Wurzburg (Institute of Inor-
ganic Chemistry) on a Carlo Erba Elemental Analyzer
M 1106 apparatus. Where organic solvents are noted as
part of the elemental analysis, they were seen in the H
NMR spectrum in proper amounts.
13
8- or 5-H); C NMR (CD OD): d 18.0 (C-4), 42.0 (C-3),
3
1
2
53.9 (q, J =34 Hz, C-1), 110.6, 112.5, 119.2, 119.4,
C,F
1
120.7, 123.3 (q, JC,F=282 Hz, CF ), 124,4, 126,4,
3
+
ꢀ
1
38.6; EI±MS m/z (rel int) 240 (53) [M ] , 171 (100)
+
[
M � CF ]. Anal. calcd for C H F N HCl: C, 52.09;
3
12 11
3
2
Materials
H, 4.37; N, 10.12; found: C, 51.76; H, 4.31; N, 9.92.
All reagents used were of commercial quality. Organic
solvents were dried and distilled prior to use. Trypta-
mine hydrochloride was purchased from Sigma (Dei-
senhofen, Germany), tribromoacetaldeyhde (bromal)
from Merck (Darmstadt, Germany), and tri¯uoro-
acetaldehyde ethyl hemiacetal from Janssen Chimica
2-Formyl-1-tribromomethyl-1,2,3,4-tetrahydro-9H-pyr-
ido[3,4-b]indole (9). A solution of tryptamine hydro-
chloride (11.9 mg, 60.7 mmol) and tribromoacetalde-
hyde (9.9 mL, 26.7 g, 95.3 mmol) in formic acid (20 mL)
was stirred at room temperature for 5 h. Subsequently,
trimethylacetic formic anhydride28 (9.93 g, 68.7 mmol)
was added to the solution, and stirring was continued
for 6 h. The reaction mixture was then poured onto ice.
The solid thus obtained was ®ltered o€ and washed with
tert-butyl methyl ether (30 mL). Crystallization from
methanol yielded 9 (14.6 g, 32.6 mmol, 51% yield) as
(
Beerse, Belgium). 1,2,3,4-Tetrahydro-9H-pyrido[3,4-b]-
indole (1) was obtained from Aldrich (Milwaukee, WI,
USA). 1-Trichloromethyl-1,2,3,4-tetrahydro-9H-pyrido-
.
3,4-b]indole hydrochloride (4 HCl) was prepared
[
from tryptamine and trichloroacetaldehyde, as described
1
0,11
previously.
2-Hexanoyl-1-tribromomethyl-1,2,3,4-
ꢁ
� 1
tetrahydro-9H-pyrido[3,4-b] indole (21) was synthesized
as reported earlier.40 Reactions were monitored by
thin-layer chromatography (TLC) on aluminum plates
coated with silica gel 60 F254 (Merck, Darmstadt,
Germany). Column chromatography was performed on
Merck silica gel (0.063±0.200 mm).
colorless crystals, mp 176 C (dec). IR (KBr, cm ) 3410
(indole NH), 2940, 2870, 2820 (CH), 1650 (CˆO), 1440
1
(CH), 745 (1,2-disubstitution); H NMR (C D O):
3
6
occurrence of two distinct rotational isomers A and B,
ratio 1.4:1. Isomer A: d 2.83-3.00 (m, 2H, 4-H, overlay
by isomer B), 3.82±4.20 (m, 1H, 3-H), 5.93 (s, 1H, 1-H),
7
.07±7.22 (m, 2H, 6-H, 7-H, overlay by isomer B), 7.45±
1-Methyl-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole (2).
The title compound was synthesized according to a
published procedure, which was slightly modi®ed: A
solution of tryptamine hydrochloride (5.0 g, 25.4 mmol)
and pyruvic acid sodium salt (17.4 g, 158.5 mmol) in
7.52 (m, 2H, 5-H, 8-H, overlay by isomer B), 8.44 (s,
1H, NCHO), 10.3 (br. s, 1H, indole NH, overlay by
isomer B). Isomer B: d 2.83±3.00 (m, 2H, 4-H, overlay
by isomer A), 4.35±4.82 (m, 1H, 3-H), 6.48 (s, 1H, 1-H),
7.07±7.22 (m, 2H, 6-H, 7-H, overlay by isomer A), 7.45±
7.52 (m, 2H, 5-H, 8-H, overlay by isomer A), 8.65 (s,
1H, NCHO), 10.3 (br. s, 1H, indole NH, overlay by
isomer A); 13C NMR [(CD ) SO]: isomer A: d 20.3 (C-
2
5
4
00 mL of 2 M sodium acetate bu€er (pH=5.2) was
ꢁ
stirred at 37 C for 4 d. 1-Methyl-1-carboxy-1,2,3,4-tet-
rahydro-9H-pyrido[3,4-b]indole was obtained as a crude
crystalline precipitate, which was ®ltered o€ and washed
with ice cold water (40 mL). For decarboxylation, this
material was then re¯uxed in 6 N HCl (150 mL) for
3
2
4), 33.5 (C-3), 61.8 (C-1), 110.4, 110.9 (CBr ), 112.0,
3
119.0, 122.3, 125.3, 126.5, 136.6, 163.1 (CHO). Isomer
B: d 21.1 (C-4), 39.2 (C-3), 67.7 (C-1), 110.4, 110.9
(CBr ), 112.0, 118.3, 119.0, 122.4, 125.3, 126.5, 136.7,
3
4
5min, with intensive stirring. After cooling, the strongly
acidic solution was adjusted to pH=12 by addition of
M NaOH, and then extracted with ethyl acetate
164.4 (CHO); EI±MS m/z (rel int) 454/452/450/448 (0.5/
+
ꢀ
+
1
1.6/1.6/0.5) [M ] , 372/370/368 (0.2/0.5/0.2) [M � HBr],
+
+
(
3Â150 mL). The combined layers were dried over
291/289 (16/18) [M � HBr-Br], 199 (100) [M � CBr ],
3
anhydrous MgSO , and concentrated under reduced
4
170 (5) [199-CHO]. Anal. calcd for C H Br N O: C,
13 11
3
2
pressure. Crystallization from ethyl acetate a€orded 2
(
34.62; H, 2.46; N, 6.21; found: C, 34.56; H, 2.45; N,
6.27.
3.0 g, 16.1 mmol, 63% yield) as beige-colored crystals:
ꢁ
41,42
ꢁ
179±180 C). The spectroscopic data
mp 180 C (lit.
are in agreement with those reported in the literature.
26,43
1-Tribromomethyl-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]-
.
indole hydrochloride (5 HCl). To a solution of 9 (7.03 g,
1
indole hydrochloride (3 HCl). The title compound was
synthesized according to a published procedure, which
was slightly modi®ed: To a solution of tryptamine
hydrochloride (2.0 g, 10.2 mmol) in water (50 mL), tri-
-Tri¯uoromethyl-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]-
.
15.7 mmol) in methanol (20 mL), concentrated HCl
(2.8 mL) was added dropwise with intensive stirring at
room temperature. The reaction mixture was then
heated at re¯ux for 7 h. After cooling, the solvent was
evaporated under reduced pressure a€ording a crude
colorless solid. Crystallization from methanol-ether
2
7
¯
was added dropwise. The reaction mixture was stirred
uoroacetaldehyde ethyl hemiacetal (4.41 g, 30.6 mmol)
.
gave 5 HCl (6.45 g, 14.2 mmol, 91% yield) as an amor-
ꢁ
ꢁ
� 1
phous solid: mp 162 C (dec). IR (KBr, cm ) 3430
at 60 C for 12 h, and then allowed to cool to room

1
474
G. Bringmann et al. / Bioorg. Med. Chem. 8 (2000) 1467±1478
(
indole NH), 2910 (CH), 1550 (NH), 1450 (CH ), 750
2
General procedure for the preparation of N-acylated
.
compounds (12±21). To a suspension of 3 HCl (for 12),
1
(
1,2-disubstitution); H NMR (C D O): d 3.10±3.16 (m,
3
6
.
.
2H, 4-H), 3.43±3.54 (m, 1H, 3-H), 3.87±3.96 (m, 2H, 3-
H), 6.02 (s, 1H, 1-H), 7.08 (dt, J =7.5 Hz, J6,8=
1
4 HCl (for 13), or 5 HCl (for 14±21) (1 mol equiv) in dry
dichloromethane (0.5 mmol dissolved in 15 mL), trie-
thylamine (4 mol equiv) was added. The mixture was
treated dropwise with the respective activated acid deri-
3
5
6,7
3
5
.2 Hz, 1H, 6-H), 7.21 (dt, J =7.6 Hz, J7,5=1.2 Hz,
7,6
H, 7-H), 7.48 (d, 3J8,7=7.9 Hz, 1H, 8-H), 7.53 (d,
J5,6=7.9 Hz, 1H, 5-H); C NMR (CD OD): d 19.5 (C-
1
3
13
ꢁ
vative (3 mol equiv) at 0 C, then stirred at room tem-
3
4
), 45.0 (C-3), 69.2 (C-3), 112.9, 113.3 (CBr ), 119.3,
perature for 3 h, and extracted with water (3Â). The
3
1
4
21.0, 124,7, 125.1, 126.5, 138.6; EI±MS m/z (rel int)
+
combined organic phases were dried over anhydrous
MgSO , ®ltered o€, and concentrated in vacuo. Puri-
ꢀ
26/424/422/420 (0.2/0.7/0.7/0.2) [M ] , 344/342/340
+
4
+
(0.3/0.9/0.5) [M � HBr], 263/261 (25/26) [M � HBr� Br],
®cation of the respective residues by crystallization from
methanol-ether provided the acyl derivatives 12±21 as
crystalline material. The data are given below.
+
1
71 (100) [M � CBr ]. Anal. calcd for C H Br N HCl:
3
12 11
3
2
C, 31.37; H, 2.63; N, 6.09; found: C, 31.53; H, 2.89; N,
6.00.
2
-Tri¯uoroacetyl-1-tri¯uoromethyl-1,2,3,4-tetrahydro-9H-
.
2-Formyl-1-dibromomethylene-1,2,3,4-tetrahydro-9H-
pyrido[3,4-b]indole (10). A suspension of 9 (5.02 g,
pyrido[3,4-b]indole (12). Starting from 3 HCl (840 mg,
3.05 mmol) and tri¯uoroacetic anhydride (1.30 mL,
1.95 g, 9.07 mmol), the title compound (950 mg,
2.83 mmol, 94% yield) was obtained as colorless crys-
tals, suitable for X-ray structure analysis: mp 211±
1
3
1.2 mmol) and sodium hydrogen carbonate (3.90 g,
2.8 mmol) in methanol (300 mL) was re¯uxed for 5 h.
Concentration of the solvent yielded the title compound
as a crude precipitate, which was ®ltered o€. Crystal-
lization from methanol a€orded 10 (4.03 g, 10.9 mmol,
ꢁ
� 1
214 C (dec). IR (KBr, cm ) 3340 (indole NH), 3060,
1
2920, 2840 (CH), 1680 (CˆO), 1260 (CF). H NMR
ꢁ
9
(
7% yield) as colorless crystals: mp 192 C (dec). IR
�
(CD OD): d 2.86±3.05 (m, 2H, 4-H), 3.78±3.89 (m, 1H,
3
1
3
KBr, cm ) 3460 (indole NH), 2920, 2880 (CH), 1670
CˆO), 1500 (NH), 1450 (CH), 750 (1,2-disubstitution);
3-H), 4.36±4.42 (m, 1H, 3-H), 6.22±6.30 (q, J1,F=
7.32 Hz, 1H, 1-H), 7.03±7.09 (m, 1H, 6- or 7-H), 7.15±
7.21 (m, 1H, 7- or 6-H), 7.38±7.41 (m, 1H, 5 or 8-H),
(
1
H NMR (CD OD): occurrence of two distinct rota-
3
1
3
tional isomers A and B, ratio 3.6:1. d 3.21±3.69 (m, 2H,
7.48±7.52 (m, 1H, 8-or 5-H); C NMR (C D O): d 22.4
3
6
2
4-H), 4.10±4.28 (m, 2H, 3-H), 7.08±7.38 (m, 2H, 6-H or
(C-4), 43.4 (C-3), 52.3 (q, J =34 Hz, C-1), 110.8,
C,F
1
7-H), 7.49±7.70 (m, 2H, 5-H or 8-H), 8.19 (s, 1H, CHO,
isomer A), 8.36 (s, 1H, CHO, isomer B); C NMR
112.8, 115.3 (CF ), 119.7, 120.8, 124.1, 124.2 (q, JC,F=
3
1
3
280 Hz, 1-CF ), 127.0, 138.5; EI±MS m/z (rel int) 336
3
+
ꢀ
+
+
[
(CD ) SO]: d 23.1 (C-4), 44.8 (C-3), 107.5 (C-1), 112.7,
(64) [M ] , 316 (76) [M � HF], 267 (100) [M � CF ],
3
2
3
1
14.2 (CBr ), 115.4, 118.8, 118.9, 119.8, 123.8, 124.0,
169 (29) [267-COCF -H]. Anal. calcd for C H F N O:
6
2
3
14 10
2
1
25.0, 126.2, 137.1, 160.6 (CHO, isomer A), 163.4
C, 50.01; H, 2.99; N, 8.33; found: C, 49.72; H, 2.70; N,
8.46.
(
3
CHO, isomer B); EI±MS m/z (rel int) 372/370/368 (1.6/
+
+
ꢀ
.2/1.5) [M ] , 291/289 (99/100) [M -Br], 210 (14)
+
[M � 2 Br], 181 (57) [210-CHO]. Anal. calcd for C H
Br N O: C, 42.19; H, 2.72; N, 7.57; found: C, 42.10; H,
2
1-Trichloromethyl-2-tri¯uoroacetyl-1,2,3,4-tetrahydro-
.
9H-pyrido[3,4-b]indole (13). Starting from 4 HCl (1.00 g,
3.07 mmol) and tri¯uoroacetic anhydride (1.08 mL,
13
10
2
2
.73; N, 7.46.
1
2.62 mmol, 85% yield) was obtained as colorless crys-
.61 g, 7.68 mmol), the title compound (1.01 g,
1
-Dibromomethylene-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]-
ꢁ
(dec). IR (KBr, cm ) 3350 (indole NH), 3045, 2940,
.
indole hydrochloride (11 HCl). Concentrated HCl
2.8 mL) was added to a solution of 10 (1.00 g,
.70 mmol) in methanol (20 mL). The mixture was
tals, suitable for X-ray structure analysis: mp 231 C
�
1
(
2
1
2900, 2840 (CH), 1675 (CˆO), 820, 800 (CCl). H NMR
re¯uxed for 8 h. The orange-colored residue obtained
after removal of the solvent was puri®ed by column
chromatography on silica gel (eluent: dichloromethane-
methanol, 95:5). Saturated methanolic HCl was added
to the combined fractions. After evaporation to dryness,
the crude product was recrystallized from dichloro-
(CDCl ): d 2.97±3.02 (m, 2H, 4-H), 4.28±4.33 (m, 2H, 3-
3
H), 6.57 (s, 1H, 1-H), 7.15±7.21 (m, 1H, 6- or 7-H),
7.26±7.32 (m, 1H, 6-or 7-H, overlay by solvent), 7.40±
7.44 (m, 1H, 5-or 8-H), 7.53±7.57 (m, 1H, 5-or 8-H),
8.45 (br. s, 1H, indole NH); 13C NMR (C D O): d 22.3
3
6
(C-4), 41.6 (C-3), 64.3 (C-1), 109.0 (CCl ), 112.2, 112.5,
3
.
methane-diethyl ether a€ording 11 HCl (417 mg,
115.0 (CF ), 119.3, 120.4, 123.9, 125.3, 126.4, 137.9; EI±
3
1
1
.22 mmol, 45% yield) as a red amorphous solid: mp
ꢁ
MS m/z (rel int) 390/388/386/384 (0.2/1.1/3.4/3.8)
+
� 1
+
ꢀ
63 C (dec). IR (KBr, cm ) 3420 (indole NH), 2920
[M ] , 352/350/348 (0.5/2.3/3.0) [M � HCl], 315/313
+
+
(
CH), 1620 (CˆC), 1540 (aryl CˆC), 750 (1,2-dis-
(6.8/3.0) [M � HCl-Cl], 267 (100) [M � CCl ], 169 (21)
3
1
ubstitution); H NMR (CD OD): d 3.17±3.63 (m, 2H,
[267-COCF -H]. Anal. calcd for C H Cl F N O: C,
2
3
3
14 10
3
3
4
-H), 4.03±4.25 (m, 2H, 3-H), 7.16±7.60 (m, 2H, 6-H,
43.61; H, 2.61; N, 7.27; found: C, 43.55; H, 2.63; N,
7.14.
13
7-H), 7.71±7.83 (m, 2H, 5-H, 8-H); C NMR (CD3
OD): d 20.1 (C-4), 44.6 (C-3), 107.7 (C-1), 114.6, 114.7
CBr ), 123.2, 123.7, 123.8, 125.9, 131.0, 132,2, 144.3;
+
(
EI±MS m/z (rel int) 344/342/340 (4.3/8.5/4.2) [M ] ,
1-Tribromomethyl-2-tri¯uoroacetyl-1,2,3,4-tetrahydro-
.
9H-pyrido[3,4-b]indole (14). Starting from 5 HCl
2
ꢀ
+
+
2
(
63/261 (12/14) [M � Br], 182 (48) [M � 2 Br], 154
(100mg, 0.22mmol) and tri¯uoroacetic anhydride
(90 mL, 134 mg, 0.63mmol), the title compound (108mg,
0.21 mmol, 96% yield) was obtained as pale yellow
100) [182-CH N]. Anal. calcd for C H Br N HCl: C,
2
12 10
2
2
3
7
8.08; H, 2.93; N, 7.40; found: C, 38.32; H, 3.11; N,
.45.
ꢁ
crystals, suitable for X-ray structure analysis: mp 163 C

G. Bringmann et al. / Bioorg. Med. Chem. 8 (2000) 1467±1478
1475
�
(
1
dec). IR (KBr, cm 1) 3450 (indole NH), 2920 (CH),
4.31±4.33 (m, 2H, 3-H), 6.89 (s, 1H, 1-H), 7.06 (dt,
3
6,8=
1
5
3
680 (CˆO). H NMR (C D O): d 2.92±3.13 (m, 2H, 4-
J6,7=7.5 Hz, J
5
1.2 Hz, 1H, 6-H), 7.18 (dt, J7,6=
3
3
6
H), 4.31±4.59 (m, 2H, 3-H), 6.70 (s, 1H, 1-H), 7.15 (dt,
3
7.6 Hz, J7 =1.2 Hz, 1H, 7-H), 7.48 (d, J =7.9 Hz,
8,7
13
,5
5
3
3
J6,7=7.95 Hz, J =0.9 Hz, 1H, 6-H), 7.29 (dt, J7,6=
6,8
1H, 8-H), 7.53 (d, J =7.9 Hz, 1H, 5-H); C NMR
5,6
5
3
0 0
2 ), 40.2 (C-3), 64.1 (C-1), 107.9, 112.3 (CBr ), 112.6,
7
1
.95 Hz, J =1.3 Hz, 1H, 7-H), 7.60 (d, J =8.1 Hz,
7
(C D O): d 14.2 (C-4 ), 19.2 (C-3 ), 22.4 (C-4), 35.9 (C-
,5
8,7
13
3
6
3
0
H, 8-H), 7.66 (d, J5,6=7.8 Hz, 1H, 5-H); C NMR
3
(
(
1
0
C D O): d 22.7 (C-4), 41.2 (C-3), 65.8 (C-1), 112.0
119.2, 120.2, 123.4, 126.7, 128.8, 137.6, 173.3 (CˆO);
+
3
6
+
CBr ), 113.0, 115.3 (CF ), 119.6, 120.7, 124.1, 126.6,
EI±MS m/z (rel int) 414/412/410 (1.2/2.6/1.0) [M �
3
3
27.1; EI±MS m/z (rel int) 522/520/518/516 (0.6/1.9/2.0/
+
HBr], 344/342/340 (7.7/14/7.6) [M � HBr-C H O], 333/
4
6
ꢀ
+
+
+
.5) [M ] , 359/357 (79/71) [M � HBr- Br], 267 (100)
331 (97/97) [M � HBr-Br], 253 (19) [M � 3 Br], 241
+
+
[M � CBr ], 169 (14) [267-COCF -H]. Anal. calcd for
C H Br F N O: C, 32.39; H, 1.94; N, 5.39; found: C,
(28) [M � CBr ], 171 (100) [241-C H O]. Anal. calcd
3
3
3
4
6
for C H Br N O: C, 38.98; H, 3.48; N, 5.68; found: C,
16 17 3 2
1
4
10
3
3
2
32.44; H, 2.01; N, 5.31.
38.88; H, 3.40; N, 5.64.
0
hydro-9H-pyrido[3,4-b]indole (18). Starting from 5 HCl
2
[
-Acetyl-1-tribromomethyl-1,2,3,4-tetrahydro-9H-pyrido-
2-(2 -Methylpropanoyl)-1-tribromomethyl-1,2,3,4-tetra-
.
.
3,4-b]indole (15). Starting from 5 HCl (100 mg,
0
0
.22 mmol) and acetyl chloride (66 mL, 72 mg,
.92 mmol), the title compound (99 mg, 0.21 mmol, 98%
(200 mg, 0.44 mmol) and isobutyryl chloride (150 mL,
151 mg, 1.42 mmol), the title compound (193 mg,
0.39 mmol, 89% yield) was obtained as colorless crys-
ꢁ
dec). IR (KBr, cm ) 3430 (indole NH), 2910, 2890
yield) was obtained as colorless crystals: mp 165 C
�
1
ꢁ
� 1
(
tals: mp 171 C (dec). IR (KBr, cm ) 3310 (indole NH),
1
1
(CH), 1660 (CˆO). H NMR (C D O): d 2.29 (s, 3H,
CH ), 2.91±2.96 (m, 2H, 4-H), 4.28±4.43 (m, 2H, 3-H),
3050, 2970, 2900, 2870 (CH), 1645 (CˆO). H NMR
3
6
(C D O): d 1.12 [dd, J =21.7 Hz, J=6.7 Hz, 6H,
3
3
6
gem
3
5
6
1
.84 (s, 1H, 1-H), 7.06 (dt, J =6.9 Hz, J6,8=1.1. Hz,
3
CH(CH ) ), 2.89±2.95 (m, 2H, 4-H), 3.17 [sept, 1H,
CH(CH ) ], 4.35±4.40 (m, 2H, 3-H), 6.93 (s, 1H, 1-H),
6,7
3 2
5
H, 6-H), 7.20 (dt, J =6.9 Hz, J =1.1 Hz, 1H, 7-
7,5
7,6
3 2
3
3
3
5
H), 7.48 (d, J =8.1 Hz, 1H, 8-H), 7.53 (d, J5,6=
,7
7.06 (dt, J =7.5 Hz, J =0.9 Hz, 1H, 6-H), 7.18 (dt,
6,7 6,8
8
1
3
3
5
3
7
2
1
4
.6 Hz, 1H, 5-H); C NMR (CD OD): d 22.8 (CH ),
J7,6=7.6 Hz, J =1.2 Hz, 1H, 7-H), 7.49 (d, J =
7,5
3
3
8,7
13
3
3.0 (C-4), 41.8 (C-3), 64.8 (C-1), 108.8 (CBr ), 113.4,
20.0, 124.3, 139.1, 171.3 (CˆO); EI±MS m/z (rel int)
8.8 Hz, 1H, 8-H), 7.51 (d, J =7.9 Hz, 1H, 5-H);
5,6
C
3
0 0
31.2 (C-2 ), 40.0 (C-3), 63.9 (C-1), 112.1 (CBr ), 112.6,
NMR (C D O): d 19.6 (C-3 ), 20.5 (C-3 ), 22.8 (C-4),
3
0
6
+
ꢀ
68/466/464/462 (0.2/0.4/0.4/0.2) [M ] , 386/384/382
+
3
+
(
0.8/1.7/0.8) [M � HBr], 305/303 (89/82) [M � HBr-
119.2, 120.2, 123.4, 126.8, 128.7, 137.6, 177.1 (CˆO);
+
+
+
Br], 213 (66) [M � CBr ], 171 (100) [213-C H O]. Anal.
EI±MS m/z (rel int) 414/412/410 (0.4/0.8/0.4) [M -
HBr], 344/342/340 (7.2/15/7.5) [M � HBr-C H O], 333/
3
2
2
calcd for C H Br N O: C, 36.16; H, 2.88; N, 6.02;
2
1
4
13
3
4
6
+
+
found: C, 35.92; H, 2.86; N, 5.98.
331 (99/99) [M � HBr-Br], 253 (31) [M � 3 Br], 241
3 4 6
+
+
(
[241-C H O]. Anal. calcd for C H Br N O: C, 38.98;
23) [M � CBr ], 182 (16) [M � 3 Br-C H O]. 171 (100)
4 6 16 17 3 2
2
pyrido[3,4-b]indole (16). Starting from 5 HCl (200 mg,
-Propanoyl-1-tribromomethyl-1,2,3,4-tetrahydro-9H-
.
H, 3.48; N, 5.68; found: C, 38.84; H, 3.52; N, 5.83.
0
1
9
.44 mmol) and propionyl chloride (120 mL, 125 mg,
.35 mmol), the title compound (192 mg, 0.40 mmol,
0
0
tetrahydro-9H-pyrido[3,4-b]indole (19). Starting from
2-(2 ,2 -Dimethylpropanoyl)-1-tribromomethyl-1,2,3,4-
ꢁ
1% yield) was obtained as colorless crystals: mp 168 C
�
1
.
5 HCl (100 mg, 0.22 mmol) and pivaloyl chloride
(
dec). IR (KBr, cm ) 3300 (indole NH), 3030, 2920,
1
2
1
890, 2820 (CH), 1640 (CˆO). H NMR (C D O): d
(55 mL, 79 mg, 0.65 mmol), the title compound (81 mg,
0.14 mmol, 63% yield) was obtained as colorless amor-
3
6
0
0
.15 (t, 3H, 3 -CH ), 2.65 (m, 2H, 2 -CH ), 2.89±2.93
3
2
ꢁ
[s, 9H, C(CH ) ], 2.91±2.94 (m, 2H, 4-H), 4.30±4.62 (m,
1
(
m, 2H, 4-H), 4.29±4.32 (m, 2H, 3-H), 6.88 (s, 1H, 1-H),
3
phous solid: mp 151 C (dec). H NMR (C D O): d 1.39
3
6
5
7
.06 (dt, J =7.5 Hz, J =1.2 Hz, 1H, 6-H), 7.18 (dt,
6,8
6
,7
3 3
3
5
3
3
J7,6=7.6 Hz, J =1.2 Hz, 1H, 7-H), 7.48 (d, J
7,5
=
8,7
13
C
2H, 3-H), 7.01 (s, 1H, 1-H), 7.05 (dt, J6,7=6.9 Hz,
5
3
5
3
7
.9 Hz, 1H, 8-H), 7.53 (d, J =7.9 Hz, 1H, 5-H);
5
J6,8=1.2 Hz, 1H, 6-H), 7.16 (dt, J =6.9 Hz, J7,5=
7,6
,6
0
C-3), 64.2 (C-1), 112.3 (CBr ), 112.6, 119.2, 120.2,
0
3
NMR (C D O): d 9.7 (C-3 ), 27.2 (C-4), 39.9 (C-2 ), 49.3
(
1.2 Hz, 1H, 7-H), 7.48 (d, J8,7=8.1 Hz, 1H, 8-H), 7.53
3
3
6
13
(d, J5,6=7.8 Hz, 1H, 5-H); C NMR (C D O): d 23.5
3
3
6
1
23.4, 126.8, 128.7, 137.6, 174.1 (CˆO); EI±MS m/z (rel
(C-3), 29.4 [C(CH ) ], 40.7 [C(CH ) ], 41.9 (C-4), 65.4
(C-1), 112.4 (CBr ), 113.3, 119.9, 120.9, 124.1, 127.3,
3 3 3 3
+
int) 400/398/396 (0.2/0.4/0.2) [M � HBr], 319/317 (89/
3
+
+
9
2) [M � HBr- Br], 227 (39) [M � CBr ], 171 (100)
129.4, 138.3, 178.4 (CˆO); EI±MS m/z (rel int) 428/426/
+
3
+
+
[227-C H O]. Anal. calcd for C H Br N O: C, 37.57;
H, 3.15; N, 5.84; found: C, 37.64; H, 3.11; N, 6.03.
424 (0.5/1.1/0.5) [M � HBr], 347/345 (49/50) [M �
3
4
15 15
3
2
HBr-Br], 255 (100) [M � CBr ], 171 (15) [255-C H O].
3
5
8
Á
Anal. calcd for C H Br N O 1/3 C H O: C, 41.38; H,
4
17
19
3
2
10
2-Butanoyl-1-tribromomethyl-1,2,3,4-tetrahydro-9H-pyr-
.
ido[3,4-b]indole (17). Starting from 5 HCl (200 mg,
4.23; N, 5.27; found: C, 41.41; H, 4.33; N, 5.08.
0
1
8
.44 mmol) and n-butyryl chloride (145 mL, 148 mg,
.39 mmol), the title compound (189 mg, 0.38 mmol,
2-Pentanoyl-1-tribromomethyl-1,2,3,4-tetrahydro-9H-
.
pyrido[3,4-b]indole (20). Starting from 5 HCl (200 mg,
ꢁ
7% yield) was obtained as colorless crystals: mp 148 C
�
0.44 mmol) and valeroyl chloride (160 mL, 159 mg, 1.31
mmol), the title compound (187 mg, 0.37 mmol, 84%
1
(
2
dec). IR (KBr, cm ) 3300 (indole NH), 3030, 2940,
1
ꢁ
IR (KBr, cm ) 3430 (indole NH), 2910, 2890 (CH),
910, 2850, 2820 (CH), 1645 (CˆO). H NMR
yield) was obtained as yellow crystals: mp 157 C (dec).
� 1
0
CH ), 2.47±2.89 (m, 2H, 2 -CH ), 2.89±2.95 (m, 2H, 4-H),
0
(
C D O): d 0.98 (t, 3H, 4 -CH ), 1.63±1.78 (m, 2H, 3 -
3
6
3
0
1
0
1660 (CˆO). H NMR (C D O): d 0.95 (t, 3 H, 5 -CH ),
2
2
3
6
3

1
476
G. Bringmann et al. / Bioorg. Med. Chem. 8 (2000) 1467±1478
0
.49-2.76 (m, 2H, 2 -CH ), 2.89-2.97 (m, 2H, 4-H), 4.31-
0
1
2
4
.34-1.49 (m, 2H, 4 -CH ), 1.63-1.82 (m, 2H, 3 -CH ),
2
determined by least-squares re®nement using 22 cen-
ꢁ
2
0
.34 (m, 2H, 3-H), 6.89 (s, 1H, 1-H), 7.06 (dt, J6,7=7.5
ꢁ
tered re¯ections within 15.3 <y< 27.5 . A total of
3973 re¯ections were collected in o-scan mode to
2
3
5
3
ꢁ
Hz, J =1.2 Hz, 1H, 6-H), 7.18 (dt, J7,6=7.6 Hz,
6
2ymax=55 (h : 0!15, k : 0!15, l :-14!13) of which
,8
5
3
J7,5=1.2 Hz, 1H, 7-H), 7.48 (d, J =7.9 Hz, 1H, 8-
8,7
3792 were unique with I>2s(I ). In re®nements, weights
3
13
2
H), 7.53 (d, J5,6=7.9 Hz, 1H, 5-H);
(
C NMR
C D O): d 14.2 (C-5 ), 22.4 (C-4 ), 23.1 (C-4), 28.0 (C-
were used according to the scheme w=1/[s (F )]. The
o
0
), 33.7 (C-2 ), 40.1 (C-3), 64.1 (C-1), 107.9, 112.3
0
re®nement converged to the ®nal agreement factors
R=0.044, and R =0.041, for 213 parameters and 2642
3
6
0
CBr ), 112.6, 119.2, 120.2, 123.4, 126.8, 128.8, 137.6,
0
3
w
(
observed re¯ections with F>3s(F ); data-to-parameter
ratio being 12.40. The electron density of the largest
3
1
0
73.4 (CˆO); EI±MS m/z (rel int) 428/426/424 (0.8/2.0/
+
+
Ê
� 3
.8) [M � HBr], 347/345 (90/92) [M � HBr- Br], 267
di€erence peak was found to be 0.28 eA , while that of
the largest di€erence hole was 0.24 eA . In the crystals
of 13, the molecules are connected by intermolecular
+
+
Ê
� 3
(22) [M � 3 Br], 255 (22) [M � CBr ], 171 (100) [255-
C H O]. Anal. calcd for C H Br N O: C, 40.27; H,
3
5
8
17 19
3
2
.
..
Ê
3.78; N, 5.52; found: C, 40.24; H, 3.68; N, 5.73.
hydrogen bonds H(12) O(15) [2.2 A] to form a zigzag
chain parallel to [100].
Single-crystal X-ray di€raction analysis of 12, 13 and
4. Unless stated otherwise, all measurements of dif-
fraction intensities were performed on a Siemens P4
1
Crystal data for 14. The crystal chosen for X-ray inves-
tigations was a clear pale yellow prism with the
approximate dimensions 0.30Â0.40Â0.15 mm. C H
di€ractometer with an incident beam graphite mono-
Ê
1
4
10
� 1
Br F N O (518.97 g mol ) crystallizes in the mono-
3 3 2
chromator (Mo-K radiation, l=0.71073 A) in o-scan
a
ꢁ
ꢁ
44
mode in the range of 1.75 <y<27.5 . The structures
were solved by direct phase determination and re®ned
by full-matrix anisotropic least-squares with the aid of
the program Shelxtl-Plus. All non-hydrogen atoms
were re®ned anisotropically. The hydrogen positions
were calculated using a riding model and were con-
sidered ®xed with isotropic thermal parameters in all
re®nements. Software used to prepare material for pub-
clinic system, space group P2 /c, with a=10.695 (9),
1
Ê
ꢁ
b=12.278 (9), c=12.766 (9) A, b=105.216 (6) ,
Ê
3
� 1
V=1617.2 (2) A , Z=4, m(Mo-K )=7.52 mm , and
a
Dcalcd=2.132 g cm . The unit cell parameters were
4
5
� 3
determined by least-squares re®nement using 60 cen-
ꢁ
ꢁ
tered re¯ections within 16.7 <y< 30.0 . A total of
4093 re¯ections were collected in o-scan mode to
ꢁ
2ꢀmax=55 (h : 0!13, k : 0!15, l :-16!16) of which
46
lication: Schakal 88.
3715 were unique with I>2s(I ). In re®nements, weights
2
were used according to the scheme w=1/[s (F )]. The
o
Crystal data for 12. The crystal chosen for X-ray inves-
tigations was a clear colorless prism with the approx-
imate dimensions 0.50Â0.60Â0.60 mm. C H F N O
re®nement converged to the ®nal agreement factors
R=0.052, and R =0.050, for 213 parameters and 2814
w
observed re¯ections with F>3s(F ); data-to-parameter
ratio being 13.21. The electron density of the largest
1
4
10
6
2
�
1
(
336.24 g mol ) crystallizes in the monoclinic system,
Ê
� 3
di€erence peak was found to be 0.65 eA , while that of
space group P2 /n, with a=9.0197 (4), b=12.036
1
Ê
ꢁ
Ê
3
Ê
� 3
(
1), c=12.944 (1) A, b=98.043 (4) , V=1391.4 (1) A ,
the largest di€erence hole was 0.60 eA . In the crystals
of 14, the molecules are connected by intermolecular
�
1
� 3
Z=4, m (Mo-K )=0.16mm , and Dcalcd=1.605g cm .
The unit cell parameters were determined by least-
a
.
..
Ê
hydrogen bonds H(12) O(15) [2.4 A] to form a zigzag
chain parallel to [010].
squares re®nement using 60 centered re¯ections within
ꢁ
ꢁ
2
5.3 <y<29.9 . A total of 3558 re¯ections were col-
ꢁ
lected in o-scan mode to 2ꢀmax=55 (h: 0!11, k:
Tables of bond distances and angles, atomic coordi-
nates, and anisotropic thermal parameters for 12
(CCDC 118762), 13 (CCDC 118764), and 14 (CCDC
118763) have been deposited with the Cambridge Crys-
tallographic Data Centre.47
0
!15, l:� 16!16) of which 3197 were unique with
I>2s(I). In re®nements, weights were used according to
2
the scheme w=1/[s (F )]. The re®nement converged to
the ®nal agreement factors R=0.051, and R =0.055,
o
w
for 213 parameters and 2633 observed re¯ections with
F>3s(F ); data-to-parameter ratio being 12.36. The
electron density of the largest di€erence peak was found
1
-Octanol/water partition coecients
Ê
� 3
to be 0.23 eA , while that of the largest di€erence hole
Partition coecients were determined at room tem-
perature by high speed counter current chromatography
(HSCCC) on a centrifugal partition apparatus (P.C.,
Potomac, MD, USA), equipped with analytical coil No.
Ê
was 0.20 eA . In the crystals of 12, the molecules are
� 3
...
connected by intermolecular hydrogen bonds H(12)
Ê
O(15) [2.1 A] to form a zigzag chain parallel to [001].
1
4 (volume 78 mL, I.D. 1.7 mm). The chromatograph
Crystal data for 13. The crystal chosen for X-ray inves-
tigations was a clear colorless prism with the approx-
imate dimensions 0.45Â0.50Â0.30 mm. Data were
collected on a Siemens R3m/V four-circle di€ractometer
using graphite monochromatic Mo-Ka radiation.
was connected to two P 700 LC pumps (Latek, Eppel-
heim, Germany). Pump A was used for the delivery of
the mobile phase (1-octanol or cyclohexane), and pump
B for the delivery of the stationary phase (0.01 M solu-
tion of 3-morpholinopropanesulfonate, pH 7.4). Both,
the organic and the aqueous phases were mutually
saturated. Concentrated or saturated solutions of each
THbC tested were prepared in the mobile phase and
introduced to the chromatograph via a 0.5-mL sample
loop (Merck, Darmstadt, Germany). The THbCs were
�
1
C H Cl F N O (385.60 g mol ) crystallizes in the
2
1
4
10
3
3
monoclinic system, space group P2 /a, with a=12.091
1
Ê
ꢁ
1), b=12.125 (1), c=10.860 (1) A, b=98.72 (1) ,
3
(
Ê
� 1
V=1573.6 (3) A , Z=4, m(Mo-K )=0.62 mm , and
a
�
3
Dcalcd=1.627 g cm . The unit cell parameters were

G. Bringmann et al. / Bioorg. Med. Chem. 8 (2000) 1467±1478
1477
detected at 254 nm using an SPD-6AV UV detector
Shimadzu, Kyoto, Japan). The chromatograms were
expressed as mean (%)ÆSEM of the peak height of the
catechol signal recorded at the contralateral side. The
paired Student's t-test was used to test the statistical
signi®cance. After the completion of the voltammetric
measurements, the rats were perfused with 4% bu€ered
formaldehyde solution. Brain slices (20 mm) were per-
formed and stained with eosin in order to verify the
exact position of the glass micro cannula and the carbon
®bre electrodes.
(
recorded with a Model SP 4290 integrator (Spectra
Physics, San Jose, CA, USA). Measurements were per-
formed using operation conditions described in litera-
4
8�50
ture,
but modi®ed in the following manner: The
rotating coil was ®rst ®lled with the mobile phase, and
then the desired volume of the stationary phase was
added to the coil by displacing the mobile phase. The
installed coil was used with a volume ratio of mobile
and stationary phase of ca. 1:2. The ¯ow-rate of the
eluent (setting 3 mL/min) was measured to be 2.5 mL/
min for the 1-octanol/water system, and 2.15 mL/min
for the cyclohexane/water system. The speed of rotation
was set to 900 rpm. The highly lipophilic anthracene was
taken as the non-retained compound to assess the col-
Inhibition of mitochondrial respiration enzymes
Rat brain homogenates and submitochondrial particles
(SMPs) from isolated rat liver mitochondria were pre-
pared as described previously.21 Enzyme activities in rat
brain homogenates and SMPs were assayed in a ®nal
ꢁ
umn dead time, t . The 1-octanol/water partition coe-
0
volume of 500 mL at 30 C using a Kontron Uvicon 943
cient P of a given THbC (at pH 7.4) was calculated
according to the equation log P=log [V /(V � V )], with
spectrophotometer. Mitochondrial respiration on SMPs
and intact mitochondria were measured polaro-
graphically using a Clark-type oxygen electrode (Rank
s
r
0
V being the dead volume (V =¯owÂt ), V being the
0
0
0
s
ꢁ
volume of the stationary phase, and V the retention
r
Brothers, England) at 30 C with 0.25 mg protein/mL.
volume of the sample (V =¯owÂt , with t being the
The IC50 respectively IC100 values, de®ned as the con-
centrations required to inhibit NADH-ubiquinone
reductase activity (complex I; EC 1.6.99.3) by 50%
respectively 100% were determined after 5 min of pre-
incubation with the inhibitors. NADH-linked oxidation
was measured by adding NADH to a concentration of
0.28 mM. All toxins were dissolved in methanol. The
total volume of the organic solvent did not exceed 5%
(v/v). At this concentration, methanol did not signi®-
cantly a€ect the enzymes tested. Protein concentrations
were determined according to the method of Lowry et
r
r
r
retention time of the sample).
In vivo pulse voltammetry after intranigral application of
the THꢀCs 1±5. Adult male Han-Wistar rats (body
weight: 280±320 g) were housed with free access to food
and water on a 12/12 light/dark cycle (room tempera-
ꢁ
ture: 23 C, humidity: 60%) for a period of 6 weeks. The
animals were anaesthetized with chloral hydrate (400
mg/kg, i.p.) prior to the stereotactic implantation of a
glass micro cannula attached to a Hamilton syringe into
the right substantia nigra pars compacta (SNpc) using
55
al. using fat-free bovine serum albumin as standard.
5
2
coordinates according to Paxinos and Watson (from
interaural zero: anterior=4.0 mm, lateral=1.9 mm, ver-
tical=2.0 mm). Intranigral injection (for THbCs: 10 mg
dissolved in 2 mL of 10% ethanol; control experiment:
Acknowledgements
This investigation was supported by the Bundesmini-
2
mL of 10% ethanol) was performed over a period of
sterium fu
Technologie (Fo
KL 9405; `Neurotoxine und Neuroprotektion: Hydro-
phile radikalische Schaden und Chloral-abgeleitete
È
r Bildung, Wissenschaft, Forschung und
10 min. The cannula was subsequently removed 2.5 min
È
rderkennzeichen 01 KL 9404/2 and 01
after the end of the toxin application, and the skin was
immediately closed with sutures. The rats were then
single housed with food and water ad libitum. One week
after toxin treatment, carbon ®bre microelectrodes were
implanted into the striata of both hemispheres (from
È
endogene Neurotoxine bei Morbus Parkinson'), and by
the Fonds der Chemischen Industrie. Our special thanks
are due to Dr. R. God for synthetic contributions to this
®eld, and to Professor K.-H. Sontag (Max-Planck-
interaural line: anterior=+8.5 mm, lateral=Æ 3.5 mm,
vertical=+4.5 mm)52 in anaesthetized rats (400 mg/kg
Institut fu
many) for fruitful and intensive discussions. Moreover,
we are indebted to Dipl.-Chem. M. Munchbach and
È
È
r Experimentelle Medizin, Gottingen, Ger-
of chloral hydrate, i.p.). Subsequently, 40 min after the
implantation procedure, the extracellular levels of
È
3,4-dihydroxyphenylacetic acid (DOPAC) were assayed
Dipl.-Chem. J. Kraus for skilful technical help. A gen-
erous gift of ubiquinone-1 by Eisai Chemical Co.
as a measure of the dopamine metabolism by pulse
voltammetry as described previously.30,31,53 The volt-
ammetric peak 2 at+100 mV was obtained by oxidation
of the catechol moiety present in dopamine and
DOPAC. The pharmacological characterization of this
peak with the monoamine oxidase inhibitor pargyline
(
Tokyo, Japan) is gratefully acknowledged.
References and Notes
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