Facile synthesis and in vitro properties of 1-alkyl- and 1-alkyl-N-propargyl-1,2,3,4-tetrahydroisoquinoline derivatives on PC12 cells

Article abstract of DOI:10.1016/j.ejmech.2009.04.035

The synthesis of several 1-alkyl-1,2,3,4-tetrahydroisoquinolines, which may play an important role in Parkinson's disease, has been achieved by modified Pictet-Spengler cyclization of the formyliminium ion. The direct cytotoxicity and preventative effects

Full text of DOI:10.1016/j.ejmech.2009.04.035

European Journal of Medicinal Chemistry 44 (2009) 4034–4043
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Facile synthesis and in vitro properties of 1-alkyl- and 1-alkyl-N-propargyl-
1,2,3,4-tetrahydroisoquinoline derivatives on PC12 cells
Michikazu Kitabatake a, Junko Nagai a, Kenji Abe b, Yukihiro Tsuchiya c, Keita Ogawa a,
,
Takashi Yokoyama a, Kunihiko Mohri a, Kyoji Taguchi a, Yoshie Horiguchi a
*
a Showa Pharmaceutical University, 3-3165 Higashitamagawagakuen, Machida, Tokyo 194-8543, Japan
b School of Pharmaceutical Sciences, Ohu University, 31-1 Tomitamachi, Koriyama, Fukushima 963-8611, Japan
c Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology
and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of several 1-alkyl-1,2,3,4-tetrahydroisoquinolines, which may play an important role in
Parkinson’s disease, has been achieved by modified Pictet–Spengler cyclization of the formyliminium ion.
The direct cytotoxicity and preventative effects towards MPPþ-mediated death of PC12 cells were esti-
mated. The cytotoxicities of 1-alkyl-TIQs were apoptotic and depended on their lipophilic properties.
Conversely, introducing the N-propargyl substituent reduced cytotoxicity. 1-Methyl-, 1-methyl-N-prop-
argyl- and 1-cyclopropyl-TIQ partially inhibited MPPþ-induced cell death, whereas relatively large alkyl
substituents at the first position did not enhance the viability of PC12 cells. In summary, our findings
suggest a crucial role for the N-propargyl functional group for the effective reduction of cytotoxicity, and
show the importance of size and lipophilicity of substituents at the 1-position of 1-alkyl-TIQs.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Received 27 August 2008
Received in revised form
18 April 2009
Accepted 23 April 2009
Available online 5 May 2009
Keywords:
Pictet–Spengler reaction
1,2,3,4-Tetrahydroisoquinoline
Structure–activity relationships
Cytotoxicity
Parkinson’s disease
Cell protective effect
Propargyl group
1. Introduction
TIQ- and 1-benzyl-TIQ-induced behavioral abnormalities in rodents
[3,5]. These findings suggest that the substituents attached to the 1-
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP)
is
position of the TIQ ring system may influence the pharmacological
properties and biological activities of TIQ derivatives.
a remarkably selective dopaminergic neurotoxin that produces
a clinical syndrome similar to Parkinson’s disease (PD) [1]. However,
because MPTP is an exogenous compound, it is possible that similar
endogenous toxins accumulate in the central nervous system and
cause PD. It has been reported that 1,2,3,4-tetrahydroisoquinoline
(TIQ) and its derivatives, which possess structural similarity to
MPTP, may act as endogenous or environmental neurotoxins
causing PD [2,3]. One TIQ derivative, 1-benzyl-TIQ, exists naturally
in animal brain tissue and was found to induce Parkinsonism in
monkeys and mice [3,4]. 1-Benzyl-TIQ has been implicated as
a neurotoxin causing PD in humans because its concentration in the
cerebro-spinal fluid of Parkinsonian patients is approximately three
times that found in healthy individuals [3]. In contrast,1-methyl-TIQ
is present in normal mouse brain, and has been shown to prevent
Methods for synthesizing the isoquinoline ring system include
the Pictet–Spengler reaction [6], the Bischler–Napieralski reaction
[7] and the Pomeranz–Fritsch reaction [8,9]. In addition to these
methods, the synthesis of 1-methyl-TIQ,1-phenyl-TIQ and 1-benzyl-
TIQ using a Pummerer-type cyclization reaction as the key step has
been previously reported [10,11]. Of these reactions, the Pictet–
Spengler reaction and Pummerer-type cyclization reaction have
proven effective for synthesizing TIQs [12–14]. The simple endoge-
nous TIQs are, in fact, proposed to be biosynthesized from imines
derived from 2-phenylethylamine and formaldehyde, acetaldehyde
or phenylacetaldehyde through a mechanism similar to that in the
Pictet–Spengler reaction [15]. This biogenetic mechanism is not
necessarily consistent with the observation that the Pictet–Spengler
cyclization is very sensitive to the aromatic substituents of the 2-
arylethylamine moiety of imines, and that an electron-donating
hydroxyl or alkoxy group at the para position of the cyclization site is
the minimum requisite for cyclization [16–18]. Cyclization of an
* Corresponding author. Tel./fax: þ81 42 721 1570.
E-mail address: horiguti@ac.shoyaku.ac.jp (Y. Horiguchi).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.04.035

M. Kitabatake et al. / European Journal of Medicinal Chemistry 44 (2009) 4034–4043
4035
O
MeO
MeO
MeO
MeO
HCOOH-
Ac2O
R1
R2
MeO
MeO
NH2
MeO
MeO
CF3COOH
+
2
N
CHO
R2
N
N
R1 R2
3
CHO
Ti(O-iPr)4
R1
R1
R2
1
5
4
Fig. 1. Synthesis of N-formyl 1,1-disubstituted 1,2,3,4-tetrahydroisoquinlines via a modified Pictet–Spengler reaction, described previously.
imine that lacks an electron-donating group in the aromatic ring
requires drastic reaction conditions. Yokoyama and coworkers
reported the use of the very strong acid, trifluoromethane sulfonic
acid (TFSA), to generate the dicationic species to promote the
cyclization reaction for the synthesis of 1-substitued TIQs [19].
Recently, we developed a highly efficient method for synthe-
sizing N-formyl 1,1-disubstituted TIQs (3) using a modified Pictet–
Spengler reaction. In this reaction, cyclization was accelerated by
the presence of the N-formyliminum ion (5) intermediate formed in
situ from imine (4) of arylethylamine (1) using acetic-formic
anhydride [20]. The high reactivity of the formyliminium ion (5)
was apparent since 5 readily caused cyclization in the presence of
the relatively weak acid, trifluoroacetic acid (TFA) (Fig. 1).
We also previously showed that the cytotoxicity of various 1-
alkyl-TIQ derivatives, and the size and electron-donating properties
of its functional groups, may influence the cytotoxic properties of
the compound [21].
In the present study, we describe a convenient protocol for the
synthesis of several 1-alkyl-TIQs (11) using a modified Pictet–
Spengler reaction, and estimate their cytotoxicity effects on PC12
cells. Selegiline, an N-propargyl-containing compound with some
structural resemblance to TIQ (12), is used as a therapeutic drug for
PD, and it has been reported that the N-propargyl functional group
plays a crucial neuroprotective role [22]. Thus, we also synthesized
1-alkyl-N-propargyl-TIQ and evaluated the effects of this and other
TIQ derivatives on improving cell viability.
Hydrolytic deprotection of the N-formyl group gave the corre-
sponding 1-alkyl-TIQ (11). Moreover, TIQs (12) were generated
from propargylamines such as Selegiline, which is a potent neu-
roprotective agent, by the propargylation of the nitrogen atom at
the 2-position with propargylbromide and cesium carbonate in
acetonitrile (Fig. 2).
The synthesized TIQs (11, 12) were biochemically evaluated for
their neuroprotective effects.
3. Biological results and discussion
3.1. Effects of 1-alkyl-TIQ on PC12 cell viability
The effect of each 1-alkyl-TIQ derivative on the viability of PC12
cells was estimated by comparing viability to that of untreated
control cells (whose viability was defined as 100%). Cell viability
was unaffected except in the highest concentration of 1-methyl-
TIQ, the smallest C-1 substituent derivative. However, potent
cytotoxicity was observed proportional to the size of the linear
extended alkyl C-1 substituent. 1-Normal (n)-hexyl-TIQ, the largest
substituent TIQ derivative investigated in the present study,
resulted in significantly reduced cell viability except at the lowest
concentration tested (Fig. 3A).
Annulatedsubstituentsatthe1-positionalsoreducedcellviability,
depending on the carbon number in the substituent. Similar to 1-n-
hexyl-TIQ,1-cyclo (cy)-hexyl-TIQ resulted in significantly reduced cell
viability except at the lowest concentration tested (Fig. 3B).
We calculated the concentration resulting in 50% toxicity (TC50
)
2. Chemistry
and computer calculated the log P value (used as a parameter for
liposolubility) for every TIQ derivative; the correlation found
between these parameters is shown in Fig. 4. This result suggests
that the cytotoxicity of a 1-alkyl-TIQ derivative is proportional to its
liposolubility.
Modified Pictet–Spengler reaction conditions were applied to
imine (8) obtained from 2-phenylethylamine (6) and aldehyde (7).
The formyliminium ion (9a) was prepared by condensation of 6 and
benzaldehyde (7a) by heating at 80 ꢀC in titanium(IV) isopropoxide
for 1 h, followed by treatment with acetic-formic anhydride
prepared from formic acid (300 eq) and acetic anhydride (100 eq)
at 70 ꢀC for 3 h. and the in situ formed 9a was treated with TFA
(200 eq) by heating at 70 ꢀC for 18 h to produce N-formyl-1-phenyl
TIQ (10a) in 99% yield. The use of formic acid instead of TFA gave
10a in only 7% yield.
3.2. Effects of 1-alkyl-N-propargyl-TIQ derivatives on PC12
cell viability
1-Alkyl-TIQ derivatives substituted with the N-propargyl group
showed weaker cytotoxicity than 1-alkyl-TIQ derivatives, and
The formyliminium ion (9b–g) and (9j, k) prepared in situ from 6
and other acyclic and cyclic aldehydes such as paraldehyde (7b),
propionaldehyde (7c), n-butylaldehyde (7d), n-pentanal (7e), n-
hexanal (7f), n-heptanal (7g), cyclopentanecarbaldehyde (7j) and
cyclohexanecarbaldehyde (7k) gave corresponding N-formyl
1-alkyl-TIQs (10b–g and 10j, k) in excellent to moderate yields
under similar reaction conditions. Product yields are summarized
in Table 1. A unique case is the reaction of 6 with cyclo-
propanecarbaldehyde (7h). This reaction yielded the cyclized
products 1-(3-hydroxypropyl) TIQ (10l) and 1-(3-acetoxypropyl)
TIQ (10m), formed by the acid catalyzed hydrolytic cleavage of the
cyclopropane ring followed by acetylation with acetic anhydride.
TIQs (10a–d) and (10j, k) obtained as described above were
identical with those obtained from the Pummerer-type cyclization
reactions described previously [21].
Table 1
Synthesis of N-formyl 1-substituted TIQs 10.
Run
Aldehyde 7
Product yields (%)
R
1
2
3
4
5
6
7
8
9
10
7a
7b
7c
7d
7e
7f
7g
7h
7j
Ph
methyl
ethyl
n-propyl
n-butyl
n-pentyl
n-hexyl
cyclopropyl
cyclopentyl
cyclohexyl
10a
10b
10c
10d
10e
10f
10g
10l
99
57
49
48
76
87
90
64
71
95
10m
33
10j
10k
7k

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M. Kitabatake et al. / European Journal of Medicinal Chemistry 44 (2009) 4034–4043
O
NH2
R
H
HCOOH-
Ac2O
7
CF3COOH
+
N
H
N CHO
N
Ti(O-iPr)4
CHO
R
6
H
R
R
10
9
8
Br
NaOH
10
N
NH
in EtOH
Cs2CO3 in CH3CN
R
12
R
11
7 12
-
for Compounds
a
b
c
d: n-propyl,e: n-butyl,f: n-pentyl, g: n-hexyl
: Ph, : methyl, : ethyl,
h: cyclopropyl, i: cyclobutyl, j: cyclopentyl, k: cyclohexyl,
l: 3-hydroxypropyl, m: 3-acetoxypropyl
Fig. 2. Synthesis of 1-alkyl-2-propargyl-1,2,3,4-tetrahydroisoquinolines 12, using a modified Pictet–Spengler cyclization reaction.
significantly reduced viability of PC12 cells was observed only at
the highest concentration tested or, for several derivatives, also at
the second highest concentration tested (Fig. 5A and B). This
suggests that the N-propargyl group helps reduce the cytotoxicity
of 1-alkyl-TIQ derivatives. This cytotoxicity-decreasing effect
became evident when N-propargyl derivatives were compared to
potent cytotoxic 1-alkyl-TIQs. For example, N-propargylation
changed the properties of two compounds that induced cell death
1-n-alkyl-TIQ
A
NH
CH3
NH
NH
NH
NH
NH
120
100
80
60
40
20
0
**
**
**
**
**
**
**
**
**
**
****
**
**
**
**
Drug concentration (mM)
1-cy-alkyl-TIQ
B
NH
NH
NH
NH
120
100
80
60
40
20
0
*
**
*
**
**
**
**
**
**
**
**
**
Drug concentration (mM)
Fig. 3. Cytotoxic effects of various 1-alkyl-TIQs on cultured PC12 cells. Cells were exposed to each compound for 48 h, and cell viability is presented as a percentage of the untreated
control cells (defined as 100% viable). Data are expressed as means ꢁ standard error (S.E., n ¼ 3–6). A: normal (n)-alkyl substituents and B: cyclo (cy)-alkyl substituents of TIQs were
estimated. Asterisks show significant differences from control cell viability (*P < 0.05, **P < 0.01).

M. Kitabatake et al. / European Journal of Medicinal Chemistry 44 (2009) 4034–4043
4037
group at the 1-position of 1-alkyl-TIQ derivatives, and the cyto-
toxicity-reducing effect of the N-propargyl substituent.
3
2
NH
3.3. Apoptotic induction of 1-alkyl-TIQ
R
At high concentrations, 1-n-hexyl- and 1-cy-hexyl-TIQ were
clearly cytotoxic (as shown in Fig. 3A and B). The ability of these
compounds and 1-methyl-4-phenylpyridinium ion (MPPþ) to
induce DNA damage in individual cells was evaluated using the
comet assay. Fig. 7A depicts untreated control cells, and typical
migrated cell nuclei with fragmented DNA caused by treating with
MPPþ. Small portions of DNA remain at the origin, but most of the
DNA has fragmented and migrated to form a ‘comet’ tail. Diffuse,
migrated DNA is indicative of apoptosis in a cell. In the present
study, a cell exhibiting such a ‘comet’ tail was considered to be in
the apoptotic state, and the whole length of the ‘comet’ was
measured to estimate the cytotoxicity of the 1-alkyl-TIQ derivatives
described above.
R
Symbol
methyl-
ethyl-
1
0
n-propyl-
n-butyl-
n-pentyl-
n-hexyl-
cy-propyl-
cy-butyl-
cy-pentyl-
cy-hexyl-
r=0.942
1
10
CLogP
-1
Fig. 4. Correlation diagram between log P values (as an indicator of liposolubility) and
50% toxicity concentration (TC50) of 1-alkyl-TIQ derivatives. n: normal, cy: cyclo-.
All of the MPPþ, 1-n-hexyl-TIQ and 1-cy-hexyl-TIQ compounds
induced DNA fragmentation and extended the length of the ‘comet’
(Fig. 7B). To estimate cytotoxicity, we documented the length of
at high concentrations, 1-n-butyl-TIQ and 1-cy-hexyl-TIQ. The
cytotoxicity of these compounds was almost completely abolished
by introducing the propargyl group at the N-position of the TIQ ring
(Fig. 6). Thus, it appears that a crucial relationship exists between
the liposolubility property (C-number) or the size of the functional
each ‘comet’ at intervals of 30
‘comets’ were between 60 and 90
1-cy-hexyl-TIQ treated cells, most ‘comets’ were 150–180
m
m. In MPPþ treated cells, most
m long. In 1-n-hexyl-TIQ and
m and
m
m
1-n-alkyl-N-propargyl-TIQ
A
N
N
N
N
CH3
120
100
80
**
60
40
20
0
Drug concentration (mM)
1-cy-alkyl-N-propargyl-TIQ
B
N
N
N
N
120
100
80
*
**
*
**
**
60
40
20
0
Drug concentration (mM)
Fig. 5. Cytotoxic effects of N-propargyl-substituted 1-alkyl-TIQ (1-alkyl-N-propagyl-TIQ) derivatives on cultured PC12 cells. Each compound was incubated for 48 h with the cells,
and cell viability is presented as a percentage of the untreated control (defined as 100% viable). Data are expressed as means ꢁ standard error (S.E., n ¼ 3–5). A: normal (n)-alkyl
substituents and B: cyclo (cy)-alkyl substituents of N-propargyl-TIQs were estimated. Asterisks show significant differences from control group viability (*P < 0.05, **P < 0.01).

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M. Kitabatake et al. / European Journal of Medicinal Chemistry 44 (2009) 4034–4043
A
B
1-cy-hexyl-TIQs
1-n-butyl-TIQs
120
100
80
60
40
20
0
120
100
80
60
40
20
0
N
N
NH
NH
0.01
0.05
0.1
0.5
1.0
0.01
0.05
0.1
0.5
1.0
Drug concentration (mM)
Drug concentration (mM)
Fig. 6. Effect of N-propargyl substituent in 1-alkyl-TIQ derivatives on PC12 cell viability. N-Propargylation clearly decreases cytotoxicity of 1-alkyl-TIQ derivatives. n: normal,
cy: cyclo-.
120–150
m
m long, respectively. Increased ‘comet’ length suggested
3.6. Effect of 1-methyl-N-propargyl- and 1-cy-propyl-N-propargyl-
TIQ on monoamine oxidase (MAO) activity in vitro
increased DNA fragmentation. Thus, these results indicate that both
1-n-hexyl-TIQ and 1-cy-hexyl-TIQ derivatives induce DNA damage
in PC12 cells. This apoptotic DNA damaging effect may be related to
the liposolubility (C-number) or size of the functional group at the
1-position, and may contribute to the cytotoxicity of TIQ derivatives.
Finally, we examined the effect of twoTIQ derivatives that improved
cell viability (1-methy-N-propargyl- and 1-cy-propyl-N-propargyl-TIQ)
Typical apoptotic comet tail
A
3.4. Effects of 1-alkyl- and 1-alkyl-N-propargyl-TIQ derivatives
on MPPþ-induced cell death
+
control
MPP
comet length
The ability of various 1-alkyl- and 1-alkyl-N-propargyl-TIQ
derivatives to protect against MPPþ-induced cell death was evalu-
ated. Results are expressed as the number of surviving cells treated
with TIQ derivatives compared to the number of cells surviving
following MPPþ treatment alone (defined as 100%). In the 1-alkyl-TIQ
(1-methyl-, ethyl-, cy-propyl- and cy-butyl-TIQ) group, only 1-
methyl-TIQ significantly increased the number of surviving cells
(Fig. 8A). The effects of N-propargyl-substituted 1-alkyl-TIQ deriva-
tives on MPPþ-induced cell death were also investigated. Statistically
significant improvements in cell viability were observed upon
treatment with 1-methyl-N-propargyl- and 1-cy-propyl-N-prop-
argyl-TIQ. When the substituent was increased by one carbon at the
1-position, i.e. 1-ethyl-N-propargyl-TIQ and 1-cy-butyl-N-propargyl-
TIQ, the cells were no longer protected (Fig. 8B). The importance of
the liposolubility properties of 1-alkyl-N-propargyl-TIQ derivatives
and their ability to reduce cytotoxicity will be investigated in future
studies. However, the present results indicate that the size of the C-1
substituent, rather than its liposolubility property, is the most
important parameter determining the ability of 1-alkyl-N-propargyl-
TIQs to protect cells against MPPþ-induced death.
100 µm
100 µm
Frequency in each of comet length
B
100
90
80
70
60
50
40
30
20
10
0
control
MPP+
1
-n-hexyl-TIQ
1-cy-hexyl-TIQ
3.5. Depressive effect of 1-alkyl- and 1-alkyl-N-propargyl-TIQ
derivatives towards caspase activity
TIQ derivatives that improved cell viability (i.e., 1-methyl-TIQ,
1-methy-N-propargyl- and 1-cy-propyl-N-propargyl-TIQ) were
investigated further. The results are expressed as a ratio of cas-
pase-3,7 activity, with untreated cells defined as 100%. Caspase-
3,7 activity in MPPþ-treated cells was significantly increased
(about 1.7 times higher) compared to untreated control cells. The
presence of 1-methyl-, 1-methyl-N-propargyl- and 1-cy-propyl-N-
propargyl-TIQ with MPPþ almost entirely prevented the increased
activity of caspase-3,7 (Fig. 9). This result indicates that MPPþ-
induced apoptosis and apoptotic inhibition by these TIQ deriva-
tives on PC12 cells are due at least in part to their effect on
caspase-3,7 activity.
<30
60-90
120-150
90-120 150-180
Range of comet length (µm)
180-210
240-270
300-330
330-360
30-60
210-240
270-300
Fig. 7. ‘Comet’ tail formation due to fragmentation of DNA in PC12 cells caused by 3 h
exposure to 1 mM MPPþ, 1-normal (n)-hexyl- and 1-cyclo (cy)-hexyl-TIQ. A: The
absence of ‘comets’ in control cells and typically observed ‘comets’ in cells treated with
MPPþ. Images were obtained using a fluorescence microscope at ꢂ200 magnification
and a wavelength of 568 nm. In ‘comets’, a small portion of the DNA remains at the
origin, but most of the DNA fragments migrate to form the ‘comet’ tail. In the present
study, damaged cells such as these are termed apoptotic damaged cells. B: The
measured length of a ‘comet’ at intervals of 30
mm in cells that produced ‘comet’ tails
upon treatment with MPPþ, 1-n-hexyl- and 1-cy-hexyl-TIQ.

M. Kitabatake et al. / European Journal of Medicinal Chemistry 44 (2009) 4034–4043
4039
A
1-alkyl-TIQ
180
160
140
120
N
H
NH
CH3
NH
NH
**
**
*
*
100
80
Drug concentration (mM)
1-alkyl-N-propargyl-TIQ
B
180
160
140
120
N
N
N
N
CH3
**
**
**
**
**
**
**
100
80
Drug concentration (mM)
Fig. 8. Inhibitory effect of 1-alkyl- and 1-alkyl-N-propargyl-TIQ derivatives on MPPþ-induced cell death. Each compound was incubated for 48 h with cells together with MPPþ
(0.25 mM), and cell viability is presented as a percentage of the cells incubated with MPPþ alone (defined as 100%). Data are expressed as means ꢁ standard error (S.E., n ¼ 3–4).
1-Alkyl-TIQs (A) and 1-alkyl-N-propargyl-TIQs (B) were estimated. Asterisks show significant differences from control cell viability (*P < 0.05, **P < 0.01).
on MAO activity in vitro. Selegiline was used as a positive control to
selectively inhibit MAO-B. Results are expressed as the ratio of MAO
activity, in which cells treated with vehicle are defined as 100% active.
Selegilinepotentlyinhibits MAO-Bactivity inthe caseofbothsubstrates
(tyramine and benzylamine) but does not affect MAO-A activity. Both 1-
methy-N-propargyl- and 1-cy-propyl-N-propargyl-TIQ did not influ-
ence MAO activity, irrespective of their subtype and substrate (Table 2).
These results show that these compounds, which help protect PC12 cell
viability from MPPþ cytotoxicity, may not inhibit dopaminergic
metabolism. Selegiline helps retain dopamine at synapses due to its
specific inhibition of MAO-B activity, and has been used as a putative
neuroprotective agent to treat PD [23]. However, since 1-methy-N-
propargyl- and 1-cy-propyl-N-propargyl-TIQ did not change MAO-A
and MAO-B activities, it is possible that the cytoprotective effectof these
compounds may follow a different mechanism(s) from Selegiline and
does not depend on inhibition of MAO-B. Designing compounds that
cytoprotect cells from cytotoxic substances without MAO inhibition
may lead to new drugs for treating PD.
phenylethylamine and aldehyde using a modified Pictet–Spengler
reaction. The cyclization reaction was accelerated by the presence
of N-formyliminium ion (9) as an intermediate, was carried out
using a one-pot procedure, and was shown to be a convenient and
effective method for preparing various TIQs.
The cytotoxic properties of 1-alkyl-TIQ compounds on PC12 cells
depended on their liposolubility properties, and introducing an
+
MPP
1-methyl-TIQ
1-methyl-N-propargyl-TIQ
1-cy-propyl-N-propargyl-TIQ
0
50
100
150
200
250
Caspase-3,7 activity (% of control)
Fig. 9. Inhibitory effect of 1-methyl-, 1-methyl-N-propargyl- and 1-cy-propyl-N-
propargyl-TIQ on MPPþ-enhanced caspase-3,7 activity. Enzyme activity in untreated
cells is defined as 100%. MPPþ significantly enhanced caspase-3,7 activity and co-
addition of the above TIQ derivatives clearly depressed this activity. Data are expressed
as means ꢁ standard error (S.E., n ¼ 3–4). Asterisks show significant differences
compared to the untreated cells (**P < 0.01) and the MPPþ treated cells (#P < 0.05).
4. Conclusions
A number of 1-alkyl- and 1-alkyl-N-propargyl-TIQ derivatives
potentially useful for treating PD were synthesized from

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M. Kitabatake et al. / European Journal of Medicinal Chemistry 44 (2009) 4034–4043
Table 2
5.1.1.1. Analytical data for the new compounds
Effect of TIQ derivatives on MAO activity.
5.1.1.1.1. 1-n-Butyl-N-formyl-1,2,3,4-tetrahydroisoquinoline (10e). Yield:
76%, asa colorless gumeluted with AcOEt:hexane ¼ 1:4. IR:1670.1H-
NMR: 0.90, 0.94 (total 3H, each t, J ¼ 7 Hz, CH3), 1.22–1.49 (4H, m, n-
butyl), 1.74–1.88 (2H, m, n-butyl), 2.75–3.15, 3.52–3.62, 3.66–3.73,
4.44–4.50 (total 4H, each m, C3-H and C4-H), 4.45–4.50, 5.38–5.43
(total 1H, each m, C1-H), 7.08–7.26, 7.36–7.49 (total 4H, each m, Ar),
8.20, 8.22 (total 1H, each s, CHO). LR-FABMS m/z: 218 (MHþ), 218
(base peak). HR-FABMS: Calcd for C14H20NO: 218.1544, found:
218.1544.
Substrate
Enzyme MAO activity (% of vehicle control)
Vehicle
Selegiline
1-methyl-N-
1-cy-propyl-N-
propargyl-TIQ propargyl-TIQ
Tyramine
MAO-B 100.0 ꢁ 0.7
4.8 ꢁ 0.1** 100.9 ꢁ 0.5
98.4 ꢁ 0.4
97.5 ꢁ 0.8
96.2 ꢁ 1.2
Benzylamine MAO-B 100.0 ꢁ 1.0 20.7 ꢁ 0.3**
99.4 ꢁ 0.8
97.5 ꢁ 1.2
Tyramine
MAO-A 100.0 ꢁ 1.1 94.2 ꢁ 1.0
Data are expressed as means ꢁ standard error (S.E., n ¼ 8). Asterisk shows significant
difference from vehicle control (**P < 0.01).
5.1.1.1.2. N-Formyl-1-n-pentyl-1,2,3,4-tetrahydroisoquinoline(10f).
Yield: 87%, as a colorless gum eluted with AcOEt:hexane ¼ 1:4. IR:
1654. 1H-NMR: 0.88, 0.90 (total 3H, each t, J ¼ 7 Hz, CH3), 1.22–1.49,
1.68–1.86 (total 8H, each m, n-pentyl), 2.75–3.15, 3.12–3.61, 3.66–
3.73, 4.45–4.51 (total 4H, each m, C3-H and C4-H), 4.45–4.51, 4.38–
5.43 (total 1H, each m, C1-H), 7.08–7.80 (4H, m, Ar), 8.21, 8.23 (total
1H, each s, CHO). LR-FABMS m/z: 232 (MHþ), 232 (base peak). HR-
FABMS: Calcd for C15H22NO: 232.1702, found: 232.1699.
5.1.1.1.3. N-Formyl-1-n-hexyl-1,2,3,4-tetrahydroisoquinoline (10g).
Yield: 90%, as a yellow oil eluted with AcOEt:hexane ¼ 1:6. IR: 1672.
1H-NMR: 0.87, 0.89 (total 3H, each t, J ¼ 7 Hz, CH3), 1.22–1.40, 1.79–
1.86 (total 10H, each m, n-hexyl), 2.74–3.15, 3.51–3.60, 3.61–3.72,
4.44–4.50 (total 4H, each m, C3-H and C4-H), 4.44–4.49, 5.38–5.42
(total 1H, each m, C1-H), 7.07–7.22 (4H, m, Ar), 8.20, 8.22 (total 1H,
each s, CHO). LR-FABMS m/z: 246 (MHþ), 246 (base peak). HR-
FABMS: Calcd for C16H24NO: 246.1858, found: 246.1855.
N-propargyl substituent reduced the cytotoxicity of 1-alkyl-TIQ. In
addition, the contribution of apoptotic DNA damage was confirmed
for 1-alkyl-TIQ-induced cytotoxicity. Although only 1-methyl-TIQ
prevented MPPþ-induced cell death, introducing the N-propargyl
group enhanced the cell-protecting effects of 1-cy-propyl-TIQ.
However, such enhancement was not observed when relatively
large substituents were introduced, showing that the size of the
functional group at the 1-position of 1-alkyl-TIQ derivatives is
important. In conclusion, these results suggest that the N-propargyl
functional group is crucial for reducing cytotoxicity, and demon-
strate the importance of the size or liposolubility of substituents at
the 1-position of 1-alkyl-TIQ derivatives. Further investigations into
the synthesis and biological activity of TIQ derivatives with N-
propargyl or different C-1 substituents may help develop new
agents for treating PD.
5.1.1.1.4. 2-Formyl-1-(3-hydroxypropyl)-1,2,3,4-tetrahydroisoquin-
oline(10l). Yield: 64% as a colorless prisms from AcOEt, mp 113–
114 ꢀC. IR: 3340, 1655. 1H-NMR: 1.57–1.98 (4H, m, –CH2CH2CH2OH),
2.75–3.03 (2H, m, C4-H), 3.07–3.17, 3.53–3.63 (total 2H, each m, C3-
H), 3.68–3.77 (2H, m, –CH2CH2CH2OH), 4.54, 5.48 (total 1H, each
dd, J ¼ 7 Hz, 7 Hz, J ¼ 5 Hz, 9 Hz, C1-H), 7.08–7.32 (4H, m, –Ar), 8.23
(1H, s, –CHO). 13C-NMR: 27.8, 28.9 (CH2CH2CH2OH), 29.5, 29.7 (C4),
33.2, 33.3 (CH2CH2CH2OH), 34.2, 41.0 (C3), 50.9, 57.4 (C1), 61.9, 62.4
(CH2CH2CH2OH),126.3,126.6 (Ar),126.8 (Ar),127.1,127.4 (Ar),129.0,
129.2 (Ar), 132.6, 133.6 (C4a), 136.5, 136.7 (C8a), 161.6, 162.0 (CHO).
LRMS m/z: 219 (Mþ), 160 (base peak). HRMS: Calcd for C13H17NO2:
219.1260, found: 219.1271.
5.1.1.1.5. Acetic acid 3-(2-formyl-1,2,3,4-tetrahydroisoquinolin-1-
yl)-propyl ester(10m). Yield: 33% as a yellow gum. IR: 1736,1670.1H-
NMR: 1.69–1.98 (4H, m –CH2CH2CH2OC(O)CH3), 2.04, 2.06 (total 3H,
each s, –CH2CH2CH2OC(O)CH3), 2.77–3.16 (3H, m, C3-H and C4-H),
3.51–3.61, 3.69–3.75 (total 1H, each m, C3-H), 4.09–4.17, 4.45–4.49
(total 2H, each m, –CH2CH2CH2OC(O)CH3), 4.50–4.55, 5.42–5.47
(total 1H, each m, C1-H), 7.09–7.33 (4H, m, –Ar), 8.23, 8.24 (total 1H,
each s, –CHO).13C-NMR: 20.9, 21.0 (CH2CH2CH2OC(O)CH3), 25.5, 25.8
(CH2CH2CH2OC(O)CH3), 27.8, 29.6 (C4), 32.9, 33.1 (CH2CH2CH2O-
C(O)CH3), 34.2, 39.9 (C3), 50.5, 57.0 (C1), 63.6, 64.0 (CH2CH2CH2O-
C(O)CH3), 126.4, 126.6 (Ar), 126.7, 126.8 (Ar), 127.2 (Ar), 129.1, 129.3
(Ar), 132.8, 133.6 (C4a), 136.3, 136.4 (C8a), 161.4, 161.8 (CHO), 171.0,
171.1 (CH2CH2CH2OC(O)CH3). LRMS m/z: 261 (Mþ), 161 (base peak).
HRMS: Calcd for C15H19NO3: 261.1366, found: 261.1396.
5. Experimental procedure
5.1. Chemistry
Unless otherwise noted, the following methods were used.
Melting points were taken on a Yanagimoto SP-M1 hot-stage melting
point apparatus and were uncorrected. Infrared spectra were
measured with a HORIBA FT-710 Fourier transform infrared spec-
trophotometer; samples were cast as films for oils and gums, and
disks were prepared for solids using KBr. The x-axis on the spectra is
cmꢃ1. Nuclear magnetic resonance spectra were measured with
a JEOL JNM-AL300 NMR spectrometer (1H: 300 MHz, 13C: 75 MHz).
Samples were dissolved in chloroform-d with tetramethylsilane as
an internal standard. Results are expressed in terms of d value. High
resolution fast atom bomb ionization mass spectra were taken on
a JEOL HX-110A mass spectrometer using m-nitrobenzyl alcohol as
a matrix and a solvent, and results are shown as m/z.
5.1.1. General procedure for the synthesis of 1-alkyl-N-formyl-TIQ
10 using a modified Pictet–Spengler reaction
A solution of phenylethylamine (6) (5.52 mmol), aldehyde (7)
(4.60 mmol) and Ti(O-iPr)4 (6.90 mmol) was heated at 80 ꢀC for 1 h
under an argon atmosphere. To this reaction mixture a solution of
acetic-formic anhydride [prepared from formic acid (1.38 mol) and
acetic anhydride (0.46 mol)] was added at 0 ꢀC and then heated at
70 ꢀC for 3 h. The reaction mixture was cooled to 0 ꢀC again, tri-
fluoroacetic acid (0.92 mol) was further added to the solution and
then the resulting mixture was stirred for 16 h. The reaction
mixture was diluted with MeOH (50 mL) and passed through
a short silica gel column to remove TiO2. The eluent was concen-
trated in vacuo and diluted with CHCl3, washed with 10% NaOH
solution and brine and then dried over Na2SO4. After removal of the
solvent in vacuo, the residue was purified by chromatography on
silica gel to give 10. Yields are summarized in Table 1.
5.1.2. General procedure for the hydrolysis of 10
A solution of 10 (1.90 mmol) in EtOH (15 mL)–20% NaOH (15 mL)
wasrefluxed for 3.0 h. The reaction mixturewas diluted with H2O and
extracted with CHCl3. The product was purified by column chroma-
tography on SiO2 (AcOEt) to give 1,2,3,4-tetrahydroisoquinoline 11.
5.1.2.1. Analytical data for the new compounds
5.1.2.1.1. 1-n-Butyl-1,2,3,4-tetrahydroisoquinoline (11e) [24–28]
. Yield: 93%, as a pale yellow oil eluted with AcOEt:hexane ¼ 1:4,
HCl salt; colorless prisms from ether–MeOH, mp 127–128 ꢀC (lit.
[27] mp 143–144 ꢀC). 1H-NMR: 0.90–0.95 (3H, m, CH3), 1.30–1.50
(4H, m, (CH2)2), 1.66–1.91 (2H, m, CH2), 2.69–2.88 (2H, m, C4-H),
For compounds 10a–d and 10j, k, please refer to our previous
papers [11,12,21].

M. Kitabatake et al. / European Journal of Medicinal Chemistry 44 (2009) 4034–4043
4041
2.98 (1H, ddd, J ¼ 12, 7, 5 Hz, C3-H), 3.24 (1H, dt, J ¼ 12, 5 Hz, C3-H),
3.96 (1H, dd, J ¼ 9, 4 Hz, C1-H), 7.05–7.21 (4H, m, Ar). 13C-NMR: 14.1
(CH3), 22.9 (n-butyl), 28.4 (n-butyl), 30.0 (C4), 36.2 (n-butyl), 41.1
(C3), 55.8 (C1), 125.7 (C7), 125.7 (C6), 126.1 (C5), 129.2 (C8), 135.1
(C8a), 139.9 (C4a). LR-FABMS m/z: 190 (MHþ), 154 (base peak). HR-
FABMS: Calcd for C13H20N: 190.1556, found: 190.1604.
5.1.2.1.2. 1-n-Pentyl-1,2,3,4-tetrahydroisoquinoline (11f). Yield:
90%, as a pale yellow oil eluted with AcOEt:hexane ¼ 1:4, HCl salt;
colorless prisms from ether–MeOH, mp 83–84 ꢀC. 1H-NMR: 0.83–
0.87 (3H, m, CH3), 1.27–1.52 (6H, m, (CH2)3), 1.65–1.89 (2H, each m,
CH2), 2.69–2.88 (2H, m, C4-H), 2.99 (1H, ddd, J ¼ 12, 7, 5 Hz, C3-H),
3.24 (1H, dt, J ¼ 12, 5 Hz, C3-H), 3.96 (1H, dd, J ¼ 9, 4 Hz, C1-H).
7.05–7.21 (4H, m, Ar). 13C-NMR: 14.1 (CH3), 22.6 (n-pentyl), 25.9 (n-
pentyl), 30.1 (C4), 32.0 (n-pentyl), 36.5 (n-pentyl), 41.1 (C3), 55.8
(C1), 125.7 (C7), 125.7 (C6), 126.1 (C5), 129.2 (C8), 135.1 (C8a), 139.9
(C4a). LR-FABMS m/z: 204 (MHþ), 154 (base peak). HR-FABMS:
Calcd for C14H22N: 204.1754, found: 204.1756.
5.1.2.1.3. 1-n-Hexyl-1,2,3,4-tetrahydroisoquinoline (11g). Yield:
82%, as a pale yellow oil eluted with AcOEt:hexane ¼ 1:2, HCl salt;
colorless needles from ether–MeOH, mp 123–126 ꢀC. 1H-NMR: 0.86
(3H, t, J ¼ 7 Hz, CH3), 1.25–1.53, 1.79–1.90 (total 10H, each m, n-
hexyl), 2.69–2.88 (2H, m, C4-H), 2.98 (1H, ddd, J ¼ 12, 8, 5 Hz, C3-H),
2.24 (1H, dt, J ¼ 12, 5 Hz, C3-H), 3.95 (1H, dd, J ¼ 9, 4 Hz, C1-H),
7.05–7.17 (4H, m, Ar). 13C-NMR: 14.1 (CH3), 22.7 (n-hexyl), 26.2 (n-
hexyl), 29.5 (n-hexyl), 30.0 (C4), 31.8 (n-hexyl), 36.5 (n-hexyl), 41.1
(C3), 55.8 (C1), 125.7 (C7), 125.7 (C6), 126.1 (C5), 129.2 (C8), 135.1
(C8a), 139.8 (C4a). LR-FABMS m/z: 218 (MHþ), 132 (base peak). HR-
FABMS: Calcd for C15H24N: 218.1909, found: 218.1886.
mp 109–110 ꢀC. 1H-NMR: 0.88 (3H, t, J ¼ 7 Hz, CH2CH2CH3), 1.16–
1.51 (2H, m, CH2CH2CH3), 1.65–1.84 (2H, m, CH2CH2CH3), 2.20 (1H,
t, J ¼ 2 Hz, C30-H), 2.79 (2H, t, J ¼ 6 Hz, C4-H) 2.95 (1H, dt, J ¼ 12,
6 Hz, C3-H), 3.12 (1H, dt, J ¼ 12, 6 Hz, C3-H), 3.45 (1H, dd, J ¼ 17,
2 Hz, C10-H), 3.58 (1H, dd, J ¼ 17, 2 Hz, C10-H), 3.87 (1H, t, J ¼ 5 Hz,
C1-H), 7.05–7.17 (4H, m, Ar). 13C-NMR: 14.3 (CH2CH2CH3), 18.4
(CH2CH2CH3), 26.7 (C4), 37.3 (CH2CH2CH3), 43.2 (C10), 45.9 (C3),
59.7 (C1), 72.4 (C30), 80.1 (C20), 125.7 (C7), 125.8 (C6), 127.3 (C5),
128.6 (C8), 134.8 (C8a), 138.3 (C4a). HR-FABMS: Calcd for C15H20N:
214.1596, found: 214.1574.
5.1.3.1.4. 1-n-Butyl-2-propargyl-1,2,3,4-tetrahydroisoquinoline 12e.
Yield: 59% as a pale yellow oil eluted with AcOEt: hexane ¼ 1:7, p-
TsOH salt; colorless needles from isopropanol-ether, mp 146–
148 ꢀC. 1H-NMR: 0.86 (3H, t, J ¼ 7 Hz, CH3), 1.09–1.44, 1.69–1.87
(each 3H, each m, n-butyl), 2.01 (1H, t, J ¼ 2 Hz, C30-H), 2.80 (2H, t,
J ¼ 6 Hz, C4-H), 2.96 (1H, dt, J ¼ 12, 6 Hz, C3-H), 3.11 (1H, dt, J ¼ 12,
6 Hz, C3-H), 3.45 (1H, dd, J ¼ 17, 2 Hz, C10-H), 3.59 (1H, dd, J ¼ 17,
2 Hz, C10-H), 3.87 (1H, t, J ¼ 5 Hz, C1-H), 7.05–7.18 (4H, m, Ar). 13C-
NMR: 14.1 (CH3), 22.9 (n-butyl), 26.8 (C4), 27.2 (n-butyl), 34.6 (n-
butyl), 43.2 (C01), 46.1 (C3), 59.8 (C1), 72.5 (C03), 80.0 (C02), 125.7
(C7), 125.8 (C6), 127.2 (C5), 128.6 (C8), 134.8 (C8a), 138.3 (C4a). HR-
FABMS: Calcd for C16H22N: 228.1751, found: 228.1773.
5.1.3.1.5. 1-n-Pentyl-2-propargyl-1,2,3,4-tetrahydroisoquino-
line 12f. Yield: 85% as a pale yellow oil eluted with AcOEt:
hexane ¼ 1:2. 1H-NMR: 0.85 (3H, t, J ¼ 7 Hz, CH3), 1.18–1.30 (5H, m,
n-pentyl), 1.37–1.44 (1H, m, n-pentyl), 1.69–1.86 (2H, each m, n-
pentyl) 2.20 (1H, t, J ¼ 2 Hz, C30-H), 2.77–2.81 (2H, m, C4-H), 2.91–
2.99 (1H, m, C3-H), 3.07–3.15 (1H, m, C3-H), 3.45 (1H, dd, J ¼ 17,
2 Hz, C10-H), 3.59 (1H, dd, J ¼ 17, 2 Hz, C10-H), 3.86 (1H, t, J ¼ 5 Hz,
C1-H), 7.04–7.18 (4H, m, Ar). 13C-NMR: 14.1 (CH3), 22.6 (n-pentyl),
24.7 (n-pentyl), 26.8 (C4), 32.1 (n-pentyl), 34.9 (n-pentyl), 43.2
(C01), 46.1 (C3), 59.9 (C1), 72.4 (C03), 80.0 (C02), 125.7 (C7), 125.8
(C6), 127.2 (C5), 128.6 (C8), 134.8 (C8a), 138.3 (C4a). HR-FABMS:
Calcd for C17H24N: 242.1909, found: 242.1917.
5.1.3.1.6. 1-n-Hexyl-2-propargyl-1,2,3,4-tetrahydroisoquinoline
12g. Yield: 37% as a pale yellow oil eluted with chloroform:
hexane ¼ 1:2. 1H-NMR: 0.83 (3H, t, J ¼ 7 Hz, CH3), 1.15–1.43, 1.67–
1.86 (total 10H, each m, n-hexyl), 2.20 (1H, t, J ¼ 2 Hz, C30-H), 2.79
(2H, t, J ¼ 6 Hz, C4-H), 2.95 (1H, dt, J ¼ 12, 6 Hz, C3-H), 3.11 (1H, dt,
J ¼ 12, 6 Hz, C3-H), 3.44 (1H, dd, J ¼ 17, 2 Hz, C10-H), 3.59 (1H, dd,
J ¼ 17, 2 Hz, C10-H), 3.86 (1H, d, J ¼ 5 Hz, C1-H), 7.04–7.20 (4H, m, Ar).
13C-NMR: 14.1 (CH3), 22.7 (n-hexyl), 25.0 (n-hexyl), 26.8 (C4), 29.6
(n-hexyl), 31.8 (n-hexyl), 34.9 (n-hexyl), 43.2 (t, C01), 46.1 (C3), 59.8
(C1), 72.5 (C03), 80.0 (C02), 125.7 (C7), 125.8 (C6), 127.2 (C5), 128.6
(C8), 134.8 (C8a), 138.3 (C4a). HR-FABMS: Calcd for C18H26N:
256.2065, found: 256.2039.
5.1.3. General procedure for the synthesis of 1-alkyl-2-propargyl-TIQ 12
A solution of propargylbromide (5.1 mmol) in acetonitrile
(5 mL) was added to a solution of 1-alkyl-TIQ 11 (3.4 mmol) and
cesium carbonate (6.8 mmol) in acetonitrile (20 mL) under an
argon atmosphere, and the mixture was refluxed for 30 min. After
cooling, the insoluble residue was removed by filtration and the
filtrate was concentrated in vacuo. The resulting mixture was
diluted with chloroform and extracted. The organic layer was
concentrated in vacuo and purified by column chromatography on
silica gel to afford 1-alkyl-2-propargyl-TIQ 12.
5.1.3.1. Analytical data for the new compounds
5.1.3.1.1. 1-Methyl-2-propargyl-1,2,3,4-tetrahydroisoquinoline 12b.
Yield: 53% as a pale yellow oil eluted with AcOEt: hexane ¼ 1:2; HCl
salt; colorless prisms from MeOH–ether, mp 120–122 ꢀC. 1H-NMR:
1.40 (3H, d, J ¼ 7 Hz, CH3), 2.22 (1H, t, J ¼ 2 Hz, C30-H), 2.76–3.01 (4H,
m, C3-H and C4-H), 3.50 (1H, dd, J ¼ 17, 2 Hz, C10-H), 3.67 (1H, dd,
J ¼ 17, 2 Hz, C10-H), 4.00 (1H, t, J ¼ 7 Hz, C1-H), 7.00–7.36 (4H, m, Ar).
13C-NMR: 20.1 (CH3), 28.5 (C4), 43.4 (C10), 46.6 (C3), 55.4 (C1), 72.8
(C30), 79.3 (C20), 125.8 (C7), 126.9 (C6), 128.3 (C5), 128.6 (C8), 134.0
(C8a), 139.5 (C4a). HR-FABMS: Calcd for C13H16N: 186.1282, found:
186.1287.
5.1.3.1.2. 1-Ethyl-2-propargyl-1,2,3,4-tetrahydroisoquinoline
12c. Yield: 80% as a pale yellow oil eluted AcOEt: hexane ¼ 1:2; HCl
salt; colorless prisms from acetone–ether, mp 120–122 ꢀC. 1H-
NMR: 0.79 (3H, t, J ¼ 7 Hz, CH2CH3), 1.73–1.88 (2H, m, CH2CH3), 2.16
(1H, t, J ¼ 2 Hz, C30-H), 2.66–2.75, 2.77–2.82, 2.84–2.94, 2.98–3.07
(each 1H, m, C3-H and C4-H), 3.42 (1H, dd, J ¼ 17, 2 Hz, C10-H), 3.58
(1H, dd, J ¼ 17, 2 Hz, C10-H), 3.84 (1H, t, J ¼ 5 Hz, C1-H), 7.04–7.15
(4H, m, Ar). 13C-NMR: 8.9 (CH2CH3), 26.9 (C4), 27.4 (CH2CH3), 43.1
(C10), 46.5 (C3), 60.4 (C1), 72.4 (C30), 79.6 (C20), 125.5 (C7), 125.6
(C6), 126.9 (C5), 128.3 (C8), 135.1 (C8a), 137.7 (C4a). HR-FABMS:
Calcd for C14H18N: 200.1441, found: 200.1453.
5.1.3.1.7. 1-Cyclopropyl-2-propargyl-1,2,3,4-tetrahydroisoquino-
line 12h. Yield: 40% as a pale yellow oil eluted with AcOEt:
hexane ¼ 1:8, HCl salt; colorless needles from isopropanal-ether,
mp 161–164 ꢀC. 1H-NMR: 0.50–0.57, 0.63–0.76 (total 4H, each m,
cyclopropyl), 0.91–1.03 (1H, m, cyclopropyl), 2.22 (1H, t, J ¼ 2 Hz,
C30-H) 2.80–3.03, 3.12–3.19 (total 4H, each m, C3-H and C4-H), 3.21
(1H, d, J ¼ 9 Hz, C1-H), 3.53 (1H, dd, J ¼ 17, 2 Hz, C10-H) 3.96 (1H, dd,
J ¼ 17, 2 Hz, C10-H) 7.07–7.18, 7.45–7.49 (total 4H, each m, Ar). 13C-
NMR: 4.0 (cyclopropyl), 4.4 (cyclopropyl), 15.5 (cyclopropyl), 27.8
(C4), 43.6 (C10), 47.3 (C3), 63.9 (C1), 73.0 (C30), 79.8 (C20), 125.6 (C7),
126.3 (C6), 127.4 (C5), 128.7 (C8), 134.3 (C8a), 138.2 (C4a). HR-
FABMS: Calcd for C15H18N: 212.1439, found: 212.1427.
5.1.3.1.8. 1-Cyclobutyl-2-propargyl-1,2,3,4-tetrahydroisoquinoline
12i. Yield: 67% as
a pale yellow oil eluted with AcOEt:
hexane ¼ 1:3; HCl salt; colorless prisms from isopropanol-ether,
mp 99–102 ꢀC. 1H-NMR: 1.64–2.06 (6H, m, cyclobutyl), 2.21 (1H, t,
J ¼ 12 Hz, C30-H), 2.50–2.68, 2.81–2.91 (total 3H, each m, cyclobutyl
and C4-H), 2.97–3.04 (1H, m, C3-H), 3.17–3.26 (1H, m, C3-H), 3.37
(1H, dd, J ¼ 16, 2 Hz, C10-H), 3.45 (1H, dd, J ¼ 16, 2 Hz, C10-H), 3.69
5.1.3.1.3. 2-Propargyl-1-n-propyl-1,2,3,4-tetrahydroisoquino-
line 12d. Yield: 57% as
a
pale yellow oil eluted with
AcoEt:hexane ¼ 1:7; HCl salt; colorless prisms from acetone–ether,

4042
M. Kitabatake et al. / European Journal of Medicinal Chemistry 44 (2009) 4034–4043
(1H, d, J ¼ 9 Hz, C1-H), 7.05–7.14 (4H, m, Ar). 13C-NMR: 18.2
(cyclobutyl), 24.7 (C4), 27.0 (cyclobutyl), 27.5 (cyclobutyl), 41.7
(cyclobutyl), 43.5 (C10), 44.5 (C3), 65.0 (C1), 72.0 (C30), 81.0 (C20),
125.4 (Ar), 126.1 (Ar), 128.1 (Ar), 128.7 (Ar), 134.0 (Ar), 136.4 (Ar).
HR-FABMS: Calcd for C16H20N: 226.1602, found: 226.1532.
5.1.3.1.9. 1-Cyclopentyl-2-propargyl-1,2,3,4-tetrahydroisoquino-
line 12j. Yield: 58% as a pale yellow oil eluted with AcOEt:
hexane ¼ 1:3. 1H-NMR: 1.25–1.68, 1.84–1.86 (total 8H, each m,
cyclopentyl), 1.97–2.11 (1H, m, cyclopentyl), 2.18 (1H, t, J ¼ 2 Hz,
C30-H), 2.61–2.70 (2H, m, C4-H), 2.98–3.06, 3.29–3.35 (total 2H,
each m, C3-H), 3.34 (1H, dd, J ¼ 17, 2 Hz, C10-H), 3.42 (1H, dd, J ¼ 17,
2 Hz, C10-H), 3.57 (1H, d, J ¼ 9 Hz, C1-H), 7.11–7.16 (4H, m, Ar). 13C-
NMR: 24.3 (cyclopentyl), 24.4 (C4), 25.1 (cyclopentyl), 30.4 (cyclo-
pentyl), 31.1 (cyclopentyl), 43.6 (C10), 44.2 (C3), 47.3 (cyclopentyl),
65.0 (C1), 71.8 (C30), 81.2 (C20), 125.3 (C7), 126.1 (C6), 128.6 (C5),
128.8 (C8), 134.3 (C8a), 137.5 (C4a). HR-FABMS: Calcd for C17H22N:
240.1753, found: 240.1761.
5.1.3.1.10. 1-Cyclohexcyl-2-propargyl-1,2,3,4-tetrahydroisoquino-
line 12k. Yield: 85% as a pale yellow oil eluted with benzene:
hexane ¼ 2:1; HCl salt; colorless prisms from MeOH–ether, mp
193–196 ꢀC. 1H-NMR: 0.82–1.86 (11H, m, cyclohexyl), 2.15 (1H, t,
J ¼ 2 Hz, C30-H), 2.75 (2H, t, J ¼ 6 Hz, C4-H), 2.91 (1H, dt, J ¼ 6, 12 Hz,
C3-H), 3.18 (1H, dt, J ¼ 6, 12 Hz, C3-H), 3.39 (1H, dd, J ¼ 17, 2 Hz, C10-
H), 3.50 (1H, dd, J ¼ 17, 2 Hz, C10-H), 3.55 (1H, d, J ¼ 6 Hz, C1-H),
7.00–7.16 (4H, m, Ar). 13C-NMR: 26.5 (cyclohexyl), 26.6 (cyclohexyl),
26.6 (cyclohexyl), 26.7 (C4), 30.3 (cyclohexyl), 30.3 (cyclohexyl),
44.6 (C10), 44.8 (C3), 46.0 (cyclohexyl), 65.9 (C1), 71.9 (C30), 80.8
(C20), 125.0 (C7), 125.8 (C6), 128.1 (C5), 128.8 (C8), 135.7 (C8a), 136.6
(C4a). LR-FABMS m/z: 254 (MHþ), 254 (base peak). HR-FABMS:
Calcd for C18H24N: 254.1909, found: 254.1922.
low-melting point agarose. The mixture (75 mL aliquots) was applied
to the comet slides, and the slides were kept horizontal at 4 ꢀC until
the agarose became completely transparent. The slides were then
treated with Lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris
base, 1% sodium laurylsarcosinate, and 1% Triton X-100) for 1 h at
4 ꢀC. The slides were immersed in alkaline solution (300 mM NaOH
and 1 mM EDTA) at room temperature for 30 min in the dark, fol-
lowed by two immersions in TBE buffer for 5 min. Electrophoresis
was conducted at 15 V at room temperature for 30 min, then the
slides were dipped in 70% EtOH for 5 min. After drying, DNA on the
slides was stained with 50 mL of SYBR Green (0.1 mL/mL) solution.
Comet images were observed through a fluorescence micro-
scope at ꢂ200 magnification and 568 nm. Routinely, 200 cells were
screened per sample. In selecting cells for measurement, straight
line scanning of a slide was begun at an arbitrary point and cells
were measured as they came into the field, provided there was no
overlap with patterns from other cells. The length of the ‘comet’
was measured using an ocular micrometer disk.
5.2.4. Inhibitory effect of TIQ derivatives on MPPþ-induced
cell death
Cell viability in the presence of 0.25 mM MPPþ and TIQ deriva-
tives was estimated according to the method described in Section
5.2.2. The effect of TIQ derivatives in preventing cell death was
calculated as a percentage of MPPþ-induced cell death in the
absence of TIQ derivatives (defined as 100%).
5.2.5. Determination of caspase activity
Cells were suspended in serum containing DMEM at a concen-
tration of 3 ꢂ 105 cells/mL and were transferred to a Petri dish. After
24 h incubation, supernatants were removed and 0.25 mM of MPPþ
or 0.01 mM of TIQ derivative solution (final concentration, prepared
by using non-serum DMEM) was added. After an additional 48 h
incubation, cells were harvested and lysed using 20 mM phosphate
buffer (pH 7.4) containing 0.05% Triton-X and 1 mM EDTA for 3 min.
Centrifuged supernatants (4 ꢀC, 10 min) were collected as cell
5.2. Biological experiments
5.2.1. Cell culture
PC12 cells were obtained from the American Tissue Type Culture
Collection (reference no. CRL 1721). Dulbecco’s modified Eagle’s
medium (DMEM) was purchased from Gibco (USA), and fetal calf
serum (FCS) was purchased from Roche Diagnostics (Switzerland).
PC12 cells were routinely maintained in DMEM containing 10%
heat-inactivated FCS, 0.5 unit penicillin/mL and 0.05 mg strepto-
mycin/mL under an atmosphere containing 5% CO2 at 37 ꢀC. The
medium was changed every 2–3 days.
extract. Enzyme assay buffer (32
(pH 7.5), 31.25% sucrose, 0.325% CHAPS), 0.5 mM Ac-DEVD-AMC
(10 L), DMSO (2 L), 100 mM dithiothreitol (10 L), H2O (36 L)
and cell extract (10
L) were mixed and incubated at 37 ꢀC for
mL, containing 312.5 mM HEPES
m
m
m
m
m
60 min. Fluorescence derived from 7-amino-4-methylcoumarin
(AMC), released by fragmentation due to caspase-3,7 activity, was
measured with a microplate reader (excitation at 360 nm and
emission at 460 nm Applied Biosystems Model CytofluorII). Data
are presented as a percentage compared to the activity of the
untreated control (defined as 100%).
5.2.2. Determination of cell viability
Cells used for experiments were in logarithmic growth. Viable
cell ratios were determined by colorimetric assay of formazan
produced by the reduction of a water-soluble tetrazolium salt,
similar to the MTT assay. Cells were suspended in serum containing
DMEM at a concentration of 3 ꢂ105 cells/mL and were transferred
to 96-well plates. After 24 h incubation, the supernatants were
removed and various concentrations of 1-alkyl- and 1-alkyl-N-
propargyl-TIQ derivatives in solution (prepared using non-serum
5.2.6. Determination of MAO activity
Quantitative analysis of MAO activity was conducted using
a fluorescence monoamine oxidase A and B detection kit (Fluoro
MAOÔ, Peninsula Laboratories, Inc.) according to the manufactur-
er’s protocol. The intensity of the fluorescent dye released in the
presence of H2O2 (upon reaction of MAO and substrate) and the kit
DMEM) were added (100
Counting Kit-8 solution (Dojindo, 10
m
L/well). After 48 h incubation, Cell
L/well) was added to each
m
peroxidase reagent was measured. MAO-A or MAO-B (10 mg/mL,
well and incubated further for 4 h at 37 ꢀC. The absorbance at
490 nm was measured with a microplate reader (Bio-Rad, Model
550), and cell viability was evaluated in terms of A490 and expressed
as a percentage of the untreated control.
Sigma) and corresponding substrate (0.5 mM tyramine for MAO-A
and MAO-B, 2.5 mM benzylamine for MAO-B) were co-incubated
with 100 nM 1-methy-N-propargyl- or 1-cy-propyl-N-propargyl-
TIQ at 37 ꢀC for 60 min. Fluorescence was measured using
a microplate reader (excitation at 530 nm and emission at 590 nm;
Applied Biosystems Model CytofluorII). Data are presented as
a percentage of the vehicle control activity (defined as 100%).
5.2.3. Determination of apoptotic induction (comet assay)
Reagents and equipment for the comet assay were purchased
from Trevigen (USA). PC12 cells (5 ꢂ105 cells/well, suspended in
PBS(ꢃ)) and MPPþ or TIQ derivative solution (1.0 mM final concen-
tration) were incubated in 24-well microplates at 37 ꢀC for 3 h. After
5.2.7. Data analysis
Data are expressed as means ꢁ standard error (S.E.) for each
exposure, 10 mL of the cell suspension was mixed with 100 mL of 1%
group. Each datum was first analyzed by Bartlett’s test for

M. Kitabatake et al. / European Journal of Medicinal Chemistry 44 (2009) 4034–4043
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