Synthesis and in vitro evaluation of N-substituted aza-trozamicol analogs as vesicular acetylcholine transporter ligands

Article abstract of DOI:10.1016/j.bmcl.2006.02.033

As dysfunction of cerebral cholinergic neurotransmission is one of the main features in patients with Alzheimer's disease, in vivo imaging of the vesicular acetylcholine transporter (VAChT) can be of great value for the early diagnosis of this disease. Tw

Full text of DOI:10.1016/j.bmcl.2006.02.033

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2654–2657
Synthesis and in vitro evaluation of N-substituted
aza-trozamicol analogs as vesicular acetylcholine
transporter ligands
Thaer Assaad,a,b Sylvie Mavel,a,b Stanley M. Parsons,c Shane Kruse,c Laurent Galineau,a,b
Hassan Allouchi,d Michael Kassiou,e,f Sylvie Chalon,a,b
Denis Guilloteaua,b and Patrick Emonda,b,
*
aUniversite´ Franc¸ois-Rabelais de Tours, 37200 Tours, France
b
ˆ
INSERM U619, 37044 Tours Cdx 09, CHRU, Hopital Bretonneau, 37044 Tours, France
cUniversity of California, Department of Chemistry and Biochemistry, Santa Barbara, CA 93106, USA
dUniversite´ Franc¸ois-Rabelais de Tours, Laboratoire de Chimie Physique, 31 av. Monge, 37200 Tours, France
eDepartment of PET and Nuclear Medicine, Royal Prince Alfred Hospital, Missenden Road,
Camperdown, NSW, Sydney 2050, Australia
fDepartment of Pharmacology, University of Sydney, NSW 2006, Australia
Received 10 January 2006; revised 10 February 2006; accepted 11 February 2006
Available online 3 March 2006
Abstract—As dysfunction of cerebral cholinergic neurotransmission is one of the main features in patients with Alzhei-
mer’s disease, in vivo imaging of the vesicular acetylcholine transporter (VAChT) can be of great value for the early
diagnosis of this disease. Two series of positional isomers of m-iodobenzyltrozamicol (MIBT): 3-hydroxy-4-(N-phenylpip-
erazinyl)piperidine and 4-hydroxy-3-(N-phenylpiperazinyl)piperidine substituted by benzyl, aryl, alkyl or vinyl groups at
the nitrogen have been synthesized. These compounds have been evaluated in vitro by competition studies and five
compounds (N-benzyl derivatives) showed high affinity for the VAChT (11 nM < IC50 < 66 nM). These compounds will
be soon radiolabeled with [125I] for further biological evaluation using single photon emission computed tomography
(SPECT).
Ó 2006 Elsevier Ltd. All rights reserved.
Cholinergic neurotransmission plays an important role
in the central nervous system which requires the synthe-
sis of acetylcholine (ACh) in the cytoplasm, accumula-
tion in presynaptic vesicles, and release from the
terminal neuron. The vesicular acetylcholine transporter
(VAChT) which is responsible for ACh accumulation
into presynaptic vesicles plays a crucial role in regulating
cholinergic neurotransmission as it concentrates and
makes ACh available. Moreover, as the VAChT is
located exclusively on cholinergic neurons, it has
emerged as a useful target to study cholinergic
function.1–3 Alzheimer’s disease (AD) is a progressive
and neurodegenerative disorder which affects the
cholinergic system and leads to cognitive deficits. More-
over, as this disease is progressive, the initial stages are
not well defined compared to normal age ‘decline’ and
it is classified as mild cognitive impairment. Exploration
of cholinergic neurotransmission and especially the
detection and quantification of the VAChT in vivo
should provide important and useful information for
the early diagnosis of this disease. As vesamicol
(Fig. 1) binds to the VAChT but also to sigma receptors,
structure–activity relationship studies (SARs) have been
employed4 to aid the development of radiopharmaceuti-
cals suitable for use in single photon emission computed
tomography (SPECT) and positron emission tomogra-
phy (PET).5 Among those radioligands, (ꢀ)-(2R,3R)-
2-hydroxy-3-(4-phenylpiperidino)-5-[123I]iodotetralin [(ꢀ)-
Keywords: Vesicular acetylcholine transporter; VAChT; Vesamicol
derivatives.
*
Corresponding author. Tel.: +33 247367242; fax: +33 247367224;
e-mail: emond@univ-tours.fr
[
123I]-5-IBVM] (Fig. 1) is the most promising SPECT
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.02.033

T. Assaad et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2654–2657
2655
O
O
O
a
b
N
CH3
, HCl
N
N
OCH2CCl3
OCH2CCl3
1
2
3
HO
HO
O
d
d
N
N
N
N
N
N
N
NH
OCH2CCl3
4
6a
c
+
HO
HO
N
N
N
NH
OCH2CCl3
O
5
7a
HO
Figure 1. Vesamicol analogs.
HO
6a
or
e
N
N
N
R
or
N
N
compound to be used in human studies. This radioiodin-
ated derivative was found to be a reliable marker of cho-
linergic integrity at 22 h postinjection, as its binding
correlates with choline acetyltransferase (ChAT) activi-
ty.6 In addition, a 3.7% reduction per decade in cortical
binding has been reported with [123I]IBVM in normal
aging and an inverse relationship between cortical
N
R
7a
6b-k
7b-k
R
R
g
h
i
j
k
b
CH2C6H5
CH2CH=CH2
CH2C(CH3)=CH2
(E)-CH2-CH=CHCH3
CH2- C CH
c
d
e
f
CH2-o-Br-C6H4
CH2-m-Br-C6H4
CH2-p-Br-C6H4
CH2CH3
(E)-CH2-CH=CHI
[
123I]IBVM binding and the severity of AD has been
reported.7 Furthermore, although its gastrointestinal
toxicity is not an issue, its slow pharmacokinetics are
far from ideal.7,8
Scheme 1. Reagents and conditions: (a) Cl3CCH2O2CCl, reflux, 3 h;
(b) m-CPBA, Et2O, rt, 5 h; (c) 1-phenylpiperazine, EtOH, reflux, 20 h;
(d) Zn/AcOH, rt, 48 h; (e) RBr (2.5 equiv), EtOH (10 mL/mmol), Et3N
(140 lL/mmol), KI, reflux, 16 h.
To improve these parameters, SARs have been
performed on vesamicol structures which revealed that
the piperidine group of vesamicol is important to obtain
compounds with high affinity for the VAChT.9 More
recently, Bando et al.10 have reported that the
replacement of the piperidine ring of IBVM by a piper-
azine ring resulting in the formation of DRC140
derivative (Fig. 1) afforded a high affinity compound
(KiDRC140 = 1.21 nM) with high selectivity for VAChT
over r receptors.
The exact regioisomer (3-OH or 4-OH) of the piperidine
ring was confirmed by X-ray diffraction measurements
carried out on compound 7a.14 Both piperidine and
piperazine rings were found to exist in the chair confor-
mation with the piperidine and piperazine rings nearly
perpendicular: 80.9° for the (S,S) derivative and
ꢀ83.5° for the (R,R) derivative. These angles conformed
to those described for (ꢀ)-vesamicol9 and a trozamicol
derivative.11
Alkylation of the trozamicol scaffold (Fig. 1) at nitrogen
by a m-iodobenzyl group afforded a highly potent deriv-
ative for the VAChT (Ki(+)-MIBT = 4.8 1.1 nM).11 Sim-
ilarly, [(ꢀ)-[18F]-FBT] (Fig. 1) has proved to be a
suitable radioligand in non-human primate studies using
PET,12 but has yet to be assessed in human studies.
N-Alkylation of 6a or 7a was performed with the appro-
priate alkyl bromide reagent in ethanol which gave the
corresponding N-substituted-4-hydroxy-3-(N-phenyl-
piperazinyl)piperidine 6b–j or N-substituted-3-hydroxy-
4-(N-phenylpiperazinyl)piperidine 7b–j in 50–71% yield.
Compounds 6k or 7k were obtained in two steps. Stan-
nyl analogs of 6k or 7k were prepared by N-alkylation
of 6a or 7a with (E)-3-chloro-1-tributylstannylprop-
2-ene in ethanol15 in 56% and 59% yield, respectively.
Treatment with iodine of these stannyl derivatives in
CHCl3 resulted in compounds 6k or 7k in 66% and
71% yield, respectively. All target compounds were char-
From these results, we hypothesized that a fused skele-
ton between DRC-140 and MIBT structures (Scheme
1, target compounds) should result in compounds with
a high affinity for the VAChT. Here, we report the syn-
thesis of new aza-trozamicol derivatives and their in vitro
evaluation on a human VAChT cell line.
1
acterized by H NMR and mass spectrometry and gave
results consistent with the assigned structure.
Two series of compounds (6a–k and 7a–k) were pre-
pared as presented in Scheme 1. The synthesis started
with 1-methyl-1,2,3,6-tetrahydropyridine hydrochloride
1, which was converted into its corresponding carba-
mate 2 by treatment with 2,2,2-trichloroethylchlorofor-
mate under reflux. The crude carbamate was then
treated with m-chloroperbenzoic acid which generated
the required epoxide9 3. The addition of phenylpiper-
azine to the epoxide 3 afforded two regioisomers 4 and
5 which were separated by flash chromatography (silica
gel, EtOH/Et3N, 10/1). The amine function was then
deprotected (6a and 7a) by reduction with zinc in acetic
acid13 in quantitative yield.
Human VAChT stably expressed in the PC12 cell line,16
which is a common model system for neurosecretory
phenomena, was used to screen target compounds.
Because the cell line expresses a relatively large amount
of human VAChT (>2 pmol/mg postnuclear superna-
tant), displacement of bound [3H]-vesamicol by compet-
ing ligands is monophasic and simple.
A low
concentration of [3H]-vesamicol was used so that the
IC50 value for displacement is essentially equal to the
Kd value. Cells were grown in quantity, harvested, and
homogenized to prepare a low-speed, postnuclear super-

2656
T. Assaad et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2654–2657
Table 1. IC50 values (nM) at the human VAChT compared to the literature data
b
b
Compound
R
Series 6a
Series 60
Series 7a
Series 70
a
b
c
d
e
f
H
CH2C6H5
ꢁ100
5000 ( 700)
83 ( 7)
>100
>100
2900 ( 400)
30 ( 7)
22 ( 6)
21 ( 8)
25 ( 10)
—
57 ( 26)
11 ( 4)
66 ( 24)
>100
CH2–o-Br–C6H4
CH2–m-Br–C6H4
CH2–p-Br–C6H4
CH2CH3
55 ( 8)
—
310 ( 130)
47 ( 18)
46 ( 20)
>100
>1000
>100
>100
—
—
—
—
—
—
>1000
>100
>100
g
h
i
CH2CH@CH2
CH2C(CH3)@CH2
(E)-CH2–CH@CHCH3
CH2C„CH
—
—
ꢁ100
>100
>100
>100
—
—
j
>1000
>100
k
(E)-CH2–CH@CHI
—
a Values are means of three experiments, standard deviation is given in parentheses. In the same conditions, (ꢀ)-vesamicol showed an IC50
=
20 ( 2.3) nM.
b Literature data obtained on VAChT isolated from the electric organ of Torpedo californica,17 ( )-vesamicol showed a Kd = 34 ( 6) nM.
natant rich in synaptic-like microvesicles containing
human VAChT. Competition was allowed to proceed
for 24 h to ensure equilibration of low concentrations
of target compounds. It could not be carried out at
37 °C due to the length of incubation. However, binding
of vesamicol is not strongly temperature dependent.
seems not to influence the binding affinity. These results
contrast with those obtained previously for trozamicol
and prezamicol congeners.17,18 For these compounds,
the 1,4 orientation of the nitrogen relative to the hydroxyl
group (trozamicol series) was found to be preferred.
Regarding the nature of the R group, substitution of
compounds 6a and 7a with alkyl, alkenyl, and alkynyl
groups was based on results obtained for hydroxylated
decahydroquinoline vesamicol derivatives.18 In this
study, a (E)-iodoprop-2-enyl group was successfully
introduced to obtain a compound with nanomolar
VAChT affinity. Contrary to these results, the N-alkyl-
ation of compounds 6a or 7a failed to obtain com-
pounds with VAChT potency. Only some of the
benzyl derivatives displayed IC50 less than 100 nM
(6b–d and 7c–d). Moreover, the position of the bromide
atom on the benzyl group induces variability in the
VAChT affinity as the para-substituted derivatives
(6e and 7e) showed low VAChT affinities. Here also,
these results contrast with those obtained for benzyl-
substituted prezamicol or trozamicol derivatives as the
biological activity was not influenced by the position
of the substituent on the ring.17
Because IBVM and MIBT are compounds with high
affinities for the VAChT; IC50 values have only been
determined for potent compounds with IC50 lower than
100 nM. Affinities of synthesized compounds are pre-
sented in Table 1.
The only structural difference between prezamicol ana-
logs (60a–e),17 and compounds 6a–e, trozamicol analogs
(70a–e)17 and compounds 7a–e is the replacement of the
4-phenylpiperidine group for a N-phenylpiperazine
group. Comparison of their in vitro affinities would pro-
vide information on the impact of an additional nitro-
gen at this part of the molecule. Even compounds 6a
(IC50 ꢁ 100 nM) and 7a (IC50 > 100 nM) displayed
higher affinities for the VAChT compared to prezamicol
(60a;
IC50 = 5000 nM)
and
trozamicol
(70a;
IC50 = 2900 nM),17 compounds 6b,6c, and 6e
(IC50 = 57, 11, and >100 nM, respectively) exhibited
similar affinities for the VAChT compared to their pre-
zamicol analogs 60b,60c, and 60e (IC50 = 83, 55, and
310 nM, respectively).17 Moreover, even compounds 7c
and 7d (IC50 = 47 and 46 nM, respectively) showed
VAChT affinities in the same range of magnitude as
their trozamicol analogs 70c and 70d (IC50 = 22 and
21 nM, respectively), while compounds 7b and 7e
(IC50 = >100 nM) are less potent compared to com-
pounds 70b and 70e (IC50 = 30 and 25 nM, respectively).
Because the substitution of the 4-phenylpiperidine group
for N-phenylpiperazine generated compounds with or
without improved affinities for the VAChT, an addition-
al nitrogen at this part of the molecule cannot be consid-
ered as essential for the binding to the VAChT.
Although it is well known that interaction at the
VAChT binding site is stereoselective,9 only racemic
mixtures have been screened. We can expect that enan-
tiomeric separation of compounds 6b–d and 7c–d would
provide compounds with improved VAChT affinity. In
parallel, as compound 6c is the most potent of this ser-
ies, its radiolabeling with bromide-76 but also with
iodine-125/123 is underway to further evaluate its in vivo
pharmacological profile.
Acknowledgments
This work was supported by Grant (82/2001 and 582/
2003) In Aid scientific research from the AECS (Atomic
Energy Commission of Syria). This work was partially
supported by DEST and the French Embassy in Austra-
lia under the FAST program FR040051. This study was
funded in part by the EC-FP6-project DiMI, LSHB-CT-
2005-512146. We thank Jeffrey D. Erickson of the
Louisiana State University Health Sciences Center,
Except for the benzyl group for which the VAChT affinity
could be considered significantly different for compounds
6b and 7b, the VAChT affinity in series 6 and 7 is in the
same range for each R substituent. In these series of com-
pounds, the orientation of the nitrogen in the piperidine
ring relative to the hydroxyl group (in position 3 or 4)

T. Assaad et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2654–2657
2657
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Supplementary data
Supplementary data associated with this article can
be found, in the online version, at doi:10.1016/j.bmcl.
2006.02.033.
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