Synthesis and in vitro evaluation of new benzovesamicol analogues as potential imaging probes for the vesicular acetylcholine transporter

Article abstract of DOI:10.1016/j.bmc.2004.10.043

Our goal was to synthesize new stereospecific benzovesamicol analogues, which could potentially be used as SPECT or PET radioligands for the vesicular acetylcholine transporter (VAChT). This paper describes the chemical synthesis, resolution and determination of binding affinity for four enantiomeric pairs of derivatives. Their intrinsic affinities were determined by competition against binding of [³H]vesamicol to human VAChT. Of the eight enantiomers, (E)-(R,R)-5-AOIBV [(R,R)-3], and (R,R)-5-FPOBV [(R,R)-4] displayed the highest binding affinities for VAChT (K_d = 0.45 and 0.77 nM, respectively), which indicated that an elongation of the chain from 5-idodo as in the case of 5-iodobenzovesamicol (5-IBVM), to a 5-(E)-3-iodoallyloxy or 5-fluoropropoxy substituent, as in 5-AOIBV and 5-FPOBV, respectively, was very well tolerated at the vesamicol binding site. The enantiomer (R,R)-4-MAIBV [(R,R)-16], which retains the basic structure of (-)-5-IBVM but possess an additional aminomethyl substituent in the 4-position of the piperidine ring, displayed lower binding affinity (K_d = 8.8 nM). Nevertheless, the result suggests that substitution at this position may be an interesting alternative to investigate for development of new benzovesamicol analogues. As expected, the corresponding (S,S) enantiomers displayed lower K_d values, they were approximately 10-fold lower in the case of (S,S)-5-FPOBV (K_d = 8.4 nM) and (E)-(S,S)-5-AOIBV (K_d = 4.3 nM). (R,R)-3, and (R,R)-4 showed the same high affinity for VAChT as (-)-5-IBVM and may be suitable as imaging agents of cholinergic nerve terminals.

Full text of DOI:10.1016/j.bmc.2004.10.043

Bioorganic & Medicinal Chemistry 13 (2005) 745–753
Synthesis and in vitro evaluation of new benzovesamicol
analogues as potential imaging probes for the
vesicular acetylcholine transporter
Yolanda Zea-Ponce,a Sylvie Mavel,a,* Thaer Assaad,a Shane E. Kruse,b
Stanley M. Parsons,b Patrick Emond,a Sylvie Chalon,a Nicolas Giboureau,a,d
Michael Kassiouc,d and Denis Guilloteaua
a
´
´
Faculte de Pharmacie, INSERM U619, Laboratoire de Biophysique Medicale, 31 Av. Monge, 37200 Tours, France
bDepartment of Chemistry and Biochemistry and the Neuroscience Research Institute, University of California,
Santa Barbara, CA, USA
cDepartment of PET and Nuclear Medicine, Royal Prince Alfred Hospital, Missenden Road, Camperdown,
NSW, Sydney 2050, Australia
dDepartment of Pharmacology, University of Sydney, NSW 2006, Australia
Received 27 August 2004; revised 12 October 2004; accepted 19 October 2004
Available online 11 November 2004
Abstract—Our goal was to synthesize new stereospecific benzovesamicol analogues, which could potentially be used as SPECT or
PET radioligands for the vesicular acetylcholine transporter (VAChT). This paper describes the chemical synthesis, resolution and
determination of binding affinity for four enantiomeric pairs of derivatives. Their intrinsic affinities were determined by competition
against binding of [3H]vesamicol to human VAChT. Of the eight enantiomers, (E)-(R,R)-5-AOIBV [(R,R)-3], and (R,R)-5-FPOBV
[(R,R)-4] displayed the highest binding affinities for VAChT (Kd = 0.45 and 0.77nM, respectively), which indicated that an elonga-
tion of the chain from 5-idodo as in the case of 5-iodobenzovesamicol (5-IBVM), to a 5-(E)-3-iodoallyloxy or 5-fluoropropoxy sub-
stituent, as in 5-AOIBV and 5-FPOBV, respectively, was very well tolerated at the vesamicol binding site. The enantiomer (R,R)-4-
MAIBV [(R,R)-16], which retains the basic structure of (À)-5-IBVM but possess an additional aminomethyl substituent in the 4-
position of the piperidine ring, displayed lower binding affinity (Kd = 8.8nM). Nevertheless, the result suggests that substitution
at this position may be an interesting alternative to investigate for development of new benzovesamicol analogues. As expected,
the corresponding (S,S) enantiomers displayed lower Kd values, they were approximately 10-fold lower in the case of (S,S)-5-
FPOBV (Kd = 8.4nM) and (E)-(S,S)-5-AOIBV (Kd = 4.3nM). (R,R)-3, and (R,R)-4 showed the same high affinity for VAChT as
(À)-5-IBVM and may be suitable as imaging agents of cholinergic nerve terminals.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
been directed towards the development of more selective
radiotracer molecules.7–10 The interest in benzovesami-
col derivatives as radioligands for AlzheimerÕs disease
(AD) studies is based on the observation that as the dis-
ease progresses, the levels of VAChT change in parallel
fashion and magnitude with other cholinergic marker
proteins, in particular choline acetyltransferase (ChAT).
Radiolabelled benzovesamicol analogues had already
been used in the past as imaging probes in single photon
emission computer tomography (SPECT) and positron
emission tomography (PET) aimed for in vitro and in
vivo studies of AD. Some of those radiotracers showed
high selectivity and high binding affinity for the VAChT,
but their pharmacokinetics and/or toxicity restricted
their use as in vivo imaging agents.10–15 Among those
Benzovesamicol (BVM) is an analogue of (À)-vesamicol
(Fig. 1), a compound known to be a stereoselective
inhibitor of acetylcholine uptake into pre-synaptic cho-
linergic vesicles. Vesamicol binds with high affinity to
the vesicular acetylcholine transporter (VAChT) at an
allosteric site.1–4 Since (À)-vesamicol also binds to a-
adrenoreceptors5 and r-receptors,6 new efforts have
Keywords: VAChT; (E)-(R,R)-5-AOIBV, (R,R)-5-FPOBV; Benzove-
samicol derivatives.
*
Corresponding author. Tel.: +33 2 47 36 72 42; fax: +33 2 47 36 72
24; e-mail: sylvie.mavel@univ-tours.fr
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.10.043

746
Y. Zea-Ponce et al. / Bioorg. Med. Chem. 13 (2005) 745–753
HO
HO
HO
R1
N
N
N
R3
R
R2
(–)-vesamicol
R= I
(–)-5-IBVM
New Analogues : R1= H, OCH3
R2= OCH2CH=CHI,
O(CH2)3F, or I
R= O(CH2)2F FEOBV
R3= H, CH2NH2
Figure 1. Chemical structure of (À)-vesamicol, (À)-5-iodobenzovesamicol, (À)-5-fluoroethoxybenzovesamicol and (R,R) new analogues.
radiotracers, (À)-(2R,3R)-2-hydroxy-3-(4-phenylpiperi-
dino)-5-(2-[18F]fluoroethoxy)tetralin [(À)-[18F]-5-FEO-
BV]14 (Fig. 1) has proved to be an excellent imaging
tool in rodent studies by PET,16 but its use in human
studies is missing so far. For SPECT studies, (À)-
(2R,3R)-2-hydroxy-3-(4-phenylpiperidino)-5-[123I]iodo-
tetralin [(À)-[123I]-5-IBVM] (Fig. 1) has been the more
promising of the radioiodinated benzovesamicol ana-
logues for the determination of VAChT density and
atrophy in humans. Its gastrointestinal toxicity is not
at issue due to the very small amount of cold compound
to be injected, but its pharmacokinetics are far from
ideal.11–13
the presence of an iodide versus fluoride atom in a sub-
stituent chain of similar length, (d) the effect of substitu-
tion in the 4-position of the piperidine ring; an
alternative not previously examined in benzovesamicol
series.
In this paper, we describe the chiral synthesis of four
enantiomeric pairs of benzovesamicol derivatives (Fig.
1), and evaluation of in vitro binding to the VAChT
radioligand binding experiments.
2. Results
For the development of new, more potent and selective
benzovesamicol derivatives as radiotracers, two consid-
erations are of uppermost importance: (a) in vivo studies
in rats of the neuronal mapping potential of iodo-
benzovezamicol derivatives have revealed that consid-
erable bulk tolerance exists at different structural
positions of the benzovesamicol structure;11,17 and (b)
the binding to the VAChT is known to be highly
enantioselective.
2.1. Chemistry
The syntheses of the four pairs of enantiomeric benzove-
samicol derivatives, reported here, were accomplished
via the routes illustrated in Schemes 1–3.
Synthesis and enantiomeric resolution of racemic 5-ami-
nobenzovesamicol (5-ABV) was achieved as previously
reported.20 Sandmeyer reaction on each of the pure
5-ABV enantiomers, produced the corresponding
5-hydroxybenzovesamicol (5-HOBV; 1) enantiomer14
(Scheme 1). The p-toluenesulfonate ester of (E)-3-
(tri-n-butylstannyl)prop-2-enol was synthesized as
reported,21 and later coupled with each enantiomer of
the phenol 1, leading to the allylstannylated precursor
(5-Bu3SnAOIBV; 2) in 71–80% yield. Iododestannyl-
ation was accomplished under mild conditions with io-
dine or N-iodosuccinimide (NIS), with chemical yields
of 67% and 77%, respectively, producing the iodoallyl-
oxy derivative (5-AOIBV; 3) (Scheme 1).
Of special consideration in the development for new
VAChT probes is the method to be used in determina-
tion of their binding affinity. The previous method of
choice utilized cholinergic synaptic vesicles purified from
the marine ray Torpedo.18 However, differences between
elasmobranch and human VAChT affinities for ana-
logues of vesamicol can be expected. Human VAChT
has now been cloned and constitutes the preferred screen
for development of clinically useful analogues.19
Our purpose was to search for new benzovesamicol
derivatives with high affinity for the VAChT, lower tox-
icity and good pharmacokinetic characteristics. Our
goal was to find analogues with potential use as radiola-
belled probes for SPECT or PET with application in the
detection of density changes at the pre-synaptic level,
useful in early detection and follow-up of AlzheimerÕs
disease in humans.
On the other hand, fluorination of propanediol ditosyl-
ate using tetra-n-butylammonium fluoride (TBAF) pro-
duced 3-fluoropropyltosylate [F(CH2)3OTs] in 35%
yield. Reactions between this tosylate and the nucleophi-
lic enantiomers of 5-HOBV, 1, in acetonitrile led to
authentic 5-fluoropropyloxybenzovesamicol (5-FPOBV;
4) in 84–90% yield (Scheme 1).
To that effect, we looked into how changes in lipophilic-
ity and structural conformation could impact on radio-
tracer characteristics in comparison to (À)-5-IBVM. We
chose to work with enantiomerically pure compounds
and to look specifically at: (a) elongation of the substit-
uent chain, (b) increase or decrease lipophilicity by plac-
ing additional substituents on the parent molecule, (c)
Scheme 2 describes the route followed for the synthesis of
the enantiomeric derivatives 8-methoxy-5-iodobenzove-
samicol (8-MOIBV; 8). Through a Sandmeyer reaction,
racemic 8-aminobenzovesamicol (8-ABV; 5) was con-
verted to racemic 8-hydroxybenzovesamicol (8-HOBV;
6), which later was used to generate racemic
8-methoxybenzovesamicol (8-MOBV; 7) in 67% yield.

Y. Zea-Ponce et al. / Bioorg. Med. Chem. 13 (2005) 745–753
747
HO
HO
b
N
N
a
(R, R)-2
(S, S)-2
(R, R)-3
(S, S)-3
O
O
HO
I
Bu3Sn
N
(R, R)-1= (-)-1
(S, S)-1= (+)-1
HO
c
HO
10
11
9
2
1
3
N
4
(R, R)-4
(S, S)-4
O
F
Scheme 1. Synthesis of the enantiomeric pairs (R,R)/(S,S)-5-AOIBV, 3 and (R,R)/(S,S)-5-FPOBV, 4. Reagents and conditions: (a) TBAOH,
CH3CN, (E)-Bu3SnCH@CH–CH2OTs, 80ꢁC; (b) from (S,S)-2: I2, CHCl3, rt, from (R,R)-2: NIS, THF, rt; (c) TBAOH, CH3CN, F(CH2)3OTs, 80ꢁC.
HO
HO
HO
NH
a
OH
OCH
3
2
b
N
N
N
7
5
6
c
MTPAO
N
HO
OCH
OCH
3
3
d
N
8
(R,R)-9
(S,S)-9
I
I
e
HO
N
OCH
3
(R,R)-8
(S,S)-8
I
Scheme 2. Synthesis of the enantiomeric pair (R,R)- and (S,S)-8-MOIBV, 8. Reagents and conditions: (a) NaNO2, H2SO4, THF, 0ꢁC then reflux; (b)
TBAOH, CH3CN, CH3I, 80ꢁC; (c) ICl, CH3CO2H, rt; (d) (R)(À)MTPA-Cl, 4-DMAP, TEA, CHCl3, rt; (e) 2N NaOH, CH3OH, THF, rt.
HO
HO
O
+
NH2
a
+
N
NH
N
NC
NC
NC
10
11
12
NHCOCF3
13
H2N
HO
HO
HO
NH2
c, d
NH2
13
N
N
N
b
NC
e
NH2
NH2
(R,R)-15
(S,S)-15
14
I
I
I
(R,R)-16
(S,S)-16
Scheme 3. Synthesis of the enantiomeric pair (R,R)- and (S,S)-MAIBV, 16. Reagents and conditions: (a) EtOH, Et3N, reflux, NaOH, rt; (b) ICl,
CH3CO2H, rt; (c) (R)(À)MTPA-Cl, 4-DMAP, TEA, CH3Cl, rt; (d) DIBAL-H, toluene, À78ꢁC, then rt; (e) H3PO2, HCl, NaNO2, 0ꢁC, then rt.
Racemic 8-methoxyiodobenzovesamicol (8-MOIBV; 8)
was then obtained by iodination of 7 with iodine mono-
chloride in glacial acetic acid, and the racemic mixture re-
solved through their MTPA esters, followed by 2N
Scheme 3 details the synthetic path used in the pre-
paration of enantiomeric 3-(4-methylamino-4-phenyl-
piperidino)-5-iodobenzovesamicol
(MAIBV;
16).
Condensation in ethanol of 4-cyano-4-phenylpiperidine
10 with N-(trifluoroacetyl)-1-amino-5,8-dihydronaph-
thalene oxide, 11, produced a mixture of racemic
NaOH
hydrolysis,
as
reported
for
similar
compounds.14,17

748
Y. Zea-Ponce et al. / Bioorg. Med. Chem. 13 (2005) 745–753
regioisomers 12 and 13. The regioisomers were sepa-
rated via silica gel chromatography. Unfortunately,
under standard conditions used for IBVM, the
diazotization of the 5-amino-derivative 12 and reaction
with potassium iodide did not lead to the desired iodo-
derivative 16 (data not shown). So, an alternate
approach was done: direct iodination of the regioisomer
13 with ICl in glacial acetic acid produced racemic 14.
During the chiral resolution, in the reductive cleavage
step using DIBAL-H, as expected, the cyano group
was reduced to aminomethyl (15). Subsequent deamina-
tion of the aryl amine with hypophosphorus acid
(H3PO2) and sodium nitrite (NaNO2) led to enantio-
meric 16 (Scheme 3).
the chiral syntheses, and the in vitro determination of
the binding affinity for the VAChT of enantiomeric
compounds 3, 4, 8 and 16. In fact, this route could also
be used to generate pure enantiomeric precursors needed
for radiolabelling: radioiodinated compounds could be
obtained by iododestannylation of the corresponding
tributyltin derivatives, which are usually prepared from
the iodo precursors.
In each pair tested, the one with structure designation
(R,R) consistently exhibit Kd value higher than the
(S,S), in agreement with data previously reported11,16,20
(Table 1).
Of the eight enantiomers synthesized, (E)-(R,R)-5-
AOIBV [(R,R)-3] and (R,R)-5-FPOBV [(R,R)-4] dis-
played the highest binding affinities for VAChT
(Kd = 0.45 and 0.77nM, respectively), which suggest
that elongation of the substituent chain from 5-idodo,
as in the case of (À)-5-IBVM (Kd = 0.30nM) to a five
member 5-O-halogenoalkyl or alkenyl chain, had lim-
ited effect on affinity for VAChT. That bulkier ether
substituents at this position does not impair the binding
affinity to VAChT has been previously shown.14,16 In
this position, the electronegativity, and the halogen
atom size does not influence the protein–ligand interac-
tion. We chose a propyl chain to compare the activity
with the fluoroethyloxy derivative (FEOBV).14 The
fluoropropyl may be more stable than the fluoromethyl
or fluoroethyl compound in an in vitro metabolic and
environment, and this type of radiotracer is hoped to
be a better ligand in vivo. Moreover, we kept a consist-
ent chain length between FEOBV and 5-AOIBV for
comparison of their receptor–ligand interaction.
In each case, chiral resolution of racemic derivatives was
achieved with (À)-(R)-a-methoxy-a-(trifluoromethyl)-
phenylacetyl chloride (MTPA-Cl)22 followed by
deprotection with either 2N NaOH or DIBAL-H (for
the ester-amide obtained with 14) as required. Reactions
carried out with the (R,R)-enantiomers were repeated
with the corresponding (S,S)-enantiomers.
2.2. Biological evaluation
Human VAChT stably expressed in the PC12 cell line,23
which is a common model system for neurosecretory
phenomena, was used to screen target compounds. Be-
cause the cell line expresses a relatively large amount
of human VAChT (>2pmol/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 sup-
ernatant rich in synaptic-like microvesicles containing
human VAChT. Competition was allowed to proceed
24h to ensure equilibration of low concentrations of tar-
get compounds. It could not be carried out at 37ꢁC due
to the length of incubation. However, binding of vesam-
icol is not strongly temperature dependent.
Substitution in the 4-position on the piperidine ring,
which has not been extensively explored, with an amino-
methyl group [(R,R)-16, Kd = 8.8nM] slightly decreased
the affinity compared to 5-IBVM, but this position
might produce potent compounds having much more
polarity.
On the other hand, (R,R)-8, which also maintains the
basic structure of (À)-5-IBVM but with an additional
methoxy group in the 8-position of the aromatic ring,
showed very low activity for the target site (Kd around
100nM); confirming the negative effect on receptor
binding when substituents are present at this position
as shown in studies by others.17,20 An unsubstituted
8-position thus seems to be critical to the binding, spe-
cially since the lipophilicity (as inferred from their calcu-
lated logP, Table 1) appears to be of similar magnitude
for both, 8-MOIBV (8) and 5-IBVM.
3. Discussion
In the development of radiohalogenated benzovesamicol
analogues as potential radiotracers for SPECT or PET,
is necessary that the radiohalogen incorporated into the
molecule does not alter the vesamicol-like inhibitory
activity of the compound. Moreover, in cases where
the binding to the receptor is enantioselective, enantio-
meric purity of the tracer is a must. Here we report
Table 1. Affinities (Kd SEM) of vesamicol, 5-IBVM, and selected analogues 3, 4, 8 and 16 for VAChT
(R,R)-3
(S,S)-3
(R,R)-4
(S,S)-4
(R,R)-8
(S,S)-8
(R,R)-16
(S,S)-16
Vesamicol
5-IBVM
Kd (nM)a
ClogPb
0.45 0.11
4.3 1.4
0.77 0.18
8.4 1.9
102 43
>100
8.8 3.0
>100
20 2.3
3.5
0.30 0.11
5.2
5.5
4.4
5.2
3.8
a Kd values were determined by competition of derivatives against bound [3H]vesamicol under equilibrium at 22ꢁC, unless stated; vesamicol and 5-
IBVM were the pure (À)-enantiomer.
b Calculated logP (cLogP, ChemDraw Ultra 6.0.1, 2000).

Y. Zea-Ponce et al. / Bioorg. Med. Chem. 13 (2005) 745–753
749
4. Conclusion
to give 44mg (71%) of the precursor (S,S)-2 as a pale
pink solid. The same procedure, starting with 37mg
(115lmol) of the enantiomer (R,R)-114 produced 60mg
(80%) of the corresponding precursor (R,R)-2 as a pale
pink solid.
We have synthesized eight new authentic optical isomer
analogues of benzovesamicol. Of these, the iodinated
derivative (E)-5-AOIBV [(R,R)-3] and the fluorinated
derivative 5-FPOBV [(R,R)-4] displayed VAChT bind-
ing affinity of comparable magnitude to that of (À)-5-
IBVM; suggesting its potential application, when
radiohalogenated, as SPECT or PET imaging agents
for studies involving cholinergic nerve terminals.
3
1H NMR (CDCl3): d 0.95 (t, 9H, J = 7.0Hz, 3CH3),
1.25–1.68 [m, 18H, (CH3C3H6)3Sn], 1.72–2.06 (m, 4H,
4H-10), 2.44–3.19 (m, 9H, H-11, H-1, 2H-4, H-3, 4H-
3
9), 3.30 (dd, 1H, J = 5.6Hz, 3J = 16Hz, H-1), 3. 88
(m, 1H, H-2), 4.60 (d, 2H, J = 4.3Hz, OCH2), 6.13–
3
6.44 (m, 2H, H-13, H-14), 6.66–6.76 (m, 2H, H-6, H-
8), 7.09 (t, 1H, 3J = 7.6Hz, H-7), 7.19–7.38 (m, 5H,
5HAr). 13C NMR: d 9.5 [(CH2)3Sn], 13.7 (3CH3), 20.1
(C-4), 27.2 [(CH3CH2C2H4)3Sn], 29.0 [(CH3CH2-
CH2CH2)3Sn], 33.9–34.4 (2C-10), 38.0 (C-1), 42.9
(C-11), 44.9, 53.6 (2C-9), 65.3 (C-2), 66.6 (C-3), 71.3
(OCH2), 108.8 (CHAr), 121.3 (CHAr), 124.1 (CHAr),
126.2 (CAr), 126.6 (CHAr), 126.8 (2CHAr), 128.4
(2CHAr), 131.1 (SnCH@), 135.3 (CAr), 143.1 (CH@),
146.1 (CAr), 156.5 (CAr).
5. Experimental
5.1. Chemistry
NMR spectra were recorded on a Bruker DPX Avance
1
200 spectrometer (200MHz for H, 50.3MHz for 13C).
CDCl3 was used as solvent; chemical shifts are expressed
in ppm relative to TMS as an internal standard. Melting
points were determined in a Buchi B540 capillary melt-
ing point apparatus (Switzerland) and are uncorrected.
Column chromatography was carried out using VWR
silica gel (230–400mesh), preparative TLC was carried
out using 20 · 20cm, glass backed silica gel F254 plates,
1mm or higher thickness, from VWR. Thin-layer chro-
matography was performed using plastic backed sheets
with silica gel F254 also from VWR. Chemicals and sol-
vents ACS grade or higher, commercially available, were
used without further purification. Elemental analyses of
new compounds were within 0.4% of theoretical val-
ues. Phenylpiperidine used in the synthesis of 5- and
8-aminobenzovesamicol (ABV),20 was obtained by alka-
line fusion from 4-cyano-4-phenylpiperidine,24 (E)-3-
(tri-n-butylstannyl)prop-2-en-1-ol p-toluene sulfonate
was prepared from (E)-3-(tri-n-butylstannyl)-prop-2-
en-1-ol and tosyl chloride in the presence of potassium
trimethylsilanolate (KOSiMe3) as described.21
5.1.2. (2S,3S)-(E)-2-Hydroxy-5-(iodoallyloxy)-3-(4-phen-
ylpiperidino)tetralin, (E)-5-AOIBV, (S,S)-(E)-3. Stannyl
derivative (S,S)-2 (34mg, 52lmol), was dissolved in
CHCl3 and cooled at À5ꢁC. A solution of iodine in
CH2Cl2 (0.1M, 532lL) was then added in aliquots, un-
der constant stirring, until a coloured solution resulted.
The reaction mixture was washed with 12% NaHSO3
(5mL), the organic layer separated and the aqueous ex-
tracted with CH2Cl2 (3 · 10mL); the combined organic
layers were dried over anhydrous Na2SO4 and rotoevap-
orated off. The crude product was purified by preparative
TLC on silica gel plates, with 30% EtOAc in hexane plus
diethyl ether (7:3) to give 17mg (67%) of (S,S)-3.
1H NMR (CDCl3): d 1.74–1.96 (m, 4H, 4H-10), 2.48–
2.75 (m, 3H, H-11, H-1, H-4), 2.78–3.15 (m, 6H, H-4,
3
3
H-3, 4H-9), 3.33 (dd, 1H, J = 5.6Hz, J = 16Hz, H-
1), 3.83–3.93 (m, 1H, H-2), 4.44 (s, OH), 4.50–4.53 (m,
2H, OCH2), 6.52–6.687 (m, 2H, CH@, ICH@), 6.78–
6.91 (m, 2H, H-6, H-8), 7.14 (t, 1H, J = 7.6Hz, H-7),
7.25–7.41 (m, 5H, 5HAr). 13C NMR: d 20.1 (C-4),
33.9–34.4 (2C-10), 37.9 (C-1), 42.8 (C-11), 44.8–53.5
(2C-9), 65.2 (C-2), 66.4 (C-3), 69.6 (OCH2), 79.2
(ICH), 108.2 (CHAr), 121.9 (CHAr), 124.1 (CAr), 126.2
(CHAr), 126.7 (2CHAr), 126.8 (2CHAr), 128.4 (CHAr),
135.7 (CAr), 140.9 (CH@), 146.2 (CAr), 155.8 (C-5).
(À)-5-IBVM was synthesized in the laboratory as previ-
ously described.11,20
5.1.1. (2R,3R)- and (2S,2S)-(E)-2-hydroxy-5-(tributyl-
stannylallyloxy)-3-(4-phenylpiperidino)tetralin,
(E)-5-
Bu3SnAOIBV, (R,R)- and (S,S)-2. A solution of (+)-5-
HOBV;14 (S,S)-1, (30.5mg, 94lmol) was dissolved in
3mL of CH2Cl2 under inert atmosphere and 100lL of
1M tetrabutylammonium hydroxide (TBAOH) in meth-
anol added; the solvent was removed by rotary evapora-
tion at room temperature and to the residue 2mL of dry
CH3CN added and the mixture rotoevaporated to dry-
ness, to azeotropically remove traces of moisture. The
process was repeated once more and then, under inert
atmosphere, the mixture was resuspended in 3mL of
dry CH3CN and (E)-3-(tri-n-butylstannyl)prop-2-en-1-
ol, p-toluenesulfonate21 (60mg, 119lmol) added. The
mixture was heated, at 75ꢁC for 4h, and then let slowly
rise to room temperature overnight. After evaporation
of the solvent, the crude product was partitioned be-
tween CH2Cl2 and water (20 and 10mL, respectively)
and the organic layer washed with 0.5N NaOH, dried
over anhydrous Na2SO4, concentrated and purified by
preparative TLC on silica gel (30% EtOAc in hexane)
5.1.3. (2R,3R)-(E)-2-Hydroxy-5-(iodoallyloxy)-3-(4-phen-
ylpiperidino)tetralin, (E)-5-AOIBV, (R,R)-(E)-3. The
(R,R)-2 precursor (38mg, 58lmol) was dissolved in de-
gassed THF (5mL) and stirred for 5min under inert
atmosphere. N-Iodosuccinimide (NIS, 13.8mg, 61lmol)
dissolved in 0.5mL degassed THF, was added and the
mixture stirred for 20min at room temperature. The sol-
vent was rotoevaporated off, and the crude product
purified by preparative TLC on silica gel, 30% EtOAc
in hexane to give 22mg (77%) of (R,R)-3.
5.1.4. Toluene-4-sulfonic acid-3-fluoropropyl ester:
F(CH2)3OTs. Under inert atmosphere, propanediol
ditosylate TsO(CH2)3OTs (2.00g, 5.20mmol) was

750
Y. Zea-Ponce et al. / Bioorg. Med. Chem. 13 (2005) 745–753
dissolved in tetrabutylammonium fluoride 1M solution
in THF (TBAF) (13.4mL). The mixture was heated,
under inert atmosphere, at 80ꢁC for 15min. The solvent
was removed by rotary evaporation. The crude product
was portioned between Et2O and 2N Na2CO3 solution,
the organic layer was washed with water, dried over
Na2SO4, concentrated and purified by flash chromato-
graphy on silica gel with 50% CH3Cl3 in hexane to give
0.432g (36%) of 3-(tosyloxy)-l-propyl fluoride
[F(CH2)3OTs] as a colourless oil. NMR spectrum data
matched the one previously reported for this
compound.25
the addition was completed and then allowed to cool
to room temperature. The solution was bought to
pH = 9 with 5N NaOH and extracted with ethyl acetate
(3 · 50mL). The combined extracts were dried over
anhydrous NaSO4 and concentrated under reduced
pressure. The residue was purified by chromatography
on silica gel with 30% EtOAc in hexane to afford race-
mic 8-HOBV; ( )-6 (181mg, 97%) as a white solid (mp
206–207ꢁC).
1H NMR (CDCl3): d 1.74–1.98 (m, 4H, 4H-10), 2.37–
3.00 (m, 9H, 4H-9, 2H-4, H-3, H-11, H-1), 3.47 (dd,
3
3
1H, J = 6.2Hz, J = 16.2Hz, H-1), 3.92–3.98 (m, 1H,
H-2), 6.63–6.67 (m, 2H, H-5, H-7), 7.07 (t, 1H,
3J = 8Hz, H-6), 7.22–7.41 (m, 5H, 5HAr). 13C NMR: d
26.2 (C-4), 31.8 (C-1), 33.6–34.3 (2C-10), 42.9 (C-11),
45.0–53.6 (2C-9), 65.8 (C-3), 66.0 (C-2), 112.4 (C-7),
121.3 (C-5), 126.2 (C-8a), 126.8 (C-6), 126.9 (2CHAr),
128.3 (2CHAr), 137.0 (C-4a), 147.1 (CAr), 157.4 (C-8).
5.1.5. (2R,3R)-2-Hydroxy-3-(4-phenylpiperidino)-5-(3-
fluoropropoxy)tetralin, 5-FPOBV, (R,R)-4. A solution
of (À)-5-HOBV; (R,R)-1 (30.5mg, 94lmol) was dis-
solved in 2mL of CH2Cl2 under inert atmosphere and
100lL of 1M tetrabutylammonium hydroxide
(TBAOH, 100lmol) in methanol added; the solvent
was removed by rotary evaporation at room tempera-
ture and the residue treated twice with 2mL of dry
CH3CN followed each time by rotoevaporation to azeo-
tropically remove traces of moisture. Under inert atmos-
phere, the phenoxide was resuspended in 4mL of dry
CH3CN, and 3-fluoropropyltosylate in 2mL dry
CH3CN was added; the reaction mixture was heated at
75ꢁC for 3h. After rotoevaporation of the solvent, the
crude product was partitioned between CH2Cl2 and
water (20 and 10mL, respectively) and the organic layer
washed with 40mL of 0.5N NaOH, dried over anhy-
drous Na2SO4, concentrated to a yellow solid and puri-
fied by preparative TLC on silica gel plate and 50%
EtOAc in hexane to give 30mg (84%) of (R,R)-4 as a
white solid with mp 142–143ꢁC. The same procedure
starting with 30mg (93lmol) of the (S,S)-1 enantiomer
produced 32mg (90%) of (S,S)-4 as a white solid.
5.1.7. ( )-trans-2-Hydroxy-8-methoxy-3-(4-phenylpiper-
idino)tetralin, 8-MOBV, ( )-7. Racemic 8-hydroxy-
benzovesamicol, ( )-6 (181mg, 0.56mmol) in 10mL
dry CH3CN plus a few drops CH2Cl2, was rotoevapo-
rated to dryness, 2mL dry CH3CN added and rotoevap-
oration repeated, to remove traces of moisture. To the
residue, dissolved in 4mL of dry CH3CN, was added un-
der inert atmosphere 70lL of 1M tetrabutylammonium
hydroxide in methanol; the reaction mixture was stirred
for 3–4min followed by solvent rotoevaporation at
room temperature. Traces of moisture were removed
by azeotropically adding 2mL of dry CH3CN and roto-
evaporation to dryness. Under inert atmosphere, the res-
idue was resuspended in 6mL of dry CH3CN and CH3I
(160mg, 1.12mmol) added. The mixture was heated, at
85ꢁC for 1h, and then let slowly rise to room tempera-
ture. After rotary evaporation of the solvent, the crude
product was partitioned between CH2Cl2 and water
(20 and 10mL, respectively) and organic layer washed
with 0.5N NaOH (40mL), dried over anhydrous
Na2SO4, concentrated and purified by chromatography
on silica gel (30% EtOAc in hexane) to give 127mg
(67%) of ( )-7, as a white solid (mp 202–203ꢁC).
1H NMR (CDCl3): d 1.65–2.05 (m, 4H, 4H-10), 2.28
(qd, J = 26.0, 5.9Hz, CH2), 2.42–3.14 (m, 9H, 4H-9,
3
3
2H-4, H-3, H-11, H-1), 3.33 (dd, 1H, J = 16.0, 5.4Hz,
H-1), 3.82 (ddd, 1H, 3J = 5.6, 10.2, 20.4Hz, H-2),
3
3
4.15 (t, J = 5.9Hz, OCH2), 4.73 (dt, J = 47.0, 5.9Hz,
CH2F), 6.70–6.81 (m, 2H, H-6, H-8), 7.16 (t, 1H,
3J = 8.0Hz, H-7), 7.25–7.41 (m, 5H, 5HAr). 13C NMR:
d 19.9 (C-4), 30.4 (CH2, 2J = 20Hz), 33.9, 34.4 (2C-
10), 38.0 (C-1), 42.1 (C-11), 44.9, 53.6 (2C-9), 63.4
1H NMR (CDCl3): d 1.75–1.94 (m, 4H, 4H-10), 2.36–
3.00 (m, 9H, 4H-9, 2H-4, H-3, H-11, H-1), 3.48
3
3
(dd, 1H, J = 6Hz, J = 17Hz, H-1), 3.78–3.82 (m, 4H,
3
(OCH2, J = 5.5Hz), 65.3–66.5 (C-3, C-2), 80.9 (CH2F,
3
1J = 165Hz), 108.0 (C-6), 121.5 (CHAr), 123.9 (C-4a),
126.2 (CHAr), 126.8 (2CHAr), 128.5 (2CHAr), 135.5 (C-
8a), 146.2 (CAr), 156.4 (C-5).
OCH3, H-2), 6.70 (t, 2H, J = 7Hz, H-5, H-7), 7.12–
7.41 (m, 6H, 5HAr, H-6). 13C NMR: d 26.1 (C-4), 32.0
(C-1), 33.9–34.3 (2C-10), 42.9 (C-11), 45.0–53.6 (2C-9),
55.6 (OCH3), 65.8 (C-3), 66.1 (C-2), 107.3 (C-7), 121.1
(C-5), 126.2 (C-8a), 126.6 (C-6), 126.8 (2CHAr), 128.4
(2CHAr), 136.3 (C-4a), 146.1 (CAr), 157.4 (C-8).
5.1.6.
( )-trans-2,8-Dihydroxy-3-(4-phenylpiperidino)-
tetralin, ( )-8-HOBV, ( )-6. A 1:2 dilution of concen-
trated sulfuric acid in water (6mL) was added to a
cooled solution of racemic 8-hydroxybenzovesamicol
(8-ABV)20 (185mg, 574lmol) in THF (5mL), then a
solution of sodium nitrite (47.5mg, 690lmol) in water
(2mL) was added dropwise while the temperature of
the reaction mixture was maintained below 5ꢁC. Stirring
was continued for 1h. The resulting diazonium solution
was then added carefully in small aliquots, to a second
dilution of sulfuric acid (2mL) in water (10mL) at its
boiling point. The mixture was boiled for 5min after
5.1.8. ( )-trans-2-Hydroxy-5-iodo-8-methoxy-3-(4-phen-
ylpiperidino)tetralin, 8-MOIBV, ( )-8. To a solution of
( )-7 (60mg, 178lmol) in glacial acetic acid (7mL) was
added dropwise a solution of iodine monochloride
(65mg, 400lmol) in acetic acid (1mL) and was heated
at reflux overnight. The solvent was evaporated under re-
duced pressure. The residue was dissolved in CHCl3
(20mL), and the solution was washed with saturated
NaHCO3 solution (20mL) and the organic layer sepa-

Y. Zea-Ponce et al. / Bioorg. Med. Chem. 13 (2005) 745–753
751
rated. The aqueous layer was extracted with additional
CHCl3 (4 · 20mL). The combined extracts were dried
over anhydrous Na2SO4 and concentrated under reduced
pressure. The crude reaction mixture was purified by pre-
parative TLC (silica gel and 30% ethyl acetate in petro-
leum ether) to afford 40mg (47%) of ( )-8 as a white solid.
5.1.11. ( )-trans-5-Amino-2-hydroxy-3-(4-cyano-4-phen-
ylpiperidino)tetralin, 5-ACBV, ( )-12, ( )-trans-8-amino-
2-hydroxy-3-(4-cyano-4-phenylpiperidino)tetralin,
8-
ACBV, ( )-13. 4-Cyano-4-phenylpiperidine hydrochlo-
ride 10 (3.3g, 14.5mmol) was dissolved in ethanol
(40mL) containing triethyl amine (3.5mL) and then
added to N-(trifluoroacetyl)-1-amino-5,8-dihydronaph-
thalene oxide 11 (1.71g, 6.66mmol) dissolved in EtOH
(20mL). The mixture was refluxed for 20h. The solvent
was evaporated and the oil residue was dissolved in
methanol (25mL) and treated with 2N NaOH (40mL)
and stirred at room temperature for 22h. The reaction
mixture was extracted with CH2Cl2 (2 · 50mL). The or-
ganic layer was dried with anhydrous sodium sulfate
and evaporated under reduced pressure. The crude
product was purified by chromatography on silica gel
with CH2Cl2/MeOH/Et3N: 195/5/1. ( )-12 is the less
polar, (Rf = 0.53, CH2Cl2/EtOH/TEA: 195/5/1) and is
obtained in 36% yield. ( )-13, the more polar,
(Rf = 0.29) is obtained in 28% yield.
1H NMR (CDCl3): d: 1.78–1.94 (m, 4H, 4H-10), 2.42–
3.00 (m, 9H, 4H-9, 2H-4, H-3, H-11, H-1), 3.33 (dd,
3
1H, J = 16.9, 6Hz, H-1), 3.78–3.87 (m, 4H, OCH3, H-
2), 6.48 (d, 1H, 3J = 6.8Hz, H-7), 7.23–7.38 (m, 5H,
5HAr), 7.67 (d, 1H, 3J = 6.8Hz, H-6). 13C NMR: d
32.2, 33.0, 33.9, 34.3 (C-4, C-1, 2C-10), 42.9 (C-11),
45.0, 53.6 (2C-9), 55.6 (OCH3), 65.4, 66.7 (C-3, C-2),
91.0 (C-5), 109.8 (C-7), 125.3 (CHAr), 125.2 (C-8a),
126.8 (2CHAr), 128.4 (2CHAr), 137.0 (C-6), 138.1 (C-
4a), 146.1 (CAr), 157.1 (C-8).
5.1.9. Diastereomeric O-(S)-a-methoxy-a-trifluorometh-
ylphenylacetyl derivatives of 8-MOIBV; (R,R)-9 and
(S,S)-9. This procedure illustrates enantiomeric resolu-
tion in small scale, for molecules possessing only one
reactive hydroxy group.
1
( )-12, H NMR (CDCl3): d 2.12–2.53 (m, 4H, 4H-10),
2.85–3.35 (m, 9H, 2H-1, 2H-4, H-3, 4H-9), 3.66 (s, 2H,
NH2), 3.93–4.15 (m, 2H, H-2, OH), 6.20–6.24 (m, 2H,
3
A mixture of racemic 8 (25mg, 54lmol), plus 4-(dimeth-
ylamino)pyridine, DMAP (18lmol) and triethylamine
(163lmol) was prepared in dry CHCl3 (2mL). To the
mixture was added in small portions and at room tem-
perature, (R)-(À)-a-methoxy-a-(trifluoromethyl)phenyl-
acetyl chloride; 18mg (71lmol). The reaction mixture
was stirred for 18h at room temperature, then poured
into ethyl acetate (10mL) and washed with saturated
NaHCO3 (15mL). After separation of the organic layer,
the aqueous layer was extracted with ethyl ether
(3 · 20mL). The combined organic extracts were dried
over anhydrous Na2SO4 and concentrated under reduced
pressure. The two diastereomeric MTPA esters of 8 were
separated by preparative TLC (silica gel; 5% EtOAc in
petroleum ether, plate developed twice). After work up,
10.3mg of the less polar (Rf = 0.25) diastereomer and
7mg of the more polar (Rf = 0.17) were obtained. The
less polar of the two MTPA esters, was presumed to be
the (S,S)-9 diastereomer and the more polar, the (R,R)-
9 diastereomer; by analogy with the TLC behaviour of
the diastereomeric MPTA esters of 5-IBVM.17
H-6, H-8), 7.04 (t, 1H, J = 7.7Hz, H-7), 7.39–7.58 (m,
5H, 5HAr). 13C NMR: d 26.5 (C-4), 33.0 (C-1), 36.8,
37.1 (2C-10), 41.9, 50.1 (2C-9), 43.0 (C-11), 65.8, 65.9
(C-3, C-2), 112.6 (CHAr), 118.6 (CN), 119.3 (CHAr),
122.2 (CAr), 125.5 (2CHAr), 126.9 (CHAr), 128.2 (CHAr),
129.0 (2CHAr), 135.4 (CAr), 140.1 (CAr), 145.7 (CAr).
1
( )-13, H NMR (CDCl3): d 2.12–2.58 (m, 4H, 4H-10),
2.64–3.03 (m, 7H, 2H-4*, H-3, 4 H-9), 3.26 (m, 2H, 2H-
1*), 3.71 (s, 2H, NH2), 3.90–4.07 (m, 2H, H-2, OH),
3
6.59–6.65 (m, 2H, H-5, H-7), 7.04 (t, 1H, J = 7.7Hz,
H-6), 7.39–7.59 (m, 5H, 5HAr). 13C NMR: d 21.2 (C-
4), 36.9, 37.3, 38.0 (2C-10, C-1), 43.0 (C-11), 41.9, 50.1
(2C-9), 65.3, 66.7 (C-3, C-2), 112.7 (CHAr), 119.1
(CAr), 119.5 (CHAr), 125.5 (2CHAr), 127.1 (CHAr),
128.3 (CHAr), 129.1 (2CHAr), 135.6 (CAr), 139.8 (CAr),
144.5 (CAr).
5.1.12. ( )-trans-8-Amino-2-hydroxy-5-iodo-3-(4-cyano-
4-phenylpiperidino)tetralin, 8-ACIB, ( )-14. To a solu-
tion of ( )-8-aminocyanobenzovesamicol (13) (0.63g,
1.82mmol) in acetic acid (18mL), at room temperature,
was added dropwise a solution of iodine monochloride
(0.3g, 1.82mmol) in acetic acid (2mL). The reaction
mixture was stirred overnight, and the solvent was evap-
orated under reduced pressure. The residue was dis-
solved in CHCl3 (20mL), washed with saturated
NaHCO3 solution (15mL) and extracted with CHCl3
(3 · 30mL). The combined extracts were dried over
anhydrous Na2SO4 and concentrated under reduced
pressure. The crude product was purified by chromato-
graphy on silica gel with (35% ethyl acetate in petroleum
ether) to afford 430mg (50%) of ( )-14 as a white solid
(mp = 250–252ꢁC).
5.1.10. Alkaline hydrolysis leading to (R,R)-8 and (S,S)-8.
In separated reactions, each MTPA-ester (S,S)-9 or
(R,R)-9, (10mg, 14.7lmol and 7mg, 10.3lmol, respec-
tively) was dissolved in THF (1mL) and a solution of
CH3OH (0.5mL) plus 2N NaOH (2mL) added. The
reaction mixture was stirred at room temperature for
16h. EtOAc (2.5mL) was added, stirring for 10min be-
fore transferring the mixture into a separatory funnel.
The reaction vessel was washed with an additional
2.5mL EtOAc and added to the same funnel. The or-
ganic layer was separated and the aqueous layer ex-
tracted with more EtOAc (2 · 5mL) plus once with
CH2Cl2 (5mL). The combined organic extracts were
dried over anhydrous Na2SO4 and concentrated under
reduced pressure. After preparative TLC purification
(30% EtOAc in petroleum ether), pure (R,R)-8 was ob-
tained in 71% yield and (S,S)-8 in 97% yield.
1H NMR (CDCl3): d 2.16–2.26 (m, 4H, 4H-10), 2.54–
3.00 (m, 7H, 2H-4*, H-3, H-1, 3H-9), 3.26 (m, 2H, H-
1, H-9*), 3.71 (s, 2H, NH2), 3.97–3.90 (m, 2H, H-2,
3
OH), 6.36 (d, 1H, J = 8.5Hz, H-7), 7.37–7.57 (m, 6H,

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Y. Zea-Ponce et al. / Bioorg. Med. Chem. 13 (2005) 745–753
5HAr, H-6). 13C NMR: d 21.6 (C-4), 36.8, 37.2 (2C-10),
41.9, 43.0 (C-1, C11), 44.4, 50.2 (2C-9), 65.9, 65.1 (C-3,
C-2), 87.7 (C-5), 114.8 (CHAr), 121.3 (CN), 122.3 (CAr),
125.5 (2CHAr), 128.3 (CHAr), 129.1 (2CHAr), 137.1
(CAr), 137.1 (CHAr), 140.0 (C-4a), 144.8 (CAr).
anhydrous Na2SO4 and concentrated under reduced
pressure. The crude product was purified by
chromatography on silica gel (CH2Cl2/EtOAc/hexane/
EtOH/Et3N: 1.1/1/1/0.1/0.02). The larger fraction col-
lected (about 110mg) was re-purified using two prepar-
ative TLC 20 · 20 glass backed silica plates (about
55mg in each) with EtOAc/hexane/EtOH/Et3N (1/1/
0.25/0.02) as mobile phase. The lowest band was
scratched, extracted (MeOH), filtered (sintered funnel)
and the solvent evaporated under reduced pressure.
Recovered 80mg of a white solid (mp = 230–231ꢁC).
The enantiomer (S,S)-8-amino-2-hydroxy-5-iodo-3-
5.1.13. Enantiomeric resolution of ( )-14, as a synthetic
route to the enantiomeric pair 8-AMAIB: (R,R)- and
(S,S)-15
5.1.13.1. Diastereomeric bis-N,O-(S)-a-methoxy-a-tri-
fluoromethylphenylacetyl derivatives 14. Racemic 14 is an
analogue possessing one hydroxy plus one amino react-
ing groups, its corresponding bis-N,O-(S)-a-methoxy-a-
trifluoromethylphenylacetyl (MTPA) derivatives can be
separated by chromatography by a similar method as
that reported for 5-ABV.14 Reductive cleavage of the
protecting MTPA groups in each of the separated deriv-
atives, using DIBAL-H, led to simultaneous reduction
of the cyano into methylamino group.
(4-methylamino-4-phenylpiperidino)tetralin;
AMAIB, (S,S)-15 was obtained in 45% yield.
(S,S)-8-
The (R,R)-15 enantiomer was obtained, by a similar
procedure, in 41% yield, starting with 260mg
(0.29mmol) of the (R,R)-bis-MTPA diastereomer.
1H NMR (CDCl3): d 1.38 (t, 2H, 3J = 7.2Hz, CH2NH2),
2.00–2.10 (m, 4H, 4H-10), 2.34–3.0 (m, 10H, H-1, H-3,
A solution of ( )-14 (0.41mg, 0.87mmol), plus 4-
(dimethylamino)pyridine (63.4mg, 0.52mmol) and tri-
ethylamine (0.5mL, 1.82mmol) was prepared in dry
CHCl3 (15mL). To the mixture was added in small por-
tions and at room temperature, via syringe, (R)-(À)-
MTPA chloride (503mg, 2mmol) in 2mL dry CHCl3.
The reaction mixture was stirred for 19h at ambient tem-
perature, then poured into ethyl acetate (30mL) and
washed with saturated NaHCO3 (30mL) added carefully
with constant stirring. After separation of the organic
layer, the aqueous layer was extracted with ethyl acetate
(2 · 40mL). The combined organic extracts were dried
over anhydrous Na2SO4 and concentrated under reduced
pressure. The two diastereomeric N,O-bis-MTPA iso-
mers of racemic 14 were separated by chromatography
(silica gel, CH2Cl2), to obtain 360mg of the less polar
compound (Rf = 0.53, EtOAc/CH2Cl2/petroleum ether:
1/2/7) and 260mg of the more polar diastereomer
(Rf = 0.36, EtOAc/CH2Cl2/petroleum ether: 1/2/7). By
analogy with the TLC behaviour of the diastereomeric
N,O-bis-MPTA derivatives of 5-ABV,14 the less polar
compound was presumed to be the (S,S)-N,O-bis-MTPA
diastereomer of 14 (46% yield) and the more polar the
(R,R)-MTPA diastereomer of 14 (33% yield).
3
2H-4, 4H-9, CH2NH2), 3.35 (dd, 1H, J = 16.7, 5.6Hz,
H-1), 3.58 (s, 2H, NH2), 3.63 (s, 1H, OH), 3.75–3.88 (m,
3
1H, H-2), 6.32 (d, 1H, J = 8.5Hz, H-7), 7.29–7.53 (m,
7H, 5HAr, H-6, H-8). 13C NMR: d 32.9 (C-1), 33.8 (C-
4), 34.8 (2C-10), 42.8 (C-11), 44.9 (2C-9), 50.9 (CH2NH2),
65.2, 67.2 (C-3, C-2), 102.9 (CI), 126.2 (CHAr), 126.8
(2CHAr), 127.8 (CHAr), 128.5 (2CHAr), 129.6 (CHAr),
135.8 (CAr), 137.1 (CHAr), 137.4 (CAr), 146.0 (CAr).
5.1.15. (R,R)- and (S,S)-2-Hydroxy-5-iodo-3-(4-amino-
methyl-4-phenylpiperidino)tetralin, MAIBV, (R,R)- and
(S,S)-16. A solution of NaNO2 (13mg, 188lmol) in
H2O (0.7mL) was added dropwise to a cooled (5ꢁC)
solution of (S,S)-15 (82mg, 172lmol) in 50% hypophos-
phorous acid (4mL) and concentrated HCl (1mL) keep-
ing the reaction temperature around 0ꢁC, stirring for
60min and then allowed to warm to room temperature
overnight, under continuous stirring. The reaction mix-
ture was poured into saturated NaCl solution (20mL)
and extracted with CH2Cl2 (3 · 20mL). The aqueous
layer was removed, carefully basified with NaHCO3
(around 60mL) and extracted with more CH2Cl2
(5 · 20mL); the combined extracts were carefully
washed with saturated NaHCO3 (20mL), and then with
water (5mL), dried over anhydrous sodium sulfate and
concentrated under reduced pressure. The residue was
placed on two preparative TLC silica gel glass backed
plates and run twice with EtOAc/hexane/EtOH/Et3N
(1.25/1.25/0.2/0.12). After work up, 73mg (92%) of the
enantiomer (S,S)-16 was obtained as a solid (from
MeOH). The same experimental conditions, starting
with 56mg (117lmol) of (R,R)-15 afforded 48mg
(88%) of the enantiomer (R,R)-16.
5.1.14. Reductive cleavage to yield (R,R)- and (S,S)-15.
Procedure was similar to the one reported for 5-ABV,14
except that the reaction vessel set up was flushed initially
with nitrogen gas for 30min. (S,S)-Diastereomer,
(360mg, 0.398mmol) was dissolved in dry toluene
(9mL) and keep under inert atmosphere, stirring for
10min and then cooled to À78ꢁC. Diisobutyl alumin-
ium hydride (DIBAL-H, 2mL of 1M solution in cyclo-
hexane; 2mmol) was added dropwise via syringe. The
resulting solution was stirred at À80ꢁC for 30min and
then 2mL more of DIBAL-H were added to assure com-
plete reduction of the cyano group. The reaction mix-
ture was allowed to slowly warm to room temperature,
quenched with 2.5N HCl (10mL) and left overnight at
room temperature. The toluene layer was separated,
the aqueous layer made alkaline (pH10) with 6N NaOH
(about 8mL) and extracted with CH2Cl2 (4 · 40mL).
The combined organic extracts were dried over
1H NMR (CDCl3): d 1.31 (t, 2H, 3J = 7.2Hz, CH2NH2),
2.06–3.12 (m, 14H, 4H-10, 4H-9, 2H-4, H-3, H-1,
3
CH2NH2), 3.38 (dd, 1H, J = 6.0Hz, J = 16.8Hz, H-
3
1), 3.61 (s, 1H, OH), 3.73–3.96 (m, 1H, H-2), 6.81 (t,
1H, J = 7.6Hz, H-7), 7.02 (d, 1H, J = 7.6Hz, H-8),
3
3
3
7.28–7.48 (m, 5H, 5HAr), 7.71 (d, 1H, J = 7.6Hz, H-
6). 13C NMR: d 27.2 (C-4), 31.5 (C-1), 42.5 (2C-10),
43.2 (C-11), 44.4 (2C-9), 47.6 (CH2NH2), 65.5, 66.6

Y. Zea-Ponce et al. / Bioorg. Med. Chem. 13 (2005) 745–753
753
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described.26 Compounds were assayed in increments of
10-fold from 0.1 to 10,000nM concentration. The sur-
faces of containers were precoated with Sigmacote. Sam-
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F glass fiber filters coated with polyethylenimine and
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liquid scintillation spectrometry. Averaged data were
fit by regression with a rectangular hyperbola to esti-
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Acknowledgements
This work was supported by INSERM, and by a grant
(82/2001 and 582/2003) in aid scientific research from
the AECS (Atomic Energy Commission of Syria). We
thank Jeffrey D. Erickson of the Louisiana State Univer-
sity Health Sciences Center, New Orleans, for PC12 cells
stably transfected with human VAChT. This work was
supported by DEST, by the French Embassy in Aus-
19. Varoqui, H.; Erickson, J. D. J. Biol. Chem. 1996, 271,
27229.
´
tralia, and by a grant Ômobilite internationaleÕ from
Franc¸ois Rabelais University. We would like to thank
SAVIT (Tours, France) for chemical analyses.
20. Rogers, G. A.; Parsons, S. M.; Anderson, D. C.; Nilsson,
L. M.; Bahr, B. A.; Kornreich, W. D.; Kaufman, R.;
Jacobs, R. S.; Kirtman, B. J. Med. Chem. 1989, 32, 1217.
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