Syntheses of R and S isomers of AF-DX 384, a selective antagonist of muscarinic M2 receptors

Article abstract of DOI:10.1016/S0968-0896(99)00307-7

Enantiomers of 5,11-dihydro-11-[2-[2-[(N,N-dipropylaminomethyl)piperidin-1-yl]ethylamino]-carbonyl]-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one (AF-DX 384) 1, have been synthesized from (S)-(+) and (R)-(-)-2-[N,N-dipropylaminomethyl]piperidine 4. The enantiomeric excess of 1 has been determined by capillary electrophoresis by using the α-highly sulphated cyclodextrin (α-HSCD) as chiral selector within the running electrolyte. (S)-(+)-(4) was prepared from (S)-(-)-pipecolic acid in a 4-step procedure (overall yield: 30%, ee: 99%) and (R)-(-)-AF-DX 384 from (R)-(+)-pipecolic acid. The (R)-(-) isomer exhibited in vitro a 23-fold higher affinity than its enantiomer (S)-(+) towards muscarinic receptors of subtype 2. Copyright (C) 2000.

Full text of DOI:10.1016/S0968-0896(99)00307-7

Bioorganic & Medicinal Chemistry 8 (2000) 591±600
Syntheses of R and S Isomers of AF-DX 384, a Selective
Antagonist of Muscarinic M2 Receptors
Juliette Martin, a Annamaria Deagostino, a Cecile Perrio, a Francois Dauphin, b
Christophe Ducandas, a Christophe Morin, c Paul-Louis Desbene c
and Marie Claire Lasne a,*
a
Â
Laboratoire de Chimie Moleculaire et Thio-organique, (CNRS UMR 6507), Institut des Sciences de la
Á
Â
Matiere et du Rayonnement, 6 Boulevard du Marechal Juin, 14050 Caen, France
bLaboratoire ``Mort Neuronale, Neuroprotection, Neurotransmission'', (CNRS UMR 6551),
Â
Universite de Caen-Basse Normandie, Boulevard H. Becquerel, 14074 Caen, France
Á
Â
Universite de Rouen, 55 rue Saint Germain, 27000 Evreux, France
c
Laboratoire d'Analyse des Systemes Organiques Complexes, (UPRES 2659- IRCOF et IFRMP),
Received 7 June 1999; accepted 4 November 1999
AbstractÐEnantiomers of 5,11-dihydro-11-[2-[2-[(N,N-dipropylaminomethyl)piperidin-1-yl]ethylamino]-carbonyl]-6H-pyrido[2,3-
b][1,4]benzodiazepin-6-one (AF-DX 384) 1, have been synthesized from (S)-(+) and (R)-( )-2-[N,N-dipropylaminomethyl]piper-
idine 4. The enantiomeric excess of 1 has been determined by capillary electrophoresis by using the a-highly sulphated cyclodextrin
(a-HSCD) as chiral selector within the running electrolyte. (S)-(+)-(4) was prepared from (S)-( )-pipecolic acid in a 4-step proce-
dure (overall yield: 30%, ee: 99%) and (R)-( )-AF-DX 384 from (R)-(+)-pipecolic acid. The (R)-( ) isomer exhibited in vitro a 23-
fold higher anity than its enantiomer (S)-(+) towards muscarinic receptors of subtype 2. # 2000 Published by Elsevier Science
Ltd. All rights reserved.
Introduction
to be involved in the cardiac pathophysiology such as
cardiomyopathies, cardiac ischemia and heart failure.
Acetylcholine mediates a number of central and periph-
eral functions such as smooth muscle contraction and
dilatation, cardiac inotropic e€ects, salivation as well as
sleep mechanisms and cognition.1 These e€ects are the
result of an interaction of acetylcholine to ®ve muscari-
nic receptor subtypes (M1±M5) (mAChR) distributed in
di€erent organs.2 Among these receptor subtypes, M2
receptors, were originally described as binding sites with
high anity for AF-DX 384 1,3±7 AF-DX 116 28±11
(Scheme 1) or methoctramine, low anity for pir-
enzepine, hexahydrosiladifenidol.12 These receptors
were found mainly in the heart of mammals, and
thought to be an homogeneous population in this
organ. Although the latter point has recently been
readdressed13 and heart muscarinic receptors proven to
correspond to a heterogeneous population of M1, M2
and M3 receptors, M2 receptors have been demonstrated
Cholinergic muscarinic receptor ligands have been radi-
olabelled and studied in vivo using positron emission
tomography (PET) or single photon emission computed
tomography (SPECT).14 However, most of the currently
used radiotracers (or their analogues), except bromo-
caramiphen15 and (R,S)-¯uoromethyl QNB,16 were
found to be nonselective with regard to the muscarinic
receptor subtypes.14,16 AF-DX 384 1 and AF-DX 116 2
have been described as selective antagonists of M2
receptors endowed with a nanomolar anity. Their tri-
tiated analogues were tested for their in vivo binding
and metabolisation characteristics in rats.17 Based on
these results, racemic AF-DX 384 has been synthesized
3,18
and labelled with carbon-11 (b+ emitter, t1/2: 20
min).19 However, knowing that the spatial arrangement
of the moieties interacting with mAChRs plays a deci-
sive role for anity to the receptor, the possibility exists
that enantiomers of 1 might demonstrate a high eudis-
mic ratio. For example, a high stereoselectivity ratio has
been reported for hyoscyamine enantiomers,20 and
many other chiral antagonists.1,21 Consequently, both
*Corresponding author. Tel.: +33-2-3145-2892; fax: +33-2-3145-
2877; e-mail: lasne@ismra.fr
0968-0896/00/$ - see front matter # 2000 Published by Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00307-7

592
J. Martin et al. / Bioorg. Med. Chem. 8 (2000) 591±600
the ee. This prompted us to develop a new route to the
optical isomers of 4 starting from the commercially
available (R)-(+) and (S)-( )-pipecolic acid.
Synthesis of (‹)-AF-DX 384 1 from (‹)-pipecolic acid
5. Due to the ¯exible nature of the piperidine ring and
the strong neighbouring group participation of the ring
nitrogen, the functional transformations of pipecolic
acid (+)- or ( )-5, without racemization, are not
necessarily straightforward.18,32 Thus, the synthesis of
AF-DX 384 1 was ®rst studied using the racemic acid
(‹)-5. Attempts to transform pipecolic acid (‹)-5 or its
methyl ester into amide (‹)-13 without protecting the
heterocyclic nitrogen failed. A three-step procedure was
®rst envisaged for the synthesis of polyamine (‹)-3
(Scheme 1). It was based on the use of a cyanomethyl
group which can act both as a protective group36,37 and
as a latent aminoethyl moiety. Moreover, it has been
shown that a cyanomethyl ester is more reactive than a
carboxylic acid in amidation reactions38±42 and that this
reaction proceeds without racemization when no car-
bonyl group is present b to the stereogenic centre.43±45
Reaction of acid (‹)-5 with chloroacetonitrile and
Et3N allowed both N-alkylation and esteri®cation of the
acid. Ester (‹)-7a was obtained in 99% yield. Its reac-
tion with an excess of dipropylamine in acetonitrile did
not give any amide (‹)-8. Deprotection of the ester was
partially observed with the formation of N,N-dipropy-
laminoacetonitrile 9 (12%).46 Using lithium dipropyla-
mide in THF±hexane, the nitrile 9 was formed with a
60% yield. The reaction of dipropylamine with ester
(‹)-7a was also attempted in the presence of sodium
cyanide, an ecient catalyst of aminolysis.47 When the
reaction was carried out in acetonitrile, amine nitrile 9
was the major product (71% isolated) together with the
N-cyanomethyl acid (‹)-6. Meanwhile, in methanol,
amide (‹)-8 was obtained in more than 60% yield. An
in situ transesteri®cation of the cyanomethyl ester could
explain this result.47 Amine (‹)-3 was obtained in 42%
yield by reduction of amide (‹)-8 with lithium alumi-
nium hydride.18 Reaction of (‹)-3 with chlor-
ocarbamate 1018 in acetonitrile led to (‹)-AF-DX 384 1
in 65% yield (Scheme 2).
Scheme 1. Structures of AF-DX 116 and AF-DX 384.
enantiomers of a muscarinic ligand are generally syn-
thesized for a comparison of their relative anities
towards mAChRs (for chiral muscarinic antagonists,
see refs. 22±25). In order to examine pharmacokinetics
and pharmacological pro®les of individual enantiomers
of AF-DX 384 1 we describe here their syntheses and
the evaluation of their in vitro anity towards mus-
carinic M2 receptors. The labelling of both enantiomers
of AF-DX 384 with carbon-11 would allow the in vivo
determination of the speci®c and nonspeci®c binding of
the more active enantiomer as it was shown for dex-
etimide, levetimide and analogues.26±28
Results and Discussion
Enantiomers of (dipropylaminomethyl)piperidine 4 are
the key chiral compounds in the synthesis of (+)- and
( )-AF-DX 384 (+)- and ( )-1. 2-(Dialkylamino-
methyl)piperidines are generally prepared by catalytic
hydrogenation of the corresponding pyridines,3,29,30 or
by hydride reduction of a piperidine carboxamide.31±33
Reductive amination of a N-protected piperidine car-
boxaldehyde29 and functional transformations of piper-
idine methanol have also been reported.18,31 The
preparation of optically active 2-(dialkylamino-
methyl)piperidines required either the resolution of
the racemic compound10 or a synthesis starting from
enantiomerically pure piperidine carboxylic acid (pipe-
colic acid) 5.32±34 An elegant approach using ( )-2-
cyano-6-phenyloxazolopiperidine has recently been
described.35
Piperidine carboxamides are usually prepared from acid
5 after protection of the amine function with a meth-
oxy,48 ethoxy,29 tert-butyloxy,31,49 benzyloxy29,32±34
group. The carbamates (‹)-11a, (‹)-11b, (‹)-11c and
(‹)-11d, respectively, were prepared in more than 75%
yield. All attempts (mixed anhydride using isobutyl
chloroformate,34 acid activation,31,50 or use of a tin
derivative51) to e€ect amidation of (‹)-11c or benzy-
loxycarbonyl (‹)-11d derivatives with N,N-dipropyla-
mine failed. Amide (‹)-12b was obtained in 81% by
reaction of N,N-dipropylamine with cyanomethyl ester
(‹)-7b or in 95% yield by activation of the acid (‹)-
11b with isobutyl chloroformate in the presence of
Et3N, followed by reaction with N,N-dipropylamine.
Amidation of N-methoxycarbonyl acid (‹)-11a was
attempted under di€erent conditions (mixed anhydride
method, thionyl chloride in the presence of DMAP).62
The highest yield was obtained when the acid (‹)-11a
was ®rst transformed into its dicyclohexylammonium
Synthesis of ( )-4 by resolution. The resolution of
piperidine (‹)-(4), according to a procedure described
for an analogue,10 was ®rst attempted to access to (+)-
and ( )-(4). Enantiomer ( )-(4) was obtained in 35%
yield by treatment of (‹)-(4) with one equivalent of L-
(+) tartaric acid and by crystallization of one diastereo-
meric salt from isopropanol. However, the enantiomeric
excess (ee) did not exceed 60%. Attempts to use other
solvents (or mixture of solvents) or to modify the molar
ratio of tartaric acid to amine (‹)-(4) did not improve

J. Martin et al. / Bioorg. Med. Chem. 8 (2000) 591±600
593
Scheme 2. Reagents and conditions: (i) ClCH2CN, MeCN: 7a (80 ꢀC, 22 h) 99%; 7b (24 h, rt) 81%; (ii) HNPr2, NaCN, MeCN, 80 ꢀC, 24 h; (iii)
HNPr2, NaCN, MeOH, 65 ꢀC, 10 h, 8: 67%; (iv) LiAlH4, 0 ꢀC, 2 h, 72%; (v) 80 ꢀC, 4 h, 65%.
1
tion of the diastereoisomeric signals in the H NMR
spectra of amides (‹)-8 and (‹)-13. Chiral HPLC was
used for the separation of enantiomers of amide 12a but
all tested columns (see experimental) failed in the cases of
polyamines 3, 4 and AF-DX 384 1. Among the large
variety of reagents and methods developed for the deter-
mination of the enantiomeric composition of amines,57±59
we ®rst chose to derivatize optically enriched amines 4
and 3 with a-methoxy-a-tri¯uoromethylphenylacetyl
chloride (Mosher reagent).60 However, no clear separa-
tion of the ¯uorine resonances were observed in 19F
NMR spectra of the corresponding amides. On the
other hand, the 19F NMR spectrum of the adducts 15,
formed from (R)-( )-a-¯uorophenylacetyl chloride61,62
and amine (‹)-4, exhibited four well separated doublets
Scheme 3. Synthesis of amine (‹)-4. Reagents and conditions: (i)
(C6H11)2NH, HNPr2, DBU, rt, 48 h, 12a: 55%; (ii) Me3SiI, 70 ꢀC, 38 h,
(each diastereoisomer with their rotamers). The dicul-
ties encountered to prepare enantiomerically pure (R)-
( )-a-¯uorophenylacetyl chloride and the kinetic reso-
lution observed when trying to determine the ee of
amide 13, limited the use of this ¯uorinated reagent.
Amine 4 was thus derivatized with (S)-( )-methyl-2-
88%; (iii) LiAlH4, 70 ꢀC, 24/h, 68%; (iv) BrCH2CN, 70 ꢀC, 5 h, 75%;
(v) LiAlH4, 0 ꢀC, 2 h, 72%; (v) 80 ꢀC, 4 h, 65%.
salt53 then treated sequentially with thionyl chloride and
N,N-dipropylamine in the presence of diazabicy-
cloundecene (DBU) which gave amide (‹)-12a in 55%
yield. Attempts to prepare (‹)-13 from (‹)-11a using
conditions previously described (SOCl2 then amine) for
similar compounds,30 gave only tars (Scheme 3).
1
isocyanato-3-methylbutanoate. In the H NMR spectra
of the diastereoisomeric mixture 16, the diastereo-
isomeric excess (de) could be easily measured by com-
parison of integration curves of the proton to the
carboxylate group (Scheme 4).
Deprotection (hydrochloric acid54 or tri¯uoroacetic acid
55) of the ethoxycarbonyl group of (‹)-12b led to the
crude amide (‹)-13 in a low yield (<40%). On the
other hand, deprotection of the N-methoxycarbonyl
group with iodotrimethylsilane,56 yielded to piperidine
carboxamide (‹)-13 in 88% yield. Reduction of the
amide function with LiAlH4 then gave the amine (‹)-4
which was transformed into (‹)-AF-DX 384 1 accord-
ing to a described procedure.18
We also analyzed the 31P NMR spectrum of the mixed
complex 17 formed between amine 4 and (S)-( )-tert-
butylphenylphosphinothioic acid 18.63 Unfortunately,
no distinct phosphorus signals were observed and no
attempts were made to search for another phosphorous
1
derivative.64 Surprisingly, in the H NMR spectrum of
17, the two methyl triplets were clearly separated and
their integration gave a good and rapid measurement
of the de. To our knowledge, it is the ®rst use of the
phosphorus derivative 18 for the determination of ee of a
chiral secondary amine using 1H NMR spectroscopy.
Table 1 summarizes the chemical shifts di€erences mea-
sured for the separated signals with the di€erent reagents.
Synthesis of (R)-( )-AF-DX 384 and (S)-(+)-AF-DX
384: determination of the optical purities. The same
method was followed for preparing both enantiomers of
AF-DX 384 1. Starting from (S)-( )-pipecolic acid,
(+)-amine 4 and (+)-AF-DX 384 1 were obtained.
Similarly, (R)-(+)-pipecolic acid gave amine ( )-4
which was transformed into ( )-1. The formation of
amine (+)-(4) and (+)-AF-DX 384 1 from (S)-( )-
pipecolic acid allowed the assignment of the absolute
con®guration S to the (+) enantiomer of 1 and thus R
to ( )-4 and ( )-1. The optical rotations of piperidines
3, 4 and AF-DX 384 being unknown, di€erent methods
were investigated for the determination of the enantio-
meric purity of these compounds. Chiral lanthanide
shift reagent ((Eu(hfc)3)57,58 did not show any separa-
Attempts to use the chiral solvating agent 18 with the
primary amine 3 failed. Finally, due to its high eciency
in the separation of enantiomers,67,68 we attempted high
performance capillary electrophoresis (HPCE) to deter-
mine the ee of AF-DX 384. We ®rst studied the intro-
duction of highly sulphated cyclodextrins (HSCD) (a-
HSCD, b-HSCD or g-HSCD) in the running electrolyte.
In these context, cyclodextrins were used both as carrier
giving a negative charge to the analysed enantiomers via
inclusion complexes, and as chiral selector, if the com-
plex formation constants of the enantiomers with the

594
J. Martin et al. / Bioorg. Med. Chem. 8 (2000) 591±600
de®ned as area/migration time ratio. Each enantiomer
of AF-DX 384 was obtained in 99% ee from (S) or (R)-
pipecolic acid, via amine (S)-(+)- or (R)-( )-4 (ee
>99% determined from 1H NMR of 17). This con-
®rmed that no racemization was observed when using
this synthetic route.
Scheme 4. Structures of the compounds used for determination of
enantiomeric purity of amine 4.
Biological Evaluation
Racemic and enantiomers of AF-DX 384, in comparison
with the nonselective, high-anity muscarinic antagonist
atropine were assayed for their anity to rat heart M2
muscarinic receptors against [3H]-N-methyl scopola-
mine. As shown in Table 2, analysis of competition
curves con®rmed that (‹)-1 has a high anity towards
muscarinic receptors albeit lower than atropine.
chiral cavity of the cyclodextrins are di€erent. In order
to increase the di€erences of electrophoretic behaviour
of the diastereoisomeric complexes ((+) AF-DX 384/
HSCD and ( )AF-DX 384/HSCD), we also analysed
the racemic (‹) AF-DX 384 in acidic medium (phos-
phate bu€er, pH 2.5). Under these conditions, the
enantiomers had a positive charge and their electro-
phoretic mobility was opposite to the electrophoretic
mobility of the cyclodextrin complexes. With such a
strategy, the selectivity obtained in HPCE for the three
HSCD studied was maximum after optimisation of the
phosphate bu€er concentration and of the HSCD con-
centration. Co-injection of each enantiomer in the race-
mic mixture leads to peak identi®cation. Whatever the
HSCD used (a, b or g), AF-DX 384 racemic mixture
was resolved, and detected at the anodic end of the
capillary. The resolution (Rs) obtained, when HSCDs
were introduced at a 5% (w/w) concentration in the
running electrolyte, increased when the cyclodextrin
internal diameter decreased: Rs (a-HSCD) > Rs (b-
HSCD) > Rs (g-HSCD). Consequently the ee of the
optically enriched AF-DX 384 was determined using a-
HSCD as chiral selector, assuming that both enantio-
mers have the same UV molecular absorption coecient
whatever their state (i.e. as complexes or not).69 The
apparent migration mobility of the two diaster-
eoisomeric complexes being di€erent, the ee determined
in HPCE were calculated with corrected areas which are
The (R)-( )-(1) isomer was found to exhibit a 23-fold
higher anity than its (S)-(+)-(1) enantiomer. Com-
pared to AF-DX 116 (selectivity (+):( ): 8)10 the
eudismic ratio is slightly higher. Its value was closer to
the one reported for analogues of QNB65 but remains
low in comparison to hyoscyamine, procyclidine or
benzetimide (anities ratio (S):(R), respectively: 330,
0.003 and >4000).1
Conclusion
In summary, enantiomers of AF-DX 384 1 have been
synthesized from (R)-(+) and (S)-( )-pipecolic acids
with a 99% ee. This method allowed the assignment of
their absolute con®gurations, the (+) enantiomer being
obtained from (S)-( )-pipecolic acid, and the ( ) one
from the (R)-(+)-acid 5. For the ®rst time, the phos-
phorus reagent 18 has been used in conjunction with 1H
NMR to measure the ee of a secondary amine (4). The
validity of the method was con®rmed by comparison of
the results with those obtained after derivatization of 4
Table 1. 1H NMR and 19F NMR nonequivalences
Compounds
Nuclei
Fa
Position
13
Solvent
CDCl3
Multiplicity, d (ppm) (con®gurations)
Ád ppm
15
d, 168.8;
d, 172.4b (2R, 13S)
dd, 4.28 (2S,14S)
t, 0.78 (2S,Sp)
d, 171.5;
2.7
3.1
0.13
0.12
d, 175.5c (2S,13S)
dd, 4.41 (2R,14S)
t, 0.66 (2R,Sp)
16
17
H
H
H-14
Me-11
CDCl3
CDCl3
a 19F NMR.
b Rotamers: ratio 52:48.
c Rotamers: ratio 65:35.
Table 2. Anities of tested compounds for M2 muscarinic receptors (rat heart tissue)
a
b
Compounds
pIC50
Compounds
pIC50
Atropine
(‹)-AF-DX 384 1
(R)-( )AF-DX 384 (R)-( )-(1)
(S)-(+)AF-DX 384 (S)-(+)-(1)
8.70‹0.33
7.36‹0.30
8.02‹0.51
6.66‹0.38
(‹)-AF-DX 116 2
(+)-AF-DX 116 (+)-(2)c
(
6.85
7.30
6.39
)-AF-DX 116 ( )-(2)c
apIC50 values represent the negative logarithm of the concentration that produces 50% inhibition of the antagonist maximal response and are
10
expressed as means‹SEM, n=3 using 5Â10
M [3H]methyl scopolamine.
bData from refs 3±7 and obtained from competition studies against 3.10
cThe absolute con®guration was not determined.3±7
M [3H]methyl scopolamine.
10

J. Martin et al. / Bioorg. Med. Chem. 8 (2000) 591±600
595
and measurements of the diastereoisomeric excesses
1
experiment, with a model È pH meter (Beckman) and
before each analysis the capillary was ¯ushed with water
(1 min) then with the electrolyte (10 min). The rinsing
pressure is equal to 20 psi. Finally the electrolytes were
systematically degassed by sonication (Brandson).
either by H NMR or HPLC. The ee of enantiomers of
AF-DX 384 has been determined by capillary electro-
phoresis using 5% (w/w) a-highly sulphated cyclodex-
trin in acidic medium (phosphate bu€er, pH 2.5).
Finally, the anities of the enantiomers of AF-DX 384
toward M2 receptors were evaluated and the (R)-( )-
enantiomer was shown to be the eutomer.
Optical rotations were measured on a Perkin±Elmer 241
LC polarimeter at l=589 nm. [a]d Values are given in
1
units of 10 deg cm2 g 1. Microanalyses were carried
out at CNRS centre (Vernaison, France). Melting
points (MP) were determined on a Gallenkamp appa-
ratus and are uncorrected. NMR spectra were recorded
on a Bruker Avance DPX 250 (1H: 250 MHz; 13C:
62 MHz; 31P: 101 MHz; 19F: 235 MHz) or a Bruker
Avance DRX 400 (400 and 100 MHz, 1H and 13C,
respectively) spectrometers. Chemical shifts in CDCl3
are reported in d units (parts per million) from tetra-
methylsilane (1H and 13C), ¯uorotrichloromethane (19F)
as internal standards or phosphoric acid (31P) as exter-
nal standard. Multiplicities are reported as singlet (s),
doublet (d), triplet (t), quartet (q), multiplet (m). Cou-
pling constants (J) are given in Hertz. All the attempts
to determine the optical purities using a chiral lantha-
nide shift reagent were made by recording the 1H NMR
spectra of compounds (racemic or optically enriched,
0.05 mmol in 0.5 mL in CDCl3) in the presence of
incremental additions (from 0.1 to 0.5 equiv) of Euro-
pium(III) tris[3-hepta¯uorobutanoyl-(1R)-camphorate]
[Eu(hfc)3]. IR spectra were recorded on a Perkin±Elmer
16 PC-FT-IR spectrometer. Electron impact (EI, 70 ev)
mass spectra were recorded with a Nermag R10 instru-
ment and high resolution mass spectra (HRMS) on a
Jeol JMSD 300 or a Jeol AX 500.
Experimental
General techniques
All reactions were carried out under a nitrogen atmo-
sphere. Tetrahydrofuran (THF) was distilled from sodium
benzophenone. (‹)-2-(N,N-Dipropylaminomethyl)piper-
idine 4,18 (R)-( )-a-¯uoro phenyl acetic acid, (R)-( )-a-
¯uoro phenyl acetyl chloride,61 (‹)-[(N-ethoxycarbonyl)
piperidine]-2-carboxylic acid 11b29 (‹)-[N-(tert-butoxy-
carbonyl)piperidine]-2-carboxylic acid 11c31,49 and (‹)-
[(N-benzyloxycarbonyl)piperidine]-2-carboxylic acid 11d,29
were synthesized according to described methods. (S)-
( )-tert-Butyl phenyl phosphinothioic acid was kindly
provided by M. Mikolajczyk (Poland). All other
reagents were used as obtained from commercial sour-
ces (purity >98% Janssen Chimica, Aldrich or Sigma).
Organic solutions were dried over anhydrous MgSO4
and evaporated at <50 ꢀC under reduced pressure.
Thin-layer chromatography was performed on silica gel
60 F-254 plates (0.1 mm, Merck) with iodine and/or UV
detection. Chromatographic separations were made on
silica gel columns (Kieselgel 60, 70±230 mesh, Merck).
Analytical high performance liquid chromatography
(HPLC) was carried out with a Waters instrument
(detector 486 and pump 510 or detector M996 (200±
300 nm) and pump 600). The following columns
(0.46Â25 cm): Daicel Chiralcel OD, OJ, DNBPGJ. T.
Baker, Nucleodex b-OH, Nucleosil chiral-2 were tested
for the chiral separations.
Resolution of (‹)-2-[N,N-dipropylaminomethyl]piperidine
4. Diamine (‹) 4 (1 g, 0.5 mmol) and (L)-(+) tartaric
22
acid (0.758 g, 0.5 mmol, ‰aŠ +12 ꢀ (c 20, H2O)) were
d
dissolved in hot isopropanol (400 mL). The mixture was
allowed to crystallize at room temperature. After 96 h,
the white and round crystals were collected (mp 132±
133 ꢀC after two crystallizations) then dissolved in
dichloromethane. Aqueous sodium hydroxide (15%)
was added and the mixture vigorously stirred for 1 h.
The organic layer was separated, washed with brine,
and dried. The solvent was removed under vacuum to
Enantiomeric resolution by capillary electrophoresis
All solutions were prepared by using the 18Mꢀ.cm water
(Millipore, Bedford, MA, USA). The highly sulphated
cyclodextrins were purchased from Beckman (Beckman,
Fullerton, CA, USA). Triethylammonium phosphate of
analytical purity was purchased from Aldrich. All these
reagents were used without further puri®cation.
yield (R)-( )-4 as a pale-yellow oil (0.350 g, 35%). Bp
0.002 mmHg 87.5 ꢀC; ‰aŠ
22
d
49 ee: 58%.
(S,R) and (R,R) 1-[2-(N,N-Dipropylaminomethyl)piperi-
din-1-yl]-2-¯uoro-2-phenyl-ethanone 15. (R)-( )-a-Fluo-
ro phenyl acetyl chloride (0.022 mL, 0.15 mmol) was
added dropwise to the amine (‹)-4 (0.015 g, 0.09 mmol)
in CH2Cl2 (2 mL) at 0 ꢀC. The mixture was stirred at
room temperature for 2 h. Aqueous sodium hydroxide
(2 mL, 15%) was added and the mixture was vigorously
stirred for 10 min. After extraction with CH2Cl2
(2Â5 mL), the organic layer was successively washed
with NaHCO3 (2Â5 mL, saturated solution) and brine
then dried. The solution was concentrated to give the
title compounds (S,R)-15 and (R,R)-15 as a pale-yellow
oil (0.015 g, 52%). IR nmax (cm 1, NaCl) 1652; 19F
All analyses were carried out on a P/ACE 2100 system
(Beckman, Fullerton, CA, USA) ®tted with an UV
detector. The acquisition and the processing of data
were performed with a Dell computer by means of P/
ACE Station software (Beckman). The samples were
systematically injected in hydrodynamic mode (injection
pressure: 0.5 psi or 3.4 kPa) and their analysis were
achieved on a fused silica capillary of 50mm id presenting
a total length of 57cm (50 cm e€ective length to detection
window). Injections were performed at the cathodic end of
the capillary. The temperature was ®xed at 22ꢀC. The pH
of the solutions was measured at the temperature of the
NMR (235 MHz, CDCl3) (S,R)-15:
d 169.1 (d,
J=51.7 Hz, 0.52 F), 172.6 (d, J=51.7 Hz, 0.48 F);

596
J. Martin et al. / Bioorg. Med. Chem. 8 (2000) 591±600
(R,R)-15: d 171.4 (d, J=51.7 Hz, 0.65 F), 175.7 (d,
J=51.7 Hz, 0.45 F); HRMS m/z calcd for C20H31N2OF:
334.24203, found 334.23559; MS (EI+) 334 (M+, 100),
319 (61), 305 (29), 286 (23), 267 (80).
48ꢀC; IR (KBr, cm 1) 2234 (CN), 1754 (COO); 1H NMR
d 4.86 and 4.75 (AB, JAB=15.7 Hz, 2H, OCH2CN), 3.78
and 3.56 (AB, JAB=17.6 Hz, 2H, NCH2CN), 3.36 (dd,
J2ax,3eq=3.6Hz, J2ax,3ax=9.1Hz, 1H, H-2ax), 2.92 (m,
1H, H-6eq), 2.59 (m, 1H, H-6ax), 2.13±2.00 (m, 1H, H-3),
1.82±1.60 (m, 4H, H-5, 1 H-4, 1 H-3), 1.49±1.41 (m, 1H,
H-4); 13C NMR d 171.0 (CO), 114.8 (NCH2CN), 114.1
(OCH2CN), 62.1 (C2), 51.5 (C6), 48.8 (OCH2CN), 44.0
(NCH2CN), 29.5 (C3), 24.9 (C5), 22.2 (C4); MS (EI) m/z
208 (M+1+, 0.3), 207 (M+, 0.5), 123 (100), 84 (18), 55
(22), 49 (30), 42 (18), 41 (34); HRMS calcd for
C10H13N3O2: 207.1008; found: 207.1012.
(S,R)- and (S,S)-[2-[2-(N,N-dipropylaminomethyl)piperi-
din-1-yl-carbonyl]amino]-3-methyl butanoic acid methyl
ester 16. The isocyanate (0.19 mmol) was added drop-
wise to a solution of amine (‹)-4 (0.038 g, 0.19 mmol) in
CDCl3 (1.5 mL) at room temperature The mixture was
stirred for 3 h and an aliquot was removed for 1H NMR
analysis. The ureas were isolated as followed. CH2Cl2
was added to the reaction mixture and the organic layer
was washed successively with HCl (3 M), NaOH (15%),
H2O then dried. After evaporation of the solvent, the
diastereoisomeric ureas 16 were isolated and character-
ized without further puri®cation. IR nmax (cm 1, NaCl)
3350, 1742, 1644 and 1520; 1H NMR (250 MHz, CDCl3)
d 6.78 (bd, 1H, J=5.9 Hz, CONHCHiPrCOOCH3),
6.18 (bd, 1H, J=5.9 Hz, CONHCHiPrCOOCH3), 4.41
(dd, 1H, J=8.0 and 4.9 Hz, CONHCHiPrCOOCH3
(R,S)), 4.28 (dd, 1H, J=8.7 and 5.7 Hz, CON-
HCHiPrCOOCH3 (S,S)), 3.97±4.20 (m, 5H), 3.72 (s,
3H, CONHCHiPrCOOCH3), 3.70 (s, 3H, CON-
HCHiPrCOOCH3), 2.56±2.87 (m, 3H), 2.31±2.52 (m,
8H), 1.92±2.30 (m, 5H), 1.35±1.80 (m, 19H), 0.81±0.98
(m, 24H); 13C NMR (100 MHz, CDCl3) d 12.3, 12.3,
18.7, 18.8, 19.4, 19.6, 19.8, 20.1, 20.1, 20.4, 25.4, 25.5,
31.7, 31.8, 39.2, 52.0, 52.1, 53.4, 57.9, 58.5; 59.2, 59.4,
63.6, 159.0, 174.2, 174.4; HRMS, m/z calcd for
C19H37O3N3: 355.28347, found 355.29143; MS (EI+)
355 (100), 343 (14), 331 (24), 312 (11), 281 (50), 269 (24).
(‹)-N-(Cyanomethyl)piperidine-2-carboxylic acid (‹)-6.
A mixture of ester (‹)-7a (0.730 g, 3.48 mmol), dipro-
pylamine (12 mL, 87 mmol) and sodium cyanide
(0.427 g, 8.7 mmol) in dry acetonitrile (30 mL) was
heated at 80 ꢀC for 20 h. After cooling to room tem-
perature and concentration, the residue was treated with
CH2Cl2 (50 mL). The organic layer was washed with
brine (2Â30 mL) and then acidi®ed at pH 3.5 by slow
addition of KHSO4 (saturated solution). The organic
layer was dried then concentrated to give (N,N-dipro-
pylamino)acetonitrile 9 (0.345 g, 71%).46 The aqueous
layer was continuously extracted with chloroform for
24 h. The residue of the organic layer was dried then
concentrated to give the title compound (‹)-6 (0.146 g,
25%). Rf 0.65 (CH2Cl2:MeOH, 85:15); mp 133 ꢀC; IR
(NaCl, cm 1) 3428 (COOH), 2514 (CN), 1718 (CO); 1H
NMR d 8.70 (s, 1H, COOH); 3.94 and 3.60 (AB,
JAB=17.6 Hz, 2H, NCH2CN), 3.25 (dd, J=3.3 and
10.0 Hz, 1H, NCHCH2), 2.94 (m, 1H, H-6eq), 2.62 (m,
1H, H-6ax), 2.12±2.06 (m, 1H, NCHCH2), 1.82±1.60
(m, 4H), 1.49±1.39 (m, 1H); 13C NMR d 176.9 (COOH),
114.8 (CN), 63.4 (NCH); 52.0 (NCH2CH2), 43.9
(NCH2CN), 29.8 (NCHCH2), 24.8 (NCH2CH2), 22.7
(NCH2CH2CH2); MS (EI) m/z: 168 (M+, 0.02); 123
(100), 55 (24), 54 (16), 42 (18), 41 (41); HRMS calcd for
C8H12N2O2: 168.0899; found: 168.0900.
(S)-(±)-tert-Butyl phenyl phosphinothioic acid±amine (‹)-
(4) complex 17. (S)-( )-tert-Butyl phenyl phosphi-
nothioic acid 18 (0.008 g, 0.037 mmol) and amine (‹)-4
(0.006 g, 0.030 mmol) in CDCl3 (0.6 mL) were mixed in
an NMR tube and the spectrum recorded after a few
1
min. IR nmax (cm 1, KBr) 3424 and 1018; H NMR
(250 MHz, CDCl3) d 7.20±7.30 (m, 10H, Ph), 4.28 (m,
1H), 4.13 (m, 1H), 3.45 (m, 2H), 3.25 (m, 1H), 3.05 (td,
1H, J=12 and 2.7 Hz), 2.51±2.95 (m, 10H), 2.45 (td,
1H, J=12.0 and 2.7 Hz), 1.20±2.05 (m, 23H), 1.10 (s,
9H, C(CH3)3), 1.00 (s, 9H, C(CH3)3), 0.78 (t, 6H,
J=7.2 Hz, CH2CH3, (S,S)), 0.66 (t, 6H, J=7.3 Hz,
CH2CH3, (S,R)); 13C NMR (62.5 MHz, CDCl3) d 11.1,
11.2, 17.4, 17.7, 21.7, 22.4, 25.2, 25.2, 27.0, 35.6, 36.8,
43.9, 44.2, 53.1, 55.5, 55.6, 56.7, 56.8, 127.0, 127.2,
129.6, 129.7, 132.8, 132.9, 138.1, 139.5; 31P NMR
(101.3 MHz, CDCl3) 83.0.
(‹)-[(N 0 -Cyanomethyl)piperidine-2-(N,N-dipropyl)car-
boxamide (‹)-8. To a solution of the ester (‹)-7a
(0.536 g, 2.59 mmol) in methanol (25 mL), dipropyla-
mine (1.77 mL, 12.93 mmol) and sodium cyanide
(0.317 g, 6.46 mmol) were added under nitrogen atmo-
sphere. The reaction mixture was heated at 80 ꢀC for
10 h. After cooling and evaporation of the volatile
compounds, the resulting oil was treated with CHCl3
(15 mL) and washed with water (15 mL). The organic
layer was dried and evaporated. The residue was pur-
i®ed by chromatography on silical gel (eluent
CHCl3:MeOH, 97:3) to give the title compound (‹)-8
(0.260 g, 67%) as a yellow oil. Rf 0.9 (CH2Cl2:MeOH,
85:15); IR (KBr, cm 1): 2224 (CN), 1670 (CO); 1H
NMR d 3.58 and 2.82 (AB, JAB=15.4 Hz, 2H,
NCH2CN), 3.63±3.42 (m, 4H, NCH2CH2), 3.20±3.13
(m, 1H, H-2), 2.88±2.84 (m, 1H), 2.48±2.44 (m, 1H),
2.30±2.24 (m, 1H), 2.08 (m, 1H), 1.83±1.78 (m, 1H),
1.62±1.18 (m, 7H); 0.90 (t, J=7.4 Hz, 3H, CH3), 0.84 (t,
J=7.4 Hz, 3H, CH3); 13C NMR d 169.0 (CO), 118.6
(CN), 55.7 (C-6), 52.0 (NCH2CN), 50.5 (C-2), 50.0
(NCH2CH2), 27.4 (C-4), 25.0 (C-3), 24.4 (C-5), 20.5
(CH2CH3), 11.4 (CH3); MS (EI) m/z: 251 (M+, 0.8), 97
Cyanomethyl N-(cyanomethyl)-2-piperidine carboxylate
(‹)-7a. A mixture of pipecolic acid (‹)-5 (0.500 g,
3.88 mmol), triethylamine (2.7 mL, 19.3 mmol) and
chloroacetonitrile (2.2 mL, 19 mmol) in acetonitrile
(30 mL) was heated at 80 ꢀC for 22 h. After cooling to
room temperature, the volatile compounds were evapo-
rated under vacuum and the residue was treated with
dichloromethane (25 mL). The organic layer was
washed with brine (50 mL), dried and evaporated.
Chromatography of the residue on silica gel (eluent
CH2Cl2:MeOH, 97:3) gave the title compound (‹)-7a
(0.830g, 99%); R=0.75 (CH2Cl2:MeOH, 85:15); mp

J. Martin et al. / Bioorg. Med. Chem. 8 (2000) 591±600
597
(64), 84 (65), 49 (87), 43 (70), 42 (54), 41 (100); HRMS
calcd for C14H25N3O: 251.1998; found: 251.2007.
1.64 mmol) were added dropwise at room temperature
under nitrogen atmosphere. The mixture was stirred at
room temperature for 30 min before addition, at this
temperature, of dipropylamine (0.18 mL, 1.05 mmol)
and DBU (0.30 mL, 2.05 mmol) in CH2Cl2 (2 mL). After
48 h of stirring, brine (5 mL) was poured into the mix-
ture, and the product was extracted with AcOEt
(2Â10 mL). The organic layers were washed successively
with HCl 3 N (3Â10 mL), NaOH 1 N (2Â10 mL), and
brine (10 mL) then dried. Evaporation of the volatile
compounds gave a crude product, which was puri®ed by
chromatography on silica gel (CH2Cl2:MeOH, 97:3) to
(‹)-Cyanomethyl [(N-ethoxycarbonyl)piperidine]-2-car-
boxylate (‹)-7b. A mixture of acid (‹)-11b (0.050 g,
0.25 mmol), triethylamine (0.069 mL, 0.5 mmol) and
chloroacetonitrile (0.031 mL, 0.25 mmol) in CH2Cl2
(1 mL) was stirred for 24 h at room temperature. After
evaporation of the volatile compounds, CH2Cl2 (20 mL)
was added. The organic layer was washed with brine
(25 mL) then dried. After concentration, the residue was
puri®ed by column chromatography (eluent CH2Cl2
then MeOH) to give the title compound (‹)-7b (0.048 g,
81%) as a yellow oil. Rf 0.85 (CHCl3:MeOH, 90:10); IR
(NaCl, cm 1): 2862 (CN), 1758 (COO), 1696 (NCO); 1H
NMR d 5.00 (s, 0.62H, H-2), 4.91 (s, 0.38H, H-2), 4.79
(AB, JAB=15.9 Hz, 2H, OCH2CN), 4.16 (q, J=7.0 Hz,
2H, CH3CH2O), 4.04±3.99 (m, 1H, H-6), 3.07±2.93 (m,
1H, H-6), 2.25±2.20 (m, 1H, H-3), 1.93±1.67 (m, 3H, H-
5, H-4, H-3), 1.53±1.42 (m, 2H, H-5, H-4), 1.28±1.25 (m,
3H, CH3); 13C NMR d 170.6 (COOCH2CN), 156.6
(COOCH2CH3), 114.3 (CN), 61.9 (CH2CH3), 54.6 (C-
2), 48.8 (CH2CN), 41.8 (C-6), 26.7 (C-3), 24.5 (C-5),
20.7 (C-4), 14.6 (CH3); MS (EI) m/z: 241 (M+1+, 6),
240 (M+, 6), 128 (100); HRMS calcd for C11H16N3O4:
240.1110; found: 240.1123.
give (S)-( )-12a (yellow oil, 0.1484 g, 55%). Rf 0.6
22
(CH2Cl2:MeOH, 97:3); ‰aŠ ±28ꢀ (c 4, CHCl3) ee >99%
d
(HPLC, Chiralcel OD, 220 nm, n-heptane:isopropanol,
95:5; 0.6 mL min 1: 12min). The spectral data of the two
rotamers (70:30) were identical with those previously
described.66 HRMS calcd for C14H26N2O3 270.1943;
found: 270.1932; anal. calcd C, 62.19; H, 9.69; N, 10.36;
O, 17.75. Found: C, 62.09; H, 9.57; N, 10.18; O, 17.75.
(‹)-N,N-Dipropyl [(N 0-ethoxycarbonyl)piperidine]-2-car-
boxamide (‹)-12b. A solution of acid (‹)-11b (1.014 g,
5.04 mmol) chloroform (12 mL) and triethylamine
(728 mL, 5.24 mmol) was cooled to 15 ꢀC. Isobutyl
chloroformate was added dropwise (654 mL, 5.04 mmol)
and the reaction mixture stirred at 15 ꢀC for 1 h.
Dipropylamine (691 mL, 5.04 mmol) was then added
dropwise and allowed to react with stirring at 15 ꢀC
for 3 h. After addition of CHCl3 (20 mL), the organic
layer was successively washed with brine (3Â20 mL),
ice-cooled citric acid (2Â15 mL, 10% solution),
NaHCO3 (10 mL, saturated solution) and ®nally with
brine (3Â20 mL) and dried. Evaporation of the volatile
compounds gave the title compound (‹)-12b (1.34 g,
95%). Rf 0.5 (CHCl3:MeOH, 97:3); IR (NaCl, cm 1):
(S)-( )-[(N-Methoxycarbonyl)piperidine]-2-carboxylic
acid (S)-( )-(11a). To a solution of pipecolic acid (S)-
( )-5 (0.183 g, 1.42 mmol) in water (3 mL, c 0.51 M, pH
7.3) at 4 ꢀC were added dropwise and simultaneously
the methyl chloroformate (1.67 mmol) and aqueous
NaOH (2 N, 1.45 mL, 2.84 mmol) in order to maintain
the reaction mixture weakly basic (pH 8±9). The mix-
ture was stirred at room temperature overnight. The
aqueous layer was washed with Et2O (10 mL) then acid-
i®ed (pH 3) with 6 N HCl. The acidic aqueous layer was
extracted with Et2O (3Â10mL) and the combined extracts
were dried and evaporated to give the protected piperidine
1
1690 (NCOO), 1644 (CON); H NMR d 5.01 (s, 0.90H,
H-2), 4.90 (s, 0.10H, H-2), 4.13 (q, J=7.0 Hz, 2H,
CH2CH3), 3.96±3.90 (m, 1H, H-6), 3.54±3.42 (m, 2H),
3.25±2.98 (m, 3H), 1.89±1.43 (m, 10H), 1.25 (t, 3 H,
J=7.0Hz, CH2CH3), 0.92 (t, J=7.4 Hz, 3H, (NCH2
(S)-( )-11a. White solid (0.257g, 97%). Rf 0.4
22
d
(CH2Cl2:MeOH, 97:3); mp 108 ꢀC; ‰aŠ ±55ꢀ (c 6.9,
CHCl3); IR (NaCl, cm 1): 3060 (COOH), 1744 (OCON),
CH2CH3)), 0.88 (t, J=7.4 Hz, 3H, (NCH2CH2CH3)); C
3
1
1666 (CO); H NMR d 9.02 (s, 1H, COOH), 4.90 (m,
NMR d 172.3 (CON), 156.7 (COO), 61.5 (CH2CH3), 50.9
(C-2), 49.6 and 47.7 (NCH2CH2), 42.0 (C-6), 27.3 (C-3),
25.0 (C-5), 21.0 (C-4), 19.5 (CH2CH2CH3), 14.7 (COO
CH2CH3), 11.4 (CH2CH2 CH3); MS (EI) m/z: 285
(M+1+, 5), 284 (M+, 7), 157 (62), 156 (100), 55 (32), 43
(49), 42 (21), 41 (52); HRMS calcd for C15H28N2O3
284.20999; found: 284.19996.
1H, H-2), 4.10 (m, 1H, H-6), 3.73 (s, 3H, CH3), 3.04 (m,
1H, H-6), 2.32 (m, 1H, H-3), 1.8±1.5 ( m, 3H, H-3, H-5,
H-4), 1.5±1.2 (m, 2H, H-4, H-5); 13C NMR d 176.6 (C-
7), 157.5 (C-9), 53.1 (C-10), 48.1 (C-2), 41.9 (C-6), 26.6
(C-3), 24.5 (C-5), 20.7 (C-4); MS (EI) m/z: 188 (M+1+,
3), 187 (M+, 5), 156 (100). Anal. calcd for C8H13NO4:
C, 51.34; H, 7.00; N, 7.48; O, 34.17. Found: C, 51.11; H,
6.83; N, 7.42 O 34.12
(S)-( )-(N,N-Dipropyl)piperidine-2-carboxamide
(S)-
( )-13. Iodotrimethylsilane (0.2 mL, 4.2 mmol) was
added dropwise to a solution of the carbamate (S)-( )-
12a (0.115 g, 0.42 mmol) in CHCl3 (15 mL) at room
temperature. The solution was then heated to re¯ux for
16 h. Methanol (15 mL) was added and the re¯ux con-
tinued overnight. The solution was concentrated. The
residue was diluted in CH2Cl2 (10 mL) and washed
with HCl 3 N (3Â10 mL). The aqueous layer was made
basic with NaOH 2 N and extracted with CH2Cl2
(3Â20 mL). The combined organic layers were dried
and concentrated. The residue was puri®ed by column
chromatography (eluent CH2Cl2:MeOH:C6H12:NH4OH
(S)-( )-N,N-Dipropyl [(N0-methoxycarbonyl)piperidine]-
2-carboxamide (S)-( )-12a. To a solution of (S)-( )-
11a (0.257 g, 1.37 mmol) in AcOEt (2 mL), was added
dicyclohexylamine (0.296 mL, 1.49 mmol) at room tem-
perature. After stirring for 1 h, the precipitate was col-
lected and recrystallised from Et2O to give a yellow
solid of [(N-methoxycarbonyl)piperidine]-2-dicyclohex-
ylammonium carboxylate (0.506 g, 100%). IR (KBr,
cm 1): 1550 (NH+2 ), 1450 (COO ). To a stirred solution
of this salt (0.506 g, 1.37 mmol) in CH2Cl2 (8 mL), pyri-
dine (0.14 mL, 1.78 mmol) and thionyl chloride (0.12 mL,

598
J. Martin et al. / Bioorg. Med. Chem. 8 (2000) 591±600
(28%), 68:15: 15:2) to give the title compound (S)-( )-
13 (0.0784 g, 88%). Rf 0.7 (CH2Cl2:MeOH:C6H12:N-
22
d
H4OH, 68:15: 15:2); ‰aŠ
11ꢀ (c 4, CHCl3), ee > 99%
determined by HPLC of the amide (S)-( )-12a formed
by reaction of (S)-( )-13 with methyl chloroformate
under the conditions described previously. IR (NaCl,
cm 1): 3502 (NH); 1642 (CON); H NMR d 3.47±3.43
1
(m, 1H, H-2), 3.33±3.25 (m, 1H), 3.16±3.06 (m, 4H,
NCH2CH2), 2.62 (dt, J=11.5 and 4.2 Hz, 1H, H-6), 2.39
(s, 1H, NH), 1.82±1.64 (m, 1H, H-4), 1.60±1.25 (m, 9H,
CH2CH2CH3, H-3, H-4, H-5), 0.89 (t, J=7.3 Hz, 3H,
CH3), 0.77 (t, J=7.3 Hz, 3H, CH3); 13C NMR d 173.3
(CON), 56.5 (C-2), 49.3 (CONCH2), 47.6 (CONCH2),
45.7 (C-6), 30.4 (C-3), 26.8 (C-5), 24.5 (C-4), 22.7
(CONCH2CH2), 20.9 (CONCH2CH2), 11.4 (CH3); m/z:
213 (M+1+, 0.38); 212 (M+, 1.0); 84 (100); HRMS
calcd for C12H24N2O 212.1883; found: 212.1889.
(S)-(+)-2-(N,N-Dipropylaminomethyl)piperidine (S)-(+)-
(4). A mixture of LiAH4 (0.120 g, 3.10 mmol) in THF
(1 mL) and amide (S)-( )-13 (0.326 g, 1.5 mmol) in THF
(4 mL) was stirred at 70 ꢀC for 2 h. After cooling to 0 ꢀC,
water (0.112 mL, 6.20 mmol), NaOH (15%, 0.112 mL)
and water (0.340 mL) were successively added and the
mixture stirred for 2 h. After ®ltration, the solution was
concentrated. Bulb-to-bulb distillation of the residue
gave the title compound (S)-(+)-(4) (0.238 g, 80%) ‰aŠ
22
d
+74ꢀ (c 2.5, CHCl3) (ee >99% see theoretical part).
Figure 1. Separation of AF-DX 384 enantiomers as a function of the
HSCD introduced into the electrolyte. Conditions: applied voltage:
15 kV; anodic detection l=200 nm; electrolyte: triethylammonium
phosphate 25Â10 M/HSCD 5% (w/w), pH=2.5; hydrodynamic
injection 1 mL of an ethanolic solution of (‹)AF-DX 384 (1.2 mg/mL).
(S)-( )-[2-(N0,N0 -Dipropylaminomethyl)piperidin-1-yl]-
acetonitrile (S)-( )-(14). Alkylation of amine (S)-(+)-4
3

J. Martin et al. / Bioorg. Med. Chem. 8 (2000) 591±600
599
Figure 2. Example of ( ) AF-DX 384 enantimoer identi®cation. Conditions: identical to those reported in Figure 1.
with bromoacetonitrile was carried out according to a
previously described method,18 and led to (S)-( )-14.
‰aŠ
(S)-( )-2-[-2-(N0,N0-Dipropylaminomethyl)piperidin-1-yl]-
ethan-1-amine (S)-( )-3. (S)-( )-14 (0.051 g, 0.22 mmol)
in THF (0.6 mL) was added dropwise to LiAH4
(0.017 g, 0.44 mmol) in THF (0.12 mL). The mixture was
stirred at 70 ꢀC for 2 h. After cooling, water (0.88 mmol,
0.016 mL), NaOH (15%, 0.016 mL) and water
(0.048 mL) were successively added and the mixture stir-
red for 2 h. The solids were ®ltered and the solution con-
genisation in 10 volumes of 50 mM Tris±HCl bu€er
(pH 7.4 at +4 ꢀC) with a Polytron (2Â30 s, maximal
setting) followed by ultracentrifugation (23,000 Â g, 60
min,+4 ꢀC). Aliquot fractions of proteins (30 mg) were
incubated for 90 min at room temperature in the pre-
sence of 0.5 nM [3H]N-methyl-scopolamine (NEN-
DuPont, France; speci®c activity: 84 Ci/mmol) and 6±8
increasing concentrations of competitors in a ®nal
volume of 200 mL of 50 mM Tris±HCl (pH 7.4) bu€er.
Incubation was stopped by rapid ®ltration over What-
man GF/B glass ®ber ®lters presoaked with 0.3% poly-
22
d
19ꢀ (c 1.7, CHCl3).
ethylenimine using
Nonspeci®c binding was determined in the presence of
a
Brandell Cell Harvester.
centrated. Bulb-to-bulb distillation of the residue gave the
61ꢀ (c 1,
22
d
6
title compound (S)-( )-3 (0.038g, 72%). ‰aŠ
10
M atropine. Anity values were calculated
CHCl3).
through the use of the EBDA/Ligand software and
expressed as pIC50‹SEM.
In Figure 1, we have shown the electropherograms in
optimised conditions, for the (‹) AF-DX 384 with each
HSCD and in Figure 2, an example of (S)-( )-AF-DX
384 enantiomer identi®cation (retention times: (S)-( )-
AF-DX 384: 11.1 min; (R)-(+)-AF-DX 384: 11.4 min)
Acknowledgements
This work was supported by the Reseau Interregional
Normand de Chimie Organique Fine (RINCOF, Con-
trat de Plan Bassin Parisien, Regions Haute et Basse
Normandie, Action 2) and the authors thank Jean-
Christophe Plaquevent, Jacques Maddaluno, Jean
Claude Combret and Lucette Duhamel (University of
Rouen) for helpful discussions.
(S)-(+)-5,11-Dihydro-11-[2-[2-[(N,N-dipropylaminome-
thyl)piperidin-1-yl]-ethylamino]-carbonyl]-6H-pyrido[2,3-
b][1,4]benzodiazepin-6-one (S)-(+)-1. (S)-(+)-1 AF-DX
22
384 ‰aŠ +12ꢀ (c 1, CHCl3) was obtained from (S)-( )-
d
3 according to the procedure previously described.18
Data from capillary electrophoresis: RT 11.4 min, ee
99%. The same reactions were used to prepare (R)-( )-
AF-DX 384 from (R)-( )-4. Data from capillary elec-
trophoresis: RT 11.1 min, ee 99%.
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