5-(2-Pyrrolidinyl)oxazolidinones and 2-(2-pyrrolidinyl)benzodioxanes: Synthesis of all the stereoisomers and α4β2 nicotinic affinity

Article abstract of DOI:10.1016/j.bmcl.2008.12.002

The four stereoisomers of 2-oxazolidinone 5-substituted with 1-methyl-2-pyrrolidinyl (1), of 1,4-benzodioxane 2-substituted with the same residue (2) and of the nor-methyl analogue of this latter (2a) were synthesized as candidate nicotinoids. Of the 12 c

Full text of DOI:10.1016/j.bmcl.2008.12.002

Bioorganic & Medicinal Chemistry Letters 19 (2009) 854–859
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
5-(2-Pyrrolidinyl)oxazolidinones and 2-(2-pyrrolidinyl)benzodioxanes:
Synthesis of all the stereoisomers and
a4b2 nicotinic affinity
a
a
c
b
Marco Pallavicini a, , Cristiano Bolchi , Matteo Binda , Antonio Cilia , Francesco Clementi ,
Rossana Ferrara a, Laura Fumagalli a, Cecilia Gotti b, Milena Moretti b, Alessandro Pedretti a,
Giulio Vistoli a, Ermanno Valoti a
*
a Istituto di Chimica Farmaceutica e Tossicologica, Università di Milano ‘Pietro Pratesi’, via Mangiagalli 25, I-20133 Milano, Italy
b CNR, Istituto di Neuroscienze, Dipartimento di Farmacologia Medica, Università di Milano, via Vanvitelli 32, I-20129 Milano, Italy
c Divisione Ricerca e Sviluppo Farmaceutico, Recordati s.p.a., via Civitali 1, I-20148 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The four stereoisomers of 2-oxazolidinone 5-substituted with 1-methyl-2-pyrrolidinyl (1), of 1,4-benzo-
dioxane 2-substituted with the same residue (2) and of the nor-methyl analogue of this latter (2a) were
synthesized as candidate nicotinoids. Of the 12 compounds, two N-methylated pyrrolidinyl-benzodiox-
ane stereoisomers, namely those with the same relative configuration at the pyrrolidine stereocentre
Received 12 September 2008
Revised 28 November 2008
Accepted 2 December 2008
Available online 6 December 2008
as (S)-nicotine, bind at
a4b2 nicotinic acetylcholine receptor with submicromolar affinity. Consistently
with the biological data, docking analysis enlightens significant differences in binding site interactions
not only between 1 and 2, but also between 2 and 2a and between the stereoisomers of 2 accounting
for the critical role played, in the case of the pyrrolidinyl-benzodioxanes, by the chirality of both the ster-
eolabile and stereostable stereogenic atoms, namely the protonated tertiary nitrogen and the two asym-
metric carbons.
Keywords:
nAChR
Nicotine
Ligand
Affinity
Ó 2008 Elsevier Ltd. All rights reserved.
Oxazolidinone
Benzodioxane
Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-
gated ion channels playing an important role in the regulation of
neurotransmission in the CNS. Their dysfunction has been related
to a number of severe brain pathologies, including Parkinson’s
and Alzheimer’s diseases, schizophrenia, anxiety and some forms
of epilepsy. Novel ligands for neuronal nAChRs, in particular for
by the only rotatable bond, prompted us to investigate also the SS
and RR enantiomers of 1 and 2. Therefore, a new synthetic strategy
was designed to obtain all the four stereoisomers of pyrrolidinyl-
oxazolidinone and of pyrrolidinyl-benzodioxane. Considering the
promising results previously obtained for one enantiomer of 2,
we prepared also the four stereoisomers of 2a, the nor-methyl
derivative of 2. In fact, it is known that the presence or the absence
of the N-methyl group at the cationic head represents a critical fea-
ture for several nicotinoids resulting in unexpectedly drastic affin-
ity changes, as well demonstrated by the opposite cases of nicotine,
whose nor-methyl analogue displays an almost 100-fold lower
the two major a4b2 and a7 brain subtypes, may have a great ther-
apeutic potential.1,2 Subtype selective nAChR ligands have been
developed over the last decade leading to the formulation of reli-
able
a4b2 and a
7 pharmacophores.3–6 Furthermore, models of
the ligand binding domain or full-length models for these nicotinic
receptor subtypes have been published.7–10 Recently, we have
reported the RS and SR enantiomers of 5-(1-methyl-2-pyrrolidi-
nyl)oxazolidinone (1) and 2-(1-methyl-2-pyrrolidinyl)-1,4-benzo-
dioxane (2) as potential nicotinoids, where the typical nicotinoid
2-pyrrolidinyl residue is linked to a cyclic hydrogen bond acceptor
affinity for
a
a4b2 nAChR, and of epibatidine, which exhibits reduced
4b2 binding when N-methylated.12
N
N
N
O
N
Me
R
Me
and p-electron rich group (HBA/p), demonstrating that one of the
two synthesized enantiomers of 2, namely that with the same rel-
2
2a
R = Me
= H
O
O
ative configuration at the pyrrolidine stereocentre as (S)-nicotine,
HN
displays a submicromolar
intriguing feature of two vicinal stereocentres, positioned on the
cationic head and on the HBA/ group, respectively, and connected
a
4b2 affinity.11 This result and the
O
(S)-nicotine
1
p
The key step of the previous syntheses of 1 and 2 was the reaction of
the diastereomeric mixture of RS and SS (1-carbobenzyloxy-2-pyr-
* Corresponding author. Tel.: +39 2 50317524; fax: +39 2 50317565.
E-mail address: marco.pallavicini@unimi.it (M. Pallavicini).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.002

M. Pallavicini et al. / Bioorg. Med. Chem. Lett. 19 (2009) 854–859
855
rolidinyl)oxiranes, epimers at the epoxide chiral centre prepared
O
O
O
a
b
from (S)-(1-carbobenzyloxy-2-pyrrolidinyl)methanol, with diben-
zylamine and 2-benzyloxyphenate, respectively.11 However, such
a reaction allows only one diastereomer of the resultant secondary
alcohol to be isolated, namely that deriving from the SS pyrrolidiny-
loxirane and having R and S configuration at the exocyclic stereo-
genic carbon of the aminoalcohol and of the phenoxyalcohol,
respectively (Scheme 1). Upon this reaction, the other oxirane, the
RS diastereomer with R configuration at epoxide chiral centre, has
N
Boc
N2
N
Boc
N
Boc
OH
Br
(S)-3
(S)-4
Scheme 2. Reagents and conditions: (a) iBuOCOCl, triethylamine, Et2O, ꢀ10 °C,
20 min; CH2N2, 0 °C, 45 min and room temperature over-night, 78%; (b) aqueous
HBr, Et2O, 0 °C, 30 min and room temperature, 1 h, 71%.
a
different fate, which is intramolecular cyclisation to tetra-
hydropyrroloxazolone, as demonstrated in the case of the synthesis
of 2 by the isolation of the o-benzyloxyphenoxymethyl substituted
tetrahydro-pyrroloxazolone shown in Scheme 1, and presumable,
but not demonstrated, in the case of the synthesis of 1.
O
OH
2'
(S)-5
a
2
(S)-4
N
Boc
N
NBn2
NBn2
b
Boc
c
Symmetrically, the specular diastereomeric mixture of SR and
RR (1-carbobenzyloxy-2-pyrrolidinyl)oxiranes, epimers at the
epoxide chiral centre prepared from (R)-(1-carbobenzyloxy-2-pyr-
rolidinyl)methanol, provides only the SR aminoalcohol and the RR
phenoxyalcohol. As a consequence, only one pair of enantiomers
of 1 and 2 could be obtained by these synthetic pathways, namely
the RS and SR enantiomers.11 The present synthesis, which yields
all the stereoisomers of 1 and 2, has 2-bromoacetylpyrrolidine in
place of 2-pyrrolidinyloxirane as a key intermediate. (S)-N-t-
Butoxycarbonyl-2-bromoacetylpyrrolidine [(S)-4] was prepared
from (S)-N-t-butoxycarbonylproline. Treatment of the N-protected
amino acid with triethylamine and isobutyl chloroformate and
then with diazomethane yielded (S)-N-t-butoxycarbonyl-2-diazo-
acetylpyrrolidine [(S)-3],13 which was converted into (S)-414 by
reaction with aqueous HBr (Scheme 2).
The nucleophilic substitution of bromide with dibenzylamine
afforded the dibenzylaminomethylketone [(S)-5]15 (Scheme 3).
Racemisation of bromoketone during the reaction was excluded
on the basis of the chiral HPLC analysis of (S)-5 and (R)-5, obtained
from (S)-4 and (R)-4, respectively. Consistently with this finding,
no significant variation of optical rotation was observed after
recrystallization of the two substitution products. The successive
carbonyl reduction with lithium aluminium hydride yielded a
60:40 mixture of secondary aminoalcohols (2S,20S)-616 and
(2R,2'S)-6
OH
OH
OH
c
2'
2'
2'
2
2
2
N
N
NBn2
NBn2
N
NBn2
CH3
d
CH3
d
Boc
(2S,2'S)-7
(2R,2'S)-7
(2S,2'S)-6
OH
OH
2'
2'
2
2
N
N
NH2
NH2
CH3
e
CH3
e
(2S,2'S)-8
(2R,2'S)-8
O
O
O
O
2'
2'
5
5
NH
NH
N
N
CH3
CH3
(5R,2'S)-1
(5S,2'S)-1
Scheme 3. Reagents and conditions: (a) dibenzylamine Et2O, 0 °C, 2 h, 80%; (b)
LiAlH4, THF, 0 °C, 1 h, 51% [(2S,20S)-6] and 35% [(2R,20S)-6]; (c) LiAlH4, THF, reflux,
4 h, 86% [(2S,20S)-7] and 85% [(2R,20S)-7]; (d) H2–Pd/C, MeOH, 5 atm, over-night,
97% [(2S,20S)-8] and 84% [(2R,20S)-8]; (e) 1,10-carbonyldiimidazole, THF, 3 h, 59%
[(2S,20S)-1] and 51% [(2R,20S)-1].
(2R,20S)-6,17 which were separated by crystallization of the major
SS diastereomer and chromatographic isolation of the minor RS
diastereomer from the crystallization mother liquors. Absolute R
configuration was assigned to the oxygen-bound asymmetric car-
bon of this latter diastereomer, second eluted in the chromato-
graphic purification, and, consequently S configuration to the
same carbon of the other diastereomer on the basis of the succes-
sive reduction of N-protecting group to methyl, which produced,
when applied to the chromatographically purified diastereomer,
diamine (2R,20S)-718 with the same 1H NMR as the RS diamine pre-
viously synthesized via pyrrolidinyloxirane and diamine (2S,20S)-
BnO
O
S
R
S
O
N
N
O
b
Cbz
a
O
S
OH
O
OH
S
S
N
Bn
Bn
R
N
N
7
19 with a quite different 1H NMR, when applied to the crystallized
O
O
Cbz
OBn
diastereomer. Hydrogenolitic debenzylation afforded the primary
amines (2S,20S)-820 and (2R,20S)-8,21 which were finally cyclised
to (5S,20S)-122 and (5R,20S)-123 by reaction with 1,10-carbonyl-
diimidazole.
Starting from (R)-N-t-butoxycarbonylproline, the same syn-
thetic route24–32 illustrated in Schemes 2 and 3 led to (5R,20R)-
133 and (5S,20R)-1.34
The absolute configuration of the oxazolidinone stereocenter of
the four stereoisomers of 1 was established by comparison with
the RS and SR enantiomers previously synthesized via prolinal
and 2-pyrrolidinyloxirane.11
The same synthetic strategy was followed to prepare the four
stereoisomers of 2 reacting (S)-4 with pyrocatechol in the presence
of a base (Scheme 4). In this case, however, suitable reaction con-
ditions had to be found to avoid the bromomethylketone racemisa-
tion, which was, for instance, complete using potassium carbonate
O
O
S
R
NH
N
O
S
(5R,2'S)-1
R
N
O
(2R,2'S)-2
Scheme 1. Reagents and conditions: (a) dibenzylamine, 2-propanol, MW irradia-
tion (150 °C, 100 W), 15 min, 48%; (b) 2-benzyloxyphenol, potassium carbonate,
2-propanol, reflux, 24 h, 42%.

856
M. Pallavicini et al. / Bioorg. Med. Chem. Lett. 19 (2009) 854–859
tional and 1H NMR analysis, recognizing the relative anti
OH
O
disposition of benzodioxane methine axial proton not only to one
of the two benzodioxane methylene protons but also to pyrrolidine
methine proton as indicative of an opposite configuration of ben-
zodioxane C2 to that of pyrrolidine stereocenter, since the anti dis-
position of the two methine protons is sterically forbidden when
the two stereocenters have the same configuration. Consistently
with this statement, which had allowed to establish the RS and
SR configurations of the two enantiomers of 2 prepared via oxirane,
the 1H NMR spectra of the two new enantiomers of 2 obtained via
bromoketone showed the anti disposition of benzodioxane
methine proton only to one of the two benzodioxane methylene
protons, but not to the pyrrolidine methine proton, thus proving
their SS and RR configurations.
The optical powers and the melting points of the RS and SR
forms of 1 and the optical powers of the RS and SR forms of 2 pre-
pared via bromoketone, as described here, were significantly high-
er than those of the same stereoisomers synthesized from prolinal,
as previously reported. This indicates that the synthesis via alde-
hyde implies partial racemisation.
O
b
a
(S)-9
(S)-4
N
Boc
OH
OH
OH
OH
O
c
2'
O
c
2'
1
1
N
N
Boc
Boc
(1S,2'S)-10
(1R,2'S)-10
O
2
O
2
2'
2'
N
N
O
O
d
e
e
Boc
Boc
d
(2R,2'S)-11
(2S,2'S)-11
O
2
O
2
2'
2'
N
N
O
O
We evaluated the affinity towards the
types present in rat cortex membranes by binding studies using,
as ligands, [3H]-epibatidine and 125I]-
Bungarotoxin, respec-
a4b2 and the a7 sub-
CH3
CH3
(2S,2'S)-2
(2R,2'S)-2
[
a
tively.53 Nicotine was included in the series for comparison as well
as the RS and SR forms of 1 and 2, considering their higher enantio-
meric purity resulting from the new preparation method. As shown
in Table 1 and as previously reported for (5R,20S)-1 and (5S,20R)-1,11
we found that the four pyrrolidinyl-oxazolidinones are virtually
devoid of nicotinic affinity, with the exception of (5S,20S)-1, which
O
2
O
2
2'
2'
N
H
N
H
O
O
(2R,2'S)-2a
(2S,2'S)-2a
Scheme 4. Reagents and conditions: (a) pyrocatechol monosodium salt, acetone,
22 °C, over-night, 66%; (b) NaBH4, THF, ꢀ10 °C, 2 h, 45% [(1R,20S)-10] and 30%
[(1S,20S)-10]; (c) triphenylphosphine, DEAD, THF, reflux, over-night, 93% [(2S,20S)-
11] and 88% [(2R,20S)-11]; (d) LiAlH4, THF, reflux, 2 h, 89% [(2S,20S)-2] and 94%
[(2R,20S)-2]; (e) TFA, DCM, 22 °C, 2 h, 83% [(2S,20S)-2a] and 93% [(2R,20S)-2a].
shows a modest micromolar a4b2 affinity. The two RS and SR ster-
eoisomers, prepared by the new method, exhibit slightly higher,
but always very low affinities compared with those previously
found11 for the same forms differently synthesized. On the con-
trary, two out of the four N-methyl pyrrolidinyl-benzodioxanes 2,
those with the same S configuration at the pyrrolidine stereocenter
in acetone, as suggested by the almost null optical activity of iso-
lated (S)-9 and then proved by its chiral HPLC analysis. The use
of preformed pyrocatechol monosodium salt in methanol, DMF,
or acetone resulted in high enantiomeric excesses of (S)-9, gener-
ally not lower than 85%. In particular, the reaction in acetone at
room temperature allowed (S)-9 to be obtained in 66% yield and
with >98% ee after 24 h. At 30 °C, the same reaction gave (S)-935
in 90% yield after 1.5 h, but with lower ee (85%). The successive
carbonyl reduction with sodium borohydride in THF afforded a
60:40 mixture of secondary alcohols (1R,20S)-1036 and (1S,20S)-
10,37 thus showing the same diastereopreference observed for
the analogous reduction of (S)-5, which had produced a (2S,20S)-
6/(2R,20S)-6 mixture in 60/40 ratio. In fact, it is to be noticed that
(1R,20S)-10 and (1S,20S)-10 have the same relative configuration
of (2S,20S)-6 and (2R,20S)-6, respectively. The two diastereomer
were separated by chromatography on silica gel and the first
eluted, solid RS diastereomer further purified by crystallization
from diethyl ether. HPLC analysis demonstrated a >99.5% diaste-
reomeric purity of the two separated secondary alcohols, while
chiral HPLC analysis allowed to ascertain their unvaried enantio-
meric excess with respect to (S)-9. The successive cyclisation by
intramolecular Mitsunobu reaction yielded the pyrrolidinyl-benzo-
dioxanes (2R,20S)-1138 and (2S,20S)-11,39 whose N-protecting group
was reduced to methyl to give (2R,20S)-240 and (2S,20S)-241 or re-
moved to give (2R,20S)-2a42 and (2S,20S)-2a,43 respectively.
as (ꢀ)-nicotine, show a moderate submicromolar affinity for
a4b2
nAChR and are 50- to 100-fold more potent than their enantio-
mers. In particular, a significant increase of enatioselectivity was
observed for the newly synthesized RS–SR enantiomeric pair in
comparison with the previously reported data11 and this increment
is consistent with the higher enantiomeric purity ensured by the
new preparation method reported here. No enantiopreference
Table 1
Nicotine and compounds 1, 2 and 2a: affinity for native receptor subtypes, present in
rat brain membranes, labelled by [3H]-epibatidine, and [125I]-
a
a
Bungarotoxin and for
2-ARs, present in rat cortex membranes, labelled by [3H]RS 79948-197
a
4b2 nAChR
a
[
7 nAChR
a
2-AR [3H]RS
79948-197 Ki (lM)
[3H]-Epi Ki (
l
M)
125I]-
aBgtx Ki (
lM)
(ꢀ)-Nicotine
(5R,20S)-1
0.004 (18)
35 (34)
37 (34)
2.7 (33)
40 (31)
0.234 (29)
100 (78)
43 (69)
21 (62)
>100
(5S,20R)-1
(5S,20S)-1
(5R,20R)-1
(2R,20S)-2ꢁHCl
(2S,20R)-2ꢁHCl
(2R,20R)-2ꢁHCl
(2S,20S)-2ꢁHCl
(2R,20S)-2aꢁHCl
(2S,20R)-2aꢁHCl
(2R,20R)-2aꢁHCl
(2S,20S)-2aꢁHCl
Kd (nM)
0.26 (32)
12.5 (17)
43.8 (16)
0.47 (30)
2.1 (30)
88 (18)
11.6 (17)
14.4 (32)
0.036 (22)
21 (44)
35 (45)
14.8 (35)
14.6 (54)
36 (41)
14.7 (42)
27 (38)
32.2 (45)
0.3 (35)
0.77 (6)
0.47 (11)
6.67 (20)
1.67 (7)
1.35(3)
14 (27)
6.33 (6)
0.64 (5)
1.01 (0.5)
Starting from (R)-N-t-butoxycarbonylproline, the same syn-
thetic route44–48 illustrated in Schemes 2 and 4 led to (2S,20R)-249
and (2R,20R)-250 and to the respective nor-methyl derivatives
(2S,20R)-2a51 and (2R,20R)-2a.52
The Kd and Ki values were derived from [3H]-epibatidine, [125I]-
aBungarotoxin and
[3H]RS 79948-197 saturation and two competition binding experiments on rat
brain membranes as described in Refs. 53–55. The curves were fitted using a
nonlinear least squares analysis program and the F test. The numbers in brackets
represent the %CV.
As previously reported,11 the absolute configuration of the ben-
zodioxane C2 was established on the basis of compared conforma-

M. Pallavicini et al. / Bioorg. Med. Chem. Lett. 19 (2009) 854–859
857
was found in the case of the lower affinities shown by 2 stereoiso-
mers for 7 nAChR.
The two stereoisomers of 2 with S configuration at pyrrolidine
C2 display a remarkable similar 4b2 versus 7 selectivity. The
N-demethylation of 2 results in both modest 4b2 and 7 affini-
ties: the Ki of all the nor-methyl derivatives are lower than
10
M, excepting (2R,20S)-2a, which shows, however, only a mod-
est micromolar 4b2 affinity.
Furthermore, the structural analogy of benzodioxane deriva-
tives to 2-adrenergic receptor ( 2-AR) ligands such as efaroxan,
imiloxan and, especially, idazoxan and the reported affinity of
2-AR ligands for receptors labelled by [3H]-epibatidine54 induced
to evaluate the binding of 2 and 2a also to 2-ARs. As previously
reported for (2R,20S)-2 and (2S,20R)-2,11 also (2S,20S)-2a proved to
compete with [3H]RS 79948-197 for rat cortex
2-ARs with submi-
imum closeness of the pyridine nitrogen of nicotine to the benzo-
dioxane O(4). The submicromolar
4b2 binding affinity of (2R,20S)-
2, significantly higher than that of (2S,20R)-2, was consistent with
those indications. Now, the additional 4b2 affinity data, submi-
a
a
a
a
a
a
a
cromolar also for (2S,20S)-2, complete the picture, while docking
results better support SAR analysis enlightening (a) similar pat-
terns of beneficial interactions of the charged N-methyl pyrrolidine
residue for the two most affinitive RS and SS isomers of 2 and for
(S)-nicotine, contrarily to the two modestly affinitive isomers with
R configuration at the pyrrolidine stereocenter, and (b) different,
but ever productive accommodations of the HBA moiety, namely
the dioxane ring, for the two most affinitive RS and SS isomers of
2. Such a different accommodation of the dioxane ring, which
seems to interact with the same amino acids of the binding site
through its O(4) in (2R,20S)-2 and through its O(1) in (2S,20S)-2,
would justify the failed prediction of the affinity of the latter by
the previous conformational analysis, which had emphasized the
maximum closeness of O(4) to pyridine nitrogen in the superposi-
tion onto nicotine undervaluing the interaction potentiality of
O(1).11 In the case of 1, the same analysis had let us hope that
(5R,20R)-1 could show a good or moderate nicotinic affinity. The
present data deny such a prediction indicating that identical rela-
tive configuration to (S)-nicotine at the pyrrolidine stereocentre
is more important than the other conformational properties we
had previously considered for 1 stereoisomers.11
l
a
a
a
a
a
a
cromolar affinity thus confirming such a coexistence of affinities
for the two different receptor systems.55,56
Figure 1 depicts the best docked complexes of
a4b2 nAChR
binding site with the two most affinitive isomers of 2, namely
(2R,20S)-2 and (2S,20S)-2, the chiral nitrogen of the ligands being
in S configuration.57 The protonated nitrogen of both isomers sta-
bilizes a reinforced H-bond with the carbonyl group of Trp147(a4),
while the carbon skeleton of their pyrrolidine ring contacts a set of
apolar residues not displayed for clarity [e.g. Val109(b2),
Phe117(b2), Leu119(b2), and Tyr195(
mainly differ in the benzodioxane interactions: H-bonds are
formed by O(1) of (2S,20S)-2 with Lys77(b2) and Tyr195(
4),
a4)]. The two stereoisomers
Finally, the poor affinities of the four nor-methyl derivatives 2a
cause to reflect over the role of the N-methyl group. It is known
a
whereas the same amino acids form H-bonds with O(4) of
(2R,20S)-2. The greater distance between mutually repulsive
Lys77 and pyrrolidine nitrogen resulting from the interaction with
O(4) instead of O(1) may explain the higher affinity of (2R,20S)-2
and the same repulsive interaction, exalted by the demethylation
of pyrrolidine nitrogen, could be responsible for the scarce affinity
of the four stereoisomers of the nor-methyl derivative 2a, even if
these conserve the H-bond with Trp147. The two less affinitive iso-
mers of 2, namely those having the pyrrolidine stereocenter in R
configuration, are unable to correctly arrange the pyrrolidine ring.
For the pyrrolidinyl-oxazolidinones 1, the docking analysis shows
that the interaction of the protonated nitrogen with Trp147, also
observed for the pyrrolidinyl-benzodioxanes, is not associated, dif-
ferently from these latter, with significant contacts of the extra-
pyrrolidine portion with Tyr195 and Lys77. Among the four oxazo-
lidinones, the best docking score was obtained for the SS form,
which is 13- to 15-fold more affinitive than the other stereoisomer.
Our previous investigations, based on the superposition of the
lowest energy conformers of the four stereoisomers of 2 onto (S)-
nicotine, had indicated the two SR and RS stereoisomers as the best
candidates to display nicotinic affinity, with a preference for
(2R,20S)-2, thanks to the same chirality of (S)-nicotine and the max-
Table 2
Docking scores (kcal/mol) for nicotine, nornicotine, 2 and 2a in a
receptor model
a4b2 nicotinic
(S)-Nicotine
ꢀ76.03
(2R,20S)-2 (2S,20S)-2 (2S,20R)-2 (2R,20R)-2
Me
ꢀ65.25
ꢀ63.25
ꢀ63.08
ꢀ59.66
(S)
N+
N+
H
Me
ꢀ47.57
ꢀ39.81
ꢀ39.22
ꢀ39.49
ꢀ34.76
(R)
H
(S)-Nornicotine (2R,20S)-2a (2S,20S)-2a (2S,20R)-2a (2R,20R)-2a
H
ꢀ55.67
ꢀ38.90
ꢀ44.22
ꢀ41.36
ꢀ39.58
N+
H
Figure 1. Main polar contacts stabilizing the putative complexes between a
4b2 nAChR binding site and (2R,20S)-2 (left) and (2S,20S)-2 (right).

858
M. Pallavicini et al. / Bioorg. Med. Chem. Lett. 19 (2009) 854–859
d 1.34–1.39 (m, 1H), 1.62–1.77 (m, 3H), 2.17–2.33 (m, 2H), 2.35 (s, 3H), 2.48 (d,
J = 6 Hz, 2H), 2.97–3.03 (m, 1H), 3.56–3.63 (m, 1H), 3.50 and 3.76 (2 d,
J = 13.5 Hz, 4H), 7.21–7.32 (m, 10H).
that N-demethylation of nicotinoids can enhance binding affinity,
especially when the ligand molecule is relatively rigid and the con-
figuration preferentially assumed by the chiral tertiary N-methyl
ammonium leads to an unfavourable disposition of the nitrogen-
bound proton and methyl.58 Here, we observe the opposite trend:
the N-demethylation of 2 reduces the binding affinity, as in the
case of nicotine. We impute the reduced affinity of 2a to a repulsive
interaction of one of its two nitrogen protons with a binding site
amino acidic residue. Interestingly, the same unfavourable interac-
tion is shown by the unique nitrogen proton of 2 stereoisomers,
when their chiral nitrogen assumes R configuration, and it is signif-
icant that 2a and 2 with N+ in R configuration have similar docking
scores, worse than 2 with N+ in S configuration (see Table 2). This
would further address the key role, often neglected, of the proton-
ation-induced chirality, whose implication in the interaction with
the binding site transcends the mere stereoelectronic effects of
N-methyl group.
20. (2S,20S)-8: Isolated as an oil by concentration of the reaction solution in
methanol after removal of the catalyst; 1H NMR (CDCl3) d 1.53–1.56 (m, 1H),
1.70–1.77 (m, 2H), 1.85–1.94 (m, 1H), 2.31–2.42 (m, 2H), 2.43 (s, 3H), 2.58 (dd,
J = 12.7, 8.3 Hz, 1H), 2.77 (dd, J = 12.7, 3.3 Hz, 1H), 3.01–3.07 (m, 1H), 3.20–3.26
(m, 1H).
21. (2R,20S)-8: Isolated as an oil by concentration of the reaction solution in
methanol after removal of the catalyst; 1H NMR spectrum as described in Ref. 11.
22. (5S,20S)-1: Isolated as a solid by chromatography on silica gel (eluent 8:2
toluene/methanol); mp 128–129 °C (dec); ½a D25
ꢂ
ꢀ7.0 (c1, methanol); 1H NMR
(CDCl3) d 1.49–1.60 (m, 1H), 1.72–1.98 (m, 3H), 2.30 (pseudo q, J = 8.7 Hz, 1H),
2.45 (s, 3H), 2.63 (pseudo q, J = 8.7 Hz, 1H), 3.05–3.11 (m, 1H), 3.33 (pseudo t,
J = 8.5 Hz, 1H), 3.57 (pseudo t, J = 8.5 Hz, 1H), 4.65 (pseudo q, J = 7.7 Hz, 1H),
5.40 (br s, 1H).
23. (5R,20S)-1: Isolated as a solid by chromatography on silica gel (eluent 8:2
toluene/methanol); mp 173–174 °C;
½
a 2D5
ꢂ
ꢀ96.0 (c1, methanol); 1H NMR
spectrum as described in Ref. 11.
24. (R)-3: Mp and 1H NMR identical to the enantiomer.
25. (R)-4: ½a 2D5
ꢂ
+66.4 (c1, acetone); 1H NMR identical to the enantiomer.
+46.1 (c1, MeOH); ee 98.8% (by HPLC on a Kromasil Amycoat
26. (R)-5: ½a 2D5
ꢂ
column; 9:1 hexane/isopropanol, 1.5 ml/min at 254 nm; tR 3.90 min); mp and
1H NMR identical to the enantiomer.
27. (2R,20R)-6:
½
a 2D5
ꢂ
+77.7 (c1, MeOH); mp and 1H NMR identical to the
Acknowledgements
enantiomer.
28. (2S,20R)-6: ½a 2D5
ꢂ
+32.8 (c1, MeOH); mp and 1H NMR identical to the enantiomer.
+74.4 (c1, MeOH); 1H NMR identical to the enantiomer.
+35.4 (c1, MeOH); 1H NMR identical to the enantiomer.
This research was financially supported by funds of the Italian
Ministry of University and Scientific Research (FIRB Grant
RBNE03FH5Y 002).
29. (2R,20R)-7: ½a 2D5
ꢂ
30. (2S,20R)-7: ½a 2D5
ꢂ
31. (2R,20R)-8: 1H NMR identical to the enantiomer.
32. (2S,20R)-8: 1H NMR identical to the enantiomer.
33. (5R,20R)-1: ½a 2D5
ꢂ
+6.9 (c1, MeOH); mp and 1H NMR identical to the enantiomer.
+94.9 (c1, MeOH); mp and 1H NMR identical to the enantiomer.
34. (5S,20R)-1: ½a 2D5
ꢂ
References and notes
35. (S)-9: Isolated as an oil by chromatography on silica gel (eluent 7:3
cyclohexane/ethyl acetate); ½a D25
ꢀ91.8 (c1, MeOH); ee 98.2% (by HPLC on a
ꢂ
1. Cassels, B. K.; Bermúdez, I.; Dajas, F.; Abin-Carriquiry, J. A.; Wonnacott, S. DDT
2005, 10, 1657.
2. Arneric, S. P.; Holladay, M.; Williams, M. Biochem. Pharmacol. 2007, 74, 1092.
3. Jensen, A. A.; Frølund, B.; Liljefors, T.; Krogsgaard-Larsen, P. J. Med. Chem. 2005,
48, 4705.
4. Tønder, J. E.; Olesen, P. H. Curr. Med. Chem. 2001, 8, 651.
5. Nicolotti, O.; Pellegrini-Calace, M.; Altomare, C.; Carotti, A.; Carrieri, A.; Sanz, F.
Curr. Med. Chem. 2002, 9, 1.
6. Mazurov, A.; Hauser, T.; Miller, C. H. Curr. Med. Chem. 2006, 13, 1567.
7. Pedretti, A.; Marconi, C.; Bolchi, C.; Fumagalli, L.; Ferrara, R.; Pallavicini, M.;
Valoti, E.; Vistoli, G. Biochem. Biophys. Res. Commun. 2008, 369, 648.
8. Bisson, W. H.; Scapozza, L.; Westera, G.; Mu, L.; Schubiger, P. A. J. Med. Chem.
2005, 48, 5123.
Chiralcel OJ column; 9:1 hexane/isopropanol, 0.6 ml/min at 276 nm; tR
13.90 min); 1H NMR (CDCl3) d 1.48 and 1.51 (2s, 9H), 1.61–2.21 (m, 4H),
3.34–3.59 (m, 2H), 3.94 (d, J = 11.0 Hz, 1H), 4.09–4.14 (m, 1.5H), 4.27–4.30 (m,
0.5H), 6.83–6.94 (m, 4H).
36. (1R,20S)-10: Isolated as a white solid by chromatography on silica gel (eluent
7:3 toluene/ethyl acetate) and crystallization from diethyl ether; mp 105–
106 °C; ½a 2D5
ꢂ
ꢀ47.8 (c1, chloroform); de 99.0% (by HPLC on a LiChroCART
LiChrospher Si 60 column, 5
lm; 65:35 hexane/ethyl acetate, 2 ml/min at
276 nm; tR 4.74 min); ee 98.8% (by HPLC on a Chiralcel OD-H column; 9:1
hexane/isopropanol, 0.5 ml/min at 276 nm; tR 12.23 min); 1H NMR (CDCl3) d
1.48 (s, 9H), 1.50–2.10 (m, 4H), 3.26–3.34 (m, 1H), 3.48–3.58 (m, 1H), 3.81 (br
s, 1H), 3.98 (dd, J = 10.5, 5.2 Hz, 1H), 4.05–4.12 (m, 2H), 6.74–6.80 (m, 1H),
6.90–6.96 (m, 3H).
9. Bisson, W. H.; Westera, G.; Schubiger, P. A.; Scapozza, L. J. Mol. Model. 2008, 14,
891.
10. Grazioso, G.; Cavalli, A.; De Amici, M.; Recanatini, M.; De Micheli, C. J. Comput.
Chem. 2008, 29, 2593.
37. (1S,20S)-10: Isolated as an oil by chromatography on silica gel (eluent 7:3
toluene/ethyl acetate); ½a D25
ꢂ
ꢀ14.2 (c1, chloroform); de 99.4% (by HPLC on a
LiChroCART LiChrospher Si 60 column, 5
l
m; 65:35 hexane/ethyl acetate, 2 ml/
11. Pallavicini, M.; Moroni, B.; Bolchi, C.; Cilia, A.; Clementi, F.; Fumagalli, L.; Gotti,
C.; Meneghetti, F.; Riganti, L.; Vistoli, G.; Valoti, E. Bioorg. Med. Chem. Lett. 2006,
16, 5610.
min at 276 nm; tR 5.95 min); ee 97.9% (by HPLC on a Chiralcel OD-H column;
9:1 hexane/isopropanol, 0.5 ml/min at 276 nm; tR 12.42 min); 1H NMR (CDCl3)
d 1.46 (s, 9H), 1.79–2.05 (m, 4H), 3.23–3.31 (m, 1H), 3.46–3.54 (m, 1H), 3.86–
3.92 (m, 1H), 4.04–4.10 (m, 3H), 6.77–6.82 (m, 1H), 6.89–6.96 (m, 3H).
12. Schmitt, J. D. Curr. Med. Chem. 2000, 7, 749.
38. (2R,20S)-11: Isolated as a viscous oil by chromatography on silica gel (eluent
13. (S)-3: Isolated as a yellow solid after chromatographic separation on silica gel
(eluent 7:3 cyclohexane/ethyl acetate); mp 45–46 °C; IR 2104 cmꢀ1
;
1H NMR
95:5 toluene/ethyl acetate); ½a D25
ꢂ
ꢀ45.1 (c1, chloroform); 1H NMR (CDCl3) d
(CDCl3) d 1.42, 1.43 and 1.46 (3s, 9H), 1.85–2.19 (m, 4 H), 3.42–3.50 (m, 2H),
4.19 and 4.28 (2br s, 1H), 5.38 and 5.47 (2br s, 1H).
1.45 (s, 9H), 1.89–2.06 (m, 4H), 3.32–3.55 (m, 2H), 3.92–3.95 (m, 1H), 4.22–
4.37 (m, 3H), 6.80–6.88 (m, 4H).
39. (2S,20S)-11: Isolated as a viscous oil by chromatography on silica gel (eluent
14. (S)-4: Isolated as a pale yellow oil by chromatography on silica gel (eluent 7:3
cyclohexane/ethyl acetate); ½a D25
ꢂ
ꢀ67.0 (c1, acetone); 1H NMR (CDCl3) d 1.41
95:5 toluene/ethyl acetate); ½a D25
ꢂ
ꢀ152.0 (c1, chloroform); 1H NMR (CDCl3) d
and 1.44 (2s, 9H), 1.87–2.03 (m, 3H), 2.12–2.29 (m, 1H), 3.41–3.56 (m, 2H),
4.02–4.17 (m, 2H), 4.45–4.55 (m, 1H).
1.47 (s, 9H), 1.91–2.06 (m, 3H), 2.16–2.22 (br s, 1H), 3.39–3.49 (m, 2H), 3.93–
4.10 (m, 3H), 4.29–4.33 (dd, J = 11.3, 1.9 Hz, 1H), 6.81–6.89 (m, 4H).
40. (2R,20S)-2: Isolated as an oil by concentration of the reaction mixture after
adding dichloromethane, washing with water and filtering through celite; 1H
NMR spectrum as described in Ref. 11. (2R,20S)-2 Hydrochloride: Mp and 1H
15. (S)-5: Isolated as a white solid after chromatographic separation on silica gel
(eluent 7:3 cyclohexane/ethyl acetate) and crystallization from hexane; mp
89–90 °C;
½
a 2D5
ꢂ
ꢀ44.6 (c1, methanol); ee 99.8% (by HPLC on
a Kromasil
NMR spectrum as described in Ref. 11; ½a D25
ꢂ
+33.3 (c2, EtOH).
AmyCoat column; 9:1 hexane/isopropanol, 1.5 ml/min at 254 nm; tR 6.35 min);
1H NMR (DMSO-d6, 100 °C) d 1.24 (s, 9H), 1.51–1.77 (m, 3H), 1.98–2.07 (m, 1H),
3.23–3.33 (m, 2H), 3.37 (s, 2H), 3.61–3.74 (2d, 4H), 4.27–4.32 (dd, 1H), 7.20–
7.36 (m, 10H).
41. (2S,20S)-2: Isolated as an oil by concentration of the reaction mixture after
adding dichloromethane, washing with water and filtering through celite; 1H
NMR (CDCl3) d 1.43–1.94 (m, 4H), 2.22–2.30 (m, 1H), 2.43 (s, 3H), 2.47–2.53
(m, 1H), 3.10–3.15 (m, 1H), 4.00 (dd, J = 11.0, 8.3 Hz, 1H), 4.10 (ddd, J = 8.3, 4.4,
1.9 Hz, 1H), 4.28 (dd, J = 11.0, 1.9 Hz, 1H), 6.79–6.96 (m, 4H). (2S,20S)-2
16. (2S,20S)-6: Isolated as a white solid by crystallization from diisopropyl ether
and furthermore from the crystallization mother liquors by chromatography
on silica gel (eluent 7:3 cyclohexane/ethyl acetate); mp 101–102 °C; ½a D25
ꢂ
Hydrochloride: Mp 144–145 °C; ½a D25
ꢂ
ꢀ107.5 (c2, EtOH); 1H NMR (DMSO-d6) d
ꢀ78.5 (c1, methanol); 1H NMR (DMSO-d6, 100 °C) d 1.37 (s, 9H), 1.57–1.77 (m,
4H), 2.39–2.53 (m, 2H), 3.00–3.05 (m, 1H), 3.29–3.36 (m, 1H), 3.57–3.70 (m,
4H), 3.76–3.79 (m, 1H), 3.95–3.98 (m, 1H), 4.17 (br s, 1H), 7.17–7.34 (m, 10H).
17. (2R,20S)-6: Isolated as a white solid by chromatography on silica gel (eluent 7:3
1.87–2.10 (m, 4H), 3.11–3.14 (m, 1H), 2.89 and 2.90 (2s, 3H), 3.50–3.60 (m,
1H), 3.70–3.80 (m, 1H), 3.94 (dd J = 11.6, 8.5 Hz, 1H), 4.42 (dd, J = 11.6, 2.2 Hz,
1H), 4.70–4.73 (m, 1H), 6.85–6.95 (m, 4H), 9.95 (br s, 1H).
42. (2R,20S)-2a: Isolated as a colourless oil by diethyl ether/aq KOH extraction from
the reaction mixture and concentration of the organic phase; 1H NMR (CDCl3) d
1.53–1.65 (m, 1H), 1.73–1.97 (m, 3H), 2.28 (br s, 1H), 2.88–2.96 (m, 1H), 3.03–
3.10 (m, 1H), 3.21–3.28 (m, 1H), 3.97–4.05 (m, 2H), 4.22–4.30 (m, 1H), 6.79–
cyclohexane/ethyl acetate); mp 70–71 °C; ½a D25
ꢂ
ꢀ34.5 (c1, methanol); 1H NMR
(DMSO-d6, 100 °C) d 1.35 (s, 6H), 1.41 (s, 3H), 1.42–1.96 (m, 4H), 2.38–2.47 (m,
2H), 3.07–3.15 (m, 1H), 3.28–3.36 (m, 1H), 3.43–3.48 (m, 2H), 3.69–3.74 (m,
2H), 3.77–3.79 (m, 1H), 4.12–4.15 (m, 1H), 4.16 (br s, 1H), 7.18–7.35 (m, 10H).
18. (2R,20S)-7: isolated as a colourless oil by chromatography on silica gel (eluent
6.91 (m, 4H). (2R,20S)-2a Hydrochloride: Mp 154–155 °C; a 2D5
½ ꢂ +68.3 (c2,
EtOH); 1H NMR (DMSO-d6) d 1.75–2.12 (m, 4H), 3.11–3.23 (m, 2H), 3.59–3.68
(m, 1H), 4.07 (dd, J = 11.8, 6.6 Hz, 1H), 4.32 (dd, J = 11.8, 2.5 Hz, 1H), 4.45–4.50
(m, 1H), 6.83–6.93 (m, 4H), 9.46 (br s, 2H).
97:3 ethyl acetate/triethylamine);
spectrum as described in Ref. 11.
½
a 2D5
ꢂ
ꢀ32.8 (c1, methanol); 1H NMR
19. (2S,20S)-7: Isolated as a colourless oil by chromatography on silica gel (eluent
43. (2S,20S)-2a: Isolated as a colourless oil by diethyl ether/aq KOH extraction from
the reaction mixture and concentration of the organic phase; 1H NMR (CDCl3) d
97:3 ethyl acetate/triethylamine); ½a D25
ꢂ
ꢀ71.4 (c1, methanol); 1H NMR (CDCl3)

M. Pallavicini et al. / Bioorg. Med. Chem. Lett. 19 (2009) 854–859
859
1
1.72–1.99 (m, 4H), 2.28 (s, 1H), 2.93–3.02 (m, 2H), 3.26–3.33 (m, 1H), 3.99 (dt,
50. (2R,20R)-2: H NMR identical to the enantiomer. (2R,20R)-2 Hydrochloride: Mp
J = 7.2, 2.2 Hz, 1H), 4.05 (dd, J = 11.0, 7.2 Hz, 1H), 4.34 (dd, J = 11.0, 2.2 Hz, 1H),
145–146 °C; ½a 2D5
ꢂ
+114.8 (c2, EtOH); 1H NMR identical to the enantiomer.
6.79–6.89 (m, 4H). (2S,20S)-2a Hydrochloride: Mp 149–150 °C; ½a 2D5
ꢂ
ꢀ83.3 (c2,
51. (2S,20R)-2a: H NMR identical to the enantiomer. (2S,20R)-2a Hydrochloride:
1
EtOH); 1H NMR (DMSO-d6) d 1.80–2.07 (m, 4H), 3.18 (br s, 2H), 3.70–3.82 (m,
1H), 4.02 (dd, J = 11.6, 7.4 Hz, 1H), 4.46 (dd, J = 11.6, 2.2 Hz, 1H), 4.52 (dt,
J = 7.4, 2.2 Hz, 1H), 6.82–6.91 (m, 4H), 9.46 (br s, 1H), 10.10 (br s, 1H).
Mp 154–155 °C; ½a D25
ꢂ
ꢀ67.6 (c2, EtOH); 1H NMR identical to the enantiomer.
1
52. (2R,20R)-2a: H NMR identical to the enantiomer. (2R,20R)-2a Hydrochloride:
Mp 150–151 °C; ½a D25
ꢂ
+86.5 (c2, EtOH); 1H NMR identical to the enantiomer.
44. (R)-9: ½a 2D5
ꢂ
+95.8 (c1, MeOH); ee 100.0% (by HPLC on a Chiralcel OJ column; 9:1
53. Carbonelle, E.; Sparatore, F.; Canu-Boido, C.; Salvano, C.; Baldani-Guerra, B.;
Terstappen, G.; Zwart, R.; Vijverberg, H.; Clementi, F.; Gotti, C. Eur. J. Pharmacol.
2003, 471, 85.
hexane/isopropanol, 0.6 ml/min at 276 nm; tR 12.11 min); 1H NMR identical to
the enantiomer.
45. (1R,20R)-10: ½a 2D5
ꢂ
+13.4 (c1, chloroform); de 99.0% (by HPLC on a LiChroCART
m; 65:35 hexane/ethyl acetate, 2 ml/min at
54. Abelson, K. S. P.; Höglund, A. U. Basic Clin. Pharmacol. Toxicol. 2004, 94, 153.
55. Diop, L.; Dausse, J. P.; Meyer, P. J. Neurochem. 1983, 41, 710.
56. Uhlén, S.; Dambrova, M.; Näsman, J.; Schiöth, H. B.; Gu, Y.; Wikberg-Matsson,
A.; Wikberg, J. E. S. Eur. J. Pharmacol. 1998, 343, 93.
57. All the ligands were built by VEGA in their protonated form and the
conformational space was explored by systematically rotating the unique
rotatable bond connecting the two rings. The docking and scoring procedures
involved extensive rigid-body sampling with the OpenEye Scientific Software
LiChrospher Si 60 column, 5
l
276 nm; tR 5.95 min); ee 98.7% (by HPLC on a Chiralcel OD-H column; 9:1
hexane/isopropanol, 0.5 ml/min at 276 nm; tR 11.20 min); 1H NMR identical to
the enantiomer.
46. (1S,20R)-10: ½a 2D5
ꢂ
+49.0 (c1, chloroform); de 100.0% (by HPLC on a LiChroCART
m; 65:35 hexane/ethyl acetate, 2 ml/min at
LiChrospher Si 60 column, 5
l
276 nm; tR 4.74 min); ee 100.0% (by HPLC on a Chiralcel OD-H column; 9:1
hexane/isopropanol, 0.5 ml/min at 276 nm; tR 13.89 min); 1H NMR identical to
the enantiomer.
package FRED, using the rat
a4b2 nicotinic model (PDB Id 1OLE). The FRED-
based sampling was performed in box defined by known residues implicated in
ligand recognition (namely, Trp55, Lys77, Trp147, and Tyr188). The docking
results were scored using the ChemGauss3 function (as compiled in Table 2)
which accounts for interactions, steric fitting and desolvation.
47. (2S,20R)-11: ½a 2D5
ꢂ
+43.3 (c1, chloroform); 1H NMR identical to the enantiomer.
+157.0 (c1, chloroform); 1H NMR identical to the enantiomer.
48. (2R,20R)-11: ½a 2D5
ꢂ
49. (2S,20R)-2: 1H NMR spectrum as described in Ref. 11. (2S,20R)-2 Hydrochloride:
Mp and 1H NMR spectrum as described in Ref. 11; ½a 2D5
ꢂ
ꢀ35.5 (c2, EtOH).
58. Kanne, D. B.; Abood, L. G. J. Med. Chem. 1988, 31, 2227.