Unichiral 2-(2′-pyrrolidinyl)-1,4-benzodioxanes: The 2 R,2′ S diastereomer of the N -methyl-7-hydroxy analogue is a potent α4β2- and α6β2-nicotinic acetylcholine receptor partial agonist

Article abstract of DOI:10.1021/jm200937t

A series of unichiral 7-substituted 2-(1′-methyl-2′- pyrrolidinyl)-1,4-benzodioxanes were synthesized and tested for the affinity for the α4β2 and α7 central nicotinic receptors; the 2R,2′S diastereomer of the 7-OH analogue [(R,S)-7], unique in the series, has a high α4β2 affinity (12nM K_i). N-Demethylation and configuration inversion of the stereocenters greatly weaken its α4β2 affinity, confirming that such a rigid molecule can be considered a new template for α4β2 ligands. Docking analysis showed how (R,S)-7 is capable of strongly and specifically interacting with the amino acidic counterpart of the α4β2 receptor binding site. Further pharmacological characterization demonstrated that (R,S)-7 also has a high affinity for the α6β2 receptor, and in vitro functional tests indicated that it is a potent α4β2 and α6β2 partial agonist, with modest affinity and potency for the α3β4 receptor. Comparison with varenicline, a well-known nicotinic partial agonist used as a smoking cessation aid, interestingly reveals similar nicotinoid profiles.

Full text of DOI:10.1021/jm200937t

ARTICLE
pubs.acs.org/jmc
Unichiral 2-(20-Pyrrolidinyl)-1,4-benzodioxanes: the 2R,20S
Diastereomer of the N-Methyl-7-hydroxy Analogue Is a Potent α4β2-
and α6β2-Nicotinic Acetylcholine Receptor Partial Agonist
Cristiano Bolchi,† Cecilia Gotti,‡ Matteo Binda,† Laura Fumagalli,† Luca Pucci,‡ Francesco Pistillo,§
Giulio Vistoli,† Ermanno Valoti,† and Marco Pallavicini*,†
†Dipartimento di Scienze Farmaceutiche “Pietro Pratesi”, Universitꢀa degli Studi di Milano, via Mangiagalli 25, I-20133 Milano, Italia
‡CNR, Istituto di Neuroscienze, via Vanvitelli 32, I-20129 Milano Italia
§Neuromed IRCCS, via Atinese 18, I-86077 Isernia, Italia
S Supporting Information
b
ABSTRACT: A series of unichiral 7-substituted 2-(10-methyl-20-
pyrrolidinyl)-1,4-benzodioxanes were synthesized and tested for
the affinity for the α4β2 and α7 central nicotinic receptors; the
2R,20S diastereomer of the 7-OH analogue [(R,S)-7], unique in
the series, has a high α4β2 affinity (12nM Ki). N-Demethylation
and configuration inversion of the stereocenters greatly weaken its
α4β2 affinity, confirming that such a rigid molecule can be
considered a new template for α4β2 ligands. Docking analysis
showed how (R,S)-7 is capable of strongly and specifically inter-
acting with the amino acidic counterpart of the α4β2 receptor binding site. Further pharmacological characterization demonstrated
that (R,S)-7 also has a high affinity for the α6β2 receptor, and in vitro functional tests indicated that it is a potent α4β2 and α6β2
partial agonist, with modest affinity and potency for the α3β4 receptor. Comparison with varenicline, a well-known nicotinic partial
agonist used as a smoking cessation aid, interestingly reveals similar nicotinoid profiles.
’ INTRODUCTION
selective ligands. On the other hand, interaction of HBA/π
with a less conserved and more heterogeneous subpocket,
involving, in the case of α4β2 subtype, polar residues of β2
subunit, could be exploited to selectively promote α4β2 vs α7
affinity.8,14 Recently, we have reported the moderate and
selective α4β2 affinity of one of the four stereoisomers of
2-(1-methyl-2-pyrrolidinyl)-1,4-benzodioxane (1), designed
as rigidified analogues of prolinol phenyl ether and conceived
as potential templates and scaffolds for nicotinic ligands
(Chart 1).15
The rigidity of 1 and the significant variations in affinity
depending on the methylation of nitrogen and on the config-
uration of the two stereocenters have allowed to well support
SAR with docking analysis.15 The results, showing the binding
potential of the benzodioxane as a HBA/π group, prompted
us to investigate if the introduction of substituents, in parti-
cular at its 7-position, can positively affect the α4β2/α7
affinity profile. Here, we report the synthesis, the pharmaco-
logical evaluation, and the SAR analysis of the unichiral
7-substituted 2-(2-pyrrolidinyl)benzodioanes 2ꢀ9 constructed as
new α4β2 nAChR ligands (Chart 2).
The key role played by α4β2 and/or α7 neuronal nicotinic
acetylcholine receptors (nAChRs) in a wide range of CNS
functions, such as learning, memory, attention, information
processing, analgesia, and nicotine addiction, makes the agonists
at these receptors good candidates for the treatment of several
CNS disorders, including cognitive dysfunction and neurode-
generative conditions, pain, and nicotine dependence.1ꢀ3 Potent
and selective α4β2 and α7 nAChR agonists are of great interest
to drug research, and recent studies have intensively focused on
the structural differences which determine the relative selectivity
for these two major brain subtypes.4,5 Representatives of the
known α4β2- or α7-ligands or newly designed nicotinoids have
been submitted to batteries of assays to assess the subtype affinity
and activity, while SAR analysis and modeling studies have
tentatively rationalized the difference in subtype activation and
subtype binding modes.4ꢀ10
Protonated or quaternarized amine and a hydrogen bond
acceptor and π-electron rich group (HBA/π) have been
established as the two key elements of the nicotinic agonist
pharmacophore.11ꢀ13 According to current models of nico-
tinic receptors and of their binding modes, the charged moiety
of the ligand would interact with the side chains of tyrosine
and tryptophan residues well-conserved across nAChR sub-
types, thus not providing much basis for designing subtype-
Received: July 14, 2011
Published: September 25, 2011
r
2011 American Chemical Society
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’ CHEMISTRY
The resultant (R,S) and (S,S) p-hydroxymethylphenyl ethers 24
were tritylated at the benzylic OH-group, separated, and cyclized
to 7-trityloxymethylbenzodioxanes (S,S)- and (R,S)-26. The final
treatment with lithium aluminum hydride, followed by acidic
hydrolysis, caused Boc reduction to methyl and detritylation,
providing (S,S)- and (R,S)-3. The (R,S)/(S,S) diastereomeric
mixture of the secondary alcohol 18, treated with hydrogen,
underwent debenzylation, N-deprotection and, after adding
formaldehyde, reductive N-methylation. The successive separa-
tion of diastereomeric p-acetylphenyl ethers 27 and the Mitsu-
nobu intramolecular cyclization gave (S,S)- and (R,S)-4.
Scheme 3 shows the routes from the resolved diastereomeric
alcohols 19ꢀ21 to the compounds 5ꢀ9. The intermediates (R,S)-
and (S,S)-19 were treated with trifluoroacetic acid to remove the
MEM protection and cyclized to (S,S)- and (R,S)-5, while (R,S)-
and (S,S)-20 were debenzylated, cyclized, and reduced to (S,S)- and
(R,S)-6. Mesylation of the secondary hydroxyl group of the o,p-
dibenzyloxyphenyl ethers (R,S)- and (S,S)-21, removal of both
benzyl protections, and nucleophilic intramolecular displacement of
mesylate afforded (S,S)- and (R,S)-33, the common intermediates
to the (S,S) and (R,S) diastereomers of 7, 7a, 8, and 9. In fact,
N-deprotection gave (S,S)-and(R,S)-7a,whichwereN-methylated to
yield (S,S)- and (R,S)-7, while O-methylation and O-isopropylation,
followed by Boc reduction, provided (S,S)- and (R,S)-8 and (S,S)-
and (R,S)-9, respectively.
All the stereoisomers of the compounds 7 and 7a and only two
diastereomers, epimers at the dioxane stereocenter, of the
compounds 2ꢀ6 and 8ꢀ9 were prepared. We applied the
synthetic strategy previously reported for 1 and 1a,15 with the
modifications made necessary by the susbstituent at the 7-posi-
tion of benzodioxane. N-Protected (S)- and (R)-2-bromoacetyl-
pyrrolidine were reacted with the suitable o,p-disubstituted
sodium phenate, prepared according to literature,16ꢀ18 or by
original methods, having OBn or OMEM at the ortho position
and the para-substituent identical to that of the target benzo-
dioxane (Br, Ph, Ac) or easily convertible into it (CHO, OBn).
The enantiomeric excess of the resultant S and R phenoxyacetyl
pyrrolidines 12ꢀ16 was determined by chiral HPLC and found
to be always more than 97%. The carbonyl reduction of the S
isomers of 12ꢀ16 with sodium borohydride or lithium alumi-
num hydride in THF afforded the (R,S)/(S,S) diastereomeric
mixture of the secondary alcohols 17ꢀ21 (Scheme 1). All of
these mixture were resolved by chromatography except for 17
and 18.
Scheme 2 reports the successive transformations of 17 and 18
leading to the compounds 2ꢀ4. Overnight hydrogenation of (R,S)/
(S,S)-17 yielded (R,S)- and (S,S)-22, which were separated
by chromatography, intramolecularly cyclized to N-Boc pro-
tected 2-pyrrolidinyl-7-methylbenzodioxanes (S,S)- and (R,S)-
23 by Mitsunobu reaction, and finally reduced to the correspond-
ing N-methyl analogues (S,S)- and (R,S)-2. Otherwise, a short
hydrogenation of (R,S)/(S,S)-17 produced only debenzylation.
Starting from the (S,R)/(R,R) diastereomeric mixture of 21,
obtained by reduction of (R)-16, the same synthetic strategy
illustrated for (S,S)- and (R,S)-7 and for (S,S)- and (R,S)-7a, gave
(R,R)- and (S,R)-7 and the corresponding normethyl analogues
(R,R)- and (S,R)-7a.
Chart 1. Design of Conformationally Restricted Prolinol
Phenyl Ethers
’ BIOLOGY
Binding Studies. The synthesized compounds were tested in
vitro on rat cerebral cortex to evaluate their affinity for the central
nicotinic receptors; [3H]-epibatidine and [125I]-αBungarotoxin
were used as radioligand to label α4β2 and α7 receptors, respec-
tively. Nicotine was included in the series for comparison. The
results are listed in Table 1, together with those previously reported
for the stereoisomers of 1 and 1a.15 The compounds with the
highest α4β2 affinity were (R,S)-7 (0.012 μM Ki) and (S,S)-7
(0.421 μM Ki), and their affinity for the α7 subtype was respectively
35-fold (0.427 μM Ki) and 47-fold (20 μM Ki) lower than that for
the α4β2 subtype. Both compounds were also tested for their
affinity for the human α3β4 subtype: (R,S)-7 showed 26-fold lower
affinity (0.310 μM Ki) than that determined for the α4β2 subtype,
whereas (S,S)-7 had very similar affinity for the α3β4 (0.698 μM Ki)
and α4β2 subtype (0.421 μMKi).As(R,S)-7had the greatest affinity
and selectivity for the α4β2 subtype, we also studied its affinity for the
Chart 2. Target 7-Substituted 2-(2-
Pyrrolidinyl)benzodioxanes
Scheme 1. Synthesis of the R,S and S,S Stereoisomers of the Intermediates 17ꢀ21a
a Reagents and conditions: (a) NaBH4 or LiAlH4, THF, rt.
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Scheme 2. Synthesis of the R,S and S,S Stereoisomers of Compounds 2ꢀ4a
a Reagents and conditions: (a) H2, Pd/C, MeOH, rt, 24 h; (b) (Ph)3P, DIAD, THF, 100 °C, 10 min, MW; (c) LiAlH4, THF, reflux, 3 h; (d) H2, Pd/C,
MeOH, rt, 1 h; (e) TrCl, TEA, DME, reflux 16 h; (f) LiAlH4, THF, reflux, 2 h then HCl 1M, MeOH, rt, 3 h; (g) H2, Pd/C, CH2O, MeOH, rt, 16 h;
(h) Ph3P, reflux, or (CH3)3P, rt, DEAD, THF, 16 h.
Scheme 3. Synthesis of the R,S and S,S Stereoisomers of Compounds 5ꢀ9a
a Reagents and conditions: (a) CF3COOH, DCM, rt, 30 min; (b) (CH3)3P, DEAD, THF, rt, 24 h; (c) H2, Pd/C, MeOH, rt, 48 h; (d) (CH3)3P,
DEAD, THF, 100 °C, 10 min, MW; (e) LiAlH4, THF, reflux, 1ꢀ2 h; (f) MsCl, TEA, DCM, rt, 5 h; (g) H2 5 atm, Pd/C, MeOH, rt, 16 h; (h) K2CO3,
DME reflux 6 h, (i) CF3COOH, DCM, rt, 2 h; (j) H2 Pd/C, CH2O, MeOH, rt, 16 h; (k) DMC, DBU, 90 °C, 16 h; (l) NaH, i-propyl bromide,
DME, 60 °C, 16 h.
native immunoimmobilized α6β2* subtype, which was 0.013 μM Ki,
and almost identical to that for the α4β2 subtype (Figure 1).
In Vitro Functional Activity at nAChR. The affinity measure-
ments indicate that (R,S)-7 has a high affinity for α4β2 and
α6β2* receptors but give no indication of its possible functional
activity. To study the functional response of (R,S)-7, we tested its
ability to induce [3H]DA release from striatal slices using an in
vitro 96-well plate filter assay19 and compared its functional
profile with that of the full agonist nicotine.
induced [3H]DA release from striatal slices in a dose-dependent
manner and with similar potency, but the intrinsic agonist efficacy of
(R,S)-7 was only 51% of that of nicotine. The α4β2 and α6β2
subtypes each contribute 50% of the nicotine-induced striatal
[3H]DA release20 and, as the binding studies showed that (R,S)-7
also binds the native α6β2* subtype, we quantified its effect on
[3H]DA release mediated by the α6β2* subtype. To this end,
striatal slices were incubated with 100 nM αconotoxin MII (α-MII),
a specific antagonist of the α6β2* subtype. Figure 2 shows that the
presence of α-MII led to a further decrease in the ability of (R,S)-7
to induce [3H]DA release with almost no change in the potency
As shown in Figure 2, both nicotine (EC50 67 nM; pEC50 ( SE,
7.17 ( 0.04) and (R,S)-7 (EC50 82 nM; pEC50 ( SE, 7.08 ( 0.07)
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Table 1. Nicotine and Compounds 1ꢀ9: Affinity for Native Receptor Subtypes, Present in Rat Brain Membranes, Labeled by
[3H]-Epibatidine, and [125I]-αBungarotoxin, and Docking Scores (kcal/mol) in a α4β2 Nicotinic Receptor Modela
α4β2 nAChR [3H]-Epi
Ki (μM)
α7 nAChR [125I]-αBgtx
Ki (μM)
energy
α4β2 nAChR
[3H]-Epi Ki (μM)
α7 nAChR
125I]-αBgtx Ki (μM)
energy
(kcal/mol)
[
(kcal/mol)
nicotine
(R,S)-1
(S,S)-1
(S,R)-1
(R,R)-1
(R,S)-1a
(S,S)-1a
(S,R)-1a
(R,R)-1a
(R,S)-2
(S,S)-2
(R,S)-3
(S,S)-3
(R,S)-4
(S,S)-4
(R,S)-5
(S,S)-5
0.004 (18)
0.26 (32)
0.47 (30)
12.5 (17)
43.8 (16)
2.1 (30)
14.4 (32)
88 (18)
0.234 (29)
21 (44)
14.6 (54)
35 (45)
14.8 (35)
36 (41)
32.2 (45)
14.7 (42)
27 (38)
126 (50)
64 (43)
60 (79)
12 (41)
110 (51)
30 (50)
22 (42)
22 (43)
ꢀ30.21
ꢀ28.45
(R,S)-6
(S,S)-6
(R,S)-7
(S,S)-7
(S,R)-7
(R,R)-7
(R,S)-7a
(S,S)-7a
(S,R)-7a
(R,R)-7a
(R,S)-8
(S,S)-8
(R,S)-9
(S,S)-9
35 (20)
3.1 (23)
0.012 (13)
0.421 (30)
3.5 (9)
36 (45)
51 (45)
0.427 (34)
20 (36)
4.5 (72)
1.3 (63)
33 (34)
53 (36)
35 (70)
3 (71)
ꢀ24.97
ꢀ28.05
ꢀ40.86
ꢀ30.43
ꢀ17.75
ꢀ21.67
ꢀ27.78
ꢀ25.00
ꢀ16.83
ꢀ19.42
ꢀ24.43
ꢀ27.56
ꢀ24.71
ꢀ27.79
3 (7)
9 (26)
11.6 (17)
42 (19)
1.2 (20)
18 (24)
1.3 (9)
ꢀ24.01
ꢀ27.99
ꢀ24.41
ꢀ27.05
ꢀ25.24
ꢀ28.91
ꢀ23.21
ꢀ28.91
5.7 (21)
51 (19)
17 (12)
4.2 (9)
32 (38)
26 (43)
6.1 (32)
43 (42)
9.5 (19)
97 (21)
9.3 (14)
3.2 (10)
11.7 (10)
8.1 (10)
3 (10)
Kd (nM)
0.036 (22)
0.3 (35)
a The Kd and Ki values were derived from [3H]-epibatidine and [125I]-αBungarotoxin saturation and two competition binding experiments on rat brain
membranes as described in ref 23. The curves were fitted using a nonlinear least-squares analysis program and the F test. The numbers in brackets
represent the %CV. The affinities of compounds 1 and 1a are those previously reported in ref 15.
Figure 2. Concentrationꢀresponse curves of agonist-induced [3H]DA
release from rat striatal slices. The slices were preloaded with 100 nM
[3H]DA and exposed to the indicated concentration of nicotine, (R,S)-7
Figure 1. Inhibition of [3H]-epibatidine binding to the α4β2, α6β2*,
alone, or in the presence of 100 nM αMII. Results were normalized and
and α3β4 subtypes and of [125I]-αBungarotoxin binding to the α7
expressed as a percentage of 10 μM nicotine-induced release. The data
subtype by increasing concentration of (R,S)-7. (R,S)-7 was preincu-
points represent the mean SEM of 3ꢀ4 separate experiments performed
bated with the indicated subtypes for 5 min, and then [3H]-epibatidine
100 pM (for α4β2 and α6β2*) or 250 pM (α3β4) or 1 nM [125I]-
αBungarotoxin (α7) was added and left overnight at 4 °C. The Ki values
were determined by fitting three separate experiments using LIGAND
program.14 In each experiment, each dilution of the drug was tested in
triplicate. All of the values are expressed in relation to [3H]-epibatidine-
specific binding to α4β2, α6β2*, and α3β4 receptors and [125I]-
αBungarotoxin to α7 receptors (considered as 100%). The curves were
generated using Prism 4.0 (GraphPad, San Diego, CA).
in triplicate.
published data,22 found that it is a partial agonist with an intrinsic
agonist efficacy that is 49% of that of 10 μM nicotine and an EC50 of
46 nM, which is very similar to that of (R,S)-7. To study the
functional effect of (R,S)-7 on the α3β4 subtype, we tested its
ability to induce [3H]NA release, which is mediated by the α3β4
subtype in rat. We found that (R,S)-7 is able to induce [3H]NA
release with an EC50 of 17.6 μM (pEC50 ( SE, 4.75 ( 0.09) and an
efficacy similar to that of 100 μM nicotine. In line with the finding of
binding studies showing low affinity for the α3β4 subtype, this
functional assay showed that (R,S)-7 is less potent on this subtype
than on β2-containing receptors but similarly efficacious as nicotine,
thus acting as a full agonist.
(EC50 of 72 nM; pEC50 ( SE, 7.14 ( 0.11). These results indicate
that (R,S)-7 induces [3H]DA release by acting as a partial agonist on
both α4β2 and α6β2* subtypes. As a control, we also tested the
effect of varenicline, a cytisine derivative recently introduced in the
treatment of smoking cessation,21 and, in agreement with previously
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can contribute to ligand potency only, while the β2 serine
residues can concur to ligand selectivity, confirming that, as a
rule, selectivity can be increased by reinforcing the contacts with
the less conserved regions of β2 subunit.
The narrowness of the subpocket harboring the hydroxyl
function prevents a suitable pose of the other selected substitu-
ents. As an example, Figure S2 (Supporting Information) reports
the best complex obtained for (R,S)-4. Although the carbonyl of
the acetyl substituent of (R,S)-4 seems to conserve a weak
contact with Ser111, the methyl of the same substituent clashes
against the side chains of the mentioned serine residues, thus
inducing a detrimental distortion of the entire binding site and
preventing the key polar contacts between ligand and receptor as
described for (R,S)-7.
Figure S2 shows what happens when the benzodioxane con-
figuration of (R,S)-4 is inverted. All the contacts described for
(S,S)-7 are conserved by (S,S)-4, while the acetyl substituent is
inserted into a less stringent region, which roughly corresponds
to the entrance of the binding cavity. Here, the acetyl does not
elicit significant interactions and at least conserves a weak
H-bond with Ser111.
Taken together, docking results confirm that the subpocket
lined by the two serine residues can be contacted only by very
small and polar 7-substituents able to both accept and donate
H-bonds and born by benzodioxane in R configuration. Con-
versely, the adjacent region, in which the 7-substituents of the S,S
isomers are harbored, is surely wider and more tolerant but
unable to stabilize strong interactions with the ligand.
Figure 3. Main polar contacts stabilizing the putative complexes
between α4β2 nAChR binding site and (R,S)-7 (upper panel) and
(S,S)-7 (lower panel).
’ DOCKING
Docking results on the other two stereoisomers of the hydro-
xyl derivative, (S,R)-7 and (R,R)-7, as well as on the four
stereoisomers of nor-methyl 7a, emphasize the critical role of
an optimal arrangement of protonated pyrrolidine to elicit the
required H-bond with Trp147(α4). Indeed, (S,R)-7 and (R,R)-7
unfavorably contact Trp147(α4) with the N-methyl group, while
a repulsive interaction of one of the two nitrogen protons of 7a
stereoisomers with Lys77 cannot be avoided. Such a detrimental
approach, exacerbated by the rigidity of these molecules, is
shown also by the N-methyl derivatives, when the protonated
nitrogen is in R configuration, but it is bypassed thanks to the
possibility of inverting the nitrogen chirality.
Docking scores, as computed by an electrostatic interaction
energy with distance-dependent dielectric constant and com-
piled in Table 1, are in line with the above observation: (a) (R,S)-
7 shows the largely best score, (b) (R,S)-1 has an intermediate
score, (c) the R,S isomers of the other 7-substituted derivatives
display energy scores markedly worse than unsubstituted (R,S)-
1, (d) all the 7-substituted S,S isomers, irrespective of the
substituent, report very similar scores, which are in turn similar
to that of unsubstituted (S,S)-1, (e) the score differences
between the four isomers of 7 are remarkable, and (f) the
docking energies of all the 7a are scarce independently on the
configurations. Finally, docking energies address the impor-
tance of protonation induced chirality because the two most
affinitive (R,S)-7 and (S,S)-7 show quite poor scores (ꢀ21.87
and ꢀ18.45 kcal/mol, respectively) when considered with the
charged nitrogen in R configuration.
As detailed under Computational Methods, the proposed
nicotinoids were investigated by a docking analysis with the
available rat α4β2 model (PDB ID: 1OLE),24 the chiral proto-
nated tertiary nitrogen of pyrrolidine being in S configuration.
The Figure 3 compares the best obtained complexes for (R,S)-7
and (S,S)-7. The protonated nitrogen atom of both stereoi-
somers stabilizes a reinforced H-bond with the carbonyl group of
Trp147(α4), while the carbon atoms of the pyrrolidine ring
contact some apolar residues not displayed for clarity [e.g.,
Val109(β2), Phe117(β2), Leu119(β2), and Tyr195(α4)]. Si-
milarly to what already observed for unsubstituted compounds
(R,S)-1 and (S,S)-1, the 7-hydroxy derivatives (R,S)-7 and (S,S)-
7 differently arrange their benzodioxane ring because the pivotal
H-bonds with Lys77(β2) and Tyr195(α4) are elicited by O(1)
in (S,S)-7 and by O(4) in (R,S)-7, thus resulting in a beneficially
greater distance between mutually repulsive Lys77 and pyrroli-
dine nitrogen in the latter isomer. In addition, the (R,S)-7 isomer
is able to insert the hydroxyl function in a very narrow subpocket
where it can elicit two additional H-bonds with the side chain of
Ser111(β2) and Ser115(β2). Conversely, the less affinitive (S,S)-
7 isomer arranges the hydroxyl function in a different region
quite distant from the mentioned serine residues, where it cannot
stabilize significant polar interactions.
The sequence alignment, as computed for rat α4, α3, α6,
α7, β2, and β4 subunits and reported in Figure S1 (Supporting
Information), shows that the main residues interacting with the
charged pyrrolidine and the benzodioxane oxygen atom (i.e.,
Trp147 and Tyr195) belong to highly conserved regions, while
Ser111 and Ser115 belong to a very variable region. In particular,
none of the aligned sequences conserves serine residues corre-
sponding to Ser111 and Ser115 of β2 subunit apart from β4,
which shows a conserved serine residue corresponding to Ser115
(i.e., Ser119 in β4 subunit). This suggests that the first residues
’ DISCUSSION
The α4β2 affinity and selectivity of (R,S)-1 assimilate benzo-
dioxane to other bicycles, which show analogous nicotinic profiles
when bearing 2-pyrrolidinyl, such as quinoline,6 furopyridine,25 and
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chroman.26 In the case of 1,4-benzodioxan-2-yl group, the presence
of the stereocenter and the interaction potential of both oxygen
atoms as HBAs are additional features, whose combined influence
has to be considered. For the unsubstituted N-methylpyrrolidinyl-
benzodioxane 1, we had previously argumented that the presence of
the two dioxane oxigens seems to obscure the role of dioxane
chirality. Indeed, the inversion of dioxane C(2) of (R,S)-1, the most
α4β2 affinitive stereoisomer of 1, having R configuration at the
dioxane stereocenter and S at the pyrrolidine C(2), slightly affects
the α4β2 affinity (cf. 0.26 μM Ki of (R,S)-1 and 0.47 μM Ki of
(S,S)-1), while inversion of pyrrolidine configuration results in
a 170-fold decrease (cf. 0.26 μM Ki of (R,S)-1 and 43.8 μM Ki of
(R,R)-1). Consistently with these data, docking analysis had shown
different, but ever productive accommodations for the dioxane ring
in both the configurations thanks to the capability of interacting with
the same amino acids through O(4) or, in alternative, through O(1),
whereas pyrrolidine ring can be correctly arranged in the binding
pocket only when its C(2) has S configuration.15
These results naturally proposed pyrrolidinylbenzodioxane for
the same investigations on the effect of substituents at the aromatic
ring on the nicotinic affinity already made for the phenyl and pyridyl
ethers of prolinol, which are well-known nicotinoids.27ꢀ29 Rigidity
of the whole molecule, on the one hand, and chirality of the HBA/π
group and presence of two potential HBAs in it, on the other hand,
made such an investigation particularly attractive. The same sub-
stituent would differently affect affinity in dependence on these
factors, increasing the chance of selecting substituted derivatives
which optimally fit into the receptor binding site. Moreover, any
ameliorating substitution would be highly informative for the
binding site structure and pharmacophore modeling. However,
consideration that four stereoisomers of a differently substituted
2-(2-phenoxy-1-hydroxyethyl)pyrrolidyne had to be synthesized for
each selected substituent or substitution pattern forced us to limit
the study to the diastereomeric pairs with S configuration at the
pyrrolidine stereocenter and to monosubstitution, in particular at
the benzodioxane C(7). The former choice was suggested by the
sensibly higher α4β2 affinities of such diastereoisomers in the case
of the unsubstituted lead 1. The latter one was inspired by the SAR
studies on the phenyl and pyridyl ethers of prolinol, indicating, on
the basis of wide and systematic investigations, the substitutions at
the meta-position to the methyleneoxy linker as the most productive
ones.28,29 Our docking analyses15 on (R,S)-1 had previously showed
the proximity of benzodioxane C(7), corresponding to the above
meta-carbon of pyridyl and phenyl ethers, to the hydroxyls of two
serine residues (Ser111 and Ser 115), belonging, moreover, to β2
subunit, which affords the minus side of the binding site. The
presence of these two functions indicated OH, a both HBA and
HBD group, as the first-choice substituent, although, quite surpris-
ingly, the reported long series of phenyl- and pyridine-modified
analogues of phenyl and pyridyl ethers of prolinol had never
enclosed any hydroxyphenyl or hydroxypyridyl ether. The increase
of α4β2 affinity from the 0.26 μM Ki value of unsubstituted (R,S)-1
to 12 nM for the 7-OH substituted analogue (R,S)-7 was consistent
with the working hypothesis. The choice of the successive substit-
uents, namely hydroxymethyl, methyloxy, isopropyloxy, acetyl, and
methyl, was mainly aimed at verifying the importance of the
postulated interactions between the benzodioxane OH and the
two serine OH. Suppression of both HBA and HBD properties of
the substituent (CH3) as well as of only its HBD character
(COCH3, OCH3, and OCH(CH3)2) with no or modest increase
of its volume resulted in dramatically lowered affinities (see
10ꢀ100 μM Ki of (R,S)-2, (R,S)-4, (R,S)-8, and (R,S)-9). Also
the simple homologation to CH2OH was highly deleterious (see
51 μM Ki of (R,S)-3). After that, two substituents substantially
different from OH, namely bromo and phenyl, were considered, but
these substitutions were pejorative as well. Halo substitution, which
is reported to enhance the affinity of prolinol phenyl-ethers29 and
not to affect that of prolinol 3-pyridyl ethers,28 when accomplished
in 3 and in 5, respectively, resulted in a 30-fold weaker affinity of
the bromobenzodioxane derivative (cf. 8.1 μM Ki of (R,S)-5 and
0.26 μM Ki of (R,S)-1). The 7-phenyl analogue (R,S)-6 had 130-
fold lower affinity compared to (R,S)-1.
Interestingly, the inversion of the configuration of the benzo-
dioxane stereocenter produced opposite effects on the 7-hydroxy
analogue compared to all the other 7-substituted analogues.
Compound (S,S)-7 possesses 35-fold weaker affinity than
(R,S)-7 and behaves as OH had no influence on the interac-
tion, because its moderate submicromolar α4β2 affinity is
equal to that of unsubstituted (S,S)-1. On the contrary, all the
S,S stereoisomers of the other substituted analogues show
modest 1ꢀ10 μM α4β2 affinities, which are however from
3- to 11-fold higher than the corresponding R,S stereoisomers.
Furthermore, the substitutions at C(7) seem to be better
tolerated than in the case of the R,S stereoisomers because
their α4β2 affinities are not so far from that of unsubstituted
(S,S)-1 as those of the R,S stereoisomers from (R,S)-1. Dock-
ing analysis well supports these results, showing how the
substituent at C(7) of benzodioxane can be profitably settled
in a narrow subpocket of the binding site only if it is small and
capable of strongly and specifically interacting with the amino
acidic counterpart and if the bicycle has R configuration at
C(2). This is the case, unique in the R,S series, of the OH of
(R,S)-7 interacting with the two β2 serine residues. When, on
the contrary, benzodioxane is in S configuration, different
substituents can be accepted. However, the absence of tight
interactions between them and the counterpart makes their
contribution negligible (OH) or slightly negative (the other
substituents), attenuating the diastereopreference. This latter
is always for the S,S isomer, with the obvious exception of 7
due to the mentioned interactions proper only to the hydroxyl
of its R,S isomer.
These observations prompted us to closely examine compound
7. We decided to characterize all its four stereoisomers and, in
addition, the four stereoisomers of its nor-methyl analogue 7a.
In line with compounds 1 and 1a, the modest α4β2 affinities of
(S,R)-7 and (R,R)-7 and of all the four stereoisomers of 7a confirm
that the ligand recognition is conditioned by the S configuration of
the pyrrolidine stereocenter and by the presence of only one proton
at positively charged nitrogen. These results, which can be satisfac-
torily rationalized with the aid of docking analysis, as discussed in
detail in the previous paragraph, stress those obtained for com-
pounds 1 and 1a. Comparison of the α4β2 affinity ratios makes
evident that the 7-OH substitution significantly enhances the
eudismic index of the R,S/S,R pair (cf. 2.46 of 7 with 1.68 of 1),
the epimeric eudismic index of the R,S/S,S and R,S/R,R pairs
(cf. 1.55 and 2.40 of 7 with 0.26 and 2.23 of 1, respectively) and
the N-methyl/N-desmethyl affinity ratio (cf. 750 of (R,S)-7/(R,S)-
7a with 8 of (R,S)-1/(R,S)-1a). These unidirectional increases
unambiguously indicate that the introduction of the 7-OH sub-
stituent produces a tighter ligandꢀreceptor interaction with more
stringent steric demand.
All the tested pyrrolidinyl benzodioxanes have poor α7
affinity; none of them reaches submicromolar values of Ki except
for (R,S)-7, which however maintains a significant α4β2 versus
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ARTICLE
α7 selectivity, similar to that of nicotine and to those of (S,S)-7,
(S,S)-7a, (R,S)-1, and (S,S)-1, namely the compounds with the
highest α4β2 affinities after (R,S)-7. The other compounds
display both α4β2 and α7 modest affinities. These data indicate
pyrrolidinyl benzodioxanes, unsubstituted or 7-OH substituted,
as new α4β2 selective nicotinoids. The presence of Ser113 in the
α7 subunit, although not aligned to Ser111 and Ser115 of the β2
subunit (Figure S1, Supporting Information), might explain the
enhancement of α7 affinity from 21 μM Ki of unsubstituted
(R,S)-1 to 0.427 μM Ki of 7-OH substituted (R,S)-7 and,
although the α3β4 affinity of (R,S)-1 is unavailable as a reference
value, also the submicromolar α3β4 affinity of (R,S)-7 might be
advantaged by interactions of benzodioxane OH with conserved
Ser119, corresponding to Ser115 of β2 subunit (Figure S1,
Supporting Information).
The further investigations on the neuropharmacological pro-
file of (R,S)-7 show that this compound has high affinity for
native β2-containing receptors, both α4β2 and α6β2, and that
this affinity is much higher not only than that for α7 subtype but
also than that for α3β4 subtype. The α7 receptor is localized in
the central and peripheral nervous systems, where it is involved in
many important physiological functions, whereas the α3β4*
subtype is predominant in the autonomic ganglia and adrenal
medulla and its distribution in the CNS is very limited. In
autonomic ganglia, α3β4* receptors are located at presynaptic
terminals, where they facilitate spontaneous and evoked Ach
release, and at postsynaptic terminals, where they mediate
ganglionic fast synaptic transmission and control the function
of organs such as heart, intestine, and bladder. The low affinity of
(R,S)-7 is a promising characteristic, as it suggests that the
compound will not have undesirable side effects due the α3β4
receptors in the peripheral nervous system.
for optimal interaction as well as N methylation. The rigidity of
the pyrrolidinylbenzodioxane scaffold allows these results to be
satisfactorily rationalized by docking in a α4β2 receptor model.
Furthermore, the high affinity of (2R,20S)-7-hydroxy-2-(10-meth-
yl-20-pyrrolidinyl)-1,4-benzodioxane also for the α6β2* nicotinic
subtype suggests that the 7-hydroxy-benzodioxane substructure
is a HBA/π group capable of selectively promoting the interac-
tion with nicotinic β2-containing receptors. The potent α4β2
and α6β2 partial agonism, together with the modest affinity and
activity at the α3β4* subtype, are worthy features of the title
compound, which substantiate such a hypothesis.
’ EXPERIMENTAL SECTION
Chemistry. 1H NMR spectra were recorded operating at 300 MHz.
Chemical shifts are given in parts per million relative to residual solvent
(CHCl3 or DMSO) as internal standard. Optical rotations were
determined by a Jasco P-1010 polarimeter. HPLC analyses were
performed on a Kromasyl Amycoat column using Hewlett-Packard
1050 instrument. Thermal analyses were performed on 2ꢀ5 mg
samples in closed pans at 5 °C/min using DSC 2010 TA INSTRU-
MENTS. Elemental analyses (CHN) are within (0.40% of theoretical
values. Purifications were performed using KP-Sil 32ꢀ63 μm 60 Å
cartridges and Merck silica gel (particle size 40ꢀ63 μm). The results of
elemental analyses indicated that the purity of all tested compounds was
higher than 95%. 2-Benzyloxyphenates p-substituted with CHO and
OBn were prepared according to refs 17 and 18 and transformed into
sodium salts by treatment of their solution in methanol with 1 M
aqueous NaOH. The preparations of the sodium salts of 2-benzylox-
yphenols p-substituted with Ph and COCH3 are reported in the
Supporting Information as well as the preparation of the sodium
2-MEM-4-bromophenate from 2-hydroxy-4-bromoacetophenone, in
turn obtained from 3-bromophenol according to ref 16.
The results of these preliminary in vitro studies show that
(R,S)-7, compared with nicotine, has similar potency and sig-
nificantly less intrinsic activity on [3H]DA release and that this is
due to reduced intrinsic activity at both the α4β2 and α6β2*
subtypes. Given the importance of DA release in the mesolimbic
dopaminergic system in determining nicotine dependence, a
partial agonist of α4β2 and α6β2 subtypes may be useful in the
treatment of nicotine addiction. The pharmacological profile of
(R,S)-7 is very similar to that of varenicline, a currently marketed
smoking cessation aid,20 in terms of subtype specificity. (R,S)-7
has a lower affinity for β2-containing receptors than varenicline,
but it resulted in having the same intrinsic partial agonist activity
and the same potency as varenicline when tested for [3H]DA
release.22,30 The difference in the binding affinity of varenicline
and (R,S)-7 and their very similar functional potency may reflect
a difference in their ability to desensitize the β2-containing
receptors measured by binding studies.22,30 Like varenicline,
(R,S)-7 may facilitate the extinction of behaviors associated with
nicotine dependence by partially stimulating β2-containing
receptors in the absence of nicotine while preventing nicotine
from triggering a full response.
In each described preparation, the moles of reagents are given for one
mole of substrate.
General Procedure for Synthesis of Compounds 12ꢀ16.
The S and R isomers of target compounds 12, 15, and 16 were
synthesized combining N-Boc protected (S)- and (R)-2-bromoacetyl-
pyrrolidine with sodium 2-benzyloxyphenate p-substituted with CHO
(12), Ph (15) and OBn (16). The S and R isomers of target compounds
13 and 14 were synthesized by combining N-Cbz protected (S)- and
(R)-2-bromoacetylpyrrolidine with sodium 2-benzyloxy-4-acetylphe-
nate (13) and 2-(2-methoxyethoxymethoxy)-4-bromophenate (14).
All the reactions were carried out in acetone at room temperature using
exceeding bromoketone (1.1 mol) with the exception of 15 (0.8 mol of
bromoketone in DMF).
(S)-N-Boc-2-(o-benzyloxy-p-formylphenoxyacetyl)pyrrolidine [(S)-
12]. Obtained as an amorphous solid in 95% yield after chromatography
on silica gel (cyclohexane/EtOAc 1:1): [α]D25 = ꢀ35.1 (c 1, CHCl3),
99.35% ee (n-hexane/ethanol 8:2). 1H NMR (CDCl3) δ 9.85 (s,
0.33H), 9.82 (s, 0.67H), 7.52ꢀ7.27 (m, 7H), 6.98 (d, 0.66H, J = 8.25
Hz), 6.90 (d, 0.34H, J = 8.25 Hz), 5.18 (s, 2H), 4.98 (s, 1.35H), 4.81 (s,
0.65H), 4.60ꢀ4.55 (m, 1H), 3.38ꢀ3.56 (m, 2H), 2.18ꢀ1.77 (m, 4H),
1.47 (s, 5.4H), 1.41 (s, 3.6H).
(S)-N-Cbz-2-(o-benzyloxy-p-acetylphenoxyacetyl)pyrrolidine [(S)-
13]. Obtained as a white solid in 82% yield after chromatography on
silica gel (cyclohexane/EtOAc 1:1): mp 91.8 °C; [α]D25 = ꢀ21.5 (c 1,
’ CONCLUSIONS
1
CHCl3); 97.51% ee (n-hexane/ethanol 8:2). H NMR (CDCl3) δ
Introduction of OH substituent to C(7) of (2R,20S)-2-
(10-methyl-20-pyrrolidinyl)-1,4-benzodioxane increases α4β2 ni-
cotinic affinity from 0.26 to 0.012 μM Ki, maintaining the good
α4β2 vs α7 selectivity of the parent unsubstituted compound.
Other substituents at this position seem to be not tolerated and R
and S configuration of the two stereocenters are strictly required
7.61ꢀ7.52 (m, 2H), 7.47ꢀ7.26 (m, 10H), 6.86 (d, 0.6H, J = 8.53 Hz),
6.54 (d, 0.4H, J = 8.26 Hz), 5.18ꢀ5.01 (m, 4H), 4.90 (s, 1.25H),
4.71ꢀ4.62 (m, 1.75H), 3.59ꢀ3.49 (m, 2H), 2.54 (s, 1.87H), 2.52 (s,
1.13H), 2.17ꢀ2.11 (m, 1H), 1.97ꢀ1.77 (m, 3H).
(S)-N-Cbz-2-(o-methoxyethoxymethoxy-p-bromophenoxyacetyl)-
pyrrolidine [(S)-14]. Obtained as an amorphous solid in 80% yield after
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Journal of Medicinal Chemistry
ARTICLE
chromatography on silica gel (cyclohexane/EtOAc 1:1): [α]D25 = ꢀ17.7
[α]D25 = ꢀ19.1 (c 1, MeOH). 1H NMR (CDCl3) δ 7.30 (d, J = 2.20 Hz,
1H), 7.06 (dd, 1H, J = 2.20 and 8.53 Hz), 6.80 (d, 1H, J = 8.53 Hz), 5.26 (s,
2H), 4.16ꢀ4.11 (m, 1H), 4.04ꢀ3.98 (m, 1H), 3.92ꢀ3.84 (m, 3H),
3.57ꢀ3.54 (m, 2H), 3.38 (s, 3H), 3.13ꢀ3.07 (m, 1H), 3.02ꢀ2.82 (bs, 1H),
2.46ꢀ2.38 (m, 1H), 2.36 (s, 3H,), 2.32ꢀ2.23 (m, 1H), 1.87ꢀ1.77 (m, 4H).
(1R,20S)-1-(10-Boc-20-pyrrolidinyl)-2-(o-benzyloxy-p-phenylphenoxy)
ethanol [(R,S)-20]. Obtained as an oil in 39% yield after chromatography
1
(c 1, MeOH); 98.62% ee (n-hexane/ethanol 8:2). H NMR (DMSO
100 °C) δ: 7.40ꢀ7.20 (m, 6H), 7.11ꢀ7.01 (m, 1.5H), 6.82ꢀ6.70 (m,
1.5H), 5.19 (s, 2H), 5.08 (s, 2H), 4.82 (s, 2H), 4.68ꢀ4.60 (m, 1H),
3.80ꢀ3.71 (m, 2H), 3.52ꢀ3.40 (m, 4H), 3.22 (s, 3H), 2.25ꢀ2.09 (m,
1H), 1.96ꢀ1.76 (m, 3H).
(S)-N-Boc-2-(o-benzyloxy-p-phenylphenoxyacetyl)pyrrolidine [(S)-
15]. Obtained as an amorphous solid in 58% yield after chromatography
25
1
on silica gel (toluene/EtOAc 8:2): [α]D = ꢀ27.3 (c 1, CHCl3). H
NMR (CDCl3) δ 7.52ꢀ7.28 (m, 10H), 7.26ꢀ7.17 (m, 2H), 7.03 (d,
1H, J = 8.26 Hz), 5.17 (s, 2H), 5.02 (bs, 1H), 4.16ꢀ4.02 (m, 3H), 4.00ꢀ
3.82 (m, 1H), 3.40ꢀ3.20 (bs, 1H), 3.22ꢀ3.19 (bs, 1H), 2.04ꢀ1.69 (m,
4H), 1.48 (s, 9H).
25
on silica gel (toluene/EtOAc 8:2): [α]D = ꢀ25.3 (c 1, CHCl3);
1
97.67% ee (n-hexane/ethanol 8:2). H NMR (CDCl3) δ 7.54ꢀ7.28
(m, 10H), 7.20ꢀ7.11 (m, 2H), 6.97ꢀ6.93 (m, 1H), 5.19 (s, 2H), 4.89
(s, 1H), 4.76 (s, 1H), 4.68ꢀ4.60 (m, 1H), 3.52ꢀ3.40 (m, 2H),
2.20ꢀ2.09 (m, 1H), 1.96ꢀ1.76 (m, 3H), 1.47 (s, 4.5 H), 1.40 (s, 4.5 H).
(S)-N-Boc-2-(o,p-dibenzyloxyphenoxyacetyl)pyrrolidine [(S)-16].
Obtained as an oil in 67% yield after chromatography on silica gel
(1S,20S)-1-(10-Boc-20-pyrrolidinyl)-2-(o-benzyloxy-p-phenylphenoxy)
ethanol [(S,S)-20]. Obtained as an oil in 50% yield after chromatography
25
1
on silica gel (toluene/EtOAc 8:2): [α]D = ꢀ43.3 (c 1, CHCl3). H
NMR (CDCl3) δ 7.57ꢀ7.29 (m, 10H), 7.18ꢀ7.14 (m, 2H), 7.01 (d,
1H, J = 7.98 Hz), 5.15 (s, 2H), 4.56 (bs, 1H), 4.20ꢀ3.90 (m, 4H), 3.48ꢀ
3.37 (m, 1H), 3.30ꢀ3.19 (m, 1H), 2.20ꢀ2.12 (m, 1H), 2.05ꢀ1.62 (m,
3H), 1.46 (s, 9H).
25
(tolune/EtOAc 95:5): [α]D = ꢀ12.7 (c 1, MeOH); 99.42% ee (n-
1
hexane/i-propyl alcohol 8:2). H NMR (CDCl3) δ 7.28ꢀ7.43 (m,
10H), 6.85 (m, 1H), 6.64 (m, 1H), 6.46 (m, 1H), 4.76 (s, 2H), 5.06 (s,
1H), 4.98 (s, 1H), 4.76 (s, 1H), 4.65 (s, 1H), 4.55ꢀ4.60 (m, 1H),
3.37ꢀ3.50 (m, 2H), 2.04ꢀ2.17 (m, 1H), 1.70ꢀ1.92 (m, 3H), 1.45 (s,
4.5H), 1.36 (s, 4.5H).
(1R,20S)-1-(10-Boc-20-pyrrolidinyl)-2-(o,p-dibenzyloxyphenoxy)ethanol
[(R,S)-21]. Obtained as an oil in 49% yield after chromatography on silica
25
1
gel (toluene/EtOAc 85:15): [α]D = ꢀ35.8 (c 1, MeOH). H NMR
(CDCl3) δ 7.24ꢀ7.42 (m, 10H), 6.86 (d, 1H, J = 8.81 Hz), 6.63 (d, 1H,
J = 2.75 Hz), 6.49 (dd, 1H, J = 8.81 and 2.75 Hz), 4.99 (s, 2H), 5.06 (s, 2H),
4.08ꢀ3.90 (m, 4H), 3.47ꢀ3.39 (m, 1H), 3.27ꢀ3.18 (m, 1H), 2.17ꢀ2.08
(m, 1H), 1.93ꢀ1.65 (m, 3H,), 1.44 (s, 9H).
(R)-N-Boc-2-(o-benzyloxy-p-formylphenoxyacetyl)pyrrolidine [(R)-
12]. [α]D25 = +35.4 (c 1, CHCl3); 97.52% ee.
(R)-N-Cbz-2-(o-benzyloxy-p-aceylphenoxyacetyl)pyrrolidine [(R)-
13]. [α]D25 = +20.1 (c 1, CHCl3); 97.42% ee.
(R)-N-Cbz-2-(o-methoxyethoxymethoxy-p-bromophenoxyace-
tyl)pyrrolidine [(R)-14]. [α]D25 = +18.2 (c 1, MeOH); 99.35% ee.
(R)-N-Boc-2-(o-benzyloxy-p-phenylphenoxyacetyl)pyrrolidine [(R)-
15]. [α]D25 = +25.2 (c 1, CHCl3); 98.01% ee.
(R)-N-Boc-2-(o,p-dibenzyloxyphenoxyacetyl)pyrrolidine [(R)-16].
[α]D25 = +12.7 (c 1, MeOH); 99.64% ee.
General Procedure for Synthesis of Compounds 17ꢀ21.
The target compounds were obtained by treatment of the S isomers of
12ꢀ16 and of (R)-16 with 2 mol of NaBH4 (17, 18, and 21), 3 mol of
NaBH4 (20), or 2 mol of LiAlH4 (19) in THF. The reactions were
carried out at temperatures between ꢀ10 and ꢀ20 °C except for 21
(room temperature) for 0.5ꢀ3 h. The diastereomeric mixture of 17 and
18 were not resolved.
(1S,20S)-1-(10-Boc-20-pyrrolidinyl)-2-(o,p-dibenzyloxyphenoxy)ethanol
[(S,S)-21]. Obtained as an oil in 27% yield after chromatography on silica gel
25
(toluene/EtOAc 85:15): [α]D = ꢀ17.4 (c 1, MeOH). 1H NMR
(CDCl3) δ 7.27ꢀ7.45 (m, 10H), 6.90 (d, 1H, J = 8.80 Hz), 6.64 (d,
1H, J = 2.75 Hz), 6.50 (dd, 1H, J = 8.80 and 2.75 Hz), 5.07 (s, 2H), 5.00 (s,
2H), 3.88ꢀ4.08 (m, 4H), 3.42ꢀ3.50 (m, 1H), 3.25ꢀ3.32 (m, 1H),
1.80ꢀ2.03 (m, 3H), 1.65ꢀ1.77 (m, 1H), 1.46 (s, 9H).
(1R,20R)-1-(10-Boc-20-pyrrolidinyl)-2-(o,p-dibenzyloxyphenoxy)ethanol
[(R,R)-21]. Obtained as an oil in 31% yield after chromatography on silica
25
1
gel (toluene/EtOAc 85:15): [α]D = +14.8 (c 1, MeOH). H NMR
identical to (S,S)-21.
(1S,20R)-1-(10-Boc-20-pyrrolidinyl)-2-(o,p-dibenzyloxyphenoxy)ethanol
[(R,S)-21]. Obtained as an oil in 49% yield after chromatography on silica
(1R,20S)- and (1S,20S)-1-(10-Boc-20-pyrrolidinyl)-2-(o-benzyloxy-p-
hydroxymethylphenoxy)ethanol [(R,S)/(S,S)-17]. Obtained as a crude
mixture of diastereoisomers in 85% yield. 1H NMR (CDCl3) δ
7.46ꢀ7.29 (m, 5H), 6.99ꢀ6.87 (m, 3H), 5.09 (s, 1H), 5.07 (s, 1H),
5.01 (bs, 1H), 4.59 (s, 1H), 4.57 (s, 1H), 4.18ꢀ3.91 (m, 4H),
3.50ꢀ3.34 (m, 1H), 3.32ꢀ3.23 (m, 1H), 2.18ꢀ1.70 (m, 4H), 1.47
(s, 4.5H), 1.45 (s, 4.5H).
25
1
gel (toluene/EtOAc 85:15): [α]D = +36.2 (c 1, MeOH). H NMR
identical to (S,R)-21.
(1R,20S)-1-(10-Boc-20-pyrrolidinyl)-2-(o-hydroxy-p-methyl-
phenoxy)ethanol [(R,S)-22]. Obtained as an oil in 35% yield by
hydrogenation (H2 1 atm, 10% Pd(C)) of (R,S)/(S,S)-17 in methanol
25
for 24 h and chromatography on silica gel (toluene/EtOAc 8:2): [α]D
=
ꢀ76.0 (c 1, CHCl3). 1H NMR (CDCl3) δ 7.88 (bs, 1H), 6.79 (d, 1H,
J = 7.90 Hz), 6.75 (s, 1H), 6.57 (d, 1H, J = 7.90 Hz), 6.27 (bs, 1H),
4.07ꢀ4.00 (m, 2H,), 3.96ꢀ3.90 (m, 1H), 3.82ꢀ3.76 (m, 1H),
3.60ꢀ3.44 (m, 1H,), 3.38ꢀ3.25 (m, 1H), 2.25 (s, 3H), 2.12ꢀ2.01
(m, 1H), 1.88ꢀ1.70 (m, 3H), 1.48 (s, 9H).
(1R,20S)- and (1S,20S)-1-(10-Cbz-20-pyrrolidinyl)-2-(o-benzyloxy-p-
acetylphenoxy)ethanol [(R,S)/(S,S)-18]. Obtained as a diastereomeric
mixture in 88% yield after removal of the p-hydroxyethyl analogue by
chromatography on silica gel (cyclohexane/EtOAc 1:1). 1H NMR
(CDCl3) δ 7.59ꢀ7.55 (m, 2H), 7.46ꢀ7.26 (m, 10H), 6.96ꢀ6.88 (m,
1H), 5.20ꢀ5.10 (m, 4H), 4.73 (bs, 1H), 4.27ꢀ3.85 (m, 3H), 3.64ꢀ3.30
(m, 2H), 2.54 (s, 3H, CH3), 2.24ꢀ2.17 (m, 1H), 2.15ꢀ1.82 (m, 2H),
1.76ꢀ1.69 (m, 1H).
(2S,20S)-2-(10-Boc-20-pyrrolidinyl)-7-methyl-1,4-benzodiox-
ane [(S,S)-23]. Obtained as an oil in 60% yield by treatment of (R,S)-22
with PPh (1.2 mol) and DIAD (1.2 mol) in THF under microwave
3
(1R,20S)-1-(10-Methyl-20-pyrrolidinyl)-2-(o-methoxyethoxymethoxy-p-
irradiation at 100 °C and 300 W for 10 min and chromatography on silica
25
1
gel (toluene/EtOAc 8:2): [α]D = ꢀ134.9 (c 1, CHCl3). H NMR
(CDCl3) δ 6.76 (d, 1H, J = 8.25 Hz,), 6.69 (s, 1H), 6.62 (d, 1H, J = 8.25
Hz), 4.28 (dd, 1H, J = 1.93 and 11.28 Hz), 4.12ꢀ3.90 (m, 3H),
3.49ꢀ3.36 (m, 2H), 2.24 (s, 3H), 2.21ꢀ2.14 (m, 1H), 2.02ꢀ1.80 (m,
3H), 1.48 (s, 9H).
bromophenoxy)ethanol [(R,S)-19]. Obtained as an oil in 35% yield after
25
chromatography on silica gel (DCM/MeOH/30%NH3 95:5:2.5): [α]D
=
ꢀ21.2 (c 1, MeOH). 1H NMR (CDCl3) δ 7.28 (d, 1H, J = 2.2 Hz), 7.06
(dd, 1H, J = 2.2 and 8.53 Hz), 6.79 (d, 1H, J = 8.53 Hz), 5.25 (s, 2H),
3.99ꢀ3.90 (m, 2H), 3.86ꢀ3.83 (m, 2H), 3.81ꢀ3.77 (m, 1H), 3.58ꢀ3.55
(m, 2H), 3.38 (s, 3H), 3.11ꢀ3.07 (m, 1H), 2.95ꢀ2.82 (bs, 1H),
2.72ꢀ2.68 (m, 1H), 2.45 (s, 3H), 2.39ꢀ2.33 (m, 1H), 1.98ꢀ1.68(m, 4H).
(1S,20S)-1-(10-Methyl-20-pyrrolidinyl)-2-(o-methoxyethoxymethoxy-
p-bromophenoxy)ethanol [(S,S)-19]. Obtained as an oil in 29% yield
after chromatography on silica gel (DCM/MeOH/30%NH3 95:5:2.5):
(2S,20S)-2-(10-Methyl-20-pyrrolidinyl)-7-methyl-1,4-benzo-
dioxane [(S,S)-2]. Obtained as an oil in 83% yield by treatment of (S,
S)-23 with LiAlH4 (2 mol) in boiling THF for 3 h: [α]D25 = ꢀ106.6 (c 1,
CHCl3). 1H NMR (CDCl3) δ 6.77ꢀ6.74 (m, 2H), 6.62 (dd, 1H, J =
1.65 Hz and 8,25 Hz), 4.25 (dd, 1H, J = 1.93 and 11.00 Hz), 4.09 (ddd,
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1H, J = 1.93 Hz, 4.40 Hz e 7.98 Hz), 3.98 (dd, 1H, J = 7.98 Hz, e 11.00
Hz), 3.16ꢀ3.08 (m, 1H), 2.53ꢀ2.46 (m, 1H), 2.43 (s, 3H), 2.30ꢀ2.22
(m, 1H), 2.24 (s, 3H), 1.93ꢀ1.74 (m, 4H). Anal. (C14H19NO2) C, H, N.
(1S,20S)-1-(10-Boc-20-pyrrolidinyl)-2-(o-hydroxy-p-methyl-
phenoxy)ethanol [(S,S)-22]. Obtained as an oil in 29% yield besides
(R,S)-22 and separated from (R,S)-22 by chromatography on silica gel
(toluene/EtOAc 8:2): [α]D25 = ꢀ13.0 (c 1, CHCl3). 1H NMR (CDCl3) δ
7.59 (bs, 1H), 6.77ꢀ6.74 (m, 2H), 6.58 (d, 1H, J = 7.97 Hz,), 5.12 (bs,
1H), 4.15ꢀ3.98 (m, 3H), 3.90ꢀ3.78 (m, 1H), 3.58ꢀ3.40 (m, 1H),
3.37ꢀ3.20 (m, 1H), 2.25 (s, 3H), 2.10ꢀ1.75 (m, 4H), 1.46 (s, 9H).
(2R,20S)-2-(10-Boc-20-pyrrolidinyl)-7-methyl-1,4-benzo-
dioxane [(R,S)-23]. Obtained as an oil in 60% yield from (S,S)-22 in
chromatography on silica gel (cyclohexane/EtOAc 8:2): [α]D25 = ꢀ6.5
(c 1, CHCl3). 1H NMR (CDCl3) δ 7.53ꢀ7.49 (m, 6H), 7.33ꢀ7.17 (m,
9H), 7.03 (d, 1H, J = 1.74 Hz), 6.86 (d, 1H, J = 8.25 Hz), 6.79 (dd, 1H,
J = 8.25 and 1.74 Hz), 5.01 (bs, 1H), 4.13ꢀ4.06 (m, 5H), 3.92ꢀ3.86 (m,
1H,), 3.58ꢀ3.46 (m, 1H), 3.32ꢀ3.26 (m, 1H), 2.36ꢀ1.79 (m, 4H),
1.75 (s, 2H), 1.47 (s, 7H).
(2R,20S)-2-(10-Boc-20-pyrrolidinyl)-7-trityloxy-1,4-benzo-
dioxane [(R,S)-26]. Obtained as an amorphous solid in 76% yield by
treatment of (S,S)-25 with PPh (2.2 mol) and DIAD (2.2 mol) in
3
THF under microwave irradiation at 100 °C and 300 W for 10 min and
25
chromatography on silica gel (cyclohexane/EtOAc 8:2): [α]D
=
ꢀ27.4 (c 1, CHCl3). 1H NMR (CDCl3) δ 7.52ꢀ7.48 (m, 6H),
7.34ꢀ7.21 (m, 9H), 6.95ꢀ6.85 (m, 1H), 6.83ꢀ6.79 (m, 2H),
4.37ꢀ4.23 (m, 3H), 4.04 (s, 2H), 3.95ꢀ3.92 (m, 1H), 3.56ꢀ3.33
(m, 2H), 2.20ꢀ2.03 (m, 3H), 1.95ꢀ1.83 (m, 1H), 1.46 (s, 9H).
(2R,20S)-2-(10-Methyl-20-pyrrolidinyl)-7-hydroxymethyl-1,4-
benzodioxane [(R,S)-3]. Obtained as an oil in 90% yield by treatment
of(R,S)-26 with LiAlH4 (3 mol) in boiling THF for 2 h and bydetritylation
of the resultant crude reduction product with 1 M aqueous HCl (7 mol) in
methanol for 3 h at room temperature: [α]D25 = +2.5 (c 1, CHCl3). 1H
NMR (CDCl3) δ 6.91ꢀ6.90 (m, 1H), 6.84ꢀ6.79 (m, 2H), 4.56 (s, 2H),
4.27 (dd, 1H, J = 10.97 and 2.19 Hz), 4.09 (dt, 1H, J = 7.31 and 2.19 Hz),
3.96 (dd, 1H, J = 10.97 and 7.31 Hz), 3.12ꢀ3.06 (m, 1H), 2.64ꢀ2.57 (m,
1H), 2.48 (s, 3H), 2.32ꢀ2.23 (pq, 1H), 2.00ꢀ1.62 (m, 4H). Anal.
(C14H19NO3) C, H, N.
25
the same manner as (S,S)-23 from (R,S)-22: [α]D = ꢀ45.4 (c 1,
1
CHCl3). H NMR (CDCl3) δ 6.69ꢀ6.48 (m, 3H), 4.32ꢀ4.03 (m,
3H), 3.85ꢀ3.75 (m, 1H), 3.52ꢀ3.28 (m, 2H), 2.16 (s, 3H), 2.03ꢀ1.73
(m, 4H), 1.40 (s, 9H).
(2R,20S)-2-(10-Methyl-20-pyrrolidinyl)-7-methyl-1,4-benzo-
dioxane [(R,S)-2]. Obtained as an oil in 80% yield from (R,S)-23 in the
same manner as (S,S)-2 from (S,S)-23: [α]D25 = ꢀ5.5 (c 1, CHCl3). 1H
NMR (CDCl3) δ 6.74 (d, 1H, J = 8.20 Hz), 6.70 (d, 1H, J = 1.47 Hz), 6.62
(dd, 1H, J = 1.47 Hz and 8,20 Hz), 4.26 (dd, 1H, J = 2.05 and 11.14 Hz),
4.11 (dt, 1H, J = 2.05 and 7.33 Hz), 3.96 (dd, 1H, J = 7.33 and 11.14 Hz),
3.13ꢀ3.07 (m, 1H), 2.56ꢀ2.66 (m, 1H), 2.49 (s, 3H), 2.32ꢀ2.26 (m,
1H), 2.24 (s, 3H), 1.97ꢀ1.65 (m, 4H). Anal. (C14H19NO2) C, H, N.
(1R,20S)- and (1S,20S)-1-(10-Boc-20-pyrrolidinyl)-2-(o-hydro-
xy-p-hydroxymethylphenoxy)ethanol [(R,S)/(S,S)-24]. Obtained
as an oil in 35% yield by hydrogenolysis (H2 1 atm, 10% Pd(C)) of (R,S)/(S,
S)-17 in methanol for 1 h and chromatography on silica gel (cyclohexane/
(1R,20S)-1-(10-Methyl-20-pyrrolidinyl)-2-(o-hydroxy-p-acetyl-
phenoxy)ethanol [(R,S)-27]. Obtained as an amorphous solid in 70%
yield by hydrogenation (H2 1 atm, 10% Pd(C)) of (R,S)/(S,S)-18 in
methanol for 2 h and, after adding an excess of formalin, for additional 16 h
and subsequent chromatography on silica gel (DCM/MeOH/TEA
1
EtOAc 1:1). H NMR (CDCl3) δ 6.92ꢀ6.76 (m, 3H), 4.57 (s, 2H),
4.15ꢀ3.93 (m, 3H), 3.89ꢀ3.83 (m, 1H), 3.55ꢀ3.34 (m, 1H), 3.32ꢀ3.26
(m, 1H), 2.09ꢀ1.69 (m, 4H), 1.48 (s, 4.5H), 1.46 (s, 4.5H).
25
1
95:5:2): [α]D = ꢀ3.9 (c 1, CHCl3). H NMR (CDCl3) δ 7.51 (d,
1H, J = 1.93 Hz), 7.45 (dd, 1H, J = 8.39 and 1.93 Hz), 6.91 (d, 1H, J = 8.39
Hz), 4.68 (bs, 2H), 4.11 (dd, 1H, J = 9.91 and 4.13 Hz), 4.03 (dd, 1H, J =
9.91 and 5.78 Hz), 3.92ꢀ3.89 (m, 1H), 3.19ꢀ3.14 (m, 1H), 2.80ꢀ2.77
(m, 1H), 2.53 (s, 3H), 2.52 (s, 3H), 2.50ꢀ2.43 (m, 1H), 2.15ꢀ1.95 (m,
1H), 1.88ꢀ1.67 (m, 3H).
(1R,20S)-1-(10-Boc-20-pyrrolidinyl)-2-(o-hydroxy-p-trityloxy-
methylphenoxy)ethanol [(R,S)-25]. Obtained as an amorphous
solid in 36% yield by treatment of (R,S)/(S,S)-24 with trityl chloride
(1.2 mol) and exceeding TEA in boiling DME overnight and chromatog-
25
raphy on silica gel (cyclohexane/EtOAc 8:2): [α]D = ꢀ23.4 (c 1,
1
CHCl3). H NMR (CDCl3) δ 7.84 (bs, 1H), 7.53ꢀ7.50 (m, 6H),
(2S,20S)-2-(10-Methyl-20-pyrrolidinyl)-7-acetyl-1,4-benzo-
dioxane [(S,S)-4]. Obtained as an oil in 60% yield by treatment of (R,
S)-27 with PMe3 (2 mol) and DEAD (1.1 mol) in THF at room
temperature for 16 h and chromatography on silica gel (DCM/MeOH/
7.33ꢀ7.17 (m, 9H), 7.03 (d, 1H, J = 1.65 Hz), 6.88 (d, 1H, J = 8.25 Hz),
6.77 (dd, 1H, J = 8.25 and 1.65 Hz), 6.19 (bs, 1H), 4.12ꢀ4.06 (m, 4H),
3.98 (dd, 1H, J =10.46 and5.23 Hz), 3.87ꢀ3.78 (m, 1H), 3.56ꢀ3.51 (m,
1H), 3.35ꢀ3.27 (m, 1H), 2.36ꢀ2.02 (m, 1H), 1.93ꢀ1.66 (m, 3H), 1.65
(s, 3H), 1.49 (s, 6H).
25
30% NH3 98:2:0.25): [α]D = +17.7 (c 0.8, CHCl3). 1H NMR
(CDCl3) δ 7.57 (d, 1H, J = 1.92 Hz), 7.48 (dd, 1H, J = 8.53 and 1.92
Hz), 6.90 (d, 1H, J = 8.53 Hz), 4.34 (d, 1H, J = 9.35 Hz), 4.12ꢀ4.02
(m, 2H), 3.15ꢀ3.09 (m, 1H), 2.56ꢀ2.50 (m, 1H), 2.51 (s, 3H), 2.43
(s, 3H), 2.32ꢀ2.23 (m, 1H), 1.94ꢀ1.75 (m, 4H). Anal. (C15H19NO3)
C, H, N.
(2S,20S)-2-(10-Boc-20-pyrrolidinyl)-7-trityloxy-1,4-benzo-
dioxane [(S,S)-26]. Obtained as an amorphous solid in 37% yield by
treatment of (R,S)-25 with PPh3 (2.2 mol) and DIAD (2.2 mol) in
THF under microwave irradiation at 100 °C and 300 W for 10 min and
25
chromatography on silica gel (cyclohexane/EtOAc 9:1): [α]D
=
(1S,20S)-1-(10-methyl-20-pyrrolidinyl)-2-(o-hydroxy-p-acetyl-
phenoxy)ethanol [(S,S)-27]. Obtained as an amorphous solid in 27%
yield besides (R,S)-27 and separated from (R,S)-27 by chromatography on
silica gel (DCM/MeOH/TEA 95:5:2): [α]D25 = ꢀ13.0 (c 1, CHCl3). 1H
NMR (CDCl3) δ7.51 (d, 1H, J = 3.30 Hz), 7.46 (dd, 1H, J= 8.52 and 3.30
Hz), 6.85 (d, 1H, J = 8.52 Hz), 5.66 (bs, 2H), 4.28ꢀ4.23 (m, 1H), 4.07
(dd, 1H, J = 9.63 and 3.58 Hz), 3.96 (dd, 1H, J = 9.63 and 7.98 Hz),
3.22ꢀ3.16 (m, 1H), 2.57ꢀ2.40 (m, 1H), 2.52 (s, 3H), 2.45 (s, 3H),
2.40ꢀ2.30 (m, 1H), 1.99ꢀ1.90 (m, 1H), 1.86ꢀ1.73 (m, 3H).
(2R,20S)- 2-(10-Methyl-20-pyrrolidinyl)-7-acetyl-1,4-benzo-
dioxane [(R,S)-4]. Obtained as an oil in 52% yield by treatment of (S,
S)-27 with PPh3 (1.1 mol) and DEAD (1.1 mol) in boiling THF for 16 h
and chromatography on silica gel (DCM/MeOH/30% NH3 98:2:0.25):
[α]D25 = ꢀ118.4 (c 1, CHCl3). 1H NMR (CDCl3) δ 7.51 (d, 1H, J = 1.93
Hz), 7.48 (dd, 1H, J = 8.25 and 1.93 Hz), 6.89 (d, 1H, J = 8.25 Hz), 4.36
(dd, 1H, J = 11.28 and 2.20 Hz), 4.14 (dt, 1H, J = 8.10 and 2.20 Hz), 4.02
(dd, 1H, J = 11.28 and 8.10 Hz), 3.14ꢀ3.07 (m, 1H), 2.68ꢀ2.61 (m, 1H),
ꢀ91.7 (c 1, CHCl3). 1H NMR (CDCl3) δ 7.61ꢀ7.45 (m, 6H),
7.38ꢀ7.00 (m, 9H), 6.98ꢀ6.91 (m, 1H), 6.86ꢀ6.79 (m, 2H), 4.31
(dd, 1H, J = 11.27 and 1.92 Hz), 4.11ꢀ3.93 (m, 4H), 3.53ꢀ3.39 (m,
3H), 2.24ꢀ2.19 (m, 1H), 2.08ꢀ1.87 (m, 3H), 1.48 (s, 9H).
(2S,20S)-2-(10-Methyl-20-pyrrolidinyl)-7-hydroxymethyl-1,4-
benzodioxane [(S,S)-3]. Obtained as an oil in 93% yield by treatment
of(S,S)-26 withLiAlH4 (3mol) in boilingTHFfor 2handbydetritylation
of the resultant crude reduction product with 1 M aqueous HCl (7 mol) in
methanol for 3 h at room temperature: [α]D25 = ꢀ91.7 (c 1, CHCl3). 1H
NMR (CDCl3) δ 6.95 (d, 1H, J = 1.90 Hz), 6.86ꢀ6.79 (m, 2H), 4.54 (s,
2H), 4.25 (dd, 1H, J = 11.00 and 1.79 Hz), 4.08 (ddd, 1H, J = 7.98 Hz,
4.40 and 1.79 Hz), 3.98 (dd, 1H, J = 11.00 and 7.98 Hz), 3.10 (ddd, 1H,
J= 8.80 Hz, 4.12 and 2.47 Hz), 2.50 (m, 1H), 2.41 (s, 3H), 2.25 (pq, 1H, J=
8.80), 2.08 (bs, 1H), 1.92ꢀ1.73 (m, 4H). Anal. (C14H19NO3) C, H, N.
(1S,20S)-1-(10-Boc-20-pyrrolidinyl)-2-(o-hydroxy-p-tritylox-
ymethylphenoxy)ethanol [(S,S)-25]. Obtained as an amorphous
solid in 22% yield besides (R,S)-25 and separated from (R,S)-25 by
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Journal of Medicinal Chemistry
ARTICLE
2.52 (s, 3H), 2.50 (s, 3H), 2.34ꢀ2.26 (pq, 1H), 1.99ꢀ1.90 (m, 1H),
1.82ꢀ1.65 (m, 3H). Anal. (C15H19NO3) C, H, N.
(1S,20S)-1-(10-Boc-20-pyrrolidinyl)-2-(o-hydroxy-p-phenyl-
phenoxy)ethanol [(S,S)-29]. Obtained as an oil in 93% yield from
25
1
(1R,20S)-1-(10-methyl-20-pyrrolidinyl)-2-(o-hydroxy-p-bro-
mophenoxy)ethanol [(R,S)-28]. Obtained as an oil in quantitative
yield by treatment of (R,S)-19 with CF3COOH (4 mol) in DCM at
room temperature for 30 min: [α]D25 = ꢀ15.4 (c 1, MeOH). 1H NMR
(CDCl3) δ 7.05 (d, 1H, J=2.20 Hz), 6.85 (dd, 1H, J = 2.20 and 8.53 Hz),
6.77 (d, 1H, J = 8.53 Hz), 4.76 (bs, 2H), 4.03ꢀ3.91 (m, 2H),
3.85ꢀ3.80 (pq, 1H), 3.17ꢀ3.12 (m, 1H), 2.77ꢀ2.71 (m, 1H), 2.48
(s, 3H), 2.45ꢀ2.39 (m, 1H), 2.04ꢀ1.68 (m, 4H).
(S,S)-20 as described for (R,S)-29: [α]D = ꢀ7.8 (c 1, CHCl3). H
NMR (CDCl3) δ 7.55 (d, 2H, J = 7.15 Hz), 7.40 (t, 2H, J = 7.15 Hz),
7.34ꢀ7.20 (m, 2H), 7.08ꢀ7.01 (m, 1H), 6.98ꢀ6.93 (m, 1H), 5.08 (bs,
1H), 4.20ꢀ4.06 (m, 3H), 4.00ꢀ3.90 (m, 1H), 3.61ꢀ3.42 (m, 1H),
3.33ꢀ3.21 (m, 1H), 2.08ꢀ1.68 (m, 4H), 1.47 (s, 9H).
(2R,20S)-2-(10-Boc-20-pyrrolidinyl)-7-phenyl-1,4-benzodiox-
ane [(R,S)-30]. Obtained as an oil in 72% from (S,S)-29 as described for
(S,S)-30: [α]D25 = ꢀ44.1 (c 1, CHCl3). 1H NMR (CDCl3) δ 7.53 (d, 2H,
J = 7.43 Hz), 7.40 (t, 2H, J = 7.43 Hz), 7.32ꢀ7.21 (m, 1H), 7.20ꢀ7.15
(m, 1H), 7.14ꢀ7.02 (m, 1H), 6.93 (d, 1H, J = 8.25 Hz), 4.50ꢀ4.18 (m,
3H), 4.06ꢀ3.90 (m, 1H), 3.65ꢀ3.39 (m, 2H), 2.20ꢀ2.00 (m, 3H),
1.98ꢀ1.86 (m, 1H), 1.47 (s, 4.5H), 1.57 (s, 4.5H).
(2S,20S)-2-(10-Methyl-20-pyrrolidinyl)-7-bromo-1,4-benzo-
dioxane [(S,S)-5]. Obtained as an oil in 69% yield by treatment of (R,
S)-28 with PMe3 (1.5 mol) and DEAD (1.1 mol) in THF at room
temperature for 24 h and chromatography on silica gel (EtOAc/TEA
98:2): [α]D25 = ꢀ133.7 (c 0.5, MeOH). 1H NMR (CDCl3) δ 7.08 (d,
1H, J = 2.20 Hz), 6.91 (dd, 1H, J = 2.2 and 8.53 Hz), 6.72 (d, 1H, J =
8.53 Hz), 4.27 (dd, 1H J = 1.93 and 11.00 Hz), 4.06 (ddd, 1H, J = 1.93
Hz, 4.40 and 7.98 Hz), 3.97 (dd, 1H, J = 7.98 and 11.00 Hz), 3.12ꢀ3.06
(m, 1H), 2.50ꢀ2.46 (m, 1H), 2.41(s, 3H), 2.30ꢀ2.24 (m, 1H),
1.88ꢀ1.75 (m, 4H). Anal. (C13H16BrNO2) C, H, N.
(2R,20S)- 2-(10-Methyl-20-pyrrolidinyl)-7-phenyl-1,4-ben-
zodioxane [(R,S)-6]. Obtained as an oil in 80% yield from (R,S)-30
25
as described for (S,S)-6: [α]D = ꢀ29.4 (c 1, CHCl3). 1H NMR
(CDCl3) δ 7.53 (d, 2H, J = 7.15 Hz), 7.40 (t, 2H, J = 7.15 Hz), 7.30 (d,
1H, J = 7.15 Hz), 7.15 (d, 1H, J = 2.20 Hz), 7.08 (dd, 1H, J = 8.53 and
2.20 Hz), 6.92 (d, 1H, J = 8.53 Hz), 4.33 (dd, 1H, J = 2.20 and 11.27
Hz), 4.19 (dt, 1H, J = 2.20 and 7.42 Hz), 4.06 (dd, 1H, J = 7.42 and
11.27 Hz), 3.15ꢀ3.09 (m, 1H), 2.71ꢀ2.64 (m, 1H), 2.52 (s, 3H, CH3),
2.39ꢀ2.23 (m, 1H), 2.20ꢀ1.92 (m, 1H), 1.84ꢀ1.64 (m, 3H). Anal.
(C19H21NO2) C, H, N.
(1S,20S)-1-(10-Methyl-20-pyrrolidinyl)-2-(o-hydroxy-p-bro-
mophenoxy)ethanol [(S,S)-28]. Obtained as an oil in quantitative
yield by treatment of (S,S)-19 with CF3COOH (4 mol) in DCM at
room temperature for 30 min: [α]D25 = ꢀ5.8 (c 1, MeOH). 1H NMR
(CDCl3) δ 7.06 (d, 1H, J = 2.2 Hz), 6.87 (dd, 1H, J = 2.2 and 8.53 Hz),
6.73 (d, 1H, J = 8.53 Hz), 6.30 (bs, 2H), 4.13ꢀ4.08 (m, 1H),
4.05ꢀ4.00 (m, 1H), 3.84ꢀ3.78 (m, 1H), 3.15ꢀ3.11 (m, 1H), 2.40 (s,
3H), 2.38ꢀ2.28 (m, 2H), 1.86ꢀ1.76 (m, 4H).
(1R,20S)-1-(10-Boc-20-pyrrolidinyl)-2-(o,p-dibenzyloxyphe-
noxy)ethyl mesylate [(R,S)-31]. Obtained as a white solid in 55%
yield from (R,S)-21 by treatment with equimolar mesyl chloride and
trimethylamine in DCM at room temperature for 5 h and subsequent
chromatography on silica gel (cyclohexane/EtOAc 7:3): mp 97.3 °C;
(2R,20S)-2-(10-Methyl-20-pyrrolidinyl)-7-bromo-1,4-ben-
zodioxane [(R,S)-5]. Obtained as an oil in 69% yield from (S,S)-28
25
1
[α]D = ꢀ61.4 (c 1, CHCl3). H NMR (CDCl3) δ 7.30ꢀ7.50 (m,
10H), 6.77 (d, 1H, J = 8.81 Hz), 6.66 (d, 1H, J = 2.75 Hz), 6.47 (dd, 1H,
J = 8.81 and 2.75 Hz), 5.10ꢀ5.30 (m, 1H), 4.91ꢀ5.00 (m, 4H),
3.99ꢀ4.11 (m, 3H), 3.36ꢀ3.50 (m, 2H), 2.82 (bs, 3H), 1.78ꢀ2.10 (m,
4H), 1.47 (s, 7H), 1.42 (s, 2H).
25
as described for (S,S)-5: [α]D = ꢀ2.8 (c 1, MeOH). 1H NMR
(CDCl3) δ 7.03 (d, 1H, J = 2.20 Hz), 6.91 (dd, 1H, J = 2.20 and 8.52
Hz), 6.74 (d, 1H, J = 8.52 Hz), 4.31 (dd, 1H, J = 2.20 and 11.28 Hz),
4.11 (dt, 1H, J = 2.20 and 7.15 Hz), 3.96 (dd, 1H, J = 7.15 and 11.28
Hz), 3.12ꢀ3.06 (m, 1H), 2.65ꢀ2.57 (m, 1H), 2.47 (s, 3H),
2.33ꢀ2.24 (pq, 1H), 1.97ꢀ1.72 (m, 4H). Anal. (C13H16BrNO2)
C, H, N.
(1R,20S)-1-(10-Boc-20-pyrrolidinyl)-2-(o,p-dihydroxyphenox-
y)ethyl mesylate [(R,S)-32]. Obtained as an oil in 87% yield by
hydrogenolysis (H2 5 atm, 10% Pd(C)) of (R,S)-31 in methanol for 16
h: [α]D25 = ꢀ49.4 (c 1, MeOH). 1H NMR (CDCl3) δ 6.59 (m, 1H), 6.49
(d, 1H, J = 2.75 Hz), 6.27 (m, 1H), 5.34 (m, 1H), 4.15ꢀ3.90 (m, 4H),
3.46ꢀ3.28 (m, 3H), 3.05 (s, 3H), 1.84ꢀ2.04 (m, 4H), 1.48 (s, 9H).
(2S,20S)-2-(10-Boc-20-pyrrolidinyl)-7-hydroxy-1,4-benzodiox-
ane [(S,S)-33]. Obtained as a white solid in 77% yield by treatment of
(R,S)-32 with equimolar K2CO3 in boiling DME for 6 h and subsequent
chromatography on silica gel (cyclohexane/EtOAc 6:4): mp 166.5 °C;
[α]D25 = ꢀ174.4 (c 1, MeOH). 1H NMR (CDCl3) δ 6.71 (d, 1H, J = 8.53
Hz), 6.40 (d, 1H, J = 2.75 Hz), 6.33 (d, 1H, J = 8.53 Hz), 5.10ꢀ4.90 (bs,
1H), 4.24 (dd, 1H, J = 11.55 and 2.20 Hz), 4.20ꢀ3.85 (m, 3H), 3.30ꢀ3.55
(m, 2H), 2.30ꢀ2.15 (m, 1H), 2.05ꢀ1.85 (m, 3H), 1.47 (s, 9H).
(2S,20S)-2-(20-Pyrrolidinyl)-7-hydroxy-1,4-benzodioxane
[(S,S)-7a]. Obtained as a white solid in 48% yield by treatment of (S,S)-
33 with CF3COOH in DCM at room temperature for 2 h and successive
crystallization from diisopropyl ether: mp 205.5 °C; [α]D25 = ꢀ76.4 (c
1, MeOH). 1H NMR (DMSO-d6) δ 8.89 (s, 1H), 6.58ꢀ6.61 (d, 1H, J =
8.25 Hz), 6.20 (d, 1H, J = 3.05 Hz), 6.16 (m, 1H), 4.21 (dd, 1H, J =
11.28 and 1.93 Hz), 3.83 (dd, 1H, J = 11.28 and 7.15 Hz), 3.70 (t, 1H,
J = 7.15 Hz), 3.04ꢀ3.08 (m, 1H), 2.71ꢀ2.82 (m, 2H), 2.53 (bs, 1H),
1.83ꢀ1.56 (m, 4H). Anal. (C12H15NO3) C, H, N.
(1R,20S)-1-(10-Boc-20-pyrrolidinyl)-2-(o-hydroxy-p-phenyl-
phenoxy)ethanol [(R,S)-29]. Obtained as an oil in 85% yield by
hydrogenolysis (H2 1 atm, 10% Pd(C)) of (R,S)-20 in methanol for 48
h: [α]D25 = ꢀ34.1 (c 1, CHCl3). 1H NMR (CDCl3) δ 7.92 (bs, 1H),
7.55 (d, 2H, J = 7.15 Hz), 7.40 (t, 2H, J = 7.15 Hz), 7.31 (d, 1H, J = 7.15
Hz), 7.20 (d, 1H, J = 1.93 Hz), 7.02 (dd, 1H, J = 1.93 and 8.26 Hz), 6.98
(d, 1H, J = 8.26 Hz), 6.23 (bs, 1H), 4.14ꢀ4.07 (m, 1H), 4.06ꢀ3.99 (m,
1H), 3.90ꢀ3.80 (m, 1H), 3.61ꢀ3.49 (m, 1H), 3.38ꢀ3.27 (m, 1H),
2.13ꢀ2.05 (m, 1H), 2.00ꢀ1.65 (m, 3H,), 1.50 (s, 9H).
(2S,20S)-2-(10-Boc-20-pyrrolidinyl)-7-phenyl-1,4-benzodiox-
ane [(S,S)-30]. Obtained as an oil in 60% yield by treatment of (R,S)-29
with PMe3 (2.2 mol) and DEAD (2.2 mol) in THF under microwave
irradiation at 100 °C and 300 W for 10 min and chromatography on silica
gel (cyclohexane/EtOAc 7:3): [α]D25 = ꢀ126.4 (c 1, CHCl3). 1H NMR
(CDCl3) δ 7.54 (d, 2H, J = 7.7 Hz), 7.40 (t, 2H, J = 7.7 Hz), 7.34ꢀ7.22
(m, 1H), 7.19ꢀ7.05 (m, 2H), 6.94 (d, 1H, J = 8.25 Hz), 4.35 (d, 1H, J =
11.27 Hz), 4.25ꢀ3.90 (m, 3H), 3.62ꢀ3.35 (m, 2H), 2.36ꢀ2.20 (m,
1H), 2.10ꢀ1.80 (m, 3H), 1.43 (s, 4.5H), 1.42 (s, 4.5H).
(2S,20S)-2-(10-Methyl-20-pyrrolidinyl)-7-phenyl-1,4-benzodiox-
ane [(S,S)-6]. Obtained as an oil in 70% yield by treatment of (S,S)-30 with
LiAlH4 (2 mol) in boiling THF for 1 h: [α]D25 = ꢀ83.4 (c 1, CHCl3). 1H
NMR (CDCl3) δ7.55 (d, 2H, J= 7.15 Hz), 7.41 (t, 2H, J= 7.15 Hz), 7.31 (d,
1H, J = 7.15 Hz), 7.24 (d, 1H, J = 2.20 Hz), 7.10 (dd, 1H, J = 8.52 and 2.20
Hz), 6.95 (d, 1H, J = 8.52 Hz), 4.31 (dd, 1H, J = 1.92 and 11.27 Hz), 4.17
(ddd, 1H, J = 1.92 Hz, 4.40 and 8.00 Hz), 4.06 (dd, 1H, J = 8.00 and 11.27
Hz), 3.18ꢀ3.13 (m, 1H), 2.58ꢀ2.52 (m, 1H), 2.46 (s, 3H), 2.33ꢀ2.25 (m,
1H), 2.03ꢀ1.77 (m, 4H). Anal. (C19H21NO2) C, H, N.
(2S,20S)-2-(10-Methyl-20-pyrrolidinyl)-7-hydroxy-1,4-ben-
zodioxane [(S,S)-7]. Obtained as a white solid in 96% yield by
treatment of (S,S)-7a with H2 (1 atm)/10% Pd(C) in methanol in the
25
presence of formalin for 16 h: mp 161.3 °C; [α]D = ꢀ95.0 (c 1,
CHCl3). 1H NMR (DMSO-d6) δ 8.91 (s, 1H), 6.61 (d, 1H, J = 8.52
Hz), 6.21 (m, 1H), 6.16 (d, 1H, J = 2.75 Hz), 4.20 (dd, 1H, J = 11.28 and
1.92 Hz), 3.92ꢀ3.97 (ddd, 1H, J = 7.70 Hz, 4.67 and 1.92 Hz),
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3.80ꢀ3.87 (dd, 1H, J = 11.28 and 7.71 Hz), 2.90ꢀ2.96 (m, 1H), 2.52
(m, 1H), 2.29 (s, 3H), 2.11ꢀ2.18 (m, 1H), 1.58ꢀ1.85 (m, 4H). Anal.
(C13H17NO3) C, H, N.
80% yield from (S,R)-21 as described for (R,S)-31; [α]D25 = +58.7 (c 1,
CHCl3). 1H NMR identical to (R,S)-31.
(1S,20R)-1-(10-Boc-20-pyrrolidinyl)-2-(o,p-dihydroxyphenox-
y)ethyl Mesylate [(S,R)-32]. Obtained as an oil in 82% yield from
(1S,20S)-1-(10-Boc-20-pyrrolidinyl)-2-(o,p-Dibenzyloxyphe-
noxy)ethyl mesylate [(S,S)-31]. Obtained as an amorphous solid in
75% yield from (S,S)-21 as described for (R,S)-31; [α]D25 = ꢀ14.5 (c 1,
MeOH). 1H NMR (CDCl3) δ 7.32ꢀ7.43 (m, 10H), 6.77 (d, 1H, J = 8.81
Hz), 6.67 (d, 1H, J = 2.75 Hz), 6.47 (dd, 1H, J = 8.81 and 2.75 Hz),
5.25ꢀ4.90 (m, 5H), 4.01ꢀ4.25 (m, 3H), 3.30ꢀ3.60 (m, 2H), 2.85 (s,
3H), 1.95ꢀ2.20 (m, 2H) , 1.90ꢀ1.75 (m, 2H), 1.54 (s, 2H), 1.46 (s, 7H).
(1S,20S)-1-(10-Boc-20-pyrrolidinyl)-2-(o,p-dihydroxyphenox-
y)ethyl mesylate [(S,S)-32]. Obtained as an oil in 82% yield from (S,
S)-31 as described for (R,S)-32; [α]D25 = ꢀ29.5 (c 1, CHCl3). 1H NMR
(CDCl3) δ 6.64 (d, 1H, J = 8.53 Hz), 6.47 (s, 1H), 6.27 (d, 1H, J = 8.53
Hz), 5.32 (bs, 1H), 5.11 (m, 1H), 4.08ꢀ4.26 (m, 3H), 3.91ꢀ3.99 (pt,
1H, J = 10.18 Hz), 3.60ꢀ3.42 (m, 2H), 3.40ꢀ3.25 (m, 2H), 3.09 (s,
3H), 1.92ꢀ2.01 (m, 4H), 1.48 (s, 9H).
25
1
(S,R)-31 as described for (R,S)-32; [α]D = +50.3 (c 1, MeOH). H
NMR identical to (R,S)-32.
(2R,20R)-2-(10-Boc-20-pyrrolidinyl)-7-hydroxy-1,4-benzodiox-
ane [(R,R)-33]. Obtained as a white solid in 83% yield from (S,R)-32 as
described for (S,S)-33; mp 165.4 °C; [α]D25 = +179.1 (c 1, MeOH). 1H
NMR identical to (S,S)-33.
(2R,20R)-2-(20-Pyrrolidinyl)-7-hydroxy-1,4-benzodioxane
[(R,R)-7a]. Obtained as a white solid in 70% yield from (R,R)-33 as
described for (S,S)-7a; mp 204.1 °C; [α]D25 = +76.8 (c 1, MeOH). 1H
NMR identical to (S,S)-7a. Anal. (C12H15NO3) C, H, N.
(2R,20R)-2-(10-Methyl-20-pyrrolidinyl)-7-hydroxy-1,4-ben-
zodioxane [(R,R)-7]. Obtained as a white solid in 75% yield from (R,
25
R)-7a as described for (S,S)-7; mp 160.9 °C; [α]D = +101.1 (c 1,
CHCl3). 1H NMR identical to (S,S)-7. Anal. (C13H17NO3) C, H, N.
(2S,20S)-2-(10-Boc-20-pyrrolidinyl)-7-methoxy-1,4-benzo-
dioxane [(S,S)-34]. Obtained as an oil in 95% yield by treatment of
(S,S)-33 with DBU (2 mol) in dimethylcarbonate at 90 °C for 16 h and
successive chromatography on silica gel (cyclohexane/EtOAc 7:3);
[α]D25 = ꢀ142.2 (c 1, CHCl3). 1H NMR (CDCl3) δ 6.78 (d, 1H, J =
8.94 Hz), 6.44 (d, 1H, J = 2.75 Hz), 6.40 (dd, 1H, J = 8.94 and 2.75
Hz), 4.26 (dd, 1H, J = 11.28 and 2.20 Hz), 4.18ꢀ4.05 (m, 1H),
4.00ꢀ3.88 (m, 2H), 3.73 (s, 3H), 3.51ꢀ3.73 (m, 2H), 2.23ꢀ2.18 (m,
1H), 2.04ꢀ1.90 (m, 3H), 1.47 (s, 9H).
(2R,20S)-2-(10-Boc-20-pyrrolidinyl)-7-hydroxy-1,4-benzodiox-
ane [(R,S)-33]. Obtained as a white solid in 79% yield from (S,S)-32 as
described for (S,S)-33; mp 164.5 °C; [α]D25 = ꢀ47.0 (c 1, MeOH). 1H
NMR (CDCl3) δ 6.70 (d, 1H, J = 8.53 Hz), 6.39 (d, 1H, J = 2.75 Hz), 6.30
(dd, 1H, J = 8.53 and 2.75 Hz), 4.21ꢀ4.30 (m, 4H), 3.82ꢀ3.89 (m, 1H),
3.45ꢀ3.31 (m, 2H), 2.04ꢀ1.98 (m, 3H), 1.95ꢀ1.85 (m, 1H), 1.45 (s, 9H).
(2R,20S)-2-(20-Pyrrolidinyl)-7-hydroxy-1,4-benzodioxane
[(R,S)-7a]. Obtained as a white solid in 30% yield from (R,S)-33 as
described for (S,S)-7a; mp 177.3 °C; [α]D25 = +30.2 (c 1, CHCl3). 1H
NMR (DMSO-d6) δ 6.73 (d, 1H, J = 3.02 Hz), 6.67 (d, 1H, J = 8.80
Hz), 6.30 (dd, 1H, J = 8.80 and 3.03 Hz), 4.16ꢀ4.21 (dd, 1H, J = 11.00
and 1.92 Hz), 3.98ꢀ4.04 (dt, 1H, J = 8.53 and 1.65 Hz), 3.81ꢀ3.87
(dd, 1H, J = 11.00 and 8.53 Hz), 3.20ꢀ3.28 (m, 1H), 3.09ꢀ3.17 (m,
1H), 2.94ꢀ3.03 (m, 1H), 2.00ꢀ1.78 (m, 3H), 1.65ꢀ1.53 (m, 1H).
Anal. (C12H15NO3) C, H, N.
(2R,20S)-2-(10-Methyl-20-pyrrolidinyl)-7-hydroxy-1,4-benzo-
dioxane [(R,S)-7]. Obtained as a white solid in 85% yield from (R,S)-7a
as described for (S,S)-7; mp 143.7 °C; [α]D25 = ꢀ7.9 (c 1, CHCl3). 1H
NMR (DMSO-d6) δ 8.91 (s, 1H), 6.60 (d, J = 8.25 Hz), 6.20ꢀ6.22 (m,
1H), 6.18 (d, 1H, J = 2.76 Hz), 4.12ꢀ4.17 (dd, 1H, J = 11.28 and 2.20
Hz), 4.02ꢀ4.07 (dt, 1H, J = 7.42 and 2.20 Hz), 3.83ꢀ3.89 (dd, 1H, J =
11.28 and 7.43 Hz), 2.90ꢀ2.95 (m, 1H), 2.50ꢀ2.56 (m, 1H), 2.34 (s,
3H), 2.10ꢀ2.15 (m, 1H), 1.84ꢀ1.75 (m, 1H), 1ꢀ70ꢀ1.57 (m, 3H).
Anal. (C13H17NO3) C, H, N.
(2S,20S)-2-(10-Methyl-20-pyrrolidinyl)-7-methoxy-1,4-ben-
zodioxane [(S,S)-8]. Obtained as an oil in 85% yield by treatment of
(S,S)-34 with LiAlH4 (4 mol) in boiling THF for 2 h; [α]D25 = ꢀ104.5
(c 1, CHCl3). 1H NMR (CDCl3) δ 6.77 (d, 1H, J = 8.80 Hz), 6.55 (d,
1H, J = 2.89 Hz), 6.40 (dd, 1H, J = 8.80 and 2.89 Hz), 4.22 (dd, 1H, J =
11.28 and 2.20 Hz), 4.13 (m, 1H), 3.95 (dd, 1H, J = 11.28 and 7.98 Hz),
3.72 (s, 3H), 3.15 (m, 1H), 2.55ꢀ2.45 (m, 1H), 2.43 (s, 3H),
2.30ꢀ2.25 (m, 1H), 1.93ꢀ1.74 (m, 3H). Anal. (C14H19NO3) C, H, N.
(2R,20S)-2-(10-Boc-20-pyrrolidinyl)-7-methoxy-1,4-benzodiox-
ane [(R,S)-34]. Obtained as an oil in 96% yield from (R,S)-33 as described
for (S,S)-34; [α]D25 = ꢀ44.9 (c 1, CHCl3). 1H NMR (CDCl3) δ 6.77 (d,
1H, J= 8.80 Hz), 6.44ꢀ6.38 (m, 2H), 4.28ꢀ4.18 (m, 3H), 3.97ꢀ3.80 (m,
1H), 3.73 (s, 3H), 3.54ꢀ3.32 (m, 2H), 2.04ꢀ1.86 (m, 4H), 1.44 (s, 9H).
(2R,20S)-2-(10-Methyl-20-pyrrolidinyl)-7-methoxy-1,4-ben-
zodioxane [(R,S)-8]. Obtained as an oil in 91% yield from (R,S)-34 as
described for (S,S)-8; [α]D25 = ꢀ15.7 (c 1, CHCl3). 1H NMR (CDCl3)
δ 6.76 (d, 1H, J = 8.80 Hz), 6.46 (d, 1H, J = 3.02 Hz), 6.40 (dd, 1H, J =
8.80 and 3.02 Hz), 4.25 (dd, 1H, J = 11.28 and 2.20 Hz), 4.13 (dt, 1H,
J = 7.15 and 2.20 Hz), 3.94 (dd, 1H, J = 11.28 and 7.15 Hz), 3.73 (s, 3H),
3.12ꢀ3.07 (m, 1H), 2.67ꢀ2.59 (m, 1H), 2.49 (s, 3H), 2.28 (pq, 1H),
1.98ꢀ1.89 (m, 1H), 1.81ꢀ1.66 (m, 3H). Anal. (C14H19NO3) C, H, N.
(2S,20S)-2-(10-Boc-20-pyrrolidinyl)-7-isopropoxy-1,4-benzo-
dioxane [(S,S)-35]. Obtained as an oil in 84% yield by treatment of (S,
S)-33withexceeding 2-bromopropaneandNaH (1mol) inDMEat60°C
for 16 h and subsequent chromatography on silica gel (cyclohexane/
EtOAc 7:3); [α]D25 = ꢀ133.2 (c 1, CHCl3). 1H NMR (CDCl3) δ 6.75
(d, 1H, J = 8.80 Hz), 6.44 (d, 1H, J = 2.75 Hz), 6.39 (dd, 1H, J = 8.80 and
2.75 Hz), 4.38 (sept, 1H, J = 6.18 Hz), 4.25 (dd, 1H, J = 11.28 and 2.20
Hz), 4.20ꢀ3.85 (m, 3H), 3.55ꢀ3.32 (m, 2H), 2.25ꢀ2.10 (m, 1H,),
2.02ꢀ1.85 (m, 3H,), 1.47 (s, 9H), 1.29 (d, 6H, J = 6.18 Hz).
(1R,20R)-1-(10-Boc-20-pyrrolidinyl)-2-(o,p-dibenzyloxyphe-
noxy)ethyl Mesylate [(R,R)-31]. Obtained as an amorphous solid in
97% yield from (R,R)-21 as described for (R,S)-31; [α]D25 = +14.1 (c 1,
MeOH). 1H NMR identical to (S,S)-31.
(1R,20R)-1-(10-Boc-20-pyrrolidinyl)-2-(o,p-dihydroxyphe-
noxy)ethyl Mesylate [(R,R)-32]. Obtained as an oil in 86% yield
from (R,R)-31 as described for (S,R)-32; [α]D25 = +29.3 (c 1, CHCl3).
1H NMR identical to (S,S)-32.
(2S,20R)-2-(10-Boc-20-pyrrolidinyl)-7-hydroxy-1,4-benzodiox-
ane [(S,R)-33]. Obtained as a white solid in 85% yield from (R,R)-32 as
described for (S,S)-33; mp 164.7 °C; [α]D = +43.9 (c 1, MeOH). 1H
25
NMR identical to (R,S)-33.
(2S,20R)-2-(20-Pyrrolidinyl)-7-hydroxy-1,4-benzodioxane
[(S,R)-7a]. Obtained as a white solid in 88% yield from (S,R)-33 as
described for (S,S)-7a; mp 166.7 °C; [α]D25 = ꢀ32.9 (c 1, CHCl3). 1H
NMR identical to (R,S)-7a. Anal. (C12H15NO3) C, H, N.
(2S,20R)-2-(10-Methyl-20-pyrrolidinyl)-7-hydroxy-1,4-ben-
zodioxane [(S,R)-7]. Obtained as a white solid in 91% yield from (S,
(2S,20S)-2-(10-Methyl-20-pyrrolidinyl)-7-isopropoxy-1,4-ben-
zodioxane [(S,S)-9]. Obtained as an oil in 78% yield by treatment of (S,
S)-35 with LiAlH4 (6 mol) in boiling THF for 2 h; [α]D25 = ꢀ88.9 (c 1,
CHCl3). 1H NMR (CDCl3) δ 6.75 (d, 1H, J = 8.80 Hz), 6.54 (d, 1H, J =
2.88 Hz), 6.38 (dd, 1H, J = 8.80 and 2.88 Hz), 4.37 (sept, 1H, J = 6.05 Hz),
4.22 (dd, 1H, J = 11.14 and 2.20 Hz), 4.13 (ddd, 1H, J = 7.98 Hz, 3.85 and
2.20 Hz), 3.94 (dd, 1H, J = 11.14 and 7.98 Hz), 3.16ꢀ3.09 (m, 1H),
25
R)-7a as described for (S,S)-7; mp 145.5 °C; [α]D = +8.7 (c 1,
CHCl3). 1H NMR identical to (R,S)-7. Anal. (C13H17NO3) C, H, N.
(1S,20R)-1-(10-Boc-20-pyrrolidinyl)-2-(o,p-dibenzyloxyphe-
noxy)ethyl Mesylate [(S,R)-31]. Obtained as an amorphous solid in
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2.54ꢀ2.48 (m, 1H), 2.43 (s, 3H), 2.30ꢀ2.21 (m, 1H), 1.93ꢀ1.73 (m,
4H), 1.28 (d, 6H, J = 6.05 Hz). Anal. (C16H23NO3) C, H, N.
the wells, and after overnight incubation at 4 °C, the wells were washed
and the binding experiments were performed as previously described.20
Heterologously Expressed α3β4 Receptors. HEK 293 cells were
grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal
bovine serum, 1% L-glutamine, 100 units/mL penicillin G, and 100 μg of
streptomycin in a humidified atmosphere containing 10% CO2. ThecDNAs
encoding α3 and β4 were transfected into the HEK 293 cells at 30%
confluency. The cell transfections were carried out in 100 mm Petri dishes
using 30 μL of JetPEI (Polypus, France) (1 mg/mL, pH 7.2) and 10 μg of
cDNAs. After 48 h transfection, the cells were collected, washed with PBS by
centrifugation, and used for binding analysis.
(2R,20S)-2-(10-Boc-20-pyrrolidinyl)-7-isopropoxy-1,4-benzo-
dioxane [(R,S)-35]. Obtained as an oil in 66% yield from (R,S)-33 as
described for (S,S)-35; [α]D25 = ꢀ38.9 (c 1, CHCl3). 1H NMR (CDCl3)
δ 6.74 (d, 1H, J = 8.52 Hz), 6.43 (d, 1H, J = 2.75 Hz), 6.38 (dd, 1H, J =
8.52 and 2.75 Hz), 4.38 (sept, 1H, J = 6.18 Hz), 4.27ꢀ4.21 (m, 3H),
3.90ꢀ3.80 (m, 1H), 3.60ꢀ3.30 (m, 2H), 2.17ꢀ1.88 (m, 4H), 1.45 (s,
5H), 1.42 (s, 4H), 1.29 (d, 6H, J = 6.18 Hz).
(2R,20S)-2-(10-Methyl-20-pyrrolidinyl)-7-isopropoxy-1,4-ben-
zodioxane [(R,S)-9]. Obtained as an oil in 72% yield from (R,S)-35 as
described for (S,S)-9; [α]D25 = ꢀ2.7 (c 1, CHCl3). 1H NMR (CDCl3) δ
6.73 (d, 1H, J = 8.80 Hz), 6.46 (d, 1H, J = 2.75 Hz), 6.39 (dd, 1H, J = 8.80
and 2.75 Hz), 4.38 (sept, 1H, J= 6.05 Hz), 4.24 (dd, 1H, J = 11.14 and 2.20
Hz), 4.12 (dt, 1H, J = 7.16 and 2.2 Hz), 3.94 (dd, 1H, J = 11.14 and 7.16
Hz), 3.13ꢀ3.07 (m, 1H), 2.66ꢀ2.59 (m, 1H), 2.49 (s, 3H), 2.28 (pq,
1H), 1.98ꢀ1.88 (m, 1H), 1.81ꢀ1.63 (m, 3H), 1.28 (d, 6H, J = 6.05 Hz).
Anal. (C16H23NO3) C, H, N.
[3H]-epibatidine was bound to α6β2* immunoimmobilised recep-
tors or HEK 293 α3β4 receptors by means of overnight incubation at
4 °C at concentrations ranging from 0.005 to 1 nM in a buffer containing
50 mM Tris-HCl, pH 7, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2,
2.5 mM CaCl2, and 2 mg/mL BSA in the presence (in the case of the
α6β2 subtype) or absence (in the case of α3β4 subtype) of 0.05%
Tween 20. Specific ligand binding was defined as total binding minus the
binding in the presence of 100 nM cold epibatidine.
Binding to Nicotinic Receptor Subtypes. Frozen cortex and
striatum specimens taken from adult male SpragueꢀDawley rats
(Charles River, Calco Italy) were homogenized using a Potter homo-
genizer in an excess of buffer A (50 mM Tris-HCl, pH 7, 120 mM NaCl,
5 mM KCl, 1 mM MgCl2 2.5 mM CaCl2, and 2 mM phenylmethylsul-
fonyl fluoride), centrifuged (60 min at 30000g), and rinsed twice. The
homogenates were resuspended in the same buffer containing 20 μg/mL
of the protease inhibitors leupeptin, bestatin, pepstatin A, and aprotinin.
[3H]-epibatidine and [125I] ]-αBungarotoxin binding to α4β2 and
α7 cortical membrane-subtypes were performed as previously
described.23 Briefly, for [3H]-epibatidine, binding cortical membranes
were first preincubated with 1 μM α-bungarotoxin in order to prevent
the binding of [3H]-epibatidine to the α7 receptors and then with
(()-[3H]-epibatidine (specific activity of 56ꢀ60 Ci/mmol, purchased
from Perkin-Elmer, Boston MA). In the [3H]-epibatidine saturation
experiments, aliquots of cortical membranes were incubated overnight at
4 °C with concentrations of [3H]-epibatidine ranging between 0.005 and
1 nM diluted in buffer A. Nonspecific binding was determined in parallel
by means of incubation in the presence of 100 nM unlabeled epibatidine.
At the end of the incubation, the samples were filtered on GFC filters
presoaked in polyethylenimine through a harvester apparatus, and the
filters were counted in a β counter. The inhibition of radioligand binding
was obtained by preincubating increasing doses (10 pM to 10 mM) of
the test compounds for 30 min at rt, followed by overnight incubation
with a final concentration of 0.1 nM [3H]-epibatidine and then
incubated overnight at 4 °C.
The inhibition of [3H]-epibatidine binding induced by (R,S)-7 and
(S,S)-7 was measured by incubating increasing concentrations for 5 min
followed by overnight incubation with 0.1 nM[3H]-epibatidine (in the
case of the α6β2* subtype) or 0.25 nM (in the case of the α3β4
subtype). After incubation, the wells containing the α6β2* subtype or
the membranes of HEK cells transfected with α3β4 subtype were
washed seven times with ice-cold PBS, and [3H]-epibatidine binding
was determined by means of liquid scintillation counting in a β counter.
Neurotransmitter Release. [3H]dopamine (DA) from striatal
slices and [3H] noradrenaline (NA) from hippocampal slices were
measured using 96-well assays as previously described.19 For each
experiment, two rats were killed by cervical dislocation, their brain
was rapidly removed, and the hippocampi and striata were dissected and
transferred to ice-cold Krebs buffer (KB: 118 mM NaCl, 2.4 mM KCl,
2.4 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4 7H2O, 25 mM
3
NaHCO3, 10 mM D-glucose, and 1 mM ascorbic acid), gassed with 95%
O2/ 5% CO2 for at least 1 h at 37 °C, and the pH was adjusted to 7.4. The
tissue was chopped three times (two 60° rotations) using a McIlwain
tissue chopper to give slices of 150 μm. After two washes with warm KB,
hippocampal and striatal slices were respectively incubated for 30 min
with 150 nM [3H]NA and 100 nM [3H]DA, in 5 mL of KB supple-
mented with 10 μM pargyline to prevent the [3H]catecholamine
degradation that occurs at 37 °C. To remove the excess tritium, four
washes with KB containing 10 μM pargyline and 0.5 μM nomifensine
were performed to prevent [3H]catecholamine reuptake. The slices were
then loaded onto 96-well filter plates (Millipore Corporation, Milan,
Italy) and incubated for 5 min with buffer in the presence or absence of
antagonists or peptides under studies. Then, the buffer was removed by
filtration (to obtain the basal value) and collected in a 96-well Optiplate
(PerkinElmer). Buffer (100 μL) containing an agonist or an antagonist
was then added to each well (in each experiment, buffer stimulation was
used to determine the fractional release of [3H]DA or [3H]NA evoked
by the buffer alone). After 5 min at 37 °C, the buffer was collected by
filtration in a 96-well Optiplate to determine the fraction of
[3H]catecholamine released. Optiphase Supermix (Perkin-Elmer)
(200 μL per well) was added to Optiplate, and the radioactivity in each
well was counted for 2 min using a Microbeta 3 counter (Wallac 1450
Microbeta Trilux; PerkinElmer) with a counting efficiency of 30%. To
determine the amount of tritium remaining in the slices, 50 μL of
Optiphase Supermix was added to each well and the radioactivity was
counted in Microbeta 3 counter. The amount of [3H]catecholamine
released was expressed as a percentage of the total radioactivity taken up
in the slices before stimulation (i.e., the amount of tritium released +
tritium remaining in the slices after stimulation). Each experiment was
performed in six replicates and repeated at least three times.
The [125I]-α-Bungarotoxin (specific activity 150 Ci/mmol, pur-
chased from Perkin-Elmer, Boston MA) saturation binding was per-
formed by incubating cortical membranes overnight with 0.1ꢀ10 nM
concentrations of [125I]-α-bungarotoxin at rt. Nonspecific binding was
determined in parallel by means of incubation in the presence of 1 μM
unlabeled α-bungarotoxin. After incubation, the samples were filtered as
described above and the bound radioactivity directly counted in a γ
counter. The inhibition of [125I]-α-Bungarotoxin binding by the test
compounds was measured by preincubating cortical membranes with
increasing concentrations (10 pM to 10 mM) of the drug to be tested for
30 min at rt, followed by overnight incubation with a final concentration
of 1 nM [125I]-α-bungarotoxin at rt.
α6β2* Subtype Immunoimmobilization. The α6β2* (*indicates that
other subunits may be present in addition to the α6 and β2 subunits)
subtype was isolated from striatal membranes by solubilizing the
membranes with 2% Triton-100 and immobilizing [3H]-epibatidine
labeled receptors using anti-α6 subunit specific antibodies (Abs).
Briefly, 10 μg/mL Abs were bound to microwells (Maxi-Sorp; Nunc,
Roskilde, Denmark) by means of overnight incubation at 4 °C. After
washing to remove the unbound Abs, rat striatal extracts were added to
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Journal of Medicinal Chemistry
ARTICLE
Data Analysis. The Ki values of all the tested compounds were
determined by means of the LIGAND program using the data obtained
from three independent saturation and competition binding experi-
ments and compared by means of the F-test. The striatal DA release data
and hippocampal NA release data were expressed as the fractional
percentage of total radioactivity for each well. Basal release was
subtracted from stimulated release for each sample, and the results were
normalized to the maximal release evoked by nicotine 10 μM (striatum)
or 100 μM (hippocampus). Each concentration of control or peptide
was applied in quadruplicate replicates. Concentrationꢀresponse para-
meters were determined by nonlinear regression analysis using Prism 4.0
(GraphPad, San Diego, CA). Pooled normalized data were analyzed in
Prism to determine EC50, IC50, and Emax as a percentage of nicotine
response.
Computational Methods. The ligand’s conformational space was
explored by systematically rotating the rotatable bond connecting the
two rings. The docking and scoring procedures were performed by
GriDock, a parallel tool based on the AutoDock4.0 engine,31 using the
rat α4β2 nicotinic model (PDB ID: 1OLE).31 In detail, the grid box was
set to include all residues within a 15 Å radius sphere around the highly
conserved Trp147 residue, thus comprising the entire binding cavity.
The resolution of the grid was 60 ꢁ 60 ꢁ 60 points with a grid spacing of
0.450 Å. For the docking simulations, the flexible bonds of the ligand
were automatically recognized by GriDock and left free to rotate so as to
account for ligand flexibility within the binding cavity. Each substrate
was docked with the Lamarckian algorithm as implemented in Auto-
Dock. The genetic-based algorithm ran 30 simulations per substrate with
2000000 energy evaluations and a maximum number of generations of
27000. The crossover rate was increased to 0.8, and the number of
individuals in each population to 150. All other parameters were left at
the AutoDock default settings. The obtained complexes were finally
minimized to favor the mutual adaptability between ligand and receptor,
and the optimized complexes were then used to recalculate AutoDock
docking scores and the VEGA energy scores.
azodicarboxylate; DME, dimethoxyethane; HBA, hydrogen
bond acceptor; HBA/π, hydrogen bond acceptor-π-electron
rich group; HBD, hydrogen bond donor; HEK, human embryonic
kidney; KB, Krebs buffer; MEM, methoxy ethoxy methyl; NA,
noradrenaline; nAChR, nicotinic acetylcholine receptor; SAR,
structureꢀactivity relationship; TEA, triethylamine
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’ ASSOCIATED CONTENT
Supporting Information. 1H NMR spectra and elemen-
S
b
tal analysis results for the final compounds 2ꢀ9 and 7a.
Preparations of 2-MEM-4-bromophenol and of 2-benzyloxyphe-
nol p-substituted with COCH3 and Ph. The alignment of six rat
nicotinic subunits (α4, α3, α6, α7, β2, β4). The complexes
between α4β2 nAChR binding site and (R,S)-4 and (S,S)-4. This
material is available free of charge via the Internet at http://pubs.
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’ AUTHOR INFORMATION
Corresponding Author
*Phone: +39 02 50319336. Fax: +39 02 50319359. E-mail:
marco.pallavicini@unimi.it.
’ ACKNOWLEDGMENT
This research was supported by the Universitꢀa degli Studi of
Milan and by the EC Neurocypres grant no. 202088 and the
TERDISMENTAL grant no. ID 16983 SAL-50 from Regione
Lombardia to Cecilia Gotti.
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’ ABBREVIATIONS USED
Boc, t-butoxycarbonyl; Cbz, carbobenzyloxy; CNS, central nervous
system; DA, dopamine; DBU, diazabicycloundecene; DCM, di-
chloromethane; DEAD, diethyl azodicarboxylate; DIAD, diisopropyl
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