(+)-Methyl (1 R, 2S)-2-{[4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl]methyl}-1-phenylcyclopropanecarboxylate [(+)-MR200] derivatives as potent and selective sigma receptor ligands: Stereochemistry and pharmacological properties

Article abstract of DOI:10.1021/acs.jmedchem.7b01584

Methoxycarbonyl-1-phenyl-2-cyclopropylmethyl based derivatives cis-(+)-1a [cis-(+)-MR200], cis-(-)-1a [cis-(-)-MR201], and trans-(±)-1a [trans-(±)-MR204], have been identified as new potent sigma (σ) receptor ligands. In the present paper, novel enantiomerically pure analogues were synthesized and optimized for their σ receptor affinity and selectivity. Docking studies rationalized the results obtained in the radioligand binding assay. Absolute stereochemistry was unequivocally established by X-ray analysis of precursor trans-(+)-5a as camphorsulfonyl derivative 9. The most promising compound, trans-(+)-1d, showed remarkable selectivity over a panel of more than 15 receptors as well as good chemical and enzymatic stability in human plasma. An in vivo evaluation evidenced that trans-(+)-1d, in contrast to trans-(-)-1d, cis-(+)-1d, or cis-(-)-1d, which behave as σ₁ antagonists, exhibited a σ₁ agonist profile. These data clearly demonstrated that compound trans-(+)-1d, due to its σ₁ agonist activity and favorable receptor selectivity and stability, provided an useful tool for the study of σ₁ receptors.

Full text of DOI:10.1021/acs.jmedchem.7b01584

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Article
(+)-Methyl (1R,2S)-2-{[4-(4-chlorophenyl)-4-hydroxypiperidin-1-
yl]methyl}-1-phenylcyclopropanecarboxylate [(+)-MR200]
Derivatives as Potent and Selective Sigma Receptor
Ligands: Stereochemistry and Pharmacological Properties
Emanuele Amata, Antonio Rescifina, Orazio Prezzavento, Emanuela Arena, Maria Dichiara, Valeria Pittalà,
Ángeles Montilla-García, Francesco Punzo, Pedro Merino, Enrique J. Cobos, and Agostino Marrazzo
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01584 • Publication Date (Web): 08 Dec 2017
Downloaded from http://pubs.acs.org on December 8, 2017
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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(
+)ꢀMethyl
(1R,2S)ꢀ2ꢀ{[4ꢀ(4ꢀchlorophenyl)ꢀ4ꢀhydroxypiperidinꢀ1ꢀyl]methyl}ꢀ1ꢀ
phenylcyclopropanecarboxylate [(+)ꢀMR200] Derivatives as Potent and Selective Sigma
Receptor Ligands: Stereochemistry and Pharmacological Properties
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†
†
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†
†
Emanuele Amata, Antonio Rescifina, Orazio Prezzavento, Emanuela Arena, Maria Dichiara,
†
‡
†
§
Valeria Pittalà, Ángeles Montilla-García, Francesco Punzo, Pedro Merino, Enrique J.
‡
†,
Cobos, Agostino Marrazzo *
†
Dipartimento di Scienze del Farmaco, Università di Catania, Viale A. Doria 6, 95125 Catania,
Italy.
‡
Institute of Neuroscience and Department of Pharmacology, Faculty of Medicine, University of
Granada, Avenida de Madrid 11, Eꢀ18012 Granada, Spain.
§
Laboratorio de Sıntesis Asimétrica, Departamento de Sıntesis y Estructura de Biomoléculas,
́
́
Instituto de Sıntesis Quımica y Catálisis Homogénea (ISQCH), Universidad de Zaragoza, CSIC,
́
́
Campus San Francisco, Eꢀ50009 Zaragoza, Aragón, Spain.
ABSTRACT
Methoxycarbonylꢀ1ꢀphenylꢀ2ꢀcyclopropylmethyl based derivatives cisꢀ(+)ꢀ1a [cisꢀ(+)ꢀMR200],
cisꢀ(–)ꢀ1a [cisꢀ(−)ꢀMR201], and transꢀ(±)ꢀ1a [trans-(±)ꢀMR204], have been identified as new
potent sigma (σ) receptor ligands. In the present paper, novel enantiomerically pure analogues
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were synthesized and optimized for their σ receptor affinity and selectivity. Docking studies
rationalized the results obtained in the radioligand binding assay. Absolute stereochemistry was
unequivocally established by Xꢀray analysis of precursor transꢀ(+)ꢀ5a as camphorsulfonyl
derivative 9. The most promising compound, transꢀ(+)ꢀ1d, showed remarkable selectivity over a
panel of more than fifteen receptors as well as good chemical and enzymatic stability in human
plasma. An in vivo evaluation evidenced that transꢀ(+)ꢀ1d, in contrast to transꢀ(–)ꢀ1d, cisꢀ(+)ꢀ1d,
or cisꢀ(–)ꢀ1d which behave as σ antagonists, exhibited a σ agonist profile.
0
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These data clearly demonstrated that compound transꢀ(+)ꢀ1d, due to its σ agonist activity and
1
favorable receptor selectivity and stability, provided an useful tool for the study of σ receptors.
1
INTRODUCTION
Sigma (σ) receptors, first introduced as subtypes of the opioid receptors, are now considered
unique class of proteins classified into two subtypes, sigmaꢀ1 (σ ) and sigmaꢀ2 (σ ) with peculiar
1
2
structure, biological functions and ligands sensitivity. Indeed, σ binding sites display high
1
affinity for dextro benzomorphan enantiomers like (+)ꢀpentazocine, while opioid receptors bind
1
ꢀ3
preferentially the levo isomers. The σ and σ receptor subtypes can be distinguished by
1
2
molecular weight (25 and 18‒21.5 kDa, respectively), tissue distribution, subcellular
4,5
localization, and ligand binding profile.
The σ receptor has been classified as a protein chaperone at the endoplasmatic reticulum (ER)ꢀ
1
2
+
6
mitochondrion interface that regulates Ca signaling and cell survival. Additional important
functions have also been well documented. Among these, the σ receptor modulates ion channel
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7
,8
activity and ER stress, having thus an important role in neurodegenerative disorders. In this
perspective, mutations of the σ receptor were linked to the establishment of amyotrophic lateral
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sclerosis in humans. In addition, it has been shown a close relationship between σ and opioid
1
receptors in pain modulation, where the σ system acts as antiopioid and deeply influences the
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0ꢀ12
sensitivity toward opioid analgesia, since σ antagonists enhance opioid antinociception.
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Ligands that specifically target the σ receptor have shown a plethora of pharmacological
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effects; in particular, σ antagonists, in addition to the modulation of opioid antinociception,
1
proved their ability in relieving neuropathic and inflammatory pain and to have antiproliferative
3
,14ꢀ23
and antiangiogenic effects, while σ agonists provided an efficacious neuroprotection.
The
1
σ1 receptor is also involved in other human diseases including schizophrenia, depression, and
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4,25
drug/alcohol addiction.
The σ receptor is overexpressed in a wide variety of cancer tissues
2
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6ꢀ28
and due to this peculiarity has been used for the generation of biomarker for cancer imaging.
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Moreover, σ receptor ligands are known to cause cancer cell death in vitro and in vivo.
2
Recently, the σ receptor was purified, revealing its identity as the transmembrane protein 97
2
(TMEM97), an endoplasmic reticulumꢀresident transmembrane protein that regulates the sterol
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transporter Niemann–Pick disease protein (NPC1). Some quantitative structure–activity
32ꢀ35
relationship (QSAR) models for the determination of σ receptors binding were setup.
Although no σ receptors ligands have been yet marketed as drugs, there are ongoing clinical
trials aimed at their evaluation. Thus, σ receptors represent attractive targets useful for
neuropathic pain management, neurodegenerative disorders, and as brand new tools for cancer
treatment and diagnosis.
Haloperidol, a typical neuroleptic agent, is a promiscuous drug showing high affinity for a panel
of targets including the main dopaminergic D , D , D , D , serotoninergic 5ꢀHT , adrenergic
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4.2
2A
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6
α , σ and σ receptors. Haloperidol has already been used for the design of selective σ
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7
receptors ligands.
Indeed, we previously reported two methoxycarbonylꢀ1ꢀphenylꢀ2ꢀ
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cyclopropylmethyl based compounds, cisꢀ(+)ꢀ1a [cisꢀ(+)ꢀMR200] and cisꢀ(–)ꢀ1a [cisꢀ(−)ꢀ
MR201], with a notable improvement of pharmacological in vitro σ receptors selectivity with
respect to haloperidol. At the same time, we reported the synthesis and the radioligand binding
3
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profile of transꢀ(±)ꢀ1a [trans-(±)ꢀMR204] (Figure 1).
Figure 1. Structures of sigma receptors ligands.
We noted that the transꢀ(±)ꢀ1a racemic mixture, while keeping a doubleꢀdigit nanomolar affinity
for σ and σ receptors, showed a similar affinity toward dopaminergic D receptor with respect
1
2
2
to compound cisꢀ(+)ꢀ1a, but a lower affinity with respect compound cisꢀ(–)ꢀ1a, [K = 3230, 378
i
and 5200 nM for compounds cisꢀ(+)ꢀ1a, cisꢀ(–)ꢀ1a, and transꢀ(±)ꢀ1a, respectively]. While
considering that these molecules share the same 4ꢀ(4ꢀchlorophenyl)piperidinꢀ4ꢀol portion, our
rationale is driven by the hypothesis that the fragment Nꢀtethered to piperidine nucleus may be a
major driver for selectivity towards σ receptors with respect to dopaminergic receptors. Thus,
here we report the synthesis and pharmacological characterization of the enantiomerically pure
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compounds transꢀ(+)ꢀ1a and transꢀ(–)ꢀ1a. Moreover, to evaluate the importance of the ester
substituent on σ receptor affinity, we synthesized the corresponding ethyl, nꢀpropyl and
isopropyl esters of both cisꢀand transꢀ1a enantiomerically pure compounds. The designed
39,40
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compounds fit known pharmacophore models for σ receptor recognition.
Finally, the
absolute stereochemistry of synthesized compounds was confirmed by a comparison with a
transꢀ(+)ꢀ5a precursor. Stereochemistry of this precursor has been in turn established by Xꢀray
analysis upon transformation of the corresponding camphorsulfonyl derivative 9 (see supporting
information).
RESULTS AND DISCUSSION
Chemistry
The synthetic pathway of compounds cisꢀ1b–d and transꢀ1a–d was pursued by coupling the
intermediates transꢀ6a–d, and cisꢀ7b–d, synthesized following the previously reported
procedures (see SI), with 4ꢀ(4ꢀchlorophenyl)piperidinꢀ4ꢀol (8) in the appropriate solvent and base
36,38,41ꢀ47
under reflux conditions (Scheme 1).
The acid derivative compound transꢀ(–)ꢀ1e was
produced by hydrolysis of the corresponding ethyl ester compound transꢀ(–)ꢀ1b (Scheme 1). All
1
13
compounds were characterized by means of their Hꢀ and CꢀNMR spectroscopic data. The
optical purity was determined by chiral HPLC and reported as percentage of enantiomeric excess
4
8
(
e.e.). Moreover its absolute stereochemistry was also confirmed by comparison with the
configuration of the camphorsulfonyl derivative of 9 (Figure 2) as specified in the Experimental
Section.
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Scheme 1. Synthetic strategy for the preparation of target compounds. Reagents and conditions:
(
i) NaNH , (S)ꢀ(+)ꢀ or (R)ꢀ(–)ꢀepichlorohydrin, benzene, rt, 2 h; (ii) KOH, EtOH, reflux, 15 h;
2
(iii) 150 °C, 30 min at atmospheric pressure, then 45 min at 15 mmHg; (iv) ROH, H SO , reflux,
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h; (v) SOBr , ROH, rt, 24 h; (vi) tosyl chloride, pyridine, CH Cl , 0 °C, 24 h; (vii) no base or
2 2 2
KHCO or NaHCO , ROH or DMF, 75–80 °C, 9 h; (viii) Transꢀ(+)ꢀ1b, LiOH, MeOH/H O, 60
3
3
2
°
C, 3 h.
Xꢀray Analysis
With the aim of determining the absolute stereochemistry, compound 9 (Figure 2) has been
crystallized in the monoclinic P21 space group with one molecule in the asymmetric unit, by
using diethyl ether. Colorless block crystals were obtained. Its unit cell refined to a = 6.395Å, b
=
16.488 Å, c = 10.488 Å, β = 107.02 °. No residual solvent accessible voids were evidenced as
49
a consequence of a fairly high (67.9%) packing index. Two linear, weak and intermolecular
hydrogen bonds are present – namely C2–H2A⋯O6 and C17–H17C⋯O6 – both involving O6 in
5
0,51
an adjacent molecule (symmetry code x,y,ꢀ1+z).
They contribute to the stabilization of the
5
2,53
whole molecular architecture.
A ring puckering analysis highlights the presence of two fiveꢀmembered rings with a typical
envelope form – C1–C4–C12–C8–C7 and C1–C4–C12–C14–C9 – as well as a boat form for the
sixꢀmembered ring – C1–C7–C8–C12–C14–C9 – resulting from the merging of the above
5
4ꢀ56
mentioned fiveꢀmembered ones.
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Figure 2. 2D and Xꢀray ortep structures of compound 9.
Radioligand Binding and σ and σ Receptor Affinities
1
2
The synthesized compounds [cisꢀ(+)ꢀ1b–d, cisꢀ(–)ꢀ1b–d, transꢀ(+)ꢀ1a–d, and transꢀ(–)ꢀ1a–d]
were evaluated for their affinities for both σ and σ receptors (Table 1 and 2). Moreover,
1
2
compounds transꢀ(–)ꢀ1a and transꢀ(+)ꢀ1a, were also evaluated for their affinity against the
dopaminergic receptors D and D . All the reported compounds show a high affinity towards σ
1
1
2
receptor and a σ /σ ratio ranging from 1.1 to 262.4.
2
1
a
Table 1. Radioligand binding assays
a
Compound
K ± SD (nM)
i
Selectivity
σ1
σ2
D1
D2
σ2/σ
1
transꢀ(+)ꢀ1a
transꢀ(–)ꢀ1a
129 ± 4.2
11 ± 0.8
142 ± 4.8
40 ± 3.8
21.9 ± 2.4
23.4 ± 2.7
61.1 ± 4.5
16 ± 2.7
>10,000
>10,000
>10,000
>10,000
>10,000
318 ± 59
4,970 ± 148
5,624 ± 164
3,230 ± 110
378 ± 49
1.1
3.6
b
cisꢀ(+)ꢀ1a
1.51 ± 0.2
5.6 ± 0.4
14.6 ± 1.3
2.2 ± 0.5
14.5
4.2
4.2
b
cis-(–)ꢀ1a
b
trans-(±)ꢀ1a
5,200 ± 241
2.1 ± 0.3
b
Haloperidol
7.3
a
b
38
Each value is the mean ± SD of three determinations. Data from following reference.
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The methyl esters transꢀ(+)ꢀ1a and transꢀ(–)ꢀ1a showed a slightly lower affinity with respect to
parent cis methyl ester compounds (+)ꢀ and (–)ꢀ1a, with K σ of 129 and 11 nM and K σ of 142
i
1
i
2
and 40 nM, respectively (Table 1). The compound transꢀ(–)ꢀ1a demonstrated a better affinity
profile with respect to its racemic mixture transꢀ(±)ꢀ1a. While the compound transꢀ(+)ꢀ1a,
showed a lower affinity towards both σ receptor subtypes in comparison with transꢀ(±)ꢀ1a and
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transꢀ(–)ꢀ1a. With regard to dopaminergic receptors D and D , compounds transꢀ(–)ꢀ1a and
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transꢀ(+)ꢀ1a evidenced a similar affinity to the racemic mixture transꢀ(±)ꢀ1a [K D >10000 nM
i
1
and K D 4970, 5624, and 5200 nM for transꢀ(–)ꢀ1a, transꢀ(+)ꢀ1a, and transꢀ(±)ꢀ1a,
i
2
respectively]. In general, the trans methyl ester compounds with respect to the cis methyl ester
compounds and haloperidol, present a better σ receptors selectivity over dopaminergic receptors.
When the ester modification takes place, cis and trans derivatives behave differently (Table 2).
Within the cis series, the elongation or branching of the alkyl chain determined a limited
reduction in both σ and σ affinity for both dextrorotatory and levorotatory series (nucleus A
1
2
and B respectively), with compound cisꢀ(+)ꢀ1b being the most active and selective with K σ of
i
1
1
.35 nM and K σ of 57 nM respectively. Thus, dextro cis methyl and ethyl ester compounds
i 2
displayed the best affinity profile, with the ethyl ester compound cisꢀ(+)ꢀ1b showing a better
selectivity (σ /σ = 14.5 and 42.2 for compounds cisꢀ(+)ꢀ1a and cisꢀ(+)ꢀ1b, respectively). In the
2
1
trans dextro series (nucleus C), the alkyl chain branching gave rise to an optimal affinity; in
particular, compound transꢀ(+)ꢀ1d, bearing an iꢀPr group, exhibited a K σ of 7 nM and K σ of
i
1
i
2
1837 nM, with a notable 262ꢀfold selectivity for σ receptor. Comparable results were observed
1
for the other trans compounds, in both dextro (nucleus C) and levo (nucleus D) configurations,
showing a good σ and a weaker σ affinity (K σ ranging from 12 to 52 nM and K σ ranging
1
2
i
1
i
2
from 73 to 1300 nM respectively). These results suggest that the alkyl chain bonded to the ester
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function may play a pivotal role in discriminating between the sigma receptor subtypes
5
7
depending on the appropriate stereochemistry of cyclopropyl skeleton. Finally, in order to
evaluate the importance of the ester function in the σ receptor binding affinity, we evaluated the
acid derivative of the trans dextro (nucleus C) series [transꢀ(+)ꢀ1e]. Results showed that the acid
compound transꢀ(+)ꢀ1e has negligible affinity at both σ receptor subtypes (Table 2).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Table 2. Radioligand binding Assays of enantiomerically pure compounds.
a
K ±SD (nM)
i
Selectivity
σ2/σ1
42.2
1
Compound
cisꢀ(+)ꢀ1b
Nucleus
R
σ1
σ2
A
A
A
B
B
B
C
C
C
C
Et
nꢀPr
iꢀPr
Et
1.35 ±0.1
14 ±2.1
20 ±3.4
45 ±2.8
33 ±4.6
67 ±5.1
52 ±4.0
13 ±1.4
7 ±0.6
57 ±3.7
cisꢀ(+)ꢀ1c
146 ±7.0
209 ±8.7
918 ±26.1
290 ±14.4
1,638 ±64.3
1,300 ±45.7
171 ±16.9
1,837 ±49.5
650 ±31
10.4
cisꢀ(+)ꢀ1d
cis-(–)ꢀ1b
10.5
20.4
cis-(–)ꢀ1c
nꢀPr
iꢀPr
Et
8.8
cis-(–)ꢀ1d
24.4
transꢀ(+)ꢀ1b
transꢀ(+)ꢀ1c
transꢀ(+)ꢀ1d
transꢀ(+)ꢀ1e
25.0
nꢀPr
iꢀPr
H
13.2
>250
1.5
445 ±21
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transꢀ(–)ꢀ1b
transꢀ(–)ꢀ1c
D
D
D
Et
45 ±2.9
12 ±0.9
43 ±4.7
107 ±9.1
73 ±8.2
2.4
6.1
5.0
nꢀPr
iꢀPr
transꢀ(–)ꢀ1d
214 ±11.2
a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Each value is the mean ±SD of three determinations.
58
Finally, we evaluated a ratio for the considered enantiomerically pure compounds. By
analyzing the ratios reported in Table 3, it can be noticed that there is a slight preference of the
pure enantiomers cis dextro and trans levo for both receptor subtypes. Within the series, the
unique exception is compound transꢀ(+)ꢀ1d, which shows an opposite profile.
Table 3. Ratios for σ and σ receptor subtypes of enantiomerically pure compounds.
1
2
K (nM)
K (nM)
i
Ratio
i
Compound
cisꢀ(+)ꢀ1a
σ1
σ2
21.9
57
Compound
cis-(–)ꢀ1a
σ1
σ2
23.4
918
290
1638
40
(–/+) σ1
3.71
(–/+) σ2
1.07
1.51
1.35
14
5.6
45
33
67
11
45
12
43
cisꢀ(+)ꢀ1b
cis-(–)ꢀ1b
33.33
2.36
16.11
1.99
cisꢀ(+)ꢀ1c
146
209
142
1300
171
1837
cis-(–)ꢀ1c
cisꢀ(+)ꢀ1d
20
cis-(–)ꢀ1d
3.35
7.84
transꢀ(+)ꢀ1a
transꢀ(+)ꢀ1b
transꢀ(+)ꢀ1c
transꢀ(+)ꢀ1d
129
52
transꢀ(–)ꢀ1a
transꢀ(–)ꢀ1b
transꢀ(–)ꢀ1c
transꢀ(–)ꢀ1d
0.09
0.28
107
73
0.87
0.08
13
0.92
0.43
7
214
6.14
0.12
Given the notable profile of compound transꢀ(+)ꢀ1d, we investigated the binding affinities
towards other pharmacological systems, its water solubility, chemical and enzymatic stability.
1
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The σ agonist or antagonist in vivo pharmacological activity for cisꢀ and transꢀ1d isopropyl
1
esters was also investigated through determination of the modulation of opioid antinociception.
0
1
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9
0
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0
Binding affinity profile
Binding studies to assess the affinities of compound transꢀ(+)ꢀ1d towards opioid, dopaminergic,
serotoninergic, and adrenergic receptors showed its very low affinity for opioid, dopamine (D1,
D , D , D ), 5HT, and α receptors compared to Haloperidol and 1,3ꢀdiꢀ(2ꢀtolyl)guanidine
2
3
4.2
1
(DTG). In addition, transꢀ(+)ꢀ1d did not show any significant affinity for other receptors or
transporters tested (see supporting information, Table S2).
Docking studies
A docking study was performed to probe and identify the key molecular interactions involved
into the protein/ligand recognition and in order to better understand the observed affinity. To this
extent, the crystal structure of the human σ receptor model bound to the highꢀaffinity and
1
selective the σ antagonist PD144418 were used. To allow each ligand to appropriately adapt to
1
the catalytic site we carried out this study utilizing a threeꢀstep sequence: i) docking of ligand
upon σ receptor; ii) 5 ns of molecular dynamic (MD) simulation of the best pose obtained for
1
ligandꢀσ receptor complex, in order to accommodate the ligand; iii) reꢀdocking of the complex
1
obtained from the last 3 ns of MD simulation averaged frames. Since compounds 1 possess a
tertiary amine functionality, by considering physiological conditions (pH = 7.4), we performed
all the studies on the Nꢀprotonated structures.
The calculated binding energies (ꢁG ) and K values to the catalytic site of the σ receptor, for
B
i
1
compounds 1a,d, haloperidol, and PD144418 are reported in Table 4. These data show that
1
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compound transꢀ(+)ꢀ1d has the lowest binding energy and, although all calculated K values are
i
overestimated by a factor of roughly 4.5ꢀ and 2.5ꢀfold for compounds 1a and 1d, respectively,
their relative magnitudes are well in accord with the experimental results. Even for haloperidol
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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9
0
1
2
3
4
5
6
7
8
9
0
and PD144418 there is an overestimation of the K values comparable to that of compounds 1.
i
Table 4. Calculated binding energies (kcal/mol) and inhibition constants (nM) to the binding site
of the σ receptor for compounds 1a,d.
1
Compound
transꢀ(+)ꢀ1a
transꢀ(–)ꢀ1a
cisꢀ(+)ꢀ1a
Calculated ꢁGB
–8.5
Calculated Ki
K calcd. – K exp.
i i
583
46
454.0
35.0
37.5
71.4
10.0
65.0
35.0
145.0
7.8
–10.0
–10.1
–9.7
39
cis-(–)ꢀ1a
77
transꢀ(+)ꢀ1d
transꢀ(–)ꢀ1d
cisꢀ(+)ꢀ1d
–10.6
–9.5
17
108
55
–9.9
cis-(–)ꢀ1d
–9.1
212
10
Haloperidol
–10.9
a
PD144418
–10.3
28
23.7
a
59
Experimental K σ 4.3 nM.
i
1
Active site analysis for each of the four stereoisomers corresponding to compounds 1a,d showed
that, for each of them, the chemical scaffold has the same orientation. Moreover, the position of
the protonated piperidine ring, for all ligands, is rather characteristic being near to the carboxylic
moiety of Glu172. In the case of the compounds transꢀ(+)ꢀ1a, transꢀ(–)ꢀ1a, transꢀ(+)ꢀ1d, transꢀ
(–)ꢀ1d, and cisꢀ(+)ꢀ1d the piperidinium proton engages a salt bridge interaction with the Glu172
(Figure 3) and, in the case of transꢀ(+)ꢀ1a, there is also be a hydrogen bond between the
1
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piperidinium hydrogen and the oxygen atom of the Tyr103 residue. However, these interactions
are not sufficient to effectively stabilize these poses with respect to the other; indeed, both cisꢀ
(+)ꢀ1a and cis-(–)ꢀ1 result more active than the transꢀ(+)ꢀ1a.
0
1
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0
1
2
3
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5
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8
9
0
1
2
3
4
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8
9
0
Figure 3. (Left) 3D superposition of the bestꢀdocked pose for transꢀ(+)ꢀ1a (blu) transꢀ(–)ꢀ1a
(red) cisꢀ(+)ꢀ1a (magenta) cis-(–)ꢀ1a (yellow). (Right) The same for transꢀ(+)ꢀ1d (blu) transꢀ(–)ꢀ
1d (red) cisꢀ(+)ꢀ1d (magenta) cis-(–)ꢀ1d (yellow).
The bestꢀdocked pose (i.e. the binding pose with the lowest energy in the binding site whose geometry
and/or orientation is consistent with that of the original ligand) of transꢀ(+)ꢀ1d, with amino acidic
interactions in the binding pocket of the σ receptor is represented in Figure 4 in both 2D and 3D
1
arrangement (top and bottom).
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Figure 4. Compound transꢀ(+)ꢀ1d complexed with σ receptor from docking. 2D Schematic
1
view of hydrophobic and hydrogen bond interactions (top). 3D analog, the ligand is represented
as stick whereas amino acids as a small stick (bottom).
Water solubility and chemical and enzymatic stability
The water solubility and chemical stability of transꢀ(+)ꢀ1d were experimentally determined
while LogP was theoretically calculated (Table 4). Compound transꢀ(+)ꢀ1d displayed moderate
water solubility (3.1 mg/mL) and a LogP value below 5 (theoretically calculated logP 4.37). The
1
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chemical stability of transꢀ(+)ꢀ1d was checked in vitro at 37 ºC in an aqueous buffer solutions at
pH 1.3 (nonꢀenzymatic simulated gastric fluid) as well as pH 7.4. The compound showed good
stability both at pH 1.3 and 7.4 (84 and 91 hours, respectively). The enzymatic stability of transꢀ
0
1
2
3
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0
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0
(+)ꢀ1d was also studied at 37 ºC in rat, mouse, and human plasma. As evidenced by data in Table
5
, the compound undergoes a rapid hydrolysis in rat plasma, whereas in mouse and human
plasma it is fairly stable.
Table 5. Water solubility, cLogP, chemical and enzymatic stabilities for compound transꢀ(+)ꢀ1d
transꢀ(+)ꢀ1d
‒
1
t½ (h)
kobs (h )
a
Chemical Stability
pH 1.3
83.6 ±3.4
91.0 ±4.3
0.38 ±0.004
1.14 ±0.009
65.2 ±1.8
0.009 ±0.001
0.009 ±0.001
3.26 ±0.09
0.51 ±0.02
0.01 ±0.001
pH 7.4
a
Enzymatic Stability
Rat plasma
Mouse plasma
Human plasma
b
Water solubility (mg/mL)
3.1 ±0.2
c
LogP
4.37
a
b
c
Values are means ±SD of three experiments. Value are means ±SD of three experiments. nꢀ
Octanol/water partition coefficients were theoretically calculated by the ChemAxon program
JChem for Excel 14.9.100.809.
In vivo pharmacological activity
Mechanical antinociception induced by the tested drugs was assessed by monitoring the increase
in the struggle response latency to the nociceptive mechanical stimulus applied to the hindpaw.
Subcutaneous (s.c.) administration of transꢀ(–)ꢀ1d (32 mg/kg) alone did not alter the struggle
response latency to the mechanical stimulus in comparison to control mice treated with HPMC
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(
Figure 5A). A low dose of s.c. morphine (4 mg/kg) was also unable to alter the behavioral
response to the nociceptive stimulation (Figure 5A). However, when we tested the association of
morphine with transꢀ(–)ꢀ1d (16‒32 mg/kg), we observed a doseꢀdependent increase in the
response latency, indicating a synergistic antinociceptive effect (Figure 5A). Very similar results
were found when testing the effects of cisꢀ(+)ꢀ1d (16‒32 mg/kg, s.c.) and cisꢀ(–)ꢀ1d (12ꢀ16
mg/kg, s.c.): these compounds were unable to induce any antinociceptive effect when
administered alone, but they both markedly enhanced morphine antinociception (Figure 5B and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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0
5
C, respectively). These results fully agree with the widely reported effects of σ antagonists on
1
1
2,60ꢀ64
opioid antinociception.
Furthermore they evidenced a σ antagonist profile of the tested
1
compounds. We then studied the effects of the σ agonist PREꢀ084 (32 mg/kg, s.c.) on the
1
synergistic antinociceptive effect induced by the association of transꢀ(–)ꢀ1d, cisꢀ(+)ꢀ1d or cisꢀ(–
)ꢀ1d with morphine. This dose of PREꢀ084 has been shown to reverse the effects of prototypical
12,60,61
σ1 antagonists on opioid antinociception.
We found that PREꢀ084 completely reversed the
potentiation of morphine antinociception induced by transꢀ(–)ꢀ1d, cisꢀ(+)ꢀ1d and cisꢀ(–)ꢀ1d.
These results further confirm that the effects induced by these three compounds on morphine
antinociception were mediated by antagonism at σ receptor.
1
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Figure 5. Effects of the administration of transꢀ(–)ꢀ1d, cisꢀ(+)ꢀ1d and cisꢀ(–)ꢀ1d on mechanical
nociception and on the antinociceptive effect induced by morphine. The results represent the
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struggle response latency during stimulation of the hindpaws with 450 g pressure. Mice were
treated subcutaneously (s.c.) with morphine (4 mg/kg) alone or associated with (A) transꢀ(–)ꢀ1d,
(B) cisꢀ(+)ꢀ1d or (C) cisꢀ(–)ꢀ1d. Animals treated with the association of morphine with transꢀ(–)ꢀ
1
1
1
1
1
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1
1
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1
d, cisꢀ(+)ꢀ1d or cisꢀ(–)ꢀ1d were administered with the σ agonist PREꢀ084 (32 mg/kg, s.c.).
1
HPMC was used as the solvent of all drugs tested. Each bar and vertical line represents the mean
SEM of values obtained at both hindpaws in 6–8 animals. Oneꢀway analysis of variance
±
followed by the Bonferroni test was used to determine statistically significant differences
between the values obtained in the groups treated with HPMC and the groups treated with
morphine associated with transꢀ(–)ꢀ1d, cisꢀ(+)ꢀ1d and cisꢀ(–)ꢀ1d (*P < 0.05; **P < 0.01) and
between the values obtained in mice treated with each of these drug combinations alone or
associated with PREꢀ084 (##P < 0.01).
On the other hand, transꢀ(+)ꢀ1d (16‒32 mg/kg, s.c.) did not potentiate the antinociceptive effect
of morphine (Figure 6). These results are in marked contrast to those obtained using transꢀ(–)ꢀ
1
d, cisꢀ(+)ꢀ1d, cisꢀ(–)ꢀ1d, and therefore suggesting that transꢀ(+)ꢀ1d does not exert σ1
antagonistic actions. We then tested whether this compound might act as a σ receptor agonist by
1
reversing the effects of the prototypical σ antagonist BDꢀ1063 on morphine antinociception.
1
BDꢀ1063 (8 mg/kg, s.c.) enhanced morphineꢀinduced antinociceptive effects (Figure 6), in
6
0,61
agreement with previous studies .
The association of transꢀ(+)ꢀ1d (32 mg/kg, s.c.) with BDꢀ
1
063 completely reversed the potentiation of morphine antinociception induced by this known σ1
receptor antagonist.
Therefore, our results suggest that transꢀ(–)ꢀ1d, cisꢀ(+)ꢀ1d or cisꢀ(–)ꢀ1d act as σ receptor
1
antagonists, while transꢀ(+)ꢀ1d acts as a σ receptor agonist.
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Figure 6. Effects of the administration of transꢀ(+)ꢀ1d on mechanical nociception and on the
antinociceptive effect induced by the association of BDꢀ1063 and morphine. The results
represent the struggle response latency during stimulation of the hindpaws with 450 g pressure.
Mice were treated subcutaneously (s.c.) with morphine (4 mg/kg) alone or associated with transꢀ
(+)ꢀ1d. Animals treated with the association of morphine with transꢀ(+)ꢀ1d were administered
with the σ antagonist BDꢀ1063 (8 mg/kg, s.c.). HPMC was used as the solvent for all tested
1
drugs. Each bar and vertical line represents the mean ± SEM of values obtained at both hindpaws
in 6–8 animals. Oneꢀway analysis of variance followed by the Bonferroni test was used to
determine statistically significant differences between the values obtained in the groups treated
with HPMC and the group treated with morphine associated with transꢀ(+)ꢀ1d (**P < 0.01) and
between the values obtained in mice treated with transꢀ(+)ꢀ1d + morphine associated with BDꢀ
1
063 (##P < 0.01).
CONCLUSIONS
The present paper deals with the synthesis and the optimization of a new series of
enantiomerically pure analogues of compound 1a in which the role of the ester function has been
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investigated. The chosen synthetic route afforded four different diastereoisomers in high purity
and the absolute stereochemistry was unequivocally established by means of Xꢀray single crystal
diffraction. Molecular docking studies highlighted for each of the four stereoisomers the same
scaffold orientation with a characteristic salt bridge interaction with Glu172. In radioligand
binding assay compound cisꢀ(+)ꢀ1b revealed to be the most potent but rather unselective over σ2
1
1
1
1
1
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receptor (σ /σ ratio ≈ 40). Compound transꢀ(+)ꢀ1d, though maintaining a strong nanomolar
2
1
affinity for σ receptor, possessed the best selectivity ratio over σ (σ /σ ratio >250) and a
1
2
2
1
remarkable selectively against a panel of more than fifteen receptors. In addition, results showed
that the acid compound transꢀ(+)ꢀ1e has negligible affinity at both σ receptor subtypes
highlighting the importance of the ester function in the σ receptor binding affinity. Compound
transꢀ(+)ꢀ1d, with the best combined pharmacological profile from the in vitro characterization,
good chemical and enzymatic stability in human plasma also performed as σ agonist in in vivo
1
studies. Conversely, compounds transꢀ(–)ꢀ1d, cisꢀ(+)ꢀ1d, and cisꢀ(–)ꢀ1d, exhibited σ antagonist
1
profile, providing evidence that stereochemistry on cyclopropane ring drives not only affinity
and selectivity but also functional activity profile at σ receptor.
1
As a whole, these studies provided the evidence that transꢀ(+)ꢀ1a may provide an important
chemical probe which will enable the investigation to further clarify σ receptor function.
1
EXPERIMENTAL SECTION
Chemistry
General Details. Reagents used for synthesis were purchased from SigmaꢀAldrich (Milan, Italy)
unless otherwise specified. The reaction was monitored by thin layer chromatography (TLC) on
precoated silica gel 60 F254 aluminum sheets (Merck, Darmstadt, Germany). Visualization of
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TLC spots was performed under ultraviolet (UV) light or in an iodine chamber. Merck silica gel
6
0, 230ꢀ400 mesh, was used for flash column chromatography. Melting points were obtained in
open capillary tubes with a Büchi 530 apparatus (Büchi Italia, Assago, Italy) and are
1
13
0
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uncorrected. H and C nuclear magnetic resonance (NMR) spectra were recorded with a Varian
Inova 200 MHz spectrometer (Varian, Leini, Italy). Chemical shifts are reported in δ values
(ppm) relative to an internal standard of tetramethylsilane. Optical rotations were determined in
MeOH or CH Cl (c = 1) with a PerkinꢀElmer 241 polarimeter. Elemental analyses (C, H, N)
2
2
were determined on an elemental analyzer, Carlo Erba model 1106 (Carlo Erba, Milan, Italy),
and the results were within 0.4% of the theoretical values. The optical purity was determined by
chiral HPLC analysis performed on a Perkin Elmer Flexar FXꢀ10 UHPLC System (software
Analyst 1.6.1) with a Daicel chemical industries chiral column ChiralcelꢀOJ, 0.46x250 mm,
using a singleꢀwavelength UV–visible detector. Compounds nomenclature were generated with
3
ChemBioDraw Ultra version 16.0.0.82. Radioactive materials. [ H]ꢀ(+)ꢀPentazocine (45
3
3
3
Ci/mmol), [ H]ꢀDTG (31 Ci/mmol), [ H]ꢀSCH23390 (75.5 Ci/mmol), [ H]ꢀSpiperone (20
3
3
3
Ci/mmol), [ H]ꢀQNB (42 Ci/mmol), [ H]ꢀKetanserin (63.3 Ci/mmol), [ H]ꢀ5HT (80 Ci/mmol),
3
3
3
3
3
3
[
[
[
[
H]ꢀPrazosin (83.8 Ci/mmol), [ H]ꢀPyrilamine (20 Ci/mmol), [ H]ꢀCGP39653 (40 Ci/mmol),
3
3
H]ꢀWIN 35,428 (86 Ci/mmol), [ H]ꢀMK801 (30 Ci/mmol), [ H]ꢀ7OHꢀDPAT (139 Ci/mmol),
3
3
H]ꢀMesulergine (86 Ci/mmol), [ H]ꢀLY278584 (84 Ci/mmol), [ H]ꢀGR113808 (81 Ci/mmol),
3
3
H]ꢀCitalopram (85 Ci/mmol), [ H]ꢀRX821002 (49 Ci/mmol), and [ H]ꢀNaloxone (55.5
Ci/mmol) were obtained from PerkinꢀElmer.
General procedure for compounds transꢀ(+)ꢀ1a, transꢀ(–)ꢀ1a: Procedure B. A mixture of
anhydrous MeOH (10 mL), 4ꢀ(4ꢀchlorophenyl)piperidinꢀ4ꢀol (2.07 mmol), trans tosylꢀester (0.83
mmol), and NaHCO (0.83mmol) was kept under magnetic stirring for 9 hours at 80 °C under
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argon atmosphere in a sealed tube (Ace pressure tube). The tube content was transferred in a
round bottom flask and the solvent evaporated under vacuum. The crude was dissolved in
CH Cl , filtered over filter paper and washed with a saturated solution of NaHCO and brine.
2
2
3
1
1
1
1
1
1
1
1
1
1
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The organic solvent was dried over Na SO and removed in vacuum. The crude product was
2
4
purified via silica gel chromatography. After purification the pure product was dissolved in
diethyl ether and a solution of oxalic acid in diethyl ether was added dropwise to obtain the
desired product as oxalic acid salt.
Methylꢀ(1S,2S)ꢀ2ꢀ((4ꢀ(4ꢀchlorophenyl)ꢀ4ꢀhydroxypiperidinꢀ1ꢀyl)methyl)ꢀ1ꢀ
phenylcyclopropaneꢀ1ꢀcarboxylate [transꢀ(+)ꢀ1a]: yield 47%; [α]ꢁꢂ +38° (c 1, MeOH); white
ꢀ
solid; mp 180–182 °C. According to general procedure B. Compound transꢀ(+)ꢀ1a was
synthesized starting from the tosyl derivative transꢀ(+)ꢀ6a and purified via silica gel
1
chromatography, eluting with 90% EtOAc in cyclohexane. HꢀNMR (200 MHz, DMSOꢀd ) (free
6
base) δ 1.58–1.80 (m, 4H), 1.85–2.05 (m, 1H), 2.05–2.30 (m, 2H), 3.00–3.42 (m, 6H), 3.55 (s,
1
3
3
H), 5.58 (bs, 5H), 7.22–7.50 (m, 9H). CꢀNMR (200 MHz, DMSOꢀd ) (free base) δ 20.92,
6
2
0.99, 32.38, 34.92, 47.80, 48.24, 52.52, 56.34, 67.91, 126.70, 127.63, 128.04, 128.27, 131.06,
131.46, 134.26, 146.99, 164.51, 173.19. Anal. calcd for: C H ClNO H C O ·0.8 H O: C,
2
3
26
3
2
2
4
2
5
9.53; H, 5.92; N, 2.78. Found: C, 59.53; H, 6.03; N, 2.86.
Methylꢀ(1R,2R)ꢀ2ꢀ((4ꢀ(4ꢀchlorophenyl)ꢀ4ꢀhydroxypiperidinꢀ1ꢀyl)methyl)ꢀ1ꢀ
phenylcyclopropaneꢀ1ꢀcarboxylate [transꢀ(–)ꢀ1a]: yield 51%; [α]ꢁꢂ –40° (c 1, MeOH); white
ꢀ
solid; mp 180–182 °C. According to general procedure B. Compound transꢀ(–)ꢀ1a was
synthesized starting from the tosyl derivative transꢀ(–)ꢀ6a and purified via silica gel
1
chromatography, eluting with 90% EtOAc in cyclohexane HꢀNMR (200 MHz, DMSOꢀd ) (free
6
base) δ 1.58–1.80 (m, 4H), 1.85–2.05 (m, 1H), 2.05–2.30 (m, 2H), 3.00–3.42 (m, 6H), 3.55 (s,
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H), 5.58 (bs, 5H), 7.22–7.50 (m, 9H). CꢀNMR (200 MHz, DMSOꢀd ) (free base) δ 20.93,
6
1.09, 32.36, 34.99, 47.82, 48.27, 52.50, 56.14, 67.95, 126.70, 127.61, 128.04, 128.26, 131.07,
131.44, 134.30, 147.04, 164.56, 173.21. Anal. calcd for: C H ClNO H C O ·0.8 H O: C,
2
3
26
3
2
2
4
2
0
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9.53; H, 5.92; N, 2.78. Found: C, 59.83; H, 6.13; N, 2.96.
Compound Synthesis. General procedure for compounds cisꢀ(+)ꢀ1b–d, cisꢀ(–)ꢀ1b–d:
Procedure A. A mixture of the anhydrous alcohol (10 mL), 4ꢀ(4ꢀchlorophenyl)piperidinꢀ4ꢀol
(
328 mg, 1.55 mmol) and the bromoꢀester (0.70 mmol), was kept under magnetic stirring for 9
hours at 80 °C under argon atmosphere in a sealed tube (Ace pressure tube). The tube content
was transferred into a round bottom flask and the solvent evaporated under vacuum. The crude
was dissolved in CH Cl and washed with a saturated solution of NaHCO and brine. The
2
2
3
organic solvent was dried over Na SO and removed in vacuum. The crude product was purified
2
4
via silica gel chromatography. After purification the pure product was dissolved in diethyl ether
and a solution of oxalic acid in diethyl ether was added dropwise to obtain the desired product as
oxalate. According to this procedure the following compounds were obtained.
Ethylꢀ(1R,2S)ꢀ2ꢀ((4ꢀ(4ꢀchlorophenyl)ꢀ4ꢀhydroxypiperidinꢀ1ꢀyl)methyl)ꢀ1ꢀ
phenylcyclopropaneꢀ1ꢀcarboxylate [cisꢀ(+)ꢀ1b]: yield 78%; [α]ꢁꢂ +26° (c 1, MeOH); light
ꢀ
yellow sticky solid. Compound cisꢀ(+)ꢀ1b was synthesized starting from the bromo derivative
cisꢀ(+)ꢀ7b in anhydrous EtOH and purified via silica gel chromatography, eluting with 90%
1
EtOAc in cyclohexane. HꢀNMR (200 MHz, CDCl ) (free base) δ 1.16 (t, 3H, J = 7.0 Hz), 1.34
3
(
dd, 1H, J = 4.2, 8.8 Hz), 1.65 (dd, 1H, J = 4.2, 7.2 Hz), 1.68–1.90 (m, 3H), 1.95–2.32 (m, 3H),
13
2
.40–3.10 (m, 6H), 4.04 (dq, 2H, J = 7.2, 10.8 Hz), 7.10–7.60 (m, 9H). CꢀNMR (200 MHz,
CDCl ) (free base) δ 14.18, 20.09, 26.36, 33.90, 38.29, 38.34, 48.89, 49.32, 55.56, 60.96, 70.77,
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26.08, 126.95, 128.06, 128.30, 129.94, 132.61, 140.59, 146.97, 172.2. Anal. calcd for:
C H ClNO ·H C O ·0.3 H O: C, 61.31; H, 6.06; N, 2.75. Found: C, 61.56; H, 5.71; N, 2.89.
24
28
3
2
2
4
2
Ethylꢀ(1S,2R)ꢀ2ꢀ((4ꢀ(4ꢀchlorophenyl)ꢀ4ꢀhydroxypiperidinꢀ1ꢀyl)methyl)ꢀ1ꢀ
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
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phenylcyclopropaneꢀ1ꢀcarboxylate [cisꢀ(–)ꢀ1b]: yield 72%; [α] ꢁꢀ ꢂ –26° (c 1, MeOH); light
yellow sticky solid. Compound cisꢀ(–)ꢀ1b was synthesized starting from the bromo derivative
cisꢀ(–)ꢀ7b in anhydrous EtOH and purified via silica gel chromatography, eluting with 90%
1
EtOAc in cyclohexane. HꢀNMR (200 MHz, CDCl ) (free base) δ 1.16 (t, 3H, J = 7.0 Hz), 1.34
3
(dd, 1H, J = 4.2, 8.8 Hz), 1.65 (dd, 1H, J = 4.2, 7.2 Hz), 1.68–1.90 (m, 3H), 1.95–2.32 (m, 3H),
1
3
2
.40–3.10 (m, 6H), 4.04 (dq, 2H, J = 7.2, 10.8 Hz), 7.10–7.60 (m, 9H). CꢀNMR (200 MHz,
CDCl ) (free base) δ 14.18, 20.09, 26.36, 33.90, 38.29, 38.34, 48.89, 49.32, 55.56, 60.96, 70.77,
3
1
26.08, 126.95, 128.06, 128.30, 129.94, 132.61, 140.59, 146.97, 172.25. Anal. calcd for:
C H ClNO ·H C O ·0.3 H O: C, 61.31; H, 6.06; N, 2.75. Found: C, 60. 65; H, 5.90; N, 2.56.
24
28
3
2
2
4
2
Propylꢀ(1R,2S)ꢀ2ꢀ((4ꢀ(4ꢀchlorophenyl)ꢀ4ꢀhydroxypiperidinꢀ1ꢀyl)methyl)ꢀ1ꢀ
phenylcyclopropaneꢀ1ꢀcarboxylate [cisꢀ(+)ꢀ1c]: yield 90%; [α] ꢁꢀ ꢂ +26° (c 1,MeOH); light
yellow sticky solid. Compound cisꢀ(+)ꢀ1c was synthesized starting from the bromo derivative
cisꢀ(+)ꢀ7c in anhydrous nꢀPrOH and purified via silica gel chromatography, eluting with 90%
1
EtOAc in cyclohexane. HꢀNMR (200 MHz, CDCl ) (free base) δ 0.80 (t, 3H, J = 7.6 Hz), 1.35
3
(dd, 1H, J = 4.2, 8.8 Hz), 1.48 (t, 2H, J = 7.2), 1.64–1.90 (m, 4H), 2.10–2.35 (m, 3H), 2.40–3.10
1
3
(m, 6H), 7.10–7.60 (m, 9H). CꢀNMR (200 MHz, CDCl ) (free base) δ 10.33, 20.06, 21.85,
3
2
1
6
6.49, 33.99, 38.37, 38.43, 48.95, 49.35, 56.58, 66.55, 70.86, 126.10, 126.95, 128.05, 128.33,
29.98, 132.66, 140.67, 146.96, 172.32. Anal. calcd for: C H ClNO ·H C O ·0.2 H O: C,
2
5
30
3
2
2
4
2
2.17; H, 62.17; N, 2.69. Found: C, 62.11; H, 5.92; N, 2.35.
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Propylꢀ(1S,2R)ꢀ2ꢀ((4ꢀ(4ꢀchlorophenyl)ꢀ4ꢀhydroxypiperidinꢀ1ꢀyl)methyl)ꢀ1ꢀ
phenylcyclopropaneꢀ1ꢀcarboxylate [cisꢀ(–)ꢀ1c]: yield 91%; [α] ꢁꢀ ꢂ –24° (c 1, MeOH); light
yellow sticky solid. Compound cisꢀ(–)ꢀ1c was synthesized starting from the bromo derivative
0
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cisꢀ(–)ꢀ7c in anhydrous nꢀPrOH and purified via silica gel chromatography, eluting with 90%
1
EtOAc in cyclohexane. HꢀNMR (200 MHz, CDCl ) (free base) δ 0.80 (t, 3H, J = 7.6 Hz), 1.35
3
(dd, 1H, J = 4.2, 8.8 Hz), 1.48 (t, 2H, J = 7.2), 1.64–1.90 (m, 4H), 2.10–2.35 (m, 3H), 2.40–3.10
1
3
(
m, 6H), 7.10–7.60 (m, 9H). CꢀNMR (200 MHz, CDCl ) (free base) δ 10.33, 20.06, 21.85,
3
2
1
6
6.49, 33.99, 38.37, 38.43, 48.95, 49.35, 56.58, 66.55, 70.86, 126.10, 126.95, 128.05, 128.33,
29.98, 132.66, 140.67, 146.96, 172.32. Anal. calcd for: C H ClNO ·H C O ·0.4 H O: C,
25 30
3
2
2
4
2
1.75; H, 6.29; N, 2.67. Found: C, 61.74; H, 5.90; N, 2.32.
Isopropylꢀ(1R,2S)ꢀ2ꢀ((4ꢀ(4ꢀchlorophenyl)ꢀ4ꢀhydroxypiperidinꢀ1ꢀyl)methyl)ꢀ1ꢀ
phenylcyclopropaneꢀ1ꢀcarboxylate [cisꢀ(+)ꢀ1d]: yield 77%; [α] ꢁꢀ ꢂ +22° (c 1, MeOH); white
solid; mp 191–193 °C. Compound cisꢀ(+)ꢀ1d was synthesized starting from the bromo derivative
cisꢀ(+)ꢀ7d in anhydrous iꢀPrOH and purified via silica gel chromatography, eluting with 90%
1
EtOAc in cyclohexane. HꢀNMR (200 MHz, CDCl ) (free base) δ 1.10 (d, 3H, J = 6.2 Hz), 1.16
3
(d, 3H, J = 6.2 Hz), 1.35 (dd, 1H, J = 4.6, 9.2 Hz), 1.63 (dd, 1H, J = 4.6, 7.0 Hz),1.68–2.35 (m,
1
3
6
H), 2.45–3.09 (m, 6H), 4.96 (ept, 1H, J = 6.2 Hz), 7.10–7.60 (m, 9H). CꢀNMR (200 MHz,
CDCl ) (free base) δ 19.86, 21.60, 21.81, 26.15, 34.08, 38.42, 49.00, 49.40, 56.73, 68.41, 70.90,
3
126.10, 126.84, 128.02, 128.34, 129.84, 132.68, 140.80, 146.94, 171.72. Anal. calcd for:
C H ClNO ·H C O ·0.5 H O: C, 61.53; H, 6.31; N, 2.66. Found: C, 61.51; H, 6.01; N, 2.88.
25
30
3
2
2
4
2
Isopropylꢀ(1S,2R)ꢀ2ꢀ((4ꢀ(4ꢀchlorophenyl)ꢀ4ꢀhydroxypiperidinꢀ1ꢀyl)methyl)ꢀ1ꢀ
phenylcyclopropaneꢀ1ꢀcarboxylate [cisꢀ(–)ꢀ1d]: yield 85%; [α] ꢁꢀ ꢂ –24° (c 1, MeOH); light
yellow solid; mp 192–194 °C. Compound cisꢀ(–)ꢀ1d was synthesized starting from the bromo
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derivative cisꢀ(–)ꢀ7d in anhydrous iꢀPrOH and purified via silica gel chromatography, eluting
1
with 90% EtOAc in cyclohexane. HꢀNMR (200 MHz, CDCl ) (free base) δ 1.10 (d, 3H, J = 6.2
3
Hz), 1.16 (d, 3H, J = 6.2 Hz), 1.35 (dd, 1H, J = 4.6, 9.2 Hz), 1.63 (dd, 1H, J = 4.6, 7.0 Hz),
13
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
.68–2.35 (m, 6H), 2.45–3.09 (m, 6H), 4.96 (ept, 1H, J = 6.2 Hz), 7.10–7.60 (m, 9H). CꢀNMR
(
200 MHz, CDCl ) (free base) δ 19.86, 21.60, 21.81, 26.15, 34.08, 38.42, 49.00, 49.40, 56.73,
3
6
8.41, 70.90, 126.10, 126.84, 128.02, 128.34, 129.84, 132.68, 140.80, 146.94, 171.72. Anal.
calcd for: C H ClNO ·H C O ·0.5 H O: C, 61.53; H, 6.31; N, 2.66. Found: C, 61.49; H, 6.25;
2
5
30
3
2
2
4
2
N, 2.45.
General procedure for compounds transꢀ(+)ꢀ1b–d, transꢀ(–)ꢀ1b–d: procedure C. A mixture
of anhydrous DMF (5 mL), 4ꢀ(4ꢀchlorophenyl)piperidinꢀ4ꢀol (0.69 mmol) and trans tosylꢀester
(
0.32 mmol) and KHCO (0.64), was kept under magnetic stirring for 9 hours at 75 °C under
3
argon atmosphere in a sealed tube (Ace pressure tube). The tube content was transferred in a
round bottom flask and the solvent evaporated under vacuum. The crude was dissolved in
CH Cl , filtered over filter paper and washed with a saturated solution of NaHCO and brine.
2
2
3
The organic solvent was dried over Na SO and removed in vacuum. The crude product was
2
4
purified via silica gel chromatography. After purification the pure product was dissolved in
diethyl ether and a solution of oxalic acid in diethyl ether was added dropwise to obtain the
desired product as oxalic acid salt.
Ethylꢀ(1S,2S)ꢀ2ꢀ((4ꢀ(4ꢀchlorophenyl)ꢀ4ꢀhydroxypiperidinꢀ1ꢀyl)methyl)ꢀ1ꢀ
phenylcyclopropaneꢀ1ꢀcarboxylate [transꢀ(+)ꢀ1b]: yield 66%; [α]ꢁꢂ +44° (c 1, MeOH); white
ꢀ
solid; mp 185–187 °C. According to general procedure C. Compound transꢀ(+)ꢀ1b was
synthesized starting from the tosyl derivative transꢀ(+)ꢀ6b and purified via silica gel
1
chromatography, eluting with 90% EtOAc in cyclohexane. HꢀNMR (200 MHz, CDCl ) (free
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1
1
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2
2
2
2
2
2
2
2
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3
3
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base) δ 1.10 (t, 3H, J = 7.0 Hz), 1.55 (dd, 1H, J = 4.6, 7.5 Hz), 1.70 (dd, 1H, J = 4.6, 8.5 Hz),
1
.77–2.20 (m, 1H), 2.30–2.92 (m, 4H), 3.18–3.70 (m, 6H), 3.90 (bs, 1H), 4.04 (dq, 2H, J = 7.0,
1
3
10.8 Hz), 6.70–7.42 (m, 9H). CꢀNMR (200 MHz, CDCl ) (free base) δ 13.95, 20.61, 21.80,
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
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9
0
3
1
6
3.33, 38.27, 51.66, 55.66, 61.93, 71.00, 124.90, 127.51, 128.50, 129.15, 136.00, 140.85, 144.48,
71.56. Anal. calcd for: C H ClNO H C O ·0.2 H O: C, 61.52; H, 6.04; N, 2.76. Found: C,
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3
2
2
4
2
1.73; H, 5.94; N, 3.01.
Ethylꢀ(1R,2R)ꢀ2ꢀ((4ꢀ(4ꢀchlorophenyl)ꢀ4ꢀhydroxypiperidinꢀ1ꢀyl)methyl)ꢀ1ꢀ
phenylcyclopropaneꢀ1ꢀcarboxylate [transꢀ(–)ꢀ1b]: yield 59%; [α] ꢁꢀ ꢂ –42° (c 1, MeOH); light
yellow solid; mp 186–189 °C. According to general procedure C. Compound transꢀ(–)ꢀ1b was
synthesized starting from the tosyl derivative transꢀ(–)ꢀ6b and purified via silica gel
1
chromatography, eluting with 90% EtOAc in cyclohexane. HꢀNMR (200 MHz, CDCl ) (free
3
base) δ 1.10 (t, 3H, J = 7.0 Hz), 1.55 (dd, 1H, J = 4.6, 7.5 Hz), 1.70 (dd, 1H, J = 4.6, 8.5 Hz),
1.77–2.20 (m, 1H), 2.30–2.92 (m, 4H), 3.18–3.70 (m, 6H), 3.90 (br s, 1H), 4.04 (dq, 2H, J = 7.0,
1
3
1
0.8 Hz), 6.70–7.42 (m, 9H). CꢀNMR (200 MHz, CDCl ) (free base) δ 13.95, 20.61, 21.80,
3
3
3.33, 38.27, 51.66, 55.66, 61.93, 71.00, 124.90, 127.51, 128.50, 129.15, 136.00, 140.85, 144.48,
171.56. Anal. calcd for: C H ClNO ·H C O ·0.3 H O: C, 61.31; H, 6.06; N, 2.75. Found: C,
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3
2
2
4
2
6
1.20; H, 6.35; N, 3.07.
Propylꢀ(1S,2S)ꢀ2ꢀ((4ꢀ(4ꢀchlorophenyl)ꢀ4ꢀhydroxypiperidinꢀ1ꢀyl)methyl)ꢀ1ꢀ
phenylcyclopropaneꢀ1ꢀcarboxylate [transꢀ(+)ꢀ1c]: yield 76%; [α] ꢁꢀ ꢂ +42° (c 1, MeOH); white
solid; mp 180–183 °C. According to general procedure C. Compound transꢀ(+)ꢀ1c was
synthesized starting from the tosyl derivative transꢀ(+)ꢀ6c and purified via silica gel
1
chromatography, eluting with 90% EtOAc in cyclohexane. HꢀNMR (200 MHz, CDCl ) (free
3
base) δ 0.98 (t, 3H, J = 7.4 Hz), 1.43 (t, 2H, J = 7.4), 1.65–2.35 (m, 7H), 2.22–2.75 (m, 6H),
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.50 (br s, 1H) 3.70–4.15 (m, 2H), 6.70–7.50 (m, 9H). CꢀNMR (200 MHz, CDCl ) (free base) δ
3
1
0.15, 20.14, 21.84, 22.42, 31.36, 38.38, 51.66, 55.63, 67.88, 70.33, 124.86, 127.58, 128.20,
128.90, 130.03, 136.33, 140.87, 145.40, 170.68. Anal. calcd for: C H ClNO ·H C O ·0.2 H O:
2
5
30
3
2
2
4
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
C, 62.17; H, 6.26; N, 2.69. Found: C, 61.52; H, 5.99; N, 3.09.
Propylꢀ(1R,2R)ꢀ2ꢀ((4ꢀ(4ꢀchlorophenyl)ꢀ4ꢀhydroxypiperidinꢀ1ꢀyl)methyl)ꢀ1ꢀ
phenylcyclopropaneꢀ1ꢀcarboxylate [transꢀ(–)ꢀ1c]: yield 75%; [α] ꢁꢀ ꢂ –40° (c 1, MeOH); white
solid; mp 183–185 °C. According to general procedure C. Compound transꢀ(–)ꢀ1c was
synthesized starting from the tosyl derivative transꢀ(–)ꢀ6c and purified via silica gel
1
chromatography, eluting with 90% EtOAc in cyclohexane. HꢀNMR (200 MHz, CDCl ) (free
3
base) δ 0.98 (t, 3H, J = 7.4 Hz); 1.43 (st, 2H, J = 7.4); 1.65–2.35 (m, 7H); 2.22–2.75 (m, 6H);
13
3
.50 (br s, 1H) 3.70–4.15 (m, 2H); 6.70–7.50 (m, 9H). CꢀNMR (200 MHz, CDCl ) (free base)
3
δ 10.15; 20.14; 21.84; 22.42; 31.36; 38.38; 51.66; 55.63; 67.88; 70.33; 124.86; 127.58; 128.20;
128.90; 130.03; 136.33; 140.87; 145.40; 170.68. Anal. calcd for: C H ClNO ·H C O ·0.4 H O:
2
5
30
3
2
2
4
2
C, 61.75; H, 6.29; N, 2.67. Found: C, 61.90; H, 5.98; N, 3.04.
Isopropylꢀ(1S,2S)ꢀ2ꢀ((4ꢀ(4ꢀchlorophenyl)ꢀ4ꢀhydroxypiperidinꢀ1ꢀyl)methyl)ꢀ1ꢀ
phenylcyclopropaneꢀ1ꢀcarboxylate [transꢀ(+)ꢀ1d]: yield 65%; [α]ꢁꢂ +36° (c 1, MeOH); white
ꢀ
solid; mp 176–178 °C. According to general procedure C. Compound transꢀ(+)ꢀ1d was
synthesized starting from the tosyl derivative transꢀ(+)ꢀ6d and purified via silica gel
1
chromatography, eluting with 90% EtOAc in cyclohexane. HꢀNMR (200 MHz, CDCl ) (free
3
base) δ 1.10 (d, 3H, J = 6.2 Hz), 1.13 (d, 3H, J = 6.2 Hz), 1.50–2.50 (m, 4H), 2.60–3.05 (m,
13
3
H), 3.20–3.60 (m, 6H), 3.80 (br s, 1H), 4.83 (ept, 1H, J = 6.2 Hz), 6.65–7.60 (m, 9H). Cꢀ
NMR (200 MHz, CDCl ) (free base) δ 19.50, 19.65, 20.84, 21.48, 33.45, 38.40, 51.61, 56.65,
3
69.83, 70.12, 124.87, 127.52, 128.63, 129.00, 130.21, 134.87, 141.86, 146.32, 171.99. Anal.
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