Chiral arylpyrrolidinols: Preparation and biological profile

Article abstract of DOI:10.1016/j.bmc.2005.02.054

The preparation and biological evaluation of a new class of arylpyrrolidinols is reported. The antinociceptive activity was evaluated in vivo with the hot plate test (HPT) and formalin test (FT), excluding any involvement on motor coordination with the rota-rod test (RRT). The nociceptive behavior in the late phase of FT (representative of chronic pain) suggests an involvement of the antiinflammatory process and it is clearly influenced by the stereochemical features, being the eutomer of phenylpyrrolidinols, the (2R,3S) enantiomer. Despite this, a specific mechanism of action is not yet clarified.

Full text of DOI:10.1016/j.bmc.2005.02.054

Bioorganic & Medicinal Chemistry 13 (2005) 3117–3126
Chiral arylpyrrolidinols: preparation and biological profile
Simona Collina,a,* Daniela Rossi,a Guya Loddo,a Annalisa Barbieri,b Enrica Lanza,b
Laura Linati,c Stefano Alcaro,d Andrea Gallellid and Ornella Azzolinaa
a
`
Dipartimento di Chimica Farmaceutica, Universita di Pavia, Viale Taramelli, 12, I-27100 Pavia, Italy
`
b
Dipartimento di Farmacologia Sperimentale ed Applicata, Universita di Pavia, Viale Taramelli, 14, I-27100 Pavia, Italy
c
`
Centro Grandi Strumenti, Universita di Pavia, Via Bassi 21, 27100 Pavia, Italy
`
d
`
Dipartimento di Scienze Farmacobiologiche, ꢀComplesso Ninı Barbieriꢁ, Universita di Catanzaro ‘Magna Graecia’,
88021 Roccelletta di Borgia (CZ), Italy
Received 14January 2005; revised 21 February 2005; accepted 25 February 2005
Abstract—The preparation and biological evaluation of a new class of arylpyrrolidinols is reported. The antinociceptive activity was
evaluated in vivo with the hot plate test (HPT) and formalin test (FT), excluding any involvement on motor coordination with the
rota-rod test (RRT). The nociceptive behavior in the late phase of FT (representative of chronic pain) suggests an involvement of the
antiinflammatory process and it is clearly influenced by the stereochemical features, being the eutomer of phenylpyrrolidinols, the
(2R,3S) enantiomer. Despite this, a specific mechanism of action is not yet clarified.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
the rota-rod test. All compounds with a different aro-
matic nucleus showed relevant antinociceptive proper-
ties, with exception for the 9-phenantrenyl derivative
(2R,3S/2S,3R)-10.
In recent years, we have focused our attention on het-
erosteroid 17-methyl-17-aza-equilenine analogues, syn-
thesizing structurally related compounds with analgesic
activity that interact with the opioidergic system.1–3
Aryl-1,2-dimethylpyrrolidinols (Fig. 1) have been stud-
ied owing to their interesting biological properties, and
a SAR study has been performed in order to investigate
the relationship between stereoidogenesis and opioid
analgesia. All compounds were found to possess inter-
esting analgesic activity,1–3 being tested in the hot plate
test (HPT), but evidenced poor affinity with opioid
receptor subtypes in vitro, as evaluated in binding as-
says. Despite this, the reversal of the antinociceptive
activity by the nonspecific opioid antagonist naloxone
(NLX) could confirm an indirect involvement of the
opioidergic system. The involvement of cholecystokinin
or neuropeptide FF analogues could be considered to
explain antinociceptive properties of these compounds.
Any effect on motor coordination3 was evidenced by
monitoring the locomotory control of the animals with
The most interesting compounds are (2R,3S/2S,3R)-1
(hit compound), (2R,3S/2S,3R)-5, (2R,3S/2S,3R)-6,
and (2R,3S/2S,3R)-8, so a more deep investigation has
been performed with the formalin test in mice, which
demonstrated a pain reduction, suggesting their involve-
ment in the antiinflammatory process.
Herein, (2R,3S)-1 and (2R,3S)-3–8 and their enantio-
mers were prepared according to the synthetic procedure
performed for the enantiomers of compound 2, previ-
ously published,1 in order to investigate the role of ste-
reochemistry on the pharmacological activity.
Moreover the aryl-1-methylpyrrolidinols (R/S)-17–19
(Fig. 2) were also synthesized according to the principles
of simplification.
The antinociceptive activity was evaluated in vivo with
the hot plate test (HPT) and formalin test (FT), exclud-
ing any involvement of motor coordination with the
rota-rod test (RRT).
Keywords: Arylpyrrolidinols; Enantioselective synthesis; Antinocicep-
tive activity; Molecular modeling.
*
A molecular modeling study of (2R,3S)-1, (2R,3S)-5,
and their simplified analogues (S)-17 and (S)-18 has
Corresponding author. Tel.: +39 382 987376; fax: +39 382 422975;
e-mail: simona.collina@unipv.it
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.02.054

3118
S. Collina et al. / Bioorg. Med. Chem. 13 (2005) 3117–3126
Compound
Ar
Compound
Ar
1
9
2
3
10
11
HO
CH3
H3CO
H3C
CH3
H
N
4
5
6
12
13
14
F
F3C
Ar
OH
F
C6H5CH2O
HO
7
8
15
16
H3CO
(H3C)2N
Cl
Figure 1. Pyrrolidinols 1–16.
added to a solution of 4-bromo-phenol in PEG 400 at
room temperature under stirring. After 24h the reaction
mixture was added with water and extracted with diethyl
ether. The combined organic phases provided the
1-benzyloxy-4-bromo-benzene, as confirmed by IR and
1H NMR analysis.
Compound
Ar
17
18
19
CH3
H
N
H
This reaction occurs via a nucleophllic attack of the aro-
matic anion at the prochiral center of the (R)-20 or (S)-
20 on the less hindered side, consequently (2R,3S)- or
(2S,3R)-1–8 enantiomers were obtained, respectively,
as evidenced by 1H NMR analysis of the crude products.
Ar
OH
The employed synthetic procedure provided compounds
with high enantiomeric excess (ee), as reported in Table
1. Liquid chromatographic procedures were attempted
using several chiral stationary phases in order to develop
a suitable direct method for the evaluation of the enan-
tiomeric excess of all enantiomeric compounds. Baseline
separation was successfully achieved on analytical Chir-
alpak AD, Chiralpak AS, and Chiralcel OD-H columns
with good a and Rs values (Table 2).
Figure 2. Pyrrolidinols 17–19.
been performed in order to consider the conformational
properties of biologically relevant compounds.
2. Results
2.1. Chemistry
Compounds (R/S)-17–19 were prepared from ketone 21,
according to the same synthetic procedure employed for
compounds 1–8 (Scheme 1).
Compounds (2R,3S)-1 and (2R,3S)-3–8, and their enan-
tiomers were synthesized (Scheme 1) by nucleophylic
addition to (R)-20 or (S)-204 (Fig. 3) of the appropriate
aromatic anion commercially available, with exception
for compound 6 whose precursor, the 1-benzyloxy-4-
bromo-benzene, has been prepared according to Bala-
subramanian et al.5 Benzilbromide and KOH were
1-Methyl-pyrrolidin-3-one 21 was prepared accord-
ing to the synthetic procedure described by Cavalla
et al.,6 with suitable modifications (Scheme 2). N-
Methyl-b-alaninonitrile and anhydrous K2CO3 were
added with a-Br-acetic acid ethyl ester affording

S. Collina et al. / Bioorg. Med. Chem. 13 (2005) 3117–3126
CH3
3119
R
N
Br
1) tert-BuLi, -30˚C, Et2O
H
Ar
2) (R)-20, (S)-20 or (R/S)-21, 0˚C
Ar
3) H2O
OH
R = CH3 (2R,3S)-1-8 and enantiomers
R = H (R/S)-17-19
Scheme 1. Synthesis of the enantiomers of compounds 1–8 and 17–19.
[(2-cyano-ethyl)-methyl-amino]-acetic acid ethyl ester
(intermediate A), and a further alcoholysis gave 3-(eth-
oxycarbonylmethyl-methyl-amino)-propionic acid ethyl
ester (intermediate B). Treatment with sodium ethano-
ate to obtain intermediate C was unsuccessful. There-
fore, a Dieckmann condensation with 60% NaH in
anhydrous toluene at 0 ꢁC was performed, obtaining 1-
methyl-4-oxo-pyrrolidine-3- carboxylic acid ethyl ester.
This not isolated intermediate (C) was hydrolyzed and
decarboxylated with HCl 6 N under heating, affording
21 as a yellow oil. The crude product has been purified
by distillation under reduced pressure and kept in the
dark at ꢀ10 ꢁC and in nitrogen atmosphere. GC analy-
sis showed a 99.9% purity of the desired product. All
isolated intermediates as well as compound 21 were
CH3
R
N
H
O
R = CH3 20
R = H 21
Figure 3. Compounds 20 and 21.
Table 1. Optical properties of compounds 1, 3–8
Compound
[a]a
Ee %
1
characterized by IR and H NMR analysis.
(2R,3S)-1
(2S,3R)-1
(2R,3S)-3
(2S,3R)-3
(2R,3S)-4
(2S,3R)-4
(2R,3S)-5
(2S,3R)-5
(2R,3S)-6
(2S,3R)-6
(2R,3S)-7
(2S,3R)-7
(2R,3S)-8
(2S,3R)-8
+22.5 (589)
ꢀ22.8 (589)
+34.6
99.9
99.9
99.9
99.7
99.5
99.9
99.9
99.9
99.2
99.7
99.9
99.9
99.9
99.9
1
The results of the elemental analyses, IR and H NMR
spectra agreed with the assigned structures for all
compounds.
ꢀ33.8
+22.9 (365.1)
ꢀ23.9 (365.1)
ꢀ24.40
2.2. Molecular mechanics calculations
+24.69
The computational work was dedicated to the characteri-
zation of biologically relevant compounds and to the
clarification of the C2 methyl substitution. Stereoisom-
ers of compounds 1, 5, 17, and 18 were considered in this
study. The (2R,3S)-1 and (2R,3S)-5 were modeled by the
Monte Carlo (MC) conformational search method
applied to torsional angles (see Experimental section).
+12.03
ꢀ12.67
+46.88
ꢀ46.62
+38.18
ꢀ38.46
a Performed on hydrochlorides, c 0.5, in MeOH; k 405 nm.
Table 2. Analytical resolution of compounds 1, 3–8
Compound
Columna
Mobile phaseb
90.0/10.0/0.1
a
Rs
Rt (min) (config.)
1ÆHCl
Chiralcel OD-H
6.5 (2R,3S)
9.3 (2S,3R)
20.2 (2S,3R)
23.1 (2R,3S)
24.1 (2S,3R)
26.1 (2R,3S)
18.6 (2R,3S)
25.9 (2S,3R)
S,3R)
3ÆHCl
4ÆHCl
5ÆHCl
6ÆHCl
7ÆHCl
8ÆHCl
Chiralpak AD
Chiralpak AD
Chiralpak AD
Chiralpak AS
Chiralpak AD
Chiralpak AD
98.0/2.0/0.1
1.18
1.11
1.47
2.23
98.0/2.0/0.1
95.0/5.0 EtOH/0.05
98.0/2.0/0.1
1.23
1.18
1.15
1.5421.7 (2
2.25
25.1 (2R,3S)
20.1 (2S,3R)
23.0 (2R,3S)
25.1
98.0/2.0/0.1
99.0/1.0/0.1
1.44
28.2
a 250 · 4.6 mm.
b Solvent mixture: n-hexane/IPA/DEA (v/v/v); flow rate: 0.5 mL/min; UV detector: k 273 nm.

3120
S. Collina et al. / Bioorg. Med. Chem. 13 (2005) 3117–3126
O
OC2H5
NC
NC
1) EtOH assol.
H2SO4 conc., ∆T
N
NH
+
K2CO3
Br
OC2H5
CH3
O
CH3
2) Na2CO3
A
C2H5O
O
O
C2H5OOC
C2H5OOC
O
1) HCl 6N
2) Na2CO3
NaH
-5˚C
N
N
N
CH3
CH3
CH3
B
C
21
Scheme 2. Synthesis of compound 21.
Table 3. Conformational distribution of stereoisomers 1, 5, 17, and 18
(data not shown). So the conformational analysis was
focused on the (2R,3S) series. Starting with these ste-
reoisomers, molecular models of simplified compounds
(S)-17 and (S)-18 were obtained and the same MC
search was applied to these compounds. The conforma-
tional distribution of the four stereoisomers is reported
in Table 3.
Compound Number of conformers
within 50 kJ/mol above
Number of conformers
with Boltzmann
the global energy minimum population > 90%
(2R,3S)-1
(2R,3S)-5
(S)-17
16
7
4
2
4
2
13
7
(S)-18
The superimposition of four of the most populated
conformers of (2R,3S)-1 and (S)-17 revealed a perfect
match of the pyrrole ring atoms, not affected at all
by the methyl substitution in C2. In few conformations,
where this moiety interacts directly to the Ar group,
the rotatable bond C3–Ar, pertinent to the orientation
of this substituent with respect to the five-member ring,
seems to be fairly influenced. A moderate effect is also ob-
served with the hydroxyl rotamer, orientating the alco-
holic hydrogen toward the nitrogen to establish an
The quality of the MC conformational exploration was
considered exhaustive as demonstrated by the consistent
average number of duplicates found during the simula-
tions, found always higher than 53.2. The conforma-
tional search was repeated also considering the (2S,3R)
stereoisomer of compounds 1 and 5. As expected, the
same conformational distribution was found with spec-
ular geometry with respect to the former stereoisomers
Figure 4. Comparison between the four most populated conformers of compounds 1 and 17 (a–d) and the two of compounds 5 and 18 (e–f)
superimposed onto the pyrrole ring.

S. Collina et al. / Bioorg. Med. Chem. 13 (2005) 3117–3126
3121
Table 4. Effect (in s) in the HPT of compounds administered at doses
of 2, 4, and 8 mg/kg
intramolecular hydrogen bond, as found in the most pop-
ulated conformers (Fig. 4). Similar considerations can be
done by the superimpositions of the two (2R,3S)-5 and
(S)-18 most populated conformers (Fig. 4).
Compounds
E (s) (2 mg/kg) E (s)
(4mg/kg)
E (s) (8 mg/kg)
(2R,3S/2S,3R)-1 31.2 3.1
35.3 6.8 41.6 5.6
44.5 5.5 34.8 4.2
39.4 5.6 47.4 4.7
27.0 5.3 37.4 6.8
23.6 5.7 34.3 5.3
27.3 5.1 35.8 5.9
34.3 4.5 50.2 3.3
49.6 3.3 51.2 4.2
48.5 4.9 52.5 4.3
28.5 4.3 47.1 4.6
28.1 6.0 35.0 6.9
26.3 4.8 45.4 5.3
35.0 3.5 40.5 4.9
45.9 4.6 38.5 5.2
45.7 6.1 35.9 5.3
54.4 3.7 49.6 4.7
35.2 5.0 40.8 4.0
39.7 5.6 39.2 5.3
41.8 4.6 48.2 4.6
38.8 4.0 47.5 4.1
44.1 5.0 31.8 4.0
43.2 4.4 37.1 4.5
42.7 4.7 45.5 5.9
48.6 4.6 45.8 5.1
45.3 5.4 42.7 4.9
43.6 6.2 38.1 5.8
36.8 4.1 36.3 5.6
2.3. Biological evaluation
(2R,3S)-1 37.2 6.4
(2S,3R)-1 34.6 7.2
(2R,3S/2S,3R)-2 25.4 4.2
The synthesized compounds were evaluated as antinoci-
ceptive agents by the in vivo HPT and FT assays (see
Experimental section). The influence on the locomotory
activity was also determined with the RRT to exclude
motor impairment associated with the analgesic effect.
(2R,3S)-2 23.5 4.6
(2S,3R)-2 25.9 3.2
(2R,3S/2S,3R)-3 28.7 3.3
(2R,3S)-3 45.8 3.9
(2S,3R)-3 30.8 4.9
(2R,3S/2S,3R)-4 26.2 4.3
(2R,3S)-4
(2S,3R)-4
n.d.
n.d.
3. Discussion
(2R,3S/2S,3R)-5 29.4 4.5
Racemic arylpyrrolidinols possess interesting analgesic
activity in the HPT,2,3 with a potency similar or superior
to that of morphine (AD50 values between 0.10 and
4.82 mg/kg). Although the biological effect is reversed
by naloxone, the mechanism of action of these com-
pounds is still to be determined, since binding affinity
for opioid receptors (Ki, lM) is too low to account for
the opioid analgesia.
(2R,3S)-5 38.1 4.7
(2S,3R)-5 38.2 8.3
(2R,3S/2S,3R)-6 43.6 7.1
(2R,3S)-6 29.4 5.8
(2S,3R)-6 39.1 5.8
(2R,3S/2S,3R)-7 36.3 4.8
(2R,3S)-7 29.5 4.3
(2S,3R)-7 39.3 4.7
(2R,3S/2S,3R)-8 49.6 4.0
(2R,3S)-8
(2S,3R)-8
(R/S)-17
(R/S)-18
(R/S)-19
36.5 3.6
46.5 4.2
45.1 5.7
34.1 6.1
30.6 4.5
After a wide, but unsuccessful in vitro screening of the
hit with several receptors7 (cannabinoid, angiotensin
type, benzodiazepinic, cholecystokinic, GABAergic,
hystaminergic, dopaminergic, adrenergic, muscarinic,
serotoninergic and ionotropic glutamate receptors, and
Ca2+ channel) we focused our attention on the in vivo
profile of novel compounds.
n.d.: not determined.
Conversely, the nociceptive behavior in the late phase of
FT is clearly influenced by the stereochemical features.
The eutomer of compounds bearing the phenyl group
(5–8) is the (2R,3S) enantiomer, which significatively
reduced the nociceptive response (p < 0.01).
Therefore, we performed an investigation with the for-
malin test, an experimental model suitable to assess
the animalꢁs response both to acute and continuous
pain. As known, the response to formalin is biphasic:
the early phase (phase I) is related to an intense pain
comparable to that obtained by thermal stimulus in
HPT and the late phase (phase II) is related to a moder-
ate and continuous pain caused by damaged tissue.
In order to confirm the relevance of the stereogenic cen-
ter C2 in the analgesic process, compounds (R/S)-17–19
were prepared and tested by HPT and FT. Experimental
data did not show noticeable differences of activity in
the acute pain if compared to the corresponding racemic
analogs (2R,3S/2S,3R)-1, (2R,3S/2S,3R)-5, and (2R,3S/
2S,3R)-7.
In this paper we have investigated the role of the stereo-
genic center C2 of the heterocyclic moiety preparing
both enantiopure compounds 1, 3–8 and the C2 unsub-
stituted analogs 17–19. First of all, compounds were
tested on mice with HPT (Table 4) and FT by subcuta-
neous administration of 2, 4, 8 mg/kg. At the doses
employed in our study, no motor impairment was
evidenced with RRT (mice remaining on the rota-rod:
P67%).
Nevertheless, the antinociceptive activity in the continu-
ous pain seems to be strictly related to the asymmetry at
C2, as suggested by the fact that compounds 17–19 did
not reduce the nociceptive response in phase II of for-
malin test.
In HPT, the dose of 4mg/kg ( Fig. 5), comparable with
the AD50 of morphine (4.18 mg/kg), was the most signi-
ficative ideal one for almost all compounds, being 2 mg/
kg sometimes not active and 8 mg/kg too high to assess
for the analgesic activity. In the FT (Fig. 6) the most sig-
nificative response was obtained at 8 mg/kg.
To better understand these findings, a molecular model-
ing study was performed to establish the role of the con-
formational distribution in the biological activity.
The molecular mechanics experiments carried out with
the biologically relevant compounds revealed that the
presence of the methyl moiety in C2 did not change sig-
nificantly the conformational profiles of compounds 1
and 5 with respect to the simplified 17 and 18. For in-
stance, distances between the Ar ring centroid and the
N-methyl group are not effected by the modification
Regarding the acute pain, experimental data (Figs. 5
and 6) clearly evidence that in both assays there are no
significative differences (p > 0.05) in the activity of the
enantiomers for all the examined compounds.

3122
S. Collina et al. / Bioorg. Med. Chem. 13 (2005) 3117–3126
Hot Plate Test
60
50
40
30
20
10
0
control
2R,3S
2S,3R
Cl
MeO
F
HO
C6H5CH2O
Hot Plate Test
60
50
40
30
20
10
0
control
R = CH3
R = H
Figure 5. HPT (4mg/kg dose).
on C2. Minor effects have been observed in the rotatable
bonds C3–Ar and C3–OH, due to the Van der Waals
repulsion induced by the methyl in C2 of compounds
1 and 5. Unfortunately these small differences cannot ex-
plain the different biological response of these com-
pounds with respect to the simplified 17 and 18.
IR spectra were recorded on a Perkin–Elmer FT-IR
1605 spectrophotometer; only noteworthy absorptions
are given.
1H NMR spectra were performed at 9.4T (TMS as
internal standard d = 0) with an ADVANCE spectro-
meter at 400 MHz, mod. Bruker Germany and a BB1
5 mm probe; chemical shifts are given in ppm.
The different biological effect should be related to the ste-
reochemistry of the C2/C3 chiral centers of the methyl-
ated analogues, even though conformational differences
between the two series are not significantly appreciable.
Gas chromatography (GC) analyses were performed on
a DANI 3800 apparatus equipped with PVT and FID.
HPLC analyses were performed on a Jasco system con-
sisting of a PU-1580 pump, a Reodyne 7125 injector
(20 lL sample loop) and a MD-1510 Diode Array
Detector (wavelength 273 nm). Experimental data were
acquired and interpreted with Borwin PDA and Borwin
chromatograph software. Chiral HPLC analyses were
performed on Chiralpak AD column (Daicel) (250 ·
4.6 mm, 10 lm), Chiralpak AS column (Daicel) (250 · 4.6
mm, 10 lm), and Chiralcel OD-H column (250 · 4.6
mm, 5 lm); elution was carried out using n-hexane,
isopropyl alcohol (IPA), ethyl alcohol (EtOH), and
diethylamine (DEA). All the solvents were purchased
by Carlo Erba and are HPLC grade. [a] measurements
were recorded at room temperature on a Jasco DIP
1000 polarimeter.
Among the investigated compounds, worth noting is
(2S,3R)-6 whose activity resides only in phase II of cro-
nic analgesia and which is suggested to be the best com-
pound for a more detailed pharmacological study to
elucidate the mechanism of action. Consequently, a
rationalization of the biological results will be possible
using molecular modeling and QSAR techniques.
4. Experimental
4.1. General methods
All reagents and solvents were purchased from commer-
cial suppliers and employed without further purification.
Analytical TLC was performed using precoated glass-
backed plates (Fluka Kieselgel 60 F254) and visualized
by ultra-violet radiation, acidic ammonium molyb-
date(IV) or potassium permanganate. Melting points
were measured on SMP3 Stuart Scientific apparatus.
Elemental analyses were performed on a Carlo Erba
1106 C, H, N analyzer.
4.2. General procedure for the preparation of 1-methyl-
pyrrolidin-3-one [21]
4.2.1. Intermediate A. N-Methyl-b-alaninonitrile (30.3 g,
360 mmol) and anhydrous K2CO3 (49.6 g, 360 mmol)
were refluxed in 100 mL of 2-butanone under stirring
and slowly added with a-Br-acetic acid ethyl ester

S. Collina et al. / Bioorg. Med. Chem. 13 (2005) 3117–3126
3123
2247, 1737, 1189, 1063, 1031, 972, 847. 1H NMR
(CD3OD): d 1.28 (t, 3H, CH3CH2, J = 7.0 Hz), 2.44 (s,
3H, CH3–N), 2.63 (t, 2H, CH2–CH2N, J = 6.8 Hz),
2.90 (t, 2H, CH2–CH2N, J = 6.8 Hz), 3.34(s, 2H,
COCH2–N), 4.18 (q, 2H, CH3CH2, J = 7.0 Hz).
FT: phase I
80
70
60
50
40
30
20
10
0
*
*
*
*
**
**
**
control
2R,3S
2S,3R
4.2.2. Intermediate B. A solution of [(2-cyano-ethyl)-
methyl-amino]-acetic acid ethyl ester (54.1 g, 320 mmol)
in absolute ethanol (270 mL) under stirring was slowly
added with H2SO4 96% (76 mL). The reaction mixture
was refluxed for 48 h. After cooling, the mixture was di-
luted with water (500 mL), added with NaHCO3 until
pH = 9 and extracted with diethyl ether (5 · 200 mL).
The combined organic phases were dried with anhy-
drous Na2SO4 and evaporated under vacuum, affording
the crude product as an orange oil, purified by fractional
distillation under reduced pressure (bp9mmHg=144–
145 ꢁC). Yield 38.3 g (54.0%). TLC (n-Hex 60/AcOEt
40), Rf = 0.51, purity = 99% (GC). IR (Nujol): cmꢀ1
C6H5CH2
O
Cl
30
25
20
15
10
5
FT: phase II
*
*
control
2R,3S
2S,3R
**
**
**
1
1736, 1459, 1374, 1298, 1183, 1051, 854, 794. H NMR
0
(CD3OD): d 1.24(m, 6H, C H3–CH2O), 2.37 (s, 3H,
CH3–N), 2.47 (t, 2H, COCH2CH2, J = 3.3 Hz), 2.85 (t,
2H, CH2CH2–N, J = 7.3 Hz), 3.27 (s, 2H, COCH2–N),
3.70 (q, 2H, CH3CH2O, J = 7.0 Hz), 4.13 (m, 2H,
CH3CH2O).
Cl
C6H5CH2
O
80
70
60
50
40
30
20
10
0
FT: phase I
*
*
4.2.3. 1-Methyl-pyrrolidin-3-one [21]. NaH (60% in min-
eral oil 5.5 g, 138 mmol) in anhydrous toluene (120 mL)
was slowly added under stirring in nitrogen atmosphere
at ꢀ5 ꢁC with a solution of intermediate B (15 g,
690 mmol) in anhydrous toluene (120 mL). The reaction
mixture was kept at the same temperature for 3 h and
then added with water (30 mL) and HCl 6 N (50 mL).
The aqueous and the organic layers were separated and
washed with toluene (1 · 80 mL) and HCl 6 N
(2 · 40 mL), respectively. The combined aqueous phases
were stirred at room temperature for 24h and then re-
fluxed for 4h. The reaction mixture was added with
Na2CO3 until pH > 9 and extracted with dichlorometh-
ane (5 · 40 mL). The combined organic phases were
dried with anhydrous Na2SO4 and evaporated under vac-
uum, affording a brown oil. The crude product was puri-
fied by distillation in nitrogen atmosphere under reduced
pressure, affording a colorless oil (bp28mmHg = 4 5ꢁC).
Yield 3.15 g (46%). TLC (n-Hex 60/AcOEt 40),
Rf = 0.48. IR (Nujol): cmꢀ1 1751, 1441, 1250, 1183,
1116, 850. 1H NMR (CD3OD): d 2.40 (t, 2H,
CH2CH2–N, J = 3.3 Hz), 2.44 (s, 3H, CH3–N), 2.86 (t,
2H, CH2CH2–N, J = 7.3 Hz), 2.91 (s, 2H, COCH2–N).
**
**
**
control
R=CH3
R=H
FT: phase II
30
25
20
15
10
5
control
*
R=CH3
R=H
**
**
**
0
Figure 6. FT (8 mg/kg dose).
(60 g, 360 mmol). The reaction mixture was refluxed for
2 h. After cooling and filtration, the evaporation of the
solvent under vacuum afforded a yellowish oil. The
crude product was added with water (100 mL) and
NaHCO3 until pH = 8, and the aqueous phase was ex-
tracted with diethyl ether (6 · 100 mL). The combined
organic phases, dried with anhydrous Na2SO4 and
evaporated under vacuum, provided the desired product
as a yellow oil. Yield 54.1 g (88.9%). TLC (n-Hex 60/
AcOEt 40), Rf = 0.35, purity = 99% (GC method:
CBWX-15N capillary column; flow rate 45–48 cm/s.
Oven temperature: 70 ꢁC for 4min then 180 ꢁC for
8 min. Injection volume: 1 lL). IR (Nujol): cmꢀ1 3619,
4.3. General procedure for the preparation of (2R,3S) or
(2S,3R)-1–8ÆHCl and (R/S)-17–19ÆDL-tartrates
The synthesis of enantiomers of 1 and 3–8 was essen-
tially accomplished according to the procedure already
described for the preparation of the racemic com-
pounds. A 1.7 M solution of t-BuLi in pentane
(49.9 mL, 84.8 mmol) in anhydrous diethyl ether was
added under stirring in nitrogen atmosphere at ꢀ30 ꢁC
to a solution of the appropriate aromatic precursor
(42.4 mmol). After stirring at ꢀ40 ꢁC for 1 h, the tem-
perature was allowed to warm to 0 ꢁC and then a solu-
25
tion of (R)-20 {½aŠ ¼ þ49:9 (c 0.5, MeOH)}, (S)-20
D

3124
S. Collina et al. / Bioorg. Med. Chem. 13 (2005) 3117–3126
25
D
{½aŠ ¼ ꢀ54:0 (c 0.5, MeOH)} or 21, respectively,
4.3.5. (2R,3S)-1,2-Dimethyl-3-hydroxy-3-(6-fluoronaph-
thalen-2-yl)-pyrrolidineÆHCl [(2R,3S)-4ÆHCl]. Yield
(35.3 mmol) in anhydrous diethyl ether (20 mL) was
added dropwise. After stirring for 3 h at 0 ꢁC, the reac-
tion mixture was quenched with water (120 mL) and,
after an acid–base work up, the combined organic
phases were evaporated under vacuum. The resulting
crude product was added with a 10% solution of HCl
(15 mL), with exception for (R/S)-17–19, which were
treated with DL-tartaric acid. The corresponding hydro-
chlorides or DL-tartrates, obtained as white solids, were
crystallized from an appropriate solvent.
71.8%; white crystals (IPA 8/H2O 2, v/v), mp 210–
212 ꢁC. For [a] and ee% values see Table 1. IR (Nujol):
cmꢀ1 3250, 2675, 1610, 1510, 1407, 1190, 1103, 969, 949,
1
897, 802. H NMR (CD3OD): d 1.15 (d, 3H, CH3–CH,
J = 6.5 Hz),
2.28
(ddd,
1H,
HCH–CH2N,
Jgem = 13.7 Hz, Jvic = 3.4–8.6 Hz), 2.75 (ddd, 1H,
HCH–CH2N, Jgem = 13.7 Hz, Jvic = 8.6–11.6 Hz), 3.42
(t, 1H, HCH–N, Jgem = 11.3 Hz, Jvic = 4Hz), 3.70 (q,
1H, CH3–CH, J = 6.5 Hz), 3.90 (dt, 1H, HCH–N,
Jgem = 11.3 Hz, Jvic = 8.4–8.4 Hz), 7.28 (dt, 1H, aro-
matic), 7.49 (d, 1H, aromatic), 7.62 (d, 1H, aromatic),
7.85 (d, 1H, aromatic), 7.90 (dd, 1H, aromatic), 8.03
(s, 1H, aromatic). Elemental analysis: C16H19NOFCl re-
quires C, 64.97; H, 6.47; N, 4.74. Found: C, 64.95; H,
6.59; N, 4.99.
4.3.1. (2R,3S)-1,2-Dimethyl-3-hydroxy-3-naphthalen-2-yl-
pyrrolidineÆHCl [(2R,3S)-1ÆHCl]. Yield 49.4%; white
crystals (IPA 9/H2O 1, v/v), mp 168–170 ꢁC. For [a]
and ee% values see Table 1. IR (Nujol): cmꢀ1 3300,
2680, 1602, 1505, 1112, 1075, 960, 900, 819, 750. H
NMR (CD3OD): d 1.15 (d, 3H, CH3–CH, J = 6.5 Hz),
2.28 (ddd, 1H, HCH–CH2N, Jgem = 14.0 Hz,
1
4.3.6. (2S,3R)-1,2-Dimethyl-3-hydroxy-3-(6-fluoronaph-
Jvic = 3.5–8.7 Hz), 2.78 (ddd, 1H, HCH–CH2N, Jgem
=
thalen-2-yl)-pyrrolidineÆHCl
70.5%; white crystals (IPA 8/H2O 2, v/v), mp 210–
211 ꢁC. For [a] and ee% values see Table 1. IR and H
NMR spectra are identical to that of the corresponding
enantiomer. Elemental analysis: C16H19NOFCl requires
C, 64.97; H, 6.47; N, 4.74. Found: C, 65.08; H, 6.34; N,
4.80.
[(2S,3R)-4ÆHCl].
Yield
14.0 Hz, Jvic = 8.3–10.5 Hz), 2.97 (s, 3H, CH3–N), 3.45
(dt, 1H, HCH–N, Jgem = 11.0 Hz, Jvic = 3.4Hz), 3.75
(q, 1H, CH3–CH, J = 6.5 Hz), 3.91 (dt, 1H, HCH–N,
Jgem = 11.0 Hz, Jvic = 8.0 Hz), 7.45 (m, 2H, aromatic),
7.61 (dd, 1H, aromatic), 7.83–8.01 (s and m, 4H, aro-
matic). Elemental analysis: C16H20NOCl requires C,
69.18; H, 7.26; N, 5.04. Found: C, 69.29; H, 7.56; N,
4.88.
1
4.3.7. (2R,3S)-1,2-Dimethyl-3-hydroxy-3-phenyl-pyrroli-
dineÆHCl [(2R,3S)-5ÆHCl]. Yield 23%; white crys-
tals (CH3COCH3 15/H2O 1, v/v), mp 233–235 ꢁC.
For [a] and ee% values see Table 1. IR (Nujol): cmꢀ1
4.3.2. (2S,3R)-1,2-Dimethyl-3-hydroxy-3-naphthalen-2-yl-
pyrrolidineÆHCl [(2S,3R)-1ÆHCl]. Yield 50.9%; white
crystals (IPA 9/H2O 1, v/v), mp 170–171 ꢁC. For [a]
1
3223, 2923, 1895, 1599, 1317, 1230, 973, 754. H NMR
1
and ee% values see Table 1. IR and H NMR spectra
(CD3OD): d 1.16 (d, 3H, CH3–CH, J = 6.6 Hz), 2.27
(ddd, 1H, HCH–CH2N, Jgem = 12.7 Hz, Jvic = 3.5–
are identical to that of the corresponding enantiomer.
Elemental analysis: C16H20NOCl requires C, 69.18; H,
7.26; N, 5.04. Found: C, 69.32; H, 7.31; N, 4.96.
8.5 Hz), 2.69 (ddd, 1H, HCH–CH2N, Jgem
=
12.7 Hz, Jvic = 2.2–8.5 Hz), 2.99 (s, 3H, N–CH3), 3.46
(dt, 1H, HCH–N, Jgem = 12.0 Hz, Jvic = 3.5 Hz), 3.68
(q, 1H, CH–CH3, J = 6.6 Hz), 3.92 (dt, 1H, HCH–
N, Jgem = 12.0 Hz, Jvic = 2.83–8.12 Hz), 7.30 (t, 1H,
aromatic), 7.40 (m, 2H, aromatic), 7.57 (m, 2H,
aromatic). Elemental analysis: C12H18NOCl requires
C, 63.29; H, 7.97; N, 6.15. Found: C, 63.38; H, 7.85;
N, 6.20.
4.3.3.
(2R,3S)-1,2-Dimethyl-3-hydroxy-3-(6-methoxy-
naphthalen-2-yl)-pyrrolidineÆHCl [(2R,3S)-3ÆHCl]. Yield
20.7%; white crystals (IPA 9/H2O 1, v/v), mp 215–
217 ꢁC. For [a] and ee% values see Table 1. IR (Nujol):
cmꢀ1 3220, 2662, 1630, 1605, 1200, 1028, 962, 905, 850,
812. 1H NMR (CD3OD): d 1.15 (d, 3H, CH3–CH,
J = 6.5 Hz),
2.28
(ddd,
1H,
HCH–CH2N,
Jgem = 13.5 Hz, Jvic = 3.5–8.5 Hz), 2.74(ddd, 1H,
HCH–CH2N, Jgem = 13.5 Hz, Jvic = 8.6–11.5 Hz), 2.95
(s, 3H, CH3–N), 3.42 (dt, 1H, HCH–N, Jgem = 11.3 Hz,
Jvic = 3.5 Hz), 3.68 (q, 1H, CH3–CH, J = 6.5 Hz), 3.85
(s, 3H, OCH3), 3.89 (dt, 1H, HCH–N, Jgem = 11.3 Hz,
Jvic = 8.5 Hz), 7.10 (dd, 1H, aromatic), 7.19 (dt, 1H, aro-
matic), 7.53 (dd, 1H, aromatic), 7.73 (d, 1H, aromatic),
7.77 (d, 1H, aromatic), 7.91 (s, 1H, aromatic). Elemental
analysis: C17H22NO2Cl requires C, 66.33; H, 7.20; N,
4.55. Found: C, 66.36; H, 7.41; N, 4.68.
4.3.8. (2S,3R)-1,2-Dimethyl-3-hydroxy-3-phenyl-pyrroli-
dineÆHCl [(2S,3R)-5ÆHCl]. Yield 22%; white crystals
(CH3COCH3 15/H2O 1, v/v), mp 234–235 ꢁC. For [a]
1
and ee% values see Table 1. IR and H NMR spectra
are identical to that of the corresponding enantiomer.
Elemental analysis: C12H18NOCl requires C, 63.29; H,
7.97; N, 6.15. Found: C, 63.11; H, 8.16; N, 5.98.
4.3.9. (2R,3S)-1,2-Dimethyl-3-hydroxy-3-(4-benzyloxy-
phenyl)-pyrrolidineÆHCl [(2R,3S)-6ÆHCl]. Yield 35%;
white crystals (CH3COCH3), mp 215–219 ꢁC. For [a]
and ee% values see Table 1. IR (Nujol): cmꢀ1 3353,
1511, 1459, 1179, 1074, 1001, 944, 724. 1H NMR
(CD3OD): d 1.19 (d, 3H, CH3–CH, J = 6.4Hz), 2.25
(ddd, 1H, HCH–CH2N, Jgem = 11.3 Hz, Jvic = 3.2–
8.1 Hz), 2.66 (ddd, 1H, HCH–CH2N, Jgem = 11.3 Hz,
Jvic = 2.8–9.2 Hz), 2.97 (s, 3H, CH3–N), 3.40 (dt, 1H,
HCH–N, Jgem = 10.6 Hz, Jvic = 3.9 Hz), 3.55 (q, 1H,
CH–CH3, J = 6.4Hz), 3.89 (dt, 1H, HCH–N,
4.3.4.
(2S,3R)-1,2-Dimethyl-3-hydroxy-3-(6-methoxy-
naphthalen-2-yl)-pyrrolidineÆHCl [(2S,3R)-3ÆHCl]. Yield
21.3%; white crystals (IPA 9/H2O 1, v/v), mp 214–
1
217 ꢁC. For [a] and ee% values see Table 1. IR and H
NMR spectra are identical to that of the corresponding
enantiomer. Elemental analysis: C17H22NO2Cl requires
C, 66.33; H, 7.20; N, 4.55. Found: C, 66.10; H, 7.32;
N, 4.89.

S. Collina et al. / Bioorg. Med. Chem. 13 (2005) 3117–3126
3125
Jgem = 10.6 Hz, Jvic = 3.5 Hz), 5.12 (s, 2H, CH2–O), 7.05
(m, 2H, aromatic), 7.33 (m, 1H, aromatic), 7.38 (m, 2H,
aromatic), 7.45 (m, 4H, aromatic). Elemental analysis:
C19H24NO2Cl requires C, 68.36; H, 7.25; N, 4.20.
Found: C, 68.01; H, 7.40; N, 3.98.
4.3.15. (R/S)-1-Methyl-3-hydroxy-3-naphthalen-2-yl-pyr-
rolidineÆDL-tartrate [(R/S)-17ÆDL-tartrate]. Yield 79%;
white solid, hygroscopic. IR (Nujol): cmꢀ1 3300, 3053,
1
2789, 1475, 1271, 1152, 947, 747. H NMR (CD3OD):
d 2.40 (dd, 1H, HCH–CH2N, J = 9.9 Hz), 2.70 (dd,
1H, HCH–CH2N, J = 9.9 Hz), 3.05 (s, 3H, CH3), 3.62
(dt, 1H, HCH–N, J = 11.3 Hz), 3.77 (dt, 1H, HCH–N,
J = 8.1 Hz), 4.47 (s, 2H, CH2–COH), 7.47 (m, 2H, aro-
matic), 7.63 (d, 1H, aromatic), 7.83 (t, 1H, aromatic),
7.87 (m, 2H, aromatic), 8.02 (s, 1H, aromatic). Elemen-
tal analysis: C19H23NO7 requires C, 60.47; H, 6.14; N,
3.71. Found: C, 60.30; H, 6.53; N, 4.08.
4.3.10. (2S,3R)-1,2-Dimethyl-3-hydroxy-3-(4-benzyloxy-
phenyl)-pyrrolidineÆHCl [(2S,3R)-6ÆHCl]. Yield 39%;
white crystals (CH3COCH3), mp 215–218 ꢁC. For [a]
1
and ee% values see Table 1. IR and H NMR spectra
are identical to that of the corresponding enantiomer.
Elemental analysis: C19H24NO2Cl requires C, 68.36; H,
7.25; N, 4.20. Found: C, 68.74; H, 7.33; N, 4.04.
4.3.16. (R/S)-1-Methyl-3-hydroxy-3-phenyl-pyrrolidineÆ
DL-tartrate [(R/S)-18ÆDL-tartrate]. Yield 33%; white
solid, hygroscopic. IR (Nujol): cmꢀ1 3200, 2945, 2787,
4.3.11. (2R,3S)-1,2-Dimethyl-3-hydroxy-3-(4-biphenyl)-
pyrrolidineÆHCl [(2R,3S)-7ÆHCl]. Yield 35%; white crys-
tals [(CH3)2CHOH 10/H2O 1, v/v], mp 260–261 ꢁC.
For [a] and ee% values see Table 1. IR (Nujol): cmꢀ1
1
1493, 1343, 1154, 962, 762. H NMR (CD3OD): d 2.33
(dd, 1H, HCH–CH2N, J = 9.9 Hz), 2.58 (ddd, 1H,
HCH–CH2N, J = 9.9 Hz), 3.01 (s, 3H, CH3), 3.55 (dt,
1H, HCH–N, J = 11.3 Hz), 3.70 (dt, 1H, HCH–N,
J = 11.3 Hz), 4.44 (s, 2H, CH2–CHOH), 7.29 (t, 1H,
aromatic), 7.37 (m, 2H, aromatic), 7.54(m, 2H, aro-
matic). Elemental analysis: C15H21NO7 requires C,
55.04; H, 6.47; N, 4.28. Found: C, 55.27; H, 6.81; N,
4.01.
1
3197, 2652, 1230, 1166, 1068, 949, 839, 729. H NMR
(CD3OD): d 1.91 (d, 3H, CH3–CH, J = 6.4Hz), 2.29
(ddd, 1H, HCH–CH2N, Jgem = 11.3 Hz, Jvic = 3.2–
8.1 Hz), 2.73 (ddd, 1H, HCH–CH2N, Jgem = 11.3 Hz,
Jvic = 2.9–9.2 Hz), 2.98 (s, 3H, CH3–N), 3.45 (dt, 1H,
HCH–N, Jgem = 10.6 Hz, Jvic = 3.9 Hz), 3.69 (q, 1H,
CH–CH3, J = 6.4Hz), 3.92 (dt, 1H, HCH–N,
Jgem = 10.6 Hz, Jvic = 3.5 Hz), 7.32 (m, 1H, aromatic),
7.42 (m, 2H, aromatic), 7.60 (m, 2H, aromatic), 7.65
(m, 4H, aromatic). Elemental analysis: C18H22NOCl re-
quires C, 71.16; H, 7.30; N, 4.61. Found: C, 71.22; H,
7.02; N, 4.87.
4.3.17. (R/S)-1-Methyl-3-hydroxy-3-(4-biphenyl)-pyrroli-
dineÆDL-tartrate [(R/S)-19ÆDL-tartrate]. Yield 86%; white
solid, hygroscopic. IR (Nujol): cmꢀ1 3300, 1290, 1237,
1
1153, 1035, 961, 842, 730. H NMR (CD3OD): d 2.25
(dd, 1H, HCH–CH2N, J = 9.9 Hz), 2.52 (dd, 1H,
HCH–CH2N, J = 9.9 Hz), 2.93 (s, 3H, CH3), 3.47 (dt,
1H, HCH–N, J = 11.3 Hz), 3.62 (dt, 1H, HCH–N
J = 11.3 Hz), 4.35 (s, 2H, CH2–COH), 7.21 (m, 1H, aro-
matic), 7.31 (m, 2H, aromatic), 7.49 (m, 2H, aromatic),
7.52 (m, 4H, aromatic). Elemental analysis: C21H25NO7
requires C, 62.52; H, 6.25; N, 3.47. Found: C, 62.56; H,
6.60; N, 3.76.
4.3.12. (2S,3R)-1,2-Dimethyl-3-hydroxy-3-(4-biphenyl)-
pyrrolidineÆHCl [(2S,3R)-7ÆHCl]. Yield 34%; white crys-
tals [(CH3)2CHOH 10/H2O 1, v/v], mp 261–262 ꢁC.
1
For [a] and ee% values see Table 1. IR and H NMR
spectra are identical to that of the corresponding enan-
tiomer. Elemental analysis: C18H22NOCl requires C,
71.16; H, 7.30; N, 4.61. Found: C, 70.82; H, 7.45; N,
4.22.
4.4. Molecular mechanics calculations
4.3.13. (2R,3S)-1,2-Dimethyl-3-hydroxy-3-(4-chlorophen-
yl)-pyrrolidineÆDL-tartrate [(2R,3S)-8ÆDL-tartrate]. Yield
56%; white solid, hygroscopic. For [a] and ee% values
see Table 1. IR (Nujol): cmꢀ1 3317, 2679, 2360, 1162,
1082, 976, 946, 721. 1H NMR (CD3OD): d 1.19 (d,
3H, CH3–CH, J = 6.4Hz), 2.29 (ddd, 1H, HC H–
CH2N, Jgem = 12.7 Hz, Jvic = 3.4–8.8 Hz), 2.70 (ddd,
1H, HCH–CH2N, Jgem = 12.7 Hz, Jvic = 2.4–8.8 Hz),
2.99 (s, 3H, CH3–N), 3.45 (dt, 1H, HCH–N,
Jgem = 10.8 Hz, Jvic = 2.5 Hz), 3.63 (q, 1H, CH–CH3,
J = 6.4Hz), 3.93 (dt, 1H, HCH–N, Jgem = 10.8 Hz,
Jvic = 2.9 Hz), 7.44 (m, 2H, aromatic), 7.57 (m, 2H, aro-
matic). Elemental analysis: C12H17NOCl2 requires C,
54.97; H, 6.54; N, 5.34. Found: C, 54.90; H, 6.33; N,
5.12.
The molecular mechanics experiments were performed
using the MacroModel package 7.28 implemented under
Linux operating systems. All calculations were carried
out on a Intel Xeon biprocessor 3.0 GHz with 1 GB of
RAM. The Maestro Graphical User Interface was used
for assembling the starting geometries of 1, 5, 17, and 18
stereoisomers. The conformational search was carried
out by a 1000 iteration MC exploration of torsional
internal degrees of freedom set adding manually the
other dihedral angles not selected by the automatic set-
up procedure. This operation detected only the hydroxyl
in C3. The flexibility of the five-member ring was consid-
ered adding a closure bond defined by the atoms C3–
C4–C5–N1 and two dihedral angles, respectively, rotat-
ing the N1–C2 and C2–C3 bonds. The Ar–C3 bond was
also added to the list of the rotatable bonds, but for the
symmetric phenyl moiety of compounds 5 and 18, atom
equivalencies were considered in order to avoid conform-
ational duplications. Compounds 1 and 5 required the
chirality check to be activated for chiral carbon atoms
C2 and C3. Only this last atom was considered for the
simplified compounds. Specular geometries were
4.3.14. (2S,3R)-1,2-Dimethyl-3-hydroxy-3-(4-chlorophen-
yl)-pyrrolidineÆDL-tartrate [(2S,3R)-8ÆDL-tartrate]. Yield
58.5%; white solid, hygroscopic. For [a] and ee% values
1
see Table 1. IR and H NMR spectra are identical to
that of the corresponding enantiomer. Elemental analy-
sis: C12H17NOCl2 requires C, 54.97; H, 6.54; N, 5.34.
Found: C, 55.07; H, 6.26; N, 5.62.

3126
S. Collina et al. / Bioorg. Med. Chem. 13 (2005) 3117–3126
obtained inverting the sign of the Z atomic coordinates
using a module of the MOLINE program.9 Another mod-
ule of this tool was considered for computing the Boltz-
mann population of each conformer at room
temperature. The force field considered was the
MMFFs10 and the water environment was simulated
by the GB/SA implicit model solvation.11 During the
MC simulations the deduplication protocol between
two generic conformations was set to the default values,
4.5.3. Rota-rod test. The integrity of motor coordination
was controlled with RRT on mice after treatment with
the investigated substances.14 Randomly selected mice,
were examined 15 min after treatment with doses corre-
sponding to 4and 8 mg/kg. Results are expressed as the
percentage of mice remaining on the rod during a 30 s
period.
4.5.4. Statistical analysis. The results of HPT, FT, and
RRT were expressed as means SEM and the means
˚
that is, RMS deviation lower than 0.25 A and energy
*
difference lower than 4.184 kJ/mol. The average number
of duplicates was considered to estimate the convergence
in the MC search. Graphical superimpositions were per-
formed by the analysis module of the Maestro GUI.
were compared using Studentꢁs t-test,
(p values
(p < 0.01) being considered as statistically
**
<0.05) or
significant or highly significant, respectively.
All the statistical analyses were performed using the sta-
tistical software package SYSTAT.
4.5. Biological assays
The experiments were carried out on male adult Swiss
mice (30 5 g). Control and experimental groups con-
sisted of 8–10 animals each. The salts of the investigated
compounds were dissolved in saline solution and admin-
istered subcutaneously within 1 h of dissolution.
References and notes
1. Ghislandi, V.; Collina, S.; Azzolina, O.; Barbieri, A.;
Lanza, E.; Tadini, C. Chirality 1999, 11, 21.
2. Collina, S.; Azzolina, O.; Vercesi, D.; Sbacchi, M.;
Scheideler, M. A.; Barbieri, A.; Lanza, E.; Ghislandi, V.
Bioorg. Med. Chem. 2000, 8, 1925.
3. Collina, S.; Azzolina, O.; Vercesi, D.; Brusotti, G.; Rossi,
D.; Barbieri, A.; Lanza, E.; Mennuni, L.; Alcaro, S.;
Battaglia, D.; Linati, L.; Ghislandi, V. Il Farmaco 2003,
58, 939.
4. Collina, S.; Gamba, V.; Ghislandi, V.; Azzolina, O.
Chirality 1995, 7, 439.
5. Balasubramanian, D.; Sukumar, P.; Chandani, B. Tetra-
hedron Lett. 1979, 37, 3543.
6. Cavalla, J. F.; Davoll, J.; Dean, M. J.; Franklin, C. S.;
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4.5.1. Hot plate test. The hot plate test was employed to
assess the antinociceptive effects. MorphineÆHCl was
used as a standard antinociceptive agent. The response
to a thermal stimulus was evaluated using a copper plate
heated to 55 ꢁC and proceeding according to a previously
reported method.12 The animal responded by sitting on
its hind legs and licking. The experiment was conducted
on mice treated with 2, 4, and 8 mg/kg of the compounds.
The reaction time to the pain stimulus was measured
20 min after the injection. The cut-off was 24.7 2.1 s.
Experimental data are reported in Table 4.
4.5.2. Formalin test (FT). Nociception was induced by
injecting 20 lL dilute formalin (1% in saline solution) un-
der the skin of the dorsal surface of the hind paw of the
mouse.13 The licking of the treated hind paw was used as
a measure of nociception. Each mouse was injected with
formalin 10 min after being pretreated with analgesic
compounds and then placed into a transparent plastic
cage and observed. The licking response was monitored
until 30 min, starting immediately after the injection of
formalin. Experiments were performed on two separate
groups of animals: the control group, receiving formalin
only, and the treated one receiving formalin and each
compound administered at 8 mg/kg. Antinociception
was defined as a statistically significant reduction in the
time spent licking, in comparison with the vehicle control
group during the early (0–5 min) and late phase (20–
25 min). Results obtained are expressed as licking time
in seconds. Experimental data are reported in Figure 6.
12. Collina, S.; Azzolina, O.; Vercesi, D.; Benevelli, F.;
Callegari, A.; Tadini, C.; Lanza, E.; Barbieri, A.; Ghis-
landi, V. Bioorg. Med. Chem. 2000, 8, 769.
13. Murray, C. W.; Porreca, F.; Cowan, A. J. Pharm.
Methods 1988, 20, 175.
14. Collina, S.; Azzolina, O.; Vercesi, D.; Barbieri, A.; Lanza,
E.; Ghislandi, V. Farmaco 2000, 55, 611.