Chemical and biological profile of racemic and optically active dialkylaminoalkylnaphthalenes with analgesic activity

Article abstract of DOI:10.1016/S0957-4166(02)00241-0

The racemic mixtures and the enantiomers of dialkylaminoalkylnaphthalenes are described here as novel analgesic agents with potencies similar, or superior to that of morphine. The synthesis and isolation of the pure enantiomers and a study of the absolute

Full text of DOI:10.1016/S0957-4166(02)00241-0

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 13 (2002) 1073–1081
Chemical and biological profile of racemic and optically active
dialkylaminoalkylnaphthalenes with analgesic activity
a,
a
a
a
b
Ornella Azzolina, * Simona Collina, Gloria Brusotti, Daniela Rossi, Athos Callegari,
c
d
a
Laura Linati, Annalisa Barbieri and Victor Ghislandi
a
Dipartimento di Chimica Farmaceutica, Universit a` di Pavia, Viale Taramelli 12, 27100, Pavia, Italy
b
Dipartimento di Scienze della Terra, Universit a` di Pavia, Via Ferrata 1, 27100 Pavia, Italy
c
Centro Grandi Strumenti, Universit a` di Pavia, Via Bassi 21, 27100 Pavia, Italy
d
Dipartimento di Farmacologia Sperimentale ed Applicata, Universit a` di Pavia, Viale Taramelli 14, 27100 Pavia, Italy
Received 24 April 2002; accepted 14 May 2002
Abstract—The racemic mixtures and the enantiomers of dialkylaminoalkylnaphthalenes are described here as novel analgesic
agents with potencies similar, or superior to that of morphine. The synthesis and isolation of the pure enantiomers and a study
of the absolute configuration are reported. The resolution of racemates was accomplished by preparative liquid chromatography
using a Chiralpak AD column. The configurational assignment was performed on the basis of the X-ray crystallographic analysis
1
of (+)-benzyl-(3-hydroxy-3-naphthalen-2-yl-butyl)dimethylammonium bromide and by comparative study of CD and NOESY H
NMR spectra of the resolved enantiomers. Pharmacological evaluation of the analgesic activity by means of the hot plate test is
described. © 2002 Elsevier Science Ltd. All rights reserved.
1
. Introduction
(Fig. 1, series II) were synthesized by us and their
pharmacological activities were investigated by hot
4–8
Our studies during recent years have focused on pain
control and have been aimed at finding new ligands
with high affinity for receptors and sub-receptors
involved in the regulation of nociceptive stimuli. The
availability of novel pharmacological compounds with
analgesic effects for the treatment of post-operative,
neurogenic and osteoarthritic pain would be very useful
in order to avoid the many side effects of morphine and
plate test (HPT) in mice.
Several compounds were
found to possess antinociceptive activity comparable or
superior to that of morphine as shown by their AD50
values. In particular, in series II it was shown that the
naphthalene nucleus influences the in vivo activity,
because substantial differences in the response to the
pain stimulus were recorded according to structural
modifications at the 6-position of the aromatic ring.
1
–3
its derivatives.
We therefore directed our research
toward the design, synthesis and pharmacological eval-
uation of possible analgesic compounds as potential
effective and safe drugs. Dialkylaminoalkylnaphthal-
enes (Fig. 1, series I) and cycloaminoalkylnaphthalenes
Herein, we report a study of the novel tertiary amino
alcohols 1–3 (Scheme 1) to investigate the role of the
substituent in the aromatic portion of the molecule,
also in the open chain series I. Due to a hypothetical
stereoselectivity of the drug–receptor interaction, we
also describe the preparation of optically active 1–3. To
the best of our knowledge neither enantioselective syn-
thesis or the resolution of open chain tertiary amino
alcohols, with only one stereogenic center, has been
reported. Therefore, on the basis of our previous expe-
riences, all of the enantiomerically pure compounds 1–3
were obtained by chiral resolution of racemates 1–3,
using a direct chromatographic method with a chiral
R1
OH
R1
N
(
CH2)n
N
OH
R2
R
R
Series I
Series II
Figure 1. Dialkylaminoalkyl- and cycloaminoalkylnaphthal-
enes.
9–11
stationary phase (CSP).
The absolute configurations
of the two enantiomers of all compounds were deter-
1
mined by X-ray crystallographic analysis, CD and H
*
Corresponding author. Tel.: 00390382507356; fax: 00390382422975;
e-mail: ornella.azzolina@unipv.it
NMR spectroscopy. Finally, a preliminary profile of
0
957-4166/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(02)00241-0

1
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O. Azzolina et al. / Tetrahedron: Asymmetry 13 (2002) 1073–1081
OH
Br
1) tert-BuLi
N
2
) CH CO(CH ) N(CH )
3
2 2
3 2
R
R
3
) H2O
R = H
R = F
R = OCH3
(RS)-1
(RS)-2
(RS)-3
Scheme 1.
the biological properties was established by measuring
the analgesic activity of the compounds with HPT
performed on mice.
logical investigation, the resolution of the racemates
1–3 was performed by semi-preparative HPLC with a
Chiralpak AD column (25×2 cm) (Table 2). Chromato-
graphic resolutions were scaled up to the maximum
tolerated level by increasing the amounts of samples
injected into the column until the maximum amount
compatible with the best yield of both enantiomers was
reached. Mixtures of n-hexane/2-propanol (IPA), con-
taining a small amount of diethylamine (DEA), were
used as eluents, at a flow rate of 8 mL/min. The
injection of the racemic samples was repeated every 45
min to optimize the efficiency of the chromatographic
resolution. In this way about 12 mg of enantiopure 1–3
was obtained from each chromatographic run. Enan-
tiomeric excess (e.e.) measurements were completed on
analytical scale and all enantiomers were obtained with
e.e. of ]99.3% with a recovery from the preparative
resolution of about 90% (Fig. 2, Table 3). The first
eluting enantiomers are (R)-(+)-1, (S)-(−)-2 and (S)-
(−)-3, highlighting the influence of the naphthalene
substituent in the stability of diastereoisomeric com-
plexes deriving from the solute–CSP interaction.
2
. Results and discussion
The synthesis of racemic 1–3 was accomplished follow-
ing the procedure that we had used for previous com-
4
pounds.
The results of elemental analyses,
1
chromatographic tests, IR and H NMR spectra agreed
with the assigned structures for all compounds. Differ-
ential scanning calorimetry (DSC) showed the absence
of crystallization solvent in these compounds.
In order to develop a rapid, direct method for the
resolution of racemic 1–3 we attempted several liquid
chromatography procedures using a number of chiral
stationary phases. Baseline separations of all com-
pounds were successfully achieved on analytical scale
with Chiralcel OB-H, Chiralpak AD and Chiralpak AS
CSPs: good separation factor values and very short
analysis times were obtained, as shown by the results
reported in Table 1.
The absolute configurations of the resolved enan-
tiomers were established on the basis of crystallo-
graphic studies as well as CD and H NMR studies.
The absolute configuration of the dextrorotatory enan-
1
So as to obtain sufficient quantities of both enan-
tiomers of 1–3 for configurational study and pharmaco-
Table 1. Analytical resolution of racemic mixtures 1–3
Compd
Column (250×4.6 mm)
Mobile phase
Flow rate (mL/min)
rt1 (min) (config.)
rt2 (min) (config.)
1
2
AD
AS
AD
OB-H
AD
95/5/0.01a
0.7
1.0
1.0
1.0
1.0
10.6 (R)
7.3 (R)
12.0 (S)
11.1 (S)
8.7 (S)
12.5 (S)
9.4 (S)
14.4 (R)
13.1 (R)
10.9 (R)
a
98.5/1.5/0.1
a
97/3/0.01
98/2/0.1
b
3
a
95/5/0.01
a
Solvent mixture: n-hexane/IPA/DEA (v/v/v).
Solvent mixture: n-hexane/EtOH/DEA (v/v/v). Detector: u=273 nm.
b
Table 2. Semi-preparative resolution of racemic mixtures 1–3 on a Chiralpak AD column (25×2 cm)
Compd
Mobile phasea
Injection volume (mL)
Conc.b (mg/mL)
rt1 (min) (config.)
rt2 (min) (config.)
1
2
3
95/5/0.01
97/3/0.01
95/5/0.01
3
3
3
11.2
12.5
13.0
17.6 (R)
20.0 (S)
20.5 (S)
21.0 (S)
24.0 (R)
26.0 (R)
a
n-hexane/IPA/DEA (v/v/v); flow rate=8 mL/min.
In the mobile phase. Detector: u=273 nm.
b

O. Azzolina et al. / Tetrahedron: Asymmetry 13 (2002) 1073–1081
1075
Table 3. Chiroptical and physico-chemical data for the
resolved enantiomers of compounds 1–3
2
4
2
05
E.e. %a
Compound
[h]
Column
Mp (°C)
(R)-1
(S)-1
(R)-2
(S)-2
(R)-3
(S)-3
+14.8b
99.9
99.5
99.3
99.9
99.9
99.9
AD
AD
AS
AS
OB-H
OB-H
85–87
85–87
89–91
90–92
108–109
108–109
b
−14.1
c
+6.4
c
−6.5
b
+42.4
−42.5b
a
b
c
Table 1 and Fig. 2.
c=1, MeOH.
c=0.5, MeOH.
Br-
OH
N
+
Figure 3. (R)-(+)-5.
tion spectroscopy because of the presence of the high
12–14
density bromine atom.
Crystalline sample of (+)-5
was obtained by slow crystallization from EtOH. A
stereoscopic view, with the atomic numbering scheme
of the compound is shown in Fig. 4 and the molecular
structure packing, obtained using the ORTEP 3 pro-
12
gram, is shown in Fig. 5. In this way compound (+)-5
was found to have (R)-configuration. Therefore, the
(
R)-configuration was also assigned to (+)-1 because its
transformation into (+)-5 did not involve reaction at
the stereogenic center.
The configurational assignment of 2 and 3 was effected
by comparative CD curve analysis of the levorotatory
isomers 1–3. The spectra show analogous profiles, with
the same Cotton effects in the spectral region between
2
00 and 250 nm (Fig. 6). Therefore, the (S)-absolute
configuration of (−)-1 may also be assigned to (−)-2 and
−)-3. Further confirmation of the configurational
(
1
1
assignment was obtained by performing H, H-COSY
and H-NOESY NMR experiments on the
1
L-tartrate
salts of (S)-(−)-2 and (S)-(−)-3. The NOESY spectra
clearly show a significant NOE effect corresponding to
the proton interactions between the methyl group
linked to the asymmetric carbon atom and the H1 of
the aromatic nucleus. This interaction is possible only
for the (S)-enantiomer, as confirmed by a molecular
15
modeling study, according to Ewig et al. Fig. 7 shows
the NOESY spectrum (A) and a molecular view (B) of
the -tartrate salt of (S)-(−)-3.
L
To establish a preliminary pharmacological profile, the
analgesic activity of the -tartrate salts of 1–3 was
investigated by HPT in mice. AD values were deter-
L
Figure 2. Analytical resolutions of racemic 1–3 and control of
the fractions collected in the semi-preparative separations.
Experimental conditions are reported in Table 1.
16
50
1
7
mined using a computer program.
tiomer of 1 was determined by crystallographic analysis
of its N-benzylammonium bromide derivative (+)-5
The experimental data (Table 4) clearly show that all
new compounds possess very interesting antinociceptive
properties with AD50 values ranging from 4.97 (4.54–
(
Fig. 3), suitable for structural study by X-ray diffrac-

1
076
O. Azzolina et al. / Tetrahedron: Asymmetry 13 (2002) 1073–1081
Figure 4. ORTEP III view of molecular structure of (R)-(+)-5.
Figure 5. ORTEP view along [c] axis of (R)-(+)-5.

O. Azzolina et al. / Tetrahedron: Asymmetry 13 (2002) 1073–1081
1077
the pain stimulus was substantially decreased by the
presence of a substituent on this hydrophobic portion.
Furthermore, the activity of the two enantiomers was
very different for compounds 2 and 3. Thus, the enan-
tioselectivity also seems to be strictly correlated with
the presence of a substituent on the aromatic ring. The
most active compounds are (R)-2 and (R)-3. On the
contrary, no significant difference was observed in the
activity of the two enantiomers of 1.
Due to their interesting analgesic activity, all com-
pounds investigated in this work and, in particular, the
most active compounds (R)-2 and (R)-3 will be further
investigated to acquire more information about their
pharmacological properties in vivo. Moreover, in vitro
tests will also be executed to investigate their possible
influence on receptor systems involved in pain control.
3
. Conclusions
Chromatographic resolution of the naphthylamino
alcohols 1–3, at the 400–500 mg scale, has been per-
formed using a Chiralpak AD column (25×2 cm). In
this way both enantiomers of all compounds examined
could be obtained with very high enantiomeric excess in
relatively short times. The derivatization of (+)-1
allowed us to obtain a compound suitable for X-ray
analysis and, consequently, to assign the (R)-configura-
tion to (+)-1. CD and NOESY NMR studies permitted
us to assign (R)-configuration to (+)-2 and (+)-3. Phar-
macological investigation highlighted the activity of
(
R)-enantiomers of compounds 2 and 3.
4
. Experimental
4
.1. General
Commercially available reagents and solvents were used
as received from the supplier. Diethyl ether was dried
1
8
and distilled according to standard procedures. Melt-
ing points were measured on SMP3 Stuart Scientific
apparatus and are uncorrected. Elemental analyses
were executed by Carlo Erba 1106 C, H, N analyzer.
TLC analyses were carried out on silica gel Kieselgel 60
F254 (Merck) and the chromatograms were detected
with UV light. UV spectra were measured on Beckman
Figure 6. CD spectra and Dm data of compounds (S)-(−)-1,
S)-(−)-2 and (S)-(−)-3.
(
5.44) to 0.22 (0.08–0.58). Their potency is similar or
superior to that of morphine [AD =4.18 (3.11–5.80)].
5
0
The naphthalene nucleus influences the in vivo activity:
Table 4. Analgesic activity in the HPT
Compound
Morphine
AD50 (mg/kg)
Conf. limits
Relative potency (versus morphine)
4.18
2.39
2.58
1.12
0.68
0.22
4.97
0.71
0.63
3.59
3.11–5.80
1.05–5.44
1.91–3.48
0.42–2.95
0.22–2.03
0.08–0.58
4.54–5.44
0.15–3.33
0.31–1.29
2.29–5.62
1.0
1.7
1.6
3.7
6.1
19.0
0.8
5.9
6.6
1.2
(
(
(
(
(
(
(
(
(
RS)-1
R)-(+)-1
S)-(−)-1
RS)-2
R)-(+)-2
S)-(−)-2
RS)-3
R)-(+)-3
S)-(−)-3

1
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O. Azzolina et al. / Tetrahedron: Asymmetry 13 (2002) 1073–1081
1
Figure 7. H NMR and NOESY spectra (A) and conformational view of (S)-(−)-3·L-tartrate.

O. Azzolina et al. / Tetrahedron: Asymmetry 13 (2002) 1073–1081
1079
DU-7000. Differential scanning calorimetry (DSC) was
carried out using a Mettler TA 4000 apparatus
equipped with DSC 20 cell and TC 10 processor. IR
spectra were recorded on a Perkin–Elmer FT-IR 1605
spectrophotometer; only noteworthy absorbtions are
given in cm . X-Ray crystallographic analysis was
performed on Philips Pw 1100 computer-controlled
four circle diffractometer, graphite-monochromated
Mo Ka radiation, v scan technique (±h, ±k, ±l), scan
width 1.5°, scan speed 0.05°/min, range 2–20° theta.
NMR spectra were performed at 9.4 T (TMS as inter-
nal standard l=0) with an AVANCE 400 spectrometer
warm to −10°C then a solution of ketone 4 in dry Et O
2
was added. Stirring was continued for 3 h at 0°C and
then the reaction mixture was treated with water. The
aqueous phase was extracted with Et O and the com-
2
bined organic phases were extracted with 5% DL-tar-
−
1
8
taric acid aqueous solution. The acid aqueous layer
was made alkaline with NaHCO to pH 8 and, after
3
extraction with CH Cl and evaporation of the solvent,
2
2
an oily product was obtained.
The crude products were crystallized from the appropri-
ate solvents and transformed into the salts (RS)-1–
3·DL-tartrate (molar ratio 1/1). DSC analysis evidenced
only an endothermic process corresponding to the melt-
ing points of the tartrates; no thermal phenomena
attributable to the evaporation of crystallization sol-
vents were observed.
(
Bruker, Germany) and a BBI 5 mm probe, chemical
shifts are given in ppm. Optical rotations were mea-
sured on a Jasco DIP-1000 photoelectric polarimeter
(
0.5 dm cell). Circular dichroism spectra were recorded
on a Jasco J-710 dichrograph. Chiral analyses were
performed by liquid chromatography on Chiralcel OB-
H, Chiralpak AS and Chiralpak AD columns (250×4.6
mm) of Daicel Chemical Industries, Tokyo, Japan. The
chiral stationary phases of these columns were cellulose
tris-4-methylbenzoate, amylose 1-phenylethylcarbamate
and amylose tris-3,5-dimethylphenylcarbamate, respec-
tively, coated on a 5 mm silica-gel substrate. Semi-
preparative resolutions of racemic 1–3 were obtained by
Chiralpak AD column (25×2 cm, 10 mm). The HPLC
system consisted of two Gilson pumps, mod. 306, a
Reodyne 7125 injector (20 mL or 3 mL sample loop,
respectively, for analytical or semi-preparative resolu-
tions) and a Gilson, mod.119, double wavelength UV
detector. Experimental data were analyzed with the
Gilson 715 HPLC software.
4.3.1. (RS)-4-Dimethylamino-2-(naphthalen-2-yl)-butan-
2-ol, (RS)-1. White solid, yield 5.37 g (62%); mp 64–
66°C (acetone 1/H O 1), R =0.29 (n-Hex 87/IPA
2
f
13/DEA 2); IR (Nujol): 3160 (broad), 3040, 1605, 1280,
1
1255, 1215, 1125, 1010, 865, 820. H NMR (400 MHz,
CDCl , TMS): l =7.87 (s, 1H, aromatic); 7.76 (m, 3H,
3
H
aromatic, J=4.8, 3.2, 5.3); 7.46 (d, 1H, aromatic, J=
8.6); 7.37 (m, 2H, aromatic J=2.8, 1.7, 4.6); 2.27 (dt,
,
1H, HCHC-OH, J=8.6, 4.9, 5.1); 2.10 (s, m, 7H,
-N(CH ) and HCHC-OH); 2.02 (m, 2H, CH -N); 1.53
3
2
2
(s, 3H, -CH ). Elemental analysis: C H NO requires
3
16 21
C, 78.97; H, 8.70; N, 5.76. Found C, 78.64; H, 9.03; N,
5.59%.
4
.3.2. (RS)-1·DL-Tartrate. White solid, mp 130–133°C.
4.2. 4-Dimethylaminobutan-2-one 4
Elemental analysis: C H NO·C H O requires C,
16
21
4
6
6
6
1.10; H, 6.92; N, 3.56. Found C, 61.24; H, 7.08; N,
The synthesis of 4-dimethylaminobutan-2-one 4 was
performed essentially according to the method reported
by Swaminathan for the preparation of 4-diethyl-
3.35%.
4.3.3.
(RS)-4-Dimethylamino-2-(6-fluoronaphthalen-2-
1
9
aminobutan-2-one. Methylvinylketone (91 mL, 1.23
mol) was added dropwise, over 50 min, to a stirred
solution of dimethylamine (1.23 mol, 220 mL) and
glacial acetic acid (3 mL) in absolute EtOH (190 mL).
Stirring was continued for 2 h and, after this time, the
solvent was removed under reduced pressure affording
a brown oil. By fractional distillation pure compound
was obtained as a colorless oil (90.0 g; yield 64%;
yl)butan-2-ol, (RS)-2. White solid, yield 1.4 g (25%), mp
98–100°C (acetone 1/H O 1), R =0.34 (n-Hex 87/IPA
2
f
13/DEA 2); IR (Nujol): 3125 (broad), 3058, 1610, 1280,
1250, 1195, 1175, 1085, 898, 870. H NMR (400 MHz,
1
CDCl , TMS): l =7.95 (s, 1H, aromatic); 7.87 (q, 1H,
3
H
aromatic, J=5.5, 3.5); 7.78 (d, 1H, aromatic, J=9.2);
7.56 (d, 1H, aromatic H, J=8.5); 7.46 (dd, 1H, aro-
matic J=9.2); 7.26 (dt, 1H, aromatic J=9.2, 2.8); 2.32
(m, 2H, CH -N); 2.15 (s, 6H, -N(CH ) ); 2.05 (m, 2H,
bp20 mmHg 60–62°C). IR (film): 2944, 2861, 2817, 1712,
2
3 2
1
1
460, 1229, 1158, 1041, 867, 812. H NMR (400 MHz,
CH C-OH); 1.57 (s, 3H, -CH ). Elemental analysis:
2
3
CDCl , TMS): l =2.57 (m, 4H, CH CH ); 2.20 (s, 6H,
C H FNO requires C, 73.53; H, 7.71; N, 5.36. Found
C, 73.64; H, 7.75; N, 5.35%.
3
H
2
2
16 20
N(CH ) ); 2.16 (s, 3H, CH CO). Elemental analysis:
3
2
3
C H NO requires C, 62.57; H, 11.38; N, 12.16. Found
C, 62.61; H, 11.29; N, 12.20%.
6
13
4.3.4. (RS)-2·DL-Tartrate. White solid, mp 149–151°C.
Elemental analysis: C H FNO·C H O requires C,
16
20
4
6
6
4
.3. General procedure for the preparation of
58.39; H, 6.37; N, 3.40. Found C, 58.52; H, 6.59; N,
3.24%.
compounds 1–3 in racemic form
The syntheses of (RS)-1–3 were essentially performed
4.3.5. (RS)-4-Dimethylamino-2-(6-methoxynaphthalen-2-
yl)butan-2-ol, (RS)-3. White solid, yield 4.10 g (69%),
mp 98–100°C (acetone 1/H O 1), R =0.38 (n-Hex 87/
as reported in the Scheme 1, according to the procedure
4
that we have already described.
2
f
IPA 13/DEA 2); IR (Nujol): 3095 (broad), 1632, 1604,
1260, 1218, 1192, 1125, 1010, 915, 865. H NMR (400
1
To a solution of the appropriate 2-bromonaphthalene
in dry Et O, cooled to −40°C, tert-BuLi was added with
MHz, CDCl , TMS): l =7.89 (s, 1H, aromatic); 7.75
(t, 2H, aromatic, J=9.2); 7.50 (d, 1H, aromatic, J=
2
3
H
stirring under N . After 1 h the mixture was allowed to
2

1
080
O. Azzolina et al. / Tetrahedron: Asymmetry 13 (2002) 1073–1081
8
.5); 7.20 (sb, 1H, aromatic); 7.11 (dd, 1H, aromatic,
structure parameter is 0.02(4). Cell parameters were
determined from a least-squares refinement of the set-
ting angles of 60 reflections. The H atoms were calcu-
lated and inserted with an isotropic displacement factor
proportional to those of their neighboring atoms and
not refined. Crystallographic data for (+)-5 reported in
this paper have been deposited with Cambridge Crys-
tallographic Centre (CCDC 182723).
J=8.5); 3.89 (s, 3H, CH O); 3.10 (q, 1H, HCHC-
N(CH ) , J=5.7); 2.86 (q, 1H, HCHC-N(CH ) , J=
3
3
2
3 2
7
.1); 2.78 (s, 6H, -N(CH ) ); 2.29 (m, 2H, CH -N); 1.66
3 2 2
(
s, 3H, -CH ). Elemental analysis: C H NO requires
3 17 23 2
C, 74.69; H, 8.48; N, 5.12. Found C, 74.82; H, 8.65; N,
5
.05%.
4
.3.6. (RS)-3·DL-Tartrate. White solid, mp 131–133°C.
4
.7. Circular dichroism analysis
Elemental analysis: C H NO ·C H O requires C,
17
23
2
4
6
6
5
3
9.56; H, 6.9; N, 3.31. Found C, 59.72; H, 7.05; N,
.28%.
Solutions of the levo-isomers 1–3 in n-hexane (c=2.5×
−
5
1
0
M; optical path 1 cm) were analyzed in the spectral
region between 200 and 250 nm. CD spectra are
reported in Fig. 6.
4
.4. (R)-(+)-Benzyl-(3-hydroxy-3-naphthalen-2-ylbutyl)-
dimethylammonium bromide, (R)-(+)-5
1
4
.8. H NMR spectroscopy
Benzyl bromide (29.2 mg, 0.171 mmol) was added to a
22
solution of (+)-1 (41.6 mg, 0.171 mmol, [h] =+14.8
D
1
1
1
H, H-COSY and H-NOESY NMR spectra of (S)-(−)-
(
c=1 MeOH), e.e. 99.9% HPLC) in absolute EtOH (1
2
and 3·L-tartrate salts were performed at 9.4 T, in
mL) and the solution was stirred for 24 h at room
temperature. After solvent evaporation and addition of
diethyl ether (1 mL), a white solid precipitated, corre-
CD OD at room temperature (TMS as internal stan-
3
dard l=0).
2
2
sponding to (+)-5 (62 mg, 88%), mp 209–211°C, [h]
=
4
05
1
4.9. Molecular modelling study
A systematic conformational search of compound (S)-
+
29.9 (c=0.6 MeOH). H NMR (400 MHz, CDCl ,
3
TMS): l =7.98 (s, 1H, aromatic); 7.85 (m, 3H, aro-
H
matic, J=5.7); 7.60 (dd, 1H, aromatic, J=8.5); 7.49 (m,
(
−)-3·L-tartrate was performed using the MMFF94
2
7
3
H, aromatic, J=4.2); 7.40 (t, 1H, aromatic, J=7.1);
force field as implemented in the MOE 2001.01 soft-
.27 (m, 4H, aromatic, J=8.5); 4.4 (s, 2H, -CH -Ph);
2
15
ware. Only those conformations with energies within
.43 (dt, 2H, -CH -N(CH ) , J =12, J =4.9); 2.92
2
3 2
gem
vic
1
0 kcal/mol of the lowest energy structure were consid-
(d, 6H, -N(CH ) , J=9.9); 2.45 (dt, 2H, CH -C-OH,
3 2 2
ered. One of the low-energy conformations obtained is
represented in Fig. 7B.
Jgem=13.4, Jgem=4.8); 1.74 (s, 3H, -CH ). Elemental
3
analysis: C H BrNO requires C, 66.67; H, 6.81; N,
2
3
28
3
.38. Found C, 66.74; H, 6.73; N, 3.34%.
4
4
.10. Pharmacology
4.5. Chiral chromatographic separation of racemic
.10.1. Antinociception activity. Male adult Swiss mice
weighing 30±5 g were used. Antinociception was esti-
compounds 1–3
1
6
mated by means of the hot plate test (HPT). Com-
pounds were dissolved in saline solution and
administered within 1 h of dissolution. The response to
the thermal stimulus was evaluated using a copper plate
heated to 55°C. The time at which the mouse displayed
a nociceptive response, by sitting on its hind legs and
licking, was measured in seconds. Once the basal ani-
mal reaction time was determined, to establish the
dose–response curve, groups of 10 mice were treated
The chromatographic resolution of (RS)-1–3 was
accomplished by semi-preparative high-pressure chro-
matography with a Chiralpak AD column (250×20 mm,
10 mm) at room temperature, with the experimental
conditions reported in Table 2. The enantiomers were
recovered by evaporating the eluate, which was parti-
tioned according to the profile of the chromatogram.
The isolated enantiopure products were characterized
by their enantiomeric excesses (e.e.) and specific rota-
tion data (Tables 1 and 3).
(
via s.c. injection) with increasing doses of the com-
pounds. Control animals received the same volume of
saline solution. The reaction time to the pain stimulus
was measured 20 min following injection. The reaction
time of the control animals (cut off time) was 23±2 s.
4.6. X-Ray crystallographic analysis
Single crystals of (+)-5 were grown by slow crystalliza-
tion from EtOH and used for data collection. A colour-
less needle-shaped crystal, (0.17×0.17×0.43 mm), was
used. The crystal structure was solved by direct meth-
ods using MULTAN 80 and refined isotropically
AD50 values and their 95% confidence intervals were
17
determined using a computer program.
Experimental data are reported in Table 4.
2
13,14
(
except Br atom) on F .
orthorhombic, Mr=414.36, orthorhombic, space group
P2 2 2 , a=10.23 (3), b=35.19 (20), c=6.02 (2) A
Crystal data: C H NOBr,
23 28
,
,
1
1 1
Acknowledgements
3
−3
V=2167.16 A , Z=4, D =1.27 g cm , v=1.908
,
x
−
1
mm , F(000)=864.00, T=293° K, reflections collected
067, unique reflections 2023 (R =0.278), data/
8
This work was supported by Grant from MIUR (Min-
istero dell’Istruzione, dell’Universit a` e della Ricerca,
Italy).
int
2
parameters=2023/113, goodness of fit on F =0.925,
R =0.0809 [(I>2s(I)] and wR =0.1094. The absolute
1
2

O. Azzolina et al. / Tetrahedron: Asymmetry 13 (2002) 1073–1081
1081
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