Favourable involvement of α2A-adrenoreceptor antagonism in the I2-imidazoline binding sites-mediated morphine analgesia enhancement

Article abstract of DOI:10.1016/j.bmc.2012.02.016

Aim of the present study was to obtain novel α₂- adrenoreceptor (α₂-AR) antagonists, possibly endowed with subtype-selectivity. Therefore, inspired by the non subtype-selective α₂-AR antagonist idazoxan, we designed 1,4-di

Full text of DOI:10.1016/j.bmc.2012.02.016

Bioorganic & Medicinal Chemistry 20 (2012) 2259–2265
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Bioorganic & Medicinal Chemistry
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Favourable involvement of a2A-adrenoreceptor antagonism in the
I2-imidazoline binding sites-mediated morphine analgesia enhancement
Valerio Mammoli a, Alessandro Bonifazi a, Fabio Del Bello a, Eleonora Diamanti a, Mario Giannella a,
a
Alan L. Hudson b, Laura Mattioli c, Marina Perfumi c, Alessandro Piergentili a, , Wilma Quaglia ,
⇑
Federica Titomanlio c, Maria Pigini a
a School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, via S. Agostino 1, 62032 Camerino, Italy
b Department of Pharmacology, 9-70 Medical Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
c School of Pharmacy, Pharmacognosy Unit, University of Camerino, via Madonna delle Carceri 9, 62032 Camerino, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Aim of the present study was to obtain novel
with subtype-selectivity. Therefore, inspired by the non subtype-selective
designed 1,4-dioxane derivatives bearing an aromatic area in position 5 or 6 and the imidazoline nucleus
in position 2. Among the novel molecules 1–6, compound 2, with a trans stereochemical relationship
between 5-phenyl and 2-imidazoline groups, was able to antagonize the sole
showed an affinity at I2-imidazoline binding sites (I2-IBS) comparable to that at
2 strongly increased morphine analgesia. This interesting behaviour appeared to be induced by the
a
2-adrenoreceptor (
a
2-AR) antagonists, possibly endowed
2-AR antagonist idazoxan, we
Received 22 December 2011
Revised 3 February 2012
Accepted 4 February 2012
Available online 13 February 2012
a
a
2A-subtype. Moreover, 2
2A-AR. In in vivo studies
a
Keywords:
a2A-Adrenoreceptor antagonists
favourable involvement of
a
2A-AR antagonism in the I2-IBS-mediated morphine analgesia enhancement.
I2-IBS morphine analgesia modulation
Morphine analgesia enhancement
1,4-Dioxane nucleus
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
strategy for the treatment of Alzheimer’s and Parkinson’s diseases.6
Nevertheless, probably due to the inability to discriminate the a2
-
a
2-Adrenoreceptors (
Gi/Go protein coupled receptors, have been classified into
2B-, and
2C-subtypes.1 They are widely distributed throughout
a
2-ARs), belonging to the superfamily of
AR subtypes, the commonly used 2-AR antagonists idazoxan,
phentholamine and yohimbine do not exhibit unequivocal
pharmacological properties. Therefore, the therapeutic potential
a
a2A-,
a
a
the peripheral and central nervous systems (PNS and CNS) and
are considered attractive therapeutic targets for the treatment of
a wide range of diseases.2 Studies on mice lacking or overexpress-
of a2-AR subtype-selective antagonists might not have yet
completely been explored.
The focus of our research over the years has been the develop-
ing
a
2-AR subtypes3 indicated that the
a
a
2A-subtype mediates
2B-subtype mediates
2C-subtype appears to be involved in
ment of ligands targeting
2C-agonists have been discovered.7 In the present study, our
aim was to obtain novel 2-AR antagonists, possibly endowed with
subtype-selectivity. Therefore, the quite planar 1,4-benzodioxane
scaffold of the
2-AR antagonist idazoxan8 was replaced by the less
a2-AR subtypes and interesting a2C- and
hypotension, sedation and analgesia, the
hypertension, while the
many CNS processes.
a2A/a
a
a
The primary use of
a2-AR agonists has long been the treatment
a
of hypertension, but the associated sedative effects have severely
limited their prescription; in contrast, they still represent valuable
tools for the relief of intractable cancer pain.4 Besides being anti-
dotes for reversing effects of agonist overdose, centrally acting
conformationally constrained 1,4-dioxane nucleus substituted in
position 5 or 6 with one phenyl group in both cis or trans stereo-
chemical relationship with the imidazoline ring in position 2
(compounds 1–4) (Chart 1). These modifications allowed us to eval-
uate the influence of different distances between the aromatic and
basic moieties on the biological effects and the stereochemical
requirements of the receptor interaction. Moreover, the effect of
the enlargement of the aromatic area was examined by the
insertion of two phenyl groups in position 5 or 6 (compounds 5
and 6, respectively). This investigation was also prompted by our
recent studies in which the 1,4-dioxane nucleus proved to be a suit-
a
2-AR antagonists have been postulated for the treatment of
depression.5 Moreover,
a2-AR antagonists may represent a rational
Abbreviations: AR, adrenoreceptor; PNS, peripheral nervous system; CNS,
central nervous system; IBS, imidazoline binding sites; CHO, Chinese hamster
ovary; (1S)-(+)-10-CSA, (1S)-(+)-10-camphorsulfonic acid; m-CPBA, m-chloroper-
benzoic acid; MPE, Maximum Possible Effect.
able scaffold for potent muscarinic,9
serotoninergic receptor ligands.10 Finally, the enantiomers of the
a1D-adrenergic and 5-HT1A
⇑
Corresponding author. Tel.: +39 0737402237; fax: +39 0737637345.
E-mail address: alessandro.piergentili@unicam.it (A. Piergentili).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.016

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V. Mammoli et al. / Bioorg. Med. Chem. 20 (2012) 2259–2265
4
O
by flash chromatography. The oxidation of (2S,5R)-(ꢀ)-15 with
O
O
5
6
3
2
KMnO4 to the corresponding acid (2R,5R)-(ꢀ)-16, followed by
esterification with methanol, yielded the methyl-ester (2R,5R)-
(ꢀ)-8, which was transformed into the corresponding imidazoline
(2S,5R)-(ꢀ)-2.
H
H
R
N
N
O
1
N
N
The enantiomer (2S,5R)-(ꢀ)-2, obtained by stereoselective syn-
thesis, showed an enantiomeric purity >98%, determined by 1H
NMR spectroscopy in the presence of the chiral shift reagent (S)-
(+)-2,2,2-trifluoro-1-(9-anthryl)-ethanol. Since the 1H NMR spectra
of this enantiomer and of (ꢀ)-2, obtained by fractional crystalliza-
tion, in the presence of the chiral shift reagent were identical, it
reasonably follows that (ꢀ)-2 has a (2S,5R) configuration.
Idazoxan
1, 5-C6H5 cis
2, 5-C6H5 trans
3, 6-C6H5 cis
4, 6-C6H5 trans
5, 5-(C6H5)2
6, 6-(C6H5)2
Chart 1. Chemical structures of idazoxan and the compounds 1–6.
3. Results and discussion
most interesting compound 2 were prepared and studied. The
affinity values and antagonist potencies of the novel compounds
were evaluated on CHO cells expressing recombinant human
The affinity values (pKi) and antagonist potencies (pKb) of the
novel compounds at
a2-AR subtypes are reported in Table 1 to-
gether with those of idazoxan7 included for useful comparison.
From these data it emerged that a general decrease of affinity
and potency was observed for all the compounds. Nevertheless,
noteworthy is the observation that 2, bearing the phenyl group
in position 5 in trans stereochemical relationship with the imidaz-
a
2-AR subtypes, according to procedures previously reported.7
Moreover, since some
a2-AR ligands are known to be able to
interact also with
a
1-ARs, 5-HT1A receptor8 and I2-imidazoline
binding sites (I2-IBS),11 the affinity values of 2 at these systems
were assessed in agreement with procedures already reported.10,12
Compound 2 was also evaluated in an algesiometric test.
oline ring, was able to antagonize the sole
a2A-subtype (pKi = 6.30;
pKb = 6.40) and was devoid of agonist activity at the other
a
2-AR
subtypes (data not shown). The fact that the corresponding cis dia-
stereomer 1 was endowed with negligible affinity and potency
highlighted the important role played by stereochemistry in the
receptor recognition. This observation was further strengthened
by the study of the biological behaviour of the enantiomers of 2. In-
deed, only the levorotatory form (2S,5R)-(ꢀ)-2 showed the same
2. Chemistry
Compounds 1–6 were prepared by treatment of methyl esters
7–12, obtained by the corresponding acids,10 with ethylenedia-
mine in the presence of Al(CH3)3 (Scheme 1). The two enantiomers
(ꢀ)-2 and (+)-2 were obtained by fractional crystallization of the
a
2A-affinity and antagonist potency of the racemic compound,
whereas the enantiomer (2R,5S)-(+)-2 was inactive up to 10
concentration. In addition, unlike idazoxan,8 2 showed no appre-
ciable affinity at 1A-ARs and 5-HT1A receptor (pKi <5).
The use of 2-AR agonists (i.e. clonidine) as adjuvants with
salts of ( )-2 with hydrogen dibenzoyl-
L- and hydrogen diben-
lM
zoyl-D-tartaric acid, respectively. The enantiomeric purity, deter-
mined by 1H NMR spectroscopy in the presence of the chiral shift
reagent (S)-(+)-2,2,2-trifluoro-1-(9-anthryl)-ethanol, was found to
be >98% (detection limit) for both enantiomers. In fact, the 1H
NMR spectrum of racemic compound ( )-2 showed two double
doublets at d 4.76 for the proton in position 5 of 1,4-dioxane nu-
cleus, whereas only one double doublet was observed for (ꢀ)-2
and (+)-2 at d 4.74 and d 4.78, respectively. The (2S,5R) absolute
configuration of the enantiomer (ꢀ)-2 was determined by
comparing the sign of its optical rotation with that of the (2S,5R)
enantiomer obtained by stereoselective synthesis (Scheme 2).
Compound (R)-(ꢀ)-13, obtained by treatment of methyl (R)-(ꢀ)-
mandelate (commercially available) with allyl bromide in presence
of Ag2O, was reduced with LiAlH4 to afford the intermediate
alcohol (R)-(ꢀ)-14. The epoxidation with m-chloroperbenzoic acid
(m-CPBA) and subsequent cyclization with (1S)-(+)-10-camphor-
sulfonic acid [(1S)-(+)-10-CSA] in CH2Cl2 afforded a cis/trans
diastereomeric mixture, from which (2S,5R)-(ꢀ)-15 was obtained
a
a
opioids continues to be the focus of clinical investigations. Never-
theless, Scheinin and co-workers reported that antinociceptive re-
sponses to partial opioid agonists were very markedly accentuated
by targeted inactivation of the
mechanisms included reversal of
tion of some components of the common intracellular signalling
pathways of 2A-ARs and -opioid receptors, and removal of con-
formational constraints imposed by hetero-oligomeric 2A-ARs to
-opioid receptor activation by partial agonists. Therefore, these
a
2A-AR gene in mice. The possible
a
2A-AR-dependent desensitiza-
a
l
a
l
authors hypothesized that selective a2A-AR antagonists might en-
hance the antinociceptive effects of partial opioid agonists.13 On
the basis of this hypothesis and with the hope of improving the
knowledge of the therapeutic potential of
a2A-AR antagonists, 2
has been evaluated in in vivo studies. Our aim was also encouraged
by some recent observations indicating that
a2-AR antagonists,
O
O
O
O
O
a)
b)
H
R
R
R
OH
O
N
CH3
O
O
O
N
5-C6H5 cis
5-C6H5 trans
6-C6H5 cis
6-C6H5 trans
5-(C6H5)2
7, 5-C6H5 cis
1, 5-C6H5 cis
2, 5-C6H5 trans
3, 6-C6H5 cis
4, 6-C6H5 trans
5, 5-(C6H5)2
6, 6-(C6H5)2
8, 5-C6H5 trans
9, 6-C6H5 cis
10, 6-C6H5 trans
11, 5-(C6H5)2
12, 6-(C6H5)2
6-(C6H5)2
Scheme 1. Reagents: (a) CH3OH/H2SO4; (b) NH2CH2CH2NH2, Al(CH3)3, toluene.

V. Mammoli et al. / Bioorg. Med. Chem. 20 (2012) 2259–2265
2261
OH
O
O
c)
d)
e)
O
O
O
O
OH
CH3
CH3
OH
O
O
R-(-)-13
R-(-)-14
(2S,5R)-(-)-15
f)
b)
a)
O
O
O
O
O
H
O
O
OH
N
CH3
O
N
O
(2S,5R)-(-)-2
(2R,5R)-(-)-8
(2R,5R)-(-)-16
Scheme 2. Reagents: for (a) and (b) see Scheme 1; (c) allyl bromide, Ag2O, Et2O; (d) LiAlH4, THF; (e) m-CPBA, CH2Cl2; (1S)-(+)-10-CSA, CH2Cl2; flash chromatography; (f)
KMnO4, KOH, H2O.
Table 1
a
b
Affinity Constants (pKi) and Antagonist Potency (pKb) on Human
a2-AR Subtypes.
Compd
pKi
pKb
a2A
a2B
a2C
a2A
a2B
a2C
Idazoxan
1
2
8.15 0.11
<5
7.64 0.09
<5
<5
<5
<5
<5
<5
7.75 0.08
<5
<5
<5
<5
<5
<5
7.73 0.09
<5
6.40 0.11
<5
6.40 0.16
<5
<5
<5
7.16 0.10
5.50 0.16
<5
<5
7.92 0.08
<5
<5
<5
<5
<5
<5
<5
<5
6.30 0.14
5.10 0.09
6.40 0.12
5.27 0.15
5.20 0.09
<5
(2R,5S)-(+)-2
(2S,5R)-(ꢀ)-2
<5
3
4
5
6
5.18 0.13
<5
<5
<5
5.55 0.09
5.35 0.13
5.13 0.11
5.21 0.14
5.42 0.10
5.37 0.10
a
pKi values were calculated from [3H]RX 821002 on membrane preparations from CHO cells expressing individually each human
pKb values were determined by applying the cytosensor microphysiometry system to the same cell models and they express the ability of the antagonist to shift the
a
2-AR subtype (a2A
, a2B, a2C).
b
agonist (clonidine) concentration-effect curve. The data are expressed as means SEM of three to six separate experiments.
though endowed with moderate affinity (pKi = 6.58) and potency
Morphine
2 (1 mg/kg) + Morph
2 (2 mg/kg) + Morph
(pKb = 6.25), were anyhow able to evoke significant in vivo re-
sponses.14 Therefore, 2 was administered to mice 15 min before
morphine and was tested in the algesiometric paradigm mouse tail
flick test. From in vivo dose–response studies (Fig. 1) it emerged
that 2, lacking antinociceptive properties alone, at low doses
(1 or 2 mg/kg) caused a significant increase of morphine analgesia.
The increased tail-flick latency was still observed 120 min after the
administration. On the contrary, confirming previous data,15 the
2 (1 mg/kg)
2 (2 mg/kg)
Yohimbine + Morph
Idazoxan + Morph
**
*
100
80
60
40
20
0
known
a2-AR non subtype selective antagonists idazoxan or
**
*
**
yohimbine,7,16 used in this test as reference compounds, were de-
void of this effect. These results, supporting Scheinin and co-work-
er’s hypothesis,13 suggested the favourable role played by the
**
*
inhibition of the sole
a2A-AR subtype in morphine analgesia
enhancement induced by 2.
Also IBS, classified into I1- and I2-IBS, represent therapeutically
interesting targets. While I1-IBS participate in the regulation of car-
diovascular function, I2-IBS appear to be involved in Parkinson’s
disease, depression and modulation of morphine analgesia as well
as tolerance and addiction to opioids.17 We have previously dem-
onstrated that ligands bearing a wholly carbon bridge between
phenyl and imidazoline moieties induced selective I2-IBS recogni-
tion. Indeed, phenyzoline, bearing an ethylene bridge, showed a
0
30
60
90
120
240
pKi value of 8.60 at I2-IBS and an I2-IBS/a2-ARs selectivity ratio of
794.18 Moreover, phenyzoline significantly enhanced morphine
analgesia, while its ortho-phenyl derivative diphenyzoline, another
selective I2-IBS ligand, decreased morphine antinociceptive effect.
Both the positive or negative morphine analgesia modulations
Figure 1. Effect of 2 (1 and 2 mg/kg, ip) pre-treatment on morphine (5 mg/kg, sc)
analgesia in the tail-flick test. The reaction latencies were expressed as a percent of
the Maximum Possible Effect (%MPE). Values are means SEM of 8–10 mice.
⁄p<0.05, ⁄⁄p <0.01 compared to morphine group; where not indicated, the difference
was not statistically significant.

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V. Mammoli et al. / Bioorg. Med. Chem. 20 (2012) 2259–2265
4. Experimental section
Idz + 2 + Morph
Idz + Morph
Morphine
2
2 + Morph
4.1. Chemistry
Melting points were taken in glass capillary tubes on a Büchi
SMP-20 apparatus and are uncorrected. IR and 1H NMR spectra
were recorded on Perkin-Elmer 297 and Varian EM-390 instru-
ments, respectively. Chemical shifts are reported in parts per
million (ppm) relative to tetramethylsilane (TMS), and spin multi-
plicities are given as s (singlet), br s (broad singlet), d (doublet), t
(triplet), q (quartet), or m (multiplet). The elemental compositions
of the compounds were performed by the Microanalytical Labora-
tory of our department and agreed to within 0.4% of the calcu-
lated value. When the elemental analysis was not included, crude
compounds were used in the next step without further purifica-
tion. Optical activity was measured at 20 °C with a Perkin–Elmer
241 polarimeter. Chromatographic separations were performed
on silica gel columns (Kieselgel 40, 0.040–0.063 mm, Merck) by
flash chromatography. Chemical names were generated using
ChemDraw Ultra (CambridgeSoft, version 9.0). The purity of the
novel tested compounds was determined by combustion analysis
and was P95%.
**
100
80
60
40
20
0
°
-20
Figure 2. Effect of idazoxan (2.0 mg/kg, ip) on enhancement of morphine (5 mg/kg,
sc) analgesia induced by in the tail-flick test. The reaction latencies were
expressed as
2
4.1.1. cis-Methyl 5-Phenyl-1,4-dioxane-2-carboxylate (7)
A solution of cis-5-phenyl-1,4-dioxane-2-carboxylic acid (1.1 g,
5.23 mmol)10 in MeOH (20 mL) and H2SO4 (1.5 mL) was heated to
reflux for 5 h. Removal of the solvent under reduced pressure gave
a residue, which was dissolved in water. The aqueous solution was
basified with 2 N NaOH and extracted with Et2O. Removal of dried
solvent gave a residue which was purified by column chromatogra-
phy, eluting with cyclohexane/EtOAc (95:5) to afford an oil: 89%
yield. 1H NMR (CDCl3): d 3.82 (dd, 1, cycle), 3.91 (s, 3, CH3), 4.08
(dd, 2, cycle), 4.31 (d, 1, cycle), 4.42 (d, 1, cycle), 4.62 (dd, 1, cycle),
7.23–7.39 (m, 5, ArH). Anal. Calcd for C12H14O4: C, 64.85; H, 6.35.
Found: C, 64.74; H, 6.60.
a
percent of the Maximum Possible Effect (%MPE). Values are
means SEM of 8–10 mice. ⁄⁄p <0.01 compared to morphine group; °p <0.05
compared to 2+morphine group.
proved to be mediated by I2-IBS, because they were completely re-
versed by the mixed
speculated that phenyzoline and diphenyzoline, interacting with
I2-IBS, might induce a perturbation of -opioid receptors to higher
a2-AR/I2-IBS antagonist idazoxan. We also
l
or lower, respectively, affinity state conformation for opioid
ligands.15
From the aforementioned study18 it also emerged that, instead,
the presence of the oxygen atom in the bridge allowed ligand to
interact with both
a2-ARs and I2-IBS. Therefore, on the basis of
4.1.2. trans-Methyl 5-Phenyl-1,4-dioxane-2-carboxylate (8)
This was obtained following the procedure described for 7 start-
ing from trans-5-phenyl-1,4-dioxane-2-carboxylic acid10 to afford
an oil: 87% yield. 1H NMR (CDCl3): d 3.58 (dd, 1, cycle), 3.77 (dd,
1, cycle), 3.80 (s, 3, CH3), 4.03 (dd, 1, cycle), 4.26 (dd, 1, cycle),
4.37 (dd, 1, cycle), 4.61 (dd, 1, cycle), 7.31–7.37 (m, 5, ArH). Anal.
Calcd for C12H14O4: C, 64.85; H, 6.35. Found: C, 64.77; H, 6.22.
these observations, the peculiar nature of 1,4-dioxane ring and
its presence as benzocondensed fragment in idazoxan, compatible
with the high I2-IBS affinity of this ligand (pKi = 8.37),19 prompted
us to assess the affinity values of 2 and its enantiomers at I2-IBS.
From the obtained data the racemic compound 2 showed an affin-
ity value (pKi = 6.35) comparable to that found for a2A-AR. Also at
this system (2S,5R)-(ꢀ)-2 was the eutomer, its affinity value
(pKi = 6.52) being significantly higher than that of (2R,5S)-(+)-2
(pKi = 5.48). These results did not allow us to exclude the I2-IBS
contribute on morphine analgesia enhancement induced by 2.
Therefore, to verify the real I2-IBS involvement, analogously to
what made for phenyzoline,15 we assessed the tail-flick latencies
after pre-treatment of the mice with idazoxan. The observation
that idazoxan partially contrasted morphine analgesia enhance-
ment (Fig. 2) supported the involvement of I2-IBS in the positive ef-
fect induced by 2. Therefore, the biological profile of 2 was
4.1.3. cis-Methyl 6-Phenyl-1,4-dioxane-2-carboxylate (9)
This was obtained following the procedure described for 7 start-
ing from cis-6-phenyl-1,4-dioxane-2-carboxylic acid10 to afford an
oil: 87% yield. 1H NMR (CDCl3): d 3.42 (dd, 1, cycle), 3.61 (dd, 1, cy-
cle), 3.79 (s, 3, CH3), 3.98 (m, 1, cycle), 4.18 (dd, 1, cycle), 4.51 (dd,
1, cycle), 4.75 (dd, 1, cycle), 7.24–7.42 (m, 5, ArH). Anal. Calcd for
C12H14O4: C, 64.85; H, 6.35. Found: C, 64.99; H, 6.58.
4.1.4. trans-Methyl 6-Phenyl-1,4-dioxane-2-carboxylate (10)
This was obtained following the procedure described for 7 start-
ing from trans-6-phenyl-1,4-dioxane-2-carboxylic acid10 to afford
an oil: 89% yield. 1H NMR (CDCl3): d 3.51 (dd, 1, cycle), 3.82 (d, 1,
cycle), 3.84 (s, 3, CH3), 3.95 (dd, 1, cycle), 4.33 (d, 1, cycle), 4.42
(d, 1, cycle), 5.16 (dd, 1, cycle), 7.31–7.37 (m, 5, ArH). Anal. Calcd
for C12H14O4: C, 64.85; H, 6.35. Found: C, 64.59; H, 6.10.
characterized by its ability both to selectively antagonize
a2A-AR
subtype and to positively modulate I2-IBS-mediated morphine
analgesia.
In conclusion, considering that (i) yohimbine and idazoxan, non
subtype selective
a2-AR antagonists, did not enhance morphine
analgesia; (ii) phenyzoline, a selective I2-IBS ligand positively mod-
ulating morphine analgesia, required significantly higher dose
(10 mg/kg)15 for producing an effect comparable to that of 2, we
4.1.5. Methyl 5,5-diphenyl-1,4-dioxane-2-carboxylate (11)
This was obtained following the procedure described for 7 start-
ing from 5,5-diphenyl-1,4-dioxane-2-carboxylic acid10 to afford an
oil: 86% yield. 1H NMR (CDCl3): d 3.75 (d, 1, cycle), 3.78 (s, 3, CH3),
3.96 (d, 1, cycle), 4.00 (d, 1, cycle), 4.40 (dd, 1, cycle), 4.62 (d, 1,
think that
I2-IBS-mediated morphine analgesia enhancement. Anyway, 2,
lacking sedative and hypotensive side effects due to its 2A-AR
antagonism, in combination with opioids, might be a novel prom-
ising tool therapeutically useful in pain management.
a2A-AR antagonism might favourably contribute to
a

V. Mammoli et al. / Bioorg. Med. Chem. 20 (2012) 2259–2265
2263
cycle), 7.19–7.52 (m, 10, ArH). Anal. Calcd for C18H18O4: C, 72.47; H,
6.08. Found: C, 72.70; H, 6.17.
3.90 (dd, 1, cycle), 4.39 (dd, 1, cycle), 4.48 (m, 1, cycle), 4.81 (dd,
1, cycle), 5.54 (br, 1, NH, exchangeable with D2O), 7.24–7.41 (m,
5, ArH). The free base was transformed into the oxalate salt, which
was crystallized from EtOH: mp 171–172 °C. Anal. Calcd for
4.1.6. Methyl 6,6-diphenyl-1,4-dioxane-2-carboxylate (12)
This was obtained following the procedure described for 7 start-
ing from 6,6-diphenyl-1,4-dioxane-2-carboxylic acid10 to afford an
oil: 90% yield. 1H NMR (CDCl3): d 3.61 (m, 2, cycle), 3.79 (s, 3, CH3),
4.08 (dd, 1, cycle), 4.37 (dd, 1, cycle), 4.62 (d, 1, cycle), 7.21–7.59
(m, 10, ArH). Anal. Calcd for C18H18O4: C, 72.47; H, 6.08. Found:
C, 72.19; H, 6.31.
C
13H16N2O2ꢁH2C2O4: C, 55.90; H, 5.63; N, 8.69. Found: C, 55.74;
H, 5.42; N, 8.47.
4.1.11. 2-(5,5-Diphenyl-1,4-dioxan-2-yl)-4,5-dihydro-1H-
imidazole (5)
This was obtained following the procedure described for 1 start-
ing from 11 to afford a white solid: 54% yield; mp 208–210 °C. 1H
NMR (CDCl3): d 3.58 (s, 4, NCH2CH2N), 3.60–3.80 (m, 2, cycle), 4.00
(dd, 1, cycle), 4.50 (dd, 1, cycle), 4.63 (d, 1, cycle), 5.98 (br, 1, NH,
exchangeable with D2O), 7.18–7.56 (m, 10, ArH). The free base
was transformed into the oxalate salt, which was crystallized from
EtOH: mp 219–220 °C. Anal. Calcd for C19H20N2O2ꢁH2C2O4: C,
63.31; H, 5.57; N, 7.03. Found: C, 63.12; H, 5.79; N, 7.25.
4.1.7. cis-2-(5-Phenyl-1,4-dioxan-2-yl)-4,5-dihydro-1H-
imidazole (1)
A solution of ethylenediamine (0.42 mL, 10.91 mmol) in dry tol-
uene (10 mL) was added dropwise to a mechanically stirred solu-
tion of 2 M trimethylaluminum (5.4 mL, 10.91 mmol) in dry
toluene (6 mL) at 0 °C in nitrogen atmosphere. The mixture was
stirred at room temperature for 1 h then, after cooling to 0 °C, a
solution of 7 (1.2 g, 5.4 mmol) in dry toluene (10 mL) was added
dropwise. The reaction mixture was heated to 70 °C for 3 h, cooled
to 0 °C, and quenched cautiously with MeOH (10 mL) followed by
H2O (0.3 mL). After addition of CHCl3 (10 mL), the mixture was left
at room temperature for 30 min to ensure the precipitation of the
aluminum salts. The mixture was filtered and the organic layer was
extracted with 2 N HCl. The aqueous layer was made basic with
10% NaOH and extracted with CHCl3. The organic layer was dried
over Na2SO4, filtered and evaporated to give an oil, which was puri-
fied by flash chromatography eluting with ciclohexane/EtOAc/
MeOH/33% NH4OH (8:2:1:0.1) to afford a solid: 57% yield; mp
175–177 °C. 1H NMR (CDCl3): d 3.65 (s, 4, NCH2CH2N), 3.89 (m, 3,
cycle), 4.00 (dd, 1, cycle), 4.38 (dd, 1, cycle), 4.69 (dd, 1, cycle),
5.78 (br, 1, NH, exchangeable with D2O), 7.22–7.40 (m, 5, ArH).
The free base was transformed into the oxalate salt, which was
crystallized from EtOH: mp 199–200 °C. Anal. Calcd for
4.1.12. 2-(6,6-Diphenyl-1,4-dioxan-2-yl)-4,5-dihydro-1H-
imidazole (6)
This was obtained following the procedure described for 1 start-
ing from 12 to afford an oil. 1H NMR (CDCl3): d 3.60–3.76 (m, 6,
NCH2CH2N and cycle), 4.12 (dd, 1, cycle), 4.40 (dd, 1, cycle), 4.61
(d, 1, cycle), 5.18 (br, 1, NH, exchangeable with D2O), 7.20–7.58
(m, 10, ArH). The free base was transformed into the oxalate salt
and crystallized from EtOH: mp 207–208 °C. Anal. Calcd for
C
19H20N2O2ꢁH2C2O4: C, 63.31; H, 5.57; N, 7.03. Found: C, 63.60;
H, 5.44; N, 6.97.
4.1.13. Resolution of trans-2-(5-phenyl-1,4-dioxan-2-yl)-4,5-
dihydro-1H-imidazole ( )-2
Racemic 2 (1 g, 4.3 mmol) in 95% EtOH (30 mL) was treated
with
a
solution of (ꢀ)-O,O0-dibenzoyl-
L-tartaric acid (1.54 g
4.3 mmol) in 95% EtOH (35 mL) and left at room temperature for
30 h. The white crystals were crystallized twice from 95% EtOH:
1.2 g yield. The salt was dissolved in water (50 mL) and the ice-
cooled solution was made basic with 2 N NaOH and extracted with
ether (3 ꢂ 30 mL). Removal of dried solvent gave (2S,5R)-(ꢀ)-2:
C
13H16N2O2ꢁH2C2O4: C, 55.90; H, 5.63; N, 8.69. Found: C, 55.76;
H, 5.88; N, 8.78.
4.1.8. trans-2-(5-Phenyl-1,4-dioxan-2-yl)-4,5-dihydro-1H-
imidazole (2)
0.4 g; ½a 2D0
ꢃ
ꢀ11.09 (c 1, CHCl3). The 1H NMR spectrum was identical
This was obtained following the procedure described for 1 start-
ing from 8 to afford a white solid: 63% yield; mp 178–180 °C. 1H
NMR (CDCl3): d 3.55 (dd, 1, cycle), 3.61 (s, 4, NCH2CH2N), 3.80
(dd, 1, cycle), 3.95 (dd, 1, cycle), 4.29 (dd, 1, cycle), 4.43 (dd, 1, cy-
cle), 4.59 (dd, 1, cycle), 6.47 (br, 1, NH, exchangeable with D2O),
7.25–7.38 (m, 5, ArH). The free base was transformed into the oxa-
late salt, which was crystallized from EtOH: mp 202–204 °C. Anal.
Calcd for C13H16N2O2ꢁH2C2O4: C, 55.90; H, 5.63; N, 8.69. Found: C,
55.69; H, 5.49; N, 8.55.
to that of the racemic compound 2. A similar treatment of 2 with
(+)-O,O0-dibenzoyl-
D
-tartaric acid gave the other enantiomer
(2R,5S)-(+)-2: ½a D20
ꢃ
+10.5 (c 1, CHCl3). The 1H NMR spectrum was
identical to that of the racemic compound 2.
4.1.14. (R)-(ꢀ)-Methyl 2-(Allyloxy)-2-phenylacetate [R-(ꢀ)-13]
Ag2O (1.28 g, 5.58 mmol) was added portionwise, under vigor-
ous stirring, to a solution of methyl (R)-(ꢀ)-mandelate (Aldrich)
(1 g, 6.02 mmol) and allyl bromide (1 g, 8.32 mmol) in anhydrous
diethyl ether (11.7 mL) at room temperature. The mixture was re-
fluxed for 2 h, then it was cooled and filtered over Celite. The evap-
oration of dried (Na2SO4) organic layer gave a residue that was
purified by flash chromatography eluting with cycloexane/EtOAc
4.1.9. cis-2-(6-Phenyl-1,4-dioxan-2-yl)-4,5-dihydro-1H-
imidazole (3)
This was obtained following the procedure described for 1 start-
ing from 9 to afford a white solid: 58% yield; mp 117–118 °C. 1H
NMR (CDCl3): d 3.48 (dd, 1, cycle), 3.62 (s, 4, NCH2CH2N), 3.63
(dd, 1, cycle), 3.85 (dd, 1, cycle), 4.18 (dd, 1, cycle), 4.61 (dd, 1, cy-
cle), 4.76 (dd, 1, cycle), 5.66 (br, 1, NH, exchangeable with D2O),
7.28–7.42 (m, 5, ArH). The free base was transformed into the oxa-
late salt, which was crystallized from EtOH: mp 177–179 °C. Anal.
Calcd for C13H16N2O2ꢁH2C2O4: C, 55.90; H, 5.63; N, 8.69. Found: C,
60.08; H, 5.82; N, 8.59.
(95:5) to give an oil: 48% yield; ½a D20
ꢃ
ꢀ112.41 (c 1, CHCl3); 1H
NMR (CDCl3): d 3.72 (s, 3, COOCH3), 4.08 (dd, 2, OCH2), 4.98 (s, 1,
CHO), 5.20–5.30 (m, 2, CH2@CH), 5.90–6.00 (m, 1, CH@CH2),
7.35–7.51 (m, 5, ArH). Anal. Calcd for C12H14O3: C, 69.88; H, 6.84.
Found: C, 69.69; H, 6.69.
4.1.15. (R)-(ꢀ)-2-(Allyloxy)-2-phenylethanol [R-(ꢀ)-14]
A solution of R-(ꢀ)-13 (0.4 g, 1.94 mmol) in anhydrous diethyl
ether (2.43 mL) was added dropwise to a suspension of LiAlH4
(0.1 g, 2.63 mmol) in anhydrous diethyl ether (3.04 mL) at 0 °C un-
der a nitrogen atmosphere and vigorous stirring. The mixture was
allowed to warm to room temperature; after 2 h it was poured
onto crushed ice and 2.5 M sodium hydroxide (5.83 mL) was
added. The precipitate was filtered off over Celite and washed with
4.1.10. trans-2-(6-Phenyl-1,4-dioxan-2-yl)-4,5-dihydro-1H-
imidazole (4)
This was obtained following the procedure described for 1 start-
ing from 10 to afford a white solid: 57% yield; mp 98–100 °C. 1H
NMR (CDCl3): d 3.60–3.68 (m, 2, cycle), 3.72 (s, 4, NCH2CH2N),

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V. Mammoli et al. / Bioorg. Med. Chem. 20 (2012) 2259–2265
diethyl ether (10 mL). The organic layer was dried over Na2SO4 and
evaporated. The residue was purified by flash chromatography
eluting with cycloexane/EtOAc (9:1) to give an oil: 65% yield;
a 12:12 h light/dark cycle (lights on at 9:00 a.m.), a temperature
of 20–22 °C and a humidity of 45–55%. They were offered free
access to tap water and food pellets (4RF18, Mucedola, Settimo
Milanese, Italy). Ethical guidelines for the investigation of experi-
mental pain in conscious animals were followed, and procedures
were carried out according to EEC ethical regulations for animal re-
search (EEC Council 86/609; D. Lgs. 27/01/1992, No. 116).
½
a 2D0
ꢃ
ꢀ92.91 (c 1, CHCl3); 1H NMR (CDCl3) d 2.40 (br s, 1, OH,
exchangeable with D2O), 3.70 (d, 2, CH2OH), 4.02 (dd, 2, OCH2),
4.42 (dd, 1, CHO), 5.18 (m, 2, CH2@CH), 5.84–6.01 (m, 1, CH@CH2),
7.20–7.42 (m, 5, ArH). Anal. Calcd for C11H14O2: C, 74.13; H, 7.92.
Found: C, 74.39; H, 7.67.
4.2.1.2. Nociceptive test.
Nociception was evaluated by the
4.1.16. (2S,5R)-(ꢀ)-(5-Phenyl-1,4-dioxan-2-yl)methanol
[(2S,5R)-(ꢀ)-15]
radiant heat tail-flick test: briefly, it consists of the irradiation
of the lower third of the tail with an I. R. source (Ugo Basile,
Comerio, Italy). The basal pre-drug latency, ranged between 2–
3 s, was calculated as the mean of two trials performed at
30 min interval. Then mice received 2 (1 and 2 mg/kg, ip) or re-
lated vehicle (distilled water), 15 min before morphine (5.0 mg/
kg, s.c.) administration or saline. Yohimbine (1.250 mg/kg, ip)
and idazoxan (2.0 mg/kg, ip) were used as reference compounds.
The antinociceptive activity was evaluated 30, 60, 90, 120 and
240 min after morphine injection. A cut-off latency of 12 s was
established to minimize tissue damage. As shown in Figure 1,
2 had no analgesic effect when given alone at all doses tested,
but significantly increased the analgesic response of morphine
at all times, as evidenced by increase reaction latencies in the
m-Chloroperbenzoic acid (50%) (1.16 g, 3.36 mmol) was added
to a solution of R-(ꢀ)-14 (0.3 g, 1.68 mmol) in CH2Cl2 (12 mL). After
20 h at room temperature under stirring the reaction mixture was
washed with 10% Na2SO3, 5% Na2CO3 and H2O. Removal of dried
solvents afforded a mixture of the two diastereomers as an oil. A
solution of this mixture (2.94 g, 15.14 mmol) and (1S)-(+)-10-cam-
phorsulfonic acid (0.33 g, 14.21 mmol) in CH2Cl2 (13.4 mL) was re-
fluxed for 8 h. The reaction mixture was then washed with NaHCO3
saturated solution and dried over Na2SO4. Removal of the solvent
afforded a residue, which was purified by column chromatography
gradient eluent, eluting with cyclohexane/EtOAc (8:2 to 6:4). The
trans diastereomer (2S,5R)-(ꢀ)-15 eluted first as a solid: 34% yield;
mp 79–81 °C; ½a 2D0
ꢃ
ꢀ64.8 (c 1, CHCl3); 1H NMR (CDCl3): d 1.93 (t, 1,
tail-flick test expressed as % MPE. The analgesic effect of
OH, exchangeable with D2O), 3.52–4.04 (m, 7, OCH2 and cycle),
4.58 (dd, 1, cycle), 7.28–7.40 (m, 5, ArH). Anal. Calcd for
morphine (5 mg/kg, s.c.) was observed after 30 min, and then it
decreased progressively. Pretreatment with 2 (1 and 2 mg/kg)
significantly increased the antinociception produced by mor-
phine until 90 and 120 min respectively. The ANOVA confirmed
a statistically significant treatment effect [F(6,54) = 17.928; p
<0.001]. Both yohimbine and idazoxan did not affect morphine
analgesia over the entire time course.
In order to evaluate the involvement of I2-IBS on morphine
analgesia, animals were pre-treated ip with idazoxan at the dose
of 2.0 mg/kg, 15 min before 2 treatment. Figure 2 shows the effect
of idazoxan on enhancement of morphine analgesia induced by 2.
Pretreatment with idazoxan slightly reduced the effect of 2 on
morphine analgesia (ꢀ24%) [F(4,36) = 38.574; p <0.001].
C11H14O3: C, 68.02; H, 7.27. Found: C, 67.84; H, 7.07.
4.1.17. (2S,5R)-(ꢀ)-5-Phenyl-1,4-dioxane-2-carboxylic acid
[(2R,5R)-(ꢀ)-16]
A solution of KMnO4 (3.25 g, 20.6 mmol) in H2O (15 mL) was
added dropwise to a stirred mixture of (2S,5R)-(ꢀ)-15 (2.16 g,
11.1 mmol) in 1 N KOH (15 mL) such that the temperature was
maintained below 10 °C. After 18 h at room temperature the mix-
ture was filtered over Celite, MeOH was added and the solvent was
concentrated under vacuum. The resulting aqueous solution was
acidified with 6 N H2SO4 and extracted with CHCl3. After evapora-
tion of the dried solvent, the residue was crystallized from EtOAc/
petroleum ether: 40% yield; mp 165–167 °C; ½a D20
ꢃ
ꢀ42.09 (c 1,
4.2.1.3. Statistical analysis.
Antinociceptive effect was ex-
CHCl3); 1H NMR (CDCl3): d 3.62 (dd, 1, cycle), 3.81 (dd, 1, cycle),
4.07 (dd, 1, cycle), 4.35 (dd, 1, cycle), 4.41 (dd, 1, cycle), 4.62 (dd,
1, cycle), 5.91 (br s, 1, COOH, exchangeable with D2O), 7.31–7.41
(m, 5, ArH). Calcd for C11H12O4: C, 63.45; H, 5.81. Found: C,
63.34; H, 5.94.
pressed as a percent of the Maximum Possible Effect (MPE) accord-
ing to the following formula: %MPE = (measured latencyꢀbasal
latency)/(cut-off timeꢀbasal latency) ꢂ 100%. Data are reported
as means SEM. Statistical evaluation of data was carried out by
analysis of variance (ANOVA) and sequential differences among
means according to Student–Newman–Keuls test. Statistical signif-
icance was set at p <0.05.
4.1.18. (2S,5R)-(ꢀ)-Methyl 5-phenyl-1,4-dioxane-2-carboxylate
[(2R,5R)-(ꢀ)-8]
This was obtained following the procedure described for 7 start-
Acknowledgments
ing from (2R,5R)-(ꢀ)-16 to afford an oil: 80% yield; ½a D20
ꢀ76.26 (c
ꢃ
1, CHCl3). The 1H NMR spectrum was identical to that of the race-
mic compound 8. Anal. Calcd for C12H14O4: C, 64.85; H, 6.35.
Found: C, 64.90; H, 6.23.
This work was supported by Grants from the MIUR (Rome),
the University of Camerino, the Monte dei Paschi di Siena
Foundation, and National Sciences and Engineering Council of
Canada (NSERC).
4.1.19. (2S,5R)-(ꢀ)-2-(5-Phenyl-1,4-dioxan-2-yl)-4,5-dihydro-
1H-imidazole [(2S,5R)-(ꢀ)-2]
References and notes
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ing from (2R,5R)-(ꢀ)-8 to afford a white solid: 57% yield. ½a D20
ꢃ
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