New isoxazole derivatives designed as nicotinic acetylcholine receptor ligand candidates

Article abstract of DOI:10.1016/S0223-5234(01)01327-7

In this work we report the synthesis and evaluation of the analgesic properties of new isosteric heterocyclic derivatives, presenting the isoxazole nucleus, designed as nicotinic acetylcholine receptor ligand candidates, analogues to alkaloid epibatidine. Compound 2-(3-methyl-5-isoxazolyl)pyridine (3) presented the best analgesic profile of this series in hot plate test, which was partially prevented by pretreatment with nicotinic receptor antagonist mecamylamine.

Full text of DOI:10.1016/S0223-5234(01)01327-7

Eur. J. Med. Chem. 37 (2002) 163–170
Laboratory note
New isoxazole derivatives designed as nicotinic acetylcholine
receptor ligand candidates
Nilda M. Silva a,b, Jorge L.M Tributino a, Ana L.P. Miranda a, Eliezer J. Barreiro a,b,
Carlos A.M. Fraga a,b,
*
a Laborato´rio de A6aliac¸a˜o e S´ıntese de Substaˆncias Bioati6as (LASSBio, http://www.farmacia.ufrj.br/lassbio), Faculdade de Farma´cia,
Uni6ersidade Federal do Rio de Janeiro, PO Box 68006, 21944-970 Rio de Janeiro, RJ, Brazil
b Instituto de Qu´ımica, Uni6ersidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
Received 6 July 2001; received in revised form 15 November 2001; accepted 15 November 2001
Abstract
In this work we report the synthesis and evaluation of the analgesic properties of new isosteric heterocyclic derivatives,
presenting the isoxazole nucleus, designed as nicotinic acetylcholine receptor ligand candidates, analogues to alkaloid epibatidine.
Compound 2-(3-methyl-5-isoxazolyl)pyridine (3) presented the best analgesic profile of this series in hot plate test, which was
´
partially prevented by pretreatment with nicotinic receptor antagonist mecamylamine. © 2002 Published by Editions scientifiques
et me´dicales Elsevier SAS.
Keywords: Isoxazole derivatives; Nicotinic receptor ligand; Analgesic activity; Molecular hybridization; Epibatidine
glionar and sympatic post-ganglionar synapses as well
as displaying several functions in central nervous sys-
tem (CNS), e.g. in the cognitive processes [1].
The receptors modulated by acetylcholine stimulation
comprehended two principal types, i.e. muscarinic
(mAchR) and nicotinic (nAchR), defined by action of
the specific alkaloid ligands muscarine and nicotine
(Fig. 1) [2].
1. Introduction
Acetylcholine is an important neurotransmitter
molecule, which is associated to parasympatic pre-gan-
The current interest in the medicinal chemistry as-
pects of nAchR ligands is due, in part, to perceived
beneficial effects of nicotine in CNS disorders, such as
Alzheimer disease [3,4], Parkinson disease [5,6] or pain
reflexes [7,8]. Indeed, recently ABT-418 [9,10] (1) and
epibatidine [11,12] (2) were described as nicotinic recep-
tor agonists which presented potent analgesic profile.
In the scope of a research programme aiming at the
synthesis of new nicotinic receptor ligands candidates,
we described in this paper the preparation and evalua-
tion of the analgesic profile of two series of substituted
isoxazole derivatives 3a–g and 4a–c (Fig. 1) [13]. The
compound 3a was planned by molecular hybridization
Fig. 1. Design concept of new isoxazole derivatives 3–4.
* Correspondence and reprints.
E-mail address: cmfraga@pharma.ufrj.br (C.A.M. Fraga).
´
0223-5234/02/$ - see front matter © 2002 Published by Editions scientifiques et me´dicales Elsevier SAS.
PII: S0223-5234(01)01327-7

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between ABT-418 (1) and epibatidine (2), by associa-
tion of the pharmacophoric 3-methylisoxazolyl sub-unit
(a) from 1 with 2-chloropyridinyl framework (b) of 2
(Fig. 1).
Compounds 3b–e were structurally designed in order
to investigate the eventual contribution of the isosteric
substitution [14] of the pharmacophoric nitrogen-con-
taining six member ring (b), e.g. 2-, 3-, 4-pyridine and
pyridazine nucleus in the analgesic activity (Fig. 1).
Additionally, we promoted the isosteric replacement of
the aromatic nitrogen of this sub-unit (b) by carba-
halogenated framework as in the compounds 3f and 3g
(Fig. 1).
Fig. 2. General method for synthesis of isoxazole derivatives 3–4
from carboxylic esters.
In addition, we decided to investigate the contribu-
tion of the distance between both heteroaromatic rings,
by introduction of unsaturated C2 spacer unit in the
derivatives of the (E)-vinylogous series 4, in the antici-
pated analgesic profile (Fig. 1).
The fit of the new isoxazole derivative 3a in the
nicotinic acetylcholine pseudo-receptor model develo-
ped in our laboratory [15] indicated the presence of all
minimal structural requirements envisioned to a nico-
tinic receptor ligand [16].
2. Chemistry
The new substituted isoxazole derivatives 3a–g and
4a–c were synthesised exploring Beam’s method [17],
involving the condensation of 1,4-dilithium salts of
acetone oxime with the corresponding methyl ester
derivative (Fig. 2).
The esters precursors of the series 3 and 4 were
obtained commercially or prepared from corresponding
aromatic aldehyde or carboxylic acid derivative, as
shown in Figs. 3 and 4.
Employing the oxidative Yamada’s procedure [18],
the esters 5, 6 and 8 were obtained from the respective
aldehyde derivative 12–14, in yields ranging from 75 to
85%, by treatment with 2.6 equiv. of KOH and 1.3
equiv. of iodine in methanol at 0 °C (Fig. 3). Additio-
nally, methyl 2-pyrazinecarboxylate (9) was prepared
quantitatively by diazomethane esterification of the 2-
pyrazine carboxylic acid (15). Finally, Fisher esterifica-
tion of 3-chlorobenzoic acid (16) and 2,4-dichloro-
benzoic acid (17) by treatment with MeOH using cata-
lytic amounts of sulphuric acid furnished the desired
methyl benzoate derivatives 10 and 11, in 90 and 85%
yield, respectively.
Fig. 3. Synthesis of aromatic or heteroaromatic methyl esters 5–6,
8–11 from the corresponding aldehydes or carboxylic acid deriva-
tives.
The diastereoselective synthesis of the a,b-unsatu-
rated ester precursors 18–20 was achieved, in yields
that varied from 81 to 98%, through Knovenagel con-
densation of corresponding aromatic aldehydes 21–23
with malonic acid monomethyl ester, using catalytic
amount of piperidine and pyridine as solvent, followed
Fig. 4. Synthesis of a,b-unsaturated aromatic or heteroaromatic
methyl esters 18–20.
1
by in situ descarboxylation (Fig. 4) [19]. The H-NMR

N.M. Sil6a et al. / European Journal of Medicinal Chemistry 37 (2002) 163–170
165
Table 1
Chemical yields and physical data of new substituted isoxazole derivatives 3a–g and 4a–c
Starting material
N
X
W
Y
Z
Isoxazole derivative
t (h)
Molecular formula b Yield (%)
Melting point (°C)
5
6
0
0
0
0
0
0
0
1
1
1
CH CCl
CH
CH CH
CH
CH CH
CH CH
N
CH
N
CH
N
CCl CH
CH
CH
CH
CH
N
3a
3b
3c
3d
3e
3f
2
2
5
3
1
2
5
1
2
3
C9H7N2OCl
C9H8N2O
C9H8N2O
C9H8N2O
C8H7N3O
C10H8NOCl
C10H7NOCl2
C11H9N2OCl
C11H10N2O
C12H10NOCl
40
20
65
30
35
47
37
45
35
40
122–4
55–7
66–8
54–6
113–5
42–4
75–7
145–7
64–6
62–4
N
7 a
8
N
9
10
11
12
13
14
CH CCl CH
CCl 3g
CH
CH
CH CCl
CH
CH CH
N
CH
CCl CH
4a
4b
4c
N
a Purchased from Aldrich Co, Milwaukee, USA.
b The analytical results for C, H, N were within 90.4% of calculated values.
spectra of methyl esters 18, 19 and 20 showed a typical
AB pattern (Jꢀ16 Hz) for the vinyl protons with the
expected (E)-configuration.
latency time of the animals, similar to that obtained for
morphine (39.5 mmol kg−1), used as a positive control
(Fig. 6A). This effect seems to be dose-dependent in
view that it was diminished, but even significant, when
The key-step to synthesize desired isoxazole deriva-
tives 3a–g and 4a–c was accomplished by treatment of
1.5 equiv. of dilithium salt of acetone oxime, prepared
from reaction of acetone oxime with n-BuLi in THF at
room temperature [17,20], with respective methyl ester
derivatives 5–11 and 18–20 as described in Table 1.
Compound 3b was obtained in poor yield, due to the
formation in 30% yield of the undesirable bipyridine-N-
oxide derivative 21 (Fig. 5), resulting from addition of
a second molecule of dilithium salt of acetoxime to the
keto-carbonyl intermediate I (Fig. 2), followed by cy-
clization and dehydratation, as previously described by
Elliot et al. [21], during the synthesis of isoxazole
compound 3b was tested at a dose of 100 mmol kg−1
.
The antinociceptive effect for both doses peaked at 40
min and still present at 80 min after i.p. administration
(Fig. 6A). In addition, after oral administration, com-
pound 3b was effective only at the first’s 20 min,
probably due to differences in bioavailability between
these two routes of administration (data not shown).
Moreover, the antinociceptive effect of compound
3b, at 100 mmol kg−1 but not at 250 mmol kg−1, was
slightly but significantly prevented by pretreatment with
the noncompetitive neuronal nicotinic acetylcholine re-
ceptor antagonist mecamylamine (5 mmol kg−1; i.p.)
(Fig. 6B).
derived from
L-proline methyl ester. Despite several
experimental variations in the number of equivalents of
dilithium salt used, we are not able to improve the
formation of the isoxazole derivative 3b in this process.
All new isoxazole derivatives described herein (3a–g
and 4a–c) were completely structurally characterized by
1H- and 13C-NMR (Tables 2 and 3, respectively) to be
subsequently submitted to pharmacological evaluation.
The antinociceptive effects of epibatidine (2) and
other nAChR ligands such ABT-594 (1) are also pre-
vented by mecamylamine [23,24], suggesting the in-
volvement of neuronal AChRs in these effects.
3. Results and discussion
The preliminary evaluation of the central analgesic
profile of isoxazole derivatives 3a–g and 4a–c in hot
plate test [22] showed that only compound 3b (250
mmol kg−1; i.p.) promotes a significant elevation of
Fig. 5. Bipyridine-N-oxide derivative 21.

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N.M. Sil6a et al. / European Journal of Medicinal Chemistry 37 (2002) 163–170
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N.M. Sil6a et al. / European Journal of Medicinal Chemistry 37 (2002) 163–170
Fig. 6. (A) Effect of i.p. administration of isoxazole derivatives (250 mmol kg−1) on the course of the latency times in the hot plate test in mice.
Results are expressed as means9S.E.M. N=8–10 mice per group. (B) Effect of mecamylamine (Mec) pretreatment (5 mmol kg−1, i.p.) on the
antinociceptive effect of compound 3b at 250 mmol kg−1 (i.p.) and 100 mmol kg−1 (i.p.), in the hot plate test. Mecamylamine (Mec) was
administered 20 min before the test compound. N=8–10 mice per group. *Significantly different from derivative 3b group (PB0.05).
ABT-594 (1) is effective in the hot plate test at 0.62
mmol kg−1 (i.p.) [24].
As concluding remarks, our results suggested that
isoxazole derivative 3b showed the best analgesic profile
of this series, probable due to its action at nicotinic
receptor level, which could be considered as a new
heterocyclic template for design of new nAchR ligands.
solutions (each 15 mL) of KOH (1.36 g, 24.31 mmol)
and iodine (3.13 g, 12.15 mmol). After stirring for 1 h
at 0 °C, small amounts of saturated NaHSO3 solution
(ca. 15 mL) were added until the disappearance of the
brown colour. Next, the methanol was almost totally
evaporated under reduced pressure. The residue was
partitioned between water (20 mL) and ethyl ether (30
mL), followed by separation of the organic phase. The
aqueous layer was further extracted with ethyl ether
(3×10 mL) and the organic extracts were combined
and submitted to the usual work-up to give the desired
methyl esters 5, 6 and 8 (see Fig. 3).
4. Experimental protocols
4.1. Chemistry
4.1.2. General procedure for synthesis of methyl esters
10 and 11 from corresponding benzoic acids 16 and 17
To a solution of the substituted benzoic acid deriva-
tive (16 or 17) (8.20 mmol) and sulphuric acid (0.8 mL)
in MeOH (14 mL), was refluxed until TLC analysis
indicated the total consumption of the starting mate-
rial. Then, solvent was concentrated and pH was ad-
justed to 7.0 with 10% aq. NaHCO3 solution, the water
phase was extracted with ethyl ether (3×15 mL), and
the combined extracts were dried with anhydrous
sodium sulphate and concentrated under reduced pres-
sure to give the desired methyl esters, as described in
Fig. 3.
Melting points were determined with a Quimis 340
apparatus and are uncorrected. H-NMR spectra were
1
determined in deuterated chloroform containing ca. 1%
tetramethylsilane as an internal standard, with Brucker
AC 200 or Varian Gemini 200 at 200 MHz. 13C-NMR
spectra were determined in the same spectrometers
described above at 50 MHz, employing the same sol-
vents. Microanalysis data was obtained with Perkin–
Elmer 240 analyser, using Perkin–Elmer AD-4 balance.
The usual work-up means that the organic extracts
prior to concentration under reduced pressure, were
treated with a saturated aqueous sodium chloride solu-
tion, referred as to brine, dried over anhydrous sodium
sulphate and filtered.
4.1.3. Methyl 2-pyrazinecarboxylate (9)
4.1.1. General procedure for synthesis of methyl esters
5, 6 and 8 from corresponding heteroaromatic
aldehydes 12, 13 and 14
To a solution of the heterocyclic aldehyde derivative
12, 13 or 14 (9.35 mmol)) in absolute MeOH (10 mL)
cooled at 0 °C, were successively added methanolic
To a stirred solution of 2-pyrazine carboxylic acid
(15) (0.50 g; 4.03 mmol) in CH2Cl2 (10 mL) was slowly
dropped an ethereal solution of diazomethane until the
reaction was finished (TLC). Then glacial acetic acid
(ca. 1.0 mL) was added and the organic phase was
dried with anhydrous K2CO3. The resulting suspension

N.M. Sil6a et al. / European Journal of Medicinal Chemistry 37 (2002) 163–170
169
was filtered and the solvent was concentrated at re-
duced pressure to afford, quantitatively, the methyl
ester 9 as a yellow solid, m.p. 55–56 °C (Fig. 3).
Animals were placed on a plate heated at 5590.1 °C
and their responses to thermal stimulation (licking or
withdraw of the hindpaw) were timed. Three control
measures were done (in the absence of the test drugs) in
intervals of 30 min to determine the control latency
mean time and the cut-off time (maximum time of
permanence of the animal in the plate), calculated as
three times the control mean value (25 s). The response
time for each mouse was registered at 20 min intervals
after drug administration for a total of 120 min. Data
are expressed as latency time and the percentage of
latency time increase (%LTI)
4.1.4. General procedure for synthesis of
h,i-unsaturated methyl esters 18, 19 and 20 from their
corresponding aromatic or heteroaromatic aldehydes 21,
22 and 23
To a solution of malonic acid monomethyl ester [25]
(0.41 g; 3.51 mmol) in pyridine (1.05 mL) containing a
catalytic amount of piperidine (0.02 mL) was added
2.34 mmol of the aldehyde derivative (21, 22 or 23).
The reaction mixture was refluxed, until TLC analysis
indicated total consumption of the aromatic aldehyde
(see below). Then, after cooling at room temperature,
water (ca. 10 mL) was added, the pH was adjusted to
7.0 with 20% aq. HCl and the aqueous phase was
extracted with ethyl ether (4×5 mL). The organic
extracts were joined, dried over anhydrous sodium sul-
phate and concentrated under reduced pressure to give
a residue, which was purified by flash chromatography
(eluent n-hexane–ethyl acetate, gradient 95/5 to 50/50
v/v). See yields in Fig. 4.
test latency−control latency
% LTI=
×100
cut-off−control latency
4.2.2. Effect of compounds on the hot plate test
Immediately after the control measures, groups of
10–13 mice were treated intraperitoneally with arabic
gum 5% (vehicle control group), isoxazole derivatives
3a–g and 4a–c (100 and 250 mmol kg−1) or morphine
(39.5 mmol kg−1) in a volume of 5 mL kg−1. Latency
times were measured as described above.
4.2.3. Effect of pretreatment with mecamylamine on the
hot plate test
4.1.5. General procedure for synthesis of new isoxazole
deri6ati6es 3a–g and 4a–c from corresponding methyl
ester deri6ati6es 5–11 and 18–20
Immediately after the control measures, groups of 10
mice were treated with mecamylamine (1 mg kg−1
;
To a solution of acetone oxyme (0.48 g; 6.62 mmol)
in dry THF (12 mL) maintained at 0 °C, was added a
solution of n-butyllithium in hexane (9.5 mL; 13.22
mmol/solution 1.39 M). The ice bath was removed and
the mixture was stirred at room temperature for addi-
tional 30 min. Then, a solution of aromatic or he-
teroaromatic methyl ester derivative (4.42 mmol) in dry
THF (3 mL) was added and the reaction was stirred at
room temperature until TLC analysis indicated the
total consumption of the starting material. Next, sul-
phuric acid (3.6 mL) was added at 0 °C, followed by
stirring at room temperature for 1 h. Then, pH was
adjusted to 7.0 with 50% aq. NaOH solution and water
(15 mL) was added. The aqueous phase was extracted
with CH2Cl2 (5×15 mL) and combined organic layers
were dried under anhydrous Na2SO4 and concentrated
under reduced pressure. The crude residue obtained was
purified by silica gel column chromatography (eluent
n-hexane–ethyl acetate, gradient 95/5 to 70/30 v/v) (see
Tables 1–3).
i.p.), 20 min prior to the administration of test
compounds.
4.2.4. Statistical analysis
Differences between responses were statistically
analysed by Student t-test and analysis of variance
(ANOVA), followed by Bonferroni’s t-test, for a signifi-
cance level of *PB0.05. Results are expressed as
means9S.E.M. of measurements.
Acknowledgements
We are grateful to the CNPq (BR.), FAPERJ (BR.)
and FUJB (BR.) for the financial support of this work
and fellowships (N.M.S., J.L.M.T., A.L.P.M.,
C.A.M.F. and E.J.B.). The authors also thank the
Analytical Center of NPPN (UFRJ, BR.) for spectro-
scopic facilities.
4.2. Pharmacological assays
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