6-Aminoalkyloxazolo[4,5-b]pyridin-2(3H)-ones: Synthesis and evaluation of antinoceptive activity

Article abstract of DOI:10.1016/S0968-0896(98)00137-0

Series of 6-aminoalkyloxazolo[4,5-b]pyridin-2(3H)-ones incorporating structural modifications both in the alkyl chain and basic amino moiety were tested for their analgesic efficacy and safety in mice and rats. Two of the synthesised compounds, 4a (3-meth

Full text of DOI:10.1016/S0968-0896(98)00137-0

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 1963±1973
6-Aminoalkyloxazolo[4,5-b]pyridin-2(3H)-ones: Synthesis and
Evaluation of Antinoceptive Activity
L. Savelon,a J. G. Bizot-Espiard,b D. H. Caignard,c B. Pfei€er,c P. Renard,c
M. C. Viauda,* and G. Guillaumeta
a
Â
Â
Â
Institut de Chimie Organique et Analytique, associe au CNRS, Universite d'Orleans, B. P. 6759, 45067 Orleans Cedex 2, France
b
Â
IRI Servier, 6, place des Pleiades, 92415 Courbevoie Cedex, France
c
Â
ADIR, 1, rue Carle Hebert, 92415 Courbevoie Cedex, France
Received 17 November 1997; accepted 25 February 1998
AbstractÐSeries of 6-aminoalkyloxazolo[4,5-b]pyridin-2(3H)-ones incorporating structural modi®cations both in the
alkyl chain and basic amino moiety were tested for their analgesic ecacy and safety in mice and rats. Two of the
synthesised compounds, 4a (3-methyl-6-[(4-phenyl-1-piperazinyl)methyl]oxazolo[4,5-b]pyridin-2(3H)-one) and 12a (3-
methyl-6{1-[2-(4-phenyl-1-piperazinyl)ethan-1-ol]}oxazolo[4,5-b]pyridin-2(3H)-one) were found to be more potent than
aspirin with ED50 values of 26 (16.1±42.4) and 15.5 (11.4±21.2) mg/kg po (mouse, phenylquinone writhing test)
respectively and 6 (3.1±9.8) and 5.5 (3.5±8.8) mg/kg po (rat, acetic acid writhing test). Compounds 4a and 12a proved
to be potent nonopioid nonantiin¯ammatory analgesics but unfortunately have sedative properties at relatively low
doses (respectively 64 and 16 mg/kg po, mice) # 1998 Published by Elsevier Science Ltd. All rights reserved.
Introduction
Two main groups of analgesics on the market are the
opioids such as morphine and codeine and the non
steroidal antiin¯ammatory agents including aspirin,
paracetamol and ibuprofen.1
The opioids produce their action by interacting with
speci®c receptors in the central nervous system but their
therapeutic use is limited by adverse side e€ects includ-
ing tolerance, constipation, respiratory depression and
physical dependence.2 The non steroidal antiin-
¯ammatory drugs act mainly by inhibiting the pros-
taglandin synthesis peripherally, but unfortunately they
induce gastrointestinal lesions.3
With regard to the bio-isostere relationships, replacing the
carbon by a heteroatom in a drug template is a common
strategy in medicinal chemistry. Thereby, in connection
with our previous studies on heterocyclic compounds
with analgesic activity,5±10 we report here the synthesis
and the biological evaluation of oxazolo[4,5-b]pyridin-
2(3H)-ones substituted at the 6-position by an ami-
noalkyl unit functionalised or not (compounds D).
Previous work have shown that benzoxazolinones sub-
stituted in position 6 by an (arylpiperazinyl)alkyl4a
(compounds A and B) or an (arylpiperazinyl)hydroxy
alkyl4b (compound C) are good analgesics with ED50
respectively of 2.8, 3.9 and 9 mg/kg po in the phe-
nylquinone induced writhing test performed with mice.
*Corresponding author: Tel.: 33 2 38 49 45 80; Fax: 33 2 38 41
70 78.
0968-0896/98/$ - see front matter # 1998 Published by Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00137-0

1964
L. Savelon et al./Bioorg. Med. Chem. 6 (1998) 1963±1973
derivative 111 using Stille's reaction12 with tetra-
Chemistry
methyltin in toluene. This compound, after introduction
of the methyl group, was treated with N-bromosuccini-
mide in carbon tetrachloride with a catalytic amount of
radical initiator. The brominated derivative 3, obtained
The synthesis of aminooxazolopyridinones 4 is illu-
strated in Scheme 1. Compound 2 was obtained with
a
satisfactory yield starting from the brominated
Scheme 1.
Scheme 2.

L. Savelon et al./Bioorg. Med. Chem. 6 (1998) 1963±1973
1965
in 83% yield, was submitted to reaction with 1-phe-
nylpiperazine or morpholine in 1,4-dioxane in the pre-
sence of triethylamine to provide products 4 in excellent
yields.
Preparation of compounds 11 and 12 is reported in
Scheme 3. Compound 9 was easily obtained from
bromo derivative 1 under Heck's conditions.13 Reaction
of 1 with butyl vinyl ether in N,N-dimethylformamide
and triethylamine as base, in the presence of tri-o-tolyl-
phosphine as ligand, gave, after acid hydrolysis,14 the
ketone 9 in 90% yield. This compound was converted
into bromo acetyl derivative 10 by treatment with bro-
mine in chloroform (73%). The amino compounds 11
were prepared in 81±84% yields by alkylation of 1-phe-
nylpiperazine or morpholine with the bromo ketone 10
in an aprotic solvent such as 1,4-dioxane in the presence
of triethylamine. Finally, the reduction of 11 with
sodium borohydride in methanol furnished the amino
alcohols 12 in excellent yields.
In Scheme 2 is reported the synthesis of compound 8.
Vinylation of bromo derivative 1 with tributyl(vinyl)tin
under palladium catalysis12 gave a reproducible isolated
yield of 72% of the expected product 3. The treatment
of this product with thexylborane in THF followed by
addition of hydrogen peroxide and aqueous sodium
hydroxide solution a€orded the alcohol 6 in 70% yield.
Tosylation of 6 with p-toluenesulfonyl chloride in the
presence of triethylamine in dichloromethane led to the
corresponding p-toluenesulfonate 7 in 81% yield. The
conversion of 7 into the ®nal compound 8a was accom-
plished in 91% yield without any diculty by reaction
of 7 with 1-phenylpiperazine in 1,4-dioxane.
The synthesis of the target oxazolopyridinones 16 is
shown in Scheme 4. The coupling reaction12 of 1 with
Scheme 3.

1966
L. Savelon et al./Bioorg. Med. Chem. 6 (1998) 1963±1973
1-tributylstannyl-3,3-diethoxyprop-1-ene15 in the pre-
sence of tetrakis(triphenylphosphine) palladium(0) and
lithium chloride in tetrahydrofuran gave, after acid
hydrolysis, the unsaturated aldehyde 13 in 72% yield.
Catalytic hydrogenation of this compound over 10%
Pd±C a€orded the saturated alcohol 14 in 92% yield and
its treatment with p-toluenesulfonyl chloride in dichloro-
methane furnished the corresponding tosylate 15 in 78%
yield. Finally, the N-alkylation of 1-phenylpiperazine or
morpholine with 15 a€orded the desired amino deriva-
tives 16 in satisfactory yields.
This preliminary evaluation shows that, whatever the
length (1±3 carbon atoms) and the nature (alkyl,
hydroxyalkyl, alkylcarbonyl) of the spacer between the
oxazolo[4,5-b]pyridin-2(1H)one and the basic amino
moiety, phenylpiperazino derivatives (4a, 11a, 12a, and
16a) are always clearly more active than their morpho-
lino counterpart (4b, 11b, 12b, and 16b).
Surprisingly, the length of the alkyl spacer (from 1 to 3
carbon atoms) has not clear in¯uence on the analgesic
properties in the PBQ writhing test. The best results are
however obtained with a 3 carbon atoms spacer (com-
pound 16a) with an activity ratio higher than 1.18 ver-
sus aspirin, compared to 1.02 for 4a (methylene spacer)
and 1.04 for 8a (ethyl spacer).
Biological Results and Discussion
Nine 6-aminoalkyloxazolo[4,5-b]pyridin-2(3H)ones were
evaluated for their analgesic activity. All were ®rst pre-
screened at a standard dose of 50 mg/kg po using the
phenylquinone (PBQ) and acetic acid writhing tests
performed respectively in mice and rats. Results were
expressed as percentages of inhibition of the writhing
and also as an activity ratio versus aspirin to correct
for slight variations in response between the same
evaluation procedures not performed simultaneously
(Table 1).
Replacement of the ethyl spacer (compound 8a) by a
hydroxyethyl spacer (compound 12a) results in a clear
increase of the analgesic activity in the PBQ writhing
test (activity ratio of 1.04 compared to 1.56), whilst its
replacement by an acetyl linker (compound 11a) leads to
a clear decrease in potency with an activity ratio of 0.79.
On the basis of the prescreening results, compounds 4a
and 12a, which were found more potent than aspirin in
Scheme 4.

L. Savelon et al./Bioorg. Med. Chem. 6 (1998) 1963±1973
1967
both the PBQ and acetid acid-included writhing test,
were selected for further investigations. Their ED50 were
of 26 (16.1±42.4) mg/kg po for 4a and 15.5 (11.4±21.2)
mg/kg po for 12a in the PBQ writhing test and of 6 (3.7±
9.8) mg/kg po 4a and 5.5 (3.5±8.8) mg/kg po for 12a in
the acetic acid writhing test compared to, respectively,
63 (56±73) and 32 (28±46) mg/kg po for aspirin. Com-
pound 12a proved to be slightly less active than its
benzo analogue C which have a ED50 of 9 mg/kg po in
the PBQ writhing test.4b
The oral general acute toxicity of 4a and 12a as well as
their behavioural e€ects were investigated via an Irwin
test performed in mice (a careful observation of the
general behaviour is also useful to avoid possible false
positives in the writhing tests). For both compounds,
the ®rst behavioural changes are sedation and hypo-
thermia that appear at 16 mg/kg po for 12a and 64 mg/
kg po for 4a. The mortality threshold dose is higher
than 512 mg/kg po for the two compounds. An oral
safety index was de®ned as the ratio of the ED50 po in
Table 1. Analgesic activity screening of 6-aminoalkyloxazola[4,5-b]pyridin-2(3H)ones
Phenylquinone(PBQ)-induced
writhing test (mice)
Acetic acid-induced
writhing test (rat)
Compda
n
Z
R2N
% inhibition at
50 mg/kg pob
activity ratio
versus aspirinc
% inhibition at
50 mg/kg pob
activity ratio
versus aspirin
4a
4b
0
0
H,H
H,H
57%**d
32%
1.02
0.57
98%***
1.38
0.47
33%*
NDe
8a
1
1
1
H,H
O
86.3%**
53%**
18.8%
97%***
20%
1.04
0.79
0.28
1.56
0.33
1.18
0.38
11a
11b
12a
12b
16a
16b
96.8%***
26%
1.01
0.28
1.52
0.68
O
1 H,OH
1 H,OH
86%**
38%
2
2
H,H
H,H
100%***
32%**
ND
23%
0.37
aThese compounds were tested as oxalate.
bFive animals were used for each compound, seven animals for the control group.
% of inhibition with the compound at 50mg=kg p:o:
cActivity ratio=
% of inhibition with aspirin at 50mg=kg p:o:
d*P<0.05; **P<0.01; ***P<0.001.
eND: not determined.

1968
L. Savelon et al./Bioorg. Med. Chem. 6 (1998) 1963±1973
mice with the dose of appearance of the ®rst beha-
vioural changes in the Irwin test. This safety index is
clearly insucient with values of 2.5 for 4a and 1 for
12a.
rotatory evaporator. Analytical thin-layer chromato-
graphy (TLC) was carried out on precoated plates (silica
gel, 60 F-254), and spots were visualised with UV light
or an alcohol solution of ammonium cerium(IV) nitrate.
Column chromatography was performed with Kieselgel
60 (70±230 mesh) silica gel (Merck) for gravity columns
and Kieselgel 60 (230±400 mesh) silica gel (Merck) for
¯ash columns. Where analyses are indicated by symbols
of the elements, analytical results obtained for those
elements were ‹0.4% of the theoretical values. All
anhydrous reactions were performed in oven-dried
glassware under an atmosphere of argon. The column
chromatography solvents employed were distilled and
solvent mixtures were reported as volume to volume
ratios. Tetrakis (triphenylphosphine) palladium(0) was
prepared by using the literature procedure.17
In order to investigate a possible morphinic mechanism
of action, studies of binding were investigated and
showed, for both compounds, an absence of anity
(Ki<10 5 M) for the opioid m, K, and d, receptors. A-
nities for the a1, a2, H1, H2, D1, D2, 5HT1A, 5HT1B
,
5HT1D, 5HT2A, 5HT2C, and 5HT3 receptors were also
investigated. Compounds 12b proved to have moderate
anity for H1 (Ki=1.4Â10 7 M), 5HT1A (Ki=9.7
Â10 8 M), 5HT1B (Ki=7.7Â10 6 M), 5HT2A (Ki=3.3
Â10 6 M) receptors whilst 4a showed same anity for
a1 (Ki=6Â10 6 M), a2 (Ki=7Â10 6 M) and 5HT1A
(Ki=8Â10 6 M). These anities could explain some of
the side e€ects observed in the Irwin test.
3,6-Dimethyloxazolo[4,5-b]pyridin-2(3H)-one (2). To a
stirred solution of 6-bromo-3-methyloxazolo[4,5-b]pyr-
idin-2(3H)-one 1 (414 mg, 1.8 mmol) in toluene (10 mL)
was added successively tetramethyltin (0.3 mL,
2.2 mmol) and bis(triphenylphosphine)palladium(II)
chloride (70 mg, 0.1 mmol). Then, the mixture was stir-
red for 8 h at re¯ux. After cooling, the solvent was eva-
porated and the residue was quenched with water. After
extraction with dichloromethane, organic layers were
washed, dried over magnesium sulfate and evaporated.
The crude product was puri®ed by silica gel column
chromatography (eluent:dichloromethane) to provide 2
(416 mg) in 72% yield.; mp 115±116 ꢀC; 1H NMR
(CDCl3) d 2.37 (s, 3H, CH3), 3.46 (s, 3H, NCH3), 7.23
(d, 1H, J=1.5 Hz, H7), 7.93 (d, 1H, J=1.5 Hz, H5); IR
(KBr) n 1780 cm 1. Anal. C8H8N2O2 (C,H,N).
The inhibitory properties of compounds 4a and 12a on
cyclooxygenase and 5-lipoxygenase were evaluated in
vitro by measuring the inhibition of the production of
PGE2 and LTB4 from rabbit granulocytes cells stimu-
lated by calcium ionophore A 23187.16 Both compounds
appeared to be devoid of any inhibitory activity at a
concentration of 10 5 M.
Conclusion
In conclusion within this class of 6-aminoalkyl-
oxazolo[4,5-b]pyridin-2(3H)-ones, we have shown that
some compounds possess potent nonopioid anti-
nociceptive activity without antiin¯ammatory proper-
ties. Two of them, 4a and 12a proved to be more active
than aspirin but unfortunately with a clearly insucient
safety index.
6-Bromomethyl-3-methyloxazolo[4,5-b]pyridin-2(3H)-one
(3). Under argon atmosphere, compound 2 (0.5 g,
3.05 mmol) was dissolved in carbon tetrachloride
(50 mL). To this solution was added N-bromosuccini-
mide (597 mg, 3.35 mmol) and a trace of benzoyl per-
oxide, then the mixture was stirred at re¯ux for 3 h.
After cooling and ®ltration, the solvent was removed
under reduce pressure and the residue was puri®ed by
¯ash chromatography (eluent:dichloromethane) to give
Experimental
Chemistry
Melting points were determined on a Ko¯er hot-stage
apparatus and are uncorrected. Proton NMR were
recorded on a Bruker 300 spectrometer. The coupling
constants are recorded in hertz (Hz) and the chemical
shifts are reported in parts per million (d, ppm) down-
®eld from tetramethylsilane (TMS), which was used as
an internal standard. Infrared spectra were obtained
with a Perkin±Elmer spectrophotometer 297. Mass
spectra were recorded on a R 10-10 C Nermag (70 eV)
apparatus. Organic solvents were puri®ed when neces-
sary by the methods described by D. D. Perrin, W. L. F.
Armarego and D. R. Perrin (Puri®cation of Laboratory
Chemicals; Pergamon: Oxford, 1986) or purchased from
Aldrich Chimie. All solutions were dried over anhy-
drous magnesium sulfate and evaporated on a Buchi
compound 3 (617 mg) in 83% yield; mp 105±106 ꢀC; H
1
NMR (CDCl3) d 3.49 (s, 3H, NCH3), 4.53 (s, 2H, CH2),
7.46 (d, 1H, J=1.5 Hz, H7), 8.13 (d, 1H, J=1.5 Hz, H5);
IR (KBr) n 1785 cm 1. Anal. C8H7BrN2O3 (C,H,N).
3-Methyl-6-[(4-phenyl-1-piperazinyl)methyl]oxazolo[4,5-b]-
pyridin-2(3H)-one (4a). Compound 3 (1.0 g, 4.1 mmol)
was dissolved, under argon atmosphere, in 1,4-dioxane
(25 mL) at room temperature, then was added succes-
sively at this same temperature 1-phenyl-piperazine
(0.70 g, 4.3 mmol) and triethylamine (0.62 g, 6.2 mmol).
After 5 h of stirring the crude mixture was con-
centrated in vacuo and water was added to the residue.

L. Savelon et al./Bioorg. Med. Chem. 6 (1998) 1963±1973
1969
The mixture was extracted with dichloromethane and
the extracts were washed with water and dried over
magnesium sulfate. After evaporation, the crude pro-
duct was puri®ed by silica gel column chromatography
(eluent: dichloromethane/methanol, 98/2) to provide 4a
(1.28 g) in 96% yield; mp 131±132 ꢀC (i-PrOH); 1H
NMR (CDCl3) d 2.61 (dd, 4H, J=5.2, 4.4 Hz, CH2piperaz),
3.19 (dd, 4H, J=5.2, 4.4 Hz, CH2piperaz), 3.48 (s, 3H,
NCH3), 3.58 (s, 2H, CH2), 6.86 (t, 1H, J=7.3 Hz,
dichloromethane. The organic layer was dried (MgSO4)
and evaporated to dryness under reduced pressure. The
residual oil was chromatographed on a silica gel col-
umn (eluent: dichloromethane/methanol, 95/5) to give 6
(679 mg) in 70% yield as a white solid; mp 143±144 ꢀC;
1H NMR (CDCl3+D2O) d 2.89 (t, 2H, J=6.6 Hz,
CH2), 3.47 (s, 3H, NCH3), 3.88 (t, 2H, J=6.6 Hz,
CH2O), 7.35 (s, 1H, H7), 8.00 (s, 1H, H5); IR (KBr) ꢀ
3400±3100, 1775 cm 1. Anal. C9H10N2O3 (C,H,N).
Harom), 6.92 (d, 2H, J=8.8 Hz, Harom), 7.20±7.28 (m,
2H, Harom), 7.51 (d, 1H, J=1.8 Hz, H7), 8.05 (d, 1H,
J=1.8 Hz, H5); IR (KBr) n 1775 cm 1; MS (IC/NH3) m/
z 325 (M+1). Anal. C18H20N4O2 (C,H,N).
3-Methyl-6-[2-(p-toluenesulfonyloxy)ethyl]oxazolo[4,5-b]-
pyridin-2(3H)-one (7). To a stirred solution of alcohol 6
(194 mg, 1.0 mmol) in dichloromethane (5 mL) were
added successively p-toluenesulfonyl chloride (286 mg,
1.5 mmol) and triethylamine (0.4 mL, 3 mmol) at 0 ꢀC
under argon atmosphere. The reaction mixture was
allowed to reach room temperature. After hydrolysis
with water, the crude was extracted with dichloro-
methane. The organic layers were washed, dried over
magnesium sulfate and concentrated in vacuo. The resi-
due was puri®ed by column chromatography (eluent:
dichloromethane) to provide 7 (282mg) in 81% yield; mp
141±142 ꢀC; 1H NMR (CDCl3) d 2.42 (s, 3H, CH3), 2.97
(t, 2H, J=6.6 Hz, CH2), 3.46 (s, 3H, NCH3), 4.23 (t,
2H, J=6.6 Hz, OCH2), 7.13 (d, 1H, J=1.5 Hz, H7), 7.27
(d, 2H, J=8.1 Hz, Harom), 7.68 (d, 2H, J=8.1 Hz,
3-Methyl-6-[(1-morpholino)methyl]oxazolo[4,5-b]pyridin-
2(3H)-one (4b). This compound was prepared according
to the procedure above from product 3 and morpholine.
After column chromatography (eluent: dichloromethane/
methanol, 98/2) the expected product 4b was obtained in
90% yield; mp 144±145 ꢀC (i-PrOH); H NMR (CDCl3)
1
d 2.46 (dd, 4H, J=5.2, 4.3 Hz, CH2morph), 3.49 (s, 3H,
NCH3), 3.52 (s, 2H, CH2), 3.71 (dd, 4H, J=5.2, 4.3 Hz,
CH2morph), 7.50 (d, 1H, J=1.8 Hz, H7), 8.03 (d, 1H,
J=1.8 Hz, H5); IR (KBr) n 1775 cm 1 MS (IC/NH3) mz
250 (M+1). Anal. C12H15N3O3 (C,H,N).
3-Methyl-6-vinyloxazolo[4,5-b]pyridin-2(3H)-one (5). To
a
Harom), 7.89 (d, 1H, J=1.5 Hz, H5); IR (KBr) ꢀ
1790 cm 1. Anal. C16H16N2O5S (C,H,N).
stirred solution of 6-bromo-3-methyloxazolo[4,5-
b]pyridin-2(3H)-one 1 (414 mg, 1.8 mmol) in toluene
(10 mL) was added successively tributyl(vinyl)tin (697 mg,
2.2 mmol), and bis(triphenylphosphine)palladium (II)
chloride (70 mg, 0.1 mmol). The mixture was stirred at
re¯ux for 8 h. After cooling, evaporation of solvent and
hydrolysis with water, the product was extracted with
dichloromethane. The organic layer was dried over
magnesium sulfate and the solvent was removed under
reduce pressure. The crude product was puri®ed by
silica gel column chromatography (eluent: dichloro-
methane) to a€ord 5 (274 mg) in 86% yield; mp 124±
3-Methyl-6-[2-(4-phenyl-1-piperazinyl)ethyl]oxazolo[4,5-b]-
pyridin-2(3H)-one (8a). To a solution of 7 (210 mg,
0.6 mmol) in 1,4-dioxane (8 mL) were added successively
1-phenylpiperazine (0.10 mL, 1.38 mmol) and triethyla-
mine (0.18 mL, 1.3 mmol) at room temperature. Then,
the mixture was stirred at the same temperature for 24 h.
After evaporation of solvent under reduced pressure, the
residue was quenched with water. The aqueous mixture
was extracted with dichloromethane and was dried over
magnesium sulfate. The organic solution was con-
centrated and the residue was puri®ed by column chro-
matography (eluent: dichloromethane/methanol, 95/5)
to give 8a (185 mg) in 91% yield; mp 81±82 ꢀC
(Et2O); 1H NMR (CDCl3) d 2.64±2.72 (m, 6H, CH2 and
CH2piperaz), 2.88 (dd, 2H, J=8.0, 7.1 Hz, CH2), 3.24 (dd,
4H, J=5.6, 4.8 Hz, CH2piperaz), 3.49 (s, 3H, NCH3), 6.88
(t, 1H, J=7.1 Hz, Harom), 6.95 (d, 2H, J=7.9 Hz,
125 ꢀC; H NMR (CDCl3) d 3.48 (s, 3H, NCH3), 5.34
1
(d, 1H, J=11.1 Hz, CH2), 5.72 (d, 1H, J=17.4 Hz,
=CH2), 6.71 (dd, 1H, J=11.1, 17.4 Hz, =CH), 7.51 (d,
1H, J=1.3 Hz, H7), 8.08 (d, 1H, J=1.3 Hz, H5); IR
(KBr) n 1790 cm 1. Anal. C9H8N2O2 (C,H,N).
6-[2-(Hydroxyethyl)]-3-methyloxazolo[4,5-b]pyridin-2(3H)-
one (6). To a solution of thexylborane, previously pre-
pared according to the literature procedure,18 was added
at 0 ꢀC the vinyl compound 5 (880 mg, 5.0 mmol)
dissolved in tetrahydrofuran (20 mL). After stirring for
2 h at 0 ꢀC, 10% aqueous NaOH (2.40 mL) then hydro-
gen peroxide (2.00 mL) were successively added and
reaction mixture stirred for an additional 1 h at room
temperature. The solvent was removed under pressure
and the product was extracted from the resulting crude
reaction mixture, after aqueous hydrolysis, with
Harom), 7.24±7.32 (m, 2H, Harom), 7.35 (d, 1H,
J=1.6 Hz, H7), 8.01 (d, 1H, J=1.6 Hz, Harom); IR
(KBr) ꢀ 1790 cm 1; MS (IC/NH3) m/z 339 (M+1).
Anal. C19H22N4O2 (C, H, N).
(3-Methyl-2-oxooxazolo[4,5-b]pyridin-6-yl)ethanone (9).
To a stirred solution of brominated compound 1
(0.50 g, 2.18 mmol) in N,N-dimethylformamide (5 mL)
were added successively triethylamine (0.44 g, 4.36 mmol),
butyl vinyl ether (1.2 g, 12 mmol), 1,2-bis(diphenyl-

1970
L. Savelon et al./Bioorg. Med. Chem. 6 (1998) 1963±1973
phosphino)ethane (24 mg, 0.06 mmol) and palladium(II)
acetate (12 mg, 0.054 mmol). The mixture was stirred at
re¯ux for 8 h under inert atmosphere. After cooling, the
solution was hydrolysed with a solution of 10% HCl
and stirring was maintained for 1 h. The solvent was
evaporated and the residue treated with water and
extracted with CH2Cl2. The combined organic extracts
were washed with water, dried over MgSO4, and eva-
porated in vacuo. The residue was puri®ed by ¯ash
chromatography (eluent: dichloromethane/ethyl acetate,
8/2) to a€ord 9 (377 mg) in 90% yield; mp 163±164 ꢀC;
1H NMR (CDCl3) d 2.53 (s, 3H, CH3), 3.63 (s, 3H,
NCH3), 7.91 (d, 1H, J=1.5 Hz, H7), 8.72 (d, 1H,
J=1.5 Hz, H5); IR (KBr) ꢀ 1790, 1670 cm 1. Anal.
C9H8N2O3 (C,H,N).
ketone 11a (352 mg, 1.0 mmol) in methanol (15 mL) was
added sodium borohydride (42 mg, 1.1 mmol) and the
mixture was stirred for 5 h at room temperature. After
hydrolysis, the resulting precipitate was collected, dried
and crystallised from diethyl ether to give the product
12a (336 mg) in 95% yield; mp 183±184 ꢀC (Et2O); 1H
NMR (CDCl3+D2O) d 2.47±2.60 (m, 2H, CH2), 2.61±
2.70 (m, 2H, CH2piperaz), 2.88±2.98 (m, 2H, CH2piperaz),
3.18±3.33 (m, 4H, CH2piperaz), 3.48 (s, 3 H, NCH3), 4.83
(dd, 1H, J=10.3, 4.4 Hz, CH), 6.88 (t, 1 H, J=7.4,
H
H
arom), 6.94 (d, 2H, J=8.1 Harom), 7.25±7.33 (m, 2H,
arom), 7.53 (d, 1H, J=1.5, H7), 8.10 (d, 1H, J=1.5,
H5); IR (KBr) ꢀ 3400 3100, 1775 cm 1; MS (IC/NH3)
m/z 355 (M+1). Anal. C19H22N4O3 (C,H,N).
3-Methyl-6-[1-(2-morpholinyl)ethan-1-ol]oxazolo[4,5-b]-
pyridin-2(3H)-one (12b). The compound 12b was pre-
pared as above from the ketone 11b in 98% yield; mp
146±147 ꢀC (Et2O); 1H NMR (CDCl3+D2O) d 2.36±
2.46 (m, 4H, CH2 and CH2morph), 2.68±2.78 (m, 2H,
CH2morph), 3.48 (s, 3H, NCH3), 3.66±3.80 (m, 4H,
CH2morph), 4.76 (dd, 1H, J=10.4, 3.9, CH), 7.47 (d, 1H,
J=1.5, H7), 8.04 (d, 1H, J=1.5, H5); IR (KBr) ꢀ 3400±
3100, 1775 cm 1; MS (IC/NH3) m/z 280 (M+1). Anal.
C13H17N3O4 (C,H,N).
2-Bromo-1-(3-methyl-2-oxooxazolo[4,5-b]pyridin-6-yl)-
ethanone (10). To a stirred solution of 9 (500 mg,
2.6 mmol) in chloroform (15 mL) was added dropwise
bromine (0.2 mL, 2.6 mmol). After stirring at room
temperature for 5 h, the solution was evaporated under
reduced pressure. The residue was chromatographed on
silica gel column (eluent: dichloromethane) to provide
10 (515 mg) in 73% yield; mp 133±134 ꢀC; 1H NMR
(CDCl3) d 3.54 (s, 3H, NCH3), 4.41 (s, 2H, CH2), 7.97
(d, 1H, J=1.5 Hz, H7), 8.81 (d, 1H, J=1.5 Hz, H5); IR
(KBr) ꢀ 1770, 1670 cm 1. Anal. C9H7BrN2O3 (C,H,N).
(E)-3-(3-Methyl-2-oxooxazolo[4,5-b]pyridin-6-yl)propenal
(13). The corresponding diethylacetal of the compound
13 was prepared according to the same experimental
procedure described for product 2 using 1-tributyl-
stannyl-3,3-diethoxyprop-1-ene15 as the reagent and
tetrahydrofuran as the solvent. The crude diethylacetal
obtained was dissolved in water and treated with few
drops of 10% HCl. The mixture was stirred for 1 h at
room temperature, then extracted several times with
dichloromethane. After drying over MgSO4 followed by
evaporation of the solvent, the residue was puri®ed by
silica gel column chromatography (eluent: dichloro-
methane/methanol, 98/2) to provide the aldehyde 13 in
72% yield; mp 211±212 ꢀC; 1H NMR (CDCl3) d 3.51 (s,
3H, NCH3), 6.68 (dd, 1H, J=15.4, 7.4 Hz, =CH), 7.49
(d, 1H, J=15.4 Hz, =CH), 7.60 (d, 1H, J=2.2 Hz, H7),
8.29 (d, 1H, J=2.2 Hz, H5), 9.72 (d, 1H, J=7.4 Hz,
CHO); IR (KBr) ꢀ 1790, 1680 cm 1. Anal. C10H8N2O3
(C,H,N).
1-(3-Methyl-2-oxooxazolo[4,5-b]pyridin-2-yl)-2-(4-phenyl-
1-piperazinyl)ethanone (11a). This compound was pre-
pared according to the same experimental procedure
described for compounds 4 using 10 and 1-phenylpiper-
azine as starting materials. Compound 11a was obtained
in 84% yield; mp 202±203 ꢀC (Et2O); 1H NMR (CDCl3)
d 2.75 (dd, 4H, J=5.1, 4.4 Hz, CH2piperaz), 3.24 (dd, 4H,
J=5.1, 4.4 Hz, CH2piperaz), 3.52 (s, 3H, NCH3), 3.78 (s,
2H, CH2), 6.85 (t, 1H, J=7.4 Hz, Harom), 6.92 (d, 2H,
J=8.1, Harom), 7.21±7.30 (m, 2H, Harom), 8.05 (d, 1H,
J=1.5, H5), 8.99 (d, 1H, J=1.5, H5); IR (KBr) ꢀ
1
1775 cm
C19H20N4O3 (C,H,N).
; MS (IC/NH3) m/z 353 (M+1). Anal.
1-(3-Methyl-2-oxooxazolo[4,5-b]pyridin-6-yl)-2-morph-
olinylethanone (11b). This compound was prepared
according to the same experimental procedure described
for compounds 4 using 10 and morpholine as starting
materials. Compound 11b was obtained in 86% yield;
mp 186±187 ꢀC (Et2O); 1H NMR (CDCl3) d 2.57 (dd,
4H, J=5.2, 4.4 Hz, CH2morph), 3.51 (s, 3H, NCH3), 3.71
(s, 2H, CH2), 3.73 (dd, 4H, J=5.2, 4.4 Hz, CH2morph),
8.01 (d, 1H, J=1.5, H7), 8.93 (d, 1H, J=1.5, H5); IR
(KBr) ꢀ 1775 cm 1; MS (IC/NH3) m/z 280 (M+1).
Anal. C13H15N3O4 (C,H,N).
3-Methyl-6-[2-(propan-1-ol)]oxazolo[4,5-b]pyridin-2(3H)-
one (14). Compound 13 (500 mg, 2.45 mmol) was dis-
solved in methanol (35 mL). Then palladium on char-
coal (10%) (50 mg) was added. The solution was kept
under hydrogen pressure in a Parr shaker at 40 psi for
6 h. The catalyst was removed by ®ltration, then the
solvent was evaporated under reduce pressure. The
crude product was puri®ed by silica gel column chroma-
tography (eluent: dichloromethane/methanol, 95/5) to
give the alcohol 14 (470mg) in 92% yield; mp 218±219 ꢀC;
3-Methyl-6-{1-[2-(4-phenyl-1-piperazinyl)ethan-1-ol]}-
oxazolo[4,5-b]pyridin-2(3H)-one (12a). To a solution of

L. Savelon et al./Bioorg. Med. Chem. 6 (1998) 1963±1973
1971
1H NMR (CDCl3+D2O) d 1.84±1.94 (m, 2H, CH2),
2.77 (dd, 2H, J=8.1, 7.0 Hz, CH2), 3.47 (s, 3H, NCH3),
3.70 (dd, 2H, J=7.0, 5.9 Hz, CH2), 7.29 (d, 1H,
J=1.5 Hz, H7), 7.98 (d, 1H, J=1.5 Hz, H5); IR (KBr) ꢀ
3400±3100, 1770 cm 1. Anal. C10H12N2O3 (C,H,N).
each mouse received an intraperitoneal injection of
0.25 mL of a solution containing 0.01% phenylquinone
in 5% ethanol. The number of writhes elicited in each
mouse during the period between the 5th and 25th minutes
after phenylquinone administration was recorded.19,20
3-Methyl-6-[3-(p-toluenesulfonyloxy)propyl]oxazolo[4,5-b]-
pyridin-2(3H)-one (15). This compound was prepared
according to the same experimental procedure described
for compound 7 using 14 as the starting material. Com-
pound 15 was obtained in 78% yield; mp 151±152 ꢀC;
1H NMR (CDCl3) 1.91±2.03 (m, 2H, CH2), 2.47 (s, 3H,
CH3), 2.77 (t, 2H, J=7.4 Hz, CH2), 2.47 (s, 3H, CH3),
2.77 (t, 2H, J=7.4 Hz, CH2), 3.46 (s, 3H, NCH3), 4.05
(t, 2H, J=7.4 Hz, CH2), 7.11 (d, 1H, J=1.5 Hz, H7),
7.36 (d, 2H, J=8.1 Hz, Harom), 7.79 (d, 2H, J=8.1 Hz,
The ratio of the mean writhes of the control animals
versus the mean writhes of the treated animals was cal-
culated, and the results are expressed as a percentage of
inhibition (Table 2). For the ED50 determination, eight
animals were used per dose and the 95% con®dence
limits were estimated using the probit method of Finney.21
Acetic acid-induced writhing in rats
Male Wistar rats in the weight range of 140±160 g were
used for the study after an overnight fast. The test
compounds were suspended in 0.5% carbomethyl cellu-
lose at each of the required doses immediately prior to
dosing. The rats were dosed orally with either the test
compound or the vehicle using a constant dose volume
of 10 mL/kg. For the screening evaluation at 50 mg/kg,
there were seven animals in the control group and ®ve
animals in each of the treated groups. Thirty minutes
after oral treatment, each rat received and intraper-
itoneal injection of 1.0 mL of a solution containing 1%
acetic acid in distilled water. The number of writhes eli-
cited in the following 25 min period was recorded.22 The
ratio of the mean writhes of the control animals versus
the mean writhes of the treated animals was calculated,
and the results are expressed as a percentage of inhibi-
tion (Table 2). For the ED50 determination, eight ani-
mals were used per dose and the 95% con®dence limits
were estimated using the probit method of Finney.21
H
arom), 7.88 (d, 1H, J=1.5 Hz, H5); IR (KBr) ꢀ
1790 cm 1. Anal. C17H18N2O5S (C,H,N).
3-Methyl-6-[3-(4-phenyl-1-piperazinyl)propyl]oxazolo[4,5-b]-
pyridin-2(3H)-one (16a). This compound was prepared
according to the same procedure described for product
8a using 15 as the starting material. Compound 16a was
obtained in 81% yield; amorphous solid; 1H NMR
(CDCl3) d 1.79±1.90 (m, 2H, CH2), 2.41 (t, 2H, J=7.4 Hz,
CH2), 2.59 (dd, 4H, J=5.1, 4.4 Hz, CH2piperaz), 2.71 (t,
2H, J=7.4 Hz, CH2), 3.21 (dd, 4H, J=5.1, 4.4 Hz,
CH2piperaz), 3.49 (s, 3H, NCH3), 6.85 (t, 1H, J=7.4 Hz,
Harom), 6.92 (d, 2H, J=8.1 Hz, Harom), 7.22±7.29 m (m,
3H, H7+Harom), 7.96 (d, 1H, J=15 Hz, H5); IR (KBr) ꢀ
1
1785 cm
C20H24N4O2 (C,H,N).
; MS (IC/NH3) m/z 353 (M+1). Anal.
3-Methyl-6-[3-(1-morpholino)propyl]oxazolo[4,5-b]pyridin-
2(3H)-one (16b). The compound 16b was prepared as
above from the tosylate 15 in 83% yield; amorphous
Evaluation of toxic, physiological, and behavioural
e€ects in mice (Irwin)
1
solid; H NMR (CDCl3) d 1.75±1.90 (q, 2H, J=7.4 Hz,
CH2), 2.37 (t, 2H, J=7.4 Hz, CH2), 2.44 (t, 4H,
J=4.4 Hz, CH2piperaz), 2.71 (t, 2H, J=7.4 Hz, CH2),
3.49 (s, 3H, NCH3), 3.74 (t, 4H, J=4.4 Hz, CHmorph),
7.28 (d, 1H, J=1.5 Hz, H7), 7.97 (d, 1H, J=1.5 Hz, H7);
IR (KBr) ꢀ 1790 cm 1; MS (IC/NH3) m/z 278 (M+1).
Anal. C14H19N3O3 (C,H,N).
Three animals per dose were treated po with the test
compound (dispersed in a 5% acacia gum suspension at
a volume of 0.25 mg/20 g) and observed through a stan-
dardised observation grid at regular intervals for up to
24 h. The presence or absence and the intensity of var-
ious symptoms were noted.23,24
Pharmacological methods
Receptor binding assays
Phenylquinone (PBQ)-induced writhing in mice. Male
CD1 mice in the weight range of 20±35 g were used for
the study after an overnight fast. The test compounds
were suspended in 0.5% carboxymethyl cellulose at each
of the required doses immediately prior to dosing. The
mice were dosed orally with either test compound or
vehicle using a constant dose volume of 10 mL/kg. For
the screening evaluation at 50 mg/kg, there were seven
animals in the control group and ®ve animals in each of
the treated groups. Thirty minutes after oral treatment,
Receptor binding assays were performed by incubating
membranes prepared from the rat central nervous sys-
tem with [3H]DAMGO, [3H]-p-Cl-Phe-DPDPE, respec-
tively, for receptors m and d.25,26 For receptors H1 and
H2, membranes were prepared from guinea pig cerebral
cortex and incubated with [3H]pyrilamine and [3H]tioti-
dine, respectively.27 For receptor K, membrane was
prepared from guinea pig cerebellum incubated with
[3H]U 69593.28 5HT1A assays used rat hippocampus

1972
L. Savelon et al./Bioorg. Med. Chem. 6 (1998) 1963±1973
membranes, [3H]-8-OH-DPAT, and buspirone for non-
speci®c binding (NSB),29 5HT1B assays used rat cortex,
4. (a) Caignard, D. H.; Couquelet, J.; Lesieur, D.; Lespagnol,
C.; Lamar, J. C.; Beaughard, M.; Leinot, M.; Tisne-Versailles,
J. Il Farmaco Ed. Sci. 1985, 40, 854. (b) Bonte , J. P. unpub-
lished results.
[
125I]CYP, and serotonin for NSB. 5HT2 assays used
calf frontal cortex, [3H]ketanserin, and spiperone for
NSB.30 5HT3 assays used NLE-115 cells, [3H]BRL
43694 for NSB.31 D1 assays used rat striatum, [3H]SCH
23390 for NSB.32 D2 assays used rat striatum, [3H]YM
09151-2 for NSB.33 For a1 and a2 receptors, membranes
were prepared from rat brain incubated respectively
with [3H]prazosin34 and [3H]rauwolscine.35
5. Flouzat, C.; Bresson, Y.; Mattio, A.; Bonnet, J.; Guillau-
met, G. J. Med. Chem. 1993, 36, 497.
6. Viaud, M. C.; Jamoneau, P.; Bizot-Espiard, J. G.; Pfei€er,
B.; Renard, P.; Caignard, D. H.; Adam, G.; Guillaumet, G.
Bioorg. Med. Chem. 1995, 3, 929.
7. Viaud, M. C.; Jamoneau, P.; Flouzat, C.; Bizot-Espiard, J.
G.; Pfei€er, B.; Renard, P.; Caignard, D. H.; Adam, G.; Guil-
laumet, G. J. Med. Chem. 1995, 38, 1278.
After the incubation period, bound and unbound radi-
oligands were separated by ®ltration. Radioactivity
bound to membranes in the absence and presence of
compounds was counted in a liquid scintillation coun-
ter. Triplicates of each compound were determined at
8. Viaud, M. C.; Jamoneau, P.; Baudin, M. L.; Savelon, L.;
Guillaumet, G. Tetrahedron 1997, 53, 5159.
9. Viaud, M. C.; Pavli, P.; Baudin, M. L.; Bizot-Espiard, J. G.;
Pfei€er, B.; Renard, P.; Guillaumet, G. Pharm. Sci. 1997, 3, 283.
10. Savelon, L.; Bizol-Espiard, J. G.; Caignard, D. H.; Pfei€er,
B.; Renard, P.; Viaud, M. C.; Guillaumet, G. Bioorg. Med
Chem. 1998, 6, 133.
7
two concentrations (10 and 10 5 M).
Cyclooxygenase and 5-lipoxygenase activities
11. Viaud, M. C.; Jamoneau, P.; Savelon, L.; Guillaumet, G.
Tetrahedron Lett. 1996, 37, 2409.
Isolated rabbit granulocytes were precubated during
15 min at 37 ꢀC with tested compound at a concentra-
tion of 10 5 M in DMSO. Calcic ionophore A 32187
(5Â10 6 M in DMSO) was added during 15 min. For
each compound, cycloxygenase and 5-lipoxygenase
inhibition were evaluated by dosing respectively PGE2
and LTB4 formation using the enzymoimmunoassay
(EIA) method.16 The reference compounds used were
indomethacine (IC50=2.7Â10 9 M) and NDGA
(IC50=4Â10 7 M), respectively, for inhibition of PGE2
and LTB4 formation.
12. Stille, J. K. Ang. Chem. Int. Ed. 1986, 25, 508.
13. Heck, R. F. Org. React. 1982, 27, 345.
14. Cabri, W.; Candiani, I.; Bedeshi, A.; Penco, S.; Santi, R. J.
Org. Chem. 1992, 57, 1481.
15. Parrain, J. L.; Duchene, A.; Quintard, J. P. J. Chem. Soc.
Perkin Trans. 1, 1990, 187.
16. Trush M. A.; Wilson, M. E.; Van Dyke, K. Methods
Enzymol. 1978, 57, 462.
17. Coulson, D. R.; Satek, L. C.; Grim, S. O. Inorg. Synth.
1978, 43, 121.
18. Musolf, M. C.; Speier, J. L. J. Org. Chem. 1964, 29, 2519.
19. Siegmund, E.; Cadmus, R.; Lu, G. Proc. Sol. Exp. Biol.
Med. 1957, 95, 729.
Acknowledgements
20. Pearl, J.; Stander, H.; McKean, D. B. J. Pharmacol. Exp.
Ther. 1968, 167, 9.
The phenylquinone- and acetic acid-induced writhing
tests were performed at the Hungtington Life Science
(Cambridgeshire, PE18 6ES, England). The receptor
binding assays were performed at CEREP (86600 Celle
l'Evescault, France). The general behaviour test was
performed at ITEM Labo (53940 Le Genest Sainte Isle,
France). The authors thank Mrs M. M. Le Floch for
typing the manuscript.
21. Finney, D. J. In the Probit Analysis; Cambridge University:
Cambridge, 1971 (3rd Edition).
22. 1. Koster, R.; Anderson, M.; Debeer, E. J. Fed. Proc. 1959,
18, 412.
23. Irwin, S. Psychopharmacologia, 1968, 13, 222.
24. Boissier, J. M.; Simon, P. Arch. Int. Pharmacodyn. 1965,
158, 212.
25. Yoburn, B. C.; Lutfy, K. Eur. J. Pharmacol. 1991, 193, 105.
26. Vaughn, L. K.; Knapp, R. J.; Toth, G.; Wan, Y. P.; Hruby,
V. J.; Yamamura, H. J. Life Sci. 1989, 45, 1001.
References
27. Gajtkowski, G. A.; Norris, D. B.; Rising, T. J.; Wood, T.
P. Nature (London) 1983, 304, 65.
1. Vaught, J. L.; Carson, J. R.; Carmoson, R. J.; Blum, P. S.;
Persico, F. J.; Hageman, W. E.; Hank, R. P.; Ra€a, R. B. J
Pharmacol. Exp. Ther. 1990, 255, 1.
28. Kinouchi, K.; Pasternak, G. W. Mol. Pharmacol. 1991,
207, 135.
2. Ja€e, J. H.; Maltin, W. R.; In The Pharmacological Basis of
Therapeutics; Goodman, L. S.; Gilman, A.; Rall, T. W.;
Nics, A. S.; Taylor, P., Eds; Pergamon: New York, 1990;
p 485.
29. Hall, M., Mestikawy, S., Emerit, M., Pichat, I., Hamon,
M., Gozlan, H. J. Neurochem. 1985, 44, 1685.
30. Leysen, J. E., Niemegeers, C. J. E., Van Nueten, J. M.,
Laduron, F. M. Mol. Pharmacol. 1982, 21, 301.
3. Clinch, D.; Banerjee, A. K.; Ostick, G.; Levy, D. W. J. R.
Coll. Physicians Lond. 1983, 17, 228.
31. Hoyer, D., Neijt, H. C. Mol. Pharmaco. 1988, 33, 303.

L. Savelon et al./Bioorg. Med. Chem. 6 (1998) 1963±1973
1973
32. Trampus, M., Ongini, E., Varani, K., Borea, A. Eur. J
Pharmacol. 1991, 194, 17.
34. Greengrass, P., Bremmer, R. Eur. J. Pharmacol. 1979, 55,
323.
33. Terai, M., Hidaka, K., Nakamura, Y. Eur. J. Pharmacol.
1989, 173, 177.
35. Diop, L., Dausse, J. P., Meyer, P. J. Neurochem. 1983, 41,
710.