Substituted pyrido[3,2-b]oxazin-3(4H)-ones: Synthesis and evaluation of antinociceptive activity

Article abstract of DOI:10.1016/S0968-0896(97)10005-0

A new series of N-substituted pyrido[3,2-b]oxazinones has been synthesized, pharmacologically evaluated, and compared with acetyl salicylic acid. The compound with the maximal combination of safety and analgesic efficacy was 4-{3-[4-(4-fluorophenyl-1-piperazinyl)propyl]}- 2H-pyrido[3,2-b]-1,4-oxazin-3(4H)-one (6c) with ED₅₀ values of 12.5 mg/kg po (mouse: phenylquinone writhing test) and 27.8 mg/kg po (rat: acetic acid writhing test), respectively. Compound 6c proved to be more active than aspirin with a safety index of 5.1.

Full text of DOI:10.1016/S0968-0896(97)10005-0

BMC 844p
BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 133±142
Substituted pyrido[3,2-b]oxazin-3(4H)-ones: synthesis and
evaluation of antinociceptive activity
L. Savelona, J. G. Bizot-Espiardb, D. H. Caignardc, B. Pfei€erc, P. Renardc,
M. C. Viauda*, G. Guillaumeta
a
Â
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 30 May 1997; accepted 6 August 1997
Abstract
A new series of N-substituted pyrido[3,2-b]oxazinones has been synthesized, pharmacologically evaluated, and com-
pared with acetyl salicylic acid. The compound with the maximal combination of safety and analgesic ecacy was 4-{3-
[4-(4-¯uorophenyl-1-piperazinyl)propyl]}-2H-pyrido[3,2-b]-1,4-oxazin-3(4H)-one (6c) with ED50 values of 12.5 mg/kg
po (mouse: phenylquinone writhing test) and 27.8 mg/kg po (rat: acetic acid writhing test), respectively. Compound 6c
proved to be more active than aspirin with a safety index of 5.1. # 1998 Elsevier Science Ltd. All rights reserved.
1. Introduction
the nitrogen atom at the 4-position or an acetyl moiety
at the 7-position.
The search for a new analgesic agents that are devoid
of side e€ects (such as tolerance, respiratory depression,
constipation, physical dependency, and fear of addic-
tion) typical of morphine-like opioid agonists, as well as
of the gastro-intestinal problems associated with
NSAIDs, has attracted signi®cant attention in recent
years. In this regard, a considerable number of benzox-
azolinone, benzoxazinone, oxazolopyridine, pyridoox-
azinone, and oxazolopyridinone derivatives, with
analgesic properties, has been reported [1±9]. Among
these types of compounds, benzoxazolinones A and B
[1,2], oxazolo[4,5-b]pyridin-2(3H)-ones C [8], and oxa-
zolo [5,4-b]pyridin-2(1H)-ones D [9] have emerged as
being of particular interest (Chart 1).
2. Chemistry
Starting materials 2 and 3, not commercially avail-
able, may be prepared by a method reported by Rufe-
nacht et al. [16] involving the condensation of 2-amino-
3-hydroxypyridine 1 and the appropriate chloroacetyl
chloride in the presence of sodium hydrogencarbonate
(Scheme 1).
Two di€erent general synthetic methods were
employed for the preparation of the desired derivatives
4, 5, 6, 10, 11, and 12 from the pyrido[3,2-b]oxazin-
3(4H)-ones 2 and 3.
In our continuing study of heteropolycyclic com-
pounds with potential biological activity [8±15], we
report here the synthesis and the biological evaluation
of a new series of substituted pyrido[3,2-b]oxazin-3(4H)-
ones having the general formula E (Chart 2). These
compounds possess a 4-arylpiperazinylalkyl chain on
The ®rst synthetic procedure (method A, Scheme 2)
was used to prepare products 4, 5, and 6 where n stands
for two or three carbons. The free nitrogen of the pyr-
ido[3,2-b]oxazin-3(4H)-ones was alkylated in anhydrous
N,N-dimethylformamide in the presence of sodium
ethoxide with (2-chloroethyl)- or (3-chloropropyl)amines,
which were prepared according to the procedure descri-
bed in the literature [17]. When the substituent at the
4-position had more than three carbons, this method
*Corresponding author.
0968-0896/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(97)10005-0

134
L. Savelon et al./Bioorg. Med. Chem. 6 (1998) 133±142
Chart 1.
Scheme 1.
Scheme 2. Method A.
was not satisfactory owing to the intramolecular cycli-
sation of the 4-(4-chlorobutyl)-1-arylpiperazines into
their spiro-fused quaternary chloride salt [18].
The second protocol (method B, Scheme 3) con-
veniently circumvented this obstacle. The anions of
compounds 2 and 3 reacted with dibromoalkanes in
DMF to provide 7, 8, and 9 in satisfactory yields.
Compounds 10, 11, and 12 were prepared in excellent
yields by alkylation of amine intermediates with the
appropriate bromo compounds 7, 8, and 9 in an aprotic
solvent such as acetonitrile in the presence of diisopropyl-
ethylamine.
Chart 2.

L. Savelon et al./Bioorg. Med. Chem. 6 (1998) 133±142
135
Preparation of acetyl derivative 15 is reported in
Scheme 4 (method C). Treatment of 2 with bromine
in DMF, as solvent, according to a procedure described
in oxazolopyridine series [19] provides with a 80% yield
compound 13. This compound was converted by reac-
tion with sodium hydride in dry N,N-dimethylforma-
mide at room temperature into its anion, which reacted
with iodomethane to provide 14 in good yield. Com-
pound 15 was obtained satisfactorily from 14 under a
variety of normal Heck conditions [20]. Reaction of 14
with butylvinyl ether in DMF and triethylamine as base,
in the presence of 1,2-bis(diphenyl phosphino)ethane as
ligand, gave, after acid hydrolysis [21], the acetyl deri-
vative 15 in 81% yield.
Table 1 summarizes the experimental and physical
data for the desired compounds. General procedures
detailing the synthesis of target stuctures are reported in
the Experimental section.
Scheme 3. Method B.
Scheme 4. Method C.

136
L. Savelon et al./Bioorg. Med. Chem. 6 (1998) 133±142
3. Biological results and discussion
studied for their analgesic activity 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 ratios of activity versus
aspirin to correct for slight variations in response
between the same evaluation procedures not performed
simultaneously (Table 2).
According to the structure±activity relationships we
previously established within the oxazolo[4,5-b]pyridin-
(3H)-ones [8] and oxazolo[5,4-b]pyridin-2(1H)-ones [9]
series, only substituted or unsubstituted 4-(4-phenylpiper-
azinylalkyl)pyrido[3,2-b)oxazin-3(4H)-ones (12 com-
pounds) were synthesized and evaluated. All were ®rst
Table 1
1-[4-Arylpiperazinyl)alkyl]pyrido[3,2-b]oxazin-3(4H)-ones
Compound
n
2
W
H
R
H
A
Methoda Yield (%)b mp (ꢀC) Solventc
Formula
Anal.d
C,H,N
4a
A
91
oil
C19H22N4O2
4b
4c
5
2
2
2
3
H
H
H
H
H
H
A
A
A
A
92
92
89
94
oil
C20H21F3N4O2
C19H21FN4O2
C25H26N4O2
C20H24N4O2
C,H,N
C,H,N
C,H,N
C,H,N
109±110 i-PrOH
Ph
H
oil
oil
6a
6b
3
3
4
H
H
H
H
H
H
A
A
B
70
89
96
93±94 i-PrOH
103±104 i-PrOH
86±87 i-PrOH
C21H23F3N4O2
C20H23FN4O2
C21H26N4O2
C,H,N
C,H,N
C,H,N
6c
10a
10b
10c
11
4
4
4
5
H
H
H
H
H
H
B
B
B
B
C
95
91
90
95
81
85±86 i-PrOH
69±70 i-PrOH
94±95 i-PrOH
71±72 i-PrOH
183±184 i-PrOH
C22H25F3N4O2
C21H25FN4O2
C27H30N4O2
C22H28N4O2
C10H10N2O3
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
Ph
H
12
15
0 CH3CO
H
CH3
aMethod A is shown in Scheme 2; method B is shown in Scheme 3; method C is shown in Scheme 4; see the Experimental section.
bIsolated yields of pure products; no e€orts were made to optimize yields. For compound 15, the yield is calculated from 14.
cSolvent of recrystallization.
dAnalytical results were within ‹0.4% of the theoretical value.

L. Savelon et al./Bioorg. Med. Chem. 6 (1998) 133±142
137
This preliminary evaluation shows that among the
unsubstituted 4-(4-phenylpiperazinylalkyl)pyrido[3,2-
b]oxazin-3(4H)-ones (4a, 6a, 10a, and 12), the maximal
activity is obtained when the alkyl spacer is a butyl
function (10a).
Only one pyrido[3,2-b]oxazin-3(4H)-one acylated at
7-position was synthesized and evaluated. This com-
pound (15), which was devoid of any basic amino moi-
ety, was found half as potent as aspirin.
Seven compounds (4a, 4b, 5, 6a, 6b, 6c, and 10a) were
found to be more potent than aspirin itself on the basis
of this preliminary evaluation. One of them, compound
6c, which is protected of metabolic hydroxylation by the
mean of the ¯uor group in para position, was selected
for further evaluation.
Its ED50 were 12.5 (8.7±18.2) mg/kg po and 27.8
(18.1±42.7) mg/kg po in the PBQ induced and the acetic
acid induced writhing tests, respectively, compared to 63
(56±75) mg/kg po and 32 (28±46) mg/kg po for acetyl
salicylic acid.
Compound 6c proved also to be signi®cantly active in
the hot-plate test performed in mice where it increases
the foot licking latency of 5.7‹0.3 s (p<0.01) at 32 mg/
kg po compared to 9.3‹1.2 s (p<0.001) at 16 mg/kg ip
for morphine.
Substitution of the phenyl ring of the phenyl piper-
azine moiety by a m-tri¯uoromethyl group (4b, 6b, 10b)
or a p-¯uorine (4c, 6c, 10c) leads to activity levels that
are surprisingly abnormally low for compounds 4c and
10b. As the results for this two compounds appear illo-
gical, and also in discord with those previously obtained
[8,9], it is reasonable not to take them into account for
the present discussion.
Once that is done, we can conclude that substitution
with a m-tri¯uoromethyl group (4b, 6b) appeared to
have only little e€ect on analgesic activity with a slight
improvement for 4b compared to the unsubstituted 4a
(n ˆ 2) and a slight decrease for 6b compared to 6a
(n ˆ 3). Substitution with a p-¯uorine (6c, 10c) results in
a slight improvement for 6c (n ˆ 3) and a clear decrease
for 10c (n ˆ 4).
Its oral general acute toxicity as well as its beha-
vioural e€ects were investigated via an IRWIN test per-
formed in mice (a careful observation of the general
behaviour is also useful to avoid possible false positives
in the writhing tests).
The ®rst behavioural changes are sedation and hypo-
thermia that appear at 64 mg/kg po while the mortality
threshold dose is higher than 1024 mg/kg po.
Direct substitution of the pyrido[3,2-b]oxazine ring
with a 2-phenyl group has almost no e€ect on analgesic
activity when n ˆ 2 (5). When n ˆ 4, compound 11
retains a good activity in the acetic acid-induced writhing
test (82% inhibition, activity ratio of 1.45) but appears
clearly less active in the PBQ test (22% inhibition, activity
ratio of 0.35). This discrepancy between the results of
the two writhing tests could be explained by di€erences
in the metabolism of compound 11 in rat and mouse.
An oral safety index was de®ned as the ratio of the
ED50 po in mice with the dose of appearance of the ®rst
Table 2
Analgesic activity screening of 1-[(4-arylpiperazinyl)alkyl]pyrido[3,2-b]oxazin-3(4H)-ones
Phenylquinone (PBQ)-induced writhing test (mice)
Compound
Acetic acid-induced writhing test (rat)
% inhibition at
50 mg/kg poa
Activity ratio
versus aspirinb
% inhibition at
50 mg/kg poa
Activity ratio
versus aspirinb
4ac
4bc
4c
60%**e
74%**
39%***
54%**
71%**
86%***
75%***
97%***
18%
1.03
1.28
0.76
1
64%**
68%**
12%
48%**
75%**
94%***
91%***
100%***
NDd
71%**
82%**
97%**
28%
1.03
1.09
0.18
1.22
1.21
1.05
1.40
>1.6
Ð
5c
6ac
6b
1.22
1.25
1.47
1.67
0.35
1
6c
10a
10b
10c
11
69%***
0.79
1.45
1.73
0.43
22%
0.35
0.85
0.57
12
15
53%*
29%
aFive animals were used for each compound, seven animals for the control group.
% of inhibition with compound at 50 mg=kp po
bActivity ratio:
% of inhibition with aspirin at 50 mg=kg po
cThese compounds were tested as oxalate.
dND : Not determined.
*p<0.05; **p<0.01; ***p<0.001.

138
L. Savelon et al./Bioorg. Med. Chem. 6 (1998) 133±142
behavioural changes in the IRWIN test. This safety
index is relatively good for 6c with a value of 5.1 if PBQ
induced writhing assay in taken into account.
Aldrich Chimie. All solutions were dried over anhy-
drous magnesium sulfate and evaporated on a Buchi
rotatory evaporator. Analytical thin-layer chromato-
graphy (TLC) was carried out on precoated plates (silica
gel, 60 F254), 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 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 ele-
ments were ‹0.4% of the theoretical values. All anhy-
drous reactions were performed in oven dried glassware
under an atmosphere of argon. The column chromato-
graphy solvents employed were distilled and solvent
mixtures were reported as volume to volume ratios.
In order to investigate possible mechanism of its
action, binding studies were performed and they showed
absence of signi®cant anity for the ꢀ, ꢁ and K opioid
receptors. A small anity was observed for the histamine
H1 and H2 receptors with inhibition of respectively 99%
at 10 5 M and 29% at 10 7 M for H1 and 36% at 10 5 M
7
and 19% at 10 M for H2 receptor. This could explain
the sedative e€ects observed in the IRWIN test.
However a moderate anity was observed for the
serotoninergic 5-HT1A and 5-HT2 receptors with inhi-
5
7
bition of respectively 97% at 10 M and 32% at 10
5
7
M for 5-HT1A and 92% at 10 M and 67% at 10 M
for 5-HT2. Similarly, a mild anity was obtained for the
adrenergic ꢂ1 and ꢂ2 receptors with inhibition of
5
7
respectively 99% at 10 M and 56% at 10 M for ꢂ1
and 100% at 10
5.1.1 2H-Pyrido[3,2-b]-1,4-oxazin-3(4H)-one (2)
2-Amino-3-hydroxypyridine 1 (5.00 g, 45 mmol) was
added to a suspension of sodium hydrogenocarbonate
(9.10 g, 108 mmol) in a mixture of water (30 ml) and
2-butanone (30 ml). After cooling the solution to 5ꢀC,
chloroacetyl chloride (4.2 ml, 51 mmol) diluted with
methylethylketone (10 ml) was added dropwise and
slowly (for 1 h) so that temperature did not exceed 10ꢀC.
The mixture was stirred at room temperature for 30 min
and then at 75ꢀC for 30 min. The mixture was cooled
and the solvent removed. The resulting precipitate was
collected by ®ltration, washed with water, and dried
over P2O5 under vacuum. The product 2 was recris-
tallized from CH3OH/H2O (1/1:v/v) and obtained in a
yield of 67%; mp 204±205ꢀC (CH3OH/H2O:1/1) [lit
[24].: 205±206ꢀC]; IR (KBr) ꢃ 3200±2400, 1700 cm 1; 1H
NMR (CDCl3+D2O) ꢁ 4.68 (s, 2H, OCH2), 6.97 (dd,
1H, H7, J=8.1, 5.2), 7.27 (dd, 1H, H8, J=8.1, 1.5), 8.05
(dd, 1H, H6, J=5.2, 1.5).
5
7
M and 31% at 10
M for ꢂ2.
According to these values, the antinociceptive activity
could perhaps result from serotoninergic [22] and/or
adrenergic [23] e€ects.
4. Conclusion
In conclusion, within this class of pyrido[3,2-b]oxazin-
3(4H)-ones, we have shown that some compounds pos-
sess potent nonopioid antinociceptive activity. One of
the most active compound of this family, 4-{3-[4-
(4-¯uorophenyl-1-piperazinyl)propyl]}-2H-pyrido[3,2-b]-
1,4-oxazin-3(4H)-one 6c proves to be more active than
aspirin with a safety index of 5.1.
Complementary safety pharmaceutical evaluation is
currently under investigation to determine if the com-
pound could be a good candidate for clinical develop-
ment.
5.1.2 2-Phenyl-2H-pyrido[3,2-b]-1,4-oxazin-3(4H)-one
(3)
5. Experimental
Compound 3 was prepared by the same procedure as
was used for 2, using 2-chloro-2-phenylacetyl chloride
(9.64 g, 51 mmol) as the starting reagent, and obtained
5.1 Chemistry
in a yield of 45%; mp 196±198ꢀC (CH3OH/H2O:1/1);
1
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 (ꢁ, 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
IR (KBr)
ꢃ
3200±2400, 1690 cm
;
1H NMR
(CDCl3+D2O) ꢁ 5.75 (s, 1H, OCH), 6.97 (dd, 1H, H7,
J=7.9, 5.4), 7.30±7.47 (m, 6H, H8+Harom), 8.02 (dd,
1H, H6, J=5.4, 1.3). Anal. C13H10N2O2 (C,H,N).
5.2 4-[n-(4-Aryl-1-piperazinyl)alkyl]-2H-pyrido[3,2-
b]-1,4-oxazin-3(4H)-ones and 4-(n-amino-alkyl)-2H-
pyrido[3,2-b]-1,4-oxazin-3(4H)-ones. General
procedure
5.2.1 Method A (Scheme 1)
Compound 2 (150 mg, 1.0 mmol) was added to a
solution of sodium ethylate in ethanol (prepared with

L. Savelon et al./Bioorg. Med. Chem. 6 (1998) 133±142
139
sodium, 28 mg, 1.2 mmol, in anhydrous ethanol, 6 ml).
The mixture was stirred at room temperature for 1 h and
then ethanol was removed under reduced pressure. The
anion was dissolved in N,N-dimethylformamide (10 ml)
and the solution treated with (2-chloroethyl)- or (3-
chloropropyl)amine (1.1 mmol) added dropwise and
then re¯uxed 2 h. After cooling, the solvent was
removed and the crude mixture was treated with water
and extracted with methylene chloride. The organic
layers were dried over magnesium sulfate, evaporated,
and chromatographied on silica gel (eluent: CH2Cl2/
CH3OH: 8/2).
4.23 (t, 2H, CONCH2, J=7.4), 4.66 (s, 2H, OCH2), 6.84
(t, 1H, Harom, J=6.6), 6.90±6.95 (m, 3H, H7+Harom),
7.19±7.32 (m, 3H, H8+Harom), 8.00 (dd, 1H, H6, J=5.3,
1.3); MS (IE) m/z 352 (M+).
5.2.1.6 4-{3-[4-(3-Trifluoromethyl)phenyl-1-piperazinyl]
propyl}-2H-pyrido[3,2-b]-1,4 -oxazin-3(4H)-one (6b).
IR (KBr) ꢃ 1690 cm 1; 1H NMR (CDCl3) ꢁ 1.94 (q, 2H,
CH2, J=7.7), 2.52 (t, 2H, CH2, J=7.7), 2.62 (t, 4H,
2ÂCH2piperaz
,
J=5.5), 3.21 (t, 4H, 2ÂCH2piperaz
,
J=5.5), 4.23 (t, 2H, CONCH2, J=7.7), 4.65 (s, 2H,
OCH2), 6.92 (dd, 1H, H7, J=8.2, 4.6), 7.01±7.11 (m,
3H, Harom), 7.21 (dd, 1H, H8, J=8.2, 1.5), 7.32 (t, 1H,
5.2.1.1
ido[3,2-b]-1,4-oxazin-3(4H)-one (4a). IR (neat)
1690 cm
4-[2-(4-Phenyl-1-piperazinyl)ethyl]-2H-pyr-
ꢃ
Harom, J=8.2), 8.01 (dd, 1H, H6, J=4.6, 1.5); MS (IC/
NH3) m/z 421 (M+1).
1
;
1H NMR (CDCl3) ꢁ 2.68±2.75 (m, 6H,
CH2+2ÂCH2piperaz), 3.15 (t, 4H, 2ÂCH2piperaz, J=5.1),
4,34 (t, 2H, CONCH2, J=6,2), 4,66 (s, 2H, OCH2), 6.83
(t, 1H, Harom, J=7.1), 6.88±6.94 (m, 3H, H7, Harom),
7.19±7.28 (m, 3H, H8+Harom), 8.01 (dd, 1H, H6, J=5.0,
1.2); MS (IE) m/z 338 (M+).
5.2.1.7 4-{3-[4-(4-Fluorophenyl-1-piperazinyl)propyl]}-
2H-pyrido[3,2-b]-1,4-oxazin-3 (4H)-one (6c). IR (KBr)
1
ꢃ 1690 cm
;
1H NMR (CDCl3) ꢁ 1.94 (q, 2H, CH2,
J=7.3), 2.51 (t, 2H, CH2, J=7.3), 2.60 (t, 4H,
2ÂCH2piperaz
,
J=5.1), 3.10 (t, 4H, 2ÂCH2piperaz
,
J=5.1), 4.22 (t, 2H, CONCH2, J=7.3), 4.65 (s, 2H,
OCH2), 6.83±6.98 (m, 5H, H7+Harom), 7.21 (dd, 1H,
H8, J=7.9, 1.4), 8.00 (dd, 1H, H6, J=4.7, 1.4); MS (IC/
NH3) m/z 371 (M+1).
5.2.1.2 4-{2-[4-(3-Trifluoromethyl)phenyl-1-piperazin-
yl]ethyl}-2H-pyrido[3,2-b]-1,4-oxazin-3(4H)-one (4b).
1
IR (neat) ꢃ 1690 cm
;
1H NMR (CDCl3) ꢁ 2.66±2.76
(m, 6H, CH2+2ÂCH2piperaz), 3.19 (t, 4H, 2ÂCH2piperaz
,
J=5.4), 4.33 (t, 2H, CONCH2, J=7.0), 4.65 (s, 2H,
OCH2), 6.92 (dd, 1H, H7, J=7.7, 4.9), 7.00±7.07 (m,
2H, Harom), 7.08 (s, 1H, Harom), 7.22 (d, 1H, H8,
J=7.7), 7.32 (t, 1H, Harom, J=7.7), 8.01 (dd, 1H, H6,
J=4.9, 0.7); MS (IE) m/z 406 (M+).
5.2.2 Method B (Scheme 3) 4-(n-Bromoalkyl)-2H-
pyrido[3,2-b]-1,4-oxazin-3(4H)-ones. General
procedure
To a stirred solution of pyrido[3,2-b]oxazin-3(4H)-
one 2 or 3 (1 mmol) in DMF (10 ml) was added NaH
(26 mg, 1.1 mmol, 60% in dispersion in oil) at room
temperature. The mixture was stirred for 1 h at the same
temperature. Then the 1,4-dibromobutane or 1,5-dibro-
mopentane (1.1 mmol) diluted with DMF was added
dropwise. The reaction mixture was heated at 110ꢀC for
2 h, cooled to room temperature, and concentrated in
vacuo. After addition of water (100 ml), the mixture was
extracted with CH2Cl2. Organic layers were dried with
anhydrous magnesium sulfate and the solvent was
removed under vacuum. Puri®cation by column chro-
matography (eluent:CH2Cl2) gave the pure product.
5.2.1.3 4-{2-[4-(4-Fluorophenyl-1-piperazinyl)ethy]}-2H-
pyrido[3,2-b]-1,4-oxazin-3(4H)-one (4c). IR (KBr) ꢃ
1
1690 cm
;
1H NMR (CDCl3) ꢁ 2.70±2.75 (m, 6H,
CH2+2ÂCH2piperaz), 3.07 (t, 4H, 2ÂCH2piperaz, J=5.2),
4.34 (t, 2H, CONCH2, J=7.1), 4.67 (s, 2H, OCH2),
6.82±6.99 (m, 5H, H7+Harom), 7.22 (dd, 1H, H8, J=7.4,
1.5), 8.01 (dd, 1H, H6, J=4.4, 1.5); MS (IC/NH3) m/z
357 (M+1).
5.2.1.4 2-Phenyl-4-[2-(4-phenyl-1-piperazinyl)ethyl]-2H-
pyrido[3,2-b]-1,4-oxazin-3(4H)-one (5). IR (neat) ꢃ
1
1690 cm
;
1H NMR (CDCl3) ꢁ 2.64±2.88 (m, 6H,
5.2.2.1 4-(4-Bromobutyl)-2H-pyrido[3,2-b]-1,4-oxazin-
1
CH2+2ÂCH2piperaz), 3.13 (t, 4H, 2ÂCH2piperaz, J=4.7),
3(4H)-one (7). Yield 83%; Oil; IR (neat) ꢃ 1690 cm
;
4.25±4.36 (m, 1H, CONCH), 4.42±4.53 (m, 1H,
CONCH), 5.77 (s, 1H, OCH), 6.82 (t, 1H, Harom
1H NMR (CDCl3) ꢁ 1.78±2.02 (m, 4H, 2ÂCH2), 3.45 (t,
2H, CH2Br, J=6.6), 4.18 (t, 2H, NCH2, J=6.6), 4.66 (s,
2H, OCH2), 6.93 (dd, 1H, H7, J=7.9, 4.8), 7.22 (dd, 1H,
H8, J=7.9, 1.5), 8.01 (dd, 1H, H6, J=4.8, 1.5). Anal.
C11H13BrN2O2 (C,H,N).
,
J=7.4), 6.87±6.95 (m, 5H, H7+Harom), 7.21±7.46 (m,
6H, H8+Harom), 8.00 (dd, 1H, H6, J=4.4, 1.3); MS (IE)
m/z 414 (M+).
5.2.1.5 4-[3-(4-Phenyl-1-piperazinyl)propyl]-2H-pyrido
[3,2-b]-1,4-oxazin-3(4H)-one (6a). IR (neat)
5.2.2.2 4-(4-Bromobutyl)-2-phenyl-2H-pyrido[3,2-b]-1,4-
oxazin-3(4H)-one (8). Yield 68%; Oil; IR (neat) ꢃ
ꢃ
1
1
1690 cm
;
1H NMR (CDCl3) ꢁ 1.94 (q, 2H, CH2,
1690 cm
;
1H NMR (CDCl3) ꢁ 1.72±2.00 (m, 4H,
J=7.4), 2.51 (t, 2H, CH2, J=7.4), 2.57 (t, 4H,
CH2piperaz, J=5.3), 3.19 (t, 4H, 2ÂCH2piperaz, J=5.3),
2ÂCH2), 3.34±3.50 (m, 2H, CH2Br), 4.05±4.30 (m, 2H,
NCH2), 5.75 (s, 1H, OCH), 6.87 (dd, 1H, H7, J=8.1,

140
L. Savelon et al./Bioorg. Med. Chem. 6 (1998) 133±142
4.4), 7.17±7.35 (m, 6H, H8,+Harom), 7.93 (dd, 1H, H6,
J=4.4, 1.5). Anal. C17H17BrN2O2 (C,H,N).
1H, H8, J=8.1, 1.5), 8.01 (dd, 1H, H6, J=5.5, 1.5); MS
(IC/NH3) m/z 385 (M+1).
5.2.2.3 4-(5-Bromopentyl)-2H-pyrido[3,2-b]-1,4-oxazin-
3(4H)-one (9). Yield 80%; mp 76±78ꢀC; IR (KBr) ꢃ
5.2.3.4 2-Phenyl-4-[4-(4-phenyl-1-piperazinyl)butyl]-2H-
pyrido[3,2-b]-1,4-oxazin-3(4H)-one (11). IR (KBr) ꢃ
1
1690 cm
;
1H NMR (CDCl3) ꢁ 1.47±1.58 (m, 2H,
1
CH2), 1.63±1.78 (m, 2H, CH2), 1.87±1.98 (m, 2H, CH2),
3.41 (t, 2H, CH2Br, J=6.6), 4.14 (t, 2H, NCH2, J=6.6),
4.65 (s, 2H, OCH2), 6.93 (dd, 1H, H7, J=8.1, 4.4), 7.21
(dd, 1H, H8, J=8.1, 1.5), 8.01 (dd, 1H, H6, J=4.4, 1.5).
Anal. C12H15BrN2O2 (C,H,N).
1690 cm
;
1H NMR (CDCl3) ꢁ 1.47±1.61 (m, 2H,
CH2), 1.66±1.78 (m, 2H, CH2), 2.38 (t, 2H, CH2,
J=7.4), 2.52 (t, 4H, 2ÂCH2piperaz, J=4.9), 3.13 (t, 4H,
2ÂCH2piperaz, J=4.9), 4.09±4.25 (m, 2H, CONCH2),
5.69 (s, 1H, OCH), 6.78 (t, 1H, Harom, J=7.1), 6.83±
6.89 (m, 3H, H7+Harom), 7.16±7.32 (m, 8H,
H8+Harom), 7.94 (d, 1H, H6, J=4.4); MS (IC/NH3) m/z
443 (M+1).
5.2.3 4-[n-(4-Aryl-1-piperazinyl)alkyl]-2H-pyrido[3,2-
b]-1,4-oxazin-3(4H)-ones and 4-(n-amino-alkyl)-2H-
pyrido[3,2-b]-1,4-oxazin-3(4H)-ones. General procedure
A mixture containing the bromo compound 7, 8, or 9
(1 mmol), the appropriate amine (1.5 mmol), diisopro-
pylethylamine (193 mg, 1.5 mmol), and CH3CN (10 ml)
was heated at 60ꢀC for 6 h. After cooling, the mixture
was concentrated in vacuo. The resulting residue was
poured into water, extracted with CH2Cl2, and dried
over anhydrous magnesium sulfate. After evaporation
of the solvent under vacuum, the desired product was
puri®ed by column chromatography (eluent: CH2Cl2/
MeOH: 95/5).
5.2.3.5
[3,2-b]-1,4-oxazin-3(4H)-one (12). IR (KBr)
4-[5-(Phenyl-1-piperazinyl)pentyl]-2H-pyrido
ꢃ
1690 cm
CH2), 1.54±1.64 (m, 2H, CH2), 1.67±1.77 (m, 2H, CH2),
1
;
1H NMR (CDCl3) ꢁ 1.38±1.46 (m, 2H,
2.39 (t, 2H, CH2, J=7.7), 2.59 (t, 4H, 2ÂCH2piperaz
,
J=5.0), 3.20 (t, 4H, 2CH2piperaz, J=5.0), 4.14 (t, 2H,
CONCH2, J=7.7), 4.65 (s, 2H, OCH2), 6.84 (t, 1H,
Harom, J=7.4), 6.89±6.94 (m, 3H, H7+Harom), 7.19±
7.28 (m, 3H, H8+Harom), 8.01 (dd, 1H, H6, J=5.2, 1.5);
MS (IC/NH3) m/z 381 (M+1).
5.2.3.1 4-[4-(4-Phenyl-1-piperazinyl)butyl]-2H-pyrido
[3,2-b]-1,4-oxazin-3(4H)-one (10a). IR (KBr)
5.2.4 Method C (Scheme 4)
ꢃ
1
1690 cm
;
1H NMR (CDCl3) ꢁ 1.54±1.68 (m, 2H,
5.2.4.1 7-Bromo-2H-pyrido[3,2-b]-1,4-oxazin-3(4H)-one
(13). To a stirred solution of 2H-pyrido[3,2-b]-1,4-oxa-
zin-3(4H)-one 2 (5.32 g, 23.25 mmol) in DMF (100 ml)
was added bromine (1.28 ml, 25.58 mmol) slowly at
room temperature and the reaction was stirred at this
temperature over 2 h. Addition of water (50 ml) allowed
the precipitation of compound 13. After ®ltration,
washing with water, and drying, 13 was isolated pure in
CH2), 1.68±1.82 (m, 2H, CH2), 2.46 (t, 2H, CH2,
J=7.6), 2.62 (t, 4H, 2ÂCH2piperaz, J=5.1), 3.22 (t, 4H,
2ÂCH2piperaz, J=5.1), 4.20 (t, 2H, CONCH2, J=7.6),
4.68 (s, 2H, OCH2), 6.85 (t, 1H, Harom, J=5.5), 6.90±
7.00 (m, 3H, H7+Harom), 7.20±7.30 (m, 3H,
H8+Harom), 8.01 (dd, 1H, H6, J=5.3, 1.3); MS (IE) m/z
366 (M+).
80% of yield; mp 224-226ꢀC; IR (KBr) ꢃ 3200±3100,
1
5.2.3.2 4-{4-[4-(3-Trifluoromethyl)phenyl-1-piperazin-
yl]butyl}-2H-pyrido[3,2-b]-1,4-oxazin-3(4H)-one
(10b). IR (KBr) ꢃ 1690 cm 1; 1H NMR (CDCl3) ꢁ 1.53±
1.67 (m, 2H, CH2), 1.67±1.79 (m, 2H, CH2), 2.43 (t, 2H,
CH2, J=7.3), 2.58 (t, 4H, 2ÂCH2piperaz, J=6.5), 3.22 (t,
4H, 2ÂCH2piperaz, J=6.5), 4.17 (t, 2H, CONCH2,
J=7.3), 4.69 (s, 2H, OCH2), 6.94 (dd, 1H, H7, J=7.7,
4.7), 7.05±7.14 (m, 2H, Harom), 7.16 (s, 1H, Harom),
1700 cm
;
1H NMR (DMSO+D2O) ꢁ 4.63 (s, 2H,
OCH2), 7.60 (d, 1H, H8, J=1.6); 7.84 (d, 1H, H6,
J=1.6); MS (IC/NH3) m/z 229 (M+1). Anal
C7H5BrN2O2 (C,H,N).
5.2.4.2 7-Bromo-2H-4-methylpyrido[3,2-b]-1,4-oxazin-
3(4H)-one (14). To a stirred solution of 7-bromo-2H-
pyrido[3,2-b]-1,4-oxazin-3(4H)-one 13 (458 mg, 2 mmol)
in DMF (10 ml) was added sodium hydride (53 mg,
2.20 mmol, 80% in dispersion in oil) at room tempera-
ture and the reaction was stirred at this temperature for
1 h. A solution of iodomethane (426 mg, 3.0 mmol) in
DMF (0.5 ml) was then added slowly to the reaction.
Then the mixture was stirred 2 h at 110ꢀC. After cooling
and concentration, the resulting residue was poured into
water, extracted with CH2Cl2, and dried over magne-
sium sulfate. After evaporation the resulting product
was puri®ed by silica gel column chromatography (elu-
ent: CH2Cl2) to provide 87% of 14 as a crystalline
7.22 (dd, 1H, H8, J=7.7, 1.2), 7.38 (t, 1H, Harom
J=8.3), 8.00 (dd, 1H, H6, J=4.7, 1.2); MS (IE) m/z 435
,
(M+).
5.2.3.3 4-{4-[4-(4-Fluorophenyl-1-piperazinyl)butyl]}-2H-
pyrido[3,2-b]-1,4-oxazin-3(4H)-one (10c). IR (KBr) ꢃ
1
1690 cm
;
1H NMR (CDCl3) ꢁ 1.55±1.67 (m, 2H,
CH2), 1.68±1.80 (m, 2H, CH2), 2.45 (t, 2H, CH2,
J=7.4), 2.58 (t, 4H, 2ÂCH2piperaz, J=4.9), 3.12 (t, 4H,
2ÂCH2piperaz, J=4.9), 4.18 (t, 2H, CONCH2, J=7.4),
4.67 (s, 2H, OCH2), 6.83±6.98 (m, 5H, Harom), 7.21 (dd,

L. Savelon et al./Bioorg. Med. Chem. 6 (1998) 133±142
141
product; mp 130±132ꢀC; IR (KBr) ꢃ 1690 cm
;
1H
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 [28].
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
[27].
1
NMR (CDCl3) ꢁ 3.44 (s, 3H, NCH3), 4.68 (s, 2H,
OCH2), 7.35 (d, 1H, H8, J=1.5), 8.04 (d, 1H, H6,
J=1.5). Anal. C8H7BrN2O2 (C,H,N).
5.2.4.3 (2H-4-Methyl-3-oxopyrido[3,2-b]-1,4-oxazin-7-
yl)ethanone (15). To a stirred solution of 6-bromo-2H-
4-methylpyrido[3,2-b]-1,4-oxazin-3(4H)-one 14 (530 mg,
2.18 mmol) in N,N-dimethylformamide (5 ml) were
added triethylamine (0.44 g, 4.36 mmol), butylvinyl
ether (1.2 g, 12 mmol), 1,2-bis(diphenylphosphino)
ethane (24 mg, 0.06 mmol), and palladium (II) acetate
(12 mg, 0.054 mmol). The mixture was heated at re¯ux,
under argon, for 8 h. After cooling to room tempera-
ture, HCl (10%) was added and after 1 h of stirring, the
mixture was concentrated under reduced pressure. The
residue was partitioned between CH2Cl2 and water. The
organic layer was dried over MgSO4 and concentrated.
Puri®cation by ¯ash chromatography (eluent: CH2Cl2/
AcOEt: 8/2) furnished 81% of 15 as a crystalline pro-
5.3.3 Hot-plate test in mice
According to the method described by Eddy [29],
mice were placed on a heated plate (55ꢀC) inside a
Plexiglass cylinder. The latency before the animals star-
ted to lick their feet was measured. If no reaction was
noted, the test was terminated after 120 s. Ten animals
were studied per dose (dispersed in a 5% acacia gum
suspension at a volume of 0.25 mg/20 g). The compound
was administered 1 h (po) or 30 min (ip) before the test.
1
duct; IR (KBr) ꢃ 1690 cm 1; H NMR (CDCl3) ꢁ 2.59
(s, 3H, CH3), 3.53 (s, 3H, NCH3), 4.72 (s, 2H, OCH2),
7.74 (d, H8, J=1.4), 8.61 (d, H6, J=1.4); MS (IC/NH3)
m/z 207 (M+1).
5.3 Pharmacological methods
5.3.4 Evaluation of toxic, physiological, and behavioural
e€ects in mice (Irwin)
5.3.1 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, each mouse received and intraper-
itoneal 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 adminis-
tration was recorded [25,26].
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 [30,31].
5.3.5 Receptor binding assay
Receptor binding assays were performed by incubat-
ing membranes prepared from the rat central nervous
system with [3H]DAMGO, [3H]-p-Cl-Phe-DPDPE,
respectively, for receptors ꢀ and ꢄ [32,33]. For receptors
H1 and H2, membranes were prepared from guinea pig
cerebral cortex and incubated with [3H]pyrilamine and
[3H]tiotidine, respectively [34]. For receptor K, mem-
brane was prepared from guinea pig cerebellum incu-
bated with [3H]U 69593 [35]. 5-HT1A assays used rat
hippocampus membranes, [3H]-8-OH-DPAT, and bus-
pirone for non speci®c binding (NSB) [36], 5-HT2 assays
used calf frontal cortex, [3H]ketanserin, and spiperone
for NSB [37]. For ꢂ1 and ꢂ2 receptors, membranes were
prepared from rat brain incubated respectively with
[3H]prazosin [38] and [3H]rauwolscine [39].
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 [27].
5.3.2 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
After the incubation period, bound and unbound
radioligands were separated by ®ltration. Radioactivity
bound to membranes in the absence and presence of
compounds was counted in a liquid scintillation coun-

142
L. Savelon et al./Bioorg. Med. Chem. 6 (1998) 133±142
ter. Triplicates of each compound were determined at
7
[12] Benarab A, Guillaumet G. Heterocycles 1993;36:2327.
[13] Besson T, Podona T, Baudin ML, Coudert G, Guillaumet
G. Bioorg Med Chem Lett 1993;3:1935.
5
two concentrations (10 and 10 M).
[14] Podona T, Guardiola-Lemaõtre B, Caignard DH, Adam
G, Pfei€er B, Renard P, Guillaumet G. J Med Chem
1994;37:1779.
Acknowledgements
[15] Besson T, Ruiz N, Coudert G, Guillaumet G. Tetra-
hedron 1995;51:3197.
[16] Rufenacht K, Kristinsson H, Mattern G. Helv Chim Acta
1976;59:1593.
[17] Bourdais J. Bull Soc Chim Fr 1968:3246.
[18] Yevich JP, New JS, Smith DW, Lobeck WG, Catt JD,
Mincelli JL, Eison MS, Taylor DP, Riblet LA, Temple
DL Jr. J Med Chem 1986;29:359.
[19] Viaud MC, Jamoneau P, Savelon L, Guillaumet G. Het-
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[20] Heck RF. Org React 1982;27:345.
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 hot-plate and general behavior
tests were performed at ITEM Labo (53940 Le Genest
Sainte Isle, France). The authors thank Mrs M. M. Le
Floch for typing the manuscript.
[21] Cabri W, Candiani I, Bedeshi A, Penco S, Santi RJ. Org
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