Synthesis and SAR of 5-, 6-, 7- and 8-aza analogues of 3-aryl-4-hydroxyquinolin-2(1H)-one as NMDA/glycine site antagonists

Article abstract of DOI:10.1016/S0968-0896(01)00115-8

A series of 5-, 6-, 7- and 8-aza analogues of 3-aryl-4-hydroxyquinolin-2(1H)-one was synthesized and assayed as NMDA/glycine receptor antagonists. The in vitro potency of these antagonists was determined by displacement of the glycine site radioligand [³H]5,7-dicholorokynurenic acid ([³H]DCKA) in rat brain cortical membranes. Selected compounds were also tested for functional antagonism using electrophysiological assays in Xenopus oocytes expressing cloned NMDA receptor (NR) 1A/2C subunits. Among the 5-, 6-, 7-, and 8-aza-3-aryl-4-hydroxyquinoline-2(1H)-ones investigated, 5-aza-7-chloro-4-hydroxy-3-(3-phenoxyphenyl)quinolin-2-(1H)-one (13i) is the most potent antagonist, having an IC₅₀ value of 110 nM in [³H]DCKA binding and a K_b of 11 nM in the electrophysiology assay. Compound 13i is also an active anticonvulsant when administered systemically in the mouse maximum electroshock-induced seizure test (ED₅₀ = 2.3 mg/kg, IP).

Full text of DOI:10.1016/S0968-0896(01)00115-8

Bioorganic & Medicinal Chemistry 9 (2001) 2061–2071
Synthesis and SAR of 5-, 6-, 7- and 8-Aza Analogues of 3-Aryl-4-
hydroxyquinolin-2(1H)-one as NMDA/Glycine Site Antagonists
Zhang-Lin Zhou,a,b James M. Navratil,b Sui Xiong Cai,a Edward R. Whittemore,a
Stephen A. Espitia,a Jon E. Hawkinson,a Minhtam Tran,a Richard M. Woodward,a
Eckard Webera and John F.W. Keanab,*
aCoCensys, Inc., 213 Technology Drive, Irvine, CA 92618, USA
bDepartment ofChemistry, University ofOregon, Eugene, OR 97403, USA
Received 3 October 2000; accepted 23 March 2001
Abstract—A series of 5-, 6-, 7- and 8-aza analogues of 3-aryl-4-hydroxyquinolin-2(1H)-one was synthesized and assayed as
NMDA/glycine receptor antagonists. The in vitro potency of these antagonists was determined by displacement of the glycine site
radioligand [3H]5,7-dicholorokynurenic acid ([3H]DCKA) in rat brain cortical membranes. Selected compounds were also tested for
functional antagonism using electrophysiological assays in Xenopus oocytes expressing cloned NMDA receptor (NR) 1A/2C sub-
units. Among the 5-, 6-, 7-, and 8-aza-3-aryl-4-hydroxyquinoline-2(1H)-ones investigated, 5-aza-7-chloro-4-hydroxy-3-(3-phenox-
yphenyl)quinolin-2-(1H)-one (13i) is the most potent antagonist, having an IC50 value of 110 nM in [3H]DCKA binding and a Kb of
11 nM in the electrophysiology assay. Compound 13i is also an active anticonvulsant when administered systemically in the mouse
maximum electroshock-induced seizure test (ED50=2.3 mg/kg, IP). # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
synthesis and SAR of 5-, 6-, 7- and 8-aza-3-aryl-4-hydro-
xyquinolin-2(1H)-ones as NMDA receptor glycine site
antagonists patterned after the prototypic quinolinone
A.3
Excessive stimulation of N-methyl-d-aspartate (NMDA)
receptors, a subclass of ionotropic glutamate receptors,
is known to cause neurodegeneration.1 Channel gating
at NMDA receptors is regulated by an allosteric
strychnine-insensitive glycine binding site. Occupation
of this site by an agonist, either glycine or d-serine, is
necessary for channel gating by the excitatory neuro-
transmitter glutamate.2 Considerable effort has gone
into the development of antagonists for the NMDA
receptor glycine site with the aim of finding therapies for
a variety of CNS disorders.
Several classes of potent glycine site antagonists have
been reported. Examples include 4-hydroxy-3-(3-phe-
noxyphenyl)quinoline-2(1H)-ones such as A,3 1,2,3,4-
tetrahydroquinoline-2,3,4-trione-3-oximes such as B,4
substituted 1,4-dihydroquinoxaline-2,3-diones (QX) such
as licostinel (C),5 7-chloro-6-methyl-5-nitro QX (D),6 4-
hydroxy-3-nitroquinolin-2(1H)-ones such as E,7 and 5-
(N-oxyaza)-7-substituted-1,4-dihydroquinoxaline-2,3-
diones such as F8 (Chart 1). Herein, we report on the
*Corresponding author. Tel.: +1-541-346-4609; fax: +1-541-346-
0487; e-mail: jkeana@oregon.uoregon.edu
Chart 1.
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00115-8

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Z.-L. Zhou et al. / Bioorg. Med. Chem. 9 (2001) 2061–2071
Chemistry
mide 15.9 Imide 15 underwent a Hofmann rearran-
gement to yield 3-aminopicolinic acid (1) in 10%
yield.10
The synthesis of aza 4-hydroxy-3-phenylquinolin-2(1H)-
ones (13a–q) is shown in Scheme 1. Treatment of aza-
amino acids 1–5 with diazomethane or refluxing ethanol
containing sulfuric acid provided the corresponding
ester 6–10. Ester 11 was prepared from ester 10 by
chlorination in HCl containing 30% H2O2. Acylation of
6–11 in turn with the appropriate acid chloride in 1,2-
dichloroethane in the presence of triethylamine afforded
the corresponding amides 12a–q. Intramolecular cycli-
zation of each amide with two equiv of potassium hex-
amethyldisilazide (KHDMS) in THF afforded the
desired azaquinolinones 13a–q.
3-Amino-5-chloropicolinic acid (2) was prepared from
commercially available 2-amino-5-chloropyridine (16)
as shown in Scheme 3. Nitration of pyridine 16 with
concentrated nitric acid and sulfuric acid at 60 ꢀC gave
2-amino-5-chloro-3-nitropyridine (17) in 67% yield.11
Diazotitation followed by bromination with hydro-
bromic acid provided 2-bromo-5-chloro-3-nitropyridine
(18) in 65% yield. Reaction of 18 with Cu(I) cyanide
afforded 5-chloro-2-cyano-3-nitropyridine (19) in 92%
yield based on recovered compound 18. Hydrogenation
of 19 in the presence of Raney Ni in 95% ethanol with
concomitant hydrolysis afforded 3-amino-5-chloro-
picolinamide (20) in 95% yield. Hydrolysis of 20 with
concentrated hydrochloric acid gave 3-amino-5-chloro-
picolinic acid (2) as its HCl salt.
3-Aminopicolinic acid (1) was prepared from commer-
cially available quinolinic acid 14 as shown in Scheme 2.
Acid 14 was heated in acetic anhydride and then
allowed to react with acetamide to form quinolini-
Scheme 1. (a) CH2N2/MeOH; (b) EtOH/H2SO4/reflux; (c) Concd HCl, 30% H2O2, 60ꢀ, 94%; (d) ArCH2COCl/Et3N/CH2Cl2/rt; (e) (i) KHMDS/
THF/ꢁ78 ꢀC to rt; (ii) H3O+
.

Z.-L. Zhou et al. / Bioorg. Med. Chem. 9 (2001) 2061–2071
2063
It is noteworthy that several attempts to hydrolyze 5-
chloro-2-cyano-3-nitropyridine (19) failed to give the
desired product. For example, treatment of compound
19 with MeOH/H2SO4 or 6 N HCl under reflux
returned only starting material. Hydrolysis of 19 in
methanol or ethanol containing aqueous sodium
hydroxide resulted in denitration giving 5-chloro-2-
cyano-3-methoxypyridine (21a) and 5-chloro-2-cyano-3-
ethoxypyridine (21b) in 65 and 55% yield, respectively.
dichlorokynurenic acid ([3H]DCKA) binding to rat brain
cortical membranes (Table 1).8,12 For selected com-
pounds, potencies at the NMDA receptor glycine site
and a-amino-3-hydroxy-5-methyl-4-isoxazole propionic
acid (AMPA) receptors were determined electro-
physiologically in Xenopus oocytes expressing recombi-
nant rat NR1A/2C receptors and rat brain poly(A)+
RNA, respectively (Table 2). Apparent antagonist dis-
sociation constants (Kb values) were estimated by
assuming competitive inhibition and assaying suppres-
sion of membrane current responses elicited by fixed
concentrations of agonist: 1 mm glycine and 100 mm glu-
tamate for NMDA receptors; 10 mm AMPA for AMPA
receptors. Anticonvulsant activity of selected com-
pounds was measured in a mouse maximum electro-
shock-induced seizure (MES) model.8
4-Aminonicotinic acid (3) and amino acid 4 were syn-
thesized as depicted in Scheme 4. Nitration of 3-pico-
line-1-oxide (22) with 70% nitric acid in sulfuric acid
gave nitro compound 23 in 46% yield. Oxidation of the
methyl groupof 23 afforded carboxylic acid 24 in 26%
yield, which was then hydrogenated to give 4-aminoni-
cotinic acid (3) in 40% yield. Amino acid 4 was pre-
pared by effecting a Hofmann rearrangement on
commercially available 3,4-pyridinedicarboximide (25).
Results and Discussion
5-Aza-7-chloro-4-hydroxy-3-nitroquinolin-2-one
(28)
The SAR of aza analogues of 3-aryl-4-hydroxyquinolin-
2(1H)-ones as antagonists at the NMDA/glycine site is
given in Table 1. Compound 13b is much more potent
than 13a, demonstrating the importance of having a
chlorine atom in the 7-position of the 5-aza analogues
for high potency. Compound 13b is 10-fold more potent
than 5-aza-3-nitro analogue 28, indicating that a phenyl
was prepared by acetylation of amine 7 with acetic
anhydride to form amide 26, cyclization of 26 with
KHDMS, and then nitration with concentrated nitric
acid in acetic acid (Scheme 5).
Pharmacology
The affinity of compounds for the NMDA receptor
glycine site was measured by displacement of [3H]5,7-
Scheme 2. (a) (i) (CH3CO)2O/120 ꢀC; (ii) CH3CONH2/reflux, 56%;
(b) 10% NaOH/NaOBr/rt to 85 ꢀC, 10%.
Scheme 4. (a) 70% HNO3/H2SO4/0–10 ꢀC, 46%; (b) concd H2SO4/
Na2Cr2O7/rt, then 20–40 ꢀC, 26%; (c) 10% Pd/C, NH4OH/H2O/H2,
40%; (d) NaOBr/10% NaOH/85 ꢀC, 79%.
Scheme 3. (a) HNO3/H2SO4/60 ꢀC, 67%; (b) NaNO2/HBr/Br2, 65%;
(c) CuCN/185 ꢀC, 92%; (d) Raney Ni/H2/95% EtOH, 95%; (e) concd
HCl, reflux, 85%; (f) ROH/10% NaOH/rt.
Scheme 5. (a) (CH3CO)2O/dioxane, 65%; (b) KHDMS/THF, then
H3O+, 78%; (c) HNO3/AcOH/60 ꢀC, 35%.

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Z.-L. Zhou et al. / Bioorg. Med. Chem. 9 (2001) 2061–2071
groupis favored over a nitro groupin the 3-position in
this series. However, analogue 28 is 20-fold less potent
than its carbocyclic analogue 29. Apparently, 5-aza
substitution is detrimental to potency in the 3-nitro
series.
for the poor binding, although the chlorine atom is also
not optimally positioned in 13p,q.
Values from the [3H]DCKA binding assay indicate that
13i is 5-fold less potent than the prototypic quinolinone
A (IC50 in a [3H]L-689,560 binding assay=2 nM3). To
directly compare these compounds and to get a measure
of the functional activity in the series, we tested selected
compounds in oocyte electrophysiological assays (Table
2). All the compounds tested were functional antago-
nists. The overall trend in potency ran in parallel with
the [3H]DCKA binding assay, that is 13i was the most
potent compound tested while 13l and 13p were the least
potent. However, apparent affinities calculated from the
electrophysiological assay ranged from 3- to 30-fold
lower than in the binding assays. In particular, the Kb
for 13i was 11 nM as compared to 110 nM in the bind-
ing assay. The prototypic compound A had a Kb of
3.5 nM, which was only three times more potent than
13i. Interestingly, the kinetics of the blockade and wash
were distinctly slow for 13i and A as compared to qui-
noxaline-2,3-dione antagonists such as licostinel (C),
taking min to equilibrate to steady-state levels and tens
of min to wash out.
Introducing a chlorine atom (13d), a methyl group
(13g), a methoxy group( 13f) or a nitro group( 13h) in
the meta-position of the 3-phenyl ring of 13b has little
effect on potency while adding a chlorine atom at either
the ortho- (13e) or para- (13c) position results in an 8-
and 4-fold drop in potency, respectively. This suggests
there may be bulk tolerance specifically at the meta-
position of the 3-phenyl ring. Consistent with this
notion and with the observations of Kulagowski et al.3
in the non-aza series, introduction of a m-phenoxy
group( 13i) increased potency 30-fold to 0.11 mM, ren-
dering 13i the most potent compound in the 5-aza ser-
ies, and among all the aza analogues of A investigated.
Either a 2- (13k) or 3-thienyl group( 13j) may be sub-
stituted for the phenyl group in 13b without much
change in potency.
Moving the nitrogen atom from the 5-postion in 13i to
the 6-position and deleting the chlorine atom gave 6-aza
analogue 13l, which has an IC50 value of 20 mM. It
seems likely that the 180-fold dropin potency results at
least partially from the change in the position of the
nitrogen atom to the 6-position. Aza analogues con-
taining a chlorine atom adjacent to the nitrogen atom
were not prepared owing to the potential lability of the
chlorine atom toward nucleophilic substitution and long
term stability concerns about such antagonists. The 7-
aza analogue 13m (IC50 5.4 mM) is more potent than 13l,
but is still significantly less potent than 13i. The 8-aza
analogues 13n–q derived from nicotinic acid all have
low potencies. Introduction of a 6-chloro substitution
(13p versus 13n and 13q versus 13o) does not improve
activity significantly. Apparently, the unshared electrons
of the nitrogen atom in the 8-position are responsible
Compounds in Table 2 were also tested for inhibition of
rat brain AMPA receptors expressed in oocytes. Unlike
a number of classes of NMDA glycine site antagonists,4ꢁ6
the aza-3-aryl-4-hydroxyquinolinones were weak or
inactive as AMPA receptor antagonists. This is consistent
with the pharmacology of the prototype compound A.3
Quinolinone A is reported to be active as an antic-
onvulsant in vivo.3 Although we have not tested com-
pound A ourselves in vivo, we have tested compound
13i administered systemically in the mouse maximum
electroshock-induced seizure (MES) test. In this assay,
compound 13i was active as an anticonvulsant
(ED50=2.3 mg/kg, IP), indicating that 5-aza derivative
13i likely penetrates the blood–brain barrier.
Table 1. SAR of 4-hydroxyquinolin-2(1H)-ones at the NMDA receptor glycine site
Compd no.
W
X
Y
Z
Ar
[3H]DCKA IC50 (mM)
A
C–H
N
N
N
N
N
N
N
N
N
N
N
C-H
C-H
C-H
C-H
C–H
C–H
N
C–H
C–H
C–H
C–H
C–H
C–H
C–H
C–H
C–H
C–H
C–H
C–H
N
C–H
C–H
C–H
C–Cl
C–Cl
C–H
C–H
C–H
C–Cl
C–H
C–Cl
C–Cl
C–Cl
C–Cl
C–Cl
C–Cl
C–Cl
C–Cl
C–Cl
C–Cl
C–H
N
C–H
C–H
C–H
C–H
C–Cl
C–Cl
C–Cl
C–H
C–H
C–H
C–H
C–H
C–H
C–H
C–H
C–H
C–H
C–H
C–H
C–H
C–H
N
m-PhOC6H4
C6H5
C6H5
0.023ꢂ0.001
>100
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
13o
13p
13q
28
3.3ꢂ0.7
p-ClC6H4
m-ClC6H4
o-ClC6H4
m-MeOC6H4
m-MeC6H4
m-NO2C6H4
m-PhOC6H4
3-thienyl
2-thienyl
m-PhOC6H4
m-PhOC6H4
m-PhOC6H4
C6H5
13ꢂ3
3.0ꢂ0.1
24ꢂ6
2.9ꢂ0.2
1.8ꢂ0.3
7.4ꢂ1.6
0.11ꢂ0.02
4.3ꢂ0.6
9.8ꢂ1.9
20ꢂ5
5.4ꢂ1.3
31ꢂ7
N
N
N
C–H
C–H
C–H
>100
m-PhOC6H4
C6H5
NO2
19ꢂ3
>100
29ꢂ4
29
E
C–H
C–Cl
NO2
NO2
1.5ꢂ0.1a
0.22ꢂ0.05a
aRef 7.

Z.-L. Zhou et al. / Bioorg. Med. Chem. 9 (2001) 2061–2071
2065
Table 2. Functional antagonism of rat NMDA receptors expressed
in Xenopus oocytesa
(87%) of the title compound as an orange solid: mp
149–151 ꢀC (lit.13 148–150 ꢀC); 1H NMR d 1.44 (t,
J=6.9 Hz, 3H), 4.45 (q, J=6.9 Hz, 3H), 5.84 (brs, 2 H),
7.05 (d, J=1.5 Hz, 1H), 7.99 (d, J=1.8 Hz, 1H).
Compd
NMDA (glycine) Kb (mM)
n
A
0.0035 (0.0032–0.0037)
0.49 (0.46–0.52)
0.46 (0.42–0.50)
0.60 (0.57–0.64)
0.011 (0.010–0.012)
5.1 (4.6–5.6)
3
4
3
4
3
3
4
13b
13c
13f
13i
13l
13p
Ethyl
3-(m-phenoxyphenyl)acetylamido-5-chloropicoli-
nate (12i). To a solution of m-phenoxyphenylacetic acid
(3.0 g, 13 mmol) in 50 mL of dichloromethane was
added 3.3 g (26 mmol) of oxalyl chloride. The resulting
solution was allowed to stir at room temperature for
3 h. The solvent was evaporated to give the acid chloride
as an oil (3.20 g, 100%), which was used in the next step
without purification. To a solution of ethyl 3-amino-5-
chloropicolinate (7) (0.353 g, 1.76 mmol) in 10 mL of
1,2-dichloroethane and 0.51 mL of triethylamine was
5.8 (5.3–6.3)
aInhibition of NMDA receptors was measured in oocytes expressing
the rat subunit combination NR1A/2C. Kb values for glycine binding
sites were determined from inhibition of currents elicited by 1 mM gly-
cine and 100 mM glutamate. Values are given to two significant figures
and numbers in parentheses are 95% confidence intervals adjusted to
the linear scale. Current ranges and mean responses for the NMDA
evoked currents were (in nA): 315–800 (570ꢂ31, n=24).
added
m-phenoxyphenylacetyl
chloride
(3.20 g,
13 mmol). The resulting solution was allowed to reflux
for 12 h. After cooling to room temperature, the solvent
was evaporated in vacuo to dryness. Water (15 mL) was
added to the residue and then it was extracted with ethyl
acetate (3ꢃ15 mL). The combined extracts were washed
with brine and dried over sodium sulfate. The solvent
was evaporated in vacuo and the product was obtained
by flash chromatography (15% EtOAc in hexane), giv-
ing 3.68 g (90%) of the product as a white solid: mp 95–
Conclusion
The
5-aza-3-aryl-7-chloro-4-hydroxyquinolin-2(1H)-
ones represent a novel class of potent, systemically
active NMDA receptor glycine site antagonists. The
corresponding 6-, 7- and 8-aza analogues appear to be
significantly less active. The most potent compound
synthesized in this series, 5-aza-7-chloro-4-hydroxy-3-
(3-phenoxyphenyl)quinolin-2(1H)-one (13i), has an IC50
of 110 nM in [3H]DCKA binding, a Kb of 11 nM for
functional antagonism of cloned NMDA receptors
expressed in oocytes and an ED50 of 2.7 mg/kg IP in a
mouse MES assay.
ꢀ
1
97 C; H NMR d 1.43 (t, J=7.2 Hz, 3H), 3.76 (s, 2H),
4.42 (q, J=7.2 Hz, 2H), 6.85 (m, 5H), 7.33 (m, 4H), 8.35
(d, J=1.2 Hz, 1H), 9.23(d, J=1.2 Hz, 1H), 11.08 (s,
1H).
5-Aza-7-chloro-4-hydroxy-3-(m-phenoxyphenyl)quino-
line-2-one (13i). To a solution of KHDMS in toluene
(42 mL, 21 mmol) in 40 mL of THF was added dropwise
a solution of ethyl 3-(m-phenoxyphenyl)acetylamido-5-
chloropicolinate (12i) (2.87 g, 7 mmol) in 40 mL of THF
at ꢁ78 ꢀC under N2. The resulting mixture was allowed
to warm to room temperature and then stirred for
additional 12 h. Water (100 mL) was added to the reac-
tion mixture, which was then extracted with ethyl ace-
tate (30 mL). The aqueous phase was acidified with 4 N
HCl to pH=2. The white solid was obtained by filtra-
tion and purified by recrystallization from DMSO-H2O,
giving 2.20 g (86%) of the product as a pale yellow
Experimental
Melting points were determined in open capillary tubes
on a Mel-Tempapparatus and are uncorrected. The 1H
NMR spectra were recorded at 300 MHz in CDCl3
unless otherwise stated. Chemical shifts are reported in
ppm (d), and J coupling constants are reported in Hz.
Elemental analyses were performed by Desert Analytics,
Tucson, AZ, USA. Mass spectra (MS) were obtained
with a VG 12-250 or VG ZAB-2FHF mass spectro-
meter. Reagent grade solvents were used without fur-
ther purification unless otherwise specified. Reverse
phase HPLC analyses were monitored at 254 nM on a
4.6ꢃ250 mM microsorb-MV C18 column, using as sol-
vents 0.1% trifluoroacetic acid in water (A) and 0.1%
trifluoroacetic acid in acetonitrile (B). The linear gra-
dient was 20% B in A to 95% B in A with a flow rate of
1 mL/min.
solid: mp248–250 ꢀC; H NMR (DMSO-d6) d 6.89 (m,
1
1H), 7.02 (m, 2H), 7.11 (m, 2H), 7.21 (d, J=7.2 Hz,
1H), 7.35 (s, 3H), 7.68 (d, J=1.5 Hz, 1H), 8.46 (d,
J=1.5 Hz, 1H), 11.00 (brs, 1H), 11.67 (s, 1H). EI-MS
m/e 364 (M+, 40), 77 (100). HR-MS calcd for
C20H13ClN2O3: 364.0603. Found: 346.0609. Anal. calcd
for C20H13ClN2O3: C, 65.85; H, 3.59; N, 7.65. Found:
C, 65.51; H, 3.59; N, 7.65.
Typical procedure for synthesis of 3-aryl-5-aza-4-
hydroxyquinoxaline-2-ones
Ethyl 3-phenylacetylamido-5-chloropicolinate (12b). Mp
120–122 ꢀC. H NMR d 1.43 (t, J=7.5 Hz, 3H), 3.79 (s,
1
Ethyl 3-amino-5-chloropicolinate (7). A mixture of 3-
amino-5-chloropicolinic acid (2, 2.30 g, 9.36 mmol) in
100 mL of absolute EtOH and 10 mL of concentrated
H2SO4 (96%, d=1.84) was refluxed for 12 h. The
resulting solution was cooled to rt and poured over ice,
and neutralized to pH 5 (pH paper) with solid
NaHCO3. The resulting mixture was extracted with
EtOAc (3ꢃ30 mL). The organic layer was dried over
Na2SO4 and rotevaporated to dryness, giving 1.64 g
2H), 4.45 (q, J=7.5 Hz, 2H), 7.37 (m, 5H), 8.32 (d,
J=2.1 Hz, 1H), 9.21 (d, J=2.1 Hz, 1H), 11.08 (s, 1H).
5-Aza-7-chloro-4-hydroxy-3-phenylquinoline-2-one (13b).
Mp328–329 ꢀC (dec.) 1H NMR (DMSO-d6) d 7.28–
7.44 (m, 5H), 7.71 (d, J=2.1 Hz, 1H), 8.48 (d,
J=2.1 Hz, 1H), 10.88 (s, 1H), 11.69 (s, 1H). Anal. calcd
for C14H9ClN2O2.0.2H2O: C, 60.86; H, 3.43; N, 10.14.
Found: C, 61.11; H, 3.18; N, 9.82.

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Z.-L. Zhou et al. / Bioorg. Med. Chem. 9 (2001) 2061–2071
Ethyl 3-(p-chloro)phenylacetylamido-5-chloropicolinate
7.70 (d, J=2.1 Hz, 1H), 8.48 (d, J=2.1 Hz, 1H), 10.82
(brs, 1H), 11.68 (s, 1H). Anal. calcd for C15H11ClN2O2: C,
62.84; H, 3.87; N, 9.77. Found: C, 62.63; H, 3.61; N, 9.61.
(12c). Mp140–142 ꢀC. H NMR d 1.44 (t, J=6.9 Hz,
1
3H), 3.75 (s, 2H), 4.47 (q, J=6.9 Hz, 2H), 7.25–7.38 (m,
4H), 8.34 (d, J=2.1 Hz, 1H), 9.18 (d, J=2.1 Hz, 1H),
11.13 (s, 1H).
Ethyl 3-(3-thienyl)ꢀphenylacetylamido-5-chloropicolinate
1
(12j). Mp116–118 C. H NMR d 1.43 (t, J=6.6 Hz,
5-Aza-7-chloro-4-hydroxy-3-(p-chloro)phenylquinoline-2-
3H), 3.83 (s, 2H), 4.44 (q, J=6.6 Hz, 2H), 7.09 (s, 1H),
7.27 (s, 1H), 7.37 (s, 1H), 8.34 (s, 1H), 9.22 (s, 1H),
11.07 (s, 1H).
one (13c). Mp342–344 ꢀC. H NMR (DMSO-d6) d 7.41
1
(d, J=8.1 Hz, 2H), 7.47 (d, J=8.1 Hz, 2H), 7.72 (d,
J=2.4 Hz, 1H), 8.49 (d, J=2.4 Hz, 1H), 11.10 (brs, 1H),
11.74 (brs, 1H). Anal. calcd for C14H8Cl2N2O2.0.4H2O:
C, 53.49; H, 2.82; N, 8.91. Found: C, 53.26; H, 2.55; N,
8.84.
5-Aza-7-chloro-4-hydroxy-3-(3-thienyl)phenylquinoline-
2-one (13j). Mp310–312 ꢀC. 1H NMR (DMSO-d6) d
7.48 (m, 1H), 7.68 (m, 2H), 7.98 (d, J=2.1 Hz, 1H), 8.48
(d, J=2.1 Hz, 1H), 11.17 (brs, 1H), 11.71 (brs, 1H).
Anal. calcd for C12H7ClN2O2S.0.15H2O: C, 51.22; H,
2.61; N, 9.95. Found: C, 51.10; H, 2.46; N, 9.72.
Ethyl 3-(m-chloro)phenylacetylamido-5-chloropicolinate
ꢀ
1
(12d). Mp123–124 C. H NMR d 1.42 (t, J=7.2 Hz,
3H), 3.81 (s, 2H), 4.42 (q, J=7.2 Hz, 2H), 6.85 (m, 3H),
7.30 (s, 1H), 8.34 (s, 1H), 9.21 (d, J=1.2 Hz, 1H), 11.08
(s, 1H).
Ethyl 3-(2-thienyl)phenylacetylamido-5-chloropicolinate
ꢀ
1
(12k). Mp141–143 C. H NMR d 1.43 (t, J=6.9 Hz,
3H), 4.01 (s, 2H), 4.43 (q, J=6.9 Hz, 2H), 7.04–7.07 (m,
2H), 7.30 (m, 1H), 8.35 (d, J=1.8 Hz, 1H), 9.22 (d,
J=1.8 Hz, 1 H), 11.15 (s, 1H).
5-Aza-7-chloro-4-hydroxy-3-(m-chloro)phenylquinoline-
2-one (13d). Mp324–326 ꢀC. H NMR (DMSO-d6) d
1
7.400 (m, 4H), 7.692 (d, J=1.5 Hz, 1H), 8.484 (d,
J=1.2 Hz, 1H), 8.463 (s, 1H), 11.158 (brs, 1H), 11.738
(s, 1H). Anal. calcd for C14H8Cl2N2O2: C, 54.75; H,
2.63; N, 9.12. Found: C, 54.52; H, 2.42; N, 9.00. (HPLC
purity >98%).
5-Aza-7-chloro-4-hydroxy-3-(2-thienyl)phenylquinoline-
2-one (13k). Mp318–320 ꢀC (dec.). H NMR (DMSO-
d6) d 7.13 (m, 1H), 7.53 (d, J=3.9 Hz, 1H), 7.73 (s, 1H),
8.22 (s, 1H), 8.53 (s, 1H), 11.89 (brs, 2H). Anal. calcd
for C12H7ClN2O2S: C, 51.71; H, 2.53; N, 10.05. Found:
C, 51.83; H, 2.53; N, 9.88.
1
Ethyl 3-(o-chloro)phenylacetylamido-5-chloropicolinate
(12e). Mp163–165 ꢀC. H NMR d 1.40 (t, J=6.9 Hz,
1
3H), 3.95 (s, 2H), 4.42 (q, J=6.9 Hz, 2H), 7.19–7.30 (m,
3H), 7.39 (d, J=10.5 Hz, 1H), 8.34 (s, 1H), 9.23 (s, 1H),
11.09 (s, 1H).
Methyl 3-aminopicolinate (6). To a solution of 3-amino-
picolinic acid (1, 0.58 g, 4.24 mmol) in 45 mL of absolute
MeOH was added an ethereal solution of diazomethane
(0.30 M, 30 mL). The solution was stirred for an addi-
tional 30 min and the solvent was removed in vacuo.
The crude residue was chromatographed on a column of
silica using CHC13–MeOH as eluent, giving 0.33 g
(52%) of the title product as a yellow solid. Mp 139–
146 C; H NMR d 3.98 (t, J=7.2 Hz, 3 H), 5.73 (d,
J=1.8 Hz, 2H), 7.05 (dd, J1=1.2 Hz, J2=8.4 Hz, 1H),
7.22 (dd, J1=4.2 Hz, J2=8.4 Hz, 1H), 8.07 (dd,
J1=1.2 Hz, J2=4.2 Hz, 1H); IR (KBr): 3455, 3295,
3160, 1689, 1617, 1408, 1335, 1244, 1115.
5-Aza-7-chloro-4-hydroxy-3-(o-chloro)phenylquinoline-2-
ꢀ
1
one (13e). Mp248–250 C. H NMR (DMSO-d6) d 7.29
(m, 1H), 7.34–7.37 (m, 2H), 7.49 (m,1H), 7.73 (d,
J=2.1 Hz, 1H), 8.50 (d, J=2.1 Hz, 1H), 11.14 (brs, 1H),
11.72 (s, 1H). Anal. calcd for C14H8Cl2N2O2: C, 54.75;
H, 2.63; N, 9.12. Found: C, 54.82; H, 2.52; N, 8.83.
ꢀ
1
Ethyl 3-(m-methoxy)phenylacetylamido-5-chloropicoli-
nate (12f). Mp108–110 ꢀC. 1H NMR d 1.42 (t,
J=7.2 Hz, 3H), 3.80 (s, 3H), 3.81 (s, 2H), 4.42 (q,
J=7.2 Hz, 2H), 6.85 (m, 3H), 7.30(s, 1H), 8.34 (d,
J=1.2 Hz, 1H), 9.21 (d, J=1.2 Hz, 1H), 11.08 (s, 1H).
Methyꢀl 3-amido-(N-phenylacetyl)picolinate (12a). Mp
64–69 C. 1H NMR d 3.78 (s, 2H), 3.97 (s, 3H), 7.38 (m,
6H), 8.39 (dd, J1=0.6 Hz, J2=3.9 Hz, 1H), 9.10 (dd,
J1=8.4 Hz, J2=8.3 Hz, 1H), 10.96 (s, 1H); EI–MS (m/
e): 270 (M+, 35), 211 (35), 179 (85), 153 (40), 147 (65),
119 (40), 94, (40), 91 (100).
5-Aza-7-chloro-4-hydroxy-3ꢀ-(m-methoxy)phenylquino-
1
line-2-one (13f). Mp256–258 C. H NMR (DMSO-d6)
d 3.74 (s, 3H), 6.80–7.00 (m, 3H), 7.28 (m, 1H), 7.71 (d,
J=2.1 Hz, 1H), 8.48 (d, J=2.1 Hz, 1H), 10.90 (brs, 1H),
11.68 (s, 1 H). Anal. calcd for C15H11ClN2O3: C, 59.52;
H, 3.66; N, 9.25. Found: C, 59.34; H, 3.57; N, 8.98.
5-Aza-4-hydroxy-3-phenylquinotin-2(lH)-one (13a). Mp
297–299 ꢀC. H NMR (DMSO-d6) d 7.29 (d, J=7.2 Hz,
1
1H), 7.37 (t, J=7.2 Hz, 2H), 7.45 (d, J=7.5 Hz, 2H),
7.60 (dd, J1=4.2 Hz, J2=8.1 Hz, 1H), 7.69 (d,
J=8.1 Hz, 1H), 8.49 (d, J=4.2 Hz, 1H), 11.62 (s, 1H).
IR (KBr): 3427, 3161, 2923, 2854, 1655, 1477, 1402,
1123, 693; EI-MS (m/z): 238 (M+, 100), 210 (10), 181
(15), 93 (20), 89 (15), 78 (15), 63 (15), 51 (10), 39 (25).
Ethyl 3-(m-Methyl)phenylacetylamido-5-chloropicolinate
(12g). Mp94–96 ꢀC. 1H NMR d 1.41 (t, J=6.9 Hz,
3H), 2.36 (s, 3H), 3.73 (s, 2H), 4.43 (q, J=6.9 Hz, 2H),
7.15–7.28 (m, 4H), 8.31 (m,1H), 9.21 (m, 1H), 11.06 (s,
1H).
5-Aza-7-chloro-4-hydroxy-3-(m-methyl)phenylquinoline-
ꢀ
Ethyl 4-aminonicotinate (8). Mp109–111 ꢀC (lit.14 109–
111 C). H NMR d 1.39 (t, J=7.0 Hz, 3H), 4.35 (q,
J=7.0 Hz, 2H), 6.27 (bm, 2H), 6.53 (d, J=3.9 Hz, 1H),
ꢀ
1
1
2-one (13g). Mp300–302 C. H NMR (DMSO-d6) d
2.31 (s, 3H), 7.09 (d, J=6.9 Hz, 1H), 7.18–7.28 (m, 3H),

Z.-L. Zhou et al. / Bioorg. Med. Chem. 9 (2001) 2061–2071
2067
8.19 (d, J=2.1 Hz, 1H), 8.92 (dd, J1=3.3 Hz,
J2=4.5 Hz, 1H).
J1=1.5 Hz, J2=4.8 Hz, 1H), 10.81 (s, 1H); IR (KBr):
3441, 3168, 1723, 1668, 1660, 1549, 1490, 1436, 1409,
1307, 1246, 1211, 1143, 1027, 980, 775, 700; HPLC
100%; EI-MS (m/z): 377 (10), 376 (M+, 45), 211 (15),
210 (100), 183 (10), 167 (15), 166 (25), 147 (35), 121 (10),
94 (35), 89 (30), 77 (15), 51 (10), 39 (10); HR-MS calcd
for C22H20N2O4: 376.1423. Found: 376.1419.
Ethyl
4-amido-(N-[3-phenoxylphenylacetyl)nicotinate
(12l). Mp92–93.5 ꢀC. 1H NMR d 1.41 (t, J=7.2 Hz,
3H), 3.77 (s, 2H), 4.37 (q, J=7.2 Hz, 2H), 7.20 (m, 9H),
8.58 (d, J=5.4 Hz, 1H), 8.66 (d, J=5.4 Hz, 1H), 9.14 (s,
1H), 11.24 (bs, 1H); IR (KBr): 3448, 3243, 3141, 2991,
1702, 1586, 1511, 1450, 1416, 1300, 1259, 1225, 1116,
802, 707, 543; HPLC 100%; EI-MS (m/z): 377 (20), 376
(M+, 80), 210 (65), 193 (40), 184 (25), 183 (35), 168
(10), 167 (65), 165 (70), 147 (100), 89 (30), 77 (20), 51
(10); HR-MS calcd for C22H20N2O4: 376.1423. Found:
376.1430.
8-Aza-4-hydroxy-3-(ꢀ30-phenoxy)phenylquinolin-2(lH)-one
1
(13n). Mp246–248 C (dec.). H NMR (DMSO-d6) d
7.09 (m, 6H), 7.23 (dd, J1=2.4 Hz, J2=7.8 Hz, 1H),
7.38 (m, 3H), 8.30 (d, J=7.8 Hz, 1H), 8.50 (d,
J=2.4 Hz, 1H), 10.52 (bm, 1H), 11.81 (s, 1H); IR (KBr)
3434, 3127, 1648, 1607, 1498, 1409, 1341, 1246, 1150,
946, 789, 761, 700, 571, 543, 475; HPLC 100%; EI-MS
(m/z): 332 (15), 331 (75), 330 (M+, 100), 121 (10), 93
(10), 77 (1 0), 51 (10), 39 (10); HR-MS calcd for
C20Hl4N2O3: 330.1004. Found: 330.1015.
6-Aza-4-hydroxy-3-(30-phenoxy)phenylquinolin-2(lH)-one
(13l). Mp165–167 ꢀC. H NMR (DMSO-d6) d 7.20 (m,
1
10 H), 8.47 (dd, J1=1.2 Hz, J2=3.6 Hz, 1H), 9.04 (s,
1H), 11.81 (s, 1H); IR (KBr): 3434, 3134, 1654, 1491,
1402, 1239, 1130, 700; HPLC 100%; LR-MS (m/z): 331
(30), 330 (M+, 100), 329 (100). 236 (20), 121 (40), 93
(40), 77 (50), 66 (20), 51 (40), 39 (25); HR-MS calcd for
C20H14N2O3: 330.1004. Found: 330.1002.
1
Ethyl 2-amido-(N-phenylacetyl)nicotinate (12o). Oil; H
NMR d 1.37 (t, J=6.9 Hz, 3H), 3.91 (s, 2H), 4.33 (q,
J=6.9 Hz, 2H), 7.04 (dd, J1=4.2 Hz, J2=7.5 Hz, 1H),
7.39 (m, 5H), 8.27 (dd, J1=0.9 Hz, J2=7.6 Hz, 1H),
8.60 (d, J=3.9 Hz, 1H), 10.76 (s, 1H).
Ethyl 3-aminoisonicotinate (9). Mp63–64 ꢀC (lit.15
65 ꢀC). 1H NMR d 1.40 (t, J=7.0 Hz, 3H), 4.37 (q,
J=7.0 Hz, 2H), 5.77 (d, J=1.2 Hz, 2H), 7.64 (d,
J=5.1 Hz, 1H), 7.91 (d, J=5.1 Hz, 1H), 8.25 (s, 1H).
8-Aza-4-hydroxy-3-phenylquinolin-2(lH)-one (13o). Mp
340–342 ꢀC. 1H NMR (DMSO-d6)
d
7.17 (dd,
J1=4.8 Hz, J2=7.8 Hz, 1H), 7.25 (m, 1H), 7.34 (t,
J=7.2 Hz, 2H), 7.42 (dd, J1=0.9 Hz, J2=7.2 Hz, 2H),
8.29 (dd, J1=1.2 Hz, J2=7.8 Hz, 1H), 8.45 (dd,
J1=1.2 Hz, J2=7.8 Hz, 1H), 11.64 (s, 1H); HPLC
99.2%; LR-MS (m/z): 239 (M+, 100), 238 (90), 181
(20), 121 (40), 118 (20), 93 (40), 91 (55), 89 (15), 77 (25),
69 (20), 63 (25), 57 (35), 55 (40), 51 (30), 43 (55), 41 (55);
HR-MS calcd for C14H10N2O2: 238.0742. Found:
238.0737.
Ethyl 3-amido-(N-[3-phenoxylphenylacetyl)isonicotinate
1
(12m). Oil; H NMR d 1.40 (t, J=7.2 Hz, 3H), 3.77 (s,
2H), 4.35 (q, J=7.2 Hz, 2H), 7.10 (m, 9H), 7.77 (d,
J=5.1 Hz, 1H), 8.41 (d, J=5.1 Hz, 1H), 10.01 (s, 1H),
10.65 (brs, 1H); IR (KBr): 3291, 3066, 2991, 1709, 1593,
1518, 1491, 1416, 1368, 1293, 1259, 1225, 1191, 1116,
1020, 973, 796, 768, 700; HPLC: 98.3%; EI–MS (m/z):
377 (15), 376 (M+, 70), 211 (15), 210 (100), 193 (25),
184 (20), 183 (45), 166 (25), 165 (15), 147 (40), 89 (20);
HR-MS calcd for C22H20N2O4: 376.1423. Found:
376.1430.
Ethyl 2-amido-[N-(3-phenoxy)phenylacetyl]-5-chloronico-
tinate (12p). Mp103–104 ꢀC. 1H NMR d 1.39 (t,
J=7.2 Hz, 3H), 3.88 (s, 2H), 4.35 (q, J=7.2 Hz, 2H),
7.10 (m, 9H), 8.24 (d, J=2.4 Hz, 1H), 8.52 (d,
J=2.4 Hz, 1H), 10.70 (d, J=2.4 Hz, 1H); IR (KBr)
3434, 3155, 2925, 1722, 1659, 1401, 1252, 1205, 1102,
775, 700; HPLC 99%; LR-MS (m/z): 41 0 (M+, 40), 211
(35), 210 (100), 200 (35), 183 (30), 181 (40), 128 (15), 89
(50), 77 (20), 51 (15); HR-MS calcd for C22Hl9ClN2O4:
410.1033. Found: 410.1026.
7-Aza-4-hydroxy-3-(30-phenoxy)phenylquinolin-2(1H)-
ꢀ
one (13m). Mp277–280 C (dec.). 1H NMR (DMSO-d6)
d 7.20 (m, 9H), 7.81 (d, J=5.4 Hz, 1H), 8.32 (d,
J=5.4 Hz, 1H), 8.62 (s, 1H), 10.65 (d, J=2.4 Hz, 1H),
11.74 (s, 1H); IR (KBr): 3427, 3148, 2936, 2861, 1661,
1614, 1498, 1402, 1307, 1252, 1225, 1198, 1136, 857,
700, 564; HPLC: 100%; LR-MS (m/z): 332 (20), 331
(90), 330 (M+, 100), 121 (15), 93 (15), 77 (10); HR-MS
calcd for C20Hl4N2O3: 330.1004. Found: 330.1018.
Anal. calcd for C20Hl4N2O3.H2O: C 69.0%, H 4.6%, N
8.0%. Found: C 69.3%, H 4.3%, N 7.7%.
8-Aza-6-chloro-4-hydroxy-3-(30-phenoxy)phenylquinolin-
2(l H)-one (13p). Mp237–240 ꢀC (dec.). 1H NMR
(DMSO-d6) d 7.10 (m, 9H), 8.33 (d, J=2.0 Hz, 1H),
8.54 (d, J=2.0 Hz, 1H), 10.70 (m, 1H), 12.04 (s, 1H); IR
(KBr): 3434, 3155, 1641, 1409, 1218, 1136, 1096; HPLC:
98.0%; LR-MS (m/z): 366 (25), 365 (45), 364 (M+, 80),
363 (100), 214 (30), 169 (15), 168 (10), 155 (15), 141 (15),
127 (15), 78 (30), 77 (30), 63 (40), 51 (30), 45 (10), 39
(10); HR-MS calcd for C20Hl3CN2O3: 364.0615. Found:
364.0606.
ꢀ
Ethyl 2-aminonicotinate (10). Mp90.5–92.5 C (lit.16 94–
95 ꢀC). 1H NMR d 1.38 (t, J=7.2 Hz, 3H), 4.34 (q,
J=7.2 Hz, 2H), 6.40 (bm, 2H), 6.62 (dd, J1=4.9 Hz,
J2=7.8 Hz, 1H), 8.14 (dd, Jl=1.0 Hz, J2=8.4 Hz, 1H),
8.21 (dd, J1=1.2 Hz, J2=3.8 Hz, 1H).
Ethyl
2-amido-[N-(3-phenoxy)phenylacetyl]nicotinate
Ethyl 2-amido-(N-phenylacetyl)-5-chloronicotinate (12q).
Mp113–114 ꢀC. 1H NMR d 1.38 (t, J=7.2 Hz, 3H),
3.90 (s, 2H), 4.34 (q, J=7.2 Hz, 2 H), 7.38 (m, 5H), 8.22
(d, J=2.4 Hz, 1H), 8.54 (d, J=2.4 Hz, 1H), 10.64 (s,
(12n). Mp90–91 ꢀC. 1H NMR d 1.39 (t, J=6.8 Hz,
3H), 3.83 (s, 2H), 4.35 (q, J=6.8 Hz, 2H), 7.10 (m, 9H),
8.29 (dd, J1=1.5 Hz, J2=7.8 Hz, 1H), 8.59 (dd,

2068
Z.-L. Zhou et al. / Bioorg. Med. Chem. 9 (2001) 2061–2071
1H); IR (KBr): 3427, 3196, 1743, 1640, 1600, 1409, 1272,
1218, 1136, 1096, 796; HPLC: 100%; EI-MS (m/z): 318
(M+, 25), 202 (20), 201 (10), 200 (60), 183 (15), 181 (40),
154 (10), 128 (10), 118 (100), 91 (80), 65 (25), 39 (10); HR-
MS calcd for C16Hl5ClN203: 318.0771. Found: 318.0772.
flask. The flask was equipped with an internal thermo-
meter, a glass funnel and stopper, and placed in a salt/
ice bath. When the internal temperature reached 5 ꢀC, 2-
amino-5-chloropyridine (16, 77.2 g, 0.60 mol) was added
over 1 h with stirring. The suspension was then stirred at
rt to dissolve the rest of the solid. The resulting solution
was heated to 55 ꢀC. Nitric acid (70%, 40.5 mL, d=1.41,
0.634 mol) was added dropwise through an addition funnel
so as to maintain the internal temperature at 57ꢂ3 ꢀC. The
reaction solution was poured over ice (1.5kg), and the
resulting mixture was partially neutralized with 40%
NaOH (ꢄ600 mL). The mixture was filtered to leave a
yellow/orange solid. This solid was washed by resuspen-
sion in water (600 mL). The mixture was filtered and the
resulting solid was dried in the oven 48 h to yield 66.3 g
(64%) of the title compound as an orange/yellow solid:
8-Aza-6-chloro-4-hydroxy-3-phenylquinolin-2(lH)-one
(13q). Mp324–326 ꢀC (dec.). H NMR (DMSO-d6) d
1
7.36 (m, 5H), 8.35 (d, J=2.1 Hz, 1H), 8.55 (d,
J=2.1 Hz, 1H), 10.64 (bm, 1H), 12.05 (s, 1H); IR
(KBr): 3434, 3134, 1661, 1559, 1402, 1341, 1232, 1143,
673, 564; HPLC: 100%; LR-MS (m/z): 274 (30), 273
(45), 272 (M+, 100), 271 (90), 215 (10), 157 (10), 155 (35),
127 (20), 89 (15), 77 (10), 63 (10), 51 (10), 39 (10); EI-MS
calcd for C14H9ClN2O2: 272.0352. Found: 272.0349.
ꢀ
Quinolinimide (15). The procedure of Crum and Fuchs-
man9 was modified. A mixture of quinolinic acid (14,
16.8 g, 101 mmol) and acetic anhydride (22 mL, d=1.08,
230 mmol) was heated at 120 ꢀC to distill off 19 mL of
distillate. To the cooled residue (100 ꢀC) was added
acetamide (13.4 g, 227 mmol) over 5 min. The stirred
mixture was heated at reflux for 2.5 h. The mixture was
then cooled to rt, filtered, and the filter cake was washed
with water (2ꢃ25 mL) to obtain a brown solid. The
solid was recrystallized in hot ethanol (95%, 50 mL) to
give 8.4 g (56%) of the title compound as a brown solid:
mp230–231 ꢀC (lit.9 230–233 ꢀC); 1H NMR (DMSO-d6)
d 7.76 (dd, J1=2.4 Hz, J2=5.1 Hz, 1H), 8.24 (dd,
J1=0.9 Hz, J2=7.2 Hz, 1H), 8.96 (dd, J1=0.9 Hz,
J2=7.5 Hz, 1H), 11.66 (brs, 1H). IR (KBr): 3484, 3189,
3100, 3083, 1735, 1704, 1086, 736.
mp191–193 C (lit.11 190–103 ꢀC); 1H NMR d 6.69 (brs,
2H), 8.33 (d, J=1.5 Hz, 1H), 8.43 (d, J=1.5 Hz, 1H).
2-Bromo-5-chloro-3-nitropyridine (18). To HBr (48%,
d=1.49, 214 mL, 1.89 mol) was added 2-amino-5-
chloro-3-nitropyridine (17, 66.0 g, 0.38 mol) in portions
at 0 ꢀC with a mechanical stirring. The mixture was
stirred until the internal temperature <0 ꢀC, and then
bromine (65 mL, d=3.102, 1.3 mol) was added drop-
wise. The resulting orange mixture was stirred <0 ꢀC
and a solution of NaNO2 (91.3 g, 1.32 mol) in 125 mL of
water was added slowly as to maintain the internal
temperature <0 ꢀC. The mixture was stirred for 45 min
at <0 ꢀC, and then a solution of NaOH (139.3 g,
3.48 mol) in 200 mL of water was added to the mixture
slowly to maintain the internal temperature <20 ꢀC.
The mixture was stirred at <20 ꢀC for an additional h,
and then filtered. The recovered brown solid was dried
at 25 ꢀC under vacuum for 6 h. It was purified by
recrystallization from 95% ethanol to give 46.0 g (52%)
of the title compound as a yellow solid: mp 73–75 ꢀC
3-Aminopicolinic acid (1). The method of Oakes et al.10
was modified. To a solution of quinolinimide (15, 8.0 g,
54 mmol) in an ice-cold 10% NaOH solution (160 mL)
was added an ice-cold aqueous sodium hypobromite
solution (prepared by adding bromine (3.0 mL, 9.3 g,
58 mmol) to 56 mL of an ice-cold 15% NaOH solution)
over 10 min. The brown solution was stirred at rt for 1 h
and at 85 ꢀC for 1 h. The solution was then cooled to rt
and the pH was adjusted to 5 (pH paper) using sulfuric
acid (50%, 50 mL). The solution was stirred at 4 ꢀC for
63 h. The resulting mixture was filtered and the mother
liquor was treated with copper (II) acetate-monohydrate
(3.2 g, 16 mmol) in 64 mL of hot water containing
1.60 mL of glacial acetic acid. The resulting mixture was
cooled to rt and filtered, and the filter cake was washed
with water (2ꢃ25 mL). The precipitate was resuspended
in water (64 mL) and saturated with hydrogen sulfide.
The mixture was filtered through a fritted filter to
remove the copper sulfide. The filtrate was rotevapo-
rated to dryness, leaving an orange solid. The orange
solid was recrystallized from water to give 0.82 g (10%)
of the title compound as light brown crystals: mp 207.5–
(lit.17 75 ꢀC); H NMR d 8.15 (d, J=2.1 Hz, 1H), 8,57
1
(d, J=2.1 Hz, 1H).
5-Chloro-3-nitropicolinonitrile (19). The procedure of
Berrie et al. was modified.17 2-Bromo-5-chloro-3-nitro-
pyridine (18, 6.0 g, 25 mmol) was mixed with copper (I)
cyanide (4.6 g, 51 mmol) in a 100 mL round-bottom
flask fitted with a condenser loosely plugged with cot-
ton. The flask was slowly heated in an oil bath. When
the temperature reached ꢄ150 ꢀC (takes 2 h), the reac-
tion mass began to turn black. When the reaction mass
turned completely black (or close to it), the pressure was
reduced to ꢄ1 mm Hg by vacuum and the oil bath
removed after 30 s. The mixture was cooled to rt, and
the sublimed solid (on the cotton) and reaction mass
were treated with hot acetone (100 mL). The resulting
mixture was filtered, and the mother liquor rotaevapo-
rated to dryness to yield the crude title compound as a
dark brown solid. This solid was purified by column
chromatography using 4:1 hexane-EtOAc (Rf=0.13) to
give 7.30 g (80%) of the title compound as a white solid:
208 ꢀC (lit.10 210 ꢀC); H NMR (DMSO-d6) d 7.26 (d,
1
J=8.4 Hz, 1H), 7.33 (dd, J1=4.2 Hz, J2=8.4 Hz, 1H),
7.82 (d, J=3.9 Hz, 1H); IR (KBr) 386, 3200, 3133, 1644,
1565, 1532, 1398, 1286, 806.
ꢀ
mp95–97 C (lit.17 98 ꢀC); 1H NMR d 8.62 (d,
J=1.5 Hz, 1H), 8.95 (d, J=1.5 Hz, 1H).
2-Amino-5-chloro-3-nitropyridine (17). The procedure of
Vaughan et al.11 was modified. Sulfuric acid (97%,
300mL) was placed in a 500 mL three-neck round-bottom
3-Amino-5-chloropicolinamide (20). The procedure of
McCaustland and Cheng was modified.18 Raney nickel

Z.-L. Zhou et al. / Bioorg. Med. Chem. 9 (2001) 2061–2071
2069
(10 g of 50% slurry in water) was washed with water
(100 mL), 5% AcOH (100 mL), water (100 mL), and
95% EtOH (3ꢃ100 mL). The slurry was added to a
solution of 5-chloro-3-nitropicolinonitrile (19, 3.0 g,
16.3 mmol) in 95% EtOH (100 mL). The mixture was
hydrogenated at 45 psi for 2.5 h. The mixture was then
filtered over a bed of Celite and washed with 95% EtOH
(2ꢃ100 mL). The red/brown filtrate was rotaevaporated
to dryness, leaving 2.7 g (95%) ofꢀthe title compound as
a light brown solid: mp162–163 C (lit.18 165–166 ꢀC);
1H NMR d 5.42 (bs, 1H), 6.05 (bs, 2H), 7.00 (d,
J=1.2 Hz, 1H), 7.71 (bs, 1H), 7.79 (d, J=1.2 Hz, 1H).
tralized with Na2CO3 (210 g). The mixture was placed in
the cold room overnight and then filtered. The filter
cake was washed with water (20 mL) and dried at 45ꢀC
for 6 h, giving 32.4 g (46%) of the title compound as yellow
ꢀ
flakes: mp134–136 C (lit.19 136–137ꢀC); 1H NMR d 2.62
(s, 3H), 8.02 (d, J=6.9 Hz, 1H), 8.10 (s, 1H), 8.13 (s, 1H).
4-Nitronicotinic acid N-oxide (24). The procedure of
Taylor and Crovetti20 was used. To concentrated H2SO4
(96%, 61 mL, d=1.84, 1.1 mol) in an ice-bath was
added 4-nitro-3-picoline-N-oxide (24, 9.0 g, 58 mmol)
slowly so as to maintain the internal temperature at 20–
25 ꢀC. To the resulting orange solution was added
ꢅ
3-Amino-5-chloropicolinic acid hydrochloride (2). Con-
centrated HCl (38%, 32 mL, d=1.20, 400 mmol) was
added to 3-amino-5-chloropicolinamide (20, 2.3 g,
13 mmol). The mixture was stirred and heated to reflux.
The resulting solution was refluxed (100 ꢀC) for 17 h.
The resulting mixture was cooled to rt, then placed in
the cold room for 3 h. The mixture was filtered, leaving
2.5 g (85%) of the title compound as its hydrochloride
Na2Cr2O7 2H2O (17.4 g, 58.4 mmol) over 45 min while
maintaining the internal temperature at 30–40 ꢀC. The
dark green mixture was stirred at 30 ꢀC for 2.5 h and
then poured onto ice (125 g). The mixture was diluted
with water (total volume: 215 mL) and placed in the
cold room overnight. The mixture was filtered and dried
in vacuo to give 2.8 (26%) of the title compound as a
ꢀ
light green solid: mp171 C (dec.) (lit.20 170–172 ꢀC); 1H
salt: mp235–236 ꢀC; H NMR (DMSO-d6) d 6.68 (brs,
NMR (DMSO-d6) d 8.13 (d, J=6.9 Hz, 1H), 8.47 (d,
J=5.1 Hz, 1H), 8.64 (s, 1H).
1
2H), 7.33 (d, J=1.8 Hz, 1H), 7.80 (d, J=2.1, 1H). calcd
for C6H6Cl2N2O2: C, 34.47; H, 2.89; N, 13.40. Found:
C, 34.19; H, 2.75; N, 13.40.
4-Aminonicotinic acid (3). The method of Herz and
Murty was modified.21 4-Nitronicotinic acid N-oxide
(24, 0.8 g, 4 mmol) was added to water (20 mL). The
slurry was stirred while concentrated NH4OH (29%,
d=0.9, 0.6 mL, 4 mmol) was added dropwise. The
resulting suspension was diluted with water (total
volume, 40 mL) and then 10% Pd/C (0.2 g) was added.
The resulting mixture was hydrogenated at 50 psi for
2 h. The mixture was filtered over a bed of Celite and
washed with 0.3% NH4OH (20 mL). The filtrate was
concentrated to 10 mL and neutralized with 4 N HCl
(four drops). The mixture was filtered leaving 0.2 g
(40%) of the title compound as a gray solid: mp 327.5–
5-Chloro-2-cyano-3-methoxypyridine (21a). To a solu-
tion of 5-chloro-3-nitropicolinonitrile (19) (183.5 mg,
1.0 mmol) in 5 mL of methanol was added 1.2 mL of
10% sodium hydroxide aqueous solution at 0 ꢀC. The
resulting solution was allowed to stir at rt for 12 h.
Methanol was evaporated in vacuo to give a suspension,
which was neutralized with 4 N HCl to pH=6. A yellow
solid was collected by filtration and dried in vacuo to
give 110 mg (65%) of the product: mp 93–95 ꢀC; 1H
NMR d 4.00 (s, 3 H), 7.36 (s, 1H), 8.25 (s, 1H). Anal.
calcd for C7H5ClN2O: C, 49.87; H, 2.99; N, 16.62.
Found: C, 49.91; H, 3.02; N, 16.5.
329.5 ꢀC (lit.21 330 ꢀC); H NMR (DMSO-d6) d 6.71 (d,
1
J=6.0 Hz, 1H), 8.54 (s, 1H).
5-Chloro-2-cyano-3-ethoxypyridine (21b). To a solution
(134 mg,
of
5-chloro-3-nitropicolinonitrile
(19)
3-Aminoisonicotinic acid (4). The procedure of Chen
and Deady was modified.15 3,4-Pyridinedicarboximide
(25, 4.95 g, 30 mmol) was added in portions to an ice-
cold solution of sodium hypobromite, prepared by
adding Br2 (1.75 mL, d=3.102, 34.0 mmol) to 100 mL of
an ice-cold 10% NaOH solution. More 10% NaOH
(60 mL) was added and the resulting yellow solution
was heated over 25 min to 85 ꢀC. The stirred solution
was maintained at 85 ꢀC for 10 min and then cooled to
rt. The solution was cooled in an ice bath and acidified
to pH 3 with 50% H2SO4 (20 mL). The mixture was
suction filtered, leaving an orange/brown solid. This
solid was dried at 45 ꢀC for 1.5 h to leave 3.7 g (79%) of
the title compound as an orange solid: mp 283–285 ꢀC
0.73 mmol) in 5 mL of ethanol was added 0.8 mL of
10% sodium hydroxide aqueous solution at 0 ꢀC. The
resulting solution was allowed to stir at rt for 12 h.
Methanol was evaporated in vacuo to give a suspension,
which was neutralized with 4 N HCl to pH=6. A yellow
solid was collected by filtration and dried in vacuo to
give 75 mg (55%) of the product: mp 53–55 ꢀC; 1H
NMR d 1.53 (t, J=6.9 Hz, 3H), 4.20 (q, J=6.9 Hz, 2H),
7.33 (s, 1H), 8.23 (s, 1H). Anal. calcd for C8H7ClN2O:
C, 52.62; H, 3.86; N, 15.34. Found: C, 52.47; H, 3.80; N,
15.35.
4-Nitro-3-picoline-l-oxide (23). The method of Taylor
and Crovetti was utilized.19 To a cooled (0–5 ꢀC) mix-
ture of concentrated HNO3 (70%, 137.5 mL, d=1.40,
2.139 mol) and concentrated H2SO4 (96%, 175 mL,
d=1.84, 3.15 mol) was slowly added so as to maintain
internal temperature <10 ꢀC 3-picoline-l-oxide (22,
50.0 g, 0.458 mol). The resulting solution was heated
slowly to 100–105 ꢀC and held there for 2.5 h. The
orange solution was cooled to rt, and then poured over ice
(500 g). The green/yellow solution was partially neu-
(lit.15 280 ꢀC); 1H NMR (DMSO-d6)
d 7.45 (d,
J=7.2 Hz, 1H), 7.71 (d, J=5.4 Hz, 1H), 8.18 (s, 1H).
Ethyl 2-amino-5-chloronicotinate (11). To a solution of
ethyl 2-aminonicotinate (10, 163 mg, 0.99 mmol) in 1 mL
of concentrated HCl (35%) was added 0.10 mL of H2O2
(30%, d=1.110, 0.98 mmol). The resulting solution was
heated at 55–60 ꢀC for 2 h. The solution was cooled to
rt, diluted with water (10 mL) and basified to pH 8 (pH

2070
Z.-L. Zhou et al. / Bioorg. Med. Chem. 9 (2001) 2061–2071
paper) with solid NaHCO3. The mixture was filtered,
washed with water (2ꢃ10 mL) and dried at 45 ꢀC for 2 h
to leave 0.186 g (94%) of the title compound as a white
213 (35), 212 (15), 211 (100), 197 (15), 196 (15), 183 (15),
181 (30), 157 (15), 155 (50), 154 (20), 153 (25), 139 (15),
128 (15), 127 (20), 126 (15), 125 (15), 114 (15), 112 (50),
103 (15), 91 (25), 76 (30), 73 (20), 65 (15), 64 (45), 52
(25), 38 (25); HR-MS calcd for C8H4ClN3O4: 240.9890.
Found: 240.9888.
solid. Mp119.5–121.5 ꢀC; 1H NMR
d 1.39 (t,
J=7.05 Hz, 3H), 4.35 (q, J=7.05 Hz, 2H), 6.44 (bm,
2H), 8.11 (d, J=2.40 Hz, 1H), 8.16 (d, J=2.4 Hz, 1H);
IR (KBr): 3448, 3441, 3284, 3161, 1709, 1641, 1402,
1307, 1232, 1150, 1109, 796; HPLC: 100%; EI-MS (m/
e): 202 (30), 200 (M+, 100), 172 (15), 156 (20), 155 (30),
154 (60), 130 (15), 129 (20), 128 (50), 127 (45), 126 (15),
100 (15), 92 (10), 73 (20), 65 (15), 64 (15); HR-MS calcd
for C8H9ClN2O2: 200.0352. Found: 200.0355.
Binding assay. The [3H]DCKA binding assay was per-
formed as described previously.8
Electrophysiology. Agonist concentration–response
curves were analyzed as described previously.8 EC50 and
slope values for glycine on NR1a/2C receptors were
0.327 mM and 1.81, respectively (n=4). Kb values were
estimated from five point concentration-inhibition
data using a generalized form of the Cheng–Prusoff
equation.
Ethyl 3-acetylamido-5-chloropicolinate (26). To a solu-
tion of ethyl 3-amino-5-chloropicolinate (7, 2.0 g,
10 mmol) in 10 mL of 1,4-dioxane was added 4 mL of
acetic anhydride. The resulting solution was allowed to
stir at 50 ꢀC for 24 h. Solvent was evaporated and water
(10 mL) was added to the residue. The mixture was
neutralized by saturated sodium bicarbonate solution to
pH=7. A pale yellow solid was collected by filtration
and dried iꢀn vacuo, giving 2.0 g (83%) oꢀf the product:
mp98–100 C; 1H NMR d 1.48 (t, J=7.2 Hz, 3H), 4.45
(q, J=7.2 Hz, 3H), 8.35 (d, J=2.1 Hz, 1H), 9.22 (d,
J=2.1 Hz, 1H), 11.09 (s, 1H).
Acknowledgements
Financial support of research at the University of
Oregon by CoCensys, Inc. is gratefully acknowledged.
References and Notes
5 - Aza - 7 - chloro - 4 - hydroxyquinolin - 2(1H) - one (27).
KHMDS (6.0 mL of 0.5 M soln in toluene) was placed
in a 25 mL round-bottom flask under nitrogen. The
solution was cooled to ꢁ78 ꢀC (acetone/dry ice). After
15 min, a solution of ethyl 3-amido-(N-acetyl)-5-chlor-
opicolinate (26, 243.0 mg, 1.00 mmol) in dry THF
(4.0 mL) was added via syringe. The resulting mixture
was stirred under nitrogen and allowed to warm to rt.
The mixture was stirred at rt overnight. The reaction
mixture was quenched with water (10 mL) and washed
with EtOAc (2ꢃ5 mL). The aqueous layer was acidified
to pH <2 by the addition of 4 N HCl. The resulting
mixture was filtered and the filter cake washed with
water. The solid was dried in vacuo to leave 152.8 mg
(78%) of the title compound as a light brown solid: mp
294–296 C; H NMR (DMSO-d6) d 5.86 (s, 1H), 7.66
(s, 1H), 8.42 (s, 1H), 11.35 (s, 1H); IR (KBr): 3434,
3134, 2929, 2854, 1682, 1470, 1396, 1239, 1218, 1164,
802; HPLC: 98.2%; EI-MS (m/z): 198 (35), 196 (M+,
100), 170 (20), 168 (60), 127 (20), 113 (15), 69 (25), 64
(15); HR-MS calcd for C8H5ClN2O2: 196.0040. Found:
196.0045.
1. Dolbe, A. Therapie 1995, 50, 319.
2. Rothman, S. M.; Olney, J. W. Trends Neurosci. 1995, 18,
57.
3. Kulagowski, J. J.; Baker, R.; Curtis, N. R.; Leeson, P. D.;
Mawer, I. M.; Moseley, A. M.; Ridgill, M. P.; Rowley, M.;
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1
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Egbuwoku, Z. O.; Hawkinson, J. E.; Woodward, R. M.;
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9. Crum, J. D.; Fuchsman, C. H. J. Heterocycl. Chem. 1966,
3, 252.
10. Oakes, V.; Pascoe, R.; Rydon, H. N. J. Chem. Soc. 1956,
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11. Vaughan, J. R.; Krapcho, J.; English, J. P. J. Am. Chem.
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12. Canton, T.; Doble, A.; Miquet, J. M.; Jimonet, P.; Blan-
chard, J. C. J. Pharm. Pharmacol. 1992, 44, 812.
13. McCaustland, D. J.; Cheng, C. C. J. Heterocycl. Chem.
1970, 7, 467.
14. Fox, H. H. J. Org. Chem. 1952, 17, 547.
15. Chen, Q.; Deady, L. W. Aust. J. Chem. 1993, 46,
987.
5-Aza-7-chloro-4-hydroxy-3-nitroquinolin-2(lH)-one (28).
To a suspension of 5-aza-7-chloro-4-hydroxyquinolin-
2(1H)-one (25, 107.0 mg, 0.54 mmol) in 2 mL of glacial
acetic acid was added 0.2 mL of HNO3 (70%, d=1.40,
3 mmol). The orange mixture was heated at 100 ꢀC for
1 h, then cooled to rt. The mixture was quenched with
water (3 mL) and the mixture was filtered, giving yellow
crystals. The crystals were dried (2 h at 45 ꢀC) to give
45.6 mg (46%) of the title compound as yellow crystals:
ꢀ
1
mp252–254 C; H NMR (DMSO-d6) d 7.74 (s, 1H),
8.58 (s, 1H), 12.01 (d, J=1.5 Hz, 1H); IR (KBr) 3441,
3141, 3004, 2930, 2861, 1675, 1546, 1470, 1402, 1293,
1239, 1157, 11 02, 932, 904, 809, 625, 468; HPLC:
100%; EI–MS (m/e): 243 (25), 241 (M+, 80), 225 (30),
16. Hirai, E. Chem. Pharm. Bull. 1966, 14, 861.

Z.-L. Zhou et al. / Bioorg. Med. Chem. 9 (2001) 2061–2071
2071
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