Discovery, SAR, and pharmacokinetics of a novel 3-hydroxyquinolin-2(1H)-one series of potent D-amino acid oxidase (DAAO) inhibitors

Article abstract of DOI:10.1021/jm900128w

3-Hydroxyquinolin-2(1H)-one (2) was discovered by high throughput screening in a functional assay to be a potent inhibitor of human DAAO, and its binding affinity was confirmed in a Biacore assay. Cocrystallization of 2 with the human DAAO enzyme defined

Full text of DOI:10.1021/jm900128w

3576
J. Med. Chem. 2009, 52, 3576–3585
Discovery, SAR, and Pharmacokinetics of a Novel 3-Hydroxyquinolin-2(1H)-one Series of
Potent D-Amino Acid Oxidase (DAAO) Inhibitors†
Allen J. Duplantier,* Stacey L. Becker, Michael J. Bohanon, Kris A. Borzilleri, Boris A. Chrunyk, James T. Downs,
Lain-Yen Hu, Ayman El-Kattan, Larry C. James, Shenping Liu, Jiemin Lu, Noha Maklad, Mahmoud N. Mansour, Scot Mente,
Mary A. Piotrowski, Subas M. Sakya, Susan Sheehan, Stefanus J. Steyn, Christine A. Strick, Victoria A. Williams,‡ and
Lei Zhang
Pfizer Global Research and DeVelopment, Groton Laboratories, Eastern Point Road, Groton, Connecticut 06340
ReceiVed February 2, 2009
3-Hydroxyquinolin-2(1H)-one (2) was discovered by high throughput screening in a functional assay to be
a potent inhibitor of human DAAO, and its binding affinity was confirmed in a Biacore assay. Cocrystallization
of 2 with the human DAAO enzyme defined the binding site and guided the design of new analogues. The
SAR, pharmacokinetics, brain exposure, and effects on cerebellum D-serine are described. Subsequent
evaluation against the rat DAAO enzyme revealed a divergent SAR versus the human enzyme and may
explain the high exposures of drug necessary to achieve significant changes in rat or mouse cerebellum
D-serine.
Scheme 1a
Introduction
D-Serine, a coagonist at the glycine site on the N-methyl
D-aspartate (NMDA) receptor,1 is synthesized from L-serine by
serine racemase and is metabolized by D-amino acid oxidase
(DAAOa). Because phencyclidine (PCP), a noncompetitive
antagonist of the NMDA receptor, has been shown to produce
effects in healthy volunteers resembling the positive, negative,
and cognitive symptoms of schizophrenia,2 it has been proposed
that increasing function of this receptor, perhaps by inhibiting
the breakdown of D-serine by DAAO, may ameliorate schizo-
phrenic symptoms.3 In support of this idea, several factors have
linked low levels of D-serine to schizophrenia: (1) D-serine levels
in serum and CSF have been reported to be decreased in
schizophrenia patients vs healthy controls,4,5 (2) in schizophrenia
patients, DAAO activity and expression have been reported to
be increased,6-8 and (3) in small controlled clinical studies, oral
administration of D-serine (30 mg/kg/day) improved positive,
negative, and cognitive symptoms of schizophrenia as add-on
therapy to typical and atypical antipsychotics,9,10 with the
exception of clozapine.11 It has been suggested that coadmin-
istration of D-serine with a DAAO inhibitor may be a more
effective means of delivering D-serine to the brain.12
a Reagents and conditions: (i) ethyl diazoacetate, diethylamine, EtOH,
RT, 2 d; (ii) 1 N HCl, RT, 12 h; (iii) LiOH (10 equiv), 1:1 MeOH/water,
150 °C, microwave, 2 h; (iv) 1 N HCl, RT; (v) (TMS)diazomethane,
diethylamine, EtOH, RT, 18 h; (vi) BBr3 (3 equiv), CH2Cl2, -78 °C to
RT, 3 h.
Chemistry. The construction of the quinolinone ring has
recently been reviewed,16 and several syntheses of compound
2 have been reported in the literature: (a) ring expansion reaction
of isatin with diazomethane,17 (b) Eistert ring expansion of isatin
with ethyl diazoacetate,18 and (c) treatment of 2-aminobenzal-
dehyde with chloroacetic anhydride.19 In addition, syntheses of
some chloro (7,17 8,20 and 919), methyl (13,19 23,21 24,22 and
2523) and pyridyl (1824) analogues of 2 have also been reported
(see Table 1 for structures). Modified versions of the literature
routes for 2 starting from the appropriate isatin were used for
the preparation of compounds 2-5, 7-12, 14-17, and 26-27
(Scheme 1). Intermediate “A” (Scheme 1) was obtained in
40-85% yields by treatment of the appropriate isatin with ethyl
diazoacetate in the presence of diethylamine over a couple of
days followed by addition of dilute aqueous HCl. The resulting
ethyl ester was saponified and decarboxylated in the microwave
at 150 °C over two hours to provide 6-, 7-, and 8- substituted
3-hydroxyquinolin-2(1H)-ones in low (<20%) to moderate
(>50%) yields. 5-Substituted analogues did not decarboxylate
under these conditions. Alternatively, substituted isatins
were treated with TMS-diazomethane in the presence of
diethylamine to provide methoxy intermediates “B” (Scheme
1), which were then demethylated with boron tribromide.25 The
7-azaisatin reagent needed for the synthesis of 27 (compound
Reported DAAO inhibitors such as benzoic acid,13 5-meth-
ylpyrazole-3-carboxylic acid (AS057278),14 6-chlorobenzo-[d]-
isoxazol-3-ol (CBIO),12 and 5-carboxyfuro[3,2-b]pyrrole (1)15
are small aryl carboxylic acids or acid-isosteres that are ionized
(pKa’s ) 4-5) at the peroxisomal pH of ∼8.2. High-throughput
screening of our compound file against the human DAAO
enzyme uncovered lead compounds with pKa’s > 8 that are not
completely ionized within the peroxisome. Herein, we report
the binding mode, SAR, and pharmacokinetics of 3-hydrox-
yquinolin-2(1H)-one (2, pKa ) 8.7) and its analogues as potent
and novel DAAO inhibitors (Chart 1).
†
Protein Data Bank: PDB ID number 3G3E.
* To whom correspondence should be addressed. Phone: 860-441-6009.
Fax: 860-715-2350. E-mail: allen.j.duplantier@pfizer.com.
‡
Current address: Westhill High School, Stamford, CT.
a Abbreviations: DAAO, D-amino acid oxidase; DDO, D-aspartate
oxidase; FAD, flavin adenine dinucleotide; HRP, horseradish peroxidase;
NMDA,N-methylD-aspartate;CSF,cerebralspinalfluid;SAR,structure-activity
relationship.
10.1021/jm900128w CCC: $40.75
2009 American Chemical Society
Published on Web 05/13/2009

3-Hydroxyquinolin-2(1H)-one Series of DAAO Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3577
Chart 1
Scheme 2a
reserved in subsequently designed compounds. Figure 2 is a
schematic diagram comparing the bonding interactions of
compound 2 with those of benzoic acid. As seen in Figures 1
and 2, the aryl ring of 2 is in contact with multiple hydrophobic
residues (Ile230, Leu215, Leu51, and Gln63), leaving no room
for large multiatom substitution off of that ring.
a Reagents and conditions: (i) NBS (4 equiv), tert-BuOH, RT, 2 h, 97%;
Structure optimization studies were initially based on the
predicted binding interactions of compound 2 with the enzyme
active site. Compound 2 was docked into a cocrystallized
h-DAAO-benzoic acid structure29 using our in-house AG-
DOCK-docking program.30 It was observed that 3-hydrox-
yquinolin-2(1H)-ones dock to DAAO in the same manner as
do small carboxylic acid inhibitors. All interactions observed
from the X-ray structure of 2 are conserved in the AGDOCK
conformation of 2 and its close-in analogues. Perhaps due to
the simplicity of the binding site and the directionality afforded
by the polar interactions, docking of 2 and benzoic acid appear
largely straightforward, with the top ranked pose nearly identical
to the observed X-ray structure. In addition, the docking score31
shows a reasonable correlation (R2 ) 0.43) to the observed
binding activity for the ligands reported in Table 1 and is better
correlated to the observed activity than either simple physical
property descriptors (molecular weight, calculated log P, or polar
surface area) or orbital parameters (electronegativity, orbital
hardness).
Using the docking score as a tool to help define which
substituents would best fit in the binding pocket pointed clearly
to smaller substituents as being most favorable. Figure 3 shows
a box-plot representation of possible monosubstituted analogues
of compound 2 ordered by heavy atom count along the x-axis.
Here, 12 heavy atoms refers to the parent compound (2) as well
as 4 possible 3-hydroxy-naphthyridin-2(1H)-one analogues
(compounds 18-21); 13 heavy atoms refers to methyl, fluoro
and chloro (compounds 3-14); 14 heavy atoms refers to ethyl,
cyano, and methoxy (compounds 15-17); 15 atoms refers to
n-propyl and iso-propyl, and 18 heavy atoms refers to phenyl
substituted compounds. Compounds with 12 and 13 heavy atoms
scored the best, with an average predicted IC50 below 10 nM,
while compounds with 14 heavy atoms were predicted to be
much less active (∼50 nM). iso-Propyl and n-propyl substituents
were all predicted to have significantly diminished potency and
were not synthesized. The phenyl substituted compounds simply
can not fit in the active site, and the resulting unreliable docking
scores reflect impossible thermodynamic interactions of overlay-
ing bonds. This analysis reinforced the prediction that larger
substitutions would be detrimental to activity and, in addition,
only monofluoro substitutions were predicted to improve
potency beyond that of compound 2.
(ii) silver trifluoroacetate (2 equiv), 5% water/CH3CN, darkness, 3 h, 5%.
Scheme 3a
a Reagents and conditions: (i) ethyl methoxyacetate (3 equiv), lithium
bis(trimethylsilyl)amide (3 equiv), THF, -78 °C, 20 min, then amino
aldehyde, -78 °C to RT over 18 h, then 6 N HCl, reflux, 2 h; (ii) BBr3 (3
equiv), CH2Cl2, -78 °C to RT, 3 h.
30) was prepared from azaindole 28 by reaction with NBS
followed by silver trifluoroacetate (Scheme 2).26 In cases where
an isatin starting material was not readily available, the
corresponding aryl amino aldehyde (or ketone) was condensed
with the enolate of ethyl methoxyacetate to provide methoxy
intermediate “C” (Scheme 3), which was subsequently dem-
ethylated with boron tribromide (compounds 6, 13, and 18-23).
Biology. Potencies of compounds were determined in a cell
free fluorescence assay using recombinant human or rat DAAO
in which the H2O2 generated from the degradation of D-serine
was linked to oxidation of Amplex Red in the presence of
horseradish peroxidase (HRP). A comparable assay using
recombinant human D-aspartate oxidase (DDO) was run to
determine selectivity against DDO,27 the closest human homo-
logue to DAAO, and to eliminate any artifact arising from
inhibition of HRP. Binding affinity and kinetics were measured
using biotinylated recombinant human DAAO bound to a
Neutravidin surface in a Biacore binding assay. In vivo activity
of selected compounds was established by LC/MS-MS quan-
titation of D-serine in cerebellum extracts from male Sprague-
Dawley rats or male 129 SVEV mice treated with inhibitors.
Structure Based Drug Design. Compound 2 was cocrys-
tallized with the h-DAAO enzyme. The X-ray structure of 2
bound to h-DAAO (Figure 1) shows the 3-hydroxyl group
involved in two hydrogen bonds, one with the Tyr228-OH and
the other with the Arg283-NH. Additional interactions to the
Arg residue are made by the 2-carbonyl group of 2, while the
lactam -NH donates a hydrogen bond to the backbone carbonyl
of Gly313. Consistent with similar structures of aryl acids bound
to DAAO,28 compound 2 forms a π-π stacking interaction with
the re-face of the flavin ring of flavin adenine dinucleotide
(FAD). The aryl chain of Tyr224 stacks on the other side of
compound 2. Because all reported DAAO inhibitors12-15 have
an aromatic moiety that is likely involved in similar π-π
stacking interactions with FAD and Tyr224, these interactions
are considered important to compound 2 binding and are
Results and Discussion
Compound 2 was reported in the literature to be a weak
inhibitor of [3H]-glycine binding to the site associated with the
NMDA receptor (29% inhibition at 10 µM)17. In our hands, 2
was found to be a potent inhibitor of h-DAAO (IC50 ) 4 nM)
with good selectivity against h-DDO (IC50 ) 855 nM). Parallel
evaluation of 2 for stability in human microsomes suggested
low metabolic clearance, and a low MDR/MDCK ratio32 (0.35)

3578 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Table 1. Human and Rat in Vitro Potencies of Compounds 1-27
Duplantier et al.

3-Hydroxyquinolin-2(1H)-one Series of DAAO Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3579
Table 1. Continued
a Human D-amino acid oxidase assay, geometric mean (95% confidence interval). b Human D-aspartate oxidase assay. c Rat D-amino acid oxidase assay,
mean ( SD.
unbound fraction of drug in the brain (Fubrain ) 0.22), and this
translated into free brain exposures of 670 nM (160× over the
h-DAAO IC50 of 4 nM) at the 4 h time point. Because of
solubility constraints in the vehicle, we were unable to increase
the dose of 2 further. With the above data in hand, our goals
for follow-up of 2 were to improve oral bioavailability and lower
clearance while retaining nM potency and low molecular weight
and ultimately to lower the dose of 2 required to see significant
changes in D-serine levels.
Initial SAR development of the 3-hydroxyquinolin-2(1H)-one
series was based on our understanding of the binding mode of
2 at the active site of the h-DAAO enzyme (Figures 1 and 2).
Knowing that substitution of multi-atom groups around the aryl
ring of 2 would not be allowed at the active site, we explored
the effect of smaller groups by systematically substituting fluoro,
chloro, methyl, and ethyl groups around the 5-, 6-, 7-, and 8-
positions of the quinolinone ring (compounds 2-17, Table 1).
As the fluoro group was well tolerated in all positions
Figure 1. Compound 2 (carbon atoms in magenta and oxygen in red)
at h-DAAO enzyme active site. Side chains of key interacting residues
(compounds 3-6), compounds substituted with the larger chloro
group displayed a range of h-DAAO activity with the 5-position
(compound 10) being most tolerated [IC50 comparison: 2 ) 10
(5-chloro) < 7 (8-chloro) < 8 (7-chloro) < 9 (6-chloro)]. The
analogous methyl analogues followed a similar trend with the
exception of the 6-methyl analogue 13 showing a more
substantial decrease in h-DAAO activity (IC50 ) 2,750 nM).
To test further the size limits of the h-DAAO active site, an
ethyl group was evaluated at the 5-, 7-, and 8-positions. The
5-ethyl analogue (15) showed potent activity (IC50 ) 8 nM),
but ethyl at the latter two positions (16 and 17) was detrimental
to activity, thus confirming the small binding site. Substitution
at the 4-position of the quinolinone ring was found to be
unforgiving, as the 4-methyl analogue 23 was >4000× less
active than 2.
are shown with carbon colored in green and nitrogen in blue. Hydrogen
bonding interactions are shown in dash. FAD is shown with carbons
in cyan. The coordinates of this structure have been deposited in the
Protein Data Bank (PDP ID number 3G3E).
for 2 meant that it was not likely a substrate for P-glycoprotein
and thus should have a reasonable chance of getting across the
blood-brain barrier (data not shown). In vivo PK studies of 2
in the rat revealed moderate clearance (46 mg/min/kg) and low
oral bioavailability (F ) 0.9%), resulting in low oral plasma
exposures (Cmax ) 5.5 ng/mL) (see Table 2). To determine the
ability of 2 to elevate cerebellum D-serine levels in the rat,
we dosed 2 subcutaneously and found that a minimum effective
dose of 56 mg/kg was required to significantly elevate D-serine
by 2-4 fold above vehicle. At this dose, compound 2 was
determined to have a brain/plasma ratio of 0.7 and a large

3580 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Duplantier et al.
Figure 2. Schematic representation of residues in the (left) DAAO-benzoic acid X-ray structure, and (right) DAAO-compound 2 X-ray structure.
Table 3. Human DAAO in Vitro Binding Data in Biacore Assay
h-DAAO
h-DAAO
kon
koff
compds IC50 nM KD nM ( SE (M-1s-1) ( SE
(s-1) ( SE
1
2
4
5
6
15
18
9
4
10
9
8
8
29 ( 2.3
13 ( 0.5
38 ( 0.9
17 ( 2.7
18 ( 3.2
25 ( 0.6
2 ( 0.2
6 ( 0.5 × 104
9 ( 0.4 × 104
9 ( 0.2 × 104
1 ( 0.1 × 105
5 ( 0.6 × 104
2 ( 0.05 × 10-3
1 ( 0.02 × 10-3
4 ( 0.02 × 10-3
2 ( 0.2 × 10-3
8 ( 1 × 10-4
1 ( 0.03 × 105 3 ( 0.03 × 10-3
8
3 ( 0.1 × 105
6 ( 0.6 × 10-4
Figure 3. Analysis of Docking Score versus number of heavy atoms
in analogues of 2.
Figure 4. Cerebellum D-serine levels in mice (normalized to vehicle),
10 mg/kg, sc, 4 h post dose.
Table 2. Rat Pharmacokinetic Data (3 mg/kg, po; 1 mg/kg, iv)
CLp
(mL/min/kg)
oral Cmax
(ng/mL)
because independently replacing either of the two nitrogen atoms
of 22 with carbon led to potent h-DAAO activity (compounds
18 and 21).
To rule out that the aforementioned analogues of 2 were not
inhibiting [3H]-glycine binding to the site associated with the
NMDA receptor, compounds 2-6, 10, 14, 15, and 18 were
evaluated33 and all were found to have <50% inhibition at 10
µM (data not shown). Therefore, we were not concerned that
this class of DAAO inhibitors would compete with D-serine at
the NMDA receptor.
Selected compounds from Table 1 were screened in rats for
plasma exposures after oral administration at 3 mg/kg (data not
shown). Of the compounds screened, the 7- and 6-fluoroquino-
linones (4 and 5) displayed the highest Cmax (754 and 216 ng/
mL, respectively), and further PK studies revealed good oral
bioavailability for both (see Table 2). In addition, 4 was shown
to have low clearance and a reasonable half-life (T1/2 ) 5.4 h).
compds
Vd (L/kg)
T1/2 (h)
%F
2
4
5
46
15
72
10.8
1.2
2.3
ND
5.4
0.9
0.9
38
44
5.5
754
216
Disruption of the key hydrogen bond interaction of 2 with
Arg283 (Figures 1 and 2) was accomplished by methylating
the 3-hydroxy group (compound 24). As predicted, 24 was
rendered inactive without the H-bond interaction with Arg283.
In a similar fashion, disrupting the interaction of 2 with Gly313
by methylating the amido nitrogen (compound 25) also greatly
reduced activity. These results led us to conclude that the amide
and 3-hydroxy moieties of 2 were critical for nM potency.
In efforts to improve solubility, we prepared the four
regiomeric naphthyridinone analogues of 2 (compounds 18-21)
as well as the pyridopyrazinone 22. Of these heterocyclic
analogues, 18 was the most active of the naphthyridinones (IC50
) 8 nM) and 22 was inactive. This latter result was surprising

3-Hydroxyquinolin-2(1H)-one Series of DAAO Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3581
Table 4. Mouse Exposure Data (10 mg/kg, sc, 4 h)
compds
[plasma] (nM)
Fu (plasma)
[plasma]free (nM)
[brain] (nM)
Fu (brain)
[brain]free (nM)
B/P
1
2
27
1340
28
1620
0.95
0.43
0.11
1270
12
178
1120
56
96
1.0
0.26
0.12
1120
15
12
0.8
2.0
0.1
With the bioavailability issue addressed, we returned to the
more potent of compounds 2-25 in efforts to obtain a better
understanding of the in vitro pharmacology of the series. As
shown in Table 1, h-DDO selectivity was not an issue because
all compounds tested had only weak h-DDO activity. Rat DAAO
enzyme (r-DAAO) activity was determined for most of the
compounds in Table 1, and r-DAAO potencies were found to
be 10-500 fold weaker than h-DAAO. Specifically, compound
2 was 54× less active at the rat enzyme (IC50 ) 215 nM),
possibly explaining the high free brain exposure (670 nM)
necessary to increase in vivo D-serine levels in the rat. We also
found the literature compound, pyrrole carboxylic acid 1, to be
50× weaker against the rat enzyme. Comparing h- and r-DAAO
enzyme, there is one deletion in r-DAAO that is near the
VAAGL hydrophobic stretch (L27 in human), and we speculate
that this may change the conformation of r-DAAO around that
area.34 SAR derived from the r-DAAO data suggested that either
halogen substitution at the 5-position of the quinolinone ring
(6 and 10) or replacement of the 8-carbon atom with nitrogen
(18) alleviated the potency differences between r- and h-DAAO.
In efforts to confirm h-DAAO binding affinity (KD), the most
potent compounds from Table 1 were evaluated in a Biacore
assay and the data is presented in Table 3. The KD for most
compounds tested was 2-4 fold weaker than the h-DAAO IC50
data, with the exception of 1,8-naphthyridinone 18, which had
a KD of 2 nM (4× more potent than its IC50). We were excited
to find that the potent binding affinity of 18 was due in part to
a slower off rate (koff ) 6 × 10-4).
carbon atoms with nitrogen did not further improve the h-DAAO
potency of 2. However, fluoro substitution at the 6- or 7-position
improved oral bioavailability, chloro substitution at the 5-posi-
tion improved r-DAAO potency, and replacement of the
8-carbon atom with nitrogen improved h-DAAO binding
affinity. The accumulation of these three SAR features led to
the design of 27, a 4 nM DAAO inhibitor against both rat and
human enzyme that elevated D-serine levels in the mouse at
free brain exposures three times its rat IC50. We conclude that
the ability of a compound to increase cerebellum D-serine levels
can be predicted based on free brain exposure versus its potency
in the same (or similar) species. The relationship of cerebellum
D-serine levels to activity in animal models will be reported in
future publications.
Experimental Section
Biology. DAAO in Vitro Activity Assay. Inhibitor compounds
are diluted in 100% DMSO starting at 4 mM in half log increments
to create an 11-point dose response curve. Each dilution is spotted
in duplicate, 0.5 µL/well, into black 384-well plates (Costar no.
3573). No inhibition control wells (ZPE) were spotted with 0.5 µL
of 100% DMSO, and 100% inhibition control wells (HPE) were
spotted with 0.5 µL of 4 mM 3-hydroxyquinolin-2(1H)-one in 100%
DMSO. Then 20 µL of assay buffer (100 mM Tris-HCL, pH 8.5)
containing 4 nM human or 40 nM rat DAAO enzyme expressed in
sf9 insect cells (produced and purified in-house), 80 uM flavin
adenine dinucleotide (Sigma no. F6625), 0.8 units horseradish
peroxidase (Sigma no. P8250), and 100 uM Amplex Red (Molecular
Probes no. A12222) were added to each well of the plate using a
Titertek MultiDrop-384 reagent addition device. Next, 20 microliters
of assay buffer containing D-serine (Sigma no. S4250) 0.4 mM for
human or 20 mM for rat DAAO were added using the MultiDrop.
The plates were spun at 1000 rpm to ensure all liquid was coalesced
to the bottom of the well. Exposure of Amplex Red to light must
be kept to a minimum. The reaction was then incubated in the dark
at ambient temperature for 10-30 min before reading the plates
on a Perkin-Elmer Envision 2103 multilabel reader using the
following settings: 10 flashes of the flash lamp, excitation filter
530 nm, emission filter 590 nm. The mean of the plate HPE and
ZPE control values was used to calculate % inhibition values for
each compound well, and nonlinear curve fitting was used to
calculate an IC50 value for each compound.
DDO in Vitro Activity Assay. The assay to measure DDO
activity was identical to the DAAO assay with the following
exceptions. Human DDO enzyme 0.2 nM, also expressed and
purified from sf9 insect cells in-house, was substituted for DAAO
and D-serine was replaced with 0.5 mM D-aspartic acid (Sigma no.
219096). The 100% inhibition control wells (HPE) were spotted
with 0.5 µL of 4 mM 1-naphthol in 100% DMSO.
Biacore Binding of DAAO Inhibitors. A custom Neutravidin
surface was generated using standard amine coupling methods from
Biacore on a CM5 sensor chip. An enzymatically biotinylated
h-DAAO was immobilized onto the Neutravidin surface to a level
of ∼9000 Ru. Binding studies were performed in 100 mM TRIS,
150 mM NaCl, 0.05% P20, 50uM FAD, 3% DMSO, pH 8.5 at 25
°C in a Biacore 3000 (GE Healthcare) instrument. Compounds were
injected at three concentrations, and binding responses were
processed using Scrubber 2 (BioLogic Software, Inc.) to align and
double reference the data. The kinetic data were fit globally to a
simple 1:1 interaction model using Biaeval (GE Healthcare) to
obtain the rate constants and affinity.
Compound 26 was designed by combining the 6-fluoro
substitution (5) that provided enhanced oral bioavailability
with the 4-chloro group (10) that allowed for improved
activity at the rat receptor. Incorporating further the 1,8-
naphthyridinone ring of 18 that improved h-DAAO binding
affinity (Biacore assay) led to 27, the first compound in this
series with single digit nM activity against both the r- and
h-DAAO enzyme (see Table 1).
In efforts to improve in vivo screening throughput and reduce
the amount of compound required for testing, we chose to use
the mouse (instead of the rat) to evaluate a compound’s ability
to increase cerebellum D-serine levels. Screening at 10 mg/kg,
sc in the mouse, Figure 4 shows the magnitude of cerebellum
D-serine changes for compounds 1, 2, 4, 5, 7, 14, 15, and 27.
Compound 27, the only analogue of 2 that increased D-serine
at this dose, is also the only compound tested with r-DAAO
IC50 < 10 nM. Free brain exposures of compounds 1, 2, and 27
in this experiment (Table 4) revealed that 1 and 27 had
exposures 2.6- and 3-fold over their r-DAAO IC50′s, whereas
compound 2 had a free brain exposure 14-fold below its
r-DAAO IC50 of 215 nM. This data suggests that a compound
with free brain concentrations greater than its IC50 in the
r-DAAO assay should increase cerebellum D-serine levels in
the mouse.
Conclusions
The 3-hydroxyquinolin-2(1H)-one 2 is a potent h-DAAO
inhibitor that binds within the small active site in a similar
manner as benzoic acid binds to h-DAAO. Placement of small
groups around the quinolinone ring and/or replacement of ring

3582 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Duplantier et al.
In Vivo Activity of DAAO Inhibitors. Male Sprague-Dawley
CD rats (Charles River Laboratories) or male 129/SVEV mice
(Taconic Laboratories, Hudson, NY) were housed under a 12 h
light-dark cycle with lights on at 0600. Food and water was
provided ad libitum, and animals were acclimated for 5-7 days.
Animals were handled and cared for according to the Guide for
the Care and Use of Laboratory Animals (National Research
Council, 1996), and all procedures were performed with the
approval of the Pfizer Animal Care and Use Committee. Rats were
dosed sc at 1-2 mL/kg and mice at 10 mL/kg with vehicle alone
or compound prepared in 5/5/90 vehicle (5% DMSO, 5% Cremo-
phor EL (Fluka), 90% 0.9% sodium chloride solution). At 4 h post
injection, animals were anesthetized using CO2 gas and cerebella
were dissected out and stored at -80 °C until processed for D-serine
measurements. Tissue was homogenized in 3 volumes PBS with a
5 mm stainless steel bead in a MixerMill 300 (Qiagen). Homogenate
was mixed with 16 volumes of HPLC grade acetonitrile and
centrifuged at 13000 rpm in refrigerated microcentrifuge for 15
min. A 0.1-0.2 mL aliquot of the supernatant was dried down in
a speed vacuum system and stored at -20 °C prior to analysis.
D-Serine Assay. Calibration Standards. Stock solutions of
D-serine (2 mg/mL, Fluka no. 84970) and 13C3, 15N DL-serine
(internal standard, 2 mg/mL, Cambridge Isotope Laboratories, no.
CNLM-4207) were prepared individually in HPLC-grade water.
Calibration standard (CS) samples were prepared by serial dilution
of D-serine stock solution with methanol/water (80/20) to yield
concentrations of 0.003, 0.006. 0.012, 0.023, 0.047, 0.094, 0.188,
0.375, 0.750, 1.50, and 3.00 µg/mL. Quality control (QC) samples
were similarly prepared to concentrations of 0.01, 0.1, and 1.0 µg/
mL. CS and QC samples were extracted with 4 volumes of
acetonitrile, centrifuged, and the supernatants evaporated to dryness
under a stream of nitrogen. Sets of dried CS and QC samples were
stored at -20 °C until needed.
were found to be g95% pure by this method. Reference compound
1 was prepared using literature procedures.35
UHPLC/MS Analysis. The UHPLC was performed on a Waters
ACQUITY UHPLC system (Waters, Milford, MA), which was
equipped with a binary solvent delivery manager, column manager,
and sample manager coupled to ELSD and UV detectors (Waters,
Milford, MA). Detection was performed on a Waters LCT premier
XE mass spectrometry (Waters, Milford, MA). The instrument was
fitted with an Acquity BEH (bridged ethane hybrid) C18 column
(30 mm × 2.1 mm, 1.7 µm particle size, Waters, Milford, MA)
operated at 60 °C.
General Procedure A. 7-Fluoro-3-hydroxyquinolin-2(1H)-one
(4). Step 1. 6-Fluoro-1H-indole-2,3-dione (200 mg, 1.2 mmol),
diethylamine (0.24 mL, 2.3 mmol), and ethyl diazoacetate (0.24
mL, 2.3 mmol) were dissolved in ethanol (15 mL) and stirred at
room temperature for 64 h. Removal of solvent in vacuo then
provided the diazo intermediate as an oil (LCMS m/z 278.0 [M-1]);
this was treated with 1 N HCl (75 mL) and allowed to react for
40 h at room temperature. Filtration of the reaction mixture yielded
ethyl 7-fluoro-3-hydroxy-2-oxo-1,2-dihydroquinoline-4-carboxylate
as an orange solid (144 mg, 48% yield). LCMS m/z 252.1 (M +
1). 1H NMR (400 MHz, CD3OD) δ 1.42 (t, J ) 7.0 Hz, 3H), 4.48
(q, J ) 7.0 Hz, 2H), 7.02 (m, 2H), 7.67 (dd, J ) 5.6, 8.9 Hz, 1H).
Step 2. Methanol (7.5 mL), water (7.5 mL), and the compound
from step 1 (100 mg, 0.4 mmol) were combined in a 30 mL
microwave tube and treated with LiOH (86 mg, 3.6 mmol). The
reaction was subjected to microwave conditions (Biotage Advancer,
150 °C, high power) for 2 h, with 30 s of prestirring. A white solid
was removed via filtration, and the filtrate was acidified to pH 0
with 1 N HCl. Filtration of the resulting precipitate provided the
title compound as a beige solid (70 mg, 68% yield). MS (APCI)
m/z 180.0 (M + 1). 1H NMR (400 MHz, CD3OD) δ 6.98 (m, 2H),
7.13 (br s, 1H), 7.51 (dd, J ) 6.0, 8.5 Hz, 1H).
General Procedure B. 3-Hydroxy-1,8-naphthyridin-2(1H)-
one Hydrobromide (18). Step 1. Ethyl methoxyacetate (0.71 mL,
6.0 mmol) was added to a -78 °C solution of lithium bis(trimeth-
ylsilyl)amide (1.0 M solution in THF, 6.0 mL, 6.0 mmol) in THF
(6 mL). After 20 min, a solution of 2-aminonicotinaldehyde (244
mg, 2.0 mmol) in THF (4 mL) was added. The resulting orange
solution was allowed to come slowly to room temperature as the
dry ice/acetone bath warmed up. After 18 h, the reaction was
quenched with 6 N HCl (2.2 mL, 13.2 mmol), providing a
precipitate; this mixture was heated to reflux for 1 h, then allowed
to stir at room temperature for 65 h. The liquid was decanted off,
and the semisolids were washed with DCM and triturated with
MeOH, providing 3-methoxy-1,8-naphthyridin-2(1H)-one as an off-
Sample Preparation. Aliquots of tissue samples, CS and QC
aliquots (previously extracted and dried), were reconstituted in LC/
MS buffer [95:5 methanol/(water + 10 mM ammonium formate
+ 0.1% formic acid)]. Internal standard DL-serine had been
previously added to the LC/MS buffer to a concentration of 0.5
µg/mL. Dried samples were reconstituted to original volume,
sonicated for 3 min, and vortexed for 1 min.
LC/MS-MS Conditions. A Shimadzu LC-10AD equipped with
a degasser and a CTC Analytics HTS PAL autosampler (Leap
Technologies) were used to inject 10 µL aliquots of the processed
samples/calibration standards/QC’s onto an Astec Chirobiotic-T
column (2.1 mm × 250 mm, 5 µm), run isocratically. The mobile
phase was a 95/5 mixture of mobile phase A (methanol) and phase
B (water + 10 mM ammonium formate + 0.1% formic acid).
Quantitation was achieved by MS/MS detection in positive ion
mode for analyte and internal standard using a Thermo Quantum
Ultra triple quadrupole mass spectrometer equipped with an ESI
source. Detection of the ions was performed in the selected reaction
monitoring (SRM) mode, monitoring the transition of m/z 106.1
f 60.1 for D-serine and m/z 110 f 63.1 for the internal standard.
The analytical data were processed by Xcalibur software (version
2). Calibration curves were acquired by plotting the peak area ratio
of analyte to internal standard against the nominal concentration
of calibration standards. Standard curves were calculated with a
4-parameter fit analysis using a 1/y weighting factor.
Chemistry. General Methods. Solvents and reagents were of
reagent grade and were used as supplied by the manufacturer.
All reactions were run under a N2 atmosphere. Organic extracts
were routinely dried over anhydrous Na2SO4. Concentration refers
to rotatory evaporaration under reduced pressure. Chromatography
refers to flash chromatography using disposable RediSepRf 4g silica
columns on a CombiFlash Companion automated system. Elemental
analyses were performed by QTI, Whitehouse, NJ (compounds 2,
4, 5, and 18). All target compounds were analyzed using ultra high
performance liquid chromatography/UV/ evaporative light scattering
detection coupled to time-of-flight mass spectrometry (UHPLC/
UV/ELSD/TOFMS). Unless otherwise noted, all tested compounds
1
white solid (167 mg, 48% yield). LCMS m/z 175.1 (M - 1). H
NMR (400 MHz, DMSO-d6) δ 3.82 (s, 3H), 7.21 (dd, J)7.7, 4.8
Hz, 1H), 7.23 (s, 1H), 8.00 (dd, J)7.8, 1.8 Hz, 1H), 8.35 (dd, J)4.8,
1.9 Hz, 1H), 12.28 (br s, 1H).
Step 2. 3-Methoxy-1,8-naphthyridin-2(1H)-one (20 mg, 0.11
mmol) was mixed with anhydrous DCM (2 mL) and cooled in a
dry ice/acetone bath. BBr3 (1.0 M solution in DCM, 0.46 mL, 0.46
mmol) was added, and the reaction was allowed to slowly warm
to room temperature. After stirring for 18 h, the reaction was cooled
to -78 °C again and quenched with methanol (5 mL). The mixture
was warmed to ambient temperature, solvents were removed under
reduced pressure, and the residue was triturated with DCM to
provide an off-white solid (23 mg, 86% yield). LCMS m/z 161.1
1
(M - 1). H NMR (400 MHz, CD3OD) δ 7.30 (s, 1H), 7.58 (dd,
J ) 7.9, 5.8 Hz, 1H), 8.46 (m, 2H). UHPLC: 94%(UV), 100%
(ELSD).
3-Hydroxyquinolin-2(1H)-one (2). General Procedure A, Step
1. Ethyl 3-hydroxy-2-oxo-1,2-dihydroquinoline-4-carboxylate was
prepared from 1H-indole-2,3-dione. Yellow solid (79% yield).
LCMS m/z 232.2 (M - 1). 1H NMR (400 MHz, CDCl3) δ 1.53 (t,
J ) 7.1 Hz, 3H), 4.60 (q, J ) 7.1 Hz, 2H), 7.30 (m, 1H), 7.37 (d,
J ) 8.3 Hz, 1H), 7.43 (m, 1H), 8.13 (dd, J ) 8.3, 1.1 Hz, 1H),
10.16 (br s, 1H), 11.28 (br s, 1H).
Step 2. Product was triturated with MeOH and EtOAc. Light-
brown solid (17% yield). LCMS m/z 160.4 (M - 1). 1H NMR (400

3-Hydroxyquinolin-2(1H)-one Series of DAAO Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3583
MHz, DMSO-d6) δ 7.08 (s, 1H), 7.12 (ddd, J ) 7.9, 6.8, 1.7 Hz,
1H), 7.27 (m, 2H), 7.49 (d, J ) 7.9 Hz, 1H), 9.46 (br s, 1H), 12.01
(br s, 1H).
dissolved in ethanol (5 mL) and stirred at 25 °C. After 18 h, the
resulting heterogeneous mixture was filtered and the solid was rinsed
with ethanol (2 × 1 mL) to provide 125 mg of 5-chloro-3-
methoxyquinolin-2(1H)-one as a gray solid. LCMS m/z 210.1 (M
+ 1).
Step 2. The title compound was prepared according to general
procedure B, step 2, except that 5-chloro-3-methoxyquinolin-2(1H)-
one was used as the starting material and the crude product was
slurried with hot methanol rather than triturated with DCM. Gray
solid (70% yield). LCMS m/z 194.0 (M - 1). 1H NMR (400 MHz,
DMSO-d6) δ 7.22 (s, 1H), 7.27 (m, 3H), 10.03 (br s, 1H), 12.24
(br s, 1H).
3-Hydroxy-8-methylquinolin-2(1H)-one (11). General Procedure
A, Step 1. Ethyl 3-hydroxy-8-methyl-2-oxo-1,2-dihydroquinoline-
4-carboxylate was prepared from 7-methyl-1H-indole-2,3-dione.
Beige solid (38% yield). LCMS m/z 246.2 (M - 1). 1H NMR (400
MHz, CDCl3) δ 1.50 (t, J ) 7.0 Hz, 3H), 2.49 (s, 3H), 4.57 (q, J
) 7.0 Hz, 2H), 7.20 (dd, J ) 8, 7.5 Hz, 1H), 7.27 (obscured by
solvent peak, presumed 1H), 7.85 (d, J ) 7.5 Hz, 1H), 9.29 (br s,
2H).
8-Fluoro-3-hydroxyquinolin-2(1H)-one (3). General Procedure
A, Step 1. Ethyl 8-fluoro-3-hydroxy-2-oxo-1,2-dihydroquinoline-
4-carboxylate was prepared from 7-fluoro-1H-indole-2,3-dione.
1
Orange solid (68% yield). LCMS m/z 250.1 (M - 1). H NMR
(400 MHz, CD3OD) δ 1.42 (t, J ) 7.1 Hz, 3H), 4.49 (q, J ) 7.1
Hz, 2H), 7.19 (m, 2H), 7.41 (m, 1H).
Step 2. Product was rinsed with pH 7 buffer, then methanol.
1
Tan solid (55% yield). LCMS m/z 178.0 (M - 1). H NMR (400
MHz, DMSO-d6) δ 7.10 (ddd, J ) 8, 8, 5.2 Hz, 1H), 7.13 (d, J )
1.6 Hz, 1H), 7.18 (ddd, J ) 10.3, 8.1, 1.4 Hz, 1H), 7.32 (br d, J )
7.8 Hz, 1H), 9.74 (br s, 1H), 12.06 (br s, 1H).
6-Fluoro-3-hydroxyquinolin-2(1H)-one (5). General Procedure
A, Step 1. Ethyl 6-fluoro-3-hydroxy-2-oxo-1,2-dihydroquinoline-
4-carboxylate was prepared from 5-fluoro-1H-indole-2,3-dione.
Gray solid (85% yield). LCMS m/z 250.2 (M - 1).
Step 2. Product was triturated with a mixture of MeOH/EtOAc.
1
Brown solid (19% yield). LCMS m/z 178.1 (M - 1). H NMR
Step 2. Purified by two sequential preparative thin layer
(400 MHz, DMSO-d6) δ 7.06 (s, 1H), 7.14 (ddd, J ) 8.7, 8.7, 2.9
Hz, 1H), 7.24 (dd, J ) 8.9, 5.2 Hz, 1H), 7.33 (dd, J ) 9.5, 2.9 Hz,
1H), 9.70 (br s, 1H), 12.06 (br s, 1H).
chromatographic separations on silica gel (1:1 ethyl acetate:
1
hexanes). Gray solid (23% yield). LCMS m/z 174.2 (M - 1). H
NMR (400 MHz, CDCl3) δ 2.50 (s, 3H), 6.83 (br s, 1H), 7.16 (dd,
J ) 7.5, 7.5 Hz, 1H), 7.20 (s, 1H), 7.24 (br d, J ) 7.5 Hz, 1H),
7.39 (br d, J ) 7.5 Hz, 1H), 9.51 (br s, 1H).
5-Fluoro-3-hydroxyquinolin-2(1H)-one (6). Step 1. Ethyl meth-
oxyacetate (253 uL, 2.15 mmol) was added to a solution of lithium
bis(trimethylsilyl)amide (1.0 M solution in tetrahydrofuran, 2.2 mL,
2.2 mmol) in THF (2 mL) at -78 °C. After 20 min, a solution of
2-amino-6-fluorobenzaldehyde (100 mg, 0.719 mmol) in THF
(2 mL) was added dropwise. The resulting yellow solution was
kept at -78 °C for 15 min and then allowed to warm to room
temperature, then 6 N HCl (360 uL, 2.2 mmol) was added and the
precipitate was collected by filtration. Purification by chromatog-
raphy (gradient: 100% DCM to 5:1 DCM:MeOH) provided
5-fluoro-4-hydroxy-3-methoxy-3,4-dihydroquinolin-2(1H)-one. White
3-Hydroxy-7-methylquinolin-2(1H)-one (12). General Procedure
1
A. Tan solid (88% yield). LCMS m/z 174.1 (M - 1). H NMR
(400 MHz, DMSO-d6) δ 2.32 (s, 3H), 6.94 (d, J ) 8.0 Hz, 1H),
6.98 (s, 1H), 7.04 (s, 1H), 7.34 (d, J ) 8.0 Hz, 1H).
3-Hydroxy-6-methylquinolin-2(1H)-one (13). General Procedure
A, Step 1. Ethyl 3-hydroxy-6-methyl-2-oxo-1,2-dihydroquinoline-
4-carboxylate was prepared from 5-methyl-1H-indole-2,3-dione.
Purification by chromatography (gradient: 3% ethyl acetate/heptane
to 100% ethyl acetate); dark-yellow solid (36% yield). LCMS m/z
248.1 (M + 1). 1H NMR (400 MHz, CDCl3) δ 1.52 (t, J ) 7.0 Hz,
3H), 2.44 (s, 3H), 4.59 (q, J ) 7.0 Hz, 2H), 7.26 (m, 2H), 7.84 (s,
1H), 9.72 (br s, 1H), 11.47 (br s, 1H).
Step 2. Purified by preparative silica gel thin layer chromatog-
raphy (70% ethyl acetate/hexanes); grayish-beige solid (25% yield).
LCMS m/z 174.2 (M - 1). 1H NMR (400 MHz, CDCl3) δ 2.42 (s,
3H), 6.85 (br s, 1H), 7.14 (s, 1H), 7.15 (m, 1H), 7.21 (m, 1H),
7.30 (s, 1H), 10.33 (br s, 1H).
1
solid (100 mg, 65% yield). MS (APCI) m/z 209.8 (M - 1). H
NMR (400 MHz, CD3OD) δ 3.64 (s, 3H), 4.11 (d, J ) 3.7 Hz,
1H), 5.24 (d, J ) 3.7 Hz, 1H), 6.73 (d, J ) 8.0 Hz, 1H), 6.80 (br
dd, apparent t, J ) 9, 9 Hz, 1H), 7.29 (ddd, apparent td, J ) 8.2,
1
8.2, 6.0 Hz, 1H). Partial H NMR of minor diastereomer: δ 3.45
(s, 3H), 3.77 (d, J ) 3.1 Hz, 1H), 5.02 (d, J ) 2.9 Hz, 1H).
General Procedure B, Step 2. Tan solid, 91% yield. LCMS
m/z 180.1 (M + 1). 1H NMR (400 MHz, CD3OD) δ 6.95 (dd, J )
10.1, 8.0 Hz, 1H), 7.12 (d, J ) 8.4 Hz, 1H), 7.27 (s, 1H), 7.32
(ddd, apparent td, J ) 8.2, 8.2, 5.8 Hz, 1H). UHPLC: 93% (UV),
100% (ELSD).
3-Hydroxy-5-methylquinolin-2(1H)-one (14). General Procedure
B, Step 1. 3-Methoxy-5-methylquinolin-2(1H)-one was prepared
from 2-amino-6-methylbenzaldehyde. Purification by chromatog-
raphy (gradient:DCM to 5:1 DCM:MeOH). The product crystallized
out of one of the fractions and was collected by filtration. White
8-Chloro-3-hydroxyquinolin-2(1H)-one (7). General Procedure
A, Step 1. Ethyl 8-chloro-3-hydroxy-2-oxo-1,2-dihydroquinoline-
4-carboxylate was prepared from 7-chloro-1H-indole-2,3-dione.
Yellow solid (78% yield). LCMS m/z 266.1 (M - 1).
Step 2. Product was triturated with EtOAc. Yellow solid (11%
yield). LCMS m/z 194.2 (M - 1). 1H NMR (400 MHz, DMSO-d6)
δ 7.14 (dd, J ) 7.8, 7.8 Hz, 1H), 7.15 (s, 1H), 7.43 (dd, J ) 7.8,
1.3 Hz, 1H), 7.50 (dd, J ) 8.0, 1.2 Hz, 1H), 9.86 (br s, 1H), 11.37
(br s, 1H).
7-Chloro-3-hydroxyquinolin-2(1H)-one (8). General Procedure
A. Purified by precipitating out of hot methanol. White amorphous
solid (51% yield). LCMS m/z 194.0 (M - 1). 1H NMR (400 MHz,
DMSO-d6) δ 6.59 (s, 1H), 7.03 (dd, J ) 8.5, 2.1 Hz, 1H), 7.20 (d,
J ) 2.1 Hz, 1H), 7.29 (d, J ) 8.5 Hz, 1H).
1
crystalline solid (17% yield). MS (APCI) m/z 187.8 (M - 1). H
NMR (400 MHz, CD3OD) δ 2.56 (s, 3H), 3.95 (s, 3H), 7.09 (d, J
) 7.2 Hz, 1H), 7.16 (d, J ) 8.2 Hz, 1H), 7.27 (m, 1H), 7.29 (s,
1H).
Step 2. Tan solid, quantitative yield. LCMS m/z 176.1 (M + 1).
1H NMR (400 MHz, CD3OD) δ 2.55 (s, 3H), 7.20 (d, J ) 7.0 Hz,
1H), 7.30 (d, J ) 7.8 Hz, 1H), 7.36 (dd, J ) 7.2, 7.2 Hz, 1H), 7.57
(s, 1H).
5-Ethyl-3-hydroxyquinolin-2(1H)-one (15). General Procedure
C, Step 1. A mixture of 5-ethyl-3-methoxyquinolin-2(1H)-one and
5-ethyl-3-hydroxyquinolin-2(1H)-one was prepared from 4-ethylin-
doline-2,3-dione. Tan solid (59% yield). Without further purifica-
tion, the mixture was carried directly to next step.
6-Chloro-3-hydroxyquinolin-2(1H)-one (9). General Procedure
A, Step 1. Ethyl 6-chloro-3-hydroxy-2-oxo-1,2-dihydroquinoline-
4-carboxylate was prepared from 5-chloro-1H-indole-2,3-dione.
Gray solid (66% yield). LCMS m/z 266.1 (M - 1).
Step 2. Brown solid (91% yield). LCMS m/z 190.1 (M + 1). 1H
NMR (400 MHz, CD3OD) δ 1.30 (tr, J ) 7.6 Hz, 3H), 2.94 (q, J
) 7.6 Hz, 2H), 7.18 (d, J ) 8.0 Hz, 1H), 7.27 (d, J ) 8.2 Hz, 1H),
7.37 (m, 1H), 7.55 (s, 1H).
Step 2. Beige solid (66% yield). LCMS m/z 196.1 (M + 1). 1H
NMR (400 MHz, DMSO-d6) δ 7.07 (s, 1H), 7.24 (d, J ) 8.7 Hz,
1H), 7.31 (dd, J ) 8.7, 2.5 Hz, 1H), 7.61 (d, J ) 2.5 Hz, 1H), 9.77
(br s, 1H), 12.13 (br s, 1H).
7-Ethyl-3-hydroxyquinolin-2(1H)-one (16). General Procedure
A, Step 1. Ethyl 7-ethyl-3-hydroxy-2-oxo-1,2-dihydroquinoline-4-
carboxylate was prepared from 6-ethyl-1H-indole-2,3-dione. Puri-
fied by chromatography (gradient: 50% EtOAc/heptane to 100%
EtOAc), 45% yield, LCMS m/z 260.2 (M - 1), containing ∼5%
6-ethyl-1H-indole-2,3-dione.
General Procedure C. 5-Chloro-3-hydroxyquinolin-2(1H)-one
(10). Step 1. 4-Chloro-1H-indole-2,3-dione (182 mg, 1.00 mmol),
diethylamine (0.207 mL, 2.0 mmol), and (trimethylsilyl)diaz-
omethane (2 M solution in hexanes, 1.0 mL, 2.0 mmol) were

3584 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Duplantier et al.
Step 2. Purified by column chromatography (gradient: 5%
EtOAc/heptane to 100% EtOAc), followed by preparative silica
gel TLC (50% EtOAc/hexanes). Brown solid (1.9 mg, 0.01 mmol,
5% yield). LCMS m/z 188.2 (M - 1). 1H NMR (400 MHz, CDCl3)
δ 1.28 (t, 3H, presumedsobscured by solvent), 2.74 (q, J ) 7.5
Hz, 2H), 6.87 (s, 1H), 7.11 (m, 2H), 7.20 (s, 1H), 7.43 (d, J ) 8.1
Hz, 1H), 11.15 (br s, 1H).
7-Hydroxypyrido[2,3-b]pyrazin-6(5H)-one (22). General Proce-
dure B, Step 1. Solid was dissolved in MeOH and filtered through
a 0.2 µm nylon membrane 13 mm syringe filter. Filtrate was
concentrated to give an amorphous solid. LCMS m/z 164.1 (M +
1
1). H NMR (400 MHz, CD3OD) δ 6.13 (s, 1H), 8.52 (d, J ) 2.2
Hz, 1H), 8.61 (d, J ) 2.0 Hz, 1H).
3-Hydroxy-4-methylquinolin-2(1H)-one (23). General Procedure
B, Step 1. 3-Methoxy-4-methylquinolin-2(1H)-one was prepared
from 1-(2-aminophenyl)ethanone. Pink solid (79%). LCMS m/z
190.1 (M + 1). 1H NMR (400 MHz, CD3OD) δ 2.50 (s, 3H), 3.88
(s, 3H), 7.33 (ddd, J ) 8.2, 7.2, 1.2 Hz, 1H), 7.37 (br d, J ) 8.3
Hz, 1H), 7.51 (ddd, J ) 8.3, 7.2, 1.4 Hz, 1H), 7.80 (br d, J ) 8.2
Hz, 1H).
8-Ethyl-3-hydroxyquinolin-2(1H)-one (17). General Procedure
C, Step 1. Purified by TLC prep plate (1:1 EtOAc/heptane and 1%
triethylamine). White gummy solid (10% yield). LCMS m/z 190.1
1
(M + 1). H NMR (400 MHz, CD3OD) δ 1.30 (tr, J ) 7.5 Hz,
3H), 2.90 (q, J ) 7.5 Hz, 2H), 7.15 (m, 2H), 7.24 (d, J ) 7.3 Hz,
1H), 7.35 (d, J ) 7.7 Hz, 1H).
Step2.Theresiduewastrituratedwithdiethylether.Pinkish-white
solid (8% yield). LCMS m/z 174.1 (M - 1). 1H NMR (400 MHz,
CD3OD) δ 2.40 (s, 3H), 7.26 (ddd, J ) 8.0, 7.0, 1.4 Hz, 1H), 7.30
(dd, J ) 8.2, 1.3 Hz, 1H), 7.37 (ddd, J ) 8.2, 7.0, 1.3 Hz, 1H),
7.68 (dd, J ) 8.0, 0.9 Hz, 1H).
5-Chloro-6-fluoro-3-hydroxyquinolin-2(1H)-one (26). General Pro-
cedure A, Step1. Ethyl 5-chloro-6-fluoro-3-hydroxy-2-oxo-1,2-
dihydroquinoline-4-carboxylate was prepared from 4-chloro-5-
fluoro-1H-indole-2,3-dione, except that the aqueous filtrate after
HCl treatment was extracted with DCM (2 × 15 mL) and the
combined organic layers concentrated in vacuo to provide additional
aliquots of intermediate (total: 220 mg, 0.77 mmol, 77% yield).
LCMS m/z 284.1 (M - 1).
3-Hydroxy-1,6-naphthyridin-2(1H)-one Hydrobromide (19). Gen-
eral Procedure B, Step 1. 3-Methoxy-1,6-naphthyridin-2(1H)-one
was prepared from 4-aminonicotinaldehyde. The residue was
triturated with ether to provide a tan solid which was subsequently
subjected to DCVC36 over silica gel (gradient: 100% DCM to 10:1
DCM:MeOH). White solid, 56% yield. APCI m/z 175.1 (M - 1).
1H NMR (400 MHz, CD3OD) δ 3.96 (s, 3H), 7.37 (s, 1H), 7.39
(m, apparent br d, J ) 6.0 Hz, 1H), 8.40 (d, J ) 6.0 Hz, 1H), 8.86
(s, 1H).
Step 2. A mixture of 3-methoxy-1,6-naphthyridin-2(1H)-one (25
mg, 0.14 mmol) in anhydrous DCM (2 mL) at -78 °C was treated
with BBr3 (1.0 M solution in dichloromethane, 1.14 mL, 1.14
mmol). After 4 d at ambient temp, the mixture was cooled to -78
°C and quenched with methanol (5 mL). The resulting mixture was
warmed to room temp, concentrated, and the residue was triturated
sequentially with DCM, EtOAc, and hot MeOH. Off-white solid
Step 2. Microwave reaction was carried out for 5 h. Beige solid
(9 mg, 0.042 mmol, 9% yield). LCMS m/z 212.1 (M - 1). 1H NMR
(400 MHz, DMSO-d6) δ 7.19 (s, 1H), 7.24 (dd, J ) 4.6, 9.1 Hz,
1H), 7.35 (dd, J ) 9.1, 9.1 Hz, 1H), 10.27 (br s, 1H), 12.27 (br s,
1H)∼.
1
(13.2 mg, 0.054 mmol, 39% yield). APCI m/z 161.1 (M - 1). H
NMR (400 MHz, CD3OD) δ 7.27 (s, 1H), 7.62 (d, J ) 6.6 Hz,
1H), 8.46 (m, apparent br d, J ) 6.6 Hz, 1H), 8.98 (s, 1H).
5-Chloro-6-fluoro-3-hydroxy-1,8-naphthyridin-2(1H)-one Hydro-
bromide (27). General Procedure C, Step 1. 5-Chloro-6-fluoro-3-
methoxy-1,8-naphthyridin-2(1H)-one was prepared from 4-Chloro-
5-fluoro-1H-pyrrolo[2,3-b]pyridine-2,3-dione (30). Beige solid (31
3-Hydroxy-1,7-naphthyridin-2(1H)-one Hydrobromide (20). Gen-
eral Procedure B, Step 1. 3-Methoxy-1,7-naphthyridin-2(1H)-one
was prepared from 3-aminoisonicotinaldehyde. Light-yellow solid,
25% yield. LCMS m/z 175.1 (M - 1). 1H NMR (400 MHz,
CD3OD) δ 3.97 (s, 3H), 7.25 (s, 1H), 7.60 (d, J ) 5.4 Hz, 1H),
8.27 (d, J ) 5.4 Hz, 1H), 8.54 (s, 1H).
1
mg, 24% yield). MS m/z 299.1 (M + 1). H NMR (400 MHz,
DMSO-d6) δ 3.29 (br s, 3H), 7.12 (br s, 1H), 8.52 (br s, 1H).
Step 2. Beige solid (16 mg, 40% yield). MS m/z 215.1 (M + 1).
1H NMR (400 MHz, DMSO-d6) δ 7.13 (s, 1H), 8.47 (m, 1H), 12.74
(br s, 1H). UHPLC: 92% (UV), 100% (ELSD).
Step 2. To a stirred mixture of methoxy-1,7-naphthyridin-2(1H)-
one (10.9 mg, 0.062 mmol) in anhydrous DCM (2 mL) at -78 °C
was added BBr3 (1.0 M solution in DCM, 1.32 mL, 1.32 mmol).
After stirring for 7 days at ambient temp, the mixture was cooled
to 0 °C and quenched with methanol (4 mL). The mixture was
warmed to room temperature, solvents were removed under reduced
pressure, and the residue was triturated with DCM and then with
20:1 DCM:MeOH. Light-gray solid (8 mg, 0.033 mmol, 53% yield).
3,3-Dibromo-4-chloro-5-fluoro-2,3-dihydro-1H-pyrrolo[2,3-b]pyri-
dine (29). To a stirred solution of 4-chloro-5-fluoro-1H-pyrrolo-
[2,3-b]pyridine (28) (2 g, 12 mmol) in tert-butanol (100 mL) was
added N-bromosuccinimide (8.35 g, 46.9 mmol). After 2 h, the
mixture was concentrated and the residue was dissolved in DCM
(100 mL), washed with water (100 mL), and concentrated to give
a brownish-beige solid (3.8 g, 94% yield). MS m/z 344.8 (M +
1
LCMS m/z 161.1 (M - 1). H NMR (400 MHz, CD3OD) δ 7.28
1
(s, 1H), 8.02 (d, J ) 6.2 Hz, 1H), 8.36 (d, J ) 6.2 Hz, 1H), 8.61
1). H NMR (400 MHz, DMSO-d6) δ 8.45 (d, J ) 2.2 Hz, 1H),
(s, 1H).
12.33 (br s, 1H).
4-Chloro-5-fluoro-1H-pyrrolo[2,3-b]pyridine-2,3-dione (30). To a
stirred solution of 3,3-dibromo-4-chloro-5-fluoro-2,3-dihydro-1H-
pyrrolo[2,3-b]pyridine (29) (3.8 g, 11 mmol) in 5% H2O-
acetonitrile (150 mL) in an aluminum foil wrapped flask was added
silver trifluoroacetate (4.87 g, 22.1 mmol). After stirring at reflux
for 1 h, the mixture was cooled to room temp and the precipitated
silver bromide was removed by filtration. The dark-green filtrate
was concentrated and the residue was triturated with ether and
DCM. The combined ether and DCM filtrates were concentrated
and purified by chromatography eluting with a gradient of 100%
n-heptane to 100% ethyl acetate to give a brown solid (114 mg,
3-Hydroxy-1,5-naphthyridin-2(1H)-one Hydrobromide (21). Gen-
eral Procedure B, Step 1. 3-Methoxy-1,5-naphthyridin-2(1H)-one
was prepared from 3-aminopyridine-2-carbaldehyde. White solid,
55% yield. LCMS m/z 175.1 (M - 1). 1H NMR (400 MHz,
CD3OD) δ 3.98 (s, 3H), 7.26 (s, 1H), 7.41 (dd, J ) 4.6, 8.3 Hz,
1H), 7.70 (br d, J ) 8.3 Hz, 1H), 8.45 (dd, J ) 1.2, 4.6 Hz, 1H).
Step 2. In a microwave vial equipped with a stir bar, finely
ground product from step 1 (10 mg, 0.06 mmol) was mixed with
anhydrous DCM (1 mL) and cooled in a dry ice/acetone bath.
BBr3 (1.0 M solution in DCM, 0.38 mL, 0.38 mmol) was added
and the reaction was warmed to room temperature over 20 min
and then heated in a microwave reactor for 10 min at 100 °C.
The tightly capped reaction was then subjected to conventional
heating at 60 °C. After 65 h, the mixture was cooled to 0 °C,
MeOH (3 mL) was added, and the resulting mixture was
concentrated in vacuo and the residue was triturated with DCM
and filtered. Gray solid (10.8 mg, 0.044 mmol, 73%). LCMS
1
5% yield). MS m/z 199.1 (M - 1). H NMR (400 MHz, DMSO-
d6) δ 8.54 (d, J ) 2.4 Hz, 1H), 11.85 (br s, 1H).
Acknowledgment. We thank Brian Rago for analytical
support measuring in vivo drug exposures, Katherine Fisher
and Jessica Adams for in vitro assay development, Nancy
Stratman for [3H]-glycine binding data, and James Valentine
and Don Tunucci for high-throughput screening of our
compound file.
1
m/z 161.0 (M - 1). H NMR (400 MHz, CD3OD) δ 7.23 (s,
1H), 7.79 (dd, J ) 5.7, 8.4 Hz, 1H), 8.20 (d, J ) 8.0 Hz, 1H),
8.53 (dd, J ) 1.2, 5.7 Hz, 1H).

3-Hydroxyquinolin-2(1H)-one Series of DAAO Inhibitors
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