Synthesis and biochemical evaluation of N-(4-phenylthiazol-2- yl)benzenesulfonamides as high-affinity inhibitors of kynurenine 3- hydroxylase

Article abstract of DOI:10.1021/jm970467t

In this paper we describe the synthesis, structure-activity relationship (SAR), and biochemical characterization of N-(4-phenylthiazol-2- yl)benzenesulfonamides as inhibitors of kynurenine 3-hydroxylase. The compounds 3,4-dimethoxy-N-[4-(3-nitrophenyl)thi

Full text of DOI:10.1021/jm970467t

4378
J . Med. Chem. 1997, 40, 4378-4385
Syn th esis a n d Bioch em ica l Eva lu a tion of
N-(4-P h en ylth ia zol-2-yl)ben zen esu lfon a m id es a s High -Affin ity In h ibitor s of
Kyn u r en in e 3-Hyd r oxyla se
Stephan Ro¨ver,* Andrea M. Cesura, Philipp Huguenin,† Rolf Kettler, and Andre Szente
Pharma Division, Preclinical Research, Nervous System Diseases and Isotope Laboratory, F. Hoffmann-La Roche Ltd.,
CH-4070 Basel, Switzerland
Received J uly 17, 1997X
In this paper we describe the synthesis, structure-activity relationship (SAR), and biochemical
characterization of N-(4-phenylthiazol-2-yl)benzenesulfonamides as inhibitors of kynurenine
3-hydroxylase. The compounds 3,4-dimethoxy-N-[4-(3-nitrophenyl)thiazol-2-yl]benzenesulfona-
mide 16 (IC50 ) 37 nM, Ro-61-8048) and 4-amino-N-[4-[2-fluoro-5-(trifluoromethyl)phenyl]-
thiazol-2-yl]benzenesulfonamide 20 (IC50 ) 19 nM) were found to be high-affinity inhibitors of
this enzyme in vitro. In addition, both compounds blocked rat and gerbil kynurenine
3-hydroxylase after oral administration, with ED50’s in the 3-5 µmol/kg range in gerbil brain.
In a microdialysis experiment in rats, 16 dose dependently increased kynurenic acid concentra-
tion in the extracellular hippocampal fluid. A dose of 100 µmol/kg po led to a 7.5-fold increase
in kynurenic acid outflow. These new compounds should allow detailed investigation of the
pathophysiological role of the kynurenine pathway after neuronal injury.
Ch a r t 1. Structures of Known Kynurenine
3-Hydroxylase Inhibitors
In tr od u ction
Pharmacological control of the cascade of toxic events
that lead to delayed neuronal cell death after stroke,
brain trauma, or microbial and viral infections remains
an unmet medical need. Research has centered on the
excitotoxin hypothesis of neuronal injury. This hypoth-
esis states that a massive release of excitotoxic amino
acid neurotransmitters, mainly glutamate, from dying
neurons aggravates the injury to the brain, in a vicious
circle of glutamate released by dying neurons and
concomitant Ca2+ influx into cells unaffected by the
primary insult but not able to cope with the metabolic
stress presented by excessively high intracellular Ca2+
levels.1,2 Excellent tools, in particular potent antago-
nists at the N-methyl-D-aspartate (NMDA) class of
glutamate receptors, have yielded insight into this
pathological cascade. These compounds have been
shown to be effective in animal models of stroke
especially when given immediately after the insult.3 At
the same time, endogenous substances were discovered
that exert neurotoxic or neuroprotective effects through
glutamate receptors, including two metabolites of the
kynurenine pathway4 for the metabolism of L-tryp-
tophan, namely kynurenic acid (KYNA) and quinolinic
acid (QUIN). KYNA is an unselective glutamate recep-
tor antagonist and has neuroprotective properties. It
also blocks the glycine site of the NMDA receptor and
has served as a lead compound in the development of
glycine site antagonists.5,6 QUIN on the other hand is
an agonist at NMDA receptors and therefore neurotoxic.
Under physiological conditions QUIN and KYNA
concentrations in brain tissues are low, possibly due to
the low expression level and heterogeneous distribution
of the kynurenine pathway enzymes. Recently, it has
been shown that cerebral QUIN concentrations rise dra-
matically under pathological conditions which are ac-
companied by brain inflammation.7,8 The rise in QUIN
concentration can be attributed to several factors: (a)
Indolamine 2,3-dioxygenase (IDO), the first enzyme of
the pathway in nonhepatic tissues, is inducible by
IFN-γ9 and enzyme activity rises up to 30-fold in in-
flammatory brain tissue.7,10 (b) Invading macrophages
bring with them their enzymatic machinery to produce
QUIN.11 (c) Microglial cells will produce QUIN from
L-tryptophan and/or kynurenine upon immune activa-
tion.12 Studies done at the National Institute of Mental
Health (NIMH) have shown that QUIN concentrations
rise dramatically in some neuroinflammatory conditions
in humans (AIDS-dementia complex) and that in dif-
ferent models of brain inflammation this rise in con-
centration closely follows the time course and severity
of neurological impairment.8,13-15 Evidence that this
rise in QUIN concentration is causative for or contrib-
utes to the delayed neurological damage has been
difficult to obtain due to the lack of potent and in vivo
active inhibitors of the kynurenine pathway enzymes.
Scientific debate has focused on QUIN concentrations
measured in inflammation models vs those reported to
be toxic in neuronal cell cultures instead. However,
QUIN concentrations reported to be toxic in culture
range from 0.1 µM after long-term exposure to 100 µM
in an acute setting.16-18
The scenario changed when in 1994 (m-nitrobenzoyl)-
alanine (m-NBA)19,20 and later FCE 2883321 (Chart 1)
were developed as inhibitors of kynurenine 3-hydrox-
ylase and reported to have neuroprotective properties.
Kynurenine 3-hydroxylase22 has been chosen as target
with the rational of decreasing QUIN concentration,
concomitantly shunting the metabolism of kynurenine
† Isotope Laboratory.
X Abstract published in Advance ACS Abstracts, November 15, 1997.
S0022-2623(97)00467-6 CCC: $14.00 © 1997 American Chemical Society

Inhibitors of Kynurenine 3-Hydroxylase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4379
Sch em e 1a
Sch em e 2a
a
Reagents: (a) pyridine, room temperature.
Sch em e 3a
a
Reagents: (a) sodium carbonate, room temperature; (b) N-
bromosuccinimide, dibenzoyl peroxide, CCl4, reflux; (c) sodium
hydroxide, 50% ethanol, reflux; (d) sodium azide, ammonium
chloride, DMF, 80 °C.
toward KYNA via kynurenine aminotransferase. Thus
the neuroprotective properties observed with m-NBA
and FCE 28833 have been ascribed, at least in part, to
elevated KYNA levels.21 However these compounds still
may not be potent enough in vivo for an unequivocal
assessment of the role of QUIN in animal models of
delayed neurological damage.
Herein, we describe our efforts to identify inhibitors
of kynurenine-3-hydroxylase using m-NBA and a screen-
ing hit, the sulfonamide 5, as lead structures. This has
resulted in the identification of 3,4-dimethoxy-N-[4-(3-
nitrophenyl)thiazol-2-yl]benzenesulfonamide 16 and
4-amino-N-[4-[2-fluoro-5-(trifluoromethyl)phenyl]thiazol-
2-yl]benzenesulfonamide 20, compounds which possess
high affinity and in vivo potency as inhibitors of
kynurenine-3-hydroxylase. These new compounds should
allow a more detailed assessment of the relevance of
QUIN and other kynurenine pathway metabolites in
animal models of neurological damage.
a
Reagents: (a) diphosgene, toluene, reflux; tert-butyl alcohol,
reflux; (b) t-BuLi, THF, Me3SnCl, -90 °C; (c) L-4-[(chlorocar-
bonyl)methyl]-5-oxooxazolidine-3-carboxylic acid benzyl ester,
[Pd2(dba)3]CHCl3, toluene, 70 °C; (d) 30% HBr in acetic acid;
propylene oxide, i-PrOH; (e) 3H2, Pd/C (10%), DMF.
ylstannanyl)phenyl]carbamic acid tert-butyl ester, which
was generated in situ from (2,6-dibromophenyl)carbamic
acid tert-butyl ester 22. The latter had to be prepared
from commercially available 2,6-dibromoaniline via the
diphosgene route. Modern protecting group chemistry
for the introduction of the Boc group failed due to the
steric bulk of the bromine atoms. L-[3-3H]kynurenine
25 was then prepared by catalytic tritiation of 24.
Ch em istr y
Resu lts a n d Discu ssion
The synthesis of the desamino analogue 1 of m-NBA
has been described previously.23 Isoxazoles 2 and 3
were prepared following the route depicted in Scheme
1. 1.3-Dipolar cycloaddition of hydroxycarboncyanidim-
ic chloride24 to 3-nitrostyrene yielded the dihydroisox-
azole 4, which was oxidized with NBS to afford upon
saponification the restricted rotation analogue 2 of
m-NBA and upon treatment with sodium azide its
restricted rotation tetrazole bioisostere 3.
Sulfonamides 5-20 were synthesized in a conven-
tional manner, condensing the appropriate arylsulfo-
chloride with 2-amino-4-arylthiazoles (Scheme 2), fol-
lowed by a deprotection step if necessary, as described
in the Experimental Section.
The synthetic access to L-3-bromokynurenine 24, a
convenient precursor for L-[3-3H]kynurenine 25, has
been inspired by the reported synthesis of L-kynure-
nine25 and is shown in Scheme 3. The key step involves
a Stille-type coupling of (S)-3-(benzyloxycarbonyl)-5-oxo-
4-oxazolidineacetyl chloride26 with [2-bromo-6-(trimeth-
The compounds described were evaluated in an in
vitro enzyme inhibition assay with rat kidney kynure-
nine 3-hydroxylase using L-[3-3H]kynurenine as sub-
strate. The results are given as IC50’s (concentration
required for 50% inhibition) and shown in Tables 1 and
2. Selected compounds were tested in an ex vivo enzyme
inhibition assay in rat liver and kidney homogenates
(Table 3, results as ED50s after oral administration of
the test compounds). The extremely low expression
level of kynurenine 3-hydroxylase in rat brain hampered
the determination of enzyme activity in this tissue.
Therefore, ex vivo kynurenine 3-hydroxylase inhibition
was also measured in gerbils after oral administration.
This species has measurable kynurenine 3-hydroxylase
activity in the brain. The results obtained in gerbils
are displayed in Table 4.
Choosing m-NBA as a lead compound our initial
attempt to design new inhibitors focused on one ques-
tion. Is the relatively low affinity of this compound (IC50
) 0.8 µM) an indication that not all functional groups

4380 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Ro¨ver et al.
Ta ble 3. Ex Vivo Inhibition of Kynurenine 3-Hydroxylase
Ta ble 1. Inhibition of Rat Kidney Kynurenine 3-Hydroxylase
by Compounds Related to m-NBAa
Activity in Kidney and Liver from Rats Treated with Various
Inhibitorsa
ED50 (µmol/kg po)
compd
kidney
liver
16
19
20
15
11
1.2 (0.8-1.3)
6.6 (5.4-8.4)
4.7 (3.6-5.6)
5.3 (4.3-6.9)
4.6 (4.2-4.9)
9.0 (5.8-13)
33.4 (28.6-41.7)
222 (105-466)
1.1 (1.0-1.2)
3.0 (2.4-3.2)
13.3 (11.0-15.8)
25.1 (17.1-36.9)
FCE 28833
a
The compounds were administered po 2 h before decapitation.
ED50 values were calculated using six different doses of the
inhibitors and four or five animals per group. Values in paren-
theses represent 95% confidence limits.
Ta ble 4. Ex Vivo Inhibition of Kynurenine 3-Hydroxylase
Activity in Brain and Peripheral Organs from Gerbils Treated
with Various Inhibitorsa
ED50 (µmol/kg po)
compd
brain
kidney
liver
16
19
20
5.5 (3.3-9.2)
5.2 (2.9-7.3)
3.0 (1.1-7.8)
0.8 (0.5-1.2)
1.8 (1.1-3.2)
0.9 (0.4-1.8)
0.4 (0.3-0.5)
0.9 (0.7-1.3)
0.7 (0.3-1.3)
FCE 28833 461 (119-1789)
22.3 (16.9-30.8)108 (68-173)
a
The compounds were administered po 2 h before decapitation.
ED50 values were calculated using six different doses of the
inhibitors and four or five animals per group. Values in paren-
theses represent 95% confidence limits.
a
IC50 values were calculated using at least nine inhibitor
concentrations. Values are the means ( SEM from three to six
experiments, each performed in triplicate.
Ta ble 2. Inhibition of Rat Kidney Kynurenine 3-Hydroxylasea
parameters for brain penetration. The finding that the
carboxylic acid can be replaced by its tetrazole bioiso-
stere, as in 3 (IC50 ) 1.2 µM), without loss of affinity,
turned our attention to sulfonamides as the other
classical carboxylic acid bioisosters and led ultimately
to the rapid identification of the sulfonamide 5 (IC50
0.1 µM) from our chemical library.
)
compd
R′
R′′
IC50 (nM)
Since sulfonamides were expected to have a superior
pharmacological profile with respect to oral bioavail-
ability,28 further optimization was done on the sulfon-
amide lead 5. Examination of the substituent effects29
in the “western” phenyl ring (compounds 6-11) revealed
a preference for lipophilic substituents, one of the best
substituents being 3,4-dichloro (10) (IC50 ) 0.06 µM).
6
7
8
9
10
11
12
13
14
15
16
17
18
H
4-Me
4-Me
4-Me
4-Me
4-Me
4-Me
H
470 ( 190
240 ( 41
56 ( 8
76 ( 14
56 ( 8
4-OMe
4-Cl
4-Me
3-Cl, 4-Cl
3-NO2
3-NO2
3-NO2
3-NO2
3-NO2
3-NO2
48 ( 12
200 ( 35
120 ( 36
84 ( 29
51 ( 16
37 ( 3
3-Cl, 4-Cl
4-Cl
4-OMe
3-OMe, 4-OMe
4-NH2
The high affinity of the 3-nitro compound (11) (IC50
)
0.05 µM), however, may indicate that these substituents
do not target a truly lipophilic site in the enzyme. The
high affinity of 3-nitro and 3,4-dichloro compound is a
surprising coincidence with the SAR of benzoylalanine-
type inhibitors of kynurenine 3-hydroxylase19,30 and may
suggest a common binding site for sulfonamide- and
benzoylalanine-type inhibitors. Support for this notion
comes from kinetic analysis of the mode of inhibition of
16 (Figure 1) and FCE 28833 (not shown), both of which
behaved as competitive inhibitors of kynurenine 3-hy-
droxylase with Ki values of 4.8 ( 2.1 and 95 ( 27 nM,
respectively.
Of the two lead compounds 10 and 11, only the latter
efficiently inhibited the enzyme after oral administra-
tion (ED50 ) 13.3 µmol/kg in the kidney, Table 3). A
somewhat higher ED50 in the liver (33.4 µmol/kg) may
reflect the fact that sulfonamides are cleared mainly via
the renal route and thus achieve highest concentration
levels in this organ,31 a property which they seem to
share with benzoylalanines such as FCE 28833 (Table
3).
3-NO2
3-NO2
2-F, 5-CF3
2-F, 5-CF3
40 ( 3
4-i-Pr
990 ( 20
39 ( 17
19 ( 2
19
20
3-OMe, 4-OMe
4-NH2
m-NBA
FCE 28833
774 ( 113
237 ( 32
a
IC50 values were calculated using at least nine inhibitor
concentrations. Values are the means(SEM from three to six
experiments, each performed in triplicate.
are involved in binding?27 The fact that the desamino
analogue 1 of m-NBA shows only 1 order of magnitude
lower affinity (IC50 ) 5.7 µM) suggested to us that the
amino group while improving affinity is not involved in
a salt bridge. Thus the binding motif of substrate type
inhibitors may constitute simply of a substituted phenyl
ring in the correct orientation and distance to a car-
boxylic acid function, with one or two heteroatoms as
electron-pair donors between the carboxylic acid and the
phenyl ring. Isoxazole 2 (IC50 ) 2.7 µM) supported this
hypothesis, providing a simplified non-amino acid struc-
ture. However, it still contained the carboxylic acid
function which limits optimization of physicochemical
Cursory investigation of the effect of substituents in
the “eastern” phenyl ring (compounds 12-17, Table 2)

Inhibitors of Kynurenine 3-Hydroxylase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4381
F igu r e 1. Kinetic analysis of the inhibition of kynurenine 3-hydroxylase in rat kidney mitochondria preparations by various
concentrations of 16 (Ro 61-8048). Panel A: saturation analysis. Panel B: double-reciprocal transformation plot of the data. The
data shown are representative of three experiments. Note that in this experiment, no preincubation period was performed and
the reaction was started by the addition of the enzyme preparation.
revealed that substituents with negative σ-values led
to the most potent compounds; however not much
difference in IC50’s were found between 4-methyl- (11,
0.05 µM), 4-methoxy- (15, 0.05 µM), 3,4-dimethoxy- (16,
0.04 µM), and 4-amino-substituted compounds (17, 0.04
µM). The reason for this poor differentiation is unclear,
although it might be due in part to a counterbalancing
effect of substituent size, which becomes more evident
in the 4-isopropyl compound (18, 1.0 µM). While 11,
15, and 16 are virtually equipotent in vitro, they differ
quite clearly in their efficacy after oral administration
(Table 3). With an ED50 of 1.2 µmol/kg po in the rat
kidney, 16 (Ro 61-8048) is about 10-fold more potent
than 11 and about 2-fold more potent than 15 after oral
administration.
While 16 achieved our initial goal of developing
kynurenine 3-hydroxylase inhibitors well suited for
assessing the relevance of the kynurenine pathway in
models of neurological damage, this compound is a
nitroaromat and thus a potential mutagen. While
searching for potential replacements of the 3-nitrophen-
yl group, we found that a 2-fluoro-5-(triflouromethyl)-
phenyl group was a suitable substitute. Compound 20,
which features this substitution pattern on the western
phenyl ring, is virtually equipotent to 16 both in vitro
(Table 2) and ex vivo (Tables 3 and 4).
We have studied the time course of kynurenine
3-hydroxylase inhibition after administration of a single
oral dose of 16 in gerbils and rats (Figure 2). In gerbils,
a dose of 30 µmol/kg po led to inhibition of the cerebral
enzyme which peaked after 2 h (∼85% inhibition) and
persisted for up to 8 h (Figure 2A). In gerbil kidney
and liver this dose of 16 led to >80% inhibition of the
enzyme for about 24 h. Recovery of enzyme activity in
rats was faster than that observed in gerbils (Figure
2B).
The effect of 16 on the cerebral outflow of KYNA was
F igu r e 2. Time course of kynurenine 3-hydroxylase inhibition
evaluated in rat hippocampus after implantation of a
in homogenates from gerbil (panel A) and rat (panel B) tissues
transversal microdialysis probe (Figure 3). The inhibi-
tor dose-dependently increased the outflow of KYNA,
with a single dose of 100 µmol/kg po (42 mg/kg po)
after oral administration of 30 µmol/kg 16 (Ro 61-8048). The
data are mean values ( SEM of four or five animals per
group: (circle) brain; (triangle) kidney; (square) liver.

4382 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Ro¨ver et al.
F igu r e 3. Effect of the kynurenine 3-hydroxylase inhibitors 16 (Ro 61-8048) (panel A) and FCE 28833 (panel B) on KYNA outflow
from rat hippocampus. Data are means ( SEM of three or four experiments as indicated in the figure. The basal level of KYNA
was 0.22 ( 0.01 pmol/sample (20 µL) corresponding to a concentration of 11.0 ( 0.5 nM (value not corrected for KYNA recovery
through the microdialysis probe). No significant changes in KYNA outflow were observed after administration of vehicle solution
to the animals.
leading to a concentration of this metabolite 7.5-fold over
basal levels. The concentration of KYNA peaked 4 h
after oral administration of the compound and remained
elevated longer than 6 h. A lower dose (50 µmol/kg po)
of 16 led only to a 4-fold increase in KYNA levels,
suggesting incomplete inhibition of kynurenine 3-hy-
droxylase at this dose. At 100 µmol/kg po FCE 28833
produced only a 3-fold increase in KYNA outflow which
returned to basal levels after 5 h (Figure 3B). Reports
of 8-12-fold increases in rat hippocampal KYNA con-
centrations have been made with m-NBA at 800 mg/kg
ip and FCE 28833 at 400 mg/kg po.19,21 Given the low
levels of kynurenine 3-hydroxylase in rat brain and the
massive increase in plasma KYNA,21 a substantial
proportion of the KYNA found in rat hippocampal
dialysate may come from the periphery.32 Thus relevant
doses, i.e., doses that might influence brain QUIN
concentrations in animal models of neurological damage,
might be higher than those needed to elevate brain
KYNA concentrations.
Exp er im en ta l Section
Ch em istr y. Gen er a l P r oced u r es. Melting points were
taken with a Bu¨chi 535 melting point apparatus and are
uncorrected. The 1H spectra were recorded on a Bruker AC250
instrument in DMSO (unless noted otherwise). Low-resolution
EI-MS spectra (EI: 70 eV) were recorded on a MS9 updated
with a VG ZAB console, Finnigan data system SS300, with
direct sample introduction. Microanalysis (C, H, N) were
performed on a Heraeus Vario EL. Halogens were determined
by standard titrimetric procedures. NMR data are reported
in parts per million (δ) relative to internal tetramethylsilane
and are referenced to the deuterium lock signal from the
sample solvent (d6-DMSO unless otherwise stated); coupling
constants (J ) are in hertz. All reactions were performed under
argon. Drying of organic solutions was with MgSO4, evapora-
tion in a rotary evaporator at 40 °C in vacuo as appropriate.
For chromatography, Merck silica gel 60 (size 70-230 mesh)
was used. Analytical TLC plates employed were Merck silica
gel 60 F-254 plates. Starting materials were high-grade
commercial products unless stated otherwise. The tritiation
apparatus was purchased from Radiumchemie AG.33 Radio-
chemical samples were counted in a Berthold BF 5020 liquid
scintillation counter using Safetron-150 as scintillation cock-
tail. Counting efficiency was determined by adding [3H]-
hexadecane standard (Amersham Radiochemicals) to each
sample. The radiochemical purity was determined on a TLC
linear analyzer LB284 from Berthold AG. The HPLC ap-
paratus consisted of a Anacomp 220 controller, T114 pump,
Uvikon 720 LC detector. The Millex-SR 0.5 µm filter car-
tridges were obtained from Millipore AG.
5-(3-Nit r op h e n yl)-4,5-d ih yd r oisoxa zole -3-ca r b on i-
tr ile (4). A solution of sodium carbonate in water (40 mL,
0.5 M) was added to a mixture of 3-nitrostyrene (11.1 mL, 80
mmol) and hydroxycarboncyanidimic chloride24 in diethyl ether
(44 mL, 0.9 M) at room temperature. Stirring was continued
overnight. The mixture was partitioned between diethyl ether
and water. The organic phases were pooled and dried with
MgSO4. Evaporation afforded 4 as colorless crystals (4.1 g,
47%); mp 101-103 °C; 1H NMR (CDCl3) 3.22 (dd, J ) 8.6, J )
17.4, 1H), 3.75 (dd, J ) 11.7, J ) 17.4, 1H), 5.97 (dd, J ) 8.6,
J ) 11.7, 1H), 7.66 (m, 2H), 8.19 (bs, 1H), 8.25 (m, 1H). Anal.
Calcd (C10H7N3O3) C, H, N.
Con clu sion
We have discovered N-(4-phenylthiazol-2-yl)benzene-
sulfonamides as inhibitors of kynurenine 3-hydroxylase.
This novel series of compounds yielded the most potent
kynurenine 3-hydroxylase inhibitors known so far,
namely 3,4-dimethoxy-N-[4-(3-nitrophenyl)thiazol-2-yl]-
benzenesulfonamide 16 (IC50 ) 37 nM, Ro 61-8048) and
4-amino-N-[4-[2-fluoro-5-(trifluoromethyl)phenyl]thiazol-
2-yl]benzenesulfonamide (20) (IC50 ) 19 nM). These
compounds were not only potent in vitro but were also
shown to inhibit kynurenine 3-hydroxylase after oral
administration, with ED50’s in the range of 3-5 µmol/
kg in gerbil brain. 16 (Ro 61-8048) increased kynurenic
acid concentration in extracellular hippocampal fluid of
rats, and appears to be more potent than FCE 28833.
The pharmacological features of the inhibitors described
should allow a more detailed investigation of the role
of the kynurenine pathway in certain neurological
disorders, in particular those associated with neuroin-
flammation.
5-(3-Nitr op h en yl)isoxa zole-3-ca r boxylic Acid (2). N-
Bromosuccinimide (2.0 g, 11 mmol) was added to a solution of
4 (2.5 g, 11 mmol) in tetrachloromethane (50 mL). Dibenzoyl
peroxide (0.14 g) was added and the mixture boiled overnight.
The mixture was cooled to room temperature and filtered and

Inhibitors of Kynurenine 3-Hydroxylase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4383
the filtrate evaporated. Chromatography on silica gel with
ethyl acetate/hexanes (1:4) yielded the 5-(3-nitrophenyl)-
isoxazole-3-carbonitrile 21 as a beige solid (1.4 g). This solid
(0.3 g, 1.4 mmol) was suspended in a mixture of water (10 mL),
ethanol (10 mL), and sodium hydroxide solution (28%, 4 mL).
The mixture was boiled for 1 h, and ethanol was evaporated.
The residue was acidified with HCl and the precipitate
collected. Stirring with diethyl ether/n-hexane (1:1) yielded
2 as a yellowish solid (0.08 g, 24%): mp >176 °C dec; 1H NMR
7.73 (s, 1H), 7.87 (t, 1H), 8.39 (t, 2H), 8.73 (s, 1H), 13.8 (bs,
1H); MS (high res) mass calcd for C10H6N2O5 (M•+) ) 234.0277;
obsd m/z at 234.0286, deviation from theoretical ) 0.9 mmu.
5-[5-(3-Nitr op h en yl)isoxa zol-3-yl]-1H-tetr a zole (3). To
a solution of 21 (0.8 g, 3.7 mmol) in DMF (10 mL) was added
sodium azide (0.3 g, 4.8 mmol) and ammonium chloride (0.26
g, 4.8 mmol). The mixture was stirred for 3 h at 80 °C and
cooled to room temperature. Solids were removed by filtration,
and the filtrate was evaporated to dryness. The residue was
dissolved in water and the product precipitated by addition of
HCl. Recrystallization from water/ethanol (1:1) afforded 3 as
a beige solid (0.75 g, 79%): mp 228-230 °C; 1H NMR 7.90 (t,
1H), 8.05 (s, 1H), 8.41 (d, 1H), 8.48 (d, 1H), 8.80 (s, 1H), 17.5
(bs, 1H). Anal. Calcd (C10H6N6O3) C, H, N.
P r epar ation of N-(4-P h en ylth iazol-2-yl)ben zen esu lfon -
a m id es (5-16, 18-19). Gen er a l P r oced u r e. A mixture of
the appropriately substituted 4-arylthiazol-2-ylamine (1.7
mmol) and arylsulfochloride (1.9 mmol) in pyridine (2 mL) was
stirred overnight at room temperature. The red solution was
poured into 1 N HCl (50 mL). The yellowish precipitate was
collected, redissolved in a mixture of ethanol (20 mL) and 2 N
NaOH (20 mL), and treated with Norite. Filtration and
neutralizing the filtrate with concentrated HCl yielded the
product as a powder which was recrystallized from EtOH/
water mixtures.
4-Ch lor o-N-[4-(3-n itr op h en yl)th ia zol-2-yl]ben zen esu l-
fon a m id e (14): 0.43 g (64%) of a yellow powder from 50%
EtOH; mp 195-197 °C; 1H NMR 7.59 (s, 1H), 7.65 (d, 2H),
7.74 (t, 1H), 7.87 (d, 2H), 8.18 (d, H), 8.23 (d, 1H), 8.61 (s, 1H),
13.6 (bs, 1H). Anal. Calcd (C15H10ClN3O4S2) C, H, N, Cl, S.
4-Met h oxy-N-[4-(3-n it r op h en yl)t h ia zol-2-yl]b en zen e-
su lfon a m id e (15): 0.31 g (47%) of a yellow powder from 50%
EtOH; mp 157-159 °C; 1H NMR 3.82 (s, 3H), 7.09 (d, 2H),
7.58 (bs, 1H), 7.73 (t, 1H), 7.81 (d, 2H), 8.19 (t, 2H), 8.61 (s,
1H), 13.3 (bs, 1H). Anal. Calcd (C16H13N3O5S2) C, H, N, S.
3,4-Dim et h oxy-N-[4-(3-n it r op h en yl)t h ia zol-2-yl]b en -
zen esu lfon a m id e (16): 0.26 g (36%) of a light yellow powder
from 70% EtOH; mp 185 °C; 1H NMR 3.81 (s, 6H), 7.10 (d,
1H), 7.32 (s 1H), 7.46 (d, 1H), 7.60 (bs, 1H), 7.73 (t, 1H), 8.19
(t, 2H), 8,60 (s, 1H), 13.3 (bs, 1H). Anal. Calcd (C17H15N3O6S2)
C, H, N, S.
4-Am in o-N-[4-(3-n itr op h en yl)th ia zol-2-yl]ben zen esu l-
fon a m id e (17). Following the general procedure a mixture
of 4-(3-nitrophenyl)thiazol-2-ylamine and 4-acetaminobenze-
nesulfochloride in pyridine yielded 0.60 g of N-[4-[[[4-(3-
nitrophenyl)thiazol-2-yl]amino]sulfonyl]phenyl]acetamide (25)
1
(84%): mp >250 °C; H NMR 2.06 (s, 3H), 7.57 (s, 1H), 7.75
(m, 5H), 8.19 (t, 2H), 8.60 (s, 1H), 10.3 (s, 1H), 13.3 (bs, 1H).
Anal. Calcd (C17H14N4O5S2) C, H, N, S. This material was
boiled for 20 h in 6 N HCl (6 mL) and gave upon cooling and
recrystallization of the solid precipitate 0.20 g (38%) of a yellow
1
powder from 40% EtOH: mp 191-193 °C; H NMR 6.05 (bs,
2H), 6.59 (d, 2H), 7.52 (d, 2H), 7.62 (bs, 1H), 7.72 (t, 1H), 8.19
(d, 2H), 8.60 (s, 1H), 13.0 (bs, 1H). Anal. Calcd (C15H12N4O4S2)
C, H, N, S.
4-Isop r op yl-N-[4-(3-n itr op h en yl)th ia zol-2-yl]ben zen e-
su lfon a m id e (18): 0.19 g (28%) of a light yellow powder from
1
50% EtOH; mp 144-145 °C; H NMR 1.20 (d, 6H), 2.95 (m,
1H), 7.44 (d, 2H), 7.58 (s, 1H), 7.70 (t, 1H), 7.79 (d, 2H), 8.19
(m, 2H), 8.60 (s, 1H), 13.4 (bs, 1H). Anal. Calcd (C18H17N3O4S2)
C, H, N, S.
N-[4-(4-Hyd r oxy-3-m eth ylp h en yl)th ia zol-2-yl]-4-m eth -
ylben zen esu lfon a m id e (5): 0.15 g (25%) of a colorless powder
1
from 50% EtOH; mp 168-170 °C dec; H NMR (DMSO) 2.11
N-[4-[2-F lu or o-5-(tr iflu or om eth yl)p h en yl]th ia zol-2-yl]-
3,4-d im eth oxyben zen esu lfon a m id e (19): 0.43 g (55%) of a
(s, 3H), 2.34 (s, 3H), 6.77 (d, 1H), 6.89 (s, 1H), 7.34 (m, 3H),
7.43 (bs, 1H), 7.71 (d, 2H), 9.75 (s, 1H), 13.00 (bs, 1H). Anal.
Calcd (C17H16N2O3S2) C, H, N, S.
1
colorless powder from 60% EtOH; mp 126-128 °C; H NMR
(CDCl3) 3.91 (s, 6H), 6.90 (d, 1H), 7.04 (s, 1H), 7.20 (t, 1H),
7.52 (s, 1H), 7.53 (m, 1H), 7.64 (dd, 1H), 7.83 (dd, 1H), 9.8 (bs,
1H). Anal. Calcd (C18H14F4N2O4S2) C, H, N, F, S.
4-Me t h yl-N -(4-p h e n ylt h ia zol-2-yl)b e n ze n e su lfon -
a m id e (6):34 0.16 g (28%) of a colorless powder from 30% Et-
OH; mp 152-153 °C. Anal. Calcd (C16H14N2O2S2) C, H, N, S.
N-[4-(Met h oxyp h en yl)t h ia zol-2-yl]-4-m et h ylb en zen e-
su lfon a m id e (7): 0.24 g (39%) of a colorless powder from 50%
4-Am in o-N-[4-[2-flu or o-5-(tr iflu or om eth yl)p h en yl]th i-
a zol-2-yl]ben zen esu lfon a m id e (20): Following the general
procedure a mixture of 4-[2-fluoro-5-(trifluoromethyl)phenyl]-
thiazol-2-ylamine and 4-acetaminobenzenesulfochloride in py-
ridine yielded 0.53 g of N-[4-[[[4-[2-fluoro-5-(trifluoromethyl)-
phenyl]thiazol-2-yl]amino]sulfonyl]phenyl]acetamide (68%). This
material was used without further purification, boiled for 36
h in 6 N HCl (6 mL), and gave upon cooling and recrystalli-
zation of the solid precipitate 0.21 g (44%) of a colorless powder
from 50% EtOH: mp 107-110 °C; 1H NMR 6.05 (bs, 2H), 6.59
(d, 2H), 7.38 (bs,1H), 7.51 (d, 2H), 7.57 (t, 1H), 7.80 (m, 1H),
8.22 (d, 1H). Anal. Calcd (C16H11F4N3O2S2) C, H, N, F, S.
(2,6-Dib r om op h en yl)ca r b a m ic Acid ter t-Bu t yl E st er
(22). Diphosgene (21.9 mL, 181 mmol) was added at room
temperature to a solution of 2,6-dibromoaniline (37.8 g, 151
mmol) in toluene (400 mL). The mixture was heated slowly
to 105 °C and maintained at that temperature for 3 h.
(Caution: the reaction is exothermic and started at 80 °C.)
After being cooled to room temperature the solution was
purged with argon, and the solvent was evaporated. The
residue was boiled with tert-butyl alcohol (400 mL) for 5 h.
The solution was then cooled to room temperature and
evaporated to dryness. Chromatography of the residue on
silicagel with diethyl ether/hexanes (1:5) afforded 22 as a
colorless solid (21.2 g, 40%): mp 103-106 °C; 1H NMR (CDCl3)
1.51 (s, 9H), 6.15 (bs, 1H), 6.99 (t, 1H), 7.57 (d, 2H). Anal.
Calcd (C11H13Br2NO2) C, H, N, Br.
1
EtOH; mp 85 °C dec; H NMR 2.36 (s,3H), 3.78 (s, 3H), 6.99
(d, 2H), 7.03 (s, 1H), 7.35 (d, 2H), 7.64 (d, 2H), 7.73 (d, 2H),
13.2 (bs, 1H). Anal. Calcd (C17H16N2O3S2) C, H, N, S.
N-[4-(4-Ch lor op h en yl)t h ia zol-2-yl]-4-m et h ylb en zen e-
su lfon a m id e (8): 0.18 g (28%) of a colorless powder from 40%
EtOH; mp 237-239 °C; 1H NMR 2.36 (s, 3H), 7.28 (s, 1H),
7.36 (d, 2H), 7.51 (d, 2H), 7.73 (d, 2H), 7.73 (d, 2H). Anal.
Calcd (C16H13ClN2O2S2) C, H, N, Cl, S.
4-Me t h y l-N -(4-p -t o ly lt h ia zo l-2-y l)b e n ze n e s u lfo n -
a m id e (9): 0.46 g (78%) of a beige powder from 50% EtOH;
mp 186 °C dec; 1H NMR 2.31 (s, 3H), 2.36 (s, 3H), 7.13 (s, 1H),
7.24 (d, 2H), 7.35 (d, 2H), 7.59 (d, 2H), 7.73 (d, 2H), 13.15 (s,
1H). Anal. Calcd (C17H16N2O2S2) C, H, N, S.
N-[4-(3,4-Dich lor op h en yl)t h ia zol-2-yl]-4-m et h ylb en -
zen esu lfon a m id e (10): 0.33 g (48%) of a colorless powder
1
from 50% EtOH; mp 205-206 °C; H NMR 2.36 (s, 3H), 7.36
(d, 2H), 7.44 (s, 1H), 7.71 (s, 2H), 7.73 (d, 2H), 8.03 (s, 1H),
13.3 (bs, 1H). Anal. Calcd (C16H12Cl2N2O2S2) C, H, N, Cl, S.
4-Me t h yl-N -[4-(3-n it r op h e n yl)t h ia zol-2-yl]b e n ze n e -
su lfon a m id e (11): 0.42 g (62%) of a light yellow powder from
50% EtOH; mp 187-189 °C; 1H NMR 2.36 (s, 3H), 7.37 (d,
2H), 7.58 (bs, 1H), 7.74 (t, 1H), 7.76 (d, 2H), 8.19 (t, 2H), 8.60
(s, 1H). Anal. Calcd (C16H13N3O4S2) C, H, N, S.
N -[4-(3-N it r o p h e n y l)t h ia z o l-2-y l]b e n z e n e s u lfo n -
a m id e (12):34 0.12 g (20%) of a light yellow powder from 50%
EtOH; mp 141-142 °C. Anal. Calcd (C15H11N3O4S2) C, H, N, S.
3,4-Dich lor o-N-[4-(3-n itr op h en yl)th ia zol-2-yl]ben zen e-
su lfon a m id e (13): 0.30 g (41%) of a light yellow powder from
50% EtOH; mp 189-191 °C; 1H NMR 7.61 (s, 1H), 7.75 (t, 1H),
7.83 (m, 2H), 8.00 (s, 1H), 8.18 (d, 1H), 8.23 (d, 1H), 8.62 (s,
1H), 13.6 (bs, 1H). Anal. Calcd (C15H9Cl2N3O4S2) C, H, N, Cl, S.
(2S)-4-[2-[3-Br om o-2-[(ter t-bu toxycar bon yl)am in o]ph en -
yl]-2-oxoeth yl]-5-oxooxa zolid in e-3-ca r boxylic Acid Ben -
zyl Ester (23). A solution of 22 (33.8 g, 96 mmol) in THF
(700 mL) was cooled to -90 °C. tert-Butyllithium (135 mL,
1.5 M) was added, so that the temperature did not rise above
-85 °C. Stirring at -90 °C was continued for 2 h, the
temperature raised to -75 °C, and trimethyltin chloride (18.7

4384 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Ro¨ver et al.
g, 94 mmol) added. Stirring at -75 °C was continued for 1.5
h and the reaction quenched with water (250 mL). The
mixture was partitioned between diethyl ether and water. The
organic phases were pooled, washed with brine, and dried with
MgSO4. Evaporation of the solvents afforded a yellow oil (33.4
g), containing 50% monotinylated species contaminated with
ditinylated compound and starting material.
counting resulted in 14.4 Ci/mmol. This value was consistent
with the analysis by mass spectrometry (approximately 50%
t1-species).
Bioch em istr y. P r ep a r a tion of Ra t Kid n ey Mitoch on -
d r ia . Rat kidneys were homogenized in 5 volumes of 0.32 M
sucrose, buffered to pH 7.4 with 5 mM Tris HCl, containing
0.5 mM phenylmethanesulfonyl fluoride, using a glass-Teflon
homogenizer. After centrifugation at 800g for 20 min at 4 °C,
the supernatant was collected and recentrifuged at 10000g for
20 min at 4 °C. The pellet, consisting of a crude mitochondrial
fraction, was rinsed three times with 50 mM potassium
phosphate buffer, pH 7.4, containing 0.5 mM EGTA, resus-
pended in the same buffer and eventually stored in 1 mL
aliquots at -80 °C until use.
This oil (33.4 g) was dissolved in toluene (500 mL), (S)-3-
(benzyloxycarbonyl)-5-oxo-4-oxazolidineacetyl chloride26 (23 g,
77 mmol) and tris(dibenzylideneacetone)dipalladium chloro-
form (0.4 g) were added. The mixture was heated to 70 °C for
4 h, cooled to room temperature, and filtered. The filtrate was
partitioned between ethyl acetate and water. The organic
phases were pooled and washed with saturated sodium
bicarbonate solution and brine. Drying with MgSO4 and
evaporation of the solvents yielded a residue which was
chromatographed on silica gel with ethyl acetate/hexanes (2:
3) to afford 23 as a yellowish foam (6.9 g, 16%): 1H NMR
(CDCl3) 1.46 (s, 9H), 3.61 (m, 2H), 4.42 (m, 1H), 5.0-5.6 (m,
4H), 7.0-7.2 (m, 2H), 7.34 (bs, 5H), 7.69 (d, 1H). [R]20D +89.2
Protein content was determined using the bicichoninic acid
BCA protein assay kit from Pierce.
Kyn u r en in e 3-Hyd r oxyla se Activity Deter m in a tion
a n d in Vitr o In h ibition . Kynurenine 3-hydroxylase activity
was measured using a microplate version of the radioenzy-
matic method described by Erickson et al.,35 based on the
release of tritiated water after hydroxylation of L-[3-3H]-
kynurenine (see above). The assay was performed in 96 well/
plate in a final volume of 100 µL of 0.1 M potassium phosphate
buffer, pH 7.4. The reaction mixture consisted of 100 µM
unlabeled L-kynurenine sulfate (Sigma), 0.1 µCi of L-[3-3H]-
kynurenine (sp act. 14.4-16.1 Ci/mmol; 1 Ci ) 37 GBq), 200
µM NADPH (Boehringer Mannheim), 0.4 unit/mL glucose
6-phosphate dehydrogenase (Boehringer Mannheim), 3 mM
glucose 6-phosphate, and 30 µg of the mitochondrial enzyme
preparation. In most experiments, a preincubation period of
15 min at 37 °C in the presence of the appropriate concentra-
tion of the inhibitor was performed before starting the reaction
by the addition of the substrate, and the incubation was
prolonged for 10 min at 37 °C. To absorb the unreacted
substrate, 150 mL of a 10% (w/v) suspension of activated
charcoal (Norit A, Serva) were added to each well, and after
shaking for 2 min, the plates were centrifuged for 10 min at
4000 rpm in a Eppendorf model 5403 centrifuge. An aliquot
(50 mL) of the samples was then transferred to the well of a
second plate, and after addition of 200 µL/well of scintillation
fluid (Microscint 40, Packard), sealing, and vigorous shaking
for 20 min, the plates were counted for radioactivity using a
Top-Count (Packard) counter.
Enzyme activity kinetic constants were calculated by fitting
the data obtained from saturation experiments to Michaelis-
Menten equation using the Ultrafit (Biosoft) computer program
or after Lineweaver-Burke double reciprocal transformation
of the data. Inhibition constants (Ki) were calculated from
replots of the apparent Km values versus the inhibitor con-
centrations used.
Determination of IC50 values was performed by fitting the
experimental data from inhibition curves using at least eight
different inhibitor concentrations to logistic sigmoid equation
using a computer program.
An im a l Tr ea tm en t a n d ex Vivo In h ibition of Kyn u r e-
n in e 3-Hyd r oxyla se Activity in Ra t a n d Ger bils. Male
albino rats (120-150 g, RORO) and female gerbils (60-80 g)
were obtained from Biological Research Laboratories (Fu¨llins-
dorf, Switzerland). Treatment of the animals was performed
by oral administration of the compounds (0.5 mL per 100 g of
animal weight) at five or six different doses (four or five
animals per dose). To ensure the compounds were adminis-
tered as an homogeneous suspension, they were resuspended
in 0.1% Tween-80 in water, homogenized in an all-glass
homogenizer, and sonicated in a water-bath sonicator. Control
animals were administered with vehicle solution. Two hours
after the treatment, the animals were killed by decapitation,
and brain, liver, and kidney were rapidly removed. The organs
were immediately frozen in dry ice and stored at -80 °C until
analysis.
(c 1, MeOH); MS (high res) mass calcd for C24H25BrN2O7 (M•+
)
) 532.0845, obsd m/z at 532.0846, deviation from theoretical
) 0.1 mmu.
(2S)-2-Am in o-4-(2-a m in o-3-b r om o-p h en yl)-4-oxo-b u -
tyr ic a cid (24). 23 (6.0 g, 11.2 mmol) was added to HBr in
acetic acid (100 mL, 33%). After 40 min of stirring at room
temperature, the mixture was poured into diethyl ether (1.3
L). The hygroscopic precipitate was collected, dried (25 °C/1
mbar), and dissolved in 2-propanol (500 mL). Addition of
propylene oxide (5 mL) and stirring for 4 h at room temper-
ature afforded 24 as yellow solid (0.8 g, 25%): mp >175 °C
1
dec; H NMR 3.3 (dd, 1H), 3.55 (dd, 1H), 3.7 (m, 1H), 6.57 (t,
1H), 7.22 (bs, 2-3H), 7.68 (d, 1H), 7.81 (d, 1H); [R]20D -36.0 (c
0.5, dioxane/water 1:1); MS (high res) mass calcd for C10H12
-
BrN2O3 (M•+) ) 287.00313, obsd m/z at 287.00324, deviation
from theoretical ) 0. 11 mmu.
(S)-3-[3H]Kyn u r en in e (25). 24 (11.4 mg, 40 µmol) was
dissolved in dimethylformamide (6 mL). Dissolution was
facilitated by heating and sonication. After filtration of the
turbid solution through a Millex-SR 0.5 µm cartridge and
addition of 3 mg of 10% Pd on charcoal, the suspension was
stirred under tritium gas for 90 min at room temperature.
Excess tritium gas was reabsorbed back into the uranium trap
and the solvent was lyophilized off by gentle heating with an
air blower. then 3 × 3.3 mL of ethanol-water (9/1) were
vacuum-transferred into the reaction flask. The suspension
was stirred briefly, and the solvent was lyophilized off to
remove all volatile tritium activity (2.8 Ci). The vacuum was
relieved by nitrogen gas, and the residue was taken up in
methanol. After filtration of the catalyst suspension through
a Millex-SR 0.5 µm cartridge, the solution was diluted with
methanol-toluene (4/1) to 100 mL and stored at -75 °C. The
total tritium activity of the crude product was 268 mCi. Half
of the crude product was prepurified by chromatography on
Amberlite XAD-4. Shortly before use the Amberlite XAD-4
column, 9 × 190 mm was rinsed with 50 mL of ethanol-water
(1/1) containing 0.1 mL of concentrated ammonium hydroxide
and then with water. Half of the crude product solution was
evaporated and redissolved in 10 mL of 0.1 N hydrochloric acid,
which was subsequently transferred onto the column. After
rinsing with 3 × 2.5 mL of 0.1 N hydrochloric acid and 70 mL
of water, 25 was eluted with ethanol-water (1/4). The total
tritium activity was 80 mCi. The product was further purified
by HPLC using the following conditions: column, Nucleosil
120 C18 5 mm, 4 × 250 mm; mobile phase, buffer-methanol,
3/2; buffer, 50 mM potassium phosphate, pH 5, containing 3
mM; cetyltrimethylammonium bromide; flow rate, 0.8 mL/min,
UV detection at 243 nm.
The eluate was lyophilized, and the residue was dissolved
in 25 mL of water. This solution was desalted by chromatog-
raphy on Amberlite XAD-4 as described above. The final
product was stored under argon in methanol-toluene (4/1) at
-75 °C. The total tritium activity was 64 mCi, and the
radiochemical purity was 98% according to TLC (silica gel,
1-butanol-acetic acid-water, 4/1/1). The specific activity was
determined by HPLC quantification. Liquid scintillation
For ex vivo kynurenine 3-hydroxylase activity, the tissues
were homogenized using a glass-Teflon homogenizer in 9
volumes of 0.32 M sucrose/5 mM Tris-HCl buffer, pH 7.4,
containing 0.5 mM phenylmethanesulfonyl fluoride. The
enzyme activity was determined using 25 µL of the tissue
homogenates as described above except that the incubation

Inhibitors of Kynurenine 3-Hydroxylase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4385
(15) Flanagan, E. M.; Erickson, J . B.; Viveros, H.; Chang, S. Y.;
Reinhard, J . F., J r. Neurotoxin Quinolinic Acid is Selectively
Elevated in Spinal Cords of Rats with Experimental Allergic
Encephalomyelitis. J . Neurochem. 1995, 64, 1192-1196.
(16) Whetsell, W. O. J .; Schwarcz, R. Prolonged Exposure to Submi-
cromolar Concentrations of Quinolinic Acid Causes Excitotoxic
Damage in Organotypic Cultures of Rat Corticostriatal System.
Neurosci. Lett. 1989, 97, 271-275.
times were 2 min for kidney and liver and 30 min for brain
homogenates. ED50 values were determined by fitting the data
to a logistic sigmoid equation as above.
Micr od ia lysis Tech n iqu e a n d Deter m in a tion of KYNA
in Dia lysa te. Dialysis probes (Filtral 12, AN 69 HF, Hospal
Industrie, France) were transversally inserted into the hip-
pocampus of male albino rats (250-300 g) as previously
described.36 After surgery, the animals were allowed to recover
for 24 h in their home cages with free access to food and water.
The dialysis probes were connected to a microinfusion pump
(CMA 100, Schmidlin, Switzerland) and to an electrically
actuated injector (Valco, model EC10W). A modified Ringer
solution (148 mM NaCl, 5 mM KCl, 1.6 mM CaCl2) was
perfused at a flow rate of 2 mL/min through the dialysis probe.
The concentration of KYNA in the dialysate fluid was
determined by HPLC with fluorescence detection.37 The
injection of the samples (40 µL) into the HPLC column
(Spherisorb ODS2, 125 × 4.6 mm, 3 mm particles; PhaseSep)
was controlled by a timer (Valco, Model DVSP4) set to remain
in the load position for 20 min and subsequently to the inject
position for 5 s. The mobile phase (50 mM sodium acetate
buffer, pH 6.2, containing 4.5% acetonitrile) flow rate was 0.75
mL/min. Zinc acetate (0.5 M) was delivered postcolumn at a
flow rate of 0.4 mL/min. The fluorescence detector (Merck-
Hitachi Model F1080) was set at excitation and emission
wavelengths of 344 and 398 nm, respectively.
(17) Schurr, A.; West, C. A.; Rigor, B. M. Neurotoxicity of Quinolinic
Acid and its Derivatives in Hypoxic Rat Hippocampal Slices.
Brain Res. 1991, 568, 199-204.
(18) Schurr, A.; Payne, R. S.; Heine, M. F.; Rigor, B. Hypoxia,
Excitotoxicity, and Neuroprotection in the Hippocampal Slice
Preparation. J . Neurosci. Methods 1995, 59, 129-138.
(19) Pellicciari, R.; Natalini, B.; Constantino, G.; Mahmoud R. M.;
Mattoli, L.; Sadeghpour, B. M.; Moroni, F.; Chiarugi, A.;
Carpenedo, R. Modulation of the Kynurenine Pathway in Search
for New Neuroprotective Agents. Synthesis and Preliminary
Evaluation of (m-Nitrobenzoyl)alanine, a Potent Inhibitor of
Kynurenine-3-Hydroxylase. J . Med. Chem. 1994, 37, 647-655.
(20) Carpenedo, R.; Chiarugi, A.; Russi, P.; Lombardi, G.; Carla, V.;
Pellicciari, R.; Mattoli, L.; Moroni, F. Inhibitors of Kynurenine
Hydroxylase and Kynureninase Increase Cerebral Formation of
Kynurenate and have Sedative and Anticonvulsant Properties.
Neuroscience 1994, 61, 237-244.
(21) Speciale, C.; Cini, M.; Wu, H.-Q.; Salvati, P.; Schwarcz, R.;
Molinari, A.; Calabresi, M.; Varasi, M. Kynurenic acid-enhancing
and anti-ischemic effects of the potent kynurenine 3-hydroxylase
inhibitor FCE 28833 in rodents. Adv. Exp. Med. Biol. 1996, 398,
221-227.
(22) Alberati-Giani, D.; Cesura, A. M.; Broger, C.; Warren, W. D.;
Ro¨ver, S.; Malherbe, P. Cloning and functional expression of
human kynurenine 3-monooxygenase. FEBS Lett. 1997, 410,
407-412.
(23) Roy, K.; Srivastava, R. P.; Tekwani, B. L.; Pandey, V. C.;
Bhaduri, A. P. Synthesis and Exploration of New Biological
Activities in Ethyl 6/7-Substituted and 6,7-Disubstituted Quino-
lin-4-One-3-Carboxylates. Bioorg. Med. Chem. Lett. 1996, 6,
121-126.
(24) Kozikowski, A. P.; Adamczyk, M. Methods for the Stereoselective
Cis Cyanohydroxylation and Carboxyhydroxylation of Olefins.
J . Org. Chem. 1983, 48, 366-372.
(25) Salituro, F. G.; MacDonald, I. A. Facile Synthesis of L-Kynure-
nine. J . Org. Chem. 1988, 53, 6138-6139.
(26) Hsiao, C.-N.; Leanna, M. R.; Bhagavatula, L.; De Lara, E.;
Zydowsky, T. M.; Horrom, B. W.; Morton H. E. Synthesis of N-(t-
Butoxycarbonyl)-3-(4-Thiazolyl)-L-Alanine. Synth. Commun. 1990,
20, 3507-3517.
(27) Andrews, P. R.; Craik, D. J .; Martin J . L. Functional Group
Contributions to Drug-Receptor Interactions. J . Med. Chem.
1984, 27, 1648-1657.
(28) Atef, M.; Youssef, A. H.; Ramadan, A.; Issa, M. Kinetic Disposi-
tion, Systemic Bioavailability and Acetylation of Some Sulphon-
amides in Goats. Arch. Int. Pharmacodyn. 1989, 302, 27-39.
(29) Topliss, J . G. A Manual Method for Applying the Hansch
Approach to Drug Design. J . Med. Chem. 1977, 20, 463-469.
(30) Giordani, A.; Corti, L.; Cini, M.; Marconi, A.; Pillan, A.; Ferrario,
R.; Schwarcz, R.; Guidetti, P.; Speciale, C.; Varasi, M. Benzoyl-
alanine Analogues as Inhibitors of Rat Brain Kynureninase and
Kynurenine 3-Hydroxylase. In Recent Advances in Tryptophan
Research; Filippini, G. A., Costa, C. V., Bertazzo, A., Eds.;
Plenum Press: New York, 1996; pp 499-505.
Ack n ow led gm en t. The skillful assistance of B. Frei,
R. Wermuth, D. Zimmerli, N. Gebhard, F. D’Agostini,
St. Henriot, C. Kuhn, and G. Py is gratefully ac-
knowledged. The authors also thank Drs. W. Arnold,
St. MY¨ ller, W. Vetter, Mr. W. Meister, and G. Oesterhelt
for spectroscopic determinations and analysis.
Refer en ces
(1) Rothman, S. M.; Olney, J . W. Excitotoxicity and the NMDA
Receptor. Trends Neurosci. 1987, 10, 299-302 and references
therein.
(2) Choi, D. W. Ionic Dependence of Glutamate Neurotoxicity. J .
Neurosci. 1987, 7, 369-379 and references therein.
(3) Monn, J . A.; Schoepp, D. D., Recent Progress in Excitatory Amino
Acid Research. Annu. Rep. Med. Chem. 1994, 29, 53-64 and
references therein.
(4) (a) Bender, D. A. Biochemistry of Tryptophan in Health and
Disease. Mol. Aspects Med. 1982, 6, 101-197 and references
therein. (b) For a discussion of the relevance of the kynurenine
pathway and its metabolites in disease see: refs 8, 19, and 20.
(5) Stone, T. W.; Perkins, M. N. Actions of Excitatory Amino Acids
and Kynurenic Acid in the Primate Hippocampus: a Preliminary
Study. Neurosci. Lett. 1984, 52, 335-340.
(6) Kessler M.; Terramani, T.; Lynch, G.; Baudry, M. A Glycine Site
Associated with N-Methyl-D-aspartic Acid Receptors. Charac-
terization and Identification of a New Class of Antagonists. J .
Neurochem. 1989, 52, 1319-1328.
(7) Saito, K.; Nowak, T. S.; Markey, S. P.; Heyes, M. P. Mechanism
of Delayed Increases in Kynurenine Pathway Metabolism in
Damaged Brain Regions Following Transient Cerebral Ischemia.
J . Neurochem. 1993, 60, 180-192.
(8) Reinhard, J . F., J r.; Erickson, J . B.; Flanagan, E. M. Quinolinic
Acid in Neurological Disease: Opportunities for Novel Drug
Discovery. Adv. Pharmacol. 1994, 30, 85-125 and references
therein.
(31) Sammes, P. G. Sulfonamides and Sulfones. In Comprehensive
Medicinal Chemistry Vol. 2; Hansch, C., Sammes, P. G., Taylor,
J . B., Eds.; Pergamon: Oxford, 1990; pp 255-270.
(32) Lou, G. L.; Pinsky, C.; Sitar, D. S. Kynurenic Acid Distribution
into Brain and Peripheral Tissues of Mice. Can. J . Physiol.
Pharmacol. 1994, 72, 161-167.
(33) Eberle, A. N.; Zeller, A. Tritiation of peptides to high specific
radioactivity. Part 1. Synthesis and Biological Properties of [13-
(3H4)Norvaline]-R-MSH and of [2,23-bis(3H2)Tyrosine)]ACTH(1-
24). Helv. Chim. Acta 1985, 68, 1880-92.
(34) Vanmaele, L. Dye Donor Element for Use in a Thermal Dye
Transfer Method. Eur. Pat. Appl., EP 687573 A1 951220.
(35) Erickson, J . B.; Flanagan, E. M.; Russo, S.; Reinhard, J . F., J r.
A Radiometric Assay for Kynurenine 3-Hydroxylase Based on
the Release of 3H2O During Hydroxylation of L-[3,5-3H]-Kynure-
nine. Anal. Biochem. 1992, 205, 257-262.
(36) Colzi, A; D’Agostini, F.; Cesura, A. M.; Borroni, E.; Da Prada,
M. Monoamine oxidase-A inhibitors and dopamine metabolism
in rat caudatus: Evidence that an increased cytosolic level of
dopamine displaces reversible monoamine oxidase-A inhibitors
in vivo. J . Pharmacol. Exp. Ther. 1993, 265, 103-111.
(37) Swartz, K. J .; During, M. J .; Freese, A.; Beal M. F. Cerebral
Synthesis and Release of Kynurenic Acid: An Endogenous
Antagonist of Excitatory Amino Acid Receptors. J . Neurosci.
1990, 10, 2965-2973.
(9) Takikawa, O.; Yoshida, R.; Kido, R.; Hayashi, O. Tryptophan
Degradation in Mice Initiated by Indoleamine 2,3-dioxygenase.
J . Biol. Chem. 1986, 261, 3648-3653.
(10) Saito, K.; Nowak, T. S.; Suyama, K.; Quearry, B. J .; Saito, M.;
Crowley, J . S.; Markey, S. P.; Heyes, M. P. Kynurenine Pathway
Enzymes in Brain: Responses to Ischemic Brain Injury Versus
Systemic Immune Activation. J . Neurochem. 1993, 61, 2061-
2070.
(11) Heyes, M. P.; Saito, K.; Markey, S. P. Human Macrophages
Convert L-Tryptophan into the Neurotoxin Quinolinic Acid.
Biochem. J . 1992, 283, 633-635.
(12) Heyes, M. P.; Achim, C. L.; Wiley: A. A.; Major, E. O.; Saito, K;
Markey, S. P. Human microglial convert L-tryptophan into the
neurotoxin quinolinic acid. Biochem. J . 1996 320, 595-597.
(13) Yoshitatsu, S.; Paul, I. A.; Saito, K.; Layar, R.; Hartley, J . W.;
Morse III, H. C.; Skolnick, P.; Heyes, M. P. Quinolinic Acid
Levels in a Murine Retrovirus-Induced Immunodeficiency Syn-
drome. J . Neurochem. 1996, 66, 296-302.
(14) Blight, A. R.; Cohen, T. I.; Saito, K.; Heyes, M. P. Quinolinic
Acid Accumulation and Functional Deficits Following Experi-
mental Spinal Cord Injury. Brain 1995, 118, 735-752.
J M970467T