126 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 1
Drysdale et al.
Human peripheral blood monocytes were isolated using
Ficoll gradients and differentiated into macrophages by ex-
posure for 1 week to granulocyte-macrophage colony-stimu-
lating factor7 before being exposed to the human interferon-γ
(100 U/mL). The compounds were added immediately after the
IFN-γ as was [2H]tryptophan and the samples were incubated
for an additional 24 h at which time aliquots of the media were
analyzed by gas chromatography/mass spectrometry for [2H]-
QUIN.7,8
Ch em istr y. Melting points were taken on either an Elec-
trothermal or Gallenkamp digital melting point apparatus and
are uncorrected. Proton NMR spectra were recorded on a
Brucker AC200 spectrometer; chemical shifts were recorded
in parts per million (ppm) downfield from tetramethylsilane.
Elemental analyses were obtained using a Carlo Erba 1106
elemental analyzer. Anhydrous solvents were used directly as
purchased from Aldrich Chemical Co. Ltd., Gillingham, En-
gland.
F igu r e 3. Inhibition of QUIN formation in human macroph-
Met h od A. Met h yl 2-H yd r oxy-4-oxo-4-p h en ylb u t -2-
en oa te (2a ). To a freshly prepared solution of sodium meth-
oxide made by dissolving sodium (1.42 g, 61.7 mmol) in dry
methanol (30 mL) under a nitrogen atmosphere was added
dimethyl oxalate (7.16 g, 60.6 mmol) at room temperature. This
solution was cooled to 0 °C and acetophenone (7.20 g, 7.07 mL,
60.6 mmol) added dropwise. The reaction was allowed to warm
to room temperature overnight and diluted with diethyl ether
(50 mL) and the resulting solid filtered off. This solid was
stirred in water (200 mL) for 0.5 h and the solution filtered
and made to pH 4 using acetic acid. After cooling to 0 °C for
1 h the resulting white solid was filtered off, washed with
water, and dried in a vacuum desiccator over P2O5 to yield
the desired ester 2a (5.13 g, 38%): mp 56-57 °C; NMR
(DMSO-d6) δ 3.88 (3H, s), 7.10 (1H, s), 7.55-7.77 (3H, m),
8.05-8.12 (2H, m).
Meth yl 4-(3,4-Diflu or op h en yl)-2-h yd r oxy-4-oxobu t-2-
en oa te (2n ). Method as for 2a except using 3,4-difluoroac-
etophenone to yield the desired ester 2n (8.22 g, 56%): mp
107-108 °C; NMR (CDCl3) δ 3.97 (3H, s), 7.00 (1H, s), 7.23-
7.37 (1H, m), 7.73-7.91 (2H, m).
Meth od B. 2-Hyd r oxy-4-oxo-4-p h en ylbu t-2-en oic Acid
(3a ). Methyl 2-hydroxy-4-oxo-4-phenylbut-2-enoate (2a ) (200
mg, 0.970 mmol) was stirred in 6 N HCl (20 mL) for 4 h at
room temperature. After this time the mixture was basified
with 10 N NaOH and washed with dichloromethane (20 mL).
The aqueous layer was filtered, cooled on ice, and acidified
with 6 N HCl. The resulting solid was filtered off, washed with
water, and dried in a vacuum desiccator over P2O5 to yield
the desired acid 3a (100 mg, 54%): mp 148-150 °C; NMR
(DMSO-d6) δ 4.55 (0.1H, s, keto form), 7.09 (0.9H, s, enol form),
7.52-7.77 (3H, m), 7.94-7.99 (0.2H, m, keto form), 8.02-8.11
(1.8H, m, enol form).
Meth od C. Meth yl 2-Am in o-4-(3,4-d iflu or op h en yl)-4-
oxobu t-2-en oa te (4n ). To methyl 4-(3,4-difluorophenyl)-2-
hydroxy-4-oxobut-2-enoate (2n ) (4.55 g, 18.8 mmol) and am-
monium acetate (1.70 g, 22 mmol) in benzene (50 mL) was
added glacial acetic acid (1.70 mL, 29.7 mmol) and the mixture
refluxed under Dean and Stark conditions for 12 h. After
cooling to room temperature the mixture was washed with
saturated sodium hydrogen carbonate solution (100 mL) and
the organic layer dried (MgSO4) and concentrated in vacuo.
The resulting solid was recrystallized from dichloromethane
and hexane to yield the desired product 4n as orange crystals
(3.325 g, 74%): mp 107-109 °C; NMR (CDCl3) δ 3.96 (3H, s),
6.10 (2H, br s), 6.55 (1H, s), 7.16-7.30 (1H, m), 7.67-7.86 (2H,
m).
Meth od D. 2-Am in o-4-(3,4-d iflu or op h en yl)-4-oxobu t-2-
en oic Acid (5n ). Methyl 2-amino-4-(3,4-difluorophenyl)-4-
oxobut-2-enoate (4n ) (500 mg, 2.07 mmol) was dissolved in
anhydrous THF (10 mL) under an atmosphere of nitrogen and
cooled to 0 °C. To this was added a solution of 0.5 N KOH
(4.67 mL, 2.34 mmol) dropwise and the mixture allowed to stir
at ambient temperature overnight. The reaction mixture was
concentrated in vacuo and the residue partitioned between
water and ethyl acetate. The aqueous layer was cooled on ice
ages.
chloro-4-fluorophenyl)-2-hydroxy-4-oxobut-2-enoic acid
(3o) (IC50 ) 0.27 µM) are the most potent inhibitors of
KH reported so far. In the 2-amino series, 3,4-dihalo-
genated analogues were considerably more potent than
the corresponding monohalogenated analogues. The
most potent of this type is the 3,4-chloro compound 5m
(IC50 ) 1.0 µM) and the 3,4-difluoro compound 5o (IC50
) 0.82 µM).
The ability of selected compounds to inhibit QUIN
production was performed using macrophage cultures
stimulated with interferon-γ as a model for QUIN
formation in inflammatory disease. Normally QUIN
levels are very low, but following activation of the type-2
interferon receptor, significant quantities of QUIN are
produced. The present study measured QUIN synthesis
(and its inhibition) directly from the conversion of [2H]-
tryptophan to [2H]QUIN by intact cells. The data in
Figure 3 reveal unexpected differences between the
3-chlorophenyl and 3-fluorophenyl matched ester/acid
pairs 2d ,3d and 2f,3f, respectively. The most potent
inhibitor, on intact cells, was 2d (IC50 ) 6 nM). The
corresponding acid 3d was 4-fold less potent (24 nM).
In contrast, the acid 3f inhibited cellular QUIN produc-
tion with an IC50 ) 40 nM, while its ester 2f was ∼150-
fold less potent (5600 nM). Such differences would not
be expected if the esters were simply prodrugs for the
acids and most likely reveal differences in binding to
the substrate site of KH. Additionally, 2d might be
expected to have improved brain penetration as the
ester compared to the corresponding acid 3d . The high
potency of 2d may reflect the fact that hydrolysis of the
ester may occur intracellularly and the active acid 3d
is retained within the cell. The lipophilicity and potency
of 2d make it an ideal candidate for neuroinflammatory
diseases in which QUIN is pathogenic.
Exp er im en ta l Section
Biologica l Assa ys. KH was isolated from rat liver as the
P2 mitochondrial pellet, resuspended in 0.32 M sucrose, and
used as the enzyme source. The assay of KH was based on
the release of 3HOH from 3-[3H]-L-kynurenine6 and was
performed at 37 °C under conditions of linearity with time and
enzyme protein at Km concentrations of substrate at 5 con-
centrations of inhibitor. Ki values were calculated from second-
ary plots of Km/[I] or estimated using the Chang-Prussoff
equation where
Ki ) IC50/(1 + [S]/Km)
Drysdale, Martin J.
