2-Amino-4-[3′-hydroxyphenyl]-4-hydroxybutanoic acid; A potent inhibitor of rat and recombinant human kynureninase

Article abstract of DOI:10.1016/S0960-894X(01)00758-2

A novel structural analogue of kynurenine, 2-amino-4-[3′-hydroxyphenyl]-4-hydroxybutanoic acid 6, was synthesised as an inhibitor of kynureninase. The compound had a significant inhibitory effect on kynureninase from both rat and human, giving a K_i

Full text of DOI:10.1016/S0960-894X(01)00758-2

Bioorganic & Medicinal Chemistry Letters 12 (2002) 361–363
2-Amino-4-[30-hydroxyphenyl]-4-hydroxybutanoic Acid; A Potent
Inhibitor of Rat and Recombinant Human Kynureninase
Harold A. Walsh, Pauline L. Leslie, Karen C. O’Shea and Nigel P. Botting*
School of Chemistry, University of St Andrews, St Andrews, Fife KY16 9ST, UK
Received 31 August 2001; accepted 7 November 2001
Abstract—A novel structural analogue of kynurenine, 2-amino-4-[30-hydroxyphenyl]-4-hydroxybutanoic acid 6, was synthesised as
an inhibitor of kynureninase. The compound had a significant inhibitory effect on kynureninase from both rat and human, giving a
Ki of 100 nM. It was thus found that removal of the aryl amino group coupled with a reduction of the carbonyl group at position 7
of the alanine side chain greatly enhanced potency of the inhibitor. # 2002 Elsevier Science Ltd. All rights reserved.
The excitotoxin quinolinic acid is implicated as an etio-
logical factor in a number of neurodegenerative diseases
such as AIDS related dementia, Alzheimer’s disease and
Parkinson’s disease as a result of its agonist activity at
the NMDA receptor.1 One method of regulating the
effects of this metabolite would involve the use of syn-
thetic inhibitors of the enzymes on the kynurenine
pathway of tryptophan metabolism, which is the
biosynthetic source of quinolinic acid.2 Kynureninase
(l-kynurenine hydrolase, E.C. 3.7.1.3), which catalyses
the b,g-hydrolytic cleavage of kynurenine 1 or 3-hydroxy-
kynurenine 2 (Scheme 1), is one potential therapeutic
target.
phinic acid analogue of kynurenine,7 which are all
competitive inhibitors of bacterial kynureninase with
varying potency. Drysdale and Reinhard8 have also
examined S-aryl-l-cysteine S,S-dioxides as inhibitors of
mammalian kynureninase from rat liver and observed
good inhibition. Recently this laboratory synthesised a
series of novel bicyclic kynurenine analogue inhibitors
of which a naphthyl analogue proved to be the most
potent against both the bacterial and recombinant
human enzymes.9 It is known that the constitutive rat
and human enzymes share an 85% amino acid sequence
homology10 and display a similar substrate-selectivity
and therefore it is possible that the potency of inhibitory
compounds in this instance could be similar.
To date there have been relatively few reported inhib-
itors for kynureninase and many lack specificity.3,4
number of inhibitors have been developed that mimic
the transition state for the kynureninase catalysed reac-
tion, including (4S)- and (4R)-dihydro-l-kynurenine,5 a
series of S-aryl-l-cysteine S,S-dioxides6 and a phos-
A
As part of our ongoing studies on kynureninase, 2-amino-
4-[30-hydroxyphenyl]-4-hydroxybutanoic acid
6 was
synthesised as a potential inhibitor for the enzyme. The
compound was designed to include a hydroxyl group at
the 7-position as a mimic of the putative tetrahedral
Scheme 1.
*Corresponding author. Tel.: +44-1334-463856; fax: +44-1334-463808; e-mail: npb@st-andrews.ac.uk
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00758-2

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H. A. Walsh et al. / Bioorg. Med. Chem. Lett. 12 (2002) 361–363
transition state, plus a hydroxyl group at C-3to give it
greater specificity for the mammalian enzymes. The
amino group at C-20 was not included, as previously, in
order to simplify the synthesis. The compound was
synthesised using a modification of our previous meth-
ods (Scheme 2),9 including a step to protect the
hydroxyl group at the 30-position as a pivaloyl deriva-
tive. The carbonyl group was reduced by catalytic
hydrogenation and all the protecting groups removed in
one step, using acid hydrolysis. Following final recrys-
tallisation, the spectral data11 indicated the presence of
a single diastereomer of the final product as a racemic
mixture. Comparison with previous literature data5
indicated that the two enantiomers were likely to be of
the (4S,2S) and (4R,2R) configuration.
using a spectrofluorimetric assay.13 The compound was
found to inhibit all three enzymes in a dose dependent
and reversible manner with equimolar affinity, giving
Ki=130 nM for the rat enzyme (Fig. 1) and Ki=100 nM
for the human enzyme (Fig. 2). However, the inhibitor
was approximately 100-fold less potent with the bac-
terial enzyme, giving a Ki of 10 mM. The inhibition was
distinctly mixed for the recombinant human enzyme
(Fig. 2). At low substrate concentrations the inhibition
was competitive whilst at higher levels the inhibition
became non-competitive. This is consistent with some
recent observations in our laboratory, which have indi-
cated the presence of a second regulatory binding site
on the human kynureninase.14 The degree of inhibition
with 5 mM 30-hydroxykynurenine as substrate was 55%
for the recombinant human enzyme (data not shown).
The inhibitory activity of the racemic 2-amino-4-[30-
hydroxyphenyl]-4-hydroxybutanoic acid 6 was then
assessed with rat liver kynureninase,9 recombinant
human kynureninase9,12 and bacterial kynureninase
Our experimental findings reveal that 2-amino-4-[30-
hydroxyphenyl]-4-hydroxybutanoic acid 6 is one of
the most potent inhibitors of human kynureninase
yet designed and synthesised. The importance of the
30-hydroxy group as a recognition element for the
mammalian enzyme is highlighted by the poorer inhibi-
tion observed with the bacterial enzyme. Indeed, recent
results in our laboratory have shown that purified
homogeneous recombinant human kynureninase only
hydrolyses 30-hydroxykynurenine and shows no activity
at all with kynurenine.14 It is possible that this com-
pound could provide a good lead for the develop-
ment of structurally related drugs with a greater
potency for inhibiting the activity of human kynur-
eninase, which may ultimately be used to regulate the
availability of quinolinate concentrations at the level of
the receptor.
Scheme 2. (a) (CH3)3CCOCl, pyridine (79%); (b) CuBr2, EtOAc/
CHCl3 (63%); (c) CH(CO2Et)2NHAc, NaH, DMF (57%); (d) H2, 5%
Pd/C, EtOAc (66%); (e) 6 N HCl (98%).
Figure 1. Kinetic data of inhibition of rat hepatic kynureninase 2-
amino-4-[30-hydroxyphenyl]-4-hydroxybutanoic acid. The inhibition
of rat kynureninase is depicted with respect to 3-hydroxykynurenine as
substrate. Data are presented as a Lineweaver–Burk plot for the fol-
lowing inhibitor concentrations: 0 mM (Á), 0.95 mM (*), 1.90 mM
(^), 3.80 mM (&). Initial rates of reaction (v=nmol/min/mg protein)
were assayed as described in ref 9. The concentration of substrate was
varied between 0 and 20 mM. An enzyme concentration of 3.4 mg/mL
was employed. The graph shown is the mean of three triplicate
experiments. Measurements were performed spectrofluorimetrically.
The inset is a secondary plot of slope against the concentration of
inhibitor, which was used to calculate Ki.
Figure 2. Kinetic data of inhibition of recombinant human kynur-
eninase 2-amino-4-[30-hydroxyphenyl]-4-hydroxybutanoic acid. The
inhibition of human recombinant kynureninase is depicted with
respect to 3-hydroxykynurenine as substrate. Data are presented as a
Lineweaver–Burk plot for the following inhibitor concentrations: 0
nM (*), 160 nM (^), 320 nM (*), 640 nM (~), 1280 nM (&), 2.56
mM (!) and 5 mM (&). Experiments were performed as described for
Figure 1 and detailed in ref 9. An enzyme concentration of 1.8 mg/mL
was employed.

H. A. Walsh et al. / Bioorg. Med. Chem. Lett. 12 (2002) 361–363
363
Acknowledgements
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Biochim. Biophys. Acta 1995, 1252, 185.
The authors gratefully acknowledge financial support
from the Wellcome Trust (for HAW) and a BBSRC
studentship (for KCO’S).
11. Mp 237 ꢀC; (found: C, 51.97; H, 6.24; N, 6.026; calcd for
ꢁ
C10H13NO4 H2O: C, 52.40; H, 6.60; N, 6.11%); dH (200 MHz,
2H2O) 2.31 (1H, m, J3a,3b=12.7, J2,3a=12.0 and J3a,4=10.7,
3a-H), 3.06 (1H, ddd, J3a,3b=12.7, J2,3b=8.2 and J3b,4=4.9,
3b-H), 4.57 (1H, dd, J2,3a=12.0 and J2,3b=8.2, 2-CH), 5.54
Referencesand Notes
(1H, dd, J3a,4=10.7 and J3b,4=4.9 Hz, 4-CH), 6.83(1H, dd,
0
J2 ,4 =2, J4 ,5 = 7, 40-H), 6.85 (1H, d, J2 ,4 =2, 2 -H), 6.93
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0
0
0
0
0
0
0
0
0
0
0
0
0
0
(1H, d, J5 ,6 =7, 6 -H), 7.29 (1H, t, J4 ,5 =J5 ,6 =7, 5 -H); dC
(75.45 MHz, H2O) 34.98 (s, 3-CH2), 49.9 (s, 2-CH), 80.0 (s,
2
4-CH), 113.1 (s, 20-C), 116.5 (s, 40-C), 118.3(s, 6 -C), 130.7 (s,
0
50-C), 138.6 (s, 10-C), 156.1 (s, 30-C), 173.6 (s, CO2H); m/z (EI)
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[MꢂNH2ꢂCO2HꢂH2O]+).
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