Inhibitors of kynureninase

The present invention provides inhibitors of kynureninase having the formula ##STR1## where X is CHOH, S, SO.sub.2, SO, SONH.sub.2, PO.sub.2 H or PONH.sub.2, R.sub.a and R.sub.b, independently of one another are H, a halogen, CF.sub.3 or a small alkyl group having one to three carbon atoms; R.sub.1 is H, NH.sub.2, NR.sub.6 RT, NO.sub.2, halogen, CF.sub.3 or a small alkyl group having from one to three carbon atoms, wherein: R.sub.6 and R.sub.7, independently of one another, are H, a formyl group or a small alkyl group having from one to three carbon atoms with the exception that only one of R.sub.6 or R.sub.7 can be a formyl group; R.sub.2 is OH, H, halogen, CF.sub.3 or a small alkyl group having from one to three carbon atoms; and R.sub.3, R.sub.4 and R.sub.5, independently of one another, are H, halogen, CF.sub.3, NO.sub.2, NH.sub.2, or small alkyl group having from one to three carbon atoms. In particular, compounds of this formula in which X is CHOH, S or SO.sub.2 are provided. In compounds of this formula in which X is CHOH, those having the (.alpha.S,.gamma.S) configuration or the (.alpha.R,.gamma.R) configuration when R.sub.A or R.sub.B is a hydrogen, are more potent inhibitors of kynureninase. Inhibitors of mammalian kynureninase are of particular use in therapy for certain neurological disorders.

BACKGROUND OF THE INVENTION 
Kynureninases are a group of pyridoxal-5'-phosphate dependent enzymes which 
catalyze the hydrolytic cleavage of aryl-substituted 
.alpha.-amino-.gamma.-keto acids, particularly L-kynurenine or 
3-hydroxy-L-kynurenine to give L-alanine and anthranilic acid or 
3-hydroxyanthranilic acid, respectively (see: K. Soda and K. Tanizawa 
(1979)Advances Enzym. 49:1-40). Kynureninase is involved in the microbial 
catabolism of L-tryptophan via the aromatic pathway. In plants and 
animals, a kynureninase is required in tryptophan catabolism and for NAD 
biosynthesis via quinolinic acid. Quinolinic acid is a relatively toxic 
metabolite which has been implicated in the etiology of neurological 
disorders, including epilepsy and Huntington's chorea (R. Schwarcz et al. 
(1988) Proc. Natl. Acad. Sci. USA 85:4079; M. F. Beal et al. (1986) Nature 
321:168-171; S. Mazzari et al. (1986) Brain Research 380:309-316; H. Baran 
and R. Schwarcz (1990) J. Neurochem. 55:738-744). Inhibitors of 
kynureninase are thus important targets for treatment of such neurological 
disorders. 
L-kynurenine (which can also be designated 
.alpha.,2-diamino-.gamma.oxobenzenebutanoic acid) is the preferred 
substrate of bacterial kynureninase, which is exemplified by that of 
Pseudomonas fluorescens (O. Hayaishi and R. Y. Stanier (1952) J. Biol. 
Chem. 195:735-740). The kynureninase of tryptophan metabolism in plants 
and animals has a somewhat different substrate specificity with 
3-hydroxy-L-kynurenine (which can be designated 
.alpha.,2-diamino-3-hydroxy-.gamma.-oxo-benzenebutanoic acid) being the 
preferred substrate (Soda and Tanizawa (1979) supra). 
The mechanism of kynureninases has been the subject of considerable 
interest due to the unique nature of this pyridoxal-5'-phosphate dependent 
reaction. Mechanisms based on redox reactions ((J. B. Longenecker and E. 
E. Snell (1955) J. Biol. Chem. 213:229-235) or transamination (C. E. 
Dalgleish et al. (1951) Nature 168:20-22) have been proposed. More 
recently mechanisms involving either a nucleophilic mechanism with an 
"acyl-enzyme" intermediate (C. Walsh (1979) "Enzymatic Reaction 
Mechanisms" W. H. Freeman and Co., San Francisco, p. 821; M. Akhtar et al. 
(1984) "The Chemistry of Enzyme Action" New Comprehensive Biochemistry, 
Vol. 6 (M. I. Page, ed.) Elsevier, N.Y., p.821) or a general 
base-catalyzed mechanism (K. Tanizawa and K. Soda (1979) J. Biochem. 
(Tokyo) 86:1199-1209) have been proposed. 
In addition to the physiological reaction, kynureninase has been shown to 
catalyze an aldol-type condensation of benzaldehyde with incipient 
L-alanine formed from L-kynurenine to give 
.alpha.-amino-.gamma.-hydroxy-.gamma.-phenylbutanoic acid (G. S. Bild and 
J. C. Morris (1984) Arch. Biochem. Biophys. 235:41-47). The 
stereochemistry of the product at the .gamma.-position was not determined, 
although the authors suggested that only a single isomer was formed. 
J. L. Stevens (1985) J. Biol. Chem 260:7945-7950 reports that rat liver 
kynureninase displays cysteine conjugate .beta.-lyase activity. This 
enzyme activity is associated with cleavage of S-cysteine conjugates of 
certain xenobiotics to give pyruvate, ammonia and a thiol, for example, 
cleavage of S-2-(benzothiazolyl)-L-cysteine to give 
2-mercaptobenzothiazole, pyruvate and ammonia. 
Several reports concerning the relative reactivities of kynurenine analogs 
with bacterial kynureninase or rat liver kynureninase are summarized in 
Soda and Tanizawa (1979) supra. Tanizawa and Soda (1979) supra reported 
that a number of ring substituted L-kynurenines, namely: 3-hydroxy-, 
5-hydroxy-, 5-methyl-, 4-fluoro-, and 5-fluoro-L-kynurenine were 
substrates of kynureninase of P. fluorescens. These authors also reported 
that dihydrokynurenine (called .gamma.-(o-aminophenyl)-L-homoserine 
therein) was a substrate for that kynureninase, yielding 
o-aminobenzaldehyde and L-alanine. The K.sub.m of dihydrokynurenine was 
reported to be 67 .mu.M compared to a K.sub.m of 35 .mu.M for L-kynurenine 
and 200 .mu.M for 3-hydroxy-L-kynurenine. N'-formyl-L-kynurenine and 
.beta.-benzoyl-L-alanine were likewise reported to be substrates (with 
K.sub.m =2.2 mM and 0.16 mM, respectively) for the bacterial kynureninase. 
Tanizawa and Soda measured relative reactivity as relative amounts of 
L-alanine formed. 
O. Hayaishi (1955) in "A Symposium on Amino Acid Metabolism" (W. D. McElroy 
and H. B. Glass, eds.) Johns Hopkins Press, Baltimore pp. 914-929 reported 
that 3-hydroxy- and 5-hydroxy-L-kynurenine, .beta.-benzoyl-L-alanine and 
.beta.-(o-hydroxybenzoyl)-L-alanine were substrates for the bacterial 
enzyme, but that N'-formyl-L-kynurenine was not a substrate. O. Hayaishi 
measured relative reactivities by determining the amount of substrate 
hydrolyzed. 
Tanizawa and Soda (1979) supra reported that S-benzoyl-L-cysteine, 
L-asparagine and D-kynurenine were not substrates of kynureninase, while 
O. Hayaishi (1955) supra reported that .beta.-(p-aminobenzoyl)-L-alanine, 
.beta.-(o-nitrobenzoyl)-L-alanine, .beta.-(m-hydroxybenzoyl)-L-alanine, 
3-methoxy-L-kynurenine, .beta.-benzoylpropanoic acid, 
and.beta.-(o-aminobenzoyl)propanoic acid do not react with bacterial 
kynureninase. Kynureninase is reported to act only on L-amino acids (M. 
Moriguchi et al. (1973) Biochemistry 12:2969-2974). 
O. Wiss and H. Fuchs (1950) Experientia 6:472 (see: Soda and Tanizawa 
(1979) supra) reported that 3-hydroxy-L-kynurenine, L-kynurenine, 
.beta.-benzoyl-L-alanine, .gamma.-phenyl-L-homoserine, 
.gamma.-methyl-L-homoserine, 2-aminolevulinic acid and 
.alpha.-amino-.gamma.-hydroxypentanoic acid reacted with rat liver 
kynureninase to produce alanine, while .beta.-(o-nitrobenzoyl)-L-alanine 
did not. 
G. M. Kishore (1984) J. Biol. Chem. 259:10669-10674 has reported that 
certain .beta.-substituted amino acids are mechanism-based inactivators of 
bacterial kynureninase. Several .beta.-substituted amino acids including 
.beta.-chloro-L-alanine, O-acetyl-L-serine, L-serine 0-sulfate, 
S-(o-nitrophenyl)-L-cysteine and .beta.-cyano-L-alanine inactivated 
kynureninase. These .beta.-substituted amino acids react with kynureninase 
to give pyruvate and ammonia. However, a portion of the turnovers of the 
enzyme lead to formation of an inactive enzyme complex. 
L-S-(o-nitrophenyl)-L-cysteine was described as the "most efficient 
suicide substrate at low concentrations" with a K.sub.i of 0.1 mM. 
Bacterial kynureninase is also strongly inhibited by o-aminobenzaldehyde 
(K.sub.i =6.5 .mu.M, non-competitive inhibition). Several other aromatics 
having "a carboxyl group on the benzene ring and an amino group at the 
ortho-position" including o-aminoacetophenone, anthranilic acid 
o-nitrobenzaldehyde and benzaldehyde were described as inhibitors 
(Tanizawa and Soda (1979) supra). It was suggested that inhibition relates 
to binding of the formyl group to the portion of the enzyme that serves as 
a binding site for the .gamma.-carboxyl of kynurenine. Anthranilate and 
3-hydroxanthranilate, the products of the kynureninase reaction, were also 
reported to inhibit the enzyme (Takeuchi et al. (1980) J. Biochem. (Tokyo) 
88:987-994). 
J. P. Whitten et al. (1989) Tetrahedron Letts. 30:3649-3652 reported the 
synthesis of 2,2-difluoro-.alpha.-benzoyl alanine 
(.alpha.-amino-.beta.,.beta.-difluoro-.gamma.-oxobenzene butanoic acid) 
which is said to be a "potential new inhibitor of kynureninase." 
Fluoroketone-containing peptides are described as capable of forming 
stable hydrates or hemiketals which are "thought to inhibit" proteolytic 
enzymes as analogs of a tetrahedral transition state. The difluoro 
compound is described as a competitive inhibitor of kynureninase, but no 
details of this inhibition are given in the reference. 
The present work is based on a reexamination of the mechanism of 
kynureninase catalysis, in particular, through an investigation of the 
stereospecificity of the retroaldol reaction catalyzed by the enzyme. 
During the course of this work, the reactivity of dihydrokynurenine with 
kynureninase was found to be significantly different than had previously 
been reported. The result of these mechanism and reactivity studies was 
the identification of a class of potent kynureninase inhibitors. The 
present invention provides kynureninase inhibitors which were designed to 
be "transition-state analogue" inhibitors. 
SUMMARY OF THE INVENTION 
It is an object of the present invention to provide means and compositions 
for inhibition of kynureninase. The inhibitors of this invention are amino 
acid derivatives of the formula: 
##STR2## 
wherein the stereochemical configuration at the .alpha. carbon is 
indicated (the same configuration as in L-kynurenine) where X is CHOH, S, 
SO.sub.2, SO, SONH, PO.sub.2 H, or PONH.sub.2, wherein R.sub.a and 
R.sub.b, independently of one another are H, halogen, CF.sub.3 or a small 
alkyl group having one to three carbon atoms; R.sub.1 is H, halogen, 
NH.sub.2, NR.sub.6 R.sub.7, NO.sub.2, CF.sub.3, or a small alkyl having 
from one to three carbon atoms; with R.sub.6 and R.sub.7, independently of 
one another, being H, CH.sub.3 or COH wherein only one of R.sub.6 or 
R.sub.7 can be COH; R.sub.2 is OH, H, halogen, CF.sub.3 or a small alkyl 
having from one to three carbon atoms; and R.sub.3, R.sub.4 and R.sub.5, 
independently of one another, are H, OH, halogen, CF.sub.3, NO.sub.2, 
NH.sub.2, or a small alkyl group having from one to three carbon atoms. 
X is preferably CHOH, S or SO.sub.2 with SO.sub.2 being generally more 
preferred than S. 
It is preferred that the halogen is fluorine, that R.sub.2 is H or OH, that 
R.sub.1 is H or NH.sub.2, and that R.sub.A, R.sub.B, R.sub.4 and R.sub.5 
are H or fluorine. It is preferred that R.sub.3 is H, NH.sub.2, NO.sub.2 
or fluorine, with H or fluorine more preferred. It is more preferred that 
R.sub.1 is NH.sub.2 and R.sub.A, R.sub.B, R.sub.3, R.sub.4 and R.sub.5 are 
H. 
For inhibition of bacterial kynureninase, it is preferred that R.sub.2 is 
H. For inhibition of plant and animal kynureninase, it is preferred that 
R.sub.2 is OH. 
It is a specific object of the present invention to provide kynureninase 
inhibitors which are derivatives of 
.alpha.-amino-.gamma.-hydroxy-.gamma.-hydroxybenzene butanoic acids of the 
formula: 
##STR3## 
wherein the stereochemical configuration at the .alpha. carbon is as 
indicated (the same configuration as L-kynurenine), wherein R.sub.a and 
R.sub.b, independently of one another are H, halogen, CF.sub.3 or a small 
alkyl group having one to three carbon atoms; R.sub.1 is H, halogen, 
NH.sub.2, NR.sub.6 R.sub.7, NO.sub.2, CF.sub.3 or a small alkyl group 
having from one to three carbon atoms, with R.sub.6 and R.sub.7, 
independently of one another, being H, CH.sub.3 or COH, wherein only one 
of R.sub.6 or R.sub.7 can be COH; R.sub.2 is OH, H, halogen, CF.sub.3, or 
a small alkyl group having from one to three carbon atoms; and R.sub.3, 
R.sub.4 and R.sub.5, independently of one another, are H, OH, halogen, 
CF.sub.3, NO.sub.2, NH.sub.2, or a small alkyl group having from one to 
three carbon atoms. It is preferred that the halogen is fluorine, that 
R.sub.2 is H or OH that R.sub.1 is NH.sub.2 or H and that R.sub.A, 
R.sub.B, R.sub.3, R.sub.4 and R.sub.5 are H or fluorine. It is more 
preferred that R.sub.1 is NH.sub.2 and that R.sub.A, R.sub.B, R.sub.3, 
R.sub.4 and R.sub.5 are H. 
For inhibition of bacterial kynureninase it is preferred that R.sub.2 is H. 
For inhibition of plant and animal kynureninase it is preferred that 
R.sub.2 is OH. 
It is a more specific object of this invention to provide kynureninase 
inhibitors which are .alpha.-amino-.gamma.-hydroxy-.gamma.-aryl butanoic 
acids having the structure: 
##STR4## 
wherein the stereochemical configuration at the .alpha. and .gamma. 
carbons is as indicated (the configuration at the .alpha. carbon being the 
same as in L-kynurenine) and wherein R.sub.1-7, R.sub.A and R.sub.B are as 
defined above for formulas I and II. For inhibition of bacterial 
kynureninase it is preferred that R.sub.2 is H. For inhibition of plant 
and animal kynureninase it is preferred that R.sub.2 is OH. 
It is a second specific object of this invention to provide S-aryl 
derivatives of L-cysteine which are inhibitors of kynureninase having the 
formula: 
##STR5## 
where the stereochemical configuration at the .alpha.-carbon is as 
indicated (the same as in L-kynurenine) where R.sub.1-7, R.sub.A and 
R.sub.B are as defined for formulas I, II and III. 
It is a further specific object of this invention to provide 
S-aryl-L-cysteine sulfones which are inhibitors of kynureninase having the 
formula: 
##STR6## 
where the stereochemical configuration at the .alpha.-carbon is as 
indicated (the same as in L-kynurenine) and R.sub.1-7, R.sub.A and R.sub.B 
are as defined for formulas I-IV. 
Salts of the compounds of formulas I-V are considered functional 
equivalents thereof with respect to inhibition of kynureninase. In 
particular, pharmaceutically acceptable salts of the compounds of formulas 
I-V are useful for the methods of the present invention and are useful in 
any therapeutic treatment of animals based on the inhibitory action of the 
compounds of formulas I-V. 
Inhibitors of the present invention include, among others, ring fluorinated 
dihydrokynurenines: (.alpha.S,.gamma.S)- or (.alpha.S, 
.gamma.R)-.alpha.,2-diamino-.gamma.-hydroxy-4-fluorobenzenebutanoicacid, 
(.alpha.S,.gamma.S)- or 
(.alpha.S,.gamma.R)-.alpha.,2-diamino-.gamma.-hydroxy-4-fluorobenzenebutan 
oic acid; ring hydroxylated dihydrokynurenines: (.alpha.S,.gamma.S)- or 
(.alpha.S,.gamma.R)-.alpha.,2-diamino-.gamma.,5-dihydroxybenzenebutanoic 
acid; ring methylated dihydrokynurenines (.alpha.S,.gamma.S)- or 
(.alpha.S,.gamma.R)-.alpha.,2-diamino-.gamma.-hydroxy- 
5-methylbenzenebutanoic acid, or ring-substituted (.alpha.S,.gamma.S)- or 
(.alpha.S,.gamma.R)-.alpha.-amino-.gamma.,2-dihydroxybenzenebutanoic acid. 
Inhibitors of kynureninase also include dihydrokynurenines: 
(.alpha.S,.gamma.S)-.alpha.,2-diamino-.gamma.-hydroxybenzenebutanoic acid 
and (.alpha.S,.gamma.R)-.alpha.,2-diamino-.gamma.-hydroxybenzenebutanoic 
acid; 3-hydroxydihydrokynurenines: 
(.alpha.S,.gamma.S)-.alpha.,2-diamino-.gamma.,3-dihydroxybenzenebutanoic 
acid and 
(.alpha.S,.gamma.R)-.alpha.,2-diamino-.gamma.,3-dihydroxylbenzenebutanoic 
acid and dihydrodesaminokynurenines: 
(.alpha.S,.gamma.S)-.alpha.-amino-.gamma.-hydroxybenzenebutanoic acid and 
(.alpha.S,.gamma.R)-.alpha.-amino-.gamma.-hydroxybenzenebutanoic acid. 
Dihydrokynurenine and dihydrodesaminokynurenine ( see Soda and Tanizawa 
(1979) Supra p. 32, Table VIII) were previously reported to be substrates 
for certain kynureninases. Alternate substrates will act as competitive 
inhibitors toward the "natural" enzyme substrate. Dihydrokynurenine 
(Tanizawa and Soda (1979) supra) was reported to react readily with 
bacterial kynureninase with a reactivity about 65% that of L-kynurenine. 
The dihydrokynurenine employed in that reference was indicated to be a 
mixture of the (.alpha.S,.gamma.S) and (.alpha.S,.gamma.R) 
dihydrokynurenine diastereomers. It was not disclosed therein and the data 
given therein do not suggest that one of the diastereomers 
(.alpha.S,.gamma.S) is not a substrate for the kynureninase but acts as a 
competitive inhibitor of the enzyme for reaction of its natural 
substrates. 
It is a further object of this invention to provide a method of inhibiting 
kynureninase in vitro and/or in vivo which comprises the step of 
contacting the enzyme with an inhibitory amount of one or more of the 
compounds of formulas I-V or salts, particularly pharmaceutically 
acceptable salts, thereof. It is well understood in the art that a 
precursor prodrug may be converted in vivo to a therapeutically active 
drug. Any such prodrug precursors of the compounds of formulas I-V are 
encompassed by this invention. 
Therapeutic applications of the methods of the present invention relate 
particularly to inhibition of animal kynureninases, particularly those of 
mammals. Inhibitors in which R.sub.1 is NH.sub.2 and R.sub.2 is OH are 
preferred for such therapeutic applications. 
Compounds of the present invention that are preferred for therapeutic 
applications of the methods of the present invention are those that have 
minimal toxic or irritant effect toward the target of the therapy. If the 
inhibitor reacts with kynureninase, it is important that the product of 
that reaction be substantially nontoxic. 
Kynureninases from different sources have different substrate preferences. 
For example, the preferred substrate of mammalian kynureninase is 
3-hydroxy-L-kynurenine rather than L-kynurenine. In general, for a 
particular kynureninase, a preferred inhibitor of formula I-V will possess 
the phenyl ring substitutions of a preferred substrate of that 
kynureninase. 
DETAILED DESCRIPTION OF THE INVENTION 
Kynureninases catalyze the hydrolysis of aryl-substituted 
.gamma.-keto-.alpha.-amino acids. Kynureninase has been identified and 
isolated from certain bacteria, fungi, yeasts as well as from mammalian 
sources. Kynureninases from different sources have been reported to have 
different substrate specificities. L-kynurenine is the preferred "natural" 
substrate of bacterial kynureninase. In contrast for mammalian, yeast and 
fungal kynureninases, 3-hydroxy-L-kynurenine is the preferred "natural" 
substrate. This preference for 3-hydroxy-L-kynurenine, as assessed by 
relative substrate K.sub.m 's, is characteristic of animal and plant 
kynureninases. The relative affinities of kynureninases for substrates 
other than L-kynurenine and 3-hydroxy-L-kynurenine can also depend on the 
source of the enzyme. Animal and plant kynureninases are sometimes called 
3-hydroxykynureninases. The term kynureninase as used herein includes both 
bacterial, plant and animal kynureninases. Bacterial kynureninases are 
exemplified by the enzyme isolated from Pseudomonas fluorescens. Mammalian 
kynureninase is exemplified by the enzyme isolated from mammalian liver, 
in particular rat liver. A bacterial kynureninase will generally display 
substrate specificity like that of the P. fluorescens kynureninase. 
Mammalian kynureninase will generally display substrate specificity like 
that of rat liver kynureninase. Kynureninases, from all sources, catalyze 
the same types of reactions and so the mechanisms of the reactions they 
catalyze should be the same. Differences in affinities for substrates is 
believed to be associated with differences in the substrate binding site. 
The present invention provides inhibitors of kynureninase. Some of these 
inhibitors are substrates of the enzyme, some are not substrates. Many of 
the inhibitors of this invention are competitive inhibitors of the enzyme 
for their natural substrates L-kynurenine and 3-hydroxy-L-kynurenine. 
Inhibition, as used herein, refers to inhibition of the hydrolysis of 
L-kynurenine and 3-hydroxy-L-kynurenine. Competitive inhibition and 
noncompetitive inhibition can be assessed by in vitro methods well-known 
in the art. Preferred inhibitors of a particular kynureninase are those 
having a K.sub.i less than or equal to the K.sub.m of the preferred 
substrate either L-kynurenine or 3-hydroxy-L-kynurenine for that 
kynureninase. In general for competitive inhibitors, it is preferred that 
the inhibitor have an affinity equal to or greater than that of the 
preferred substrate for the enzyme. The level of inhibition that is 
achieved is dependent on the concentration of inhibitor in the vicinity of 
the enzyme. In general, the higher the affinity of the enzyme for the 
inhibitor, the more potent an inhibitor is. For applications of the 
methods of inhibition of kynureninase, particularly therapeutic 
applications, it is generally preferred to employ high affinity (low 
K.sub.i) inhibitors to minimize the amount of inhibitor that must be 
administered. 
Kynureninases are known to catalyze other reactions, for example, cysteine 
conjugate .beta.-lyase activity. Inhibition of kynureninases can also be, 
at least qualitatively, assessed employing in vitro assays for such 
alternate kynureninase activities. 
The aldol reaction of L-kynurenine and benzaldehyde catalyzed by 
kynureninase was found to proceed to give predominantly (80%) the 
(.alpha.S, .gamma.R) diastereomer of 
.alpha.-amino-.gamma.-hydroxybenzenebutanoic acid. 
The stereospecificity of the aldol reaction, as well as the results of Bild 
and Morris, Arch. Biochem. Biophys. (1984) 235:41-47, supports a general 
base mechanism for kynureninase, as shown in Scheme I. The 
stereospecificity for cleavage of the (4R)-isomer is likely a reflection 
of favorable orientation for the active site general base to initiate the 
retro-aldol cleavage by proton abstraction (Scheme IA). 
##STR7## 
The basic group involved is probably the carboxylate that Kishore (1984) 
supra reported is modified by suicide substrate inhibitors. Although 
Kishore proposed that this carboxylate is responsible for .alpha.-proton 
abstraction, stereochemical studies by Palcic et al., J. Biol. Chem. 
(1985) 260:5248-5251, found that a .alpha.-proton of kynurenine is 
scrambled between the .alpha. and .beta.-positions of the L-alanine 
product, and thus the proton abstraction at the .alpha.-C is probably due 
to a polyprotic base, most likely a lysine .epsilon.-amino group. In the 
hydrolysis of L-kynurenine, the second general base would be required to 
assist in hydration of the ketone, by abstraction of a proton from a water 
molecule (Scheme IB). The observed stereochemistry of the aldol-reactions 
suggests that the water attacks on the reface of the carbonyl group, 
giving the (S)-gem-diolate anion. Subsequent rapid collapse of this 
tetrahedral intermediate is likely and would generate the enzyme-bound 
enamine of PLP-L-alanine and anthranilic acid (Scheme IB). In the case of 
the (4S)-isomer, the carbinol group would mimic this gem-diol tetrahedral 
intermediate, but is not oriented in a position favorable for the 
retro-aldol reaction to occur. Thus, this compound is a "transition-state 
analogue," and would be expected to bind to kynureninase very tightly. 
As an extension of these mechanistic studies, the reactivities of 
dihydrokynurenine diastereomers were examined. 
(.alpha.S,.gamma.)-Dihydrokynurenine 
(.alpha.S,.gamma.)-.alpha.,2-diamino-.gamma.-hydroxybenzenebutanoic acid) 
was found to be a slow substrate for the retro-aldol cleavage reaction 
catalyzed by kynureninase, while the analogous (.alpha.S,.gamma.S) 
diastereomer was unreactive. When these compounds were included in 
reaction mixtures of enzyme and L-kynurenine, the reaction was strongly 
inhibited. Analysis of the kinetic data in the presence of various 
concentrations of the dihydrokynurenines demonstrated that they act as 
competitive inhibitors with respect to kynurenine, and the data indicate 
that (.alpha.S,.gamma.S)-dihydrokynurenine binds more tightly than does 
L-kynurenine. This increased affinity of 
(.alpha.S,.gamma.S)-dihydrokynurenine is characteristic of 
mechanism-based, or "transition-state analogue" inhibitors. 
The design of the kynureninase inhibitors of the present invention was 
based on the results of the inhibition studies on the diastereomers of 
dihydrokynurenine in combination with what is known of substrate 
specificity of kynureninases. 
Although not wishing to be bound by any specific theory, it is believed 
that the inhibitors of the present invention represent "transition-state 
analogue" inhibitors of kynureninase in view of the newly proposed 
mechanism of Scheme I. Based on this proposed mechanism 
.alpha.-amino-.gamma.-hydroxybenzenebutanoic acids having electron 
withdrawing groups, including but not limited to, CF.sub.3, halogen, 
NO.sub.2, CN etc. appropriately substituted on the benzene ring to 
stabilize the proposed "transition state" will act as inhibitors of the 
kynureninase. 
The kynureninase inhibitors of the present invention can be prepared as 
exemplified for the preparation of the dihydrokynurenine diastereomers by 
selective reduction of the keto group of an appropriate .gamma.-keto-amino 
acid or by other methods well known in the art. Kynurenines, including 
various ring-substituted kynurenines, can be prepared by ozonolysis of 
tryptophans. Alternatively, kynurenine analogs with desired ring 
substitution can be prepared enzymatically from appropriate tryptophans as 
described in Tanizawa and Soda (1979) supra and O. Hayaishi (1953) in 
Biochemical Preparations (E. E. Snell, ed.) Vol. 3, John Wiley & Sons, 
Inc., New York, pp. 108-111. The .gamma.-keto amino acid, 
.beta.-benzoyl-DL-alanine can be prepared in several ways (for example, C. 
E. Dalgleish (1952) J. Chem. Soc. 137-141 and F. M. Veronese et al. (1969) 
Z. Naturforsch. 24:294-300) including amination of .beta.-benzoylacrylate 
(Tanizawa and Soda (1979) supra). .beta.-Benzoyl alanines having various 
desired ring substitution can be prepared using analogous methods starting 
with appropriately substituted starting materials. Hayaishi (1955) supra 
and Wiss and Fuchs (1950) supra also provide sources of .gamma.-keto amino 
acids useful for preparation of the compounds of the present invention. 
.beta.-Benzoyl alanines can be selectively reduced by means known to the 
art to produce the inhibitors of the present invention. 
Similarly, .beta.-substituted .gamma.-keto amino acids can serve as 
precursors to the .beta.-(or 2-)substituted .gamma.-hydroxy amino acids of 
the present invention. Whitten et al. (1990) supra, provides a synthesis 
of 2,2-difluoro-2-benzoyl alanine which can be selectively reduced to give 
.alpha.-amino-.gamma.,.gamma.-difluoro-.gamma.-hydroxybenzenebutanoic 
acid. Analogous methods can be employed to prepare .beta.-substituted, 
phenyl-ring substituted .gamma.-hydroxybenzenebutanoic acids of the 
present invention. 
As will be appreciated by those in the art, reduction of a chiral 
nonracemic .gamma.-keto amino acid, preferably an L-amino acid will 
generally result in a mixture of diastereomers. Techniques are available 
and well known in the art for the separation of diastereomers (HPLC, 
preparative TLC, etc.). 
S-(nitrophenyl)-L-cysteines (IV (NO.sub.2)), were synthesized by 
nucleophilic aromatic substitution of fluoronitrobenzenes with L-cysteine 
in DMF in the presence of triethylamine (Phillips et al. (1989) Enzyme 
Microb. Techno. 11:80-83). The unsubstituted S-phenyl-L-cysteine (IV (H)) 
was synthesized enzymatically following a procedure by Soda et al. (1983) 
47(12):2861 (Scheme II). This method involves incubating thiophenol with 
L-serine in the presence of tryptophan synthase at 37.5.degree. C. for 48 
hrs. Reduction of S-(nitrophenyl)-L-cysteines was accomplished by stirring 
with Zn dust and acetic acid. The oxidation of thioethers to sulfones was 
achieved by using a procedure described by Goodman et al. (1958) J. Org. 
Chem 23:1251, with slight modifications. This method produced good results 
when the aryl cysteines were treated with a mixture of formic acid (98%) 
and hydrogen peroxide (30%). However, when 88% formic acid was used for 
this reaction a slightly impure product was obtained and the yields were 
also lower. The ease of formation of the sulfone depends on the position 
of nitro group on the ring. When a nitro group is present at the ortho 
position the completion of reaction took 48 hours or more, whereas, when 
there is no nitro group on the ring or when the nitro group is at the 
4-position, the reaction is complete in 12 hours. Reduction of the nitro 
sulfones was performed by catalytic hydrogenation using acetic acid or 
formic acid as solvent (Scheme III). 
Sulfoxide derivatives (I where X=SO) of the present invention can be 
prepared from known and readily available starting materials by means 
well-known to the art, for example, by oxidation of corresponding 
thioethers as described in Example 7 in the presence of a limiting amount 
of hydrogen peroxide. 
Sulfoxamide derivatives of this invention can also be prepared from known 
and readily available starting materials by means well-known to the art. 
Phosphinate and phosphinamide derivatives (I where X =PO.sub.2 H or 
PONH.sub.2) can be prepared from known and readily available starting 
materials by means well-known in the art, for example, by the Arbuzov 
reaction (Arbuzov. (1964) Pur Appl. Chem. 9:307-335) or routine 
modifications thereof. 
The results of competitive inhibition of certain thioether and sulfone 
compounds are shown in Table 1. Among all the compounds tested, the 
unsubstituted S-phenyl-L-cysteine was found to be a very weak inhibitor, 
with K.sub.i value of 0.7 mM, however, its oxidized analog, 
S-phenyl-L-cysteine sulfone, showed a 180 fold decrease in K.sub.i value 
to 3.9 .mu.M. Similarly, substitution of an ortho-amino group in the 
S-(2-aminophenyl)-L-cysteine showed a 318 fold decrease in K.sub.i to 2.2 
.mu.M. The compound which combined both of these structural features, 
S-(2-aminophenyl)-L-cysteine sulfone turned out to be the most potent 
inhibitor of kynureninase, with K.sub.i of about 70 nM. A similar, but 
less significant improvement in the activity of the compounds were 
observed by sulfone formation in the cases of 2-nitro,4-nitro and 4-amino 
compounds. The results discussed above on the potent inhibition by 
dihydrokynurenines indicate that the kynureninase reaction proceeds via a 
gem-diol intermediate. The results of Table 1 indicate that the oxygens on 
the sulfur mimic the gem-diol tetrahedral transition state in the reaction 
of L-kynurenine with kynureninase. Therefore, these compounds are 
additional examples of transition state analogs. The presence of amine 
group and its position on the aromatic ring plays an important role in the 
activity of the compound. When the amine group is moved from the 
2-position to the 4-position of the ring, the activity of the compound 
drops 50-fold in case of cysteines and 120-fold in case of the respective 
sulfones. This regiospecificity is expected, since the 
2-aminophenyl-L-cysteines are closer structural analogues of kynurenine. 
The presence of the nitro group on the ring decreases the activity of all 
the compounds by several fold, possibly due to unfavorable steric 
interactions. 
As has been described herein, one of the pair of diastereomers in cases in 
which diastereomers can exist, will be a preferred kynureninase inhibitor. 
It will be appreciated, however, that inhibition can be obtained by use of 
a mixture of the diastereomers. In order to obtain maximal inhibition for 
the amount of inhibitor employed, it will be preferable to maximize the 
amount of the more inhibitory diastereomer in the mixture. 
TABLE 1 
______________________________________ 
Competitive Inhibition of Kynureninase. 
##STR8## 
M K.sub.i (.mu.M) 
______________________________________ 
##STR9## 700 
##STR10## 3.9 
##STR11## 100 
##STR12## 23 
##STR13## 2.5 
##STR14## 0.07.sup.1 
##STR15## -- 
##STR16## 12 
##STR17## 140 
10. 
##STR18## 8.5 
______________________________________ 
.sup.1 This value is an upper limit. K.sub.i here is approximately the 
same order of magnitude as the concentration of enzyme in the assay, so 
that the steadystate approximation may not apply.

EXAMPLES 
Example 1 
Investigation of the Mechanism of Kynureninase-catalyzed adol-reactions. 
Bacterial kynureninase was prepared from cells of Pseudomonas fluorescens 
(ATCC 11250, for example) essentially as described by Hayaishi and Stanier 
(1952) J. Biol. Chem. 195:735-740. Cells were grown on a minimal medium 
containing 0.1% L-tryptophan as the sole carbon and nitrogen source. 
From 100 l of medium, grown for 18 h at 30.degree. C., 230 g of wet cell 
paste was obtained. The cells were suspended in 1 l of 0.01M potassium 
phosphate, pH 7.0, and disrupted by 2 passages through a Manton-Gaulin 
homogenizer. After centrifugation of the cell extract for 1 h at 10000 g, 
the enzyme was partially purified by ion-exchange chromatography on 
DEAE-cellulose and ammonium sulfate precipitation. The preparation used in 
the results of Table 1 exhibited a specific activity of 0.2 .mu.mol 
min.sup.-1 mg.sup.-1. 
L-kynurenine and benzaldehyde (in excess) were incubated with kynureninase 
under the conditions described by Bild and Morris (1984) Arch. Biochem. 
Biophys. 235:41-47, which is incorporated by reference herein. The product 
of this reaction was purified by preparative HPLC and identified as 
.alpha.-amino-.gamma.-hydroxybenzenebutanoic acid. This product was 
produced in quantitative yield based on L-kynurenine. 
The .alpha.-amino-.gamma.-hydroxybenzenebutanoic acid produced in the 
kynureninase reaction exhibited a negative CD (circular dichroism) 
extremum at 260 nm, with vibronic splitting characteristic of a chirally 
substituted benzoyl alcohol chromophore. Based on a comparison of the CD 
spectra of the product with those of (R)- and (S)-mandelic acids, the 
predominant chiral product was determined to have the same absolute 
configuration as (S)-mandelic acid and thus to have the 
(.gamma.R)-configuration. (The terms R and S are employed as is 
conventional according to the Cahn-Ingold-Prelog rules.) NMR analysis (300 
MH.sub.z .sup.1 H) of the product demonstrates that it is an 80:20 mixture 
of (.alpha.S,.gamma.R):(.alpha.S,.gamma.S) diastereomers of 
.alpha.-amino-.gamma.-hydroxybenzene butanoic acid. 
Example 2 
Reactivity of Dihydrokynurenine with Kynureninase. 
L-kynurenine (from commercial sources) was reduced with NaBH.sub.4 in 
H.sub.2 O to give dihydrokynurenine 
[.alpha.,2-diamino-.gamma.-hydroxybenzenebutanoic acid]. The progress of 
reaction was monitored by the disappearance of the 360 nm UV absorption 
band of L-kynurenine. The reduction resulted in a 60:40 mixture of 
diastereomers. The diastereomers were separated by preparative HPLC on a 
20.times.250 mm C18 column (Rainin, Dynamax) eluting with 0.1% acetic acid 
(5 ml/min). The first peak to elute from the HPLC column was identified by 
.sup.1 H NMR analysis to be the (.alpha.S,.gamma.S)-diastereomer. The 
second peak to elute was identified by .sup.1 H NMR analysis to be the 
(.alpha.S,.gamma.R)-diastereomer. 
The CD spectra of the separated dihydrokynurenine diastereomers were 
consistent with this identification. 
The reactivity of the two dihydrokynurenines with kynureninase in 0.1M 
potassium phosphate buffer, pH 8.0, at 25.degree. was examined. Reaction 
was followed by the appearance of o-aminobenzaldehyde, as determined 
spectrophotometrically by the increase in absorbance at 360 nm (See 
Tanizawa and Soda (1979) Biochem. (Tokyo) 86:1199-1209, which is 
incorporated by reference herein). 
The (.alpha.S,.gamma.R)-dihydrokynurenine diastereomer reacted slowly with 
kynureninase to produce o-aminobenzaldehyde. No significant reaction of 
the (.alpha.S,.gamma.S)-diastereomer was detected. Tanizawa and Soda 
(1979) supra had reported that dihydrokynurenine reacted with kynureninase 
with a Vmax of about 65% that of L-kynurenine. In contrast, the present 
work indicates that only the (.alpha.S,.gamma.R)-diastereomer of 
dihydrokynurenine reacts, only at about 5% of the rate of L-kynurenine. 
Under the conditions employed and with the bacterial kynureninase prepared 
as described in Example 1, K.sub.m of the reaction of L-kynurenine was 
determined to be 25 .mu.M. This value is similar to the K.sub.m of 35 
.mu.M for L-kynurenine obtained by Tanizawa and Soda. 
Example 3 
Inhibition of Kynureninase by Dihydrokynurenine. 
Inhibition of kynureninase by dihydrokynurenine was measured by including 
the potential inhibitor in the enzyme assay mixture (see Example 1 and 
Tanzawa and Soda (1979) supra) and determining the apparent Km for 
L-kynurenine (the preferred substrate of bacterial kynurenine) in the 
absence and presence of the potential inhibitor. K.sub.i values were then 
calculated using the standard equation: 
EQU (K.sub.m).sub.app =K.sub.m (1+[I]/K.sub.i) 
where [I] is the molar concentration of inhibitor and K.sub.m =25 .mu.M. 
Inhibition of kynureninase by the (.alpha.S,.gamma.R)- and 
(.alpha.S,.gamma.S)-diastereomers of dihydrokynurenine was examined and 
K.sub.i 's were determined. Both compounds strongly inhibited the reaction 
of kynureninase with L-kynurenine. The K.sub.i value for the (.alpha.S, 
.gamma.S)-diastereomer was lower than for the 
(.alpha.S,.gamma.R)-diastereomer. Both compounds were found to be 
competitive inhibitors of kynureninase. 
Inhibition of mammalian kynureninase can be measured using several 
different assays for enzyme activity. Rat liver kynureninase is obtained 
from homogenization of rat liver, followed by precipitation with 
(NH.sub.4).sub.2 SO.sub.4, as described by Steven, J. L., J. Biol. Chem. 
(1985) 260:7945-7950, which is incorporated by reference herein. The 
activity of rat liver kynureninase was assessed by measurement of the 
cysteine conjugate .beta.-lyase activity, as described by Steven (supra), 
with S-(2-benzothiazolyl)cysteine, a nonphysiological chromophoric 
substrate. Inhibition of kynureninase by the dihydrokynurenine 
diastereomers was assessed with respect to reaction with that substrate. 
Both the (.alpha.S,.gamma.R) and (.alpha.S,.gamma.S) diastereomers of 
dihydrokynurenine were found to inhibit the reaction of rat liver 
kynureninase. The (.alpha.S,.gamma.S) diastereomer was found to be the 
stronger competitive inhibitor with K.sub.i under the assay conditions of 
about 690 .mu.M. 
Example 4 
Synthesis of S-(phenyl)-L-cysteines (IV (H)). 
A mixture containing 1.23 ml (12 mM) of thiophenol, 0.525 g (5 mM) of 
L-serine, 10 .mu.M of potassium phosphate buffer, pH 7.8, 0.13 mg (20 nM) 
of pyridoxal-5'-posphate and 5 mg of tryptophan synthase in a total volume 
of 25 ml was stirred at 37.5.degree. C. After 48 hours the reaction 
mixture was cooled, the thick white precipitate was filtered and washed 
with water and ethanol to give 0.31 g of white crystals of 
S-(phenyl)-L-cysteine. 
Tryptophan synthase was purified from cells of E. coli CB149 with plasmid 
pSTB7 containing the trpA and trpB genes from Salmonella typhimurium, as 
described by Miles et al. (1989) J. Biol. Chem. 264:6280. 
Example 5 
Synthesis of S-(nitrophenyl)-L-cysteines (IV(NO.sub.2). 
To a flask containing 5 g of L-cysteine, 4.47 g of fluoronitrobenzene and 
20 ml of DMF was added 7.84 ml of triethylamine. After stirring at room 
temperature for 3-4 hours, the contents of the flask solidifed into a 
thick yellow cake. This solid was mixed with 15-20 ml of water and 
filtered to give crude S-nitrophenyl-L-cysteine. Recrystalization from hot 
water gave lemon yellow crystals of the product. 
Example 6 
Synthesis of S-(aminophenyl)-L-cysteines (IV(NH.sub.2)). 
0.4 g of the S-nitrophenyl compound was dissolved in 50 mL of acetic acid, 
2.0 g of zinc dust was added, and the mixture stirred at room temperature 
overnight. After completion of the reaction, the solid was filtered on 
celite and the filtrate was concentrated in vacuo to give an oil. This oil 
was triturated with water and methanol to give an off white solid of the 
reduced compound. 
Example 7 
Synthesis of S-phenyl and S-nitrophenyl-L-cysteine sulfones (V(H) and 
V(NO.sub.2)). 
0.65 g of S-phenyl or S-nitrophenyl compound was dissolved in 20 ml of 98% 
formic acid and 4 ml of 30% hydrogen peroxide, and the mixture stirred at 
room temperature for 12-48 hours, depending on the compound as discussed 
above. After completion of the reaction, the solvent was carefully 
evaporated in vacuo at 25.degree.-30.degree. C. to give a white solid of 
the desired product. 
Example 8 
Synthesis of S-(aminophenyl)-L-cysteine sulfones (V(NH.sub.2)). 
0.4 g of nitrophenyl sulfone was dissolved in 50 ml of formic acid, 0.045 g 
of 10% Pd--C was added, and the mixture hydrogenated for 30 minutes. The 
charcoal was removed by filtration on celite and the filtrate was 
concentrated in vacuo to give a light tan oil, which upon trituration with 
methanol gave a light tan solid of the aminophenyl sulfone. 
Example 9 
Competitive Inhibition of Kynureninase by Compounds (IV and V). 
Kynureninase activity was measured at 25.degree. C. by following the 
decrease in absorbance at 360 nm (.epsilon.=-4500 M.sup.-1 cm.sup.-1). A 
typical assay mixture contained 0.4 mM L-kynurenine in 0.04M potassium 
phosphate, pH 7.8, containing 40 .mu.M pyridoxal-5'-phosphate, at 
25.degree. C. The reactions of S-aryl cysteines and S-aryl cysteine 
sulfones with kynureninase were performed using a spectrophotometric 
coupled assay with lactate dehydrogenase and NADH, by monitoring a 
decrease in absorbance due to pyruvate formation. A typical assay mixture 
contained 30 .mu.l lactate dehydrogenase solution (2 mg/ml), 0.1 mM NADH, 
40 .mu.M pyridoxal-5'-phosphate, 0.04M Tris.HCl buffer, pH 7.8, with 
varying concentrations of the compounds. The competitive inhibition of 
these compounds was measured by variation of L-kynurenine concentrations 
at several fixed values of the inhibitor. K.sub.m and V.sub.max values 
were calculated by fitting of initial rate data to the Michaelis-Menten 
equation with ENZFITTER (Elsevier) on a Z-286 personal computer. KI values 
were determined from the equation: 
EQU v=V.sub.max [S]/(K.sub.m (1+[I]/K.sub.i)+[S] 
Results for certain compounds of formulas IV-IX are given in Table 1.