Synthesis and QSAR of substituted 3-hydroxyanthranilic acid derivatives as inhibitors of 3-hydroxyanthranilic acid dioxygenase (3-HAO)

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Abstract

Novel 4,5-, 4,6-disubstituted and 4,5,6-trisubstituted 3-hydroxyanthranilic acid derivatives were synthesized and their ability to reduce the production of the excitotoxin quinolinic acid (QUIN) by inhibition of brain 3-hydroxyanthranilic acid dioxygenase (3-HAO) was subsequently investigated. The potency of the compounds to inhibit 3-HAO was assayed in rat brain homogenate, while chemical stability of certain compounds was studied by HPLC. The data were used to generate quantitative structure-activity relationship (QSAR) models for potency of 3-HAO inhibition and compound stability. Compounds with longer half-lives were obtained when the difference between the HOMO and LUMO was increased, while electron withdrawing groups in the 4- and 5-positions increased the potency of 3-HAO inhibition. Selected compounds that showed high potency in vitro were also found to be efficacious inhibitors in vivo after cerebral administration in rats.

Introduction

The cytosolic, non-haem ferrous (Fe2+) enzyme 3-hydroxyanthranilic acid dioxygenase (3-HAO; EC 1.13.11.6) plays an important role in the metabolic transformation of L-tryptophan to nicotinamide in the kynurenine pathway (figure 1). The enzyme oxidizes and ring opens 3-hydroxyanthranilic acid (3-HANA) to produce α-amino-β-carboxymuconic acid ω-semialdehyde, which subsequently, spontaneously cyclizes and forms quinolinic acid (QUIN) [1]. 3-HAO seems mainly to be localized to hepatic tissue, but it has also been demonstrated that the enzyme is expressed both in the brain [2], [3] and in inflammatory cells [2], [4] where the production of QUIN has been shown to be stimulated by certain cytokines [5], [6], [7], [8], [9]. Biochemical and immunological analysis in the rat suggest that the brain and liver 3-HAO are identical proteins [10]. Moreover, there appears to be a high degree of homology (94% similarity) between the rat and the human 3-HAO amino acid sequence [11].

QUIN is an NMDA receptor agonist and an excitotoxin [12] that has been reported to cause convulsions [13] or neurodegeneration [14] after intracerebral administration. It has been shown that intrastriatal injections of QUIN in rats induce biochemical and morphological alterations similar to those observed in Huntington's disease [15], [16]. Furthermore, increases in neuronal Huntingtin immunoreactivity [17] have recently been reported to occur after administration of QUIN into the striatum of mice, providing further support for a role of QUIN in the pathogenesis of Huntington's disease. It is also possible that QUIN is involved in epilepsy. For example, the levels of QUIN are higher in epilepsy-prone mice in a transgenic model of epilepsy than in the corresponding wild type mice [18]. Moreover, increased QUIN levels, or enhanced kynurenine pathway activity, have been implicated in inflammatory diseases of the central nervous system. For instance, QUIN has been associated with neurological dysfunction occurring following various viral or bacterial infections [19], [20], [21], [22], [23].

To elucidate the possible role of 3-HAO-mediated formation of QUIN in different neurological conditions, inhibitors of the enzyme are valuable tools, and may also find therapeutic utility. In this report, the synthesis of several analogues of 3-HANA and their structure-activity relationship as inhibitors of 3-HAO are presented. The main objective was to develop inhibitors with improved chemical and pharmacological properties compared to previously described 4-halogenated 3-HANA analogues [24]. A particular problem associated with 3-HANA analogues is the propensity of 3-HANA for auto-oxidation leading to its degradation and the formation of oxidized cinnabarinic acid [25], [26]. Hence, the stability of some of the synthesized compounds was studied by HPLC. The potency of the compounds to inhibit 3-HAO was assessed in rat brain tissue homogenates, while the ability of selected compounds to inhibit 3-HAO in vivo was also studied following intracerebral administration.

Section snippets

Chemistry

A series of 4,5-, 4,6-disubstituted and 4,5,6-trisubstituted 3-hydroxyanthranilic acids were prepared. The 4,5-dihalo substituted compounds were synthesized from the known 4-chloro- and 4-bromo-3-hydroxyanthranilic acids. The strong directing effect of the amino substituent gives access to derivatives 11 and 12 after reaction with bromine and chlorine, respectively. When the 5-position is substituted, the 4-substituted halogens can be obtained by a regio-selective reaction at the 4-position.

QSAR

A quantitative structure-activity relationship was developed for the 16 compounds presented in  table I. The σ of the R1, R2 and R3 substituents, the π and MR for the R4, R5 and R6 substituents were obtained from the literature [27]. The pKa values for the CO2H, NH2 and OH substituents and logP for the molecules were calculated using the Pallas software [28]. Semi-empirical calculations using AM1 (Spartan) [29] provided values for HOMO and LUMO energies, e-neg, hardness, heat of formation,

Pharmacology

The compounds were tested for their ability to inhibit 3-HAO in homogenates of rat brain tissue according to the method of Foster et al. [18]. The production of radioactive QUIN was measured after addition of [1-14C]3-HANA to determine inhibition of the enzyme. Test compound concentrations resulting in a 50% inhibition of the enzyme (IC50) are reported.

Also, the ability of selected compounds to inhibit cerebral 3-HAO in vivo was studied following intracerebroventricular (i.c.v.) administration

Results and discussion

A PLS analysis# (Codex, AP Scientific Service) of potency resulted in a four component model, accounting for 97% of the variance in pIC50 (cross-validated Q2 = 0.85) (figure 10). All the components in the model were statistically significant according to cross-validation. The two most important components accounted for 86% of the variance. A plot of the PLS-weights for these components indicated that the most important

Chemical methods

Melting points were determined on a Büchi SMP-20 apparatus. 1H and 13C NMR spectra were recorded at ambient temperature on a Varian Unity 400 or Varian Gemini 300 instrument. Chemical shifts are given in ppm from internal standards. For 1H NMR and 13C NMR spectra the internal references were tetramethylsilane (0.0 ppm), CDCl3 (δ 7.26 or δ 77.0 ppm), CD3OD (δ 3.38 or δ 49.3 ppm) or DMSO-d6 (δ 2.49 or δ 39.5 ppm), respectively. Coupling constants are given in Hertz, and the splitting patterns are

Acknowledgements

The authors are grateful for the analysis of QUIN by Mr Göran Fredriksson, for surgical assistance by Ms Eva Vänerman and to Mr Göran Stening for skillful technical assistance. 4-Halogenated 3-HANA analogues were generously provided by Dr Barry Carpenter, Cornell University.

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