Synthesis of New C-3 Substituted Kynurenic Acid Derivatives.

The application of kynurenic acid (KYNA) as an electron-rich aromatic system in the modified Mannich reaction has been examined. The extension possibility of the reaction was tested by using amines occurring in a number of bioactive products, such as morpholine, piperidine, or N-methylpiperazine and aldehydes of markedly different reactivities, like formaldehyde and benzaldehyde. The influence of substituents attached to position 3 on the aminoalkylation was also investigated. Thus, reactions of 3-carbamoyl-substituted precursors with tertiary amine containing side-chains were also tested to afford new KYNA derivatives with two potential cationic centers. By means of NMR spectroscopic measurements, supported by DFT calculations, the dominant tautomer form of KYNA derivatives was also determined.

Behavioural studies with a newly developed neuroprotective KYNA-amide.

The neuroactive properties and neuroprotective potential of endogenous L: -kynurenine, kynurenic acid (KYNA) and its derivatives are well established. KYNA acts as an antagonist on the obligatory co-agonist glycine site, and has long been at the focus of neuroprotective trials. Unfortunately, KYNA is barely able to cross the blood-brain barrier. Accordingly, the development and synthesis of KYNA analogs which can readily cross the BBB have been at the focus of research interest with the aim of neuroprotection. Earlier we reported a new KYNA-amide crosses the BBB and proved neuroprotective in several experiments. In the present study, we investigated the locomotor activity, working memory performance, and also the long-lasting, consolidated reference memory of animals treated intraperitoneally (i.p.) with the novel analog. The effects of the novel analog on the spatial orientation and learning ability of rats were assessed in the Morris water maze (MWM) paradigm. The effects on locomotor activity of mice was assessed in the open field (OF) paradigm, and those on the spatial orientation and learning ability of mice were investigated in the radial arm maze (RAM) paradigm. It emerged that there is a dose of this KYNA-amide which is neuroprotective, but does not worsen the cognitive function of the brain. This result is significant in that a putative neuroprotectant without adverse cognitive side-effects is of great benefit.

Synthesis and biological effects of some kynurenic acid analogs.

The overactivation of excitatory amino acid receptors plays a key role in the pathomechanism of several neurodegenerative disorders and in ischemic and post-ischemic events. Kynurenic acid (KYNA) is an endogenous product of the tryptophan metabolism and, as a broad-spectrum antagonist of excitatory amino acid receptors, may serve as a protective agent in neurological disorders. The use of KYNA is excluded, however, because it hardly crosses the blood-brain barrier. Accordingly, new KYNA analogs which can readily cross this barrier and exert their complex anti-excitatory activity are generally needed. During the past 6 years, we have developed several KYNA derivatives, among others KYNA amides. These new analogs included one, N-(2-N,N-dimethylaminoethyl)-4-oxo-1H-quinoline-2-carboxamide hydrochloride (KYNA-1), that has proved to be neuroprotective in several models. This paper reports on the synthesis of 10 new KYNA amides (KYNA-1-KYNA-10) and on the effectiveness of these molecules as inhibitors of excitatory synaptic transmission in the CA1 region of the hippocampus. The molecular structure and functional effects of KYNA-1 are compared with those of other KYNA amides. Behavioral studies with these KYNA amides demonstrated that they do not exert significant nonspecific general side-effects. KYNA-1 may therefore be considered a promising candidate for clinical studies.

Kynurenic Acid and Its Synthetic Derivatives Protect Against Sepsis-Associated Neutrophil Activation and Brain Mitochondrial Dysfunction in Rats.

Background and Aims: The systemic host response in sepsis is frequently accompanied by central nervous system (CNS) dysfunction. Evidence suggests that excessive formation of neutrophil extracellular traps (NETs) can increase the permeability of the blood-brain barrier (BBB) and that the evolving mitochondrial damage may contribute to the pathogenesis of sepsis-associated encephalopathy. Kynurenic acid (KYNA), a metabolite of tryptophan catabolism, exerts pleiotropic cell-protective effects under pro-inflammatory conditions. Our aim was to investigate whether exogenous KYNA or its synthetic analogues SZR-72 and SZR-104 affect BBB permeability secondary to NET formation and influence cerebral mitochondrial disturbances in a clinically relevant rodent model of intraabdominal sepsis. Methods: Sprague-Dawley rats were subjected to fecal peritonitis (0.6 g kg-1 ip) or a sham operation. Septic animals were treated with saline or KYNA, SZR-72 or SZR-104 (160 µmol kg-1 each ip) 16h and 22h after induction. Invasive monitoring was performed on anesthetized animals to evaluate respiratory, cardiovascular, renal, hepatic and metabolic parameters to calculate rat organ failure assessment (ROFA) scores. NET components (citrullinated histone H3 (CitH3); myeloperoxidase (MPO)) and the NET inducer IL-1ß, as well as IL-6 and a brain injury marker (S100B) were detected from plasma samples. After 24h, leukocyte infiltration (tissue MPO) and mitochondrial complex I- and II-linked (CI-CII) oxidative phosphorylation (OXPHOS) were evaluated. In a separate series, Evans Blue extravasation and the edema index were used to assess BBB permeability in the same regions. Results: Sepsis was characterized by significantly elevated ROFA scores, while the increased BBB permeability and plasma S100B levels demonstrated brain damage. Plasma levels of CitH3, MPO and IL-1ß were elevated in sepsis but were ameliorated by KYNA and its synthetic analogues. The sepsis-induced deterioration in tissue CI-CII-linked OXPHOS and BBB parameters as well as the increase in tissue MPO content were positively affected by KYNA/KYNA analogues. Conclusion: This study is the first to report that KYNA and KYNA analogues are potential neuroprotective agents in experimental sepsis. The proposed mechanistic steps involve reduced peripheral NET formation, lowered BBB permeability changes and alleviation of mitochondrial dysfunction in the CNS.

Synthesis and pharmacological evaluation of some 4-oxo-quinoline-2-carboxylic acid derivatives as anti-inflammatory and analgesic agents.

The synthesis and the pharmacological activity of a series of 1-aroyl derivatives of kynurenic acid methyl ester (4-oxo-quinolin-2-carboxy methyl (KYNA) esters), structurally related to NSAID indomethacin are described. The derivatives were screened in vivo for anti-inflammatory and analgesic activities. Most of the compounds exhibited good anti-inflammatory and analgesic activities. An automatic docking of the synthesized compounds was performed using X-ray structures of COX-1 and COX-2. Docking results are in good accordance with the experimental biological data.

Multifunctional Analogs of Kynurenic Acid for the Treatment of Alzheimer's Disease: Synthesis, Pharmacology, and Molecular Modeling Studies.

We report the synthesis and pharmacological investigation of analogs of the endogenous molecule kynurenic acid (KYNA) as multifunctional agents for the treatment of Alzheimer's disease (AD). Synthesized KYNA analogs were tested for their N-methyl-d-aspartate (NMDA) receptor binding, mGluR5 binding and function, acetylcholinesterase (AChE) inhibition, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, interference with the amyloid ß peptide (Aß) fibrillation process, and protection against Aß-induced toxicity in transgenic Caenorhabditis elegans strain GMC101 expressing full-length Aß42. Molecular modeling studies were also performed to predict the binding modes of most active compounds with NMDAR, mGluR5, and Aß42. Among the synthesized analogs, 3c, 5b, and 5c emerged as multifunctional compounds that act via multiple anti-AD mechanisms including AChE inhibition, free radical scavenging, NMDA receptor binding, mGluR5 binding, inhibition of Aß42 fibril formation, and disassembly of preformed Aß42 fibrils. Interestingly, 5c showed protection against Aß42-induced toxicity in transgenic C. elegans strain GMC101. Moreover, 5b and 5c displayed high permeability in an MDR1-MDCKII cell-based model of the blood-brain barrier (BBB). Compound 3b emerged with specific activity as a micromolar AChE inhibitor, however it had low permeability in the BBB model. This study highlights the opportunities that exist to develop analogs of endogenous molecules from the kynurenine pathway for therapeutic uses.

Tricyclic quinoxalinediones, aza-kynurenic acids, and indole-2-carboxylic acids as in vivo active NMDA-glycine antagonists.

This review article describes the development of in vivo active antagonists for the glycine binding site of the N-Methyl-D-Aspartate (NMDA) receptor. There were several difficulties in identifying a class of antagonists with in vivo efficacy and only a few compounds succeeded in emerging with activity in vivo. A series of tricyclic quinoxalinediones was highly potent glycine antagonists in vitro and the derivatives having a zwitterionic moiety including SM-18400 indeed showed in vivo activity. Similarly, tricyclic indole-2-carboxylic acids having a zwitterionic moiety such as SM-31900 were also active in vivo. In fact, SM-18400 and SM-31900 exhibited efficacy in several animal stroke models using intravenous infusion protocols. The practical syntheses of SM-18400 and SM-31900 as well as the novel synthesis of moderately active glycine antagonists, tricyclic azakynurenic acids, were also developed.

Novel kynurenic acid analogues in the treatment of migraine and neurodegenerative disorders: preclinical studies and pharmaceutical design.

Though migraine and neurodegenerative disorders have a high socioeconomic impact, their therapeutic management has not been fully addressed. Their pathomechanisms are not completely understood, but glutamateinduced excitotoxicity, mitochondrial disturbances and oxidative stress all seem to play crucial roles. The overactivation of glutamate receptors contributes to the hyperexcitability observed in migraine and also to the neurodegenerative process. The kynurenine pathway of the tryptophan metabolism produces the only known endogenous Nmethyl- D-aspartate receptor antagonist, kynurenic acid, which has been proven in different preclinical studies to exert a neuroprotective effect. Influencing the kynurenine pathway might be beneficial in migraine and neurodegenerative diseases, and in the normalization of glutamatergic neurotransmission and the prevention of excitotoxic neuronal damage. The synthesis of kynurenic acid analogues may offer a valuable tool for drug development.

Kynurenic acid derivatives. Structure-activity relationships for excitatory amino acid antagonism and identification of potent and selective antagonists at the glycine site on the N-methyl-D-aspartate receptor.

Derivatives of the nonselective excitatory amino acid antagonist kynurenic acid (4-oxo-1,4-dihydroquinoline-2-carboxylic acid, 1) have been synthesized and evaluated for in vitro antagonist activity at the excitatory amino acid receptors sensitive to N-methyl-D-aspartic acid (NMDA), quisqualic acid (QUIS or AMPA), and kainic acid (KA). Introduction of substituents at the 5-, 7-, and 5,7-positions resulted in analogues having selective NMDA antagonist action, as a result of blockade of the glycine modulatory (or coagonist) site on the NMDA receptor. Regression analysis suggested a requirement for optimally sized, hydrophobic 5- and 7-substituents, with bulk tolerance being greater at the 5-position. Optimization led to the 5-iodo-7-chloro derivative (53), which is the most potent and selective glycine/NMDA antagonist to date (IC50 vs [3H]glycine binding, 32 nM; IC50's for other excitatory amino acid receptor sites, greater than 100 microM). Substitution of 1 at the 6-position resulted in compounds having selective non-NMDA antagonism and 8-substituted compounds were inactive at all receptors. The retention of glycine/NMDA antagonist activity in heterocyclic ring modified analogues, such as the oxanilide 69 and the 2-carboxybenzimidazole 70, suggests that the 4-oxo tautomer of 1 and its derivatives is required for activity. Structurally related quinoxaline-2,3-diones are also glycine/NMDA antagonists, but are not selective and are less potent than the 1 derivatives, and additionally show different structure-activity requirements for aromatic ring substitution. On the basis of these results, a model accounting for glycine receptor binding of the 1 derived antagonists is proposed, comprising (a) size-limited, hydrophobic binding of the benzene ring, (b) hydrogen-bond acceptance by the 4-oxo group, (c) hydrogen-bond donation by the 1-amino group, and (d) a Coulombic attraction of the 2-carboxylate. The model can also account for the binding of quinoxaline-2,3-diones, quinoxalic acids, and 2-carboxybenzimidazoles.

Synthesis, pharmacokinetics and anticonvulsant activity of 7-chlorokynurenic acid prodrugs.

7-Chlorokynurenic acid 1 is a potent glycine-N-methyl-D-aspartate (NMDA) receptor antagonist, but it shows weak activity after systemic administration. In order to overcome the Blood-brain barrier (BBB), we synthetized three new esters 2-4 of 1 obtained by chemical conjugation with essential nutrients such as glucose and galactose, that are actively transported across the BBB. These compounds were assayed to evaluate their in vitro chemical and enzymatic hydrolysis. In addition the prodrugs 2-4 were tested for their ability to protect mice against NMDA-induced seizures after systemic administration. All the prodrugs 2-4 appeared moderately stable in pH 7.4 buffered solution and were susceptible to in vitro enzymatic hydrolysis. Intraperitoneal administration of either esters 2 or 4 was highly protective against seizures induced by NMDA in mice, with the latter prodrug showing the highest anticonvulsive activity. In addition, ester 4 undergoes a time-dependent extracellular hydrolysis into 1 when applied to mixed cultures of mouse cortical cells, a model that reproduces in vitro the cellular milieu encountered by the prodrugs once they penetrate the brain parenchyma.

Synthesis and in vivo evaluation of 7-chloro-5-[123I]iodo-4-oxo-1,4-dihydroquinoline-2-carboxylic acid.

As a possible radioligand for SPECT visualization of the NMDA receptors in the central nervous system, 7-chloro-5-[123I]iodokynurenic acid was prepared. This paper presents the synthesis of both the radioactive and the non-radioactive product, starting from 5-bromo-7-chlorokynurenic acid and using a non-isotopic nucleophilic halogen exchange reaction in the presence of iodide (Na123I or KI). Under the best labelling conditions, the radiochemical yield was 85%. The specific activity based on UV detection was found to be higher than 1 Ci/mumol (= 37 GBq/mumol) and the chemical and radiochemical (> 95%) purity of the tracer was checked by RP-HPLC.

Kynurenic acid and 3-hydroxykynurenine production from D-kynurenine in mice.

Kynurenic acid (KYNA), an antagonist of the α7 nicotinic acetylcholine receptor and the N-methyl-D-aspartate receptor, and 3-hydroxykynurenine (3-HK), a generator of reactive oxygen species, are neuroactive metabolites of the kynurenine pathway of tryptophan degradation. In the mammalian brain as elsewhere, both compounds derive from a common bioprecursor, L-kynurenine (L-KYN). Recent studies in rats demonstrated that D-kynurenine (D-KYN), a metabolite of the bacterial amino acid D-tryptophan, can also function as a bioprecursor of brain KYNA. We now investigated the conversion of systemically administered D-KYN to KYNA in mice and also explored the possible production of 3-HK in the same animals. Thirty min after an injection of D-KYN or L-KYN (30 mg/kg, i.p.), newly produced KYNA and 3-HK were recovered from plasma, liver, forebrain and cerebellum in all cases. Using a new chiral separation method, 3-HK produced from D-KYN was positively identified as D-3-HK. L-KYN was the more effective precursor of KYNA in all tissues and also exceeded D-KYN as a precursor of brain 3-HK. In contrast, D-KYN was more potent as a precursor of 3-HK in the liver. The production of both KYNA and 3-HK from D-KYN was rapid in all tissues, peaking at 15-30 min following a systemic injection of D-KYN. These results show that biosynthetic routes other than those classically ascribed to L-KYN can account for the synthesis of both KYNA and 3-HK in vivo. This new insight may be of significant physiological or pathological relevance.

Syntheses, transformations and pharmaceutical applications of kynurenic acid derivatives.

The syntheses and transformations of 4-hydroxyquinoline-2-carboxylic acid, kynurenic acid, are reviewed, and special attention is paid to the pharmacological activities and pharmaceutical applications of its derivatives.

Kynurenic acid amides as novel NR2B selective NMDA receptor antagonists.

A novel series of kynurenic acid amides, ring-enlarged derivatives of indole-2-carboxamides, was prepared and identified as in vivo active NR2B subtype selective NMDA receptor antagonists. The synthesis and SAR studies are discussed.

Anticonvulsant activity of 4-urea-5,7-dichlorokynurenic acid derivatives that are antagonists at the NMDA-associated glycine binding site.

Twelve 4-urea-5,7-dichlorokynurenic acid derivatives were synthesized by reacting the 4-tosylimino derivative of 5,7-dichlorokynurenate methyl ester first with triphosgene and then with a secondary amine. Compounds were screened in mice for anticonvulsant activity using maximal electroshock (MES), subcutaneous pentylenetetrazole (Met), and threshold tonic extension (TTE) tests. A rotorod test was used to determine neurotoxicity. Seven of the derivatives had anticonvulsant activity in TTE testing at 100 mg/kg. One compound, 2-methyl carboxylate-5,7-dichloro-4-([¿diphenylamino¿-carbonyl]amino)-quino line, had an ED50 value of 134 mg/kg (95% conf. int.: low-78.5, high-205.7; slope 1.9, SE = 0.44) in TTE testing. Two derivatives had MES activity. Only one compound, an N,N-diethylamino derivative, was neurotoxic in the rotorod test. Compounds were screened at a 10-microM concentration for activity in displacing 5,7-dichlorokynurenic acid from synaptosomal membrane fragments. Since 9 of the 12 compounds synthesized and tested have demonstrated anticonvulsant activity, this class of chemicals offers promise for the production of useful therapeutic agents.