The Impact of C-3 Side Chain Modifications on Kynurenic Acid: A Behavioral Analysis of Its Analogs on Motor Domain

1. Introduction

The central nervous system (CNS), which comprises the brain and spinal cord, regulates vital processes, including cognition, motion, and emotion [1,2,3]. Neurological conditions such as Alzheimer's disease (AD), Parkinson's disease (PD), epilepsy, stroke, brain tumors, and psychiatric conditions including major depressive disorders and schizophrenia are examples of disorders that can affect the CNS [4,5,6]. To treat these conditions, medications must reach the cells and tissues of interest in the CNS. However, this is challenging because the blood–brain barrier (BBB) protects the CNS [7,8,9]. In addition to endothelial cells that are interconnected via junctional proteins, the BBB comprises structurally and functionally supporting cells including astrocytes, pericytes, and microglia. The BBB is a natural defense mechanism that prevents toxins, pathogens, and foreign substances from entering the brain where they can potentially cause harm [10,11,12]. However, it also restricts the delivery of most therapeutic agents because only small, lipophilic, and uncharged molecules can passively diffuse across the BBB [13,14,15]. Consequently, because it reduces the bioavailability and efficacy of numerous drugs, the BBB is a significant barrier to drug delivery to the brain.

The BBB restricts the passage of highly polar molecules, such as sugars, amino acids, peptides, and nucleosides, isolating the brain from many essential compounds [16,17,18,19]. Various modifications of their side chains have been explored to facilitate the penetration of these impermeable molecules [16,20,21,22]. One strategy involves the use of hydrocarbon "staples" to link the side chains of polar molecules, and the other is the use of N-methyl phenylalanine-rich peptides, which have been investigated as highly versatile BBB shuttles [23,24,25]. These modifications aim to enhance the ability of highly polar molecules to traverse the BBB, thereby enabling their access to the CNS for potential therapeutic and research applications. Modifying the polarity of molecules by altering their side chains can be an effective approach for enhancing their BBB permeability and improving their CNS efficacy [20,26,27,28].

Tryptophan (Trp) is an essential amino acid involved in various biological processes, such as the synthesis of protein, neurohormone such as serotonin and melatonin, and various indole derivatives [29,30,31,32,33]. Over 90% of Trp in the body is metabolized through the kynurenine (KYN) pathway, which generates several bioactive metabolites that have diverse effects on the central nervous and immune systems [34,35,36,37]. Dysregulation of the KYN metabolism has been implicated in various neuropsychiatric disorders such as depression, schizophrenia, AD, and PD [38,39,40,41,42,43]. The KYN degradation take place through two main branches of metabolism: the neurotoxic and neuroprotective branches [44,45,46]. The balance between the neurotoxic and neuroprotective metabolic branches is crucial for maintaining homeostasis and function of the CNS [47,48,49]. However, this balance can be disturbed by various factors, resulting in the accumulation of neurotoxic KYNs and depletion of neuroprotective KYNs in the CNS [50,51,52]. This imbalance can cause neuronal damage, synaptic dysfunction, and cognitive impairment and contribute to the pathogenesis of neuropsychiatric disorders [53,54,55]. Therefore, KYN metabolism is a potential therapeutic target for neuropsychiatric disorders (Figure 1) [56,57,58].

Kynurenic acid (KYNA) is metabolized by the Trp-KYN metabolic pathway and functions as a neuroprotective metabolite [59,60,61]. Its antioxidant properties and antagonistic activity against ionotropic glutamate receptors, including N-methyl-D-aspartate (NMDA) receptors, are responsible for its neuroprotective effects [62,63,64]. KYNA has been implicated in various neuropsychiatric and neurodegenerative disorders, and KYNA levels in the brain and body are influenced by factors such as inflammation, stress, aging, and genetic variation [62,65,66,67,68,69]. Furthermore, recent studies have shown promising connections between KYNA and emotional learning, shedding light on its potential role in modulating affective motor function and emotional responses [70,71,72,73,74,75,76,77]. The neural substrates involved in emotional learning, particularly KYNA, suggest a plausible impact on the limbic system, including structures such as the amygdala and prefrontal cortex, which are known to be involved in emotional regulation and associative learning [74,78,79,80,81,82,83,84,85,86]. For example, it has been shown that KYNA and its synthetic analogs, such as SZR-72 and SZR-104, possess the ability not only to influence motor domains of behavior but also to potentially modulate emotional responses [87]. Therefore, KYNA appears to be a potential drug candidate for the treatment of neuropsychiatric disorders because it can regulate the balance between neurotoxicity and neuroprotection [70,88,89,90,91,92,93,94,95,96].

However, more research is needed to evaluate its safety and efficacy and to consider its interactions with other metabolic pathways of Trp. KYNA binds to the receptor strychnine-insensitive glycine-binding site of NMDA receptor [97,98]. At millimolar concentrations, KYNA inhibits the postsynaptic ionotropic glutamate receptor, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, kainate receptor, and the interaction with the G protein-coupled receptor 35 [99,100]. NMDA receptors are present in the mammalian brain in the post- and extrasynaptic membranes of glutamatergic neurons (Figure 2) [101,102]. We understand the various NMDA receptor subtypes associated with the gamma-aminobutyric acidergic and dopaminergic systems, as well as their functions [103,104,105,106].

Synthetic analogs of KYNA investigated in this study include N-(2-(dimethylamino)ethyl)-4-oxo-1,4-dihydroquinoline-2-carboxamide (SZR-72) as a KYNA amide derivative, N-(2-(dimethylamino)ethyl)-3-(morpholinomethyl)-4-oxo-1,4-dihydroquinoline-2-carboxamide (SZR-104) as an aminoalkylated amide derivative with the new function at C–3 position, and N-(2-(dimethylamino)ethyl)-3-methyl-4-oxo-1,4-dihydroquinoline-2-carboxamide (SZRG-21) that contains an alkyl group in C–3 position as a transitional derivative between SZR-72 and SZR-104 (Figure 3). They are able to mimic the pharmacological actions of KYNA, including the antagonistic effects on glutamate receptors [107]. They can also affect the morphology and function of microglia, which are brain immune cells, as well as the expression and methylation of histone H3 a protein that controls gene transcription [107,108]. These analogs may have potential therapeutic applications for neuroinflammatory and neurodegenerative disorders.

Given their ability to mimic KYNA's pharmacological actions of KYNA, particularly its antagonistic effects on glutamate receptors, these analogs have the potential to alter the delicate balance within neural circuits implicated in conditions such as schizophrenia, bipolar disorder, and major depressive disorder, all of which are associated with glutamatergic dysregulation [89,109,110,111]. Moreover, the influence of these analogs on microglial morphology and function, along with their impact on histone H3 expression and methylation, suggests their broader implications in neuroinflammatory processes mediated by brain-heart interactions [73,112,113]. By modulating microglial behavior and epigenetic regulation, these analogs may exert neuroprotective effects, potentially attenuating the progression of debilitating disorders [114,115,116,117]. The multifaceted actions of these analogs on both the immune response and epigenetic regulation highlight their promise as a novel class of compounds for addressing the complex pathophysiological mechanisms underlying various neuropsychiatric and neurologic conditions [118,119,120].

The main objective of this study was to investigate the effects of KYNA and its analogs on the motor domain of behavior in mice. This study aimed to synthesize KYNA analogs, SZR-72, SZR-104, and SZRG-21, with different side chain modifications that may affect their permeability to the BBB and administer them intracerebroventricularly (i.c.v.) at two different doses (0.04 and 0.1 mol/4 L) to mice (Figure 3), in order to get the most objective and immediate feedback about the effects of analog molecules in vivo on individual brain regions and thus on memory and motor functions. The study also aimed to measure the exploratory and affective motor function and motor skills of the mice using the OF and RR tests and compare the behavioral outcomes of KYNA and its analogs. The study further opens avenues to analyze the possible mechanisms underlying their effects on the motor domain of behavior and evaluate their potential as novel therapeutic agents for neuropsychiatric disorders involving motor impairments.

2. Results

2.2. Behavioral Tests

2.2.1. Pilot Study

The 1-methyl-8-azabicyclo[3.2.1]octane (Dizocilpine aka. MK-801) molecule (Figure 4) in lower dose, 0.04 μmol/4 μL caused ataxia symptoms in mice. However, KYNA did not cause any side effects at this dose (Figure 5).

Figure 5. Effects of MK-801 on pilot stereotype/ataxia tests. MK-801 caused massive stereotypes and ataxia symptoms in mice after i.c.v. injection. (A) Stereotype symptoms score of MK-801 treatment, 0.04 μmol/4 μL MK-801 vs. control (Ctrl) group, 0.1 μmol/4 μL MK-801 vs. Ctrl group (P=0.017). The statistical analysis was Kruskal-Wallis Non-parametric test (P<0.05) (B) Ataxia symptoms score of MK-801 treatment, 0.04 μmol/4 μL MK-801 vs. Ctrl group, 0.1 μmol/4 μL MK-801 vs. Ctrl group (P=0.001). The statistical analysis was Kruskal-Wallis Non-parametric test (P <0.05). We show the number of animals and the score category. *: 0.05; **: 0.01; ***: 0.001, N(Ctrl)=5, N(0.04 μmol/4 μL MK-801)=5, and N(0.1 μmol/4 μL MK-801)=5.

Figure 5. Effects of MK-801 on pilot stereotype/ataxia tests. MK-801 caused massive stereotypes and ataxia symptoms in mice after i.c.v. injection. (A) Stereotype symptoms score of MK-801 treatment, 0.04 μmol/4 μL MK-801 vs. control (Ctrl) group, 0.1 μmol/4 μL MK-801 vs. Ctrl group (P=0.017). The statistical analysis was Kruskal-Wallis Non-parametric test (P<0.05) (B) Ataxia symptoms score of MK-801 treatment, 0.04 μmol/4 μL MK-801 vs. Ctrl group, 0.1 μmol/4 μL MK-801 vs. Ctrl group (P=0.001). The statistical analysis was Kruskal-Wallis Non-parametric test (P <0.05). We show the number of animals and the score category. *: 0.05; **: 0.01; ***: 0.001, N(Ctrl)=5, N(0.04 μmol/4 μL MK-801)=5, and N(0.1 μmol/4 μL MK-801)=5.

On the other hand, when the animals were treated with the higher dose, 0.1 μmol/4 μL, the ataxia and stereotype scores were higher in the MK-801 treated group than in the lower dose group. We observed that the 0.1 μmol/4 μL dose induced ataxia symptoms in the KYNA group (Figure 6).

2.2.2. Stereotype/Ataxia Test

In our previous experiments, we observed that MK-801 had a more pronounced effect at both doses, whereas the effect of KYNA has more pronounced at higher doses. Therefore, we sought to investigate the effects of KYNA and its analog molecules at both doses in further experiments. Specifically, we treated the right lateral brain ventricles of mice with KYNA, SZR-72, SZR-104, and SZRG-21 at 0.04 and 0.1 μmol/4 μL. We did not observe a significant difference in stereotype between the treated groups at the 0.04 μmol/4 μL dose (Figure 7), as this dose did not cause ataxia. However, at the 0.1 μmol/4 μL dose, we did observe a significantly higher ataxia score in the KYNA group (P=0.004) than in the Control, SZR-72, and SZRG-21 groups (Figure 8B), but we did not observe a significant difference in stereotype between the treated groups (Figure 8A).

2.2.3. Open Field (OF) Test

Ten minutes after that we treated i.c.v. the animals with both doses (0.04 and 0.1 μmol/4 μL) of KYNA and its analog molecules and after the stereotype/ataxia behavior test was monitoring, was observed the spontaneous locomotor and exploration activities of mice. The mice were inserted into the center of the open-field box, and their behavior was measured within 15 min. The results of experiment were significant difference in the horizontal motion (ambulation distance) between the 0.04 μmol/ 4 μL dose of KYNA and its analogs treated groups. The ambulation distance was significantly higher in the mice treated with SZR-104 than in the control (P=0.01), KYNA (P=0.001), and SZR-72 (P=0.026) groups (Figure 9A). The vertical motion (rearing count) was significantly different between the SZR-72 and control (P=0.019), SZR-104 (P=0.015), and SZRG-21 (P=0.02) groups (Figure 9B).

When we treated the animals with a 0.1 μmol/4 μL dose of KYNA and analog molecules, there was no significant difference in the horizontal motion (ambulation distance) between the treated groups (Figure 10A). However, in the vertical motion (rearing count), there was a significant difference (P=0.04) between the 0.1 μmol/4 μL dose of SZR-72 and the Control group (Figure 10B).

2.2.4. Rotarod (RR) Test

Based on data from previous experiments, it was confirmed that KYNA did not accumulate in the extracellular space, it eluted rapidly [121], and the KYNA concentration in the mouse serum and CNS samples decrease after 30-40 minutes [122]. Therefore, we were interested in the effect of KYNA on motor coordination and balance in mice 25-30 minutes after i.c.v. treatment. During our investigations, we found that the 0.04 and 0.1 mol/4 L dose of KYNA and its analogs after i.c.v. injection did not significantly affect the locomotion skills of the mice (Figure 11 and Figure 12).

3. Discussion

The CNS plays a critical role in regulating essential functions such as cognition, motion, and emotion [123,124,125,126]. However, they are susceptible to various disorders that impair their function [4,5,6,127,128,129,130,131]. One of the major challenges in treating these disorders is the BBB, a semi-permeable membrane that protects the CNS from harmful substances but also limits the delivery of therapeutic agents to the brain [15,132,133,134]. The BBB is composed of various cells, junctions, and transporters that regulate the transport of molecules between the blood and brain [135,136,137,138]. Strategies have been developed to overcome this barrier by modifying the polarity of highly polar molecules such as sugars, amino acids, peptides, and nucleosides, which are essential for the CNS but cannot cross the BBB [139,140,141]. These modifications enhance the BBB permeability of these molecules, enabling their access to the CNS for potential therapeutic and research applications [20,142,143,144].

Trp is metabolized into neurotoxic and neuroprotective KYNs [44,45,46]. The balance of these KYNs affects the CNS and the immune system, and its dysregulation is linked to neuropsychiatric disorders [38,39,40,145,146,147]. Neurotoxic and neuroprotective branches of KYN, the equilibrium of which is critical for maintaining homeostasis and function in the CNS, have been implicated in its degradation [47,48,49,148,149]. On the contrary, KYN metabolites demonstrate an extensive array of bioactive characteristics, including but not limited to immunomodulating, oxidant, antioxidant, anti-inflammatory, and neurotoxin action [150,151,152]. The specific effects of these metabolites are contingent upon their concentration and the cellular milieu [35,153,154]. Furthermore, the metabolic system operates within intricate positive and negative feedback loops [66,155,156,157]. Furthermore, a critical aspect is the absence of consensus concerning the functionalities of KYN metabolites.

KYNA is a neuroprotective KYN that possess antioxidant property, antagonizes glutamate receptors, and modulates immunity and digestion [158,159,160]. KYNA is involved in neurological disorders, affected by various factors, and is a potential drug candidate for neuropsychiatric conditions [91,161,162,163,164]. The KYNA analog SZR-72 has been demonstrated to attenuate the severity of acute necrotizing pancreatitis in experimental settings, regulate body weight and home-cage activity in mice, and inhibit nitroglycerol-induced enhancement in c-Fos immunoreactivity within the rat caudal trigeminal nucleus [165,166,167]. Its effects are comparable to those of KYNA. Similarly, SZR-104 has been shown to modulate the immune systems, and inhibit glutamate receptors, with effects similar to KYNA [168]. In animal models, SZR-104 has been found to inhibit epileptiform seizures [169]. Furthermore, SZR-104 alters the intracellular distribution and methylation patterns of histone H3, a protein that regulates gene expression [108]. These findings indicate the potential therapeutic applications of these compounds in neurological and psychiatric disorders. However, further studies are required to evaluate its safety and efficacy. SZRG-21, the C-3 alkyl group-transitional derivative of SZR-72 and SZR-104, is a recently synthesized analog whose biological functions have yet to be investigated.

Firstly, the pilot study showed that the effects of KYNA and MK-801, two NMDA receptor antagonists, on mice behavior. MK-801 caused more ataxia and stereotype than KYNA at both doses (0.04 and 0.1 μmol/4 μL). KYNA causes ataxia at a higher dose. Our previous study showed that KYNA elicits antidepressant-like effects and improve learning and memory [163,170]. We investigated the effects of KYNA and its analogs (SZR-72, SZR-104, and SZRG-21) on spontaneous locomotor and exploratory activities of mice after i.c.v. injections. The horizontal and vertical motions of the mice were measured in an OF box for 15 min. The lower dose (0.04 μmol/4 μL) of SZR-104 increased the horizontal motion, while the lower dose of SZR-72 decreased the vertical motion, compared to the control and other groups. The higher dose (0.1 μmol/4 μL) of SZR-72 decreased the vertical motion compared to the control group. The other groups did not show any significant differences at the higher doses. It was also observed that KYNA and its analogs did not affect the motor coordination and balance of mice 25-30 min after i.c.v. injections.

Studying the effects of MK-801, KYNA, and KYNA analogs on mice behavior presents several challenges and requires specific knowledge and technology. The research revealed that MK-801 caused more ataxia and stereotypes than KYNA at both doses, whereas KYNA caused ataxia at a higher dose. Additionally, the study tested KYNA analogs and found that they did not cause significant behavioral changes. To achieve these results, researchers need a deeper understanding of NMDA receptor antagonists, mice behavior, and the specific effects of KYNA and its analogs. The technology required for this study includes precise dosing and administration methods for i.c.v. injections, as well as behavioral testing equipment to measure ataxia, stereotype, and spontaneous locomotor and exploration activities in mice [171,172,173]. Additional research is warranted in this field, as these results supplement current knowledge that specific dosages of KYNA enhance cognition and memory in addition to its antidepressant-like properties and pain modulation [163,170,174]. Additionally, KYNA analogs have exhibited promising results in animal models of various neurological and psychiatric disorders, although their mechanisms of action, pharmacokinetics, and safety warrant further investigation [165,166,167,168,175]. Therefore, KYNA analogs represent a novel class of drugs with the potential for future clinical applications.

Preclinical research, including in vitro and in vivo studies, can provide invaluable data, which are not feasible to investigate in humans [176,177,178,179,180,181,182,183,184,185,186,187,188,189,190]. This study showed that incorporation of the C-3 side chain elicits subtle differences in curiosity and emotion in animal models of motor function. Ongoing clinical studies have advanced our understanding of the behavioral domains in neuropsychiatric conditions and have sought their potential management [184,191,192,193,194,195,196,197,198,199]. Furthermore, computational strategies accelerate the advancement in comprehending their pathology as well as developing novel strategies for the management of neurological and psychiatric disorders, including neurotropic computer-assisted drug design [200,201,202,203,204,205,206,207,208]. Integrating these interdisciplinary approaches further adds impetus and optimizes strategies, including drug development research, leading to the testing and assessment of potential lead compounds. These outcomes enable researchers to evaluate the effects of novel interventional approaches such as drug-assisted brain stimulation [106,209,210,211,212,213,214]. These methods demonstrate promise for the development of new and more effective treatments, including novel drugs [215]. Furthermore, advanced imaging techniques have played a significant role in brain research [216,217,218]. Neuroimaging studies have uncovered structural and functional brain changes that are associated with neuropsychiatric disorders and therapeutics [219,220,221,222]. These imaging techniques can aid in identifying unique clinical cases and provide valuable insights into the pathophysiology of these disorders and novel therapeutic strategies [214,223,224,225]. Further research is needed to determine the optimal concentration for neuroprotection and the threshold for neurotoxicity. The findings of this study suggest that KYNA analogs represent a new class of drugs with potential clinical applications in neurological and psychiatric disorders.

4. Materials and Methods

4.1. Materials

We utilized a solution containing 0.9% saline (B Braun Melsungen AG, Hessen, Germany) in a 4 μL volume, as well as MK-801 (Sigma-Aldrich Ltd., Budapest, Hungary) and KYNA (Sigma-Aldrich Ltd., Budapest, Hungary) molecules in a pilot study. In our experiment, we employed 0.9% saline and KYNA (Sigma-Aldrich Ltd., Budapest, Hungary) in a 0.1 μmol/4 μL dose, as well as KYNA analogs (SZR-72, SZR-104, SZRG-21) in equal doses to KYNA. The new analogs were synthesized at the Faculty of Pharmacy, Institute of Pharmaceutical Chemistry, University of Szeged using the procedures described in Section 4.9. Fresh solutions of MK-801, KYNA, and its analogs were prepared by dissolving them in 0.9% aqueous saline and adjusting the pH to 7.4.

4.2. Kynurenic Acid Analogs Synthesis

The synthesis of KYNA analogs SZR-72 and SZR-104 was carried out in accordance with previously reported methods [175]. The synthesis of compound SZRG-21 commenced with aniline 1 (1.00 g, 10.70 mmol) which was reacted with diethyl oxalpropionate (4 mL, ρ = 1.073 g/mL, 21.2 mmol) in ethanol (20 mL) under reflux conditions. The progress of the reaction was monitored by TLC (eluent = n-hexane:ethyl acetate, 4:1), and once the reaction was complete, it was cooled to room temperature. Fewer side products were observed compared to the reaction run in acetic acid, which is commonly used as a solvent in the literature for synthesizing this compound [226,227,228,229]. After evaporation of the ethanol under reduced pressure, column chromatography was employed for purification using n-hexane:ethyl acetate (4:1) as the eluent. Diethyl 2-methyl-3-(phenylamino)maleate (2) was obtained as a yellow oil, yield = 0.81 g (65 %; Figure 13).

¹H (500 MHz, DMSO), δ(ppm): 1.06 (3H, t, J = 7.2 Hz); 1.25 (3H, t, J = 6.9 Hz); 1.77 (3 H, s); 4.12-4.21 (4H, m); 6.99 (2H, d, J = 7.2 Hz); 7.07 (1H, t, J = 7.5 Hz); 7.30 (t, 2H, J = 7.6 Hz, 1H); 10.01 (1H, s), ¹³C (125 MHz, DMSO), δ(ppm): 13.6, 13.9, 14.7, 60.4, 62.2, 95.9, 120.7, 124.2, 129.8, 140.5, 146.9, 164.7, 169.9 (Figure S1 and S2).

The solvents used during the ring closure process were modified from those reported in the literature, including PPA, mineral oil, and diphenyl ether; instead, 1,2-dichlorobenzene (DCB, 20 mL) was employed as the solvent. The reaction was carried out at reflux temperature for a total of 24 h, resulting in almost complete conversion of the starting material to compound 3, with only minor side products evident (as determined by TLC with the eluent DCM:MeOH 19:1). After the removal of the solvent under reduced pressure, crystallization was induced using Et₂O (3 mL), yielding a beige crystal with a mass of 0.64 g (95 %; Figure 13).

The amidation process was carried out under neat conditions, starting with 3 (0.60 g, 2.59 mmol) and employing an excess of N¹,N¹-dimethylethane-1,2-diamine at room temperature. The progress of the reaction was assessed by TLC (eluent: DCM:MeOH 19:1), and upon completion, 5 mL of DCM was added. The resulting precipitate was filtered and washed with diethyl ether in two separate instances (2 × 10 mL). The final product (4) was a white crystal, yield = 0.59 g (83 %; Figure 13), and exhibiting a melting point of 192-194 °C.

¹H (500 MHz, DMSO), δ(ppm): 1.99 (3H, s); 2.20 (6H, s); 2.43 (2H, t, J = 6.5 Hz); 3.33-3.43 (2H, m); 7.29 (1H, t, J = 7.5 Hz); 7.57-7.65 (2H, m); 8.08 (1H, d, J = 7.6 Hz); 8.72-8.79 (1H, m); ¹³C (125 MHz, DMSO), δ(ppm): 11.7, 37.7, 45.7, 58.3, 113.5, 118.7, 123.3, 123.8, 125.4, 132.0, 139.6, 143.6, 163.9, 187.7 (Figure S3, S4).

SZRG-21 was prepared by initiating from 4 (0.55 g, 2.01 mmol) in Et₂O (10 mL). HCl/EtOH (22%) was then slowly added until the pH reached 1-2. The resulting crystals were filtered and washed with Et₂O (2 × 10 ml). The yield of SZRG-21 was 0.59 g (94 %). During melting point determination SZRG-21 decomposed over 300 °C (Figure 13).

¹H (500 MHz, DMSO), δ(ppm): 2.00 (3H, s); 2.85 (6H, d, J = 4.8 Hz); 3.31 (2H, q, J = 6.1 Hz); 3.66 (2H, q, J = 5.9 Hz), 7.32 (1H, t, J = 7.6 Hz); 7.66 (1H, t, J = 7.2 Hz); 7.77 (1H, d, J = 8.1 Hz); 8.11 (1H, d, J = 7.8 Hz); 9.18 (1H, t, J = 5.5 Hz); 10.43 (1H, brs); 12.45 (1H, brs); ¹³C (125 MHz, DMSO), δ(ppm): 11.6, 34.8, 42.8, 55.7, 113.9, 118.8, 123.6, 123.7, 125.3, 132.2, 139.5, 142.8, 164.3 (Figure S5, S6).

4.3. Animals

The study utilized male C57BL6/J mice (Mus musculus, Charles River Laboratories, Germany) that weighed between 25-30 grams. These animals were 10-12 weeks old and housed in cages containing a maximum of five mice per cage. The mice were kept under standard laboratory conditions, including access to tap water and regular mouse chow, and were maintained on a 12-hour light-dark cycle at a temperature of 24±1 °C and humidity of 50±10%. The animals were handled in accordance with the Regulations of the Faculty of Medicine, University of Szeged, Ethical Committee for the Protection of Animals in Research. This study was approved by the Ethical Committee for the Protection of Animals in Research at the University of Szeged (XVII/275/2023) and the Hungarian Health Committee (40/2013 (II.14.)), and the European Community Council Directive (2010/63/EU).

4.4. Surgery

Mice were anesthetized with 4% chloral hydrate (Sigma-Aldrich Ltd., Budapest, Hungary) at a dose of 0.4 g/kg body weight. A polyethylene cannula (Fisher Scientific, Intramedic Clay Adams polyethylene tube, Budapest Hungary) was inserted into the right lateral brain ventricle and fixed to the skull using cyanoacrylate (Ferrobond, Budapest Hungary). The stereotaxic coordinates were set at anterior-posterior 0.2 mm, medial-lateral 1.09 mm to the bregma, with the cannula extending 2.3 mm deep into the skull surface. After a recovery period of five days, the mice were used for the experiments. On the 8th day following surgery, KYNA or its analogs or saline (4 µL) were injected into the right lateral brain ventricle of the mice using an infusion pump (KD Scientific, Holliston, Massachusetts, USA) at a rate of 8 µL/min. The correct location of the inner i.c.v. cannula was confirmed by injecting 1% methylene blue solution after the experiment [230].

4.5. Behavioral Tests

The tests were carried out at the same time of the day to minimize variations in the diurnal rhythm of the animals. Prior to assessing each animal, the equipment was thoroughly cleaned with 70% alcohol to remove lingering scents [231,232].

4.5.1. Pilot Study

To determine the most effective dose of KYNA that did not cause severe side effects, we compared KYNA with an NMDA receptor antagonist molecule, MK-801. We treated the animals with 0.9 % of saline in 4 μL volumen, MK-801 in 0.04 and 0.1 μmol/4 μL dose and KYNA in equal dose of MK-801 (Figure 4). These molecules were administered to mice in the right lateral brain ventricle. We used five mice per group and had 5 groups during the pilot experiment. We observed the effect of MK-801 and KYNA in the Stereotype/Ataxia behavior test after i.c.v. injections. The duration of the observation was 10 min.

4.5.2. Stereotype Behavior and Ataxia

Behavioral changes, including ataxia and stereotyped behavior, were observed and recorded 10 min after treatment administration. The mice were then monitored for an additional 10 min, during which their behavior was rated using the scale described by Sturgeon et al. (1979) [233] and Contreras (1990) [234]. The scale measures five categories of stereotyped behavior, including sniffing, grooming, and rearing behavior, as well as reciprocal forepaw treading or undirected head movement, backward walking, head weaving, circling behavior, continuous head weaving, circling, or backward walking, and dyskinetic extensions or flexion of the limbs, head, and neck, or weaving greater than four. For ataxia, the scale measures awkward and jerky movements, stumbling or awkward posture, falling, inability to move beyond a small area or support weight on the stomach or haunches, and inability to move, except for twitching movements.

4.5.3. Open Field (OF) Test

Spontaneous locomotor and exploratory activities were assessed using an automated tracking system within the activity chamber. The chamber was linked to a computer that recorded the exploration and locomotor activity of the subjects [235,236,237]. Each animal was placed individually at the center of a black box measuring 60 × 60 × 70 cm, which was equipped with automated infrared photocells for precise measurements. The box platform is divided into nine equal squares. The animals were allowed to move freely for 15 min and divided into three distinct sessions. The movement signals were analyzed using Conducta 1.0. We quantified ambulation distance and duration of rearing, which serve as indicators of horizontal and vertical movement, spontaneous locomotor activity, and exploratory behavior.

4.5.4. Rotarod (RR) Test

The rotarod test was used to evaluate motor coordination and balance. The rod, which rotated about its longitudinal axis, was positioned horizontally, and the animals were required to walk forward to maintain equilibrium and prevent falling off. The time taken for the mice to fall from the rotating rod was measured using an infrared sensor and the latency to fall was automatically scored [238,239]. Motor coordination was assessed by comparing the latency to fall between the treatment groups, whereas motor learning was evaluated by comparing the first trial to subsequent trials after training, which demonstrated an increase in latency to fall over time. Prior to training, each mouse was acclimatized to the device for one hour at rest. On the first and second days, the animals were trained at a constant speed of 5 revolutions per minute (rpm) on a rotating rod, with three trials lasting five minutes each, and 15-minute intervals between trials. On the third day, 25 min after intraventricular (i.c.v.) injection, the latency at which each mouse fell off the rod in standard mode (5 rpm within 5 min) was recorded.

4.6. Statistical Analysis

In our study, we utilized Microsoft SPSS software (version 2.0) for the statistical analysis of our data. To evaluate our results, we employed a One-way ANOVA test that was adjusted using both the LSD and Bonferroni post hoc tests, as well as the Kruskal-Wallis non-parametric test.

5. Conclusion

The BBB hinders drug delivery to the CNS, where many neuropsychiatric disorders arise. KYNA, a Trp metabolite, regulates CNS functions such as cognition, mood, and motor activity. Abnormal KYNA levels are linked to disorders, such as schizophrenia, depression, and AD. However, KYNA cannot cross the BBB owing to its polarity. In this study, we prepared three KYNA analogs with different side chains, SZRG-21, SZR-72, and SZR-104, to improve their lipophilicity and BBB permeability. We tested their effects on mice behavior using the OF and RR tests. We found that SZR-104 increased horizontal exploration, whereas SZR-72 decreased the vertical motion. Neither KYNA nor its analogs affected motor skills. These results show that side-chain modification alters KYNA’s behavioral effects of KYNA and its interactions with its receptors in the CNS. Our study offers new insights into the pharmacological properties of KYNA-based drugs for neuropsychiatric disorders and their pharmacological properties. We also emphasize the need for further research on KYNA and its analogs in the CNS.