Development towards a novel screening method for nipecotic acid bioisosteres using molecular imprinted polymers (MIPs) as alternative to in vitro cellular uptake assays.

Impaired expression of GABA transporters (GATs) is closely related to the pathogenesis of among others Parkinson's disease and epilepsy. As such, lipophilic nipecotic acid analogs have been extensively studied as GAT1-addressing drugs and radioligands but suffer from limited brain uptake due to the zwitterionic properties of the nipecotic acid moiety. Bioisosteric replacement of the carboxylic acid group is a promising strategy to improve the brain uptake, though it requires knowledge on the binding of these isosteres to GAT1. To screen nipecotic acid isosteres for their affinity to GAT1 in a time- and cost-effective manner, this research aims to develop a molecular imprinted polymer (MIP) that mimics the natural binding site of GAT1 and can act as an alternative screening tool to the current radiometric and mass spectrometry cellular-based assays. To this end, a nipecotic acid MIP was created using the electropolymerization of ortho-phenylenediamine (oPD) by cyclic voltammetry (CV). The optimization of the generated receptor layer was achieved by varying the scan rate (50-250 mV/s) and number of CV cycles (5-12), yielding an optimized MIP with an average imprinting factor of 2.6, a linear range of 1-1000 nm, and a theoretical LOD of 0.05 nm, as analyzed by electrical impedance spectroscopy (EIS). Selectivity studies facilitated the investigation of major binding interactions between the MIP and the substrate, building an experimental model that compares characteristics of various analogs. Results from this model indicate that the substrate carboxylic acid group plays a more important role in binding than an amine group, after comparing the binding of cyclohexanecarboxylic acid (average IF of 1.7) and piperidine (average IF of 0.46). The research culminates in a discussion regarding the feasibility of the in vitro model, comparing the synthetic system against the biological performance of GAT1. Thus, evaluating if it is possible to generate a synthetic GAT1 mimic, and if so, provide directions for follow-up research.

Design, synthesis, molecular docking and pharmacological evaluation of some thiadiazole based nipecotic acid derivatives as a potential anticonvulsant and antidepressant agents.

In our continuous effort to develop novel antiepileptic drug, a new series of nipecotic acid derivatives having1,3,4-thiadiazole nucleus were designed and synthesized. This study aims to improve the lipophilicity of nipecotic acid by attaching some lipophilic anchors like thiadiazole and substituted aryl acid derivatives. In our previous study, we noticed that the N-substituted oxadiazole derivative of nipecotic acid exhibited significant antiepileptic activity in the rodent model. The synthesized compounds were characterized by FT-IR, 1H-NMR, 13C-NMR, Mass, and elemental analysis. The anticonvulsant activity was evaluated by using the maximal electroshock-induced seizure model in rats (MES) and the subcutaneous pentylenetetrazol (scPTZ) test in mice. None of the compounds were found to be active in the MES model whereas compounds (TN2, TN9, TN12, TN13, and TN15) produced significant protection against the scPTZ-induced seizures model. The compounds showing antiepileptic activity were additionally evaluated for antidepressant activity by using the forced swim test, 5-hydroxytryptophan (5-HTP)-induced head twitch test, and learned helplessness test. All the molecules that showed anticonvulsant activity (TN2, TN9, TN12, TN13, and TN15), also exerted significant antidepressant effects in the animal models. The selected compounds were subjected to different toxicity studies. Compounds were found to have no neurotoxicity in the rota-rod test and devoid of hepatic and renal toxicity in 30 days repeated oral toxicity test. Further, a homology model was developed to perform the in-silico molecular docking and dynamics studies which revealed the similar binding of compound TN9 within the active binding pocket and were found to be the most potent anti-epileptic agent. The market expectation for newly developed antiepileptic thiadiazole-based nipecotic acid derivatives is significant, driven by their potential to offer improved therapeutic outcomes and reduced side effects, addressing a critical need in epilepsy treatment. These innovative compounds hold promise for meeting the demand for more effective and safer antiepileptic medications. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03897-1.

Synthesis and biological evaluation of new antiseizure compounds derived from valproic acid.

Background: New hybrid compounds were synthesized by linking the valproic acid (VPA) structure with other anticonvulsant/anti-inflammatory scaffolds. Materials & methods: The chemistry involved the incorporation of the linker oxymethyl ester into VPA, followed by reaction with the second scaffold. The antiseizure effects were investigated by the maximal electroshock seizure test, and the most active compound was additionally evaluated in the 6 Hz test and pentylenetetrazol test in mice. Results: The compounds showed protection against seizures. The hybrid structure with the butylparaben scaffold exhibited an ED50 of 8.265 mg/kg (0.0236 mmol/Kg) in the maximal electroshock seizure test and 50.00 mg/kg (0.147 mmol/kg) in the 6 Hz test. Conclusion: The antiseizure activity of the synthesized compounds highlighted the potential of hybrid structures to treat multifactorial diseases such as epilepsy.

This article focuses on the design of new anticonvulsant compounds that combine the chemical structure of valproic acid with other interesting scaffolds with anticonvulsant or anti-inflammatory properties. These compounds protected against in vivo acute seizure models (mice). The results revealed the capacity of combining known scaffolds into a single structure to generate new active compounds with multitarget purposes.

Nipecotic Acid Derivatives as Potent Agents against Neurodegeneration: A Preliminary Study.

Alzheimer's Disease (AD) is a common neurodegenerative disorder characterized by memory loss and cognitive impairment. Its pathology has not been fully clarified and therefore highly effective treatments have not been obtained yet. Almost all the current treatment options aim to alleviate only the symptoms and not to eliminate the disease itself. Acetylcholinesterase inhibitors are the main therapeutic agents against AD, whereas oxidative stress and inflammation have been found to be of great significance for the development and progression of neurodegeneration. In this work, ethyl nipecotate (ethyl-piperidine-3-carboxylate), a heterocyclic carboxylic acid derivative, which acts as a GABA reuptake inhibitor and has been used in research for diseases involving GABAergic neurotransmission dysfunction, was amidated with various carboxylic acids bearing antioxidant and/or anti-inflammatory properties (e.g., ferulic acid, sinapic acid, butylated hydroxycinnamic acid). Most of our compounds have significant antioxidant potency as lipid peroxidation inhibitors (IC50 as low as 20 µΜ), as oxidative protein glycation inhibitors (inhibition up to 57%), and act as DPPH reducing agents. Moreover, our compounds are moderate LOX inhibitors (up to 33% at 100 µΜ) and could reduce rat paw edema induced by carrageenan by up to 61%. Finally, some of them possessed inhibitory activity against acetylcholinesterase (IC50 as low as to 47 µΜ). Our results indicate that our compounds could have the potentiality for further optimization as multi-targeting agents directed against AD.

Nipecotic acid as potential lead molecule for the development of GABA uptake inhibitors; structural insights and design strategies.

Gamma-Aminobutyric Acid (GABA) inhibitory neurotransmitter departs an energetic role in brain signalling system. Levels of GABA in the brain influence human behaviour, diminishes in the degree of GABA can cause seizures, depression, Parkinson's. To put it plainly, it plays a basic part in the significant issues of mind. It is exceptionally important to cure the issues linked to GABA. Writing overview proposed that nipecotic acid is an intense GABA reuptake inhibitor. This scaffold is likewise present in one of the promoted anticonvulsant drugs 'Tiagabine'. Tiagabine is only drug in the market which works through this mechanism however the medication is regulated with one more prescription for the synergistic impact. Nipecotic acid has several disadvantages such as it can't cross the blood-brain barrier because of its hydrophilic and zwitterionic nature. To avoid this problem nipecotic acid scaffold hybrids with the different aromatic groups can enhance the physical (lipophilicity) as well as biological properties of the resultant compound. So, there is a dire requirement for compounds that work through this mechanism. Several medicinal chemists and researchers are already working in this field and developed outstanding newer molecules. This review article compiles these developed new hybrids along with design strategies, structure-activity relationship, and biological activity as well as in silico studies. This review also demonstrates the synthesis of nipecotic acid and the core mechanism through which nipecotic acid acts as a GABA reuptake inhibitor.

Synthesis and Biological Evaluation of Nipecotic Acid and Guvacine Derived 1,3-Disubstituted Allenes as Inhibitors of Murine GABA Transporter mGAT1.

A new class of nipecotic acid and guvacine derivatives has been synthesized and characterized for their inhibitory potency at mGAT1-4 and binding affinity for mGAT1. Compounds of the described class are defined by a four-carbon-atom allenyl spacer connecting the nitrogen atom of the nipecotic acid or guvacine head with an aromatic residue. Among the compounds investigated, the mixture of nipecotic acid derivatives rac-{(Ra )-1-[4-([1,1':2',1''-terphenyl]-2-yl)buta-2,3-dien-1-yl](3R)-piperidine-3-carboxylic acid} and rac-{(Sa )-1-[4-([1,1':2',1''-terphenyl]-2-yl)buta-2,3-dien-1-yl](3R)-piperidine-3-carboxylic acid} (21 p), possessing an o-terphenyl residue, was identified as highly selective and the most potent mGAT1 inhibitor in this study. For the (R)-nipecotic acid derived form of 21 p, the inhibitory potency in [3 H]GABA uptake assays was determined as pIC50 =6.78±0.08, and the binding affinity in MS Binding Assays as pKi =7.10±0.12. The synthesis of the designed compounds was carried out by a two-step procedure, generating the allene moiety via allenylation of terminal alkynes which allows broad variation of the terminal phenyl and biphenyl subunit.

Generation and screening of pseudostatic hydrazone libraries derived from 5-substituted nipecotic acid derivatives at the GABA transporter mGAT4.

The γ-aminobutyric acid (GABA) transporter mGAT4 represents a promising drug target for the treatment of epilepsy and other neurological disorders; however, the lack of highly potent and selective inhibitors for mGAT4 still retards its pharmacological elucidation. Herein, the generation and screening of pseudostatic combinatorial hydrazone libraries at the murine GABA transporter mGAT4 for the search of novel GABA uptake inhibitors is described. The hydrazone libraries contained more than 1100 compounds derived from nipecotic acid derivatives substituted at the 5-position instead, as common, at the 1-position of the core structure. Two hits were found and evaluated, which display potencies in the lower micromolar range at mGAT4 and its human equivalent hGAT3. These compounds possess a lipophilic moiety derived from a biphenyl residue attached to the 5-position of the hydrophilic nipecotic acid moiety via a three-atom spacer. Thus, the novel structures with potencies close to that of the bench mark mGAT4 inhibitor (S)-SNAP-5114 add new insights into the structure-activity relationship of mGAT4 inhibitors and could provide a promising starting point for the development of new mGAT4 inhibitors with even higher potencies.

Synthesis and biological evaluation of novel N-substituted nipecotic acid derivatives with a trans-alkene spacer as potent GABA uptake inhibitors.

Our study presents the synthesis and structure-activity relationship (SAR) of novel N-substituted nipecotic acid derivatives closely related to DDPM-1457 [(S)-2a], a chemically stable analog of (S)-SNAP-5114 (1), in the pursuit of finding new and potent mGAT4 selective inhibitors. Iminium ion chemistry served as key step for the preparation of the desired, new N-substituted nipecotic acid derivatives containing a variety of different heterocycles attached to the nipecotic acid moiety via a trans-alkene spacer. The target compounds were characterized with regard to their potency at and subtype selectivity for the GABA transporters mGAT1-mGAT4.

Synthesis and biological evaluation of novel N-substituted nipecotic acid derivatives with an alkyne spacer as GABA uptake inhibitors.

In this study, we present the synthesis and structure-activity relationships (SAR) of novel N-substituted nipecotic acid derivatives closely related to (S)-SNAP-5114 (2) in the pursuit of finding new and potent mGAT4 selective inhibitors. By the use of iminium ion chemistry, a series of new N-substituted nipecotic acid derivatives containing a variety of heterocycles, and an alkyne spacer were synthesized. Biological evaluation of the prepared compounds showed, how the inhibitory potency and subtype selectivity for the murine GABA transporters (mGATs) were influenced by the performed modifications.

Design, Synthesis, Evaluation and Computational Studies of Nipecotic Acid-Acetonaphthone Hybrids as Potential Antiepileptic Agents.

BACKGROUND: Nipecotic acid is considered to be one of the most potent inhibitors of neuronal and glial γ-aminobutyric acid (GABA) uptake in vitro. However, nipecotic acid does not readily cross the blood-brain barrier (BBB) following peripheral administration, owing to its hydrophilic nature. OBJECTIVE: A series of substituted acetonaphthones tethered nipecotic acid derivatives were designed and synthesized with an aim to improve the lipophilicity and the blood-brain barrier (BBB) permeation. METHODS: Synthesized compounds were tested in mice models of PTZ, pilocarpine, and DMCM induced epilepsy, in vivo. The rota-rod test was performed to determine the acute neurotoxicity of the potential leads (4a, 4b, and 4i). These potential hybrids were also evaluated for their ability to cross the BBB by an in vitro parallel artificial membrane permeability BBB assay (PAMPA-BBB). The leads were subjected to in silico molecular docking and dynamics studies on homology modelled protein of human GABA (γ-amino butyric acid) transporter 1 (GAT1) and prediction of their pharmacokinetic properties. RESULT: Amongst the synthesized derivatives, compounds 3a, 3b, 3i, 4a, 4b, and 4i exhibited increased latency of seizures against subcutaneous pentylenetetrazole (scPTZ) induced seizures in mice. Derivatives 4a, 4b, 4i were more effective compared to nipecotic acid ester counterparts 3a, 3b and 3i placing the importance of the presence of free carboxyl group in the centre. The findings revealed that 4i was comparatively more permeable (Pe= 8.89) across BBB than the standard tiagabine (Pe= 7.86). In silico studies proved the consensual interactions of compound 4i with the active binding pocket. CONCLUSION: Some nipecotic acid-acetonaphthone hybrids with considerable anti-epileptic activity, drug like properties and the ability to permeate the BBB have been successfully synthesized.

GABA Uptake Inhibition Reduces In Vivo Extraction Fraction in the Ventral Tegmental Area of Long Evans Rats Measured by Quantitative Microdialysis Under Transient Conditions.

Inhibitory signaling in the ventral tegmental area (VTA) is involved in the mechanism of action for many drugs of abuse. Although drugs of abuse have been shown to alter extracellular γ-aminobutyric acid (GABA) concentration in the VTA, knowledge on how uptake mechanisms are regulated in vivo is limited. Quantitative (no-net-flux) microdialysis is commonly used to examine the extracellular concentration and clearance of monoamine neurotransmitters, however it is unclear whether this method is sensitive to changes in clearance for amino acid neurotransmitters such as GABA. The purpose of this study was to determine whether changes in GABA uptake are reflected by in vivo extraction fraction within the VTA. Using quantitative (no-net-flux) microdialysis adapted for transient conditions, we examined the effects of local perfusion with the GABA uptake inhibitor, nipecotic acid, in the VTA of Long Evans rats. Basal extracellular GABA concentration and in vivo extraction fraction were 44.4 ± 1.9 nM (x-intercepts from 4 baseline regressions using a total of 24 rats) and 0.19 ± 0.01 (slopes from 4 baseline regressions using a total of 24 rats), respectively. Nipecotic acid (50 µM) significantly increased extracellular GABA concentration to 170 ± 4 nM and reduced in vivo extraction fraction to 0.112 ± 0.003. Extraction fraction returned to baseline following removal of nipecotic acid from the perfusate. Conventional microdialysis substantially underestimated the increase of extracellular GABA concentration due to nipecotic acid perfusion compared with that obtained from the quantitative analysis. Together, these results show that inhibiting GABA uptake mechanisms within the VTA alters in vivo extraction fraction measured using microdialysis and that in vivo extraction fraction may be an indirect measure of GABA clearance.

Development of Highly Potent GAT1 Inhibitors: Synthesis of Nipecotic Acid Derivatives with N-Arylalkynyl Substituents.

A new scaffold of highly potent and mGAT1-selective inhibitors has been developed. Compounds in this class are characterized by an alkyne-type spacer connecting nipecotic acid with an aromatic moiety. Preliminary evaluations made it apparent that a nipecotic acid derivative with an N-butynyl linker and a terminal 2-biphenyl residue exhibiting a binding affinity (pKi ) of 7.61±0.03 to mGAT1 and uptake inhibition (pIC50 ) of 7.00±0.06 selective for mGAT1 could serve as a hit compound. Docking calculations for compounds based on this structure in an hGAT1 homology modeling study indicated binding affinities similar to or even higher than that of the well-known mGAT1 inhibitor tiagabine. Synthesis of the designed compounds was readily carried out by two consecutive cross-coupling reactions, giving flexible access to variously substituted biphenyl subunits. With an appropriate substitution pattern of the biphenyl moiety, the binding affinity of enantiopure (R)-nipecotic acid derivatives to mGAT1 increased to pKi =8.33±0.01, and the uptake inhibitory potency up to pIC50 =7.72±0.02.

Synthesis of 4-substituted nipecotic acid derivatives and their evaluation as potential GABA uptake inhibitors.

In this study, we disclose the design and synthesis of novel 4-susbtituted nipecotic acid derivatives as inhibitors of the GABA transporter mGAT1. Based on molecular modeling studies the compounds are assumed to adopt a binding pose similar to that of the potent mGAT1 inhibitor nipecotic acid. As substitution in 4-position should not cause an energetically unfavorable orientation of nipecotic acid as it is the case for N-substituted derivatives this is expected to lead to highly potent binders. For the synthesis of novel 4-substituted nipecotic acid derivatives a linear synthetic strategy was employed. As a key step, palladium catalyzed cross coupling reactions were used to attach the required biaryl moieties to the ω-position of the alkenyl- or alkynyl spacers of varying length in the 4-position of the nipecotic acid scaffold. The resulting amino acids were characterized with respect to their binding affinities and inhibitory potencies at mGAT1. Though the biological activities found were generally insignificant to poor, two compounds, one of which possesses a reasonable binding affinity for mGAT1, rac-57, the other a notable inhibitory potency at mGAT4, rac-84, both displaying a slight subtype selectivity for the individual transporters, could be identified.

Development of Highly Potent GAT1 Inhibitors: Synthesis of Nipecotic Acid Derivatives by Suzuki-Miyaura Cross-Coupling Reactions.

A new series of potent and selective mGAT1 inhibitors has been identified, featuring a nipecotic acid residue and an N-butenyl linker with a 2-biphenyl residue at the ω-position. Docking, combined with MD calculations, revealed a binding mode for the new compounds similar to that of tiagabine, the only mGAT1 inhibitor currently approved as antiepileptic drug. For the synthesis, a Suzuki-Miyaura cross-coupling reaction was used as a key step by which variously substituted biaryl subunits were assembled. Biological evaluation revealed several compounds that possess binding affinities and inhibitory potencies toward mGAT1, together with subtype selectivities against mGAT2-mGAT4 that were similar to or even higher than those for tiagabine. A derivative carrying the 2',4'-dichloro-2-biphenyl moiety attached to N-but-3-enylnipecotic acid at the terminal position of the linker chain was found to be the most potent binder, with the racemic form of the compound displaying a binding affinity of 8.05±0.13 (pKi ), while the R enantiomer exhibited an affinity value of 8.33±0.06 (pKi ).

Hairpin formation promoted by the heterochiral dinipecotic acid segment: A DFT study.

Conformational preferences for the turn and ß-hairpin structures of Ala-based peptides [Ac-Ala(n)-(R)-Nip-(S)-Nip-Ala(n)-X (n = 0-2; X = NHMe or NMe2)] containing nipecotic acid (Nip) residues were carried out using the density functional M06-2X and the implicit solvation model SMD in CH2Cl2 and/or water. The turn structure of the (R)-Nip-(S)-Nip segment with a C10 H-bond between two terminal groups was found to be most preferred (populated at 98.9%) in CH2Cl2; this structure is consistent with IR and (1)H NMR results. The stabilities of the ß-hairpins containing the (R)-Nip-(S)-Nip segment as a turn motif relative to the extended structures increased with peptide sequence length. The relative strengths of the H-bonds between the carbonyl oxygen and the amide hydrogen appeared to be responsible for stabilizing the turn and ß-hairpin structures in CH2Cl2. In addition, the (R)-Nip-(S)-Nip segment exhibited the capability to be incorporated into one of the two ß-turn motifs of gramicidin S (GS). The structure of this GS derivative (GS-Nip2 ) was generally similar to the native peptide but was less hydrophobic and it is therefore expected to exhibit lower hemolytic activity; however, further experiments are needed to evaluate its antimicrobial activity. The structure of GS-Nip2 was somewhat more flexible than GS in solvents of higher polarity. Thus, our calculated results regarding the turn and ß-hairpin motifs of the (R)-Nip-(S)-Nip segment indicate that this structure might be useful for the design of bioactive macrocyclic peptides containing ß-hairpin mimics as well as binding epitopes in protein-protein and protein-nucleic acid recognitions.

Discovery of a subtype selective inhibitor of the human betaine/GABA transporter 1 (BGT-1) with a non-competitive pharmacological profile.

The γ-aminobutyric acid (GABA) transporters (GATs) are essential regulators of the activity in the GABAergic system through their continuous uptake of the neurotransmitter from the synaptic cleft and extrasynaptic space. Four GAT subtypes have been identified to date, each displaying different pharmacological properties and expression patterns. The present study focus on the human betaine/GABA transporter 1 (BGT-1), which has recently emerged as a new target for treatment of epilepsy. However, the lack of selective inhibitors of this transporter has impaired the exploration of this potential considerably. With the objective of identifying novel compounds displaying selectivity for BGT-1, we performed a screening of a small compound library at cells expressing BGT-1 using a [(3)H]GABA uptake assay. The screening resulted in the identification of the compound N-(1-benzyl-4-piperidinyl)-2,4-dichlorobenzamide (BPDBA), a selective inhibitor of the human BGT-1 transporter with a non-competitive profile exhibiting no significant inhibitory activity at the other three human GAT subtypes. The selectivity profile of the compound was subsequently confirmed at cells expressing the four mouse GAT subtypes. Thus, BPDBA constitutes a potential useful pharmacological tool compound for future explorations of the function of the BGT-1 subtype.