Advances in drug design and therapeutic potential of selective or multitarget 5-HT1A receptor ligands.

5-HT1A receptor (5-HT1A-R) is a serotoninergic G-protein coupled receptor subtype which contributes to several physiological processes in both central nervous system and periphery. Despite being the first 5-HT-R identified, cloned and studied, it still represents a very attractive target in drug discovery and continues to be the focus of a myriad of drug discovery campaigns due to its involvement in numerous neuropsychiatric disorders. The structure-activity relationship studies (SAR) performed over the last years have been devoted to three main goals: (i) design and synthesis of 5-HT1A-R selective/preferential ligands; (ii) identification of 5-HT1A-R biased agonists, differentiating pre- versus post-synaptic agonism and signaling cellular mechanisms; (iii) development of multitarget compounds endowed with well-defined poly-pharmacological profiles targeting 5-HT1A-R along with other serotonin receptors, serotonin transporter (SERT), D2-like receptors and/or enzymes, such as acetylcholinesterase and phosphodiesterase, as a promising strategy for the management of complex psychiatric and neurodegenerative disorders. In this review, medicinal chemistry aspects of ligands acting as selective/preferential or multitarget 5-HT1A-R agonists and antagonists belonging to different chemotypes and developed in the last 7 years (2017-2023) have been discussed. The development of chemical and pharmacological 5-HT1A-R tools for molecular imaging have also been described. Finally, the pharmacological interest of 5-HT1A-R and the therapeutic potential of ligands targeting this receptor have been considered.

New potent muscarinic receptor ligands bearing the 1,4-dioxane nucleus: Investigation on the nature of the substituent in position 2.

A new series of muscarinic acetylcholine receptor (mAChR) ligands obtained by inserting different substituents in position 2 of the potent 6,6-diphenyl-1,4-dioxane antagonists 4 and 5 was designed and synthesized to investigate the influence of steric bulk on the mAChR affinity. Specifically, the insertion of a 2-methyl group, affording compounds 6 and 9, resulted as the most favorable modification in terms of affinity for all muscarinic subtypes. As supported by computational studies performed on the hM1 receptor, this substituent may contribute to stabilize the ligand within the binding site by favoring the formation of stable interactions between the cationic head of the ligand and the residue D105. The increase of steric bulk, obtained by replacing the methyl group with an ethyl (7 and 10) and especially a phenyl substituent (8 and 11), caused a marked decrease of mAChR affinity, demonstrating the crucial role played by the steric bulk of the 2-substituent in the mAChR interaction. The most intriguing result was obtained with the tertiary amine 9, which, surprisingly, showed two different pKi values for all mAChRs, with preferential subpicomolar affinities for the M1, M3, and M4 subtypes. Interestingly, biphasic curves were also observed with both the eutomer (S)-(-)-9 and the distomer (R)-( + )-9.

Targeting orexin receptors: Recent advances in the development of subtype selective or dual ligands for the treatment of neuropsychiatric disorders.

Orexin-A and orexin-B, also named hypocretin-1 and hypocretin-2, are two hypothalamic neuropeptides highly conserved across mammalian species. Their effects are mediated by two distinct G protein-coupled receptors, namely orexin receptor type 1 (OX1-R) and type 2 (OX2-R), which share 64% amino acid identity. Given the wide expression of OX-Rs in different central nervous system and peripheral areas and the several pathophysiological functions in which they are involved, including sleep-wake cycle regulation (mainly mediated by OX2-R), emotion, panic-like behaviors, anxiety/stress, food intake, and energy homeostasis (mainly mediated by OX1-R), both subtypes represent targets of interest for many structure-activity relationship (SAR) campaigns carried out by pharmaceutical companies and academies. However, before 2017 the research was predominantly directed towards dual-orexin ligands, and limited chemotypes were investigated. Analytical characterizations, including resolved structures for both OX1-R and OX2-R in complex with agonists and antagonists, have improved the understanding of the molecular basis of receptor recognition and are assets for medicinal chemists in the design of subtype-selective ligands. This review is focused on the medicinal chemistry aspects of small molecules acting as dual or subtype selective OX1-R/OX2-R agonists and antagonists belonging to different chemotypes and developed in the last years, including radiolabeled OX-R ligands for molecular imaging. Moreover, the pharmacological effects of the most studied ligands in different neuropsychiatric diseases, such as sleep, mood, substance use, and eating disorders, as well as pain, have been discussed. Poly-pharmacology applications and multitarget ligands have also been considered.

The neuromedin U system: Pharmacological implications for the treatment of obesity and binge eating behavior.

Neuromedin U (NMU) is a bioactive peptide produced in the gut and in the brain, with a role in multiple physiological processes. NMU acts by binding and activating two G protein coupled receptors (GPCR), the NMU receptor 1 (NMU-R1), which is predominantly expressed in the periphery, and the NMU receptor 2 (NMU-R2), mainly expressed in the central nervous system (CNS). In the brain, NMU and NMU-R2 are consistently present in the hypothalamus, commonly recognized as the main "feeding center". Considering its distribution pattern, NMU revealed to be an important neuropeptide involved in the regulation of food intake, with a powerful anorexigenic ability. This has been observed through direct administration of NMU and by studies using genetically modified animals, which revealed an obesity phenotype when the NMU gene is deleted. Thus, the development of NMU analogs or NMU-R2 agonists might represent a promising pharmacological strategy to treat obese individuals. Furthermore, NMU has been demonstrated to influence the non-homeostatic aspect of food intake, playing a potential role in binge eating behavior. This review aims to discuss and summarize the current literature linking the NMU system with obesity and binge eating behavior, focusing on the influence of NMU on food intake and the neuronal mechanisms underlying its anti-obesity properties. Pharmacological strategies to improve the pharmacokinetic profile of NMU will also be reported.

Investigating the role of the central melanocortin system in stress and stress-related disorders.

The melanocortinergic neural circuit, known for its influence on energy expenditure and feeding behavior, also plays a role in stress and stress-induced psychiatric disorders, including anxiety and depression. The major contribution is given by the melanocortin-4 receptor (MC4R) subtype, highly expressed in brain regions involved in the control of stress responses. Furthermore, the MC4R appears to profoundly affect the activity of the hypothalamic-pituitary-adrenal (HPA) axis, and it has been also highlighted a functional and anatomical interaction with the corticotropin-releasing factor (CRF), an important mediator of stress and stress-related behaviors. The MC4R agonists seem to exacerbate stress-inducing anxiety- and depressive-like behavior, while MC4R antagonists have been demonstrated to mitigate such disorders, as shown in several preclinical behavioral tests. The evidence collected in the present review suggests that the melanocortin system, through the MC4R, could possibly modulate behavioral responses to stress, suggesting the use of MC4R antagonists as a possible novel treatment for anxiety and depression induced by stress.

Cu(I) and Cu(II) Complexes Based on Lonidamine-Conjugated Ligands Designed to Promote Synergistic Antitumor Effects.

Bis(pyrazol-1-yl)- and bis(3,5-dimethylpyrazol-1-yl)-acetates were conjugated with the 2-hydroxyethylester and 2-aminoethylamide derivatives of the antineoplastic drug lonidamine to prepare Cu(I) and Cu(II) complexes that might act through synergistic mechanisms of action due to the presence of lonidamine and copper in the same chemical entity. Synchrotron radiation-based complementary techniques [X-ray photorlectron spectroscopy and near-edge X-ray absorption fine structure (NEXAFS)] were used to characterize the electronic and molecular structures of the complexes and the local structure around the copper ion (XAFS) in selected complexes. All complexes showed significant antitumor activity, proving to be more effective than the reference drug cisplatin in a panel of human tumor cell lines, and were able to overcome oxaliplatin and multidrug resistance. Noticeably, these Cu complexes appeared much more effective than cisplatin against 3D spheroids of pancreatic PSN-1 cancer cells; among these, PPh3-containing Cu(I) complex 15 appeared to be the most promising derivative. Mechanistic studies revealed that 15 induced cancer cell death by means of an apoptosis-alternative cell death.

Assessing the role of ghrelin and the enzyme ghrelin O-acyltransferase (GOAT) system in food reward, food motivation, and binge eating behavior.

The peripheral peptide hormone ghrelin is a powerful stimulator of food intake, which leads to body weight gain and adiposity in both rodents and humans. The hormone, thus, increases the vulnerability to obesity and binge eating behavior. Several studies have revealed that ghrelin's functions are due to its interaction with the growth hormone secretagogue receptor type 1a (GHSR1a) in the hypothalamic area; besides, ghrelin also promotes the reinforcing properties of hedonic food, acting at extra-hypothalamic sites and interacting with dopaminergic, cannabinoid, opioid, and orexin signaling. The hormone is primarily present in two forms in the plasma and the enzyme ghrelin O-acyltransferase (GOAT) allows the acylation reaction which causes the transformation of des-acyl-ghrelin (DAG) to the active form acyl-ghrelin (AG). DAG has been demonstrated to show antagonist properties; it is metabolically active, and counteracts the effects of AG on glucose metabolism and lipolysis, and reduces food consumption, body weight, and hedonic feeding response. Both peptides seem to influence the hypothalamic-pituitary-adrenal (HPA) axis and the corticosterone/cortisol level that drive the urge to eat under stressful conditions. These findings suggest that DAG and inhibition of GOAT may be targets for obesity and bingeing-related eating disorders and that AG/DAG ratio may be an important potential biomarker to assess the risk of developing maladaptive eating behaviors.

Synthesis and Cytotoxic Activity Evaluation of New Cu(I) Complexes of Bis(pyrazol-1-yl) Acetate Ligands Functionalized with an NMDA Receptor Antagonist.

In the present article, copper(I) complexes of bis(pyrazol-1-yl) carboxylic acid (LH), bis(3,5-dimethylpyrazol-1-yl) carboxylic acid (L2H), and bis(pyrazol-1-yl) acetates conjugated with an N-methyl-d-aspartate (NMDA) receptor antagonist (LNMDA or L2NMDA) and phosphane ligands (triphenylphosphine or 1,3,5-triaza-7-phosphaadamantane) were synthesized. The selection of an NMDA antagonist for the coupling with LH and L2H was suggested by the observation that NMDA receptors are expressed and play a role in different types of cancer models. All the new complexes showed a significant antitumor activity on a panel of human tumor cell lines of different histology, with cisplatin-sensitive, cisplatin-resistant, or multi-drug-resistant phenotype. Their half maximal inhibitory concentration (IC50) values were in the low- and sub-micromolar range and, in general, significantly lower than that of cisplatin. Interestingly, the fact that all the complexes proved to be significantly more active than cisplatin even in three-dimensional (3D) spheroids of H157 and BxPC3 cancer cells increased the relevance of the in vitro results. Finally, morphological analysis revealed that the most representative complex 8 induced a massive swelling of the endoplasmic reticulum (ER) membrane, which is a clear sign of ER stress.

Synthesis, Biological, and Computational Evaluations of Conformationally Restricted NAD-Mimics as Discriminant Inhibitors of Human NMN-Adenylyltransferase Isozymes.

Nicotinamide adenine dinucleotide (NAD) cofactor metabolism plays a significant role in cancer development. Tumor cells have an increased demand for NAD and ATP to support rapid growth and proliferation. Limiting the amount of available NAD by targeting critical NAD biosynthesis enzymes has emerged as a promising anticancer therapeutic approach. In mammals, the enzyme nicotinamide/nicotinic acid adenylyltransferase (NMNAT) catalyzes a crucial downstream reaction for all known NAD synthesis routes. Novel nicotinamide/nicotinic acid adenine dinucleotide (NAD/NaAD) analogues 1-4, containing a methyl group at the ribose 2'-C and 3'-C-position of the adenosine moiety, were synthesized as inhibitors of the three isoforms of human NMN-adenylyltransferase, named hNMNAT-1, hNMNAT-2, and hNMNAT-3. An NMR-based conformational analysis suggests that individual NAD-analogues (1-4) have distinct conformational preferences. Biological evaluation of dinucleotides 1-4 as inhibitors of hNMNAT isoforms revealed structural relationships between different conformations (North-anti and South-syn) and enzyme-inhibitory activity. Among the new series of NAD analogues synthesized and tested, the 2'-C-methyl-NAD analogue 1 (Ki = 15 and 21 µM towards NMN and ATP, respectively) emerged as the most potent and selective inhibitor of hNMNAT-2 reported so far. Finally, we rationalized the in vitro bioactivity and selectivity of methylated NAD analogues with in silico studies, helping to lay the groundwork for rational scaffold optimization.

Copper-Based Complexes with Adamantane Ring-Conjugated bis(3,5-Dimethyl-pyrazol-1-yl)acetate Ligand as Promising Agents for the Treatment of Glioblastoma.

The new ligand L2Ad, obtained by conjugating the bifunctional species bis(3,5-dimethylpyrazol-1-yl)-acetate and the drug amantadine, was used as a chelator for the synthesis of new Cu complexes 1-5. Their structures were investigated by synchrotron radiation-induced X-ray photoelectron spectroscopy (SR-XPS), near-edge X-ray absorption fine structure (NEXAFS) spectroscopy, and by combining X-ray absorption fine structure (XAFS) spectroscopy techniques and DFT modeling. The structure of complex 3 was determined by single-crystal X-ray diffraction analysis. Tested on U87, T98, and U251 glioma cells, Cu(II) complex 3 and Cu(I) complex 5 decreased cell viability with IC50 values significantly lower than cisplatin, affecting cell growth, proliferation, and death. Their effects were prevented by treatment with the Cu chelator tetrathiomolybdate, suggesting the involvement of copper in their cytotoxic activity. Both complexes were able to increase ROS production, leading to DNA damage and death. Interestingly, nontoxic doses of 3 or 5 enhanced the chemosensitivity to Temozolomide.

New flurbiprofen derivatives: synthesis, membrane affinity and evaluation of in vitro effect on ß-amyloid levels.

Alzheimer's disease (AD) is characterized by irreversible and progressive loss of memory and cognition and profound neuronal loss. Current therapeutic strategies for the treatment of AD have been directed to a variety of targets with the aim of reversing or preventing the disease but, unfortunately, the available treatments often produce no significant clinical benefits. During the last decades compounds that inhibit or modulate γ-secretase, reducing ß amyloid (Aß) levels, have been considered as potential therapeutics for AD. Among these the (R)-enantiomer of flurbiprofen (FLU) seems to be very promising, but it shows low brain penetration. In this study, in order to improve the properties of FLU against Alzheimer's pathogenesis we synthesized some novel FLU lipophilic analogues. Lipophilicity of the new molecules has been characterized in terms of clogP, log K(C18/W) and log K(IAM/W) values. Permeability has been determined in both gastrointestinal PAMPA (PAMPA-GI) at different pH values and in brain blood barrier PAMPA (PAMPA-BBB) models. They were also tested for their ability to inhibit in vitro γ-secretase activity using rat CTXTNA2 astrocytes. Interestingly, the investigated molecules demonstrated to reduce Aß 42 levels without affecting the amyloid precursor protein APP level in a clear concentrations-dependent manner.

Properly Substituted Benzimidazoles as a New Promising Class of Nicotinate Phosphoribosyltransferase (NAPRT) Modulators.

The prevention of nicotinamide adenine dinucleotide (NAD) biosynthesis is considered an attractive therapeutic approach against cancer, considering that tumor cells are characterized by an increased need for NAD to fuel their reprogrammed metabolism. On the other hand, the decline of NAD is a hallmark of some pathological conditions, including neurodegeneration and metabolic diseases, and boosting NAD biosynthesis has proven to be of therapeutic relevance. Therefore, targeting the enzymes nicotinamide phosphoribosyltransferase (NAMPT) and nicotinate phosphoribosyltransferase (NAPRT), which regulate NAD biosynthesis from nicotinamide (NAM) and nicotinic acid (NA), respectively, is considered a promising strategy to modulate intracellular NAD pool. While potent NAMPT inhibitors and activators have been developed, the search for NAPRT modulators is still in its infancy. In this work, we report on the identification of a new class of NAPRT modulators bearing the 1,2-dimethylbenzimidazole scaffold properly substituted in position 5. In particular, compounds 24, 31, and 32 emerged as the first NAPRT activators reported so far, while 18 behaved as a noncompetitive inhibitor toward NA (Ki = 338 µM) and a mixed inhibitor toward phosphoribosyl pyrophosphate (PRPP) (Ki = 134 µM). From in vitro pharmacokinetic studies, compound 18 showed an overall good ADME profile. To rationalize the obtained results, docking studies were performed on the NAPRT structure. Moreover, a preliminary pharmacophore model was built to shed light on the shift from inhibitors to activators.

Advances in the Development of Nonpeptide Small Molecules Targeting Ghrelin Receptor.

Ghrelin is an octanoylated peptide acting by the activation of the growth hormone secretagogue receptor, namely, GHS-R1a. The involvement of ghrelin in several physiological processes, including stimulation of food intake, gastric emptying, body energy balance, glucose homeostasis, reduction of insulin secretion, and lipogenesis validates the considerable interest in GHS-R1a as a promising target for the treatment of numerous disorders. Over the years, several GHS-R1a ligands have been identified and some of them have been extensively studied in clinical trials. The recently resolved structures of GHS-R1a bound to ghrelin or potent ligands have provided useful information for the design of new GHS-R1a drugs. This perspective is focused on the development of recent nonpeptide small molecules acting as GHS-R1a agonists, antagonists, and inverse agonists, bearing classical or new molecular scaffolds, as well as on radiolabeled GHS-R1a ligands developed for imaging. Moreover, the pharmacological effects of the most studied ligands have been discussed.

Highly Potent and Selective Dopamine D4 Receptor Antagonists Potentially Useful for the Treatment of Glioblastoma.

To better understand the role of dopamine D4 receptor (D4R) in glioblastoma (GBM), in the present paper, new ligands endowed with high affinity and selectivity for D4R were discovered starting from the brain penetrant and D4R selective lead compound 1-(3-(4-phenylpiperazin-1-yl)propyl)-3,4-dihydroquinolin-2(1H)-one (6). In particular, the D4R antagonist 24, showing the highest affinity and selectivity over D2R and D3R within the series (D2/D4 = 8318, D3/D4 = 3715), and the biased ligand 29, partially activating D4R Gi-/Go-protein and blocking ß-arrestin recruitment, emerged as the most interesting compounds. These compounds, evaluated for their GBM antitumor activity, induced a decreased viability of GBM cell lines and primary GBM stem cells (GSC#83), with the maximal efficacy being reached at a concentration of 10 µM. Interestingly, the treatment with both compounds 24 and 29 induced an increased effect in reducing the cell viability with respect to temozolomide, which is the first-choice chemotherapeutic drug in GBM.

Recent findings leading to the discovery of selective dopamine D4 receptor ligands for the treatment of widespread diseases.

Since its discovery, the dopamine D4 receptor (D4R) has been suggested to be an attractive target for the treatment of neuropsychiatric diseases. Novel findings have renewed the interest in such a receptor as an emerging target for the management of different diseases, including cancer, Parkinson's disease, alcohol or substance use disorders, eating disorders, erectile dysfunction and cognitive deficits. The recently resolved crystal structures of D4R in complexes with the potent ligands nemonapride and L-745870 strongly improved the knowledge on the molecular mechanisms involving the D4R functions and may help medicinal chemists in drug design. This review is focused on the recent development of the subtype selective D4R ligands belonging to classical or new chemotypes. Moreover, ligands showing functional selectivity toward G protein activation or ß-arrestin recruitment and the effects of selective D4R ligands on the above-mentioned diseases are discussed.

Scaffold Hybridization Strategy Leads to the Discovery of Dopamine D3 Receptor-Selective or Multitarget Bitopic Ligands Potentially Useful for Central Nervous System Disorders.

In the search for novel bitopic compounds targeting the dopamine D3 receptor (D3R), the N-(2,3-dichlorophenyl)piperazine nucleus (primary pharmacophore) has been linked to the 6,6- or 5,5-diphenyl-1,4-dioxane-2-carboxamide or the 1,4-benzodioxane-2-carboxamide scaffold (secondary pharmacophore) by an unsubstituted or 3-F-/3-OH-substituted butyl chain. This scaffold hybridization strategy led to the discovery of potent D3R-selective or multitarget ligands potentially useful for central nervous system disorders. In particular, the 6,6-diphenyl-1,4-dioxane derivative 3 showed a D3R-preferential profile, while an interesting multitarget behavior has been highlighted for the 5,5-diphenyl-1,4-dioxane and 1,4-benzodioxane derivatives 6 and 9, respectively, which displayed potent D2R antagonism, 5-HT1AR and D4R agonism, as well as potent D3R partial agonism. They also behaved as low-potency 5-HT2AR antagonists and 5-HT2CR partial agonists. Such a profile might be a promising starting point for the discovery of novel antipsychotic agents.

Design, synthesis, and preliminary pharmacological evaluation of new imidazolinones as L-DOPA prodrugs.

L-DOPA, the immediate biological precursor of dopamine, is still considered the drug of choice in the treatment of Parkinson's disease. However, therapy with L-DOPA is associated with a number of acute problems. With the aim to increase the bioavailability after oral administration, we designed a multi-protected L-DOPA prodrugs able to release the drug by both spontaneous chemical or enzyme catalyzed hydrolysis. The new compounds have been synthesized and preliminarily evaluated for their water solubility, log P, chemical stability, and enzymatic stability. The results indicate that the incorporation of the amino acidic moiety of L-DOPA into an imidazoline-4-one ring provides prodrugs sufficiently stable to potentially cross unchanged the acidic environment of the stomach, and to be absorbed from the intestine. They also might be able to release L-DOPA in human plasma after enzymatic hydrolysis. The ability of prodrugs 6a-b to increase basal levels of striatal DA, and influence brain neurochemistry associated with dopaminergic activity following oral administration, as well as the radical-scavenging activity against DPPH for compounds 6a-b and 15a are also reported.

Novel imidazoline compounds as partial or full agonists of D2-like dopamine receptors inspired by I2-imidazoline binding sites ligand 2-BFI.

Based on the well known biological versatility of the imidazoline nucleus, we prepared the novel derivatives 3a-k inspired by 2-BFI scaffold to assess imidazoline molecules as D(2)-like dopamine receptor ligands. Conservative chemical modifications of the lead structure, such as the introduction of an hydroxy group in the aromatic ring alone or associated with N-benzyl substitution, provided partial (3f) or nearly full (3e and 3h) agonists, all endowed with D(2)-like potency comparable to that of dopamine.

Evaluation of polymer mucoadhesiveness by the use of acoustic spectroscopy.

An innovative and simple methodology has been developed and used for the evaluation of mucoadhesive properties of several polymers by means of sound speed measurements using high-resolution acoustic spectroscopy. In systems made of polymers in water, variations in hydration shell of polymeric chains determine changes of dispersions compressibility, and this phenomenon can be monitored by sound speed measurements. Four different polymers have been selected, namely PEG 6000, Carbopol 974, HPMC K4M, and Pectin 200/USP, all characterised by very different mucoadhesive properties. Samples made of each polymer alone (0.3-1.0% w/w) or in mixture with mucin (mucin fixed at 1.0% w/w) in water were investigated while using high-resolution ultrasonic spectrometer at two different frequencies (5.2 and 8.2 MHz). Polymer-mucin interaction was evaluated comparing experimental sound speed values of polymer-mucin samples with their theoretical values derived from the addition of sound speeds obtained while analysing each component alone. Results demonstrated the ability of the acoustic method to discriminate between mucoadhesive and no mucoadhesive polymer-mucin dispersions and allowed also the comparison between their mucoadhesive strengths. The study has therefore demonstrated the potential of using high-resolution ultrasonic spectroscopy to evaluate the polymers' mucoadhesiveness, with the great advantage of testing small amount of samples even if opaque.

Novel Potent Muscarinic Receptor Antagonists: Investigation on the Nature of Lipophilic Substituents in the 5- and/or 6-Positions of the 1,4-Dioxane Nucleus.

A series of novel 1,4-dioxane analogues of the muscarinic acetylcholine receptor (mAChR) antagonist 2 was synthesized and studied for their affinity at M1-M5 mAChRs. The 6-cyclohexyl-6-phenyl derivative 3b, with a cis configuration between the CH2N+(CH3)3 chain in the 2-position and the cyclohexyl moiety in the 6-position, showed pKi values for mAChRs higher than those of 2 and a selectivity profile analogous to that of the clinically approved drug oxybutynin. The study of the enantiomers of 3b and the corresponding tertiary amine 33b revealed that the eutomers are (2S,6S)-(-)-3b and (2S,6S)-(-)-33b, respectively. Docking simulations on the M3 mAChR-resolved structure rationalized the experimental observations. The quaternary ammonium function, which should prevent the crossing of the blood-brain barrier, and the high M3/M2 selectivity, which might limit cardiovascular side effects, make 3b a valuable starting point for the design of novel antagonists potentially useful in peripheral diseases in which M3 receptors are involved.