Exploring Biginelli-based scaffolds as A2B adenosine receptor antagonists: Unveiling novel structure-activity relationship trends, lead compounds, and potent colorectal anticancer agents.

Antagonists of the A2B adenosine receptor have recently emerged as targeted anticancer agents and immune checkpoint inhibitors within the realm of cancer immunotherapy. This study presents a comprehensive evaluation of novel Biginelli-assembled pyrimidine chemotypes, including mono-, bi-, and tricyclic derivatives, as A2BAR antagonists. We conducted a comprehensive examination of the adenosinergic profile (both binding and functional) of a large compound library consisting of 168 compounds. This approach unveiled original lead compounds and enabled the identification of novel structure-activity relationship (SAR) trends, which were supported by extensive computational studies, including quantum mechanical calculations and free energy perturbation (FEP) analysis. In total, 25 molecules showed attractive affinity (Ki < 100 nM) and outstanding selectivity for A2BAR. From these, five molecules corresponding to the new benzothiazole scaffold were below the Ki < 10 nM threshold, in addition to a novel dual A2A/A2B antagonist. The most potent compounds, and the dual antagonist, showed enantiospecific recognition in the A2BAR. Two A2BAR selective antagonists and the dual A2AAR/A2BAR antagonist reported in this study were assessed for their impact on colorectal cancer cell lines. The results revealed a significant and dose-dependent reduction in cell proliferation. Notably, the A2BAR antagonists exhibited remarkable specificity, as they did not impede the proliferation of non-tumoral cell lines. These findings support the efficacy and potential that A2BAR antagonists as valuable candidates for cancer therapy, but also that they can effectively complement strategies involving A2AAR antagonism in the context of immune checkpoint inhibition.

Exploring structural determinants of neuroprotection bias on novel glypromate conjugates with bioactive amines.

Neurodegenerative disorders of the central nervous system (CNS) such as Alzheimer's and Parkinson's diseases, afflict millions globally, posing a significant public health challenge. Despite extensive research, a critical hurdle in effectively treating neurodegenerative diseases is the lack of neuroprotective drugs that can halt or reverse the underlying disease processes. In this work, we took advantage of the neuroprotective properties of the neuropeptide glycyl-l-prolyl-l-glutamic acid (Glypromate) for the development of new peptidomimetics using l-pipecolic acid as a proline surrogate and exploring their chemical conjugation with relevant active pharmaceutical ingredients (API) via a peptide bond. Together with prolyl-based Glypromate conjugates, a total of 36 conjugates were toxicologically and biologically evaluated. In this series, the results obtained showed that a constrained ring (l-proline) at the central position of the peptide motif accounts for enhanced toxicological profiles and biological effects using undifferentiated and differentiated human neuroblastoma SH-SY5Y cells. Additionally, it was shown that biased biological responses are API-dependent. Conjugation with (R)-1-aminoindane led to a 38-43% reduction of protein aggregation induced by Aß25-35 (10 µM), denoting a 3.2-3.6-fold improvement in comparison with the parent neuropeptide, with no significative difference between functionalization at α and γ-carboxyl ends. On the other hand, the best-performing neuroprotective conjugate against the toxicity elicited by 6-hydroxydopamine (6-OHDA, 125 µM) was obtained by conjugation with memantine at the α-carboxyl end, resulting in a 2.3-fold improvement of the neuroprotection capacity in comparison with Glypromate neuropeptide. Altogether, the chemical strategy explored in this work shows that the neuroprotective capacity of Glypromate can be modified and fine-tuned, opening a new avenue for the development of biased neurotherapeutics for CNS-related disorders.

Exploring the Effect of Halogenation in a Series of Potent and Selective A2B Adenosine Receptor Antagonists.

The modulation of the A2B adenosine receptor is a promising strategy in cancer (immuno) therapy, with A2BAR antagonists emerging as immune checkpoint inhibitors. Herein, we report a systematic assessment of the impact of (di- and mono-)halogenation at positions 7 and/or 8 on both A2BAR affinity and pharmacokinetic properties of a collection of A2BAR antagonists and its study with structure-based free energy perturbation simulations. Monohalogenation at position 8 produced potent A2BAR ligands irrespective of the nature of the halogen. In contrast, halogenation at position 7 and dihalogenation produced a halogen-size-dependent decay in affinity. Eight novel A2BAR ligands exhibited remarkable affinity (Ki < 10 nM), exquisite subtype selectivity, and enantioselective recognition, with some eutomers eliciting sub-nanomolar affinity. The pharmacokinetic profile of representative derivatives showed enhanced solubility and microsomal stability. Finally, two compounds showed the capacity of reversing the antiproliferative effect of adenosine in activated primary human peripheral blood mononuclear cells.

A2B adenosine receptor antagonists rescue lymphocyte activity in adenosine-producing patient-derived cancer models.

BACKGROUND: Adenosine is a metabolite that suppresses antitumor immune response of T and NK cells via extracellular binding to the two subtypes of adenosine-2 receptors, A2ARs. While blockade of the A2AARs subtype effectively rescues lymphocyte activity, with four A2AAR antagonists currently in anticancer clinical trials, less is known for the therapeutic potential of the other A2BAR blockade within cancer immunotherapy. Recent studies suggest the formation of A2AAR/A2BAR dimers in tissues that coexpress the two receptor subtypes, where the A2BAR plays a dominant role, suggesting it as a promising target for cancer immunotherapy. METHODS: We report the synthesis and functional evaluation of five potent A2BAR antagonists and a dual A2AAR/A2BAR antagonist. The compounds were designed using previous pharmacological data assisted by modeling studies. Synthesis was developed using multicomponent approaches. Flow cytometry was used to evaluate the phenotype of T and NK cells on A2BAR antagonist treatment. Functional activity of T and NK cells was tested in patient-derived tumor spheroid models. RESULTS: We provide data for six novel small molecules: five A2BAR selective antagonists and a dual A2AAR/A2BAR antagonist. The growth of patient-derived breast cancer spheroids is prevented when treated with A2BAR antagonists. To elucidate if this depends on increased lymphocyte activity, immune cells proliferation, and cytokine production, lymphocyte infiltration was evaluated and compared with the potent A2AAR antagonist AZD-4635. We find that A2BAR antagonists rescue T and NK cell proliferation, IFNγ and perforin production, and increase tumor infiltrating lymphocytes infiltration into tumor spheroids without altering the expression of adhesion molecules. CONCLUSIONS: Our results demonstrate that A2BAR is a promising target in immunotherapy, identifying ISAM-R56A as the most potent candidate for A2BAR blockade. Inhibition of A2BAR signaling restores T cell function and proliferation. Furthermore, A2BAR and dual A2AAR/A2BAR antagonists showed similar or better results than A2AAR antagonist AZD-4635 reinforcing the idea of dominant role of the A2BAR in the regulation of the immune system.

Optimization of 2-Amino-4,6-diarylpyrimidine-5-carbonitriles as Potent and Selective A1 Antagonists.

We herein document a large collection of 108 2-amino-4,6-disubstituted-pyrimidine derivatives as potent, structurally simple, and highly selective A1AR ligands. The most attractive ligands were confirmed as antagonists of the canonical cyclic adenosine monophosphate pathway, and some pharmacokinetic parameters were preliminarilly evaluated. The library, built through a reliable and efficient three-component reaction, comprehensively explored the chemical space allowing the identification of the most prominent features of the structure-activity and structure-selectivity relationships around this scaffold. These included the influence on the selectivity profile of the aromatic residues at positions R4 and R6 of the pyrimidine core but most importantly the prominent role to the unprecedented A1AR selectivity profile exerted by the methyl group introduced at the exocyclic amino group. The structure-activity relationship trends on both A1 and A2AARs were conveniently interpreted with rigorous free energy perturbation simulations, which started from the receptor-driven docking model that guided the design of these series.

Design, Synthesis, and Biological Evaluation of Hybrid Glypromate Analogues Using 2-Azanorbornane as a Prolyl and Pipecolyl Surrogate.

Neurodegenerative disorders of the central nervous system are a class of heterogeneous pathologies affecting millions of people worldwide and represent a global health burden in developed and developing countries. Without restorative treatments currently available, research on neuroprotective drugs is considered a health priority. In this study, new analogues of the glycyl-l-prolyl-l-glutamic acid (Glypromate) neuropeptide were designed, synthesized, and biologically evaluated using (1R,3S,4S)-2-azanorbornane-3-carboxylic acid as a hybrid construct of l-proline and l-pipecolic acid. Neuroprotection assays carried out in human neuroblastoma SH-SY5Y cells using 6-hydroxydopamine as a stress inducer showed great percentage of recovery (29.7-40.0%) at 100 µM. Among this series, [(1R,3S,4S)-2-glycyl-2-azanorbornane-3-carbonyl]-l-aspartic acid (2a) stands out with a remarkable percentage of recovery (40.0%, at 100 µM) and safe toxicological profile in SH-SY5Y and human adipose mesenchymal stem cells.

Potent and Subtype-Selective Dopamine D2 Receptor Biased Partial Agonists Discovered via an Ugi-Based Approach.

Using a previously unexplored, efficient, and versatile multicomponent method, we herein report the rapid generation of novel potent and subtype-selective DRD2 biased partial agonists. This strategy exemplifies the search for diverse and previously unexplored moieties for the secondary/allosteric pharmacophore of the common phenyl-piperazine scaffold. The pharmacological characterization of the new compound series led to the identification of several ligands with excellent DRD2 affinity and subtype selectivity and remarkable functional selectivity for either the cAMP (22a and 24d) or the ß-arrestin (27a and 29c) signaling pathways. These results were further interpreted on the basis of molecular models of these ligands in complex with the recent DRD2 crystal structures, highlighting the critical role of the secondary/allosteric pharmacophore in modulating the functional selectivity profile.

3,4-Dihydropyrimidin-2(1H)-ones as Antagonists of the Human A2B Adenosine Receptor: Optimization, Structure-Activity Relationship Studies, and Enantiospecific Recognition.

We present and thoroughly characterize a large collection of 3,4-dihydropyrimidin-2(1H)-ones as A2BAR antagonists, an emerging strategy in cancer (immuno) therapy. Most compounds selectively bind A2BAR, with a number of potent and selective antagonists further confirmed by functional cyclic adenosine monophosphate experiments. The series was analyzed with one of the most exhaustive free energy perturbation studies on a GPCR, obtaining an accurate model of the structure-activity relationship of this chemotype. The stereospecific binding modeled for this scaffold was confirmed by resolving the two most potent ligands [(±)-47, and (±)-38 Ki = 10.20 and 23.6 nM, respectively] into their two enantiomers, isolating the affinity on the corresponding (S)-eutomers (Ki = 6.30 and 11.10 nM, respectively). The assessment of the effect in representative cytochromes (CYP3A4 and CYP2D6) demonstrated insignificant inhibitory activity, while in vitro experiments in three prostate cancer cells demonstrated that this pair of compounds exhibits a pronounced antimetastatic effect.

Nitrogen-Walk Approach to Explore Bioisosteric Replacements in a Series of Potent A2B Adenosine Receptor Antagonists.

A systematic exploration of bioisosteric replacements for furan and thiophene cores in a series of potent A2BAR antagonists has been carried out using the nitrogen-walk approach. A collection of 42 novel alkyl 4-substituted-2-methyl-1,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrimidine-3-carboxylates, which contain 18 different pentagonal heterocyclic frameworks at position 4, was synthesized and evaluated. This study enabled the identification of new ligands that combine remarkable affinity (Ki < 30 nM) and exquisite selectivity. The structure-activity relationship (SAR) trends identified were substantiated by a molecular modeling study, based on a receptor-driven docking model and including a systematic free energy perturbation (FEP) study. Preliminary evaluation of the CYP3A4 and CYP2D6 inhibitory activity in optimized ligands evidenced weak and negligible activity, respectively. The stereospecific interaction between hA2BAR and the eutomer of the most attractive novel antagonist (S)-18g (Ki = 3.66 nM) was validated.

X-Ray Crystallography and Free Energy Calculations Reveal the Binding Mechanism of A2A Adenosine Receptor Antagonists.

We present a robust protocol based on iterations of free energy perturbation (FEP) calculations, chemical synthesis, biophysical mapping and X-ray crystallography to reveal the binding mode of an antagonist series to the A2A adenosine receptor (AR). Eight A2A AR binding site mutations from biophysical mapping experiments were initially analyzed with sidechain FEP simulations, performed on alternate binding modes. The results distinctively supported one binding mode, which was subsequently used to design new chromone derivatives. Their affinities for the A2A AR were experimentally determined and investigated through a cycle of ligand-FEP calculations, validating the binding orientation of the different chemical substituents proposed. Subsequent X-ray crystallography of the A2A AR with a low and a high affinity chromone derivative confirmed the predicted binding orientation. The new molecules and structures here reported were driven by free energy calculations, and provide new insights on antagonist binding to the A2A AR, an emerging target in immuno-oncology.

Trifluorinated Pyrimidine-Based A2B Antagonists: Optimization and Evidence of Stereospecific Recognition.

We report the identification of two subsets of fluorinated nonxanthine A2B adenosine receptor antagonists. The novel derivatives explore the effect of fluorination at different positions of two pyrimidine-based scaffolds. The most interesting ligands combine excellent hA2B affinity (Ki < 15 nM) and remarkable subtype selectivity. The results of functional cAMP experiments confirmed the antagonistic behavior of representative ligands. The compounds were designed on the basis of previous molecular models of the stereoselective binding of the parent scaffolds to the hA2B receptor, and we herein provide refinement of such models with the fluorinated compounds, which allows the explanation of the spurious effects of the fluorination at the different positions explored. These models are importantly confirmed by a synergistic study combining chiral HPLC, circular dichroism, diastereoselective synthesis, molecular modeling, and X-ray crystallography, providing experimental evidence toward the stereospecific interaction between optimized trifluorinated stereoisomers and the hA2B receptor.

Perturbation Theory Machine Learning Modeling of Immunotoxicity for Drugs Targeting Inflammatory Cytokines and Study of the Antimicrobial G1 Using Cytometric Bead Arrays.

ChEMBL biological activities prediction for 1-5-bromofur-2-il-2-bromo-2-nitroethene (G1) is a difficult task for cytokine immunotoxicity. The current study presents experimental results for G1 interaction with mouse Th1/Th2 and pro-inflammatory cytokines using a cytometry bead array (CBA). In the in vitro test of CBA, the results show no significant differences between the mean values of the Th1/Th2 cytokines for the samples treated with G1 with respect to the negative control, but there are moderate differences for cytokine values between different periods (24/48 h). The experiments show no significant differences between the mean values of the pro-inflammatory cytokines for the samples treated with G1, regarding the negative control, except for the values of tumor necrosis factor (TNF) and Interleukin (IL6) between the group treated with G1 and the negative control at 48 h. Differences occur for these cytokines in the periods (24/48 h). The study confirmed that the antimicrobial G1 did not alter the Th1/Th2 cytokines concentration in vitro in different periods, but it can alter TNF and IL6. G1 promotes free radicals production and activates damage processes in macrophages culture. In order to predict all ChEMBL activities for drugs in other experimental conditions, a ChEMBL data set was constructed using 25 biological activities, 1366 assays, 2 assay types, 4 assay organisms, 2 organisms, and 12 cytokine targets. Molecular descriptors calculated with Rcpi and 15 machine learning methods were used to find the best model able to predict if a drug could be active or not against a specific cytokine, in specific experimental conditions. The best model is based on 120 selected molecular descriptors and a deep neural network with area under the curve of the receiver operating characteristic of 0.904 and accuracy of 0.832. This model predicted 1384 G1 biological activities against cytokines in all ChEMBL data set experimental conditions.

Synthesis, Pharmacological, and Biological Evaluation of MIF-1 Picolinoyl Peptidomimetics as Positive Allosteric Modulators of D2R.

This work describes the synthesis and pharmacological evaluation of picolinoyl-based peptidomimetics of melanocyte stimulating hormone release inhibiting factor 1 (MIF-1) as dopamine modulating agents. Eight novel peptidomimetics were tested for their ability to enhance the maximal effect of tritiated N-propylapomorphine ([3H]-NPA) at dopamine D2 receptors (D2R). Methyl picolinoyl-l-valyl-l-alaninate (compound 6b) produced a statistically significant increase in the maximal [3H]-NPA response at 0.01 nM (11.9 ± 3.7%), which is close to the effect of MIF-1 in this assay at same concentration (18.3 ± 9.1%). Functional assays measuring cAMP mobilization in the presence of dopamine corroborate the activity of peptidomimetic 6b as a positive allosteric modulator (PAM) of D2R. In this assay, 6b produced a typical bell-shaped dose-response curve similar to that of the parent neuropeptide (18.3 ± 7.1% for 6b vs 15.4 ± 5.5% for MIF-1, both at 0.1 nM). Dose-response curves for dopamine in the presence of 6b show EC50 (0.33 ± 0.21 µM for 6b vs 0.17 ± 0.07 µM for MIF-1) and Emax (86.0 ± 5.4% for 6b vs 93.6 ± 4.4% for MIF-1) comparable to those of MIF-1, both at 0.01 nM. Furthermore, peptidomimetic 6b was tested for agonist activity at the human D2R and the results show that it displays no intrinsic agonism effect, endorsing its activity as a PAM of D2R. Cytotoxic and neurotoxic assays were performed for peptidomimetic 6b using HEK 293T cells and cortex neurons from 19 day old Wistar-Kyoto rat embryos, respectively, suggesting this analogue displays no toxicity effect in these assays up to 100 µM. Conformational energy minimization for 6b shows that this peptidomimetic cannot adopt the postulated type-II ß-turn bioactive conformation, endorsing the possibility of an extended bioactive conformation as claimed by other researchers as a second bioactive conformation of MIF-1. Overall, the pharmacological and toxicological profile of peptidomimetic 6b together with its favorable druglike properties and structural simplicity makes it a potential lead compound for further development and optimization.

Enantiospecific Recognition at the A2B Adenosine Receptor by Alkyl 2-Cyanoimino-4-substituted-6-methyl-1,2,3,4-tetrahydropyrimidine-5-carboxylates.

A novel family of structurally simple, potent, and selective nonxanthine A2BAR ligands was identified, and its antagonistic behavior confirmed through functional experiments. The reported alkyl 2-cyanoimino-4-substituted-6-methyl-1,2,3,4-tetrahy-dropyrimidine-5-carboxylates (16) were designed by bioisosteric replacement of the carbonyl group at position 2 in a series of 3,4-dihydropyrimidin-2-ones. The scaffold (16) documented herein contains a chiral center at the heterocycle. Accordingly, the most attractive ligand of the series [(±)16b, Ki = 24.3 nM] was resolved into its two enantiomers by chiral HPLC, and the absolute configuration was established by circular dichroism. The biological evaluation of both enantiomers demonstrated enantiospecific recognition at A2BAR, with the (S)-16b enantiomer retaining all the affinity (Ki = 15.1 nM), as predicted earlier by molecular modeling. This constitutes the first example of enantiospecific recognition at the A2B adenosine receptor and opens new possibilities in ligand design for this receptor.

Discovery of Potent and Highly Selective A2B Adenosine Receptor Antagonist Chemotypes.

Three novel families of A2B adenosine receptor antagonists were identified in the context of the structural exploration of the 3,4-dihydropyrimidin-2(1H)-one chemotype. The most appealing series contain imidazole, 1,2,4-triazole, or benzimidazole rings fused to the 2,3-positions of the parent diazinone core. The optimization process enabled identification of a highly potent (3.49 nM) A2B ligand that exhibits complete selectivity toward A1, A2A, and A3 receptors. The results of functional cAMP experiments confirmed the antagonistic behavior of representative ligands. The main SAR trends identified within the series were substantiated by a molecular modeling study based on a receptor-driven docking model constructed on the basis of the crystal structure of the human A2A receptor.

Synthesis by microwave-assisted 1,3-dipolar cycloaddition of 1,2,3-triazole 1'-homo-3'-isoazanucleosides and evaluation of their anticancer activity.

Racemic 1'-homo-3'-isoazanucleosides have been obtained by microwave-assisted 1,3-dipolar cycloaddition of 3,5-disubstituted proline derivative (±)-2 with different alkynes. The compounds obtained were evaluated for their cytotoxic activities in vitro against human breast carcinoma cell lines (MCF-7), human ovary carcinoma cell lines (A2780) and human lung carcinoma cell lines (NCI-H460).

Structure-based design of new KSP-Eg5 inhibitors assisted by a targeted multicomponent reaction.

An integrated multidisciplinary approach that combined structure-based drug design, multicomponent reaction synthetic approaches and functional characterization in enzymatic and cell assays led to the discovery of new kinesin spindle protein (KSP) inhibitors with antiproliferative activity. A focused library of new benzimidazoles obtained by a Ugi+Boc removal/cyclization reaction sequence generated low-micromolar-range KSP inhibitors as promising anticancer prototypes. The design and functional studies of the new chemotypes were assessed by computational modeling and molecular biology techniques. The most active compounds-20 (IC50 =1.49 µM, EC50 =3.63 µM) and 22 (IC50 =1.37 µM, EC50 =6.90 µM)-were synthesized with high efficiency by taking advantage of the multicomponent reactions.

Model for high-throughput screening of drug immunotoxicity--study of the anti-microbial G1 over peritoneal macrophages using flow cytometry.

Quantitative Structure-Activity (mt-QSAR) techniques may become an important tool for prediction of cytotoxicity and High-throughput Screening (HTS) of drugs to rationalize drug discovery process. In this work, we train and validate by the first time mt-QSAR model using TOPS-MODE approach to calculate drug molecular descriptors and Linear Discriminant Analysis (LDA) function. This model correctly classifies 8258 out of 9000 (Accuracy = 91.76%) multiplexing assay endpoints of 7903 drugs (including both train and validation series). Each endpoint correspond to one out of 1418 assays, 36 molecular and cellular targets, 46 standard type measures, in two possible organisms (human and mouse). After that, we determined experimentally, by the first time, the values of EC50 = 21.58 µg/mL and Cytotoxicity = 23.6% for the anti-microbial/anti-parasite drug G1 over Balb/C mouse peritoneal macrophages using flow cytometry. In addition, the model predicts for G1 only 7 positive endpoints out 1251 cytotoxicity assays (0.56% of probability of cytotoxicity in multiple assays). The results obtained complement the toxicological studies of this important drug. This work adds a new tool to the existing pool of few methods useful for multi-target HTS of ChEMBL and other libraries of compounds towards drug discovery.

Selective and potent adenosine A3 receptor antagonists by methoxyaryl substitution on the N-(2,6-diarylpyrimidin-4-yl)acetamide scaffold.

The influence of diverse methoxyphenyl substitution patterns on the N-(2,6-diarylpyrimidin-4-yl)acetamide scaffold is herein explored in order to modulate the A(3) adenosine receptor antagonistic profile. As a result, novel ligands exhibiting excellent potency (K(i) on A(3) AR < 20 nM) and selectivity profiles (above 100-fold within the adenosine receptors family) are reported. Moreover, our joint theoretical and experimental approach allows the identification of novel pharmacophoric elements conferring A(3)AR selectivity, first established by a robust computational model and thereafter characterizing the most salient features of the structure-activity and structure-selectivity relationships in this series.

Discovery of 3,4-Dihydropyrimidin-2(1H)-ones As a Novel Class of Potent and Selective A2B Adenosine Receptor Antagonists.

We describe the discovery and optimization of 3,4-dihydropyrimidin-2(1H)-ones as a novel family of (nonxanthine) A2B receptor antagonists that exhibit an unusually high selectivity profile. The Biginelli-based hit optimization process enabled a thoughtful exploration of the structure-activity and structure-selectivity relationships for this chemotype, enabling the identification of ligands that combine structural simplicity with excellent hA2B AdoR affinity and remarkable selectivity profiles.