Cytotoxic and Antiproliferative Activity of LASSBio-2208 and the Attempts to Determine Its Drug Metabolism and Pharmacokinetics In Vitro Profile.

Inappropriate expression of histone deacetylase (HDAC-6) and deregulation of the phosphatidylinositol 3-kinase (PI3K) signalling pathway are common aberrations observed in cancers. LASSBio-2208, has been previously described as a dual inhibitor in the nanomolar range of HDAC-6 and PI3Kα and is three times more potent in inhibiting HDAC-6. In this paper we described the cytotoxic and antiproliferative potency of LASSBio-2208 on different tumour cell lines, its possible synergism effect in association with PI3K and HDAC-6 inhibitors, and its drug metabolism and pharmacokinetics (DMPK) in vitro profile. Our studies have demonstrated that LASSBio-2208 has moderate cytotoxic potency on breast cancer cell line MCF-7 (IC50 = 23 µM), human leukaemia cell line CCRF-CEM (IC50 = 8.54 µM) and T lymphoblast cell line MOLT-4 (IC50 = 7.15 µM), with no cytotoxic effect on human peripheral blood mononuclear cells (hPBMC). In addition, it has a good antiproliferative effect on MCF-7 cells (IC50 = 5.44 µM), low absorption by parallel artificial membrane permeability-gastrointestinal tract (PAMPA-GIT) and low permeation by parallel artificial membrane permeability-blood-brain barrier (BBB) (PAMPA-BBB), exhibiting high metabolic stability in rat plasma. Moreover, LASSBio-2208 exhibited synergism when combined with getadolisib and tubastatin A, using the concentrations corresponding to their CC50 values on MOLT-4 and CCRF-CEM cells.

2-Arylpropionic Acid Pyrazolamides as Cannabinoid CB2 Receptor Inverse Agonists Endowed with Anti-Inflammatory Properties.

Among the most recent proposals regarding the mechanism of action of dipyrone, the modulation of cannabinoid receptors CB1 and CB2 appears to be a promising hypothesis. In this context, the present work describes a series of five novel pyrazolamides (7-11) designed as molecular hybrids of dipyrone metabolites and NSAIDs, such as ibuprofen and flurbiprofen. Target compounds were obtained in good overall yields (50-80%) by classical amide coupling between 4-aminoantipyrine and arylacetic or arylpropionic acids, followed in some cases by N-methylation of the amide group. The compounds presented good physicochemical properties in addition to stability to chemical (pH 2 and 7.4) and enzymatic (plasma esterases) hydrolysis and showed medium to high gastrointestinal and BBB permeabilities in the PAMPA assay. When subjected to functional testing on CB1- or CB2-transfected cells, compounds demonstrated an inverse agonist profile on CB2 receptors and the further characterization of compound LASSBio-2265 (11) revealed moderate binding affinity to CB2 receptor (Ki = 16 µM) with an EC50 = 0.36 µM (Emax = 63%). LASSBio-2265 (11) (at 1, 3, and 10 mg/kg p.o.) was investigated in the formalin test in mice and a remarkable analgesic activity in the late inflammatory phase was observed, suggesting it could be promising for the treatment of pain syndromes associated with chronic inflammatory diseases.

Multitarget Inhibition of Histone Deacetylase (HDAC) and Phosphatidylinositol-3-kinase (PI3K): Current and Future Prospects.

The discovery of histone deacetylase (HDAC) inhibitors is a hot topic in the medicinal chemistry community regarding cancer research. This is related primarily to two factors: success in the clinic, e. g., the four FDA-approved HDAC inhibitors, and strong versatility to combine their pharmacophoric features to design new hybrid compounds with multitarget profiles. Thus, the selection of adequate pharmacophores to combine, i. e., combining targets that can result in a synergistic effect, is desirable, as it increases the probability of discovering a new useful therapeutic strategy. In this work, we highlight the design of multitarget HDAC/PI3K inhibitors. Although this approach is still in its early stages, many significant works have described the design and pharmacological evaluation of this new promising class of multitarget inhibitors, where compound CUDC-907, which is already in clinical trials, stands out. Therefore, the question emerges of whether there still space for the design and evaluation of new multitarget HDAC/PI3K inhibitors. When considering the selectivity profile of the described multitarget compounds, the answer appears to be in the affirmative, especially since the first examples of compounds with a certain selectivity profile only recently appeared in 2020.

Histone deacetylases as targets for the treatment of neurodegenerative disorders: Challenges and future opportunities.

Despite the applicability of histone deacetylase inhibitors (HDACis) for cancer treatment, several works in the literature have shown that these inhibitors can be used in several other diseases, such as neurodegenerative diseases (NDs). This review begins by discussing the signaling pathways of HDACs, focused on the context of NDs, presenting a discussion about the pharmacophoric features of HDACis and crystal structure analysis and discussing interesting case studies from the literature about the development of HDACis. Additionally, a discussion about the consequences of isoform-selective inhibition vs pan-HDACis on neurotoxic effects and clinical trial investigations of HDACis for NDs is also presented. Finally, we describe our perspective related to the future use of these inhibitors in the pharmacotherapy of NDs.

Synthesis and trypanocidal activity of novel pyridinyl-1,3,4-thiadiazole derivatives.

Herein, we present the design, synthesis and trypanocidal evaluation of sixteen new 1,3,4-thiadiazole derivatives from N-aminobenzyl or N-arylhydrazone series. All derivatives were assayed against the trypomastigote form of Trypanosoma cruzi, showing IC50 values ranging from 3 to 226 µM, and a better trypanocidal profile was demonstrated for the 1,3,4-thiadiazole-N-arylhydrazones (3a-g). In this series, the 2-pyridinyl fragment bound to the imine subunit of the hydrazine moiety presented pharmacophoric behavior for trypanocidal activity. Compounds 2a, 11a and 3e presented remarkable activity and excellent selectivity indexes. Compound 2a was also active against the intracellular amastigote form of T. cruzi. Moreover, its corresponding hydrochloride, compound 11a, showed the most promising profile, producing phenotypic changes similar to those caused by posaconazole, a well-known inhibitor of sterol biosynthesis. Thus, 1,3,4-thiadiazole derivative 11a could be considered a good prototype for the development of new drug candidates for Chagas disease therapy.

Design, Synthesis, and Pharmacological Evaluation of First-in-Class Multitarget N-Acylhydrazone Derivatives as Selective HDAC6/8 and PI3Kα Inhibitors.

Targeting histone deacetylases (HDACs) and phosphatidylinositol 3-kinases (PI3Ks) is a very promising approach for cancer treatment. This manuscript describes the design, synthesis, in vitro pharmacological profile, and molecular modeling of a novel class of N-acylhydrazone (NAH) derivatives that act as HDAC6/8 and PI3Kα dual inhibitors. The surprising selectivity for PI3Kα may be related to differences in the conformation in the active site. Cellular studies showed that these compounds act in HDAC6 inhibition and the PI3/K/AKT/mTOR pathway. The compounds that are selective for inhibition of HDAC6/8 and inhibit PI3Kα show potential for the treatment of cancer.

Identification of Novel Functionalized Carbohydrazonamides Designed as Chagas Disease Drug Candidates.

BACKGROUND: Although several research efforts have been made worldwide to discover novel drug candidates for the treatment of Chagas disease, the nitroimidazole drug benznidazol remains the only therapeutic alternative in the control of this disease. However, this drug presents reduced efficacy in the chronic form of the disease and limited safety after long periods of administration, making it necessary to search for new, more potent and safe prototypes. OBJECTIVE: We described herein the synthesis and the trypanocidalaction of new functionalized carbohydrazonamides (2-10) against trypomastigote forms of Trypanosoma cruzi. METHODS: These compounds were designed through the application of molecular hybridization concept between two potent anti-T. cruzi prototypes, the nitroimidazole derivative megazol (1) and the cinnamyl N-acylhydrazone derivative (14) which have been shown to be twice as potent in vitro as benznidazole. RESULTS: The most active compounds were the (Z)-N'-((E)-3-(4-nitrophenyl)-acryloyl)-1-methyl-5- nitro-1H-imidazol-2-carbohydrazonamide (6) (IC50=9.50 µM) and the (Z)-N'-((E)-3-(4- hydroxyphe-nyl)-acryloyl)-1-methyl-5-nitro-1H-imidazol-2-carbohydrazonamide (8) (IC50=12.85 µM), which were almost equipotent to benznidazole (IC50=10.26 µM) used as standard drug. The removal of the amine group attached to the imine subunit in the corresponding N-acylhydrazone derivatives (11-13) resulted in less potent or inactive compounds. The para-hydroxyphenyl derivative (8) presented also a good selectivity index (SI = 32.94) when tested against mammalian cells from Swiss mice. CONCLUSION: The promising trypanocidal profile of new carbohydrazonamide derivatives (6) and (8) was characterized. These compounds have proved to be a good starting point for the design of more effective trypanocidal drug candidates.

The Chalcone Lonchocarpin Inhibits Wnt/ß-Catenin Signaling and Suppresses Colorectal Cancer Proliferation.

The deregulation of the Wnt/ß-catenin signaling pathway is a central event in colorectal cancer progression, thus a promising target for drug development. Many natural compounds, such as flavonoids, have been described as Wnt/ß-catenin inhibitors and consequently modulate important biological processes like inflammation, redox balance, cancer promotion and progress, as well as cancer cell death. In this context, we identified the chalcone lonchocarpin isolated from Lonchocarpus sericeus as a Wnt/ß-catenin pathway inhibitor, both in vitro and in vivo. Lonchocarpin impairs ß-catenin nuclear localization and also inhibits the constitutively active form of TCF4, dnTCF4-VP16. Xenopus laevis embryology assays suggest that lonchocarpin acts at the transcriptional level. Additionally, we described lonchocarpin inhibitory effects on cell migration and cell proliferation on HCT116, SW480, and DLD-1 colorectal cancer cell lines, without any detectable effects on the non-tumoral intestinal cell line IEC-6. Moreover, lonchocarpin reduces tumor proliferation on the colorectal cancer AOM/DSS mice model. Taken together, our results support lonchocarpin as a novel Wnt/ß-catenin inhibitor compound that impairs colorectal cancer cell growth in vitro and in vivo.

The Use of Conformational Restriction in Medicinal Chemistry.

During the early preclinical phase, from hit identification and optimization to a lead compound, several medicinal chemistry strategies can be used to improve potency and/or selectivity. The conformational restriction is one of these approaches. It consists of introducing some specific structural constraints in a lead candidate to reduce the overall number of possible conformations in order to favor the adoption of a bioactive conformation and, as a consequence, molecular recognition by the target receptor. In this work, we focused on the application of the conformational restriction strategy in the last five years for the optimization of hits and/or leads of several important classes of therapeutic targets in the drug discovery field. Thus, we recognize the importance of several kinase inhibitors to the current landscape of drug development for cancer therapy and the use of G-protein Coupled Receptor (GPCR) modulators. Several other targets are also highlighted, such as the class of epigenetic drugs. Therefore, the possibility of exploiting conformational restriction as a tool to increase the potency and selectivity and promote changes in the intrinsic activity of some ligands intended to act on many different targets makes this strategy of structural modification valuable for the discovery of novel drug candidates.

Evaluation of Functional Selectivity of Haloperidol, Clozapine, and LASSBio-579, an Experimental Compound With Antipsychotic-Like Actions in Rodents, at G Protein and Arrestin Signaling Downstream of the Dopamine D2 Receptor.

LASSBio-579, an N-phenylpiperazine antipsychotic lead compound, has been previously reported as a D2 receptor (D2R) ligand with antipsychotic-like activities in rodent models of schizophrenia. In order to better understand the molecular mechanism of action of LASSBio-579 and of its main metabolite, LQFM 037, we decided to address the hypothesis of functional selectivity at the D2R. HEK-293T cells transiently coexpressing the human long isoform of D2 receptor (D2LR) and bioluminescence resonance energy transfer (BRET)-based biosensors were used. The antagonist activity was evaluated using different concentrations of the compounds in the presence of a submaximal concentration of dopamine (DA), after 5 and 20 min. For both signaling pathways, haloperidol, clozapine, and our compounds act as DA antagonists in a concentration-dependent manner, with haloperidol being by far the most potent, consistent with its nanomolar D2R affinity measured in binding assays. In our experimental conditions, only haloperidol presented a robust functional selectivity, being four- to fivefold more efficient for inhibiting translocation of ß-arrestin-2 (ß-arr2) than for antagonizing Gi activation. Present data are the first report on the effects of LASSBio-579 and LQFM 037 on the ß-arr2 signaling pathway and further illustrate that the functional activity could vary depending on the assay conditions and approaches used.

Duvelisib: A 2018 Novel FDA-Approved Small Molecule Inhibiting Phosphoinositide 3-Kinases.

Duvelisib (Copiktra®) is a dual inhibitor of phosphoinositide 3-kinases (PI3Kδ and PI3Kγ). In 2018, duvelisib was first approved by the Food and Drug Administration (FDA) for the treatment of adult patients with relapsed or refractory chronic lymphocytic leukaemia (CLL)/ small lymphocytic lymphoma (SLL) after at least two prior therapies. Duvelisib has also been approved under accelerated track for relapsed or refractory follicular lymphoma (FL) after at least two prior systemic therapies. In this review, we provide a series of information about duvelisib, such as the development of clinical trials for LLC/SLL and FL and the steps used for its synthesis.

Design, Synthesis, Experimental and Theoretical Characterization of a New Multitarget 2-Thienyl-N-Acylhydrazone Derivative.

Pulmonary arterial hypertension (PAH) is a chronic cardiovascular disease that displays inflammatory components, which contributes to the difficulty of adequate treatment with the available therapeutic arsenal. In this context, the N-acylhydrazone derivative LASSBio-1359 was previously described as a multitarget drug candidate able to revert the events associated with the progression of PAH in animal models. However, in spite of having a dual profile as PDE4 inhibitor and adenosine A2A receptor agonist, LASSBio-1359 does not present balanced potencies in the modulation of these two targets, which difficult its therapeutic use. In this paper, we describe the design concept of LASSBio-1835, a novel structural analogue of LASSBio-1359, planned by exploiting ring bioisosterism. Using X-ray powder diffraction, calorimetric techniques, and molecular modeling, we clearly indicate the presence of a preferred synperiplanar conformation at the amide function, which is fixed by an intramolecular 1,5-N∙∙∙S σ-hole intramolecular interaction. Moreover, the evaluation of LASSBio-1835 (4) as a PDE4 inhibitor and as an A2A agonist confirms it presents a more balanced dual profile, being considered a promising prototype for the treatment of PAH.

N-Acylhydrazones as drugs.

Over the last two decades, N-acylhydrazone (NAH) has been proven to be a very versatile and promising motif in drug design and medicinal chemistry. Herein, we discuss the current and future challenges in the emergence of bioactive NAH-based scaffolds and to developing strategies to overcome the failures in drug discovery. The NAH-related approved drugs nitrofurazone, nitrofurantoin, carbazochrome, testosterone 17-enanthate 3-benzilic acid hydrazine, nifuroxazide, dantrolene, and azumolene are already used as therapeutics in various countries. PAC-1 is an NAH-based therapeutic agent that entered clinical trials in 2015. Another NAH-derived scaffold, LASSBio-294, is in preclinical trials. This review highlights the detailed comprehensive assessment and therapeutic landscape of bioactive NAH motif scaffolds in preclinical and clinical studies published to date and their promise and associated challenges in current and future drug discovery of NAH-based drugs that will progress to clinical use.

Cardioprotection Induced by Activation of GPER in Ovariectomized Rats With Pulmonary Hypertension.

Pulmonary hypertension (PH) is a disease of women (female-to-male ratio 4:1), and is associated with cardiac and skeletal muscle dysfunction. Herein, the activation of a new estrogen receptor (GPER) by the agonist G1 was evaluated in oophorectomized rats with monocrotaline (MCT)-induced PH. Depletion of estrogen was induced by bilateral oophorectomy (OVX) in Wistar rats. Experimental groups included SHAM or OVX rats that received a single intraperitoneal injection of MCT (60 mg/kg) for PH induction. Animals received s.c. injection of either vehicle or G1, a GPER agonist, (400 µg/kg/day) for 14 days after the onset of disease. Rats with PH exhibited exercise intolerance and cardiopulmonary alterations, including reduced pulmonary artery flow, biventricular remodeling, and left ventricular systolic and diastolic dysfunction. The magnitude of these PH-induced changes was significantly greater in OVX versus SHAM rats. G1 treatment reversed both cardiac and skeletal muscle functional aberrations caused by PH in OVX rats. G1 reversed PH-related cardiopulmonary dysfunction and exercise intolerance in female rats, a finding that may have important implications for the ongoing clinical evaluation of new drugs for the treatment of the disease in females after the loss of endogenous estrogens.

Different mol-ecular conformations in the crystal structures of three 5-nitro-imidazolyl derivatives.

The crystal structures of (E)-1-methyl-5-nitro-1H-imidazole-2-carbaldehyde O-benzyl-oxime, C12H12N4O3, (I), (E)-1-methyl-5-nitro-1H-imidazole-2-carb-alde-hyde O-(4-fluoro-benz-yl) oxime, C12H11FN4O3, (II), and (E)-1-methyl-5-nitro-1H-imidazole-2-carbaldehyde O-(4-bromo-benz-yl) oxime, C12H11BrN4O3, (III), are described. The dihedral angle between the ring systems in (I) is 49.66 (5)° and the linking Nm-C-C=N (m = methyl-ated) bond shows an anti conformation [torsion angle = 175.00 (15)°]. Compounds (II) and (III) are isostructural [dihedral angle between the aromatic rings = 8.31 (5)° in (II) and 5.34 (15)° in (III)] and differ from (I) in showing a near-syn conformation for the Nm-C-C=N linker [torsion angles for (II) and (III) = 17.64 (18) and 8.7 (5)°, respectively], which allows for the occurrence of a short intra-molecular C-H⋯N contact. In the crystal of (I), C-H⋯N hydrogen bonds link the mol-ecules into [010] chains, which are cross-linked by very weak C-H⋯O bonds into (100) sheets. Weak aromatic π-π stacking inter-actions occur between the sheets. The extended structures of (II) and (III) feature several C-H⋯N and C-H⋯O hydrogen bonds, which link the mol-ecules into three-dimensional networks, which are consolidated by aromatic π-π stacking inter-actions. Conformational energy calculations and Hirshfeld fingerprint analyses for (I), (II) and (III) are presented and discussed.

NF-κB-IKKß Pathway as a Target for Drug Development: Realities, Challenges and Perspectives.

BACKGROUND: Nuclear factor κB (NF-κB) comprises a family of proteins that act as transcription factors promoting the expression of many genes. Activation of NF-κB biochemical cascades is associated with the regulation of innate and adaptive immune responses and inflammation, among other physiological responses. However, genetic abnormalities and continuous stimulation of the NF- κB-IKKß pathway are directly related to many types of inflammatory and autoimmune diseases, as well as to the genesis and survival of tumor cells. OBJECTIVES: Inhibition of the NF-κB-IKKß cascade can be considered an attractive therapeutic method for the genesis of new prototypes to combat these chronic multifactorial diseases. RESULTS: This review describes some prototypes and drugs that act to inhibit the NF-κB-IKKß pathway, highlighting the realities, challenges and perspectives for therapeutic use. CONCLUSION: Although only proteasome inhibitors, such as bortezomib and carfilzomib, are a reality as therapeutically useful drugs among the known modulators of possible targets in the NF-κB-IKKß pathway, some other prototypes described as IKKß inhibitors have entered clinical stages as drug candidates for the control of inflammatory diseases. It is important to note that some classical drugs available on the pharmaceutical market, such as acetylsalicylic acid, were also described more recently as NF-κB pathway modulators as IKKß inhibitors.

Discovery of naphthyl-N-acylhydrazone p38α MAPK inhibitors with in vivo anti-inflammatory and anti-TNF-α activity.

Protein kinases constitute attractive therapeutic targets for development of new prototypes to treat different chronic diseases. Several available drugs, like tinibs, are tyrosine kinase inhibitors; meanwhile, inhibitors of serine/threonine kinases, such as mitogen-activated protein kinase (MAPK), are still trying to overcome some problems in one of the steps of clinical development to become drugs. So, here we reported the synthesis, the in vitro kinase inhibitory profile, docking studies, and the evaluation of anti-inflammatory profile of new naphthyl-N-acylhydrazone derivatives using animal models. Although all tested compounds (3a-d) have been characterized as p38α MAPK inhibitors and have showed in vivo anti-inflammatory action, LASSBio-1824 (3b) presented the best performance as p38α MAPK inhibitor, with IC50  = 4.45 µm, and also demonstrated to be the most promising anti-inflammatory prototype, with good in vivo anti-TNF-α profile after oral administration.

LASSBio-897 Reduces Lung Injury Induced by Silica Particles in Mice: Potential Interaction with the A2A Receptor.

Silicosis is a lethal fibro-granulomatous pulmonary disease highly prevalent in developing countries, for which no proper therapy is available. Among a small series of N-acylhydrazones, the safrole-derived compound LASSBio-897 (3-thienylidene-3, 4-methylenedioxybenzoylhydrazide) raised interest due to its ability to bind to the adenosine A2A receptor. Here, we evaluated the anti-inflammatory and anti-fibrotic potential of LASSBio-897, exploring translation to a mouse model of silicosis and the A2A receptor as a site of action. Pulmonary mechanics, inflammatory, and fibrotic changes were assessed 28 days after intranasal instillation of silica particles in Swiss-Webster mice. Glosensor cAMP HEK293G cells, CHO cells stably expressing human adenosine receptors and ligand binding assay were used to evaluate the pharmacological properties of LASSBio-897 in vitro. Molecular docking studies of LASSBio-897 were performed using the genetic algorithm software GOLD 5.2. We found that the interventional treatment with the A2A receptor agonist CGS 21680 reversed silica particle-induced airway hyper-reactivity as revealed by increased responses of airway resistance and lung elastance following aerosolized methacholine. LASSBio-897 (2 and 5 mg/kg, oral) similarly reversed pivotal lung pathological features of silicosis in this model, reducing levels of airway resistance and lung elastance, granuloma formation and collagen deposition. In competition assays, LASSBio-897 decreased the binding of the selective A2A receptor agonist [3H]-CGS21680 (IC50 = 9.3 µM). LASSBio-897 (50 µM) induced modest cAMP production in HEK293G cells, but it clearly synergized the cAMP production by adenosine in a mechanism sensitive to the A2A antagonist SCH 58261. This synergism was also seen in CHO cells expressing the A2A, but not those expressing A2B, A1 or A3 receptors. Based on the evidence that LASSBio-897 binds to A2A receptor, molecular docking studies were performed using the A2A receptor crystal structure and revealed possible binding modes of LASSBio-897 at the orthosteric and allosteric sites. These findings highlight LASSBio-897 as a lead compound in drug development for silicosis, emphasizing the role of the A2A receptor as its putative site of action.

LASSBio-1422: a new molecular scaffold with efficacy in animal models of schizophrenia and disorders of attention and cognition.

Aiming to identify new antipsychotic lead-compounds, our group has been working on the design and synthesis of new N-phenylpiperazine derivatives. Here, we characterized LASSBio-1422 as a pharmacological prototype of this chemical series. Adult male Wistar rats and CF1 mice were used for in-vitro and in-vivo assays, respectively. LASSBio-1422 [1 and 5 mg/kg, postoperatively (p.o.)] inhibited apomorphine-induced climbing as well as ketamine-induced hyperlocomotion (1 and 5 mg/kg, p.o.), animal models predictive of efficacy on positive symptoms. Furthermore, LASSBio-1422 (5 mg/kg, p.o.) prevented the prepulse impairment induced by apomorphine, (±)-2,5-dimethoxy-4-iodoamphetamine, and ketamine, as well as the memory impairment induced by ketamine in the novel object-recognition task at the acquisition, consolidation, and retrieval phases of memory formation. Potential extrapyramidal side-effects and sedation were assessed by catatonia, rota-rod, locomotion, and barbiturate sleeping time, and LASSBio-1422 (15 mg/kg, p.o.) did not affect any of the parameters observed. Binding assays showed that LASSBio-1422 has a binding profile different from the known atypical antipsychotic drugs: it does not bind to AMPA, kainate, N-methyl-D-aspartate, glicine, and mGluR2 receptors and has low or negligible affinity for D1, D2, and 5-HT2A/C receptors, but high affinity for D4 receptors (Ki=0.076 µmol/l) and, to a lesser extent, for 5-HT1A receptors (Ki=0.493 µmol/l). The antagonist action of LASSBio-1422 at D4 receptors was assessed through the classical GTP-shift assay. In conclusion, LASSBio-1422 is effective in rodent models of positive and cognitive symptoms of schizophrenia and its ability to bind to D4 and 5-HT1A receptors may at least in part explain its effects in these animal models.