New Smoothened ligands based on the purine scaffold as potential agents for treating pancreatic cancer.

Aberrant activation of the Hedgehog (Hh) signalling pathway has been associated with the development and progression of pancreatic cancer. For this reason, blockade of Hh pathway by inhibitors targeting the G protein-coupled receptor Smoothened (SMO) has been considered as a therapeutic target for the treatment of this cancer. In our previous work, we obtained a new SMO ligand based on a purine scaffold (compound I), which showed interesting antitumor activity in several cancer cell lines. In this work, we report the design and synthesis of 17 new purine derivatives, some of which showed high cytotoxic effect on Mia-PaCa-2 (Hh-dependent pancreatic cancer cell lines) and low toxicity on non-neoplastic HEK-293 cells compared with gemcitabine, such as 8f, 8g and 8h (IC50 = 4.56, 4.11 and 3.08 µM, respectively). Two of these purines also showed their ability to bind to SMO through NanoBRET assays (pKi = 5.17 for 8f and 5.01 for 8h), with higher affinities to compound I (pKi = 1.51). In addition, docking studies provided insight the purine substitution pattern is related to the affinity on SMO. Finally, studies of Hh inhibition for selected purines, using a transcriptional functional assay based on luciferase activity in NIH3T3 Shh-Light II cells, demonstrated that 8g reduced GLI activity with a IC50 = 6.4 µM as well as diminished the expression of Hh target genes in two specific Hh-dependent cell models, Med1 cells and Ptch1-/- mouse embryonic fibroblasts. Therefore, our results provide a platform for the design of SMO ligands that could be potential selective cytotoxic agents for the treatment of pancreatic cancer.

A New Smoothened Antagonist Bearing the Purine Scaffold Shows Antitumour Activity In Vitro and In Vivo.

The Smoothened (SMO) receptor is the most druggable target in the Hedgehog (HH) pathway for anticancer compounds. However, SMO antagonists such as vismodegib rapidly develop drug resistance. In this study, new SMO antagonists having the versatile purine ring as a scaffold were designed, synthesised, and biologically tested to provide an insight to their mechanism of action. Compound 4s was the most active and the best inhibitor of cell growth and selectively cytotoxic to cancer cells. 4s induced cell cycle arrest, apoptosis, a reduction in colony formation and downregulation of PTCH and GLI1 expression. BODIPY-cyclopamine displacement assays confirmed 4s is a SMO antagonist. In vivo, 4s strongly inhibited tumour relapse and metastasis of melanoma cells in mice. In vitro, 4s was more efficient than vismodegib to induce apoptosis in human cancer cells and that might be attributed to its dual ability to function as a SMO antagonist and apoptosis inducer.

New 2,6,9-trisubstituted purine derivatives as Bcr-Abl and Btk inhibitors and as promising agents against leukemia.

Bcr-Abl and Btk kinases are among the targets that have been considered for the treatment of leukemia. Therefore, several strategies have focused on the use of inhibitors as chemotherapeutic tools to treat these types of leukemia, such as imatinib (for Bcr-Abl) or ibrutinib (for Btk). However, the efficacy of these drugs has been reduced due to resistance mechanisms, which have motivated the development of new and more effective compounds. In this study, we designed, synthesized and evaluated 2,6,9-trisubstituted purine derivatives as novel Bcr-Abl and Btk inhibitors. We identified 5c and 5d as potent inhibitors of both kinases (IC50 values of 40 nM and 0.58/0.66 µM for Abl and Btk, respectively). From docking and QSAR analyses, we concluded that fluorination of the arylpiperazine system is detrimental to the activity against two kinases, and we also validated our hypothesis that the substitution on the 6-phenylamino ring is important for the inhibition of both kinases. In addition, our studies indicated that most compounds could suppress the proliferation of leukemia and lymphoma cells (HL60, MV4-11, CEM, K562 and Ramos cells) at low micromolar concentrations in vitro. Finally, we preliminarily demonstrated that 5c inhibited the downstream signaling of both kinases in the respective cell models. Therefore, 5c or 5d possessed potency to be further optimized as anti-leukemia drugs by simultaneously inhibiting the Bcr-Abl and Btk kinases.

New aryloxy-quinone derivatives with promising activity on Trypanosoma cruzi.

Continuing with a program to develop new quinone derivatives as biologically active compounds, we designed and synthesized a new series of aryloxy-quinones, which were evaluated in vitro against Trypanosoma cruzi in epimastigote form. Chemical modifications in three specific moieties on the aryloxy-quinone core were considered for developing new anti-T. cruzi agents. The majority of our new quinones showed higher potency (IC50 values of <0.70 µM) than nifurtimox, a known pharmaceutical used as a baseline drug (IC50 values of 7.00 µM); however, only two of them elicited higher selectivity than nifurtimox against Vero cells. A structure-activity relationship analysis provided information about the stereoelectronic features of these compounds, which are responsible for an increase in trypanosomicidal activity. Using a pharmacophore model, we mapped the substitution patterns of the five pharmacophoric features of trypanosomicidal activity. We chose the Epc1 compounds and found no relationship with the trypanosomicidal effects. These results provided useful information about the structural characteristics for developing new aryloxy-quinones with higher potency against the protozoan parasite T. cruzi.

Nature-inspired pyrrolo[2,3-d]pyrimidines targeting the histamine H3 receptor.

Inspired by marine compounds the derivatization of the natural pyrrolo[2,3-d]pyrimidine lead scaffold led to a series of novel compounds targeting the histamine H3 receptor. The focus was set on improved binding towards the receptor and to establish an initial structure-activity relationship for this compound class based on the lead structure (compound V, Ki value of 126 nM). As highest binding affinities were found with 1,4-bipiperidines as basic part of the ligands, further optimization was focused on 4-([1,4'-bipiperidin]-1'-yl)-pyrrolo[2,3-d]pyrimidines. Related pyrrolo[2,3-d]pyrimidines that were isolated from marine sponges like 4-amino-5-bromopyrrolo[2,3-d]pyrimidine (compound III), showed variations in halogenation pattern, though in a next step the impact of halogenation at 2-position was evaluated. The chloro variations did not improve the affinity compared to the dehalogenated compounds. However, the simultaneous introduction of lipophilic cores with electron-withdrawing substitution patterns in 7-position and dehalogenation at 2-position (11b, 12b) resulted in compounds with significantly higher binding affinities (Ki values of 7 nM and 6 nM, respectively) than the initial lead structure compound V. The presented structures allow for a reasonable structure-activity relationship of pyrrolo[2,3-d]pyrimidines as histamine H3 receptor ligands and yielded novel lead structures within the natural compound library against this target.

Promising 2,6,9-Trisubstituted Purine Derivatives for Anticancer Compounds: Synthesis, 3D-QSAR, and Preliminary Biological Assays.

We designed, synthesized, and evaluated novel 2,6,9-trisubstituted purine derivatives for their prospective role as antitumor compounds. Using simple and efficient methodologies, 31 compounds were obtained. We tested these compounds in vitro to draw conclusions about their cell toxicity on seven cancer cells lines and one non-neoplastic cell line. Structural requirements for antitumor activity on two different cancer cell lines were analyzed with SAR and 3D-QSAR. The 3D-QSAR models showed that steric properties could better explain the cytotoxicity of compounds than electronic properties (70% and 30% of contribution, respectively). From this analysis, we concluded that an arylpiperazinyl system connected at position 6 of the purine ring is beneficial for cytotoxic activity, while the use of bulky systems at position C-2 of the purine is not favorable. Compound 7h was found to be an effective potential agent when compared with a currently marketed drug, cisplatin, in four out of the seven cancer cell lines tested. Compound 7h showed the highest potency, unprecedented selectivity, and complied with all the Lipinski rules. Finally, it was demonstrated that 7h induced apoptosis and caused cell cycle arrest at the S-phase on HL-60 cells. Our study suggests that substitution in the purine core by arylpiperidine moiety is essential to obtain derivatives with potential anticancer activity.

New lead elements for histamine H3 receptor ligands in the pyrrolo[2,3-d]pyrimidine class.

This work describes the microwave assisted synthesis of twelve novel histamine H3 receptor ligands. They display pyrrolo[2,3-d]pyrimidine derivatives with rigidized aliphatic amines as warheads. The compounds were screened for H3R and H4R binding affinities in radioligand displacement assays and the most potent compounds were evaluated for H3R binding properties in vitro and in docking studies. The combination of a rigidized H3R warhead and the pyrrolo[2,3-d]pyrimidine scaffold resulted in selective activity at the H3 receptor with a pKi value of 6.90 for the most potent compound. A bipiperidine warhead displayed higher affinity than a piperazine or morpholine motif, while a naphthyl moiety in the arbitrary region increased affinity compared to a phenyl derivative. The compounds can be starting points for novel, simply synthesized histamine H3 receptor ligands.

Fluorescence properties of aurone derivatives: an experimental and theoretical study with some preliminary biological applications.

In this paper, we explored the fluorescence properties of eight aurone derivatives bearing methoxy groups and bromine atoms as substituents in the benzene rings. All derivatives showed strong solvatochromic absorption and emission properties in solvents of different polarities. Some of them showed high fluorescence quantum yields, which make them potential compounds for sensing applications. The position of the methoxy groups in the benzofuranone moiety and the presence of bromine atoms in the benzene ring had a strong influence on the fluorescence behaviour of the aurones. DFT calculations allowed us to explain the emission properties of aurones and their solvatochromism, which was related to an excited state with strong charge-transfer character. Aurone 4 has the most promising characteristics showing a large difference in the quantum yields and large Stokes shifts depending on the solvent polarities. These results prompted us to explore some preliminary biological applications for aurone 4 such as the sensing of hydrophobic pockets of a protein and its thermotropic behaviour in liposomes.

Design, synthesis, biological evaluation and binding mode modeling of benzimidazole derivatives targeting the cannabinoid receptor type 1.

A series of N-acyl-2,5-dimethoxyphenyl-1H-benzimidazoles were designed based on a CoMFA model for cannabinoid receptor type 1 (CB1) ligands. Compounds were synthesized and radioligand binding affinity assays were performed. Eight novel benzimidazoles exhibited affinity for the CB1 receptor in the nanomolar range, and the most promising derivative compound 5 displayed a K(i) value of 1.2 nM when compared to CP55,940. These results confirm our previously reported QSAR model on benzimidazole derivatives, providing new information for the development of small molecules with high CB1 affinity.

Synthesis and pharmacophore modelling of 2,6,9-trisubstituted purine derivatives and their potential role as apoptosis-inducing agents in cancer cell lines.

A series of 2,6,9-trisubstituted purine derivatives have been synthesized and investigated for their potential role as antitumor agents. Twelve compounds were obtained by a three step synthetic procedure using microwave irradiation in a pivotal step. All compounds were evaluated in vitro to determine their potential effect on cell toxicity by the MTT method and flow cytometry analysis on four cancer cells lines and Vero cells. Three out of twelve compounds were found to be promising agents compared to a known and effective anticancer drug, etoposide, in three out of four cancer cell lines assayed with considerable selectivity. Preliminary flow cytometry data suggests that compounds mentioned above induce apoptosis on these cells. The main structural requirements for their activity for each cancer cell line were characterized with a preliminary pharmacophore model, which identified aromatic centers, hydrogen acceptor/donor center and a hydrophobic area. These features were consistent with the cytotoxic activity of the assayed compounds.

Synthesis and biological characterization of new aryloxyindole-4,9-diones as potent trypanosomicidal agents.

A new indole-4,9-dione and their phenoxy derivatives were synthesized and evaluated in vitro against the epimastigote form of Trypanosoma cruzi, Y strain. All of these novel compounds were found to be extremely potent and selective that the standard drug nifurtimox. Interestingly, phenoxyindole-4,9-dione 9d displayed excellent nanomolar inhibitory activity, IC50=20 nM, and high selectivity index, SI=625. In silico studies using MOE program were performed to generate a preliminary pharmacophore model.

2-Phenylaminonaphthoquinones and related compounds: synthesis, trypanocidal and cytotoxic activities.

A series of new 2-aminonaphthoquinones and related compounds were synthesized and evaluated in vitro as trypanocidal and cytotoxic agents. Some tested compounds inhibited epimastigote growth and trypomastigote viability. Several compounds showed similar or higher activity and selectivity as compared with current trypanocidal drug, nifurtimox. Compound 4l exhibit higher selectivity than nifurtimox against Trypanosoma cruzi in comparison with Vero cells. Some of the synthesized quinones were tested against cancer cells and normal fibroblasts, showing that certain chemical modifications on the naphthoquinone moiety induce and excellent increase the selectivity index of the cytotoxicity (4g and 10). The results presented here show that the anti-T. cruzi activity of 2-aminonaphthoquinones derivatives can be improved by the replacement of the benzene ring by a pyridine moiety. Interestingly, the presence of a chlorine atom at C-3 and a highly lipophilic alkyl group or aromatic ring are newly observed elements that should lead to the discovery of more selective cytotoxic and trypanocidal compounds.

New Inhibitors of Bcr-Abl Based on 2,6,9-Trisubstituted Purine Scaffold Elicit Cytotoxicity in Chronic Myeloid Leukemia-Derived Cell Lines Sensitive and Resistant to TKIs.

Bcr-Abl is an oncoprotein with aberrant tyrosine kinase activity involved in the progression of chronic myeloid leukemia (CML) and has been targeted by inhibitors such as imatinib and nilotinib. However, despite their efficacy in the treatment of CML, a mechanism of resistance to these drugs associated with mutations in the kinase region has emerged. Therefore, in this work, we report the synthesis of 14 new 2,6,9-trisubstituted purines designed from our previous Bcr-Abl inhibitors. Here, we highlight 11b, which showed higher potency against Bcr-Abl (IC50 = 0.015 µM) than imatinib and nilotinib and exerted the most potent antiproliferative properties on three CML cells harboring the Bcr-Abl rearrangement (GI50 = 0.7-1.3 µM). In addition, these purines were able to inhibit the growth of KCL22 cell lines expressing Bcr-AblT315I, Bcr-AblE255K, and Bcr-AblY253H point mutants in micromolar concentrations. Imatinib and nilotinib were ineffective in inhibiting the growth of KCL22 cells expressing Bcr-AblT315I (GI50 > 20 µM) compared to 11b-f (GI50 = 6.4-11.5 µM). Molecular docking studies explained the structure-activity relationship of these purines in Bcr-AblWT and Bcr-AblT315I. Finally, cell cycle cytometry assays and immunodetection showed that 11b arrested the cells in G1 phase, and that 11b downregulated the protein levels downstream of Bcr-Abl in these cells.

Photoaged polystyrene nanoplastics exposure results in reproductive toxicity due to oxidative damage in Caenorhabditis elegans.

The increase of plastic production together with the incipient reuse/recycling system has resulted in massive discards into the environment. This has facilitated the formation of micro- and nanoplastics (MNPs) which poses major risk for environmental health. Although some studies have investigated the effects of pristine MNPs on reproductive health, the effects of weathered MNPs have been poorly investigated. Here we show in Caenorhabditis elegans that exposure to photoaged polystyrene nanoplastics (PSNP-UV) results in worse reproductive performance than pristine PSNP (i.e., embryonic/larval lethality plus a decrease in the brood size, accompanied by a high number of unfertilized eggs), besides it affects size and locomotion behavior. Those effects were potentially generated by reactive products formed during UV-irradiation, since we found higher levels of reactive oxygen species and increased expression of GST-4 in worms exposed to PSNP-UV. Those results are supported by physical-chemical characterization analyses which indicate significant formation of oxidative degradation products from PSNP under UV-C irradiation. Our study also demonstrates that PSNP accumulate predominantly in the gastrointestinal tract of C. elegans (with no accumulation in the gonads), being completely eliminated at 96 h post-exposure. We complemented the toxicological analysis of PSNP/PSNP-UV by showing that the activation of the stress response via DAF-16 is dependent of the nanoplastics accumulation. Our data suggest that exposure to the wild PSNP, i.e., polystyrene nanoplastics more similar to those actually found in the environment, results in more important reprotoxic effects. This is associated with the presence of degradation products formed during UV-C irradiation and their interaction with biological targets.

Design, synthesis, binding and docking-based 3D-QSAR studies of 2-pyridylbenzimidazoles--a new family of high affinity CB1 cannabinoid ligands.

A series of novel 2-pyridylbenzimidazole derivatives was rationally designed and synthesized based on our previous studies on benzimidazole 14, a CB1 agonist used as a template for optimization. In the present series, 21 compounds displayed high affinities with Ki values in the nanomolar range. JM-39 (compound 39) was the most active of the series (KiCB1 = 0.53 nM), while compounds 31 and 44 exhibited similar affinities to WIN 55212-2. CoMFA analysis was performed based on the biological data obtained and resulted in a statistically significant CoMFA model with high predictive value (q2 = 0.710, r2 = 0.998, r2pred = 0.823).

Substituted Purines as High-Affinity Histamine H3 Receptor Ligands.

Continuing with our program to obtain new histamine H3 receptor (H3R) ligands, in this work we present the synthesis, H3R affinity and in silico studies of a series of eight new synthetically accessible purine derivatives. These compounds are designed from the isosteric replacement of the scaffold presented in our previous ligand, pyrrolo[2,3-d]pyrimidine ring, by a purine core. This design also considers maintaining the fragment of bipiperidine at C-4 and aromatic rings with electron-withdrawing groups at N-9, as these fragments are part of the proposed pharmacophore. The in vitro screening results show that two purine derivatives, 3d and 3h, elicit high affinities to the H3R (Ki values of 2.91 and 5.51 nM, respectively). Both compounds are more potent than the reference drug pitolisant (Ki 6.09 nM) and show low toxicity with in vitro models (IC50 > 30 µM on HEK-293, SH-SY5Y and HepG2 cell lines). Subsequently, binding modes of these ligands are obtained using a model of H3R by docking and molecular dynamics studies, thus determining the importance of the purine ring in enhancing affinity due to the hydrogen bonding of Tyr374 to the N-7 of this heterocycle. Finally, in silico ADME properties are predicted, which indicate a promising future for these molecules in terms of their physical−chemical properties, absorption, oral bioavailability and penetration in the CNS.

New Amino Naphthoquinone Derivatives as Anti-Trypanosoma cruzi Agents Targeting Trypanothione Reductase.

To develop novel chemotherapeutic alternatives for the treatment of Chagas disease, in this study, a set of new amino naphthoquinone derivatives were synthesised and evaluated in vitro on the epimastigote and trypomastigote forms of Trypanosoma cruzi strains (NINOA and INC-5) and on J774 murine macrophages. The design of the new naphthoquinone derivatives considered the incorporation of nitrogenous fragments with different substitution patterns present in compounds with activity on T. cruzi, and, thus, 19 compounds were synthesised in a simple manner. Compounds 2e and 7j showed the lowest IC50 values (0.43 µM against both strains for 2e and 0.19 µM and 0.92 µM for 7j). Likewise, 7j was more potent than the reference drug, benznidazole, and was more selective on epimastigotes. To postulate a possible mechanism of action, molecular docking studies were performed on T. cruzi trypanothione reductase (TcTR), specifically at a site in the dimer interface, which is a binding site for this type of naphthoquinone. Interestingly, 7j was one of the compounds that showed the best interaction profile on the enzyme; therefore, 7j was evaluated on TR, which behaved as a non-competitive inhibitor. Finally, 7j was predicted to have a good pharmacokinetic profile for oral administration. Thus, the naphthoquinone nucleus should be considered in the search for new trypanocidal agents based on our hit 7j.

Design, Synthesis, In Silico Studies and Inhibitory Activity towards Bcr-Abl, BTK and FLT3-ITD of New 2,6,9-Trisubstituted Purine Derivatives as Potential Agents for the Treatment of Leukaemia.

We report 31 new compounds designed, synthesized and evaluated on Bcr-Abl, BTK and FLT3-ITD as part of our program to develop 2,6,9-trisubstituted purine derivatives as inhibitors of oncogenic kinases. The design was inspired by the chemical structures of well-known kinase inhibitors and our previously developed purine derivatives. The synthesis of these purines was simple and used a microwave reactor for the final step. Kinase assays showed three inhibitors with high selectivity for each protein that were identified: 4f (IC50 = 70 nM for Bcr-Abl), 5j (IC50 = 0.41 µM for BTK) and 5b (IC50 = 0.38 µM for FLT-ITD). The 3D-QSAR analysis and molecular docking studies suggested that two fragments are potent and selective inhibitors of these three kinases: a substitution at the 6-phenylamino ring and the length and volume of the alkyl group at N-9. The N-7 and the N-methyl-piperazine moiety linked to the aminophenyl ring at C-2 are also requirements for obtaining the activity. Furthermore, most of these purine derivatives were shown to have a significant inhibitory effect in vitro on the proliferation of leukaemia and lymphoma cells (HL60, MV4-11, CEM, K562 and Ramos) at low concentrations. Finally, we show that the selected purines (4i, 5b and 5j) inhibit the downstream signalling of the respective kinases in cell models. Thus, this study provides new evidence regarding how certain chemical modifications of purine ring substituents provide novel inhibitors of target kinases as potential anti-leukaemia drugs.

Hepatoprotective species from the Chilean medicinal flora: Junellia spathulata (Verbenaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Chilean population relies on medicinal plants for treating a wide range of illnesses, especially those of the gastrointestinal system. Junellia spathulata (Gillies & Hook.) Moldenke var. spathulata (Verbenaceae), called as "verbena-azul-de-cordilleira", is a medicinal plant native to Argentina and Chile traditionally used for treating digestive disorders. Although the species of the genus are important as therapeutic resources for the Andean population, the plants are very scarcely studied. AIMS OF THE STUDY: The purpose of the present study was to find out the main constituents and investigate the protective effect of J. spathulata against oxidative stress induced by the potent oxidant 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) in human hepatoblastoma cells. MATERIALS AND METHODS: The crude methanol extract of J. spathulata and an iridoid obtained by chromatographic processes were tested to access the hepatoprotective effect and cytotoxicity in HepG2 cell. In addition, the reducing power of the samples and their ability to scavenge free radicals were evaluated using FRAP and ORAC assay systems. RESULTS: The iridoid asperuloside, the main compound of the crude methanol extract of J. spathulata, was isolated and identified by means of NMR analysis. The crude methanol extract of J. spathulata and asperuloside protected HepG2 cells against oxidative damage triggered by AAPH-derived free radicals. This effect can be credited to the ability of the extract and asperuloside to protect the liver cells from chemical-induced injury, which might be correlated to their free radical scavenging potential. CONCLUSIONS: This study experimentally evidenced the ethnopharmacological usefulness of J. spathulata as a treatment of digestive disorders. Our result could stimulate further investigations of hepatoprotective agents in other Chilean Junellia species.

State of the art of Smo antagonists for cancer therapy: advances in the target receptor and new ligand structures.

Since the Hedgehog signaling pathway has been associated with cancer, it has emerged as a therapeutic target for cancer therapy. The main target among the key Hedgehog proteins is the GPCR-like Smo receptor. Therefore, some Smo antagonists that have entered clinical trials, including the US FDA-approved drugs vismodegib and sonidegib, to treat basal cell carcinoma and medulloblastoma. However, early resistance of these drugs has spawned the need to understand the molecular bases of this phenomena. We therefore reviewed details about Smo receptor structures and the best Smo antagonist chemical structures. In addition, we discussed strategies that should be considered to develop new, safer generations of Smo antagonists that avoid current clinical limitations.