Synthesis and Anti-Diabetic Activity of an 8-Purine Derivative as a Novel DPP-4 Inhibitor in Obese Diabetic Zücker Rats.

Type 2 diabetes mellitus (T2DM) is one of the world's principal metabolic diseases characterized by chronic hyperglycemia. The gut incretin hormones, glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP), which has been proposed as a new treatment for T2DM, are extensively metabolized by Dipeptidyl peptidase 4 (DPP-4). Inhibitors of DPP-4 block the degradation of GLP-1 and GIP and may increase their natural circulating levels, favoring glycemic control in T2DM. A novel and potent selective inhibitor of DPP-4 with an 8-purine derived structure (1) has been developed and tested in vitro and in vivo in Zücker obese diabetic fatty (ZDF) rats, an experimental model of the metabolic syndrome and T2DM to assess the inhibitory activity using vildagliptin as reference standard. ZDF rats were subdivided into three groups (n = 7/group), control (C-ZDF), and those treated with compound 1 (Compound1-ZDF) and with vildagliptin (V-ZDF), both at 10 mg/kg/d rat body weight, in their drinking water for 12 weeks, and a group of lean littermates (ZL) was used. ZDF rats developed DM (fasting hyperglycemia, 425 ± 14.8 mg/dL; chronic hyperglycemia, HbA1c 8.5 ± 0.4%), compared to ZL rats. Compound 1 and vildagliptin reduced sustained HbAl1c (14% and 10.6%, P < 0.05, respectively) and fasting hyperglycemia values (24% and 19%, P < 0.05, respectively) compared to C-ZDF group (P < 0.001). Compound 1 and vildagliptin have shown a potent activity with an IC50 value of 4.92 and 3.21 µM, respectively. These data demonstrate that oral compound 1 administration improves diabetes in ZDF rats by the inhibitory effect on DPP-4, and the potential to be a novel, efficient and tolerable approach for treating diabetes of obesity-related T2DM, in ZDF rats.

Preclinical evaluation of bozepinib in bladder cancer cell lines: modulation of the NPP1 enzyme.

Bladder cancer (BC) is the most common cancer of the urinary tract. Bozepinib (BZP), a purine-derived molecule, is a potential compound for the treatment of cancer. Purinergic signaling consists of the activity of nucleosides and nucleotides present in the extracellular environment, modulating a variety of biological actions. In cancer, this signaling is mainly controlled by the enzymatic cascade involving the NTPDase/E-NPP family and ecto-5'-nucleotidase/CD73, which hydrolyze extracellular adenosine triphosphate (ATP) to adenosine (ADO). The aim of this work is to evaluate the activity of BZP in the purinergic system in BC cell lines and to compare its in vitro antitumor activity with cisplatin, a chemotherapeutic drug widely used in the treatment of BC. In this study, two different BC cell lines, grade 1 RT4 and the more aggressive grade 3 T24, were used along with a human fibroblast cell line MRC-5, a cell used to predict the selectivity index (SI). BZP shows strong antitumor activity, with notable IC50 values (8.7 ± 0.9 µM for RT4; 6.7 ± 0.7 µM for T24), far from the SI for cisplatin (SI for BZP: 19.7 and 25.7 for RT4 and T24, respectively; SI for cisplatin: 1.7 for T24). BZP arrests T24 cells in the G2/M phase of the cell cycle, inducing early apoptosis. Moreover, BZP increases ATP and ADP hydrolysis and gene/protein expression of the NPP1 enzyme in the T24 cell line. In conclusion, BZP shows superior activity compared to cisplatin against BC cell lines in vitro.

N-aryltetrahydroisoquinoline derivatives as HA-CD44 interaction inhibitors: Design, synthesis, computational studies, and antitumor effect.

Hyaluronic acid (HA) plays a crucial role in tumor growth and invasion through its interaction with cluster of differentiation 44 (CD44), a non-kinase transmembrane glycoprotein, among other hyaladherins. CD44 expression is elevated in many solid tumors, and its interaction with HA is associated with cancer and angiogenesis. Despite efforts to inhibit HA-CD44 interaction, there has been limited progress in the development of small molecule inhibitors. As a contribution to this endeavour, we designed and synthesized a series of N-aryltetrahydroisoquinoline derivatives based on existing crystallographic data available for CD44 and HA. Hit 2e was identified within these structures for its antiproliferative effect against two CD44+ cancer cell lines, and two new analogs (5 and 6) were then synthesized and evaluated as CD44-HA inhibitors by applying computational and cell-based CD44 binding studies. Compound 2-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroisoquinolin-5-ol (5) has an EC50 value of 0.59 µM against MDA-MB-231 cells and is effective to disrupt the integrity of cancer spheroids and reduce the viability of MDA-MB-231 cells in a dose-dependent manner. These results suggest lead 5 as a promising candidate for further investigation in cancer treatment.

Selective Anticancer Therapy Based on a HA-CD44 Interaction Inhibitor Loaded on Polymeric Nanoparticles.

Hyaluronic acid (HA), through its interactions with the cluster of differentiation 44 (CD44), acts as a potent modulator of the tumor microenvironment, creating a wide range of extracellular stimuli for tumor growth, angiogenesis, invasion, and metastasis. An innovative antitumor treatment strategy based on the development of a nanodevice for selective release of an inhibitor of the HA-CD44 interaction is presented. Computational analysis was performed to evaluate the interaction of the designed tetrahydroisoquinoline-ketone derivative (JE22) with CD44 binding site. Cell viability, efficiency, and selectivity of drug release under acidic conditions together with CD44 binding capacity, effect on cell migration, and apoptotic activity were successfully evaluated. Remarkably, the conjugation of this CD44 inhibitor to the nanodevice generated a reduction of the dosis required to achieve a significant therapeutic effect.

Development of bozepinib-loaded nanocapsules for nose-to-brain delivery: preclinical evaluation in glioblastoma.

Aim: To develop and characterize bozepinib-loaded lipid-core nanocapsules (BZP-LNC+) as a potential treatment for glioblastoma (GBM). Methods: Characterization of nanocapsules was performed by diameter, polydispersity index, Zeta potential, pH and encapsulation efficiency. GBM cell viability, cell cycle and Annexin/PI were evaluated after BZP-LNC+ treatment. Synergism between BZP-LNC+ and temozolomide (TMZ) was performed by CompuSyn software and confirmed in vitro and in vivo. Results: BZP-LNC+ showed adequate particle sizes, positive Zeta potential, narrow size distribution and high encapsulation efficiency. BZP-LNC+ reduces GBM growth by inducing apoptosis. BZP-LNC+ and TMZ showed synergistic effect in vitro and reduced the in vivo glioma growth by approximately 81%. Conclusion: The present study provides proof-of-principle insights for the combination of these drugs for GBM treatment.

Design, synthesis, HER2 inhibition and anticancer evaluation of new substituted 1,5-dihydro-4,1-benzoxazepines.

A series of 11 new substituted 1,5-dihydro-4,1-benzoxazepine derivatives was synthesised to study the influence of the methyl group in the 1-(benzenesulphonyl) moiety, the replacement of the purine by the benzotriazole bioisosteric analogue, and the introduction of a bulky substituent at position 6 of the purine, on the biological effects. Their inhibition against isolated HER2 was studied and the structure-activity relationships have been confirmed by molecular modelling studies. The most potent compound against isolated HER2 is 9a with an IC50 of 7.31 µM. We have investigated the effects of the target compounds on cell proliferation. The most active compound (7c) against all the tumour cell lines studied (IC50 0.42-0.86 µM) does not produce any modification in the expression of pro-caspase 3, but increases the caspase 1 expression, and promotes pyroptosis.

New insights into cytotoxic mechanisms of bozepinib against glioblastoma.

Glioblastoma (GBM) is the most frequent and aggressive brain tumor in adults and the current treatments only have a modest effect on patient survival. Recent studies show that bozepinib (BZP), a purine derivative, has potential applications in cancer treatment. The aim of this study was to evaluate the effect of BZP against GBM cells, specially concerning the purinergic system. Thus, GBM cells (C6 and U138 cell lines) were treated with BZP and cell viability, cell cycle, and annexin/PI assays, and active caspase-3 measurements were carried out. Besides, the effect of BZP over the purinergic system was also evaluated in silico and in vitro. Finally, we evaluate the action of BZP against important markers related to cancer progression, such as Akt, NF-κB, and CD133. We demonstrate here that BZP reduces GBM cell viability (IC50 = 5.7 ± 0.3 µM and 12.7 ± 1.5 µM, in C6 and U138 cells, respectively), inducing cell death through caspase-dependent apoptosis, autophagosome formation, activation of NF-κB, without any change in cell cycle progression or on the Akt pathway. Also, BZP modulates the purinergic system, inducing an increase in CD39 enzyme expression and activity, while inhibiting CD73 activity and adenosine formation, without altering CD73 enzyme expression. Curiously, one cycle of treatment resulted in enrichment of GBM cells expressing NF-κB and CD133+, suggesting resistant cells selection. However, after another treatment round, the resistant cells were eliminated. Altogether, BZP presented in vitro anti-glioma activity, encouraging further in vivo studies in order to better understand its mechanism of action.

Recent advances in the design of choline kinase α inhibitors and the molecular basis of their inhibition.

Upregulated choline metabolism, characterized by an increase in phosphocholine (PCho), is a hallmark of oncogenesis and tumor progression. Choline kinase (ChoK), the enzyme responsible for PCho synthesis, has consequently become a promising drug target for cancer therapy and as such a significant number of ChoK inhibitors have been developed over the last few decades. More recently, due to the role of this enzyme in other pathologies, ChoK inhibitors have also been used in new therapeutic approaches against malaria and rheumatoid arthritis. Here, we review research results in the field of ChoKα inhibitors from their synthesis to the molecular basis of their binding mode. Strategies for the development of inhibitors and their selectivity on ChoKα over ChoKß, the plasticity of the choline-binding site, the discovery of new exploitable binding sites, and the allosteric properties of this enzyme are highlighted. The outcomes summarized in this review will be a useful guide to develop new multifunctional potent drugs for the treatment of various human diseases.

Enhancement of Tumor Cell Death by Combining gef Gene Mediated Therapy and New 1,4-Benzoxazepin-2,6-Dichloropurine Derivatives in Breast Cancer Cells.

New treatment modalities are urgently needed to better manage advanced breast cancer. Combination therapies are usually more effective than monotherapy. In this context, the use of cyclic and acyclic O,N-acetals derivative compounds in combination with the suicide gef gene shown a potent anti-tumor activity and represent a new generation of anticancer agents. Here, we evaluate the use of the gef gene to promote and increase the anti-tumor effect of cyclic and acyclic O,N-acetals purine derivatives and elucidate their mechanisms of action. Among all compounds tested, those with a nitro group and a cyclic pattern structures (FC-30b2, FC-29c, and bozepinib) are the most benefited from the gef gene effect. These compounds, in combination with gef gene, were able to abolish tumor cell proliferation with a minimal dose leading to more effective and less toxic chemotherapy. The effect of this combined therapy is triggered by apoptosis induction which can be found deregulated in the later stage of breast cancer. Moreover, the combined therapy leads to an increase of cell post-apoptotic secondary necrosis that is able to promote the immunogenicity of cancer cells leading to a successful treatment. This data suggests that this novel combination therapy represents a promising candidate for breast cancer treatment.

Lysophosphatidylcholine Regulates Sexual Stage Differentiation in the Human Malaria Parasite Plasmodium falciparum.

Transmission represents a population bottleneck in the Plasmodium life cycle and a key intervention target of ongoing efforts to eradicate malaria. Sexual differentiation is essential for this process, as only sexual parasites, called gametocytes, are infective to the mosquito vector. Gametocyte production rates vary depending on environmental conditions, but external stimuli remain obscure. Here, we show that the host-derived lipid lysophosphatidylcholine (LysoPC) controls P. falciparum cell fate by repressing parasite sexual differentiation. We demonstrate that exogenous LysoPC drives biosynthesis of the essential membrane component phosphatidylcholine. LysoPC restriction induces a compensatory response, linking parasite metabolism to the activation of sexual-stage-specific transcription and gametocyte formation. Our results reveal that malaria parasites can sense and process host-derived physiological signals to regulate differentiation. These data close a critical knowledge gap in parasite biology and introduce a major component of the sexual differentiation pathway in Plasmodium that may provide new approaches for blocking malaria transmission.

1-(Benzenesulfonyl)-1,5-dihydro-4,1-benzoxazepine as a new scaffold for the design of antitumor compounds.

AIM: Bozepinib is a potent and selective anticancer compound which chemical structure is made up of a benzofused seven-membered ring and a purine moiety. We previously demonstrated that the purine fragment does not exert antiproliferative effect per se. METHODOLOGY: A series of 1-(benzenesulfonyl)-4,1-benzoxazepine derivatives were synthesized in order to study the influence of the benzofused seven-membered ring in the biological activity of bozepinib by means of antiproliferative, cell cycle and apoptosis studies. RESULTS & CONCLUSION: Our results show that the methyleneoxy enamine sulfonyl function is essential in the antitumor activity of the structures and thus, it is a scaffold suitable for further modification with a view to obtain more potent antitumor compounds.

Antitumoral activity of 1,2-diaminocyclohexane derivatives in breast, colon and skin human cancer cells.

AIM: Cancer is among the leading causes of death worldwide. Medical interest has focused on macrocyclic polyamines because of their properties as antitumor agents. Results/Methodology: We have designed and synthesized a series of 1,2-diaminocyclohexane derivatives with notable in vitro antiproliferative activities against the MCF-7, HCT-116 and A375 cancer cell lines. Cell cycle and apoptosis analyses were also carried out. Our results show that all the compounds are potent cytotoxic agents, especially against the A375 cell line. CONCLUSION: The selective activity of the macrocyclic derivative against A375, via apoptosis, supposes a great advantage for future therapeutic use. This exemplifies the potential of 1,2-diaminocyclohexane derivatives to qualify as lead structures for future anticancer drug development due to their easy syntheses and noteworthy bioactivity.

Plasmodium falciparum Choline Kinase Inhibition Leads to a Major Decrease in Phosphatidylethanolamine Causing Parasite Death.

Malaria is a life-threatening disease caused by different species of the protozoan parasite Plasmodium, with P. falciparum being the deadliest. Increasing parasitic resistance to existing antimalarials makes the necessity of novel avenues to treat this disease an urgent priority. The enzymes responsible for the synthesis of phosphatidylcholine and phosphatidylethanolamine are attractive drug targets to treat malaria as their selective inhibition leads to an arrest of the parasite's growth and cures malaria in a mouse model. We present here a detailed study that reveals a mode of action for two P. falciparum choline kinase inhibitors both in vitro and in vivo. The compounds present distinct binding modes to the choline/ethanolamine-binding site of P. falciparum choline kinase, reflecting different types of inhibition. Strikingly, these compounds primarily inhibit the ethanolamine kinase activity of the P. falciparum choline kinase, leading to a severe decrease in the phosphatidylethanolamine levels within P. falciparum, which explains the resulting growth phenotype and the parasites death. These studies provide an understanding of the mode of action, and act as a springboard for continued antimalarial development efforts selectively targeting P. falciparum choline kinase.

In vitro antiplasmodial and cytotoxic activities of asymmetrical pyridinium derivatives.

An in vitro investigation of the antiplasmodial and cytotoxic activities of a series of human choline kinase inhibitors against Plasmodium falciparum is reported. Structure-activity relationship analyses have allowed us to determine the essential parameters for the antimalarial effect of these asymmetrical pyridinium derivatives. One of the compounds meets the World Health Organization's criteria for hit identification against P. falciparum exhibiting an IC50 of 0.0016 µg/ml and a selectivity index of >3000.

HER2-signaling pathway, JNK and ERKs kinases, and cancer stem-like cells are targets of Bozepinib small compound.

Identification of novel anticancer drugs presenting more than one molecular target and efficacy against cancer stem-like cells (CSCs) subpopulations represents a therapeutic need to combat the resistance and the high risk of relapse in patients. In the present work we show how Bozepinib [(RS)-2,6-dichloro-9-[1-(p-nitrobenzenesulfonyl)-1,2,3,5-tetrahydro-4,1-benzoxazepin-3-yl]-9H-purine], a small anti-tumor compound, demonstrated selectivity on cancer cells and showed an inhibitory effect over kinases involved in carcinogenesis, proliferation and angiogenesis. The cytotoxic effects of Bozepinib were observed in both breast and colon cancer cells expressing different receptor patterns. Bozepinib inhibited HER-2 signaling pathway and JNK and ERKs kinases. In addition, Bozepinib has an inhibitory effect on AKT and VEGF together with anti-angiogenic and anti-migratory activities. Moreover, the modulation of pathways involved in tumorigenesis by Bozepinib was also evident in microarrays analysis. Interestingly, Bozepinib inhibited both mamo- and colono-spheres formation and eliminated ALDH+ CSCs subpopulations at a low micromolar range similar to Salinomycin. Bozepinib induced the down-regulation of c-MYC, ß-CATENIN and SOX2 proteins and the up-regulation of the GLI-3 hedgehog-signaling repressor. Finally, Bozepinib shows in vivo anti-tumor and anti-metastatic efficacy in xenotransplanted nude mice without presenting sub-acute toxicity. These findings support further studies on the therapeutic potential of Bozepinib in cancer patients.

Anti-proliferative activity of 2,6-dichloro-9- or 7-(ethoxycarbonylmethyl)-9H- or 7H-purines against several human solid tumour cell lines.

As leads we took several benzo-fused seven- and six-membered scaffolds linked to the pyrimidine or purine moieties with notable anti-proliferative activity against human breast, colon and melanoma cancerous cell lines. We then decided to maintain the double-ringed nitrogenous bases and change the other components to the ethyl acetate moiety. This way six purine and two 5-fluorouracil derivatives were obtained and evaluated against the MCF-7, HCT-116, A-375 and G-361 cancer cell lines. Two QSARs are obtained between the anti-proliferative IC50 values for compounds 26-33 and the clog P against the melanoma cell lines A-375 and G-361. Our results show that two of the analogues [ethyl 2-(2,6-dichloro-9H- or 7H-purine-9- or 7-yl)acetates (30 and 33, respectively)] are potent cytotoxic agents against all the tumour cell lines assayed, showing single-digit micromolar IC50 values. This exemplifies the potential of our previously reported purine compounds to qualify as lead structures for medicinal chemistry campaigns, affording simplified analogues easy to synthesize and with a noteworthy bioactivity. The selective activity of 30 and 33 against the melanoma cell line A-375, via apoptosis, supposes a great advantage for a future therapeutic use.

Discovery of a new binding site on human choline kinase α1: design, synthesis, crystallographic studies, and biological evaluation of asymmetrical bispyridinium derivatives.

Human choline kinase α (CKα) is a validated drug target for the treatment of cancer. In recent years, a large number of CK inhibitors have been synthesized, and one of them is currently being evaluated in Phase I clinical trials as a treatment for solid tumors. Here we have evaluated a new series of asymmetrical biscationic CK inhibitors by means of enzymatic, crystallographic, and antitumor studies. We demonstrate that one of these structures adopts a completely new binding mode not observed before inducing the aperture of an adjacent binding site. This compound shows antiproliferative and apoptotic effects on cancer cells through activation of caspase-3. Therefore, this study not only provides fruitful insights into the design of more efficient compounds that may target different regions in CKα1 but also explains how these compounds induce apoptosis in cancer cells.

Bozepinib, a novel small antitumor agent, induces PKR-mediated apoptosis and synergizes with IFNα triggering apoptosis, autophagy and senescence.

Bozepinib [(RS)-2,6-dichloro-9-[1-(p-nitrobenzenesulfonyl)-1,2,3,5-tetrahydro-4,1-benzoxazepin-3-yl]-9H-purine] is a potent antitumor compound that is able to induce apoptosis in breast cancer cells. In the present study, we show that bozepinib also has antitumor activity in colon cancer cells, showing 50% inhibitory concentration (IC50) values lower than those described for breast cancer cells and suggesting great potential of this synthetic drug in the treatment of cancer. We identified that the double-stranded RNA-dependent protein kinase (PKR) is a target of bozepinib, being upregulated and activated by the drug. However, p53 was not affected by bozepinib, and was not necessary for induction of apoptosis in either breast or colon cancer cells. In addition, the efficacy of bozepinib was improved when combined with the interferon-alpha (IFNα) cytokine, which enhanced bozepinib-induced apoptosis with involvement of protein kinase PKR. Moreover, we report here, for the first time, that in combined therapy, IFNα induces a clear process of autophagosome formation, and prior treatment with chloroquine, an autophagy inhibitor, is able to significantly reduce IFNα/bozepinib-induced cell death. Finally, we observed that a minor population of caspase 3-deficient MCF-7 cells persisted during long-term treatment with lower doses of bozepinib and the bozepinib/IFNα combination. Curiously, this population showed ß-galactosidase activity and a percentage of cells arrested in S phase, that was more evident in cells treated with the bozepinib/IFNα combination than in cells treated with bozepinib or IFNα alone. Considering the resistance of some cancer cells to conventional chemotherapy, combinations enhancing the diversity of the cell death outcome might succeed in delivering more effective and less toxic chemotherapy.