Design and Synthesis of Potent in Vitro and in Vivo Anticancer Agents Based on 1-(3′,4′,5′-Trimethoxyphenyl)-2-Aryl-1H-Imidazole

We acknowledge “Proyecto de Excelencia de la Consejeria de Innovacion y Ciencia de la Junta de Andalucia, Spain ref. P12-CTS-696” for its financial support. We also acknowledge the support of the Life Science Research Net- work Wales grant no. NRNPGSep14008, an initiative funded through the Welsh Government’s Ser Cymru program.A novel series of tubulin polymerization inhibitors, based on the 1-(3′,4′,5′-trimethoxyphenyl)-2-aryl-1H-imidazole scaffold and designed as cis-restricted combretastatin A-4 analogues, was synthesized with the goal of evaluating the effects of various patterns of substitution on the phenyl at the 2-position of the imidazole ring on biological activity. A chloro and ethoxy group at the meta- and para-positions, respectively, produced the most active compound in the series (4o), with IC50 values of 0.4-3.8 nM against a panel of seven cancer cell lines. Except in HL-60 cells, 4o had greater antiproliferative than CA-4, indicating that the 3′-chloro-4′-ethoxyphenyl moiety was a good surrogate for the CA-4 B-ring. Experiments carried out in a mouse syngenic model demonstrated high antitumor activity of 4o, which significantly reduced the tumor mass at a dose thirty times lower than that required for CA-4P, which was used as a reference compound. Altogether, our findings suggest that 4o is a promising anticancer drug candidate that warrants further preclinical evaluation.Junta de Andalucía, Spain P12-CTS-696Welsh Government’s NRNPGSep1400

Choline kinase inhibitors EB-3D and EB-3P interferes with lipid homeostasis in HepG2 cells

A full understanding of the molecular mechanism of action of choline kinase α (ChoKα) inhibitors at the cell level is essential for developing therapeutic and preventive approaches for cancer. The aim of the present study was to evaluate the effects of the ChoKα inhibitors EB-3D and EB-3P on lipid metabolism in HepG2 cells. We used [methyl-14C]choline, [1,2-14C]acetic acid and [2-3H]glycerol as exogenous precursors of the corresponding phospholipids and neutral lipids. [Methyl-14C]choline was also used to determine choline uptake. Protein levels were determined by Western blot. Ultrastructural alterations were investigated by transmission electron microscopy. In this work, we demonstrate that EB-3D and EB-3P interfere with phosphatidylcholine biosynthesis via both CDP-choline pathway and choline uptake by the cell. Moreover, the synthesis of both diacylglycerols and triacylglycerols was affected by cell exposure to both inhibitors. These effects were accompanied by a substantial decrease in cholesterol biosynthesis, as well as alterations in the expression of proteins related to cholesterol homeostasis. We also found that EB-3D and EB-3P lowered ChoKα protein levels. All these effects could be explained by the modulation of the AMP-activated protein kinase signalling pathway. We show that both inhibitors cause mitochondrial alteration and an endoplasmic reticulum stress response. EB-3D and EB-3P exert effects on ChoKα expression, AMPK activation, apoptosis, endoplasmic reticulum stress and lipid metabolism. Taken together, results show that EB-3D and EB-3P have potential anticancer activity through the deregulation of lipid metabolism.This work was aided by the Andalusian Regional Government (P11-CVI-7859). The inhibitors were developed under the Cei-Biotic Project CEI2013-MP-1 (University of Granada)

Choline Kinase Alpha Inhibition by EB-3D Triggers Cellular Senescence, Reduces Tumor Growth and Metastatic Dissemination in Breast Cancer

Choline kinase (ChoK) is the first enzyme of the Kennedy pathway leading to the biosynthesis of phosphatidylcholine (PtdCho), the most abundant phospholipid in eukaryotic cell membranes. EB-3D is a novel choline kinase 1 (ChoK 1) inhibitor with potent antiproliferative activity against a panel of several cancer cell lines. ChoK 1 is particularly overexpressed and hyperactivated in aggressive breast cancer. By NMR analysis, we demonstrated that EB-3D is able to reduce the synthesis of phosphocholine, and using flow cytometry, immunoblotting, and q-RT-PCR as well as proliferation and invasion assays, we proved that EB-3D strongly impairs breast cancer cell proliferation, migration, and invasion. EB-3D induces senescence in breast cancer cell lines through the activation of the metabolic sensor AMPK and the subsequent dephosphorylation of mTORC1 downstream targets, such as p70S6K, S6 ribosomal protein, and 4E-BP1. Moreover, EB-3D strongly synergizes with drugs commonly used for breast cancer treatment. The antitumorigenic potential of EB-3D was evaluated in vivo in the syngeneic orthotopic E0771 mouse model of breast cancer, where it induces a significant reduction of the tumor mass at low doses. In addition, EB-3D showed an antimetastatic effect in experimental and spontaneous metastasis models. Altogether, our results indicate that EB-3D could be a promising new anticancer agent to improve aggressive breast cancer treatment protocols.This work was supported by funds from Istituto di Ricerca Pediatrica (IRP)-Città della Speranza and Cassa di Risparmio di Padova e Rovigo—CARIPARO Foundation (project IRP13/05) and by the University of Granada, (Cei-Biotic project CEI2013-MP-1), and Associazione Italiana per la Ricerca sul Cancro (AIRC) MFAG 18459 grant (R.R.). E.M. was supported by AIRC (21101) and V.S. by FIRC (16616) fellowships

Biological Evaluation of New Thienopyridinium and Thienopyrimidinium Derivatives as Human Choline Kinase Inhibitors

This research was funded by Convocatoria 2019 Proyectos de I + D + i − RTI Tipo B “Ministerio de Innovación y Ciencia” grant number PID2019-109294RB-I00 and “Convocatoria 2020 Proyectos I + D + i del Programa Operativo FEDER 2020”, grant number B-CTS-216-UGR20. E.P thanks the European Regional Development Fund (ERDF) project BioDrug (No. 1.1.1.5/19/A/004) and the Latvian Council of Science (grant No. lzp-2020/2-0013) for financial support.Due to its role in lipid biosynthesis, choline kinase α1 (CKα1) is an interesting target for the development of new antitumor agents. In this work, we present a series of 41 compounds designed based on the well-known and successful strategy of introducing thienopyridine and pyrimidine as bioisosteres of other heterocycles in active antitumor compounds. Notwithstanding the fact that some of these compounds do not show significant enzymatic inhibition, others, in contrast, feature substantially improved enzymatic and antiproliferative inhibition values. This is also confirmed by docking analysis, whereby compounds with longer linkers and thienopyrimidine cationic head have been identified as the most compelling. Among the best compounds is Ff-35, which inhibits the growth of different tumor cells at submicromolar concentrations. Moreover, Ff-35 is more potent in inhibiting CKα1 than other previous biscationic derivatives. Treatment of A549, Hela, and MDA-MB-231 cells with Ff-35 results in their arrest at the G1 phase of the cell cycle. Furthermore, the compound induces cellular apoptosis in a concentration-dependent manner. Altogether, these findings indicate that Ff-35 is a promising new chemotherapeutic agent with encouraging preclinical potential.Ministerio de Innovación y Ciencia PID2019-109294RB-I00FEDER 2020 B-CTS-216-UGR20European Regional Development Fund (ERDF) 1.1.1.5/19/A/004Latvian Council of Science lzp-2020/2-00

New Compounds with Bioisosteric Replacement of Classic Choline Kinase Inhibitors Show Potent Antiplasmodial Activity

This research was funded by Convocatoria 2019 Proyectos de I + D + i - RTI Tipo B “Ministerio de Ciencia e Innovación” grant number PID2019–109294RB-I00, University of Granada, Cei-BioticProject grant number CEI2013-MP-1, the Instituto de Salud Carlos III Subdirección General de Redes y Centros de Investigación Cooperativa-Red de Investigación Cooperativa en Enfermedades Tropicales (RICET: RD16/0027/0014), the Plan Nacional (SAF PID2019-109623RB-I002016-79957-R) and the Junta de Andalucía (BIO-199).Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3390/pharmaceutics13111842/s1, Figure S1. Pf CKIC50 Curves Inhibition of Pf CK. Figure S2. IC50Curves. Figure S3. Spectra.In the fight against Malaria, new strategies need to be developed to avoid resistance of the parasite to pharmaceutics and other prevention barriers. Recently, a Host Directed Therapy approach based on the suppression of the starting materials uptake from the host by the parasite has provided excellent results. In this article, we propose the synthesis of bioisosteric compounds that are capable of inhibiting Plasmodium falciparum Choline Kinase and therefore to reduce choline uptake, which is essential for the development of the parasite. Of the 41 bioisosteric compounds reported herein, none showed any influence of the linker on the antimalarial and enzyme inhibitory activity, whereas an effect of the type of cationic heads used could be observed. SARs determined that the thienopyrimidine substituted in 4 by a pyrrolidine is the best scaffold, independently of the chosen linker. The decrease in lipophilicity seems to improve the antimalarial activity but to cause an opposite effect on the inhibition of the enzyme. While potent compounds with similar good inhibitory values have been related to the proposed mechanism of action, some of them still show discrepancies and further studies are needed to determine their specific molecular target.Ministerio de Ciencia e Innovación PID2019–109294RB-I00University of Granada CEI2013-MP-1Plan Nacional (SAF PID2019-109623RB-I002016-79957-R)Junta de Andalucía (BIO-199)Instituto de Salud Carlos III (RICET: RD16/0027/0014

Design, synthesis, crystallization and biological evaluation of new symmetrical biscationic compounds as selective inhibitors of human Choline Kinase α1 (ChoKα1)

The authors gratefully acknowledge the ‘Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía’ (Excellence Research Project: P07-CTS-03210), the ‘Diputación General de Aragón (B89)’ and the ‘Ministerio de Ciencia e Innovación’ (SAF2009-11955, BFU2010-19504 and CTQ2013-44367-C2-2-P) for the financial support, the award of grants from ‘Ministerio de Educación’ to P.R.-M. and S.S.-E. is gratefully acknowledged, and the ‘Centro de Servicios de Informática of the University of Granada (Spain) for the use of their computers and scientific software. G.V.,R.B., R.M. and G.B. We thanks also the Fondazione Cariparo by the “Progetto Ricerca Pediatrica”. We thank synchrotron radiation sources ALBA (Barcelona), and in particular the beamline XALOC. The research leading to these results has also received funding from the FP7 (2007–2013) under BIOSTRUCTX-7687.A novel family of compounds derivative of 1,1′-(((ethane-1,2-diylbis(oxy))bis(4,1-phenylene))bis(methylene))-bispyridinium or –bisquinolinium bromide (10a-l) containing a pair of oxygen atoms in the spacer of the linker between the biscationic moieties, were synthesized and evaluated as inhibitors of choline kinase against a panel of cancer-cell lines. The most promising compounds in this series were 1,1′-(((ethane-1,2-diylbis(oxy))bis(4,1-phenylene))bis(methylene))bis(4-(dimethylamino)pyridinium) bromide (10a) and 1,1′-(((ethane-1,2-diylbis(oxy))bis(4,1-phenylene))bis(methylene))-bis(7-chloro-4-(pyrrolidin-1-yl)quinolinium) bromide (10l), which inhibit human choline kinase (ChoKα1) with IC50 of 1.0 and 0.92 μM, respectively, in a range similar to that of the previously reported biscationic compounds MN58b and RSM932A. Our compounds show greater antiproliferative activities than do the reference compounds, with unprecedented values of GI50 in the nanomolar range for several of the cancer-cell lines assayed, and more importantly they present low toxicity in non-tumoral cell lines, suggesting a cancer-cell-selective antiproliferative activity. Docking studies predict that the compounds interact with the choline-binding site in agreement with the binding mode of most previously reported biscationic compounds. Moreover, the crystal structure of ChoKα1 with compound 10a reveals that this compound binds to the choline-binding site and mimics HC-3 binding mode as never before.Junta de Andalucía P07-CTS-03210Diputación General de Aragón (B89)Ministerio de Ciencia e Innovación (SAF2009-11955, BFU2010-19504, CTQ2013-44367-C2-2-P)Ministerio de EducaciónUniversity of Granada (Spain)Fondazione CariparoBIOSTRUCTX-7687 FP7 (2007–2013

Biological Evaluation of New Thienopyridinium and Thienopyrimidinium Derivatives as Human Choline Kinase Inhibitors

Due to its role in lipid biosynthesis, choline kinase α1 (CKα1) is an interesting target for the development of new antitumor agents. In this work, we present a series of 41 compounds designed based on the well-known and successful strategy of introducing thienopyridine and pyrimidine as bioisosteres of other heterocycles in active antitumor compounds. Notwithstanding the fact that some of these compounds do not show significant enzymatic inhibition, others, in contrast, feature substantially improved enzymatic and antiproliferative inhibition values. This is also confirmed by docking analysis, whereby compounds with longer linkers and thienopyrimidine cationic head have been identified as the most compelling. Among the best compounds is Ff-35, which inhibits the growth of different tumor cells at submicromolar concentrations. Moreover, Ff-35 is more potent in inhibiting CKα1 than other previous biscationic derivatives. Treatment of A549, Hela, and MDA-MB-231 cells with Ff-35 results in their arrest at the G1 phase of the cell cycle. Furthermore, the compound induces cellular apoptosis in a concentration-dependent manner. Altogether, these findings indicate that Ff-35 is a promising new chemotherapeutic agent with encouraging preclinical potential

Nuevos derivados pirrólicos y pirazólicos: síntesis y evaluación biológica frente a óxido nítrico sintasa

Tesis Univ. Granada. Departamento de Química Farmacéutica y Orgánica. Leída en 200