Synthesis of Novel Acyl Derivatives of 3-(4,5,6,7-Tetrabromo-1H-benzimidazol-1-yl)propan-1-ols—Intracellular TBBi-Based CK2 Inhibitors with Proapoptotic Properties

Protein kinase CK2 has been considered as an attractive drug target for anti-cancer therapy. The synthesis of N-hydroxypropyl TBBi and 2MeTBBi derivatives as well as their respective esters was carried out by using chemoenzymatic methods. Concomitantly with kinetic studies toward recombinant CK2, the influence of the obtained compounds on the viability of two human breast carcinoma cell lines (MCF-7 and MDA-MB-231) was evaluated using MTT assay. Additionally, an intracellular inhibition of CK2 as well as an induction of apoptosis in the examined cells after the treatment with the most active compounds were studied by Western blot analysis, phase-contrast microscopy and flow cytometry method. The results of the MTT test revealed potent cytotoxic activities for most of the newly synthesized compounds (EC₅₀ 4.90 to 32.77 µM), corresponding to their solubility in biological media. We concluded that derivatives with the methyl group decrease the viability of both cell lines more efficiently than their non-methylated analogs. Furthermore, inhibition of CK2 in breast cancer cells treated with the tested compounds at the concentrations equal to their EC₅₀ values correlates well with their lipophilicity since derivatives with higher values of logP are more potent intracellular inhibitors of CK2 with better proapoptotic properties than their parental hydroxyl compounds.     

Derivatives of Di-O-octanoylglycerol and mono-O-octylglycerol as modulators of protein kinase C and diacylglycerol kinase activities

Twelve analogs of 1,2-di-O-octanoylglycerol modified at C-3 and three quaternaryN-alkyl-ammonium derivatives of glycerol were synthesized. The compounds were testedin vitro as potential modulators of the calcium activated, phospholipid dependent protein kinase C (PKC) and diacylglycerol (DAG) kinase activities in order to understand the molecular interactions of these enzymes with their natural activators, inhibitors, or substrates. PKC activity was assayed by measuring histone H₁ phosphorylation, and the compounds synthesized were tested either in the presence (inhibitors) or in the absence (activators) of 1,2-di-O-octanoyglycerol analogs with the phosphatidylserine/Ca²⁺ mixture. DAG kinase activity was measured by the incorporation of phosphate into 1,2-di-O-oleoyl-sn-glycerol in the presence of the various analogs synthesized. In regard to PKC activity, the assays revealed that 1,2-di-O-octanoylglycerol analogs are inactive when modified at C-3 with groups which do not permit hydrogen bonding. Under our conditions, di-O-octanoylthioglycerol, which has been reported as inactive, was able to activate PKC in the presence of phosphatidylserine. It has been shown to give a synergistic activation with diacylglycerol and had no affinity for the phorbol ester receptor binding site, suggesting thatO-octanoylthioglycerol interacts with the enzyme at a different site from the phorbol ester receptor binding site. PKC and DAG kinase activities are inhibited byN-alkyl-ammonium compounds (IC₅₀ 24 μM) only when either two 8-carbon alkyl or acyl chains are present at the 1- and 2-positions of the glycerol backbone. The fact that these compounds have a strong effect on the binding of [³H]phorbol 12,13-dibutyrate to protein kinase C, and also inhibit DAG kinase, may suggest binding to the DAG site of the regulatory domain of PKC.     

Structure-function relationships of alkyl-lysophospholipid analogs in selective antitumor activity

This investigation was initiated in order to delineate the structure-function relationship of the anticancer alkyllysophospholipids and assess their degree of selective cytotoxicity toward neoplastic cells. A series of glycerol phosphocholine analogs with varying substitutions in thesn-1 andsn-2 position were tested for their inhibitory activity as measured by thymidine incorporation, clonogenic assays and effects on protein kinase C activity against a series of human leukemic cell lines and healthy bone marrow progenitor cells. The IC₅₀ was determined for each of the compounds in each cell line and healthy bone marrow cells following a 4-h incubation. The data indicated that a 16–18 carbon chain at thesn-1 coupled with a short substitution atsn-2 had the broadest antitumor activity and was the least toxic to normal bone marrow cells. The results provide a number of useful leads toward the design and development of potentially more active phospholipid compounds.     

Synthesis and SAR of Tetracyclic Inhibitors of Protein Kinase CK2 Derived from Furocarbazole W16

The serine/threonine kinase CK2 modulates the activity of more than 300 proteins and thus plays a crucial role in various physiological and pathophysiological processes including neurodegenerative disorders of the central nervous system and cancer. The enzymatic activity of CK2 is controlled by the equilibrium between the heterotetrameric holoenzyme CK2α₂β₂ and its monomeric subunits CK2α and CK2β. A series of analogues of W16 ((3aR,4S,10S,10aS)-4-{[(S)-4-benzyl-2-oxo-1,3-oxazolidin-3-yl]carbonyl}-10-(3,4,5-trimethoxyphenyl)-4,5,10,10a-tetrahydrofuro[3,4-b]carbazole-1,3(3aH)-dione ((+)- 3 a )) was prepared in an one-pot, three-component Levy reaction. The stereochemistry of the tetracyclic compounds was analyzed. Additionally, the chemically labile anhydride structure of the furocarbazoles 3 was replaced by a more stable imide ( 9 ) and N-methylimide ( 10 ) substructure. The enantiomer (−)- 3 a (K_i=4.9 μM) of the lead compound (+)- 3 a (K_i=31 μM) showed a more than sixfold increased inhibition of the CK2α/CK2β interaction (protein-protein interaction inhibition, PPII) in a microscale thermophoresis (MST) assay. However, (−)- 3 a did not show an increased enzyme inhibition of the CK2α₂β₂ holoenzyme, the CK2α subunit or the mutated CK2α′ ^C336S subunit in the capillary electrophoresis assay. In the pyrrolocarbazole series, the imide (−)- 9 a (K_i=3.6 μM) and the N-methylimide (+)- 10 a (K_i=2.8 μM) represent the most promising inhibitors of the CK2α/CK2β interaction. However, neither compound could inhibit enzymatic activity. Unexpectedly, the racemic tetracyclic pyrrolocarbazole (±)- 12 , with a carboxy moiety in the 4-position, displays the highest CK2α/CK2β interaction inhibition (K_i=1.8 μM) of this series of compounds.     

Synthesis of Novel Pyrido[1,2-c]pyrimidine Derivatives with 6-Fluoro-3-(4-piperidynyl)-1,2-benzisoxazole Moiety as Potential SSRI and 5-HT1A Receptor Ligands

Two series of novel 4-aryl-2H-pyrido[1,2-c]pyrimidine (6a–i) and 4-aryl-5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine (7a–i) derivatives were synthesized. The chemical structures of the new compounds were confirmed by ¹H and ¹³C NMR spectroscopy and ESI-HRMS spectrometry. The affinities of all compounds for the 5-HT_1A receptor and serotonin transporter protein (SERT) were determined by in vitro radioligand binding assays. The test compounds demonstrated very high binding affinities for the 5-HT_1A receptor of all derivatives in the series (6a–i and 7a–i) and generally low binding affinities for the SERT protein, with the exception of compounds 6a and 7g. Extended affinity tests for the receptors D₂, 5-HT_2A, 5-HT₆ and 5-HT₇ were conducted with regard to selected compounds (6a, 7g, 6d and 7i). All four compounds demonstrated very high affinities for the D₂ and 5-HT_2A receptors. Compounds 6a and 7g also had high affinities for 5-HT₇, while 6d and 7i held moderate affinities for this receptor. Compounds 6a and 7g were also tested in vivo to identify their functional activity profiles with regard to the 5-HT_1A receptor, with 6a demonstrating the activity profile of a presynaptic agonist. Metabolic stability tests were also conducted for 6a and 6d.     

Design of CK2β-Mimicking Peptides as Tools To Study the CK2α/CK2β Interaction in Cancer Cells

The ubiquitously expressed Ser/Thr kinase CK2 is a key regulator in a variety of key processes in normal and malignant cells. Due to its distinctive anti-apoptotic and tumor-driving properties, elevated levels of CK2 have frequently been found in tumors of different origin. In recent years, development of CK2 inhibitors has largely been focused on ATP-competitive compounds; however, targeting the CK2α/CK2β interface has emerged as a further concept that might avoid selectivity issues. To address the CK2 subunit interaction site, we have synthesized halogenated CK2β-mimicking cyclic peptides modified with the cell-penetrating peptide sC18 to mediate cellular uptake. We investigated the binding of the resulting chimeric peptides to recombinant human CK2α using a recently developed fluorescence anisotropy assay. The iodinated peptide sC18-I-Pc was identified as a potent CK2α ligand (K_i=0.622 μm ). It was internalized in cells to a high extent and exhibited significant cytotoxicity toward cancerous HeLa cells (IC₅₀=37 μm ) in contrast to non-cancerous HEK-293 cells. The attractive features and functionalities of sC18-I-Pc offer the opportunity for further improvement.     

Design,Synthesis, and Evaluation of 2‐Amino‐6‐nitrobenzothiazole‐Derived Hydrazones as MAO Inhibitors: Role of the Methylene Spacer Group

A series of 2‐amino‐6‐nitrobenzothiazole‐derived extended hydrazones were designed, synthesized, and investigated for their ability to inhibit monoamine oxidase A and B (MAO‐A/MAO‐B). The compounds were found to exhibit inhibitory activities in the nanomolar to micromolar range. Some of the compounds showed excellent potency and selectivity against the MAO‐B isoform. N′‐(5‐Chloro‐2‐oxoindolin‐3‐ylidene)‐2‐(6‐nitrobenzothiazol‐2‐ylamino)acetohydrazide (compound 31 ) showed the highest MAO‐B inhibitory activity (IC₅₀=1.8±0.3 nm , selectivity index [SI]=766.67), whereas compound 6 [N′‐(1‐(4‐bromophenyl)ethylidene)‐2‐(6‐nitrobenzothiazol‐2‐ylamino)acetohydrazide] was found to be the most active MAO‐A inhibitor (IC₅₀=0.42±0.003 μm ). Kinetic studies revealed that compounds 6 and 31 exhibit competitive‐type reversible inhibition against both MAO‐A and MAO‐B, respectively. Structure–activity relationship (SAR) studies disclosed several structural aspects significant for potency and the contribution of the methylene spacer toward MAO‐B inhibitory potency, with minimal or no neurotoxicity. Molecular modeling studies yielded a good correlation between experimental and theoretical inhibitory data. Binding pose analysis revealed the significance of cumulative effects of π–π stacking and hydrogen bond interactions for effective stabilization of virtual ligand–protein complexes. Further optimization studies of compound 31 , including co‐crystallization of inhibitor–MAO‐B complexes, are essential to develop these compounds as potential therapeutic agents for MAO‐B‐associated neurodegenerative diseases.     

Control of TRPM3 Ion Channels by Protein Kinase CK2-Mediated Phosphorylation in Pancreatic β-Cells of the Line INS-1

In pancreatic β-cells of the line INS-1, glucose uptake and metabolism induce the openings of Ca²⁺-permeable TRPM3 channels that contribute to the elevation of the intracellular Ca²⁺ concentration and the fusion of insulin granules with the plasma membrane. Conversely, glucose-induced Ca²⁺ signals and insulin release are reduced by the activity of the serine/threonine kinase CK2. Therefore, we hypothesized that TRPM3 channels might be regulated by CK2 phosphorylation. We used recombinant TRPM3α2 proteins, native TRPM3 proteins from INS-1 β-cells, and TRPM3-derived oligopeptides to analyze and localize CK2-dependent phosphorylation of TRPM3 channels. The functional consequences of CK2 phosphorylation upon TRPM3-mediated Ca²⁺ entry were investigated in Fura-2 Ca²⁺-imaging experiments. Recombinant TRPM3α2 channels expressed in HEK293 cells displayed enhanced Ca²⁺ entry in the presence of the CK2 inhibitor CX-4945 and their activity was strongly reduced after CK2 overexpression. TRPM3α2 channels were phosphorylated by CK2 in vitro at serine residue 1172. Accordingly, a TRPM3α2 S₁₁₇₂A mutant displayed enhanced Ca²⁺ entry. The TRPM3-mediated Ca²⁺ entry in INS-1 β-cells was also strongly increased in the presence of CX-4945 and reduced after overexpression of CK2. Our study shows that CK2-mediated phosphorylation controls TRPM3 channel activity in INS-1 β-cells.     

Mutation of recombinant catalytic subunit {alpha} of the protein kinase CK2 that affects catalytic efficiency and specificity

In order to understand better the structural and functionalrelations between protein kinase CK2 catalytic subunit, thetriphosphate moiety of ATP, the catalytic metal and the peptidicsubstrate, we built a structural model of Yarrowia lipolyticaprotein kinase CK2 catalytic subunit using the recently solvedthree-dimensional structure of the maize enzyme and the structureof cAMP-dependent protein kinase peptidic inhibitor (1CDK) astemplates. The overall structure of the catalytic subunit isclose to the structure solved by Niefind et al. It comprisestwo lobes, which move relative to each other. The peptide usedas substrate is tightly bound to the enzyme, at specific locations.Molecular dynamic calculations in combination with the studyof the structural model led us to identify amino acid residuesclose to the triphosphate moiety of ATP and a residue sufficientlyfar from the peptide that could be mutated so as to modify thespecificity of the enzyme. Site-directed mutagenesis was usedto replace by charged residues both glycine-48, a residue locatedwithin the glycine-rich loop, involved in binding of ATP phosphatemoiety, and glycine-177, a residue close to the active site.Kinetic properties of purified wild-type and mutated subunitswere studied with respect to ATP, MgCl₂ and protein kinase CK2specific peptide substrates. The catalytic efficiency of theG48D mutant increased by factors of 4 for ATP and 17.5 for theRRRADDSDDDDD peptide. The mutant G48K had a low activity withATP and no detectable activity with peptide substrates and wasalso inhibited by magnesium. An increased velocity of ADP releaseby G48D and the building of an electrostatic barrier betweenATP and the peptidic substrate in G48K could explain these results.The kinetic properties of the mutant G177K with ATP were notaffected, but the catalytic efficiency for the RRRADDSDDDDDsubstrate increased sixfold. Lysine 177 could interact withthe lysine-rich cluster involved in the specificity of proteinkinase CK2 towards acidic substrate, thereby increasing itsactivity.     

Study of third‐order optical nonlinearities of substituted hydrazones in PMMA

We investigated the third‐order nonlinear optical properties of donor–acceptor substituted hydrazones doped into PMMA matrix using single beam Z‐scan technique at 532 nm. The magnitude of third‐order susceptibility, χ⁽³⁾ is of the order of 10⁻¹³esu. The nonlinear refractive index, n₂ and the molecular second order hyperpolarisability, γ_h are of the order of 10⁻¹¹esu and 10⁻³²esu, respectively. The compounds exhibit larger third‐order NLO properties in PMMA host when compared to the pure compounds. The study reveals that these hydrazones are a good candidate of nonlinear optical materials for photonic applications. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Experimental and Computational Evaluation of Piperonylic Acid Derived Hydrazones Bearing Isatin Moieties as Dual Inhibitors of Cholinesterases and Monoamine Oxidases

A set of piperonylic acid derived hydrazones with variable isatin moieties was synthesized and evaluated for their inhibitory activity against the enzymes acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and monoamine oxidases A and B (MAO-A/B). The results of in vitro studies revealed IC₅₀ values in the micromolar range, with the majority of the compounds showing selectivity for the MAO-B isoform. N-[2-Oxo-1-(prop-2-ynyl)indolin-3-ylidene]benzo[d][1,3]dioxole-5-carbohydrazide ( 3 ) was identified as a lead AChE inhibitor with IC₅₀=0.052±0.006 μm . N-[(3E)-5-chloro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-2H-1,3-benzodioxole-5-carbohydrazide ( 2 ) was the lead MAO-B inhibitor with IC₅₀=0.034±0.007 μm , and showed 50 times greater selectivity for MAO-B over MAO-A. The kinetic studies revealed that compounds 2 and 3 displayed competitive and reversible inhibition of AChE and MAO-B, respectively. The molecular docking studies revealed the significance of hydrophobic interactions in the active site pocket of the enzymes under investigation. Further optimization studies might lead to the development of potential neurotherapeutic agents.     

SOLVENT EXTRACTION OF SOME TERVALENT LANTHAN01DS WITH 5-BR0M0-, 3,5-DIBROMO- AND 5-NITROSALICYLALDEHYDE ACETOHYDRAZONES

ABSTRACT

5-Bromo-, 3,5-dibromo- and 5-nitrosalicylaldehyde acetohydrazones (BSAH, DBSAH and NSAH, respectively, or H₂L) were synthesized. The extraction of lanthanoid ions (Ln3+) including praseodymium, europium and ytterbium ions into 1,2-dichloroethane with the synthesized hydrazones in the presence of both tri-n-butylphos-phate (TBP) and perchlorate has been investigated. The extracted species were (Ln³⁺)(HLP^?)₂(TBP)₃(CI₄ ^?) for all the hydrazones. The acid dissociation constants, Ka, and the partition coefficients, K_o, of the hydrazones and the extraction constants, K_ex, of the lanthanoid complexes were determined. The introduction of electron-withdrawing bromo or nitro groups to the 3- and/or 5-position of the benzene ring in salicylaldehyde acetohydrazone (SAH), the mother compound of the synthesized hydrazones, was very effective for increasing not only the K_ex value of SAH but also its K_D value and the K_ex values of its lanthanoid complexes. Taking into consideration the above-mentioned three constants, of the synthesized hydrazones DBSAH was the most balanced and recommend-able extractant for the lanthanoids.     

Hydrosoluble Benzo[e]pyridoindolones as Potent Inhibitors of Aurora Kinases

Aurora kinases play an essential role in mitotic progression and are potentially druggable targets in cancer therapy. We identified benzo[e]pyridoindoles (BePI) as powerful aurora kinase inhibitors. Their efficiency was demonstrated both in enzymatic inhibition studies and in cell culture assays. New BePI molecules were synthesized, and a structure–activity relationship study was conducted with the aim of improving the activity and solubility of the lead compound. Tetracyclic BePI derivatives are characterized by a particular curved shape, and the presence of an oxo group on the pyridine ring was found to be required for aurora kinase B inhibition. New hydrosoluble benzo[e]pyridoindolones were subsequently designed, and their efficacy was tested by a combination of cell‐cycle analysis and time‐lapse experiments in live cells. The most active BePI derivative, 13 b , inhibited the cell cycle, drove cells to polyploidy, and eventually induced apoptosis. It exhibited high antiproliferative activity in HeLa cells with an IC₅₀ value of 63 nM . Relative to compounds tested in clinical trials, this antiproliferative potency places 13 b among the top 10 aurora kinase inhibitors. Our results justify further in vivo evaluation in preclinical animal models of cancer.     

Synthesis and Biological Evaluation of Benzo[b]furans as Inhibitors of Tubulin Polymerization and Inducers of Apoptosis

A series of benzo[b]furans was synthesized with modification at the 5‐position of the benzene ring by introducing C‐linked substituents (aryl, alkenyl, alkynyl, etc.). These compounds were evaluated for their antiproliferative activities, inhibition of tubulin polymerization, and cell‐cycle effects. Some compounds in this series displayed excellent activity in the nanomolar range against lung cancer (A549) and renal cell carcinoma (ACHN) cancer cell lines. (6‐Methoxy‐5‐((4‐methoxyphenyl)ethynyl)‐3‐methylbenzofuran‐2‐yl)(3,4,5‐trimethoxyphenyl)methanone ( 26 ) and (E)‐3‐(6‐methoxy‐3‐methyl‐2‐(1‐(3,4,5‐trimethoxyphenyl)vinyl)benzofuran‐5‐yl)prop‐2‐en‐1‐ol ( 36 ) showed significant activity in the A549 cell line, with IC₅₀ values of 0.08 and 0.06 μM , respectively. G₂/M cell‐cycle arrest and subsequent apoptosis was observed in the A549 cell line after treatment with these compounds. The most active compound in this series, 36 , also inhibited tubulin polymerization with a value similar to that of combretastatin A‐4 (1.95 and 1.86 μM , respectively). Furthermore, detailed biological studies such as Hoechst 33258 staining, DNA fragmentation and caspase‐3 assays, and western blot analyses with the pro‐apoptotic protein Bax and the anti‐apoptotic protein Bcl‐2 also suggested that these compounds induce cell death by apoptosis. Molecular docking studies indicated that compound 36 interacts and binds efficiently with the tubulin protein.     

2‐Substituted 6‐(Het)aryl‐7‐deazapurine Ribonucleosides: Synthesis,Inhibition of Adenosine Kinases,and Antimycobacterial Activity

A series of 6‐(hetero)aryl‐ or 6‐methyl‐7‐deazapurine ribonucleosides bearing a substituent at position 2 (Cl, F, NH₂, or CH₃) were prepared by cross‐coupling reactions at position 6 and functional group transformations at position 2. Cytostatic, antiviral, and antimicrobial activity assays were performed. The title compounds were observed to be potent and selective inhibitors of Mycobacterium tuberculosis adenosine kinase (ADK), but not human ADK; moreover, they were found to be non‐cytotoxic. The antimycobacterial activities against M. tuberculosis, however, were only moderate. The reason for this could be due to either poor uptake through the cell wall or to parallel biosynthesis of adenosine monophosphate by the salvage pathway.     

Discovery of Akt Kinase Inhibitors through Structure-Based Virtual Screening and Their Evaluation as Potential Anticancer Agents

Akt acts as a pivotal regulator in the PI3K/Akt signaling pathway and represents a potential drug target for cancer therapy. To search for new inhibitors of Akt kinase, we performed a structure-based virtual screening using the DOCK 4.0 program and the X-ray crystal structure of human Akt kinase. From the virtual screening, 48 compounds were selected and subjected to the Akt kinase inhibition assay. Twenty-six of the test compounds showed more potent inhibitory effects on Akt kinase than the reference compound, H-89. These 26 compounds were further evaluated for their cytotoxicity against HCT-116 human colon cancer cells and HEK-293 normal human embryonic kidney cells. Twelve compounds were found to display more potent or comparable cytotoxic activity compared to compound H-89 against HCT-116 colon cancer cells. The best results were obtained with Compounds a46 and a48 having IC₅₀ values (for HCT-116) of 11.1 and 9.5 µM, respectively, and selectivity indices (IC₅₀ for HEK-293/IC₅₀ for HCT-116) of 12.5 and 16.1, respectively. Through structure-based virtual screening and biological evaluations, we have successfully identified several new Akt inhibitors that displayed cytotoxic activity against HCT-116 human colon cancer cells. Especially, Compounds a46 and a48 may serve as useful lead compounds for further development of new anticancer agents.     

New 3-Aryl-2-(2-thienyl)acrylonitriles with High Activity Against Hepatoma Cells

New 2-(thien-2-yl)-acrylonitriles with putative kinase inhibitory activity were prepared and tested for their antineoplastic efficacy in hepatoma models. Four out of the 14 derivatives were shown to inhibit hepatoma cell proliferation at (sub-)micromolar concentrations with IC₅₀ values below that of the clinically relevant multikinase inhibitor sorafenib, which served as a reference. Colony formation assays as well as primary in vivo examinations of hepatoma tumors grown on the chorioallantoic membrane of fertilized chicken eggs (CAM assay) confirmed the excellent antineoplastic efficacy of the new derivatives. Their mode of action included an induction of apoptotic capsase-3 activity, while no contribution of unspecific cytotoxic effects was observed in LDH-release measurements. Kinase profiling of cancer relevant protein kinases identified the two 3-aryl-2-(thien-2-yl)acrylonitrile derivatives 1b and 1c as (multi-)kinase inhibitors with a preferential activity against the VEGFR-2 tyrosine kinase. Additional bioinformatic analysis of the VEGFR-2 binding modes by docking and molecular dynamics calculations supported the experimental findings and indicated that the hydroxy group of 1c might be crucial for its distinct inhibitory potency against VEGFR-2. Forthcoming studies will further unveil the underlying mode of action of the promising new derivatives as well as their suitability as an urgently needed novel approach in HCC treatment.     

Identification,SAR Studies,and X‐ray Co‐crystallographic Analysis of a Novel Furanopyrimidine Aurora Kinase A Inhibitor

Herein we reveal a simple method for the identification of novel Aurora kinase A inhibitors through substructure searching of an in‐house compound library to select compounds for testing. A hydrazone fragment conferring Aurora kinase activity and heterocyclic rings most frequently reported in kinase inhibitors were used as substructure queries to filter the in‐house compound library collection prior to testing. Five new series of Aurora kinase inhibitors were identified through this strategy, with IC₅₀ values ranging from ～300 nM to ～15 μM , by testing only 133 compounds from a database of ～125 000 compounds. Structure–activity relationship studies and X‐ray co‐crystallographic analysis of the most potent compound, a furanopyrimidine derivative with an IC₅₀ value of 309 nM toward Aurora kinase A, were carried out. The knowledge gained through these studies could help in the future design of potent Aurora kinase inhibitors.