Advanced preparation of fragment libraries enabled by oligonucleotide-modified 2',3'-dideoxynucleotides.

The ever-growing demand for inexpensive, rapid, and accurate exploration of genomes calls for refinement of existing sequencing techniques. The development of next-generation sequencing (NGS) was a revolutionary milestone in genome analysis. While modified nucleotides already were inherent tools in sequencing and imaging, further modification of nucleotides enabled the expansion into even more diverse applications. Herein we describe the design and synthesis of oligonucleotide-tethered 2',3'-dideoxynucleotide (ddONNTP) terminators bearing universal priming sites attached to the nucleobase, as well as their enzymatic incorporation and performance in read-through assays. In the context of NGS library preparation, the incorporation of ddONNTP fulfills two requirements at once: the fragmentation step is integrated into the workflow and the obtained fragments are readily labeled by platform-specific adapters. DNA polymerases can incorporate ddONNTP nucleotides, as shown by primer extension assays. More importantly, reading through the unnatural linkage during DNA synthesis was demonstrated, with 25-30% efficiency in single-cycle extension.

CKT0353, a novel microtubule targeting agent, overcomes paclitaxel induced resistance in cancer cells.

Microtubule targeting agents (MTAs) are extensively used in cancer treatment and many have achieved substantial clinical success. In recent years, targeting microtubules to inhibit cell division has become a widespread pharmaceutical approach for treatment of various cancer types. Nevertheless, the development of multidrug resistance (MDR) in cancer remains a major obstacle for successful application of these agents. Herein, we provided the evidence that CKT0353, α-branched α,ß-unsaturated ketone, possesses the capacity to successfully evade the MDR phenotype as an MTA. CKT0353 induced G2/M phase arrest, delayed cell division via spindle assembly checkpoint activation, disrupted the mitotic spindle formation and depolymerized microtubules in human breast, cervix, and colorectal carcinoma cells. Molecular docking analysis revealed that CKT0353 binds at the nocodazole binding domain of ß-tubulin. Furthermore, CKT0353 triggered apoptosis via caspase-dependent mechanism. In addition, P-glycoprotein overexpressing colorectal carcinoma cells showed higher sensitivity to this agent when compared to their sensitive counterpart, demonstrating the ability of CKT0353 to overcome this classic MDR mechanism involved in resistance to various MTAs. Taken together, these findings suggest that CKT0353 is an excellent candidate for further optimization as a therapeutic agent against tumors with MDR phenotype.

Improving the Hsp90 Inhibitors Containing 4-(2,4-Dihydroxyphenyl)-1,2,3-thiadiazole Scaffold: Synthesis, Affinity and Effect on Cancer Cells.

BACKGROUND: Human Hsp90 chaperone inhibitors are known to be potential anticancer drugs. Previously we have shown a couple of 5-aryl-4-(2,4-dihydroxyphenyl)-1,2,3-thiadiazoles to be promising anticancer agents. OBJECTIVE: To improve the compounds containing 4-(2,4-dihydroxyphenyl)-1,2,3-thiadiazole scaffold as human Hsp90 inhibitors. METHOD: We employed chemical synthesis to obtain new compounds and assayed their binding to Hsp90 using the fluorescence thermal shift assay and used MTT assays to determine their effect on cancer cells. RESULTS: A series of compounds containing the 4-(2,4-dihydroxyphenyl)-1,2,3-thiadiazole scaffold were synthesized as Hsp90 inhibitors. Analysis of their binding to the recombinant N-terminal domain of Hsp90 revealed that four of these compounds bound to Hsp90 with Kd of 0.6 to 0.8 nM. The compounds fully inhibited the growth of all tested cancer cell lines: A549 (lung adenocarcinoma), IGR39 (melanoma), and U87 (glioblastoma), with the effective antiproliferative concentration (EC50) of the compounds reaching 0.35 µM. CONCLUSION: This series of 14 novel and efficient Hsp90 inhibitors provided additional information on the structure-activity relationship of Hsp90 inhibitors and may be further developed into drugs targeting Hsp90.

Synthesis and antiproliferative activity of α-branched α,ß-unsaturated ketones in human hematological and solid cancer cell lines.

A series of α-branched α,ß-unsaturated ketones were prepared via boron trifluoride etherate mediated reaction between arylalkynes and carboxaldehydes. The evaluation of the antiproliferative activity over hematological (NB4) and solid cancer (A549, MCF-7) cell lines provided a structure-activity relationship. 5-Parameter QSAR equations were built which were able to explain 80%-92% of the variance in activity. The resulting selective lead compound showed IC50 value 0.6 µM against the hematological cell line and did not cause apoptosis, but blocked cell cycle in G0/G1. Moreover, it was demonstrated that this compound enhances and accelerates retinoic acid induced granulocytic differentiation.

Divergent pathways and competitive mechanisms of metathesis reactions between 3-arylprop-2-ynyl esters and aldehydes: an experimental and theoretical study.

Mechanistic studies of the reaction between 3-arylprop-2-ynyl esters and aldehydes catalyzed by BF3 ⋅Et2 O were performed by isotopic labeling experiments and quantum chemical calculations. The reactions are shown to proceed by either a classical alkyne-carbonyl metathesis route or an unprecedented addition-rearrangement cascade. Depending on the structure of the starting materials and the reaction conditions, the products of these reactions can be Morita-Baylis-Hillman (MBH) adducts that are unavailable by traditional MBH reactions or E- and Z-α,ß-unsaturated ketones. (18) O-Labeling studies suggested the existence of two different reaction pathways to the products. These pathways were further examined by quantum chemical calculations that employed the DFT(wB97XD)/6-311+G(2d,p) method, together with the conductor-like screening model for realistic solvation (COSMO-RS). By using the wB97XD functional, the accuracy of the computed data is estimated to be 1-2 kcal mol(-1) , shown by the careful benchmarking of various DFT functionals against coupled cluster calculations at the CCSD(T)/aug-cc-pVTZ level of theory. Indeed, most of the experimental data were reproduced and explained by theory and it was convincingly shown that the branching point between the two distinct mechanisms is the formation of the first intermediate on the reaction pathway: either the four-membered oxete or the six-membered zwitterion. The deep mechanistic understanding of these reactions opens new synthetic avenues to chemically and biologically important α,ß-unsaturated ketones.

Formation of condensed 1H-pyrrol-2-ylphosphonates and 1,2-dihydropyridin-2-ylphosphonates via Kabachnik-Fields reaction of acetylenic aldehydes and subsequent 5-exo-dig or 6-endo-dig cyclizations.

Kabachnik-Fields reactions of various carbocyclic or heterocyclic acetylenic aldehydes together with subsequent Lewis acid catalyzed cyclizations have been studied. It was found that 5-exo-dig versus 6-endo-dig cyclization mode strongly depends on the structure of starting materials. Thus, nonaromatic acetylenic α-anilinomethylphosphonates underwent gold(III)-catalyzed or iodine-mediated 5-exo-dig cyclization to 1H-pyrrol-2-ylphosphonates. In contrast, electron-withdrawing heteroaromatic substrates formed 1,2-dihydropyridin-2-ylphosphonate ring containing materials via an exclusive 6-endo-dig ring-closure process. The dual mode of cyclization is possible only for α-amino (2-alkynylphenyl)methylphosphonates containing a benzene ring.

Rearrangements of propargylic esters can be induced by some electrophiles.

An electrophile-induced rearrangement of propargylic esters without need of transition catalysis is possible. In particular, this observation provides a mild, economic, and effective method for the introduction of benzyl ether derivatives to access functionalized α,ß-unsaturated ketones. Preliminary mechanistic studies suggest that this rearrangement undergoes an intramolecular 1,3-acyloxy shift.

Synthesis and antiproliferative activity of α-branched α,ß-unsaturated ketones.

A series of α-branched α,ß-unsaturated ketones were prepared in a straightforward manner by the acid catalyzed coupling between arylalkynes and carbaldehydes. The method also allows producing as side product chalcone analogs bearing an additional α,ß-unsaturated arylketone in the molecular scaffold. The evaluation of the antiproliferative activity in the human solid tumor cell lines HBL-100 (breast), HeLa (cervix), SW1573 (non-small cell lung), T-47D (breast) and WiDr (colon) provided a structure-activity relationship. Overall, the compounds presented active against the resistant cancer cells T-47D. The resulting lead, displaying an unprecedented chalcone scaffold, showed GI50 values in the range 0.32-0.53 µM against all cell lines tested. The methoxy group present in the lead might play an important role in the activity.

The first example of the Fischer-Hepp type rearrangement in pyrimidines.

A N-nitroso moiety can be used for the activation of chloropyrimidines toward a nucleophilic substitution reaction with amines. The subsequent treatment of the obtained products with aq H2SO4 can lead to either N-denitrosation to obtain 4,6-pyrimidinediamines or to a Fischer-Hepp type rearrangement to obtain 5-nitroso-4,6-pyrimidinediamines. It was found that the outcome of the reaction strongly depends on the structure of the pyrimidines. Activation of the pyrimidine ring by three groups with a positive mesomeric effect is crucial for the intramolecular nitroso group migration.

A unique cascade reaction between 3-arylprop-2-inylcarboxylates and benzaldehydes leading to the formation of Morita-Baylis-Hillman adducts.

During an alkyne-carbonyl metathesis reaction between electron-rich 3-arylprop-2-inylcarboxylates and electron-poor benzaldehydes, a smooth migration of carboxylate groups takes place. This unique cascade reaction allows the formation of Morita-Baylis-Hillman (MBH) adducts unavailable via a traditional MBH reaction.

Co-crystalization and in vitro biological characterization of 5-aryl-4-(5-substituted-2-4-dihydroxyphenyl)-1,2,3-thiadiazole Hsp90 inhibitors.

A potential therapeutic strategy for targeting cancer that has gained much interest is the inhibition of the ATP binding and ATPase activity of the molecular chaperone Hsp90. We have determined the structure of the human Hsp90α N-terminal domain in complex with a series of 5-aryl-4-(5-substituted-2-4-dihydroxyphenyl)-1,2,3-thiadiazoles. The structures provide the molecular details for the activity of these inhibitors. One of these inhibitors, ICPD 34, causes a structural change that affects a mobile loop, which adopts a conformation similar to that seen in complexes with ADP, rather than the conformation generally seen with the pyrazole/isoxazole-resorcinol class of inhibitors. Competitive binding to the Hsp90 N-terminal domain was observed in a biochemical assay, and these compounds showed antiproliferative activity and induced apoptosis in the HCT116 human colon cancer cell line. These inhibitors also caused induction of the heat shock response with the upregulation of Hsp72 and Hsp27 protein expression and the depletion of Hsp90 clients, CRAF, ERBB2 and CDK4, thus confirming that antiproliferative activity was through the inhibition of Hsp90. The presence of increased levels of the cleavage product of PARP indicated apoptosis in response to Hsp90 inhibitors. This work provides a framework for the further optimization of thiadiazole inhibitors of Hsp90. Importantly, we demonstrate that the thiadiazole inhibitors display a more limited core set of interactions relative to the clinical trial candidate NVP-AUY922, and consequently may be less susceptible to resistance derived through mutations in Hsp90.

A modular approach to trim cellular targets in anticancer drug discovery.

A Phenotypic Drug Discovery strategy was applied to study a set of pyrimidine analogs prepared by means of intramolecular oxidation-reduction reactions of N-substituted-N-(2,6-disubstituted-5-nitro-4-pyrimidinyl)aminoacetic acid methyl esters in basic media. The combined and rational use of specific assays allowed in short time reducing from all possible cellular targets to those involved in metaphase to anaphase transition.

Antiproliferative activity of novel benzo[b][1,6]naphthyridines in human solid tumor cell lines.

A series of 2-substituted 1,2-dihydro-3-phenyl-1-(trichloromethyl)benzo[b][1,6]naphthyridines were synthesized and their in vitro antiproliferative activities were examined against human solid tumor cell lines and relevant strains of bacteria and Candida. The compounds induced considerably growth inhibition in all cancer cell lines, whilst showed inactive against microbial strains. Furthermore, we found analog 2-ethoxy-1H-pyrano[4,3-b]quinoline as selective inhibitor of microbial strains.

Synthesis and antiproliferative activity of 2,4-disubstituted 6-aryl-7H-pyrrolo[3,2-d]pyrimidin-7-one 5-oxides.

A series of 2,4-disubstituted 6-aryl-7H-pyrrolo[3,2-d]pyrimidin-7-one 5-oxides were synthesized and in vitro antiproliferative activities were examined in the human solid tumor cell lines A2780, HBL-100, HeLa, SW1573, T-47D, and WiDr. The most potent analog induced considerably growth inhibition in the range 0.35-2.0microM. Cell cycle studies in the breast and lung cancer cells revealed arrest in the G(2)/M compartment. The results showed that the title compounds bearing alkylamino or dialkylamino moieties in position 2 of the pyrimidine ring are more active than those bearing hydrogen or methylthio groups.

5-Aryl-4-(5-substituted-2,4-dihydroxyphenyl)-1,2,3-thiadiazoles as inhibitors of Hsp90 chaperone.

A series of 5-aryl-4-(5-substituted-2,4-dihydroxyphenyl)-1,2,3-thiadiazoles were synthesized and their binding to several constructs of human Hsp90 chaperone measured by isothermal titration calorimetry (ITC). The most potent compound bound Hsp90 with the dissociation constant of about 5 nM.