The potential of thienoguanosine (thG), an isomorphic fluorescent analogue of guanosine, was evaluated as a reporter for probing the structure and dynamics of hTel22, a human telomeric G-quadruplex (G4) sequence. Nine of the 12 guanines directly participating in G-tetrads were individually substituted with thG, and their impact on the hTel22 structure was examined by circular dichroism, thermal melting, and 1H NMR spectroscopy. In Na+ buffer, substitutions at external tetrads maintained the native antiparallel topology and thermal stability, while substitutions within the middle tetrad significantly disrupted the G4 structure. Molecular dynamics simulations supported that thG incorporation in external tetrads is well tolerated, whereas it alters base stacking and Na+ coordination in middle tetrad positions. In K+ buffer, external substitutions favored the antiparallel over the hybrid topology, resulting in a modest destabilization. thG-labeled sequences at external tetrads in both Na+ and K+ buffers exhibited high fluorescence quantum yields, long fluorescence lifetimes, and high sensitivity to local conformation. As an application, thG fluorescence signal proved instrumental in accurately characterizing the kinetics of Na+-induced G4 folding, enabling all kinetically resolved folding steps to be captured from a single fluorescence observable. Overall, thG serves as a remarkably sensitive, minimally perturbing fluorescent guanosine analogue for structural and dynamic studies of G4s. Moreover, its longest lifetime (18.5 to 28.5 ns), at least twice that in DNA duplexes, is suitable for time-gated detection with high signal-to-noise ratio and easy distinction of G4s from duplexes, offering a key advantage for G4-targeting drug discovery.

To fully exploit the potential of isothiazologuanosine (tzG), an isomorphic and isofunctional fluorescent analogue of guanosine, as a probe for DNA and RNA, we characterized its photophysics and in particular its excited-state reactions over a wide pH range (-0.6 to 12) and time scale (100 fs-100 ns) by combining transient absorption and time-correlated single photon counting measurements with quantum mechanical calculations. At acidic pH, the dominant ground-state species tzG-H1-H3+, where the N atoms in positions 1 and 3 are protonated, rapidly converts to the more stable tautomer tzG-H1-H7+ in its excited state. The latter then deprotonates to form the tzG-H1 neutral species with an excited-state pKa* value that differs by three pH units from the ground-state pKa value. The rate constants governing the excited-state reactions and the fluorescence lifetime of each species were all determined. With the exception of intramolecular and solvent relaxations, no excited-state reactions in the femtosecond to nanosecond time scale were, however, observed between the dominant tzG-H1 and tzG-H3 tautomers in equilibrium at neutral pH or for tzG-H1 deprotonation at high pH. Because of the distinct spectra, fluorescence quantum yields and lifetimes of its different protonated and deprotonated forms, tzG is highly responsive over a wide range of acidic (0-5) and basic pH values (8-10). The mechanisms revealed herein will be instrumental for tzG-labelled oligonucleotides in order to interpret proton transfer reactions as well as interactions with specific protein domains, which, due to local electrostatic changes and water exclusion effects, may shift the pKa values of tzG to a more physiologically relevant range.

Supplementary Materials and Methods. Figure S1: MALDI-TOF spectrum of the single-stranded oligonucleotide products of primer extension experiment with human DNA polymerase γ, AB61-TP, primer Prim248 and template (bio)-OligoAterm.

Abstract 7-(2-Thienyl)-7-deazaadenosine (AB61) showed nanomolar cytotoxic activities against various cancer cell lines but only mild (micromolar) activities against normal fibroblasts. The selectivity of AB61 was found to be due to inefficient phosphorylation of AB61 in normal fibroblasts. The phosphorylation of AB61 in the leukemic CCRF-CEM cell line proceeds well and it was shown that AB61 is incorporated into both DNA and RNA, preferentially as a ribonucleotide. It was further confirmed that a triphosphate of AB61 is a substrate for both RNA and DNA polymerases in enzymatic assays. Gene expression analysis suggests that AB61 affects DNA damage pathways and protein translation/folding machinery. Indeed, the formation of large 53BP1 foci was observed in nuclei of AB61-treated U2OS-GFP-53BP1 cells indicating DNA damage. Random incorporation of AB61 into RNA blocked its translation in an in vitro assay and reduction of reporter protein expression was also observed in mice after 4-hour treatment with AB61. AB61 also significantly reduced tumor volume in mice bearing SK- OV-3, BT-549, HT-29 and MDA-MB231 xenografts. The results indicate that AB61 is a promising compound with the unique mechanism of action and deserves further development as an anticancer agent. This work was supported by the Ministry of Education of the Czech Republic (LO1304). Citation Format: Petr Dzubak, Marian Hajduch, Pavla Perlikova, Gabriela Rylová, Petr Naus, Tomas Elbert, Eva Tloustova, Aurelie Bourderioux, Lenka Slavetinska, Kamil Motyka, Dalibor Dolezal, Pawel Znojek, Alice Nova, Monika Harvanova, Michal Siler, Jan Hlavac, Michal Hocek. AB61, a new potent nucleoside cytostatic: Molecular mechanisms of action and preclinical activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5100. doi:10.1158/1538-7445.AM2017-5100

Abstract7-(2-Thienyl)-7-deazaadenosine (AB61) showed nanomolar cytotoxic activities against various cancer cell lines but only mild (micromolar) activities against normal fibroblasts. The selectivity of AB61 was found to be due to inefficient phosphorylation of AB61 in normal fibroblasts. The phosphorylation of AB61 in the leukemic CCRF-CEM cell line proceeds well and it was shown that AB61 is incorporated into both DNA and RNA, preferentially as a ribonucleotide. It was further confirmed that a triphosphate of AB61 is a substrate for both RNA and DNA polymerases in enzymatic assays. Gene expression analysis suggests that AB61 affects DNA damage pathways and protein translation/folding machinery. Indeed, formation of large 53BP1 foci was observed in nuclei of AB61-treated U2OS-GFP-53BP1 cells indicating DNA damage. Random incorporation of AB61 into RNA blocked its translation in an in vitro assay and reduction of reporter protein expression was also observed in mice after 4-hour treatment with AB61. AB61 also significantly reduced tumor volume in mice bearing SK-OV-3, BT-549, and HT-29 xenografts. The results indicate that AB61 is a promising compound with unique mechanism of action and deserves further development as an anticancer agent. Mol Cancer Ther; 15(5); 922–37. ©2016 AACR.

The performance of tert-butanesulfinamides as nitrogen nucleophiles in Pd(0)-catalyzed allylic substitution reactions has been investigated. Metalated N-alkyl and N-acetyl sulfinamides have been identified as suitable partners for the reaction with pi-allyl-palladium complexes. The cross-coupling of N-acetyl tert-butanesulfinamide with 2- or 3-substituted linear allylic carbonates is achieved in the presence of Pd(OAc)(2) (5 mol%) and dppe (7.5 mol%) and does not require an additional base. The reaction proceeds in high yields (59-98%) to produce the corresponding E-configured linear allylic sulfinamides in a totally regioselective and highly diastereoselective manner. The sulfur atom remains configurationally stable throughout the allylation process, and thus the coupling products are obtained in enantiomerically pure form. (C) 2013 Elsevier B. V. All rights reserved.

Adenosine kinase (ADK) from Mycobacterium tuberculosis (Mtb) was selected as a target for design of antimycobacterial nucleosides. Screening of 7-(het)aryl-7-deazaadenine ribonucleosides with Mtb and human (h) ADKs and testing with wild-type and drug-resistant Mtb strains identified specific inhibitors of Mtb ADK with micromolar antimycobacterial activity and low cytotoxicity. X-ray structures of complexes of Mtb and hADKs with 7-ethynyl-7-deazaadenosine showed differences in inhibitor interactions in the adenosine binding sites. 1D (1)H STD NMR experiments revealed that these inhibitors are readily accommodated into the ATP and adenosine binding sites of Mtb ADK, whereas they bind preferentially into the adenosine site of hADK. Occupation of the Mtb ADK ATP site with inhibitors and formation of catalytically less competent semiopen conformation of MtbADK after inhibitor binding in the adenosine site explain the lack of phosphorylation of 7-substituted-7-deazaadenosines. Semiempirical quantum mechanical analysis confirmed different affinity of nucleosides for the Mtb ADK adenosine and ATP sites.

A series of novel sugar-modified derivatives of cytostatic 7-hetaryl-7-deazaadenosines (2'-C-methylribonucleosides, 2'-deoxy-2'-fluoroarabinonucleosides, arabinonucleosides and 2'-deoxyribonucleosides) was prepared and screened for biological activity. The synthesis consisted of preparation of the corresponding sugar-modified 7-iodo-7-deazaadenine nucleosides and their aqueous-phase Suzuki-Miyaura cross-coupling reactions with (het)arylboronic acids or Stille couplings with hetarylstannanes in DMF. The synthesis of 7-iodo-7-deazaadenine nucleosides was based on a glycosidation of 6-chloro-7-iodo-7-deazapurine with a suitable sugar synthon or on an interconversion of 2'-OH stereocenter (for arabinonucleosides). Several examples of 2'-C-Me-ribonucleosides showed moderate anti-HCV activities in a replicon assay accompanied by cytotoxicity. Several 7-hetaryl-7-deazaadenine fluoroarabino- and arabinonucleosides exerted moderate micromolar cytostatic effects. The most active was 7-ethynyl-7-deazaadenine fluoroarabinonucleoside which showed submicromolar antiproliferative activity. However, all the sugar-modified derivatives were less active than the parent ribonucleosides.

A series of 7-aryl- and 7-hetaryl-7-deazaadenosines were prepared by the cross-coupling reactions of unprotected or protected 7-iodo-7-deazaadenosines with (het)arylboronic acids, stannanes, or zinc halides. Nucleosides bearing 5-membered heterocycles at the position 7 exerted potent in vitro antiproliferative effects against a broad panel of hematological and solid tumor cell lines. Cell cycle analysis indicated profound inhibition of RNA synthesis and induction of apoptosis in treated cells. Intracellular conversion to triphosphates has been detected with active compounds. The triphosphate metabolites showed only a weak inhibitory effect on human RNA polymerase II, suggesting potentially other mechanisms for the inhibition of RNA synthesis and quick onset of apoptosis. Initial in vivo evaluation demonstrated an effect of 7-(2-thienyl)-7-deazaadenine ribonucleoside on the survival rate in syngeneic P388D1 mouse leukemia model.

A new route to the first N-methyloxoarcyriaflavin was designed. The compound was obtained by a palladium-catalyzed Stille cross-coupling reaction, followed by an electrophilic cyclization onto a C-2 indolic position as a key step.

In the course of a program aimed at designing new antitumor agents, we were interested in the synthesis of new substituted benzo and pyrido carbazoles. The synthesis was performed through an efficient four-step sequence from a 2-trimethylstannylindole derivative and via C-2 (het)arylindoles. The synthetic sequence was developed using two palladium mediated reactions including, at the end of the synthesis, a direct (het)arylannulation, which led to the desired heterocycles.

We report the synthesis and biological evaluation of new oxophenylarcyriaflavins designed as potential anticancer agents. An efficient synthesis involving palladium-catalyzed Suzuki and Stille reactions is presented, without any indolic protective group. The central ring closure of the scaffold was performed through an electrophilic reaction on the position C-2 of the indole ring. The use of indole and 5-benzyloxyindole, along with substituted phenyl rings, generated three different scaffolds, which were successively exploited to modulate the structure. The cytotoxicity of the newly designed compounds on four cancer cell lines and activities against three kinases (CDK1, CDK5 and GSK3) were evaluated. Several compounds showed a marked cytotoxicity with IC(50) values in the sub-micromolar range, and induced important cell cycle perturbations, with a G2/M arrest. Some compounds revealed DNA binding properties and were found to inhibit topoisomerase-mediated DNA relaxation of supercoiled DNA, but these properties are not mandatory for a cytotoxic action. A novel lead compound (32) has been identified and warrants further investigations.

Le cancer, qui est la deuxieme cause de mortalite en France, est aujourd'hui un probleme de sante publique majeur et fait l'objet de multiples recherches. De nombreuses molecules ont ete synthetisees dans l'optique de trouver des medicaments plus efficaces, plus selectifs et surtout presentant moins d'effets secondaires. Parmi ces molecules se trouve la famille des indolocarbazoles, dont la rebeccamycine et la staurosporine sont les representants les plus connus. Les relations structure-activite (RSA) de cette famille ont ete etudiees. Dans le cadre de la recherche de nouveaux agents cytotoxiques et d'inhibiteurs de kinases toujours plus selectifs, la structure principale des phenylcarbazoles, appartenant a la famille des indolocarbazoles, a ete modifiee par introduction d'une tropone centrale, cycle a 7 chainons porteur d'une fonction carbonyle. Les differentes voies de synthese permettant d'acceder a cette nouvelle famille de composes appeles oxophenylarcyriaflavines ont ete etudiees. La methode de choix retenue pour l'etape finale de la synthese est la cyclisation electrophile en position 2 de l'indole. Cette synthese a ensuite ete generalisee aux composes substitues par des groupements hydroxyles en position 5 de l'indole d'une part et en position 4' et 5' du noyau phenyle d'autre part. Les 17 molecules finales ainsi synthetisees ont subi divers tests biologiques permettant d'etablir les RSA de cette nouvelle famille de composes. Finalement, la methodologie mise au point pour les oxophenylarcyriaflavines a ete etendue a la synthese de la toute premiere famille de composes bisindoliques possedant egalement une tropone centrale.

In the course of a program aimed at designing new antitumor agents, we were interested in the synthesis of mixed structures of maleimidophenyl carbazoles and natural product caulersine as potential CDK inhibitors. This was performed through an efficient four-step sequence starting from indole or 3-formyl-N-Boc indole. 5H-Benzocycloheptaindol-6-one derivatives equipped with a fused maleimide (oxophenylarcyriaflavins) or a methyl ester (benzo analog of caulersine) on the central tropone ring were thus obtained.

New oxophenylarcyriaflavins were synthesized in a few efficient steps. The key steps involved at first a palladium cross-coupling between the 3-bromo-4-(1H-indol-3-yl)1-methylpyrrole-2,5-dione and the 2-formylphenylboronic acid or a methyl 2-trialkylstannylbenzoate, followed by an intramolecular acylation in a C-2 indolic position. All the sequence was carried out without any indolic protective group.

Myrosinase, a thioglucoside glucohydrolase, is the only enzyme able to hydrolyse glucosinolates, a unique family of molecules bearing an anomeric O-sulfated thiohydroximate function. Non-hydrolysable myrosinase inhibitors have been devised and studied for their biological interaction. Diverse modifications of the O-sulfate moiety did not result in a significant inhibitory effect, whereas replacing the D-glucopyrano residue by its carba-analogue allowed inhibition to take place. X-Ray experiments carried out after soaking allowed for the first time inclusion of a non-hydrolysable inhibitor inside the enzymatic pocket. Structural tuning of the aglycon part in its pocket is being used as a guide for the development of simplified and more potent inhibitors.