Petr Nauš

6-(Het)aryl-7-Deazapurine Ribonucleosides as Novel Potent Cytostatic Agents

A series of novel 7-deazapurine ribonucleosides bearing an alkyl, aryl, or hetaryl group in position 6 and H, F, or Cl atom in position 7 has been prepared either by Pd-catalyzed cross-coupling reactions of the corresponding protected 6-chloro-(7-halogenated-)7-deazapurine ribonucleosides with alkyl- or (het)arylorganometallics followed by deprotection, or by single-step aqueous phase cross-coupling reactions of unprotected 6-chloro-(7-halogenated-)7-deazapurine ribonucleosides with (het)arylboronic acids. Significant cytostatic effect was detected with a substantial proportion of the prepared compounds. The most potent were 7-H or 7-F derivatives of 6-furyl- or 6-thienyl-7-deazapurines displaying cytostatic activity in multiple cancer cell lines with a geometric mean of 50% growth inhibition concentration ranging from 16 to 96 nM, a potency comparable to or better than that of the nucleoside analogue clofarabine. Intracellular phosphorylation to mono- and triphosphates and the inhibition of total RNA synthesis was demonstrated in preliminary study of metabolism and mechanism of action studies.

Synthesis and Significant Cytostatic Activity of 7-Hetaryl-7-deazaadenosines

A series of 7-aryl- and 7-hetaryl-7-deazaadenosines was 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.

Synthesis, Cytostatic, Antimicrobial, and Anti-HCV Activity of 6-Substituted 7-(Het)aryl-7-deazapurine Ribonucleosides

A series of 80 7-(het)aryl- and 7-ethynyl-7-deazapurine ribonucleosides bearing a methoxy, methylsulfanyl, methylamino, dimethylamino, methyl, or oxo group at position 6, or 2,6-disubstituted derivatives bearing a methyl or amino group at position 2, were prepared, and the biological activity of the compounds was studied and compared with that of the parent 7-(het)aryl-7-deazaadenosine series. Several of the compounds, in particular 6-substituted 7-deazapurine derivatives bearing a furyl or ethynyl group at position 7, were significantly cytotoxic at low nanomolar concentrations whereas most were much less potent or inactive. Promising activity was observed with some compounds against Mycobacterium bovis and also against hepatitis C virus in a replicon assay.

CycloSal-phosphate Pronucleotides of Cytostatic 6-(Het)aryl-7-deazapurine Ribonucleosides: Synthesis, Cytostatic Activity, and Inhibition of Adenosine Kinases

A series of cycloSal‐phosphate prodrugs of a recently described new class of nucleoside cytostatics (6‐hetaryl‐7‐deazapurine ribonucleosides) was prepared. The corresponding 2′,3′‐isopropylidene 6‐chloro‐7‐deazapurine nucleosides were converted into 5‐O′‐cycloSal‐phosphates. These underwent a series of Stille or Suzuki cross‐couplings with diverse (het)arylstannanes or ‐boronic acids to yield the protected 6‐(het)aryl‐7‐deazapurine pronucleotides that were subsequently deprotected to give 12 derivatives of free pronucleotides. The in vitro cytostatic effect of the pronucleotides was compared with parent nucleoside analogues. In most cases, the activity of the pronucleotide was similar to or somewhat lower than that of the corresponding parent nucleosides, with the exception of 7‐fluoro pronucleotides 13 a, 13 b, and 13 d, which had exhibited GIC50 values that were improved by one order of magnitude (to the low nanomolar range). The presence of a cycloSal‐phosphate group also influenced selectivity toward various cell lines. Several pronucleotides were found which strongly inhibit human adenosine kinase but only weakly inhibit the MTB adenosine kinase.

Structural Basis for Inhibition of Mycobacterial and Human Adenosine Kinase by 7-Substituted 7-(Het)aryl-7-deazaadenine Ribonucleosides

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.

7-(2-Thienyl)-7-Deazaadenosine (AB61), a New Potent Nucleoside Cytostatic with a Complex Mode of Action

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, 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.

6-Alkyl-, 6-aryl- or 6-hetaryl-7-deazapurine ribonucleosides as inhibitors of human or MTB adenosine kinase and potential antimycobacterial agents

Title 6-alkyl-, 6-aryl- and 6-hetaryl-7-deazapurine ribonucleosides previously known as nanomolar cytostatics were found to be potent inhibitors of either human or mycobacterial (MTB) adenosine kinase (ADK). Several new derivatives bearing bulky substituents at position 6 were non-cytotoxic but selectively inhibited MTB ADK. However, most of the nucleosides (ADK inhibitors) as well as their octadecylphosphate prodrugs were inactive in the whole cell assay of inhibition of Mycobacterium bovis growth. 6-Methyl-7-deazapurine ribonucleoside was found to be a potent antimycobacterial agent.

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.

Abstract 5100: AB61, a new potent nucleoside cytostatic: Molecular mechanisms of action and preclinical activity

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 Rylova, 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