Sugar-modified derivatives of cytostatic 7-(het)aryl-7-deazaadenosines: 2'-C-methylribonucleosides, 2'-deoxy-2'-fluoroarabinonucleosides, arabinonucleosides and 2'-deoxyribonucleosides.

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.

The acyclic 2,4-diaminopyrimidine nucleoside phosphonate acts as a purine mimetic in HIV-1 reverse transcriptase DNA polymerization.

The acyclic pyrimidine nucleoside phosphonate (ANP) phosphonylmethoxyethoxydiaminopyrimidine (PMEO-DAPym) differs from other ANPs in that the aliphatic alkyloxy linker is bound to the C-6 of the 2,4-diaminopyrimidine base through an ether bond, instead of the traditional alkyl linkage to the N-1 or N-9 of the pyrimidine or purine base. In this study, we have analyzed the molecular interactions between PMEO-DAPym-diphosphate (PMEO-DAPym-pp) and the active sites of wild-type (WT) and drug-resistant HIV-1 reverse transcriptase (RT). Pre-steady-state kinetic analyses revealed that PMEO-DAPym-pp is a good substrate for WT HIV-1 RT: its catalytic efficiency of incorporation (k(pol)/K(d)) is only 2- to 3-fold less than that of the corresponding prototype purine nucleotide analogs PMEA-pp or (R)PMPA-pp. HIV-1 RT recognizes PMEO-DAPym-pp as a purine base instead of a pyrimidine base and incorporates it opposite to thymine (in DNA) or uracil (in RNA). Molecular modeling demonstrates that PMEO-DAPym-pp fits into the active site of HIV-1 RT without significant perturbation of key amino acid residues and mimics an open incomplete purine ring that allows the canonical Watson-Crick base pairing to be maintained. PMEO-DAPym-pp is incorporated more efficiently than (R)PMPA-pp by mutant K65R HIV-1 RT and is not as efficiently excised as (R)PMPA by HIV-1 RT containing thymidine analog mutations. Overall, the data revealed that PMEO- DAPym represents the prototype compound of a novel class of pyrimidine acyclic nucleoside phosphonates that are recognized as a purine nucleotide and should form the rational basis for the design and development of novel purine nucleo(s)(t)ide mimetics as potential antiviral or antimetabolic agents.

Inhibition of SAH-hydrolase activity during seed germination leads to deregulation of flowering genes and altered flower morphology in tobacco.

Developmental processes are closely connected to certain states of epigenetic information which, among others, rely on methylation of chromatin. S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are key cofactors of enzymes catalyzing DNA and histone methylation. To study the consequences of altered SAH/SAM levels on plant development we applied 9-(S)-(2,3-dihydroxypropyl)-adenine (DHPA), an inhibitor of SAH-hydrolase, on tobacco seeds during a short phase of germination period (6 days). The transient drug treatment induced: (1) dosage-dependent global DNA hypomethylation mitotically transmitted to adult plants; (2) pleiotropic developmental defects including decreased apical dominance, altered leaf and flower symmetry, flower whorl malformations and reduced fertility; (3) dramatic upregulation of floral organ identity genes NTDEF, NTGLO and NAG1 in leaves. We conclude that temporal SAH-hydrolase inhibition deregulated floral genes expression probably via chromatin methylation changes. The data further show that plants might be particularly sensitive to accurate setting of SAH/SAM levels during critical developmental periods.

8-Aza-7,9-dideazaxanthine acyclic nucleoside phosphonate inhibitors of thymidine phosphorylase.

3- and 8-(8-phosphonooctyl)-8-aza-7,9-dideazaxanthine, and 1,8-bis(8-aza-7,9-dideazaxanthin-8-yl)octane were prepared and found to inhibit thymidine phosphorylase from Escherichia coli, human recombinant TP expressed in V79, and TP purified from human placenta. The IC(50) values ranged from 3.5 to 27µM.

Synthesis and biological activity of desmethoxy analogues of coruscanone A.

A series of simple desmethoxy analogues of coruscanone A was prepared via a novel version of Ti(iPrO)(4)-mediated Knoevenagel condensation of cyclopentenedione with substituted benzaldehydes and cinnamic aldehydes, and the compounds were evaluated for antifungal activity and cytotoxicity. The most potent 2-benzylidenecyclopent-4-ene-1,3-dione possessed antifungal effect comparable to coruscanone A and a somewhat broader spectrum of activity against Candida species. The compound was also superior to fluconazole against several non-albicans Candida sp. Evaluation of the ability of the compound to influence cell proliferation using two different assays showed that 2-benzylidenecyclopent-4-ene-1,3-dione has lower cytotoxicity compared to the natural product.

Phosphoramidate pronucleotides of cytostatic 6-aryl-7-deazapurine ribonucleosides.

A series of O-phenyl methyl-, ethyl- and benzylalanyl phosphoramidate pronucleotides derived from cytostatic 6-aryl-7-deazapurine ribonucleosides were prepared by the cross-coupling reactions of the 2',3'-isopropylidene protected 6-chloro-7-deazapurine ribonucleoside phosphoramidates with (het)arylboronic acids or -stannanes followed by deprotection. Most of the prepared prodrugs exerted in vitro cytostatic effects against both solid tumor and lymphoid cancer cells within low micromolar range of concentrations. These activities were in general weaker or comparable to the activities of the parent nucleosides. Additional testing of selected prodrugs suggests that the lack of activity improvement over parent nucleosides is not due to the lack of permeability or inefficient catabolism of alanyl-ester by intracellular hydrolases. More likely, active efflux of prodrugs may play a role in their weak cytotoxic activity.

Synthesis and antiviral activity of N9-[3-fluoro-2-(phosphonomethoxy)propyl] analogues derived from N6-substituted adenines and 2,6-diaminopurines.

An efficient method for the synthesis of N(9)-[3-fluoro-2-(phosphonomethoxy)propyl] (FPMP) derivatives of purine bases has been developed. Both (R)- and (S)-enantiomers of the N(6)-substituted FPMP derivatives of adenine and 2,6-diaminopurine were prepared and their anti-human immunodeficiency virus (HIV) and anti-Moloney murine sarcoma virus (MSV) activity was evaluated. Whereas none of the 6-substituted FPMPA derivatives showed any antiviral activity, several FPMPDAP derivatives had a moderate antiretroviral activity. Moreover, the data obtained from the study of the substrate activity of the active derivatives towards N(6)-methyl-AMP aminohydrolase support the notion that the studied N(6)-substituted FPMPDAP derivatives act as prodrugs of the antiretroviral FPMPG analogues.

3,5-disubstituted pyranone analogues of highly antifungally active furanones: conversion of biological effect from antifungal to cytostatic.

A series of 3-aryl-5-acyloxymethyl-5,6-dihydro-2H-pyran-2-ones, related to highly antifungally active butenolides, was synthesized via cyclization of substituted δ-hydroxy acids as the key step, and evaluated for their in vitro antifungal activity and cytostatic activity. While the extension of the furanone ring to pyranone led to a complete loss of the antifungal effect, some of the compounds displayed promising effect against several cell lines, including the resistant colorectal carcinoma cells.

Structural diversity of nucleoside phosphonic acids as a key factor in the discovery of potent inhibitors of rat T-cell lymphoma thymidine phosphorylase.

Structurally diverse, sugar-modified, thymine-containing nucleoside phosphonic acids were evaluated for their ability to inhibit thymidine phosphorylase (TP, EC 2.4.2.4) purified from spontaneous T-cell lymphomas of an inbred Sprague-Dawley rat strain. From a large set of tested compounds, among them a number of pyrrolidine-based derivatives, 10 nucleotide analogues with IC(50) values below 1 microM were selected. Out of them, four compounds strongly inhibited the enzyme with IC(50) values lying in a range of 11-45 nM. These most potent compounds might be bi-substrate analogues.

Antifungal 3,5-disubstituted furanones: From 5-acyloxymethyl to 5-alkylidene derivatives.

5-Acetoxymethyl-3-(4-bromophenyl)-2,5-dihydrofuran-2-one previously described as highly antifungally active was found to provide the corresponding 5-methylene derivative via an unusual DMSO-promoted elimination of the ester group at C5 under antifungal assay conditions. Since the latter possessed nearly the same antifungal effect as that originally reported for the former, the 5-acetoxymethyl furanone just served as a precursor of the actual antifungally active species. A few series of compounds with alkyloxy, aryloxy and alkylidene substituents at C5 of the parent furanone structure were therefore prepared and evaluated. In line with the ease of elimination of the substituent from C5, low activities of the 5-alkoxy compounds were observed. On the other hand, their 5-aryloxymethyl congeners were found to be capable of liberating the antifungally active 5-methylene furanone into the testing medium. The antifungal effect of the 5-alkylidene derivatives was highly sensitive to substitution of the alkylidene moiety; a substituent in the allylic position was necessary for a compound to retain high activity. Parallel evaluation of cytostatic activity showed moderate activities of the antifungally active derivatives against HeLa S3 and CCRF-CEM lines. Cell cycle analysis of CCRF-CEM cells following the treatment with 5-methylene-3-(4-bromophenyl)-2,5-dihydrofuran-2-one revealed that this compound is a necrotic agent.

Lasioglossins: three novel antimicrobial peptides from the venom of the eusocial bee Lasioglossum laticeps (Hymenoptera: Halictidae).

Three novel structurally related pentadecapeptides, named lasioglossins, were isolated from the venom of the eusocial bee Lasioglossum laticeps. Their primary sequences were established as H-Val-Asn-Trp-Lys-Lys-Val-Leu-Gly-Lys-Ile-Ile-Lys-Val-Ala-Lys-NH(2) (LL-I), H-Val-Asn-Trp-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Ala-Lys-NH(2) (LL-II) and H-Val-Asn-Trp-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Val-Lys-NH(2) (LL-III). These lasioglossins exhibited potent antimicrobial activity against both Gram-positive and Gram-negative bacteria, low haemolytic and mast cell degranulation activity, and a potency to kill various cancer cells in vitro. The lasioglossin CD spectra were measured in the presence of trifluoroethanol and sodium dodecyl sulfate solution and indicated a high degree of alpha-helical conformation. NMR spectroscopy, which was carried out in trifluoroethanol/water confirmed a curved alpha-helical conformation with a concave hydrophobic and convex hydrophilic side. To understand the role of this bend on biological activity, we studied lasioglossin analogues in which the Gly in the centre of the molecule was replaced by other amino acid residues (Ala, Lys, Pro). The importance of the N-terminal part of the molecule to the antimicrobial activity was revealed through truncation of five residues from both the N and C termini of the LL-III peptide. C-terminal deamidation of LL-III resulted in a drop in antimicrobial activity, but esterification of the C terminus had no effect. Molecular modelling of LL-III and the observed NOE contacts indicated the possible formation of a bifurcated H-bond between hydrogen from the Lys15 CONH peptide bond and one H of the C-terminal CONH(2) to the Ile11 oxygen atom. Such interactions cannot form with C-terminal esterification.

In vivo modulation of angiogenic gene expression by acyclic nucleoside phosphonates PMEDAP and PMEG.

Acyclic nucleoside phosphonates PMEDAP and PMEG modulate expression of selected proangiogenic genes in SD-lymphoma bearing rats. Antiangiogenic efficacy of PMEDAP is relatively weak and is manifested mainly by down-regulation of vascular endothelial growth factor (VEGF) and its receptor VEGFR detectable 24 hours after treatment. Compound PMEG (an active metabolite of the prodrug GS-9219) down-regulates selected proangiogenic genes EGF, FGF, PDGF, VEGF, EGFR, FGFR, PDGFR and VEGFR much more efficiently. Its antiangiogenic potency persists and is more intensive 48 hours after treatment. Findings show that in vivo antitumour efficacy of both antimitotic acyclic nucleoside phosphonates PMEDAP and PMEG consequently affect the angiogenesis in T-cell lymphoma.

Syntheses of N3-substituted thymine acyclic nucleoside phosphonates and a comparison of their inhibitory effect towards thymidine phosphorylase.

A series of N(3)-substituted thymine acyclic nucleoside phosphonates bearing a number of (phosphonomethoxy)alkyl groups were synthesized and investigated for their ability to inhibit the human thymidine phosphorylase expressed in V79 Chinese hamster cells, as well as thymidine phosphorylase from SD-lymphoma, Escherichia coli and human placenta. In comparison to N(1)- substituted analogues which possess a considerable inhibitory activity towards thymidine phosphorylase from SD-lymphoma, the results showed a marginal inhibitory effect of these compounds. None of the presented N(3)-substituted derivatives possess a significant cytostatic activity.

Synthesis and photophysical properties of 7-deaza-2'-deoxyadenosines bearing bipyridine ligands and their Ru(II)-complexes in position 7.

The synthesis of the title 7-deazaadenine 2'-deoxyribonucleosides bearing bipyridine, phenanthroline or terpyridine ligands linked to position 7 via an acetylene or phenylene spacer is reported based on aqueous cross-coupling reactions of unprotected 7-iodo-7-deaza-2'-deoxyadenosine with ligand-functionalized acetylenes or boronic acids. The aqueous cross-coupling with acetylene or boronate building blocks containing the Ru(bpy)(3)-type of complex gave the corresponding Ru-containing nucleosides. Photophysical and electrochemical properties were studied and the most efficient type of complex was selected for future luminescent and redox labelling of DNA. The title nucleosides also showed some cytostatic and anti-HCV activities.

Synthesis, cytostatic and anti-HCV activity of 6-(N-substituted aminomethyl)-, 6-(O-substituted hydroxymethyl)- and 6-(S-substituted sulfanylmethyl)purine nucleosides.

An efficient and facile synthesis of a large series of diverse 6-(N-substituted aminomethyl)-, 6-(O-substituted hydroxymethyl)- and 6-(S-substituted sulfanylmethyl)purine nucleosides (55 examples of both ribo- and 2'-deoxyribonucleosides), aimed at identifying novel homologues of natural nucleosides, was developed. The key transformation involved nucleophilic substitutions of Tol-protected 6-(mesyloxymethyl)purine nucleosides by primary or secondary amines, alcoholates or thiolates. While the 2'-deoxyribonucleosides were inactive, the ribonucleosides exerted considerable cytostatic effects and some anti-HCV activity with low selectivity.

Synthesis, cytostatic, and antiviral activity of novel 6-[2-(dialkylamino)ethyl]-, 6-(2-alkoxyethyl)-, 6-[2-(alkylsulfanyl)ethyl]-, and 6-[2-(dialkylamino)vinyl]purine nucleosides.

An efficient and facile synthesis of a large series of diverse 6-[2-(dialkylamino)vinyl]-, 6-[2-(dialkylamino)ethyl]-, 6-(2-alkoxyethyl)-, and 6-[2-(alkylsulfanyl)ethyl]purine nucleosides (35 examples of both ribo- and 2'-deoxyribonucleosides) was developed. The key transformations involved conjugate nucleophilic additions of amines, alcoholates, or thiolates to Tol-protected 6-alkylylpurine or 6-vinylpurine nucleosides. 6-[(2-Dialkylamino)vinyl]- and some 6-[(2-dialkylamino)ethyl]purine ribonucleosides exerted significant cytostatic effects and some anti-HCV activity with low selectivity.

Discovery of 5-substituted-6-chlorouracils as efficient inhibitors of human thymidine phosphorylase.

Thymidine phosphorylase plays an important role in angiogenesis, which is an attractive target for therapy of cancer and other diseases. In our continuous effort to develop novel inhibitors of thymidine phosphorylase, we have discovered that 6-halouracils substituted at position C5 by certain hydrophobic groups exhibit significant inhibitory activity against this enzyme. The most potent compounds bear a five- or six-membered cyclic substituent containing a pi-electron system at C5 and a chlorine atom attached at C6. 6-Chloro-5-cyclopent-1-en-1-yluracil 7a is the most efficient derivative in this study, with Ki = 0.20 +/- 0.03 microM (Ki/dThdKm = 0.0017) for thymidine phosphorylase expressed in V79 cells and Ki = 0.29 +/- 0.04 microM (Ki/dThdKm = 0.0024) for the enzyme purified from placenta.

Syntheses of pyrimidine acyclic nucleoside phosphonates as potent inhibitors of thymidine phosphorylase (PD-ECGF) from SD-lymphoma.

In the present study, we synthesized a series of pyrimidine acyclic nucleoside phosphonates bearing a number of substituents in C-5 position of uracil moiety and in the N-1-side chain. In addition, we have investigated in particular the novel syntheses of fluorinated derivatives substituted in the N-1-side chain and uracil C-5 position because fluorine-containing substituents are often powerful modifiers of chemical and biological properties. The obtained compounds exhibit a considerable inhibitory potency of thymidine phosphorylase from SD-lymphoma. In contrast, the synthesized phosphonates are not efficient inhibitors of E. coli and human thymidine phosphorylase.

N6-methyl-AMP aminohydrolase activates N6-substituted purine acyclic nucleoside phosphonates.

In this study we present the identification and characterization of the enzyme involved in the N6-cyclopropyl-2,6-diamino-9-[2-(phosphonomethoxy)ethyl]purine (N6-cyclopropyl-PMEDAP) conversion to biologically active 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG) as well as abacavir 5'-phosphate to carbovir 5'-phosphate. This enzyme was purified from rat liver to homogeneity; it appears to be composed from six 42 kDa subunits and its native form has the molecular weight 260 kDa. This so far unknown enzyme catalyzes conversion of both N6-methyl-AMP and N6-methyl-dAMP to IMP and/or dIMP, respectively. The enzyme acts as 6-(N-substituted amino)purine 5'-nucleotide aminohydrolase with the reaction mechanism very similar to AMP deaminase. The enzyme does not deaminate AMP and dAMP, or the corresponding nucleosides. It is inhibited by deoxycoformycin 5'-phosphate but not by deoxycoformycin or erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA).

Cytostatic 6-arylpurine nucleosides. 6. SAR in anti-HCV and cytostatic activity of extended series of 6-hetarylpurine ribonucleosides.

Significant anti-HCV activity of 6-hetarylpurine ribonucleosides has been discovered and is reported here for the first time and compared with cytostatic effect. An extended series of 6-hetarylpurine nucleosides has been prepared by heterocyclizations in position 6 of purine nucleosides or by cross-couplings of 6-chloropurine nucleosides with hetarylboronic acids, -stannanes, or -zinc halides. The most anti-HCV active were purine ribonucleosides bearing pyrrol-3-yl or 2-furyl groups exerting EC(90) = 0.14 and 0.4 microM, respectively.