Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery.

Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.

Two novel classes of fused azaisocytosine-containing congeners as promising drug candidates: Design, synthesis as well as in vitro, ex vivo and in silico studies.

Searching for new less toxic anticancer drug candidates is a big challenge from a medical point of view. The present investigation was aimed at describing two independent synthetic approaches based on isosteric replacements, spectroscopic characteristics, in vitro anticancer and ex vivo antihaemolytic activities of novel molecules (9-22) and correlations between their standardised lipophilicity indices, computed log Paverage values and pharmacokinetic descriptors. Two novel protocols for annelation of the triazinone template on hydrazinylideneimidazolidines (1-8) (showing a high reactivity towards electrophilic reagents, such as ethyl trifluoropyruvate and ethyl 3-methyl-2-oxobutyrate) were developed for the first time, giving rise to two original classes of highly conjugated azaisocytosine-containing molecules (9-16 and 17-22). Both syntheses proceeded under basic conditions to yield the most probable intermediates (e.g. hemiaminals and imines), which in refluxing two-component solvent mixtures or a suitable solvent cyclised through closing the triazinone ring on functionalised imidazolidines in both cases. All fused azaisocytosine-containing congeners were investigated with the purpose of preselecting possible drug candidates with a better selectivity that could be suitable for further more detailed drug development studies. The majority of test molecules revealed strong antiproliferative effects in most tumour cell cultures and they were more cytotoxic against tumour cells than anticancer drug - pemetrexed. These cytotoxicities may be associated with the activation of initiator and executioner caspases (confirmed for compound 12) which are inducers of apoptosis. Simultaneously, three bioisosteres bearing the trifluoromethyl moiety at the C-3 and the ortho substitution at the phenyl ring (10, 12 and 13) proved to be the most promising in terms of selectivity as they were less or equally toxic to normal cells as pemetrexed. It was shown that isosteric replacement of the ethyl group in antitumour active congeners by the trifluoromethyl or isopropyl group was favourable for the selectivity of the designed drug-like molecules. Almost all new compounds revealed the protective effects in an ex vivo model of oxidatively stressed rat erythrocytes (better or comparable than that of ascorbic acid/Trolox), proving that they are safe to red blood cells. The statistically significant and predictive QSAR equations were derived that describe relationships between some pharmacokinetic descriptors (such as log Ka, HSA, fu, brain, Caco-2, log Kp) and lipophilicity parameters of test molecules. Among all molecules with anticancer profile, the possible drug candidates seem to be 10, 12, 13, 19 and 21 which are the least toxic for normal cells, deprived of haemolytic effects on oxidatively-stressed red blood cells and have the optimum pharmacokinetic descriptors in terms of their lipophilicity parameters. Because of a high development potential they should be utilised in further more extended in vivo investigations aimed at developing novel less toxic anticancer agents.

Binding investigation and preliminary optimisation of the 3-amino-1,2,4-triazin-5(2H)-one core for the development of new Fyn inhibitors.

Fyn tyrosine kinase inhibitors are considered potential therapeutic agents for a variety of human cancers. Furthermore, the involvement of Fyn kinase in signalling pathways that lead to severe pathologies, such as Alzheimer's and Parkinson's diseases, has also been demonstrated. In this study, starting from 3-(benzo[d][1,3]dioxol-5-ylamino)-6-methyl-1,2,4-triazin-5(2H)-one (VS6), a hit compound that showed a micromolar inhibition of Fyn (IC50 = 4.8 µM), we computationally investigated the binding interactions of the 3-amino-1,2,4-triazin-5(2H)-one scaffold and started a preliminary hit to lead optimisation. This analysis led us to confirm the hypothesised binding mode of VS6 and to identify a new derivative that is about 6-fold more active than VS6 (compound 3, IC50 = 0.76 µM).

Crystal Structure of Mistletoe Lectin I (ML-I) from Viscum album in Complex with 4-N-Furfurylcytosine at 2.85 Å Resolution.

BACKGROUND: Viscum album (the European mistletoe) is a semi-parasitic plant, which is of high medical interest. It is widely found in Europe, Asia, and North America. It contains at least three distinct lectins (i.e. ML-I, II, and III), varying in molecular mass and specificity. Among them, ML-I is in focus of medical research for various activities, including anti-cancer activities. To understand the molecular basis for such medical applications, a few studies have already addressed the structural and functional analysis of ML-I in complex with ligands. In continuation of these efforts, we are reporting the crystal structure of ML from Viscum album in complex with the nucleic acid oxidation product 4-N-furfurylcytosine (FC) refined to 2.85 Å resolution. FC is known to be involved in different metabolic pathways related to oxidative stress and DNA modification. METHODS: X-ray suitable hexagonal crystals of the ML-I/FC complex were grown within four days at 294 K using the hanging drop vapor diffusion method. Diffraction data were collected up to a resolution of 2.85 Å. The ligand affinity was verified by in-silico docking. RESULTS: The high-resolution structure was refined subsequently to analyze particularly the active site conformation and a binding epitope of 4-N-furfurylcytosine. A distinct 2Fo-Fc electron density at the active site was interpreted as a single FC molecule. The specific binding of FC is achieved also through hydrophobic interactions involving Tyr76A, Tyr115A, Glu165A, and Leu157A of the ML-I A-chain. The binding energy of FC to the active site of ML-I was calculated as well to be -6.03 kcal mol-1. CONCLUSION: In comparison to other reported ML-I complexes, we observed distinct differences in the vicinity of the nucleic acid base binding site upon interaction with FC. Therefore, data obtained will provide new insights in understanding the specificity, inhibition, and cytotoxicity of the ML-I A-chain, and related RIPs.

Direct conjugation of peptides and 5-hydroxymethylcytosine in DNA.

Recent discovery of functional 5-hydroxymethylcytosine in vertebrate genomes prompted for elaboration of methods to localize this modification at the nucleotide resolution level. Among several covalent modification-based approaches, atypical activity of cytosine-5 DNA methyltransferases to couple small molecules to 5-hydroxymethylcytosine stands out for acceptance of broad range of ligands. We went further to explore the possibility for methyltransferase-maintained coupling of compounds possessing autonomous functions. Functionalization of DNA was achieved by direct conjugation of chemically synthesized peptides of regular structure. Sequence, residue, and position-specific coupling of DNA containing 5-hydroxymethylcytosine and different peptides has been demonstrated, with the nature of the resulting conjugates confirmed by protease treatment and mass spectrometry. Coupling products were compatible with affinity-driven separation from the unmodified DNA. This approach highlights an emerging avenue toward the enzymatic, sequence-specific DNA functionalization, enabling a single step merge of the DNA and peptide moieties into a bifunctional entity.

Synthesis and complementary self-association of novel lipophilic π-conjugated nucleoside oligomers.

A series of lipophilic nucleosides comprising natural and non-natural bases that are π-conjugated to a short oligophenylene-ethynylene fragment has been synthesized. These bases comprise guanosine, isoguanosine, and 2-aminoadenosine as purine heterocycles, and cytidine, isocytosine and uridine as complementary pyrimidine bases. The hydrogen-bonding dimerization and association processes between complementary bases were also studied by (1)H NMR and absorption spectroscopy in order to obtain the relevant association constants.

New cytosine derivatives as inhibitors of DNA methylation.

DNA cytosine methylation catalyzed by DNA methyltransferase 1 (DNMT1) is an epigenetic method of gene expression regulation and development. Changes in methylation pattern lead to carcinogenesis. Inhibition of DNMT1 activity could be a good strategy of safe and efficient epigenetic therapy. In this work, we present a novel group of cytosine analogs as inhibitors of DNA methylation. We show new methods of synthesis and their effect on in vitro reaction of DNA methylation. Almost all of analyzed compounds inhibit DNA methyltransferase activity in the competitive manner. K(i) values for the most potent compound 4-N-furfuryl-5,6-dihydroazacytosines is 0.7 µM. These compounds cause also a decrease of 5-methylcytosine (m(5)C) level in DNA of mammalian HeLa and HEK293 cells.

Cytosine derivatives form hemiprotonated dimers in solution and the gas phase.

Hemiprotonated dimers of cytosine derivatives, implicated in the formation of the i-motif of DNA, have been created in solution and the gas phase. The mechanism of dimerization has been analyzed by mass spectrometry and multidimensional NMR spectroscopy.

The new 5- or 6-azapyrimidine and cyanuric acid derivatives of l-ascorbic acid bearing the free C-5 hydroxy or C-4 amino group at the ethylenic spacer: CD-spectral absolute configuration determination and biological activity evaluations.

We report on the synthesis of the novel types of cytosine and 5-azacytosine (1-9), uracil and 6-azauracil (13-18) and cyanuric acid (19-22) derivatives of l-ascorbic acid, and on their cytostatic activity evaluation in human malignant tumour cell lines vs. their cytotoxic effects on human normal fibroblasts (WI38). The CD spectra analysis revealed that cytosine (5 and 6), uracil (14-16), 6-azauracil (17) and cyanuric acid (21) derivatives of l-ascorbic acid bearing free amino group at ethylenic spacer existed as a racemic mixture of enantiomers, whereas L-ascorbic derivatives containing the C-5 substituted hydroxy group at the ethylenic spacer were obtained in (4R, 5S) enantiomeric form. The stereochemistry of 6-azauracil derivative of l-ascorbic acid (13) was confirmed by X-ray crystal structure analysis. The molecules are self-assembled by one N-H⋯O hydrogen bond, two C-H⋯O hydrogen bonds and two C-H⋯π interactions into three-dimensional framework. Cytostatic activity evaluation indicated that compounds did not show distinctive antiproliferative effects on tested cell line panel. However, the cytosine derivative of l-ascorbic acid (1) containing the C4-C5 double bond conjugated with the lactone moiety produced rather marked growth inhibitory effect on hepatocellular carcinoma (HepG2), metastatic breast epithelial carcinoma (MCF-7) and cervical carcinoma (HeLa) cell lines at micromolar concentrations, but also exerted strong cytostatic effect on WI38. 5-Azacytosine derivative of l-ascorbic acid (2) with a double bond at the C4-C5 conjugated with the lactone moiety displayed potent antitumour activity against tested tumour cell lines with meanIC(50) values ranging from 0.92 to 5.91 µM. However, this compound also exhibited pronounced cytotoxicity towards WI38. Flow cytometric analysis of the cell cycle revealed that compound 2 triggers S phase arrest, which clearly demonstrates its interference with DNA replication, a key event of cell proliferation. Marked anticancer efficacy of compound 2 supports further in vivo investigation into its possible clinical utility.

Synthesis, metabolic stability and antiviral evaluation of various alkoxyalkyl esters of cidofovir and 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine.

Alkoxyalkyl esters of cidofovir (CDV) are orally active agents which inhibit the replication of a variety of double stranded DNA (dsDNA) viruses including variola, vaccinia, ectromelia, herpes simplex virus, cytomegalovirus, adenovirus and others. One of these compounds, hexadecyloxypropyl-CDV (HDP-CDV, CMX001) is in clinical development for prevention and treatment of poxvirus infection, vaccination complications, and for infections caused by cytomegalovirus, adenovirus, herpesviruses and other dsDNA viruses. This class of lipid analogs is potentially prone to undergo omega oxidation of the alkyl moiety which can lead to a short chain carboxylic acid lacking antiviral activity. To address this issue, we synthesized a series of alkoxyalkyl or alkyl glycerol esters of CDV and (S)-HPMPA having modifications in the structure of the alkyl residue. Antiviral activity was assessed in cells infected with vaccinia, cowpox or ectromelia viruses. Metabolic stability was determined in S9 membrane fractions from rat, guinea pig, monkey and human liver. All compounds had substantial antiviral activity in cells infected with vaccinia, cowpox or ectromelia. Metabolic stability was lowest in monkey liver S9 incubations where rapid disappearance of HDP-CDV and HDP-(S)-HPMPA was noted. Metabolic stability in monkey preparations increased substantially when a ω-1 methyl group (15-methyl-HDP-CDV) or a terminal cyclopropyl residue (14-cyclopropyl-tetradecyloxypropyl-CDV) was present in the alkyl chain. The most stable compound was 1-O-octadecyl-2-O-benzyl-sn-glycero-3-CDV (ODBG-CDV) which was not metabolized extensively by monkey liver S9. In rat, guinea pig or human liver S9 incubations, most of the modified antiviral compounds were considerably more stable.

Synthesis of 4,6-dideoxy-3-fluoro-2-keto-beta-D-glucopyranosyl analogues of 5-fluorouracil, N6-benzoyl adenine, uracil, thymine, N4-benzoyl cytosine and evaluation of their antitumor activities.

The synthesis of the unsaturated 4,6-dideoxy-3-fluoro-2-keto-beta-d-glucopyranosyl nucleosides of 5-fluorouracil (6a), N(6)-benzoyl adenine (6b), uracil (6c), thymine (6d) and N(4)-benzoyl cytosine (6e), is described. Monoiodination of compounds 1a,b, followed by acetylation, catalytic hydrogenation and finally regioselective 2'-O-deacylation afforded the partially acetylated dideoxynucleoside analogues of 5-fluorouracil (5a) and N(6)-benzoyl adenine (5b), respectively. Direct oxidation of the free hydroxyl group at the 2'-position of 5a,b, with simultaneous elimination reaction of the beta-acetoxyl group, afforded the desired unsaturated 4,6-dideoxy-3-fluoro-2-keto-beta-D-glucopyranosyl derivatives 6a,b. Compounds 1c-e were used as starting materials for the synthesis of the dideoxy unsaturated carbonyl nucleosides of uracil (6c), thymine (6d) and N(4)-benzoyl cytosine (6e). Similarly a protection-selective deprotection sequence followed by oxidation of the free hydroxyl group at the 2'-position of the dideoxy benzoylated analogues 9c-e with simultaneous elimination reaction of the beta-benzoyl group, gave the desired nucleosides 6c-e. None of the compounds was inhibitory to a broad spectrum of DNA and RNA viruses at subtoxic concentrations. The 5-fluorouracil derivative 6a was more cytostatic (50% inhibitory concentration ranging between 0.2 and 12 microM) than the other compounds.

Discovery of a new template for anticancer agents: 2'-deoxy-2'-fluoro-4'-selenoarabinofuranosyl-cytosine (2'-F-4'-seleno-ara-C).

The first synthesis of 2'-deoxy-2'-fluoro-4'-selenoarabinofuranosyl pyrimidines as potent anticancer agents was accomplished using the DAST fluorination as a key step. It was first revealed that selenium atom participated in the DAST fluorination of 4'-selenonucleosides and that conformational bias induced by bulky selenium acted as a decisive factor in the DAST fluorination. Among compounds tested, 2'-F-4'-seleno-ara-C (4a) exhibited highly potent anticancer activity in all cancer cell lines tested and was more potent than ara-C (1).

Dideoxy fluoro-ketopyranosyl nucleosides as potent antiviral agents: synthesis and biological evaluation of 2,3- and 3,4-dideoxy-3-fluoro-4- and -2-keto-beta-d-glucopyranosyl derivatives of N(4)-benzoyl cytosine.

The synthesis of the dideoxy fluoro ketopyranonucleoside analogues, 1-(2,3-dideoxy-3-fluoro-6-O-trityl-beta-d-glycero-hexopyranosyl-4-ulose)-N(4)-benzoyl cytosine (7a), 1-(3,4-dideoxy-3-fluoro-6-O-trityl-beta-d-glycero-hexopyranosyl-2-ulose)-N(4)-benzoyl cytosine (13a) and their detritylated analogues 8a and 14a, respectively, is described. Condensation of peracetylated 3-deoxy-3-fluoro-D-glucopyranose (1) with silylated N(4)-benzoyl cytosine, followed by selective deprotection and isopropylidenation afforded compound 2. Routine deoxygenation at position 2', followed by a deprotection-selective reprotection sequence afforded the partially tritylated dideoxy nucleoside of cytosine 6, which upon oxidation of the free hydroxyl group at the 4'-position, furnished the desired tritylated 2,3-dideoxy-3-fluoro ketonucleoside 7a in equilibrium with its hydrated form 7b. Compound 2 was the starting material for the synthesis of the dideoxy fluoro ketopyranonucleoside 13a. Similarly, several subsequent protection and deprotection steps as well as routine deoxygenation at position 4', followed by oxidation of the free hydroxyl group at the 2'-position of the partially tritylated dideoxy nucleoside 12, yielded the desired carbonyl compound 13a in equilibrium with its hydrated form 13b. Finally, trityl removal from 7a/b and 13a/b provided the unprotected 2,3-dideoxy-3-fluoro-4-keto and 3,4-dideoxy-3-fluoro-2-ketopyranonucleoside analogues 8a and 14a, in equilibrium with their gem-diol forms 8b and 14b. None of the compounds showed inhibitory activity against a wide variety of DNA and RNA viruses at subtoxic concentrations, except 7a/b that was highly efficient against rotavirus infection. Nucleoside 7a/b also exhibited cytostatic activity against cells of various cancers. BrdU-cell cycle analysis revealed that the mechanism of cytostatic activity may be related to a delay in G1/S phase and initiation of programmed cell death.

Serine peptide phosphoester prodrugs of cyclic cidofovir: synthesis, transport, and antiviral activity.

Cidofovir (HPMPC, 1), a broad-spectrum antiviral agent, is currently used to treat AIDS-related human cytomegalovirus (HCMV) retinitis and has recognized therapeutic potential for orthopox virus infections, but is limited by its low oral bioavailability. Cyclic cidofovir (2) displays decreased nephrotoxicity compared to 1, while also exhibiting potent antiviral activity. Here we describe in detail the synthesis and evaluation as prodrugs of four cHPMPC dipeptide conjugates in which the free POH of 2 is esterified by the Ser side chain alcohol group of an X-L-Ser(OMe) dipeptide: 3 (X=L-Ala), 4 (X=L-Val), 5 (X=L-Leu), and 6 (X=L-Phe). Perfusion studies in the rat establish that the mesenteric permeability to 4 is more than 20-fold greater than to 1, and the bioavailability of 4 is increased 6-fold relative to 1 in an in vivo murine model. In gastrointestinal and liver homogenates, the cHPMPC prodrugs are rapidly hydrolyzed to 2. Prodrugs 3, 4, and 5 are nontoxic at 100 microM in HFF and KB cells and in cell-based plaque reduction assays had IC 50 values of 0.1-0.5 microM for HCMV and 10 microM for two orthopox viruses (vaccinia and cowpox). The enhanced transport properties of 3-6, conferred by incorporation of a biologically benign dipeptide moiety, and the facile cleavage of the Ser-O-P linkage suggest that these prodrugs represent a promising new approach to enhancing the bioavailability of 2.

Unsaturated fluoro-ketopyranosyl nucleosides: synthesis and biological evaluation of 3-fluoro-4-keto-beta-d-glucopyranosyl derivatives of N(4)-benzoyl cytosine and N(6)-benzoyl adenine.

The protected beta-nucleosides 1-(2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-beta-d-glucopyranosyl)-N(4)-benzoyl cytosine (2a) and 9-(2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-beta-d-glucopyranosyl)-N(6)-benzoyl adenine (2b), were synthesized by the coupling of peracetylated 3-deoxy-3-fluoro-d-glucopyranose (1) with silylated N(4)-benzoyl cytosine and N(6)-benzoyl adenine, respectively. The nucleosides were deacetylated and several subsequent protection and deprotection steps afforded the partially acetylated nucleosides of cytosine 7a and adenine 7b, respectively. Finally, direct oxidation of the free hydroxyl group at 4'-position of 7a and 7b, and simultaneous elimination reaction of the beta-acetoxyl group, afforded the desired unsaturated 3-fluoro-4-keto-beta-d-glucopyranosyl derivatives. These newly synthesized compounds were evaluated for their potential antitumor and antiviral activities. Compared to 5FU, the newly synthesized derivatives showed to be more efficient as antitumor growth inhibitors and they exhibited direct antiviral effect toward rotavirus.

Atypical cytostatic mechanism of N-1-sulfonylcytosine derivatives determined by in vitro screening and computational analysis.

We have previously shown that N-1-sulfonylpyrimidine derivatives have strong antiproliferative activity on human tumor cell lines, whereby 1-(p-toluenesulfonyl)cytosine showed good selectivity with regard to normal cells and was easily synthesized on a large scale. In the present work we have used an interdisciplinary approach to elucidate the compounds' mechanistic class. An augmented number of cell lines (11) has allowed a computational search for compounds with similar activity profiles and/or mechanistic class by integrating our data with the comprehensive DTP-NCI database. We applied supervised machine learning methodology (Random Forest classifier), which offers information complementary to unsupervised algorithms commonly used for analysis of cytostatic activity profiles, such as self-organizing maps. The computational results taken together with cell cycle perturbation and apoptosis analysis of the cell lines point to an unusual mechanism of cytostatic action, possibly a combination of nucleic acid antimetabolite activity and a novel molecular mechanism.

Troxacitabine prodrugs for pancreatic cancer.

Troxacitabine is a cytotoxic deoxycytidine analogue with an unnatural L-configuration, which is activated by deoxycytidine kinase (dCK). The configuration is responsible for differences in the uptake and metabolism of troxacitabine compared to other deoxynucleoside analogues. The main drawback in the use of most nucleoside anticancer agents originates from their hydrophilic nature, which property requires a high and frequent dosage for an intravenous administration. To overcome this problem several troxacitabine prodrugs modified in the aminogroup with a linear aliphatic chain with a higher lipophilicity were developed. To determine whether these prodrugs have an advantage over Troxacitabine pancreatic cancer cell lines were exposed to Troxacitabine and the lipophilic prodrugs. The addition of linear aliphatic chains to troxacitabine increased sensitivity of pancreatic cancer cell lines to the drug > 100-fold, possibly due to a better uptake and retention of the drug.

Ester prodrugs of cyclic 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine: synthesis and antiviral activity.

Reaction of 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine (1) with dicyclohexylcarbodiimide and N,N,-dicyclohexyl-4-morpholinocarboxamidine in dimethylformamide at elevated temperature afforded the corresponding cyclic phosphonate 2, that is, 1-{[(5S)-2-hydroxy-2-oxido-1,4,2-dioxaphosphinan-5-yl]methyl}-5-azacytosine. Compound 2 exerts strong in vitro activity against DNA viruses, comparable with activity of parent compound 1. Transformation of 2 to its tetrabutylammonium salt followed by reaction with alkyl or acyloxyalkyl halogenides enabled us to prepare a series of structurally diverse ester prodrugs: alkyl (octadecyl), alkenyl (erucyl), alkoxyalkyl (hexadecyloxyethyl), and acyloxyalkyl (pivaloyloxymethyl) (3-6). The introduction of an alkyl, alkoxyalkyl, or acyloxyalkyl ester group to the molecule resulted in an increase of antiviral activity; the most active compound was found to be the hexadecyloxyethyl ester 5. The relative configuration of the diastereoisomer trans-6 was determined using H,H-NOESY NMR.

In vitro optimization of non-small cell lung cancer activity with troxacitabine, L-1,3-dioxolane-cytidine, prodrugs.

l-1,3-Dioxolane-cytidine, a potent anticancer agent against leukemia, has limited efficacy against solid tumors, perhaps due to its hydrophilicity. Herein, a library of prodrugs were synthesized to optimize in vitro antitumor activity against non-small cell lung cancer. N4-Substituted fatty acid amide prodrugs of 10-16 carbon chain length demonstrated significantly improved antitumor activity over l-1,3-dioxolane-cytidine. These in vitro results suggest that the in vivo therapeutic efficacy of l-1,3-dioxolane-cytidine against solid tumors may be improved with prodrug strategies.

Antiviral activity of triazine analogues of 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (cidofovir) and related compounds.

Treatment of 5-azacytosine sodium salt with diisopropyl [(2-chloroethoxy)methyl]phosphonate followed by removal of ester groups with BrSi(CH3)3 afforded 1-[2-(phosphonomethoxy)ethyl]-5-azacytosine (3). Reaction of 5-azacytosine with [(trityloxy)methyl]-(2S)-oxirane followed by etherification with diisopropyl (bromomethyl)phosphonate and removal of ester groups gave 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine (1). The synthesis of 6-azacytosine congener 2 was analogous using N4-benzoylated intermediates. Compound 1 was shown to exert strong activity against a broad spectrum of DNA viruses including adenoviruses, poxviruses, and herpesviruses (i.e., herpes simplex viruses, varicella zoster virus, and human cytomegalovirus). Decomposition of 1 in alkaline solutions resulted in products 17 and 18. While the N-formylguanidine derivative 17 proved active, the carbamyolguanidine derivative 18 was devoid of antiviral activity.