Utility of green chemistry for feasible spectrofluorometric determination of trifluridine via metal complexation: Application to dosage forms.

The goal of the current research was to establish a quick and practical fluorometric technique for trifluridine analysis. The approach relied on the drug's complex formation with the zinc ion to produce a high-fluorescence product. The fluorescence was further enhanced by adding sodium dodecyl sulfate, and it was observed at 450 nm following excitation at 400 nm. With a determination coefficient of 0.9994, the association between emission intensity and trifluridine concentration was linear between 1 and 125 ng mL-1. The quantitation limit was 0.987 ng mL-1 while 0.333 ng mL-1 was the detection limit. The buffer type, pH and concentration, type of surfactant and concentration, and finally the diluting solvent were among the reaction conditions that were closely examined. With great precision and reliability, the drug in question was quantified using this method in dosage formulations. The proposed method's level of greenness was assessed using two methodologies: the analytical greenness metric (AGREE) and the Green Analytical Procedure Index (GAPI).

Virtual screening, ADME/T, and binding free energy analysis of anti-viral, anti-protease, and anti-infectious compounds against NSP10/NSP16 methyltransferase and main protease of SARS CoV-2.

Recently, a pathogen has been identified as a novel coronavirus (SARS-CoV-2) and found to trigger novel pneumonia (COVID-19) in human beings and some other mammals. The uncontrolled release of cytokines is seen from the primary stages of symptoms to last acute respiratory distress syndrome (ARDS). Thus, it is necessary to find out safe and effective drugs against this deadly coronavirus as soon as possible. Here, we downloaded the three-dimensional model of NSP10/NSP16 methyltransferase (PDB-ID: 6w6l) and main protease (PDB-ID: 6lu7) of COVID-19. Using these molecular models, we performed virtual screening with our anti-viral, inti-infectious, and anti-protease compounds, which are attractive therapeutics to prevent infection of the COVID-19. We found that top screened compound binds with protein molecules with good dock score with the help of hydrophobic interactions and hydrogen bonding. We observed that protease complexed with Cyclocytidine hydrochloride (anti-viral and anti-cancer), Trifluridine (anti-viral), Adonitol, and Meropenem (anti-bacterial), and Penciclovir (anti-viral) bound with a good docking score ranging from -6.8 to -5.1 (Kcal/mol). Further, NSP10/NSP16 methyltransferase complexed with Telbivudine, Oxytetracycline dihydrate (anti-viral), Methylgallate (anti-malarial), 2-deoxyglucose and Daphnetin (anti-cancer) from the docking score of -7.0 to -5.7 (Kcal/mol). In conclusion, the selected compounds may be used as a novel therapeutic agent to combat this deadly pandemic disease, SARS-CoV-2 infection, but needs further experimental research.HighlightsNSP10/NSP16 methyltransferase and main protease complex of SARS CoV-2 bind with selected drugs.NSP10/NSP16 methyltransferase and protease interacted with drugs by hydrophobic interactions.Compounds show good DG binging free energy with protein complexes.Ligands were found to follow the Lipinski rule of five.

TAS-102: a novel antimetabolite for the 21st century.

TAS-102, a novel antimetabolite combination chemotherapy agent, consists of a rediscovered antimetabolite agent, trifluorothymidine (trifluridine) combined with the metabolic inhibitor of thymidine phosphorylase, tipiracil, in a 1:0.5 molar ratio. Mechanism of action studies suggest that this agent works by incorporation into DNA. Both preclinical and clinical studies demonstrate that this agent is noncross-resistant with 5-fluorouracil. Tipiracil may also have antiangiogenic effects through inhibition of thymidine phosphorylase. Recent randomized Phase II and III trials demonstrate clinical activity (improved progression-free survival, time to decrease in performance status, prolonged overall survival) in metastatic colorectal cancer refractory to all standard agents. Monotherapy with TAS-102 has now been approved for this indication in Japan and in the USA.

Ligand-directed acid-sensitive amidophosphate 5-trifluoromethyl-2'-deoxyuridine conjugate as a potential theranostic agent.

Herein, we report a novel strategy to engineer an acid-sensitive anticancer theranostic agent using a vector-drug ensemble. The ensemble was synthesized by directly conjugating the linoleic acid (LA)-modified branched polyethyleneimine with a chemotherapeutic drug trifluorothymidine. Linoleic acid residues were grafted onto 25 kDa polyethyleneimine (PEI) by treating PEI with linoleic acid chloroanhydride. 5-Trifluoromethyl-2'-deoxyuridine (trifluorothymidine, TFT) was introduced into LA-PEI conjugate by phosphorylating the conjugate with amidophosphate of trifluorothymidine 5'-monophosphate (pTFT), which had been activated by its conversion into the N,N-dimethylaminopyridine derivative. The extent of mononucleotide analog incorporation in the polymer was regulated by the ratio of pTFT to the polymer during the synthesis. Samples containing 20-70 TFT residues per PEI molecule were obtained. The cytotoxicity of PEI-LA-pTFT conjugates decreased with increasing nucleotide content, as examined using the MTT method. Due to the presence of fluorine atoms, TFT-based conjugates could be detected directly in the animals by (19)F magnetic resonance imaging. In addition, the presence of the amidophosphate group in PEI-LA-pTFT conjugates allowed their detection by in vivo(31)P NMR spectroscopy. Indeed, the (31)P NMR signal of a phosphoramide (δ ~ 12 ppm) was observed in the mouse muscle tissue treated with PEI-LA-pTFT conjugate along with the signals from endogenous phosphorus-containing compounds. At the same time, the use of PEI-LA-pTFT conjugate for chemotherapeutic drug delivery is limited due to the low release of pTFT from the carrier. To enhance the release of the drug from the conjugate in the endosomes, PEI-LA polymer was coupled with urocanic acid (UA), which bears imidazole ring and thus can form an acid-labile P-N bond with pTFT. The PEI-LA-UA-pTFT conjugate containing 30 residues of UA and 40 residues of pTFT was tested against the murine Krebs-II ascites carcinoma, grown as an ascetic tumor. The intraperitoneal injection of the conjugates resulted in prolongation of the animals' life and to the complete disappearance of the tumor after three injections.

Inhibitory Effect of Multimodal Nanoassemblies against Glycative and Oxidative Stress in Cancer and Glycation Animal Models.

In recent years, there has been a progress in the study of glycation reaction which is one the possible reason for multiple metabolic disorders. Glycation is a nonenzymatic reaction between nucleic acids, lipids, and proteins resulting into the formation of early glycation products that may further lead to the accumulation of advanced glycation end products (AGEs). The precipitation of AGEs in various cells, tissues, and organs is one of the factors for the initiation and progression of various metabolic derangements including the cancer. The AGE interaction with its receptor "RAGE" activates the inflammatory pathway; yet, the downregulation of RAGE and its role in these pathways are not clear. We explore the effect of anticancer novel nanoassemblies on AGEs to determine its role in the regulation of the expression of RAGE, NFƙB, TNF-α, and IFN-γ. This paper is based on the in vivo and in vitro study in glycation and lung cancer model systems. Upon the treatment of nanoassemblies in both the model systems, we observed a protective effect of nanoassemblies over the inhibition of glycative and oxidative stress via mRNA expression analysis. The mRNA expression results corroborated with the reactive oxygen species (ROS), carboxy-methyl-lysine (CML), and fluorescence studies. In this study, we found that the presence of common factors for glycation and lung cancer is oxidative and glycative stress. This oxidation and glycation might be responsible for the initiation of inflammation which may further lead to uncontrolled growth of cells leading to cancer. This can be a strong association between lung cancer and glycation reaction. The intervention of the anticancer and antiglycation effects of multimodal nanoassemblies throughout the study promises a new pathway for cancer research.

Theoretical analyses and experimental validation of the effects caused by the fluorinated substituent modification of DNA.

Halogen-modified nucleic acid molecules, such as trifluorothymidine (FTD) and 5-fluorouracil, are widely used in medical science and clinical site. These compounds have a very similar nucleobase structure. It is reported that both of these compounds could be incorporated into DNA. The incorporation of FTD produces highly anti-tumor effect. However, it is not known whether to occur a significant effect by the incorporation of 5-fluorouracil. Nobody knows why such a difference will occur. To understand the reason why there is large differences between trifluorothymidine and 5-fluorouracil, we have performed the molecular dynamics simulations and molecular orbital calculations. Although the active interaction energy between Halogen-modified nucleic acids or and complementary adenine was increased, in only FTD incorporated DNA, more strongly dispersion force interactions with an adjacent base were detected in many thermodynamic DNA conformations. As the results, the conformational changes occur even if it is in internal body temperature. Then the break of hydrogen bonding between FTD and complementary adenine base occur more frequently. The double helix structural destabilization of DNA with FTD is resulted from autoagglutination caused by the bonding via halogen orbitals such as halogen bonding and the general van der Waals interactions such as CH-[Formula: see text], lone pair (LP)-[Formula: see text], and [Formula: see text]-[Formula: see text] interactions. Therefore, it is strongly speculated that such structural changes caused by trifluoromethyl group is important for the anti-tumor effect of FTD alone.

Direct Analysis of Incorporation of an Anticancer Drug into DNA at Single-Molecule Resolution.

Identifying positions at which anticancer drug molecules incorporate into DNA is essential to define mechanisms underlying their activity, but current methodologies cannot yet achieve this. The thymidine fluorine substitution product trifluridine (FTD) is a DNA-damaging anticancer agent thought to incorporate into thymine positions in DNA. This mechanism, however, has not been directly confirmed. Here, we report a means to detect FTD in a single-stranded oligonucleotide using a method to distinguish single molecules by differences in electrical conductance. Entire sequences of 21-base single-stranded DNAs with and without incorporated drug were determined based on single-molecule conductances of the drug and four deoxynucleosides, the first direct observation of its kind. This methodology may foster rapid development of more effective anticancer drugs.

Synthesis of nucleoside-based antiviral drugs in ionic liquids.

Nucleoside-based antiviral drugs have been synthesized using imidazolium-based ionic liquids as reaction medium. The ionic liquids were proved to be better solvents for all the nucleoside in terms of solubility and reaction medium as compared to conventional molecular solvents.

Collision induced dissociation studies of alkali metal adducts of tetracyclines and antiviral agents by electrospray ionization, hydrogen/deuterium exchange and multiple stage mass spectrometry.

The collision induced dissociation (CID) mass spectra were obtained for the X(+)-adducts (X=Na(+) or Li(+)) of five tetracyclines, four pyrimidine and three purine derivatives and their fully D-exchanged species in which the labile hydrogens were replaced by deuterium by either gas phase or liquid phase exchange. The CID spectra were obtained for [M + Na](+) and [M + Li](+) and the exchanged analogs, [M(D) + Na](+) and [M(D) + Li](+), and compositions of product ions and mechanisms of decomposition were determined by comparison of the MS(n) spectra of the undeuterated and deuterated species. Metal ions are bound to the base of purine and pyrimidine antiviral agents and dissociate primarily to give the metal complexes of the base [B + X](+). For vidarabine monophosphate, however, the metal ions are bound to the phosphate group, resulting in unique and characteristic cleavage reactions not observed in the uncomplexed system, and dissociate through the loss of phosphate and/or phosphate metal ion complex. The [B + X](+) of these antiviral agents are relatively stable and show no or little fragmentation compared to [B + H](+). The CID of [B + X](+) of guanine derivative occurs mainly through elimination of NH(3) and that of trifluoromethyl uracil dissociates primarily through the loss of HF. For tetracyclines, metal ions are bound to ring A at the tricarbonylmethyl group and dissociate initially by the loss of NH(3)/ND(3) from [M(H) + X](+) and [M(D) + X](+). The CID spectra of [M + X](+) of tetracyclines are somewhat similar to those of [M + H](+). The dominant fragments from the metal complexes of these compounds are charge remote decompositions involving molecular rearrangements and the loss of small stable molecules. Additionally, tetracyclines and the antiviral agents show more selectivity towards Li+ ion than the corresponding complexes with Na(+) or K(+).

Collisionally-induced dissociation of substituted pyrimidine antiviral agents: mechanisms of ion formation using gas phase hydrogen/deuterium exchange and electrospray ionization tandem mass spectrometry.

ESI and CID mass spectra were obtained for four pyrimidine nucleoside antiviral agents and the corresponding compounds in which the labile hydrogens were replaced by deuterium using gas-phase exchange. The number of labile hydrogens, x, was determined from a comparison of ESI spectra obtained with N(2) and with ND(3) as the nebulizer gas. CID mass spectra were obtained for [M + H](+) and [M - H](-) ions and the exchanged analogs, [M(D(x)) + D](+) and [M(D(x)) - D](-), produced by ESI using a SCIEX API-III(plus) mass spectrometer. Protonated pyrimidine antiviral agents dissociate through rearrangement decompositions of base-protonated [M + H](+) ions by cleavage of the glycosidic bonds to give the protonated bases with a sugar moiety as the neutral fragment. Cleavage of the glycosidic bonds with charge retention on the sugar moiety eliminates the base moiety as a neutral molecule and produces characteristic sugar ions. CID of protonated pyrimidine bases, [B + H](+), occurs through three major pathways: (1) elimination of NH(3) (ND(3)), (2) loss of H(2)O (D(2)O), and (3) elimination of HNCO (DNCO). Protonated trifluoromethyl uracil, however, dissociates primarily through elimination of HF followed by the loss of HNCO. CID mass spectra of [M - H](-) ions of all four antiviral agents show NCO(-) as the principal decomposition product. A small amount of deprotonated base is also observed, but no sugar ions. Elimination of HNCO, HN(3), HF, CO, and formation of iodide ion are minor dissociation pathways from [M - H](-) ions.

Photo-cross-linking using trifluorothymidine and 3-cyanovinylcarbazole induced a large shifted (19)F MR signal.

Photo-cross-linking of trifluorothymidine ((TF)T) using 3-cyanovinylcarbazole ((CNV)K) clearly shifted its (19)F nuclear magnetic resonance (NMR) signal 8 ppm. This (CNV)K mediated ultrafast photo-cross-linking-induced shift can be utilized for miRNA detection by hybridization chain reaction (HCR) to detect 10 nM of a target in a sequence-specific manner.

A novel antimetabolite, TAS-102 retains its effect on FU-related resistant cancer cells.

TAS-102 is a new oral anti-tumor drug preparation, composed of a 1:0.5 mixture (on a molar basis) of alpha,alpha,alpha-tri-fluorothymidine (FTD) and thymidine phosphorylase inhibitor (TPI). TAS-102 is currently undergoing clinical trials, and has been demonstrated to have at least 2 mechanisms; inhibition of thymidylate synthase (TS) and incorporation into DNA. 5-FU is widely used in the treatment of solid tumor, but the inherent or acquired resistance of certain tumors to 5-FU therapy is a major clinical problem. In the present study, we investigated FTD in vitro and in vivo comparing with 5-FU and using FU-resistant cells. There was no relationship between FTD and 5-FU growth inhibition effect in vitro. A different sensitivity pattern was observed by the log-mean graph. We next investigated the anti-tumor activity of TAS-102 in a FU-resistant xenograft model. Comparative efficacy was observed between FU-resistant cell and its parent cell. We also studied the influence of TAS-102 on liver metastasis in a mouse model of human colorectal cancer, because liver metastasis of colorectal cancer is associated with patient survival. Human cancer DNA was detected by PCR, and TAS-102 markedly inhibited the number of liver metastasis. A novel angiogenic factor, platelet-derived endothelial cell growth factor (PD-ECGF), was shown to be identical to a previously characterized intracellular enzyme, thymidine phosphorylase, TAS-102 can be expected to have not only anti-tumor cytocidal effects but also antiangiogenesis activity and may inhibit liver metastasis. Our findings suggested that TAS-102 is a promising candidate for clinical use and can be expected to decrease minimal residual disease.

Highly regioselective synthesis of 3'-O-acyl-trifluridines catalyzed by Pseudomonas cepacia lipase.

3'-O-Acyl-trifluridines were prepared successfully through an enzymatic approach for the first time. Among the ten commercially available lipases tested, Pseudomonas cepacia lipase displayed the highest regioselectivity towards the acylation of 3'-hydroxyl of trifluridine. Furthermore, the effects of some crucial factors on the enzymatic myristoylation of trifluridine were examined. The optimal reaction medium, molar ratio of trifluridine to vinyl myristate and reaction temperature were found to be anhydrous THF, 1:7 and 50 °C, under which the reaction rate, substrate conversion, and 3'-regioselectivity were 63.9 mM/h, >99.0%, and 99%, respectively. Additionally, the enzyme recognition of the chain length of the acyl donors was investigated. The results showed that 3'-regioselectivity of the enzyme maintained 99% with the increment of acyl chain length (C6, C10, and C14). The reason might derive from the strong hydrophobic interaction between 5-CF(3) group of the base moiety and Leu 287 located in the medium-sized pocket of the active site.

Simple and rapid enzymatic method for the synthesis of single-strand oligonucleotides containing trifluorothymidine.

To investigate the mechanism of trifluorothymidine (TFT)-induced DNA damage, we developed an enzymatic method for the synthesis of single-strand oligonucleotides containing TFT-monophosphate residues. Sixteen-mer oligonucleotides and 14-mer 5'-phosphorylated oligonucleotides were annealed to the template of 25-mer, so as to empty one nucleotide site. TFT-triphosphate was incorporated into the site by DNA polymerase and then ligated to 5'-phosphorylated oligonucleotides by DNA ligase. The synthesized 31-mer oligonucleotides containing TFT residues were isolated from the 25-mer complementary template by denaturing polyacrylamide electrophoresis. Using these single-strand oligonucleotides containing TFT residues, the cleavage of TFT residues from DNA, using mismatch uracil-DNA glycosylase (MUG) of E.coli origin, was compared with that of 5-fluorouracil (5FU) and 5-bromodeoxyuridine (BrdU). The TFT/A pair was not cleaved by MUG, while the other pairs, namely, 5FU/A, 5FU/G, BrdU/A, BrdU/G, and TFT/G, were easily cleaved from each synthesized DNA. Thus, this method is useful for obtaining some site-specifically modified oligonucleotides.

Trifluorothymidine resistance is associated with decreased thymidine kinase and equilibrative nucleoside transporter expression or increased secretory phospholipase A2.

Trifluorothymidine (TFT) is part of the novel oral formulation TAS-102, which is currently evaluated in phase II studies. Drug resistance is an important limitation of cancer therapy. The aim of the present study was to induce resistance to TFT in H630 colon cancer cells using two different schedules and to analyze the resistance mechanism. Cells were exposed either continuously or intermittently to TFT, resulting in H630-cTFT and H630-4TFT, respectively. Cells were analyzed for cross-resistance, cell cycle, protein expression, and activity of thymidine phosphorylase (TP), thymidine kinase (TK), thymidylate synthase (TS), equilibrative nucleoside transporter (hENT), gene expression (microarray), and genomic alterations. Both cell lines were cross-resistant to 2'-deoxy-5-fluorouridine (>170-fold). Exposure to IC(75)-TFT increased the S/G(2)-M phase of H630 cells, whereas in the resistant variants, no change was observed. The two main target enzymes TS and TP remained unchanged in both TFT-resistant variants. In H630-4TFT cells, TK protein expression and activity were decreased, resulting in less activated TFT and was most likely the mechanism of TFT resistance. In H630-cTFT cells, hENT mRNA expression was decreased 2- to 3-fold, resulting in a 5- to 10-fold decreased TFT-nucleotide accumulation. Surprisingly, microarray-mRNA analysis revealed a strong increase of secretory phospholipase-A2 (sPLA2; 47-fold), which was also found by reverse transcription-PCR (RT-PCR; 211-fold). sPLA2 inhibition reversed TFT resistance partially. H630-cTFT had many chromosomal aberrations, but the exact role of sPLA2 in TFT resistance remains unclear. Altogether, resistance induction to TFT can lead to different mechanisms of resistance, including decreased TK protein expression and enzyme activity, decreased hENT expression, as well as (phospho)lipid metabolism. Mol Cancer Ther; 9(4); 1047-57. (c)2010 AACR.

Inhibition of in vitro DNA chain elongation of 5-trifluoromethyl-2'-deoxyuridine residue in the template.

5-Trifluoromethyl-2'-deoxyuridine (CF3dUrd) is incorporated into the DNA of mammalian cells in culture. We have synthesized oligonucleotides that allows site specific introduction of CF3dUrd residue into synthetic DNA oligonucleotide. We described here the utilization of these oligonucleotides as template for in vitro DNA synthesis. When CF3dUrd residue located at an internucleotide site in the template, the chain elongation was partially arrested one nucleotide after or before the CF3dUrd residue of template using Escherichia coli polymerase I (Klenow fragment) or human polymerase alpha (pol alpha). These results suggested that a mechanism of antitumor activity of CF3dUrd is inhibition of DNA replication.