Synergistic Combination Chemotherapy of Camptothecin and Floxuridine through Self-Assembly of Amphiphilic Drug-Drug Conjugate.

Combination chemotherapy has been widely applied in cancer treatment; however, the cocktail administration of combination chemotherapy could cause the nonuniform biodistribution of anticancer agents, thus impairing the therapeutic efficacy. In the present study, to address this concern, we proposed a novel strategy of preparing self-assembled nanoparticles from amphiphilic drug-drug conjugate for synergistic combination chemotherapy. The conjugate was synthesized by two-step esterification of hydrophobic camptothecin (CPT) and hydrophilic floxuridine (FUDR) through a linker compound. Because of its amphiphilic nature, the CPT-FUDR conjugate self-assembled into stable nanoparticles which could simultaneously release fixed dosage of the two drugs in cancer cells. In vitro studies demonstrated synergistic anticancer efficacy of the CPT-FUDR nanoparticles including improved cell apoptosis, varied cell cycle arrest, as well as effective inhibition of cancer cell proliferation.

Rigid 2',4'-difluororibonucleosides: synthesis, conformational analysis, and incorporation into nascent RNA by HCV polymerase.

We report on the synthesis and conformational properties of 2'-deoxy-2',4'-difluorouridine (2',4'-diF-rU) and cytidine (2',4'-diF-rC) nucleosides. NMR analysis and quantum mechanical calculations show that the strong stereoelectronic effects induced by the two fluorines essentially "lock" the conformation of the sugar in the North region of the pseudorotational cycle. Our studies also demonstrate that NS5B HCV RNA polymerase was able to accommodate 2',4'-diF-rU 5'-triphosphate (2',4'-diF-rUTP) and to link the monophosphate to the RNA primer strand. 2',4'-diF-rUTP inhibited RNA synthesis in dinucleotide-primed reactions, although with relatively high half-maximal inhibitory concentrations (IC50 > 50 µM). 2',4'-diF-rU/C represents rare examples of "locked" ribonucleoside mimics that lack a bicyclic ring structure.

Reductive activation of 5-fluorodeoxyuridine prodrug possessing azide methyl group by hypoxic X-irradiation.

We prepared a 5-fluorodeoxyuridine (5-FdUrd) derivative possessing azide methyl group (N(3)-FdUrd) as a novel radiation-activated prodrug. The parent antitumor agent, 5-FdUrd, was released efficiently from N(3)-FdUrd by hypoxic X-irradiation. On the other hand, the activation of N(3)-FdUrd was suppressed upon X-irradiation under aerobic conditions. A biological assay using A549 cells revealed that the cytotoxicity of N(3)-FdUrd was significantly enhanced by hypoxic X-irradiation.

Design, synthesis and biological evaluation of 2'-deoxy-2',2'-difluoro-5-halouridine phosphoramidate ProTides.

We report the synthesis of a series of novel 2'-deoxy-2',2'-difluoro-5-halouridines and their corresponding phosphoramidate ProTides. All compounds were evaluated for antiviral activity and for cellular toxicity. Interestingly, 2'-deoxy-2',2'-difluoro-5-iodo- and -5-bromo-uridines showed selective activity against feline herpes virus replication in cell culture due to a specific recognition (activation) by the virus-encoded thymidine kinase.

Glycosyl ortho-(1-phenylvinyl)benzoates versatile glycosyl donors for highly efficient synthesis of both O-glycosides and nucleosides.

Both of O-glycosides and nucleosides are important biomolecules with crucial rules in numerous biological processes. Chemical synthesis is an efficient and scalable method to produce well-defined and pure carbohydrate-containing molecules for deciphering their functions and developing therapeutic agents. However, the development of glycosylation methods for efficient synthesis of both O-glycosides and nucleosides is one of the long-standing challenges in chemistry. Here, we report a highly efficient and versatile glycosylation method for efficient synthesis of both O-glycosides and nucleosides, which uses glycosyl ortho-(1-phenylvinyl)benzoates as donors. This glycosylation protocol enjoys the various features, including readily prepared and stable donors, cheap and readily available promoters, mild reaction conditions, good to excellent yields, and broad substrate scopes. In particular, the applications of the current glycosylation protocol are demonstrated by one-pot synthesis of several bioactive oligosaccharides and highly efficient synthesis of nucleosides drugs capecitabine, galocitabine and doxifluridine.

Efficient regioselective synthesis of 3'-O-crotonylfloxuridine catalysed by Pseudomonas cepacia lipase.

Pseudomonas cepacia lipase-catalysed preferential acylation of the secondary hydroxy group of FUdR (floxuridine) with vinyl crotonate was carried out in spite of the presence of the primary hydroxy group, and 3'-O-crotonylfloxuridine was prepared successfully for the first time. The isomerization of the double bond of crotonate, which occurs in conventional organic synthesis, could be effectively avoided during the enzymatic acylation. The effects of some key factors such as reaction medium, initial a(w) (water activity), molar ratio of vinyl crotonate to FUdR, FUdR concentration and reaction temperature on the reaction were examined. Under the optimized reaction conditions, the initial reaction rate, substrate conversion and 3'-regioselectivity of the reaction were as high as 24 mM/h, 98% and 85% respectively.

Growth inhibition of Mycobacterium bovis, Mycobacterium tuberculosis and Mycobacterium avium in vitro: effect of 1-beta-D-2'-arabinofuranosyl and 1-(2'-deoxy-2'-fluoro-beta-D-2'-ribofuranosyl) pyrimidine nucleoside analogs.

The resurgence of tuberculosis and the emergence of multiple-drug-resistant strains of Mycobacteria necessitate the search for new classes of antimycobacterial agents. We synthesized a series of 1-beta-D-2'-arabinofuranosyl and 1-(2'-deoxy-2'-fluoro-beta-D-ribofuranosyl) pyrimidine nucleosides possessing diverse sets of alkynyl, alkenyl, alkyl, and halo substituents at the C-5 position of the uracil and investigated their effect on activity against M. tuberculosis, M. bovis, and M. avium. Among these molecules, 5-alkynyl-substituted derivatives emerged as potent inhibitors of M. bovis, M. tuberculosis, and M. avium. Nucleosides 1-beta-D-2'-arabinofuranosyl-5-dodecynyluracil (5), 1-(2'-deoxy-2'-fluoro-beta-D-ribofuranosyl)-5-dodecynyluracil (24), and 1-(2'-deoxy-2'-fluoro-beta-D-ribofuranosyl)-5-tetradecynyluracil (25) showed the highest antimycobacterial potency against M. bovis and M. tuberculosis. The MIC90 exhibited by compounds 5, 24, and 25 was similar or close to that of the reference drug rifampicin. The most active compounds 5, 24, and 25 were also found to retain sensitivity against a rifampicin-resistant strain of M. tuberculosis H37Rv at similar concentrations. Some of these analogs also revealed in vitro antimicrobial effect against several other gram-positive pathogens.

In vivo evaluation of 5-[(18)F]fluoro-2'-deoxyuridine as tracer for positron emission tomography in a murine pancreatic cancer model.

We used a murine tumor progression model for the evaluation of potential proliferation markers using positron emission tomography (PET). 5-[(18)F]-2'-deoxyuridine ([(18)F]FdUrd) was synthesized with >98% radiochemical purity and investigated in a pancreatic cancer model, transforming growth factor alpha transgenic mice crossbred to p53 deficient mice. Thymidylate synthase was increased already in premalignant lesions, whereas thymidine kinase 1 mRNA levels were up-regulated 4-fold in the pancreatic cancer specimen of these mice. PET imaging was performed after injection of 1 MBq of [(18)F]FdUrd and 1 MBq of [(18)F]fluoro-deoxyglucose. Animals with pancreatic cancer displayed focal uptake of both tracers. The [(18)F]FdUrd uptake ratio closely correlated with the proliferation index as evaluated in morphometric and fluorescence-activated cell sorter analysis. These results indicate the potential of our tumor model for the evaluation of PET tracers and suggest [(18)F]FdUrd as a tracer for the assessment of proliferation in vivo.

3'-O- and 5'-O-Propargyl Derivatives of 5-Fluoro-2'-Deoxyuridine: Synthesis, Cytotoxic Evaluation and Conformational Analysis.

A series of new 3'-O- and 5'-O-propargyl derivatives of 5-fluoro-2'-deoxyuridine (1-4) was synthesized by means of propargyl reaction of properly blocked nucleosides (2,4), followed by the deprotection reaction with ammonium fluoride. The synthesized propargylated 5-fluoro-2'-deoxyuridine analogues (1-4) were evaluated for their cytotoxic activity in three human cancer cell lines: cervical (HeLa), oral (KB) and breast (MCF-7), using the sulforhodamine B (SRB) assay. The highest activity and the best SI coefficient in all of the investigated cancer cells were displayed by 3'-O-propargyl-5-fluoro-2'-deoxyuridine (1), and its activity was higher than that of the parent nucleoside. The other new compounds exhibited moderate activity in all of the used cell lines.

Studies on antitumor agents. 8. Antitumor activities of O-alkyl derivatives of 2'-deoxy-5-(trifluoromethyl)uridine and 2'-deoxy-5-fluorouridine.

O-Benzyl and O-ethyl derivatives of 2'-deoxy-5-(trifluoromethyl)uridine (F3Thd) and 2'-deoxy-5-fluorouridine (FUdR) were synthesized. The oral antitumor activity of the compounds against sarcoma 180 in mice was examined. The 5'-O-ethyl (3b), 3'-O-ethyl (3c), 5'-O-benzyl (3e), and 3'-O-benzyl (3f) derivatives of F3Thd were 4-fold more active than F3Thd itself. Among the substituted-benzyl derivatives of F3Thd, 3'-O-(p-chlorobenzyl)-F3Thd (3h) showed the highest activity, with an ED50 less than one-tenth of that of F3Thd. The activities of 5'-O-benzyl (7c) and 3'-O-benzyl (7d) derivatives of FUdR were equal to those of the effective O-alkyl derivatives of F3Thd.

Antineoplastic activity of 3'-(chloroethyl)nitrosourea analogues of 2'-deoxyuridine and 2'-deoxy-5-fluorouridine.

The (chloroethyl)nitrosourea analogues of 2'-deoxyuridine and 2'-deoxy-5-fluorouridine, 3'-[3-(2-chloroethyl)-3-nitrosoureido]-2',3'-dideoxyuridine (3'-CdUNU, 7) and 3'-[3-(2-chloroethyl)-3-nitrosoureido]-2,3'-dideoxy-5-fluorouridine (3'-CFdUNU, 8), have been synthesized by treatment of the corresponding 3'-amino nucleosides with chloroethyl isocyanate, followed by nitrosation of the resulting ureas. Nucleoside nitrosoureas 7 and 8 exhibited marked anticancer activity against L1210 leukemia in tumor-bearing mice. At an optimum dosage level of 40 mg/kg, 7 and 8 produced 90% and 60% "cures" (greater than 60-day survivors), respectively. The structure-activity relationships are discussed.

Synthesis of 2'-deoxyuridine and 5-fluoro-2'-deoxyuridine derivatives and evaluation in antibody targeting studies.

Derivatives of 2'-deoxyuridine and of the anticancer agent 5-fluoro-2'-deoxyuridine (FdUR) were linked indirectly via a human serum albumin carrier (HSA) to the murine antiosteosarcoma monoclonal antibody 791T/36. Starting from the 2'-deoxyuridines 1a and 1b, the new nucleosides containing 5'-succinamic acid 7 and 5'-maleamic acid 8 spacers were synthesized from the key intermediate 5'-aminonucleoside 4, and the ribofuronamidobenzoic acid 13 from ribofuranuronic acid 10. These nucleosides were linked via their spacer functionality to HSA. High molar substitution ratios (MSR: moles of drug/mole of HSA) of 25-40 for these derivative-HSA conjugates were achieved. All derivatives were less cytotoxic than the parent drug against both antigen positive osteogenic sarcoma 791T and antigen negative bladder carcinoma T24 cell lines; no IC50 was achieved with any derivative against 791T cells. The fluorodeoxyuridine-HSA conjugates were then further linked via a stable thioether bond to the mouse monoclonal antibody 791T/36. The optimum fluorinated 5'-succinamic acid immunoconjugate exhibited an IC50 of 1 microM against 791T and T24 cells, slightly better than that of fluorodeoxyuridine. The unconjugated derivative 7 was much less cytotoxic than immunoconjugate, with an IC50 of 62 microM on T24 cells, and failed to reach 50% inhibition of 791T cell growth at 290 microM concentration. Derivative 7-HSA conjugate was 10-fold less cytotoxic than the immunoconjugate against both cell lines. Immunoconjugates synthesized with the other 5-fluoro derivatives were unable to effect 50% inhibition of growth of cell lines. Nonfluorinated derivatives and their HSA conjugates and immunoconjugates exhibited no cytotoxicity.

2'-Fluorinated arabinonucleosides of 5-(2-haloalkyl)uracil: synthesis and antiviral activity.

The synthesis of 5-(2-fluoroethyl)-2'-deoxyuridine (FEDU, 4b), its 2'-fluoro analogue 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-(2-fluoroethyl)-1H,3H- pyrimidine-2,4-dione (FEFAU, 4k), and the 2'-fluoro analogue of the potent antiherpes virus compound 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), 5-(2-chloroethyl)-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-1H,3H-pyr imidine - 2,4-dione (CEFAU, 4i), is described. The antiviral activities of these compounds were determined in cell culture against herpes simplex virus (HSV) types 1 and 2 and varicella zoster virus (VZV). All compounds were shown to possess significant and selective antiviral activity. FEDU proved less potent than CEDU against VZV replication; however, it was more active against HSV-2. CEFAU showed marked activity against HSV-1, HSV-2, and VZV. The compound containing fluorine at both positions, FEFAU, exhibited the strongest antiviral potency against HSV-1, HSV-2, and VZV. It inhibited HSV-1 at a concentration of 0.03-0.2 microgram/mL, HSV-2 at 0.1-0.3 microgram/mL, and VZV at 0.03 microgram/mL. Neither FEDU nor CEFAU or FEFAU exerted a significant inhibitory effect on cell proliferation at a concentration of 100 micrograms/mL. Thus, the cytotoxicity of these compounds is as low as that of CEDU and compares favorably to that of previously described 2'-fluoroarabinosyl nucleoside analogues.

Synthesis and tumor uptake of 5-82Br- and 5-131I-labeled 5-halo-1-(2-fluoro-2-deoxy-beta-D-ribofuranosyl)uracils.

A synthesis of 5-bromo- and 5-iodo-1-(2-fluoro-2-deoxy-beta-D-ribofuranosyl)uracil (3 and 4) and their 5-82Br and 5-131I analogues has been developed. The tissue distribution of the radiolabeled compounds in BDF1 mice bearing Lewis lung tumors has been investigated. After injection of the radiolabeled analogues of compounds 3 and 4 there was a rapid initial excretion of activity. Compound 3 was excreted unchanged in the urine. Residual activity in mice after 4 h showed a distribution characteristic of bromide (Br-). Compound 4 was excreted mainly as unchanged starting material with increasing amounts of iodide (I-) detected at later time periods, in addition to 5-iodouridine and unidentified metabolites at shorter time periods. Both 3 and 4 demonstrated a remarkable in vivo stability relative to related 5-substituted nucleosides that do not contain the 2'-fluoro group. The tumor uptake was minimal, with only the 5-bromo analogue demonstrating a slight elevation in tumor to blood ratios relative to other tissues. Compounds 3 and 4 were shown to compete with thymidine for the same binding site in the transport of nucleosides across the cell membrane in mouse erythrocytes. The inhibition constants (Ki) show that the compounds were weak competitors of thymidine binding to pyrimidine nucleoside transporter compared to physiological nucleosides. Other evidence indicates that compounds 3 and 4 are not substrates for mammalian kinase enzymes.

Synthesis and biological activity of 5-fluoro-2',3'-dideoxy-3'-fluorouridine and its 5'-phosphate.

5-Fluoro-2',3'-dideoxy-3'-fluorouridine (3'-FFdUrd) and 5-fluoro-2',3'-dideoxy-3'-fluorouridine 5'-phosphate (3'-FFdUMP) have been synthesized, and their interactions with thymidine (dThd) phosphorylase and thymidylate (dTMP) synthetase, respectively, have been examined. 3'-FFdUrd is not a substrate for dThd phosphorylase, but is a weak, noncompetitive inhibitor (Ki = 1.7 mM). 3'-FFdUMP inhibits dTMP synthetase competitively with deoxyuridylate (Ki = 0.13 mM) when both the substrate and inhibitor are present simultaneously. However, in the presence of 5,10-methylenetetrahydrofolate, the inhibition increases with time in a first-order manner (konobsd = 0.029 s-1). A complex is formed between [6-3H]3'-FFdUMP and dTMP synthetase, which is isolable on nitrocellulose filters, and has a dissociation rate (koffobsd = 1.4 X 10(-2) min-1) similar to that of the potent inhibitor 5-fluoro-2'-deoxyuridylate (koffobsd = 1.3 X 10(-2) min-1) from its ternary complex with dTMP synthetase. These results are explained in terms of a two-stage model involving the initial formation of a reversible adsorption complex, followed by a slow conversion to a tight-binding catalytic complex.

Synthesis and biological evaluation of 5-fluoro-2'-deoxyuridine phosphoramidate analogs.

A series of alkylating phosphoramidate analogs of 5-fluoro-2'-deoxyuridine has been prepared and their growth inhibitory activity evaluated against murine L1210 leukemia and B16 melanoma cells in vitro. These compounds were designed to undergo intracellular release of the phosphoramidate anions, which it was hoped would function as irreversible inhibitors of thymidylate synthase. The expectation was that binding of the nucleoside moiety would be followed by alkylation of the enzyme via the phosphoramidate. The chloride, bromide, iodide, and tosylate analogs were highly potent inhibitors of L1210 cell proliferation, with increased inhibition observed at both higher drug concentrations and longer exposure times. Addition of thymidine completely reversed the inhibition for all compounds, suggesting that these compounds are acting via inhibition of thymidylate synthase. Although the nonalkylating morpholine analog 1f was ca. 50-fold less potent than the methyl(chloroethyl)amino compound, the piperidine analog 1g was only 2-fold less potent, confirming that nitrogen basicity may be as important as the presence of an alkylating group. Addition of thymidine reversed the growth inhibition of the morpholine and piperidine analogs, suggesting that these compounds may also undergo intracellular conversion to 5-fluoro-2'-deoxyuridine 5'monophosphate. The thymidine and deoxyuridine derivatives 2 and 3 showed minimal growth inhibition in the L1210 assay. The alkylating analogs showed modest cytotoxicity against B16 melanoma cells, and the potency of the analogs was more dependent upon the alkylating moiety than on the 5-substituent.

Synthesis and tumor uptake of 5-halo-1-(2'-fluoro-2'-deoxy-beta-D-ribofuranosyl)[2-14C]uracils.

A synthesis of 5-chloro- and 5-fluoro-1-(2'-fluoro-2'-deoxy-beta-D-ribofuranosyl)uracil (4a and 4b) and their 2-14C analogues has been developed. The tissue distribution of these radiolabeled compounds in BDF1 mice bearing Lewis lung tumors has been investigated. Compounds 4a and 4b undergo rapid blood clearance and urinary excretion. Selective retention of radioactivity was observed in tumor tissue, spleen, and intestine and with compound 4b also in the bone. Maximum tumor to blood ratios of 4.2 for the 5-chloro compound 4a and 10.3 for the 5-fluoro compound 4b were observed at 4h. These compounds were resistant to phosphorylytic cleavage and dehalogenation as indicated by the metabolic products observed in the urine and the absence of radioactivity in the liver. The interaction of 4b with the mouse erythrocyte transporter system was compared with physiological nucleosides in respect to ability to effect zero-trans influx of thymidine. The results show a competitive inhibition between 4b and the natural nucleoside. Evidence is presented for the direct metabolic defluorination of 5-fluorouracil to form uracil.

Synthesis and interaction with uridine phosphorylase of 5'-deoxy-4',5-difluorouridine, a new prodrug of 5-fluorouracil.

5'-Deoxy-4',5-difluorouridine (4'-F-5'-dFUrd) (10) has been synthesized on the basis of the rationale that the labilization of the glycosidic linkage caused by the 4'-fluoro substituent might allow this compound to be a better prodrug form of the anticancer drug 5-fluorouracil (FUra) than is the widely studied fluoropyrimidine 5'-deoxy-5-fluorouridine (5'-dFUrd). The rate of solvolytic hydrolysis of the glycosidic linkage of 4'-F-5'-dFUrd at pH 1 was about 500-fold faster than that of 5'-dFUrd. Since uridine phosphorylase is thought to be the enzyme that causes degradation of 5'-dFUrd in vivo to generate FUra, we compared the substrate interactions of 5'-dFUrd and 4'-F-5'-dUrd with this enzyme. The Vmax for hydrolysis of 4'-F-5'-dFUrd to FUra by uridine phosphorylase was about 5-fold greater than that of 5'-dFUrd, whereas the Km value of 4'-F-5'-dFUrd was 10-fold lower. The combination of these two factors results in 4'-F-5'-dFUrd having a 50-fold higher value of V/K than does 5'-dFUrd. Against L1210 cells in culture, the IC50 value for growth inhibition by 4'-F-5'-dFUrd was 3 X 10(-7) compared to 3 X 10(-6) for 5'-dFUrd.

Synthesis and biological activity of novel 5-fluoro-2'-deoxyuridine phosphoramidate prodrugs.

A series of novel haloethyl and piperidyl phosphoramidate FdUMP prodrug analogues has been synthesized, and the growth inhibitory activity of these compounds has been evaluated against L1210 mouse leukemia cells. All compounds exhibited potent inhibition of L1210 cell proliferation with IC(50) values in the nanomolar range. Growth inhibition was reversed by the addition of 5 microM thymidine, suggesting a mechanism of action involving the intracellular release of FdUMP. (31)P NMR studies carried out on model haloethyl phosphoramidates confirm the release of nucleotide via cyclization of the phosphoramidate anion to the aziridinium ion intermediate followed by hydrolysis of the P-N bond. The data suggests that <50% of the prodrug is converted to FdUMP intracellularly by this pathway. Piperidyl phosphoramidate analogues are also converted to nucleotide intracellularly, presumably by the action of an endogenous phosphoramidase.

Synthesis of [76Br]bromofluorodeoxyuridine and its validation with regard to uptake, DNA incorporation, and excretion modulation in rats.

UNLABELLED: This investigation aimed to validate 5-[76Br]bromo-2'-fluoro-2'-deoxyuridine (BFU) as a proliferation marker using PET. METHODS: Five megabecquerels 76Br-BFU were injected into the tail vein of Sprague-Dawley rats. At 6 or 16 h after injection, the rats were killed and the radioactivity concentration was measured in 6 different organs and blood. The fraction of radioactivity incorporated into DNA was determined for the spleen and small intestine. In parallel experiments, the animals were pretreated with hydroxyurea. In a few experiments, the urinary excretion of radioactivity was measured from administration of 76Br-BFU until 6 h. A sample of urine was analyzed with HPLC. In separate experiments, rats were given different doses of cimetidine, and the organ uptake and the fraction of radioactivity in DNA were determined at 24 h. RESULTS: The highest organ uptake of radioactivity was found in the spleen, followed by the small intestine. Approximately 90% of the radioactivity in these organs was incorporated into DNA, and inhibition by hydroxyurea was pronounced. Intact tracer constituted more than 95% of the radioactivity in urine. With cimetidine, the uptake of radioactivity increased approximately 2-5 times at different doses, whereas the urine radioactivity decreased markedly. CONCLUSION: 76Br-BFU was predominantly incorporated into DNA after administration in vivo in rats. If cimetidine was given in combination with the tracer, an increased contrast of radioactivity concentration between organs of high proliferation and organs of low proliferation was observed. The investigation suggested that 76Br-BFU has good potential as a PET tracer for the assessment of proliferation in vivo.